The caspase (cysteinyl-aspartate protease) family represents a class ofintracellular proteases playing a critical role in apoptotic cell death pathwaysand activation of pro-inflammatory cytokines. Their enzymatic properties aregoverned by a nearly absolute specificity for substrates containing asparticacid at the P1 site and by the use of a cysteine side-chain for peptide-bondhydrolysis. To date, 14 human caspases have been identified. Since activationof caspase-dependent apoptotic cell death has been implicated in the etiologyof many harmful human disorders, such as immunodeficiency, Alzheimer′s,Parkinson′s, and Huntington′s diseases, as well as ischemia, brain trauma, andamyotrophic lateral sclerosis, inhibitor of caspase is believed to be a valuabletherapeutic approach.There are now many designed potent and selective protease inhibitors.Although caspase inhibitors display a diverse range of biological properties,To be effective and practical drugs, they still have some deficiencies to beovercome, such as instability, low bioavailability and poor pharmacologicalprofiles. Therefore to be effective drugs, protease inhibitors need to haveminimal peptide characters, high stability to nonselective proteolyticdegradation, good membrane permeability, long lifetimes in the bloodstreamand in cells, low susceptibility to elimination, high selectivity for 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 biologicalhalf-lives. Examples of modifications introduced in peptides are the placementof L-amino acid residues by D-amino acids (retro-inverso transformation) orby unnatural residues (e.g., sarcosine and β-alanine) and the modification ofpeptide bonds. These changes provide pseudopeptides or peptidomimeticswith a higher metabolic stability, since most natural proteases cannot cleaveD-amino acid residues and nonpeptide bonds. At present, the retro-inversotransformation remains an important pseudopeptidic modification undertakenby numberous bioorganic chemists. Many partially modification retro-inverso(PMRI) analogs of peptides were synthesized and tested. It was reported thatreversed peptide not only displayed stability toward enzymatic degradation butalso improved bioavailability and potency. The remarkable resistance of PMRIanalogs to proteolytic degradation combined with retention of high biologicalactivity following oral or intravenous administration provides a strong impetusfor the continuing efforts on this modification.In this paper, we designed the novel PMRI caspase-3 inhibitors based on theinhibitor Z-VAD-CHO. We replaced L-amino acid with D-amino acid, whichresulted in all the amide bonds reversed. However, C-terminal P1 siteremained. Our synthesis progress consisted of three major parts, C-terminal P1site synthesis, coupling and deprotection of the inhibitor and completion of theinhibitor. Among them, C-terminal compound synthesis is the key part. Part1: In the first step of our experiment ,succinic anhydride reacted with benzylalcohol to give monobenzyl succinate. Second, the product was treated withLithium 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 inthe presence of p-toluene sulfonic acid as the catalyst to provide. Part 2: thebenzyl group was removed by catalytic hydrogenation using 5% Pd/c. Peptidealdehyde derivatives were formed by using the BOP method. Part 3 : Finally,we removed the protective group of aldehyde using 5% aqueous trifluoroaceticacid. In order to stabilize aldehyde group, we transformed them into itsbisulfite adduct by adding saturated solution of sodium bisulfite. Now, wehave already successfully synthesized the analogues of Z-VAD-CHO andevery product have been purified and characterized by 1~H NMR or MS.The analogues have been tested for their ability to inhibit caspase-3catalyzed proteolytic breakdown of its fluorogenic substrate, DEVD-AFC. Inour experiment, we made use of the product of Biovision company, Caspase-3Inhibitor Drug Screening Kit. We have demonstrated inhibitory activity ( IC50 )of analogues are 71μM and 43μM,according to the method provided by kit.Parallel experiments demonstrated that the IC50 value for Z-VAD-FMK, apotent tripeptide inhibitor of caspase-3 was equal to 1.7μM under the sameexperimental conditions.In summary, we have developed a general procedure for preparation ofretro-inverso peptidyl aldehydes, and synthesized PMRI caspase-3 inhibitorwhich has never been reported before using the new method. Bioassays showsthe inhibitor has moderate activity compared with that of the inhibitorsreported previously. This is only our primary work, and the result isencouraging, suggesting that caspase-3 inhibitors built on this novel scaffoldhave the potential for future drug development.