Drug Discovery and Design: Medical Aspects (Biomedical and Health Research, 55)

DRUG DISCOVERY AND DESIGN: MEDICAL ASPECTS

Biomedical and Health Research Volume 55 Earlier published in this series Vol. 22. J. Matsoukas and T. Mavromoustakos (Eds.), Bioactive Peptides in Drug Discovery and Design: Medical Aspects Vol. 23. M. Hallen (Ed.), Human Genome Analysis Vol. 24. S.S. Baig (Ed.), Cancer Research Supported under BIOMED 1 Vol. 25. N.J. Gooderham (Ed.), Drug Metabolism: Towards the Next Millennium Vol. 26. P. Jenner (Ed.), A Molecular Biology Approach to Parkinson's Disease Vol. 27. P.A. Frey and D.B. Northrop (Eds.), Enzymatic Mechanisms Vol. 28. A.M.N. Gardner and R.H. Fox, The Venous System in Health and Disease Vol. 29. G. Pawelec (Ed.), EUCAMBIS: Immunology and Ageing in Europe Vol. 30. J.-F. Stoltz, M. Singh and P. Riha, Hemorheology in Practice Vol. 31. B.J. Njio, A. Stenvik, R.S. Ireland and B. Prahl-Andersen (Eds.), EURO-QUAL Vol. 32. B.J. Njio, B. Prahl-Andersen, G. ter Heege, A. Stenvik and R.S. Ireland (Eds.), Quality of Orthodontic Care - A Concept for Collaboration and Responsibilities Vol. 33. H.H. Goebel, S.E. Mole and B.D. Lake (Eds.), The Neuronal Ceroid Lipofuscinoses (Batten Disease) Vol. 34. G.J. Bellingan and G.J. Laurent (Eds.), Acute Lung Injury: From Inflammation to Repair Vol. 35. M. Schlaud (Ed.), Comparison and Harmonisation of Denominator Data for Primary Health Care Research in Countries of the European Community Vol. 36. F.F. Parl, Estrogens, Estrogen Receptor and Breast Cancer Vol. 37. J.M. Ntambi (Ed.), Adipocyte Biology and Hormone Signaling Vol. 38. N. Yoganandan and F.A. Pintar (Eds.), Frontiers in Whiplash Trauma Vol. 39. J.-M. Graf von der Schulenburg (Ed.), The Influence of Economic Evaluation Studies on Health Care Decision-Making Vol. 40. H. Leino-Kilpi, M. Valimaki, M. Arndt, T. Dassen, M. Gasull, C. Lemonidou, P.A. Scott, G. Bansemir, E. Cabrera, H. Papaevangelou and J. Mc Parland, Patient's Autonomy, Privacy and Informed Consent Vol. 41. T.M. Gress (Ed.), Molecular Pathogenesis of Pancreatic Cancer Vol. 42. J.-F. Stoltz (Ed.), Mechanobiology: Cartilage and Chondrocyte Vol. 43. B. Shaw, G. Semb, P. Nelson, V. Brattstrom, K. M01sted and B. Prahl-Andersen, The Eurocleft Project 1996-2000 Vol. 44. R. Coppo and Dr. L. Peruzzi (Eds.), Moderately Proteinuric IgA Nephropathy in the Young Vol. 45. L. Turski, D.D. Schoepp and E.A. Cavalheiro (Eds.), Excitatory Amino Acids: Ten Years Later Vol. 46. I. Philp (Ed.), Family Care of Older People in Europe Vol. 47. H. Aldskogius and J. Fraher (Eds.), Glial Interfaces in the Nervous System - Role in Repair and Plasticity Vol. 48. H. ten Have & R. Janssens (Eds.), Palliative Care in Europe - Concepts and Policies Vol. 49. T. Reilly (Ed.), Musculoskeletal Disorders in Health-Related Occupations Vol. 50. R. Busse, M. Wismar and P.C. Berman (Eds.), The European Union and Health Services Vol. 51. G. Lebeer (Ed.), Ethical Function in Hospital Ethics Committees Vol. 52. J.-F. Stoltz (Ed.), Mechanobiology: Cartilage and Chondrocyte, Vol. 2 Vol. 53. In production Vol. 54. I.M. Shapiro, B.D. Boyan and H.C. Anderson (Eds.), The Growth Plate ISSN: 0929-6743

Drug Discovery and Design Medical Aspects Edited by

J. Matsoukas Department of Chemistry, University of Patras, Patras, Greece

and

T. Mavromoustakos Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Athens, Greece

IOS

Press

•11 Ohrrisha

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© 2002, The authors mentioned in the Table of Contents All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior written permission from the publisher. ISBN 1 58603 261 5 (IOS Press) ISBN 4 274 90522 5 C3047 (Ohmsha) Library of Congress Control Number: 2002106945

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Preface This volume is a potpourri of review articles and research articles related to the "Bioactive Drugs in Drug Discovery and Design". It can be regarded as a continuation of Volume 22 published by IOS Press in the series Biomedical and Health Research. Most of the articles are part of the lectures given in the course of "Medicinal Chemistry" for graduate students. The book, like the first volume, is divided into four different topics in order to help the reader locate easily the manuscript of his interest. The first topic covers the synthesis and properties of bioactive molecules and enzymes. The second part examines the role of membranes in drug activity and formulation. The book continues with the third part that covers the conformational analysis of bioactive drugs. The final part mainly touches aspects of the molecular targets and drug design. This part is actually broader in scope and covers biological aspects of medicinal chemistry. The logic of classification of these parts is related to the route that medicinal chemistry follows. Novel compounds are synthesized which their conformations, and their interactions with the active site are studied. Finally, these compounds are undertaken for biological test. Meanwhile, new aspects like proteomics are developing in parallel to enhance the knowledge of the molecular basis of drug action. While the major part of the book is written by Greek specialists in their field several other articles come from researchers from different countries in order to assist the reader to get a global understanding of the recent activities in the field of Drug Discovery and Design. The editors wish to express their deep appreciation to all contributors in this book. In particular, to Rafarm Pharmaceutical Company, to the authors for their collaboration, to Panagiotis Zoumpoulakis for his assistance in editing the book, to George Zoumpoulakis for his artistic cover and IOS Press for providing the suitable and high-standard forum through which important findings of this research will become available to the scientific community. The Editors J. Matsoukas T. Mavromoustakos

The cover illustration was painted by the artist G. Zoumpoulakis. The revival of the plant is a metaphor for the healing of humans with the help of drugs.

Contents Preface

v

Part I: Synthesis and Properties of Bioactive Compounds Design, Synthesis and Biological Evaluation of Cyclic Angiotensin II Analogues with 3,5 Side-Chain Bridges: Role of C-Terminal Aromatic Residue and Positions 3,5 for Activity, L. Polevaya, P. Roumelioti, T. Mavromoustakos, P. Zoumpoulakis, N. Giatas, I. Mutule, T. Keivish, A. Zoga, D. Vlahakos, E. Iliodromitis, D. Kremastinos and J. Matsoukas 3 Synthesis of RGD Analogs Incorporating Moiety of Salicylic Acid Derivatives and their Effect on Human Platelet Aggregation In Vitro, Y. Sarigiannis, G. Stavropoulos and M. Liakopoulou-Kyriakides 13 Synthesis of Substance P C-terminal Analogs Incorporating D-Amino Acids and Studies of their Antineoplastic Properties in Vitro, P. Vakalopoulou, G. Stavropoulos, M. Liakopoulou-Kyriakides, Z. lakovidou and E. Mioglou 20 Synthesis of Chiral Tetramic Acids Using Activated Derivatives of N-protected a-Amino Acids as Precursors. Structural Characterization of Tetramic Acid Complexes with Metal Ions, J. Markopoulos, A. Detsi, E. Gavrielatos, G. Athanasellis and O. Igglessi-Markopoulou 25 L-Asparaginase: Structure, Properties and Anti-Tumor Activity, A.A. Pritsa and D.A. Kyriakidis 33 Growth Inhibitory and Apoptotic Effects of Gonadotropin-Releasing Hormone (GnRH) on Human Hepatocellular Carcinoma, C. V. Andreu-Vieyra and H.R. Habibi 41 Recent Applications of the Amide Approach to the Synthesis of Medicinally Interesting Polyamine Analogues, N. Tsiakopoulos, S. Vassis, M. Militsopoulou, C. Damianakos, P. Gatos, K. Voyiatzi, E. Pantazaka and D. Papaioannou 53 Design and Synthesis of Fluorescent Steroidal Lipopolyamine Conjugates for Monitoring Gene Delivery, I.S. Blagbrough and A.P.Neal 64 Synthesis of (S)-2-Amino-oleic Acid and Other Bioactive Molecules through Glutamate Semialdehydes, V. Constantinou-Kokotou, V. Magrioti, T. Makridis and G. Kokotos 76 Development of New Methods for Fluorometric Assays of Enzymes, C. Tzougraki 83 Enhancement of the Cytogenetic and Cytostatic Activity of Chlorambucil after its Esteric Connection with a Hecogenin Lactam, V. Karayianni, Ch. Camoutsis, V. Soulakis, D. Mourelatos, E. Mioglou and Z. lakovidou 97 In Searching for New Anti-Influenza A Drags: Heterocyclic and Carbocyclic Aminoadamantanes Bearing a 2-Adamantyl Group with Potent Activity Against Influenza A Virus, N. Kolocouris, A. Kolocouris, G.B. Foscolos, G. Fytas, E. Padalko, J. Neyts and E. De Clercq 103 Design, Synthesis and Conformational Properties of Linear Analogues of Human Myelin Basic Protein Epitope MBP1-11, S. Deraos, T. Tselios, I. Daliani, P. Zoumpoulakis, L. Probert, A. Troganis, P. Papathanassopoulos, T. Mavromoustakos and J. Matsoukas \ 16

Part II: Role of Membranes in Drug Activity The Effects of Vinblastine Sulfate on Dipalmitoylphosphatidylcholine Single and Multiple Bilayer Membranes, H. Maswadeh, C. Demetzos, I. Daliani, T. Mavromoustakos, G. Nounesis and A. Tsortos Stealth Liposomal Vinca Alkaloids (Vinorelbine and Vincrastine) and In Vitro Studies on Release by Buffer and Rat Plasma, C. Demetzos Structural Elucidation, Conformational Properties and Effects in Membrane Bilayers of the Toxin Paralysin, -Ala-Tyr from Larvae of the Gray Flesh Fly, /. Kyrikou, T. Mavromoustakos and C. Poulos Effects of NSAIDs in Membrane Bilayers, T. Mavromoustakos, I. Kyrikou, A. Kapou and D. Kovala-Demertzi

\ 25 131

142 150

Part III: Conformational Analysis of Bioactive Compounds The Structure of Endotoxin-Neutralizing Peptides Bound to LPS, P. Pristovsek and J. Kidric Synthesis and Conformational Analysis of Steroidal Carriers of Alkylating Agents with the Use of Two-Dimensional Nuclear Magnetic Resonance, A. Kapou, T. Mavromoustakos, S.G. Grdadolnik and S. Nikolaropoulos Structure Elucidation and Conformational Properties of Irbesartan, P. Zoumpoulakis, M. Zervou, S.G. Grdadolnik and T. Mavromoustakos 1 H and 13CNMR Assignment and Conformational Studies of a Myelin Basic Protein Epitope 74–85 Implicated in Multiple Sclerosis, A. Tzakos, A.N. Troganis, T. Tselios, A. Bonvin, N. van Nuland, S. Deraos, J. Matsoukas and IP. Gerothanassis Conformational Analysis of Biomolecules in Solution: NMR - 3D Structures Biological Implications, G.A. Spyroulias, S. Papazacharias, A. Galanis and P. Cordopatis Synthesis and Structural Investigation of a Synthetic LHRH Analogue in Solution, A.A. Zompra, G.A. Spyroulias, V. Magafa and P. Cordopatis Synthesis, Biological Evaluation & NMR Solution Structural Models of New Oxytocin Analogues, M. Fragiadaki, S. Koumentakos, D. Raptis, G.A. Spyroulias, V. Magafa, J. Slaninova and P. Cordopatis

161 167 174

180 188 205 217

Part IV. Molecular Targets and Drug Design Molecular Aspects of Human Abdominal Aortic Aneurysm: Differential Expression of Genes Coding for Extracellular Matrix Proteoglycans, A.D. Theocharis and N.K. Karamanos Advances in Botulinum Neurotoxin Inhibitors Based on the SNARE Motif, G.J. Moore, D. Moore, S. Roy, L.J. Hayden and G. Murray Effect of Linear and Cyclic Peptide Analogues of Myelin Basic Protein Epitope MBP72-85 on Human T-cell Activation, S. Thymianou, K. Chatzantoni, M. Karakantza, T. Tselios, P. Papathanassopoulos, J. Matsoukas and A. Mouzaki Use of Synthetic Peptides for the Identification of the Biologically Active Domains of the Growth Factor HARP, E. Papadimitriou, A. Polykratis, E. Karestou, J. Courty and P. Katsoris Unusual Interactions between MHC, Peptide and the T cell Receptor, V. Apostolopoulos and M. Plebanski

229 236

241 251 258

Effects of Angiotensin II, III and IV on Memory Retention of Rats: Interaction with Adenosine A1 Receptor Related Ligands, J. Tchekalarova and V. Georgiev Molecular Targets and Compounds for Anti-HIV Therapy, E. De Clercq Proteomics in Drug Discovery: Potential and Limitations, M. Fountoulakis Evaluation of Tumor Capabilities for Recurrence in Patients with Larynx and Pharynx Malignancies on the Basis of DNA Criterion, S. Andreychenko Significance of G-protein-coupled Receptor Polymorphisms: The Case of tt2-adrenergic Receptor, A. S. Manolis, A. Lymperopoulos, E.A. Bouga, M. Scheinin and C.S. Flordellis Computer Graphics Applications on Molecular Biology and Drug Design, K. Perdikuri and A. Tsakalidis Investigation of Novel DNA Gyrase Inhibitors Using the High Resolution NMR Spectroscopy, S.G. Grdadolnik, M. Oblak, T. Solmajer and R. Jerala Author Index

267 272 279 294 299 305 312 323

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Part I.

Synthesis and Properties of Bioactive Compounds

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Drug Discovery and Design: Medical Aspects J. Matsoukas and T. Mavromoustakos (Eds.) IOS Press, 2002

Design, Synthesis and Biological Evaluation of Cyclic Angiotensin II Analogues with 3,5 Side-Chain Bridges: Role of C-Terminal Aromatic Residue and Positions 3,5 for Activity L Polevaya1, P. Roumelioti2, T. Mavromoustakos3, P. Zoumpoulakis3, N. Giatas2, I. Mutule 1 , T. Keivish1, A. Zoga4, D. Vlahakos4, E. Iliodromitis4, D. Kremastinos4, J. Matsoukas2 Laboratory of Peptide Chemistry, Latvian Institute of Organic Synthesis, Riga, LV-1006, Latvia 2 Department of Chemistry, University of Patras, 26500 Patras, Greece 3 Section of Organic and Pharmaceutical Section, National Research Institute, Athens, Greece 4 Onassis Cardiac Surgery Center, 17674 Athens, Greece Abstract. The novel amide linked Angiotensin II cyclic analogues: y, e - cyclo(3, 5) -[Sar1Glu3-Lys5] ANG II (1) and y, e - cyclo(3, 5) -[Sar1-Glu3-Lys5-Ile8] ANG II (2) have been designed, synthesized and bioassayed in anesthetized rabbits in order to further unravel structural ring cluster characteristics important for receptor activation. The two analogues were synthesized in particular to investigate differences in activity and conformation upon 1) replacement of aromatic residue Phe with aliphatic Ile at position 8 and 2) reversing the position of the Lys, Glu linking residues at positions 3 and 5. Design was based on previous SAR and NMR studies in which residues 3 and 5 are the least important for activity and therefore could be used to create a link without disturbing the ring cluster conformation needed for activity (4, 5). The constrained cyclic analogues with a lactam amide bridge linking Glu, Lys residues at positions 3 and 5 and with Phe or Ile at position 8, were synthesized by solution procedure using the maximum protection strategy. Analogue 1 with Phe at position 8 was found to be a potent agonist while analogue 2 with He at position 8 was found to be an inhibitor of Angiotensin II. It appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo- electronic feature for Angiotensin II to exert its

L. Polevaya et al. / Cyclic Angiotensin II Analogues

biological activity. A 15th membered ring in the central X3-Tyr4-*5 core (X=Glu, Lys, vF=Lys, Glu) allows this cluster to exert its receptor action.

Introduction The octapeptide Angiotensin II (ANG II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is the main pressor component of the Renin-Angiotensin System (RAS) [1,2]. Accumulated experimental evidence for AngiotensinII supports a bioactive conformation characterized by a charge relay system between Tyr hydroxyl, His imidazole and Phe carboxylate, analogous to that found in serine proteases [3], as well as a ring cluster of the triad key aminoacids Tyr4-His6- Phe8 which appears to be responsible for activity [4,5]. Thus conformational analysis using modern 2D NMR techniques in receptor-simulating environments has shown proximity of the three key aminoacids sidechains and the formation of tyrosinate has been demonstrated by nanosecond time resolved tyrosinate fluorescence studies [6-7]. Comparative nuclear magnetic resonance studies of the backbone structure between peptide agonists and antagonists have shown that only agonists display ring clustering and form a change relay system [8]. In addition the proposed conformation overlays the recently discovered nonpeptide ANG II receptor antagonist Losartan and analogs when molecular modeling techniques and superimposition studies are applied [9,10]. Furthermore, the ring cluster conformation was recently supported by the design and synthesis of a novel constrained ANG II cyclic analogue, [Sar1-Lys3-Glu5] ANG anesthesized rabbits [5]. This potent cyclic analog was designed to have as a major molecular feature the integrity of the ring cluster. Other structure- activity studies have illustrated the importance of the C- terminal aromatic residue Phenylalanine (Phe) for agonist activity. Replacement of residue Phe at position 8 with an aliphatic one, as Ile, results in an antagonist [Sar1-Ile8] ANG II (Sarilesin) [1,2,11]. In this work two cyclic ANG II analogues 1, 2 with Glu, Lys residues at positions 3 and 5 and with Phe or Ile respectively at position 8 have been synthesized. The aims of this work were to investigate furthermore the role of a ring cluster receptor conformation in agonist activity and shed light to intriguing differences in activity and conformation upon 1) replacement of aromatic residue Phe with aliphatic Ile 2) reversing the position of the Lys-Glu sequence in the central amide bridge. While replacement of Phe with Ile at position 8 produces an antagonist, the reverse of the Glu-Lys order to a Lys-Glu one has a minimal effect in the agonist or antagonist activity of cyclic ANG II analogues. A 15th membered ring in the central core X-Tyr-^F allows a ring cluster and activity regardless of the Glu-Tyr-Lys or Lys-Tyr-Glu sequence in the agonist cyclic peptide. Based on these structure activity relationships which demand the presence of Phe, Tyr and His residues in ANG II as well as in linear and constrained ANG II analogues to possess biological activity, it can be inferred that the ability to form a ring cluster and consequently a charge relay system may be the key stereoelectronic molecular features of ANG II for exerting biological activity. These studies show also that cyclization at 3 and 5 positions does not affect the activity of the counterparts linear peptides Results and discussion The limited stability of peptides often severely restricts their medical and industrial application. Therefore, the engineering of stable proteins is of great technological and economic importance. If designed carefully without causing drastic changes in the

L. Polevaya et al. / Cyclic Angiotensin II Analogues

5

conformation of active peptides, the rigid geometry of the cyclic peptides enhances the binding affinity towards a selected target molecule compared to their linear counterparts [12, 13]. Furthermore cyclic analogues are important intermediates in the design and synthesis of non-peptide mimetics with the potential to be used as drugs [14,15]. With this aim, our group has been involved for several years in the design and synthesis of cyclic analogues for important peptides such as, Angiotensin [5, 16, 33], Thrombin Receptor Peptides [17] and Myelin Basic Protein [15] involved respectively in hypertension, cancer and multiple sclerosis. So far, a limited number of conformationally restricted via cyclization Angiotensin II analogues have been reported by us and others. In these studies cyclization was achieved either by the disulfide method using cysteine moieties at various locations of the peptide molecule or by the amide-linkage method [18-30]. Our interest in the conformational model of Angiotensin II, which could be used as a basis for the synthesis of non-peptide receptor antagonists, prompted us to design and synthesize two novel cyclic amide linked Angiotensin II analogues cyclo(3, 5)-[Sar1-Glu3Lys5] ANG II (1) and cyclo(3, 5)-[Sar1-Glu3-Lys5-Ile8] ANG II (2) (Fig. 1) that differ only in residue at position 8. The two analogues furthermore differ from cyclo(3, 5)-[Sar1-Lys3Glu 5 -X 8 ] ANG II (X=Phe or Ile) [31] only in the sequence of the Lys-Glu bridging residues. Both analogues can-potentially pertain the Angiotensin II conformational characteristics (i.e. backbone bend and aromatic ring clustering). It seems that reversal of the Lys-Glu order to Glu-Lys does not affect activity. The synthesis of counterpart novel analogue 2 with Ile of position 8 was prompted to further explore the role of the aromatic residue at position 8 for agonist activity. Cyclization was achieved by forming an amidelinkage between the -NH2 and -COOH side chain groups of Glu and Lys residues at positions 3 and 5, respectively, which are the least important for activity. With this study a confirmation of our Angiotensin II model was sought and further evidence to support the importance of the suggested ring cluster for receptor activation in which the Phe residue at position 8 plays a key role. In addition, it is studied the role of 3,5 aminoacids to affect the agonist or antagonist activity. Our findings indicate the importance of a Phe ring at position 8 for integration of a ring cluster receptor conformation and activity. On the contrary, the positioning of bridging residues Lys-Glu at positions 3 and 5 or 5 and 3 does not affect substantially the degree of potency in the two agonist and two antagonist cyclic peptides. Synthesis of central Glu3 -Tyr 4 -Lys 5 rings: The two cyclic analogues of angiotensin II (AIIand cyclo (3, 5)-Sar-Arg Glu-Tyr-Lys-His-Pro-Ile(I)) and cyclo (3, 5)-Sar-Arg-GluTyr-Lys-His-Pro-Phe (II) were synthesized by solution methods (Figure 1). Design of compounds was based on previous SAR and NMR studies, which has shown that residues 3 and 5 are the least important for activity and therefore could be used to create an amide link without disturbing the ring cluster conformation (4, 5). Peptide Synthesis: In particular, we have prepared two conformationally constrained analogues of AII with y,e-amide bridge between side chains of the glutamic acid and lysine residues at positions 3 and 5, containing an aromatic phenylalanine or aliphatic isoleucine residues in the position 8, respectively. The 3,5-cyclic AII analogues were synthesized by the solution procedure in combination with the maximum protection strategy. These compounds were constructed from N- and C-terminal linear fragments and the central a 15mer cyclic (Glu-Tyr-Lys) part, to produce the fully protected octapeptides, as it is shown in Scheme 1 for cyclo (3, 5)-Sar-Arg-Glu-Tyr-Lys-His-Pro-Ile (I). The same procedure was used to synthesize peptide cyclo (3, 5)-Sar-Arg-Glu-Tyr-Lys-His-Pro-Phe (II) except that tripeptide H-His(Bzl)-Pro-PheONb.HCI (18) was used (see Scheme 1), to obtain the

6

L.

Polevaya et al. / Cyclic Angiotensin II Analogues

protected octapeptide cyclo (3, 5)-Z-Sar-Arg(NO2)-Glu-Tyr-Lys-His(Bzl)-Pro-Phe-ONb (19). The key step in the preparation of each of these analogues was cyclization performed at the tripeptide stage by connecting the side chains of lysine and glutamic acid residues separated by tyrosine residue in the linear Glu3-Tyr4-Lys5 resulting in the formation of a cyclic amide (lactam) bond. The protected linear tripeptide 7 was synthesized stepwise using commercially available HCl.Lys(Z)-OtBu as the starting material.

H3C—NH H

O

HA

O

V

*

H3C—NH

Figure 1. Chemical structures of the Sar-Arg-{y,£-cyclo(Glu-Tyr-Lys)]-His-Pro-Phe (I), Sar-Arg-(y,£-cyclo(Glu-Tyr-Lys)]-His-Pro-Ile (II) analogues of AII, Sar-Arg-[y,e-cyclo(Lys-Tyr-Glu)]-His-Pro-Phe (III) and Sar-Arg-[y,E-cyclo-(Lys-Tyr-Glu)]-His-Pro-Ile(II)

L. Polevaya et al. / Cyclic Angiotensin II Analogues

CENTRAL FRAGMENT C-TERMINAL FRAGMENT

HCl .Lys(Z)-OtBu I Boc-Tyr(Bzl)-OH DCC/HOBt/DMF 1 DIPEA / EtOAc

Boc-Pro-OH HONB / EtOAc DCC HCl, H-Ile-Onb DIPEA

I

AcOH / tert-ButOH 3.7M HCl / tert-ButOH

Z-Sar-Arg(NO2)-Ome (11)

Boc-Pro-Ile-ONb (1) I

Z-Sar-OH

Tyr(Bzl)-Lys(Z)-OtBu (5)

H-Tyr(Bzl)-Lys(Z)-OtBu.HCl (6) I DMF/EtOAc I DIPEA / DMF * Boc-Glu(Bzl)-OPfp Boc-Glu(OBzl>Tyr(Bzl)-Lys(Z)-OtBu (7)

TFA

H-Pro-Ile-ONb.TFA (2)

Boc-His(Bzl)-OH / HOBt D1C/DIPEA

I 1.0M NaOH Z-Sar-Arg(NO)-OH (12)

MeOH / AcOH / H2O Pd black / H2

Boc-Glu-Tyr-Lys-OtBu.AcOH (8) Boc-His(Bzl)-Pro-Ile-ONb (3)

I HOBt / DIC / DIPEA

2.6M HCl / EtOAc

Boc-[ cyclo-(Glu-Tyr-Lys) ]-OtBu (9) Boc-His(Bzl)-Pro-Ile-ONb.HCl (4)

3M HCl / dioxane

[ cyclo-(Glu-Tyr-Lys) ]-OtBu (10) II

12

DIC / HOBt / DIPEA

Z-Sar-Arg(NO2)-[ cyclo-(Glu-Tyr-Lys) ]-OtBu (13) I TFA / DCM

Z-Sar-Arg(NO2)-[ cyclo-(Glu-Tyr-Lys) ]-OH (14) T DCC/HATU / DIPEA Z-Sar-Arg(NO2)-[ cyclo-(Glu-Tyr-Lys) ]-His(Bzl)-Pro-IIe-ONb (15) MeOH / AcOH / H2O H2 / Pd black 1M HCl / dioxane

H-Sar-Arg-[ cyclo-(Glu-Tyr-Lys) ]-His-Pro-Ile-OH.2HCl (I)

L. Polevaya et al. / Cyclic Angiotensin II Analogues Boc-His(Bzl)-OH (16) HBr. H-Pro-Phe-ONb

I

Boc-His(Bzl)-Pro-Phe-ONb (17) 2.9M HCl / EtOAc

I

H-His(Bzl)-Pro-Phe-ONb.HCl (18) 14 , DCC / HATU DIPEA Z-Sar-Arg(NO2)-[ cyclo (Glu-Tyr-Lys)]-His(Bzl)-Pro-Phe-ONb (19) MeOH / AcOH / H2O H2 / Pd black 1M HCl / dioxane

H-Sar-Arg-[ cyclo (Glu-Tyr-Lys)]-His-Pro-Phe-OH.2HCl (II)

Scheme 1. Synthetic routes for the peptides I and II.

The protected cyclic tripeptide Boc-[cyclo(Glu-Tyr-Lys)]-OtBu (9) was prepared after de-protection of the side chains of the bridgehead residues from 7 by coupling the linear precursor 8 via N-hydroxybenzotriazole active ester in situ using DIC/HOBt/ DIPEA. Cyclic dimer (-10%), which formed as a by-product during the cyclization, was removed by HPLC chromatography in the last stage of purification. Selective acidolysis of the N-terminal tert-butyl protecting group in compounds 5 and 9 and de-protection was monitored by regular TLC control in solvent system D. The tert-butyloxycarbonyl group present in 5 was selectively removed by treatment with 3.7 M HCl/tert-ButOH for 25 min at 45 °C in AcOH/tert-ButOH (5:3), whereas tertbutyloxycarbonyl group of 9 was cleaved during treatment with 3M HCl/dioxane for 25 min at room temperature to give the cyclic peptide H-[cyclo(Glu-Tyr-Lys)]-OtBu.HCl (10) in moderate yield (56,7%). tert-Butyloxycarbonyl group de-protection under these conditions, resulted in the formation of small amount of by-product as a result of the simultaneous acidolysis of N- tert-butyloxycarbonyl and tert-butyl ester protections. The N- and C-terminal fragments were synthesized step by step from their respective C-amino acid esters. Z-Sar-Arg(NO2)-OH (12) was obtained by saponification from methyl ester 11. The protected tripeptides Boc-His(Bz)-Pro-Ile-ONb (3) and Boc-His (Bz)-Pro-Phe-ONb (17) were synthesized with p-nitrobenzyl ester protection for the Cterminals and Boc group for the a-amino functions. After tert-butyloxycarbonyl group acidolytic cleavage by HCl/EtOAc, hydrochlorides of the C-terminal tripeptides 4 and 18 were used for preparation of the protected octapeptides 15 and 19, respectively. The fully protected cyclo (3,5)-Z-Sar-Arg(NO2)-Glu-Tyr-Lys-OtBu (13) was prepared by fragment condensation between Z-Sar-Arg(NO2)-OH (12) and cyclo(Glu-TyrLys)-OtBu.HCl (10) using DIC/HOBt carboxyl activation induced by DIPEA. The tertbutyl ester group of pentapeptide 13 was then converted to the free carboxyl by treatment with TFA in DCM for 40 min; The resulting peptide cyclo (3,5)-Z-Sar-Arg(NO2)-Glu-Tyr-

L. Polevaya et al. / Cyclic Angiotensin II Analogues

9

Lys-OH (14) reacted with either H-His(Bz)-Pro-Ile-ONb. HC1 (4) to generate the protected octapeptide 15 or with H-His(Bz)-Pro-Phe-ONb. HC1 (18) to produce octapeptide 19. The preparations of the Y,£-cyclo(glutamyl-tyrosyl-lysyl)-containing analogues I and II were completed by simultaneous catalytic hydrogenolysis of all protection groups from 15 and 19, further purification by reversed-phase HPLC and conversation of the hygroscopic trifluoracetate salts to the hydrochlorides. Crystallization from iso-propanol gave a desired compound cyclo (3, 5)-Sar-Arg-Glu-Tyr-Lys-His-Pro-Ile (I) and cyclo (3, 5)-Sar-Arg-GluTyr-Lys-His-Pro-Phe (II), whereas I was additionally purified by double re-precipitation from rnethanol-diethyl ether. Biological activity: The constrained amide linked cyclic Angiotensin II analogues were found to be active in both ex vivo and in vivo experiments. In the rat uterus assay, the agonist activity of the cyclic analogues were determined by matching its response with the equivalent response to human Angiotensin II and it was found to be -15% of that observed with the intact octapeptide. In anesthetized rabbits, previous testing had revealed that Angiotensin II infused at a rate of 2 mg/min exerts a significant and submaximal hypertensive response (+105 ± 13 mmHg). Thus, in each animal a comparison was made between blood pressure changes produced by 2 (ug/min of Angiotensin II with those produced by 10, 20, 40 and 80 ug/min of the cyclic analogues. As shown in Figure 2, c-[Sar1-Lys3-Glu5] ANG II and c-[Sar1Glu3-Lys5] ANG II are potent agonists producing significant and dose-dependent blood pressure increases. The blood pressure started rising within approximately 1 min after the commencement of drug infusion, reached a plateau within 1-2 min and returned to preinjection levels within 2-4 min after discontinuation of the drug. Of note, the magnitude of the blood pressure elevation was much higher with Angiotensin II than with equimolar quantities of the two potent cyclic analogues. In contrast, the other two cyclic Angiotensin II analogues with Ile at position 8 namely [Sar1-Lys3-Glu5-Ile8] ANG II and [Sar1-Glu3Lys5-Ile80] ANG II failed as agonists but caused a dose-dependent antagonism of the hypertensive response induced by Angiotensin II infusion.

Figure 2. Changes of Blood Pressure in mmHg for the four cyclic peptides under study.

10

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Polevaya et al. / Cyclic Angiotensin II Analogues

Conclusion This research was aiming at establishing differences between Angiotensin II agonist and antagonist peptides and at confirming the aromatic ring clustering conformational model for Angiotensin II which has been recently proposed on the basis of structureactivity relationships, NMR and fluorescence life time studies. A strong Tyr4-Ile5-His6 bend exists in cyclic agonist and antagonist cyclic analogues which both bear the same central Glu-Tyr-Lys or Lys-Tyr-Glu ring moiety, indicating that the difference in activity is resulting mainly from the nature of the C-terminal residue. An aromatic residue such as Phe allowing the formation of a ring cluster is necessary for exertion of agonist activity in ANG II. The constrained cyclic amide linked Angiotensin II analogues c-[Sar1-Lys3-Glu5] ANG II and c-[Sar1-Glu3-Lys5] ANG II were designed to keep intact the clustering and backbone bend characteristics of the peptide hormone Angiotensin II. On the contrary, the cyclic antagonists [Sar1-Lys3-Glu5-Ile8] ANG II and [Sar1-Glu3-Lys5-Ile8] ANG II without an aromatic residue at position 8, lack the ring cluster conformation. The molecules were designed with the hypothesis that residues 3 and 5 are not important for activity and exist on the other side of the molecule from the functionally important aromatic side chains and this structure can be accommodated in the charge relay conformation proposed for Angiotensin II. The lower potency of both agonist and antagonist cyclic analogues compared to ANG II and Sarilesin points out that the achieved conformation by cyclic analogues is not the optimal in inducing or suppressing ANG II induced hypertension. In the potent constrained agonist analogues the three rings are closely spaced at the same side of the cyclic ring and this has been shown by NOE interactions and molecular modeling. Activity is similar regardless of Lys-Tyr-Glu or Glu-Tyr-Lys order in cyclic agonist or antagonist peptides. The obtained data confirm our hypothesis that the aromatic side chains together with the C-terminal carboxylate are the essential pharmacophoric groups for receptor activation. These data also emphasize the role of closely spaced residues 4, 6 and 8 to form a possible relay system, which is not possible in Sarilesin and cyclic ANG II antagonists, lacking a C-terminal aromatic residue. In addition, the data show that the way of cyclisation at 3 and 5 positions does not interfere with the activity of the resulting peptide. The following figure shows models of the four peptides under study. These were derived using a combination of NMR spectroscopy and Molecular Modeling.

L. Polevaya et al. / Cyclic Angiotensin II Analogues

Figure 3. Models of the four analogs analogs. The models show proximity of the three aminoacids (Tyr, His and Phe or Ile) and that cyclization through Lys3-Glu5 or Glu5-Lys3 does not affect the activity of the peptides.

Acknowledgments This work was supported by the Ministry of Development, General Secretariat of Research and Technology of Greece (EPET II/115, PENED 1999), a NATO Linkage Grant (974548), the Latvian Science Council grant (96.0726) and the Latvian National Scientific Programme (ZP-10). References [1] H. Gavras, H.R. Brunner, J.H. Laraph, J.E. Sealey, I. Gavras, R.A. Vukovich, N. Engl. J. Med. 291 (1974) 817. [2] H. Gavras, H.R. Brunner, G.A. Turini, G.R. Kershaw, C.P. Tifft, S. Cuttelod, I. Gavras, R.A. Vukovich, D.N. McKinstry, N. Engl. J. Med. 298 (1978) 991. (3] Blow et. al., Nature 221 (1969) 337. [4] J. Matsoukas, J. Hondrelis, M. Keramida, T. Mavromoustakos, A. Makriyannis, R. Yamdagni, Q. Wu and G.J. Moore, 7. Biol. Chem. 269 (1994) 5303. [5] J. Matsoukas, J. Ancans, T. Mavromoustakos, P. Roumelioti, D.V. Vlahakos, R. Yamdagni, Q. Wu, G.J. Moore, Bioorg. Med. Chem. 8 (2000) 1. J. Matsoukas, G. Bigam, N. Zhou and G. Moore, Peptides 11 (1990) 359. J. Matsoukas, R. Yamdagni, G. Moore, Peptides 11 (1990) 367. J. Matsoukas, G. Agelis, A. Wahhab, J. Hondrelis, D. Panagiotopoulos, R. Yamdagni, Q. Wu, T. Mavromoustakos, H. Maia, R. Ganter, G.J. Moore, J. Med. Chem. 38 (1995) 4660. [9] T. Mavromoustakos, A. Kolocouris, M. Zervou, P. Roumelioti, J. Matsoukas, R. Weisemann, J. Med. Chem. 42 (1999) 1714. [10] P. Roumelioti, T. Tselios et. al., Bioorg. And Med. Chem. Letters 10 (2000) 1. [ 1 1 ] H . Gavras and I. Gavras, Bioactive Peptides in Drug Discovery and Design: Medical Aspects J. Matsoukas and T. Mavromoustakos (Eds.) IOS Press. 1999. [ 12]K. Alexopoulos, P. Fatseas, E. Melissari, D. Vlahakos, J. Smith, T. Mavromoustakos, M. Saiteddine, G. Moore, M. Hollenberg, J. Matsoukas, Bioorganic and Medicinal Chemistry 1 (1999) 1033.

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[13] A Wahhab, J.R. Smith, R.C. Ganter, D.M. Moore, J. Hondrelis, J. Matsoukas and G.J. Moore, Drug Research 43 (1993) 1157. [14]J. Matsoukas, D. Panagiotopoulos, M. Keramida, T. Mavromoustakos, R. Yamdagni, Q. Wu, G.J. Moore, M. Saifeddine, M.D. Hollenberg, J. Med. Chem. 39 (1996) 3585. [15]T. Tselios, L Probert, I. Daliani, E. Matsoukas, A. Troganis, I. Gerothanassis, T. Mavromoustakos, G. Moore and J. Matsoukas, 7. Med. Chem. 42 (1999) 170. [16]J. Matsoukas, J. Hondrelis, G. Agelis, K. Barlos, R. Ganter, D. Moore and G.J. Moore, J. Med. Chem. 37 (1994) 2958. [17]K. Alexopoulos, D. Panagiotopoulos, T. Mavromoustakos, P. Fatseas. MC. Paredes-Carbajal, D. Mascher, S. Mihailescu, J. Matsoukas, J Med Chem. 44 (2001) 328. [18]K,L. Spear, M.S. Brown, E.J. Reinhard, E.G. McMahon, G.M. Olins, M.A. Palomo, D.R. Patton, J. Med. Chem. 33 (1990) 1935. [19]E. Sugg, C. Dolan, A. Patchett, R. Chang, K. Faust, V. Lotti, In Peptides: Chemistry, Structure and Biology, ESCOM Science: Leiden, The Netherlands, 1990, pp 305-306. [20] J. Matsoukas, M. Scanlon, G.J. Moore, J. Med. Chem. 27 (1984) 404. [21]G.V. Nikiforovich, G.R. Marshall, Biochem. Biophys. Res. Commun. 195 (1993) 222. [22]G.V. Nikiforovich, J.L.-F. Kao, K. Plucinska., W.J Zhang, G.R. Marshall, Biochemistry 33 (1994) 3591. [23] K. Plucinska, T. Kataoka, M. Yodo, W.L. Cody, J.X. He, C. Humblet, G.H. Lu, E. Lunney, T.C. Major, R.L. Panek, P. Schelkun, R. Skeen, G.R. Marshall, 7. Med. Chem. 36 (1990) 1902. [24] W.J. Zhang, G.V. Nikiforovich, J. Perodin, D.E. Richard, E. Escher, G.R. Marshall, J. Med. Chem. 39 (1996) 2738. [25] E. Jorgensen, W. Patton, J. Med. Chem. 12 (1969) 935. [26] J. De Coen, E. Ralston, J. Durieux, In Peptides: Chemistry, Structure and Biology, Ann Arbpr. Ml, 1975, pp.553-558. [27] J.Ancans, D. Biseniece, N. Myshliakova, E. Porunkevich, Bioorg. Khim. 12 (1986) 118. [28] J. Ancans, D. Biseniece, N. Myshliakova, G. Chipens, Bioorg. Khim. 16 (1990) 358. [29] J. Matsoukas, J. Hondrelis, G. Agelis, K. Barlos, D. Gatos, R. Ganter, D. Moore, G.J. Moore, 7. Med. Chem. 37 (1994) 2958. [30] E. Gross, R. Meienhofer, J., G. Eds. Chipens, G. Nikiforovich, F. Mutulis, N. Veretennikova, I. Vosekalna, A. Sosnov, L. Polevaya, J. Ancans, N. Mishlyakova, E. Liepinsh, I. Sekacis, M Breslav, Cyclic analogs of linear peptides. In Peptides. Structure and Biological Function. Proceeding of the Sixth American Peptide Symposium, Pierce Chemical Company: Rockford, IL, 1979 pp. 567-570. [31]L. Polevaya, T. Mavromoustakos et. al., Bioorg. Med. Chem. 9 (2001) 1639.

Drug Discovery and Design: Medical Aspects J. Matsoukas and T. Mavromoustakos (Eds.) IOS Press, 2002

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Synthesis of RGD AnalogsIncorporating Moiety of Salicylic Acid Derivatives and their Effect on Human Platelet Aggregation in vitro Y. Sarigiannis1, G. Stavropoulos1, M. Liakopoulou-Kyriakides2 1

2

Dept. of Chemistry, University of Patras, Patras, Greece, Dept. of Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece

Abstract. A series of RGD (Arg-Gly-Asp) analogs incorporating salicylic acid derivatives at their N-terminal amino group have been synthesized by conventional solution techniques and/or by solid phase synthesis. The synthesized compounds were tested for their inhibitory activity on human platelet aggregation in vitro. They exert their inhibitory activity by adding the aggregation reagent to citrated platelet rich plasma from venous blood. The analog 2-HO-C6H4-CO-Arg-Gly-Asp-NH2 (1) showed higher inhibitory activity than the parent peptide Arg-Gly-Asp-NH2, which means that the combination of salicylic acid and RGD-NH2 in the same molecule increases the antiplatelet activity. The analog 2-HO-C6H4-CO-Arg-Gly-Asp(OBzl)NH2 (3) was found to be the most potent inhibitor of platelet aggregation with IC50 0.05 mM. This ensures our previous observations about the requirements of a lipophile group at the C-terminal side of the peptidic sequence.

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Y. Sarigiannis et al. / Synthesis of RGD Analogs

Introduction The development of new antithrombotic agents has been stimulated by clinical needs as long as the major cause of death and disablement, in developed countries, are cardiovascular diseases, mainly unstable angina, stroke and acute myocardial infraction. There are two classes of antithrombotic agents -anticoagulants and antiplatelet agentsthat are used in antithrombotic therapy. Anticoagulants inhibit thrombin generation and fibrin formation and antiplatelet agents block platelet aggregation. Aspirin, coumarins, low-molecular weight heparins and direct inhibitors of thrombin have been used as antithrombotic agents for many years with effectiveness but they have their limitations. Antagonists of platelet glycoprotein IIb/IIIa (GP IIb/IIIa) represent a new therapeutic approach in inhibiting platelet aggregation, thus providing a powerful form of antithrombotic therapy [1-4]. The minimum sequence on fibrinogen recognized by GP IIb/IIIa receptor is Arg-Gly-Asp (RGD) [5-7]. Extensive research on structure-activity as well as conformational studies have been performed on the pharmacofore groups [810] and have led to the development of peptidic [11,12] and not peptidic analogs [13,14]. Other researchers isolated RGD-containing peptides from snake venoms with effective activity on platelet aggregation [15-17]. In order to get more stable molecules, researchers synthesized cyclic analogs [18-21]. We have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of RGD sequence with salicylic or acetylsalicylic acid moiety 2-RO-C6H4-CO-, [where R: H or Ac] at their N-terminal amino group have shown inhibitory activity on human platelet aggregation stimulated by collagen, ADP or adrenaline [22-24]. Here, with this report we carry out our investigational effort. All the synthesized compounds contain amino acid(s) of RGD sequence incorporating moiety of salicylic acid or derivatives. Experimental Part Materials and Analytical Methods: Solid Phase Peptide Synthesis (SPPS) was carried out using the 2-chlorotrityl chloride resin [25] (CBL, Patras, Greece) as solid support on which the Rink linker was coupled. All amino acids and derivatives used in this work are of S-configuration and are purchased from CBL (Patras, Greece), while the aggregating agents collagen and ADP were purchased from Sigma Chem. Co (Germany). The used solvents were of analytical grade. Capillary melting points were determined on a Electrothermal apparatus and are reported uncorrected. IR spectra were recorded as KBr pellets on a Perkin-Elmer 16PC FT-IR spectrophotometer. 1H and 13C NMR spectra were obtained at 400 MHz on a Bruker (Avance) instrument using CDCl3 or DMSO-d6 as solvent and TMS as internal standard. Chemical shifts are reported as 6 values in parts per million. ESI-MS were recorded on a Micromass (Platform LC) mass spectrometer. Analysis by TLC was performed on precoated plates of silica gel 60 F254 (Merck) with the following solvent systems (ratio by volume) AcCN/H2O (5:1) (1), nBuOH/AcOH/H20 (4:1:1) (2), n-BuOH/AcOH/Pyr/H2O (4:1:1:2) (3). Spots on TLC plates were detected by UV light, with ninhydrin solution and/or by chlorination followed by solution of 1% starch/ 1% KI (1:1 v/v). For the cleaning of the synthesized crude compounds was used Flash Chromatography with silica gel 60, 230-400 mesh

Y. Sarigiannis et al. / Synthesis of RGD Analogs

(ASTM), whereas the collection was done with the Ultrorac 7000 fraction collector (LKB). Biological Assays: Platelet aggregation was studied by a conventional photometric technique with a Dual Channel Coulter Electronic Aggregometer, at 37°C, with continuous recording of light transmission, according to the method of Born [26]. The aggregation agents used were at a final concentration of 60u,g/ml for collagen and 10mM for ADP. Platelets were obtained from venous blood of healthy donors who had not taken any medication during the week preceding blood collection. The blood was immediately mixed at a 9:1 ratio with 3.8% sodium citrate solution and was centrifuged at 200g for 5min to yield platelet rich plasma. Chemistry Activation and coupling procedure: Fmoc amino acid (3mmol, threefold excess of the resin with linker substitution) and 1-hydroxybenzotriazole (HOBt, 4.5mmol) were dissolved in DMF (4 ml). Diisopropylcarbodiimide (DIC, 3.3mmol) was added and the solution was transferred to the solid phase reactor. After 3h, the mixture was washed (3x DMF, 2x 2-propanol, 2x DMF, 3x 2-propanol and 2x ether). The progress of the reaction was checked by Kaiser's test. In case of uncompleted reaction, the coupling was repeated by using half of the quantities of the Fmoc-AA-OH and the rest reagents.

H3CO

Rink Amide Linker

O-CH2COOH

2-CLTR

Cleavage of the Fmoc-group: The cleavage was performed at room temperature with 20% piperidine in DMF for 5 min followed by a second treatment for 25 min. After that, the mixture was washed with the same washing procedure, as above. Coupling of the salicylic-acid and its derivatives with the tripeptides:Salicylic-acid or its derivatives (3 mmol, three-fold excess of the resin with linker substitution) and PyBOP (3.63 mmol) were dissolved in DMF (4 ml). DIPEA (4.5 mmol) was added and the solution was transferred to the solid phase reactor. After 5h, the progress of the reaction was checked by Kaiser's test. Subsequently, the above washing procedure was applied. Cleavage of the salicyl-peptides from 2-Chlorotrityl resin: The salicylic-peptide-linker resin ester was suspended in a mixture of 25ml of DCM/TFE/AcOH (7:2:1) for 2.5h at room temperature. The resin was filtered off and the filtrate was concetrated in vacuo. The crude peptide was precipitated by addition of ether, collected by filtration, washed with ether on the filter and dried in vacuo.

15

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Y. Sarigiannis et al. / Synthesis of RGD Analogs

Removal of the side-protected groups and linker's moiety: The salicylic-peptide-linker was treated in a solution of TFA/DCM/scavengers for 2.5h at room temperature.The solution was concetrated in vacuo. The crude salicylic-peptide was precipitated by addition of ether, collected by filtration, washed with ether on the filter and dried in vacuo

H3CO

I.Fmoc-X-OH

1

X1:Glyorl_eu

2. Fmoc-X'-OH

[oiC/HOBt

X : AspCOBut). Asp(OBzl).

3. Fmoc-Arg(Pbf)-OH Deprotection with 20% piperidine in DMF

Asp(OMe)

*• H-Arg(Pbf)-X1-X-Linker-O-Q PyBOP DIPEA, 5h

CO-Arg-X1-X-NH2 X1: GlyorLeu X : Asp, Asp(OBzl), Aspry of Organic Chemistry, Department of Chemistry, University of Athens, Greece Laboratory

b

Abstract. 2-Amino-oleic acid is of great interest, because it is used as a tissue treatment for advanced generation bioprostheses. Efficient methodology for the synthesis of enantiopure 2-amino-oleic acid was developed using either tert-butyl (2S)-2[bis(fert-butoxycarbonyl)amino]-5-oxopentanoate or methyl (2S)-2-[bis(tertbutoxycarbonyl)amino]-5-oxopentanoate as key-intermediates. Furthermore, a route to enantiopure (0-functionalized a-amino acids was developed.

Introduction In recent years, the field of amino acids and peptides has gained enormous popularity and relevance, particularly with the emergence of unnatural analogues as components of compounds with therapeutic potential [1]. The need to replace natural amino acids in peptides with non-proteinogenic counterparts in order to obtain new medicinal agents, exhibiting better binding to specific receptors and more potent inhibition of target enzymes, has stimulated a great deal of innovation on synthetic methods. Lipidic a-amino acids (LAAs) is a class of unnatural amino acids with long aliphatic side chains. LAAs and lipidic peptides combine the structural features of lipids with those of amino acids and peptides [2]. These compounds are lipophilic due to the long carbon chains, yet show polar, chemical and conformational behaviour characteristics of amino acids and peptides. Amides and esters of saturated LAAs as well as lipopeptides present interesting biological properties [3–5]. Racemic LAAs and their oligomers have been used as drug delivery systems [6,7] and for the construction of a lipid-core-peptide system for the development of adjuvant semisynthetic vaccines [8]. Long chain 2-amino alcohols and 1,2-diamines obtained from LAAs exhibit interesting cytotoxic [9–11] and anti-inflammatory activity [12]. The enantiomeric synthesis of saturated LAAs, based on the oxidative cleavage of amino diols, obtained by the regioselective opening of enantiomerically enriched 2,3-epoxy alcohols, has been presented [13]. Also, the synthesis of optically active 8,E-unsaturated LAAs, including a-amino arachidonic acid, has been achieved through a Wittig reaction on the suitable glutamate semialdehyde [ 14].

V. Constantinou-Kokotou et al. / Synthesis of (S)-2-Amino-oleic Acid

77

(S)-2-Amino-oleic Acid Efficient methodology for the synthesis of optically pure 2-amino-oleic acid (1) (Figure 1) has been developed by our group using either tert-butyl (2S)-2-[bis(tertbutoxycarbonyl)amino]-5-oxopentanoate or methyl (2S)-2-[bis(tertbutoxycarbonyl)amino]-5-oxopentanoate as key-intermediates.

o OH NH2

J

/

Figure 1. (S)-2-Amino-oleic acid

Applications of 2-Amino-oleic Acid: Racemic 2-amino-oleic acid is used as a tissue treatment for advanced generation bioprostheses [15]. Valvular defects are among the most frequent causes of circulatory insufficiency. Surgical treatment, which usually consists in the implantation of prosthetic valve, improves haemodynamic conditions through anatomic correction. Heart valve prosthesis implanted for that purpose are divided into mechanical and biological valves, ( valve leaflets derived from human or animal tissues-bioprosthesis) [16]. The introduction of tissues crossed-linked with formaldehyde or glutaraldehyde into heart valve production ensured valve sterility. Calcification is a frequent cause of the clinical failures of glutaraldehyde-pretreated bioprosthetic heart valves fabricated from glutaraldehyde-cross-linked porcine aortic valves. Racemic 2-amino-oleic acid has been shown in in vivo studies to be a promising anti-calcification agent. In fact, the incubation of pericardial valve cross-linked with glutaraldehyde with the admixture of 2-amino-oleic acid revealed reduced calcium ions diffusion through such tissue and a nearly 20-fold decrease in calcium deposition [17,18]. The presence of the amino group is very important, because it seems that it binds with aldehyde residues of glutaraldehyde [19]. The existing synthetic method [15,20] starts from oleic acid and through a number of modifications at the exposition leads to the racemic 2-amino-oleic acid. However, this sequence of reactions may be applied only for the synthesis of racemic product. On the contrary, the proposed, by our group, methodology permits the synthesis of either (S)- or (R)- enantiomer depending on the chirality of the starting material, prepared from (S)- or (R)-glutamic acid. (S)-2-Aminooleic acid is expected to find a lot of applications for the construction of either peptide or lipid mimetics. Synthesis of (S)-2-Amino-oleic Acid Through tert-Butyl (2S)-2-[bis(tertButoxycarbonyl)amino]-5-oxopentanoate: The natural amino acid L-glutamic acid was used as inexpensive starting material. A strategy combining N-Boc group and a-tert-butyl ester group was chosen so that both groups may be removed simultaneously at the last step of the synthesis. Thus, (S)-Boc-Glu(OMe)-OBu1 (2) was treated with Boc2O in the presence of DMAP to afford derivative 3 (Figure 2). Selective reduction of the Y-methyl ester of compound 3 using DIBALH afforded aldehyde 4 [21].

78

V. Constantinou-Kokotou et al. / Synthesis of(S)-2-Amino-oleic Acid

u

u o

Jl

MeO.A^^AoBu' NHBoc

DIBALH Et

BOC2

°-DMAP

' *°

-78°C

i u N(Boc)2

N(Boc)2

Figure 2. Synthesis of re/t-butyl (2S)-2-[bis(ferr-butoxycarbonyl)amino]-5-oxopentanoate

Treatment of the key intermediate aldehyde 4 with the ylide, generated from EtOOC(CH2)3P*Ph3 Br and KHMDS yielded the Z-olefin 5 (Figure 3). The corresponding saturated compound 6, obtained from 5 after catalytic hydrogenation, was selectively reduced under controlled conditions by DBBALH to afford aldehyde 7. Wittig reaction of this aldehyde with the ylide generated from CrtyCHWsP^PhB Br", followed by subsequent removal of Boc and Bu' protecting groups, gave (5)-2-amino-oleic acid [21]. EtOOC(CH2)3PPh3Br. ^ KHMDS. THF, -78 °C

N(Boc)2 •OBu1

EtO

N(Boc)2

N(Boc)2

EtO

H2.10%Pd/C

OBu1

DIBALH, Et2O

r o

KHMDS, toluene, -78 C

-78 °C

N(Boc)2

1.TFA/CH2CI250%, 2 - neutralization

8

1

Figure 3. Synthesis of (5)-2-amino-oleic acid using aldehyde 4

Synthesis of (S)-2-Amino-oleic Acid Through Methyl (2S)-2-[bis(tertButoxycarbonyl)amino]-5-oxopentanoate: An improved route to (5)-2-amino-oleic acid used methyl (25)-2-[bis(/e/t-butoxycarbonyl)amino]-5-oxopentanoate (11) as key intermediate. The synthesis of this aldehyde [14] is depicted in Figure 4. Suitably protected glutamic acid semialdehyde 11 and related compounds have been proven useful synthons for the synthesis of biologically interesting compounds [22-25]. L-Glu

2. BoczO, Et3N, MeOH

MeO

-

O

0 U

.. T

OMe

N(Boc)2 10

g

DIBALH. Et2O .78oc

O

H^kx-XjAoMe NfBocfe 11

Figure 4. Synthesis of methyl (2S)-2-[bis(fe/-r-butoxycarbonyl)amino]-5-oxopentanoate

79

V. Constantinou-Kokotou et al. /Synthesis of(S)-2-Amino-oleic Acid

The Horner-Wadsworth-Emmons olefination reaction of aldehyde 11 with the phosphonate anion generated from triethyl-4-phosphonocrotonate by treatment with LiOH afforded compound 12 (Figure 5). After hydrogenation of 12, compound 13 was selectively reduced by DIBALH to afford aldehyde 14. Wittig reaction of 14 with the ylide generated by treatment of CH3(CH2)8P+Ph3 Bf with KHMDS produced compound 15. This fully protected a-amino acid was identified as Z-olefin after !H NMR analysis. o II

H2, 10%Pd/C

EtOOCCH=CHCH2P(OEt)2

11

OMe N(Boc)2

EtO

LiOH, THF

12

DIBALH, Et2O EtO

;5

OMe N(Boc)2

-78°C

j OMe N(Boc)2

C9H19PPhfB® KHMDS, toluene -78°C

14

13

s

Y OMe N(Boc)2

15

Figure 5. Synthesis of protected (S)-2-amino-oleic acid

Free 2-amino-oleic acid and derivatives were prepared as depicted in Figure 6. Both Boc groups of 15 were removed by treatment with HC1 in Et2O to produce compound 17. The saponification of 15 by treatment with 1 N NaOH was proved a very slow reaction. One out of the two Boc groups of 15 was removed selectively by treatment with Mg(ClO4)2 [26]. In the case of Af-monoprotected compound 16 the saponification was not complete after 24 h. Since racemization may occur after prolonged treatment under alkaline conditions, to prepare free 2-amino-oleic acid 1, it is recommended first to remove both Boc groups and then the methyl ester group by treatment with NaOH. Mg(CIO4)2, MeCN

is

.

°

^^r^fV™8 NHBoc 16

4N HCI/Et2O

OMe NH2.HCI

INNaOH, MeOH

5

I

OH

NH2

17

Figure 6. Free (S)-2-amino-oleic acid and derivatives

Both proposed procedures to 2-amino-oleic acid are free of racemization. To verify that point, (5)-2-amino-oleic acid obtained as depicted in Figure 3 was converted into its methyl ester. This compound as well as compound 17 were converted into their corresponding amides with (+)- and (-)-Mosher acid [27]. !H and I9F NMR analysis of all Mosher amides indicated absence of any diastereomeric signal. Both methods for the synthesis of (5)-2-amino-oleic acid, are simple and efficient. However, the latter presents higher overall yield.

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V. Constantinou-Kokotou et al. / Synthesis of (S)-2-Amino-oleic Acid

Synthesis of Enantiopure -Functionalized a-Amino Acids The stereoselective synthesis of a-amino acids and the construction of bioactive chiral molecules using amino acids has been achieved through a variety of methods [28]. Approaches using regioselective functionalization of readily available chiral building blocks, e.g. aldehydes obtained from amino acids, are especially attractive for the synthesis of unnatural amino acids. An efficient method to prepare enantiopure co-functionalized ctamino acids using the protected glutamic acid y-aldehyde 11 as key-intermediate was developed. Our attention was focused on long chain derivatives, because LAAs are of great importance for the synthesis of drug delivery systems and bioactive lipid mimetics. The Wittig olefination of aldehyde 11 with oxido, carboxy or amino ylides may directly lead to a-amino acids containing a free functional group in the side chain. Maryanoff [29] has studied the reaction of hexanal and benzaldehyde with triphenylphosphonium ylides containing anionic nucleophilic groups in their side chain. Following Maryanoff's procedure the reactions of 11 with the ylides generated from phosphonium salts of 10-bromo-decan-l-ol and 11-bromo-undecanoic acid were tested. In each case, the desired product was not obtained. Thus, we decided to use an ylide containing an co-protected hydroxy group. To prepare such ylides, a,to-alkane diols 20a,b were treated with TrtCl and DMAP in pyridine to produce mono-protected diols 21a,b (Figure 7). The free hydroxy group of 21a,b was activated by conversion to mesylate and the methanesulphonyloxy group was replaced by iodide ion. Iodides 22a,b were treated with PPh3 in MeCN under reflux to produce triphenylphosphonium salts 23a,b. HCL.OH TrtCl. DMAP TrtOv^OH Tin pyridine "" ^ 20a,b 21a,b

1 MsCI. Et3N

2Nal

TrtCL^I ™1 22a,b

PPh3. MeCN

TrtCX.PPhs*! *'n 23a,b

b 10 Figure 7. Synthesis of triphenylphosphonium salts 23a,b

Aldehyde 11 reacted with the ylides generated by treatment of 23a,b with KHMDS in toluene at 0 °C (Figure 8). The reactions were carried out at -78 °C and the protected amino acids 24a,b were produced in high yields. Both compounds were identified as Zolefins, by 'H NMR analysis. Methyl ester of amino acid 25 was obtained almost quantitatively by treatment of 24b with HC1 in THF. To check the enantiomeric purity, compound 25 was converted into the corresponding Mosher amides [27] and studied by I9F NMR analysis. An enantiomeric excess > 95% was indicated for 25 by the absence of any diastereomeric signal in the 19F NMR spectrum of each MTP amide.

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V. Constantinou-Kokotou et al. / Synthesis of (S)-2-Amino-oleic Acid

OMe 23aor23b. . KHMDS, toluene N(Boc)2

OMe

4N HCI/THF

N(Boc)2 24a, n=4 24b, n=10

11

"OMe

H2, 10%Pd/C

NH2.HCI 25

"OMe N(Boc)2 26

Figure 8. Synthesis of o>hydroxy a-amino acid derivatives

Catalytic hydrogenation of 24b produced the saturated oo-hydroxy a-amino acid 26 (Figure 8). This derivative was chosen as a model compound and was converted to co-carboxy, a>oxo and co-amino a-amino acids as depicted in Figure 9. Oxidation of 26 using 2.5 equivalents of NaOCl in the presence of 4-acetamido-2,2,6,6-tetramethyl-l-piperidinyloxy free radical (AcNH-TEMPO) and Aliquat 336 [30] produced the to-carboxy amino acid 27. In the absence of the phase transfer catalyst and using 1.1 equivalent of NaOCl [31], the 00oxo derivative 28 was isolated. To prepare diamino acids, the hydroxy group of 26 was activated as its methanesulphonate and was converted directly into azide 29 by treatment with sodium azide in DMF at 60 °C in high yield. Catalytic hydrogenation of 29 produced the free o>-amino functionalized derivative 30, whereas in the presence of N-(9fluorenylmethoxycarbonyloxy)succinimide the selectively protected derivative 31 was isolated. NaOCl, AcNH-TEMPO, Aliquat, KBr, NaHCO3, CHjClj, H2O

NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc, toluene, HSO

1 MsCI, Et3N 2 NaN3, DMF

Figure 9. Synthesis of protected co-functionalized a-amino acids

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In conclusion, an efficient route for the synthesis of enantiopure o>-hydroxy, co-carboxy and (D-amino a-amino acids was developed. The length of the side chain depends on the starting alkanediol used to prepare the phosphonium salts. Both enantiomers of CDfunctionalized a-amino acids may be prepared, because the chirality of the final product depends on the chirality of the key-intermediate glutamic acid y-aldehyde 11 obtained from the available in both enantiomeric forms Glu. References [I] For reviews see: (a) J. Game, Angew. Chem., Int. Ed. Engl. 33 (1994) 1699. (b) A. E. P. Adang et al., Rec. Trav. Chim. Pays Bas 113 (1994) 63-78. (c) G. L. Olson et al., J. Med. Chem. 36 (1993) 3039. (d) A. Giannis and T. Roller, Angew. Chem., Int. Ed. Engl. 32 (1993) 1244. [2] W.A.Gibbons et al., Liebigs Ann. Chem. (1990) 1175. [3] G. Kokotos et al.. Int. J. Peptide Protein Res. 48 (1996) 160. [4] G. Kokotos et al., Amino Acids 11 (1996) 329. [5] A. Nicolaou et al., J. Pep. Sci. 3 (1997) 291. [6] I. Toth, J. Drug Targeting 2 (1994) 217. [7] I. Toth et al., J. Med. Chem. 42 (1999) 4010. [8] I. Toth et al., Tetrahedron Lett. 34 (1993) 3925. [9] G. Kokotos et al., Bioorg. Med. Chem. Lett. 8 (1998) 1525. [10] V. Constantinou-Kokotou et al., Anticancer Res. 18 (1998) 3439. [II] T. Markidis et al., Anticancer Res. 21 (2001)2835. [ 12] G. Kokotos et al, Lipids 34 (1999) 307. [ 13] G. Kokotos et al., Tetrahedron: Asymmetry 7 (1996) 857. [14] G. Kokotos et al., J. Org. Chem. 63 (1998) 3741. [15] J. M. Girardot, Prevention of Prosthesis Calcification, US Patent No. 4,976,733, 1990. [16] J. K. Nozynski et al., Med. Sci. Monit. 7 (2001) 550. [17] S. K. Khanna et al.. Thorax 36 (1981) 330. [18] J. P. Gott et al., Ann. Thorac. Surg. 53 (1992) 207. [19] W. Chen et al.. Circulation, 90 (1994) 323. [20] F. Amat Guerri, Grasas Aceites (Seville) 26 (1975) 90. [21] V. Constantinou-Kokotou et al., J. Peptide Res. 58 (2001) 325. [22] N. Hernandez and V. Martin, J. Org. Chem. 66 (2001) 4934. [23] T. Markidis and G. Kokotos, J. Org. Chem. 66 (2001) 1919. [24] A. Sutherland et al., Chem. Commun. 6 (1999) 555. [25] J. M. Padron et al.. Tetrahedron: Asymmetry 9 (1998) 3381. [26] J.A. Stafford et al.. Tetrahedron Lett. 34 (1993) 7873. [27] J. A. Dale et al., J. Org. Chem. 34 (1969) 2543. [28] (a) R. H. Williams, The Synthesis of Optically Active a-Amino Acids, Pergamon, New York, 1989. (b) G. M. Coppola and H. F. Schuster, Asymmetric Synthesis: Construction of Chiral Molecules Using Amino Acids, Wiley, New York, 1987. [29] B. E. Maryanoff et al, J. Am. Chem. Soc. 107 (1985) 217. [30] P. L. Anelli et al., J. Org. Chem. 52 (1987) 2559. [31 ] M. R. Leanna et al.. Tetrahedron Lett. 33 (1992) 5029.

Drug Discovery and Design: Medical Aspects J. Matsoukas and T. Mavromoustakos (Eds.) IOS Press, 2002

83

Development of New Methds for

Fluorometric Assays of Enzymes

C. Tzougraki Department of Chemistry, University of Athens, Panepistimiopolis, Zografou, Athens 15771, Greece

Abstract. This article describes the development of new fluorescent markers and their use in the solution or solid phase synthesis of fluorogenic and intramolecularly quenched fluorogenic substrates (IQFS) for proteases or Upases. The determination and study of several proteases, such as chymotrypsin, oxytokinase, prolylendopeptidase, aminopeptidase M, elastase and trypsin are reviewed. The results of an extensive study on the donoracceptor combination and their application in the synthesis of very sensitive IQFS, as well as in the conversion of p-nitroanilide substrates into IQFS, are given. A new general and convenient method for the direct synthesis of any fluorogenic substrate or IQFS by using exclusively solid phase peptide synthesis is also described.

Introduction Proteases play essential roles in the regulation of important biological processes and serve as possible indicators for the evaluation of various diseases [1]. Synthetic peptide substrates are widely used for studies of proteolytic enzymes and determination of their activities. These substrates must have the appropriate amino acid sequence for interaction with the active sites of enzymes. A number of convenient methods exist for studying the hydrolysis of these substrates, such as potentiometry, spectroscopy, etc. [2–5]. Spectroscopic methods offer the advantage of the small quantity of the sample required for the measurement. In particular, synthetic fluorogenic substrates have found a wide application in basic and clinical research for the determination of proteases, because the assays based on them are convenient and more sensitive than those based on chromogenic substrates. The fluorogenic substrates are of the general form P-A n -A n .| ...A2-A|-F, while the estimation of the enzyme activity is based on the increase of fluorescence observed upon enzymatic liberation of the fluorophore HF.

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Several fluorescent compounds have been used as fluorophores HF, mostly coumarin derivatives, such as 7-amino-4-methylcoumarin (AMec) (1) [6], 7-amino-4(trifluoromethyl)coumarin (AFMec) (2) [7], 7-amino-3-phenylcoumarin [8], 7-amino3,4-benzocoumarin [8], 7-aminocoumarin-4-methanesulphonic acid [9], etc. (Scheme 1). AMec is the most common and widely used leaving group in fluorometric assays. A considerable number of proteases, such as chymotrypsin [6, 10], trypsin [10, 11], elastase [10], ami nopeptidase [12], thrombin [13, 14], renin [15], yglutamyltransferase [16] and oxytokinase [17], have been studied by using fluorogenic substrates, N-acyl-derivatives of AMec, and many of the latter are commercially available. However, the tremendous number and diversity of enzymes in nature, their appearance in impure form in coexistence with others and their low concentrations are the main reasons that demand more specific and sensitive analytical methods. Some years ago, while working mainly in the field of peptide chemistry, following the tradition of our laboratory, we were also engaged in the synthesis of heterocyclic compounds, and more specifically in the synthesis and study of coumarin and quinolinone derivatives [18]. Having found that some of them had very good fluorescence properties, we decided to examine them as potential markers in synthetic peptide substrates. Thus, we became involved in the field of enzymatic analysis, hoping to develop more sensitive and specific methods of enzymatic assays. In this review, some results from our efforts in the above field will be summarized. Coumarin and quinolinone derivatives as fluorescent markers: In order to evaluate new fluorescent markers, several substrates for chymotrypsin, Glt-Phe-NHRx, were prepared. [19], where RXNH2 are the compounds shown in Scheme 1, 7-amino-4methyl-2-quinolinone (3, AMeq), 7-amino-4-trifluoromethyl-2-quinolinone (4, AFMeq), 3-aminocoumarin (5, 3-Ac), 6-aminocoumarin (6, 6-Ac), 3-acetamido-6aminocoumarin (7, 3-AcNH-6-Ac) and 3-acetamido-8-aminocoumarin (8, 3-AcNH-8Ac). The synthesis of compounds 5, 8 and 9 has been described in Ref. 18 and that of 4 in Ref. 20. AMeq was prepared according to the literature [21]. The fluorescence properties of the free amines and those of the corresponding substrates were examined [19] and compared with those of others known from the literature.

H,N

AMec AFMec AMeq AFMeq

X = NH X = NH

= CH3 . = CF3 = CH3 = CF3

5 6 7 8

3-Ac 6-Ac 3-AcNH-6-Ac 3-AcNH-8-Ac

R, R, R, R,

= NH2 =H = NHCOCH, = NHCOCH,

Scheme 1: Coumarin and quinolinone derivatives used as fluorescent markers.

R2 = R} = H R2 = NH2, R, = H R2 = NH2. R, = H R2 = H. R, = NH2

C. Tzougraki / New Methods for Fluorometric Assays of Enzymes

85

Table 1. Fluorescence properties of substituted coumarins and quinolinonesa Rel. Fluor.b

Maxima

Compound

1

0.98 345 445 AMec 365 0.23 490C AFMec 1.00 344 418 AMeq 3 0.31 360 470 AFMeq 4 0.14 370 333 9 Glt-Phe-NH-Meq 0.07 340 Glt-Phe-NH-FMeq 390 10 1.0 uM solution in 0.05 M TES, pH 8, containing 1% DMSO. D The fluorescence intensities are relative to the intensity of AMeq on an equimolar basis. c 495 in Ref.[7a]. 2

i (b) lOr

s

350

40O

450

500

Wavelength (nm)

400

450

500

*

Figure 1. Fluorescence spectra of AMeq ( ), a: X^x = 344 nm; P: XeX = 360 nm, and Glt-Phe-NH-Meq (—-), a: Xex = 333 nm; (3: A^ = 360 nm. 1.0 uM solution in 0.05 M TES, pH 8, containing 1% DMSO.

Among the compounds examined, AMeq and AFMeq had the best fluorescence properties. Their excitation and emission maxima and their relative fluorescence intensities are distinctly different than those of their N-acyl derivatives, as shown in Table 1. Compounds AMec and AFMec are also listed for comparison. As expected, the replacement of the methyl group of AMeq by the electron withdrawing trifluoromethyl group in AFMeq produced a red shift of 52 nm and a decrease in fluorescence intensity [22]. Both 2-quinolinone derivatives AMeq and AFMeq exhibited a blue shift of approximately 25 nm and greater fluorescence intensity than their coumarin analogs AMec and AFMec respectively [20]. At maximum wavelengths the relative fluorescence intensities of AMeq and AFMeq are only 7- and 4.5-fold greater than those of their corresponding N-acyl derivatives respectively (Figure la). However, at XeX = 360 nm and A«m = 435 nm AMeq has a relative fluorescence approximately 1000-fold greater than that of an equimolar solution of the substrate, retaining 28% of its maximum fluorescence (Figure Ib).

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C. Tzougraki / New Methods for Fluorometric Assays of Enzymes

Similarly, the relative intensity of AFMeq at A«x = 380 nm and Xem = 480 nm is 850fold greater than that of its N-acyl derivative, retaining 41% of its maximum. At the mentioned wavelengths the presence of the substrate in each case does not interfere, and enzymatic hydrolyses can be followed by measuring the development of fluorescence. As has been reported by Zimmerman et al. [6], AMec possessed a relative fluorescence approximately 700-fold greater than that of an equi molar amount of the substrate, retaining 22% of the maximum fluorescence. The results of the performed study showed that the 2-quinolinone derivatives AMeq and AFMeq would be exceptionally useful fluorophores in synthetic peptide substrates for proteases, because they are highly fluorescent, easily prepared in one step synthesis and they can be acylated by amino acids in high yield. Having proved the utility of the proposed fluorescent markers, we further proceeded to their application for the determination and study of several proteases. Studies of several proteases Determination of chymotrypsin: Chymotrypsin (CT) and CT-like enzymes play an important role in metabolism by participating in many processes, such as digestion, fertilization of ova, inhibition of thrombin-induced platelet aggregation, peptide hormone generation, and catabolism, etc. Table 2. Kinetic parameters of several fluorogenic substrates' Compound

1, V -1 Detection limit Km Kcat f^-m [mM] [M-'s1] [ng/ml] 47 9 Glt-Phe-NH-Meq 0.50 10 10 Glt-Phe-NH-FMeq 99 0.28 35 11 0.67 Glt-Phe-NH-Mec 78 500 12 Glt-Leu-Phe-NH-Meq 0.62 0.7 710 13 Glt-Leu-Phe-NH-FMeq 0.33 367 10 14 Glt-Tyr-NH-Meq 0.13 630 Glt-Leu-Tyr-NH-Meq 0.22 15 2100 16 TFA-H-Ala-Ala-Phe-NH-Mec 0.50 1660 25 a 1.0 uM solution in 0.05 M TES, pH 8, containing 1% DMSO.

Ref. [19] [20] [6, 10] [20] [25] [25] [3, 10]

From the above mentioned study, conducted on the model compounds, it was already concluded that the compound Glt-Phe-NH-Meq (9) was a suitable substrate for chymotrypsin. Its detection limit was 10 ng/ml, 50 times less than that obtained by using the corresponding substrate of AMec, Glt-Phe-NH-Mec (11) (Table 2). A concentration of 10 ng/ml of the enzyme chymotrypsin has been found in the adults' serum by radioimmunoassay [23]. This finding requires a higher sensitivity assay. Therefore, we tried further to increase the sensitivity of the assay by extending the peptide backbone, in order to take advantage of the role of the amino acid at the P2 position on the binding affinity between substrate and enzyme. It is known that chymotrypsin hydrolyses long peptide amides more efficiently than short ones and that bulky non-polar residues at the P2 position interact with the side chain of isoleucine-99 of chymotrypsin [24]. Thus the substrates Glt-Leu-Phe-NH-Meq (12) and Glt-Leu-PheNH-FMeq (13) with leucine at P2 position were prepared and their kinetic parameters

C. Tzougraki / New Methods for Fluorometric Assays of Enzymes

87

were measured. The results of their hydrolysis catalyzed by chymotrypsin are given in Table 2, along with others for comparison. All four substrates 9, 10, 12 and 13 were specific for chymotrypsin showing no detectable hydrolysis when incubated with porcine pancreatic or leukocyte elastase. As expected, the presence of the amino acid leucine at the P2 position increased the kcat/Km value, while it had no considerable effect on the Km value. It also increased the sensitivity of the assays. As little as 0.7 ng/ml of enzyme can be detected using the substrate Glt-Leu-Phe-NH-Meq, which was the most sensitive fluorogenic substrate reported. The assays using the substrates containing the fluorescent marker AMeq were more sensitive than those using the corresponding substrates containing AFMeq. However, as the assays using the latter substrates were conducted at longer wavelengths, the background fluorescence exhibited by most biological matter, when excited in the spectral region 330–360 nm, could be avoided. Later, the substrates Glt-Tyr-NH-Meq (14) and Glt-Leu-Tyr-NH-Meq (15) with tyrosine at P1 position were synthesized [25]. The improved kinetic parameters of 14 and 15 compared to those of 9 and 12 proved that the presence of tyrosine instead of phenylalanine increases the affinity to the enzyme. The substrate 15 had the highest kcat/K.m value among the fluorogenic ones reported. All six substrates 9, 10, 12–15, are suitable for a sensitive and convenient determination of chymotrypsin. Determination of Oxytokinase: The determination of the activity of serum oxytocinasecystine aminopeptidase (CAP - EC 3.4.11.3) - which progressively increases during the course of normal pregnancy [26, 27], is very useful in the evaluation of the placental insufficiency [28–30]. Increased CAP activity has been found in the serum of patients with serious liver diseases and in seminal plasma [31]. CAP activity has been detected in the brain synaptic membranes [32] and in a haemolysate of human erythrocytes [33]. Recently, it has been suggested that placental oxytocinase may have an intriguing possibility in a variety of events not restricted to the regulation of pregnancy induced phenomena [34]. Several colorimetric assays using cystyl or cysteinyl p-naphthylamides or pnitroanilides have been developed for the determination of CAP activity in human serum. However, the utilization of these substrates is limited by their low solubility in water, and an affinity to oxytocinase (0.5 - 2.0- 10-3 mol 1-1) by 2-3 orders lower than the affinity of the natural substrate - oxytocin. The required amount of organic solvent for their solubilization influences adversely the course of the enzymic reaction. These drawbacks led to the application of a fluorogenic substrate H-Cys(Bzl)-NH-Mec [35] for ultramicrodetermination CAP in clinical practice. Since we had shown that in case of chymotrypsin substrates containing AMeq permit a more sensitive enzyme determination than the corresponding ones containing AMec, we prepared the fluorogenic substrate H-Cys(Bzl)-NH-Meq (17) and we used it for the determination of oxytocinase isolated from human retroplacental serum [36]. The enzymatic release of Ameq was followed at XeX = 340 nm and Xem = 430 nm, where its relative intensity is 100-fold greater than that of an equimolar solution of the substrate. Michael is constants (Km) of CAP for H-Cys(Bzl)-NH-Meq were determined at various pH values. The Km value at optimum pH (7.0-7.05) was 2.3 umol 1-1. The affinity of this substrate to oxytocinase was by two orders higher than that of chromogenic ones and greater than the affinity of the aminocoumarin substrate, as was evident from the comparison of the Km values: H-Cys(Bzl)-NH-N p , Km = 360 umol 1-1

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c. Tzougraki / New Methods for Fluorometric Assays of Enzymes

[37], H-Cys(Me)-NH-Np, Km = 440 umol 1-1, H-Cys(Bzl)-NH-Mec, Km = 5.4 umol 1-1 [35]. The measurement of the relative activity of serum oxytocinase with several concentrations of DMSO at various pH values, showed that already 5% of DMSO in the reaction mixture caused an approximately 50% decrease in activity. However, due to the high affinity of the substrate 17, in the range of its concentrations used for the kinetic studies, the concentration of DMSO (