<p>This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an <em>N</em>-acyl-(4<em>R</em>)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated.</p> / Report code: LIU-TEK-LIC-2006:46.

Hepatitis C is a blood-borne disease affecting 130-170 million people worldwide. The causative agent, hepatitis C virus (HCV), infects the liver and is the major reason for chronic liver disease worldwide. The HCV NS3 protease, a key enzyme in the virus replication cycle, has been confirmed to be an important target for drug development. With the recent release of two HCV NS3 protease inhibitors onto the market and an arsenal of inhibitors in clinical trials, there are now hopes of finally combating the disease. However, the success of treatment relies heavily on the ability to overcome the emergence of drug-resistant forms of the protease. The main focus of this thesis was on designing and synthesizing novel inhibitors of the NS3 protease with a unique resistance profile. Efforts were also made to decrease the peptide character of the compounds, with the long-term goal of making them into more drug-like compounds. Special attention was devoted to developing inhibitors based on a phenylglycine in the P2 position, instead of the highly optimized and commonly used P2 proline. Around ninety acyclic and macrocyclic inhibitors have been synthesized and biochemically evaluated. P2 pyrimidinyloxy phenylglycine was successfully combined with an aromatic P1 moiety and alkenylic P1´ elongations, yielding a distinct class of HCV NS3 protease inhibitors. Macrocyclization was performed in several directions of the inhibitors via ring-closing metathesis. Only the macrocyclization between the P3-P1´ residues was successful in terms of inhibitory potency, which suggests that the elongated P1-P1´ residue is oriented towards the P3 side chain. The metathesis reaction was found to be significantly more dependent on the substrate than on the reaction conditions. It was also found that the P3 truncated inhibitors were able to retain good inhibitory potency, which initiated the synthesis and evaluation of a series of P2-P1´ inhibitors. The potential of the P3-P1´cyclized inhibitor and the smaller, acyclic P2-P1´ as new potential drug leads remains to be determined through pharmacokinetic profiling. Gratifyingly, all the inhibitors evaluated on A156T and D168V substituted enzyme variants were able to retain inhibitory potency towards these as compared to wild-type inhibition.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated. / Report code: LIU-TEK-LIC-2006:46.

Hepatitis C is a global health problem that affects approximately 120–180 million people. This viral infection causes serious liver diseases and the therapy available suffers from low efficiency and severe side effects. Consequently, there is a huge unmet medical need for new therapeutic agents to combat the hepatitis C virus (HCV). Inhibition of the viral NS3 protease has recently emerged as a promising approach to defeat this infection, and the first HCV NS3 protease inhibitors have now entered clinical trials. In this project, several novel HCV NS3 protease inhibitors have been designed, synthesized and biochemically evaluated. Inhibitors with various P1 C-terminal functional groups intended as potential bioisosteres to the carboxylic acid found in product-based inhibitors have been revealed. Special focus has been placed on establishing structure–activity relationships of inhibitors containing the promising P1 C-terminal acyl sulfonamide group. The properties of the acyl sulfonamide functionality that are important for producing potent inhibitors have been identified. In addition, the advantages of the acyl sulfonamide group compared to the carboxylic acid have been demonstrated in both enzymatic and cell-based assays. Besides the acyl sulfonamide functionality, the acyl cyanamide and the acyl sulfinamide groups have been identified as new carboxylic acid bioisosteres in HCV NS3 protease inhibitors. The synthetic work included the development of a fast and convenient methodology for the preparation of aryl acyl sulfonamides. The use of microwave heating and Mo(CO)6 as a solid carbon monoxide source provided aryl acyl sulfonamides from aryl halides in excellent yields. This method was subsequently used in the decoration of novel HCV NS3 protease inhibitors comprising a non-natural P1 moiety. This new class of compounds can be used as lead structures in a future optimization process aimed at producing more drug-like HCV NS3 protease inhibitors.

The hepatitis C virus (HCV), with a global prevalence of roughly 2%, is among the most serious diseases today. Among the more promising HCV targets is the NS3 protease, for which several drug candidates have entered clinical trials. In this work, computational methods have been developed and applied to the design of inhibitors of the HCV NS3 protease. Comparative molecular field analysis (CoMFA) modeling and molecular docking are the two main computational tools used in this work. CoMFA is currently the most widely used 3D-QSAR method. Methodology for improving its predictive performance by evaluating 6120 combinations of non-default parameters has been developed. This methodology was tested on 9 data sets for various targets and found to consistently provide models of enhanced predictive accuracy. Validation was performed using q2, r2pred and response variable randomization. Molecular docking was used to develop SARs in two series of inhibitors of the HCV NS3 protease. In the first series, preliminary investigations indicated that replacement of P2 proline with phenylglycine would improve potency. Docking suggested that phenylglycine-based inhibitors may participate in two additional interactions but that the larger, more flexible phenylglycine group may result in worse ligand fit, explaining the loss in potency. In the second series, β-amino acids were explored as α-amino acid substitutes. Although β-amino acid substitution may reduce the negative attributes of peptide-like compounds, this study showed that β-amino acid substitution resulted in reduced potency. The P3 position was least sensitive to substitution and the study highlighted the importance of interactions in the oxyanion hole. Finally, docking was used to provide the conformations and alignment necessary for a CoMFA model. This CoMFA model, derived using default settings, had q2 = 0.31 and r2pred = 0.56. Application of the optimization methodology provided a more predictive model with q2 = 0.48 and r2pred = 0.68.

Infection by the hepatitis C virus (HCV) leads to inflammation of the liver, i.e. hepatitis. The acute infection often progresses to a chronic phase during which the liver function is gradually impaired. Approximately 20% of these chronic cases develop liver cirrhosis, with an ensuing increased risk of liver cancer. Global estimates of the total number of chronic cases range from 123–170 million. Yet, neither specific anti-HCV drugs nor vaccines are available. When drugs become available for daily clinical use, rapid development of drug-resistant strains is expected, making resistance an important issue. One of the most studied targets for specific anti-HCV drugs is the NS3 protease. The main objectives of the work presented in this thesis were to design and synthesise peptidomimetic inhibitors of this enzyme, and to establish the structure–activity relationships (SARs) regarding the inhibition of the wild type as well as of the known resistant variants A156T and D168V. Substituted prolines are common P2 residues in HCV NS3 protease inhibitors. To decrease the peptide character of the inhibitors, the non-coded phenylglycine was evaluated as a proline replacement in combination with known and novel P3 and P1 residues and P2 substituents. The results confirmed that phenylglycine is a promising P2 scaffold, with a possible π-stacking interaction with histidine 57 of the active site. However, to benefit from its full potential, additional optimisation is required. A 2(1H)-pyrazinone-based scaffold was introduced as P3 residue. Utilising the scope of the method developed for the pyrazinone scaffold synthesis, the phenylglycine side-chain was transferred to the scaffold. In combination with an aromatic P1 building-block, this design yielded achiral, peptidomimetic inhibitors, three times more potent than the tripeptide lead. The SARs for the inhibition of the resistant variants A156T and D168V were investigated for compounds based on either P2 proline or phenyl­glycine. It was concluded that the vulnerability of the inhibitors to alterations in the enzyme depends on the P2 and the P1 residue, not only on the P2 as previously suggested. These results provide important information for the design of a new generation of inhibitors with improved properties.

This thesis focuses on the design and synthesis of inhibitors targeting Mycobacterium tuberculosis ribonucleotide reductase (RNR) and hepatitis C virus (HCV) NS3 protease; enzymes that have been identified as potential drug targets for the treatment of tuberculosis and hepatitis C, respectively. Small peptides have been recognized as inhibitors of these enzymes. However, the use of peptides as drugs is limited due to their unfavorable properties. These can be circumvented by the development of less peptidic molecules, often referred to as peptidomimetics. When this work was initiated, only a few inhibitors targeting M. tuberculosis RNR had been identified, whereas the HCV NS3 protease was an established drug target. Therefore, early peptidomimetic design strategies were applied to inhibitors of RNR while the NS3 protease inhibitors were subjected to modifications in a later stage of development. It has previously been shown that peptides derived from the C-terminus of the small subunit of M. tuberculosis RNR can compete for binding to the large subunit, and thus inhibit enzyme activity. To investigate the structural requirements of these inhibitors, different series of peptides were evaluated. First, peptides from an N-terminal truncation, an alanine scan and a designed library were synthesized and evaluated to examine the importance of the individual amino acid residues. Then, a set of N-terminally Fmoc-protected peptides was evaluated, and it was found that the N-terminal group improved the affinity of the peptides even when the length of the compounds was reduced. Furthermore, potential inhibitors of less peptidic character were generated by the introduction of a benzodiazepine-based scaffold. To further reduce the peptidic character and investigate the binding properties of HCV NS3 protease inhibitors, a series of tripeptides incorporating a β-amino acid was synthesized. Inhibition was evaluated and docking studies were performed to understand how the structural changes affected inhibitory potency. The results illustrated the importance of preserving the hydrogen bonding network and retaining electrostatic interactions in the oxyanion hole between inhibitor and protein.

Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes. The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities. Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.

Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions.

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