Stereospecific Synthesis of Peptidyl .alpha.-Keto Amides as Inhibitors of Calpain

Abstract

Peptidyl alpha-keto amides have been synthesized and tested as inhibitors of the cysteine protease calpain. A stereospecific synthesis was devised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxidation 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 stereochemistry 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 polar groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Studies of the stability of these alpha-keto amides showed that absolute stereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacent to the ketone occurred rapidly. The alpha-hydroxy precursors were inactive as inhibitors of calpain, which supports the hypothesis that the alpha-keto compounds reversibly form an enzyme-bound tetrahedral species that results from the nucleophilic addition of the catalytic thiol of calpain to the electrophilic ketone of the inhibitor.

BACE1- and BACE2-expressing Human Cells

“…The peptide backbone of the compounds was stepwise assembled by classical methods, using Boc as the ␣-amino protecting group and benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate as the coupling reagent, either in homogeneous phase or on solid phase with methylbenzhydrylamine resin, necessitating a final HF cleavage procedure. Published protocols were followed for the formation of the peptide bond isostere moieties, reduced amide bond (Leu-⌿(CH s NH)-Asp in JMV963) (21,22), norstatine (Norsta-containing compounds) (23,24), and statine and analogs (Sta-, AHPPA-, ACHPA-containing compounds) (25,26). All synthetic inhibitors were purified on C18 reverse-phase HPLC, and their purity and identity were assessed by reverse-phase HPLC and electrospray mass spectrometry.…”

Inhibitors of human heart chymase based on a peptide library.

“…Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found K1 values were 1 nM and 1 ,uM, respectively. The corresponding K1 values for chymotrypsin were 10 nM and 100 ,uM.…”

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

“…Till recently, most of the reported cysteine protease inhibitors have been irreversible inhibitors like fluromethyl ketones [21], vinylsulfones [22] or epoxysuccinates [23]. Numerous warheads [24] have been utilized in designing of reversible inhibitors of cysteine proteases including peptidic aldehydes [25], nitriles [26], cyclopropanones [27], diamino ketones [28] and a-ketoamides [29]. There has also been a report on the use of non-covalent amides as cathepsin K inhibitors [30].…”