Synthesis, spectroscopic characterization, and anticancer activity of new 10-substituted 1,6-diazaphenothiazines

New phenothiazine derivatives as 10-substituted dipyridothiazines of the 1,6-diazaphenothiazine structure were obtained in the cyclization reaction of 3-amino-3′-nitro-2,2′-dipyridinyl sulfide and 3,3′-dinitro-2,2′-dipyridinyl disulfide, and in the reaction of 2-chloro-3-ntropyridine with sodium 3-amino-2-pyridinethiolate followed by various alkylation and arylation reactions. The reaction of the thiazine ring formation ran via the Smiles rearrangement of the S-N type. As the alkylation reactions could proceed at the thiazine, azine or both nitrogen atoms, the product structure elucidation was based on the 2D NMR (Rotating-frame Overhauser Effect Spectroscopy, Correlated Spectroscopy, Heteronuclear Single Quantum Coherence, and Heteronuclear Multiple Bond Correlation) spectra of the N-methylated product. Some 10-substituted 1,6-diazaphenothizines (5, 10, 12, 13) were at least anticancer active against melanoma C-32 and breast cancer MCF-7 cell lines as a reference drug – cisplatin. The monoazaphenothiazine drug, prothipendyl, turned out to be less active than least 6 derivatives of the 1,6-diazaphenothiazine structure.


Introduction
Tricyclic phenothiazines (dibenzo-1,4-thiazines) are important class of heterocycles possessing significant biological activities and interesting chemical features. Classical 10-substituted phenothiazines with the aminoalkyl groups at the nitrogen atom have been for many years valuable drugs exhibiting neuroleptic, antihistaminic, antitussive, and antiemetic activities (Gupta and Kumar, 1988). They are relatively easy-obtainable, inexpensive, and low toxic, and they can be valuable source for searching new drugs of other biological activities. The chemical structure modifications of these compounds were carried out mainly by introduction of new substituents at the thiazine nitrogen atom and substitution of one or two benzene rings with homoaromatic and heteroaromatic rings. Such modifications are expected to change not only potency but also types of activities. Both classical and modified phenothiazines are found to exhibit very promising anticancer, antibacterial, antifungal, anti-inflammatory, and multidrug resistance reversal activities, summarized recently in the review articles and chapters in monographs (Motohashi et al., 2000(Motohashi et al., , 2006Mitchell, 2006;Dasgupta et al., 2008;Aaron et al., 2009;Sudeshna and Parimal, 2010;Pluta et al., 2011;Wesołowska, 2011;Jaszczyszyn et al., 2012). They show also a potential benefit in treatment of Alzheimer's, Creutzfeldt-Jakob's, and AIDS-associated diseases (Mosnaim et al., 2006;González-Muñoz et al., 2010).
It is well known that the synthesis of phenothiazine and azaphenothiazine ring system may proceed via 1,4-thiazine ring formation with the use of diphenyl sulfides, phenyl azinyl sulfides or diazinyl sulfides directly in the Ullmann cyclization or indirectly through the Smiles rearrangement of the S-N type to diphenylamines, phenylazinylamines, and diazinylamines followed by cyclization. During the rearrangement, the phenyl or azinyl part migrates from the sulfur atom to the nitrogen atom Silberg et al., 2006). There only two reports of double Smiles rearrangement during those syntheses (Morak et al., 2002;Morak-Młodawska et al., 2012).
The synthesis of 10-substituted derivatives from 10Hdiazaphenothiazines by the alkylation of the thiazine nitrogen atom can be disturbed by alkylation of the azine nitrogen atom.
For those reasons, the unquestioned elucidation of the structure of the direct product, NH-azaphenothiazine, and its N-substituted derivatives is crucial.
In continuation of those studies we have worked out an efficient synthesis of another type of dipyridothiazines, 10H-1,6-diazaphenothiazine, and the transformation of this parent compound into 10-substituted derivatives, possessing alkyl, arylalkyl, heteroaryl and dialkylaminoalkyl, and imidoalkyl groups. In this work, we discuss the synthesis and the structure elucidation of the NH-and N-alkyl-1,6diazaphenothiazines, and their anticancer activity.

Chemistry
The possibility of the Smiles rearrangement depends on the sulfide structure and reaction conditions. The most often the rearrangement proceeds under basic conditions (sodium hydroxide in ethanol), rarely under neutral or acidic media. It is sometimes difficult to state if the rearrangement took place as the rearranged and non-rearranged products can have the same or similar structure. In the last case, the structure difference is in the location of a substituent or a nitrogen atom (in the azaphenothiazine structure) .
The next step was transformation of compound 7 into N-substituted derivatives mainly by alkylation. Although the alkylation of phenothiazines proceeds mainly at the thiazine nitrogen atom, there are a few reports on the alkylation of azaphenothiazines at the azine nitrogen atom giving azaphenothiazinium salts and neutral N-alkylazaphenothiazines (Clarke et al., 1961;Werle et al., 1962;Pappalardo et al., 1973;Carter and Cheeseman, 1977;Saari et al., 1983). We studied the reaction of compound 7 with methyl iodide in dry DMF in the presence of sodium hydride. The methylated product possessed only one methyl group (observed in the 1 H NMR spectrum) without the ammonium function what could point at the structure 8. To exclude alternative neutral N-methylazaphenothiazine structure i.e. 1-methyl-1,6-diazaphenothiazine 8A, we recorded 2D NMR (Rotating-frame Overhauser Effect Spectroscopy (ROESY), Correlated Spectroscopy (COSY), Heteronuclear Single Quantum Coherence (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC)) spectra of the N-methyl product. The ROESY experiment with irradiation of the methyl protons at 3.40 ppm showed the proximity of the methyl group to the proton at 6.98 ppm (γ-pyridinyl proton) and pointed at the structure 8. Only spatial 1 H-1 H connectivity with a proton at about 8 ppm (αpyridinyl proton) could point at the structure 8A. The full proton signal assignment was achieved by study of other proton spatial proximity (ROESY) and 1 H-1 H connectivities (COSY). The signal at 6.98 ppm was assigned as H-9 proton. The confirmation of the proton assignment came from the 13 C NMR spectrum which was solved by the use of HSQC and HMBC spectra indicating the 13 C-1 H relationship. The HSQC spectrum showed which proton was bonded to the carbon atom (the C-H relationship through one bond, 1 J C,H connectivity) and the HMBC spectrum indicated the C-H relationship through three (predominantly), two and four (exceptionally) bonds ( 3 J C,H , 2 J C,H and 4 J C,H connectivities). Selected spatial protonproton proximity, proton-proton and proton-carbon connectivities for compound 8 were shown in Scheme 2. The all 1 H-1 H and 1 H-13 C connectivities were included in Table 1. The resulted product was identified as 10methyldipyrido[2,3-b;2′,3′-e][1,4]thiazine 8. The 1 H and 13 C NMR spectra of the rest compounds were solved using the HSQC and HMBC experiments.

Anticancer activity
The anticancer activity of 1,6-diazaphenothiazines 7-19 was investigated in vitro using cultured glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cell lines. Normal human fibroblast (HFF-1) cell line was used as a control and cisplatin as a reference drug. To compare the influence of the nitrogen atoms in the azaphenothiazine system on the anticancer activity, the classical monoazaphenothiazine drug, prothipendyl (10-dimethylaminopropyl-1-azaphenothiazine), was also tested. The tested compounds exhibited different activities against the cell lines. The MCF-7 cell line was found as very sensitive for most compounds. Eight derivatives exhibited good anticancer activity with IC 50 o 10 μg/mL ( Table 2). The most active (IC 50 o 5 μg/ mL) were compounds 7, 10 and 12 with the hydrogen atom, and the propargyl and nitropyridinyl groups. Those compounds were more active than cisplatin. Compound 19 (with the methylpiperazinylbutynyl group) was as active as cisplatin and compounds 13, and 17 (with the diethylaminoethyl and phthalimidopropyl groups) were slightly less active.
The parent compound 7 and derivative 13 (with the diethylaminoethyl group) were as active as cisplatin against melanoma C-32 cell line. The SNB-19 cell line was the most resistant for the tested compounds. The most active derivative 9 (with the allyl group) exhibited IC 50 close to 20 μg/mL.
Prothipendyl, containing only one pyridine ring, was moderately active only against the MCF-7 cell line but about 5-6 times less active than the most active diazaphenothiazines. It worth noting that the most active compounds 7, 12 and 13 (together with six other compounds) were nontoxic against normal fibroblasts (HFF-1) with the IC 50 4 50 μg/mL whereas cisplatin turned out to be toxic. Scheme 3 Synthesis of 10-substituted 1,6-diazaphenothiazines

Conclusion
We report here synthesis of new 10-substituted 1,6-diazaphenothiazines. Parent compound, 10H-1,6-diazaphenothiazine 7, was obtained in three ways from appropriate dipyridinyl sulfide and disulfide, and a pair of 2,3-disubstituted pyridines. The thiazine ring formation ran via the Smiles rearrangement of the S-N type. The parent compound was transformed into 10-subtituted derivatives with the alkyl, heteroaryl, dialkylaminoalkyl, dialkylaminoalkynyl and imidoalkyl groups in the alkylation and heteroarylation reactions. As the alkylation reactions could proceed at the thiazine, azine or both nitrogen atoms, the product structure elucidation was based on the 2D NMR (ROESY, COSY, HSQC, and HMBC) spectra of the Nmethylated product. Some 1,6-diazaphenothiazines (7, 10, 12, 13) were at least anticancer active against melanoma C-32 and breast cancer MCF-7 cell lines as a reference drugcisplatin. Monoazaphenothiazine drug, prothipendyl, turned out to be less active than at least six derivatives of 1,6diazaphenothiazines against all three cancer cell lines.

Experimental Chemistry
Melting points were determined in open capillary tubes on a Boetius melting point apparatus and are uncorrected. The 1 H, 13 C NMR, COSY, NOESY, HSQC, HMBC spectra were recorded on a Bruker Ascend TM 600 spectrometer at 600 MHz in deuteriochloroform with tetramethylsilane as the internal standard. The 13 C NMR spectra were recorded at 150 MHz. Electron impact mass spectra (EI MS) and fast atom bombardment mass spectra (FAB MS, in glycerol) were run on a Finnigan MAT 95 spectrometer at 70 eV. The thin layer chromatography was performed on silica gel 60 F 254 (Merck 1.05735) with CHCl 3 -EtOH (5:1 and 10:1 v/v) and on aluminum oxide 60 F 254 neutral (type E) (Merck 1.05581) with CHCl 3 -EtOH (10:1 v/v) as eluents.

Cell proliferation and viability
In recent years tetrazolium salts have been described to be used for the measurement of cell proliferation and viability. The tetrazolium salts are cleaved to formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is quantified by a scanning enzyme-linked immunosorbent assay reader by measuring the absorbance of the dye solution at appropriate wavelengths (λ = 420-480 nm with a reference wavelength λ = 600 nm).

WST-1 assay
The WST-1 assay (Roche Diagnostics, Mannheim, Germany) was used to evaluate the effect of compounds on the number of cells in cultures, which as the cytotoxic effect of the tested compounds and their influence on the proliferation of cells. After exposure to tested compounds (at concentrations between 0 and 100 μg/mL) for 72 h, cells were incubated with WST-1 (10 μL) for 1 h, and the absorbance of the samples against a background control was read at 450 nm with a reference wavelength λ = 600 nm using a microplate reader UVM340 (Biogenet). Results are expressed as means of at least two independent experiments performed in triplicate.