STEREOSPECIFIC SYNTHESIS OF PEPTIDYL ALPHA-KETO AMIDES AS INHIBITORS OF CALPAIN

Peptidyl alpha-keto amides have been synthesized and tested as inhibit ors of the cysteine protease calpain. A stereospecific synthesis was d evised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxida tion was accomplished in good yields and without epimerization of the chiral center adjacent to the ketone. The potent inhibition of porcine calpain I by the L,L diastereomers, combined with the poor inhibition by the L,D diastereomers, established the requirement for the all-L s tereochemistry of the active inhibitor. The early lead inhibitors were very hydrophobic and, therefore, poorly soluble in aqueous solutions. Using the stereospecific route, new compounds were prepared with pola r groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Stud ies of the stability of these alpha-keto amides showed that absolute s tereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacen t to the ketone occurred rapidly. The alpha-hydroxy precursors were in active as inhibitors of calpain, which supports the hypothesis that th e alpha-keto compounds reversibly form an enzyme-bound tetrahedral spe cies that results from the nucleophilic addition of the catalytic thio l of calpain to the electrophilic ketone of the inhibitor.