Design, synthesis, and evaluation of tricyclic, conformationally constrained small-molecule mimetics of second mitochondria-derived activator of caspases

Abstract

A series of tricyclic, conformationally constrained Smac mimetics have been designed, synthesized, and evaluated. The most potent compound 6 (WS-5) binds to XIAP, cIAP-1, and cIAP-2 with K(i) of 18, 1.1, and 4.2 nM, respectively. Compound 6 antagonizes XIAP in a functional assay, induces cIAP-1 degradation, inhibits cell growth with an IC(50) of 68 nM in the MDA-MB-231 cancer cell line, and effectively induces cancer cells to undergo apoptosis.

Figure 1

Examples of reported Smac mimetics.

Figure 2

Chemical structures of new Smac mimetics.

Figure 3

Predicted binding models of compounds 1 and 5 in complex with XIAP BIR3, in superposition on the crystal structure of Smac in complex with XIAP BIR3. Protein is displayed in surface model with key binding residues shown and labeled. Compounds 1, 5, and AVPI peptide are shown in stick. Carbon atoms of AVPI peptide are depicted in green and carbon atoms of compounds 1 and 5 are depicted in yellow. Oxygen and nitrogen atoms in these compounds are shown in red and blue, respectively.

Figure 4

Inhibition of cell growth by Smac mimetics in the MDA-MB-231 human breast cancer cell line. Cells were treated for 4 days and cell growth was determined using a WST-8 assay.

Figure 5

Induction of cell death by compounds 5, 6 and 8 in the MDA-MB-231 breast cancer cell line. Cells were treated with different concentrations of the compounds for 48 hours. Cell viability was determined using a trypan blue exclusion assay.

Figure 6

Functional antagonism of Smac mimetics against XIAP BIR3 in a cell-free functional assay. Addition of dATP and cyctochrome c into the MDA-MB-231 cell lysates induced activation of caspase-3/-7 and XIAP BIR3 at 500 nM completely inhibited the caspase-3/-7 activity. Compounds 1, 5, 6, 7 and 8 dose-dependently recovered the caspase activity.

Figure 7

Western blot analysis of the levels of cIAP-1, caspase-8, pro- and cleaved PARP. MDA-MB-231 breast cancer cells were treated with different concentrations of Smac mimetics for 24 hours and proteins were probed with specific antibodies. GAPDH was used as the loading control.

Scheme I

Synthesis of compounds. 5-7. Reagents and conditions: (a) i. H₂, 10% Pd-C, methanol; ii. N-phthaloyl-L-phenylpropanoic acid, EDC, HOBt, N-methylmorpholine, CH₂Cl₂-DMF 1:1, 0°C - rt, overnight, 92% over two steps; (b) CF₃COOH, 4 Å molecular sieve, CHCl₃, reflux, 88%; (c) trifluorosulfonic acid, trifluorosulfonic anhydride, CH₂Cl₂, 98%; (d) i. hydrazine hydrate, methanol, 3 days; ii. L-N-Boc-N-methyl-alanine, EDC, HOBt, N-methylmorpholine, CH₂Cl₂-DMF 1:1, 0 °C - rt, overnight, 92% over two steps; (e) i. 2 N LiOH, then 1 N HCl; ii. amine, EDC, HOBt, N-methylmorpholine, CH₂Cl₂-DMF 1:1, 0 °C - rt, overnight; iii. ZnBr₂, CH₂Cl_2.