Cytotoxic Activity of Benzo[b]cyclohept[e][1,4]oxazines

BACKGROUND: Although numerous articles have dealt with the biological activities of azulenes, studies of benzo[b]cyclohept[e][1,4]oxazines are limited. In the present study, we investigated a total of 14 newly-synthesized benzo[b]cyclohept[e][1,4]oxazines for their growth stimulation at low concentrations (so-called 'hormesis'), cytotoxicity at higher concentrations and apoptosis-inducing activity. MATERIALS AND METHODS: Cytotoxicity of these compounds against human normal gingival fibroblast (HGF) and human oral squamous cell carcinoma cell lines derived from gingival tissue (Ca9-22), was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The tumor specificity (TS) was determined by the ratio of the 50% cytotoxic concentration (CC₅₀) value for HGF cells to that for Ca9-22 cells. Apoptosis induction was evaluated by DNA fragmentation and caspase-3 activation. RESULTS: Compounds 10-(2-methoxyethylamino)benzo[b] cyclohept[e][1,4]oxazine and 10-(3-methoxypropylamino) benzo[b]cyclohept[e][1,4] oxazine, but not other compounds, induced hormesis only in HGF cells. Compound 10-(6-hydroxyhexylamino)benzo[b] cyclohept[e][1,4]oxazine [4] showed the highest cytotoxicity against Ca9-22 cells, followed by 10-(4-hydroxybutylamino) benzo[b]cyclohept[e] [1,4]oxazine and 10-(5-hydroxypentylamino)benzo[b]cyclo-hept[e][1,4]oxazine. Compound [4] did not induce apoptosis markers, but rather induced necrotic cell death (characterized by a smear pattern of DNA fragmentation). CONCLUSION: The present study suggests that the OH group and a certain length of methylene group are necessary for maximal cytotoxicity, and substitution of fluoride in the benzene ring enhances cytotoxicity.