The replacement of the P

The replacement of the P-butyl urea cap with sulfonamide derived cap had a profound effect on binding. Compound obtained by the replacement of -butyl urea P cap of compound with sulfonamide demonstrated a =34nM, a fifteen folds improvement in potency. Similarly replacement of P-butyl cap in the P cyclobutylmethyl derived compound resulted in inhibitor (=500pM); a twenty fold improvement in activity over . This was the first time we had identified a compound that bound to NS3 protease in the picomolar range. We next evaluated the boronic angiotensin receptor blockers analogs of compounds and . The corresponding boronic acid analogs of compound , compound demonstrated a =52nM; whereas the boronic acid derivative of compound , compound had a =200pM. Comparison of binding activities of the pinene diol esters – to their corresponding boronic acid inhibitors – suggested that the boronate esters were readily hydrolyzed in the assay conditions and the activity of the boronate esters probably arose from the corresponding boronic acid derivatives. The X-ray structure of inhibitor bound to HCV NS3 protease was solved and is shown in . From the structure of bound to protease, it was clear that the cyclobutyl group at P occupy the S pocket. The P (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane ring adopted a bent conformation that allowed maximum overlap of the methylenes of proline and cyclopropyl ring to Ala-156. The conformation adopted by cyclopropyl group allowed the methyl group proximal to the carbonyl to interact with His-57 and the methyl group distal to carbonyl to interact with Ala-156 and Arg-155. The butyl group of P sulfonamide capping occupied the S region of space making excellent van der Waals contact with the protein and the oxygens of the sulfonamide group formed a hydrogen bond with Cys-159. The oxygen of Ser-139 is bound to the lewis acidic boron to form a tetrahedral boronate derivative. In addition to van der Waals contacts, inhibitor formed a series of specific hydrogen bonds with the protein surface. Mapping out the various hydrogen bonding interactions that existed between inhibitor and NS3 protease it is evident that the urea nitrogens donated two hydrogen bonds to alanine-157 thus improving potency and HCV specificity. Additionally, the nitrogen of the P residue donated a hydrogen bond to Arg-155 and oxygen of P carbonyl group accepted a hydrogen bond from the Ala-157. We next evaluated the activity of these potent inhibitors in the replicon based cellular assay. Evaluation of these compounds demonstrated an EC>5.0μM suggesting that cell permeability may be limiting and be responsible for poor cellular activity. In search of a possible second generation compound to Sch 503034 () we evaluated the possibility of replacements of the ketoamide with a boronic acid electrophile. The replacement of this group with boronic acid resulted in inhibitor which was equipotent to our first generation compound . We next evaluated the replacement of P capping with modified P cap derived such as -(2-amino-3,3-dimethylbutyl)--methylmethanesulfonamide which resulted in compound that had markedly improved activity (=200pM). This was the first time we identified compounds in the picomolar range. Analysis of X-ray structure of bound to the protease revealed binding of the boronic acid to Ser-139, where the boronic acid forms a tetrahedral borate. Evaluation of these inhibitors in the replicon based cellular assay showed EC>5μM suggesting that the cell penetration was probably an issue. Further studies are on way to improve cellular activity of these inhibitors.