Design, synthesis and antiproliferative activity against colorectal cancer cells of methyl 2-(3-(2-oxo-3-phenylquinoxalin-1(2H)-yl)propanamido)alkanoates and related compounds

This work describes the design and synthesis of a series of new 2-oxo-3-phenylquinoxalines and their testing against colorectal cancer (HCT-116) cells designed for mixed MMP-2 and MMP-9 inhibition. Thirty new 2-oxo-3-phenylquinoxalines conjugated to amino acid esters via a propanoyl spacer were synthesized using chemo selective Michael reaction, DCC, and azide coupling methods. The derivatives, specifically methyl 2-(3-(2-oxo-3-phenylquinoxalin-1(2H)-yl)propanamido)alkanoates, reacted with hydroxylamine and hydrazine to produce N-hydroxypropanamides and hydrazides, respectively. Eleven compounds were evaluated for their anticancer activity against colorectal cancer (HCT-116) cells over 48 h using an in vitro MTT assay, with nine demonstrating significant inhibitory effects. DAPI staining revealed chromatin condensation and apoptotic body formation, confirming apoptosis induced by compound 8d Image analysis further quantified the impact of compound 8d, which showed the highest activity. In untreated control cells, 359 nuclei were detected, with a median diameter of 19.5 pixels and a total nuclear coverage of 44.1 %, while 321 nuclei were detected in treated cells, with a median diameter of 13.8 pixels and a reduced nuclear coverage of 20.6 %. Similarly, cell analysis showed a reduction in the number and size of cells, with untreated cells exhibiting a median cell diameter of 19.5 pixels and 44.3 % cell coverage, whereas treated cells had a reduced median diameter of 14.4 pixels and 22.0 % coverage. The results were further supported by molecular docking which substantiated a mixed MMP-2 and MMP-9 inhibitory activity and which could be correlated with the exhibited apoptotic pathway.