Antineoplastic multi-targeted spiro[indoline-3,2'-pyrrolidines] incorporating urea function: Rational design,...

The research study is dedicated towards synthesis of novel spiro[indoline-3,2'-pyrrolidines] incorporating a urea functionality. Crucially, the study seeks to elucidate their anticipated multi-targeted mechanism of action, moving towards the final objective of producing multi-functional antineoplastic agents. 1,3-Dipolar cycloaddition was used to construct the final compounds, the spiro-analogs 15a-m. 2-Propen-1-ones 12a-j provided a crucial part of the structure (the urea group). Isatins 13a,b and sarcosine 14, reacted together during the process to form in situ the necessary dipole, azomethine ylide. This synthetic protocol produced the targeted agents 15a-m with high yields. Biological evaluation identified compound 15l as a highly potent agent against the HCT116 colon cancer line, outperforming sunitinib and 5-fluorouracil by 3.7-fold and 7.9-fold, respectively. It is also identified with efficacy against MCF7 (breast cancer) close to that of 5-fluorouracil. Additionally, compound 15h exhibited exceptional potency against A431 skin squamous carcinoma surpassing 5-fluorouracil by 9.1-fold. Mechanistic studies confirmed the dual-action of rational design. Compound 15h demonstrated VEGFR-2 inhibition comparable to sorafenib, while 15g emerged as a dual MDM2 inhibitor and p53 activator, significantly outperforming doxorubicin. The correlation between MDM2 inhibition and p53 activation suggests that the disruption of the MDM2-p53 protein-protein interaction, alongside VEGFR-2 inhibition, drives the antiproliferative effects. Furthermore, compounds 15g, 15h, 15j, and 15k induced apoptosis through the upregulation of caspase-3 and BAX and the downregulation of Bcl-2. Cell cycle (flow cytometry) studies of 15h and 15j confirmed G0/G1 phase arrest. Moreover, the Annexin V-FITC/PI dual staining assay evidenced the apoptotic potential of compounds 15h and 15j. CAM assays supported the anti-angiogenic potential of 15g, 15h, 15j, and 15k. Finally, molecular docking (PDB ID: 5LAW) and dynamic simulations validated the binding stability and mechanism within the MDM2 pocket.