Annual Reports in
MEDICINAL CHEMISTRY VOLUME
44 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in-Chief
JOHN E. MACOR Neuroscience Discovery Chemistry Bristol-Myers Squibb Wallingford, CT, United States Section Editors ROBICHAUD STAMFORD BARRISH MYLES PRIMEAU LOWE DESAI
Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo Academic Press is an imprint of Elsevier
ACADEMIC PRESS
Academic Press is an imprint of Elsevier Linacre House, Jordan Hill, Oxford OX2 8DP, UK 84 Theobald’s Road, London WC1X 8RR, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA
First edition 2009 Copyright r 2009 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. ISBN: 978-0-12-374766-2 ISSN: 0065-7743 For information on all Academic Press publications visit our web site at www.elsevierdirect.com
Printed and bound in USA 09 10 11 12 13
10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Neel K. Anand
339
James N. Leonard
149
Jan D. Andersson
501
Shawn Maddaford
27
Subhash C. Annedi
27
Ju¨rgen Maibaum
105
Gregory S. Bisacchi
379
Carl L. Manthey
211
Robert M. Borzilleri
301
Dennis J. McCarthy
501
Bruce D. Car
555
Nicholas A. Meanwell
397
Karen Miller-Moslin
323
Terry W. Moore
443
Bernard P. Murray
535
379
John M. Nuss
339
Charles S. Elmore
515
Brian T. O’Neill
David L. Feldman
105
Stefan Peukert
323
William R. Foster
555
M. Edward Pierson
501
Robert G. Gentles
3
William J. Pitts
247
Christer Halldin
501
Mark R. Player
211
Sangdon Han
227
Mimi L. Quan
189
51
Suman Rakhit
27
Jotham W. Coe John Gaetano D’Angelo Gene M. Dubowchik Jacques Dumas
Simon N. Haydar
71 359 3
71
Shridhar Hegde
577
Jailall Ramnauth
27
Christopher Paul Hencken
359
Hans Rollema
71
Warren D. Hirst
51
Stefan U. Ruepp
555
Shuanghua Hu
3
Thomas Ryckmans
129
Arthur T. Sands
475
397
Michelle Schmidt
577
Alvin Solomon Kalinda
359
Paul M. Scola
397
Rao Kalla
265
Joanne M. Smallheer
189
John A. Katzenellenbogen
443
Roland G.W. Staal
Robert M. Jones John F. Kadow
149, 227
51
xv
xvi
Contributors
Nicole van Straten
171
Dolatrai M. Vyas
281
Jayant Thatte
227
Stephen T. Wrobleski
247
Thomas N. Thompson
459
Mark D. Wittman
281
Marco Timmers
171
Heedong Yun
51
Amy Lew Tsuhako
339
Jeff Zablocki
265
Upender Velaparthi
281
Brian P. Zambrowicz
475
Gregory D. Vite
301
PREFACE
Annual Reports in Medicinal Chemistry has reached Volume 44. I hope that it continues to be the review resource for medicinal chemists. Volume 44 continues the traditions of Annual Reports in Medicinal Chemistry with 28 chapters covering the themes of central nervous system disease, cardiovascular and metabolic diseases, inflammation/pulmonary/gastrointestinal (GI), oncology, infectious disease, topics in biology, topics in drug design and discovery and finally our review of new drugs introduced in 2008 in the ‘‘To Market, To Market’’ section. I am particularly pleased that Volume 44 contains three case histories detailing the discovery and development of varenicline for smoking cessation, aliskerin for hypertension and ixabepilinone for cancer. We will continue to seek case histories for future volumes because I believe these successes are some of the most instructive stories in medicinal chemistry. It is also gratifying to see the breadth of the sources of the reviews in Volume 44. My colleagues at Bristol-Myers Squibb continue to embrace the series with seven contributions in Volume 44. Scientists from Pfizer and AstraZeneca each contributed three chapters to Volume 44, while Arena Pharmaceuticals and Novartis each contributed two chapters. Wyeth, Neuraxon, Schering-Plough, Johnson&Johnson, CV Therapeutics, Exelixis, Lexicon Pharmaceuticals and Gilead scientists each contributed one chapter to Volume 44. Finally, chapters from Alfred University, the University of Illinois and an individual consultant completed the line up for Volume 44. I will continue to look to increase the diversity of contributing organizations and urge those organizations with significant medicinal chemistry resources who have not contributed recently to ‘‘step up to the plate’’ and contribute to this community resource known as Annual Reports in Medicinal Chemistry. Although we all worry about the mergers and resulting contractions of medicinal chemistry departments, we remain a vibrant science, and I am confident that Annual Reports in Medicinal Chemistry will continue to receive the quality contributions that have defined this series for over 40 years. Putting together Annual Reports in Medicinal Chemistry is a team effort of volunteers, starting with the chapter authors themselves. I thank the contributors to Volume 44 for their dedication and talent. Helping bring
xvii
xviii
Preface
this all together are the Section Editors: Joel Barrish, Manoj Desai, John Lowe, David Myles, John Primeau, Albert Robichaud and Andrew Stamford. I thank them for yet another seamless quality effort. Helping them and me were a team of reviewers/proof readers that have done a spectacular job behind the scenes as well. I acknowledge these reviewers/proof readers by listing their names below as a demonstration of our appreciation for their time and effort. AstraZeneca — Mike Barbachyn and Brian Sherer Bristol-Myers Squibb — Stephen Adams, Joanne Bronson, James Corte, Andrew Degnan, Murali Dhar, Douglas Dischino, James Duan, Carolyn Dzierba, Rick Ewing, Matthew Hill, John Hynes, George Karageorge, Lawrence Marcin, Ivar McDonald, Harold Mastalerz, Michael Miller, Natesan Murugesan, Richard Olson, Kenneth Santone, Michael Sinz, Lawrence Snyder, John Starrett, Drew Thompson, Dolatrai Vyas, Michael Walker and Christopher Zusi Gilead Sciences — Randall Halcomb, Richard Mackman and Will Watkins Novartis — Lawrence Hamann Pfizer — Joe Brady and Joel Morris Schering-Plough — Brian McKittrick Wyeth — Jonathan Gross, Steven O’Neil and Dane Springer Finally, I also acknowledge Shridhar Hedge and Michelle Schmidt for compiling the ‘‘To Market, To Market’’ review once again. I see that section as one of the consistent highlights of the book. And last, I also thank Ms. Catherine Hathaway, my Administrative Assistant, who always keeps things on an even keel. In summary, I hope that Volume 44 of Annual Reports in Medicinal Chemistry continues to be a key reference for your medicinal chemistry pursuits. As Editor-in-Chief, I continue to look for ways to optimize, improve and evolve the series. Please do not hesitate to contact me with suggestions for improving the series (
[email protected]). Thank you. John E. Macor Bristol-Myers Squibb, R&D, Wallingford, CT, USA
CHAPT ER
1 Recent Advances in the Discovery of GSK-3 Inhibitors and a Perspective on their Utility for the Treatment of Alzheimer’s Disease Robert G. Gentles, Shuanghua Hu and Gene M. Dubowchik
Contents
1. 2. 3. 4.
Introduction Etiology of Alzheimer’s Disease Principal Functions of GSK-3 Additional Neuroprotective Indications for GSK-3 Inhibition 5. Selective Functional Inhibition of GSK-3 6. GSK-3 Isoforms 7. GSK-3 Crystal Structures 8. Challenges in the Development of Effective GSK-3 Inhibitors 9. Advances in GSK-3 Inhibitors 9.1 Indirubins 9.2 Maleimides 9.3 Organometallic GSK-3 inhibitors 9.4 Thiadiazolidinones: noncompetitive GSK-3 inhibitors 9.5 Established drugs with GSK-3 inhibitory properties 9.6 Miscellaneous chemotypes 10. Conclusion References
3 4 5 7 8 9 9 9 10 11 13 14 18 19 20 22 22
Bristol Myers Squibb Company, 5 Research Parkway, Wallingford, CT 06492, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04401-7
r 2009 Elsevier Inc. All rights reserved.
3
4
Robert G. Gentles et al.
1. INTRODUCTION Alzheimer’s disease (AD) is the seventh leading cause of death in the United States and the fifth leading cause of mortality in people over the age of 65. Currently more than 5 million suffer from the condition and this number is expected to grow significantly with progressive aging of the population, rising to between 11 and 16 million affected individuals by 2050 [1]. It is estimated that the cost of treating AD and other dementias currently exceeds $148 billion annually in direct and indirect costs, and this is expected to increase dramatically with projected demographic changes. Current therapies are mostly palliative [2], and there exists a significant unmet medical need in the treatment of this devastating condition. AD is a dementia characterized symptomatically by progressive agerelated memory loss and cognitive impairment. Histopathologically, the disease is defined by neuronal loss, the presence of intracellular neurofibrillary tangles (NFTs), the formation of extracellular senile plaques [3], and the occurrence of cerebral amyloid angiopathy (CAA) [4]. NFTs are composed of aggregated hyperphosphorylated forms of the microtubule-associated protein tau [5]. This aggregation is thought to impair intracellular transport mechanisms [6] and may result in neuronal dystrophy [7]. Extracellular plaques are composed of precipitated amyloid beta-peptide (Ab) and are frequently associated with activated microglia, inflammation, and neuronal atrophy [8]. In CAA, Ab is deposited in cerebral arteries where it is associated with increased risk of hemorrhagic stroke [9,10].
2. ETIOLOGY OF ALZHEIMER’S DISEASE The most long-considered theory on the cause of AD is the ‘‘amyloid cascade hypothesis’’ [3,11]. This posits that Ab overproduction leads directly to the formation of senile plaques and CAA [12,13]. The presence of significant degenerative neuronal processes in the area of plaques, and the death of smooth muscle cells in the vicinity of the Ab deposits in cerebral arteries, is highly suggestive of the toxicity of the Ab peptide [14,15]. Additionally, areas of the brain most significantly affected in AD show co-localization between Ab plaques and neuronal cell death [16]. There has been much discussion on the exact form of Ab that may be noxious [17]. Experiments have shown that under some conditions fibrillation of Ab is required to observe neurotoxic effects [18,19]. However, at present, the exact toxic species of Ab has yet to be unambiguously identified.
Recent Advances in the Discovery of GSK-3 Inhibitors
5
An additional aspect of Ab pathology is its induction of hyperphosphorylation of tau [20]. This is thought to be primarily mediated through the activation of glycogen synthase kinase-3 (GSK-3), which has been shown to be responsible for the phosphorylation of key tau epitopes known to be relevant in AD [21,22]. Hyperphosphorylated tau disengages from microtubules, resulting in their structural destabilization with concomitant negative effects on intracellular structures and transport mechanisms [6]. In addition, the uncomplexed hyperphosphorylated tau assembles into paired helical filaments (PHFs) that aggregate to produce the stereotypic intracellular NFTs associated with AD [23,24]. It should be noted that other kinases such as CDK5 are also known to hyperphosphorylate tau and have also been pursued as therapeutic targets [25]. A recently proposed alternative theory to the amyloid cascade hypothesis postulates that GSK-3 may play a more instigative role in the etiology of AD [26]. It has been suggested that aberrant wnt or insulin signaling results in increased GSK-3 function [27–29] and this is responsible for the observed hyperphosphorylation of tau and the formation of PHFs and NFTs. In addition, elevated GSK-3 activity may induce increased Ab formation through its action on g-secretase [30,31], and thereby give rise to the primary stereotypic pathology observed in AD. GSK-3 has also been demonstrated to be involved in mechanisms underlying memory and learning, and dysregulation of GSK-3 function may explain some of the early cognitive deficiencies observed in AD [26]. However, some recent research has suggested the potential for the induction of behavioral deficits if constitutive GSK-3 activity is overly suppressed [32]. While these hypotheses continue to be challenged, refined, and even reconsidered, there are currently no definitive refutations of either theory. Consequently, it seems reasonable to pursue therapeutic approaches focused both on the direct modulation of Ab levels and on the inhibition of GSK-3. Ultimately, the successful treatment of AD may require that multiple treatment options be explored in different patient populations, and that differently targeted therapeutics be exploited either sequentially or in combination.
3. PRINCIPAL FUNCTIONS OF GSK-3 GSK-3 is a proline-directed serine/threonine kinase. It effects the phosphorylation of a range of substrates and is involved in the regulation of numerous diverse cellular functions, including metabolism [33,34], differentiation, proliferation, and apoptosis [35]. GSK-3 is constitutively active, with its basal level of activity being positively modulated by
6
Robert G. Gentles et al.
phosphorylation on Tyr216 [36]. GSK-3 has a unique substrate selectivity profile that is distinguished by the strong preference for the presence of a phosphorylated residue optimally located four amino acids C-terminal to the site of GSK-3 phosphorylation [37]. Most commonly, GSK-3 activity is associated with inducing a loss of substrate function, such that GSK-3 inhibition will frequently result in increased downstream activity. GSK-3 is widely distributed in the brain [38,39] and as discussed above, a principal consequence of its dysregulation is the hyperphosphorylation of the microtubule-associated protein tau [20], as depicted in the bottom-right panel of Figure 1. This function of GSK-3 has been demonstrated both in cell culture [40] and in in vivo studies looking at tau phosphorylation [41] and NFT formation [22,42]. The exact form of GSK3 (complexed or free) responsible for the phosphorylation of tau has not been characterized.
GSK-3: Discrete Cellular Pools Wnt Pathway Non-Stimulated
P
P
P
P
P
P
β-Catenin P
IRS
P
P
AXIN
P
P
CK1
GSK-3
P P
β-Catenin β-Catenin β-Catenin
Z
Glycogen Z Synthase
APC
AXIN
P
GSK-3 Glycogen Synthase
Tau
PKB
Z Phosphatases
Phosphorylation of Tau
Microtubule
P
GSK-3 FRAT
PDK1
PI3K
APC
GSK-3
P
Wnt Pathway Stimulated
Insulin Signaling Pathway
Insulin
β-Catenin
GSK-3
P
Microtubule
Microtubule
P
P
CK1 Disassembles
PHF’s
NFT’s
Figure 1 Key elements in the primary cellular pathways involving GSK-3 discussed in this review. APC, adenomatous polyposis coli; CK1, casein kinase 1; IRS, insulin receptor substrate; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PDK1, phosphoinositide-dependent kinase-1. (See Color Plate 1.1 in Color Plate Section.)
Recent Advances in the Discovery of GSK-3 Inhibitors
7
GSK-3 is also known to play a key role in glucose metabolism, and was first identified as the enzyme responsible for effecting the inhibitory phosphorylation of glycogen synthase [43], the result of which is to reduce the rate of conversion of glucose to glycogen, giving rise to elevations in blood glucose levels. This function of GSK-3 is controlled by insulin, operating through the signaling pathway [44] shown in the topright panel of Figure 1. The net effect of this cellular cascade is inhibition of GSK-3 by a process that exploits features of the substrate selectivity mechanism described above. Binding of insulin to its receptor leads indirectly to the activation of protein kinase B (PKB) and subsequent phosphorylation of a key serine residue in the N-terminal domain of GSK-3 [45]. The phosphorylated N-terminus functions as a pseudosubstrate, folding in a manner such that it occupies the substrate-binding cleft [46,47]. This conformation is stabilized by interactions between the phosphorylated serine and residues on the enzyme that constitute the substrate selectivity pocket. An unrelated mechanism of GSK-3 inhibition operates in the wntsignaling cascade, a cellular pathway involved in controlling cell-fate, differentiation, and proliferation [48,49]. In this system, GSK-3 is complexed with APC, axin, and b-catenin, as well as other proteins [50], as depicted in the top-left panel of Figure 1. This protein assembly is occasionally termed the ‘‘destruction complex’’. When the wnt system is in a non-stimulated state, GSK-3 phosphorylates axin and APC, the effect of which is to create a more tightly associated complex. CK1, which is also associated with this protein assembly, functions in one capacity as a priming kinase for GSK-3 by performing an initial phosphorylation of b-catenin [51]. This protein is then poly-phosphorylated by GSK-3, resulting in its dissociation from the complex and its subsequent ubiquitination and destruction by the proteosome [52], thereby regulating cellular levels of b-catenin [53,54]. However, following binding of wnt ligands to their receptors, axin is displaced from the complex with GSK-3 as a result of binding of the latter to FRAT (frequently rearranged in advanced T-cell lymphomas) peptide [55], see bottom-left panel of Figure 1. This leads to the dissociation of the destruction complex, the consequence of which is that b-catenin is no longer effectively phosphorylated and degraded. Consequently, intracellular concentrations of the protein rise, and there is increased trafficking of b-catenin to the nucleus [56] where it interacts with transcription factors that induce increased expression of wnt regulated genes, which, among other things, are operative in certain neoplastic transformations. It is interesting to note that no crossover in these inhibition mechanisms is thought to occur: GSK-3 mediated phosphorylation of axin and b-catenin is not inhibited by a phosphorylated hexapeptide that is effective at inhibiting GSK-3 phosphorylation of primed substrates
8
Robert G. Gentles et al.
[37], suggesting that N-terminal serine phosphorylation is not operative in controlling GSK-3 activity within the destruction complex. In addition, wnt signaling does not affect the phosphorylation state of uncomplexed GSK-3 [57]. Correspondingly, the effects of insulin or wnt signaling would appear to be limited to discrete cellular pools of GSK-3, the significance of which is discussed below.
4. ADDITIONAL NEUROPROTECTIVE INDICATIONS FOR GSK-3 INHIBITION GSK-3 has been demonstrated to be involved in apoptosis regulation, and it has been suggested that its inhibition can have neuroprotective effects distinct from prevention of tau hyperphosphorylation. Mitochondria, for example, have been found to be susceptible to unregulated GSK-3 activity, resulting in Parkinson’s disease (PD)-like effects [58]. In addition, GSK-3 inhibition can attenuate apoptosis resulting from MPP+ and rotenone challenges that mimic PD neuronal pathology [58]. GSK-3 inhibition has also been implicated as a target for amyotrophic lateral sclerosis (ALS). Highly phosphorylated tau protein, along with upregulated GSK-3 has been observed in ALS with cognitive impairment [59]. A recent study has shown that chronic lithium dosing (vide infra) to ALS patients (2 150 mg/day) significantly delayed disease progression and increased survival time in comparison with those taking riluzole, a neuroprotective agent. In addition, lithium was shown by the same group to be protective in a mutant superoxide dismutase 1 (SOD1) model of ALS [60]. Clinical trials with lithium (alone, and in combination with riluzole) in ALS are ongoing [61].
5. SELECTIVE FUNCTIONAL INHIBITION OF GSK-3 It is apparent that inhibition of GSK-3 may be associated with significant mechanism-based toxicities, potentially ranging from hypoglycemia to tumorigenesis. To successfully develop a GSK-3 inhibitor it may be necessary to identify agents that can selectively inhibit specific cellular functions of GSK-3. How might this be realized? One potential mechanism could involve targeting or obviating the inhibition of the discrete cellular pools described above. It is conceivable that this could be achieved by exploiting subtle differences in the structures of GSK-3 that might arise when the enzyme is associated within different protein assemblies, or when the enzyme is in an uncomplexed form. Alternatively, if inhibitors can be identified that interact in the vicinity of
Recent Advances in the Discovery of GSK-3 Inhibitors
9
the ATP-binding site it may be possible to achieve substrate-selective inhibition as has been observed in the development of gamma secretase inhibitors [62]. Hypothetically, the identification of allosteric inhibitors [63] could afford very different and potentially unique functionally selective inhibition profiles [63], as could the development of isoform-selective GSK-3 inhibitors (vide infra). To date, the primary literature is devoid of such information but it might safely be assumed that acquisition of data of this type is a feature of many preclinical programs.
6. GSK-3 ISOFORMS GSK-3 exists in two isoforms, GSK-3a (51 kDa) and GSK-3b (47 kDa), that share 84% overall identity and greater than 98% identity within their respective catalytic domains [64,65]. A minor splice variant of GSK-3b, denoted as GSK-3b2, has been reported [66]. This isoform has a 13-amino-acid insert within its kinase domain and displays reduced activity toward tau protein. It has been found by immunohistochemistry to be localized predominantly in cell soma, unlike GSK-3b that is found extensively in neuronal processes. Both primary isoforms are ubiquitously expressed [67,68], with particularly high levels observed in the brain and testes. In most brain areas, GSK-3b is the predominant isoform and has become of primary interest as a CNS target. However, it should be noted that some studies suggest that GSK-3a and GSK-3b share very similar, if not entirely redundant functions in a number of cellular processes [69]. Therefore, the utility of an isoform-selective inhibitor could be determined by the particular cellular function targeted, as well as the relative isoform expression levels in the tissue of interest.
7. GSK-3 CRYSTAL STRUCTURES The crystal structure of GSK-3 has been reported [46], as have structures of GSK-3 complexed to components of the wnt-signaling pathway [50]. All these data have provided insight into the mechanism of action of this kinase, the nature of its substrate selectivity, and the unique mechanism of inhibition effected through phosphorylation of the N-terminal serine residue [70]. In addition, a number of co-crystal structures have been published of GSK-3 bound to a diverse set of active site inhibitors [71–73]. These data have been exploited in improving the intrinsic potency of these ligands, as well as suggesting pathways for improved kinase selectivity profiles. More recently, these crystal structures have
10
Robert G. Gentles et al.
been employed in a variety of virtual screening protocols directed at the identification of novel GSK-3 inhibitors [74,75].
8. CHALLENGES IN THE DEVELOPMENT OF EFFECTIVE GSK-3 INHIBITORS In common with all kinase-targeted therapeutics, a key issue to be addressed relates to selectivity. This must be sufficient to avoid off-target toxicities, and this may be more important for CNS-targeted drugs than compounds designed for use in oncology, where targeting multiple kinases can occasionally be advantageous. In general, the broader the kinase counter screen used in determining selectivity, the greater the confidence that this problem can be adequately assessed. However, in the extant GSK-3 literature, limited selectivity data are disclosed beyond that related to phylogenetically related kinases. Historically, this can be attributed to technological limitations in large-scale parallel kinase screening. However, recent developments [76] have largely eliminated such restrictions and more extensive kinase counter-screening data are now beginning to be disclosed [77]. A specific issue for the use of GSK-3 inhibitors in AD is the need to access the CNS compartment. Given the polar nature of many kinase inhibitors it would be desirable to include early assessments of blood brain barrier (BBB) permeability and P-glycoprotein (Pgp) substrate potential. In addition, having suitable brain-to-plasma ratios may be a determinant in achieving an adequate therapeutic index by limiting the peripheral exposure required to drive CNS efficacy. Again, few data in this area have been offered, and most reports are limited to the discussion of biochemical activities with limited disclosures of in vivo studies. With regard to mechanism-based toxicities, the primary concern for GSK-3-targeted therapeutics relates to their potential to induce transformation of nonmalignant cells or exacerbate preexisting malignancies through their actions on b-catenin (vide supra). It is worth recalling however, that lithium has been used as a standard therapeutic for the treatment of bipolar disorder since the 1950’s. This agent is a weak GSK-3 inhibitor, exerting its effect through a mixed mechanism of direct inhibition [78] and activation of PKC-a [79], the latter of which leads to increased GSK-3 Ser9/21 phosphorylation. At therapeutic doses, lithium is estimated to achieve B25% inhibition of total GSK-3 activity, and this degree of inhibition has not yet been associated with increased levels of tumorigenesis or deaths from cancer. This is perhaps the most compelling argument for the potential safety of GSK-3 inhibitors.
Recent Advances in the Discovery of GSK-3 Inhibitors
11
9. ADVANCES IN GSK-3 INHIBITORS Over the past 20 years a number of chemical classes of GSK-3 inhibitors have been discovered [80]. More recently, the structural diversity of compounds reported in this area has expanded considerably, especially in the patent literature. However, in light of the preceding discussion we limit ourselves here to a review of GSK-3 inhibitors on which additional data beyond that of the primary enzyme activity have been disclosed, such that the utility of these compounds in the treatment of AD might be better assessed.
9.1 Indirubins The indirubins are a class of natural product bis-indoles that can be isolated from a variety of plant and animal sources, or alternatively, can be relatively easily synthesized from appropriately functionalized indoles and isatins. They have been demonstrated to possess potent kinase inhibition activities, and considerable efforts over a number of years have resulted in the identification of GSK-3-selective analogs [81]. An issue constraining their biological evaluation is their characteristically low aqueous solubility. Recent work has focused on addressing this limitation while attempting to maintain required potency and selectivity profiles. A number of co-crystal structures of indirubins complexed with various kinases have been reported. These have proved valuable in understanding the molecular features that determine affinity and selectivity, and have contributed to the prioritization of vectors that could be exploited for the modulation of off-target and physicochemical properties. In the case of the co-crystal structure of 6-bromo-indirubin-3u-oxime (6BIO) with GSK-3, the principal interactions between the ligand and the enzyme have been identified [82,83]. From the extant structure activity relationship (SAR) data, it is known that bromine at the 6-position of the indirubin core can be a key determinant of selectivity for GSK-3 over the phylogenetically related cyclin-dependent kinases, CDK1 and CDK5. From the co-crystal data this can be rationalized by noting that bulky substituents at position 6 take advantage of the sterically more accommodating ‘‘gatekeeper’’ residue of GSK-3 (Leu132) compared to the more demanding related residue (Phe80) for the cyclin-dependent kinases (CDKs). Furthermore, from this structure it is apparent that the 3u-oxime projects out from a solvent-accessible cavity and would appear to offer a vector from which to extend solubilizing functionality. This supposition was supported in a series of analogs that incorporated a basic moiety appended with a hydrocarbon linker bound to the oxygen atom of the oxime [84]. A diverse range of groups were examined and the molecules were evaluated for potency against GSK-3, CDK1/cyclinB, and CDK5/p25 and for cytotoxicity
12
Robert G. Gentles et al.
in an SH-SY5Y cell line. Compound 1 (GSK-3, IC50 5 nM; CDK1/cyclinB, IC50 600 nM; and CDK5/p25, IC50 500 nM), compound 2 (GSK-3, IC50 11 nM; CDK1/cyclinB, IC50 2,800 nM, and CDK5/p25, IC50 300 nM), and compound 3 (GSK-3, IC50 14 nM; CDK1/cyclinB, IC50 900 nM; and CDK5/ p25, IC50 310 nM), all displayed similar or improved potency and selectivity profiles relative to 6BIO (GSK-3, IC50 5 nM; CDK1/cyclinB, IC50 320 nM; and CDK5/p25, IC50 83 nM), and all were significantly less cytotoxic against an SH-SY5Y cell line. N Br
HO
N
Br
O
N
N HO NH
N H
N H
O 6BIO
O
(1)
N
N N
NH
Br
O
N
Br
O N
N O
O N H (2)
NH O
OH
N H
NH O
(3)
The cellular activity of this compound class was confirmed in a b-catenin reporter assay where 1 (IC50 700 nM) was demonstrated to be approximately equipotent to 6BIO (IC50 300 nM). Additionally, selected compounds also displayed activity in a cell culture luminescence assay of GSK-3-mediated circadian rhythmicity. Importantly, all of the above analogs were dramatically more soluble (0.1–4.2 mg/mL) than the parent bromo-indirubin (o5 mg/mL), a feature that should significantly facilitate the biological assessment of this compound class. In an unrelated study, a series of indirubins were prepared that were independently functionalized at positions 5- and 3u-, as depicted in the structures given below [85].
13
Recent Advances in the Discovery of GSK-3 Inhibitors
4′
O
HNAc
O2N
5
HO
6
4
O
N 7
5′
3′
6′ 7′
N H
NH 1
O
(4)
N H (5)
NH O
N H
NH O
(6)
Of the many reported analogs, only compounds 5 (GSK-3, IC50 2 nM; CDK1/cyclinB, IC50 19 nM; and CDK5/p25, IC50 6 nM) and 6 (GSK-3, IC50 7 nM; CDK1/cyclinB, IC50 50 nM; and CDK5/p25, IC50 18 nM) displayed single-digit nanomolar activity, and none of the analogs were particularly selective for GSK-3. Interestingly however, there appeared to be interdependent SARs at the positions explored, with the optimal group at one vector being dependent on the functionality incorporated at the other positions explored. This would argue that a matrix-based optimization of this chemotype could lead to further enhancements in potency and selectivity.
9.2 Maleimides The natural product staurosporine (7) [86] is a broadly promiscuous polycyclic kinase inhibitor. The related bis(indolyl)maleimide ruboxistaurin (8), although less conformationally constrained, is relatively specific in its inhibition of PKC-b, having a reported IC50 of 5 nM and no other significant activity in a broad kinase counter screen [87]. This selectivity has been attributed in part to the nonplanar disposition of the two indole moieties in 8, and recently reported work [73] has focused on the synthesis of analogs of 8 that exploit a cyclophane structure, as shown in compounds 9 through 14. These molecules also present a nonplanar arrangement of the bis-indolyl moiety, but in a more constrained ‘‘pyridinophane’’ cycle, and it was anticipated that this might favorably impact the potency and selectivity profiles previously observed with other macrocyclic bis(indolyl)maleimides.
14
Robert G. Gentles et al.
H N
H N
O
O
O R1
N
N
N
N
O R3
Ru
R2 O
MeO HN (7)
(15) R1 = R2 = R3 = H (16) R1 = OH, R2 = Br, R3 = CO2CH3
Expansion of the 14-membered macrocycle in ruboxistaurin to 16–22membered rings has previously been shown to not only retain excellent PKC-b inhibitory activity but also introduce significant activity against GSK-3. Contrastingly, the larger ring pyridinophanes 9 through 14 all display potent GSK-3 activity and are highly selective against PKC-b. For example, compound 13 inhibited GSK-3b and PKC-bII with IC50 values of 3 and 1,400 nM, respectively. This potency and selectivity profile had not previously been reported for any of the known bis(indolyl)maleimides. To further assess the broader kinase selectivity of this novel series of pyridinophanes, compounds 9, 10, and 12 were screened against a panel of 100 kinases. With the exception of the GSK-3a and GSK-b isozymes, the only other significant activities observed were against MSK1, PKC-y, and Rsk3. More specifically, compound 13 had IC50 values against these kinases of 510, 98, and 48 nM, respectively. A co-crystal structure of 13 with GSK-3b was obtained from which it was noted that the maleimide moiety makes key hydrogen bonds with the hinge residues, Asp133 and Val135, and an additional hydrogen bond through a bridging water molecule to Asp200. A number of hydrophobic interactions were observed, but interestingly, the 2-dimethylaminopyridine group is largely solvent exposed and does not interact directly with the enzyme, suggesting a site for further modification [73].
9.3 Organometallic GSK-3 inhibitors Organometallics have been used for a considerable time as pharmaceutical agents, especially in the oncology arena [88]. More recently, interest has grown in the use of transition metal complexes in chemical genetics, where it is believed that they may access unexplored chemical space, and thereby present opportunities for the design of small molecules with novel biological properties [89]. More specifically, given their unique
Recent Advances in the Discovery of GSK-3 Inhibitors
15
geometries, these compounds may be useful additions to the tools available to address some of the unanswered questions raised in the introduction of this review. In the area of kinase inhibitors, metal complexes are an expanding area of interest, and several highly potent and selective GSK-3 inhibitors have recently been disclosed. In all of the complexes discussed, the metals are thought to be biologically inert and function solely by preorganizing their ligands to present a complementary surface to cognate proteins. This can be appreciated by comparing the structurally complex staurosporine 7, with the simpler ruthenium complex 15, in which the indolocarbazole alkaloid scaffold has been replaced with a somewhat simpler bidentate pyridocarbazole ligand that retains the main features of the indolocarbazole aglycone [90]. The remaining metal ligands project into areas occupied by the glycoside element of staurosporine, and both structures have related overall topologies. Importantly, these molecular geometries are significantly easier to explore with transition metal complexes than they are with many organic macrocycles that are typically accessed by extended syntheses. Additionally, the complexes reviewed here have been shown to be stable in air and aqueous solution, and are tolerant of millimolar concentrations of thiols. H N
O
N O
Ru
O
N Cl NH
(17)
The racemate of the ruthenium complex 15 has IC50 values for GSK-3a and GSK-3b of 20 and 50 nM (100 mm ATP), respectively. In addition, it was demonstrated to inhibit Pim-1 kinase with an IC50 of 3 nM, as well as MSK1 (IC50 120 nM), nMRsk1 (IC50 300 nM), and TrkA (IC50 70 nM), but did not significantly inhibit other kinases in a panel of 50 isozymes explored, including the phylogenetically related CDKs [90]. To address these cross-reactivities, a limited SAR study resulted in the identification of compound 16 that was shown to be selective for GSK-3
16
Robert G. Gentles et al.
when screened in a panel of 57 kinases. This compound was resolved by chiral HPLC and the R isomer was highly potent against GSK-3a and GSK-3b (IC50 350 and 550 pM, respectively), and the previously noted activities against Pim-1, MSK1, nMRsk1, and TrkA were all significantly attenuated. Interestingly, the S isomer has the reverse selectivity for Pim1 over GSK-3 [90]. The complex 16 is configurationally stable in solution when stored in the freezer, but can be expected to slowly epimerize under physiological conditions. Of note is the fact that the uncomplexed pyridocarbazole ligand of 16 is a more potent Pim-1 inhibitor (IC50 10 nM) than GSK-3a (IC50 30 nM) or GSK-3b inhibitor (IC50 50 nM). Consequently, complexation of this ligand to the ruthenium half-sandwich fragment of 16 increases the potency for GSK-3 by more than an order of magnitude, and reverses the selectivity profile for GSK-3 over Pim-1 [89]. The cellular activity of (R)-16 was determined in a b-catenin-dependent luminescence reporter assay, in which the degree of luminescence relates to cellular b-catenin levels. Activity was observed at 30 nM and a luminescence increase factor of 700 was determined, indicative of significant b-catenin stabilization. This level of potency was significantly greater than that observed with the longer-studied GSK-3 inhibitor 6BIO, and confirmed the ability of these complexes to permeate cells and inhibit GSK-3, including the fraction of GSK-3 bound in the b-catenin destruction complex. Zebrafish embryos that were exposed to micromolar concentrations of (R)-16 demonstrated a number of developmental abnormalities, strongly suggesting that effective functional inhibition of GSK-3 had been achieved in vivo [89]. In an interesting extension to this work, a series of octahedral ruthenium complexes incorporating a bidentate pyridocarbazole in combination with a diversity of other ligands were explored for activity against GSK-3a, Pim-1, MSK1, and CDK2/cyclinA [91]. When bound to a target kinase, the pyridocarbazole makes key H-bond contacts with the target enzyme, and the metal and its remaining ligands occupy the approximately spherical ribose pocket of the enzyme’s active site. In the case of octahedral complexes, the choices of substituents on the four valences projecting into the sugar pocket are key determinants of selectivity and, as such, achieve this in a different fashion compared to many organic inhibitors. Of the octahedral complexes explored, compound 17 was identified as a potent GSK-3a inhibitor (IC50 8 nM) and was 10-fold selective over Pim-1.
Recent Advances in the Discovery of GSK-3 Inhibitors
H N
O
17
O
HO F O
N
N Ru
NH
O
O OH (18)
A space-filling model of 17 showed that the ruthenium atom is buried within the complex and is not able to directly interact with target proteins. As previously noted, the metal functions primarily to organize its coordinated ligands into specific and well-defined geometries. In addition, complexes such as 17 are relatively rigid and kinetically inert under physiological conditions. The most interesting feature of compound 17 is its observed selectivity for GSK-3a (IC50 8 nM) over GSK3b (IC50 50 nM). Selectivity between these isozymes is somewhat surprising given the high homology of their active sites (W97%) and has not been previously reported. Understanding the structural features contributing to this differential activity would be a valuable addition to the field. More recent studies directed at the further optimization of ruthenium-based GSK-3 inhibitors resulted in the identification of an extremely high-affinity GSK-3 inhibitor [92]. The complex (RRu)-NP549 (18) inhibits GSK-3b with a Ki of 5 pM and is one of the most potent kinase inhibitors yet reported. H N
O
O
H N
O
O
OH N
N Ru
N
N Os
O
(19)
OH
O
(20)
A co-crystal of (RRu)-NP549 and GSK-3b has been obtained and shows a remarkable complementarity between the surfaces of the
18
Robert G. Gentles et al.
ruthenium complex and the ATP-binding site of GSK-3. Nearly all of the polar functionality in 18 makes productive H-bonds with the enzyme. Interestingly, the CO ligand contacts the glycine-rich loop and is buried in a small pocket, contributing to the affinity and selectivity of this compound. In an unrelated study, the activities of a pair of ruthenium 19 and osmium 20 congeners of 18 were compared [93]. Both analogs were evaluated in a variety of biochemical and cell assays and had essentially indistinguishable properties. Both 19 and 20 were highly active at GSK-3b (IC50 1.4 and 0.6 nM, respectively), and both induced strong and essentially identical apoptotic effects in 1205 Lu melanoma cells. This effect is thought to be attributable to GSK-3 inhibition that produces p53-induced apoptosis. The equivalence of these ruthenium and osmium complexes in a cytotoxicity assay using 1205 Lu cells is noteworthy, as such exchange of one metal with its higher periodic table congener is not normally tolerated in many classes of cytotoxics. R2 N
S O
O
N R1
(21) R1 = Bn, R2 = Me: NP031112 (22) R1 = Ph, R2 = Et: NP01138 (23) R1 = Bn, R2 = Bn: NP00111
A co-crystal structure of the osmium analog 20 complexed to Pim-1 was obtained and displayed a binding mode similar to that for related ruthenium complexes. Not surprisingly, when 19 was modeled into this structure, the two analogs were essentially indistinguishable. This is consistent with their almost identical biological properties and with the hypothesis that the metals in these complexes function primarily as structural components and do not participate in direct interactions with target proteins.
9.4 Thiadiazolidinones: noncompetitive GSK-3 inhibitors Kinase inhibitors that are non-ATP-competitive are potentially attractive for several reasons. By not having to compete with endogenous ATP, they may show better cellular and in vivo potency in comparison with competitive inhibitors having comparable ADME properties. In addition, much better kinase selectivity may be expected from inhibitors that bind outside of the ATP pocket. This may be especially attractive for CNS
Recent Advances in the Discovery of GSK-3 Inhibitors
19
indications where target overlap is probably less desirable than it can be for anticancer therapies. A series of 1,2,4-thiadiazole-3,5-diones (TDZDs) have been described, examples of which appear to be noncompetitive with ATP [63]. The initial lead compound in this series, TDZD-8 (NP031112, 21), showed modest GSK-3b inhibition (IC50 2 mM), noncompetitive enzyme kinetics, and good selectivity against a limited set of other kinases (CDK1, CK2, PKA, and PKC). Putative binding sites, both inside and outside the ATP pocket, have been suggested through a combination of 3D-SAR and molecular docking analyses [94]. Dose-dependent reduction of tau phosphorylation in human SHSY5Y neuroblastoma cells has been reported in conference presentations (IC50 ¼ 51 mM). NP031112 was tested in GSK-3b conditional, and hAPPx tau transgenic mouse models (100–200 mg/kg/day, PO for 3 weeks and 3 months, respectively). Following these prolonged treatments, cognitive function was significantly improved and neuronal loss, amyloid deposition, tau phosphorylation, and neuroinflammation were reduced [95]. O H3CO H3CO N (24)
Further studies suggest that NP031112, as well as related compounds NP01138 (22) and NP00111 (23), may have more pronounced neuroprotective effects that may operate through alternate mechanisms, such as peroxisome proliferator-activated receptor g (PPARg) modulation under excitotoxic conditions [96]. Company reports state that NP031112 began phase I clinical trials for AD in April 2006.
9.5 Established drugs with GSK-3 inhibitory properties Donepezil (Aricepts, 24) is an acetylcholinesterase inhibitor that is widely used for the treatment of cognitive and behavioral symptoms of mild-to-moderate AD. Although its effect on disease progression has been a source of controversy, a clinical study has suggested that it may have benefit in severe AD patients [97]. A recent study has demonstrated GSK-3 inhibition in primary cortical neuron cultures challenged with Ab (20 mM) and treated with donepezil, resulting in neuroprotection (dosedependent, from 0.1 to 10 mM) and reduced tau phosphorylation (10 mM) [98]. The presence of a PI3 kinase inhibitor (LY294002) abolished activity,
20
Robert G. Gentles et al.
while a nicotinic acetylcholine blocker, mecamylamine, only partially reversed neuroprotection, suggesting that GSK-3 inhibition results from blockade of inhibitory phosphorylation. CN N
S
N NH N
NH
N
S
HN
N
N (25)
(26)
Olanzapine (25) is an atypical antipsychotic that is associated with weight gain and disturbances in glucose metabolism. A recent study demonstrated GSK-3 inhibition in brains of mice treated with atypical antipsychotics [99]. One group decided to look at direct inhibition of GSK-3 by 25, first by in silico molecular docking experiments utilizing the published co-crystallized structure of the known GSK-3 inhibitor AR-A014418, and then by enzyme assay [100]. Modeling suggested a good fit in the ATP pocket and, indeed, olanzapine was a fairly potent inhibitor of GSK-3b (IC50 91 nM). O F
OH
OH
N
HN
N
N
N
O Cl
N
NH2
(27)
O
(28)
N
Recent Advances in the Discovery of GSK-3 Inhibitors
21
O O HN S
NH2 N N
O
N
(29)
The same group carried out wider in silico docking studies on a structural database of established drugs and identified the following as potent inhibitors [101]: cimetidine (26) (IC50 13 nM), hydroxychloroquine (27) (IC50 33 nM), and gemifloxacin (28) (IC50 88 nM).
9.6 Miscellaneous chemotypes 9.6.1 Furopyrimdines A furopyrimidine scaffold that had previously yielded potent VEGFR2 and Tie-2 inhibitors was modified to provide a potent series of GSK-3 inhibitors [102]. This was done by rational design following analysis of the published binding modes (revealed by X-ray crystallography) of analogous aza- and diazaindazoles. An exemplary compound 29 demonstrated good binding affinity (IC50 30 nM), with good-to-excellent selectivity against a panel of nine other kinases including CDK2. Critical to GSK-3 selectivity and potency was the 3-pyridyl group whose nitrogen is expected to form a hydrogen bond with K85.
CN
F
H3CO
OCH3
S
O O
S
N
O
O
N
N N
N N (30)
(31)
9.6.2 1,3,4-Oxadiazoles A high-throughput screening effort resulted in the identification of a 2-aryl-1,3,4-oxadiazole hit, 30, with double-digit nanomolar potency against GSK-3b (IC50 65 nM) [71]. Subsequent optimization, guided by an
22
Robert G. Gentles et al.
X-ray structure of 30 in the GSK-3 ATP pocket, led to 31, a highly potent inhibitor (IC50 2.3 nM) with W1,000-fold selectivity against a panel of 23 other kinases, including CDK2. In a rat oral PK study, compound 31 showed low oral bioavailability (F : 1.7%), reportedly due to limited intestinal absorption.
F3C HN
N N
NH
HO OH (32)
9.6.3 2,5-Diaminopyrimidines One group’s recent screening efforts led to identification of a purinone scaffold that proved difficult to progress. However, investigation of synthetic intermediates led to a small series of adamantylaminopyrimidines that showed good GSK-3 inhibition. Compound 32 (GSK-3b IC50 41 nM) also showed good oral bioavailability in rats (F : 34%) [103]. N N
H N
Cl
N H
OH
N
N
(33)
9.6.4 Amino-1,3,5-triazines In a recent study, a series of biheteroaryl triazine CDK inhibitors were prepared and examined as potential antitumor agents [104]. The most potent compound in this class, 33 (CDK1, IC50 21 nM; CDK2, IC50 7 nM; and CDK5, IC50 3 nM), was similarly active against GSK-3b (IC50 20 nM), but fairly selective against a panel of 12 other kinases.
Recent Advances in the Discovery of GSK-3 Inhibitors
23
10. CONCLUSION In addition to the compounds discussed above, the patent literature is replete with examples of structures reported to be active at GSK-3, and it would appear reasonable to assume that compounds with sufficient potency, kinase-selectivity, and appropriate physicochemical properties can be identified to evaluate their use in the treatment of AD. However, the key outstanding issue to be addressed remains mechanism-based toxicity, and whether this can be avoided. This is unlikely to be known until a number of analogs (with a variety of inhibitory profiles) are comprehensively tested in vivo or significant clinical progress is reported [80].
REFERENCES [1] [2] [3] [4]
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
2009 Alzheimer’s Disease Facts and Figures. Alzheimer’s & Dementia, 2009, 5, 30. R. Gilstad John and E. Finucane Thomas, J. Am. Geriatr. Soc., 2008, 56, 1556. J. Hardy, J. Alzheimer’s Dis., 2006, 9(Suppl. 3), 151. J. Zaghi, B. Goldenson, M. Inayathullah, A. S. Lossinsky, A. Masoumi, H. Avagyan, M. Mahanian, M. Bernas, M. Weinand, M. J. Rosenthal, A. Espinosa-Jeffrey, J. Vellis, D. B. Teplow and M. Fiala, Acta Neuropathol., 2009, 117, 111. I. Grundke-Iqbal, K. Iqbal, M. Quinlan, Y. C. Tung, M. S. Zaidi and H. M. Wisniewski, J. Biol. Chem., 1986, 261, 6084. H. Stebbings, Cytoskeleton, 1996, 2, 113. M. L. Michaelis, S. Ansar, Y. Chen, E. R. Reiff, K. I. Seyb, R. H. Himes, K. L. Audus and G. I. Georg, J. Pharmacol. Exp. Ther., 2005, 312, 659. K. Vehmas Anne, H. Kawas Claudia, F. Stewart Walter and C. Troncoso Juan, Neurobiol. Aging, 2003, 24, 321. C. G. Dotti and B. De Strooper, Nat. Cell Biol., 2009, 11, 114. K. Jellinger, J. Neurol., 1977, 214, 195. J. A. Hardy and G. A. Higgins, Science, 1992, 256, 184. G. G. Glenner and C. W. Wong, Biochem. Biophys. Res. Commun., 1984, 120, 885. C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald and K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 4245. J. W. Geddes, K. J. Anderson and C. W. Cotman, Exp. Neurol., 1986, 94, 767. M. Kawai, P. Cras and G. Perry, Brain Res., 1992, 592, 278. J. Rogers and J. H. Morrison, J. Neurosci., 1985, 5, 2801. J. B. Standridge and E. M. Welsh (eds), Trends in Alzheimer’s Disease Research, Nova Science Publishers, Hauppauge, NY, 2006, pp. 53–96. B. A. Yankner, Nat. Med., 1996, 2, 850. A. Lorenzo and B. A. Yankner, Ann. N.Y. Acad. Sci., 1996, 777, 89, Neurobiology of Alzheimers Disease. A. Ferreira, Q. Lu, L. Orecchio and K. S. Kosik, Mol. Cell. Neurosci., 1997, 9, 220. P. Delobel, S. Flament, M. Hamdane, A. Delacourte, J. P. Vilain and L. Buee, FEBS Lett., 2002, 516, 151. K. Leroy, Z. Yilmaz and J. P. Brion, Neuropathol. Appl. Neurobiol., 2007, 33, 43. K. Iqbal and I. Grundke-Iqbal, J. Alzheimer’s Dis., 2006, 9(Suppl. 3), 219.
24
Robert G. Gentles et al.
[24] C. M. Wischik, M. Novak, P. C. Edwards, A. Klug, W. Tichelaar and R. A. Crowther, Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 4884. [25] M. A. Glicksman, G. D. Cuny, M. Liu, B. Dobson, K. Auerbach, R. L. Stein and K. S. Kosik, Curr. Alzheimer Res., 2007, 4, 547. [26] C. Hooper, R. Killick and S. Lovestone, J. Neurochem., 2008, 104, 1433. [27] G. J. Biessels and L. J. Kappelle, Biochem. Soc. Trans., 2005, 33, 1041. [28] G. V. De Ferrari, A. Papassotiropoulos, T. Biechele, F. W. De-Vrieze, M. E. Avila, M. B. Major, A. Myers, K. Saez, J. P. Henriquez, A. Zhao, M. A. Wollmer, R. M. Nitsch, C. Hock, C. M. Morris, J. Hardy and R. T. Moon, Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 9434. [29] E. M. Reiman, J. A. Webster, A. J. Myers, J. Hardy, T. Dunckley, V. L. Zismann, K. D. Joshipura, J. V. Pearson, D. Hu-Lince, M. J. Huentelman, D. W. Craig, K. D. Coon, W. S. Liang, R. H. Herbert, T. Beach, K. C. Rohrer, A. S. Zhao, D. Leung, L. Bryden, L. Marlowe, M. Kaleem, D. Mastroeni, A. Grover, C. B. Heward, R. Ravid, J. Rogers, M. L. Hutton, S. Melquist, R. C. Petersen, G. E. Alexander, R. J. Caselli, W. Kukull, A. Papassotiropoulos and D. A. Stephan, Neuron, 2007, 54, 713. [30] C. J. Phiel, C. A. Wilson, V. M. Y. Lee and P. S. Klein, Nature (London), 2003, 423, 435. [31] X. Sun, S. Sato, O. Murayama, M. Murayama, J. M. Park, H. Yamaguchi and A. Takashima, Neurosci. Lett., 2002, 321, 61. [32] S. Hu, N. Begum Aynun, R. Jones Mychica, S. Oh Mike, K. Beech Walter, H. Beech Beverly, F. Yang, P. Chen, J. Ubeda Oliver, C. Kim Peter, P. Davies, Q. Ma, M. Cole Greg and A. Frautschy Sally, Neurobiol. Dis., 2009, 33, 193. [33] G. I. Welsh, C. M. Miller, A. J. Loughlin, N. T. Price and C. G. Proud, FEBS Lett., 1998, 421, 125. [34] G. I. Welsh and C. G. Proud, Biochem. J., 1993, 294(Pt 3), 625. [35] S. Kaku, S. Chaki and M. Muramatsu, Curr. Signal Transduct. Ther., 2008, 3, 195. [36] R. V. Bhat, J. Shanley, M. P. Correll, W. E. Fieles, R. A. Keith, C. W. Scott and C.-M. Lee, Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 11074. [37] S. Frame, P. Cohen and R. M. Biondi, Mol. Cell, 2001, 7, 1321. [38] J. J. Pei, T. Tanaka, Y. C. Tung, E. Braak, K. Iqbal and I. Grundke-Iqbal, J. Neuropathol. Exp. Neurol., 1997, 56, 70. [39] J.-J. Pei, E. Braak, H. Braak, I. Grundke-Iqbal, K. Iqbal, B. Winblad and R. F. Cowburn, J. Neuropathol. Exp. Neurol., 1999, 58, 1010. [40] B. R. Sperber, S. Leight, M. Goedert and V. M. Y. Lee, Neurosci. Lett., 1995, 197, 149. [41] K. Spittaels, C. Van den Haute, J. Van Dorpe, H. Geerts, M. Mercken, K. Bruynseels, R. Lasrado, K. Vandezande, I. Laenen, T. Boon, J. Van Lint, J. Vandenheede, D. Moechars, R. Loos and F. Van Leuven, J. Biol. Chem., 2000, 275, 41340. [42] T. Engel, P. Goni-Oliver, J. J. Lucas, J. Avila and F. Hernandez, J. Neurochem., 2006, 99, 1445. [43] N. Embi, D. B. Rylatt and P. Cohen, Eur. J. Biochem., 1980, 107, 519. [44] D. R. Alessi, Biochem. Soc. Trans., 2001, 29, 1. [45] D. A. E. Cross, D. R. Alessi, P. Cohen, M. Andjelkovich and B. A. Hemmings, Nature (London), 1995, 378, 785. [46] R. Dajani, E. Fraser, S. M. Roe, N. Young, V. Good, T. C. Dale and L. H. Pearl, Cell, 2001, 105, 721. [47] A. R. Saltiel and C. R. Kahn, Nature, 2001, 414, 799. [48] M. Ilyas, J. Pathol., 2005, 205, 130. [49] T. C. Dale, Biochem. J., 1998, 329(Pt 2), 209. [50] R. Dajani, E. Fraser, S. M. Roe, M. Yeo, V. M. Good, V. Thompson, T. C. Dale and L. H. Pearl, EMBO J., 2003, 22, 494. [51] V. H. Bustos, A. Ferrarese, A. Venerando, O. Marin, J. E. Allende and L. A. Pinna, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 19725.
Recent Advances in the Discovery of GSK-3 Inhibitors
[52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71]
[72]
[73] [74] [75] [76]
[77]
[78] [79]
25
H. Aberle, A. Bauer, J. Stappert, A. Kispert and R. Kemler, EMBO J., 1997, 16(13), 3797. J. Huelsken and J. Behrens, J. Cell Sci., 2002, 115, 3977. R. Nusse, Cell Res., 2005, 15, 28. G. M. Thomas, S. Frame, M. Goedert, I. Nathke, P. Polakis and P. Cohen, FEBS Lett., 1999, 458, 247. E.-K. Suh and B. M. Gumbiner, Exp. Cell Res., 2003, 290, 447. V. W. Ding, R.-H. Chen and F. McCormick, J. Biol. Chem., 2000, 275, 32475. T. D. King, B. Clodfelder-Miller, K. A. Barksdale and G. N. Bijur, Neurotox. Res., 2008, 14, 367. W. Yang, C. Leystra-Lantz and M. J. Strong, Brain Res., 2008, 1196, 131. N. Vanacore and F. Galeotti, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, E35, author reply E36 Clinicaltrials.gov, Identifiers: NCT00790582 and NCT00818389. T. Yang, D. Arslanova, Y. Gu, C. Augelli-Szafran and W. Xia, Mol. Brain, 2008, 1, 15. A. Martinez, M. Alonso, A. Castro, C. Perez and F. J. Moreno, J. Med. Chem., 2002, 45, 1292. J. R. Woodgett, EMBO J., 1990, 9, 2431. J. R. Woodgett, Methods Enzymol., 1991, 200, 564. F. Mukai, K. Ishiguro, Y. Sano and S. C. Fujita, J. Neurochem., 2002, 81, 1073. B. Schaffer, M. Wiedau-Pazos and D. H. Geschwind, Gene, 2003, 302, 73. K. F. Lau, C. C. J. Miller, B. H. Anderton and P. C. Shaw, J. Pept. Res., 1999, 54, 85. B. W. Doble, S. Patel, G. A. Wood, L. K. Kockeritz and J. R. Woodgett, Dev. Cell, 2007, 12, 957. E. ter Haar, J. T. Coll, D. A. Austen, H.-M. Hsiao, L. Swenson and J. Jain, Nat. Struct. Biol., 2001, 8, 593. M. Saitoh, J. Kunitomo, E. Kimura, Y. Hayase, H. Kobayashi, N. Uchiyama, T. Kawamoto, T. Tanaka, C. D. Mol, D. R. Dougan, G. S. Textor, G. P. Snell and F. Itoh, Bioorg. Med. Chem., 2009, 17, 2017. D. A. Heerding, N. Rhodes, J. D. Leber, T. J. Clark, R. M. Keenan, L. V. Lafrance, M. Li, I. G. Safonov, D. T. Takata, J. W. Venslavsky, D. S. Yamashita, A. E. Choudhry, R. A. Copeland, Z. Lai, M. D. Schaber, P. J. Tummino, S. L. Strum, E. R. Wood, D. R. Duckett, D. Eberwein, V. B. Knick, T. J. Lansing, R. T. McConnell, S. Y. Zhang, E. A. Minthorn, N. O. Concha, G. L. Warren and R. Kumar, J. Med. Chem., 2008, 51, 5663. H.-C. Zhang, L. V. R. Bonaga, H. Ye, C. K. Derian, B. P. Damiano and B. E. Maryanoff, Bioorg. Med. Chem. Lett., 2007, 17, 2863. I. M. Withers, M. P. Mazanetz, H. Wang, P. M. Fischer and C. A. Laughton, J. Chem. Inf. Model., 2008, 48, 1448. H.-J. Kim, H. Choo, Y. S. Cho, K. T. No and A. N. Pae, Bioorg. Med. Chem., 2008, 16, 636. M. A. Fabian, W. H. Biggs, D. K. Treiber, C. E. Atteridge, M. D. Azimioara, M. G. Benedetti, T. A. Carter, P. Ciceri, P. T. Edeen, M. Floyd, J. M. Ford, M. Galvin, J. L. Gerlach, R. M. Grotzfeld, S. Herrgard, D. E. Insko, M. A. Insko, A. G. Lai, J.-M. Lelias, S. A. Mehta, Z. V. Milanov, A. M. Velasco, L. M. Wodicka, H. K. Patel, P. P. Zarrinkar and D. J. Lockhart, Nat. Biotechnol., 2005, 23, 329. M. W. Karaman, S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atteridge, B. T. Campbell, K. W. Chan, P. Ciceri, M. I. Davis, P. T. Edeen, R. Faraoni, M. Floyd, J. P. Hunt, D. J. Lockhart, Z. V. Milanov, M. J. Morrison, G. Pallares, H. K. Patel, S. Pritchard, L. M. Wodicka and P. P. Zarrinkar, Nat. Biotechnol., 2008, 26, 127. M. Hong, D. C. R. Chen, P. S. Klein and V. M. Y. Lee, J. Biol. Chem., 1997, 272, 25326. N. Kirshenboim, B. Plotkin, B. Shlomo Shani, O. Kaidanovich-Beilin and H. EldarFinkelman, J. Mol. Neurosci., 2004, 24, 237.
26
Robert G. Gentles et al.
[80] M. Medina and A. Castro, Curr. Opin. Drug Discov. Dev., 2008, 11, 533. [81] L. Meijer,, N. Guyard,, L. A. Skaltsounis, and G. Eisenbrand (eds), Life in Progress, Station Biologique, Roscoff, France, 2006, p. 297. [82] P. Polychronopoulos, P. Magiatis, A.-L. Skaltsounis, V. Myrianthopoulos, E. Mikros, A. Tarricone, A. Musacchio, S. M. Roe, L. Pearl, M. Leost, P. Greengard and L. Meijer, J. Med. Chem., 2004, 47, 935. [83] L. Meijer, A.-L. Skaltsounis, P. Magiatis, P. Polychronopoulos, M. Knockaert, M. Leost, X. P. Ryan, C. A. Vonica, A. Brivanlou, R. Dajani, C. Crovace, C. Tarricone, A. Musacchio, S. M. Roe, L. Pearl and P. Greengard, Chem. Biol., 2003, 10, 1255. [84] K. Vougogiannopoulou, Y. Ferandin, K. Bettayeb, V. Myrianthopoulos, O. Lozach, Y. Fan, C. H. Johnson, P. Magiatis, A.-L. Skaltsounis, E. Mikros and L. Meijer, J. Med. Chem., 2008, 51, 6421. [85] M. J. Moon, S. K. Lee, J.-W. Lee, W. K. Song, S. W. Kim, J. I. Kim, C. Cho, S. J. Choi and Y.-C. Kim, Bioorg. Med. Chem., 2006, 14, 237. [86] T. Tamaoki and H. Nakano, Biotechnology, 1990, 8, 732. [87] M. R. Jirousek, J. R. Gillig, C. M. Gonzalez, W. F. Heath, J. H. McDonald, III, D. A. Neel, C. J. Rito, U. Singh, L. E. Stramm, et al., J. Med. Chem., 1996, 39, 2664. [88] J. Respondek and J. Engel, Drugs Future, 1996, 21, 391. [89] E. Meggers, G. E. Atilla-Gokcumen, H. Bregman, J. Maksimoska, S. P. Mulcahy, N. Pagano and D. S. Williams, Synlett, 2007, 1177. [90] G. E. Atilla-Gokcumen, D. S. Williams, H. Bregman, N. Pagano and E. Meggers, ChemBioChem., 2006, 7, 1443. [91] J. Maksimoska, L. Feng, K. Harms, C. Yi, J. Kissil, R. Marmorstein and E. Meggers, J. Am. Chem. Soc., 2008, 130, 5764. [92] G. E. Atilla-Gokcumen, N. Pagano, C. Streu, J. Maksimoska, P. Filippakopoulos, S. Knapp and E. Meggers, ChemBioChem., 2009, 10, 198. [93] J. Maksomiska, D. S. Williams, G. E. Atilla-Gokcumen, K. S. M. Smalley, P. J. Carroll, R. D. Webster, P. Filippakopoulos, S. Knapp, M. Herlyn and E. Meggers, Chem. Eur. J., 2008, 14, 4816. [94] A. Martinez, M. Alonso, A. Castro, I. Dorronsoro, J. L. Gelpi, F. J. Luque, C. Perez and F. J. Moreno, J. Med. Chem., 2005, 48, 7103. [95] E. Martin-Aparicio, A. Fuertes, M. J. Perez-Puerto, D. Perez-Navarro, M. Alonso, A. Martı´nez and M. Medina, Neurobiol. Aging, 2004, 25(Suppl. 2), Abst P4–428. [96] R. Luna-Medina, M. Cortes-Canteli, S. Sanchez-Galiano, J. A. Morales-Garcia, A. Martinez, A. Santos and A. Perez-Castillo, J. Neurosci., 2007, 27, 5766. [97] B. Winblad, L. Kilander, S. Eriksson, L. Minthon, S. Batsman, A. L. Wetterholm, C. Jansson-Blixt and A. Haglund, Lancet, 2006, 367, 1057. [98] M. Y. Noh, S. H. Koh, Y. Kim, H. Y. Kim, G. W. Cho and S. H. Kim, J. Neurochem., 2009, 108, 1116. [99] X. Li, K. M. Rosborough, A. B. Friedman, W. Zhu and K. A. Roth, Int. J. Neuropsychopharmacol., 2007, 10, 7. [100] M. K. Mohammad, I. M. Al-masri, M. O. Taha, M. A. S. Al-Ghussein, H. S. AlKhatib, S. Najjar and Y. Bustanji, Eur. J. Pharmacol., 2008, 584, 185. [101] M. O. Taha, Y. Bustanji, M. A. Al-Ghussein, M. Mohammad, H. Zalloum, I. M. Al-Masri and N. Atallah, J. Med. Chem., 2008, 51, 2062. [102] Y. Miyazaki, Y. Maeda, H. Sato, M. Nakano and G. W. Mellor, Bioorg. Med. Chem. Lett., 2008, 18, 1967. [103] C. Lum, J. Kahl, L. Kessler, J. Kucharski, J. Lundstrom, S. Miller, H. Nakanishi, Y. Pei, K. Pryor, E. Roberts, L. Sebo, R. Sullivan, J. Urban and Z. Wang, Bioorg. Med. Chem. Lett., 2008, 18, 3578.
CHAPT ER
2 Advancements in the Development of Nitric Oxide Synthase Inhibitors Shawn Maddaford, Subhash C. Annedi, Jailall Ramnauth and Suman Rakhit
Contents
1. Introduction 2. NOS — Structure and Function 2.1 Introduction 2.2 Isoform-selective inhibitors — structural basis 3. Selective NOS Inhibitors 3.1 Introduction 3.2 Selective nNOS inhibitors 3.3 Selective iNOS inhibitors 4. Clinical Findings with NOS Inhibitors 5. Future Directions — Dual Action NOS Inhibitors 5.1 Introduction 5.2 Dual action nNOS inhibitors 5.3 Dual action iNOS inhibitors 6. Conclusions References
27 28 28 29 32 33 33 38 41 43 43 44 45 46 47
1. INTRODUCTION Nitric oxide (NO) is perhaps one of the most intensively studied neurotransmitters since its discovery by Ignarro [1] and Furchgott [2] in the early 1980s as a mediator of vascular tone. This early work revealed NeurAxon Inc, 480 University Ave, Suite 900, Toronto, ON M5G 1V2, Canada Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04402-9
r 2009 Published by Elsevier Inc.
27
28
Shawn Maddaford et al.
that endothelium-derived relaxing factor (EDRF), the agent found in the endothelium that was responsible for dilation of blood vessels, was in fact NO. Since then, the unraveling of the diverse biological functions of NO has continued unabated as evidenced by more than 80,000 publications on nitric oxide synthase (NOS) and NO described in the literature. In addition to its role in regulating blood pressure, NO is important in platelet aggregation, bone remodeling, inflammation, and neurotransmission, wherein NO initiates changes in neuronal excitability and synaptic strength by acting at pre- and/or post-synaptic locations. The reduction in pathophysiological levels of NO through inhibition of NOS has the potential to be therapeutic in a multitude of indications including the treatment of septic shock, stroke, neurodegenerative disorders (e.g., Parkinson’s, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS)), and in the treatment of pain [e.g., migraine, chronic tension-type headache (CTTH), visceral, and neuropathic] [3]. However, the therapeutic control of NO synthesis has, until recently, been unattainable due to the difficulties in achieving isoform-selective inhibition. The selective inhibition of the neuronal NOS (nNOS) enzyme and/or the inducible (iNOS) over the endothelial NOS (eNOS) enzymes for the treatment of pain or migraine would be required to avoid the cardiovascular liabilities associated with eNOS inhibition [4]. Earlier reviews in this publication have focused on the enzymology and synthesis of NOS inhibitors [5,6]. This review focuses on recent advances in the design of selective inhibitors of NOS and some of the emerging preclinical and clinical developments of these newer inhibitors.
2. NOS — STRUCTURE AND FUNCTION 2.1 Introduction NO is synthesized by three isoforms of NOS, which catalyzes the fiveelectron oxidation of L-arginine to L-citrulline [7]. The neuronal or brain NOS (nNOS or NOS1) and endothelial form (eNOS or NOS3) are constitutively expressed, whereas the inducible form (iNOS or NOS2) is expressed under conditions of stress or upon the release of inflammatory mediators such as tumor necrosis factor-a, interleukin-1, or lipopolysaccharides (LPS). These homodimeric proteins consist of a C-terminal reductase domain that transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) through the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) prosthetic groups to the Nterminal oxygenase domain that binds the arginine substrate, (1uR, 2uR, 6R)-5,6,7,8-tetrahydrobiopterin (BH4) and heme [8]. The two constitutive forms are activated by increasing Ca2+ concentration and the binding of a Ca2+/calmodulin complex while the activity of iNOS appears to be
29
Advancements in the Development of NOS Inhibitors
independent of Ca2+ concentration due to the tight binding of the complex at the dimer interface [9]. The active dimeric form of NOS is stabilized by a structural zinc binding at the oxygenase dimer interface [10], which forms the BH4-binding site. The closest related enzymes are the cytochrome (CYP) P450 enzymes, wherein both the function and amino acid sequence of the reductase domains and the function but not the sequence of the oxygenase domains are similar. Reduction of the BH4 and heme iron allows for the activation of O2 and subsequent CYP-like oxidation of L-arginine to No-hydroxy-L-arginine and finally to citrulline, ultimately releasing NO. In the absence of BH4, NOS reduces oxygen to form superoxide [11]. The inhibitor design reviewed herein has primarily focused on targeting the arginine or BH4-binding sites to prevent the overproduction of NO or the uncoupled reduction of O2 to superoxide, thus minimizing their deleterious effects.
2.2 Isoform-selective inhibitors — structural basis The three NOS isoforms possess approximately 50% overall sequence homology and identical structural architecture. With the exception of the human nNOS enzyme, the crystal structures for all three isoforms have been determined allowing for the application of structure-based inhibitor design [10,12–17]. However, analysis of the oxygenase domains reveals striking active site structural conservation across the three isoforms with ˚ of the substrate-binding site being identical 16 of 18 residues within 6 A [18]. Despite the high similarity of the binding pockets, inhibitors 1–4 derived from L-NG-nitroarginine (L-NNA) show selectivity for rat nNOS versus bovine eNOS (Table 1). These compounds make interactions with the L-amino acid-specific binding pocket demonstrating hydrogen (H) bonds to Gln249, Tyr359, Glu363, and Asn368 (rat nNOS) [19]. In all cases, the guanidine and aminopyridine groups exhibit similar bifurcated H-bonds with the conserved glutamate (e.g., Glu363 or 592) across the three isoforms. However, the nitro group of inhibitors 1–3 provides Table 1 Peptide, reduced amide, and aminopyridine-based substrate inhibitor selectivity for 1–4 Compound Rat nNOS
1 2 3 4
0.30 0.10 0.15 0.38
Note: Ki values are in mM.
Bovine eNOS
Murine iNOS
e/n
i/n
D597N nNOS
N368D eNOS
107 110 80 434
25 29 39 58
1538 1280 2617 1114
192 290 325 150
67 34 21 –
9.5 4.6 5.1 –
30
Shawn Maddaford et al.
additional hydrogen bonding and non-bonded contacts with the protein backbone, which results in enhanced binding affinity over L-arginine [20]. Inspection of the X-ray complexes suggests that the selectivity of 1–3 for nNOS over eNOS arises from a single amino acid substitution of a neutral Asn368 (eNOS) residue for a charged Asp597 (nNOS) residue (Figure 1). These inhibitors adopt a curled conformation in the nNOS active site placing the a-amino group between Glu592 and Asp597, ˚ ) and potentially a so that it makes a direct H-bond to Glu592 (2.8 A favorable electrostatic interaction with the charged Asp597 (nNOS). In contrast, the inhibitors bound to eNOS were in a fully extended conformation with the a-amino group in 1, reorienting to make a H-bond with Gln249 rather than Glu592. Mutagenesis studies confirmed that this single residue difference was responsible for the two orders of magnitude difference in potency between the two isoforms (Table 1). NO2 HN
H2N
NO2
NH
HN
NO2 NH
HN
N
NH
NH NH
NH
NH
O H N H2N
NH2 O
H N
O
H2N
H2N O
1
NH2
NH
HN
H N NH2
NH2
NH2
4
3
2
ASP597 GLN478 ASN368 ASN569
GLU592
GLN249
ASN340
GLU363
Figure 1 Active site poses of dipeptide amide 1 in the active site of nNOS (left) and eNOS (right). The a-amino group makes hydrogen bonding and electrostatic interaction with Glu592 in nNOS, whereas 1 displays an extended conformation in eNOS and makes an H-bond with Gln249 as opposed to Glu363. (See Color Plate 2.1 in Color Plate Section.)
31
Advancements in the Development of NOS Inhibitors
The D597N mutation in nNOS resulted in a drop in potency (1; w.t. ¼ 0.3 mM, D597 N ¼ 67 mM), whereas the corresponding eNOS mutant N368D resulted in an increase in potency (1; w.t. ¼ 107 mM, N368D ¼ 9.5 mM). Similar trends were observed for compounds 2 and 3. Finally, a crystal structure of the nNOS D597N mutant complexed with 1 revealed an extended conformation, whereas the eNOS mutant N368D complexed to 1 adopted a curled conformation [19]. Recently, it has been suggested that the selectivity for a series of quinazoline and aminopyridine inhibitors for the iNOS enzyme is due to an isozyme-specific residue plasticity, wherein residues distant from the active site modulate conformational changes of invariant residues in contact with the inhibitors [17]. In this ‘‘anchored plasticity approach,’’ an inhibitor core anchored in a conserved binding pocket and a rigid bulky substituent extend into remote specificity pockets upon conformational changes of ‘‘plastic residues.’’ This approach is conceptually different to the approach described earlier where the ligand changes its conformation to interact with variant residues within the immediate active site. Thus, non-selective ligands such as S-ethylisothiourea (SEITU), aminopyridine 5, or the selective aminopyridine 6 bound to human iNOS (Figure 2) are anchored through bidendate hydrogen bonds with Glu377. The bulky side chain of 6 induces an opening of invariant
Phe
Val Cα
Phe286
Val305 Cα
Leu288 Cα O
O Asn H2N
H2N
Asn
H2N
Asn
O Arg
HN
Ile269 Cα
NH
H HN N
Arg
Arg
NH2
NH2
H2N
H HN N
O
O O H2N hiNOS Gln-closed (A)
Gln
H2N hiNOS Gln-open
Gln
H2N
Gln
heNOS Gln-open
(B)
Figure 2 (A) X-ray crystal structures of human iNOS in the Gln-closed form (gray carbons-SEITU bound) and Gln-open form (green carbon atoms; compound 6). The bulky inhibitor 6 avoids steric clashes by creation of a new pocket as a result of rotation of first shell residue Gln263 about its w1 and w2 torsion angles. (B) Schematic representation of the movement of residues in iNOS. In eNOS, the Gln-open form is not possible due to the bulky third shell Leu and Ile residues. (See Color Plate 2.2 in Color Plate Section.)
32
Shawn Maddaford et al.
first-shell residues Gln263 and Arg373 inducing a cascading effect of second-shell residue Asn283 toward third-shell residues Phe286 and Val305. Small non-selective inhibitors do not induce this conformational change and thus bind to a first-shell Gln-closed state, whereas the bulky ligand 6 binds to the Gln-open state in a newly created binding pocket. Compound 6 binds weakly to eNOS in a Gln-closed state. In human eNOS, the invariant first (Gln246 and Arg249) and second shell residues (Asn266) are unable to undergo this conformational change required for binding since the final third shell residues are the much bulkier isoleucine (Ile269) and leucine residues (Leu288) compared with the smaller Phe and Val residues found in human iNOS.
O N N
NH2
N
N H
5: iNOS: 0.12 μM 6: iNOS: 0.37 μM eNOS: 0.32 μM eNOS: 100 μM nNOS: 0.16 μM nNOS: 23 μM
O
O
OEt
O
O N
N N
N H 7: iNOS: 0.52 μM eNOS: >100 μM nNOS: 15 μM
N
N H NH2 8: iNOS: 24 μM eNOS: >100 μM nNOS: 39 μM
Although the use of crystal structures can provide valuable insights into the understanding of drug–ligand interactions, caution must be used when interpreting structural data. In general, modelers and medicinal chemists make several assumptions about the protein crystal structure: 1) the protein structure and amino acid sequence is correct; 2) the structure of the ligand and its interactions with the protein are correct; and 3) the protein–ligand structure is relevant for drug discovery. Issues often arise when the resolution of the structure is insufficient to determine the identity or orientation of a ligand in the active site or the position of side chain residues. In addition, crystal structures do not provide information on the thermodynamics of ligand binding and desolvation contributions to the overall binding. A recent example illustrates these limitations of X-ray crystallography in the design of iNOS inhibitors related to structures 6 and 7 [21]. The co-crystal structure of mouse iNOS with 7 revealed a single amino acid difference in contact with the inhibitor (Asp376 in iNOS substituted for Asn in eNOS). Compound 8 was designed in attempts to exploit this difference through an interaction of the basic nitrogen with the Asp residue and displacement of a bound water molecule H-bonded to this residue. Both docking studies and a ˚ ) seemed to confirm this interaction. low resolution X-ray structure (3.3 A However, the in vitro NOS assay revealed a substantial reduction in potency for iNOS (24 mM). It is possible that the loss of hydrogen bonds between the displaced water molecule with the protein or an energy
Advancements in the Development of NOS Inhibitors
33
penalty associated with desolvation of the primary amine group accounts for this loss in potency. In another example, Silverman and co-workers [22] designed an N-hydroxy analog of 3 to displace a structural water molecule hydrogen-bonded between the heme propionate groups of the nNOS enzyme. A crystallographic analysis revealed that indeed the water molecule was successfully displaced, but this change failed to improve the in vitro potency. The successful use of NOS X-ray structures in drug design are discussed in Section 3.2.
3. SELECTIVE NOS INHIBITORS 3.1 Introduction Numerous attempts have been made toward the design of isozymeselective NOS inhibitors targeting both the L-arginine and BH4-binding sites. Early inhibitors were based on modifications of mono- and dipeptides, with subsequent inhibitor design and synthesis utilizing co-crystal structures of all NOS isozymes. Several review articles covering progress toward the development of selective nNOS inhibitor syntheses [23,24], selective iNOS inhibitor syntheses [25], and computational studies [26] have already appeared in the literature. The application of selective NOS inhibitors for various therapeutic indications such as the treatment of shock [27], asthma [28], migraine, tension-type and cluster headache [29], neurological diseases [30], and inflammatory joint diseases [31] were recently reviewed. Many early NOS inhibitors were modified substrate (L-arginine) or product (L-citrulline) analogs. First-generation arginine-based inhibitors such as NG-methyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NNA) and its L-nitroarginine methyl ester (L-NAME), NG-amino-L-arginine (LNAA), NG-allyl-L-arginine (L-ALA), NG-cyclopropyl-L-arginine (L-NCPA), and NG-propyl-L-arginine (L-NPA) are reasonably potent but are poorly selective toward the NOS isoforms [23]. The most commonly used arginine-based NOS inhibitors for in vitro and in vivo assays are L-NMMA, L-NNA, and L-NAME, a consequence of commercial availability, chemical stability, water solubility, and low toxicity. Owing to the peptide nature and poor drug-like properties of these inhibitors, non-peptidic small molecule drug design and synthesis are warranted.
3.2 Selective nNOS inhibitors 3.2.1 Peptide-based inhibitors The amino group appears to be very important, whereas the carboxylic group seems unnecessary for enzyme inhibition. In an attempt to
34
Shawn Maddaford et al.
increase isozyme selectivity, the carboxylic acid can be functionalized with amino acids to form dipeptides. Most of the arginine-based dipeptides display excellent potency and selectivity (such as compounds 1 and 2) for rat nNOS over bovine eNOS and murine iNOS [24]. However, the activity of these compounds with human isoforms has not been reported. In an attempt to improve brain penetration, various aminopyridine analogs have been prepared using a de novo design strategy called ‘‘fragment hopping’’ and demonstrate a significant improvement in nNOS activity and selectivity over eNOS [32]. This strategy is based on pharmacophore requirements such as hydrophobic and steric interactions, as demonstrated with compound 4, which revealed lipophilic pockets near the 4-position of the aminopyridine ring and the terminal aminomethyl group. Compounds 9 and 10, derived from 4, are much more potent at nNOS (Ki ¼ 0.08 and 0.01 mM) and selective over eNOS (1000- and 2000-fold) than the corresponding unsubstituted parent compounds [18]. Both compounds prevented hypoxia-ischemia-induced death in a rabbit model for cerebral palsy, also reducing the number of newborn kits exhibiting symptoms of cerebral palsy without affecting blood pressure. Maternal administration of compounds 9 or 10 showed 83% and 69% normal kits, respectively, compared to 9% of saline-treated kits. H N
N H
R
H N
N n
H2N 9: n = 1, R = 4-Cl 10: n = 2, R = 3-F
3.2.2 Non-peptide-based inhibitors 7-Nitroindazole (7-NI, 11) and 1-(2-trifluoromethylphenyl)imidazole (TRIM) (12) [33] are two of the most extensively studied non-peptidic NOS inhibitors, due to a high in vivo selectivity for nNOS despite modest selectivity in vitro (7-NI; rat nNOS IC50 ¼ 0.47 mM and bovine eNOS IC50 ¼ 0.7 mM) [23]. The inhibitory mechanism and selectivity is the result of a reversible competitive interaction with both the L-arginine and the BH4-binding sites in nNOS [34], which is limited to the pterin cofactor site with iNOS and eNOS. In addition to NOS knockout studies, these early ‘‘brain selective’’ inhibitors provided much of the foundation for
35
Advancements in the Development of NOS Inhibitors
elucidating the pharmacological role of nNOS including nociception [35,36], antidepressant and anxiolytic properties [37], modification of opioid-induced side effects, and synergistic enhancement of serotonergic antidepressants [38].
CF3
H N
N N H
Cl N
NH
N
N H
NO2
11
12
S
13
Compound 13 (ARL17477) is a selective nNOS inhibitor with IC50 values of 0.035, 5.0, and 3.5 mM for human nNOS, iNOS, and eNOS, respectively [39]. Evaluation of ARL17477 in a global ischemia model produced a significant reduction (52% protection) in ischemia-induced hippocampal damage following global ischemia dosed immediately (50 mg/kg i.p.) post-occlusion [40]. In addition, ARL17477 was reported to provide greater neuroprotection (44%) than L-NAME (19%), 7-NI (22%), or TRIM (8%) in a gerbil model of global cerebral ischemia. In an endothelin-1 model of focal ischemia, a 1 mg/kg i.v. administration (0, 1, or 2 h post-endothelin-1) of ARL17477 significantly attenuated the infarct volume when compared to pre-treatment with the NMDA antagonist MK-801, post-treatment with the iGluR5 antagonist LY377770, or the immunophilin FK-506. In another study, a combination of ARL17477 with MK-801 or LY293558 provided 78% and 71% greater neuroprotection than the calculated additive effects of the individual treatments [41]. ARL17477 also inhibited electrically induced nitrergic relaxations in pig gastric fundus strips and gastric fundic compliance in conscious pigs in a dose-dependent manner [42]. On the basis of the knowledge that incorporation of a basic side chain could improve potency and selectivity in amidine compounds such as ARL17477, a series of related fused 2-aminodihydroquinoline compounds were designed [43]. Thus, incorporation of the amine side chain into 14a to give 15a improved the potency and selectivity (nNOS over eNOS) up to 224-fold. To improve brain penetration through a reduction in basicity, fluoro analogs 14b and 15b were synthesized. Incorporation of fluorine into the unsubstituted analog 14a decreased the pKa from 9.7 to 7.9 (14a and 14b, respectively) without affecting the potency or selectivity (nNOS ¼ 0.16 mM, 0.1 mM, eNOS ¼ 3.3 mM, 2.7 mM). However, fluorination of the corresponding amine side chain–containing compound 15a to give 15b led to a similar reduction in pKa (9.9 to 8.0) but
36
Shawn Maddaford et al.
was accompanied by a loss in potency relative to the parent compound 15a [44].
H
H X
Cl
X N H
NH
NHR N
NH 16a R = 1-(4-F-Benzylpiperidin-4-yl) 16b R = 2-(1H-Imidazol-5-yl)ethyl
15a: X = H, 15b: X = F
14a: X = H,14b: X = F
S
NH
HN N H
H N
S
A series of substituted 2-aminobenzothiazole inhibitors were shown to be selective toward nNOS over eNOS and iNOS [45]. By varying substitution at the 2-position, up to 40–50-fold selectivity was achieved for human nNOS over eNOS (16a; nNOS ¼ 0.2 mM, eNOS ¼ 8 mM, e/n ¼ 40). It is notable that substantial species differences were observed in some cases such as 16b (IC50 values for human nNOS ¼ 0.3 mM, eNOS ¼ 0.7 mM and iNOS ¼ 6 mM; rat nNOS ¼ 4.7 mM, bovine eNOS ¼ 362 mM). Compound 16b showed neuroprotection in a concentrationdependent manner, when preincubated for 60 min before NMDA challenge in rat cortical cultures. At 50 and 100 mM concentration, increases in cell survival rates of 80% and 90%, respectively, were observed, compared to 30% in NMDA-treated controls [46].
NH
N S H N
S NH
N
N H
NH
N
N
N H 17
H N
S
N 18
19
Various di-substituted indole compounds were investigated for their activity in all three human NOS isozymes and showed submicromolar potencies for nNOS and good selectivity for nNOS over both eNOS and iNOS [47,48]. In this study, the basic amine side chain was varied at both the 1- and the 3-positions on the indole and the amidine group between the 5- and the 6-positions. The most potent compound in the series was obtained with the basic amine group at the 1-position and with the amidine at the 5-position (IC50: human nNOS ¼ 0.02 mM; eNOS ¼ 1.92; iNOS ¼ 17 mM). Alternatively, the best selectivity was achieved with a quinuclidine-based system and the basic side chain at the 3-position
Advancements in the Development of NOS Inhibitors
37
(17; human IC50: nNOS ¼ 0.76 mM, eNOS ¼ 103 mM, iNOS ¼ 89; e/n ¼ 136, i/n ¼ 117). In further studies, compound 18 (human IC50: nNOS ¼ 1.2 mM, eNOS ¼ 15 mM, iNOS ¼ 60; e/n ¼ 12.5, i/n ¼ 50) was highly neuroprotective and demonstrated efficacy in multiple pain models. Pre-incubation of rat cortical cells at 25 mM concentration of 18 followed by an NMDA challenge resulted in 95% cell survival when compared to only 50% in NMDA-treated controls. In a separate experiment, 18 was able to reduce hippocampal cell death up to 95% in a concentration-dependent manner after oxygen–glucose deprivation. The antinociceptive effect of compound 18 was measured by formalininduced hyperalgesia and inflammation in an experimental model of sustained inflammatory nociception associated with long-term intracellular changes of nociceptive processing at the level of the spinal cord. Pre-administration of compound 18 (5 and 10 mg/kg i.p.) or 7-NI (2.5 and 5 mg/kg i.p.) reduced paw licking in a concentration-dependent manner for both compounds indicating efficacy for the treatment of inflammatory pain. In the Chung Spinal Nerve Ligation (SNL) neuropathic pain model, 18 demonstrated a complete reversal of both thermal and tactile hyperalgesia in rats after 3 and 20 mg/kg i.p. administration, respectively. A series of indoline, oxindole, tetrahydroquinoline, quinolone, and benzazepine derivatives were explored in an attempt to identify selective human nNOS inhibitors [49]. In this study, nanomolar potency for human nNOS (IC50 ¼ 0.01 mM) and more than 1,000-fold selectivity over eNOS (IC50 ¼ 14.2 mM) was achieved. Compound 19 (nNOS ¼ 0.34 mM, eNOS ¼ 48 mM, e/n ¼ 141) was able to completely reverse tactile allodynia in the sciatic nerve cuff model of neuropathic pain after i.p. administration at 30 mg/kg. At the same time, compound 19 was able to reduce the frequency of paw lifts up to 50% on a cold platform in the same model after multiple dose administration. The 2-aminopyridine group serves as an effective isosteric replacement of the guanidinium group and even simple derivatives such as 4-methyl-2-aminopyridine exhibit potent but non-selective inhibition of NOS. Analogs such as 20 are potent nNOS inhibitors with modest selectivity over eNOS (nNOS ¼ 140 nM, eNOS ¼ 887 nM, e/n ¼ 6) with minimal off-target activity at m2 and m4 receptors (Ki ¼ 0.32 mM and 0.59 mM, respectively) [50]. Pharmacokinetic studies with 20 after subcutaneous administration (24 mg/kg) gave a Cmax of 1.7 mg/mL in plasma, 0.45 mg/mL in cerebrospinal fluid (CSF), and 11 mg/g in whole brain, indicating sufficient brain levels to inhibit nNOS in vivo. Although the compound did not increase blood pressure in rats at doses of up to 100 mg/kg, the rat eNOS values were not reported. Further modifications of this scaffold by incorporating bulk onto the phenyl ring further increased the selectivity for nNOS (e.g., 21, e/n ¼ 50) [51]. Compound 21
38
Shawn Maddaford et al.
was active in the harmaline-induced cGMP model in rat cerebellum (ED50 ¼ 7 mg/kg s.c.). In contrast to 20, compound 21 inhibited phencyclidine (PCP)-induced hypermotility (2.8 mg/kg s.c.) in rats, a model used to assess antipsychotic agents.
N O
NH2
N
NH2
N
N
O
N Ph
20
21
A set of 41 pteridine nNOS-selective inhibitors employing four scaffolds was developed by a combination of ligand- and structure-based design [52]. The X-ray crystal structure for rat NOS-dimeric oxygenase domain with BH4 and L-arginine was used to develop a human isoform homology model. Substitution on the 4-, 5-, 6-, and 7-positions are necessary for both affinity and selectivity. Bulky and hydrophobic substituents at the 5- or 6-position increased the selectivity for nNOS over iNOS and eNOS. The tetrahydro antipterins were more active and selective for nNOS over eNOS; up to 58-fold selectivity was observed with compound 22. Ph Cl 5 H N
HN 4 3N
6
2 H2N
N H 8
N 1
7
22
3.3 Selective iNOS inhibitors Inducible NOS has been implicated in a number of inflammatory diseases such as septic shock, multiple sclerosis, rheumatoid and osteoarthritis, ulcerative colitis, and asthma. Not surprisingly, many research institutions have developed programs to design inhibitors of iNOS. In addition to
Advancements in the Development of NOS Inhibitors
39
potency, selectivity against the other two isoforms, especially eNOS, is required since they are important in normal physiology. Selective inhibitors of iNOS have been reviewed previously [25]. Early inhibitors of iNOS were simple analogs of the substrate L-arginine in which the guanidine group or the side-chain had been modified. Compound 23 (GW274150), a selective and modestly potent inhibitor of human iNOS (IC50 ¼ 1.4 mM) has completed a phase II clinical trial for the acute and prophylactic treatment of migraine headache and asthma [53]. HN
O S
HO NH2
O N
N H N H H
23
O
H
24
NH
N
N H
CN 25
In general, due to their physicochemical properties, amino acid arginine-like inhibitors will rely on active transport processes for their absorption and distribution. Consequently, a great deal of effort has been invested in discovering NOS inhibitors with more drug-like properties. Amidines and isothiourea derivatives have been developed to replace the guanidino group, a key anchor point in the active site. Generally, the isothioureas are more selective for nNOS while amidines tend to be selective for iNOS. Considerable effort has been expended in the design and synthesis of selective amidine iNOS inhibitors. Notably, the fused bicyclic amidine, 24, has been reported to inhibit human iNOS with an IC50 ¼ 22 nM and greater than 30-fold selectivity over eNOS, but only two-fold over nNOS [54]. The ability of compound 24 to inhibit iNOS activity in vivo was measured in a rat endotoxin assay. This model measures the plasma nitrate/nitrite (NOx) levels due to the induction of iNOS following LPS administration. An oral dose of 24 (10 mg/kg) given at 1 or 16 h before LPS challenge showed complete inhibition or 40% reduction of NOx respectively, at the 5-h time points. This demonstrates that efficacious levels of 24 can be maintained in circulation for more than 16 h. The 2-aminopyridine group used as an isosteric replacement for amidines is generally less basic and should therefore be more membrane permeable. The N-alkylated aminopyridine 25 is a potent (71 nM) and selective (W1,000- and 100-fold selective against eNOS and nNOS, respectively) inhibitor of iNOS [55]. Reported in a later publication, compound 25 was tested in a rat model of LPS-induced NO production and showed an oral dose-dependent inhibition of elevated plasma NOx levels (IC50 ¼ 1.8 mM) measured 4 h after LPS administration [17].
40
Shawn Maddaford et al.
Most competitive NOS inhibitors possess a minimum pharmacophore of a basic cis-amidine (a guanidine isostere) required for a bidentate interaction with the carboxyl group of Glu371. The imidazopyridine 26 and the spirocyclic quinazoline 27 represent novel classes of potent and selective iNOS inhibitors bearing a masked cis-amidine moiety. Compound 26, a potent iNOS arginine-competive inhibitor (86 nM) exhibiting more than 1,500-fold selectivity over eNOS and 80-fold selectivity over nNOS, was identified by screening a corporate compound library [56]. Inhibition of iNOS-derived NOx formation in various intact cells following LPS treatment was observed for compound 26: nitrite generation after induction of iNOS was inhibited with IC50 values of 3.1 mM in murine macrophage cell line RAW, 33 mM in rat mesangial cell line RMC, and 13 mM in a human HEK293/iNOS assay. The spirocyclic quinazoline 27 inhibits purified recombinant human iNOS (IC50 ¼ 37 nM) and NO synthesis in a whole cell assay (human DLD-1 cells, IC50 ¼ 0.9 mM) [57]. The compound was shown to be selective over nNOS (25-fold) and eNOS (W2,000-fold). When given orally to rats, 27 produced a dose-dependent inhibition of NO production induced by LPS (ID50 ¼ 3 mmol/kg, 4 h post-dose). In addition, 27 showed efficacy in the Freund’s complete adjuvant-induced polyarthritis model in rats. Administration of 27 (10–100 mmol/kg p.o., twice daily) commenced on the day of challenge delayed the onset of observable symptoms (inflammation, edema, pain, and joint destruction) and reduced their severity in a dose-dependent manner (ED50 at 20 d timepoint B10 mmol/kg). At a higher dose (100 mmol/kg), the compound completely abolished all indications of developing arthritis for the full 20 days of the experiment. O
O F N
N
N
F
N N
N
HN
26
NH2 N
H N
S
CN HN
O
O
NH2
27
28
The structure-based design of compound 28 was based on the ‘‘anchored plasticity’’ approach (vide ante) [17]. Compound 28, with a cyclic amidine, inhibits human iNOS with an IC50 ¼ 0.4 mM and greater than 100-fold selectivity over eNOS. However, the selectivity over nNOS was only 2-fold. Compound 29 is an L-arginine competitive human iNOS inhibitor (IC50 ¼ 90 nM) that was optimized from a high-throughput screening (HTS) hit [58]. The compound does not contain a guanidine mimic that
Advancements in the Development of NOS Inhibitors
41
distinguishes it from other compounds described in the literature. It appears to interact primarily through p-stacking and non-bonded interactions. Compound 29 inhibits rat nNOS (IC50 ¼ 0.56 mM) and bovine eNOS (IC50 ¼ 11.1 mM). A new series of aminopiperidines with potent human iNOS activity (IC50 ¼ 0.33 mM; e/iW25, n/i ¼ 16) represented by compound 30 was reported recently [59]. Again as in compound 29, the optimized HTS hit 30 does not contain a guanidine-like functionality, and docking studies reveal that 30 p-stacks with the heme.
CN O
NH2
Cl
O
H N
O
N O
N N
O
29
O
NH
Cl
N
N
30
N
31
A unique class of iNOS inhibitors was obtained by screening a combinatorial library using a whole-cell assay [60]. These compounds inhibit the dimerization of iNOS monomers, thus preventing the formation of the active dimeric form of the enzyme. Optimization led to the identification of compound 31 (IC50 ¼ 14 nM) with selectivity greater than 500 for eNOS and 300 for nNOS. This compound is orally available and was shown to significantly ameliorate adjuvant-induced arthritis in a rat model.
4. CLINICAL FINDINGS WITH NOS INHIBITORS Early clinical trials [61] shed light on the role of NO in the mechanism of migraine pain. In several double-blind studies [62–64], it was shown that after intravenous administration of nitroglycerine (GTN), non-migraineurs rapidly developed a headache that subsided after removal of GTN, whereas migraineurs developed an initial headache, followed by a delayed (B5–6 h) migraine attack [65], suggesting that NO may be partially or completely responsible for migraine pain. A similar observation was made by using sublingual isosorbide dinitrate, another NO donor [65]. The first clinical trial with a NOS inhibitor was conducted using the non-selective NOS inhibitor L-NMMA (32) for the treatment of migraine headache. In a randomized double-blind study, 15 migraine patients with
42
Shawn Maddaford et al.
single spontaneous attacks were infused with 6 mg/kg L-NMMA or placebo (5% dextrose) over a period of 15 min and monitored at 0, 30, 60, and 120 min post-dosing for headache severity, clinical disability (scored on a scale of 0–3), nausea, photophobia, and phonophobia. Heart rate, blood pressure, and ECG were also monitored continuously. A significant antimigraine effect was observed in patients relative to placebo, 67% vs. 25%, albeit with an increase in blood pressure by 17% and decrease in heart rate by 21%. The antimigraine effect was treatment related, most likely the result of specific inhibition of NOS [66–68] and not due to a direct vasoconstriction effect. Migraine relief associated with vasoconstriction was ruled out because of a lack of effect of L-NMMA observed on transcranial Doppler-determined velocity in the middle cerebral artery of the patients. A similar study in the reduction of CTTH by the infusion of L-NMMA was reported [69,70], although the effect was not as pronounced as in the case of migraine headache. NH N H
N H
CO2H NH2
32
In a recent study [71], the effect of infusion of 6 mg/kg L-NMMA on basal and acetazolamide induced changes in cerebral arteries and regional blood flow, indicating that the non-selective NOS inhibitor decreased regional cerebral blood flow only to a minor degree. The authors inferred that in migraine with aura where cerebral blood flow is reduced, treatment of migraine with aura with non-specific inhibitors of NOS might be problematic. Most recently, at the European Headache and Migraine Trust International Congress 2008, three companies presented the results of the effect of NOS inhibitors for the treatment of either acute migraine or migraine prophylaxis. In a study conducted with the selective iNOS inhibitor GW274150 (23) using up to 120 mg daily for 12 weeks for the prophylactic treatment of migraine, the drug was found to be no more effective than the placebo although it did not manifest any intolerable side effects [72]. In a related study utilizing an adaptive clinical trial of GW274150 in the treatment of acute migraine, no significant differences between any of the doses, ranging from 5 to 180 mg, and placebo were observed in the proportion of subjects who became pain free at 2 h after treatment [73]. In addition, no treatment-related benefit was observed for
Advancements in the Development of NOS Inhibitors
43
other migraine symptoms including nausea, photophobia, phonophobia, and allodynia. Since GW274150 is known to reduce exhaled NO levels in patients [74], these studies indicate that the antimigraine effect of L-NMMA is not related to inhibition at the iNOS isoform. In a placebocontrolled double-blind study, the non-selective NOS inhibitor S-alkyl isothiourea MTR-106 was found to be effective in female migraine patients without aura [75]. The study showed statistically significant pain reduction after 2 h with 23% of subjects remaining pain free after 24 h. Although there was no dose-dependent relationship (25, 50, 75, and 100 mg), the drug exhibited linear pharmacokinetics and more adverse effects with higher doses. Recent phase I clinical trials with NXN-188, a novel dual action nNOS inhibitor with 5-HT1B/D agonism, assessed the safety and tolerability of the drug after once daily or multiple twice daily oral doses [76]. The compound exhibited an initial rapid absorption, followed by a more prolonged absorption phase with a Tmax of 4–5 h. The drug displayed near linear pharmacokinetics over the dose range studied (0.027–16 mg/kg). Of the 168 patients that received active drug, no serious adverse events were reported up to the maximally administered dose of 800 mg. Importantly, it was noted that NXN-188 had no effect on blood pressure in the treated patients unlike the non-selective NOS inhibitor L-NMMA. NXN-188 was also studied for the treatment of acute migraine in a phase II study wherein the drug produced extended relief in patients [77]. Future studies will be required to fully elucidate the pharmacological effects of combined nNOS inhibition and 5HT1D/B agonism on the pharmacological profile relative to the existing triptans. Besides inhibition of NOS, another strategy to reduce the amount of NO in migraine patients is to use a NO scavenger. In an open trial, the NO scavenger hydroxocobalamin was assessed in migraine prophylaxis. Daily treatment with intranasal hydroxocobalamin (1 mg) for a period of 3 months demonstrated W50% reduction in the frequency of migraine attacks in 10 of 19 patients. Given the percentage of responders was 35–40%, it is unlikely that this was a placebo effect [78].
5. FUTURE DIRECTIONS — DUAL ACTION NOS INHIBITORS 5.1 Introduction Before the development of molecular biology, traditional drug discovery relied on the use of in vivo animal models of disease to seek out new medicines, often without knowledge of the molecular mechanisms of
44
Shawn Maddaford et al.
their action. Many of the older ‘‘dirty’’ drugs discovered by this means, albeit very efficacious, suffered from undesirable off-target-related side effects. This resulted in a paradigm shift in drug discovery toward optimization of compounds against a single molecular target. Although this approach has delivered many new drugs with potentially fewer side effects, many complex, multifactorial diseases such as cancer, diabetes, and pain are still inadequately treated with single-action drugs. It is likely that complex diseases arise from a complicated network of interdependent biological changes occurring in multiple organs over a period of time. Still, prediction of efficacy or off-target side effects for a single mechanism compound must be put in the context of interspecies differences in target potency and selectivity and the usefulness of acute animal models of disease for evaluating complex, chronic human diseases. To address this biological complexity, there is an increasing readiness to develop agents that modulate multiple targets simultaneously (polypharmacology) and to develop new animal models of chronic disease, with the aim of enhancing efficacy or improving safety relative to drugs that address only a single target.
5.2 Dual action nNOS inhibitors It has been shown that a combination of a nNOS inhibitor and an antioxidant in a model of focal ischemia was synergistic in reducing neuronal damage [79]. A novel strategy for the treatment of stroke consists of designing hybrid molecules such as compound 33, which possess a NOS inhibitor pharmacophore linked to an antioxidant fragment to scavenge reactive oxygen species (ROS). This compound displayed potent nNOS inhibition (Ki ¼ 0.12 mM) and inhibition of lipid peroxidation induced by ROS (IC50 ¼ 0.4 mM). Scavenging ROS and inhibiting NOS simultaneously has been shown to enhance neuronal survival after cerebral ischemia in animal models [80]. Given that NO reacts with O2 to form peroxynitrite, it is perhaps not surprising that the two mechanisms work synergistically. The inhibition of nNOS is known to enhance opioid analgesia and reduce the development of tolerance. It has been shown that the development of morphine-induced hyperalgesia after chronic administration in rats can be delayed or reversed by the addition of a selective nNOS inhibitor [46]. Thus, a dual nNOS inhibitor and m-opioid receptor agonist would be beneficial in the treatment of various pain states. Benzimidazole 34 inhibits nNOS (IC50 ¼ 0.44 mM) and binds to the m-opioid receptor with an IC50 ¼ 13 nM [81]. In a functional assay, the compound displayed much weaker m-opioid receptor agonist activity (EC50 ¼ 0.44 mM).
45
Advancements in the Development of NOS Inhibitors NEt2
N S
H N N H HO
NH
S
S
N
NH
H N
N
N H
NH
N H
OEt
33
34
35
NO derived from nNOS appears to modify the activity of many neurotransmitter systems including glutamate (NMDA), opioid, GABA, and serotonin, likely through spatiotemporal interactions of the nNOS protein with these systems. As an example, nNOS appears to co-localize with the serotonin transporter (SERT) in serotonergic neurons through PDZ-binding domains (PDZ is an acronym combining the first letters of three proteins — post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1)). This negatively regulates SERT activity, and SERT-mediated 5-HT uptake enhances nNOS activity. Conversely, administration of selective 5-HT reuptake inhibitors or tricyclic antidepressants into the hippocampus reduces nNOS activity. In an effort to reduce the cardiovascular side effect of the triptans, hybrid compounds with both selective nNOS inhibition and 5HT1D/1B agonism were designed. The triptan-like 35 inhibits nNOS (IC50 ¼ 0.89 mM, 40-fold selective over eNOS) and binds to both the 5HT1D and the 5HT1B receptors (5HT1D IC50 ¼ 130 nM and 5HT1B IC50 ¼ 310 nM) [48].
5.3 Dual action iNOS inhibitors In an inflammatory setting or in the presence of endotoxin and cytokines, the inducible NOS isoform is expressed in numerous cell types. Cyclooxygenase (COX), an enzyme which converts arachidonic acid to prostaglandins (PG), is another critical enzyme in many inflammatory diseases. Studies have shown that production of high levels of PG can be augmented in the presence of NO. Early amino acid–selective inhibitors of iNOS such as N-iminoethyl-L-lysine (L-NIL) have been shown to not only block NO formation but also attenuate the elevated release of prostaglandins [82]. The c-Src kinase was identified as a proto-oncogene, and in some abnormal cases, such as mutation or over-expression, these enzymes can become hyperactivated, resulting in uncontrolled cell proliferation. The role of iNOS during tumor development is highly complex and incompletely understood, but there is evidence to show that the malignant transformation, angiogenesis, and metastasis effects of tumors are all modulated by iNOS. A potential anticancer strategy involves the dual inhibition of c-Src and iNOS, two key enzymes in tumorigenesis.
46
Shawn Maddaford et al.
Modification of a protein tyrosine kinase inhibitor to incorporate an NOS pharmacophore led to compound 36. This compound potently inhibits c-Src kinase (IC50 ¼ 9.2 nM) and is a modest inhibitor for iNOS of mouse macrophage ANA-1 (IC50 ¼ 2.2 mM) [83]. Studies have shown that iNOS and peroxisome proliferator-activated receptor g (PPARg) play important roles in neuroinflammation. For instance, the PPARg agonists Rosiglitazone and Pioglitazone, which are approved for the treatment of diabetes, preserve cognitive function in early Alzheimer’s patients and attenuate learning and memory deficits in transgenic Alzheimer mouse models. In a rat spinal cord injury model, treatment of rats with thiazolidinedione PPARg agonists prevented neuronal damage, motor dysfunction, myelin loss, and the development of neuropathic pain [84]. Similarly, increased iNOS function is important in neuroinflammation and pain. A series of 2-phenyl-ethenesulfonic acid phenyl esters were synthesized and shown to suppress NO production in LPS/interferon g-stimulated RAW 264.7 cells and activate PPARg in a cell-based transactivation assay [85]. Compound 37 inhibits iNOS activity (IC50 ¼ 1.8 mM) in a whole cell assay and binds competitively to the PPARg receptor (IC50 ¼ 1.4 mM).
O
Cl
N
O
N
O S
N O
CN
O Cl
NH
S
Cl
N
36
37
6. CONCLUSIONS The preceding sections of this review exemplify the connection between NO and various central nervous system (CNS) disorders. Therefore, targeting isoforms of NOS may be a valid strategy to produce novel mechanism-based therapeutics. Despite the enormous efforts by multiple groups to obtain isoform-selective compounds, it is surprising that no candidates have progressed into the clinic until recently. One of the reasons may be that in most cases, the enzymes chosen for primary assay were of rat, mouse, or bovine origin and not of human origin. Although
Advancements in the Development of NOS Inhibitors
47
there is considerable homology between the enzymes from different species, differences in the inhibitory potencies exist between species (e.g., compound 16b). Therefore, a rat isoform-selective compound showing the desired pharmacological effect in rats (usual animal species for initial screening) may not translate into humans because of low potency or non-selectivity in human forms of the enzymes. Still, tremendous progress has been achieved in the design and synthesis of selective nNOS or iNOS inhibitors using various techniques including structure-based design and HTS. These recent compounds are more drug-like as demonstrated by preclinical animal studies and recent clinical trials. Novel chemical entities that combine several selective and additive mechanisms into one molecule (designed multiple ligands) [86] may also address the failure of current analgesics to control hyperalgesia and allodynia. It can be hoped that NOS inhibitors with appropriate potency and selectivity, and dual action selective nNOS or iNOS small molecule inhibitors incorporating additional mechanisms of action will ultimately improve the current standard of care for pain management [87] and the treatment of CNS disorders.
REFERENCES [1] L. J. Ignarro, G. M. Buga, K. S. Wood, R. E. Byrns and G. Chaudhuri, Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 9265. [2] R. F. Furchgott, in Mechanisms of Vasodilation (ed. P. M. Vanhoutte), Raven, New York, 1988, pp. 401–404. [3] P. Vallance and J. Leiper, Nat. Rev. Drug. Disc., 2002, 1, 939. [4] A. J. Cayatte, J. J. Palacino, K. Horten and R. A. Cohen, Arterioscler. Thromb., 1994, 14, 753. [5] J. M. Fukuto and Y. Komori, Annu. Rep. Med. Chem., 1994, 29, 83. [6] J. E. MacDonald, Annu. Rep. Med. Chem., 1996, 31, 221. [7] D. J. Stuehr, Annu. Rev. Pharmacol. Toxicol., 1997, 37, 339. [8] D. S. Bredt, P. M. Hwang, C. E. Glatt, C. Lowenstein, R. R. Reed and S. H. Snyder, Nature, 1991, 351, 714. [9] H. J. Cho, Q. W. Xie, J. Calaycay, R. A. Mumford, K. M. Swiderek, T. D. Lee and C. Nathan, J. Exp. Med., 1992, 176, 599. [10] T. O. Fischmann, A. Hruza, X. D. Niu, J. D. Fossetta, C. A. Lunn, E. Dolphin, A. J. Prongay, P. Reichert, D. J. Lundell, S. K. Narula and P. C. Weber, Nat. Struct. Biol., 1999, 6, 233. [11] S. Pou, L. Keaton, W. Surichamorn and G. M. Rosen, J. Biol. Chem., 1999, 274, 9573. [12] B. R. Crane, A. S. Arvai, R. Gachhui, C. Wu, D. K. Ghosh, E. D. Getzoff, D. J. Stuehr and J. A. Tainer, Science, 1997, 278, 425. [13] B. R. Crane, A. S. Arvai, D. K. Ghosh, C. Wu, E. D. Getzoff, D. J. Stuehr and J. A. Tainer, Science, 1998, 279, 2121. [14] C. S. Raman, H. Li, P. Marta´sek, V. Kral, B. S. S. Masters and T. L. Poulos, Cell, 1998, 95, 939. [15] H. Li, C. S. Raman, C. B. Glaser, E. Blasko, T. A. Young, J. F. Parkinson, M. Whitlow and T. L. Poulos, J. Biol. Chem., 1999, 274, 21276.
48
Shawn Maddaford et al.
[16] H. Li, H. Shimizu, M. Flinspach, J. Jamal, W. Yang, M. Xian, T. Cai, E. Z. Wen, Q. Jia, P. G. Wang and T. L. Poulos, Biochemistry, 2002, 41, 13868. [17] E. D. Garcin, A. S. Arvai, R. J. Rosenfeld, M. D. Kroeger, B. R. Crane, G. Andersson, G. Andrews, P. J. Hamley, P. R. Mallinder, D. J. Nicholls, S. A. St-Gallay, A. C. Tinker, N. P. Gensmantel, A. Mete, D. R. Cheshire, S. Connolly, D. J. Stuehr, A. Aberg, A. V. Wallace, J. A. Tainer and E. D. Getzoff, Nat. Chem. Biol., 2008, 4, 700. [18] J. Haitao, L. Huiying, P. Marta´sek, L. J. Roman, T. L. Poulos and R. B. Silverman, J. Med. Chem., 2009, 52, 779. [19] M. L. Flinspach, H. Li, J. Jamal, W. Yang, H. Huang, J.-M. Hah, J. A. Go´mez-Vidal, E. A. Litzinger, R. B. Silverman and T. L. Poulos, Nat. Struct. Mol. Biol., 2004, 11, 54. [20] C. S. Raman, H. Li, P. Marta´sek, G. Southan, B. S. S. Masters and T. L. Poulos, Biochemistry, 2001, 40, 13448. [21] A. M. Davis, S. A. St-Gallay and G. J. Kleywegt, Drug. Disc. Today, 2008, 13, 831. [22] J. Seo, J. Igarashi, H. Li, P. Marta´sek, L. J. Roman, T. L. Poulos and R. B. Silverman, J. Med. Chem., 2007, 50, 2089. [23] E. P. Erdal, E. A. Litzinger, J. Seo, Y. Zhu, H. Ji and R. B. Silverman, Curr. Top. Med. Chem., 2005, 5, 603. [24] R. B. Silverman, Acc. Chem. Res., 2009, 42, 439. [25] A. C. Tinker and A. V. Wallace, Curr. Top. Med. Chem., 2006, 6, 77. [26] A. Tafi, L. Angeli, G. Venturini, M. Travagli, F. Corelli and M. Botta, Curr. Med. Chem., 2006, 13, 1929. [27] L. G. Howes and D. G. Brillante, Exp. Opin. Invest. Drugs, 2008, 17, 1573. [28] G. Folkerts and F. P. Nijkamp, Curr. Pharm. Des., 2006, 12, 3221. [29] J. Olesen, Pharmacol. Ther., 2008, 120, 157. [30] P.-E. Chabrier, C. Demerle-Pallardy and M. Auguet, Cell. Mol. Life Sci., 1999, 55, 1029. [31] D. O. Stichtenoth and J. C. Frolish, Br. J. Rheumatol., 1998, 37, 246. [32] H. Ji, B. Z. Stanton, J. Igarashi, H. Li, P. Marta´sek, L. J. Roman, T. L. Poulos and R. B. Silverman, J. Am. Chem. Soc., 2008, 130, 3900. [33] R. L. C. Handy, P. Wallace, Z. A. Gaffen, K. J. Whitehead and P. K. Moore, Br. J. Pharmacol., 1995, 116, 2349. [34] R. L. C. Handy and P. K. Moore, Life Sci., 1997, 60, 389. [35] M. Boulouard, P. Schumann-Bard, S. Butt-Gueulle, E. Lohou, S. Stiebing, V. Collot and S. Rault, Bioorg. Med. Chem. Lett., 2007, 17, 3177. [36] B. Cottyn, F. Acher, B. Ramassamy, L. Alvey, M. Lepoivre, Y. Frapart, D. Stuehr, D. Mansuy, J.-L. Boucher and D. Vichard, Bioorg. Med. Chem., 2008, 16, 5962. [37] V. Volke, G. Wegener, M. Bourin and E. Vasar, Behav. Brain Res., 2003, 140, 141. [38] G. Ulak, O. Mutlu, F. Y. Akar, F. I. Komsuog˘lu, P. Tanyeri and B. F. Erden, Pharmacol. Biochem. Behav., 2008, 90, 563. [39] L. Salerno, V. Sorrenti, C. Di Giacomo, G. Romeo and M. A. Siracusa, Curr. Pharm. Des., 2002, 8, 177. [40] M. J. O’Neill, T. K. Murray, D. R. McCarty, C. A. Hicks, C. P. Dell, K. E. Patrick, M. A. Ward, D. J. Osborne, T. R. Wiernicki, C. R. Roman, D. Lodge, J. H. Fleisch and J. Singh, Brain Res., 2000, 871, 234. [41] C. A. Hicks, M. A. Ward, J. B. Swettenham and M. J. O’Neill, Eur. J. Pharmacol., 1999, 381, 113. [42] R. A. Lefebvre, J. M. C. Dick, S. Guerin and C.-H. Malbert, Eur. J. Pharmacol., 2005, 525, 143. [43] S. Jaroch, P. Holscher, H. Rehwinkel, D. Sulzle, G. Burton, M. Hillmann and F. M. McDonald, Bioorg. Med. Chem. Lett., 2003, 13, 1981. [44] S. Jaroch, H. Rehwinkel, P. Holscher, D. Sulzle, G. Burton, M. Hillmann, F. M. McDonald and H. Miklautz, Bioorg. Med. Chem. Lett., 2004, 14, 743. [45] J. Patman, N. Bhardwaj, J. Ramnauth, S. C. Annedi, P. Renton, S. P. Maddaford, S. Rakhit and J. S. Andrews, Bioorg. Med. Chem. Lett., 2007, 17, 2540.
Advancements in the Development of NOS Inhibitors
49
[46] J. Ramnauth, N. Bhardwaj, S. Rakhit and S. Maddaford, US Patent 7141595 B2, 2006. [47] S. Maddaford, J. Ramnauth, S. Rakhit, J. Patman, P. Renton and S. C. Annedi, PCT Patent WO 2007118314 A1, 2007. [48] S. Maddaford, J. Ramnauth, S. Rakhit, J. Patman, P. Renton and S. C. Annedi, US Patent US2006258721 A1, 2006. [49] S. Maddaford, J. Ramnauth, S. Rakhit, J. Patman, S. C. Annedi, J. Andrews, P. Dove, S. Silverman and P. Renton, PCT Patent WO 2008116308 A1, 2008. [50] J. A. Lowe III, W. Qian, R. A. Volkmann, S. Heck, J. Nowakowski, R. Nelson, C. Nolan, D. Liston, K. Ward, S. Zorn, C. Johnson, M. Vanase, W. S. Faraci, K. A. Verdries, J. Baxter, S. Doran, M. Sanders, M. Ashton, P. Whittle and M. Stefaniak, Bioorg. Med. Chem. Lett., 1999, 9, 2569. [51] J. A. Lowe III, W. Qian, S. E. Drozda, R. A. Volkmann, D. Nason, R. B. Nelson, C. Nolan, D. Liston, K. Ward, S. Faraci, K. Verdries, P. Seymour, M. Majchrzak, A. Villalobos and W. F. White, J. Med. Chem., 2004, 47, 1575. [52] H. Matter, H. S. A. Kumar, R. Fedorov, A. Frey, P. Kotsonis, E. Hartmann, L. G. Froehlich, A. Reif, W. Pfleiderer, P. Scheurer, D. K. Ghosh, I. Schlichting and H. H. H. W. Schmidt, J. Med. Chem., 2005, 48, 4783. [53] R. J. Young, R. M. Beams, K. Carter, H. A. R. Clark, D. M. Coe, C. L. Chambers, P. I. Davies, J. Dawson, M. J. Drysdale, K. W. Franzman, C. French, S. T. Hodgson, H. F. Hodson, S. Kleanthous, P. Rider, D. Sanders, D. A. Sawyer, K. J. Scott, B. G. Shearer, R. Stocker, S. Smith, M. C. Takley and R. G. Knowles, Bioorg. Med. Chem. Lett., 2000, 10, 597. [54] R. N. Guthikonda, S. K. Shah, S. G. Pacholok, J. L. Humes, R. A. Mumford, S. K. Grant, R. M. Chabin, B. G. Green, N. Tsou, R. Ball, D. S. Fletcher, S. Luell, D. E. MacIntyre and M. MacCoss, Bioorg. Med. Chem. Lett., 2005, 15, 1997. [55] S. Connolly, A. Aberg, A. Arvai, H. G. Beaton, D. R. Cheshire, A. R. Cook, S. Cooper, D. Cox, P. Hamley, P. Mallinder, I. Millichip, D. J. Nicholls, R. J. Rosenfeld, S. A. St-Gallay, J. Tainer, A. C. Tinker and A. V. Wallace, J. Med. Chem., 2004, 47, 3320. [56] A. Strub, W.-R. Ulrich, C. Hesslinger, M. Eltze, T. Fuchss, J. Strassner, S. Strand, M. D. Lehner and R. Boer, Mol. Pharmacol., 2006, 69, 328. [57] A. C. Tinker, H. G. Beaton, N. Boughton-Smith, T. R. Cook, S. L. Cooper, L. Fraser-Rae, K. Hallam, P. Hamley, T. McInally, D. J. Nicholls, A. D. Pimm and A. V. Wallace, J. Med. Chem., 2003, 46, 913. [58] S. A. Jackson, S. Sahni, L. Lee, Y. Luo, T. R. Nieduzak, G. Liang, Y. Chiang, N. Collar, D. Fink, W. He, A. Laoui, J. Merrill, R. Boffey, P. Crackett, B. Rees, M. Wong, J.-P. Guilloteau, M. Mathieu and S. S. Rebello, Bioorg. Med. Chem., 2005, 13, 2723. [59] B. Le Bourdonnec, L. K. Leister, C. A. Ajello, J. A. Cassel, P. R. Seida, H. O’Hare, M. Gu, G.-H. Chu, P. A. Tuthill, R. N. DeHaven and R. E. Dolle, Bioorg. Med. Chem. Lett., 2008, 18, 336. [60] D. D. Davey, M. Alder, D. Arnaiz, K. Eagen, S. Erickson, W. Guilford, M. Kenrick, M. M. Morrissey, M. Ohlmeyer, G. Pan, V. M. Paradkar, J. Parkinson, M. Polokoff, K. Saionz, C. Santos, B. Subramanyam, R. Vergona, R. G. Wei, M. Whitlow, B. Ye, Z. Zhao, J. J. Devlin and G. Phillips, J. Med. Chem., 2007, 50, 1146. [61] J. Olesen, H. K. Iversen and L. L. Thomsen, NeuroReport, 1993, 4, 1027. [62] J. Olesen, L. L. Thomsen and H. Iversen, Trends in Pharmacol. Sci., 1994, 15, 149. [63] L. L. Thomsen, H. K. Iversen, T. A. Brinck and J. Olesen, Cephalalgia, 1993, 13, 395. [64] S. K. Afridi, H. Kaube and P. J. Goadsby, Pain, 2004, 110, 675. [65] P. Bellantonio, G. Micieli, M. G. Buzzi, S. Marcheselli, A. E. Castellano, F. Rossi and G. Nappi, Cephalalgia, 1997, 17, 183. [66] L. H. Lassen, M. Ashina, I. Christiansen, V. Ulrich and J. Olesen, Lancet, 1997, 349, 401. [67] L. H. Lassen, H. K. Iversen and J. Olesen, Eur. J. Clin. Pharmacol., 2003, 59, 499.
50
Shawn Maddaford et al.
[68] L. H. Lassen, M. Ashina, I. Christiansen, V. Ulrich, R. Grover, J. Donaldson and J. Olesen, Cephalalgia, 1998, 18, 27. [69] M. Ashina, L. H. Lassen, L. Bendtsen, R. Jensen and J. Olesen, Lancet, 1999, 353, 287. [70] M. Ashina, Exp. Opin. Pharmacother., 2002, 3, 395. [71] L. H. Lassen, B. Sperling, A. R. Andersen and J. Olesen, Cephalalgia, 2005, 25, 344. [72] K. Hoye, B. E. Laurijssens, L. O. Harnisch, C. K. Twomey, R. M. Dixon, A. Kirkham, P. M. Williams and A. L. Wentz, Cephalalgia, 2009, 29, 132. [73] J. E. Palmer, F. L. Guillard, B. E. Laurijssens, A. L. Wentz, R. M. Dixon and P. M. Williams, Cephalalgia, 2009, 29, 124. [74] D. Singh, D. Richards, R. G. Knowles, S. Schwartz, A. Woodcock, S. Langley and B. J. O’Connor, Am. J. Respir. Crit. Care Med., 2007, 176, 988. [75] A. Mosek, S. Groppa and R. Barkan, Cephalalgia, 2009, 29, 118. [76] D. Vaughan, J. Speed, R. Medve and J. S. Andrews, unpublished results. [77] R. A. Medve and J. S. Andrews, Cephalalgia, 2009, 29, 126. [78] P.-H. M. van der Kuy, F. W. H. M. Merkus, J. J. H. M. Lohman, J. W. M. ter Berg and P. M. Hooymans, Cephalalgia, 2002, 22, 513. [79] S. Auvin, M. Auguet, E. Navet, J. J. Harnett, I. Viossat, J. Schulz, D. Bigg and P.-E. Chabrier, Bioorg. Med. Chem. Lett., 2003, 13, 209. [80] P.-E. Chabrier, M. Auguet, B. Spinnewyn, S. Auvin, S. Cornet, C. Demerle-Pallardy, C. Guilmard-Favre, J.-G. Marin, B. Pignol, V. Gillard-Roubert, C. Roussillot-Charnet, J. Schulz, I. Viossat, D. Bigg and S. Moncada, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 10824. [81] P. Renton, S. Maddaford, S. Rakhit and J. Andrews, PCT Int. Appl., WO2007017764 A2, 2007. [82] D. Salvemini, P. T. Manning, B. S. Sweifel, K. Seibert, J. Connor, M. G. Currie, P. Needleman and J. L. Masferrer, J. Clin. Invest., 1995, 96, 301. [83] X. Cao, Q.-D. You, Z.-Y. Li, Q.-L. Guo, J. Shang, M. Yan, J.-W. Chern and M.-L. Chen, Bioorg. Med. Chem., 2008, 16, 5890. [84] S.-W. Park, J.-H. Yi, G. Miranpuri, I. Satriotomo, K. Bowen, D. K. Resnick and R. Vemuganti, J. Pharm. Exp. Ther., 2007, 320, 1002. [85] Y.-Z. Lee, C.-W. Yang, I.-J. Kang, S.-H. Wu, Y.-S. Chao, J.-H. Chern and S.-J. Lee, Bioorg. Med. Chem. Lett., 2008, 18, 5676. [86] R. Morphy and Z. Rankovic, J. Med. Chem., 2005, 48, 1. [87] J. Woodcock, J. Witter and R. A. Dionne, Nat. Rev. Drug Discovery, 2007, 6, 703.
CHAPT ER
3 Small-Molecule Protein–Protein Interaction Inhibitors as Therapeutic Agents for Neurodegenerative Diseases: Recent Progress and Future Directions Simon N. Haydar*, Heedong Yun*, Roland G.W. Staal** and Warren D. Hirst**
Contents
1. Introduction 2. Ab Aggregation and Oligomers in Alzheimer’s Disease 2.1 Ab aggregation and neurotoxic oligomers 2.2 Small-molecule inhibitors of Ab aggregation 3. Tau Aggregation in Alzheimer’s Disease 3.1 Tau pathophysiology 3.2 Small-molecule inhibitors of tau aggregation 4. a-Synuclein Aggregation in Parkinson’s Disease 4.1 Biochemistry of a-synuclein aggregation 4.2 Small-molecule inhibitors of a-synuclein aggregation 5. Conclusion References
51 52 52 53 58 58 59 63 63 64 65 66
* Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543 ** Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543 Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04403-0
r 2009 Elsevier Inc. All rights reserved.
51
52
Simon N. Haydar et al.
1. INTRODUCTION Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common chronic, progressive neurodegenerative diseases affecting an estimated 10% and 1%, respectively, of the elderly population [1,2], with financial costs of hundreds of billions of dollars per year. AD is characterized by the presence of extracellular parenchymal and vascular amyloid deposits containing b-amyloid peptide (Ab) and intracellular neuronal tangles composed of hyperphosphorylated tau. a-Synuclein containing Lewy bodies, spherical inclusions found in the cytoplasm of surviving neurons, are the cardinal hallmark of PD. Despite their distinct pathologies, these neurodegenerative diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. Compelling evidence strongly supports the hypothesis that accumulation of misfolded proteins leads to synaptic dysfunction, neuronal apoptosis, brain damage, and disease. However, the mechanisms by which protein misfolding and aggregation trigger neurodegeneration and the identity of the neurotoxic structures are still unclear. Current hypotheses propose that, in the aggregation process, there is an accumulation of small soluble oligomeric intermediates, which leads to the neuropathology, whereas the large insoluble deposits that make up the inclusion bodies might function as reservoirs of these toxic, soluble oligomers [3]. As we increase our knowledge of the role of oligomeric, fibrillar, and higher-order molecular entities of the misfolded proteins in neurodegenerative diseases, new approaches may offer themselves for therapeutic intervention. Over the past few years, there has been significant interest in developing therapeutics and chemical probes that inhibit these specific protein–protein interactions. This effort has been hampered by the size and the geometry of the protein interaction interface, which are devoid of defined ‘‘pockets’’ into which a small molecule can bind in an energetically favorable manner [4]. Despite the challenges of developing compounds that are capable of specifically inhibiting protein–protein interactions, there are a number of examples of small molecules that achieve this with reasonable potency [5]. This was made possible because of the discovery of ‘‘hot spots’’ on the protein interaction surfaces [6]. These ‘‘hot spots’’ are small regions on the protein interaction interface that are responsible for a disproportionate contribution to the binding energy of the two proteins. This review will highlight the recent progress in the development of small-molecule protein–protein interaction inhibitors that have applications in furthering the mechanistic understanding of neurodegenerative diseases and will potentially lead to the development of rational therapeutics.
Therapeutic Agents for Neurodegenerative Diseases
53
2. Ab AGGREGATION AND OLIGOMERS IN ALZHEIMER’S DISEASE 2.1 Ab aggregation and neurotoxic oligomers Substantial genetic and physiological evidence suggest that the Ab plays a central role in AD pathogenesis. Ab is a 39- to 42-amino acid peptide derived from the proteolytic processing of the amyloid precursor protein (APP) by secretases [7]. Gradual changes in the steady-state levels of Ab in the brain are thought to initiate the amyloid cascade [8,9]. Since the elucidation of the Ab sequences [10–12], investigators have used synthetic Ab to examine aggregation and its effects on physiology. Many in vitro studies have suggested that aggregation of Ab is essential for toxicity, but characterization of the Ab species that formed was limited. However, amyloid plaque number does not correlate well with severity of dementia [13–15], and instead, there is a stronger link between soluble Ab levels and the extent of synaptic loss and the severity of cognitive impairment [16–18]. Therefore, more recent studies have focused on various soluble forms of synthetic Ab1-40 and Ab1-42, ranging from monomeric to protofibrils [19]. A number of reports have described the biochemical characterization of the soluble Ab extracted from human AD brain. The presence of sodium dodecyl sulfate (SDS)-stable dimers and trimers in the soluble fraction of human brain and in extracts of amyloid plaques suggests that SDS-stable, low n oligomers of Ab are the fundamental building blocks of insoluble amyloid deposits and could be the earliest mediators of neuronal dysfunction [20]. Recent studies have described the physiological characterization of Ab dimers isolated from AD brains that inhibit long-term potentiation (LTP), a physiological correlate of memory, and reduce dendritic spine density in normal rodent hippocampus [21]. In addition, the dimers disrupt memory of a learned behavior when directly injected into the brains of normal rats. As outlined earlier, a number of Ab assemblies have been proposed to exert neurotoxic effects, but with evidence only recently emerging on which forms that are the pathological species in vivo, and the scarcity of structural data on the oligomers complicates a rational search for compounds that could inhibit Ab aggregation and toxicity. Despite these obstacles, a number of different compounds that interfere with Ab aggregation, in one way or another, have been described; these are reviewed in the following sections.
2.2 Small-molecule inhibitors of Ab aggregation 2.2.1 Scyllo-inositol Recent in vitro studies with scyllo-inositol (1) have shown that it can interact with Ab42 peptide promoting a conformational change from
54
Simon N. Haydar et al.
random coil to b-sheet structure and stabilized it in small, nonfibrillar complexes, blocking fibril formation [22]. These stabilized complexes were significantly less toxic to neuronal cell lines and primary neuronal cultures than untreated Ab42 or chiro-inositol-treated Ab42 [22]. Moreover, in in vivo studies in the TgCRND8 mouse model of AD, it was shown that compound 1 inhibited Ab aggregation, decreased Ab-induced impairments in spatial memory, reduced the cerebral Ab pathology, and attenuated the rate of mortality [23]. To explore the molecular details of the inositol–Ab42 interaction, a series of scylloinositols were prepared in which one or two hydroxyl groups were replaced with fluoro, chloro, methoxy, or hydrogen substituents [24]. After incubation with Ab42 for 7 days, the activity of the derivatives was measured by electron microscopy to monitor the formation of Ab42 fibers. Despite the synthesis of numerous analogs, only single hydroxyl substitutions such as 1-deoxy-1-fluoro-scyllo-inositol (2) and the disubstituted analog 1,4-dimethoxy-scyllo-inositol (3) were shown to have similar activity to the parent compound 1. Compound 3 was shown to exhibit the most pronounced effect on Ab42 aggregation, in that it produced a more homogenous population of small amorphous aggregates and no fibers were detected [24]. OH
HO HO HO
OH OH 1
OH
HO HO HO
OH F 2
OH
MeO HO HO
OH OMe 3
2.2.2 Tricyclic pyrones and pyridinones Hua et al. [25] used a neuronal cell line overexpressing a C-terminal fragment of APP (MC65 cells) to identify inhibitors of toxicity related to intracellular Ab and discovered a class of tricyclic pyrones (TP). In particular, a TP 4 that contains an adenine moiety (at N-3u) attached at the C7-alkyl side chain of the ring system showed significant protection in the MC65 cell assay with an EC50 value of 0.31 mM [25]. Further characterization of compound 4 using surface plasmon resonance (SPR) spectroscopy, atomic force microscopy (AFM), and protein quantification studies showed it binds to Ab42 oligomers, inhibits Ab aggregation, and disaggregates Ab42 oligomers and protofibrils [26]. Transgenic mice treated for 2 weeks with compound 4, administered i.c.v., resulted in 40% and 50% decreases in non-fibrillar and fibrillar Ab species, respectively. [26]. To further investigate the structure activity relationship, various heterocycles and nitrogen-containing TP were prepared [27]. It was concluded that attachment of N3u-adenine at C7 side chain in compound
Therapeutic Agents for Neurodegenerative Diseases
55
4 provides the strongest MC65 protective activity with an EC50 of 0.31 mM. However, tricyclic pyranoisoquinolinones lacking the adenine moeity as in compounds 5 and 6 still possess protective activity with EC50 values of 2.49 and 1.25 mM, respectively. The 6u-amino group in compound 4 enhances potency but is not required for activity. O
O 1
10
H
O N
7
NH2
N 14
12
N
3′
N 9′
4
OMe O
O
O H O
N
N 5
6
Among the various C7 side chain heterocycles prepared, none showed better protective activity than the adenine in compound 4. It is noteworthy that the most potent analog prepared in this class was a 2-aminopurine derivative 7 with EC50 of 0.86 mM. Replacement of the oxygen at position 2 with a nitrogen, as in compound 8, provides similar protective activity (EC50 ¼ 0.35 mM) as that of compound 4 [27]. O
O
OMe
1 10
H
O
O
H O
N 7
N
N
N 14
12
3′
N 7
N NH2
N
N N
NH2 8
56
Simon N. Haydar et al.
2.2.3 Indoles Highly ordered p-stacking interactions between aromatic ring systems are important in self-assembly of complex biological and chemical supramolecular structures [28]. Several studies showed that aromatic interactions may play a critical role in early-stage amyloid formation by providing stability, as well as order and directionality in the formation of amyloid fibrils, presumably facilitated by restricted geometry of interaction between planar aromatic systems [29]. In particular, in amyloid peptide fragments, a high frequency of aromatic residues was noted. When these aromatic residues were replaced with hydrophobic amino acids, a decrease in the amyloidogenic propensity was observed [30]. Gazit et al. screened various indole derivatives for their ability to prevent formation of amyloid fibrils using fluorescence spectroscopy, AFM, and electron microscopy. Three inhibitors were identified: indole-3-carninol (9), 3hydroxyindole (10), and 4-hydroxyindole (11) with IC50 values of 85, 100, and 200 mM, respectively. These simple indoles effectively inhibited Ab fibril formation and prevented cell death induced by 5 mM Ab40 in PC12 cells in culture. Curiously, analog 9 inhibited Ab fibrillization only at high concentration, and it did not show a dose dependency on inhibition of fibril formation. The inhibitory mechanism of these compounds remains unclear; however, the authors suggest that the hydroxyl group interacts with the backbone of the peptides preventing the ability of the Ab peptide to create a p-stacking interaction, which limit the fibrillogenesis process. OH
OH
HO
N H
N H 9
N H 10
11
Recent adsorption studies of Ab solutions on poly(tetrafluoroethylene) surfaces showed that the fluorinated surface strongly promoted a-helix re-formation [31]. A similar effect was observed with solution of Ab in CF3-containing solvents [32]. On the basis of these findings, Torok et al. [33] demonstrated the design and application of a new class of trifluoroethylindoles against amyloid fibrillogenesis. Analogs 12a–c showed significant inhibitory effect with IC50 values of 0.53, 0.23, and 0.36 molinhibitor/molAb. Further structure–activity relationship demonstrated that the CF3 and OH groups are necessary for binding to Ab peptide. It was suggested that the acidity of the hydroxyl group plays a key role in binding to one or both lysine residues of the Ab peptide. Interestingly, removing the ester group slightly diminished the inhibitory activity.
Therapeutic Agents for Neurodegenerative Diseases
57
HO CO Et 2 F3C X N H 12a, b, c a; X = Cl b; X = Br c; X = I
Tramiprosate (3-amino-1-propanesulfonic acid; 3APS; Alzhemedt) (13) was found to maintain Ab in a non-fibrillar form, decrease Ab42-induced cell death in neuronal cell cultures, and also inhibit amyloid deposition [34]. Treatment of TgCRND8 mice with Tramiprosate resulted in significant reduction (B30%) in the brain amyloid plaque load and a significant decrease in the cerebral levels of soluble and insoluble Ab40 and Ab42 (B20–30%) [34]. Although Tramiprosate ultimately failed in a phase III clinical trial, it provided a proof of concept that small-molecule inhibitors of Ab protofibril formation may be a viable approach to AD treatment [5].
H2N
O O S OH
13, Tramiprosate
Another recent development in Ab aggregation inhibitors was the development of Memoquin (14). This compound was shown to be a multifunctional therapy to AD, acting as an acetyl cholinesterase (AchE) inhibitor (Ki ¼ 2.6 nM), a free radical scavenger, and an inhibitor of Ab aggregation [35,36].
N O
O
H N
N H
O
N
O 14, Memoquin
Additional small molecules such as Congo red, curcumin, and galantamine have been described in the literature as inhibitors of Ab aggregation, recently reviewed by Hawkes et al. (2009) [37]. These molecules were also found to be inhibitors of a-synuclein, and we will report on their activity in Section 4.2.
58
Simon N. Haydar et al.
3. TAU AGGREGATION IN ALZHEIMER’S DISEASE 3.1 Tau pathophysiology Tau is predominantly expressed in neurons where its main function is thought to be stabilizing microtubules, particularly in axons. The tau gene consists of 16 exons and alternative splicing results in six isoforms of tau protein ranging in size from 352–441 amino acids [38]. Tau stabilizes microtubules by binding to them through an interaction with the three or four microtubule-binding domains at the C-terminus of the protein. Stabilization of microtubules by tau in neurons is important for maintenance of cellular morphology and transport of molecules and organelles over long distances [39]. Binding of tau to microtubules is also regulated post-translationally, primarily through phosphorylation, although other modifications such as glycosylation, ubiquitylation, and proteolysis have been reported for the tau protein [40]. Tau contains W80 serine and threonine residues, which are potential phosphorylation sites. The phosphorylation state, which is controlled by a balance of kinase and phosphatase activity, affects the microtubule-binding affinity. Hyperphosphorylation of tau at many sites, as seen in tauopathies, of which AD is the most common, leads to reduced affinity for microtubules, which causes disruption of cellular trafficking leading to degeneration of synaptic terminals. This loss of function may be exacerbated by a toxic gain of function, where higher than normal concentrations of tau increase the chances of pathogenic conformational changes, which in turn lead to the aggregation and fibrillization, which might block transport and cause cell death [40]. Phosphorylation of certain residues on tau, specifically S396 and S404, has been shown to increase the fibrillogenic nature of tau and contribute to its accumulation into paired helical filaments [41–43]. Similarly, the removal of the C-terminus of the protein increases tau aggregation [44,45]. It is thought that tau aggregation occurs in a multi-step process whereby tau is phosphorylated and dissociates from microtubules. The unbound hyperphosphorylated tau abnormally localizes to the somatodendritic compartment of the cell, undergoes conformational changes and further phosphorylation. Finally, the hyperphosphorylated tau forms fibrils that aggregate into neurofibrillary tangles (NFTs) [46]. Tau aggregates also form in axons and dendrites, called neuropil threads. Both NFTs and neuropil threads are postulated to have a toxic gain of function. In cell models, tau aggregation in the cell body causes cell death [47]. In axons, neuropil threads are thought to be toxic because they might physically impair transport, which would be toxic to synaptic terminals [48]. Similar to Ab, oligomeric forms of tau, which are promoted by phosphorylation [49] and are observed in aging and early AD [50], could
59
Therapeutic Agents for Neurodegenerative Diseases
also be the toxic species leading to neurodegeneration. In animal models of tauopathy, there is indirect evidence that soluble, not aggregated, forms of tau are toxic: flies expressing human tau can exhibit neurodegeneration without fibril formation [51], and in some mouse models of tauopathy, overexpression of tau causes neuronal loss in areas without extensive neurofibrillary pathology [52]. If oligomeric tau is toxic, formation of large aggregates could be viewed as protective because oligomers are sequestered into insoluble neurofibrillary pathology [3,49]. The two principal current strategies targeting tau in neurodegenerative disease are (i) reducing tau phosphorylation through inhibition of specific protein kinases [53] and (ii) anti-aggregation approaches [54]. The issue of whether phosphorylation of tau precedes or follows tau aggregation remains a subject of debate, but reducing tau phosphorylation is regarded by many as the preferred target, and some transgenic animal studies have shown this to be a valid strategy [53]. In the following sections, structurally diverse small-molecule inhibitors of tau aggregation are described.
3.2 Small-molecule inhibitors of tau aggregation 3.2.1 Phenylthiazolyl hydrazides In an effort to develop small-molecule inhibitors of tau aggregation, Mandelkow et al. [55,56] identified compounds related to structure 15 from a high throughput screen of a collection of 200,000 compounds. To establish the structure–activity relationship (SAR), a series of thiazolylhydrazides were prepared by synthetic derivatization of R1, R2, R3, and R4 [56]. Structure–activity relationship of phenylthiazolyl hydrazides demonstrated that two aromatic rings at R1 and R4, a hydrophobic region on the thiazole ring, and a hydrogen bonding acceptor on the carboxyl amide are essential for inhibitory effect of tau aggregation. Notably, compound 16 showed superior potency with IC50 value of 1.6 mM for inhibiting tau aggregation and reduced toxicity when tested in an N2A cell model of tau aggregation [57]. The potency of compound 16 is believed to be due to the hydrogen bonding capacity of the nitro group and to the p-stacking interactions with the indazole group as confirmed by saturation transfer difference (STD) NMR spectroscopy experiments. O
R2 R1
N H
H N
N
N
R4
S R3 15
N H
H N
N S
N H 16
NO2
60
Simon N. Haydar et al.
3.2.2 Rhodanine-based inhibitors Mandelkow’s group has also described the SAR of substituted rhodanines (2-thioxothiazolidin-4-ones) [58]. Extensive SAR studies resulted in the preparation of an interesting biphenyl rhodanine derivative 17, which displayed an IC50 of 170 nM for assembly inhibition and DC50 of 130 nM for disassembly induction. It is noteworthy that the presence of an aromatic side chain appeared necessary, supporting hydrophobic p-stacking interactions of this fragment [59]. Unfortunately, compound 17 and many other analogs showed a large discrepancy between in vitro and cell-based activity. Poor physiochemical properties were implicated as the likely cause of poor cellular results, reflecting a need for further optimization of this series. O
Ph
S
HO
N
S
O
O
17
3.2.3 Cationic thiacarbocyanine dye Thiacarbocyanine dye N744 (compound 18) has been shown to inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers with an IC50 value of 0.3 mM [60]. In an effort to increase potency, a cyclic bis-thiacarbocyanine 19 was synthesized and characterized with respect to tau fibrillization inhibition by electron microscopy and ligand aggregation state by absorbance spectroscopy [61]. Data showed that the inhibitory activity of the bis-thiacarbocyanine 19 was similar to a monomeric cyanine dye, but was more potent with an IC50 of 80 nM. Data reported for these two compounds further suggest that the inhibitory activity of bis-thiacarbocyanine 19 results from multivalency. This finding might offer an new mode of interaction for design of tau aggregation inhibitors [61]. S
S Br
O
S
O
S
N
N
N
Br
N N
N
HO
OH 18
S
S 19
61
Therapeutic Agents for Neurodegenerative Diseases
3.2.4 N-Phenylamines and anthraquinones Additional small molecule tau aggregation inhibitors were reported in the literature derived from N-phenylamines (20) and anthraquinones such as daunorubicin (21) and adriamycin (22) [55,62].
NO2
O 2N
O
OH
A
B
C
D
O
O
OH O
H N N
O OH
OH
20
OH
A
B
C
O
O
OH O
NH2
21
D
O
O
O
O
HO OH
O OH
OH
NH2
22
Compared to the two compound classes discussed earlier (rhodamines and phenylthiazolylhydrazides), the N-phenylamines displayed far lower potencies in vitro and in cells, which precluded their use in in vivo models [54]. The b-hydroxyenone moiety in anthraquinone, also observed in other inhibitor classes, such as flavonoids and naphthoquinones, may play a significant role in the inhibitory potency of these chemotypes. Compounds 21 and 22 were able to inhibit the aggregation of the K19 tau construct and induced the disaggregation of preformed aggregates [55]. Substitution on the ring A in compound 21 does not appear to play a critical role of inhibitory activity. Ring D in compounds 21 and 22 bearing the sugar moiety is moderately sensitive to the substitutions on that ring, which indicates further opportunity for structural modifications to improve the inhibitory potency. Despite the observed activity of anthraquinones, it should be noted that they are known cytostatics and present a hazardous toxicological profile, which preclude them from being desired therapeutics for the chronic treatment of AD.
3.2.5 Polyphenols, phenothiazines, and porphyrins Taniguchi et al. [63] reported three classes of compounds (phenols, phenothiazines, and porphyrins) that were able to inhibit aggregation of human tau 46. Polyphenols such as compound 23 shared the SAR described in the previously section for anthraquinones with characteristic b-hydroxyenone moieties. Phenothiazines possess a tricyclic core that incorporates sulfur and nitrogen atoms on the central ring system. They possess positively charged atoms and hydrophobic
62
Simon N. Haydar et al.
aromatic groups similar to the benzothiazole inhibitors described previously. The charge-neutral phenothiazines are reported to have good blood–brain barrier permeability. The planarity and aromaticity of the central heterocyclic core appears to play a critical role of tau aggregation inhibitory activity. In particular, compound 24 (MTC, methylthioninium chloride) also known as methylene blue was reported as a potent in vivo tau aggregation inhibitor with sub-micromolar potency in cells [64,65]. Data from the phase II clinical trial with methylene blue reported a significantly lower rate of decline of cognitive functions compared with a placebo (81%, po0.0001). Although these data are preliminary and require further confirmation, it does present a promising approach for the management of AD and a potential proof of concept for the strategy of inhibiting tau aggregation.
OH HO
N O
HO
OH N
S
N
HO O
OH
23
24
Porphyrins such as compound 25 are the only organometallic compounds that bind differently than other reported tau aggregation inhibitors. The inhibitory activity depends on a central metal (iron or zinc) as phthalocyanine compound lacking the central metal has a weaker inhibitory potency [63]. It has been suggested that the coordination of the metal center with histidine on the protein–protein interface plays a significant role in tau aggregation inhibition [66]. HOOCC2H4
HOOCC2H4
N N Fe N
25
N
Therapeutic Agents for Neurodegenerative Diseases
63
4. a-SYNUCLEIN AGGREGATION IN PARKINSON’S DISEASE 4.1 Biochemistry of a-synuclein aggregation a-Synuclein is a small 140 amino acid, natively unfolded protein with little secondary or tertiary structure whose aberrant aggregation has been linked to the etiology of PD. a-Synuclein can assume either an a-helical conformation upon binding to lipids and membranes or a b-sheet conformation upon aggregation at high concentrations, elevated temperature, agitation, or in the presence of metals, simple alcohols, or detergents [67,68]. Increases in b-sheet content are associated with formation of small soluble oligomers, larger protofibrils, and the macromolecular fibrils. Dimers are the smallest oligomeric aggregates with limited b-sheet structure and have been proposed to act as seeds in the nucleation-dependent aggregation of a-synuclein [69,70]. Both oligomers and protofibrils have increased amounts of b-sheet structure compared with monomeric or lipid bound a-synuclein. The predominantly observed structure of the protofibrils is globular, although they are also able to form rod-like filaments as well as annular rings that can insert into lipid membranes enabling leakage of small molecules [67]. These annular protofibrils have been implicated in the pathogenesis of PD by the virtue that the A53T and A30P mutations either increase the propensity of a-synuclein to form annular protofibrils or stabilize them. The annular protofibrils can insert into lipid membranes and enable leakage of ions, neurotransmitters, and small dyes [67]. Although these properties make the oligomers and protofibrils attractive targets, their size and morphological continuum present a tremendous logistical hurdle for assay development and structure activity relationship analysis. Eventually, a-synuclein aggregates into insoluble fibrils that have a very high b-sheet content. It is the insoluble fibril that is deposited in Lewy bodies in the brains of patients suffering from PD, which are the defining pathological hallmark of the disease. A long-standing issue has been whether Lewy bodies are markers of a neurodegenerative process or a protective mechanism, serving as a means to sequester toxic oligomers and protofibrils [3]. Still, fibrils, as the end point of aggregation and the hallmark of pathological PD, have been targeted extensively in attempts to discover small-molecule inhibitors of a-synuclein aggregation as a treatment to slow down or halt the progression of PD. Many molecules have been shown to inhibit fibril formation (curcumin, Congo red, epigallocatechin gallate, peptide-mimetics, and non-steroidal anti-inflammatories) including the flavonoid, baicalein, and the neurotransmitter, dopamine [71–77]. More detailed studies with the latter two molecules reveal that while these compounds are inhibiting
64
Simon N. Haydar et al.
fibril formation, they are also stabilizing oligomers/protofibrils [77]. In the case of dopamine, the aggregates are much more stable than the oligomers/protofibrils produced under more ‘‘conventional’’ conditions, in that they are SDS stable. Caution should also be exercised when targeting disruption of fibril b-sheet as the tertiary structure of many proteins contains b-sheets. If inhibitors of fibril formation are to be developed as a therapeutic for PD, appropriate screens should be developed to assess their ability to stabilize soluble oligomers/protofibrils, to assess the toxicity of any stabilized species and the ability to disrupt the b-sheet structure in other proteins.
4.2 Small-molecule inhibitors of a-synuclein aggregation Conway et al. [77] have shown that catecholamines such as compound 26 can inhibit the formation of a-synuclein fibrils by stabilizing oligomeric intermediates. Li et al. [78] reported that the oxidation state of catecholamine affected the inhibitory activity. As such, dopaminochrome (27), one of the oxidation products of dopamine, was shown to be more potent at inhibiting a-synuclein fibril formation than the parent dopamine [78,79]. This also raises the possibility that the protein may be covalently modified by the dopaminochrome or dopamine under oxidative conditions. H
N O
HO
N H
O
HO 26
27
Various polyphenolic compounds such as flavonoids have also been shown to be effective inhibitors of a-synuclein aggregation [80]. One of these inhibitors is baicalein, 28, isolated from the Chinese skullcap plant (Scutellaraia baicalensis), has been shown to directly bind to a single site on a-synuclein with submicromolar affinity and, as such, inhibit formation of fibrils through the stabilization of oligomers [71,81]. It has been suggested that the quinone oxidation by-product (29) of baicalein is responsible for the observed inhibitory activity. HO
O
HO
O
O
O OH O
OH O
28
29
Therapeutic Agents for Neurodegenerative Diseases
65
Other small-molecule inhibitors of a-synuclein have been identified in the literature such as Congo red.[80]. Polyphenolic compounds such as rifampicin, curcumin, and tetracycline are capable of inhibiting both a-synuclein [79] and Ab aggregation [82] in a concentration-dependent manner with some potency (IC50o10 mM). Li et al. [83] point out that many of these polyphenolic anti-fibrillogenic compounds have antioxidant activities and readily oxidize in the presence of atmospheric oxygen to form quinones and thus may all act through oxidative modification of peptides. This modification, formation of quinone adducts or formation of Schiff-base, may act to inhibit fibril formation by constraining the peptides to a conformation not compatible with the tight orderly packing of b-sheets found in fibrils, but at the same time stabilizing soluble oligomers. Although some of these compounds have been demonstrated to protect cells against a-synuclein overexpression, in agreement with the biochemistry of a-synuclein aggregation [84], many reports either attribute the efficacy of the compounds to metal chelation and antioxidant activity or do not show the mechanism to be inhibition of protein aggregation. Furthermore, caution in pursuing these polyphenolic and catecholamine compounds as aggregation inhibitors is urged, however, as one long-standing hypothesis postulates that it is actually the soluble oligomers, not the insoluble fibrils that are neurotoxic in PD. Still, these polyphenolic/ anti-oxidant types of compounds appear to share a common mechanism: oxidation-dependent modification of a-synuclein, which inhibits fibril formation. If the issues, raised earlier, are resolved in the drug discovery process, then small-molecule inhibitors of a-synuclein aggregation could be key therapeutics for PD [82]. However, in general, it has been noted from the current literature that there are few reports and limited efforts to develop structural activity relationship around the compounds listed earlier, suggesting that drug discovery strategies to identify small-molecule inhibitors of a-synuclein are still evolving.
5. CONCLUSION In summary, the literature reviewed in this chapter imply two general assumptions regarding the inhibition mechanism of amyloid protein fibril formation by small molecules: (a) specific structural conformation is necessary for b-sheet interaction and stabilization of the inhibition– protein complex; (b) aromatic interaction between the inhibitor molecule and the aromatic residues in the amyloidogenic sequence, potential ‘‘hot spots,’’ may direct the inhibitor to the amyloidogenic core blocking
66
Simon N. Haydar et al.
the protein–protein interaction. These assumptions are highly relevant for future design of small-molecule inhibitors as therapeutic agents for the treatment of amyloid-associated diseases. In conclusion, drug development for AD and PD remains highly active, and there is a realistic expectation that new therapies will be evaluated in clinical trials in the near future.
REFERENCES [1] B. L. Plassman, K. M. Langa, G. G. Fisher, S. G. Heeringa, D. R. Weir, M. B. Ofstedal, J. R. Burke, M. D. Hurd, G. G. Potter, W. L. Rodgers, D. C. Steffens, R. J. Willis and R. B. Wallace, Neuroepidemiology, 2007, 29, 125. [2] L. M. Lau and M. M. Breteler, Lancet Neurology, 2006, 5, 525. [3] C. Haass and D. J. Selkoe, Nat. Rev. Mol. Cell. Biol., 2007, 8, 101. [4] M. R. Arkin and J. A. Wells, Nat. Rev. Drug Discov., 2004, 3, 301. [5] L. L. Blazer and R. R. Neubig, Neuropsychopharmacology, 2009, 34, 126. [6] A. A. Bogan and K. S. Thorn, J. Mol. Biol., 1998, 280, 1. [7] D. J. Selkoe, Physiol. Rev., 2001, 81, 741. [8] D. J. Selkoe, Neuron, 1991, 6, 487. [9] J. A. Hardy and G. A. Higgins, Science, 1992, 256, 184. [10] G. G. Glenner and C. W. Wong, Biochem. Biophys. Res. Commun., 1984, 120, 885. [11] C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald and K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 4245. [12] D. J. Selkoe, C. R. Abraham, M. B. Podlisny and L. K. Duffy, J. Neurochem., 1986, 46, 1820. [13] R. Katzman, N. Engl. J. Med., 1986, 314, 964. [14] R. D. Terry, E. Masliah, D. P. Salmon, N. Butters, R. DeTeresa, R. Hill, L. A. Hansen and R. Katzman, Ann. Neurol., 1991, 30, 572. [15] D. W. Dickson, H. A. Crystal, C. Bevona, W. Honer, I. Vincent and P. Davies, Neurobiol. Aging, 1995, 16, 285. [16] L. F. Lue, Y. M. Kuo, A. E. Roher, L. Brachova, Y. Shen, L. Sue, T. Beach, J. H. Kurth, R. E. Rydel and J. Rogers, Am. J. Pathol., 1999, 155, 853. [17] C. A. McLean, R. A. Cherny, F. W. Fraser, S. J. Fuller, M. J. Smith, K. Beyreuther, A. I. Bush and C. L. Masters, Ann. Neurol., 1999, 46, 860. [18] J. Wang, D. W. Dickson, J. Q. Trojanowski and V. M. Lee, Exp. Neurol., 1999, 158(2), 328. [19] M. A. Findeis, Pharmacol. Ther., 2007, 116, 266. [20] D. M. Walsh and D. J. Selkoe, J. Neurochem., 2007, 101, 1172. [21] G. M. Shankar, S. Li, T. H. Mehta, A. Garcia-Munoz, N. E. Shepardson, I. Smith, F. M. Brett, M. A. Farrell, M. J. Rowan, C. A. Lemere, C. M. Regan, D. M. Walsh, B. L. Sabatini and D. J. Selkoe, Nature Med., 2008, 14, 837. [22] J. McLaurin, R. Golomb, A. Jurewicz, J. P. Antel and P. E. Fraser, J. Biol. Chem., 2000, 275, 18495. [23] J. McLaurin, M. E. Kierstead, M. E. Brown, C. A. Hawkes, M. H. L. Lambermon, A. L. Phinney, A. A. Darabie, J. E. Cousins, J. E. French, M. F. Lan, F. Chen, S. S. N. Wong, H. T. J. Mount, P. E. Fraser, D. Westaway and P. George-Hyslop, Nat. Med., 2006, 12, 801.
Therapeutic Agents for Neurodegenerative Diseases
67
[24] Y. Sun, G. Zhang, C. A. Hawkes, J. E. Shaw, J. McLaurin and M. Nitz, Bioorg. Med. Chem., 2008, 16, 7177. [25] D. H. Hua, X. Huang, M. Tamura, Y. Chen, M. Woltkamp, L.-W. Jin, E. M. Perchellet, J.-P. Perchellet, P. K. Chiang, I. Namatame and H. Tomoda, Tetrahedron, 2003, 59, 4795. [26] H.-S. Hong, S. Rana, L. Barrigan, A. Shi, Y. Zhang, F. Zhou, L.-W. Jin and D. H. Hua, J. Neurochem., 2009, 108, 1097. [27] S. Rana, H.-S. Hong, L. Barrigan, L.-W. Jin and D. H. Hua, Bioorg. Med. Chem. Lett., 2009, 19, 670. [28] A. Aggeli, M. Bell, N. Boden, J. N. Keen, P. F. Knowles, T. C. McLeish, M. Pitkeathly and S. E. Radford, Nature, 1997, 386, 259. [29] E. Gazit, FASEB J., 2002, 16, 77. [30] T. Cohen, A. Frydman-Marom, M. Rechter and E. Gazit, Biochemistry, 2006, 45, 4727. [31] C. E. Giacomelli and W. Norde, Biomacromolecules, 2003, 4, 1719. [32] E. P. Vieira, H. Hermel and H. Mohwald, Biochim. Biophys. Acta, Proteins Proteomics, 2003, 1645, 6. [33] M. Torok, M. Abid, C. Mhadgut Shilpa and B. Torok, Biochemistry, 2006, 45, 5377. [34] F. Gervais, J. Paquette, C. Morissette, P. Krzywkowski, M. Yu, M. Azzi, D. Lacombe, X. Kong, A. Aman, J. Laurin, W. A. Szarek and P. Tremblay, Neurobiol. Aging, 2007, 28, 537. [35] A. Cavalli, M. L. Bolognesi, S. Capsoni, V. Andrisano, M. Bartolini, E. Margotti, A. Cattaneo, M. Recanatini and C. Melchiorre, Angew. Chem., Int. Ed., 2007, 46, 3689. [36] A. Cavalli, M. L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini and C. Melchiorre, J. Med. Chem., 2008, 51, 347. [37] C. A. Hawkes, V. Ng and J. McLaurin, Drug Dev. Res., 2009, 70, 111. [38] A. Andreadis, W. M. Brown and K. S. Kosik, Biochemistry, 1992, 31, 10626. [39] P. W. Baas and L. Qiang, Trends Cell Biol., 2005, 15, 183. [40] C. Ballatore, V. M. Lee and J. Q. Trojanowski, Nat. Rev. Neurosci., 2007, 8, 663. [41] J. Leger, M. Kempf, G. Lee and R. Brandt, J. Biol. Chem., 1997, 272, 8441. [42] J. Eidenmuller, T. Fath, T. Maas, M. Pool, E. Sontag and R. Brandt, Biochem. J., 2001, 357(Pt 3), 59. [43] T. Fath, J. Eidenmuller and R. Brandt, J. Neurosci., 2002, 22, 9733. [44] A. Abraha, N. Ghoshal, T. C. Gamblin, V. Cryns, R. W. Berry, J. Kuret and L. I. Binder, J. Cell Sci., 2000, 113(Pt 21), 3737. [45] R. W. Berry, A. Abraha, S. Lagalwar, N. LaPointe, T. C. Gamblin, V. L. Cryns and L. I. Binder, Biochemistry, 2003, 42, 8325. [46] J. Kuret, E. E. Congdon, G. Li, H. Yin, X. Yu and Q. Zhong, Microsc. Res. Tech., 2005, 67, 141. [47] I. Khlistunova, J. Biernat, Y. Wang, M. Pickhardt, M. Bergen, Z. Gazova, E. Mandelkow and E. M. Mandelkow, J. Biol. Chem., 2006, 281, 1205. [48] O. Katsuse, W. L. Lin, J. Lewis, M. L. Hutton and D. W. Dickson, Neurosci. Lett., 2006, 409, 95. [49] W. Chun and G. V. Johnson, J. Biol. Chem., 2007, 282, 23410. [50] S. Maeda, N. Sahara, Y. Saito, S. Murayama, A. Ikai and A. Takashima, Neurosci. Res., 2006, 54, 197. [51] C. W. Wittmann, M. F. Wszolek, J. M. Shulman, P. M. Salvaterra, J. Lewis, M. Hutton and M. B. Feany, Science, 2001, 293, 711.
68
Simon N. Haydar et al.
[52] T. L. Spires, J. D. Orne, K. SantaCruz, R. Pitstick, G. A. Carlson, K. H. Ashe and B. T. Hyman, Am. J. Pathol., 2006, 168, 1598. [53] M. P. Mazanetz and P. M. Fischer, Nat. Rev. Drug Disc., 2007, 6, 464. [54] B. Bulic, M. Pickhardt, B. Schmidt, E.-M. Mandelkow, H. Waldmann and E. Mandelkow, Angew. Chem., Int. Ed., 2009, 48, 1740. [55] M. Pickhardt, Z. Gazova, M. Von Bergen, I. Khlistunova, Y. Wang, A. Hascher, E.-M. Mandelkow, J. Biernat and E. Mandelkow, J. Biol. Chem., 2005, 280, 3628. [56] G. Larbig, M. Pickhardt, D. G. Lloyd, B. Schmidt and E. Mandelkow, Curr. Alzheimer Res., 2007, 4, 315. [57] M. Pickhardt, G. Larbig, I. Khlistunova, A. Coksezen, B. Meyer, E.-M. Mandelkow, B. Schmidt and E. Mandelkow, Biochemistry, 2007, 46, 10016. [58] B. Bulic, M. Pickhardt, I. Khlistunova, J. Biernat, E.-M. Mandelkow, E. Mandelkow and H. Waldmann, Angew. Chem. Int. Ed. Engl., 2007, 46, 9215. [59] M. L. Waters, Curr. Opin. Chem. Biol., 2002, 6, 736. [60] E. E. Congdon, M. Necula, R. D. Blackstone and J. Kuret, Arch. Biochem. Biophys., 2007, 465, 127. [61] N. S. Honson, J. R. Jensen, M. V. Darby and J. Kuret, Biochem. Biophys. Res. Commun., 2007, 363, 229. [62] M. Pickhardt, J. Biernat, I. Khlistunova, Y. P. Wang, Z. Gazova, E. M. Mandelkow and E. Mandelkow, Curr. Alzheimer Res., 2007, 4, 397. [63] S. Taniguchi, N. Suzuki, M. Masuda, S.-I. Hisanaga, T. Iwatsubo, M. Goedert and M. Hasegawa, J. Biol. Chem., 2005, 280, 7614. [64] C. M. Wischik, P. C. Edwards, R. Y. Lai, M. Roth and C. R. Harrington, Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 11213. [65] C. M. Wischik, J. E. Rickard, C. R. Harrington, D. Horsley, J. M. D. Storey, C. Marshall and J. P. Sinclair, Patent Application WO2007110630, 2007. [66] D. Howlett, P. Cutler, S. Heales and P. Camilleri, FEBS Lett., 1997, 417, 249. [67] M. J. Volles and P. T. Lansbury Jr., Biochemistry, 2003, 42, 7871. [68] V. N. Uversky, J. Neurochem., 2007, 103, 17. [69] S. Krishnan, E. Y. Chi, S. J. Wood, B. S. Kendrick, C. Li, W. Garzon-Rodriguez, J. Wypych, T. W. Randolph, L. O. Narhi, A. L. Biere, M. Citron and J. F. Carpenter, Biochemistry, 2003, 42, 829. [70] V. N. Uversky, J. Biomol. Struct. Dyn., 2003, 21, 211. [71] M. Zhu, S. Rajamani, J. Kaylor, S. Han, F. Zhou and A. L. Fink, J. Biol. Chem., 2004, 279, 26846. [72] S. Mandel, G. Maor and M. B. Youdim, J. Mol. Neurosci., 2004, 24, 401. [73] M. Hirohata, K. Ono, A. Morinaga and M. Yamada, Neuropharmacol, 2008, 54, 620. [74] J. N. Rao, V. Dua and T. S. Ulmer, Biochemistry, 2008, 47, 4751. [75] A. M. Bodles, O. M. El-Agnaf, B. Greer, D. J. Guthrie and G. B. Irvine, Neurosci. Lett., 2004, 359, 89. [76] K. Ono and M. Yamada, J. Neurochem., 2006, 97, 105. [77] K. A. Conway, J. C. Rochet, R. M. Bieganski and P. T. Lansbury Jr., Science, 2001, 294, 1346. [78] J. Li, M. Zhu, A. B. Manning-Bog, D. A. Monte and A. L. Fink, FASEB J., 2004, 18, 962. [79] J. Li, M. Zhu, S. Rajamani, V. N. Uversky and A. L. Fink, Chem. Biol., 2004, 11, 1513. [80] Y. Porat, A. Abramowitz and E. Gazit, Chem. Biol. Drug Des., 2006, 67, 27. [81] D. P. Hong, A. L. Fink and V. N. Uversky, J. Mol. Biol., 2008, 383, 214.
Therapeutic Agents for Neurodegenerative Diseases
69
[82] T. Tomiyama, A. Shoji, K.-i. Kataoka, Y. Suwa, S. Asano, H. Kaneko and N. Endo, J. Biol. Chem., 1996, 271, 6839. [83] H. T. Li, D. H. Lin, X. Y. Luo, F. Zhang, L. N. Ji, H. N. Du, G. Q. Song, J. Hu, J. W. Zhou and H. Y. Hu, FEBS J., 2005, 272, 3661. [84] C. L. Kragh, L. B. Lund, F. Febbraro, H. D. Hansen, W. P. Gai, O. El-Agnaf, C. RichterLandsberg and P. H. Jensen, J. Biol. Chem., 2009, 284, 10211.
CHAPT ER
4 Case History: ChantixTM/ChampixTM (Varenicline Tartrate), a Nicotinic Acetylcholine Receptor Partial Agonist as a Smoking Cessation Aid Jotham W. Coe, Hans Rollema and Brian T. O’Neill
Contents
1. Introduction 2. Partial Agonists at Nicotinic ACh Receptors 3. The Search for Partial Agonists: Cytisine as a Key Starting Point 4. Semi-Synthetic Analogs of Cytisine 5. Cytisine Synthesis and Early Template Expansion 6. Discovery of the Bicyclic Benzazepine Core 7. Fused Bicyclic Benzazepines 8. In vivo Efficacy of Partial Agonists 9. Properties of Varenicline 9.1 Pharmacology 9.2 Absorption, distribution, metabolism, excretion (ADME) 10. Clinical Studies 11. Conclusions References
71 74 77 80 84 87 91 93 95 95 97 98 98 99
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04404-2
r 2009 Elsevier Inc. All rights reserved.
71
72
JOTHAM W. COE et al.
1. INTRODUCTION Serious attention to the public health impact of smoking emerged in the United Kingdom in the late 1950s after a prospective study, begun in 1951, focused on the frequency of lung cancer among doctors with known smoking habits [1]. Early data demonstrated a link between smoking tobacco and lung cancer, which was born-out over the 50-year span of the study and grew to include many other causes of death. The overall conclusions were that tobacco use will kill at least half of all smokers and that smokers lose on average 10 years of life compared with non-smokers. Smokers who were able to quit smoking when they were between 30 and 40 years of age decreased their risk of premature mortality to that of a non-smoker [2]. In the United States, the Surgeon General’s 1964 report concluded that ‘‘lung cancer and chronic bronchitis are causally related to cigarette smoking’’ [3]. The report also noted that there was suggestive evidence, if not definite proof, for a causative role of smoking in other illnesses. Surprisingly, it was not until the 11th Surgeon General’s report in 1979 that smoking was defined as a ‘‘nicotine addiction’’ [4]. In 2007, the U.S. Senate proposed removing the pejorative term ‘‘abuse’’ from the name of the National Institute of Drug Abuse (NIDA) and the National Institute on Alcohol Abuse and Alcoholism (NIAAA) replacing it with the words ‘‘diseases’’ and ‘‘addiction’’ [5]. Evidence that profound neurological changes result from smoking has led experts to characterize nicotine addiction as a disease, a clear shift in perception from the view that this behavior was a habit one simply stops. Awareness of smoking’s health toll has been beneficial in the United States; cigarette smoking rates in U.S. men have dropped steadily in the past 50 years, from more than 42% to the current rate of B21%. Despite this trend, cigarette smoking, still the leading cause of preventable illness and mortality in the United States, contributes to the death of 443,000 people each year. More than 5 million people die annually in the developed world from smoking-related illness, a statistic on course to double by 2030 [6]. As health care costs have risen, worldwide government awareness of the problem has intensified. The World Health Organization (WHO) effort to define targets for smoking rate reductions highlights this international shift [7]. Still, smoking remains a ritual ingrained in the societal fabric throughout the developed world. Recognition that nicotine addiction is initiated by a pharmacological substance that profoundly impacts the physiology of the patient was a large step forward. This tenet stimulated research toward therapies that could address nicotine addiction by acting directly at the receptors that mediate the addictive effects. In the late 1980s, the only available treatment was nicotine replacement therapy (NRT), introduced in the late 1970s, which
Case History: Chantixt/Champixt (Varenicline Tartrate)
73
increased quit rates in clinical trials [8]. Later, in 1997, after our entry into smoking cessation research, bupropion (Zybans), first marketed as the anti-depressant (Wellbutrins), was found to be efficacious for smoking cessation [9]. Behavioral therapy and counseling have been offered in various ‘‘quit smoking’’ settings, and although common, they have been generally underutilized, their success has been limited by commitment and finances of the smoker [10]. All of these treatments approximately doubled the chance of continuous abstinence compared with placebo controls, highlighting the need for more efficacious therapies. NH N N
varenicline (1)
Varenicline (1) is the first medicine targeting nicotinic acetylcholine receptors (nAChRs), other than nicotine (2) as replacement therapy (NRT), approved as an aid to smoking cessation by both the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) in 2006. The effort to discover and develop varenicline began at Pfizer in 1993, with the objective from the outset to find a partial agonist to treat nicotine dependence. A partial agonist was theorized to provide significant but reduced nicotinic reward that an abstinent smoker craves while simultaneously blocking the reward from relapse smoking. Prior pharmacological approaches to treat addiction were limited to opioid treatment for heroin addiction. Our nAChR partial agonist program was seeded in the growing appreciation of the effectiveness of buprenorphine as an aid to opioid addiction [11]. Buprenorphine, available today as Buprenexs (Suboxones, Subutexs), a semisynthetic analgesic, was shown in 1976 to be a m-opioid receptor partial agonist and was studied in the 1970s and 1980s as a treatment for heroin (and cocaine) addiction. Compared to methadone, a m-opioid receptor full agonist and naloxone, a m-opioid receptor competitive antagonist, buprenorphine is more effective, produces less physical dependence, has a reduced risk of respiratory suppression resulting from overdose, and fewer side effects. Rose and Levin [12] proposed the concept of a combined agonist and antagonist for smoking cessation in a series of publications in 1989–1992. They originally suggested that NRT (nicotine (2) as the nAChR agonist) when combined with mecamylamine (3) (nAChR antagonist) would theoretically improve quit rates. The effects of this combination were studied in clinical trials that demonstrated the dual agent approach provided higher quit rates than NRT alone. This was an attractive concept,
74
JOTHAM W. COE et al.
but suffered from the challenges of co-administration of two agents with different pharmacokinetic (PK) profiles, and the unfavorable side effect profile of mecamylamine (3). The dual agonist/antagonist approach could be better achieved with a single molecule, a partial agonist. In 1993, it was agreed at Pfizer to pursue a partial agonist as a novel smoking cessation agent, in part because one of the key challenges, how to measure partial agonist responses in vitro, was in principle achievable using cloned nAChRs for in vitro electrophysiological studies of ligand effects [13]. However, at that time, it was still much less evident how to identify or design partial agonist chemical structures. Herein we describe our efforts as they unfolded over time from the beginning of this project in 1993. Interestingly, the considerable synthetic and pharmacologic effort in the opioid field in the 1970s played a pivotal role in the discovery of varenicline.
2. PARTIAL AGONISTS AT NICOTINIC ACh RECEPTORS ‘‘Partial agonists are ligands that produce a smaller than maximal response compared with the natural ligand at full receptor occupation’’ [14]. Acetylcholine (4) is the natural full agonist at two known receptor subclasses important for neurotransmission: the muscarinic (mAChRs) and nicotinic (nAChRs) receptors, which are distributed throughout the central nervous system (CNS) and the periphery. The nicotinic receptor subtypes are ligand-gated ion channels that mediate fast synaptic transmission upon acetylcholine binding and also mediate the full agonist effect of nicotine. These ion channels are formed from pentameric assemblies of subunits creating a central aqueous pore. There are 12 neuronal nAChR subunits divided into a and b subtypes (a2a10, b2b4) differentiated by the presence or absence of a key ‘‘cys-cys loop’’ near the acetylcholine-binding site respectively. The known combinations of subunits form a broad range of nAChR subtypes with significantly different characteristics of ligand pharmacology (Figure 1) and cation permeability [15]. H N H N
N HN
N
N
O O
nicotine, 2
Figure 1
O
mecamylamine, 3
Nicotinic ligands.
acetylcholine, 4
cytisine, 5
Case History: Chantixt/Champixt (Varenicline Tartrate)
N
O O
N
acetylcholine, 4
A • O N
O O
5.9 Å
HN
75
• C • B
cation center (-)-cytisine, 5 H-bond acceptor
polarizing group
Figure 2 Nicotinic pharmacophore (Beers and Reich [17]).
Nicotine (2) binds tightly to the a4b2 heteromeric nAChR, which is the most abundant subtype in the CNS [16], and it acts as a full agonist at this nAChR subtype. The pharmacophore and binding requirements of the nAChR site were first explored and described by Beers and Reich in 1970 [17]. Their model is still useful today as it identifies a spatial relationship for key interactions within the nicotinic-binding site (Figure 2) and differentiates those interactions from the mAChrR-binding site (not shown). The defined distance between the cation/amine functionality and the hydrogen bond acceptor region has been verified with dozens of known receptor ligands, including nicotine (2), acetylcholine (4), and cytisine (5), but the connection between structural and functional activity has been less well understood. However, the level of polarization of the hydrogen bond and the electron density of the polarizable group make important but not well-defined contributions to functional activity. The complex functional characteristics of the receptor, as defined by Changeux [18], are a consequence of at least three distinguishable states: activated, desensitized, and inactivated during a response to drug binding (Figure 3) [19]. The functional outcome due to ligand binding over the dynamic course of all three receptor states is not well defined: it is likely that the scope of behavioral responses can be linked to the dynamic relationship among multiple nicotinic states whether activated or desensitized. Receptor binding is measured at the high affinity desensitized state, whereas functional activity is of limited duration at high concentrations during channel opening and ion flow in the activated state [20]. The 1980–1990s witnessed the development of in vitro functional assays of nicotinic receptors in Xenopus oocytes, which made it possible to assess the inherent agonist activity of compounds, as well as their antagonism of nicotine [13]. Despite this, we felt that in vivo models of behavior or specific biochemical endpoints could give us a more meaningful picture of the consequences of ligand binding.
76
JOTHAM W. COE et al.
Figure 3 Schematic representation of the transitions among the functional states of nAChRs, indicating the effects of agonists (open squares) and antagonists (black squares) on the equilibrium between the resting, active, and desensitized state of the nAChR (J. Med. Chem., 2005, 48, 4705, reprinted with permission).
At the outset of our program, stable human cell lines expressing nicotinic receptor subtypes were not readily available to screen our sample collection nor were high throughput functional screens (such as Fluorescent Imaging Plate Reader (FLIPR) platforms). The most informative biological tools available were animal models of the rewarding effects of nicotine [21]. These consisted of quantitative measurements of effects on dopamine (DA) release in brain or in brain slices, and nicotine selfadministration and drug discrimination in rats, mirroring subjective effects experienced by smokers. Nicotine discrimination was pioneered by Morrison and Stephenson [22] in an experiment that demonstrated that rats could detect and respond to nicotine’s presence. The exact receptor that mediated this effect was not known at the time; however, discrimination could be blocked by a central nicotinic antagonist such as mecamylamine but not by chlorisondamine, a peripheral antagonist. Later studies by Romano [23], Rosecrans and coworkers [24], and Stolerman and coworkers [25] identified central nAChR stimulation as the likely source of the nicotine cue rather than mAChRs. Self-administration of nicotine in rats had been achieved by Corrigall and Coen [26] mimicking many of the behavioral responses displayed by humans. As with the discrimination model, the response was dose-dependently reduced by pre-treatment with either nicotine or mecamylamine but not with hexamethonium (10), a peripheral nicotinic antagonist. The dependence-producing effects of nicotine are believed to be mediated through its action as a full agonist at a4b2 nAChRs [27,28]. Activation of a4b2 receptors in the ventral tegmental area by nicotine
Case History: Chantixt/Champixt (Varenicline Tartrate)
77
increases the release of DA in the nucleus accumbens and prefrontal cortex [29], an effect shared by most substances of abuse, although each through distinct neurochemical pathways. We measured the change in DA metabolites in response to activation by partial agonist ligands and after co-administration with nicotine. We sought compounds that would provide a reduced reward relative to that of nicotine while simultaneously blocking the full effect of nicotine itself. These models were assembled for our program and validated with known nicotinic receptor ligands.
3. THE SEARCH FOR PARTIAL AGONISTS: CYTISINE AS A KEY STARTING POINT Early in our drug discovery program, we sought information on the level of partial agonist efficacy that would be effective for smoking cessation and any available chemical tools to distinguish a partial agonist from the effects of the full agonist nicotine. We required a substance that would displace nicotine from its neuronal binding site and reduce the subjective experience of nicotine administration by simultaneously attenuating the dopaminergic response to nicotine. Evidence for partial agonism would include lower efficacy than nicotine and antagonism of nicotine’s effects in vitro and in vivo. The de novo design of a single agent nAChR partial agonist was and still is hampered by the lack of a clear understanding of structural requirements needed for the appropriate functionality and selectivity. Fortunately, nature’s portfolio in the nicotinic area is substantial, and we directed our focus there as a source of medicinal leads, rather than to an artificial compound sample collection [30]. Many of these natural structural classes are presumed to posses a favorable pharmacokinetic profile, but CNS penetration and potency remained the key issues to be resolved. We first profiled nicotinic agonists with lower functional efficacy than nicotine (o70%) such as anabaseine (6), GTS-21 (7) [31], anabasine (8) [32], lobeline (9), and cytisine (5) (Figure 4) in nicotinic assays. These included drug discrimination and self-administration behavioral models and ex vivo biochemical endpoints such as DA release. Early debate in the team focused on the perceived advantages of anabasine and derivatives, which had been shown to display partial agonist activity in rat neuronal a4b2 nAChRs expressed in Xenopus oocytes (EC50 ¼ 30 mM vs ACh EC50 ¼ 2 mM), but full agonist activity at muscle receptors [33]. Reports by Stolerman and coworkers [34] had indicated that the nicotinic agent cytisine (5) was 5-fold more potent than nicotine (2) in binding to CNS receptors but 10% less potent in producing the nicotine cue. The latter response was determined to be centrally mediated since mecamylamine (3, Figure 1) blocked the effect, but the non-CNS
78
JOTHAM W. COE et al.
H N
OMe
MeO
OH N
N
N
HN
N N
Anabaseine, 6 Anabasine, 8
O
O
GTS-21, 7
cytisine, 5
Lobeline, 9
N N Hexamethonium, 10
Figure 4
Early nAChR lead ligands and the peripheral antagonist hexamethonium.
penetrant antagonist hexamethonium (10) did not. Anabasine (8), which was 30- to 60-fold less potent than cytisine in discrimination assays, as well as cytisine, appeared to be reasonable partial agonist templates. They both produced a maximal B60% response to a nicotine cue at their highest tolerated dose without reducing the control response rate. An in vivo nicotine-like effect was considered crucial as the drug must be ‘‘experienced’’ as nicotine-like to the smoker, and so the weak agonist responses of anabasine and cytisine pointed to platforms from which a partial agonist therapeutic could be built. Lobeline (9), despite displacing [3H]-nicotine in cells and achieving high brain penetration, did not produce nicotinic-like behavioral effects, possibly due to PK issues such as free fraction concentrations below the IC50, and so we moved away from this lead. In vivo, anabasine and cytisine were reported initially to be full agonists in DA release in the striatum; however, our work and that reported later by Sibia confirmed partial agonist activity in the striatum and on DA turnover (DATO) in rat nucleus accumbens [35]. Although anabasine (8) was shown to have partial agonist activity, its weak effect in drug discrimination models required doses that were close to those that impaired control responding for food, suggesting a limited therapeutic index. We were also concerned by anabasine’s full agonist profile at the muscle receptor. Anabaseine (6) possessed less attractive structural features such as potentially unstable imine functionality and was not pursued further. (–)-Cytisine (5), a nicotinic agent isolated from natural sources more than 100 years ago [36], had garnered little attention from a medicinal chemistry standpoint despite its use as a natural smoking cessation aid in Eastern Europe [37]. Cytisine’s unusual properties as a high affinity, but low efficacy, ligand for the neuronal nAChR may have restricted interest in this compound as a therapeutic agent [38]. We hypothesized that cytisine’s rigid structure and polarizable pyridone ring could potentially
Case History: Chantixt/Champixt (Varenicline Tartrate)
79
contribute to its partial agonist profile at the receptor level, and we targeted derivatives of this compound as a starting point. At about the same time, Papke and Heinemann [39] demonstrated that cytisine behaves as a partial agonist relative to acetylcholine at a4b2 nAChRs in vitro with an EC50 of 9 mM. In the discussion that follows, we describe structure–activity relationships (SARs) based on cytisine and analogs that were aimed to maintain or improve its partial agonist activity in vitro and to substantially improve its in vivo pharmacokinetic profile, as part of our efforts to discover novel partial agonist platforms. Commercial supplies of (–)-cytisine were limited and expensive, but we made the most of small quantities while pursuing local sources of the natural product. Commercial material had been derived from extracts of natural plant sources and we engaged our suppliers to locate a source of bulk material to support analog studies. We were not able to readily obtain the natural product from Sopharma, the Bulgarian firm that sells cytisine under the trade name Tabex. Material was eventually secured by working through the Austin Chemical Co., USA, and the Specs-Biospecs Company in the Netherlands. They contracted with Chinese and Russian farms that could produce plant material from Thermopsis lupinoides (L Link), a legume species from which (–)-cytisine would be readily extractable. Precise timing was critical to intercept the bean pods at peak production of the natural product in mid-July before the pod fell from the plants. From these sources, we obtained 2 kg of W98% pure (–)-cytisine; an early batch was procured for $30,000/kg. This legume species was outside of the normal medicinal herb target population, but reasonable cost estimates put the eventual price of production quantities of (–)-cytisine at $150/kg. We were also able to estimate that quantities of 13–18 kg of purified cytisine could be produced per acre. While we were awaiting harvest, we initiated a parallel collaboration with Smith College Botanical Garden in Northampton Massachusetts. Cytisine was known to be a constituent of the Maackia amurensis (M. amurensis) species and material from tree pruning was made available by the college. Tree material, including bark, leaves, roots, and stems, was milled and extracted with methanol for up to 20 h followed by concentration and chromatographic separation from which we obtained 5.5 g of W95% pure (–)-cytisine from 64 kg of tree material. These small quantities allowed us to prepare several close analogs that we hoped would provide access to a library of molecules with good brain penetration and hopefully better potency. Cytisine was profiled [40] in neurochemical models, ex-vivo DATO in rat nucleus accumbens and [3H]-DA release in rat striatum. We found that cytisine functions as a partial agonist, producing lower than the maximal nicotine-induced increases in [3H]-DA release in striatal slices and in DATO. Cytisine also antagonized the effect of nicotine in these
80
JOTHAM W. COE et al.
assays, displaying the dual effects expected of a partial agonist. At 1 mg/ kg subcutaneous administration (s.c.), nicotine maximally increased DATO in nucleus accumbens to 170% of control (ED50 ¼ 0.156 mg/kg s.c.), whereas the maximal cytisine-induced increase was 130% of controls (ED50 ¼ 1 mg/kg). In addition, cytisine reversed the effect of 1 mg/kg s.c. nicotine on DATO in rat nucleus accumbens with an ID50 of 1.77 mg/kg. These results met our criteria for a partial agonist profile, except for the rather low in vivo potency, and validated our thoughts around a suitable structural framework for developing a partial agonist portfolio. The high cytisine dose required for inhibiting nicotineinduced DATO was eventually linked to low central exposure. Cytisine has low molecular weight and low polar surface area with moderate basicity, but it is hydrophilic, as shown by its partition coefficients in octanol/water (log P ¼ 0.01; log D7.4 ¼ 0.2), which may partially explain its limited brain penetration. The measured brain-to-plasma ratio of cytisine, which does not bind to plasma or brain proteins, was 0.1 and the cerebral spinal fluid (CSF)-to-free-plasma ratio was 0.27 resulting in low observed brain concentrations, ranging from 10 to 50 ng/g over the course of 6 h after a 1 mg/kg p.o. dose.
4. SEMI-SYNTHETIC ANALOGS OF CYTISINE Semi-synthetic analogs of cytisine were targeted once supplies of the natural product were secured [40]. Early on we established significant decreases in nAChR binding activity with N-group substitution (e.g., Me, Bn, Allyl, COR, SO2CF3), a result later confirmed in related templates. Medicinal chemistry enabling synthetic studies targeted aromatic ring modifications as well as total synthesis options, which would require the synthesis of novel templates. Although cytisine was the foundation for further nicotinic partial agonists, we were aware that improvements in cytisine’s profile might not succeed or be thwarted by issues of toxicity, CNS penetration, or other PK issues. Furthermore, novel rigid templates that offered receptor interactions complementary to cytisine would diversify the approach into novel matter. Cytisine binds with high affinity at the a4b2 nAChR with an in vitro agonist EC50 of 9 mM, but its poor brain penetration contributes to its reduced in vivo potency. Therefore, sufficient in vivo efficacy would either require a bold potency improvement within the cytisine structural class, or a physical property improvement that allowed CNS penetration from a novel template outside of the cytisine structural domain. Since we did not want to overplay the benefits of a particular series such that high doses would be required to achieve efficacy, it seemed a better strategy to look to novel series based on the cytisine framework for partial agonist activity to
Case History: Chantixt/Champixt (Varenicline Tartrate)
81
maintain and improve the profile we had from nature. We planned to move in parallel from cytisine to novel rigid templates that possessed similar receptor interactions, while monitoring partial agonist properties using electrophysiology, neurochemistry, drug discrimination, and selfadministration models. Early cytisine derivatives were intended primarily as intermediates for further analog synthesis. (–)-Cytisine (5), obtained from natural sources, was protected as the N-t-Boc derivative under standard conditions. Halogenation gave access to novel cytisine intermediates, with bromination of N-protected cytisine affording a mixture of monobromo and dibromo adducts. Although mono-halogenation of 11 using N-bromosuccinimide (NBS) or iodine monochloride (ICl) suffered from poor selectivity, reaction of 11 with N-chlorosuccinimde (NCS) gave a 1:9 mixture of regioisomeric compounds 15 and 18, respectively. The opposite regioisomer predominated when trifluoroacetamide 12 was iodinated with ICl in buffered methanol (Scheme 1, Table 1). Binding affinities of our analogs, including synthetic intermediates such as the halogenated cytisine derivatives, were routinely evaluated in nAChR-binding screens. We were gratified to see a large potency shift in moving from cytisine to 3-bromocytisine 14. This derivative was more potent than either the 5-bromo-derivative 17 or 3,5-dibromocytisine with a B100-fold shift in agonist efficacy (EC50 ¼ 95 nM compared to cytisine EC50 ¼ 9 mM). Thus, with a small change in molecular weight, the inherent potency of cytisine could be improved. Comparison of physical properties suggested that 3-bromocytisine’s enhanced potency could be partially ascribed to increased lipophilicity (3-bromocytisine: log P ¼ 0.99, log D ¼ 0.35 vs cytisine: log P ¼ 0.01, log D ¼ 0.2), without decreasing either ligand efficiency (the binding energy per atom) [41], which remained at nearly 0.90, or lipophilic lipid efficiency [42] (LLE ¼ pIC50log P) which is B12. These high values can be partially attributed to the small volume of the nAChR binding pocket. We also postulated that the electronic effect of halogens either improved the X O
O
O N
halogenate
N
N
+
deprotect N P 11. P = t-Boc 12. P = CF3CO−
Scheme 1
N H 13. X = I 14. X = Br 15. X = Cl
Halogenated cytisine derivatives.
N H 16. Y = I 17. Y = Br 18. Y = Cl
Y
82
JOTHAM W. COE et al.
Table 1
Halogenation of Cytisine Derivatives.
Condition
Protection
Product
Ratio
ICl CaCO3 ICl CaCO3 NBS CH2Cl2 NCS CH2Cl2
t-Boc TFA t-Boc t-Boc
13/16 13/16 14/17 15/18
1:1 9:1 3:2 1:9
H N
H N
H N 3-Br-cyt
(-)-cytisine
O 5
5
N C 3
Figure 5
13
C-NMR 163.6 ppm
H N
O 14
N C
3-Cl-cyt
13
C-NMR 160.1 ppm
Br
O 15
N C
N
13
C-NMR 159.0 ppm
Cl
O 19
X
Halide effects on N-C(O) polarization by 13C-NMR.
hydrogen bond acceptor properties of the pyridone carbonyl or favorably contributed to the aromatic polarizability by increasing the double-bond character of the pyridine C–N bond, as depicted in pyridinium oxide 19 in Figure 5. Pharmacokinetic measurements indicated that the increase in lipophilicity of 3-bromocytisine 14 improved CNS penetration (B/P ¼ 1.4) with a symmetric CSF/free plasma ratio of 1 and virtually no protein binding (Cytisine (5), B/P ¼ 0.1 and CSF/free plasma ratio 0.27). Parallel in vivo studies demonstrated that 3-bromocytisine 14 was a potent partial agonist in DATO with a B70-fold shift in agonist activity (ED50 ¼ 0.032 mg/kg) relative to cytisine and potent antagonist activity (ID50 ¼ 0.179 mg/kg). Compared to cytisine, 3-bromocytisine fully substituted for nicotine in a drug discrimination model showing a B70-fold leftward shift of the dose–response curve. Importantly, 3bromocytisine 14 also potently inhibited nicotine self-administration in rats with an ED50 of 0.05 mg/kg. Close in analogs of cytisine demonstrated additional examples of potency enhancement but none exceeded that of 3-bromocytisine 14. Synthetic chemistry efforts gave access to multiple derivatives beginning from 14 as shown in Scheme 2. The 3-chlorocytisine derivative 15 was also a potent partial agonist as were the 3-cyano and 3-methyl derivatives, 23 and 26, respectively. It has been theorized that electronic effects play a crucial and complementary role in binding and functional activity at the nicotinic receptor, leading to the proposal of a three-point nicotinic pharmacophore
83
Case History: Chantixt/Champixt (Varenicline Tartrate)
O Br N
N
O
O
N
N
HN
N
O HN
N
t-BOC
t-BOC
(-)-cytisine (5)
11
Boc-14
20 Acylation
plus regioisomer
Me
B(OH)2 N
N
O N
CN
N t-BOC N
21
N
O
O
26 Stille
25 Buchwald Hartwig CO2Me
23 Cyanation
X O N
HN
O
HN
22 Suzuki
Scheme 2
O
HN
HN
HN
N
O
24 Carbonylation
Cytisine analog semi-synthesis through Pd catalysis.
providing two contacts with the protein and one locus of polarizability [17]. We therefore explored isomeric changes that maintain the physical separation between the carbonyl and basic amine within a rigid framework directly related to cytisine. Nitrogen was repositioned across the ring to evaluate the effect of this structural change on biological activity (Figure 6) [43]. The isomeric cytisine analog, racemic N-methyl isocytisine (27), displayed a B100-fold drop in binding potency and was a functional antagonist. The alkyl substituent on nitrogen was critical for maintaining any nicotinic binding activity in this series. Although isosteric with H N
O
H N
N
O
27
N
H N
H N
N
N
O
O
28 isocytisine
(-)-3-methylcytisine, 26
Figure 6 Methyl-cytisine/isocytisine resonance forms.
84
JOTHAM W. COE et al.
3-methylcytisine (26), the subtle structural change and corresponding shift in efficacy point to a delicate balance between electronic structure and functional activity. Perhaps, building positive charge on nitrogen (the oxo-pyridinium tautomeric form, 28) in that region of the binding pocket is detrimental to the binding and functional activity, despite maintaining carbonyl H-bond acceptor capacity presumed to be similar to cytisine. The unsubstituted isocytisine pyridone (N-H) derivative was inactive, likely due to repulsion of the hydrogen bond donor in that binding pocket. The cytisine template served to initiate an essential SAR study for the nicotinic partial agonist program. Our early work demonstrated that CNS penetration could be improved compared with the natural product without sacrificing the desired pharmacodyamic parameters. We further showed that cytisine-derived partial agonists could be discriminated as nicotinic agents and would provide a reduced nicotinic-like reward while blocking the effect of nicotine. We uncovered a range of functional activities by modifying ring substitutions or by subtle changes in the electronic character of the polarizable heteroaromatic ring. Unfortunately, the overall profile of these ligands was marred by genetic toxicology findings within the substituted cytisine class. Our original plan to create novel scaffolds using the chemistry that follows opened up a broad and entirely novel range of partial agonist templates.
5. CYTISINE SYNTHESIS AND EARLY TEMPLATE EXPANSION With the advances provided by studies of cytisine and derivatives, access to compounds not available by direct analog generation became more important. The cost of cytisine as a raw material had originally been a concern (B$1.7 M/kg, Aldrich, B1995), but had been addressed through potential agricultural harvest. To support further derivatization and SAR development, synthetic approaches became essential, as they offered the ability to make not only cytisine but also novel cytisine-like derivatives inaccessible from cytisine itself. These synthetic efforts began in earnest within our group in 1995. Of the strategies we considered for cytisine’s construction, two novel synthetic designs were ultimately demonstrated and later published. The most concise strategy is exemplified by biaryl coupling chemistry of pyridine precursors 29 and 30 to access both the pyridone and the piperidine ring atoms of cytisine (5) (Scheme 3). This step-efficient approach requires a nucleophilic nitrogen component in 31 to reveal the cytisine pyridone in the bicycle formation step [44] and allows the introduction of a range of substituents on the final pyridone ring [45].
Case History: Chantixt/Champixt (Varenicline Tartrate)
H
N
Z
O
O
O
29
Scheme 3
N
N
N
X
N
X
Y
N
85
30
(+/-)-cytisine 5
31
Biaryl coupling approach to cytisine [44].
A second strategy employed a palladium-mediated Heck cyclization of a glutarimide derived enol triflate intermediate (33) to access the bicyclic ring core (34), ultimately providing racemic cytisine in six steps from cyclopent-3-enyl-methanol (32) (Scheme 4) [46]. In this approach, the N-C bond of the pyridone ring was established first, with the piperidine synthesis finalized after bicycle formation (34-5). The construction of alternative templates not containing a nitrogen ring fusion atom was possible utilizing the same strategic approach (32-37, Scheme 5). Soon after the successful syntheses of racemic cytisine were established, we targeted non-pyridone-containing derivatives and the efforts rapidly yielded active compounds [43,47]. An early anisole derivative (38) had high affinity (Ki ¼ 1.4 nM) and displayed weak partial agonist activity at the receptor in vitro in oocytes (30% relative to nicotine, Figure 7). In vivo this analog displayed weak activity in the DATO assay, in part due to rapid demethylation in vivo to the less active phenol (40, Ki ¼ 90 nM, partial agonist). These promising early results launched an 18-month effort of intensive chemical synthesis, producing B100 novel carbon analogs of cytisine. Despite the targeted chemical exploration, derivatives from this series generally displayed either reduced affinity or antagonist activity in oocytes (e.g., 39 and 41, o20% efficacy relative to nicotine). Compounds worthy of further pursuit as partial agonists did not emerge from the effort. The most potent non-pyridone cytisine
H N X N
N HO 32
Scheme 4
N
O
O 33
O 34
(+/-)-cytisine 5
Palladium mediated heck approach to cytisine [46].
86
JOTHAM W. COE et al.
H N X R2
R2
HO
32
Scheme 5
R2
R1
R1 35
R1 non-pyridone derivatives 37
36
Approach to non-pyridone cytisine derivatives.
derivatives generally were compounds with antagonist action at the receptor, such as the difluoroderivative 42 [48]. One aspect of the pharmacophore brought into question by SAR developed in this series was the role of the cytisine carbonyl group. Most published binding motifs suggested the existence of a hydrogen bond between the cytisine carbonyl and the receptor [17,49]. In contrast, we saw enhanced potency of compounds possessing electron withdrawing functionality, the most potent being the difluoro derivative 42, which possesses H-bond acceptor interactions that are arguably weak [50]. These data suggested that dipole or other electronic interactions are important for binding and functional efficacy at the a4b2 receptor. These results were consistent with the profound changes we observed between cytisine (5) and isocytisine (27) activity, wherein H-bonding alone is insufficient to explain the pharmacophore interactions. Furthermore, 42 was almost a full antagonist (3% agonist), clearly demonstrating
H N
H In vitro N Ki α 4β2 1.4 nM 30% ag. / 51% antag. vs. nicotine In vivo active short half-life H3CO
OCH3 38 H N
In vitro Ki α4β2 0.4 nM 3% ag. / 85% antag. vs. nicotine In vivo active antagonist
F
39
antagonists
In vitro Ki α4β2 2.9 nM
3
R O
2
H
Figure 7
40
41
1 C
H
HO OH
42
F
HN
H N In vitro Ki α4β2 90 nM partial agonist
H N
In vitro Ki α4β2 >500 nM
H
H
O H
H
binding site
Selected SAR and H-bond pharmacophore probes.
untolerated
43
87
Case History: Chantixt/Champixt (Varenicline Tartrate)
H N
HN
O 44
H N
H N
O
N
N O
H N
H N
O 45
O 46
N 27
N R 47
48
Figure 8 Additional templates.
non-parallel responses of ligand binding and functional receptor responses. With no rational explanation regarding control of functional efficacy, structural guidance from SAR was for the most part empirically derived. A brief exploration of heterocyclic derivatives (e.g., 48, Figure 8) [43], accessed by the same synthetic route design, provided compounds with either weak functional activity or minimal selectivity over the nicotinic muscle receptor, another undesirable property.
6. DISCOVERY OF THE BICYCLIC BENZAZEPINE CORE The weak in vitro and in vivo responses of non-pyridone cytisine derivatives (Figure 7) drove our expanded search for alternative structural scaffolds targeting improved potency and partial agonist activity (Figure 8). We synthesized various reasonable assemblies of the critical functional groups found in many natural nicotinic agents, probing alternative locations and orientations of necessary functionality. Many of these approaches proved disappointing, since they did not provide improvements in nAChR activity. The avenue that defined much of our future work came from the opioid literature, as we recognized that morphine and cytisine both possess embedded [3.3.1]-bicyclic core structures. Paul Mazzocchi’s work from the 1970s explored various simplified versions of morphine 49, the complex alkaloid from the opium poppy, Papaver somniferum. Bicyclic substructures of morphine were evaluated, including the [3.3.1]-bicyclic benzomorphan 50, and Mazzocchi and coworkers found that N-alkyl derivatives of 50 possessed morphine-like anti-nociceptive effects mediated through m-opioid receptors. Additional publications compared modified bicyclic frameworks of 50, including 51 and 52, all of which produced analgesia with particular nitrogen substituents (Figure 9) [51]. In 1979 Mazzocchi published the synthesis and anti-nociceptive activity of derivatives of an N-positional isomer of 52, namely [3.2.1]-bicyclic benzazepine 53 [51]. Analogs of 53 with N-alkyl substitution were found to have greatly reduced anti-nociceptive activity. Importantly, Mazzocchi
88
JOTHAM W. COE et al.
antinociceptive
OH
OH
N
HN O N
H
OH morphine
49
?
HN
H N
HN
O OH
50
51
52
53
Figure 9 Mazzocchi’s benzomorphane modifications [52]. (See Color Plate 4.9 in Color Plate Section.)
generalized and emphasized his key finding in the last sentence of the discussion: ‘‘Clearly, the change in nitrogen position in proceeding from ‘52 to 53’ manifests itself by an almost total loss of anti-nociceptive activity and a marked increase in toxicity.’’ The toxic nature of nicotinic agents derived from plants – for example nicotine (2), anatoxin a (54), cytisine (5), and epibatidine (55) – is to protect the host from microbial, insect, and animal predators. We wondered whether the non-pyridone cytisine scaffold 37 and the bicyclic benzazepine 53, both 3,5-disubstituted piperidines, possessed nicotinic pharmacology that could explain the increased toxicity of derivatives of 53 reported by Mazzocchi [52]. Truncated analogs 53 and 52 were prepared [53] (Figure 9), tested in opioid and nicotinic-binding assays and found to be highly selective, 53 for the a4b2 nAChR, and 52 for opioid receptors [51] (53 Ki m-opioid W2 mM; 52 Ki a4b2 W5 mM); no cross-reactivity was observed. Furthermore, benzazepine 53 was equipotent at the a4b2 nAChR to the unsubstituted parent non-pyridone cytisine derivative 37 R1 and R2 ¼ H (Ki ¼ 20 vs 34 nM, 53 and 37 respectively; Scheme 5). Both unsubstituted parent compounds were antagonists; however, the bridged benzazepine 53 provided an achiral symmetric template, greatly simplifying SAR development. Our studies expanded into this fertile area and yielded promising results. Electron-deficient and sp2-hybridized groups are common functionalities in nicotinic natural products, for example, acetylcholine (4), anatoxin a (54), cytisine (5), nicotine (2), and epibatidine (55), all contain acyl or pyridyl groups (Figure 10). We initially targeted similar structures to explore bridged benzazepine SAR. Our initial attempts to functionalize the bicyclic benzazepine by electrophilic substitution chemistry were unsuccessful, as the parent structure and typical N-protected versions proved inert to substitution. Usually nitration readily introduces functionality for subsequent SAR development, but early attempts at nitration reactions with both the N-carbamate-protected and the free
89
Case History: Chantixt/Champixt (Varenicline Tartrate)
HN
N
HN
O O acetylcholine, 4
anatoxin a, 54
N
N
N
O
H
Cl
HN
O
N epibatidine, 55
(-)-cytisine, 5
CH3
(-)-nicotine, 2
Figure 10 Naturally occurring nicotinic ligands.
benzazepine 53 did not progress, even under more forcing conditions. We were surprised to find that even nitronium triflate [54], a powerful nitrating agent, failed to derivatize the carbamate-protected benzazpine. We suspected that interactions of electrophilic reagents with the amine or protected amines would generate non-productive cationic piperidinium intermediates that served to insulate the aryl ring against electrophilic chemistry. To test this hypothesis, the trifluoroacetamide (TFA) group was chosen to mask the nitrogen functionality from competing for electrophilic attack. With the TFA group, aromatic substitution of 56 progressed smoothly (Scheme 6), with a breakthrough achieved while employing the combination of N-TFA protection and nitronium triflate in methylene chloride to afford the mononitrated derivative 57 in 78% yield. After deprotection, 58 was found to exhibit potent partial agonist activity in vitro and in vivo in DATO after both subcutaneous and oral administration in rats. This result established that nicotinic agents derived from 53 were excellent partial agonist targets. We then returned to the original non-pyridone cytisine [3.3.1]-bicyclic structure to introduce nitro functionality. Three isomers were generated (59, 60, and 61) under the nitration conditions, and each displayed weaker binding affinity than the nitrobenzazepine, but more importantly, all displayed weaker functional agonist activity than the nitro-benzazepine derivative 58, in line with findings of greater antagonist behavior observed with earlier non-pyridone-based cytisine analogs [47] (Scheme 5, 38). Although these templates differ by only a single core carbon atom, the consequent effect on potency and efficacy is remarkable (Figure 11) from 58. Given the relative ease of preparation of bicyclic
CF3 O
N
H N
CF3 2.6 equiv CF3SO3H 1.3 equiv HNO3
O
N
OH-
78% O
56
Scheme 6
NO2 N
O
SO3CF3
57
58 NO2
Nitro benzazepine: potent partial agonist revealed.
In vitro Ki α4β2 0.75 nM 64% ag. / 36% antag. vs. nicotine In vivo active 50% partial agonist in DATO s.c. and p.o.
90
JOTHAM W. COE et al.
H N
H N In vitro Ki α4β2 4.5 nM 20% ag. / 40% antag. vs. nicotine
59
NO2
Figure 11
H N
In vitro Ki α4β2 6.5 nM 2% ag. /50% antag. vs. nicotine NO2
60
In vitro NO2 Ki α4β2 14 nM 0% ag. /100% antag. vs. nicotine
61
Nitrated non-pyridone cytisines.
benzazepine analogs from a common intermediate, the partial agonist activity of suitably substituted compounds at a4b2 nAChRs, and the promising early in vivo efficacy signals, our active pursuit moved to this series. During the scale up of the nitration reaction with a slight excess of nitronium triflate (56-57), we discovered that a dinitrated by-product was formed. This reaction gave the desired mono-nitrated product 57 after crystallization in high yield (Scheme 6), but we re-examined the residues for a doubly nitrated by-product that had been observed in low yield by gas chromatography with mass spectrometry (GCMS) in the crude material, now enriched in the mother liquors. After isolation, a 9:1 mixture of two dinitro derivatives were identified, with the major isomer assigned the structure resulting from unexpected vicinal dinitration, compound 62, the minor was the expected meta isomer 63 (Scheme 7). Upon exposure to greater than two equivalents of nitronium triflate, the conversion to the dinitrated benzazepine 62 proceeded in 77% yield. This discovery fueled much of our future chemistry effort by facilitating rapid access to fused rings [55]. As SAR developed within this series (Figure 12), we found that monofunctionalized products (65, 66) were uniformly active at the a4b2 nAChR, with electron-deficient functional groups displaying subnanomolar affinity. Electron-donating groups (R1) conferred weaker binding affinity, and functional activity and peri-substitution further reduced activity (R2 position in 67), consistent with the SAR
O
O
CF3 N
CF3 N
> 2 eq NO2.OSO2CF3
9:1 NO2
O 2N
56
Dinitration of benzazepine 56.
CF3 N
-78 - 20 °C, 24 h
Scheme 7
O
NO2
62
O2N
63
Case History: Chantixt/Champixt (Varenicline Tartrate)
H N
H N
H N
H N
N O R Cytisine Derivatives 64
91
R2 R1
R1
Benzazapines less active enantiomer
Benzazapines more active enantiomer
Benzazapines less active regioisomer
65
66
67
Figure 12 Benzazepine SAR trends.
observed within the non-pyridone analog series. Electrophysiological measurements at a4b2 nAChRs expressed in oocytes showed that the partial agonist activity within mono-substituted benzazepines in racemic form ranged from 0 to 86%, with most electron withdrawing groups (R1) imparting partial agonist profiles worthy of further evaluation in optically pure form [56]. Most pharmacophore models for high-affinity nAChR ligands identify two critical ligand components: an ammonium ion and a Hbond acceptor [17,49]. The resolved enantiomers revealed an unexpected SAR trend. We expected that substituents on mono-functionalized compounds (R1) would align with cytisine’s carbonyl to establish Hbonding interactions with the receptor. We were surprised to find these isomers were less active i.e. 65 (Figure 12). The more potent enantiomers 66 positioned the electron withdrawing group away from the cytisine carbonyl orientation (typically the more potent isomers were B10-fold higher binding affinity with greater agonist functional efficacy, Figure 12). In addition, electron-withdrawing groups of widely varying H-bond acceptor ability were generally equipotent and efficacious, although each presents dissimilar H-bonding orientations into the receptor pocket (R1 ¼ NO2, Ac, CN, halo, CF3, etc.). These findings have led us to question the validity of assertions that H-bonding is a predominant feature within the a4b2 nAChR pharmacophore. Instead, these results suggest that a key feature governing affinity and possibly functional efficacy may be an interface between the ligand sp2-hybridized component and key receptor residues through productive p-electron interactions. This notion may extend our understanding of the welldocumented p-cation interaction of the ammonium head group common to all nicotinic agents [57] by including productive interactions of the ligand p-system and receptor aryl groups in addition to the ammonium p-cation interaction.
92
JOTHAM W. COE et al.
7. FUSED BICYCLIC BENZAZEPINES As mentioned earlier, the dinitration reaction of TFA-protected benzazepine (56-62) led to a number of important fused heterocycles. SAR development in the ‘‘6,6-fused’’ derivatives revealed the importance of the ‘‘quinoline nitrogen’’ (5-position) to affinity and functional activity (Figure 13). Quinoxaline derivatives (68), directly accessed from dinitro intermediates in two steps, displayed optimal partial agonist activity with hydrogen substitution (e.g., varenicline (1)). Small groups were welltolerated at C-6 and 7 (H, CH3, OH, OCH3, etc.) but reduced the functional potency of these derivatives. Aryl appendages generally decreased binding affinities and reduced partial agonist efficacy. Both quinoline (69) and quinazoline (70) derivatives with small groups displayed high affinity, but were uniformly full agonists, being more efficacious at 10 mM compared with the effect of 10 mM nicotine in oocytes. 3-Substitutied quinolines with groups larger than methyl (7-position of 69, Figure 13) displayed decreased agonist activity and affinity. Isoquinolines (71) were 10-fold less potent compared with similar quinoline (69) and quinazoline (70) derivatives. Electronic and steric changes at the ‘‘5-position,’’ which occupies a position similar to the ‘‘cytisine carbonyl’’ in molecular modeling overlays, considerably reduced activity and revealed the importance of this site. 6,5-Fused heterocyclic analogs also exhibited a broad range of functional activities in oocytes from full agonist to antagonist at 10 mM relative to 10 mM nicotine (Figure 14). With the exception of larger groups in the 6-position, most compounds based on this general construction (72–75) had Ki values o1 nM. The functional efficacy of analogs within this 6,5-fused heterocyclic series was sensitive to structural changes at the 6-position, as illustrated by benzimidazole (72) and benzisoxazole (73) derivatives. All C-6 hydrogen-substituted analogs are partial agonists with high affinity, whereas C-6 methyl substituted analogs were consistently found to be agonists with greater efficacy in oocytes than nicotine itself when measured at 10 mM. The 2-methyl benzothiazole (74) and benzisoxazole (75) derivatives were also particularly efficacious agonists, but increases in C-2 substituent size beyond methyl (in 72 and 73) appeared to inversely affect functional efficacy and binding affinity of
8 N
R′
R′
HN
1
3
68
N 5
N
HN
HN R
N
N
R
69
Figure 13 6,6-Fused heterocyclic benzazepines.
70
HN
N
R
71
93
Case History: Chantixt/Champixt (Varenicline Tartrate)
R′ N 7 HN N
3
1
72
S
O R
R
HN
73
HN
N
N
N
5
O R
74
75
Figure 14 6,5-Fused heterocyclic benzazepines.
analogs. Benzimidazoles (72) with R7-substituents were uniformly potent presumably by accessing a pocket not found to be productive within the 6,6-fused heterocycles of Figure 13. An available binding pocket for substitution appended to the aryl group is an SAR point observed with other known nicotinic agents, but the orientational distinctions between the effect of 6,6- and the effect of 6,5-fused heterocycle substitution on activity is striking [49].
8. IN VIVO EFFICACY OF PARTIAL AGONISTS The in vivo efficacy of analogs was determined by measuring effects on mesolimbic DATO, which reflects a change in postmortem tissue concentration ratios of DA and its metabolites in the rat nucleus accumbens. Compounds were evaluated subcutaneously (s.c.) and, if active, were re-examined after oral administration. In this assay, nicotine (2) produces a maximal DATO response at 1 mg/kg s.c. of B180% of control levels. The partial agonist activity of an agent alone was determined by comparison of the effect of maximum well-tolerated doses with that of 1 mg/kg s.c. nicotine. These partial agonist efficacies at the maximal well-tolerated dose of the compounds are represented as black bars in Figure 15, showing a wide range of efficacies relative to nicotine. Compounds with sufficient potency at well-tolerated doses were then examined for another hallmark of partial agonist activity, that is, the ability to inhibit nicotine-induced increases in DATO. This property was uncovered in the ‘‘antagonist mode’’ of DATO, in which attenuation of the maximal nicotine response was evaluated after co-administration of the compound and nicotine, and is represented by the shaded bars in Figure 15. Although many compounds act as partial agonists alone and produce sub-maximal DATO increases relative to nicotine, few compounds fully antagonized nicotine’s effect at their maximal welltolerated dose. Only compounds that effectively blocked nicotine’s effect with sufficient tolerability displayed the desired partial agonist dual action to warrant further pursuit. Figure 15 shows that many cytisine analogs have an optimal profile as efficacious partial agonists that can completely block nicotine-induced DATO increases. 3-Bromocytisine is particularly efficacious, displaying
94
JOTHAM W. COE et al.
110
agent alone
100
agent + 1 mg/kg s.c. nicotine
Dopamine turnover
% nicotine response + SEM
90 80 +
70
*
+
+
60
+
++
**
* *
50
*
*
*
40
*
*
**
*
**
+
++
30 20 10 0 -10 0
2
5.6
5
0.178
14
5.6
20
8.0
38
5.0
42
5.6
72
1.78
73
3.2
74
5.6
75
5.6 mg/kg sc
1
Figure 15 Effects of (–)-nicotine, (–)-cytisine, and selected agents on DATO in rat nucleus accumbens 1 h post-dose. All values are expressed as percentages of the effect of 1.0 mg/kg s.c. nicotine (100%)7SEM (N ¼ 510). Each compound was administered at the indicated dose (mg/kg s.c.) alone (black bars) and together with 1 mg/kg s.c. nicotine (shaded bars). *p o.05 agent alone vs vehicle; **p o.01 agent alone vs vehicle; +p o.05 and ++p o.01: agent with nicotine vs nicotine alone (oneway ANOVA with post hoc Dunnett’s test).
high partial agonist efficacy and fully antagonizing the effect of nicotine when co-administered at a very low dose of 0.178 mg/kg. This in vivo potency is consistent with 3-bromocytisine’s potent functional agonist activity in vitro in oocytes (EC50 ¼ 90 nM). However, this dose also represents the maximum tolerated dose in animals, which translates to a very narrow therapeutic index. The non-pyridone cytisine analogs failed to potently elevate DATO or to block nicotine’s effect, consistent with their weaker binding and presumably poor brain exposure. The bicyclic aryl piperidines (benzazepines) showed a wide range of agonist activities, with some analogs being highly active as antagonists of nicotine’s effect. Potent benzazepine analogs that were particularly efficacious were also well-tolerated at the high doses used in this assay. Comparing the in vivo effects of compounds on DATO greatly aided our triage for suitable clinical candidates and limited the selection to cytisine derivatives (e.g., 5, 14, 20), mono-substituted bicyclic benzazepine derivatives (e.g., 72, 73) and fused bicyclic benzazepine derivatives (e.g., 74, 75, varenicline (1)). Fused heterocyclic compounds were eliminated from further evaluation if they displayed functional efficacy
Case History: Chantixt/Champixt (Varenicline Tartrate)
95
either too low or too high and fell outside the in vitro window of acceptable partial agonism. We had targeted in vitro functional efficacy of 25–80% of nicotine (10 mM in oocytes), but many compounds that were progressed and evaluated in DATO lacked adequate partial agonist activity in vivo, which we attributed to insufficient free brain exposure necessary to exhibit robust agonist and antagonist efficacy. For example, benzimidazole derivatives of benzazepine (6-propyl and 6-butyl derivatives 74 and 75, respectively) are partial agonists when given alone, but failed to fully antagonize nicotine at tolerated doses, whereas the ‘‘nonpyridone’’ cytisine analogs (e.g., 38, 42) were less efficacious in vivo and poor antagonists of the nicotine response. Unlike cytisine derivatives, which were uniformly potent in vivo as agonists and as nicotine antagonists (e.g., 5, 14, 20), the other series displayed a broader range of in vivo efficacies and potencies.
9. PROPERTIES OF VARENICLINE Varenicline (1) was selected from the compounds described in this study as the primary development candidate. It is a low molecular weight (221.27 g/mol) achiral alkaloid with high CNS penetration in rat brain (B/P ¼ 3.5), a symmetric CSF/free plasma ratio of 1 and low protein binding in blood (B80% free) and brain (B67% free). Varenicline’s properties are highlighted by a ligand efficiency of 0.82 and LLE (pIC50log P) of 11.
9.1 Pharmacology Receptor binding studies [55,58] demonstrated that varenicline (1) has high affinity only for the a4b2 neuronal nicotinic receptor subtype in rat and human cortex (Ki B0.1–0.4 nM). Varenicline did not bind with significant affinity to various other neurotransmitter receptors and transporters, enzymes, modulatory binding sites, and ion channels, in membranes derived from relevant tissues and cell lines (Ki values W1,000 nM). In vitro functional patch clamp studies in Xenopus oocytes and HEK cells expressing human nAChRs showed that varenicline (1) is a partial agonist with 45% of nicotine’s maximal effect at the a4b2 nAChR that can fully antagonize the effect of simultaneously applied nicotine [55,58]. In neurochemical models, varenicline (1) displayed significantly lower efficacy (40–60%) than nicotine in stimulating [3H]-DA release from rat brain slices in vitro and in increasing DATO in rat nucleus accumbens, while it inhibited nicotine-induced DATO, as shown earlier [55,58]. In vivo microdialysis was used to examine the effects of varenicline (1) on the concentration of extracellular DA levels in the nucleus accumbens
96
JOTHAM W. COE et al.
225 nicotine Dopamine release in n. accumbens % of basal ± SEM
200
175 varenicline 150
125 nicotine + varenicline 100 Nicotine 0.32 mg/kg
Varenicline 1 mg/kg
75 -120
-60
0
60
120
180
240
300
360
Time (minutes)
Figure 16 Effects of 0.32 mg/kg s.c. nicotine alone (curve without symbols), 1 mg/kg p.o. varenicline alone (gray curve with triangles) and combined administration of 1 mg/kg p.o. varenicline with 0.32 mg/kg s.c. nicotine (black curve with open squares) on DA release in rat n. accumbens. Data are expressed as percentage of basal levels7SEM (N ¼ 35) (adapted from Coe et al., 2005) [55].
of conscious rats, to directly assess varenicline’s effects on the mesolimbic DA system. Dose–response curves indicated that varenicline maximally increased DA release to 153% of baseline with an ED50 of 0.032 mg/kg p.o. The maximal response of varenicline is about 63% of the full agonist nicotine (2), which produces a maximal increase in DA release at 0.32 mg/kg s.c. to 184% of basal levels. When administered together, varenicline reduced the peak effect of nicotine to the level of its own maximal effect on DA release, that is, about 60% of the maximal nicotine increase (Figure 16) [55,58]. In behavioral animal models, varenicline (1) reduces nicotine selfadministration in rats and supports lower self-administration break points than nicotine. In a progressive ratio self-administration paradigm, animals must work at increasingly harder levels (more bar presses) to receive their next drug infusion [58]. The lower breakpoint found for varenicline than for nicotine is consistent with the notion that a partial agonist is less reinforcing and less dependence producing than nicotine. Neurochemical support for the lack of abuse liability is provided by the
Case History: Chantixt/Champixt (Varenicline Tartrate)
97
time courses for the effects on DA release that demonstrate the slow onset and long-lasting effect of varenicline compared with nicotine’s rapid steep rise and fall in accumbens DA (Figure 16), thought to be characteristic of drugs of abuse. These animal studies, as well as data from a later abuse liability study in human subjects confirm that varenicline has no abuse potential [58]. Taken together, all data support the hypothesis that the partial agonist varenicline (1) will have utility as a smoking cessation aid. Through submaximal activation of the mesolimbic dopaminergic system, it can reproduce to some extent the subjective effects of smoking to reduce cravings and withdrawal symptoms without nicotine’s abuse liability. In addition, since varenicline effectively reduces the rapid and robust mesolimbic DA response to nicotine, the antagonist action will prevent the reinforcing and rewarding effects of nicotine to protect against smoking relapse. On the basis of these findings, varenicline (1) was advanced into clinical development.
9.2 Absorption, distribution, metabolism, excretion (ADME) As a small hydrophilic, weak base (log P ¼ 1.1; pKa ¼ 9.9; measured Elog D7.4 0.28, MW ¼ 211), varenicline (1) is well absorbed, as indicated by preclinical disposition studies that found 89% to be recovered in the urine of carbon-14-labeled material after oral administration (B1% in feces). Protein binding of varenicline is low (human blood free unbound B80%) and it has a moderate volume of distribution (1.9 L/kg). Varenicline is virtually not metabolized, comprising 90% of circulating drug-related material and is excreted mostly unchanged in the urine [59]. Four minor metabolites of varenicline were observed, with two minor urinary metabolites comprising less than 5% of the dose. Metabolites observed in excreta arose through N-carbamoyl glucuronidation and oxidation. These metabolites were also observed in the circulation, in addition to metabolites that arose through N-formylation and formation of a novel hexose conjugate. Unbound renal clearance is in slight excess of glomerular filtration rate. Varenicline is neither a substrate nor an inhibitor of cytochrome P450 enzymes. It is expected to be neither the cause of nor subject to drug interactions through alterations of P450 activities [59]. As such, drug–drug interactions are generally limited to those induced by decreased nicotine intake upon quitting smoking, as nicotine is a cytochrome P450 2A6 substrate [60]. In phase 1 clinical trials, varenicline (1) was found to have an excellent PK profile, with a half-life of 24 h, steady-state levels achieved in 4 days, no food effects, and with the drug essentially completely absorbed and not metabolized (W89% of C-14-labeled material accounted for in urine) [61,62]. The long half-life is a particularly
98
JOTHAM W. COE et al.
beneficial property for protecting smokers from smoking relapse, as nicotine is rapidly absorbed and displays a short 1–2 h half-life in human subjects. Toleration at higher doses was limited by nausea and vomiting. The maximum well-tolerated dose of 1 mg BID p.o. (twice daily, orally) was chosen to minimize the nausea signal and to maximize efficacy in smoking cessation.
10. CLINICAL STUDIES Results of phase 2 and phase 3 studies have been described in detail and have been reviewed elsewhere [63]. Efficacy was established in phase 2 studies for three different doses [1 mg QD (once daily), 0.5 mg BID and 1 mg BID] given for 6 or 12 weeks [64] One milligram BID for 12 weeks was chosen as the most efficacious treatment and used for all further clinical trials. In two identically designed phase 3 clinical trials, varenicline (1) was compared to placebo and the active comparator, bupropion SR (Zybans sustained release). In these studies smokers were treated for 12 weeks pharmacotherapy with 40 weeks post-treatment follow-up of smoking status. At the end of the 12 week treatment period, the CO-confirmed 4-week abstinence rates were 44.0% for smokers who received varenicline (N ¼ 696) compared with 29.7% in the bupropion SR group (N ¼ 671) and 17.7% in the placebo group (N ¼ 685). At the end of 1 year, 22.4% in the varenicline group, 15.4% in the bupropion group, and 9.3% in the placebo group remained completely abstinent from smoking. In pooled analyses, varenicline was statistically superior to both bupropion SR and placebo at the end of the treatment period and at the 1-year follow-up [65–68]
11. CONCLUSIONS When smokers quit tobacco, they typically experience considerable side effects [69] and are highly susceptible to relapse from re-exposure to inhaled tobacco smoke, especially during quit attempts. Properties of nicotine make this particularly true, as it is completely absorbed in the lungs and delivered to the brain within 7–10 s, making nicotine addiction one of the most challenging addictions to overcome. We sought a medicine that not only decreased nicotine craving and withdrawal symptoms but effectively and safely reduced the neurochemical reinforcement produced by smoking. Partial agonists theoretically address both of these primary physiologic aspects of nicotine dependence, but required the identification of compounds with in vivo features that effectively competed with inhaled nicotine. These objectives helped
Case History: Chantixt/Champixt (Varenicline Tartrate)
99
define the testing parameters necessary to identify an effective partial agonist as a smoking cessation aid, with long duration to reduce craving and withdrawal symptoms while effectively shielding smokers in the event they smoke during quit attempts. Varenicline met these stringent criteria, not only in preclinical models, but later in clinical trials. Many important medicinal agents originated from work on smallmolecule natural product progenitors. Cytisine helped us establish preclinical parameters for a discovery program and to elucidate a starting point for a chemical and medicinal journey. Natural products not only provided a starting point, they inspired total synthesis strategies that helped access unnatural analogs in the search for novel templates with the desired activity. This ultimately led us to studies of structurally related opioid natural products. In retrospect, it is fitting that a partial agonist approach for opioid addiction sparked the conceptual foundation of this approach and that opioid research should later provide new inspiration leading to varenicline. Disclosure: Jotham W. Coe, Hans Rollema, and Brian T. O’Neill are employees of Pfizer Inc.
REFERENCES [1] R. Doll and A. B. Hill, Br. Med. J., 1954, 1451. [2] R. Doll, R. Peto, J. Boreham and I. Sutherland, Br. Med. J., 2004, 328, 1519. [3] ‘‘Smoking and Health: Report of the Advisory Committee to the Surgeon General of the Public Health Service,’’ 1964. [4] Smoking and Health: A Report of the Surgeon General, 1979. [5] The ‘‘Recognizing Addiction as a Disease Act of 2007,’’ SB-1011. [6] B. Adhikari, J. Kahende, A. Malarcher, T. Pechacek and V. Tong, Morb. Mortal. Wkly. Rep., 2008, 57, 1226. [7] WHO Report on the Global Tobacco Epidemic, 2008 – The MPOWER package. http:// www.who.int/tobacco/mpower/en/index.html [8] K.-O. Fagerstro¨m, J. Behav. Med., 1982, 5, 343. [9] J. R. Hughes et al., Antidepressants for smoking cessation. Cochrane Database of Systemic Reviews 1, CD000031. DOI: 10.1002/14651858.CD000031.pub3, 2007. [10] (a) C. A. Hughes, Jr., Cancer J. Clin., 2000, 50, 143; (b) M. C. Fiore, et al., J. Am. Med. Assoc., 2000, 283, 3244.(c) M. C. Fiore et. al., U.S. Public Health Service, U.S. DHHS, Rockville, MD, May 2008. [11] S. Wakhlu, J. Opioid Manag., 2009, 5, 59. [12] (a) J. E. Rose and E. D. Levin, Pharmacol. Biochem. Behav., 1991, 41, 219; (b) J. E. Rose, F. M. Behm, E. C. Westerman, E. D. Levin, R. M. Stein and G. V. Ripka, Clin. Pharmacol. Ther., 1994, 56, 86. [13] J. Boulter, J. Connolly, E. Deneris, D. Goldman, S. Heinemann and J. Patrick, Proc. Nat. Acad. Sci. U.S.A., 1987, 84, 7763. [14] R. Lape, D. Colquhoun and L. G. Sivilotti, Nature, 2008, 454, 722. [15] A. A. Jensen, B. Frolund, T. Liljefors and P. Krogsgaard-Larsen, J. Med. Chem., 2005, 48, 4705. [16] D. Paterson and A. Nordberg, Neurobiology, 2000, 61, 75.
100
JOTHAM W. COE et al.
[17] (a) W. H. Beers and E. Reich, Nature, 1970, 228, 917; (b) R. P. Sheridan, J. Med. Chem., 1986, 29, 899. [18] J. P. Changeux, A. Devillers-Thiery and P. Chemouilli, Science, 1984, 225, 1335. [19] M. R. Picciotto, N. A. Addy, Y. S. Mineur and D. H. Brunzell, Prog. Neurobiol., 2008, 84, 329. [20] R. C. Hogg and D. Bertrand, Curr. Drug. Targets. CNS Neurol. Disord., 2004, 3, 123. [21] M. E. M. Benwell and D. J. K. Balfour, Br. J. Pharamacol., 1992, 105, 849. [22] C. F. Morrison and J. A. Stephenson, Psychopharmacologia (Berl.), 1969, 15, 351. [23] C. Romano, A. Goldstein and N. P. Jewell, Psychopharmacology, 1981, 74, 310. [24] W. T. Chance, M. D. Kallman, J. A. Rosecrans and R. M. Spencer, Br. J. Pharmac., 1978, 63, 609. [25] J. A. Pratt, I. P. Stolerman, H. S. Garcha, V. Giardini and C. Feyerabend, Psychopharmacology, 1983, 81, 54. [26] W. A. Corrigall and K. M. Coen, Psychopharmacology, 1989, 99, 473. [27] (a) M. R. Picciotto, M. Zoli, R. Rimondini, C. Lena, L. M. Marubio, E. M. Pich, K. Fuxe and J. P. Changeux, Nature, 1998, 391, 173; (b) S. S. Watkins, M. P. Epping-Jordan, G. F. Koob and A. Markou, Pharmacol. Biochem. Behav., 1999, 62, 743. [28] A. R. Tapper, S. L. McKinney, R. Nashmi, J. Schwarz, P. Deshpande, C. Labarca, P. Whiteaker, M. J. Marks, A. C. Collins and H. A. Lester, Science, 2004, 5, 1029. [29] (a) G. Di Chiara, Eur. J. Pharmacol., 2000, 393, 295; (b) J. A. Dani and M. De Biasi, Pharmacol. Biochem. Behav., 2001, 70, 439. [30] R. M. Wilson and S. J. Danishefsky, J. Org. Chem., 2006, 71, 8329. [31] F. van Haaran, K. G. Anderson, S. C. Haworth and W. R. Kem, Pharmacol. Biochem. Behav., 1999, 64, 439. [32] W. R. Kem, V. M. Mahnir, R. L. Papke and J. L. Lingel, J. Pharmacol. Exp. Ther., 1997, 283, 979. [33] W. R Kem and R. L. Papke, Soc. Neurosci., 1992, 18, 1358. [34] C. Reavill, B. Walther, I. P. Stolerman and B. Testa, Neuropharmacology, 1990, 29, 619. [35] A. I. Sacaan, J. L. Dunlop and G. K. Lloyd, J. Pharmacol. Exp. Ther., 1995, 274, 224. [36] A. Partheil, Arch. Pharm., 1894, 232, 161. [37] G. Scharfenberg, S. Benndorf and G. Kempe, Dtsch Gesundheitsw., 1971, 26, 463. [38] C. W. Luetje and J. Patrick, J. Neurosci., 1991, 11, 837. [39] R. L. Papke and S. F. Heinemann, Mol. Pharmacol., 1994, 45, 142. [40] (a) R. Mansbach, F. D. Tingley, C. Rovetti, T. Davis, L. Chambers, C. Fox, S. Sands, E. Arnold, A. Elder, J. Huang, D. Schulz and B. T. O’Neill, Soc. Neurosci., 1998, 294, 10. (b) B. T. O’Neill, PCT Int. Appl. WO 98 18,798, Abstr. 1998, 119, 4774k, 1998. [41] A. L. Hopkins, C. R. Groom and A. Alex, Drug Discov. Today, 2004, 9, 430. [42] P. D. Leeson and B. Springthorpe, Nat. Rev. Drug Discov., 2007, 6, 881. [43] S. Demers, H. Stephenson, J. Candler, C. G. Bashore, E. P. Arnold, B. T. O’Neill and J. W. Coe, Tetrahedron Lett., 2008, 49, 3368. [44] (a) B. T. O’Neill, D. Yohannes, M. W. Bundesmann and E. P. Arnold, Org. Lett., 2000, 2, 4201; (b) E. Marriere, J. Rouden, V. Tadino and M.-C. Lasne, Org. Lett., 2000, 2, 1121; (c) O. Nicolotti, C. Canu Boido, F. Sparatore and A. Carotti, Farmaco, 2002, 57, 469; (d) C. Canu Boido, A. Carotti and F. Sparatore, Farmaco, 2003, 58, 265. [45] (a) A. P. Kozikowski, S. K. Chellappan, Y. Xiao, K. M. Bajjuri, H. Yuan, K. J. Kellar and P. l. A. Petukhov, Chem. Med. Chem., 2007, 2, 1157; (b) S. K. Chellappan, Y. Xiao, W. Tueckmantel, K. J. Kellar and A. P. Kozikowski, J. Med. Chem., 2006, 49, 2673. [46] J. W. Coe, Org. Lett., 2000, 2, 4205. [47] J. W. Coe, M. G. Vetelino, C. G. Bashore, M. C. Wirtz, P. R. Brooks, E. P. Arnold, L. A. Lebel, C. B. Fox, S. B. Sands, T. I. Davis, D. W. Schulz, H. Rollema, F. D. Tingley, III and B. T. O’Neill, Bioorg. Med. Chem. Lett., 2005, 15, 2974.
Case History: Chantixt/Champixt (Varenicline Tartrate)
101
[48] C. G. Bashore, M. G. Vetelino, M. C. Wirtz, P. R. Brooks, H. N. Frost, R. E. McDermott, D. C. Whritenour, J. A. Ragan, J. L. Rutherford, T. W. Makowski, S. J. Brenek and J. W. Coe, Org. Lett., 2006, 8, 5947. [49] R. A. Glennon and M. Ducat, Pharm. Acta. Helv., 2000, 74, 103. [50] E. Carosati, S. Sciabola and G. Cruciani, J. Med. Chem., 2004, 47, 5114. [51] (a) P. H. Mazzocchi and B. C. Stahly, J. Med. Chem., 1979, 22, 455; (b) M. Mokotoff and A. E. Jacobson, J. Het. Chem., 1970, 7, 773; (c) P. H. Mazzocchi and A. M. Harrison, J. Med. Chem., 1978, 21, 238. [52] T. Eisner and J. Meinwald (eds), Chemical Ecology: The Chemistry of Biotic Interaction, National Academy of Sciences, Washington, DC, 1995. [53] (a) P. R. Brooks, S. Caron, J. W. Coe, K. K. Ng, R. A. Singer, E. Vazquez, M. G. Vetelino, H. H. Watson, Jr., D. C. Whritenour and M. C. Wirtz, Synthesis, 2004, 11, 1755; (b) R. A. Singer, J. D. McKinley, G. Barbe and R. A. Farlow, Org. Lett., 2004, 6, 2357; (c) C. J. O’Donnell, R. A. Singer, J. D. Brubaker and J. D. McKinley, J. Org. Chem., 2004, 69, 5756. [54] C. L. Coon, W. G. Blucher and M. E. Hill, J. Org. Chem., 1973, 38, 4243. [55] J. W. Coe, P. R. Brooks, M. G. Vetelino, M. C. Wirtz, E. P. Arnold, J. Huang, S. B. Sands, T. I. Davis, L. A. Lebel, C. B. Fox, A. Shrikhande, J. H. Heym, E. Schaeffer, H. Rollema, Y. Lu, R. S. Mansbach, L. K. Chambers, C. C. Rovetti, D. W. Schulz, F. D. Tingley, III and B. T. O’Neill, J. Med. Chem., 2005, 48, 3474. [56] J. W. Coe, P. R. Brooks, M. C. Wirtz, C. G. Bashore, K. E. Bianco, M. G. Vetelino, E. P. Arnold, L. A. Lebel, C. B. Fox, F. D. Tingley, III., D. W. Schulz, T. I. Davis, S. B. Sands, R. S. Mansbach, H. Rollema and B. T. O’Neill, Bioorg. Med. Chem. Lett., 2005, 15, 4889. [57] J. P. Gallivan and D. A. Dougherty, Proc. Nat. Acad. Sci. U.S.A., 1999, 96, 9459. [58] H. Rollema, L. K. Chambers, J. W. Coe, J. Glowa, l. R. S. Hurst, L. A. Lebel, Y. Lu, R. S. Mansbach, R. J. Mather, C. C. Rovetti, S. B. Sands, E. Schaeffer, D. W. Schulz, F. D. Tingley, III and K. E. Williams, Neuropharmacol, 2007, 52, 985. [59] R. S. Obach, A. E. Reed-Hagen, S. S. Krueger, B. J. Obach, T. N. O’Connell, K. S. Zandi, S. A. Miller and J. W. Coe, Drug Metab. Dispos., 2006, 34, 121. [60] J. Hukkanen, P. Jacob, III and N. L. Benowitz, Pharmacol. Rev., 2005, 57, 79. [61] H. M. Faessel, B. J. Smith, M. A. Gibbs, J. S. Gobey, D. J. Clark and A. H. Burstein, J. Clin. Pharmacol., 2006, 46, 991. [62] H. M. Faessel, M. A. Gibbs, D. J. Clark, K. Rohrbacher, M. Stolar and A. H. Burstein, J. Clin. Pharmacol., 2006, 46, 1439. [63] K. I. Cahill, L. F. Stead, T. Lancaster, Cochrane Database of Systematic Reviews 1, 2007, Art. No.: CD006103, in The Cochrane Library, Issue 1, Wiley, Chichester, UK. doi:101002/ 14651858.CDC006103.pub2, 2007. [64] M. Nides, C. Oncken, D. Gonzales, S. Rennard, E. J. Watsky, R. Anziano and K. R. Reeves, Arch. Intern. Med., 2006, 166, 1547. [65] D. Gonzales, S. I. Rennard, M. Nides, C. Oncken, S. Azoulay, C. B. Billing, E. J. Watsky, J. Gong, K. E. Williams and K. R. Reeves, J. Am. Med. Assoc., 2006, 296, 47. [66] D. E. Jorenby, J. T. Hays, N. A. Rigotti, S. Azoulay, E. J. Watsky and K. E. Williams, J. Am. Med. Assoc., 2006, 296, 56. [67] S. Tonstad, P. Tonnesen, P. Hajek, K. E. Williams, C. B. Billing and K. R. Reeves, J. Am. Med. Assoc., 2006, 296, 64. [68] M. Nides, E. D. Glover, V. I. Reus, A. G. Christen, B. J. Make, C. B. Billing and K. E. Williams, Am. J. Health. Behav., 2008, 32, 664. [69] M. J. Jarvis, Bri. Med. J., 2004, 32, 277.
CHAPT ER
5 Case History on Tekturnas/ Rasilezs (Aliskiren), a Highly Efficacious Direct Oral Renin Inhibitor as a New Therapy for Hypertension Ju¨rgen Maibaum* and David L. Feldman**
Contents
1. 2. 3. 4.
Introduction Rationale for the Use of Direct Renin Inhibitors Pre-Clinical Models to Study Direct Renin Inhibitors Medicinal Chemistry Evolution — The Early Renin Inhibitor Program at Ciba-Geigy 4.1 The emerging novel topology design concept 4.2 Macrocyclic renin inhibitors 4.3 (P3-P1)-Tethered hydroxyethylene transition-state mimetics 4.4 SAR in the THQ series — early pre-clinical leads 4.5 Modification of the transition-state mimetic portion 4.6 SAR in the ‘Phenoxy’ series 4.7 Challenges of a multiple chemotype approach 5. First Convergent and Scalable Synthesis Development 6. Pre-Clinical Properties of Aliskiren 7. Effects of Aliskiren in Disease Models 8. Clinical Studies with Aliskiren 9. Conclusions References
105 106 107 108 110 111 112 113 115 116 119 120 122 122 123 124 124
Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland Novartis Institutes for BioMedical Research, East Hanover, NJ, USA
Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04405-4
r 2009 Elsevier Inc. All rights reserved.
105
106
Ju¨rgen Maibaum and David L. Feldman
1. INTRODUCTION Hypertension is a major risk factor for cardiovascular diseases, affecting more than 25% of adults worldwide [1]. The high percentage of patients with insufficiently controlled blood pressure (BP) levels suggests a continued need for improved antihypertensive drug therapies. Direct renin inhibition is a promising new type of treatment for high BP. The renin-angiotensin-aldosterone system (RAAS) is a key physiological regulator of BP and fluid homeostasis. The first and rate-limiting step (i.e., point of activation) of this cascade is catalyzed by the highly specific aspartic protease renin [2]. Direct blockade of this step has long been recognized as most attractive for therapeutic intervention. This triggered extensive research activities initiated in the late 1970s in the quest for identifying clinically efficacious orally active direct renin inhibitors. However, various challenges persisted in this area for more than two decades despite all the efforts made in the pharmaceutical industry. Recently, an unprecedented topological structure-based drug design strategy at Ciba-Geigy (now Novartis) enabled the discovery of novel, highly potent and selective non-peptide transition-state mimetic (TSM) renin inhibitors. These efforts culminated in the discovery of TEKTURNAs/RASILEZs (aliskiren, 1, Figure 1) recently approved as a new therapy for the treatment of hypertension. As a first-in-class direct renin inhibitor, aliskiren has demonstrated orally active anti-hypertensive efficacy and end-organ protection. Thus, more than 10 years after the introduction of the angiotensin (Ang) AT1 receptor blockers (ARBs), a therapy directed at renin is available to patients.
2. RATIONALE FOR THE USE OF DIRECT RENIN INHIBITORS Blockade of the RAAS is a primary treatment in many cardiovascularrenal diseases including hypertension, congestive heart failure and renal disease [3]. The ultimate goal of such therapy is to block the formation or action of Ang II, the principal effector of this pathway. Until the approval of aliskiren, the RAAS could be inhibited either at the step of conversion of Ang I to Ang II with Ang I converting enzyme inhibitors (ACEi) or at CH3O
OH
H N
H2N O CH3O
Figure 1
TEKTURNAs/RASILEZs (aliskiren).
O 1
CONH2
Case History on Tekturnas/Rasilezs (Aliskiren)
107
the Ang II receptor level with ARBs. Such interventions have yet to halt the progression of cardiovascular diseases. That is, tissue damage may continue in the heart, blood vessels and kidney, possibly because the RAAS is not comprehensively blocked with ACEi or ARB. In contrast, inhibiting the RAAS at the point of its activation (i.e., renin) may lead to superior downstream RAAS inhibition as compared to existing therapies. Moreover, formation of Ang II by alternative pathways (e.g., chymase) is limited with renin inhibition because Ang I formation is blocked. A unique feature of renin inhibition is the ability to block the increase in plasma renin activity (PRA) that accompanies the rise in the plasma levels of renin during RAAS blockade. Thus, although ACEi and ARB actually raise PRA, renin inhibitors reduce PRA despite the high levels of circulating renin. This action leads to effective inhibition of Ang II formation and potentially greater organ protection.
3. PRE-CLINICAL MODELS TO STUDY DIRECT RENIN INHIBITORS The road towards developing human renin inhibitors has been challenging with respect to establishing appropriate in vivo models in which to test the efficacy of these compounds. This difficulty relates, at least in part, to the prominent species specificity displayed by renin and the high primate specificity of potent human renin inhibitors. Thus, most inhibitors of human renin are only weakly effective in rats. Consequently, although rats are the most commonly used species in hypertension efficacy studies, such models have been of limited use to study human renin inhibitors [4]. Potent inhibitors of rat renin have allowed in vivo studies in this species; however, the design of dual human–rat renin inhibitors has been extremely challenging [5,6]. Normotensive nonhuman primates such as cynomolgus, squirrel, rhesus and marmosets [7–9], and dogs [10,11], in which the RAAS is stimulated by a low salt diet and/or a diuretic, have been used to demonstrate the efficacy of renin inhibition on BP, cardiac function and plasma RAAS components. We initially established a model of sodium-depletion in restrained conscious marmosets (Callithrix jacchus). In this model, BP and heart rate (HR) measurements were performed invasively and thereby allowed to determine only short-term hemodynamic effects [12]. Subsequently, the application of radio-telemetry for continuous measurement of BP, HR and changes in the electrocardiogram in freely moving animals permitted the assessment of the effects of drug treatment under less stressful conditions over a longer time range [13]. We also measured the 24 h timecourse of PRA ex vivo as a surrogate marker to indicate that BP-lowering effects were specifically related to renin inhibition.
108
Ju¨rgen Maibaum and David L. Feldman
The ability to manipulate the genome of rodents has permitted the development of several transgenic rat and mouse models, in which renin is expressed. This eliminated the issue of species specificity of renin inhibitors in vivo. Indeed, beneficial effects of renin inhibition, which are considered to be of important clinical relevance, have been demonstrated in these models [14]. Double transgenic rats (dTGR) express human genes for renin and angiotensinogen [15]. The high expression of this ‘human RAAS’ leads to high levels of Ang II and consequently to renal and cardiac damage [16]. These animals are highly responsive to inhibition of their RAAS with human renin inhibitors. Aliskiren, 1, has been intensively studied in dTGRs and other transgenic rodents for its effects on BP and organ protection.
4. MEDICINAL CHEMISTRY EVOLUTION — THE EARLY RENIN INHIBITOR PROGRAM AT CIBA-GEIGY Tremendous resources have been devoted to the design of orally efficacious renin inhibitors, starting in the late 1970s [17–20], and a vast number of potent and selective peptide-like TSM inhibitors have been reported. Several candidates progressed into the clinic but suffered from poor intestinal absorption or high liver first-pass elimination, high synthetic complexity and hence high cost of goods, and/or insufficient clinical efficacy. The renin drug discovery project at Ciba-Geigy started in 1980 and transitioned through the evolution over three generations of renin inhibitors, ranging from modified linear peptides initially, to more drug-like peptidomimetics and ultimately to completely novel chemical structures that still function as TSM inhibitors. Computer-aided molecular modeling along with X-ray crystallography of human renin with and without bound inhibitors enabled us to adopt a systematic direct targetstructure based strategy. This eventually culminated in the discovery of aliskiren, 1, representing a unique class of topological peptidomimetic renin inhibitors, as described in more detail by this review. The large peptide inhibitor CGP29287, 2 (Figure 2), derived from the N-terminal sequence of angiotensinogen by incorporation of the statine TSM at the renin cleavage site and with modification of both N- and Cterminal sites, was the first proteolytically stable, long-acting renin inhibitor shown to be active after intravenous and oral administration to monkeys [12]. Although not considered ‘drug-like’ due to its peptide characteristics, this first-generation inhibitor proved its importance as a pharmacological tool for studying the endocrine, hemodynamic, renal and cardiac responses to tissue and systemic renin inhibition in vivo in primates [21]. Subsequently, reduction of molecular size by truncation at both the N- and the C-terminus and incorporation of a hydroxyethylene
Case History on Tekturnas/Rasilezs (Aliskiren)
109 O
O
O
H N
N
H N O
NH
N
NH
HN
O
H N
NH2
HN
O
H N
N H
O
OH
O N H
O
O
H N
O
N H
O
O
N
O N H
NH HN
NH2
Figure 2 First generation peptide renin inhibitor — CGP29287 (2).
N
NH
O S O O
N H
H N
N OH
O
H N
O
S
H N
N H
O O
O
3 N
O N
S
O O
N H
NH
H N O
OH
O
OH
4
N
N
S
O N
OH
OH
S
O O
5
N H
H N O
OH OH
6
Figure 3 Second-generation peptide-based renin inhibitors.
(HE) isostere provided inhibitors spanning the S4-S2u enzyme recognition sites. Further structure refinement identified CGP38560, 3 (Figure 3) [22], a renin inhibitor of the second generation and the first selected at CibaGeigy for clinical investigations. This potent and selective inhibitor of human and marmoset plasma renin in vitro (IC50 ¼ 0.7 nM) reduced mean arterial pressure (MAP) and suppressed PRA in Na-depleted restrained marmosets (10 mg/kg oral dose) [23]. Owing to its short duration of action in hypertensive patients and low (o1%) oral bioavailability in humans, 3 was not considered to be a clinically viable drug [24,25]. Remikiren (RO-42-5892, 4), with a plasma human renin IC50 ¼ 0.8 nM, combined the N-terminal tert-butylsulfonyl of 3 with a shorter C-terminal diol TSM [8] and was investigated by Hoffmann–La Roche in phase II trials. RO-42-5892 caused significant BP lowering in hypertensive
110
Ju¨rgen Maibaum and David L. Feldman
patients, but antihypertensive responses were only short-lasting at the maximal tolerated dose [26]. Oral bioavailability of 4 was low in monkeys, rats, dogs and humans (B1%) due to rapid liver first-pass metabolism and biliary excretion of unchanged drug [27,28]. It is noteworthy that 4 was proposed to act primarily by inhibiting an extra-plasma renin pool [29]. RO-42-5892 is the first renin inhibitor for which extensive partitioning to, and retention by, the kidney has been reported [30]. More water soluble second generation inhibitors with superior pharmacokinetics in animal species were subsequently disclosed. SC56525, 5 (IC50 ¼ 1.2 nM), was reported by Searle to exert potent oral BP lowering effects in both Na-depleted and renal hypertensive dogs [11]. Oral bioavailability of 5 was 66% in dogs (30 mg/kg po); however, nonlinear pharmacokinetics and a short elimination half-life (1.3 h) were observed [31]. Zankiren (A-72517, 6, IC50 ¼ 1.1 nM) culminated from a structure–oral absorption optimization strategy at Abbott [7,32–34]. Oral bioavailability in cynomolgus monkey, dog and rat was 8, 53 and 24%, respectively, with the dog predicting best the human bioavailability [33]. Remarkably, distribution studies in animals showed selective uptake of 6 into the kidneys, which could explain its favorable effects on renal hemodynamics [34]. In patients, 6 showed rapid oral absorption and significant antihypertensive efficacy [35]. Zankiren has been considered a hallmark second-generation renin inhibitor, although the drug was not developed further [33].
4.1 The emerging novel topology design concept During the late 1980s, the need became apparent to identify a structurally novel concept that could lead to the development of orally active renin inhibitors with improved properties. Such improvements included reduced molecular weight, increased aqueous solubility and other factors considered to impact oral efficacy. In view of the experience with CGP38560, 3, it was deliberately decided to move away in a radical fashion from the classical peptidomimetic approach. Re-direction of the design strategy and medicinal chemistry resources towards ‘conceptually different inhibitors’, the title of the new project, gave birth to the topological (P3-P1-P0) working hypothesis during 1987–1988 (Figure 4). No validated hits resulted from a small ‘random screening’ campaign that was based on a collection of B20,000 compounds delivered by our newly established New Lead Discovery Unit. Our new design concept was entirely target structure-based and evolved through several major steps: i) using a human renin homology model, exploration of aryl and bulky alkyl P3 fragments linked to diverse cycloalkyl P1 templates bearing polar head groups as putative truncated TSMs ; ii) starting from P1u-P2u extended HE isostere TSMs and growing
Case History on Tekturnas/Rasilezs (Aliskiren)
P4
P3
P2 N
O S O O
N H
P1
P1′
P2′
NH H N
OH
P0 Truncated transition-state mimetic OH *H2N
H N
P1′
111 P2′
OH* *optional
O
O
3
P3
P1
Figure 4 The initial (P3-P1-P0) topology approach.
towards P3 from a rigid P1 residue, providing sub-micromolar leads; iii) introduction of H-bond acceptors/donors to optimized (P3-P1)pharmacophores, leading to a significant increase in potency and in vivo activity; iv) discovery of a distinct non-substrate pocket S3sp by X-ray crystallography as a canonical binding pocket for different sub-series and experimental confirmation of the topology design concept; v) structurebased lead optimization of key series and the search for novel (P3-P1)chemotypes. The conceptual innovation was based on the hypothesis that both the S3 and the S1 renin recognition sites constitute a contiguous ‘hydrophobic super-pocket’ — or ‘hydrophobic hot spot’ — similar to fungal aspartyl proteases with strong preference for lipophilic ligands positioned in spatial proximity. This notion provoked the search for hydrophobic scaffolds composed of directly linked P1 and P3 motifs. Initially, a diverse array of novel conformationally more or less constrained mono-, bi- and tricyclic core structures was intensively investigated, which were modeled on optimized SAR for P1 and P3 of peptide-derived renin inhibitors. These compounds were further characterized by one or more OH and NH2 groups of an acyclic or cyclic terminal scaffold envisaged to target the catalytic aspartates as simplified versions of a TSM truncated at its prime-site (P3-P1-P0 hypothesis, Figure 4). Despite the tremendous efforts and difficult chemical syntheses involved, none of these molecules was found to inhibit renin up to 100 mM in the enzymatic assay.
4.2 Macrocyclic renin inhibitors The lack of success in identifying even very low-affinity inhibitors initially was disappointing and raised major concerns about the validity of the design concept. Up to this point, our modeling depended on overlaps with the crystal structures of pepstatin or related peptides complexed with non-mammalian aspartyl proteases such as penicillopepsin and rhizopuspepsin [36]. In the course of our work, a more refined homology model of human renin derived from the endothiapepsin X-ray structure [37] was used to predict the active site conformation of 3 [38]. The first apo-crystal structure of human renin
112
Ju¨rgen Maibaum and David L. Feldman
N O O
N H O O
Figure 5
NH H N
OH
H N O
7
(P3-P1)-linked macrocyclic peptidomimetic renin inhibitor.
was reported only in 1989 [39]. In our group, the human renin/3 complex was resolved shortly thereafter, confirming the accuracy of the computational model [40]. At this point, interest arose in the design of P3-P1 macrocyclic inhibitors for probing the feasibility of covalently linking the P1 and P3 residues of an open-chain congener inhibitor. It was furthermore envisaged that freezing such constrained peptidomimetics in their enzyme-bound conformation would lead to a gain in entropy thereby possibly allowing elimination of peripheral elements (e.g., P4 and/or P2u residues), and hence diminishing molecular size. The in vitro activity of the non-optimized (P3-P1)-macrocycle 7 (Figure 5) against human renin (IC50 ¼ 2 mM), although moderate, supported the notion that the renin S1/S3 specificity sites are not separated from each other but form a large hydrophobic cavity. More potent macrocyclic inhibitors incorporating directly linked P1 and P3 side chains were reported after we had concluded our limited efforts [41]. The proximal topology of other recognition sites of renin inspired the design of various classes of potent macrocyclic inhibitors [9].
4.3 (P3-P1)-Tethered hydroxyethylene transition-state mimetics As the original approach targeting truncated transition-state surrogates had failed to deliver any starting points for further lead optimization, the emphasis of the exploratory design was redirected. Additional binding interactions to the P1u and/or P2u sites of renin were conceived to be likely more important than was initially acknowledged for such putative (P3P1)-pharmacophores lacking interactions to the P2-P4 sites. The cyclohexyl-substituted HE isostere 8 (IC50 ¼ 30 mM, Figure 6) was selected as a starting ‘fragment’ based on the well-established preference of human renin for bulky hydrophobic P1 residues, providing selectivity against other human aspartyl proteases [42]. Inhibitor 9 was one of the most active (IC50 ¼ 0.3 mM) of several tethered 1,3- or 1,4-cis and transdisubstituted cyclohexyl analogues [43]. The 100-fold potency increase for 9 vs. 8 strengthened the evidence for the feasibility of the tethered P3-P1 topological inhibitor design.
Case History on Tekturnas/Rasilezs (Aliskiren)
113
10: R = H H2N
OH CH3 H N
R
O
H2N
R
OH CH3 H N
11: R =
O
trans (rac)
12: R =
8: R = H Cyclohexyl- 9: R = ‘optimized’ P1
Gem-dimethyl ‘symmetrical’ P1
N O
13: R = CH2CO2H
Figure 6 Growing the TSM ‘fragment’ — growing the evidence.
The synthetic complexity of cycloalkyl-derived inhibitors such as 9 restricted further optimization. Symmetrization and hence avoiding any stereocenter at P1 by tethering the geminal-dimethyl position of the TSM 10 (IC50 W100 mM; Figure 6) afforded 11 (IC50 ¼ 0.7 mM). This and subsequently the incorporation of a functionalized spacer were major breakthrough steps towards more simplified early leads with impressively improved in vitro potencies. The tetrahydroquinoline (THQ) derivative 12 (IC50 ¼ 0.05 mM) was W2,000-fold more potent than 10, which further increased our confidence in the design concept. The advanced carboxylic acid 13, appropriately N,O-protected [44], enabled a systematic and rapid SAR exploration of the THQ moiety (vide infra). Variation of the P3-P1 motif was explored intensively with the aim to optimize the interactions to the spacious S1/S3 sites. Hydrophobic van der Waals contacts to this hydrophobic ‘hot-spot’ were considered as key contributing factors for strong ligand binding. We therefore envisaged to maximize the contacts of an optimally designed lipophilic and conformationally rigid P3-P1 pharmacophore to the large surface of the S3/S1 cavity, as exemplified by inhibitors 14 (IC50 ¼ 2.7 mM; Figure 7) and 15 (IC50 ¼ 2.6 mM). Less constrained analogues of 15 with improved in vitro potencies resulted from P1 mono-substitution, and hence reintroduction of a stereocenter with a preferred configuration [45]. Enlarging the size of P1 from methyl (16, IC50 ¼ 2 mM) to isopropyl afforded inhibitor 17 (IC50 ¼ 0.1 mM). Sterically demanding residues such as tert-butyl (18, IC50 ¼ 1.5 mM) and phenyl (19, IC50 ¼ 39 mM) resulted in decreased enzyme affinities. During this work, advances in synthetic chemistry facilitated the practical access to HE isosteres by starting from the corresponding enantiomeric amino acids [45–47]. Yet, early SAR optimization often remained cumbersome due to the non-convergent, lengthy and only partially stereo-controlled reaction sequences required for each single structural modification of the P3-P1 scaffolds. This was particularly evident for inhibitors 16–19, which raised some controversy about the chances to progress this series with reasonable efforts. Incorporation of functionalized spacers into the (P3-P1)-motif of 17, allowing more rapid
114
Ju¨rgen Maibaum and David L. Feldman
H2N
OH CH3 H N
H2N
OH CH3 H N
O
O
14
15 H 2N
OH CH3 H N O
R
16: R=CH3 17: R=CH(CH3)2 18: R=tert-Butyl 19: R=Phenyl
Figure 7
Morphing the (P3–P1)-pharmacophore as a hydrophobic ‘hot spot’.
SAR studies similar to those for 12, were evaluated later in the fully elaborated ‘phenoxy’ series; however, these efforts resulted only in inferior inhibitors.
4.4 SAR in the THQ series — early pre-clinical leads Despite all efforts to optimize the (P3-P1)-scaffold by the ‘hydrophobic approach’, a plateau for in vitro activity apparently had been reached, exemplified by the moderate activities of 12 and 17. Next, we focused our attention on additional design strategies to achieve a further substantial gain in potency. Close examination of SAR data for second-generation renin inhibitors, as well as X-ray crystal structures of peptide inhibitors bound to fungal aspartyl proteases, emphasized the importance of a canonical H-bond between the P3/P2 amide carbonyl and the backbone NH of Ser219 for strong binding affinity. Intriguingly, computational docking of 11 and related analogues into the renin homology model suggested the ‘upper’ portion of the P3 moiety to be positioned in proximity to Ser219 [48,49]. Introduction of H-bond acceptors/donors at the 3- or 4-position of the naphthalene in 11 was extensively probed; however, these early attempts did not result in any affinity increase [43]. In contrast, substitution of the THQ heterocycle of 12 with a carboxylic ester at C-3 afforded inhibitor 20 (IC50 ¼ 0.8 nM, Figure 8) with a dramatic boost in potency. The 3(R)-configured 20 was 50-fold more potent than its (S)-isomer and 12, which indicated a highly specific interaction to the enzyme requiring the proper spatial orientation of the ester group [48]. The sequential discovery of 12 and 20 occurred within a few weeks of each other and represented a hallmark event at about the same time when the renin project was under close scrutiny. The ester in 20 could be replaced with amides, hydroxylated alkyl and short-chained alkyl ethers without a significant drop in the IC50s. THQ modifications were investigated by substitution, incorporation of
Case History on Tekturnas/Rasilezs (Aliskiren)
O
O H2N N
HN
OH CH3 H N
OH
N
O O
20
O O
NH
OH
21 (2′ R or S)
H N
H2N N
H N
H 2N
S
O O
O
115
O O 22
Figure 8 SAR of the THQ series — early pre-clinical lead candidates.
heteroatoms and changing the ring size. The 1,4-benzthiazine 21 (Figure 8; absolute stereochemistry not assigned), bearing a P1u isopropyl at the TSM portion (vide infra), was found to be a potent inhibitor of human and marmoset plasma renin (IC50 ¼ 3 and 16 nM) with selectivity towards bovine cathepsin D and porcine pepsin (IC50s W50 mM). It is noteworthy that 21 and similarly analogue 22 showed excellent aqueous solubility (1.74 and 3.44 g/L, pH 7.4) and low lipophilicity (logPoct 1.71 and 1.70). In restrained Na-depleted marmosets, oral administration of 21 (10 mg/kg) lowered BP significantly by 30 mmHg at peak, while PRA was completely blocked over 6 h. This first early pre-clinical lead was not progressed further, as more attractive analogues emerged from both this and other sub-series. THQ analogue 20 was the first from a total of six novel renin inhibitors from different classes, for which X-ray structures in complex with human renin were resolved [50]. Importantly, this crystal structure confirmed both the S1 and the S3 sites to be fully occupied by the geminaldimethyl group and the aryl portion of the THQ, providing the first structure proof for the computational-based topographical design. An additional H-bond interaction of the spacer carbonyl of 20 to the flexible enzyme flap domain was observed. The ester carbonyl of 20 formed H-bonds with the Ser219 NH, as was predicted, and in addition with the side chain OH. Most surprisingly, the X-ray revealed the methoxy group pointing into the direction of a solvent-shielded non-substrate binding site. This Ssp 3 pocket extends towards the center of renin and is flanked partly by hydrophobic amino acids. Given these results, focused lead optimization then targeted substitutions that would penetrate more deeply into the Ssp 3 pocket [50]. Late-stage SAR advanced towards in vivo potent THQ analogues while pre-clinical development of inhibitor 30 from the ‘phenoxy’ class was well under way (vide infra). Inhibitor 22 showed a remarkable improvement in potency compared to 21 (plasma
116
Ju¨rgen Maibaum and David L. Feldman
renin IC50 ¼ 0.5 nM vs. 3 nM) and excellent dose-dependent oral efficacy over 24 h in telemetered Na-depleted marmosets (peak DMAP of 25 mmHg at 3 mg/kg), accompanied by suppression of PRA for up to 24 h. However, 22 did not demonstrate a superior profile as compared to 30 in exploratory in vivo cardiovascular studies.
4.5 Modification of the transition-state mimetic portion Replacements of the classical HE isostere by shortening the C-terminal portion and eliminating stereocenters have been investigated intensively as a strategy towards simpler peptide-derived inhibitors with improved oral activity and bioavailability [17]. We too explored a small set of such TSM analogues early in view of the synthetic challenges we encountered during initial SAR work. The norstatine ester (23, IC50 ¼ 2.2 mM), the Nethyl oxazolidinone (24, IC50 ¼ 0.64 mM) [51], and the ‘Roche’ erythro-1,2diol (25, IC50 ¼ 4.2 mM) were at least three orders of magnitude less potent than parent 20 (Figure 9). Remarkably, the truncated amino alcohol 26 was almost equipotent (IC50 ¼ 1.0 mM) compared to 23–25, which indicated only minor binding contributions by the S1u extended TSM portion relative to that of the (P3-P1)-scaffold. Similar findings were subsequently observed for related analogues of 30. SAR by substitution of the TSM primary amine revealed, in general, a significant affinity loss even for small N-alkyl or N-acyl residues in all sub-series. These efforts, however, were limited since retaining the peptide backbone was not part of our strategy, although this approach was being used by others [20]. These results clearly emphasized that for this class of topological renin inhibitors interactions to the extended prime site of human renin are required to induce strong binding affinities. As a consequence, we restricted all our subsequent SAR optimization to the classical HE isostere, as this was thought to provide the optimal trajectory into the S2u pocket. Retrospectively, the weak potency of 26 bearing an ‘optimized’ (P3-P1)-scaffold is noteworthy in view of the expectations for hit identification at the outset of our conceptual design program (Figure 4). At the time, sensitive biophysical methods such as protein NMR to detect very low-affinity ligands were not available [52].
OH H2N N
MeO2C
O O 26: R = Me, Q = H
H2N
O
Q 24: R = Et, Q =
O
Figure 9
O
23: R = Et, Q =
RO2C
N O
Modifying the transition-state mimetic (TSM).
N
OH OH
O 25 (2′ R/S)
Case History on Tekturnas/Rasilezs (Aliskiren)
117
4.6 SAR in the ‘Phenoxy’ series The concept of incorporating suitable H-bond acceptors/donors into the (P3-P1)-scaffold was successfully applied across different series. Starting from 17 (Figure 7) and tethering a terminal ethyl carboxylate to the ortho position relative to the tert-butyl group of the phenyl spacer resulted in a 20-fold potency increase (27, purified human renin IC50 ¼ 6 nM, Figure 10) [45]. Modelling had suggested the ester carbonyl to be in binding distance to Ser219 with the methoxy group pointing towards the P4 recognition site of renin. Carboxamide 28 (IC50 ¼ 20 nM) showed only a slight drop in affinity against purified renin. However, inhibitor 28 (plasma IC50 ¼ 460 nM) and most other tert-butyl analogues were very poor inhibitors in vitro in the human plasma renin assay with angiotensinogen as the substrate. The significantly reduced potency in plasma generally observed in this series was attributed to the high intrinsic local lipophilicity of the tert-butyl group, yet the actual underlying mechanism for the potency loss under the physiological assay conditions remained unclear [53]. An important milestone was the replacement of tert-butyl by the smaller and more polar methoxy group without compromising binding affinity. This afforded inhibitors such as 29 (purified renin IC50 ¼ 42 nM, plasma IC50 ¼ 95 nM) that were equipotent under the different assay conditions. This ‘rule breaking’ finding merits emphasis in the light of the perceived preference of human renin for hydrophobic aryl or bulky alkyl residues at P3, as was suggested by ample previous SAR data. Extensive SAR optimization of 29 then focussed on modifications of the phenoxy side chain, starting from a common advanced intermediate [53]. Shortening or lengthening of the flexible linear alkoxy substituent and placing one or two oxygen atoms at different positions afforded a
O O H2N
R
MeHN
OH CH3 H N
O
O 29: Q = CH3O MeO
O 27: OMe 28: NH2
30: Q =
O
MeO MeO
OH
H N
H2N O MeO
O 31
Figure 10 Milestone SAR of the ‘Phenoxy’ series.
H2N Q
OH CH3 H N O
118
Ju¨rgen Maibaum and David L. Feldman
library of potent inhibitors, the most prominent analogue of which was 30 (purified renin/plasma IC50 ¼ 1 nM, Figure 10). The X-ray structure of human renin-bound 30, resolved shortly after that of the THQ 12, confirmed the close hydrophobic interactions of the P1 isopropyl, the phenyl spacer and the P3 methoxy to the S3-S1 ‘superpocket’ [50]. Again unpredicted by modeling, and most striking, was the finding that the methoxypropoxy residue occupied the non-substrate Ssp 3 pocket by forming a H-bond to Tyr14 at the bottom of the cavity. It is quite remarkable that optimization of the phenoxy side chain in 30, permitting a perfect fit into Ssp 3 of renin, was completed before its true binding interactions to this site were uncovered by X-ray. In telemetered Nadepleted marmosets, 30 induced pronounced BP reductions (peak DMAP 20–25 mmHg), which were still significant after 24 h (at 30 mg/kg) [53]. No changes in HR were observed over the 1–30 mg/kg oral dose range. Bioavailability in non-depleted marmosets was moderate (16%) with a short terminal half-life (2 h). Pre-clinical cardiovascular safety pharmacology, however, revealed 30 to induce a dose-dependent transient reduction in HR and changes in cardiac conduction (intravenous dose range 0.3–10 mg/kg) to anesthetized sub-primates, which were unrelated to in vivo renin inhibition. Owing to the insufficient therapeutic margin, pre-clinical investigation of 30 was terminated. Ongoing lead optimization activities aimed to identify analogues that were more efficacious in vivo with longer duration of action. An increasing body of evidence emerged within this and other series that P1u isopropyl substitution of the HE isostere gave inhibitors with similar in vitro, but markedly superior in vivo potency as compared to their P1u methyl congeners. This increased potency may have been due to as yet unknown beneficial pharmacokinetic properties. For example, 31 (purified/plasma renin IC50 ¼ 1/4 nM) showed significantly stronger depressor effects (peak DMAP of 20 mmHg) as compared to 30 over 4–5 h (3 mg/kg) [54]. Considerable efforts targeting P2u modifications identified several N-terminal carboxamides, which demonstrated tight binding in vitro to plasma renin and markedly increased oral efficacy with prolonged duration of action in Na-depleted marmosets. From this sub-series, 1 (plasma renin IC50 ¼ 0.6 nM) emerged as one of the most potent inhibitors of human renin that we had identified at that time. In vitro potency was less against plasma renin from marmoset (IC50 ¼ 2 nM) and non-primates (mouse, rat, dog, rabbit and guinea pig IC50s ¼ 4, 80, 7, 11 and 63 nM, respectively). The promising drug candidate 1 was intensively studied in vivo, first in Na-depleted marmosets [54–56]. The dose-dependent and sustained decrease in BP following oral administration (0.3–30 mg/kg) was accompanied by a decrease in PRA and an increase in plasma renin levels. Maximal efficacy of 30 mmHg DMAP was achieved at 3 mg/kg, which corresponded to the maximal BP
Case History on Tekturnas/Rasilezs (Aliskiren)
119
depressor effect induced by ACEi and ARB in this model [56]. Inhibitor 1 was markedly more effective than equivalent oral doses of remikiren, 4, and zankiren, 6. Repeated once-daily oral dosing (10 mg/kg/day) over 8 days caused effective BP lowering over 24 h without altering HR and without rebound increase in BP after termination of treatment [55]. On the basis of its promising pharmacology and safety profile, 1, which came to be known later as SPP100 and then aliskiren, was taken forward to clinical development.
4.7 Challenges of a multiple chemotype approach In the course of our novel chemotype renin inhibitor program, at least four structurally diverse major lead series emerged from the initial topological design approach providing sub-micromolar inhibitors [49]. Principal conceptual considerations such as the introduction of suitable H-bond acceptors/donors to the distinct P3-P1 scaffolds could be successfully transferred between sub-series, leading to breakthroughs in in vitro potency in each case. Although key in vitro SAR were consistent between the various sub-series, lead optimization for in vivo efficacy and refining of other key inhibitor profiles proceeded in a less parallel fashion, hence involving different sets of lead SAR. An additional complication, particularly during the late stage of the project, was the need to develop separate efficient synthetic paradigms for candidate inhibitors from different sub-series. As an example, the potent indole inhibitor 32 (purified/plasma renin IC50 ¼ 1/4 nM, Figure 11) was derived from weakly active 11 by incorporation of a synthetically flexible spacer bridging a mono-alkyl P1 residue and a P3 aryl residue [48]. This lead demonstrated a more pronounced BP reduction (DMAP 20 mmHg) but was significantly shorter acting than 30 (10 mg/kg po) in Na-depleted marmosets. Unexpectedly, X-ray analysis of an analogue of 32 in complex with human OH O N
OH
H N
H2N
O
O
N H
N
H N
H2N
O
O
N H 33
32 MeO
MeO OH O
O
N H
Figure 11 Multiple
OH
H N
H2N O
NH2
O
O
N H
34
(P3-Psp 3 )-pharmacophore
H N
O H 2N
approach.
O 35
N O
120
Ju¨rgen Maibaum and David L. Feldman
renin showed that the indole moiety was posed in a flipped binding conformation with the N-benzyl substituent positioned within the same Ssp 3 pocket, that had been identified shortly before by X-ray of 20 and 30 [50]. As a consequence, the indole was in close hydrophobic contact with the S3 pocket instead of mimicking the peptide backbone as proposed by modeling. This suggested a promiscuous character of the rigid Ssp 3 pocket accommodating both hydrophobic aryl groups and more polar heteroaliphatic side chains. Psp 3 phenyl-substituted analogues in both the THQ [48] and the ‘phenoxy’ series [53] were found to be highly potent, although IC50s dropped markedly against plasma renin due to the local lipophilicity of the Psp 3 aryl. Vice versa, polar N-substitution in the ‘indole series’ resulted in the potent inhibitor 33 (purified/plasma renin IC50 ¼ 9/16 nM). Remarkably, the preferred configuration of the P1 isopropyl of 32 and 33 was opposite to that seen in two other lead series exemplified by 30 and 35, requiring elaboration of, at least in part, independent synthesis routes. In view of these hurdles and the lack of apparent benefits, further SAR in the indole series was discontinued. Work was not resumed even when the potential of P1u isopropyl substitution for improved in vivo potency [54] as well as simplified synthetic accessibility (vide infra) became fully recognized for more advanced candidate series. In the quest for more readily accessible inhibitors of less synthetic complexity, a promising series of O-alkylated salicylamides (34, 35; Figure 11) evolved from a computational fragment-based approach starting from the high-affinity compound 30 [57]. Independent docking of the components of 30 generated by disconnecting the spacer bond between P1 and the phenyl moiety suggested a three-atom carboxamide linker as being optimal to re-connect the P3 aryl fragment through its ortho position to P1 of the TSM. Initial SAR resulted in potent and selective inhibitors [20,57], thus supporting the binding mode predicted by modelling. Subsequently, this prediction was confirmed by X-ray [50]. Since the phenyl ring is positioned more deeply into S3, extension of the ortho-alkoxy side chain by one carbon was required for inducing an Hbond with Tyr14 of the Ssp 3 cavity. Strikingly, the in vivo SAR of P2u optimization did not parallel that of other sub-series. For example, 34 (plasma IC50 ¼ 0.5 nM) bearing the same P2u residue as 1 showed an inferior pharmacological profile despite comparable in vitro potencies. The most attractive salicylamide, 35, was highly potent towards primate plasma renin (IC50 ¼ 0.3, 2, 40 and 2,000 nM for human, marmoset, dog and rat) and selective against other human aspartyl proteases. Excellent oral potency was observed in Na-depleted marmosets with a pronounced dose-dependent (1–10 mg/kg) reduction in MAP similar to the effects seen with 1, and complete blockade of PRA over 24 h (3 mg/kg). No effects on the action potential measures in isolated Langendorff rabbit heart were seen at 30 mM, the highest concentration tested, suggesting an attractive
Case History on Tekturnas/Rasilezs (Aliskiren)
121
cardiac safety potential. The failure to identify a crystalline salt of 35 suitable for drug formulation terminated its pre-clinical development.
5. FIRST CONVERGENT AND SCALABLE SYNTHESIS DEVELOPMENT The continued interest in the clinical candidate 1 required a practical stereoselective synthesis for scale-up to support comprehensive evaluation in pharmacology and animal safety studies, as well as subsequent early clinical investigations. The initial linear research route was of high complexity (W25 steps, multiple chromatography purifications) and hence was not suitable to produce larger quantities [54]. Therefore, the drug discovery team in research took the initiative to elaborate a highly efficient, convergent and stereoselective synthesis of 1. Early and tight collaborative integration of the medicinal chemistry and the process chemistry groups was a key factor for effective knowledge transfer, further optimization and eventually successful transition into the first process synthesis. The structure of 1 is unique due to the four acyclic stereocenters, which rendered cost-effective synthesis extremely challenging. Retrosynthetic analysis (Figure 12) revealed the pseudo-symmetric features of the center portion of the all-carbon skeleton, comprising a geminal synamino alcohol as well as a 1,4-diisopropyl substitution motif of identical absolute configuration. We also took advantage of knowledge previously established for CGP38560, 3, enabling the stereoselective preparation of HE isosteres [58]. Intermediate 36 displayed all stereocenters of desired configuration and had the proper functionalities for further elaboration in place. On the basis of a highly stereo-controlled bromo-lactonization/ azide displacement sequence, 36 could be prepared in four steps with each step affording a crystalline intermediate [59]. Further key reactions involved Grignard-coupling of the 3,4-dialkoxy-aryl fragment to the aldehyde derived from 36 to afford 37, followed by direct lactoneopening with the P2u amine, and finally concomitant reduction of the activated benzylic alcohol and the azide through hydrogenation. This convergent route afforded 1 in nine linear steps overall and was MeO
MeO O
O OH
H N
O MeO
NH2
O
Pseudo-symmetric core structure (aliskiren, 1)
OH
O CONH2
O
O
HO2C N3
36
N3
MeO
37
Figure 12 Retrosynthetic concept towards aliskiren, 1, and key intermediates.
122
Ju¨rgen Maibaum and David L. Feldman
amenable to a multi-kilogram scale-up for phase I clinical development. An alternative and elegant process synthesis, again involving a bromolactonization protocol, at acceptable manufacturing cost-of-goods was subsequently developed [60,61].
6. PRE-CLINICAL PROPERTIES OF ALISKIREN Comprehensive reviews on aliskiren, 1, describing its pre-clinical pharmacology and safety profile, its pre-clinical in vitro/in vivo ADME properties, as well as clinical pharmacokinetics have appeared [54–56, 61–65]. Oral bioavailability of 1 was low to moderate in rats, dogs and marmosets (2.4%, 32% and 16–30%) [54,56]. Elimination of 1 in all species involves predominantly biliary/fecal excretion of unchanged parent drug [65]. In vitro studies showed poor substrate-affinity for 1 to CYP3A4 (Km ¼ 24.3 mM), no significant interactions with a large panel of human cytochrome P450 isozymes (e.g., IC50s W200 mM for CYPs 1A2, 2C8, 2C9, 2C19, 2D6, 2E1) [65,66] and low hepatic metabolic clearance in human liver microsomes. Aliskiren is a relatively high-affinity substrate for P-glycoprotein (MDR-1)-mediated transport, which may play a role in the hepatobiliary/intestinal excretion of the drug. The drug is not an inhibitor of MDR-1 and only weakly inhibited other transporters (BCRP, OATP2B1, hOCT1) at high concentrations. Further in vivo studies supported a low likelihood of drug–drug interactions through interference of 1 with CYP enzymes or efflux transporters [62,63]. Aliskiren showed an excellent cardiac safety profile in vitro, indicating a very low risk of impacting cardiac repolarization and conduction time and a low risk of torsade de pointes (TdP). The human hERG channel was very weakly inhibited in a cellular patch-clamp assay (IC25 ¼ 671 mM). No effects were observed on the action potential measures in the isolated rabbit heart up to 100 mM, suggesting a lack of interaction of 1 with cardiac ion channels in whole organ tissues at clinically relevant concentrations [64]. In normal rats, aliskiren distributed to the kidney, possibly to tissue structures that are involved in BP control [67]. Moreover, the compound showed a markedly prolonged renal residence time, being present in the kidneys of dTGR even after a 3-week wash-out period [68]. This finding suggested the potential for sustained inhibition of the intra-renal RAAS, as confirmed in preliminary studies in TG(mRen-2)27 rats [69]. Interestingly, 1 appears to incorporate into renin granules in cultured cells, which, when stimulated appropriately, secrete renin that is already inhibited [70]. Taken together, the earlier findings indicate that aliskiren may be able to access intra-renal renin, resulting in enzyme inhibition even before secretion to the extracellular compartment.
Case History on Tekturnas/Rasilezs (Aliskiren)
123
7. EFFECTS OF ALISKIREN IN DISEASE MODELS As indicated previously, dTGR, which express the human genes for renin and angiotensinogen [15], are very responsive to inhibition with a human renin inhibitor. Single oral dosing of 1 to chronically catheterized dTGR resulted in a dose-dependent, rapid and marked lowering of MAP with a long duration of action [71]. The first evidence that 1 exhibits potent cardio-renoprotective effects was determined in dTGR after chronic treatment [16]. Left ventricular hypertrophy associated with hypertension in dTGR was prevented by 1, and this was accompanied by reductions in various markers of inflammation in cardiac tissue. In a mouse model of myocardial infarction following coronary artery ligation, cardiac function was improved and remodeling was inhibited by a dose of 1 that did not lower BP [72]. These findings suggested a benefit in hypertensive diseases in humans. Hypertensive TG(mRen-2)27 rats expressing mouse renin, which efficiently cleaves rat angiotensinogen, develop type 1 diabetes and nephropathy when pre-treated with streptozotocin. In this model of hypertensive diabetic renal damage, 1 showed renoprotective effects as determined by attenuated albuminuria and renal structural changes [67,73]. The reduction of tubulo-interstitial damage to a greater extent in aliskiren vs. ACEi treated diabetic TG(mRen-2)27 rats is of potential importance, as it may indicate tissue protective effects beyond BP control [73]. The RAAS has been recognized as a major contributor to hypercholesterolaemia-induced atherosclerosis. In two models of atherosclerotic mice [74,75], and in Watanabe heritable hyperlipemic rabbits [76], aliskiren greatly attenuated the development of atherosclerosis without affecting plasma total cholesterol levels. Moreover, evidence for the ability of 1 to improve endothelial function was obtained in rabbits [76]. Finally, in a 2-week study in spontaneously hypertensive rats (SHR), co-administration of sub-maximal effective doses of 1 with either valsartan (ARB) or benazepril (ACEi) demonstrated a significant synergistic BP-lowering effect. This effect was attributed to RAAS inhibition at multiple sites [56].
8. CLINICAL STUDIES WITH ALISKIREN Results from an extensive clinical development plan for aliskiren continue to become available [61,77]. This renin inhibitor has a very long half-life (B40 h) and consequently provides 24 h BP control [78], despite its low absolute oral bioavailability of 2.6% in human [62]. Aliskiren consistently shows strong anti-hypertensive activity, at least as effective as ACEi or ARB, in patients with mild-moderate hypertension. The side effect profile is comparable to placebo. It is noteworthy that this
124
Ju¨rgen Maibaum and David L. Feldman
BP-lowering effect is long-lasting after stopping treatment [79], an effect that may have a basis in the renal retention of aliskiren observed in preclinical studies. Moreover, evidence that aliskiren attenuates albuminuria, independently of BP control, in patients with diabetic nephropathy [80], and lowers brain natriuretic peptide (BNP) in patients with congestive heart failure [81] indicates renal and cardio-protective effects of the drug. When combined with valsartan [82], the diuretic hydrochlorothiazide [83] or amlodipine [84], aliskiren showed additional BP lowering effects vs. either monotherapy. Long renal residence time may explain the prolonged anti-hypertensive action of aliskiren in patients.
9. CONCLUSIONS Novel non-peptide topological renin inhibitors have been designed and further optimized based on computational modeling and X-ray crystallography. The unprecedented binding interaction to a non-substrate binding pocket was crucial for the tight enzyme affinity of these TSMs. Elaboration of several major lead series culminated in aliskiren, a remarkably potent and highly selective renin inhibitor. Aliskiren is orally efficacious, safe and well-tolerated in pre-clinical and clinical trials. TEKTURNAs/RASILEZs (aliskiren) received marketing approval in 2007 by the FDA and in Europe as the first direct renin inhibitor for the treatment of hypertension. The extent to which this new mechanism of action will provide improved organ protection will be determined by ongoing clinical trials with this direct renin inhibitor.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9]
B. W. Van Tassell and M. Munger, Ann. Pharmacother., 2007, 41, 456. V. J. Dzau, D. W. Burt and R. E. Pratt, Am. J. Physiol., 1988, 255, F563. U. C. Brewster, J. F. Setaro and M. A. Perazella, Am. J. Med. Sci., 2003, 326, 15. D. T. Pals, J. A. Lawson and S. J. Couch, J. Pharmacol. Meth., 1990, 23, 239. J. M. Wood, S. C. Mah, H. P. Baum, M. De Gasparo, F. Cumin, H. Ru¨eger and J. Nussbaumer, J. Pharmacol. Exp. Ther., 1990, 253, 513. M. L. Mangiapane, J. T. MacAndrew, S. S. Ellery, A. H. Smith, I. M. Purcell, E. F. Kleinman, W. F. Holt and W. R. Murphy, Clin. Exper. Hypertens., 1994, 16, 507. H. D. Kleinert, S. H. Rosenberg, W. R. Baker, H. H. Stein, V. Klinghofer, J. Barlow, K. Spina, J. Polakowski, P. Kovar, J. Cohen and J. Denissen, Science, 1992, 257, 1940. J.-P. Clozel and W. Fischli, Arzneim.-Forsch./Drug Res., 1993, 43, 260. A. E. Weber, A. G. Steiner, P. A. Krieter, A. E. Colletti, J. R. Tata, T. A. Halgren, R. G. Ball, J. J. Doyle, T. W. Schorn, R. A. Stearns, R. R. Miller, P. K. S. Siegl, W. J. Greenlee and A. A. Patchett, J. Med. Chem., 1992, 35, 3755.
Case History on Tekturnas/Rasilezs (Aliskiren)
125
[10] J. L. Wessale, H. D. Kleinert, S. V. Calzadilla, P. J. Kovar and S. H. Rosenberg, Am. J. Hypertens., 1993, 6, 514. [11] E. G. McMahon, P.-C. Yang, M. A. Babler, S. E. Bittner, O. D. Suleymanov, K. J. CainJanicki, L. J. Bedell, G. J. Hanson and C. S. Cook, Hypertension, 1995, 26, 95. [12] J. M. Wood, N. Gulati, P. Forgiarini, W. Fuhrer and K. G. Hofbauer, Hypertension, 1985, 7, 797. [13] C. R. Schnell and J. M. Wood, Am. J. Physiol., 1993, 33, H1509. [14] D. N. Mu¨ller, W. Derer and R. Dechend, J. Mol. Med., 2008, 86, 659. [15] J. Bohlender, A. Fukamizu, A. Lippoldt, T. Nomura, R. Dietz, J. Me´nard, K. Murakami, F. C. Luft and D. Ganten, Hypertension, 1997, 29, 428. [16] B. Pilz, E. Shagdarsuren, M. Wellner, A. Fiebeler, R. Dechend, P. Gratze, S. Meiners, D. L. Feldman, R. L. Webb, I. M. Garrelds, A. H. J. Danser, F. C. Luft and D. N. Muller, Hypertension, 2005, 46, 569. [17] W. J. Greenlee, Med. Res. Rev., 1990, 10, 173. [18] S. H. Rosenberg and S. A. Boyd, in Antihypertensive Drugs (eds P. A. Van Zwieten and W. J. Greenlee), Harwood, Amsterdam, The Netherlands, 1997, pp. 77–111. [19] S. H. Rosenberg, Prog. Med. Chem., 1995, 32, 37. [20] C. M. Tice, Ann. Rep. Med. Chem., 2006, 41, 155. [21] J. M. Wood, F. Cumin and J. Maibaum, Pharmacol. Ther., 1994, 61, 325. [22] P. Bu¨hlmayer, A. Caselli, W. Fuhrer, R. Go¨schke, V. Rasetti, H. Ru¨eger, J. L. Stanton, L. Criscione and J. M. Wood, J. Med. Chem., 1988, 31, 1839. [23] J. M. Wood, L. Criscione, M. De Gasparo, P. Bu¨hlmayer, H. Ru¨eger, J. L. Stanton, R. A. Jupp and J. Kay, J. Cardiovasc. Pharmacol., 1989, 14, 221. [24] M. De Gasparo, F. Cumin, J. Nussberger, T. T. Guyenne, J. M. Wood and J. Me´nard, Br. J. Clin. Pharmac., 1989, 27, 587. [25] X. Jeunemaıˆtre, J. Me´nard, J. Nussberger, T. T. Guyene, H. R. Brunner and P. Corvol, Am. J. Hypertens., 1989, 2, 819. [26] A. H. van den Meiracker, P. J. J. Admiraal, F. H. M. Derkx, C. Kleinbloesem, A. J. Man in ‘t Veld, P. Van Brummelen, P. Mulder and M. A. D. H. Schalekamp, J. Hypertens., 1993, 11, 831. [27] P. Coassolo, W. Fischli, J.-P. Clozel and R. C. Chou, Xenobiotica, 1996, 26, 333. [28] C. Weber, H. Birnbo¨ck, J. Leube, I. Kobrin, C. H. Kleinbloesem and P. Van Brummelen, Br. J. Clin. Pharmac., 1993, 36, 547. [29] W. Fischli, J.-P. Clozel, K. E. Amrani, W. Wostl, W. Neidhart, H. Stadler and Q. Branca, Hypertension, 1991, 18, 22. [30] W. F. Richter, B. R. Whitby and R. C. Chou, Xenobiotica, 1996, 26, 243. [31] P.-C. Yang, M. Babler, S. Bittner, O. Suleymanov, M. Perez, K. Cain-Janicki, L. Bedell, G. Hanson, C. Cook, J. Ottinger, J. Baran and E. McMahon, FASEB J., 1994, 8, A882. [32] S. H. Rosenberg, K. P. Spina, S. L. Condon, J. Polakowski, Z. Yao, P. Kovar, H. H. Stein, J. Coehen, J. L. Batlow, V. Klinghofer, D. A. Egan, K. A. Tricarico, T. J. Perun, W. R. Baker and H. D. Kleinert, J. Med. Chem., 1993, 36, 460. [33] S. H. Rosenberg and H. D. Kleinert, Pharm. Biotech., 1998, 11, 7. [34] H. D. Kleinert, Cardiovasc. Drugs Ther., 1995, 9, 645. [35] R. S. Boger, H. N. Glassman, R. Thys, S. K. Gupta, R. L. Hippensteel and H. D. Kleinert, Am. J. Hypertens., 1993, 6, 103A. [36] K. Suguna, E. A. Padlan, A. Eduardo, C. W. Smith, W. D. Carlson and D. R. Davies, Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 7009. [37] B. L. Sibanda, T. Blundell, P. M. Hobart, M. Fogliano, J. S. Bindra, B. W. Dominy and J. M. Chirgwin, FEBS Lett., 1984, 174, 102. [38] N. C. Cohen, in Trends in Medicinal Chemistry’88 (eds H. van der Goot, G. Doma´ny, L. Pallos, and H. Timmerman), Elsevier, Amsterdam, The Netherlands, 1989, pp. 13–28.
126
Ju¨rgen Maibaum and David L. Feldman
[39] A. R. Sielecki, K. Hayakawa, M. Fujinaga, M. E. P. Murphy, M. Fraser, A. K. Muir, C. T. Carilli, J. A. Lewicki, J. D. Baxter and M. N. G. James, Science, 1889, 243, 1346. [40] J. Rahuel, J. P. Priestle and M. G. Gru¨tter, J. Struct. Biol., 1991, 107, 227. [41] C.E. Brotherton-Pleiss, S.R. Newman, L.D. Waterbury and M.S. Schwartzberg, Pept.: Chem. Biol., Proc. Am. Pept. Symp., 12th (1992), Meeting Date 1991, pp. 816–817. [42] M.G. Bock, R.M. DiPardo, B.E. Evans, R.M. Freidinger, W.L. Whitter, L.S. Payne, J. Boger, E.H. Ulm, E.H. Blaine, et al., Pept.: Struct. Funct., Proc. Am. Pept. Symp., 9th, 1985, p751. [43] V. Rasetti, N. C. Cohen, H. Ru¨eger, R. Go¨schke, J. Maibaum, F. Cumin, W. Fuhrer and J. M. Wood, Bioorg. Med. Chem. Lett., 1996, 6, 1589. [44] V. Rasetti, H. Ru¨eger, J. K. Maibaum, R. Mah, M. Gru¨tter and N. C. Cohen, Eur. Pat. EP 702004-A2, 1996, 2. [45] R. Go¨schke, N. C. Cohen, J. M. Wood and J. Maibaum, Bioorg. Med. Chem. Lett., 1997, 7, 2735. [46] M. W. Holladay, F. G. Salituro and D. H. Rich, J. Med. Chem., 1987, 30, 374. [47] D. J. Kempf, J. Org. Chem., 1986, 51, 3921. [48] J. Maibaum, V. Rasetti, H. Ru¨eger, N. C. Cohen, R. Go¨schke, R. Mah, J. Rahuel, M. Gru¨tter, F. Cumin and J. M. Wood, in Medicinal Chemistry: Today and Tomorrow, Proceedings of the AFMC International Medicinal Chemistry Symposium, Tokyo, September 3–8, 1995 (ed. M. Yamazaki), Blackwell Science, United Kingdom, 1997, pp. 155–162. [49] N. C. Cohen, Chem. Biol. Drug Des., 2007, 70, 557. [50] J. Rahuel, V. Rasetti, J. Maibaum, H. Ru¨eger, R. Go¨schke, N. C. Cohen, S. Stutz, F. Cumin, W. Fuhrer, J. M. Wood and M. G. Gru¨tter, Chem. Biol., 2000, 7, 493. [51] S. H. Rosenberg, J. F. Dellaria, D. J. Kempf, C. W. Hutchins, K. W. Woods, R. G. Maki, E. de Lara, K. P. Spina, H. H. Stein, J. Cohen, W. R. Baker, J. J. Plattner, H. D. Kleinert and T. J. Perun, J. Med. Chem., 1990, 33, 1582. [52] S. B. Shuker, P. J. Hajduk, R. B. Meadows and S. W. Fesik, Science, 1996, 274, 1531. [53] R. Go¨schke, S. Stutz, V. Rasetti, N. C. Cohen, J. Rahuel, P. Rigollier, H.-P. Baum, P. Forgiarini, C. R. Schnell, T. Wagner, M. G. Gru¨tter, W. Fuhrer, W. Schilling, F. Cumin, J. M. Wood and J. Maibaum, J. Med. Chem., 2007, 50, 4818. [54] J. Maibaum, S. Stutz, R. Go¨schke, P. Rigollier, Y. Yamaguchi, F. Cumin, J. Rahuel, H.-P. Baum, N. C. Cohen, C. R. Schnell, W. Fuhrer, M. G. Gru¨tter, W. Schilling and J. M. Wood, J. Med. Chem., 2007, 50, 4832. [55] J. M. Wood, J. Maibaum, J. Rahuel, M. G. Gru¨tter, N. C. Cohen, V. Rasetti, H. Ru¨eger, R. Go¨schke, S. Stutz, W. Fuhrer, W. Schilling, P. Rigollier, Y. Yamaguchi, F. Cumin, H.-P. Baum, C. R. Schnell, P. Herold, R. Mah, C. Jensen, E. O’Brien, A. Stanton and M. P. Bedigian, Biochem. Biophys. Res. Commun., 2003, 308, 698. [56] J. M. Wood, C. R. Schnell, F. Cumin, J. Menard and R. L. Webb, J. Hypertens., 2005, 23, 417. [57] J. Maibaum, N.C. Cohen, J. Rahuel, C. Schnell, H.-P. Baum, P. Rigollier, W. Schilling and J.M. Wood, XVth EFMC International Symposium on Medicinal Chemistry, Edinburgh, United Kingdom, 6–10 September 1998, Abstract Book, P229. [58] P. Herold, R. Duthaler, G. Rihs and C. Angst, J. Org. Chem., 1989, 54, 1178. [59] R. Go¨schke, J.K. Maibaum, W. Schilling, S. Stutz, P. Rigollier, Y. Yamaguchi, N.C. Cohen, P. Herold, Eur Patent EP 678503-A1, 1995. [60] J. Maibaum and D. L. Feldman, Expert. Opin. Ther. Patents, 2003, 13, 589. [61] C. Jensen, P. Herold and H. R. Brunner, Nat. Rev. Drug Discov., 2008, 7, 399. [62] S. Vaidyanathan, V. Jarugula, H. A. Dieterich, D. Howard and W. P. Dole, Clin. Pharmacokinet., 2008, 47, 515. [63] F. Waldmeier, U. Glaenzel, B. Wirz, L. Oberer, D. Schmid, M. Seiberling, J. Valencia, G. J. Riviere, P. End and S. Vaidyanathan, Drug Metab. Dispos., 2007, 35, 1414. [64] S. Ayalasomayajula, C.-M. Yeh, S. Vaidyanathan, B. Flannery, H. A. Dieterich, D. Howard, M. P. Bedigian and W. P. Dole, J. Clin. Pharmacol., 2008, 48, 799.
Case History on Tekturnas/Rasilezs (Aliskiren)
127
[65] S. Vaidyanathan, G. Camenisch, H. Schuetz, C. Reynolds, C.-M. Yeh, M.-N. Bizot, H. A. Dieterich, D. Howard and W. P. Dole, J. Clin. Pharmacol., 2008, 48, 1323. [66] S. Vaidyanathan, Y. Jin, H. Schiller and C. Jensen, Basic Res. Pharmacol. Toxicol., 2005, 97(Suppl 1), 230. [67] D. L. Feldman, L. Jin, H. Xuan, A. Contrepas, Y. Zhou, R. L. Webb, D. N. Mueller, S. Feldt, F. Cumin, W. Maniara, E. Persohn, H. Schuetz, A. H. J. Danser and G. Nguyen, Hypertension, 2008, 52, 130. [68] D. L. Feldman, E. Persohn, H. Schuetz, L. Jin, R. Miserindino-Moltini, H. Xuan, S. Zhuang and W. Zhou, J. Clin. Hypertens., 2006, 8, A80–A81, P-178. ˇ ervenka, L. Kopkan, Z. Vanˇourkova´ and Z. Huskova´, J. Hypertens., [69] I. Vaneˇcˇkova´, L. C 2008, 26, S521. [70] M. Krop, I. M. Garrelds, R. J. de Bruin, J. M. van Gool, N. D. Fisher, N. K. Hollenberg and A. H. J. Danser, Hypertension, 2008, 52, 1076. [71] D. F. Rigel, F. Fu, S. Li, W. Maniara, D. N. Mu¨ller and F. C. Luft, J. Am. Coll. Cardiol., 2004, 43(Suppl. A), 483A. [72] D. Westermann, A. Riad, O. Lettau, A. Roks, K. Savvatis, P. M. Becher, F. Escher, A. H. J. Danser, H. P. Schultheiss and C. Tschoepe, Hypertension, 2008, 52, 1068. [73] D. J. Kelly, Y. Zhang, G. Moe, G. Naik and R. E. Gilbert, Diabetologia, 2007, 50, 2398. [74] J. Nussberger, J. F. Aubert, K. Bouzourene, M. Pellegrin, D. Hayoz and L. Mazzolai, Hypertension, 2008, 51, 1306. [75] H. Lu, D. L. Rateri, D. L. Feldman, R. J. Jr., A. Fukamizu, J. Ishida, E. G. Oesterling, L. A. Cassis and A. Daugherty, J. Clin. Invest., 2008, 118, 984. [76] T. Imanishi, H. Tsujioka, H. Ikejima, A. Kuroi, S. Takarada, H. Kitabata, T. Tanimoto, Y. Muragaki, S. Mochizuki, M. Goto, K. Yoshida and T. Akasaka, Hypertension, 2008, 52, 563. [77] H. Siragy, J. Huang and D. C. Lieb, Expert Opin. Emerg. Drugs., 2008, 13, 417. [78] A. H. Gradman and R. Kad, J. Am. Coll. Cardiol., 2008, 51, 519. [79] B. H. Oh, J. Mitchell, J. R. Herron, J. Chung, M. Khan and D. L. Keefe, J. Am. Coll. Cardiol., 2007, 49, 1157. [80] H. H. Parving, F. Persson, J. B. Lewis, E. J. Lewis and N. K. Hollenberg, N. Engl. J. Med., 2008, 358, 2433. [81] A. Seed, R. Gardner, J. McMurray, C. Hillier, D. Murdoch, R. MacFadyen, A. Bobillier, J. Mann and T. McDonagh, Eur. J. Heart Fail., 2007, 9, 1120. [82] S. Oparil, S. A. Yarows, S. Patel, H. Fang, J. Zhang and A. Satlin, Lancet, 2007, 370, 221. [83] A. Villamil, S. G. Chrysant, D. Calhoun, B. Schober, H. Hsu, L. Matrisciano-Dimichino and J. Zhang, J. Hypertens., 2007, 25, 217. [84] M. A. Munger, W. Drummond, M. R. Essop, M. Maboudian, M. Khan and D. L. Keefe, Eur. Heart J., 2006, 27(Suppl), 117P–784.
CHAPT ER
6 Advances in Vasopressin Receptor Agonists and Antagonists Thomas Ryckmans
Contents
1. Introduction 1.1 Emerging roles of V1a and V1b receptors in anxiety, depression and sociality 1.2 Challenges in the development of small-molecule peptidergic GPCR modulators. 2. V1a Receptor Antagonists 2.1 Azepines 2.2 Lactams 2.3 Spirocyclic piperidines 3. V1b Receptor Antagonists 3.1 Indolones and benzimidazolones 3.2 Lactams 3.3 Quinazolinones and isoquinolinones 3.4 V1b receptors and pain 4. V2 Receptor Agonists 4.1 Azepines and diazepines 4.2 Emerging chemotypes 5. V2 Receptor Antagonists 6. Dual V1a-V2 Receptor Antagonists 6.1 Azepines 6.2 Triazolones 7. Summary References
129 130 131 131 132 132 133 134 134 136 137 138 139 139 140 140 141 142 143 143 144
Pfizer PGRD, Sandwich, Kent, UK Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04406-6
r 2009 Elsevier Inc. All rights reserved.
129
130
Thomas Ryckmans
1. INTRODUCTION Arginine vasopressin (AVP) is a 9–amino-acid cyclic peptide that exerts its effect through the V1a, V1b, V2 and oxytocin (OT) family of G-proteincoupled receptors (GPCRs). V1a receptors are expressed mainly in the liver (glycogenolysis regulation), the vascular smooth muscle cells (vasoconstriction and contractility regulation) and the brain (regulation of memory formation, stress adaptation, temperature and circadian rhythm). These receptors are also involved in platelet aggregation [1]. Accordingly, V1a receptor antagonists have been progressed to the clinic for a range of indications, including Raynaud’s syndrome (vasoconstriction) and dysmenorrhea (uterine blood flow and contraction). V1b receptors (also known as V3 receptors) are expressed in the limbic system and the pituitary gland, where they regulate the hypothalamus-pituitaryadrenal (HPA) axis, which is responsible for response to stress. The V1b receptor antagonist SSR149415 has been progressed to phase II clinical trials for depressive disorders. V2 receptors are mainly expressed in the kidney and are involved in the regulation of water and sodium excretion [2–5], and several V2 receptor antagonists and mixed V1a-V2 receptor antagonists have been progressed to the clinic. It has been suggested that there is a distinct advantage in blocking both V1a and V2 receptors for indications such as congestive heart failure [6]. This review covers recent advances in the development of non-peptidic vasopressin receptor ligands, focusing on new series, structure–activity relationship (SAR) and preclinical data.
1.1 Emerging roles of V1a and V1b receptors in anxiety, depression and sociality There is growing evidence that AVP, through its action at V1a and V1b receptors, is involved in the modulation of several higher brain functions such as response to stress, mood, memory formation, aggressivity and sociality. AVP and corticotropin-releasing factor (CRF) synergistically modulate the release of adrenocorticotropic hormone (ACTH) by the pituitary gland, a key step in the HPA axis cascade, which controls response to stress. Although a detailed description of the HPA axis is beyond the scope of this review, the reader is referred to recent publications on the subject [3,7,8]. HPA axis dysfunction has been linked to anxiety, posttraumatic stress disorder (PTSD) and depression [7,9,10], and both preclinical and clinical evidence point at a key role of AVP in these conditions [10]. For example, rats bred for high anxiety-related behaviour have higher levels of AVP mRNA than rats bred for low anxiety-related behaviour. The Brattleboro rat strain, in which AVP secretion is impaired,
Advances in Vasopressin Receptor Agonists and Antagonists
131
displays reduced stress response at both the behavioural and the endocrine levels [3]. Clinical evidence has shown that both depressed and PTSD patients have higher levels of systemic AVP and that single-nucleotide polymorphism of the V1b receptor is correlated with a lower probability of developing major depression [11]. Both V1a and V1b receptors have been implicated in the modulation of aggression in rodents [12–14]. AVP has emerged as a key regulator of complex social behaviours [13,15–17]. Pair bonding in voles is modulated by the action of AVP on the V1a receptor [18–20], and a recent study demonstrated the association of genetic variation of the V1a receptor with pair bonding in humans [21]. OT has also been implicated in such processes, but this is beyond the scope of this review [22–25]. The possible implication of AVP dysfunction in conditions where sociality is impaired, such as autism and schizophrenia, has been put forward, but more evidence is clearly needed [2,26,27]. A recent review of the patent literature covering the anxiolytic and antidepressant potential of AVP antagonists is available [28], and we have therefore focused on the more recent data in the field.
1.2 Challenges in the development of small-molecule peptidergic GPCR modulators. The development of small-molecule ligands of peptidergic receptors (with the exception of opioid receptor ligands) is still considered a challenge by medicinal chemists. The average molecular weight, total polar surface area (TPSA) and cLogP of peptidergic ligands are significantly higher than those of ligands of other targets such aminergic GPCRs, ion channels and transporters [29]. Since high molecular weight, high TPSA and high cLogP are associated with poor drug-like properties [30], increased risk of offtarget toxicity [31] and poor CNS penetration, small-molecule peptidergic GPCR ligands have suffered from high attrition between the bench and the clinic. It is therefore not surprising that despite significant efforts from the pharmaceutical industry, a relatively small number of AVP antagonists have been approved for marketing.
2. V1A RECEPTOR ANTAGONISTS Relcovaptan (SR49059, 1) has shown clinical efficacy for indications modulated by the peripheral V1a receptor including Raynaud’s syndrome, dysmenorrhea and preterm labour [5], but its development has been interrupted as a result of possible interference with cytochrome P450 [32]. The development of OPC-21268 (2), another V1a receptor antagonist, has been discontinued in Europe and in the United States
132
Thomas Ryckmans
[33]. Nevertheless, the positive proof-of-concept studies with relcovaptan have triggered the interest of the pharmaceutical industry, resulting in the discovery of several classes of V1a receptor antagonists. Critically, a biomarker for V1a receptor antagonism in healthy volunteers has been recently developed [34], which could facilitate clinical development.
Cl
O OH O
O
N
O
O NH2
N
S
N N
O
O
O
O N H O
2
1
O
2.1 Azepines The activity of JNJ-17308616 (3) at V1a, V1b, V2 and OT human, rat and mouse receptors has been reported. Although JNJ-17308616 is a potent and selective antagonist of human V1a receptors (hV1a Ki ¼ 5 nM, hV1b KiW10 mM, hV2 Ki ¼ 421 nM, hOT Ki ¼ 6.5 mM), its affinity and selectivity for rat V1a receptors is lower (rV1a Ki ¼ 216 nM, rV1b KiW10 mM, rV2 Ki ¼ 276 nM, rOT Ki ¼ 1.8 mM). The compound was found to be active in several rat models of anxiety [35]. Since the kidney-localized rat V2 receptors are not believed to be associated with anxiety, the anxiolytic effect of JNJ-17308616 appears to be mediated by the V1a receptor. O
H N
O
O
O
N
O N
N O O
N
N N
O
N
N
O
F
N O
F
O
N H
F
O F
N
N
N H
O
3
4
5
133
Advances in Vasopressin Receptor Agonists and Antagonists
2.2 Lactams SRX251 (4) is a potent and selective V1a receptor antagonist (hV1a Ki ¼ 0.7 nM, hV1b KiW1 mM). The compound is orally bioavailable in rats (t1/2 ¼ 4 h), is claimed to be CNS penetrant and inhibited aggression in a hamster model when administered orally at a dose of 20 mg/kg [28,36,37]. Interestingly, non-aggressive behaviours such as locomotor activity, olfactory communication and sexual motivation were not affected. The ability of SRX251 to selectively block aggression in rats was confirmed by functional magnetic resonance imaging [12]. SRX251 has completed phase I trials and is scheduled for a phase II study targeting dysmenorrhea [38]. SRX246 (5) has a profile similar to that of SRX251 (hV1a Ki ¼ 0.3 nM, hV1b KiW10 mM, rat t1/2 ¼ 2 h) and an Investigational New Drug filing for stress-related disorders is pending [38].
2.3 Spirocyclic piperidines A range of small, low lipophilicity, selective and potent triazoles were disclosed as V1a receptor antagonists, exemplified by 6 (hV1a Ki ¼ 7 nM) [39,40]. In a related chemotype in which the triazole is replaced by an amide (as in OPC-21268), piperidines such as 7 and 8 were shown to be moderately potent V1a antagonists (hV1a Ki ¼ 25 and 31 nM respectively), but no selectivity data was disclosed.
N N
O
O
N
H N
N
N
N O
O
S
N
O
Cl Cl
6
7
8
Several patents describe related amides, with variations of the spirocyclic and heteroaryl moieties, and combinations thereof [41–47]. Spirocyclic groups and aryl substituents are preferred at the piperidine 4-position, whereas glycine amides and 2- or 3-indolecarboxamides are preferred derivatives of the piperidine nitrogen. Compounds 9–13 (hV1a Kio10 nM) are examples of such combinations.
134
Thomas Ryckmans O
O
H N
N
Ph
O
N
O
O
N HN
HN
Cl
Cl
O N
Cl
N
N
HN
O
O
O
O
O
O
O
11
10
9
Cl
N
N
O N
Cl
N
O
HN
O N 12
13
F O
3. V1B RECEPTOR ANTAGONISTS 3.1 Indolones and benzimidazolones SSR149415 (14), a potent, selective V1b receptor antagonist was progressed to phase II trials for major depression [48,49] but appears to have been discontinued [50]. The compound was extensively studied in a range of anxiety and depression models [3]. For example, SSR149415 was recently demonstrated to have efficacy similar to that of imipramine in the rat olfactory bulbectomy-induced (OBX) hyperactivity depression model [35]. The compound (at 10 and 30 mg/kg i.p.) completely inhibited OBXinduced hyperactivity upon chronic and subchronic but not acute dosing. Although the mechanism of these effects of SSR149415 is unclear, it does increase norepinephrine (but not serotonin or dopamine) levels in the prefrontal cortex and appears to act as an antidepressant rather than an anxiolytic [51]. The V1b vs. OT receptor selectivity of SSR149415 has been discussed [52], but a recent report on the ex vivo binding of radiolabelled SSR149415 appears to confirm the selectivity of this compound for V1b receptors [53]. Pituitary V1b receptors have been
135
Advances in Vasopressin Receptor Agonists and Antagonists
suggested to play a role in the anxiolytic effect of SSR149415, while the antidepressant effects appear to be mediated through central V1b receptors [7]. OH
R3
O R1
N
Cl
R3
N
N O
N O
S
R1 O
O N
R4
R4
N
S R2
O
S
O
O
R2
O
O
O
15
14
O
16
R1 = F, Cl, CN R2 = 4-CN or 4-OMe or 2-OMe, 4-OMe-phenyl or 8-quinolyl R3 = aromatic or heteroaromatic R4 = most variable group
O
Since the disclosure of SSR149415, a limited number of V1b receptor antagonists that can be distributed into three classes have appeared in the patent literature. Although indolones 15 and benzimidazolones 16 are both related to SSR149415 with clear structural overlap, new classes of compounds with beta-lactam and quinazolinone cores have also been reported. In a series of indolones related to SSR149415, 17–19 exemplify the substitution pattern with variations of R3, R2 and R4. Although urea 17 is reported to be a potent and selective V1b receptor antagonist (hV1b Kio10 nM, W50- and W100-fold selectivity over V1a and OT receptors, respectively), no selectivity data has been published for urea 18 (hV1b Kio10 nM) and carbamate 19 (hV1b Kio50 nM) [54–57].
N
O
O O
H N
N
N O
N
N N
O O
O N O O N
N
O N
17
18
N N
S
O O
N
O
N N
S
O
O N
O N
S
O O
H N
19
N
136
Thomas Ryckmans
Related high-affinity analogues (hV1b Kio1 nM) in which the R4 group is a cyclic amine have also been disclosed, such as 20 and 21 [56,58,59].
N
O
N O
O
( )n N
N
R1
O
N
O N S O O
O N S O O O
O
N
N
O
O
N
N
20
Cl
N
N
O
N
N S
21
O
(R1=F, Cl, n=1-2)
O
22
O
Compound 22, illustrating the benzimidazolone chemotype, has been reported as a V1b receptor antagonist (hV1b Kio100 nM), but no selectivity data was reported [60].
3.2 Lactams Beta-lactams 23–24 represent a new class of V1b receptor antagonists (23: rV1b Ki ¼ 70 rM, 24: rV1b Ki ¼ 30 nM; no selectivity data against the V1a receptor reported). Compound 24 was found to restore both biochemical (testosterone/cortisol levels) and behavioural (seed finding) markers in hamster at the dose of 1 mg/kg i.p. with activity comparable to that of fluoxetine, buspirone and chlordiazepoxide [61]. Interestingly, this chemotype is related to a previously discussed class of selective V1a receptor antagonists represented by 4 and 5.
O
O
O
O N
N
O
O
N
N O
O
O
N H
N H O N N
23
N N
24
137
Advances in Vasopressin Receptor Agonists and Antagonists
3.3 Quinazolinones and isoquinolinones Several V1b receptor antagonists from the chemotype 25 have been disclosed. Binding of selected compounds in CHO cells expressing hV1b receptors or in an ex vivo rat anterior pituitary cell assay was reported. Quinazolinones 26 and 27 are potent antagonists of the human receptor (hV1b Kio10 nM). Compound 28 (Y ¼ CH) is described as a rat V1b receptor antagonist (V1b Ki ¼ 10–100 nM) [62]. A very similar series of azaanalogues exemplified by 28 (Y ¼ N) has been reported (hV1b IC50 1 nM) [63]. R3
H N
H N
O
O
O
O R
2
O
N
N N X
R1
N N X=N, CH R2=OR, Het R3=iPr, tBu, nPr
25
H N
O
26
H N
O
O O
O
O
O N
N
N
N N
N
Cl
27
O
28
Y Y=CH Y=N
A potent and selective V1b receptor antagonist ORG 52186 (hV1b Ki ¼ 4 nM; hV1a, V2 and OTW10 mM) is reported to be active when administered at a dose of 5 mg/kg p.o. in a rat model of CRF-mediated release of ACTH [64]. The structure of this compound has not been disclosed, but it may belong to the quinazolinone class [8,33,62]. Compounds related to 25 (X ¼ N) with an R2 nitrogen substituent have been disclosed, exemplified by 29 (hV1b Kio10 nM) and 30 (hV1b Ki ¼ 10–100 nM), which demonstrates that an aromatic group at the 6-position is not required to achieve potency. Quinazolinone 31 (rV1b Ki ¼ 10–100 nM, ex vivo anterior pituitary cells) is disclosed as a rat V1b receptor antagonist [65]. Isoquinolinone analogues 25 (X ¼ CH) have been disclosed with a similar substitution pattern; 32 is reported to be an antagonist of human V1b receptors with a Kio10 nM.
138
Thomas Ryckmans
H N
H N
O O
O O
NH N
N
N
N
29
Cl
H N
NH N
N
30
H N
O
O O
O
N
O
N
N
N
N N
31
O
32
3.4 V1b receptors and pain Compounds 33 and 34 were shown to be active in the Chung model of neuropathic pain in rats at a dose of 10 mg/kg p.o. In the same model, SSR149415 was active at a comparable dose (6 mg/kg). Compound 34 was also active in a model of chronic inflammatory pain in rats (CFA) at 10 mg/kg p.o. [66]. Neither 33 nor 34 was active in the mouse tail-flick model of acute thermal pain. Interestingly, V1b receptor knockout mice (but not V1a receptor knockout mice) showed a hyponociceptive response in the hot plate model [67]. SSR149415 (14) was found to be efficacious in a rat model of chronic stress-induced visceral hyperalgesia, but not in an acute model. Taken together, these results indicate the potential of V1b receptor antagonists for the treatment of pain and the effect of stress on pain response.
H N
H N
O
O O
O
O N
N
N
N
N
N F
33 O
Cl
34
Advances in Vasopressin Receptor Agonists and Antagonists
139
4. V2 RECEPTOR AGONISTS The V2 receptor is mainly localized in the kidney and is responsible for fluid homeostasis. V2 receptor agonists increase the expression of aquaporin-2 water channels, which increase water reabsorption by the kidney, thus concentrating the urine (increased osmolality) and decreasing its volume. Inactivating mutations of the V2 receptor are linked to nephrogenic diabetes insipidus, in which polyuria and excessive thirst is observed. Desmopressin, a V2 receptor agonist, is indicated for the treatment of polyuria, but since the compound is also a V1b receptor agonist, and has low bioavailability, more selective agents with improved oral bioavailability have been actively sought [68].
4.1 Azepines and diazepines OPC-51803 (35), a partial V2 receptor agonist, and VNA-932 (WAY151932, 36) are both in phase II clinical trials for enuresia [33]. O N H N
N
N O
O
Cl
Cl N 35
N
N
36
Diazepine 37 was active (10 mg/kg p.o.) in a rat model of polyuria, decreasing urine volume by nearly 90%. The compound was selective for V2 receptors (rV2 binding 490 nM, rV2 EC50 1.7 nM) over V1a receptors (rV1a binding 6 mM, ex vivo blockade of AVP-induced contractions of rat tail artery IC50W30 mM) and was found to be a weak antagonist of rOT receptors (ex vivo blockade of OT-induced contractions of rat uterine strips IC50W1260 nM) [69]. The structurally related azepine 38 (hV2 Ki ¼ 5 nM, rV2 Ki ¼ 19 nM, rEC50 2 nM) was also active in a similar rat model when administered at the dose of 0.26 mg/kg p.o. [70]. Diazepine 39 (hV2 EC50 4 nM, intrinsic efficacy relative to AVP 0.85) and its analogues are also potent V2 receptor agonists [71]. Compound 40 was
140
Thomas Ryckmans
recently reported to be a potent full V2 receptor agonist (hV2 EC50 24 nM, intrinsic efficacy relative to AVP 1.0; V1a, V1b EC50W10 mM; OT KiW10 mM) and to show robust efficacy in the Brattleboro rat model (3 mg/kg p.o.), with almost full inhibition of urinary output for 2 h [72]. N HN H N
O
N
N
F F
O N
O N
N
O
N O
O
H N
O
F
N
Cl
F
O
Cl
N
F
O
N
N
37
38
O
N
39
N
40
4.2 Emerging chemotypes Phthalamide 41 (pEC50 9.4 intrinsic efficacy relative to AVP 0.27) and tetrazole 42 (pEC50 5.1 intrinsic efficacy relative to AVP 0.68) were identified as selective, partial agonists of the human V2 receptor. Compound 41 (10 mg/kg, p.o.) showed efficacy in the Brattleboro rat model, reducing urine output by 60% [68].
F HO
F F
O
N
O
N N N
N N
41
42
5. V2 RECEPTOR ANTAGONISTS Several V2 receptor antagonists, including tolvaptan 43, lixivaptan 44 and satavaptan 45, have entered clinical trials for the treatment of hyponatremia, liver cirrhosis, chronic heart failure and polycystic kidney disease and have been extensively reviewed elsewhere [5,33].
141
Advances in Vasopressin Receptor Agonists and Antagonists
Mozavaptan 46 was approved in Japan in 2006 for hyponatremia and tolvaptan was recommended for approval by an FDA advisory board for hyponatremia in June 2008 [5]. In a clinical trial aimed at evaluating the efficacy of tolvaptan in preventing heart failure, tolvaptan modestly relieved the congestive symptoms of heart failure, but failed to reduce morbidity or mortality when added to standard therapy [73]. O HO
O
N
N
N O N
O
N O
O S O
O N H
N H
O
F
Cl
O
N
N
O
O
NH
O O
HN
43
44
45
46
Compounds 47 and 48 related to satavaptan have been reported recently (47 hV2 Ki ¼ 1 nM, 48 hV2 Ki ¼ 2 nM; selectivity over OT, V1a and V1b W100 for both) but no in vivo data was disclosed [74]. In yet another variation of the beta-lactam chemotype, compounds such as 49 were reported as potent V2 receptor antagonists (hV2 Ki ¼ 0.4 nM). Activity of such compounds at the OT and neurokinin receptors was also claimed in this disclosure [75]. O Cl
N H
Cl
O
O NH
O
Cl
N
O
O
O N
O N
O
S
O O O
HN
HN O
47
N H
O
S
O
N O
N N
O
48
F
F F
49
6. DUAL V1A-V2 RECEPTOR ANTAGONISTS Conivaptan (YM-087, 50) is currently the only approved V1a-V2 receptor antagonist, and is used to treat hyponatremia. Chronic blockade of both the V1a and the V2 receptors may be advantageous in the treatment of
142
Thomas Ryckmans
congestive heart failure [6] and has prompted vigorous research in this area.
6.1 Azepines
O HN
H N
O
OH
N
N
N
N
N
O
F
O
O
Cl N H
N H
O
50
O
N H
O
51
O
52
Spirobenzazepine RWJ-339489 (51) was identified as a potent, balanced functional V1a-V2 receptor antagonist (hV1a IC50 45 nM, hV2 IC50 36 nM) with adequate rat PK (F ¼ 22%, t1/2 ¼ 6.5 h) and produced robust aquaresis when administered at the dose of 10 mg/kg p.o. [76]. Because of unexpected toxicology findings with 51, RWJ-676070 (52) was identified as a back-up candidate. Compound 52 has a functional antagonist profile at V1a and V2 receptors similar to that of 51 (hV1a IC50 14 nM, hV2 IC50 13 nM), has a favourable rat pharmacokinetic profile (F ¼ 68%, t1/2 ¼ 8.7 h) and did not display the toxicity of 51 [1,77]. The compound entered phase I clinical trials and was well-tolerated in healthy volunteers [34]. O H N
N O
HN
N
N
O
N
O
O F
N H
53
N H
O
54
N H
O
55
O
Advances in Vasopressin Receptor Agonists and Antagonists
143
Another spirobenzazepine (53) was reported to be a potent dual functional antagonist of V1a and V2 receptors (hV1a IC50 29 nM, hV2 IC50 64 nM) [78]. The subtle SAR of the balance between V1a and V2 receptor antagonism is illustrated by lactams 54 (hV1a IC50 50 nM, hV2 IC50 60 nM) and 55 (hV1a IC50 50 nM, hV2 IC50 W 3 mM) [79].
6.2 Triazolones A new series of triazolones exemplified by 56, 57 and 58 has been reported. These analogues range from selective V1a or V2 receptor antagonists or are mixed V1a-V2 receptor antagonists. For example, although 56 (hV1a IC50 42 nM, hV2 IC50 12 nM) is a balanced dual antagonist, the fluoro analogue 57 (hV1a IC50 960 nM, hV2 IC50 6 nM) is a V2 receptor antagonist with 160-fold selectivity over V1a receptors. Compound 58 (hV1a IC50 40 nM, hV2 IC50 W10 mM) is a selective V1a receptor antagonist [80]. O
R
O
O
O N
N
N
HN
N
N
HN
F F F
N
F F S
F
56 R=OMe 57 R=F
58
Cl
Cl
7. SUMMARY The historical development and the complexity of AVP pharmacology is clearly captured by the title of a recent review ‘‘The vasopressin system – from antidiuresis to psychopathology’’ [2]. V2 receptor antagonists and agonists are being evaluated in the clinic for the modulation of renal function and cardiovascular conditions. V1a receptor antagonists are being actively investigated for indications such as dysmenorrhea, given the unmet medical need and the large patient population. On the contrary, the involvement of the V1a and V1b receptors in depression, anxiety and stress disorders has been clearly demonstrated in preclinical models; however, the discovery of an orally bioavailable, well-tolerated
144
Thomas Ryckmans
and CNS-penetrant antagonist of these receptors is still a formidable challenge. To date, no V1a or V1b receptor antagonists have been approved, but the growing evidence of the key role of AVP in mood disorders is leading to renewed interest in this venue of research. The fascinating role of AVP in higher brain function and behaviour, such as sociality and bonding, has emerged in recent years, suggesting potential new indications for V1a and V1b receptor antagonists.
REFERENCES [1] J. W. Gunnet, P. Wines, M. Xiang, P. Rybczynski, P. Andrade-Gordon, L. de Garavilla, T. J. Parry, W.-M. Cheung, L. Minor, K. T. Demarest, B. E. Maryanoff and B. P. Damiano, Eur. J. Pharmacol., 2008, 590, 333. [2] E. Frank and R. Landgraf, Eur. J. Pharmacol., 2008, 583, 226. [3] A. Surget and C. Belzung, Eur. J. Pharmacol., 2008, 583, 340. [4] F. Ali, M. Guglin, P. Vaitkevicius and J. K. Ghali, Drugs, 2007, 67, 847. [5] G. Decaux, A. Soupart and G. Vassart, Lancet, 2008, 371, 1624. [6] S. R. Goldsmith, Am. J. Med., 2006, 119, S93. [7] F. Thomson and M. Craighead, Neurochem. Res., 2008, 33, 691. [8] J. Roper, A.-M. O’Carroll1, E. J. Grant and S. J. Lolait, Proc. Physiol. Soc., 2007, 5, C3. [9] B. E. Leonard, Drugs Today, 2007, 43, 705. [10] L. Q. Stewart, J. A. Roper, W. Scott Young, A.-M. O’Carroll and S. J. Lolait, Psychoneuroendocrinology, 2008, 33, 405. [11] D. van West, J. Del-Favero, Y. Aulchenko, P. Oswald, D. Souery, T. Forsgren, S. Sluijs, S. Bel-Kacem, R. Adolfsson, J. Mendlewicz, C. Van Duijn, D. Deboutte, C. Van Broeckhoven and S. Claes, Mol. Psychiatry, 2004, 9, 287. [12] C. F. Ferris, T. Stolberg, P. Kulkarni, M. Murugavel, R. Blanchard, D. C. Blanchard, M. Febo, M. Brevard and N. G. Simon, BMC Neurosci., 2008, 9, 111. [13] S. R. Wersinger, H. K. Caldwell, M. Christiansen and W. S. Young, Jr., Genes Brain Behav., 2007, 6, 653. [14] S. R. Wersinger, J. L. Temple, H. K. Caldwell and W. S. Young, Jr., Endocrinology, 2008, 149, 116. [15] A. H. Veenema and I. D. Neumann, Prog. Brain Res., 2008, 170, 261. [16] O. J. Bosch and I. D. Neumann, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 17139. [17] B. C. Nephew and R. S. Bridges, Pharmacol. Biochem. Behav., 2008, 91, 77. [18] J. T. Winslow, N. Hastings, C. S. Carter, C. R. Harbaugh and T. R. Insel, Nature, 1993, 365, 545. [19] M. M. Lim, Z. Wang, D. E. Olazabal, X. Ren, E. F. Terwilliger and L. J. Young, Nature, 2004, 429, 754. [20] M. M. Lim, E. A. D. Hammock and L. J. Young, J. Neuroendocrinol., 2004, 16, 325. [21] H. Walum, L. Westberg, S. Henningsson, J. M. Neiderhiser, D. Reiss, W. Igl, J. M. Ganiban, E. L. Spotts, N. L. Pedersen, E. Eriksson and P. Lichtenstein, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 14153. [22] T. Baumgartner, M. Heinrichs, A. Vonlanthen, U. Fischbacher and E. Fehr, Neuron, 2008, 58, 639. [23] M. R. Delgado, Neuron, 2008, 58, 470. [24] A. J. Guastella, P. B. Mitchell and M. R. Dadds, Biol. Psychiatry, 2008, 63, 3. [25] P. Kirsch, C. Esslinger, Q. Chen, D. Mier, S. Lis, S. Siddhanti, H. Gruppe, V. S. Mattay, B. Gallhofer and A. Meyer-Lindenberg, J. Neurosci., 2005, 25, 11489.
Advances in Vasopressin Receptor Agonists and Antagonists
145
[26] H. K. Caldwell, H.-J. Lee, A. H. Macbeth and W. S. Young, Prog. Neurobiol., 2008, 84, 1. [27] E. A. D. Hammock and L. J. Young, Philos. Trans. R. Soc., B, 2006, 361, 2187. [28] N. G. Simon, C. Guillon, K. Fabio, N. D. Heindel, S.-f. Lu, M. Miller, C. F. Ferris, M. J. Brownstein, C. Garripa and G. A. Koppel, Recent Pat. CNS Drug Discov., 2008, 3, 77. [29] R. Morphy, J. Med. Chem., 2006, 49, 2969. [30] P. D. Leeson and B. Springthorpe, Nat. Rev. Drug Discov., 2007, 6, 881. [31] J. D. Hughes, J. Blagg, D. A. Price, S. Bailey, G. A. DeCrescenzo, R. V. Devraj, E. Ellsworth, Y. M. Fobian, M. E. Gibbs, R. W. Gilles, N. Greene, E. Huang, T. Krieger-Burke, J. Loesel, T. Wager, L. Whiteley and Y. Zhang, Bioorg. Med. Chem. Lett., 2008, 18, 4872. [32] V. Bernier, J.-P. Morello, A. Zarruk, N. Debrand, A. Salahpour, M. Lonergan, M.-F. Arthus, A. Laperrier, R. Brouard, M. Bouvier and D. G. Bichet, J. Am. Soc. Nephrol., 2005, 17, 232. [33] M. Manning, S. Stoev, B. Chini, T. Durroux, B. Mouillac and G. Guillon, Prog. Brain Res., 2008, 170, 473. [34] L. Coltamai, M. Bucher, M. P. Maillard, U. Shukla, N. Bohidar, L. Haskell, K. Bertelsen, M. Fedgchin, B. Vogt and M. Burnier, Clin. Pharmacol. Ther., 2009, 85, 145. [35] C. J. Bleickardt, D. E. Mullins, C. P. MacSweeney, B. J. Werner, A. J. Pond, M. F. Guzzi, F. D. C. Martin, G. B. Varty and R. A. Hodgson, Psychopharmacology, 2009, 202, 711. [36] C. F. Ferris, S.-F. Lu, T. Messenger, C. D. Guillon, N. Heindel, M. Miller, G. Koppel, F. R. Bruns and N. G. Simon, Pharmacol. Biochem. Behav., 2006, 83, 169. [37] C. D. Guillon, G. A. Koppel, M. J. Brownstein, M. O. Chaney, C. F. Ferris, S.-F. Lu, K. M. Fabio, M. J. Miller, N. D. Heindel, D. C. Hunden, R. D. G. Cooper, S. W. Kaldor, J. J. Skelton, B. A. Dressman, M. P. Clay, M. I. Steinberg, R. F. Bruns and N. G. Simon, Bioorg. Med. Chem., 2007, 15, 2054. [38] http://www.azevan.com/pipeline/default.asp, 2009. [39] J. S. Bryans, M. E. Bunnage, P. S. Johnson, H. J. Mason, L. R. Roberts, T. Ryckmans, A. Stobie and T. J. Underwood, WO Patent Application 2006114706, 2006. [40] J. S. Bryans, P. S. Johnson, L. R. Roberts and T. Ryckmans, WO Patent Application 2006123242, 2006. [41] C. Bissantz, E. Goetschi, C. Grundschober, R. Masciadri, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2008084005, 2008. [42] C. Bissantz, C. Grundschober, R. Masciadri, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2008068185, 2008. [43] C. Bissantz, C. Grundschober, R. Masciadri, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2008068183, 2008. [44] C. Bissantz, C. Grundschober, R. Masciadri, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2008068184, 2008. [45] C. Bissantz, C. Grundschober, R. Masciadri, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2008068159, 2008. [46] C. Bissantz, C. Grundschober, H. Ratni, M. Rogers-Evans and P. Schnider, US Patent Application 2007155761, 2007. [47] C. Bissantz, C. Grundschober, H. Ratni, M. Rogers-Evans and P. Schnider, WO Patent Application 2007006688, 2007. [48] http://clinicaltrials.gov/ct2/show/NCT00361491, 2006. [49] http://clinicaltrials.gov/ct2/show/NCT00358631, 2006. [50] Sanofi-Aventis, half-year financial report, 2008. [51] C. Louis, C. Cohen, R. Depoortere and G. Griebel, Neuropsychopharmacology, 2006, 31, 2180. [52] C. Griffante, A. Green, O. Curcuruto, C. P. Haslam, B. A. Dickinson and R. Arban, Br. J. Pharmacol., 2005, 146, 744.
146
Thomas Ryckmans
[53] C. Serradeil-Le Gal, D. Raufaste, S. Derick, J. Blankenstein, J. Allen, B. Pouzet, M. Pascal, J. Wagnon and M. A. Ventura, Am. J. Physiol., 2007, 293, R938. [54] W. Lubisch, T. Oost, W. Wernet, W. Hornberger, L. Unger and H. Geneste, WO Patent Application 2006100081, 2006. [55] W. Lubisch, T. Oost, W. Wernet, W. Hornberger, L. Unger and H. Geneste, WO Patent Application 2006100080, 2006. [56] T. Oost, W. Lubisch, W. Wernet, W. Hornberger and L. Unger, WO Patent Application 2006100082, 2006. [57] T. Oost, W. Lubisch, W. Wernet, W. Hornberger, L. Unger, H. Geneste and A. Netz, WO Patent Application 2007063123, 2007. [58] H. Geneste, T. Oost, C. W. Hutchins, W. Wernet, L. Unger, W. Hornberger, W. Lubisch and A. Netz, WO Patent Application 2008107399, 2008. [59] T. Kuwata, H. Nozawa and S. Hayashi, JP Patent Application 2008050354, 2008. [60] T. Arndt, T. Oost, W. Lubisch, W. Wernet, W. Hornberger, L. Unger and J. Ruiz Caro, WO Patent Application 2008025736, 2008. [61] G. A. Koppel, WO Patent Application 2006102308, 2006. [62] J. Letourneau, C. Riviello, K.-K. Ho, J.-H. Chan, M. Ohlmeyer, P. Jokiel, I. Neagu, J. R. Morphy and S. E. Napier, WO Patent Application 2006095014, 2006. [63] T. Kuwada, D. Nozawa, T. Ishizaka, M. Yoshinaga and K. Yoshikawa, WO Patent Application 2009017236, 2009. [64] M. Craighead, R. Milne, L. Campbell-Wan, L. Watson, J. Presland, F. J. Thomson, H. M. Marston and C. P. MacSweeney, Prog. Brain Res., 2008, 170, 527. [65] J. Letourneau, P. Jokiel, E. Napier, K.-K. Ho, M. Ohlmeyer, D. R. McArthur, J. Fiona, P. D. Ratcliffe and S. Jurgen, WO Patent Application 2008033764, 2008. [66] I. Neumann, WO Patent Application 2008071779, 2008. [67] K. Honda and Y. Takano, J. Pharmacol. Sci. (Tokyo, Jpn.), 2009, 109, 38. [68] A. L. Del Tredici, K. E. Vanover, A. E. Knapp, S. M. Bertozzi, N. R. Nash, E. S. Burstein, J. Lameh, E. A. Currier, R. E. Davis, M. R. Brann, N. Mohell, R. Olsson and F. Piu, Biochem. Pharmacol., 2008, 76, 1134. [69] A. A. Failli, J. S. Shumsky, R. J. Steffan, T. J. Caggiano, D. K. Williams, E. J. Trybulski, X. Ning, Y. Lock, T. Tanikella, D. Hartmann, P. S. Chan and C. H. Park, Bioorg. Med. Chem. Lett., 2006, 16, 954. [70] I. Tsukamoto, H. Koshio, S. Akamatsu, T. Kuramochi, C. Saitoh, T. Yatsu, H. YanaiInamura, C. Kitada, E. Yamamoto, S. Sakamoto and S.-i. Tsukamoto, Bioorg. Med. Chem., 2008, 16, 9524. [71] M. Kanbara, T. Suzuki, T. Kondo, K. Yokoyama and H. Kobayashi, JP Patent 2007308391, 2007. [72] C. M. Yea, C. E. Allan, D. M. Ashworth, J. Barnett, A. J. Baxter, J. D. Broadbridge, R. J. Franklin, S. L. Hampton, P. Hudson, J. A. Horton, P. D. Jenkins, A. M. Penson, G. R. W. Pitt, P. Riviere, P. A. Robson, D. P. Rooker, G. Semple, A. Sheppard, R. M. Haigh and M. B. Roe, J. Med. Chem., 2008, 51, 8124. [73] J. L. Cavalcante, S. Khan and M. Gheorghiade, Expert Rev. Cardiovasc. Ther., 2008, 6, 1331. [74] L. Foulon, P. Rochard, C. Serradeil Le Gal and G. Valette, FR Patent Application 2909668, 2008. [75] G. A. Koppel and N. D. Heindel, WO Patent Application 2007109615, 2007. [76] M. A. Xiang, R. H. Chen, K. T. Demarest, J. Gunnet, R. Look, W. Hageman, W. V. Murray, D. W. Combs, P. J. Rybczynski and M. Patel, Bioorg. Med. Chem. Lett., 2004, 14, 3143. [77] M. A. Xiang, P. J. Rybczynski, M. Patel, R. H. Chen, D. F. McComsey, H.-C. Zhang, J. W. Gunnet, R. Look, Y. Wang, L. K. Minor, H. M. Zhong, F. J. Villani, K. T. Demarest, B. P. Damiano and B. E. Maryanoff, Bioorg. Med. Chem. Lett., 2007, 17, 6623.
Advances in Vasopressin Receptor Agonists and Antagonists
147
[78] M. A. Xiang, M. Patel, P. Rybczynski, J. Gunnet, K. T. Demarest, R. Look, B. Maryanoff, M. J. Costanzo and S. C. Yabut, WO Patent Application 2008036755, 2008. [79] M. A. Xiang, M. Patel, P. Rybczynski, J. Gunnet, K. T. Demarest, R. Look, B. Maryanoff, M. J. Costanzo and S. C. Yabut, WO Patent Application 2008036759, 2008. [80] H. Meier, E. Bender, U. Brueggemeier, I. Flamme, D. Karthaus, P. Kolkhof, D. Meibom, D. Schneider, V. Voehringer, C. Fuerstner, J. Keldenich, D. Lang, E. Pook and C. Schmeck, WO Patent Application 2007134862, 2007.
CHAPT ER
7 The Emergence of GPR119 Agonists as Anti-Diabetic Agents Robert M. Jones and James N. Leonard
Contents
1. Introduction 2. Discovery and Characteristics of GPR119 3. The Biology of GPR119 3.1 Proposed endogenous regulators of GPR119 3.2 GPR119 regulation of insulin release 3.3 GPR119 regulation of GLP-1 and GIP release 4. GPR119 Agonists: Medicinal Chemistry 4.1 Six-membered heterocyclic ring–based agonists 4.2 Five-membered heterocyclic ring–based agonists 4.3 Bicyclic core–based agonists 4.4 Linear core–based agonists 5. Clinical Trial Status and Future Prospects References
149 150 150 150 152 153 154 154 159 161 164 166 167
1. INTRODUCTION Type 2 diabetes mellitus (T2DM) is emerging as a disease of staggering proportions in the twenty-first century, with an estimated 300 million cases worldwide projected by 2020 [1]. The physiological hallmarks of T2DM are severe insulin resistance, inappropriate hepatic glucose production in the hyperglycemic state, and insufficient insulin production Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA 92121, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04407-8
r 2009 Elsevier Inc. All rights reserved.
149
150
Robert M. Jones and James N. Leonard
from pancreatic b cells. Historically, treatment regimens for T2DM have shown significant effectiveness for improving glucose homeostasis; however, after about 2 years of therapy, increasing failure to maintain glycemic control is observed [2]. Recent approaches based on the biology of glucagon-like peptide-1 (GLP-1) have offered new hope for T2DM patients, due to the ability of this gut hormone to (1) elicit insulin release in a glucose-dependent fashion, (2) maintain and perhaps even enhance b-cell mass, and (3) promote satiety and thus weight loss [3]. These features suggest that GLP-1 could promote improved glycemic control acutely and, furthermore, might have a chronic, disease-modifying effect by virtue of prevention or reversal of b-cell failure. GLP-1-based therapies have so far consisted of stabilized peptide agonists of the GLP-1 receptor and small molecule inhibitors of dipeptidyl peptidase-IV (DPP-IV), the enzyme primarily responsible for inactivation of endogenously produced GLP-1 [4]. DPP-IV inhibitors also prevent degradation of glucose-dependent insulinotropic peptide (GIP), the other major gutderived insulinotropic hormone, or ‘‘incretin.’’ Agonists of GPR119 have emerged from pharmaceutical discovery efforts to identify an improved GLP-1 therapeutic by combining the convenience offered by oral dosage of DPP-IV inhibitors and the pharmacological robustness of GLP-1 receptor agonists. Mounting data support this hypothesis and moreover indicate that GPR119 agonists may also spearhead a new class of anti-diabetic therapies through modulation of intestinal endocrine cells that sequester key regulators of energy homeostasis.
2. DISCOVERY AND CHARACTERISTICS OF GPR119 After the discovery of GPR119 in 1999 using data afforded by the Human Genome Project, it was subsequently described in the peer-reviewed literature as a Class A receptor with no close relatives [5]. The receptor possesses distant similarity to biogenic amine and cannabinoid receptors (B40% identity in the transmembrane regions). The complete understanding of GPR119 expression is rather recent due to the complexity of its distribution in rare endocrine cell types. Several laboratories eventually showed that GPR119 is highly expressed in pancreatic islets and in some regions of the gut, particularly, the colon [6–8]. There is some discrepancy regarding the GPR119-expressing cell types within the islet [9], but the most convincing data firmly demonstrate that GPR119 is present in the great majority of pancreatic b cells [7]. Heterologous expression of GPR119 causes substantial increases in 3’,5’-cyclic adenosine monophosphate (cAMP) levels, which are further elevated
151
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
by agonists of the receptor [6,7,10]. Therefore, like the GLP-1 receptor, GPR119 is both b-cell-expressed and Gas-coupled.
3. THE BIOLOGY OF GPR119 3.1 Proposed endogenous regulators of GPR119 Lysophosphatidylcholine (LPC, 1) was the first proposed endogenous ligand for GPR119, based on its ability to stimulate glucose-dependent insulin release and increase cAMP in GPR119-transfected cells [6]. However, subsequent studies showed that the efficacy of 1 is poor, relative to other GPR119 modulators [10–11]. 1 also mediates insulin release in insulinoma cell lines that lack GPR119, shedding further doubt on the likelihood that this lipid is a true endogenous regulator of GPR119 [11].
Me3N HO O
O P H3C
O
HO
O
O O
1
OH
2
NH
NH
O
O
3
In contrast, the satiety factor oleoyl ethanolamide (OEA, 2) is a significantly more efficacious agonist of GPR119 [10]. The in vitro efficacy of 2 is similar to that of synthetic GPR119 agonists [11]. 2 is also capable of eliciting biological responses in GLUTag cells that endogenously express GPR119, but this response is greatly attenuated when receptor levels are reduced by siRNA treatment [12]. Furthermore, levels of 2 in the gut are nutrient-regulated, consistent with the proposed role of GPR119 as a mediator of energy homeostasis [13]. On the contrary, some data cast doubt on the likelihood that 2 is a true endogenous regulator of GPR119. Nutrient regulation of 2 levels is seen primarily in the jejunem, which is not a site of significant GPR119 expression [13]. Additionally, 2 is known to modulate other molecular targets governing energy homeostasis, including the nuclear receptor PPARa [14]. It is therefore challenging to assess the relative contribution of GPR119 activation to the
152
Robert M. Jones and James N. Leonard
known functions of 2. Data from GPR119 knockout (KO) mice shed at least some light on this issue. Weight loss mediated by 2 is not seen in mice lacking PPARa, but remains fully intact in mice lacking GPR119 [14,15]. Moreover, the ability of 2 to trigger insulin release is at least partly GPR119-independent [16]. Therefore, at least some aspects of the function of 2 in vivo are not mediated through GPR119.
OH
HO
O NH
HN OH
O
4
O
5
Regardless of its putative role as an endogenous ligand, the identification of 2 as a GPR119 agonist suggests that fatty acyl amides warrant further exploration as possible ligands for the receptor. Neither endocannabinoids nor shorter chain fatty acyl amides (e.g., capsaicin) are active in this regard. However, the endovallinoids N-oleoyl dopamine (OLDA, 3) and olvanil 4 have emerged as a third class of lipidic signaling molecules proposed to be endogenous modulators of GPR119 [11]. Interestingly, close relatives of these endovallinoids, such as (R)-Noleoyltyrosinol 5, are the most potent fatty acyl amide agonists of GPR119 identified to date [11]. 3 is essentially indistinguishable from 2 in terms of its ability to stimulate cAMP in GPR119-transfected cells. 3 also stimulates insulin release in vitro, elevates the incretin hormone GIP in vivo, and improves glucose tolerance, all in a largely GPR119-dependent manner and to an extent similar to that of synthetic GPR119 agonists [11]. Synthesis of 3 in the gut and pancreas has not been assessed in the literature. However, it is noteworthy that fatty acyldopamines may be produced by direct conjugation of fatty acids and dopamine [17], both of which are highly regulated by nutrient challenge in the gut [18–19].
3.2 GPR119 regulation of insulin release Potent, selective GPR119 agonists have been used to demonstrate that the receptor mediates robust glucose-dependent insulin release in rodent islets, insulinoma cell lines, and in vivo [7]. By using the agonist AR231453
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
153
6, these effects were shown to be completely GPR119-dependent in GPR119-deficient mice and in a mouse model using siRNA to depress GPR119 levels. The hamster insulinoma cell line HIT-T15 expresses GPR119 at levels similar to those seen in pancreatic islets, which likely accounts for its particularly robust response to GPR119 agonists [7]. It is therefore a convenient in vitro model of GPR119 function. When measuring both cAMP and insulin release, the efficacy of 6 is similar to that achieved with forskolin. These data suggest that the insulinotropic effects of GPR119 agonists are highly significant. The GPR119 agonist tools, PSN375963 7, PSN632408 8, and 2, have been assessed in MIN6c4 cells, where a comparatively poor cAMP response was observed [16]. This is most likely due to relatively low levels of GPR119 in MIN6 cells when compared with HIT-T15 cells, although recent reports cast doubt on the GPR119 specificity of 7 and 8 (see Section 4.2).
N
O
N O
N
O NO2
N N
O
N
O N
NH
N
O N
N
F N
N
SO2Me AR231453 6
PSN375963 7
PSN632408 8
In rat and mouse islets, 6 stimulates insulin release at glucose concentrations of 8 mM or higher [7]. Thus, the actions of GPR119 are glucose-dependent, as would be expected from a Gas-coupled b-cell receptor. The agonist effects are robust, achieving efficacy similar to that of GLP-1. They are also GPR119-dependent, since 6 is inactive when incubated with islets from GPR119-deficient mice. These data suggest that oral GPR119 agonists should improve insulin release and glucose tolerance in vivo, and indeed, this has been shown to be the case in normoglycemic and diabetic rodent models [7]. Collectively, these data demonstrate the feasibility of developing orally active GPR119 modulators with a GLP-1-like ability to improve b-cell function.
154
Robert M. Jones and James N. Leonard
3.3 GPR119 regulation of GLP-1 and GIP release Aside from pancreatic islets, sub-regions of the gut are the only other sites of robust GPR119 expression identified to date. Cellular expression studies have extended these observations by showing that most GLP-1producing L cells in the ileum and colon also contain GPR119 [8]. This is consistent with data showing high GPR119 expression in most in vitro L-cell models [8,12]. GIP, the other major insulinotropic hormone of the gut, is produced primarily in the duodenal K cells. Here, the cellular expression of GPR119 is less clear. In one study, detectable levels of GPR119 were not seen in GIP-positive cells [8], but others have reported that primary K-cell cultures contain significant amounts of GPR119 [20]. Nevertheless, it is clear that GPR119 agonists such as 6 increase plasma levels of both GLP-1 and GIP in vivo [8]. In concordance with these data, GPR119 agonists stimulate GLP-1 release from primary colonic crypt cultures [21], fetal rat intestinal cultures [12], and GLUTag cells [8], an immortalized mouse L-cell line. GPR119 therefore enhances incretin release, whereas DPP-IV inhibitors mitigate incretin inactivation. Such joint therapeutic approaches will function synergistically with regard to GLP-1 secretion and glycemic control. In support of this hypothesis, combined administration of 6 and a DPP-IV inhibitor acutely increases plasma GLP-1 levels and improves glucose tolerance to a significantly greater degree than either agent alone [8]. Combination treatment also elevates GLP-1 release in a nutrientindependent fashion and thus may offer a more effective means to maintain elevated GLP-1 levels between meals in diabetic patients.
4. GPR119 AGONISTS: MEDICINAL CHEMISTRY 4.1 Six-membered heterocyclic ring–based agonists Following a cyclase-based high throughput screening (HTS) campaign, pyrimidine 9 was identified as an inverse agonist of hGPR119 (IC50 ¼ 84 nM) [22]. In structure activity relationship (SAR) studies related to 9, the trifluoromethyl pyrazole motif was replaced with a series of aryl ethers leading to the identification of agonists 10 (EC50 ¼ 1 mM) and 11 (EC50 ¼ 1.7 mM). Further extensive parallel SAR work led to the identification of multiple additional agonist trigger moieties, including sulfones, sulfonamides, and various five-membered heterocyclic substituents. The best activity was seen when such a group was attached to the 4-position of the phenyl ring. In addition, with the appropriate trigger in place, agonist activity was maintained with either an ether or aniline linker to the pyrimidinyl core.
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
155
Me
Me O
O
O
O
N
N
N NO2
N N
O
N NO2
N N
N O
O
NO2
N N
NH
N N F F F
X R
9
SO2Me
O
10. X = CO, R = OMe 11. X = CH2, R = Me
12
In further SAR work, the aniline sulfone group was fixed, and conservative ester replacements afforded potent GPR119 agonists. The SAR of the alkyl substituent on the oxadiazole ring was particularly remarkable. Increasing the size of this substituent from methyl to ethyl provided a more than 25-fold improvement in agonist potency. The inclusion of a-branching in this group again gave a significant improvement in potency, as exemplified by 12 (hGPR119 EC50 ¼ 5.8 nM, melanophore dispersion assay). Fluoro substituents on the aryl ring further improved potency, with 6 being the first agonist with subnanomolar potency (EC50 ¼ 0.68 nM) observed. Standard profiling of 6 (CEREP) and exhaustive counter screens against other receptors indicated that 6 is a highly selective GPR119 agonist [22–24]. In addition, 6 had no activity against DPP-IV in a fluorescent-based assay using diprotin. Importantly for a pharmacological tool, 6 had GPR119 agonist activity across species, albeit with somewhat lower potency for rodent GPR119 (cynomolgus monkey GPR119 EC50 ¼ 0.4 nM; mouse GPR119 EC50 ¼ 12 nM; rat GPR119 EC50 ¼ 14 nM; dog GPR119 EC50 ¼ 1.6 nM: melanophore dispersion assay). Notably, across all species, 6 displayed a significantly greater level of efficacy in these assays than the putative endogenous ligand 2. Importantly, 6 stimulated cAMP production through endogenously expressed GPR119 in the b-cell-like hamster insulinoma cell line HIT-T15, with an EC50 of 4.7 nM, a value comparable to the EC50 observed in cell lines over-expressing the cloned rodent receptors [24]. Notably, the EC50 for insulin release from this cell line was 3.5 nM.
156
Robert M. Jones and James N. Leonard
Oxadiazole 6 did not significantly inhibit any of the major cytochrome P450 enzymes and had no activity up to 10 mM at the human Ether-ago-go Related Gene (hERG) channel in a patch clamp assay. Furthermore, excellent exposure was observed after oral administration to C57/bl6J mice (10 mg/kg po: tmax ¼ 0.5 h, Cmax ¼ 9.84 mM). The in vivo effect of 6 with respect to % inhibition of glucose area under the curve (AUC) in an oral glucose tolerance test (oGTT) was similar in C57/bl6J mice and Sprague–Dawley (SD) rats at a dose of 3 mg/kg ip despite a clear species difference in the magnitude of the glucose excursion. This similarity may be expected in light of the similar potency and efficacy of 6 at both mouse and rat GPR119. In addition, 6 improved oral glucose tolerance in wildtype C57/bl6J mice after oral administration. Importantly, 6 also enhanced glucose-dependent insulin release and improved oral glucose tolerance in wild-type C57/bl6J mice but not in GPR119-deficient mice at a dose of 20 mg/kg po, clearly demonstrating the receptor dependence of the observed effects [23,24]. However, a statistically significant effect on glucose excursion in an oGTT following oral administration of 6 was not observed in rats, although a trend toward efficacy was seen at a dose of 30 mg/kg. The lack of a consistent effect was believed to result from the combination of a narrower window for measuring the decrease in glucose excursion in rats as well as the significantly poorer exposure of 6 in rats compared to mice following oral administration (rat, 10 mg/kg, po: tmax ¼ 1 h, Cmax ¼ 0.250 mM, F ¼ 12%). A key aspect of b-cell Gas-coupled receptors is their ability to enhance insulin release in a glucose-dependent fashion, and 6 has been used to show this is characteristic of GPR119. In both rat and mouse islets, 6 had no effect on insulin release at basal (5 mM) glucose concentrations but had GLP-1-like efficacy when the glucose concentration was W8 mM. Also, unlike the sulfonyl urea glyburide, 6 did not stimulate insulin release or cause hypoglycemia during a drug challenge to fasted mice. As noted earlier, 6 (10 mg/kg po) also stimulates both GLP-1 and GIP release during OGTTs in C57BL/6J mice [8,12,21]. No effect was seen on GIP release in GPR119 KO mice. Moreover, co-administration of 6 with a DPP-IV inhibitor leads to synergistic increases in GLP-1 levels, which results in inhibition of the glucose excursion during an OGTT by the 6/DPP-IV inhibitor combination that is greater than that achieved by either agent alone [8]. The potential of GPR119 agonists to exert b-cell protective effects through increased cAMP levels has also been demonstrated. In MIN6 pancreatic b cells expressing GPR119, 6 induced Akt phosphorylation and IRS-2 expression, key measures of islet mass protection [24,25].
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
157
O
O O
O
N
N
O
O N
N
R′
Me N
N
NH
O
Me
Me N
N R
13
14. R′ = Me, R = SO2Me 15. R′ = MeO, R = 1,2,4-triazolo
Several patent applications published during the past 24 months disclosed additional GPR119 agonists bearing six-membered ring heterocyclic core motifs. Picoline 13 was described as a selective agonist of human, dog, cynomolgus monkey, mouse, and rat GPR119 expressed in melanophores with EC50s of 2, 1, 35, 41, and 44 nM respectively [26]. Furthermore, 13 possessed significant aqueous solubility at pH 7, with no appreciable inhibition of five cytochrome P450 enzymes. Picoline 13 also elicited a dose-responsive mean inhibition of glucose excursion of 22, 24, and 70% when orally administered to normoglycemic male SD rats at doses of 0.3, 3, and 30 mg/kg respectively. Related pyrimidines including sulfone 14 and 1,2,4-triazole 15 have also been described [27,28]. Sulfone 14 displayed agonist activity at GPR119 from different species in melanophores (EC50 ¼ 2, 8, 43, and 42 nM; human, dog, cynomolgus monkey, mouse, and rat respectively). Upon dose escalation in rats, 14 afforded linearly increasing plasma exposure (AUC ¼ 4.73, 55.9, and 515.32 mg h/mL at per oral doses of 3, 30, and 300 mg/kg respectively). The ability of 14 to affect glucose homeostasis in male SD rats was assessed using an oGTT. 14 exhibited a 38% mean inhibition of glucose excursion following an oral 10 mg/kg dose. Similarly, triazole 15 showed agonist activity in GPR119-transfected melanophores (EC50 ¼ 2, 4, 57, and 81 nM at human, dog, cynomolgus monkey, mouse, and rat GPR119 respectively). It also exhibited linear plasma exposure upon dose escalation in rats (AUC ¼ 3.59, 79.82, and 285.99 mg.h/mL at 3, 30, and 300 mg/kg po), in addition to displaying oral efficacy in an oGTT.
158
Robert M. Jones and James N. Leonard
O
O
N
N
N O
O
N
O
O N
O
N
O N N
N F
F
SO2Me
SO2Me
SO2Me
16
17
18
A series of 3,6-substituted pyridines exemplified by 16 and 17 have been described as GPR119 agonists in a patent application [29]. The compounds, their salts, and their use for the treatment of a condition mediated through GPR119 such as metabolic disorders, diabetes, and obesity are claimed. Test compounds from this patent application were assayed for their effects on GLP-1 levels in vivo. Treatment of male C57/ B16 mice with 16 (30 mg/kg po) caused an increase in the total GLP-1 level to 7.572.0 pg/mL, compared to 2.371.2 pg/mL for vehicle-treated mice. A related pyridazine series has been independently disclosed exemplified by 18 [30]. Pyridazine 18 was reported to have a hGPR119 EC50 value of 0.046 mM. Assays to determine the effect of the compounds on weight gain and glucose-stimulated insulin release in mice are described, but no specific data are given. Additional GPR119 agonists bearing six-membered ring cores exemplified by pyridine 19 and bispiperidine 20 have recently been disclosed [31,32]. F O O
N
N
N N
O
N
O
N N
MeO2S SO2Me 19
20
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
159
4.2 Five-membered heterocyclic ring–based agonists In a seminal paper that described deorphanization of GPR119, the pharmacological profile of the 1,2,4-oxadiazole carbamate 8, identified by optimization of the HTS hit 7, was also disclosed as a GPR119 agonist [10]. Like the putative endogenous ligand 2, oxadiazole 8 produced a concentration-dependent increase in intracellular cAMP levels in a human embryonic kidney (HEK) cell line expressing GPR119 with an EC50 of 1.9 mM. Oral administration of 8 to rats at a dose of 100 mg/kg po reduced 24 h cumulative food intake. The reduction in food intake was not associated with drug-induced malaise, as no effects were seen on locomotor activity or in conditioned taste aversion and kaolin consumption tests. The acute anorectic effect translated into chronic effects on body weight. In dietinduced obese mice and in growing, high-fat diet-fed SD rats, attenuation of body weight gain produced by 8 (100 mg/kg/day po) was comparable to that of the prescribed anorectic drug sibutramine hydrochloride hydrate (5 mg/kg/day po) [33]. More recently, it was reported that 8 possessed similar potency and efficacy at hGPR119 (EC50 ¼ 5.6 mM, Emax ¼ 110%) relative to the putative endogenous ligand 2 (EC50 ¼ 3.2 mM, Emax ¼ 100%) in a yeast/b-galactosidase fluorescence reporter assay [34]. However, in MIN6c4 cells expressing endogenous GPR119, the synthetic agonists 7 and 8 differ from 2 in their effects on intracellular cAMP and calcium levels and insulin secretion [16]. While the endogenous GPR119 ligand 2 signals through GPR119 in a manner similar to GLP-1 and its receptor to elicit increased insulin secretion and increased intracellular Ca2+ and cAMP levels, oxadiazoles 7 and 8 produced divergent effects on these parameters. These studies suggest that 7 and 8, although they do weakly activate GPR119, may also modulate GPR119-independent pathways and thus may be unsuitable as GPR119-specific pharmacological tools [16]. These observations are somewhat corroborated by a recent report in which 8 was shown to possess an EC50 W20 mM in a CHO6CRE luciferase reporter assay [35].
O
O
O
O
O
R
O N
N
SO2Me 21
F
O O N
N
SO2Me 22 (PSN119-2)
O N
N
N
N
O N
N
O
F
N
SO2Me 23
160
Robert M. Jones and James N. Leonard
Exchange of the 4-pyridyl motif present in 7 and 8 with a 3-fluoro-4(methanesulfonyl)phenyl moiety led to agonist 21 (R ¼ F) possessing EC50 and Emax values of 0.9 mM and 375% respectively in a yeast reporter assay expressing recombinant human GPR119. Focused SAR modifications of the aromatic moiety in which the R-substituent was either replaced by other substituents (21, R ¼ –H, –Cl, –Me, –OMe) or moved to the adjacent 2-position led to reduced potency or to inactivity. Subsequently, installation of an (R)-methyl group to the methyleneoxy linker of 21 afforded PSN119-2 22 (EC50 ¼ 0.4 mM, Emax ¼ 358%). The antipode of 22 was substantially less active (10-fold) and less efficacious (twofold), as were the enantiomeric ethyl homologues. 22 was shown to stimulate insulin secretion from the hamster-derived insulinoma HITT15 cell line (EC50 ¼ 18 nM) and GLP-1 release from the immortalized entero-endocrine murine GLUTag cell line (EC50 ¼ 8 nM). In male SD rats, 22 orally administered at doses of 10 and 30 mg/kg achieved a Cmax of B2 and B5.1 mM, respectively, and significantly attenuated the glucose excursion in an oGTT. At the same doses, 22 significantly reduced cumulative 24 h food intake levels in SD rats compared to vehicle-treated control rats, but these effects were not as robust as those observed for sibutramine (5 mg/kg, po). Analogs of 22, resulting from replacement of the terminal tert-butyl carbamate with an N-linked 5-isopropyl-1,2,4-oxadiazole, 23, are the subject of a recent patent application [36]. Bioisosteric replacement of a carbamate by an oxadiazole is a successful GPR119 agonist SAR theme previously disclosed in earlier patent applications [37–41]. Additional five-membered ring core agonists have been independently reported in several recent patent applications [42–44]. In one disclosure, compounds were evaluated in cAMP stimulation assays, and thiazole 24 (10 mM) demonstrated a 69% change in Forster resonance energy transfer (FRET) signals relative to control [43]. Derivative 24 was also shown to stimulate insulin secretion by 1.66-fold in a SD rat–derived islet perfusion assay following a 16 mM glucose treatment [43]. Thiazole 24 (30 mg/kg, po) also facilitated a 52% reduction in glucose AUC in 8- to 10-week-old C57/6J fasted mice following a 2 g/kg oral glucose bolus challenge. In a more recent disclosure, a closely related series of triazolyl piperidine–based GPR119 agonists was described [43]. In vitro GPR119 agonism was again determined by measurement of the decrease in FRET signaling in response to changes in intracellular cAMP levels. Many of these compounds were reported to cause a significant reduction in FRET signaling relative to positive control (e.g., 25, 87.61% reduction). Additionally, triazole 25 (30 mg/kg po) was shown to reduce mean glucose excursion in male C57/6J mice by 40.8%.
161
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
F3C N
O O
N
N
S N
O
O
N
N
F N N N
O
N O N
O F
N F
N N 24
N
N N N
SO2Me
25
26
The characterization of the potent, selective, and orally bioavailable GPR119 agonist, MBX-3152 (structure not disclosed), has been reported [45]. In CHO cells stably expressing hGPR119, MBX-3152 afforded an EC50 value of 1.8 nM, and in immortalized murine GLUTag cells, it increased GLP-1 secretion 1.5-fold at concentrations of 200 nM and above with an EC50 value of 82 nM. When administered chronically to high fat– fed female Zucker diabetic fatty (ZDF) rats, MBX-3152 (30 mg/kg, po, 35 days) was shown to concomitantly delay disease onset and reduce food intake, body weight gain, and plasma triglycerides, thereby mitigating the profound increase in insulin resistance that develops in this animal model. In wild-type mice, but not in GPR119 KO mice, the compound [30 mg/kg, po] also improved glucose tolerance and increased incretin release in the presence of the DPP-IV inhibitor sitagliptin (1 mg/kg, po]. In rats, MBX-3152 (30 mg/kg, po) increased insulin secretion and glucose infusion rate under hyperglycemic clamp conditions implying a direct action of the agonist at pancreatic b cells. Pyrrolidinyl 1,3,4-oxadiazole 26 is representative of another chemical series of GPR119 modulators [44]. These analogs are similarly potent at stimulating cAMP in Flp-In-CHO-hGPR119 cells and are claimed to be useful for the treatment or prevention of disorders associated with GPR119.
4.3 Bicyclic core–based agonists The structure of GSK252A 27a has recently been disclosed [21]. This GPR119 agonist possesses a novel pyrrolopyrimidine-based bicyclic core and bears a striking resemblance to GPR119 agonists in which a pyrazolo
162
Robert M. Jones and James N. Leonard
[3,4-d]pyrimidine was utilized as the core scaffold, exemplified by 28, disclosed in an earlier application by an independent group [41]. Compound 27a was reported to have good pharmacokinetic properties in SD rats. At a dose of 30 mg/kg po, 27a achieved a Cmax ¼ 14 mM, a half life of 3.1 h, and absolute oral bioavailability of 60%. In a CHO6CRE reporter assay, 27a afforded an EC50 ¼ 40 nM compared to an EC50 ¼ 200 nM for 28 [21,41]. R N O
O O
O
O
N
N
N
N O O
O
N
N
N N N
N
N
N
N
N
F
F
SO2Me 27a. R = H = GSK252A 27b. R = Me
F MeO2S 28
NC 29
The effects of 27a on incretin, Peptide YY (PYY), glucagon, and insulin secretion both in vitro and in vivo were disclosed. The compound stimulated GLP-1 release from both GLUTag (EC50 ¼ 22 nM) and primary mouse colonic crypt cell cultures in vitro. In addition, 27a augmented glucose-stimulated insulin secretion from rat islet cells in the presence of 12 mM glucose but did not affect insulin output in the presence of 3 mM glucose, consistent with similar glucose-dependent effects of 6. Oral administration of 27a (10 mg/kg, po) to mice fasted overnight resulted in a fourfold increase in GLP-1 levels and a twofold increase in GIP levels when compared to vehicle control, with no increase seen in GPR119 KO mice. In addition, there was also a threefold increase in PYY levels and a twofold increase in glucagon levels in wild-type, but not in GPR119 KO mice. Augmentation of insulin secretion by 27a (10 mg/kg, po) was assessed in rats using the hyperglycemic clamp model. In the hyperglycemic state targeting a glucose concentration of 200 mg/dL, both glucose infusion rate and C-peptide levels were significantly increased indicating a robust enhancement of insulin release. Consistent with these data, 27a decreased the glucose AUC by 43% and increased the peak insulin levels by 1.4-fold during the intravenous glucose tolerance test (IVGTT) and improved glucose tolerance during the oGTT by 38% with no significant effect on the
163
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
insulin response. This effect was not significantly different from that of the DPP-IV inhibitor LAF237. A hyperglycemic euglycemic clamp was also used to show that GPR119 activation may also impact glucose utilization independent of insulin secretion. The glucose infusion rate needed to maintain euglycemia was approximately threefold higher in rats treated with 27a compared to vehicle-treated rats, suggesting that 27a promoted significantly enhanced glucose utilization [21]. Tert butyl carbamate 27b and related bicyclic pyrrolo[2,3-d]pyrimidines, including 1,2,4-oxadiazole 29 together with indoline core congeners, are claimed in two recent patent applications [46,47]. To test the effect of 27b on insulin sensitization in male SD rats, a hyperinsulinemic euglycemic clamp was initiated 90 min after oral administration of 27b (10 mg/kg po) or vehicle. As observed for 27a, the glucose infusion rate required to maintain euglycemia in rats given 27b was three times that of vehicle-treated rats. Br O
O O O
O N
N
O N N O
N N
N N N
N N O
N
N
NH
NH
F
Cl MeO2S 30
31
MeO2S 32
More recent patent filings describe selective GPR119 modulators comprised of a fused pyrimidinone core 30, specifically, 6-substituted 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4(3H)-ones and 7-substituted 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4(3H)-ones [48,49]. Both series of GPR119 modulators are described as being useful for treating or preventing obesity, diabetes, metabolic disease, cardiovascular disease, or a disorder related to the activity of GPR119 in a patient. Undisclosed compounds from these applications are purported to activate GPR119 and stimulate cAMP production in transfected HEK293 cells, exhibiting EC50 values ranging from about 50 nM to 14,000 nM. A mouse oGTT protocol is described for assessing the activity of these compounds in vivo, and various undisclosed fused pyrimidinone examples were reported to be effective in lowering blood glucose levels after glucose challenge.
164
Robert M. Jones and James N. Leonard
Furthermore, illustrative examples were found to produce a sustained decrease in blood glucose in a non-genetic mouse model of T2DM. Building on the existing knowledge base of the SAR of bicyclic-based GPR119 agonists, two series of azabicyclic agonists have been described, exemplified by 31 and 32 [50,51]. An in vitro luciferase assay using HEK 293 cells expressing hGPR119 was conducted to assess the efficacy of the compounds. Bicyclic triazole 31 exhibited a hGPR119 EC50 value of 19.49 nM, and the pyrimidine fused oxazine 32 exhibited an EC50 value of 2.78 nM.
4.4 Linear core–based agonists Removal of the oxadiazolo core unit of 8 has been the focus of recent SAR optimization work [52]. The linear ether derivative 33a was equipotent with 8 but was a partial agonist in a yeast reporter assay expressing recombinant human or mouse GPR119. Repositioning the ether linkage as in pyridyl ether, 33b produced a marginal improvement in potency (EC50 ¼ 0.5 mM) without increasing in vitro efficacy. Exchange of the pyridine ring with a 3-fluoro-4-(methanesulfonyl)phenyl moiety produced PSN119-1M 35, which afforded a dramatic increase in efficacy (EC50 ¼ 0.2 mM, Emax ¼ 392%), implicating the hydrogen bond acceptor feature as a critical driver of potency and efficacy at human GPR119.
O
O
O
O
O
N
N
O N
Y O
X
N
F
F SOMe
33a. Y = O, X = CH2 33b. Y = CH2, X = O
O
34
SO2Me 35
Extensive pharmacological characterization of racemic sulfoxide PSN119-1 34 (EC50 ¼ 0.5 mM; Emax ¼ 407% yeast reporter assay) has been reported [52–54]. Unlike the glucose-independent insulin secretagogue repaglinide (1 mg/kg, po), 34 (30 mg/kg, po) did not cause hypoglycemia when evaluated in an oGTT in high fat diet–fed (10 days) SD rats. The anti-hyperglycemic effects of 34 were confirmed in 13-week-old diabetic ZDF rats, where its effect on glucose tolerance was similar to that
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
165
of the DPP-IV inhibitor P32/98. In contrast, its glucose-lowering effect was drastically reduced during an IVGTT, pointing to a significant contribution from gastrointestinal tract (GI)-derived incretins. Like the prescribed anorectic agent sibutramine (5 mg/kg, po), 34 (50 mg/kg, po) facilitated a statistically significant reduction in cumulative food intake at both the 6 and 24 h time points post dose. Following oral administration to SD rats, sulfoxide 34 was reported to be extensively metabolized to sulfone 35 [52]. Further pharmacological characterization demonstrated that sulfoxide 34 elicited a dose-responsive effect on GLP-1 secretion in murine GLUTag cells (EC50 ¼ 153770 nM). This was confirmed in a meal tolerance test using P32/98 (50 mg/kg, po) in combination with 34 (100 mg/kg) in rats. In addition, 34 (30 mg/kg, po) inhibited gastric emptying in rats in a manner similar to the antiobesic agent HMR1426 (50 mg/kg, po). Subchronic daily dosing of 34 to high fat diet–fed rats for 21 days (100 mg/kg/qd po) significantly lowered plasma glucose and insulin levels during an oGTT test performed on day 21 and also decreased fat pad mass and plasma leptin levels [53,54]. In addition, subchronic once daily oral administration (100 mg/kg/day) slowed the progression of diabetes in a study in young db/db mice, as highlighted by the ability of 34 to maintain normal fed blood glucose levels and glucose tolerance following an oGTT on day 21 of the experiment. Further optimization of 34 afforded development candidate PSN821 [54,55]. Although the structure of PSN821 has not been disclosed, it may be encompassed by generic structure 36, which captures the essential features of a series of GPR119 agonists disclosed in five recent patent applications [56–60]. Little biological and pharmacological characterization of these analogs is provided. However, the methods associated with the biological assays used to characterize the examples are given; a yeast-based reporter assay expressing human and mouse GPR119, a cAMP AlphascreenTM, an in vivo feeding study, HIT-T15 cAMP and insulin secretion assays, and an oGTT performed in either SD rat, male C57Bl/6, or male ob/ob mice.
N
N O
N
Me O E R1
Q R2
36
36a. E = CH, Q = CH, R2 = F, R1 = SO2CH3 36b. E = CH, Q = CH, R2 = F, Me, R1 = CONHR 36c. E = CH, Q = CH, R2 = F, R1 = CH2SO2CH3 36d. E = CH, Q = N, R2 = Me, R1 = CONHR 36e. E = N, Q = CH, R2 = Me, R1 = NHCMeCH2OH
166
Robert M. Jones and James N. Leonard
Other linear core–based agonists include a recently disclosed series of [[1,2,3,4-tetrahydro-2-(methylsulfonyl)-6-isoquinolinyl]oxy]propyl derivatives exemplified by 37 [61]. The compounds of this genus were characterized using Flp-In-CHO-hGPR119 cells wherein compound 37 showed a concentration-dependent increase in cAMP levels with an EC50 o100 nM. In addition, it was reported that the simple linear thioester 38 attenuated fed blood glucose levels in diabetic db/db mice [62]. Separately, it was reported that 38, which raised intracellular cAMP levels with an EC50 of 3.2 mM in 293-EBNA cells, stimulated insulin secretion and improved glucose tolerance in an oGTT when administered at the dose of 100 mg/kg ip to SD rats [63]. GPR119 agonist 38 also demonstrated antihyperglycemic effects in diabetic Goto–Kakizaki rats following oral administration at a dose of 100 mg/kg. O N
O
O
N SO2Me 37
S
O
N 38
5. CLINICAL TRIAL STATUS AND FUTURE PROSPECTS To date, four GPR119 agonists have been reported to be in the early stages of clinical development. The structures of these compounds have not been revealed. The first compound advanced to the clinic, APD-668, was co-developed by Arena Pharmaceuticals and Ortho McNeil [64–67]. Following two initial phase I trials, it was reported in January 2008 that APD-668 may improve glucose control in diabetic patients [68]. Subsequently, further development of APD-668 was halted and APD597 was advanced into a phase I trial in December 2008 [69]. Metabolex initiated a phase I trial with MBX-2982 in March 2008 [70], and in November 2008, phase 1a data were presented [71]. The placebocontrolled trial evaluated 10–1,000 mg of MBX-2982 in healthy volunteers. The drug was rapidly absorbed, had good exposure, and
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
167
demonstrated a half-life consistent with once-daily dosing. MBX-2982 also caused dose-dependent reductions in glucose and increases in GLP1 in these individuals following a mixed meal. All doses were well tolerated with no drug-related adverse events reported; at that time, a multiple-dose, pharmacokinetics, and pharmacodynamics phase Ib trial was under way [72]. In September 2008, OSI Pharmaceuticals began a phase I trial of PSN-821 [55]. To date, no results have been reported for this compound. It is clear from these reports that there are presently insufficient data to confirm the promise of GPR119 agonists suggested by pre-clinical studies. Nevertheless, the early reports are encouraging. Based on the robust activity around this target in the patent literature, GPR119 agonists are very likely to undergo vigorous clinical testing over the next few years, and the therapeutic value of this approach should clarify significantly. Clearly, the paramount consideration will be to determine the robustness of this mechanism with regard to the lowering of fasting plasma glucose and HbA1c in diabetic patients. The effects of GPR119 agonists on incretin and insulin levels will also be extremely valuable information to clinicians, who ultimately need to determine where GPR119 agonists belong in the treatment paradigm for T2DM.
REFERENCES [1] [2] [3] [4] [5] [6]
[7]
[8]
[9] [10]
[11]
J. C. Seidell, Br. J. Nutr., 2000, 83(Suppl. 1), S5. UK Prospective Diabetes Study Group., Lancet, 1998, 352, 854. D. J. Drucker, Cell Metab, 2006, 3, 153. D. J. Drucker and M. A. Nauck, Lancet, 2006, 368, 1696. R. Fredriksson, P. J. Ho¨glund, D. E. I. Gloriam, M. C. Lagerstrom and H. B. Schio¨th, FEBS Lett, 2003, 554, 381. T. Soga, T. Ohishi, T. Matsui, T. Saito, M. Matsumoto, J. Takasaki, S. Matsumoto, M. Kamohara, H. Hiyama, S. Yoshida, K. Momose, Y. Ueda, H. Matsushime, M. Kobori and K. Furuichi, Biochem. Biophys. Res. Commun., 2005, 326, 744. Z.-L. Chu, R. M. Jones, H. He, C. Carroll, V. Gutierrez, A. Lucman, M. Moloney, H. Gao, H. Mondala, D. Bagnol, D. Unett, Y. Liang, K. Demarest, G. Semple, D. P. Behan and J. Leonard, Endocrinology, 2007, 148, 2601. Z.-L. Chu, C. Carroll, J. Alfonso, V. Gutierrez, H. He, A. Lucman, M. Pedraza, H. Mondala, H. Gao, D. Bagnol, R. Chen, R. M. Jones, D. P. Behan and J. Leonard, Endocrinology, 2008, 149, 2038. Y. Sakamoto, H. Inoue, S. Kawakami, K. Miyawaki, T. Miyamoto, K. Mizuta and M. Itakura, Biochem. Biophys. Res. Commun., 2006, 351, 474. H. A. Overton, A. J. Babbs, S. M. Doel, M. C. T. Fyfe, L. S. Gardner, G. Griffin, H. C. Jackson, M. J. Proctor, C. M. Rasamison, M. Tang-Christensen, P. S. Widdowson, G. M. Williams and C. Reynet, Cell Metab, 2006, 3, 167. Z. Chu, R. Jones, R. Chen, C. Carroll, V. Gutierrez, A. Lucman, D. P. Behan and J. Leonard, Keystone Symposium. Diabetes: Molecular Genetics, Signalling Pathways and Integrated Physiology, 2007, Abstract 230.
168
Robert M. Jones and James N. Leonard
[12] L. Lauffer, R. Iakoubov and P. L. Brubaker, Diabetes, 2009 Feb 17. [Epub ahead of print] PMID: 19208912. [13] J. Fu, G. Astarita, S. Gaetani, J. Kim, B. F. Cravatt, K. Mackie and D. Piomelli, J. Biol. Chem., 2007, 282, 1518. [14] J. Fu, S. Gaetani, F. Oveisi, J. Lo Verme, A. Serrani, F. Rodriguez de Fonseca, A. Rosengarth, H. Luecke, B. Di Giacomo, G. Tarzia and D. Piomelli, Nature, 2003, 425, 90. [15] H. Lan, G. Vassileva, A. Corona, L. Liu, H. Baker, A. Golovko et al., Keystone Symposium. Diabetes: Molecular Genetics, Signalling Pathways and Integrated Physiology, 2007, Abstract 253. [16] Y. Ning, K. O’Neill, H. Lan, L. Pang, L. X. Shan, B. E. Hawes and J. A. Hedrick, Br. J. Pharmacol., 2008, 155, 1056. [17] S. M. Huang, T. Bisogno, M. Trevisani, A. Al-Hayani, L. De Petrocellis, F. Fezza, M. Tognetto, T. J. Petros, J. F. Krey, C. J. Chu, J. D. Miller, S. N. Davies, P. Geppetti, J. M. Walker and V. Di Marzo, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 8400. [18] G. Eisenhofer, A. Aneman, P. Friberg, D. Hooper, L. Fa˚ndriks, H. Lonroth, B. Hunyady and E. Mezey, J. Clin. Endocrinol. Metab., 1997, 82, 3864. [19] E. Eldrup and E. A. Richter, Am. J. Physiol. Endocrinol. Metab., 2000, 279, E815. [20] H. E. Parker, A. M. Habib, G. J. Rogers, F. M. Gribble and F. Reimann, Diabetologia, 2009, 52, 289. [21] C. Ammala, S. Bullard, J. Kashatus, S. Katamreddy, J. Way and A. Carpenter, Keystone Symposium, Islet and b-Cell Biology, 2008, Abstract #102. [22] G. Semple, B. Fioravanti, G. Pereira, I. Calderon, J. Uy, K. Choi, Y. Xiong, A. Ren, M. Morgan, V. Dave, W. Thomsen, D. J. Unett, C. Xing, S. Bossie, C. Carroll, Z. L. Chu, A. J. Grottick, E. K. Hauser, J. N. Leonard and R. M. Jones, J. Med. Chem., 2008, 51(17), 5172. [23] G. Semple, Abstracts of Papers, 41st Western Regional Meeting of the American Chemical Society, San Diego, CA, United States, October 9–13 2007, GEN-033. [24] R. M. Jones, Abstracts of Papers, 232nd ACS National Meeting, San Francisco, CA, United States, Sept. 10–14, 2006, MEDI-275. [25] Z. Chu, C. Carroll, V. Gutierrez, A. Lucman, M. Moloney, H. Gao et al., Keystone Symposium. Diabetes: Molecular Genetics, Signalling Pathways and Integrated Physiology, Keystone, Colorado, USA, 14–19 January 2007, Abstract 117. [26] R. M. Jones and J. Lehmann, WO Patent Application 2007035355 A2, 2007. [27] R. M. Jones and J. Lehmann, WO Patent Application 2008005576 A1, 2008. [28] R. M. Jones, J. Lehmann and A. Siu-Ting Wong, WO Patent Application 2008005569 A2, 2008. [29] J. Fang, J. Tang, A. J. Carpenter, G. Peckham, C. R. Conlee, S. K. Du and S. R. Katamreddy, WO Patent Application 2008070692, 2008. [30] P. Brandt, G. Johansson, L. Johansson, T. Koolmeister, B. M. Nilsson and T. Sandvall, WO Patent Application 2008025799, 2008. [31] D. A. Wacker, K. A. Rossi and Y. Wang, WO Patent Application 2009012277 A1, 2009. [32] H. B. Wood, A. D. Adams, S. Freeman, J. W. Szewczyk, C. Santini and Y. Huang, WO Patent Application 2008085316 A1, 2008. [33] M. C. T. Fyfe, H. Overton, J. White, R. Jones, R. V. Sorensen and C. Reynet, Diabetes, 2006, 55(Suppl. 1), 346-3OR. [34] M. C. T. Fyfe, A. J. Babbs, L. S. Bertram, S. E. Bradley, S. M. Doel, S. Gadher, W. T. Gattrell, R. P. Jeevaratnam, J. F. Keily, J. G. McCormack, H. A. Overton, C. M. Rasamison, C. Reynet, C. P. Sambrook Smith, V. K. Shah, D. F. Stonehouse, S. A. Swain, J. R. White, P. S. Widdowson, G. M. Williams and M. J. Procter, Abstracts of Papers, 236th ACS National Meeting, Philadelphia, PA, United States, August 17–21, MEDI-197, 2008.
The Emergence of GPR119 Agonists as Anti-Diabetic Agents
169
[35] A. Carpenter, C. Ammala, C. Briscoe, S. Bullard, J. Kashatus, S. Katamreddy, R. Mertz and S. Ross, Symposium, Islet and b-cell biology, 2008, Abstract #102. [36] L. S. Bertram, M. C. T. Fyfe, M. J. Procter and G. M. Williams, WO Patent Application 2007116229 A1, 2007. [37] R. M. Jones, G. Semple, Y. Xiong, Y-J Shin, A. S. Ren, I. Calderon, K. Choi, B. Fioravanti, J. Lehmann and M. A. Bruce, WO Patent Application 2005007647A1, 2005. [38] R. M. Jones, G. Semple, Y. Xiong, Y-J Shin, A. S. Ren, I. Calderon and K. Choi, WO Patent Application 2005121121A2, 2005. [39] R. M. Jones, J. Lehmann, A. Wong, D. Hirst and Y-J Shin, US Patent Application 2006155128 A1, 2006. [40] R. M. Jones, J. Lehmann, A. Wong, D. Hirst and Y-J Shin, US Patent Application 2007167473 A1, 2007. [41] R. M. Jones, G. Semple, Y. Xiong, Y-J Shin, A. S. Ren, I. Calderon, K. Choi, B. Fioravanti and C. R. Sage, WO Patent Application 2005007658 A2, 2005. [42] X. Chen, P. Cheng, E.L. Clemens, J.D. Johnson, J. Ma, A. Murphy, I. Nashashibi, C.J. Rabbat, J. Song, M.E. Wilson, Y. Zhu and Z. Zhao, WO Patent Application 2008083238 A2, 2008. [43] J. Ma, C. J. Rabbat, J. Song, X. Chen, I. Nashashibi, Z. Zhao, A. Novack, D.-F. Shi, P. Cheng, Y. Zhu and A. Murphy, WO Patent Application 2009014910 A2, 2009. [44] P. B. Alper, G. Lelais, R. Epple and D. Mutnick, WO Patent Application 2008109702 A1, 2008. [45] M. Wilson, F. Gregoire, B. Pandey, A. Chandalia, P. Zhang, O. Abdel-aleem, K. Marlen, E. Clemens, J. Johnson, X. Chen, J. Ma, I. Nashashibi, C. Rabbat, J. Song, A. Novack, D-F. Shi, S. Zhao and B. Lavan, Keystone Symposium, Type II Diabetes and Insulin Resistance, Banff, Canada, Abstract #369, January 2009. [46] S. R. Katamreddy, R. D. Caldwell, D. Heyer, V. Samano, J. B. Thompson, A. J. Carpenter, C. R. Conlee, E. E. Boros and B. D. Thompson, WO Patent Application 2008008887 A2, 2008. [47] C. Ammala and C. Briscoe, WO Patent Application 2008008895 A1, 2008. [48] C. D. Boyle and B. R. Neustadt, WO Patent Application 2008130615 A1, 2008. [49] C. D. Boyle, S. Chackalamannil, C. M. Lankin, U. Shah, B. R. Neustadt, H. Liu and A. W. Stamford, WO Patent Application 2008130584 A1, 2008. [50] J. M. Fevig and D. A. Wacker, WO Patent Application 2008137436 A1, 2008. [51] J. M. Fevig and D. A. Wacker, WO Patent Application 2008137435 A1, 2008. [52] M. C. T. Fyfe, A. Babbs, L. S. Bertram, S. E. Bradley, S. M. Doel, S. Gadher, W. T. Gattrel, J. G. Horswill, R. P. Jeevaratnam, J. F. Keily, J. G. McCormack, H. A. Overton, C. M. Rasamison, C. Reynet, P. J. Rushworth, C. P. Sambrook Smith, V. K. Shah, D. F. Stonehouse, S. A. Swain, J. R. White, P. S. Widdowson, G. M. Williams and M. J. Proctor, Abstracts of Papers, 234th ACS National Meeting, Boston, MA, United States, August 19–23, 2007, MEDI-062. [53] M. C. T. Fyfe, J. White, P. Widdowson, H. Overton and C. Reynet, Diabetes, 2007, 56(Suppl. 1), 532-P. [54] M. C. T. Fyfe, J. White, P. Widdowson, H. Overton and C. Reynet, 67th Sessions ADA Meeting, Chicago, Illinois, USA, 2007. [55] OSI Pharmaceuticals Initiates Clinical Development Program for Anti-Diabetes Candidate, PSN821, Company Press Release, September 3, 2008. [56] M. C. T. Fyfe, J. Keily and S. A. Swain, WO Patent Application 2008081204 A1, 2008. [57] L. S. Bertram, M. C. T. Fyfe, R. P. Jeevaratnam, J. Keily and S. A. Swain, WO Patent Application 2008081205 A1, 2008. [58] M. C. T. Fyfe, J. Keily, M. Proctor, D. F. Stonehouse and S. A. Swain, WO Patent Application 2008081206 A1, 2008.
170
Robert M. Jones and James N. Leonard
[59] M. C. T. Fyfe, R. P. Jeevaratnam, J. Keily and S. A. Swain, WO Patent Application 2008081207 A1, 2008. [60] M. C. T. Fyfe, R. P. Jeevaratnam, J. Keily and S. A. Swain, WO Patent Application 2008081208 A1, 2008. [61] P. Alper, M. Azimioara, C. Cow, R. Epple, S. Jiang, G. Leleais, P. Michellys, T. N. Nguyen, L. Westcott-Baker and B. Wu, WO Patent Application 2008097428 A2, 2008. [62] T. Ohishi, J. Takasaki, M. Matsumoto, T. Saito, M. Kamohara, T. Soga, S. Yoshida and Y. Ueda, WO Patent Application 2002044362 A1, 2002. [63] T. Ohishi, J. Takasaki, M. Matsumoto, T. Saito, M. Kamohara, T. Soga, S. Yoshida and Y. Ueda, EP Patent Application 1338651 A1. [64] Arena Pharmaceuticals Announces Global Diabetes Collaboration with Ortho-McNeil Pharmaceutical, December 21, 2004, http://invest.arenapharm.com/releasedetail.cfm? ReleaseID ¼ 320780 [65] Arena Pharmaceuticals Announces Selection of Two Arena-Discovered Compounds for Preclinical Development by Ortho-McNeil, December 23, 2004, http://invest. arenapharm.com/releasedetail.cfm?ReleaseID ¼ 320778 [66] Arena Pharmaceuticals, Inc. Announces Initiation of Phase 1 Clinical Trial of Arena Type 2 Diabetes Drug Candidate in Collaboration With Ortho-McNeil, February 7, 2006, http://invest.arenapharm.com/releasedetail.cfm?ReleaseID ¼ 320321 [67] Arena Pharmaceuticals Announces That Ortho-McNeil Extends Research Term Under Partnership to Develop Drugs to Treat Type 2 Diabetes, September 27, 2006, http:// invest.arenapharm.com/releasedetail.cfm?ReleaseID ¼ 320291. [68] Arena Pharmaceuticals Announces APD668 Initial Clinical Study Results Suggest Glucose-Dependent Insulinotropic Receptors May Improve Glucose Control in Patients With Type 2 Diabetes, January 7, 2008, http://invest.arenapharm.com/releasedetail. cfm?ReleaseID ¼ 320208 [69] Arena Pharmaceuticals Announces Initiation of Phase 1 Clinical Trial of Type 2 Diabetes Drug Candidate in Partnership with Ortho-McNeil-Janssen Pharmaceuticals, December 15, 2008, http://invest.arenapharm.com/releasedetail.cfm?ReleaseID ¼ 354391 [70] Metabolex initiates Phase 1 Trial of MBX-2982, March 26, 2008, http://www.metabolex. com/news/mar262008.html [71] S. Zhao, F. Gregoire, E. Clemens, D. Karpf, X. Chen, B. Lavan, J. Johnson and M. Wilson, The World Congress on Controversies to Consensus in Diabetes, Obesity and Hypertension (CODHy). October 30–November 2, 2008, Barcelona, Spain. [72] Metabolex Announces Positive Results from Phase 1a Clinical Trial of MBX-2982, November 12, 2008, http://www.metabolex.com/news/nov122008.html
CHAPT ER
8 Non-Peptide Ligands for the Gonadotropin Receptors Nicole van Straten and Marco Timmers
Contents
1. Introduction 2. Non-Peptide Small Molecule Gonadotropin Receptor Ligands 2.1 LHR agonists 2.2 LHR antagonists 2.3 FSHR agonists 2.4 FSHR antagonists 3. Therapeutic Indications and Clinical Findings 3.1 Infertility 3.2 Contraception and disorders of the reproductive tract 4. Conclusions and Future Prospects References
171 173 173 175 175 179 182 182 183 184 185
1. INTRODUCTION The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play fundamental roles in the complex process of human reproduction. LH and FSH belong to a family of glycoprotein hormones, also including choriogonadotropin (CG) and thyroid-stimulating hormone (TSH). These glycoproteins are large, non-covalently linked heterodimers consisting of a common a-subunit and a hormone-specific Schering-Plough Research Institute, Department of Medicinal Chemistry, PO Box 20, 5340BH Oss, The Netherlands Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04408-X
r 2009 Elsevier Inc. All rights reserved.
171
172
Nicole van Straten and Marco Timmers
b-subunit. The a-subunit contains 92 amino acids, whereas the b-subunit incorporates 121 (LH) and 111 (FSH) amino acids [1]. LH induces ovulation in females and controls testosterone production in males. FSH is responsible for ovarian follicle growth in females and is involved in spermatogenesis in males. LH and FSH are secreted from the anterior pituitary gland and transported to the gonads to activate the G-proteincoupled LH receptor (LHR) and FSH receptor (FSHR) respectively. LHRs are present on theca cells (women) and Leydig cells (men), whereas FSHRs can be found on granulosa cells (women) and Sertoli cells (men). Binding of LH stimulates the theca and Leydig cells to produce testosterone. Subsequently, testosterone is converted into estradiol in the female granulosa cell by the cytochrome P450 enzyme aromatase, which is generated after activation of the FSHR. CG is produced during pregnancy and is involved in maintaining early pregnancy. Interestingly, CG and LH both bind and activate the LHR with similar potency. H2N
LH
Testosterone
HOOC LH receptor GnRH (Theca cells) H2N
FSH
Aromatase
Estradiol
Follicle growth and ovulation
HOOC FSH receptor (granulosa cells)
FSH and CG are applied clinically for the treatment of infertility during assisted reproductive therapy (ART). In in vitro fertilization (IVF) protocols, multiple oocytes are generated by controlled ovarian stimulation (COS) with FSH. After downregulation of the hypothalamicpituitary-gonadal axis with a gonadotropin-releasing hormone (GnRH) receptor agonist or antagonist, FSH is used during the first half of the cycle to induce follicle growth. CG is then administered to induce the final oocyte maturation, after which the oocytes can be retrieved for fertilization. In ovulation induction protocols, FSH is used to induce
Non-Peptide Ligands for the Gonadotropin Receptors
173
monofollicular growth and CG is applied to effect oocyte maturation and ovulation. The currently marketed hormones are obtained either from urinary sources or produced using recombinant biotechnology and are administered parenterally through multiple injections. The development of orally administered non-peptide LH and FSH mimics would be a more convenient approach to infertility treatment and may improve patient compliance. Moreover, synthetically prepared low-molecular-weight (LMW) gonadotropin mimics should exhibit improved stability, consistency and homogeneity compared to their glycoprotein counterparts. Since chemical synthesis does not involve the use of human- or animalderived materials (from urinary origin or serum required for recombinant protein expression), the danger of viral contamination or transmissible spongiform encephalopathy would be eliminated. The risk for ovarian hyperstimulation syndrome that is associated with the long half-life of CG may also be reduced. Conversely, antagonists of the LHR and/or FSHR may also prove to be valuable as additions to the currently available palette of contraceptive methods. The discovery and development of non-peptide, orally available LHR or FSHR antagonists would open the way to innovative non-steroidal and gonad-specific contraceptive methods. In a review by Guo in 2005 [2], an excellent assessment of non-peptide ligands for the gonadotropin receptors was presented. In 2008, Heitman and IJzerman [3] reviewed the literature on ligands for the hypothalamicpituitary-gonadal axis with selective coverage of the patent literature related to FSHR ligands. The current review will briefly summarize the patent literature on gonadotropin ligands up to 2005 and new literature on compound classes and biological data that has appeared during the past 3–4 years.
2. NON-PEPTIDE SMALL MOLECULE GONADOTROPIN RECEPTOR LIGANDS The discovery of LMW agonists for G-protein-coupled receptors (GPCRs) that are normally activated by large proteins has met with limited success. Attempts to mimic the action of FSH with small peptide-like molecules have turned out to be unsuccessful [4]. Non-peptide antagonists for peptide-binding GPCRs are more often documented in literature. Since the late 1990s, several breakthrough starting points for both non-peptide agonists and antagonists of the gonadotropin receptors have emerged as a result of high-throughput screening. However, optimization of these initial hits into suitable drug candidates has proven to be a challenging task. Several factors are responsible for this, the most
174
Nicole van Straten and Marco Timmers
important of which is the difficulty in reducing lipophilicity while retaining potency. The relatively high lipophilicity reflects the assumed binding mode of these non-peptide ligands. Whereas the endogenous ligand is known to bind to the large extracellular domain of the receptor, which is hydrophilic in nature, LMW compounds are believed to bind to the lipophilic transmembrane domain of the receptor [5,6].
2.1 LHR agonists To date, two distinct classes of LHR agonists have been reported in the literature. This low number can partly be explained by the difficulty in finding non-peptide agonists for protein-binding GPCRs. On the contrary, LHR ligands have applications in limited market segments that are currently explored only by a relatively small number of laboratories. 1 R = OMe (Org 41841) R 2R=
H N N
NH2
O
O
N S
S
N
H N
HN
3R=
N O
O
N
(Org 43553) O
OH
N
NH N NH2
O
N O
N
N H
O
NH2
OH
N
4
5
The first compound class is characterized by a bicyclic thieno-[2,3-d]pyrimidine scaffold in 2000 [7]. Org 41841 (1, human LHR: EC50 ¼ 20 nM)
Non-Peptide Ligands for the Gonadotropin Receptors
175
was reported as the first orally active LMW agonist of the LHR. A model where immature mice were primed with FSH and then treated with Org 41841 resulted in ovulation in 40% of the animals at a dose of 50 mg/kg [8]. It was established that replacement of the pyrimidine scaffold by a pyridine scaffold was tolerated, as was substitution of the five-membered thiophene ring by a six-membered ring (4) [9]. Moreover, the thiophene ring could be replaced by a furan or pyrrole moiety. Methylation of the t-butylamide functionality was reported to yield a combined LHR/TSH receptor (TSHR) agonist [10]. Using a model of the Org 41841/TSHR complex, Org 41841 was later used as a starting point to identify TSHR antagonists in a rational design approach [11]. Lead optimization of Org 41841 yielded Org 43553 (2, human LHR: EC50 ¼ 3.7 nM), in which the –NH2 substituent of the 4-phenyl group is derivatized as a morphilinoacetamide [12]. 3H-labelled Org 43553 was used to assess the relationship between LHR binding affinity (Ki) and functional activity (EC50) of a series of related analogues, which was found to be linear [13]. Org 43553 was active in an ex vivo proof-ofprinciple ovulation induction assay and showed promising pharmacokinetic properties (rat, p.o.: Cmax ¼ 3.8 mg/L, T1/2 ¼ 4.5 h, F ¼ 79%). It is the first compound reported to induce complete ovulation in cyclic rats after single-dose oral administration. Fertilization of the Org 43553induced oocytes resulted in normal implantation of healthy embryos [14]. Based on its in vivo activity and bioavailability, Org 43553 was selected as a development candidate and progressed into phase 1 clinical studies. Interestingly, Org 43553 was reported to have some FSHR agonistic activity (human FSHR: EC50 ¼ 110 nM) in addition to its LHR agonistic activity. This phenomenon was further exemplified in so-called open chain analogues such as 3 (human LHR: EC50 ¼ 2.7 nM, human FSHR: EC50 ¼ 5.7 nM) [15]. A series of 1-phenylpyrazoles was disclosed in 2001 [16]. Solid-phase synthesis of focused libraries around the original hit and subsequent lead optimization yielded compound 5 (human LHR: EC50 ¼ 20 nM). When administered intraperitoneally, 5 induced increased testosterone levels in male rats in a dose-dependent manner [17]. As with the thieno[2,3-d] pyrimidine series, the pyrazoles also showed activity at the FSHR (human FSHR: EC50 ¼ 62 nM).
2.2 LHR antagonists A set of previously disclosed compounds, some of which are also phosphodiesterase inhibitors, were claimed as LHR antagonists for the treatment of estrogen deficiencies. Contraceptive applications were also claimed, but no pharmacological data were presented to support this [18].
176
Nicole van Straten and Marco Timmers
2.3 FSHR agonists In the field of non-peptide FSHR agonists, significantly more literature on discrete compound classes has appeared when compared to the nonpeptide LHR agonists. Piperidinecarboxamide 6, which was claimed for infertility treatment, was the first FSH mimetic described (human FSHR: EC50 ¼ 3.9 nM). Compound 6 induced estradiol production in a primary rat granulosa cell assay (EC50 ¼ 1.2 mM). No pharmacokinetic or in vivo efficacy data were revealed [19]. A series of 5-alkylated thiazolidinones displayed FSHR agonistic activity [20]. The thiazolidinone derivatives were prepared using solid-phase combinatorial chemistry methods [21]. A major drawback of structure 7 (human FSHR: EC50 ¼ 14 nM) proved to be the lack of control over the relative stereochemistry at the 2- and 5-positions of the thiazolidinone ring during synthesis. Furthermore, this compound was prone to isomerization after chiral purification, possibly caused by the activated hydrogen at position 5. To overcome the chemical instability, a methyl group was introduced at position 5 [22], furnishing compound 8 (human FSHR: EC50 ¼ 51 nM). In a second stabilization approach, the thiazolidinone scaffold was replaced by a g-lactam unit [23], exemplified by compound 9 (human FSHR: EC50 ¼ 25 nM). These compounds were shown to interact within the seven-transmembrane region of the FSHR in studies using FSH/TSH receptor chimeras [24]. O H N
N O O
R H N
N
H2N
X O
O
O
O O
N
O
O
6
7 X = S, R = H 8 X = S, R = CH3 9 X = CH2, R = CH3
R1 R2
O
N R3
N N
H N
N O
O
S
N
OO
N
S
10 R1 = R2 = R3 = OMe
12 N
11 R1 = R2 = H, R3 = O
N H
N
177
Non-Peptide Ligands for the Gonadotropin Receptors
Optimization of a micromolar hit obtained from high-throughput screening of an encoded combinatorial library of diketopiperazines yielded biaryl derivatives 10 and 11 as leads [25,26]. Trimethoxy derivative 10 and aminomethylurea 11 were reported to activate the FSHR with EC50 values of 13 and 1.2 nM respectively. The compounds were claimed for use in controlling fertility, for contraception or for treatment of hormone-dependent disorders [27].
O N O CF3 H 2N H N
N O
O
N
13
O
14
O N
S N S
N N O
N N
NH
15
N-Alkylated sulfonylpiperazines such as compound 12 were claimed as FSHR agonists (human FSHR: EC50 ¼ 13 nM), useful for the treatment of infertility [28]. In 2003, tetrahydroquinoline 13 (human FSHR: EC50 ¼ 4.4 mM) was identified in a screening campaign [29,30]. It is noteworthy to mention that hit optimization resulted in an agonist-toantagonist switch. Derivatization of the 6-amino substituent with lipophilic groups resulted in FSHR antagonists with IC50 values as low
178
Nicole van Straten and Marco Timmers
as 5 nM [30]. Depending on the nature of the 6-amino substituent and on the substituent on the phenyl ring at position 4 (see, e.g., compound 14), analogues showed partial agonistic or partial antagonistic activity with EC50 or IC50 values of less than 100 nM [31,32]. Since the various representatives in this series appear to be able to activate and/or inactivate the receptor, they were claimed for use in fertility regulation, including both infertility treatment and contraception. A series of thiazolyl-isoxazoles such as 15 (human FSHR: EC50 ¼ 1.1 mM) were claimed in a patent application for the treatment of infertility and related disorders [33]. In 2005 and 2006, a series of richly decorated hexahydroquinolines was claimed for the treatment of infertility disorders including COS and in vitro fertilization procedures. Compounds of this series such as 16 were described to have FSHR IC50s of less than 1 nM [34]. A Hantzschtype cyclocondensation reaction was used for the preparation of analogues in this series resulting in a mixture of four diastereoisomers that could be separated using a chiral auxiliary [35]. For example, compound 17 (human FSHR: EC50o1 nM) was obtained in 99.2% diastereomeric excess.
F OMe
F O S N H
O MeO
O OMe
O
Br
O
O CN
CN
N H 16
O
N H 17
In an attempt to reduce the lipophilicity of compounds within this series, pyridyl- and sulfonamide-substituted hexahydroquinolines such as 18 (human FSHR: EC50o10 nM) were prepared [36]. Substitution of a phenyl ring with a less lipophilic aliphatic sulfonamide was also tolerated (19, human FSHR: EC50o10 nM) [37].
179
Non-Peptide Ligands for the Gonadotropin Receptors
O N
HN
O S NH
O
H N
Br
Br
S O
O
O
O
O CN
CN
N H
N H
18
19
2.4 FSHR antagonists In 1992, suramine was reported to inhibit binding of FSH to its receptor [38]. Since suramin is a large heavily sulfonated compound and a notoriously promiscuous binder to a plethora of (glyco)proteins, it is not suitable for use as a non-hormonal gonad-specific contraceptive method. Still, it was the starting point for a more selective FSHR antagonistic sulfonic acid series [39,40]. Although stilbene bis-sulfonic acid 20 was only active in the micromolar range in an in vitro cAMP (cyclic AMP, adenosine 3’, 5’-cyclic monophosphate) assay (human FSHR: IC50 ¼ 1.3 mM), it turned out to be equipotent in an in vitro proof-of-principle assay using human granulosa cells (IC50 ¼ 2.7 mM) [41]. In contrast to other non-peptide ligands of the gonadotropin receptors, stilbene 20 was able to inhibit binding of 125 I-labelled human FSH to the truncated extracellular domain of the human FSHR [42], indicating that this relatively hydrophilic compound may exert its biological action by binding to the extracellular part of the receptor. SMe HO3S
O
H N
O S O O
N
N
O O
S O
N H
SO3H
O
MeS 20
It is known that within a single structural class, both FSHR agonists and FSHR antagonists can be identified, depending on the nature of their
180
Nicole van Straten and Marco Timmers
pharmacophores. This phenomenon can be seen in the thiazolidinone series [43], where compound 21 (human FSHR: IC50 o11 mM) showed antagonistic properties, as opposed to the earlier mentioned analogues 7–9 that showed agonist activity. This can also be seen in the tetrahydroquinoline series (see FSHR agonist 13 and 14). Derivative 22 was reported to be a FSHR antagonist (human FSHR: IC50 ¼ 28 nM), active in an ex vivo mouse follicle culture model, in which it significantly inhibited follicle growth, resulting in 78% inhibition of ovulation [30]. O H N
N
H2N
S
H N
O
H N
O
O
S
N O
21
22
The biphenyl FSHR pharmacophore present in the tetrahydroquinolines (22), diketopiperazines (10) and thiazolyl-isoxazoles (15) is also an essential element of aminoalkylamide FSHR antagonists exemplified by compound 23 (human FSHR: IC50 ¼ 40 nM) [44]. This compound inhibited cAMP production in primary rat granolusa cells (IC50 ¼ 20 nM), and its in vivo effects on progesterone levels in female cyclic rats (p.o. dosing, 20 mg/kg BID) and on the number of sperm cells in the testes of male rats were measured (p.o. dosing, 20 mg/kg BID for 25 days). The series was claimed for contraceptive use and reproductive disorders such as endometriosis, uterine fibroids, polycystic ovary syndrome and breast and ovarian cancers. N H N
O NH2
O N
H N
O S
N Cl N
NH
O O
23
24
N H
OH
181
Non-Peptide Ligands for the Gonadotropin Receptors
Pyrrolobenzodiazepines have also been claimed as FSHR antagonists for contraception [45–48]. Here, again, the biphenyl unit is present as an important pharmacophore element. Compound 24 was found to inhibit cAMP production (human FSHR: IC50 ¼ 70 nM), but in vitro or in vivo efficacy data were not provided. It is of interest to note that the same structural core is present in a series of oxytocin receptor antagonists earlier claimed by the same research group [49]. Acyltryptophanols such as compound 25 were claimed as FSHR antagonists for control of fertility in both men and women or for the prevention and/or treatment of osteoporosis [50]. Inhibition of cAMP production by 25 was reported to be in the submicromolar range (25, human FSHR: IC50 ¼ 100 nM).
OMe OH
25 R1 = nBu-CN, R2 = H, X =
F
R1 N NH X O
26 R1 = Et, R2 = O-iPr, X = R2
OMe
It is tempting to classify the biphenyl substituent in the acyltryptophanol series as another example of this frequently observed substructure in FSHR antagonists. However, it was shown [51] that separation of the two phenyl units by an acetylene linkage provided nanomolar active compounds, demonstrated by 26 (human FSHR: IC50 ¼ 20 nM). In the acetylene series, non-aromatic substituents were also tolerated [52]. Furthermore, it was established that the hydroxymethyl group could be replaced by a cyanomethyl group [53] and that a-substitution of the tryptophanol entity was tolerated [54]. Given the lipophilicity of the compounds, it is likely that representatives from this series bind to the seven helical transmembrane domain of the receptor. The plasticity of the transmembrane part of the receptor is illustrated by structures 27–29. Incorporation of a tetrahydroisoquinoline substituent (27, human FSHR: IC50 ¼ 6 nM) [55], a chromene core (28, human FSHR: IC50 ¼ 14 nM) [56,57] or a tetrahydrocarbazole group (29, human FSHR: IC50 ¼ 70 nM) [58,59] are well tolerated. Interestingly, the indole moiety seems to be the optimal FSHR pharmacophore [60]. No in vivo efficacy data has been reported for these acyltryptophanols.
182
Nicole van Straten and Marco Timmers
H N
F
H N
NH
NH
HO
NH
HO
O
O
O
HO O
O
NH O
F
HN O
HN O
27
28
HN
Cl O 29
3. THERAPEUTIC INDICATIONS AND CLINICAL FINDINGS 3.1 Infertility Infertility is generally defined as ‘the inability to conceive after 12 months of having regular unprotected sexual intercourse’. Women reach their maximum ability to conceive around the age of 24, while the average age of women seeking medical help to conceive is 32. Today 1 out of 6 couples of reproductive age experiences fertility problems [61]. A substantial percentage (10–15%) of infertility is unexplained, but known causes in women are of hormonal (oligo- or amennorhoea) and tubal (blocked fallopian tubes) origin and are related to age. Female infertility is often the result of a disorder of the reproductive tract such as endometriosis, polycystic ovarian syndrome or uterine fibroids. Tubal and pelvic pathology is responsible for 30% of female infertility, and ovulatory dysfunction affects 20% of the patient population. Although male infertility accounts for the remaining 35% of all infertility cases, treatment is focused on the female. This may be explained by the development of novel fertilization techniques, such as intracytosolic sperm injection, allowing in vitro fertilization by male partners with low sperm count and/or mobility. Infertility treatment does not aim at solving the cause of infertility (if known) but rather focuses on ART to become pregnant. Several forms of ART are available
Non-Peptide Ligands for the Gonadotropin Receptors
183
to help couples. In the first instance, anti-estrogens, such as clomiphene citrate, may be used to accomplish ovulation induction. A second-line option is the use of gonadotropins for ovulation induction. This can be followed by intrauterine insemination to instigate a natural, monofollicular conception. To establish multifollicular fertilization in the laboratory, gonadotropins are used for COS and subsequent in vitro fertilization, after which one or more embryos are transferred. The fertility market is growing, and the recombinant and urinary gonadotropins account for the majority of sales. It is envisaged that replacement of these agents by orally active LMW mimics will improve patient convenience and even compliance, reduce the risk of side effects, improve the ‘time-to-pregnancy’ and ultimately may lead to higher ‘takehome-baby-rates’. The current success rates for IVF treatment are about 30%, which represents a major need for improvement. On the contrary, scientific developments in human reproductive biology and medicine have the potential to positively affect pregnancy outcomes [62]. Potential additional indications for LMW LHR and FSHR agonists include male indications such as male infertility, late onset hypogonadism in elderly men, and delayed puberty and mal-descendent testes in boys. Since the majority of the LMW non-peptide agonists for the gonadotropin receptors have only been identified in the past decade, almost no clinical data are available. Moreover, for those in development, clinical proof-of-concept has yet to be reported. For LHR agonist Org 43553 (2), phase I clinical data have been published [63]. Tolerability and pharmacokinetic effects of Org 43553 were assessed in sterilized women of reproductive age following single oral administration of seven escalating doses (5–2,700 mg). Mean elimination half-life varied between 30 h and 47 h, and exposure was dose proportional up to 1,800 mg. Pharmacodynamic evaluation included progesterone measurements and ultrasound to confirm ovulation. Ovulation was observed in 83 and 75% of the women in the 300 and 900 mg groups respectively. No serious adverse events occurred and those reported were mild in severity.
3.2 Contraception and disorders of the reproductive tract In principle, all women of reproductive age are potential users of contraceptive agents. In the western world, there is high penetration of contraceptives in the target population but a declining number of women of childbearing age. Special attention is needed for the developing countries, where thousands of maternal deaths are related to unintended pregnancies every year [64]. The significant population growth in the developing world further emphasizes the importance of broadly available and effective contraceptive methods.
184
Nicole van Straten and Marco Timmers
Female contraception includes core benefits related to pregnancy prevention (the need for which is well met), menstruation disorders and menstruation management. Current contraception methods rely on the prevention of follicle maturation, ovulation and implantation by the action of estrogenic and/or progestagenic steroids [65]. The active pharmaceutical ingredients can be delivered orally, as in the contraceptive pill, or through alternative routes. Most recent advances are developments of new delivery systems such as intrauterine devices [66], vaginal ring [67] or implantable polymeric rod [68,69] containing known steroidal compounds. Several non-steroidal progestins have been reported in the literature [70], and one non-steroidal progestin has thus far progressed into clinical development [71]. The only effective contraceptive in the marketplace still relies on the administration of steroidal drugs. The impact of genomics and proteomics for the discovery of new, validated non-steroidal targets for female contraception is still in the discovery stage [72]. A second drawback is the fact that currently available contraception methods are solely available for women. A safe, 100% effective male contraceptive is regarded as desirable [73]; yet, no male contraceptive pill is on the market. As more and more knowledge of the genes involved in male fertility is generated, new opportunities for male contraception may arise. The currently available palette of male fertility-associated genes may contain good target candidates for discovery of male contraceptives [74]. Thus far, the most obvious and well-validated target in this respect is the FSHR, since mutations may lead to subinfertility or complete infertility in men [75]. Vaccination against FSH-b chain leads to infertility in male primates [76], supporting the concept that the FSHR is a validated target for male contraception. In this light, the discovery of orally available non-peptide antagonists for the gonadotropin receptors may be a novel alternative to contraceptive steroids. Such a discovery would open the way for a new, non-steroidal, gonad-specific approach for contraception in both women and men. Furthermore, numerous disorders of the female reproductive tract are estrogen-related. Since the action of FSH at its receptor influences the levels of estrogens, it is suggested that FSHR antagonists may become valuable drugs to treat, for example, endometriosis, uterine fibroids, polycystic ovarian syndrome and even hormone-dependent breast cancer and ovarian cancer. These assumptions remain speculative since none of the published gonadotropin receptor antagonists has progressed into clinical development.
4. CONCLUSIONS AND FUTURE PROSPECTS The pioneering work in the late 1990s to find non-peptide ligands for protein-binding gonadotropin receptors using high-throughput
Non-Peptide Ligands for the Gonadotropin Receptors
185
screening technologies has induced a paradigm shift in the science of reproduction. Small heterocyclic molecules were identified that completely mimic the action of the endogenous glycoproteins but effect cellular signaling through allosteric receptor-binding sites. At the same time, turning these interesting starting points into bioavailable drug candidates presents a challenging task that requires years of intensive optimization. The past 4–5 years have yielded several new interesting series of LMW ligands for both the LHR and the FSHR that show promise as drug candidates. For LHR agonists, clinical results with Org 43553 indicate that it is possible to mimic the action of the endogenous hormone with an orally available small molecule drug candidate. Although ovulation induction can be regarded as proof of principle, it remains to be established whether this will indeed result in the ultimate proof of concept, for example, induction of pregnancy and live birth. FSHR agonists and antagonists are still early in the clinical development process. Whereas for the LHR agonists in vivo activity has been confirmed in both animal models and humans, translation of the available in vitro and ex vivo results for the FSHR ligands to more relevant animal and clinical models is still a subject of discovery research. In summary, strategies employing screening of large chemical libraries and subsequent hit and lead optimization have resulted in the identification of new chemical entities that mimic or modulate the activity of endogenous gonadotropins. The availability of orally active drugs for the gonadotropin receptors will certainly be of great added value to the current portfolio of parenteral infertility drugs and hormonal, non-gonad-specific contraceptives. The coming decade will reveal whether these scientific promises can be realized.
REFERENCES [1] [2] [3] [4] [5]
[6] [7] [8]
[9]
J. G. Pierce and T. F. Parsons, Ann. Rev. Biochem., 1981, 50, 465. T. Guo, Expert Opin. Ther. Patents, 2005, 15, 1555. L. H. Heitman and A. P. IJzerman, Med. Res. Reviews, 2008, 28, 975. M. Hage-van Noort, W. C. Puijk and R. H. Meloen, WO Patent Application 1992/02542, 1992. C. J. van Koppen, G. J. R. Zaman, C. M. Timmers, J. Kelder, S. Mosselman, R. v. d. Lagemaat, M. J. Smit and R. G. J. M. Hanssen, Naunyn Schmiedeberg’s Arch. Pharmacol., 2008, 378, 503. B. J. Arey, Endocrine, 2008, 34, 1. G. G. Gerritsma, N. C. R. van Straten and A. E. P. Adang, WO Patent Application 2000/ 61586, 2000. N. C. R. van Straten, G. G. Schoonus-Gerritsma, R. G. van Someren, J. Draaijer, A. E. P. Adang, C. M. Timmers, R. G. J. M. Hanssen and C. A. A. van Boeckel, ChemBioChem, 2002, 210, 1023. C. M. Timmers and W. F. J. Karstens, WO Patent Application 2002/24703, 2002.
186
Nicole van Straten and Marco Timmers
[10] M. C. Gershengorn, S. Neumann, C. Thomas, H. Jaeschke, S. Moore, G. Krause, B. Raaka and R. Paschke, WO Patent Application 2007/136776, 2007. [11] S. Neumann, G. Kleinau, S. Costanzi, S. Moore, J. K. Jiang, B. M. Raaka, C. J. Thomas, G. Krause and M. C. Gershengorn, Endocrinology, 2008, 149, 5945. [12] R. G. J. M. Hanssen and C. M. Timmers, WO Patent Application 2003/020726, 2003. [13] L. H. Heitman, J. Oosterom, K. M. Bonger, C. M. Timmers, P. H. G. Wiegerinck and A. P. IJzerman, Mol. Pharm., 2008, 73, 518. [14] R. v. d. Lagemaat, C. M. Timmers, J. Kelder, C. van Koppen, S. Mosselman and R. G. J. M. Hanssen, Hum. Reprod., 2009, 24, 640. [15] R. G. J. M. Hanssen, C. M. Timmers and J. Kelder, WO Patent Application 2003/020727, 2003. [16] H. Shroff, A. P. Reddy, N. El Tayer, N. Brugger and C. Jorand-Lebrun, WO Patent Application 2001/87287, 2001. [17] C. Jorand-Lebrun, B. Brondyk, J. Ling, S. Magar, R. Murray, A. Reddy, H. Shroff, G. Wands, W. Weiser, Q. Xu, S. McKenna and N. Brugger, Bioorg. Med. Chem. Lett., 2007, 17, 2080. [18] C. Grøndahl, WO Patent Application 1999/20223, 1999. [19] N. El tayer, A. Reddy, D. Buckler and S. Magar, WO Patent Application 2000/08015, 2000. [20] R. A. Scheuerman, S. D. Yanofsky, C. P. Holmes, D. Maclean, B. Ruhland, R. W. Barrett, J. E. Wrobel, W. Kao, A. Gopalsamy and F. Sum, WO Patent Application 2002/09706, 2002. [21] D. MacLean, F. Holden, A. M. Davis, R. A. Scheuerman, S. Yanofsky, C. P. Holmes, W. L. Fitch, K. Tsutsui, R. W. Barrett and M. A. Gallop, J. Comb. Chem., 2004, 6, 196. [22] J. Wrobel, J. Jetter, W. Kao, J. Rogers, L. Di, J. Chi, M. C. Pere´z, G.-C. Chen and E. S. Shen, Bioorg. Med. Chem., 2006, 14, 5729. [23] J. C. Pelletier, J. Rogers, J. Wrobel, M. C. Pere´z and E. S. Chen, Bioorg. Med. Chem., 2005, 13, 5986. [24] S. D. Yanofsky, E. S. Shen, F. Holden, E. Whitehorn, B. Aguilar, E. Tate, C. P. Holmes, R. Scheuerman, D. MacLean, M. M. Wu, D. E. Frail, F. J. Lo´pez, R. Winneker, B. J. Arey and R. W. Barrett, J. Biol. Chem., 2006, 19, 13226. [25] T. Guo, A. E. P. Adang, R. E. Dolle, G. Dong, D. Fitzpatrick, P. Geng, K.-K. Ho, S. G. Kultgen, R. Liu, E. McDonald, B. F. McGuinness, K. W. Saionz, K. J. Valenzano, N. C. R. van Straten, D. Xie and M. L. Webb, Bioorg. Med. Chem. Lett., 2004, 14, 1713. [26] T. Guo, A. E. P. Adang, G. Dong, D. Fitzpatrick, P. Geng, K.-K. Ho, C. H. Jilibian, S. G. Kultgen, R. Liu, E. McDonald, K. W. Saionz, K. J. Valenzano, N. C. R. van Straten and M. L. Webb, Bioorg. Med. Chem. Lett., 2004, 14, 1717. [27] T. Guo, K.-K. Ho, E. McDonald, R. E. Dolle, K.W. Saionz, S. G. Kultgen, R. Liu, G. Dong, P. Geng, A. E. P. Adang and N. C. R. van Straten, WO Patent Application 2002/070493, 2002. [28] S. Magar, A. Goutopoulos, M. Schwarz and T. J. Russell, WO Patent Application 2004/ 031182, 2004. [29] N. C. R. van Straten, R. G. van Someren and J. Schulz, WO Patent Application 2003/ 004028, 2003. [30] N. C. R. van Straten, T. H. J. van Berkel, D. R. Demont, W. F. J. Karstens, R. Merkx, J. Oosterom, J. Schulz, R. G. van Someren, C. M. Timmers and P. M. van Zandvoort, J. Med. Chem., 2005, 48, 1697. [31] C. M. Timmers and W. F. J. Karstens, WO Patent Application 2004/056779, 2004. [32] C. M. Timmers and W. F. J. Karstens, WO Patent Application 2004/056780, 2004. [33] V. J. Santora, J. A. Covel, R. Hayashi and R. R. Webb, WO Patent Application 2005/ 087765, 2005.
Non-Peptide Ligands for the Gonadotropin Receptors
187
[34] N. C. R. van Straten, G. G. Gerritsma and L. A. van der Veen, WO Patent Application 2006/117023, 2006. [35] P. M. Grima Poveda, W. F. J. Karstens and C. M. Timmers, WO Patent Application 2006/ 117368, 2006. [36] C. M. Timmers, W. F. J. Karstens and P. M. Grima Poveda, WO Patent Application 2006/ 117370, 2006. [37] W. F. J. Karstens and C. M. Timmers, WO Patent Application 2006/117371, 2006. [38] R. L. Daugherty, A. T. K. Cockett, S. R. Schoen and P. M. Sluss, J. Urol., 1992, 147, 727. [39] J. E. Wrobel, J. F. Rogers and W. Kao, WO Patent Application 2000/58276, 2000. [40] J. E. Wrobel, J. F. Rogers, D. M. Green, W. Kao and J. W. Winfield, WO Patent Application 2000/58277, 2000. [41] J. Wrobel, D. Green, J. jetter, W. Kao, J. Rogers, M. C. Pe´rez, J. Hardenburg, D. C. Deecher, F. J. Lo´pez, B. J. Arey and E. S. Shen, Bioorg. Med. Chem., 2002, 10, 639. [42] B. J. Arey, D. C. Deecher, E. S. Shen, P. E. Stevis, E. H. Meade, Jr., J. Wrobel, D. E. Frail and F. J. Lo´pez, Endocrinology, 2002, 143, 3822. [43] R. A. Scheuerman, S. D. Yanofsky, C. P. Holmes, D. Maclean, B. Ruhland, R. W. Barrett, J. E. Wrobel and A. Gopalsamy, WO Patent Application 2002/09705, 2002. [44] S. J. Coats, D. J. Hlasta, C. L. Carolina, M. J. Macielag, R. Rivero, L. J. Fitzpatrick and K. Pan, WO Patent Application 2001/47875, 2001. [45] A. Failli, G. D. Heffernan, A. A. Santilli, D. A. Quagliato, R. D. Coghlan, P. M. Andrae, S. C. Croce, E. S. Shen and E. J. Trybulski, WO Patent Application 2006/135687, 2006. [46] A. A. Failli, D. Quagliato, P. Andrae, G. D. Heffernan, R. D. Coghlan and E. S. Shen, US Patent Application 2006/0199806, 2006. [47] A. A. Failli, A. A. Santilli, D. A. Quagliato and E. S. Shen, US Patent Application 2006/ 0258644, 2006. [48] A. A. Failli, D. A. Quagliato, G. Heffernan, R. D. Coghlan and E. S. Shen, US Patent Application 2006/0258645, 2006. [49] A. A. Failli, J. S. Shumsky, T. J. Caggiano, J. P. Sabatucci, K. A. Memoli and E. J. Trybulski, WO Patent Application 2002/083683, 2002. [50] L. Wortmann, A. Cleve, H. P. Muhn, G. Langer, A. Schrey, R. Ku¨hne, B. Menzenbach, M. Koppitz and D. Kosemund, WO Patent Application 2007/017289, 2007. [51] L. Wortmann, H. P. Muhn, B. Menzenbach, A. Schrey, R. Ku¨hne, D. Kosemund and M. Koppitz, EP Patent Application 1932831A1, 2008. [52] L. Wortmann, M. Koppitz, H.-P. Muhn, T. Frenzel, F. P. Liesener, A. K. Schrey and R. Ku¨hne, WO Patent Application 2009/013333, 2009. [53] L. Wortmann, M. Koppitz, H.-P. Muhn, T. Frenzel, F. P. Liesener, A. K. Schrey and R. Ku¨hne, WO Patent Application 2009/016253, 2009. [54] L. Wortmann, M. Koppitz, H.-P. Muhn, A. K. Schrey and R. Kuehne, WO Patent Application 2009/021980, 2009. [55] L. Wortmann, H. P. Muhn, B. Menzenbach, A. Schrey, R. Ku¨hne, D. Kosemund and M. Koppitz, WO Patent Application 2008/071453, 2008. [56] L. Wortmann, B. Menzenbach, M. Koppitz, D. Kosemund, H. P. Muhn, A. Schrey, R. Ku¨hne, T. Frenzel and F. P. Liesener, WO Patent Application 2008/071455, 2008. [57] L. Wortmann, B. Menzenbach, M. Koppitz, D. Kosemund, H. P. Muhn, A. Schrey, R. Ku¨hne, T. Frenzel and F. P. Liesener, EP Patent Application 1956016A1, 2008. [58] L. Wortmann, M. Koppitz, H. P. Muhn, T. Frenzel, F. P. Liesener, A. Schrey and R. Ku¨hne, WO Patent Application 2008/0116671, 2008. [59] L. Wortmann, M. Koppitz, H. P. Muhn, T. Frenzel, F. P. Liesener, A. Schrey and R. Ku¨hne, EP Patent Application 1975159A1, 2008. [60] L. Wortmann, M. Koppitz, B. Menzenbach, D. Kosemund, N. Schmees, H.-P. Muhn, T. Frenzel, F. P. Liesener, A. K. Schrey and R. Ku¨hne, WO Patent Application 2009/ 013354, 2009.
188 [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76]
Nicole van Straten and Marco Timmers
Datamonitor, Commercial and pipeline insight: Infertility, October, 2007. M. M. Matzuk and D. J. Lamb, Nat. Med., 2008, 14, 1197. B. Mannaerts, in Proc. 4th World Congress on Ovulation (ed. M. Filicori), p. 157. Global Health Council, 2008, http://www.globalhealth.org/view_top.php3?id ¼ 225. M. A. Economidis and D. R. Mishell, Jr., Exp. Opin. Invest. Drugs, 2005, 14, 449. ESHRE Capri Workshop Group., Hum. Reprod. Update, 2008, 14, 197. F. J. M. E. Roumen, D. Apter, T. M. T. Mulders and T. O. M. Dieben, Human Reprod, 2001, 16, 469. H. B. Croxatto, Contraception, 2002, 65, 15. O. Graesslin and T. Korver, Eur. J. Contracept. Reprod. Health Care, 2008, 13(S1), 4. P. H. H. Hermkens, S. Kamp, S. Lusher and G. H. Veeneman, IDrugs, 2006, 9, 488. J. Bapst, G. Orczyk and J. Ermer, Clin. Pharm. Ther., 2005, 77(Suppl. S(44)). R. J. Aitken, M. A. Baker, G. F. Doncel, M. M. Matzuk, C. K. Mauck and M. J. K. Harper, J. Clin. Invest., 2008, 118, 1330. S. T. Page, J. K. Amory and W. J. Bremmer, Endocrine Rev, 2008, 29, 465. M. M. Matzuk and D. J. Lamb, Nat. Med, 2002, 8, S1–S49. I. Huhtaniemi, J. Reprod. Fert., 2000, 119, 173. N. R. Moughdal, M. Jeyakumar, H. N. Krishnamurthy, S. Sridhar, H. Krishnamurthy and F. Martin, Hum. Reprod. Update, 1997, 3, 335.
CHAPT ER
9 Recent Advances in Coagulation Serine Protease Inhibitors Joanne M. Smallheer and Mimi L. Quan
Contents
1. Introduction 2. Factor Xa Inhibitors 2.1 Compounds in late-stage clinical trials 2.2 Compounds in early clinical trials 2.3 Preclinical compounds 3. Thrombin Inhibitors 3.1 Compounds in clinical trials 3.2 Preclinical compounds 3.3 Dual thrombin/factor Xa inhibitors 4. Factor VIIa/TF Inhibitors 5. Factor IXa and XIa Inhibitors 6. Conclusions References
189 190 190 192 193 195 195 196 198 199 201 203 203
1. INTRODUCTION As orally bioavailable thrombin and factor Xa inhibitors advance through late-stage clinical trials and begin to gain regulatory approvals, a new era in treatment options for thromboembolic disorders may be on the horizon [1–3]. It is thought that these new highly selective inhibitors may eliminate food and drug–drug interactions and obviate the need Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, NJ 08543 Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04409-1
r 2009 Elsevier Inc. All rights reserved.
189
190
Joanne M. Smallheer and Mimi L. Quan
for therapeutic monitoring and dose titration that go hand-in-hand with current oral anticoagulant therapy with warfarin [4]. In addition to thrombin and factor Xa inhibitors, research continues to identify potential new therapeutic agents that target other key serine proteases in the coagulation cascade. This review will summarize recent results for several compounds in clinical trials and survey new small-molecule inhibitors reported in the literature over the past 2–3 years.
2. FACTOR Xa INHIBITORS Factor Xa has been a major focus of pharmaceutical research directed at novel antithrombotics during the past decade because of its central and unique position in the coagulation cascade. Factor Xa is a serine protease located at the junction of the intrinsic and extrinsic pathways, and its inhibition is considered to be an ideal approach to achieve strong efficacy and an improved therapeutic index compared to current therapies. Significant progress has been made in the discovery and development of orally bioavailable small-molecule factor Xa inhibitors. A few inhibitors have been advanced to phase III clinical studies, and the first factor Xa inhibitor was recently approved in Europe and Canada for the prophylaxis of venous thromboembolism (VTE) in patients after elective hip and knee replacement. Several comprehensive reviews have been published on Factor Xa inhibitors [5–7].
2.1 Compounds in late-stage clinical trials Rivaroxaban (BAY 59-7939, 1) is an oxazolidinone derivative with a Xa Ki of 0.4 nM and W10,000-fold selectivity against other related serine proteases [8]. The oral bioavailability of rivaroxaban was found to be 57–66% in rats and 60–86% in dogs with a relatively short half-life (2.3 and 0.9 h, respectively) in both species [9]. In rabbit models of thrombosis, rivaroxaban was effective in prevention and treatment of venous thrombosis [10]. In clinical studies, rivaroxaban was rapidly absorbed and displayed an oral bioavailability of 80% and a half-life of 5–9 h [11]. In a series of phase IIb studies, a range of rivaroxaban doses were found to have efficacy similar to that of standard treatment [12]. The efficacy and safety data from four phase III studies, including two randomized doubleblind trials in patients undergoing total knee arthroplasty, and two trials in patients after hip arthroplasty, have been published [13–16]. Rivaroxaban has been approved in the EU and Canada for the above indications. Phase III trials in chronic indications are ongoing, including treatment of VTE, stroke prevention in patients with atrial fibrillation (AF), and secondary prevention of acute coronary syndrome (ACS).
191
Recent Advances in Coagulation Serine Protease Inhibitors O Cl
S
HN
N O
O
N N
N
N
O
N
O HN
O NH HN
O
O
1
CONMe2
Cl
H2NOC
N O
O
S N NMe
OMe
2
3
Apixaban (BMS-562247, 2) is a second-generation pyrazole-based Xa inhibitor derived from an earlier clinical candidate, razaxaban [17]. The core structures differ in that the pyrazole ring of razaxaban is cyclized onto the amide nitrogen to form the apixaban bicyclic core [18]. Apixaban is a potent and selective Xa inhibitor with a Xa Ki of 0.08 nM and W30,000-fold selectivity over other relevant proteases. It has an excellent pharmacokinetic (PK) profile in dogs with low clearance (0.02 L/kg/h) and a low volume of distribution (0.2 L/kg). Apixaban is effective in rabbit models of thrombosis alone or in combination with aspirin, or aspirin and clopidogrel, without excessive increases in bleeding time [19,20]. Initial studies in humans suggest that apixaban is safe and well tolerated [21] and is rapidly absorbed, with a mean elimination half-life of 8–15 h and an oral bioavailability of 66% [22]. In phase II studies in patients with total knee replacement or for treatment of proximal deep vein thrombosis (DVT), apixaban had efficacy and safety similar to that of standard therapy [23,24]. Both indications are being evaluated in phase III studies, and data from one phase III trial in patients undergoing knee replacement surgery has been presented [25]. Phase II studies have also been completed in patients with recent ACS [26], and for prevention of thromboembolic events in patients with advanced cancer. Additional phase III trials are ongoing for treatment of VTE, stroke prevention in AF, and secondary prevention of ACS. The structure of edoxaban (DU-176b, 3) contains an N,N-dimethylcyclohexane carboxamide scaffold and has a chloropyridine moiety at the P1 position. Edoxaban is a potent and selective Xa inhibitor with a Ki of 0.56 nM and W10,000-fold selectivity over other relevant serine proteases [27]. Edoxaban was shown to produce a dose-dependent inhibition of thrombus formation in rat and rabbit models of thrombosis and did not significantly prolong bleeding time at an antithrombotic dose. Preliminary results from a phase II study show that edoxaban was superior to dalteparin in patients with total hip replacement in terms of incidence of VTE, with little bleeding, and could be dosed once daily [28]. In patients with total knee replacement, edoxaban produced a significant, dosedependent reduction in VTE [29]. A phase II trial of edoxaban in patients with AF showed a similar safety profile with once daily dosing compared
192
Joanne M. Smallheer and Mimi L. Quan
to standard treatment with warfarin [30]. A phase III trial in AF patients was initiated in early 2009.
2.2 Compounds in early clinical trials Eribaxaban (PD 0348292, 4) has a Xa IC50 of 0.32 nM with W1,000-fold selectivity over relevant serine proteases [31]. The oral bioavailability and half-life were 82% and 2.0 h in rats, and 41% and 4.9 h in dogs. Clearance in rats and dogs was 12 and 2.6 mL/kg/min, respectively. Eribaxaban showed a dose-dependent reduction in thrombus weight with an EC50 of 40 ng/mL in the rabbit arteriovenous (A-V) shunt model. A dose-ranging study to determine a dose of eribaxaban equivalent to enoxaparin 30 mg b.i.d. for prevention of VTE in patients undergoing total knee replacement has been completed [32]. Betrixaban (PRT54021, 5) has a Xa Ki of 0.12 nM with W86,000-fold selectivity against related serine proteases [33]. Oral bioavailability and half-life, respectively, were 24% and 8.8 h in rats, 52% and 21.2 h in dogs, and 59% and 9.6 h in monkeys. The antithrombotic efficacy of betrixaban was studied in rabbit, rat, and baboon thrombosis models to determine the target concentration required for clinical trials [34]. Early clinical studies showed that betrixaban was well tolerated across a wide range of doses. A phase II open-label VTE prevention trial showed that betrixaban was effective in preventing VTE after total knee replacement surgery [35]. A phase II trial for prevention of thromboembolic complications in patients with AF has been initiated. LY517717 (6, Xa KiB6 nM; 1,000-fold selectivity over related serine proteases) was non-inferior to enoxaparin in a phase II trial for prevention of VTE after hip or knee replacement surgery and showed similar rates of bleeding [36,37]. YM150 (Xa Ki ¼ 31 nM; low micromolar affinity for other relevant serine proteases), whose structure was not disclosed, is also in multiple phase II trials [38]. In a dose escalation study for VTE in patients undergoing hip replacement surgery, YM150 was effective, safe, and well-tolerated [39]. TAK-442 is another orally bioavailable Xa inhibitor currently in phase II clinical trials, but neither the structure nor any trial results have been disclosed.
MeO
H N N
HN
O O
MeO
F N
N H HN
NH
O N
N
4
5 Cl
Cl
N
O
O
H N
N
O
N N H
O
6
193
Recent Advances in Coagulation Serine Protease Inhibitors
2.3 Preclinical compounds Several series of pyrazole-based inhibitors have been reported. Compound 7 was derived from razaxaban [17] and was designed to prevent the potential formation of a primary aniline by replacing the amide linker with a ketone moiety [40]. Compound 7 has a Xa Ki of 0.11 nM with EC2x values of 5.2 and 5.1 mM in the prothrombin clotting time (PT) and activated partial thromboplastin time (aPTT) assays, respectively. It was found to have lower clearance and higher oral bioavailability in dogs than razaxaban. Compound 8 has a bicyclic core similar to that of apixaban with the P4 group replaced with an a-substituted phenylcyclopropyl moiety [41]. This analog has a Xa Ki of 0.035 nM and PT EC2x of 1.3 mM. Replacement of the fused piperidinone ring of apixaban with fluorophenyl afforded compounds with a 7-fluoroindazole core exemplified by 9, which has a Xa Ki of 4.4 nM and a twofold aPTT EC50 of 4.4 mM [42]. F
H2NOC N
O N
N
O
F3C N
F
H2NOC N N
N
NMe2 O
N
NMe2 N
F NH2
OMe
OMe
7
O N
8
9
Additional Xa inhibitors related to betrixaban possessing the anthranilamide scaffold and a chloropyridine P1 moiety have been reported. This is exemplified by anthranilamide 10, which also employed the P4 piperidinone moiety of apixaban [43]. Compound 10 has a Xa Ki of 0.057 nM and displayed efficacy comparable to that of apixaban in a rabbit A-V shunt thrombosis model. In compound 11 (Xa Ki ¼ 0.005 nM), the P4 phenylpiperidinone of 10 was replaced with a chlorothiophene substituted by a (methylamino(imidazol-1-yl)methyl) moiety [44]. Although this resulted in about a 10-fold improvement in Ki, the PT EC2x values of the two compounds were similar (1.5 mM for 10 and 1.2 mM for 11). Compound 11 has low clearance (4.4 mL/kg/min) and high oral bioavailability (98%) in dogs. The estimated EC50 of 11 in an intravenous (iv) rat vena cava stasis model was 0.36 mg/kg.
N H HN
O
OMe O
Cl
OMe O
Cl
N
S
N H
O HN
O
NHMe Cl
N
N 10 Cl
Cl
11
N
N
194
Joanne M. Smallheer and Mimi L. Quan
Several series of Xa inhibitors have been reported that contain a pyrrolidine core structure and a 5-chlorothiophene P1 moiety similar to that of rivaroxaban. For example, compound 12 has a Xa Ki of 3.0 nM and PT EC2x of 1.7 mM [45]. Compound 13, where a longer linker to the P1 thiophene residue was employed, is comparable in affinity (Xa IC50 ¼ 5.5 nM) [46]. Factor Xa affinity was restored in compound 14 (Kio1 nM) by incorporation of a pyrrolidinone scaffold and the basic biaryl P4 moiety present in razaxaban [47]. This analog showed clearance of 13 mL/kg/min and good oral bioavailability (52%) in rats.
H N Cl
S
H N
N
F O
O O
O
N
S
N
OMe
O
O S N H
N
Cl
12
N
13
Cl S H S N O O
N N
O
O
CH2OH S
O
NMe2
15
N
16
O N
N Me
14
Cl
N O
Cl
N
O
O O S N
F
O O S N
N
F
N
O O S
N
N
N
N N
Cl
O
17
Xa inhibitors containing piperidine and piperazine cores have also been reported. Compound 15 has a chloronaphthyl P1 group appended through a sulfonamide linkage to a 3-aminopiperidinone core to provide a potent inhibitor of Xa (Ki ¼ 0.02 nM) with PT EC2x of 1.7 mM [48]. Substitution on the nitrogen of the sulfonamide improved Xa affinity. The 6-chloronaphthylsulfonyl P1 residue of 15 in combination with a fused spirocyclic core afforded 16 that displayed a Xa IC50 of 1.2 nM [49]. Compound 16 was effective in a rat venous thrombosis model, but was
195
Recent Advances in Coagulation Serine Protease Inhibitors
less efficacious than warfarin. Another series of Xa inhibitors containing a chloronaphthylsulfonyl P1 moiety is exemplified by 17 that has a Xa IC50 of 4.8 nM with PT EC2x value of 1.0 mM and was reported to be orally bioavailable in rats and monkeys [50]. Several edoxaban analogs have been reported as Xa inhibitors. Replacement of the 5-chloropyridine P1 moiety of edoxaban with a 5-chloroindole gave 18 (Xa IC50 ¼ 2.3 nM) resulting in 10-fold loss of Xa affinity [51]. As illustrated by 19 (Xa IC50 ¼ 8.4 nM), it was possible to remove a chiral center and retain most of the Xa affinity by replacement of the cyclohexyl ring with a piperidine [52]. Cyclopentyl compound 20 includes a 4-chlorothiophene at the P1 position and a pyridone as the P4 moiety and has a Xa Ki of 0.43 nM and PT EC2x of 1.7 mM [53]. CONMe2 X O
Cl
N H
N NH H O
O
Cl S N
N
NH
S O
N H
O N 20
18, X = CH (S) 19, X = N
3. THROMBIN INHIBITORS 3.1 Compounds in clinical trials Direct inhibitors of thrombin have been the target of anticoagulant research for many years, based on the key role played by thrombin in the formation of fibrin clots [54,55]. Ximelagatran, 21, was the first orally bioavailable, active site-specific serine protease inhibitor to be approved for human use. A double prodrug, 21 is rapidly converted after oral absorption to melagatran, 22, a potent and selective direct thrombin inhibitor [56]. The data from clinical trials have been extensively reviewed [57,58]. Ximelagatran was approved in Europe in 2003 for the prevention of DVT, but failed to receive approval in the United States due to concerns relating to potential liver toxicity. This toxicity ultimately led to the withdrawal of the drug from all markets and discontinuation of ongoing clinical trials in February 2006 [59,60]. A structurally related compound, 23 (AZD0837), which is also a benzamidine prodrug, is currently in phase II clinical trials [55]. At a 150 mg b.i.d. dose in AF patients, the safety and tolerability of 23 was shown to be comparable to that of warfarin [61]. A phase III trial with an extended release
196
Joanne M. Smallheer and Mimi L. Quan
formulation for once daily dosing in patients with AF is projected to start later this year [62]. Dabigatran etexilate, 24, is an orally bioavailable double prodrug, which is rapidly metabolized by non-specific esterases in the blood to dabigatran, 25, a potent, reversible, and highly selective direct thrombin inhibitor [63]. Several recent reviews of the clinical findings with dabigatran etexilate are available [64–67]. The half-life in humans was 7–9 h for a single dose and 14–17 h after t.i.d. dosing [68]. Oral bioavailability of dabigatran after administration of a single dose of the prodrug was B6% [69]. The results of three phase III clinical trials for the prevention of VTE after knee or hip surgery have been published [70–72]. Dabigatran etexilate has been approved in Europe and Canada for the prevention of VTE in patients undergoing total hip or total knee replacement surgeries [63]. A phase II dose-finding study in patients with ACS is ongoing, as are phase III studies for prevention of stroke in patients with AF and in treatment of VTE.
O O
O R1 NH2
O
HN
OMe
NH2
O
OH
N
N H
N
N
N H
N
R2 O
OCHF2 23
Cl
21 R1 = OH, R2 = Et 22 R1 = R2 = H
O O R1 N H2N
H N
OR2 N
N N Me
N
24 R1 = CO2-n-hex, R2 = Et 25 R1 = R2 = H
3.2 Preclinical compounds Much of the recent research to identify orally active direct thrombin inhibitors has focused on the replacement of the highly basic amidinecontaining P1 groups with less basic aminoheterocycles, as has been successfully achieved with the Xa inhibitors described earlier. P1
197
Recent Advances in Coagulation Serine Protease Inhibitors
imidazole and 2-aminothiazole groups were introduced as amidine replacements into a 3-aminopyridone series of thrombin inhibitors [73]. Single-digit nanomolar thrombin affinity was maintained with 26 (Ki ¼ 8 nM) and 27 (Ki ¼ 10 nM), but these compounds were not orally bioavailable. The introduction of the aminomethylimidazole P1 group into a 3-aminopyrazinone core series resulted in 28 which, although less potent (Ki ¼ 53 nM), was the first compound with this P1 moiety to show improved PK parameters [73]. When 28 was administered to dogs at an oral dose of 4 mg/kg, a 2-h half-life and Cmax of 1.4 mM were observed. Further optimization of the methylimidazole P1 group by introduction of an acetamide substituent on the imidazole nitrogen provided 29, with a thrombin Ki of 1.2 nM and aPTT EC2x of 0.7 mM [74]. Compound 29 was efficacious in the rat ferric chloride efficacy assay at an iv dose of 10 mg/kg/min, and some exposure was achieved after a 5 mg/kg oral dose to both dogs (Cmax ¼ 1.46 mM; t½ ¼ 1.6 h) and rhesus monkeys (Cmax ¼ 0.36 mM; t½ ¼ 1.1 h). Thrombin inhibitors with a 2-(2-chloro-6-fluorophenyl)acetamide central core exemplified by 30 (Ki ¼ 0.7 nM) and 31 (Ki ¼ 47 nM) have been reported [75]. A crystal structure of 31 bound to thrombin shows the aromatic F substituent in proximity to the backbone NH of Gly 216, suggesting that this F forms a F–H bond that contributes to the affinity of this compound. Further optimization of compounds retaining the oxyguanidine P1 moiety of 31 provided 2-cyano-6-fluoroacetamide analog 32, which had a thrombin Ki of 1.2 nM and aPTT EC2x of 0.36 mM and good oral bioavailability in dogs, but not in rats (49% and 2%, respectively) [76]. Various substitutions on the P3 pyridine moiety were made without loss of thrombin affinity; however, methyl and chloro substitution resulted in increased plasma protein binding as compared to 32. Efficacy was observed with this compound in a dog A-V shunt model when administered at a dose of 3 mg/kg p.o. O O S N H
O N
R
N H
O
H N N
N H
N O
26 R =
N H
S N
O N H
N
O
N N
28
H2N
27 R =
N
HN
N
O
H N
N
N H
O
29
N H
198
Joanne M. Smallheer and Mimi L. Quan
O
Cl
N H H2N
Cl N H
F
N
N
NH H2N
F F
O N H
O
R
N H
30
F
N H F F
X
31 R = Cl, X = CH 32 R = CN, X = N
3.3 Dual thrombin/factor Xa inhibitors It has been proposed by several groups that a dual-acting Xa/thrombin inhibitor might provide a better overall profile than either mechanism alone by the prevention of thrombin formation through the inhibition of Xa and simultaneous direct inhibition of thrombin activity [77–79]. A dual thrombin/Xa inhibitor, tanogitran (BIBT 986, 33), which has Xa and thrombin Ki values of 26 nM and 2.7 nM, respectively, was evaluated in a phase II clinical trial by iv administration in a human model of endotoxin-induced coagulation [80]. Tanogitran prolonged plasma aPTT and reduced in vivo thrombin generation in a dose-dependent manner and was safe and well tolerated. OH O NH
O N
N H2N NH
O
N
S
S
H N
O N
O
O
Cl
HN
N O
33
34
Vinyl sulfonamide 34 is a dual inhibitor of thrombin and Xa that has equal affinity for both enzymes (Ki ¼ 2 nM), whereas the analog lacking the vinyl methyl group is a selective Xa inhibitor. Compound 34 displayed aPTT and PT EC1.5x of 32 mM and 0.54 mM, respectively, and oral bioavailability in both rats and dogs [81]. Modification of a series of selective Xa inhibitors by the introduction of a proline residue in place of a glycine moiety at the P2 position provided compounds such as 35 which were potent dual inhibitors of Xa (Ki ¼ 1.5 nM) and thrombin (Ki ¼ 3.5 nM) [82]. The ratio of Xa to thrombin affinity could be reversed by the modification of the P3 pyridyl to a 4-hydroxyphenyl to provide a compound with Xa Ki of 8.3 nM and thrombin Ki of 3.2 nM. Compound 35 displayed good in vitro anticoagulant activity and moderate Caco-2 permeability (Papp ¼ 50 nm/s). Bioavailability in rats after an oral dose of 5 mg/kg was o5%.
Recent Advances in Coagulation Serine Protease Inhibitors
N
Cl
H2N
O N HN Cl
O
O
199
N N
N H
CO2H
N
N
HN
HN
N 35
36
In a series of oxazolopyridine-based thrombin inhibitors, replacement of the P3 pyridine moiety with a piperidine provided dual thrombin/Xa inhibitors exemplified by 36 with good in vitro and in vivo anticoagulant efficacy [83]. Racemic compound 36 has thrombin and Xa Ki values of 0.04 and 3.9 nM, respectively, and aPTT EC2x of 0.07 mM. In an iv PK study in dogs, 36 had a 4.2 h half-life, high clearance, and moderate volume of distribution. The compound completely blocked occlusion in a rat FeCl3 arterial thrombosis model but was not orally bioavailable.
4. FACTOR VIIa/TF INHIBITORS The high-affinity complex formed between the serine protease factor VIIa and the cell-associated glycoprotein tissue factor (VIIa/TF) has long been considered an ideal point of intervention by virtue of its unique role in the initiation of the coagulation cascade in response to an injury at the vessel wall [84]. Despite much research to identify orally bioavailable inhibitors of VIIa/TF, no orally bioavailable small-molecule inhibitors have progressed into clinical trials to date. A phase I clinical trial with PCI-27483, a parenteral VIIa inhibitor of undisclosed structure, was completed in late 2008. This compound will be further evaluated in a phase Ib/II trial in pancreatic cancer patients [85]. The fluorinated phenylglycine derivative 37 was reported to be an inhibitor of VIIa (Ki ¼ 81 nM), displayed good selectivity over thrombin, Xa, and trypsin, and had a PT EC2x of 2.0 mM [86]. In PK studies in rats, the compound had a half-life of B6 h with low clearance and volume of distribution. Oral administration of a 3 mg/kg dose of the ethyl ester, amidoxime double prodrug of 37 to rats resulted in 20% bioavailability. In a guinea pig model of arterial thrombosis, oral administration of the prodrug resulted in dose-dependent plasma concentrations of 37. Antithrombotic efficacy was achieved in this model with minimal effect on bleeding time, in contrast to a dual VIIa/Xa inhibitor, where bleeding time was significantly increased at higher doses.
200
Joanne M. Smallheer and Mimi L. Quan
Further optimization of previously reported amidinobenzimidazole VIIa inhibitors provided potent inhibitor 38 (VIIa Ki ¼ 4 nM), wherein the pendant fluorophenol moiety imparted good selectivity over Xa, and incorporation of the succinate side chain led to improvement in PK parameters in rats (clearance ¼ 0.3 mL/min/kg; mean residence time ¼ 9 h) [87,88]. Urea analog 39 was also a potent and highly selective VIIa inhibitor (Ki ¼ 2 nM) with much-improved ex vivo coagulation efficacy (PT EC2x ¼ 1.9 mM) [89]. Other acid, amide, and amine functionalities were also well tolerated in place of the urea at this position. With the additional interaction afforded by the urea substituent, it was found that the succinic acid side chain could be replaced by methyl, fluoro, or hydrogen without loss of VIIa affinity, anticoagulant efficacy, or selectivity versus other proteases [90]. HO2C
CO2H
HO
HO
OEt HO2C H2N
F
HN
NH
O HN
37
OH
N
NH
O
H2N
OH
R
NH C5H11
NH
O
HN
38 R = F 39 R = CH2NHCONH2
NH2
40
Additional modification of this scaffold led to indole 40 (VIIa Ki ¼ 13 nM), which has a hexanamide functionality in place of the urea of 39 [90]. Some loss of selectivity, particularly versus Xa, was observed on replacement of the urea with amide substituents, but a desirable improvement in PK parameters after iv dosing to rats was obtained with 40 (mean residence time ¼ 89 min). To assess the potential to achieve oral bioavailability, 40 was converted to its hydroxyamidine prodrug. After oral administration to rats at a dose of 10 mg/kg, the prodrug was orally absorbed (F ¼ 11%), but the parent compound 40 was not detected in vivo. A related analog 41 was reported wherein the 5-amidinobenzimidazole and indole P1 groups of 39 and 40 were successfully replaced with the less basic 5-amino-4-azaindole, maintaining good VIIa affinity (Ki ¼ 20 nM) [91]. The extended P3 urea substitution conferred B15-fold improvement in affinity compared to a nitro substituent at the same position of the biphenyl core. Introduction of 2,6-difluoro substitution on the urea phenyl substituent of 5-azaindole 42 (VIIa Ki ¼ 33 nM) provided a twofold improvement in affinity compared to its unsubstituted phenylurea counterpart [92]. This moiety was also effective in
201
Recent Advances in Coagulation Serine Protease Inhibitors
combination with a 4-amino-5-azaindole moiety as the benzamidine mimic, as exemplified by 43 (Ki ¼ 2.6 nM, PT EC2x ¼ 2.4 mM), which had improved VIIa affinity and ex vivo anticoagulant activity [93]. All three of these latter compounds have good selectivity for VIIa over other relevant serine proteases, but no PK data was reported. R2
CO2H
R1 O
N NH OH
H2N
N H
O F
N
NH HO
N H
N H
N H
F 42 = =H 43 R1 = NH2; R2 = C(CH3)2CO2H R1
41
R2
5. FACTOR IXa AND XIa INHIBITORS Both factor IXa and factor XIa are primarily associated with the intrinsic or propagation phase of coagulation and offer an alternative strategy for therapeutic intervention [94,95]. TTP889 is an orally bioavailable, selective, small-molecule partial inhibitor of factor IXa whose structure has not been disclosed. Antithrombotic efficacy was demonstrated with TTP889 in rat and porcine AV shunt models with no effect on bleeding times [96]. In phase I clinical trials, TTP889 was safe and well tolerated with a half-life suitable for once daily dosing [97]. Phase II clinical trial results for extended prevention of VTE in patients undergoing hip surgery were disappointing. At a 300-mg dose administered daily over 3 weeks following 1 week of standard treatment with low-molecularweight heparin, TTP889 was ineffective in reducing biomarkers for thrombin generation or the incidence of VTE compared to placebo [98,99]. A second phase II trial in patients with vascular assist devices is planned.
NH H2N
N H HO
44
O
NH
N H2N
N N H
45
B
O
N NH HO
H2N
H N NH
O O
46
N
202
Joanne M. Smallheer and Mimi L. Quan
HN
NH2 HN
NH
N H
NH
R
O
H N
N H
O
Br
NH2
O
S N H
N O
OH H N
Cl
N H
O 47 R = 4-hydroxyphenyl 48 R = 3-pyridyl
S
O
N O
49
Biarylbenzimidazole 44 has a IXa Ki of 99 nM and was identified through screening of small-molecule libraries [100]. This compound showed good selectivity with respect to thrombin, but lacked selectivity over Xa and VIIa and had poor efficacy in ex vivo clotting assays. Replacement of the central phenol with a 4-hydroxypyrazole moiety provided 45 (IXa Ki ¼ 50 nM) with B2-fold improved IXa affinity and also imparted improved selectivity over thrombin, VIIa, and Xa. Compound 45 also had improved potency in the aPTT assay, with an EC2x of 2.64 mM compared to W10 mM for 44. Several small-molecule inhibitors of factor XIa have also been reported. Racemic boronate 46 has an XIa IC50 of 1.4 mM with 30- and 8-fold selectivity over Xa and thrombin, respectively [101]. A series of potent keto-arginine-based peptidomimetics, exemplified by 47, are irreversible inhibitors of XIa and form a covalent bond to the catalytic serine of the enzyme [102]. Compound 47 has a XIa IC50 of 6 nM and aPTT EC2x of 2.4 mM and shows good selectivity over VIIa, Xa, and thrombin. In an iv PK study in rats, 47 had high clearance (32 mL/kg/ min), a short half-life (45 min), and a small volume of distribution (B236 mL/kg). In an iv rat vena cava model of venous thrombosis, 47 showed efficacy comparable to that of heparin. Pyridyl analog 48 (XIa IC50 ¼ 12 nM) was evaluated in a rat mesenteric bleeding model, and at fourfold the efficacious dose (1 mg/kg, continuous iv infusion) did not alter bleeding time. Modifications to reduce the peptidic character and molecular weight of these inhibitors led to 49 with XIa IC50 of 0.116 mM [103]. Br NH H2N
O
CO2H N
N H
N O 50
N
NH2 N H
HN
O
O NH
N H
OH Br HO2C
O 51
Recent Advances in Coagulation Serine Protease Inhibitors
203
The in vitro and in vivo antithrombotic profile of BMS-262084, 50, a potent irreversible inhibitor of XIa with IC50 of 2.8 nM and aPTT EC2x of 2.2 mM was reported [104]. Efficacy in FeCl2 models of arterial and venous thrombosis was observed in rats dosed iv with 50 (12 mg/ kg+12 mg/kg/h), whereas cuticle, mesenteric, or renal bleeding times were unchanged compared to vehicle controls. Clavatadine A, 51, a natural product isolated from a marine sponge was also reported to be an irreversible inhibitor of XIa with an IC50 of 1.3 mM [105].
6. CONCLUSIONS Potent and selective inhibitors of factor Xa and thrombin are well established. Several of these are undergoing advanced clinical trials, and rivaroxaban and dabigatran recently gained regulatory approval. In addition, prototype, selective inhibitors of factors VIIa, IXa, and XIa have been identified. From the perspective of the medicinal chemist, the biggest challenge inherent to these serine proteases as drug targets lies in the design of compounds that have good oral bioavailability and are suitable for b.i.d. or q.d. dosing. In the final analysis, it is likely that differentiation between these serine protease targets may ultimately require human clinical trials in multiple thromboembolic disorders.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
S. Haas, J. Thromb. Thrombolysis, 2008, 25, 52. B. I. Eriksson, D. J. Quinlan and J. I. Weitz, Clin. Pharmacokinet., 2009, 48, 1. M. Hammwo¨hner and A. Goette, J. Cardiovasc. Pharmacol., 2008, 52, 18. A. C. Spyropoulos, Thromb. Res., 2008, 123, S29. J. Harenberg, Therapy, 2008, 5, 177. J. P. Piccini, M. R. Patel, K. W. Mahaffey, K. A. A. Fox and R. M. Califf, Expert Opin. Investig. Drugs, 2008, 17, 925. J. Carreiro and J. Ansell, Expert Opin. Investig. Drugs, 2008, 17, 1937. E. Perzborn, J. Strassburger, A. Wilmen, J. Pohlmann, S. Roehrig, K. H. Schlemmer and A. Straub, J. Thromb. Haemost., 2005, 3, 514. C. Weinz, U. Buetehorn, H. P. Daehler, C. Kohlsdorfer, U. Pleiss, S. Sandmann, K. H. Schlemmer, T. Schwarz and W. Steinke, Xenobiotica, 2005, 35, 891. B. J. Biemond, E. Perzborn, P. W. Friederich, M. Levi, U. Buetehorn and H. R. Bu¨ller, Thromb. Haemost., 2007, 97, 471. D. Kubitza, M. Becka, B. Voith, M. Zuehlsdorf and G. Wensing, Clin. Pharmacol. Ther., 2005, 78, 412. H. R. Buller, A. W. Lensing, M. H. Prins, G. Agnelli, A. Cohen, A. S. Gallus, F. Misselwitz, G. Raskob, S. Schellong and A. Segers, Blood, 2008, 112, 2242. M. R. Lassen, W. Ageno, L. C. Borris, J. R. Lieberman, N. Rosencher, T. J. Bandel, F. Misselwitz and A. G. G. Turpie, N. Engl. J. Med., 2008, 358, 2776.
204
Joanne M. Smallheer and Mimi L. Quan
[14] A. G. G. Turpie, K. A. Bauer, B. Davidson, M. Gent, L. Kwong, M. R. Lassen, F. Cushner, P. Lotke, S. D. Berkowitz, T. J. Bandel, F. Misselwitz and W. Fisher, Blood (ASH Annual Meeting Abstracts), 2008, 112, Abstract 35. [15] B. I. Eriksson, L. C. Borris, R. J. Friedman, S. Haas, M. V. Huisman, A. K. Kakkar, T. J. Bandel, H. Beckmann, E. Muehlhofer, F. Misselwitz, W. Geerts and M. Levine, N. Engl. J. Med., 2008, 358, 2765. [16] A. K. Kakkar, B. Brenner, O. E. Dahl, B. I. Eriksson, P. Mouret, J. Muntz, A. G. Soglian, ´ . F. Pap, F. Misselwitz and S. Haas, Lancet, 2008, 372, 31. A [17] M. L. Quan, P. Y. S. Lam, Q. Han, D. J. P. Pinto, M. Y. He, R. Li, C. D. Ellis, C. G. Clark, C. A. Teleha, J. H. Sun, R. S. Alexander, S. Bai, J. M. Luettgen, R. M. Knabb, P. C. Wong and R. R. Wexler, J. Med. Chem., 2005, 48, 1729. [18] D. J. P. Pinto, M. J. Orwat, S. Koch, K. A. Rossi, R. S. Alexander, A. Smallwood, P. C. Wong, A. R. Rendina, J. M. Luettgen, R. M. Knabb, K. He, B. Xin, R. R. Wexler and P. Y. S. Lam, J. Med. Chem., 2007, 50, 5339. [19] P. C. Wong, C. A. Watson and E. J. Crain, J. Thromb. Haemost., 2008, 6, 1736. [20] P. C. Wong, E. J. Crain, B. Xin, R. R. Wexler, P. Y. S. Lam, D. J. Pinto, J. M. Luettgen and R. M. Knabb, J. Thromb. Haemost., 2008, 6, 820. [21] C. Frost, Z. Yu, S. Nepal, R. Mosqueda-Garcia and A. Shenker, J. Thromb. Haemost., 2007, 5(Suppl. 2), P-M-665. [22] C. Frost, Z. Yu, S. Nepal, A. Bragat, K. Moore, A. Shenker, Y. Barrett and F. Lacreta, J. Clin. Pharmacol., 2008, 48, 1099, Abstract 142. [23] H. Buller, D. Deitchman, M. Prins and A. Segers, J. Thromb. Haemost., 2008, 6, 1313. [24] M. R. Lassen, B. L. Davidson, A. Gallus, G. Pineo, J. Ansell and D. Deitchman, J. Thromb. Haemost., 2007, 5, 2368. [25] M. R. Lassen, A. S. Gallus, G. F. Pineo and G. E. Raskob, Blood (ASH Annual Meeting Abstracts), 2008, 112, Abstract 31. [26] J. Alexander, Annual Congress European Society of Cardiology, Munich, Germany, August 30 to September 3, 2008, Abstract 3208. [27] T. Furugohri, K. Isobe, Y. Honda, C. Kamisato-Matsumoto, N. Sugiyama, T. Nagahara, Y. Morishima and T. Shibano, J. Thromb. Haemost., 2008, 6, 1542. [28] G. Raskob, Annual Congress European Cardiology Society, Munich, Germany, August 30 to September 3, 2008, Abstract 3712. [29] T. Fuji, S. Fujita, S. Tachibana and Y. Kawai, Blood (ASH Annual Meeting Abstracts), 2008, 112, Abstract 34. [30] J. I. Weitz, S. J. Connolly, S. Kunitada, J. Jin and I. Patel, Blood (ASH Annual Meeting Abstracts), 2008, 112, Abstract 33. [31] J. T. Kohrt, C. F. Bigge, J. W. Bryant, A. Casimiro-Garcia, L. Chi, W. L. Cody, T. Dahring, D. A. Dudley, K. J. Filipski, S. Haarer, R. Heemstra, N. Janiczek, L. Narasimhan, T. McClanahan, J. T. Peterson, V. Sahasrabudhe, R. Schaum, C. A. Van Huis, K. M. Welch, E. Zhang, R. J. Leadley and J. J. Edmunds, Chem. Biol. Drug Des., 2007, 70, 100. [32] A. T. Cohen, D. Armstrong, T. Gazdzik, C. Ryge, R. Pak, J. Mandema, R. Boyd, S. McBride and L. A. DiCarlo, Blood (ASH Annual Meeting Abstracts), 2008, 112, Abstract 980. [33] P. Zhang, H. Wenrong, L. Wang, L. Bao, Z. J. Jia, S. M. Bauer, E. A. Goldman, G. D. Probst, Y. Song, T. Su, J. Fan, Y. Wu, W. Li, J. Woolfrey, U. Sinha, P. W. Wong, S. T. Edwards, A. E. Arfsten, L. A. Clizbe, J. Kanter, A. Pandey, G. Park, A. Hutchaleelaha, J. L. Lambing, S. J. Hollenbach, R. M. Scarborough and B. Y. Zhu, Bioorg. Med. Chem. Lett., 2009, 19, 2179. [34] K. Abe, G. Siu, S. Edwards, P. H. Lin, B. Y. Zhu, U. Marzec, S. Hanson, Y. Pak, S. Hollenbach and U. Sinha, Blood (ASH Annual Meeting Abstracts), 2006, 108, Abstract 901.
Recent Advances in Coagulation Serine Protease Inhibitors
205
[35] A. G. G. Turpie, K. A. Bauer, B. L. Davidson, W. D. Fisher, M. Gent, M. H. Huo, U. Sinha and D. D. Gretler, Thromb. Haemost., 2009, 101, 68. [36] G. Agnelli, S. Haas, J. S. Ginsberg, K. A. Krueger, A. Dmitrienko and J. T. Brandt, J. Thromb. Haemost., 2007, 5, 746. [37] F. Kuo, D. K. Clodfelter, T. R. Priest and D. L. K. Kau, J. Label. Compd. Radiopharm., 2004, 47, 599. [38] Y. Iwatsuki, T. Shigenaga, Y. Moritani, M. Suzuki, T. Ishihara, F. Hirayama and T. Kawasaki, Blood (ASH Annual Meeting Abstracts), 2006, 108, Abstract 911. [39] B. I. Eriksson, A. G. G. Turpie, M. R. Lassen, M. H. Prins, G. Agnelli, P. Ka¨lebo, M. L. Gaillard and L. Meems, J. Thromb. Haemost., 2007, 5, 1660. [40] J. G. Varnes, D. A. Wacker, D. J. P. Pinto, M. J. Orwat, J. P. Theroff, B. Wells, R. A. Galemo, J. M. Luettgen, R. M. Knabb, S. Bai, K. He, P. Y. S. Lam and R. R. Wexler, Bioorg. Med. Chem. Lett., 2008, 18, 749. [41] J. X. Qiao, D. L. Cheney, R. S. Alexander, A. M. Smallwood, S. R. King, K. He, A. R. Rendina, J. M. Luettgen, R. M. Knabb, R. R. Wexler and P. Y. S. Lam, Bioorg. Med. Chem. Lett., 2008, 18, 4118. [42] Y.-K. Lee, D. J. Parks, T. Lu, T. V. Thieu, T. Markotan, W. Pan, D. F. McComsey, K. L. Milkiewicz, C. S. Crysler, N. Ninan, M. C. Abad, E. C. Giardino, B. E. Maryanoff, B. P. Damiano and M. R. Player, J. Med. Chem., 2008, 51, 282. [43] J. R. Corte, T. Fang, D. J. P. Pinto, W. Han, Z. Hu, X.-J. Jiang, Y.-L. Li, J. F. Gauuan, M. Hadden, D. Orton, A. R. Rendina, J. M. Luettgen, P. C. Wong, K. He, P. E. Morin, C.-H. Chang, D. L. Cheney, R. M. Knabb, R. R. Wexler and P. Y. S. Lam, Bioorg. Med. Chem. Lett., 2008, 18, 2845. [44] B. Ye, D. O. Arnaiz, Y.-L. Chou, B. D. Griedel, R. Karanjawala, W. Lee, M. M. Morrissey, K. L. Sacchi, S. T. Sakata, K. J. Shaw, S. C. Wu, Z. Zhao, M. Adler, S. Cheeseman, W. P. Dole, J. Ewing, R. Fitch, D. Lentz, A. Liang, D. Light, J. Morser, J. Post, G. Rumennik, B. Subramanyam, M. E. Sullivan, R. Vergona, J. Walters, Y.-X. Wang, K. A. White, M. Whitlow and M. Kochanny, J. Med. Chem., 2007, 50, 2967. [45] K. G. Zbinden, L. Anselm, D. W. Banner, J. Benz, F. Blasco, G. De´coret, J. Himber, B. Kuhn, N. Panday, F. Ricklin, P. Risch, D. Schlatter, M. Stahl, S. Thomi, R. Unger and W. Haap, Eur. J. Med. Chem., 2009, 44, 2787. [46] Y. Shi, D. Sitkoff, J. Zhang, W. Han, Z. Hu, P. D. Stein, Y. Wang, L. J. Kennedy, S. P. O’Connor, S. Ahmad, E. C. K. Liu, S. M. Seiler, P. Y. S. Lam, J. A. Robl, J. E. Macor, K. S. Atwal and R. Zahler, Bioorg. Med. Chem. Lett., 2007, 17, 5952. [47] R. J. Young, A. D. Borthwick, D. Brown, C. L. Burns-Kurtis, M. Campbell, C. Chan, M. Charbaut, M. A. Convery, H. Diallo, E. Hortense, W. R. Irving, H. A. Kelly, N. P. King, S. Kleanthous, A. M. Mason, A. J. Pateman, A. N. Patikis, I. L. Pinto, D. R. Pollard, S. Senger, G. P. Shah, J. R. Toomey, N. S. Watson, H. E. Weston and P. Zhou, Bioorg. Med. Chem. Lett., 2008, 18, 28. [48] J. M. Smallheer, S. Wang, M. L. Laws, S. Nakajima, Z. Hu, W. Han, I. Jacobson, J. M. Luettgen, K. A. Rossi, A. R. Rendina, R. M. Knabb, R. R. Wexler, P. Y. S. Lam and M. L. Quan, Bioorg. Med. Chem. Lett., 2008, 18, 2428. [49] F. Saitoh, H. Nishida, T. Mukaihira, N. Kosuga, M. Ohkouchi, T. Matsusue, I. Shiromizu, Y. Hosaka, M. Matsumoto and I. Yamamoto, Chem. Pharm. Bull., 2007, 55, 317. [50] Y. Imaeda, T. Kuroita, H. Sakamoto, T. Kawamoto, M. Tobisu, N. Konishi, K. Hiroe, M. Kawamura, T. Tanaka and K. Kubo, J. Med. Chem., 2008, 51, 3422. [51] T. Nagata, T. Yoshino, N. Haginoya, K. Yoshikawa, M. Nagamochi, S. Kobayashi, S. Komoriya, A. Yokomizo, R. Muto, M. Yamaguchi, K. Osanai, M. Suzuki and H. Kanno, Bioorg. Med. Chem., 2009, 17, 1193. [52] A. Mochizuki, Y. Nakamoto, H. Naito, K. Uoto and T. Ohta, Bioorg. Med. Chem. Lett., 2008, 18, 782.
206
Joanne M. Smallheer and Mimi L. Quan
[53] J. X. Qiao, C.-H. Chang, D. L. Cheney, P. E. Morin, G. Z. Wang, S. R. King, T. C. Wang, A. R. Rendina, J. M. Luettgen, R. M. Knabb, R. R. Wexler and P. Y. S. Lam, Bioorg. Med. Chem. Lett., 2007, 17, 4419. [54] H. Bounameaux, Seminars Thromb. Hemostasis., 2008, 34(Suppl. 1), 12. [55] A. Schwienhorst, Cell. Mol. Life Sci., 2006, 63, 2773. [56] D. Gustafsson and M. Elg, Thromb. Res., 2003, 109, S9. [57] A. Choudhury, D. Goyal and G. Y. H. Lip, Drugs Today, 2006, 42, 3. [58] S.-J. Ho and T. A. Brighton, Vasc. Health Risk Manag., 2006, 2, 49. [59] AstraZeneca PLC, Press Release, February 14, 2006, http://www.astrazeneca.com/ media/latest-press-releases/2006/5217?itemId ¼ 3891692 (last accessed 25/02/09). [60] G. Agnelli, B. I. Eriksson, A. T. Cohen, D. Bergqvist, O. E. Dahl, M. R. Lassen, P. Mouret, N. Rosencher, M. Andersson, A. Bylock, E. Jensen and B. Boberg, Thromb. Res., 2009, 123, 488. [61] B. Olsson, L. H. Rasmussen, A. Tveit, E. Jensen, P. Wessman, S. Panfilov, H. D. Ekdal and K. Wa˚hlander, J. Thromb. Haemost., 2007, 5(Suppl. 2), O-W-053. [62] AstraZeneca PLC, Press Release, January 29, 2009, http://www.astrazeneca.com/ _mshost3690701/content/resources/media/investors/AZN-Q4-2008/q4-results2008-narrative.pdf (last accessed 25/02/09). [63] B. I. Eriksson, H. Smith, U. Yasothan and P. Kirkpatrick, Nat. Rev. Drug Discov., 2008, 7, 557. [64] B. E. Baetz and S. A. Spinler, Pharmacotherapy, 2008, 28, 1354. [65] M. Ieko, Curr. Opin. Investig. Drugs, 2007, 8, 758. [66] H. Nishio, M. Ieko and T. Nakabayashi, Expert Opin. Pharmacother., 2008, 9, 2509. [67] M. Sanford and G. L. Plosker, Drugs, 2008, 68, 1699. [68] J. Stangier, K. Rathgen, H. Sta¨hle, D. Gansser and W. Roth, Br. J. Clin. Pharmacol., 2007, 64, 292. [69] S. Blech, T. Ebner, E. Ludwig-Schwellinger, J. Stangier and W. Roth, Drug Metab. Dispos., 2008, 36, 386. [70] B. I. Eriksson, O. E. Dahl, N. Rosencher, A. A. Kurth, C. N. van Dijk, S. P. Frostick, P. Ka¨lebo, A. V. Christiansen, S. Hantel, R. Hettiarachchi, J. Schnee and H. R. Bu¨ller, J. Thromb. Haemost., 2007, 5, 2178. [71] B. I. Eriksson, O. E. Dahl, N. Rosencher, A. A. Kurth, C. N. van Dijk, S. P. Frostick, M. H. Prins, R. Hettiarachchi, S. Hantel, J. Schnee and H. R. Bu¨ller, Lancet, 2007, 370, 949. [72] J. S. Ginsberg, B. L. Davidson, P. C. Comp, C. W. Francis, R. J. Friedman, M. H. Huo, J. R. Lieberman, J. E. Muntz, G. E. Raskob, M. L. Clements, S. Hantel, J. M. Schnee and J. A. Caprini, J. Arthroplasty, 2009, 24, 1. [73] R. C. A. Isaacs, M. G. Solinsky, K. J. Cutrona, C. L. Newton, A. M. Naylor-Olsen, J. A. Krueger, S. D. Lewis and B. J. Lucas, Bioorg. Med. Chem. Lett., 2006, 16, 338. [74] R. C. A. Isaacs, M. G. Solinsky, K. J. Cutrona, C. L. Newton, A. M. Naylor-Olsen, D. R. McMasters, J. A. Krueger, S. D. Lewis, B. J. Lucas, L. C. Kuo, Y. Yan, J. J. Lynch and E. A. Lyle, Bioorg. Med. Chem. Lett., 2008, 18, 2062. [75] L. Lee, K. D. Kreutter, W. Pan, C. Crysler, J. Spurlino, M. R. Player, B. Tomczuk and T. Lu, Bioorg. Med. Chem. Lett., 2007, 17, 6266. [76] K. D. Kreutter, T. Lu, L. Lee, E. C. Giardino, S. Patel, H. Huang, G. Xu, M. Fitzgerald, B. J. Haertlein, V. Mohan, C. Crysler, S. Eisennagel, M. Dasgupta, M. McMillan, J. C. Spurlino, N. D. Huebert, B. E. Maryanoff, B. E. Tomczuk, B. P. Damiano and M. R. Player, Bioorg. Med. Chem. Lett., 2008, 18, 2865. [77] E. U. Graefe-Mody, U. Schu¨hly, K. Rathgen, H. Sta¨hle, J. M. Leitner and B. Jilma, J. Thromb. Haemost., 2006, 4, 1502. [78] A. Kranjc and D. Kikelj, Curr. Med. Chem., 2004, 11, 2535.
Recent Advances in Coagulation Serine Protease Inhibitors
207
[79] H. Nar, M. Bauer, A. Schmid, J.-M. Stassen, W. Wienen, H. W. M. Priepke, I. K. Kauffmann, U. J. Ries and N. H. Hauel, Structure, 2001, 9, 29. [80] J. M. Leitner, B. Jilma, F. B. Mayr, F. Cardona, A. O. Spiel, C. Firbas, K. Rathgen, H. Sta¨hle, U. Schu¨hly and E. U. Graefe-Mody, Clin. Pharm.Ther., 2007, 81, 858. [81] R. J. Young, D. Brown, C. L. Burns-Kurtis, C. Chan, M. A. Convery, J. A. Hubbard, H. A. Kelly, A. J. Pateman, A. Patikis, S. Senger, G. P. Shah, J. R. Toomey, N. S. Watson and P. Zhou, Bioorg. Med. Chem. Lett., 2007, 17, 2927. [82] D. Do¨nnecke, A. Schweinitz, A. Stu¨rzebecher, P. Steinmetzer, M. Schuster, U. Stu¨rzebecher, S. Nicklisch, J. Stu¨rzebecher and T. Steinmetzer, Bioorg. Med. Chem. Lett., 2007, 17, 3322. [83] J. Z. Deng, D. R. McMasters, P. M. A. Rabbat, P. D. Williams, C. A. Coburn, Y. Yan, L. C. Kuo, S. D. Lewis, B. J. Lucas, J. A. Krueger, B. Strulovici, J. P. Vacca, T. A. Lyle and C. S. Burgey, Bioorg. Med. Chem. Lett., 2005, 15, 4411. [84] R. A. Shirk and G. P. Vlasuk, Arterioscler. Thromb. Vasc. Biol., 2007, 27, 1895. [85] Pharmacyclics, Company Website, 2009, http://www.pharmacyclics.com/wt/page/ pci_27483 (last accessed 05/03/09). [86] K. G. Zbinden, D. W. Banner, K. Hilpert, J. Himber, T. Lave´, M. A. Riederer, M. Stahl, T. B. Tschopp and U. Obst-Sander, Bioorg. Med. Chem., 2006, 14, 5357. [87] A. Kolesnikov, R. Rai, W. B. Young, J. Mordenti, L. Liu, S. Torkelson, W. D. Shrader, E. M. Leahy, H. Hu, E. Gjerstad, J. Janc, B. A. Katz and P. A. Sprengeler, Bioorg. Med. Chem. Lett., 2006, 16, 2243. [88] W. D. Shrader, A. Kolesnikov, J. Burgess-Henry, R. Rai, J. Hendrix, H. Hu, S. Torkelson, T. Ton, W. B. Young, B. A. Katz, C. Yu, J. Tang, R. Cabuslay, E. Sanford, J. W. Janc and P. A. Sprengeler, Bioorg. Med. Chem. Lett., 2006, 16, 1596. [89] W. B. Young, J. Mordenti, S. Torkelson, W. D. Shrader, A. Kolesnikov, R. Rai, L. Liu, H. Hu, E. M. Leahy, M. J. Green, P. A. Sprengeler, B. A. Katz, C. Yu, J. W. Janc, K. C. Elrod, U. M. Marzec and S. R. Hanson, Bioorg. Med. Chem. Lett., 2006, 16, 2037. [90] D. Vijaykumar, R. Rai, M. Shaghafi, T. Ton, S. Torkelson, E. M. Leahy, J. R. Riggs, H. Hu, P. A. Sprengeler, W. D. Shrader, C. O’Bryan, R. Cabuslay, E. Sanford, E. Gjerstadt, L. Liu, J. Sukbuntherng and W. B. Young, Bioorg. Med. Chem. Lett., 2006, 16, 3829. [91] R. Rai, A. Kolesnikov, P. A. Sprengeler, S. Torkelson, T. Ton, B. A. Katz, C. Yu, J. Hendrix, W. D. Shrader, R. Stephens, R. Cabuslay, E. Sanford and W. B. Young, Bioorg. Med. Chem. Lett., 2006, 16, 2270. [92] J. R. Riggs, H. Hu, A. Kolesnikov, E. M. Leahy, K. E. Wesson, W. D. Shrader, D. Vijaykumar, T. A. Wahl, Z. Tong, P. A. Sprengeler, M. J. Green, C. Yu, B. A. Katz, E. Sanford, M. Nguyen, R. Cabuslay and W. B. Young, Bioorg. Med. Chem. Lett., 2006, 16, 3197. [93] H. Hu, A. Kolesnikov, J. R. Riggs, K. E. Wesson, R. Stephens, E. M. Leahy, W. D. Shrader, P. A. Sprengeler, M. J. Green, E. Sanford, M. Nguyen, E. Gjerstad, R. Cabuslay and W. B. Young, Bioorg. Med. Chem. Lett., 2006, 16, 4567. [94] E. L. Howard, K. C. D. Becker, C. P. Rusconi and R. C. Becker, Arterioscler. Thromb. Vasc. Biol., 2007, 27, 722. [95] D. Gailani and T. Renne´, J. Thromb. Haemost., 2007, 5, 1106. [96] R. Rothlein, J. M. Shen, N. Naser, D. R. Gohimukkula, T. B. Caligan, R. C. Andrews, A. M. Schmidt, E. A. Rose and A. M. M. Mjalli, Blood (ASH Annual Meeting Abstracts), 2005, 106, Abstract 1886. [97] TransTech Pharma, Press Release, 2004, http://www.ttpharma.com/press_releases/ first_phase_i.html (last accessed 28/02/09). [98] B. I. Eriksson, O. E. Dahl, M. R. Lassen, D. P. Ward, R. Rothlein, G. Davis and A. G. G. Turpie, J. Thromb. Haemost., 2008, 6, 457.
208
Joanne M. Smallheer and Mimi L. Quan
[99] O. E. Dahl, B. I. Eriksson, M. R. Lassen, D. P. Ward, R. Rothlein, G. Davis and A. G. G. Turpie, J. Thromb. Haemost., 2007, 5(Suppl. 2), P-T-065. [100] D. Vijaykumar, P. A. Sprengeler, M. Shaghafi, J. R. Spencer, B. A. Katz, C. Yu, R. Rai, W. B. Young, B. Schultz and J. Janc, Bioorg. Med. Chem. Lett., 2006, 16, 2796. [101] T. I. Lazarova, L. Jin, M. Rynkiewicz, J. C. Gorga, F. Bibbins, H. V. Meyers, R. Babine and J. Strickler, Bioorg. Med. Chem. Lett., 2006, 16, 5022. [102] J. Lin, H. Deng, L. Jin, P. Pandey, J. Quinn, S. Cantin, M. J. Rynkiewicz, J. C. Gorga, F. Bibbins, C. A. Celatka, P. Nagafuji, T. D. Bannister, H. V. Meyers, R. E. Babine, N. J. Hayward, D. Weaver, H. Benjamin, F. Stassen, S. S. Abdel-Meguid and J. E. Strickler, J. Med. Chem., 2006, 49, 7781. [103] H. Deng, T. D. Bannister, L. Jin, R. E. Babine, J. Quinn, P. Nagafuji, C. A. Celatka, J. Lin, T. I. Lazarova, M. J. Rynkiewicz, F. Bibbins, P. Pandey, J. Gorga, H. V. Meyers, S. S. Abdel-Meguid and J. E. Strickler, Bioorg. Med. Chem. Lett., 2006, 16, 3049. [104] W. A. Schumacher, S. E. Seiler, T. E. Steinbacher, A. B. Stewart, J. S. Bostwick, K. S. Hartl, E. C. Liu and M. L. Ogletree, Eur. J. Pharmacol., 2007, 570, 167. [105] M. S. Buchanan, A. R. Carroll, D. Wessling, M. Jobling, V. M. Avery, R. A. Davis, ¨ ster, T. Fex, J. Deinum, J. N. A. Hooper and R. J. Quinn, J. Med. Y. Feng, Y. Xue, L. O Chem., 2008, 51, 3583.
CHAPT ER
10 Advances in the Discovery of Anti-Inflammatory FMS Inhibitors Carl L. Manthey and Mark R. Player
Contents
1. Introduction 1.1 Pivotal driver of the macrophage lineage 1.2 Role in tumor biology 1.3 Role in skeletal pathophysiology 1.4 Role in inflammation 2. Recent FMS Inhibitors 2.1 Aryl- and heteroarylamides 2.2 Quinazolines, quinolines, and quinolones 2.3 Other chemotypes 2.4 Multitargeted kinase inhibitors 3. Conclusion References
211 211 212 213 213 215 215 217 218 220 221 222
1. INTRODUCTION 1.1 Pivotal driver of the macrophage lineage The tyrosine kinase receptor, FMS, is emerging as an attractive drug target to control macrophage numbers in multiple disease settings. The biology of FMS and its ligand, colony stimulating factor-1 (CSF-1), have been the subject of recent reviews [1–3]. Robust expression of FMS is restricted primarily to the cells of the macrophage lineage including Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Spring House, PA 19477, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04410-8
r 2009 Elsevier Inc. All rights reserved.
211
212
Carl L. Manthey and Mark R. Player
monocytes, tissue macrophages, dendritic cells, and osteoclasts. FMS dimerization and autophosphorylation of the cytoplasmic domain occurs when CSF-1 binds to the extracellular domains of FMS. Small molecule inhibitors of the FMS kinase domain block autophosphorylation and the signals that would otherwise ensure macrophage survival, prime expression of cytokines, activate proliferation of macrophage progenitors, and differentiate some macrophages into osteoclasts. Mice, nullizygous for either CSF-1 or FMS, exhibit severe deficits in osteoclasts and in several subpopulations of tissue macrophages including those found in the synovium, skin, kidney, and gastric mucosa [4,5]. Dendritic cells are also reduced. For the aforementioned reasons, FMS inhibitors are being optimized and may be useful in treating diseases such as cancer, rheumatoid arthritis (RA), and other autoimmune or remodeling diseases that are characterized by pathogenic macrophages, dendritic cells, and osteoclasts.
1.2 Role in tumor biology Treatment of malignancy by targeting of tumor-associated macrophages is an exciting and emerging concept. Direct correlations have been made between macrophage numbers, angiogenesis and tumor progression [6]. In some tumors the microenvironment appears to activate macrophage expression of soluble factors that: (i) promote angiogenesis, (ii) support tumor cell survival, proliferation, drug resistance, and motility, and (iii) suppress anti-tumor immunity (reviewed in Ref. [7]). CSF-1 is overexpressed by many cancers, where it is a negative prognostic factor [8]. Preclinical studies have identified a role for CSF-1dependent macrophages in tumor angiogenesis. Tumor angiogenesis is blunted in mice that are nullizygous for CSF-1, whereas forced overexpression of CSF-1, by either the host or the tumor, caused remarkable increases in tumor infiltrating macrophages and tumor vessel densities, as well as enhanced oncogenicity [9,10]. Pharmacological intervention studies corroborate a role for CSF-1 in tumor growth. CSF-1 antisense oligonucleotides, CSF-1 antibody, FMS siRNA, and a selective FMS kinase inhibitor have reduced the growth rates of diverse xenografts [11–13]. In these studies, tumor-associated macrophages and macrophage-derived pro-angiogenic factors were depleted in association with marked reductions in tumor microvasculature. Anti-CSF-1 reversed the chemoresistance of MCF-7 xenografts, which identified an unanticipated role for CSF-1 in chemoresistance. Additionally, the siRNA, Mi160 was determined to inhibit CSF-1 synthesis and was, itself, down-regulated in several drug-resistant cancer lines [14]. Macrophages also mediate hormone resistance in prostate cancers by a nuclear receptor derepression pathway [15]. CSF-1-dependent macrophages also mediate tumor
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
213
cell egress. In fact, spontaneous metastasis of mammary carcinomas of the lung is profoundly reduced in CSF-1-nullizygous mice [16,17]. High expression of FMS has been documented in several types of cancers [8]. The contribution of FMS to oncogenesis of these tumors remains speculative, but FMS inhibitors may, in some instances, have direct anticancer activity. Nonetheless, by targeting macrophages, FMS inhibitors may have broad utility in blocking critical tumor–stroma interactions.
1.3 Role in skeletal pathophysiology The skeletal microenvironment activates a program of macrophage fusion and differentiation whereby osteoclasts are formed and endowed with the ability to resorb bone. Osteoclast differentiation requires two factors, CSF-1 and RANK ligand, that act interdependently [18]. Metastatic bone disease and RA are characterized by increased local expression of CSF-1. Osteoclasts provide the resorbing edge of the tumor/pannus tissue that erodes bone and leads to deformity and fracture in the case of cancer. Bone protection is a salient feature of FMS inhibitor pharmacology. Anti-FMS antibody blocked completely the otherwise fulminate osteoclastogenesis and bone erosion in a murine KRN-serum transfer arthritis model [19]. Four structurally unrelated oral FMS inhibitors have provided dramatic protection to bone in murine models of collagen- or adjuvant-induced arthritis [20–23]. FMS inhibitors, tested in models of metastatic bone disease, reduced radiographic lesions [24]. These data provide the preclinical rationale for evaluating FMS inhibitors in RA and metastatic bone disease. Pfizer has advanced an anti-CSF-1 antibody into Phase I safety testing in RA [25]. Completion of this trial, anticipated in 2009, might provide the first human bone biomarker data for CSF-1/FMS inhibition. Preclinical studies suggest that FMS inhibitors may provide oral alternatives to zoledronate, currently, the first-line therapy for the prevention of skeletal events in cancer patients, and to the RANK ligand antibody, denosumab, an experimental agent with efficacy as a bone protective agent in RA [26].
1.4 Role in inflammation Macrophages amplify and resolve inflammation and remodel tissue in auto-immune or auto-inflammatory diseases [2]. Of cell populations present in the rheumatoid synovium, macrophage numbers provide the strongest correlation with clinical symptoms, and successful therapy is highly associated with declines in this cell population [27]. Immunohistochemical studies identified macrophages as the dominant
214
Carl L. Manthey and Mark R. Player
source of tumor necrosis factor-alpha (TNFa) and interleukin-1b (IL-1b) in rheumatoid pannus [28]. In addition to activating fibroplasia and vascular inflammation, TNFa and IL-1b induce CSF-1 expression by many cell types [29]. CSF-1 levels are elevated markedly in rheumatoid plasma, synovial fluid, and synovium [30,31], where, presumably, it contributes to monocyte recruitment and differentiation, as well as macrophage survival, proliferation, and further expression of IL-1b and TNFa. Inhibition of CSF-1/FMS provides a tractable intervention point in this apparent positive feedback loop. Indeed, CSF-1-nullizygous mice were resistant to collagen-induced arthritis, while recombinant CSF-1 exacerbated clinical signs in this classic mouse model of RA. Additionally, neutralizing anti-CSF-1 and two unrelated oral FMS kinase inhibitors inhibited clinical signs of disease [21,23,32]. In one study, FMS inhibition reduced disease-associated splenic CD11b macrophages to normal levels and reduced type II collagen-induced expression of TNFa and IL-1b. A FMS inhibitor was also effective at reducing ankle swelling in a streptococcal cell wall–induced model of arthritis in rats [22]. These data provide preclinical rationale for evaluating FMS inhibitors in RA. However, anti-FMS antibody and oral FMS inhibitors exerted less influence on inflammation in mouse KRN-serum transfer arthritis and in rat adjuvant arthritis, despite robust bone protection [19,20,22]. One FMS inhibitor provided anti-inflammatory activity in adjuvant arthritis only when combined with methotrexate [22]. It is not presently known why these latter models are less responsive to the anti-inflammatory activity of FMS inhibitors. Many forms of immune-mediated nephritis in humans, including lupus nephritis, are associated with an expanded population of renal macrophages and increased tubular and glomerular expression of CSF-1. A negative correlation exists between renal CSF-1 and creatinine clearance [33]. Plasma CSF-1 is elevated in patients with systemic lupus [34], and persistently high levels of CSF-1 in urine after initial remission, is a predictor of renal flares [35]. MRL-Faslpr mice exhibit spontaneous lupus-like disease including nephritis and cutaneous lesions together with increased local and systemic levels of CSF-1. Transgenic overexpression of CSF-1 exacerbates disease, and CSF-1-nullizygous mice demonstrate resistance [36,37]. Because UVB light triggers cutaneous lesions in wild-type but not in CSF-1-deficient MRL-Faslpr mice [37], FMS inhibitors might find additional application treating the subset of patients with cutaneous disease. Elevated CSF-1 or FMS expression, together with increased macrophage numbers, has been associated with coronary artery disease [38], inflammatory bowel diseases [39], sarcoidosis [40], and obesity [41]. CSF-1 deficiency conferred resistance to diet-induced atherogenesis in mice [42]. Anti-CSF-1 or CSF-1 deficiency reduced dextran sodium
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
215
sulfate colitis in mice [43]. CSF-1 deficiency also conferred resistance to peripheral demyelinating disease [44], and a FMS kinase inhibitor reduced markedly the loss of motor function in a MOG peptide–induced model of multiple sclerosis [45]. However, dinitrobenzene sulfonic acid–induced colitis was exacerbated in CSF-1-deficient mice [46]. In some settings, CSF-1-dependent macrophages may be a source of antiinflammatory molecules and may mediate tissue repair [2]. Although much remains to be learned regarding the long-term consequences of FMS inhibition in man, FMS inhibition in rats is well tolerated [20], and FMS provides a tractable and exciting target to control the macrophage lineage across a spectrum of human disease.
2. RECENT FMS INHIBITORS 2.1 Aryl- and heteroarylamides From a simple arylamide high-throughput screening (HTS) hit (IC50 ¼ 400 nM), a series of 2-cyano-5-carboxamide inhibitors were identified with FMS kinase IC50s of about 5 and 100 nM in a cellular assay of CSF-1stimulated bone marrow–derived macrophage (BMDM) proliferation [47–49]. A co-crystal structure of an early analog (1) was obtained with FMS. Interaction occurs in the hinge region of the adenosine triphosphate (ATP) pocket and involves a hydrogen bond between the arylamide carbonyl and the backbone NH of Cys666. The ring oxygen of the furan is not involved in direct binding to a specific FMS residue; instead, it shares an intramolecular hydrogen bond with the amide NH, thereby stabilizing a flat conformation of the arylamide core. The methylpiperidine occupies the ATP sugar pocket and the hydroxymethyl extends away from this region, making a hydrogen bond with the phenol of Tyr665 [50]. In a 20-kinase selectivity panel, 1 inhibited only TrkA greater than 50% at 1 mM. Further optimization afforded 2,4-disubstituted arylamides that demonstrated IC50s in the 1 nM range in enzyme and cellular assays [23,51]. Compound 2 had a FMS kinase IC50 of 800 pM but inhibited several other receptor tyrosine kinases (c-Kit, Axl, TrkA, Flt-3, and IRKb) at IC50s less than 100 nM. This compound, at doses of 30 and 5 mg/kg, p.o. BID, reduced clinical scores by 65 and 35%, respectively, in a murine collagen–induced arthritis model. Histological examination revealed that pannus growth and bone destruction were reduced by 80%, a level of efficacy comparable to anti-TNF strategies in collagen-induced arthritis models. Additional optimization efforts replaced the phenylenediamine moiety with carbon-based linkers, maintaining potency and mitigating the potential for idiosyncratic drug reactions as evidenced by the loss of the compounds’ ability to conjugate glutathione in vitro [52].
216
Carl L. Manthey and Mark R. Player
For example, 3 demonstrated IC50s of 400 and 360 pM in kinase and BMDM cellular assays respectively.
N
N
H N
O
CN
O N
1
CN N H O
N HO
N
H N
2
N
H N
CN N H O
HN
3
A series of 2-(a-methylbenzylamino)pyrazines exemplified by CYC10268 (4) have been recently disclosed [53]. Pyrazines, such as 4, have been proposed to bind with the 4-N hydrogen bonding to the Cys666 amide NH and the 1-N and 2-NH hydrogen bonding to the Thr663 hydroxyl (gatekeeper residue) [54]. There are also potential interactions with the DFG motif, which is in the ‘‘out’’ configuration. Compound 4 is a 15 nM FMS inhibitor with the ability to block CSF-1mediated survival of murine BMDM [55]. It is also a moderately potent PDGFRb and c-Kit inhibitor with IC50s of 89 and 210 nM respectively. Compound 4 also inhibited both CSF-1-induced ERK1/2 and Akt phosphorylation, and macrophage gene expression. In addition, 4 prevented CSF-1 from priming the macrophages for increased lipopolysaccharide (LPS)-induced production of TNFa, IL-6, and IL-12. In contrast to BMDM, thioglycollate-elicited peritoneal macrophages (TEPM) do not require CSF-1 for survival but behave as CSF-1-primed macrophages. After overnight treatment of TEPM with 4, LPS-induced TNFa, IL-6, and IL-12 production were markedly impaired (regardless
217
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
of CSF-1 pretreatment), and this effect was not a result of decreased survival. Pyridyl and thiazolyl bisamides have also been prepared; an example is compound 5, which possesses a FMS IC50 of 7 nM. In an assay of CSF1-driven proliferation of 3T3 cells engineered to express human fulllength FMS, 5 inhibited growth with an IC50 of 110 nM [56–58]. In rats, 5 had oral bioavailability of 31%, low clearance (2 mL/kg/min) and a t½ of over 7 h. A mouse pharmacodynamic (PD) model was constructed using 3T3 cells engineered to express human mutant full-length FMS in which the kinase activity was constitutively on. After implantation in nude mice and tumor growth to W250 mm3, phosphoFMS (pFMS) was measured by ELISA. Compound 5, when dosed at 50 mg/kg p.o., resulted in 100% inhibition of pFMS at 2 and 6 h after dosing. O HO N 1 4 N
O
H N
F3C
N H
2
Cl
O
H N
N H
O
N
4
F
S N
5
2.2 Quinazolines, quinolines, and quinolones Early FMS inhibitors were derived from the widely used 4-aminoaryl6,7-dimethoxyquinazoline chemotype [59,60]. Moderate potency was obtained by methylation of the 4-aminoaryl moiety (IC50 ¼ 500 nM) as well as by bioisosteric replacement of the 6,7-dimethoxyquinazoline with a pyrazolo[3,4-d]pyrimidine (IC50 ¼ 180 nM). A quinoline urea derivative, Ki20227 (6) with kinase IC50 of 2 nM as well as modest selectivity versus VEGFR2 and c-Kit (IC50s ¼ 12 and 451 nM respectively), has been shown to inhibit CSF-1-dependent growth of M-NFS-60 cells in vitro [24,61]. Compound 6 also inhibits osteoclast development both in vitro and in vivo and suppresses metastatic tumorinduced osteolysis. Compound 6 inhibited CSF-1-dependent LPSinduced TNF production in BMDM in vitro [26]. Administration of 0.02% 6 in food suppressed joint inflammatory cell infiltration as measured by F4/80 immunostaining and improved the clinical score from day 8 to 30 in a mouse collagen–induced arthritis model (p ¼ 0.0032) [21].
218
Carl L. Manthey and Mark R. Player
A series of 3,4,6-trisubstituted 2-quinolones such as 7 were developed from a quinolone 3-carboxylate ester HTS hit through isosteric replacement. Compound 7 was shown to have FMS kinase and cellular IC50s of 2.5 and 5.0 nM respectively [62]. CSF-1-induced c-fos mRNA elevation in a PD mouse model was reduced to control levels with a dose of 50 mg/kg p.o., demonstrating that FMS signaling was completely inhibited. A co-crystal structure of an analog bound to the inactive form of FMS was obtained, and the N-1 and the O-2 of the keto tautomer were shown to form hydrogen bonds to the backbone of hinge residues Cys666 and Glu664 [50]. A series of 3-amido-4-arylamino-7-arylquinolines has been identified with enzyme IC50s of 8–100 nM, along with 100 nM–3 mM IC50s for growth inhibition of FMS-expressing cell lines [63]. These compounds were generally cleared rapidly, and no kinase selectivity data was reported. A related series of 3-amido-4-arylaminoquinolines, with small 6- and 7-position alkoxy and alkylamino substitutions, has also been identified [64–67]. The series was optimized for reduced clearance and modest cellular potency to achieve 8, which attained an enzyme IC50 of 6 nM, and in an assay based on inhibiting the CSF-1-driven proliferation of huFMS-expressing 3T3 cells, possessed an IC50 of 230 nM. Compound 8 had good pharmacokinetic properties in rats (%F ¼ 100, Cl ¼ 12 mL/ kg/min, t1/2 ¼ 2.1 h). In addition, 8 showed excellent selectivity, only showing appreciable inhibitory activity versus Ark5 (51% at 1 mM) when evaluated in a panel of 85 kinases. In a mouse PD model [56,67], 8 resulted in 90 and 65% inhibition of pFMS at 2 and 6 h after dosing at 25 mg/kg p.o. Homologous 4-aminoarylcinnolines have also recently been described [68].
O
H N
H N O
O
S N
N NC
N H
O O
N H N
6
7
O
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
219
2.3 Other chemotypes Oxyheteroaryl benzthiazoles, such as 9, have been found to be potent inhibitors of FMS kinase activity (100% inhibition at 1 mM) while offering good selectivity versus PDGFRb and c-Kit [69]. Tyrosine phosphorylation of FMS in HEK293H cells, transfected to express full-length hFMS, was completely inhibited by 9. In addition, 9 completely inhibited the proliferation of CSF-1-dependent M-NFS-60 cells in vitro at a 1 mM concentration. F
F
N
NH
O
O
N
N S
N
N
O
NH2 7
O
N
8
S
O NH
N
N
9
Structure-based optimization of pyrido[2,3-d]pyrimidin-5-ones led to hydroxamate 10, which possessed an IC50 of 400 pM in an enzyme assay and 600 pM in a BMDM cellular assay [22,70–73]. The compound interacts with the hinge region of FMS through hydrogen bonding interactions mediated through the C-2 anilino NH and the pyrimidine N-3. A water-bridged interaction is also present between the C-5 carbonyl oxygen and the hydroxyl group of Thr663. Compound 10 effectively blocked CSF-1-induced c-fos mRNA elevation in a PD mouse model with an ED90 value of 3 mg/kg p.o. During the chronic phase of streptococcal cell wall–induced arthritis in rats, 10 (10, 3, and 1 mg/kg p.o. BID) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 10 partially prevented joint swelling at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. However, at a low dose (0.33 mg/kg), 10 demonstrated good anti-inflammatory activity when given in combination with a sub-maximally effective dose of methotrexate. The diaminopyrimidine GW2580 (11) has been reported to completely inhibit FMS at a concentration of 60 nM [74]. Compound 11 binds to the hinge region of the kinase where the pyrimidine 2-NH2 and 3-N hydrogen bond to the carbonyl and backbone NH of Cys666, while the pyrimidine 4-NH2 forms a bidentate hydrogen bond with the carbonyl of Glu664 and the side chain of Thr663 [75]. Contacts are also made with the backbone NH of Asp796 in the phosphate region with the ortho ether
220
Carl L. Manthey and Mark R. Player
oxygens of 11. The ability of CSF-1 to induce growth of mouse M-NFS-60 myeloid cells and human monocytes in vitro was completely blocked by 11. Compound 11 was selective versus 26 kinases, and this inactivity was confirmed in multiple cell-based assays relevant to those kinases. In vitro, 11 inhibited bone degradation by human osteoclasts and in rat calavaria and rat fetal long bone assays by 80–100% at 1 mM. Dosing of 11 at 40 mg/kg p.o. blocked the ability of a subsequent injection of recombinant CSF-1 to prime mice for enhanced LPS-induced IL-6 production. Compound 11 also inhibited LPS-induced TNFa production with or without CSF-1 priming. In an adjuvant-induced arthritis model, 11 dose-dependently inhibited joint connective tissue and bone destruction measured by radiological, histological, and bone mineral content criteria, while not affecting ankle swelling [20]. N
O N N H
HN
O O
N
NH2
N
N H2N
O
N O
10
O
11
Numerous patent filings also identify 3-substituted-7-azaindoles as selective FMS inhibitors, though many compounds are c-Kit inhibitors as well. Neither discrete IC50s nor in vivo PD or efficacy data have been reported [76–82].
2.4 Multitargeted kinase inhibitors Multitargeted kinase inhibitors have been reported in the literature to include FMS in their target profile. Examples include sunitinib (12), imatinib (13), and ABT-869 (14).
221
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
N HN
N
N
O N H
N HN
N H
F
O
N N
O N H 13
12
H N
NH2
H N
N HN
O
F
14
Compounds 12 and 14 potently inhibit VEGFR2, PDGFRb, c-Kit, FLT3, and FMS [83], and 12 is approved for the treatment of renal cell carcinoma and gastrointestinal stromal tumors (GIST). The phosphorylation of FMS in a 3T3-hFMS cell line was inhibited by 12 with an IC50 between 50 nM and 100 nM [84,85], and in vitro differentiation of BMDM into osteoclasts was inhibited half maximally at a concentration between 10 nM and 100 nM. A MDA-MB-435-HAL-luc breast cancer xenograft model was used to demonstrate that 12 at 80 mg/kg/day inhibited tumor growth in bone by 89% as measured by bioluminescence imaging. Compound 13 is approved for the treatment of chronic myelogenous leukemia and GIST. In cellular assays, 13 inhibits Abl (IC50 ¼ 250 nM), c-Kit (IC50 ¼ 100 nM), PDGFRb (IC50 ¼ 250 nM), and FMS (IC50 ¼ 1.4 mM). Therapeutic responses in chronic myelogenous leukemia and GIST are based on inhibition of Abl and Kit respectively. Nonetheless, longterm administration of 13 promotes bone formation, a possible consequence of either PDGFR or FMS inhibition, or both [86]. Furthermore, 13 is efficacious in mouse collagen–induced arthritis [87], and case reports indicate potential utility in RA [88]. At present, there is no published data on inhibition of FMS phosphorylation by 12, 13, or 14 in dosed individuals, and it is unclear
222
Carl L. Manthey and Mark R. Player
to what extent FMS is inhibited at prescribed doses. FMS inhibition may contribute to the ability of 12 to inhibit solid tumor growth and of 13 to increase bone formation and to relieve symptoms in RA. However, tolerance issues may limit the widespread use of these agents outside of oncology.
3. CONCLUSION A significant investment has been made in the discovery of a richly diverse set of oral small molecule FMS inhibitors. It can be anticipated that at least some of these compounds will advance to clinical trials and reveal the human pharmacology of selective FMS inhibition. The association of CSF-1 expression and macrophage numbers with diverse human diseases, and the preclinical efficacy of FMS inhibitors in models of cancer, arthritis, and multiple sclerosis, presage a broad range of potential therapeutic applications for these compounds.
REFERENCES [1] V. Chitu and E. R. Stanley, Curr. Opin. Immunol., 2006, 18, 39. [2] J. Hamilton, Nat. Rev. Immunol., 2008, 8, 533. [3] W. Yu, J. Chen, Y. Xiong, F. J. Pixley, X. M. Dai, Y. G. Yeung and E. R. Stanley, J. Leukoc. Biol., 2008, 84, 852. [4] M. G. Cecchini, M. G. Dominguez, S. Mocci, A. Wetterwald, R. Felix, H. Fleisch, O. Chisholm, W. Hofstetter, J. W. Pollard and E. R. Stanley, Development, 1994, 120, 1357. [5] X. M. Dai, G. R. Ryan, A. J. Hapel, M. G. Dominguez, R. G. Russell, S. Kapp, V. Sylvestre and E. R. Stanley, Blood, 2002, 99, 111. [6] L. Bingle, N. J. Brown and C. E. Lewis, J. Pathol., 2002, 196, 254. [7] S. K. Biswas, A. Sica and C. E. Lewis, J. Immunol., 2008, 180, 2011. [8] B. Kascinski, Cancer Treat. Res., 2002, 107, 285. [9] L. Wang, G.-G. Zheng, C.-H. Ma, Y.-M. Lin, H.-Y. Zhang, Y.-Y. Ma, J.-H. Chang and K.-F. Wu, Cancer Res., 2008, 68, 5639. [10] E. Y. Lin, J.-F. Li, L. Gnatovskiy, Y. Deng, L. Zhu, D. A. Grzesik, H. Qian, X. N. Xue and J. W. Pollard, Cancer Res., 2006, 66, 11238. [11] S. Aharinejad, P. Paulus, M. Sioud, M. Hofmann, K. Zins, R. Scha¨fer, E. R. Stanley and D. Abraham, Cancer Res., 2004, 64, 5378. [12] P. Paulus, E. R. Stanley, R. Schafer, D. Abraham and S. Aharinejad, Cancer Res., 2006, 66, 4349. [13] Y. Kubota, K. Takubo, T. Shimizu, H. Ohno, K. Kishi, M. Shibuya, H. Saya and T. Suda, J. Exp. Med., 2009, 206, 1089. [14] A. Sorrentino, C.-G. Liu, A. Addario, C. Peschle, G. Scambia and C. Ferlini, Gynecol. Oncol., 2008, 111, 478. [15] P. Zhu, S. H. Baek, E. M. Bourk, K. A. Ohgi, I. Garcia-Bassets, H. Sanjo, S. Akira, P. F. Kotol, C. K. Glass, M. G. Rosenfeld and D. W. Rose, Cell, 2006, 124, 615. [16] J. B. Wyckoff, Y. Wang, E. Y. Lin, J. F. Li, S. Goswami, E. R. Stanley, J. E. Segall, J. W. Pollard and J. Condeelis, Cancer Res., 2007, 67, 2649.
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
223
[17] E. Y. Lin, A. V. Nguyen, R. G. Russell and J. W. Pollard, J. Exp. Med., 2001, 193, 727. [18] S. L. Teitelbaum, Am. J. Pathol., 2007, 170, 427. [19] H. Kitarua, P. Zhow, H.-J. Kim, D. V. Novack, F. P. Ross and S. L. Teitelbaum, J. Clin. Invest., 2005, 12, 3418. [20] J. G. Conway, H. Pink, M. L. Bergquist, B. Han, S. Depee, S. Tadepalli, P. Lin, R. C. Crumrine, J. Binz, R. L. Clark, J. L. Selph, S. A. Stimpson, J. T. Hutchins, S. D. Chamberlain and T. A. Brodie, J. Pharmacol. Exp. Ther., 2008, 326, 41. [21] H. Ohno, Y. Uemura, H. Murooka, H. Takanashi, T. Tokieda, Y. Ohzeki, K. Kubo and I. Serizawa, Eur. J. Immunol., 2008, 38, 283. [22] H. Huang, D. A. Hutta, J. M. Rinker, H. Hu, W. H. Parsons, C. Schubert, R. L. DesJarlais, C. S. Crysler, M. A. Chaikin, R. R. Donatelli, Y. Chen, D. Cheng, Z. Zhou, E. Yurkow, C. L. Manthey and M. R. Player, J. Med. Chem., 2009, 52, 1081. [23] C. R. Illig, J. Chen, M. J. Wall, K. J. Wilson, S. K. Ballentine, J. M. Rudolf, R. J. DesJarlais, Y. Chen, C. Schubert, I. P. Petrounia, C. M. Crysler, C. J. Molloy, M. A. Chaikin, C. L. Manthey, M. R. Player, B. E. Tomczuk and S. K. Meegalla, Bioorg. Med. Chem. Lett., 2008, 18, 1642. [24] H. Ohno, K. Kubo, H. Murooka, Y. Kobayashi, T. Nishitoba, M. Shibuya, T. Yoneda and T. Isoe, Mol. Cancer Ther., 2006, 5, 2634. [25] http://www.clinicaltrials.gov/ct2/show/NCT00550355 [26] P. Durez, J. Malghem, A. N. Toukap, G. Depresseux, B. R. Lauwerys, R. Westhovens, F. P. Luyten, L. Corluy, F. A. Houssiau and P. Verschueren, Arth. & Rheum., 2008, 58, 1299. [27] J. J. Haringman, D. M. Gerlag, A. H. Zwinderman, T. J. M. Smeets, M. C. Kraan, D. Baeten, I. B. McInnes, B. Bresnihan and P. P. Tak, Ann. Rheum. Dis., 2005, 64, 834. [28] C. Q. Chu, M. Field, S. Allard, E. Abney, M. Feldmann and R. N. Maini, Br. J. Rheumatol., 1992, 31, 653. [29] I. K. Campbell, G. Ianches and J. A. Hamilton, Biochim. Biophy. Acta, 1993, 1182, 57. [30] K. Nakano, Y. Okada, K. Saito, R. Tanikawa, N. Sawamukai, Y. Sasaguri, T. Kohro, Y. Wada, T. Kodama and Y. Tanaka, Rheumatol. (Oxford), 2007, 46, 597. [31] I. Rioja, F. J. Hughes, C. H. Sharp, L. C. Warnock, D. S. Montgomery, M. Akil, A. G. Wilson, M. H. Binks and M. C. Dickson, Arth. & Rheum., 2008, 58, 2257. [32] I. K. Campbell, M. J. Rich, R. J. Bischof and J. A. Hamilton, J. Leukoc. Biol., 2000, 68, 144. [33] N. M. Isbel, D. J. Nikolic-Paterson, P. A. Hill, J. Dowling and R. C. Atkins, Nephrol. Dial. Transplant., 2001, 16, 1638. [34] P. T. Yang, W. G. Xiao, L. J. Zhao, J. Lu, L. M. He, H. Kasai and M. Ito, Ann. Rheum. Dis., 2008, 67, 429. [35] S. Tian, J. Li, L. Wang, T. Liu, H. Liu, G. Cheng, D. Liu, Y. Deng, R. Gou, Y. Wan, J. Jia and C. Chen, Inflamm. Res., 2007, 56, 304. [36] D. M. Lenda, E. R. Stanley and V. R. Kelley, J. Immunol., 2004, 173, 4744. [37] J. Menke, M.-Y. Hsu, K. T. Byrne, J. A. Lucus, W. A. Rabcal, B. P. Croker, X.-J. Zong, E. R. Stanley and V. R. Kelley, J. Immunol., 2008, 181, 7367. [38] L. S. Rallidis, M. G. Zolindaki, P. C. Pentzeridis, K. P. Poulopoulos, A. H. Velissaridou and T. S. Apostolou, Heart, 2004, 90, 25. [39] F. H. Klebl, J. E. Olsen, S. Jain and W. F. Doe, J. Pathol., 2001, 195, 609. [40] H. Kreipe, H. J. Radzun, K. Heidorn, J. Barth, J. Kiemle-Kallee, W. Petermann, J. Gerdes and M. R. Parwaresch, Lab. Invest., 1990, 62, 697. [41] I. Harman-Boehm, M. Bluher, H. Redel, N. Sion-Vardy, S. Ovadia, E. Avinoach, I. Shai, N. Kloting, M. Stumvoll, N. Bashan and A. Rudich, J. Clin. Endocrinol. & Metabol., 2007, 92, 2240. [42] T. Rajavashisth, J. H. Qiao, S. Tripathi, J. Tripathi, N. Mishra, M. Hua, X. P. Wang, A. Loussararian, S. Clinton, P. Libby and A. Lusis, J. Clin. Invest., 1998, 101, 2702.
224
Carl L. Manthey and Mark R. Player
[43] D. Marshall, J. Cameron, D. Lightwood and A. D. Lawson, Inflamm. Bowel Dis., 2007, 13, 219. [44] M. Mu¨ller, M. Berghoff, I. Kobsar, R. Kiefer and R. Martini, Exp. Neurol., 2007, 203, 55. [45] Y. Uemura, H. Ohno, Y. Ohzeki, H. Takanashi, H. Murooka, K. Kubo and I. Serizawa, J. Neuroimmunol., 2008, 195, 73. [46] J.-E. Ghia, F. Galeazzi, D. C. Ford, C. M. Hogaboam, B. A. Vallance and S. Collins, Am. J. Physiol. Gastrointest. Liver Physiol., 2008, 294, G770. [47] R. J. Patch, B. M. Brandt, D. Asgari, N. Baindur, N. K. Chadha, T. Georgiadis, W. S. Cheung, I. P. Petrounia, M. A. Chaikin and M. R. Player, Bioorg. Med. Chem. Lett., 2007, 17, 6070. [48] M. R. Player, N. Baindur, B. Brandt, N. Chadha, R. J. Patch, D. Asgari and T. Georgiadis, US Patent 7,427,683, 2008. [49] M. R. Player, N. Baindur, B. Brandt, N. Chadha, R. J. Patch, D. Asgari and T. Georgiadis, US Patent 7,429,603, 2008. [50] C. Schubert, C. Schalk-Hihi, G. T. Struble, H.-C. Ma, I. P. Petrounia, B. Brandt, I. C. Deckman, R. J. Patch, M. R. Player, J. C. Spurlino and B. A. Springer, J. Biol. Chem., 2007, 282, 4094. [51] C. R. Illig, S. K. Ballentine, J. Chen, R. L. DesJarlais, S. K. Meegalla, M. Wall and K. Wilson, US Patent 7,414,050, 2008. [52] S. K. Meegalla, J. Chen, M. J. Wall, K. J. Wilson, S. K. Ballentine, R. J. DesJarlais, C. Schubert, R. Donatelli, C. M. Crysler, Y. Chen, C. J. Molloy, M. A. Chaikin, C. L. Manthey, M. R. Player, B. E. Tomczuk and C. R. Illig, Bioorg. Med. Chem. Lett., 2008, 18, 3632. [53] C. J. Burns, M. F. Harte and J. T. Palmer, WO Patent Application 2008/058341-A1, 2008. [54] C. J. Burns, M. F. Harte, X. Bu, E. Fantino, M. Giarusso, M. Joffe, M. Kurek, F. S. Legge, P. Razzino, S. Su, H. Treutlein, S. S. Wan, J. Zeng and A. F. Wilks, Bioorg. Med. Chem. Lett., 2009, 19, 1206. [55] K. M. Irvine, C. J. Burns, A. F. Wilks, S. Su, D. A. Hume and M. J. Sweet, FASEB J., 2006, 20, 1921. [56] D. A. Scott, B. M. Aquila, G. A. Bebernitz, D. J. Cook, L. A. Dakin, T. L. Deegan, M. M. Hattersley, S. Ioannidis, P. D. Lyne, C. A. Omer, M. Ye and X.-K. Zheng, Bioorg. Med. Chem. Lett., 2008, 18, 4794. [57] B. Aquila, D. Cook L. Dakin, S, Ioannidis, P. Lyne, D. Scott and X. Zhang, WO Patent Application 2007/071955-A1, 2007. [58] L. Almeida, B. Aquila, D. Cook, S. Cowen, L. Daikan, J. Ezhuthachan, S. Ioannidis, S. Lee, P. Lyne, T. Pontz, D. Scott, M. Su and X. Zheng, WO Patent Application 2006/ 067445-A2, 2006. [59] M. R. Myers, N. N. Setzer, A. P. Spada, P. E. Persons, C. Q. Ly, M. P. Maguire, A. L. Zulli, D. L. Cheney, A. Zilberstein, S. E. Johnson, C. F. Franks and K. J. Mitchell, Bioorg. Med. Chem. Lett., 1997, 7, 421. [60] M. R. Myers, A. P. Spada, M. P. Maguire, P. E. Persons, A. Zilberstein, C.-Y. J. Hou and S. E. Johnson, US Patent 5,714,493, 1998. [61] K. Kubo, H. Ohno, T. Isoe and T. Nishitoba, WO Patent Application 2006/0235033-A1, 2006. [62] M. J. Wall, J. Chen, S. Meegalla, S. Ballentine, K. Wilson, R. J. DesJarlais, C. Schubert, M. A. Chaikin, C. Crysler, R. R. Donatelli, E. J. Yurkow, M. R. Player, R. J. Patch, C. L. Manthey, C. Molloy, B. Tomczuk and C. Illig, Bioorg. Med. Chem. Lett., 2008, 18, 2097. [63] T. L. Smalley, S. D. Chamberlain, W. Y. Mills, D. L. Musso, S. A. Randhawa, J. A. Ray, V. Samano and L. Frick, Bioorg. Med. Chem. Lett., 2007, 17, 6257. [64] D. Cook, L. Dakin, D. Delvalle, T. Gero, D. Scott and X. Zheng, WO Patent Application 2007/119046-A1, 2007.
Advances in the Discovery of Anti-Inflammatory FMS Inhibitors
225
[65] L. Dakin, K. Daly, D. Delvalle, T. Gero, C. A. Ogoe, D. Scott and X. Zheng, WO Patent Application 2008/056148-A1, 2008. [66] D. A. Scott, C. L. Balliet, D. J. Cook, A. M. Davies, T. W. Gero, C. A. Omer, S. Poondru, M.-E. Theoclitou, B. Tyurin and M. J. Zinda, Bioorg. Med. Chem. Lett., 2009, 19, 697. [67] D. A. Scott, K. J. Bell, C. T. Campbell, D. J. Cook, L. A. Dakin, D. J. Del Valle, L. Drew, T. W. Gero, M. M. Hattersley, C. A. Omer, B. Tyurin and X. Zheng, Bioorg. Med. Chem. Lett., 2009, 19, 701. [68] L. Dakin, C. A. Ogoe, D. Scott and X. Zheng, WO Patent Application 2009/0012084-A1, 2009. [69] J. C. Sutton, M. Wiesmann, W. Wang, M. Lindvall, J. Lan, S. Ramurthy, A. Sharma, E. J. Mieuli, L. M. Klivansky, W. Lenahan, S. Kaufman, H. Yang, S. C. Ng, K. Pfister, A. S. Wagman, V. Sung and M. Sendzik, WO Patent Application 2008/0045528-A1, 2008. [70] H. Huang, D. A. Hutta, H. Hu, R. L. DesJarlais, C. Schubert, I. P. Petrounia, M. A. Chaikin, C. L. Manthey and M. R. Player, Bioorg. Med. Chem. Lett., 2008, 18, 2355. [71] M. R. Player, H. Huang and D. A. Hutta, WO Patent Application 2007/0060577-A1, 2007. [72] M. R. Player, H. Huang and D. A. Hutta, WO Patent Application 2007/0060578-A1, 2007. [73] M. R. Player, W. H. Parsons, H. Huang, D. A. Hutta, H. Hu and J. Rinker, WO Patent Application 2008/0114007-A1, 2008. [74] J. G. Conway, B. McDonald, J. Parham, B. Keith, D. W. Rusnack, E. Shaw, M. Jansen, P. Lin, A. Payne, R. M. Crosby, J. H. Johnson, L. Frick, M. Jasmine Lin, S. Depee, S. Tadepalli, B. Votta, I. James, K. Fuller, T. J. Chambers, F. C. Kull, S. D. Chamberlain and J. T. Hutchins, Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 16078. [75] L. M. Schewchuk, A. M. Hassell, W. D. Holmes, J. M. Veal, H. K. Emmerson, D. L. Musso, S. D. Chamberlain and G. E. Peckham, US Patent Application 2004/ 0002145-A1, 2004. [76] P. N. Ibrahim, D. R. Artis, R. Bremer, S. Mamo, M. Nespi, C. Zhang, J. Zhang, Y.-L. Zhu, J. Tsai, K.-P. Hirth, G. Bollag, W. Spevak, H. Cho, S. Gillette, G. Wu, H. Zhu and S. Shi, WO Patent Application 2007/002325-A1, 2007. [77] P. N. Ibrahim, D. R. Artis, R. Bremer, G. Habets, S. Mamo, M. Nespi, C. Zhang, J. Zhang, Y.-L. Zhu, R. Zuckerman, B. West, Y. Suzuki, J. Tsai, K.-P. Hirth, G. Bollag, W. Spevak, H. Cho, S. Gillette, G. Wu, H. Zhu and S. Shi, WO Patent Application 2007/ 002433-A1, 2007. [78] C. Zhang, J. Zhang, P. N. Ibrahim, C. R. Hurt, R. Zuckerman, D. R. Artis, R. Bremer, W. Spevak, G. Wu and H. Zhu, WO Patent Application 2007/013896-A2, 2007. [79] C. Zhang, J. Zhang, P. N. Ibrahim, D. R. Artis, R. Bremer, G. Wu, H. Zhu and M. Nespi, WO Patent Application 2008/063888-A2, 2008. [80] C. Zhang, J. Zhang, P. N. Ibrahim, D. R. Artis, R. Bremer, G. Wu, H. Zhu and M. Nespi, WO Patent Application 2008/064255-A2, 2008. [81] C. Zhang, J. Zhang, P. N. Ibrahim, D. R. Artis, R. Bremer, G. Wu, H. Zhu and M. Nespi, WO Patent Application 2008/064265-A2, 2008. [82] P. N. Ibrahim, R. Bremer, C. Zhang, J. Zhang, K.-P. Hirth, G. Wu and H. Zhu, WO Patent Application 2008/080001-A2, 2008. [83] J. Guo, P. A. Marcotte, J. O. McCall, Y. Dai, L. J. Pease, M. R. Michaelides, S. K. Davidsen and K. B. Glaser, Mol. Can. Ther., 2006, 5, 1007. [84] L. Murray, T. Abrams, K. R. Long, T. J. Ngai, L. M. Olson, W. Hong, P. K. Keast, J. A. Brassard, A. M. O’Farrell, J. M. Cherrington and N. K. Pryer, Clin. Exp. Met, 2003, 20, 757. [85] L. Murray, A.-M. O’Farrell and T. Abrams, US Patent Application 2004/0209937-A1, 2004. [86] S. Fitter, A. L. Dewar, P. Kostakis, L. B. To, T. P. Hughes, M. M. Roberts, K. Lynch, B. Vernon-Roberts and A. C. W. Zannettino, Blood, 2008, 111, 2538.
CHAPT ER
11 Recent Advances in the Discovery of CB2 Selective Agonists Sangdon Han, Jayant Thatte and Robert M. Jones
Contents
1. Introduction 2. Evaluation of CB2 Agonists In Preclinical Models 2.1 Pain 2.2 Experimental autoimmune encephalomyelitis 2.3 Other potential indications 3. Medicinal Chemistry 3.1 Monocyclic core-based CB2 agonists 3.2 Bicyclic core-based CB2 agonists 3.3 Tricyclic core-based CB2 agonists 4. Clinical Trials Status 5. Conclusions References
227 228 228 231 232 232 233 237 241 241 242 242
1. INTRODUCTION The CB2 receptor is a member of a family of G-protein-coupled receptors (GPCRs) that mediate the effects of several structurally related endocannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) [1]. The endocannabinoid system is important in an array of biological processes involving the central nervous system, the immune system, and metabolism. CB2 appears to be a promising therapeutic target for the treatment of pain and inflammation. However, the therapeutic utility of Arena Pharmaceuticals Inc., 6166 Nancy Ridge Dr., San Diego, CA 92121, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04411-X
r 2009 Elsevier Inc. All rights reserved.
227
228
Sangdon Han et al.
non-selective, brain penetrable cannabinoid agonists is limited by undesirable psychotropic effects associated with activation of the CB1 receptor. Such side effects are not apparent upon CB2 receptor activation. Selectivity for CB2 activation over CB1 activation is thus an essential feature needed for therapeutic uses directed at CB2. CB2 receptor activation results in the inhibition of adenylyl cyclase [2] and mitogenactivated protein (MAP) kinase activation [3] through coupling to the asubunit of Gi/o proteins. Although expression of CB2 was initially thought to be predominantly restricted to immune cells in the periphery [4], recent data suggest that this receptor is also expressed centrally in perivascular microglial cells [5] and in brainstem neurons [6]. CB2 is up-regulated in both dorsal root ganglia and peripheral neurons following injury [7,8]. Preclinical data using CB2 agonists and receptor knockout mice have validated the receptor as a potential target for the treatment of pain and neuro-inflammation. However, the challenge for this field has remained the identification of highly selective CB2 agonists that maintain functional activity across species. Despite these difficulties, several candidate molecules have advanced into clinical development. In this chapter, we describe pharmacological studies using novel CB2 agonists, which suggest utility for these agents in the treatment of pain and other disorders. We also describe the key medicinal chemistry features of this class of compounds, their current clinical trial status and future prospects as therapeutics.
2. EVALUATION OF CB2 AGONISTS IN PRECLINICAL MODELS 2.1 Pain CB2 agonists such as GW405833 (1) have been found to be efficacious in several preclinical models for inflammatory, neuropathic, and postoperative pain. GW405833 is a selective CB2 agonist with a binding affinity of 3.9271.58 nM for human CB2 versus 477271676 nM for CB1 (W1,000-fold selectivity) [9]. Selectivity for CB2 was diminished for rat receptors, with only a 78-fold selectivity ratio for CB2 over rat CB1. Functionally, this compound appeared to be a partial agonist at the human CB2 receptor, showing maximum inhibition of forskolinmediated cAMP production of only 44.673.4% [9]. Interestingly, 1 has also been reported to be a partial inverse agonist at both human and rat CB2 receptors [10], suggesting that it might be a protean agonist, properties that were previously reported for a structurally similar compound, AM1241 [11]. The i.v. pharmacokinetic profile of 1 in rats included a plasma half-life of 3.68 h; clearance rate of 1.9670.06 L/h/kg, volume of distribution 10.470.2 L/kg, and brain-to-plasma ratio of 5.1570.43.
Recent Advances in the Discovery of CB2 Selective Agonists
229
In a neuropathic pain model utilizing partial ligation of sciatic nerve, 1 showed 63.3% reversal of pain at 1 h following a 10 mg/kg i.p. dose with an ED50 ¼ 0.077 mg/kg. In the hind paw surgical incision model and the Freund’s complete adjuvant (FCA)-induced inflammatory pain model, 1 displayed a dose-dependent reversal of hyperalgesia with an ED50 ¼ 2.58 mg/kg and 0.17 mg/kg, respectively, at the 1 h time point. The on-target effect of this compound was confirmed in CB2 knockout mice where it did not reverse inflammatory pain. Central effects of 1 were evaluated in the catalepsy assay, the rotorod assay for motor function, and tail flick and hot plate tests for acute analgesia. The highest dose, 100 mg/kg, induced catalepsy and decreased rotorod performance, suggesting CB1-associated activity at this dose, but not at the lower doses [9]. Abbott has published data for two CB2 agonists, A-796260 (2) [10] and A-836339 (3) [12]. In the [3H]CP-55,940 competitive binding assay, A-796260 was 193-fold selective for human CB2 with Ki ¼ 845 nM for hCB1 versus 4.37 nM for hCB2, and 30-fold selective for rat CB2 with Ki ¼ 395 nM for rat CB1 compared to 13 nM for rat CB2. Functional selectivity was 1,380-fold for hCB2 and 175-fold for rat CB2 as determined using the adenylate cyclase assay. Although the pharmacokinetic properties for A-796260 have not been described, it was administered intraperitoneally in rats in specific pharmacological models. In the FCA-induced model of thermal hyperalgesia, A-796260 displayed dose-dependent efficacy (ED50 ¼ 2.8 mg/kg). In this model, specificity was confirmed using two CB2-receptor-selective antagonists: SR144528 [13] and AM630 [14], both of which blocked the analgesic effects of A-796260. In contrast, the CB1-receptor-selective antagonist SR141716A had no effect on A-796260-induced analgesia. In the skin incision model of postoperative pain, A-796260, administered i.p. 1.5 h post-surgery dose-dependently attenuated tactile allodynia with an ED50 ¼ 18 mg/kg and maximal efficacy of 6877%. Tolerance to A-796260 did not develop in this model after repeat administration, b.i.d. for 5 days. In the chronic constriction injury of sciatic nerve model of neuropathic pain, A-796260 dose-dependently attenuated tactile allodynia with an ED50 ¼ 15 mg/kg and 6679% efficacy. A-796260 is the only CB2 agonist reported to date to have an efficacy in mono-iodoacetate-induced osteoarthritis pain. Efficacy was comparable to celecoxib (56% efficacy at 35 mg/kg dose of A-796260 versus 62% efficacy at 38 mg/kg dose of celecoxib). This compound did not show significant effects in locomotor activity up to a dose of 35 mg/kg. A-836339 exhibited sub-nanomolar potencies in competition binding assays (Ki values of 0.64 nM for hCB2 and 0.76 nM for rat CB2), with 425- and 189-fold selectivity for CB2 receptor over the human and rat CB1 receptors, respectively. Functionally, the potency of A-836339
230
Sangdon Han et al.
(EC50 ¼ 1.6 nM) was similar to A-796260 (EC50 ¼ 0.71 nM). A-836339 demonstrated full agonist activity (Emax ¼ 102%) at the CB2 receptor and also showed weaker but full agonist activity at both human and rat CB1 receptors (EC50 ¼ 740 nM and 1,200 nM, respectively). A-796260, as well as A-836339, showed minimal off-target (o50% inhibition) activities in the CEREP binding platform containing a panel of 74 GPCRs and ion channels. The only activity observed with A-796260 was on the d-opioid receptor (67% displacement) and with 10 mM A-836339 on the A3 and 5-HT2C receptors (55% and 53%, respectively). In the FCA-induced inflammatory pain model, A-836339 dose-dependently reversed thermal hyperalgesia with an ED50 ¼ 1.96 mmol/kg. A-836339 was also found to be efficacious in the skin incision model, the capsaicin-induced mechanical hyperalgesia model and in the chronic constriction injury model of neuropathic pain with ED50 ¼ 12 mmol/kg, 10.4 mmol/kg, and 12.9 mmol/kg, respectively. The activities of both A-796260 and A-836339 were found to be m-opioid receptor-independent since naloxone did not antagonize their anti-hyperalgesic effects. Despite the high in vitro selectivity of A-836339 for the CB2 receptor over the CB1 receptor (189fold), it clearly showed a CB1-associated reduction in horizontal locomotor activity at the 15 and 45 mmol/kg doses, which was blocked by treatment with the CB1 receptor antagonist SR141716A.
O N O O
O
N N
S
N
N
O N
Cl
O
Cl 1
O
2
3
GlaxoSmithKline’s (GSK) clinical candidate GW842166X (4) was reported to be fully selective on human CB2 receptor with EC50 ¼ 63 nM and 91 nM for human and rat CB2 receptors, respectively [15]. It had no significant agonist activity at concentrations up to 30 mM on either human or rat CB1 receptors. Oral bioavailability was 58% with a 3 h half-life and a brain:plasma ratio of 0.8. In the FCA model of inflammatory pain, it showed high potency with oral ED50 ¼ 0.1 mg/kg and full reversal of
Recent Advances in the Discovery of CB2 Selective Agonists
231
hyperalgesia at the 0.3 mg/kg dose. In 4-day studies, development of tolerance was not observed. MDA7 (5), a compound developed at the MD Anderson Cancer Center, was shown to dose-dependently attenuate tactile allodynia produced by spinal nerve ligation or paclitaxel in rats [16]. In a [3H]CP55,840 radioligand displacement assay, 5 showed no affinity for the human CB1 receptor at concentrations up to 10 mM, with a 4227123 nM Ki on human CB2. On rat receptors, the mean Ki values were 25657695 nM and 2387143 nM for CB1 and CB2 receptors, respectively. In the GTPgS functional assay, this compound showed weak partial agonist activity on human CB1 receptor at concentration of W1 mM. A transient thermal anti-nociceptive effect was reported in naı¨ve rats at the 10 mg/kg dose administered intraperitoneally. PF-03550096 (6), another cross-species-selective agonist from Pfizer, was reported to significantly suppress visceral hypersensitivity in the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colonic pain model in rats [17,18]. SR144528 reversed the inhibitory effects of 6 indicating that the compound is functionally active on the rat CB2 receptor. O
H2N O
O
O
N
NH O
F3C N
N
N
O
O N
NH Cl
Cl 4
OH 5
6
2.2 Experimental autoimmune encephalomyelitis In this preclinical animal model for multiple sclerosis (MS), two key sets of data lend support for a potential role for CB2 agonists in treatment. First, experimental autoimmune encephalomyelitis (EAE) is exacerbated in CB2 knockout mice upon disease induction compared to wild-type littermates [19,20]. Second, therapeutic treatment of mice at the peak of disease with the CB2 agonist HU-308 (7, Ki human CB2 ¼ 22.7+3.9 nM; human CB1 KiW10 mM; Ki for rodent CB2 not reported) [21] show a significant reduction in disease score compared to the vehicle-treated
232
Sangdon Han et al.
animals [20]. This effect was attributed to decreased microglial and infiltrating myeloid cell proliferation. HO CF3 O O N Cl O 7
N H
N H N
N
8
2.3 Other potential indications CB2 agonists have shown promise in other indications at the preclinical level. GW833972A (8), a 1,000-fold CB2-selective compound, was shown to inhibit citric acid-induced cough in a conscious guinea-pig model [22]. The CB2 antagonist SR144528 but not the CB1 antagonist SR141716A, reversed the effect of 8, demonstrating involvement of the CB2 receptor. A potential role for CB2 agonists in the treatment of osteoporosis has been proposed based on in vitro suppression of trabecular osteoclastogenesis due to inhibition of proliferation of osteoclast precursors and receptor activator of NFkB ligand, RANKL, by the CB2 agonist 7 [23]. In the same report, 7 was tested in an ovarectomized mouse model of postmenopausal osteoporosis. The CB2 agonist HU-308 attenuated trabecular bone loss from 41% in the untreated mice to 27% in this model. A potential role for CB2 in treatment of atherosclerosis in apolipoprotein E knockout mice has been reported using the non-selective cannabinoid agonist D9-tetrahydrocannabinol (THC) [24]. Involvement of CB2 was demonstrated in this report using CB2 antagonist SR144528, which completely abolished efficacy of THC.
3. MEDICINAL CHEMISTRY This section is focused on advances that have occurred in the medicinal chemistry of CB2 agonists since the publication of an earlier review [25]. With the recent entry of several CB2-selective agonists into the clinic, there has been an increase in the number of reports of novel selective agonists with demonstrable efficacy in both acute and chronic pain models. Pharmacophore-based de novo virtual screening methods [26] and high-throughput hit-based optimization have been the two main
233
Recent Advances in the Discovery of CB2 Selective Agonists
strategies adopted for the discovery of new ligands. Broadly speaking, the majority of the reported selective CB2 agonists are characterized by an aromatic heterocyclic core connected to a bulky alkyl or an aryl amide motif. In general, these highly lipophilic molecules possess high CB2 receptor affinity with tandem selectivity over the CB1 subtype.
3.1 Monocyclic core-based CB2 agonists 3.1.1 Six-membered aromatic cores
O Cl
N H Cl
N H
N
Cl
N
O Cl
9
N H
O
N 10
Following the disclosure of the chemical structure of the GSK clinical candidate (4), a number of alternate six-membered ring aromatic corebased CB2 ligands have appeared in the literature. Motivated in part by the poor aqueous solubility of 4 (2.0 mg/mL at pH 7.4), replacement of the pyrimidine core with pyridine was scrutinized [27]. The nature of the aniline group appears to be important for aqueous solubility of the pyridine series. Replacement of the 2,4-dichloro aniline in 9 with 3-methoxy aniline afforded improved aqueous solubility (168 mg/mL at pH 7.4), although this modification reduced affinity for the CB2 receptor. Compound 9 was found to be selective over CB1 (hCB2/hCB1 EC50 ¼ 79 nM/W30 mM) in a yeast reporter assay. Although 9 had low aqueous solubility (7.0 mg/mL at pH 7.4), it exhibited good exposure and oral bioavailability in rat pharmacokinetic studies (AUC(0t)/dose ¼ 15 min g/L, %F ¼ 39, 3.0 mg/kg, p.o.). Compound 9 was evaluated in the FCA model of inflammatory pain (ED50 ¼ 0.07 mg/kg, p.o.) and demonstrated full reversal of hyperalgesia (0.3 mg/kg, p.o.) [27]. New reports describing a conformationally restricted morpholinyl motif, exemplified by 10, have appeared [28–30]. Compound 10 retained in vitro activity and selectivity for CB2 (hCB2 EC50 ¼ 10 nM, selectivity (hCB1) W2,000-fold); however, it was not competitive with [3H]-CP55,940 in a binding assay (hCB2 KiW10 mM) possibly indicating an interaction with a different binding pocket within the CB2 receptor. Interestingly, this (R)-phenyl morpholine analog was reported to function as an inverse agonist. Compound 10 demonstrated oral efficacy in a mouse zymosan-induced paw inflammation (ZIPI) model (85%
234
Sangdon Han et al.
inhibition at 100 mg/kg, p.o.). Its ZIPI model efficacy confirmed an antiinflammatory activity at CB2 [28,29].
R1
O S O
N
O S O
O N
R1 O
O HN
O S O
NH
N O
11 R1 = morpholine
12
13
Several sulfamoyl benzamide-based CB2-selective agonists have emerged, composed of a central phenyl ring with a bulky lipophilic cycloalkyl carboxamide [31–34]. Saturated cyclic amines such as morpholine, pyrrolidine, and piperidine at R1 displayed similar in vitro profiles, with 11 being slightly more selective for CB2 [31]. Amide 11 exhibited 120-fold selectivity over CB1 in the [35S]GTPgS functional assay (hCB2/hCB1 EC50 ¼ 4.6 nM/550 nM) and did not produce catalepsy at doses of 6 and 10 mg/kg, i.p. Compound 11 reversed the nerve injuryinduced tactile allodynia in the L5 SNL rat model of neuropathic pain (3 mg/kg, i.p.) and also produced a significant anti-allodynic effect (10 mg/kg, i.p.) comparable to the effect produced by morphine (3 mg/ kg, s.c.) in the hind paw incision model of post-operative pain. However, 11 had no oral efficacy in the incision pain model due to poor oral bioavailability [31]. Structure–activity relationship studies of a closely related genus of sulfamoyl benzamides have also been reported [32], wherein, 12 (R1 ¼ CH3) is described as the preferred compound in this series (hCB2/ hCB1 EC50 ¼ 4 nM/W8.9 mM in the adenylate cyclase assay) [33]. The desmethyl analog (R1 ¼ H), or derivatives, composed of a large alkyl group (R1 ¼ cyclohexyl) resulted in a significant loss of potency (hCB2 EC50 W20 mM). The anti-inflammatory effect of 12 (R1 ¼ cyclohexyl) was evaluated in a mouse zymosan-induced paw inflammation (ZIPI) model [33]. A retro-amide modification of 11 led to a new series of agonists exemplified by 13 (hCB2 EC50 ¼ 11 nM). These compounds possessed improved selectivity over CB1 while maintaining good binding affinity for CB2 [34]. Compound 13 demonstrated anti-allodynic activity in the hind paw incision model of post-operative pain when delivered intraperitoneally
235
Recent Advances in the Discovery of CB2 Selective Agonists
(30 mg/kg). However, 13 was not orally efficacious in the incision pain model due to metabolic instability (RLM ¼ 2% remaining at 30 min). Oral efficacy in the skin incision model from the aminobenzotriazole (ABT, the cytochrome P450 suicide inhibitor) pretreated animals also confirmed a rapid metabolism of 13 in the liver [34].
3.1.2 Five-membered heterocyclic cores N
N Cl
N H
Cl
Cl
N
Cl N
N F
O N
O N
14
F
15
1,2,4-Oxadiazole-based CB2 agonists have been identified following examination of a number of alternative five-membered heterocycles derived from an initial HTS hit [35]. The amino quinoline analog 14 (hCB2/hCB1 EC50 ¼ 2.2 nM/550 nM in the adenylate cyclase assay) displayed improved physicochemical properties [simulated intestinal fluid (SIF) solubility ¼ 87 mg/mL] and a good pharmacokinetic profile (AUC ¼ 43.1 mg.h/mL, Cmax ¼ 8.4 mg/mL, t1/2 ¼ 5.1 h, and %FW100 at 10 mg/kg, p.o.) in rats [35]. Structural modification of 14 led to the conformationally constrained N-arylpiperidine oxadiazole 15 with improved metabolic stability (Clint ¼ 38 mL/min/mg in HLM) and in vitro selectivity (hCB2/hCB1 EC50 ¼ 11 nM/W2.0 mM) in the cyclase assay. Oxadiazole 15 displayed reasonable exposure despite poor oral bioavailability (AUC024h ¼ 5240 ng h/mL, Cmax ¼ 682 ng/mL, t1/2 ¼ 8.9 h, and %F ¼ 2 at 10 mg/kg, p.o.) in rats [36,37]. O CF3
CF3
N N
N O
S
N N
N O
F O
N NH
S N
16
17
18
F F
236
Sangdon Han et al.
For thiazolylidene benzamide 16 [38,39], the cyclopropylmethyl group at the 3-position and a bulky lipophilic tert-butyl group at 5position of the thiazole were important for obtaining high affinity and selectivity for the CB2 receptor (16, hCB2 IC50 ¼ 13 nM, CB2 selectivity ¼ 270-fold in the [3H]-CP-55,940 binding assay). Amide 16 also possessed good stability in HLM (93% remaining at 15 min) and an acceptable pharmacokinetic profile (AUC024h ¼ 326 ng h/mL, Cmax ¼ 43.1 ng/mL, t1/2 ¼ 4.8 h, %F ¼ 52, 3 mg/kg) in rats [39]. A report describing the synthesis and SAR of a related pyrazolylidene derivative CBS0550, 17, has also appeared [40]. Although 16 was poorly soluble (o0.01 mg/100 mL in water), the pyrazole derivative 17 exhibited improved solubility in water (5.9 mg/100 mL) presumably due to its increased basicity. Compound 17 also displayed an improved in vitro profile in the [3H]-CP-55,940 binding assay (hCB2 IC50 ¼ 2.9 nM, CB2 selectivity ¼ 1400-fold). The pharmacokinetic properties of 17 were evaluated in rats (AUC08h ¼ 2160 ng h/mL, Cmax ¼ 545 ng/mL, t1/2 ¼ 2.5 h at 10 mg/kg, p.o.), where it demonstrated dose-dependent reversal of mechanical hyperalgesia in the Randall-Selitto model of inflammatory pain (10 mg/kg and 30 mg/kg, p.o.), whereby the antinociceptive effect lasted at least 3 h after administration [40]. A new series of CB2 receptor modulators based on a thiazole core have also been reported [41]. Analog 18 is representative (hCB2 EC50 ¼ 0.7 nM in the cAMP assay). The thiazole appears to be optimal, as replacement with an isoxazole or a phenyl ring led to a decrease in potency [41].
S O O
Cl
H N O 19
O N
S O O
H N O
N N
20
Two closely related a-amidosulfone series have been independently described by two different research groups [42,43]. Alkylsulfone 19 exhibited potent CB2 agonist activity (hCB2 EC50 ¼ 0.04 nM) in the adenylate cyclase assay, and cyclo-alkyl derivatives (cyclohexane, cycloheptane, tetrahydropyran, etc.) also retained sub-nanomolar CB2 activity, but no further biological data was given [42]. Arylsulfone 20 appears to be another promising chemotype as evidenced by the reported affinity for CB2 versus CB1 (hCB2/hCB1 EC50 ¼ 25 nM/W2.0 mM) in the adenylate cyclase assay [43]. SAR studies revealed that the five-membered heterocycles had strict size requirements for their substituents, with only the tert-butyl group maintaining good potency
Recent Advances in the Discovery of CB2 Selective Agonists
237
and selectivity for the CB2 receptor. Compound 20 had low intrinsic clearance in both human and rat liver microsomes (HLM CLint ¼ 14 mL/ min/mg and RLM CLint ¼ 20 mL/min/mg) and good pharmacokinetic properties (AUC0inf ¼ 3570 ng h/mL, Cmax ¼ 971 ng/mL, %F ¼ 43 at 10 mg/kg, p.o.) in rats and was therefore selected for further in vivo evaluation [43].
3.2 Bicyclic core-based CB2 agonists 3.2.1 Benzimidazole cores
X
N N R1
S O O
N
N
N O O
21 R1 = 3-ethoxypyridin-4-yl, X = O 22 R1 = Et, X = O 23 R1 = 2,6-dichloropyridin-4-yl, X = O 24 R1 = Cyclopentyl, X = N-COCH3
25
Two distinct classes of benzimidazole-derived selective CB2 modulators have also emerged recently. Compound 21 exhibited potent agonist activity (hCB2 EC50 ¼ 0.3 nM) and high selectivity over the CB1 receptor (W4,200-fold). Furthermore, this compound displayed good pharmacokinetic properties in male SD rats (AUC0inf ¼ 2501 ng h/mL, Cmax ¼ 731 ng/mL, t1/2 ¼ 3.4 h, %F ¼ 43 at 10 mg/kg, p.o.). SAR studies revealed that the size of the substituent at the 2-position of the benzimidazole was a key determining factor of in vitro efficacy (Emax) and agonist potency. Of note, ethyl analog 22 is potent and selective (hCB2 EC50 ¼ 1.2 nM, CB1/CB2 B500), but induced the prototypical CB1mediated psychotropic effects when dosed in rats [44]. By contrast, 21, circumvented the CB1-mediated side effects due to limited blood–brain barrier (BBB) penetration (brain/plasma ¼ 0.1 in rat) and good selectivity. The major reported liabilities associated with 21 include poor solubility and inhibition of both CYP2C9 and CYP2C19 [44]. A closely related analog 23, wherein the ethoxy group was replaced with two chlorine substituents, displayed improved in vitro selectivity, up to W13,000-fold, over CB1 in the cyclase assay (hCB2/hCB1 EC50 ¼ 0.74
238
Sangdon Han et al.
nM/W10 mM) [45]. Replacement of the pyran with the N-acetyl piperidine afforded compound 24 which retained single nanomolar agonistic CB2 activity in the cyclase assay (hCB2 EC50 ¼ 3.23 nM). CB1related side effects of 24 were examined in a hypothermia experiment [lowest acceptable dose (LAD) W40 mg/kg] [46]. In the second benzimidazole-derived series [47], bis-alkyl amides in place of the sulfone were well tolerated, suggesting the presence of a large hydrophobic binding pocket at the receptor site. The mono-ethyl amide or primary amide resulted in loss of activity at CB2. The N-1benzimidazole position can accommodate a wide variety of groups, with a preference for alkyl substituents. However, selectivity versus CB1 diminished with increasing steric bulk. A representative compound, 25, had good in vitro potency and selectivity in the [3H]-CP55,940 binding assay (hCB2/hCB1 Ki ¼ 4.5 nM/W5.0 mM) [47] but was not desirable for in vivo evaluation due to poor metabolic stability, oral bioavailability, and solubility [48].
3.2.2 Imidazo bicyclic cores O
F
NH
F
O
N HN
N
O N
N
N
HN O
N
N O
N
N
N n
N
N
O F3C 26
27
28
29 n = 1
Imidazopyridine-based CB2 agonists have also been disclosed and assessed in both the FCA model of inflammatory pain and iodoacetate model of osteoarthritis [49]. Compound 26 (hCB2/hCB1 EC50 ¼ 5.2 nM/ W17 mM in cAMP) is representative of this series. The structurally related 27 was found to retain good potency for CB2 but was somewhat less selective over CB1 (hCB2/hCB1 EC50 ¼ 2.2 nM/878 nM) [49]. In a subsequent PCT filing by the same group, removal of carbonyl group from the 1 or 3 substituent at the imidazopyridine core was described. Compound 28 (hCB2 EC50 ¼ 17 nM in cAMP) likely afforded an improvement in solubility due to its basic nitrogen [50]. Insertion of a five-membered heterocyclic spacer (pyrazole, imidazole, etc.) between
239
Recent Advances in the Discovery of CB2 Selective Agonists
the core and the phenyl was also explored as part of the SAR in this application; however, this modification did not improve potency for CB2 [50]. A presumed water soluble CB2 series based on imidazo diazepine, 29 (n ¼ 1) and its imidazo piperazine (n ¼ 0) congener, is the subject of a separate disclosure [51]. Compound 29 (hCB2/hCB1 EC50 ¼ 10 B 100 nM/W10 mM in cAMP) exhibited analgesic activity in the murine acetic acid-induced writhing of model visceral pain (3, 10, and 30 mg/kg, s.c.), in the carrageenan model of acute inflammation in rats (30 mg/kg, s.c.), and exhibited an anti-allodynic response in the spinal nerve ligation model of neuropathic pain in rats (30 mg/kg, p.o.) [51].
3.2.3 Miscellaneous bicyclic cores
O
HN N
O
N
O
N
N S O O
N
N Cl
Cl 30
Cl
31
32
Various 6,6-fused heterocycles were reported as selective CB2 modulators. SAR studies of a series of quinolone-3-carboxamides have been described [52]. The nature of the 3-carboxamide substituent seems to be important for both receptor affinity and selectivity, since the highly lipophilic adamantylamide is the key feature for governing excellent in vitro profiles. Compound 30 exhibited W190-fold selectivity over CB1 in the [3H]CP-55,940 binding assay (hCB2/hCB1 Ki ¼ 6.3 nM/1220 nM). Moreover, this compound displayed significant analgesic effects in the formalin test of acute peripheral and inflammatory pain in mice (3.0 mg/ kg i.p.). Specificity was confirmed by full reversal of the anti-nociceptive response by AM630 (3.0 mg/kg i.p.), a selective CB2 antagonist [52]. A new chemical series based on tetrahydronaphthyridinone scaffolds were reported. CB2-selective compound 31 is representative of this series (hCB2/hCB1 EC50 o100 nM/W10 mM in cAMP assay). Replacement of the methyl sulfone with acetyl led to a loss of selectivity [53]. A novel series of aminoquinazolines were also disclosed as selective CB2 receptor modulators and are exemplified by 32. The successful
240
Sangdon Han et al.
replacement of the piperidine group with pyrrolidine, morpholine, thiomorpholine, or piperazine was demonstrated in this disclosure [54].
R1 HN
CN
HN
O
O
N N
O
NH S
O H
F
NH N H
Cl 33 R1 = CH3 34 R1 = CH2OH
H
O
H 35
36
Glenmark has reported a series of CB2 agonists based on a bridged bicyclic pyrazole core motif [55] and has initiated phase I clinical trials with GRC-10693 (structure not disclosed) for inflammatory and neuropathic pain [56]. GRC-10693 showed W80-fold selectivity (hCB2/hCB1 Ki ¼ 11.8 nM/985.2 nM) over CB1 in [3H]CP-55,940 binding studies in human CHO cells, and B20-fold selectivity [rCB2 (spleen) Ki ¼ 12.8 nM and rCB1 (brain) Ki ¼ 253.5 nM] in rat tissues. GRC-10693 had a good pharmacokinetic properties in SD rats (t½ ¼ 5.78 h, %F ¼ 48, 10 mg/kg, p.o.). GRC-10693 demonstrated good oral efficacy in the FCA model of inflammatory pain (ED50 ¼ 1.67 mg/kg) and also significantly reversed chronic construction injury in male SD rats (ED50 ¼ 2.15 mg/kg, p.o.) [57]. In a subsequent PCT filing by Glenmark, ethanol-amide 34 displayed a significant improvement in pharmacokinetic parameters (Cmax ¼ 1,949 ng/mL, AUC0inf ¼ 11,970 ng h/mL, t1/2 ¼ 4.8 h) relative to compound 33 (Cmax ¼ 131.3 ng/mL, AUC0inf ¼ 1,553 ng h/mL, t1/2 ¼ 7.3 h) [58]. Recently, a new chemical series composed of a mono or a bicyclic aromatic core with vicinal bis-amides has appeared. Thieno pyran 35 was reported to be a potent and selective CB2 modulator (hCB2/hCB1 EC50 ¼ 21 nM/W1.5 mM). Intraperitoneal administration of 35 demonstrated analgesia in the rat FCA-induced mechanical hyperalgesia, skin incision, and spinal nerve ligation pain models [59,60]. Since the initial discovery of an aminoalkylindole cannabinoid ligand, WIN-55,212-2 [61], the indole has been repeatedly reported as a CB2-biased moiety. Indole 2-indane amides have been described as a new class of CB2 receptor modulators [62]. Unlike previous indole series, the indole 2-carboxamide chemotype recognizes the CB2 receptor
241
Recent Advances in the Discovery of CB2 Selective Agonists
without an N-1 alkyl substituent (e.g., compound 36, hCB2 binding Ki ¼ 0.24 nM) [62].
3.3 Tricyclic core-based CB2 agonists
O
O
O N N N
O O
N S
O
37
38
O
O OO S N H N H
O O 39
Several groups have disclosed CB2 ligands composed of a conformationally restricted tricyclic core [63–65]. Sch35966 37 was reported to be a potent CB2 agonist for both human and monkey receptors (human CB2 Ki ¼ 6.872.3 nM, monkey CB2 Ki ¼ 5.470.4 nM) and for rodent receptors (rat CB2 Ki ¼ 2.470.5 nM, mouse CB2 Ki ¼ 4.871.6 nM). Furthermore, this compound exhibited W450-fold selectivity over the CB1 receptor [63]. A new class of CB2 agonists based on the tetrahydropyrrolo indole (e.g., 38) moiety was also identified and assessed in vitro [64]. SAR of the tetrahydropyran greatly impacted the CB2 receptor affinity and selectivity. Pyrrolo tetrahydropyran 38 was described as a potent and full CB2 agonist (hCB2 EC50 ¼ 1.7 nM, Emax ¼ 107% relative to WIN55212-2) as determined in the GTPg[35S] assay and possessed the best selectivity ratio (hCB2/hCB1 Ki ¼ 17.6 nM/6183 nM) in this series. Further biological evaluation of several examples from within this series is currently underway [64]. A PCT application describing the synthesis and SAR of a series of octahydrophenanthridine core-based CB2 modulators, exemplified by 39 (hCB2/hCB1 EC50 ¼ 36.7 nM/W10 mM in cAMP), has been reported [65]. In the mouse acetic-acid-induced writhing assay, 39 demonstrated % maximum possible effect of 41% at 10 mg/kg, s.c., along with reduction of body temperature change of 0.21C [65].
4. CLINICAL TRIALS STATUS Three pharmaceutical companies have reported entering clinical trials with CB2 agonists for treatment of pain. Pharmos tested their candidate
242
Sangdon Han et al.
Cannabinor [PRS-211375 (40), a 37-fold selective CB2 agonist] i.v. in a third molar dental extraction phase IIa trial. Results of this trial were confounding. The lowest 12-mg dose showed significant effect, whereas two higher doses of 24 and 48 mg did not achieve significance. Pharmos also ran a separate phase IIa study for induced-pain in healthy volunteers. The 48-mg dose of Cannabinor was not effective in reducing capsaicin-induced pain, but did show activity in mechanical and thermal hyperalgesia in normal skin [66]. Pharmos has discontinued future development efforts with this compound [67]. O
O OH
O O HO 40
GSK has completed a phase II trial for dental pain (third molar tooth extraction) and two phase II trials for osteoarthritis pain with their clinical candidate GW842166. The results of these trials have not been disclosed thus far. Glenmark pharmaceutical’s recent press release indicated successful completion of a phase I trial in Europe for their clinical candidate GRC10693 for neuropathic pain, osteoarthritis, and inflammatory pain disorders [68]. The structure of this candidate has not been disclosed.
5. CONCLUSIONS Novel pain therapeutic alternatives with minimal adverse side effects and abuse potential are highly desired by patients and healthcare professionals. The preclinical data that have emerged so far with CB2 agonists has been promising and suggestive that therapies directed at this target could fulfill this unmet therapeutic need. Numerous CB2 agonists have now shown efficacy in multiple animal pain models without apparent CB1-associated psychotropic effects. Whether this promise will be fulfilled in the clinic will become apparent as clinical trial data emerges.
Recent Advances in the Discovery of CB2 Selective Agonists
243
REFERENCES [1] G. T. Whiteside, G. P. Lee and K. J. Valenzano, Curr. Med. Chem., 2007, 14, 917. [2] D. M. Slipetz, G. P. O’Neill, L. Favreau, C. Dufresne, M. Gallant, Y. Gareau, D. Guay, M. Labelle and K. M. Metters, Mol. Pharmacol., 1995, 48, 352. [3] (a) M. Bouaboula, C. Poinot-Chazel, J. Marchand, X. Canat, B. Bourrie´, M. RinaldiCarmona, B. Calandra, G. Le Fur and P. Casellas, Eur. J. Biochem., 1996, 237, 704; (b) V. Di Marzo and L. De Petrocellis, Ann. Rev. Med., 2006, 57, 553. [4] (a) S. Munro, K. L. Thomas and M. Abushaar, Nature, 1993, 365, 61; (b) S. Galiegue, S. Mary, J. Marchand, D. Dussossoy, D. Carriere, P. Carayon, M. Bouaboula, D. Shire, G. Lefur and P. Casellas, Eur. J. Biochem., 1995, 232, 54. [5] E. Nunez, C. Benito, M. R. Pazos, A. Barbachano, O. Fajardo, S. Gonzalez, R. M. Tolon and J. Romero, Synapse, 2004, 53, 208. [6] M. D. Van Sickle, M. Duncan, P. J. Kingsley, A. Mouihate, P. Urbani, K. Mackie, N. Stella, A. Makriyannis, D. Piomelli, J. S. Davison, L. J. Marnett, V. Di Marzo, Q. J. Pittman, K. D. Patel and K. A. Sharkey, Science, 2005, 310, 329. [7] U. Anand, W. R. Otto, D. Sanchez-Herrera, P. Facer, Y. Yiangou, Y. Korchev, R. Birch, C. Benham, C. Bountra, I. P. Chessell and P. Anand, Pain, 2008, 138, 667. [8] G. Wotherspoon, A. Fox, P. McIntyre, S. Colley, S. Bevan and J. Winter, Neuroscience, 2005, 135, 235. [9] K. T. Valenzano, L. Tafesse, G. Lee, J. E. Harrison, J. M. Boulet, S. L. Gottshall, L. Mark, M. S. Peason, W. Miller, S. Shan, L. Rabadi, Y. Rotshteyn, S. M. Chaffer, P. I. Turchin, D. A. Elsemore, M. Toth, L. Koetzner and G. T. Whiteside, Neuropharmacology, 2005, 48, 658. [10] B. B. Yao, G. C. Hsieh, J. M. Frost, Y. Fan, T. R. Garrison, A. V. Daza, G. K. Grayson, C. Z. Zhu, M. Pai, P. Chandran, A. K. Salyers, E. J. Wensink, P. Honore, J. P. Sullivan, M. J. Dart and M. D. Meyer, Br. J. Pharmacol., 2008, 153, 390. [11] B. Bingham, P. G. Jones, A. J. Uveges, S. Kotnis, P. Lu, V. A. Smith, S. C. Sun, L. Resnick, M. Chlenov, Y. He, B. W. Strassle, T. A. Cummons, M. J. Piesla, J. E. Harrison, G. T. Whiteside and J. D. Kennedy, Br. J. Pharmacol., 2007, 151, 1061. [12] B. B. Yao, G. Hsieh, A. V. Daza, Y. Fan, G. K. Grayson, T. F. Garrisob, O. E. Kouhen, B. A. Hooker, M. Pai, E. J. Wensink, A. K. Salyers, P. Chandran, C. Z. Zhu, C. Zhong, K. Ryther, M. E. Gallagher, C. L .Chin, A. E. Tovcimak, V. P. Hradil, G. B. Fox, M. J. Dart, P. Honore and M. D. Meyer, J. Pharmacol. Exp. Therap., 2009, 328, 141. [13] M. Rinaldi-Carmona, F. Barth, J. Millan, J. M. Derocq, P. Casellas, C. Congy, D. Oustric, M. Sarran, M. Bouaboula, B. Calandra, M. Portier, D. Shire, J. C. Brelie`re and G. L. Fur, J. Pharmacol. Exp. Therap., 1998, 284, 644. [14] Y. Hosohata, R. M. Quock, K. Hosohata, A. Makriyannis, P. Consroe, W. R. Roeske and H. I. Yamamura, Eur. J. Pharmacol., 1997, 321(1), R1. [15] G. M. P. Giblin, C. T. O’shaughnessy, A. Naylor, W. L. Mitchell, A. J. Eatherton, B. P. Slingsby, D. A. Rawlings, P. Goldsmith, A. J. Brown, C. P. Haslam, N. M. Clayton, A. W. Wilson, I. P. Chessell, A. R. Wittington and R. Green, J. Med. Chem., 2007, 31, 2597. [16] M. Naguib, P. Diaz, J. J. Xu, F. Astruc-Diaz, S. Craig, P. Vivas-Mejia and D. L. Brown, Br. J. Pharmacol., 2008, 155, 1104. [17] A. Kikuchi, K. Ohashi, Y. Sugie, H. Sugimoto and H. Omura, J. Pharmacol. Sci., 2008, 106, 219. [18] H. Omura, M. Kawai, A. Shima, Y. Iwata, F. Ito, T. Masuda, A. Ohta, N. Makita, K. Omoto, H. Sugimoto, A. Kikuchi, H. Iwata and K. Ando, Bioorg. Med. Chem. Lett., 2008, 18, 3310. [19] K. Maresz, G. Pryce, E. D. Ponomarev, G. Marsicano, J. L. Croxford, L. P. Shriver, C. Ledent, X. Cheng, E. J. Carrier, M. K. Mann, G. Giovannoni, R. G. Pertwee, T.
244
[20]
[21]
[22] [23]
[24] [25] [26]
[27]
[28]
[29] [30] [31]
[32]
[33]
[34]
[35]
[36]
[37]
Sangdon Han et al.
Yamamura, N. E. Buckley, C. J. Hillard, B. Lutz, D. Baker and B. N. Dittel, Nat. Med., 2007, 13, 492. J. Palazuelos, N. Davoust, B. Julien, E. Hatterer, T. Aguado, R. Mechoulam, C. Benito, J. Romero, A. Silva, M. Guzman, S. Nataf and I. Galve-Roperh, J. Biol. Chem., 2008, 283, 13320. L. Hanus, A. Breuer, S. Tchilibon, S. Shiloah, D. Goldenberg, M. Horowitz, R. G. Pertwee, R. A. Ross, R. Mechoulam and E. Fride, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 14228. M. G. Belvisi, H. J. Patel, V. Freund-Michel, D. J. Hele, N. Crispino and M. A. Birrell, Br. J. Pharmacol., 2008, 155, 547. O. Ofek, M. Karsak, N. Leclerc, M. Fogel, B. Frenkel, K. Wright, J. Tam, M. AttarNamdar, V. Kram, E. Shohami, R. Mechoulam, A. Zimmer and I. Bab, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 696. S. Steffens, N. R. Veillard, C. Arnaud, G. Pelli, F. Burger, C. Staub, A. Zimmer, J. Frossard and F. Mach, Nature, 2005, 434, 782. E. K. Ho¨genauer, Exp. Opin. Ther. Patents, 2007, 17(12), 1457. P. Markt, C. Feldmann, J. M. Rollinger, S. Raduner, D. Schuster, J. Kirchmair, S. Distinto, G. M. Spitzer, G. Wolber, C. Laggner, K.-H. Altmann, T. Langer and J. Gertsch, J. Med. Chem., 2009, 52, 369. W. L. Mitchell, G. M. P. Giblin, A. Naylor, A. J. Eatherton, B. P. Slingsby, A. D. Rawlings, K. S. Jandu, C. P. Haslam, A. J. Brown, P. Goldsmith, N. M. Clayton, A. W. Wilson, I. P. Chessell, R. H. Green, A. R. Whittington and I. D. Wall, Bioorg. Med. Chem. Lett., 2009, 19, 259. R. Zindell, D. Reither, T. Bosanac, A. Berry, M. J. Gemkow, A. Ebneth, S. Lo¨bbe, E. L. Raymond, D. Thome, D.-T. Shih and D. Thomson, Bioorg. Med. Chem. Lett., 2009, 19, 1604. D. Reither, D. Thomson and R. Zindell, WO Patent Application 2008/48914, 2008. C. Leung, M. Tomaszewski and S. Woo, WO Patent Application 2004/060882, 2004. K. Worm, Q. J. Zhou, C. T. Saeui, R. C. Green, J. A. Cassel, G. J. Stabley, R. N. DeHaven, N. Conway-James, C. J. LaBuda, M. Koblish, P. J. Little and R. E. Dolle, Bioorg. Med. Chem. Lett., 2008, 18, 2830. M. Ermann, D. Riether, E. R. Walker, I. F. Mushi, J. E. Jenkins, B. Noya-Marino, M. L. Brewer, M. G. Taylor, P. Amouzegh, S. P. East, B. W. Dymock, M. J. Gemkow, A. F. Kahrs, A. Ebneth, S. Lo¨bbe, K. O’Shea, D.-T. Shih and D. Thomson, Bioorg. Med. Chem. Lett., 2008, 18, 1725. M. Emann, D. Riether, I. Mushi, J. E. Jenkins, E. Walker, B. Noya-Marino, M. Brewer, M. Taylor, P. Amouzegh, S. P. East, B. Dymock, M. Gemkow, S. Lo¨bbe, A. Ebneth, A. F. Kahrs, D. Thome, K. O’Shea, R. Dinallo, E. Raymond, D.-T. Shih and D. Thomson, Molecular Medicine Tri-Conference, San Francisco, CA, March 2008. A. J. Goodman, C. W. Ajello, K. Worm, B. L. Bourdonnec, M. A. Savolaninen, H. O’Hare, J. A. Cassel, G. J. Stabley, R. N. DeHaven, C. J. LaBuda, M. Koblish, P. J. Little, B. L. Brogdon, S. A. Smith and R. E. Dolle, Bioorg. Med. Chem. Lett., 2009, 19, 309. Y. Cheng, B. K. Albecht, J. Brown, J. L. Buchanan, W. H. Buckner, E. F. DiMauro, R. Emkey, R. T. Fremeau, J.-C. Harmange, B. J. Hoffman, L. Huang, M. Huang, J. H. Lee, F.-F. Lin, M. W. Martin, H. Q. Nguyen, V. F. Patel, S. A. Tomlinson, R. D. White, X. Xia and S. A. Hitchcock, J. Med. Chem., 2008, 51, 5019. E. F. DiMauro, J. L. Buchanan, A. Cheng, R. Emkey, S. A. Hitchcock, L. Huang, M. Y. Huang, B. Janosky, J. H. Lee, X. Li, M. W. Martin, S. A. Tomlinson, R. D. White, X. M. Zheng, V. F. Patel and R. T. Fremeau,, Bioorg. Med. Chem. Lett., 2008, 18, 4267. E. F. DiMauro, J. L. Buchanan and A. Cheng, XXth International Medicinal Chemistry Symposium, Vienna, Austria, September 2008.
Recent Advances in the Discovery of CB2 Selective Agonists
245
[38] H. Ohta, T. Ishizaka, M. Yoshinaga, A. Morita, Y. Tomishima, Y. Toda and S. Saito, Bioorg. Med. Chem. Lett., 2007, 17, 5133. [39] H. Ohta, T. Ishizaka, M. Tatsuzuki, M. Yoshinaga, I. Iida, Y. Tomishima, Y. Toda and S. Saito, Bioorg. Med. Chem. Lett., 2007, 17, 6299. [40] H. Ohta, T. Ishizaka, M. Tatsuzuki, M. Yoshinaga, I. Iida, T. Yamaguchi, Y. Tomishima, N. Futaki, Y. Toda and S. Saito, Bioorg. Med. Chem., 2008, 16, 1111. [41] P. F. Cirillo, D. S. Thomson, D. Reither, A. Berry, L. Wu and E. R. Hickey, WO Patent Application 2008/064054, 2008. [42] A. Berry, P. F. Cirillo, E. R. Hickey, D. Reither, D. Thomson, R. M. Zindell, M. Ermann, J. E. Jenkins, I. Mushi, M. Taylor, C. Chowdhury, C. F. Palmer and N. Blumire, WO Patent Application 2008/039645, 2008. [43] I. E. Marx, E. F. DiMauro, A. Cheng, R. Emkey, S. A. Hitchcock, L. Huang, M. Y. Huang, J. Human, J. H. Lee, X. Li, M. W. Martin, R. D. White, R. T. Fremeau, Jr. and V. F. Patel, Bioorg. Med. Chem. Lett., 2009, 19, 31. [44] B. M. P. Verbist, M. A. Cleyn, M. Surkyn, E. Fraiponts, J. Aerssens, M. J. M. A. Nijsen and H. J. M. Gijsen, Bioorg. Med. Chem. Lett., 2008, 18, 2574. [45] H. J. M. Gijsen, M. A. J. De Cleyn, M. Surkyn and B. M. P. Verbist, WO Patent Application 2008/003665, 2008. [46] H. J. M. Gijsen, M. A. J. De Cleyn and M. Surkyn, WO Patent Application 2008/119694, 2008. [47] D. Page, E. Balaux, L. Boisvert, Z. Liu, C. Milburn, M. Tremblay, Z. Wei, S. Woo, X. Luo, Y.-X. Cheng, H. Yang, S. Srivastava, F. Zhou, W. Brown, M. Tomaszewski, C. Walpole, L. Hodzic, S. St-Onge, C. Godbout, D. Salois and K. Payza, Bioorg. Med. Chem. Lett., 2008, 18, 3695. [48] Z. Liu, H. Yang, J. Ducharme, D. Page, Z.-Y. Wei, M. Tremblay, S. Srivastava, R. Dolaine, C. J. Milburn, E. Lessard, P. E. Morin, S. St-Onge, K. Payza and C. Walpole, Abs 240, The 31st National Medicinal Chemistry Symposium, Pittsburgh, PA, June 2008. [49] M. T. Bilodeau, C. S. Burgey, Z. J. Deng, J. C. Hartnett, N. R. Kett, J. Melamed, P. M. Munson, K. K. Nanda, W. Thomson, B. W. Trotter and Z. Wu, WO Patent Application 2008/085302, 2008. [50] Z. Wu, A. I. Green and J. C. Hartnett, WO Patent Application 2009/025785, 2009. [51] R. P. Beckett, R. Foster, C. Henault, J. L. Ralbovsky, C. M. Gauss, G. R. Gustafson, Z. Luo, A.-M. Campbell and T. E. Shelekhin, WO Patent Application 2008/157751, 2008. [52] S. Pasquini, L. Botta, T. Semeraro, C. Mugnaini, A. Ligresti, E. Palazzo, S. Mainoe, V. Marzo and F. Corelli, J. Med. Chem., 2008, 51, 5075. [53] G. R. Gustafson and R. P. Beckett, WO Patent Application 2008/079316, 2008. [54] T. C. Gahman, D. J. Thomas, H. Lang and M. E. Massari, WO Patent Application 2008/ 157500, 2008. [55] M. Muthuppalanippan, G. Balasubramanian, S. Gullapalli, N. Khairatkar-Joshi and S. Narayanan, WO Patent Application 2006/129178, 2006. [56] http://www.glenmarkpharma.com/media/pdf/releases/glenmark_molecule_GRC_ 10693.pdf [57] S. Narayanan, M. Muthuppalanippan, A. Thomas, N. Khairatkar-Joshi, K. Varanasi, S. Gullapalli and S. K. V. S. Vakkalanka, Society for Neuroscience, Atlanta, Georgia, Oct. 14, 2006. [58] M. Muthuppalanippan, K. Sukeerthi, G. Balasubramanian, S. Gullapalli, N. KhairatkarJoshi, S. Narayanan and P. V. Karnik, WO Patent Application 2008/053341, 2008. [59] W. A. Carroll, D. W. Nelson and A. Perez-Medrano, WO Patent Application 2009/ 009550, 2009. [60] D. W. Nelson, M. E. Gallagher, K. Ryther, Abs 220, The 31st National Medicinal Chemistry symposium, Pittsburgh, PA, June 2008.
246
Sangdon Han et al.
[61] D. R. Compton, L. H. Gold, S. J. Ward, R. L. Balster and B. R. Martin, J. Pharmacol. Exp. Therap, 1992, 263(3), 1118. [62] C. Liu, S. T. Wrobleski, K. Leftheris, G. Wu, P. M. Sher and B. A. Ellsworth, WO Patent Application 2009/015169, 2009. [63] W. Gonsiorek, C. A. Lunn, X. Fan, G. Deno, J. Kozlowski and R. W. Hipkin, Br. J. Pharmacol., 2007, 151, 1261. [64] D. Page, H. Yang, W. Brown, C. Walpole, M. Fleurent, M. Fyfe, F. Gaudreault and S. St-Onge, Bioorg. Med. Chem. Lett., 2007, 17, 6183. [65] J. L. Ralbovsky and P. R. Beckett, WO Patent Application 2008/109007, 2008. [66] http://www.pharmoscorp.com/news/pr/pr042407.html: Company press release. [67] http://integrity.prous.com/integrity/servlet/xmlxsl/pkpipeline.xmlProductMielstone? pentryNumber ¼ 352376 [68] http://www.glenmarkpharma.com/media/pdf/releases/Glenmarks_molecule_ neuropathicpain_osteoarthritis_GRC10693_succes.pdf. Company press release.
CHAPT ER
12 Advances in the Discovery of Small Molecule JAK3 Inhibitors Stephen T. Wrobleski and William J. Pitts
Contents
1. Introduction 2. Rationale for Selective Targeting of JAK3 in Inflammatory Diseases 3. Clinical Trials and Supporting Preclinical Data 3.1 Clinical candidates 3.2 Organ transplantation 3.3 Rheumatoid arthritis 3.4 Psoriasis 4. Challenges in Designing Selective JAK3 Inhibitors 5. Recent Medicinal Chemistry Efforts 5.1 Pyrrolopyrimidines 5.2 Pyrrolopyridines 5.3 Purines and purinones 5.4 Monocyclic pyridines and pyrimidines 5.5 Other heterocycles 6. Conclusions References
247 248 249 250 251 252 253 253 255 255 257 258 259 260 261 261
1. INTRODUCTION JAnus Kinase 3 (JAK3) is a member of the JAK family of non-receptor protein tyrosine kinases (PTKs) that include the closely related isoforms JAK1, JAK2 and tyrosine kinase 2 (TYK2). In the early 1990s, TYK2 was identified as the prototypical member of this new kinase family [1]. Bristol-Myers Squibb R&D, Route 206 & Province Line Rd., Princeton, NJ 08543, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04412-1
r 2009 Elsevier Inc. All rights reserved.
247
248
Stephen T. Wrobleski and William J. Pitts
Subsequent to this initial discovery, a second group of researchers independently identified two additional family members, which they noted contained two adjacent kinase domains: the catalytically active JH1 domain and a non-typical, pseudokinase domain JH2 that appears to be catalytically inactive [2]. The presence of the dual kinase domains led the researchers to name these kinases as JAK1 and JAK2 after the two-faced Roman god, Janus. The function of these kinases was not known at the time of their discovery, and JAK was also referred to as ‘Just Another Kinase’. Ironically, it was subsequently discovered that these kinases played a critical role in the signal transduction of the cytokine receptor superfamily [3]. Once the importance of this kinase family became known, a third group of researchers used a polymerase chain reaction (PCR) strategy to identify JAK3 as the last family member [4]. Since inhibition of the JAK kinases would be expected to block cytokine signaling, members of this family have become attractive targets for small-molecule drug discovery [5]. The realization that human defects in JAK3 signaling result in the clinical manifestation of a severe combined immunodeficiency (SCID) phenotype has suggested that selective JAK3 inhibitors may be useful as therapeutic agents in the areas of organ transplantation and autoimmune diseases.
2. RATIONALE FOR SELECTIVE TARGETING OF JAK3 IN INFLAMMATORY DISEASES The JAK signal transducer and activator of transcription (STAT) signaling pathway has been extensively studied but specific details regarding cascade regulation remain unelucidated [6]. In cells, JAK3 associates with cytokine receptors, which homodimerize and heterodimerize upon binding of the ligand. JAK3 binds specifically to the gamma common chain (gc) that is shared by the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-21 [5]. Upon ligand binding, JAK3 becomes activated in concert with JAK1 through transphosphorylation which in turn results in phosphorylation of the receptor. Receptor phosphorylation then results in binding and phosphorylation of an associated STAT protein (e.g., STAT5 in the case of IL-2 stimulation and STAT6 in the case of IL-4 stimulation). After STAT dimerization, the complex is thought to translocate to the nucleus, interact with DNA, and initiate transcription of target genes (Figure 1). Because JAK3 plays a specific role in regulating gc cytokine signaling and is primarily expressed in lymphoid tissues, it appears to be a selective regulator of lymphoid development and function within the immune system [7]. This is consistent with the SCID phenotype that has been identified in a subpopulation of patients that harbor abnormalities associated with JAK3 based on genetic analysis [8]. These abnormalities
Advances in the Discovery of Small Molecule JAK3 Inhibitors
249
IL-2, IL-4, IL-7, IL-9, IL-15, IL-21
γc
JAK1
JAK3
STAT
STAT STAT
STAT
P
P Cytoplasm P
Nucleus
STAT STAT STAT STAT P DNA
Gene transcription
Figure 1 Schematic of gamma common chain cytokine receptor and JAK3 signaling pathway.
include a ‘kinase dead’ mutant, a mutant incapable of binding the gc of the IL-2 family of receptors, or a lack of detectable JAK3 protein, each of which is associated with a human SCID phenotype [9]. This disorder is characterized by a significant decrease in the number of circulating T and NK cells with normal numbers of B cells (albeit with compromised B-cell function). Hematopoetic stem cell transplantation results in the normalization of the T-cell population in these patients [10]. Since patients with defects in JAK3 show symptoms that are restricted to the immune system, it follows that a selective inhibitor of JAK3 could function as a well-tolerated immunosuppressant for use in a number of autoimmune disorders [11]. In comparison with JAK3, the other JAK family members, JAK1, JAK2 and TYK2, are known to be more ubiquitous in their expression patterns. In addition to participating in concert with JAK3 in the signaling of the IL-2 family of cytokine receptors, JAK1 has been shown to be a regulator of IL-6 and gp130 cytokine signaling. It has therefore been suggested that dual inhibition of JAK1 and JAK3 might lead to enhanced cellular potency and broader immunosuppressive effects, but may also increase the risk of viral and bacterial infections [12]. Moreover, JAK1 has been linked to tumor surveillance [13], and JAK1 knockout mice do not thrive, as the pups fail to nurse because of presumed neurological defects [14]. Despite these potential concerns, no adverse clinical outcome has been conclusively linked to JAK1 inhibition
250
Stephen T. Wrobleski and William J. Pitts
(vide infra). These observations make it difficult to assess the risk to benefit ratio of dual inhibition of JAK1 and JAK3 a priori. JAK2 is classically associated with interferon-g (IFN-g) production through the IL-12 pathway; however, it also mediates the signaling of important hematopoietic growth factors such as erythropoietin (EPO), thrombopoietin (TPO) and granulocyte macrophage colony-stimulating factor (GM-CSF). As a result, JAK2 inhibitors are being examined for oncological applications, and this is beyond the scope of this review. It has been suggested that JAK2 inhibition may result in adverse hematopoietic effects such as anemia, thrombocytopenia and generalized leukopenia in the clinic [12].
3. CLINICAL TRIALS AND SUPPORTING PRECLINICAL DATA 3.1 Clinical candidates
N
N
CN O
N N
N H 1
The clinical candidate CP-690,550 (1) is one of the most extensively studied JAK3 inhibitor to date. A review on CP-690,550 has recently been published [15]. This compound was originally reported to be a selective JAK3 inhibitor within the JAK family (JAK3 IC50 ¼ 1 nM, JAK2 IC50 ¼ 20 nM, JAK1 IC50 ¼ 112 nM) with generally high selectivity against other tested kinases [16]. However, a recent disclosure based on Ki values has indicated that CP-690,550 is less selective within the JAK family than originally reported (JAK3 Ki ¼ 0.2 nM, JAK2 Ki ¼ 1.0 nM, JAK1 Ki ¼ 0.7 nM, Tyk2 Ki ¼ 4.4 nM) [17]. Other researchers have also reported a lower degree of JAK3 selectivity versus JAK2 and JAK1 [18,19]. However, a high degree of selectivity against greater than 300 non-JAK family kinases has been confirmed [20]. Despite its potent activity against the JAK2 enzyme, CP-690,550 shows modest selectivity for inhibition of JAK1/3 signaling pathways compared to JAK2 in cellular assays. CP-690,550 in human whole blood stimulated with anti-CD3/CD28 in the presence of IL-2 gave potent inhibition of the JAK1/3 pathway (IC50 ¼ 34 nM) and was B15-fold more selective relative to the inhibition
Advances in the Discovery of Small Molecule JAK3 Inhibitors
251
of the JAK2 pathway as measured by IL-12 stimulation in human whole blood (IC50 ¼ 501 nM) [17]. In separate assays using fluorescenceactivated cell sorting (FACS) analysis in human whole blood, the inhibition of IL-15 activation of pSTAT5 in CD8+ T cells (JAK1/3 pathway) relative to GM-CSF activation of pSTAT3 in CD14+ monocytes (JAK2 pathway) was assessed. In these assays, CP-690,550 was determined to be B24-fold selective for JAK1/3 relative to JAK2 (IC50 ¼ 56 and 1377 nM, respectively) [17]. To date, phase II clinical studies in rheumatoid arthritis with CP-690,550 have been completed, and patients for phase III studies are being actively recruited. Phase II studies in kidney transplant have also been completed with additional phase II studies recruiting patients in psoriasis (oral and topical administration), Crohn’s disease and dry-eye syndrome (topical) [21]. Two additional JAK3 inhibitors, R-348 and VX-509 (structures undisclosed), have been reported to have entered clinical trials. R-348 has entered phase I trials for rheumatoid arthritis, psoriasis and other immunerelated disorders [22]. A subsequent report disclosed that R-348 acts as a prodrug that is converted to an active metabolite, R-333. The latter inhibits both the JAK1/3 and Syk pathways in cellular assays (IC50 ¼ 180 and 140 nM, respectively) [23]. The JAK3 and Syk enzyme IC50 values for R-348 or R-333 have not been reported. The JAK3 inhibitor VX-509 has recently completed phase I studies in healthy volunteers with phase II studies in rheumatoid arthritis expected to begin in the second half of 2009 [24]. The potency and selectivity profile of VX-509 has not been reported to date. CN
N N
N N H
N 2
In addition to these JAK3 inhibitors, a reported JAK1/JAK2 inhibitor, INCB18424 (2), is being studied for oncological indications and in rheumatoid arthritis and psoriasis clinical trials [25]. This compound has been reported to give potent inhibition of both JAK1 and JAK2 with selectivity versus JAK3 and TYK2 (IC50 values for JAK1, JAK2, JAK3 and TYK2 are 2.7, 4.5, 332 and 19 nM, respectively) [26]. The paradox of a
252
Stephen T. Wrobleski and William J. Pitts
JAK1/2 inhibitor with selectivity versus JAK3 demonstrating efficacy in autoimmune disease may be explained by the fact that all receptors which use JAK3 also use JAK1 for signaling [27]. INCB28050 (structure undisclosed) has also been reported as a potent JAK1/JAK2 inhibitor in phase I clinical trails as a backup clinical compound to INCB18424 [28].
3.2 Organ transplantation As discussed earlier, patients with deletions or mutations in JAK3 are severely immunocompromised. As a result, the prevention of organ rejection is a logical indication for JAK3 inhibitors. CP-690,550 monotherapy has been reported to prevent kidney allograft rejection in nonhuman primates [29] and decrease T cell and NK cell populations. In combination with mycophenolate mofetil [30], significant improvement in allograph survival was noted. CP-690,550 has also been shown to prevent allograph vasculopathy in a rodent model of aortic transplantation [31]. CP-690,550 has been examined in a phase I trial (dosed at 5, 15 and 30 mg BID for 28 days) in a population of stable renal allograft recipients maintained on mycophenolate mofetil and, in the case of the 5 and 15 mg dose, a calcinurin inhibitor (cyclosporin or tacrolimus). In this study CP-690,550 demonstrated an acceptable safety and tolerability profile. The most frequent adverse events were related to infections and GI tolerability [32]. Slight decreases in hemoglobin and reticulocyte counts (reversible) at the higher doses were also noted. Examination of the immune system of these patients showed a decrease in the number of NK cells and T-reg cells (other T-cell subpopulations appeared unchanged), an increase in the number of B cells and a decrease in IL-2stimulated IFN-g production [33]. The JAK3/Syk inhibitor R-348 has also been reported to be effective in a rat cardiac allograft model [23].
3.3 Rheumatoid arthritis CP-690,550 has been shown to be effective at reducing clinical scores and related histological inflammatory changes when administered by minipump with an ED50 of B1.5 mg/kg/day in both a mouse collagen– induced arthritis model and a rat adjuvant–induced arthritis model [34]. Analysis of dendritic cells from the synovial tissue of patients with rheumatoid arthritis showed high levels of expression of JAK3, STAT4 and STAT6 [35]. CP-690,550 was evaluated in a randomized placebocontrolled phase IIa study of patients with moderate to severe active rheumatoid arthritis at doses of 5, 15 and 30 mg BID. At week 6, 13–28% of patients achieved an ACR70 score compared to placebo (3%), 33–54% of patents achieved an ACR50 score compared to placebo (6%), and 70–81% of patients achieved an ACR20 score compared to placebo (29%)
Advances in the Discovery of Small Molecule JAK3 Inhibitors
253
[36]. In a separate study, patients (n ¼ 509, 80% women) with active disease while maintained on a background of methotrexate were randomized to placebo, or CP,690-550 dosed at 1, 3, 5, 10, 15 mg, all BID, or 20 mg QD [37]. At week 12, doses of 3 mg BID or higher were efficacious as measured by ACR20, ACR50 and ACR70 scores. The most frequent adverse events were nausea (2.4%), headache (2.2%) and increase in alanine aminotransferase (ALT) levels (2.0%). Five serious infections were reported with no apparent dose-related pattern. Other observations included minor dose-related decreases in hemoglobin, and dose-dependent increases in low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol. The JAK1/JAK2 inhibitor, INCB18424, has been reported to demonstrate preliminary efficacy in a small clinical trial (n ¼ 41) of rheumatoid arthritis. ACR 20, 50 or 70 scores were achieved in up to 83, 50 or 30% of patients when dosed at 15, 25 or 50 mg BID for 28 days, respectively [38].
3.4 Psoriasis CP-690,550 has been reported to produce dose-dependent improvements in mPASI scores (28 at 5 mg BID, 52 at 30 mg BID) when dosed for 14 days in patients with psoriatic lesions [39]. A decrease in keratin 16 expression, as measured by immunohistochemistry, was observed in three of four skin biopsies in the 30 mg BID group. This was consistent with a reversal of hyperplasia and other disease-associated pathology. INCB18424 has been studied as a topical agent (1.5% cream applied BID) in patients with psoriatic lesions and was shown to produce a similar level of efficacy to topical steroids [40].
4. CHALLENGES IN DESIGNING SELECTIVE JAK3 INHIBITORS Selective inhibition of specific isoforms within a kinase family represents a formidable challenge in drug discovery since small-molecule kinase inhibitors most commonly target the highly conserved adenosine triphosphate (ATP)-binding domain. The JAK kinase family consists of four members, JAK1, JAK2, JAK3 and TYK2, which contain B1,100 amino acids that constitute the seven homology domains JH1–JH7. The JH1 domain (kinase domain) is highly conserved across the family, with JAK2 having the highest sequence identity compared to JAK3 (62% homologous) closely followed by JAK1 (52%) and TYK2 (50%). X-ray crystallographic structures of the kinase domain of JAK1, JAK2 and JAK3 in complex with inhibitors have been reported [18,41,42]. These structures have elucidated the JAK1 and JAK2 binding modes of the clinical candidate CP-690,550 (1), the JAK3 binding mode of the staurosporine analog AFN941 (3) and the JAK2 binding mode of the
254
Stephen T. Wrobleski and William J. Pitts
Glycine Loop
Glycine Loop H3C
CN
Glycine Loop
NH
O
Me Me
H3C
O N
CH3
O
N
H HN
Me
N
N N Me N
N
N H O
H
N
1 H
N Hinge region
3 O
H N Hinge region
F
N
O
O
H
4 O
H N
Hinge region
Figure 2 Schematic of compounds 1, 3 and 4 illustrating key hydrogen bonds to hinge region in a generic JAK family active site.
tetracyclic pyridone 4 (Figure 2). In all cases, the inhibitors bind within the ATP-binding site and form dual H-bonds to the hinge region of the protein. In addition, the N-terminal portion of the activation loop that contains the highly conserved Asp-Phe-Gly (DFG) motif adopts the ‘inward’ conformation characteristic of active kinases. A patent application disclosing a crystal structure of a JAK3 kinase domain complexed with adenylyl-imidodiphosphate (AMP-PNP) has also been published [43]. At present, there are no reported crystal structures for TYK2 or any fulllength JAK family member. On the basis of the JAK3 X-ray structure of 3, a sequence alignment analysis of the active site residues that are in proximity to the complexed inhibitor revealed only two differences that the authors believed could be exploited to provide selectivity between JAK3 and its most closely related isoform JAK2 [41]. They speculated that JAK2-selective compounds could be designed to exploit the extra space afforded by the difference between an active site glycine (JAK2) and Ala966 (JAK3). A subsequent report disclosing the JAK2 structure with 4 also revealed that the JAK2 glycine carbonyl is in a flipped conformation relative to that found in all other tyrosine kinase structures including the JAK3 structure with 3 [42]. In addition, exploiting the residue difference between Cys909 (JAK3) and an analogous serine residue in JAK2 has been suggested as a potential strategy for obtaining JAK3 selectivity over JAK2. Several JAK3 inhibitors have been reported, which might interact with Cys909.
255
Advances in the Discovery of Small Molecule JAK3 Inhibitors
Mannich base NC1153, and has been shown to be a selective inhibitor of JAK3 capable of preventing allograft rejection in a rodent transplantation model [44]. A patent has disclosed irreversible Bruton’s tyrosine kinase (BTK) inhibitors that react with the free thiol of an active site cysteine and are relatively selective [45]. Compound 6 inhibited both BTK and JAK3, whereas 7 did not inhibit JAK3. JAK2 inhibition data for these compounds were not disclosed. OPh HCl Me
Me
Me
N
N
NH2
Me
5
O
N
HCl
O
O
N H
N
N N
N
6 BTK IC50 = 0.5 nM JAK3 IC50 = 10.4 nM
7 BTK IC50 = 1.0 nM JAK3 IC50 > 10,000 nM
A recent comparison of the binding modes of 4 and CP-690,550 in JAK1 and JAK2 shows the protein structures to be very similar [18]. As a result the authors suggest that designing highly selective inhibitors for a specific JAK family member may represent a challenge. Three JAK1 residues Phe958, Arg879 and His885 differ when compared to JAK2, although these residues are in proximity to the active site. The authors also postulate that subtle variations in the electrostatic potential differences in the active site might also be useful to consider in the design of more selective compounds. Finally, a recent report using a JAK1 homology model has suggested that two residue differences between JAK3 and JAK1 [Glu(JAK1)-Asp(JAK3) and Phe(JAK1)-Tyr(JAK3)] may be important for obtaining selectivity between JAK3 and JAK1 [46].
5. RECENT MEDICINAL CHEMISTRY EFFORTS 5.1 Pyrrolopyrimidines The pyrrolopyrimidine ring system that is contained in the two clinical candidates, CP-690,550 and INCB18424, has been the most useful for generating potent JAK family inhibitors. Because of the potent activity and promising clinical efficacy reported for CP-690,550, knowledge of the SAR that was generated in its development would be of interest in medicinal chemistry efforts at optimizing JAK3 inhibition. Extensive disclosure of this SAR has not appeared to date, but some of the SAR of the chiral piperidine residue has been reported. This includes a recent patent application that provided JAK3 IC50 values for CP-690,550 and 30
256
Stephen T. Wrobleski and William J. Pitts
analogs using a glutathione S-transferase (GST)-tagged JH1 kinase domain [47]. CP-690,550 was the most potent analog with a JAK3 IC50 of 2.4 nM. Replacement of the cyanoacetyl group in CP-690,550 with the ethyl and propyl sulfonyl groups gave the less potent analogs 8 and 9 (JAK3 IC50 ¼ 14.3 and 17.3 nM, respectively). Importantly, preference for the 3R, 4R absolute configuration on the piperidine ring was illustrated by the loss in potency for the corresponding cis, racemic analogs 10–12 (JAK3 IC50 of 3.4, 137 and 59.5 nM, respectively). A further reduction in potency was observed for the C-4 des-methyl racemates 13 and 14 (IC50 ¼ 625 and 459 nM, respectively), and this suggests that the methyl group makes a significant contribution to JAK3 potency in this series. The des-methyl analog of CP-690,550 was not reported. The remaining analogs were significantly less potent than the enantiopure analogs 1, 8 and 9 and were mainly derivatives with modified piperidine N-substitutions. enantiopure
cis, racemates NH
NH 3R
R N
NH
N
N 4R
C-4 des-methyl, racemates
N
N
R N
1, R = -C(O)CH2CN 8, R = -SO2Et 9, R = -SO2CH2CH2CH3
N N
N
R N 4
10, R = -C(O)CH2CN 11, R = -SO2Et 12, R = -SO2CH2CH2CH3
N
N
13, R = -SO2Et 14, R = -SO2CH2CH2CH3
NH O NC
3
N
N 4
N
N
15, 3(S),4(S)-isomer 16, 3(R),4(S)-isomer 17, 3(S),4(R)-isomer
A recent report also confirmed the preference for the 3R, 4R configuration in 1 by independently synthesizing and testing the other three possible stereoisomers 15–17 (Ambit JAK3 Kd values were 0.7, 190, 180 and 150 nM, respectively) [48]. Additional Kd values for 1 indicated potent affinity for JAK2 (2 nM) and JAK1 (3 nM) and less for TYK2 (250 nM). These Kd values are more consistent with the Ki values that had been recently reported [17] and are significantly more potent than the JAK2 IC50 of 20 nM and the JAK1 IC50 of 112 nM that were originally
257
Advances in the Discovery of Small Molecule JAK3 Inhibitors
reported [16] for 1. Interestingly, despite its potent affinity for the JAK2 enzyme, 1 was shown to be ineffective at inhibiting JAK2 or TYK2mediated STAT4 phosphorylation in IL-12-stimulated human CD4+ T cells at concentrations up to 500 nM. In comparison, 1 was shown to be effective at significantly inhibiting JAK3/JAK1-mediated phosphorylation of STAT5 in IL-2-stimulated CD4+ cells at 10-fold lower concentrations (50 nM). On the basis of these results, the authors speculate that 1 may be capable of selectively inhibiting JAK3 without disrupting the functions of JAK2 or TYK2 in a cellular environment at the concentrations tested. These results are consistent with the previous studies of 1 in IL-2-stimulated versus IL-12-stimulated human whole blood, which showed IC50 of 34 and 501 nM, respectively, indicating B15-fold selectivity with respect to the inhibition of the JAK3/JAK1 relative to the JAK2/Tyk2 pathway [17]. As previously mentioned, the binding mode of 1 in the active site of JAK1 and JAK2 has been determined by recent X-ray crystallography studies [18]. Similar binding modes were observed, and this is consistent with the nearly equipotent activity of 1 that has been reported recently. A conformational analysis of 1 and molecular docking studies using the available crystal structures of JAK3 and JAK2 revealed similar binding modes for both JAK3 and JAK2 consistent with their similar Ki values against these enzymes. In the case of JAK3, the proposed model suggests the pyrrolopyrimidine core as the hinge-binding element forming key hydrogen bonding interactions between the N3 and N9 nitrogen atoms and residues Glu903 and Leu905. Furthermore, the chiral piperidine was proposed to bind within the phosphate-binding region with the cyano substituent forming a H-bond interaction with Arg953 of the activation loop. Other JAK3 inhibitors containing a pyrimidine-based 5,6-ring system have recently been reported in the patent literature. This includes substituted pyrrolopyrimidines such as 18 that have been claimed to be JAK and CDK inhibitors with particular embodiments around inhibiting JAK3 and CDK4 [49]. Pyrrolopyrimidines containing mainly saturated monocyclic and bicyclic amines linked to the pyrrolopyrimidine core as in 19 have also been claimed as JAK3 inhibitors [50].
O
N
CH3O N
N N H 18
N
O N
N H
F
N H 19
N
N H
258
Stephen T. Wrobleski and William J. Pitts
5.2 Pyrrolopyridines Analogs based on a pyrrolopyridine ring system have also been claimed as potent JAK3 inhibitors. Pyrrolopyridine 20 functionalized with a chiral 1-amino-2-methyl cyclohexane moiety was disclosed as having a JAK3 IC50 ¼ 3 nM [51]. A closely related tricyclic variant 21 with a dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2(1H)-one ring system was also reported to have similar potency (JAK3 IC50 ¼ 3 nM). Its ethyl analog, 22, still maintained respectable JAK3 potency (JAK3 IC50 ¼ 5.1 nM). Replacement of the chiral cyclohexane residue with other pharmacophores provided analogs with improved JAK3 inhibition, for example, the adamantyl analog 23 (JAK3 IC50 ¼ 0.65 nM) and the piperidine analog 24 (JAK3 IC50 ¼ 0.3 nM) [52,53]. R O HN
O
N HN
H2N N
N H
N H
N
21, R = Me 22, R = Et
20
CN F
N
O
O N
O
HN
CN N H
N 23
N
HN
H2N N
N H
24
5.3 Purines and purinones A purine ring system was utilized as a scaffold to generate JAK3 inhibitors such as compounds 25–27 [54]. They were claimed to have oral
259
Advances in the Discovery of Small Molecule JAK3 Inhibitors
activity in a mouse delayed-type hypersensitivity (DTH) model. A series of substituted 2-benzimidazoyl purine and purinone derivatives has also been recently disclosed as a new class of JAK3 inhibitors [55–58]. This structural class is unique among reported JAK3 inhibitors since the benzimidazoyl group can be recognized as the hinge binding motif [59] rather than the purine core. PS020613 (28) has been reported to be comparable in JAK3 potency and selectivity to CP-690,550 and has been shown to be orally active in a mouse DTH model [60]. Additional analogs closely related to 28, including 29–31, were also disclosed. In general, N-substitution at the 8-position of the purine ring, as in the case of 29, was found to improve JAK3 selectivity with respect to Aurora A (58-fold) [58]. Modification of the fluorine substitution pattern on the chromene moiety and replacement of the fluorine substituent on the benzimidazole ring with a cyano group gave 30, which displayed a further enhancement in JAK3 selectivity versus Aurora A (73-fold). An N,N-dimethylamino ethylene group at the 8-position of the purinone ring gave 31, which had a JAK3 selectivity versus Aurora A of 724-fold. R′
N
R′′ F
F
O
N
O
N
O N
N N H
N
N H
25, R′ = Me, R′′ = Pr 26, R′ = Et, R′′ = Pr 27, R′ = R′′ = Et
N N
N
N N
F
N
N
N
O N
N R
28, R = H 29, R = Me
NC
N
F O
N R
30, R = H 31, R = CH2CH2N(CH3)2
5.4 Monocyclic pyridines and pyrimidines In addition to bicyclic and tricyclic chemotypes, JAK3 inhibitors with monocyclic pyridine or pyrimidine cores such as 32 and 33 have been claimed [61,62]. They carry a potentially reactive acrylamide group like those used in irreversible, covalent inhibitors of BTK and HER2. JAK3 and these kinases contain an active site cysteine residue with a nucleophilic thiol functionality that can react with acrylamides to form a covalent attachment that irreversibly inhibits the enzyme. As previously mentioned, irreversible inhibition of JAK3 by targeting this cysteine residue has been postulated as a viable strategy for designing inhibitors that are selective against JAK3 since the JAK1 and JAK2 isoforms contain
260
Stephen T. Wrobleski and William J. Pitts
a much less nucleophilic serine residue at this location. Compounds 32 and 33 are claimed as selective JAK3 inhibitors that are at least 10-fold more potent against JAK3 relative to JAK1 and JAK2. A series of 2,4-diaminopyrimidines has also been claimed as selective inhibitors of the JAK pathway relative to the Syk pathway and as being effective in treating immune-related diseases [63]. Inhibition of the Syk pathway was determined using mast cell degranulation assay, whereas inhibition of the JAK pathway was determined using an IL-4-activated Ramos B-cell assay. For example, pyrimidine 34 was claimed to be W207-fold selective for the JAK pathway (IC50o0.240 nM) compared to the Syk pathway (IC50W50 uM) in these cellular assays [63]. As previously mentioned, the clinical candidate R-348 has been reported to inhibit both the JAK3 and the Syk pathways. H N H N
O
O
O
S
O N
N
N H N
N
N H 33
32
N HN F
N N
N N
N H
Cl 34
5.5 Other heterocycles Finally a report on the use of virtual screening to identify JAK3 inhibitors appeared [64]. Starting with the pan-JAK inhibitor 4 [41], iterative similarity searches identified 35 as a promising starting point for further
Advances in the Discovery of Small Molecule JAK3 Inhibitors
261
optimization. A pharmacophore search identified indazole 36. JAK family selectivity data for either lead were not presented. F
N NH
O NH
N
Ph N
O
N N H
HN N
H N
NH
N N
CF3
N N
4 JAK3 IC50 = 5 nM
35 JAK3 IC50 = 98 nM
36 JAK3 IC50 = 2,640 nM
6. CONCLUSIONS The pharmaceutical industry continues to invest in the discovery and development of orally active small molecules that may offer the potential to effectively treat significant inflammatory and immune-related diseases with minimal or no undesirable side effects. Targeted inhibition of the tyrosine kinase JAK3 has appeared as an attractive strategy in this regard primarily due to its localized tissue expression and specific effects within the immune system. The promising clinical efficacy reported for the JAK3 inhibitor CP-690,550 in rheumatoid arthritis patients is noteworthy and suggests that obtaining efficacy comparable to, or perhaps better than, the current marketed biologic therapies in this disease may be possible with a small molecule. Although highly selective inhibition of JAK3 for immunosuppression is particularly attractive from a safety perspective, it remains to be convincingly demonstrated in the clinic. While CP-690,550 does potently inhibit JAK3, it has been shown to inhibit to some extent other JAK family members, namely JAK1 and JAK2, which may contribute to enhanced efficacy in the clinic relative to purely selective JAK3 inhibition [27]. Subsequent medicinal chemistry efforts in generating inhibitors with alternative JAK family selectivity profiles, although challenging, may allow for the dissection of the pharmacology associated with the inhibition of JAK3 as well as the other members of this important family of enzymes.
REFERENCES [1] J. J. Krowelski, R. Lee, R. Eddy, T. B. Shows and R. Dalla-Favera, Oncogene, 1990, 5, 277. [2] A. F. Wilks, A. G. Harpur, R. R. Kurban, S. J. Ralph, G. Zurcher and A. Ziemiecki, Mol. Cell. Biol., 1991, 11, 2057.
262
Stephen T. Wrobleski and William J. Pitts
[3] J. N. Ihle, Trends Endocrinol. Metab., 1994, 5, 137. [4] S. G. Rane and E. P. Reddy, Oncogene, 1994, 9, 2415. [5] M. Pesu, A. Laurence, N. Kishore, S. H. Zwillich, G. Chan and J. J. O’Shea, Immunol. Rev., 2008, 223, 132. [6] P. J. Murray, J. Immunol., 2007, 178, 2623. [7] M. Aringer, S. R. Hofmann, D. M. Frucht, M. Chen, M. Centola, A. Morinobu, R. Visconti, D. L. Kastner, J. S. Smolen and J. J. O’Shea, J. Immunol., 2003, 170, 6057. [8] J. L. Roberts, A. Lengi, S. M. Brown, M. Chen, Y.-J. Zhou, J. J. O’Shea and R. H. Buckley, Blood, 2004, 103, 2009. [9] C. M. Mjannes, R. W. Hendershot, R. R. Quinones and E. W. Gelfand, J. Allergy Clin. Immunol., 2007, 119, 1542. [10] M. A. Slatter, K. Brigham, A. M. Dickinson, H. L. Harvey, D. Barge, A. Jackson, N. Bown, T. J. Flood, A. J. Cant, M. Abinun and A. R. Gennery, J. Allergy Clin. Immunol., 2008, 121, 316. [11] J. J. O’Shea, M. Husa, D. Li, S. R. Hofmann, W. Watford, J. L. Roberts, R. H. Buckley, P. Changelian and F. Candotti, Mol. Immunol., 2004, 41, 727. [12] L. L. Rokosz, J. R. Beasley, C. D. Carroll, T. Lin, J. Zhao, K. C. Appell and M. L. Webb, Expert Opin. Ther. Targets, 2008, 12, 883. [13] V. Sexl, B. Kovacic, R. Piekorz, R. Moriggl, D. Stoiber, A. Hoffmeyer, R. Liebminger, O. Kudlacek, E. Weisz, K. Rothammer and J. N. Ihle, Blood, 2003, 101, 4937. [14] S. J. Rodig, M. A. Meraz, J. M. White, P. A. Lampe, J. K. Riley, C. D. Arthur, K. L. King, K. C. F. Sheehan, L. Yin, D. Pennica, E. M. Johnson, Jr. and R. D. Schreiber, Cell, 1998, 93, 373. [15] L. A. Sorbera, N. Serradell, J. Bolos, E. Rosa and J. Bozzo, Drugs Future, 2007, 32, 674. [16] P. S. Changelian, M. E. Flanagan, D. J. Ball, C. R. Kent, K. S. Magnuson, W. H. Martin, B. J. Rizzuti, P. S. Sawyer, B. D. Perry, W. H. Brissette, S. P. McCurdy, E. M. Kudlacz, M. J. Conklyn, E. A. Elliott, E. R. Koslov, M. B. Fisher, T. J. Strelevitz, K. Yoon, D. A. Whipple, J. Sun, M. J. Munchhof, J. L. Doty, J. M. Casavant, T. A Blumenkopf, M. Hines, M. F. Brown, B. M. Lillie, C. Subramanyam, S.-P. Chang, A. J. Milici, G. E. Beckius, J. D. Moyer, C. Su, T. G. Woodworth, A. S. Gaweco, C. R. Beals, B. H. Littman, D. A. Fisher, J. F. Smith, P. Zagouras, H. A. Magna, M. J. Saltarelli, K. S. Johnson, L. F. Nelms, S. G. Des Etages, L. S. Hayes, T. T. Kawabata, D. Finco-Kent, D. L. Baker, M. Larson, M.-S. Si, R. Paniagua, J. Higgins, B. Holm, B. Reitz, Y.-J. Zhou, R. E. Morris, J. J. O’Shea and D. C. Borie, Science, 2003, 302, 875. [17] X. Li, M. Jesson, J. Lee, J. Hirsch, M. Saabye, S. Bonar, N. Venkatraman, J. Zhang, L. Kahn, S. Ghosh, C. Sommers, D. Meyer and N. Kishore, Poster A164, The 15th International Inflammation Research Association Conference, Chantilly, VA, September 2008. [18] N. K. Williams, R. S. Bamert, O. Patel, C. Wang, P. M. Walden, A. F. Wilks, E. Fantino, J. Rossjohn and I. S. Lucet, J. Mol. Biol., 2009, 387, 219. [19] M. P. Clark, K. M. George, R. G. Bookland, J. Chen, S. K. Laughlin, K. D. Thakur, W. Lee, J. R. Davis, E. J. Cabrera, T. A. Brugel, J. C. VanRens, M. J. Laufersweiler, J. A. Maier, M. P. Sabat, A. Golebiowski, V. Easwaran, M. E. Webster, B. De and G. Zhang, Bioorg. Med. Chem. Lett., 2007, 17, 1250. [20] M. W. Karaman, S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atteridge, B. T. Campbell, K. W. Chan, P. Ciceri, M. I. Davis, P. T. Edeen, R. Faraoni, M. Floyd, J. P. Hunt, D. J. Lockhart, Z. V. Milanov, M. J. Morrison, G. Pallares, H. K. Patel, S. Pritchard, L. M. Wodicka and P. P. Zarrinkar, Nature Biotech 2008, 26, 127 (supplemental Info). [21] www.clinicaltrials.gov as of March 7, 2009. [22] www.rigel.com as of March 7, 2009. [23] T. Deuse, J. B. Velotta, G. Hoyt, J. A. Govaert, V. Taylor, E. Masuda, E. Herlaar, G. Park, D. Carroll, M. P. Pelletier, R. C. Robbins and S. Schrepfer, Transplantation, 2008, 85, 885.
Advances in the Discovery of Small Molecule JAK3 Inhibitors
263
[24] www.vpharm.com as of March 7, 2009. [25] Q. Lin, D. Meloni, Y. Pan, M. Xia, J. Rodgers, S. Shepard, M. Li, L. Galya, B. Metcalf, T.-Y. Yue, P. Liu and J. Zhou, Org. Lett., 2009, 11, 1999. [26] S. Verstovsek, H. Kantarjian, A. Pardanani, D. Thomas, J. Cortes, R. Mesa, J. Redman, C.-M. Staschen, J. Fridman, K. Vaddi and A. Tefferi, Presentation 558, American Society of Hematology Meeting, Atlanta, GA, December 2007. [27] K. Ghoreschi, A. Laurence and J. J. O’Shea, Nat. Immunol., 10, 356. [28] J. Fridman, P. Scherle, R. Collins, T. Burn, Y. Li, J. Li, M. Covington, B. Thomas, M. Favata, J. Shi, R. McGee, S. Shepard, J. Rodgers, S. Yeleswaram, G. Hollis, R. Newton, B. Metcalf, S. Friedman and K. Vaddi, American College of Rheumatology Scientific Meeting, San Francisco, CA, October 2008, Poster Abstract 352. [29] R. Paniagua, M.-S. Si, M. G. Flores, G. Rousvoal, S. Zhang, O. Aalami, A. Campbell, P. S. Changelian, B. A. Reitz and D. C. Borie, Transplantation, 2005, 80, 1283. [30] D. C. Borie, M. J. Larson, M. G. Flores, A. Campbell, G. Rousvoal, S. Zhang, J. P. Higgins, D. J. Ball, E. M. Kudlacz, W. H. Brissette, E. A. Elliott, B. A. Reitz and P. A. Changelian, Transplantation, 2005, 80, 1756. [31] G. Rousvoal, M.-S. Si, M. Lau, S. Zhang, G. J. Berry, M. G. Flores, P. S. Changelian, B. A. Reitz and D. C. Borie, Transpl. Int., 2006, 19, 1014. [32] E. Van Gurp, W. Weimar, R. Gaston, D. Brennan, R. Mendez, J. Pirsch, S. Swan, M. D. Pescovitz, G. Ni, C. Wang, S. Krishnaswami, V. Chow and G. Chan, Am. J. Transplant., 2008, 8, 1711. [33] E. A. F. J. Van Gurp, W. Schoordijk-Vershoor, M. Klepper, S. S. Korevaar, G. Chan, W. Weimar and C. C. Baan, Transplantation, 2009, 87, 79. [34] A. J. Milici, E. M. Kudlacz, L. Audoly, S. Zwillich and P. Changelian, Arthritis Res. Ther., 2008, 10, R14, (http://arthritis-research.com/content/10/1/R14). [35] J. G. Walker, M. J. Ahern, M. Coleman, H. Weedon, V. Papangelis, D. Beroukas, P. J. Roberts-Thomson and M. D. Smith, Ann. Rheum. Dis., 2007, 66, 992. [36] T. Hampton, JAMA, 2007, 297, 28. [37] J. Kremer, S. Cohen, B. Wilkinson, C. Connell, J. French, J. Gomez Reino, D. Gruben, K. Kanik, S. Krishnaswami, V. Pascual-Ramos, G. Wallenstein and S. Zwillich, American College of Rheumatology Meeting Abstract, San Francisco, CA, October 2008, Presentation L13. [38] W. Williams, P. Scherle, J. Shi, R. Newton, E. McKeever, J. Fridman, T. Burn, K. Vaddi, R. Levy and L. Moreland, American College of Rheumatology Meeting Abstract, San Francisco, CA, October 2008, Presentation 714. [39] B. Wilkinson, A. Gaweco, P. Changelian, M. Boy, C. Wang, V. Chow, G. Chan, J. Herron, S. Zwillich and J. Krueger, Ann. Rheum. Dis., 2007, 66 (Suppl. 2, EULAR meeting), Barcelona, Spain, June 2007, Abstract THU0099. [40] N. Punwani, W. Williams, P. Scherle, J. Shi, R. Newton, R. Flores, S. Friedman, K. Vaddi, R. Levy, A. VanVorhees and A. Gottlieb, Presentation, European Academy of Dermatology Venerology meeting, Paris, France, September 2008. [41] T. J. Boggon, Y. Li, P. W. Manley and M. J. Eck, Blood, 2005, 106, 996. [42] I. S. Lucet, E. Fantino, M. Styles, R. Bamert, O. Patel, S. E. Broughton, M. Walter, C. J. Burns, H. Treutlein, A. F. Wilks and J. Rossjohn, Blood, 2006, 107, 176. [43] H. Zuccola, M. Jacobs, L. Swenson and K. Saxena, WO Patent Application 2005/105988, 2005. [44] S. M. Stepkowski, J. Kao, M.-E. Wang, N. Tejpal, H. Podder, L. Furian, J. Dimmock, A. Jha, U. Das, B. D. Kahan and R. A Kirken, J. Immunol., 2005, 175, 4236. [45] L. Honigberg, E. Verner, J. J. Buggy, D. Loury and W. Chen, WO Patent Application 2008/121742, 2008. [46] X. Zhang, Y. Hu and Z. Yuan, Biochem. Biophys. Res. Comm., 2008, 370, 72. [47] P. S. Changelian and S. H. Zwillich, WO Patent Application 2008/029237, 2008.
264
Stephen T. Wrobleski and William J. Pitts
[48] J.-K. Jiang, K. Ghoreschi, F. Deflorian, Z. Chen, M. Perreira, M. Pesu, J. Smith, D.-T. Nguyen, E. H. Liu, W. Leister, S. Costanzi, J. J. O’Shea and C. J. Thomas, J. Med. Chem., 2008, 51, 8012. [49] C. T. Brain, G. Thoma, M. J. Sung and L. T. McNally, WO Patent Application 2007/ 140222, 2007. [50] J. Salas Solana, C. Almansa Rosales, R. Soliva, M. Fontes Ustrell and M. Vendrell Escobar, WO Patent Application 2008/119792, 2008. [51] T. Inoue, T. Tojo, M. Morita, Y. Nakajima, K. Hatanaka, S. Shirakami, H. Sasaki, A. Tanaka, F. Takahashi, K. Mukoyoshi, Y. Higashi, A. Okimoto, T. Hondo and H. Sawada, WO Patent Application 2007/007919, 2007. [52] T. Inoue, A. Tanaka, K. Nakai, H. Sasaki, F. Takahashi, S. Shirakami, K. Hatanaka, Y. Nakajima, K. Mukoyoshi, H. Hamaguchi, S. Kunikawa and Y. Higashi, WO Patent Application 2007/077949, 2007. [53] S. Shirakami, T. Inoue, K. Mukoyoshi, Y. Nakajima, H. Usuda, H. Hamaguchi, Y. Higashi and K. Hatanaka, WO Patent Application 2008/084861, 2008. [54] J. Salas Solana, C. Almansa Rosales, R. Soliva Soliva, M. Fontes Ustrell, M. Virgili Bernado, J. Comelles Espuga and J. J. Pastor Porras, WO Patent Application 2008/ 090181, 2008. [55] M. Ohlmeyer, A. Bohnstedt, C. Kingsbury, K.-K. Ho, J. Quintero, M. You, H. Park and Y. Lu, WO Patent Application 2006/108103, 2006. [56] Y. Lu, C. Kingbury, A. Bohnstedt, M. Ohlmeyer and V. Paradkar, WO Patent Application 2008/043019, 2008. [57] I. Neagu, D. Dillar, C. Kingsbury, A. Bohnstedt, M. Ohlmeyer, V. Paradkar and N. Ansari, WO Patent Application 2008/043031, 2008. [58] M. Ohlmeyer, A. Bohnstedt, C. Kingsbury, K.-K. Ho and J. Quintero, WO Patent Application 2008/060301, 2008. [59] M. Sabat, J. C. VanRens, M. J. Laufersweiler, T. A. Brugel, J. Maier, A. Golebiowski, B. De, V. Easwaran, L. C. Hsieh, R. L. Walter, M. J. Mekel, A. Evdokimov and M. J. Janusz, Bioorg. Med. Chem. Lett., 2006, 16, 5973. [60] M. Sills, K. Appell, A. Bohnstedt, et al., Inflamm. Res., 2006, 55, S118. [61] M. R. Kling and C. J. Burns, WO Patent Application 2007/062459, 2007. [62] D. G. Bourke, X. Bu, C. J. Burns, A. N. Cuzzupe, J. T. Trill, T. L. Nero, M. B. Blannin, J. Zeng and S. P. Gaynor, WO Patent Application 2008/092199, 2008. [63] B. Wong, US Patent Application 2006/0270694, 2006. [64] X. Chen, L. J. Wilson, R. Malaviya, R. L. Argentieri and S.-M. Yang, J. Med. Chem., 2008, 51, 7015.
CHAPT ER
13 Recent Advances in Adenosine Receptor (AR) Ligands in Pulmonary Diseases Rao Kalla and Jeff Zablocki
Contents
1. Introduction 2. A1 Adenosine Receptor Antagonists: L-97-1, BG-9928, FK-838, and WRC-0571 3. A2A Adenosine Receptor Agonists: CGS-21680, UK-371104, and GW-328276 4. A2B Adenosine Receptor Antagonists: CVT-6883, MRE 2029-F20, LAS-38096, and OSIP-339391 5. A3 Adenosine Receptor Antagonists: MRS-1523, KF-26777, and MRE-3008-F20 6. Summary References
265 266 268 270 272 274 275
1. INTRODUCTION Adenosine is an endogenous ligand that has a short half-life due to its rapid conversion to inosine by adenosine deaminase [1,2]. When generated locally in various tissues of the body, adenosine binds to one of its four P1 family of G-protein receptors: A1 and A3 that are coupled to Gi and lower cyclic adenosine monophosphate (cAMP) levels and A2A and A2B that are coupled to Gs and increase cAMP levels [1]. Adenosine has been implicated in both the pro-inflammatory and immunomodulatory Department of Medicinal Chemistry, CV Therapeutics, 3172 Porter Drive, Palo Alto, CA 94304, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04413-3
r 2009 Elsevier Inc. All rights reserved.
265
266
Rao Kalla and Jeff Zablocki
pathogenic mechanisms of asthma and chronic obstructive pulmonary disease (COPD) [3–5]. For instance, adenosine levels are elevated in the bronchoalveolar lavage fluid (BALF) of asthmatics relative to healthy volunteers [6], and adenosine is found in the exhaled breath condensate of asthmatics [7]. In addition, when adenosine monophosphate (AMP) is administered to asthmatics and healthy, normal individuals, it provides a source of adenosine that leads to bronchoconstriction in asthmatics, but not normals [8]. Cigarette smokers with COPD demonstrate a higher prevalence of hyper-responsiveness to AMP than non-smokers with COPD [9]. Furthermore, AMP induces bronchoconstriction in COPD patients [9]. In addition, an adenosine uptake blocker, dipyridamole, can precipitate asthma [10]. Thus, substantial evidence supports the role of adenosine inducing bronchoconstriction in asthma and COPD. The bronchoconstrictor effect is predominantly through mast cell–mediated histamine release [11–13] and the release of prostaglandins, leukotrienes, and interleukin (IL)-8 [14,15]. The role of adenosine in asthma and COPD will be further delineated in the description of therapeutic ligands by each receptor subtype.
2. A1 ADENOSINE RECEPTOR ANTAGONISTS: L-97-1, BG-9928, FK-838, AND WRC-0571 Activation of A1 adenosine receptors (ARs) may play a role in mucus production by human epithelial cells [16], human bronchial smooth muscle contraction [17], and neutrophil chemotaxis [18,19]. However, activation of A1 ARs on macrophages has the following effects: inhibits the production of several pro-inflammatory cytokines, including tumor necrosis factor-a (TNF-a), IL-6, and IL-8, and enhances the release of the anti-inflammatory cytokine, IL-10 [20–22]. Several compounds from different classes of A1 AR antagonists are noteworthy for either demonstrating efficacy in animal models of asthma or entering clinical trials for asthma or other indications. Xanthines are considered classical antagonists for ARs. For example, the non-selective xanthine AR antagonists, theophylline (1,3-dimethylxanthine) and caffeine (1,3,7-trimethylxanthine), display micromolar affinity at various AR subtypes, with some affinity at the A1 AR [23]. Over the past 20 years, multiple research groups contributed to the structure activity relationships (SAR) for substitution at the 1-, 3-, 7-, and 8-positions of the xanthine core to provide selective antagonists for the AR subtypes [24–26]. Compound 1 (L-97-1) is a result of such SAR optimization and was described as a water-soluble, small molecule, A1 AR antagonist with high affinity (580 nM) and W100-fold selectivity over the other AR subtypes
267
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
[27]. In an allergic rabbit model, compound 1 blocked early and late allergic responses and bronchial hyper-responsiveness to histamine [27]. Also, in the same rabbit model, it reduced the number of eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage. Following oral dosing in rats, L-97-1 displayed good plasma concentrations, potentially supporting its use as an oral anti-asthma treatment [27]. In addition, allergen-sensitized rabbits that were treated with A1 AR antisense required a W10-fold increase in adenosine to induce a 50% decrease in dynamic compliance of the lung, thus supporting the role of the A1 AR in the rabbit [28].
N
OH O
O N
N O
N
N
NH2
H N
N O
N
COOH
N
BG-9928 (2)
L-97-1 (1)
HO N
N NH N
N
N
N O
N
N
N
O HO FK-838 (3)
WRC-0571 (4)
Substitution of the xanthine core, at the 8-position, with a bicyclo[2.2.2]octyl group produced compound 2 (BG-9928) that has high affinity (7.4 nM) for the A1 AR and displays good selectivity (915-fold)
268
Rao Kalla and Jeff Zablocki
over the A2A AR [29]. BG-9928 has high oral bioavailability in rat (99%), dog (78%), and cynomolgus monkey (94%), and it demonstrated excellent oral efficacy (ED50 ¼ 0.01 mg/kg) in a rat diuresis model [29]. Compound 2 was well tolerated when dosed to rats and cynomolgus monkeys through either intravenous or oral administration for a period of 3 months. Compound 2 (BG-9928) is in phase III clinical trails for the treatment of the edema associated with congestive heart failure [30]. FK-838 (3) represents a structurally unique class of A1 AR antagonists, pyrazolo[1,5-a]pyridines [31]. Compound 3 displayed high affinity (120 nM) and selectivity (W50-fold over A2A AR) for A1 AR and had very good aqueous solubility (10 mg/mL) and high oral availability (78%). Compound 3 is in phase II clinical trials as a diuretic antihypertensive agent [31]. Compound 4 (WRC-0571) is an adenine-based A1 antagonist that has high affinity (1.7 nM) and W100-fold selectivity over other AR subtypes [32]. Compound 4 was demonstrated to have good oral bioavailability across species and is in preclinical development as both a diuretic agent and for the treatment of renal failure [30,32].
3. A2A ADENOSINE RECEPTOR AGONISTS: CGS-21680, UK-371104, AND GW-328276 Activation of A2A AR has the following anti-inflammatory effects on various cell types: inhibition of the release of histamine and tryptase from mast cells [33] and reduction of chemotaxis, in addition to activation/degranulation of neutrophils [19,34,35]. Activation of A2A AR also increases the production of anti-inflammatory IL-10 from monocytes and macrophages [36]. Therefore, various A2A AR agonists have been evaluated in animal models of asthma and have even advanced into clinical trials [37]. Early on, compound 5 was synthesized by exploring the SAR studies on N-ethyl carboxamide adenosine (NECA), a non-selective AR agonist [38]. Substitution at the 2-position of NECA with a phenethyl amine derivative provided compound 5 (CGS-21680) that displayed 144-fold selectivity for the A2A AR over the A1 AR [38]. When compound 5 was given intratracheally, it inhibited both the early and the late inflammatory reactions in allergen-challenged Brown Norway rats [39]. Compound 5 inhibited both inflammatory cell influx of BALF in asthmatic mice and neutrophil activation in a mouse model of COPD [40]. However, cardiovascular side effects were observed with similar doses of CGS-21680, thus demonstrating no apparent separation of the anti-inflammatory effects of CGS-21680 from its cardiovascular effects [39]. Since cardiovascular effects, in particular,
269
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
hypotension, were observed in laboratory animals when A2A agonists were administered systemically [39], inhaled administration of an A2A agonist was targeted. The compounds were designed to have inhaled local lung effects by optimizing the physiochemical properties of the compound by increasing the molecular weight and hydrophilicity for high systemic clearance. After extensive SAR, N6-(2,2-diphenylethyl)-2[N-[2-(1-piperidinyl)ethyl]carbamoyl] adenosine compound 6 (UK-371, 104) displayed good A2A AR affinity (65 nM), and it also inhibited the release of inflammatory mediators from isolated human neutrophils [41]. Compound 6 inhibited the capsaicin-induced bronchoconstriction in an anaesthetized guinea pig model without affecting blood pressure [37]. The cardiovascular side effects of compound 6, following the intratracheal administration at a dose of 1 mg/kg, are much less compared to CGS-21680 because of the low systemic exposure of the drug. UK432,097, an analog of UK-371,104, had a similar pharmacological profile in the anaesthetized guinea pig model, and it was moved into clinical trials [37]. In 2008, the clinical trials were discontinued for the development of UK-432,097 because of lack of efficacy [42]. Another A2A AR agonist, which targets the lung, compound 7 (GW-328267X), was found to have good A2A and A3 AR affinity (16 nM) with good selectivity over A1 and A2B AR subtypes (W60- and W300-fold respectively) [43]. Treatment of non-smoking, atopic asthmatics, who underwent an inhaled allergen challenge, did not provide significant protection against the allergen-induced early and late asthmatic reaction [44]. In the same study, compound 7 did not inhibit the accompanying inflammatory response, as measured by sputum total cell counts, number of EG2+ cells, and the concentrations of IL-8 and eosinophil cationic protein [44,45]. Compound 7, which was in phase II clinical trials for COPD, has been dropped from development because of lack of efficacy [30] that might be due to the low dose used (25 mg, twice daily), limited by prohibitive cardiovascular side effects [45].
NH2 N O
N O
N H
NH2 N
N O
N
N O
N N
N H
N H HO
OH
NECA
HO
OH
O
CGS-21680 (5)
OH
270
Rao Kalla and Jeff Zablocki Ph
HN N N O
N
Ph H N
N O
HO HO
NH2 N
N
N
N N O
N
UK-371, 104 (6)
N
N H
N HO
OH
OH
N
OH
GW-328276X (7)
4. A2B ADENOSINE RECEPTOR ANTAGONISTS: CVT-6883, MRE 2029-F20, LAS-38096, AND OSIP-339391 There is substantial evidence to indicate that A2B ARs contribute to airway inflammation in asthma and that a compound specifically inhibiting the A2B AR may have beneficial effects [46–49]. Activation of A2B ARs on human bronchial smooth muscle cells (BSMCs) has been shown to induce the release of the inflammatory cytokines IL-6 and monocytic chemotactic protein-1 [50]. A2B AR activation on human bronchial epithelial cells (HBECs) releases IL-19, which in turn activates human monocytes to induce the release of TNF-a, thereby upregulating A2B AR expression on human bronchial endothelial cells [51]. The above evidence supports the hypothesis that adenosine plays a role in asthma, and its effects may be at least, in part, mediated through the A2B AR. Several high-affinity and selective antagonists of A2B AR have been reported recently by several groups. These examples can be used to fully understand the therapeutic potential of A2B AR antagonists as anti-inflammatory and anti-angiogenic agents [24–26,52]. The structural approach taken by these groups can be divided into two classes of compounds, xanthines and non-xanthine derivatives. Exploration of 8-heteroaryl substitution on xanthines led to the identification of 8-(pyrazol-4-yl) xanthines as high-affinity and selective A2B AR antagonists [53]. Differential substitution at the N-1 and N-3 positions of the xanthine core with propyl and ethyl groups, respectively, led to compound 8 (CVT-6883), which displays high affinity (22 nM) for the A2B AR with good selectivity (88-, 148-, and 48-fold over A1, A2A, and A3 ARs respectively) [54]. Compound 8 antagonized the NECA-induced cAMP accumulation in HEK-A2B cells and NIH 3T3 cells (KB values of 6 and 2 nM respectively) [55] and has also been shown to completely abolish the NECA-induced cAMP accumulation in BSMCs [50]. When dosed orally to rats at 2 mg/kg, compound 8 had excellent systemic exposure with a t1/2 of 4 h, Cmax W1100 ng/mL, and a dose-adjusted area
271
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
under curve (dAUC) of 6500 ngh/mL [54]. In a mouse model of asthma, compound 8 demonstrated a dose-dependent (0.42.5 mg/kg) blocking effect on NECA-induced increases in airway reactivity [56]. Also, in this same model, compound 8 significantly reduced both the late allergic airway response and the recruitment of inflammatory cells in BALF [56]. In the adenosine deaminase–deficient mouse model, compound 8 attenuated pulmonary inflammation, fibrosis, airway enlargement, and lung injury [55]. CVT-6883 is currently being developed as an oral treatment for asthma and has completed phase I clinical trials [30,57].
O
O H N
N O
N
N
N
H N
N
N
N
N
O
O N
N
O
NH
CF3 O
MRE 2029-F20 (9)
CVT-6883 (8)
O
H N
N NH
H N
N
O
O
N
N N N
N
N
N H
O
N
LAS-38096 (10)
OSIP-339391 (11)
In another series of compounds, substitution at the 8-position of xanthine, with a 5-pyrazole group, led to compound 9 that displayed high affinity and selectivity for the A2B AR [58]. The 5-pyrazolyl derivative, 9 (MRE 2029-F20), displayed good A2B AR affinity (5.5 nM) and selectivity (W180-fold over all AR subtypes). Compound 9 blocks NECA-induced cAMP accumulation with IC50 values in the nanomolar range [58]. The tritium-labeled derivative of 9 displayed a Kd value of 1.6570.10 nM in Chinese Hamster Ovary (CHO) cells that express hA2B receptors, and it can be useful as a pharmacological tool in competitive binding studies [59]. Two series of compounds, 2-aminopyridines and 2aminopyrimidines, were explored as A2B AR antagonists. This work led
272
Rao Kalla and Jeff Zablocki
to the identification of N-heteroaryl 4u-furyl-4,5u-bipyrimidin-2u-amines as high-affinity and selective A2B AR antagonists [60]. The lead compound, 2u-amino(3-pyridyl) derivative 10 (LAS-38096) has a A2B AR affinity of 17 nM and very good selectivity (W58-, W147-, and W58-fold over A1, A2A, and A3 ARs respectively). Compound 10 inhibited the NECAinduced cAMP levels in HEK-293 expressing human A2B AR (IC50 of 321 nM) and CHO cells (IC50 of 349 nM) that were transfected with mouse A2B AR [60]. Compound 10 displayed good systemic exposure, with a Cmax of 11 mM and an AUC of 16 mM/h, when dosed to rats, and also displayed good exposure following oral dosing in mice and dogs [60]. After treatment of an allergic mouse (female Balb/c mice) with compound 10, the mouse showed decreased bronchial hyperresponsiveness, mucus production, and a slight decrease in eosinophil infiltration and Th2 cytokine levels [61]. Compound 11 (OSIP-339391) is a representative of the deaza-adenine class of A2B AR antagonists [62]. A 2-phenyl-7-deazaadenine analog, compound 11, demonstrated excellent A2B AR affinity (0.5 nM) and good selectivity (74-, 656-, and 900-fold over A1, A2A, and A3 ARs respectively). The tritium-labeled analog of 11 displayed a Kd value of 0.4170.06 nM for binding to human A2B AR expressed in HEK-293 cells [62]. This selective and high-affinity radioligand can be a useful tool in further characterization of the pharmacology of the A2B AR. There is now considerable evidence that A2B ARs are involved in both airway inflammation and the process of airway remodeling. The availability of high-affinity and selective A2B AR antagonists allows for the clinical evaluation of the role of the A2B AR in asthma and COPD.
5. A3 ADENOSINE RECEPTOR ANTAGONISTS: MRS-1523, KF-26777, AND MRE-3008-F20 There are major differences among species in the expression and function of the A3 AR subtype, which complicates the understanding of the functional significance of this receptor subtype in the pathogenesis of chronic inflammatory airway diseases [63]. It has been shown that the A3 AR mediates the adenosine-induced mast cell degranulation in rats, mice, and guinea pigs [64], whereas A3 receptors have not been identified on human mast cells [65]. A3 ARs have been shown to play an important role in eosinophilia and mucus production in animal models [65]. Also, A3 ARs are found on human eosinophils, and elevated levels of A3 receptors are observed in lung biopsies of patients with asthma or COPD [66].
273
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
O
O
S
N H N
N
O N
O
N
N
MRS-1523 (12)
KF-26777 (13)
O
CN
O N H
NH N
N
N
O
N NH
N
N N
MRE-3008-F20 (14)
N
S
N
N
N Compound (15)
The classical xanthine antagonists of ARs, theophylline and caffeine, typically have low binding affinities for the A3 AR, and therefore, the search for A3 AR antagonists has been focused on novel heterocyclic systems. Initially, a dihydropyridine derivative that has good affinity for the A3 AR was discovered by screening diverse chemical libraries. Further optimization led to the corresponding pyridine derivative, compound 12 (MRS-1523), that displayed high affinity (18.9 nM for human and 113 nM for rat) and selectivity (W130-fold for rA1 AR) for the A3 AR [67]. Treatment of adenosine deaminase (ADA)-deficient mice with selective A3 AR antagonist 12 reduced the number of airway eosinophils and decreased mucus production in the airways. Similar findings were also observed in the lungs of ADA/A3 knockout mice, supporting the important role of A3 AR mediation of lung eosinophilia and mucus hyperplasia in pulmonary disorders triggered by elevated adenosine levels [65]. A series of tricyclic imidazo[2,1-i]purinones have been prepared as A3 AR antagonists by modifications of the xanthine
274
Rao Kalla and Jeff Zablocki
core [68]. An analog of the series is compound 13 (KF-26777) that displays high affinity (0.20 nM) and selectivity (1,000-fold) for the A3 AR. Compound 13 inhibited the binding of [35S]GTPgS, which was stimulated by 2-chloro-N6-(3-iodobenzyl)adenosine-5-N-methyluronamide (Cl-IB-MECA) with an IC50 value of 270785 nM. It also antagonized the [Ca2+]I mobilization, induced by Cl-IB-MECA, with a KB value of 0.4270.14 nM, suggesting that it is a highly potent and selective A3 AR antagonist [68]. A tricyclic series, illustrated by lead compound 14 (MRE-3008-F20), displayed high affinity (0.28 nM) and selectivity (W30,000-fold) for the A3 AR [69]. Compound 14 blocked the IB-MECA-induced cAMP production in CHO cells with an IC50 of 4.5 nM, confirming that it is a functional antagonist [69]. The tritiumlabeled analog of 14 bound to hA3 ARs, expressed in CHO cells, with a Kd value of 0.82 nM, suggesting that it can be a useful tool for characterization of A3 ARs in both normal and pathological conditions [70]. In recent reports, a series of 5-heterocyclic-substituted aminothiazoles have been identified as dual antagonists for both the A2B and the A3 ARs [71]. Of these analogs, trisubstituted aminothiazole 15 displayed high affinity for both A2B (3 nM) and A3 (10 nM) ARs and gave selectivity against the A1 (W20-fold) and A2A (W160-fold) ARs. Compound 15 showed good oral bioavailability (30%) in Wistar rats and displayed good absorption distribution metabolism excretion (ADME) properties [71]. Some of the compounds in this series also inhibited the p38 mitogen-activated protein (MAP) kinases a and b and the phosphodiesterase 4D (PDE4D) isoenzyme with IC50 values in the low nanomolar range [72]. This compound represents a new series that inhibits both A2B and A3 ARs and can potentially be used as a new tool in testing the therapeutic potential of dual inhibition in allergic diseases.
6. SUMMARY Adenosine has been strongly implicated in asthma and COPD based on supporting studies in animal models, AMP (adenosine precursor) effects in humans, and elevated adenosine levels in BALF of asthmatic patients. The major biological effect of adenosine is thought to occur through histamine released from activated mast cells through A2B AR activation. However, additional effects are mediated through multiple ARs including inflammation (A1, A2A, and A2B), mucus production (A1), and inflammatory cell recruitment (A1, A2A, A2B, and A3). Selective AR antagonists have been discovered for each AR isoform through optimization of substituents on each core scaffold. This optimization has been driven by a mixture of classical medicinal chemistry, ligand-based modeling, and initially homology AR modeling based on the rhodopsin X-ray structure
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
275
[24–26]. Recently, the A2A AR X-ray has been solved with an A2A AR antagonist ZM-241385 that may prove useful in the design of subsequent subtype selective AR antagonists and AR agonists [73]. Although many selective A1 antagonists have been discovered, the principal focus of these compounds has been on treatment of the edema associated with CHF and not asthma or COPD. This may be due to an early bias in animal models (e.g., rabbit) where the A1 AR may play a more critical role in asthma than in humans. Selective A2A agonists are available for local delivery to the lung, but initial asthma clinical studies were not promising. Certainly, a number of A2B antagonists possess very high affinity and selectivity and demonstrate activity in animal models of asthma (8 and 10). Compound 8 is the most advanced A2B antagonist and is currently in phase I clinical trials for asthma. Some early animal data with respect to the role of ARs in asthma and COPD is under scrutiny. For instance, the studies with MRS1523 in rodents suggest that the activation of A3 AR may contribute to airway obstruction by inducing hypersecretion of the mucus or by migration of eosinophils to the airways. However, the distribution of A3 ARs in rodents does not match the distribution in humans; therefore, the clinical relevance of these animal models is unclear. The consequence of A3 AR activation in humans must be further investigated due to conflicting animal and human data with respect to the possible role of A3 receptors. The availability of selective antagonists of AR subtypes will help define the role of adenosine in asthma and COPD in future clinical trials.
REFERENCES [1] C. E. Muller, Farmaco, 2001, 56, 77. [2] B. Pavan and A. P. Ijzerman, Biochem. Pharmacol., 1998, 56, 1625. [3] D. Zeng, R. Polosa, I. Biaggioni and L. Belardinelli, in Asthma: Modern Therapeutic Targets (eds R. Polosa and S. T. Holgate), Clinical Publishing, Oxford, UK, 2007, pp. 1–13. [4] R. A. Brown, D. Spina and C. P. Page, Br. J. Pharmacol., 2008, 153, S446. [5] M. van der Berge, M. N. Hylkema, M. Versluis and D. S. Postma, Drugs RD, 2007, 8, 13. [6] A. G. Driver, C. A. Kukoly, S. Ali and S. J. Mustafa, Am. Rev. Respir. Dis., 1993, 148, 91. [7] E. Huszar, G. Vass and E. Vizi, Eur. Respir. J., 2002, 20, 1393. [8] M. J. Cushley, A. E. Tattersfield and S. T. Holgate, Am. Rev. Respir. Dis., 1984, 129, 380. [9] Y. Oosterhoff, J. W. de Jong and M. A. Jansen, Am. Rev. Respir. Dis., 1993, 147, 553. [10] J. R. Fozard and C. Carth, Curr. Opin. Investig. Drugs, 2002, 3, 69. [11] G. D. Phillips, P. Rafferty and S. T. Holgate, Thorax, 1987, 42, 939. [12] G. D. Phillips and S. T. Holgate, Am. Rev. Respir. Dis., 1989, 139, 463. [13] S. R. Rutgers, G. H. Koetger and T. W. van der Mark, Clin. Exp. Allergy, 1999, 29, 1287. [14] M. K. Church, S. T. Holgate and P. J. Hughes, Br. J. Pharmacol., 1983, 80, 719. [15] P. J. Hughes, S. T. Holgate and M. K. Church, Biochem. Pharmacol., 1984, 33, 3847. [16] N. McNamara, M. Gallup, A. Khong, A. Sucher, I. Maltseva, J. Fahy and C. Basbaum, FASEB J., 2004, 18, 1770. [17] M. F. Ethier and J. M. Madison, Am. J. Respir. Cell Mol. Biol., 2006, 35, 496.
276
Rao Kalla and Jeff Zablocki
[18] B. N. Cronstein, L. Daguma, D. Nicholas, A. J. Hutchison and M. Williams, J. Clin. Invest., 1990, 85, 1150. [19] B. N. Cronstein, R. I. Levin, M. Philips, R. Hirschhorn, S. B. Abramson and G. Weissmann, J. Immunol., 1992, 148, 2201. [20] F. G. Sajjadi, K. Takabayashi, A. C. Foster, R. C. Domingo and G. S. Firestein, J. Immunol., 1996, 156, 3435. [21] G. Hasko, C. Szabo, Z. H. Nemeth, V. Kvetan, S. M. Pastores and E. S. Vizi, J. Immunol., 1996, 157, 4634. [22] O. Monie, P. Stordeur, L. Schandene, A. Marchant, D. de Groote, M. Goldman and J. Deviere, J. Immunol., 1996, 156, 4408. [23] K. A. Jacobson, A. P. Ijzerman and J. Linden, Drug. Dev. Res, 1999, 47, 45. [24] R. Akkari, J. C. Burbiel, J. Hockemeyer and C. E. Muller, Curr. Top. Med. Chem., 2006, 6, 1375. [25] K. A. Jacobson and Z.-G. Gao, Nat. Rev. Drug Discov., 2006, 5, 247. [26] P. G. Baraldi, M. A. Tabirizi, S. Gessi and P. A. Borea, Chem. Rev., 2008, 108, 238. [27] P. C. M. Obiefuna, V. K. Batra, A. Nadeem, P. Borron, C. N. Wilson and S. J. Mustafa, J. Pharmacol. Exp. Therp., 2005, 315, 329. [28] J. W. Nyce and W. J. Metzger, Nature, 1997, 385, 721. [29] W. F. Kiesman, J. Zhao, P. R. Conlon, J. E. Dowling, R. C. Petter, F. Lutterodt, X. Jin, G. Smits, M. Fure, A. Jayaraj, J. Kim, G. Sullivan and J. Linden, J. Med. Chem., 2006, 49, 7119. [30] http://integrity.prous.com [31] A. Akahane, H. Katayama, T. Mitsunaga, T. Kato, T. Kinoshita, Y. Kita, T. Kusunoki, T. Terai, K. Yoshida and Y. Shiokawa, J. Med. Chem., 1999, 42, 779. [32] P. L. Martin, R. J. Wysocki, Jr., R. J. Barrett, J. M. May and J. Linden, J. Pharmacol. Exp. Therp., 1996, 276, 490. [33] H. Suzuki, M. Takei, T. Nakahata and H. Fukamachi, Biochem. Biophys. Res. Commun., 1998, 242, 697. [34] A. Wollner, S. Wollner and J. B. Smith, Am. J. Respir. Cell. Mol. Biol., 1993, 9, 179. [35] B. B. Fredholm, Y. Zhang and I. van der Ploeg, Naunyn Schmiedebergs Arch. Pharmacol., 1996, 354, 262. [36] S. Gessi, K. Varani, S. Merighi, E. Ongini and P. A. Borea, Br. J. Pharmacol., 2000, 129, 2. [37] M. A. Trevethick, S. J. Mantell, E. F. Stuart, A. Barnard, K. N. Wright and M. Yeadon, Br. J. Pharmacol., 2008, 155, 463. [38] A. J. Hutchison, R. L. Webb, H. H. Oel, G. R. Ghai, M. B. Zimmerman and M. Williams, J. Pharmacol. Exp. Ther., 1989, 251, 47. [39] J. R. Fozard, K. M. Ellis, M. F. V. Dantas, B. Tigani and L. Mazzoni, Eur. J. Pharmacol., 2002, 438, 183. [40] O. Bonneau, D. Wyss, S. Ferretti, C. Blaydon, C. S. Stevenson and A. Trifilieff, Am. J. Physiol. Lung Cell Mol. Physiol., 2006, 290, L1036. [41] S. J. Mantell, P. T. Stephenson, S. M. Monaghan, G. N. Maw, M. A. Trevethick, M. Yeadon, R. F. Keir, D. K. Walker, R. M. Jones, M. D. Selby, D. V. Batchelor, S. Rozze, H. Chavaroche, T. J. Hobson, P. G. Dodd, A. Lemaitre, K. N. Wright and E. F. Stuart, Bioorg. Med. Chem. Lett., 2008, 18, 1284. [42] http://www.clinicaltrials.gov. Safety and efficacy of UK-432,097 in chronic obstructive pulmonary disease, study was terminated as of March 17, 2009. [43] N. Bevan, P. R. Butchers, R. Cousins, J. Coates, E. V. Edgar, V. Morrison, M. J. Sheehan, J. Reeves and D. J. Wilson, Eur. J. Pharmacol., 2007, 564, 219. [44] B. Luijk, M. van der Berge, H. A. M. Kerstjens, D. S. Postma, L. Cass, A. Sabin and J.-W. J. Lammers, Allergy, 2008, 63, 75. [45] J. Rimmer, H. L. Peake, C. M. C. Santos, M. Lean, P. Bardin, R. Robson, B. Haumann, F. Loehrer and M. L. Handel, Clin. Exp. Allergy, 2007, 37, 8.
Recent Advances in Adenosine Receptor Ligands in Pulmonary Diseases
277
[46] I. Feoktistov and I. Biaggioni, Pharmacol. Rev., 1997, 49, 381. [47] I. Feoktistov, R. Polosa, S. T. Holgate and I. Biaggioni, Trends Pharmacol. Sci., 1998, 19, 148. [48] S. Ryzhov, A. E. Goldstein, A. Matafonov, D. Zeng, I. Biaggioni and I. Feoktistov, J. Immunol., 2004, 172, 7726. [49] S. T. Holgate, Br. J. Pharmacol., 2005, 145, 1009. [50] H. Zhong, L. Beladinelli, T. Maa, I. Feoktistov, I. Biaggioni and D. Zeng, Am. J. Respir. Cell Mol. Biol., 2004, 30, 118. [51] H. Zhong, Y. Wu, L. Belardinelli and D. Zeng, Am. J. Respir. Cell Mol. Biol., 2006, 35, 587. [52] J. Zablocki, E. Elzein and R. Kalla, Expert. Opin. Ther. Patents, 2006, 16, 1347. [53] R. V. Kalla, E. Elzein, T. Perry, X. Li, V Palle, V. Varkhedkar, A. Gimbel, T. Maa, D. Zeng and J. Zablocki, J. Med. Chem., 2006, 49, 3682. [54] E. Elzein, R. V. Kalla, X. Li, T. Perry, A. Gimbel, D. Zeng, D. Lustig, K. Leung and J. Zablocki, J. Med. Chem., 2008, 51, 2267. [55] C.-X. Sun, H. Zhong, A. Mohsenin, E. Morschi, J. L. Chunn, J. G. Molina, L. Belardinelli, D. Zeng and M. Blackburn, J. Clin. Invest., 2006, 116, 2173. [56] S. J. Mustafa, A. Nadeem, M. Fan, H. Zhong, L. Belardinelli and D. Zeng, J. Pharmacol. Exp. Ther., 2007, 320, 1246. [57] www.cvt.com press releases. [58] P. G. Baraldi, T. M. Aghazadeh, D. Preti, A. Bovero, R. Romagnoli, F. Fruttarolo, A. N. Zaid, A. R. Moorman, K. Varani, S. Gessi, S. Merighi and P. A. Borea, J. Med. Chem., 2004, 47, 1434. [59] P. G. Baraldi, T. M. Aghazadeh, D. Preti, A. Bovero, R. Romagnoli, F. Fruttarolo, A. R. Moorman, K. Varani and P. A. Borea, Bioorg. Med. Chem. Lett., 2004, 14, 3607. [60] A. Carotti, M. I. Cadavid, N. B. Centeno, C. Esteve, M. I. Loza, A. Martinez, R. Nieto, E. Ravina, F. Sanz, V. Segarra, E. Sotelo, A. Stefanachi and B. Vidal, J. Med. Chem., 2006, 49, 282. [61] M. Aparici, A. Nueda, J. Beleta, N. Prats, R. Fernandez and M. Miralpeix, Proceedings of the Symposium of the Collegium International Allergologicum, 2006, Poster 162. [62] M. Stewart, A. G. Steinig, C. Ma, J.-P. Song, B. McKibben, A. L. Castelhano and S. J. MacLennan, Biochem. Pharmacol., 2004, 68, 305. [63] J. Linden, Trends Pharmacol. Sci., 1994, 15, 298. [64] J. R. Fozard and J. P. Hanon, Clin. Exp. Allergy, 2000, 30, 1213. [65] H. W. J. Young, J. G. Molina, D. Dimina, H. Zhong, M. Jacobson, L.-N. L. Chan, T.-S. Chan and M. R. Blackburn, J. Immunol., 2004, 173, 1380. [66] B. A. Walker, M. A. Jacobson, D. A. Knight, C. A. Salvatore, T. Weir, D. Zhou and T. R. Bai, Am. J. Respir. Cell Mol. Biol., 1997, 16, 531. [67] A.-H. Li, S. Moro, N. Melman, X.-D. Ji and K. A. Jacobson, J. Med. Chem., 1998, 41, 3186. [68] M. Saki, H. Tsumuki, H. Nonaka, J. Shimada and M. Ichimura, Eur. J. Pharmacol., 2002, 444, 133. [69] P. G. Baraldi, B Cacciari, R. Romagnoli, G. Spalluto, K.-N. Koltz, E. Leung, K. Varani, S. Gessi, S. Merighi and P. A. Borea, J. Med. Chem., 1999, 42, 4473. [70] K. Varani, S. Merighi, S. Gessi, K.-N. Koltz, E. Leung, P. G. Baraldi, B. Cacciari, R. Romagnoli, G. Spalluto and P. A. Borea, Mol. Pharmacol., 2000, 57, 968. [71] N. J. Press, R. J. Taylor, J. D. Fulletron, P. Tranter, C. McCarthy, T. H. Keller, L. Brown, R. Cheung, J. Christie, S. Haberthuer, J. D. I. Hatto, M. Keenan, M. K. Mercer, N. E. Press, H. Sahri, A. R. Tuffnell, M. Tweed and J. R. Fozard, Bioorg. Med. Chem. Lett., 2005, 15, 3081. [72] A. Trifilieff, T. H. Keller, N. J. Press, T. Howe, P. Gedeck, D. Beer and C. Walker, Br. J. Pharmacol., 2005, 144, 1002. [73] V.-P. Jaakola, M. T. Griffith, M. A. Hanson, V. Cherezov, E. Y. T. Chien, J. R. Lane, A. P. Ijzerman and R. C. Stevens, Science, 2008, 322, 1211.
CHAPT ER
14 Recent Progress in the Development of Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase Mark D. Wittman, Upender Velaparthi and Dolatrai M. Vyas
Contents
1. Introduction 2. IGF-1R Signaling 3. ATP-Competitive Inhibitors 3.1 5,7-Disubstititued pyrrolopyrimidines 3.2 5-7-Disubstituted pyrrolopyrimidine isosteres 3.3 2,4-Disubstituted pyrimidines 3.4 2,4-Disubstituted pyrimidine isosteres 3.5 Miscellaneous heterocyclic systems 4. Non ATP-Competitive Inhibitors 4.1 Catechols 4.2 Natural lignans 5. Conclusions References
281 282 283 283 284 286 288 290 293 293 295 295 296
1. INTRODUCTION Cancer therapeutics that target growth factor receptors are gaining prominence in the successful treatment of a wide variety of malignancies. Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492-7660, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04414-5
r 2009 Elsevier Inc. All rights reserved.
281
282
Mark D. Wittman et al.
Kinase activation or overexpression is often pivotal in triggering the aberrant signaling pathways typical of the malignant cellular phenotype. The identification of appropriate kinase targets for inhibition with small molecules remains an active area of research for oncology drug discovery. Since the early observations of the mitogenic properties of insulin [1], interest in the insulin-like growth factor-1 receptor (IGF-1R) signaling pathway has been increasing. The convergence of efficacy data for an IGF-1R-specific antibody [2] and epidemiological findings [3] have prominently positioned IGF-1R among the emerging cell signaling pathways currently being explored for cancer therapy. Several approaches to suppress IGF-1R signaling are actively being investigated. These include the use of monoclonal antibodies (mAb) directed against the extracellular ligand-binding domain of the receptor, modulation of the circulating levels of ligand (IGF-1 and IGF-2) through the IGF-binding proteins (IGFBPs), and small molecule kinase inhibitors of IGF-1R. The mAb approach has yielded the first IGF-1R inhibitors to enter clinical development [4–6]. The advantage of the mAbs is their inherent selectivity for IGF-1R over the closely related insulin receptor (IR). However, the lack of cross-reactivity with IR may adversely affect antitumor efficacy since the IR-A isoform exhibits high affinity for IGF-2 and is expressed at high levels in some breast cancers [7]. In addition, the downregulation of IGF-1R by siRNA in breast tumor cell lines sensitizes cells to insulin activation of downstream signaling pathways [8]. The most advanced mAb, CP-751871, is currently in phase III trials. Responses have been reported in advanced adrenocortical cancer and various sarcomas [9]. CP-751871 has also shown responses in non-small cell lung cancer (NSCLC) in combination with paclitaxel and carboplatin [10]. These studies provide proof of concept for the importance of inhibiting IGF-1R signaling in cancer therapy. Several earlier reviews of IGF-1R inhibitors have appeared [11–15]. This chapter will focus on the most recent advances in small molecule inhibitor design and specifically highlight those inhibitors that are entering the early stages of clinical development. Small molecule inhibitors of IGF-1R fall into two sub-categories: those that target the ATP-binding pocket of IGF-1R kinase (ATP-competitive) and those that target the substrate-binding site (non ATP-competitive). The most advanced small molecules are ATP-competitive kinase inhibitors. The greatest challenge for ATP-competitive inhibitors is achieving selectivity versus other kinases, including the closely related IR which shares high overall sequence homology (84%) and complete homology among the residues that contact ATP in the kinase-binding domain [16].
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
283
2. IGF-1R SIGNALING IGF-1R is a heterotetramer composed of two extracellular a-subunits that contain the ligand-binding domain and two b-subunits that contain the cytoplasmic kinase domain. Binding of the ligands IGF-1 and IGF-2 to the extracellular domain of the receptor leads to autophosphorylation of the cytoplasmic b-subunit and activation of the intrinsic kinase activity of the receptor. Activation results in the phosphorylation of insulin receptor substrates (IRS-1-4) and Src-homology containing adapter protein (Shc). These in turn activate the PI-3K/Akt/mTOR survival pathway and the mitogenic RAS/Raf/MAPK pathway respectively [17,18]. IGF1R signaling has pleiotropic effects ranging from cell proliferation, differentiation, and migration to regulation of the apoptotic machinery. The crosstalk observed between epidermal growth factor receptor (EGFR) and IGF-1R signaling suggests wide potential for using IGF-1R inhibitors in combination therapy with other targeted agents, cytotoxics, and radiation therapy. IGF-1R activation has also been implicated in the development of resistance toward trastuzumab treatment in breast cancer [19] and lung cancer [20]. mAbs
Insulin
IGF-I
IGF-I IGF-II
pp p Insulin R
p p
pp p
IGF-1R/IR
IGF-1R
IRS PI3K Akt mTOR Survival
Shc Grb2/SOS Ras MAPK Proliferation
284
Mark D. Wittman et al.
3. ATP-COMPETITIVE INHIBITORS 3.1 5,7-Disubstititued pyrrolopyrimidines The 5,7-disubstituted pyrrolopyrimidines were among the first chemotypes described as inhibitors of IGF-1R. NVP-AEW-541, 1 [21], and NVPADW742, 2 [22], are the most studied members of this class. To date, no structure-activity relationship (SAR) studies have been published for this series, but additional analogs are exemplified in a patent application [23]. Pyrrolopyrimidine 1 is a 150 nM(IC50) inhibitor of IGF-1R kinase and is equipotent versus IR. Despite the lack of selectivity in the in vitro kinase assay, 1 is 27-fold selective over IR in a cellular context. The authors suggest that the cellular selectivity arises from conformational differences that exist between the native forms of the enzymes in the cellular context, which are not present in the recombinant enzymes [21]. It remains to be seen if the observed cellular selectivity will translate into a clinical benefit compared to other non-selective inhibitors of IGF-1R/IR signaling. This compound has been extensively studied in vitro and in preclinical animal models [24–26] and was reported to have entered phase I clinical trials in 2004, but nothing has been published to date on the clinical findings.
O NH2
R=
N 1
N N
N
R=
N 2
R
3.2 5-7-Disubstituted pyrrolopyrimidine isosteres 3.2.1 Imidazopyrazines Several isosteric replacements of the pyrrolopyrimidine core have been investigated by various groups. In general, these isosteric scaffolds possess the same cellular selectivity described for the pyrrolopyrimidines
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
285
(Section 3.1). Included among these scaffolds are the imidazopyrazines [27–29]. Initial lead optimization efforts focused on replacing the benzyl ether present in 1 and 2 to improve metabolic stability and potency. These studies identified an advanced lead, PQIP, 3 (IC50 ¼ 24 nM) [30]. In a Geo colon carcinoma model, 3 is able to inhibit 80% of IGF-1R phosphorylation within the tumor resulting in 70–80% inhibition of tumor growth (%TGI) after a daily oral dose of 25 mg/kg [31]. Further absorption, distribution, metabolism, and excretion (ADME) optimization led to the clinical candidate OSI-906, 4 (IC50 ¼ 35 nM) with a high degree of selectivity over other kinase targets. Imidazopyrazine 4 shows a wide range of anti-proliferative effects in colorectal, NSCLC, breast, pancreatic, and rhabdomyosarcoma with TGI ranging from 53% to W100% in preclinical models at oral doses of 30–60 mg/kg [32].
N
N NH2
NH2 N
N
N
N
N
N
3
4 OH
N
N
3.2.2 Pyrazolopyrimidines From a series of pyrazolopyrimidine inhibitors, A-928605, 5, was identified with both IGF-1R (IC50 ¼ 35 nM) and EGFR activity (IC50 ¼ 65 nM) [33]. The dual IGF-1R/EGF activity was further optimized to take advantage of the crosstalk between IGF-1R and EGFR, leading to compound 6, which represents the optimal balance of IGF-1R (IC50 ¼ 81 nM), EGF (IC50 ¼ 58 nM), and ErB-2 (IC50 ¼ 54 nM) activity, cellular activity, and pharmacokinetics [34].
286
Mark D. Wittman et al.
3.2.3 Pyrrolotriazines The pyrrolotriazine scaffold, 7, has also been used to optimize for IGF-1R potency. A patent application specifically claiming these compounds as IGF-1R inhibitors has appeared, although no IGF-1R inhibitory data is disclosed [35].
R1 HN
HN
HN N
NH2
NH2
NH2
N
N
N N N
N
N MeO
Cl
N
N
N
N
N
N R2
5
6 N
7 HN
O
OMe
3.3 2,4-Disubstituted pyrimidines A number of inhibitors utilize the pyrimidine scaffold with various substitutions at the 2 and 4 positions. Pyrimidine 8 is representative of one such series with IGF-1R activity (IC50o50 nM) [36,37]. From this series, XL-228 (structure not disclosed) has advanced into the clinic. XL-228 is a multi-targeted protein kinase inhibitor with singledigit nanomolar activity reported for IGF-1R, IR, Src, AurA/B, Fak, FGFR1,2,3 (fibroblast growth factor receptor 1,2,3), and BCR-Abl. Ph+CML and Ph+ALL patients were administered a 1-h intravenous infusion of XL-228 at a dose of 10.8 mg/kg on a once-weekly schedule. The dose-limiting toxicities observed included hyperglycemia and syncope [38]. A patent has been filed specifically claiming compound 9, which has an IC50 of 4.3 nM versus IGF-1R with eightfold selectivity over the IR [39]. The aminopyrazole element is common to both 8 and 9 and most likely forms a hydrogen bond triad with the hinge region of the kinase.
287
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
NH
N
N
NH N
HN
HN
N O
N HN
N
N
N O
N H
MeO
N
N
N
8
9
N
3-Aminoquinoline-containing pyrimidines are also claimed as IGF-1R inhibitors. Pyrimidine 10 is equipotent against IGF-1R and IR (IC50 ¼ 25 nM) and demonstrated 33% tumor growth inhibition at 100 mg/kg in a Calu6 tumor xenograft model. Despite the lack of in vitro selectivity over IR, no significant effects on blood glucose levels were observed following insulin and glucose challenge [40]. The closely related compound 11 is a 120 nM (IC50) inhibitor of IGF-1R [41]. N N
O
HN
H N
O R=
N N
N H
R
N H
10
11
The 2-aminoimidazole-substituted pyrimidine, 12, has modest IGF-1R activity (IC50 ¼ 150 nM) [42]. The related pyrimidine, TEA 226, 13, is described as a dual inhibitor of IGF-1R and FAK with inhibitory effects on mTOR signaling in esophageal cancer cells indicating a potential application in esophageal cancer [43]. O N
N H N H
HN
O H N
N N
HN
O
Cl
N N
N H
N 12
N H 13
O
288
Mark D. Wittman et al.
Imidazo[1,2-a]pyridine inhibitor 14 was identified as a screening hit with modest IGF-1R activity (IC50 ¼ 180 nM). C-2 optimization coupled with the reversal of the amide bond connectivity culminated in GSK1904529A, 15, which is equipotent against IGF-1R and IR in a receptor autophosphorylation assay. The compound has 35–124 nM (IC50) potency in cell lines representing multiple tumor types and is orally bioavailable in rat, dog, and monkey [44,45].
F
F O
N
N
H N
N N H F
F
N
H
N
O MeO
N H
H N
N
N
14
N OMe
15
N N
N
N O
S
O
3.4 2,4-Disubstituted pyrimidine isosteres The pyrrolotriazine BMS-754807, 16, has recently been presented as a 2 nM (IC50) inhibitor of IGF-1R with no selectivity over IR and is orally active in a transgenically derived, IGF-1R-driven, IGF-1R Sal tumor model at a dose of 3 mg/kg [46]. The compound is also orally active at 3 mg/kg in the IGF-1R-driven sarcoma model, Rh41, and the Geo colon carcinoma model at 12 mg/kg. The combination of 16 plus cetuximab (EGFR inhibitor) is therapeutically synergistic. Initial single ascending dose studies in normal healthy volunteers demonstrated good bioavailability and tolerability. Further clinical evaluation is ongoing [47]. A similarly substituted triazine, 17, is described in the patent literature which inhibits 96% of tumor growth in the IGF-Sal tumor model at a 3 mg/kg oral dose [48].
289
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
N
NH
N
HN
NH
HN O
H N
N N
O N N
N
N
H N
N
N F
N
N
N N
O
16
17
OMe
Several pyrrolopyrimidine-based inhibitors of IGF-1R have also been described. The 4,6-bis-anilino-1H-pyrrolo[2,3,-d]pyrimidine, 18, has an IC50 of 5 nM in an enzymatic assay and an IC50 of 109 nM in a cellular assay of IGF-1R phosphorylation [49]. Further optimization at C-5u provided compounds with single-digit nanomolar inhibition of IGF-1R in enzymatic and cellular assays. Pyrrolopyrimidine 19 is a potent IGF-1R inhibitor (enzyme IC50 ¼ 2 nM, cellular IGF-1R phosphorylation IC50 ¼ 85 nM), W1,000-fold selective over the JNK1 and JNK3 kinases, and has 98% oral bioavailability in rats [50]. The N,N,-dimethyl glycinamide forms a hydrogen bond with the backbone NH of Asp1056. Indoline 20 was designed to prevent the acid-mediated cyclization of the pyrimidine moiety onto the pendant carboxamide observed with 18 and 19. This compound maintains enzymatic and cellular potency while improving chemical stability (t1/2 ¼ W1,000 h at 231C in 0.1N HCl) [51].
NH2
F
NH2
O
O R= R
HN
HN
N
N
19 HN MeO
N H
N
18
HN
N H
N
MeO R=
O
N N N
N
NMe2
20
290
Mark D. Wittman et al.
3.5 Miscellaneous heterocyclic systems 3.5.1 Benzimidazoles Benzimidazole-pyridones were among the early small molecule chemotypes described as ATP-competitive inhibitors of IGF-1R. The initial screening hit was optimized for potency and Cytochrome p-450 (CYP) inhibition to provide an early lead structure, BMS-536924, 21 [52–60]. This compound inhibits both IGF-1R and IR with equal potency (IC50 ¼ 120 nM), is selective versus other kinases, inhibits the phosphorylation of Akt and MAP kinase (MAPK) in cells, and blocks proliferation in a wide variety of human cancer cell lines including colon, breast, lung, pancreas, prostate, sarcoma, and multiple myeloma (IC50’s of 110–460 nM). Tumor growth inhibition is observed in vivo when dosed orally in the IGF-1R Sal tumor model [46] and in a broad range of human tumor xenografts such as Colo205, Geo, and RD1 (50–100 mg/kg). Oral bioavailability is observed across all species, and a twofold window between antitumor efficacy and glucose elevation at the efficacious dose was reported [55]. Benzimidazole 21 reverses IGF-1R-induced transformation of mammary epithelial cells, blocks proliferation, and restores apical-basal polarity in MCF-7 cells [56]. The potential for CYP inhibition, time-dependent CYP inhibition, and pregnane X receptor (PXR) transactivation was reduced by replacing the morpholine ring in 21 with a C-linked piperidine. Combining this modification with the chloropyrazole side chain [57] led to the discovery of BMS-695735, 22, which demonstrates in vivo efficacy in the IGF-Sal, Colo205, Geo colon carcinoma, and JJN3 multiple myeloma models when administered orally at doses between 50 mg/kg and 100 mg/kg [59]. O N
H N
F
O
N
O
H N
NH
N
NH
N HN
HN
HO N N Cl
21
Cl
22
3.5.2 Bicyclic pyrazoles The patent literature also describes bicyclic pyrazole inhibitors of IGF-1R. These structures represent a significant departure from other reported
291
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
leads. Limited IGF-1R activity is presented in these patents with 23 being the most potent example reported (IC50 ¼ 49 nM) [61–63].
N N H N
N HN
O
HN
N H O
N
O S O
23
F
F
3.5.3 Ureas One of the most promising diarylurea (DAU) inhibitors is PQ401, 24, which inhibits the autophosphorylation of IGF-1R in human cultured MCF-7 cells with an IC50 of 12 mM. Treatment of MCNeuA cells implanted into mice with 24 reduced tumor growth when dosed three times per week intraperitoneally [64]. Lead optimization around a series of 3,5-disubstituted 1H-pyrrolo[2,3-b]pyridines led to the identification of compound 25, which shows potent in vitro kinase activity (IC50 ¼ 21 nM) and inhibits IGF-1R phosphorylation in cells (IC50 ¼ 68 nM) [65].
O H N
H N
N
O
O
H N
H N
N O
O
24
Cl
25 N
N H
292
Mark D. Wittman et al.
3.5.4 Pyrrolocarboxaldehydes Pyrrolocarboxaldehydes have been disclosed as monocyclic ATPcompetitive inhibitors of IGF-1R [66]. Aldehyde 26 is modestly selective versus IR in enzymatic and cell-based assays (IGF-1R, IC50 ¼ 490 nM; IR, IC50 ¼ 2 mM) and forms a reversible, covalent adduct with the kinase active site. CHO HN
O
OEt O O
26
3.5.5 Quinolines IGF-1R inhibitors have also been built around the quinoline core structure. Optimization of the cyanoquinoline template provided 27 with potent IR (IC50 ¼ 2 nM) and IGF-1R (IC50 ¼ 9 nM) activity as well as activity in a cellular myloid assay with an IC50 of 90 nM [67]. Cl S
N
H N
R2
N HN MeO
R1
O
CN NH
O
N
O R1
Br
28
N
R2
N
N
29 27
O
N
N
N H
The isoquinolinedione 28 was identified as an initial micromolar hit that binds at the ATP-binding site in a similar mode to the
293
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
benzimidazoles described above (Section 3.5.1). Optimization of R1 and R2 culminated in compound 29 (IGF-1R, IC50 ¼ 319 nM), which is equipotent against IR and has improved selectivity over cyclindependent kinase (CDK)-4 [68].
4. NON ATP-COMPETITIVE INHIBITORS The discovery and development of IGF-1R selective, non ATP-competitive inhibitors has been slow relative to ATP-competitive inhibitors due to the inherent medicinal chemistry challenges involved in optimizing potency for the more open substrate-binding site, requiring the use of peptide or peptidomimetic elements. To date, the two main classes of non ATP-competitive inhibitors are catechols and naturally occurring lignans.
4.1 Catechols A systematic discovery effort to design more potent and selective IGF-1R substrate inhibitors commenced with the screening of the ‘‘tyrphostins’’ (Tyrosine Phosphorylation Inhibitors) that are synthetic catechols previously shown to inhibit EGFR [69–71]. These efforts identified biscatechols 30 [72,73] and 31 [74]. Tyrphostin 30 has activity against IGF-1R (IC50 ¼ 61 nM), IR (IC50 ¼ 113 nM), and EGFR (IC50 ¼ 370 nM) receptor kinases. Based on the published X-ray structure of the kinase domain of IR, the catechol rings in 30 function as phosphate bioisosteres of phosphotyrosines 1158 and 1162 [71]. The novel tertiary amine catechol 31 inhibited the IGF-1R receptor with an IC50 of 170 nM in a cell-free kinase assay (inhibition of polyTyrGlu (pGT) phosphorylation catalyzed by IGF-1 receptor) and exhibited inhibitory IC50 values in the range 4–6 mM in a colony formation assay in soft agar for three cell lines (MCF-7, LNCap, PC-3). HO
O
N
OH
HO
HO
OH OH
CN OH
HO
O O
30
31
SAR and lead optimization efforts to mitigate metabolic liabilities due to the catechol rings led to some moderately potent benzoxazolone
294
Mark D. Wittman et al.
analogs 32a–c [73]. No selectivity over IR was observed. Replacement of both catechols with benzoxazolone rings led to inactive compounds.
R1
32a
R2
32b
CN 32
IGF-1R IC50
O
OH
N H
OH
~370 nM
O
O R1
R2
H N
OH
O
OH
~430 nM
O H N
HO 32c
~600 nM O
O
HO
To date, none of the synthetic catechols or their close analogs discussed above have progressed into clinical development. The naturally occurring bis-catechol, nordihydroguaiaretic acid, 33 (NDGA, INSM-18), has advanced to phase II clinical trials using continuous oral dosing for the treatment of prostate cancer [75–77]. NDGA inhibits IGF1R phosphorylation of a synthetic nonspecific tyrosine kinase substrate and proliferation of MCF-7 breast cell line with an IC50 of 0.9 and 24.6 mM respectively. Some early positive clinical data is now emerging from the prostate trial with respect to reduction in prostate-specific antigen (PSA) levels and delay in PSA doubling time in patients. Doses up to 2,500 mg/ day are tolerated with minimal toxic effects. NDGA has Her-2 receptor kinase and 15-lipoxygenase inhibitory activity. Although there is no direct mention of 33 being a non ATP-competitive IGF-1R inhibitor, it is a non ATP-competitive inhibitor of FGFR3 tyrosine kinase [78].
OH HO OH HO 33
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
295
4.2 Natural lignans The naturally occurring lignan picropodophyllotoxin (PPP) or AXL-1717, 34, has recently advanced into human clinical trials (oral dosing) [79,80]. The epimeric podophyllotoxin 35 (PPT), a cytotoxic agent known for its potent antimitotic activity, is also reported to be an IGF-1R selective substrate inhibitor. Both 34 and 35 are reported to inhibit IGF-1R catalyzed substrate phosphorylation of pGT with an IC50 value of 6 nM. Antiproliferative IC50 values against 11 cell lines for both compounds are between 20 nM and 25 nM. In preclinical models, 34 is efficacious when dosed intraperitoneally in a wide range of tumor models such as breast, prostate, malignant melanoma, and multiple myeloma. Results from cell culture studies using IGF-1R-deficient cell lines, mouse embryonic fibroblasts (MEFs), and HepG2 cells have called into question whether the observed antitumor activity is due to IGF-1R inhibition [81,82]. OH
OH
O
O O
O
O O
O
H3CO
OCH3
O
H3CO
OCH3
OCH3
OCH3
34
35
5. CONCLUSIONS Significant progress has been made in developing small molecule inhibitors of IGF-1R for use in the clinic. Coupled with the advances being made toward exploiting and validating the IGF-1R target using mAbs, the stage is set to determine the clinical potential of IGF-1R inhibitors. It will be interesting to follow the clinical development of molecules such as AXL-1717, BMS-754807, INSM-18, OSI-906, and XL-228 with respect to efficacy, safety, and tolerability. The hope remains that small molecule inhibitors will provide a complementary approach to mAb therapeutics in terms of efficacy and dosing flexibility, particularly, in combination studies with cytotoxics and EGFR antagonists.
296
Mark D. Wittman et al.
REFERENCES [1] J. J. Elias, Proc. Soc. Exp. Biol. Med., 1959, 101, 500. [2] C. L. Arteaga, L. J. Kitten, E. B. Coronado, S. Jacobs, F. C. Kull, Jr., D. C. Allred, C. K. Osborne and C. Kent, J. Clin. Invest, 1989, 84, 1418. [3] J. M. Chan, M. J. Stempfer, E. Giovannucci, P. H. Gann, J. Ma, P. Wilkinson, C. H. Hennekens and M. Pollak, Science, 1998, 279, 563. [4] J. Rodon, V. De Santos, R. J. Ferry, Jr. and R. Kurzrock, Mol. Cancer Ther., 2008, 7, 2575. [5] M. Pollak, Nat. Rev. Cancer, 2008, 8, 915. [6] M. Pollak, Curr. Opin. Pharm., 2008, 8, 384. [7] F. Frasca, G. Pandini, P. Scalia, L. Sciacca, R. Mineo, A. Costantino, I. D. Goldfine, A. Belfiore and R. Vigneri, Mol. Cell Biol., 1999, 19, 3278. [8] H. Zhang, A. M. Pelzer, D. T. Kiang and D. Yee, Cancer Res., 2007, 67, 391. [9] D. Olmos, R. Molife, S. Okuno, F. Worden, G. Hammer, T. Yap, H. Shaw, S. Schuetze, L. Roberts, A. Gualberto, J. de-Bono and P. Haluska, AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, San Francisco, CA, October 22–26, 2007, Abstract A63. [10] D. D. Karp, L. G. Paz-Ares, L. J. Blakely, H. Kreisman, P. D. Eisenberg, R. B. Cohen, L. Garland, C. J. Langer, C. L. Melvin and A. Gualberto, 43rd American Society of Clinical Oncology, June, 2007, Abstract 7506. [11] M. Hewish, I. Chau and D. Cunningham, Recent Pat. Anticancer Drug Discov., 2009, 4, 54. [12] P. K. S. Sarma, R. Tandon, G. Praful, S. G. Dastidar, A. Ray, B. Das and I. A. Cliffe, Expert Opin. Ther. Patents, 2007, 17, 25. [13] R. D. Hubbard and J. L. Wilsbacher, Chem. Med. Chem., 2007, 2, 41. [14] C. Garcia-Echeverria, Idrugs, 2006, 9, 415. [15] F. Hofmann and C. Garcia-Echeverria, Drug Discov. Today, 2005, 10, 1041. [16] S. Favelyukis, J. H. Till, S. R. Hubbard and W. T. Miller, Nat. Struct. Biol., 2001, 8, 1058. [17] E. Y. Skolnik, C. H. Lee, A. Batzer, L. M. Vicentini, M. Zhoa, R. Daly, M. J. Myers, Jr., J. M. Becker, A. Ulrich, et al., EMBO J., 1993, 12, 1929. [18] B. Vanhaesebroeck and D. R. Alessi, J. Biochem., 2000, 346, 561. [19] A. Camirand, M. Zakikhani, F. Young and M. Pollak, Breast Cancer Res., 2005, 7, R570. [20] F. Morgillo, J. K. Woo, E. S. Kim, W. K. Hong and H. Y. Lee, Cancer Res., 2006, 66, 10100. [21] C. Garcia-Echeverria, M. A. Pearson, A. Marti, T. Meyer, J. Mestan, J. Zimmermann, J. Gao, J. Brueggen, H.-G. Capraro, R. Cozens, D. B. Evans, D. Fabbro, P. Furet, D. G. Porta, J. Liebetanz, G. Martiny-Baron, S. Ruetz and F. Hofmann, Cancer Cell, 2004, 5, 231. [22] C. S. Mitsiades, N. S. Mitsiades, C. J. McMullan, V. Poulaki, R. Shringarpure, M. Akiyama, T. Hideshima, D. Chauhan, M. Joseph, T. A. Libermann, C. Garcia-Echeverria, M. A. Pearson, F. Hofmann, K. C. Anderson and A. L. Kung, Cancer Cell, 2004, 5, 221. [23] H.-G. Capraro, P. Fuuret and C. Garcia-Echeverria, WO Patent Application 2004/043962, 2004. [24] K. Scotlandi, M. C. Manara, G. Nicoletti, P. L. Lollini, S. Lukas, S. Benini, S. Croci, S. Perdichizzi, D. Zambelli, M. Serra, C. Garcia-Echeverria, F. Hofmann and P. Picci, Cancer Res., 2005, 65, 3868. [25] B. Tanno, C. Mancini, R. Vitali, M. Mancuso, H. P. McDowell, C. Dominici and G. Raschella, Clin. Cancer Res., 2006, 12, 6772. [26] M. C. Manara, L. Landuzzi, P. Nanni, G. Nicoletti, D. Zambelli, P. L. Lollini, C. Nanni, F. Hofmann, C. Garcia-Echeverria, P. Picci and K. Scotlandi, Clin. Cancer Res., 2007, 13, 1322. [27] M. J. Mulvihill, Q.-S. Ji, D. Werner, P. Beck, C. Cesario, A. Cooke, M. Cox, A. Crew, H. Dong, L. Feng, K. W. Foreman, G. Mak, A. Nigro, M. O’Connor, L. Saroglou,
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
[28]
[29] [30]
[31]
[32] [33]
[34]
[35]
[36]
[37] [38]
[39] [40] [41] [42] [43]
[44]
[45]
297
K. M. Stolz, I. Sujka, B. Volk, Q. Weng and R. Wilkes, Bioorg. Med. Chem. Lett., 2007, 17, 1091. L. D. Arnold, C. Cesario, H. Coate, A. P. Crew, H. Dong, K. Foreman, A. Honda, R. Laufer, A.-H. Li, K. M. Mulvihill, M. J. Mulvihill, A. Nigro, B. Panicker, A. G. Steinig, Y. Sun, Q. Weng, D. S. Werner, M. J. Wyle and T. Zhang, WO Patent Application 2005/ 097800, 2005. A. P. Crew, M. J. Mulvihill and D. S. Werner, US Patent Application 2006/019957, 2006. M. J. Mulvihill, Q.-S. Ji, H. R. Coate, A. Cooke, H. Dong, L. Feng, K. Foreman, M. Rosenfeld-Franklin, A. Honda, G. Mak, K. M. Mulvihill, A. I. Nigro, M. O’Connor, C. Pirrit, A. G. Steinig, K. Siu, K. M. Stolz, Y. Sun, P. A. R. Tavares, Y. Yao and N. W. Gibson, Bioorg. Med. Chem., 2008, 16, 1359. Q.-S. Ji, M. J. Mulvihill, M. Rosenfeld-Franklin, A. Cooke, L. Feng, G. Mak, M. O’Connor, Y. Yao, C. Pirritt, E. Buck, A. Eyzaguirre, L. D. Arnold, N. W. Gibson and J. A. Pachter, Mol. Cancer Ther., 2007, 6, 2158. M. Mulvihill, ACS Prospectives Conference PK/PD for Medicinal Chemists, Boston, MA, September 8, 2008. R. D. Hubbard, N. Y. Bamaung, S. D. Fidanze, S. A. Erickson, R. A. Manteil, P. Kovar, G. T. Wang, J. Wang, G. S. Sheppard and R. L. Bell, Abstract 566, Eur. J. Cancer Suppl., 2008, 6(October), 176. R. D. Hubbard, N. Y. Bamaung, S. D. Fidanze, S. A. Erickson, F. Palazzo, J. L. Wilbacher, Q. Zhang, L. A. Tucker, X. Hu, P. Kovar, D. J. Osterling, E. F. Johnson, J. Bouska, J. Wang, S. K. Davidsen, R. L. Bell and G. S. Sheppard, Bioorg. Med. Chem. Lett., 2009, 19, 1718. S. J. O’Connor, J. Dumas, W. Lee, J. Dixon, D. Cantin, D. Gunn, J. Burke, B. Phillips, D. Lowe, T. Shelekhin, G. Wang, X. Ma, S. Ying, A. Mcclure, F. Achebe, M. Lobell, F. Ehrgott, C. Iwuagwu and K. Parcella, WO Patent Application 2007/056170, 2007. J. Chen, L. E. Dalrymple, S. Epshteyn, T. P. Forsyth, T. P. Huynh, M. A. Ibrahim, J. W. Leahy, G. L. Lewis, G. Mann, L. W. Mann, R. T. Noguchi, B. H. Ridgway, J. C. Sangalang, K. L. Schnepp, X. Shi, C. S. Takeuchi, M. A. Williams, J. Nuss and A. K. Cheung, WO Patent Application 2006/074057, 2006. W. Zhang, WO Patent Application 2008/005538, 2008. J. Cortes, R. Paquette, M. Talpaz, J. Pinilla-Ibarz, K. El-Shami, M. Wetzler, J. Lipton, C. Kasap, L. A. Bui, P. Woodard, O. O Clary and N. Shah, 50th Annual Meeting of the American Society of Hematology, December 6–9, 2008, Abstract 3232. T. Nowak, S. C. Purkiss and A. P. Thomas, WO Patent Application 2008/117051, 2008. J.-C. Harmange, J. L. Buchanan, S. Chaffee, P. M. Novak, S. Van Der Plas and X. Zhu, US Patent 2005/6939874, 2005. T. Heinrich, A. Blaukat, and M. Kordowicz, WO Patent Application 2006/108487, 2006. T. Heinrich, A. Blaukat and M. Kordowicz, US Patent Application 2008/0194605, 2008. Z. G. Wang, T. Fukazawa, T. Nishikawa, N. Watanabe, K. Sakurama, T. Motoki, M. Takaoka, S. Hatakeyama, O. Omori, T. Ohara, S. Tanabe, Y. Fujiwara, Y. Shirakawa, T. Yamatsuji, N. Tanaka and Y. Naomoto, Oncol. Rep., 2008, 20, 1473. K. A. Emmitte, B. J. Wilson, E. W. Baum, H. K. Emerson, K. W. Kuntz, K. E. Nailor, J. M. Salovich, S. C. Smith, M. Cheung, R. M. Gerding, K. L. Stevens, D. E. Uehling, R. A. Mook, Jr., G. S. Moorthy, S. H. Dickerson, A. M. Hassell, M. A. Leesnitzer, L. M. Shewchuk, A. R. Groy, J. L. Rowand, K. Anderson, C. L. Atkins, J. Yang, P. Sabbatini and R. Kumar, Bioorg. Med. Chem. Lett., 2009, 19, 1004. P. Sabbatini, J. L. Rowand, A. Groy, S. Korenchuk, Q. Liu, C. Atkins, M. Dumble, J. Yang, K. Anderson, B. J. Wilson, K. A. Emmitte, S. K. Rabindran and R. Kumar, Clin. Cancer Res., 2009, 15, 3058.
298
Mark D. Wittman et al.
[46] J. M. Carboni, A. V. Lee, D. L. Hadsell, B. R. Rowley, F. Y. Lee, D. K. Bol, A. E. Camuso, M. Gottardis, A. F. Greer, C. P. Ho, W. Hurlburt, A. Li, M. Saulnier, U. Velaparthi, C. Wang, M.-L. Wen, R. A. Westhouse, M. Wittman, K. Zimmermann, B. A. Rupnow and T. W. Wong, Cancer Res., 2005, 65, 3781. [47] M. Wittman, J. Carboni, Z. Yang, F. Lee, G. Cantor, M. Antman, R. Attar, P. Balimane, C. Chen, S. Cheng, L. Discenza, C. Fairchild, F. G. Finckenstein, D. Frennesson, M. Gottardis, A. Greer, X. Gu, W. Hurlburt, A. Li, J. Li, P. Liu, W. Johnson, D. Langley, H. Mastalarz, A. Mathur, K. Menard, K. Patel, J. Sack, X. Sang, M. Saulnier, K. Stefanski, S. Traeger, G. Trainor, U. Velaparthi, S. Yeola, G. Zhang, K. Zimmerman and D. Vyas, MEDI-163, 235th ACS National Meeting, Salt Lake City, UT, March, 2009. [48] U. Velaparthi, P. Liu, M. D. Wittman and D. R. Langley, WO Patent Application 2009/ 015254, 2009. [49] S. D. Chamberlain, J. W. Wilson, F. Deanda, S. Patnaik, A. M. Redman, B. Yang, L. Shewchuk, P. Sabbatini, M. A. Leesnitzer, A. Groy, C. Atkins, R. Gerding, M. Hassell, H. Lei, R. A. Mook, Jr., G. Moorthy, J. L. Rowand, K. L. Stevens, R. Kumar and J. B. Shotwell, Bioorg. Med. Chem. Lett., 2009, 19, 469. [50] S. D. Chamberlain, A. M. Redman, J. W. Wilson, F. Deanda, J. B. Shotwell, R. Gerding, H. Lei, B. Yang, K. L. Stevens, A. M. Hassell, L. M. Shewchuk, M. A. Leesnitzer, J. L. Smith, P. Sabbatini, C. Atkins, A. Groy, J. L. Rowand, R. Kumar, R. A. Mook, Jr., G. Moorthy and S. Patnaik, Bioorg. Med. Chem. Lett., 2009, 19, 360. [51] S. D. Chamberlain, A. M. Redman, S. Patnaik, K. Brickhouse, Y. C. Chew, F. Deanda, R. Gerding, H. Lei, G. Moorthy, M. Patrick, K. L. Stevens, J. W. Wilson and J. B. Shotwell, Bioorg. Med. Chem. Lett., 2009, 19, 373. [52] M. D. Wittman, B. Balasubramanian, K. Stoffan, U. Velaparthi, P. Liu, S. Krishnananthan, J. Carboni, A. Li, A. Greer, R. Attar, M. Gottardis, C. Chang, B. Jacobson, Y. Sun, S. Hansel, M. Zoeckler and D. Vyas, Bioorg. Med. Chem. Lett., 2007, 17, 974. [53] U. Velaparthi, M. Wittman, P. Liu, K. Stoffan, K. Zimmermann, X. Sang, J. Carboni, A. Li, R. Attar, M. Gottardis, A. Greer, C. Y. Chang, B. L. Jacobsen, J. S. Sack, Y. Sun, D. R. Langley, B. Balasubramanian and D. Vyas, Bioorg. Med. Chem. Lett., 2007, 17, 2317. [54] U. Velaparthi, P. Liu, B. Balasubramanian, J. Carboni, R. Attar, M. Gottardis, A. Li, A. Greer, M. Zoeckler, M. D. Wittman and D. Vyas, Bioorg. Med. Chem. Lett., 2007, 17, 3072. [55] M. Wittman, J. Carboni, R. Attar, B. Balasubramanian, P. Balimane, P. Brassil, F. Beaulieu, C. Chang, W. Clarke, J. Dell, J. Eummer, D. Frennesson, M. Gottardis, A. Greer, S. Hansel, W. Hurlburt, B. Jacobson, S. Krishnanathan, F. Y. Lee, A. Li, T. A. Lin, P. Liu, C. Ouellet, X. Sang, M. G. Saulnier, K. Stoffan, Z. Yang, K. Zimmermann, M. Zoeckler and D. Vyas, J. Med. Chem., 2005, 48, 5639. [56] B. C. Litzenburger, H. J. Kim, I. Kuiaste, J. M. Carboni, R. M. Attar, M. M. Gottardis, C. R. Fairchild and A. V. Lee, Clin. Cancer Res., 2009, 15, 226. [57] M. G. Saulnier, D. B. Frennesson, M. D. Wittman, K. Zimmermann, U. Velaparthi, D. R. Langley, C. Struzynski, X. Sang, J. Carboni, A. Li, A. Greer, Z. Yang, P. Balimane, M. Gottardis, R. Attar and D. Vyas, Bioorg. Med. Chem. Lett., 2007, 18, 1702. [58] K. Zimmermann, M. D. Wittman, M. G. Saulnier, U. Velaparthi, D. R. Langley, X. Sang, D. Frennesson, J. Carboni, A. Li, A. Greer, M. Gottardis, R. M. Attar, Z. Yang, P. Balimane, L. N. Discenza and D. Vyas, Bioorg. Med. Chem. Lett., 2008, 18, 4075. [59] U. Velaparthi, M. Wittman, P. Liu, J. M. Carboni, F. Y. Lee, R. Attar, P. Balimane, W. Clarke, M. W. Sinz, W. Hurlburt, K. Patel, L. Discenza, S. Kim, M. Gottardis, A. Greer, A. Li, M. Saulnier, Z. Yang, K. Zimmermann, G. Trianor and D. Vyas, J. Med. Chem., 2008, 51, 5897. [60] P. Haluska, J. M. Carboni, D. A. Loegering, F. Y. Lee, M. Wittman, M. G. Saulnier, D. B. Frennesson, K. R. Kalli, C. A. Conover, R. M. Attar, S. H. Kaufmann, M. Gottardis and C. Erlichman, Cancer Res., 2006, 66, 362.
Small Molecule Inhibitors of Insulin-Like Growth Factor-1 Receptor Kinase
299
[61] T. Bandiera, A. L. Borgia, P. Polucci, M. Villa, M. Nesi, M. Angiolini and M. Varsi, WO Patent Application 2007/068619, 2007. [62] T. Bandiera, A. L. Borgia, S. C. Orrenius, E. Perrone, I. Beria, D. Fancelli and A. Galvani, WO Patent Application 2007/068637, 2007. [63] T. Bandiera, E. Perrone, A. L. Borgia and M. Varasi, WO Patent Application 2007/099166, 2007. [64] K. L. Gable, B. A. Maddux, C. Penaranda, M. Zavodovskaya, M. J. Campbell, M. Lobo, L. Robinson, S. Schow, J. A. Kerner, I. D. Goldfine and J. F. Youngren, Mol. Cancer Ther., 2006, 5, 1079. [65] S. Patnaik, K. L. Stevens, R. Gerding, F. Deanda, J. B. Shotwell, J. Tang, T. Hamajima, H. Nakamura, M. A. Leesnitzer, A. M. Hassell, L. M. Shewchuck, R. Kumar, H. Lei and S. D. Chamberlain, Bioorg. Med. Chem. Lett., 2009, 19, 3136. [66] I. M. Bell, S. M. Stirdivant, J. Ahern, J. C. Culberson, P. L. Darke, C. J. Dinsmore, R. A. Drakas, S. N. Gallicchio, S. L. Graham, D. C. Heimbrook, D. L. Hall, J. Hua, N. R. Kett, A. S. Kim, M. Kornienko, L. C. Kuo, S. K. Munshi, A. G. Quigley, J. C. Reid, B. W. Trotter, L. H. Waxman, T. M. Williams and C. B. Zartman, Biochemistry, 2005, 44, 9430. [67] L. M. Miller, S. C. Mayer, D. M. Berger, D. H. Boschelli, F. Boschelli, L. Di, X. Du, M. Dutia, M. B. Floyd, M. Johnson, C. H. Kenny, G. Krishnamurthy, F. Moy, S. Petusky, D. Tkach, N. Torres, B. Wu and W. Xu, Bioorg. Med. Chem. Lett., 2009, 19, 62. [68] S. C. Mayer, A. L. Banker, F. Boschelli, L. Di, M. Johnson, C. H. Kenny, G. Krishnamurthy, K. Kutterer, F. Moy, S. Petusky, M. Ravi, D. Tkach, H. Tsou and W. Xu, Bioorg. Med. Chem. Lett., 2008, 18, 3641. [69] A. Gazit, P. Yaish, C. Gilon and A. Levitzki, J. Med. Chem., 1989, 32, 2344. [70] A. Gazit, N. Osherov, I. Posner, P. Yaish, E. Poradosu, C. Gilon and A. Levitzki, J. Med. Chem., 1991, 34, 1896. [71] G. Blum, A. Gazi and A. Levitzki, Biochemistry, 2000, 39, 15705. [72] A. Levitzki and E. Mishani, Ann. Rev. Biochem., 2006, 75, 93. [73] G. Blum, A. Gazit and A. Levitzki, J. Biol. Chem., 2003, 42, 40442. [74] L. Steiner, G. Blum, Y. Friedmann and A. Levitzki, Eur. J. Pharmacol., 2007, 562, 1. [75] J. F. Youngren, K. Gable, C. Penaranda, B. A. Maddux, M. Zavodovskaya, M. Lobo, M. Campbell, J. Kerner and I. D. Goldfine, Breast Cancer Res. Treat., 2005, 94, 37. [76] J. E. Blecha, M. O. Anderson, J. M. Chow, C. C. Guevarra, C. Pender, C. Penaranda, M. Zavodovskaya, J. F. Youngren and C. E. Berkman, Bioorg. Med. Chem. Lett., 2007, 17, 4026. [77] C. J. Ryan, A. H. Harzstark, J. Rosenberg, A. Lin, C. Claros, I. D. Goldfine, J. F. Kerner and E. J. Small, Brit. J. Urol. Int., 2008, 101, 436. [78] A. N. Meyer, C. W. McAndrew and D. J. Donoghue, Cancer Res., 2008, 68, 7362. [79] O. Larsson and M. Axelson, WO PCT Publication 2002/102804. [80] A. Girnita, L. Girnita, F. Del Prete, A. Bartolazzi, O. Larsson and M. Axelson, Cancer Res., 2004, 64, 236. [81] S. Linder, M. C. Shoshan and R. S. Gupta, Cancer Res., 2007, 67, 2899. [82] O. Larsson and M. Axelson, Cancer Res., 2007, 67, 2899.
CHAPT ER
15 Case History: Discovery of Ixabepilone (IXEMPRATM), a First-in-Class Epothilone Analog for Treatment of Metastatic Breast Cancer Robert M. Borzilleri and Gregory D. Vite
Contents
1. Introduction 2. Epothilone Natural Products 2.1 Fermentation and isolation 2.2 Biosynthesis 2.3 Biological properties and mechanism of action 2.4 Evaluation of epothilones as drug leads 3. Approaches to Identify Drug Candidates 3.1 General strategy 3.2 Total synthesis 3.3 Semisynthesis 4. Preclinical Pharmacology 4.1 Cytotoxicity 4.2 In vivo efficacy 4.3 Profiling 5. Clinical Results 5.1 Phase I/II highlights 5.2 Phase II/III registrational trials 5.3 Pharmacogenomics 6. Future Directions 7. Conclusions References
301 302 302 303 303 304 304 304 306 307 310 311 311 314 314 314 315 316 317 318 319
Bristol-Myers Squibb R&D, P.O. Box 4000, Princeton, NJ 08543-4000 Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04415-7
r 2009 Elsevier Inc. All rights reserved.
301
302
Robert M. Borzilleri and Gregory D. Vite
1. INTRODUCTION Since Food and Drug Administration (FDA) approval in 1991, Taxols (paclitaxel) has been a mainstay of chemotherapy for various cancers including breast, ovarian, and lung cancers [1,2]. Despite the clinical success, shortcomings of this treatment were noted early on, and even today, our understanding of factors that determine whether patients will respond to the drug is still evolving. For these reasons, intense research has been directed toward discovering other drug modalities that take advantage of and improve upon paclitaxel’s unique mechanism of action (i.e., microtubule stabilization). These research efforts include development of new paclitaxel formulations and delivery systems, synthesis of taxane analogs, and exploration of new classes of natural products that kill tumor cells by the same mechanism [3–5]. At Bristol-Myers Squibb (BMS), the initial discovery of promising novel taxane analogs eventually gave way to an in-depth study of two newly discovered natural products, eleutherobin and epothilone [6]. Comparisons of early preclinical activities and the feasibility of re-supply for these two agents led to a decision to pursue the epothilone class. The main objective of this effort was to discover an agent with a broader spectrum of antitumor activity, especially against paclitaxel-resistant tumors and preferably with a reduction of undesired side effects. Accordingly, this account describes the drug discovery path from epothilone natural product to semisynthetic analog ixabepilone (IXEMPRATM), currently FDAapproved for the treatment of metastatic and locally advanced breast cancer.
2. EPOTHILONE NATURAL PRODUCTS 2.1 Fermentation and isolation German chemist Gerhard Ho¨fle and microbiologist Hans Reichenbach of Gesellschaft fu¨r Biotechnologische Forschung (GBF, now the Helmholtz Center for Infection Research) spent much of their careers studying myxobacteria, a unique class of microorganisms. They discovered that a particular strain of the species Sorangium cellulosum (So ce90), cultured from a soil sample collected in South Africa, produced the epothilone natural products as secondary metabolites [7,8]. In general, S. cellulosum bacteria can be readily found in soil and decaying plant material, but culturing these bacteria in the laboratory is not trivial. Epothilone production was found to be highly dependent on strain selection and fermentation media. To facilitate recovery of the epothilones, the fermentation was carried out with XAD-16 resin. Extraction with
Discovery of Ixabepilone (IXEMPRATM)
S
R
R
O
S
12 OH
15
N
303
OH
N
17 O 1 O
O OH
1 (R = H) 2 (R = Me)
O
O
OH
O
3 (R = H) 4 (R = Me)
Figure 1 Epothilones AD.
methanol followed by purification using reverse-phase chromatography and recrystallization afforded the major metabolites epothilones A (1) and B (2) in a 2:1 ratio. A typical fermentation provided approximately 120 mg/L of epothilone A. Strain and process improvements resulted in higher yield and enhanced recovery of epothilone B, which was required for semi-synthesis and manufacture of ixabepilone (vide infra). The molecular structures of epothilones A and B were confirmed through single crystal X-ray analysis by Ho¨fle and co-workers (Figure 1) [8]. In addition, Ho¨fle’s careful analysis of the So ce90 fermentation broth revealed the presence of more than 30 other epothilone-related, minor metabolites [9].
2.2 Biosynthesis The German group established early on that the epothilone biosynthesis occurs through the action of a distinct polyketide synthase (PKS), because a radiolabel (13C) could be incorporated into the natural product through addition of isotope-enriched acetate or propionate to the fermentation medium [10,11]. Furthermore, the thiazolyl starter unit required for initiation of the biosynthesis was reported to be derived from cysteine. Shortly thereafter, the epothilone PKS gene cluster was independently cloned by researchers at Novartis and Kosan Biosciences [12,13]. The latter group demonstrated successful heterologous expression of the PKS in Streptomyces coelicolor and validated production of epothilones in this organism. This work demonstrated that chain extension by the PKS is followed by macrocyclization and release from the PKS to give the 12,13-olefinic epothilones C (3) and D (4) (Figure 1). Subsequently, post-PKS oxidation by a cytochrome P450 affords the corresponding 12,13-oxiranes [14]. Discovery of the P450 gene epoK by Kosan Biosciences led to the development of an epoK-mutant strain of Myxococcus xanthus that could be used for improved recovery of olefinic epothilones [15]. Interestingly, the entire genome of S. cellulosum (closely
304
Robert M. Borzilleri and Gregory D. Vite
related strain So ce56) was sequenced recently and consequently was claimed to be the largest bacterial genome sequencing to date [16].
2.3 Biological properties and mechanism of action At the time of the initial discovery of the epothilones, it was determined that these natural products possess potent antifungal activity. This biological activity and implications for agricultural applications were briefly explored by GBF and collaborators. However, Bollag and co-workers later reported that the epothilones were potent cytotoxic agents that induce G2-M cell-cycle arrest [17]. He further demonstrated that this activity against cancer cells was maintained in paclitaxelresistant cell lines, likely due to less susceptibility to drug efflux pumps. The epothilones were found to inhibit the binding of radiolabeled paclitaxel to b-tubulin in a competitive binding assay, suggesting both a common mechanism of action and a common binding site for the two molecules. This was further supported by structural studies by Nettles and co-workers [18]. Accordingly, electron diffraction studies using zincinduced, two-dimensional sheets of a,b-tubulin suggested a common binding site for epothilone A and paclitaxel, although the epothilone was shown to make unique interactions with the protein. There is controversy regarding the accuracy of this model because this work does not recapitulate the three-dimensional characteristics of a- and b-tubulin protein interactions that are present in intact microtubules [19]. Still, a fair amount of the structure-activity relationships (SAR) for the epothilones can be rationalized by the Nettles model, suggesting that it is not far from the true binding mode.
2.4 Evaluation of epothilones as drug leads Initial studies at BMS confirmed the potent cytotoxicity of the epothilone natural products. Therefore, there was considerable enthusiasm to assess the antitumor activity of these agents in preclinical models of cancer. In a xenograft model (Pat-7) derived from a breast cancer patient, epothilones A and B were not efficacious when dosed intravenously (iv) at their respective maximum tolerated doses (MTD) [6,20]. Similarly, iv bolus administration of epothilone B in a murine allograft model (M5076) was inactive. However, slow iv infusion of the compound was efficacious, suggesting that epothilone B was metabolically unstable in mice. This hypothesis was confirmed by in vitro studies in both murine plasma and liver microsomal fractions. Higher stability was observed in the corresponding human in vitro assays (vide infra). Additional in vitro and in vivo studies in the presence of a general esterase inhibitor, bisdinitrophenyl phosphate, revealed that the macrolactone is susceptible to
Discovery of Ixabepilone (IXEMPRATM)
305
esterase-mediated cleavage. Demonstration of activity in murine xenograft models was an arbitrary prerequisite to advancement of a drug candidate at BMS. Therefore, it was deemed necessary to identify an epothilone analog that demonstrated activity in rodent xenograft models which might be predictive of efficacy in humans.
3. APPROACHES TO IDENTIFY DRUG CANDIDATES 3.1 General strategy The initial disclosure of the absolute stereochemistry of epothilones A and B in 1996 [8] spawned a highly competitive research endeavor to discover biologically active drug analogs. The pioneering total synthesis routes developed by the Danishefsky [21,22], Nicolaou [23,24], and Schinzer [25] groups provided the foundation for the preparation and evaluation of a myriad of diverse epothilone analogs [26–29]. Complementary to these efforts, several additional academic laboratories and industrial research organizations obtained epothilones through fermentation, biosynthesis, biotransformation, total synthesis, semisynthesis, and combinatorial synthesis methods [26,30–34]. Medicinal chemistry efforts at BMS initially focused on utilizing total synthesis approaches to access epothilone analogs. However, semisynthesis approaches were quickly adopted to drive the SAR, once multi-gram quantities of several natural epothilones became available through the collaboration with GBF. Structural modifications were readily accessible at three regions of the molecule, as outlined in Figure 2. The primary objective of the BMS drug discovery program was to identify epothilone analogs with improved: (1) metabolic stability, (2) chemical stability (for oral administration), (3) physicochemical/ thiazole derivatization and C-21 methyl substitution tolerated S
O
isosteric replacements of epoxide allowed
R
OH
N
modifications to lactone and C2−C3 positions allowed
O O
OH
5
Figure 2 Summary of BMS medicinal chemistry efforts.
O
306
Robert M. Borzilleri and Gregory D. Vite
pharmaceutical properties, and (4) pharmacokinetic parameters relative to the natural products [30–31]. Moreover, selection of a suitable development candidate from this unique structural class would require robust in vivo efficacy in multiple preclinical human tumor xenograft models, particularly those resistant to the taxanes, and an acceptable safety profile (therapeutic index). The intrinsic activity of the epothilone analogs or their ability to induce the formation of hyperstable tubulin polymers was measured spectrophotometrically as a function of changes in turbidity. The rate of change in the proportion of polymerized tubulin was expressed as the effective concentration of drug capable of inducing an initial slope of 0.01 when plotting absorbance (A280 nm/min rate) versus time (EC0.01). Preliminary in vitro antitumor activity was assessed using the taxanesensitive human colon carcinoma cell line HCT-116 (non-P-gp expressing line) and expressed as the concentration required for 50% growth inhibition (IC50).
3.2 Total synthesis To address the metabolic instability associated with the epothilones and evaluate the practicality of a total synthesis strategy, researchers at BMS initiated a synthesis of the corresponding lactam analogs using a ring closing metathesis (RCM) route based on published syntheses of epothilones A and C (Figure 3) [27,28]. The key olefin metathesis precursor 8, derived from coupling of allylic amine 6 and polypropionate
OH
S
EDCI, HOBt DMF, 77%
+ N
HO2C NH2
OTBS O
6
7
S 7 OH
N 3
HN O
RuBnCl2(PCy3)2
O
N
benzene 41% (~5:1 E/Z)
OTBS O
HN O
8 TFA, CH2Cl2 0 °C, 68%
Figure 3
OH
S
OP
9a (E-isomer, P = TBS) 9b (Z-isomer, P = TBS) 10 (P = H)
Initial work on the total synthesis of lactam analogs of Epo C.
Discovery of Ixabepilone (IXEMPRATM)
307
acid 7, underwent a ruthenium-catalyzed RCM reaction to afford the C12,13-E-isomeric macrolactam 9a, predominantly [35]. Subsequent deprotection of the silyl ether 9a gave E-epothilone C-lactam 10, which was found to be inactive in the tubulin polymerization assay (EC0.01 W 1,100 mM). The stereochemical outcome of the RCM process was shown to be highly substrate dependent in the context of epothilone research [27–29], and subsequent studies revealed that an additional silyl protecting group on the C7-alcohol produced 1:1 mixtures of the E-and Z-olefinic macrocycles [36]. While the initial total synthesis work demonstrated that the critical RCM reaction was operative, the yields of the desired cis-olefinic macrocycle 9b were unsatisfactory. In addition, the lengthy synthetic routes subsequently developed by others to prepare the lactam analogs [37] were deemed limiting in terms of the quantities of material necessary to sustain a competitive medicinal chemistry effort. To circumvent these issues, efficient and reliable semisynthetic methods were explored to access epothilone derivatives, including the desired macrolactam analogs.
3.3 Semisynthesis The natural olefin-containing epothilones C (3) and D (4) (Figure 1) were found to retain potent activity in the tubulin polymerization assay with EC0.01 values of 3.7 and 0.6 mM respectively [20]. These data suggest that the epoxide oxygen does not engage in critical non-bonded interactions with the tubulin protein. To explore bioisosteric replacements of the C12,13-epoxide moiety and identify more stable epothilone derivatives, larger quantities of the minor metabolites 3 and 4 were required. BMS scientists developed stereoselective titanium- and tungsten-promoted deoxygenation reactions of the more abundant epothilones A and B to gain access to substantial supplies of 3 and 4, respectively [38]. The Z-12,13-cyclopropyl analogs 11 and 12 obtained from direct stereospecific cyclopropanation of the olefinic epothilone derivatives were found to be equipotent in the tubulin polymerization (EC0.01 ¼ 1.42.1 mM) and HCT-116 cell line cytotoxicity (IC50 ¼ 0.701.4 nM) assays (Figure 4) [38]. Unfortunately, high plasma protein binding (W99%) precluded further development of these analogs [20]. Initial attempts to prepare 12a,13a-aziridine analogs by direct aziridination of 3 or 4 using nitrene cycloaddition chemistry were unsuccessful. However, the aziridine analog of epothilone A (13) was prepared using a short synthetic sequence involving double inversion of stereochemistry at C12 and C13 of 1 (Figure 4) [39]. The parent aziridine 13 was approximately sevenfold less potent than epothilones A (1) and B (2) (Figure 1) in the tubulin polymerization assay.
308
Robert M. Borzilleri and Gregory D. Vite
R
R
N
S
S OH
N O
O O
OH
O
O
O
O S OH
N O O
OH
17 (R = OH) 18 (R = NH2)
Figure 5
O
Epothilone cyclopropane and aziridine analogs.
S R
OH
13 (R = H) 14 (R = Me) 15 (R = Ac) 16 (R = SO2NMe2)
11 (R = H) 12 (R = Me)
Figure 4
OH
N
O
N+ O-
OH O O
OH
O
19
Thiazole ring SAR.
However, the N-methyl, N-acyl, N-sulfonylureido derivatives 1416 demonstrated comparable in vitro potency relative to parent macrolides (EC0.01 ¼ 1.02.6 mM) [39]. More importantly, 14 was found to be six times more potent than 2 in inhibiting the proliferation of HCT-116 cells (IC50 ¼ 0.13 nM). Structural permutations of the C15-thiazolylethenyl side chain of the epothilone backbone have been extensively investigated [26–34]. Numerous analogs were found to be active in vitro; however, only a few examples provided significant potential for further development. Because epothilone F (17) was initially isolated in relatively low abundance from fermentation, GBF and BMS pursued alternative routes such as semisynthesis [40,41] to secure sufficient amounts of material for in-depth preclinical testing (Figure 5). Epothilone F was found to be equipotent to 2 in the tubulin polymerization (EC0.01 ¼ 1.8 mM) and HCT-116 cytotoxicity (IC50B0.28 nM) assays [20]. The corresponding 21-amino analog 18 (BMS-310705) was approximately three- to fourfold less potent than 2 in the primary in vitro biochemical and cellular assays. However, the rate of metabolism of 18 in mouse S9 liver fraction was significantly (W15-fold) improved relative to
Discovery of Ixabepilone (IXEMPRATM)
309
epothilone B [20]. Based on its robust preclinical pharmacology and pharmacokinetic profile, the second-generation epothilone 18 was advanced into phase I trials [42]. The interesting N-oxide derivative 19, isolated as an intermediate in the semisynthesis of 17 and 18, also performed well in the tubulin polymerization assay (EC0.01 ¼ 3.90 mM) and inhibited the proliferation of colon (HCT-116), lung (A549), and cervical (KB-3.1) carcinoma cell lines with IC50s of 12 nM [20,40]. However, the compound was found to be considerably less potent against cell lines that express the multi-drug resistant (MDR) phenotype, such as the HCT116/VM46 variant [20]. Structural modifications at the C1C3 positions of the polypropionate-derived portion of the macrocycle have been relatively scarce with few analogs demonstrating significant in vitro activity. The transenoates 20 and 21 (Figure 6) were prepared through semisynthetic routes involving chemoselective dehydration of the 3-hydroxyl groups of epothilones A and B, respectively [43]. While 21 was equipotent to epothilone B in the tubulin polymerization assay, the enoate was approximately 10-fold less potent than the natural product in cell culture [20]. Efforts to address the metabolic stability of the lactone moiety remained a high priority within the research group at BMS. Although the total synthesis efforts to prepare the lactam analogs of the epothilones were met with challenges, a straightforward semisynthetic approach to directly convert the lactone oxygen to nitrogen was realized (Figure 7) [35]. Since the macrolactone moiety of the epothilones is allylic, it was postulated that it may be susceptible to a palladium-catalyzed ring opening to form a p-allylpalladium complex, which could then be trapped by a nitrogen nucleophile. Indeed, unprotected epothilones A and B undergo Pd(0)-catalyzed azidation in the presence of sodium
O
R
S OH
N O O 20 (R = H) 21 (R = Me)
Figure 6 Enoate analogs of 1 and 2.
O
310
Robert M. Borzilleri and Gregory D. Vite
O
R
O
S OH
N
Pd(PPh3)4, NaN3 O OH
HO2C N3
THF-H2O, 45°C 65−70% O
OH
N
O
OH
1 (R = H) 2 (R = Me)
O
O
22 (R = H) 23 (R = Me)
"one-pot" 20−25%
PMe3, THF-H2O 53−89% R
O
R
S
S OH
N HN O
OH
O
DPPA, DMF 4 °C, 24 h or
N
EDCI, HOBt MeCN-DMF 40−65%
26 (R = H) 27 (R = Me)
Figure 7
R
S
OH HO2C NH2 OH
O
24 (R = H) 25 (R = Me)
Semisynthesis of esterase-resistant lactam analogs.
azide to produce 22 and 23, respectively, as single diastereomers (Figure 7) [35]. These intermediates were formed with complete retention of configuration, presumably through stereospecific anti-attack of the palladium and subsequent regioselective reaction of azide from the opposite face of the p-allylpalladium intermediate. Chemoselective reduction of the azide derivatives with trimethylphosphine, followed by cyclization of the corresponding amino acid intermediates 24 and 25, gave the desired macrolactam analogs 26 and 27 (BMS-247550, ixabepilone). The entire three-step sequence can be telescoped into a remarkably efficient and practical ‘‘one pot’’ process that can be carried out over a 24-h period without isolation of intermediates or use of protecting groups. Epothilone F-lactam was synthesized in similar fashion, whereas the macrolactam analogs of epothilones C and D were obtained from 3 and 4, respectively, using the aforementioned stereoselective metal-mediated deoxygenation chemistry [35]. In general, the tubulin-polymerizing and cytotoxic potencies of the lactam analogs were reduced compared to their natural epothilone counterparts. One important exception to this trend, however, was 27. Although the lactam analog of epothilone B (EC0.01 ¼ 3.8 mM) is approximately 1.5-fold to 3-fold less potent than epothilones A and B at inducing tubulin polymerization, the semisynthetic analog is comparable to paclitaxel in both in vitro assays (HCT-116, IC50 ¼ 3.4 nM).
Discovery of Ixabepilone (IXEMPRATM)
311
As anticipated, the metabolic stability of 27 in mouse S9 liver fraction was found to be superior to that of the other natural epothilone analogs with a rate of hydrolysis 100-fold lower than that of epothilone B [20]. Furthermore, 27 demonstrated dramatically lower plasma protein binding in mice (79%) and only slightly higher MDR susceptibility relative to the natural epothilones [20]. Since several of these in vitro parameters serve as predictors of in vivo efficacy in mice, 27 was positioned as the lead development candidate.
4. PRECLINICAL PHARMACOLOGY 4.1 Cytotoxicity In preliminary experiments, 27 was found to possess broad spectrum cytotoxic activity (IC50s ¼ 1.435 nM) against a diverse panel of 21 tumor cell lines characterized as either paclitaxel-sensitive (e.g., ovarian: A2780/TAX; breast: MCF-7; prostate: LNCaP, PC3; colon: HCT-116, LS174T; lung: A549, LX-1) or paclitaxel-resistant (A2780/TAX-R, HCT116/VM46) [44]. Additional profiling across panels of specific tumor cell lines revealed that 23 human lung carcinoma cell lines were highly sensitive to 27 (IC50 ¼ 2.319.2 nM) [45]. Only 4 of 35 human breast and 2 of 20 human colon cell lines were found to be significantly resistant to this agent (IC50s W 100 nM). Collectively, the cytotoxicity data suggested that 27 addressed the known mechanisms associated with resistance to taxanes, that is, MDR-mediated efflux due to P-gp overexpression (HCT-116/VM46), expression of specific b-tubulin mutations (A2780/ TAX-R), and overexpression of the bIII-tubulin isoform (LX-1) [46]. Based on IC90 values from clonogenic cell survival (colony formation) assays, 27 was significantly more effective at killing HCT-116/VM46 and A2780/TAX-R cells relative to paclitaxel following a 16-h drug exposure [44,45]. Mechanistically, lactam 27 was as effective as epothilone B in its ability to arrest proliferating HCT-116 human colon carcinoma cells during the mitotic phase of the cell cycle (G2-M transition) as measured by flow cytometry [44]. Moreover, the concentration of 27 needed to arrest cells in mitosis corresponded to the concentration required to kill cells over the same treatment duration. Subsequent studies with the HCT-116 line revealed 27 affects multiple apoptotic pathways. The compound induced apoptosis of this BAX-positive cell line in a p53-dependent manner through upregulation of the pro-apoptotic protein PUMA [47], although a transcriptionindependent pathway may also be operative in the BAX activation response to 27.
312
Robert M. Borzilleri and Gregory D. Vite
4.2 In vivo efficacy Consistent with its in vitro cytotoxicity profile, 27 demonstrated robust in vivo efficacy in a wide array (W 90%) of the paclitaxel-sensitive and paclitaxel-resistant human tumor xenograft models (Figure 8) [44–46,48,49]. These models were derived from subcutaneous implants of tumor fragments in athymic mice. Antitumor activity was determined at the MTD or the dose level immediately below where excessive toxicity (W one death) was observed. Responses of Z1 tumor log cell kill (LCK), defined as the tumor growth delay in days (TC) divided by the time for a log increase in tumor volume (calculated as 3.32 times the tumor volume doubling time (TVDT)) were considered active. In general, compound 27 was administered iv as a solution in ethanol/water or Cremophort/ethanol/water to nude mice bearing staged tumors at its optimal dose of 616 mg/kg on an intermittent q4d 3 schedule. For example, significant antitumor effects (W four LCK) were observed with 27 in paclitaxel-sensitive tumor models such as HCT-116 and A2780. Similar to paclitaxel at its optimal dose and schedule, this agent was found to be curative (no detectable disease for W 10 times the TVDT) in W50% of the animals bearing HCT-116 tumors [44]. More importantly, 27 demonstrated robust efficacy against several paclitaxel-resistant tumor models. For instance, the MDR variant of HCT-116 (HCT-116/VM46) was equally sensitive to 27 (LCK ¼ 2.4) as compared to the parent tumor line. In addition, 27 demonstrated activity against the P-gp expressing Pat-7 xenografts. This model was established directly from an ovarian cancer patient who was treated with multiple chemotherapeutic agents, including Taxols, but ultimately, this patient’s cancer became resistant. >4 3.5 3 2.5 2 1.5
Active
1 0.5
BT 47 KP 4 L M M -4 M CF7 CF D /A 7 A M -M DR D B A- -2 M 31 B4 Pa 35 t Pa -14 t-2 A5 1 C 49 al u L2 -6 98 7 LX Pa -1 t-2 Pa 4 tPa 25 tPa 26 A2 A t-2 78 27 7 0/ 80 TA s X C -R D M 22 W 8 38 PA 7 35 Pa 4 Pa t-7 tPa 18 CW t-2 2 R M LuC -22 D A- ap PC 35 a2 PCb 3 G H H E C T- CT O 11 -1 6/ 16 VM 4 H 6 N T2 C 9 I-H 69 N 8 A4 7 31
0
Breast
Lung
Pancreatic
Ovarian
Prostate
Colon
SCLC Gastric Squamous
Figure 8 Antitumor efficacy of 27 in several human tumor xenograft models.
Discovery of Ixabepilone (IXEMPRATM)
313
Since 27 demonstrated good oral bioavailability in the presence of a phosphate buffer (pH 8.0), the compound was evaluated in Pat-7 and HCT-116 xenograft models, comparing oral (po) and iv dosing regimens [44]. In these tumor models, equivalent antitumor efficacy (Z2.4 LCK) was obtained following oral administration (6080 mg/kg) of the compound in the phosphate buffer on an every other day (five times) schedule or through the 10 mg/kg iv dose (q4d 3). In fact, 27 cured 7 of 8 mice bearing the HCT-116 tumors when administered orally at 90 mg/kg on the q2d 5 schedule. To better understand the ability of 27 to overcome paclitaxel resistance, additional tumor models were evaluated. For instance, 27 was equally effective against A2780/TAX-R, a non-MDR-related tumor model with acquired paclitaxel resistance attributed to b-tubulin mutations [44]. However, it has been suggested that specific tubulin point mutations may not be relevant to clinical resistance. Therefore, additional tumor models were developed from direct implantation of patient biopsies into immunocompromised mice. Gratifyingly, 27 elicited superior responses in the Pat-21 (2.3 LCK) and Pat-26 (1.2 LCK) human breast and pancreatic carcinoma xenograft models relative to optimally dosed paclitaxel (r0.4 LCK) [45]. Pat-21 tumor cells do not overexpress the efflux transporters (P-gp or MDR-1) or carry tubulin mutations but rather have elevated levels of bIII-tubulin [46]. Selective overexpression of the bIII-tubulin isotype has been reported in various advanced tumors (breast, lung, ovarian) of patients treated with taxanes, and in some cases, this mechanism for resistance has been associated with aggressive disease and lower probability of a response to chemotherapy [46]. The mechanism of resistance for Pat-26 xenograft model, which was derived from a metastatic pancreatic cancer patient innately resistant to paclitaxel, is still not well understood. Childhood malignancies, particularly advanced metastatic disease, represent another area of high unmet medical need and potential opportunities for new chemotherapeutic agents. The tubulin depolymerization agents vincristine and vinblastine (Vinca alkaloids) represent the mainstay of multimodality therapy for many pediatric cancers, whereas the taxanes are considerably less effective. Interestingly, 27 induced objective responses (Z50% volume regression) in several pediatric solid tumor models, including human rhabdomyosarcomas (3/3), neuroblastomas (3/5), osteosarcomas (2/6), and Wilms’ tumor models (6/7) when administered iv at 6.6 and 10 mg/kg on an every 4 day 3 schedule in mice [48]. Attempts to develop tumor models resistant to 27 have been challenging. In fact, resistance to 27 has not been observed following passage of the A2780 xenografts for more than 3 years in the presence of
314
Robert M. Borzilleri and Gregory D. Vite
the drug, whereas resistance to paclitaxel in this ovarian tumor model emerged in o6 months following continuous exposure to the taxane [20]. Therapeutic concentrations of drug are likely maintained in the tumor cells, since 27 exhibits reduced susceptibility to MDR-related efflux and the compound does not readily induce tumor cells to overexpress drug efflux pumps (e.g., ATP-binding cassette transporters P-gp or MRP-1). In addition to robust in vivo efficacy as a single agent, 27 demonstrated synergistic antitumor effects when combined with the targeted antiangiogenic agent bevacizumab in models derived from breast (Pat21, KPL4), colon (HCT-116/VM46, WiDr, GEO), lung (L2987), and kidney (151 b) cancers [49]. Statistically superior efficacy (synergy) as measured by growth delay (LCK) and tumor volume reduction was observed with the 27-bevacizumab combination regardless of the tumor model sensitivity (or resistance) to either of the single agents alone at their optimal doses and schedules. Even in the case of the 151b renal model, which was resistant to both single agents (r0.6 LCK), the combination resulted in significant tumor growth delay (1.5 LCK). Similar results were obtained with another antiangiogenic agent, sunitinib in the 151b model. Importantly, the 27-bevacizumab combination was statistically more effective at inhibiting the growth of the GEO and the MDRexpressing HCT-116/VM46 tumor models compared to the combination of paclitaxel and bevacizumab. It has been postulated that the enhanced in vivo efficacy of 27 relative to paclitaxel in combination of bevacizumab may be due to higher antiangiogenic activity (killing of tumor-associated endothelial cells) and/or reduced susceptibility to drug efflux proteins [49].
4.3 Profiling Pharmacokinetic analysis of 27 in female nude mice revealed rapid clearance (4.35.1 L/h/kg), extensive tissue distribution (Vss ¼ 2137 L/ kg), and a mean half-life of 1316 h following a single iv dose of either 10, 6, or 4 mg/kg [45]. Additional in vitro studies indicated that 27 is primarily metabolized by CYP3A4 to many inactive metabolites [20]. In human liver microsomes, 27 did not inhibit a representative panel of CYP450 isozymes (1A2, 3A4, 2B6, 2C8, 2C9, 2C19, 2D6) or induce the activity of CYP3A4, CYP1A2, CYP2B6, or CYP2C9 in cultured human hepatocytes at clinically relevant concentrations [20,50]. Due to its impressive in vivo efficacy profile across a range of taxane-resistant xenograft models and favorable ADME properties, 27 (ixabepilone) was selected for clinical development.
Discovery of Ixabepilone (IXEMPRATM)
315
5. CLINICAL RESULTS 5.1 Phase I/II highlights Based on the robust preclinical activity of ixabepilone, multiple clinical trials across a broad range of tumor types were initiated, including some in collaboration with the National Cancer Institute (NCI). Early phase I studies explored dose, schedule, method of administration, and tolerability [51,52]. Weekly iv infusions (25 mg/m2) did not appear to offer significant advantages over a standard 3-week dosing schedule. Dose escalation for the latter schedule reached 50 mg/m2, but the recommended phase II dose was set at 40 mg/m2 (3 h iv infusion) due to high incidence of peripheral neuropathy at the higher dose. Daily dosing (6 mg/m2, QD 5, every 3 weeks) demonstrated dose-limiting neutropenia, but neuropathies were reduced with this schedule [53]. This daily dosing regimen provided a promising 57% response rate (5.5 months time-to-progression) in a phase II breast cancer trial where patients had not received prior taxane therapy (i.e., taxane naı¨ve) (Table 1) [54]. Another chemotherapy-naı¨ve phase II trial, in hormone refractory prostate cancer, showed an ixabepilone response rate of 32%, which increased to 48% when ixabepilone was combined with estramustine (EMP) [55]. Prostate-specific antigen (PSA) declines of W50% were observed for 48% of patients in the single-agent arm, while 69% of patients had W50% PSA decline in the drug combination arm of the trial. Efficacy was also observed in patients with chemoresistant renal carcinomas, lymphomas, and drug-resistant lung cancers, whereas
Table 1 Select clinical data in taxane-naı¨ve patients and clinical data in heavily pre-treated patients that supported NDA filing N
Dose
Schedule
Response rate (%)
6 mg/m2 35 mg/m2 35 mg/m2 + 280 mg EMP
Daily 5, Q 3weeks 57 Q 3weeks 32 Q 3weeks 48 TID 5, po
Phase II Breast [54] Prostate [55]
23 45 47
Phase II/III Registrational Breast [50,57] 126 Breast [58] 752
Q 3weeks 40 mg/m2 40 mg/m2 Q 3weeks + 1000 mg/m2 CAPE BID 14, po
12 35
316
Robert M. Borzilleri and Gregory D. Vite
results obtained with other tumor types, such as colorectal cancer, were less impressive [51]. The pharmacokinetics in humans at the recommended phase II dose of 40 mg/m2 iv can be characterized as rapid and extensive tissue distribution (large Vdss) and a favorable half-life of 35 h [45,56]. Appreciable oral bioavailability (Fpo ¼ 54%) was achieved with ixabepilone at a 25 mg/m2 dose in a phase I dose finding study [30].
5.2 Phase II/III registrational trials Following on the promising phase I/II studies in breast cancer, ixabepilone was studied in a phase II trial with patients referred to as ‘‘triple-refractory’’ [57]. These patients received prior treatment with an anthracycline, a taxane, and capecitabine (CAPE), and all experienced disease progression before entering the ixabepilone trial. Patients were dosed every 3 weeks with 40 mg/m2 of ixabepilone (3-h iv infusion). In this highly drug-resistant population, the objective response rate as determined by independent radiological review was 12%, with an additional 50% of patients experiencing stable disease. Investigator-determined response rate was 18% with 44% of patients characterized as having stable disease (Table 1). A phase III trial enrolling W750 metastatic breast cancer patients was conducted to compare the combination of ixabepilone and CAPE versus CAPE alone in a setting where patients had progressive disease following treatment with an anthracycline or a taxane [58]. The dose of ixabepilone and schedule of administration were the same as noted above, along with orally dosed CAPE (1,000 mg/m2, bid 14) in the drug combination arm. An approximate twofold improvement in objective response rate and a progression-free survival benefit of 5.8 months (vs. 4.2 months for CAPE) was observed for the drug combination. Detailed retroanalysis suggested that certain subgroups showed preferential benefit, including those patients referred to as triple negative (i.e., no or low expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER), and progesterone receptor (PR)). Grade 3/4 peripheral neuropathy (23%) and neutropenia (68%) were notably higher in the combination arm of the trial. Taken together, these two studies demonstrated the effectiveness of ixabepilone against breast cancer and served as the basis for submitting a New Drug Application to the FDA.
5.3 Pharmacogenomics In the post-genomic era, identifying subsets of patients that are likely to be responders to chemotherapy as well as targeted treatments is a high priority. Accordingly, retrospective analysis of clinical data from ixabepilone trials has been used in attempt to identify genomic
Discovery of Ixabepilone (IXEMPRATM)
317
signatures that predict positive outcomes [59]. Candidate genes include those encoding microtubule-associated proteins (MAPs), HER2, and ER. Low expression of ER correlated with cytotoxicity of ixabepilone in vitro and with tumor response in patients, whereas expression level of tau protein (a MAP) was less predictive [60]. The relevance of tubulin isotype expression as an indicator of drug sensitivity is emerging. For instance, overexpression of the bIII-tubulin isotype is associated with resistance to taxanes. As noted above, preclinical studies in cancer cells and taxaneresistant xenograft models that overexpress bIII-tubulin suggest that ixabepilone’s clinical activity in a refractory setting may be related to the drug’s indifference to overexpression of this isoform [46]. Clearly, further studies and analyses are required to develop a better understanding of predictive biomarkers for ixabepilone.
6. FUTURE DIRECTIONS In addition to ongoing clinical trials across a wide range of malignancies (e.g., prostate, NSCLC, endometrial), ixabepilone is being investigated in combination with targeted agents such as bevacizumab and trastuzumab [61]. These pivotal phase II trials are supported by the impressive synergistic activity observed with the drug combinations in preclinical models and the manageable safety/tolerability profile of ixabepilone observed in patients (vide supra). There are several other epothilone analogs being evaluated in the clinic (Figure 9). Novartis continues to develop one of the initially isolated natural products, epothilone B (2, patupilone, EPO-906) [62]. Clinical activity has been observed with 2 in the drug-resistant disease setting, including patients with renal cell carcinoma (RCC) and relapsed/ refractory ovarian cancer. In contrast to ixabepilone, diarrhea is the doselimiting toxicity (DLT), which requires careful monitoring and management. Epothilone B is currently being investigated in phase III trials involving patients with platinum-resistant ovarian cancer [61,62]. Kosan, in collaboration with Roche, initiated clinical studies on epothilone D (4) but later discontinued development of the drug to pursue a secondgeneration epothilone D analog, KOS-1584 (28, dehydelone) [63]. Based on disease stabilization observed in various advanced malignancies and a confirmed partial response in NSCLC, KOS-1584 was advanced into phase II trials (NSCLC). BMS is conducting preclinical studies on thirdgeneration analogs, such as KOS-1803 (29), for oncology. Other groups are evaluating brain penetrant analogs for potential neurodegenerative disease indications (e.g., Alzheimer’s) [64]. The completely synthetic epothilone analog 30 (sagopilone, ZK-EPO) is being developed by Bayer Schering Pharma AG [65,66]. Confirmed partial responses have been
318
Robert M. Borzilleri and Gregory D. Vite
R1
R2
O S O
O
OH OH
N O OH
O O
28 (R1 = CH3, R2 = 2-methylthiazole) 29 (R1 = CF3, R2 = 5-methylisoxazole)
OH
O
30
HN
NH2 NH
CO2H
O N H
O N
HN H2N
N
N H
H N O
O N H O CO2H
H N
O
CO2H
N H CO2H
S
O
O
S O
N
N S
OH
N
31
O O
Figure 9
OH
O
Additional epothilone analogs of interest.
observed in taxane-pretreated or resistant breast cancer, uterine cancer, platinum-resistant ovarian cancer, NSCLC, and melanoma [66]. Similar to ixabepilone, peripheral neuropathy has been defined as the most common drug-related adverse event (principal DLT). In an alternative approach to selectively target tumors that overexpress the membrane-bound folate receptor alpha (FRa) (e.g., ovarian, endometrial, breast, renal, and lung cancers), BMS and collaborators at Endocyte Corporation designed the aziridine conjugate 31 (BMS-753493) [67–69]. The exquisitely potent hydroxyethyl-substituted aziridine (HCT-116, IC50 ¼ 0.34 nM) was appended to vitamin folic acid through a cleavable disulfide linker followed by a short ionizable peptide sequence (Figure 9) [68,69]. After delivery and subsequent internalization of the folate-drug conjugate into tumor cells through FR-mediated endocytosis, the cytotoxic aziridine can be released by reduction of the disulfide bond. As predicted, 31 demonstrated in vivo efficacy against the FR-positive human KB nasopharyngeal, IGROV ovarian, HeLa cervical, and murine 98M109 lung models but was found to be less active against FR-deficient tumors [70]. Moreover, the epothilone-folate conjugate demonstrated synergistic antitumor activity against FR-positive human tumor xenografts when combined with ixabepilone, bevacizumab, and cisplatin. Phase I studies are underway to assess the safety and tolerability of 31 in cancer patients [61].
Discovery of Ixabepilone (IXEMPRATM)
319
7. CONCLUSIONS The discovery of the epothilones by Ho¨fle and Reichenbach galvanized global research and development efforts to identify drug candidates from this class with potential to provide clinical benefit when patients fail to respond to existing chemotherapies. While these polyketide-derived macrolides provided potent antineoplastic activity in vitro, these effects were not observed in murine tumor xenograft models in our laboratories, presumably due to esterase-mediated inactivation of the compound in vivo. Consequently, a collaboration between BMS and GBF was established to identify metabolically stable epothilone derivatives. A highly efficient semisynthetic sequence was developed to access the macrolactam analogs of the natural macrolides. Ixabepilone, the lactam analog of epothilone B, demonstrated potent tubulin polymerization activity and robust cytotoxicity versus several paclitaxel-resistant cell lines. Moreover, improved metabolic stability and pharmacokinetic properties were achieved with this analog relative to the natural macrolides. In vivo, the novel microtubule-stabilizing agent demonstrated broad spectrum antitumor activity against various human tumor xenograft models, including those resistant to paclitaxel. The robust preclinical efficacy was recapitulated in early human clinical trials for several cancers, including metastatic breast and prostate cancer patients with multidrug-resistant disease. Ixabepilone (IXEMPRAt) was approved by the FDA in October 2007 as a first-in-class agent for treatment of drug-resistant/refractory metastatic or locally advanced breast cancers in combination with CAPE or as monotherapy following failure of an anthracycline, a taxane, and CAPE.
REFERENCES [1] M. C. Wani, H. L. Taylor, M. E. Wall, P. Coggon and A. T. McPhail, J. Am. Chem. Soc., 1971, 93, 2325. [2] P. B. Schiff, J. Fant and S. B. Horwitz, Nature, 1979, 277, 665. [3] C. Ferlini, D. Gallo and G. Scambia, Expert Opin. Investig. Drugs, 2008, 17, 335. [4] L. He, G. A. Orr and S. B. Horwitz, Drug Discov. Today, 2001, 6, 1153. [5] D. C. Myles, Annu. Rep. Med. Chem., 2002, 37, 125. [6] J. T. Hunt, Mol. Cancer Ther., 2009, 8, 275. [7] K. Gerth, N. Bedorf, G. Ho¨fle, H. Irschik and H. Reichenbach, J. Antibiot., 1996, 49, 560. [8] G. Hoefle, N. Bedorf, H. Steinmetz, D. Schomburg, K. Gerth and H. Reichenbach, Angew. Chem., Int. Ed. Engl., 1996, 35, 1567. [9] I. Hardt, H. Steinmetz, K. Gerth, F. Sasse, H. Reichenbach and G. Hofle, J. Nat. Prod., 2001, 64, 847. [10] K. Gerth, H. Steinmetz, G. Ho¨fle and H. Reichenbach, J. Antibiot., 2000, 53, 1373. [11] K. Gerth, H. Steinmetz, G. Ho¨fle and H. Reichenbach, J. Antibiot., 2001, 54, 144.
320
Robert M. Borzilleri and Gregory D. Vite
[12] I. Molna´r, T. Schupp, M. Ono, R. E. Zirkle, M. Milnamow, B. Nowak-Thompson, N. Engel, C. Toupet, A. Stratmann, D. D. Cyr, J. Gorlach, J. M. Mayo, A. Hu, S. Goff, J. Schmid and J. M. Ligon, Chem. Biol., 2000, 7, 97. [13] L. Tang, S. Shah, L. Chung, J. Carney, L. Katz and C. Khosla, Science, 2000, 287, 640. [14] B. Julien, S. Shah, R. Ziermann, R. Goldman, L. Katz and C. Khosla, Gene, 2000, 249, 153. [15] B. Julien and S. Shah, Antimicrob. Agents Chemother., 2002, 46, 2772. [16] S. Schneiker, O. Perlova, O. Kaiser, K. Gerth, A. Alici, M. O. Altmeyer, D. Bartels, T. Bekel, S. Beyer, E. Bode, H. B. Bode, C. J. Bolten, J. V. Choudhuri, S. Doss, Y. A. Elnakady, B. Frank, L. Gaigalat, A. Goesmann, C. Groeger, F. Gross, L. Jelsbak, J. Kalinowski, C. Kegler, T. Knauber, S. Konietzny, M. Kopp, L. Krause, D. Krug, B. Linke, T. Mahmud, R. Martinez-Arias, A. C. McHardy, M. Merai, F. Meyer, S. Mormann, J. Munoz-Dorado, J. Perez, S. Pradella, S. Rachid, G. Raddatz, F. Rosenau, C. Rueckert, F. Sasse, M. Scharfe, S. C. Schuster, G. Suen, A. Treuner-Lange, G. J. Velicer, F.-J. Vorhoelter, K. J. Weissman, R. D. Welch, S. C. Wenzel, D. E. Whitworth, S. Wilhelm, C. Wittmann, H. Bloecker, A. Puehler and R. Mueller, Nat. Biotechnol., 2007, 25, 1281. [17] D. M. Bollag, P. A. McQueney, J. Zhu, O. Hensens, L. Koupal, J. Liesch, M. Goetz, E. Lazarides and C. M. Woods, Cancer Res., 1995, 55, 2325. [18] J. H. Nettles, H. Li, B. Cornett, J. M. Krahn, J. P. Snyder and K. H. Downing, Science, 2004, 305, 866. [19] D. W. Heinz, W.-D. Schubert and G. Hoefle, Angew. Chem. Int. Ed. Engl., 2005, 44, 1298. [20] F. Y. F. Lee, R. Borzilleri, C. R. Fairchild, A. Kamath, R. Smykla, R. Kramer and G. Vite, Cancer Chemother. Pharmacol., 2008, 63, 157. [21] A. Balog, D. Meng, T. Kamenecka, P. Bertinato, D.-S. Su, E. J. Sorensen and S. J. Danishefsky, Angew. Chem. Int. Ed. Engl., 1996, 35, 2801. [22] D.-S. Su, D. Meng, P. Bertinato, A. Balog, E. J. Sorensen, S. J. Danishefsky, Y.-H. Zheng, T.-C. Chou, L. He and S. B. Horwitz, Angew. Chem. Int. Ed. Engl., 1997, 36, 757. [23] Z. Yang, Y. He, D. Vourloumis, H. Vallberg and K. C. Nicolaou, Angew. Chem. Int. Ed. Engl., 1997, 36, 166. [24] K. C. Nicolaou, F. Sarabia, S. Ninkovic and Z. Yang, Angew. Chem. Int. Ed. Engl., 1997, 36, 525. [25] D. Schinzer, A. Limberg, A. Bauer, O. M. Bohm and M. Cordes, Angew. Chem. Int. Ed. Engl., 1997, 36, 523. [26] K.-H. Altmann, B. Pfeiffer, S. Arseniyadis, B. A. Pratt and K. C. Nicolaou, Chem. Med. Chem., 2007, 2, 396. [27] K. C. Nicolaou, F. Roschangar and D. Vourloumis, Angew. Chem. Int. Ed. Engl., 1998, 37, 2014. [28] K. C. Nicolaou, A. Ritze´n and K. Namoto, Chem. Commun., 2001, 17, 1523. [29] C. R. Harris and S. J. Danishefsky, J. Org. Chem., 1999, 64, 8434. [30] R. M. Borzilleri and G. D. Vite, Drugs Fut., 2002, 27, 1149. [31] G. D. Vite, R. M. Borzilleri, S.-H. Kim, A. Regueiro-Ren, W. G. Humphreys and F. Y. F. Lee, in Anticancer Agents: Frontiers in Cancer Chemotherapy, ACS Symposium Series 796 (eds I. Ojima, G. D. Vite, and K.-H. Altmann), American Chemical Society, Washington, D.C., 2001, p. 97. [32] K.-H. Altmann, M. J. J. Blommers, G. Caravatti, A. Florsheimer, K. C. Nicolaou, T. O’Reilly, A. Schmidt, D. Schinzer and M. Wartmann, in Anticancer Agents: Frontiers in Cancer Chemotherapy, ACS Symposium Series 796 (eds I. Ojima, G. D. Vite, and K.-H. Altmann), American Chemical Society, Washington, D.C., 2001, p. 112. [33] U. Klar, W. Skuballa, B. Buchmann, W. Schwede, T. Bunte, J. Hoffmann and R. B. Lichtner, in Anticancer Agents: Frontiers in Cancer Chemotherapy, ACS Symposium Series 796 (eds I. Ojima, G. D. Vite, and K.-H. Altmann), American Chemical Society, Washington, D.C., 2001, p. 131.
Discovery of Ixabepilone (IXEMPRATM)
321
[34] K. C. Nicolaou, N. Winssinger, J. Pastor, S. Ninkovic, F. Sarabia, Y. He, D. Vourloumis, Z. Yang, T. Li, P. Giannakakou and E. Hamel, Nature, 1997, 387, 268. [35] R. M. Borzilleri, X. Zheng, R. J. Schmidt, J. A. Johnson, S.-H. Kim, J. D. DiMarco, C. R. Fairchild, J. Z. Gougoutas, F. Y. F. Lee, B. H. Long and G. D. Vite, J. Am. Chem. Soc., 2000, 122, 8890. [36] D. Schinzer, K.-H. Altmann, F. Stuhlmann, A. Bauer and M. Wartmann, Chem. Bio. Chem., 2000, 1, 67. [37] S. J. Stachel, M. D. Chappell, C. B. Lee, S. J. Danishefsky, T.-C. Chou and S. B. Horwitz, Org. Lett., 2000, 2, 1637. [38] J. A. Johnson, S.-H. Kim, M. Bifano, J. DiMarco, C. Fairchild, J. Gougoutas, F. Lee, B. Long, J. Tokarski and G. Vite, Org. Lett., 2000, 2, 1537. [39] A. Regueiro-Ren, R. M. Borzilleri, X. Zheng, S.-H. Kim, J. A. Johnson, C. R. Fairchild, F. Y. F. Lee, B. H. Long and G. D. Vite, Org. Lett., 2001, 3, 2693. [40] G. Ho¨fle, N. Glaser, M. Kiffe, H.-J. Hecht, F. Sasse and H. Reichenbach, Angew. Chem. Int. Ed. Engl., 1999, 38, 1971. [41] G. Ho¨fle, N. Glaser, T. Leibold and M. Sefkow, Pure Appl. Chem., 1999, 71, 2019. [42] A. V. Kamath, M. Chang, F. Y. Lee, Y. Zhang and P. H. Marathe, Cancer Chemother. Pharmacol., 2005, 56, 145. [43] A. Regueiro-Ren, K. Leavitt, S.-H. Kim, G. Ho¨fle, M. Kiffe, J. Z. Gougoutas, J. D. DiMarco, F. Y. F. Lee, C. R. Fairchild, B. H. Long and G. D. Vite, Org. Lett., 2002, 4, 3815. [44] F. Y. F. Lee, R. Borzilleri, C. R. Fairchild, S.-H. Kim, B. H. Long, C. Reventos-Suarez, G. D. Vite, W. C. Rose and R. A. Kramer, Clin. Cancer Res., 2001, 7, 1429. [45] F. Y. F. Lee, R. Smykla, K. Johnston, K. Menard, K. McGlinchey, R. W. Peterson, A. Wiebesiek, G. Vite, C. R. Fairchild and R. Kramer, Cancer Chemother. Pharmacol., 2009, 63, 201. [46] C. Dumontet, M. A. Jordan and F. F. Y. Lee, Mol. Cancer Ther., 2009, 8, 17. [47] H. Yamaguchi, J. Chen, K. Bhalla and H.-G. Wang, J. Biol. Chem., 2004, 279, 39431. [48] J. K. Peterson, C. Tucker, E. Favours, P. J. Cheshire, J. Creech, C. A. Billups, R. Smykla, F. Y. F. Lee and P. J. Houghton, Clin. Cancer Res., 2005, 11, 6950. [49] F. Y. F. Lee, K. L. Covello, S. Castaneda, D. R. Hawken, D. Kan, A. Lewin, M.-L. Wen, R.-P. Ryseck, C. R. Fairchild, J. Fargnoli and R. Kramer, Clin. Cancer Res., 2008, 14, 8123. [50] FDA-Approved Patient Label for IXEMPRAt (ixabepilone), Bristol-Myers Squibb Company, Princeton, NJ, October 2007. [51] N. Denduluri and S. M. Swain, Expert Opin. Investig. Drugs, 2008, 17, 423. [52] M. Harrison and C. Swanton, Expert Opin. Investig. Drugs, 2008, 17, 523. [53] J. Abraham, M. Agrawal, S. Bakke, A. Rutt, M. Edgerly, F. M. Balis, B. Widemann, L. Davis, B. Damle, D. Sonnichsen, D. Lebwohl, S. Bates, H. Kotz and T. Fojo, J. Clin. Oncol., 2003, 21, 1866. [54] N. Denduluri, J. A. Low, J. J. Lee, A. W. Berman, J. M. Walshe, U. Vatas, C. K. Chow, S. M. Steinberg, S. X. Yang and S. M. Swain, J. Clin. Oncol., 2007, 25, 3421. [55] J. J. Lee and W. K. Kelly, Nat. Clin. Pract. Oncol., 2009, 6, 85. [56] S. Mani, H. McDaid, A. Hamilton, H. Hochster, M. B. Cohen, D. Khabelle, T. Griffin, D. E. Lebwohl, L. Liebes, F. Muggia and S. B. Horwitz, Clin. Cancer Res., 2004, 10, 1289. [57] E. Thomas, J. Tabernero, M. Fornier, P. Conte, P. Fumoleau, A. Lluch, L. T. Vahdat, C. A. Bunnell, H. A. Burris, P. Viens, J. Baselga, E. Rivera, V. Guarneri, V. Poulart, J. Klimovsky, D. Lebwohl and M. Martin, J. Clin. Oncol., 2007, 25, 3399. [58] E. S. Thomas, H. L. Gomez, R. K. Li, H.-C. Chung, L. E. Fein, V. F. Chan, J. Jassem, X. B. Pivot, J. V. Klimovsky, F. Hurtado de Mendoza, B. Xu, M. Campone, G. L. Lerzo, R. A. Peck, P. Mukhopadhyay, L. T. Vahdat and H. H. Roche´, J. Clin. Oncol., 2007, 25, 5210.
322
Robert M. Borzilleri and Gregory D. Vite
[59] J. J. Lee and S. M. Swain, Clin. Cancer Res., 2008, 14, 1618. [60] H. Lee, L. Xu, S. Wu, B. Paul, J. Baselga, A. Llombart, G. G. Steger, S. Galbraith and E. Clark, Abstract # 3011, 42nd ASCO Annual Meeting, Atlanta, Georgia, June, 2006. [61] U.S. National Institutes of Health, ClinicalTrials.gov. [62] J. M. Larkin, Drugs Fut., 2007, 32, 323. [63] Y. S. Cho, K.-D. Wu, M. A. S. Moore, T.-C. Chou and S. J. Danishefsky, Drugs Fut., 2005, 30, 737. [64] D. Huryn, C. Ballatore, K. Brunden, E. Hyde, R. F. Deiches, V. M. Y. Lee, J. Trojanowski, J. Potuzak and A. B. Smith, MEDI-200, 235th ACS National Meeting, New Orleans, LA, April, 2008. [65] U. Klar, B. Buchmann, W. Schwede, W. Skuballa, J. Hoffmann and R. B. Lichtner, Angew. Chem. Int. Ed. Engl., 2006, 45, 7942. [66] U. Klar, J. Hoffmann and M. Giurescu, Expert Opin. Investig. Drugs, 2008, 17, 1735. [67] C. P. Leamon, Curr. Opin. Invest. Drugs, 2008, 9, 1277. [68] G. D. Vite, F. Y. F. Lee, C. P. Leamon and I. R. Vlahov, US Patent Application 2007/ 275904-A1, 2007. [69] G. D. Vite, ACS Princeton Fall Organic Chemistry Symposium, September 19, 2008. [70] K. Covello, C. Flefleh, K. Menard, A. Wiebesiek, K. McGlinchey, M.-L. Wen, R. Westhouse, J. Reddy, I. Vlahov, J. Hunt, W. Rose, C. Leamon, G. Vite and F. Lee, Abstract # 2326, 99th AACR Annual Meeting, San Diego, CA, April, 2008.
CHAPT ER
16 Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics Stefan Peukert and Karen Miller-Moslin
Contents
1. Introduction 2. Mechanism of Hedgehog Pathway Signaling 3. Inhibitors of the Hedgehog Pathway 3.1 Smoothened inhibitors 3.2 Non-Smoothened inhibitors 4. Hedgehog Inhibitors in Clinical Trials 5. Conclusions References
323 324 325 325 331 332 333 334
1. INTRODUCTION Targeting fundamental molecular signaling pathways that control growth and cell death have been suggested as a promising new paradigm for the discovery of drugs [1]. Of particular interest is the Hedgehog (Hh) pathway, initially discovered in Drosophila by Wieschaus and Nu¨ssleinVollhard [2], which directs the development of multiple tissues during embryonic development and contributes to tissue homeostasis in adults [3–5]. Inactivation of the pathway during embryonic development causes birth defects [6,7], whereas abnormal activation is linked to tumorigenesis in several cancers [3]. Genetic validation of this pathway in human tumors comes from observations that patients with a germline mutation in Patched (Ptch1), a component of the Hh pathway, have activated Hh signaling and develop Gorlin syndrome, also known as nevoid basal cell Novartis Institutes for Biomedical Research Inc., Cambridge, MA, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04416-9
r 2009 Elsevier Inc. All rights reserved.
323
324
Stefan Peukert and Karen Miller-Moslin
carcinoma (BCC) [8]. Gorlin syndrome patients present with skeletal and dental abnormalities due to developmental patterning defects, show high incidence of sporadic BCC, and an elevated incidence of medulloblastoma, ovarian cysts, and ovarian carcinoma. Moreover, somatic mutations in Patched and Smoothened (Smo), another Hh pathway component leading to constitutive pathway activation, have been found in 20% of pediatric medulloblastomas [9,10] and in more than 70% of sporadic BCCs [11,12]. Furthermore, aberrant activation of the Hh pathway without known mutation is implicated in a number of additional tumor types, including small cell lung cancer, gut-related tumors, pancreatic and prostate cancer [3]. Inhibition of the Hh pathway in either tumor cells directly or nonmalignant stromal cells, which, as part of the tumor microenvironment, support tumor growth [13], has emerged as an attractive target in anticancer therapy [14–16]. Recent positive results from the topical and systemic treatment of BCC patients with Hh antagonists provide the first evidence for the therapeutic benefit resulting from inhibition of this signaling pathway [17,18].
2. MECHANISM OF HEDGEHOG PATHWAY SIGNALING Although a detailed description of the components of the Hh pathway and their interactions is described in recent reviews [19,20], some information is necessary to understand the mechanism of action of the Hh inhibitors described herein. The Hh signaling pathway (Figure 1) received its name from a small family of secreted proteins which comprises Sonic hedgehog (Shh), Inhibitors of Hh protein
Smo inhibitors PTCH1 HH
SMO SUFU Gli
Inhibitors of Gli-Transcription
Gli
Target genes
Figure 1 Components of the Hh signaling pathway and molecular sites targeted by Hh pathway inhibitors. Source: From Ref. 16 with permission of the publisher. (See Color Plate 16.1 in Color Plate Section.)
325
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
Indian hedgehog (Ihh), and Desert hedgehog (Dhh) in mammals. The Shh gene was discovered in 1993 and was named after Sonic the Hedgehog, a popular video game hero at this time, since mutations of this gene in fruit flies gave rise to spiky hairs. The other related Hh genes are named after species of living hedgehogs [21]. In the absence of Hh ligands, the 12-pass transmembrane receptor Ptch1 inhibits activity of the downstream receptor Smoothened, which resembles G-protein-coupled receptors (GPCR) in general topology. The interactions of the components of the Hh pathway can occur in the cilium of cells [22]. In the absence of Hh protein, Ptch1 is localized in the primary cilium and seems to exclude ciliary localization of Smo. Binding of Shh to Ptch1 causes Smo, stored in intracellular vesicles, to move to the cilium and activate signal transduction. Active Smo signals through a cytosolic complex of proteins including Suppressor of Fused (SuFu). This leads to activation of the glioma (Gli) family of transcription factors and their translocation to the nucleus, where they trigger the expression of specific genes that promote cell proliferation and differentiation. On the basis of the current understanding of the pathway, several druggable nodes have been identified, and assays have been developed that can detect small molecules able to alter the activity of these targets.
3. INHIBITORS OF THE HEDGEHOG PATHWAY Small-molecule modulators of the Hh signaling pathway have been the subject of recent reviews [15,23–26], and the last few years have brought a tremendous increase in reports of novel inhibitors. Although the majority of such inhibitors target the Smoothened receptor, a few reports of small molecules targeting other members of this pathway have also appeared.
3.1 Smoothened inhibitors The natural product alkaloid cyclopamine (1) was among the first smallmolecule inhibitors of the Hh pathway to be reported in the literature [7]. It was later established that cyclopamine achieves this inhibition through direct binding to the heptahelical bundle of the Smoothened receptor [27]. HN H
H
O
H N
H O
H H
H H
HO
H
H
O
1: cyclopamine
2: IPI-269609/IPI-609
H
326
Stefan Peukert and Karen Miller-Moslin
H
H N
O
H
O
H
H O
O
H
H
H H
S N H
H N
H
H
HN N
H
H
3: IPI-926
4
A recent report described the semi-synthesis of the D-homocyclopamine analog 2 (IPI-269609/IPI-609) from cyclopamine through an oxidation/cyclopropanation/ring expansion sequence [28]. Compound 2 was then further functionalized through acylation, reductive amination, or direct alkylation. Hh pathway inhibition was assessed in murine progenitor C3H10T1/2 cells. When the Hh pathway is active, these cells differentiate into osteoblasts and produce high levels of alkaline phosphatase (AP). A reduction in AP production in the presence of compound is indicative of Hh pathway inhibition. Several of these analogs showed an EC50 o1 mM in this assay. Relative to cyclopamine (EC50 ¼ 0.17 mM), 2 (EC50 ¼ 0.20 mM) showed improved aqueous solubility at pH 7.4 (W20-fold), enhanced stability in simulated gastric fluid, and good oral exposure (80% oral bioavailability in mice). Compound 2 was also shown to block Hh signaling in pancreatic cancer cells in vitro and in vivo and to abolish distant metastases in orthotopic murine xenografts of human pancreatic cancer [29]. Further modifications to the D-homocyclopamine skeleton have been exemplified in recent patent applications [30,31]. For example, methyl sulfonamide 3 (IPI-926) and fused pyrazole 4 were each reported to have EC50 o20 nM in the AP assay. Both compounds were evaluated in in vivo models of pancreatic cancer and medulloblastoma. IPI-926 showed regression of medulloblastoma and no tumor regrowth upon dosing at 40 mg/kg for 50 days [32]. Proline derivatives such as 5 (Cur61414) have been identified as Hh pathway inhibitors that act through binding to Smo [33,34]. Compound 5 was able to suppress proliferation and induce apoptosis in in vitro models of BCC. Phenyl quinazolinone ureas such as 6 are also reported to be potent inhibitors of Hh signaling [35,36]. Compounds were evaluated in a cellbased reporter gene assay (to be abbreviated Gli-Luc RGA), which quantified Hh pathway activity based on downstream activation of GliLuciferase. Some structure-activity relationship (SAR) within this series has been reported. Replacement of the 4-fluorophenyl ring of 6 (IC50 ¼ 70 nM)
327
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
with an isopropyl group led to a significant loss in activity (B6-fold), and replacement of the quinazolinone by a pyrimidinone ring was not tolerated. Replacement of the para-chloro substituent on the aniline ring with a hydrogen led to a decrease in potency (B10-fold). Structurally related quinazolines (7) and pyridopyrimidines (8) have also been reported as potent Hh inhibitors in the patent literature (IC50 of 2.8 and 3.6 nM, respectively, in a Gli-Luc RGA) [37].
F O
O N
N
O
NH F
N
N N
F
H N
HN
F
O
O
O Cl O
5: Cur61414
6
O N H
N N
O
N H
N
7
N H
N N
N H
8
Various Smoothened inhibitors have been reported in the patent literature that contain either an N-(4-chloro-phenyl)-benzamide or an N-(4-chloro-phenyl)-nicotinamide core. This core has been substituted at the 3-position of the aniline with benzimidazoles to provide compounds such as 9 (Hh-Antag691) and 10 [38,39]. Compound 9 is reported to have IC50 o0.001 mM in a Gli-Luc RGA and has been shown to eliminate medulloblastoma in Ptch1+/ p53/ mice [40]. Substitution at the 3-position with quinoxalines provided compounds such as 11 (IC50 o1 mM in Gli-Luc RGA) [41]. Pyrazines (e.g., 12, IC50 ¼ 0.01 mM in an AP assay) [42] and pyridines (e.g., 13, IC50 ¼ 3 nM in a Gli-Luc RGA) [43,44] have also been exemplified at the 3-position. Amide substitution at the
328
Stefan Peukert and Karen Miller-Moslin
3-position has also been reported, affording inhibitors such as 14 [45]. N-phenyl-benzamides that lack the 4-chloro substituent, such as 15 (IC50 o0.003 mM in an AP assay), have also been reported [46]. Cyclization of the amide to afford 1-aminoisoquinolines such as 16 has been described in a recent patent application. Such compounds afforded EC50o 500 nM in a Gli-Luc RGA [47]. Cl O O
N
N H NH O
9: Hh-Antag691
N
Cl
O
Cl O N
O N
N H
N H
N N F
NH
F
10
Cl
Cl
O N
O
N
N H
H2N
Cl
N H
F
N
O
11
F
O
N
S
F F
12
O
13: GDC-0449
Cl O HN
N H
O N
N
N
N
O
N H O
NH
14
15
N
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
329
Cl N H N
N H N
N 16
O
A related series of biphenylcarboxamides, represented by 17, has also appeared in the recent patent literature [48,49]. When dosed orally for 10 days at 50 mg/kg/day, 17 completely blocks the expansion of luciferased lymphoma cells in vivo. Migration of the carboxamide substituent in these compounds to the ortho position is tolerated, as demonstrated by ortho-biphenyl carboxamides such as 18, which have been reported as potent antagonists of Smoothened [50,51]. Compound 18 showed excellent activity in a Gli-Luc RGA (IC50 ¼ 10 nM), as well as direct binding assays using either mouse or human Smo membranes (IC50 ¼ 9 and 7 nM, respectively). SAR showed that a -CH2- linkage between the aminoindane moiety, and the heteroaryl ring was superior to an amide or sulfonamide linkage, and that the (S)-aminoindane enantiomer afforded a 50-fold improvement in binding relative to the (R)-enantiomer. Various aryl and heteroaryl substituents were tolerated on the methylene unit. F O
F F F
F
F
H N
O H N O
N H
N
18
N 17
S
O
Benzamides substituted with a saturated ring also are potent Smo inhibitors, as demonstrated by the appearance of compounds such as 19 in the patent literature [52]. Compound 19 afforded 114.1% inhibition at 2 mM in a Gli-Luc RGA. The cyclohexane core can also be successfully replaced with nitrogen-containing heterocycles, for example, compound 20 (114.0% inhibition at 2 mM) [53].
330
Stefan Peukert and Karen Miller-Moslin F
F F
N
N
N
N H
O
N H
N H
19
O N H
20
N H
O
O
Another distinct class of Smo inhibitors that appeared in the recent patent literature are heteroaryl-piperazines such as 21 [54] and 22 [55,56]. Compound 21 showed dose-related anti-tumor activity and Gli1 inhibition when administered orally in a Ptch+/ p53/ mouse medulloblastoma allograft model. SAR revealed that the phthalazine was superior to several other heterocyclic systems (e.g., isoquinoline, indole) and that substitu;tion at the 5-position of the pyridine ring had a significant impact on binding affinity [57]. Oral dosing of 22 at 10 mg/kg/day for 6 days in a Ptch+/ p53/ mouse medulloblastoma allograft model afforded a W99% reduction in tumor size relative to vehicle control. Triazole derivatives such as 23 have also been reported as Smoothened antagonists [58]. An unspecified compound from this patent application led to tumor shrinkage in a Ptch+/ mouse medulloblastoma allograft model when dosed orally at 80 mg/kg bid. OH
F F
O O
N N
N
N
N
N
N
N
N N
N N N
N F F F
F F
21
22
F 23
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
331
3.2 Non-Smoothened inhibitors 3.2.1 Hedgehog protein The extracellular protein Shh that binds to the transmembrane receptor Ptch, reversing its inhibitory effect on Smo, is the target of the macrocycle robotnikinin (24) [59]. A small-molecule microarraybased screen of a bacterially expressed biologically active Shh N-terminal fragment (ShhN) provided a macrocyclic hit. Optimization of this hit resulted in the identification of robotnikinin. The compound binds to ShhN with a Kd of 3.1 mM and inhibits Hh signaling in a Gli-luciferase reporter gene cell line, in human primary keratinocytes, and in a synthetic model of the human skin in a dose-dependent fashion. The authors suggest a mechanism involving inhibition of the actions of Shh, either directly or indirectly, by interfering with a precursor complex.
3.2.2 Gli-mediated transcription Two low-molecular weight compounds, 25 (GANT58) and 26 (GANT61), were identified, which inhibit Hh signaling downstream of Smo and SuFu at the level of Gli with an IC50 of B5 mM in a Gli-luciferase cellular assay [60]. Mechanistically, both inhibitors act at the nucleus to block Gli function, and one of them, 26, interferes with DNA binding of Gli1. Both compounds display selectivity for the Hh pathway over several unrelated signal transduction pathways such as the TNF/NFkB signaling, glucocorticoid receptor gene transactivation, and the Ras-Raf-MekMapk cascade. Subcutaneous application of 26, dosed at 25 mg/kg/day for 18 days, in a human prostate cancer xenograft mouse model induced growth regression with concurrent strongly reduced mRNA levels of Ptch. Screening of natural products in a cell-based reporter assay of Gli1-mediated transcription provided several hits. The physalins F (27) and B (28) were the most potent inhibitors of Gli1-mediated activity (IC50 values of 0.66 mM and 0.62 mM, respectively) [61]. Additionally, these two compounds also reduced Gli2-mediated transcription in a separate cell assay (IC50 values of 1.5 mM and 1.4 mM, respectively). Compounds 27 and 28 were cytotoxic in a PANC1 human pancreatic cancer cell line (IC50 values of 2.6 and 5.3 mM) and decreased the mRNA expression of Gli1, 2 and Ptch genes. The authors did not identify the molecular targets but point out that physalins are known modulators of the NFkB cascade, and the mechanism of their Gli-mediated transcriptional inhibition might include a path of PKC inhibition.
332
Stefan Peukert and Karen Miller-Moslin O O
N
O
N
N H
N
S
N
N
O N
N
HN N
24: robotnikinin
N
25: GANT58
Cl
N
O
O O HO H
N
H
H
O
26: GANT61
H
O O
O
O
HO
O
H
H
O
OEt
N
O
H
H
S
O
N H
O
27: physalin F
28: physalin B
29: JK184
3.2.3 Microtubule depolymerization JK184 (29) potently inhibited Hh signaling in a Gli-luciferase cellular assay (IC50 ¼ 30 nM); this inhibition was confirmed by measuring the mRNA levels of Gli1 and Ptch1 by quantitative reverse transcription–polymerase chain reaction (RT–PCR) in this cell line [62]. Compound 29 was not found to bind to Smo, but rather binds directly to alcohol dehydrogenase 7 (Adh 7) with a Kd of 348 nM, and was shown to inhibit the oxidation of retinol by this enzyme with similar potency. The oxidation of retinol by Adh7 is part of the retinoic acid (RA) signaling pathway, which in mouse embryos affects Hh signaling. However, recent studies demonstrated that compound 29 exerts its effects on the Hh pathway by destabilizing microtubules, and its inhibition of Adh7 is an ancillary activity. In this model, 29 disrupts microtubule-dependent processes that are required for the conversion of endogenous full-length Gli proteins into functional transcriptional activators [63].
4. HEDGEHOG INHIBITORS IN CLINICAL TRIALS The first Hh inhibitor studied in humans was the natural product cyclopamine (1) for the topical treatment of BCC. Tas- and Avci reported
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
333
rapid regression of facial BCC in four out of four patients without adverse effects [17]. In 2005, a phase 1 clinical trial was initiated with Cur61414 (5) for the topical treatment of BCC [64]. The trial was halted a year later, as the compound did neither produce significant clinical changes nor downregulate the pharmacodynamic marker Gli1, leading to speculations that the drug candidate may not have adequately penetrated the human skin [65]. A second compound, GDC-0449 (13), provided proof-of-concept in 2008 in a first-in-human study in patients with metastatic or locally advanced BCC. In eight of nine patients, the compound caused either partial responses or stable disease upon oral administration, and mRNA levels of Gli1 were reduced in skin biopsies. The compound seemed to have minimal toxicity and was well tolerated at doses of up to 540 mg/ day. The long half-life of the drug caused marked accumulation and provided a steady-state plasma concentration of around 20–30 mM at all three doses tested (150, 270 and 540 mg) [18,66]. A pivotal phase 2 study is underway to evaluate the efficacy of this compound in patients with either metastatic BCC or locally advanced, inoperable BCC [67]. Additional phase 2 clinical trials will also investigate the anti-tumor effects of 13 in patients with metastatic colorectal and ovarian cancers [68,69]. Furthermore, the safety and pharmacokinetics of this compound are being evaluated in children with medulloblastoma in a clinical trial sponsored by the National Cancer Institute [70]. IPI-926 (3), a cyclopamine derivative with better biophysical and pharmacokinetic properties than the natural product, entered a phase 1 study in 2008 in patients with solid tumor malignancies [71]. Around the same time, BMS-833923 (XL-139, structure not disclosed) entered phase 1 clinical trials in patients with solid tumors, including sporadic BCC [72]. The latest Hh/Smo inhibitor to enter clinical trials is LDE225 (structure not disclosed), which started phase 1 studies in early 2009, again in patients with solid tumors [73].
5. CONCLUSIONS The search for Hh inhibitors of therapeutic benefit has resulted in Smoothened antagonist 13, which provided proof-of-concept in a phase 1 study in advanced and metastatic BCC patients and was well tolerated at efficacious doses [18]. At least three additional and structurally distinct compounds have entered clinical trials. The clinical results in BCC patients and the impressive tumor regression observed in murine medulloblastoma models with various compounds provide promise that Smoothened inhibitors will become useful monotherapy agents against
334
Stefan Peukert and Karen Miller-Moslin
these two genetically driven tumor types. In other tumor types, which either show a paracrine requirement for Hh signaling in tumorigenesis [13] or where cancer stem cells play an important role [74], Hh inhibitors may be more appropriate as an adjunct therapy. Combination treatment might also be a suitable therapeutic strategy for cases in which Hh inhibitors show efficacy against metastatic spread but only modest growth inhibition against the primary tumor [29]. Because the Hh signaling pathway is essential in the development and tissue homeostasis, great care should be taken to evaluate the safety profile of these drugs. Phase 1 clinical results for 13 indicate that the compound is well tolerated [75], but a study looking at the effects of 9 in young mice (10–14 days) showed permanent defects in bone growth [76]. This effect was not seen in adult mice [40]. Although it is unclear whether and how these bone toxicities in young mice may translate into effects in children, the cost versus benefit needs to be carefully determined for a treatment in a pediatric population such as medulloblastoma cancer patients.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]
[9] [10]
[11]
[12]
[13]
[14] [15] [16]
M. C. Fishman and J. A. Porter, Nature, 2005, 437, 491. C. Nu¨sslein-Vollhard and E. Wieschaus, Nature, 1980, 287, 795. M. Pasca di Magliano and M. Hebrok, Nat. Rev. Cancer, 2003, 3, 903. P. A. Beachy, S. S. Karhadkar and D. M. Berman, Nature, 2004, 432, 324. L. Lum and P. A. Beachy, Science, 2004, 304, 1755. J. P. Incardona, W. Gaffield, R. P. Kapur and H. Roelink, Development, 1998, 125, 3553. M. K. Cooper, J. A. Porter, K. E. Young and P. A. Beachy, Science, 1998, 280, 1603. R. L. Johnson, A. L. Rothman, J. Xie, L. V. Goodrich, J. W. Bare, J. M. Bonifas, A. G. Quinn, R. M. Myers, D. R. Cox, E. H. Epstein, Jr. and M. P. Scott, Science, 1996, 272, 1668. Y. Lee, H. L. Miller, P. Jensen, R. Hernan, M. Connelly, C. Wetmore, F. Zindy, M. F. Roussel, T. Curran, R. J. Gilbertson and P. J. McKinnon, Cancer Res., 2003, 63, 5428. M. C. Thompson, C. Fuller, T. L. Hogg, J. Dalton, D. Finkelstein, C. C. Lau, M. Chintagumpala, A. Adesina, D. M. Ashley, S. J. Kellie, M. D. Taylor, T. Curran, A. Gajjar and R. J. Gilbertson, J. Clin. Oncol., 2006, 24, 1924. M. R. Gailani, M. Stahle-Backdahl, D. J. Leffell, M. Glynn, P. G. Zaphiropoulos, C. Pressman, A. B. Unden, M. Dean, D. E. Brash, A. E. Bale and R. Toftga˚rd, Nat. Genet., 1996, 14, 78. J. Xie, M. Murone, S.-M. Luoh, A. Ryan, Q. Gu, C. Zhang, J. M. Bonifas, C.-W. Lam, M. Hynes, A. Goddard, A. Rosenthal, H. E. Epstein, Jr. and F. J. de Savage, Nature, 1998, 391, 6662. R. L. Yauch, S. E. Gould, S. J. Scales, T. Tang, H. Tian, C. P. Ahn, D. Marshall, L. Fu, T. Januario, D. Kallop, M. Nannini-Pepe, K. Kotkow, J. C. Marsters, Jr., L. L Rubin and F. J. de Sauvage, Nature, 2008, 455, 405. J. Xie, Curr. Oncol. Rep., 2008, 10, 107. L. L. Rubin and F. J. de Savage, Nat. Rev. Drug Disc., 2006, 5, 1026. E. H. Epstein, Jr., Nat. Rev. Cancer, 2008, 8, 743.
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
[17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
[29]
[30] [31] [32]
[33]
[34] [35] [36] [37]
[38] [39] [40] [41] [42] [43]
335
S. Tas- and O. Avci, Eur. J. Dermatol., 2004, 14, 96. D. D. Van Hoff, Proc. 99th Annu. Meeting Am. Assoc. Cancer Res., 2008, Abstract LB-138. R. Rohatgi and M. P. Scott, Nat. Cell Biol., 2007, 9, 1005. M. Varjosalo and J. Taipale, J. Cell. Sci., 2007, 120, 3. J. Rennie, Sci. Am., 1994, 270, 4. R. Rohatgi, L. Milnekovic and M. P. Scott, Science, 2007, 317, 372. N. Mahindroo, C. Punchihewa and N. Fujii, J. Med. Chem., 2009, 52, 3829. A. S. Kiselyov, Anti-Canc. Agents Med. Chem., 2006, 6, 445. G. V. Borzillo and B. Lippa, Curr. Top. Med. Chem., 2005, 5, 147. R. W. King, J. Biol., 2002, 1, 8. J. K. Chen, J. Taipale, M. K. Cooper and P. A. Beachy, Genes Dev., 2002, 16, 2743. M. R. Tremblay, M. Nevalainen, S. J. Nair, J. R. Porter, A. C. Castro, M. L. Behnke, L.-C. Yu, M. Hagel, K. White, K. Faia, L. Grenier, M. J. Campbell, J. Cushing, C. N. Woodward, J. Hoyt, M. A. Foley, M. A. Read, J. R. Sydor, J. K. Tong, V. J. Palombella, K. McGovern and J. Adams, J. Med. Chem., 2008, 51, 6646. G. Feldmann, V. Fendrich, K. McGovern, D. Bedja, S. Bisht, H. Alvarez, J.-B. M. Koorstra, N. Habbe, C. Karikari, M. Mullendore, K. L. Gabrielson, R. Sharma, W. Matsui and A. Maitra, Mol. Cancer Ther., 2008, 7, 2725. M. J. Grogan and M. Tremblay, WO Patent Application 2008/109184 A1, 2008. B. Austad, S. Janardanannair, A. Lescarbeau, M. Tremblay, D. Grayzel and W. Matsui, WO Patent Application 2008/083252 A2, 2008. M. R. Tremblay, A. Lescarbeau, M. J. Grogan, E. Tan, G. Lin, B. C. Austad, L.-C. Yu, M. L. Behnke, S. J. Nair, M. Hagel, K. White, J. Conley, J. D. Manna, T. M. Alvarez-Diez, J. Hoyt, C. N. Woodward, J. R. Sydor, M. Pink, J. MacDougall, M. J. Campbell, J. Cushing, J. Ferguson, M. S. Curtis, K. McGovern, M. A. Read, V. J. Palombella, J. Adams and A. C. Castro, J. Med. Chem., 2009, 52, 4400. J. A. Williams, O. M. Guichert, B. I. Zaharian, Y. Xu, L. Chai, H. Wichterle, C. Kon, C. Gatchalian, J. A. Porter, L. L. Rubin and F. Y. Wang, Proc. Nat. Acad. Sci. U.S.A., 2003, 100, 4616. A. D. Baxter, E. A. Boyd, O. M. Guichert, S. Price and L. Rubin, Patent Application WO 01/26644 A2, 2001. S. A. Brunton, J. H. A. Stibbard, L. L. Rubin, L. I. Kruse, O. M. Guicherit, E. A. Boyd and S. Price, J. Med. Chem., 2008, 51, 1108. A. D. Baxter, E. A. Boyd, O. M. Guichert, S. Price and L. D. Rubin, WO Patent Application 01/19800 A2, 2001. S. Bahceci, W. Bajjalieh, J. Chen, S. Epshteyn, T. P. Forsyth, T. P. Huynh, B. G. Kim, J. W. Leahy, M. S. Lee, G. L. Lewis, M. B. Mac, G. Mann, C. K. Marlowe, B. H. Ridgway, J. C. Sangalang, X. Shi, C. S. Takeuchi and Y. Wang, WO Patent Application 2008/112913 A1, 2008. L. Rubin, O. M. Guichert, S. Price and E. A. Boyd, WO Patent Application 03/011219 A2, 2003. O. M. Guichert, E. A. Boyd, S. A. Brunton, S. Price, J. H. A. Stibbard and C. H. MacKinnon, Patent Application WO 2006/050506 A1, 2006. J. T. Romer, H. Kimura, S. Magdaleno, K. Sasai, C. Fuller, H. Baines, M. Connelly, C. F. Stewart, S. Gould, L. L. Rubin and T. Curran, Cancer Cell, 2004, 6, 229. M. F. T. Koehler, R. Goldsmith and D. P. Sutherlin, WO Patent Application 2006/078283 A2, 2006. B. Fauber, A. Hird, J. Janetka, D. J. Russell and B. Yang, WO Patent Application 2009/ 030952 A2, 2009. J. Gunzner, D. Sutherlin, M. Stanley, L. Bao, G. Castanedo, R. Lalonde, S. Wang, M. Reynolds, S. Savage, K. Malesky and M. Dina, WO Patent Application 2006/028958, 2006.
336
Stefan Peukert and Karen Miller-Moslin
[44] D. P. Sutherlin, Abstracts of Papers, 237th ACS National Meeting, Salt Lake City, UT, March 22–26, 2009, MEDI-177. [45] R. A. Goldsmith, D. P. Sutherlin, K. D. Robarge and A. G. Olivero, WO Patent Application 2007/059157 A1, 2007. [46] L. Dakin, B. Fauber, A. Hird, J. Janetka, D. J. Russell, Q. Su, B. Yang and X. Zheng, WO Patent Application 2009/027746 A1, 2009. [47] D. Cheng, D. Han, W. Gao, J. Jiang, S. Pan and Y. Wan, WO Patent Application 2008/ 014291 A2, 2008. [48] W. Gao, J. Jiang, Y. Wan, D. Cheng, D. Han, X. Wu and S. Pan, WO Patent Application 2007/131201 A2, 2007. [49] C. Dierks, M. Warmuth and X. Wu, WO Patent Application 2008/154259 A1, 2008. [50] S. Peukert, R. K. Jain, A. Geisser, Y. Sun, R. Zhang, A. Bourret, A. Carlson, J. DaSilva, A. Ramamurthy and J. F. Kelleher, Bioorg. Med. Chem. Lett., 2009, 19, 328. [51] R. K. Jain, J. Kelleher, S. Peukert and Y. Sun, WO Patent Application 2007/120827 A2, 2007. [52] M. J. Munchhof, L. A. Reiter, A. Shavnya, C. S. Jones, Q. Li and R. G. Linde, WO Patent Application 2008/075196 A1, 2008. [53] M. J. Munchhof, L. A. Reiter, S. D. La Greca, C. S. Jones and Q. Li, US Patent Application 2009/0005416-A1, 2009. [54] M. Dai, F. He, R. K. Jain, R. Karki, J. Kelleher, J. Lei, L. Llamas, M. A. McEwan, K. Miller-Moslin, L. B. Perez, S. Peukert and N. Yusuff, WO Patent Application 2008/ 110611 A1, 2008. [55] R. J. Austin, J. Kaizerman, B. Lucas, D. L. McMinn and J. Powers, WO Patent Application 2009/002469 A1, 2009. [56] J. Kaizerman, B. Lucas, D. L. McMinn and R. Zamboni, WO Patent Application 2009/ 035568 A1, 2009. [57] K. Miller-Moslin, S. Peukert, R. K. Jain, M. A. McEwan, R. Karki, L. Llamas, N. Yusuff, F. He, Y. Li, Y. Sun, M. Dai, L. Perez, W. Michael, T. Sheng, H. Lei, R. Zhang, J. Williams, A. Bourret, A. Ramamurthy, J. Yuan, R. Guo, M. Matsumoto, A. Vattay, W. Maniara, A. Amaral, M. Dorsch and J. F. Kelleher, J. Med. Chem., 2009, 52, 3954. [58] J. M. Balkovec, R. Thieringer and S. T. Waddell, US Patent Application 2008/262051 A1, 2008. [59] B. Z. Stanton, L. F. Peng, N. Maloof, K. Nakai, X. Wang, J. L. Duffner, K. M. Taveras, J. M. Hyman, S. W. Lee, A. N. Koehler, J. K. Chen, J. L. Fox, A. Mandinova and S. L. Schreiber, Nat. Chem. Biol., 2009, 5, 154. ˚ . Bergstro¨m, T. Shimokawa and R. Toftga˚rd, Proc. Natl. Acad. Sci. U.S.A., [60] M. L. Lauth, A 2007, 104, 8455. [61] T. Hosoya, M. A. Arai, T. Koyano, T. Kowithayakorn and M. Ishibashi, Chem. Bio. Chem., 2008, 9, 1082. [62] J. Lee, X. Wu, M. P. di Magliano, E. C. Peters, Y. Wang, J. Hong, M. Hebrok, S. Ding, C. Y. Cho and P. G. Shultz, Chem. Bio. Chem., 2007, 8, 1916. [63] T. Cupido, P. G. Rack, A. J. Firestone, J. M. Hyman, K. Han, S. Sinha, C. A. Ocasio and J. K. Chen, Angew. Chem. Int. Ed., 2009, 48, 2321. [64] Curis press release, June 8, 2005, http://phx.corporate-ir.net/phoenix.zhtml?c ¼ 123198&p¼ irol-newsArticle_print&ID ¼ 718277&highlight ¼ [65] Curis press release, July 11, 2006, http://phx.corporate-ir.net/phoenix.zhtml?c ¼ 123198&p¼ irol-newsArticle&ID ¼ 881050&highlight ¼ [66] F. de Sauvage, Targeting the Hedgehog pathway in cancer: From bench to clinic, DanaFarber Cancer Institute, October 28, 2008, Boston. [67] A study evaluating the efficacy and safety of GDC-0449 (Hedgehog pathway inhibitor) in patients with advanced basal cell carcinoma, http://clinicaltrials.gov/ct2/show/ NCT00833417?term ¼ GDC-0449&rank ¼ 1
Hedgehog Signaling Pathway Inhibitors as Cancer Therapeutics
337
[68] A study of GDC-0449 (Hedgehog pathway inhibitor) with concurrent chemotherapy and bevacizumab as first-line therapy for metastatic colorectal cancer, http:// clinicaltrials.gov/ct2/show/NCT00636610?term ¼ GDC-0449&rank ¼ 4 [69] A study of GDC-0449 (Hedgehog pathway inhibitor) as maintenance therapy in patients with ovarian cancer in a second or third complete remission, http:// clinicaltrials.gov/ct2/show/NCT00739661?term ¼ GDC-0449&rank ¼ 5 [70] GDC-0449 in treating young patients with medulloblastoma that is recurrent or did not respond to previous treatment, http://clinicaltrials.gov/ct2/show/NCT00822458?term¼ GDC-0449&rank ¼ 2 [71] A phase 1 study of IPI-926 in patients with advanced and/or metastatic solid tumor malignancies, http://clinicaltrials.gov/ct2/show/NCT00761696?term¼ IPI-926 &rank¼ 1 [72] A phase 1 study of BMS-833923 (XL139) in subjects with advanced or metastatic cancer, http://clinicaltrials.gov/ct2/show/NCT00670189?term ¼ XL-139&rank ¼ 1 [73] Novartis new molecules presentation, AACR 100th Annual Meeting, Denver, CO, April 18–22, 2009. [74] I. Ischenko, H. Seeliger, M. Schaffer, K.-W. Jauch and C. J. Bruns, Curr. Med. Chem, 2008, 15, 3171. [75] P. M LoRusso, C. M. Rudin, M. J. Borad, L. Vernillet, W. C. Darbonne, H. Mackey, J. F. DiMartino, F. de Savage, J. A. Low and D. D. Von Hoff, J. Clin. Oncol. (Meet. Abstr.), 2008, 26, 3516. [76] H. Kimura, J. M. Y. Ng and T. Curran, Cancer Cell, 2008, 13, 249.
CHAPT ER
17 Emerging Therapies Based on Inhibitors of PhosphatidylInositol-3-Kinases John M. Nuss, Amy Lew Tsuhako and Neel K. Anand
Contents
1. Introduction 2. Inhibitors of PI3Ks: Early Studies 3. Clinically Investigated Pan-Active Class I PI3K Inhibitors 4. Additional Pan-Active Class I PI3K Inhibitors 5. Clinically Investigated Isozyme-Selective PI3K Inhibitors 6. Pre-Clinical Isozyme-Selective PI3K Inhibitors 7. Conclusions References
339 341 343 347 347 349 350 351
1. INTRODUCTION The superfamily of lipid kinases collectively referred to as phosphoinositide kinases (PIKs, including PI3K, PI4K, and others) has been an area of intense investigation due to the crucial role of members of this class in various signal transduction–mediated events [1–5]. As the basic biology of the PIKs has become better understood, their roles in various pathophysiologies such as cancer, inflammation, and cardiovascular disease are also becoming clearer, leading to the development of agents targeting the function of these kinases as new modalities for the treatment of a number of disease states. The PIKs catalyze the conversion of phosphatidyl inositol (PI) to various phosphoinositides [1–5]. Of particular importance is the Exelixis, Inc., South San Francisco, CA 94080, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04417-0
r 2009 Published by Elsevier Inc.
339
340
John M. Nuss et al.
phosphoinositide 3-kinase (PI3K) catalyzed formation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2); this second messenger is a critical regulator of cellular functions such as metabolism, cell growth, differentiation, and chemotaxis. A simplified view of PI3K signaling is shown in Figure 1. Activation of PI3Ks, either by growth factor (e.g., insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF)) receptor binding or by G-proteincoupled receptor (GPCR) activation, results in the formation of PIP3, which recruits the kinases 3-phosphoinositide dependent kinase (PDK) and protein kinase B (PKB, aka AKT) to the cell membrane; AKT is activated through phosphorylation by the membrane-bound PDK. Activation of AKT effects a myriad of downstream processes, including modulation of numerous proteins such as mTOR, GSK3, forkhead, NFkB transcription factors, and eNOS, which are involved in processes such as metabolism, cell growth, survival, and angiogenesis [6]. Dysregulation of these processes is crucial for the pathophysiology of several diseases, as attenuated signaling of the insulin receptor is a major contributor to type-2 diabetes and mutations that lead to amplification of PI3K signaling are among the most common mutations in human cancers [7,8]. PI3K signaling is also negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10); this phosphatase is responsible for the dephosphorylation of PIP3 to PIP2 [9]. PTEN has been shown to be mutated or absent in many human cancers, further implicating aberrant PI3K signaling in cancer.
Figure 1
Schematic of PI3K signaling. (See Color Plate 17.1 in Color Plate Section.)
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
341
PI3Ks can be categorized into three classes, I, II, and III [1–5,10]. The most thoroughly studied class, the class I PI3Ks, are heterodimeric proteins, with each member of this family containing a smaller regulatory domain and a larger 110-kD catalytic domain in each of the four isoforms, p110a, p110b, p110g, and p110d. These have been further divided into two sub-classes, the class Ia (p110a, p110b, p110d) and the class Ib (p110g). The class Ia catalytic domains combine with SH2-domain containing 85-kD regulatory subunits and are predominantly activated by growth factor receptor tyrosine kinases such as the insulin receptor tyrosine kinase, whereas the class Ib isoforms generally combine with a 101-kD regulatory domain and are activated as a result of GPCR signaling. Structurally, the catalytic domains of the PI3Ks display the highly conserved domain structure of the protein kinases, having two lobes, an N-terminal lobe composed of five b-sheets and three a-helices and a C-terminal lobe consisting of mainly a-helices with a flexible linker joining the two lobes [11]. The class II and III PI3Ks have not garnered much attention from the perspective of inhibitor design and pharmacological study and will not be discussed in detail here [12]. Additionally, a number of related kinases, including the serine/threonine kinases mTOR, DNA dependent protein kinase (DNA-PK), and ataxia telangiectasia mutated kinase (ATM), are classified as PI3K-related kinases, or class IV PI3Ks, due to gross structural similarities to the PI3Ks. This similarity often impacts inhibitor design, as cross-reactivity with these kinases is often observed [4–6]. In fact, the intentional design of mTOR/PI3K dual inhibitors is an area of intense investigation, and several compounds having this profile are currently under clinical evaluation as cancer therapeutics (vide infra). Functional elucidation of the class I PI3K isozymes is an extremely active area of investigation. Studies have linked p110g and p110d to chronic inflammation [13], p110b to thrombosis [14], and p110a to many tumor types including ovarian, breast, gastric, and colon cancers [15]. PI3Ka and -b are distributed in many tissues, in contrast to the more narrowly distributed PI3Kg and -d, which are mainly found in leukocytes, endothelial cells, and smooth muscle cells [16]. Importantly, genetic studies suggest that mutation in the p110a gene is oncogenic [17,18]. Consequently, p110a is considered to be a highly validated target for human cancers and has been an extremely active area of drug discovery.
2. INHIBITORS OF PI3Ks: EARLY STUDIES Given the crucial role that the dysregulation of PI3Ks, and in particular, the amplification of PI3K-derived signaling, plays in various pathophysiologies, it is not surprising that considerable effort has gone into the
342
John M. Nuss et al.
Figure 2 Interaction of LY294002 with ATP-binding site of PI3Kg. (See Color Plate 17.2 in Color Plate Section.)
design and pharmacological characterization of both pan-active and isozyme-selective inhibitors of the PI3Ks [3–6]. As with a majority of protein kinase inhibitors, most reported inhibitors of the PI3Ks target the ATP-binding pocket in the catalytic domain. Some of the early inhibitors that have been reported include LY294002, 1 [19], and wortmannin, 2 [20]. Many inhibitors of the PI3Ks have their genesis in the quercetin derivative 1, a promiscuous kinase inhibitor that was found to be an ATP-competitive, pan-active inhibitor of class I PI3Ks [19]. The morpholine oxygen forms the critical H-bonding interaction with the Val882 (PI3Kg notation) residue on the flexible hinge region of the kinase in the ATP-binding pocket; the carbonyl group stabilizes the binding further by forming two hydrogen bonds, one with the backbone NH of Asp964 in the activation loop and one with the Lys833 of the salt bridge (Figure 2) [21]. Many groups have attempted to optimize these interactions to design ‘‘morpholine’’-based inhibitors of PI3Ks having enhanced potency and selectivity [22]. O
O O MeO
O
O
17
N O
O
H O
O 20 1
O 2
The steroidal furan 2 and its derivatives have been found to be PI3K inhibitors [20]. It potently inhibits all of the class I PI3Ks with IC50o10 nM; this natural product also inhibits the PI3K-related kinases
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
343
mTOR and DNA-PK, inter alia. Structural and mechanistic studies have shown that 2 binds to the ATP-catalytic site, the critical interaction being the formation of a H-bond between the hinge residue Val882 and the C-17 carbonyl in the D-ring (steroid notation) [23,24]. In contrast to 1, 2 binds irreversibly to PI3Kg by forming a covalent adduct between Lys833 in the catalytic site and the electrophilic site C-20 in 2, in turn leading to destruction of the catalytic ability of the kinase. Despite the general reluctance of the pharmaceutical industry to develop irreversible enzyme inhibitors, derivatives of 2 have been investigated clinically (vide infra). Structural studies to provide insight into ligand–enzyme interactions to facilitate inhibitor design are just emerging. Of the B20 PI3K structures that have been reported in the Protein Data Base Repository (PDB) as of April 2009 [25], the only ligand-bound structures disclosed involve PI3Kg structures. The PI3Ka apo-enzyme structure has been solved [26]; however, no ligand-bound structures have been reported. Structures of the other isozymes have not been disclosed. It is expected that intensified structural efforts in this area will greatly facilitate the design of both pan-active and isozyme-selective inhibitors. As a result of extensive efforts in the design of kinase inhibitors and screening, a diverse array of scaffolds have also been reported to inhibit the lipid PI3Ks. The number and types of PI3K inhibitors continues to increase rapidly. Both pan-active and isozyme-selective inhibitors have been described and are discussed in the next section.
3. CLINICALLY INVESTIGATED PAN-ACTIVE CLASS I PI3K INHIBITORS SF1126 (3) – Although 1 emerged as an invaluable tool in establishing the biological role of PI3K in human cancer, poor physicochemical properties and short plasma half-life made it unsuitable for further development. Attempts to improve the pharmacokinetic profile of 1 have led to the development of a water soluble prodrug 3 (Figure 3), currently in phase I clinical trials for cancer. Prodrug 3 is a peptide conjugate of 1 that specifically targets cell-surface integrins within the tumor. Although 3 demonstrates only B100–500 nM activity against various PI3K isoforms, significant inhibition of tumor growth was observed in various xenograft models (U87MG, PC-3, U251, and U251vIII) due to the high levels of accumulation of the drug in the tumor tissue [27]. It should also be noted that not only does 3 inhibit all forms of PI3Ks, but it also inhibits mTOR, DNA-PK, PLK-1, PIM-1, and CK2. This drug is reported to be well tolerated in patients with solid tumors treated twice a week for 4 weeks by i.v. infusion. In this study, 3 of the 12 patients experienced stable disease [28].
344
John M. Nuss et al.
O
RGDS
O MeO
O O
O
H O
O
N+
O
O N
O O
N
O
N
N N
OH O
N
RGDS=Ar g-Gly -Glu-Ser
3
4
O
5 O
SO2Me N
N N HN
N N
S
N
O N S
N HO
N
N
6
N N
7 N
O
N HO
8
N
F
OMe N
O
F
S N
EtHN N
N
O
N
N
O
9
OMe
N
HN S
N N H
N
N
N H
OMe
O
10
11 O
O HN
N
S N
R1HN
O
N
N H
X
12 HN N
R1
R2
N
R1
R2
N
15
Figure 3
N N
Pan-active PI3K inhibitors.
N
R3
N
N
R2
13
R1HN
N
14
OR2
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
345
PX-866 (4) – As previously mentioned, the irreversible inhibitor 2 is one of the earliest discovered inhibitors of the PI3K pathway [20,29,30]; multiple groups have attempted to make derivatives with an improved pharmacokinetic profile and therapeutic index. The wortmannin derivative 4 (Figure 3) is currently in phase I clinical trials [31,32]. This broad spectrum PI3K inhibitor demonstrated improved stability and hepatotoxicity compared to 2 [33,34]. In addition, 4 showed tumor regression in human ovarian and colon carcinoma xenograft models, as well as nonsmall-cell lung cancer resistant to gefitinib. However, in mice treated with 4, increases in both blood insulin and glucose are observed. This hyperglycemic effect can be moderated with pioglitazone, a PPARg agonist approved for the treatment of diabetes [35]. In July 2008, clinical trials were initiated in patients with advanced solid tumors [36]. This is currently the only irreversible PI3K inhibitor in clinical trials. NVP-BEZ235 (5) and BGT226 (structure undisclosed) – BGT226 and 5 (Figure 3) are two dual PI3K/mTOR inhibitors currently undergoing trials for breast and other solid tumors [37]. Both are reported to be reversible, ATP competitive inhibitors of, inter alia, the class I PI3Ks as well as the PIK-related kinase, mTOR. 5 inhibits PI3K a, b, g, d at 4, 76, 7, and 5 nM respectively, as well as mTOR (21 nM), but not DNA-PK [38,39]. In addition, 5 potently inhibits cellular proliferation for both wild-type and oncogenic p110-a mutated cells and in multiple myeloma [40]. This potent antiproliferative effect has been shown to translate into tumor regression in xenograft models, where the regressions are wellcorrelated with the inhibition of 437S-p-AKT and other PI3K pathway markers [41–43]. In addition, recent data suggest that hyperactivation of the PI3K pathway leads to lapatinib resistance, which can be reversed by 5 [44]. Of concern for anti-PI3K therapy is the potential induction of insulin resistance. However, although 5 is a pan PI3K/mTOR inhibitor, no effect on insulin or glucose levels were reported in vivo in rodents given efficacious dosages for 13 weeks, indicating that attaining isozymeselective PI3K inhibitors may not be necessary for developing PI3K inhibitors with manageable effects on glucose regulation [45]. GDC-0941 (6) – This compound (Figure 3) is currently being investigated in multiple phase I trials. It is a pan-selective PI3K inhibitor (3, 33, 75, 3 nM against a, b, g, d) that does not potently inhibit mTOR (580 nM) or DNA-PK (1230 nM) [46,47]. The compound was developed from modifications of previously reported pyrimidine derivatives (Figure 3, 7, 8-aka PI-103) that had PI3Ka inhibition IC50o10 nM, but poor pharmacokinetic profiles [48,49]. Attempts to improve the pharmacokinetic profile took advantage of the available crystal structure of p110g and a p110a homology model [11]. 6 shows broad antiproliferative effects associated with inhibition of pAKT, the downstream signal of PI3K
346
John M. Nuss et al.
inhibition. In animal models (U87MG human glioblastoma xenografts), oral doses of 6 reduces tumor growth by 83%. Of particular note, reduction of pAKT was also observed in patients with solid tumors dosed daily for 3 weeks with 6 [50,51]. Thus far, no reports have emerged regarding tumor regression in humans, but in general, the drug has been reported to be well tolerated. Several patent applications have published describing thienopyrimidines structurally related to 6 [52–55]. XL147 (structure undisclosed) – This compound is currently in phase I clinical trials as a class I PI3K inhibitor (IC50: 39, 383, 23, 36 nM for p110a, b, g, d, respectively) and importantly has no mTOR or DNA-PK activity [56–58]. In pre-clinical studies, XL147 is reported to block PI3K signaling in cultured tumor cells vascular endothelial growth factor (VEGF)induced tubule formation in cultured endothelial cells, and hepatocyte growth factor (HGF)-stimulated migration of B16 melanoma cells. In mouse xenograft models, XL147 showed strong tumor growth inhibition. This antitumor efficacy correlates in a dose-dependent manner with increased apoptosis and inhibition of angiogenesis. In clinical trials, XL147 at doses of up to 600 mg has generally been well tolerated in patients with metastatic or unresectable solid tumors. At the 600 mg daily oral dose, reductions of 70–80% in the phosphorylation of the PI3K pathway markers such as pAKT, pPRAS40, and pS6 have been observed in the patient tumor tissues. To date, 8 of the 23 patients tested in this phase I trial showed prolonged stable disease (W3 months). In addition, glucose levels were stable in the 23 patients, although insulin levels did increase in some patients [59]. XL765 (structure undisclosed) – In contrast to XL147, XL765 inhibits both class I PI3Ks and mTOR (IC50: 39, 113, 43, 9,157 nM for p110a, b, g, d, and mTOR, respectively). In preclinical studies, XL765 potently inhibits PI3K pathway signaling in cultured cells as well as various xenograft tumor models [60]. Data reported from the phase I dose escalation trial of XL765 showed that in 28 patients with metastatic or unresectable solid tumors dosed orally b.i.d., XL765 was well tolerated at 30 mg [61,62]. As with XL147, dosing of XL765 in patients led to an increase in plasma insulin levels, but no change in blood glucose levels. Pharmacodynamic analyses showed significant reductions of 80–90% in pAKT, p4EBP1, and pS6 levels in patient tumor tissue [63], with 5 of the 28 patients enrolled showing stable disease (W3 months) [64]. GSK-1059615 or GSK-615 (9) – The PI3K inhibitor 9 (Figure 3) inhibits AKT phosphorylation in various tumor cell lines, and especially in T47-D breast carcinoma cell (IC50: 34 nM)[65]. In in vivo studies with xenograft models such as BT474 breast tumor, and HCC1954 breast cancer cell, 9 shows dose-dependent tumor growth inhibition without significant
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
347
body weight loss or toxicity. GSK-615 is currently in phase I clinical trials for lymphoma, solid tumors, metastatic breast cancer, and endometrial cancer [66].
4. ADDITIONAL PAN-ACTIVE CLASS I PI3K INHIBITORS ZSTK474 (10) – A potent, pan-active, ATP competitive PI3K inhibitor 10 (Figure 3) has been identified (IC50: 16, 44, 46, 49 nM for a, b, g, d, respectively) with considerably lower activity for mTOR and DNA-PK [67,68]. Although 10 is a long way from proving that isoform nonspecific inhibitors can achieve acceptable therapeutic indices, this triazine analog shows strong antiproliferative effects in cells and antitumor activity in in vivo xenograft models without body weight loss [69–72]. Disclosures of compounds structurally related to 10 have recently appeared [73–80]. AEZS-126 (11) – This compound (Figure 3) has been reported to be a potent, orally bioavailable pan-class I PI3K inhibitor [81,82]. It inhibits the class I PI3Ks (51, 3000, 177, 139 nM for a, b, d, g, respectively), but does not inhibit mTOR (12.9 uM) or DNA-PK (W31 uM). In pre-clinical xenograft studies, 11 demonstrated up to 30–50% tumor growth inhibition in HCT116 and PC3 tumor models. In addition to these compounds, many other class I pan-active compound have recently been reported. Although there is a paucity of data associated with many of these reports, a sampling of these structures are given in Figure 3, 12–15, and the reader is referred to the original publications for further information [83–86].
5. CLINICALLY INVESTIGATED ISOZYME-SELECTIVE PI3K INHIBITORS The development of isozyme-selective inhibitors of PI3K has started to attract great interest as this may enable the possibility of targeting specific therapeutic areas while reducing off-target effects [6]. This vision has the potential to become reality due to recent research advances culminating in availability of data regarding tissue distribution, functional elucidation, and structural variation of the individual isozymes [3,6,87]. CAL-101(structure undisclosed) – This compound is a PI3Kd-selective inhibitor with an IC50 of 1–10 nM with a reported W30-fold selectivity over other isoforms, mTOR and DNA-PK [88]. It is likely that the structure of CAL-101 derives from a series of quinazolinone derivatives that yielded the inhibitor IC-87114 (aka D-030) (Figure 4, 16) [89–91]. The d-isozyme
348
John M. Nuss et al.
O NH2
N
N
N
N
N N
H2N
N
OH OH
N
N
N NH2 16
Figure 4
17
Isozyme-selective PI3K inhibitors.
selectivity of these compounds (and also the PIK-39, PIK-293, PIK-294 analogs) is postulated to arise due to a conformational rearrangement of Met752 (Met804 in PI3Kg), which is distal to the highly homologous adenine binding pocket [22]. CAL-101 is being evaluated as an oral therapeutic in two phase I clinical trials: in a recently completed trial as a treatment for allergic rhinitis (presumably as an anti-inflammatory agent) [92] and in an on-going trial as a treatment for cancer, focused on patients with hematologic malignancies [88,93–95]. CAL-101 inhibits p110dmediated basophil activation in whole blood with an IC50 of 30–70 nM and has demonstrated well-tolerated, sustained plasma concentrations of 500–5000 nM in a 7-day multidose healthy human volunteer study, indicating a viable therapeutic index [88,94]. Although data on preclinical PI3Kd-selective inhibitors other than IC-87114 is limited, recent patent disclosures suggest that this area remains active [96–98]. TG100-115 (17) – The dual d/g-selective ATP-competitive inhibitor 17 (Figure 4) (PI3K IC50: 1300, 1200, 83, 235 nM for a, b, g, d, respectively) has been the subject of clinical evaluation in a number of cardiovascular indications [99,100]. The design of the pteridine 17 involved pharmacophore-based modeling starting from known PI3K inhibitors, 2, and quercetin [99]. Initial SAR was developed using an in vivo Miles assay evaluating vascular permeability, with in vitro assays being subsequently introduced to determine the molecular target and the cellular signaling pathway for the demonstrated inhibition of vascular permeability [99]. In contrast to pan-active PI3K inhibitors, 17 showed no effect on mitogenesis [99]. Pre-clinically, 17 exhibited well-behaved pharmacokinetic properties when administered intravenously and demonstrated cardioprotective attributes in animal models [99,101,102]. A phase I/II clinical trial to evaluate intravenously administered 17 in terms of safety in patients who suffer a heart attack and then undergo angioplasty to restore blood flow and also to evaluate its potential efficacy to reduce heart muscle damage related to this ischemia/reperfusion injury, was completed in 2008, with no further clinical development reported [103].
349
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
Compound 17 is also being investigated pre-clinically for the treatment of inflammatory diseases [104]. AZD6482 (structure undisclosed) – No pre-clinical data has been disclosed for AZD6482, a PI3Kb-selective inhibitor that is being investigated as an antithrombotic agent in multiple phase I clinical trials [105,106]. The first clinical trial, completed in May 2008, was a 5-month trial that investigated the safety and tolerability of a single escalating intravenously administered dose of AZD6482 alone, and co-administered with acetylsalicylic acid (ASA) [105]. In February 2009, a second phase I clinical trial was opened for patient recruitment to evaluate the effect of AZD6482, compared with clopidogrel on bleeding times in healthy volunteers receiving low dose ASA (75 mg aspirin) [106].
6. PRE-CLINICAL ISOZYME-SELECTIVE PI3K INHIBITORS A number of compounds that exhibit PI3K isozyme selectivity have been disclosed, which have yet to enter the clinic. Generally, these pre-clinical compounds are selectively potent for a sub-set of the PI3K isozymes (i.e., they are not specific inhibitors), but for clarity in this review, they will be classified by the isozyme which is most potently inhibited. TGX-221 (18) – Compound 18 (Figure 5) (PI3K IC50: W5000, 5–10, W3500, 100–200 nM for a, b, g, d, respectively) is the lead compound among a series reported to show potent, selective inhibition of PI3Kb and is also being investigated as an antithrombotic agent [14,107,108]. 18 utilizes key structural characteristics of the core scaffold of 1; specifically the 2-morpholine moiety, the 4-carbonyl, and the 8-aryl substituents. From docking studies in a homology model of PI3Kb, it is postulated that 18 resides in the ATP-binding pocket of PI3Kb where it forms a critical hydrogen bond interaction with Val854 in the hinge through the morpholine oxygen and forms additional contacts with Lys805 O
N NH
O
NO2
O
O
N
S
N
N O
O
NH
S
NH
O
S
N N
Br
O
O
NH S
O
HO
N
N
F O N
F O
F
18
Figure 5
19
20
Additional isozyme-selective PI3K inhibitors.
21
22
O
350
John M. Nuss et al.
(salt bridge) and Asp937 (DFG loop) through the carbonyl group and exocyclic aniline, respectively [109]. In the absence of X-ray crystal structure of PI3Kb, the origin of the isozyme selectivity has also been investigated by mutagenesis, resulting in a number of interesting hypotheses regarding binding modes and ways to improve potency against PI3Kb [110]. Owing to their selective PI3Kb inhibitory profile, 18 and related analogs may serve as very important tools for elucidating the role of PI3Kb signaling in thrombosis as well as cancer [14,111–116]. PIK-75 (19) – Compound 19 (Figure 5) (PI3K IC50: 3–6, 850–1300, 40–76, 510 nM for a, b, g, d, respectively, mTOR IC50: W1000 nM, and DNA-PK IC50: 2 nM) was identified as a leading compound from a series of imidazopyridines that exhibit potent inhibition of PI3Ka [22,114,117–119]. This analog has been used as a reference compound for further exploring SAR for selective PI3Ka inhibition [120]. Compound 19 potently inhibits tumor cell proliferation in vitro (IC50o100 nM) in various PI3K-driven cell lines and has demonstrated tumor growth inhibition in a xenograft model [121]. Owing to its PI3Ka inhibitory profile, 19 has served as a tool compound for elucidating the role of PI3Ka signaling [22,111]. However, as 19 has demonstrated generalized cytotoxicity likely unrelated to PI3K inhibition [114,122], it is unlikely to be developed further. As aberrant PI3Ka signaling has been strongly implicated in various cancers, particularly through activating mutations [114], interest in PI3Ka-selective inhibitors remains very strong as evidenced by recently disclosed inhibitors in the open and patent literature [48,49,123–125]. AS-252424 (20)/AS-605240 (21)/AS-604850 (22) – The PI3Kg-selective inhibitors (Figure 5) are exemplified by the thiazolidinedione derivatives 20 (PI3K IC50: 940, W20,000, 30, W20,000 nM for a, b, g, d, respectively) [113,126], 21 (PI3K IC50: 60, 270, 8, 300 nM for a, b, g, d, respectively) [127– 130] and 22 (PI3K IC50: 4,500, W20,000, 250, W20,000 nM for a, b, g, d, respectively) [130]. The crystal structures in PI3Kg of 20 [126] and 22 [130] have been disclosed, and initial docking studies to elucidate differences in key interactions between the thiazolidinedione derivatives and various isozymes have been conducted [109]. Furthermore, these derivatives have been used to investigate the role of PI3Kg signaling [13,113,131] and are reported to demonstrate anti-inflammatory effects [130–133]. There is a lot of interest in PI3Kg-selective inhibitors as evidenced by recently disclosed inhibitors in the literature and patents [134–138].
7. CONCLUSIONS Recent research has clearly shown that PI3K signaling is involved in many pathways and that the isozymes do not have identical roles.
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
351
Currently, the optimal PI3K isoform inhibitory profile (with or without mTOR or DNA-PK inhibition) for a successful oncology, thrombosis, inflammation, or cardiac therapeutic is undetermined as there is insufficient clinical data to form hard conclusions. Early concerns that all panactive PI3K inhibitors may suffer from an unacceptable therapeutic index due to issues such as metabolic toxicity may be overstated. Additionally, it may be hypothesized that drug resistance due to mutations in certain PI3K isozymes may be more easily overcome by inhibiting multiple PI3K isozymes along with PIK-related kinases. Conversely, inhibiting a specific isozyme (or group of isozymes) may lead to a potent indication-focused therapy with an improved therapeutic index due to minimized off-critical isozyme effects. It is still to be determined whether intervention in these pathways with PI3K inhibitors will have therapeutic benefit in humans, although admittedly the story is just beginning. Only with more time and more pre-clinical and clinical results will we be able to determine whether the incredible therapeutic promise of PI3K inhibitors will come to fruition.
REFERENCES [1] J. A. Engelman, J. Luo and L. C. Cantley, Nat. Rev. Genet., 2006, 7, 606. [2] B. T. Hennessy, D. L. Smith, P. T. Ram, Y. Lu and G. B. Mills, Nat. Rev. Drug Disc., 2005, 4, 988. [3] R. Marone, V. Cmiljanovic, B. Giese and M. P. Wymann, Biochim. Biophys. Acta, 2008, 1784, 159. [4] T. J. Sundstrom, A. C. Anderson and D. L. Wright, Org. Biomol. Chem., 2009, 7, 840. [5] C. Garcia-Echeverria and W. R. Sellers, Oncogene, 2008, 27, 5511. [6] N. T. Ihle and G. Powis, Mol. Cancer Ther., 2009, 8, 1. [7] I. Sansal and W. R. Sellers, J. Clin. Oncol., 2004, 22, 2954. [8] T. F. Franke, Oncogene, 2008, 27, 6473. [9] S. Kang, A. G. Bader and P. K. Vogt, Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 802. [10] M. P. Wymann and L. Pirola, Biochim. Biophys. Acta, 1998, 1436, 127. [11] E. H. Walker, O. Perisic, C. Reid, L. Stephens and R. Williams, Nature, 1999, 402, 313. [12] M. Falasca and T. Maffucci, Biochem. Soc. Trans., 2007, 35, 211. [13] T. Ruckle, M. K. Schwarz and C. Rommel, Nat. Rev. Drug Discov., 2006, 5, 903. [14] S. P. Jackson, S. M. Schoenwaelder, I. Goncalves, W. S. Nesbitt, C. L. Yap, C. E. Wright, V. Kenche, K. E. Anderson, S. M. Dopheide, Y. Yuan, S. A. Sturgeon, H. Prabaharan, P. E. Thompson, G. D. Smith, P. R. Shepherd, N. Daniele, S. Kulkarni, B. Abbott, D. Saylik, C. Jones, L. Lu, S. Giuliano, S. C. Hughan, J. A. Angus, A. D. Robertson and H. H. Salem, Nat. Med., 2005, 11, 507. [15] L. Zhao and P. K. Vogt, Oncogene, 2008, 27, 5486. [16] B. Vanhaesbroeck, S. J. Leevers, K. Ahmadi, J. Timms, R. Katso, P. C. Driscoll, R. Woscholski, P. J. Parker and M. D. Waterfield, Annu. Rev. Biochem., 2001, 70, 535. [17] Y. Samuels, Z. Wang and A. Bardelli, Cell, 2007, 129, 957. [18] J. J. Zhao, Z. Liu, L. Wang, E. Shin, M. F. Loda and T. M. Roberts, Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 18443. [19] C. J. Vlahos, W. F. Matter, K. Y. Hui and R. F. Brown, J. Biol. Chem., 1994, 269, 5241. [20] A. Arcaro and M. P. Wymann, Biochem. J., 1993, 296, 297.
352
John M. Nuss et al.
[21] E. H. Walker, M. E. Pacold, O. Perisic, L. Stephens, P. T. Hawkins, M. P. Wymann and R. L. Williams, Mol. Cell, 2000, 6, 909. [22] Z. A. Knight, B. Gonzalez, M. E. Feldman, E. R. Zunder, D. D. Goldenberg, O. Williams, R. Loewith, D. Stokoe, A. Balla, B. Toth, T. Balla, W. A. Weiss, R. L. Williams and K. M. Shokat, Cell, 2006, 125, 733. [23] B. H. Norman, C. Shih, J. E. Toth, J. E. Ray, D. W. Dodge, D. W. Johnson, P. G. Rutherford, R. M. Schultz, J. F. Worzall and C. J. Vlahos, J. Med. Chem., 1996, 39, 1106. [24] S. Stoyanovea, G. Bulgarelli-Leva, C. Kirsch, T. Hanck, R. Klinger, R. Wetzker and M. P. Wymann, Biochem. J., 1997, 324, 489. [25] RCSB Protein Structure Database, http://www.rcsb.org/pdb/home/home.do [26] C. H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V. E. Velculescu, K. W. Kinzler, B. Vogelstein, S. B. Gabelli and L. M. Amzel, Science, 2007, 318, 1744. [27] J. R. Garlich, P. De, N. Dey, N. Dey, J. D. Su, X. Peng, A. Miller, R. Murali, Y. Lu, G. B. Mills, V. Kundra, H. K. Shu, Q. Peng and D. L. Durden, Cancer Res., 2008, 68, 206. [28] http://www.semaforepharma.com/pdfs/news/Semafore_ASCO_June-04-2008.pdf [29] M. P. Wymann, G. Bulgarelli-Leva, M. J. Zvelebil, L. Pirola, B. Vanhaesebroeck, M. D. Waterfield and G. Panayotou, Mol. Cell Biol., 1996, 16, 1722. [30] H. Yano, S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui, Y. Nonomura and Y. Matsuda, J. Biol. Chem., 1993, 268, 25846. [31] J. W. Millard, L. D. Kirkpatrick, L. A. Pestano and G. Powis, 19th Rocky Mountain Regional Meeting of the American Chem. Soc., Tucson, AZ, October 2006. [32] P. Wipf, L. Kirkpatrick and G. Powis, US Patent US20090087441, 2009. [33] N. T. Ihle, R. Williams, S. Chow, W. Chew, M. I. Berggren, G. Paine-Murrieta, D. J. Minion, R. J. Halter, P. Wipf, R. Abraham, L. Kirkpatrick and G. Powis, Mol. Cancer Ther., 2004, 3, 763. [34] A. L. Howes, G. G. Chiang, E. S. Lang, C. B. Ho, G. Powis, K. Vuori and R. T. Abraham, Mol. Cancer Ther., 2007, 6, 2505. [35] N. T. Ihle, R. Lemos, D. Schwartz, J. Oh, R. J. Halter, P. Wipf, L. Kirkpatrick and G. Powis, Mol. Cancer Ther., 2009, 8, 94. [36] www.clinicaltrials.gov/ct2/show/NCT00726583 [37] http://www.novartisoncology.com/research-innovation/pipeline/bgt226.jsp [38] F. Stauffer, S.-M. Maira, P. Furet and C. Garcia-Echeverria, Bioorg. Med. Chem. Lett., 2008, 18, 1027. [39] S.-M. Maira, F. Stauffer, J. Brueggen, P. Furet, C. Schnell, C. Fritsch, S. Brachmann, P. Chene, A. De Pover, K. Schoemaker, D. Fabbro, D. Gabriel, M. Simonen, L. Murphy, P. Finan, W. Sellers and C. Garcia-Echeverria, Mol. Cancer Ther., 2008, 7, 1851. [40] P. Baumann, S. Mandl-Weber, F. Oduncu and R. Schmidmaier, Exp. Cell Res., 2009, 315, 485. [41] V. Serra, B. Markman, M. Scaltriti, P. J. A. Eichhorn, V. Valero, M. Guzman, M. L. Botero, E. Llonch, F. Atzori, S. Di Cosimo, M. Maira, C. Garcia-Echeverria, J. L. Parra, J. Arribas and J. Baselga, Cancer Res., 2008, 68, 8022. [42] P. Cao, S.-M. Maira, C. Garcia-Echeverria and D. W. Hedley, Br. J. Cancer, 2009, 100, 1267. [43] O. Dorigo, C. Santiskulvong, M. Fekete, A. Karam, D. Mulholland, C. Engl and H. Wu, 17, 40th Annual Meeting Women’s Cancer, San Antonio, TX, February, 2009. [44] P. J. A. Eichhorn, M. Gili, M. Scaltriti, V. Serra, M. Guzman, W. Nijkamp, R. L. Beijersbergen, V. Valero, J. Seoane, R. Bernards and J. Baselga, Cancer Res., 2008, 68, 9221. [45] E. Marrer, et al., 215, Proceedings of the American Association for Cancer Research (AACR), San Diego, CA, April, 2008. [46] A. J. Folkes, K. Ahmadi, W. K. Alderton, S. Alix, S. J. Baker, G. Box, I. S. Chuckowree, P. A. Clark, P. Depledge, S. A. Eccles, L. Friedman, A. Hayes, T. C. Hancox,
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
[47]
[48] [49]
[50]
[51]
[52] [53]
[54]
[55]
[56] [57] [58] [59] [60] [61]
[62] [63]
[64] [65]
353
A. Kugendradas, L. Lensun, P. Moore, A. Olivero, J. Pang, S. Patel, G. Pergl-Wilson, F. Raynaud, A. Robson, N. Saghir, L. Salphati, S. Sohal, M. Ultsch, M. Valenti, H. Wallweber, N. Wan, C. Weisman, P. Workman, A. Zhyvoloup, M. Zvelebil and S. Shuttleworth, J. Med. Chem., 2008, 51, 5522. T. P. Heffron, M. Berry, G. Castanedo, C. Chang, I. Chuckowree, J. Dotson, A. Folkes, J. Gunzner, J. Lesnick, C. Lewis, K. Malesky, S. Mathieu, J. Nonomiya, A. Olivero, J. Pang, D. Peterson, L. Salphati, D. Sampath, D. Sutherlin, V. Tsui, M. Ultsch, N. Wan, S. Wang, C. Weismann, S. Wong and B. Zhu, MEDI-220, American Chemical Society National Meeting (ACS), Salt Lake City, UT, March 2009. M. Hayakawa, H. Kaizawa, H. Moritomo, T. Koizumi, T. Ohishi, M. Okada, M. Ohta, S. Tsukamoto, P. Parker, P. Workman and M. Waterfield, Bioorg. Med. Chem., 2006, 14, 6847. M. Hayakawa, H. Kaizawa, H. Moritomo, T. Koizumi, T. Ohishi, M. Yamano, M. Okada, M. Ohta, S. Tsukamoto, F. Raynaud, P. Workman, M. Waterfield and P. Parker, Bioorg. Med. Chem. Lett., 2007, 17, 2438. S. Patel, D. Sampath, M. Belvin, A. Brown, K. Edgar, L. Lensun, P. Moore, L. Salphati, H. Stern and L. Friedman, 217, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008. P. LoRusso, D. Sarker, D. Von Hoff, R. Tibes, M. K. Derynck, J. A. Ware, Y. Yan, G. D. Demetri, J. S. deBono and A. J. Wagner, 223, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008. T. C. Hancox, A. N. Pegg, C. M. Beswick, J. T. Blench, A. E. Dechaux, J. J. Kulagowski, J. A. Nadin and S. Price, WO Patent WO2008152390, 2008. G. Castanedo, R. Goldsmith, J. Gunzner, T. Heffron, S. Mathieu, A. Olivero, S. Staben, D. Sutherlin, V. Tsui, S. Wang, B. Zhu, T. Bayliss, I. Chuckowree, A. Folkes and N. Wan, WO Patent WO2008073785, 2008. T. Bayliss, I. Chuckowree, A. Folkes, S. Oxenford, N. Wan, G. Castanedo, R. Goldsmith, J. Gunzner, T. Heffron, S. Mathieu, A. Olivero, S. Staben, D. Sutherlin and B. Zhu, WO Patent WO2008070740, 2008. A. Folkes, S. Shuttleworth, I. Chuckowree, S. Oxenford, N. Wan, G. Castanedo, R. Goldsmith, J. Gunzner, T. Heffron, S. Mathieu, A. Olivero, S. Staben, D. Sutherlin and B. Zhu, WO Patent WO2007127175, 2007. P. Foster, C199, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, San Francisco, CA, October 2007. G. Shapiro, G. Edelman and E. Calvo, C205, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, San Francisco, CA, October 2007. W. Bajjalieh, L. C. Bannen, S. D. Brown, P. Kearney, M. Mac, C. K. Marlowe, J. M. Nuss, Z. Tesfai, Y. Wang and W. Xu, WO Patent WO2007044729, 2007. http://www.exelixis.com/pipeline_xl147.shtml D. Laird, B250, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, San Francisco, CA, October 2007. B. Markman, P. M. LoRusso, A. Patnaik, E. Heath, A. D. Laird, B. Van Leeuwen, K. P. Papadopoulos and J. Baselga, 216, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008. A. Patnaik, P. M. LoRusso and J. Tabernero, B265, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, San Francisco, CA, October 2007. K. P. Papadopoulos, B. Markman, J. Tabernero, A. Patnaik, E. I. Heath, A. DeCillis, D. Laird, S. K. Aggarwal, L. Nguyen and P. M. LoRusso, 3510, American Society of Clinical Oncology (ASCO), Chicago, IL, May 2008. http://www.exelixis.com/pipeline_xl765.shtml K. R. Auger, L. Luo, S. Knight, G. Van Aller, P. J. Tummino, R. A. Copeland, M. Diamond, D. Sutton, J. R. Jackson and D. Dhanak, 221, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008.
354 [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81]
[82]
[83] [84] [85] [86] [87] [88]
[89] [90] [91] [92] [93] [94]
[95]
[96]
John M. Nuss et al.
http://www.clinicaltrials.gov/ct2/show/NCT00695448 D. Kong and T. Yamori, Cancer Sci., 2007, 98, 1638. D. Kong, S. Yaguchi and T. Yamori, Biol. Pharm. Bull., 2009, 32, 297. D. Kong, M. Okamura, H. Yoshimi and T. Yamori, Eur. J. Cancer, 2009, 45, 857. S. Dan, H. Yoshimi, M. Okamura, Y. Mukai and T. Yamori, Biochem. Biophys. Res. Comm., 2009, 379, 104. T. Yamori and M. Okamura, 225, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008. D. Kong, H. Yoshimi and T. Yamori, 226, EORTC-NCI-AACR International Conference Molecular Targets Cancer Therapy, Geneva, October 2008. S. Butterworth, J.E. Griffen, B.G. Hill and M. Pass, WO Patent WO2008032036, 2008. S. Butterworth, J.E. Griffen, B.G. Hill and M. Pass, WO Patent WO2008032027, 2008. S. Butterworth, J.E. Griffen and M. Pass, WO Patent WO2008032077, 2008. S. Butterworth, J.E. Griffen and M. Pass, WO Patent WO2008032064, 2008. S. Butterworth, J.E. Griffen and M. Pass, WO Patent WO2008032091, 2008. S. Butterworth, J.E. Griffen, B.G. Hill and M. Pass, WO Patent WO2008032033, 2008. S. Butterworth, J.E. Griffen and M. Pass, WO Patent WO2008032028, 2008. S. Butterworth, J.E. Griffen and M. Pass, WO Patent WO2008032086, 2008. I. Seipelt, M. Gerlach, L. Blumenstein, G. Mueller, M. Teifel, E. Polymeropoulos and E. Guenther, 3706, Proceedings of the American Association for Cancer Research (AACR), Denver, CO, April 2009. I. Seipelt, S. Baasner, M. Gerlach, M. Teifel, J. Fensterle, L. Blumenstein, G. Mueller and E. Guenther, 3705 Proceedings of the American Association for Cancer Research (AACR), Denver, CO, April 2009. M. G. Buckley, T. Morgan and M.V. Sabin, WO Patent WO2008044022, 2008. P. Imbach, G. Stauffer, P. Furet and H-G. Capraro, WO Patent WO2008037477, 2008. S. Pecchi, Z. Ni, M. Burger, A. Wagman, G. Atallah, S. Bartulis, S. Ng, K. B. Pfister, A. Smith, Y, Zhang, H. Merritt and C. Voliva, WO Patent WO2008098058, 2008. S. D. Knight, C. A. Parrish, L. H. Ridgers, M. A. Sarpong and A. M. Chaudhari, WO Patent WO2009021083, 2009. L. M. Amzel, C.-H. Huang, D. Mandelker, C. Lengauer, S. B. Gabelli and B. Vogelstein, Nat. Rev. Cancer, 2008, 8, 665. B. J. Lannutti, S. A. Meadows, A. Kashishian, B. Steiner, S. May, A. J. Johnson, R. G. Ulrich, A. Yu, M. l. W. Gallatin, J. C. Byrd, K. D. Puri and N. A. Giese, Blood, 2008, 112(ASH Annual Meeting Abstracts), 16. C. Sadhu, K. Dick, J. Treiberg, C. G. Sowell, E. A. Kesicki and A. Oliver, US Patent, US6,518,277, 2003. C. Sadhu, K. Dick, J. Treiberg, C. G. Sowell, E. A. Kesicki and A. Oliver, US Patent US6,667,300, 2003. C. Billottet, V. L. Grandage, R. E. Gale, A. Quattropani, C. Rommel, B. Vanhaesebroeck and A. Khwaja, Oncogene, 2006, 25, 6648. http://www.clinicaltrials.gov/ct2/show/NCT00836914 http://www.clinicaltrials.gov/ct2/show/study/NCT00710528 S. E. May, A. Kashishian, T. S. Lin, J. A. Jones, J. M. Flynn, R. G. Ulrich, H. Chen, A. S. Yu, K. D. Puri, B. J. Lannutti, N. A. Giese, J. C. Byrd and A. J. Johnson, Blood, 2008, 112(ASH Annual Meeting Abstracts), 3165. H. Ikeda, T. Hideshima, Y. Okawa, S. Vallet, S. Pozzi, L. Santo, G. Gorgun, M. Fulciniti, N. S. Raje, G. Perrone, N. Munshi, P. Richardson, B. J. Lannutti, K. D. Puri, N. A. Giese and K. C. Anderson, Blood, 2008, 112(ASH Annual Meeting Abstracts), 2753. Y. Chen, T. D. Cushing, X. Hao, X. He, A. Reichelt, R. M. Rzasa, J. Seganish, Y. Shin and D. Zhang, WO Patent WO2008118454, 2008.
Emerging Therapies Based on Inhibitors of Phosphatidyl-Inositol-3-Kinases
355
[97] Y. Chen, T. D. Cushing, X. Hao, X. He, A. Reichelt, R. M. Rzasa, J. Seganish, Y. Shin and D. Zhang, WO Patent WO2008118455, 2008. [98] Y. Chen, T. D. Cushing, J. A. Duquette, F. Gonzalez Lopez de Turiso, X. Hao, X. He, B. Lucas, L. R. McGee, A. Reichelt, R. M. Rzasa, J. Seganish, Y. Shin and D. Zhang, WO Patent WO2008118468, 2008. [99] M. S. S. Palanki, E. Dneprovskaia, J. Doukas, R. M. Fine, J. Hood, X. Kang, D. Lohse, M. Martin, G. Noronha, R. M. Soll, W. Wrasidlo, S. Yee and H. Zhou, J. Med. Chem., 2007, 50, 4279. [100] W. Wrasidlo, J. Doukas, I. Royston, G. Noronha, J. D. Hood, E. Dneproyskaia, X. Gong, U. Splittgerber and N. Zhao, US Patent, US7,208,493, 2007. [101] J. Doukas, W. Wrasidlo, G. Noronha, E. Dneprovskaia, J. Hood and R. Soll, Biochem. Soc. Trans., 2007, 35, 204. [102] J. Doukas, W. Wrasidlo, G. Noronha, E. Dneprovskaia, R. Fine, S. Wels, J. Hood, A. DeMaria, R. Soll and D. Cheresh, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 19866. [103] http://www.clinicaltrials.gov/ct2/show/NCT00103350 [104] J. Doukas, L. Eide, K. Stebbins, A. Racaneli-Layton, L. Dellamary, M. Martin, E. Dneprovskaia, G. Noronha, R. Soll, W. Wrasidlo, L. M. Acevedo and D. A. Cheresh, J. Pharmacol. Exp. Ther., 2009, 328, 758. [105] http://clinicaltrials.gov/show/NCT00688714 [106] http://clinicaltrials.gov/show/NCT00853450 [107] S. P. Jackson, A. D. Robertson, V. Kenche, P. Thompson, H. Prabaharan, K. Anderson, B. Abbott, I. Goncalves, W. Nesbitt, S. Schoenwaelder, D. Saylik, WO Patent WO2004/ 016607, 2004. [108] S. A. Sturgeon, C. Jones, J. A. Angus and C. E. Wright, Eur. J. Pharmacol., 2008, 587, 208. [109] M. J. Zvelebil, M. D. Waterfield and S. J. Shuttleworth, Archiv. Biochem. Biophys., 2008, 477, 404. [110] M. Frazzetto, C. Suphiolu, J. Zhu, O. Schmidt-Kittler, I. G. Jennings, S. L. Cranmer, S. P. Jackson, K. W. Kinzler, B. Vogelstein and P. E. Thompson, Biochem. J., 2008, 414, 383. [111] C. Chaussade, G. W. Rewcastle, J. D. Kendall, W. A. Denny, K. Cho, L. M. Gronning, M. L. Chong, S. H. Anagnostou, S. P. Jackson, N. Daniele and P. R. Shepherd, Biochem. J., 2007, 404, 449. [112] J. Guillermet-Guibert, K. Bjorklof, A. Salpekar, C. Gonella, F. Ramadani, A. Bilancio, S. Meek, A. J. H. Smith, K. Okkenhaug and B. Vanhaesebroeck, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 8292. [113] R. Heller, Q. Chang, G. Ehrlich, S. N. Hsieh, S. M. Schoenwaelder, P. J. Kuhlencordt, K. T. Preissner, E. Hirsch and R. Wetzker, Cardiovasc. Res., 2008, 80, 96. [114] N. E. Torbett, A. Luna-Moran, Z. A. Knight, A. Houk, M. Moasser, W. Weiss, K. M. Shokat and D. Stokoe, Biochem. J., 2008, 415, 97. [115] P. K. Vogt, M. Gymnopoulos and J. R. Hart, Curr. Opin. Genet. Dev., 2009, 19, 12. [116] C. Benistant, H. Chapuis and S. Roche, Oncogene, 2000, 19, 5083. [117] M. Hayakawa, H. Kaizawa, K.-I. Kawaguchi, N. Ishikawa, T. Koizumi, T. Ohishi, M. Yamano, M. Okada, M. Ohta, S.-I. Tsukamoto, F. I. Raynaud, M. D. Waterfield, P. Parker and P. Workman, Bioorg. Med. Chem., 2007, 15, 403. [118] M. Hayakawa, K.-I. Kawaguchi, H. Kaizawa, T. Koizumi, T. Ohishi, M. Yamano, M. Okada, M. Ohta, S.-I. Tsukamoto, F. I. Raynaud, P. Parker, P. Workman and M. D. Waterfield, Bioorg. Med. Chem., 2007, 15, 5837. [119] M. Hayakawa, H. Kaizawa, K.-I. Kawaguchi, K. Matsuda, N. Ishikawa, T. Koizumi, M. Yamano, M. Okada and M. Ohta, US Patent US6,403,588, 2001. [120] J. D. Kendall, G. W. Rewcastle, R. Frederick, C. Mawson, W. A. Denny, E. S. Marshall, B. C. Baguley, C. Chaussade, S. P. Jackson and P. R. Shepherd, Bioorg. Med. Chem., 2007, 15, 7677.
356
John M. Nuss et al.
[121] M. Hayakawa, K.-I. Kawaguchi, H. Kaizawa, T. Koizumi, T. Ohishi, M. Yamano, M. Okada, M. Ohta, S.-I. Tsukamoto, F. I. Raynaud, P. Parker, P. Workman and M. D. Waterfield, Bioorg. Med. Chem., 2007, 15, 5837. [122] J. S. Chen, L. J. Zhou, M. Entin-Meer, X. Yang, M. Donker, Z. A. Knight, W. Weiss, K. M. Shokat, D. Haas-Kogan and D. Stokoe, Mol. Cancer Ther., 2008, 7, 841. [123] N. D. Adams, J. L. Burgess, A. M. Chaudhari, S. D. Knight and C. A. Parrish, WO Patent WO2008150827, 2008. [124] N. D. Adams, J. L. Burgess, M. G. Darcy, S. D. Knight, K. A. Newlander, L. H. Ridgers and S. J. Schmidt, WO Patent WO2008157191, 2008. [125] N. D. Adams, D. Dhanak, S. D. Knight, L. Schaller and J. Tang, WO Patent WO2008014219, 2008. [126] V. Pomel, J. Klicic, D. Covini, D. D. Church, J. P. Shaw, K. Roulin, F. Burgat-Charvillon, D. Valognes, M. Camps, C. Chabert, C. Gillieron, B. Franon, D. Perrin, D. Leroy, D. Gretener, A. Nichols, P. A. Vitte, S. Carboni, C. Rommel, M. K. Schwarz and T. Ru¨ckle, J. Med. Chem., 2006, 49, 3857. [127] Z. A. Knight and K. M. Shokat, Biochem. Soc. Trans., 2007, 35, 245. [128] T. Rueckle, X. Jiang, P. Gaillard, D. Church and T. Vallottton, WO Patent WO04007491, 2004. [129] G. De Luca, WO Patent WO04006916, 2004. [130] M. Camps, T. Ru¨ckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, C. Ferrandi, C. Chabert, C. Gillieron, B. Franc- on, T. Martin, D. Gretener, D. Perrin, D. Leroy, P.-A. Vitte, E. Hirsch, M. P. Wymann, R. Cirillo, M. K. Schwarz and C. Rommel, Nat. Med., 2005, 11, 936. [131] A. M. Condliffe, K. Davidson, K. E. Anderson, C. D. Ellson, T. Crabbe, K. Okkenhaug, B. Vanhaesebroeck, M. Turner, L. Webb, M. P. Wymann, E. Hirsch, T. Ru¨ckle, M. Camps, C. Rommel, S. P. Jackson, E. R. Chilvers, L. R. Stephens and P. T. Hawkins, Blood, 2005, 106, 1432. [132] K. Ito, G. Caramori and I. M. Adcock, J. Pharmacol. Exp. Ther., 2007, 321, 1. [133] D. F. Barber, A. Bartolome, C. Hernandez, J. M. Flores, C. Redondo, C. FernandezAria, M. Camps, T. Ru¨ckle, M. K. Schwarz, S. Rodrı´guez, C. Martinez-A, D. Balomenos, C. Rommel and A. C. Carrera, Nat. Med., 2005, 11, 933. [134] T. B. Lanni, Jr., K. L. Greene, C. N. Kolz, K. S. Para, M. Visnick, J. L. Mobley, D. T. Dudley, T. J. Baginski and M. B. Liimatta, Bioorg. Med. Chem. Lett., 2007, 17, 756. [135] T. Crabbe, Biochem. Soc. Trans., 2007, 35, 253. [136] N. C. Barvian, C. N. Kolz, K. S. Para, W. C. Patt and M. Visnick, WO Patent WO2004052373, 2004. [137] R. D. Gogliotti, K. L. Muccioli, K. S. Para and M. Visnick, WO Patent WO2004056820, 2004. [138] M. Shimada, T. Murata, K. Fuchikami, H. Tsujishita, N. Omori, I. Kato, M. Miura, K. Urbahns, F. Gantner and K. Bacon, WO Patent WO04029055, 2004.
CHAPT ER
18 The Anti-Infective and Anti-Cancer Properties of Artemisinin and its Derivatives Christopher Paul Hencken*, Alvin Solomon Kalinda* and John Gaetano D’Angelo**
Contents
1. Introduction 2. Antiparasitic Uses of Artemisinin 2.1 Antimalarial activity 2.2 Anti-toxoplasmosis activity 2.3 Anti-leishmaniasis activity 2.4 Anti-shistosomiasis activity 2.5 Anti-trypanosomiasis activity 3. Other Therapeutic Uses of Artemisinin 3.1 Antiviral activity 3.2 Antifungal activity 3.3 Anticancer activity 4. Toxicity 4.1 Evidence in favor of safety 4.2 Evidence of neurotoxicity 5. Conclusion References
359 360 360 365 367 368 369 370 370 371 372 373 374 374 375 375
1. INTRODUCTION Artemisinin, a 1,2,4-trioxane sesquiterpene lactone with an atypical endoperoxide moiety, was isolated as the active component from the * Johns Hopkins University, 3400N. Charles Street, Baltimore, MD 21218, USA ** Alfred University, 1 Saxon Drive, Alfred, NY 14802, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04418-2
r 2009 Elsevier Inc. All rights reserved.
359
360
Christopher Paul Hencken et al.
Chinese medicinal plant commonly known as Qinghaosu (Artemisia annua) [1,2] and was found to be extremely active against the deadly cerebral form of malaria. While it has become the new cornerstone of antimalarial treatment, it has biological applications that reach far beyond this one disease. These applications include activity against toxoplasmosis, leishmaniasis, viral infections, certain bacterial infections, shistosomiasis, and cancer. The use of artemisinin and its derivatives as a broad-spectrum anti-infective agents was recently patented [3]. It has been shown that the active pharmacophore is the 1,2,4-trioxane, specifically (and perhaps strangely), the peroxide unit [4]. In this chapter, we have paid particular attention to the activity of artemisinin-based compounds beyond the well-documented area of malaria, while providing a cursory overview of their antimalarial properties. For the interested reader, we suggest some recent reviews on the antimalarial activity of artemisinin and its derivatives [5–7]. For the purposes of saving space, we will use the following short hand for the structural core of artemisinin (1) throughout the chapter. H O H
O O O C-10
O C-10
O
O
1
1
2. ANTIPARASITIC USES OF ARTEMISININ The use of herbal teas brewed from A. annua against fever and chills dates to AD 340; however, the first mention of its use dates as far back as 168 BC [8]. Since that time, artemisinin has been identified as the active constituent of A. annua in 1972 [4]. More recently, the uses of artemisinin have been shown to be quite vast. The wide therapeutic applications of artemisinin make it difficult to attribute artemisinin’s effectiveness to a common mechanism. Since intra-cellular/parasitic metabolic degradation of artemisinin is known to induce the formation of oxygen and carbon-centered radicals, radical-induced cell death is certainly one attractive possibility. Despite the increasing known breadth of activity, the most popular uses
Anti-Infective and Anti-Cancer Properties of Artemisinin
361
remain the antiparasitic uses. These uses, though, are not necessarily limited to antimalarial (Section 2.1 for which artemisinin has been made famous), anti-toxoplasmosis (Section 2.2), anti-leishmaniasis (Section 2.3) anti-shistosomiasis (Section 2.4), and anti-trypanosomiasis (Section 2.5).
2.1 Antimalarial activity Malaria (from Italian origin, ‘‘aria male,’’ meaning bad air) is caused by an erythrocytic protozoan parasite first identified in 1880 by Alfonse Laveran [7]. Every year, between 300 and 500 million people are infected with malaria in the endemic areas (Africa, India, southeast Asia, the Middle East, Oceania, and Central/South America) with 1–2 million of these infected people dying each year and a child dying of malaria approximately every 30 s. Human malaria is caused by four species of the genus Plasmodium; vivax, ovale, malariae, and falciparum. The falciparum species is responsible for the majority of human deaths from malaria. Humans contract malaria when bitten by the female of any one of the 60 species of Anopheles mosquito [9]. The life cycle of the parasite from mosquito to human blood, to the human liver, back to the blood, and back to another mosquito is well-known [9,10]. The antimalarial mechanism of action for the artemisinin class of compounds is the most extensively studied of its mechanisms of action. It is possible, though unproven, that all of the potential mechanisms, two prevailing hypotheses presented below, are at work. It is now widely believed that the 1,2,4-trioxane class of antimalarials, including artemisinin, exert their activity on the erythrocytic stage of the parasite life cycle. Additionally, the fact that the endoperoxide is vital for artemisinin’s antimalarial activity has been well-established [4]. The most widely agreed-upon view is that liberated heme acts as the source of FeII responsible for the activation of the endoperoxide bridge of artemisinin (1) to produce cytotoxic radical species. The liberated heme results from the breakdown of hemoglobin that Plasmodium uses as a food source. The hemoglobin is transported to the parasitic food vacuole where it is digested becoming amino acids essential to parasite life and the aforementioned free heme that is toxic to the parasite. The parasite then detoxifies the free heme by converting it to hemazoin, a polymeric form of heme. It is believed that this potentiated heme in the form of hemazoin is what activates the artemisinin family of compounds. The activation cascade is initiated by reduction of the peroxide bond with heme FeII to give an oxy radical and oxidized heme FeIII that can lead to a number of subsequent radical entities, all of which may be lethal to the parasite. This mechanism has been called into question by others who believe artemisinins disrupt parasitic calcium homeostasis by targeting sarco/endoplasmic reticulum Ca+2-ATPases (SERCAs) [11,12]. Artemisinin
362
Christopher Paul Hencken et al.
and thapsigargin, a sesquiterpene lactone with structural similarities to artemisinin and known SERCA inhibitor, were shown to be similarly potent as inhibitors of a P. falciparum SERCA ortholog (PfATP6) in Xenopus oocytes. However, this has been contested by the Posner lab with their demonstration that enantiomers of a fully synthetic trioxane had the same level of activity [13]. Were the SERCA enzymes the primary target, one would expect only one of the enantiomers to be active as is seen in other enzyme-based mechanisms. Additionally, the epimers of artemether (3) showed nearly equal in vivo activity (1.02 mg/kg of body weight for the a epimer vs. 1.42 mg/kg of body weight for the b epimer) [14] furthering the heme-based mechanism over the SERCA-based mechanism where a greater disparity in the activities would be expected. Some final points regarding the mechanism bear mentioning here. First, it was recently shown [15] that there exists a strong correlation between a set of fully synthetic trioxolanes’ ability to alkylate heme in the presence of FeII and their respective in vitro antimalarial activity. Curiously, artemisinin-derived derivatives were less potent heme-alkylators, suggesting possible alternative modes of action for the more structurally complex artemisinin derivatives. Another study [16] found that endoperoxides tagged with a fluorescent label localized within the parasitic digestive vacuole. It was proposed that heme-iron activation then followed, allowing parasite membrane damage to occur. Despite this promising progress, more work must be done to fully elucidate the mechanism of these compounds. For a more detailed discussion, we direct the interested reader to work done by the Posner lab and others [13,16], which supports this accepted mechanism, especially evidence of the alkylation of heme by artemisinin [17], even in malaria-infected mice [18], and those that argue against it, especially the work by the Haynes lab [5,6,19–21]. Despite the debate that still surrounds the mechanism of action, this family of compounds has displayed and continues to display an exceptional safety profile and very rapid, high levels of activity. However, artemisinin and dihydroartemisinin (DHA) (2) do suffer drawbacks such as low bioavailability and recrudescence of the disease. Even with these drawbacks, they are especially effective against severe cerebral malaria, one of the most lethal forms of the disease. Arteether (4), the ethyl ether of DHA, was designed with the intent on optimizing the lipophilicity of artemisinin to improve passive permeability through cell membranes and improve bioavailability. This is especially important in the treatment of cerebral malaria where the treatment needs to permeate the bloodbrain barrier (BBB). The BBB requires compounds to be fairly lipophilic with an octanol-water partition coefficient, or logP, value of W2. That is to say that the compound in question is two times more soluble in octanol than it is in water. Arteether, logP of 3.99, is more lipophilic than the parent artemisinin, logP of 2.94, and therefore is expected to be more effective against cerebral malaria. Arteether is also fast-acting and effective
Anti-Infective and Anti-Cancer Properties of Artemisinin
363
against drug-resistant strains of Plasmodium. Notably, the World Health Organization selected arteether as an emergency treatment for individuals infected with malaria. Sodium artesunate, the sodium salt of artesunic acid (5), is well-tolerated and less toxic than (1) and along with (5) represents an example of one of the few water-soluble derivatives [22].
O C-10 O
O C-10
O OR 2 R=H 3 R = CH3 4 R = CH2CH3
HO O 5
To combat the shortcomings of first generation endoperoxides such as metabolic instability, short plasma half-life, poor bioavailability, and chemical instability, a new generation of molecules has been designed. One new generation of molecules, the C-10 carba analog class, replaces the exocyclic C-10 carbon–oxygen bond with a carbon–carbon bond, resulting in derivatives with no exocyclic oxygen group at C-10. The range of C-10 analogs is large and varies from fluorinated substituents [23] to amino sulfones [24] to differing-length linked dimers [25]. Begue’s lab sought to improve the activity of artemisinin by the addition of fluorinated alkyl groups to a variety of artemsinins. The most active of these derivatives, (6), substitutes a trifluoromethyl group for the C-10 hydrogen of (2) creating a trifluoromethyl alcohol moiety. When tested in vitro against the D6 and W2 strains of falciparum malaria, (6) was active at very low levels, 2.6 and 0.9 nM, respectively. When (6) was given subcutaneously to P. bergei-infected mice, ED50 was 0.7 mg/kg and ED90 was 1.8 mg/kg, but when given orally, the ED50 and ED90 values were 4.3 and 13.0 mg/kg respectively. The oral values were not significantly better than those for sodium artesunate (5.4 mg/kg for ED50 and 15.3 mg/kg for ED90) while those for the subcutaneous route of administration were appreciably better than those for sodium artesunate (2.8 mg/kg for ED50 and 10.4 mg/kg for ED90) [23].
O C-10
OH
F3C 6
The Haynes lab prepared and tested several C-10 nitrogen derivatives against P. bergei of which (7) demonstrated the best mix of lipophilicity
364
Christopher Paul Hencken et al.
(logP of 2.49), ease of preparation (crystalline compound that is easily purified), safety parameters (considered ‘‘nontoxic’’ by the author), and activity. The activity of (7) was tested against chloroquine-sensitive P. bergei-infected and chloroquine-resistant P. yoelii-infected mice. When administered subcutaneously, the ED90 values were 1.5 and 3.9 mg/kg, respectively. When given orally to the mice the values were 3.1 and 5.0 mg/kg, respectively [24].
O
C-10
N
S O
O 7
The in vivo mouse activity results for several C-10 isobutylene-linked dimers prepared by the Posner lab showed these analogs to be orally ‘‘curative’’ 30 days post-infection at doses of 3 30 mg/kg. Eight derivatives were shown to double the lifetime of the animals relative to the control animals at 3 10-mg/kg doses. Shown below are the most active derivatives prepared and tested by the Posner lab to date. Both (8, 9) were curative at 3 30 mg/kg, while 3 10 mg/kg dosing of (8) displayed 17.7 days average survival post-infection and 3 10 mg/kg dosing of (9) displayed 16.3 days average survival post-infection. Control animals (drug-delivery vehicle only) survived 6–7 days in both dosing groups. Animals given (1) survived 7.2 days at 30 mg/kg and 6.5 days at 10 mg/kg [25].
O C-10
O C-10
O C-10
O C-10
O O O
N O
N H N
8
9
365
Anti-Infective and Anti-Cancer Properties of Artemisinin
Curiously, a family of hybrid analogs related to (10), which incorporate a traditional antimalarial drug into a hybrid molecule with (1), showed moderate increases in activity compared to (1), but less activity than (3) [26]. This is somewhat surprising when it is considered that these drugs should have the potential for multiple mechanisms of action. It is possible that the parasite was able to pump the compound out of the cell due to the presence of the chloroquine unit based on the accepted mechanism of malaria resistance to chloroquine.
O C-10 O O N O C-10
Cl
HN
H N
H N
HO
N 3
3
N
O N
O
10
Cl
N chloroquine
11
In another recent study, an artemisinin-quinine hybrid was found to have potent antimalarial activity with IC50 values ranging from 8.95 nm to 10.4 nM against various strains of P. falciparum [27]. Notably, hybrid analog (11) was found to have more potent activity than quinine or artemisinin alone and, even more importantly, more potent activity than a 1:1 molar concentration of artemisinin and quinine combination treatment. Together, these studies make the important point that artemisininbased compounds can be chemically combined with other drugs leading to increased biological activity compared to combined treatment with two separate drugs. Due to the fact that combination therapy has become the recommended form of treatment for malaria, HIV, cancer, and many other diseases, artemisinin’s tolerance of such incorporation is highly important. Furthermore, with the fact that combination therapy often involves a complicated pill-taking regimen, a molecule that incorporates both drugs is an important simplification of treatment that will lead to less drug failure due to poor patient compliance. As an added benefit, as with (11), these hybrid analogs have been shown to possess better activity than even a 1:1 molar concentration treatment of the two drugs alone.
366
Christopher Paul Hencken et al.
2.2 Anti-toxoplasmosis activity Toxoplasma gondii is an apicomplexan protozoan parasite that infects humans and has been linked to chronic neuropsychiatric diseases and behavioral abnormalities [28]. Toxoplasmosis is most commonly transmitted by consumption of undercooked, contaminated meats; contaminated water; or contact with feces from an infected cat. In most immuno-competent humans, the disease is asymptomatic; however, there are serious fetal complications if acquisition of the disease occurs near conception. In immunocompromised individuals, such as HIV patients or organ transplant patients, systemic infection and widespread organ damage can occur [29]. The first reported activity of artemisinin against T. gondii was in the early 1990s by Ou-Yang and co-workers [30]. More recently, new C-10 carba derivatives such as (12) have been prepared by the Posner lab demonstrating an IC50 ¼ 1.2 mM [31]. An additional study by the same lab revealed multiple potential mechanisms of action. In this work [32], derivatives of artemisinin (13, 14) were found to not only inhibit growth (IC50 ¼ 1.0 and 1.7 mM, respectively) of the parasite but also result in parasite death and prevent entry of the parasite into the cells. Of particular note, a derivative lacking the endoperoxide (15) was found to be more effective at inhibiting entry into the cell by the parasite than its peroxide-containing analog. This result suggests, especially when it is considered that this same nonperoxide derivative showed no growth inhibition and no parasite death, that entry inhibition may not be dependent on the peroxide while growth inhibition clearly is. H no peroxide O
O C-10
H
O O
Br
O C-10
O C-10
O C-10 O S
N
S
N
O
12
13
14
15 inactive
At this time, no mechanism of action for the observed T. gondii activity has been proposed. However, recent studies have implicated calcium homeostasis as a possible mechanism of action of artemisinin against apicomplexa through its interaction with SERCA-type Ca2+
Anti-Infective and Anti-Cancer Properties of Artemisinin
367
ATPase [33], similar to what has been proposed for malaria. However, it is also believed that activation of the peroxide by ferrous iron is essential and that the likely source of this ferrous iron is heme. Given the close proximity of the molecule to heme upon activation, it has also been argued that heme alkylation may be the mechanism of action, especially with respect to malarial activity, as described earlier. This is a less likely mechanism in the case of T. gondii, which perhaps is explained by the SERCA-based mechanism. To date, no study has been done that investigates the relative effectiveness of two enantiomers for T. gondii like was done for malaria disallowing for the same counterargument made with malaria. More work must be done to fully elucidate the mechanism of action against this target. It is possible, though thus far unproven, that these two similar parasites share a biological target for artemisinin. It is appropriate to point out here that artemisinin is not the only peroxide-containing molecule that has shown activity against T. gondii or malaria. For example, see the work by Chang and co-workers [34] and the work of Vennerstrom [15] who demonstrated that fully synthetic peroxides (16, 17) also possess activity against Toxoplasma and malaria, respectively. The results of Chang’s study [34] suggest that 1,2,4-trioxanes were able to block nucleotide synthesis of intracellular parasites. While it is possible that artemisinin-derived analogs have the same mode of action; there is currently no proof this is the case. O
Ph
Ph
O
O
Ph
O
O
Ph
O
O
16
17
IC50 = 5.98 µM
IC50 = 71.7 µM
2.3 Anti-leishmaniasis activity Leishmaniasis is a widespread disease that takes three major forms in humans: cutaneous leishmaniasis, mucocutaneous leishmaniasis, and the potentially lethal visceral leishmaniasis. All of these forms are caused by various protozoan parasites of the genus Leishmania and are transmitted by the female sand fly. Most of the visceral leishmanaiasis cases have been related to HIV infections [35]. Similar to the antimalarial activity, a wide variety of derivatives of artemisinin have been prepared to target
368
Christopher Paul Hencken et al.
leishmaniasis, with substitutions on different regions of the artemisinin parent molecule. All of the derivatives presented here are known to also possess antimalarial activity. CF3 Cl
O C-10
O C-10
CF3
Cl O
19
18
CF3
O C-10
O 20
Several derivatives (18–20) have been reported to have quite potent activity against Leishmania, notably (19), whose IC50 is a very impressive 0.3 mM [36].
2.4 Anti-shistosomiasis activity Schistosoma mansoni and other schistosoma flatworms are the causative agents for schistosomiasis. The schistosomas are ordinarily located in the blood vessels of the human host. Although S. mansoni is the most widespread schistosoma, others such as Schistosoma japonicum and Schistosoma haematobia are also known. The parasite is contracted by humans through contact with infected water by way of direct penetration of the human skin. After in vivo maturation, severe cases give rise to fibrosis of the liver and hepatosplenomegaly. Artemisinin derivatives, especially (3), are known to have activity against all of the schistosomas [37]. A recent clinical study in Sudan, where artesunate–sulfamethoxypyrazine–pyrimethamine or artemether-lumefantrine combinations were administered to patients co-infected with P. falciparum and S. mansoni, showed complete clearance of both parasites [38]. Curiously,
Anti-Infective and Anti-Cancer Properties of Artemisinin
369
a separate study found a lower cure rate for artesunate–sulfadoxide– pyrimethamine combinations compared to praziquantel alone [39]. In another study where schoolchildren infected with Schistosoma haematobium were treated with artesunate or praziquantel, both drugs demonstrated reduced egg counts in patients at a single dose of 40 mg/kg of praziquantel and 20 mg/kg artesunate. However, praziquantel was found to perform better [40], consistent with the aforementioned results [39].
2.5 Anti-trypanosomiasis activity The two most common human-afflicting trypanosomal infections are American and African trypanosomiasis. The American type, Trypanosoma cruzi, more commonly known as Chagas disease (named for Carlos Chagas, its discoverer in 1909), is passed from the feces of triatomine bugs to humans and affects 8–11 million people in South and Central America and Mexico. Many infected individuals are unaware of their infection and if left untreated will be lifelong and possibly fatal. The triatomine bugs are commonly found in earthen-made structures common in the rural regions of the affected countries. The African type, more commonly known as African sleeping sickness, is further distinguished based on the region of Africa where the infection was contracted. Trypanosoma brucei rhodesiense is responsible for East African sleeping sickness and Trypanosoma brucei gambiense is responsible for the West African form. Both types of African sleeping sickness are contracted from the painful bite of the honeybee-sized tsetse fly with 50,000–70,000 cases reported annually. T. b. gambiense is responsible for the majority of the African cases [41,42]. A recent report demonstrated the efficacy of (1, 2, 7, 21) against T. b. rhodesiense, T. cruzi, and L. donovani in vitro.
O
C-10
O
C-10
N
S O
O
7
F
21
370
Christopher Paul Hencken et al.
Both (7) and (21) exhibited IC50 values similar to that of (1) and (2) against both trypanosomes tested. The demonstrated IC50 values for (1), (2), (7), and (21) against T. cruzi were 13.4, 12.8, 23.3, and 17.9 mM, respectively. Likewise, the values against T. b. rhodesiense were 20.4, 24.6, 22.5, and 15.7 mM, respectively [43]. Recently, hybrid derivatives have been reported that incorporate either a moiety that permits targeted delivery of the drug (22, 23) or a second active unit with an ideally different mechanism of biological activity (24, 10). MeO NH2 N
O
N
O C-10
O N
N H2N
N
N H
O
X
22 X = O 23 X = NH
O
N
C-10 O
24 Cl
Chollet et al. recently demonstrated the superior activity of (22) and (23), compared to artesunate against all evaluated strains of Trypanosoma brucei [44]. T. brucei is known to take up diamidine, thus the superior biological activity of the diamidine-containing (22) and (23) was speculated to be due to targeted delivery of the artemisinin-based drug. However, no direct evidence of such an increase in delivery was provided.
3. OTHER THERAPEUTIC USES OF ARTEMISININ Although the antiparasitic uses of artemisinin are much more common, there are other uses that are becoming more prevalent. For example, antiviral (Section 3.1), antifungal (Section 3.2), and anticancer (Section 3.3) properties are discussed here. Although the anticancer activity is mentioned rather briefly here, the anticancer properties of this family of compounds are at least as well-documented as each of the antiinfective properties, with the only exception being malaria.
3.1 Antiviral activity One of the earliest reports of artemisinin’s antiviral activity came in the early 1980s [45]. Sodium artesunate has been shown to inhibit the human
Anti-Infective and Anti-Cancer Properties of Artemisinin
371
cytomegalovirus (HCMV) (IC50 ¼ 5.9 mM against the AD169 strain with 91% inhibition at 15 mM) [34] and the Herpes simplex virus type 1 (93% inhibition at 15 mM for a clinical isolate, no IC50 reported) [46]. It was found that sodium artesunate inhibited central regulatory processes of HCMV-infected cells such as activation pathways dependent on NF-kB or Sp1 [46]. This was suggested to interfere with critical host-cell-type interactions and metabolism requirements for viral replication. However, since other sesquiterpene lactones have shown similar anti-HCMV activity [47,48], this suggests it may not be the endoperoxide that is responsible for the activity. It should be stated, however, that no mechanism of action has been elucidated to date and that although artemisinin does contain a lactone, sodium artesunate does not. Sodium artesunate has also been found to inhibit the Epstein–Barr virus [49]. Furthermore, sodium artesunate was found to be active at 600 nM concentration against both the M-tropic and the T-tropic HIV-1 strains [46]. Although this concentration is quite high, given the relative safety of the artemisinin family of drugs, even treatment at this high concentration may be well-tolerated. Considering that this is thus far an unused method of HIV treatment, the possibility becomes more attractive. Curiously, a separate study investigating patients with HIV/malaria co-infection not only showed delayed clearance of P. falciparum but no report of anti-HIV activity for artemisinin [50]. It was also found that hepatitis B virus (HBV) DNA release was inhibited by sodium artesunate at an IC50 of 0.5 mM with host cell viability being reduced at 20 mM, and the compound also showed activity against HBV at W10 mM. Meanwhile, artemisinin was found to inhibit hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two HCV subgenomic replicon constructs at concentrations that did not affect the host cells. The reader is referred to recent broad reviews [47–49] and a recent patent [51], describing the antiviral activity of artemisinin and its semisynthetic derivatives, for more information and additional references.
3.2 Antifungal activity There have been several accounts of artemisinin derivatives possessing antifungal activity. For example [52], it has been shown that a-arteether (4) inhibits various genotypes of the EG-1-103 and F-400 strains of Saccharomyces cerevisiae at minimal inhibitory concentrations of 2.0 and 1.0 mg/mL respectively. Furthermore in another study [53], a wide variety of artemisinin-derived compounds (25–27) displayed activity against the yeasts Canadida albicans and Cryptococcus neoformans at IC50 values ranging from 0.045 mg/mL to 30 mg/mL. Curiously, a deoxo derivative (27) related to 15 also displayed marginal activity, suggesting
372
Christopher Paul Hencken et al.
the peroxide may not be the active pharmacophore in this application. Generally, the activity against C. albicans was far weaker than C. neoformans and in some cases was completely absent.
O
H
O
O
O
O
H
O H
O
HO HO 25
26
27
It was also recently discovered [54] that artemisinin displayed growth inhibitory properties against a set of isogenic S. cerevisiae strains that carried disruptions of the major multidrug ABC transporter genes in the multidrug-resistant PDR1-3 background. In this study, a synergistic effect was observed between artemisinin and ketoconazole where genotypes (PDR1-3 and PDR1) that displayed minimum inhibitory concentrations (MICs) W200 mg/mL with artemisinin alone displayed MICs of 25 and 3 mg/mL, respectively, when artemisinin was employed to potentiate ketoconazole. Against two other strains (D5 and D1D2D5), artemisinin alone possessed an MIC of 50 mg/mL. To date, there is no clear explanation that accounts for the antifungal activity of the artemisinin family of compounds.
3.3 Anticancer activity Nearly 1.5 million Americans were diagnosed with cancer in 2008 with more than one-third of them dying from the disease. Cancer (sarcoma, carcinoma, leukemia, lymphoma/myeloma, and central nervous system cancer) treatments have come to include radiation therapy, chemotherapy, surgery, and other treatment methods including anti-angiogenesis therapy, gene therapy, and hyperthermia [55]. The anticancer properties of artemisinin have been under in vivo investigation since the 1980s. Anfosso et al. demonstrated a correlation between angiogenesis-related genes and the cellular response to artemisinins [56]. They found many angiogenesisregulating factors among their panel such as, vascular endothelial growth factor-C (VEGFC), fibroblast growth factor-1 (FGF1), matrix metalloproteinase-9 (MMP9), thrombospondin-1 (THBS1), hypoxia-inducing factor-a (HIF1A), and angiogenin (ANG) [57–59]. Artemisinin has also been found
Anti-Infective and Anti-Cancer Properties of Artemisinin
373
to inhibit proliferation, migration, and tube formation in human umbilical vein endothelial cells (HUVEC); inhibit VEGF binding to surface receptors on HUVEC; and reduce expression of VEGF receptors on HUVECs [60– 62]. Artesunate has been shown to play other roles in the control of cancer such as inducing apoptosis and oncogene deactivation/tumor suppressor gene activation [63]. The reader is encouraged to read the review by Krishna for further discussion of this topic [64].
O C-10
O C-10 O O
O P OR
28 R = Me 29 R = Ph
Two of a series of bis-trioxane dimers (28, 29) were shown to be more potent against cancer, in vitro, than doxorubicin, a currently used cancer chemotherapy agent. It was noted that all the cell lines sensitive to (28) and (29) all overexpressed transferrin receptors [65]. Based on that information, nuclear iron-dependent activation to free radical species that damage DNA was proposed to be involved in the tumoricidal mechanism of action [66]. To further the idea that transferrin receptors are a likely target, a series of artemisinin–transferrin conjugates were prepared and tested against the prostate cancer cell line DU 145. These conjugates were found to be cytotoxic to the cell line through a transferrin receptor–dependent induction of apoptosis [67]. Currently, it is unknown whether the anticancer properties of artemisinin derivatives are due to cytotoxicity against the cancer cells, antiangiogenesis properties, or some combination of both. Other recent work has shown that artemisinin-acridine hybrids such as the aforementioned (23) were found to have a cytotoxicity profile against several cancer cell lines, with IC50 values four times lower than DHA in several derivatives [68], demonstrating the widespread applicability of this concept of hybrid drug analogs.
4. TOXICITY Great debate has raged on the topic of toxicity for this compound class. While generally characterized as having excellent tolerability and
374
Christopher Paul Hencken et al.
safety [69], some adverse affects, particularly in non-human mammals, have been observed.
4.1 Evidence in favor of safety A review of clinical trials [70] showed that together, 9% of patients showed adverse drug reactions. These reactions included neutropenia, reduced reticulocyte count, anemia, eosinophilia, acute haemolysis, elevated aspartate aminotransferase, ECG abnormalities (w/o clinical effect), transient bradycardia, prolongation of the QTc interval, prolonged PR interval (first-degree atrioventricular block), atrial extrasystoles, and non-specific T-wave changes. In all cases, the effects were independent of the artemisinin derivative and route of administration. An additional study [71] showed that when treating uncomplicated falciparum malaria with artemisinin derivatives alone, substantially fewer side effects were observed than with mefloquine-containing combination therapies. Notably, there was significantly less nausea, vomiting, anorexia, and dizziness. Furthermore, studies have shown no adverse effects on mother or fetus if artesunate or artemether are used to treat acute falciparum at various stages of gestation [72] or during breast feeding [69]. However, this matter can hardly be considered closed. Although several studies have been done and no evidence of adverse effects have been observed, the sample size has thus far been small [73], and complications (most notably, a high percentage of post-implantation losses) have been observed at doses of 35 and 75 mg/kg in Wistar rats, especially when administered early in the pregnancy [74]. Meanwhile, another study using Wistar rats and considerably lower doses of artemether (3.5 and 7 mg/kg) during blastogenesis, organogenesis and fetal period had no adverse effects other than reduction in fetal body weight and pre-term delivery in 3/10 rats at the 7-mg/kg dose. Some fetal growth retardation without incidence of malformations was also noted in the study [75].
4.2 Evidence of neurotoxicity It is important to note that dogs [76], mice, rats, and Rhesus monkeys [77–79] have all demonstrated evidence of neurotoxicity apparently caused by artemisinin derivatives. Specifically, gait disturbances, loss of spinal and pain response reflexes, cardio respiratory depression, and even death have been documented. In mice, balance was damaged irreversibly and death also occurred. Importantly, it was found that intramuscular administration was found to be more toxic than oral [69]. In humans, even when adverse neurological effects have been documented, the effects resolved with time after the treatment was
Anti-Infective and Anti-Cancer Properties of Artemisinin
375
stopped [71]. These effects included problems with co-ordination, fine finger dexterity, hearing, nystagmus, and balance. For more information concerning the toxicity of this important class of compounds, we direct the interested reader to a relatively recent review [80]. An explanation for the apparent lack of a correlation between animal toxicity and human toxicity is still lacking and is urgently needed.
5. CONCLUSION The artemisinin family of compounds has become more important in recent years. The very diverse biological activity and relative safety of this family of drugs make it important in the global fight against many diseases. In the coming years, it is anticipated that this exotic natural product will become even more important to the chemotherapy of various diseases, perhaps even above and beyond those mentioned here.
REFERENCES [1] J. M. Liu, M. Y. Ni, J. F. Fen, Y. Y. Tu, Z. H. Wu, Y. L. Wu and W. S. Zhou, Huaxue Xueabo, 1979, 37, 129. [2] D. L. Klayman, A. J. Lin, W. Acton, J. P. Scovill, J. M. Hoch, W. K. Milhous and A. D. Theoharides, J. Nat. Prod., 1984, 47, 715. [3] M. A. Avery and K. M. Muraleedharan, International patent 03/095444A1 2003. [4] D. L. Klayman, Science, 1985, 228, 1049. [5] R. K. Haynes and S. Krishna, Microbes Infect., 2004, 6, 1339. [6] S. Krishna, L. Bustamante, R. K. Haynes and H. M. Staines, Trends Pharmacol. Sci., 2008, 29, 520. [7] N. J. White, Science, 2008, 320, 330. [8] T. Efferth, Planta Med., 2007, 73, 299. [9] www3.niaid.nih.gov/topics/Malaria/understandingMalaria/facts.htm [10] www.cdc.gov/malaria/faq.htm [11] R. K. Haynes, D. Monti, D. Taramelli, N. Basilico, S. Parapini and P. Olliaro, Antimicrob. Ag. Chemother, 2003, 47, 1175. [12] U. Eckstein-Ludwig, R. J. Webb, I. D. A. vanGoethem, J. M. East, A. G. Lee, M. Kimura, P. M. O’Neill, P. G. Bray, S. A. Ward and S. Krishna, Nature, 2003, 424, 957. [13] P. M. O’Neill, S. L. Rawe, K. Borstnik, A. Miller, S. A. Ward, P. G. Bray, J. Davies, C. H. Oh and G. H. Posner, ChemBioChem, 2005, 6, 2048. [14] Chinese-Cooperative-Research-Group, J. Trad. Chin. Med., 1982, 2, 31. [15] D. J. Creek, W. M. Charman, F. C. K. Chiu, R. J. Prankerd, Y. Dong, J. L. Vennerstrom and S. A. Charman, Antimicrob. Ag. Chem., 2008, 52, 1291. [16] C. L. Hartwig, A. S. Rosenthal, J. D’Angelo, C. F. Griffin, G. H. Posner and R. A. Cooper, Biochem. Pharmacol., 2009, 77, 322. [17] P. L. Olliaro, R. K. Haynes, B. Meunier and Y. Yuthavong, Trends Parasitol., 2001, 17, 122. [18] F. B.-E. Garah, C. Claparols, F. Benoit-Vical, B. Meunier and A. Robert, Antimicrob. Ag. Chem., 2008, 52, 2966. [19] R. K. Haynes, W. C. Chan, C.-M. Lung, A.-C. Uhlemann, U. Eckstein, D. Taramelli, S. Parapini, D. Monti and S. Krishna, ChemMedChem, 2007, 2, 1480.
376
Christopher Paul Hencken et al.
[20] S. Krishna, A.-C. Uhlemann and R. K. Haynes, Drug Resist. Updat., 2004, 7, 233. [21] A.-C. Uhlemann, A. Cameron, U. Eckstein-Ludwig, J. Fischbarg, P. Iserovich, F. A. Zuniga, M. East, A. Lee, L. Brady, R. K. Haynes and S. Krishna, Nat. Struct. Mol. Biol., 2005, 12, 628. [22] A. Ryde´n and O. Kayser, Top. Heterocycl. Chem., 2007, 9, 1. [23] J. Be´gue´ and D. Bonnet-Delpon, ChemMedChem, 2007, 2, 608. [24] R. K. Haynes, B. Fugmann, J. Stetter, K. Rieckmann, H.-D. Heilmann, H.-W. Chan, M.-K. Cheung, W.-L. Lam, H.-N. Wong, S. L. Croft, L. Vivas, L. Rattray, L. Stewart, W. Peters, B. L. Robinson, M. D. Edstein, B. Kotecka, D. E. Kyle, B. Beckermann, M. Gerisch, M. Radtke, G. Schmuch, W. Steinke, U. Wollborn, K. Schmeer and A. Ro¨mer, Angew. Chem. Int. Ed., 2006, 45, 2082. [25] G. H. Posner, W. Chang, L. Hess, L. Woodard, S. Sinishtaj, A. R. Usera, W. Maio, A. S. Rosenthal, A. S. Kalinda, J. G. D’Angelo, K. S. Peterson, R. Stohler, J. Chollet, J. Santo-Tomas, C. Snyder, M. Rottmann, S. Wittlin, R. Brun and T. A. Shapiro, J. Med. Chem., 2008, 51, 1035. [26] N. C. P. Arauju, V. Barton, M. Jones, P. A. Stocks, S. A. Ward, J. Davies, P. G. Brat, A. E. Shone, M. L. S. Cristiano and P. M. O’Neill, Bioorg. Med. Chem. Lett., 2009, 19, 2038. [27] J. J. Walsh, D. Coughlan, N. Hehghan, G. Gaynor and A. Bell, Bioorg. Med. Chem. Lett., 2007, 17, 3599. [28] S. Bachmann, J. Schro¨der, C. Bottmer, E. F. Torrey and R. H. Yolken, Psychopathology, 2005, 38, 87. [29] A. M. Tenter, A. R. Keckeroth and L. M. Weiss, Int. J. Parasitol., 2000, 30, 1217. [30] K. Ou-Yang, E. C. Krug, J. J. Marr and R. L. Berens, Antimicrob. Ag. Chem., 1990, 34, 1961. [31] L. Jones-Brando, J. D’Angelo, G. H. Posner and R. Yolken, Antimicrob. Ag. Chem., 2006, 50, 4206. [32] J. G. D’Angelo, C. Bordo´n, G. H. Posner, R. Yolken and L. Jones-Brondo, J. Antimicrob. Chem., 2009, 63, 146. [33] K. Nagamune, W. L. Beatty and L. D. Sibley, Eukaryot. Cell, 2007, 6, 2147. [34] H. R. Chang, C. W. Jefford and J.-C. Peche`re, Antimicrob. Ag. Chem., 1989, 33, 1748. [35] J. Moreno, C. Caravate, C. Chamizo, F. Laguna and J. Alvar, Tran. R. Soc. Trop. Med. Hyg., 2000, 94, 328. [36] M. A. Avery, K. M. Muraleedharan, P. V. Desai, A. K. Bandyopadhyaya, M. M. Furtado and B. L. Tekwani, J. Med. Chem., 2003, 46, 4244. [37] X. Shuhua, M. Tanner, E. K. N’Goran, J. Utzinger, J. Chollet, R. Benquist, C. Minggang and Z. Jiang, Acta Trop., 2002, 82, 175. [38] I. Adam, O. A. Elhardello, M. O. Elhadi, E. Abdalla, K. A. Elmandi and F. H. Jansen, Ann. Trop. Med. Parasitol., 2008, 102, 39. [39] A. A. Mohamed, H. M. Mahgoub, M. Magzoub, G. I. Gasim, W. N. Eldein, A. Ahmed and I. Adam, Trans. Roy. Soc. Trop. Med. Hyg., 2009, in press. [40] D. DeClercq, J. Vercruysse, A. Kongs, P. Verlei, J. P. Dompnier and P. C. Faye, Acta Trop., 2002, 82, 61. [41] http://wwwn.cdc.gov/travel/yellowbookCh4-AfricanSleepingSickness.aspx [42] http://wwwn.cdc.gov/travel/yellowBookCh4-Chagas.aspx [43] Y. V. Mishina, S. Krishna, R. K. Haynes and J. C. Meade, Antimicrob. Ag. Chem., 2007, 51, 1852. [44] C. Chollet, A. Baliani, P. E. Wong, M. P. Barrett and I. H. Gilbert, Bioorg. Med. Chem., 2009, 17, 2512. [45] R. S. Qian, Z. L. Li, J. L. Yu and D. J. Ma, J. Tradit. Chin. Med., 1982, 2, 271. [46] T. Efferth, M. Marschall, X. Wang, S.-M. Huong, I. Hauber, A. Olbrich, M. Kronschnabl, S. Stamminger and E.-S. Huang, J. Mol. Med., 2002, 80, 233. [47] P. M. Bork, M. L. Schmitz, M. Kuhnt, C. Escher and M. Heinrich, FEBS Lett., 1997, 402, 85.
Anti-Infective and Anti-Cancer Properties of Artemisinin
377
[48] B. Siedle, A. J. Garcia-Pineres, R. Murillo, J. Schulte-Mo¨nting, V. Castro, P. Ru¨ngeler, C. A. Klaas, F. B. DaCosta, W. Kisiel and I. Merfort, J. Med. Chem., 2004, 47, 6042. [49] T. Efferth, M. R. Romero, D. G. Wolf, T. Stamminger, J. J. G. Marin and M. Marschall, Clin. Infect. Dis., 2008, 47, 804. [50] Y. Binku, E. Mekonnen, A. Bjorkman and D. Wolday, Ethiop. Med. J., 2002, 40, 17. [51] J. Vandenkerckhov, E. B. Sas, E. Pets and J. VanHemel, WO International Patent 2004/ 041176A2, 2004. [52] S. Kumar, S. P. S. Khanuja, T. R. S. Kumar, D. C. Jain, S. Srivastava, A. K. Bhattacharya, D. Saikia, A. K. Shasany, M. P. Darokar and R. P. Sharma, US Patent 6,127,405, 2000. [53] A. M. Galal, S. A. Ross, M. Jacob and M. A. ElSohly, J. Nat. Prod., 2005, 68, 1274. [54] M. Kolaczkowski, A. Kolaczkowska and F. R. Stermitz, Microb. Drug Resist., 2009, 15, 11. [55] http://seer.cancer.gov/statfacts/html/all.html [56] L. Anfosso, T. Efferth, A. Albini and U. Pfeffer, Pharmacogenomics J., 2006, 6, 269. [57] J. Folkmann, J. Natl. Cancer Inst., 1990, 82, 4. [58] A. F. Karamysheva, Biochemistry (Moscow), 2008, 73, 751. [59] M. Shibuya, J. Biochem. Mol. Biol., 2006, 39, 469. [60] H.-H. Chen, H.-J. Zhou, W.-Q. Wang and G.-D. Wu, Cancer Chemother. Pharmacol., 2004, 53, 423. [61] H.-H. Chen, H.-J. Zhou, G.-D. Wu and X.-E. Lou, Pharmacology, 2004, 71, 1. [62] H.-H. Chen, H.-J. Zhou and X. Fang, Pharmacol. Res., 2003, 48, 231. [63] T. Efferth, G. Ruecker, M. Falkenberg, D. Manns, A. Olbrich and U. Fabry, Arzneimittelforschung, 1996, 46, 196. [64] S. Krishna, L. Bustamante, R. K. Haynes and H. M. Staines, Trends Pharmacol. Sci., 2008, 29, 520. [65] G. H. Posner, J. D’Angelo, P. M. O’Neill and A. Mercer, Expert Opin. Ther. Patents, 2006, 16, 1665. [66] J. P. Jeyadevan, P. G. Bray, J. Chadwick, A. E. Mercer, A. Byrne, S. A. Ward, B. K. Park, D. P. Williams, R. Cosstick, J. Davies, A. P. Higson, E. Irving, G. H. Posner and P. M. O’Neill, J. Med. Chem., 2004, 47, 1290. [67] I. Nakase, B. Gallis, T. Takatani-Nakase, S. Oh, E. Lacoste, N. P. Singh, D. R. Goodlett, S. Tanaka, S. Futaki, H. Lai and T. Sasaki, Cancer Lett., 2009, 274, 290. [68] M. Jones, A. E. Mercer, P. A. Stocks, L. J. I. LaPense´e, R. Cosstick, B. K. Park, M. E. Kennedy, I. Piantanida, S. A. Ward, J. Davis, P. G. Bray, S. L. Rawe, J. Baird, T. Charidza, O. Janneh and P. M. O’Neill, Bioorg. Med. Chem. Lett., 2009, 19, 2033. [69] W. R. J. Taylor and N. J. White, Drug Saf., 2004, 27, 25. [70] I. R. Ribeiro and P. Olliaro, Med. Trop. (Mars), 1998, 58(3 suppl.), 50. [71] R. Price, M. vanVugt, L. Phaipun, C. Luxemburger, J. Simpson, R. McGready, F. TerKuile, A. Kham, T. Chongsuphajaisiddhi, N. J. White and F. Nosten, Am. J. Trop. Med. Hyg., 1999, 60, 547. [72] R. McGready, T. Cho, N. K. Keo, K. L. Thwai, L. Villegas, S. Looareesuwan, N. J. White and F. Nosten, Clin. Infect. Dis., 2001, 33, 2009. [73] S. Dellicour, S. Hall, D. Chandramohan and B. Greenwood, Malar. J., 2007, 6, 15. [74] A. C. Boareto, J. C. Muller, A. C. Bufalo, G. G. K. Botelho, S. L. deAraujo, M. A. Foglio, R. N. deMorais and P. R. Dalsenter, Reprod. Toxicol., 2008, 25, 239. [75] M. H. El-Dakdoky, Food Chem. Toxicol., 2009, 47, 1437. [76] China-Cooperative-Research-Group-On-Qinghaosu-and-its-Derivatives-as-Antimalarials, J. Tradit. Chin. Med., 1982, 2, 45. [77] R. F. Genovese, D. B. Newman, J. M. Petras and T. G. Brewer, Pharmacol. Biochem. Behav., 1998, 60, 449.
378
Christopher Paul Hencken et al.
[78] J. M. Petras, G. D. Young, R. A. Bauman, D. E. Kyle, M. Gettayacamin, H. K. Webster, K. D. Corcoran, J. O. Peggins, M. A. Vane and T. G. Brewer, Anat. Embryol. (Berl), 2000, 201, 383. [79] A. Nontprasert, S. Pukrittayakamee, A. M. Dondorp, R. Clemens, S. Looareesuwan and N. J. White, Am. J. Trop. Med. Hyg., 2002, 67, 423. [80] T. Gordi and E.-I. Lepist, Toxicol. Lett., 2004, 147, 99.
CHAPT ER
19 Recent Advances in the Inhibition of Bacterial Type II Topoisomerases Gregory S. Bisacchi and Jacques Dumas
Contents
1. Introduction 2. Inhibition at the ATP-Binding Site 2.1 Novobiocin/clorobiocin analogs 2.2 Cyclothialidine analogs 2.3 Ethyl-ureas 2.4 Lead compounds derived from fragment-based approaches 2.5 Miscellaneous compounds or inhibitors 3. Inhibition Outside of the ATP-Binding Site 3.1 Classical quinolone inhibitors 3.2 Quinolone-like structures 3.3 Quinolines 3.4 Quinoline pyrimidine triones 4. Conclusion References
379 381 381 382 383 385 387 388 388 390 391 392 393 393
1. INTRODUCTION The natural product antibiotic novobiocin (1) was reported in 1955 [1], and in 1960, synthetic antibacterial compounds having a 3-carboxyquinolone core (e.g., 2) were first disclosed [2]. These discoveries were made AstraZeneca Pharmaceuticals LP, 35 Gatehouse Drive, Waltham, MA 02451, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04419-4
r 2009 Elsevier Inc. All rights reserved.
379
380
Gregory S. Bisacchi and Jacques Dumas
during the so-called golden age of antibiotics [3] and were based on inhibition of bacterial growth in culture. Target-based discovery of antibacterials was still several decades in the future. By the mid-to-late 1970s, the molecular mechanism of novobiocin and the quinolones was elucidated to be the inhibition of bacterial topoisomerase, specifically DNA gyrase [4–5]. In 1994, a target-based screen of fermentation broths led to the discovery of cyclothialidine (7) as an inhibitor of DNA gyrase [6]. From the 1990s to the present day, bacterial topoisomerase has steadily gained prominence as a target of choice for the discovery of antibacterial agents [7–10].
OH O H2N
O
O
O
O
O OH
OH
O
H N O
CO2H O
Me
X
N
Me
1
2 X = CH 3 X=N
Bacterial topoisomerases, like their eukaryotic counterparts, are divided into two main groups: Type I, which involves DNA singlestrand processing and is typically ATP-independent, and Type II, which processes double-stranded DNA and requires ATP. Topoisomerase function is absolutely required by both prokaryotic and eukaryotic cells to allow, among other processes, proper unwinding and topological display of the DNA strands before replication [11]. To date, no advanced inhibitors of bacterial Type I topoisomerases have been reported [12]. In contrast, useful inhibitors of Type II topoisomerases are abundant, from the well-established fluoroquinolone class to a variety of emerging classes in various stages of clinical or preclinical evaluation. The two principal subclasses of bacterial Type II topoisomerase are DNA gyrase and topoisomerase IV. DNA gyrase exists as a tetramer, consisting of two subunits of GyrA and two subunits of GyrB [13]. The GyrB subunit contains the ATP-binding pocket. Tetrameric topoisomerase IV is closely related to DNA gyrase and consists of two subunits of ParC and two subunits of ParE, with ParE containing the ATP-binding pocket (Figure 1). Novobiocin and cyclothialidine inhibit DNA gyrase and topoisomerase IV by binding to the ATP sites in GyrB and ParE, respectively, while the quinolone class inhibits these enzymes by binding to a site in GyrA and ParC near the intersection of the subunits and the associated DNA strand. Medicinal chemistry programs targeting inhibitors of the ATP-binding pocket of GyrB/ParE have the benefit of
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
381
ATP binding sites novobiocin cyclothialidine ethyl-ureas X-ray structure-guided 2GyrB
2ParE Other binding sites quinolones isothiazoloquinolone 3-aminoquinazolinedione quinolines quinoline pyrimidine triones emerging structural information
2GyrA
DNA Gyrase
2ParC
Topoisomerase IV
Figure 1 Schematic diagram of bacterial type II topoisomerases.
crystallographic structural information for analog design [14]. However, outside the ATP sites, details of how inhibitors bind are only emerging [15]. This chapter broadly organizes small-molecule inhibitors of bacterial Type II topoisomerase into those that bind at the ATP sites and those that bind at other sites within the enzyme tetramers. Because the quinolone class of GyrA/ParC inhibitors has been employed therapeutically for decades, many bacteria species have developed substantial, and ever-increasing, resistance to this class, as they have to varying degrees for all other classes of antibacterials. The pharmaceutical industry and medical community response to the problem of increasing resistance to marketed quinolone antibacterial drugs has been, in part, both to investigate further variants of this established class, which may possess incremental advantages against resistant pathogens, and to discover and develop entirely different classes of inhibitors of bacterial topoisomerase II, which are not susceptible to pre-established resistance mechanisms. Both of these approaches are described in this chapter.
2. INHIBITION AT THE ATP-BINDING SITE 2.1 Novobiocin/clorobiocin analogs Antibiotics of the coumarin class, such as novobiocin (1) or clorobiocin (4), bind to the ATP pocket of GyrB. These analogs are produced by Streptomyces strains; for the most part, the gene clusters responsible for their biosynthesis have been elucidated and characterized [16]. A large body of work has been devoted to the generation of novel analogs through directed mutagenesis of Streptomyces strains. Deletion of the cloQ
382
Gregory S. Bisacchi and Jacques Dumas
and cloL genes in Streptomyces coelicolor M512 suppresses the biosynthesis of clorobiocin [17]. As a second step, introduction of the couL sequence (an amide synthase with broad specificity, involved in the biosynthesis of coumermycin) resulted in the identification of three novel analogs with a modified substitution pattern on the coumarin core, for example, ferulobiocin (5). A similar two-stage mutagenesis approach produced analogs with significant anti-bacterial potencies against Gram-positive organisms [18,19]. For example, coumarin 6 inhibits the growth of Staphylococcus aureus ATCC 29213 with a minimum inhibitory concentration (MIC) of o0.06 mg/mL. Despite this, activity against wild-type Gram-negative organisms remains elusive for this compound class. OH OH O O
H N
O
H N
O O
O
H3C
O
O
Cl
OH
4
OH OH H N
O O H N
O
OCH3 O
O
O
H3C
O
OH
O
Cl 5
OH OH H N
O O H N H3C
O O O
O
O
OH
CH3 6
O
383
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
2.2 Cyclothialidine analogs Cyclothialidine (7), a 12-membered ring lactone isolated from Streptomyces filipinensis, is a potent inhibitor of GyrB and, like novobiocin, binds to the ATP site [20]. Its structure, albeit complex, allows the preparation of analogs through total synthesis. The binding mode of cyclothialidine with GyrB is known and suggests that one of the phenolic groups is required for activity, while other functional groups in the molecule can tolerate derivatization. A medicinal chemistry approach has focused on simplified macrolactones and modifications of the C-terminal chain [21]. Macrolactone 8 is a potent inhibitor of Escherichia coli DNA gyrase in a supercoiling assay, exhibiting a maximum non-effective concentration (MNEC) of 2 ng/mL. This analog shows a potent MIC against S. aureus Smith (0.25 mg/mL) and in vivo efficacy in a murine septicaemia model using the same pathogen (ED50 ¼ 8.5 mg/kg). The introduction of a thioamide functionality and the enlargement of the macrolactone ring significantly raised potency. Higher plasma free fraction and improved pharmacokinetics have been attributed to the aminomethyl group on the oxadiazole ring.
HN OH
S
O
COOH OH
O HN
O
O
O
N
N H
HO O
O
HN OH
NH2
N
S
NH2
N
S
O MeO O
HO
7
8
OH
Cl OH
N N H
N MeO Br
Cl
O
9
O 10
Simplified analogs conserve the key phenolic residue of cyclothialidine as a minimum pharmacophore. The same team used 9, an high throughput screen (HTS) hit, as a starting point for further modification [22].
384
Gregory S. Bisacchi and Jacques Dumas
Quinoline 10 shows broad Gram-positive activity and comes close to novobiocin in terms of potency. The introduction of the dehydro-indolone core was a critical step in improving anti-bacterial activity. In addition, the authors suggest that the acidity of the phenol group, and/or the overall lipophilicity of the molecule seem to play an important role in penetration of the bacterial membrane.
2.3 Ethyl-ureas A new class of GyrB inhibitors, built around an ethyl-urea pharmacophore, has recently emerged in the literature. Carbamate 11 was first identified in a high-throughput assay targeting the ATP-ase activity of GyrB [23]. The optimization of 11 into VRT-752586 (12) was aided by docking into a crystal structure of novobiocin complexed with S. aureus enzyme. Three major steps led to 12: The carbamate oxygen of 11 is replaced by an NH, improving an interaction with Asp-73, an amino acid residue bound to the adenine of ATP. The 3-pyridyl substitution in 12 takes advantage of a conserved arginine residue, Arg-136 (E. coli numbering), creating an interaction already present in the complex between novobiocin and GyrB. The 3-fluoro-pyridine-2-yl substituent fills available space in the carbohydrate-binding pocket of the enzyme and makes favorable lipophilic interactions, while locking the inhibitor in its most productive conformation with the use of an internal hydrogen bond. N OH
O
O O
HN N
N HN
HN
N H
N H
12 F N
11
N
N HN
N
O
O HN
N
N
N HN
HN N
N
N N
14
13 N
N
N
385
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
N
N
O HN
O N
HN N
HN N 15 S
N HN
N
S
16 S
N
Two publications devoted to 12 report a robust antibacterial profile. VRT-752586 acts as a dual inhibitor of GyrB and ParE, and each of these enzymes may be responsible for its anti-bacterial activity, depending on the bacterial species [24]. In addition, its dual targeting leads to very low resistance frequencies (o5.2 1010 at 4 MIC measured in Enterococcus faecalis). This analog shows broad activity against Gram-positive organisms (MIC90 equal of better than 0.12 mg/mL in all organisms tested) [25]. VRT-752586 is, however, less potent against fastidious Gramnegative pathogens, Mycobacterium tuberculosis, two atypical pathogens (Legionella pneumophila and Mycoplasma pneumoniae), and a panel of anaerobes. Evidence of Gram-negative efflux was reported against E. coli (no wild-type activity, but MIC of 0.13 mg/mL against a tolC mutant). Finally, VRT-752586 shows in vivo activity in two models of infection in mice (S. aureus/thigh and Streptococcus pneumoniae/lung), at doses ranging from 25 mg/kg to 50 mg/kg i.v. [23]. The potent activity of this class generated much interest and led several pharmaceutical companies into the field. Scaffold-hopping initiatives have conserved the urea moiety, the adjacent heteroatom, and both heteroaryl substituents, resulting in triazolo-pyridines such as 13. While this analog defines new proprietary space and shows useful anti-bacterial potencies against Gram-positive organisms, it appears less potent than its benzimidazole counterpart [26]. Additional triazolo-pyridines [27], as well as the related imidazo-pyridines [28] and benzothiazoles [29], have been described in the patent literature (compounds 14–16 are representative examples). Overall, the chemical space around the ethyl-ureas is now becoming increasingly crowded.
2.4 Lead compounds derived from fragment-based approaches The ATP-binding site of GyrB appears well-suited for fragment-based approaches. The adenine of ATP binds to a conserved aspartate residue (Asp-73 in S. aureus) and an active-site water molecule in a bi-dentate fashion. These key interactions can be mimicked with a variety of adenine-like cores, some of them not dissimilar to donor-acceptor kinase
386
Gregory S. Bisacchi and Jacques Dumas
inhibitor fragments. In addition, the X-ray structures of novobiocin and clorobiocin complexed with DNA gyrase provide a hint that these fragments could be grown toward the solvent interface and point at two conserved arginine residues (Arg-76 and Arg-136) to deliver additional binding energy. A first example of such an approach makes use of a triazine core [30]. Triazine 17 was identified as a screening hit in a DNA gyrase assay (IC50 ¼ 0.75 mM), and its binding mode was elucidated by X-ray crystallography. Fine-tuning of the triazine substitution, and incorporation of a coumarin fragment to mimic novobiocin, led to the identification of 18, a potent DNA gyrase inhibitor (IC50 ¼ 70 nM). H N
H N
N N
F
H N
Cl
N
N
F
HN
O
N OH
NH2
Cl
17
O
N
18 F
H N N
H N N
O
S
O2N O
19
20 O
NH2
OH
O H N
N
O
N N
N
O
O
O 22
21
Cl Cl
Br
O
387
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases H2N
H N
O
H N
O
OH
O S
HN
Br
N
HN
Cl
N
Cl
N
23
O
N
24
Cl
In a similar way, indazole 19, identified in a ‘‘needle screening’’ effort [31], effectively binds to DNA gyrase in the high micromolar range, through a contact at Asp-73. As seen previously, addition of a moiety targeted at Arg-76 and Arg-136 led to 20, a much more potent analog (MNEC ¼ 0.03 mg/mL). A similar approach from the same team [32] culminated in pyrazolo-pyrimidine 21, also a potent inhibitor of DNA gyrase (MNEC ¼ 0.06 mg/mL). Recently, an NMR screen directed at the ATP pocket of GyrB led to pyrrole 22, with a Kd of 160 mM [33]. Iterative, structure-based design [34,35] optimized the pyrrole substitution and introduced additional interactions with both conserved arginines, as in pyridine 23, which shows an IC50 of 10 nM in an E. coli ATPase assay. Productive interactions of 23 with a 24-kDa subunit of GyrB were confirmed by X-ray crystallography. The pyrrolamide series demonstrates promising activity across a broad range of pathogens; its mode of action is bactericidal and consistent with DNA synthesis inhibition. Thiazole-carboxylic acid 24, a representative example, shows dose-dependent, oral activity in a murine S. pneumoniae lung infection [36,37].
2.5 Miscellaneous compounds or inhibitors In the past few years, several GyrB inhibitor structures have been published in the literature. All three compounds below bind in the ATP pocket of DNA gyrase, albeit with modest potencies. OH
H N O
HO
O OH
H N OH 25
N
OH
O
26
388
Gregory S. Bisacchi and Jacques Dumas
OH OH HO
O OH O OH
OH O 27 OH OH
The indolinone core 25 binds to GyrB through its donor–acceptor pair, in a tyrosine-kinase-like fashion [38]. The natural flavinoid quercetin (26) and its analog ()-epigallocathechin gallate (27) also bind in the ATP pocket of GyrB with affinities in the double-digit micromolar range [39,40].
3. INHIBITION OUTSIDE OF THE ATP-BINDING SITE 3.1 Classical quinolone inhibitors Following the launch of nalidixic acid (3) in 1962, research into variants of the classic quinolone/napthyridone structures has continued unabated to this day [15,41]. With only a few notable exceptions (covered in Section 3.2), replacement of the critical 3-carboxy substituent has resulted in inactive or less useful agents. The vast majority of useful antibacterial agents in this class rely upon variation of peripheral substituents, leaving the heterocyclic cores largely intact. First-generation analogs such as 2 and 3 had narrow activity against Gram-negative pathogens, mainly E. coli. Second- and third-generation analogs culminated in the commercialization of a number of 6-fluoroquinolones, notably, ciprofloxacin (1987), levofloxacin (1993), and moxifloxacin (1999), whose spectrum and potencies encompassed most Gram-positive and Gramnegative species, as well as anaerobes and atypical bacterial pathogens. The structure of moxifloxacin (28) is shown to exemplify this later, broadspectrum class. Yet, many other quinolones launched during this more recent period were beset by unexpected toxicological issues of varying severity and were withdrawn from a number of markets. Notable examples include trovafloxacin (hepatoxicity), temafloxacin (hemolytic reactions), grepafloxacin (cardiotoxicity), and gatifloxacin (glucose
389
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
homeostasis abnormalities) [15,42]. Other side effects associated with fluoroquinolones have included phototoxicity, central nervous system (CNS) effects, and tendonitis. To date, the fluoroquinolone field is challenged with providing drugs that are predictably safe, maintain or further expand pathogen spectrum, and show therapeutically useful potency against current quinolone-resistant strains. Delafloxacin (29) and nemonoxacin (30), two of several quinolones now in clinical development, seem to fit this profile. Delafloxacin is at least 32-fold more potent than comparators such as levofloxacin and moxifloxacin against many quinolone-resistant Gram-positive pathogens including methicillinresistant s. aureus (MRSA) while maintaining good Gram-negative potency. [43]. Likewise, nemonoxacin, which lacks the traditional 6-fluoro substituent, exceeds comparators (ciprofloxacin, levofloxacin, moxifloxacin) with regard to in vitro potency against quinolone-resistant MRSA and retains a good Gram-negative spectrum [44]. Additionally, it appears to be less prone to bacterial resistance development, as assessed in vitro using clinical isolates of S. pneumoniae, compared to standard quinolone comparators [45]. Both 29 and 30 have recently completed phase II clinical trials for community-acquired pneumonia. O
O
O F
F
CO2H
H N
N
N
H3C
N Cl
OCH3
N
N OCH3
F N
HO
NH H
CO2H
CO2H
NH2
H2N F
28
29
30
O F O N
O
O CO2H
F
F
CO2H
CO2H
HN N
N
N
N
N
N O
OCH3
F
F
NH2
31
32
N
F
NH2
33
Analogs 31–33 are representative of recent preclinical efforts. Compound 31 was evaluated in vitro against clinical strains of S. aureus having defined mutations in the quinolone resistance-determining regions (QRDRs) of its GyrA and ParC [46]. Double and triple mutant
390
Gregory S. Bisacchi and Jacques Dumas
strains are resistant to ciprofloxacin and other standard quinolone comparators, but still susceptible to 31 (MICs ¼ 0.25–0.5 mg/mL). Analog 32 has shown potent in vitro activity against quinolone-susceptible and quinolone-resistant S. aureus and S. pneumoniae clinical isolates, showing superiority in all cases to moxifloxacin [47]. This compound also shows superiority to moxifloxacin in an in vivo model of quinolone-resistant S. aureus infection. Analog 33, similar to 31 and 32, displays a moxifloxacin-like spectrum of in vitro antibacterial activity, but with enhanced Gram-positive potency. In particular, 33 shows an MIC90 of 0.5 mg/mL versus MRSA, which is eightfold more potent than moxifloxacin [48]. Its MIC90 versus quinolone-resistant S. pneumoniae (0.25 mg/mL) is 32-fold more potent than moxifloxacin [49]. Considering the relative diversity of substitution patterns at N1, C6, C7, and C8 within this set of analogs (29–33), it is remarkable that all achieve a rather similar antibacterial profile, that is, enhanced Gram-positive spectrum, with distinct advantages against quinolone-resistant strains. Although the medicinal chemistry of quinolone antibacterials has over the past 50 years generated vast SAR guidance with which to frame current and future analog work, it still apparently has room to surprise. The recent clinical need for more effective Gram-positive agents, especially those with excellent potency against MRSA, has undoubtedly led to a focussed interest in, and bias toward, recent agents such as 29–33, having those qualities. However, one major challenge for future quinolone research remains the discovery of analogs with enhanced potency against multidrug-resistant Gram-negative pathogens, especially Klebsiella, Acinetobacter, and Pseudomonas aeruginosa species [50].
3.2 Quinolone-like structures Two separate medicinal chemistry efforts have developed scaffold series containing viable replacements for the otherwise canonic quinolone 3-carboxy group. The 3-aminoquinazolinediones, represented by lead structure 34, evolved from an early tricyclic hit series [51]. Based on analysis of resistant mutants in S. pneumoniae, 34 and related analogs apparently target primarily GyrB and ParE, unlike quinolones [52]. This series is not cross-resistant to established fluoroquinolone-resistant pathogenic strains. Sequencing of resistant mutants grown in Neisseria gonorrhoeae identified mutations in or near the QRDR suggesting that the binding site is near or overlapping that of the fluoroquinolones. Against a levofloxacin-resistant strain of S. pneumoniae, 34 exhibits an MIC90 of 0.06 mg/mL, with fluoroquinolone comparators in the 2- to 16-mg/mL range. In general, activity against Gram-positive pathogens is the strength of this series, with potencies against Gram-negatives either equal to or less than current fluoroquinolone comparators.
391
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
The isothiazolo-quinolone series originated in the 1980s as part of a quinolone carboxylic acid replacement program. Subsequently, the compounds synthesized at that time were found to carry significant eukaryotic topoisomerase inhibitory liability [53,54] and further interest in the series waned. O
O
F N
NH2
O
F
H
NH
H N
N
H2N
O
N
N
H2N
34
S
OCH3 35
Recently, however, the series was revived and progress has been made to enhance inhibition of prokaryotic versus eukaryotic topoisomerase Type II [55]. Compound 35, a representative of the series, shows MIC90s of 0.5, 0.5, and 2 mg/mL against MRSA, E. faecalis, and Enterococcus faecium, respectively [56]. In contrast, levofloxacin has MIC90s of W 16 mg/mL against all of these Gram-positive pathogens. Furthermore, Gram-negative activity is on a par with levofloxacin. Mutation analysis indicated that 35 targets both GyrA and ParC, with first-step mutations occurring in GyrA only [57].
3.3 Quinolines Nearly 150 years following Pasteur’s chemical fragmentation of quinine [58,59] to provide d-quinotoxine (36), certain synthetic derivatives of that scaffold were found to possess broad-spectrum antibacterial activity [60], which provided a fresh starting point for a potential new class of broadspectrum antibacterial agents. Analogs 37–39 are variations within this class, which acts by inhibition of bacterial gyrase and topoisomerase IV. Compound 37 retains activity in multiple fluoroquinolone-resistant strains of S. aureus having a variety of mutations in the QRDR, suggesting that it targets topoisomerases in a manner distinct to that of fluoroquinolones [61,62]. Quinoline 37 is efficacious against a strain of fluoroquinolone-resistant S. pneumoniae in a mouse lung infection model. When dosed orally at 100 mg/kg b.i.d., 37 resulted in 70% survival, compared to 10% for moxifloxacin at the same dose [63]. This compound advanced to phase I clinical trials but was discontinued due to QTc (the electrocardiogram QT interval corrected for heart rate) prolongation [64].
392
Gregory S. Bisacchi and Jacques Dumas
Compound 38 and related analogs were shown to inhibit DNA gyrase and topoisomerase IV in S. aureus, S. pneumoniae, and E. coli and displays single-digit or sub-micromolar MICs against all three pathogens [65]. This series also retains excellent potency against a panel of fluoroquinolone-resistant S. aureus species. For example, 39 shows MICs ranging from 0.007 mg/mL to 0.5 mg/mL, compared to 4 to W128 mg/mL for three fluoroquinolone comparators. Yet another sub-series in this class, exemplified by 39, was reported to have potencies of o2 mg/mL against a panel of Gram-positive and Gram-negative pathogens [66]. Other research groups have also reported their own scaffold entrants to this general class [67–69]. HO2C NH
O MeO
MeO N
S
N
HO
S
F
36
37 N
OMe
N N
O H N
N
38
O
N H
N
H N
S MeO
N
F
N
O
O
39
N
3.4 Quinoline pyrimidine triones A new class of antibiotics, exemplified by PNU-286607 (40), has recently been reported [70,71]. PNU-286607 was identified by whole cell screening for MICs, followed by a reverse-genomics approach to elucidate its mode of action, and features an unusual quinoline pyrimidine trione scaffold. Its structure was fully elucidated by X-ray crystallography [70]. Quinoline pyrimidine trione 40 has significant antimicrobial activity against a wide range of Gram-positive and to a lesser extent against Gram-negative pathogens. This compound acts through inhibition of the
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
393
DNA gyrase complex [71], does not show cross-resistance with fluoroquinolones, and is orally active in lethal systemic infection model in mice, using an MRSA strain (ED50 ¼ 19.5 mg/kg). Overall, 40 represents a promising lead structure, targeting a new binding site of the GyrA/GyrB complex.
O
H N
O
O2N
NH H
O CH3
N O 40 CH3
4. CONCLUSION Over 50 years have passed since the first discovery of DNA gyrase inhibitors. What started as a classical, MIC-driven drug discovery approach (novobiocin and first-generation quinolones) has now become a multidisciplinary field expanding at the frontiers of medicinal chemistry, microbiology, genomics, and structural biology. The ‘‘classical’’ quinolone family still produces extremely potent, broad-spectrum analogs, and year after year proves to be extremely flexible with regard to structural modifications. In addition, in the past decade, a number of new lead structures targeting DNA gyrase have been reported in the literature. Many of these already show useful MICs against Grampositive pathogens and a complete lack of cross-resistance against the widely used fluoroquinolones. Based on this progress, it appears probable that DNA gyrase inhibition will continue to produce novel, life-saving antibiotics for many years to come.
REFERENCES [1] R. W. Fairbrother and B. L. Williams, Lancet, 1956, 1177. [2] N. Barton, A. F. Crowther, W. Hepworth, D. N. Richardson and G. W. Driver, BR Patent Application, 830,832, 1960. [3] P. B. Fernandes, R. Menzel, D. J. Hardy, Y.-C. Tse-Ding, A. Warren and D. A. Elsemore, Med. Res. Rev., 1999, 19, 559.
394
Gregory S. Bisacchi and Jacques Dumas
[4] M. Gellert, M. H. O’Dea, T. Itoh and J.-I. Tomizawa, Proc. Natl. Acad. Sci. U.S.A., 1976, 73, 4474. [5] A. Sugino, C. L. Peebles, K. N. Kreuzer and N. R. Cozzarelli, Proc. Natl. Acad. Sci. U.S.A., 1977, 74, 4767. [6] J. Watanabe, N. Nakada, S. Sawairi, H. Shimada, S. Oshima, T. Kamiyama and M. Arisawa, J. Antibiot., 1994, 47, 32. [7] B. J. Bradbury and M. J. Pucci, Curr. Opin. Pharmacol., 2008, 8, 574. [8] M. Oblak, M. Kotnik and T. Solmajer, Curr. Med. Chem., 2007, 14, 2033. [9] Y. C. Tse-Dinh, Infect. Disord. Drug Targets, 2007, 7, 3. [10] A. Maxwell and D. M. Lawson, Curr. Top. Med. Chem., 2003, 3, 283. [11] J. C. Wang, Annu. Rev. Biochem., 1996, 65, 635. [12] B. Cheng, I.-F. Liu and Y.-C. Tse-Dinh, J. Antimicrob. Chemother., 2007, 59, 640. [13] C. Levine, H. Hiasa and K. J. Marians, Biochim. Biophys. Acta., 1998, 1400, 29. [14] S. Bellon, J. D. Parsons, Y. Wei, K. Hayakawa, L. L. Swenson, P. S. Charifson, J. A. Lippke, R. Aldape and C. H. Gross, J. Antimicrob. Chemother., 2004, 48, 1856. [15] I. Laponogov, M. K. Sohi, D. A. Veselkov, X. S. Pan, R. Sawhney, A. W. Thompson, K. McAuley, L. M. Fisher and M. R. Sanderson, RCSB Protein Databank, 3fof. [16] L. Heide, B. Gust, C. Anderle and S.-M. Li, Curr. Top. Med. Chem., 2008, 8, 667. [17] C. Anderle, S.-M. Li, B. Kammerer, B. Gust and L. Heide, J. Antibiot., 2007, 60, 504. [18] C. Anderle, M. Stieger, M. Burrell, S. Reinelt, A. Maxwell, M. Page and L. Heide, Antimicrob. Agents Chemother., 2008, 52, 1982. [19] C. Anderle, S. Hennig, B. Kammerer, S.-M. Li, L. Wessjohann, B. Gust and L. Heide, Chem. Biol., 2007, 14, 955. [20] N. Nakada, H. Shimada, T. Hirata, Y. Aoki, T. Kamiyama, J. Watanabe and M. Arisawa, Antimicrob. Agents Chemother., 1993, 37, 2656. [21] P. Angehrn, S. Buchmann, C. Funk, E. Goetschi, H. Gmuender, P. Hebeisen, D. Kostrewa, H. Link, T. Luebbers, R. Masciadri, J. Nielsen, P. Reindl, F. Ricklin, A. Schmitt-Hoffmann and F.-P. Theil, J. Med. Chem., 2004, 47, 1487. [22] T. Luebbers, P. Angehrn, H. Gmuender and S. Herzig, Bioorg. Med. Chem. Lett., 2007, 17, 4708. [23] P. S. Charifson, A.-L. Grillot, T. H. Grossman, J. D. Parsons, M. Badia, S. Bellon, D. D. Deininger, J. E. Drumm, C. H. Gross, A. LeTiran, Y. Liao, N. Mani, D. P. Nicolau, E. Perola, S. Ronkin, D. Shannon, L. L. Swenson, Q. Tang, P. R. Tessier, S.-K. Tian, M. Trudeau, T. Wang, Y. Wei, H. Zhang and D. Stamos, J. Med. Chem., 2008, 51, 5243. [24] T. H. Grossman, D. J. Bartels, S. Mullin, C. H. Gross, J. D. Parsons, Y. Liao, A.-L. Grillot, D. Stamos, E. R. Olson, P. S. Charifson and N. Mani, Antimicrob. Agents Chemother., 2007, 51, 657. [25] N. Mani, C. H. Gross, J. D. Parsons, B. Hanzelka, U. Mueh, S. Mullin, Y. Liao, A.-L. Grillot, D. Stamos, P. S. Charifson and T. H. Grossman, Antimicrob. Agents Chemother., 2006, 50, 1228. [26] S. P. East, C. B. White, O. Barker, J. Bennett, D. Brown, E. A. Boyd, C. Brennan, C. Chowdhury, I. Collins, E. Convers-Reignier, B. W. Dymock, R. Fletcher, D. J. Haydon, M. Gardiner, S. Hatcher, P. Ingram, P. Lancett, P. Mortenson, K. Papadopoulos, C. Smee, H. B. Thomaides-Brears, H. Tye, J. Workman and L. Czaplewski, Bioorg. Med. Chem. Lett., 2009, 19, 894. [27] D. C. D. Butler, H. Chen, V. R. Hedge, C. Limberakis, R. M. Rasne, R. J. Sciotti and J. T. Starr, WO Patent Application 2006/038116 A2, 2006. [28] R. J. Sciotti, J. T. Starr, C. Richardson, G. W. Rewcastle, B. D. Palmer, H. S. Sutherland, J. A. Spicer and H. Chen, WO Patent Application 2005/089763 A1, 2005. [29] D. R. Haydon, L. G. Czaplewski, N. J. Palmer, D. R. Mitchell, J. F. Atherall, C. R. Steele and T. Ladduwahetty, WO Patent Application 2007/148093 A1, 2007. [30] M. H. Block and W. W. Nichols, Mol. Med. Microbiol., 2002, 1, 609.
Recent Advances in the Inhibition of Bacterial Type II Topoisomerases
395
[31] H. J. Boehm, M. Boehringer, D. Bur, H. Gmuender, W. Huber, W. Klaus, D. Kostrewa, H. Kuehne, T. Luebbers, N. Meunier-Keller and F. Mueller, J. Med. Chem., 2000, 43, 2664. [32] T. Luebbers, P. Angehrn, H. Gmuender, S. Herzig and J. Kulhanek, Bioorg. Med. Chem. Lett., 2000, 10, 821. [33] O. Green, H. Ni, A. Singh, G. Walkup, D. Timms, N. Hales, A. Breeze and A. E. Eakin, F1-2025, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [34] B. Sherer, G. Basarab, K. Hull, S. Hauck, S. Bist and A. Eakin, F1-2026, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [35] K. Hull, O. Green, A. Singh, S. Bist, J. Demeritt, J. Loch, G. Mullen, S. Hauck, B. Sherer, H. Ni and A. Eakin, F1-2027, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [36] R. N. Illingworth, M. Uria-Nickelsen, J. Bryant and A. Eakin, F1-2028, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [37] M. R. Uria-Nickelsen, A. Blodgett and A. Eakin, F1-2029, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [38] M. Oblak, S. Golic Grdadolnik, M. Kotnik, A. Poterszman, R. A. Atkinson, H. Nierengarten, D. Desplancq, D. Moras and T. Solmajer, Biochem. Biophys. Res. Commun., 2006, 349, 1206. [39] A. Plaper, M. Golob, I. Hafner, M. Oblak, T. Solmajer and R. Jerala, Biochem. Biophys. Res. Commun., 2003, 306, 530. [40] H. Gradisar, P. Prisovsek, A. Plaper and R. Jerala, J. Med. Chem., 2007, 50, 264. [41] A. S. Wagman and M. P. Wentland, in Comprehensive Medicinal Chemistry II (eds J. B. Taylor and D. J. Triggle), Elsevier Ltd, Oxford, UK, 2006, p. 567. [42] A. J. Mehlholm and D. A. Brown, Ann. Pharmacother., 2007, 41, 1859. [43] L. S. Almer, J. B. Hoffrage, E. L. Keller, R. K. Flamm and V. D. Shortridge, Antimicrob. Agents Chemother., 2004, 48, 2771. [44] G. A. Pankuch, K. Kosowska-Shick, C. R. King and P. C. Appelbaum, C1-189, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [45] C. R. King, L. Lin and R. Leunk, C1-1971, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [46] H.-J. Yun, Y.-H. Min, Y. W. Jo, M.-J. Shim and E.-C. Choi, Int. J. Antimicrob. Agents, 2005, 25, 334. [47] Y. Asahina, K. Kobayasi, M. Takadoi, A. Jojima, K. Ohata, S. Katayama, T. Komine, A. Nakamura, K. Yokota, O. Nagae, T. Sato, T. Shibue, E. Nagata, H. Takano and Y. Fukuda, F1-2121, 47th Annual ICAAC Meeting, Chicago, IL, September, 2007. [48] W. He, K. Amsler, K. Bush and B. J. Morrow, F1-2035, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [49] B. J. Morrow, B. Foleno, R. Goldschmidt, W. He, K. Amsler, M. Maceielag and K. Bush, F1-2052, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [50] H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards, D. Gilbert, L. B. Rice, M. Scheld, B. Spellberg and J. Bartlett, Clin. Infect. Dis., 2009, 48, 1. [51] T. P. Tran, E. L. Ellsworth, M. A. Stier, J. M. Domagala, H. D. Hollis Showalter, S. J. Gracheck, M. A. Shapiro, T. E. Joannides and R Singh, Bioorg. Med. Chem. Lett., 2004, 14, 4405. [52] M. D. Huband, M. A. Cohen, M. Zurack, D. L. Hanna, L. A. Skerlos, M. C. Sulavic, G. W. Gibson, J. W. Gage, E. Ellsworth, M. A. Stier and S. J. Gracheck, Antimicrob. Agents Chemother., 2007, 51, 1191. [53] D. T. W. Chu, P. B. Fernandes, A. K. Claiborne, L. Shen and A. G. Pernet, Drugs Exp. Clin. Res., 1988, 14, 379. [54] W. E. Kohnbrenner, N. Wideburg, D. Weigl, A. Saldivar and D. T. W. Chu, Antimicrob. Agents Chemother., 1992, 36, 81.
396
Gregory S. Bisacchi and Jacques Dumas
[55] Q. Wang, E. Lucien, A. Hashimoto, G. C. G. Pais, D. M. Nelson, Y. Song, J. A. Thanassi, C. W. Marlor, C. L. Thoma, J. Cheng, S. D. Podos, Y. Ou, M. Deshpande, M. J. Pucci, D. D. Buechter, B. J. Bradbury and J. A. Wiles, J. Med. Chem., 2007, 50, 199. [56] M. J. Pucci, C. L. Thoma, S. D. Podos, J. Cheng, J. A. Thanassi, B. J. Bradbury and M. Despande, F1-2021, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [57] J. A. Thanassi, J. Cheng, S. D. Podos, B. J. Bradbury, M. Deshpande and M. J. Pucci, F1-2022, 48th Annual ICAAC Meeting, Washington, DC, October, 2008. [58] L. Pasteur and C. R. Hebd, Seances Acad. Sci., 1853, 37, 162. [59] J. I. Seeman, Angew. Chem. Int. Ed. Engl., 2007, 46, 1378. [60] W. J. Coates, M. N. Gwynn, I. K. Hatton, P. J. Masters, N. D. Pearson, S. S. Rahman, B. Slocombe and J. D. Warrack, WO Patent Application 99/37635 A1, 1999. [61] I. Morrissey, J. Lowther, M. Soltani and J. Northwood, O 457, 17th European Congress of Clinical Microbiology and Infectious Disease, Munich, 2007. [62] M. T. Black, T. Stachyra, D. Platel, A.-M. Girard, M. Claudon, J.-M. Bruneau and C. Miossec, Antimicrob. Agents Chemother., 2008, 52, 3339. [63] P. Levasseur, A. M. Girard and J. Lowther, F1-2125, 47th Annual ICAAC Meeting, Chicago, IL, September, 2007. [64] Press release, June 30, 2008; http://www.novexel.com/ [65] J. J. M. Wiener, L. Gomez, H. Venkatesan, A. Santillan, B. D. Allison, K. L. Schwarz, S. Shinde, L. Tang, M. D. Hack, B. J. Morrow, S. T. Motley, R. M. Goldschmidt, K. J. Shaw, T. K. Jones and C. A Grice, Bioorg. Med. Chem. Lett., 2007, 17, 2718. [66] J. M. Axten, G. Brooks, P. Brown, D. Daview, T. F. Gallager, R. E. Markwell, W. H. Miller, N. D. Pearson and M. Seefeld, WO Patent Application 2004/058144 A1, 2004. [67] T. Kiyoto, J. Ando, T. Tanaka, Y. Tsutsui, M. Yokotani, T. Noguchi, F. Ushiyama, H. Urabe and H. Horikiri, WO Patent Application 2007/138974, 2007. [68] C. Hubschwerlen, G. Rueedi, J.-P. Surivet and C. Zumbrunn Acklin, WO Patent Application 2008/152603, 2008. [69] M. Cronin, B. Geng and F. Reck, WO Patent Application 2009/001126, 2009. [70] J. C. Ruble, A. R. Hurd, T. A. Johnson, D. A. Sherry, M. R. Barbachyn, P. L. Toogood, G. L. Bundy, D. R. Graber and G. M. Kamilar, J. Am. Chem. Soc., 2009, 131, 3991. [71] A. A. Miller, G. L. Bundy, J. E. Mott, J. E. Skepner, T. P. Boyle, A. E. Hromockyj, K. R. Marotti, G. E. Zurenko, J. B. Munzner, M. T. Sweeney, G. F. Bammert, J. C. Hamel, C. W. Ford, W.-Z. Zhong, D. R. Graber, G. E. Martin, F. Han, L. A. Dolak, E. P. Seest, J. C. Ruble, G. M. Kamilar, J. R. Palmer, L. S. Banitt, A. R. Hurd and M. R. Barbachyn, Antimicrob. Agents Chemother., 2008, 52, 2806.
CHAPT ER
20 Progress towards the Discovery and Development of Specifically Targeted Inhibitors of Hepatitis C Virus Nicholas A. Meanwell, John F. Kadow and Paul M. Scola
Contents
1. Introduction 2. Specifically Targeted Inhibitors of HCV 2.1 HCV NS2 protease inhibitors 2.2 HCV NS3/4A protease inhibitors 2.3 HCV NS3 helicase inhibitors 2.4 HCV NS4B replication factor inhibitors 2.5 HCV NS5A replication factor inhibitors 2.6 HCV NS5B polymerase inhibitors 2.7 Emerging mechanisms 3. Conclusions References
397 398 398 400 407 408 409 412 420 427 428
1. INTRODUCTION The incidence of hepatitis C virus (HCV) infection in the United States (U.S.) is estimated to be approaching 5 million, against a backdrop of 150–200 million worldwide, with the majority of individuals unaware of infections that are not sufficiently advanced to cause overt liver disease [1]. Department of Chemistry, Bristol-Myers Squibb Pharmaceutical Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04420-0
r 2009 Elsevier Inc. All rights reserved.
397
398
Nicholas A. Meanwell et al.
The current standard of care (SOC) comprises weekly subcutaneous injections of pegylated interferon-a2a (PEG-IFN-a2a) (Pegasyss) or -a2b (Pegintrons) in combination with twice daily doses of ribavirin, a regimen poorly tolerated due to the association with a significant incidence of side effects. Moreover, this regimen is ineffective in achieving a sustained virologic response (SVR), defined as the absence of viremia 6 months after completion of therapy, in the majority of patients infected with genotype 1 HCV, which is most prevalent in the U.S. [2,3]. Several IFN derivatives designed to improve tolerability are in clinical development [4], including albinterferon-a2b (Albuferons), a protein that fuses IFN-a2b with human albumin to enhance the in vivo half-life and reduce dosing frequency [5], pegylated IFN-l (interleukin 29), which offers increased specificity towards the liver in vivo [6], and Locterons, a sustained release formulation of IFN-a2b [7]. However, the antiviral activity of IFNs is indirect, and ribavirin is a non-specific antiviral agent with inhibitory activity towards some host cell proteins, a circumstance that has contributed to the considerable effort being expended to identify and develop specifically targeted antiviral therapies for HCV (STAT-C) [8–15]. Despite disappointments with the failure of several early candidates in clinical trials [16,17], there is currently a robust and expanding pipeline of specific HCV inhibitors in clinical trials or late-stage preclinical testing, which target a range of viral proteins and RNA, summarized in Table 1 [8–15,18–20]. Of particular concern with STAT-C is the potential for resistance to emerge during or as a consequence of therapy based on the high rate and poor fidelity of replication of HCV [21–27], an anxiety that anticipates combination therapy with two or more antiviral agents [28,29]. However, whether a combination of STAT-C will be able to achieve a SVR in the absence of immune stimulation awaits the results of well-controlled clinical trials. The first clinical assessment of a combination of small molecules was initiated in late 2008 with a 14-day study designed to evaluate the safety and efficacy of an NS3 protease inhibitor and a polymerase inhibitor in treatment-naı¨ve patients infected with genotype 1 virus [30]. In this review, we provide a synopsis of progress made towards identifying and developing inhibitors of HCV during the period January 2007 to April 2009 with the most advanced compounds summarized in Table 1 and discussed in the individual sections.
2. SPECIFICALLY TARGETED INHIBITORS OF HCV 2.1 HCV NS2 protease inhibitors The NS2 protein is essential for the production of infectious virus, and a crystal structure of the protease catalytic domain has revealed it to be a
Table 1
The HCV inhibitor clinical landscape NS3/4A
Phase III Phase II
NS5A
Telaprevir Boceprevir BI-201335
R-7128 — nucleoside analogue active site PF-868554 — thumb domain site 2
ABT-450
BMS-790052
VBY-106
A-831/AZD-2836 A-689/AZD-7295
IDX-136 IDX-316
Albuferon albumin/IFN-a fusion protein Locteron extended release formulation of IFN-a 2b
NIM-811 cyclosporin analogue DEBIO-025 cyclosporin analogue SCY-635 cyclosporin analogue Nitazoxanide host cell target Celgosovir a-glucosidase inhibitor Silibinin VCH-222 — thumb domain site 2 PEG-IFN l (IL-29) liver-targeted IFN derivative MK-3281 — thumb domain site 1 IDX-184 — liver targeting nucleoside prodrug IDX-375 — palm domain site 1 ABT-333 — palm domain site 1 ABT-072 — palm domain site 1 GL-60667 (LDI-133) — thumb domain site 1
399
Preclinical
VCH-759 — thumb domain site 2 VCH-916 — thumb domain site 2 ANA-598 — palm domain site 2 GS-9190 — replication complex inhibitor
Miscellaneous
Discovery and Development of Inhibitors of HCV
ITMN-191/ R-7227 TMC-435350 MK-7009 SCH-900518
Phase I
NS5B
400
Nicholas A. Meanwell et al.
cysteine protease that functions as a dimer [31,32]. The two composite active sites interact cooperatively, with the catalytic histidine and glutamate residues from one domain working in concert with the catalytic cysteine from the second domain. Interestingly, although the protease domain of NS2 is essential for the in vitro production of infectious virus, its enzymatic activity is not required [31,33]. Additional insights into the biological functions of NS2 have been described [34–37], and assays designed to screen for inhibitors of the protease activity have also been disclosed [38,39], but no well-characterized inhibitors of this enzyme have been described to date.
2.2 HCV NS3/4A protease inhibitors Proof-of-concept for NS3 protease inhibitors as antiviral agents with potential for the treatment of HCV was provided by clinical studies conducted with BILN-2061 (1). This compound was administered to patients infected with genotype 1 virus using a dosing regimen of 200 mg BID for 2 consecutive days, which produced a greater than 2–3 log10 reduction in plasma HCV RNA in all patients treated [40]. Since this seminal study, a significant number of protease inhibitors have progressed into clinical trials and results from these investigations continue to shape and refine treatment strategies designed to optimize long-term efficacy (SVR) and minimize the emergence of resistant virus [8,41–45]. Telaprevir (VX-950, 2) is the most advanced protease inhibitor, having recently entered into phase III clinical trials. Telaprevir (2) is a potent and selective, mechanism-based inhibitor of the NS3/4A enzyme that forms a covalent, but reversible complex with the enzyme [46]. Binding studies conducted with 2 and a genotype 1a HCV protease indicate that dissociation of the enzyme–inhibitor complex occurs slowly, with a halflife of approximately 1 h. Telaprevir (2) exhibits low to moderate oral bioavailability in both rat (25%) and dog (41%), with liver exposure in the rat exceeding plasma levels by a factor of 35. Me S MeO
Me
N
N
N
H
H
O H H N
O
N
N O
O OH
N
O N
O
1
H H
O H N H
N
N O Me
O Me Me
2
O
H
N O
N O
Me
Discovery and Development of Inhibitors of HCV
401
In a seminal phase I clinical study, genotype 1 patients receiving a 750-mg TID dose of telaprevir (2) experienced a median 4.4 log10 reduction in HCV RNA after 14 days of treatment, with some patients achieving undetectable levels of HCV RNA [47]. Subsequent clinical studies have confirmed the antiviral activity of telaprevir (2) while also providing evidence for the rapid emergence of resistance during the course of monotherapy [48,49]. In a phase II study, genotype 1 patients were randomly divided into three treatment groups and dosed for 14 days with polyethyleneglycol (PEG)-IFN, telaprevir (750 mg, TID) or telaprevir+PEGIFNa before optionally evolving to a SOC regimen. The median change in HCV RNA observed in each of these dosing groups was 1.09 log10, 3.99 log10 and 5.49 log10, respectively. Importantly, viral breakthrough was observed in four of eight patients dosed with telaprevir (2) alone while, in contrast, all eight patients treated with telaprevir+PEG-IFNa experienced a continuous or sustained drop in viral load. Interestingly, samples of virus from patients who displayed viral rebound were shown to vary in their genetic constitution as a function of time. For example, on day 4, wild-type virus was dominant with the single mutants V36/A/M, R155K/T and A156V/T observed exclusively but at low levels (5–20%). The population of these variants increased in samples isolated on days 8 and 12, but by day 15, these single mutants were replaced with high-level resistant double mutants (V36/R155). A subset of samples from patients receiving both telaprevir (2) and PEG-IFNa showed the presence of a A156T variant that demonstrates high-level resistance in vitro but breakthrough was not observed. In separate studies, the double viral mutant V36/R155 produced on therapy was shown to be slightly more fit than single mutants such as R155K/T [50]. Although these early clinical studies demonstrate the acute antiviral activity of telaprevir (2) and its potential as a therapeutic agent in combination with IFN, they also reveal the limitation of the drug as monotherapy for the treatment of HCV due to the rapid emergence of resistant virus. These general observations have been upheld in subsequent clinical trials with a series of protease inhibitors (vide infra). In a phase II clinical study, the safety and efficacy of telaprevir (2) in combination with PEG-IFNa, with or without ribavirin, in chronic HCV patients was evaluated [51]. The results from this study demonstrate that the addition of telaprevir (2) to PEG-IFNa/ribavirin (RBV) may allow for a significant reduction in overall treatment time from 48 to 24 weeks. Specifically, treatment-naı¨ve genotype 1-infected patients upon administered telaprevir+PEG-IFN/RBV for 12 weeks followed by PEG-IFNa/RBV for an additional 12 weeks (24 weeks total therapy) collectively experienced a SVR rate of 69%, which compared to a 46% SVR in the 48 week SOC arm. In addition, ribavirin was shown to be a critical component of this dosing triad since patients receiving telaprevir+PEGIFNa but not ribavirin experienced significantly greater viral breakthrough
402
Nicholas A. Meanwell et al.
at week 12 when compared to patients receiving telparevir+PEG-IFNa/ RBV. The profile of telaprevir (2) will be confirmed and expanded upon in the ongoing PROVE 3 study [52]. Boceprevir (SCH-503034, 3) has also entered phase III clinical trials. Analogous to telaprevir (2), boceprevir (3) is a mechanism-based inhibitor of the NS3/4A protease that demonstrates a Ki* of 14 nM [53,54]. The selectivity of boceprevir (3) for HCV NS3/4A over human neutrophil elastase is thought to be derived, in part, from the P1 cyclobutylalanine moiety [53,54]. Boceprevir (3) demonstrates an EC90 of 350 nM in a replicon assay and exhibits an oral bioavailability of 34, 26, 30 and 4% in the mouse, rat, dog and monkey, respectively. In rat, liver levels of the compound exceeded that of plasma by a factor of 30. In clinical trials, boceprevir (3) has been studied as add-on therapy to PEG-IFNa/RBV [55,56]. In the SPRINT-1 trial, boceprevir (3) administered to genotype 1 patients at a dose of 800 mg TID in combination with SOC for 28 or 48 weeks produced SVRs of 55 and 66%, respectively, an improvement over the 38% SVR observed in the control group who received PEG-IFNa/RBV for 48 weeks. Two additional cohorts in this study were dosed with a leadin regimen of PEG-IFNa/RBV for 4 weeks, which was followed by treatment with boceprevir (800 mg TID)+PEG-IFNa/RBV for 24 or 44 weeks. The SVRs reported in these studies were 56 and 74%, respectively, which again provided a more favorable outcome than the 38% SVR observed with SOC. The lead-in dosing with SOC appears to reduce the incidence of viral breakthrough in both cohorts. Phase III studies are ongoing to further evaluate treatment options with boceprevir (3). A second-generation protease inhibitor, SCH-900518, that demonstrates improved potency [57] has advanced into clinical trials, and while the structure of this compound has not been released, more general efforts to further optimize this chemotype have been described [58–61]. Me MeO Me
Me Me
Me
N
N O Me
N
Me
O
O
N H
Me
Me H
H
S N
N O O
Me
H
NH2
N
O Me
O N
O
O O S N H
O
Me
3
4
TMC-435350 (4) is a macrocyclic tripeptide derivative that incorporates a functionalized cyclopentyl moiety as a replacement for the P2 proline found in most of the highly potent HCV NS3/4A inhibitors. In addition,
403
Discovery and Development of Inhibitors of HCV
this compound employs an acylsulfonamide as a carboxylic acid isostere that interacts with the catalytic elements and allows the cyclopropyl ring to project into the S1u pocket of the protease [62,63]. In preclinical studies, TMC-435350 (4) demonstrated potent inhibition of a genotype 1b replicon, EC50 ¼ 8 nM, an oral bioavailability of 11–44% and up to 100% in rat and dog, respectively, and a liver to plasma area under the curve (AUC) ratio of greater than 35 in the rat [62,63]. In a phase IIa clinical trial designated OPERA-1, patients infected with genotype 1 virus were dosed daily with either 25 or 75 mg of TMC-435350 (4) for 4 weeks in conjunction with PEG-IFNa/RBV and compared to an arm in which there was a 7-day lead in with TMC-435350 (4) as monotherapy [64]. At day 7, viral loads were reduced by 3.47 and 4.55 log10 in the 25 and 75 mg triple therapy arms, respectively, whereas monotherapy was less efficacious, producing a 2.63 and 3.43 log10 reduction in viremia at the 25 and 75 mg doses, respectively [64]. Further evaluation of TMC-435350 (4) in clinical trials is ongoing. ITMN-191 (R-7227, 5), a tripeptidic acylsulfonamide incorporating the P1 and P3 moieties into a 15-membered macrocyclic ring, is currently in phase II clinical trials. ITMN-191 (5) displays enzyme kinetics that appear to be unique for this structural class, indicative of a slow/tight binding mechanism with slow dissociation, which may suggest a complementary conformational change within the enzyme subsequent to the initial binding event [65,66]. ITMN-191 (5) exhibits excellent potency in a genotype 1b replicon, EC50 ¼ 2 nM, and the concentration of compound in the liver exceeds that of plasma after oral dosing, with an AUCobs ratio of B10 in the rat and B127 in the monkey. In clinical studies, genotype 1 HCV-infected patients were treated with ITMN-191 (5) BID or TID for a period of 14 days, with the most significant antiviral response observed in the 200 mg TID cohort where a 3.9 log10 mean reduction in viral load was observed [67]. Ongoing clinical studies are evaluating this compound as add-on to SOC, whereas a pioneering study initiated in Australia and New Zealand late in 2008 is evaluating the safety and efficacy of ITMN-191 (5) and the polymerase inhibitor R-7128 (vide infra) in treatment-naı¨ve patients infected with genotype 1 virus over 14 days, with promising initial results [30].
N
F
O
N
O
O H
Me Me
H N
O Me
O
N
N O
O
O
O
O O S N H
H Me Me
O
H N O Me
5
N
N O Me Me
6
O
O
O O S
N H
Me
404
Nicholas A. Meanwell et al.
MK-7009 (6), also in phase II clinical trials, incorporates a structurally unique P2-P4 macrocycle designed to improve potency compared to analogous acyclic compounds [68]. MK-7009 (6) is a rapidly reversible enzyme inhibitor that is potently active in a genotype 1b replicon, EC50 ¼ 4 nM. In phase 1 clinical trials conducted in genotype 1 HCVinfected patients, QD and BID dosing regimens of the compound were examined for a duration of 8 days, with the optimal antiviral response observed in the 700 mg BID cohort, which produced a 4.6 log10 mean reduction in HCV RNA [69,70]. Phase II studies are ongoing to determine the efficacy of MK-7009 in combination with SOC. Clinical data have been recently reported for BI-201335 (structure not disclosed) administered QD to genotype-1-infected patients for 14 days at doses of 20, 48, 120 and 240 mg [71]. In each cohort, the maximal viral response was observed between day 2 and day 4, with the greatest effect on mean viral load seen in the 240 mg cohort where HCV RNA declined by 4 log10. However, virologic breakthrough was observed during treatment in the majority of patients at all doses. In a separate study, BI-201335 was administered QD at doses of 48, 120 and 240 mg in combination with PEG-IFNa/RBV for 28 days to treatment-experienced genotype 1 patients, producing median changes in HCV RNA of 5.0 log10, 5.2 log10 and 5.3 log10, respectively [72]. Virologic breakthrough was observed in two of six patients in the 48 mg dose cohort and one of seven in the 120 mg dose group but no breakthroughs were observed in the 240 mg cohort during the 28 days of treatment. At higher doses, an increased incidence of unconjugated hyperbilirubinemia was noted. Several additional HCV NS3 protease inhibitors have entered early clinical development or are in late-stage preclinical studies, including ABT-450, VBY-106, IDX-136 and IDX-316, the structures of which have not been disclosed. HCV NS3 protease inhibition continues to be an area of considerable interest with a number of publications and patent applications describing potent compounds that advance structure–activity relationships. A survey of acylsulfonamides as isosteres of the terminal carboxylic acid moiety revealed that the significantly increased potency in both enzymatic and cell-based assays was dependent on the pKa and implicated a hydrogen bond between one of the sulfone oxygen atoms and the amide NH of Q41 and backbone NH of G137 of the enzyme [73]. Optimal activity was observed with compounds bearing a P1 cyclopropyl amino acid moiety, with a vinyl substituent imparting a significant increase in potency, as demonstrated by the comparison between 7 and 8.
405
Discovery and Development of Inhibitors of HCV MeO
N
Ph Me
Me
O H
O
N H Me
O
N O
N
O
Me Me
O Me
H O O S Ph N
N
H
O
H
N
O
H
H
N
N
R
N
N O
O
O
O
O Me
7, R = H, Ki = 0.055 µM 8, R = CH = CH2, Ki = 0.00076 µM
9
Optimization of ketoamide-based NS3/4A inhibitors has focused on the P4 capping element with the 4,4-dimethyl-substituted glutarimide derivative 9 significantly more potent than a simple tert-butyl urea cap [58,60]. This compound demonstrated an EC90 in the HCV replicon screen of 30 nM, which represents a 12-fold increase in cellular potency compared to boceprevir (3) and appears to be a function of an additional hydrogen bond between one of the imide carbonyl groups and C159 of NS3, observed in the X-ray structure of 9 bound to the enzyme. Further studies in this series have explored P1–P3 macrocycles [58,60]. Tripeptide derivatives that present a boronic acid as the electrophilic trap for the catalytic serine of NS3 have been described, with 10 demonstrating excellent intrinsic inhibitory activity towards HCV NS3, Ki ¼ 200 pM [61]. An X-ray structure of 10 bound to the enzyme revealed a Lewis acid complex engaging the boron atom and the oxygen of S139 of the enzyme. However, the cellular activity of this compound is limited, with an EC90 of W5.0 mM, indicative of poor membrane penetration [61]. Additional patent applications claim acyclic compounds with P2 moieties based on 4-hydroxy proline [74,75]. Me
O O S Me N Me
H
H
N
Me
N
N O
H
OH
N
B
OH
O O
Me Me Me
10
Several novel P2 elements have been elucidated in acyclic tripeptide inhibitors, including the biphenylated proline incorporated in 11, with
406
Nicholas A. Meanwell et al.
optimal compounds claimed to demonstrate enzyme inhibition with EC50 of o50 nM [76,77]. Additional P3 caps have also been described in which a phenyl ring or a heterocycle replaces the more common amide or carbamoyl moiety, with compounds similar to 12 exhibiting NS3 inhibitory activity at concentrations o100 nM [78]. OMe Ph N
Cl OMe H H Me Me
O Me
O Me
N
N
N
O
O
O
O O O S N H
H H MeO
N
N
Me Me
O
Me
O
N
O O S
N
O
H
Me Me
11
12
Efforts to replace the P2 proline group in the tripeptide series have been attempted in the context of the macrocycles 13 and 14, with the former exhibiting a Ki of 76 nM [79,80]. Although these compounds appear to be less potent than the analogous proline derivatives, they may serve as useful starting points for further efforts in this area. MeO
N
Ph
Me
S
Me MeO
N
N
Me
O O H O
Me Me
O
H
N
N N
Me
O
H
13
O
O
O O S N H
O Me N
H N
N H
O
O
O O Me S
N H
14
Interesting macrocyclic inhibitors that explore a tether between the phenyl moiety of an acylsulfonamide P1u and either P1, as exemplified by 15, or P3 have been described although the success of this strategy is not immediately apparent [81,82].
407
Discovery and Development of Inhibitors of HCV
Me
H N S MeO
Me N
N
O H
O
N H Me
N O
N
O O S N H
O O Me
HN
Me 15
ACH-806 (16) has been described as a novel inhibitor of NS3/4A activity with resistance mapping to NS4A [83]. This compound is a potent inhibitor in replicons, fully retaining activity towards replicons resistant to active site inhibitors of NS3. In HCV genotype-1-infected patients, a dose of 300 mg BID of ACH-806 (16) for 5 days produced a 0.9 log10 reduction in HCV RNA, providing clinical proof-of-concept for an NS4A inhibitor [84]. However, development of ACH-806 (16) was terminated due to an elevation of serum creatinine levels, a marker of kidney toxicity [84]. More recently, a second compound, ACH-1095 (undisclosed structure), has entered clinical trials. O O
O
S N H
N H
Me
Ph
O
OH
CF3
N
Me F
N
16
17
2.3 HCV NS3 helicase inhibitors It is well established that the helicase function of NS3 is required for in vivo replication of infectious virus, which is dependent, in part, on the RNA unwinding activity of the enzyme [85]. Although the precise role of this enzyme in the HCV life cycle is not fully understood, the extent of
408
Nicholas A. Meanwell et al.
helicase binding and hydrolysis of adenosine triphosphate (ATP) have been linked to RNA translocation while unwinding studies are suggestive of an inchworm-type mechanism [86]. The NS3 protease and NS3 helicase activities have been shown to be interdependent, with the helicase domain enhancing serine protease activity and the protease domain enhancing helicase activity [87]. Although these findings reflect significant gains in the understanding of NS3 helicase function and mechanism, there are only limited reports of small molecule inhibitors of this essential enzyme despite the availability of an X-ray crystallographic structure [88]. A recent patent application claims a series of indole-based inhibitors, represented by 17, as inhibitors of NS3 helicase function with an IC50 of less than 10 mM [89].
2.4 HCV NS4B replication factor inhibitors HCV NS4B is a protein integral to the endoplasmic reticulum membrane that is predicted to be composed of four transmembrane domains with a topology that projects the N- and C-termini into the cytoplasm of the host cell [18]. Two C-terminal cysteine residues have been shown to be palmitoylated and the protein appears to be capable of forming oligomers. HCV NS4B is critical for viral replication and is postulated to form a web in the membrane that functions as a scaffold for the assembly of the virus replication complex, which includes NS5B and NS3 [90,91]. The HCV NS4B protein has been shown to hydrolyze both ATP and guanosine triphosphate (GTP), with the former the better substrate based on a 25-fold kinetic advantage, and express adenylate kinase activity, properties that map to a Walker nucleotide-binding domain [91]. This domain is required for viral replication and also appears to mediate cellular transformation, providing the basis for a cell-based assay to detect inhibitors of NS4B [92,93]. Small-molecule inhibitors of HCV NS4B are just beginning to emerge. A hypothesis that NS4B binds RNA was substantiated with the development of a binding assay based on microfluidic technology, which revealed a specific and high-affinity interaction with the 3u-terminus of the viral negative RNA strand, Kd ¼ 3.4 nM [94]. The binding sites on the protein mapped to an arginine-rich motif in the last 71 amino acids of the cytoplasmic C terminus. A high-throughput screen identified the histamine H1 antagonist clemizole (18) as a potent inhibitor of NS4BRNA binding, IC50 ¼ 24 nM, that demonstrated activity in a replicon, EC50 ¼ 8 mM. Resistance was mapped to W55R located in the cyctoplasmic amino terminus and R214Q in the C-terminal cytoplasmic domain. Mutant replicons were less fit than wild-type and the individually mutated 4B proteins demonstrated higher affinity for viral RNA, Kd ¼ 0.75 nM for W55R and Kd ¼ 0.6 nM for R214Q. The introduction of methyl substituents at C-5 and C-6 of the benzimidazole leads to increased inhibitory potency in the replicon, EC50 ¼ 1 mM [95,96].
409
Discovery and Development of Inhibitors of HCV N N
H3C N
N N N
Cl CH3
F
N
N N
CH3
N
18
19
O S HN
N CH3
Cl
F
N
O
20
A U.S. patent application discloses a range of structurally diverse compounds claimed to be inhibitors of NS4B, which were discovered by a screening campaign that exploited a binding assay monitoring changes in the intrinsic fluorescence of NS4B [97]. The triazinoindole 19 was active in a 1b replicon assay with an EC50 ¼ 1.13 mM. Resistance mapped to G120V in the second transmembrane domain of NS4B and K52R and A210S located in the N- and C-terminus cytoplasmic domains of the protein. The activity of the pyrazolopyridine anguizole (20), also disclosed in this patent application, has been confirmed [98]. This compound inhibits both 1a and 1b replicons with EC50 of 560 and 310 nM, respectively, with resistance mapped to H94A of NS4B, a residue immediately preceding the first transmembrane domain of the protein.
2.5 HCV NS5A replication factor inhibitors HCV NS5A is a 447 residue phosphoprotein that plays a critically important role in virus replication, assembly and egress, but the function of this protein is extraordinarily complex as a consequence of extensive interactions with both viral and host cell proteins [20,99–101]. HCV NS5A is composed of three domains: domain I, composed of residues 1–213, which incorporates an amphipathic N-terminus helix thought to be involved in membrane association [102], a zinc-binding motif and binding regions for both HCV NS4A and NS5B; domain II that encompasses residues 250–342 and contains the putative IFN sensitivitydetermining region (ISDR) [103] in addition to sites of interaction with HCV NS5B and several host cell factors; domain III, residues 356–447, which has recently been associated with an important role in virus assembly and is regulated by phosphorylation [104–106]. Although HCV NS5A does not express enzymatic function, targeted efforts have focused on disrupting activity associated with the N-terminal helix [102,107] while screening campaigns using HCV replicons have surfaced several classes of inhibitor for which resistance has been mapped to domain I [99]. SWLRDIWDWICEVLSDFK, an 18 mer peptide derived from residues 3–20 of the amphipathic a-helical N-terminus membrane anchor domain of a genotype 1a virus and designated C5A, inhibits replication of JFH1
410
Nicholas A. Meanwell et al.
infectious virus in cell culture with an EC50 of 0.79 mM [107]. The D-amino acid analogue performs similarly but scrambled peptides are inactive. A series of biochemical studies led to the suggestion that these peptides bind to viral membranes and compromise integrity, leading to exposure of the viral genome to exonucleases [107]. Consistent with this hypothesis, C5A also inhibits human immunodeficiency, dengue, respiratory syncytial, West Nile and measles viruses with similar potency [107,108]. Small-molecule inhibitors of HCV NS5A that encompass several structural classes have been disclosed with three compounds, A-831 (AZD-2836), A-689 (AZD-7295) and BMS-790052 (structures not disclosed), advanced into clinical studies [109,110]. However, A-831 was recently withdrawn from clinical evaluation pending reformulation, whereas data on A-689 have not been released. In genotype 1 HCV-infected patients, single 1, 10 and 100 mg doses of BMS-790052 produced impressive 1.8 log10, 3.2 log10 and 3.6 log10 mean reductions in viral load, respectively, with efficacy at the 100 mg dose persisting for 144 h [110]. BMS-790052 is a potent inhibitor of genotype 1a and 1b RNA replication in replicons, EC50 ¼ 50 and 6 pM, respectively, and infectious genotype 2a virus, EC50 ¼ 12 pM [20,110]. Several recent patent applications claim a series of biphenyl-based inhibitors of HCV NS5A, with 21 representative [111–114], whereas an alternative patent estate focuses on two general themes that have been explored broadly, exemplified by the quinazoline 22 and amide 23 [115–120]. NH2 N CH3
N
H3C H3CO2CHN
NH
N
O
O
HN
H N
N
O
CH3 N
N
CH3
21
N F H3C
O
O
N
HN N
H3C
O
N N
N CH3
N
22
CH3
N
N H
NH
O
N
N
O
23
CH3
S O
411
Discovery and Development of Inhibitors of HCV
A series of NS5A inhibitors based on proline has been extensively explored with GL-101267 (24) representative of the basic chemotype, a molecule that demonstrates an EC50 of 80 nM in the replicon assay [99,121–124]. However, the most recent patent application discloses a series of amides, represented by 25, that bear some structural resemblance to 23 [125]. O
N N H
H N
N S
N O
24
O O
Ph
CH3 O
O
S
N
O
N N
N H
N
O S
O
N
NH
H3C S
O 25
A series of HCV NS5A inhibitors based on homoproline and its isosteres have been claimed, with 26 a representative example that demonstrates an EC50 of o1 mM in a genotype 1b HCV replicon assay [126]. However, resistance mapping identified mutations not only in NS5A (Y93H) but also in NS3, NS4A and NS4B.
CH3
H N
HO
O
N O
H N
O
O N
Boc
N
O
HN
OH
Ph
N N
26
27
H3C
Using a replicon assay, a series of piperazine-based inhibitors of replication was identified from which 27 is the most potent, EC50 ¼ 160 nM, with
412
Nicholas A. Meanwell et al.
activity dependent on the presence of the phenol moiety [127]. Resistance was mapped to A92V, Y93H and R157W of NS5A, residues that are located at the dimer interface of domain 1 in the X-ray crystallographic structure, suggesting that these molecules may modulate the dimerization process either by interfering with protein association or exerting a stabilizing effect on the dimer [127,128].
2.6 HCV NS5B polymerase inhibitors HCV NS5B is the virus-encoded RNA-dependent, RNA polymerase (RdRp), possessing enzymatic activity that is responsible for the synthesis of viral RNA, a fundamental and critical step in replication. Inhibitors of NS5B that target the active site (nucleoside derivatives) or one of four distinct allosteric binding sites have been characterized, and representative inhibitors of each site have been clinically validated [8,129–131]. Reflecting the classical nature of this enzyme as an antiviral target and the multiple biochemical opportunities for intervention, studies in the NS5B inhibitor field have been vigorous, spawning a number of clinical candidates [8,16,17]. However, this mechanistic class has been associated with considerable attrition in the early stages of development [8,16,17]. Nevertheless, multiple compounds continue to progress through clinical studies, although the structures of several remain proprietary (Table 1). Panels of sensitive and resistant viruses from multiple HCV genotypes have been used to characterize the binding sites of NS5B inhibitors and understand interactions between the different classes or with other HCV inhibitors in vitro [132,133]. In a comparative study, the intrinsic potency of NS5B inhibitors representing several classes was found to be similar to that of protease inhibitors, IFN or cyclophilinbinding molecules [133]. On the basis of in vitro studies, a failure to detect pre-existing insensitive variants or the S282T mutant that arises in response to several nucleoside inhibitors in clinical studies, resistance to active site NS5B inhibitors may be much less likely to arise from therapy than for allosteric NS5B or NS3 protease inhibitors [27,134]. Moreover, studies in replicons have demonstrated that resistant mutants arising in response to nucleoside inhibitors exhibit reduced fitness compared with replicons resistant to allosteric NS5B inhibitors [27,134]. This raises the specter of clinical resistance with allosteric NS5B inhibitors and the overall consensus of multiple in vitro studies is that, despite excellent potency, the pre-existence or development of relatively fit resistant mutants will dictate that these molecules be used in combination therapy [24]. In vitro studies with combinations of two or three drugs that contain at least one replicase inhibitor have shown synergistic interactions [27,28].
413
Discovery and Development of Inhibitors of HCV
2.6.1 Nucleoside inhibitors The most advanced active site NS5B inhibitor in clinical development is R-1728 (28), the bis-isobutyl ester prodrug of 2u-deoxy-2u-fluoro-2u-Cmethylcytidine (PSI-6130). Interestingly, metabolism of PSI-6130 proceeds through a bifurcated pathway, with HCV inhibitory activity believed to be a function of the triphosphate of both PSI-6130 and the corresponding uridine analogue [135–137]. The latter compound, PSI-7851 (29, precise structure not disclosed), is being developed independently as a phosphoramidate prodrug of the monophosphate [138]. As monotherapy at a dose of 1500 mg BID for 14 days, R-1728 (28) produced a 2.7 log10 reduction in viral RNA, whereas 88% of patients taking 1000 mg BID of the drug in combination with SOC for 28 days followed by SOC for 44 weeks achieved undetectable RNA levels compared to 19% in the placebo group, with viremia reduced by 5.0 log10 at 28 days [139]. NH2 O
O
N
O
N O
R2 O
R3OOC
NH
O N P O H OR1
N
CH3 O O
F 28
O
O CH3 HO
F 29
In preclinical studies, PSI-7851 demonstrated 15–20-fold greater potency than PSI-6130, with an EC50 of 0.31 mM in the replicon compared to 4.8 mM for PSI-6130, and the compound effectively targets the liver in vivo, resulting in high levels of the active triphosphate [138]. Dosing of PSI-7851 (29) in a phase I SAD trial in healthy volunteers was initiated in March 2009. Development of two cytidine-based nucleoside analogues, R-1626 (30) and valopicitabine (31), both being developed as prodrugs, were discontinued due to toxicity [17,140–142]. R-1626 (30) was associated with an unacceptable level of grade 4 neutropenia observed during a 4-week study of the triple combination of the compound (1500 mg BID) in conjunction with SOC while valopicitabine (31) caused gastrointestinal side effects [17]. MK-0608 (32) has also been abandoned for undisclosed reasons despite showing good efficacy in HCV-infected chimpanzees [17,143]. Nevertheless, this area continues to be of interest, and a liver-targeted nucleoside prodrug designated IDX-184 has recently
414
Nicholas A. Meanwell et al.
advanced into phase I clinical studies [144] while R-1728 (28) is being investigated in combination with the NS3 protease inhibitor ITMN-191 (5) [30]. NH3+Cl−
NH2 O
N
N O
N
O
NH2
N
HO
O
N O
HO
N
O
O
O
O N3
O O
O
CH3
CH3
H2N
O
30
N
OH
HO
31
OH
32
2.6.2 Allosteric, non-nucleoside inhibitors 2.6.2.1 Thumb site I inhibitors. Thumb site 1 inhibitors bind to an allosteric pocket normally occupied by the polymerase finger loop, an interaction critical to the initiation of RNA synthesis, and are characterized by resistance arising by mutation at P495, P496 or V499. Clinical proof-ofconcept for inhibitors that target this site was obtained with BILB-1941, a compound of undisclosed structure that was terminated due to gastrointestinal side effects [145]. Inhibitors that target this site have been extensively investigated and are based on chemotypes that incorporate a 6,5 fused heterocyclic core, typically an indole or a benzimidazole, with a cyclohexyl substituent at the one position of the five-membered ring, a polar carboxamide-based substituent at C-6 and an aromatic ring at C-2, as represented by 33 [8,129–131,146,147]. JTK-109 (34) was advanced into clinical studies and is representative of early inhibitors based on this chemotype that have been the focus of further optimization [148]. Cl O
R2 6
Y
R3
X
2
R1
F HO2C
N O N N
33
34
415
Discovery and Development of Inhibitors of HCV
This chemotype has been pursued with some vigor leading to compounds 35 and 36 as representative of a broad patent and literature estate [149–151]. MK-3281 (structure not disclosed) has emerged as a potent inhibitor of a 1b replicon, EC90 in 50%HS ¼ 241 nM, which has been advanced into clinical studies [152]. MK-3281 was generally well tolerated in humans, exhibiting a pharmacokinetic profile predictive of a BID dosing regimen in infected patients, consistent with observations in HCV-infected chimps that experienced a 3.8 log10 (1b) and a mean 1.4 log10 (1a) reduction in viral RNA when dosed at 10 mg/kg BID, with no rapid emergence of resistance observed [153]. NEt2
NMe2 H
O O Me2N
O S
N H
H
HO2C
N
N
N
O
OMe
35
36
GL-60667 (LDI-133, 37), a potent inhibitor of HCV replication in vitro, EC50 ¼ 75 nM (1b) and 130 nM (1a), has been reported to have a preclinical profile suitable for development [154]. A series of patent applications claim polycyclic NS5B inhibitors with structures consistent with a thumb site 1 target, with 38 representative of a cyclopropylamide-based chemotype [155–161]. More recent applications claim compounds in which the amide moiety is replaced by a heterocycle, as exemplified by 39 [162]. O
O O
N O HO2C
O
O H3C
N N
N S
37
H3C
N
S
O N
HN
CH3 CH3
38
N
416
Nicholas A. Meanwell et al.
H3CO N N
H3C O H3C
O
CH3
CH3
CH3 O
S O
N
N
N H
OCH3
39
2.6.2.2 Thumb site II inhibitors. The second thumb site is a hydrophobic pocket at the base of this domain that is characterized by resistant mutations emerging at M423 or L419 in response to exposure of replicons to these inhibitors. The discovery and preclinical profile of filibuvir (40), currently in phase II clinical studies with SOC, has been described in some detail [163,164]. Filibuvir (40) is a potent 1b replicon inhibitor, EC50 ¼ 41 nM, that demonstrates good aqueous solubility, 2.5 mg/mL, and no cytochrome P (CYP) inhibition liabilities [163]. In HCV-infected individuals, filibuvir (40) administered as monotherapy for 8 days, reduced mean plasma viral RNA by 0.97, 1.84, 1.74 and 2.13 log10 at doses of 100, 300, 450 mg BID and 300 mg TID, respectively [165]. A number of subjects experienced a plateau or rebound in HCV RNA after an initial rapid reduction, attributed to mutations arising at M423 [165]. HO OH N Et
O
O
N
CH3
CH3
N
N
N
N
CH3 Et 40
H3C H3C CH 3
S
O COOH 41
The second major class of NS5B inhibitor binding to thumb site II is based on a thiophene carboxylic acid core with 41 selected as representative from a recent patent application [166]. Three compounds
Discovery and Development of Inhibitors of HCV
417
from this series, VCH-916, VCH-759 and VCH-222 (structures not disclosed), have been advanced into clinical trials, with the latter two apparently remaining in active development. Doses of VCH-916 at 200 mg TID for 14 days or 300 or 400 mg BID for 3 days as monotherapy in genotype 1 subjects produced greater than a 1.2 log10 reduction in HCV RNA [167]. Resistant mutants, primarily at L419 or M423, emerged in the 14-day study but not during the 3-day regimen [167]. VCH-222 exhibits improved efficacy with a 750-mg BID dose reducing plasma viral RNA levels by 3.7 log10 at the end of a 3-day study [168]. VCH-222 is a potent inhibitor of genotype 1a and 1b replication in vitro, EC50 ¼ 23 and 12 nM, respectively, and displays good oral bioavailability in rats and dogs, with a liver to plasma ratio of 5 in rats [169,170].
2.6.2.3 Palm site I inhibitors. Palm site I is located at the junction of the thumb and palm domains, proximal to the active site, and inhibitors were originally discovered with a benzothiadiazine chemotype. Resistance to benzothiadiazines mapped to M414T as a signature mutation along with alterations in nearby amino acids, including N411. Several compounds targeting this site have been advanced into clinical studies, including GSK-625433 (42), evolved from a series of pyrrolidine derivatives but discontinued due to hepatotoxicity in preclinical species [171], ANA-598 (structure not disclosed), which has demonstrated efficacy in phase I clinical trials [172], and ABT-072 and ABT-333 (structures not disclosed) [173]. ANA-598 is presumably derived from a series of thiadiazines that have been extensively studied by Anadys with the 5,6-dihydropyridone 43 representing the most recent refinement of the chemotype in the context of optimizing its preclinical profile, particularly oral bioavailability [174]. This compound is a potent inhibitor of HCV polymerase, IC50 o10 nM, that is effective in a replicon, EC50 ¼ 16 nM, with activity dependent on the absolute configuration. The compound exhibits good metabolic stability in monkey liver microsomes and is permeable across a confluent layer of Caco-2 cells, properties that translate into good exposure following oral administration to cynomolgus monkeys [174]. OMe N N HO2C
O
N H
N
OH
N
S
NHSO2Me
OH
N
H
N
O
O H3C
CH3 CH3
H3C CH OMe 3 F
42
43
O S N H
N H
O H3C
O
O S
R
44: R = H 45: R = CH3
NHSO2Me
418
Nicholas A. Meanwell et al.
Another compound from this general chemotype is A-837093 (44), which has been the subject of detailed profiling [175]. The saturated dimethyl butane side chain found in 44 provides improved potency compared to an unsaturated isobutylene moiety, and activity is dependent on chirality since enantiomers are typically 30–40-fold less potent. The introduction of the chiral carbon atom in the ring enhances solubility compared to earlier compounds that incorporate a nitrogen atom at this site and confer a more planar disposition. The tert-butyl homologue 45 shows greater metabolic stability than A-837093 (44), which translates into considerably improved PK characteristics in multiple species following IV dosing, although the advantage was not as apparent after oral dosing [175]. Resistance to A-837093 (44), generated in 1b replicons, mapped to S368A, Y448H, G554D, Y555C and D559G, but these mutants retained sensitivity to a protease inhibitor, a thumb site II non-nucleoside inhibitor and IFN-a [176]. A-837093 (44) produced a maximal viral RNA reduction of 1.4 log10 in a chimpanzee infected with genotype 1a HCV following dosing at 30 mpk BID for 14 days [177]. However, the compound was more efficacious in a 1b-infected animal where a 2.5 log10 reduction in plasma RNA was measured. Viral rebound and partial viral rebound occurred in the 1b- and 1a-infected chimps, respectively, and many of the same resistance mutations observed in in vitro studies were characterized in these animals along with a C316Y mutation in the 1b-infected chimpanzee [177]. Of the two clinical candidates, ABT-333 appears to be the most advanced, but ABT-072 is the more potent [173,178–180]. ABT-333 displays EC50 of 2–7 nM in replicons, with a 10–14-fold shift in the presence of 40% human serum, whereas the corresponding data for ABT072 are EC50 of 0.3–5.3 nM, with an 8–17-fold shift when 40% human serum is added [175,178]. Both compounds elicited resistant mutations at positions C316Y, M414T, Y448H/C or S556G in vitro but most resistant replicons displayed reduced replication capacity. Results from SAD and MAD studies with ABT-333 at doses of 200–400 mg BID to healthy volunteers were encouraging, with exposure of the compound minimally affected by co-dosing with the CYP 3A4 inhibitor ketoconazole [179,180].
2.6.2.4 Palm site II inhibitors. The palm site II pocket partially overlaps with palm site I and is fully formed by a conformational change in the R200 region of the enzyme, which occurs upon binding of inhibitors. Inhibitors that bind to this site select for mutations at C316, I363 or S365. Nesbuvir (HCV 796, 46) is the most prominent palm site II inhibitor that potently blocks HCV genome replication in vitro with EC50 of 5 and 9 nM for 1a and 1b constructs, respectively. In a chimeric mouse model of HCV infection, nesbuvir (46) produced a W2 log10 reduction in viremia, which,
Discovery and Development of Inhibitors of HCV
419
along with a good preclinical PK profile, provided confidence to advance this compound into clinical studies [181]. As monotherapy at a dose of 1000 mg BID, this compound reduced viral RNA by 1.4 log10 at day 4 [43,181]. However, viral rebound occurred during continued treatment, potentially attributed to a C316Y mutant, with the result that the reduction in viral load was only 0.8 log10 at day 14 [43,181]. Co-administration of nesbuvir (46) with PEG-IFNa and ribavirin reduced HCV viral load by up to 3.5 log10 at day 14, an improvement over the 1.7 log10 reduction seen with PEG-IFNa alone [181]. The C316Y mutant has been shown to be 138–166-fold less sensitive to nesbuvir (46) and the molecular mechanisms of resistance and effect on viral fitness in vitro have been thoroughly studied [182,183]. Unfortunately, the discontinuation of nesbuvir (46) was announced in 2007 following the emergence of severe hepatotoxicity in 8% of HCV-infected subjects following 8 weeks of treatment with the compound in combination with SOC [184]. H3C
NH
O
F
HO
O
N O
S O CH3 46
GS-9190 has been advanced into clinical studies and, although the structure has not been disclosed, the compound appears to be derived from a series of imidazo[4,5-c]pyridines of which 47 is prototypical [185]. Compound 47 exhibits an EC50 of 0.010 mM and a CC50 of 108 mM in a replicon system [185]. Compounds from this class of NS5B inhibitor are thought to interact with a fifth allosteric site that is very close to palm site II and which has been referred to as a palm or finger binding site and is characterized by mutations at C445 (F or Y), Y448 and Y452 (H) [128–130]. However, these compounds are somewhat cryptic in their inhibitory activity since they are inactive towards the isolated NS5B enzyme in vitro. GS-9190 displays an EC50 of 0.7 nM towards a genotype 1b replicon and in clinical studies produced peak viral load reductions of 1.4 log10 at 40 mg BID and 1.7 log10 at 120 mg BID following an 8-day treatment regimen [186]. However, cardiac monitoring revealed possible abnormalities leading to a temporary suspension of clinical studies while the QT risk was more fully assessed. GS-9190 is currently in phase IIb trials where it is being evaluated at a dose of 40 mg BID in
420
Nicholas A. Meanwell et al.
conjunction with SOC. Although the structure of GS-9190 has not been revealed, two recent patent applications focus specifically on compound 48 [187,188]. CF3 F F3C
F F
N N
N
47
N
F3C N
N N
N
48
2.7 Emerging mechanisms 2.7.1 HCV entry inhibitors Elucidation of the biochemical steps associated with HCV entry and identification of the cellular proteins involved is a dynamic area of study that has the potential to provide new opportunities for therapeutic intervention [189–193]. An important and apparently final piece in the puzzle of the full repertoire of cellular factors required for viral entry is the identification of the tight junction protein occludin as a co-factor [194,195]. Occludin appears to function in concert with another tight junction protein, claudin 1 [196–200], the tetraspanin CD81 [201,202] and the scavenger receptor (SR) B1 [203–208]. However, although CD81 is a determinant of infectivity both in vitro and in vivo, it does not appear to play a critical role in cell–cell transmission of virus [209,210]. The host cell factor EWI-2wint has been identified as an endogenous partner of CD81 that blocks infection when co-expressed, perhaps an important element in viral tropism since EWI-2wint is not expressed in hepatocytes [211]. Plasma membrane enriched in ceramide leads to CD81 downregulation by ATP-independent internalization [212]. Claudin-1 is internalized by increases in cellular cAMP and protein kinase A activity while increased fatty acid synthase activity reduces claudin-1 expression [213,214]. High avidity antibodies to SR-B1 block infection in the presence of LDL, whereas IFN-a has been shown to downregulate this cellular receptor [215,216]. The heavily glycosylated HCV surface proteins E1 and E2 mediate viral entry, although the precise role of each protein in the carefully choreographed events associated with membrane fusion remains to be determined [217,218]. Nevertheless, the entry process can be studied in the context of pseudoparticles [219] or replicating virus and screens of this type are being used to identify effective inhibitors [220], with the
421
Discovery and Development of Inhibitors of HCV
first drug candidates advancing into clinical studies. Arbidol (49) is a broad-spectrum antiviral agent that has been shown to inhibit pseudoparticle-mediated infection with an IC50 of 11.3 mM by a mechanism that is not specific, since it inhibits HCV replication in a replicon by a mechanism that remains to be elucidated [221]. Benzyl salicylate acid and terfenadine have been characterized as inhibitors of E2-CD81 association, but SAR studies with each molecule failed to significantly improve potency [222,223].
CO2CH3 N
CH3
O
N CH 3
H3C N
N
HO
S Ph N
Br
N
N
Cl
CF3
S
CH3 OCH3
H3CO
49
50
51
PRO-206 (structure not disclosed) is an inhibitor of HCV entry optimized from a lead identified in a library of over 370,000 compounds, with structural refinement affording molecules with favorable antiviral activity and PK properties [224]. PRO-206 inhibits the entry of HCV pseudoparticles expressing a range of viral envelopes with EC50 in the 1–50 nM range for the more sensitive sequences. However, several viral envelopes demonstrated reduced sensitivity with EC50 ranging from 0.186 to W10 mM [224]. Phase 1 clinical trials with the HCV entry inhibitor ITX-5061, a picomolar inhibitor of genotype 1 and 2 HCV entry, have been completed, and the compound is undergoing proof-of-concept trials in HCV-infected patients [225]. Neither the structure nor the mode of action of ITX-5061 has been disclosed but a patent application claims a range of compounds based on variants of a linear tricyclic scaffold that are claimed to block HCV entry, with 50 a representative structure [226]. A patent application claims a broad series of HCV entry inhibitors, with 51 representative, that exhibits an EC50 of o100 nM in an in vitro infection assay in HepG2 cells [227]. JTK-652 (structure not disclosed), an HCV entry inhibitor discovered by screening using a pseudovirus assay, was evaluated in 10 genotype 1 HCV-infected patients at a dose of 100 mg TID as monotherapy for 28 days but failed to exhibit any significant effect on viral load [228].
422
Nicholas A. Meanwell et al.
2.7.2 HCV internal ribosome entry site inhibitors The HCV viral mRNA incorporates an internal ribosome entry site (IRES) that recruits ribosomes and initiation factors directly to the translation start site, avoiding 5u-cap-dependent protein synthesis [229,230]. The functional roles of the domains of the HCV IRES have been mapped, and gross structural information is available from cryo-electron microscopy studies, whereas X-ray crystallographic and NMR structural data have recently been obtained for domain II, the smaller of the two major domains [231–232]. The HCV IRES is viewed as a potentially interesting target for therapeutic intervention, but studies described in detail to date have been largely restricted to RNA-based technologies, including RNA aptamers [233–236], small interfering RNAs (siRNAs) [237–239] and antisense RNA [240,241]. Delivery of an antisense RNA 17 mer to replicon cells was markedly enhanced by conjugation with lipid, octadecanol or cholesterol, to which an alkyne moiety was introduced to allow participation in a copper-catalyzed cycloaddition with an azide at the 5u-position of the oligonucleotide [241]. A bicyclic peptide aptamer with high affinity and good selectivity for the HCV IRES was discovered after multiple rounds of selection from a library [242]. A 27 residue peptide containing three cysteines was exposed to dibromoxylene to afford a bicyclic peptide that bound to the HCV IRES with a Kd of 0.7 nM, close to 10-fold more potent than the acyclic peptide. The IRES recognition element was mapped to the 8N-terminus residues, KCSRGIRC, which demonstrated a Kd of 17.5 nM in the linear form and improved to a Kd of 3.7 nM on cyclization with dibromoxylene [242]. The pokeweed antiviral protein, a 262 residue peptide, and mutated derivatives devoid of cytotoxicity have been claimed to bind to stem loop domains II or IIId of the HCV IRES with single-digit nanomolar affinity, thereby interfering with access by the 40S ribosomal subunit [243]. A series of patent applications claim indole-based inhibitors of the HCV IRES, discovered using proprietary gene-expression modulation by small molecules (GEMS) technology, which inhibit HCV replication in a replicon [244–246]. PS-102123 (52, SCH-1383646) is highlighted as a compound that demonstrates an EC50 of o500 nM in a replicon assay and interacts in a synergistic fashion with the HCV protease inhibitor SCH-446211 (SCH-6) [246,247]. CN
O
N N
CH3
O NH
O
N Cl 52
CH3
423
Discovery and Development of Inhibitors of HCV
2.7.3 HCV assembly and egress inhibitors HCV virion production in human hepatocytes is dependent on the assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL) and vesicles in which HCV replicates are enriched in proteins involved in VLDL assembly, including apolipoprotein B (apoB), apoE, and microsomal triglyceride transfer protein (MTP). MTP is a chaperone protein and BMS-201038 (53) and CP-346086 (54), potent MTP inhibitors, block VLDL assembly and reduce HCV secretion by up to 80% at 100 nM without reducing HCV RNA production, indicating that secretion but not replication depends on VLDL assembly [248,249]. Although dose-dependent effects were seen in both studies, inhibition was incomplete with B20% of HCV still released, possibly by a VLDL-independent pathway [248,249]. siRNA directed towards apoB also blocks HCV secretion [248]. CF3
CF3 O
CF3
O
NH
N
N HN
NH
O
N N
HN
53
54
Narigenin (55), a known inhibitor of VLDL secretion that reduces the expression and activity of MTP and acyl CoA cholesteryl acyl transferase (ACAT), dose dependently reduces HCV virion secretion with an 80% reduction observed at 200 mM [250]. OH HO
O
OH
O 55
O
N
HO
H
CH3
O OH OH 56
2.7.4 a-Glucosidase inhibitors a-Glucosidase inhibitors interfere with virus morphogenesis by reducing protein glycosylation in the ER, leading to the misfolding of viral
424
Nicholas A. Meanwell et al.
proteins that, as a consequence, are targeted for destruction. In addition, protein complex formation is impaired with the result that prebudding complexes of viral proteins are reduced, leading to inhibition of virion production and secretion. As might be anticipated, a-glucosidase inhibitors exhibit broad-spectrum antiviral properties, interfering with the replication of a wide range of viruses including HCV [251,252]. Iminosugars have emerged as the most prominent and effective class of a-glucosidase inhibitors with celgosovir (56), the 6-butanoyl ester prodrug of castanospermine, advanced into clinical trials in HCVinfected patients. In a phase II study, celgosivir, in combination with IFNa-2b and ribavirin, produced a mean HCV viral load reduction of 1.2 log10, which compared with a 0.4 log10 reduction in patients receiving only the IFN-a-2b/ribavirin combination [251].
2.7.5 Inhibitors of host cell targets 2.7.5.1 Cyclosporins. The peptidyl-prolyl cis-trans isomerase cyclophilin B associates with HCV NS5B to stimulate RNA binding, an interaction that is critical for viral replication and represents an interesting host cell target for therapeutic intervention [253–255]. Cyclosporin A (CsA, 57) and non-immunosuppressive cyclosporins inhibit HCV replication, apparently functioning by binding to cyclophilin B and effectively competing with the polymerase. However, cyclophilin A has also been implicated, although with a distinct mechanism [256,257]. The cyclophilin B binding site has been mapped to the C-terminal residues 521–591 of NS5B, with P450 a key amino acid since the P450A mutation eliminates protein–protein association and compromises HCV replication [253–255]. Three non-immunosuppressive cyclosporin derivatives have been examined as inhibitors of HCV – NIM-811 (58) [258,259], DEBIO-025 (59) [260–262] and SCY-635 (60) [263,264]. Resistance to CsA (57) and SCY-635 (60) maps to both HCV NS5B and NS5A, with SCY-635 (60) eliciting T77K and I432V mutations in NS5B and T17A and E295K changes in NS5A [263,265,266]. The I432V change, although located outside of the cyclophilin-binding domain, appears to enhance the NS5Bcycophilin B interaction, and interestingly, this change can rescue the lethal P450A mutation [265]. CsA (57) elicits a different range of mutations, with P538T and S556G in NS5B and several in NS5A [266]. The three non-immunosuppressive cyclosporin analogues are potent inhibitors of HCV genotype 1b replication in vitro, with EC50 less than 1 mM, approximately 10-fold more potent than CsA (57), and interact in an additive to synergistic fashion with IFNa or ribavirin [259,261,263]. In addition, they are not cross-resistant with several STAT-C agents. A genotype 4 replicon is similarly susceptible to NIM-811 (58), but the JFH1 genotype 2a replicon is less sensitive to both NIM-811 and CsA since this replicon does not depend on cyclophilin B for replication [266].
Discovery and Development of Inhibitors of HCV
425
In clinical trials in patients co-infected with HIV, DEBIO-025 (59), at a dose of 1200 mg BID for 15 days, effected a mean 3.63 log10 reduction in viral load in those infected with HCV genotypes 1, 3 and 4 [260,267]. Genotype-3-infected patients were more responsive, achieving a 4.46 log10 reduction in viremia. In a phase IIa dose range–finding study conducted in 90 treatment-naı¨ve patients, DEBIO-025 (59) (1000 mg BID for 7 days as a loading dose followed by 1000 mg QD for 21 days) in conjunction with PEG-IFNa-2a produced a 4.75 log10 reduction in viral load in genotype 1 and 4 patients measured at week 4 [260,268]. This result was superior to either agent alone, with efficacy more pronounced in genotype 2 and 3 patients. SCY-635 (60), which exhibits high affinity for both cyclophilin A and B, IC50 ¼ 7 and 10 nM, respectively, reduced plasma HCV RNA by 2.20 log10 at day 11 and 1.82 log10 at day 15 when administered at a dose of 300 mg TID for 15 days, with lower doses of 100 and 200 mg TID producing inconsistent effects on viremia [269]. CH3 N
N CH3 O HO CH3 O N
N
N
O
O
H N
CH3 N
CH3 O
58
N
CH3 O
O N CH3
O
H N O
57
N H
CH3 O
CH3 N O
N
N
O
N H
O
59 N S
CH3 N
N CH3 O
OH
60
2.7.5.2 Nitazoxanide (Alinias). Nitazoxanide (61) is a nitrothiazole derivative and a prodrug of tizoxanide (62) that was originally developed as a treatment for parasitic infections [270]. However, these compounds have been characterized as inhibitors of both HBV and HCV replication in vitro [271–274] following the observation of clinical efficacy at reducing viral load as monotherapy in HCV genotype-4-infected patients [275]. In HCV replicons, nitazoxanide (61) and tizoxanide (62) inhibit viral RNA replication with EC50 of 210 and 150 nM, respectively, with good therapeutic indexes, CC50 ¼ 38 and 15 mM, respectively [272].
426
Nicholas A. Meanwell et al.
HBV viral DNA production in an in vitro replication assay was inhibited with similar potency, results consistent with the notion that nitazoxanide (61) and tizoxanide (62) modulate a host cell process and supported by resistance studies in HCV replicons [273]. In replicons and Huh-7 cells infected with a genotype 2a virus, nitazoxanide (61) increased the levels of phosphorylated elF2a and its dsRNA-activated protein kinase (PKR), key components of the host antiviral defenses, an effect augmented by IFN [274]. Collectively, these results suggest that nitazoxanide (61) modulates an innate antiviral pathway. In a placebo-controlled clinical trial, 7 of 23 genotype-4-infected patients administered nitazoxanide (61) as monotherapy at a dose of 500 mg BID for 24 weeks had a response at the end of treatment while 4 of 23 patients with a baseline viral load of o400,000 IU/mL had a SVR compared to none in the placebo group [275]. In a follow-up study, also conducted in genotype-4-infected individuals but not blinded, a 12-week lead-in with nitazoxanide (61) (500 mg BID) followed by PEG-IFNa2a/ribavirin or PEG-IFNa2a/ ribavirin/nitazoxanide for 36 weeks was compared with 48 weeks of PEG-IFNa2a/ribavirin [276]. SVR rates at 24 weeks post-treatment were 50% in the control group, 61% in the PEG-IFNa2a/ribavirin group and 79% in those who received triple therapy for 36 weeks [276]. A recent patent application claims analogues of nitazoxanide that broadly explores SAR associated with both the nitro and phenol moieties [277].
OR
O
HO
N N H
NO2 S
H3CO
O
HO 61: R = Ac 62: R = H
OH
O HO 63
O
OH
2.7.5.3 Silymarin and silibinin. A standardized preparation of silymarin, an extract of milk thistle (Silybum marianum) with antioxidant activity that has a history of use as self-medication for liver disease spanning several centuries [278,279], inhibits HCV JFH1 viral infection of Huh 7.5.1 cells in culture in a dose-dependent fashion, with 20 mg/mL demonstrating efficacy comparable to IFN-a [280]. A cocktail therapy comprising silymarin along with seven additional antioxidants demonstrated benefit in HCV-infected individuals who had not responded to IFN therapy, with normalization of liver enzymes in 40–50% of patients but modest
Discovery and Development of Inhibitors of HCV
427
reductions in viral load in only 25% of the cohort [281,282]. Silibinin (63) is one of the six major flavonolignans in silymarin, and this compound showed dose-dependent reductions in viremia when administered intravenously to HCV-infected individuals unresponsive to full doses of PEG-IFNa/ribavirin [283]. A dose of 20 mg/kg infused daily for 7 days was associated with a 3.02 log10 decrease in viral load, with a further reduction to 4.85 log10 below baseline after an additional 7 days in combination with PEG-IFNa and ribavirin [282]. However, oral dosing of silibinin (63) was not effective in maintaining the antiviral effect following the 7 days of infusion [283].
3. CONCLUSIONS The identification and development of specifically targeted inhibitors of HCV has advanced considerably over the past 2 years, and despite setbacks in clinical studies with several early candidates, there is currently a robust pipeline of potential drugs that target a range of mechanisms. The advent of HCV replicons in 1999 was an important catalyst to drug discovery, providing a screening tool to not only confirm the activity of NS3 and NS5B inhibitors but also identify compounds acting at alternative targets. The development of infectious virus offers similar promise for the identification of HCV inhibitors that interfere with virus entry and the process of assembly and egress. Although the majority of compounds in clinical studies are still in the early stages of development, the first steps towards developing a combination of specific antiviral agents to treat HCV unresponsive to SOC, either as addon or replacement therapy, have been taken. With compounds acting at multiple sites in the virus replication cycle in development, there is reason to be optimistic about the potential to identify effective combinations that offer higher rates of therapeutic cure of HCV infection resistant to the current SOC.
NOMENCLATURE AUC PEG RBV ATP GTP CYP VLDL
area under the curve polyethyleneglycol ribavirin adenosine triphosphate guanosine triphosphate cytochrome P (CYP 450 = cytochrome P450) very low density lipoprotein
428
Nicholas A. Meanwell et al.
REFERENCES [1] M. J Alter, World J. Gastroenterol., 2007, 13, 2436. [2] K. Neukam, J. Macı´as, J. A. Mira and J. A. Pineda, Expert. Opin. Pharmacother., 2009, 10, 417. [3] G. Foster and P. Mathurin, Antiviral Ther., 2008, 13(Suppl. 1), 3. [4] S. Chevaliez and J.-M. Pawlotsky, Adv. Drug Deliv. Rev., 2007, 59, 1222. [5] L. A. Sorbera, Drugs Future, 2007, 32, 937. [6] Data available on the Zymogenetics web site, http://www.zymogenetics.com/ products/Interleukin29ProductBackgrounder.htm [7] L. G. J. Leede, J. E. Humphries, A. C. Bechet, E. J. Hoogdalem, R. Verrijk and D. G. Spencer, J. Interferon Cytokine Res., 2008, 28, 113. [8] V. Soriano, M. G. Peters and S. Zeuzem, Clin. Infect. Dis., 2009, 48, 313. [9] A. Thompson, K. Patel, H. Tillman and J. G. McHutchison, J. Hepatol., 2009, 50, 184. [10] R. E. Stauber and H. H. Kessler, Drugs, 2008, 68, 1347. [11] V. Sorianao, A. Madejohn, E. Vispo, P. Labarga, J. Garcia-Samaniego, L. MartinCarbonero, J. Sheldon, M. Bottecchia, P. Tuma and P. Barreiro, Expert. Opin. Emerg. Drugs, 2008, 13, 1. [12] M. P. Manns, G. R. Foster, J. K. Rockstroh, S. Zeuzem, F. Zoulim and M. Houghton, Nat. Rev. Drug Discov., 2007, 6, 991. [13] E. B. Keeffe, Antiviral Ther., 2007, 12, 1015. [14] S. A. Harrison, Am. J. Gastroenterol., 2007, 102, 2332. [15] J.-M. Pawlotsky, S. Chevaliez and J. G. McHutchison, Gastroenterol., 2007, 132, 1979. [16] K. Garber, Nat. Biotechnol., 2007, 25, 1379. [17] N. A. Meanwell and G. Koszalka, Curr. Opin. Investig. Drugs, 2008, 9, 128. [18] D. Moradpour, F. Penin and C. M. Rice, Nat. Rev. Microbiol., 2007, 5, 453. [19] T. L. Tellinghuisen, M. J. Evans, T. Hahn, S. You and C. M. Rice, J. Virol., 2007, 81, 8853. [20] T. P. Holler, T. Parkinson and D. C. Pryde, Expert Opin. Drug Discov., 2009, 4, 293. [21] J. Timm and M. Roggendorf, World J. Gastroenterol., 2007, 13, 4808. [22] G. Koev and W. Kati, Expert. Opin. Investig. Drugs, 2008, 17, 303. [23] B. H. McGovern, B. K. Abu Dayyeh and R. T. Chung, Hepatology, 2008, 48, 1700. [24] T. Kuntzen, J. Timm, A. Berical, N. Lennon, A. M. Berlin, S. K. Young, B. Lee, D. Heckerman, J. Carlson, L. L. Reyor, M. Kleyman, C. M. McMahon, C. Birch, J. Schulze zur Wiesch, T. Ledlie, M. Koehrsen, C. Kodira, A. D. Roberts, G. M. Lauer, H. R. Rosen, F. Bihl, A. Cerny, U. Spengler, Z. Liu, A. Y. Kim, Y. Xing, A. Schneidewind, M. A. Madey, J. F. Fleckenstein, V. M. Park, J. E. Galagan, C. Nusbaum, B. D. Walker, G. V. Lake-Bakaar, E. S. Daar, I. M. Jacobson, E. D. Gomperts, B. R. Edlin, S. M. Donfield, R. T. Chung, A. H. Talal, T. Marion, B. W. Birren, M. R. Henn and T. M. Allen, Hepatology, 2008, 48, 1769. [25] D. J. Bartels, Y. Zhou, E. Z. Zhang, M. Marcial, R. A. Byrn, T. Pfeiffer, A. M. Tigges, B. S. Adiwijaya, C. Lin, A. D. Kwong and T. L. Kieffer, J. Infect. Dis., 2008, 198, 800. [26] M. Cubero, J. I. Esteban, T. Otero, S. Sauleda, M. Bes, R. Esteban, J. Guardia and J. Quer, Virology, 2008, 370, 237. [27] S. Pogam, A. Seshaadri, A. Kosaka, S. Chiu, H. Kang, S. Hu, S. Rajyaguru, J. Symons, N. Cammack and I. Na´jera, J. Antimicrob. Chemother., 2008, 61, 1205. [28] D. L. Wyles, K. A. Kaihara, F. Vaida and R. T. Schooley, J. Virol., 2007, 81, 3005. [29] C. Gru¨nberger, D. L. Wyles, K. A. Kaihara and R. T. Schooley, J. Infect. Dis., 2008, 197, 42. [30] E. J. Gane, S. K. Roberts, C. Stedman, P. W. Angus, B. Ritchie, R. Elston, D. Ipe, L. Baher, P. Morcos, I. Najera, M. Mannino, B. Brennan, M. Berrey, W. Bradford, E. Yetzer, N. Shulman and P. F. Smith, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009,
Discovery and Development of Inhibitors of HCV
[31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
[41] [42] [43] [44] [45] [46]
[47]
[48] [49] [50]
[51]
[52]
[53] [54]
429
Abstract 1046 available at http://www.abstractserver.com/easl2009/planner/sp. php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 2661&EASL2009 ¼ o39t1tihqm 4c4mhf7mp4d7udr1&EASL2009 ¼ o39t1tihqm4c4mhf7mp4d7udr1 C. T. Jones, C. L. Murray, D. K. Eastman, J. Tassello and C. M. Rice, J. Virol., 2007, 81, 8374. I. C. Lorenz, J. Marcotrigiano, T. G. Dentzler and C. M. Rice, Nature, 2006, 442, 831. V. Jirasko, R. Montserret, N. Appel, A. Janvier, L. Eustachi, C. Brohm, E. Steinmann, T. Pietschmann, F. Penin and R. Bartenschlager, J. Biol. Chem., 2008, 283, 28546. C. L. Murray, C. T. Jones, J. Tassello and C. M. Rice, J. Virol., 2007, 81, 10220. V. Schregel, S. Jacobi, F. Penin and N. Tautz, Proc. Natl. Acad. U.S.A., 2009, 106, 5342, early edition, http://www.pnas.org/cgi/reprint/0810950106v1 Y. She, Q. Liao, X. Chen, L. Ye and Z. Wu, Arch. Virol., 2008, 153, 1991. J.-K. Oem, C. Jackel-Cram, Y.-P. Li, H.-N. Kang, Y. Zhou, L. A. Babiuk and Q. Liu, Arch. Virol., 2008, 153, 293. R. Maurice and D. Thibeault, WO Patent Application 2007/048254-A1, 2007. L. Lamarre, L. Lagace´ and D. Thibeault, US Patent Application 2007/0141701-A1, 2007. D. Lamarre, P. C. Andreson, M. Bailey, P. Beaulieu, G. Bolger, P. Bonneau, M. Bo¨s, D. R. Cameron, M. Cartier, M. G. Cordingley, A. M. Faucher, N. Goudreau, S. H. Kawai, G. Kukolj, L. Legace´, S. R. LaPlante, H. Narjes, M. A. Poupart, J. Rancourt, R. E. Sentjens, R. St. George, B. Simoneau, G. Steinmann, D. Thibeault, Y. S. Tsantrizos, S. M. Weldon, C. L. Yong and M. Llina`s-Brunet, Nature, 2003, 426, 186. T. Asselah, Y. Benhamou and P. Marcellin, Liver Int., 2009, 29, 57. Y. Tsantrizos, Acc. Chem. Res., 2008, 41, 1252. A. D. Kwong, L. McNair, I. Jacobson and S. George, Curr. Opin. Pharmacol., 2008, 8, 522. R. Francesco and A. Carfi, Adv. Drug Del. Rev., 2007, 59, 1242. B. Kronenberger and S. Zeuzem, Curr. Gastroenterol. Rep., 2009, 11, 15. R. B. Perni, S. J. Almquist, R. A. Byrn, G. Chandorkar, P. R. Chaturvedi, L. F. Courtney, C. J. Decker, K. Dinehart, C. A. Gates, S. L. Harbeson, A. Heiser, G. Kalkeri, E. Kolaczkowski, K. Lin, Y.-P. Luong, B. G. Rao, W. P. Taylor, J. A. Thomson, R. D. Tung, Y. Wei, A. D. Kwong and C. Lin, Antimicrob. Agents Chemother., 2006, 50, 899. H. W. Reesink, S. Zeuzem, C. J. Weegink, N. Forestier, A. Van Vliet, J. Van de wetering de Rooij, L. Mcnair, S. Purdy, R. Kauffman, J. Alam and P. L. M. Jansen, Gastroenterol., 2006, 131, 997. T. L. Kieffer, C. Sarrazin, J. S. Miller, M. W. Welker, N. Forestier, H. W. Reesink, A. D. Kwong and S. Zeuzem, Hepatology, 2007, 46, 631. N. Forestier, H. W. Reesink, C. J. Weegink, L. McNair, T. L. Kieffer, H.-M. Chu, S. Purdy, P. L. M. Jansen and S. Zeuzem, Hepatology, 2007, 46, 640. C. Sarrazin, T. L. Kieffer, D. Bartels, B. Hanzelka, U. Muh, M. Welker, D. Wincheringer, Y. Zhou, H.-M. Chu, C. Lin, C. Weegink, H. Reesink, S. Zeuzem and A. D. Kwong, Gastroenterol., 2007, 132, 1767. S. Zeuzem, C. Hezode, P. Ferenci, G. M. Dusheiko, K. Alves, L. Bengtsson, S. Gharakhanian, R. Kauffman, J. J. Alam and J.-M. Pawlotsky, Hepatology, 2008, 48(S1), A243. J. G. McHutchison, M. L. Schiffman, N. Terrault, M. P. Manns, A. M. DiBisceglie, I. M. Jacobson, N. H. Afdhal, E. Heathcote, S. Zeuzem, H. W. Reesink, S. George, N. Adda and A. J. Muir, Hepatology, 2008, 48(S1), 431, A269. F. G. Njoroge, K. X. Chen, N.-Y. Shih and J. J. Piwinski, Acc. Chem. Res., 2008, 41, 50. A. J. Prongay, Z. Guo, N. Yao, J. Pichardo, T. Fischmann, C. Strickland, J. Myers, P. C. Weber, B. M. Beyer, R. Ingram, Z. Hong, W. W. Prosise, L. Ramanathan, S. S. Taremi, T. Yarosh-Tomaine, R. Zhang, M. Senior, R.-S. Yang, B. Malcolm, A. Arasappan, F. Bennett, S. L. Bogen, K. Chen, E. Jao, Y.-T. Liu, R. G. Lovey, A. K. Saksena,
430
[55] [56]
[57]
[58]
[59]
[60]
[61] [62]
[63]
[64]
[65] [66]
[67] [68]
[69]
Nicholas A. Meanwell et al.
S. Venkatraman, V. Girijavallabhan, F. G. Njoroge and V. Madison, J. Med. Chem., 2007, 50, 2310. C. Sarrazin, R. Rouzier, F. Wagner, N. Forestier, D. Larrey, S. K. Gupta, M. Hussain, A. Shah, D. Cutler, J. Zhang and S. Zeuzem, Gastroenterol., 2007, 132, 1270. P. Kwo, E. J. Lawitz, J. McCone, E. R. Schiff, J. M. Vierling, D. Pound, M. Davis, J. S. Galati, S. C. Gordon, N. Ravendhran, L. Rossaro, F. H. Anderson, I. M. Jacobson, R. Rubin, K. Koury, E. I. Chaudhri and J. K. Albrecht, Hepatology, 2008, 48(S1), 268A, LB16. X. Tong, A. Arasappan, F. Bennett, R. Chase, B. Feld, Z. Guo, A. Hart, V. Madison, B. Malcolm, J. Pichardo, A. Prongay, R. Ralston, A. Skelton, E. Xia and F. G. Njoroge, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 967 available at http://www.abstractserver.com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 595&EASL2009 ¼ lb58m4anr8hk3eaqos5519g1d7 &EASL2009 ¼ lb58m4anr8hk3eaqos5519g1d7 S. Venkatraman, F. Velazquez, W. Wu, M. Blackman, K. X. Chen, S. Bogen, L. Nair, X. Tong, R. Chase, A. Hart, S. Agrawal, J. Pichardo, A. Prongay, K.-C. Cheng, V. Girijavallabhan, J. Piwinski, N.-Y. Shih and F. G. Njoroge, J. Med. Chem., 2009, 52, 336. F. Velazquez, S. Venkatraman, M. Blackman, P. Pinto, S. Bogen, M. Sannigrahi, K. Chen, J. Pichardo, A. Hart, X. Tong, V. Girijavallabhan and F. G. Njoroge, J. Med. Chem., 2009, 52, 700. K. X. Chen, L. Nair, B. Vibulbhan, W. Yang, A. Arasappan, S. L. Bogen, S. Venkatraman, F. Bennett, W. Pan, M. L. Blackman, A. I. Padilla, A. Prongay, K.-C. Cheng, X. Tong, N.-Y. Shih and F. G. Njoroge, J. Med. Chem., 2009, 52, 1370. S. Venkatraman, W. Wu, A. Prongay, V. Girijavallabhan and F. G. Njoroge, Bioorg. Med. Chem. Lett., 2009, 19, 180. P. Raboisson, H. Kock, A. Rosenquist, M. Nilsson, L. Salvador-Oden, T.-I. Lin, N. Roue, V. Ivanov, H. Wahling, K. Wickstrom, E. Hamelink, M. Edlund, L. Vrang, S. Vendeville, W. Vreken, D. McGowan, A. Tahri, L. Hu, C. Boutton, O. Lenz, F. Delouvroy, G. Pille, D. Surleraux, P. Wigerinck, B. Samuelsson and K. Simmen, Bioorg. Med. Chem. Lett., 2008, 18, 4853. T.-I. Lin, O. Lenz, G. Fanning, T. Verbinnen, F. Delouvroy, A. Scholliers, K. Vermeiren, A. Rosenquist, M. Edlund, B. Samuelsson, L. Vrang, H. Kock, P. Wigerinck, P. Raboisson and K. Simmen, Antimicrob. Agents Chemother., 2009, 53, 1377. M. P. Manns, H. W. Reesink, C. Moreno, T. Berg, Y. Benhamou, Y. J. Horsmans, G. M. Dusheiko, R. Flisiak, P. Meyvisch, O. Lenz, K. Simmen and R. Verloes, Hepatology, 2008, 48(S1), 2687, LB8. R. Rajagopalan, S. Misialek, S. K. Stevens, D. G. Myszka, B. J. Brandhuber, J. A. Ballard, S. W. Andrews, S. D. Seiwert and K. Kossen, Biochemistry, 2009, 48, 2559. S. D. Seiwert, S. W. Andrews, Y. Jiang, V. Serebryany, H. Tan, K. Kossen, P. T. Ravi Rajagopalan, S. Misialek, S. K. Stevens, A. Stoycheva, J. Hong, S. R. Lim, X. Qin, R. Rieger, K. R. Condroski, H. Zhang, M. Geck Do, C. Lemieux, G. P. Hingorani, D. P. Hartley, J. A. Josey, L. Pan, L. Beigelman and L. M. Blatt, Antimicrob. Agents Chemother., 2008, 52, 4432. N. Forestier, D. G. Larrey, D. Guyader, P. Marcellin, R. Rouzier, A. A. Patat, W. Z. Bradford, S. Porter and S. Zeuzem, Hepatology, 2008, 48(S1), 1132A, A1847. N. J. Liverton, M. K. Holloway, J. A. McCauley, M. T. Rudd, J. W. Butcher, S. S. Carroll, J. DiMuzio, C. Fandozzi, K. F. Gilbert, S.-S. Mao, C. J. McIntyre, K. T. Nguyen, J. J. Romano, M. Stahlhut, B.-L. Wan, D. B. Olsen and J. P. Vacca, J. Am. Chem. Soc., 2008, 130, 4607. D. H. Wright, J. L. Miller, I. Verlinden, C. Cilissen, J. Valentine, P. Sun, M. De Smet, J. de Hoon, M. Depre, L. Cavens, J. Chodakewitz and J. A. Wagner, Hepatology, 2008, 48(S1), 1165A, A1910.
Discovery and Development of Inhibitors of HCV
431
[70] E. J. Lawitz, M. S. Sulkowski, I. M. Jacobson, S. Faruqui, W. K. Kraft, B. Maliakkal, M. Al-Ibrahim, R. H. Ghalib, S. C. Gordon, P. Kwo, J. Rockstroh, M. Miller, P. Hwang, J. Gress and E. Quirk, Hepatology, 2008, 48(S1), 203A, A211. [71] M. P. Manns, M. Bourliere, Y. Benhamou, S. Pol, M. Bonacini, T. Berg, C. Trepo, D. Wright, G. Steinmann, D. B. Huang, J. Mikl, G. Kukolj and J. O. Stern, Hepatology, 2008, 48(S1), 1133A, A1849. [72] M. P. Manns and M. Bourlie`re, Hepatology, 2008, 48(S1), 1151A, A1882. [73] R. Ronn, Y. A. Sabnis, T. Gossas, E. Akerblom, U. H. Danielson, A. Hallberg and A. Johansson, Bioorg. Med. Chem., 2006, 14, 544. [74] D. A. Campbell, D. T. Winn, J. M. Betancort and M. E. Hepperle, WO Patent Application 2007/016476-A2, 2007. [75] D. A. Campbell, M. E. Hepperle, D. T. Winn and J. M. Betancort, WO Patent Application 2007/089618-A2, 2007. [76] A. X. Wang, B. Z. Zheng, S. D’Andrea, Q. Zhao and P. M. Scola, WO Patent Application 2008/064066-A1, 2008. [77] M. D. Bailey, F. Bilodeau, P. Forgione, V. Gorys, M. Llina`s-Brunet, J. Naud, J. O’Meara and M.-A. Poupart, WO Patent Application 2008/098368-A1, 2008. [78] N. Sin, B. L. Venables, L.-Q. Sun, S.-Y. Sit, Y. Chen and P. M. Scola, WO Patent Application 2008/060927-A2, 2008. [79] P. Ortqvist, S. D. Peterson, E. Kerblom, T. Gossas, Y. A. Sabnis, R. Fransson, G. Lindeberg, U. H. Danielson, A. Karlen and A. Sandstrom, Bioorg. Med. Chem., 2007, 15, 1448. [80] C. C. Parsy, F.-R. Alexandre and D. Surleraux, WO Patent Application 2009/014730-A1, 2009. [81] T. Brandl, S. Cottens, C. Ehrhardt, J. Fu, S. Karur, D. T. Parker, M. A. Patane, P. Raman, S. A. Randl, P. Rigollier, M. Seepersaud and O. Simic, WO Patent Application 2008/ 033389-A2, 2009. [82] S. D. Britt, J. Fu, D. T. Parker, M. A. Patane, P. Raman, B. Radetich, M. Seepersaud, A. Yifru, R. Zheng, T. Brandl, S. Cottens, C. Ehrhardt, S. A. Randl, P. Rigollier, N. Schiering and O. Simic, WO Patent Application 2008/101665-A1, 2008. [83] W. Yang, Y. Zhao, J. Fabrycki, X. Hou, X. Nie, A. Sanchez, A. Phadke, M. Deshpande, A. Agarwal and M. Huang, Antimicrob. Agents Chemother., 2008, 52, 2043. [84] M. Huang, Y. Sun, W. Yang, H. Hou, J. Fabrycki, X. Nie, A. Sanchez, Y. Zhao, A. Phadke and M. Deshpande, 42nd Annual Meeting of the European Association for the Study of the Liver, Barcelona, Spain, April 11–15, 2007, http://www.easl.ch/ easl2007/Program/ViewAbstract.asp [85] A. M. I. Lam and D. N. Frick, J. Virol., 2006, 80, 404. [86] S. Dumont, W. Cheng, V. Serebrov, R. K. Beran, I. Tinoco, A. M. Pyle and C. Bustamante, Nature, 2006, 439, 105. [87] R. K. F. Beran and A. M. Pyle, J. Biol. Chem., 2008, 283, 29929. [88] J. L. Kim, K. A. Morgenstern, J. P. Griffith, M. D. Dwyer, J. A. Thomson, M. A. Murcko, C. Lin and P. R. Caron, Structure, 1998, 6, 89. [89] L. Biegelman, B. Buckman, G. Wang, J. Matulic-Adamic, A. Stoycheva, S. W. Andrews, S. M. Misialek, P. T. Ravi Rajagopalan, A. M. Fryer, I. Gunawardana, J. Haas, L. Huang, M. R. Madduru, G. Zhang, K. Kossen and V. Serebryany, WO Patent Application 2008/100867-A2, 2008. [90] A. M. Paredes and K. J. Blight, J. Virol., 2008, 82, 10671. [91] A. A. Thompson, A. Zou, J. Yan, R. Duggal, W. Hao, D. Molina, C. N. Cronin and P. A. Wells, Biochemistry, 2009, 48, 906. [92] S. Einav, E. H. Sklan, H. M. Moon, E. Gehrig, P. Liu, Y. Hao, A. W. Lowe and J. S. Glenn, Hepatology, 2008, 47, 827. [93] J. S. Glenn and S. Einav, WO Patent Application 2009/005615-A1, 2009.
432
Nicholas A. Meanwell et al.
[94] S. Einav, D. Gerber, P. D. Bryson, E. H. Sklan, M. Elazar, S. J. Maerkl, J. S. Glenn and S. R. Quake, Nat. Biotechnol., 2008, 26, 1019. [95] S. R. Quake, S. Einav, J. S. Glenn, R. Mcdowell, W. Yang, D. Gerber and H. DvorySobol, WO Patent Application 2009/039246-A2, 2009. [96] S. Einav, J. S. Glenn, R. Mcdowell and W. Yang, WO Patent Application 2009/039248A2, 2009. [97] S. K. Chunduru, C. A. Benatatos, T. J. Nitz and T. R. Bailey, US Patent Application 2007/ 0269420-A1, 2007. [98] P. D. Bryson, N.-J. Cho, S. Einav, C. Lee, V. Tai, J. Bechtel, M. Sivaraja, C. Roberts, U. Schmitz and J. S. Glenn, 15th International Symposium on Hepatitis C Virus and Related Viruses, San Antonio, TX, USA, October 5–9, 2008, Abstract 213. [99] U. Schmitz and S.-L. Tan, Recent Pat. Antiinfect. Drug Discov., 2008, 3, 77. [100] Y. Huang, K. Staschke, R. Francesco and S.-L. Tan, Virology, 2007, 364, 1. [101] M. Binder, D. Quinkert, O. Bochkarova, R. Klein, N. Kezmic, R. Bartenschlager and V. Lohmann, J. Virol., 2007, 81, 5270. [102] N.-J. Cho, K. H. Cheong, C.-H. Lee, C. W. Frank and J. S. Glenn, J. Virol., 2007, 81, 6682. [103] R. Brillet, F. Penin, C. Hezode, P. Chouteau, D. Dhumeaux and J.-M. Pawlotsky, J. Infect. Dis., 2007, 195, 432. [104] N. Appel, M. Zayas, S. Miller, J. Krijnse-Locker, T. Schaller, P. Friebe, S. Kallis, U. Engel and R. Bartenschlager, PLoS Pathog., 2008, 4, e1000035. [105] T. L. Tellinghuisen, K. L. Foss and J. Treadaway, PLoS Pathog., 2008, 4, e1000032. [106] T. Masaki, R. Suzuki, K. Murakami, H. Aizaki, K. Ishii, A. Murayama, T. Date, Y. Matsuura, T. Miyamura, T. Wakita and T. Suzuki, J. Virol., 2008, 82, 7964. [107] G. Cheng, A. Montero, P. Gastaminza, C. Whitten-Bauer, S. F. Wieland, M. Isogawa, B. Fredericksen, S. Selvarajah, P. A. Gallay, M. R. Ghadiri and F. V. Chisari, Proc. Natl. Acad. Sci. U.S.A, 2008, 105, 3088. [108] M. D. Bobardt, G. Cheng, L. Witte, S. Selvarajah, U. Chatterji, B. E. Sanders-Beer, T. B. H. Geijtenbeek, F. V. Chisari and P. A. Gallay, Proc. Natl. Acad. Sci. U.S.A, 2008, 105, 5525. [109] Arrow Therapeutics’ web site: http://www.arrowt.co.uk/product-hcv.asp [110] R. Nettles, C. Chien, E. Chung, A. Persson, M. Gao, M. Belema, N. A. Meanwell, M. DeMicco, T. C. Marbury, R. Goldwater, P. Northup, J. Coumbis, W. K. Kraft, M. R. Charlton, J. C. Lopez-Talavera and D. M. Grasela, Hepatology, 2008, 48(S1), 267A, LB12. [111] C. Bachand, M. Belema, D. H. Deon, A. C. Good, J. Goodrich, C. A. James, R. Lavoie, O. D. Lopez, A. Martel, N. A. Meanwell, V. N. Nguyen, J. L. Romine, E. H. Ruediger, L. B. Snyder, D. R. St. Laurent, F. Yang, D. R. Langley and L. G. Hamann, WO Patent Application 2008/021928-A2, 2008. [112] C. Bachand, M. Belema, D. H. Deon, A. C. Good, J. Goodrich, C. A. James, R. Lavoie, O. D. Lopez, A. Martel, N. A. Meanwell, V. N. Nguyen, J. L. Romine, E. H. Ruediger, L. B. Snyder, D. R. St. Laurent, F. Yang, D. R. Langley and L. G. Hamann, WO Patent Application 2008/021936-A2, 2008. [113] C. Bachand, M. Belema, D. H. Deon, A. C. Good, J. Goodrich, L. G. Hamann, C. A. James, D. R. Langley, R. Lavoie, O. D. Lopez, A. Martel, N. A. Meanwell, V. N. Nguyen, J. L. Romine, E. H. Ruediger, L. B. Snyder, D. R. St. Laurent, F. Yang and G. Wang, WO Patent Application 2008/144380-A1, 2008. [114] C. Bachand, M. Belema, D. H. Deon, A. C. Good, J. Goodrich, C. A. James, R. Lavoie, O. D. Lopez, A. Martel, N. A. Meanwell, V. N. Nguyen, J. L. Romine, E. H. Ruediger, L. B. Snyder, D. R. St. Laurent, F. Yang, D. R. Langley, G. Wang and L. G. Hamann, WO Patent Application 2008/021927-A2, 2008. [115] C. J. Wheelhouse, A. J. F. Thomas, D. J. Bushnell, J. Lumley, J. I. Salter, M. C. Carter, N. Mathews, C. J. Pilkington and R. M. Angell, WO Patent Application 2007/031791A1, 2007.
Discovery and Development of Inhibitors of HCV
433
[116] N. Mathews, A. J. F. Thomas, K. C. Spencer, H. Dennison, M. C. Barnes, S. S. Chana, L. Jennens and C. J. Pilkington, WO Patent Application 2007/042782-A1, 2007. [117] N. Mathews, A. J. F. Thomas, K. C. Spencer, N. Tiberghien, C. J. Pilkington, L. Jennens, S. Chana and I. J. Fraser, WO Patent Application 2007/080401-A1, 2007. [118] J. Lumley, J. I. Salter, M. C. Carter, N. Mathews, C. J. Pilkington, A. J. F. Thomas and I. Fraser, WO Patent Application 2007/138242-A1, 2007. [119] S. Chana, WO Patent Application 2008/056149-A1, 2008. [120] M. C. Carter, S. Cockerill, S. S. Flack and C. J. Wheelhouse, WO Patent Application 2009/034390-A1, 2009. [121] F. U. Schmitz, C. D. Roberts, A. D. M. Abadi, R. C. Griffith and M. R. Leivers, WO Patent Application 2007/070556-A2, 2007. [122] F. U. Schmitz, C. D. Roberts, A. D. M. Abadi, R. C. Griffith, M. R. Leivers, I. Slobodov and R. Rai, WO Patent Application 2007/070600-A2, 2007. [123] M. R. Leivers, F. U. Schmitz, R. C. Griffith, C. D. Roberts, A. D. M. Abadi, S. A. Chan, R. Rai, I. Slobodov and T. L. Ton, WO Patent Application 2008/064218-A2, 2008. [124] M. R. Leivers, F. U. Schmitz, C. D. Roberts and A. Dehghani Mohammad Abadi, WO Patent Application 2008/070447-A2, 2008. [125] R. Rai, F. U. Schmitz, C. D. Roberts, I. Slobodov and M. R. Leivers, WO Patent Application 2008/154601-A1, 2008. [126] G. Li, R. Fathi, Z. Yang, Y. Liao, Q. Zhu, A. Lam, A. Sandrasagra, K. Nawoschik, H.-J. Cho, J. Cao, W. Ruoqiu and R. C. Wobbe, WO Patent Application 2008/048589-A2, 2008. [127] I. Conte, C. Giuliano, C. Ercolani, F. Narjes, U. Koch, M. Rowley, S. Altamura, R. De Francesco, P. Neddermann, G. Migliaccio and I. Stansfield, Bioorg. Med. Chem. Lett., 2009, 19, 1799. [128] R. A. Love, O. Brodsky, M. J. Hickey, P. A. Wells and C. N. Cronin, J. Virol., 2009, 83, 4395. [129] P. L. Beaulieu, Exp. Opin. Ther. Pat., 2009, 19, 145. [130] E. Tramontano, Mini Rev. Med. Chem., 2008, 8, 1298. [131] U. Koch and F. Narjes, Curr. Topics Med. Chem., 2007, 7, 1302. [132] F. Pauwels, W. Mostmans, L. M. M. Quirynen, L. Helm, C. W. Boutton, A.-S. Rueff, E. Cleiren, P. Raboisson, D. Surleraux, O. Nyanguile and K. A. Simmen, J. Virol., 2007, 81, 6909. [133] J. Paeshuyse, I. Vliegen, L. Coelmont, P. Leyssen, O. Tabarrini, P. Herdewijn, H. Mittendorfer, J. Easmon, V. Cecchetti, R. Bartenschlager, G. Puerstinger and J. Neyts, Antimicrob. Agents Chemother., 2008, 52, 3433. [134] M. F. McCown, S. Rajyaguru, S. Pogam, S. Ali, W.-R. Siang, H. Kang, J. Symons, N. Cammack and I. Najera, Antimicrob. Agents Chemother., 2008, 52, 1604. [135] H. Ma, W.-R. Jiang, N. Robledo, V. Leveque, S. Ali, T. Lara-Jaime, M. Masjedizadeh, D. B. Smith, N. Cammack, K. Klumpp and J. Symons, J. Biol. Chem., 2007, 282, 29812. [136] E. Murakami, H. Bao, M. Ramesh, T. R. McBrayer, T. Whitaker, H. M. Micolochick Steuer, R. F. Schinazi, L. J. Stuyver, A. Obikhod, M. J. Otto and P. A. Furman, Antimicrob. Agents Chemother., 2007, 51, 503. [137] E. Murakami, C. Niu, H. Bao, H. M. Micolochick Steuer, T. Whitaker, T. Nachman, M. J. Sofia, P. Wang, M. J. Otto and P. A. Furman, Antimicrob. Agents Chemother., 2008, 52, 458. [138] P. A. Furman, P. Wang, C. Niu, D. Bao, W. Symonds, D. Nagarathnam, H. M. Steuer, S. Rakakonda, B. S. Ross, M. J. Otto and M. J. Sofia, Hepatology, 2008, 48(S1), 1161A, A1901. [139] M. Rodriguez-Torrez, J. Lazelari, E. J. Gane, E. DeJesus, D. R. Nelson, G. T. Everson, I. M. Jacobson, K. R. Reddy, J. G. McHutchson, A. Beard, S. Walker, W. Symonds and M. M. Berrey, Hepatology, 2008, 48(S1), 1160A, A1899.
434
Nicholas A. Meanwell et al.
[140] D. B. Smith, J. A. Martin, K. Klumpp, S. J. Baker, P. A. Blomgren, R. Devos, C. Granycome, J. Hang, C. J. Hobbs, W.-R. Jiang, C. Laxton, S. Le Pogam, V. Leveque, H. Ma, G. Maile, J. H. Merrett, A. Pichota, K. Sarma, M. Smith, S. Swallow, J. Symons, D. Vesey, I. Najera and N. Cammack, Bioorg. Med. Chem. Lett., 2007, 17, 2570. [141] P. Toniutto, C. Fabris, D. Bitetto, E. Fumolo, E. Fornasiere and M. Pirisi, IDrugs, 2008, 11, 738. [142] P. Toniutto, C. Fabris, D. Bitetto, E. Fornasiere, R. Rapetti and M. Pirisi, Curr. Opin. Investig. Drugs, 2007, 8, 150. [143] S. S. Carroll, S. Ludmerer, L. Handt, K. Koeplinger, N. Rena Zhang, D. Graham, M.-E. Davies, M. MacCoss, D. Hazuda and D. B. Olsen, Antimicrob. Agents Chemother., 2009, 53, 926. [144] Idenix press release, http://www.idenix.com/hepc/drug/ [145] A. Erhardt, K. Deterding, Y. Benhamou, M. Reiser, X. Forns, S. Pol, J. L. Calleja, S. Ross, H. C. Spangenberg, J. Garcia-Samaniego, M. Fuchs, J. Enrı´quez, J. Wiegand, J. Stern, K. Wu, G. Kukolj, M. Marquis, P. Beaulieu, G. Nehmiz and J. Steffgen, Antiviral Ther., 2009, 14, 23. [146] P. D. Patel, M. R. Patel, N. Kaushik-Basu and T. T. Talele, J. Chem. Inf. Model., 2008, 48, 42. [147] H. Cao, R. Cao, H. Zhang, X. Zheng and D. Gao, Curr. Med. Chem., 2008, 15, 1462. [148] S. Hirashima, T. Oka, K. Ikegashira, S. Noji, H. Yamanaka, Y. Hara, H. Goto, R. Mizojiri, Y. Niwa, T. Noguchi, I. Ando, S. Ikeda and H. Hashimoto, Bioorg. Med. Chem. Lett., 2007, 17, 3181. [149] I. Stansfield, M. Pompei, I. Conte, C. Ercolani, G. Migliaccio, M. Jairaj, C. Giuliano, M. Rowley and F. Narjes, Bioorg. Med. Chem. Lett., 2007, 17, 5143. [150] I. Stansfield, C. Ercolani, A. Mackay, I. Conte, M. Pompei, U. Koch, N. Gennari, C. Giuliano, M. Rowley and F. Narjes, Bioorg. Med. Chem. Lett., 2009, 19, 627. [151] J. Habermann, E. Capito, M. D. R. R. Ferreira, U. Koch and F. Narjes, Bioorg. Med. Chem. Lett., 2009, 19, 633. [152] D. Brainard, D. H. Wright, K. Sneddon, C. Cummings, P. Sun, J. Valentine, M. Anderson, S. Warrington, B. Sanderson, J. Chodakewitz and J. Wagner, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 937, http://www.abstractserver.com/easl2009/ planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 464&EASL2009 ¼ grnhh65l25p1sdrskl81trcq53&EASL2009 ¼ grnhh65l25p1sdrskl81trcq53 [153] R. De Francesco, G. Paonessa, D. Olsen, M. Rowley, B. Crescenzi, J. Habermann, F. Narjes and R. Laufer, HepDart, Lahaina, Hawaii, December 9–13, 2007, http:// www.informedhorizons.com/hepdart2007/pdf/hepdart07_presentations/Tues_07_ RDeFrancesco_HepDart_2007export.pdf [154] J. Pouliot, R. N. Pagila, K. Fung, G. Deng, G. Chenail, D. R. Latour, J. Zhang, S. KooMcCoy, M. Sivaraja, J. Bechtel, K. Lin, J. Gralapp, S. G. Alvarez, M. Leivers, U. Schmitz, T. Compton and R. Griffith, Hepatology, 2008, 48(S1), 1165A, A1911. [155] C. P. Bergstrom, J. A. Bender, R. G. Gentles, P. Hewawasam, T. W. Hudyma, J. F. Kadow, S. W. Martin, A. Regueiro-Ren, K.-S. Yeung, Y. Tu, K. A. Grant-Young and X. Zheng, WO Patent Application 2007/033175-A1, 2007. [156] T. W. Hudyma, X. Zheng, F. He, M. Ding, C. P. Bergstrom, P. Hewawasam, S. W. Martin and R. G. Gentles, WO Patent Application 2007/092000-A1, 2007. [157] N. A. Meanwell, R. G. Gentles, M. Ding, J. A. Bender, J. F. Kadow, P. Hewawasam, T. W. Hudyma and X. Zheng, WO Patent Application 2008/111978-A1, 2008. [158] R. G. Gentles, X. Zheng, M. Ding, Y. Tu, Y. Han, P. Hewawasam, J. F. Kadow, J. A. Bender, K.-S. Yeung, K. A. Grant-Young and T. W. Hudyma, WO Patent Application 2008/112473-A1, 2008. [159] J. A. Bender, R. G. Gentles, Y. Han, Y. Tu, Z. Yang, K.-S. Yeung and K. A. Grant-Young, WO Patent Application 2008/112841-A1, 2008.
Discovery and Development of Inhibitors of HCV
435
[160] R. G. Gentles, X. Zheng, M. Ding, Y. Tu, Y. Han, P. Hewawasam, J. F. Kadow, J. A. Bender, K.-S. Yeung, K. A. Grant-Young and T. W. Hudyma, WO Patent Application 2008/112848-A1, 2008. [161] K.-S. Yeung, J. A. Bender, R. G. Gentles, Z. Yang, M. Ding, Y. Tu, P. Hewawasam, Y. Han and J. F. Kadow, WO Patent Application 2008/112851-A1, 2008. [162] S. W. Martin, C. P. Bergstrom, R. G. Gentles and K.-S. Yeung, WO Patent Application 2009/029384-A2, 2009. [163] H. Li, J. Tatlock, A. Linton, J. Gonzalez, T. Jewell, L. Patel, S. Ludlum, M. Drowns, S. V. Rahvendran, H. Skor, R. Hunter, S. T. Shi, K. J. Herlihy, H. Parge, M. Hickey, X. Yu, F. Chau, J. Nonomiya and C. Lewis, J. Med. Chem., 2009, 50, 3969. [164] S. T. Shi, K. J. Herlihy, J. P. Graham, J. Nonomiya, S. V. Rahavendran, H. Skor, R. Irvine, S. Binford, J. Tatlock, H. Li, J. Gonzalez, A. Linton, A. K. Patick and C. Lewis, Antimicrob. Agents Chemother., 2009, 53, 2544. [165] P. Troke, M. Lewis, P. Simpson, E. van der Ryst, J. Hammond, C. Craig, M. Perros and M. Westby, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 968, http:// www.abstractserver.com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_ iplanner&absno ¼ 1795&EASL2009 ¼ n2c2gteknasbmv8149gnu3obj3&EASL2009 ¼ n2c2gteknasbmv8149gnu3obj3 [166] L. Chan Chung Kong, S. Kumar Das, C. G. Yannopoulos, G. Falardeau, L. Vaillancourt and R. Denis, WO Patent Application 2008/058393-A1, 2008. [167] O. Nicolas, I. Boivin, A. Berneche-D’Amours, P. Fex, F. Denis, S. Selliah and J. Bedard, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 961, http://www.abstractserver. com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 943&EASL2009 ¼ 5qubar83ml3599p7d1vqhmcr21&EASL2009 ¼ 5qubar83ml3599p7 d1vqhmcr21 [168] C. Cooper, R. Larouche, B. Bourgault, N. Chauret and L. Proulx, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 940, http://www.abstractserver.com/easl2009/ planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 1504&EASL2009 ¼ lvfhdsnju362ukbm4c5sv96994&EASL2009 ¼ lvfhdsnju362ukbm4c5sv96994 [169] J. Bedard, O. Nicolas, D. Bilimoria, L. L’Heureux, P. Fex, M. David and L. Chan, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 935, http://www.abstractserver.com/easl2009/ planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 937&EASL2009 ¼ 5qubar83ml3599p7d1vqhmcr21&EASL2009 ¼ 5qubar83ml3599p7d1vqhmcr21 [170] N. Chauret, C. Chagnon-Labelle, M. Diallo, J. Laquerre, J. Laterreur, S. May and L. SteMarie, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 939, http://www.abstractserver. com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 928&EASL2009 ¼ 2e4ntr9scpfap06bi90l8knv01&EASL2009 ¼ 2e4ntr9scpfap06bi90l8 knv01 [171] M. J. Slater, E. M. Amphlett, D. M. Andrews, G. Bravi, G. Burton, A. G. Cheasty, J. A. Corfield, M. R. Ellis, R. H. Fenwick, S. Fernandes, R. Guidetti, D. Haigh, C. D. Hartley, P. D. Howes, D. L. Jackson, R. L. Jarvest, V. L. H. Lovegrove, K. J. Medhurst, N. R. Parry, H. Price, P. Shah, O. M. P. Singh, R. Stocker, P. Thommes, C. Wilkinson and A. Wonacott, J. Med. Chem., 2007, 50, 897. [172] E. Lawitz, M. Rodriguez-Torres, M. DeMicco, T. Nguyen, E. Godofsky, J. Appleman, M. Rahimy, C. Crowley and J. Freddo, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 1055, http://www.abstractserver.com/easl2009/planner/sp.php?go ¼ abstract
436
[173]
[174]
[175]
[176]
[177]
[178]
[179]
[180]
[181]
[182]
Nicholas A. Meanwell et al.
&action ¼ abstract_iplanner&absno ¼ 2687&EASL2009 ¼ v90uv7ab1nsl3simolr28m6n u2&EASL2009 ¼ v90uv7ab1nsl3simolr28m6nu2 G. Koev, R. Mondal, J. Beyer, T. Reisch, S. Masse, W. Kati, D. Hutchinson, C. Flentge, J. Randolph, P. Donner, A. Krueger, R. Wagner, P. Yan, T. Lin, C. Maring and A. Molla, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 953, http://www.abstractserver. com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 678&EASL2009 ¼ lam2cg9t20hujsupclpiietmt1&EASL2009 ¼ lam2cg9t20hujsupclpii etmt1 F. Ruebsam, C. V. Tran, L.-S. Li, S. H. Kim, A. X. Xiang, Y. Zhou, J. K. Blazel, Z. Sun, P. S. Dragovich, J. Zhao, H. M. McGuire, D. E. Murphy, M. T. Tran, N. Stankovic, D. A. Ellis, A. Gobbi, R. E. Showalter, S. E. Webber, A. M. Shah, M. Tsan, R. A. Patel, L. A. LeBrun, H. J. Hou, R. Kamran, M. V. Sergeeva, D. M. Bartkowski, T. G. Nolan, D. A. Norris and L. Kirkovsky, Bioorg. Med. Chem. Lett., 2009, 19, 451. R. Wagner, D. P. Larson, D. W. A. Beno, T. D. Bosse, J. F. Darbyshire, Y. Gao, B. D. Gates, W. He, R. F. Henry, L. E. Hernandez, D. K. Hutchinson, W. W. Jiang, W. M. Kati, L. L. Klein, G. Koev, W. A. Kolbrenner, A. C. Krueger, J. Liu, Y. Liu, M. A. Long, C. J. Maring, S. V. Masse, T. Middleton, D. A. Montgomery, J. K. Pratt, P. Stuart, A. Molla and D. J. Kempf, J. Med. Chem., 2009, 52, 1659. L. Lu, T. Dekhtyar, S. Masse, R. Pithawalla, P. Krishnan, W. He, T. Ng, G. Koev, K. Stewart, D. Larson, T. Bosse, R. Wagner, T. Pilot-Matias, H. Mo and A. Molla, Antiviral Res., 2007, 76, 93. C.-H. Chen, Y. He, L. Lu, H. B. Lim, R. L. Tripathi, T. Middleton, L. E. Hernandez, D. W. A. Beno, M. A. Long, W. M. Kati, T. D. Bosse, D. P. Larson, R. Wagner, R. E. Lanford, W. E. Kohlbrenner, D. J. Kempf, T. J. Pilot-Matias and A. Molla, Antimicrob. Agents Chemother., 2007, 51, 4290. C. Maring, R. Wagner, D. Hutchinson, C. Flentge, W. Kati, G. Koev, Y. Liu, D. Beno, J. Shen, Y. Y. Lau, Y. Gao, J. Fischer, S. Vaidyanathan, B. H. Lim, J. Beyer, R. Mondal and A. Molla, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 955, http://www.abstract server.com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 681&EASL2009 ¼ lam2cg9t20hujsupclpiietmt1&EASL2009 ¼ lam2cg9t20 hujsupclpiietmt1 R. Menon, D. Cohen, A. Nada, E. Olson Dumas, Y.-L. Chiu, T. Podsadecki, W. Awni, B. Bernstein and C. Klein, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 956, http:// www.abstractserver.com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_ iplanner&absno ¼ 2046&EASL2009 ¼ lam2cg9t20hujsupclpiietmt1&EASL2009 ¼ lam 2cg9t20hujsupclpiietmt1 R. Menon, D. Cohen, A. Nada, Y.-L. Chiu, T. Podsadecki, W. Awni, B. Bernstein and C. Klein, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009, Abstract 957, http://www.abstractserver. com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 2067&EASL2009 ¼ lam2cg9t20hujsupclpiietmt1&EASL2009 ¼ lam2cg9t20hujsupclpi ietmt1 N. M. Kneteman, A. Y. M. Howe, T. Gao, J. Lewis, D. Pevear, G. Lund, D. Douglas, D. F. Mercer, D. L. J. Tyrrell, F. Immermann, I. Chaudhary, J. Speth, S. A. Villano, J. O’Connell and M. Collett, Hepatology, 2009, 49, 745. A. Y. M. Howe, H. Cheng, S. Johann, S. Mullen, S. K. Chunduru, D. C. Young, J. Bard, R. Chopra, G. Krishnamurthy, T. Mansour and J. O’Connell, Antimicrob. Agents Chemother., 2008, 52, 3327.
Discovery and Development of Inhibitors of HCV
437
[183] M. Flint, S Mullen, A. M. Deatly, W. Chen, L. Z. Miller, R. Ralston, C. Broom, E. A. Emini and A. Y. M. Howe, Antimicrob. Agents Chemother., 2009, 53, 401. [184] A. Feldstein, D. Kleiner, D. Kravetz and M. Buck, J. Clin. Gastroenterol., 2009, 43, 374. [185] G. Puerstinger, J. Paeshuyse, S. Heinrich, J. Mohr, N. Schraffl, E. Clercq and J. Neyts, Bioorg. Med. Chem. Lett., 2007, 17, 5111. [186] I. Vliegen, J. Paeshuyse, E. Mabery, B. Peng, I.-H. Shih, L. S. Lehman, H. Dutartre, B. Selisko, B. Canard, S. Bondy, W. Tse, H. Reiser, E. De Clerq, W. A. Lee, G. Pu¨rstinger, W. Zhong and J. Neyts, Hepatology, 2007, 46(S1), 222A, A1385. [187] E. D. Dowdy, K. M. Kent, N. J. Tom and V. Zia, WO Patent Application 2009/009001-A1, 2009. [188] S. S. Bondy, T. C. Dahl, D. A. Oare, R. Oliyai, W. C. Tse and V. Zia, WO Patent Application 2008/005519-A2, 2008. [189] P. Poumbourios and H. E. Drummer, Antiviral Chem. Chemother., 2007, 18, 169. [190] J. M. Timpe and J. A. McKeating, Gut, 2008, 57, 1728. [191] J. Dubuisson, F. Helle and L. Cocquerel, Cell. Microbiol., 2008, 10, 821. [192] F. Helle and J. Dubuisson, Cell. Mol. Life Sci., 2008, 65, 100. [193] T. Hahn and C. M. Rice, J. Biol. Chem., 2008, 283, 3689. [194] S. Liu, W. Yang, L. Shen, J. R. Turner, C. B. Coyne and T. Wang, J. Virol., 2009, 83, 2011. [195] A. Ploss, M. J. Evans, V. A. Gaysinskaya, M. Panis, H. You, Y. P. Jong and C. M. Rice, Nature, 2009, 457, 882. [196] M. J. Evans, T. Hahn, D. M. Tscherne, A. J. Syder, M. Panis, B. Woelk, T. Hatziioannou, J. A. McKeating, P. D. Bieniasz and C. M. Rice, Nature, 2007, 446, 801. [197] M. R. Beard and F. J. Warner, Hepatology, 2007, 46, 277. [198] A. Zheng, F. Yuan, Y. Li, F. Zhu, P. Hou, J. Li, X. Song, M. Ding and H. Deng, J. Virol., 2007, 81, 12465. [199] L. Meertens, C. Bertaux, L. Cukierman, E. Cormier, D. Lavillette, F.-L. Cosset and T. Dragic, J. Virol., 2008, 82, 3555. [200] W. Yang, C. Qiu, N. Biswas, J. Jin, S. C. Watkins, R. C. Montelaro, C. B. Coyne and T. Wang, J. Biol. Chem., 2008, 283, 8643. [201] G. Koutsoudakis, E. Herrmann, S. Kallis, R. Bartenschlager and T. Pietschmann, J. Virol., 2007, 81, 588. [202] M. Brazzoli, A. Bianchi, S. Filippini, A. Weiner, Q. Zhu, M. Pizza and S. Crotta, J. Virol., 2008, 82, 8316. [203] J. Grove, T. Huby, Z. Stamataki, T. Vanwolleghem, P. Meuleman, M. Farquhar, A. Schwarz, M. Moreau, J. S. Owen, G. Leroux-Roels, P. Balfe and J. A. McKeating, J. Virol., 2007, 81, 3162. [204] Z. S. Jia, D. W. Du, Y. F. Lei, X. Wei, W. Yin, L. Ma, J. Q. Lian, P. Z. Wang, D. Li and Y. X. Zhou, J. Int. Med. Res., 2008, 36, 1319. [205] M. Regeard, M. Trotard, C. Lepere, P. Gripon and J. Seyec, J. Viral Hepat., 2008, 15, 865. [206] M. B. Zeisel, G. Koutsoudakis, E. K. Schnober, A. Haberstroh, H. E. Blum, F.-L. Cosset, T. Wakita, D. Jaeck, M. Doffoel, C. Royer, E. Soulier, E. Schvoerer, C. Schuster, F. StollKeller, R. Bartenschlager, T. Pietschmann, H. Barth and T. F. Baumert, Hepatology, 2007, 46, 1722. [207] S. B. Kapadia, H. Barth, T. Baumert, J. A. McKeating and F. V. Chisari, J. Virol., 2007, 81, 374. [208] H. J. Harris, M. J. Farquhar, C. J. Mee, C. Davis, G. M. Reynolds, A. Jennings, K. Hu, F. Yuan, H.-K. Deng, S. G. Hubscher, J. H. Han, P. Balfe and J. A. McKeating, J. Virol., 2008, 82, 5007. [209] P. Meuleman, J. Hesselgesser, M. Paulson, T. Vanwolleghem, I. Desombere, H. Reiser and G. Leroux-Roels, Hepatology, 2008, 48, 1761.
438
Nicholas A. Meanwell et al.
[210] J. Witteveldt, M. J. Evans, J. Bitzegeio, G. Koutsoudakis, A. M. Owsianka, A. G. N. Angus, Z.-Y. Keck, S. K. H. Foung, T. Pietschmann, C. M. Rice and A. H. Patel, J. Gen. Virol., 2009, 90, 48. [211] C. Schuster and T. F. Baumert, J. Hepatol., 2009, 50, 222. [212] C. Voisset, M. Lavie, F. Helle, A. Op De Beeck, A. Bilheu, J. Bertrand-Michel, F. Terce, L. Cocquerel, C. Wychowski, N. Vu-Dac and J. Dubuisson, Cell. Microbiol., 2008, 10, 606. [213] M. J. Farquhar, H. J. Harris, M. Diskar, S. Jones, C. J. Mee, S. U. Nielsen, C. L. Brimacombe, S. Molina, G. L. Toms, P. Maurel, J. Howl, F. W. Herberg, S. C. D. IJzendoorn, P. Balfe and J. A. McKeating, J. Virol., 2008, 82, 8797. [214] W. Yang, B. L. Hood, S. L. Chadwick, S. Liu, S. C. Watkins, G. Luo, T. P. Conrads and T. Wang, Hepatology, 2008, 48, 1396. [215] M. T. Catanese, R. Graziani, T. Hahn, M. Moreau, T. Huby, G. Paonessa, C. Santini, A. Luzzago, C. M. Rice, R. Cortese, A. Vitelli and A. Nicosai, J. Virol., 2007, 81, 8063. [216] K. Murao, H. Imachi, X. Yu, W. M. Cao, T. Nishiuchi, K. Chen, J. Li, R. A. M. Ahmed, N. C. W. Wong and T. Ishida, Gut, 2008, 57, 664. [217] E. Falkowska, F. Kajumo, E. Garcia, J. Reinus and T. Dragic, J. Virol., 2007, 81, 8072. [218] F. Helle, A. Goffard, V. Morel, G. Duverlie, J. McKeating, Z.-Y. Keck, S. Foung, F. Penin, J. Dubuisson and C. Voisset, J. Virol., 2007, 81, 8101. [219] B. Bartosch and F.-L. Cosset, Methods Mol. Biol., 2009, 510, 279. [220] J.-P. Yang, D. Zhou and F. Wong-Staal, Methods Mol. Biol., 2009, 510, 295. [221] E.-I. Pecheur, D. Lavillette, F. Alcaras, J. Molle, Y. S. Boriskin, M. Roberts, F.-L. Cosset and S. J. Polyak, Biochemistry, 2007, 46, 6050. [222] M. Holzer, S. Ziegler, A. Neugebauer, B. Kronenberger, C. D. Klein and R. W. Hartmann, Arch. Pharm. Chem. Life Sci., 2008, 341, 478. [223] M. Holzer, S. Ziegler, B. Albrecht, B. Kronenberger, A. Kaul, R. Bartenschlager, L. Kattner, C. D. Klein and R. W. Hartmann, Molecules, 2008, 13, 1081. [224] G. Coburn, A. Q. Han, J. de Muys, C. Gauss, K. Provoncha, M. Canfield, D. Paul, S. Mohamed, S. Moorji, D. Fisch, J. D. Murga, Y. Rotshteyn, D. Qian, P. J. Maddon and W. C. Olson, Hepatology, 2008, 48(S1), 1162A, A1904. [225] iTherX, press release Febraury 3, 2009, http://www.itherx.com/press.html [226] T. J. Cuthbertson, M. Ibanez, C. A. Rijnbrand, A. J. Jackson, G. K. Mittapalli, F. Zhao, J. E. MacDonald and F. Wong-Staal, WO Patent Application 2008/021745-A2, 2008. [227] H. Ueno, T. Shimada, K. Aoyagi, S. Katoh, H. Shinkai, T. Motomura, Y. Komoda, T. Otsubaki, Y. Soejima and I. Kawahara, WO Patent Application 2007/058392 A1, 2007. [228] J. de Bruijne, J. Bergmann, C. Weegink, K. van Nieuwkerk, R. de Knegt, J. van de Wetering de Rooij, A. van Vliet, R. Molenkamp, J. Schinkel, H. Reesink and H. Janssen, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009. Abstract 822, http://www.kenes.com/ easl2009/posters/Abstract822.htm [229] C. S. Fraser and J. A. Doudna, Nat. Rev. Microbiol., 2007, 5, 29. [230] P. J. Lukavsky, Virus Res., 2009, 139, 166. [231] S. M. Dibrov, H. Johnston-Cox, Y.-H. Weng and T. Hermann, Angew. Chem. Intl. Ed., 2007, 46, 226. [232] Q. Zhao, Q. Han, C. R. Kissinger, T. Hermann and P. A. Thompson, Acta Crystallogr. D Biol. Crystallogr., 2008, D64, 436. [233] C. Alotte, A. Martin, S. A. Caldarelli, A. Di Giorgio, R. Condom, F. Zoulim, D. Durantel and O. Hantz, Antiviral Res., 2008, 80, 280. [234] C. Romero-Lopez, R. Diaz-Gonzalez and A. Berzal-Herranz, Cell. Mol. Life Sci., 2007, 64, 2994. [235] K. Konno, S. Fujita, M. Iizuka, S. Nishikawa, T. Hasegawa and K. Fukuda, Nucleic Acids Symp. Ser., 2008, 52, 493.
Discovery and Development of Inhibitors of HCV
439
[236] K. Konno, S. Nishikawa, T. Hasegawa and K. Fukuda, Nucleic Acids Symp. Ser., 2007, 51, 393. [237] C. Chevalier, A. Saulnier, Y. Benureau, D. Flechet, D. Delgrange, F. Colbere-Garapin, C. Wychowski and A. Martin, Mol. Ther., 2007, 15, 1452. [238] V. Vlassov, B. Korba, K. Farrar, S. Mukerjee, A. A. Seyhan, H. Ilves, R. L. Kaspar, D. Leake, S. A. Kazakov and B. H Johnston, Oligonucleotides, 2007, 17, 223. [239] T. Kanda, R. Steele, R. Ray and R. B. Ray, J. Virol., 2007, 81, 669. [240] V. Guerniou, R. Gillet, F. Berree, B. Carboni and B. Felden, Nucleic Acids Res., 2007, 35, 6778. [241] G. Godeau, C. Staedel and P. Barthelemy, J. Med. Chem., 2008, 51, 4374. [242] A. Litovchick and J. W. Szostak, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 1529. [243] A. Bhattacharyya, C. R. Trotta and S. W. Peltz, Drug Discov. Today, 2007, 12, 553. [244] R. Di and N. E. Tumer, WO Patent Application 2008/088875-A2, 2008. [245] G. M. Karp, WO Patent Application 2007/084413-A2, 2007. [246] G. M. Karp, P. S. Hwang, J. J. Takasugi, H. Ren, R. G. Wilde, A. A. Turpoff, A. Arefolov, G. Chen and J. A. Campbell, WO Patent Application 2007/084435-A2, 2007. [247] F. C. Lahser and G. M. Karp, WO Patent Application 2007/106317-A2, 2007. [248] H. Huang, F. Sun, D. M. Owen, W. Li, Y. Chen, M. Gale and J. Ye, Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 5848. [249] V. Icard, O. Diaz, C. Scholtes, L. Perrin-Cocon, C Ramie`re, R. Bartenschlager, F. Penin, V. Lotteau and P. Andre´, PLoS ONE, 2009, 4, e4233. [250] Y. Nahmias, J. Goldwasser, M. Casali, D. Poll, T. Wakita, R. T. Chung and M. L. Yarmush, Hepatology, 2008, 47, 1437. [251] D. Durantel, C. Alotte and F. Zoulim, Curr. Opin. Investig. Drugs, 2007, 8, 125. [252] C. Chapel, C. Garcia, B. Bartosch, P. Roingeard, N. Zitzmann, F.-L. Cosset, J. Dubuisson, R. A. Dwek, C. Trepo, F. Zoulim and D. Durantel, J. Gen. Virol., 2007, 88, 1133. [253] R. Flisiak, J.-M. Dumont and R. Crabbe´, Exp. Opin. Investig. Drugs, 2007, 16, 1345. [254] K. Watashi and K. Shimotohno, Rev. Med. Virol., 2007, 17, 245. [255] K. Moriishi and Y. Matsuura, Rev. Med. Virol., 2007, 17, 343. [256] F. Yang, J. M. Robotham, H. B. Nelson, A. Irsigler, R. Kenworthy and H. Tang, J. Virol., 2008, 82, 5269. [257] S. Chinnaswamy, I. Yarbrough, S. Palaninathan, C. T. R. Kumar, V. Vijayaraghavan, B. Demeler, S. M. Lemon, J. C. Sacchettini and C. C. Kao, J. Biol. Chem., 2008, 283, 20535. [258] M. A. El-Farrash, H. H. Aly, K. Watashi, M. Hijikata, H. Egawa and K. Shimotohno, Microbiol. Immunol., 2007, 51, 127. [259] J. E. Mathy, S. Ma, T. Compton and K. Lin, Antimicrob. Agents Chemother., 2008, 52, 3267. [260] R. Crabbe´, G. Vuagniaux, J.-M. Dumont, V. Nicolas-Metral, J. Marfurt and L. Novaroli, Exp. Opin. Investig. Drugs, 2009, 18, 211–220. [261] L. Coelmont, S. Kaptein, J. Paeshuyse, I. Vliegen, J.-M. Dumont, G. Vuagniaux and J. Neyts, Antimicrob. Agents Chemother., 2009, 53, 967. [262] R. G. Ptak, P. A. Gallay, D. Jochmans, A. P. Halestrap, U. T. Ruegg, L. A. Pallansch, M.D. Bobardt, M.-P. Be´thune, J. Neyts, E. Clercq, J.-M. Dumont, P. Scalfaro, K. Besseghir, R. M. Wenger and B. Rosenwirth, Antimicrob. Agents Chemother., 2008, 52, 1302. [263] S. Hopkins, B. Scorneaux, S. M. Mosier, Z. Huang, M. G. Murray and R. R. Harris, Hepatology, 2008, 48(S1), 1117A, A1814. [264] M. Peel, R. Harris, Z. Huang, S. Hopkins, K. Li, M. Peel, T. E. Richardson, B. Scorneaux, A. Scribner and S. Wring, Hepatology, 2008, 48(S1), 1167A, A1915. [265] J. M. Robida, H. B. Nelson, Z. Liu and H. Tang, J. Virol., 2007, 81, 5829.
440
Nicholas A. Meanwell et al.
[266] F. Fernandes, D. S. Poole, S. Hoover, R. Middleton, A.-C. Andrei, J. Gerstner and R. Striker, Hepatology, 2007, 46, 1026. [267] R. Flisiak, A. Horban, P. Gallay, M. Bobardt, S. Selvarajah, A. Wiercinska-Drapalo, E. Siwak, I. Cielniak, J. Higersberger, J. Kierkus, C. Aeschlimann, P. Grosgurin, V. Nicolas-Me´tral, J.-M. Dumont, H. Porchet, R. Crabbe´ and P. Scalfaro, Hepatology, 2008, 47, 817. [268] R. Flisiak, S. V. Feinman, M. Jablowski, A. Horban, W. Kryczka, M. Pawlowska, J. E. Heathcote, G. Mazzella, C. Vandeli, V. Nicolas-Me´tral, P. Grosgurin, J. S. Liz, P. Scalfaro, H. Porchet and R. Crabbe´, Hepatology, 2009, 49, 1460. [269] S. Hopkins, D. Heuman, E. Gavis, J. Lalezari, E. Glutzer, B. DiMassimo, P. Rusnak, S. Wring, C. Smitley and Y. Ribeill, 44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22–26, 2009. Abstract 90, http://www.abstractserver.com/easl2009/planner/sp.php?go ¼ abstract&action ¼ abstract_iplanner&absno ¼ 2400&EASL2009 ¼ plp2pf2nk7dajqfdc38tkjf1l2&EASL 2009 ¼ plp2pf2nk7dajqfdc38tkjf1l2 [270] V. R. Anderson and M. P. Curran, Drugs, 2007, 67, 1947. [271] J. F. Rossignol and E. B. Keeffe, Future Microbiol., 2008, 3, 539. [272] B. E. Korba, A. B. Montero, K. Farrar, K. Gaye, S. Mukerjee, M. S. Ayers and J.-F. Rossignol, Antiviral Res., 2008, 77, 56. [273] B. E. Korba, M. Elazar, P. Lui, J.-F. Rossignol and J. S. Glenn, Antimicrob. Agents Chemother., 2008, 52, 4069. [274] M. Elazar, M. Liu, S. McKenna, P. Liu, E. A. Gehrig, A. Elfert, J. Puglisi, J.-F. Rossignol and J. S. Glenn, Hepatology, 2008, 48(S1), 1151A, A1881. [275] J.-F. Rossignol, S. M. Kabil, Y. El-Gohary, A. Elfert and E. B. Keeffe, Aliment. Pharmacol. Ther., 2008, 28, 574. [276] J.-F. Rossignol, A. Elfert, Y. El-Gohary and E. B. Keeffe, Gastroenterol., 2009, 136, 856. [277] J. E. Semple and J.-F. Rossignol, WO Patent Application 2009/035788-A1, 2009. [278] R. Gazak, D. Walterova and V. Kren, Curr. Med. Chem., 2007, 14, 315. [279] M. Y. Parmar and T. R. Gandhi, Phcog. Rev., 2008, 2, 102. [280] S. J. Polyak, C. Morishima, M. C. Shuhart, C. C. Wang, Y. Liu and D. Y.-W. Lee, Gastroenterol., 2007, 132, 1925. [281] A. Melhem, M. Stern, O. Shibolet, E. Israeli, Z. Ackerman, O. Pappo, N. Hemed, M. Rowe, H. Ohana, G. Zabrecky, R. Cohen and Y. Ilan, J. Clin. Gastroenterol., 2005, 39, 737. [282] E. Gabbay, E. Zigmond, O. Pappo, N. Hemed, M. Rowe, G. Zabrecky, R. Cohen and Y. Ilan, World J. Gastroenterol., 2007, 13, 5317. [283] P. Ferenci, T.-M. Scherzer, H. Kerschner, K. Rutter, S. Beinhardt, H. Hofer, M. Schoeniger-Hekele, H. Holzmann and P. Steindl-Munda, Gastroenterol., 2008, 135, 1561.
CHAPT ER
21 Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions Terry W. Moore and John A. Katzenellenbogen
Contents
1. Introduction 2. Coregulators of Nuclear Hormone Receptors 3. Coactivator Binding Inhibitors 3.1 Peptidic coactivator binding inhibitors 3.2 Small-molecule coactivator binding inhibitors 4. Summary References
443 445 445 445 449 455 455
1. INTRODUCTION Nuclear hormone receptors (NRs) function as transcription factors, and many NRs are activated by endogenous hormones (e.g., estrogens, androgens, and thyroid hormones) as well as by synthetic analogs of these hormones. Upon binding of an agonist to a NR, several structural and functional changes occur; typically, these include receptor dimerization, nuclear translocation, and DNA binding at gene regulatory sites. Another important change is a conformational restructuring, described more fully later, which allows the NR to interact with its associated coregulatory proteins, both coactivators and corepressors. The NR– coregulator complex then modifies chromatin structure and either aids in (coactivates) or blocks (corepresses) the recruitment of the basal transcription machinery for RNA polymerase II (RNA pol II), which Department of Chemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 S. Mathews Ave., Urbana, IL 61801, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04421-2
r 2009 Elsevier Inc. All rights reserved.
443
444
Terry W. Moore and John A. Katzenellenbogen
leads to alterations in the level of gene expression. Although there are 48 members of the human NR superfamily, there are nearly 300 recognized coregulators [1,2]. In addition, coregulatory proteins have various enzymatic activities that can themselves be regulated by post-translational modifications. This complexity, combined with NR dimerization partners and tissue-specific expression, serves to mediate the vast array of physiological functions and tissue specificity of the NRs. Activating or inhibiting the NRs has profound physiological consequences, and, as a result, the NRs have been exploited as therapeutic targets across a number of diseases and physiological states. NR modulators currently used as therapeutic antagonists, such as the breast cancer drug tamoxifen, 1, and the prostate cancer drug flutamide, 2, bind to the ligand-binding pocket of the NR and induce a conformation of the receptor that either disfavors coactivator binding or favors corepressor binding. The inhibitory mechanism of these antagonists is indirect or allosteric, because ligand binding at one site affects protein–protein interaction at a second site. This conventional antagonist therapy is not without drawbacks, however, because many diseases and physiological states affected by an NR often become refractory to the effects of conventional antagonists, examples being the resistance to tamoxifen and flutamide that develops in breast and prostate cancer, respectively. Because it is well-established that blocking the recruitment of coactivators to the NR through this indirect, conventional antagonist mechanism is sufficient to block the transcription of NR-regulated genes, it is logical to assume that directly blocking the NR–coactivator interaction would yield comparable or perhaps superior results and could provide a second line of defense when resistance to conventional antagonist therapies develops. Me N Me O
Me F3C
H N Me O
H
Me 1
O2N 2
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
445
Coactivator binding inhibitors (CBIs) are molecules that block the interaction of NRs with coactivators by a direct, competitive mechanism. Such compounds are not without precedent in the literature, and both peptidic and small-molecule CBIs have been reported. From a discovery viewpoint, peptidic CBIs can yield much information about the requirements for disrupting the NR/coactivator interaction, but from a practical standpoint, small-molecule CBIs that would avoid the liabilities of peptides, such as cellular impermeability and metabolic instability, are preferred. Historically, interrupting protein–protein interactions with small molecules has posed significant challenges in drug discovery; however, there have been numerous recent reports of small molecules inhibiting the interaction of two proteins both specifically and potently [3–5]. Herein we outline recent advances in developing NR CBIs.
2. COREGULATORS OF NUCLEAR HORMONE RECEPTORS The NR coregulators constitute a large family of proteins, diverse in size and function, the best known of which are the coactivators of the p160 class (SRC1, 2, and 3) and the corepressors NCoR-1 and SMRT, although there are many others [2]. Additionally, those mentioned earlier are known to interact with other, non-NR transcription factors, a reflection of the complexity of the interactome [6,7]. The coregulator sequence elements, typically LXXLL motifs (NR boxes) in coactivators [8,9] and LXXXIXXXL motifs (corepressor nuclear receptor [CoRNR] boxes) in corepressors [10,11], occur over two and three turns of an a-helix, respectively, and make contacts with the NR by placing the denoted nonpolar residues in a short but deep hydrophobic groove formed by residues from helices 3, 4, 5, 11, and 12 of the NR ligand-binding domain, often termed the activation function 2 (AF2) region. The p160 coactivators, when bound to NR-ligand complexes, function as scaffolds onto which a series of large multi-protein complexes assemble, leading to chromatin modifications, the recruitment of RNA polymerase II, and alteration of gene expression.
3. COACTIVATOR BINDING INHIBITORS 3.1 Peptidic coactivator binding inhibitors Although many studies have focused on disrupting the NR–coactivator interaction with peptides derived from endogenous coactivators, there have been relatively few studies of unnatural or modified peptides that perturb this interaction. Because these latter studies delineate
446
Terry W. Moore and John A. Katzenellenbogen
opportunity space not accessed by the natural coactivators, they can suggest designs for CBIs that might exhibit increased potency and selectivity.
3.1.1 Phage display peptides Various peptides generated by phage-display were shown to bind to estrogen receptor a (ERa) and/or ERb, in the presence of 17b-estradiol (E2), tamoxifen, or in the absence of any ligand [12,13]. In particular, the peptide a/bI (SSNHQSSRLIELLSR) was found to interact with either ERa or ERb in the presence of E2. Interestingly, these probes, obtained from a very large (W109 phage) random library, contain an LXXLL motif, further establishing this as an important consensus sequence for the ER/coactivator interaction. Another peptide, aII (SSLTSRDFGSWYASR), bound to ERa, but not ERb, in the presence of either tamoxifen or E2. Subsequent structural studies showed that this peptide did not bind at the coactivator binding groove, but consistent with the ability of both E2 and tamoxifen to recruit the probe, at a previously unknown binding site on the opposite side of the receptor [14]. A final class of peptide probes, a/bV ([S/M]X[D/E][W/F][W/F]XXXL), was found to bind to ERa or ERb in the presence of tamoxifen, but not E2; however, based on competition experiments, these peptides were believed to bind at the AF2 region, unlike aII. After further rounds of focused phage display, another peptide (BT1) was found to bind to the tamoxifen–ER complex with a higher potency, particularly when helix 12 was removed from ER [15]. The H12 sequence (LYDLLLEML) is a pseudo-CoRNR box motif believed responsible for the poor recruitment of corepressors by antagonist-liganded ER in reconstituted in vitro systems, because it competes with their binding to AF2; deleting this sequence allows these putative corepressor mimics to bind to ER [16]. The sequence of BT1 (ELFDAFQLRQLILRGLQDDIPYH) is also reminiscent of the CoRNR box consensus motif (LXXXIXXXL), so it is believed that a/bV and BT1 are peptidomimetics of corepressors. The respective co-crystal structures of each compound bound to a hydroxytamoxifen–ERa complex, expressed without H12, show that the peptides bind in the coactivator binding area, evidence that corepressors and coactivators bind at overlapping areas of the ER ligand-binding domain [16]. This same approach has also been used to find non-natural peptidic inhibitors of other NRs [17–21].
3.1.2 Synthetic peptides Beyond studies on the random peptides generated by phage display, efforts have been made to rationally design short peptides that might be more potent in inhibiting the NR/SRC interaction. Approaches have involved introduction of constraints on the peptide to enforce the
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
447
a-helical structure of the coactivator, as well as incorporation of nonnatural amino acids. A macrolactam constraint between E691 and K695 (Ac-EKHKILcyclo(E691RLLK695)DS-OH) of the coactivator SRC2 (also known as GRIP1) gave a peptide that was 15 times more potent than the unconstrained peptide in disrupting the interaction between thyroid hormone receptor b (TRb) and a peptide derived from NR Box 2 of the coactivator SRC2 [22]. The IC50 value of 0.62 mM for the cyclized peptide was determined by a fluorescence polarization assay. The same constraint (cyclo(E691–K695)), used together with incorporation of non-natural amino acids at the L690, L693, or L694 positions [23,24], gave peptides that were more potent and showed selectivity among three different NRs (ERa, ERb, and TRb). The SRCs themselves bear flanking residues that impart some selectivity, but this study demonstrated that a great degree of selectivity among NRs can be obtained simply by manipulating the side chains of the LXXLL motif itself. These peptides were most often selective for ERa (12 of 37), with one (L690W) showing a W600-fold selectivity for ERa (0.144 mM); one was ERb-selective (0.824 mM; o-Cl-phenylalanine at L693); and one was TRb selective (7.02 mM; p-F-phenylglycine at L690). ERa appeared more capable of binding large and a-branched substituents. Because docking suggested that cooperativity could not be achieved by mutating more than one residue, compounds containing multiple mutations were not examined. This constrained-peptide library was also used to study differential agonist ligand-dependent effects (E2, diethylstilbestrol, and genistein) on peptide recruitment to the AF2 region of ERa and ERb [25]. On the basis of analysis of crystal structures, the authors note differences in the size and shape of the grooves of TRb, ERb, and ERa: a ridge between the leucine-binding subpockets is more substantial in TRb than in ERa, and extra space unique to each NR is also found. For instance, in the ˚ 3 not available in TRb/SRC2-2/triiodothyronine complex, there is 268 A 3 ˚ ERa/SRC2-2/E2, and in ERa there is 357 A of space that is not available in TRb. Lack of available structures made the comparison across other NRs/ligand pairs more difficult. Of 37 inhibitors, 19 were more than 10-fold selective for ERa over ERb when liganded with E2; fifteen and six were ERa-selective when liganded with diethylstilbestrol and genistein, respectively. Only two peptides were selective for ERb, one each for E2 and genistein. Generally, ERa can accommodate replacement of the leucines with aromatic amino acids when an appropriately folded helical scaffold is used. Interestingly, experimental results reveal aspects of binding that the computational studies could not predict; this was attributed to the plasticity of the surface of ERa, which can readily expose new subsites for binding.
448
Terry W. Moore and John A. Katzenellenbogen
In a related report, a panel of 32 unconstrained 20 mers with sequences from NR boxes from various coactivators was assayed against TRb bound with three different ligands. The most potent was the SRC2-2 box sequence bound to a triiodothyronine-liganded TRb (0.7 mM). Most interestingly, ligand structure did influence which coregulator sequences were preferentially recruited, implying that the NR conformation induced by each agonist is sufficiently distinct to target a different coregulator/NR complex. This is likely a molecular determinant of the well-known tissue-selective pharmacology of the NRs. In a related approach, modified peptides were constrained by incorporation of a disulfide bond at various positions. Using a fluorescence-based assay [26], the researchers found that the sequence H-Lys-cyclo(D-Cys-IleLeu-Cys)-Arg-Leu-Leu-Gln–NH2 bound to E2-ERa with an IC50 of 25 nM, but about 15 times more weakly to E2-ERb (390 nM). An X-ray crystal structure of this peptide, referred to as peptidomimetic ER modulator 1 (PERM-1), showed it bound to the AF2 region of ERa [27]. Other disulfideand amide-linked peptides did not bind as well (high nanomolar to micromolar IC50s), but all peptides reported were between 2.4- and 64-fold selective for ERa over ERb. Building on this approach, disulfide-constrained peptides having exquisite affinities for ERa were synthesized, the most potent of which (H-Arg-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Npg-Leu-Gln-NH2, where Npg ¼ neopentylglycine) exhibited a Ki of 0.07 nM for ERa and 1.2 nM for ERb [28]. Almost all peptides published in this report were selective for ERa, but one was fivefold selective for ERb (H-Arg-cyclo(D-Cys-IleLeu-Cys)-Arg-tLeu-Leu-Gln-NH2, where tLeu is t-butylglycine): ERa Ki/ERb Ki ¼ 7/1.2 nM. In a finding consistent with the crystal structure [29], the authors assert that L2 of the LXXLL motif seems less important for binding to the receptor than are L1 and L3, based on the observation that Npg in the L2 position binds much more tightly than Leu (0.07 nM vs. 11 nM) but that Npg would not make any more contacts with the receptor than would Leu (the third methyl points toward the solvent). It is hypothesized that the extra methyl group in Npg stabilizes the conformation through a hydrophobic interaction with Ile (i1). Thus, L2 is more important for stabilizing the helix than for interacting with the receptor. The disulfide linkage in these peptides would likely subvert their use in the reducing environment of the cell. To address this issue, heterodimeric peptides were synthesized that were more likely to be cell permeable by appending, through a disulfide bond, a group that might facilitate uptake (i.e., a decanoyl or hepta-Arg group) [30]. Inside the cell, the disulfide bond would be cleaved, leaving the active monomeric peptide, a compound having a Ki of 60 nM on ERa (1850 nM on ERb) [28]. In a related approach, the stabilizing disulfide of another constrained peptide (H-Arg-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln–NH2; ERa
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
449
Ki/ERb Ki ¼ 11/77 nM) was replaced with a thioether linkage (ERa Ki/ERb Ki ¼ 6.9/64 nM), so that if it were cell-permeable, it would not be reduced by excess glutathione present in the cell [31]. Owing to the most unfortunate, premature death of the principal investigator, Arno Spatola, no further studies have been conducted to date to see whether peptides prepared by either of these approaches have activity in cell-based assays.
3.2 Small-molecule coactivator binding inhibitors Peptidic CBIs are useful as high-affinity probes for investigating NR action, but small molecule CBIs will likely be required for any therapeutic use. There are, however, a number of hurdles that must be overcome in developing high-affinity small-molecule CBIs: Small molecules are typically much smaller than the peptides thus far studied, and their smaller footprint can be an intrinsic disadvantage when inhibiting a high-affinity protein–protein interaction that spans a large surface area. The residues flanking the LXXLL sequence in the coactivator proteins also bolster potency in interaction with NRs; it would be difficult for a small molecule to reach these more distant interaction sites. The LXXLL sequence shared among the three SRCs and other coactivators is used for interaction with many, perhaps all, of the NRs. Therefore, issues regarding the selectivity that a small molecule might have for a particular NR are a concern: Are the slight differences in the coactivator interaction sites of different NRs sufficient so that receptor-selective small-molecule CBIs can be developed? A significant practical challenge in assaying small molecules for NR CBI activity is the need to demonstrate that they have little to no affinity for the ligand-binding pocket of the NR; if they do, they might displace the agonist and block coactivator binding indirectly by functioning as conventional antagonists. Fortunately, with most of the small-molecule CBIs described later, relatively simple control experiments have been used to distinguish between inhibition of coactivator binding by direct and indirect means. Because there are few sites for hydrogen bonding within the coactivator binding region of receptors, small molecules must rely largely on van der Waals and other hydrophobic interactions; they must, however, also maintain sufficient aqueous solubility. Because most high-throughput screening libraries are highly enriched in low molecular weight (i.e., o500 Da) compounds, following the Lipinski specifications for drug-like properties, finding even modest affinity hits for blocking a protein–protein interaction from a highthroughput screen of such libraries — a popular modus operandi for hit identification — can be difficult [32].
450
Terry W. Moore and John A. Katzenellenbogen
Although daunting, these challenges are not impossible to address, and significant work has been done to undertake these and other problems inherent in small molecule CBIs. Published small-molecule CBIs are listed below by NR.
3.2.1 Estrogen receptor The first non-peptidic CBIs for any of the NRs were reported in 2004 [33]. These tri-substituted pyrimidines, although exhibiting only modest affinities (KiB30 mM in a fluorescence polarization assay), provided evidence that small molecules could disrupt the ERa/SRC1 Box 2 interaction without binding in the ligand-binding pocket of the receptor. Each of the iso-propyl-terminated alkyl groups in 3 is thought to mimic one of the three leucines of the LXXLL motif. A library of compounds has been generated around this initial hit, producing compounds that show activity in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay and in an ERa-mediated transcription assay using luciferase as a reporter gene. The structure– activity relationship (SAR) around this hit shows that: (a) substituents at the 2- and 4-positions of the pyrimidine ring need to be linked through an N-atom, and there is a preference for smaller substituents (e.g., isobutyl) at the 2-position; (b) substituents can be linked to the pyrimidine ring at the 6-position through either carbon or nitrogen (but not sulfur or oxygen); (c) the CBI binding pocket in ERa can accommodate no more than two phenyl substituents and no more than one naphthyl substituent on the tri-substituted pyrimidine; and (d) to satisfy what are likely either hydrogen bonding or electrostatic requirements, one of the substituents must be linked through an –NHlinkage (not –NMe-). The best of these compounds have inhibition constants of 2–3 mM [34]. The guanylhydrazone 4, also described in 2004, inhibited the interaction of ERa or ERb (but not progesterone receptor) with SRC1, 2, and 3 with IC50 values B25 mM in an ELISA; moreover, the compound was active in a mammalian two-hybrid assay (Gal4 DNA-binding domain/human ERa ligand-binding domain fusion and SRC1, SRC3, or SRC3/VP16 fusion), exhibiting an IC50 of 5.5 mM [35]. At 10–20 mM, 4 was found to inhibit expression of the ERa-controlled gene pS2 in the ERpositive MCF-7 breast cancer cell line, although cell death occurred at concentrations W20 mM by an unknown, presumably ER-independent mechanism. The best of a small library of guanylhydrazone analogs had IC50 values in reporter gene and mammalian two-hybrid assays of 0.9 and 4.6 mM, respectively [36]. Observations from this study are that: (a) the most potent compounds possessed a chloro substituent at the 1-position; (b) substitution at the phenyl ring had little effect; (c) the second ring was not required (i.e., acyclic compounds showed good
451
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
activity), but, if present, it must be saturated; and (d) a ketonic hydrazone, rather than an aldehydic one, was completely inactive.
Me
Me
Me
Me O
HN HN Cl
N Me
N
N H
Me
3
Me
N
NHCOR
Me
H N
NH2
CO2H
Me
NH
Me
Me
4
5
Me
N H2N Me
NH2
Me
N
Me
Me O NH2
OH
6
7
In a TR-FRET assay, a series of bicyclo-[2.2.2]-octanes developed using structure-based design were only partial antagonists of the ERa/SRC3 interaction, with Kis ranging from 7 (i.e., bicyclooctane 5) to 40 M [37]. Given the great structural similarity between the LXXLL motif and the modeled bicyclooctane 5, the weak activity of these molecules illustrates that the precise placement of substituents mimicking the LXXLL leucine residues is certainly not the sole determinant of CBI activity. In a fluorescence polarization assay, the pyridylpyridone 6 was found to inhibit the interaction of ERa/SRC1 NR Box 2 peptide with a Ki of 4.2 mM [38]. A crystal structure of a related analog overlaid with the three substituents of the LXXLL motif shows how 6 is predicted to bind to ER. A series of amphipathic benzenes (e.g., 7) displaying alternating hydrophobic and hydrophilic substituents produces facially amphipathic
452
Terry W. Moore and John A. Katzenellenbogen
molecules that mimic the nonpolar leucine residues of the LXXLL motif on one face of the benzene ring and have polar groups to interact with water on the other [39,40]. Aminoethyl and pentyl groups gave the best combination of polar and hydrophobic substituents, respectively, for inhibiting the ERa/SRC3 protein fragment interaction in a TR-FRET assay (IC50 ¼ 1.7 mM) and the ERa/SRC protein interaction in reporter gene (IC50 ¼ 3.2 mM) and mammalian two-hybrid assays (IC50 ¼ 3.2 mM). In a surprising finding, two molecules of the selective ER modulator hydroxytamoxifen 8 were found in an ERb crystal structure, one in the ligand-binding pocket, but the other bound at the AF2 surface [41]. The unsubstituted phenyl of 8 was buried deep within the coactivatorbinding groove, but no polar interactions to keep the inhibitor in place were evident. Although this crystal structure is consistent with previous biochemical studies suggesting a second binding site for 8 [42], it is not clear whether this second-site binding occurs under physiological conditions.
3.2.2 Thyroid hormone receptor In a high-throughput screen of 138,000 compounds, some Mannich bases (i.e., 9) were found to inhibit the interaction of TRb with a peptide fragment from SRC2 NR Box 2 in a fluorescence polarization assay (IC50 ¼ 2 mM) [43]. The compounds eliminate amines, giving a,bunsaturated ketones that react with cysteine sulfhydryl groups on the TRb surface [44]. A crystal structure showing enone 10 bound noncovalently to the surface of TRb suggests that after elimination of dimethylamine, the compound binds irreversibly with the S-H group ˚ from the electrophilic center. of either C298 or C308, which are B7 A Mutation, fragmentation, and pull-down studies suggest that upon alkylation at C298, both coactivator and corepressor recruitment are irreparably impaired. Through further SAR studies, it was found that p-hexyl or p-heptyl substituents on the core hydrophobic ring were essential for good activity, with highly reactive enones (i.e., lacking large b-substituents) being most potent [45]. Other thiol-reactive electrophilic groups showed similar activity (e.g., a-haloketones, keto-epoxides, b-bromoketones). These changes, however, did not significantly improve compound potency. When assayed against a TR-positive thyroid cancer cell line (ARO) and a TR-negative osteosarcoma epithelial cell line (U2OS), no clear trends in cytotoxicity were seen. Thus, the compounds might also be working at targets other than the TRs. Interestingly, in in vitro assays, all compounds showed up to 18-fold selectivity for TRb over TRa.
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
453
Me N Me Me
O R
N
8
Me
Me
O
O
10
9 Me
HO
3.2.3 Androgen receptor After four off-patent drugs were discovered as hits in a highthroughput fluorescence polarization screen for compounds that block androgen receptor (AR)/SRC 2 Box 3 interaction, an X-ray screen of a smaller subset of compounds yielded crystal structures of seven molecules (flufenamic acid, 11; triiodothyronine, T3, 12; Triac, 13; 14; 15; 2-methylindole; and indole-3-carboxylic acid) bound to a previously unrecognized surface of AR [46]. This ‘‘Binding Function 3’’ (BF3) region on AR is near the junction of H1, the H3–H5 loop, and H9 and is adjacent to and nearly as large as AF2. The binding of these molecules to the BF3 region causes a slight restructuring of residues within both BF3 and AF2 regions, and a major repositioning of Arg726 at the AF2 boundary, which induces an AF2 conformation that disfavors SRC binding. Although the in vitro potencies of these molecules are quite modest (i.e., 50 mM), they are active as inhibitors in AR-regulated reporter gene assays at 10–30 mM. Also, although their mode of action is fundamentally different from that of the other CBIs mentioned earlier (i.e., not direct inhibition but allosteric inhibition originating from a site different from the AR ligand-binding pocket), their functional outcomes are similar. Some of these ligands (i.e., T3 and Triac) would cross-react by binding strongly to the ligand-binding pocket of TR; the competing activity of others on their established targets could also be problematic.
HO2C
I H N
CF3 HO2C
11
NH2
I O
I
I
OH
12
HO2C
O
I
I
OH
13
454
Terry W. Moore and John A. Katzenellenbogen
Me NH2
NH2 Me
N
OH
N
N
N
N
N
N
N Me
Me
Me Me
Me
14
Me
15
3.2.4 Pregnane X receptor (or steroid xenobiotic receptor) Xenobiotic activation of the pregnane X receptor (PXR) is known to upregulate genes involved in metabolism, often including metabolism of the inducing xenobiotic itself. Therefore, inhibiting this interaction could enhance the pharmacokinetic profile of a drug that is inducing its own degradation. The antifungal ketoconazole (16) has been reported to inhibit both the PXR/SRC1 and the constitutive androstene receptor (CAR)/SRC1 interactions [47], and it has been reported that the phytoestrogen coumestrol (17) also inhibits the PXR/SRC1 interaction [48]. Ketoconazole exhibited an IC50 of 74 mM in a pulldown assay and similar potency in a mammalian two-hybrid assay. Ketoconazole also delayed the metabolism of the anesthetic tribromoethanol in mice. The Ki of coumestrol in a fluorescence polarization assay (PXR ligand-binding domain/SRC1 peptide) was 1.2–1.3 mM, but this number is believed to be high because of poor compound solubility; lower coumestrol concentrations (i.e., 25 mM) antagonized rifampicin-activated PXR in mammalian two-hybrid assays. Mutational studies suggest that coumestrol is binding to a surface on PXR outside of the ligand-binding pocket. Coumestrol is also an agonist of PXR at 13 mM. O
Cl N
N
O
Cl
O
OH
Me
O
O N N
16
HO O O
17
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
455
4. SUMMARY Directly blocking the interaction of NRs and their coactivators could have profound implications in treating a number of different NR-regulated diseases. The work done thus far in developing these CBIs has focused on both peptidic and small-molecule inhibitors. Many peptidic CBIs are potent, and seemingly selective, but, despite work to enhance their cellular uptake and stability, they have not been active in cellular assays. Small-molecule CBIs, thus far, are not as potent as their peptidic counterparts, but many are active in cell-based assays of transcription and gene expression at low micromolar concentrations. Although more work remains to be done, these results provide a promising starting point for developing potent and selective small-molecule CBIs that could provide a valuable and necessary alternative approach to the modulation of NR activity.
REFERENCES ˚ . Gustafsson and V. Laudet, Nat. Rev. Drug Discov., 2004, 3, 950. [1] H. Gronemeyer, J-A [2] D. M. Lonard, R. B. Lanz and B. W. O’Malley, Endocr. Rev., 2007, 28, 575. [3] H. Yin, G. Lee, K. A. Sedey, O. Kutzki, H. S. Park, B. P. Orner, J. T. Ernst, H.-G. Wang, S. M. Sebti and A. D. Hamilton, J. Am. Chem. Soc., 2005, 127, 10191. [4] H. Yin, G. Lee, H. S. Park, G. A. Payne, J. M. Rodriguez, S. M. Sebti and A. D. Hamilton, Angew. Chem. Int. Ed., 2005, 44, 2704. [5] L. T. Vassilev, B. T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong, U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi and E. A. Liu, Science, 2004, 303, 844. [6] S-K. Lee, H-J. Kim, S-Y. Na, T. S. Kim, H-S. Choi, S.-Y. Im and J. W. Lee, J. Biol. Chem., 1998, 273, 16651. [7] S. Werbajh, I. Nojek, R. Lanz and M. A. Costas, FEBS Lett., 2000, 485, 195. [8] D. M. Heery, E. Kalkhoven, S. Hoare and M. G. Parker, Nature, 1997, 387, 733. [9] H. Y. Mak, S. Hoare, P. M. A. Henttu and M. G. Parker, Mol. Cell. Biol., 1999, 19, 3895. [10] J. D. Chen and R. M. Evans, Nature, 1995, 377, 454. [11] A. J. Ho¨rlein, A. M. Na¨a¨r, T. Heinzel, J. Torchia, B. Gloss, R. Kurokawa, A. Ryan, Y. Kamei, M. So¨derstro¨m, C. K. Glass and M. G. Rosenfeld, Nature, 1995, 377, 397. [12] J. D. Norris, L. A. Paige, D. J. Christensen, C.-Y. Chang, M. R. Huacani, D. Fan, P. T. Hamilton, D. M. Fowlkes and D. P. McDonnell, Science, 1999, 285, 744. [13] L. A. Paige, D. J. Christensen, H. Grfn, J. D. Norris, E. B. Gottlin, K. M. Padilla, C.-Y. Chang, L. M. Ballas, P. T. Hamilton, D. P. McDonnell and D. M. Fowlkes, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3999. ˚ . Gustafsson, E. Treuter, R. E. Hubbard and A. C. W. Pike, [14] E. H. Kong, N. Heldring, J.-A Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 3593. [15] H.-J. Huang, J. D. Norris and D. P. McDonnell, Mol. Endocrinol., 2002, 16, 1778. ˚ . Gustafsson and A. C. W. Pike, [16] N. Heldring, T. Pawson, D. McDonnell, E. Treuter, J.-A J. Biol. Chem., 2007, 282, 10449. [17] C. Chang, J. Abdo, T. Hartney and D. P. McDonnell, Mol. Endocrinol., 2005, 19, 2478.
456
Terry W. Moore and John A. Katzenellenbogen
[18] C.-Y. Chang, J. D. Norris, H. Grfn, L. A. Paige, P. T. Hamilton, D. J. Kenan, D. Fowlkes and D. P. McDonnell, Mol. Cell. Biol., 1999, 19, 8226. [19] J. M. Hall, C. Chang and D. P. McDonnell, Mol. Endocrinol., 2000, 14, 2010. [20] N. B. Mettu, T. B. Stanley, M. A. Dwyer, M. S. Jansen, J. E. Allen, J. M. Hall and D. P. McDonnell, Mol. Endocrinol., 2007, 21, 2361. [21] J. W. Pike, P. Pathrose, O. Barmina, C.-Y. Chang, D. P. McDonnell, H. Yamamoto and N. K. Shevde, J. Cell. Biochem., 2003, 88, 252. [22] T. R. Geistlinger and R. K. Guy, J. Am. Chem. Soc., 2001, 123, 1525. [23] T. R. Geistlinger and R. K. Guy, J. Am. Chem. Soc., 2003, 125, 6852. [24] T. R. Geistlinger and R. Kiplin Guy, in Methods in Enzymology (eds D. W. Russell and D. J. Mangelsdorf), Vol. 364, Elsevier, San Diego, 2003, p. 223. [25] T. R. Geistlinger, A. C. McReynolds and R. K. Guy, Chem. Biol., 2004, 11, 273. [26] K. S. Bramlett, Y. F. Wu and T. P. Burris, Mol. Endocrinol., 2001, 15, 909. [27] A-M. Leduc, J. O. Trent, J. L. Wittliff, K. S. Bramlett, S. L. Briggs, N. Y. Chirgadze, Y. Wang, T. P. Burris and A. F. Spatola, Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 11273. [28] A. K. Galande, K. S. Bramlett, J. O. Trent, T. P. Burris, J. L. Wittliff and A. F. Spatola, Chembiochem, 2005, 6, 1991. [29] A. C. W. Pike, A. M. Brzozowski and R. E. Hubbard, J. Steroid Biochem. Mol. Biol., 2000, 74, 261. [30] A. K. Galande and A. F. Spatola, Org. Lett., 2003, 5, 3431. [31] A. K. Galande, K. S. Bramlett, T. P. Burris, J. L. Wittliff and A. F. Spatola, J. Pept. Res., 2004, 63, 297. [32] J. A. Wells and C. L. McClendon, Nature, 2007, 450, 1001. [33] A. L. Rodriguez, A. Tamrazi, M. L. Collins and J. A. Katzenellenbogen, J. Med. Chem., 2004, 47, 600. [34] A. A. Parent, J. R. Gunther and J. A. Katzenellenbogen, J. Med. Chem., 2008, 51, 6512. [35] D. Shao, T. J. Berrodin, E. Manas, D. Hauze, R. Powers, A. Bapat, D. Gonder, R. C. Winneker and D. E. Frail, J. Steroid Biochem. Mol. Biol., 2004, 88, 351. [36] A. L. LaFrate, J. R. Gunther, K. E. Carlson and J. A. Katzenellenbogen, Bioorg. Med. Chem., 2008, 16, 10075. [37] H.-B. Zhou, M. L. Collins, J. R. Gunther, J. S. Comninos and J. A. Katzenellenbogen, Bioorg. Med. Chem. Lett., 2007, 17, 4118. [38] J. Becerril and A. D. Hamilton, Angew. Chem. Int. Ed., 2007, 46, 4471. [39] J. R. Gunther, T. W. Moore, M. L. Collins and J. A. Katzenellenbogen, ACS Chem. Biol., 2008, 3, 282. [40] K. V. Kilway and J. S. Siegel, Tetrahedron, 2001, 57, 3615. [41] Y. Wang, N. Y. Chirgadze, S. L. Briggs, S. Khan, E. V. Jensen and T. P. Burris, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 9908. [42] Y. Berthois, M. Pons, C. Dussert, A. C. de Paulet and P. M. Martin, Mol. Cell. Endocrinol., 1994, 99, 259. [43] L. A. Arnold, E. Este´banez-Perpin˜a´, M. Togashi, N. Jouravel, A. Shelat, A. C. McReynolds, E. Mar, P. Nguyen, J. D. Baxter, R. J. Fletterick, P. Webb and R. K. Guy, J. Biol. Chem., 2005, 280, 43048. [44] E. Este´banez-Perpin˜a´, L. A. Arnold, N. Jouravel, M. Togashi, J. Blethrow, E. Mar, P. Nguyen, K. J. Phillips, J. D. Baxter, P. Webb, R. K. Guy and R. J. Fletterick, Mol. Endocrinol., 2007, 21, 2919. [45] L. A. Arnold, A. Kosinski, E. Este´banez-Perpin˜a´, R. J. Fletterick and R. K. Guy, J. Med. Chem., 2007, 50, 5269.
Inhibitors of Nuclear Hormone Receptor/Coactivator Interactions
457
[46] E. Este´banez-Perpin˜a´, A. A. Arnold, P. Nguyen, E. D. Rodrigues, E. Mar, R. Bateman, P. Pallai, K. M. Shokat, J. D. Baxter, R. K. Guy, P. Webb and R. J. Fletterick, Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 16074. [47] H. Huang, H. Wang, M. Sinz, M. Zoeckler, J. Staudinger, M. R. Redinbo, D. G. Teotico, J. Locker, G. V. Kalpana and S. Mani, Oncogene, 2007, 26, 258. [48] H. Wang, H. Li, L. B. Moore, M. D. L. Johnson, J. M. Maglich, B. Goodwin, O. R. R. Ittoop, B. Wisely, K. Creech, D. J. Parks, J. L. Collins, T. M. Willson, G. V. Kalpana, M. Venkatesh, W. Xie, S. Y. Cho, J. Roboz, M. Redinbo, J. T. Moore and S. Mani, Mol. Endocrinol., 2008, 22, 838.
CHAPT ER
22 Safety Testing of Drug Metabolites Thomas N. Thompson
Contents
1. Introduction 1.1 Importance of metabolites in safety and efficacy 1.2 Brief chronology of events leading up to issuance of guidance 2. Evolution of the MIST Guidance 2.1 The MIST issues are defined and debated 2.2 Key points of the 2005 draft guidance 2.3 Areas of concern after issuance of 2005 draft guidance 2.4 Issuance of the final MIST guidance 3. Potential Issues Related to Implementation of a Sound MIST Strategy 4. Implications of Metabolism in Safety Testing of New Drugs 5. Strategy for Implementation of Best Practices 5.1 What metabolites should be measured? 5.2 What kind of information is needed and when is it needed? 6. Role of the Medicinal Chemist 7. Summary References Appendix: Decision Tree Flow Diagram
459 459 460 461 461 462 463 463 464 466 467 467 468 471 472 472 474
R&D Services Pharma Consulting, 663N. 132nd St. #126, Omaha, NE 68154, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04422-4
r 2009 Elsevier Inc. All rights reserved.
459
460
Thomas N. Thompson
1. INTRODUCTION 1.1 Importance of metabolites in safety and efficacy In the course of modern drug discovery and development, the clear mandate for introducing new drugs is that they be both effective and safe. As such, the safety evaluation program is of paramount importance in the development of any new drug. Indeed, the observance of toxicity in animal tests is a leading cause of drug withdrawal. Safety evaluation of potential new drugs has historically focused on the unchanged form of that drug in various species under varying experimental protocols. Metabolites have not been tested per se because they were assumed to be present during animal testing, and any toxicity attributable to metabolites was accounted for in the overall safety profile of the parent. In addition, the ability to understand the routes and extent of metabolism early enough in development to allow for proper safety evaluation of metabolites has been limited in the past by available analytical methods. With the advent of new and powerful analytical and biochemical techniques, the potential toxicity of metabolites in the overall profile of a drug candidate can now be better considered. In principle, the logic to consider safety testing of metabolites is compelling. After all, metabolites represent new chemicals to which a human is exposed [1]. Administration of a new drug to animals or humans sometimes results in no new metabolites, but more often, there is generation of one to a few to over a dozen metabolites. The concern is that 1) some of these metabolites could have inherent toxicity; 2) they might be formed in humans; and 3) they would not be generated at appreciable levels in animals during toxicity testing. Under a worst case scenario, a metabolite-related toxicity occurs in humans but not in animal species used in non-clinical safety evaluation [2]. Even though the role of metabolites as mediators of toxicity has not always been considered in safety assessment, there have been numerous examples of drugs (e.g., acetaminophen, mephenytoin) that have been known for decades to give rise to toxic metabolites [3]. Indeed, it is often the case that when a drug shows an unexpected off-target adverse effect in clinical trials, there is speculation that the toxicity is attributable to a metabolite. It was against this backdrop that scientists in both the drug industry and the United States Food and Drug Administration (FDA) came to conclude that some form of risk assessment of metabolites is both prudent and necessary. Yet, despite recognizing its importance, significant concerns were raised as to how a policy would be implemented. Some of these concerns were 1) the significant additional cost and development time to implement metabolite studies, and 2) whether such
Safety Testing of Drug Metabolites
461
a program would truly be useful enough in avoiding human toxicity to justify the increased time and cost. On a more practical level, other questions were raised: 1) What degree of metabolism would be the threshold for concern and how would it be measured, for example, by a fraction of circulating drug related material, or by total mass basis? 2) Would these regulations apply to both reactive and stable metabolites, which have different mechanisms of toxicity (e.g., covalent binding in the case of the former versus excessive on or off target pharmacology in the case of the latter)? [2]; 3) What would be the timing of these metabolism studies? and 4) For ‘‘major’’ active metabolites, what toxicity studies would be appropriate and would administration of preformed metabolites really produce meaningful safety data relative to when the metabolites are formed in situ [4]?
1.2 Brief chronology of events leading up to issuance of guidance The initial drug industry position paper on the subject of Metabolites in Safety Testing (MIST) was published by representatives of the Pharmaceutical Research and Manufacturers of America (PhRMA) in 2002 in Toxicology and Applied Pharmacology (TAP) [5]. The FDA response to the PhRMA position paper and the industry group’s response to FDA’s comments were published in 2003 as letters to the editor in TAP [6,7]. In June 2005, the FDA issued their Draft Guidance on Safety Testing of Drug Metabolites issued and requested comments, a compilation of which was completed in August 2005. In December 2005, representatives from PhRMA and FDA attended a joint DruSafe discussion to share feedback on the draft guidance and industry comments. This was followed by several years of ad hoc commentary in the literature by scientists from the industry and FDA [2–4,8–10] and at other meetings such as an open forum sponsored by the Analysis and Pharmaceutical Quality (APQ) section of the American Association of Pharmaceutical Scientists (AAPS) entitled ‘‘MIST and beyond: the role of bioanalysis in the assessment of drug metabolites in safety testing,’’ following the 2006 AAPS annual meeting to further discuss the proposed guidelines. The discussion and commentary culminated in February 2008 when the FDA issued the final version of the ‘‘Guidance for Industry: Safety Testing of Drug Metabolites’’ (herein referred to as the ‘‘MIST Guidance’’) [11]. However, despite the issuance of a final MIST Guidance, the debate and commentary continues [12–14] as the drug industry and the FDA discuss how best to balance the safety concerns potentially posed by metabolites with the enormous strategic and financial costs involved in
462
Thomas N. Thompson
safety testing. In November 2008, in a reprise of the 2006 meeting, the AAPS presented a short course entitled ‘‘Metabolites Data as a Requisite for Regulatory Submissions: Current Practice, Challenges and Case Studies.’’ Finally, as recently as February 2009, Chemical Research in Toxicology devoted an issue largely to commentary, interpretation of the guidance and new methodology on how to best address the crucial issues in the MIST Guidance.
2. EVOLUTION OF THE MIST GUIDANCE 2.1 The MIST issues are defined and debated The initial industry position, as outlined in the 2002 MIST document [5], was intended to be a proactive attempt to provide a framework for when to consider MIST of new molecular entities. Within this framework, a logical sequence of decision points and laboratory studies was outlined that would both address important scientific issues as well as offer a strategy for when and where to direct resources to the problem. One of the main goals was to define a process that was scientifically driven, not merely a rules- or check-box-based list of studies [14]. In essence, the MIST document suggested that if one or more circulating metabolites (i.e., metabolites found in plasma, not excreta) were identified in humans in the definitive radiolabeled Absorption/ Distribution/Metabolism/Excretion (ADME) study, which were major, active, and either unique or disproportionate, then separate toxicology studies that assess the potential adverse effects of such metabolite(s) should be considered. Moreover, such metabolite(s) should be monitored using a validated bioanalytical assay in these special toxicology studies. In this document, ‘‘major’’ human metabolites were defined as those which constituted W25% drug-related material in the human ADME study, whereas unique or disproportionate metabolites are those found to be absent in animals or only present at relatively low concentrations. In addition, it was proposed that the structure of the metabolite should be taken into account in decision making. For example, metabolites that possessed a moiety with a known structural alert should also be considered for evaluation even if they are below the 25% threshold. On the contrary, phase II conjugates are generally not pharmacologically active or chemically reactive, so safety evaluation would not normally be needed (acyl glucuronides being an exception). In addition, the MIST document gave a thorough list of points to consider regarding separate geno-, general, and reproductive toxicity testing of metabolites. Regarding the subject of carcinogenicity testing, the point was made that if carcinogenicity testing is not necessary for
Safety Testing of Drug Metabolites
463
approval of the parent compound, then testing of the metabolite likewise should not be required.
2.2 Key points of the 2005 draft guidance In the initial draft guidance issued in June 2005, FDA was in essential agreement with the industry’s position on many if not most points. However, there were key areas of difference. With regard to the definition of ‘‘major,’’ the draft guidance made the point that 10% of administered dose or systemic exposure, whichever is less, should be the action point rather than the 25% mentioned in the MIST document. The draft guidance advocated the 10% level ostensibly to be consistent with other guidelines and guidances currently used by United States and foreign regulatory agencies. In addition, they cited several cases in which it has been determined that the toxicity of a drug could be attributed to one or more metabolites present at less than 25% of the administered dose, including halothane, felbamate, cyclophosphamide, and acetaminophen. In general, the draft guidance recommended that in vitro-in vivo metabolite correlation studies be conducted as ‘‘early’’ as possible in drug development, both to identify potential species differences and, should qualitative interspecies differences in metabolism be detected, to assist in the selection of the appropriate animal species for toxicological assessments. If the metabolite profile in animals is qualitatively similar and animals form the metabolites in equal or greater amounts than those formed in humans, then the standard toxicology testing program for parent drug is adequate. However, if humans form the metabolite(s) exclusively, or to a greater extent than animals, then other considerations apply. If the unique or disproportionate human metabolite is o10%, then again, the standard toxicology testing program for the parent drug will apply. However, if the human metabolite is W10%, then special toxicity testing is warranted, including a ‘‘bridge’’ general toxicity study, and evaluation of genotoxicity and reprotoxicity. Depending on the genotoxicity results, discrete carcinogenicity testing may be warranted for some metabolites.
2.3 Areas of concern after issuance of 2005 draft guidance After the issuance of the draft guidance and a period for public comments, months of commentary at meetings and in the literature followed. Perhaps the most significant and recurring comment had to do with the definition of ‘‘major’’ and whether it should be based on absolute abundance rather than relative abundance as specified in the draft guidance [8,10]. Another significant point raised was that when a toxicity study on a metabolite is specifically warranted, complications
464
Thomas N. Thompson
may arise due to potential differences in disposition between exogenously and endogenously formed metabolites, which could render the resultant data difficult to interpret [4]. It was also claimed that metabolite levels in excreta should not be relied upon to accurately reflect systemic exposure. Because sensitive analytical methods exist today that allow quantitation in drug-related material in plasma, only circulating metabolites should be of concern and need safety assessment. Other miscellaneous comments included 1) confusion over definition of ‘‘unique’’ and ‘‘major’’; 2) what is meant by saying human ADME studies should be done ‘‘early’’? 3) should the ‘‘free’’ (e.g., unbound to plasma proteins) or total (bound plus unbound to plasma proteins) metabolite concentrations be considered? and 4) should known quantitative structure–activity relationships (QSAR) for reactive metabolites (aka ‘‘structural alerts’’) be considered?
2.4 Issuance of the final MIST guidance In February 2008, nearly 3 years after the draft guidance was issued, the final MIST Guidance was published [11]. The term ‘‘disproportionate’’ drug metabolite was defined in the guidance as a metabolite present only in humans or present at higher plasma concentrations in humans than in the animals used in non-clinical studies. Consistent with the 2005 draft, a ‘‘major’’ metabolite was still defined as one with W10% systemic exposure with the added proviso that exposure should be determined at steady state. Generally speaking, only stable metabolites can be detected in circulation. If reactive metabolites are detected, they may require special consideration, although a strategy for reactive metabolites was not specifically addressed in the MIST Guidance. If at least one of the two species used in toxicology studies of parent drug forms the metabolite at levels that approach the expected human concentrations, then additional toxicity tests are not necessary. However, if exposure to the metabolite in animals is lacking entirely, or it does not form at levels that approach expected human exposure, then additional toxicity tests may be required. This can be accomplished either by administration of parent drug to a species that forms the metabolite or, if this is not possible, by direct administration of the metabolite itself. It should be emphasized that the steps outlined in the MIST Guidance are recommendations, not requirements. The agency encourages active dialogue on a case-by-case basis, especially if the new drug candidate is intended for serious or life-threatening diseases that lack an approved effective therapy. A schema for metabolite testing is shown in the Appendix.
Safety Testing of Drug Metabolites
465
3. POTENTIAL ISSUES RELATED TO IMPLEMENTATION OF A SOUND MIST STRATEGY Regarding the issue of safety testing of drug metabolites, it is the patient’s safety and well-being that are of paramount importance to the drug industry, to the FDA and to all parties involved in drug discovery and development. Accordingly, the MIST Guidance as written charts a conservative action plan and invites pharmaceutical companies into dialogue on a ‘‘case by case’’ basis. Thus, it is very important that the sponsor company provides context regarding the contribution of metabolite(s) in the overall toxicity of their drug candidate. Given the formidable technical capabilities available today, this ownership will ensure an action plan that includes the most appropriate studies, not just those that are technically feasible. It is important to ensure not only the safety of important new medicines but also that the time and resources required to fulfill regulations add value proportionate to their cost [2,9,12]. Taken at face value, the MIST Guidance may appear to call for extensive efforts that may or may not significantly improve human safety of drugs. Obach [14] and others have opined that the regulations unintentionally may have created ‘‘conundrums’’ that both pharmaceutical companies and the FDA must still sort out. These issues will continue to challenge us to better understand the mechanisms of toxicity and to develop better testing procedures and technology to address them. Some of the more challenging issues raised by the MIST Guidelines include the following: 1)
2)
3)
4)
Very potent compounds are a special challenge because they are administered at very low doses. In that case, metabolites that meet the definition of ‘‘major’’ might be present at very low concentrations and not actually cause toxicity owing to their relatively low body burden. Extensively metabolized compounds are also a special challenge. In this case, metabolites in circulation might be ‘‘major’’ relative to parent drug and meet the definition in the MIST Guidance criteria, but in fact represent a negligible percentage of total drug-related material. The requirement to measure metabolites at steady state is technically very challenging as definitive human radiolabeled studies are not normally conducted under steady-state conditions [14]. Reactive metabolites, while mentioned only in passing in the MIST Guidance, can frequently contribute to toxicity [14]. Metabolites in excreta may indicate a body-burden of chemically reactive intermediary metabolites, which can result in toxicity through complex,
466
5)
Thomas N. Thompson
non-specific mechanisms commonly associated with ‘‘covalent binding’’ (e.g., carcinogenicity, immunoallergic response) [10]. In those situations where toxicity studies on specific metabolites are warranted, can we assume that administration of synthetic (e.g., preformed) metabolite to animals will give us meaningful information on the safety of that compound as it is formed in vivo? Several authors have questioned whether such studies are reliable indicators of true toxicity of metabolites [1,4,15–17]. For example, what if new secondary metabolites are formed after direct administration of the metabolite of interest, but these secondary metabolites are not formed in vivo after administration of the parent? If new target organ toxicity ensues from this situation, how do we put that into perspective [1,4]? Pang and others [15–17]have demonstrated that kinetic differences can be observed between metabolites formed in vivo and those that are synthesized and then orally administered.
These are but some of the vexing issues that remain as we attempt to comply with the MIST Guidance. Some insightful new strategies and new technologies have been and are being developed to meet these challenges head on and will be discussed subsequently in this review.
4. IMPLICATIONS OF METABOLISM IN SAFETY TESTING OF NEW DRUGS A circulating metabolite that is observed in human clinical studies but is either absent or only present in animals at very low concentrations can make extrapolation of safety in animals to humans quite complex. Under these circumstances, the 2008 MIST Guidance specifies that either a new species must be identified which forms the metabolite or the metabolite must be administered directly to animals in separate toxicity studies. The MIST Guidance offers the following points to consider when designing such studies [11]. First, the drug metabolite for testing must be not only synthesized but also characterized under good laboratory practice (GLP) conditions as would any new chemical entity destined for GLP toxicology studies. The ensuing toxicology studies must also be conducted under GLP conditions. In planning for these studies, the following factors should also be considered:
Similarity of the metabolite to the parent molecule. Pharmacological or chemical class. Solubility. Stability in stomach pH.
Safety Testing of Drug Metabolites
467
Phase I versus phase II metabolite. Relative amounts detected in humans versus the amounts detected in animals. The following types of studies are all part of a standard battery of toxicology tests. The MIST Guidance has commented on each of these as they may apply to safety testing of metabolites. General toxicity studies. The preferred route of administration would be the same as the parent drug’s intended clinical route of administration. However, if necessary, alternate routes can be used to achieve sufficient exposure to the disproportionate metabolite. The metabolite should be administered at multiples of the human exposure or at least at levels comparable to those measured in humans. The duration of the studies should follow appropriate International Committee on Harmonization (ICH) guidelines. As in all GLP studies, ‘‘it is crucial to gather toxicokinetic data from this type of study using a validated bioanalytical method to ensure adequate exposure.’’ Genotoxicity studies. ‘‘The potential genotoxicity of the drug metabolite should be assessed in an in vitro assay that detects point mutations and in another assay that detects chromosomal aberrations.’’ Embryo-fetal development toxicity studies. ‘‘When a drug is intended for use in a population that includes women of childbearing potential, embryo-fetal development toxicity studies should be performed with the drug metabolite.’’ Carcinogenicity studies. ‘‘Carcinogenicity studies should be conducted on metabolites of drugs that are administered continuously for at least 6 months, or that are used intermittently in the treatment of chronic or recurrent conditions when the carcinogenic potential of the metabolite cannot be adequately evaluated from carcinogenicity studies conducted with the parent drug.’’ It is also important to note that the FDA is willing to negotiate with the sponsor as to the exact plan of studies to expedite drug development for serious or life-threatening diseases other than cancer (e.g., amyotrophic lateral sclerosis (ALS), stroke, human immunodeficiency virus) for those drugs with major beneficial therapeutic advances, and are intended for illnesses that lack an approved effective therapy.
5. STRATEGY FOR IMPLEMENTATION OF BEST PRACTICES 5.1 What metabolites should be measured? Although metabolites that comprise ‘‘10% of systemic exposure at steady state’’ is the action threshold specified in MIST Guidance, it is really up to the sponsor to make a case if this number is not scientifically warranted.
468
Thomas N. Thompson
As was pointed out earlier, there are many instances where ‘‘10%’’ does not make sense based on the particular circumstance, and some of these were discussed in Section 3. FDA has said in both the MIST Guidance and public presentations that it is willing to enter a dialogue with sponsors on a ‘‘case by case’’ basis. It is worth noting here that the MIST Guidance offers no specific recommendations on the subject of reactive metabolites, even though the cases of metabolite-induced toxicity cited by the FDA as justification for the ‘‘10% of systemic exposure’’ action level in their 2005 draft guidance are due to reactive rather than stable metabolites [3,6,14]. Furthermore, in a recent review, the authors considered 14 drugs removed from the market because of human toxicity in which metabolites may have played a role. Of these 14 cases, 11 likely involved reactive metabolites whereas only three involved stable metabolites [10]. Clearly, more work is needed to define an appropriate action plan to evaluate toxicity of potentially reactive metabolites, which may be held to a higher standard of safety than a typical major human metabolite [18]. The issue of reactive metabolites poses a formidable technical challenge. As was pointed out earlier, these metabolites are not going to be detected in plasma as they most likely will not leave the organ in which they were formed, often the liver. In other cases, the presence of a reactive metabolite can be inferred from stable secondary metabolites (e.g., glutathione conjugates) that have been excreted in urine/feces [18]. The following table from a recent review on chemical mechanisms of toxicity lists some of the common structural moieties that are alerts to the possibility of reactive metabolites [19]. Hydrazines and hydrazides Arylacetic or aryl propionic acids Thiophenes, furans, pyrroles Anilines and anilides (Structures that yield) quinones and quinoneimines Medium chain fatty acids Halogenated hydrocarbons and some halogenated aromatics (BrWClWF) Nitroaromatics Moieties that form a,b-unsaturated enol-like structures Thiols, thiono compounds, thiazolidinediones, thioureas Aminothiazoles Although the subject of reactive metabolites will not be treated in detail in this review, the reader can refer to several recent reviews on the role of reactive metabolites in toxicity and methods to detect them [20–27]
Safety Testing of Drug Metabolites
469
5.2 What kind of information is needed and when is it needed? The discovery of a human metabolite not previously seen in animal studies can cause a major disruption in the development program for the parent drug. Once such a metabolite is discovered, not only will safety testing in animals have to be carefully considered, but a validated analytical method may be necessary to monitor the metabolite in subsequent clinical studies. Given these implications, metabolism should be investigated ‘‘as early as possible’’ in drug development, as the MIST Guidance points out. However, ‘‘as early as possible’’ is very subjective and depends on drug class, the potential for filling an unmet need in the market, and the innovator’s risk tolerance. Nevertheless, there are trends regarding the types and timing of studies to generate metabolite information. These are discussed later, as along with some emerging trends and technologies in metabolite profiling. In vitro metabolite profiling. These studies are conducted using unlabeled drug incubated with liver microsomes or hepatocytes from rat, dog, human, and any other appropriate species with unlabeled drug. It is now common to conduct these studies early in the discovery process because they offer a first look at any species differences between animals and human and can help guide selection of the appropriate species for toxicology studies. Modern liquid chromatography-tandem mass spectrometry (LC/MS/MS) methodology has emerged as an indispensable technology for preliminary identification of metabolites before reference standards have been prepared [12]. However, it must be noted that LC/MS/MS methodology alone provides either qualitative or, at best, relatively quantitative information. Certainly, the presence of a unique human metabolite would be noticeable and important information. Moreover, it is possible to infer relative quantitative differences between animals and humans in the formation of a given metabolite. However, it is not possible to quantify absolutely the amount of metabolite formed relative to other metabolites and/or parent drug without synthetic metabolite standards, which are typically not available at this stage. Furthermore, in the absence of corroborating in vivo metabolite profiling data, investigators must always be wary that in vitro studies do not accurately forecast human metabolism [28,29]. The same kinds of in vitro studies may be repeated later once the radiolabeled parent (at a metabolically stable position) is available, usually late in preclinical or early in clinical development. The presence of radiolabel permits absolute quantification, so this not only is another opportunity to verify to what degree animals form the metabolite relative to humans but also provides quantitative data about the abundance of the metabolite relative to the total amount of drug-related material.
470
Thomas N. Thompson
In addition, it is becoming more common to use in vitro methods to produce metabolite ‘‘standards’’ for use in subsequent metabolite profiling. This can be done either by direct isolation of the metabolite or by pooling samples from incubations with labeled drug with samples from studies where unlabeled drug has been administered. In vivo metabolite profiling. These studies are conducted in mice, rats, dogs, or other appropriate species of interest. In principle, samples from any animal pharmacokinetic (PK) or toxicokinetic (TK) study could be analyzed for the presence of a suspected metabolite using LC/MS/MS. Indeed, with the advent of more powerful instrumentation, metabolite profiling of PK and TK studies is becoming more commonplace. However, in practice, it is probably still more common to conduct metabolite profiling as part of the mass balance studies with radiolabeled drug. Timing of these studies varies widely among companies and even programs within companies. Typically, these studies are now conducted in parallel with early clinical development, although in some cases they may be conducted late in preclinical development. In addition to the mass balance studies, it is often common practice to conduct special in vivo studies such as in bile-duct cannulated or even genetically engineered [30] animals to help isolate expected metabolites. It should be noted that the combination of in vitro studies that evaluate animal and human metabolism plus the in vivo metabolite profiles in animals can provide greater clarity in forecasting human metabolites in vivo versus either group of studies alone. Several authors have recently addressed this issue [13,28,29]. Human mass balance study. The human mass balance study with radiolabeled drug has long been considered the ‘‘gold standard’’ when it comes to definitive measurement of human metabolites [5]. The design, strategy, and retrospective analysis of human mass balance studies have been the subject of two recent reviews [31,32]. Typically, this study is conducted in phase 2A since it is highly desirable to have information on human metabolism in place before definitive phase 3 studies are underway. However, it is also common to defer this study to phase 3 to be more certain of the likelihood of success for the program before committing to the expense of the human mass balance study. This approach requires the sponsor to accept the risk that disproportionate human metabolites may be an issue. Although human mass balance studies have typically been conducted using radiolabeled drug at doses up to as much as 100 mCi, with the advent of accelerator mass spectrometry (AMS), a new paradigm is emerging. It is now not only possible but also becoming more common to conduct the human mass balance study with trace levels (E200 nCi or
Safety Testing of Drug Metabolites
471
less) of radiolabeled drug such that the study is not regulated as a radiolabeled study. As such, the time and expense of the preliminary animal dosimetry and the extra clinical costs associated with a radiolabed study can be waived [33–35]. These savings help to offset the added expense of AMS analysis, which is more costly than conventional LC/MS/ MS methods. Early human in vivo metabolite profiling. To avoid late development surprises, it is now more common to conduct metabolite profiling in early clinical studies (e.g., single and multiple ascending dose phase 1 studies), although unlabeled drug was administered. This early profiling can be qualitative, ‘‘semi’’-quantitative, or even quantitative depending on the technique employed. As mentioned earlier, this trend has been facilitated by the introduction of powerful new analytical techniques. For example, recent reviews have described use of a combination of state-of-the-art hardware (e.g., ultra performance liquid chromatography–mass spectrometry analysis, high-resolution time-of-flight mass spectrometer) and software (fractional mass filtering algorithm and computer-assisted structure elucidation software routines) to enable drug metabolites to be identified in plasma samples from a first-in-human study [36,37]. Although this metabolite profiling is still qualitative in nature, it can be made at least ‘‘semiquantitative’’ by use of metabolite standards derived from in vitro or in vivo metabolite profile studies with radiolabeled drugs. The radiolabeled metabolite is added to the ‘‘cold’’ sample and is used to ‘‘calibrate’’ the MS response [36]. Another exciting new area of research that can provide semiquantitative or even quantitative metabolite data is the use of nuclear magnetic resonance (NMR). Improvements in software and hardware have facilitated the use of NMR to determine actual concentrations of isolated metabolites and even measure metabolites from in vivo samples [38,39]. Synthesis of metabolite standards. These exciting bioanalytical techniques notwithstanding, at some point a decision must be made whether synthesis of (a) metabolite standard(s) should be undertaken. The exact timing of this step depends on many factors, including size and risk tolerance of the company, the resources available, and the degree of difficulty of the synthesis. In many cases, metabolites that are relatively easy to synthesize may be prepared early in preclinical development. However, in most cases, the synthesis of metabolite standards are deferred as late as possible, typically until after it has been established unequivocally that the metabolite(s) are deemed ‘‘significant’’ in the context of the MIST Guidance and, therefore, should be monitored in subsequent toxicology and/or clinical studies.
472
Thomas N. Thompson
6. ROLE OF THE MEDICINAL CHEMIST With respect to the MIST Guidance, informed decisions and strategies are best made with full understanding and thoughtful discussion of drug metabolism in the context of the mechanisms of toxicity [40]. Accordingly, the issues presented by the MIST Guidance have now brought drug metabolism and bioanalytical scientists into dialogue with toxicologists. However, it is important to consider that the medicinal chemist can also play a pivotal role in this dialogue at all stages of discovery and development. First, the medicinal chemist’s inherent understanding of the art and science of chemical structure elucidation may be invaluable to help identify metabolite structure. Next, once an understanding of structure is available, the chemist may be in the best position to judge whether the metabolite structure is likely to render it more or less pharmacologically active. Alternately, the chemist’s knowledge of structural alerts will be valuable to help properly predict a metabolite’s risk for toxicity. For example, hidden within the structure of certain metabolites may lie evidence that a reactive intermediate either has been formed or might form by subsequent secondary metabolism. The importance of these structural evaluations cannot be overstated. The MIST Guidance is clear that both abundance and activity (pharmacological or chemical reactivity) of metabolites drive the decision for further safety evaluation. Finally, chemists may be needed either to synthesize a given metabolite(s) for early evaluations or to provide input and/or oversight into batch scale synthesis to support metabolite toxicity testing.
7. SUMMARY That drug metabolites can play an important role in overall drug toxicity is now widely accepted as a significant issue in drug development. Advances in drug metabolism and bioanalytical chemistry have facilitated our ability to detect and quantify metabolites as never before. Given this ability, early identification of human metabolites is a logical approach for a successful drug development program. Although the FDA has recently outlined a generic course of action in their 2008 MIST Guidance, the issues are complex. Accordingly, the agency appears willing to discuss individual cases with sponsors to ensure timely and cost–effective development of important new medicines. However, though dialogue is welcome, the innovator should drive the understanding of metabolism issues and suggest an action plan. New technology gives many options, and sponsors should be ever mindful of
Safety Testing of Drug Metabolites
473
conducting those metabolism studies that should be done, and not necessarily all that can be done. In conclusion, it is clear that gaining a more thorough understanding of how metabolism may affect the toxicity of drugs is not only prudent, but necessary. It is incumbent upon sponsor companies, the FDA, and even academicians to work together using a flexible, science-driven approach to investigate these issues in a timely and cost–effective manner to ensure that the new medicines developed are safe.
REFERENCES [1] J. Polli, Presented in the AAPS Short Course on ‘‘Metabolites Data as a Requisite for Regulatory Submissions: Current Practice, Challenges and Case Studies’’ at the AAPS Annual Meeting, Atlanta GA, November 2008. [2] F. P. Guengerich, Chem. Res. Toxicol., 2006, 19, 1559. [3] K. L. Davis-Bruno and A. Atrakchi, Chem. Res. Toxicol., 2006, 19, 1561. [4] T. Prueksaritanont, J. H. Lin and T. Baillie, Toxicol. Appl. Pharmacol., 2006, 217, 143. [5] T. Baillie, M. N. Cayen, H. Fouda, R. J. Gerson, J. D. Green, S. J. Grossman, L. J. Klunk, B. LeBlanc, D. C. Perkins and L. A. Shipley, Toxicol. Appl. Pharmacol., 2002, 182, 188. [6] K. L. Hastings, J. El-Hage, A. Jacobs, J. Leighton, D. Morse and R. E. Osterberg, Toxicol. Appl. Pharmacol., 2003, 190, 91. [7] T. Baillie, M. N. Cayen, H. Fouda, R. J. Gerson, J. D. Green, S. J. Grossman, L. J. Klunk, B. LeBlanc, D. C. Perkins and L. A. Shipley, Toxicol. Appl. Pharmacol., 2003, 190, 93. [8] D. A. Smith and R. S. Obach, Drug Metab. Dispos., 2005, 33, 1409. [9] W. G. Humphreys and S. E. Unger, Chem. Res. Toxicol., 2006, 19, 1564. [10] D. A. Smith and R. S. Obach, Chem. Res. Toxicol., 2006, 19, 1570. [11] Food and Drug Administration, 2008, Guidance for Industry: Safety Testing of Drug Metabolites, http://www.fda.gov/CDER/GUIDANCE/6897fnl.pdf [12] T. A. Baillie, Chem. Res. Toxicol., 2008, 21, 129. [13] D. Luffer-Atlas, Drug Metab. Rev., 2008, 40, 447. [14] R. S. Obach, Presented in Plenary Session 1 ‘‘Minimizing Drug Toxicities during Drug Discovery and Development, at the 13th North American Regional ISSX Meeting, San Diego, CA, October 2008. [15] K. S. Pang, M. E. Morris and H. Sun, J. Pharm. Pharmacol., 2008, 60, 1247. [16] H. Sun and K. S. Pang, Drug Metab. Dispos., 2009, 37, 187. [17] K. S. Pang, Chem. Biol. Interact., 2009, 179, 45. [18] D. Luffer-Atlas, Presented in the APQ Open Forum ‘‘MIST and Beyond: The Role of Bioanalysis in the Assessment of Drug Metabolites in Safety Testing,’’ at the AAPS Annual Meeting, San Antonio, TX, November 2006. [19] F. P. Guengerich and J. S. MacDonald, Chem. Res. Toxicol., 2007, 20, 344. [20] A. S. Kalgutkar, I. Gardner, R. S. Obach, C. L. Shaffer, E. Callegari, K. R. Henne, A. E. Mutlib, D. K. Dalvie, J. S. Lee, Y. Nakai, J. P. O’Donnell, J. Boer and S. P. Harriman, Curr. Drug Metab., 2005, 6, 161. [21] A. S. Kalgutkar and J. R. Soglia, Expert Opin. Drug Metab. Toxicol., 2005, 1, 91. [22] K. Park, D. P. Williams, D. J. Naisbitt, N. R. Kitteringham and M. Pirmohamed, Toxicol. Appl. Pharmacol., 2005, 207, S425. [23] T. A. Baillie, Chem. Res. Toxicol., 2006, 19, 889. [24] J. C. L. Erve, Expert Opin. Drug Metab. Toxicol., 2007, 2, 923. [25] R. S. Obach, A. S. Kalgutkar, J. R. Soglia and S. X. Zhao, Chem. Res. Toxicol., 2008, 21, 1814.
474
Thomas N. Thompson
[26] H. Takakusa, H. Masumoto, H. Yukinaga, C. Makino, S. Nakayama, O. Okazaki and K. Sudo, Drug Metab. Dispos., 2008, 36, 1770. [27] J. N. Bauman, J. M. Kelly, S. Tripathy, S. X. Zhao, W. W. Lam, A. S. Kalgutkar and R. S. Obach, Chem. Res. Toxicol., 2009, 22, 332. [28] S. Anderson, D. Luffer-Atlas and M. P. Knadler, Chem. Res. Toxicol., 2009, 22, 243. [29] D. Dalvie, R. S. Obach, P. Kang, C. Prakash, C. M. Loi, S. Hurst, A. Nedderman, L. Goulet, E. Smith, H. Z. Bu and D. A. Smith, Chem. Res. Toxicol., 2009, 22, 357. [30] M. W. Powley, C. B. Frederick, F. D. Sistare and J. J. DeGeorge, Chem. Res. Toxicol., 2009, 22, 257. [31] J. H. Beumer, J. H. Beijnen and J. H. M. Schellens, Clin. Pharmacokinet., 2006, 45, 33. [32] S. J. Roffey, R. S. Obach, J. I. Gedge and D. A. Smith, Drug Metab. Rev., 2007, 39, 17. [33] G. Lappin and R. C. Garner, Expert Opin. Drug Metab. Toxicol., 2005, 1, 23. [34] G. Lappin, M. Rowland and R. C. Garner, Expert Opin. Drug Metab. Toxicol., 2006, 2, 419. [35] G. Lappin and L. Stevens, Expert Opin. Drug Metab. Toxicol., 2008, 4, 1021. [36] T. A. Baillie, Chem. Res. Toxicol., 2009, 22, 263. [37] L. Leclercq, F. Cuyckens, G. S. Mannens, R. de Vries, P. Timmerman and D. C. Evans, Chem. Res. Toxicol., 2009, 22, 280. [38] R. Espina, L. Yu, J. Wang, Z. Tong, S. Vashishtha, R. Talaat, J. Scatina and A. Mutlib, Chem. Res. Toxicol., 2009, 22, 299. [39] K. Vishwanathan, K. Babalola, J. Wang, R. Espina, L. Yu, A. Adedoyin, R. Talaat, A. Mutlib and J. Scatina, Chem. Res. Toxicol., 2009, 22, 311. [40] D. A. Smith and R. S. Obach, Chem. Res. Toxicol, 2009, 22, 267.
APPENDIX: DECISION TREE FLOW DIAGRAM Disproportionate Drug Metabolite >10% parent systemic exposure (AUC)