Discovery of Hepatitis B Virus Surface Antigen Suppressor GS-8873

Abstract

Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.

Figure 1

Oral nucleos(t)ide analogues for CHB treatment are the standard of care.

Figure 2

In vivo PK comparison of DHQ and DPP inhibitors of HBsAg suppression. A) Rat IV PK time vs plasma concentration for 4, 7, and 8. B) Rat PO PK time vs plasma concentration for 4, 7, and 8. C) In vitro and in vivo PK parameters for 4, 7, and 8.

Figure 3

HBsAg inhibitors do not suppress HBV cccDNA. PHH cells were plated and infected with HBV for 2 days and then treated with compound 8 for 12 days, with compound replenishment every 2 to 3 days. Intracellular DNA samples were analyzed by Southern blot. A) Representative Southern blot showing no effects on cccDNA and an increase in relaxed circular DNA (RC-DNA) with compound 6 or 8 treatment. Combination treatment with compound 8 and tenofovir prevented the accumulation of RC-DNA. B) Quantification of the Southern blot is shown. Error bars represent the SD.

Figure 4

DPP 8 reduced intracellular HBsAg protein in infected PHH as analyzed by immunofluorescence microscopy. PHH cells were infected with HBV for 3 days and then treated with 250 nM compound 8 for 14 days, with compound replenishment every 2 to 3 days. Cells were fixed and stained with anti-HBsAg monoclonal antibody (red), DAPI for nucleus (blue), and phalloidin for actin (white). Two independent experiments were performed. Representative immunofluorescence confocal microscopy images are shown.

Figure 5

Prelinical PK studies with GS-8873. A) Concentration–time profiles of GS-8873 (46) in plasma across species following administration by intravenous infusion (n = 3/study). B) Pharmacokinetic parameters following intravenous infusion and oral administration of GS-8873 (46).

Scheme 1. Synthesis of GS-8873

Reagents and conditions: a) BnBr, K₂CO₃, DMF; b) i-PrMgCl·LiCl, MeTHF; c) Zn, THF/H₂O; d) NaNO₂, AcOH, H₂O/THF; e) Zn, THF/H₂O; f) TFA, H₂O, EtOH; g) Pd(OAc)_2, (oxidi-2,1-phenylene)bis(diphenylphosphine), DMA; h) potassium trifluoro(methoxymethyl)borate, Pd RuPhos G3, Cs₂CO₃, PhMe/H₂O; (i) chiral supercritical fluid chromatography; j) H₂, Pd/C, EtOH; k) 1-bromo-3-methoxypropane, K₂CO₃, DMF, then LiOH, H₂O.