As a possible back up to Sch

As a possible back-up to Sch 503034 (), we explored inhibitors that contained alternative electrophiles to ketoamides. Boronic acids have been extensively investigated as possible electrophiles that could react with nucleophilic serine in the context of developing inhibitors of thrombin receptor and other serine proteases including HCV NS3 protease. Valcade, a boronic Edoxaban mg derived dipeptide is an inhibitor of 26S proteosome that has been successfully developed into a drug for treatment of multiple myeloma. We therefore decided to investigate the replacement of ketoamide group in with a boronic acid derivative. From our previous SAR exploration of inhibitors spanning from P–P, it was determined that an ethyl group at P was well tolerated. We therefore decided to explore compound as our first boronic acid target (). Intermediates for the synthesis of the inhibitors of type were accomplished using methods outlined in . Deprotonation of dichloromethane with BuLi at −100°C, followed by treatment with trimethylborate resulted in compound . Methyl ester was converted to the pinene diol ester by transesterification with (+)-pinenediol. Treatment of with ethyl or cyclobutyl Grignard reagent resulted in compounds of type in excellent yields. Further displacement of the chloride of with LiHMDS followed by treatment with HCl yielded amine salts of type . Completion of synthesis of inhibitor was accomplished using reactions outlined in . Thus, coupling of Boc protected -leucine with 3,4-dimethylcyclopropyl fused proline using HATU and NMM resulted in dipeptide in 60% yield. The Boc group of the dipeptide was deprotected using 4M HCl in dioxane and further treatment of the resultant amine salt with -butyl isocyanate yielded -butyl urea . Hydrolysis of methyl ester of compound with aq LiOH, followed by coupling of the resulting acid with the amine salt using isobutyl chloroformate and EtN yielded inhibitor precursor , which was converted to by reaction with NaIO. Compound was tested for its inhibitory activity in a HCV protease continuous assay. Its ability to prevent the NS3 protease catalyzed hydrolysis of chromogenic 4-phenylazophenyl [PAP] ester from peptide fragment Ac-DTEDVVP(Nva)-O-4-PAP was measured using a spectrophotometric assay. The binding constant for the inhibitor was determined as =380nM. We also evaluated the pinene ester for its ability to inhibit HCV NS3 protease and determined its binding constant =500nM. Since these compounds were less potent than the corresponding first generation compound , we decided to synthesize the corresponding cyclobutylmethyl containing amino boronic acid and incorporate it into the inhibitor. The synthesis of cyclobutyl derived amino acid was accomplished in a similar manner to that of except that the cyclobutylmethyl was introduced using the appropriate Grignard reagent. The synthesis of subsequent inhibitors was also similar to that of and is as shown in . The introduction of a lipophilic cyclobutyl group at P enhanced binding of both the boronate ester compound (=10nM) as well as the boronic acid derivative (=10nM), a 50-fold improvement in binding over . Our studies in the ketoamide series of inhibitors had previously demonstrated that the replacement of the Pbutyl urea cap with various modified urea moieties enhanced binding of ketoamide inhibitors. To evaluate the effect of this novel capping group we decided to synthesize the sulfonamide derived compound . The synthesis of inhibitor is outlined in . summarizes the activity of these compounds. The coupling of Boc protected -leucine with ammonium chloride using EDCI resulted in Boc protected -leucine amide, which was further reduced to amine using BH·SMe. The amine was reacted with methanesulfonyl chloride and the resulting sulfonamide was methylated with methyl iodide and CsCO to yield N-methylated sulfonamide . Deprotection of Boc group of followed by treatment of formed amine salt with nitrophenyl carbamate of intermediate yielded compounds . The methyl ester of intermediate was hydrolyzed with aq lithium hydroxide and the intermediate acid was coupled with amine salt of type using mixed anhydride method with IBCF and EtN. The formed pinene diol ester was once again hydrolyzed to the corresponding boronic acid using NaIO to yield compounds of type .