Yuhua Huang

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.

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.

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.

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.

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.

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.

Pyridofuran substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC(50)) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC(50)) was low.

Concise syntheses and HCV NS5B polymerase inhibition of (2′R)-3 and (2′S)-2′-ethynyluridine-10 and related nucleosides

(2R)-Ethynyl uridine 3, and its (2S)-diastereomer 10, are synthesised in a divergent fashion from the inexpensive parent nucleoside. Both nucleoside analogues are obtained from a total of 5 simple synthetic steps and 3 trivial column chromatography purifications. To evaluate their effectiveness against HCV NS5B polymerase, the nucleosides were converted to their respective 5-O-triphosphates. Subsequently, this lead to the discovery of the 2-β-ethynyl 18 and -propynyl 20 nucleotides having significantly improved potency over Sofosbuvir triphosphate 24.