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The activity of the newly synthesized azaphenothiazines: tricyclic 10-substituted dipyridothiazines 1-9, pentacyclic 6-substituted diquinothiazines 10-22 and hexacyclic diquinothiazinium salt 23 was tested on 55-60 in vitro cell lines. The cell lines included nine types of cancer: leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer (National Cancer Institute, Bethesda, MD, USA). The features of the chemical substituent at the thiazine nitrogen atom confer the anticancer activity of diquinothiazines 10-23. Unexpectedly, the most active of the dipyridothiazines 1-9 was the unsubstituted compound 1 (the substituent is a hydrogen atom). The most cytotoxic compound was the half-mustard derivative 18. The GI(50) value of this compound was -7.06 (corresponding to 40 ng/ml) when tested on the melanoma cell line SK-MEL-5 and -6.0 - -6.62 using cell lines from various cancers including: leukemia (CCRF-CEM), the MOLT-4 cell line, colon cancer (HCT-116), central nervous system cancer (SNB-75 and SF-295), prostate cancer (PC-3), non-small cell lung cancer (NCI-H460 and HOP-92), ovarian cancer (IGROV1 and OVCAR-4) and breast cancer (MDA-MB-460). The ethylene group in the aminoalkylazaphenothiazines is as a good linker and is similar to the propylene and butylene linkers in aminoalkylphenothiazines. To our knowledge, this is the first demonstration of significant azaphenothiazine anticancer activity.

in Wiley InterScience (www.interscience.wiley.com).The review surveys the chemistry of diaza-, triaza-, and tetraazaphenothiazines and their benzo and dibenzo derivatives consisting of over 30 different heterocyclic systems.

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 μg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.

3,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3'-nitro-2,4'-dipyridinyl sulfide and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed by alkylation and heteroarylation. The thiazine ring formation ran via the Smiles rearrangement. The structure elucidation was based on 2D NMR and X-ray analysis of N-methylated product. 3,6-Diazaphenothiazines were investigated for antitumor activity using glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cells. 10H-3,6-diazaphenothiazine was 10 times more active (IC50 < 0.72 μg/mL) than cisplatin. Two diazaphenothiazines with the 2-pyrimidinyl and dimethylaminopropyl substituents were selectively active against MCF-7 and C-32 cells. The expressions of H3 (proliferation marker), TP53, CDKN1A (cell cycle regulators), BAX and BCL-2 (proapoptopic and antiapoptopic genes) were detected by RT-QPCR method. The expression analysis suggests the cell cycle arrest and the mitochondrial apoptosis pathway activation in MCF-7 and SNB-19 cells.