A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

We report here a series of 27 10-(4-phenylpiperazin-1-yl)methanones derived from tricyclic heterocycles which were screened for effects on tumor cell growth, inhibition of tubulin polymerization, and induction of cell cycle arrest. Several analogues, among them the 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-10H-phenoxazine-3-carbonitrile (16o), showed excellent antiproliferative properties, with low nanomolar GI values (16o, mean GI of 3.3 nM) against a large number (93) of cancer cell lines. Fifteen compounds potently inhibited tubulin polymerization. Analysis of cell cycle by flow cytometry revealed that inhibition of tumor cell growth was related to an induction of G2/M phase cell cycle blockade. Western blotting and molecular docking studies suggested that these compounds bind efficiently to β-tubulin at the colchicine binding site. Our studies demonstrate the suitability of the phenoxazine and phenothiazine core and also of the phenylpiperazine moiety for the development of novel and potent tubulin polymerization inhibitors.