Nonpeptidic and potent small-molecule inhibitors of cIAP-1/2 and XIAP proteins

Abstract

A series of compounds were designed and synthesized as antagonists of cIAP-1/2 and XIAP based upon our previously identified lead compound SM-122 (1). The most potent of these (7) binds to XIAP, cIAP-1, and cIAP-2 proteins with K(i) values of 36, <1, and <1.9 nM, respectively. Consistent with its potent binding affinities to IAPs, 7 effectively antagonizes XIAP in a cell-free caspase-9 functional assay, efficiently induces cIAP-1 degradation in cells at concentrations as low as 10 nM, and triggers activation of caspases and PARP cleavage in the MDA-MB-231 breast cancer cell line. Compound 7 potently inhibits cell growth in the MDA-MB-231 cancer cell line with an IC(50) value of 200 nM and is 9 times more potent than compound 1.

Figure 1

Structures of Smac mimetics.

Figure 2

Smac mimetics antagonize XIAP BIR3 in a cell-free caspase-9 functional assay. 500 nM of XIAP BIR3 protein achieves 80% inhibition of caspase-9 activity in Caspase-Glo 9 assay kit and Smac mimetics dose-dependently restore the activity of caspase-9. Caspase-9 activity was measured after incubation with the caspase-9 specific substrate for 1 h.

Figure 3

Inhibition of cell growth by Smac mimetics in the MDA-MB-231 cancer cell line. Cells were seeded in 96-well flat bottom cell culture plates at a density of 3-4×10² cells/well and grown overnight, then incubated with Smac mimetics for 4 days, cell growth was determined using a WST-based assay.

Figure 4

Induction of cIAP-1 degradation, cleavage of PARP, and processing of caspase-3 by compounds 1, 2, 5 and 7 in the MDA-MB-231 cell line. Cells were treated with different concentrations of the compounds for 24 h and levels of cIAP-1, cleaved PAPR (CL PARP), and cleaved caspase-3 (CL C3) were probed by Western blot analysis.

Figure 5

Crystal structure of the key intermediate 9.

Scheme 1

Synthesis of key intermediates.

Scheme 2

Synthesis of Smac mimetics 3 - 7.