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New potent biaryl sulfate-based hepatitis C virus inhibitors

https://doi.org/10.1016/j.ejmech.2016.09.031Get rights and content

Highlights

  • New compounds containing biaryl sulfone or sulfate cores were synthesized as potent HCV NS5A protein inhibitors.

  • The new inhibitors exhibit excellent inhibitory potencies against both genotype-1b and genotype-2a.

  • The compounds showed high plasma stability and no mutagenic potential.

  • The compounds had additive or synergistic effect upon co-administration with sofosbuvir.

Abstract

The discovery of a new series of potent hepatitis C virus (HCV) NS5A inhibitors containing biaryl sulfone or sulfate cores is reported. Structure-activity relationship (SAR) studies on inhibitors containing various substitution patterns of the sulfate or sulfone core structure established that m-,m′- substituted biaryl sulfate core-based inhibitors containing an amide moiety (compound 20) or an imidazole moiety (compound 24) showed extremely high potency. Compound 20 demonstrated double-digit pM potencies against both genotype 1b (GT-1b) and 2a (GT-2a). Compound 24 also exhibited double-digit pM potencies against GT-1b and sub nM potencies against GT-2a. Furthermore, compounds 20 and 24 exhibited no cardiotoxicity in an hERG ligand binding assay and showed acceptable plasma stability and no mutagenic potential in the Ames test. In addition, these compounds showed distinctive additive effects in combination treatment with the NS5B targeting drug sofosbuvir (Sovaldi®). The results of this study showed that the compounds 20 and 24 could be effective HCV inhibitors.

Introduction

Hepatitis C virus (HCV) affects essential liver health leading to infectious hepatitis [1], [2]. Since the first identification of HCV in 1989, approximately 160–200 million people have been infected worldwide. Nearly 20% of those patients developed cirrhosis and hepatocellular carcinoma (HCC) [3], [4]. In 2014, the World Health Organization (WHO) announced that hepatitis C-related liver diseases kill 350–500 thousand people each year [5].

HCV is a positive sense RNA virus about 50 nm in size. It encodes a total of approximately three thousand amino acids [6]. Polyproteins translated from HCV RNA include structural proteins and nonstructural (NS) proteins such as NS2, NS3, NS4A, NS4B, NS5A, and NS5B. These proteins are associated with the production of new virion particle undergoing replication, transcription, and translation processes [1], [7], [8]. In particular, NS5A proteins are expected to be involved in replication, reorganized membrane assembly, and secretion of HCVs from infected cells [9].

HCV is classified into 7 genotypes (1a, 1b, 2, 3, 4, 5, and 6) comprising 67 subtypes [10]. According to the WHO report, HCV patients infected with genotypes 1a (GT-1a) and 1b (GT-1b) are the most predominant and represent 60% of all cases, worldwide [11], [12].

In accordance with international therapy guidelines, the first standard of care (SOC) against HCV comprised a combination of the pegylated interferon (PEG-IFN) α series (α2a or α2b) and ribavirin (RBV), a nucleoside-type drug [13], [14]. Nevertheless, this remedy has not only 50% cure rate for the common GT-1 patients, but also other severe adverse effects such as insomnia, depression, neutropenia, flu-like symptoms, fatigue, lymphopenia, and other unexpected drug interactions [15], [16]. Therefore, Direct Acting Antivirals (DAAs) have been widely studied to overcome drawbacks of conventional therapy. The first generation of DAAs included NS3/NS4A protease inhibitors such as telaprevir ans boceprevir. These were approved by the U.S. Food and Drug Administration (USFDA) in 2011 [17]. However, these drugs still require the use of RBV and PEG-IFN combinations, and have a 54–63% sustained virologic response (SVR) in GT-1 patients [18].

Recently, a new NS5A inhibitor, daclatasvir (BMS790052, Daklinza®), was approved in Europe in 2014 (Fig. 1). As a suppressor of the NS5A replication complex, daclatasvir showed impressive results at single-digit picomolar effective concentrations (EC50) (9 pM and 50 pM against GT-1b and GT-1a, respectively). It effected 2-fold decreases in levels of viral RNA within 6 h of oral administration in clinical trials [19], [20]. Consequently, the NS5A phosphoprotein became one of the most desirable drug targets of HCV for many pharmaceutical companies [20].

Daclatasvir has a C-2 symmetric structure that consists of a biphenyl core linked to a 2-(1H-imidazol-2-yl)-pyrrolidine moiety, and an N-(methoxycarbonyl)-l-valine capping group (Fig. 1). In particular, the imidazole and carbamate groups play an important role in the interaction of daclatasvir with the HCV NS5A complex [21], [22]. Although daclatasvir had no reported serious side-effects or drug-drug interactions (DDI), resistance to the drug appeared in patients infected with GT-1a and GT1-b HCV [23]. The main amino acid mutations were Y93H and L31V in GT-1b, which resulted in 19-fold and 23-fold increases in resistance to daclatasvir, respectively [24]. When there was combination of both Y93H and L31V mutations in GT-1b, resistance to daclatasvir increased 8336-fold [25]. To address this issue, many research groups have developed HCV NS5A targeting drug candidates such as ledipasvir (combination with sofosbuvir and called Harvoni, FDA approval in 2014), ombitasvir (combination with ritonavir and paritaprevir and called Technivie, FDA approval in 2015), elbasvir (MK-8742), samatasvir (IDX-719), and AV4025 (Fig. 1) [26].

Recently, our laboratory reported a series of NS5A inhibitors represented by BMK-20113 (EC50 against GT-1b and GT-2a strains: 28 pM and 260 pM, respectively), which comprises a benzidine prolinamide skeleton and an N-(methoxycarbonyl)-d-phenylglycine based capping group (Fig. 2) [27]. By considering the structural features of other known inhibitors, we hypothesized that a longer biaryl core could improve inhibition of the HCV NS5A replication complex as shown with ombitasvir (EC50 of GT-1b and GT-2a: 5 pM and 12 pM, respectively) and AV4025 (EC50 of GT-1b and GT-2a: 3.4 pM and 51 pM, respectively) [28]. Herein, we report the discovery of new NS5A inhibitors embedding biaryl sulfone or sulfate core structures (Arsingle bondSO2single bondAr, ArOsingle bondSO2single bondOAr) with imidazole or amide connections [29], [30].

Section snippets

Chemistry

Compounds containing the biaryl sulfone core were prepared following a general amide synthesis procedure as shown in Scheme 1 [31]. Coupling of 4,4'-diaminodiphenyl sulfone 1 with N-Boc-l-proline in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) provided N-Boc-protected intermediate 2 in moderate yield (44%). After removal of the Boc group, free amine moieties were coupled with d-phenylglycine, d-valine, or l-valine, each of which was capped with an N-(methoxycarbonyl)

Results and discussion

To determine whether the compounds with sulfone or sulfate core structures are effective HCV inhibitors, we investigated the inhibitory activities of the synthesized compounds using the GT-1b replicon assay and the GT-2a HCV cell culture system (HCVcc) [37], [38], [39]. For the HCV replicon systems (GT-1b), Huh 7.5.1 cells were employed to investigate the HCV inhibitory activities of the compounds [40]. These Huh 7.5.1 cells contain a bicistronic HCV replicon NK5.1-Gluc that encodes secretory

Conclusion

In summary, we investigated new compounds containing biaryl sulfone or sulfate cores as inhibitors of the hepatitis C virus NS5A protein. Through SAR study, we discovered that two compounds containing an m-,m′-disubstituted biaryl sulfate core, 20 and 24, exhibit excellent inhibitory potencies against both GT-1b and GT-2a. In hERG ligand binding inhibition assays, no inhibition activity was detected for either 20 or 24. These compounds showed high plasma stability and no mutagenic potential as

General chemical methods

The 1H and 13C NMR-spectra were measured with a Varian/Oxford As-500 (500 MHz), and an Agilent 400-MR DD2 Magnetic Resonance System (400 MHz) spectrophotometer. Chemical shifts were measured as part per million (δ values) from Tetramethylsilane (TMS) as an internal standard at probe temperature in CD3OD or CDCl3 or DMSO-D6 for neutral compounds. Coupling constants are provided in Hz, with the following spectral pattern designations: s, singlet; d, doublet; t, triplet; q, quartet; quint,

Acknowledgment

This study was supported by the Bio R&D Program (No. 2012M3A9A9054974) through the National Research Foundation funded by the MEST, Republic of Korea.

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    These two individuals contributed equally to this work.

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