Molecular hybridization design and synthesis of novel spirooxindole-based MDM2 inhibitors endowed with BCL2 signaling attenuation; a step towards the next generation p53 activators

Abstract

Despite the achieved progress in developing efficient MDM2-p53 protein-protein interaction inhibitors (MDM2 inhibitors), the acquired resistance of tumor cells to such p53 activators posed an argument about the druggability of the pathway. Combination studies disclosed that concomitant inhibition of MDM2 and BCL2 functions can sensitize the tumor cells and synergistically induce apoptosis. Herein, we employed a rapid combinatorial approach to generate a novel series of hybrid spirooxindole-based MDM2 inhibitors (5a-s) endowed with BCL2 signaling attenuation. The adducts were designed to mimic the thematic features of the chemically stable potent spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-ones MDM2 inhibitors while installing a pyrrole ring on the core via a carbonyl spacer inspired by the natural product marinopyrrole A that efficiently inhibits BCL2 family functions by various mechanisms. NCI 60 cell-line panel screening revealed their promising broad-spectrum antiproliferative activities. The NCI-selected derivatives were screened for cytotoxic activities against normal fibroblasts, MDA-MB 231, HepG-2, and Caco-2 cells via MTT assay, subjected to mechanistic apoptosis studies for assessment of p53, BCL2, p21, and caspase 3/7 status, then evaluated for potential MDM2 inhibition utilizing MST assay. The most balanced potent and safe derivatives; 5i and 5q were more active than 5-fluorouracil, exhibited low μmrange MDM2 binding (K_D=1.32and 1.72 μm, respectively), induced apoptosis-dependent anticancer activities up to 50%, activated p53 by 47-63%, downregulated the BCL2 gene to 59.8%, and reduced its protein level (13.75%) in the treated cancer cells. Further downstream p53 signaling studies revealed > 2 folds p21 upregulation and > 3 folds caspase 3/7 activation. Docking simulations displayed that the active MDM2 inhibitors resided well into the p53 binding sites of MDM2, and shared key interactions with the co-crystalized inhibitor posed by the indolinone scaffold (5i, 5p, and 5q), the halogen substituents (5r), or the installed spiro ring (5s). Finally, in silico ADMET profiling predicted acceptable drug-like properties with full accordance to Lipinski's, Veber's, and Muegge's bioavailability parameters for 5i and a single violation for 5q.

Keywords: BCL2; MDM2 inhibitors; MDM2-p53 interaction; Spirooxindole; [3+2] cycloaddition reaction (32CA).