Progress in Medicinal Chemistry, Volume 45 (Progress in Medicinal Chemistry) (Progress in Medicinal Chemistry)

v Preface The success of any drug discovery project relies upon the quality of the lead that initiates the lead optimi...

Author: F.D. King | G. Lawton

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Preface The success of any drug discovery project relies upon the quality of the lead that initiates the lead optimization process. What defines a ‘quality lead’, where these ‘quality leads’ come from and how one discovers them has been the subject of intense debate within the pharmaceutical industry, and relies upon defining those properties that historically have led to successful drug discovery. In Chapter 1, the processes for the discovery of ‘quality leads’ are discussed and three recent examples, MCH-1 agonists, DPPIV inhibitors and CDK1/CDK2 inhibitors are included to demonstrate the application of these processes. Diabetes is a major and increasing health problem encountered across the globe and is the fourth most significant cause of mortality in the majority of developed countries. New approaches to its treatment consume a great deal of resource in drug discovery organizations. Current classes of drugs used in diabetes therapy have limitations in both efficacy and tolerability. The next major class of oral drugs to reach the market will be inhibitors of dipeptidyl peptidase IV. All of the tools of medicinal chemistry have been brought to the task and many efforts in the field are reaching the advanced stages of clinical evaluation. Following on from the use of this target as an exemplar for lead discovery in Chapter 1, the topic is thoroughly reviewed in Chapter 2. Several drug candidates offer the probability of real progress in treating diabetes and reducing the economic and social consequences of the disease. A contributor to the rising prevalence of diabetes is undoubtedly the vast increase in obesity in the developed world. The melanocyte-stimulating hormones have been shown to be important regulators of several biological functions including energy homeostasis. They act via the G-protein coupled melanocortin (MC) receptors. Efforts to develop potent and selective agonists and antagonists of the human MC4 receptor have led to the discovery of several classes of peptide and non-peptide ligands which have high potency and selectivity over other melanocortin receptor subtypes. Medicinal chemistry efforts on this target are reviewed in Chapter 3. It is anticipated that drugs emerging from these studies will have value in treating obesity, erectile dysfunction (agonists) and cachexia, anxiety/depression and pain (antagonists).

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PREFACE

Despite the advent of chemotherapy against tuberculosis (TB) in 1944, the disease remains a global health priority. The causative organism, Mycobacterium tuberculosis, is a tremendously successful coloniser of the human host and is estimated to have latently infected approximately onethird of humanity. This disease is no longer a third-world disease, but is increasingly common in the western world, particularly in immuno-compromised individuals. The most recent figures suggest that 8.9 million new cases of TB and 1.8 million deaths were reported worldwide in 2004. Problems with the treatment of TB include latency and drug resistance. Chapter 4 provides an overview of the issues in treating TB, the drugs that are currently in development and the new targets that have been identified. September 2006

Dr. F. D. King Dr. G. Lawton

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List of Contributors Gurdyal S. Besra School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Veemal Bhowruth School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Chen Chen Department of Medicinal Chemistry, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA Lynn G. Dover School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Paul Gillespie Discovery Chemistry, Roche Research Center, Nutley, NJ 07110-1199, USA Robert A. Goodnow, Jr. Discovery Chemistry, Roche Research Center, Nutley, NJ 07110-1199, USA Paul E. Wiedeman Abbott Laboratories, Abbott Park, IL 60064-6113, USA

1 Hit and Lead Identification: Efficient Practices for Drug Discovery ROBERT A. GOODNOW JR. and PAUL GILLESPIE Discovery Chemistry, Roche Research Center, Nutley, NJ 07110-1199, USA

INTRODUCTION

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HIT AND LEAD IDENTIFICATION PRACTICES Sources of Initial Hits and Leads The Hit-to-Lead Process: General Themes and Considerations

2 2 7

LEAD IDENTIFICATION AS REPORTED FOR SPECIFIC TARGETS Melanin-Concentrating Hormone-1 Receptor Antagonist Dipeptidyl Peptidase IV Inhibitors CDK1/CDK2 Inhibitors

11 11 27 36

CONCLUSION

49

REFERENCES

50

INTRODUCTION Despite great focus and much recently published discussion on the ways to enhance process efficiencies and application of new technologies, the number of new molecular entities approved by the US FDA has continued to fall during the past 10 years from 53 in 1996 to 18 in 2005 [1, 2]. Because of the usually long and expensive discovery and development cycle times relative to other industries (10–15 years), most large pharmaceutical companies pursue multiple research projects in parallel. One can define segments of the drug discovery process that will have positive impact on the overall efficiency of the entire process. A few published studies concerning

Progress in Medicinal Chemistry – Vol. 45 Edited by F.D. King and G. Lawton DOI: 10.1016/S0079-6468(06)45501-6

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r 2007 Elsevier B.V. All rights reserved.

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HIT AND LEAD IDENTIFICATION

pre-clinical attrition rates going from target validation to clinical candidate selection have provided estimates of success rates for finding leads and for optimizing those leads as 60 and 63%, respectively [3]. It has been proposed that success rates in clinical development are correlated with stricter preclinical compound selection criteria and for projects in which more than one compound can be taken forward [4]. If true, this observation highlights the opportunity to enhance the success rates in drug discovery by providing more and better leads as starting points for lead optimization without assuming an extraordinary increase in the success rates in the pre-clinical lead optimization phase [5]. Furthermore, the similarity of initial hit or lead to the eventual drug in some cases is striking, thus highlighting the significance of lead generation as a starting point in drug discovery. To this end, best processes for lead identification (LI) also known as lead generation are worthy of comment and consideration. In undertaking this analysis of LI and looking for such examples in drug discovery literature, it is helpful to define the start and stop points of the LI process. Usually, projects consider the phase of LI to begin with the availability of some biochemical assay to measure the potency of a particular molecule having a desired effect on a therapeutic target. For example, many examples (vide infra) begin the LI process with a high-throughput screen (HTS). The LI process is successfully completed with the identification of a potent compound series, which meets a desired, pre-determined set of criteria appropriate for the specific target. The transition of a series out of LI and into lead optimization (LO) is often signalled by the shift in focus from efforts to find an ‘active’ to efforts aimed at: (a) demonstrating the level of in vivo efficacy in animal models; (b) addressing any outstanding issues with respect to drug-like properties and (c) understanding the toxicity thresholds associated with specific chemical entities. Of course, these transition points vary with different organizational strategies, terminology and the availability of resources.

HIT AND LEAD IDENTIFICATION PRACTICES SOURCES OF INITIAL HITS AND LEADS

In the past few years, the terminology of ‘hit-to-lead’ has emerged in drug discovery publications [6–9]. Many excellent reviews have appeared, which

R.A. GOODNOW, JR. AND P. GILLESPIE

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discuss systematization of the process that follows the initial identification of a set of active compounds and leads to the commencement of full LO efforts [10–16] (with clear exemplification in some cases [12, 17, 18]). Defining a rational and scalable workflow for the prioritization of compounds that show some signs of the desired potency in an HTS campaign (so-called primary hits) has prompted much of the evolution of these processes. In addition to confronting a potentially large number of structures that may result from an HTS campaign, there is a growing perception that simply working with the most active compound may not be enough to justify the efforts to advance a compound for LO. In other words, good potency is just not enough. Rather, a potent primary hit represents a potential starting point to develop an understanding of a series of compounds that have a balance of properties appropriate for a particular therapeutic target. Although a highly potent compound in a primary assay is difficult to ignore, there are many cases where such compounds fail in LO due to issues that might have been detected earlier and before the expenditure of much effort [19]. Sources of Hits and Leads As stated above, the hit-to-lead process has evolved largely due to the need to sort and prioritize large primary hit sets resulting from HTS campaigns. However, it is helpful to consider other possible sources of leads (Table 1.1) to which concepts and practices of lead identification and the hit-to-lead process may be applied. Thus, for the purposes of lead identification, one can consider multiple strategies for identification of primary hits, but these hits are then evolved according to a common set of prioritization criteria. HTS Perhaps the most commonly reported source of leads is HTS of compound collections. Many pharma organizations spend large sums of money to Table 1.1 GENERAL SOURCES FOR HITS AND LEADS Lead identification strategy

Pertinent references

HTS of random and focused sets Natural products Peptides/endogenous ligands, peptidomimetics De novo structure-based design Literature and patent-based innovations Chemogenomics and virtual HTS Fragment screening and assembly

[20–22] [23] [24,25] [26,27] [28–30] [31–34]

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acquire, maintain and renew compound collections [35, 36]. The screening of compound collections greater than one million compounds is no longer remarkable; so-called high-content screening has advanced the types of cellular screens that can be run routinely. Except in the case of screens that identify almost no hits, the number of primary hits is often determined by the activity threshold (e.g., the most potent 0.3% of compounds). Thus, it is important to consider that the active structures that one can identify from an HTS campaign are dependent on the compounds that were assembled to create that collection and the definition of the threshold of ‘active’. Faced with many primary hits for some screens, some organizations have evolved defined hit-to-lead processes to guide and organize LI. The properties that define the focus on and synthesis of compounds for hit-to-lead efforts are also applicable to some extent to the selection of compounds in order to build better, more drug-like compound collections [37]. A part of the strategy of lead identification by HTS methods is the screening of focused sets of compounds. Such subsets of compounds can be focused either by simply creating subsets [38] within corporate collections according to certain chemoinformatic criteria or by the design and synthesis of target-biased libraries [39]. One advantage of these approaches is the integration of drug discovery knowledge and expectations about the structures necessary for a particular target. Creating focused libraries enhances the density of coverage around a particular chemistry space, which is often sparsely populated in a random collection of compounds. The success of deriving leads from libraries has been covered in detailed reviews [21, 40]. Another advantage of running assays on focused sub-sets is the possibility to run smaller screens more conveniently and iteratively as more compounds become available. A part of the strategy of lead identification by HTS methods is the screening of sets of diverse compounds created by diversity-oriented synthesis (DOS) [41]. DOS is the creation of diverse compounds as the result of complex, multi-step or multi-component chemistry. The result is often a combinatorial array of structures, which are not necessary target biased but are unique, potentially useful starting points for lead optimization [42, 43].

Natural products Natural products have provided a large fraction of the leads and starting points for drug discovery [23]. There is some perception that they are often too complex in a hit-to-lead process. Moreover, there are limitations concerning the quantity of material available and isolation of single pure compounds that may prevent the distribution and HTS of these compounds in


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the same manner as for small molecules produced by organic synthesis in multi-milligram quantities. It is for this reason that natural product screening is often classified as a different strategy from HTS of small molecule collections. In any case, there are a few examples of hit-to-lead with natural products as well as a resurgence in interest in this source of leads [44]. Peptides Peptides, whether synthetic or derived as fragments of the endogenous ligands, have served as a basis for peptide-mimetic research for many years. The convenient and rapid synthesis of peptide analogues facilitates the identification of peptides having attractive biological properties. There are concerns about the availability and stability of these starting points for drug discovery research. As a means to address these problems, significant efforts have been made to transform peptides into small molecules, with mixed success. Many peptides are too large to be expected to have good absorption using basic hit-to-lead criteria (vide infra). Structure-based design When structural information is available concerning a binding site or substrate ligand, efforts have been made to develop leads using structure-based design. This strategy was vigorously pursued with renin and HIV protease inhibitors [45], matrix metalloprotease inhibitors [46] as well as more recently with PTP1B inhibitors [47], for example. In cases where the mechanistic information is well understood for endogenous ligands, it has been possible to create new starting points for leads (e.g., DPP IV – vide infra). Literature- and patent-based innovations The easy availability of patents and scientific literature provides one of the richest veins for new ideas for lead identification. For obvious reasons of protecting intellectual property, organizations may be unlikely to provide full details about how leads are evolved based on information release by competitors. However, simply noting the similarities of many drugs for the same or similar targets hints at this practice. The advantage of basing lead identification on this method is assuring a greater likelihood of starting with a drug-like lead. Alternatively, the disadvantage of this approach is the extra burden of creating and protecting a new intellectual property space in a timely fashion while competitors may be taking the same strategy.

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Chemogenomics and virtual HTS In silico methods for LI are appealing because they offer a simple and rapid method to identify structures, which may accelerate the process of finding a lead. For many years, chemists have accepted the principle that similar structures are likely to have similar activity [48]. An underlying concept of chemogenomics is the integration of target sequence similarity, molecular similarity and biological activity; the intent is to create a predictive tool for identifying and designing a novel set of compounds that are likely to be active for a novel target [49, 50]. Virtual screening is a somewhat similar process in which sets of virtual compounds are ranked according to some scoring criteria. An excellent review of this practice as applied to docking structures into a protein structure derived from crystallography is available [51]. An interesting publication illustrates the application of the chemogenomic concept in the kinase family, leading to the discovery of new hits and potential leads by scientists at Ambit [52–54].

Fragment screening and assembly Perhaps because of dissatisfaction at finding only primary hits of large size and modest binding affinity in some project assays, scientists have taken an approach to start the LI process by identifying small fragments having a modest level of binding potency (e.g., 100 mM). With such fragments in hand, efforts then shift to linking up multiple fragments to create a highaffinity interaction [31, 55]. Typically, fragments are smaller in size (100–250 Da). There are several anticipated advantages. (a) It has been proposed that a diversity of o10,000 fragments will represent most possible binding modes; therefore, a fewer number of compounds need to be assayed given the focused starting points [55]. (b) Active fragments are presumed to have a higher proportion of atoms involved in the binding event (i.e., greater binding efficiency). (c) Owing to the lower complexity of these starting points, their binding interactions with the target protein may be better understood, thereby facilitating optimization [56]. Part of the fragment-screening concept includes the idea of privileged structures, fragments which are thought to confer affinity for a particular target [56].


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THE HIT-TO-LEAD PROCESS: GENERAL THEMES AND CONSIDERATIONS

The formalization of the hit-to-lead process has led to a growing understanding of the concepts of drug-likeness. This is particularly true of leads and consideration of how their quality correlates to potential resistance to attrition in the drug discovery process [57–59]. Many publications by Lipinski have resulted in the common acceptance of the ‘Rule-of-Five’ [60, 61]. These guidelines have been so widely quoted in publications and presentations that they have been reconsidered in a subsequent publication [62]. In effect, these guidelines exemplify common practices of filtering and prioritizing compounds according to objective criteria in drug discovery [63]. This thinking has been further elaborated to guide early exploratory medicinal chemistry of initial leads in evaluation of their potential success in LO. In this manuscript, the intent is to review the thought process and specific criteria that highlight good leads and then to summarize examples of lead generation for three biological targets of medicinal chemistry research. The use of specific numerical criteria allows a move away from the subjectivity that often results from potential leads evaluated in the absence of validated comparators (Table 1.2). Others have attempted to provide specific numbers Table 1.2 STAGES AND GENERAL CRITERIA FOR THE HIT-TO-LEAD PROCESS Assessing hits

Validating hits

Identification of high quality hits

‘A good lead’

Structure and purity confirmed

Activity confirmed with powder sample Prioritizing feasible chemistry for analogue synthesis Potency o10 mM

Resolution and assay of chiral isomers

Elucidation of kinetics, mode of action NMR or X-ray of structure-target complex Potency o1 mM

Not a frequent hitter Minimum toxicity alerts Minimum Lipinski rules violations Solubility, permeability, log P calculated

Appropriate target selectivity Solubility, permeability, log D measured Intellectual property issues assessed Acquiring similar commercial or historical analogues

Synthesis amenable to HTC Plausible SAR in 50–100 analogues No Lipinski rules violation Relative stability in microsomal and hepatocyte assays log D: 0–3 Permeability (Caco2, MDCK, PAMPA): high

Encouraging preliminary PK Low hERG channel binding liability Aqueous solubility >100 mg/ml Low Cyp450, PGP liabilities Selectivity in enzyme and receptor panel assays

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that serve as useful guides [11] as well as diagrams of their decision-making process [10]. Comparison of these criteria and decision-making is strikingly similar. Of course, specific numerical criteria may vary to some extent from project to project. In this discussion, the lead generation process is organized into general actions, which work towards the identification and/or generation of a ‘good lead series’. For such a lead series, there are clear advantages of potency and selectivity as well as drug-like properties; equally, outstanding issues that require resolution have been clearly identified and medicinal chemistry strategies have been developed to address and resolve such problems. In short, a ‘good lead series’ is one where one can clearly envision a successful pathway through LO by the synthesis of a number of specific structures.

Assessing hits The start of a hit-to-lead effort is an assessment stage, which applies not only to HTS primary hit sets but also to hits from other lead sources. In reviewing and sorting primary hits by potency, it is important to confirm the structure and purity of the samples. The decay of HTS solution samples, necessary for the plating and manipulation of hundreds of thousands of samples, is well-documented [64]. Such confirmation is best done by spectroscopic analysis of the batch sample used for assay, where possible. Another important step in hit assessment is identifying and discarding socalled frequent hitters. These are compounds which are detected as actives in assays but for reasons other than by reversible non-covalent binding with the target protein (e.g., interference with the readout of the assay, protein denaturation, protein aggregation or aggregation of sparingly soluble compounds themselves). Since effort to optimize such interactions is almost always futile, it is import to eliminate such structures rapidly from consideration. The issue of frequent hitters has been studied and reported on in several publications [65, 66]; a prediction tool has been reported to identify them by in silico techniques [67]. Another important step in this phase of the hit-to-lead process is the identification of false positives. Some have reported that the number of false positives often equals the number of true positives [68, 69]. It is important to have a strategy to distinguish between the true and false positives. Often a re-assay of the original hits, assay of structurally similar compounds and assessment in a secondary assay will provide information to effectively cull false positives. A complementary in silico tool that identifies potentially toxic fragments is DEREK [70]. An assessment of the Lipinski Rule-of-Five violation count is an objective way to rank compounds for consideration.


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Calculation of drug-like properties such as c log P, solubility and permeability may also help to highlight compounds with extreme values [71]. Calculated drug-like properties, however, should be used with caution as they vary with the in silico tools used and have differing degrees of accuracy. Compiling this data into spreadsheets facilitates simultaneous review of structure, potency and drug-like property information. Whenever possible, structures should be reviewed and commented upon by medicinal chemists to assess and prioritize structures of interest for further investigation.

Validating hits A second general step in this process is the validation of the hits of higher priority interest by re-assay and confirmation of the purity of interesting compounds from dry powder samples. This ensures that a sample of interest is indeed the structure presumed, that the biological activity is related to the predominant activity of a pure sample and is at the assumed concentration. It has been discussed in detail that low-temperature storage of organic compounds in DMSO (dimethylsulfoxide) can result in precipitation or ‘oiling out’ of a compound, resulting in a sample concentration less than assumed [72]. Re-assay of freshly prepared solutions from powder obviates possible doubts along these lines. Depending on the number of hits to sort at this point, a potency cut-off of 10 mM is often established. Assay of the sample in available and appropriate target selectivity assays is also timely at this point. Analogues should be assayed not only for potency and selectivity but also in a suite of ADMET assays [73, 74]. Such assays give information concerning drug-like properties, indicating the potential for metabolic stability (microsomal and hepatocyte assays), solubility (kinetic solubility), distribution (log D), permeability (PAMPA and Caco-2 protocols) and protein binding. Testing of the samples of interest in ADMET assays provides measured data for comparison with and validation of predicted values. Finally, one should determine the intellectual property space that may exist around a particular series. Naturally, structures which can be more easily and rapidly synthesized are appealing when considering the need to synthesize compounds for SAR purposes. It is also helpful to search databases of existing collections of commercially available compounds [75] as well as any collections that may exist due to an organization’s historical research efforts. There are many reported similarity-searching algorithms [76]. A common practice has been to select compounds for which their Daylight fingerprints are similar according to a Tanimoto similarity value >0.85 [77] based on the premise that compounds of similar structure are likely to have

10


similar biological activities. The cost and speed of acquiring commercially available compounds often compares favourably with the cost and rate of in-house synthesis. Identification of high quality hits The intent of the first two steps in the hit-to-lead process is the collection and validation of datasets necessary to prioritize hits based on data-driven and informed decision-making. The next two phases of the hit-to-lead process are aimed at organizing chemistry efforts to identify the hits of highest quality or potential and then to perform some exploratory medicinal chemistry in order to develop a truly good lead series that may then go on to a rapid and smooth LO. As noted in Table 1.2, one looks to establish a preliminary SAR understanding by the synthesis of some 50–100 compounds. Assay of these analogues compounds in the ADMET suite of assays now provides an orthogonal SAR with respect to drug-like properties, a highly valuable, complementary set of information. Drug safety assays such as hERG channel binding and CYP450 subtype assays should indicate any liabilities in this area. A good lead It is helpful to have two or more series of compounds to progress in this phase of characterization. In that way, it is possible to make a choice for the better series with respect to the panel of activities noted in Table 1.2. A selection of compounds for a series which has shown good properties as noted above can be further profiled in in vivo pharmacokinetic experiments to understand the potential for exposure and clearance. A series which shows encouraging exposure should be strongly prioritized. Identification of in vivo metabolites may be a means to rescue an otherwise good lead despite high in vivo clearance rates. Other aspects noted in the fourth column of Table 1.2 are features which distinguish a truly ‘good lead’, which are unfortunately rarely found in a screening hit. Aqueous solubility of organic molecules is encountered as a problem for many series [78]. The nature of organic synthesis seems to favour the synthesis of compounds of limited solubility. Although the aqueous solubility of compounds may not be limiting in in vitro assays, sparingly soluble (BCS Class II and IV [79]) compounds present formulation problems when proceeding to in vivo experiments. Solubility may be difficult to increase for some series; therefore, starting with a series of compounds that has good aqueous solubility is a significant advantage.


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For a given molecule, failure to meet some hit-to-lead criteria does not necessarily eliminate the molecule from further consideration as much as it helps to direct synthesis of other molecules to understand the potential to remediate the problem. Unlike lead optimization, the focus of hit-to-lead efforts is not to identify a single, best compound but rather to provide data around a series of compounds that justifies the decision to advance a given series of compounds to the lead optimization phase. An early identification of liabilities significant enough to disqualify a series of compounds for further work is of greatest value before expending precious LO medicinal chemistry resources that might be directed to more successful efforts. The successful conclusion of LI is not only the discovery of an active compound series but also a summary of information relating to its SAR, drug-like properties and preliminary evidence of any safety liabilities. Successful LI should also be complemented with a plan for optimization of any outstanding issues related to drug-like properties and drug safety that may have been detected during the hit-to-lead process. It is useful to consider the generalized strategies and aspects of lead identification that have been discussed in the preceding analysis, in terms of specific examples that have been published in the scientific literature. To that end, the following sections treat reported activities for the identification of lead molecules for three targets: melanin-concentrating hormone-1 receptor (MCH-R) antagonists, dipeptidyl peptidase IV (DPP IV) inhibitors and cyclin-dependent kinase (CDK1/CDK2) inhibitors. It is important to state from the outset that these examples are not presented as an exhaustive analysis of the reported medicinal chemistry for those targets. Because patent publications usually do not describe the initial strategies or hit-to-lead activities for identification of lead compounds, the examples noted here have been limited to medicinal chemistry publications where such discussion exists.

LEAD IDENTIFICATION AS REPORTED FOR SPECIFIC TARGETS MELANIN-CONCENTRATING HORMONE-1 RECEPTOR ANTAGONIST

Numerous patents and publications have appeared describing the identification and optimization of antagonists for the melanin-concentrating hormone receptor-1 and -2 (MCH-R1 and MCH-R2) as part of a therapy for the treatment of obesity. The endogenous MCH ligand is a 19-amino acid residue found in the brains of vertebrates and has been shown to serve as a mediator of food intake. It is believed that a small molecule of appropriate antagonist potency against the MCH-R with safety and brain penetrability will be useful for anti-obesity therapy. There are two receptor subtypes,

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MCH-R1 and MCH-R2; the former is found in all mammals and the latter is found in humans among other mammals, but not in rodents. Both receptors are co-expressed in many tissues [80]. Of the two receptor subtypes, MCH-R1 has been the primary target for obesity research. The role of MCH-R1 as a drug target is supported by the phenotypic characterization of mice in which its expression has been eliminated [81]. Progress in the discovery of MCH-R antagonists has been well reviewed [82–85]. An aminotetralin, T-226296 (1), was the first reported small molecule MCH-R1 antagonist [86]. Scientists at Takeda reported the identification of the negative enantiomer of T-22696 after a screen of their in-house focused compound collection specially synthesized for Gi/o-protein-coupled receptors. No hit-to-lead or other optimization for this molecule is reported. Oral administration of this molecule (30 mg/kg) almost completely suppressed food intake induced by intracerebroventricular (icv) injection of the MCH peptide. Shortly following the discovery of (1), there appeared information relating to the inhibition of MCH-R1 by SNAP-7941 (2) by scientists at Synaptic [87]. It is reported that (2) was identified by screening a GPCR-biased compound collection. No details are given as to optimization of the original hit that may have led to (2). When dosed i.p., (2) slowed weight gain through the suppression of feeding throughout the duration of treatment. Both of these structures provided a basis of target validation and benchmarking for subsequently published efforts to discover potent, selective and safe MCHR1 antagonists. F F NMe2

O

O

N H

MeO2C MeO

N H

N N

N

O

F

(1) hMCH-R1 IC50 = 5.5 nM Ca+2 release IC50 = ~100 nM MW: 403

(2) hMCH-R1 Ki = 0.11 nM MW: 614

O

O S

N

OMe N

Cl N

(3) hMCH-R1 pIC50 = 9.1 MW: 482

NHAc


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Cl

Cl

Cl HN

Cl

O HN

HN

O N N

N

CN

(4) hMCH-R1 Ki = 3.9 nM Muscarinic Ki = 532 nM MW: 537

CN

(5) hMCH-R1 Ki = 2.6 nM MW: 508

The highly potent MCH-R antagonist (3) appears to be in clinical development by GlaxoSmithKline [88]. No details are available as to the lead generation activities that rendered this structure. Although the details of lead generation are not available at the time of preparation of this manuscript, these structures provide a benchmarking perspective for success in generating new starting points for MCH-R antagonists. Researchers at Schering-Plough report the discovery of a series of biaryl ureas (4) after high-throughput screening of the corporate compound collection [89]. The initial hit-to-lead work focused on evolution of the SAR resulting in (4), a potent, but non-selective MCH-R1 antagonist. Hit-to-lead exploratory chemistry showed that it was possible to replace the tertiary carbon linker with a urea linkage as shown for (5) while retaining potency. Chemistry was developed to permit the convenient generation of a diverse set of analogues around the general structure (6). The results of two sublibraries were described in which pairs of diversity vectors were held constant while scanning the third vector (e.g., R1 ¼ m-CN, R3 ¼ 3,5-diCl- while varying R2). This resulted in the identification of the potent compound (7). Rat PK exposure levels were investigated for compounds which have various replacements of the hydroxy-pyrrolidine, indicating that this part of the molecule is useful for modulating in vivo exposure. Such understanding at an early stage is an important component of the hit-to-lead process. This hit-tolead study included evaluation of the receptor selectivities as well as an assessment of oral efficacy of (7) in a chronic rodent obesity model. Significant reduction in food intake and weight gain were early signs of the potential of this series of compounds.

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HIT AND LEAD IDENTIFICATION CF3 F R3 HN HN

O

O N N N

N

OH

R2 CN

R1

(6)

(7) hMCH-R1 Ki = 8.9 nM MW: 512

F Cl

CF3 O

Cl

N H

HN N

N

O N

N

Me

N

O OH CN

(8) hMCH-R1 Ki = 3.7 nM MW: 551

NC

(9) hMCH-R1 Ki = 2.7 nM MW: 544

Further investigation of this series of compounds was undertaken by scientists at Schering due to concerns about the formation through metabolism of 4-phenylaniline, which predicts a liability for geno-toxicity [90]. Scientists turned their attention to re-construction of the central linkage scaffold to eliminate the potential for forming a 4-phenylaniline. It was found that an introduction of one or two methylene groups between the biaryl group and the nitrogen bifurcation point was tolerated. In this way, there was reported a short series of compounds having potent MCH-R1 inhibition (e.g., (8)). The attention to potential toxic metabolites and resolution of the problem through template modification are other important features, which require consideration during the lead identification process. Schering scientists have also illustrated a solution to potential toxic liabilities for compounds containing 4-phenylanilines by the identification of a suitable alternative template, in a process known as ‘template hopping’ [91]. It was found that the central phenyl ring of structures such as compound (7) could be replaced with a trans-substituted bicyclo[3.1.0]hexane, such as (9). A similar SAR was found for this new series of compounds as for the


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4-phenylaniline-containing series. Substantial selectivities (h-MCH-R1 vs. h-MCH-R2) for (7) and (9) were observed. Scientists at Taisho reported on the discovery by HTS of hits from a GPCR-directed library and the subsequent hit-to-lead evaluation of structures such as (10) [92]. The length and regiochemistry of the spacer were investigated along with the possibility of replacing the sulfonamide linkage. As part of a routine process to monitor for any potential metabolic liabilities, it was found that the sulfonamide derivatives, in general, for this series were less stable than amide-linked derivatives in rat and human liver microsome preparations [93]. This type of general information is highly useful at an early stage in the lead identification process. An SAR investigation of amides related to (10) resulted in substantial increases in potency with respect to MCH-R1 inhibition and improved selectivity relative to Y5, and alpha2A as exemplified by (11). NMe2 N N

F N H

F H N

SO2 OCF3

O

NMe2

NH N

Br

(10) hMCH-R1 IC50 = 160 nM Y5 IC50 = 2.7 nM α2A IC50 = 7.7 nM MW: 496

N

N H

(11) hMCH-R1 IC50 = 3.4 nM Y5 IC50 = 2700 nM α2AIC50 = 260 nM MW: 425

Scientists at Pharmacopeia reported on the discovery of substituted piperidine analogues with a scintillation proximity assay using radiolabelled MCH peptide binding to MCH-R1-expressing CHO cell membrane preparations [94]. An exploration of the SAR of this initial hit series (e.g., (12)) for MCH-R1 binding was expedited with the synthesis and assay of two sublibraries comprising more than 500 compounds. It was found that >10-fold increase in binding resulted from the substitution of the dichloro-benzamide with di-halophenylureas. The information derived from this library was applied to similar bi-aryl series of compounds in which the biaryl and bipiperidyl were optimized to result in (13). Separation of (13) into its enantiomers did not result in enhancement of MCH-R1 binding potency. Pharmacopeia scientists also reported on the identification of 84 active structures found from the assay of a 19,740-member library [95]. A typical first hit, (14), is shown. Again, optimization of the synthetic modules of this

16


series resulted in a 10-fold increase in binding potency with a concomitant drop in molecular weight to give (15). These structures are good examples of rapid optimization of potency for modular HTS hits found from combinatorial libraries. CN Cl Cl

Cl H N

Cl

N

H N

Cl

O

H N

NH

N O Cl

(12) hMCH-R1 Ki = 4.1 uM MW: 557

(13) hMCH-R1 Ki = 3.0 nM MW = 506

N

F

H N

N

H N O

N

H N

Cl

N N

H N O

N

Me

Me Cl

(14) hMCH-R1 Ki = 98 nM MW: 530

(15) hMCH-R1 Ki = 3.1 nM MW: 469

Scientists at Abbott have reported the discovery, through HTS of their corporate compound collection, of (16) having sub-micromolar potency in an MCH-R binding assay and single-digit activity in an MCH-R functional cell assay [96]. Initial chemistry efforts were aimed at developing an SAR of this indole system having two potentially basic sites. It was found that removal of the tertiary amine was deleterious to MCH-R inhibitory activity. Removal of the glycine spacer and transformation of the phenyl ether to a benzyl ether substantially increased the potency while moderately reducing molecular weight. The results of the investigation of CNS penetration and PK properties were less positive for (17) than for (16). In an attempt to improve upon the drug-like properties, substitution of the indole nucleus with benzimidazole and indazole surrogates was investigated. From these efforts there emerged an understanding of several permissible modifications with respect to potency, which were further evaluated for brain and plasma exposure following oral dosing in DIO mice. Compound (18) demonstrated


17

the best balance of binding affinity, functional antagonism and CNS exposure and was, therefore, selected for efficacy study in DIO mice. It is interesting to note that small changes sometimes result in improved PK properties. When dosed orally in DIO mice, (18) caused a dose-dependent decrease in body weight during treatment by as much as 15% (30 mg/kg). The discrepancy between increased functional activity and lower potency in the binding assay was not explained.

N O

N

H N

N O

N

N X

NH

PhCH2O

OPh

(16) hMCH-R1 IC50 = 0.299 µM Ca2+ release IC50 = 1.81 µM MW: 468

N H N

O NH

(17) X = CH hMCH-R1 IC50 = 0.022 µM Ca2+ release IC50 = 0.470 µM MW: 454 (18) X = N hMCH-R1 IC50 = 1.40 µM Ca2+ release IC50 = 0.11 µM MW: 454

N N

PhO


The SAR of this series was further explored by replacing the amide with other linkers as well as synthesizing other compounds containing amines other than pyrrolidine [97]. Among those analogues reported, pyrrolidine seemed optimal, but the amide linker was replaceable with a urea link, resulting in (19). This potent compound was profiled in vivo during which time no reduction in body weight was observed despite a reduction in food intake. These results are in contrast to the results observed in in vivo experiments with (18). Such results provide a good example of the usefulness of early characterization of compounds in in vivo settings in order to measure their potential success in oral dosing experiments as well as for understanding the therapeutic potential of the biological target.

18


Scientists at Abbott have also reported on another chemotype, which was explored for MCH-R1 inhibition. The start point, (20), was also identified through HTS [98]. Compound (20) is notably smaller than many other MCH-R1 antagonists; as a result, the sub-micromolar potency of such a small molecule is an attractive starting point for hit-to-lead efforts. Abbott scientists reported the rapid exploration of the alkoxy substitution using a pre-determined set of alcohol reagents to encompass typical medicinal chemistry analogues. In this way, it was possible to identify alkoxy substituents containing alpha-branching, (21), as an important feature for good potency. The amino group was derivatized through reductive amination with a diverse set of aldehydes. Most of these analogues had reduced binding potency. Resolution of (21) resulted in one enantiomer that is approximately twice as potent as the racemate in both binding and cellular activity. The more potent enantiomer was then dosed (10 mg/kg) in an in vivo experiment in DIO mice. A brain-to-plasma ratio of greater than 1 was observed, indicating penetration of the blood–brain barrier and a T1/2 of the order of 2 h. Abbott scientists also reported the exploration of amide- and aminecontaining extensions of the C-8 alkoxy substituents, as in (22) [99]. Good brain-to-plasma ratios and brain exposure were observed for (22). However, the divergences of binding and functional potency remained a concern, making this a worthy candidate for further exploration. A strategy to constrain the propane linker between the quinoline and aniline moieties was adopted with the use of 3-hydroxypyrroline as shown in (23). It was shown early on in this investigation that the (S) configuration is five-fold more potent that the (R). Furthermore, the meta- and para-substitutions on the benzyl ring were rapidly identified to enhance potency and reduce the difference between binding and functional antagonism. Compound (23) was shown to have excellent CNS penetration and sustained high exposure levels (>17,000 ng/ml) in murine brain after oral dosing. The progress of this quinoline series from HTS hit to candidate for in vivo efficacy studies is a good example of rapid elaboration of a small, orally bioavailable molecule.

H2N H2N

N

N O

t-Bu

O Me




19 F O

H2N

N

H N

O

F O

H2N

CF3

N O N

Cl



Abbott scientists published a series of hit-to-lead efforts resulting in a class of potent 3-aminoindazoles (24) [100]. According to published literature, (24) was evolved in an interesting two-stage template-hopping exercise from initial HTS hit (25) to ortho-substituted benzamides (26) and eventually to the indazole (24). These structures and the details of their evolution represent good examples of information-driven template hopping (vide infra).

O

MeO N

HN

O

N H

O


(25) hMCH-R1 IC50 = 1.84 µM MW: 370

N

H N

Ph

N

N

H N

H N

O

Cl O NH

N O

Cl

O N

S

O NH

O Oi-Pr

N


(26) hMCH-R1 IC50 = 0.002 µM Ca2+ release IC50 = 0.016 µM MW: 485 OMe Me H N

MeO O

O N

O


20


Compound (25) was detected in an HTS campaign having micromolar affinity for MCH-R1 [101]. The modular nature of the hit (i.e., naphthyl, 4-aminopiperidyl and cinnamyl fragments and amide connector) made the follow-up strategy of examining various replacements fairly obvious. The combination of potent fragments resulted in (28), which had a good balance of binding affinity and functional potency. Replacement of the amide linker with other connectors such as urea and sulfonamides or with other templates all resulted in substantial reduction of binding potency. Compound (28) was then dosed orally in DIO mice, showing good levels and duration of brain and plasma exposure. Compound (28) was also profiled in dogs, where it was also well exposed and had low clearance; oral bioavailability was calculated as F ¼ 84%. Following this encouraging result, (28) was dosed for 2 weeks in DIO mice where significant weight loss was observed, potentially through alteration of energy expenditure levels. During the hit-to-lead effort that led to (28), it was noted that the introduction of hydrophobic substitutions at the ortho position of the benzamide ring caused reduction of MCH-R1 inhibition. Consideration of similarities to another chemotype active for MCH-R1 inhibition ((29) vide infra) hinted at the possibility of an internal hydrogen bond forming an apparent ring-like structure. To this end, substituted amines were installed at the ortho position of structures such as that shown for (26). Various amines, both alkyl and heteroaryl-alkyl were explored, but amino-methylthiazole provided the optimal balance of binding and functional potency. Installation of a heteroatom into the central template was considered as a means to modulate the brain to plasma distribution. To this end, a series of nicotinamide analogues was synthesized, resulting in the identification of (27). In this system, a greater diversity of 2-amino substitutions was tolerated with respect to binding and functional potency. Both (26) and (27) were evaluated for exposure in mice and for efficacy in DIO mice. Compound (26) demonstrated greater plasma and brain exposures and caused significant weight loss at a lower dose than (27). The indazole template of (24) resulted from a cyclization of compounds such as (26) [102]. The substitution of the piperidine moiety of (24) resulted from a scan of various substituted benzyl groups; the piperonal moiety was found to be optimal [103]. Attempts to functionalize the N-1 indazole nitrogen also produced potent MCH-R1 inhibitors, but assessment of plasma and brain AUCs was generally better for unsubstituted indazoles. In in vivo efficacy experiments with DIO mice, (24) when dosed orally, caused significant weight loss.

R.A. GOODNOW, JR. AND P. GILLESPIE O

N

N

O

HN

21

Ph

HN

R X

O

O

(29) X = O, NH, NR1

N

O


N

Ph

HN

O

N

Cl

O

Cl O

O


O

O


Scientists at Abbott and Millennium have published together a thorough characterization of compounds related to (29) [104]. During an HTS, (30) was identified as having moderate affinity for MCH-R1. Potency was rapidly gained by substitution of the coumarin ring resulting in (31), which was characterized for its PK and brain penetration properties in mice. Given these encouraging exposure results, efforts were directed to replacement of the cinnamyl group. The cinnamyl group was replaced with a series of aryl–alkyl groups; piperonyl was found to be a good substitute (32). Compound (32) was extensively characterized for its metabolic stability (stable), brain and plasma exposure after oral dosing (good) and the degree of protein binding (96%). Compound (32) was also evaluated for selectivity for MCH-R1 relative to 61 enzymes and receptors at Novascreen [105], where it was found to have moderate potency for receptors known to bind to biogenic amines (e.g., adrenergic-a1: 75% inhibition at 10 mM). Binding to the hERG channel was also measured as IC50 ¼ 2.25 mM, indicating the need to monitor cardiovascular risk for this type of compound. In an in vivo DIO mouse feeding study, it was again found that (32) caused weight loss but not reduction of food intake; such results hint at an effect of energy expenditure mechanism by MCH-R1 reduction or other off-target effects. Compound (32) was also dosed in a dog cardiovascular function model, where it was found to cause a significant increase of cardiac contractility, a potentially serious adverse event. Another two close analogues of (32), one a

22


potent MCH-R1 antagonist and the other a relatively inactive MCH-R1 antagonist, were also dosed in the dog cardiovascular function model and were observed to cause similar cardiac effects as (32). These results indicate that MCH-R1 inhibition is not likely the cause of the cardiac effect, but that this class of compound suffers from such a liability. The reports of these hit-to-lead efforts demonstrate the potential utility of diversifying a single hit into multiple and distinct template series and then characterizing each series based on early assessment of the potential for good pharmacokinetics and efficacy. The authors do not report a comparison of pharmacokinetic and efficacy properties among the different templates. Should serious problems be encountered with one series (e.g., cardiac contractility effects) then it is possible to focus lead generation efforts on a series with fewer liabilities. Scientists at 7TM report an interesting approach to identify MCH-R1 antagonist ligands based on the integration of information of the putative MCH-R1 binding site and its classification according to so-called physicogenetics [106] (i.e., a type of chemical genomics). Assuming that similar binding sites may also bind to ligands which share similar fragments, molecular modelling was used to guide the construction of small molecule libraries that contained features of T-226296 (1), a reported MCH-R1 antagonist. Dopamine D2 receptor ligands, for example clebopride (33), feature functionality which has the potential for forming an internal hydrogen bond. The assay of these libraries led to the identification of (34) [107]. The selectivity for (34) was greater than 1,000-fold with respect to hMCH-R2. No information for (34) was given with respect to D2 receptors. However, for related compound (35), D1 and D2 binding affinity at 10 mM was reported as 78 and 79%, respectively. Greater affinity was reported for 5-HT2A (0.67 mM), H2 (2.2 mM) and 5-HT2C (0.31 mM) receptors. O N H

O O Cl H2N

N N H

OMe

(33) Clebopride MW: 374

Ph

PhX

N H

N H

N

OMe

(34) X = NH hMCH-R1 IC50 = 0.008 µM IP3 IC50 = 0.025 µM MW: 502 (35) X = O hMCH-R1 IC50 = 0.008 µM IP3 IC50 = 0.025 µM MW: 503

Subsequently, scientists at 7TM described an efficient means to change the chemotype represented by (35) [108]. MCH-R1 inhibitors from the benzamide series were used to generate four- and five-point pharmacophore


23

models, which were used in a virtual screen of commercially available compounds. The pharmacophore models were used to perform in silico screening of databases of commercially available small molecules. From this screening effort, a series of 20 aminoquinolines was identified. Synthesis and assay of analogues led to (36). In the consideration of this and other analogues of this type, molecular modelling using the putative structure of MCH-R1 was used to rationalize the SAR information that was developed in order to improve on potency and solubility, resulting in compounds such as (37). Cl

Cl N

Cl

N

O O

N H

N

Me

Me O

Cl O

Me

(36) hMCH-R1 IC50 = 0.024 µM solubility = 25 µM MW: 459

N

NMe2

N

N Me

(37) hMCH-R1 IC50 = 0.006 µM solubility = 80 µM MW: 461

It is interesting and reassuring to note that a similar series was developed by scientists at Argenta, who have reported a template for potent MCH-R inhibition based on a virtual HTS approach [109]. In this report, 11 structures were chosen from the scientific literature (mostly patents) based on their MCH-R1 antagonist potencies. These structures served as the basis for several virtual screens using different similarity algorithms: 2-D substructure, 2-D similarity, 3-D similarity and 3-D substructure. The 11 structures were also manually docked into an MCH-R1 homology model in order to create pharmacophore-based search queries, which reflect bound conformations. A database of compounds available from 24 compound vendors was compiled and edited to remove duplicates and undesirable compounds as described by Rishton [69, 110] and Hann [111]; the database was further culled of compounds which did not meet the criteria as defined in this report by Argenta scientists Clark et al. (e.g., minimum and maximum limits to molecular weight, number of rings, ring size, heavy atoms, etc.). In this manner, the structures identified through a virtual screening approach would be those that would be of interest to progress as potential hits. Such filtering of compound structures led to a database of 615,000 from which 3,015 structures were identified by the named in silico techniques. These structures were reviewed by medicinal chemists in order to assess their druglikeness and synthetic feasibility. Assessment of these structures in terms of calculated properties such as molecular weight, c log P and polar surface area was also an important part of the selection process that resulted in a total of 1,490 unique compounds highlighted for purchase from the multiple

24


in silico methods. Clustering of these compounds yielded 874 clusters leading to 877 compounds identified for purchase, of which 795 were obtained. It is useful to note the attrition in numbers of structures after application of the various filtering and screening techniques. From the assay, MCH binding assay measuring the displacement of radiolabelled MCH peptide of the 795 compounds, 62 primary hits (40% inhibition of binding at 10 mM) were identified. Dose–response curves were measured for 19 structures; four compounds were found to have IC50s in the range 1–10 mM. The most potent compound was (38). Further biochemical characterization indicated that the compound is a competitive antagonist with respect to the MCH peptide. Cl

CF3 N N

O O

N H

Me

N O

Me

(38) hMCH-R1 Ki = 55 nM MW: 425

N

O N H

NHi-Pr

Me

(39) hMCH-R1 Ki = 11 nM MW: 384

At this point, a hit-to-lead process was begun with the identification of other commercially available analogues considered similar with respect to 2D similarity and substructure comparisons. A preliminary SAR was constructed around this hit with the acquisition and assay of seven additional compounds. This is a good example of the potential convenience of early SAR development with the assay of similar commercially available compounds. Manual docking of (38) into an MCH-R homology model identified putative key interactions, which further guided the synthesis of analogues. A preliminary exploration of the SAR of the template of (38) was reported in which the amino substitution, the phenol ring substitution and the linker between the quinoline and the aryl ring were explored [112]. From a small number of compounds, improvement in the IC50 by five-fold was found (e.g., (39)). The selectivity for these types of structures from MCH-R binding relative to 5-HT2A, 5-HT2B, 5-HT2C, D2 and a1A was also investigated; binding to the D2 receptor seemed most pronounced of the receptors identified. Scientists at Neurocrine Biosciences have published a series of interesting scaffold modifications for MCH-R1 inhibition. During investigation of the MCH-R1 pharmacophoric nature of T-226296, g-amino-butyric acid containing structure (40) was discovered to have potent MCH-R1 inhibitor activity [113]. The flexible nature of the 3-carbon spacer in (40) was of concern with


25

respect to further optimization of potency and selectivity of this series. Some have argued that fewer degrees of rotational freedom in a molecule is advantageous for the optimization of pharmacokinetic parameters [114]. There are also arguments that do not support such widely applied generalizations [115]. To that end, replacement of the g-amino-butyric acid moiety with 3-aminopyrrolidine led to structures such as (41), having significant enhancement in the binding potency. It had been observed previously that restriction in the number of rotatable bonds can lead to increases in binding potency [116]. Synthesis and assay of the four possible stereoisomers, highlighted binding affinity changes of nearly three orders of magnitude, the (3R, 30 S) was optimal. This report provides useful lessons for constraining flexible structures as well as defining stereochemistry as a means to enhance binding affinity. Scientists at Neurocrine have described other efforts related to enhancing the metabolic stability. Some molecules similar to (40) were shown to have low metabolic stability as predicted by the results after incubation with human liver microsomes. It was thought that the high lipophilicity of such structures may adversely affect their metabolic stability. Correlations of high lipophilicity and high affinity for metabolizing enzymes have been described previously [117, 118]. In order to enhance binding affinity and metabolic stability, the distal aryl ring of structures similar to (41) was chosen for modification and exploration; this moiety was chosen due to some metabolism information that hinted that this part of the molecule may be subject to metabolism [119]. It was found that (42) was slightly more potent in the binding assay as well as more stable in human liver microsome assays. In general, para-substitution to the aryl ring enhanced binding affinity; this affinity was further enhanced with ortho-substitution, a fact which provided some information about how this part of the molecule binds to the target receptor. It was also shown that (42) had only weak inhibitory effect on CYP2D6 and CYP3A4 (IC50>20 mM). On the basis of the preceding information, scientists at Neurocrine went on to describe a related set of compounds, exemplified by (43) [120]. It was found that the central pyrrolidine ring could be replaced with 2, 5-diaminopyridine linker, creating (43) having potent binding affinity and functional antagonism to MCH-R1. Oral dosing of the HCl salt of (43) resulted in 32% oral bioavailability with T1/2 of 2 h. Given the exposure levels possible with (43), the compound was dosed in acute and chronic Wistar rat feeding models. Suppression of food intake was noted in both studies. In addition, a decrease in body weight was seen in the latter study. Interpretation of the in vivo data suggests weight loss not only occurring as a function of the suppression of food intake but also resulting from metabolic changes occurring as a result of MCH-R1 antagonism. This possibility has

26


been commented on by other researchers of MCH-R1 antagonism summarized in this manuscript. Me O

O

Me N Me

N

3 N

S Ph

(40) hMCH-R1 Ki = 142 nM

Me

N N Me

N

N N 3' Me

Me

(41) hMCH-R1 Ki = 1.8 nM functional IC50 = 12.0 nM MW: 522

O

O S

O

O

Me

Ph

Me

N

Me N

N

O

N Me

N

NHMe

N

Cl Ph

(42) hMCH-R1 Ki = 1.0 nM Intrinsic clearance (HLM) = 19 mL/min/kg MW: 475

(43) hMCH-R1 IC50 = 2.3 nM functional IC50 = 122.0 nM MW: 451

Upon review of these published structures, several common, general features with respect to lead identification for MCH-R1 antagonism can be noted. The compound structures share a general linearity. The MCH pharmacophore contains hydrophobes, an aromatic region and a charge. Most of the structures noted herein conform to this general model. MCH-R1 binding and antagonism is generally tolerant to many modifications; the binding pocket does not appear restrictive. These general observations bode well for the invention and discovery of other MCH-R1 antagonists. Several groups have reported on early in vivo studies, which provide valuable information concerning the potential utility of a series as a therapeutic agent. However, notably absent in many of these publications is the affinity of these structures for the hERG channel. Binding to hERG is thought to be a predictor of the liability for a compound to cause unacceptable cardiovascular risk in human dosing [121]. Many of these compounds possess the classical hERG pharmacophore: two or more aromatic rings in the proximity of a charged amine [122]. Many of these publications may have appeared when the hERG channel binding affinity was found to be an insurmountable obstacle to advancing these molecules for clinical development. A recent publication by Abbott scientists discusses a method for screening multiple MCH-R antagonist templates for cardiovascular effect in an anesthetized rat model [123]. Based on those findings, further optimization efforts on multiple templates (including several of those noted in this manuscript) were


27

suspended. Also notable is the ease with which MCH-R1 antagonists are found; HTS campaigns seem to have produced highly potent antagonists of diverse structures. Many publications focus on improving potency, functional activity, in vivo exposure levels and detection of early signs of in vivo activity; the optimization of other hit-to-lead data (i.e., solubility, metabolic stability, CYP450 inhibition) has not been reported.

DIPEPTIDYL PEPTIDASE IV INHIBITORS

Dipeptidyl peptidase IV (DPP IV) is a serine protease that cleaves the Nterminal dipeptide from peptides and proteins with the sequence H2N-XPro-Y and H2N-X-Ala-Y, where X is an amino acid and Y is any amino acid other than proline. DPP IV was discovered in 1966 [124] and has been pursued as a promising therapeutic target for many years for a number of different indications. Recent attention has focused on its role in the degradation of the incretin hormones GLP-1 and GIP, and on the potential of DPP IV inhibitors in the treatment of type 2 diabetes mellitus. Several inhibitors have advanced to the clinic, and clinical proof of concept has been achieved [125–128]. Many excellent reviews have been published recently on DPP IV inhibitors [129–137] (and see also the review in this volume). It is not our intention to reproduce the material found in these reviews, but rather to focus on the sources of the leads, and the issues that arose. DPP IV is an interesting case study for an evaluation of lead identification because a variety of sources were used to identify the leads and a variety of issues were encountered in the process. The strategies utilized to overcome these issues are instructive and can be applied to many other projects. Among the issues that have arisen in DPP IV programmes are chemical stability, selectivity, pharmacokinetic issues, hERG channel inhibition, CYP 450 inhibition and phospholipidosis. Selectivity is an issue since DPP IV is a member of the prolyl oligopeptidase family of serine proteases and there are a number of closely related family members where inhibition is not desirable. For example, it is known that inhibition of DPP8 and DPP9 leads to toxicity [138, 139]. Ph Et

Me

H2N O

O

N

H2N

N

O

O

NH

NH Me

HOOC Et NO2

(44) DPP IV Ki = 3.5 µM MW: 341

(45) DPP IV Ki = 350 µM MW: 382

28

HIT AND LEAD IDENTIFICATION CONHCH2COOEt

Me N

H2N O

NO2 NH

O

N

N H

O

O O P O O

O

CONHCH2COOEt

(46) DPP IV Ki = 30 µM MW: 350

(47) DPP IV IC50 = 23 nM MW: 658

Early DPP IV inhibitors were substrates or substrate analogues (see (44)–(47)) [140–146]. Some of these compounds (e.g., (46) and (47)) are irreversible inhibitors of the enzyme and were designed with a leaving group to be displaced by the active site serine to give a covalent adduct. This approach has led to very potent compounds, including compounds that show activity in vivo [147]. i-Pr

Me N

H2N O

HO

N

N H

B OH

B

O

(48) DPP IV Ki = 2 nM MW: 186

N

H2N

HO

O

OH

(49) DPP IV Ki = 2 nM MW: 186

HO

B OH

(50) DPP IV IC50 = 16 nM MW: 214

Other early inhibitors include the proline–boronic acid derivatives, such as compounds (48)–(50). These compounds were tested for inhibition of DPP IV, because boronic acids were known to inhibit related enzymes [148]. Two issues with this series are chemical stability (the compounds cyclize to give inactive boron amides [149, 150]) and selectivity [151, 152]. Me

Et N

H2N

S

1

R

O

O

(51) DPP IV IC50 = 18 µM MW: 202 Me

2

R

S O

NH

Me N

H2N O

(52) DPP IV Ki = 2 µM MW: 170

H

N

H2N

(53) R1 = CF3CH2SO2NH-; R2 = H DPP IV IC50 = 3 nM MW: 526 (54) R1 = R2 = F DPP IV IC50 = 88 nM MW: 401

O

Acylthiazolidides and acylpyrrolidides are further examples of substrate mimetics [153, 154]. Compound (51), P32/98, has been tested in the clinic


29

and it shows efficacy in improving glucose tolerance in diabetic patients [155]. The crystal structure of valine–pyrrolidide (52) bound to the extracellular region of DPP-IV shows that the pyrrolidine ring lies in the hydrophobic S1 pocket, while the valine side chain points into a large S2 cavity without making contact with the enzyme [156]. Structure-guided efforts to improve potency led to (53). This inhibitor fills the S2 pocket much better and is more potent as a result. However, (53) is very polar and has a poor PK profile. The PK issue can be overcome at the cost of some potency by reducing polarity, and (54) has a much better PK profile [157]. Me R

O

Et

O

O N

NH2

NH2

N H

S

(55) R = Me DPP IV IC50 = 420 nM MW: 202 (56) R = Me DPP IV IC50 = 460 nM MW: 202 Me

N

O

N

F

(57) DPP IV IC50 = 3 nM hERG IC50 = 28 µM F (rat) = 4% MW: 410

Me

O

N

N

R

NH2 F F

(58) DPP IV IC50 = 64 nM hERG IC50 = 1.1 µM F (rat) = 85% MW: 344

O

N

NH2 F

O

(59) R = H DPP IV IC50 = 25 nM hERG IC50 = 8 µM F (rat) = 10% MW: 343 (60) R = Me DPP IV IC50 = 34 nM hERG IC50 > 100 µM F (rat) = 34% MW: 357

As mentioned above, selectivity against DPP8 and DPP9 is very important for a human therapeutic agent for safety reasons. One approach to highly selective compounds began with the observation that (55) and (56), while equipotent as DPP IV inhibitors, had different selectivity profiles against DPP8 and DPP9, with the threo-diastereomer (55) showing 5–10-fold better selectivity than the erythro-diastereomer (56). This observation led to the preparation of (57), a very potent DPP IV inhibitor (IC50 ¼ 3 nM) with good selectivity against DPP II, DPP8 and DPP9, and no inhibition of the hERG

30


channel. It was noted in this series that compounds bearing an acidic proton in the P2 moiety, such as (57), have poor pharmacokinetic profiles but do not inhibit the hERG channel, while compounds such as (58) that lack the acidic proton have good pharmacokinetic profiles, but inhibit hERG [158]. High protein binding has also been noted as an issue in this series [159]. Pyridone (59) had promising properties and selectivity, and an SAR study of related pyridones was initiated [160]. The optimized compound (60) has good activity against DPP IV and good selectivity against DPP II (IC50 ¼ 8 mM), DPP8 and DPP9 (IC50>100 mM). It does not inhibit hERG and has good pharmacokinetic properties in three species: rat, dog and monkey. In lean mice, (60) showed activity in an oral glucose tolerance test (OGTT) at 1 mg/ kg. This series has several interesting lessons. First, the observation that the different isomers (55) and (56) have different selectivities against DPP8 and DPP9 was the key starting point and it is important not to miss the significance of results such as this. Second, pursuing this series led to one sub-series of compounds with poor pharmacokinetics but no cardiac safety concern, while a second sub-series had good pharmacokinetics but a potential cardiac safety issue. In such situations, it is not always clear that it will be possible to resolve both issues in a single molecule. The third lesson is that persistence is important. In this case, the pursuit of the series despite the pharmacokinetic and cardiac safety problems did lead to a very promising outcome. R N

H2N O

N CN NC

H N

N H

N O

CN

(63) DPP IV IC50 = 7 nM MW: 298

(61) R = Me DPP IV Ki = 200 nM MW: 167 (62) R = c-pentyl DPP IV Ki = 1 nM MW: 221 OH

N H

N O

CN

(64) DPP IV IC50 = 3 nM MW: 303

Cyanopyrrolidides, such as (61) and (62), were originally tested as DPP IV inhibitors because compounds in this class were known to inhibit the related enzyme prolyl oligopeptidase [161, 162]. The inhibitors bind reversibly but form a covalent attachment to Ser-630 at the active site of the


31

enzyme, as shown in X-ray crystallographic studies [163, 164]. Extensive optimization of this series at Novartis led to the identification of two clinical candidates (63) and (64) [165, 166]. This is one of a number of examples in the literature where lead series emerge from the testing of compounds that are known to act against other members of a protein family. Sometimes this arises when a compound is tested in a counter-screen as a selectivity assay. In other cases, a conscious decision is made to test compounds because of the known activity against related targets. This is certainly a productive approach to finding hits, which deserves to be included in the range of options considered at the outset of a new medicinal chemistry programme. Cl

NH2 O Ph

O

Ph N

Ph

H N

O

NH

NH2

O

N

(65) DPP IV IC50 = 1.9 µM MW: 512

N H

NHSO2Me

(66) DPP IV IC50 = 11 µM MW: 535

(67) R =

N

F F

O N

DPP IV IC50 = 119 nM MW: 304

S

Ph NH2 O

(68) R = N

R

NH

DPP IV IC50 = 19 nM MW: 391

F

N

(69) R =

N

N

N CF3

DPP IV IC50 = 18 nM MW: 407

Compounds (65) (DPP IV IC50 ¼ 1.9 mM) and (66) (DPP IV IC50 ¼ 11 mM) were identified as screening hits at Merck. Optimization of (65) gave (67) (DPP IV IC50 ¼ 119 nM), which has low oral bioavailability in rat (3%), while optimization of (66) led to (68), which also has poor oral bioavailability [167, 168]. Metabolism studies with microsomes indicated that the problem was oxidation of the piperazine ring. Modifications to the piperazine ring led to compounds such as (69) (MK-0431, Sitagliptin, DPP IV IC50 ¼ 18 nM), which has good selectivity against DPP II, DPP8 and DPP9, and oral bioavailability of 76% in rats. This compound recently has been approved for the treatment of diabetes. The crystal structure of (69) bound to DPP IV shows an unexpected orientation of the amide carbonyl with the phenyl ring in the S1 pocket [169].

32

HIT AND LEAD IDENTIFICATION O F NH2 O

O

CF3

COOH

i-Pr

NH

O

NH2 O

N

NH

N

F

F

(70) DPP IV IC50 = 0.5 nM hERG IC50 = 76 µM F = 1%; MW: 517

(71) DPP IV IC50 = 720 nM hERG IC50 = 0.9 µM F = 38%; MW: 451

Compound (70) (DPP IV IC50 ¼ 0.5 nM) was also derived from the screening hit (65). It is inactive in the hERG assay but has poor oral bioavailability due to poor absorption. On the other hand, (71) (DPP IV IC50 ¼ 720 nM) has good oral bioavailability but is a hERG inhibitor (hERG IC50 ¼ 870 nM) [170]. NH2 NH2

NH2

N

N Ph

NH2

MeO

N

O

N

Cl O

(72) DPP IV IC50 = 10 µM MW: 320

(73) DPP IV IC50 = 0.1 nM MW: 404

NH2 NH2

NH2 MeO

N Ph

Cl

Cl

(74) DPP IV IC50 = 10 nM CYP 3A4 IC50 = 5 µM MW: 344

NH2

N N

N

Cl

MeO

Me

N Cl

Cl

(75) DPP IV IC50 = 9 nM CYP 3A4 IC50 = 30 µM MW: 355

Compound (72) was a high-throughput screening hit with an IC50 of 10 mM. Optimization of potency led to compound (73), with an IC50 of 0.1 nM, which represents a 100,000-fold improvement in potency. This is an example of the enormous effect on potency that can result from subtle changes in structure. The co-crystal structure of (73) bound to DPP-IV was solved [171]. The related compound (74) (IC50 ¼ 10 nM) has high solubility, high membrane permeability and high metabolic stability in human liver microsomes, but it was found to induce phospholipidosis in cultured fibroblasts and to inhibit CYP 3A4 (IC50 ¼ 5.4 mM). It was postulated that these issues could be overcome by reducing the lipophilicity (log D: 3.0) and


33

free energy of amphiphilicity of the molecule. This indeed proved to be the case. Compound (75) (IC50 ¼ 9 nM) has no phospholipidosis issue and the IC50 for CYP 3A4 is 30 mM. The molecule retains the favourable physicochemical attributes of compound (74) [172]. OH MeO HO

H N

N O

HN

NH2

COOH

O

N

OH

NH2

OH

(76) DPP IV IC50 = 8 µM MW: 554

NHSO2OH

(77) DPP IV IC50 = 22 nM MW: 272

O

COOEt MeO MeO

O

HN N

N NH2

(78) DPP IV IC50 = 17 µM MW: 290

O

P O

H2N

N H

O

O N H

Ph

O2N

(79) DPP IV IC50 = 13 µM MW: 369

A number of compounds have been identified as DPP IV inhibitors by screening, without published descriptions of extensive hit-to-lead work. TMC-2A (76) (DPP IV IC50 ¼ 8 mM) was identified by screening 20,000 extracts from screening broths [173, 174]. Sulfostin (77) (DPP IV IC50 ¼ 22 nM) was found in a screen of natural products [175]. Compound (78) (DPP IV IC50 ¼ 17 mM) was found in a high-capacity screen at Novartis [176]. Undisclosed compounds related to (79) (DPP IV IC50 ¼ 13 mM) were found in a screen of 32,000 compounds from the Korea Chemical Bank [177]. A great deal of structural information is available regarding the binding of DPP IV inhibitors to the enzyme [156, 163, 164, 169, 171, 178–180] and this structure-based design has been used extensively to optimize hits and leads. There is much less information in the literature on the use of structural information to generate novel inhibitors of DPP IV. There are two examples of virtual screening, one where a subset of a compound library was selected based on a pharmacophore approach followed by docking and the second where a fragment library was docked.

34

HIT AND LEAD IDENTIFICATION Cl

Me

O

N Me

O

Cl

NH N

N H

N H

Cl

N N

Me

(80) 82% INHIB @ 30 µM MW: 289

Cl

O

(81) 82% INHIB @ 30 µM MW: 368

Me OH

HO

MeO

Me

NH2 MeO

O

NH

Ph

N H

(82) 81% INHIB @ 30 µM MW: 232

(83) 70% INHIB @ 30 µM MW: 285

Following a high-throughput screen at Astra-Zeneca that gave a very low hit rate, where many of the inhibitors were irreversible, and others were not considered to be synthetically tractable, a virtual screening approach was implemented [181]. The aim of the study was to identify attractive compounds that were missed in the high-throughput screen because of relatively low potency or other reasons. A docking study was considered and rejected because of practical and logistical reasons and because a pharmacophore approach was considered viable. Two three-point pharmacophores were designed based on the crystal structure of (52) bound to DPP IV and a library of 500,000 compounds was run against the pharmacophores. The top 20,000 hits for each pharmacophore were docked into the crystal structure, and the resulting top ranked 8,000 compounds were clustered and visually inspected to select 4,000 compounds for screening. Fifty-one compounds were identified with >30% inhibition at 30 mM. The structures of four of the hits are shown (80)–(83).

O H2N

N

Ph

Cl

Cl

Cl

H2N

(84) IC50 = 40 µM MW: 175

N

(85) IC50 = 30 µM MW: 240

H2N

(86) IC50 = 2 µM MW: 217

H2N

(87) IC50 = 3 µM MW: 218


(88) R =

R F

(90) R =

IC50 = 23 nM MW: 355

N

N

(89) R =

NH2 O

NHCOPh CN

N O

c-PrNHCO

(91) R =

35

N N

IC50 = 90 nM MW: 383 IC50 < 100 nM MW: 381 IC50 < 100 nM MW: 396

In the fragment-based approach, a set of 10,000 fragments selected from the Available Chemicals Directory and an in-house collection were docked into DPP IV using FlexX. Visual inspection of a number of compounds for which the binding mode was known suggested that this approach was selecting the right pose. A number of fragments were assayed and compounds (84)–(87) were found to have IC50 values in the range 2–40 mM. It was noted that the FlexX scores did not correlate closely with measured activity [182]. A crystal structure showed that fragment (84) binds with the phenyl ring in the S1 pocket, reminiscent of the findings at Merck. This led to the identification of novel structural types, such as (88) and (89), which are inhibitors of human DPP IV with IC50 values of 23 and 90 nM, respectively [183]. Compounds (90) and (91) are also disclosed as sub-100 nM inhibitors in patent applications [184, 185]. Some lessons that can be drawn from the literature on DPP IV inhibitors are as follows. (1) Leads can come from many sources, including substrate mimicry, inhibitors of related enzymes, high-throughput screening and virtual screening. (2) Ligands for a given protein can be structurally diverse. This suggests that high-throughput screening of a diverse library may lead to novel chemotypes that are distinct from prior art in the field. (3) Ligands do not always bind in the expected mode. Crystal structures can give very valuable insights into novel modifications even in the case of scaffolds that have been extensively studied. (4) Multidimensional optimization is the key to discovering a drug. In many cases, optimizing one parameter (e.g., pharmacokinetics) will lead to deterioration in another parameter (e.g., hERG). In some series, it is possible to identify compounds where all parameters can be optimized in a single compound, but in other series this is not possible. It is important to design the hit-to-lead process to avoid excessive investment in series that are not optimizable.

36


(5) In some projects it is relatively easy to find a lead, and the challenge lies in optimizing the potency, selectivity and properties of the lead in order to discover a drug. In other projects, it can be difficult to find a starting point. DPP IV is an example of the former: leads have been found through multiple approaches including the protein family approach, high-throughput screening and virtual screening, and there is some diversity among the structures of the hits identified through these different methods. (6) In some projects, the target is tolerant of considerable modifications of the hit structure before activity is lost. In other projects, very small changes in structure will lead to loss of activity. DPP IV is an example of the former case, and clearly this is the most favourable situation since extensive modification may be necessary in order to optimize the properties of the inhibitor. If the target is sensitive to small changes in ligand structure, then it may be difficult to optimize potency and properties at the same time, and the prognosis for the project may be poor. CDK1/CDK2 INHIBITORS

CDK1 and CDK2 are two of the cyclin-dependent kinases, a family of serine–threonine kinases that participate in the regulation of the cell cycle. Because of their role in the control of cellular proliferation, they have been the focus of intense interest for more than 10 years with the aim of discovering and developing new medicines for the treatment of cancer. Co-crystal structures of CDK2 bound to a variety of inhibitors have been solved, and these have guided many lead identification and optimization processes. Recent research has suggested that inhibition of CDK2 alone will not be sufficient to treat cancer [185, 186] since CDK1 can substitute for CDK2. However, combined CDK1/CDK2 inhibitors or pan-CDK inhibitors may be effective. Me N

(92) CDK1 IC50 = 300 nM CDK2 IC50 = 280 nM MW: 402

OH O

HO

Cl OH

O

There have been many excellent reviews published in this area [187–197] and we will not reproduce this material here. Flavopiridol ((92), alvocidib), the most advanced of the CDK inhibitors, was identified through a cell-based screen of about 72,000 compounds against a panel of 60 human tumour cell lines at the National Cancer Institute. It inhibits CDK1 (IC50 ¼ 300 nM) and CDK2 (IC50 ¼ 280 nM), with selectivity against non-cyclin-dependent kinases [188, 198]. The bioavailability of flavopiridol in mice and dogs is approximately 20%,


37

and the clinical route of administration is intravenous [199]. Flavopiridol is moving into Phase III studies for the treatment of chronic lymphocytic leukaemia. This is a very unusual case of a compound identified by screening, making it to clinical trials without extensive modification. H N

O H N

(94) R = H

O

(95) R = (CH2)2CN CDK1 IC50 = 0.2 nM MW: 335

R

HN

HN

Br

NO2

(93) CDK1 IC50 = 400 nM MW: 327

CDK1 IC50 = 35 nM MW: 293

(96) R = (CH2)3NH2 CDK1 IC50 = 27 nM MW: 350

In an early example of a chemogenomics approach, the COMPARE algorithm was used to identify compounds with activity profiles similar to Flavopiridol (92) among the 72,000 compounds in the NCI dataset. The COMPARE algorithm was developed by Ken Paull and colleagues at the National Cancer Institute, and it calculates the linear correlation coefficient between the data over all cell lines for the query compound and all sets of data in the database. Sorting the results by the correlation coefficient identifies those compounds whose profile most closely matches the profile of the query compound [200]. Four of the compounds with profiles most similar to (92) were tested in a CDK1 inhibition assay and the synthetic compound kenpaullone (93) [201] was the most potent with an IC50 value of 400 nM [202]. Iterative cycles of modelling, design, synthesis and testing gave potent inhibitors such as (94) (CDK1 IC50 ¼ 35 nM) and (95) (CDK1 IC50 ¼ 0.2 nM) [191, 203–205]. Compound (93) has poor aqueous solubility, but this issue should be tractable since (96) is equipotent and should have better solubility because of the presence of a primary amine. Kinase selectivity is probably not a major concern: compound (95) inhibits a number of tyrosine kinases in the micromolar range. R Ph

HO

N

N

R N H

N

NH

Cl

NH

N H

i-Pr HO

N

N N H

N

N i-Pr

(97) Olomoucine R = H CDK1 IC50 = 7 µM MW: 284

(99) Purvalanol A R = H CDK1 IC50 = 4 nM MW: 388

(98) Roscovitine R = Et CDK1 IC50 = 0.45 µM MW: 312

(100) Purvalanol B R = COOH CDK1 IC50 = 6 nM MW: 432

38


6-Dimethylaminopurine (CDK1 IC50 ¼ 120 mM) was used in the 1980s as a non-specific kinase inhibitor. Other purines were screened to find a more potent inhibitor. Commercially available olomoucine (97) was found to be a 7 mM inhibitor and further work in this series led to roscovitine (98), (Seliciclib, CDK1 IC50 ¼ 450 nM), which is in Phase II clinical trials for the treatment of cancer [206]. A combinatorial chemistry approach to SAR exploration of olomoucine led to the identification of Purvalanol A (99) and Purvalanol B (100) as potent cyclin-dependent kinase inhibitors [207, 208]. Crystal structures of purines bound to CDK2 showed that different compounds bind with different binding modes. For example, the purine rings of olomoucine and ATP bind in different orientations [209]. N AcNH

O S

S

COOEt

N H

HN

(101) CDK1 IC50 = 1.9 µM CDK2 IC50 = 170 nM MW: 260

N

N H

S

Me Me

O

S

t-Bu N


S

O

S

t-Bu N

(102) CDK1 IC50 = 480 nM CDK2 IC50 = 48 nM MW: 380 HO

N

N

N H

N N

N H

S

O

S

t-Bu N


Compound (101) was found through high-throughput screening to be an inhibitor of CDK2/cyclin E and CDK1/cyclin B with IC50 values of 0.17 and 1.9 mM, respectively. It is inactive in a cell-based assay, probably due to hydrolysis of the ester to the inactive carboxylic acid. This problem was solved by replacing the ester with the stable isostere 5-tert-butyl-oxazole [210]. Crystal structure-guided optimization led to (102) (CDK2 IC50 ¼ 48 nM), which has good selectivity against other kinases. It has reasonable protein binding, a good pharmacokinetic profile and shows activity in a xenograft model in mice [211]. This compound is currently in Phase I studies for the treatment of cancer. The 2-arylamino-thiazole (103) is also a potent CDK inhibitor (CDK1 IC50 ¼ 4 nM, CDK2 IC50 ¼ 2 nM) and is active in the cell-based assay (IC50 10 nM), but it is inactive in a murine leukaemia model. The lack of activity in vivo is attributed to high protein binding and poor solubility. The crystal structure of (103) bound to CDK2 shows that a substituent attached to the 5-position of the pyridine ring will project towards solvent and allow the introduction of a solubilizing group. Compound (104) (CDK1 IC50 ¼ 18 nM, CDK2 IC50 ¼ 3 nM) is active in


39

the cell-based assay (IC50 ¼ 3 nM) and is only 90% protein bound. In the murine leukaemia model, (104) showed a 56% increase in survival time relative to untreated control [212]. SO2NMe2 N HN

HN N

Br

N

H2N

N

N

N H

(105) CDK2 IC50 = 1.8 µM GSK-3ß = 1 µM MW: 380

N

N i-Pr

(106) CDK2 IC50= 59 nM GSK-3ß = 40 µM MW: 471

Compound (105) (CDK2 IC50 ¼ 1.8 mM; GSK-3b IC50 ¼ 1 mM) was identified by screening a 3,341-member commercial kinase-directed library. It is active in a cell-based assay with a GI50 value of 8 mM. X-ray crystallography was used to optimize (105) to improve potency and selectivity to give (106) (CDK2 IC50 ¼ 59 nM, GSK-3b IC50 ¼ 40 mM, HCT116 GI50 ¼ 0.58 mM). However, (106) has poor bioavailability in rats [213]. H2NO2S

Me

N N

N N CONH2

CONH2

N

N N H

(107) CDK2 IC50 = 7 nM MW: 358 O

1

N N

(108) CDK2 IC50 = 87 nM MW: 321

SMe

HN Me

Me Me

N H

N

O

R Cl

2

R

Et

HN

N

Cl N CF3

(109) CDK4 IC50 = 8 µM MW: 408

N

Cl

Cl

Cl

(110) R1 = H, R2 = -NHCOCH2NMe2 CDK1 IC50 = 100 nM CDK2 IC50 = 18 nM CDK4 IC50 = 590 nM MW: 532 (111) R1 = R2 = OH CDK1 IC50 = 240 nM CDK2 IC50 = 20 nM CDK4 IC50 = 44 nM MW: 464

Compound (107) (CDK2 IC50 ¼ 7 nM) was a high-throughput screening hit against CDK2. Although very potent in the enzyme assay, it lacks

40


activity in the cell-based assay, presumably due to poor permeability. The properties of the compound were improved through increasing the lipophilicity and reducing the number of hydrogen bond donors. Compound (108) (CDK2 IC50 ¼ 87 nM) is active in the cell-based assay with an IC50 of 1.7 mM [214]. Compound (109) (CDK4 IC50 ¼ 8 mM) was found in a high-throughput screen of 160,000 compounds for CDK4 inhibitors. SAR studies led to (110) (CDK1 IC50 ¼ 100 nM; CDK2 IC50 ¼ 18 nM; CDK4 IC50 ¼ 590 nM), which is very potent in anti-proliferative assays in cancer cell lines (e.g., HCT116 IC50 ¼ 34 nM) and shows 41% tumour growth inhibition in a xenograft model at 10 mg/kg. The co-crystal structure of (111) (CDK2 IC50 ¼ 20 nM) bound to CDK2 was solved [215].

H N

Ph H N O

O

N

N

N H

N H

(112) CDK2 IC50 = 290 nM MW: 227

O

Me H N

(113) CDK2 IC50 = 37 nM MW: 291

N O

N

H N Br

SO2NH2

N N H

N H

(114) CDK2 IC50 = 37 nM MW: 338

(115) CDK2 IC50 = 23 nM MW: 393

Compound (112) (CDK2 IC50 ¼ 290 nM) was one of a number of 3-aminopyrazoles identified by high-throughput screening. Crystal structure-guided optimization gave (113), which has good activity in vitro, a good PK profile and is active in a mouse xenograft model. However, it has poor solubility (6 mg/ml) and high protein binding [216]. Compound (114) has better solubility (>75 mg/ml) and lower protein binding. It blocks cells in G0/G1 phase and shows efficacy in a xenograft model (73% tumour growth inhibition at 7.5 mg/kg dosed orally twice a day) [217]. A related series of amino-pyrazoles including (115) (CDK2 IC50 ¼ 23 nM) was identified through a systems-based approach at GlaxoSmithKline [218].


41

(116) R = H CDK2 IC50 = 10 µM MW: 262

R O

(117) R = SO3H CDK2 IC50 = 55 nM MW: 342 NH

N H

O

(118) R = SO2NMe2 CDK2 IC50 = 40 nM MW: 369 NMe2

O N O S Me

O S

OH

N

Me OH NH

N H

OH

O

N H

O

(119) CDK2 IC50 = 40 nM MW: 468

NH O

(120) CDK2 IC50 = 1.6 µM MW: 441

Danggui Longhui Wan, a mixture of herbal medicines used in Traditional Chinese Medicine to treat leukaemia, contains indirubin (116) as the active ingredient. Indirubin has been found to inhibit CDK1 with an IC50 of 10 mM [219]. Indirubin-5-sulfonic acid (117) has IC50 values of 55 nM against CDK1/cyclin B and 35 nM against CDK2/cyclin A. The crystal structure of (117) bound to CDK2 was solved and it shows hydrogen bonds between the lactam NH and carbonyl to the hinge region of the enzyme. However, (117) is not active in a cell-based assay, probably due to low permeability [220]. Compound (118) has good CDK2 potency (IC50 ¼ 40 nM) but poor aqueous solubility at pH 7.4 (o1 mg/ml). Solubility was improved both by reducing lipophilicity and by breaking up the planarity of the molecule. Compound (119) has solubility of 418 mg/ml and is active in enzyme (CDK2 IC50 ¼ 40 nM) and cell-based (MCF7 IC50o100 nM) assays. Optimization of this series was guided by the crystal structure of CDK2 with (120) [221]. H N

N

Cl

N Y X

N H

OH

(121) X = N, Y = CH CGP 60474 CDK1 IC50 = 20 nM MW: 355 (122) X = CH, Y = N CDK1 IC50 = 20 nM MW: 356

CGP 60474 (121) was found to be a potent CDK1 inhibitor by testing compounds in the phenylamino-pyrimidine series, which had previously been prepared as inhibitors of the serine–threonine kinase PKC, the tyrosine

42


kinases PDGFR and BCR-Abl kinase [222–224]. Scaffold-hopping gave (122), which is a potent inhibitor of CDK1 and GSK-3b, is active in the cell-based assay and gives a survival benefit in a xenograft model in nude mice [225]. N

N N

Me

OH Me2N

N H2N

N

Me O

N N H

N

(123) CDK2 IC50 = 4 µM MW: 225

N

(124) CDK2 IC50 = 32 nM MW: 418

N

N N

Me H2NO2S

S NH

N Me2N N H

N N

O

O

N N H

N

(125) CDK2 IC50 = 3 nM MW: 380

N

(126) CDK2 IC50 < 3 nM MW: 452

Compound (123) (CDK2 IC50 ¼ 4 mM; CDK4 IC50 ¼ 8 mM) was found in a high-throughput screen for CDK4 inhibitors and the binding mode was elucidated through a crystal structure of the N-acetyl derivative with CDK2. Addition of an aromatic ring gave (124) and (125), which are significantly more potent [226]. Compound (126) has an additional nitrogen in the bicyclic heterocycle, and crystal structures of CDK2 bound to this compound and to the analogous imidazo[1,2-a]pyridine show that the orientations of the bicycles are different [227]. Cl

Me

Me N

S S

Cl

N

N H2N

N S

Me

N

H2N

N OH

(127) CDK2 IC50 = 17 µM MW: 245

N

H N

(128) CDK2 IC50 = 13 µM MW: 206

Me

N H

N

(129) CDK2 IC50 = 2 µM MW: 249

R.A. GOODNOW, JR. AND P. GILLESPIE Me

Me

H2N S

Me

S

Me Me2N

N

N PhNH

N

N

N S

43

O2N

N

(130) CDK2 IC50 = 80 nM MW: 282

N H

O2N

N

(131) CDK2 IC50 = 2 nM MW: 328

Me

N N H

N

(132) CDK2 IC50 = 20 nM MW: 370

In a virtual screening approach, 50,000 commercially available compounds were docked into a CDK2 crystal structure and 176 compounds were selected for screening. Three of these compounds (127), (128) and (129) are 4-thiophenyl- or 4-thiazolyl-pyrimidines, and have IC50 values against CDK2/cyclin E of 2–17 mM [228]. In (130), the amino group is substituted with a phenyl ring and a crystal structure shows that the binding mode has changed, with the NH acting as a hydrogen bond donor to the carbonyl of Leu83 rather than to the backbone carbonyl of Glu81. Compound (130) is significantly more potent in the enzyme assay (CDK2 IC50 ¼ 80 nM) and is also active in the MTT anti-proliferative assay (IC50 ¼ 5 mM). Further modifications led to (131), which has IC50 values of 2 and 53 nM against CDK2 and CDK4, respectively, and also has high potency as an inhibitor of CDK9. Compound (131) shows good activity in the MTT assay (IC50 ¼ 0.3 mM) and blocks the cell cycle in G1/S phase. In general, the correlation of enzyme activity with cellular activity was poor, while compounds in the series were relatively lipophilic and had poor aqueous solubility. The physicochemical properties were improved in compounds such as (132) (CDK2 IC50 ¼ 20 nM, MTT IC50 ¼ 0.6 mM) with a dimethylamino group para to the aniline nitrogen [229]. SO2NH2

N NH

SO2NH2

i-Pr

N NH

Br O N H


O N H


Oxindoles were known to act as inhibitors of tyrosine kinases such as EGFR. A series of hydrazones was prepared and tested for activity against

44


CDK2. Compound (133) proved to be a potent inhibitor with an IC50 of 60 nM and a set of analogues was prepared, guided by the crystal structure of (133) bound to the inactive form of CDK2. A virtual library was generated by replacing the sulfonamido-aniline substructure in (133) with a set of 410 anilines and the library members were docked into the ATP binding site of the CDK2/cyclin A structure. Compounds were prioritized for synthesis based on a combination of docking score and visual inspection. A number of potent CDK1/CDK2 inhibitors were found using this approach, including (134) (CDK1 IC50 ¼ 37 nM; CDK2 IC50 ¼ 2.5 nM). This compound is active in a cell-based anti-proliferative assay in several cell lines including RKO and SW620 [230]. H O N

O

O

N N H O

MeO

N H O

HOOC

N H O

MeHNO2S

N H

N H

(135) SU9516 CDK2 IC50 = 110 nM MW: 241

N H

(136) CDK2 IC50 = 4 nM MW: 324

(137) CDK2 IC50 = 9 nM MW: 372

SU9516 (135) was found by screening to be an inhibitor of CDK2 with an IC50 of 110nM. Structure-guided SAR studies led to the identification of (136) and (137) as potent CDK2 inhibitors [231]. Compounds (138) and (139) were found as screening hits with IC50 values against CDK2 of 39 and 850 nM, respectively. Optimization was guided by a model, which suggested that additional potency could be gained by attaching a ribose mimic to the 4-position of the oxindole ring system. Compound (140) has an IC50 value of 3 nM in the CDK2/cyclin E assay and is active in anti-proliferative assays in cancer cell lines. A crystal structure of (140) bound to CDK2 shows hydrogen bonds between the secondary hydroxyl groups and Asp145 and Glu12 in addition to the hydrogen bonds to the hinge region [232, 233]. N H O

R N H

(138) R = NO2 CDK2 IC50 = 39 nM MW: 255 (139) R = F CDK2 IC50 = 850 nM MW: 228

NH2 OH

HO Me

N H O

F N H

(140) CDK2 IC50 = 3 nM MW: 355


CDK2 IC50 = 26 µM MW: 276 CDK2 IC50 = 38 nM CDK4 IC50 = 480 nM MW: 424 CDK2 IC50 = 7 nM CDK4 IC50 = 66 nM MW: 334 Sol < 1 µg/mL CDK2 IC50 = 44 nM CDK4 IC50 = 900 nM MW: 376 Sol = 720 µg/mL

(141) R = H OMe

(142) R = O NH R

N

45

(143) R = -NHCONH2

(144) R = -NHCOCH2NMe2

Compound (141) was found in a high-throughput screen at the DuPont Pharmaceuticals Company. It has an IC50 value of 26 mM against CDK2 and is selective for the CDK family. Optimization provided (142), with IC50 values of 38 and 480 nM against CDK2 and CDK4, and activity in the cellbased assay in HCT116 cells (IC50 ¼ 400 nM) [234]. Polar substituents at the 5-position of the indenopyrazole can be used to modulate the physicochemical properties of the CDK inhibitors. For example, the potent urea derivative (143) has poor solubility of less than 1 mg/ml (measured in buffered 5% mannitol solution at pH 3.5) while the less potent glycine derivative (144) has much better solubility [235]. Et2N(CH2)2O

Cl

N H

N N

H2N

N

O

Cl

Ph

N N

N

N Ph

O

Et

N H

N

N

O

Et

Me

(145) c-SRC IC50 = 260 nM MW: 320

Me

(146) c-SRC IC50 = 31 nM CDK4 IC50 = 2.4 µM MW: 457

(147) CDK1 IC50 = 1 µM CDK2 IC50 = 129 nM CDK4 IC50 = 620 nM MW: 266

HO(CH2)3

N N

N

N N H

N

N

(148) CDK1 IC50 = 79 nM CDK2 IC50 = 15 nM CDK4 IC50 = 4 nM MW: 418

O

N N H

N

N

(149) CDK1 IC50 > 40 µM CDK2 IC50 = 209 nM CDK4 IC50 = 8 nM MW: 473

O

46


The pyrido-pyrimidine class of CDK inhibitors represents another class where a protein family approach led successfully to the identification of a new lead series. Compound (145) was found at Parke-Davis by screening for tyrosine kinase inhibitors. It inhibits a number of tyrosine kinases including c-SRC (IC50 ¼ 260 nM), FGFR (IC50 ¼ 1.3 mM) and PDGFR (IC50 ¼ 3.8 mM). Optimization led to compounds such as (146), which are potent inhibitors of tyrosine kinases but much weaker inhibitors of the cyclin-dependent kinases (c-SRC IC50 ¼ 31 nM; CDK4 IC50 ¼ 2.4 mM) [236, 237]. Molecular modelling suggested that the 6-aryl substituent lies in a deep pocket adjacent to the ATP binding site in the tyrosine kinases and that this pocket is blocked in the cyclin-dependent kinases. As a result, compound (147) lacking the 6-aryl substituent was made and it does have activity against CDK4. Optimization gave compounds such as (148), which is an inhibitor of CDK1 (IC50 ¼ 79 nM), CDK2/cyclin A (IC50 ¼ 15 nM), CDK4 (IC50 ¼ 4 nM) and also the tyrosine kinase FGFR (IC50 ¼ 51 nM). The binding mode of these compounds to CDK2 was elucidated in a crystal structure [238]. Mechanistically, it was shown that the anti-proliferative activity of (149) is due to inhibition of CDK4, with cell-cycle arrest in G1 phase in cells expressing the pRb protein. Interestingly, while (149) is a potent inhibitor of CDK4 and CDK6, it is inactive against CDK1 [239]. H N

H N O

N NMe2

(150) CDK4 IC50 = 36 µM MW: 340

Cl O

H N

H N

N

O

(151) CDK4 IC50 = 100 nM MW: 315 Sol < 0.01µg/mL

The de novo design program LEGEND was used to design 1,000 novel ligands for a CDK4 homology model. The core of each ligand was extracted and used as a seed to search for commercially available compounds. Of the 4,884 compounds matching the queries, 382 were selected for screening. When these compounds were tested in the CDK4 assay, 18 compounds with IC50 values less than 500 mM were found. Five of the most potent compounds, including (150), belonged to the diarylurea class, and this was selected for further study. Independent modification of the two aryl rings led to a significant increase in potency. For example, (151) has an IC50 value of 100 nM against CDK4, while (152) inhibits CDK1, CDK2 and CDK4 with IC50 values of 120, 78 and 42 nM, respectively. Compound (151) has very poor solubility (o0.01 mg/ml at pH 7.4) and was unsuitable for X-ray crystallographic analysis. Compound (152) has better solubility (7 mg/ml at pH


47

7.4) and its co-crystal structure with CDK2 was solved, revealing hydrogen bonds to Leu83, with a binding mode consistent with the mode proposed from modelling [240, 241]. N

H N

O

H N

N

O

(152) CDK4 IC50 = 42 nM CDK1 IC50 = 120 nM CDK2 IC50 = 78 nM MW: 308 Sol = 7µg/mL Me Me Ph

NH2

N

N N

Ph

N

N H

N

(153) CDK1 IC50 = 6 µM MW: 287

On-Bu

O

HN

Ph

N

CCl3

(154) CDK2 IC50 = 13.5 µM MW: 327

N

N

H

(155) CDK2 IC50 = 110 nM MW: 295

A number of compounds have been identified as CDK1 or CDK2 inhibitors by screening, without published descriptions of extensive hit-to-lead work. The following compounds were found by screening: (153) (CDK1 IC50 ¼ 6 mM) [242], (154) (CDK2 IC50 ¼ 13.5 mM) [243], (155) (CDK1 IC50 ¼ 150 nM; CDK2 IC50 ¼ 110 nM) [244] and Aloisine A (156) (CDK1 IC50 ¼ 150 nM) [245]. The following compounds were found by screening natural products: Meridianin E (157) (CDK1 IC50 ¼ 180 nM) [246] and Hymenialdisine (158) (CDK2 IC50 ¼ 40 nM) [247]. H2N

Br H N

n-Bu N

N O

HN

OH N

OH

N H

(156) Aloisine A CDK1 IC50 = 150 nM MW: 267

N H2N

Br N H

N

(157) CDK1 IC50 = 180 nM MW: 305

NH O

(158) CDK2 IC50 = 40 nM MW: 324

Some lessons can be drawn from the literature on CDK inhibitors. (1) Leads can come from many sources, including high-throughput screening, screening of natural products, de novo design and testing of inhibitors of related enzymes such as tyrosine kinases.

48


(2) Crystal structure-directed optimization has proven very valuable for many CDK programs. (3) All of the inhibitors described above bind in the ATP pocket. This has two consequences: (a) given the high concentration of ATP in the cellular environment, this means that relatively high concentrations of drug are often required to show cellular activity and (b) selectivity against other kinases is often an issue. Many scientists have found ways to increase the potency of kinase screening hits by about 1,000-fold. Improvements in other drug-like parameters (such as solubility and selectivity) are often not described in detail in journal articles. Me Me

S

S N

N N

SC5H11-n

(159) MW: 270

F

N

H2N

N

H2N

OH

HN

OH

N

S

F

(160) MW: 397

Given that many publications noted in the previous target-specific lead identification discussions do not provide a more detailed description of the characterization and optimization of properties other than potency, it is useful to highlight briefly the hit-to-lead studies for a CXCR2 receptor antagonist as reported by scientists at Astra-Zeneca [12]. Compound (159) was identified in a scintillation proximity assay of recombinant human CXCR2 expressed in HEK 293 membranes. As shown below, the properties of this hit are compared to a pre-established hit-to-lead generic lead target profile (Table 1.3). Also shown in Table 1.3 are some of the properties of the HTS hit, compound (159) and the compound which resulted from hit-tolead efforts, (160). In SAR exploration, a small, focused library was constructed to explore effect of this substitutions and the replacement of the 7oxo substituent. Significant potency improvements were found by replacement of the thio-pentyl moiety with substituted benzyl sulfides, while the combination of a benzyl sulfide with replacement of the 7-hydroxy group with a substituted ethanolamine gave a substantial increase in potency. In addition to clear enhancements in binding and functional potencies, (160) is appealing as a lead because of its modest molecular weight as well as the evidence that metabolic liabilities can be improved as measured by the stability in microsomal preparations and in in vivo PK experiments in rat. The presentation of this balance of properties makes a compelling case for


49

Table 1.3 COMPARISON OF COMPOUND PROPERTIES RELATIVE TO THE PROJECT GOALS FOR A PROJECT HTS HIT MOLECULE (159) AND THAT RESULTING FROM HIT-TO-LEAD OPTIMIZATION (160) Property

Goal

(159)

(160)

Potency

IC50o0.1 mM

Stability (rat hepatocytes) Human liver microsomes Rat i.v. PK

CLo14 ml/min/106 cells

10 mMa 2.0 mMb 49

0.014 mMa 0.04 mMb 4

CLo23 ml/min/106 cells

18

31

270 27 2.0 2.9

CL ¼ 25 Vss ¼ 1.9 T1/2 ¼ 1.2 h 15% 98.4% 397 0.5 3.1 3.4

Oral bioavailability % protein binding MW Solubility c log P log D Additional

a b

6

CLo35 ml/min/10 cells Vss>0.5 l/kg T1/2>0.5 h F >10% o99.5% o450 >10 mg/ml o3.0 o3.0 Clear SAR, appropriate selectivity, patentable chemical series, multiple opportunities for different series

Binding assay. Ca flux assay.

further work to optimize (160). This balanced presentation of properties and the improvement in multiple properties is also an excellent example of hitto-lead medicinal chemistry in action. CONCLUSION The identification of high-quality leads has become an important part of the process of drug discovery. It has been pointed out that the historical focus on potency in the early stages of drug discovery has given way to a more balanced process where additional factors such as physicochemical properties, pharmacokinetics and even safety are addressed before significant resources are committed to a chemical series [248–250]. This early focus on drug-likeness is partly responsible for the reduction in the number of clinical trials that fail because of poor pharmacokinetics. The process of lead identification has been formalized to a greater or lesser extent in different companies, and publications have started to appear describing the various strategies that have been implemented. However,

50


practice in the medicinal chemistry literature still reflects the historical focus on potency: most publications describing new chemical series describe the origin of the hit series and the initial SAR studies, especially where there has been a significant increase in potency. Relatively few papers describe how issues such as poor solubility or a poor pharmacokinetic profile were improved. This is unfortunate, since SAR information is generally specific to a particular series and target, while strategies for improving poor physicochemical properties or a safety flag may have more general applicability. From a survey of the literature related to three specific targets, it is clear that leads can originate from many different sources including screening (both actual and virtual), information from competitors and endogenous ligands. High-throughput screening of large and structurally diverse libraries is a good source of novel chemotypes, which in some cases will access novel binding sites. Where crystal structures are available, they tend to be used most often (and to great effect) to guide the optimization of an existing series, rather than to design a completely new series. De novo structurebased drug design is likely to become more important in the future as force fields improve, and as understanding develops of how best to treat solvation and entropy. As best practices for lead identification mature and become adopted throughout the pharmaceutical industry, the chances are good that the recent fall in the number of approvals of new chemical entities will be reversed and that patients will benefit from many new safe and efficacious medicines.

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2. DPPIV Inhibition: Promising Therapy for the Treatment of Type 2 Diabetes PAUL E. WIEDEMAN Abbott Laboratories, Department R4CP, Building AP9B, 100 Abbott Park Road, Abbott Park, IL 60064-6113, USA

INTRODUCTION Type 2 Diabetes Pandemic Inhibition of DPPIV as a Strategy to Enhance the Incretin Effect Dipeptidyl Peptidase IV Potential for Disease Modification with DPPIV Inhibitors Importance of DPPIV Selectivity Structural Biology

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MEDICINAL CHEMISTRY AND PRECLINICAL STUDIES Early DPPIV Inhibitors Covalent DPPIV Inhibitors: P2 Secondary Amine Analogues Covalent DPPIV Inhibitors: P2 Primary Amine Analogues Non-Covalent DPPIV Inhibitors: P2 Primary Amine Analogues Peptidic DPPIV Inhibitors that Extend into P10 Non-Peptidic/Non-Covalent DPPIV Inhibitors

71 71 74 80 84 89 90

CLINICAL DATA FOR ORAL DPPIV INHIBITORS

96

CONCLUSIONS

101

AUTHORS NOTE

102

REFERENCES

102


63


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DPPIV INHIBITION

INTRODUCTION TYPE 2 DIABETES PANDEMIC

Diabetes is a major health problem encountered across the globe. Nearly 200 million individuals worldwide suffer from diabetes of which 90–95% are type 2 diabetics [1]. Some staggering figures include India, home to 35.5 million diabetics, China with 23.8 million, the United States with 16 million, Russia with 9.7 million and Japan with 6.7 million. The incidence of diabetes has not peaked. By 2025, the diabetic population of Africa, the Eastern Mediterranean, the Middle East and Southeast Asia is expected to increase by 100%. Rises are anticipated in other regions as well, including Central and South America (85%), the Western Pacific (75%), North America (50%) and Europe (20%). Already diabetes is the fourth main cause of mortality in the majority of developed countries. Healthcare systems will certainly be strained to meet the growing demand of this pandemic, as even now 50% of patients are undiagnosed. The growing diabetic population requires additional therapies with alternative mechanisms of action and improved tolerability. There are currently five main classes of oral antidiabetics, each limited in one way or other by the degree of efficacy and side effects [2]. Concerns with sulfonylureas centre on hypoglycemia and weight gain. Non-sulfonylurea secretagogues have the same issues along with a more complex dosing schedule. Patients on thiazolidinediones are prone to weight gain and oedema. Biguanides and a-glucosidase inhibitors often produce significant gastrointestinal distress. Inhibition of dipeptidyl peptidase IV (DPPIV) cleavage of glucagon-like peptide-1 (GLP-1) is a highly validated target for the treatment of type 2 diabetes. Several DPPIV inhibitors are in clinical development, and the first requests for regulatory review have been filed. The reported clinical data have established proof of concept in man, confirming the possibility that DPPIV inhibitors will be the next major new class of oral antidiabetic drug. INHIBITION OF DPPIV AS A STRATEGY TO ENHANCE THE INCRETIN EFFECT

The phenomenon of increased insulin secretion following oral administration of glucose compared to intravenous administration is known as the incretin effect. An agent responsible for this effect is the incretin hormone, GLP-1. In response to the oral ingestion of nutrients, proglucagon is processed and GLP-1 is released from enteroendocrine L-cells in the distal small intestine and colon. Binding of GLP-1 to its G-protein-coupled receptor on pancreatic b-cells increases glucose-stimulated insulin secretion [3, 4]. Additional desirable effects of GLP-1 include increased insulin gene

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expression [5], and increased pancreatic b-cell proliferation and islet neogenesis [6]. By contrast and also of benefit are the inhibition of glucagon secretion [7, 8] and decreased gastric emptying that results in the slowed rate of nutrient absorption [9,10]. GLP-1 receptors are also expressed in hypothalamic nuclei responsible for modulating feeding behaviour, and peripheral administration of GLP-1 promotes satiety and inhibits food intake in man [11, 12]. In total these effects demonstrate that GLP-1 (7–36) amide has multiple biological effects that contribute to glucose homeostasis and promotes normalization of post-meal glucose levels. It is important to recognize that GLP-1 augments insulin secretion in a glucose-dependent manner. Unlike sulfonylurea drugs or insulin, enhancing endogenous GLP-1 levels does not increase the risk of hypoglycemia. Infusion of active GLP-1 and GLP-1 (7–36) amide reduces post-meal and fasting glycemia in patients with non-insulin-dependent diabetes mellitus; thus establishing the potential of GLP-1-based therapy for the treatment of type 2 diabetes [13–15]. This effect occurs despite a blunting of the incretin effect in type 2 diabetics [16]. The key problem, however, is that active GLP1 (7–36) amide is rapidly converted to inactive GLP-1 (9–36) amide by the action of DPPIV via the cleavage of the N-terminal dipeptide (His-Ala) of GLP-1 (7–36) amide [17, 18]. The short half-life of GLP-1 (7–36) amide in the circulation (o2 min) makes it impractical as a therapeutic agent and has led to the development of alternative strategies to enhance the antidiabetogenic activity of GLP-1. One successful approach that will not be covered in this chapter is the development of GLP-1 receptor agonists that are resistant to DPPIV cleavage [19]. This approach remains an active area of research and development [20]. Exenatide (Byetta) is the first marketed GLP-1 receptor agonist. It is effective in reducing glycosylated haemoglobin, HbA1c (biomarker for glycemic control) levels in type 2 diabetics but is a twice-a-day injectable peptide with nausea as a prominent side effect [21]. Another strategy that is the focus of this chapter is to increase the circulating half-life of endogenous GLP-1 by inhibiting its enzymatic degradation by DPPIV [17] (Fig. 2.1). Another incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), is also degraded by DPPIV [18]. Similar to GLP-1, GIP is a 42-amino acid peptide secreted by endocrine K cells of the duodenum in response to ingestion of nutrients [22]. The physiological actions of GIP include glucosedependent potentiation of insulin secretion and regulation of insulin gene transcription. In contrast to GLP-1, glucose tolerance is not improved in type 2 diabetics treated with exogenous GIP [23]. However, it has been reported that the response to GIP improves in diabetic patients treated with glyburide to reduce fasting glucose levels [22]. Studies with hyperglycemic diabetic rats or normal rats made hyperglycemic by glucose clamp have

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DPPIV INHIBITION

Fig. 2.1 Inhibition of DPPIV sustains endogenous GLP-1 and modulates the incretin effect.

decreased expression of pancreatic GIP receptor, which in turn causes a loss of insulinotropic response to GIP [24]. Taken together, these data support the notion that the initial effect of DPPIV inhibition in human diabetes is primarily by GLP-1, but raises the possibility that in the long run, when glucose levels fall, treatment with DPPIV inhibitors could also improve the insulinotropic action of GIP.

DIPEPTIDYL PEPTIDASE IV

DPPIV (EC 3.4.14.5; also known as lymphocyte cell surface protein CD26) was first described in 1967 [25]. DPPIV is a post-proline cleaving serine protease with a catalytic triad of Ser-Asp-His oriented inversely to classical serine proteases and with significant homology to other a,b-hydroxylases. DPPIV is expressed as a 110 kDa glycoprotein on the surface of cells of most tissues including kidney, liver, intestine, placenta, prostate, skin, lymphocytes and endothelial cells. DPPIV is catalytically active as a dimer. Proteolytic cleavage of DPPIV from cell surfaces results in a soluble circulating form with a monomeric mass of approximately 100 kDa. In addition to cleaving GLP-1, DPPIV may play a role in the cleavage of other substrates with accessible amino-terminal Xaa-Pro- or Xaa-Ala-dipeptide sequences, resulting in their inactivation or alteration in their biological activities. Potential DPPIV substrates include growth hormone releasing hormone, GIP, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), substance P, bradykinin, gastrin releasing peptide, neuropeptide Y, peptide YY,

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certain chemokines such as RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokines [26]. Long-term safety concerns have arisen because these possible DPPIV substrates include chemokines, vasoactive peptides, neuropeptides and gastrointestinal peptides. Despite in vitro cleavage of these peptides by DPPIV, many of the activities associated with these peptides appear not to be physiologically regulated in vivo by DPPIV action. Those that appear to be regulated by DPPIV peptidase activity are described below. GLP-1 and GIP have been validated as in vivo substrates of DPPIV in DPPIV knockout (DPPIV KO) mice and DPPIV-deficient Fisher rats [27, 28]. The importance of GLP-1 and GIP in the gluco-regulatory action of DPPIV inhibitors has been shown in double incretin receptor (i.e., GLP-1R and GIPR) knockout mice where the glucose-lowering effect of DPPIV inhibitors is abolished [29]. DPPIV-deficient mice are healthy, have normal blood glucose levels in the fasted state but reduced glucose excursion after a glucose challenge [27]. The active, insulinotropic form of GLP-1 and glucose-dependent insulin levels are both increased in DPPIV KO mice compared to wild-type littermates. Similarly, DPPIV-deficient Fisher rats have a phenotype of improved glucose tolerance and enhanced glucose-dependent insulin secretion [28]. Similarly, PACAP38 has been validated as an additional target of in vivo DPPIV peptidase activity. PACAP38 is a neuropeptide that is involved with signalling to pancreatic nerves and is therefore associated with neural regulation of islet function. Preservation of endogenous levels of PACAP38 with a DPPIV inhibitor may be an additional way that the inhibitors enhance antidiabetic effects. PACAP38 was administered exogenously to both wild-type and DPPIV-deficient mice [30]. In the DPPIV-deficient mice, the rate of PACAP38 clearance was reduced and little of the DPPIV metabolite, PACAP(3–38) was observed. In another study in mice, PACAP38 was administered intravenously with glucose following previous administration of a DPPIV inhibitor [31]. As was observed in the same study with GLP-1, the PACAP38-treated animals showed increased insulin levels and a greater rate of glucose elimination. Although DPPIV clearly regulates GLP-1 (and probably GIP and PACAP38) action in vivo, the DPPIV enzyme may have a broader role in metabolic control. In this regard, DPPIV KO mice show resistance to dietinduced obesity with a concomitant reduction in adiposity compared to wild-type mice [32]. Consequently, long-term inhibition of DPPIV may have the potential to decrease the body weight. However, DPPIV inhibitors currently in clinical development have not demonstrated weight reduction, but several trials have shown weight neutrality.

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DPPIV INHIBITION POTENTIAL FOR DISEASE MODIFICATION WITH DPPIV INHIBITORS

GLP-1 has trophic effects on b-cells [33]. Not only does it stimulate b-cell proliferation but it also inhibits apoptosis of b-cells [34]. GLP-1 is capable of enhancing the differentiation of new b-cells from pancreatic ductal epithelium [6, 35]. DPPIV-mediated preservation of endogenous GLP-1 may also enhance the effects of GLP-1 on b-cell rescue/prevention of apoptosis. Indeed, chronic treatment with DPPIV inhibitors has been shown to preserve islet function in diabetic mice and to improve b-cell survival and islet cell neogenesis in streptozotocin-induced diabetic rats [36, 37]. It has been reported that humans with type 2 diabetes have a b-cell deficit most likely due to increased b-cell apoptosis [38]. Consequently, by increasing GLP-1 levels, DPPIV inhibitors may be capable of providing new b-cells in patients with type 2 diabetes and thus, prevent the worsening of the disease. Furthermore, the treatment of insulin-resistant individuals (impaired glucose-tolerant pre-diabetics) with DPPIV inhibitors may delay or prevent the onset of diabetes. This would be a major medical breakthrough in the treatment of type 2 diabetes and impaired glucose tolerance. However, clinical studies to date have not provided conclusive evidence that the b-cell sparing activity observed with DPPIV inhibitors or GLP-1 analogues in diabetic animal models will also occur in man. The diagnostics to directly measure b-cell mass in the clinical setting simply do not exist yet. However, improvements in b-cell function can and have been assessed. On an optimistic note, an apparent decrease in the rate of progression of type 2 diabetes has been observed with a DPPIV inhibitor in combination with metformin in a long-term clinical study [39]. IMPORTANCE OF DPPIV SELECTIVITY

DPPIV is a member of a family of closely related enzymes that share DPPIV-like catalytic activity [40]. Consequently, there is potential for adverse events or toxicity associated with non-selective DPPIV inhibitors. Two members of the DPPIV family, DPP8 [41] and DPP9 [42, 43], were recently discovered and characterized. Their exact physiologic role remains under investigation. However, profound toxicities in rodent and adverse side effects in dog have been reported with a DPP8/9 inhibitor [44]. Merck scientists reported in 2-week rat toxicology studies that a selective dual DPP8/ DPP9 inhibitor (1), IC50 ¼ 38 and 55 nM, respectively, caused alopecia, thrombocytopenia, anaemia, enlarged spleen, multiple histological pathologies and death. In an acute dog study, the DPP8/9 inhibitor produced bloody diarrhea, emesis and tenesmus. The same toxicity was observed in both models with a DPPIV inhibitor (2) non-selective against DPP8/9, but was not observed with a selective DPPIV inhibitor MK-0431 (3) (DPPIV

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Me

O

Me

F N

N N N F N (3) CF3 DPPIV IC50 = 27 nM DPP8 IC50 > 69000 nM DPP9 IC50 > 100000 nM

NH2 (1) DPPIV IC50 = 30000 nM DPP8 IC50 > 38 nM DPP9 IC50 > 55 nM Me

O

Me

Me N

S

NH2

O

Me

NH2 O

B(OH)2

O2N

N NH2

(2) (allo-isomer) DPPIV IC50 = 460 nM DPP8 IC50 = 220 nM DPP9 IC50 = 320 nM

(4) DPPIV IC50 < 4 nM DPP8 IC50 =4 nM DPP9 IC50 = 11 nM

H N

O O

O N NH2

(5) DPPIV IC50 = 1300 nM DPP8 IC50 = 154 nM DPP9 IC50 = 165 nM

Fig. 2.2 Selectivity for DPPIV over related peptidases.

IC50 ¼ 27 nM, DPP8 ¼ 69mM, DPP9>100 mM). Importantly, several DPPIV inhibitors reported in earlier studies were found to lack selectivity for DPPIV over DPP8/9. Some of the compounds such as (4) and (5) with poor selectivity were active in in vitro models of T-cell activation while DPPIV selective compounds exhibited no response. This data supports the premise that non-selective DPPIV inhibitors likely exhibit off-target toxicities. Additionally, skin lesions have been observed in monkeys treated with DPPIV inhibitors. Although the mechanism of action has yet to be determined, the U.S. Food and Drug Administration has insisted that all DPPIV inhibitors be tested in monkeys [45]. This data is relatively recent and a significant amount of the medicinal chemistry efforts described herein occurred prior to this understanding. It has been placed here to put the subsequent research in context and because the selectivity between prolylpeptidases may be a differentiating factor for drug candidates. Regardless of the outcome of all these evaluations, it would be prudent to remain cautious as development of DPPIV inhibitors for the treatment of type 2 diabetes proceeds (Fig. 2.2).

STRUCTURAL BIOLOGY

DPPIV has proven to be a drug target that has benefited from structural biology guidance, in particular crystallography. Initially, several homology models were constructed that provided preliminary insight into possible structural features [46–48]. Following was an extensive effort that provided

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DPPIV INHIBITION

X-ray crystal structures of recombinant human DPPIV [49–53], natural source porcine DPPIV [54], natural source rat DPPIV [55] and bacterial DPPIV [56]. Both the apo structure and those with bound inhibitors were examined. Many features were found as previously predicted by comparison with related peptidases. However, there were certainly some surprises. To summarize, DPPIV exists as a dimer with two domains, an a/b hydrolase domain and a rather rare eight-bladed propeller domain [57] with a 30–45 A˚ cavity between them. Inhibitors bind within the cavity next to the catalytic triad. Two openings access the cavity, a funnel-shaped opening through the b-propeller and a larger opening between the hydrolase and propeller domains that is believed to be the entry route of substrates. Both are negatively charged, which attract the positively charged amine moiety found in all inhibitors. Glutamic acid residues 205 and 206 form a salt bridge to the amine group in the P2 portion of inhibitors [58] and this interaction is responsible for orienting the N-terminal portion of peptide substrates for cleavage. Steric constraints limit the enzyme to dipeptide cleavage. Asparagine 710 and arginine 125 hydrogen bond to the carbonyl of a P2 amino acid. A small hydrophobic pocket is optimally filled with small flat rings, such as pyrrolidine or phenyl of the inhibitor, and the oxyanion hole is formed by two tyrosines. Tyr547 was found to be as critical as any of the catalytic triad for full enzymatic activity for its ability to stablilize the oxyanion intermediate [59]. Interactions for specific inhibitors will be elaborated in the medicinal chemistry discussions of those compounds (Fig. 2.3). As previously mentioned, the active form of the enzyme is a dimer and once formed, there is no equilibration back to the monomeric species [60]. Hydrophobic forces were determined to be responsible for dimer formation with residues Phe713, Trp734 and Tyr735, all found to be critical. The extent of glycosylation was determined not to be a factor affecting dimer formation [61]. DPPIV is an amazingly dynamic enzyme. As mentioned above, there are two channels which access the catalytic region, and analogy to the similar enzymes tricorn protease and prolyl oligopeptidase suggested that substrate entrance and cleavage products’ exit could be through the b-propeller tunnel

X

R2 R1

N H P2

R3

H N

N O

O P1

N H

O P1'

Fig. 2.3 Schematic of DPPIV substrate.

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[54]. More recent studies have determined that the side channel more likely acts as both entrance and exit, with an expansion of the channel to allow both ready access and egress [62]. Conformational change was also noted for Tyr547 (Tyr548 rat) in the active site that accommodated various inhibitor types [55, 62]. The flexibility of this tyrosine is specific to DPPIV and is likely a selectivity determinant over the similar enzymes DPP8 and DPP9. Further selectivity may be achieved with polar residues in the P10 position [63]. The firm footing on structural understanding of DPPIV has allowed virtual docking exercises to suggest novel inhibitors [64, 65]. Subsequent evaluation in in vitro assays established which chemotypes were suitable starting points for future structure-based design efforts.

MEDICINAL CHEMISTRY AND PRECLINICAL STUDIES Inhibition DPPIV has proven to be a drug target that has successfully brought together all the drug discovery disciplines. Many institutions and companies have contributed to the wealth of information both in the published and patent literature. A quick search of the patent literature will find in excess of 300 patents filed by more than 60 companies and institutions. That body of information is too extensive to be included in this chapter. The study of DPPIV inhibition has been quite active over the past decade and the medicinal chemistry effort has been reviewed at time points throughout [66–71]. Here we will concentrate on journal publications, some meeting abstracts and the occasional patent application to illustrate the drug discovery story of DPPIV inhibition. A brief section will describe some of the early ground breaking studies that set the stage for the explosive research development in this field. Structural class will loosely collect the more contemporary medicinal chemistry and preclinical information.

EARLY DPPIV INHIBITORS

Inhibitors of DPPIV have long been sought as tools to elucidate the functional significance of the enzyme. The first inhibitors were characterized in the late 1980s and 1990s. Each was important in establishing an early structure–activity relationship (SAR) for subsequent investigation. It should be noted that the inhibitors fall into two main classes, those that interact covalently with DPPIV and those that do not. Since DPPIV is a protease, it is not unexpected that inhibitors would likely have a peptidic nature and this theme has carried through to

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contemporary research. A number of non-covalent inhibitors were characterized in the same paper [72]. The tripeptides diprotin A (Ile-Pro-Ile (6)) and diprotin B (Val-Pro-Leu (7)) were identified as modest inhibitors with IC50s of 8 and 920 mM, respectively. Although these simple peptides were not anticipated to be metabolically stable, the recognition element of proline in the penultimate position was established. Lys[Z-4-NO2]-pyrrolidide (5) indicated that a simple, rather flat heterocycle was sufficient to occupy the P1 site where DPPIV favours proline and alanine. The IC50 was again modest at 2 mM, and the compound showed significant toxicity. Two other compounds with five-membered heterocycles mimicking proline in the P1 pocket were Val-pyrrolidide (9) and Ile-thiazolidide ((10), P32/98) with IC50s of 6.0 and 2.8 mM, respectively. These compounds would become two of the more extensively studied inhibitors. A natural product isolate, TMC-2 (11), from Aspergillus orzae A374 was identified as a specific inhibitor (7.7 mM) of DPPIV [73]. A solid-phase combinatorial chemistry effort reduced the size and determined the critical core structure, TSL-225 (12), that maintained equivalent potency (5.7 mM) (Fig. 2.4).

O

O

H2N

N

Et

O

H N

Me

H2N OH

iPr

O OH

O Et

Me

iPr diprotin B (7)

O2N

O

O

H N

H2N N

O

NH2

N

Et (9)

OMe HO

O H2N

iPr

(5) H N

H N

O

diprotin A (6)

O

N

OH N

H2N

OH

N

H2N O

O

N H

TMC-2 (11)

OH

O

S

Me P32/98 (10)

H N

OH

N

O

O TSL-225 (12)

Fig. 2.4 Peptidic and non-covalent inhibitors.

OH

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Inhibitors that interacted covalently with DPPIV were also investigated at an early stage. N-Peptidyl-O-aroyl hydroxylamines exemplified by (13) were weak, irreversible inhibitors found to suffer from hydrolytic instability [74]. A series of dipeptide phosphonates were likewise irreversible inhibitors [75]. Although, the potency was weak, Ki ¼ 236 mM for (14), the compounds addressed the issue faced by many transition-state inhibitors, the intramolecular cyclization of the N-terminus to the electrophilic moiety requisite for the active site serine. Phosphonate esters react poorly with nitrogen nucleophiles and the members of this series were stable in pH 7.8 buffer for hours to days. Selectivity was observed for DPPIV over the proteases and esterases chymotrypsin, trypsin, human leukocyte elastase, porcine pancreatic elastase, acetylcholinesterase, papain and cathepsin B. Applying work established previously with inhibitors of another class of post-proline-cleaving serine proteases, the IgA proteases, dipeptides incorporating the a-amino boronic acid analogue boroPro were the first truly potent inhibitors with Ki values in the low nanomolar range [76]. The SAR established in this work confirmed the requirement of a basic primary or secondary amine at the N-terminus. The boronic acid electrophile exhibited slow binding kinetics characteristic of many transition state DPPIV inhibitors. Unfortunately, the compounds were extremely unstable to intramolecular cyclization in a neutral aqueous environment with Pro-boroPro (15) having a t1/2 of 1.5 h. There has been some recent work extending the boroPro motif into a P2 N-alkyl-glycine that becomes a common group to be described below [77]. A real breakthrough occurred with the discovery of 2-cyanopyrrolidines as the first potent, selective and chemically stable inhibitors [78–80]. These competitive inhibitors were presumed to react with the active site serine, thus forming an imidate adduct. 2-Cyanopyrrolidine (16) had a Ki ¼ 200 nM while showing weak inhibition against the related enzymes DPPII (110 mM) and prolyl oligopeptidase (22% inhibition at 400 mM). Little loss of activity was observed after incubation of solutions at 37 1C for 20 h. The cyanopyrrolidine was to become the sought serine hook that provided sufficient selectivity and solution stability for drug development. Ferring There were two papers by this Research Institute in 1996 that firmly established the 2S-cyanopyrrolidide as a potent pharmacophore for DPPIV inhibition. These studies were directed at finding modulators of T-cell activity, a reflection of most DPPIV research at that time. The first paper established the superiority of the cycloalkyl side chain in the L-configuration in the P2 position delivering both potency and stability, as exemplified by

74

DPPIV INHIBITION Cl

Ph

O

O

O

H N

H N

O O

O

N

O

H2N

NH2

NO2

O H2N

N O

HO

B OH

Pro-boroPro (15)

O

CN N

H2N

N

Cl (14)

O

CN N

Me (16)

O P O

Me

(13)

N H

O

H2N Et

(17)

CN N

Me

S (18)

Fig. 2.5 Covalent inhibitors.

(17) [79]. The second provided an exploration of the SAR associated with the P1 position. Various five- and six-membered saturated heterocycles containing a combination of nitrogen, sulfur and oxygen were prepared, each having a nitrile at the 2-position [80]. The most potent compound was the thiazolidide (18). To reinforce a key difference, the DPPIV inhibitors discussed herein are divisible into two classes, those that form a covalent interaction with the enzyme and those that do not. This distinction will persist throughout the remaining part of this chapter (Fig. 2.5).

COVALENT DPPIV INHIBITORS: P2 SECONDARY AMINE ANALOGUES

Novartis These investigators were aware of the potency and selectivity achieved with the dipeptide mimetic cyanopyrrolidides having a primary a-amino acid in the P2 binding site described above. They also knew that N-methylglycine was accepted by DPPIV in the P2 site [81]. Combining these two features established the N-alkylated-glycylcyanopyrrolidides as a novel DPPIV inhibitor chemotype and established DPPIV as a drug target for large pharma [82]. Libraries of 2-(S)-pyrrolidinecarbonitriles were produced efficiently with either five-step solid-phase or three-step solution-phase sequences. When the P2 glycine was N-alkylated with 2-aminoethyl-5-nitropyridine, a potent (IC50 ¼ 8 nM) inhibitor (19) in this new chemotype was achieved. SAR exploration led to NVP-DPP728 (20) (IC50 ¼ 22 nM). It proved to

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be chemically stable against intramolecular cyclization and selective against other closely related peptidases known at the time. Oral administration of (20) (1 mmol/kg) produced a successful result in an oral glucose-tolerance test (OGTT) in cynomolgus monkey. DPPIV activity was inhibited by 89% while both peak plasma glucose levels and glucose area under the curve (AUC) were reduced. Oral bioavailability was >70% in both rat and monkey. Steady-state volumes of distribution suggested that the drug was primarily distributed to body fluids. Clearance was moderate, and the halflife of 0.85 h in both monkey and man suggested that (20) might be best dosed in conjunction with a meal. Early published results had reported several key SAR and biochemical features of NVP-DPP728 (20) [83]. The D-antipode (Ki ¼ 5.6 mM), descyano (Ki ¼ 15.6 mM) and amide (Ki ¼ 320 mM) analogues of NVP-DPP728 (Ki ¼ 0.11 mM) were compared. There was 3.9 kcal/mol of binding energy associated with the cyano group in the L-configuration. The inhibition kinetics was consistent with a two-step mechanism where an initial loose inhibitor–enzyme complex slowly isomerizes to a tight complex. SAR features included an understanding of hydrophobic or van der Waals interactions of the pyrrolidine in the P1 pocket. H-bonding and ionic interactions stabilized the P2 carbonyl and amine groups, respectively. P2 site side-chain interactions were hydrophobic in the S2 pocket. The nitrile was either considered to form a transient imidate with catalytic site serine or form some type of dipole–hydrogen bond interaction, either explained the binding energy. Additional SAR of NVP-DPP728 and the path to LAF237 (21) was combined in one paper [84]. Within NVP-DPP728, the P2 glycine was greatly preferred over the b-alanine analogue. The diamine chain length of two carbons was optimal. In addition to the nitro group, chlorine and nitrile were accepted on the terminal pyridine. Replacement of the 2-aminopyridine with pyrimidine, aniline, or phenyl was deleterious to binding potency. Based on selectivity considerations, NVP-DPP728 became the first development candidate. SAR continued with larger aliphatic side chains and rings in the P2 site, with good potency maintained with a fully substituted carbon adjacent to the P2 amine. This progressed to the investigation of multiple cyclic systems and culminated with the discovery that adamantyl groups furnished low nanomolar inhibitors. Studies on metabolites indicated that hydroxylation of the adamantyl groups was well tolerated. This led to the synthesis of the 3-hydroxylated-1-aminoadamantane analogue (21) (LAF237, IC50 ¼ 3.5 nM), which avoided the introduction of a chiral centre. LAF237 had the greatest solution stability to intramolecular cyclization of any transition-state mimetic synthesized at that time. Further functionalization of

76

DPPIV INHIBITION

Table 2.1 PEPTIDASE SELECTIVITY OF P2 PRIMARY AND SECONDARY AMINE INHIBITORS Compound

DPPIV IC50 (nM)

DPP8 IC50 (nM)

DPPII IC50 (nM)

P32/98 (10) (17) NVP-DPP728 (20) LAF237 (21)

1,660 12 53 51

2,283 27 4,573 14,219

68,985 39,873 26,520 >100,000

O2N

N

O

H N

N H

CN

NC

N

N

N H

HO

O

LAF237, vildagliptin (21) DPPIV IC50 = 3.5 nM

CN N

O Me Me

CN N

O

NVP-DPP728 (20) DPPIV IC50 = 22 nM

(19) DPPIV IC50 = 8 nM

H N

H N

N

N H

N O

CN

(22) DPPIV IC50 = 49 nM DPP8 IC50 >100000 nM

Fig. 2.6 N-alkylated glycyl-2-cyanopyrrolidides.

the adamantyl hydroxyl group decreased potency. LAF237 was selective over similar peptidases screened at the time and a recent report stated LAF237 to be a 9 mM (IC50) inhibitor of DPP8 [85]. [Note: The IC50 value reported here for DPPIV and DPP8 inhibition differs from that reported in Table 2.1. as a reflection of variance in procedures between laboratories. The constant, Ki, is preferred for comparison of data when available.] The pharmacokinetics of LAF237 was similar to NVP-DPP728, except the halflife was 2.6-fold longer. Using obese Zuckerfa/fa rats as a model of type 2 diabetes in OGTT, and administration of LAF237 (10 mmol/kg) resulted in 90% DPPIV inhibition over the 90-min study, a 60% higher level of GLP-1, a decrease in glucose excursion and a doubling of peak insulin levels. Comparable studies in cynomologus monkey dosed with LAF237 (1 mmol/kg) showed >50% DPPIV inhibition for at least 10 h compared to 4–5 h for NVP-DPP728. LAF237 was brought forward as a development candidate suitable for once daily dosing (Fig. 2.6).

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National Health Research Institutes, Taiwan These researchers used a selectivity comparison of known DPPIV inhibitors as their starting premise. The key comparison was between DPPIV and DPP8, as other proteases were not inhibited to a significant degree. It was found that P2 primary amino analogues (P32/98 (10) and (17)) were not selective over DPP8 while two P2 N-substituted glycine analogues were (Table 2.1) [86]. Based on these results, several amide-linked series were prepared. The b-alanine spacer with gem-dimethyl substitution adjacent to the P2 amine seemed optimal for potency and selectivity. A terminal isoquinoline resulted in (22), which was aqueously soluble, stable to intramolecular cyclization and pharmacologically comparable to LAF237 (21) in OGTT studies. P2 pyrrolidides: conformationally restricted analogues Mitsubishi. Introduction of a substituted proline in the P2 position essentially acted as a rigidification of N-alkyl group of NVP-DPP728 (20). Accordingly a series of 1-(g-substituted prolyl)-(S)-2-cyanopyrrolides was prepared and evaluated [87]. The various diastereomeric possibilities were prepared from hydroxyprolines with displacement chemistry or from the corresponding ketone via reductive amination. The (S)-cis-isomer of the P2 prolyl group enhanced potency against DPPIV while the (R)-trans form had less inhibitory potency than the open-chain parent. Investigation of spacers between the amine and aromatic group did not yield any improvement. Substitution of the phenyl ring produced (23) as a subnanomolar inhibitor of the enzyme. Unfortunately, the above series was determined to be quite prone to intramolecular cyclization, a problem observed with this structural motif in the earlier described Pro-boroPro (15). In response to this effect, the 2-cyanopyrrolidine was replaced with a thiazolidine [88]. Although less potent than the corresponding 2-cyanopyrrolides, the expected stability was obtained as shown with (24). Thiazolidide (24) had a good pharmacokinetic profile with a half-life of 5.27 h and oral bioavailability of 84%. Half-inhibition of DPPIV was obtained 9 h following a 100 mmol/kg oral dose. Abbott Laboratories. An amide methylene spacer between the P2 pyrrolidine and the aromatic group was found to supply potent compounds [89]. This series consisted primarily of P1 thiazolidides, but several P1 groups were evaluated inclusive of 2-cyanopyrrolidine. Thiazole (25) was exemplary and exhibited a good pharmacokinetic profile (t1/2 ¼ 5 h, F ¼ 57%), had greater than 200-fold selectivity over DPP7, DPP8 and FAP-a, inhibited

78

DPPIV INHIBITION

more than 90% of DPPIV activity for more than 2 h when dosed at 3 mg/kg and reduced the glucose excursion by 33% at the same dose. A related series has also been reported that has substitution at the C5 or d position of the P2 pyrrolidide [90]. This series produced subnanomolar Kis while having enhanced chemical stability against intramolecular cyclization. Surprisingly, the substitution on the pendant aromatic had great influence on stability and those compounds with bulky substituents in the ortho position exhibited longer half-lives. Compound (26) was exemplified and was greater than 20-fold selective for DPPIV over DPP7, DPP8, DPP9, POP and FAP-a. Other analogues in the series were >1,000-fold selective. Pharmacokinetic parameters were favourable, highlighted by an AUC of 4,110 ng h/kg when dosed orally at 5 mg/kg. A reduction in glucose exposure of 30% was noted following an OGTT. National Health Research Institute, Taiwan. A series in which a carbonyl linked the P2 pyrrolidine to an isoindoline was recently reported [91]. The isoindoline as found in (27) was the optimal P2 side chain. P1 was optimized with either a 2-cyanopyrrolidine or thiazolidine. Selectivity was noted over DPP8, DPPII and FAP-a. Oral dosing at 10 mg/kg produced maximal DPPIV inhibition in 30 min and more than 50% inhibition was observed for 12 h. Bristol– Myers Squibb. These researchers considered the Pro–Pro motif as an under-explored scaffold and concentrated on evaluation of the N-terminal amino acid coupled with a P1 cis-4,5-methano-2-cyanopyrrolidine derived from investigations discussed below [92]. P2-Piperidinyl and azetidinyl analogues were less potent than the corresponding pyrrolidinyl analogues. The L-prolyl analogue was confirmed as the desired configuration as was observed in earlier work with acyclic analogues. SAR of fusedcyclopropyl analogues suggested the importance of positions 3 and 4 on the P2 pyrrolidine. Position 3 was found to be sterically limited due to an inverse relationship between substituent size and potency. Larger groups were readily accommodated at position 4 in the cis configuration where aromatic substituents were favoured and aniline (28) was the most potent with a Ki ¼ 3.6 nM, 100-fold more potent than the parent compound with an unsubstituted P2 L-proline. Co-crystallization studies indicated that position-4 substituents were best positioned to fill the S2 pocket, which was lined primarily but not exclusively with hydrophobic groups with a considerable number of aromatic side chains. These compounds were found to have reduced solution stability relative to other series explored by these investigators described below (Fig. 2.7). Fluoro-olefin peptidomimetics, University of Antwerp. Some additional studies have recently been completed on fluoro-olefin as peptidomimetics

P. E. WIEDEMAN

NH

NC

79

NH

NC

N

N N H

N

Cl

N H

CN

O

CN

O

(23) IC50 =0.13 nM NC

O

S

NH N N H

N

S

N

N H

N N H

O

(24) IC50 = 25.4 nM

O

(25) Ki = 5 nM

O

iPr O HO2C

S

N

N N H

N

CN

O

N H

(26) Ki = 3.1 nM

O

CN

(27) IC50 = 1.7 nM

H N

NC Cl

N N H

O

CN

(28) Ki = 3.6 nM Fig. 2.7 Conformationally restricted analogues, P2 pyrrolidides.

inhibitors of dipeptidyl peptidases [93] that build upon previous investigations that sought to provide chemical stability [94, 95]. The majority of analogues prepared were N-substituted Gly-c[CF ¼ C]pyrrolidines and piperidines that exhibited selectivity for DPPII over DPPIV. Introduction of a Gly-c[CF ¼ C](2-cyano)pyrrolidines resulted in DPPIV selectivity for

80

DPPIV INHIBITION

Z(trans)

E(cis) CN H N

F

F

(29) DPPIV IC50 = 12 µM DPPII IC50 > 100 µM

CN H N

(30) DPPIV IC50 = 15 µM DPPII IC50 > 100 µM

H N

CN

N O

(31) DPPIV IC50 = 0.35 µM DPPII IC50 > 100 µM

Fig. 2.8 Fluoro-olefin peptidomimetic inhibitors.

select analogues. However, there was a potency loss of more than 40-fold. The authors attribute this to the poorer hydrogen bond accepting ability of the fluoro-olefin compared to the parent amide bond compound (31), which highlights the importance of that amide bond interaction. Interestingly, both the E (cis)-isomer (29) and Z (trans)-isomer (30) had the same IC50 when it had been presumed that the Z-isomer would be favoured for enzyme recognition as it mimics the trans-amide bond. Aqueous stability studies were carried out in a buffer solution (pH 7.5) at 37 1C. The half-lives of both Z- and E-fluoro-olefin carbonitriles was greater than 200 h. With a long incubation, the E-isomer does eventually intramolecularly cyclize, while the Z-isomer only slowly loses HF (Fig. 2.8).

COVALENT DPPIV INHIBITORS: P2 PRIMARY AMINE ANALOGUES

Bristol– Myers Squibb Substitution with a cyclopropyl group fused to the P1 cyanopyrrolidine was the focus of this initial study [96]. Several of the possible geometric combinations were prepared and compared for their influence on potency and chemical stability. Specifically, the isomers synthesized included the cis-4,5, trans-4,5, cis-3,4 and trans-2,3. The end result was that the cis-4,5 methano bridged inhibitors, exemplified by (32), maintained potency while improving chemical stability to intramolecular cyclization. For example, (32) had a Ki ¼ 7 nM and a stability t1/2 ¼ 42 h. By comparison, the parent compound lacking the methano bridge had a Ki ¼ 8 nM and a stability t1/2 ¼ 27 h. An extensive computational analysis was carried out on the series. Both bbranching of the a-side chain and the 4,5-cis methano bridge were found to be factors influencing the conformation, which disfavoured intramolecular cyclization. In vivo analysis was performed with fasted male Zuckerfa/fa rats.

P. E. WIEDEMAN

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The animals were dosed at 3 mmol/kg with inhibitor and subsequently challenged with glucose (2 g/kg) 30 min later. Inhibitor (32) produced a 30–35% blunting of the peak glucose response at 60 min when the untreated control animals were at their peak response. At the same time point, insulin levels were significantly enhanced. Meanwhile, DPPIV activity was depressed 35–60% throughout the course of the 2-h experiment with maximal suppression occurring 30 min post dosing. Building on this knowledge, additional analogues of b-quaternary Nterminal amino acids were prepared [97]. A vinyl group was incorporated into the quaternary centre to allow for further functionalization. This became necessary when (33) showed poor bioavailability (5%) and significant metabolism in the presence of rat liver microsomes despite in vitro potency and ex vivo durability. Although never directly correlated, oxidation of the vinyl group of (33) was a suspected site of metabolism and oxygenated analogues were prepared such as (34). This hydroxymethyl analogue maintained potency both in vitro and ex vivo. Important improvements were made on the metabolic front, where turnover rate was reduced in the presence of rat liver microsomes, and in the pharmacokinetic profile, where bioavailability was increased (59%). Taking the b-branching theme through to a conclusion, rigid polycyclic analogues were synthesized of which adamantyl was described. Similar to the vinyl analogue (33), the parent adamantyl compound was very potent (Ki ¼ 0.9 nM) but suffered pharmacokinetically (F ¼ 2%) and metabolically when exposed to microsomes. Oxidation at the adamantyl bridgehead position furnished (35). Again, the issues of metabolism and pharmacokinetics were addressed by oxidation at this likely metabolically unstable position. Potency was identical to the parent (Ki ¼ 0.6 nM), a lower turnover rate to liver microsomes was observed and the bioavailability was dramatically increased (F ¼ 75%). Additional hydroxylation or fluorination on the adamantyl group resulted in a return to poor oral exposure. Adamantyl analogue (35) was tested in two rodent models of diabetes, the Zuckerfa/fa and the ob/ob mouse. In the Zuckerfa/fa study, maximal antihyperglycemic effect was noted at 60% DPPIV inhibition. Higher levels of enzyme inhibition were not correlated with additional glucose level lowering. Durability of DPPIV inhibition was illustrated by performing the OGTT 4 h after dosing in the dose range of 0.3–3 mmol/kg. In the ob/ob mouse OGTT, insulin levels were raised and postprandial glucose levels were reduced. Based on the extended pharmacodynamic response, (35) was anticipated to be suitable for once daily dosing in humans. This adamantyl analogue ((35), BMS-477118, saxagliptin) has been advanced into human clinical trials. Although DPPIV recognizes peptides containing either proline or alanine at the N-terminal penultimate position, cyanopyrrolidides have been the

82

DPPIV INHIBITION

HO

tBu N

H 2N

N

H2N CN

O

O

CN

O

(33) Ki = 3.9 nM F = 5%

(32) Ki = 7 nM stability t1/2 = 42 h

N

H2N

HO

CN

(34) Ki = 7.4 nM F = 59%

HO Me N

N

H2N

H2N O

CN

(35), BMS-477118, saxagliptin Ki = 0.6 nM F = 75% no CYP3A4 inhibion Fig. 2.9

L-cis-4,5-Methanoprolinenitrile

CN

O (36) Ki = 18 nM stability t1/2 = 10 h inhibitors.

motif almost exclusively explored. A series of seco-proline nitriles was prepared to determine if an alanine-like P1 had therapeutic potential [98]. Compound (36) summarized the study. The SAR of P2 followed earlier studies and the most potent compounds were obtained with the 3-hydroxyadamantyl side chain. Generally, a glycine-derived P1 was superior with regard to potency over a corresponding alanine analogue. A small substituent such as methyl, ethyl or allyl was beneficial on the P1 amine. Although no improvement was noted in comparison to P1 proline analogues, sufficient in vitro and in vivo results were obtained to warrant further investigation (Fig. 2.9). GlaxoSmithKline This report focused on two design aspects of a cyanopyrrolidine series [99]. The P2 fragment was derived from D-penicillamine, with the geminal dimethyl moiety responsible for retarding intramolecular cyclization. The side chain was further functionalized by alkylation of the thiol. Although the corresponding sulfides were most often found to have superior in vitro potency, pharmacokinetic studies indicated that the sulfoxide and sulfone

P. E. WIEDEMAN

83

F

Me Me O

MeO

S O O

NH2 F

(37) Ki = 53 nM

H Me

Et

O

O

CN

CN N

NH2

NH2

F

(38), denagliptin IC50 = 22 nM

H Me

CN

Et

O

F

CN N

NH2 F

(39) IC50 = 0.6 nM

F

F

(40) IC50 = 0.8 nM

Fig. 2.10 Fluorocyanopyrrolidide inhibitors.

were rapidly formed in vivo and, therefore, sulfones were selected for additional evaluation. Also in this series, a fluorine was incorporated at C4 of the cyanopyrrolidine. Comparison with the des-fluoro analogue showed that the C4 fluoro analogue (37), was more potent, had longer duration of action, longer exposure, enhanced selectivity over both DPPII and seprase and greater chemical stability to intramolecular cyclization. It should be noted that a p-cyano analogue of (37) exhibited gastrointestinal toxicity when dosed orally at 10 mg/kg. Based on the corporate website (www.gsk.com), (38) (denagliptin), was selected for clinical development. Little information has been released, but the compound was reported to be modestly potent (IC50 ¼ 22 nM) and stable to intramolecular cyclization [100] (Fig. 2.10). Taisho An evaluation of 3- and 4-substituted-2-cyanopyrrolidines was carried out [101]. In brief, oxygen functional groups (hydroxy, methoxy and ketone) were all detrimental to potency at either the C3 or C4 pyrrolidine positions regardless of stereochemistry. However, fluorine at the C4 position in the S-configuration (39) enhanced potency several fold. This effect was not

84

DPPIV INHIBITION

observed with the R-isomer, chlorine, or C3 analogue. Somewhat surprisingly, the C4 geminal difluoro analogue (40) had potency comparable to (39). When dosed orally, the fluoro analogue (39) had a Cmax 2.5 times greater than the des-fluoro parent. Hyperglycemia was suppressed in an OGTT in Zucker fatty rats with dosing at 4 mg/kg. DPPIV was completely inhibited for more than 2 h at this dose.

NON-COVALENT DPPIV INHIBITORS: P2 PRIMARY AMINE ANALOGUES

Merck The Merck investigators followed an ambitious two-pronged approach in pursuit of suitable DPPIV inhibitors. The first approach involved rational design seemingly based on earlier work described in the literature. Second, a screening strategy furnished a unique series that was ultimately developed into a clinical candidate. The rational design approach will be described first, although there was overlap and influence between the two approaches. The rational design effort was initiated with the decision to pursue inhibitors lacking a serine hook in the P1 region. A review of the literature at that time indicated that P32/98, (S)-isoleucine thiazolidide (10) [72], while although only modestly potent against DPPIV (IC50 ¼ 420 nM), had shown clinical efficacy [102]. A related analogue, the (S)-cyclohexylglycine pyrrolidide (41) [79], was viewed as having a side chain more amenable to modification. To this end, an amino group was installed at the 4-position on the cyclohexyl side chain for further functionalization [103]. These investigators were amongst the most rigorous in stressing selectivity against related dipeptidyl peptidases and proline-specific enzymes throughout their research. This was an evolving issue as several new dipeptidyl peptidases were discovered during the course of the research and eventually greatly influenced which compound was advanced into clinical trials. The initial selectivity panel consisted of prolyl endopeptidase, amino peptidase P, prolidase, DPPII (quiescent prolyl peptidase, DPP7) and seprase (fibroblast activation protein). Basically, no activity was noted in the inhibition of any enzyme except DPPII and selectivity will only be discussed against that enzyme at this time. NVP-DPP728 (20) and LAF237 (21), the lead cyanopyrrolidide compounds demonstrated at least 300-fold selectivity. P32/98 (10) and (41) exhibited a more modest selectivity of 30- to 60-fold. Both the pyrrolidide and thiazolidide of 4-aminocyclohexylglycine were prepared and further functionalized as aryl sulfonamides, amides, carbamates and ureas. Two trends developed, the thiazolidides were generally several fold more potent than the corresponding pyrrolidides. However, the

P. E. WIEDEMAN

85

pyrrolidides had greater selectivity against DPPII. A breakthrough was achieved with (42), a single digit nanomolar inhibitor devoid of a serine hook that maintained selectivity. Off-target effects were assessed as minimal, with no inhibition of the hERG channel, but the pharmokinetic profile was completely unacceptable with a half-life of 0.1 h and no bioavailability. Modification of the sulfonamide moiety supplied (43), while although not as potent, had an improved pharmacokinetic (PK) profile (rat t1/2 ¼ 1.8 h, %F ¼ 36; dog t1/2 ¼ 6 h, %F ¼ 100). This compound was used to demonstrate the potential for glucose control in an OGTT in lean mice with a reduction in glucose excursion of 36% when dosed at 3 mg/kg. Two following manuscripts continued the exploration of the cycloalkylglycyl compounds. One explored the fusion of a heterocycle to the cyclohexyl side chain that produced some potent analogues of which (44) is representative [104]. The fluoropyrrolidide was introduced without any comment. The other was a thorough investigation of stereochemistry of the cyclohexyl or cyclopentyl side chains [105]. A cyclopentyl analogue (45) is shown with inverse stereochemistry relative to the cyclohexyl analogue (42). At this point, thiazolidides were considered a metabolic liability and were de-emphasized. Following this, an investigation of P1 replacements was undertaken and fluoropyrrolidides were prepared [106]. Fluoropyrrolidides had been shown earlier to function as DPPIV inhibitors [107]. To summarize, the (3S,4S)and (3R,4R)-3,4-difluoropyrrolidides were found to decrease potency while the 3,3-difluoropyrrolidides exhibited potencies similar to their thiazolidide counterparts. The (S)-3-fluoropyrrolidides were found to have good potency against DPPIV, somewhat superior to the (R)-3 analogues. Pharmacokinetic parameters were encouraging with a half-life of 1.6 h and 53% bioavailability in rats for compound (46). An OGTT in lean mice produced a 42% reduction in glucose excursion compared to control animals (Fig. 2.11). However, a metabolism study of tritiated (46) and a related compound showed time- and NADPH-dependent irreversible binding in rat liver microsomes [108]. Addition of glutathione (GSH) lowered the in vitro irreversible binding and was suggestive of nucleophilic scavenging of an electrophilic intermediate. Analysis of a series of LC/MS experiments established a pathway of pyrrolidine oxidation, HF elimination and nucleophilic attack. Semicarbazide was used to trap and verify aldehyde/ hemiaminal intermediates. In vitro experiments identified CYP3A1 and CYP3A2 as the cytochrome P450 isoforms responsible for the oxidation that starts the cascade. Concern was expressed regarding the potential to modify off-target proteins resulting in idiosyncratic drug reactions and depletion of cellular GSH causing oxidative stress (Scheme 2.1). Fluoropyrrolidide (46) was subsequently found to have an additional flaw that halted any chance of development. During the development of the

86

DPPIV INHIBITION O O

H2N H

N

O H2N

N

H

NH2

H N

(41) DPPIV IC50 = 320 nM

CH2CF3 S O O

N

H

S O O

F

O H

S O O

N

H

O H2N

N

H

N

F

SO2Me F

S N

F

(43) DPPIV IC50 = 88 nM DPPII IC50 = 8800 nM hERG Ki = 35000 nM t1/2 = 1.8 h %F = 36

O H2N

F H


F3C

N

H

(42) DPPIV IC50 = 2.6 nM DPPII IC50 = 15000 nM hERG Ki > 100000 nM t1/2 = 0.1 h %F = 0

H2N

N

N

S O O

H

(45) DPPIV IC50 = 13 nM

N

S O O

F

(46) DPPIV IC50 = 48 nM DPP8 IC50 = 993 nM DPP9 IC50 = 2720 nM

Fig. 2.11 P2-substituted cyclohexylalanine inhibitors.

O

O N

O H NH

F

O

O H NH

O

H NH

N F

(46)

O

OH

-HF

F

O

O

OH N

S-G G-SH Scheme 2.1 Metabolism of fluorinated pyrrolidides.

S-G

P. E. WIEDEMAN

87

dipeptidyl peptidase field, additional members of the enzyme family were discovered and the implications were discussed in an earlier section. Suffice it to say that selectivity against DPP8 and DPP9 is a desirable attribute. Fluoropyrrolidide (46) was found to have less than 100-fold selectivity against both of the enzymes [109]. At the same time, it was discovered that the isoleucine thiazolidide isomers (47) and (48), despite having essentially identical IC50s for DPPIV, showed different selectivity profiles indicating that the stereochemistry at the b-position influenced enzyme selectivity. The threo-isomer (47) was the more selective for DPPIV over DPP8 and DPP9. The corresponding b-methylphenylalanine analogue conferred even greater selectivity (not shown). Knowing from previous work that the P2 area was amenable to modification, a series consisting primarily of functionalized biphenyls was prepared. This biphenyl series furnished a number of potent and reasonably selective compounds that may be summarized with two compounds (49) and (50). The more lipophilic (49) exhibited a preferred PK profile while suffering from hERG binding. By contrast, the more polar (50) showed a poor PK profile while interacting to a lesser extent with the hERG channel. Attempts were made to combine these features to attain the desired profile. One study maintained the lipophilic biphenyl in the P2 position while substituting a more polar amide moiety for the b-methyl [110]. This combination did supply the desired in vitro profile in (51), but there was a lack of efficacy that was subsequently attributed to serum protein binding. Introducing polarity back into the P2 region with a heterocyclic group attached to the phenyl ring of the b-phenylalanine also attained the sought profile as shown with (52) [111]. The compound was metabolized by Ndemethylation leading to a compound with an insufficient safety margin indicated by hERG binding (IC50 ¼ 8.3 mM). Greater potency and metabolic stability were required in future research in this series (Fig. 2.12). Pfizer Extending earlier work on fluorine-substituted pyrrolidines, the cis-3,4-difluoropyrrolidine and 3,4-tetrafluoropyrrolidine analogues (53) and (54) were found to be potent inhibitors of DPPIV with Kis of 61 and 81 nM, respectively [112]. These inhibitors did show in vivo activity during an OGTT in which 10 mg/kg of the drug was administered 15 min prior to a 1 g/ kg oral glucose load. Blood glucose levels were measured 30 min later, and the results were expressed as a percentage reduction of the drugged animals’ glucose concentration over the glucose concentration in untreated animals. The observed reduction in glucose concentration was 60 and 59% for (53) and (54), respectively.

88

DPPIV INHIBITION

Me

O

Et

Me N

Et

S

NH2

N

S

NH2 (48) (allo-isomer) DPPIV IC50 = 460 nM DPP8 IC50 = 220 nM DPP9 IC50 = 320 nM

(47) (threo -isomer) DPPIV IC50 = 420 nM DPP8 IC50 = 2170 nM DPP9 IC50 = 1600 nM Me

O

O

Me

O

O N

N O

NH2 F

N NH2

N H

F

F (49) DPPIV IC50 = 64 nM DPPII IC50 = 2700 nM DPP8 IC50 = 8800 nM DPP9 IC50 = 8600 nM t1/2 = 2.2 h %F = 85 hERG IC50 = 1100 nM

Me2N

O

(50) DPPIV IC50 = 3 nM DPPII IC50 = 1100 nM DPP8 IC50 > 100000 nM DPP9 IC50 > 100000 nM t1/2 = 1.1 h %F = 4.2 hERG IC50 =28000 nM

Me

O

O N

N

Me

NH2 F F (51) DPPIV IC50 = 12 nM DPPIV IC50 = 387 nM (50% human serum) DPPII IC50 = 45000 nM DPP8 IC50 > 100000 nM DPP9 IC50 = 6900 nM t1/2 = 3.5 h %F = 67 hERG IC50 = 4600 nM

NH2

N

F

O (52) DPPIV IC50 = 34 nM DPPII IC50 = 8000 nM DPP8 IC50> 100000 nM DPP9 IC50 > 100000 nM t1/2 = 1.4 h %F = 34 hERG IC50 >100000 nM

Fig. 2.12 P2 b-methylbiphenylalanine inhibitors.

P. E. WIEDEMAN

89

O F

F F

N

H2N

N

H2N

O

O

(53) Ki = 61 nM

(54) Ki = 81 nM

H2N

F

N

F F

F

S

(55) Ki = 170 nM

Fig. 2.13 Fluorinated pyrrolidide and thiazolidide inhibitors.

Johnson and Johnson The premise of this study was to optimize P2 starting with a P1 2-cyanopyrrolidine, and then use that SAR to achieve a P1 group without a covalent interaction. Structural studies suggested a linear hydrophobic pocket in the P2 region by Phe357 and Arg358. This was achieved with a biaryl P2 side chain and a P1 thiazolidide that was associated with improved permeability and human liver microsome stability as shown with (55) [113] (Fig. 2.13).

PEPTIDIC DPPIV INHIBITORS THAT EXTEND INTO P10

Guilford Alternative serine hooks, heteroaryl ketopyrrolidines and ketoazetidines were investigated [114]. Heteroaryl groups containing nitrogen in the 2position such as 2-thiazole, 2-benzothiazole and 2-pyridyl proved required for potency. A model study indicated that the 2-thiazole nitrogen formed a hydrogen bond to the side chain of Arg125. The SAR in the P2 pocket was predictable. Stereochemistry was much more important for ketopyrrolidines than for ketoazetidines. Dosing with ketoazetidine (56) at 50 mg/kg, greater than 60% of DPPIV activity was inhibited for 6 h. Korea Research Institute of Chemical Technology A P1-pyrazoline was investigated building on some earlier studies of modestly potent inhibitors [115]. First established was the enhanced potency with the 2S-cyanopyrazoline analogue (360 nM) of NVP-DPP728 (20) [116]. This was followed by the synthesis of aryl [117] and heteroaryl [118] pyrazoline ureas. Isoxazole (57) was active in an OGTT in ob/ob mice [119] (Fig. 2.14).

90

DPPIV INHIBITION

O N S N N

O O

NH

(56) DPPIV IC50 = 115 nM

Me

O

Et

O N

Me

NH N

NH2 (57) DPPIV IC50 = 2200 nM

Fig. 2.14 Peptidic inhibitors extending to P10 .

NON-PEPTIDIC/NON-COVALENT DPPIV INHIBITORS

Novartis In a medicinal chemistry manuscript that was specifically targeting the importance of DPPIV inhibition in modulating the incretin effect in type 2 diabetics, an isoquinoline screening hit was developed into a modestly potent compound (58) (IC50 ¼ 320 nM) [120]. The importance of the primary amine moiety was emphasized along with the substitution about the heterocycle that established the potential for non-peptidic DPPIV inhibitors. Osaka University The uniquely structured sulphostin (59) was isolated from Streptomyces sp. MK251-43F3 in the course of a screening exercise [121]. The total synthesis established that the absolute configuration at phosphorous was principally important for potency [122]. A pyrrolidone analogue was the most potent in an SAR study. Docking in a model did show the interaction of the amino group with Glu205 and Glu206. Hydrogen bonds were formed between the C2 carbonyl and Tyr662, the phosphinyl group and Tyr547 and the primary amino group of the amino(sulfoamino)phosphinyl moiety with Ser630 and Tyr662. The sulfonic acid group forms an ionic interaction with Arg125 [123]. Xanthines This represents another non-peptidic core with DPPIV inhibitory activity. No publications have appeared outside of the patent literature and a few presentations give insight into the series. Both Novo Nordisk [124, 125] and Boehringer Ingelheim [126] entered this particular field early and (60) is a

P. E. WIEDEMAN

91

CO2Et

NH2

MeO

OMe

N O O P NHSO3H NH2

NH2

(58) DPPIV IC50 = 320 nM

Me

NH2

O

N

N O

CN

Me

O

O

(59) DPPIV IC50 = 21 nM

N N Me

N


NH2 N

Me N

N N

O (61) DPPIV Ki = 2 nM DPP8 > 3000 nM DPP9 > 3000 nM

Fig. 2.15 Inhibitors derived from screening hits.

representative compound. Several other companies have followed either adjusting the pendant functionality or the bicyclic heterocyclic core. One example of this was presented by Abbott Laboratories [127]. The maleimide (61) was both potent and selective. Co-crystallization within the enzyme indicated that the benzyl group filled the P1 pocket, the pendant amine on the piperidine interacts ionically with Glu205 and Glu206, while the maleimide was observed in a p-stacking interaction with Tyr548. Similar interactions were expected from other representatives of this series (Fig. 2.15). Hoffmann-La Roche A series of papers published in 2004 highlight the SAR and medicinal chemistry development of a series 5-aminomethylpyrimidines. The initial high-throughput screening hit (62) had a modest inhibitory activity (IC50 ¼ 10 mM) [128]. Preliminary investigation with substitution about the 6-phenyl ring indicated that both ortho and para substituents enhanced potency. Combining chlorine substituents at both positions resulted in a 1,000-fold potency improvement relative to the screening hit.

92

DPPIV INHIBITION

A similar exploratory effort followed with the 2-phenyl ring. Fluorine substitution at either the meta or para position resulted in picomolar activity. Somewhat confoundingly, a single meta methoxy substituent furnished an IC50 ¼ 340 nM, but inclusion of a second methoxy moiety at the other meta position produced the most potent compound of the series (63) (IC50 ¼ 0.1 nM), a five orders of magnitude increase in potency again compared to the screening hit. An elegant structural study of the binding of this compound in the DPPIV active site is described below. A subsequent study investigated conformational restriction [129]. To this end, a pyridine series with SAR corresponding to the pyrimidines described above was prepared. The thrust of this study involved tying the equivalent of the 6-phenyl ring to the pyridine ring with alkylidene chains of between one and three carbons in length. As expected, the torsion angle decreases with chain length, and the methylene linker with a torsion angle of 01 provided the most potency. Once again, a dimethoxy-substituted analogue, proved to be the most active (64) (IC50 ¼ 7 nM). This pyrimidine series of DPPIV inhibitors, exemplified by (65), exhibited a number of positive properties including good solubility, high membrane permeability and significant metabolic stability as determined by exposure to human liver microsome preparations [130]. However, upon further investigation of the pyrimidines, some issues arose. Namely cytochrome P450 3A4 (CYP3A4) was inhibited and phospholipidosis was induced in a concentrationdependent manner. A closer examination of (65) offers suggestions for these behaviours. Pyrimidine (65) is a lipophilic molecule with a pKa ¼ 7.8. Therefore, it is going to exist as a cationic amphiphilic molecule under physiologic conditions. Lipophilicity is a recognition element for CYP 3A4, and amphiphilicity is predictive of phospholipidosis potential in cationic compounds. Accordingly, the adjustment of the physicochemical properties was in order. Structural biology showed that the 6-dichlorophenyl ring and the amine moieties were optimal. This left the solvent exposed 2-phenyl group as the site amenable to physicochemical property manipulation. N-Alkylated analogues were selected and evaluated using computational tools to suggest both decreased lipophilicity (KOW_ClogP) and decreased amphiphilicity (CAFCA). Synthesis of a series of N-alkylated pyrimidines yielded (66) that gratifyingly addressed the faults uncovered in (65) while maintaining nanomolar potency. No phospholipidosis was observed up to the highest test concentration (20 mM) and CYP 3A4 inhibition was greatly reduced. This is likely due to the decrease in lipophilicity (log D 7.4 ¼ 1.6) (Fig. 2.16). Co-crystallization of the pyrimidine inhibitor (63) provided much insight to the binding of this and related types of molecules to DPPIV [128]. The 6-dichlorophenyl group fits nicely into the hydrophobic P1 pocket explaining its SAR. The 5-aminomethyl moiety symmetrically hydrogen bonds with

P. E. WIEDEMAN NH2 NH2

93

NH2 NH2

NH2 NH2

N

N MeO

O

N

N MeO

N

O

Cl

Cl

MeO

OMe

(62) IC50 = 10000 nM

(63) IC50 = 0.1 nM

Cl

Cl

(64) IC50 = 7 nM

NH2 NH2

NH2 NH2 N

N MeO

N Cl

Cl

(65) IC50 = 10 nM logD7.4 = 3.0 IC50 (CYP 3A4) = 5.4 µM phospholipidosis induction

N Me

N Cl

Cl

(66) IC50 = 9 nM logD7.4 = 1.6 IC50 (CYP 3A4) = 30 µM no phospholipidosis induction

Fig. 2.16 Aminomethylpyrimidine inhibitors.

Tyr662, Glu205 and Glu206, thus mimicking the N-terminus of the substrate motif. The 4-amino group hydrogen bonds to the backbone amide carbonyl of Glu205. The pyrimidine core is involved with a cationic pinteraction with Arg 125. The 2-dimethoxyphenyl is predominantly exposed to solvent, but one methoxy group is near the side chain of Tyr547. Lacking an electrophilic group found in the 2-cyanopyrrolidines, there is no interaction observed with the catalytic site Ser630. Merck A screening approach was the second and complementary approach to the work already described. The initial lead discovered in the corporate collection was (67) [131]. A preliminary SAR study determined that the left-hand portion of the hit compound was sufficient for maintaining binding potency as illustrated with thiazolidide (68). Other SAR findings included that the distance between the amino moiety and phenyl group was crucial as was the absolute stereochemistry of the amine. Alicyclic and heterocylic substitutions for the phenyl ring resulted in a loss of potency. Turning to substituents on the phenyl ring, small groups such as fluorine and methyl gave a potency boost while trifluoromethyl and nitrile did not. Multiple fluorine substituents enhanced potency and the 2,4,5-trifluorophenyl (69) proved to be optimal. However, a poor pharmacokinetic profile hampered any additional development of (69).

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DPPIV INHIBITION

More SAR exploration of (67) led eventually to phenoxyacetic acid analogue (70), an extremely potent and selective DPPIV inhibitor [132]. Compared to the amide lead, the terminal carboxylic acids increased binding affinity and decreased hERG channel interaction. Steric bulk alpha to the acid was found to be particularly beneficial in the S-configuration. The (R)-b-homophenylalanine was determined to be essential for selectivity as replacement with cyclohexylalanine resulted in both loss of potency and selectivity. Once again, poor pharmacokinetics eliminated (70) from further consideration. A second screening hit (71) also contained the b-phenethylamine pharmacophore seen on the earlier hit. A thorough SAR evaluation led eventually to (72), a potent and selective inhibitor likewise with a poor pharmacokinetic profile, blamed primarily on metabolism of the piperazine ring [133]. In hopes of solving the pharmacokinetic and metabolism problems that were plaguing the b-homophenylalanine series, fused heterocycles were installed as replacements for the piperazines described above [134]. Focusing specifically on triazolopiperazines was rewarded with stability demonstrated in the presence of hepatocytes. The SAR of the substituent pattern around the phenyl ring of the b-homophenylalanine was maintained as established in the earlier work. Substitution on the triazolopiperazine was limited, and trifluoromethyl was optimal, which supplied the clinical candidate MK-0431 ((3), sitagliptin). Removal of the trifluoromethyl group abolished bioavailability. X-ray crystal structure confirmed the binding interactions of (3). The trifluorophenyl group fills the S1 pocket establishing a reverse orientation relative to a-amino acid containing inhibitors. The amino moiety binds to the side chains of Glu205, Glu206 and Tyr662. The amide carbonyl bridges through a water molecule to Tyr547. Other water-mediated interactions occur between the protein and heterocycle nitrogens. The trifluoromethyl group fits into a small pocket and is near the side chains of Arg358 and Ser209. The triazolopiperazine stacks against Phe357. MK-0431 (3) showed the sought pharmacokinetic profile with an acceptable half-life and good bioavailability. Evaluation in vivo was first assessed in lean mice where an OGTT showed a dose-dependent suppression of the glucose excursion. In a separate OGTT experiment also on lean mice, DPPIV activity was inhibited by 84% and GLP-1 levels raised two- to threefold with a 3 mg/kg dose. A final experiment with diet-induced obese mice as a model of diabetes showed near normalization to a dextrose challenge again with a 3 mg/kg dose. An exploration of related heterocyclic replacements for piperazine was carried out. Piperidines fused to pyrazole, isoxazole, oxazole and thiazole were examined and their SAR described [135]. Thiazole (73) is exemplary with an in vitro and pharmacokinetic profile similar to MK-0431 (3) (Fig. 2.17).

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Cl

NH2 O Ph

O

H N

NH2 O Ph

O

N

O

(67) DPPIV IC50 = 1900 nM

NH

F

O

NH2 O

NH2 O N

CO2H iPr

F

(69) DPPIV IC50 = 119 nM

(70) DPPIV IC50 = 0.48 nM

Ph

F F

N

O N

N H

Ph

NH2 O

O O S N Me H

N NH

F

(71) DPPIV IC50 = 11000 nM

(72) DPPIV IC50 = 19 nM

F

F NH2 O

NH2 O N

N F

O

H N

N

S

F

F

S

(68) DPPIV IC50 = 3000 nM F

F

H2N

N

N

(3) MK-0431, sitagliptin DPPIV IC50 = 18 nM DPP8 IC50 = 48000 nM DPP9 IC50 > 100000 nM t1/2 = 1.7 h %F = 76

N CF3

N F

N F S

(73) DPPIV IC50 = 26 nM

Fig. 2.17 b-Homophenylalanine inhibitors.

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DPPIV INHIBITION

Cl O Me O

NH2 N

N

O F

CN N

N N Me

N

N

(74)

(75)

O Me

N

NH2

O

N

CN N N

NH2

(76) SYR-322 DPPIV IC50 = 7 nM

Fig. 2.18 Dioxo-dihydropyrimidine inhibitors.

Takeda/Syrrx Recently, a brief account was presented that described a structural chemistry guided rational design approach [136]. Impressively, the programme in a mere 4 years has positioned Syr-322 in late phase III clinical trials. Starting from a known xanthine lead (74) [137], the heterocyclic core was altered first to a 4-oxoquinazoline. This compound (75) was potent, selective and had a desirable PK profile (F ¼ 85%). However, significant CYP 3A4 inhibition (IC50 ¼ 2 nM) and significant hERG binding precluded further development. Substituting a dioxo-dihydropyrimidine core alleviated these issues. Compound (76) did not exhibit the metabolic or cardiovascular liabilities of (75) while potency and a favorable PK profile were maintained [138]. Specifically, (76) was a potent inhibitor of DPPIV (IC50 ¼ 7 nM) and selective against closely related enzymes (IC50s>100 mM). Oral bioavailabilities ranged from 45 to 87% across the species studied (rat, dog, monkey). Oral half-lives in rat, dog and monkey were 2.3, 3.0 and 5.7 h, respectively. Peak inhibition (>85%) of DPPIV was obtained with a 10 mg/kg oral dose. After 12 h, 43% inhibition of enzymatic activity was still maintained. In monkey, a single oral dose of 2–30 mg/kg resulted in >80% inhibition over a 24-h period (Fig. 2.18). CLINICAL DATA FOR ORAL DPPIV INHIBITORS Numerous major pharmaceutical manufacturers are currently undergoing human clinical trials with DPPIV inhibitors for the treatment of type 2 diabetes. Novartis (LAF237, vildagliptin, Galvus, (21)) and Merck (MK-0431, sitagliptin, Januvia, (3)) are both under regulatory review with expected commercial launch in 2007. Takeda (Syr-322, (76)) has reported that it is in Phase III trials. BMS (BMS-477118, saxagliptin, (35)), OSI

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Table 2.2 CLINICAL STATUS OF ADVANCED DPPIV INHIBITORS Code

Product

Generic name

Company

Phase

MK-0431 (3) LAF237 (21) Syr-322 (76) BMS-477118 (35) 823093 (38) PSN 9301

Januvia Galvus

Sitagliptin Vildagliptin

Merck Novartis Takeda Bristol–Myers Squibb GlaxoSmithKline OSI

Phase Phase Phase Phase Phase Phase

Saxagliptin Denagliptin

III III III II II II

Table 2.3 CLINICAL PARAMETERS NVP-DPP728 (20) GLP-1 levels Glucose levels Glucagon levels Insulin levels HbA1c Lipid levels Body weight

k 2 k 2 2

LAF237 (21)

MK-0431 (3)

m k k 2/m k 2 2

m k k m k 2

Symbols: k, decrease; m, increase; 2, little change.

Pharma (PSN 9301) and GlaxoSmithKline (denagliptin, (38)) are currently in Phase II. Additionally, more than 15 companies have DPPIV inhibitors in either Phase I or preclinical development. The majority of human clinical data has been released on three compounds from two companies, Novartis and Merck (Table 2.2). NVP-DPP728 (20) was an early DPPIV inhibitor advanced into clinical trials (Table 2.3). This modestly potent and shorter acting inhibitor was pivotal in establishing the utility of a DPPIV inhibitor in improving metabolic control [139]. The outcomes reported in the cited 4-week study were both encouraging and provocative in demonstrating the feasibility of DPPIV inhibition for the treatment of early-stage type 2 diabetes. Briefly, 93 patients with mild type 2 diabetes (BMI ¼ 27.3 kg/m2, HbA1c ¼ 7.4%) were treated with placebo or NVP-DPP728 at 100 mg t.i.d. or 150 mg b.i.d. for 4 weeks. Prior to treatment and then again at the end of the study period, patients were administered standardized meals, and then glucose and insulin levels were monitored for 24 h. Glycemic control was improved as determined by a reduction in fasting glucose levels, reduction in 4-h prandial glucose excursion and reduction in the 24-h glucose profile. Although not an efficacy parameter of the study, HbA1c levels were reduced by 0.6% over the 4-week treatment. Interestingly, 24-h insulin levels were also reduced in both

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DPPIV INHIBITION

treatment groups. To rationalize this apparent disparity between glycemic control and insulin levels, the study authors speculate that GIP and GLP-1 may have enhanced b-cell sensitivity to insulin. Alternative mechanisms resultant from enhanced GLP-1 activity included slowed gastric emptying or inhibited glucagon secretion that result in lower plasma glucose levels. A minor reduction in total cholesterol, VLDL cholesterol and triglycerides was observed. Adverse events observed during the study appeared to be predominantly short-lived with continued therapy. This study showed the potential of DPPIV inhibition as monotherapy for the treatment of early type 2 diabetes. Novartis has turned to the development of a more potent longer acting DPPIV inhibitor, LAF237 (vildagliptin, (21)) [140]. The first study was similar in design to the one just described above. Following a 4-week run-in period, 37 patients (BMI ¼ 20–32 kg/m2, HbA1c ¼ 6.3–10%) were randomized into either a placebo group or a group receiving 100 mg q.i.d. of (21) 30 min prior to breakfast. As described above, the patients were evaluated at weeks 0 and 4 over a 24-h period while consuming a standardized meal. DPPIV activity was greatly diminished by (21). Nearly complete abolition (98%) of enzymatic activity was observed 45 min after drug administration. DPPIV enzymatic activity remains low for nearly 12 h and then rebounded to approximately 60% of baseline activity after 24 h. GLP-1 levels were increased after 4 weeks of treatment both at baseline and postprandially. (Peak levels approximately doubled compared to placebo 30 min after a meal.) As observed with NVP-DPP728, (21) also showed enhanced glycemic control with reduced fasting glucose levels, reduced mean 4-h prandial glucose and reduced peak glucose levels. This improvement in glycemic control resulted in an HbA1c reduction of 0.38% relative to the placebo group. Again as observed with NVP-DPP728, there was no effect on insulin levels at fasting, in response to a meal, or over the 24-h study period. However, glucagon reduction with response to meals was observed with a correlation noted between the 1-h glucagons levels and the 2-h glucose levels after 4 weeks of (21) treatment. Neither lipid profiles nor body weight changed significantly over the course of the study and (21) was well tolerated. No hypoglycemic events occurred, and other adverse events were sufficiently mild so that no treatment was discontinued. Once again, the study demonstrated the efficacy of a DPPIV inhibitor as monotherapy in the treatment of early type 2 diabetes with reduced levels of glucose and glucagon, and stable levels of insulin, lipids and body weight. Continued evaluation of (21) in combination therapy with metformin for up to 1 year has solidified DPPIV inhibition as a potential treatment for early-stage type 2 diabetics [141]. In brief, 107 patients (BMI ¼ 20–35 kg/m2, HbA1c ¼ 7.0–9.5%) on stable metformin therapy (1.5–3) were randomized

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after a run-in period and received either placebo or LAF237 (50 mg, q.i.d.) for 12 weeks in addition to their continued metformin. At that time, approximately 90% of the patients agreed to participate in an extension study of 40 additional weeks. At the end of the 12-week study, both fasting plasma glucose and prandial glucose levels were reduced in the group receiving (21) versus the placebo control. HbA1c levels decreased by 0.7%. Insulin levels, lipid profiles and body weight did not change significantly over the 12-week study. At the end of the extension period, glycemic control was maintained in the (21) treatment group. Both fasting plasma glucose levels and 4-h AUCs following a meal challenge continued to decline in the (21) group while the placebo group values increased over the same period. Perhaps most interesting, the HbA1c levels of the placebo group rose between weeks 12 and 52 at a rate of 0.0656% per month while the (21) treatment group exhibited a rate of only 0.0128% per month, resulting in very little change in HbA1c over the 40 weeks. The difference in HbA1c between the placebo plus metformin and (21) plus metformin groups was 1.1%. Prandial insulin for the (21) treatment group increased somewhat compared to baseline while the placebo group decreased. Lipid profiles remained unchanged with the exception of a slight rise in total cholesterol, and body weight decreased very slightly with both the (21) and placebo groups losing 0.2 kg. Control of body weight may be an important contribution of incretin modulation therapy. Certainly, this study of patients receiving (21) and metformin gave a body weight neutral result. Similar co-therapy studies with the GLP-1 receptor agonist, Exenatide, and either metformin or sulfonylureas showed a dosedependent sustained weight loss over the 30-week study period [142]. The adverse events reported with (21) and metformin 52-week co-therapy included some mild hypoglycemic episodes and one case of peripheral oedema. Other reported adverse events were not believed to be drug related. With no deterioration in efficacy over time, the study authors suggest that the (21) and metformin co-therapy may be modifying the course of disease progression, but further studies are required to verify this. A dose-ranging study was carried out on LAF237 (21) [143]. In this study, a 4-week placebo run-in period was followed by patient randomization into one of five groups: 25 mg of (21) twice daily; 25, 50 or 100 mg once daily; or placebo. The dosing regimen was then followed for 12 weeks. Similar reductions of HbA1c were noted in the 50 and 100 mg groups relative to placebo controls. Four-hour postprandial glucose reduction was seen with the 50 mg dose. At the 100 mg dose, an increase in insulin level was noted, as was an increase in b-cell function (HOMA-B). The patients with the highest baseline HbA1c level showed the greatest improvement over the study period. Few side effects were noted with the greatest variance from placebo being a greater number of headaches in the 100 mg dose group. As

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DPPIV INHIBITION

in earlier studies, lipid levels and weight were unchanged over the course of the study. Two studies address the apparent disconnect between improved responses to nutrient exposure, while insulin levels remain essentially unchanged in type 2 diabetic patients on (21). The first of these was a study similar to the one previously described in which type 2 diabetic patients on metformin were treated either with placebo or (21) for an initial core period of 12 weeks followed by a voluntary 40-week extension [144]. The study conclusion was that b-cell function was enhanced most significantly in the initial 12-week exposure to the DPPIV inhibitor, but continued out through the yearlong study. In support of this, meal-related insulin secretion was enhanced over the initial 12 weeks and then maintained over the course of study. Insulin sensitivity improved throughout as well. An adaption index analysis suggested that b-cell function was improved independently of changes in insulin sensitivity. A similar study over a 4-week period in which patients were dosed twice daily with 100 mg of (21) observed a decrease in day-long glucose levels, a decrease in glucagon and an increase in plasma levels of both GLP-1 and GIP [145]. The study conclusion was that b-cell function was improved as noted by increased insulin secretion at any given glucose level. Merck has also advanced an orally active DPPIV inhibitor into human clinical trials. Results of a proof-of-concept study of MK-0431 (3) were released [146]. Fifty-six type 2 diabetic patients (mean HbA1c 8.3%) on diet and exercise treatments were randomized into a three-period crossover study. After an overnight fast, each patient received a single oral dose of either placebo or 25 or 200 mg of (3). Two hours later an OGTT was administered. All results were reported relative to the placebo controls. Incremental glucose AUCs decreased by 22 and 26% for the 25 and 200 mg dose groups, respectively. Plasma insulin AUCs increased by 22 and 23% while glucagon decreased by 8 and 14% in the 25 and 200 mg groups, respectively. Plasma C-peptide AUCs also increased. Levels of active GLP-1 doubled, as did the active to total GLP-1. The lack of observed dose responsiveness was attributed to the study being conducted at or near the Cmax. Certainly, this study confirmed the potential of (3) for the treatment of type 2 diabetes. A study of the pharmacokinetics and pharmacodynamics of (3) was performed on healthy male patients [147]. The patients received single oral doses ranging between 1.5 and 600 mg.MK-0431 (3) was well absorbed and subsequently nearly 80% was renally excreted unchanged. The renal clearance of 388 ml/min was in excess of the normal rate of 125 ml/min and was suggestive of an active renal clearance mechanism. The AUC increased in a dose-dependent manner, and the half-life ranged from 8 to 14 h, suitable for once daily administration. Little effect was noted with meals. A 50 mg or greater dose was required to inhibit 80% of DPPIV activity for 12 h, while a

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dose of 100 mg or more produced the same level of inhibition for a 24-h period. GLP-1 levels were doubled following a meal at doses at or above 12.5 mg. There was no effect noted on the levels of glucose, insulin, glucagon or C peptide over a 2-h period following a meal in these healthy patients. Adverse events were considered mild to transient and were resolved without treatment. Notably, no hypoglycemia was observed. The route of excretion of (3) is primarily renal. Type 2 diabetic patients with renal insufficiency would be expected to have increased drug exposure. This is in fact the case with an increase of 2.3-, 3.8- and 4.5-fold for patients with moderate renal impairment, severe renal impairment or on dialysis, respectively [148]. A reduction in the daily dose to 50 mg (moderate) or 25 mg (severe or dialysis) produced clinical results comparable to patients with normal renal function and receiving daily 100 mg doses. Adverse events were somewhat higher for the drug versus placebo groups. A second 10-day multiple dosing study in healthy male subjects confirmed the above results [149]. Steady state was achieved in approximately 3 days, and minimal accumulation was noted. Again, a daily dose of 100 mg or greater inhibited DPPIV enzymatic activity by at least 80% over a 24-h interval. Following a standardized meal, GLP-1 levels were doubled consistent with maintenance of endogenous GLP-1 rather than increase of GLP-1 secretion. Adverse events were mild and transient, and again, no hypoglycemic episodes were seen. Several similar durability studies were recently reported involving (3) over 12, 18 and 24 weeks, respectively [150–152]. To cite the data from the 24week study, type 2 diabetic patients were administered either 100 or 200 mg daily. The HbA1c reductions at study end were 0.79 and 0.94% for the two respective dose groups. Two-hour post-meal glucose response was reduced by approximately 50 mg/dl in the two groups. Increases were observed in prandial insulin, C-peptide AUC, ratio of insulin AUC/glucose AUC and HOMA-B. By contrast, fasting insulin, C-peptide and proinsulin did not vary from the placebo group. There was no effect on body weight.

CONCLUSIONS To bring this discussion full circle, the introduction defined diabetes as a major health issue in the developed and developing countries. DPPIV inhibitors will be the next new class of drug approved to treat type 2 diabetes. This represents both the culmination and continuation of research by numerous institutions in every continent. Nearly every large pharma and multiple biotechs have had or continue to have research directed to inhibition of this enzymatic target. Structural biology, particularly through

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DPPIV INHIBITION

co-crystallization studies, has sped the iterative drug discovery process. Biochemistry knowledge and understanding within this enzymatic family has continued to grow. Certainly, the issue of selectivity continues to be a differentiator between inhibitors. In several instances, it was noted that toxicity associated with early inhibitors is quite likely associated with offtarget enzymatic inhibition. In particular, selectivity against DPP8 and DPP9 appears to be important to achieve. The remaining question is how much selectivity is required for safety in what will be a chronic therapy. Such issues are and will be sorted out in clinical trials. It is an exciting time in the field of DPPIV research. Two drug candidates, vildagliptin (21) and sitagliptin (3), are currently undergoing regulatory review. These represent the inhibitors that interact covalently and noncovalently with the target enzyme, respectively. They each appear suitable for once-a-day dosing. Clinical targets have been met throughout the clinical trials. Fasting and prandial glucose levels have been lowered. HbA1c levels, the principal biomarker for glucose regulation, accordingly have also been lowered as required for treatment of type 2 diabetes. The DPPIV inhibitors are capable of co-therapy, at least with metformin and thiazolidinediones. A differentiator with other therapies is the weight neutrality so far observed in long-term studies. Perhaps most encouraging for this new therapy class is that it continues to show promise of disease modification. This appears primarily in the health or tone of the b-cells that improves over time with treatment. The understanding of the mechanisms of incretin action continues to evolve. The drugs appear to be well tolerated with few side effects. We eagerly look forward to the commercial launch of the first dipeptidyl peptidase IV inhibitors as well as continued research in this exciting field.

AUTHORS NOTE Merck’s sitagliptin ((3), Januvia) was approved for the treatment of type 2 diabetes by the U.S. Food and Drug Administration in October 2006. Sitagliptin may be used as monotherapy or in cotherapy with either metformin or thiazolidinediones. REFERENCES [1] International Diabetes Federation Diabetes Atlas, http://www.idf.org/home/ (Accessed 5/28/06). [2] Inzucchi, S.E. (2002) JAMA 287, 360–372. [3] Mojsov, S., Weir, G.C. and Habener, J.F. (1987) J. Clin. Invest. 79, 616–619.

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[4] Kreymann, B., Williams, G., Ghatei, M.A. and Bloom, S.R. (1987) Lancet 2(8571), 1300–1304. [5] Fehmann, H.C. and Habener, J.F. (1992) Endocrinology 130, 159–166. [6] Xu, G., Stoffers, D.A., Habener, J.F. and Bonner-Weir, S. (1999) Diabetes 48, 2270–2276. [7] Weir, G.C., Mojsov, S., Hendrick, G.K. and Habener, J.F. (1989) Diabetes 38, 338–342. [8] Larsson, H., Holst, J.J. and Ahren, B. (1997) Acta Physiol. Scand. 160(4), 413–422. [9] Schirra, J., Katschinski, M., Weidmann, C., Schaefer, T., Wank, U., Arnold, R. and Goeke, B. (1996) J. Clin. Invest. 97, 92–103. [10] Wettergren, A., Schjoldager, B., Mortensen, P.E., Myhre, J., Christiansen, J. and Holst, J.J. (1993) Dig. Dis. Sci. 38, 665–673. [11] Zander, M., Madsbad, S., Madsen, J.L. and Holst, J.J. (2002) Lancet 359(9309), 824–830. [12] Turton, M.D., O’Shea, D., Gunn, I., Beak, S.A., Edwards, C.M.B., Meeran, K., Choi, S.J., Taylor, G.M., Heath, M.M., Lambert, P.D., Wilding, J.P.H., Smith, D.M., Ghatei, M.A., Herbert, J. and Bloom, S.R. (1996). Nature (London) 379(6560), 69–72. [13] Gutniak, M.K., Svartberg, J., Hellstrom, P.M., Holst, J.J., Adner, N. and Ahren, B. (2001) J. Intern. Med. 250, 81–87. [14] Rachman, J., Barrow, B.A., Levy, J.C. and Turner, R.C. (1997) Diabetologia 40, 205–211. [15] Gutniak, M., Orskov, C., Holst, J.J., Ahren, B. and Efendic, S. (1992) N. Engl. J. Med. 326, 1316–1322. [16] Nauck, M., Sto¨ckmann, F., Ebert, R. and Creutzfeldt, W. (1986) Diabetologia 29, 46–52. [17] Deacon, C.F., Nauck, M.A., Toft-Nielsen, M., Pridal, L., Willms, B. and Holst, J.J. (1995) Diabetes 44, 1126–1131. [18] Mentlein, R., Gallwitz, B. and Schmidt, W.E. (1993) Eur. J. Biochem. 214, 829–835. [19] Egan, J.M., Meneilly, G.S. and Elahi, D. (2003) Am. J. Physiol. 284, E1072–E1079. [20] Riddle, M.C. and Drucker, D.J. (2006) Diabetes Care 29, 435–449. [21] Byetta patient information: http://www.byetta.com. [22] Meier, J.J., Nauck, M.A., Schmidt, W.E. and Gallwitz, B. (2002) Regul. Pept. 107, 1–13. [23] Nauck, M.A., Heimesaat, M.M., Orskov, C., Holst, J.J., Ebert, R. and Creutzfeldt, W. (1993) J. Clin. Invest. 91, 301–307. [24] Lynn, F.C., Pamir, N., Ng, E.H.C., McIntosh, C.H.S., Kieffer, T.J. and Pederson, R.A. (2001) Diabetes 50, 1004–1011. [25] Hopsu-Havu, V.K. and Sarimo, S.S. (1967) Hoppe-Seyler’s Z. Physiol. Chem. 348, 1540–1550. [26] Mentlein, R. (1999) Reg. Pept. 85, 9–24. [27] Marguet, D., Baggio, L., Kobayashi, T., Bernard, A.-M., Pierres, M., Nielsen, P.F., Ribel, U., Watanabe, T., Drucker, D.J. and Wagtmann, N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 6874–6879. [28] Nagakura, T., Yasuda, N., Yamazaki, K., Ikuta, H., Yoshikawa, S., Asano, O. and Tanaka, I. (2001) Biochem. Biophys. Res. Commun. 284, 501–506. [29] Hansotia, T., Baggio, L.L., Delmeire, D., Hinke, S.A., Yamada, Y., Tsukiyama, K., Seino, Y., Holst, J.J., Schuit, F. and Drucker, D.J. (2004) Diabetes 53, 1326–1335. [30] Zhu, L., Tamvakopoulos, C., Xie, D., Dragovic, J., Shen, X., Fenyk-Melody, J.E., Schmidt, K., Bagchi, A., Griffin, P.R., Thornberry, N.A. and Roy, R.S. (2003) J. Biol. Chem. 278, 22418–22423. [31] Ahreń, B. and Hughes, T.E. (2005) Endocrinology 146, 2055–2059.

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[135] Ashton, W.T., Sisco, R.M., Dong, H., Lyons, K.A., He, H., Doss, G.A., Leiting, B., Patel, R.A., Wu, J.K., Marsilio, F., Thornberry, N.A. and Weber, A.E. (2005) Bioorg. Med. Chem. Lett. 15, 2253–2258. [136] Gwaltney, S.L., Aertgeerts, K., Feng, J., Kaldor, S.W., Kassel, D.B., Manuel, M., Navre, M., Prasad, G.S., Shi, L., Skene, R.J., Stafford, J.A., Wallace, M., Xu, R., Ye, S., Zhang, Z. and Webb, D.R. (2006) 231st A.C.S. National Meeting, Atlanta, GA,USA, MEDI-018. [137] Kanstrup, A.B., Sams, C.K., Lundbeck, J.M., Christiansen, L.B. and Kristiansen, M. (2003) PCT Int. Appl. WO 03 004496. [138] Christopher, R.J., Davenport, J.M., Gwaltney, S., Kaldor, S., Kassel, D., Lee, B., Navre, M., Shi, L., Stafford, J., Xu, R. and Zhang, Z. (2006) 66th American Diabetes Association Scientific Sessions, Washington, DC, USA, Poster 452-P. [139] Ahreń, B., Simonsson, E., Larsson, H., Landin-Olsson, M., Torgeirsson, H., Jansson, P., Sandqvist, M., Bavenholm, P., Efendic, S., Eriksson, J.W., Dickinson, S. and Holmes, D. (2002) Diabetes Care 25, 869–875. [140] Ahreń, B., Landin-Olsson, M., Jansson, P., Svensson, M., Holmes, D. and Schweizer, A. (2004) J. Clin. Endocrin. Metab. 89, 2078–2084. [141] Ahreń, B., Gomis, R., Standl, E., Mills, D. and Schweizer, A. (2004) Diabetes Care 27, 2874–2880. [142] Buse, J.B., Henry, R.R., Han, J., Kim, D.D., Fineman, M.S. and Baron, A.D. (2004) Diabetes Care 27, 2628–2635. [143] Ristic, S., Byiers, S., Foley, J. and Holmes, D. (2005) Diabetes, Obes. Metab. 7, 692–698. [144] Ahreń, B., Pacini, G., Foley, J.E. and Schweizer, A. (2005) Diabetes Care 28, 1936–1940. [145] Mari, A., Sallas, W.M., He, Y.L., Watson, C., Ligueros-Saylan, M., Dunning, B.E., Deacon, C.F., Holst, J.J. and Foley, J.E. (2005) J. Clin. Endocrin. Metab. 90, 4888–4894. [146] Herman, G.A., Zhao, P.-L., Dietrich, B., Golor, G., Schrodter, A., Keymeulen, B., Lasseter, K.C., Kipnes, M.S., Hilliard, D., Tanen, M., De Lepeleire, I., Cilissen, C., Stevens, C., Tanaka, W., Gottesdiener, K.M. and Wagner, J.A. (2004) Diabetes 53(Suppl. 2), A82 (Abstract 353-OR) [147] Herman, G.A., Stevens, C., Van Dyck, K., Bergman, A., Yi, B., De Smet, M., Snyder, K., Hilliard, D., Tanen, M., Tanaka, W., Wang, A.Q., Zeng, W., Musson, D., Winchell, G., Davies, M.J., Ramael, S., Gottesdiener, K.M. and Wagner, J.A. (2005) Clin. Pharmacol. Ther. 78, 675–688. [148] Scott, R., Hartley, P., Luo, E., Yee, J., Davies, M., Ferreira, J.C.A. and WilliamsHerman, D. (2006) 66th American Diabetes Association Scientific Sessions, Washington, DC, USA, Poster 1997-P. [149] Bergman, A.J., Stevens, C., Zhou, Y., Yi, B., Laethem, M., De Smet, M., Snyder, K., Hilliard, D., Tanaka, W., Zeng, W., Tanen, M., Wang, A.Q., Chen, L., Winchell, G., Davies, M.J., Ramael, S., Wagner, J.A. and Herman, G.A. (2006) Clin. Ther. 28, 55–72. [150] Nonaka, K., Kakikawa, T., Sato, A., Okuyama, K., Fujimoto, G., Hayashi, N., Suzuki, H., Hirayama, Y. and Stein, P. (2006) 66th American Diabetes Association Scientific Sessions, Washington, DC, USA, Poster 537-P. [151] Raz, I., Hanefeld, M., Xu, L., Caria, C., Davies, M., Williams-Herman, D. and Khatami, H. (2006) 66th American Diabetes Association Scientific Sessions, Washington, DC, USA, Poster 1996-P. [152] Aschner, P., Kipnes, M., Lunceford, J., Mickel, C., Davies, M. and Williams-Herman, D. (2006) 66th American Diabetes Association Scientific Sessions, Washington, DC, USA, Poster 1995-P.

3 Recent Progress Toward Nonpeptide Ligands for the Melanocortin-4 Receptor CHEN CHEN Department of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA

INTRODUCTION Melanocortin Receptors

112 112

CYCLOHEXYLPIPERIDINES AS POTENT MC4R AGONISTS Tetrahydroisoquinoline (THIQ) and Analogues MB243 and Related Compounds Other THIQ-Related Compounds

115 115 121 125

CYCLOHEXYLPIPERAZINES AND PHENYLPIPERIDINES

126

PHENYLPIPERAZINES AS MC4R AGONISTS

128

OTHER CLASSES OF MC4R AGONISTS

133

COMPOUNDS MIMICKING a-MSH AND ITS PEPTIDE ANALOGUES

135

FROM AGONISTS TO ANTAGONISTS

137

SMALL MOLECULE MC4R ANTAGONISTS FROM INITIAL SCREEN HITS

141

OTHER NONPEPTIDE MC4R LIGANDS

144

INTERACTION OF THE MC4R WITH LIGANDS Structural Information of Melanocortin Peptides Small Peptide Ligands with HFRW Variants Conformations of Peptide Ligands Receptor Structure Information through Mutagenesis Studies Computational Modeling of the Human MC4R

145 145 146 148 149 152

Progress in Medicinal Chemistry – Vol. 45 Edited by F.D. King and G. Lawton DOI: 10.1016/S0079-6468(06)45503-X

111


112

NONPEPTIDE LIGANDS FOR THE MELANOCORTIN-4 RECEPTOR

Possible Interaction of Nonpeptide Ligands with the MC4-Receptor

153

MEDICINAL CHEMISTRY

155

CONCLUSION

158

REFERENCES

159

INTRODUCTION Recent studies have demonstrated that melanocortin-4 receptor (MC4R) plays a major role in regulating some important biological functions including energy homeostasis. MC4 receptors are widely expressed in the brain and are regulated by the endogenous peptide agonists, i.e., melanocyte-stimulating hormones (a-, b- and g-MSH), and the antagonist/inverse agonist, i.e., agouti-related protein (AgRP). Peptide or protein ligands of this receptor have difficulty in crossing the blood–brain barrier (BBB) to be delivered to the site of action. Efforts to develop potent and selective agonists and antagonists of the human MC4 receptor (hMC4R) have led to the discovery of several classes of dipeptide and nonpeptide ligands that have high potency and selectivity over other melanocortin receptor subtypes. Because of their low molecule weights, some of these compounds are able to penetrate into the brain. These selective ligands also provide powerful tools for further delineating the role of the MC4 receptor, in feeding behaviour, erectogenic activity, pain, and anxiety/depression. In addition, several key amino acid residues of the receptor, which are involved in the interaction with peptide and nonpeptide ligands, have been identified by mutagenesis studies. Understanding these interactions should help medicinal chemists to design ligands with desirable physicochemical, pharmacological, and pharmacokinetic (PK) properties, and, eventually, safe compounds for clinical studies to test biological activity in human diseases. This review focuses on recent progress in the discovery of nonpeptide agonists and antagonists, including their structure–activity relationships (SARs), PK properties, and interactions with the receptor. The biological activity in in vivo animal models associated with some key compounds will also be discussed.

MELANOCORTIN RECEPTORS

The melanocortin system is composed of five subtypes of melanocortin receptors, MC1-5R, that belong to the class A G-protein-coupled receptor (GPCR) superfamily [1, 2]. All five receptors from several species including

C. CHEN

113

human (Figure 3.1) have been identified and cloned [3]. The MC1R is known for regulating skin pigmentation and the immune system; the MC2R (ACTH receptor) controls cortico-steroid production; the MC3R might be involved in the regulation of sexual behaviour; the MC4R controls feeding and energy homeostasis; and the MC5R has a role in regulating exocrine gland secretion [4]. The melanocortin peptides are the endogenous agonists for the melanocortin receptors and consist of MSHs (a-, b-, and g-MSH) and adrenocorticotropin (ACTH), a family of peptides produced from the posttranslational processing of pro-opiomelanocortin (POMC) [5]. Interestingly, the melanocortin system has two additional members: agouti-protein [6] and AgRP [7], which are structurally different from the melanocortin peptides and function as antagonists or inverse agonists at the melanocortin receptors. All the melanocortin peptides possess a ‘‘His-Phe-Arg-Trp’’ (HFRW) motif (Figure 3.2), which has been proven to be crucial for the activation of the melanocortin receptors [8], whereas agouti-protein and AgRP have an ArgPhe-Phe unit located in a loop known to interact with the melanocortin receptors [9], and believed to be important for functional antagonism [10]. Although strong evidence suggests that MC4R activation by a-MSH and synthetic agonists such as NDP-MSH and MT-II (Figure 3.2) has anorexigenic effects [11–13], and may have application in the treatment of obesity [14–16], recent studies have shown that MC4R agonists could also promote erectogenic activity [17, 18]. For example, clinical data have demonstrated that PT-141, a peptide melanocortin agonist, is efficacious in erectile dysfunctional patients [19]. The physiological importance of a balance within the central melanocortin system has been further demonstrated in MC4R knockout [20] and AgRP overexpression mouse [21], where hyperphagia and obesity are the most obvious aspects of the phenotypes observed. Studies have showed that MC4R antagonists effectively prevent reduction of food intake and weight loss in tumour-bearing mice [22], and MC4R knockout mice resist the tumour-induced loss of lean body mass [23]. These results in animal models indicate the potential utility of MC4R antagonists for the clinical treatment of cachexia [24–27], which would be very significant since this wasting syndrome occurs in a large population of cancer patients [28]. Other studies have also showed that blockade of the MC4R signaling pathway may have effects on anxiety [29], cocaine reward [30], morphine tolerance [31], ethanol intake [32], and pain [33, 34]. Therefore, MC4R antagonism may have an important role in related clinical disorders [35]. Research efforts from academic institutes and pharmaceutical companies have led to the discovery of potent and selective nonpeptide MC4R agonists and antagonists from several chemical classes. In addition, small peptide ligands with good selectivity have also been identified. SAR studies of

114


OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R OPSD MC1R MC2R MC3R MC4R MC5R

43

80

120

164

187

238

282

N-Term MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPW LGSLNSTPTAIPQLGLAANQTGARCL MKHIINSYENINNTARNNSDCP QPTLPNGSEHLQAPFFSNQSSSAFCE NRSSYRLHSNASESLGKGYSDGGCYE FLDLNLNATEGNLSGPNVKNKSSPCE TM-1 IL-1 QFSMLAAYMFLLIMLGFPINFLTLYVTV QHKKLRTPL EVSISDGLFLSLGLVSLVENALVVATIA KNRNLHSPM RVVLPEEIFFTISIVGVLENLIVLLAVF KNKNLQAPM QVFIKPEVFLSLGIVSLLENILVILAVV RNGNLHSPM QLFVSPEVFVTLGVISLLENILVIVAIA KNKNLHSPM DMGIAVEVFLTLGVISLLENILVIGAIV KNKNLHSPM TM-2 EL-1 NYILLNLAVADLFMVFGGFTTTLYTSLH GYFVFGP YCFICCLALSDLLVSGSNVLETAVILLL EAGALVARAAVLQ YFFICSLAISDMLGSLYKILENILIILR NMGYLKPRGSFET YFFLCSLAVADMLVSVSNALETIMIAIV HSDYLTFEDQFIQ YFFICSLAVADMLVSVSNGSETIVITLL NSTDTDAQSFTV YFFVCSLAVADMLVSMSSAWETITIYLL NNKHLVIADAFVR TM-3 IL-2 TGCNLEGFFATLGGEIALWSLVVLAIERYVVV CKPMSNFRFGE QLDNVIDVITCSSMLSSLCFLGAIAVDRYISI FYALRYHSIVTL TADDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVTM HMDNIFDSMICISLVASICNLLAIAVDRYVTI FYALRYHSIMTV NIDNVIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMTV HIDNVFDSMICISVVASMCSLLAIAVDRYVTI FYALRYHHIMTA TM-4 EL-2 NHAIMGVAFTWVMALACAAPPLVG WSRYIPEGMQCSCGIDYYTPHEETN PRARRAVAAIWVASVVFSTLFIAY Y RRTVVVLTVIWTFCTGTGITMVIF S RKALTLIVAIWVCCGVCGVVFIVY S KRVGIIISCIWAACTVSGILFIIY S RRSGAIIAGIWAFCTGCGIVFILY S TM-5 IL-3 NESFVIYMFVVHFIIPLIVIFFCYGQLV FTVKEAAAQQQESATTQ DHVAVLLCLVVFFLAMLVLMAVLYVHML ARACQHAQGIARLHKRQRPVHQ HHVPTVITFTSLFPLMLVFILCLYVHMF LLARSHTRKISTLP ESKMVIVCLITMFFAMMLLMGTLYVHMF LFARLHVKRIAALPPADGVAPQQ DSSAVIICLITMFFTMLALMASLYVHMF LMARLHIKRIAVLPGTGAIRQ ESTYVILCLISMFFAMLFLLVSLYIHMF LLARTHVKRIAALPGASSARQ TM-6 EL-3 KAEKEVTRMVIIMVIAFLICWLPYAGVAFYIF THQGSDFGP GFGLKGAVTLTILLGIFFLCWGPFFLHLTLIV LCPEHPTCGCIF RANMKGAITLTILLGVFIFCWAPFVLHVLLMT FCPSNPYCACYM HSCMKGAVTITILLGVFIFCWAPFFLHLVLII TCPTNPYCICYT GANMKGAITLTILIGVFVVCWAPFFLHLIFYI SCPQNPYCVCFM RTSMQGAVTVTMLLGVFTVCWAPFFLHLTLML SCPQNLYCSRFM TM-7 C-Term IFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTL CCGKNPLGDDEASTTVSKTETSQVAPA KNFNLFLALIICNAIIDPLIYAFHSQELRRTLKEVL TCSW SLFQVNGMLIMCNAVIDPFIYAFRSPELRDAFKKMI FCSR AHFNTYLVLIMCNSVIDPLIYAFRSLELRNTFREIL CGCNGMNLG SHFNLYLILIMCNSIIDPLIYALRSQELRKTFKEII CCYPLGGLCDLSSRY SHFNMYLILIMCNSVMDPLIYAFRSQEMRKTFKEII CCRGFRIACSFPRRD

Fig. 3.1 Alignment of human melanocortin receptors (MC1-5R) with the bovine rhodopsin. The conserved residues in the transmembrane domains are highlighted.

C. CHEN α-MSH:

115

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

NDP-MSH: Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2

NHAc O

H N

O

NH O

O

O HN

H2N HN

HN

O

O

N H HN H2N

H N

N

HN

HN

H2 N

O

HN

HN

NH

O

O

N H

O H N

HN O

Ar

H2N

HN

O

O

NH

NH

MTII: Ar = Phenyl SHU9119: Ar = 2'-Naphthyl

MBP10

Fig. 3.2 Some important peptide MC4R ligands.

peptide ligands from the last 30 years have been comprehensively reviewed by Holder and Luevano [36]. Recent progress in the SAR of nonpeptide ligands is also summarized in recent review articles [37–40], and the therapeutic potential of MC4R ligands is discussed by Getting [41]. This review will focus on the properties of some key nonpeptide ligands, including their pharmacology, PK, and in vivo activities. In addition, progress on the understanding at the molecular level of ligand-receptor interactions based on receptor modeling and mutagenesis will also been discussed.

CYCLOHEXYLPIPERIDINES AS POTENT MC4R AGONISTS TETRAHYDROISOQUINOLINE (THIQ) AND ANALOGUES

Potent and selective dipeptide MC4R agonists exemplified by (1c) were first reported by Nargund and co-workers at Merck in 2001 [42]. Compound (1c) is an optimized analogue of the initial lead compounds (1a) and (1b) which were designed by using a privileged structure spiro-sulfonylindoline to mimic the Arg-Trp unit of HFRW motif [43, 44]. Interestingly, (1a) binds to the MC4R with a Ki of 108 nM, but without the ability to stimulate cAMP

116


release in CHO cells expressing the hMC4R. Thus, the profile of (1a) matches with that of SHU9119, a peptide antagonist that also has a naphthyl group at a key position (Figure 3.2). On the other hand, the phenyl analogue (1b) has moderate potency and high efficacy in stimulating cAMP production, which resembles the property of peptide agonists such as MT-II (Figure 3.2). The 4-methoxyphenylalanine compound (1d) is slightly less potent than its 4-chloro analogue (1c) (Table 3.1). It is worth noting that privileged structures similar to sulfonylindoline at the left-side of (1) have been used in ligands for other GPCRs such as the somatostatin [45] and ghrelin receptors [46]. X H N

Ar

Me

S N O O

N

O N O

Me O

N H

S N O

O N

(1a) Ar = 2-naphthyl (1b) Ar = phenyl

(1c) X = Cl, Ki = 10 nM (1d) X = MeO, Ki = 17 nM, EC50 = 59 nM

X

Cl

O N N

N O

N

1 N H

O 3

N N

HN

N

N O

Cl

HN

F

O N N N R

N H

(2a) X = MeO (2b) X = F;

(3a) 1R, 3R (THIQ); (3b) 1R, 3S (3c) 1S, 3R; (3d) 1S, 3S

N N

N H HN

O

NH2

O

N H

HN

(4a) R = H (4b) R = 1-Me; (4c) R = 2-Me

O tBu

O NH

2

N O

1 N H

N HN

(5a) 1R, 2S (MB243) (5b) 1S, 2S; 5c: 1R, 2R

Me

C. CHEN

117

Table 3.1 PHARMACOLOGICAL PROPERTIES OF CYCLOHEXYPIPERIDINES Compound

IC50 (nM)

EC50 (nM)

IA (%)a

1a 1b 1c 1d 2a 2b 3a (THIQ) 3b 3c 3d 4a 4b 4c 5a (MB243) 5b 5c 6a 6b 6c 6d 6e 7a 8a (RY764) 8b 9a 9b 10a 11 12

108 n/a 10 17 0.8 0.77 1.2 0.5 3,050 127 238 0.3 1.2 16 6,500 52 18 20 13 8 30 6 8 6 0.37 1.1 0.62 14 305

— 500

97

a b

59 14 27 2.1 8.7 — — 771 0.6 9.6 11 — 82 13 12 52 34 270 8 11 34 1.9 1.5 5.8 2 9.5

48 37 97 67 9 34 79 103 87 87 29 92 99 90 50 55 64 79 81 58 81 108 74 104

Ref.

No.b

42 42 42 42 47 47 47 47 47 47 47 47 47 56 56 56 56 56 56 56 56 59 60 60 61 61 63 64 65

3 4 n/a n/a n/a n/a 1 13 14 15 5 6 7 MB243 10 11 9 13 14 15 16 8 3 22 2 15 27 n/a 4

Intrinsic activity (% of a-MSH level). Compound number in original publications.

A well-known MC4R agonist THIQ (3a) is evolved from (1c) by replacing the sulfonylindoline with the 4-cyclohexyl-4-(1-triazolylmethyl)piperidine group. In vitro, (3a) possesses an IC50 value of 1.2 nM, which is 634- and 272-fold selective over the MC3 and MC5 receptors, respectively, in the binding assays. The agonist potency of (3a) is determined in cAMP release assays using CHO cells expressing the relevant receptors and the EC50 values are 2,487, 2.1 and 736 nM, respectively, for the MC3, MC4, and MC5 receptors, exhibiting high selectivity [47]. Two of its close analogues display low agonist efficacy but similar binding affinity. Thus, 4-methoxyphenylalanine derivative (2a) has an IC50 of 0.8 nM and an EC50 of 14 nM, but its intrinsic activity (IA) is only 48% of the a-MSH level. The 4-fluoro analogue (2b) is even less efficacious (IA ¼ 37%) despite its high affinity

118


(IC50 ¼ 0.77 nM), suggesting that the phenyl ring of the D Phe variant of (2) and (3) is critically involved in receptor activation. Although (3a) possesses the (R)-chirality in both Phe- and Tic amino acids, its three stereoisomers (3b–d) are either less potent or less efficacious. The (R)-(4-Cl)Phe derivatives (3a) and (3b) exhibit much higher binding affinity than their antipodes (3c) and (3d), indicating stereo-preference at this site. The SAR of the tetrazole analogues (4) reported by Sebhat et al. [47] is quite interesting. Although the acidic tetrazole (4a) is much less potent in binding to the MC4 receptor than the neutral N-methylated tetrazoles (4b) and (4c), which have similar or better potency than the triazole (3a), its agonist efficacy is not significantly different from that of (4b) and (4c), suggesting a unique role for the tetrazole in its interaction with the receptor. The PK properties of (3a) have been characterized in rats and dogs, and the results are summarized in Table 3.2. After 1 mg/kg intravenous (i.v.) injection to rats, (3a) exhibits a high total plasma clearance (CL) of 84 ml/ min kg and a moderate volume of distribution at steady state (Vdss ¼ 3.6 L/ kg) considering its high lipophilicity (c log P ¼ 5.3 based on ACD/cLogP software, Table 3.3), which results in a relative short half-life (t1/2) of 0.6 h in this species. Oral absorption of (3a) is fast, time (Tmax) to reach the maximal concentration (Cmax) is 1 h at a 10 mg/kg dose, and its oral bioavailability of 14% is moderate, which could be caused by a first-pass effect due to its high clearance by the liver. In dogs, (3a) also exhibits a high plasma CL of 27.6 ml/min kg, and its volume of distribution (Vdss ¼ 2.8 L/kg) is similar to that in rats. Compound (3a) has 16% oral bioavailability after a 0.5 mg/kg dose (Table 3.2). It is well known that peptide MC4R agonists such as a-MSH and MT-II stimulate erectile activity in a variety of species including man [48, 49]. However, since none of the peptides used in those studies discriminates among melanocortin receptors, it was unclear which subtype mediates these pro-erectile effects. Systemic administration of the selective agonist THIQ (3a) enhances intracavernosal pressure and stimulates erectile activity in rats ex copula. THIQ dose-dependently (1–5 mg/kg, i.v.) increases the total number of erections to an extent comparable or greater than that produced by the D2 agonist apomorphine subcutaneously (s.c.) administered at a 25 mg/kg dose [50]. Intracerebroventricular (i.c.v.) administration of 20 mg THIQ increases the number of reflexive penile erections, whereas administration of both the nonselective endogenous AgRP (5.5 mg, i.c.v.) and the MC4R-selective antagonist MPB10 (1 mg/kg, i.v.) blocks THIQ-induced erectogenesis [51]. These results provide the evidence that the MC4 receptor is responsible for the erectile activity. Cone and co-workers have reported that THIQ is capable of inhibiting feeding behaviour in mice after an i.c.v. injection [52]. Acute (1 h

Table 3.2 PHARMACOKINETIC PROPERTIES OF MC4R LIGANDS Species

i.v.dose (mg/kg)

CL (ml/min.kg)

t1/2 (h)

Vd or Vdss (L/kg)

p.o.dose (mg/kg)

Cmax (mM)

Tmax (h)

3a 3a 5a 5a 5a 8a 8a 8a 9a 9a 11 11 17 19c 22a

Rat Dog Rat Dog Monkey Rat Dog Monkey Rat Dog Rat Dog Mouse Rat Mouse

1 0.2 1 0.5 1 1 0.5 0.5 1 0.5

84 27.6 48.7 5.6 15.7 46.9 6.6 3.4 35 11 33 13 110 43

0.6 1.2 1.6 4.1 1.3 3.2 4.9 4.6 2 3.4 1.4 8.9 1.2 1.7 (0.44)

3.6 2.8 5.4 1.2 1.2 9.6 2.3 0.8 3.9 2.9 3.5 19 8.2 4.5 (19)

10 0.5 5 2 2 20 2 2 4 2

—

1 1.5 1.54 0.75 5 3 1.1 1.3 1.1 2

30 50

1,440

42a 43 45a

Rat Rat Rat

10 5 5

4.6 1.4 5.8

9.5 6.2 29

10 10 10

18 47 117

6.8 mmol 5

24 52 57

0.87 0.91 0.85

AUC (mM.h)

0.196/kg 2.7 1.3

0.1/kg 0.1/kg 3

0.5 6 2.8

F (%)

Formulation

14 16 9 17 11 32 50 16 11 42 13 6

EtOH:PEG:saline ¼ 1:4:5 EtOH:PEG:saline ¼ 1:4:5 Water Water Water Water Water Water EtOH:PEG:saline ¼ 1:4:5 EtOH:PEG:saline ¼ 1:4:5 Unknown Unknown

30 20 51a 250b 366c

1 8 12

C. CHEN

Compound

PBS, 1%HMBC, 1% Tween 80 Water Water Water

a

ng/ml h. ng/ml h, Cbrain ¼ 75 ng/g at 1.5 h, b/p ratio ¼ 1. c ng/ml h, Cbrain ¼ 158 ng/g at 1 h, b/p ratio ¼ 1.8. b

119

120

NONPEPTIDE LIGANDS FOR THE MELANOCORTIN-4 RECEPTOR Table 3.3 CALCULATED PHYSICOCHEMICAL PROPERTIESa

Compound

M.W.

clogP

clogD

pKa

PSA

FRB

HD

HA

3a 5a 6a 8a 9a 11 17 19c 22a 32a 42a 43 45a 53a 53b

589.19 557.76 543.73 582.81 604.82 530.16 565.76 596.15 638.23 530.55 588.60 535.52 527.50 519.46 519.46

5.33 4.37 3.38 5.44 5.95 6.01 4.25 2.96 3.66 6.00 4.15 3.96 3.72 5.54 5.31

4.93 3.96 2.31 3.61 4.34 4.41 4.01 2.54 3.24 4.27 1.39 2.55 2.56 3.21 3.03

7.9 7.9 8.5 9.4 9.0 9.3 7.4 7.9 7.9 9.5 9.0 8.8 8.9 9.6 9.6

92 94 103 82 105 53 120 119 110 69 119 74 105 77 77

8 8 8 8 9 6 13 7 10 6 13 11 11 9 8

2 3 4 2 4 0 3 3 2 3 4 2 3 3 3

8 8 8 7 7 6 9 9 9 6 7 7 8 6 6

a

Using ACD software; PSA, polar surface area; FRB, freely rotable bond; HD, hydrogen-bond donor; HA, hydrogen-bond acceptor.

following an overnight fast) or long-term (greater than 6 h under normal nocturnal feeding conditions) feeding inhibition is observed following THIQ administration. However, no effect on long-term feeding inhibition is observed with this compound in MC4R knockout mice, supporting a central MC4R mediated mechanism. In comparison, central administration of this compound has no effect on either metabolic rate or insulin release. There is no information reported on THIQ brain PK, which could be important since the pharmacological activity discussed above is evidently associated with central MC4R activation. Additional studies, such as the possible involvement of P-glycoprotein (P-gp) and other efflux mechanisms [53] and plasma protein binding [54], are also needed to understand the in vivo biological activity associated with this compound. An X-ray study performed on crystals of THIQ sulfate salt reveals two closely related conformations, which resemble the shape of the letter ‘‘Y’’, where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, and the triazole is highly exposed to the environment [55]. Although the solid conformation may not directly reflect low-energy conformations in solution, this structural information could provide some insight about the possible interaction of THIQ with the target in a receptor model.

C. CHEN

121

MB243 AND RELATED COMPOUNDS

A series of piperazinecarboxamides exemplified by MB243 (5a) has been reported by Palucki et al. as potent and selective MC4R agonists [56]. In comparison with the structure of (3a), the 1-triazolemethyl is replaced by an N-tert-butyl carboxamide in (5a), which probably serves as a hydrogenbond acceptor. The large and lipophilic 1, 2, 3, 4-tetrahydroisoquinolinecarbonyl (Tic) group of (3a) is now replaced by a much more hydrophilic 2-piperazinecarbonyl moiety. Interestingly, the 4-chlorophenylalanine of (3a) is switched to a 4-fluorophenylalanine in this molecule. MB243 is less potent than THIQ in vitro (EC50 of 11 nM versus 2.1 nM) as a MC4R agonist, and its selectivity over other melanocortin receptors (MC3R/ 4R ¼ 101, MC5R/4R ¼ 147) is also lower than that of THIQ based on their binding affinities. However, (5a) (clogD ¼ 3.96) is much less lipophilic than THIQ (clogD ¼ 4.93) based on the calculation using ACD software (Table 3.3), which should be more desirable since THIQ might be too lipophilic as a potential central nervous system (CNS) agent due to its large size (MW ¼ 589). The two stereoisomers (5b) and (5c) are less potent than (5a), suggesting an S-chiral preference at the piperazine group. The N-desmethyl analogue (6a) (IC50 ¼ 18 nM) of (5a) possesses similar potency to its parent at the MC4R and its selectivity over MC3R (57-fold) or MC5R (53-fold) is slightly lower than that of (5a). Otherwise, (6a) (c log D ¼ 2.31) might be a good compound due to its low lipophilicity and lack of demethylation-metabolic liability. Moreover, the high basicity of its secondary amine could facilitate brain tissue distribution. The N-ethyl analogue (6b) has an in vitro profile similar to (5a). The more lipophilic N-trifluoroethyl (6c) and isopropyl (6d) derivatives are less efficacious than (5a), whereas their binding affinities are not different (Table 3.1). Finally, the N-alanine derivative (6e) is less potent and less efficacious than (5a), suggesting that the basicity of the piperazine nitrogen has an important role in receptor interactions. PK properties (Table 3.2) of (5a) have been characterized in rats, dogs, and monkeys. In rats, it has a moderate plasma CL of 48.7 ml/min kg and a moderate Vdss of 5.4 L/kg. Its t1/2 of 1.6 h is longer than that of THIQ due to the combination of lower clearance and slightly higher volume of distribution. After an oral dose of 5 mg/kg, (5a) reaches a maximal concentration at 1.5 h, and the absolute bioavailability is calculated to be 9%. There is no information available on the brain exposure of this compound. In dogs, (5a) displays a low plasma CL of 5.6 ml/min kg, which results in a relatively long t1/2 of 4.1 h despite its low Vdss of 1.2 L/kg. Its oral bioavailability of 17% is similar to those of THIQ. The PK parameters of

122


MB243 in monkeys are quite similar to that in dogs, except for its slow oral absorption reflected by a large Tmax of 5 h. The low oral bioavailability might be in part attributed to its incomplete absorption, because low plasma clearances are observed in all species. The fact that the volume of distribution in dogs and monkeys is similar may suggest that the higher Vdss value of (5a) in rats might be caused by its lower plasma protein binding in this species [57]. Metabolic stability from in vitro liver microsomal incubation predicts a low hepatic clearance of this compound in human. Compound (5a) has been further investigated in vivo in an animal obesity model. After oral doses of 20 mg/kg twice a day for 4 days to SpragueDawley rats, the body weights of the animals treated with (5a) showed an overall decrease of 7 g relative to the vehicle group. Compound (5a) was also evaluated for its erectogenic activity in vivo using an established rodent model [17]. Sixty minutes following an oral administration of (5a) (5–40 mg/ kg), a dose-dependent increase in erections is observed. The maximal increase in the number of erections (35%) is detected at 20 and 40 mg/kg doses. Statistically significant increase in erectile responses with a mean increase of 21% is also observed after an i.v. injection at a 3 mg/kg dose. Compound (5a) has been found by Doss et al. to bind covalently to liver microsomal proteins of rats and humans in vitro [58]. In the presence of glutathione, two thioether adducts are detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts are also formed when bile duct-cannulated rats are dosed with (5a). These two adducts have been isolated and their structures are determined by accurate mass MS/MS and NMR analyses. The proposed structures result from a novel contraction of the piperazine ring to yield a substituted imidazoline (M1, and M2, Figure 3.3). A possible mechanism has been

O F

X

HN

Me

S N N

O tBu

NH

N O

N H

O

M1: X = NHCH2COOHMe M2: X = OH

Fig. 3.3 M1 and M2 structures.

C. CHEN

123

proposed and this understanding of the mechanism of bioactivation leads to the design of (5a) analogues that exhibit reduced covalent protein binding. F

F

O tBu

O NH

N O

N H

O N

O

R tBu

HN

R NH

HN

(7) (7a) R = 6-(i-Pr)

F

F

O tBu

N H

O

(6a) R = H; (6b) R = Et; (6c) R = CH2CH2F (6d) R = i-Pr; (6e) R = D-Ala

O

O NH

N

NH

N O

N H

N

(8a) 1R, 4S, 6R, 1'R (RY764) (8b) 1S, 4R, 6S, 1'R

Me tBu

O H 2N NH

N O

N H

R

A

(9) (9a) A= CH2CH2, R = H (9b) A = CH2CH2, R = Me

An SAR study focusing on the liver microsomal binding has been reported by Palucki et al. By adding a small alkyl group at the piperazine ring to prevent the formation of a reactive imine moiety, a series of (5a) analogues (7) displays lower potential for this liability [59]. For example, the alkyl substitution on the C-5 of the piperazine ring dramatically reduces covalent binding in comparison to (5a). Thus, compound (7a) displays an IC50 of 6 nM and an EC50 of 8 nM with IA of 79% at the MC4R, which is comparable to MB243. More importantly, (7a) provides a 40-fold decrease in covalent protein binding. Therefore, alkylation at either C-5 or C-6 of the piperazine ring blocks the oxidation and further bioactivation. Isoquinuclidine has been used by Ye et al. to replace the piperazine of (5a) in their efforts to attenuate the oxidative metabolism and subsequent binding to liver microsomal protein. Thus, RY764 (8a) is identified as the most promising agonist among other close analogues including stereoisomers [60]. Compound (8a) exhibits high binding affinity (IC50 ¼ 8 nM) and agonist

124


potency (EC50 ¼ 11 nM, IA ¼ 81%) at the MC4R and good selectivity over other melanocortin receptor subtypes. Its stereoisomers including (8b) are less potent. Compound (8a) also shows high potency at the rat, mouse, and dog MC4 receptors with EC50 values of 5, 6, and 11 nM, respectively. In PK studies, it has been found that the oral bioavailability of (8a) is good in rats and dogs, but only moderate in monkeys (Table 3.2). It is worth noting that the volume of distribution in rats (Vdss ¼ 9.6 L/kg) is much larger than that in dogs (2.3 L/kg) and monkeys (0.8 L/kg), indicating species difference possibly in plasma or tissue protein binding. Compound (8a) has been further evaluated in vivo. Its food intake effect has been studied in Sprague-Dawley rats in a fasting induced refeeding paradigm. Significant food intake reduction is observed at 6-h (39%) and 18-h (37%) time points after oral doses of 2, 6, and 20 mg/kg in 0.5% methylcellulose vehicle. In an overnight food intake study in MC3/4R knockout and wild-type mice, (8a) displays a significant food intake reduction in wild-type (28%) but not in knockout mice (8.6%). (8a) also augments the magnitude and duration of electrically stimulated erectile activity following i.v. administration (1 mg/kg) in mice using an established rodent model of erectile function. At the same dose, (8a) significantly augments post-stimulation by 34 and 26% at 15 and 45 min, respectively, judged by the area under curve (AUC). At a 0.1 mg/kg dose, however, (8a) fails to augment electrically evoked changes in erectile activity. [3H]-(8a) has been evaluated for irreversible protein binding in vitro, and the isoquinuclidine appreciably reduces the metabolic activation potential to relatively low levels following incubation with rat and human liver microsomes. Irreversible protein binding has also been measured in vivo in rats dosed orally at 2 and 20 mg/kg, and the study reveals that protein binding in plasma and liver at 2, 6, and 24 h post-dose is o1 pM equiv./mg of protein. The synthesis and biological profiles of the Tic mimicking compounds (9) that incorporate novel Tic surrogates to address some of the issues associated with (3a) have been reported recently by Bakshi et al. using the MB243 (5a) template [61]. One of the chiral compounds (9a) (IC50 ¼ 0.37 nM, EC50 ¼ 1.9 nM, IA ¼ 81%) has been found to be a potent and selective agonist. Its PK parameters are similar to (3a) in rats, but improved in dogs (Table 3.2). Compound (9a) has also been investigated in diet-induced obese (DIO) Sprague-Dawley rats for its effect on overnight food intake and body weight. After the compound is administered orally at 10 mg/kg by gavage 1 h before lights off, food intake and body weight are measured from 18 h post-dosing, and (9a) is efficacious in reducing both food intake 20% and 7 g.

C. CHEN

125

Erectogenic activity of (9a) has been evaluated in a rat ex copula model, and the mean number of erections elicited over a 15-min period is determined by a visual count of videotaped events. While increasing the number of erectile events at 2 and 5 mg/kg, i.v. (57 and 94%) is observed, (9a) fails to elicit significant pro-erectile activity at oral doses up to 20 mg/kg. The reason for this discrepancy is unclear. Studies on the in vitro metabolism of (9a) in human liver microsomes, however, indicate that it is both a reversible and a time-dependent inhibitor of CYP3A4, which may cause potential drug–drug interactions. Further studies show that the oxidation of the amino group of the tetralin is involved in the time-dependent inhibition of CYP3A4. Therefore, introducing a methyl group at the carbon connecting the basic amine, as shown in (9b), might attenuate the oxidation. The human MC4R receptor binding and functional activity of (9b) is similar to that of (9a). Unlike (9a), however, (9b) is not a potent reversible inhibitor of human CYP3A4 and it is also not a timedependent inhibitor of that enzyme. These results suggest that introduction of a methyl group at the benzylic position of the tetralin not only blocks the potential oxidation, but also reduces the interaction of the basic amino group with the CYP3A4 enzyme, possibly due to a steric effect. A series of b-Ala-D(4-Cl)Phe dipeptide derivatives has been studied by Ruel et al. in their efforts to improve the physicochemical properties of the lipophilic THIQ compound [62]. Although the simple b-alanine derivative (15) (IC50 ¼ 16 nM) and THIQ (IC50 ¼ 7 nM in this study) possess similar binding affinity, (15) is much less potent than THIQ (EC50 ¼ 170 nM versus 3.6 nM) in cAMP assay. Incorporation of an amide group at the b-alanine, i.e., compound (16) (IC50 ¼ 21 nM), does not significantly modify the binding affinity. On the basis of these results, the authors have proposed that this site of the molecule of (15) or (16) is solvent-exposed in the receptor; therefore, a polar group is tolerated.

OTHER THIQ-RELATED COMPOUNDS Cl

O X

N O

N H

(10a) X= HN

(10) X = N-containing heterocycle

O O S N Me Me

126

NONPEPTIDE LIGANDS FOR THE MELANOCORTIN-4 RECEPTOR Cl

F F O

N

N tBu

N

O

O (11)

NMe2

N

N N

O

N (12)

In an effort to gain further insights into the MC4R privileged structure presented in THIQ, a series of piperidine compounds (10), which mimic the triazole of THIQ and carboxamide of MB243, has been synthesized and evaluated in vitro by Bakshi et al. [63]. The pharmacological properties of one representative compound (10a) is included in Table 3.1. A novel class of pyrrolidine derivatives, exemplified by (11) as potent MC4R agonists, has been disclosed recently by Ujjainwalla et al. [64]. Compound (11) has an IC50 of 14 nM in binding assay, and as a functional agonist, it possesses an EC50 of 2 nM with 104% IA. Compound (11) exhibits IC50 values of 8.8 and 1.7 mM at the MC3R and MC5R, respectively, demonstrating high selectivity among the melanocortin receptors. Compound (11) is also a weak agonist at other subtypes with Emax (EC50, mM) values of 47%, 63% (1.4), and 72% (3.0) at the MC1, MC3, and MC5 receptors, respectively. The PK properties of this compound have been studied in rats and dogs (Table 3.2). This compound has a large volume of distribution (19 L/kg) in dogs, which results in a long half-life of 8.9 h. This property might be associated with the high basicity of the pyrrolidine functionality (calculated pKa ¼ 9.4, Table 3.3), which should favor wide tissue distribution, including the brain. An aminocyclopentyl analogue (12) has been reported as a MC4R agonist in a mutagenesis study by Hogan et al. [65]. Although this compound displays potent agonist activity (EC50 ¼ 9.5 nM), its binding affinity (Ki ¼ 305 nM) is only moderate, which is about 45-fold lower than that of THIQ (Ki ¼ 6.7 nM in this study). Since there are three chiral centers in the cyclopentane ring, it is unknown if the other stereoisomers are more potent.

CYCLOHEXYLPIPERAZINES AND PHENYLPIPERIDINES Several cyclohexylpiperazines bearing the Tic-D(4-Cl)Phe dipeptide have been synthesized and studied as MC4 ligands by Mutulis et al. [55]. A

C. CHEN

127

triazole substitution at the 2-position (14) of the cyclohexane ring of (13b) improves binding affinity. Although the 1-triazolemethyl substituted compound (13a) (Ki ¼ 1.0 mM) is only slightly better than its parent, the 2-isomer (14a) (Ki ¼ 390 nM) shows about threefold improvement in binding affinity (Table 3.4). The 2-triazole analogue (14b) displays a Ki Table 3.4 PHARMACOLOGICAL PROPERTIES AT THE MC4R Compound

IC50 (nM)

13a 13b 14a 14b 15 16 17 18 19a 19b 19c

Ki: Ki: Ki: Ki: 16 21

20a 20b 20c 21a 21b 22a 22b 23a 23b 23c 24a 24b 24c 25a 25b 25c 26a 26b 26c 27a 27b 28a 28b a b

EC50 (nM)

IA (%)a

1,000 1,400 390 260

Ki: 220 340 110 60 Ki: 30,000 Ki: 6,600 Ki: 11 Ki: 6.4 25 35 340 410 110 9.5 3.3 o0.1 2 5.8 19 Ki: 280 Ki: 4,200 Ki: 70 Ki: 40 Ki: 11 Ki: 95.6 Ki: 5.8

170 n/a 2600 2.9 80 24 380 16 11 14 7 360 357 4.7 0.44 o0.1 11 18 8.6 >10,000 >10,000 >10,000 >10,000 15 340

Intrinsic activity (% of a-MSH level). Compound number in original publications.

92 39 95 100 100 100 >80 ? >80 >80 >80 105

0 0 45 0 100 90

Ref.

No.b

55 55 55 55 62 62 66 66 67 67 67 68 72 68 68 55 68 70 70 72 72 72 72 72 73 73 73 73 73 73 74 74 74 75 75 76 76

23a 8b 23b 23c 2 5b 2 6 21 26 3 30 2 50 44 8a 26 11h 2c 6f 6e 2 7a 8b 14b 14f 14j 20f 20e 20b 22k 22o 22t 36 57 47 49

128


value of 260 nM, which is less active than THIQ (Ki ¼ 80 nM in this study). The crystal structure of (R,R)-(14b) dinitrate salt has been resolved. In comparison to THIQ, it shows a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. However, molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-(14b), suggests a possible ‘‘bioactive’’ conformation that is similar to the crystal conformation of THIQ. Phenylpiperidines with a His-DPhe dipeptide have been found to be potent MC1R agonists by Herpin and co-workers [66]. For example, compound (17) has an IC50 of 120 nM and an EC50 of 28 nM with 92% IA at the MC1R. This compound shows efficacy in an acute inflammation model. Intravenous injection of (17) in Balb/c mice at a 6.8 mmol/kg (3.85 mg/kg) dose shows that this compound has a high plasma CL of 110 ml/min kg, a high volume of distribution (Vdss ¼ 8.2 L/kg), and a short half-life of 1.2 h due to high clearance. Although (17) displays low potency at the MC4R (EC50 ¼ 2.6 mM), its close analogue (18) is a potent MC4R agonist (EC50 ¼ 2.9 nM, IA ¼ 95%), and it is still more selective at the MC1R (EC50 ¼ 0.19 nM, IA ¼ 98%).

PHENYLPIPERAZINES AS MC4R AGONISTS Cl

R

Cl

N

O N O

N H

O N N

HN

(13a) R = 1-triazol-1-ylmethyl; (13b) R = H (14a) R = 2-triazol-1-ylmethyl (14b) R = 2-triazol-1-yl

N

1 2

N O

N H

A

NR R

(15) A = CH2CH2, NR1R2 = NH2 (16) A = CH2CH2, NR1R2 = NHCOCH2OMe

Phenylpiperazines with the Tic-D(4-Cl)Phe dipeptide or its variants have been reported by several research groups as potent and selective MC4R ligands. The phenylpiperazines with a 1-triazolemethyl (19a) (EC50 ¼ 80 nM) or 4-triazolemethyl moiety (19b) (EC50 ¼ 24 nM) substituted at the 2-position of the phenyl ring were reported first by Dyck et al. to be potent

C. CHEN

129

and selective MC4R agonists, whereas other substituents provide less potent analogues [67]. For example, the methylsulfonamide (19c) has an EC50 of 300 nM with 100% IA. In comparison, the 1-imidazolemethyl analogue (20a), reported by Richardson et al. [68], has a Ki of 110 nM and an EC50 of 14 nM, and the N,N-dimethylaminomethyl derivative (20b) is slightly more potent as a MC4R agonist (Ki ¼ 60 nM, EC50 ¼ 7 nM). Interestingly, all the compounds reported by Richardson et al. have high IA (>80% of the a-MSH levels), regardless of their binding affinity and potency, suggesting this polar group at the phenyl ring is not critical for receptor activation. In this study (19c) displays a Ki of 220 nM and an EC50 of 16 nM, which is much lower than the EC50 value reported by Dyck et al. It should be noted that in the study by Richardson, an SPA-based competition cAMP assay is used, and the 20-fold discrepancy in potency might be caused by different assay conditions. As a reference, the unsubstituted piperazinebenzene is only moderately active in both binding affinity and cAMP function (20c) (IC50 ¼ 6.6 mM, EC50 ¼ 360 nM), demonstrating the important role of a polar group in contributing energy to the binding complex. Compound (19c) has been studied for its oral bioavailability in Fischer 344 rats. After a 5 mg/kg i.v. injection, it displays a relatively high plasma CL of 43 ml/min kg, a moderate volume of distribution (Vd ¼ 4.5 L/kg), and a half-life of 1.7 h. After a 30 mg/kg dose via oral gavage, the maximal concentration appears at 3-h time point, and the oral bioavailability is 30%. Cl

MeO

N R

N O

N R2

O N

nPr

N H

O 1

2

HN

(17) R = H; R = Me 1 2 (18) R = PhCO; R = H

1

R

O N O

N H

HN

(19a) R = 1-triazoleCH2; (19b) R = 4-triazoleCH2; (19c) R = MeSO2NH (20a) R = imidazoleCH2; (20b) R = Me2NCH2; (20c) R = H (21a) R = 2-thienylCH2CH2NHCH2; (21b) R =

NH N H (22a) R = MeSO2(Pr)N; (22b) R = MeSO2(NH2CH2CH2)N

130

NONPEPTIDE LIGANDS FOR THE MELANOCORTIN-4 RECEPTOR Cl

Cl

N Me

O

N

N

S O O

O

N H

N Me

R

N

S O O

(23) (23a) R = 3-tetrahydroisoquinolinyl (23b) R = 2-quinolinyl (23c) R = 1-ethyl-4-piperidinyl

Me

N

R

(24) (24a) R = Et (24b) R = Boc (24c) R = COCH2tBu Cl

O

N N Ac

N O

Cl

Et

O

N

N

R O

(25) (25a) R = 4-Boc-piperazinyl (25b) R = cyclobutylamino (25c) R = azetidinyl

N m(H2C) A (CH2)n

O N O

N H

HN

(26) (26a) m= 2, n = 1, A = NMe (26b) m= 2, n = 1, A = CH2 (26c) m= 2, n =1, A = CHNMe2

A polar group at the 2-position of the piperazinephenyl ring clearly plays a role in the interaction with the receptor. The sulfonamide (19c) is designed based on the opening of the spiroindoline of (1c) [67]. Although the triazole group of (19a) and (19b) and the imidazole of (20a) seem to mimic the triazole in the THIQ molecule, the high MC4R potency of the dimethylaminomethyl (20b) suggests that this basic group may interact with an acidic residue of the MC4 receptor. This hypothesis is strongly supported by the results from the tetrazoles (4), in which the acidic (4a) is much less potent than the neutral or weakly basic methyltetrazoles (4b) and (4c) (Table 3.1). A receptor model built by Chen et al. [69] predicts a possible interaction between the methylsulfonamide of (19c) and the Asp-122 residue at the top part of transmembrane helix-3 (TM-3) of the hMC4R. A series of piperazinebenzylamines bearing the Tic-D(4-Cl)Phe dipeptide has been synthesized and studied by Pontillo et al. [70]. One of these compounds with a 2-thienylethyl side-chain attached to the benzylamine (21a) shows high binding affinity (Ki ¼ 11 nM). Interestingly, despite its moderate potency, (21a) is still a full agonist (EC50 ¼ 357 nM, IA ¼ 105%). In comparison, an

C. CHEN

131

analogue bearing a piperazine side chain (21b) (Ki ¼ 6.4 nM, EC50 ¼ 4.7 nM) is highly potent in both binding and function [71]. The N-alkyl sulfonamide (22a) (Ki ¼ 25 nM, EC50 ¼ 0.44 nM) has also been reported as a potent and selective MC4R agonist by Fotsch et al. [72]. Interestingly, the N-(aminoethyl) sulfonamide (22b) displays an EC50 of o0.1 nM, whereas its binding affinity (IC50 ¼ 35 nM) is similar to that of (22a), suggesting that the amino group of (22b) contributes to its agonist potency. It is worth noting that (19c) is reported to have an IC50 of 340 nM and EC50 of 11 nM in this study, and these values match the data reported by Richardson et al. Compound (22a) has been further studied in mice. In vitro, (22a) binds to the mouse MC4R with an IC50 of 0.1 nM, and functions as a potent agonist (EC50 ¼ 0.5 nM). After an oral dose of 50 mg/kg (formulated in PBS with 1% HMBC and 1% Tween 80) administered to fed male CD-1 mice, (22a) reaches a maximal concentration of 1.4 mg/ml at 0.25 h. The brain maximal concentration is 130 ng/ml. Its apparent clearance (Cl/F ¼ 30 ml/min kg) is moderate and the reported volume of distribution (Vz ¼ 19 L/kg) is high. This compound has a short half-life of 0.44 h in this species. The oral bioavailability F% of 20% is given in the publication, although the information of i.v. administration is not clear. Based on the high volume of distribution in this species, the moderate brain concentration of (22a) seems to be impaired by either poor permeation through the BBB or efflux mechanism. In an acute feeding model, (22a) shows effects on cumulative food intake in fasted male C57BL/6 mice in a dose-dependent manner. Thus, the animals treated with 50 mg/kg of (22a) via oral administration show a significant reduction in food intake for 4 h, and the 100 mg/kg group shows significant reduction in feeding that lasts for 6 h. There seems to be a misconnection between the PK and the pharmacodynamics for this compound based on its high in vitro potency (EC50 ¼ 0.5 nM at the mouse MC4 receptor). One possible explanation for these results is that (22a) might have very high plasma protein binding, which dramatically reduces the free drug concentration in the blood circulation and brain. A brief SAR study on (23) has been conducted by Fotsch et al. on the Tic group, and the results show that the 3-quinoline (23b) is less active than the tetrahydroquinoline (23a), whereas the 4-piperidine (23c) is two- to threefold more potent in both binding and function (Table 3.4) [72]. A series of phenylpiperazines bearing a succinamide core has been studied as MC4R ligands by Xi et al. [73]. Interestingly, a subseries of compounds (24) having a sulfonamide substituent at the 2-position of the phenyl group exhibits high binding affinity, but low potency in cAMP stimulation. For example, the N-ethyl compound (24a) has an IC50 value of 9.5 nM in binding affinity, but is unable to stimulate cAMP release. Similarly, the N-Boc analogue (24b) displays high binding affinity without cAMP stimulation. In

132


comparison, the N-neopentylcarbonyl derivative (24c) has a very low IC50 value (o0.1 nM), and it is also a partial agonist (EC50 ¼ 74 nM, IA ¼ 45%). In contrast, members of the amide subseries (25) apparently show better cAMP stimulation than the sulfonamide analogues. Although (25a) (IC50 ¼ 2 nM, IA ¼ 0%), a close analogue of (24b), possesses high binding affinity without agonist activity, the simple cyclobutylamine (25b) is a potent agonist. Interestingly, the azetidine derivative (25c) (IC50 ¼ 19 nM, EC50 ¼ 340 nM, IA ¼ 90%) exhibits high binding affinity but moderate agonist potency with high efficacy (Table 3.4). Cl

Cl

R

F O

N Et2N

N O

N H

N Et2N

O N

HN

(27) (27a) R = H (27b) R = F(single isomer)

O

N H

R

(28) (28a) R = 3-quinolinyl (28b) R =

HN Me Me

Because of the important role of a polar group at the 2-position of the phenyl ring, a series of constrained molecules (26) has been investigated by Fisher et al. [74]. The tetrahydroisoquinoline (26a) displays a Ki value of 280 nM, about 15-fold better than the tetralin derivative (26b) (Ki ¼ 4.2 mM). Incorporating a dimethylamino group at the 2-position of the tetralin (26c) (Ki ¼ 70 nM) increases its binding affinity by 60-fold. These results demonstrate the important contribution of a basic group at this site for the interaction with the MC4 receptor. However, it is unclear if these compounds are agonists or antagonists. A series of compounds derived from the 2-aminomethylphenylpiperazines by migrating the piperazine moiety from the phenyl ring of (20b) to the benzylic position has been synthesized and studied by Fisher et al. as MC4R ligands [75]. These compounds have comparable binding affinity to (20b). For example, (27a) has a Ki value of 40 nM, whereas its 2-fluorinated analogue (27b) as a single isomer (Ki ¼ 11 nM) has slightly improved binding affinity. This series of compounds has been further elaborated by changing the Tic group (28). Thus, the compound with a quinoline (28a) (Ki ¼ 95.6 nM) is less potent than (27b), whereas the 2,2-dimethyltetrahydroisoquinolinyl derivative (28b) (Ki ¼ 5.8 nM) exhibits high binding affinity [76]. There is no information on functional activity of these compounds.

C. CHEN

133

OTHER CLASSES OF MC4R AGONISTS Studies on a series of pyridazinones based on a screening lead (29) have been reported by Ujjainwalla et al. [77]. Although (29) exhibits moderate binding affinity (IC50 ¼ 144 nM, EC50 ¼ 3,060 nM, IA ¼ 34%) and low agonist potency at the hMC4R, its derivatives such as (30a) display better activity (IC50 ¼ 33 nM, EC50 ¼ 177 nM, IA ¼ 77%). Further SAR studies reveal that partially saturated pyridazinones with similar side-chains are also potent MC4R agonists. For example, (30b) has an IC50 of 1.5 nM and an EC50 of 40 nM with IA of 67% (Table 3.5) [78]. Cl

Cl S

S N N

H N O

MeO

N N

O

O

1

Cl

Cl

R

+ N S Br

R1= 1

Ph

O

N R

Me O

(30a) A-B = CH=C (30b) A = CH2, B= CH

(29)

2

H N

O

MeO

N H

H N

A B

N H

3

N

R

R2 2

N H 3

H, = 4-Me; R = 4-MeO (31a) (31b) R = H, R = 2-MeO; R3 = 4-Me 1 2 (31c) R = R = 2-MeO; R3 = 4-Me

(32a) R = Me (32b) R = H

MeO

O

Me

N

NH R

Me

Me

Me

N

N N

N H

N

Me

N A

NH

(33a) A = S-CHMe (33b) A = C=O

Using a high-throughput screening assay based on [125I]-NDP-MSH binding to the hMC4R, a series of 2,3-diaryl-5-anilino[1,2,4]thiadiazoles (31)

134

NONPEPTIDE LIGANDS FOR THE MELANOCORTIN-4 RECEPTOR Table 3.5 PHARMACOLOGICAL PRPOERTIES AT MC4R

Compound

IC50 (nM)

EC50 (nM)

IA (%)a

29 30a 30b 31a 31b 31c 32a 32b 33a 33b 34 35 36 37 38 39 40 41a 42a 42b 43 44a 44b 45a 45b 46a 46b 47a 47b 47c 48 49a 49b 50a 50b 50c 51 52a 52b 53a 53b

144 33 1.5 174 22 4.4 KI: 590 Ki: 840

3,060 177 40

34 77 67

4,000 2,700 pEC50: 8.05 pEC50: 7.63 4

76 37

180 >10,000

85

22 142

89 76 15

a

7.7 Ki: 79 Ki: 797 Ki: 29 Ki: 13 Ki: 76 Ki: 21 Ki: 1.8 Ki: 3.2 Ki: 6.9 Ki: 11 Ki: 4.2 Ki: 6.3 Ki: 53 33 4.5 36 200 41 612 249 217 594 Ki: 7.9 124 160 180 340 3.2 13

IC50 (nM)b

93 n/a n/a n/a n/a n/a n/a 90 Pa2: 7.9 d

0.4 0.6

87 33

30 0 62 0 –27

170 530 150 12 n/a n/a n/a n/a n/a n/a n/a

e

1,000 103 51 590 330 31

Ref.

No.c

77 77 78 79 79 79 80, 81 81 82 82 85 86 87 88 89 90 90 93 93 94 96 97 97 99 99 101 101 102 102 102 104 105 105 106 107 110 111 113 113 114 114

2 36 26 3a 3b 3c 13 10 n/a n/a 2a n/a n/a 18 12 23 27 11c 5b 14c 7b 13e 14t 12i 13 3 17 1b 2b 2r 6 7f 7d 4aa MCL0129 MCL0042 7 15 21 13 22

Intrinsic activity (% of a-MSH level). Inhibition of a-MSH-stimulated cAMP release. c Compound number in original publications. d Suppression of Emax in Schild analysis. e Downward shift AGRP inhibition curve on a-MSH-stimulated cAMP release. b

C. CHEN

135

has been found to be potent and selective MC4 receptor agonists by Pan et al. [79]. The initial lead (31a) (IC50 ¼ 174 nM) is characterized as a functional agonist by increasing 35S-GTPgS binding. It shows 12-fold selectivity over the MC3 receptor. After an SAR study on the three aryl side chains, compounds with better binding affinity have been discovered. For example, (31c) has an IC50 of 4.4 nM, and it also increases 35S-GTPgS binding. When delivered intraperitoneally (i.p.) to rats, compounds (31a) and (31c) significantly decreased food intake in a fasting-induced feeding model. However, oral administration failed to show similar efficacy, presumably due to the salt structure that limits absorption. Phenylguanidines, exemplified by (32a), have been reported by Duhl et al. as MC4R agonists and compounds from this series have demonstrated efficacy in obesity animal models [80]. Some compounds from this series were later found to be very potent and selective antagonists of the melanocortin-5 receptor. For example, (32a) has a Ki value of 590 nM and EC50 of 4.0 mM with 76% activation at the MC4R. At the human MC5 receptor, this compound exhibits a Ki value of 11 nM and functions as an antagonist with an IC50 value of 89 nM for dose-dependent inhibition of a-MSH-stimulated cAMP release in cells expressing the MC5R [81]. Its close analogue (32b) (Ki ¼ 3.2 nM, IC50 ¼ 72 nM) possesses even higher binding affinity at the MC5R. Recently, a structurally related series, exemplified by compound (33a) (pEC50 ¼ 8.05), has been disclosed by Speaker et al. [82]. Analogues from this class show efficacy in feeding models, but the piperazineguanidine structure in (33a) seems to cause some PK problems for these compounds. For example, some compounds display elimination half-lives as long as weeks. Further studies show that lysosomal trapping is the main cause for this extremely slow elimination, which might be associated with the high basicity of the guanidine functionality (pKa10) [83]. Attempts to mask the second basic amine of the piperazine group (pKa ¼ 8.5), for example by using a carbonyl moiety (33b) (pEC50 ¼ 7.34), result in substantial reduction in potency. In addition, these guanidine compounds generally have high plasma protein binding and low brain penetration, partly due to their large molecular sizes and high lipophilicities (Table 3.3).

COMPOUNDS MIMICKING a-MSH AND ITS PEPTIDE ANALOGUES Efforts have been made to directly mimic the pharmacophore of the HFRW motif of melanocortin peptides in order to address the metabolic liability of peptides. [84] The cyclohexane (34) has been designed and synthesized by Fotsch et al. based on the solution structure of the cyclic peptide

136


Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH2 determined by NMR [85]. Compound (34) is a potent and selective MC4R agonist (IC50 ¼ 7.7 nM, EC50 ¼ 4 nM, IA ¼ 93%). In this molecule, the His residue of melanocortin core motif is eliminated, suggesting a minimal role of this amino acid. Cl NHAc O N

N HN

N

O N NH

Ac

NH2 O

NH HN

(34)

NH2

N H

H2N

(35)

OMe Ph O

NH2

PhO

O

NH N

O

O

NH H2N

H N

N H

NH

N

(37)

(36)

NH HN H2N

H N

O O N

NH Ph (38)

A O

Ph

B O NH2 N

NHCH2Ph

N H

PhCH2O (39) A = NH, B = O (40) A =CH2, B =H2

NH

C. CHEN

137

Using piperazinone as a core template, a series of compounds with the Phe-Arg-Trp side chain functionality has been studied by Wu et al. [86]. One of these compounds (35) (Ki ¼ 79 nM) possesses good binding affinity to the MC4R. Similarly, piperazindione has also been used in the design and synthesis of MC4R agonists to mimic the cyclic peptide MT-II. For example, one derivative (36) has been reported to possess an EC50 of 180 nM with IA of 80% [87]. Tetrahydropyran has also been used as a template in the synthesis of MC4R ligands by Mazur et al. [88]. One of these compounds shows moderate binding affinity (37) (IC50 ¼ 797 nM). In comparison, Tian and co-workers have been very successful in applying proline as a core structure and compounds with high binding affinity have been identified [89]. For example, compound (38) possesses an IC50 value of 29 nM, but is inactive in an agonist assay, indicating that it might be an antagonist. Interestingly, while a different order of decorations is applied, potent MC4R agonists have been identified from this series [90]. Thus, compound (39) has an IC50 of 13 nM and an EC50 of 22 nM with IA of 89% at the MC4R. Further studies show that replacement of the NH-moiety of the Tic-DPhe dipeptide with a CH2 unit causes only a moderate reduction in potency. For example, compound (40) has an IC50 of 76 nM and an EC50 of 142 nM with IA of 76%, suggesting that this NH-unit is not critically involved in the interaction with the receptor. All of these templates might mimic the b-turn conformation of the melanocortin peptides [91].

FROM AGONISTS TO ANTAGONISTS Although MC4R agonists may have an important role in suppressing food intake and promoting erectogenic activity as discussed above, MC4R-selective antagonists could have a potential application in promoting feeding, therefore, treating anorexia [92] or cancer cachexia [24]. A series of piperazinebenzylamines has been designed and synthesized based on the THIQ pharmacophore [69]. Starting from the benzylamine (21a), SAR studies by Pontillo et al. show that agonist potency and efficacy reduce and eventually diminish when the Tic-group, which is known to be important for agonist activity, is replaced by a less lipophilic amino acid such as b-alanine (41) [93]. Thus, (41) possesses high binding affinity (Ki ¼ 21 nM), but only stimulates cAMP production to 14% of the maximal level of a-MSH at 10 mM concentration. More importantly, it behaves as a functional antagonist by dosedependent inhibition of a-MSH-stimulated cAMP release with an IC50 of 90 nM. This functional switch is different from the THIQ series. For example, replacing the Tic group of (3a) with b-alanine results in a derivative (15) with

138


a reduction of agonist potency about 50-fold (EC50 from 3.6 to 170 nM) but not much change in IA (from 99 to 92%) [62]. Introducing an additional chlorine at the 2-position of the (4-Cl)Phe group of (41) completely abolishes its residual agonist activity [94]. Thus, compound (42a) has a Ki of 1.8 nM in binding affinity and is inactive in stimulating cAMP (o3%). In addition, (42a) displays a pA2 value of 7.9 based on a Schild analysis in a cAMP assay, demonstrating the functional antagonism of this compound. Cl

Cl 1

R N

Cl O

N

HN

O

N H

N

2

R

O N

HN OMe

Me

Et

N H

O

S (41) (41a) R1 = H; R2 = CH2CH2NH2 (42a) R1 = Cl; R2 = CH2CH2NH2 (42b) R1 = Cl; R2 = OCH2CH2F

(43)

Cl Cl

Cl Cl

N

N

N

EtHN

N H

N O

NH2

O (44) (44a) R = CH2CH2Ph (44b) R = COCH2C6H4OMe-4

NH2

N H

(45a) cis (45b) trans

Cl

Cl

O tBuHN

O

EtO2C

O

O N O

O R

N H

(46) (46a) R = H (46b) R = OPr

O

O

O N

N

* O

O

N H

R O

(47) (47a) * = D, R = H (47b) * = L, R = H (47c) * = L, R = 6-OPr

C. CHEN

139

Compound (42a) has a measured logD value of 1.1, which seems to be desirable. After a 10 mg/kg i.v. injection to rats, (42a) has a moderate plasma CL of 24 ml/min kg, and a high volume of distribution (Vd ¼ 9.5 L/ kg), resulting in a long half-life of 4.6 h in this species. Compound (42a) also seems to penetrate into the brain. At 1.5 h post-dosing, the brain/plasma ratio is unity. However, (42a) exhibits very low oral bioavailability mainly due to its poor absorption judged by portal vein sampling, which might be associated with the dibasic nature of the molecule. Its close analogue (42b) (Ki ¼ 3.2 nM) is a monobasic molecule, which also exhibits high selectivity over the other melanocortin receptor subtypes. In addition, (42b) binds to the mouse MC4 receptor with high affinity (Ki ¼ 3.7 nM). After i.c.v. administration, a 10 nmol dose of (42b) significantly increases food consumption at all the time points measured in female CD-1 mice, demonstrating the orexigenic effect of this potent and selective MC4R antagonist [95]. Lead optimization of this series of compounds resulted in the identification of compound (43), which has a small methoxy isopropyl group at the benzylamine [96]. Compound (43) possesses good binding affinity (Ki ¼ 6.9 nM) at the MC4R and is selective over the other melanocortin receptors. This compound has a measured log D value of 1.8, which is an ideal value for a CNS-penetrating compound. After i.v. injection of a 5 mg/kg dose to rats, (43) has a high plasma CL of 52 ml/min kg, and a moderate volume of distribution (6.2 L/kg), which results in a short half-life of 1.4 h in this species. Brain concentration sampled at 1-h time point is 280 ng/g, which is about 50% of that in the plasma. After oral dosing at 10 mg/kg, (43) reaches a maximal plasma concentration (Cmax ¼ 49 nM) at 6-h time point, indicating slow absorption. Oral bioavailability in this species is 8%. In comparison of piperazinebenzene (20c) with piperazinecyclohexane (13b) bearing the Tic-D(4-Cl)Phe dipeptide, the latter compound is much more potent in binding affinity [55]. With the success in identifying MC4R antagonists from the b-Ala-D(2,4-Cl)Phe dipeptide in the benzylamine series, Tran et al. have applied this unit to a series of piperazinecyclohexanes (44) [97, 98]. Thus, (44a) with a N-benzylaminomethyl side chain at the 1-position of the cyclohexane has a Ki of 11 nM in binding affinity and an IC50 of 430 nM in functional activity. Similarly, the 4-methoxyphenylacetamidomethyl (44b) has a Ki of 4.2 nM and an IC50 of 150 nM. A closely analogous series to (44) with an ethoxycarbonyl group at the 2-position of the cyclohexane (45) has also been reported by Tucci et al. as potent MC4R antagonists [99]. Thus, the cis-isomer (45a) has a Ki of 6.3 nM in binding affinity and an IC50 of 12 nM in functional activity. Compound (45a) is about nine times more potent than its trans-isomer (45b) (Ki ¼ 53 nM), and is also highly selective over the other melanocortin receptor subtypes. Compound (45a) has a measured log D value of 1.8. The

140


PK parameters of this compound have been characterized in rats. After i.v. administration of 5 mg/kg, (45a) has a high plasma CL of 57 ml/min kg, and a very high volume of distribution (Vd ¼ 29 L/kg), resulting in a long half-life of 5.8 h in this species. This compound is able to penetrate into the brain. Thus, at 1-h time point post-dosing, the whole brain concentration is 158 ng/g, compared to 88 ng/ml in the plasma. After an oral dose of 10 mg/ kg, (45a) reaches its maximal plasma concentration of 117 ng/ml at 2.8 h, and its oral bioavailability is 12%. Compound (45a) has a Ki of 9.6 nM at the mouse MC4 receptor. The effect of a single oral administration of (45a) on normal food intake in male C57BL/6 mice over a 24 h period has been examined. In this study, vehicle or 10 mg/kg of compound (45a) were orally administered at the onset of the dark phase of the light/dark cycle, and food intake was then measured 1, 2, 4, 6, and 24 h after lights-out. Compound (45a) significantly increased cumulative food intake at the 6- and 24-h measurement intervals. In a series of experiments, Markison et al. have demonstrated that peripheral administration of (45a) could effectively stimulate daytime (satiated) food intake as well as decrease basal metabolic rate in normal animals. This compound attenuates cachexia and preserved lean body mass of tumour-bearing animals in a murine cancer model. These data clearly demonstrate the potential of small molecule MC4R antagonists in the treatment of cachexia and underscore the importance of melanocortin signaling in the development of this metabolic disorder [100]. A series of compounds (46) based on the MB243 (5a) template has been reported by Soeberdt and co-workers in their efforts to identify functional MC4R antagonists [101]. Replacing the 2-piperazine of (5a) by a 2-chromone reduces the binding affinity by about ninefold (46a) (Ki ¼ 33 nM, IA ¼ 30%), but this change almost abolishes its IA. Introducing a 6-propoxy at the chromone of (46a) not only increases binding affinity but also completely eliminates its IA (46b) (IC50 ¼ 4.5 nM, IA ¼ 0%). Compound (46a) is able to prevent acute lipopolysaccharide (LPS)-induced anorexia in rats with an oral 10 mg/kg dose. In addition, (46a) reverses the decrease in lean body mass in tumour-bearing mice. Derived from the chromone, a series of phenylpiperidines (47) bearing a cyclic amide has been studied by Weyermann et al. as MC4R antagonists [102]. In this series, the D(4-Cl)Phe derivatives such as (47a) (IC50 ¼ 36 nM, EC50 ¼ 87 nM, IA ¼ 62%) are MC4R agonists, whereas compounds from L-4-chlorophenylalanine, such as (47b) (Ki ¼ 200 nM, IA ¼ 0%), are unable to stimulate cAMP release despite only about tenfold reduction in binding affinity. Optimization at the chromone leads to the discovery of compound (47c), which has an IC50 of 41 nM in binding affinity. Moreover, in functional assay, (47c) causes about 27% reduction of the basal cAMP level with an

C. CHEN

141

EC50 of 33 nM, demonstrating an inverse agonistic activity of this compound. This property could be very important since in vitro studies have shown that the endogenous AgRP might be a functional inverse agonist [103].

SMALL MOLECULE MC4R ANTAGONISTS FROM INITIAL SCREEN HITS High-throughput screening efforts from pharmaceutical companies have provided medicinal chemists some small molecule lead compounds as MC4R antagonists. A 2-(4-bromophenyl)indole (48) has been identified from a GPCR combinatorial library by Willoughby and co-workers at Merck. This compound binds to the MC4 receptor with an IC50 of 612 nM [104]. There is no further information on this lead. Arasasingham et al. at Amgen have identified a series of piperazines (49) based on an initial screening lead (49a) [105]. For example, compound (49b) inhibits a-MSH binding to the MC4R with an IC50 of 217 nM, and its binding affinity in competition with AgRP is slightly better (IC50 ¼ 52 nM). In addition, it reduces a-MSH-stimulated cAMP production at high concentrations, demonstrating functional antagonism of this compound. A similar screening lead (50a) (IC50 ¼ 594 nM) has also been found by Nakazato et al. at Taisho Pharmaceuticals, and the SAR of its analogues has been reported [106]. Optimization of the initial lead has resulted in the discovery of compound MCL0129 (50b). This compound inhibits [125I]-NDP-MSH binding to the MC4R with a Ki value of 7.9 nM without showing affinity for the MC1 and MC3 receptors. Compound (50b) at 1 mM has no apparent affinity for other receptors, transporters, and ion channels related to anxiety and depression except for a moderate affinity for the s1 receptor, serotonin transporter, and a1-adrenoceptor, demonstrating that (50b) is selective for the MC4 receptor. Compound (50b) attenuates the a-MSH-stimulated cAMP formation in COS-1 cells expressing the MC4 receptor, whereas it alone does not affect basal cAMP levels, indicating that the compound acts as a functional antagonist at the MC4 receptor. F

NHc-hexyl O N

Ph

Me Br N H

Me

N

N N

(48)

(49) (49a) R = Ph (49b) R = Me

R

142


R1

F OMe N

(CH2)n

NH

N N

Br

N N

R1 R1 1

F

R2

2

(50a) n = 0, = H, R = Me (50b) n = 3, = MeO, R2 = iPr (50c) n = 3, R = H, R2 = Me

(51)

R1 OMe O OMe N

H N

N R2

NH

Br Br

NHR

F F (52) (52a) R1 = R2 = Me (52b) R1-R2 = (CH2)4

(53) (53a) R = -(CH2)4 N (53b) R = -CH2

NEt

Swim stress markedly induces anxiogenic-like effects in both the light/ dark exploration task in mice and the elevated plus-maze task in rats, and (50b) has been shown to reverse the stress-induced anxiogenic-like effects. Under nonstress conditions, (50b) prolongs time spent in the light area in the light/dark exploration task and suppresses marble-burying behaviour. Compound (50b) shortens immobility time in the forced swim test and reduces the number of escape failures in inescapable shocks in the learned helplessness test, thus indicating an antidepressant potential. In contrast, (50b) has negligible effects on spontaneous locomotor activity, rotarod performance, and hexobarbital-induced anesthesia. These observations indicate that MCL0129 is a potent and selective MC4R antagonist with anxiolyticand antidepressant-like activities in various rodent models [107]. Melanocortins such as a-MSH and ACTH have anxiogenic effects in the social interaction test, which is an animal behavioural model of anxiety. Although MT-II dose-dependently and significantly reduces the time spent in social interaction, acute administration of (50b) has no effect on the result of this test. In contrast, when given repeatedly for 1 week by peripheral administration (oral or i.p.), (50b) significantly and dose-dependently increases the time spent in social interaction without affecting locomotor activity. These results suggest that MC4 receptor is involved in social interaction, and that

C. CHEN

143

(50b) as an MC4 receptor antagonist has an anxiolytic-like effect in this model [108] and are in agreement with the similar effects produced from i.c.v. administration of a small peptide antagonist MCL0020 [109]. MCL0042 (50c), a close analogue of MCL0129, has recently been identified by Chaki et al. for its activity in both MC4 receptor antagonism and serotonin transporter inhibition. Compound (50c) shows an IC50 of 125 and 42 nM, respectively, at the MC4 receptor and serotonin transporter determined by binding assays. Functionally, (50c) attenuates NDP-MSH-increased cAMP formation in MC4 receptor expressing cells, and inhibits [3H]serotonin uptake by rat brain synaptosomes, demonstrating that it is an MC4 receptor antagonist and serotonin transporter inhibitor (IC50 ¼ 5.1 mM and 264 nM, respectively) [110]. Compound (50c) has been examined for its anxiolytic and antidepressant effects in rats. Subcutaneous administration of (50c) significantly increases the number of licks in a Vogel punished drinking test. In addition, it significantly attenuates swim stressinduced reduction in time spent in open arms in an elevated plus-maze task, showing the anxiolytic-like potential. Moreover, repeated administration of (50c) for 14 days attenuates olfactory bulbectomy-induced locomotor hyperactivity, indicating the antidepressant-like potential. These studies show that (50c) has unique properties of both the MC4 receptor antagonist and serotonin transporter inhibitor and produces anxiolytic and antidepressant activity in rats. Therefore, blockade of both the MC4 receptor and serotonin reuptake sites might represent a useful approach in the treatment of anxiety and depression [29]. High-throughput screening has facilitated the identification of several benzamidines with low micromolar binding affinity at the MC4 receptor by Vos et al. [111], and optimized compounds with moderate affinity have been characterized. Among them ML00253764 (51) shows a Ki value of 160 nM in binding affinity, and an IC50 of 100 nM in inhibition of a-MSH-stimulated cAMP production. Compound (51) has been administered to C57B1/ 6 mice subcutaneously at 30 mg/kg to evaluate plasma and brain concentration profiles. The area under the curve in brain (29.9 mM h) is significantly higher than that in plasma (8.8 mM h). The authors have speculated that the high brain penetration of (51) is partly due to its moderate pKa of 9.6, which is substantially lower than that of its close analogue without a fluorine atom (pKa ¼ 10.4). This compound has been further evaluated for effects on body weight at a CT-26-derived mouse xenograft model. At s.c. doses of 16 mg/kg twice daily, the tumour-bearing animals, which received (51), weigh 2.5 g more than the untreated vehicle-dosed group, and 2 g more than naive controls on day 10. These results suggest that the MC4R antagonist (51) is effective in reducing the magnitude of cancer-induced weight loss in this model.

144


In a more detailed study, Nicholson et al. have shown that (51) displaces NDP-MSH binding with an IC50 of 0.32 mM in HEK-293 cells expressing the hMC4R. At concentrations more than 1 mM, (51) decreases cAMP accumulation (Emax ¼ 20%), indicating inverse agonist activity. In a Lewis lung carcinoma tumour model, the tumour-bearing mice treated with (51) maintain their lean body mass, in contrast to the loss of mass in the control animals during the 21 days of the experiment [112]. A series of imidazoles (52) such as (52a) (Ki ¼ 180 nM, IC50 ¼ 51 nM) has also been identified as MC4R antagonists by Marsilje et al. based on a metabolite of the initial benzamidines such as (51) [113]. In addition, cyclic analogues such as (52b) (Ki ¼ 340 nM, IC50 ¼ 590 nM) also display good binding affinity and antagonist potency. In comparison with the amidine series, these compounds are less basic, therefore, could have improved oral absorption as the authors have speculated. The benzamidines seem to inhibit hERG (ether-a-go-go-related gene), which encodes an ion channel subunit underlying IKr, a potassium current required for the normal repolarization of ventricular cells in the human heart. To address this liability, a series of acylguanidines has been synthesized and evaluated as MC4R antagonists by Vos et al. [114]. These compounds possess high binding affinity at the MC4 receptor and also dosedependently inhibit a-MSH-stimulated cAMP release. For example, (53a) and (53b) have an IC50 of 3.2 and 13 nM, respectively, in a binding assay. Functionally, (53a) possesses an IC50 of 330 nM, whereas (53b) (IC50 ¼ 31 nM) exhibits high potency. The reason for this discrepancy between binding affinity and functional activity is unclear. PK profiles of compounds (53a) and (53b) have been studied briefly to compare with that of (51). When dosed in rats, the oral bioavailability of 6.3% for (53a) is similar to that of (51) (4.6%), however, (53a) exhibits much longer half-life of 31 h than (51) (t1/2 ¼ 1 h) in this species. Compounds from this series, as exemplified by (53b), also have longer brain half-lives than the amidine (51). However, the plasma half-life of (53b) seems to be very short (o1 h) based on the published graph; therefore, this compound may be trapped in the brain tissues [83], which could cause the discrepancy in half-life between brain and plasma. It is worth noting that the PK results for (51) and (53a) are extrapolated based on four time points; therefore, substantial errors may be apparent.

OTHER NONPEPTIDE MC4R LIGANDS Some peptide mimetics based on the AgRP core element Arg-Phe-Phe have been synthesized by Thompson et al. in their efforts to identify MC4R

C. CHEN

145

antagonists [115]. Compounds from this set exhibit low micromolar binding affinities. Some small amines were published in 2002, which show weak binding affinity to the MC4R [116, 117]. In addition, a series of urea compounds based on the tripeptide Phe-Trp-Lys has been synthesized and pharmacologically characterized at the mouse melanocortin receptors by Joseph et al. [118]. This effort resulted in the identification of some novel melanocortin receptor agonists with potencies ranging from nanomolar to micromolar.

INTERACTION OF THE MC4R WITH LIGANDS G-protein-coupled receptors (GPCRs) are a superfamily of structurally related membrane-bound proteins that play a central role in the recognition and signal transduction of hormones and neurotransmitters. GPCRs share general structural motifs, including seven transmembrane (TM) helices connected by intra- and extracellular loops, an extracellular amino terminus, and a cytoplasmic carboxyl terminus [1, 2]. GPCRs are activated by light, small molecules such as histamine, peptides, and proteins [119, 120]. Although many GPCRs are considered to be drug targets, for those activated by peptides and proteins no detailed structural information is available [121, 122]. It has therefore been particularly challenging for medicinal chemists to design nonpeptide small-molecule agonists or antagonists for these targets. The recent resolution of the crystal structure of the bovine rhodopsin receptor provides the first three-dimensional structure of any GPCR [123], thereby making it possible to analyze experimental data of other GPCRs from a more informed and structural perspective [124]. Because of several conserved residues in each of the seven TM domains across class A GPCRs [125], the crystal structure of rhodopsin provides a good template for homology modeling of these receptors using computational tools [126–128]. In addition, site-directed mutagenesis has made it possible to characterize the ligand interaction sites of GPCRs [129, 130]. A binding pocket in the TM domain of several class A GPCRs, including the adrenergic and neurokinin receptors [131–133], has been identified via these studies.

STRUCTURAL INFORMATION OF MELANOCORTIN PEPTIDES

The HFRW motif is present in all melanocortin peptides and it has been proven to be crucial for the activation of melanocortin receptors [26, 134]. One common feature for the potent synthetic melanocortin agonists is the replacement of the L-Phe7 residue of a-MSH with its D-isomer. NDP-MSH

146


(Figure 3.2) is one of the most widely used nonselective melanocortin agonists. It has high binding affinity (Ki ¼ 1.32 nM) and agonist potency (EC50 ¼ 0.27 nM) at the human MC4R. In comparison, SHU9119 is a very close analogue of the cyclic peptide superagonist MT-II (Figure 3.2), and it has a D-(20 )Nal residue at the L-Phe7 position. SHU9119 is a potent antagonist (IC50 ¼ 1.8 nM, pA2 ¼ 9.3) at the human MC4R [135]. Chemically, the only difference between these cyclic peptide agonist and antagonist is the 0 D-(2 )Nal residue of SHU9119 and the D-Phe of MT-II. These results demonstrate a key role of the L-Phe7 moiety of a-MSH peptides in receptor activation. An alanine-scan of NDP-MSH has revealed that D-Phe7 is the most important residue of this peptide in both binding and function, followed by Arg8. Thus, D-alanine replacement of D-Phe7 results in an over 1,100-fold reduction in binding (Ki ¼ 1.6 mM) and function (EC50 ¼ 320 nM). Norleucine replacement of Arg8 gives a peptide with a 200-fold decrease in binding affinity (Ki ¼ 270 nM) and about 55-fold loss in agonist potency (EC50 ¼ 15 nM). Alanine replacement of the Trp9 residue also results in a 100-fold decrease in binding (Ki ¼ 130 nM), but much less reduction in function (EC50 ¼ 3.45 nM, Table 3.6) [136]. Many peptide ligands such as a-MSH, NDP-MSH, MT-II, and SHU9119 are not selective among the melanocortin receptors. Recently, a MC4R selective cyclic peptide antagonist MBP10 (Figure 3.2) has been discovered by Bednarek et al. [137]. MBP10 possesses an IC50 value of 0.5 nM in binding, and has 125-fold selectivity over the MC3R and >300-fold over the MC1R. This peptide has no agonist activity at the MC3R or MC4R, and only weakly at the MC5R. The deletion of the His-residue of the HisDPhe-Arg-Trp motif in MBP10 seems to be the cause of this MC3R selectivity.

SMALL PEPTIDE LIGANDS WITH HFRW VARIANTS

Structure–activity relationships of a small His-DPhe-Arg-Trp peptide and its variants have been extensively studied by several groups [26]. A detailed SAR study at the DPhe position of a series of Tic-D(Ar)Ala-Arg-Trp-NH2 core by Ye et al. provides some very interesting results (Table 3.7) [138]. Thus, a lipophilic aromatic ring increases potency but generally reduces efficacy at the MC4 receptor. In contrast, a hydrogen-bond donor or a moderate electron-withdrawing group reduces potency but increases efficacy. These results might suggest that the receptor-binding site for the 7 D-Phe group of melanocortin peptides is very lipophilic in general, but the residues that interact with this phenyl group for receptor activation might

C. CHEN

147

Table 3.6 EFFECTS OF ALA-REPLACEMENT ON NDP-MSH ACTIVITYa Entry

Peptide

Ki (nM)

Fold

EC50 (nM)

Fold

1 2 3 4 5 6 7 8

NDP-MSH Ala5[NDP-MSH] Ala6[NDP-MSH] Ala7NDP-MSH] Ala8[NDP-MSH] Ala9[NDP-MSH] Phe9[NDP-MSH] DPhe-Arg-Trp-NH2

1.32 7.1 20.3 1,592 269.7 130 8.6 6,135.5

1 5 15 1,206 204 98 7 4,648

0.27 0.98 1.1 321 15 3.45 0.31 17.7

1 4 4 1,189 56 13 1 66

a

Ref. [136].

Table 3.7 SAR OF THE TIC-D(AR)ALA-ARG-TRP-NH2 PEPTIDE AT THE D(AR)ALA POSITIONa Entry

Ar

IC50 (nM)

EC50 (nM)

IA (%)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Ph 4-MePh 4-CF3Ph 4-CNPh 4-NH2Ph 4-NO2Ph 4-OHPh 4-MeOPh 4-FPh 3-FPh 2-FPh 4-ClPh 4-BrPh 4-IPh 4-tBuPh 4-PhPh 10 -Naph 20 -Naph

31 0.93 0.92 6.8 230 5.7 1,500 3.6 11 11 27 0.89 0.51 0.3 11 6.5 6.7 57 330

32 11

81 27 1 38 66 20 64 38 88 62 110 44 22 2 0 17 41 3 94

c

27 1,200 66 8,000 17 14 35 36 3 1.2

47 170

a

Ref. [138]. Intrinsic activity. His-DPhe-Arg-Trp-NH2.

b c

be an electron-rich aromatic ring and hydrogen-bonding acceptor. For example, one of these peptides, Tic-D(4-HO)Phe-Arg-Trp-NH2, has an IC50 value of 1.5 mM in binding. It functions as a weak agonist (EC50 ¼ 8 mM, IA ¼ 64%) in cAMP assay. In comparison, its O-methyl analogue [Tic-D(4MeO)Phe-Arg-Trp-NH2] is a much more potent agonist with low efficacy

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(IC50 ¼ 3.6 nM, EC50 ¼ 17 nM, IA ¼ 38%). This later tetrapeptide is at least 50-fold selective over the other melanocortin receptor subtypes based on its binding affinity. When this potent partial agonist is administered to Sprague-Dawley rats by an i.c.v. route at a dose of 1.0 mg, significant inhibition of food intake is observed. The cumulative overnight food intake is reduced by 24% compared with the control rats. In addition, feeding duration is significantly reduced over 18 h and the effect is sustained throughout the entire test session. A synthetic tripeptide Ac-D-(20 )Nal-Arg-(20 )Nal-NH2 (MCL0020) has been discovered by Chaki et al. and shown to be a potent and selective MC4R antagonist [109]. In comparison with the HFRW peptide, the Hismoiety is deleted in MCL0020 and the Trp-group is replaced by 20 -naphthylalanine. The key Phe residue is substituted by 20 -naphthylalanine for functional antagonism, similar to that in SHU9119 and MBP10. MCL0020 possesses high affinity for the MC4 receptor with an IC50 value of 11.6 nM, and negligible affinities for the MC1 and MC3 receptors. The elimination of the imidazole of His-residue might be responsible for its selectivity over MC3R, which has been observed in MBP10. MCL0020 significantly attenuates the cAMP release induced by a-MSH in COS-1 cells expressing the MC4 receptor without affecting basal cAMP levels. The involvement of the MC4 receptor in stress and stress-related behaviour is also investigated using MC4R agonists and this selective antagonist. Thus, a-MSH and MT-II dose-dependently and significantly reduce the number of licking periods in the rat Vogel-conflict test, suggesting that stimulation of the MC4 receptor causes anxiogenic-like activity in rats. Although restraint stress significantly reduces food intake in rats, i.c.v. administration of MCL0020 dose-dependently and significantly attenuates restraint stress-induced anorexia without affecting food intake. In addition, MCL0020 significantly prevents swim stress-induced reduction in the time spent in the light area in the mouse light/dark exploration test.

CONFORMATIONS OF PEPTIDE LIGANDS

Conformation analyses of melanocortin peptide ligands using nuclear magnetic resonance (NMR) spectroscopy have been conducted to determine the geometrical locations of the residues of HFRW sequence [139, 140]. For example, comparison of the structures by NMR and molecular modeling of SHU9119, MT-II, and their close analogues indicates that, even though they exist as an average of several conformations in solution, there are distinctions in their structures. The NHi-NHi+1 NOE cross peaks and the temperature coefficients of the amide protons around the essential core

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Nal/Phe7-Arg8-Trp9 are indicative of a possible turn structure for these compounds but with differences in their NOE strengths and temperature coefficient values. Molecular modeling of these compounds based on their NMR data shows that the essential core appears as a ‘‘V’’ shape with two different orientations [140]. A conformational study conducted by Sun et al. has shown that for a series of MT-II analogues a b-turn structure is conserved. An exceptionally well-defined pharmacophore model has been derived based on these rigid analogues. This model has been used to perform a virtual screen using a library of 1,000 drug-like compounds, to which a small set of known potent ligands has been intentionally added. The utility of the model is validated by its ability to identify the known ligands from this large library [141].

RECEPTOR STRUCTURE INFORMATION THROUGH MUTAGENESIS STUDIES

The MC4R belongs to the class A GPCR family that is characterized by a short N-terminal, a sulfur-sulfur bond connecting the top TM-3 and extracellular loop-2 (EL-2), and a DRY motif at the bottom of TM-3, which is critical for receptor activation, and key conserved amino acids in each of the TM domains. However, the MC4R is slightly different from conventional class A GPCRs: it has a very short EL-2 and as such, the disulfide bridge connecting the TM-3 and EL-2 does not exist in this receptor (Figure 3.4). Mutagenesis studies on the hMC4R have been conducted by several research groups. In an effort to identify receptor residues that might interact with amino acids in DPhe7-Arg8-Trp9 sequence, 24 residues in the hMC4R TM domain have been mutated by Yang et al. [136]. Mutation of TM-3 residues Asp-122 and Asp-126, and TM-6 residues Phe-261 and His-264 decreases the binding affinity of NDP-MSH by fivefold or greater. By combining the binding affinities and potencies of the NDP-MSH peptide variants (Table 3.6) at the receptor mutants, it has been found at a molecular level that Arg8 of NDP-MSH interacts with Asp-122. In addition, mutation of Asp-122 or Asp-126 decreases the binding affinity of antagonist SHU9119 (Figure 3.2). SHU9119 is a potent antagonist of the hMC4R but an agonist at the hMC1R. In their efforts to gain insight into the molecular determinants of the MC4R in the selectivity of SHU9119, Yang et al. have constructed chimeras and mutants of the hMC1R and the hMC4R [142]. A region containing TM-3 of the hMC4R is required for selective SHU9119 antagonism. Further mutagenesis studies of this region demonstrate that the amino acid residue Leu-133 of TM-3 is critical for the selective antagonist activity of SHU9119. The single mutation of Leu-133 to methionine does not affect

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Fig. 3.4 Snake-plot of the human MC4 receptor. The amino acid residues which may interact with peptide and nonpeptide ligands are highlighted. The conserved residues are marked with a white letter and black background.

SHU9119 binding to the MC4R. However, this mutant receptor converts SHU9119 from an antagonist to an agonist. A residue Phe-284 in the TM-7 of the hMC4R has been identified through mutagenesis by Nickolls et al. have as a potential site of ligand interaction. [143]. Thus, mutation of Phe-284 to alanine reduces the binding affinity and potency of melanocortin peptides containing LPhe7 by up to 71-fold but does not appreciably affect binding of peptides containing DPhe7, suggesting an interaction between the Phe7 of a-MSH and the Phe-284 residue. Ile137Thr mutation in TM-3, a naturally occurring mutant that is linked to obesity, decreases affinity and potency of cyclic and rigid peptides, but not that of more flexible peptides, consistent with an indirect effect of the mutation on the tertiary structure of the receptor. Mutation of Ile-125 in TM-3 to phenylalanine, the equivalent residue of the hMC3R, selectively decreases affinity and potency of hMC4R-selective ligands. This effect is mirrored by the reciprocal Phe157Ile hMC3R mutant. This mutation may affect the orientation of Asp126 of the hMC4R, which has been identified as a major determinant of ligand binding affinity.

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A study to compare the binding mechanism to the hMC4R between agonist and antagonist ligands has been conducted by Fleck et al. [144]. Nonpeptide ligand interactions are affected by mutations that reduce peptide ligand binding (Asp122Ala, Asp126Ala, Ser190Ala, Met200Ala, Phe261Ala, and Phe284Ala), confirming overlapping binding determinants for peptide and nonpeptide ligands. The common halogenated phenyl group of nonpeptide ligands such as (41a) is a determinant of Phe261Ala and Phe284Ala mutations, implying that this group interacts with the aromatic side chains of these two amino acid residues. The mutations reduce the binding of 2,4-dichlorophenyl compounds such as (42a) more than the 4-chloro analogues, and Phe284Ala mutation eliminates the affinityenhancing effect of 2-chloro-substitution. Phe261Ala and Phe284Ala mutations also reduce the affinity of antagonists more than agonists, suggesting that the stronger antagonist interaction with these residues, directly or indirectly. Supporting this hypothesis, Phe261Ala mutation increases the efficacy of nonpeptide antagonist and partial agonist ligands. In addition, Asp122Ala and Asp126Ala mutations reduce the nonpeptide ligand interaction, and removing the amido group on the 4-chlorophenylalanine eliminates the effect of the mutations. Agonists, which bear an amine within this amido group, are strongly reduced by Asp126Ala mutation (550- to 3,300fold), suggesting an electrostatic interaction between this amine and the acidic group of Asp-126. These postulated interactions with aromatic and acidic regions of the hMC4R are consistent with a molecular model of the receptor. Furthermore, the strength of interaction with the aromatic pocket, and potentially the acidic pocket, controls the signaling efficacy of the ligand. Specific interactions of the hMC4R with nonpeptide and peptide agonists have also been studied by Pogozheva et al. using alanine-scanning mutagenesis [145]. The binding affinities and potencies of two synthetic smallmolecule agonists THIQ and MB243 are strongly affected by substitutions in TM-2, -3, -6, and -7 (Glu-100, Asp-122, Asp-126, Phe-261, His-264, Leu-265, and Leu-288). In addition, an Ile129Ala mutation primarily affects the binding and potency of THIQ, whereas Phe262Ala, Trp258Ala, Tyr268Ala mutations impair interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH are substantially reduced only in Asp126Ala and His264Ala mutants. Hogan and co-workers have found that Ile-125, Ile129, Tyr-268, Tyr-287, and Ile-291 residues of TM-3, TM-6, and TM-7 line the putative binding site and are likely to have direct contacts with MC4R agonists. Particularly, residues Ile-125, Ile-129, and Ile-291 form a hydrophobic pocket where Ile-291, on top of the NPXXY motif of TM-7, is likely to act as a rotamer switch for the activation of the hMC4R [65].

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Interestingly, Chen et al. have found that Asp-154 and Asp-158 in TM-3, and His-298 in TM-6 of the human MC3 receptor with alanine replacement dramatically reduce NDP-MSH binding and receptor signaling [146]. These residues align with Asp-122, Asp-126, and His-264 of the hMC4R (Figure 3.1). Substitutions of aromatic amino acids Phe-295 and Phe-296 in TM-6 of the hMC3R, which align with Phe-261 and Phe-262 of the hMC4, with alanine also significantly decrease NDP-MSH binding and receptor activation. Substitution of Leu-165 in TM-3 with methionine or alanine switches antagonist SHU9119 into an agonist, which is similar to the Leu133Met mutation in the hMC4R [143]. Site-directed mutagenesis studies by Frandberg et al. on the human MC1 receptor have indicated the importance of Asp-117 and His260 amino acid residues, which are equivalent to Asp-126 and His-264 of the hMC4R, for the binding of a-MSH, but not NDP-MSH [147]. However, a double mutant (Asp117Ala/His260Ala) displays a major loss in affinity also for NDPMSH. Although the authors conclude that the Asp117Ala and the His260Ala mutations may cause conformational changes in the receptor instead of direct interaction with any specific amino acid in the MSHpeptides, these results indicate an important role of these amino acids [148]. The molecular interactions between human MC1 and MC4 receptors and their endogenous antagonists, agouti-signaling protein (ASIP) and AgRP, have been assessed by studying the effects of site-directed mutagenesis. Mutations of homologous residues from TM-3 and TM-6 and EL3 (Asp126Ala, Ile129Ala, Phe261Ala, and Met281Phe in hMC4R) impair binding of both antagonists to hMC4R. The dependence of agonist binding on the dithiothreitol concentration follows a monophasic curve for wild-type hMC4R and its Cys40Ala, Cys271Ala, and Cys279Ala mutants, suggesting the presence of at least one structurally and functionally essential disulfide bond in both the wild-type receptor and the hMC4R mutants [149].

COMPUTATIONAL MODELING OF THE HUMAN MC4R

Because of the similarity among class A GPCRs, and highly conserved amino acid residues across different receptors, homology modeling with computational software has been a powerful tool for studying the interaction of a ligand with its receptor [128]. The crystal structure of the bovine rhodopsin has been widely used as the template for GPCR models at an inactive state of the receptor. Combined with mutagenesis, this method greatly helps medicinal chemists in understanding the mechanism of ligand– receptor interaction at the molecular level.

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153

Bondensgaard et al. have studied the docking of several privileged structures of GPCR ligands to their receptors, and found a good correlation between conservation patterns of residues in the ligand-binding pocket and the privileged structure fragments for a set of class A GPCRs. These authors believe that some regions, typically located deeper into the binding pocket, are more conserved and retain a predominantly hydrophobic and aromatic character, which is reflected in the chemical nature of most GPCR privileged structures. They propose that this common feature is recognized by the privileged structure. Three pairs of ligands recognizing widely different receptor types dock into the 3D models of their target receptors utilizing available SARs and mutagenesis data. On the basis of these results, some contacts are established between the privileged structure and residues in the binding pocket. This implies that any one particular privileged structure can target only a subset of receptors [150]. However, since this docking model for the hMC4R puts the privileged structure of THIQ near the TM-6 region in the binding pocket, it would be difficult to explain how the DPhe or its equivalent can interact with TM-6 residues such as Phe-261 and His-264, as has been established by the mutagenesis studies discussed above. Three-dimensional models of receptor–ligand complexes with their agonists have also been generated by distance-geometry using the experimental, homology-based, and other structural constraints of the hMC4R by Pogozheva et al. In these models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His6-DPhe7-Arg8-Trp9) of the linear peptide. Their charged amine groups interact with acidic residues from TM-2 and TM-3, similar to His6 and Arg8 of NDP-MSH, the substituted piperidines mimic Trp9 of the peptide and interact with TM-5 and TM-6, while the DPhe aromatic rings of all three agonists contact with Leu-133, Trp-258, and Phe-261 residues at the bottom of the binding pocket [145].

POSSIBLE INTERACTION OF NONPEPTIDE LIGANDS WITH THE MC4-RECEPTOR

Mutagenesis studies have identified several key residues on the TM-3, TM-6, and TM-7 of the hMC4R, which are important for either ligand binding or ligand-induced activation. These residues are highlighted in a 3D receptor model shown in Figure 3.5. Briefly, Asp-122, Phe-262, and Phe-284, Ile291 are important for ligand binding, whereas Asp-126, Phe-261, and His-264 are critical for receptor activation. Several different binding modes have been proposed as discussed above. However, it is known that the DPhe residue of peptide ligands, or the D-4-chlorophenylalanine of THIQ and its

154


TM-7

TM-6

Y267

TM-5

F284

F262

H264

F261

TM-1 I289

L288

D126

I125 I129

D122

TM-2

TM-3

TM-4

Fig. 3.5 Graphic representative of the key amino acid residues of the human MC4 receptor in the putative binding pocket.

analogues is the major determinant for these ligands, respectively, based on SAR studies. This functionality should have a very strong interaction with the receptor, most likely a pocket suitable for this lipophilic aromatic group. One possible candidate is the cavity near the middle TM-6, which has several aromatic residues, such as Trp-258, Phe-261, Phe-262, and His-264, which have been proven to be of importance. Similarly, three acidic residues Glu-100, Asp-122, and Asp-126 are clustered at the top of TM-2 and TM-3, possibly forming a negatively charged cage to host a polar or basic group of the ligand, such as Arg8 of a-MSH. These acids may possess different pKa depending on the environment [151], and it might be that only Asp-122 is negatively charged in principle under physiological conditions since this residue is most likely solvent-exposed based on its location at the top TM-3. The extent of the contact and the approach of a ligand to these residues may determine if it is able to activate the receptor [152]. These possible interactions based on mutagenesis and receptor modeling are summarized in Figure 3.6.

C. CHEN

TM-7 Ile-291 Phe-284

TM-6 Tyr-267 His-264 Phe-262 Phe-261

155

TM-5 Phe-196

Cl

O N

N N

N

TM-3 Asp-122 Ile-129 Ile-125

O

N H

TM-3 Asp-126

TM-5 Met-200

HN TM-4 Phe-184

Fig. 3.6 Illustration of ligand interactions with key amino acid residues of the human MC4 receptor.

MEDICINAL CHEMISTRY MC4 receptors are widely expressed in the brain and central effects are evidenced by the i.c.v. injection of melanocortin peptides such as MT-II [153]. However, systemic administration of MT-II also elicits a profound effect on food intake as well as an increase in energy expenditure in rodents [154]. A study to investigate the penetration of MT-II and iodo-MT-II, which is also effective at suppressing appetite in rats following i.v. administration into brain parenchyma, shows that significant radioactivity cannot be detected in various brain regions by autoradiography except for a group of circumventricular organs (CVOs) upon i.v. administration of [125I]-MT-II [155]. Since CVOs are anatomically situated outside the BBB, the degree of MT-II penetration into the brain therefore is negligible. Direct measurement of MT-II in the brain and plasma by LC-MS-MS following i.v. injection confirms this conclusion. These results indicate that MT-II has a very limited brain penetration capability, and its effect on feeding behaviour following systemic administration may be mediated by either the brain regions in close proximity to the CVOs or sites outside the BBB, including CVOs or other peripheral systems. A six amino acid peptide HS131, with about 20-fold higher affinity for the MC4R than for the MC3R and proved to be a functional antagonist in a cAMP assay, potently and dose-dependently increases food intake after s.c. administration (1.0 mg/kg), suggesting a possible peripheral mechanism,

156


although the authors speculate that this peptide may be able to pass the BBB [156]. Although some experiments have demonstrated a peripheral effect of a MC4R ligand, strong evidence suggests that central involvement is critical, and either agonists or antagonists need to be delivered into the brain for MC4R-related biological activities. For example, Wessells et al. have studied the possible site of the proerectile action by injecting MT-II intracerebroventribularly, intrathecally, and intravenously, and the results provide insight into the central melanocortinergic pathways that mediate penile erection [157]. One of the most important features of the brain is that it is completely separated from the blood by the BBB [158], which exists within the 400 miles of capillaries that course through the brain and spinal cord and is formed by a complex network of endothelial cells, astroglia, pericytes, perivascular macrophages, and a basal lamina [159]. While most body organs are perfused by capillaries lined with endothelial cells that have small pores to allow for the rapid movement of small molecules into the interstitial fluid from the circulation, the capillary endothelium of the brain and spinal cord lacks these pores because the endothelial cells of brain capillary are sealed together by continuous tight junctions, produced by the interaction of several transmembrane proteins that project into and seal the paracellular pathway [160]. The interaction of these junctional proteins effectively blocks the free diffusion of polar solutes from blood along these potential paracellular pathways and so denies access to brain interstitial fluid. To get into the brain, a lipophilic molecule has to go the transcellular pathways, which are gated by a variety of transporter proteins that work to extrude compounds from the brain [161]. There are several known active efflux mechanisms in the BBB, the most prominent of which is P-glycoprotein (P-gp), a 170-kDa member of the ATP-binding cassette superfamily of membrane transporters, which in humans is encoded by multidrug resistance gene 1 (MDR1) [162]. P-gp is located on the apical surface of the endothelial cells of the brain capillaries toward the vascular lumen and contributes to the poor BBB penetration of a number of lipophilic drugs [163, 164]. The interrelationship between the unbound drug fractions in blood and brain homogenate, passive membrane permeability, P-gp efflux ratio, and log octanol/water partition coefficients (c log P) in determining the extent of CNS penetration observed in vivo has been studied recently by Summerfield et al. [165]. Their results demonstrate that compounds often considered to be P-gp substrates in rodents (efflux ratio greater than 5 in multidrug resistant Madin-Darby canine kidney cells) with poor passive permeability may still exhibit reasonable CNS penetration in vivo; i.e., where the unbound

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fractions and nonspecific tissue binding act as a compensating force. In these instances, the efflux ratio and in vitro blood–brain partition ratio may be used to predict the in vivo blood–brain ratio. This relationship may be extended to account for the differences in CNS penetration observed in vivo between mdr1a/1b wild type and knockout mice [mdr1a/1b(-/-,-/-)]. In some instances, cross-species differences that might initially seem to be related to differing transporter expression can be rationalized from knowledge of unbound fractions alone. Therefore, information that provides a coherent picture of the nature of CNS penetration will allow the full understanding of CNS efficacy. There is limited information on the brain penetration of known MC4R ligands so far. Nonpeptide ligands such as THIQ and MB243 possesss high in vitro potency and selectivity, and have shown effects in several animal models associated with the MC4 receptor, but it is unclear if these results are caused by central receptor activation since brain PK profile has not been reported. THIQ has high molecular weight and MB243 possesses several hydrogen-bond donors, these features usually are undesirable for a CNS agent. The lipophilic and highly basic RY764 exhibits good PK properties in rats and dogs, including high oral bioavailability and reasonably long halflife in these species. Its high basicity could favor brain tissue binding, which is reflected by a high volume of distribution in rats (Vd ¼ 9.6 L/kg) [166]. However, the Vd values (2.3 and 0.8 L/kg, respectively) in dogs and monkeys are low, suggesting possible species difference in plasma protein binding of this compound. The high molecular weight might cause slow permeation through the BBB and therefore trigger efflux mechanism such as the P-gp pump. The plasma protein binding of these compounds, which possess one or more secondary amide functionality, is unclear. A ghrelin agonist CP464709-18 with some similarity in structure and molecular weight has been demonstrated to have low plasma protein binding in rats (unbound fraction fu ¼ 0.39), although CP-464709-18 is much less lipophilic (c log D ¼ 1.6) than THIQ and RY764 [167]. Compound (9a) has the MB243 core and a THIQ-like group. Like THIQ, it has high molecular weight and high lipophilicity (Table 3.3). In addition, this compound has higher polar surface area (PSA ¼ 105 A˚2) than THIQ, partly due to a primary amine group, which is undesirable for CNS penetration, especially for a compound with high molecular weight [168]. The pyrrolidine (11) is relative small and has less free rotatable bonds and much lower polar surface area (PSA ¼ 53 A˚2) than THIQ and others (Table 3.3). In addition, its high basicity (calculated pKa ¼ 9.3) should facilitate brain tissue binding. Compound (11) has a large volume of distribution (19 L/kg) in dogs, implying low plasma protein binding and/or high tissue binding in this species.

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There is very limited information on PK properties of MC4R antagonists, including brain penetration. The PK of compounds (42a), (43), and (45a) in rodents has been reported. Compound (42a) has low lipophilicity due to its dibasic nature. This compound has a brain/plasma (b/p) ratio of 1.8, indicating good brain penetration. However, its very low oral absorption suggests poor permeation. Therefore, the brain penetration of this compound might be caused by high brain tissue binding, even lysosomal trapping due to the two basic amine groups [83]. Compound (43) seems to have physicochemical properties suitable for brain exposure, including moderate lipophilicity and relatively low PSA. It has a moderate volume of distribution (Vd ¼ 6.2 L/kg), its brain/plasma ratio of 0.75 in rats could be adequate for molecule with such characteristic as a CNS agent depending on the extent of plasma protein binding. The MC4R antagonists derived from the THIQ agonist still possess high molecular weights and some peptide properties. Although highly potent and selective, these compounds have only poor to moderate PK properties including brain penetration. Small molecule MC4R antagonists such as ML00253764 show only moderate potency and selectivity; however, they seem to penetrate well into the brain in rodents. It is worth noting that compounds possessing a basic amidine functionality could have high brain tissue binding and even lysosomal trapping. There is no information on plasma protein binding of these small molecules, although their high basicity and lipophilicity (51, pKa ¼ 9.6, c log P ¼ 2.45) would facilitate plasma and tissue protein binding, therefore, low free drug concentration. One advantage for all the compounds reported is that they possess at least one basic amine as a pharmacophore element of both MC4R agonists and antagonists, which should be desirable as potential CNS agents. The challenges for medicinal chemists are to find compounds with a balance of molecular weight (below o450), lipophilicity (log P ¼ 1–3), high potency, and selectivity. Many of the known MC4R agonists also have peptide characteristics (hydrogen-bond donor). Capromorelin (CP-424391, MW ¼ 505, c log D ¼ 1.7), a ghrelin agonist with structural similarity to THIQ, is known to have low CNS penetration [169]. Although there is no report on the plasma protein binding of the MC4R ligands so far, this is a very important parameter for understanding the in vivo efficacy related to the MC4R, as well as PK such as free drug concentration, volume of distribution, and brain penetration.

CONCLUSION Studies have shown that MC4R plays a major role in regulating some important biological functions including energy homeostasis. MC4 receptors

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are widely expressed in the brain and modulated by the endogenous peptide agonist a-MSH and the protein antagonist AgRP. Both a-MSH and AGRP are nonselective, and because of the BBB, a synthetic peptide or protein ligand of this receptor is not suitable for this centrally active target. Efforts to develop potent and selective agonists and antagonists of the hMC4R have led to the discovery of several classes of dipeptide and nonpeptide compounds. Some of these compounds have been demonstrated to possess good PK properties in animals, including good oral bioavailability, because of their relatively low molecule weights. However, information on brain penetration is still very limited. These selective ligands have provided powerful tools for further delineating the role of the MC4 receptor in feeding behaviour, erectogenic activity, pain, and anxiety/depression. In addition, several key amino acid residues of the receptor, which are involved in the interaction with peptide and nonpeptide ligands, have been identified by mutagenesis studies and computational modeling. Understanding these interactions should help medicinal chemists to design ligands with desirable physicochemical, pharmacological, and PK properties, and eventually, safe compounds for clinical studies to test the biological activity for certain disease states in human, such as obesity, erectile dysfunction for an agonist, or cachexia, anxiety/depression, and pain for an antagonist.

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4 Tuberculosis Chemotherapy: Recent Developments and Future Perspectives VEEMAL BHOWRUTH, LYNN G. DOVER and GURDYAL S. BESRA School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

INTRODUCTION Drug Resistance and Development of Modern Anti-TB Chemotherapy

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TB DRUG DEVELOPMENT PIPELINE Diarylquinolone TMC207 n-Octanesulfonylacetamide (OSA) Phenothiazines Oxazolidinones Quinolones Azoles Nitroimidazopyran PA-824 Dihydroimidazo-oxazole OPC 67683 Sudoterb (Pyrrole LL-4858) Peptide Deformylase Inhibitor BB-3497 Acetohydroxyacid Synthase Inhibitor KHG20612 Ethambutol Analogue SQ109 Thiolactomycin Siderophore Biosynthesis Purine Analogues

173 173 174 176 176 178 179 180 181 181 182 182 183 183 184 186

POTENTIAL NEW DRUG TARGETS Targeting Mycobacterial Persistence The Stringent Response Unexplored Targets in Cell Wall Biosynthesis

187 187 190 192

CONCLUDING COMMENTS

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TUBERCULOSIS CHEMOTHERAPY

INTRODUCTION Despite the advent of chemotherapy against tuberculosis (TB1) in 1944 [1], the disease remains a global health priority. The causative organism, Mycobacterium tuberculosis, is a tremendously successful coloniser of the human host and is estimated to have latently infected approximately onethird of humanity [2]. The most recent figures suggest that 8.9 million new cases of TB and 1.8 million deaths due to it were reported worldwide in 2004 [3]. TB remains a disease firmly associated with poverty and under-developed healthcare infrastructure [4, 5] with around 95% of global TB and 98% of TB-associated mortality borne by countries of the developing world. In developed countries, TB notifications increased alarmingly towards the end of the last century driven by increasing globalisation and immigration [2]. The global incidence of TB rises by around 1% annually with much of this increase concentrated in sub-Saharan Africa with the major exacerbating factor being the HIV epidemic [3, 6]. The retroviral infection compromises host defences rendering the individual sufferers more susceptible to infection and markedly increases the risk of reactivation of latent TB infection. The estimated annual risk of reactivation among those co-infected with HIV and TB is about 5–8% compared to a cumulative lifetime risk of 5–10% in HIV-negative adults [7]. One of the major problems associated with the treatment of TB is the issue of latency. Antibiotics are most effective against actively growing M. tuberculosis rather than those cultured in stationary phase [8]. This phenotypic resistance is associated with the metabolic state of the bacteria rather than any genetic change and they are described as persistent or dormant bacilli, the latter requiring special treatments and passage through liquid culture before they can be cultured on agar plates. The impact of this phenomenon is well illustrated in the Cornell model of TB infection in mice [9, 10]. Briefly, when mice experimentally infected with M. tuberculosis are treated with the anti-tubercular agents, isoniazid (INH) and pyrazinamide (PZA) for 3 months, TB bacilli cannot be detected in mouse tissue via growth on agar plates. However, around one-third of these culture-negative 1 Abbreviations: ACP, acyl carrier protein; AG, arabinogalactan; DARQ, diarylquinolone; DNA, deoxyribonucleic acid; EMB, ethambutol; ETH, ethionamide; f, furanose; FAS, fatty acid synthase; Gal, galactose; GAT, gatifloxacin; GlcNAc, Nacetylglucosamine; ICL, isocitrate lyase; INH, isoniazid; mAGP, mycolyl-arabinogalactan-peptidoglycan; MDR-TB, multi-drug resistant TB; MIC, minimal inhibitory concentration; MXF, moxifloxacin; ORF, open reading frame; p, pyranose; PAS, p-aminosalicylic acid; PZA, pyrazinamide; RIF, rifampicin; Rha, rhamnose; TA, toxin-antitoxin; TB, tuberculosis; TLM, thiolactomycin; TPZ, trifluoroperazine.

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animals will relapse with TB after a further 3 months during which drug treatment is withdrawn and, if immuno-suppressed using steroid treatments, almost all relapse. In these cases, recovered M. tuberculosis cells are as susceptible to the drugs as the original infecting strain [9, 10], their survival being attributed to the development of phenotypic resistance rather than developing a stable genetically defined drug resistance.

DRUG RESISTANCE AND DEVELOPMENT OF MODERN ANTI-TB CHEMOTHERAPY

The ‘golden age’ of TB chemotherapy began with the discovery of the aminoglycoside streptomycin (SM) by Waksman and colleagues in 1944 [1]. Two years later, the potent anti-tubercular activity of p-aminosalicylic acid (PAS) was discovered [11]. In 1952, the investigation of nicotinamide activity on M. tuberculosis in vivo [12] led to the simultaneous discovery of INH by three drug discovery teams [13–15] and PZA [16]. Four years later, further development of the same lead gave rise to ethionamide (ETH) and prothionamide (PTH) [17]. In 1961, investigation of the anti-TB activity of polyamines and diamines prompted the production of a series of diamine analogues that gave rise to ethambutol (EMB) [18]. Several other anti-TB agents have been discovered from the screening of compounds produced by soil organisms including cycloserine [19], kanamycin [20], viomycin [21], capreomycin [22], rifamycin [23] and its derivative rifampicin (RIF) [24]. Using this array of agents, modern TB therapy was developed with the only significant introduction to the anti-TB arsenal since the 1960s being the introduction of the broad range quinolones to TB therapy in the late 1980s [25, 26]. Currently, the recommended standard chemotherapeutic regime for TB treatment is prescribed under DOTS (directly observed treatment, shortcourse). Initially, the acronym described the chemotherapy regime but has latterly become the term used to describe a broader public health strategy with five principal elements: political commitment; case detection by sputum smear microscopy; standard short-course chemotherapy with supportive patient management, including DOT; a system to ensure regular drug supplies; and a standard recording and reporting system, including the evaluation of treatment outcomes [27]. The chemotherapeutic regimen consists of an initial 2-month phase of treatment with INH, RIF, PZA and EMB followed by a continuation phase of treatment lasting 4 months with INH and RIF [2]. The need for a long therapeutic regimen involving four drugs stems from several contributing factors: bacterial dormancy and phenotypic resistance to antimicrobials are certainly important, as is the issue of exposure to drugs, the structure and pathology of necrotic tuberculous lesions

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vary and the intracellular location of many of the bacteria might limit exposure. Variations in TB lesions effectively define the metabolic status of the bacteria in vivo and give rise to at least four sub-populations: active growers, that can be killed by INH; those with sporadic metabolic bursts that can be killed by RIF; a population with low metabolic activity that also experience acidic surroundings killed by PZA; and finally dormant bacilli that are not killed by any current agents [28]. Poor patient compliance can promote the emergence of drug resistance and this is particularly true in TB chemotherapy. The complex nature and length of the treatment, combined with logistical problems concerning drug supply and the tendency for patients to feel well long before safe completion of the prescribed course, led to unacceptable rates of non-compliance that promoted the development of drug resistance and prompted the development of DOTS [29, 30]. INH and RIF represent the cornerstones of anti-TB therapy; INH is the most powerful mycobactericidal drug available, normally ensuring early sputum conversion and consequently decreasing the transmission of TB. RIF, by its mycobactericidal and sterilising activities, is crucial for preventing relapses. Strains of M. tuberculosis resistant to both INH and RIF, regardless of profiles of sensitivity/resistance to other drugs, have been termed multidrug-resistant strains. Multidrug-resistant tuberculosis (MDR-TB) is a major concern as TB patients that fail treatment have a high risk of death. While resistance to either drug may be managed with other first-line drugs, MDR-TB requires treatment with second-line drugs under DOTS-Plus [2], these possess limited sterilising capacity and are not suitable for short course treatment necessitating prolonged treatment with drugs that are less effective and more toxic [30–32]. Recently, the emergence of extensively drug-resistant TB (XDR-TB) strains with resistance to at least three of the six classes of second-line drugs (aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine and PAS) has been monitored [31]. In some regions approaching 20% of MDR-TB cases were classified as XDR-TB raising concerns over a future epidemic of virtually untreatable TB [31]. Given this backdrop, the need for rapid and continued progress in the development of new antimicrobials active against M. tuberculosis and the identification and characterisation of novel drug targets to engage medicinal chemists is clearly evident. Importantly, enhanced penetration of infection sites such as lung cavities and long biological half-lives might represent advances towards shortening therapy and lead to simpler treatment regimens with improved patient compliance [29]. Another important consideration is their effectiveness in co-administration with antiviral agents used to treat AIDS [29, 33, 34]; RIF activates cytochrome P450 enzymes that metabolise the antiviral agents significantly reducing their plasma concentrations [34].


173

TB DRUG DEVELOPMENT PIPELINE Several promising anti-TB agents are currently at various stages of development. The available information concerning these drugs will now be considered individually. DIARYLQUINOLONE TMC207

The discovery of the new TB drug diarylquinolone (DARQ) TMC207 (1) (formerly R207910) was recently reported by Johnson & Johnson [35]. The drug was identified via screening prototypes of different chemical series for inhibition of the growth of the fast-growing saprophyte Mycobacterium smegmatis. Chemical optimisation of a lead compound generated a series of DARQs expressing potent in vitro activity against several mycobacteria including M. tuberculosis. Twenty DARQs possessed a minimum inhibition concentration (MIC) below 0.5 mg/ml against M. tuberculosis H37Rv with three compounds showing encouraging in vivo antimycobacterial activity. Extensive studies have been carried out on the most active compound of the class, (1), which has a MIC of 0.003 mg/ml for M. smegmatis and 0.03 mg/ml for M. tuberculosis [35]. The drug is less active against other bacterial species, such as Staphylococcus aureus and Escherichia coli (MIC>32 mg/ml). Compound (1) demonstrated similar in vitro efficacy against M. tuberculosis clinical isolates resistant to the TB agents INH, RIF, SM, EMB, PZA and moxifloxacin (MXF) (13). This lack of cross-resistance with currently used anti-TB agents suggests that (1) retains activity against multidrug-resistant (MDR) strains and exerts it anti-mycobacterial effect through a different cellular target. Ph OH

Br

NMe2

N

(1) TMC207

TMC207 (1) did not inhibit purified M. tuberculosis DNA gyrase, the target for other quinolones, further suggesting that it operates via a unique mechanism. Furthermore, selection of spontaneous mutants resistant to (1) in M. tuberculosis demonstrated that no cross-resistance to other anti-TB agents developed. Genomic sequencing of resistant strains implicated atpE, the gene encoding a component of the F0 subunit of mycobacterial F1F0

174


proton ATP synthase, in resistance to DARQs [35]. Complementation studies verified that the mutations in the membrane-spanning region of the atpE product atpE gene were responsible for resistance to TMC207 and implying that the atpE gene product is the target of TMC207 in mycobacteria. When considering (1) monotherapy, a potent early bactericidal activity comparable to that of INH was observed in the non-established infection murine TB model. Furthermore, a late bactericidal effect was also observed in the established infection murine TB model exceeding that of RIF. As resistant mutants emerged at rates comparable with RIF mutants, DARQ anti-TB monotherapy does not appear appropriate but further studies demonstrated the potential of (1) as a component of combination therapy. Substitution of each of the three first-line drugs with (1) resulted in a significant increase in potency, leading to complete culture conversion of the lungs in some animals after 1 rather than 2 months indicating potential for reducing the length of combined therapy [35]. Human studies have shown (1) to be well tolerated, at least during a limited exposure period, with plasma levels 8 times those associated with potent in vivo activity in mice. In the first single dose clinical study subjects experienced only mild or moderate adverse effects [35]. Given that the F1F0 ATPase is common to bacteria and mitochondria, the degree of tolerance observed and the selectivity for mycobacteria displayed for these drugs is a little surprising and may reflect subtle structural differences pertinent to the mycobacterial target. N-OCTANESULFONYLACETAMIDE (OSA)

b-Sulfonylcarboxamide compounds (2), among others, were designed as potential inhibitors of b-ketoacyl-ACP synthases (KAS) of pathogenic mycobacteria by acting as mimics (both geometrically and electronically) of the putative transition state (4) in the Claisen-type condensation reaction catalysed by these enzymes/domains involved in the biosynthesis of fatty acids, the mycolic acid components of the cell wall and various polyketides in M. tuberculosis [36]. The most active compounds discovered through this strategy were amide derivatives of 3-sulfonyl fatty acids bearing alkyl chains of between 8 and 10 carbons in length, with MICs as low as 0.75–1.5 mg/ml and comparable with frontline anti-TB agents. Surprisingly, the compounds were particularly species specific, showing no activity against other bacteria even non-pathogenic fast-growing mycobacteria [36]. One of these, n-octanesulfonylacetamide (OSA) (3), was tested further against several slow-growing pathogenic and drug resistant mycobacteria including MDRTB strains [37]. Furthermore, analysis of mycobacterial lipids revealed a marked inhibition of all sub-types of mycolic acids in Mycobacterium bovis BCG without affecting the panoply of polar and non-polar extractable lipids generated by the bacterium; mycolic acid biosynthesis in the relatively


175

insensitive M. smegmatis was unaffected [37]. Analyses of treated sensitive bacteria using electron microscopy revealed dysfunction in cell wall biosynthesis and incomplete septation [37]. Further analyses of the effects of OSA using proteomic technologies revealed the over-production of two small proteins (18 kDa) in M. bovis BCG; the b-subunit of ATP synthase (F1F0 ATPase) and a small heat shock protein encoded by atpF and hsp (Rv0251c), respectively [38]. The over-expression of the atpF suggested the possible involvement of ATP synthase, either in a direct or indirect manner. Consistent with this idea, cellular ATP levels were shown to decrease upon treatment with OSA [38] mimicking the effect of the known ATP synthase inhibitor, dicyclohexylcarbodimide [39]. None of the other anti-mycobacterial-tested agents elicited the same response. Reminiscent of the DARQ series, this OSA-mediated decrease in cellular ATP may inhibit the operation of the F1F0 complex or may target other unidentified regulatory components involved in the energy production. O R

O CONH2

S

(2) R = alkyl (3) OSA R = (CH2)7CH3

Recently, the developers of (3), FASgen Inc., announced the discovery of FAS20013, their lead proprietary compound for the treatment of TB and MDR-TB [40]. The potent killing activity of FAS20013 is directed specifically towards the slow growing mycobacteria that cause disease. FAS20013 proved to be powerfully bactericidal against anaerobically adapted M. bovis BCG in the Wayne Model, i.e. against anaerobically non-replicating persistence. FAS20013 killed anaerobically adapted BCG at concentrations ranging from 1.5 mg/ml (60% killing activity) to 50 mg/ml concentration (>99% killing activity). FAS20013 also demonstrated potent sterilising activity against RIF-tolerant persisters, with an average reduction in viable cells of 1.5 log at all concentrations tested. To date, FAS20013 has shown no resistance in clinical isolates [40]. FASgen have surrounded FAS20013 with a series of worldwide intellectual property rights and issued a patent for the drug. O

S

R

O S Cys

ACP R

NH2 2+ S O

O

O

(4)

176

TUBERCULOSIS CHEMOTHERAPY PHENOTHIAZINES

M. tuberculosis is considered an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension [41]. Analysis of the M. tuberculosis genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system [42]. Both chains can be initiated with type II NADH: menaquinone oxidoreductase (NDH-2). The essential role of NDH-2 in M. tuberculosis is supported by extensive evidence from biochemical, transcriptional studies, gene deletion analysis, investigations of bacterial growth in various culture conditions and animal experiments [42, 43]. The antimycobacterial activity of phenothiazines has been sporadically reported over the past 40 years [44–46]. One such phenothiazine, trifluoroperazine (TPZ) (5) exhibited a significant effect on in vitro ATP synthesis in Mycobacterium leprae [47]. Cellular ATP levels of M. leprae were dramatically reduced in the presence of 5 mg/ml TPZ in the growth media. TPZ was used to identify NDH-2 as a target in the oxidative phosphorylation system of M. tuberculosis [42]. TPZ and its analogue chlorpromazine are effective against M. tuberculosis H37Rv in a macrophage model of infection, and are reported as synergistic with both INH and RIF [48, 49]. The in vitro concentration required for bactericidal activity is >100-fold greater than the clinical concentration of TPZ in vivo, hence the need to screen for analogues with greater potency. Fifty phenothiazine analogues were screened and three compounds showed potential inhibition activity with the most active compound, (6) having a MIC of 1.11 mg/ml in comparison to those for TPZ (19.2 mg/ml), RIF (0.5 mg/ml) and INH (0.15 mg/ml). Encouraging activity in a mouse model of acute infection reducing the bacterial load within the lungs established it as a potential lead for drug development [42]. N

Me

Me

N N

CF3

N

Me +

Ph

N

Cl

S

S

(5)

(6)

OXAZOLIDINONES

Oxazolidinones possess antimicrobial activity against a variety of Grampositive bacteria including M. tuberculosis, with a MIC of 2–4 mg/ml and are also active against tubercle bacilli in mice [50–52]. They inhibit protein


177

synthesis at the initial phase of translation in bacteria by selectively and uniquely binding to the central loop domain of 23S rRNA of the 50S ribosomal subunit [53]. Researchers at Pharmacia identified three clinical candidates, eperezolid and linezolid (10) and a thiomorpholine analogue of linezolid PNU-100480 (7), as potential antimycobacterial agents [54]. The in vitro activity of (7) was tested against M. tuberculosis strains (ATCC 27294 and clinical isolates 1104) and a panel of drug resistant strains of M. tuberculosis. Compound (7) gave an MIC50 and MIC90 of 0.1 and 0.9 mM and 0.5 and 2.5 mM against ATCC 27294 and the clinical isolate 1104, respectively [54]. Compound (7) was shown to have activity against M. tuberculosis comparable to that of INH and RIF in a murine model [55]. More recently, the antimycobacterial activity of 3-(1H-pyrrol-1-yl)2-oxazolidinones analogues was demonstrated [56]. From these studies, RBx 7644 (8) and RBx 8700 (9) were identified as active agents against MDR-TB. The activity of (8) and (9) was investigated against sensitive and MDR isolates of M. tuberculosis and for activity against bacteria within macrophages [57]. Compound (9) demonstrated good in vitro activity against sensitive as well as MDR-TB strains with MIC50 and MIC90 values of 0.032 and 0.25 mg/ml (sensitive) and 0.25 and 1.0 mg/ml (MDR strains), respectively. The MIC50 and MIC90 values for (9) and linezolid (10) were found to be 8 and 16 and 32 and 64 mg/ml, respectively [57]. Compound (9) has been considered for further development because it showed promising activity and a good correlation between in vitro MIC values and in a macrophage system. Further studies of (9) must be performed in experimental models of M. tuberculosis to determine the full potential of this drug as a possible antiTB drug. O X

N

N

O O2N

O

F

(7) PNU-100480 X = S (8) RBx 7644 X = O

X N

N

N

NHAc

O

NHAc

(9) RBx 8700 (10) linezolid

X=S X=O

Linezolid (10) has recently been approved by the FDA for the treatment of multi-drug resistant Gram-positive bacterial infections [53]. However, recent clinical studies have shown that prolonged use of (10) in the treatment of MDR-TB caused significant toxicity, including anemia and peripheral neuropathy [58, 59]. More extensive clinical studies are required to evaluate the efficacy and toxicity of oxazolidinones and the possible development of resistance against these possible anti-TB drugs.

178

TUBERCULOSIS CHEMOTHERAPY QUINOLONES

Quinolones target bacterial type II topoisomerases, DNA gyrase and topoisomerase IV [60, 61]. These ATP-dependent enzymes cooperate to facilitate DNA replication and other key DNA transactions [62]. DNA gyrase is involved in catalysing the negative supercoiling of DNA, is essential for DNA replication, recombination and transcription and appears to be the sole topoisomerase target for quinolones in M. tuberculosis. Of the series of developed quinolones derivatives, fluoroquinolones (FQs) have shown a broad-spectrum of antibacterial activity. FQs have had been used sparingly due to frequent emergence of resistance to the readily available derivatives, ofloxacin (11) and ciprofloxacin (12) [63]. New FQs containing a C-8 methoxy moiety exhibit greater activity and two compounds in particular, MXF (13) and gatifloxacin (GAT) (14), showed good activity in murine models of TB [64–66]. Compound (13) is more active than (11) and (12), with MICs of 0.125, 2 and 4 mg/ml, respectively [66]. In a mouse model, (13) was active against M. tuberculosis, with comparable results to INH [65, 66] and encouragingly, (13) also appeared to kill a sub-population of RIF-persistors [67]. A regime containing (13) has also been shown to greatly reduce time to culture conversion in murine tuberculosis [68, 69]. A combination of RIF+PZA and (13) killed tubercule bacilli in mice more effectively than the INH+RIF+PZA regime [68, 69]. The higher activity of RIF+ PZA+(13) has generated much interest as it is hoped that (13) could replace INH in a combination regime to shorten TB therapy in humans. There are, however, concerns over the potential toxicity of a RIF+PZA+(13) combination in the absence of INH as shown in the treatment of latent TB infections with RIF+PZA [70]. Compound (13) is currently in clinical trails for treatment of TB in combination with RIF and PZA. Preliminary human studies have shown that (13) has early bactericidal activity against tubercle bacilli comparable to INH and is well tolerated [71]. Recently, sixteen 7-substituted derivatives of GAT (14) were tested for antimycobacterial activity in vitro and in vivo against M. tuberculosis H37Rv and multi-drug resistant M. tuberculosis (MDR-TB) [72]. They were also tested for their ability to inhibit the supercoiling activity of DNA gyrase from M. tuberculosis. Four compounds were more active (MICo0.2 mg/ml) and five compounds were equipotent (MIC ¼ 0.2 mg/ml) to (14) against M. tuberculosis. The most active compound, (15), had an in vitro MIC of 0.0125 mg/ml against M. tuberculosis and MDR-TB. In the in vivo animal model, (15) decreased the bacterial load in lung and spleen tissues with 3.26- and 3.76-log10 protection, respectively. It was also found to be as active as GAT in the inhibition of the supercoiling activity of wild-type M. tuberculosis DNA gyrase with an IC50 of 3.0 mg/ml indicating that


179

increasing the length of the lipophilic side chain at C-7 improves the antimycobacterial activity in vitro. With this in mind, appropriate modification of (13), which is more effective than (14), could provide more effective inhibitors of DNA gyrase with improved efficacy. O

R

CO2H Me

N

N

F

O

CO2H

N

N

(12) ciprofloxacin R = F

(11) ofloxacin

Me

(13) moxifloxacin MFX R =

N N

O

CO2H

Me

(14) gatifloxacin GAT R = H

F

N N

Me

NH

N

O

OMe

NNHCONH2

(15)

R=

O N

N R

AZOLES

Azole drugs are commonly used antifungal agents, some of which have been shown to have antimycobacterial activity [73]. The recent recognition of a plethora of cytochrome P450 s, known cellular targets of azoles in fungi [74, 75], encoded in the M. tuberculosis genome [76] drove studies to examine the correlation between the presence of P450 and the susceptibility to azole drugs in M. tuberculosis [77–79]. Purified M. tuberculosis cytochrome P450 (MTCYP51) was found to bind azoles tightly providing biochemical evidence that azole drugs might also inhibit mycobacterial P450 enzymes. Two azoles, clotrimazole (16) and econazole (17) were tested for their antimycobacterial activity against M. tuberculosis H37Rv [80]. The MIC90 was 0.12 mg/ml whereas the minimum bactericidal concentration and effective concentration was 0.125 mg/ml for both drugs. Ex vivo studies on mice splenocytes following exposure to clotrimazole and econazole confirmed the

180


good tolerance and synergistic effects of these drug [81]. Further in vivo analysis is required to assess whether these azole drugs can be used for the treatment of TB. The crystal structure of MTCYP51 with bound azoles has recently been elucidated and is being probed for development of novel drug therapies against TB [82, 83].

NITROIMIDAZOPYRAN PA-824

The nitroimidazopyran PA-824 (18) represents a promising new compound for the treatment of TB that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, (18) is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect [84]. Compound (18) is very potent, with MICs as low as 0.015–0.025 mg/ml against M. tuberculosis and MDR-TB. The bioreductive activation of PA-824 requires the bacterial F240-dependent glucose-6-phosphate dehydrogenase (FGD1) and nitroreductase to activate components that then inhibit bacterial mycolic acid and protein synthesis. In preclinical testing against a broad panel of multidrug resistant clinical isolates in vitro, (18) was found to be highly active against all isolates with MICs o1 mg/ml [85]. In a short-course mouse infection model, the efficacy of (18) at 50, 100 and 300 mg/kg body weight formulated in methylcellulose or cyclodextrin/lectin after nine oral treatments were comparable with those of isoniazid, rifampin and moxifloxacin. Compound (18) also demonstrated potent activity during the continuation phase of therapy, during which it targeted bacilli that had persisted through an initial 2-month intensive phase of treatment with RIF, INH and PZA [86]. More recently, a novel nitroimidazo-oxazine specific protein has been identified and shown to be involved in (18) resistance in M. tuberculosis [87]. Resistance to (18) is most commonly mediated by a loss of the FGD1 or its deazaflavin cofactor F240 involved in reductive activation of this class of molecules. Although FGD1 and F240 are necessary, they are not sufficient for activation of (18), which requires the involvement of additional accessory proteins that directly interact with the nitroimidazole. To fully understand the mechanism of activation an extensive panel of PA-824R mutants were characterised and revealed a small class of mutants with lesions in Rv3547 that possessed normal FGD1 and F240 capabilities. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of M. tuberculosis to metabolise (18) [87]. The function of this novel protein has yet to be determined.


181

O2N N N Ph

N

Cl

N

O

Ph N

O

Cl N

Cl

O

Cl OMe

(16) clotrimazole

(17) econazole

(18) PA-824

DIHYDROIMIDAZO-OXAZOLE OPC 67683

A further azole-based lead compound OPC 67683 (19) from Otsuka Pharmaceuticals is currently in Phase I studies. The company reports that (19) has potent in vitro and in vivo activity against M. tuberculosis [88–90] and may shorten the duration of therapy against TB and MDR-TB. Currently, very little information regarding the compound has been published. O N Me N

O2N

O

OCF3

O

N

(19) OPC 67683

SUDOTERB (PYRROLE LL-4858)

Lupin has identified a lead compound, Sudoterb (LL-4858), which has activity against sensitive and resistant strain of M. tuberculosis. LL-4858 was reported to have potent anti-TB activity in vitro and in vivo (mice and guinea pig) studies. In vitro, LL-4858 has bactericidal activity similar to INH and is synergistic with RIF [91]. The combination of LL-4858 with INH, RIF and PZA led to complete sterilisation of sensitive and MDR-TB strains in infected mice within 2 months. In combination with RIF and PZA, LL-4858 also cured TB in all animals after 3 months of treatment. LL-4858 could potentially cut the time of TB treatment to 2 or 3 months [92].

182

TUBERCULOSIS CHEMOTHERAPY PEPTIDE DEFORMYLASE INHIBITOR BB-3497

Bacterial peptide deformylase (PDF) is a metallo-protease that removes the N-terminal formyl group from newly synthesised proteins. Various PDF inhibitors have activity against several pathogens including E. coli and S. aureus in vitro [93–95]. Six PDF inhibitors were screened against two isolates of M. tuberculosis and initial testing showed that three compounds, BB-3497 (20), BB-84518 and BB-83698 gave MICs in the range of 0.06–2 mg/ml [96]. These inhibitors were further tested against 17 isolates of M. tuberculosis and (20) were found to be the most active with a median MIC of 0.25 mg/ml. Further in vivo evaluation is required to fully determine the potency of (20) and clinical tests must be carried out which address whether the drug is toxic in man. A recent study suggested that PDF inhibitors had no detectable effect on two different human cell lines in vitro [97]. OH n-Bu

O

O

H N

N

H

i-Pr

O t-Bu

(20) BB-3497 ACETOHYDROXYACID SYNTHASE INHIBITOR KHG20612

Acetohydroxyacid synthase (AHAS) is a thiamine diphosphate (THDP-) and FAD-dependent enzyme that catalyses the first common step in the biosynthetic pathway of branched amino acids such as leucine, isoleucine and valine [98, 99]. The AHAS encoded by ilvB and ilvN in M. tuberculosis was over-produced in E. coli and purified to homogeneity [100]. A microplate-based enzyme assay was developed to screen small molecules as potential inhibitors of the catalytic subunits of AHAS from a chemical library composed of 5,600 compounds. Library screening identified four structurally related hit compounds, KHG20612 (21), KHG20614, KHG20613 and KHG20616 that inhibited AHAS activity by more than 90% at 40 mM. These compounds were functionally related bearing a disulfide bond and containing a phenyl or 1-substituted triazolyl groups. A range of IC50 values of 1.8–2.6 mM was reported for these four compounds. Compound (21) also showed growth inhibition activity against various strains including drugresistant strains of M. tuberculosis. N Ph

S

S

N

CONHPh

N

(21) KHG20612


183

ETHAMBUTOL ANALOGUE SQ109

Ethambutol ([S,S0 ]-2,2-[ethylenediimino]di-1-butanol) (EMB) is one of the front line drugs used against most strains of M. tuberculosis [101–103]. EMB is implicated in the inhibition of arabinan biosynthesis in both arabinogalactan [102, 104] and the cell wall associated lipoglycan, lipoarabinomannan [102]. Recently, an EMB analogue, SQ109 (22) has shown good activity against M. tuberculosis compared to the parent compound EMB with an MIC of 0.5 and 5.0 mg/ml, respectively [105–107]. Compound (22) is also more active against mycobacteria within macrophages and mice than EMB, but is less active than INH [105]. Despite its low oral bioavailability [106], (22) is currently in clinical trials. Analysis of transcriptional profiles after several different drug treatments on M. tuberculosis H37Rv revealed that despite many similarities in transcriptional profiles, (22) and EMB differentially affected a regulon containing genes within the FAS-II pathway as well as a regulon that contained genes implicated in fatty acid modification. This potential mechanistic divergence was confirmed by the observation that M. tuberculosis cells treated with EMB rapidly lost acid-fastness, whereas cells treated with (22) did not. Furthermore, cells treated with EMB contained significantly less arabinose than controls, whereas cells treated with (22) did not [43]. The in vitro and in vivo data from (22) have made it an attractive compound for further drug development [105].

Me Me

Me N H

H N

(22) SQ109

THIOLACTOMYCIN

Thiolactomycin (TLM) (23) is a thiolactone antibiotic isolated from a soil Nocardia spp [108]. Compound (23) exhibits potent in vitro activity against many pathogenic bacteria including Gram-negative and Gram-positive bacteria and M. tuberculosis. Compound (23) inhibits the b-ketoacyl-ACP synthase condensing enzymes mtFabH and KasA of the FAS-II system in M. tuberculosis [109, 110] that is involved in the synthesis of the essential mycolic acids of the cell wall. In vitro and in vivo inhibition of mtFabH and KasA leads to the inhibition of mycolic acid biosynthesis. The total synthesis of (23) was first reported by Wang and Salvino [111] and was improved to yield the active 5R stereoisomer [112]. Several analogues have

184


since been designed and tested against M. tuberculosis [113–118]. Preliminary studies showed that modifications to the thiolactone core in the C-5 position with a 5-tetrahydrogeranyl substituent (24) gave an MIC90 of 29 mM and 92% inhibition in extracts of M. smegmatis, as compared to 125 mM and 54%, respectively for (23) [118]. As a continuation to these studies, a series of C-5 substituted biphenyl and acetylene analogues were developed and two compounds, (25) and (26), showed a marked increase in an in vitro assay against mtFabH [115, 116]. Compounds (25) and (26) gave 4-fold and 18-fold increase in activity with IC50 values of 4 and 17 mM, respectively, compared to 74.9 mM for (23). In contrast, others have suggested that C5 substitutions of the TLM core render the analogues inactive against M. tuberculosis [113]; this matter awaits clarification. However, the recent determination of the crystal structure of M. tuberculosis KasB and subsequent homology modelling of KasA, using the KasB structure as a template, supports the potential for C-5 derivatisation of the TLM scaffold towards the design of improved antimycobacterial KAS inhibitors (S. Sridharan, A.K. Brown, L.G. Dover, G.S. Besra and J.C. Sacchettini, unpublished results). (23) Thiolactomycin TLM R = Me Me R HO

Me S

O

Me

(24) R =

i-Pr OCH2Ph

(25) R = (26) R =

Ac

SIDEROPHORE BIOSYNTHESIS

Siderophores are low molecular mass ferric ion-specific chelating agents elaborated by various aerobic bacteria to assimilate iron [119]. Although the metal might be plentiful, as in the tissues of pathogen-infected host organisms, it is sequestered and hence readily not available to them. These tremendously powerful chelating agents, which can remove iron from insoluble inorganic polymers or from eukaryotic proteins, are commonly recognised as virulence factors of important bacterial pathogens, although some pathogens use alternate acquisition mechanisms. M. tuberculosis produces two biosynthetically-related siderophores: carboxymycobactins that operate in


185

the normal extracellular mode, returning chelated iron to the bacterium from its environment; and mycobactins that are unique among bacterial siderophores in remaining cell bound. Both types of siderophores have identical iron-binding centres and differ only in their long acyl chains; the water-soluble carboxymycobactins bear a terminal carboxylic acid group absent in the cell-bound mycobactins. Several of the genes associated with the synthesis of the mycobactin core have been identified in the 10-gene mbt cluster [120] and a further cluster recently designated mbt2 [121, 122]. Component genes of both clusters are essential for the in vivo growth of M. tuberculosis [123–125] and expression is regulated by iron availability. The first committed steps towards the biosynthesis of the iron binding centre of the (carboxy)mycobactins is the MbtA-catalysed adenylation of salicylic acid which activates the aryl acid for its subsequent transfer to a phosphopantetheinyl-serine residue of MbtB for its subsequent ligation with serine or threonine [120]. Recently two groups designed analogues of the salicyloyl–AMP intermediate (27) incorporating stable bioisosteres to replace the labile phosphate moiety. These showed good activity against M. tuberculosis grown under iron-limiting conditions [126, 127] at low micromolar concentrations. Especially impressive was an analogue in which the phosphoryl moiety was replaced with a sulfamide (28) that exhibited an MIC for M. tuberculosis of 0.19 mM [126]. NH2

NH2 OH

O

O

N

O

O P

O

N O

N N

OH

O

O N H

N H

N

N N

O OH

OH OH

(27)

N

O S

OH

(28)

The crystal structure of Rv1347c, a product of the mbt2 cluster, identified the protein as belonging to the GCN5-related N-acetyltransferase (GNAT) family [128], to which amino glycoside N-acetyltransferase (ACC) belongs [129]. Rv1347c ligates the long acyl group to the N-hydroxylysine side chain of mycobactin [121]. The structure has been refined to 2.2 A˚ resolution [130]. The protein is monomeric and contains an acyl–CoA binding site conserved with other GNAT family members and an adjacent hydrophobic channel leading to the surface that could accommodate long acyl chain groups. Modelling studies showed the N-hydroxylysine side chain of mycobactin

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into the acceptor substrate binding groove which identifies two residues at the active site, His130 and Asp168, that have been suggested to play roles in substrate binding and catalysis [130]. Siderophore biosynthesis represents a highly attractive target for antimicrobial development and the recent availability of structural data will facilitate this goal. PURINE ANALOGUES

Recently, the screening of novel compounds has highlighted modified purines as potential antimycobacterial drugs [131–134]. Screening of a library of 6-substituted and 2,6-di-substituted 9-benzyl-purines revealed several compounds with encouraging activity against M. tuberculosis, with a general trend suggesting that 6-substituted varieties were more effective than their di-substituted counterparts, although chlorination at the 2-position did tend to increase activity [131]. 2-Mono-substituted library members possessed no anti-TB activity. High inhibitory activity was found for those carrying phenylethynyl-, trans-stryryl or aryl substituents in the 6-position; the most active bears a furyl substituent (29) with MIC as low as 0.78 mg/ml, comparable to that for RIF, and representing a good lead. In order to optimise the N-9 substituent, a library of 6-aryl, 9-alkyl-purines was synthesised and screened confirming that the N-9 benzyl substituent used in the previous study showed greatest activity [133]. More extensive testing revealed that the 6-furyl derivative exhibited low cytotoxicity for VERO cells, retained activity against several singly drug-resistant strains of M. tuberculosis and was active (MIC 8.46 mg/ml) against M. tuberculosis Erdman in bone marrow macrophages [133]. O N

N Cl

N

N Ph

(29)

Further active 6,9-di-substituted purines with significant activity against M. tuberculosis were generated by reacting 6-mercaptopurine with sulfonyl/ sulfenyl halides [132]. Several compounds had low MICs between 0.39 and 3.9 mg/ml for M. tuberculosis H37Rv. Screening of two classes of derivative against M. tuberculosis strains resistant to nine of the main clinically used antimycobacterial agents. Although most displayed cross-resistance with


187

several of these agents, one compound (30) showed little cross-resistance and thus was identified as having great potential as a lead compound [132]. S N

HN

N

N

S

NO2

(30)

Screening of another library of 6-thiopurines incorporating 6-thioaryl/ alkyl-urines, 2-thioaryl/alkyl-pyrimidines and 2- and 4-thioaryl/alkyl-pyridines identified (31) and (32) with MICs of 1.56 and 0.78 mg/ml respectively against M. tuberculosis H37Rv, with (31) showing good activity against the Erdman strain in bone marrow macrophages [134]. Together these various studies demonstrate that these purine derivatives represent interesting antiTB leads. SR N

N N

N CO2Et

(31) R = n-C10H21 (32) R = n-C12H25

POTENTIAL NEW DRUG TARGETS TARGETING MYCOBACTERIAL PERSISTENCE

The glyoxylate shunt During the chronic stages of the infection in TB, the bacterium is thought to undergo a metabolic shift to efficiently utilise C2 substrates generated by b-oxidation of fatty acids as a carbon source [135–137]. Under these conditions, glycolysis is repressed and the glyoxylate shunt is significantly upregulated allowing anaplerotic maintenance of the tricarboxylic acid cycle [138]. The glyoxylate shunt requires two enzymes; the first, isocitrate lyase (ICL), converts isocitrate to succinate and glyoxylate while the second,

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malate synthase, catalyses the Mg2+-dependent condensation of glyoxylate and the acetyl moiety of acetyl–CoA to yield malate and CoA. Expression of icl is up-regulated under certain growth conditions [139] and during infections of macrophages by Mycobacterium spp. [140]. ICL is also important for the survival of M. tuberculosis in the lungs of mice during the persistent phase of infection (2–16 weeks), but not essential during the acute phase of infection (0–2 weeks) [136]. Also an icl mutant was markedly attenuated for survival in activated but not resting macrophages suggesting that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection [136]. As this shunt is thought to critical for M. tuberculosis survival in the persistent phase of infection and is not thought to be utilised in human metabolism [141], both enzymes represent excellent therapeutic targets. Determination of the high-resolution crystal structures of ICL in complex with inhibitors, 3-bromopyruvate and 3-nitropropionate (the latter with glyoxylate) revealed a large conformational change upon ligand binding. The enzyme adopted a ‘closed conformation’ upon binding 3-nitropropionate and glyoxylate [142, 143]. 3-Bromopyruvate formed a covalent adduct with the active site nucleophile Cys191 trapping the enzyme in a catalytic conformation in which the active site was completely inaccessible to solvent. GlaxoSmithKline currently have ICL inhibitors in the discovery phase of development [144]. Some strains of M. tuberculosis carry aceA, which encodes a second isocitrate lyase [139, 141]. In M. tuberculosis H37Rv, this ORF has a frame shift resulting in two ORFs (aceAa/Rv1915 and aceAb/Rv1916) [76] and probably represents a non-expressed pseudogene [139, 141], while it is read as one continuous ORF in the CDC1551 strain [145]. The continuous gene from the latter strain has been cloned, expressed, its product AceA, which is larger than common ICL enzymes [139, 141], purified. The enzyme was shown to possess ICL activity that is sensitive to known ICL inhibitors, 3-nitropropionate and 3-bromopyruvate [139]. AceA has 23% identity with both AceA from E. coli and ICL from M. tuberculosis and contains the signature catalytic motif KKCGH rather than that for the 2-methylisocitrate lyases [139]. As the deletion of icl cannot be compensated for by the presence of AceA [136], which is dispensable in the H37Rv strain, it appears that the metabolic roles of these two ICL enzymes differ and AceA might not participate in the glyoxylate shunt at all [139]. Elucidation of a highresolution crystal structure for AceA might facilitate the development of an inhibitor that is potent against both enzymes. A single gene, glcB, which encodes malate synthase, has been identified in M. tuberculosis. The structure of malate synthase from M. tuberculosis in complex with the substrate glyoxylate has been resolved to 2.1 A˚ resolution


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[139]. The determination of crystal structure of malate synthase complexed with its products malate and CoA revealed that two conserved residues Asp633 and Arg339 appear to play a vital role in the acid/base chemistry. High-throughput inhibitor screens and structure-based drug design are currently underway to identify a potential lead that targets malate synthase. Mycolic acid cyclopropane synthases Cyclopropanated mycolic acids are the major components in the cell wall of pathogenetic mycobacteria [146]. Recent studies have shown that these modifications have a profound effect on the fluidity and permeability of the cell wall [147–149], resistance of the mycobacteria to oxidative stress [150] and persistence in mice [151]. The crystal structures of all three of the Sadenosyl-methionine (SAM)-dependent methyltransferases, PcaA, CmaA1 and CmaA2, implicated in mycolate cyclopropanation have been determined [152]. PcaA is believed to catalyse the transfer of a methyl group to a cis double bond at the proximal position of a-mycolates [151]. However, the roles of CmaA1 and CmaA2 [148, 150] inferred from heterospecific overexpression studies do not agree with the structures of mycolates derived from knockout mutants, although they are all undoubtedly involved in mycolic acid modification [153, 154]. CmaA2 has also been suggested to act at the proximal position [148] but converts a trans double bond to a cyclopropane ring in distally oxygenated mycolates [154], while CmaA1 is thought to act at the distal position, giving rise to the cyclopropane ring of a-mycolates [150]. All three enzymes contain a tunnel approximately 15 A˚ 10 A˚ extending from the surface of the protein to the SAM co-factor binding site, which is believed to be the binding site of the acyl substrate and is lined with hydrophobic residues. Co-crystallisation of CmaA1 and CmaA2 in complex with their S-adenosylhomocysteine product and lipid-like detergents showed that Ile169, Leu192, Phe200, Leu205, Try232, Leu236, Phe273 and Leu278 were important residues for protein and acyl chain interactions and also provided insight into the orientation of the reactive group within the active site and the length of the acyl chain that the enzymes will accept [152]. The discovery of the precise geometry and topologies of the binding pockets of the enzyme will certainly aid development of potential inhibitors of cyclopropane synthases in M. tuberculosis. DosR-dependent transcriptional regulation The transcriptional regulator DosR is believed to mediate the transition of M. tuberculosis into dormancy [155–157], which may contribute to latency.

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Factors contributing to the onset of dormancy are hypoxia and nitroxide exposure [158, 159]. A DosR-dependent regulon comprising 47 M. tuberculosis genes is up-regulated in response to reduced oxygen tension and nitric oxide (NO) exposure [160, 161]. Although disruption of dosR was reported to increase virulence of M. tuberculosis in mice, it led to its attenuation in guinea pigs [162, 163]; the former observation leading authors to suggest that the regulon included a novel class of genes whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms [162]. The crystal structure of the DosR C-terminal domain and its complex with the DNA of a consensus hypoxia-induced gene promoter sequence has recently been elucidated and is unique among response regulators with known structures [164]. Its C-terminal domain forms tetramers and makes numerous protein–DNA base contacts using only three amino acid residues per subunit – Lys179, Lys182 and Asn183. The structure of DosR–DNA complex showed that each DosR C-terminal domain in a dimer places it DNA-binding helix deep into the major groove, causing the two bends in the DNA. The available structural data will help facilitate the development of DosR inhibitors, which may present potential therapies against latent TB.

THE STRINGENT RESPONSE

The relmtb gene encodes a protein that mediates a global regulation of protein synthesis known as the stringent response [165]. This dual-functional protein catalyses both the synthesis and hydrolysis of the hyper-phosphorylated guanosine, (p)ppGpp, the effector of the stringent response that acts as a signalling molecule to control bacterial gene expression involved in longterm survival under starvation conditions [166]. During amino acid starvation, the RelMtb protein catalyses the transfer of the 5-b-g-pyrophosphate group from ATP to the 30 -OH group of GTP or GDP [165]. As amino acid levels return to normal the stringent response is reversed by RelMtb, which like other Gram-positive Rel proteins, also catalyses the hydrolysis of the pyrophosphate group (PPi) from the 30 -OH of both pppGpp and ppGpp, yielding GTP or GDP [165]. The suggestion that these phosphorylation and dephosphorylation reactions occurred at different active sites [167] has recently been confirmed [168]. Mutations resulted in loss of synthetic activity and the retention of hydrolysis (G241E and H344Y) or loss of hydrolysis and retention of synthesis activity (H80A and D81A). Differential regulation of the opposing activities of RelMtb is dependent on the ratio of free and


191

amino acyl-tRNA and the association of RelMtb with a complex containing tRNA, ribosomes and mRNA [167]. Structural analyses and investigations of tRNA and ribosome binding by RelMtb will help elucidate the regulatory mechanism and identify other important residues that will aid the development of drug therapies targeting RelMtb. Prokaryotic toxin– antitoxin stress response systems Bacterial genomes contain genetic units called toxin–antitoxin (TA) modules that consist of paired genes; the second gene encodes a stable toxin and the first encodes a labile antitoxin that interferes with the lethal action of the toxin [169]. Bacterial cell death has been attributed to either a decrease in expression of the antitoxin or uncontrolled expression of the toxin. Originally these were thought to be involved in Programmed Cell Death (PCD), which can refer to any form of cell death mediated by an intracellular death program, no matter what triggers it and whether or not it displays all the characteristics of apoptosis [170]. Recently however, TA modules have been proposed to provide a control mechanism that helps free-living prokaryotes cope with nutritional stress rather than functioning in PCD [170]. Eight families of TA module have now been recognised and are found on plasmids, where they are involved in plasmid maintenance, and occupying chromosomal loci [169, 171]. Interestingly, accumulation of TA modules may be beneficial for organisms characterised by slow growth but obligate hostassociated pathogens, like M. leprae do not retain TA modules [171]. Consistent with this M. tuberculosis H37Rv and CDC1551 contain 38 and 36 TA modules respectively whereas the fast-growing M. smegmatis only has two TA modules [171]. These include three homologues of E. coli relBE, in which the toxin is a global translational inhibitor that acts by cleaving the coding regions of mRNA complexed with ribosomes [172–174], and nine homologues of E. coli mazEF loci, in which MazF cleaves mRNA at ACA sites independently of ribosomes [175, 176]. Several well-known antibiotics have been found to cause cell death through inhibition of transcription, translation and folic acid metabolism, resulting in thiamine starvation. RIF, chloramphenicol and spectinomycin inhibit translation and transcription leading to the expression of E. coli MazF, the toxin component of the MazEF TA module [177]. This indicates that targeting the MazEF homologues in M. tuberculosis may allow for the development of drugs that either induce the production of the toxin or inhibit the expression of the antitoxin. The recent elucidation of the MazEF structure from E. coli should help facilitate this goal [178].

192

TUBERCULOSIS CHEMOTHERAPY UNEXPLORED TARGETS IN CELL WALL BIOSYNTHESIS

The crucial arabinogalactan component of the M. tuberculosis cell wall core underpins the covalent tethering of the mycolic acid-based ‘pseudo’ outer membrane to its peptidoglycan sacculus and is referred to as the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. The mAGP structure is essential and provides the bacterium with a formidable protective barrier against toxic insult, such as antibiotics and components of the macrophage’s bactericidal arsenal [179]. The importance of the mAGP to the persistence of M. tuberculosis in vivo is underscored by the fact that several front-line anti-TB agents target essential components of the mycobacterial cell wall [102, 104, 180–182]. For instance, INH and ETH are both potent inhibitors of mycolic acid biosynthesis, targeting the enoyl-ACP reductase InhA [180, 183, 184]. Also, administration of EMB led to cessation of mycolic acid transfer to the cell wall [182, 185]. Subsequent studies have shown that EMB disrupts the synthesis of the arabinan component of arabinogalactan by targeting arabinosyltransferases EmbA and EmbB [102, 104, 181, 186–188]. The mycobacterial mAGP core is composed of a covalently linked complex of mycolic acids, D-arabinan and D-galactan domains that are attached to peptidoglycan via an a-L-Rhap-(1-3)-a-D-GlcNAc linkage unit (LU) [189, 190]. The galactan component is a linear alternating Galf polymer of around 30 residues possessing both 5-linked b-D-Galf and 6-linked b-D-Galf glycosyl residues from which three arabinan chains are attached to C-5 of the 6-linked b-D-Galf glycosyl residues [190]. These 5,6-linked Galf residues have recently been shown to be the 4th, 8th and 12th Galf residues of the galactan core in Corynebacterium glutamicum [188] and are likely similarly located in M. tuberculosis. The galactan is linked to the C-6 of some of the N-glycolymuramic acid residues of peptidoglycan via the LU. The mycolic acids are esterified to a terminal hexa-arabinofuranoside motif to form the inner leaflet of an asymmetric bilayer [190, 191]. A number of recent studies have defined the genetics and enzymology surrounding LU synthesis, notably, wbbL (Rv3265c) as the rhamnosyltransferase involved in the synthesis of lipid intermediate 2 [192]. A novel bifunctional galactofuranosyltransferase, GlfT, that possesses both b-D-(15)-Galf and b-D-(1-6)Galf transferase activities has been identified [193] that intuitively must be responsible for the bulk of galactan deposition in the mycobacterial cell wall. In addition, hetero-specific expression M. tuberculosis aftA has recently been shown to complement an arabinan-deficient mutant in Corynebacterium glutamicum by priming the galactan core with single Araf residues for further arabinosylation by EmbA and EmbB [194];


193

corynebacteria are recognised as good models for mycobacterial cell wall biosynthesis [195]. Based on the findings that wbbL is an essential enzyme [196], and ETH and INH target later steps involved in arabinan and mycolic acid biosynthesis, we suggest that the intermediate steps in mAGP synthesis, notably galactan polymerisation, represent extremely attractive novel drug targets. Reynolds and co-workers have developed a series of octyl-substituted disaccharides to mimic elements of AG and LU structure [197, 198]. These have proven useful as neoglycolipid acceptors for assaying mycobacterial glycosyltransferases and also have modest anti-mycobacterial activity. Compound (33), that mimics the junction of LU and AG, showed activity against M. tuberculosis H37Ra with an MIC of >12.8r128 mg/ml [198]. Similar MICs were reported for (34) and (35), based around disaccharides emulating the (b, 1-5) and (b, 1-6) linked di-Galf units [197]. Interestingly, however, neither of these compounds acted as substrates or inhibitors of mycobacterial galactofuranosyltransferases in vitro, most likely due to the size of the benzyl protecting groups, which might be removed in vivo. The potential for promotion of a non-specific cellular lysis through action as uncharged surfactants remains to be resolved [197]. Additionally, the L-rhamnosyl residue of the LU is an essential component, fundamental to the structural integrity of the cell wall as it links the peptidoglycan to galactan [199, 200]. The L-rhamnosyl component is provided by a sugar donor, dTDP-rhamnose from a four-step reaction [201]. The four enzymes, a-D-glucose-1-phosphate thymidylyltransferase (RmlA), dTPD-D-glucose-4,6-dehydratase (RmlB), dTPD-4-keto-6-deoxyglucose-3,5-epimerase (RmlC) [202] and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyse the conversion of a-D-glucose-1-phosphate and TTP to dTDP-rhamnose. As rhamnose is absent from human glycobiology then this pathway also has outstanding potential in terms of lead development. Crystallisation of RmlC has been achieved, crystal structure resolved to 1.7 A˚ [203] allowing an in silico structure-guided library approach to inhibitor design [204]. The commercially available 4-thiazolidinone scaffold, based on the precedent of the sugar nucleotide utilising MurB [205], was used to mimic the di-phosphate moiety of the activated sugar donor. Seven of these inhibitors, exemplified by (36), displayed modest activity in inhibiting growth of M. tuberculosis, but unfortunately the best inhibitors of a cell-free Rml enzyme assay showed no activity against whole cells [204]. The reasons behind this disparity are unknown, but may reflect poor penetration into the mycobacterial cells or metabolic inactivation of the inhibitors [204].

194

TUBERCULOSIS CHEMOTHERAPY O(CH2)7CH3

O(CH2)7CH3 Me O

OH O HO

O O

OH Me

HO

Me O

OH O O

Me

HO

O OH Me

HO

(33) OBn O OH O HO HO

H

O O Me

(34) O(CH2)7CH3

HO Me

S

O

N

OMe

O H OH

OBn

OBn

i-Pr EtO2C

(35)

(36)

CONCLUDING COMMENTS A slightly controversial issue is the discovery of new drugs that inhibit M. tuberculosis cell wall synthesis and whether they are going to have a major impact on TB control. It is felt that adding new drugs that act on actively growing tuberculosis organisms will only help with treating multidrug resistant organisms (admittedly an increasing problem) but may do little to crack the main problem, which is the length of time of treatment. For this, drugs that kill non-multiplying persistent organisms are needed. Some argue that cell wall synthesis may be already defective or unnecessary in these organisms (they are not stainable or otherwise detectable microscopically). We would strongly argue in the favour for the need for new drug targets and inhibitors that target the mycobacterial cell wall. Historically, inhibitors of cell wall synthesis since their discovery in the 1950s have been the mainstay of many chemotherapeutic regimens. Agents such as INH, an inhibitor of mycolic acid biosynthesis, when implemented into directly observed therapies have proved highly successful in combating ‘drug-sensitive’ tuberculosis. However, a key issue is the spread of MDR-TB. As a result the search for new drug targets and cheap alternative agents is greatly needed, which represents a central theme in tuberculosis drug development. While drugs that act on non-replicating forms appear desirable on theoretical grounds we are not aware of any direct evidence that their use would actually accelerate tuberculosis therapy. We simply do not know how new drugs will impact on time to cure and we need to await animal and clinical


195

trial data before drawing any such conclusions. Also, the challenge of drug resistance, as mentioned above, and adverse drug reactions means that in a significant number of cases we are searching for cheap effective alternative agents. As a result there will always be a place for new drugs in these areas, with cell wall inhibitors being highly profitable in the past. The view that cell wall synthesis in persistent organisms may be already defective or unnecessary based on staining or microscopy is not entirely correct. For instance, Stover et al. [84] reported that nitroimidazopyran PA-824 (18) was effective against static forms (i.e. cultures grown under anaerobic conditions) of M. tuberculosis by inhibiting both lipid and protein synthesis. Compound (18) was found to produce an accumulation of hydroxymycolic acids, a biosynthetic precursor of organic extractable and cell wall bound ketomycolic acids. Compound (18) was proposed to inhibit an enzyme or delete a co-factor responsible for the oxidation of hydroxymycolic acids to ketomycolic acids. As a result the study reports one of the first drugs to be active against persistent organisms and the targeting of the synthesis of mycolic acids and the cell wall. Therefore, a basal cell wall core, mycolyl-arabinogalactan-peptidoglycan is always maintained, and cell wall turnover possibly modulated through autolytic activity as found for other bacilli, which enter a dormant or persistent state. It is also interesting to note that we have observed through micro-array experiments (unpublished results) up-regulation of DesA3 (a target of isoxyl) and others have reported that CmaA2 [154] and KasB [179], involved in mycolic acid biosynthesis, are up-regulated under anaerobic conditions following proteome analyses [206]. The latter is an interesting finding suggesting a level of metabolic activity related to mycolic acid biosynthesis under anaerobic conditions that may be possibly linked, resulting in modulation of mycolic acid chain length during a dormant or persistent anaerobic state. CmaA2 would cyclopropanate unsaturated meromycolic acid precursors [154], and KasB would modulate mycolic acid chain length [179] through FAS-II. It would appear that enzymes that function to provide key substrates, unsaturated meromycolic acid precursors and enzymes linked to the elongation process via FAS-II, are relevant under a dormant or persistent anaerobic state.

ACKNOWLEDGMENTS GSB acknowledges support from Mr James Bardrick in the form of a Personal Chair, the Lister Institute as a former Jenner Research Fellow, the Medical Research Council (UK) and the Wellcome Trust.

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[32] [33] [34] [35]

[36] [37] [38] [39] [40] [41]

[42]

[43] [44] [45] [46] [47] [48] [49] [50] [51]

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Subject Index Acetohydroxyacid synthase 182 Acylpyrrolidides 28 Acylthiazolidides 28 Adrenocorticotropin 113 Agouti-protein 113 AgRP 113 Aloisine A 47 a-L-Rhap-(13)-a-D-GlcNAc linkage unit 192 Alvocidib see flavopiridol g-Amino-butyric acid 24 Aminoglycoside N-acetyltransferase 185 Aminoindazole 19 3-Aminopyrazole 40 Aminosalicyclic acid see p-aminosalicyclic acid Anorexia 137 Antifungals 179 Antidepressant 142 Anxiety 112 Anxiolytic 142 2-Arylaminothiazole 38 Azoles 179

Chemogenomics 6 Ciprofloxacin 178 Clebopride 22 Clotrimazole 179 CNS penetration 156 COMPARE 37 Computational modeling 152 Conformational analysis 148 Coumarin 21 CP-464709-18 157 Cyanopyrrolidides 30, 73 Cyclin-dependent kinase-1 36 ff Cyclin-dependent kinase-2 36 ff Cyclohexylpiperazines 126 ff Cyclohexylpiperidines 114 cycloserine 171 DARQ see diarylquinoline Denagliptin 83, 97 Depression 112 DEREK 8 Diarylquinoline 173 Dimethylaminopurine 38 Dipeptidyl peptidase IV 27 ff, 63 ff structural biology 69 Diprotin A 72 Diprotin B 72 Disaccharides 193 Diversity-orientated synthesis 4 DNA gyrase 180 DosR-dependent transcriptional regulation 189 DPP IV see dipeptidyl peptidase IV

BB-3497 182 BB-83698 182 BB-84518 182 Benzamide 19, 22 Benzamidines 143 Biaryl ureas 13 b-Ketoacyl-ACP synthase 174, 183 Blood brain barrier 156 BMS-477118 see saxagliptin Boronic acids 28, 72 3-Bromopyruvate 188

Econazole 179 EMB see ethambutol Energy homeostasis 112 Eperezolid 177 Erectogentic activity 112, 118, 122, 125 Ethambutol 171, 183 Extensive drug-resistant TB 172

Cachexia 137 Capreomycin 171 CDK1 see cyclin-dependent kinase Cell wall biosynthesis 192 CGI-17341 180 CGP 60474 41 205

206

SUBJECT INDEX

FAS20013 175 Feeding 112 Ferric ion-specific chelating agents 184 Flavopiridol 36 FlexX 35 Fluoro-olefins 80 Fluoropyrrolidides 85 Fragment assembly 6 Fragment screening 6 Galvus see vildagliptin gatifloxacin 178 GCN5-related N-acetyltransferase 185 GIP see glucose-dependent insulinotropic polypeptide GLP-1 see glucagon-like peptide-1 Glucagon-like peptide-1 64 Glucose-dependent insulinotropic polypeptide 65 Glyoxylate shunt 187 GPCR – see G-protein coupled receptor G-protein coupled receptor – directed library 15 HFRW motif 145 High throughput screening 3 Hits- assessing 8 Hits- validating 9 HS131 155 HTS see high throughput screening Hymenialdisine 49 Imidazoles 144 Incretin 64 Indirubin 41 Indoles 141 INH see isoniazid Isocitrate lyase 187 Isoniazid 170 ff Isoxazole 89 Januvia see sitagliptin kanamycin 171 KAS see b-Ketoacyl-ACP synthase Ketoazetidine 89 KHG20612 182

KHG20613 182 KHG20614 182 KHG20616 182 LAF237 see vildagliptin Latency 170 Lead – good 10 Lead generation 2 ff Lead identification 2 ff LEGEND 46 Linezolid 177 Literature-based innovation 5 LL-4858 see sudoterb Lymphocyte cell surface protein CD26 see dipeptidyl peptidase IV Malate synthase 187 MB243 121, 157 MBP10 114, 146 MC4R see melanocortin-4 receptor MCHR see melanin-concentrating hormone receptor MCL0020 148 MCL0042 143 MCL0129 141 MDR-TB see multidrug-resistant tuberculosis Melanin-concentrating hormone receptor 11 ff Melanocortin-4 receptor 111 ff mutagenesis 149 modeling 152 structure 150 Melanocortin-4 receptor antagonists 137 ff Meridianin E 47 Metronidazole 180 MK-0431 see sitagliptin ML00253764 143 Moxifloxacin 178 MSH 113, 115 MTII 114, 155 Multidrug-resistant tuberculosis 172 Mutagenesis 149 MXF see moxifloxacin Mycobacterial persistence 187 Mycobacterium tuberculosis 170 cytochrome P450 179 Mycobactin 188 Mycolic acid cyclopropane synthases 189

SUBJECT INDEX Mycolyl-arabinogalactan-peptidoglycan complex 192 Natural products 4 NDP-MSH 114 Nitroimidazopyran 180 NVP-DPP728 74 Octanesulfonylacetamide 174 Ofloxacin 178 Olomoucine 38 OPC 67683 181 OSA see octanesulfonylacetamide Oxazolidinones 176 Oxindoles 43 PA-824 182 PACAP38 see pituatary adenylate cyclaseactivating polypeptide 38 Pain 112 p-Aminosalicylic acid 171 PAS see p-aminosalicylic acid Patent-based innovation 5 PDF see peptide deformylase Peptide deformylase 182 Peptides 5, 146 conformational analysis 148 Phenylguanidines 135 Phenylpiperidines 126 ff, 140 Pituatary adenylate cyclase-activating polypeptide 38 67 PNU-100480 179 Prokaryotic toxin-antitoxin stress response 191 Pro-opiomelanocortin 115 PSN 9301 97 Purines 186 Purvalanol A 38 Purvalanol B 38 Pyrazinamide 170 ff Pyridazones 133 ff Pyrido-pyrimidine 46 Pyrimidines 43, 91 Pyrrolidines 126 PZA see pyrazinamide Quinolones 178

R207910 see TMC207 RBx 7644 177 RBx 8700 177 RIF see rifampicin Rifampicin 171 Rifamycin 171 Rm1A 193 Rm1B 193 Rm1C 193 Rm1D 193 Rv1347c 185 RY764 123 RY764 157 S-adenosyl-methionine-dependent methyl tranferases 189 Saxagliptin 81, 96 SHU9119 114, 146, 149 Siderophores 184 Sitagliptin 31, 68, 94, 96, 102 Site-directed mutagenesis 155 SM see streptomycin SNAP-7941 12 SQ109 185 Streptomycin 171 Stringent response 190 Structural biology 68 Structure-base design 5 SU9516 44 Sudoterb 181 Sulfonylcarboxamides 176 Sulphostin 33, 90 Syr-322 96 T-226296 12, 22 Tetrahydroisoquinolines 114, 132, 153 Tetrahydropyran 137 Tetrazoles 118 Thiadiazoles 133 Thiazole 77 Thiazolidide 84, 93 Thiazolidine 77 Thiolactomycin 183 THIQ see tetrahydroisoquinolines TMC-2 33, 72 TMC207 173 Topoisomerase 178 TPZ see trifluoroperazine

207

208 Trifluoroperazine 176 TSL-255 72 Tuberculosis 169 ff extensive drug-resistant 172 multidrug-resistant 172

SUBJECT INDEX Vildagliptin 75, 96 Viomycin 171 Virtual HTS 6 XDR-TB see extensive drug-resistant TB

Cumulative Index of Authors for Volumes 1– 45 The volume number, (year of publication) and page number are given in that order. Belliard, S., 34 (1997) 1 Benfey, B.G., 12 (1975) 293 Bentue´-Ferrer, D., 34 (1997) 1 Bernstein, P.R., 31 (1994) 59 Besra, G.S., 45 (2007) 169 Bhowruth, V., 45 (2007) 169 Binnie, A., 37 (2000) 83 Bischoff, E., 41 (2003) 249 Black, M.E., 11 (1975) 67 Blandina, P., 22 (1985) 267 Bond, P.A., 11 (1975) 193 Bonta, I.L., 17 (1980) 185 Booth, A.G., 26 (1989) 323 Boreham, P.F.I., 13 (1976) 159 Bo¨s, M., 44 (2006) 65 Bowman, W.C., 2 (1962) 88 Bradner, W.T., 24 (1987) 129 Bragt, P.C., 17 (1980) 185 Brain, K.R., 36 (1999) 235 Branch, S.K., 26 (1989) 355 Braquet, P., 27 (1990) 325 Brezina, M., 12 (1975) 247 Brooks, B.A., 11 (1975) 193 Brown, J.R., 15 (1978) 125 Brunelleschi, S., 22 (1985) 267 Bruni, A., 19 (1982) 111 Buckingham, J.C., 15 (1978) 165 Bulman, R.A., 20 (1983) 225

Aboul-Ela, F., 39 (2002) 73 Adam, J., 44 (2006) 209 Adams, J.L., 38 (2001) 1 Adams, S.S., 5 (1967) 59 Afshar, M., 39 (2002) 73 Agrawal, K.C., 15 (1978) 321 Albrecht, W.J., 18 (1981) 135 Allain, H., 34 (1997) 1 Allen, M.J., 44 (2006) 335 Allen, N.A., 32 (1995) 157 Allender, C.J., 36 (1999) 235 Altmann, K.-H., 42 (2004) 171 Andrews, P.R., 23 (1986) 91 Ankersen, M., 39 (2002) 173 Ankier, S.I., 23 (1986) 121 Appendino, G., 44 (2006) 145 Arrang, J.-M., 38 (2001) 279 Armour, D., 43 (2005) 239 Aubart, K., 44 (2006) 109 Badger, A.M., 38 (2001) 1 Bailey, E., 11 (1975) 193 Ballesta, J.P.G., 23 (1986) 219 Banting, L., 26 (1989) 253; 33 (1996) 147 Barbier, A.J., 44 (2006) 181 Barker, G., 9 (1973) 65 Barnes, J.M., 4 (1965) 18 Barnett, M.I., 28 (1991) 175 Batt, D.G., 29 (1992) 1 Beaumont, D., 18 (1981) 45 Beckett, A.H., 2 (1962) 43; 4 (1965) 171 Beckman, M.J., 35 (1998) 1 Beddell, C.R., 17 (1980) 1 Beedham, C., 24 (1987) 85 Beeley, L.J., 37 (2000) 1 Beher, D., 41 (2003) 99 Beisler, J.A., 19 (1975) 247 Bell, J.A., 29 (1992) 239

Camaioni, E., 42 (2004) 125 Carman-Krzan, M., 23 (1986) 41 Carruthers, N.I., 44 (2006) 181 Cassells, A.C., 20 (1983) 119 Casy, A.F., 2 (1962) 43; 4 (1965) 171; 7 (1970) 229; 11 (1975) 1; 26 (1989) 355 Casy, G., 34 (1997) 203 Caton, M.P.L., 8 (1971) 217; 15 (1978) 357 Chambers, M.S., 37 (2000) 45 209

210

CUMULATIVE AUTHOR INDEX

Chang, J., 22 (1985) 293 Chappel, C.I., 3 (1963) 89 Chatterjee, S., 28 (1991) 1 Chawla, A.S., 17 (1980) 151; 22 (1985) 243 Chen, C., 45 (2007) 111 Cheng, C.C., 6 (1969) 67; 7 (1970) 285; 8 (1971) 61; 13 (1976) 303; 19 (1982) 269; 20 (1983) 83; 25 (1988) 35 Cherry, M., 44 (2006) 1 Clark, R.D., 23 (1986) 1 Clitherow, J.W., 41 (2003) 129 Cobb, R., 5 (1967) 59 Cochrane, D.E., 27 (1990) 143 Corbett, J.W., 40 (2002) 63 Costantino, G., 42 (2004) 125 Coulton, S., 31 (1994) 297; 33 (1996) 99 Cowley, P.M., 44 (2006) 209 Cox, B., 37 (2000) 83 Crossland, J., 5 (1967) 251 Crowshaw, K., 15 (1978) 357 Cushman, D.W., 17 (1980) 41 Cuthbert, A.W., 14 (1977) 1 Dabrowiak, J.C., 24 (1987) 129 Daly, M.J., 20 (1983) 337 D’Arcy, P.F., 1 (1961) 220 Daves, G.D., 13 (1976) 303; 22 (1985) 1 Davies, G.E., 2 (1962) 176 Davies, R.V., 32 (1995) 115 De Clercq, E., 23 (1986) 187 De Gregorio, M., 21 (1984) 111 De Luca, H.F., 35 (1998) 1 De, A., 18 (1981) 117 Deaton, D.N., 42 (2004) 245 Demeter, D.A., 36 (1999) 169 Denyer, J.C., 37 (2000) 83 Derouesne´, C., 34 (1997) 1 Dimitrakoudi, M., 11 (1975) 193 Donnelly, M.C., 37 (2000) 83 Dover, L.G., 45 (2007) 169 Draffan, G.H., 12 (1975) 1 Drewe, J.A., 33 (1996) 233 Drysdale, M.J., 39 (2002) 73 Dubinsky, B., 36 (1999) 169 Duckworth, D.M., 37 (2000) 1 Duffield, J.R., 28 (1991) 175 Durant, G.J., 7 (1970) 124 Dvorak, C.A., 44 (2006) 181

Eccleston, J.F., 43 (2005) 19 Edwards, D.I., 18 (1981) 87 Edwards, P.D., 31 (1994) 59 Eglen, R.M., 43 (2005) 105 Eldred, C.D., 36 (1999) 29 Ellis, G.P., 6 (1969) 266; 9 (1973) 65; 10 (1974) 245 Evans, B., 37 (2000) 83 Evans, J.M., 31 (1994) 409 Falch, E., 22 (1985) 67 Fantozzi, R., 22 (1985) 267 Feigenbaum, J.J., 24 (1987) 159 Ferguson, D.M., 40 (2002) 107 Feuer, G., 10 (1974) 85 Finberg, J.P.M., 21 (1984) 137 Fletcher, S.R., 37 (2000) 45 Flo¨rsheimer, A., 42 (2004) 171 Floyd, C.D., 36 (1999) 91 Franc- ois, I., 31 (1994) 297 Frank, H., 27 (1990) 1 Freeman, S., 34 (1997) 111 Fride, E., 35 (1998) 199 Gale, J.B., 30 (1993) 1 Ganellin, C.R., 38 (2001) 279 Garbarg, M., 38 (2001) 279 Garratt, C.J., 17 (1980) 105 Gerspacher, M., 43 (2005) 49 Gill, E.W., 4 (1965) 39 Gillespie, P., 45 (2007) 1 Ginsburg, M., 1 (1961) 132 Glennon, R.A., 42 (2004) 55 Goldberg, D.M., 13 (1976) 1 Goodnow, Jr. R.A., 45 (2007) 1 Gould, J., 24 (1987) 1 Graczyk, P.P., 39 (2002) 1 Graham, J.D.P., 2 (1962) 132 Green, A.L., 7 (1970) 124 Green, D.V.S., 37 (2000) 83; 41 (2003) 61 Greenhill, J.V., 27 (1990) 51; 30 (1993) 206 Griffin, R.J., 31 (1994) 121 Griffiths, D., 24 (1987) 1 Griffiths, K., 26 (1989) 299 Groenewegen, W.A., 29 (1992) 217 Groundwater, P.W., 33 (1996) 233 Guile, S.D., 38 (2001) 115 Gunda, E.T., 12 (1975) 395; 14 (1977) 181

CUMULATIVE AUTHOR INDEX Gylys, J.A., 27 (1990) 297 Hacksell, U., 22 (1985) 1 Haefner, B., 43 (2005) 137 Hall, A.D., 28 (1991) 41 Hall, S.B., 28 (1991) 175 Halldin, C., 38 (2001) 189 Halliday, D., 15 (1978) 1 Hammond, S.M., 14 (1977) 105; 16 (1979) 223 Hamor, T.A., 20 (1983) 157 Haning, H., 41 (2003) 249 Hanson, P.J., 28 (1991) 201 Hanus, L., 35 (1998) 199 Hargreaves, R.B., 31 (1994) 369 Harris, J.B., 21 (1984) 63 Harrison, T., 41 (2003) 99 Hartley, A.J., 10 (1974) 1 Hartog, J., 15 (1978) 261 Heacock, R.A., 9 (1973) 275; 11 (1975) 91 Heard, C.M., 36 (1999) 235 Heinisch, G., 27 (1990) 1; 29 (1992) 141 Heller, H., 1 (1961) 132 Henke, B.R., 42 (2004) 1 Heptinstall, S., 29 (1992) 217 Herling, A.W., 31 (1994) 233 Hider, R.C., 28 (1991) 41 Hill, S.J., 24 (1987) 30 Hillen, F.C., 15 (1978) 261 Hino, K., 27 (1990) 123 Hjeds, H., 22 (1985) 67 Holdgate, G.A., 38 (2001) 309 Hooper, M., 20 (1983) 1 Hopwood, D., 13 (1976) 271 Hosford, D., 27 (1990) 325 Hu, B., 41 (2003) 167 Hubbard, R.E., 17 (1980) 105 Hudkins, R.L., 40 (2002) 23 Hughes, R.E., 14 (1977) 285 Hugo, W.B., 31 (1994) 349 Hulin, B., 31 (1994) 1 Humber, L.G., 24 (1987) 299 Hunt, E., 33 (1996) 99 Hutchinson, J.P., 43 (2005) 19 Ijzerman, A.P., 38 (2001) 61 Imam, S.H., 21 (1984) 169 Ince, F., 38 (2001) 115

211

Ingall, A.H., 38 (2001) 115 Ireland, S.J., 29 (1992) 239 Jacques, L.B., 5 (1967) 139 James, K.C., 10 (1974) 203 Jameson, D.M., 43 (2005) 19 Ja´szbereńyi, J.C., 12 (1975) 395; 14 (1977) 181 Jenner, F.D., 11 (1975) 193 Jennings, L.L., 41 (2003) 167 Jewers, K., 9 (1973) 1 Jindal, D.P., 28 (1991) 233 Jones, B.C., 41 (2003) 1 Jones, D.W., 10 (1974) 159 Jorvig, E., 40 (2002) 107 Judd, A., 11 (1975) 193 Judkins, B.D., 36 (1999) 29 Kadow, J.F., 32 (1995) 289 Kapoor, V.K., 16 (1979) 35; 17 (1980) 151; 22 (1985) 243; 43 (2005) 189 Kawato, Y., 34 (1997) 69 Kelly, M.J., 25 (1988) 249 Kendall, H.E., 24 (1987) 249 Kennis, L.E.J., 33 (1996) 185 Khan, M.A., 9 (1973) 117 Kiefel, M.J., 36 (1999) 1 Kilpatrick, G.J., 29 (1992) 239 Kindon, N.D., 38, (2001) 115 King, F.D., 41 (2003) 129 Kirst, H.A., 30 (1993) 57; 31 (1994) 265 Kitteringham, G.R., 6 (1969) 1 Kiyoi, T., 44 (2006) 209 Knight, D.W., 29 (1992) 217 Kobayashi, Y., 9 (1973) 133 Koch, H.P., 22 (1985) 165 Kopelent-Frank, H., 29 (1992) 141 Kramer, M.J., 18 (1981) 1 Krause, B.R., 39 (2002) 121 KrogsgaardLarsen, P., 22 (1985) 67 Kulkarni, S.K., 37 (2000) 135 Kumar, K., 43 (2005) 189 Kumar, M., 28 (1991) 233 Kumar, S., 38 (2001) 1; 42 (2004) 245 Kwong, A.D., 39 (2002) 215 Lambert, P.A., 15 (1978) 87 Launchbury, A.P., 7 (1970) 1 Law, H.D., 4 (1965) 86

212


Lawen, A., 33 (1996) 53 Lawson, A.M., 12 (1975) 1 Leblanc, C., 36 (1999) 91 Lee, C.R., 11 (1975) 193 Lee, J.C., 38 (2001) 1 Lenton, E.A., 11 (1975) 193 Lentzen, G., 39 (2002) 73 Letavic, M.A., 44 (2006) 181 Levin, R.H., 18 (1981) 135 Lewis, A.J., 19 (1982) 1; 22 (1985) 293 Lewis, D.A., 28 (1991) 201 Lewis, J.A., 37 (2000) 83 Li, Y., 43 (2005) 1 Lien, E.L., 24 (1987) 209 Ligneau, X., 38 (2001) 279 Lin, T.-S., 32 (1995) 1 Liu, M.-C., 32 (1995) 1 Livermore, D.G.H., 44 (2006) 335 Llinas-Brunet, M., 44 (2006) 65 Lloyd, E.J., 23 (1986) 91 Lockhart, I.M., 15 (1978) 1 Lord, J.M., 24 (1987) 1 Lowe, I.A., 17 (1980) 1 Lucas, R.A., 3 (1963) 146 Lue, P., 30 (1993) 206 Luscombe, D.K., 24 (1987) 249 Mackay, D., 5 (1967) 199 Main, B.G., 22 (1985) 121 Malhotra, R.K., 17 (1980) 151 Malmstro¨m, R.E., 42 (2004) 207 Manchanda, A.H., 9 (1973) 1 Mander, T.H., 37 (2000) 83 Mannaioni, P.F., 22 (1985) 267 Maroney, A.C., 40 (2002) 23 Martin, I.L., 20 (1983) 157 Martin, J.A., 32 (1995) 239 Masini, F., 22 (1985) 267 Matassova, N., 39 (2002) 73 Matsumoto, J., 27 (1990) 123 Matthews, R.S., 10 (1974) 159 Maudsley, D.V., 9 (1973) 133 May, P.M., 20 (1983) 225 McCague, R., 34 (1997) 203 McFadyen, I., 40 (2002) 107 McLelland, M.A., 27 (1990) 51 McNeil, S., 11 (1975) 193

Mechoulam, R., 24 (1987) 159; 35 (1998) 199 Meggens, A.A.H.P., 33 (1996) 185 Megges, R., 30 (1993) 135 Meghani, P., 38 (2001) 115 Merritt, A.T., 37 (2000) 83 Metzger, T., 40 (2002) 107 Michel, A.D., 23 (1986) 1 Middlemiss, D.N., 41 (2003) 129 Miura, K., 5 (1967) 320 Moncada, S., 21 (1984) 237 Monkovic, I., 27 (1990) 297 Montgomery, J.A., 7 (1970) 69 Moody, G.J., 14 (1977) 51 Mordaunt, J.E., 44 (2006) 335 Morris, A., 8 (1971) 39; 12 (1975) 333 Morrison, A.J., 44 (2006) 209 Mort, C.J.W., 44 (2006) 209 Mortimore, M.P., 38 (2001) 115 Munawar, M.A., 33 (1996) 233 Murchie, A.I.H., 39 (2002) 73 Murphy, F., 2 (1962) 1; 16 (1979) 1 Musallan, H.A., 28 (1991) 1 Musser, J.H., 22 (1985) 293 Natoff, I.L., 8 (1971) 1 Neidle, S., 16 (1979) 151 Nicholls, P.J., 26 (1989) 253 Niewo¨hner, U., 41 (2003) 249 Nodiff, E.A., 28 (1991) 1 Nordlind, K., 27 (1990) 189 Nortey, S.O., 36 (1999) 169 O’Hare, M., 24 (1987) 1 O’Reilly, T., 42 (2004) 171 Ondetti, M.A., 17 (1980) 41 Ottenheijm, H.C.J., 23 (1986) 219 Oxford, A.W., 29 (1992) 239 Paget, G.E., 4 (1965) 18 Palatini, P., 19 (1982) 111 Palazzo, G., 21 (1984) 111 Palfreyman, M.N., 33 (1996) 1 Palmer, D.C., 25 (1988) 85 Parkes, M.W., 1 (1961) 72 Parnham, M.J., 17 (1980) 185 Parratt, J.R., 6 (1969) 11 Patel, A., 30 (1993) 327

CUMULATIVE AUTHOR INDEX Paul, D., 16 (1979) 35; 17 (1980) 151 Pearce, F.L., 19 (1982) 59 Peart, W.S., 7 (1970) 215 Pellicciari, R., 42 (2004) 125 Perni, R.B., 39 (2002) 215 Petrow, V., 8 (1971) 171 Picard, J.A., 39 (2002) 121 Pike, V.W., 38 (2001) 189 Pinder, R.M., 8 (1971) 231; 9 (1973) 191 Poda, G., 40 (2002) 107 Ponnudurai, T.B., 17 (1980) 105 Powell, W.S., 9 (1973) 275 Power, E.G.M., 34 (1997) 149 Price, B.J., 20 (1983) 337 Prior, B., 24 (1987) 1 Procopiou, P.A., 33 (1996) 331 Purohit, M.G., 20 (1983) 1 Ram, S., 25 (1988) 233 Rampe, D., 43 (2005) 1 Reader, J., 44 (2006) 1 Reckendorf, H.K., 5 (1967) 320 Reddy, D.S., 37 (2000) 135 Redshaw, S., 32 (1995) 239 Rees, D.C., 29 (1992) 109 Reitz, A.B., 36 (1999) 169 Repke, K.R.H., 30 (1993) 135 Richards, W.G., 11 (1975) 67 Richardson, P.T., 24 (1987) 1 Roberts, L.M., 24 (1987) 1 Rodgers, J.D., 40 (2002) 63 Roe, A.M., 7 (1970) 124 Rose, H.M., 9 (1973) 1 Rosen, T., 27 (1990) 235 Rosenberg, S.H., 32 (1995) 37 Ross, K.C., 34 (1997) 111 Roth, B., 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Roth, B.D., 40 (2002) 1 Russell, A.D., 6 (1969) 135; 8 (1971) 39; 13 (1976) 271; 31 (1994) 349; 35 (1998) 133 Ruthven, C.R.J., 6 (1969) 200 Sadler, P.J., 12 (1975) 159 Sampson, G.A., 11 (1975) 193 Sandler, M., 6 (1969) 200 Saporito, M.S., 40 (2002) 23 Sarges, R., 18 (1981) 191

213

Sartorelli, A.C., 15 (1978) 321; 32.(1995) 1 Saunders, J., 41 (2003) 195 Schiller, P.W., 28 (1991) 301 Schmidhammer, H., 35 (1998) 83 Scho¨n, R., 30 (1993) 135 Schunack, W., 38 (2001) 279 Schwartz, J.-C., 38 (2001) 279 Schwartz, M.A., 29 (1992) 271 Scott, M.K., 36 (1999) 169 Sewell, R.D.E., 14 (1977) 249; 30 (1993) 327 Shank, R.P., 36 (1999) 169 Shaw, M.A., 26 (1989) 253 Sheard, P., 21 (1984) 1 Shepherd, D.M., 5 (1967) 199 Silver, P.J., 22 (1985) 293 Silvestrini, B., 21 (1984) 111 Singh, H., 16 (1979) 35; 17 (1980) 151; 22 (1985) 243; 28 (1991) 233 Skotnicki, J.S., 25 (1988) 85 Slater, J.D.H., 1 (1961) 187 Sliskovic, D.R., 39 (2002) 121 Smith, H.J., 26 (1989) 253; 30 (1993) 327 Smith, R.C., 12 (1975) 105 Smith, W.G., 1 (1961) 1; 10 (1974) 11 Solomons, K.R.H., 33 (1996) 233 Sorenson, J.R.J., 15 (1978) 211; 26 (1989) 437 Souness, J.E., 33 (1996) 1 Southan, C., 37 (2000) 1 Spencer, P.S.J., 4 (1965) 1; 14 (1977) 249 Spinks, A., 3 (1963) 261 Sta˚hle, L., 25 (1988) 291 Stark, H., 38 (2001) 279 Steiner, K.E., 24 (1987) 209 Stenlake, J.B., 3 (1963) 1; 16 (1979) 257 Stevens, M.F.G., 13 (1976) 205 Stewart, G.A., 3 (1963) 187 Studer, R.O., 5 (1963) 1 Subramanian, G., 40 (2002) 107 Sullivan, M.E., 29 (1992) 65 Suschitzky, J.L., 21 (1984) 1 Swain, C.J., 35 (1998) 57 Swallow, D.L., 8 (1971) 119 Sykes, R.B., 12 (1975) 333 Szallasi, A., 44 (2006) 145 Talley, J.J., 36 (1999) 201 Taylor, E.C., 25 (1988) 85

214


Taylor, E.P., 1 (1961) 220 Taylor, S.G., 31 (1994) 409 Tegne´r, C., 3 (1963) 332 Terasawa, H., 34 (1997) 69 Thomas, G.J., 32 (1995) 239 Thomas, I.L., 10 (1974) 245 Thomas, J.D.R., 14 (1977) 51 Thompson, E.A., 11 (1975) 193 Thompson, M., 37 (2000) 177 Tilley, J.W., 18 (1981) 1 Timmerman, H., 38 (2001) 61 Traber, R., 25 (1988) 1 Tucker, H., 22 (1985) 121 Tyers, M.B., 29 (1992) 239 Upton, N., 37 (2000) 177 Valler, M.J., 37 (2000) 83 Van de Waterbeemd, H., 41 (2003) 1 Van den Broek, L.A.G.M., 23 (1986) 219 Van Dijk, J., 15 (1978) 261 Van Muijlwijk-Koezen, J.E., 38 (2001) 61 Van Wart, H.E., 29 (1992) 271 Vaz, R.J., 43 (2005) 1 Vincent, J.E., 17 (1980) 185 Volke, J., 12 (1975) 247 Von Itzstein, M., 36 (1999) 1 Von Seeman, C., 3 (1963) 89 Von Wartburg, A., 25 (1988) 1 Vyas, D.M., 32 (1995) 289 Waigh, R.D., 18 (1981) 45 Wajsbort, J., 21 (1984) 137 Walker, R.T., 23 (1986) 187 Walls, L.P., 3 (1963) 52 Walz, D.T., 19 (1982) 1 Ward, W.H.J., 38 (2001) 309 Waring, W.S., 3 (1963) 261 Wartmann, M., 42 (2004) 171 Watson, N.S., 33 (1996) 331 Watson, S.P., 37 (2000) 83

Wedler, F.C., 30 (1993) 89 Weidmann, K., 31 (1994) 233 Weiland, J., 30 (1993) 135 West, G.B., 4 (1965) 1 White, P.W., 44 (2006) 65 Whiting, R.L., 23 (1986) 1 Whittaker, M., 36 (1999) 91 Whittle, B.J.R., 21 (1984) 237 Wiedling, S., 3 (1963) 332 Wiedeman, P.E., 45 (2007) 63 Wien, R., 1 (1961) 34 Wikstro¨m, H., 29 (1992) 185 Wikstro¨m, H.V., 38 (2001) 189 Wilkinson, S., 17 (1980) 1 Williams, D., 44 (2006) 1 Williams, D.R., 28 (1991) 175 Williams, J., 41 (2003) 195 Williams, J.C., 31 (1994) 59 Williams, K.W., 12 (1975) 105 Williams-Smith, D.L., 12 (1975) 191 Wilson, C., 31 (1994) 369 Wilson, H.K., 14 (1977) 285 Witte, E.C., 11 (1975) 119 Wold, S., 25 (1989) 291 Wood, A., 43 (2005) 239 Wood, E.J., 26 (1989) 323 Wright, I.G., 13 (1976) 159 Wyard, S.J., 12 (1975) 191 Wyman, P.A., 41 (2003) 129 Yadav, M.R., 28 (1991) 233 Yates, D.B., 32 (1995) 115 Youdim, M.B.H., 21 (1984) 137 Young, P.A., 3 (1963) 187 Young, R.N., 38 (2001) 249 Zalacain, M., 44 (2006) 109 Zee-Cheng, R.K.Y., 20 (1983) 83 Zon, G., 19 (1982) 205 Zylicz, Z., 23 (1986) 219

Cumulative Index of Subjects for Volumes 1– 45 The volume number, (year of publication) and page number are given in that order. Antiarthritic agents, 15 (1978) 211; 19 (1982) 1; 36 (1999) 201 Anti-atherosclerotic agents, 39 (2002) 121 Antibacterial agents, 6 (1969) 135; 12 (1975) 333; 19 (1982) 269; 27 (1990) 235; 30 (1993) 203; 31 (1994) 349; 34 (1997) resistance to, 32 (1995) 157; 35 (1998) 133 Antibiotics, antitumour, 19 (1982) 247; 23 (1986) 219 carbapenem, 33 (1996) 99 X-lactam, 12 (1975) 395; 14 (1977) 181; 31 (1994) 297; 33 (1996) 99 macrolide, 30 (1993) 57; 32 (1995) 157 mechanisms of resistance, 35 (1998) 133 polyene, 14 (1977) 105; 32 (1995) 157 resistance to, 31 (1994) 297; 32 (1995) 157; 35 (1998) 133 Anticancer agents — see Antibiotics, Antitumour agents Anticonvulsant drugs, 3 (1963) 261; 37 (2000) 177 Antidepressant drugs, 15 (1978) 261; 23 (1986) 121 Antidiabetic agents, 41 (2003) 167; 42 (2004) 1 Antiemetic action of 5-HT3 antagonists, 27 (1990) 297; 29 (1992) 239 Antiemetic drugs, 27 (1990) 297; 29 (1992) 239 Antiepileptic drugs, 37 (2000) 177 Antifilarial benzimidazoles, 25 (1988) 233 Antifolates as anticancer agents, 25 (1988) 85; 26 (1989) 1 Antifungal agents, 1 (1961) 220 Antihyperlipidemic agents, 11 (1975) 119

ACAT inhibitors, 39 (2002) 121 Adamantane, amino derivatives, 18 (1981) 1 Adenosine A3 receptor ligands, 38 (2001) 61 Adenosine triphosphate, 16 (1979) 223 Adenylate cyclase, 12 (1975) 293 Adipose tissue, 17 (1980) 105 Adrenergic agonists, b3-, 41 (2003) 167 Adrenergic blockers, a-, 23 (1986) 1 X-, 22 (1985) 121 a2-Adrenoceptors, antagonists, 23 (1986) 1 Adrenochrome derivatives, 9 (1973) 275 Adriamycin, 15 (1978) 125; 21 (1984) 169 AIDS, drugs for, 31 (1994) 121 Aldehyde thiosemicarbazones as antitumour agents, 15 (1978) 321; 32 (1995) 1 Aldehydes as biocides, 34 (1997) 149 Aldose reductase inhibitors, 24 (1987) 299 Allergy, chemotherapy of, 21 (1984) 1; 22 (1985) 293 Alzheimer’s disease, chemotherapy of, 34 (1997) 1; 36 (1999) 201 M1 agonists in, 43 (2005) 113 Amidines and guanidines, 30 (1993) 203 Aminoadamantane derivatives, 18 (1981) 1 Aminopterins as antitumour agents, 25 (1988) 85 8-Aminoquinolines as antimalarial drugs, 28 (1991) 1; 43 (2005) 220 Analgesic drugs, 2 (1962) 43; 4 (1965) 171; 7 (1970) 229; 14 (1977) 249 Anaphylactic reactions, 2 (1962) 176 Angiotensin, 17 (1980) 41; 32 (1995) 37 Anthraquinones, antineoplastic, 20 (1983) 83 Antiallergic drugs, 21 (1984) 1; 22 (1985) 293; 27 (1990) 34 Antiapoptotic agents, 39 (2002) 1 Antiarrhythmic drugs, 29 (1992) 65 215

216

CUMULATIVE SUBJECT INDEX

Anti-inflammatory action of cyclooxygenase-2 (COX-2) inhibitors, 36 (1999) 201 of thalidomide, 22 (1985) 165 of 5-lipoxygenase inhibitors, 29 (1992) 1 of p38 MAP kinase inhibitors, 38 (2001) 1 Anti-inflammatory agents, 5 (1967) 59; 36 (1999) 201; 38 (2001) 1; 39 (2002) 1 Antimalarial agents, 43 (2005) 189 Antimalarial 8-aminoquinolines, 28 (1991) 1 Antimicrobial agents for sterilization, 34 (1997) 149 Antineoplastic agents, a new approach, 25 (1988) 35 anthraquinones as, 20 (1983) 83 Anti-osteoporosis drugs, 42 (2004) 245 Antipsychotic drugs, 33 (1996) 185 Ami-rheumatic drugs, 17 (1980) 185; 19 (1982) 1; 36 (1999) 201 Antisecretory agents, 37 (2000) 45 Antithrombotic agents, 36 (1999) 29 Antitumour agents, 9 (1973) 1; 19 (1982) 247; 20 (1983) 83; 23 (1986) 219; 24 (1987) 1, 129; 25 (1988) 35, 85; 26 (1989) 253, 299; 30 (1993) 1; 32 (1995) 1, 289; 34 (1997) 69; 42 (2004) 171 Antitussive drugs, 3 (1963) 89 Anti-ulcer drugs, of plant origin, 28 (1991) 201 ranitidine, 20 (1983) 67 synthetic, 30 (1993) 203 Antiviral agents, 8 (1971) 119; 23 (1986) 187; 36 (1999) 1; 39 (2002) 215 Anxiety neurokinin receptors in, 43 (2005) 53 Anxiolytic agents, CCK-B antagonists as, 37 (2000) 45 Anxiolytic agents, pyrido[l,2-a]benzimidazoles as, 36 (1999) 169 Aromatase inhibition and breast cancer, 26 (1989) 253; 33 (1996) 147 Arthritis neurokinin receptors in, 43 (2005) 53 Aspartic proteinase inhibitors, 32 (1995) 37, 239 Asthma, drugs for, 21 (1984) 1; 31 (1994) 369, 409; 33 (1996) 1; 38 (2001) 249 neurokinin receptors in, 43 (2005) 53

Atorvastatin, hypolipidemic agent, 40 (2002) 1 ATPase inhibitors, gastric, H+/K+-31 (1994) 233 Azides, 31 (1994) 121 Bacteria, mechanisms of resistance to antibiotics and biocides, 35 (1998) 133 Bacterial and mammalian collagenases: their inhibition, 29 (1992) 271 1-Benzazepines, medicinal chemistry of, 27 (1990) 123 Benzimidazole carbamates, antifilarial, 25 (1988) 233 Benzisothiazole derivatives, 18 (1981) 117 Benzodiazepines, 20 (1983) 157; 36 (1999) 169 Benzo[b]pyranol derivatives, 37 (2000) 177 Biocides, aldehydes, 34 (1997) 149 mechanisms of resistance, 35 (1998) 133 British Pharmacopoeia Commission, 6 (1969) 1 Bronchodilator and antiallergic therapy, 22 (1985) 293 Calcium and histamine secretion from mast cells, 19 (1982) 59 Calcium channel blocking drugs, 24 (1987) 249 Camptothecin and its analogues, 34 (1997) 69 Cancer, aromatase inhibition and breast, 26 (1989) 253 azides and, 31 (1994) 121 camptothecin derivatives, 34 (1997) 69 endocrine treatment of prostate, 26 (1989) 299 retinoids in chemotherapy, 30 (1993) 1 Cannabinoid drugs, 24 (1987) 159; 35 (1998) 199; 44 (2006) 207 Carbapenem antibiotics, 33 (1996) 99 Carcinogenicity of polycyclic hydrocarbons, 10 (1974) 159 Cardiotonic steroids, 30 (1993) 135 Cardiovascular system, effect of azides, 31 (1994) 121 effect of endothelin, 31 (1994) 369

CUMULATIVE SUBJECT INDEX 4-quinolones as antihypertensives, 32 (1995) 115 renin inhibitors as antihypertensive agents, 32 (1995) 37 Caspase inhibitors, 39 (2002) 1 Catecholamines, 6 (1969) 200 Cathepsin K inhibitors, 42 (2004) 245 CCK-B antagonists, 37 (2000) 45 CCR5 Receptor antagonists, 43 (2005) 239 Cell membrane transfer, 14 (1977) 1 Central nervous system, drugs, transmitters and peptides, 23 (1986) 91 Centrally acting dopamine D2 receptor agonists, 29 (1992) 185 CEP-1347/KT-7515, inhibitor of the stress activated protein kinase signalling pathway (JNK/SAPK), 40 (2002) 23 Chartreusin, 19 (1982) 247 Chelating agents, 20 (1983) 225 tripositive elements as, 28 (1991) 41 Chemotherapy of herpes virus, 23 (1985) 67 Chemotopography of digitalis recognition matrix, 30 (1993) 135 Chiral synthesis, 34 (1997) Cholesterol-lowering agents, 33 (1996) 331; 40 (2002) 1 Cholinergic receptors, 16 (1976) 257 Chromatography, 12 (1975) 1, 105 Chromone carboxylic acids, 9 (1973) 65 Clinical enzymology, 13 (1976) 1 Collagenases, synthetic inhibitors, 29 (1992) 271 Column chromatography, 12 (1975) 105 Combinatorial chemistry, 36 (1999) 91 Computers in biomedical education, 26 (1989) 323 Medlars information retrieval, 10 (1974) 1 Copper complexes, 15 (1978) 211; 26 (1989) 437 Coronary circulation, 6 (1969) 11 Corticotropin releasing factor receptor antagonists, 41 (2003) 195 Coumarins, metabolism and biological actions, 10 (1974) 85 Cyclic AMP, 12 (1975) 293 Cyclooxygenase-2 (COX-2) inhibitors, 36 (1999) 201

217

Cyclophosphamide analogues, 19 (1982) 205 Cyclosporins as immunosuppressants, 25 (1988) 1; 33 (1996) 53 Data analysis in biomedical research, 25 (1988) 291 Depression neurokinin receptors in, 43 (2005) 53 Diaminopyrimidines, 19 (1982) 269 Digitalis recognition matrix, 30 (1993) 135 Dipeptidyl peptidase IV inhibitors, 45 (2007) 63 Diuretic drugs, 1 (1961) 132 DNA-binding drugs, 16 (1979) 151 Dopamine D2 receptor agonists, 29 (1992)185 Doxorubicin, 15 (1978) 125; 21 (1984) 169 Drug-receptor interactions, 4 (1965) 39 Drugs, transmitters and peptides, 23 (1986) 91 Elastase, inhibition, 31 (1994) 59 Electron spin resonance, 12 (1975) 191 Electrophysiological (Class III) agents for arrhythmia, 29 (1992) 65 Emesis neurokinin receptors in, 43 (2005) 53 Enantiomers, synthesis of, 34 (1997) 203 Endorphins, 17 (1980) 1 Endothelin inhibition, 31 (1994) 369 Enkephalin-degrading enzymes, 30 (1993) 327 Enkephalins, 17 (1980) 1 Enzymes, inhibitors of, 16 (1979) 223; 26 (1989) 253; 29 (1992) 271; 30 (1993) 327; 31 (1994) 59, 297; 32 (1995) 37, 239; 33 (1996) 1; 36 (1999) 1, 201; 38 (2001) 1; 39 (2002) 1, 121, 215; 40 (2002) 1, 23, 63; 41 (2003) 99, 249; 42 (2004) 125, 245 Enzymology, clinical use of, 10 (1976) 1 in pharmacology and toxicology, 10 (1974) 11 Epothilones A and B and derivatives as anticancer agents, 42 (2004) 171 Erythromycin and its derivatives, 30 (1993) 57; 31 (1994) 265

218


Feverfew, medicinal chemistry of the herb, 29 (1992) 217 Fibrinogen antagonists, as antithrombotic agents, 36 (1999) 29 Flavonoids, physiological and nutritional aspects, 14 (1977) 285 Fluorescence-based assays, 43 (2005) 19 Fluoroquinolone antibacterial agents, 27 (1990) 235 mechanism of resistance to, 32 (1995) 157 Folic acid and analogues, 25 (1988) 85; 26 (1989) 1 Formaldehyde, biocidal action, 34 (1997) 149 Free energy, biological action and linear, 10 (1974) 205 GABA, heterocyclic analogues, 22 (1985) 67 GABAA receptor ligands, 36 (1999) 169 Gas-liquid chromatography and mass spectrometry, 12 (1975) 1 Gastric H+/K+-ATPase inhibitors, 31 (1994) 233 Genomics, impact on drug discovery, 37 (2000) 1 Glutaraldehyde, biological uses, 13 (1976) 271 as sterilizing agent, 34 (1997) 149 Gold, immunopharmacology of, 19 (1982) 1 Growth hormone secretagogues 39 (2002) 173 Guanidines, 7 (1970) 124; 30 (1993) 203 Halogenoalkylamines, 2 (1962) 132 Heparin and heparinoids, 5 (1967) 139 Hepatitis C virus NS3-4 protease, inhibitors of, 39 (2002) 215 Hepatitis C virus NS3/NS4A protease inhibitors, 44 (2006) 65 Herpes virus, chemotherapy, 23 (1985) 67 Heterocyclic analogues of GABA, 22 (1985) 67 Heterocyclic carboxaldehyde thiosemicarbazones, 16 (1979) 35; 32 (1995) 1 Heterosteroids, 16 (1979) 35; 28 (1991) 233 High-throughput screening techniques, 37 (2000) 83; 43 (2005) 43 Histamine, H3 ligands, 38 (2001) 279; 44 (2006) 181

Hit identification, 45 (2007) 1 H2-antagonists, 20 (1983) 337 receptors, 24 (1987) 30; 38 (2001) 279 release, 22 (1985) 26 secretion, calcium and, 19 (1982) 59 5-HT1A receptors, radioligands for in vivo studies, 38 (2001) 189 Histidine decarboxylases, 5 (1967) 199 HIV CCR5 antagonists in, 43 (2005) 239 proteinase inhibitors, 32 (1995) 239 HMG-CoA reductase inhibitors, 40 (2002) 1 Human Ether-a-go-go (HERG), 43 (2005) 1 Hydrocarbons, carcinogenicity of, 10 (1974) 159 Hypersensitivity reactions, 4 (1965) 1 Hypocholesterolemic agents, 39 (2002) 121; 40 (2002) 1 Hypoglycaemic drugs, 1 (1961) 187; 18 (1981) 191; 24 (1987) 209; 30 (1993) 203; 31(1994) 1 Hypolipidemic agents, 40 (2002) 1 Hypotensive agents, 1 (1961) 34; 30 (1993) 203; 31 (1994) 409; 32 (1995) 37, 115 Immunopharmacology of gold, 19 (1982) 1 Immunosuppressant cyclosporins, 25 (1988) 1 India, medicinal research in, 22 (1985) 243 Influenza virus sialidase, inhibitors of, 36 (1999) 1 Information retrieval, 10 (1974) 1 Inotropic steroids, design of, 30 (1993) 135 Insulin, obesity and, 17 (1980) 105 Ion-selective membrane electrodes, 14 (1977) 51 Ion transfer, 14 (1977) 1 Irinotecan, anticancer agent, 34 (1997) 68 Isothermal titration calorimetry, in drug design, 38 (2001) 309 Isotopes, in drug metabolism, 9 (1973) 133 stable, 15 (1978) 1 Kappa opioid non-peptide ligands, 29 (1992) 109; 35 (1998) 83 Lactam antibiotics, 12 (1975) 395; 14 (1977) 181 X-Lactamase inhibitors, 31 (1994) 297

CUMULATIVE SUBJECT INDEX Lead identification, 45 (2007) 1 Leprosy, chemotherapy, 20 (1983) 1 Leukocyte elastase inhibition, 31 (1994) 59 Leukotriene D4 antagonists, 38 (2001) 249 Ligand-receptor binding, 23 (1986) 41 Linear free energy, 10 (1974) 205 Lipid-lowering agents, 40 (2002) 1 5-Lipoxygenase inhibitors and their antiinflammatory activities, 29 (1992) 1 Literature of medicinal chemistry, 6 (1969) 266 Lithium, medicinal use of, 11 (1975) 193 Local anaesthetics, 3 (1963) 332 Lonidamine and related compounds, 21 (1984) 111 Macrolide antibiotics, 30 (1993) 57; 31 (1994) 265 Malaria, drugs for, 8 (1971) 231; 19 (1982) 269; 28 (1991) 1; 43 (2005) 189 Manganese, biological significance, 30 (1993) 89 Manufacture of enantiomers of drugs, 34 (1997) 203 Mass spectrometry and glc, 12 (1975) 1 Mast cells, calcium and histamine secretion, 19 (1982) 59 cholinergic histamine release, 22 (1985) 267 peptide regulation of, 27 (1990) 143 Medicinal chemistry, literature of, 6 (1969) 266 Medlars computer information retrieval, 10 (1974) 1 Melanocortin receptor 4 ligands, 45 (2007) 111 Membrane receptors, 23 (1986) 41 Membranes, 14 (1977) 1; 15 (1978) 87; 16 (1979) 223 Mercury (II) chloride, biological effects, 27 (1990) 189 Methotrexate analogues as anticancer drugs, 25 (1988) 85; 26 (1989) 1 Microcomputers in biomedical education, 26 (1989) 323 Migraine neurokinin receptors in, 43 (2005) 53

219

Molecular modelling of opioid receptorligand complexes, 40 (2002) 107 Molecularly imprinted polymers, preparation and use of, 36 (1999) 235 Molybdenum hydroxylases, 24 (1987) 85 Monoamine oxidase inhibitors, 21 (1984) 137 Montelukast and related leukotriene D4 antagonists, 38 (2001) 249 Multivariate data analysis and experimental design, 25 (1988) 291 Muscarinic Receptors, 43 (2005) 105 Neuraminidase inhibitors, 36 (1999) 1 Neurokinin receptor antagonists, 35 (1998) 57; 43 (2005) 49 Neuromuscular blockade, 2 (1962) 88; 3 (1963) 1; 16 (1979) 257 Neuropeptide Y receptor ligands, 42 (2004) 207 Neurosteroids, as psychotropic drugs, 37 (2000) 135 Next decade [the 1970’s], drugs for, 7 (1970) 215 NFkB, 43 (2005) 137 Nickel(II) chloride and sulphate, biological effects, 27 (1990) 189 Nicotinic cholinergic receptor ligands, a4b2, 42 (2004) 55 Nitriles, synthesis of, 10 (1974) 245 Nitrofurans, 5 (1967) 320 Nitroimidazoles, cytotoxicity of, 18 (1981) 87 NMR spectroscopy, 12 (1975) 159 high-field, 26 (1989) 355 Non-steroidal anti-inflammatory drugs, 5 (1967) 59; 36 (1999) 201 Non-tricyclic antidepressants, 15 (1978) 39 C-Nucleosides, 13 (1976) 303; 22 (1985) 1 Nutrition, total parenteral, 28 (1991) 175 Obesity and insulin, 17 (1980) 105 Ondansetron and related 5-HT3 antagonists, 29 (1992) 239 Opioid peptides, 17 (1980) 1 receptor antagonists, 35 (1998) 83 receptor-specific analogues, 28 (1991) 301

220


receptor-ligand complexes, modelling of, 40 (2002) 107 Oral absorption and bioavailability, prediction of, 41 (2003) 1 Organophosphorus pesticides, pharmacology of, 8 (1971) 1 Oxopyranoazines and oxopyranoazoles, 9 (1973) 117 Oxytocin antagonists, 44 (2006) 331 Poly(ADP-ribose)polyrmerase (PARP) inhibitors, 42 (2004) 125 P2 Purinoreceptor ligands, 38 (2001) 115 p38 MAP kinase inhibitors, 38 (2001) 1 Paclitaxel, anticancer agent, 32 (1995) 289 Pain neurokinin receptors in, 43 (2005) 53, 55 Parasitic infections, 13 (1976) 159; 30 (1993) 203 Parasympathomimetics, 11 (1975) 1 Parenteral nutrition, 28 (1991) 175 Parkinsonism, pharmacotherapy of, 9 (1973) 191; 21 (1984) 137 Patenting of drugs, 2 (1962) 1; 16 (1979) 1 Peptides, antibiotics, 5 (1967) 1 enzymic, 31 (1994) 59 hypoglycaemic, 31 (1994) 1 mast cell regulators, 27 (1990) 143 opioid, 17 (1980) 1 Peptide deformylase inhibitors, 44 (2006) 109 Peroxisome proliferator-acrtvated receptor gamma (PPARg) ligands, 42 (2004) 1 Pharmacology of Alzheimer’s disease, 34 (1997) 1 Pharmacology of Vitamin E, 25 (1988) 249 Phosphates and phosphonates as prodrugs, 34 (1997) 111 Phosphodiesterase type 4 (PDE4) inhibitors, 33 (1996) 1 Phosphodiesterase type 5 (PDE5) inhibitors, 41 (2003) 249 Phospholipids, 19 (1982) 111 Photodecomposition of drugs, 27 (1990) 51 Plasmodium, 43 (2005) 190 Plasmodium flaciparum dihydrofolate reductase (PfDHFR), 43 (2005) 226

Platelet-aggregating factor, antagonists, 27 (1990) 325 Platinum antitumour agents, 24 (1987) 129 Platelet aggregration, inhibitors of, 36 (1999) 29 Polarography, 12 (1975) 247 Polycyclic hydrocarbons, 10 (1974) 159 Polyene antibiotics, 14 (1977) 105 Polypeptide antibiotics, 5 (1967) 1 Polypeptides, 4 (1965) 86 from snake venom, 21 (1984) 63 Positron emission tomography (PET), 38 (2001) 189 Prodrugs based on phosphates and phosphonates, 34 (1997) 111 Prostacyclins, 21 (1984) 237 Prostaglandins, 8 (1971) 317; 15 (1978) 357 Proteinases, inhibitors of, 31 (1994) 59; 32 (1995) 37, 239 Proteosome inhibitors, 43 (2005) 155 Pseudomonas aeruginosa, resistance of, 12 (1975) 333; 32 (1995) 157 Psychotomimetics, 11 (1975) 91 Psychotropic drugs, 5 (1967) 251; 37 (2000) 135 Purines, 7 (1970) 69 Pyridazines, pharmacological actions of, 27 (1990) 1; 29 (1992) 141 Pyrimidines, 6 (1969) 67; 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Quantum chemistry, 11 (1975) 67 Quinolines, 8-amino-, as antimalarial agents, 28 (1991) 1 4-Quinolones as antibacterial agents, 27 (1990) 235 as potential cardiovascular agents, 32 (1995) 115 QT interval, 43 (2005) 4 Radioligand-receptor binding, 23 (1986) 417 Ranitidine and H2-antagonists, 20 (1983) 337 Rauwolfia alkaloids, 3 (1963) 146 Recent drugs, 7 (1970) 1 Receptors, adenosine, 38 (2001) 61

CUMULATIVE SUBJECT INDEX adrenergic, 22 (1985) 121; 23 (1986) 1; 41 (2003) 167 cholecystokinin, 37 (2000) 45 corticotropin releasing factor, 41 (2003) 195 fibrinogen, 36 (1999) 29 histamine, 24 (1987) 29; 38 (2001) 279 neurokinin, 35 (1998) 57 neuropeptide Y, 42 (2004) 207 nicotinic cholinergic, 42 (2004) 55 opioid, 35 (1998) 83 peroxisome proliferator-activated receptor gamma (PPARg), 42 (2004) 1 purino, 38 (2001) 115 Rerin inhibitors, 32 (1995) 37 Reverse transcriptase inhibitors of HIV-1, 40 (2002) 63 Serotonin, 41 (2003) 129 Ricin, 24 (1987) 1 RNA as a drug target, 39 (2002) 73 Schizophrenia Neurokinin receptors in, 43 (2005) 53 M1 agonists in, 43 (2005) 113, 117 M2 antagonists in, 43 (2005) 121 M4 antagonists in, 43 (2005) 129 Screening tests, 1 (1961) 1 Secretase inhibitors, g-, 41 (2003) 99 Serine protease inhibitors, 31 (1994) 59 Serotonin 5-HT1A radioligands, 38 (2001) 189 Serotonin (5-HT)-terminal autoreceptor antagonists, 41 (2003) 129 Single photon emission tomography (SPET), 38 (2001) 189 Snake venoms, neuroactive, 21 (1984) 63 Sodium cromoglycate analogues, 21 (1984) 1 Sparsomycin, 23 (1986) 219 Spectroscopy in biology, 12 (1975) 159, 191; 26 (1989) 355 Statistics in biological screening, 3 (1963) 187; 25 (1988) 291 Sterilization with aldehydes, 34 (1997) 149

221

Steroids, hetero-, 16 (1979) 35; 28 (1991) 233 design of inotropic, 30 (1993) 135 Stress activated protein kinase inhibitors, 40 (2002) 23 Structure-based lead generation, 44 (2006) 1 Synthesis of enantiomers of drugs, 34 (1997) 203 Tachykinins, 43 (2005) 50 Tetrahydroisoquinolines, X-adrenomimetic activity, 18 (1981) 45 Tetrazoles, 17 (1980) 151 Thalidomide as anti-inflammatory agent, 22 (1985) 165 Thiosemicarbazones, biological action, 15 (1978) 321; 32 (1995) 1 Thromboxanes, 15 (1978) 357 Tilorone and related compounds, 18 (1981) 135 Time resolved energy transfer (TRET), 43 (2005) 40 Toxic actions, mechanisms of, 4 (1965) 18 Tranquillizers, 1 (1961) 72 1,2,3-Triazines, medicinal chemistry of, 13 (1976) 205 Tripositive elements, chelation of, 28 (1991) 41 Trypanosomiasis, 3 (1963) 52 Tuberculosis chemotherapy, 45 (2007) 169 Ubiquitinylation, 43 (2005) 153 Vanilloid receptors, TRPV1 antagonists, 44 (2006) 145 Venoms, neuroactive snake, 21 (1984) 63 Virtual screening of virtual libraries, 41 (2003) 61 Virus diseases of plants, 20 (1983) 119 Viruses, chemotherapy of, 8 (1971) 119; 23 (1986) 187; 32 (1995) 239; 36 (1999) 1; 39 (2002) 215 Vitamin D3 and its medical uses, 35 (1998) 1 Vitamin E, pharmacology of, 25 (1988) 249

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