An array of 4H-chromene derivatives have been reported for anticancer properties but their selectivity and mode of anticancer activity are unexplored. In this context, we have investigated a biologically active synthetically designed 4H-Chromene carbonitrile derivative, 2-amino-6-nitro-4-(4-oxo-2-thioxothiazolidin-5-yl)-4H-chromene-3-carbonitrile (ANC) that is strongly and selectively inhibited Bcl-2 over expressing human leukemic (HL-60 and K562) cells for its interaction and elucidated the mode of action. The interaction of ANC was investigated against the antiapoptotic proteins such as Bcl-2, Bax, Bcl-xL and Bcl-w that were overexpressed in leukemic cells using in silico and fluorescent spectroscopic studies. Fluorescent spectroscopic based interaction studies showed that the derivative had strong interaction with Bcl-xL followed by Bcl-2/Bax and least interaction with Bcl-w. Based on the results, the ANC had strong interactions with antiapoptotic Bcl-2 and Bax proteins than the Bcl-xL and Bcl-w proteins. The in vitro biological validation of ANC treated leukemic cells showed downregulation of Bcl-xL than Bcl-2 but least effect on Bcl-w proteins. Furthermore, the ANC had possible four isomers as RR, RS, SR and SS isomers. Among them, RS isomer of ANC had shown more active that correlated with biological interactions and gene expression studies of ACN with oncoproteins. These results confirmed the induction of apoptosis by RS-ACN isomer through inhibition of antiapoptotic machineries of leukemic cells confirming the antiapoptotic Bcl-2 inhibitory activities. Communicated by Ramaswamy H. Sarma. © 2022 Elsevier B.V., All rights reserved.