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

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.

Figure 1.

FDA-approved NAs for treating chronic HBV infection. 1: lamivudine (3TC); 2: telbivudine (LdT); 3: entecavir (ETV); 4: adefovir (ADV); 5: tenofovir disoproxil fumarate (TDF); and 6: tenofovir alafenamide (TAF).

Figure 2.

Major CAE chemotypes reported. 7 (BAY-7690) represents the HAP chemotype; 8 (DVR-56) represents the SBA chemotype.

Figure 3.

Our CAE hit. Compound 11 strongly bound to Cp, inhibited viral DNA production without cytotoxicity, and disrupted capsid assembly. 11 also demonstrated favorable ADME properties in vitro and decent oral bioavailability in vivo. These properties, combined with its low MW and high LE, render 11 a high quality lead.

Figure 4.

SAR design for hit 11. Sites for structural variation include the C5 amino group (subtype 12), the C4 carboxamide moiety (subtypes 13—16), and the C2 carboxamide phenyl ring (subtype 19). In addition, 4,5-cyclized analogues (subtype 17) and 2,3-cyclized analogues (subtype 18) are also designed.

Figure 5.

Characterization of compound 19h. (A) TSA curve (dotted line). Solid line depicts the curve of DMSO control; (B) dose-response antiviral testing; (C) TEM images of HBV capsid. HBV capsid aggregation observed in the presence of compound 19h as compared to the DMSO control.

Figure 6.

Lead optimization from 19h to 19o. The highlighted -CH₃ group in 19h is a metabolic liability which likely accounts for the low F. Replacing the -CH₃ group with a -CF₃ group substantially reduced antiviral potency (19m), whereas a -CHF₂ substituent enhanced antiviral activity while improving metabolic stability (19o).

Figure 7.

Molecular modeling of 19o. (A) Predicted binding mode of 19o within the crystal structure of HBV Cp (PDB code: 5T2P). (B) Structures of SBA analogue (29) and HAP analogue (30) in reported co-crystal structures. (C) Structural overlay of predicted binding mode of 19o and 29 within the HBV capsid. (D) Structural overlay of predicted binding mode of 19o and 30 within the HBV capsid. Key residues are highlighted in yellow sticks. H-bond interactions are depicted as black dotted lines. Alkyl-π interactions is represented as double headed arrow in black.

Scheme 1

^a Analogue synthesis for hit 11