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Design and Synthesis of HIV-1 Protease Inhibitors

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Design and Synthesis of HIV-1 Protease Inhibitors Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 245 _____________________________ _____________________________ Design and Synthesis of HIV-1 Protease Inhibitors BY MATHIAS ALTERMAN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Organic Pharmaceutical Chemistry presented at Uppsala University in 2001 ABSTRACT Alterman, M. 2001. Design and Synthesis of HIV-1 Protease Inhibitors. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 245. 70 pp. Uppsala. ISBN 91-554-4906-9. Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome (AIDS). The C2-symmetric HIV-1 protease is one of the prime targets for chemotherapy in the treatment of the HIV infection. Inhibition of HIV-1 protease leads to immature and non-infectious viral particles. The design and synthesis of a number of C2-symmetrical C-terminal duplicated HIV-1 protease inhibitors and subsequent biological evaluation is presented in this thesis. A versatile three step synthetic route has been developed using a carbohydrate as an inexpensive chiral starting material thus allowing inhibitors with the desired stereochemistry to be obtained. By this efficient method a series of tailor-made P2/P2' modified inhibitors were synthesized, and these were evaluated on purified HIV-1 protease and in HIV-1 infected cell assays. Highly active HIV-1 protease inhibitors were identified among the tested compounds. Analyses of the X-ray crystal structures of two of the most active compounds, as complexes with the protease, guided the further design of P1/P1' elongated inhibitors. Substitutions in the para-position of the P1/P1' benzyl groups were promoted efficiently by microwave-irradiated of palladium- catalyzed reactions. Particular modifications in the P1/P1' region of the inhibitors resulted in a 40-fold increase of the anti-viral activity on HIV-1 infected cells. Furthermore, a fast, efficient, and general one-pot microwave enhanced synthesis protocol for transformations of organo-bromides to tetrazoles was developed and applied on the inhibitor scaffold. Attachment of linker molecules to the P1/P1' benzyl groups of one inhibitor was used to develop of sensitivity enhancer tools in surface plasmon resonance biosensor assays. These new assays enable the evaluation of low- molecular weight compounds as HIV-1 protease inhibitors. Mathias Alterman, Organic Pharmaceutical Chemistry, Department of Pharmaceutical Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala Sweden © Mathias Alterman 2001 ISSN 0282-7484 ISBN 91-554-4906-9 Printed in Sweden by Lindbergs Grafiska HB, Uppsala 2001 PAPERS DISCUSSED This thesis is based on the following papers. I. Alterman, M.; Björsne, M.; Mühlman, A.; Classon, B.; Kvarnström, I.; Danielson, H.; Markgren, P. O.; Nillroth, U.; Unge, T.; Hallberg, A.; Samuelsson, B. Design and Synthesis of New Potent C2-Symmetric HIV-1 Protease Inhibitors. Use of L-Mannaric Acid as a Peptidomimetic Scaffold. J. Med. Chem. 1998, 41, 3782-3792. II. Alterman, M.; Andersson, H. O.; Garg, N.; Ahlsén, G.; Lövgren, S.; Classon, B.; Danielson, U. H.; Kvarnström, I.; Vrang, L.; Unge, T.; Samuelsson, B.; Hallberg, A. Design and Fast Synthesis of C-Terminal Duplicated Potent C2- Symmetric P1/P1'-Modified HIV-1 Protease Inhibitors. J. Med. Chem. 1999, 42, 3835-3844. III. Alterman, M.; Hallberg, A. Fast Microwave-Assisted Preparation of Aryl and Vinyl Nitriles and the Corresponding Tetrazoles from Organo-halides. J. Org. Chem. 2000, 65, 7984-7989. IV. Alterman, M.; Sjöbom, H.; Säfsten, P.; Markgren, P. O.; Danielson, U. H.; Hämäläinen, M.; Löfås, S.; Hultén, J.; Classon, B.; Samuelsson, B.; Hallberg, A. P1/P1' Modified HIV Protease Inhibitors as Tools in Two New Sensitive Surface Plasmon Resonance Biosensor Screening Assays. Eur. J. Pharm. Sci. 2001, Accepted. Reprints were made with permission from the publishers. Contents CONTENTS ABBREVIATIONS 6 1. INTRODUCTION 7 1.1 Acquired Immune Deficiency Syndrome (AIDS) 7 1.2 Viruses 9 1.3 Human Immunodeficiency Virus (HIV) 10 1.4 Replicative Cycle of HIV 11 1.5 Targets for Anti-HIV Chemotheraphy 12 1.6 Reverse Transcriptase Inhibitors 12 1.7 HIV Protease Inhibitors 14 1.8 HIV-1 Protease 15 1.9 Paradigms for Drug Discovery 19 2. AIMS OF THE PRESENT STUDY 20 3. DESIGN OF HIV PROTEASE INHIBITORS 21 3.1 Design of a New C-Duplicated Scaffold 23 4. SYNTHESIS OF THE 1,6-RETRO AMIDE 25 4.1 A New Three-Step Synthesis 27 4.2 Structure-Activity Relationship of P2/P2' Modifications 29 4.3 X-Ray Crystallographic Data 31 5. SYNTHESIS OF P1/P1' SUBSTITUTED INHIBITORS 33 5.1 Structure-Activity Relationships of P1/P1' Modifications 35 5.2 X-Ray Crystallographic Data 38 6. MICROWAVE PROMOTED PREPARATION OF ORGANO- NITRILES AND THE CORRESPONDING TETRAZOLES 40 6.1 Microwave-Promoted Cyanation Reactions 41 6.2 Microwave-Promoted Cycloaddition Reactions 42 6.3 One-Pot Reactions 43 7. SURFACE PLASMON RESONANCE BIOSENSOR ASSAYS 46 7.1 Synthesis of the “Assay Tools” 48 7.2 Assay Evaluation 49 7.3 Comparison Between the One and Two Linker Strategies 50 CONCLUDING REMARKS 51 ACKNOWLEDGEMENTS 53 REFERENCES 55 5 Abbreviations ABBREVIATIONS Ac acetyl Arg arginine Asp aspartic acid 9-BBN 9-borabicyclo[3.3.1]nonane Cbz carbobenyloxy CD4 receptor on the surface of cells with in the immune system CSA (±)-camphorsulfonic acid DCM dichloromethane DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DIEA N,N-diisopropylethylamine DNA deoxyribonucleic acid DMAP 4-(dimethylamino)pyridine DMF dimethylformamide DSC N,N'-disuccinimidyl carbonate ED50 50% inhibitory concentration in cell-assay EDC 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride FDA US food and drug administration Gag polyprotein containing structural proteins gag-gene genome for Gag polyprotein Gln glutamine Gly glycine gp120,41 glycoprotein 41 and 120 HOBT 1-hydroxybenzotriazole hydrate HOAc acetic acid IC50 concentration of the inhibitor resulting in 50% inhibition Ile isoleucine IN integrase Ki inhibitory constant M46I methionine in position 46 of the protease is mutated to isoleucin MT-4 CD4+ lymphoblastoid cells NHS-LC-Biotin succinimidyl-6-(biotinamido)hexanoate NNRTI non-nucleoside reverse transcriptase inhibitor NRTI nucleoside reverse transcriptase inhibitor p7,17,24 protein 7, 17, and 24 Phe phenylalanine Pol polyprotein containing functional enzymes Pol-gene genome for Pol polyprotein PR HIV protease Rf retardation factor RNA ribonucleic acid RT reverse transcriptase RU resonance unit, arbitrary unit in SPR measurement SPR surface plasmon resonance TBDMSCl tert-butyldimethylsilyl chloride TEMPO 2,2,6,6-tetramathyl-1-piperidyloxy, free radical TFA triflouroacetic acid THF tetrahydrofuran Thr threonine V32I valine in position 32 of the protease is mutated to isoleucin V82I,A valine in position 32 of the protease is mutated to alanine or phenylalanine V84I valine in position 84 of the protease is mutated to isoleucin Val valine XTT 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xanilide 6 Introduction 1. INTRODUCTION 1.1 Acquired Immune Deficiency Syndrome (AIDS) In 1981 an increased occurrence of unusual cases of Pneumocystis carinii pneumonia and Kaposi´s cancer together with other opportunistic infections, was observed among previously healthy homosexual men and intravenous drug abusers in the USA.1,2 An underlying immunosuppression was found to be promoter of these rare diseases. This syndrome became known as Acquired Immune Deficiency Syndrome (AIDS).3 In 1983 the causative agent of AIDS was identified as a human retrovirus, first isolated in France from a patient with multiple lymphadenopathies,4 a condition linked to AIDS, and subsequently in 1984, from AIDS patients.5,6 Initially, three different names were given to the virus isolated from AIDS patients; human T lymphotropic virus III (HTLV- III),5 lymphadenopathy-associated virus (LAV),7 and AIDS-associated retrovirus (ARV).6 Eventually the AIDS-causing virus was in 1986 given an alternative name, human immunodeficiency virus (HIV).8 A few years later a second similar virus, HIV-2, was isolated from patients in West Africa.9 Both HIV subtypes can lead to AIDS, although the pathogenic course with HIV-2 might be longer. The genome homology of HIV-1 and HIV-2 are approximately 40%.10 Retrospective studies indicate that the first documented case of AIDS occurred in Central Africa in 1959 and the source of the virus is proposed to come from the same geographic area.11 The origin of the two viruses has now been shown to be derived from two African monkeys, the chimpanzee (Pan troglodytes troglodytes) for HIV-112 and the sooty mangabay (Cercocebus atys) for HIV-2.13 A striking and somewhat unique feature of HIV is that the virus infects the helper T- lymphocytes, which exert a central role in the regulating of the immune response.5,6,14- 16 Since HIV infection causes depletion of helper T-lymphocytes, AIDS patients demonstrate a weakened immune system. Thus, the gradual depletion of these cells makes the patient increasingly susceptible to opportunistic infections of bacterial, viral or fungal origin and to certain cancers, which are key features of the final stage of the HIV infection, i.e. AIDS.17 The helper T-lymphocytes were the first cell types to be identified as targets for HIV. Viral infections in macrophages and monocytes were recognized shortly after. Moreover, it was discovered that the helper T-lymphocyte surface marker, CD4, was the receptor for the HIV viral surface glycoprotein gp120.18,19 Since then, however, many 7 Introduction other cell types have been shown to be infected by the virus, including the cells in the brain, and the nervous system. The HIV virus enters the central nervous system at an early stage of the infection and forms a reservoir in the brain as evidenced by the presence of large quantities of unintegrated viral DNA in the brains of HIV infected individuals.20 The course of the HIV infection is reflected by the concentration of the CD4+ helper T- lymphocytes in the blood (Figure 1).
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