Design and Synthesis of HIV-1 Protease Inhibitors

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 245

______

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

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). Normally the concentration is 800-1200 cells/mm3 but in the final phase of the disease the number is < 200 cells/mm3 of blood.21

CD4+ cells Plasma viral load AIDS

4-8 weeks up to 12 years 1-2 years

Figure 1. The natural history of HIV infection.

The primary infection, defined as the period during which the HIV infection is established in the host, is characterized in 30 – 70% of the patients by a systemic illness including fever, headache, rash, pharyngitis, gastrointestinal disturbance, and lymphadenophathy.22,23 Subsequently, an asymptomatic phase follows that varies in length but has a mean of 10 years, prior to the final phase of the disease, when the individuals become highly vulnerable to infections by other viruses and microbial organisms (opportunistic infections).24 The HIV infection in muscles and the central nervous system results in muscular wastage and AIDS-related dementia.25,26 The average life expectancy without therapy from the appearance of AIDS is 1-2 years in developed countries.24

A decade ago, HIV/AIDS was primarily regarded as a serious health crisis. It was predicted in 1991, that in sub-Sahara Africa, by the end of the decade, nine million people would be infected and five million would die. This was later shown to be a threefold underestimate. Today, AIDS is undoubtedly a development crisis, and in some parts of the world also a security crisis. In December 1999 it was estimated that 34.3 million people were suffering from HIV/AIDS and of those, 5.4 million people were

8 Introduction infected under 1999. Altogether more than 18 million people have died since the beginning of the epidemics.27

Eastern Europe Western Europe & Central Asia 520 000 North America 420 000 900 000 East Asia & Pacific North Africa 530 000 Caribbean & Middle East 220 000 360 000 South & sub-Saharan South-East Asia Latin America Africa 5.6 million 1.3 million 24.5 million Australia & New Zealand 15 000

Figure 2. Number of people living with HIV/AIDS – total: 34.3 million (December 1999).

1.2 Viruses

Microorganisms The unicellular microorganism (like bacteria) however small and simple, are cells. Unicellular microorganisms always contain DNA as the repository of genetic information, and in addition, also RNA. These cells have their own machinery for producing energy and macromolecules. Thus, the unicellular microorganisms grow by synthesizing their own macromolecular constituents (nucleic acids, proteins, carbohydrates, and lipids), and in the majority of cases, multiply by binary fission.21

Viruses Viruses, on the other hand, are not cells. They are completely dependent on their cellular hosts for the machinery of energy production and synthesis of macromolecules. The virus particle contains only one type of nucleic acid, either DNA or RNA, never both, and differs from non-viral organisms by having two clearly defined phases in their life cycle. In the first phase (the transmission phase) outside a susceptible cell, the virus particle is metabolically inert. In the second phase (the reproductive phase) inside the cell, the viral genome exploits the metabolic pathways of the host to produce progeny genomes and viral proteins that assemble to form new infectious virus particles called virions.21

The primary criteria for the delineation of virus families are; (1) the kind of nucleic acid that constitutes the genome, either DNA or RNA, with DNA viruses the predominant group, (2) the mechanism of viral replication, (3) and the morphology of the virion.21

9 Introduction

1.3 Human Immunodeficiency Virus (HIV)

HIV-1 and HIV-2 are RNA viruses and belong to the family of retroviruses, Retroviridae (retro, backwards). The genome of retroviruses consists of duplicate copies of positive single-stranded RNA. Once a cell has become infected with a retrovirus the viral genetic information will be transformed from RNA to DNA, catalyzed by viral enzyme reverse transcriptase. The name retrovirus is derived from this unique event, which is completely opposite to the normal process where RNA is transcribed from DNA. Retroviruses are divided into seven genera, where the genus Lentivirus (lenti, slow), is characterized by the slow development of disease after infection. HIV is a typical lentivirus, since it usually has a disease latency of several years.21

A schematic drawing of the mature HIV virion is shown in Figure 3. The virion is almost spherical and is about one ten-thousandth of a millimeter across (ca. 100 nm).28 The virus is enveloped by a lipid bilayer that is derived from the infected host cell. The outer surface is studded with surface glycoproteins (gp120) that are anchored to the virus via interactions with the transmembrane protein (gp41). These surface proteins play a crucial role when HIV binds to and enters the host cells. A shell of the matrix protein (p17) lines the inner surface of the viral membrane, and a conical capsid core particle constructed out of the capsid protein (p24) is located in the center of the virus. The capsid particle encapsulates two copies of the viral genome, stabilized by the nucleocapsid protein (p7), and also contains three essential virally encoded enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN).29

Lipid bilayer gp120

gp41

IN p17

p24

RNA p7

100 nm

Figure 3. Schematic drawing of the mature HIV-virion.

10 Introduction

1.4 Replicative Cycle of HIV

The attachment of the viral surface protein (gp 120) to the CD4-receptor, located on various cells within the immune system, initiates the replicative cycle of HIV (Figure 4).30 Attached virions utilize several additional cell-surface proteins to promote the fusion of the viral and host cellular membranes.31-34 Membrane fusion is followed by a poorly understood uncoating event of the capsid that allows the release of the viral content into the host-cell cytosol. The single-stranded viral RNA complexes with reverse transcriptase, which catalyses the reverse transcription to yield a double- stranded DNA molecule.35-37 The double-stranded viral DNA is then transported into the cell nucleus and is permanently integrated into the host genome by the catalytic activity of the viral integrase. The integrated viral DNA is designated provirus.38

2 3

7 5

Figure 4. Schematic drawing of the replicative cycle: 1. Attachment to the host cell CD4-receptor, 2. Viral fusion and uncoating, 3. Reverse transcription, 4. Integration of viral DNA to the host genome, 5. Translation, 6. Viral budding, 7. Maturation via protease activities.

By an unknown activation process the cell initiates the transcription of the proviral DNA by the host cellular RNA polymerase II. Initially, short spliced RNA species that encode the regulatory proteins Tat, Rev, and Nef are synthesized. Tat acts as a stimulator of the transcription of the proviral DNA to enhance the production of viral RNA.39-41 Full-length and singly spliced RNA is needed in the cytoplasm for the synthesis of Gag and Gag-Pol polyproteins, and for packing into new virions. Rev binds to the full length and singly spliced RNA in the nucleus and protects it from further splicing and actively transports it to the cytosol. In this manner, Rev acts as a switch between the early synthesis of highly spliced RNAs and the later synthesis of unspliced

11 Introduction and singly spliced RNAs.42-44 Nef acts as a down-regulator of the number of CD4 receptors on the surface of the infected cell.45

Translations of the unspliced RNA by the ribosomes produce the polyproteins Gag and Gag-Pol. These polyproteins are transported to the plasma membrane with two molecules of viral RNA. They assemble together with the envelope protein to form an immature virus particle that is released from the cell by budding from the cell surface. To become infectious, the virion has to pass through a maturation process where the enzyme HIV protease cleaves the polyproteins into functional enzymes and structural proteins. The mature HIV virion is now ready to infect a new cell and start a new replicative cycle.29,38

1.5 Targets for Anti-HIV Chemotheraphy

In principal, every step in the HIV replication cycle can be considered as a potential target for anti-viral chemotherapy. However, the number of practical targets for drug interventions is reduced due to the fact that the virus is an intracellular parasite, which relies on the metabolic pathways of the host cell. Hence, most agents that block the replication of the virus are also lethal to the host cell. The key in selective anti-viral therapy is therefore to identify any process that is essential for the replication of the virus, but not for the survival of the cell.21,46 The gained knowledge about the replicative cycle of the HIV-virus has led to the extraction of virus-specific processes. Predominantly, scientists have focused their attentions on the following processes: a) viral binding to target cells, b) virus cell fusion, c) virus uncoating, d) reverse transcription of genomic RNA, e) viral integration, f) gene expression, and g) protease activity. So far, the two strategies d and g have been proven to be the most successful in the search for drugs that can be used for treatment of AIDS.47-50 These two targets will be described briefly in the following sections.

1.6 Reverse Transcriptase Inhibitors

Virally encoded HIV reverse transcriptase (RT) catalyses the replication of single- stranded viral RNA to a double-stranded DNA. Inhibition of RT prevents the formation of this double-stranded DNA that can be integrated in the host DNA. Reverse transcriptase inhibitors can be divided into two categories, nucleoside (NRTI) and nonnucleoside reverse transcriptase inhibitors (NNRTI).51

The nucleoside analogues are prodrugs, which have to be phosphorylated by cellular kinases to form triphosphates that mimic the natural substrate, the nucleotides.50,52,53 The phosphorylated NRTI might then be incorporated into the growing DNA chain and terminate elongation or act as a competitive inhibitor.51 As early as 1985, zidovudine

12 Introduction

(3'-azido-3'-deoxythymidine, AZT) was found to be active against HIV replication in cell culture and was approved for treatment of HIV.54 Today, there are six NRTI approved (Figure 5). Unfortunately, all of these substances are associated with side effects, such as bone marrow suppression, peripheral neuropathy and acute pancreatitis.55,56 In addition, prolonged treatment with these compounds gives rise to clinical resistance.47,57

O NH2 O N NH N NH HO N O HO N O HO N O O O N

N3 Zidovudine58 Zalcitabine59 Didanosine60

O NH2 HN N NH N N HO N O HO N O HO N O O O N NH2

S Stavudine61 Lamivudine62 Abacavir63

Figure 5. Nucleotide Reverse Transcriptase Inhibitors (NRTI) approved by the FDA.

The NNRTIs are a diverse group of compounds, which non-competitively interact with an allosteric site of HIV-1 RT and thereby inactivate the enzyme without need for pre- activation of the drugs. NNRTIs bind in a highly hydrophobic pocket of the enzyme and exhibit grater affinity for the enzyme-substrate complex than for the free enzyme.46 The hydrophobic allosteric site is unique to HIV-1 RT and is not found in other RTs or DNA polymerases. This results in a high selectivity index and a low toxicity of the NNRTs. However, rapid eliciting resistance is a major problem with this type of inhibitor as well.51 Currently, three NNRTIs are used in clinic for the treatment of HIV infection (Figure 6).

O S O HN HN N N N F3C Cl O NN NH N N O N O H H O 64 Nevirapine Efavirenz65 Delavirdine66

Figure 6. Non Nucleotide Reverse Transcription Inhibitors (NNRTI) approved by the FDA.

13 Introduction

1.7 HIV Protease Inhibitors

HIV protease was first suggested as a potential target for AIDS therapy by Kramer et al. after it was shown that a frameshift mutation in the protease region of the pol-gene prevented cleavage of the Gag polyprotein precursor, which is essential for the maturation of the HIV particles.67 Blockage of HIV protease leads to the formation of immature non-infectious virions.68 Compounds, having the ability to inhibit this protease have been studied intensively during the last decade and numerous reports of potent HIV-1 protease inhibitors have been published.69-74

O NH2 H O OH S OOH N H H H H N NN N N N O N N N S H O CONH-t-Bu O H O

75 Saquinavir Ritonavir87

H OH H H O CON-t-Bu NN N HO N N O CONH-t-Bu HO H OH N S

Indinavir88 Nelfinavir89

O O O O OO H N O S HN N N ON N O OH H H OH NH2

Amprenavir90 Lopinavir91

Figure 7. Protease Inhibitors approved by the FDA.

Saquinavir was the first approved protease inhibitor and has been in clinical use since 1995.75 Presently, there are six clinically approved protease inhibitors (Figure 7). Although the inhibitors on the market are highly selective they induce side effects such as lipodystrophy, hyperlipidaemia, insulin resistance,76-82 and emergence of resistant mutants upon prolonged use.83-86 Therefore there will probably be a constant demand for new HIV protease inhibitors.

14 Introduction

1.8 HIV-1 Protease

Enzymes, such as HIV protease, are nature’s own catalysts. Proteases are a diverse class of enzymes that catalyze the cleavage of peptides or proteins. The interaction of a substrate (peptide or protein) with the protease lowers the activation energy of the peptide bond cleavage reaction by stabilizing the transition state, which is the molecular arrangement with the highest free energy.92 The enzymes thus enable fast reactions at room temperature that otherwise would have required extended reactions times. The half-life of a peptide bond at room temperature at neutral pH has been estimated to be about 500 years.93

Based on the presence of the characteristic signature amino acid sequence, Asp-Thr- Gly, it was suggested by Toh et al. in 1985 that the protease of HIV might belong to the family of aspartic proteases.94 This was confirmed through pepstatine A inhibition, an aspartic protease selective inhibitor,95 and by site-directed mutagenesis of the active site Asp 25, which led to abolition of the catalytic activity.96 The aspartic proteases are well-characterized group of enzymes that can be found in vertebrates, plants, in addition to in fungi. Examples of proteases from the aspartic protease class are pepsin, cathepsin D, renin, chymosin, penicillopepsin, and Rhizopus pepsin, which all are two-domain enzymes with more that 300 residues in length and contain the Asp-Thr-Gly sequence in each domain that forms the active site, which effectuate the cleavage reaction.97,98 Since the HIV protease sequence is no more than 99 amino acids and contains only one of the required triad Asp-Thr-Gly it was suggested that the active form of the HIV protease was a homo-dimer of 198 amino acids.98 This hypothesis was later confirmed by X-ray crystallographic determinations.99-101

The function of the dimeric structure of the protease is probably more sophisticated than simply to enabling the virus to be parsimonious in its genetic baggage.102 A regulatory mechanism that controls activation of the enzyme is derived during the dimerization process, since it is reversible. In a concentrated solution the protease is activated (in the budded viral particle), and in a highly diluted solution the protease is inactivated (in the host cell). This regulatory mechanism seems to be important for the virus in order to prevent premature breakdown of the polyproteins and to minimize damage of the host- cellular proteins (Figure 8).69

15 Introduction

Highly diluted Concentrated In the host cell In the viral particle Monomers Dimer INACTIVE ACTIVE

Figure 8. Dimerisation of the HIV protease.

Some general feature of the HIV-1 protease structure can be described (Figure 9): (i)

The two monomers are identical and form a C2-symmetric elliptical-shaped enzyme. (ii) The N- and C- termini of each monomer are juxtaposed in a four-stranded β-sheet that serves to hold the dimer together (the dimer interface). (iii) Each monomer has a hydrophobic core consisting of two loops, one of which includes the active site aspartic acid. (iv) The dimers come together to create an extended substrate-binding cleft capable of interacting with a minimum of seven consecutive amino acids in the substrate. (v) Each monomer contributes a flexible flap that folds down to make important contacts with the bound substrate.103

Flap Flap

Active-site Dimer interface

Figure 9. Structure of native HIV-1 protease.99

The HIV-1 protease processes the Gag and Gag-Pol polyproteins proteolytically at specific cleavage sites as shown in Figure 10.73,104 The HIV-1 protease is specific for cleavage of these sites in vivo, although the general sequence homologies among these are small.

16 Introduction

1234

Gag p17 p24 p7 p6

PR RT/RNaseH IN Pol

56 78

Site Sequence

1 -Ser-Gln-Asn-Tyr Pro-Ile-Val-Gln- 2 -Ala-Arg-Val-Leu Ala-Glu-Ala-Met- 3 -Ala-Thr-Ile-Met Met-Gln-Arg-Gly- 4 -Pro-Gly-Asn-Phe Leu-Gln-Ser-Arg- 5 -Ser-Phe-Asn-Phe Pro-Gln-Ile-Thr- 6 -Thr-Leu-Asn-Phe Pro-Ile-Ser-Pro- 7 -Ala-Glu-Thr-Tyr Phe-Val-Asp-Gly- 8 -Arg-Lys-Ile-Leu Phe-Leu-Asp-Gly- Figure 10. Cleavage sites of HIV protease in the Gag and Pol polyproteins.

Several studies, experimental and ab initio calculations, of the protein cleavage mechanism have been performed. A schematic representation of the mechanism is outlined in Figure 11.105-109 Hydration of the amide carbonyl group, with a water molecule accommodated between the two side-chains of the aspartic acid residues 25/125, gives a putative tetrahedral intermediate that is suggested to be an approximate representation of the transition state of the proteolytic reaction.

Substrate Tetrahedral Intermediate

R1 H O R1 H O N N N N H H OR2 O O R2 H H H O H O H O O O H O O O O

Asp 25 Asp 125 Asp 25 Asp 125

Products

R1 O R1 H O OH H N N N 2 N H H O R2 O O R2 H

H H H O O O O

O O O O

Asp 25 Asp 125 Asp 25 Asp 125 Figure 11. Schematic representation of the HIV protease cleavage mechanism.

17 Introduction

Following the unambiguous determination of the native enzyme structure, several structures of various inhibitor complexes of the protease have been reported.110-115 Upon binding of an inhibitor, the protease undergoes significant structural changes. The most dramatic changes are observed in the flap region. The two flaps fold over the inhibitor to form a tunnel-shaped active site, which runs diagonally across the dimer interface. The flaps are held in this closed position by hydrogen bonding from the flap residues Ile 50/150 to a water molecule, which in turn is hydrogen bonded to two carbonyls in the inhibitor (Figure 12).

Figure 12. Structure of HIV-1 protease with bound inhibitor (Stereoview).

Starting from the central aspartates, in the active site tunnel, there are distinct subsites named S1, S2, S3, and S4, with corresponding S1', S2', S3', and S4' subsites according to the convention of Schechter and Burger.116 The corresponding side-chains of the substrate or of the inhibitor are named P1 to Pn outwards from the scissile peptide bound toward the amino terminus and P1' to Pn' towards the carboxyl terminus (Figure 13). All subsites in the HIV-1 protease are bounded by mostly aliphatic side-chains and have hydrophobic character, with the exception of S4/S4', which are exposed to water.117

S3 S1 S2'

P3 H O P1 H O P2' H O N N N H2N N N OH H H O P2 O P1' O P3'

S2 S1' S3'

Figure 13. Nomenclature of subsites in the enzyme (Sn, Sn') and of the substrate (Pn, Pn') according to Schechter and Burger.

18 Introduction

1.9 Paradigms for Drug Discovery

A molecule that binds with high affinity to HIV protease is not necessarily suitable as a drug for the treatment of HIV infection. A drug must be endowed with a number of qualities that are not easily accomplished. For example, a high specificity of the drug is crucial, since significant inhibition of other host aspartyl proteases may lead to toxicity. A drug must be able to conquer a large set of viral genotypes, otherwise insurgence of drug resistance might appear. A drug should work in synergy with other chemical entities used for HIV treatment and be compatible with other therapies against opportunistic infections. The concentrations of the drug in the cells and in the circulation must be able to remain at levels far above the inhibitory constant (Ki) in order secure an effective treatment. Oral bioavailability is highly desirable to enable easy use of the drug. To reach the virus hiding in the brain the drug should be able to penetrate the blood-brain barrier. All of these criteria can be met by manipulating the structure of the molecules, but do not always go hand in hand, which makes the drug discovery process a laborious but challenging task. Finally, HIV infection requires a lifetime of treatment with a combination of several drugs, and therefore the drug must be inexpensive in order to be available to all those millions of people in need of therapy.69

19 Aims of the Present Study

2. AIMS OF THE PRESENT STUDY

This investigation is part of a research project aimed at the discovery of novel, selective HIV-1 protease inhibitors, with the ultimate objective of developing simple and cost- efficient, generic synthetic routes. The specific objectives of this study have been:

(i) To design a novel HIV-1 protease inhibitor scaffold and to develop a short, diverse, and cost-efficient synthetic route to inhibitors, using readily available carbohydrates as chiral starting materials.

(ii) To utilize fast microwave promoted palladium-catalyzed reactions for the optimization of identified leads.

(iii) To establish structure-activity relationships and to utilize X-ray structural determinations to guide further inhibitor design by an iterative process, with the proviso that target compounds of acceptable biological activity would be essential to enable this.

During the course of these studies, two new objectives arose:

(iv) To develop a fast microwave promoted method for the transformation of organo- bromides to tetrazoles.

(v) To modify the HIV-1 protease inhibitors into tools for surface plasmon resonance biosensor assays thus enhancing assay sensitivity.

20 Design of HIV Protease Inhibitors

3. DESIGN OF HIV PROTEASE INHIBITORS

In order for a molecule to inhibit HIV protease and thereby prevent cleavage of polyproteins, it must possess binding properties and be stable to proteolytic processing. Strategies required to find such molecules could be random, semi-random or by rational design. The most obvious strategy, besides random high throughput screening, is to utilize the peptidic substrate as a template, with the scissile peptide bond substituted with a non-cleavable bond. An example of a non-cleavable peptide bond mimic is the reduced amide introduced into a peptide and used in the earliest inhibitors of HIV protease.118 Only modest potency was achieved with most of the reduced amide compounds. Nevertheless, MVT-101 constituted the first inhibitor to be co-crystallized with the HIV-1 protease and provided valuable three-dimensional structural information of the HIV protease in its inhibited form.110

Ac Thr Ile Gln Arg NH N 2 H O

MVT-101 Figure 14. Example of a reduced amid inhibitor.

The transition-state of the substrate often possesses thousand-folds higher affinity for the enzyme than the substrate.92 Since the cleavage mechanism of the enzyme was known and a tetrahedral intermediate was proposed as being closely related to the transition-state, the design was shifted toward the preparation of transition state analogues. Vast knowledge of transition-state analogue inhibitors was gained through many years of research of the human aspartic protease renin, which is involved in the regulation of high blood pressure.119-121 Some examples of transition state analogues are depicted in Figure 15.

R1 R1 O N N H OH O H OH Statine Norstatine

R1 O R1 OH O R1 R2 N N N N H H H H OH R2 OH R2 OH O

Hydroxyethylene Dihydroxyethylene Hydroxyethylamine

R O R1 X X 1 X X N N H H OO OR2 Statone X = H Ketone X = H Difluorostatone X =F DifluoroKetone X =F

Figure 15. Examples of transition state analogues.

21 Design of HIV Protease Inhibitors

The statine skeleton, comprising five atoms, was employed as a dipeptide (six atoms) mimic and was incorporated in peptides to serve as inhibitors of HIV protease.122-124 The mediocre potency that was achieved with this fragment was considered to be attributable to the lack of a P1' side-chain.71 However, inhibitors with the norstatine moiety, which more closely resembles a single amino acid residue carrying an extra carbon, provided increased activity against HIV protease when incorporated into inhibitors.125-127 Hydroxyethylene isosteres constituted the basis for highly potent inhibitors and provided the first inhibitors to have activity toward HIV in cell-based assays.128-130 The improvement achieved with hydroxyethylene isosteres encouraged investigation of dihydroxyethylene isoteres. It was found that the contribution from the second hydroxyl group was negligible, as suggested by similar binding affinities of hydroxyethylene and dihydroxyethylene based transition state analogues.71 The hydroxyethylamine isosteres are perhaps most successful amongst the previously known transition state analogues used in HIV protease inhibitors. This transition state analogue was utilized in the first clinically approved HIV protease inhibitor, saquinavir (Figure 7).75

P1 O HO OH O P1 H O P1' N N - Terminal duplication H2N OH C - Terminal duplication P1 O P1' NH2 H2N P1'

Figure 16. N- or C- terminal duplication strategy.

C2-Symmetric inhibitors

The C2-symmetric nature of the HIV protease homodimer provides a unique property of the enzyme that could be exploited in the design process. X-ray data revealed that both the N- and C-termini of the asymmetric substrates and the inhibitors bind to identical subsites. The design of N- or C-duplicated inhibitors therefore was thought to be beneficial in terms of novelty, potency, and selectivity (Figure 16). Abbot employed the N-terminal duplication strategy at an early stage and presented the pseudosymmetric diaminoalcohol core unit of A-74704 (Figure 17).131 A highly symmetric binding of this inhibitor was confirmed by X-ray crystallographic studies.113 The concept of N- terminal duplication was further developed by Abbot into the diaminodiol core, exemplified by A-77003, which was studied clinically as an intravenous agent.132 Systematic improvements of this class of compounds resulted in the non-symmetric clinically approved compound ritonavir.133 The N-terminal duplication strategy has also been utilized by several other groups, e.g. Budt et al., Mo et al., and Ettmayer et al.134- 136

22 Design of HIV Protease Inhibitors

H OH H Cbz ValN N Val Cbz H OH O N Val N N N N Val N O OH H

A-74704 A-77003

Figure 17. N-Terminal duplicated inhibitors.

Examples of C2-symmetric inhibitors based on C-terminal duplication have also been reported, although this strategy has been studied less extensively.72 Bone et al. were the first to report a high-affinity inhibitor, L-700,417, exploiting this concept (Figure 18).137 A more peptide like inhibitor, which also exhibited a relatively high potency, was designed and synthesized by Babine et al. independently.138 Analysis of the crystal structure of L-700,417 reveled highly symmetric binding.137

HO H OH H OH N N NH H OH H HN N Ile Ile N O O N N O O

L-700,417 Babine et al.

Figure 18. C-Terminal duplicated inhibitors.

3.1 Design of a New C-Duplicated Scaffold

We wanted to explore the concept of C-terminal duplication further. Computer aided molecular modeling was used as a tool in the search for potential scaffolds. A large number of models of potential inhibitors were built, geometrically optimized in silico and compared to reported inhibitor/HIV protease X-ray data for the evaluation of distances and positions of the different side-chains, hydroxyl groups and scaffold backbone. Our hypothesis was to introduce a dihydroxyethylene C-terminal duplicated core structure, that had been described in literature previously as an unpublished result by Norbeck et al.72 Among our modeled substances evaluated, a scaffold accommodating a 1,6-retro amide motif with a six-carbon chain emerged as an attractive core structure (Figure 19).

23 Design of HIV Protease Inhibitors

O OH O H R N N R H O OH O

Figure 19. 1,6-Retro amide scaffold.

In order to synthesize our modeled in silico inhibitors with the desired stereochemistry, a chiral scaffold was needed. Carbohydrates have been utilized previously, by us139-141 and others groups,134,142-146 as a chiral pool for the synthesis of inhibitors with desired stereochemistry. Hexoses provide a commercially available source of six carbon scaffolds with a defined stereochemistry. The modeled stereochemistry and conformation needed for complementary interaction with HIV protease was suggested to be 2R, 3R, 4R, and 5R, a stereo configuration that could be derived from L-mannitol. The distance between the two carbonyl groups in the 1,6-retro amide, designed to interact with the structural water that is hydrogen bonded to the flap residues Ile 50/150, is notably only six atoms compared to seven atoms in the natural substrates. The suggested synthesis would deliver elongated the P1/P1' arms as a result of the insertion of oxygen into the benzyl side-chains designed to mimic the Phe amino acid.

24 Synthesis of the 1,6-Retro Amide

4. SYNTHESIS OF THE 1,6-RETRO AMIDE

L-Mannitol, which is expensive compared to the naturally occurring D-mannitol, could be obtained by reduction of the less expensive oxidized form, L-mannonic-γ-lactone, by reduction with lithium borohydride.146 To enable introduction of the P1/P1' substituents, protection of the hydroxyl groups at the 1,3,4, and 6 positions was necessary (Scheme 1).

Scheme 1.

HO OH OH O O O LiBH , MeOH O 4 OH O O O HO HO O CSA, Acetone OH OH HO OH 78% O O

O OH O OH TBDMSCl 70% HOAc OH Si O HO O Si 64% DMAP OH O Pyridine OH O 2 96% 3

Br O O O O Si O N(Bu)4F, THF OH O Si HO NaH, THF 98% 76% O O O O

L-mannitol was first reacted with 2,2-dimethoxypropane to achieve the triacetonide 1, which was subsequently treated with 70% acetic acid to deprotect the terminal isopropylidene groups to furnish the 3,4-monoacetonide 2 in good yield.146 A selective silylation of the primary hydroxyl groups was performed using t-butylchlorodimethyl silane in pyridine, in the presence of a catalytic amount of dimethylaminopyridine.147 No product derived from silylation of the secondary alcohol was detected in the reaction mixture. The 2- and 5- hydroxyl groups were now available for the introduction of the P1/P1' side-chains. Benzylation of the 2,5-diol in tetrahydrofuran using benzyl bromide, sodium hydride and a catalytic amount of tetrabutylammonium iodide gave the fully protected compound 4 in 76% yield. Desilylation of the primary alcohols was performed with tetrabutylammonium fluoride in tetrahydrofuran to give the compound 5 in 98% yield.

25 Synthesis of the 1,6-Retro Amide

In order to complete the synthesis of the inhibitor scaffold the primary hydroxyl groups had to be oxidized. There are not many examples of diol oxidations, and most methods rely on the use of potassium permanganate or nitric acid.148-150 TEMPO (2,2,6,6- tetramethyl-1-piperidyloxy, free radical) with sodium hypochlorite has been used for the direct oxidation of alcohols to corresponding acids under two-phase conditions.151 Oxidation of 1,4- and 1,5-diols with this system was reported to give the lactones in good yields while oxidation of the 1,6-diol was discouraging and delivered a mixture of polymeric compounds.152 The same reagents had also been used previously for the synthesis of hydroxyaldehydes from 1,3-, 1,4-, and 1,6-diols.153

We first tried to oxidize the diol 5 with pyridinium dichromate-acetic anhydride, but the desired compound was not obtained.154 More successfully, we applied 5 mol% of TEMPO and sodium hypochlorite at 0 °C, which resulted in the dicarboxylic acid 6 in 65-72% yield within 1.5 hours (Scheme 2). Attempts to optimize the reaction by varying the amount of TEMPO or the temperature in the reaction only resulted in lower yields. However, an experiment performed by us more recently, with twice the amount of solvent resulted in 92% yield of the dicarboxylic acid 6. Apparently, the conformational stabilization by the 3,4-O-isopropylidene group might be crucial for the formation of the 1,6-diacid, since when we performed a control experiment with non- cyclic protecting groups on the central hydroxyl groups, the desired product was not obtained.

Scheme 2.

O O N O O O OH O OH HO HO NaOCl, KBr O O O O O N(Bu)4Br NaHCO2(sat.) DCM, 0 °C 5 6 67%

To introduce the P2/P2' substituents, the diacid was preactivated, using the reagent system EDC, HOBT and triethylamine in dichloromethane and tetrahydrofuran.155 To react the diacid with N,N'-disuccinimidyl carbonate to give disuccinimidyl ester 7 provided an alternative route.156 The active ester 7 that was isolated in 88% yield could be stored prior to use. The desired amine was then added to form the compounds 8-12 in moderate to good yields (Scheme 3).

To emancipate the central hydroxyls from the isopropylidene group the conditions had to be chosen carefully since the diol-product is slightly sensitive to acid. The usage of a TFA-water mixture or p-toluenesulfonic acid in dioxane-water gave complex product

26 Synthesis of the 1,6-Retro Amide mixtures. More successfully, hydrochloric acid in dry methanol was used, which gave the desired products in about 70% yields. An alternative method was to use 2,3- dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in a 9:1 mixture of acetonitrile/water.157

Scheme 3.

O O O O O N O DSC O N Pyridine O O O O RNH2 MeCN O CH ClCH Cl 88% 2 2 7

O O O O O O H OH RNH2 R N HO N R HOBT,EDC O O O H O O O N(Et)3 THF-DCM 61-73% 6 8-12

O OH O H 4% HCl R N N R MeOH H O OH O

15-18, 28-33

4.1 A New Three-Step Synthesis

Encouraged by the biological results (vide infra) and since we anticipated that a very large number of compounds would have to be synthesized during the lead optimization process, we felt it essential to improve this nine-step synthesis. A literature procedure describing the direct oxidation of D-mannonic-γ-lactone to the bicyclic D-mannaro- 1,4:3,6-dilactone using nitric acid, caught our interest.158 This reaction would furnish a compound with; protected/activated 1.6-diacids, protected 3,4-hydroxyl groups, and free 2,5-diols, which serve as handles for the direct introduction of P1/P1' substituents, and this in only one step from the starting material. Thus, we envisioned that this new strategy would significantly reduce the number of synthetic steps needed and provide a versatile route for the introduction of the P1/P1' and P2/P2' substituents.

Hence, oxidizing the L-mannonic-γ-lactone to the L-mannaro-1,4:3,6-dilactone was easily performed by heating to reflux in nitric acid for 16 hours, and resulted in a 60%

27 Synthesis of the 1,6-Retro Amide yield. Several alkaline reagents were used in combination with benzyl bromide for the introduction of the benzyl substituents, all with discouraging results, since the dilactone was subsequently proven to be alkali labile. Meanwhile, a serendipitous experiment with a related molecule resulted in the desired dilactone under acidic conditions. We therefore tried the alkylation using alternative acidic conditions, with benzyl-2,2,2- trichloroacetimidate as the benzyl source.159 Reacting the L-mannaro-1,4:3,6-dilactone with benzyl-2,2,2-trichloroacetimidate in dry dioxane with trifluoromethylsulfonic acid as catalyst gave the 2,5-di-O-benzyl-L-mannaro-1,4:3,6-dilactone in 72% yield (Scheme 4).

Scheme 4. NH

HO OH O CCl3 O O O HNO3 HO O O reflux O CF3SO3H 60% HO OH HO Dioxane 72% 13

O O OH O O H RNH2 R N O O N R O DCM H O OH O O

14 19, 20-26, 34-28

Thus far, the planned synthetic route had worked satisfactory. The next step would be to open the lactones with an appropriate nucleophile to afford the target compounds. Aware of the instability of the dilactone 14 to alkalis, the conditions had to be carefully tuned in order to minimize the elimination products formed due to the basicity of the applied amine. By adding six equivalents of the selected amines in dichloromethane (acetonitrile or chloroform) to the dilactone 14 and refluxing overnight, the target compounds formed in moderate to good yields. In more polar solvents the elimination reaction was predominant. To confirm the structure of the elimination product the dilactone 14 was treated with sodium hydroxide. This gave, as presumed, the α,β- unsaturated lactone 39 in high yield.

Scheme 5.

O O O OH 1M NaOH O O O O O Dioxan O O 95% O

14 39 28 Synthesis of the 1,6-Retro Amide

4.2 Structure-Activity Relationship of P2/P2' Modifications

Purified HIV-1 protease was then used in a standardized spectrophotometric assay to determine the IC50 values (concentration of the inhibitor resulting in 50% inhibition) of the tested compounds.160 A more sensitive fluorometric assay was also used to 161 determine the Ki values (inhibitory constant).

Table 1. Structures, yields, and enzyme inhibition of the protease inhibitors.

O OH O R R O OH O

a a Cmpd. no.R-groupYield (%) Ki (nM) Cmpd. no. R-group Yield (%) Ki (nM)

H O b N 15 N 69 5000 21 N 46 0.9 O H O H

H O N 16 N 70 ni 22 N 31 2.3 O H O H F

H Ph O N 17 N 71 ni 23 N 20 81 O H O H HO

H O N 18 N 73 ni 24 N 60 5000 O H O H

HO H H N N 500- 19 N 70 0.4 25 N 61 1500 O H O H

H H N N 20 N 76 140 26 N 73 660 O H O H

a b ni = no inhibition at 10 µM. IC50 value.

29 Synthesis of the 1,6-Retro Amide

The first set of compounds to be evaluated, 15-18, all contained lipophilic amino acid ester side-chains in the P2/P2' position (Table 1). None of the compounds exhibited any activity for the HIV-1 protease, with the exception of 15, containing valine residues, that showed modest activity (IC50 = 5 µM). The above observation was not so surprising considering that many of the inhibitors reported contain a valine moiety in P2/P2'.71 More interestingly, exchanging the ester functionality to an amide resulted in gained activity. A 300-fold activity increase was observed going from compound 15 to 19.

Table 2. Structures, yields, and enzyme inhibition of the protease inhibitors.

O OH O R R O OH O

a a Cmpd. no.R-groupYield (%) Ki (nM) Cmpd. no. R-group Yield (%) Ki (nM)

HO b 27 N 74 ni 33 N 38 ni H H

Cl N b 28 N 29 ni 34 N 59 2000 H H F

29 30b ni 35 65 7100 N N N N H S H

b HO 30 N 31 ni 36 N 49 ni N H H

b 31 HO N 25 ni 37 N 35 0.2 H H

b 32 N 32 ni 38 N 22 ni OH H H

a ni = no inhibition at 10 µM. b overall yields from 6.

30 Synthesis of the 1,6-Retro Amide

A series of methyl amide compounds with different properties was therefore synthesized and evaluated (20-26). The closely related isoleucine containing compound

21 showed a high inhibitory activity (Ki = 0.9 nM) and the slightly larger compound 22 also exhibited also good potency (Ki = 2,3 nM). Introduction of hydrogen bond accepting or donating groups resulted in decreased activity (23-26). In order to reduce the peptidic character of our inhibitors a different series of compounds was synthesized (27-36, Table 2). Mediocre potency was achieved with compounds 34 and 35 but the majority of the compounds were found to be inactive. 1(S)-amino-2(R)-indanol is a moiety that has been used in many reported inhibitors including the clinically approved indinavir. Introduction of this substituent to our scaffold gave a highly potent inhibitor,

37 (Ki = 0.2 nM), while applying the 1(R)-amino-2(S)-indanol afforded an inactive compound.

For the determination of the in vitro anti-HIV activity of the high affinity inhibitors 19, 21, and 37, an assay in MT-4 cells was used. The cytopathogenic effect was quantified using the vital dye XTT.161,162 From the cytoprotection of the tested compounds the

50% inhibitory concentration (ED50) was calculated. All three compounds exhibited anti-HIV activity, although compound 37 (ED50 = 0.09 µM) showed 17 times higher potency as compared to 19 (ED50 = 1.5 µM) and 21 (ED50 = 1.6 µM). Noteworthy, compound 37 exhibited similar potency to the clinically approved ritonavir (ED50 = 0.06

µM) and indinavir (ED50 = 0.06 µM) which were evaluated in the same assay. At this point we found it essential to obtain 3D structure data to guide the further design of our inhibitor scaffold.

4.3 X-Ray Crystallographic Data

Compounds 21 and 37 complexed with HIV-1 protease were crystallized and the X-ray structures were determined. Analyses of the structural data reveled that both of the inhibitors bound to the protease in a similar fashion (Figure 20). The central diol- moiety, designed to mimic the transition-state, was bound with one of the hydroxyls pointing toward the active site Asp 25/125 residues and formed hydrogen bonds to both the carboxyl oxygens. The second hydroxyl group points away from the active site, but was close enough to form a hydrogen bond to one of the active site Asp residues. The P1/P1' substituents, elongated with one oxygen atom, were nicely positioned in the S1/S1' pockets of the enzyme. In a distorted tetrahedral arrangement a structural water was hydrogen bonded to the amide nitrogens of the residues 50/150, positioned in the flap regions of the enzyme, and to the carbonyl oxygens of the mannaric acid scaffold, even though the scaffold is one atom shorter than the natural substrate. The X-ray data of compound 21 revealed that the isobutyl side-chains in the P2/P2' position did not completely fill the S2/S2' subsites, although compounds comprising larger side-chains in this position gave lower activity in the HIV-1 protease assay. Apparently, a larger

31 Synthesis of the 1,6-Retro Amide group must be structurally constrained to fit into the S2 pocket, since the larger aromatic part of the aminoindanole in compound 37 was accommodated nicely into the S2 pocket as shown by the X-ray data.

Figure 20. X-ray data for compound 21 and 37 (Steroview).

The next step in the iterative process of refining this class of compounds was to study the X-ray structures to decide which positions to modify next. In the extension of the para-position of the P1/P1' benzyl groups a tunnel-shaped cavity reaching towards the surface of the enzyme could be observed. There are reports describing the concept of P1 or P1' elongation and also some examples where the P1' substituent has been connected to the P3' side-chain.10,114,115,163-174 Elongation of the para-position in the P1/P1' substituents often gave inhibitors where no improvement of affinity for the HIV-1 protease was observed. However, elongation seems to provide a tool for manipulation of the anti-viral activity in cells. Our strategy was to modify the P1/P1' substituents with the aim of improving the antiviral activity.

32 Synthesis of P1/P1' Substituted Inhibitors

5. SYNTHESIS OF P1/P1' SUBSTITUTED INHIBITORS

For the synthesis of the P1/P1' elongated inhibitors we wanted to utilize the short synthetic route via the dilactone. For the introduction of the para-benzyl substituents two synthetic strategies were considered: 1) Convert para-substituted benzyl alcohols into trichloroacetimidates, and react these with the L-mannaro-1,4:3,6-dilactone. 2) Utilize synthetic-handles in the para-position of the P1/P1' benzyl groups, for subsequent introduction of substituents. The second strategy was more appealing, since the substituents could be introduced in the last step of the synthesis. The synthetic- handle of our choice was a bromide, which would be applicable to our synthetic route and which could be substituted easily with a plethora of groups through palladium- catalyzed couplings without interference of the other inhibitor functionalities.

Palladium-catalyzed reactions Palladium-catalyzed reactions provide facile, catalytic, chemoselective, and non-toxic ways of forming carbon-carbon bonds. The Heck,175 Suzuki,176 and Stille177 reactions are of particular importance to medicinal chemistry since a very large variety of functional groups can be introduced smoothly to suitable precursor fragments.

Microwave enhanced reactions For a long time microwave ovens have been standard equipment in most of our kitchens to heat and cook food quickly. In preparative organic chemistry the development has proceeded more slowly and the first reports of microwave assisted reactions were in 1986.178,179 Initially, major problems were encountered with this technique due to lack of control and reaction reproducibility. The development of controllable single-mode microwave cavities brought the technique into another level of usefulness. Nowadays, the technique is gaining popularity among organic chemist and several reports have been published the last few years,180-182 including microwave assisted palladium- catalyzed Heck, Suzuki, and Stille reactions.183

Synthesis To form the (4-bromobenzyl)-2,2,2-trichloroacetimidate (40), 4-bromobenzylalcohol was deprotonated and reacted with trichloroacetonitrile.159 Compound 40 was thereafter reacted with the L-mannaro-1,4:3,6-dilactone, synthesized as previously described (vide supra), to give the 2,5-bis-O-(4-bromobenzyl)-L-mannaro-1,4:3,6-dilactone, 41, in 88% yield (Scheme 6). The bulk of the product precipitated in the reaction vessel and could be isolated from the reaction mixture by filtration.

33 Synthesis of P1/P1' Substituted Inhibitors

Scheme 6. OH NH O CF3SO3H O O + O CCl3 O Dioxane HO Br 88% 13 40 Br Br H O N NH2 O OH O O O H H O N N O O N N O CH ClCH Cl 2 2 O H O OH O H O 45 °C, 64%

Br 41Br 42

The dilactone 41 was subsequently reacted with valine methylamide in 1,2- dichloroethane to give the dibrominated inhibitor 42, which also precipitated from the reaction mixture and was collected by filtration in 64% yield. A common precursor was thereby obtained, which could be utilized for palladium-catalyzed coupling reactions. Substitutes were introduced in the para-position of the P1/P1' benzyl groups.

Nevertheless, the precursor compound 42 was proven to be a good inhibitor, Ki = 0.3 nM, ED50 = 0.8 µM.

The dibromo-compound 42 was reacted with aryl- and heteroarylboronic acids with sodium carbonate as base in the presence of palladium tetrakis(triphenylphosphine)184 as catalyst (Scheme 7). The reaction vessels were sealed with teflon-septa as a pressure control device and microwave irradiated at 45 W for 4 min to furnish the Suzuki coupled products 43-46 in high yields (Table 3). To introduce an ethyl spaced phenyl group we utilized a 9-BBN coupling.185 An in situ hydroboration reaction of styrene with 9-BBN gave the phenylethyl-9-BBN, which was reacted with the bromo precursor 42, palladium tetrakis(triphenylphosphine), and sodium carbonate under microwave irradiation (60 W) for 2 min. No Heck coupling derived product (stilbene derivative) was detected in the reaction mixture and the compound 47 was isolated in a moderate yield (38 %).

Scheme 7. Br R

H O OH O H O H O OH O H O N N Reactant N N N N N N H H Reaction H H O O OH O medium O O OH O

Br 42 R 43-52

34 Synthesis of P1/P1' Substituted Inhibitors

The introduction of the 2-, 3-pyridyl or 2-thiazolyl substituents was performed with the trimethyl- or tributylheteroaryl tin under Stille reaction conditions, with dimethylformamide as solvent and palladium tetrakis(triphenylphosphine) as catalyst. To improve the reactions of the pyridyl couplings, cupric oxide was used as additive and in the case of thiazolyl coupling, silver(I) oxide was employed.186 The reaction mixtures were conducted at 60 W for 2 min to give the target compounds 48-50 in moderate yields (Table 3). To simplify the purification procedure, the compounds were dissolved in acetonitrile and washed with isohexane to minimize the contamination from tin residues.

Both of the Heck reactions were conducted under microwave irradiation for 2 min (60 W). The products derived from reaction with methyl acrylate and 1,2- 187 cyclohexandione both exhibit the same Rf-value as the starting material 42. With these experiments we felt prompted to force the reactions to full completion. This was easy accomplished with methyl acrylate, which gave compound 51 in 76 % yield. With the 1,2-cyclohexandione every attempt to achieve full conversion of the starting material e.g. employing increased power, prolonged reaction time, or adding additional portions of the catalyst, only resulted in increased formation of degradation products. We therefore performed the reaction under traditional thermal heating conditions at 100 °C for 48 h and were able to isolate pure compound 52 in moderate yield (Table 3).

5.1 Structure-Activity Relationships of P1/P1' Modifications

All of the tested compounds, 42-52, proved to have Ki values in the nanomolar range (determined as described earlier, vide supra), in agreement with our molecular modeling and with previously reported data. Remarkably, only minor deviations in Ki values were observed with this series of inhibitors (Table 3). The variance between the compound with the highest (51) and the lowest (47) affinity for HIV-1 protease are only a factor of 40. This should be compared to the sensitivity in P2/P2', where an exchange of a methyl group in compound 19 to a hydroxyl in compound 25 resulted in a 2500 times lower affinity for the protease. The HIV-1 protease seems be able to accommodate a variety of different substituents in the extension of the P1/P1', with a slight preference for small substituents containing hydrogen bond accepting groups.

As the next step antiviral effects of substituents in the para-position of the P1/P1' benzyl groups were considered (Table 3). We found that substitution of the para-benzyl hydrogen in the parent compound 19 with phenyl groups led to a 40-fold increase of anti-viral activity (19 ED50 = 1.5 µM, 43 ED50 = 0.04 µM). Thus compound 43 exhibited ED50 values comparable to the clinically approved inhibitors tested in the same assay system.

35 Synthesis of P1/P1' Substituted Inhibitors

Table 3. Reagents, time/effect, structures, yields, enzyme inhibitions, and anti-viral activities in cell cultures of the protease inhibitors.

a b c Cmpd. Time (min)/ Yield K ED50 ED50mutants ED50hs Reactant R-group i no. Effect (W) (%) (nM) (µM) (µM) (µM)

d e 19 0.4 1.5 nd , 20 20

d e 42 0.3 0.8 1.2 , 1.0 2.1

d e 43 4/45 93 0.7 0.04 0.4 , 0.7 0.8 (HO)2B

S S d e 44 4/45 96 1.2 0.04 0.3 , 0.2 0.8 (HO)2B

S S d e 45 4/45 86 1.2 0.04 0.3 , 0.2 1.0 (HO)2B

d e 46 4/45 85 1.4 0.05 1.1 , 0.04 1.1 (HO)2BNO2 NO2

47 2/60 38 3.8 2.1 nd nd B

N N 48 2/60 54 0.6 0.8 nd 3.6

(Me)3Sn

N N d e 49 2/60 50 0.3 2.5 >3 , >3 20 (Me)3Sn

S S d e 50 2/60 53 0.6 0.3 >3 , >3 0.4 (Bu)3Sn N N

O O 51 2/60 76 0.09 0.8 nd 5.7 O O

O O

HO HO 52 48 h/100°Cf 52 0.3 3.7 nd 20

a ED50 for reference substances tested in the same assay: ritonavir (0.06 µM), indinavir (0.06 µM), saquinavir (0.01 µM), nelfinavir (0.04 µM). b nd, not determined. c Compounds tested with 50% human AB+ serum. d Mutations in MT4/HIV-1: V32I, M46I, V82A. e Mutations in MT4/HIV-1: M46I, V82F, V84I. f Executed with traditional thermal heating.

36 Synthesis of P1/P1' Substituted Inhibitors

Similar effects were also encountered with the two electron-rich thienyl containing compounds 44, 45 and the nitro containing compound 46, despite the fact that they exhibited only half the affinity for the HIV-1 protease as compared to 43. This observed gain in anti-HIV activity might be attributable to the increased lipophility of the compounds, which could enhance the cell-membrane penetration. Contrary to this, the more lipophilic compound 47 (ED50 = 2.1 µM) showed a 50-fold decrease in anti-HIV activity compared to 43. Introduction of the electron-deficient, weakly basic 2- and 3- pyridyl heterocycles was predicted to preserve the positive effects obtained with the phenyl derivative 43 whilst in addition improving the solubility of the compounds.

Unfortunately, both of the compounds exhibited lower anti-HIV activity (48 ED50 = 2.5

µM; 49 ED50 = 0.8 µM) as compared to 43. Similar anti-HIV activities were achieved with the thiazolyl compound 50 (ED50 = 0.3 µM) and with the high affinity methyl acrylate compound 51 (ED50 = 0.8 µM). The poorest anti-HIV activity was observed with the acidic 1,2-cyclohexandione containing compound 52 (ED50 = 3.7 µM).

Mutant HIV-1 protease Viral resistance to HIV protease inhibitors is a major problem due to the error-prone nature of reverse transcriptase and the lack of proof reading functions in the virus, which causes mutations in the viral proteins and enzymes.188-190 At first this was thought to constitute a minor issue in the case of the HIV protease since the size of the enzyme is small (198 residues). However, mutations were indeed observed in many positions of the enzymes. Some of the most common mutations of the HIV protease are highlighted in Figure 21.191 To evaluate our compounds against a mutant virus, MT4- cell cultures with mutant viruses were used. The mutants were selected by growth with increased concentrations of ritonavir. The first assay contained HIV-1 protease with the mutations V32I, M46I, and V82A and in the second assay the mutations were M46I, V82F, and V84I. Compound 46 was the single exception in the series of the highly active compounds 43-46 that also retained antiviral activity against one of the viral mutants (Table 3). The other three compounds exhibited a 10-fold decrease in antiviral activity in both assays. This loss of activity can in fact be seen as a 20-fold gain of activity compared to the parent compound 19 tested against one of the mutants. The compounds 42, 49, and 50 were also evaluated against both of the two mutant viruses. The bromo compound 42 was able to maintain antiviral activity against both of the mutant viruses, but for the pyridyl 49 and the thiazolyl 50 compounds the antiviral activity against the mutants was low. In summary, although 46 exhibited a favorable inhibitory effect in one of the assay systems, and the nitro-group is bound to the same area affected by the mutation (V82F), it is difficult to draw any conclusions on a molecular level since the 3D structure of 46 complexed with the mutant protease is missing.

37 Synthesis of P1/P1' Substituted Inhibitors

Figure 21. Common mutations in the HIV-1 protease.

Non-specific protein binding The blood contains a large amount of proteins. Consequently, non-specific protein binding of the HIV protease inhibitors can occur and as a result the inhibitor might not reach the therapeutic concentration needed. To evaluate the non-specific binding of our compounds, a cell-based assay containing 50% of human serum was used. A 20-fold drop in antiviral activity was encountered for the four most active inhibitors (43-46, Table 3). The other compounds (48-52) were also affected but to a lesser extent. The thiazole derivative 50 was the only compound that was able to maintain antiviral activity in the presence of human serum.

Oral absorption Until this point the evaluations of the inhibitors were performed entirely in vitro. We therefore were interested in investigating whether or not the carbohydrate-based inhibitors exhibited bioavailability after oral administration. The inhibitors 43-45, and 49 were orally administrated to rats at a concentration of 30-40 mg/kg. Unfortunately, all of the compounds failed to attain measurable blood levels in the rats (<0.02 µg/mL). It is not clear whether poor absorption or fast metabolism accounts for the low bioavailability.

5.2 X-Ray Crystallographic Data

The 3D-structures of the 3-thienyl and 3-pyridyl compounds (44 and 49) co-crystallized with HIV-1 protease were determined with X-ray crystallography. Analyses of these two new structures in comparison to the old X-ray data from compound 21 revealed an almost identical mode of binding of the backbone structure of the inhibitors (Figure 22), in agreement with our modeled structures. The P1/P1' aromatic side-chains of compounds 44 and 49 were stretched along the backbone of the P2/P2' residues, in resemblance to the P1/P1' benzyl groups of compound 21. The elongated P1/P1' arms filled the entrance completely, although the electron density of the thiophene ring did not uniquely define the position of the sulfur atom (Figure 22). As a result of the

38 Synthesis of P1/P1' Substituted Inhibitors interactions between the protease and the elongated P1/P1' arms, the entrance was slightly contracted around the inhibitor. In the complex of compound 21, four water molecules were bound and linked together in a hydrogen bond network also involving the protease residue Arg8/108 at the common entrance of S1/S1' and S3/S3'. Upon binding of the larger compounds 44 and 49 the thienyl and pyridyl groups displaced three of these water molecules, but in the case of the thienyl compound two new water molecules bound to the entrance in slightly different positions. The similarity in Ki values of the three compounds suggest that net enthalpy and entropy, from binding the larger compounds 44 and 49 with the displacement of the hydrogen bound water as a consequence, balance the net effect of the affinity to the HIV-1 protease.192,193

21 44 49

Figure 22. X-ray data for compound 21, 44 and 49.

So far, an increased antiviral activity was obtained with the P1/P1' elongated C-terminal duplicated scaffold but still the problem with oral bioavailability had to be solved. Guided by the acceptance of acidic structures in the elongation of P1/P1', as seen in compound 52 and from published data,10,163,165 we wanted to explore the possibility of introducing a different carboxylic acid bioisostere to increase the water solubility and hopefully also the oral availability. The strategy was to use the dibromo compound 42 as starting material for the introduction of the commonly used carboxyl bioisostere, tetrazole and to explore the possibility of promoting the reactions by microwave irradiation.

39 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

6. MICROWAVE PROMOTED PREPARATION OF ORGANO- NITRILES AND THE CORRESPONDING TETRAZOLES

In 1885 J. A. Bladin, a Swedish chemist at the University of Uppsala, investigated the reactions of dicyanophenylhydrazine, the condensation product of cyanogen and phenylhydrazine. He observed that the action of nitrous acid on dicyanophenylhydrazine led to the formation of a compound, C8H5N5, to which he ascribed the structure depicted in Figure 23.194 Bladin proposed the name tetrazole for the new ring structure.195 The tetrazole moiety is now found in a large variety of compounds spanning from explosives196 to drugs197 (e.g. Losartan198). In medicinal chemistry the tetrazole moiety is a commonly used bioisostere for the carboxyl group. The metabolically stable tetrazole mimics the carboxyl group in acidity (pKa ∼ 5) and size, and when used as a bioisostere a retained pharmacological effect and a more favorable pharmacokinetic profile is often observe.197 There are several synthetic approaches to tetrazoles, although in general the most commonly used method is to react a nitrile with an azide source (e.g. hydrogen azide or trialkyltin azide).199

N N NC N N

C8H5N5

Figure 23. Structure proposed by Bladin.

Nitriles, which are used as precursors to tetrazoles, also constitute valuable synthetic intermediates for further transformations into a variety of functionalities, e.g., thiazoles, oxazolidones, triazoles, and amines.200-205 Many reports have been published on the direct transition metal-catalyzed conversion of aryl halides to aryl nitriles,206-215 and in addition several other methods are available for the preparation of nitriles.216-219

To obtain the desired tetrazole decorated inhibitor we needed a mild procedure that allowed nitrile introduction by displacement of the bromo atoms of 42. Tschaen et al. has reported an improvement of the palladium-catalyzed cyanation of aryl bromides.209 The success of the previously described palladium-catalyzed reaction on the inhibitor scaffold guided the selection of this procedure. The reaction times of the cyanation were reported to be 5-7 h and the subsequent transformation to tetrazoles also needed several hours for completion. We thought it likely that reduction of the reaction times might be possible by using microwave heating. Since these reactions have not been performed previously with microwave heating, a training set of small diverse compounds was used to optimize the reaction conditions.

40 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

6.1 Microwave-Promoted Cyanation Reactions

209 In the procedure by Tschaen et al., Zn(CN)2 was used, which is easier to handle than more commonly used, but highly poisonous sodium or potassium cyanides. To the organo-bromides 53a-h dissolved in DMF, 1 equiv Zn(CN)2 and 3 mol% of the catalyst palladium tetrakis(triphenylphosphine) was added. The reaction mixtures were microwave irradiated for 2 min at 60 W (Table 4). Full conversions were achieved in all the reactions, with the exception of the thiophene 53g that needed an extra 30 seconds of irradiation to be completed. The temperature profiles of the reactions were recorded with a fluoroptic probe placed in the reaction vessel (Figure 24). The final temperature was 175 ºC after 2.5 min. The nitriles were purified and isolated in good yields as presented in Table 4. The slightly lower yields from some of the reactions are most probably due to the volatile nature of the compounds. The microwave-promoted cyanide reaction was general and easily performed.

Table 4. Microwave-promoted cyanation reactions with organo-bromides.

Cmpd. Organo Time (min)/ Cmpd. Isolated Thermal heating no. Bromide Effect (W) no. Product Yield (%) Time (h) Yield (%)

Br CN 53a 2/60 54a 81 7 95207 O O Br CN 53b 2/60 54b 78 6 92207

O2N O2N

Br CN 53c 2/60 54c 90 6 84207

Br CN

53d 2/60 54d 90 4 97209

Br CN 53e 2/60 54e 95

Br CN 53f 2/60 54f 88 4 71209 N N Br CN

53g 2.5/60 54g 80 16 89211 S S Br CN 53h 2/60 54h 93 2 94220

41 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

6.2 Microwave-Promoted Cycloaddition Reactions

For cycloaddition reactions sodium azide was used as the azide source despite the fact that sublimation of explosive ammonium azide might be a troublesome factor. The reason for the choice of sodium azide was to allow an easy purification of the formed tetrazoles and to also eliminate the deprotection step needed when alkyltin or alkylsilicon azides are employed. The tetrazoles 55a-h were prepared by adding a large excess of sodium azide and ammonium chloride to the nitriles dissolved in DMF and subsequent irradiation of the reaction mixtures at a magnetron power of 20 W. The reaction times needed varied from 10 − 25 min. The results are summarized in Table 5. Attempts to complete the reactions more quickly by increasing the magnetron power merely resulted in the formation of side-products. The yields were generally good with the exception of the sterically hindered, ortho-substituted biphenyl 54e, which we were unable to force to completion.

Table 5. Microwave-promoted cycloaddition reactions with organo-nitriles.

Cmpd. Organo Time (min)/ Cmpd. Isolated Thermal heating no. Nitrile Effect (W) no. Product Yield (%) Time (h) Yield (%)

HN N CN N N 54a 25/20 55a 96 24 68221 O O HN N CN N N 54b 10/20 55b 95 3 97222

O2N O2N HN N CN N N 54c 15/20 55c 91 7 97223

N CN HN N N 54d 15/20 55d 48 96 35224

N HN N CN N 54e 25/20 55e 36 72 72225

HN N CN N N 54f 15/20 55f 75 7 75221 N N N HN N CN N 54g 15/20 55g 98 S S HN N CN N N 54h 15/20 55h 60

42 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

The high salt concentration in the reaction mixture led to a high reaction temperature despite the relatively low power used. After 10 min the temperature stabilized at 220 ºC (Figure 24), which demonstrates a remarkable superheating of the solvent (b.p. of DMF = 153 ºC).

6.3 One-Pot Reactions

Both of the described flash heated reactions were very clean and high yielding. However the nitriles required chromatographic purification to be isolated. Hence, we wanted to eliminate the chromatographic isolation of the intermediate nitriles. Since both reactions were performed in DMF, a sequential one-pot procedure to obtain the easily purified tetrazole was considered feasible.

Scheme 8. 1. Zn(CN)2 Pd(PPh3)4 HN N DMF Br N 2 min 60 W N

2. NaN3 NH4Cl, DMF 56 15 min 20 W 96%

Bromobenzene was reacted with Zn(CN)2 and Pd(PPh3)4, as described (vide supra). After the microwave irradiation sodium azide and ammonium chloride was added to the reaction mixture and a second microwave heating period was commenced. The resulting 5-phenyltetrazole was isolated pure in 96% yield by extraction (Scheme 8). This procedure could also be carried out on a solid support using an aryl iodide, linked to a tentaGel resin,226 with a good result (Scheme 9).

Scheme 9. 1. Zn(CN)2 H Pd(PPh3)4 H O O N O DMF N N N I 2 min 60W TFA, H O N 2 N NH N H N N NH 2. NaN 2 3 72% NH4Cl, DMF 15 min 20W 57

43 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

Temperature-time Profiles

250 B 200 A C 150

100 Temp (°C)

0 0 1020304050 Time (min)

Figure 24. Curve A: 60 W, 2.5 min, DMF, 4-Bromoanisole, Zn(CN)2, Pd(PPh3)4; Curve B: 20 W, 25 min, DMF, 4-Methoxybenzonitrile, NaN3, NH4Cl; Curve C: 13 W, 40 min, DMF, 42, NaN3, NH4Cl.

Synthesis of the tetrazole HIV protease inhibitor

The dibromo compound 42 dissolved in DMF was mixed with Zn(CN)2 and Pd(PPh3)4 and microwave irradiated for 2 min at 60W. Thereafter, the reaction vessel was charged with sodium azide and ammonium chloride and heated a second time. The inhibitor scaffold was unstable in the temperature reached upon applying 20W magnetron power, therefore the power was decreased to 13W. The reaction time was prolonged to 40 min to compensate for the lower microwave power. The temperature now only reached 150 ºC and the tetrazole inhibitor 58 was obtained in a good yield. The intermediate nitrile inhibitor was isolated in 84% yield (Scheme 10).

Scheme 10. HN N Br N 1. Zn(CN)2 N Pd(PPh3)4 H O OH O H O DMF H O OH O H O N N 2 min 60 W N N N N N N H H O H O OH O H 2. NaN3 O O OH O NH4Cl DMF N Br 40 min 13 W N 42 58 82% N NH

Pharmacological results

Both of the tested inhibitors exhibited high affinities for the HIV-protease (58 Ki = 0.6 nM, Nitrile inhibitor Ki = 0.2 nM). However, only modest antiviral activity of the tetrazole inhibitor 58 was observed and only a slightly better results were obtained with the nitrile encompassing compound (58 ED50 = >67 µM, Nitrile inhibitor ED50 = 30 µM).

44 Microwave Promoted Preparation of Organo-Nitriles and the Corresponding Tetrazoles

While we were working with the P1/P1' elongated inhibitors, other members of our research group had developed a new surface plasmon resonance based biosensor assay for the determination of HIV-1 protease activity.227 The highly atomized biosensor assay allowed determination of the binding and dissociation constants of the inhibitors.

However, the moderate sensitivity for low affinity binders (Ki > 100 nM), which made this method less practical for screening constituted a drawback. Two questions arose: 1) How can our protease inhibitors be transformed into tools that are useful in the development of biosensor assays with high sensitivity? 2) Can the para positions of P1/P1' be used as anchors for attachment to a support, as an alternative to attachment by a common backbone linking (Figure 25)? As disclosed from X-ray data, the para- positions of the P1/P1' benzyl groups were close to the surface of the enzyme, and substituents in this position did not seem to disturb the affinity to the enzyme (as demonstrated earlier). These data, together with the successful use of the palladium- mediated introduction of substituents to the bromo-precursor 42, guided the selection of strategy for the development of sensitivity enhancer tools.

Backbone linking

H O OH O H O N N N N O H O OH O H

Side-chain linking

H O OH O H O N N N N O H O OH O H

Figure 25. Linking strategies.

45 Surface Plasmon Resonance Biosensor Assays

7. SURFACE PLASMON RESONANCE BIOSENSOR ASSAYS

A biosensor is a devise, which converts a physical parameter of a biological interaction into a measurable signal, e.g. the conversion of the inhibitor/protease interaction into a curve on the computer screen.228 Surface plasmon resonance (SPR) is an optical technique that measures the refractive index of a medium near a thin film of metal on a glass substrate.229-234 In more details (Figure 26.): a glass chip surface is coated with a thin gold film (~50 nm) and a dextran matrix (~100 nm) is covalently bound to the gold film. A molecule of interest (e.g. HIV protease) is immobilized on the dextran matrix. A buffer solution is thereafter passed over the dextran surface with a continuos flow. On the glass side of the chip is a light beam pointed at a certain angle to obtain total internal reflectance. When an analyte (e.g. inhibitor), which binds to the dextran-immobilized molecule, is added to the buffer, a difference in the angle of the reflected light can be measured, as a result of binding. The difference in the reflected light angle is expressed in arbitrary units called resonance units (RU). The SPR signal is proportional to the mass of the analyte interacting with the immobilized molecule.

Light- source Optical detection unit

Prism

Sensor chip with θ gold film Dextran Immobilized matrix molecule Analyte

Flow channel

Figure 26. Schematic representation of the SPR technique.

In the previously developed SPR direct binding assay,227 HIV protease was immobilized on the surface and the binding of the inhibitor passed over the surface was monitored (Figure 27). The low molecular weight of the inhibitors resulted in a small

SPR signal and did not permit characterization of inhibitors with Ki < 100 nM.

The new assays By attaching linker molecules to the para position in P1/P1' of our inhibitor two new assay systems could be developed (Figure 27). In the first assay a biotin moiety was

46 Surface Plasmon Resonance Biosensor Assays attached via the linker to the inhibitor to obtain an “enhancer molecule”. The enhancer molecule was then mixed with the tested compound and passed over the HIV protease- immobilized surface. To enhance the SPR signal, a high molecular weight anti-biotin antibody, which binds to the biotin moiety of the enhancer molecule, was passed over the surface. A strong SPR signal could be monitored due to the large molecular weight of the antibody/enhancer molecule complex. The affinity of the tested compound is inversely proportional to the SPR signal (Figure 28).

In the second assay, our inhibitor was immobilized to the biosensor surface via the linker (Figure 27). The tested compound was mixed and pre-incubated with the HIV protease before being passed over the surface. The non-inhibited HIV protease in the solution then binds to the surface immobilized inhibitor with a strong SPR signal as a result. The affinity of the tested compound is inversely proportional to the SPR signal (Figure 28).

a) Direct Binding Assay

HIV Protease Immobilized to the Surface

Inhibitor

b) Assay 1 Compound 63

Anti-Biotin Antibody

Compound 65 Immobilized c) Assay 2 to the Surface

HIV Protease Figure 27. Schematic picture of the three different assay formats.

Assay 1 450 Assay 2 350

250

150 Response (RU) Response 50 Direct Binding Assay

-500.01 0.1 1 10 100 1000 Concentration (nM) Figure 28. Dose-response curves for nelfinavir in the three assay formats. RU = Resonance Unit.

47 Surface Plasmon Resonance Biosensor Assays

7.1 Synthesis of the “Assay Tools”

The α,β-unsaturated amide linker 60 was afforded in a good yield, by the reaction of the mono protected diamine 59235 with acryloyl chloride (Scheme 11).

Scheme 11. O O O Cl H O N O NH2 ON O ON O DIEA H H O CH2Cl2 59 92% 60 Scheme 12. Br R1 60 Pd(OAc)2 H O OH O H O P(o-Tol)3 O OH O O DIEA H H N N N N N N N N H H ° O O OH O 80 C, 8h O H O OH O H

42 Br R2 O 61 (74%) R = R = H 1 2 N O O N O 64 (15%) R1 = Br, R2 = O H

H O OH O H O 50% TFA N N N N CH Cl 2 2 O H O OH O H

R2 62 (95%) R = R = H 1 2 N O O NH2 65 (98%) R1 = Br, R2 = O

H O OH O H O NHS-LC-Biotin N N N N DIEA H H DMF O O OH O

H N O R2 O NH O O 63 (86%) R = R = 1 2 H O O N H 66 (96%) R1 = Br, R2 = N HN H H S

48 Surface Plasmon Resonance Biosensor Assays

The linker was then attached to the para-position of the dibromo inhibitor 42 by a classic Heck reaction (Scheme 12).175 When applying a large excess of the linker 60 both of the bromides were substituted yielding compound 61 (Ki = 1.3 nM). By using only 1.3 equiv of the linker the mono substituted compound 64 could be isolated, but in a low yield. Both the di- and mono linker substituted inhibitors (61 and 64) contained a small amount of the cis-isomer that was difficult to remove by chromatographic purification. Compounds 61 and 64 were treated with TFA to give the free amines 62 and 65 in high yields. The biotinylated compounds 63 and 66 were thereafter obtained in good yields by a reaction with pre-activated biotinamidocaproic acid in the presence of a base.

Immobilization The carboxyl groups of the surface matrix was activated with EDC and N- hydroxysuccinimide and reacted with the amino groups of 65 and 62 to immobilize the compounds to the biosensor surface.

7.2 Assay Evaluation

The two new assays and the previously reported direct binding assay were used with four of the clinically approved inhibitors, indinavir, nelfinavir, ritonavir, and sacquinavir. The response as a function of the inhibitor concentration was measured (Figure 29). Assay 1, with the dibiotinylated compound 63, provided good data and was able to rank the commercial inhibitors with respect to their affinities for the surface bound HIV protease. Assay 2, with the surface bound inhibitor 65, showed data with less disturbance from instrumental noise compared to the direct binding assay, although neither the direct binding nor the assay 2 enabled ranking of the affinities of the inhibitors

a) Direct Binding Assay b) Assay 1 c) Assay 2

1.2 120 120

1 100 100

0.8 80 80

0.6 60 60

0.4 40 40 % inhibition % 0.2 inhibition % 20 20 norm. response norm. 0 0 0 0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 1000 -0.2 concentration (nM) -20 concentration (nM) -20 concentration (nM)

Figure 29. The dose-response curves for indinavir •, nelfinavir ■, ritonavir !, saquinavir ×.

The monitoring of the concentration series is time consuming and therefore not practical for screening of compounds. To overcome this problem single runs at fixed concentrations were used in all of the three assays. Seventeen compounds were tested in three consecutive series and evaluated against data obtained from a standardized fluorometric assay (Figure 30).161 Once again, assay 1 provided the best results. A good

49 Surface Plasmon Resonance Biosensor Assays

correlation to the Ki values was observed over the tested range. Correlation with the Ki values was also exhibited by assay 2 although with a narrower detection range. In both the assays the deviations in the test results were small when compared to those obtained by the standardized fluorometric assay. a) Direct Binding Assay b) Assay 1 c) Assay 2

1.4 100 100 1.2 80 80 1 60 0.8 R2 = 0.93 60 0.6 40 40 %inhibtion

0.4 % inhibtion 20 20 norm. response 0.2 0 0 0 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Ki (nM) Ki (nM) Ki (nM)

Figure 30. The normal responses or percent inhibition for 17 compounds plotted against their Ki values.

7.3 Comparison Between the One and Two Linker Strategies

In assay 1, the bis-biotinylated compound 63 can bind to either one or two antibodies. To evaluate the importance of the second biotin-subunit, for enhancement of the SPR- signal, the mono-functionalized compound 66 was used in assay 1. With compound 66 as an enhancer molecule in the assay, a 30% decrease in the SPR-signal was encountered. This clearly demonstrates the importance of the second biotin unit, although the signal was not reduced by 50%, as would have been expected if both the arms of 63 were engaged simultaneously in antibody binding.

The mono substituted amino compound 65 was designed for attachment to the dextran surface of the biosensor, to be used in assay 2. The synthetically more accessible bis- functionalized compound 62 could also be attached to the dextran surface, although cross-linking to the dextran might be a problem. To evaluate this the diamino compound 62 was immobilized to the surface and used in assay 2. The second linker arm did not exert any pronounced effect on the SPR-signal. Thus both the mono and the bis-linker arm strategy could be employed in this assay.

In summary, the antibody amplifying system, assay 1, provided a highly sensitive system that could be used for the identification of low molecular HIV protease inhibitors. The immobilized inhibitor surface, assay 2, gave a very durable biosensor surface that could be used for many cycles without any decline in the capacity of the surface.

50 Concluding remarks

8. CONCLUDING REMARKS

Structure based design was used to discover a potent C2-symmetric C-terminal duplicated scaffold. The carbohydrate chiral pool was exploited enabling the ready preparation of a stereochemically pure 1,6-retroamide scaffold. Using a L-mannaro- 1,4:3,6-dilactone as a key intermediate, a synthetically simple three-step route was developed that allowed for an easy introduction of a variety of P1/P1' and P2/P2' substituents. Amino acid derivatives and amines were introduced as P2/P2' substituents to obtain high affinity inhibitors of HIV-1 protease.

The palladium-catalyzed reactions, Heck, Suzuki, and Stille couplings, provided good methods for the chemo-selective introduction of various aromatic, heterocyclic, alifatic, and olefinic groups to the P1/P1' substituents of the inhibitors. The microwave flash heating technique was successfully applied on these complex structures, rapidly providing pure materials in high yields.

The structure-activity relationships of the new class of C2-symmetric C-terminal duplicated inhibitors were evaluated as a function of modifications in P2/P2' and in the elongated P1/P1' substituents. Evaluations were made with the purified HIV-1 protease, HIV infected cells, mutant HIV infected cells, and using vivo rat models. Compounds with Ki values in the nano-molar range and with antiviral effects in cell-assays comparable to the clinically approved inhibitors were identified.

A microwave heated one-pot procedure for the direct conversion of organo-bromides to tetrazoles was developed with a notable reduction of the reaction times. This procedure was applied to the inhibitor scaffold and to a solid support bound aryl iodide with good results.

Two new sensitive surface plasmon resonance biosensor assays have been developed. The valine encompassing HIV-1 protease inhibitor was modified, by attachment of linkers to the P1/P1' benzyl groups, to serve as sensitive enhancer tools in these two assay formats.

51 Concluding remarks

P1/P1'-Elongation

P2/P2'-Modifications Acceptance of a large variety of R' substituents. Sensitive to modifications.

Small difference in Ki-values. Preference for lipophilic O OH O H side-chains. R N Increased anti-viral activity in the N R cell-based assay with Anti-viral activity in the H O OH O cell-based assay were lipophilic aromatic substituents. obtained with MeNHVal , MeNHIle, and m-Nitrophenyl substitution 1(S)-amino-2(R)-indanole. R' provided a compound with favorable effect against a mutant virus (M46I, V82F, V82I).

Figure 31. Graphic summary of structure-activity relationship for the inhibitors presented in this thesis.

O OH O H R N N R H O OH O R

O OH O O I H H r e N N p a N N P O H O OH O H II N er N Pap N N X R H

O H O OH O H O Pa O per III N N O O N N O O H O OH O H O H P N X a p e N r N IV N O O HN O NH O

HN O S O O OH O O HN H H H H N N NH N N H HN H H H NH O O OH O O S O NH

OHN ONHO O

Figure 32. Graphic summary of the synthetic work performed in this thesis.

52 Acknowledgements

ACKNOWLEDGEMENTS

This investigation was carried out at the Department of Pharmaceutical Chemistry, Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Uppsala University.

I wish to express my sincere gratitude to:

Professor Anders Hallberg, an excellent supervisor and good friend, for good and learning discussions and lots of fun, and for everlasting enthusiasm throughout this study.

My co-supervisors Docent Björn Classon and Dr. Anders Karlén for introducing me to the field of medicinal chemistry and computational chemistry, respectively, and for all the help and good advice.

All the present and former members of the chemistry groups in Stockholm and Linköping, for good collaboration and chemistry discussions.

Docent U. Helena Danielson and her co-workers for evaluations of the compounds on the HIV-1 protease assay.

Dr. Torsten Unge and his co-workers in the crystallographic group, who made it possible to “see” my molecules inside the HIV-1 protease.

The staff at Medivir AB for a pleasant atmosphere during my nine-months at your lab and afterwards, especially thanks to the super-chemist Jussi Kangasmetsä for all the good advice.

Dr. Johan Hultén, Mr. HIV protease, for interesting discussions concerning structure- activity and for sharing your enormous collection of references.

Dr. Mats Larhed, for sharing all your vast knowledge concerning palladium- and microwave chemistry.

Dr. Kristofer Olofsson and Dr. Nicholas Bonham for excellent linguistic revision of the thesis. All linguistic mistakes are without doubt my own last minute changes.

Marianne Åström for skillful secretarial assistance and Arne Andersson and Björn Carlsson for excellent technical assistance. Lotta Wahlberg for always being able to force the chemical-companies to provide me with the chemicals when I needed them.

53 Acknowledgements

Prof. Kristina Luthman, Docent Anette Johansson, and Docent Uno Svensson for good help and for providing a good educational atmosphere. Docent Eva Åkerblom and Docent Gunnar Lindeberg for guidance within the field of solid-phase synthesis. Wesley Schaal for helping me with all the computational-chemistry problems.

Anja Johansson, for constructive criticism of the thesis and for being the best snowboard partner. Anna Karlsson for good comments on the thesis.

Malin Graffner Nordberg, Petra Johannesson and all other lab-friends in B305. Jenny Hallgren for excellent performed diploma work. All former and present members of the department for making these years so enjoyable.

Dr. Susanna Lindman for being a good friend and helping me with all the paperwork needed to become a doctor. Hans Sjöbom for being patient with the Biacore manuscript and for being the best windsurfing partner.

My mother, Kerstin, and my father, Mats, for all your love, support and for always letting me choose my own ways in life. Johanna and Gabriella, for the all fun and the fights we have shared and for being such wonderful sisters.

To all my friends and especially Micke, Kalle, Johan, and Patrik for showing me wonderful times and being the best of friends.

My best friend, Per, for all the fun during the years and for all the good discussions at Kvarnen.

Anki for bringing joy with all your love and wonderful smiles.

The Swedish Academy of Pharmaceutical Sciences, the Foundation Anna Maria Lundin Scholarship Foundation (Smålands Nation in Uppsala), and European University Consortium for Pharmaceutical Research (ULLA) for making it possible for me to attend at conferences and courses.

Financial support was obtained from the Swedish Foundation for Strategic Research (SFF), the Swedish Research Council for Engineering Sciences (TFR), Medivir AB, Huddinge, Sweden, the Swedish Natural Sciences Research Council (NFR).

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