Synthesis and Antiviral Properties of Novel Tetracyclic Nucleoside Inhibitors of Hepatitis C NS5B Polymerase

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2′-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC₅₀ = 0.1 μM, a level of intrinsic potency similar to that of previous and current clinical candidates.     

Identification of [(naphthalene-1-carbonyl)-amino]-acetic acid derivatives as nonnucleoside inhibitors of HCV NS5B RNA dependent RNA polymerase

A novel series of HCV NS5B RNA dependent RNA polymerase inhibitors containing a naphthalene carboxamide scaffold were identified by high throughput screening. Optimization of substituents by parallel synthesis and the iterative design towards understanding structure-activity relationship to improve potency are described. Tetra substituted naphthalene 31 displayed potent activity with IC(50) of 120 nM against HCV NS5B enzyme and was selective over a panel of polymerases.     

Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate

The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher K(m)) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of productive RNA binding. The characterization of specific benzimidazole-5-carboxamide-based inhibitors, identified in a screening campaign, revealed that this class of compounds was non-competitive with regard to NTP incorporation and had no effect on processive elongation, but inhibited an initiation phase of the HCV polymerase activity. The potency of these compounds versus a panel of different NS5B polymerase constructs was inversely proportional to the enzymes' affinities for template/primer substrate. The benzimidazole-5-carboxamide compounds also inhibited the full-length, untagged NS5B de novo initiation reaction using HCV 3'-UTR substrate RNA and expand the diversifying pool of potential HCV replication inhibitors.     

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.     

Potent bisimidazole-based HCV NS5A inhibitors bearing annulated tricyclic motifs

This Letter describes the synthesis and biological evaluation of a number of functionalized bisimidazoles bearing annulated tricyclic motifs as potent inhibitors of HCV NS5A protein. Compound 4h, which contains a substituted tricyclic 6-6-6 xanthene, demonstrated broad genotypic spectrum, compelling potency, and good oral bioavailability with dose-dependent drug exposure level in multiple animal species.     

Structure-based virtual screening,synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening, synthesis and structure–activity relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1), sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for productive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC₅₀ values ranging from 7.7 to 68.0 μM were developed. Docking analysis of lead compound 28 within the tetracyclic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed topological similarities between these two pockets. Compound 28, a novel rhodanine analog with NS5B inhibitory potency in the low micromolar level range may be a promising lead for future development of more potent NS5B inhibitors.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides

The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in Huh-7 cells.     

Identification of thieno[3,2-b]pyrroles as allosteric inhibitors of hepatitis C virus NS5B polymerase

Thieno[3,2-b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently blocks subgenomic HCV RNA replication in HUH-7 cells with an EC50 of 2.9 microM.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-1-benzyl and N-1-[3-methylbutyl]-4-hydroxy-1,8-naphthyridon-3-yl benzothiadiazine analogs containing substituents on the aromatic ring

A series of non-nucleoside HCV NS5B polymerase inhibitors based on the N-1-benzyl or N-1-[3-methylbutyl]-4-hydroxy-1,8-naphthyridon-3-yl benzothiadiazine core substituted in the D-ring aromatic moiety have been prepared and evaluated. Aromatic substituents extending from position 7 of the D-ring exhibited excellent potency against both genotypes 1a and 1b.     

Identification of aryl dihydrouracil derivatives as palm initiation site inhibitors of HCV NS5B polymerase

Aryl dihydrouracil derivatives were identified from high throughput screening as potent inhibitors of HCV NS5B polymerase. The aryl dihydrouracil derivatives were shown to be non-competitive with respect to template RNA and elongation nucleotide substrates. They demonstrated genotype 1 specific activity towards HCV NS5B polymerases. Structure activity relationships and genotype specific activities of aryl dihydrouracil derivatives suggested that they bind to the palm initiation nucleotide pocket, a hypothesis which was confirmed by studies with polymerases containing mutations in various inhibitor binding sites. Therefore, aryl dihydrouracil derivatives represent a novel class of palm initiation site inhibitors of HCV NS5B polymerase.     

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.     

Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors

Hepatitis C virus (HCV) is a major health burden, with an estimated 170 million chronically infected individuals worldwide, and a leading cause of liver transplantation. Patients are at increased risk of developing liver cirrhosis, hepatocellular carcinoma and even liver failure. In the past two decades, several approaches have been adopted to inhibit non-structural viral proteins. The RNA-dependent RNA polymerase (NS5B) of HCV is one of the attractive validated targets for development of new drugs to block HCV infection. In this review, we report the recent progress made towards identifying and developing benzothiadiazines as HCV NS5B polymerase inhibitors. The substituted benzothiadiazine class was identified by HTS in 2002 as an NS5B inhibitor. Further optimization and modification of the core has improved the potency and pharmacokinetic properties of substituted benzothiadiazines. Research on palm site-binding benzothiadiazine analogs and related derivatives and analogs is discussed in this article.     

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.     

Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid

A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.     

Substituted benzothiadizine inhibitors of Hepatitis C virus polymerase

The synthesis and optimisation of HCV NS5B polymerase inhibitors with improved potency versus the existing compound 1 is described. Substitution in the benzothiadiazine portion of the molecule, furnishing improvement in potency in the high protein Replicon assay, is highlighted, culminating in the discovery of 12h, a highly potent oxyacetamide derivative.     

Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics

The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC(50)=79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.     

Design and evaluation of novel tetracyclic benzofurans as palm site allosteric inhibitors of HCV NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B non-nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and evaluated. Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and 2) and C-linked (compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-replicon potency profiles and demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters across the three preclinical animal species. To better understand the importance of tetracyclic structures related to pan genotypic potency profiles especially against clinically relevant GT1a variants, the teracycles with different ring size were prepared and in vitro evaluations suggested compounds with six number ring have better overall potency profiles.