Design, Synthesis And Biological Activity Of Novel Caspase Inhibitors

The caspase (cysteinyl-aspartate protease) family represents a class ofintracellular proteases playing a critical role in apoptotic cell death pathways andactivation of pro-inflammatory cytokines. Their enzymatic properties are governedby a nearly absolute specificity for substrates containing aspartic acid at the P1 siteand by the use of a cysteine side-chain for peptide-bond hydrolysis. To date, 11human caspases have been identified. Since activation of caspase-dependentapoptotic cell death has been implicated in the etiology of many harmful humandisorders, such as immunodeficiency, Alzheimer′s, Parkinson′s, and Huntington′sdiseases, as well as ischemia, brain trauma, and amyotrophic lateral sclerosis,inhibitor of caspase is believed to be a valuable therapeutic approach.There are now many designed potent and selective protease inhibitors. Althoughcaspase inhibitors display a diverse range of biological properties, As effective andpractical drugs, they still have some deficiencies to be overcome, such asinstability, low bioavailability and poor pharmacological profiles. Therefore theseprotease inhibitors need to have minimal peptide characters, high stability tononselective proteolytic degradation, good membrane permeability, long lifetimesin the bloodstream and in cells, low susceptibility to elimination, high selectivityfor a protease, and good bioavailability (preferably by oral delivery).For many years intense work has been focused on the synthesis of peptideanalogues in the search for mimics with enhanced activity and biological half-lives.Examples of modifications introduced in peptides are the placement of L-aminoacid residues by D-amino acids (retro-inverso transformation) or by unnaturalresidues (e.g., sarcosine and β-alanine) and the modification of peptide bonds.These changes provide pseudopeptides or peptidomimetics with a higher metabolicstability, since most natural proteases cannot cleave D-amino acid residues andnonpeptide bonds. At present, the retro-inverso transformation remains animportant pseudopeptidic modification undertaken by numberous bioorganicchemists. Many partially modification retro-inverso (PMRI) analogs of peptideswere synthesized and tested. It was reported that reversed peptide not onlydisplayed stability toward enzymatic degradation but also improved bioavailabilityand potency. The remarkable resistance of PMRI analogs to proteolyticdegradation combined with retention of high biological activity following oral orintravenous administration provides a strong impetus for the continuing efforts onthis modification.In this paper, we designed the novel PMRI caspase-3 inhibitors based on theinhibitor Z-VAD-CHO. We replaced L-amino acids with D-amino acids, whichresulted in all the amide bonds reversed. However, C-terminal P1 site remained.Our synthesis progress consisted of three major steps, C-terminal synthesis,coupling and deprotection of the inhibitor and completion of the inhibitor. Amongthem, C-terminal compound synthesis is the key step. Part 1: succinic anhydridereacted with benzyl alcohol to give monobenzyl succinate. the product was treatedwith Lithium bis(trimethyl-silyl) amide in dry tetrahydrofurane. The ethyl formatewas added to afford the α-formyl α-benzyl succinate . Then, the aldehyde andcarboxyl groups were protected by reacting with the trimethyl orthoformate in thepresence of p-toluene sulfonic acid as the catalyst. Part 2: the benzyl group wasremoved by catalytic hydrogenation using 5% Pd/c. Peptide aldehyde derivativeswere formed by using the BOP method. Part 3 : the protective group of aldehydewas removed using 50% aqueous trifluoroacetic acid. In order to stabilizealdehyde group, we transformed them into its bisulfite adduct by adding saturatedsolution of sodium bisulfite. Now, we have already successfully synthesized theanalogues of Z-VAD-CHO and every product have been purified and characterizedby 1~H NMR or MS.The analogues have been tested for their ability to inhibit caspase-3 catalyzedproteolytic breakdown of its fluorogenic substrate, DEVD-AFC. In our experiment,we made use of the product of Biovision company, Caspase-3 Inhibitor DrugScreening Kit. We have demonstrated inhibitory activity ( IC50 ) of analogues are71μM and 43μM,according to the method provided by kit. Parallel experimentsdemonstrated that the IC50 value for Z-VAD-FMK, a potent tripeptide inhibitor ofcaspase-3 was equal to 1.7μM under the same experimental conditions.In summary, we have developed a general procedure for preparation ofretro-inverso peptidyl aldehydes, and synthesized PMRI caspase-3 inhibitor whichhas never been reported before. Bioassays shows the inhibitors have moderateactivity compared with that of the inhibitors reported previously. This is only ourprimary work, and the result is encouraging, suggesting that caspase-3 inhibitorsbuilt on this novel scaffold have the potential for future drug development.