Exploring pyrrolidinyl-spirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/CDK2 inhibitors for halting breast cancer cells

Abstract

Cancer represents a global challenge, and the pursuit of developing new cancer treatments that are potent, safe, less prone to drug resistance, and associated with fewer side effects poses a significant challenge in cancer research and drug discovery. Drawing inspiration from pyrrolidinyl-spirooxindole natural products, a novel series of spirooxindoles has been synthesized through a one-pot three-component reaction, involving a [3 + 2] cycloaddition reaction. The cytotoxicity against breast cancer cells (MCF-7 and MDA-MB-231) and safety profile against WISH cells of the newly developed library were assessed using the MTT assay. Compounds <b>5l</b> and <b>5o</b> exhibited notable cytotoxicity against MCF-7 cells (IC₅₀ = 3.4 and 4.12 μM, respectively) and MDA-MB-231 cells (IC₅₀ = 8.45 and 4.32 μM, respectively) compared to Erlotinib. Conversely, compounds <b>5a-f</b> displayed promising cytotoxicity against MCF-7 cells with IC₅₀ values range (IC₅₀ = 5.87-18.5 μM) with selective activity against MDA-MB-231 cancer cells. Compound <b>5g</b> demonstrated the highest cytotoxicity (IC₅₀ = 2.8 μM) among the tested compounds. Additionally, compounds <b>5g</b>, <b>5l</b>, and <b>5n</b> were found to be safe (non-cytotoxic) against WISH cells with higher IC₅₀ values ranging from 39.33 to 47.2 μM. Compounds <b>5g</b>, <b>5l</b>, and <b>5n</b> underwent testing for their inhibitory effects against EGFR and CDK-2. Remarkably, they demonstrated potent EGFR inhibition, with IC₅₀ values of 0.026, 0.067, and 0.04 μM and inhibition percentages of 92.6%, 89.8%, and 91.2%, respectively, when compared to Erlotinib (IC₅₀ = 0.03 μM, 95.4%). Furthermore, these compounds exhibited potent CDK-2 inhibition, with IC₅₀ values of 0.301, 0.345, and 0.557 μM and inhibition percentages of 91.9%, 89.4%, and 88.7%, respectively, in contrast to Roscovitine (IC₅₀ = 0.556 μM, 92.1%). RT-PCR analysis was performed on both untreated and <b>5g</b>-treated MCF-7 cells to confirm apoptotic cell death. Treatment with <b>5g</b> increased the gene expression of pro-apoptotic genes P53, Bax, caspases 3, 8, and 9 with notable fold changes while decreasing the expression of the anti-apoptotic gene Bcl-2. Molecular docking and dynamic simulations (100 ns simulation using AMBER22) were conducted to investigate the binding mode of the most potent candidates, namely, <b>5g</b>, <b>5l</b>, and <b>5n</b>, within the active sites of EGFR and CDK-2.