Thiazolides as novel antiviral agents. 1. Inhibition of hepatitis B virus replication

We report the syntheses and activities of a wide range of thiazolides [viz., 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide, NTZ] 1 is a broad spectrum antiinfective agent effective against anaerobic bacteria, viruses, and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent, and selective inhibitor of hepatitis B replication (EC(50) = 0.33 μm) but is inactive against anaerobes. Several 4'- and 5'-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1; viz., the O-acetate is an effective prodrug, and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC(90) for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.     

Benzimidazole derivatives bearing substituted biphenyls as hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors: structure-activity relationship studies and identification of a potent and highly selective inhibitor JTK-109

Following the discovery of a new series of benzimidazole derivatives bearing a diarylmethyl group as inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase (HCV NS5B RdRp),1,2 we extended the structure-activity relationship (SAR) study to analogues bearing a substituted biphenyl group and succeeded in a significant advancement of activity. Starting from compound 1, optimization of the A, B, and C rings afforded potent inhibitors with low nanomolar potency against genotype 1b NS5B. The compounds, which have a substituent with a carbonyl function at the 4-position of the B-ring, efficiently blocked subgenomic viral RNA replication in the replicon cell assay at low submicromolar concentrations. Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.     

Synthesis and pharmacokinetics of valopicitabine (NM283), an efficient prodrug of the potent anti-HCV agent 2'-C-methylcytidine

In our search for new therapeutic agents against chronic hepatitis C, a ribonucleoside analogue, 2'-C-methylcytidine, was discovered to be a potent and selective inhibitor in cell culture of a number of RNA viruses, including the pestivirus bovine viral diarrhea virus, a surrogate model for hepatitis C virus (HCV), and three flaviviruses, namely, yellow fever virus, West Nile virus, and dengue-2 virus. However, pharmacokinetic studies revealed that 2'-C-methylcytidine suffers from a low oral bioavailability. To overcome this limitation, we have synthesized the 3'-O-l-valinyl ester derivative (dihydrochloride form, valopicitabine, NM283) of 2'-C-methylcytidine. We detail herein for the first time the chemical synthesis and physicochemical characteristics of this anti-HCV prodrug candidate, as well as a comparative study of its pharmacokinetic parameters with those of its parent nucleoside analogue, 2'-C-methylcytidine.     

Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope

In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.     

2-(2-Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as inhibitors of the hepatitis C virus NS5B polymerase: discovery, SAR, modeling, and mutagenesis

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently disclosed dihydroxypyrimidine carboxylates 2 as novel, reversible inhibitors of the HCV NS5B polymerase. This series was further developed into 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acids such as 34 (EC50 9.3 microM), which now show activity in the cell-based HCV replication assay. The structure-activity relationship of these inhibitors is discussed in the context of their physicochemical properties and of the polymerase crystal structure. We also report the results of mutagenesis experiments which support the proposed binding model, which involves pyrophosphate-like chelation of the active site Mg ions.     

丙型肝炎病毒NS5A抑制剂——达卡他韦

达卡他韦(Daklinza)是由美国百时美-施贵宝公司(BMS)研制的口服丙型肝炎病毒(HCV)NS5A抑制剂。继2014年欧盟批准其与联合其他抗病毒药用于基因型1-4慢性丙型肝炎成人患者的治疗后,2015年7月美国FDA批准其与索非布韦[sofosbuvir]联合用于慢性HCV基因型3感染的治疗。达卡他韦是第一个无需同时给予干扰素或利巴韦林即可有效治疗基因型3 HCV感染的药物,为该类患者提供了一个新选择,包括那些不能耐受利巴韦林患者。笔者就达卡他韦的基本信息、作用机制、药代动力学、药物相互作用、临床试验及应用等研发动态作一概述,以期能为医院临床用药及药物研究开发提供参考。     

Synthesis of new benzimidazole-coumarin conjugates as anti-hepatitis C virus agents

Nineteen new conjugated compounds were successfully synthesized by a one-flask method from benzimidazole and coumarin derivatives. A methylenethio linker was used to connect these two kinds of derivatives. In addition, substituted benzimidazol-2-thiones were also coupled with beta-D-glucose peracetate; the resultant glucosides were further converted to the corresponding 2-(methylthio)coumarin derivatives. Their activity against the hepatitis C virus was tested; two of the most potent compounds 2-[(6'-bromocoumarin-3'-yl)methylenethio]-5-fluorobenzimidazole (4i) and its derivative 1-[(2',3',4',6'-tetra-O-acetyl)glucopyranos-1'-yl]-2-[(6'-bromocoumarin-3'-yl)methylenethio]benzimidazole (7c) showed EC(50) values of 3.4 microM and 4.1 microM, respectively. At a concentration of 5.0 microM, compound 7c inhibited HCV RNA replication by 90% and had no effect on cell proliferation. Given these data, a structure-activity relationship was established.     

Application of the phosphoramidate ProTide approach to 4'-azidouridine confers sub-micromolar potency versus hepatitis C virus on an inactive nucleoside

We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4'-azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4'-Azidouridine did not inhibit HCV, although 4'-azidocytidine was a potent inhibitor of HCV replication under similar assay conditions. However 4'-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively deliver 4'-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy.     

治疗慢性丙型肝炎新药Sofosbuvir

鉴于丙型肝炎病毒（HCV）感染全球高发趋势,目前治疗方案因禁忌、严重不良反应等导致其使用的局限性,因此需要一种更有效、安全、简便、不适宜干扰素治疗的治疗药物.Sofosbuvir是具有这些特性的直接抗病毒药物,为HCV NS5 B聚合酶的尿苷核苷酸类似物抑制药,可有效对抗多种基因型HCV感染,具有良好的安全性和耐受性.本文综述Sofosbuvir的作用机制、药动学、不良反应、药物相互作用及临床试验.     

Hepatitis C virus therapy: No one will be left behind

The advent of oral direct-acting antiviral agents (DAAs) has dramatically improved the hepatitis C treatment landscape in the last 4 years, providing cure rates over 95% with shorter duration of treatment and a very good safety profile. This gave access to treatment to almost all Hepatitis C virus (HCV)-infected patients. The launch of two pangenotypic fixed-dose combinations (FDCs) in 2017 was a step forward in hepatitis C treatment, by slightly increasing efficacy and more importantly allowing the treatment of patients without HCV genotyping, and in some cases without fibrosis assessment. New triple regimens have solved the issue of retreatment of the few patients who present failure to DAAs therapy. In the present review we describe the current HCV landscape that allows almost all HCV-infected patients to be cured.     

Novel nucleoside analogues targeting HCV replication through an NS5A‐dependent inhibition mechanism

A series of new tricyclic nucleosides were synthesized and evaluated as hepatitis C virus (HCV) replication inhibitors. Initial screening in a HCV replicon system, derived from a genotype 1b isolate, identified 9‐benzylamino‐3‐(β‐D‐ribofuranosyl)‐3H‐imidazo[4′,5′:5,6]pyrido[2,3‐b]pyrazine ( 15d ) as the most potent analogue. Comparative assessment of 15d activity against HCV full‐length viruses or subgenomic replicons derived from genotypes 1 to 4 revealed a specificity of the compound for genotypes 1 and 3. Surprisingly, resistance mutations selected against 15d were mapped to domains II and III of the non‐structural protein 5A (NS5A), but not to the RNA‐dependent RNA polymerase residing in NS5B. These results argue that compound 15d might represent a lead for the development of a novel class of NS5A inhibitors.     

2'-Deoxy-2'-spirocyclopropylcytidine revisited: a new and selective inhibitor of the hepatitis C virus NS5B polymerase

The current therapy for hepatitis C virus (HCV) infection has limited efficacy, in particular against the genotype 1 virus, and a range of side effects. In this context of high unmet medical need, more efficacious drugs targeting HCV nonstructural proteins are of interest. Here we describe 2'-deoxy-2'-spirocyclopropylcytidine (5) as a new inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC(50) of 7.3 μM measured in the Huh7-Rep cell line and no associated cytotoxicity (CC(50) > 98.4 μM). Computational results indicated high similarity between 5 and related HCV inhibiting nucleosides. A convenient synthesis was devised, facilitating synthesis of multigram quantities of 5. As the exposure measured after oral administration of 5 was found to be limited, the 3'-mono- and 3',5'-diisobutyryl ester prodrugs 20 and 23, respectively, were evaluated. The oral dosing of 23 led to substantially increased exposure to 5 in both rats and dogs.     

Design, synthesis, and antiviral activity of adenosine 5'-phosphonate analogues as chain terminators against hepatitis C virus

A series of adenosine 5'-phosphonate analogues were designed to mimic naturally occurring adenosine monophosphate. These compounds (1-5) were synthesized and evaluated in a cellular hepatitis C virus (HCV) replication assay. To improve cellular permeability and enhance the anti-HCV activity of these phosphonates, a bis(S-acyl-2-thioethyl) prodrug for compound 5 was prepared, and its cellular activity was determined. To elucidate the mechanism of action of these novel adenosine phosphonates, their diphosphate derivatives (1a-5a) were synthesized. Further nucleotide incorporation assays by HCV NS5B RNA-dependent RNA polymerase revealed that 2a and 3a can serve as chain terminators, whereas compounds 1a, 4a, and 5a are competitive inhibitors with ATP. Additional steady-state kinetic analysis determined the incorporation efficiency of 2a and 3a as well as the inhibition constants for 1a, 4a, and 5a. The structure-activity relationships among these compounds were analyzed, and the implication for nucleoside phosphonate drug design was discussed.     

Hepatitis C disease among injection drug users: knowledge, perceived risk and willingness to receive treatment

We surveyed 306 former injection drug users receiving methadone maintenance treatment in 1997-1998 in Providence, RI regarding, (1) knowledge of hepatitis C transmission; (2) the concordance of self-knowledge of hepatitis C virus (HCV) status versus actual status; (3) perceived risk of cirrhosis; and (4) willingness to receive therapy for hepatitis C. The seroprevalence of HCV was 87%. While 77% of participants knew that HCV could be sexually transmitted, 30% did not know that condoms are protective against transmission. Thirty of 45 persons who reported they were HCV seronegative were actually seropositive; 51 of 62 persons (82%) who reported they had never been HCV tested or did not know their HCV status were serologically HCV-positive. Over half of respondents (53%) would "definitely" or "probably" use interferon therapy for viral hepatitis when informed of the risks and benefits of treatment. We found significant gaps in knowledge about HCV among IDUs. Serologic confirmation of HCV status is important among drug users, as self-report of HCV infection is often unreliable. This population, with its high prevalence of HCV, may be interested in treatments that include interferon.     

GB virus C/hepatitis G virus infection in chronic hepatitis C patients with and without interferon-alpha therapy.

GB virus C/hepatitis G virus (GBV-C/HGV) RNA, detected by polymerase chain reaction, and antibodies to the GBV-C/HGV envelope protein (anti-E2), detected by an enzyme-linked immunosorbent assay, were used to evaluate both the impact of GBV-C/HGV on the coexistent hepatitis C virus (HCV) infection and the course of GBV-C/HGV infection in chronic hepatitis C patients with and without interferon-alpha (IFN-alpha) treatment. Of the 162 chronic hepatitis C patients treated with INF-alpha, 17.9% were GBV-C/HGV RNA-positive and 18.5% anti-E2-positive (total exposure, 35.2%). Neither present nor past GBV-C/HGV infection had impact on the clinical features, HCV virological characteristics and response to IFN-alpha treatment in chronic hepatitis C patients. Among patients with ongoing HCV/GBV-C/HGV coinfection, 20.7% (6/29) in IFN-alpha-treated patients lost GBV-C/HGV RNA concomitant with anti-E2 seropositivity, which was significantly higher than 4.8% (2/42) in patients without INF-alpha treatment (P<0.05). Based on multivariate analyses, the significant factors associated with clearance of GBV-C/HGV viremia combined with anti-E2 seropositivity were baseline anti-E2 seropositivity and IFN-alpha treatment. In summary, GBV-C/HGV did not alter the course of coexistent HCV. IFN-alpha treatment was effective in some patients against GBV-C/HGV and might facilitate anti-E2 seroconversion in chronic hepatitis C patients with GBV-C/HGV viremia.     

Non-nucleoside inhibitors of NS5B polymerase binding to allosteric sites: 3D- QSAR and molecular docking studies

Infection caused by hepatitis C virus (HCV) is a significant world health problem for which novel therapies are in urgent demand. Nonstructural (NS5B) viral proteins have emerged as an attractive target for drug discovery efforts toward antiviral for hepatitis C virus. Toward this target several series of NS5B inhibitors that showed activity in the replicon assay have been reported. In this article, we gave a report of the NS5B allosteric sites and the corresponding non-nucleoside inhibitors, which belong to different chemical classes. Then using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods, 3-dimension quantitative structure-activity relationships (3D-QSAR) models have been built with more than two hundred benzimidazole/indole derivative inhibitors. These studies indicated that the QSAR models were statistically significant and had high predictabilities (CoMFA: q(2)=0.823, r(2)=0.942; CoMSIA: q(2)=0.817, r(2)=0.935). The flexible docking method, which was performed by the DOCK6.0 software, positioned all of the inhibitors into the allosteric site to determine the probable binding conformation. The CoMFA and CoMSIA models based on the docking conformations also yielded statistically significant and high predictive QSAR models (CoMFA: q(2)=0.509, r(2)=0.768; CoMSIA: q(2)=0.582, r(2)=0.854). Our models would offer help to better comprehend the structure-activity relationships existent for this class of compounds and also facilitate the design of new inhibitors with good chemical diversity.     

Synthesis and biological evaluation of sophocarpinic acid derivatives as anti-HCV agents

Chronic hepatitis C virus (HCV) infection has become a major public health burden worldwide. Twenty-two sophocarpinic acid or matrine derivatives were synthesized and their anti-HCV activities were evaluated in vitro. The structure-activity analysis revealed that (i) sophocarpinic acids with a D-seco 3-ring structure scaffold were more favorable than matrines with a 4-ring scaffold; (ii) the introduction of an electron-withdrawing group on the phenyl ring in 12-N-benzenesulfonyl Δ^βγ sophocarpinic acids was beneficial for the antiviral activity against HCV. Among them, compounds 9h and 9j exhibited the most potent inhibitory activities on HCV replication with selectivity indies of 70.3 and 30.9, respectively. Therefore, both were selected as antiviral candidates for further investigation.KEY WORDS: Sophocarpinic acid, Matrine, Anti-HCV, Antiviral activity, Structure−activity relationship     

In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons

Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.