Design and Synthesis of Inhibitors Targeting the Hepatitis C Virus NS3 Protease

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 53

Focus on C-Terminal Acyl Sulfonamides

ROBERT RÖNN

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Gunde Svan

Papers Included in this Thesis

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I Rönn, R.; Sabnis, Y. A.; Gossas, T.; Åkerblom, E.; Danielson, U. H.; Hallberg, A.; Johansson, A. Exploration of Acyl Sulfona- mides as Carboxylic Acid Replacements in Protease Inhibitors of the Hepatitis C Virus Full-Length NS3. Bioorg. Med. Chem. 2006, 14, 544–559.

II Rönn, R.; Gossas, T.; Sabnis, Y. A.; Daoud, H.; Åkerblom, E.; Danielson, U. H.; Sandström, A. Evaluation of a Diverse Set of Potential P1 Carboxylic Acid Bioisosteres in Hepatitis C Virus NS3 Protease Inhibitors. Bioorg. Med. Chem. Accepted.

III Wu, X.; Rönn, R.; Gossas, T.; Larhed, M. Easy-to-Execute Car- bonylations: Microwave Synthesis of Acyl Sulfonamides Using Mo(CO)6 as a Solid Carbon Monoxide Source. J. Org. Chem. 2005, 70, 3094–3098.

IV Rönn, R.; Lampa, A.; Peterson, S. D.; Gossas, T.; Åkerblom, E.; Danielson, U. H.; Karlén, A.; Sandström, A. Hepatitis C Virus NS3 Protease Inhibitors Comprising a Novel Aromatic P1 Moi- ety. Submitted.

Reprints are presented with permission from the publishers.

Contents

1 Introduction...... 11 1.1 Hepatitis C Virus...... 11 1.1.1 Prevalence and Transmission of HCV...... 11 1.1.2 Outcome and Symptoms of an HCV Infection...... 12 1.1.3 The Virus and its Life Cycle...... 13 1.1.4 The Viral Genome and its Translational Products...... 15 1.1.5 HCV Variability...... 17 1.1.6 Current Treatment of Hepatitis C ...... 17 1.1.7 HCV Drug Discovery ...... 18 1.2 The HCV NS3 Protease...... 19 1.2.1 Proteases in General and Serine Proteases in Particular...... 19 1.2.2 Structure and Function of the HCV NS3 Protease...... 21 1.2.3 HCV NS3 Protease Inhibitors...... 22 2 Aims of the Present Study ...... 31 3 Carboxylic Acid Bioisosteres in HCV NS3 Protease Inhibitors (Papers I and II)...... 32 3.1 Bioisosteres in Medicinal Chemistry ...... 32 3.2 Exploring the Acyl Sulfonamide Group (Paper I)...... 32 3.2.1 Preparation of Tetrapeptides...... 34 3.2.2 Preparation of Tripeptides ...... 35 3.2.3 Structure–Activity Relationship ...... 37 3.3 Potential P1 Carboxylic Acid Bioisosteres (Paper II)...... 42 3.3.1 Chemistry...... 42 3.3.2 Structure–Activity Relationship ...... 44 3.3.3 pH Study...... 46 4 Synthesis of Aryl Acyl Sulfonamides (Paper III) ...... 49 4.1 Palladium-Catalyzed Carbonylation...... 49 4.2 Carbon Monoxide Sources ...... 50 4.3 Microwaves as a Tool in Drug Discovery...... 50 4.4 Method Development...... 51 4.5 A Medicinal Chemistry Application ...... 54 5 A Novel Aromatic P1 Moiety in HCV NS3 Protease Inhibitors (Paper IV) ...... 56 5.1 Chemistry ...... 57 5.2 Structure–Activity Relationship...... 58 6 Concluding Remarks ...... 62 7 Acknowledgements ...... 64 8 References...... 66 Abbreviations and Definitions

ACCA 1-amino-cyclopropanecarboxylic acid ACE angiotensin-converting enzyme ADME absorption, distribution, metabolism and excretion Ala alanine Arg arginine Asp aspartic acid ATP adenosine triphosphate Bn benzyl Boc tert-butoxycarbonyl CDI 1,1´-carbonyldiimidazole Cha cyclohexylalanine Chg cyclohexylglycine CMV cytomegalovirus CNS central nervous system DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DIC N,N´-diisopropylcarbodiimide DIEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethylsulfoxide DPPIV dipeptidyl peptidase IV EC50 inhibitor concentration giving 50% inhibition of replication in a cell-based system ER endoplasmic reticulum Fmoc 9-fluorenylmethoxycarbonyl Gln glutamine Glu glutamic acid Gly glycine HATU N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1- yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide HBTU N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]- N-methylmethanaminium hexafluorophosphate N-oxide HCV hepatitis C virus His histidine HIV human immunodeficiency virus HLE human leukocyte elastase HSV herpes simplex virus HTS high-throughput screening IC50 inhibitor concentration giving 50% inhibition IFN interferon Ile isoleucine IRBM Istituto di Ricerche di Biologia Molecolare IRES internal ribosome entry site Ki inhibition constant or dissociation constant for inhibitor binding Ki* overall inhibition constant for covalent inhibitors Leu leucine Lys lysine 2-Nal 2-naphthylalanine NMM N-methylmorpholine NTP nucleotide triphosphate NTR non-translated region Nva norvaline PEG polyethylene glycol Ph phenyl ptol para-tolyl RdRp RNA-dependent RNA polymerase SAR structure–activity relationship Ser serine SPPS solid phase peptide synthesis Suc succinic acid SVR sustained virological response TBTU N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]- N-methylmethanaminium tetrafluoroborate N-oxide TES triethylsilane TFA trifluoroacetic acid THF tetrahydrofuran Thr threonine TMP 2,4,6-trimethylpyridine Val valine vinyl-ACCA (1R,2S)-1-amino-2-vinyl-cyclopropanecarboxylic acid 1 Introduction

1.1 Hepatitis C Virus In the mid 1970s, it was proposed that a viral agent apart from hepatitis type A or B caused transfusion-associated hepatitis (inflammation of the liver).1 In 1989, several years after its recognition, this viral agent causing non-A, non-B hepatitis was identified and termed hepatitis C virus (HCV).2

1.1.1 Prevalence and Transmission of HCV Today, approximately three decades after the recognition of HCV, the virus is considered to be a new global epidemic. Estimates of the global prevalence of HCV reveal that the virus affects ~120–180 million people, repre- senting ~2–3% of the world’s population.3,4 HCV is recognized as a major cause of chronic liver disease as well as the leading cause of liver transplan- tations in developed countries.5 Furthermore, mathematical models predict that HCV-related mortality will rise during the coming decades.6-9 There are large geographical differences in the prevalence of HCV; the countries with the highest prevalence rates being found in Africa and Asia (Figure 1). Egypt is by far the most affected country in the world, with reported prevalence rates up to 22%.10

Figure 1. Estimated global prevalence of HCV.3

11 HCV is a bloodborne infection with three major routes of transmission. In- jection drug use is the primary route of transmission in the developed countries while unsafe therapeutic injections are a major route of HCV transmission in the developing countries where, for example, sterile syringes may be in short supply. The extraordinary spread of HCV in Egypt is believed to be the consequence of contaminated syringes used in a nationwide schistosomi- asis treatment campaign between 1961 and 1986.10 The third major route of HCV transmission has been blood transfusions. However, this transmission route has been virtually eliminated in developed countries since the development of a screening test for HCV in 1990.11 Transfusion-related HCV transmission is still believed to be a major route in developing countries, where donated blood is not screened for the presence of HCV.5 Other possible routes of transmission are: sexual intercourse with an infected partner, perinatal transmission, tattooing, body piercing and acupuncture. It is, however, not clear to what degree these risk factors contribute to the overall transmission of HCV.5

1.1.2 Outcome and Symptoms of an HCV Infection HCV primarily infects hepatocytes in the liver causing immune-mediated inflammation. The clinical outcome of an infection is highly individual and there is no single typical course of the disease. Anything from rapid clear- ance of the virus from the body to the worst-case scenario, liver failure, can occur. A schematic representation of possible outcomes of an HCV infection is presented in Figure 2.

Recovery Exposure Acute End-stage to HCV HCV infection liver disease Chronic Cirrhosis HCV infection Hepatocellular carcinoma Figure 2. Schematic representation of potential outcomes of an HCV infection.

The initial phase after being infected by HCV is called acute hepatitis C and the infected patient may experience symptoms such as jaundice, nausea, malaise, dark urine and liver pain. However, only one third of those infected develop symptoms, making early diagnosis difficult.12 Approximately 15–25% of patients having acute hepatitis C recover spon- taneously from the virus, but the majority (~75–85%) develop a chronic infection (defined as detectable blood levels of HCV RNA for at least six months). Once the infection has become chronic, patients are less likely to have symptoms, but fatigue, nausea, liver pain, dark urine and itching may occur.13

12 The most common serious complication resulting from a chronic HCV infection is the development of cirrhosis. In cirrhosis, the normal liver tissue is replaced by scar tissue, resulting in liver dysfunction. Approximately 10– 15% of patients with chronic hepatitis C will eventually develop cirrhosis;14 the time period from infection to cirrhosis varies between 1 and 30 years.12 Further progression of the infection can lead to end-stage liver disease and hepatocellular carcinoma, conditions that require liver transplantation.12 One question that has puzzled researchers over the years is why the HCV is cleared by some patients and not by others. The outcome of the disease is largely determined by the efficiency of the host’s antiviral immune response, and the presence of viral escape mutants has been suggested to contribute to the persistence of the virus.15 Moreover, it is well documented that certain patient groups are more efficient in clearing the virus, and factors such as male sex, older age at infection, alcohol consumption, human immunodeficiency virus (HIV) coinfection and hepatitis B coinfection seem to accelerate the progression of the disease.5,16,17 However, we still do not have the complete answer to what dictates the clinical outcome of an HCV infection.

1.1.3 The Virus and its Life Cycle The hepatitis C virus is a Hepacivirus belonging to the Flaviviridae family. The virus particles are spherical with a diameter of 55–65 nm18 and both a nucleocapsid layer and an envelope surround and protect the viral RNA genome.19 HCV particles circulate in the serum of an infected patient, either in a free form or bound to lipoproteins or immunoglobulins.19 Replication of the HCV takes place mainly in the hepatocytes in the liver,13 but can also occur to some extent in the small intestine20 and the central nervous system (CNS).21 A schematic representation of the various stages in the proposed life cycle of HCV is shown in Figure 3 and a brief description is given below. Cell entry: It is believed that the virus recognizes the host cell through interactions between the viral envelope glycoproteins E1 and E2, and one or more receptors on the host cell. For example, the human CD81 receptor has been shown to be necessary for viral entry. Once bound to the surface of the host cell, endocytosis, pH-dependent fusion of the membranes and uncoating release the viral genome into the host cell cytoplasm.22 Translation and polyprotein processing: Once the viral RNA has entered the host cell it is translated by the ribosomes on the rough endoplasmic reticulum (ER). Translation yields a polyprotein that is proteolytically cleaved into 10 mature viral proteins (see Section 1.1.4).19

13 Figure 3. A schematic representation of the proposed life cycle of HCV.

RNA replication: Several of the viral proteins form a replication complex that is situated on the ER membrane (Figure 4). This replication complex synthesizes minus-strand RNA from the original plus-strand RNA which then serves as a template for the production of excess amounts of plus-strand RNA, also performed by the replication complex. In addition to the viral proteins, several host cell factors are important for RNA replication.23 Virion assembly and release: The new virions are presumably formed by budding into the ER and leave the cell through the secretory pathway.18

Figure 4. A schematic illustration of the viral proteins believed to compose the replication complex and their topology with respect to the ER membrane, as described by Lindenbach and Rice.24

14 1.1.4 The Viral Genome and its Translational Products The HCV genome is a single stranded, positive sense RNA molecule with a length of approximately 9600 nucleotides. It contains a single open reading frame flanked by 5´ and 3´ non-translated regions (NTRs). The 5´ NTR contains an internal ribosome entry site (IRES) required for RNA translation (Figure 5).23

Figure 5. The HCV genome (above) and its translational products (below).

Translation of the viral RNA takes place in the ER producing a polyprotein of approximately 3000 amino acids that is proteolytically processed into the structural proteins (C, E1 and E2), p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Figure 5). Processing of the viral polyprotein is mediated by different proteolytic enzymes, as indicated in Figure 5: signal peptide peptidase (open arrow), signal peptidase (closed arrows), the NS2/3 protease (single curved arrow) and the NS3 protease (multiple curved arrows).24 A general description of the viral proteins is given below. C: The core protein (C) forms the nucleocapsid surrounding the genetic material.19 E1 and E2: E1 and E2 are highly glycosylated envelope proteins inte- grated in the lipid membrane of the viral envelope.19 p7: Protein p7 is a membrane protein believed to provide an ion channel, crucial for HCV infectivity.25 However, the exact role of p7 in the viral life cycle is not fully understood. NS2/3 protease: The NS2 region and the N-terminal one third of NS3 en- code the NS2/3 protease that is responsible for polyprotein cleavage between

15 NS2 and NS3.26,27 It was early suggested that the NS2/3 protease was either a zinc-dependent metalloprotease or a cysteine protease.26,28 However, a recently determined structure of the catalytic domain of the NS2/3 protease, using X-ray crystallography, revealed a dimeric cysteine protease with two composite active sites.29 NS3: The NS3 protein is a bifunctional enzyme with a helicase/NTPase domain and a protease domain. It has been suggested that the NS3 helicase initiates RNA replication by unwinding RNA duplexes fueled by adenosine triphosphate (ATP) and that it removes stable secondary structures from the template RNA strand. However, the molecular mechanism behind the RNA unwinding and its exact role in the replication complex are not yet fully understood. The NS3 serine protease is a key actor in the processing of the viral polyprotein, responsible for cleavage at four sites.23 NS4A: NS4A is an essential cofactor to the NS3 protease. Upon complex binding to the protease it enables efficient catalytic activity of the protease and anchors the protease on the ER membrane (Figure 4).23 NS4B: NS4B is a highly hydrophobic integral membrane protein predicted to contain four transmembrane domains (Figure 4). It is believed to be involved in the formation of membrane structures in the ER, serving as a scaffold for the formation of the HCV replication complex. However, its exact role in the replication remains to be elucidated.23 NS5A: NS5A is a phosphorylated metalloprotein comprising three domains and an D-helix that serves as an anchor to the ER membrane (Figure 4).30-32 The structure of domain I of NS5A was recently revealed by X-ray crystallography showing the presence of a zinc atom coordinated by four cysteine residues.31 Mutational studies suggest that both the membrane an- choring D-helix and the zinc atom are required for HCV RNA replication.32,33 Moreover, the phosphorylation state of NS5A seems to be an important regulator in viral replication.34 Huang et al. discovered the ability of NS5A to bind viral RNA, a result that has led to the suggestion that NS5A oligomerizes to form an extended channel serving as a pathway for viral RNA transport during RNA replication.23,35 NS5B: The RNA-dependent RNA polymerase (RdRp) NS5B constitutes the catalytic core of the HCV replication complex. It is responsible for the generation of the template minus-strand RNA and the plus-strand RNAs.36 Its C-terminal transmembrane domain is crucial for HCV RNA replication and it anchors the enzyme to the ER membrane (Figure 4).37 The different protein subdomains generate a fully encircled active site where the template RNA and the nucleotide triphosphates (NTPs) enter through two distinct tunnels.38 Although our understanding of the viral life cycle and the function of the viral proteins is increasing, several important questions remain to be answered.

16 1.1.5 HCV Variability There are six different HCV genotypes, 1 to 6, which differ by 30–35% of their nucleotide sequence. These genotypes are further divided into a series of subtypes, identified by lower-case letters (e.g. 1a), which differ by 20– 25% of their nucleotide sequence. Genotype 1b is the most common genotype throughout the world. Due to the high replication rate of the virus (~1012 particles per day) and an error-prone polymerase, the virus further diversifies within an infected individual over time producing so-called quasispecies.39

1.1.6 Current Treatment of Hepatitis C In 1986, it was reported that the naturally occurring immune agent interferon-D (IFN-D) could be used to treat patients infected with non-A, non-B hepatitis.40 Since then, the medical treatment of HCV has relied on this 165- amino-acid peptide possessing antiviral, antiproliferative and immunomodu- lating activities. The initial monotherapy using IFN-D had only limited success with sustained virological response (SVR) rates of 6–20%. SVR is defined as the absence of detectable serum HCV RNA 24 weeks after treatment comple- tion. The combination of the broad-spectrum antiviral agent ribavirin (Figure 6) with IFN-D had major effects on SVR rates, which increased to 35–40%. The SVR rates were further improved (54–56%) through the covalent link- age of a polyethylene glycol (PEG) chain to IFN. In addition, the pegylation of IFN resulted in a prolonged half-life that reduced the number of injections required.41 The combination therapy of PEG-IFN and ribavirin for 24 or 48 weeks is currently considered the standard therapy for the treatment of chronic HCV infection.42

N O O N HO N NH2 HO OH Figure 6. Chemical structure of ribavirin.

The main determinant for the success of the medical treatment of HCV is the viral genotype. For patients infected with the most prevalent genotype 1, the SVR rates are only 42–44%, whereas SVR rates are 78–80% for genotype 2 and 3 patients.43 Factors such as race, age, body weight and viral level will also influence whether the treatment is successful or not. Interestingly, if medical treatment with IFN-D or PEG-IFN-D is given during the acute phase of an HCV infection, the development of chronic infection can be prevented in almost all patients.44,45

17 Despite the improvements associated with the interferon-based therapy, SVR rates are still only ~55%, meaning that the remaining ~45% of patients do not respond to the therapy. Furthermore, interferon-based therapy is associated with severe adverse events such as neuropsychiatric symptoms, influ- enza-like symptoms and hematological abnormalities.42 Consequently, there is a huge unmet medical need for new therapeutic agents to combat HCV.

1.1.7 HCV Drug Discovery Historically, the medical approach to defeating viral diseases has been the use of vaccines, since the availability of antiviral drugs has been very limited. Viral diseases such as smallpox, polio and measles have been successfully defeated by vaccination.46 However, the development of vaccines against a number of viral infections such as HIV, herpes simplex virus (HSV) and HCV has so far not been successful. During the past two decades, the availability of antiviral drugs has been dramatically changed, primarily due to a better understanding of viruses and their life cycles. Today, effective antiviral drugs, predominantly nucleoside analogues, are available for several viral infections, e.g. HIV, HSV and cytomegalovirus (CMV).47 Due to the inability of HCV to propagate in cell culture, early HCV drug development was hampered by the absence of a cell-based assay for the identification of promising drug candidates. In addition, a robust small- animal model for screening of anti-HCV drugs was not available; a problem that still has not been solved. The only animal that can be readily infected with HCV is the chimpanzee. Its use in drug discovery is, however, limited due to factors such as availability, price and ethics. Nevertheless, the chimpanzee has played a critical role in the study of HCV.48 A major breakthrough in HCV drug discovery was achieved when Loh- mann and coworkers introduced the cell-based subgenomic HCV replicon system in 1999.49 The subgenomic HCV replicon consists of the nonstructural proteins NS3-NS5B, which replicate in human hepatoma cell lines (Huh-7 cells). Although this subgenomic system does not exploit the complete viral genome and does not produce infectious viral particles, it has had considerable impact on HCV drug development.50 Another recent milestone in HCV research was the reporting by several groups of the successful replication of full-length viral RNA and the production of infectious viral particles in cell culture.51-53 These systems will not only be important tools for the identification of potential anti-HCV agents, but also for improving our understanding of the viral life cycle. Several drug targets have arisen from the progress in our understanding of the HCV life cycle. In principle, inhibition of any stage in the HCV life cycle can be considered in the search for new therapeutic agents. The virally encoded proteins have all been considered as drug targets and several virus- specific inhibitors are currently being evaluated in clinical trials. Apart from

18 agents acting directly on the viral proteins, other therapeutic approaches have also been considered, e.g. human toll-like receptor (TLR) agonists and human glucosidase inhibitors, as well as viral IRES inhibitors. In addition, considerable effort has been devoted to the development of vaccines and further optimization of the current treatment (IFN and ribavirin).54-56 Inspection of the drug pipeline for HCV treatment reveals that more than 50 different agents are currently under clinical investigation.57 Among the agents acting directly on the viral proteins, the NS3 protease inhibitors and the NS5B polymerase inhibitors have been most intensively studied. For further details regarding NS3 protease inhibitors currently undergoing clinical trials, please see Section 1.2.3.

1.2 The HCV NS3 Protease 1.2.1 Proteases in General and Serine Proteases in Particular Proteases are proteolytic enzymes that catalyze the cleavage of peptides and proteins through the hydrolysis of peptide bonds. Approximately 500 different proteases have been identified in the human genome58 and they are involved in numerous physiological processes.59 In addition, proteases play a crucial role in many infectious diseases, such as HIV, HCV and malaria.60 Consequently, proteases are considered very important drug targets and they constitute an attractive means of treating a variety of diseases. A huge number of protease inhibitors are currently being evaluated in either preclinical or clinical trials.60 More importantly, several protease inhibitors are currently on the market, e.g. angiotensin-converting enzyme (ACE) inhibitors for the treatment of cardiovascular conditions, HIV protease inhibitors for the treatment of HIV infection, thrombin inhibitors as anticoagulants, and a dipeptidyl peptidase IV (DPPIV) inhibitor for the treatment of type 2 diabe- tes.59,61 Based on the enzyme residue responsible for hydrolysis, proteases can be classified as serine, cysteine, threonine, aspartic, metallo or glutamic proteases.59 Another classification system of proteases is based on their amino acid sequence (family) and their three-dimensional structure (clan).62 For a substrate or inhibitor that binds to a protease, the Schechter and Berger nomenclature is used to designate the substrate/inhibitor side chains (P3, P2, P1, P1´, P2´ and P3´) that bind to the corresponding subsites (S3, S2, S1, S1´, S2´ 63 and S3´) of the protease (Figure 7).

19 S2 S1' S3'

H O P2 H OP1' H OP3' N N N N N N H H H P3 O P1 OP2' O

S3 S1 Scissile S2' bond Figure 7. A schematic representation of a substrate/inhibitor backbone and the side chains (P) that bind to the corresponding subsites (S) in the protease. The scissile amide bond is indicated.

The HCV NS3 protease, which is of great importance to the work presented in this thesis, is a serine protease. A serine protease utilizes three amino acids during the hydrolysis of a peptide bond: a histidine, an aspartic acid and a serine. These amino acids are recognized as the catalytic triad, and are num- bered according to the HCV numbering system, His57, Asp81 and Ser139. The general catalytic mechanism for a serine protease can be seen in Fig- ure 8.

Asp81 Asp81 O O O O (A) H (B) H N His57 N His57 N N P1 HN H P1 N H P ' 1 H O P1' O O O H H H H N N N N

Gly137 Ser139 Gly137 Ser139

Asp81 Asp81 Asp81 O O O O O O (C) H (D) H (E) H N His57 N His57 N His57 N N N P P1 H H P 1 H 1 H2N O OH O P ' H O H 1 O O O O O H H H H H H N N N N N N

Gly137 Ser139 Gly137 Ser139 Gly137 Ser139 Figure 8. General catalytic mechanism for serine proteases. (A) The peptide substrate binds to the active site of the serine protease exposing the carbonyl of the scissile amide bond to nucleophilic attack by the Ser139 hydroxyl group. His57 acts as a base and abstracts the proton from the Ser139 hydroxyl group. (B) The negatively charged tetrahedral intermediate is stabilized through hydrogen bonding to the backbone NHs of Gly137 and Ser139, i.e. the oxyanion hole. The tetrahedral intermediate breaks down and proton transfer from His57 releases the C-terminal product of the substrate (the amine). (C) A water molecule attacks the acyl-enzyme complex via base-assisted catalysis of His57. (D) A second tetrahedral intermediate is formed and collapses via acid-assisted catalysis of His57. (E) Ser139 is regenerated and the N-terminal product of the substrate (carboxylic acid) is released.64

20 1.2.2 Structure and Function of the HCV NS3 Protease Shortly after the HCV genome was cloned, it was suggested that the NS3 region encoded a protein with several enzymatic activities.65 This hypothesis was later confirmed when researchers identified a serine protease located in the N-terminal one-third of the NS3 protein66-68 and a helicase/NTPase in the remaining C-terminal two-thirds.69,70 The NS3 protease was found to be responsible for cleavage of the HCV polyprotein at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions.66- 68 The cleavage of the NS3/4A site occurs in cis, i.e. intramolecularly, whereas the remaining cleavages occur in trans, i.e. intermolecularly. In addition to the protease domain of NS3, the NS4A cofactor is necessary for efficient cleavage of the substrates contained in the HCV polyprotein.71 This cofactor helps anchor the protease to the ER membrane72 and stabilizes it against proteolytic degradation.73 The exact molecular mechanism behind the catalytic enhancement of the protease is not unequivocal. It has been proposed that complex binding of NS4A causes rearrangement of the protease active site, leading to an optimal positioning of the catalytic triad and thus an increase in proteolytic efficiency74 or, that NS4A binding causes conformational changes in the protease, leading to more efficient binding of the substrate, which will increase the substrate turnover.75 X-ray determined structures of the protease domain of NS3 were presented in 1996, both with76 and without77 the NS4A cofactor. These crystal structures were long awaited and confirmed previous suggestions that the NS3 protease has a chymotrypsin-like fold. In addition, they revealed a zinc- binding site suggested to play a structural role in the enzyme. Compared with other members of the chymotrypsin family, the NS3 protease was found to differ in certain loop regions, resulting in a relatively shallow and solvent- exposed substrate-binding channel.76 It was not until 1999 that Yao and colleagues managed to solve the crystal structure of the complete NS3 protein.78 This structure comprised the 631- residue bifunctional NS3 enzyme with both protease and helicase domains, and with the protease activating part of NS4A in a single polypeptide chain (Figure 9). From this structure, it could be seen that the enzymatic domains are separated and connected by a single strand. Moreover, the C-terminal of the helicase domain was located in the protease active site, which provides an idea of how the cleavage product binds after cis-cleavage of the NS3/4A junction. Interestingly, the protease active site was found to be oriented towards the molecular interior, resulting in a less solvent-exposed active site compared with that observed in the structure of the NS3 protease domain only. Exactly how the enzymatic domains of NS3 are positioned during substrate trans-cleavages in vivo remains unclear.

21 Figure 9. Connolly surface representation of the full-length NS3 protein and the protease activation part of NS4A (pdb code 1CU1).78 The helicase domain is depicted in blue, the protease domain in green and NS4A in magenta. The six C- terminal residues of the helicase occupy the protease substrate binding site and are depicted in yellow. The protease catalytic triad is colored red.

Recent advances in the understanding of how HCV evades the antiviral immune response have identified the NS3 protease as a crucial factor in an HCV infection becoming chronic. It has been demonstrated in vitro that the NS3 protease interferes with several pathways involved in the host antiviral immune response.79-81 More importantly, these pathways were restored upon treatment with an NS3 protease inhibitor. As a result, inhibition of the NS3 protease may not only block viral replication but also restore the host cell’s antiviral defense, making the NS3 protease an even more attractive drug target.

1.2.3 HCV NS3 Protease Inhibitors In the middle of the 1990s, the NS3 protease emerged as the most promising anti-HCV drug target, mainly due to its crucial role in the viral life cycle. Moreover, protease inhibitors were successfully used to combat HIV, which increased the hope that the NS3 protease would prove to be a useful drug target. However, initial attempts to identify NS3 protease inhibitors revealed unusual structural requirements of the protease since general serine protease

22 inhibitors such as phenylmethylsulfonylfluoride and 3,4- dichloroisocoumarin performed poorly against this enzyme.82 Although some non-peptidic NS3 protease inhibitors were identified, the selectivity towards other serine proteases was usually low.83,84 It soon became clear that the most fruitful approach to find potent NS3 protease inhibitors was to start with peptides derived from the natural substrates of the protease. Today, approximately a decade after the first NS3 protease inhibitors were examined, a huge number of inhibitors have been developed by the pharmaceutical community.85-87 This progress in the development of NS3 protease inhibitors has been made possible almost exclusively through rational drug design with only minor contributions from high-throughput screening (HTS) cam- paigns. A brief overview of some classes of NS3 protease inhibitors presented over the years is given below.

Non-Covalent Peptidic Inhibitors

Product-Based Inhibitors with a C-Terminal Carboxylic Acid Two research groups independently recognized that the NS3 protease was subject to substantial product inhibition. Hexapeptides derived from the N- terminal cleavage products of the NS3 substrates were identified as potent NS3 protease inhibitors. Llinàs-Brunet and coworkers presented hexapeptide 88 A (P6-P1), derived from the NS5A/5B cleavage site, and Steinkühler and colleagues presented hexapeptide B (P6-P1), derived from the NS4A/4B cleavage site (Figure 10).89

O OH HO S HO N O O O O O NH H O H O H O H N N O O N N N O H OH N N N OH H H H H N SH O O O O SH N H O HO O HO O O A, IC50=28 PM OH B, Ki=0.6 PM Figure 10. Chemical structures of hexapeptides A and B.

Initial structure–activity relationship (SAR) studies on hexapeptides A and B revealed several structural properties important for the potencies of the inhibitors.88-91 Both groups found a strong preference for a cysteine residue in P1. This had previously been observed during substrate-specificity studies of the NS3 protease,92 and could possibly be explained by favorable interactions between the sulfhydryl group and the aromatic side chain of Phe154 76,77 defining the bottom of the S1 pocket. Both groups showed that the potencies of their hexapeptides were crucially dependent on the P1 C-terminal carboxylic acid. Substitution of the C-terminal carboxylic acid in B with the primary amide, or the reduction of the carboxylate to the corresponding al-

23 cohol resulted in inhibitors with approximately 150 times lower potencies. Molecular modeling studies suggested a network of hydrogen bonds between the C-terminal carboxylate of B and the active site of the protease.89 Another very important observation was that the inhibitors containing a C-terminal carboxylic acid were selective for the NS3 protease.90 The discovery of hexapeptide A by the researchers at Boehringer Ingel- heim was the start of an extensive lead optimization process. This ultimately led to the first HCV NS3 protease inhibitor entering clinical trials in humans for the treatment of hepatitis C. Briefly, the key modifications of their initial lead compound A were the development of chemically stable and potent cysteine replacements, and the extension of the P2 proline residue. These modifications enabled N-terminal truncation of the compounds resulting in tripeptide inhibitors with more drug-like properties. Finally, macrocycliza- tion of the P3 and P1 residues led to the discovery of ciluprevir (also called BILN 2061) (Figure 11).93 Ciluprevir was the first HCV NS3 protease inhibitor to enter clinical trials in humans for the treatment of hepatitis C. A significant reduction of the HCV RNA plasma levels was observed in HCV- infected patients when treated with ciluprevir, and the results established proof-of-concept in humans for an HCV NS3 protease inhibitor.94 The ciluprevir family of compounds has served as templates for the discovery of other promising NS3 protease inhibitors by several other pharmaceutical companies (see Figures 13 and 14).

O HO N O O NH O H N N N O O O O H OH NH O H N H N O O N O H OH N H O O O C, IC50=17 PM D, IC50=3.5 PM OH

O O H N N N N S O O

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

E, Ki=0.020 PM Ciluprevir, Ki=0.00030PM EC50=0.004 PM Figure 11. A brief overview of the lead optimization process performed by Boe- hringer Ingelheim leading to the discovery of the clinical candidate ciluprevir.

24 One important finding revealed during the development of ciluprevir was the elucidation of the interaction between the C-terminal carboxylic acid and the protease. Through X-ray analysis of a macrocyclic analogue of compound E in complex with the NS3 protease, it was verified that the carboxylate was engaged in hydrogen bonding to the oxyanion hole (NHs of Gly137 and Ser139) as well as the catalytic His57.95 This finding is in accordance with product binding after substrate hydrolysis (see Figure 8E in Section 1.2.1).

Product-Based Inhibitors with a C-Terminal Acyl Sulfonamide

The promising characteristics of inhibitors containing a P1 C-terminal carboxylic acid initiated studies on the possibility of bioisosteric replacement of this crucial group. Our research group investigated the tetrazole and the phenyl acyl sulfonamide as potential carboxylic acid bioisosteres. The introduction of the acyl sulfonamide group led to inhibitors with improved potencies compared to those containing the carboxylic acid, and the acyl sulfonamide was identified as a very promising structural fragment (Figure 12).96 Moreover, the acyl sulfonamide-containing inhibitors proved to be highly selective for the HCV NS3 protease.97

N N N HO O N H

G, Ki = 0.16 PM O H O H O H O HO N N N N N N OH O H O H O H O O OO S N HO O H

F, Ki = 0.22 PM H, Ki = 0.014 PM Figure 12. The impact on inhibitory potencies when the C-terminal carboxylic acid was replaced with a tetrazole or a phenyl acyl sulfonamide.

At the same time, Campbell and coworkers at Bristol-Myers Squibb released a patent application covering potent tripeptide inhibitors containing various acyl sulfonamide groups (Figure 13).98 The discovery of the acyl sulfonamide functionality has had considerable impact on the pharmaceutical community. During the course of this work, a huge number of patent applications covering NS3 protease inhibitors with P1 C-terminal acyl sulfonamides have been filed, and numerous pharmaceutical companies are currently exploring this functional group. Some examples of compounds presented in the patent literature can be found in Figure 13 (linear compounds) and Figure 14 (macrocyclic compounds).

25 O

O O

O N O N N NH O N NH O O H O O O O H O O O HN S O HN S O N Bristol-Myers Squibb Boehringer Ingelheim

S S

O N O N N H NH O NH O N N N H O O O N H O O O O H O N HN S HN S O Vertex Schering-Plough Figure 13. Linear acyl sulfonamide-containing HCV NS3 protease inhibitors presented by Bristol-Myers Squibb,98 Boehringer Ingelheim,99 Vertex100 and Schering- Plough.101

N N N N O N

O O O H O O O O N H O N O N S O N S N N H O N O H O N O H H Chiron O Enanta

H S N F N N N O O O O O O O O N H O N S H O O O N O N H N S O N O N N H O H O O H InterMune/Roche (ITMN-191) Merck IC50<0.25 nM, EC50=2.1 nM Figure 14. Macrocyclic acyl sulfonamide-containing HCV NS3 protease inhibitors presented by Chiron,102 Enanta,103 Merck104 and InterMune/Roche.105

The anticipation of the acyl sulfonamide group as a promising structural fragment in NS3 protease inhibitors has now been further strengthened. In collaboration with Roche, InterMune has recently begun phase I clinical

26 trials with their acyl sulfonamide-containing inhibitor ITMN-191 for the treatment of hepatitis C (Figure 14).106

Phenethylamides

During the search for a replacement for the P1 C-terminal carboxylic acid found in product-based inhibitors, the research group at Istituto di Ricerche di Biologia Molecolare (IRBM) discovered the phenethylamide as a promis- 107 ing P1 C-terminal functionality in NS3 protease inhibitors (Figure 15). According to molecular modeling of compound I, the phenethylamide moiety extends into the prime side of the enzyme interacting with the side chain of Lys136. In addition, it was suggested that the para-carboxylate had close contacts with both Lys136 and Arg109 in the enzyme. The inhibitors did not inhibit human leukocyte elastase (HLE) and the P1 amide group was found to be stable against cleavage by the NS3 protease. Inhibitor I contains a 4,4- difluoro-2-aminobutyric acid in P1, a cysteine mimic previously designed by the same group.108 Subsequent work on these inhibitors rendered capped dipeptide inhibitors with more drug-like properties.109 However, only mod- erate potencies were observed for the dipeptides, as exemplified by compound J (Figure 15).

O O H O H O OH F O N N H O OH N N N N OClH O H N H CHF2 O F O CHF HO O HO 2 I, Ki=0.60 PM J, IC50=13 PM Figure 15. Phenethylamide-containing HCV NS3 protease inhibitors.

Covalent Peptidic Inhibitors A common approach to the inhibition of serine proteases is the use of inhibitors containing an electrophilic functional group (serine trap) capable of forming a covalent bond to the catalytic serine residue of the enzyme. De- pending on the electrophilic group, such inhibitors can either be irreversible (e.g. chloromethyl ketones and sulfonyl fluorides) or reversible (e.g. aldehydes, D-ketoacids and D-ketoamides). One limitation on the use of electrophilic groups in drugs is the potential problems associated with selectivity, since the electrophilic moiety can react covalently with various nucleophiles in the body.64

D-Ketoacids Researchers at IRBM have investigated NS3 protease inhibitors with a C- terminal D-ketoacid group. Highly potent inhibitors such as compound K have been presented (Figure 16).110 During efforts to reduce the peptide

27 character of their inhibitors they designed a C2-alkylated indoline ring as a peptidomimetic replacement for the P3 residue, as can be seen in compound L (Figure 16).111 Through X-ray analysis of these inhibitors in complex with the NS3 protease, they observed a different binding mode for the D-ketoacid group compared with other serine protease-D-ketoacid inhibitor complexes. After nucleophilic attack by serine at the electrophilic keto moiety, the carbonyl oxygen did not bind in the oxyanion hole as anticipated, but was engaged in hydrogen bonding with the catalytic histidine. Instead, the carboxylate group was found to occupy the oxyanion hole (Figure 16).

O H O H O O N N OH His57 O N H N Asp81 O H O O N CHF2 HO O H K, Ki*=0.027 PM O IC50=0.33 PM O O O H O O H N NH H N OH Gly137 N N H O O Ser139 O CHF2 L, IC =0.7 M S OH 50 P Figure 16. D-Ketoacid-based HCV NS3 protease inhibitors (left) and the binding mode of their C-terminal moiety with the active site of the protease (right).

D-Ketoamides Compounds containing an D-ketoamide serine trap have been thoroughly investigated as NS3 protease inhibitors. Early studies on hexapeptide inhibitors with different serine traps identified the D-ketoamide group to be a more powerful serine trap than D-ketoesters and D-diketones.112 As for the D- ketoacid inhibitors mentioned above, it has been found that D-ketoamide inhibitors bind to the active site of the NS3 protease in the same unusual way, i.e. the electrophilic keto moiety is attacked by serine and the adjacent amide carbonyl occupies the oxyanion hole.113 In contrast to the D-ketoacids, D-ketoamides are neutral with the possibility to interact with the prime side of the protease. Progress in the development of D-ketoamide NS3 protease inhibitors has been made mainly by Vertex and Schering-Plough.114 Their work in the field has generated two clinical candidates, VX-950 (telaprevir)114 and SCH 503034115 (Figure 17).

28 N H N O N O O NH O NH NH O O NH O O NH N HN NH 2 O O N O SCH 503034 VX-950 (telaprevir) Ki*=0.014 PM, EC50=0.20 PM Ki=0.04 PM, EC50=0.35 PM Figure 17. Chemical structures of the D-ketoamide-containing HCV NS3 protease inhibitors telaprevir and SCH 503034.

Clinical results from phase I trials have shown that SCH 503034 is well tolerated and exhibits potent antiviral activity in HCV genotype 1 patients, both as monotherapy116 and in combination with PEG-IFN-D.117 SCH 503034 is currently in phase II trials where it is being evaluated in combination with PEG-IFN-D.118 Telaprevir is in the frontline of NS3 protease inhibitors in clinical studies. The inhibitor was well tolerated and demonstrated substantial antiviral activity in HCV genotype 1 patients during phase I trials.119 Telaprevir is currently being evaluated in a large phase II study, PROVE 1, where it is being evaluated in combination with PEG-IFN-D and ribavirin. Initial results from PROVE 1 demonstrated that 65 of 74 patients (88%) receiving telaprevir + PEG-IFN-D + ribavirin had undetectable HCV RNA after 12 weeks of treatment, compared with 17 of 33 patients (52%) receiving placebo + PEG- IFN-D + ribavirin.120 Although adverse effects such as gastrointestinal disor- ders and rashes were observed in patients receiving telaprevir, its powerful antiviral effect provides hope for future HCV therapy containing NS3 protease inhibitors.

Non-Peptidic Inhibitors Finding non-peptidic inhibitors of the HCV NS3 protease has been very challenging, and only a few compounds have been reported in the literature. One example is compound M, which was discovered by structure-based NMR screening (Figure 18).121

O OH I O HO HO O O

I N O O H M, Ki=0.8 PM Figure 18. A non-peptidic HCV NS3 protease inhibitor.

Resistance Antiviral drugs targeting viral proteins run the risk of becoming ineffective due to the development of resistance.46 This is a significant clinical problem

29 for HIV protease inhibitors122 and the development of resistance against NS3 protease inhibitors has already been observed for several inhibitors in cell- based HCV replicon assays. The Asp168Val or the Asp168Ala mutation confers resistance to ciluprevir,123,124 and more importantly, the Ala156Thr mutation confers cross-resistance to all three clinical candidates ciluprevir,123,125 telaprevir125 and SCH 503034.126 Resistant HCV variants were recently observed in vivo, during treatment with telaprevir.119 These findings are alarming and strongly motivate the development of novel NS3 protease inhibitors. Results from in vitro studies indicate that combinations of different drugs with different mechanisms of action (NS3 protease inhibitor + IFN-D126 and NS3 protease inhibitor + NS5B polymerase inhibitor127) are effective in reducing the emergence of viral resistance. This approach is currently being practiced in clinical trials with NS3 protease inhibitors (vide supra). In conclusion, a combination of drugs with different mechanisms of action will probably be required to combat HCV, and the development of novel compounds within every mechanistic class, displaying different resistance profiles, must be pursued.

30 2 Aims of the Present Study

This study was part of an ongoing medicinal chemistry project with the overall objectives of designing and synthesizing potent inhibitors of the HCV NS3 protease.

The specific objectives of this study were:

x To establish structure–activity relationships of HCV NS3 protease inhibitors comprising potential P1 C-terminal carboxylic acid bioisosteres in general, and the acyl sulfonamide group, in particular.

x To explore novel structural motifs in HCV NS3 protease inhibitors aimed at providing more drug-like compounds.

x To develop synthetic methods that allow for efficient preparation of the designed inhibitors.

31 3 Carboxylic Acid Bioisosteres in HCV NS3 Protease Inhibitors (Papers I and II)

3.1 Bioisosteres in Medicinal Chemistry The term bioisostere is used to describe groups or molecules that have chemical and physical similarities producing broadly similar biological properties.128 When making a bioisosteric replacement, parameters such as size, shape, solubility, pKa, chemical reactivity and hydrogen bonding capacity are considered. The bioisostere concept is widely used in the drug discovery process to optimize properties such as potency, selectivity or the ADME (absorption, distribution, metabolism and excretion) profile of a lead compound.128-133 It should be noted that a good bioisosteric replacement in one series of compounds is not necessarily useful in another. One well- known example of a successful bioisosteric replacement is the replacement of the carboxylic acid in compound N by a tetrazole, a group that is as acidic as a carboxylic acid but much more lipophilic (Figure 19).129 This replacement resulted in the antihypertensive compound losartan, which was more potent and exhibited better oral bioavailability than its forerunner, N.134

Cl Cl N N OH OH N N N N OOH N NH

N losartan Figure 19. The bioisosteric replacement of the carboxylic acid in N resulted in losartan, a compound with improved potency and oral bioavailability.

3.2 Exploring the Acyl Sulfonamide Group (Paper I) The acyl sulfonamide group (Figure 20) has been used as a carboxylic acid bioisostere in various medicinal chemistry projects.134-141 It is as acidic as a 138 carboxylic acid with a pKa of approximately 5. An acyl sulfonamide, in

32 contrast to a carboxylic acid, can be structurally elongated using various R- groups while retaining its acidity. This property can be used in a lead optimization process to gain additional interactions between a ligand and a protein/receptor, as well as altering the lipophilicity of the compound.

O O OO S OH N R H Figure 20. The chemical structures of a carboxylic acid (left) and an acyl sulfonamide (right).

The discovery that the phenyl acyl sulfonamide group was a powerful P1 C- terminal carboxylic acid bioisostere in HCV NS3 protease inhibitors initiated this project (see Figure 12 in Section 1.2.3).96 Although it had been shown that the phenyl acyl sulfonamide group provided potent inhibitors when introduced into hexa- and pentapeptides, several important questions remained to be answered. How well would the group act when used in shorter peptides? Are the potencies of the inhibitors dependent on the sulfonamide substituent? What properties of the group contribute to the potency of the inhibitor? How is the potency affected by changes in other parts of the inhibitor, e.g. the P1 side chain? How do acyl sulfonamide-based inhibitors perform in a cell-based assay? In an attempt to answer the above questions, we em- barked on a thorough investigation of the acyl sulfonamide group in NS3 protease inhibitors. Three peptide scaffolds were chosen for this evaluation: two tetrapeptides originating from our tetrapeptide library142 and a tripeptide scaffold originally developed by Boehringer Ingelheim (Figure 21).143,144 The inhibitors were designed to contain various amino acid side chains as well as analogues of the acyl sulfonamide group, as illustrated in Figure 21. To enable comparisons, the corresponding inhibitors with a C-terminal carboxylic acid were included in the study.

N O O O O O O O HN S S R R O O O H OP2 H O O OO O N O HO N N S S N N OH N R NH O N HN R R O H OPH O H BocHN O O 3 OH P1 Tetrapeptide scaffolds Tripeptide scaffold

HO O Figure 21. Overview of the compounds presented in Paper I.

33 3.2.1 Preparation of Tetrapeptides The tetrapeptides 1 and 2, with a C-terminal carboxylic acid, were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc)/tBu solid phase peptide synthesis (SPPS) employing a 2-chloro-tritylchloride resin (Scheme 1).145 The first amino acid (P1) was attached to the linker using N,N- diisopropylethylamine (DIEA). Peptide coupling was performed in the presence of N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium tetrafluoroborate N-oxide (TBTU) and DIEA in N,N- dimethylformamide (DMF). Piperidine was used for Fmoc deprotection of the amino acids between the coupling steps. The P4 N-terminal was acety- lated using succinic anhydride before the peptides were cleaved from the resin with trifluoroacetic acid (TFA), H2O and triethylsilane (TES) which also deprotected the tBu group on the Glu side chain in 1.96

Scheme 1.

O O 1. FmocHN 1. FmocHN OH OH P -P P 2 4 1 O TBTU, DIEA, DMF Cl DIEA, CH2Cl2 H N 2. 25% piperidine/DMF Cl 2 O-linker- 2. 25% piperidine/DMF P 1 repeated steps

2-chloro-tritylchloride linker 1. O O O

DIEA, DMF Suc-Chg-Glu-2-Nal-Nva-OH (1) H2N-P4-P3-P2-P1-linker- Suc-Chg-Ile-Cha-Nva-OH (2) 2. TFA/H2O/TES

Preparation of inhibitors 3–6, containing C-terminal acyl sulfonamide groups, required the use of an inverse SPPS method utilizing tri-tert- butoxysilyl/9-fluorenylmethyl-ester-protected amino acids and a TentaGel resin with a photolabile linker (Scheme 2).146 Succinic acid (Suc) was attached to the solid support using succinic anhydride, followed by the introduction of the amino acids using N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5- b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) and 2,4,6-trimethylpyridine (TMP) as base. Cleavage of the silyl ester protection groups was enabled by the use of 5% TFA in CH2Cl2. The final peptide coupling was conducted between the resin-bound tripeptide (P4–P2) and the preformed P1–P1´ building block containing the acyl sulfonamide group (see Scheme 4 in Section 3.2.2 for synthesis of the P1–P1´ building blocks). The fluorenylmethyl-protected side chain of Glu was deprotected with piperidine and the piperidine salt formed was removed by acidic washing. The tetrapeptides were finally released from the solid support by photolysis at 350 nm.96

34 Scheme 2.

1. HClxH2N OSi(O-tBu)3 1. OOO P4-P2 O HATU, TMP, DMF/CH Cl H OH O 2 2 N DIEA, CH2Cl2 OH 2. 5% TFA/CH2Cl2 O NO2 -linker-O 2. 2.5% TFA/CH2Cl2 O O repeated steps

photolabile linker O O O 1. H N S 2 N R H P1 12b or 12c (see Scheme 4) Suc-Chg-Glu-2-Nal-Nva-NHSO2iPr (3) HATU, TMP, DMF Suc-Chg-Ile-Cha-Nva-NHSO2iPr (4) -linker-Suc-P4-P3-P2-OH 2. Removal of Glu side- Suc-Chg-Glu-2-Nal-Nva-NHSO2Ph (5) chain protection Suc-Chg-Ile-Cha-Nva-NHSO2Ph (6) (i. 25% piperidine/DMF ii. 5% TFA/CH2Cl2) 3. hQ

3.2.2 Preparation of Tripeptides Synthesis of the tripeptide inhibitors included in the study was performed via different routes depending on the nature of the P1 amino acid. Inhibitors containing Nva in P1 were prepared via a convergent route in order to sup- press racemization at the P1 D-carbon. Thus, the P3–P2 and the P1–P1´ building blocks were prepared (see Schemes 3 and 4, respectively) for ultimate attachment via amide bond formation (Scheme 5). Inhibitors containing a P1 residue devoid of an D-hydrogen were prepared in a linear fashion (Scheme 6). 98 The P3–P2 dipeptide 9 (Scheme 3), served as a key intermediate for the synthesis of the tripeptide inhibitors. Nucleophilic aromatic substitution between tert-butoxycarbonyl (Boc)-protected trans-4-hydroxyproline (7) and 4-chloro-7-methoxy-2-phenylquinoline followed by esterification using me- thyliodide provided compound 8. Boc deprotection, amide coupling with Boc-Val-OH and ester hydrolysis furnished the key intermediate, 9, in 63% yield over 5 steps.

Scheme 3.

O O 1. HCl in 1,4-dioxane (100%) N N NO 2.

1. OH OH O BocHN O Cl O KOtBu, DMSO (89%) HBTU, DIEA, DMF (78%) BocN BocN N OH 2. MeI, Cs2CO3, DMF (93%) O 3. LiOH, THF, MeOH, H2O (97%) OH O O BocHN O O 78 9

35 The P1–P1´ building blocks containing acyl sulfonamide, N-methylated acyl sulfonamide, benzylamide and E-ketosulfone groups were prepared from Boc-Nva-OH (10) (Scheme 4). 1,1´-Carbonyldiimidazole (CDI) was used as the activating agent in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to enable formation of the acyl sulfonamides 11a-e. Although the acyl sulfonamide bond was formed using a Boc-protected amino acid (Boc- Nva-OH), partial racemization of the Nva D-carbon occurred ( 10%). N- Methylation of the acyl sulfonamide group in 11c provided compound 11f. Boc deprotection, and for 12b and 12d, removal of the hydrochloride salt, furnished the desired P1–P1´ building blocks. The benzylamide building block 15 was prepared from Boc-Nva-OH using N-[(1H-benzotriazole-1-yl)- (dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU) and DIEA, followed by Boc deprotection. Boc-Nva-OMe (16) was successfully reacted with methyl phenyl sulfone in the presence of n-butyl lithium which, after Boc deprotection, provided the desired E- ketosulfone 18. Partial racemization (~20%) of the Nva D-carbon occurred during the synthesis of 18.

Scheme 4.

RSO2NH2, O O O O O O propylene O O O o CDI, DBU BocHN S HCl HClxH NSoxide, 50 C H NS N R 2 N R 2 N R THF EtOAc or EtOH or X 1,4-dioxane X EtOH/MeOH X

11a, X=H, R=Me (80%) 12a, X=H, R=Me (95%) 11b, X=H, R=iPr (82%) 12b, X=H, R=iPr (100%) 13b, X=H, R=iPr (99%) 11c, X=H, R=Ph (58%) 12c, X=H, R=Ph (97%) MeI, Cs2CO3, 11d, X=H, R=Bn (58%) 12d, X=H, R=Bn (98%) 13d, X=H, R=Bn (99%) DMF, 65 oC 11e, X=H, R=phenethyl (36%) 12e, X=H, R=phenethyl (93%) 11f, X=Me, R=Ph (81%) 12f, X=Me, R=Ph (94%)

O benzylamine, O O BocHN HBTU, DIEA BocHN HCl HClxH N OH N 2 N DMF H EtOAc H

10 14 (90%) 15 (100%)

O MeSO2Ph, O OO O OO o MeI, Cs CO BocHN n-BuLi, -78 C BocHN S HCl HClxH N S 2 3 O Ph 2 Ph DMF THF EtOAc

16 (86%) 17 (85%) 18 (100%)

Synthesis of the tripeptide inhibitor 19 with a C-terminal carboxylic acid was achieved by reacting 9 with H-Nva-OMe, followed by ester hydrolysis (Scheme 5). The tripeptide inhibitors 20–27 were finalized by amide bond formation between the P3–P2 key intermediate 9 and the appropriate P1–P1´ building block (Scheme 5).

36 Scheme 5.

O O O 1. HClx H N N 2 O N

O O HBTU, DIEA, 12a, 13b, 12c, 13d, DMF (95%) 12e, 12f, 15 or 18 N 9 N NH O 2. LiOH, THF, MeOH, HBTU or HATU, NH O BocHN O O H2O (92%) DIEA, DMF BocHN O O OH X Y R 19

20-24, X=NH, Y=SO2; R=Me (68%), iPr (18%), Ph (79%), Bn (45%), phenethyl (83%) 25, X=NMe, Y=SO2, R=Ph (61%) 26, X=NH, Y=CH2, R=Ph (79%) 27, X=CH2, Y=SO2, R=Ph (86%)

Inhibitors 28–32, comprising P1 amino acids devoid of D-hydrogens, could be synthesized in a linear fashion since there is no risk of racemization at the P1 D-carbon during formation of the acyl sulfonamide bond (Scheme 6). Amide bond formation between the key intermediate 9 and ester-protected 1- amino-cyclopropanecarboxylic acid (ACCA) or (1R,2S)-1-amino-2-vinyl- cyclopropanecarboxylic acid (vinyl-ACCA) followed by hydrolysis of the esters provided carboxylic acid inhibitors 2898 and 29.147 Formation of the acyl sulfonamides was enabled by the use of a protocol based on HATU as the activating agent148 that provided inhibitors 30–32.

Scheme 6.

O O O 1. HClx H N 2 OMe N N or O HClxH N 2 OEt O O

N R'SO2NH2, HATU, N NH O NH O HBTU, DIEA, DIEA, 4-dimethylamino- O DMF (84%, 76%) BocHN O O pyridine, DBU BocHN O O O 9 OH HN S 2. LiOH, THF, R DMF R R' MeOH, H O 2 28, R=H (96%) 30, R=H, R'=Ph (75%) 29, R=vinyl (85%) 31, R=vinyl, R'=Ph (60%) 32, R=vinyl, R'=Bn (63%)

3.2.3 Structure–Activity Relationship The tetra- and tripeptide inhibitors 1–6 and 19–32 were biochemically evaluated in a protease activity assay comprising the full-length NS3 protein, 142,149 and the Ki values are presented in Tables 1–3. The tripeptide inhibitors were also evaluated in a cell-based, subgenomic HCV replicon assay, and 49 their EC50 values are presented in Tables 2 and 3.

37 In analogy with previous findings on the hexa- and pentapeptides,96 introduction of the acyl sulfonamide group into the two tetrapeptide scaffolds provided inhibitors approximately five times more potent than the corresponding carboxylic acid-based inhibitors (Table 1).

Table 1. Biochemical evaluation of tetrapeptide HCV NS3 protease inhibitors.

O H O H O H O H HO N N R HO N N R N N N N O H O H O O H O H O

A HO O B

Compound Peptide R Ki ± SD (PM) 1 A O 46 ± 3 2 B OH 28 ± 3 3A O OO 12 ± 1 S N 4 B H 9.7 ± 0.8 5a A O OO 8.4 ± 0.8 S N 6 B H 5.3 ± 1.0 a 60:40 mixture of diastereomers at Nva. SD = standard deviation.

A more thorough investigation of the acyl sulfonamide functionality was performed on the tripeptide scaffold (Table 2). Also in this series, introduction of the acyl sulfonamide group was beneficial in terms of potency, although the methyl acyl sulfonamide 20 was equipotent to 19. Based on the outcome of inhibitors 20–24, having a methyl, isopropyl, phenyl, benzyl or phenethyl sulfonamide substituent, there seems to be a preference for an aromatic substituent. The most potent inhibitor, 23, with a benzyl sulfonamide, had a Ki value of 0.18 PM. Moreover, 23 was the only inhibitor exhib- iting activity in the replicon assay, with an EC50 value of 4.6 PM. The preference for an aromatic sulfonamide substituent could be explained by favorable pi-stacking interactions between the aromatic ring and the side chain of Gln41 in the enzyme, according to molecular modeling on 22 (Figure 22).

38 Table 2. Biochemical evaluation of tripeptide HCV NS3 protease inhibitors. O

N NH BocHN O O R

a Compound R Ki ± SD (PM) EC50 (PM) O 19 0.76 ± 0.10 >10 OH O O O 20 HN S 0.77 ± 0.05 >10 O O O 21 HN S 0.58 ± 0.07 >10

O O O 22 HN S 0.30 ± 0.04 >10

O O O 23 HN S 0.18 ± 0.01 4.6

O O O HN S 24 0.42 ± 0.10 >10

O O O 25b N S 3.7 ± 0.4 n.d.

26 HN 5.2 ± 1.0 >10

O O O 27c S 2.7 ± 0.6 >10

a Mean value of two determinations. b Did not bind irreversibly to the enzyme. c 80:20 mixture of diastereomers at Nva. SD = standard deviation. n.d., not determined.

Comparing compounds 25–27, lacking parts of the acyl sulfonamide functionality, with the intact acyl sulfonamide 22, reveals information on the properties required for good potencies of the acyl sulfonamide inhibitors. The acidic NH of the acyl sulfonamide is crucial since all three non-acidic

39 analogues 25–27 are significantly poorer inhibitors than the acidic inhibitor 22. In addition, the sulfonyl oxygens in the N-methylated acyl sulfonamide 25 and the E-ketosulfone 27 seem to be engaged in interactions with the enzyme since these inhibitors are slightly more potent than benzylamide inhibitor 26. Molecular modeling on inhibitor 22 suggests that the acyl sulfonamide group is engaged in a network of hydrogen bonds with the active site of the protease (Figure 22). The carbonyl oxygen is seen occupying the oxyanion hole (NHs of Gly137 and Ser139), and both sulfonamide oxygens are engaged in hydrogen bonds, one with the NH of Gly137 and the other with the side chain of Gln41.

Figure 22. Possible interactions between the acyl sulfonamide functionality of in- 78 hibitor 22 and the full-length NS3 protein. The P1 carbonyl is seen occupying the oxyanion hole (NHs of Gly137 and Ser139), the sulfonamide oxygens bind to the oxyanion hole and the side chain of Gln41, and the phenyl ring pi-stacks with the side chain of Gln41.

The influence of the P1 residue on the potencies of the tripeptide inhibitors was also addressed (Table 3). Introduction of the ACCA side chain was beneficial for the potencies of both the carboxylic acid inhibitor 28 and the acyl sulfonamide inhibitor 30. Inhibitors 28 and 30 were two and five times more potent than their Nva analogues 19 and 22. Even more potent inhibitors 150 were obtained when vinyl-ACCA, an elegantly designed P1 residue, was used in the P1 position. The carboxylic acid inhibitor 29 with a P1 vinyl- ACCA is 25 times more potent than the Nva counterpart 19. The combination of the vinyl-ACCA in P1 and the C-terminal phenyl acyl sulfonamide group was exceptionally favorable, and provided inhibitor 31 with a Ki of 0.76 nM and an EC50 of 40 nM. Inhibitor 31 is 40 times more potent than its carboxylic acid counterpart 29, whereas inhibitors 22 and 30 are three and six times more potent than their carboxylic acid counterparts 19 and 28, re-

40 spectively. Introduction of the benzyl substituent that was optimal in the tripeptide with a P1 Nva into inhibitor 32 with a vinyl-ACCA resulted in an inhibitor more potent than the carboxylic acid 29 but less potent than the corresponding phenyl acyl sulfonamide 31.

Table 3. Biochemical evaluation of tripeptide HCV NS3 protease inhibitors. O

N NH BocHN O O R' R a Compound R R´ Ki ± SD (PM) EC50 (PM) O 28 -H 0.32 ± 0.03 >10 OH O O O 30 -H HN S 0.055 ± 0.007 3.2

O 29 -vinyl 0.030 ± 0.006 1.1 OH O O O 31 -vinyl HN S 0.00076 ± 0.00026 0.040

O O O 32 -vinyl HN S 0.018 ± 0.003 0.28

a Mean value of two determinations. SD = standard deviation.

The much-improved potency of the inhibitors comprising the P1 vinyl- ACCA instead of Nva can probably be attributed to favorable interactions between the vinyl group and the aromatic ring of Phe154, which constitutes 150 the bottom of the S1 pocket. Moreover, the vinyl-ACCA can be seen as a rigidified Nva analogue with a terminal double bond. According to our modeling studies, the P1 vinyl-ACCA lies more inside the S1 pocket than the P1 Nva, thereby packing its inhibitors more tightly with the enzyme. In summary, a thorough SAR study of HCV NS3 protease inhibitors comprising the acyl sulfonamide group as a P1 C-terminal carboxylic acid bioisostere has been performed. The presence of both the NH and the SO2 of the acyl sulfonamide group is important in producing potent inhibitors. Moreover, the structures of the P1 side chain and the sulfonamide substituent strongly influence the potency of inhibitors containing the acyl sulfonamide group.

41 3.3 Potential P1 Carboxylic Acid Bioisosteres (Paper II) Although it was now well established that the acyl sulfonamide group was a very efficient carboxylic acid bioisostere in HCV NS3 protease inhibitors, the search for other potential carboxylic acid bioisosteres has not been pursued. We felt encouraged to perform an extended investigation of potential P1 carboxylic acid bioisosteres on a tripeptide scaffold. Thus, a diverse set of P1 C-terminal functional groups was explored. Common carboxylic acid bioisosteres, e.g. the hydroxamic acid, as well as groups previously not considered to be carboxylic acid bioisosteres, e.g. hydrazides, were investigated, as illustrated in Figure 23. Properties such as acidity, number of heteroatoms and hydrogen bonding capacity were considered during the selection of the functional groups. Inhibitors with a P1 C-terminal carboxylic acid and acyl sulfonamides were included to enable comparisons. In an attempt to obtain information about the interactions between the C-terminal functional group of the inhibitors and the protease active site, their inhibition constants were determined at different pH values.

O O O e.g. N HN OH HN NH R' O O O HN CN HN NH R' N O O NH N N O BocHN N O O N OH N HN H S Figure 23. Some examples of the compounds presented in Paper II.

3.3.1 Chemistry The dipeptide carboxylic acid 3398 served as a key intermediate for inhibitor synthesis and was prepared according to the same procedure as described for compound 9, with the exception of the P3 amino acid (see Scheme 3 in Sec- tion 3.2.2). The tripeptide inhibitors 34–43 were prepared via a linear route starting from 33 (Scheme 7). The P1 vinyl-ACCA was coupled to dipeptide 33, after which the ester was hydrolyzed to provide compound 34.144 The formation of the hydrazides 35 and 36 as well as the diacyl hydrazines 37 and 38 was enabled by the use of standard peptide coupling conditions between 34 and phenylhydrazine, benzylhydrazine, benzhydrazide and phenylacetic hydrazide, respectively, employing HBTU or HATU as activating agents together with DIEA as base. Carboxylic acid 34 was activated using CDI prior to coupling with (S)-(+)-p-toluenesulfinamide in the presence of DBU, which resulted in acyl sulfinamide 39 as a 2:1 mixture of diastereomers. The preparation of hydroxamic acid 40 was enabled by activa-

42 tion of 34 with isobutyl chloroformate and N-methylmorpholine (NMM) at -15 oC followed by the addition of hydroxylamine hydrochloride. The acyl cyanamide 41 was successfully prepared by the reaction between 34 and cyanamide in the presence of HATU and DIEA at 40 oC. 2,2,2- Trifluoroethylamine was straightforwardly coupled to 34 using HATU and DIEA to produce amide 42. The poor nucleophile 2-aminothiazole required the use of HATU and DIEA at 45 oC for coupling to 34. However, the heterocyclic amide 43 was obtained as a 3:1 mixture of isomers, possibly due to acylation of both the exocyclic and the endocyclic nitrogen of 2- aminothiazole.151,152

Scheme 7.

O O O O 1. HClxH N 2 OEt N N N

O O O HATU, DIEA, DMF Nucleophile, 2. LiOH, THF, MeOH, coupling reagent, H2O base N N N OH NH O NH BocHN O O BocHN O O BocHN O O R OH 33 34 (75%) 35, R=CONHNHPh (44%) 36, R=CONHNHBn (43%) 37, R=CONHNHCOPh (32%) 38, R=CONHNHCOBn (67%) 39, R=CONHSOptol (47%) 40, R=CONHOH (67%) 41, R=CONHCN (39%) 42, R=CONHCH2CF3 (82%) 43, R=CONH-2-thiazolyl (80%)

The P1–P1´ building blocks used in the synthesis of inhibitors 53–55 (Scheme 9) were prepared from Boc-vinyl-ACCA-OH (44) as described in Scheme 8. The diacyl hydrazine 45 was prepared using amide coupling conditions and successfully dehydrated using Burgess reagent to give the 1,3,4- oxadiazole, 46. Activation of 44 with N,N´-diisopropylcarbodiimide (DIC) enabled coupling with benzene sulfonylhydrazide to provide building block 48. The carboxylic acid 44 was converted to the corresponding primary amide, which was subsequently dehydrated to give the nitrile 50.153 The use of sodium azide and triethylamine hydrochloride enabled synthesis of the tetrazole 51.154 Boc deprotection furnished the desired building blocks, 47, 49 and 52, which were coupled to carboxylic acid 33 without further characterization to give inhibitors 53–55 (Scheme 9).

43 Scheme 8.

N N Benzhydrazide, O Burgess N N H o HATU, DIEA BocHN NPhreagent, 75 C BocHN Ph HCl HClxH N Ph N O 2 O CH Cl 2 2 H O THF 1,4-dioxane 45 (83%) 46 (86%) 47

O Benzenesulfonyl O H O H BocHN hydrazide, DIC BocHN N Ph HCl HClxH N N Ph OH N S 2 N S CH2Cl2 H O O 1,4-dioxane H O O

44 48 (60%) 49

o N N 1. i. CDI, THF, 70 C NaN3, HClxEt3N N N o N o N N ii. NH , 0 C to rt BocHN C rt to 50 C BocHN HCl HClxH N 3 N 2 N 2. TsCl, pyridine, toluene H 1,4-dioxane H CH2Cl2

50 (71%) 51 (63%) 52

Scheme 9.

47, 49 or 52 (see Scheme 8) O HBTU or HATU, DIEA, DMF 53, R=5-phenyl-1,3,4-oxadiazol-2-yl (71%) 33 N 54, R=CONHNHSO2Ph (59%) NH 55, R=5-tetrazolyl (41%) BocHN O O R

The acyl sulfonamide-based inhibitors 5698 and 57,98 containing a phenyl and a para-tolyl sulfonamide substituent, respectively, were prepared according to the procedure described for compounds 30–32 (see Scheme 6 in Section 3.2.2). The cyclopropyl-based inhibitor 5898 was prepared as described for inhibitors 20–27 (see Scheme 5 in Section 3.2.2).

3.3.2 Structure–Activity Relationship The inhibitors were biochemically evaluated using the enzymatic full-length NS3 assay (Ki values) as well as the subgenomic replicon assay (EC50 values); the results are summarized in Table 4. The hydrazide-based compounds, 35–38, containing both H-bond- donating and H-bond-accepting atoms, were mediocre inhibitors of the protease, possibly due to their non-acidic properties. Ring closure of the diacyl hydrazine moiety in 37 resulted in the equipotent inhibitor 53. Replacement of one of the two carbonyls in the diacyl hydrazine 37 with a sulfonyl group was beneficial and the acyl sulfonylhydrazide 54 was approximately 10 times more potent than its carbonyl counterpart. In addition, 54 showed some effect in the replicon assay with an EC50 of 1.6 PM.

44 The acyl sulfinamide group was identified as an efficient carboxylic acid bioisostere, exemplified by compound 39, which was more potent than the corresponding carboxylic acid inhibitor 34 in the enzyme assay, showing a Ki of 9 nM. However, 39 was not as potent as the sulfonyl analogue 57, which could be attributed to the somewhat lower acidity of the acyl sulfina- 136 mide group (pKa~6.5) than the acyl sulfonamide group (pKa~5). More- over, as hypothesized for compound 22, this could reflect engagement of both sulfonamide oxygens in binding to the protease (see Figure 22 in Sec- tion 3.2.3). Disappointingly, 39 was substantially less potent than 34 in the cell-based replicon assay.

Table 4. Biochemical evaluation of tripeptide HCV NS3 protease inhibitors. O

N NH BocHN O O R

Ki ± SD EC50 Ki ± SD EC50 Comp. R Comp. R (nM) (PM)a (nM) (PM)a O O 1690 ± 34 20 ± 4 0.14 42 HN >2 OH CF3 330 O 560 ± O >10 43c N 35 HN NH 97 HN 821 ± 71 >10 Ph S O 1100 ± N N 2200 ± 36 NH >10 53 >10 HN 60 O Ph Bn 700 O O 1600 ± 37 HN NH >10 54 HN NH 140 ± 7 1.6 Ph 140 S Ph O O O O 880 ± N N HN NH >10 55 N 38 Bn N 214 ± 53 2.8 130 H O O O O O 0.34 ± b O 39 HN S 9 ± 3 1.8 56 HN S 0.006 ptol Ph 0.05 O O O 0.22 ± 425 ± O 40 3.8 57 S 0.006 OH 36 HN HN ptol 0.02 O O O O 0.058 ± 41 6 ± 1 1.2 58 HN S 0.0009 HN CN 0.007 a Mean value of two determinations. b 2:1 mixture of diastereomers. c 3:1 mixture of isomers. SD = standard deviation.

Two commonly used carboxylic acid bioisosteres are the hydroxamic acid and the tetrazole. Introduction of these two groups at the C-terminal of the tripeptide scaffold provided inhibitors 40 and 55, which were significantly less potent than the reference carboxylic acid inhibitor, 34. The acyl cyana-

45 155 mide moiety (pKa~4) has been proposed to be a carboxylic acid bioisostere,128 although its presence in the literature is very limited. Nevertheless, inhibitor 41 encompassing the acyl cyanamide group proved to be a very potent NS3 protease inhibitor, with a Ki of 6 nM. However, in analogy with the acyl sulfinamide inhibitor 39, compound 41 displayed a weak inhibitory effect in the replicon assay. Amides 42 and 43, with electron-withdrawing substituents, were modest inhibitors of the NS3 protease. Comparing the inhibitory effects of inhibitors 56–58 emphasizes the supe- riority of the acyl sulfonamide group as a carboxylic acid bioisostere in HCV NS3 protease inhibitors. The cyclopropyl acyl sulfonamide group is an exceptionally powerful C-terminal, and inhibitor 58 displays subnanomolar potencies in both the enzymatic and the cell-based assays. Comparing compounds 29 and 31 (Paper I) with compounds 34 and 56 reveals the positive effect of replacing the P3 Val with a tert-Leu. This effect is most pronounced in the cell-based assay, where the EC50 values were improved almost 10 times.

3.3.3 pH Study A subset of the inhibitors was subjected to a pH study, in which the inhibition constants were determined at different pH values (6.0, 7.0 and 8.0) in the enzyme assay (Table 5). Note that the Ki values presented in Table 5 cannot be compared to those presented in Table 4 due to the use of different assay buffers. The pH values were chosen to cover the pKa of the catalytic 156 His57, determined to be 6.85, in order to investigate the effect on the Ki values when changing the protonation state of His57. It may thus be possible to obtain information regarding interactions between the P1 C-terminal group of the inhibitors and His57 of the protease.

Table 5. Inhibition constants at different pH values. K ± SD (nM) Ratio Compound i pH 6.0 pH 7.0 pH 8.0 Ki (pH 8.0)/Ki (pH 6.0) 34 0.12 r 0.015 0.68 r 0.11 8.7 r 1.0 73 35 28 r 5.8 113 r 10 287 r 23 10 41 0.14 r 0.02 0.51 r 0.08 10.8 r 1.04 77 42 71 r 6 349 r 43 734 r 72 10 43 7.7 r 0.7 294 r 41 343 r 51 45 57 0.006 r 0.002 0.056 r 0.009 0.39 r 0.06 65 SD = standard deviation.

By comparing the Ki values at pH 8.0 and 6.0 for compound 34, comprising a C-terminal carboxylic acid, it can be seen that the inhibitory effect is highly pH dependent. This observation is in line with the established binding

46 mode for the P1 C-terminal carboxylic acid, reflecting a stronger interaction between the negatively charged carboxylate and the protonated His57 (pH 6.0) than with the non-protonated His57 (pH 8.0). Similar pH dependences were observed for inhibitors 41 and 57, comprising the acyl cyanamide group and the acyl sulfonamide group, respectively. Therefore, it seems reasonable to assume that these C-terminals bind in a similar mode to the active site of the protease as the carboxylic acid in 34, i.e. the carbonyl occupies the oxyanion hole, whereas the adjacent nitrogen points towards His57 allowing for a possible H-bond (Figure 24).

Figure 24. Compound 57 docked in the protease binding site of the full-length NS3 protein.78 Enzyme residues shown are: His57, Asp81, Gly137, Ser138 and Ser139 from the protease domain and His528 from the helicase domain.

During the course of this work, the first X-ray crystallography structure of an inhibitor with a P1 C-terminal acyl sulfonamide group (ITMN-191, Figure 105 14) in complex with the NS3 protease was presented. The P1 carbonyl was found in the oxyanion hole and the adjacent nitrogen interacted with His57; findings that strengthen our hypothesis regarding the binding mode for inhibitors 34, 41 and 57 (Figure 24). A minor pH dependency was also observed for the hydrazide 35 and the trifluoroethylamide 42, although these compounds are assumed to be uncharged at all pH values studied (Table 5). These results are difficult to in- terpret assuming that the P1 carbonyl of 35 and 42 occupies the oxyanion hole and that the adjacent nitrogen is uncharged. In addition, these results are not in agreement with previous findings on an inhibitor with a neutral P1 C- terminal primary amide.89 In that study, the inhibition constant was unaf- fected by similar changes in pH when evaluated against the truncated NS3

47 protein (i.e. the protease domain only). One possible explanation of the contradictory results could be that another histidine residue, besides His57, engaged in interactions with our inhibitors, is also affected by the changes in pH. Examining the available crystal structure of the full-length NS3 protein, the only histidine residue, apart from His57, in the vicinity of the protease binding site, is His528 in the helicase domain (Figure 24). Although the possible influence of the helicase domain on the binding of protease inhibitors remains to be proven, the presence of His528 in our full-length NS3 assay may be responsible for the contradictory results. On the other hand, it cannot be ruled out that the C-terminal in inhibitors 35 and 42 possess a different binding mode than the C-terminals in inhibitors 34, 41 and 57. Surprisingly few replacements to the P1 C-terminal carboxylic acid in product-based HCV NS3 protease inhibitors have been previously studied (acyl sulfonamides and amides). Herein, a wide variety of P1 C-terminal functionalities have been investigated and two new carboxylic acid bioisosteres have been identified.

48 4 Synthesis of Aryl Acyl Sulfonamides (Paper III)

Palladium-catalyzed carbonylation reactions can be used for the production of compounds containing aryl-carbonyl-nucleophile fragments. We predicted that aryl acyl sulfonamides could be rapidly and smoothly obtained by the use of a palladium(0)-catalyzed carbonylative protocol employing microwave irradiation and a solid carbon monoxide source (Figure 25).

OO O OO S [Pd], Mo(CO)6 S Ar X + H2N R Ar N R Microwaves H X = Br, I Figure 25. The reaction described in Paper III.

4.1 Palladium-Catalyzed Carbonylation In 1974, Heck and co-workers discovered that esters could be obtained from aryl, vinyl or benzyl halides and alcohols under carbon monoxide in the presence of a palladium catalyst.157 Since then, a number of different benzoic acid derivatives have been obtained from aryl halides by carbonylative methods, e.g. aromatic acids, amides, aldehydes and ketones.158 A simplified catalytic mechanism for the formation of aromatic esters or amides from aryl halides can be seen in Figure 26.158 Pd0 forms an oxidative addition complex with the aryl halide. Carbon monoxide is inserted between PdII and the aryl moiety. The acylpalladium complex is attacked by the nucleophile (NuH = RR´NH or ROH) forming the corresponding benzoic acid derivative and Pd0 is regenerated.

49 Figure 26. A simplified catalytic mechanism for carbonylation of aryl halides.

4.2 Carbon Monoxide Sources A serious limitation of carbonylation chemistry is the use of gaseous carbon monoxide. Handling and storing gaseous carbon monoxide is troublesome and a potential safety problem as it is a flammable and highly toxic gas. However, alternative carbon monoxide sources that deliver the gas in situ have been developed, e.g. methyl formate, formamides, aldehydes and metal carbonyls.159 This has enabled a more convenient use of small-scale carbonylation chemistry. Mo(CO)6, a solid carbon monoxide source, has been successfully used in our own laboratory in various carbonylation reactions.160-168

4.3 Microwaves as a Tool in Drug Discovery Heating of chemical reactions has traditionally been performed using Bunsen burners, hot plates or oil baths. In 1986, a new heating technique was introduced into organic chemistry with the presentation of the first examples of microwave-heated organic reactions.169 Since then, the use of microwaves in organic synthesis has increased considerably and nowadays microwave heating is widely used in both the pharmaceutical industry and academia.170 The use of microwaves as a means of accelerating organic transforma- tions has shown tremendous benefits compared to classical heating tech- niques. Significant rate enhancement, improved yield and cleaner reactions are commonly reported advantages of using microwaves. In contrast to the domestic microwave ovens used in the early days of microwave-assisted organic synthesis, today’s microwave reactors are dedicated to organic synthesis and offer stirring of the reaction mixture, explosion-proof cavities and measurement of time, temperature and pressure. Although microwave- irradiated reactions can be performed in open vessels, the majority of reac-

50 tions are performed in sealed vessels. The use of sealed reaction vessels allows a solvent to be heated well above its boiling point, which is sometimes an advantage.170 Microwaves are electromagnetic waves that consist of two components: an electric component and a magnetic component. The electric component is responsible for heating a substance by two different mechanisms, i) dipolar polarization: dipoles try to align with the oscillating electric field causing molecular friction, and ii) ionic conduction: ions follow the electric field resulting in an increased collision rate.170 The ability of a material to absorb microwave energy is determined by the so-called loss tangent value (tan G), which is related to the material’s dielec- tric properties. Based on their tan G values, solvents are classified as high- (tan G>0.5, e.g. ethanol), medium- (tan G 0.1-0.5, e.g. water) or low- (tan G<0.1, e.g. hexane) microwave-absorbing. The use of polar reaction components allows low-absorbing solvents to be used in microwave-heated reactions.170

4.4 Method Development Although one example of the carbonylative synthesis of an aryl acyl sulfonamide had been presented,171 in which gaseous carbon monoxide and conventional heating were employed, we wished to find a fast and easy protocol for the preparation of various aryl acyl sulfonamides. In our initial attempts at the carbonylative synthesis of aryl acyl sulfonamides we examined the conditions previously used for the formation of diacylhydrazines from aryl iodides and hydrazides.162 Consequently, the reaction between various aryl iodides and p-tolylsulfonamide was studied using Pd(OAc)2 as the precatalyst, Mo(CO)6 as the carbon monoxide source, DBU as the base and 1,4-dioxane as the solvent. The reactions were performed under air in sealed vessels with microwave heating at 110 oC for 15 minutes (Table 6). Gratifyingly, both electronically and sterically diverse aryl iodides, as well as heterocyclic iodides, provided the corresponding aryl acyl sulfonamides in good yields (Entries 1–9). To expand the scope of our protocol, we investigated the reaction of p-tolyliodide with various primary sulfonamides, and both aromatic and aliphatic sulfonamides gave satisfactory results (Entries 10–13). Notably, 4-bromobenzenesulfonamide reacted selectively with p- tolyliodide to provide 59k in a good yield (Entry 11). The secondary N- methylbenzenesulfonamide did not provide the desired product in as good a yield as the primary sulfonamides, but 47% of 59n was isolated (Entry 14).

51 Table 6. Microwave- heated carbonylation of aryl iodides with primary and secondary sulfonamides.

OO Pd(OAc)2, Mo(CO)6, O OO S DBU, 1,4-dioxane S Ar I+ H2N R Ar N R Microwaves H 110 oC, 15 min 59a-n Entry Aryl iodide Sulfonamide Product Yield (%) O I OO OO S S 1 H2N N 59a 88 MeO H MeO OO O OO I S S 2 H2N N 59b 87 H

OO O OO I S S 3 H2N N 59c 88 H

OO O OO I S S 4 H2N N 59d 80 H

O OO I OO S S N 5 H2N H 59e 74

O OO I OO S S N Ph H N 6 2 Ph H 59f 70 O O O I OO OO S S N 7 H2N 59g 76 H F3C F3C OO O OO S I S S S 8 H2N N 59h 65 H

S OO O OO S S H N N 9 2 S 59i 79 I H OO O OO I S S 10 H2N N 59j 88 H

OO O OO I S S 11 H2N N 59k 84 H Br Br O OO I OO S 12 S N 59l 72 H2N H

O OO I OO S 13 S N CF3 59m 71 H2N CF3 H

OO O OO I S S 14 HN N 59n 47

Reaction conditions: 0.40 mmol aryl iodide, 3.0 equiv. sulfonamide, 0.10 equiv. Pd(OAc)2, o 1.0 equiv. Mo(CO)6, 3.0 equiv. DBU, 1.0 mL 1,4-dioxane, microwave heating at 110 C for 15 min in sealed vessels.

52 Table 7. Microwave- heated carbonylation of aryl bromides with primary sulfonamides. OO Herrmann's palladacycle, Fu salt, O OO S Mo(CO)6, DBU, 1,4-dioxane S Ar Br+ H2N R Ar N R Microwaves H 140 oC, 15 min 60a-k Entry Aryl bromide Sulfonamide Product Yield (%) O Br OO OO S S 1 H2N N 60a 93 MeO H MeO O Br OO OO S S 2 H2N N 60b 94 OMe H OMe OO O OO Br S S 3 H2N N 60c 91 H

OO O OO Br S S 4 H2N N 60d 93 H

OO O OO Br S S 5 H2N N 60e 95 H

O OO Br OO S S N 6 H2N H 60f 96

O Br OO OO S S N 7 H2N 60g 95 H F3C F3C O Br OO OO S S 8 H2N N 60h 83 NC H NC OO O OO S Br S S S 9 H2N N 60i 79 H

O OO Br OO S 10 S N 60j 88 H2N H

O OO Br OO S 11 S N CF3 60k 80 H2N CF3 H Reaction conditions: 0.40 mmol aryl bromide, 3.0 equiv. sulfonamide, 0.05 equiv. Herrmann’s palladacycle, 0.10 equiv. Fu salt, 1.0 equiv. Mo(CO)6, 3.0 equiv. DBU, 1.0 mL 1,4-dioxane, microwave heating at 140 oC for 15 min in sealed vessels.

The next step was to investigate aryl bromides instead of aryl iodides as coupling partners in our sulfonamidocarbonylation reactions. Aryl bromides are generally less reactive in Pd0-catalyzed reactions and they often require higher temperatures than aryl iodides.172 However, when we used the ther- mostable catalytic combination between Herrmann’s palladacycle173 and Fu salt,174 the aryl bromides provided the desired aryl acyl sulfonamides, 60a–k, in excellent yields when microwave-heated at 140 oC (Table 7). In fact, the

53 aryl bromides provided the desired products in higher yields than the aryl iodides. As for the aryl iodides, various aryl bromides (Entries 1–9) as well as sulfonamides (Entries 10–11) reacted well using this sulfonamidocarbonylation protocol.

4.5 A Medicinal Chemistry Application One important feature of a reaction protocol, especially if to be used in medicinal chemistry projects, is its tolerability to various functional groups. We decided to ascertain if our sulfonamidocarbonylation would work on more complex aryl halides and thus represent a useful reaction protocol. We hypothesized that the usefulness of our carbonylation protocol could be validated by using 61 as the starting aryl bromide since this molecule contains a Boc group, a stereocenter and an acyl sulfonamide group (Scheme 10). If we succeeded in replacing the bromide in 61 with an acidic acyl sulfonamide group, it would be possible to obtain a new type of HCV NS3 protease inhibitor with two distinct acyl sulfonamide groups and it would thus be possible to expand the series of inhibitors presented in Paper I. Thus, the aryl bromide 61 (prepared as described in Scheme 4, Section 3.2.2) served as the starting material in our validation reaction. A slightly modified reaction protocol, where 5 equivalents of methyl sulfonamide was used, afforded the desired product 62 in 52% yield. Although the yield was not as good as for the simpler aryl bromides (Table 7), the outcome of the reaction was still satisfactory. Boc deprotection and peptide coupling to key intermediate 9 (see Scheme 3 in Section 3.2.2) gave compound 63. Notably, no racemization of the Nva D-carbon was observed during the carbonylation reaction, as evidenced by the presence of only one diastereomer of 63.

Scheme 10.

O OO MeSO2NH2 O OO BocHN S Herrmann's palladacycle, Fu salt, BocHN S N N Mo(CO)6, DBU, 1,4-dioxane H H H N Br Microwaves, 140 oC, 15 min S O OO 61O 62 (52%)

1. HCl in 1,4-dioxane O 2. 9, HBTU, DIEA, DMF

N NH O O BocHN O O O HN S 63 (76%)

NH S O O O

54 Compound 63 was subjected to biochemical evaluation in the full-length NS3 protease assay and proved to be a potent HCV NS3 protease inhibitor, with a Ki value of 0.085 PM (Table 8). In comparison to its unsubstituted analogue, 22, inhibitor 63 is almost four times more potent. Inhibitor 64, comprising the bromide instead of the terminal acyl sulfonamide group, was not as potent as 63, suggesting that the terminal acyl sulfonamide group is engaged in interactions with the enzyme (unpublished results). Although 63 displayed a low Ki value in the enzymatic assay, it was not effective in the subgenomic replicon assay (Table 8).

Table 8. Biochemical evaluation of tripeptide HCV NS3 protease inhibitors. O

N NH O O BocHN O O O HN S

R a Compound R Ki ± SD (PM) EC50 (PM) 22 -H 0.30 ± 0.04 >10

63 -CONHSO2Me 0.085 ± 0.007 >10 64 -Br 0.144 ± 0.008 >10 a Mean value of two determinations. SD = standard deviation.

In conclusion, a fast and convenient protocol for the synthesis of aryl acyl sulfonamides has been established. The usefulness of this synthetic protocol was demonstrated by the preparation of an HCV NS3 protease inhibitor.

55 5 A Novel Aromatic P1 Moiety in HCV NS3 Protease Inhibitors (Paper IV)

The extensive search for HCV NS3 protease inhibitors has generated a vast number of potent compounds. However, the majority of inhibitors revealed so far rely on a scaffold comprising D-amino acids, i.e. a peptide scaffold (see Section 1.2.3). It is well known that peptides suffer from deficiencies that limit their use as drugs, e.g. rapid metabolic degradation and poor oral bioavailability.175 In our endeavor to reduce the peptide character of HCV NS3 protease inhibitors we decided to investigate if the D-amino acid in P1 could be replaced by a non-natural aromatic moiety (Figure 27). Such a substitution would not only reduce the peptide character of the compounds, but would also enable the use of synthetic methods not applicable to non- aromatic moieties. In addition, a structural modification like this would generate a new class of compounds that could ultimately exhibit a resistance profile different from that of inhibitors comprising D-amino acids in P1. Considering the crucial interactions between the inhibitor’s P1 C-terminal functionality and the enzyme active site, we predicted that acidic substituents on the aromatic moiety would be favorable. Hence, we aimed at synthesizing inhibitors comprising an aromatic P1 moiety substituted either with a carboxylic acid or an acyl sulfonamide, as illustrated in Figure 27.

O O

N OH NH O BocHN O O R R: O O S N R' H Figure 27. The scaffold used for the evaluation of inhibitors comprising a non- natural aromatic P1 moiety.

56 5.1 Chemistry Once again, the dipeptide 3398 (see Section 3.3.1) served as the starting point for inhibitor synthesis. Preparation of inhibitors 65–67, comprising the aminobenzoic acid fragment, required coupling of dipeptide 33 with the ester- protected aminobenzoic acid moiety (Scheme 11). Several different coupling conditions were evaluated and a protocol utilizing HATU as the activating agent in combination with DIEA as base was found to be optimal. Notably, the use of CH2Cl2 as the solvent was crucial since the reactions failed when DMF was used instead. Coupling was performed under dry conditions at 45 oC, which provided the desired ortho-, meta- and para-products 65–67 in reasonable to good yields (Scheme 11).

Scheme 11.

O O O

N N N

O O O O H2N OR LiOH, THF, n=0,1 MeOH, H2O N N N OH HATU, DIEA, NH NH CH Cl , 45 oC BocHN O O 2 2 BocHN O O O BocHN O O O OR OH n=0,1 n=0,1 33 65, n=0, ortho (45%) 69, n=0, ortho (78%) 66, n=0, meta (92%) 70, n=0, meta (71%) 67, n=0, para (81%) 71, n=0, para (72%) 68, n=1, ortho (80%) 72, n=1, ortho (90%)

The same conditions were used for the preparation of compound 68, but 2- aminophenylacetic acid methyl ester176 was used as the coupling partner. The esters 65–68 were then hydrolyzed to yield the desired carboxylic acids 69– 72 (Scheme 11). For the synthesis of the inhibitors containing the acyl sulfonamide group we relied on our sulfonamidocarbonylation protocol, presented in Paper III. Consequently, the aryl bromides 73–77 had to be prepared (Scheme 12). For the coupling of 2-, 3- or 4-bromoaniline to dipeptide 33, the protocol described above based on HATU was found successful, and compounds 73–75 were obtained in good yields. However, 2-bromo-6-methylaniline and 2- bromo-5-(trifluoromethyl)aniline failed to couple to 33 under the same conditions. Thus, these more demanding anilines required different coupling conditions, and a method developed for the coupling of the weak nucleophile para-nitroaniline to carboxylic acids was investigated.177 This method, employing the use of POCl3 in pyridine, was found very successful for the preparation of compounds 76 and 77 (Scheme 12).

57 Scheme 12.

O O

N N

O RSO NH O Br 2 2 H2N Herrmann's palladacycle, Fu salt, Mo(CO) , DBU, 6 O N 1,4-dioxane N O O NH NH S R HATU, DIEA, Br Microwaves N o BocHN O O o BocHN O O CH2Cl2, 45 C 140 C, 15 min H 73, ortho (77%) 78, ortho, R=phenyl (35%) 74, meta (63%) 79, meta, R=phenyl (44%) 75, para (82%) 80, para, R=phenyl (35%) 81, ortho, R=methyl (50%) 82, ortho, R=cyclopropyl (62%) 83, ortho, R=benzyl (56%) 84, ortho, R=4-methoxyphenyl (43%) 85, ortho, R=4-(trifluoromethyl)phenyl (42%) 33 86, ortho, R=2-thienyl (37%)

O O

Br N N H2N PhSO2NH2 O Herrmann's palladacycle, O R1 O Fu salt, Mo(CO)6, DBU, R O S Ph 2 1,4-dioxane O N Br N H NH POCl3, pyridine, NH Microwaves N -15 oC to rt 140 oC, 15 min BocHN O O BocHN O O

R1 R1

76, R1=CH3, R2=H (76%) 87, R1=CH3, R2=H (45%) R2 R2 77, R1=H, R2=CF3 (88%) 88, R1=H, R2=CF3 (35%)

The aryl bromides 73–77 then served as starting materials for the sulfonamidocarbonylation process (Scheme 12). The target compounds 78–88 were obtained in reasonably good yields (35-62%), which further exemplifies the usefulness of this carbonylation protocol.

5.2 Structure–Activity Relationship The compounds 69–75 and 78–88 were biochemically evaluated in both the enzymatic assay (Ki values) and the cell-based replicon assay (EC50 values) and the results are presented in Tables 9 and 10. Examining the inhibitory potencies of the ortho-, meta- and para- carboxylic acids 69–71 reveals that they are poor inhibitors of the NS3 protease with Ki values of 5.0, 6.8 and 12 PM, respectively (Table 9). Although the inhibitors harbor a C-terminal carboxylic acid, it appears that they do not interact optimally with the active site of the protease. An attempt to introduce a spacer group between the aromatic moiety and the carboxylate gave compound 72, which was even worse at inhibiting the protease. The fact that the aryl bromides 73–75 are more potent than the carboxylic acid inhibitors,

58 69–72, suggests that the carboxylate is not engaged in interactions with the protease. However, introduction of the acyl sulfonamide group instead of the carboxylic acid yielded submicromolar inhibitors (compounds 78–80, Table 9). From docking studies of inhibitor 78 in the NS3 protease binding site it was suggested that the aromatic P1 moiety was positioned above Phe154 of the enzyme which constitutes the bottom of the S1 pocket (Figure 28). More- over, it was found that the aromatic carbonyl group did not occupy the 95 oxyanion hole, as it does in inhibitors with D-amino acids in P1. The lack of interactions with the oxyanion hole could explain the low potency observed for the carboxylic acid inhibitor 69. On the other hand, it appears that both sulfonamide oxygens in 78 are engaged in interactions with the side chains of Lys136 and Gln41. These interactions, together with possible interactions between the sulfonamide substituent and the protease, may explain why the acyl sulfonamide 78 is six times more potent than the carboxylic acid 69.

Table 9. Biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P1 moiety. O

N NH R BocHN O O

a Compound R Position Ki ± SD (PM) EC50 (PM) 69 -COOH ortho 5.0 ± 1.5 >2 70 -COOH meta 6.8 ± 2.5 >2 71 -COOH para 12 ± 5 >2

72 -CH2COOH ortho 17 ± 5 >10 73 -Br ortho 3.9 ± 0.6 >10 74 -Br meta 1.4 ± 0.3 >10 75 -Br para 1.8 ± 0.3 >10

78 -CONHSO2Ph ortho 0.83 ± 0.11 >2

79 -CONHSO2Ph meta 0.78 ± 0.15 >2

80 -CONHSO2Ph para 1.1 ± 0.2 >2 a Mean value of two determinations. SD = standard deviation.

59 Figure 28. Compound 78 docked in the NS3 protease binding site of the full-length NS3 protein.78 The enzyme residues Gln41, Lys136 and Phe154 are displayed as sticks. The aromatic P1 moiety of 78 is positioned above the aromatic ring of Phe154 and the sulfonamide oxygens are hydrogen-bound to the side chains of Gln41 and Lys136.

Although the ortho-, meta- and para-acyl sulfonamides were equipotent inhibitors, we decided to further investigate the ortho-compounds based on molecular modeling studies. A small series of inhibitors with different sulfonamide substituents were prepared (compounds 81–86, Table 10). Com- pared with our starting inhibitor 78, comprising a phenyl substituent, inhibitor 81 with a methyl substituent exhibited a significantly lower Ki value. The cyclopropyl group has been widely used as an acyl sulfonamide substituent in various HCV NS3 protease inhibitors (see Figures 13 and 14 in Section 1.2.3) and provided the extremely potent inhibitor 58 (see Table 4 in Section 3.3.2). Interestingly, compound 82, comprising the cyclopropyl group, was not a very potent inhibitor, with a Ki value of 2.0 PM. This could reflect a different binding of the acyl sulfonamide group in inhibitors with an aromatic P1 moiety compared with inhibitors containing an D-amino acid in P1, as was indicated by our modeling studies. The benzyl group in compound 83 provided an inhibitor slightly more potent than 78. Various aromatic substituents were evaluated, compounds 84–86, and the introduction of a 4- (trifluoromethyl)phenyl substituent provided the most potent inhibitor, 85, with a Ki value of 0.31 PM.

60 Although the aromatic P1 moiety has the ability to interact with Phe154, it lacks a P1 side chain. Therefore, we hypothesized that the introduction of additional substituents on the aromatic moiety could be beneficial, filling the S1 pocket more optimally. A methyl group ortho to the aniline nitrogen, as in compound 87, was not helpful, but a trifluoromethyl group para to the acyl sulfonamide group, as in compound 88, was useful. Compared with the unsubstituted analogue 78, compound 88 is almost three times more potent in the enzyme assay, and it also showed some effect in the replicon assay (EC50=5.2 PM).

Table 10. Biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P1 moiety. O

O O O S R3 O N NH NH BocHN O O

R2 a Compound R1 R2 R3 Ki ± SD (PM) EC50 (PM)

78 -H -H 0.83 ± 0.11 >2

81 -H -H 5.9 ± 1.1 >2 82 -H -H 2.0 ± 0.3 >2

83 -H -H 0.61 ± 0.09 >10 O 84 -H -H 1.2 ± 0.2 >10

CF3 85 -H -H 0.31 ± 0.05 >10

S 86 -H -H 0.92 ± 0.15 >10

87 -CH3 -H 3.2 ± 0.4 >10

88 -H -CF3 0.35 ± 0.05 5.2 a Mean value of two determinations. SD = standard deviation.

In summary, we have been able to introduce a novel aromatic P1 moiety into HCV NS3 protease inhibitors. Encouragingly, a diverse SAR was found both for the sulfonamide substituent and for the substituent on the aromatic P1 moiety. These inhibitors represent a new class of promising compounds that can be used as lead compounds for further optimization.

61 6 Concluding Remarks

The work presented in this thesis has provided knowledge about HCV NS3 protease inhibitors and their structure–activity relationships. The utility of the acyl sulfonamide group has been demonstrated in various molecular scaffolds. The major results and conclusions are summarized below.

x Several functional groups have been evaluated as replacements for the P1 C-terminal carboxylic acid in HCV NS3 protease inhibitors. The acyl sulfonamide is superior to all the groups examined, but the acyl cyanamide and the acyl sulfinamide groups were identified as new carboxylic acid bioisosteres.

O O O O O O O N HN S HN CN HN S NH O BocHN O O Ki = 6 nM OH

Ki = 0.22 nM Ki = 9 nM Ki = 20 nM

x A thorough study of the structure–activity relationship of acyl sulfonamide based HCV NS3 protease inhibitors has been performed.

> Both oxygens P3 N important NH O O BocHN O O O P HN S 1 R Substituent important >> NH important

62 x The advantages of the acyl sulfonamide group compared to the carboxylic acid in HCV NS3 protease inhibitors have been demonstrated in both enzymatic and cell-based assays.

N NH O O O BocHN O O O OH HN S

Ki = 20 nM Ki = 0.058 nM EC50 = 140 nM EC50 = 0.9 nM x A convenient and robust carbonylative method for the preparation of aryl acyl sulfonamides has been presented. This protocol provides aryl acyl sulfonamides in excellent yields from both aryl iodides and aryl bromides.

OO O OO S [Pd], Mo(CO)6 S Ar X + H2N R Ar N R Microwaves H x The first account of HCV NS3 protease inhibitors comprising a non-natural aromatic P1 moiety has been reported. These inhibitors represent a new class of more drug-like inhibitors that may serve as lead compounds in future optimization processes.

O O O S CF3 N O NH NH BocHN O O

Ki = 310 nM P1 moiety

63 7 Acknowledgements

I would like to express my sincere gratitude to the following people:

Dr. Anja Sandström, my co-supervisor. You are always in a good mood and have always taken time to discuss my problems. It has been a true pleasure working closely with you and your help has been invaluable!

Prof. Anders Hallberg, my supervisor, for accepting me as a PhD student. Your vast knowledge in medicinal chemistry and your extraordinary ability to inspire and enthuse others has made these past years very instructive and enjoyable.

Dr. Eva Åkerblom, my “third supervisor”, for all our discussions regarding synthesis and inhibitor-potencies, and for keeping track of and organizing all the HCV-related patents. It is just a matter of time before we find our low- nanomolar inhibitors!

Dr. Mats Larhed and Dr. Xiongyu Wu, for good collaboration. You introduced me to the world of palladium catalysis and taught me what effective- ness is.

The computer team, Dr. Yogesh Sabnis, Dr. Anders Karlén and Shane Peter- son, for fruitful discussions regarding docking solutions, conformations and hydrogen bonds, and for your contributions to our papers. The biochemists, Thomas Gossas and Prof. Helena Danielson, for nice collaborations and your willingness to test my compounds. The staff at Medivir for fruitful discussions.

My MSc students, Hanna Daoud and Anna Lampa – you did a great job.

Gunilla Eriksson, for always taking care of everything! Sorin Srbu for quick and effective help with all my computer-related problems. Dr. Uno Svensson for answering all my nomenclature questions.

All the past and present members of the HCV group: Anja, Eva, Yogesh, Pernilla, Johanna, Shane, Anders H, Anders K and Anna.

64 Anja, Eva, Jonas L, Pernilla, Anna and Jonas S, for constructive criticism and comments on my thesis, and Helen Sheppard for outstanding linguistic revision of the text.

My room- and lab-mates, and all past and present colleagues at the depart- ment, especially, PA, Máté, Charlotta, Johan W, Karl V, Jennie G, Riina, Olle, Gopal, Hanna, Daniel M, Peter, Anna A and Ulrika R.

The LC-MS team, especially Gunnar Lindeberg and Anna Arefalk for teach- ing me the instruments.

Mr. Martin “Bobby Colby” Granlund for all your help with the figures. They are spectacular!

Mr., soon to be Dr., Christian Sköld. To have such a close friend in the pro- fessional world is a real privilege! Our common interest in chemistry has kept us together for, I don’t know how long. The past months have only been half as difficult as I imagined because I was able to share my anxiety with you. Thanks!

My sporting partners Jonas S, Jonas L and Johan G: skiing, cycling, running, snowmobiles (damn, that’s an expensive sport) and so on. Invaluable time! Sävmarker, many great moments, and our discussions regarding lap times, tire choice and suspension settings are of the utmost importance!

The Swedish Research Council (VR) and the Swedish Foundation for Stra- tegic Research (SFF) are acknowledged for financial support of this project. Anna Maria Lundin’s Stipendiefond is acknowledged for making my atten- dance at international conferences possible.

Det finns ju en massa människor utanför BMC som förgyller mitt liv också.

Mina underbara föräldrar för att ni alltid har stöttat mig!

Mina härliga systrar Sussi och Ewa, för att ni ordnade så att jag fick Tomi, Martin, Jocke, Julia och Kasper.

Yvonne, Börje, Veronica, Thomas, Basse och Wille.

New gang: Jimmy, Christian, Petter, Pelle, Jon och Ivar, Old gang: Jocke, Håkan, Anders, Kjell och Håkan, och alla brudarna. Tack för allt!

Camilla, min solstråle, jag älskar dig! Robert 070323

65 8 References

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A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy”.)

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