Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators

Abstract

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC₅₀ values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant in vivo hydrodynamic injection mouse model of HBV infection, with 12e effecting a 3 log₁₀ decline in serum HBV DNA levels at a once daily dose of 1 mg/kg. Additionally, maintenance of activity was observed in clinically relevant HBV core protein variants T33N and I105T.

Figure 1

Clinical level examples of reported CAM-E HBV capsid assembly modulators, from diverse chemical classes: sulfamoyl benzamide NVR 3-778, sulfamoyl pyrrole amide JNJ-56136379, 4-carbamoyl-1-aminoindane carbamate AB-506, and isoquinolinone urea AB-836.

Figure 2

Design rationale for conformationally constrained isoquinolinones: (A) representation of the X-ray structure of 1a (PDB ID 9C9V) at the B–C interface of Y132A Cp, (B) overlay of the bound structures of 1a in magenta (X-ray) and 2a in cyan (model) at the B–C interface, (C) design of the conformationally constrained isoquinolinone 2a.

Figure 3

X-ray diffractometry analysis of 9a (ORTEP diagram).

Figure 4

Log₁₀ reduction of HBV DNA levels in serum after 7 days of treatment with the indicated doses of CAMs 9a, 9d, 12e, and 12g (^#levels below the lower limit of quantitation). **** p < 0.0001 by One-way ANOVA with Dunnett’s multiple comparisons test.

Scheme 1. (a) CuI, K₃PO₄, 1,4-Dioxane, 90 °C, or CuI, L-Pro, K₂CO₃, DMSO, 90 °C; (b) NH₄OAc, DCE, 120 °C, Sealed Tube; (c) i. R-NH₂, Ti(OiPr)₄, 1,4-Dioxane, 70 °C, ii. NaBH₄, 1,4-Dioxane/MeOH, −12 to 0 °C; (d) for 2–9, 11: R²PhNCO, CH₂Cl₂, 0 °C or R²PhNH(CO)OPh, Et₃N, THF, for 10a: i. 3-Chloro-4-fluorophenyl Isocyanate, CH₂Cl₂, 0 °C, ii. TMSOTf, CH₂Cl₂, 0 °C; (e) for 12a–e,g: R³-1H-Indole-2-carboxylic Acid, HATU, NMO or ⁱPr₂NEt, DMF, r.t.; (f) X = N-Boc, TMSOTf, CH₂Cl₂, 0 °C; (g) for 10b: Aqueous 37% CH₂O, NaCNBH₃, MeOH, 0 °C to r.t., for 10c: TBSOCH₂CHO, NaCNBH₃, AcOH, MeOH, r.t.; (h) for 10b: 3-Chloro-4-fluorophenyl Isocyanate, CH₂Cl₂, 0 °C, for 10c: 3-Chloro-4-fluorophenyl Isocyanate, CH₂Cl₂, 0 °C, ii. TBAF, THF, r.t.

Scheme 2. (a) i. Ti(OiPr)₄, 1,4-Dioxane, 70 °C, ii. NaBH₄, 1,4-Dioxane/MeOH, −12 to 0 °C, 65%; (b) Aqueous 37% CH₂O, Na(OAc)₃BH, AcOH, DCE, 0 °C to r.t., 80%; (c) TFA, CH₂Cl₂, r.t., Quant.; (d) for 9a: R²PhNCO, CH₂Cl₂, 0 °C, for 9c_,d: R²PhNH(CO)OPh, Et₃N, THF, 50 °C; (e) for 12e–g: R³-1H-Indole-2-carboxylic Acid, HATU, NMO or ⁱPr₂NEt, DMF, r.t.