Nicotinyl Aspartyl Ketones as Inhibitors of Caspase-3 Elise Isabel, a, * W.CameronBlack, a Christopher I. Bayly, a ErichL.Grimm, a MarcK.Janes, a DanielJ.McKay, a Donald W. Nicholson, a DitaM.Rasper, a Johanne Renaud, a Sophie Roy, a JohnTam, a Nancy A. Thornberry, b John P. Vaillancourt, a Steven Xanthoudakis a and Robert Zamboni a a Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada & Co., PO Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8 b Merck Research Laboratories, Rahway, NJ 07065, USA Received 15 May 2002; accepted 8 April 2003 Abstract—Caspase-3isacysteinylproteasethatmediatesapoptoticcelldeath.Itsinhibitionmayhaveanimportantimpactinthe treatment of several degenerative diseases. Since P 1 aspartic acid is a required element of recognition for this enzyme, a library of capped aspartyl aldehydes was synthesized using solid-phase chemistry. The 5-bromonicotinamide derivative of the aspartic acid aldehyde was identified to be an inhibitor of caspase-3. Substitution at the 5-position of the pyridine ring and conversion of the aldehydetoketonesledtoaseriesofpotentinhibitorsofcaspase-3. # 2003ElsevierScienceLtd.Allrightsreserved. Thehumancaspasesareafamilyofatleast11cysteinyl- aspartate-specific proteinases that are central compo- nents in the molecular pathways that result in the apoptosisofcells. 1 Theseenzymesaredividedintothree groups.GroupIcaspases(1,4,and5)mediatecytokine maturation and are implicated in the inflammatory response. Group II caspases (2, 3 and 7) are the major effectors of cell death. Group III caspases (6, 8, 9, and 10)areupstreamactivatorenzymesofthegroupIIcas- pases. Caspase-14 is a heratinocyte specific caspase that has not yet been classified. Caspase-3 appears to be a critical participant in apoptosis in neurons. Prototype peptidyl inhibitors of caspase-3 have shown efficacy in models such as stroke, traumatic brain/spinal cord injury, hypoxic brain damage, and cardiac ischemia/ reperfusion injury. 2 DEVD-CHO is a tetrapeptide inhi- bitor based on the preferred amino acid sequence recognized by caspase-3. The aspartic acid is an essen- tial element of recognition of the enzyme and was used as the basis to develop new inhibitors. Here we report nicotinyl aspartyl ketones as potent and selective inhibitors of caspase-3. In order to generate libraries of aspartyl aldehydes, rapid analogues synthesis was performed using solid- phase chemistry (Scheme 1). Fmoc-aspartyl aldehyde 1 was reacted with a semi-carbazone linked resin 33. 3,4 The Fmoc protecting group was then cleaved under standard conditions to afford the polymer 2. The free amine was submitted to peptidic couplings with a wide variety of carboxylic acids. Reactions were routinely conducted on a 100-mg scale of resin. Subsequent treatment with 9:1 TFA/H 2 O led to cleavage from the resin with concomitant t-butyl ester removal to give final compounds with a good level of purity. Two alde- hyde replacements were considered: alkyl and thioether ketones. Alkyl ketones were prepared from Fmoc aspartatealcohol 9 (Scheme2),whichweresubmittedto Swern oxidation followed by an in situ addition of Grignard reagents. Resulting alcohols were oxidized to ketones using the Dess–Martin periodinane. These ketones were then attached to resin 33. The Fmoc pro- tecting group was cleaved and the free amine was cou- pled to the 5-bromonicotinic acid. Cleavage from the resin with wet TFA afforded ketones such as 10–12. Thioether ketones were synthesized using the Fmoc- aspartic acid b-t-butyl ester 13 (Scheme 3). After for- mation of the mixed anhydride, treatment with CH 2 N 2 and addition of a 1:1 mixture of aqueous 48% HBr/ CH 3 COOH, the a-bromoketone 14 wasobtained.Asin 0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-894X(03)00390-1 Bioorganic & Medicinal Chemistry Letters 13 (2003) 2137–2140 *Corresponding author. Tel.: +1-514-428-3655; fax: +1-514-428- 4939; e-mail: elise_isabel@merck.com