New phenothiazine conjugates as apoptosis inducing agents: Design, synthesis, In-vitro anti-cancer screening and 131I-radiolabeling for in-vivo evaluation

[Display omitted] •Design and synthesis of seventeen phenothiazine conjugates were performed and showed promising broad spectrum anti-proliferative activity.•Compounds (4b, 4 h, 4 g and 6e) showed potent induction of apoptosis with cell cycle arrest in sub-molecular phase of G0-G1 confirmed by modulation of key apoptotic genes (Casp-3, Casp-9, Bax and Bcl-2).•Radiolabeled 4b (131I −4b) showed promising anti-cancer activity with better localization and biodistribution in Ehrlich carcinoma mice model.•In-silico study suggested NADPH oxidases (NOXs) as potential molecular target. Phenothiazines (PTZs) are a group of compounds characterized by the presence of the 10H-dibenzo-[b,e]-1,4-thiazine system. PTZs used in clinics as antipsychotic drugs with other diverse biological activities. The current aim of the study is to investigate and understand the effect of potent PTZs compounds using a group of In-vitro and In-vivo assays. A total of seventeen novel phenothiazine derivatives have been designed, synthesized, and evaluated primarily in-vitro for their ability to inhibit proliferation activity against NCI-60 cancer cell lines, including several multi-drug resistant (MDR) tumor cell lines. Almost all compounds were active and displayed promising cellular activities with GI50 values in the sub-micromolar range. Four of the most promising derivatives (4b, 4h, 4g and 6e) have been further tested against two selected sensitive cancer cell lines (colon cancer; HCT-116 and breast cancer; MDA-MB231). The apoptosis assay showed that all the selected compounds were able to induce early apoptosis and compound 6e was able to induce additional cellular necrosis. Cell cycle assay showed all selected compounds were able to induce cell cycle arrest at sub-molecular phase of G0-G1 with compound 6e induced cell cycle arrest at G2M in HCT-116 cells. Accordingly, the apoptotic effect of the selected compounds was extensively investigated on genetic level and Casp-3, Casp-9 and Bax gene were up-regulated with down-regulation of Bcl-2 gene suggesting the activation of both intrinsic and extrinsic pathways. In-vivo evaluation of the antitumor activity of compound 4b in solid tumor bearing mice showed promising therapeutic effect with manifestation of dose and time dependent toxic effects at higher doses. For better evaluation of the degree of localization of 4b, its 131I-congener (131I-4b) was injected intravenously in Ehrlich solid tumor bearing mice that showed good localization