5-Aminothiophene-2,4-dicarboxamide analogues as hepatitis B virus capsid assembly effectors

Highlights

• Validation and optimization of an ATDC analogue as a CAE hit.

• SAR from a μM hit led to 6 CAEs active at nM.

• SPR led to improved metabolic stability and oral bioavailability.

• Molecular modeling study revealed a binding mode.

• The best CAEs from this series are better than reported CAEs.

Abstract

Chronic hepatitis B virus (HBV) infection represents a major health threat. Current FDA-approved drugs do not cure HBV. Targeting HBV core protein (Cp) provides an attractive approach toward HBV inhibition and possibly infection cure. We have previously identified and characterized a 5-amino-3-methylthiophene-2,4-dicarboxamide (ATDC) compound as a structurally novel hit for capsid assembly effectors (CAEs). We report herein hit validation through studies on absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK), and hit optimization via analogue synthesis aiming to probe the structure-activity relationship (SAR) and structure-property relationship (SPR). In the end, these medicinal chemistry efforts led to the identification of multiple analogues strongly binding to Cp, potently inhibiting HBV replication in nanomolar range without cytotoxicity, and exhibiting good oral bioavailability (F). Two of our analogues, 19o (EC₅₀ = 0.11 μM, CC₅₀ > 100 μM, F = 25%) and 19k (EC₅₀ = 0.31 μM, CC₅₀ > 100 μM, F = 46%), displayed overall lead profiles superior to reported CAEs 7–10 used in our studies.

Introduction

HBV chronically infects an estimated 250 million people worldwide and remains a major health threat [1]. Despite a successful vaccine, there are still around 1 million people newly infected each year. Chronic HBV infection is often associated with severe liver diseases which typically progress through fibrosis, cirrhosis and eventually develop into hepatocellular carcinoma (HCC) [2]. It is estimated that HBV-associated liver diseases result in approximately 660,000 deaths annually. Current FDA-approved HBV drugs include the immunomodulatory agent pegylated interferon alpha (IFN-α) [3] and direct acting nucleos(t)ide analogues (NAs) which target reverse transcriptase (RT) of the HBV P protein complex [4]. These NAs are based on all four endogenous nucleosides with distinct approaches with respect to mimicking the ribose moiety (Fig. 1), including β-L-nucleosides [5,6] lamivudine (1, 3 TC, analogue of dC) and telbivudine (2, LdT, analogue of T), the carbocyclic nucleoside entecavir (3, ETV, analogue of dG) [7], the acyclic nucleoside phosphonate (ANPs) [8] adefovir (4, ADV, analogue of dA) and two forms of tenofovir (TFV, analogue of dA): tenofovir disoproxil fumarate (5, TDF) and tenofovir alafenamide (6, TAF). While NAs are generally well tolerated [9] and largely effective [10,11] in suppressing viral load, they do not cure HBV infection presumably because they do not eliminate HBV covalently closed circular DNA (cccDNA) which is the main viral reservoir for persistent infection [12]. HBV cure calls for novel antiviral approaches to completely eliminate or functionally inactivate cccDNA [[13], [14], [15]]. Otherwise, clinical management of chronic HBV infection requires lifetime treatment with current drugs.

The HBV replication cycle [16] entails a critical step where Cp dimers are assembled into functional viral capsids which encapsulate both viral pregenomic RNA (pgRNA) and polymerase complex for active reverse transcription to generate genomic, partially double-stranded relaxed circular DNA (RC-DNA). Interestingly, RC-DNA-containing mature nucleocapsids are either enveloped then secreted as new infectious virions, or enter the nucleus to replenish the cccDNA pool, constituting the intracellular amplification pathway of cccDNA. Disrupting the proper assembly of capsid can therefore inhibit HBV by blocking the production of infectious viruses and depleting cccDNA replenishment, and potentially contribute to HBV cure [17]. Therefore, targeting HBV Cp represents an attractive approach for treating chronic HBV infection. A few chemotypes have been reported as potent CAEs (Fig. 2) [14,18,19]. Amongst these, the heteroaryldihydropyrimidine (HAP) [[20], [21], [22], [23], [24]] chemotype as represented by compound 7 and the sulfamoylbenzamide (SBA) chemotype [25,26] as represented by compound 8 are particularly well studied with analogues in clinical development [[27], [28], [29]]. Mechanistically, all known CAEs accelerate capsid assembly, albeit with different assembled products. The HAP series constitutes a class of its own by inducing misassembly to form aberrant nonfunctional capsid particles, whereas SBA and other chemotypes, such as phenylpropenamide (9) [30] and NZ-4 (10) [31], promote the formation of empty, morphologically normal capsids [32].

Recently we conducted a high-throughput screening (HTS) of commercial libraries in a thermal shift assay (TSA) that measures binding to HBV Cp and identified compound 11 as a strong binder (Fig. 3) [33]. In the subsequent antiviral assay 11 inhibited HBV total DNA production (EC₅₀ = 3.4 μM) with no cytotoxicity observed (CC₅₀ > 100 μM). Mechanistically, compound 11 promoted the formation of large Cp aggregates and prevented Cp from entering nucleus [33]. These results confirmed 11 as a valid CAE hit. We report herein the optimization of CAE hit 11 through analogue synthesis, SAR as well as ADME and PK studies.