Synthesis of dual action Smac/Zinc-Chelator conjugates as putative proapoptotic agents

Apoptosis, or programmed cell death, is a critical cell process in normal development and homeostasis of multicellular organisms. It is now recognized that dysfunction of the apoptosis machinery is a hallmark of cancer. Accordingly, targeting critical apoptosis regulators is an attractive approach for the development of new classes of therapies for the treatment of cancer and other human diseases. The X-linked inhibitor of apoptosis protein (XIAP) is a member of IAP proteins that potently inhibit apoptosis[1]. XIAP contains three baculovirus IAP repeat (BIR) domains. The mechanism of action of XIAP entails its binding with initiator and effector caspases through its BIR domains. In cells, the anti-apoptotic function of XIAP is antagonized by Smac/DIABLO (second mitochondria-derived activator of caspases or direct IAP binding protein with low pI). Despite the rather complex structure of Smac, its short N-terminal AVPI sequence is sufficient to trigger the inactivation of anti-apoptotic XIAP[2]. Our research group has shown how small monomeric, AVPI-inspired Smac mimics can bind XIAP on its BIR3 domain with sub-micromolar potency[3,4]. Executioner caspase-3, -6 and -7 exist within the cytosol as inactive zymogens (procaspases) activated by limited proteolysis within their inter-domain linker, carried out by an initiator caspaseThe essential executioner caspase-3 is proteolytically activated by either caspase-8 or -9. Zinc ions co-localize with procaspase-3/caspase-3 and inhibit its enzymatic activity in the cell by direct interaction with an Asp-Asp-Asp (DDD) “safety catch” region[5]. Thus, in this work we coupled a zinc chelator moiety based on di(picolylamide)amine (DPA) and its N,N-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (BPEN) derivative to pro-apoptotic Smac mimetics, synthesized starting from known intermediates 1 and 2. Dual action Smac mimetic-zinc chelators 3 and 4 were prepared from compounds 1 and 2 as shown in Scheme 1 and characterized in vitro, using cell-free and cellular assays[6,7]. Their ability to bind XIAP BIR3 domain, to process pro-caspase-3 to caspase-3 and their cytotoxicity have been experimentally determined, and favorably compared with those of a potent Smac mimic compound, especially for the most potent, tribasic dual action compound 4. Furthermore, the Zinc affinity for both compounds was confirmed by fluorescence measurements. References 1. Q. L. Deveraux, J. C. Reed. Genes & Dev. 1999, 13, 239-252 2. J. Chai, C. Du, J. W. Wu, S. Kyin, X. Wang, Y.Shi. Nature 2000, 406, 855-862 3. P. Seneci, A. Bianchi, C. Battaglia, L. Belvisi, M. Bolognesi, A. Caprini, F. Cossu, E. de Franco, M. de, D. Delia, C. Drago, A. Khaled, D. Lecis, L. Manzoni, M. Marizzoni, E. Mastrangelo, M. Milani,I. Motto, E. Moroni, D. Potenza, V. Rizzo, F. Servida, E. Turlizzi, M. Varrone, F. Vasile, C. Scolastico. Bioorg. Med. Chem. 2009, 17, 5834–5856. 4. L. Manzoni, D. Arosio, L. Belvisi, A. Bracci, M. Colombo, D. Invernizzi, C. Scolastico. J. Org. Chem. 2005, 70, 4124-4132. 5. K. S. Putt, G. W. Chen, J. M. Pearson, J. S Sandhorst, M. S. Hoagland, J. Kwon, S. Hwang, H. Jin, M. I. Churchwell, M. Cho, D. R. Doerge, W. G. Helferich, P. J. Hergenrother. Nat. Chem. Biol. 2006, 2, 543, 550 6. Z. Nikolovska-Coleska, R. Wang, X. Fang, H. Pan, Y. Tomita, P. Li, P.P. Roller, K. Krajewski, N.G. Saito, J.A. Stuckey, S. Wang, Anal. Biochem. 2004, 332, 261-273. 7. Z. Nikolovska-Coleska, J.L. Meagher, S. Jiang, S.A. Kawamoto, W. Gao, H. Yi, D. Qin, P.P. Roller, J.A. Stuckey, S. Wang, Anal. Biochem. 2008, 374, 87-98.