Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics.

HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.

Discovery of an irreversible HCV NS5B polymerase inhibitor.

The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 µM), highly selective, and safe in in vitro and in vivo assays.

Discovery of novel tricyclic indole derived inhibitors of HCV NS5B RNA dependent RNA polymerase.

The characterization of HCV genome has identified various vital functional proteins involved in the life cycle of hepatitis C virus. This has resulted in many novel enzymatic targets that are potential for development of therapeutic agents. The HCV RNA dependent RNA polymerase (HCV NS5B) is one such essential enzyme for HCV replication that has been well characterized and studied by various groups to develop novel therapies for hepatitis C. In this paper, we describe our efforts towards the identification and structure-activity relationship (SAR) of novel tricyclic indole derivatives that bind close to the palm site of the NS5B polymerase. X-ray crystal structure of an inhibitor bound to the polymerase is also described.

II. Novel HCV NS5B polymerase inhibitors: discovery of indole C2 acyl sulfonamides.

Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC(50)=0.053µM, replicon EC(50)=4.8µM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC(50)=0.039µM, replicon EC(50)=0.011µM) with >100-fold improved replicon activity.

Synthesis and SAR of geminal substitutions at the C5' carbosugar position of pyrimidine-derived HCV inhibitors.

The installation of geminal substitution at the C5' position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5' position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.

Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors.

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.

5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors.

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.

I. Novel HCV NS5B polymerase inhibitors: discovery of indole 2-carboxylic acids with C3-heterocycles.

SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 µM, replicon EC(50)>100 µM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 µM, replicon EC(50)=1.4 µM) and 7r (NS5B IC(50)=0.017 µM, replicon EC(50)=0.3 µM) with improved enzyme and replicon activity.

Structure-activity relationship (SAR) development and discovery of potent indole-based inhibitors of the hepatitis C virus (HCV) NS5B polymerase.

Starting with the indole-based C-3 pyridone lead HCV polymerase inhibitor 2, extensive SAR studies were performed at different positions of the indole core. The best C-5 groups were found to be compact and nonpolar moieties and that the C-6 attachments were not affecting potency. Limited N-1 benzyl-type substituent studies indicated that the best substitutions were fluoro or methyl groups at 2' or 5' positions of the benzyl group. To improve pharmacokinetic (PK) properties, acylsulfonamides were incorporated as acid isosteres at the C-2 position. Further optimization of the combination at N-1, C-2, C-5, and C-6 resulted in the identification of compound 56, which had an excellent potency in both NS5B enzyme (IC(50) = 0.008 µM) and cell-based replicon (EC(50) = 0.02 µM) assays and a good oral PK profile with area-under-the curve (AUC) of 14 and 8 µM·h in rats and dogs, respectively. X-ray structure of inhibitor 56 bound to the enzyme was also reported.

A novel class of highly potent irreversible hepatitis C virus NS5B polymerase inhibitors.

Starting from indole-based C-3 pyridone HCV NS5B polymerase inhibitor 2, structure-activity relationship (SAR) investigations of the indole N-1 benzyl moiety were performed. This study led to the discovery of irreversible inhibitors with p-fluoro-sulfone- or p-fluoro-nitro-substituted N-1 benzyl groups which achieved breakthrough replicon assay potency (EC(50) = 1 nM). The formation of a covalent bond with adjacent cysteine-366 thiol was was proved by mass spectroscopy and X-ray crystal structure studies. The C-5 ethyl C-2 carboxylic acid derivative 47 had an excellent oral area-under-the-curve (AUC) of 18 µM·h (10 mg/kg). Its oral exposure in monkeys and dogs was also very good. The NMR ALARM assay, mass spectroscopy experiments, in vitro counter screening, and toxicology assays demonstrated that the covalent bond formation between compound 47 and the protein was highly selective and specific. The overall excellent profile of 47 made it an interesting candidate for further investigation.