10H-1,9-diazaphenothiazine and its 10-derivatives: synthesis, characterisation and biological evaluation as potential anticancer agents

Abstract 10H-1,9-diazaphenothiazine was obtained in the sulphurisation reaction of diphenylamine with elemental sulphur and transformed into new 10-substituted derivatives, containing alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenothiazine ring system was identified with advanced 1H and 13C NMR techniques (COSY, NOESY, HSQC and HMBC) and confirmed by X-ray diffraction analysis of the methyl derivative. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB-231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza- and 3,6-diazaphenothiazine).


Introduction
In the last decades, cancer has been one of the main causes of death worldwide affecting millions of people per year. The main forms of a curative treatment for tumours are surgery, radiation, chemotherapy and biotherapy 1,2 . The goal of chemotherapeutic agents is to cure the tumour, to prolong survival and to reduce the tumour burden to alleviate symptoms. In recent years, a lot of effort has been applied to the synthesis of potential anticancer drugs with better selectivity and minor or no side effects [1][2][3][4] .
Synthetical and natural bioactive compounds with heterocyclic ring systems play an important role for the development of novel scaffolds in medicinal chemistry 5,6 . One of the most active heterocyclic rings is a 1,4-thiazine ring, containing the nitrogen and sulphur atoms 7 . This ring fused with two benzene rings forms a dibenzothiazine system, present in one of most valuable drugsphenothiazines. Classical phenothiazines with the dialkylaminoalkyl groups at the nitrogen atom (and additional simple group at the carbon atom in position 2) have still been recognised as neuroleptic, antihistaminic, antitussive and antiemetic drugs 8 . Recently, many papers were published revealing new activities for these compounds, for example, thioridazine, one of the most known phenothiazines, exhibits promising properties for multidrug-resistant tuberculosis treatment 9 and lung cancer therapy through targeting lung cancer stem cells, due to its efficacy and safety 10 .
On the other hand, the phenothiazine structure has been modified mainly by introduction of new substituents at the thiazine nitrogen atom and by replacement of one or two benzene rings with various azine rings (leading to azaphenothiazines). Recent numerous original reports, reviews and chapters in monographs describe new promising biological activity of both classical and modified phenothiazines such anticancer, anti-plasmid, antiviral, anti-inflammatory and antibacterial activities, reversal of multi-drug resistance 8,[11][12][13][14][15][16][17][18][19][20] . They are promising candidates for further studies directed to the development of new drugs useful in the treatment of Creutzfeldt-Jakob's, Alzheimer's and other neurodegenerative diseases, like amyotrophic lateral sclerosis, Parkinson's and Huntington's diseases 8,[21][22][23] .
The aim of this paper is elaboration of efficient synthesis of 10H-1,9-diazaphenothiazine, transformation this compound into 11 varied 10-substituted derivatives and determination of their anticancer activity against selected tumour cell lines. Since the synthesis of the parent compound was only mentioned in a patent 40 without any details, the elaboration of efficient method of preparation is crucial challenge.

Chemistry
Melting points were determined in open capillary tubes on a Boetius melting point apparatus and are uncorrected. The 1 H NMR, COSY, ROESY, HSQC, HMBC spectra were recorded on an Ascend TM 600 spectrometers at 600 MHz in deuteriochloroform with tetramethylsilane as the internal standard. The 13 C NMR spectrum was recorded at 75 MHz. Electron impact mass spectra (EI MS), fast atom bombardment mass spectra (FAB MS, in glycerol), chemical ionisation (CI MS) were run on a Finnigan MAT 95 spectrometer at 70 eV and HR MS was run on a Brucker Impact II. The thin layer chromatography was performed on silica gel 60 F 254 (Merck 1.05735) with CHCl 3 -EtOH (10:1 v/v) and on aluminium oxide 60 F 254 neutral (type E) (Merck 1.05581) with CHCl 3 -EtOH (10:1 v/v) as eluents.

Proliferation assay
The antiproliferative effect of the compounds obtained from both the cancer and the normal cells was determined using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). This colorimetric assay is based on the viable cell's ability to cause the bright red-coloured stable tetrazolium salt (2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) to cleave to the dark red soluble 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. An increase in the amount of formazan dye formed correlates to the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is quantified by a scanning ELISA reader that measures the absorbance of the dye solution at appropriate wavelengths. The examined cells were exposed to the tested compounds for 72 h at various concentrations between 0.1 and 100 mg/ml (prepared initially at a concentration of 1 mg/ml in DMSO). The control was performed in order to check that DMSO has no effect on the cells at the concentration used. The cells were incubated with WST-1 (10 ml) for one hour and the absorbance of the samples was measured against a background control at 450 nm using a microplate reader with a reference wavelength at 600 nm. The results are expressed as the means of at least two independent experiments performed in triplicate. The antiproliferative activity of the tested compound was compared to cisplatin. The IC 50 values (a concentration of a compound that is required for 50% inhibition) were calculated from the dose-response relationship with respect to control.
The RT-QPCR method Genes trancriptional activity (H3, TP53, CDKN1A, BCL-2, BAX) was evaluated by the real-time RT-QPCR method with OPTICON TM DNA Engine (MJ Research, Watertown, MA) and QuantTect V R SYBR V R Green RT-PCR Kit (Quiagen, Valencia, CA). Cells were exposed to compounds 5 and 8 at a concentration of 0.5 mg/ml for 24 h. The RNA extraction was made by using Quick-RNA TM Kit MiniPrep (ZYMO RESEARCH). Total RNA integrity was analysed in 1.2% agarose electrophoresis with added ethidium bromide compound. The quantity and purity of extracted total RNA were determined by using spectrophotometric analysis with HP845 (Hewlett Packard, Waldbronn, Germany) spectrophotometer. The statistical analysis was performed using the Statistica 8.0 software (StatSoft, Tulsa, OK). All values were expressed as means ± SE.

Apoptosis antibody detection array
The Proteome Profiler Human Apoptosis Array (R&D Systems) kit simultaneously detects the relative expression level of 35 apoptosis-related proteins. In short, the SNB-19 cells were treated with compounds 5 and 9 at a concentration of 0.5 mg/ml. All immunodetection steps were performed in accordance with the manufacturer's instruction. The blots were detected using an enhanced chemiluminescence system using LI-COR C-Digit Blot Scanner.

Annexin-V apoptosis detection assay
Apoptosis was analysed via Annexin-V-FLUOS Staining kit (Roche, Germany) according to the manufacture's instruction. Briefly, after 24 h incubation of the SNB-19 cells with compounds 5 and 8 (0.5 mg/ml), the samples were collected, washed with phosphatebuffered solution and treated with annexin and propidium iodide. The stained cells were analysed by a flow cytometry (LSR II Becton Dickinson flow cytometer). Viable, early apoptopic, late apoptopic and necrotic cells were determined by staining Annexin V À /PI À , Annexin V þ /PI À , Annexin V þ /PI þ Annexin V À /PI þ , respectively.
Our sulphurisation of 2,2 0 -dipyridinylamine (1) with elemental sulphur at 250 C for 1 h in open air conditions (without a solvent) gave 10H-1,9-diazaphenothiazine (2) in 28% yield. Sulphurisation in an autoclave using dioxane as a solvent was less efficient than in open air giving the desired product in 17% yield. Atempted sulphurisations in other solvents (ethylene glycol in open air conditions and water, chloroform, DMF and monomethyl ether of diethylene glycol in an autoclave) were not satisfactory. The best result (42%) was achieved when the reaction (without a solvent) was carried in a microwave reactor.

Spectroscopic analysis
The Rath product was not characterised at all 40 . Our spectroscopic study revealed a molecular formula of C 10 H 7 N 3 S from HR MS spectrum and only 3 pyridine proton signals of an AMX system in the 1 H NMR spectrum which pointed at a symmetrical structure of the sulphurisation product. To find the location of the azine nitrogen atoms in the dipyridothiazine system, the sulphurisation product was methylated with methyl iodide in dry DMF in the presence of sodium hydride. The simple 1 H NMR spectrum revealed four signals: the methyl group and three pyridine protons confirming that the methyl group is attached to the thiazine nitrogen atom.
To find the pyridine nitrogen atoms 2D NMR ROESY spectrum was recorded. An irradiation of the methyl protons at 3.45 ppm did not show any proximity of the methyl group to the protons confirming the pyridine nitrogen atoms to be in positions 1 and 9. The full assignment of the proton and carbon signals came from other 2D NMR spectra showing 1 H-1 H (COSY) and 13 C-1 H connectivities (HSQC and HMBC, in Supplementary Material). Those last two spectra showed the C-H relationship through one bond ( 1 J C,H connectivity) and three bonds ( 3 J C,H connectivity).

X-ray diffraction study
Such a high temperature process could lead to many stable and unstable compounds and some rearrangements cannot be neglected. The NMR analysis is an indirect and subtle method of the structure elucidation, a single-crystal X-ray diffraction analysis of 10-methyl derivative (3) was performed (the direct sulphurisation product did not give crystals good enough for Xray diffraction measurement).
The X-ray diffraction study confirmed the product structure concluded from the 1 H NMR spectra as 10-methyl-1,9-diazaphenothiazine and revealed a spatial arrangement in the molecule in a solid state (Figure 1).
In all known studies of N-methyl dipyridothiazine crystals, the tricyclic ring systems are planar or folded depending on the methyl group location 35,47,52 . The ring system in compound (3) is also folded along the S-N axis with the butterfly angle of 36.22(4) between two pyridine ring planes. The central thiazine ring is in boat conformation with the angle between two halves (SCCS) of 41.28 (6) . The methyl group is located in equatorial position with the S5ÁÁÁN10-C11 angle of 170.1(1) .

Apoptosis assay
Compounds 5 and 8 were selected as the most promising 1,9-diazaphenothiazines to study the mechanism of anticancer action using the RT-QPCR method. This method analysed the gene transcriptional activities of proliferation marker (H3) cell cycle regulator (TP53 and CDKNIA) and intracellular apoptosis pathway (BACL-2 and BAX). The obtained results on three cancer cell lines are collected in Table 2.
The growth, division and eventual death of the cells in the body are processes that are controlled by hundreds of genes working together. The gene encoding the histone H3 is involved in the cell cycle progression and is considered as an indicator of proliferation in molecular studies. It plays an important role in regulation of the expression of the genetic information encoded in DNA 53,54 . Both compounds reduced considerably (5 is more potent) the number of mRNA copies in all cancer lines what can be a result of a modification of the chromatin structure.
Tumour protein p53 is TP53 gene product, one of the most known tumour suppressor genes, which is involved in anticancer action by various mechanisms. The p53 protein is activated by a variety of cell stresses, such as DNA damage, oncogene activation, spindle damage and hypoxia. Activated p53 transactivates a number of target genes, many of which are involved in DNA repair, cell cycle arrest and apoptosis [55][56][57] . Compounds 8 and 5 decreased mRNA copies in 3 or 2 cancer lines, respectively (only an increase in mRNA copies was observed in C-32 line). The cell cycle inhibitor CDKN1A (p21) tightly controlled by the p53 protein is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main and well-known function of protein p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. This protein is also involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions were analysed 58,59 . Compound 8 induced increase in the expression of CDKN1A in all cancer lines but compound 5 only in breast cancer lines.
P53 protein is also responsible for keeping the right balance between expression of a proapoptotic BAX gene and an antiapoptotic BCL-2 gene. These proteins have special significance since they can determine if the cell commits to apoptosis or aborts the process. It is thought that the main mechanism of action of the BCL-2 family of proteins is the regulation of cytochrome c release from the mitochondria via alteration of mitochondrial membrane permeability 60 . In general, compounds 5 and 8 reduced (with some exception) the mRNA expression of BCL-2. In contrast to this, the compounds enhanced or reduced the expression of BAX. The ratio of BAX/BCL-2 can determine whether cells will die via apoptosis or be protected from it. In comparison with the control, the BAX/BCL-2 ratio was found to be greater for both compounds in relation to SNB-19 and MDA-MB-231 cell lines.
In summary, the analysis of the gene expression revealed that To further understand the mechanism of action of compounds 5 and 8, we performed a determination of apoptosis-related proteins using Proteome Profiler Human Apoptosis Array. We identified 12 expressed proteins in the response to the compounds in the SNB-19 cells (Supplementary Material). Proteins such as phospho-p53 (S15, phosphorylation at ser15), phospho-p53 (S46) and phospho-p53 (S392) play an important role in cell proliferation as DNA damage response, induction of apoptosis and growth suppression 61 and all three proteins were found in the protein array. We found proteins which implicated in apoptosis: BAX, pro-caspase-3, cytochrome c and SMAC/Diablo. The last protein is a proapoptogenic mitochondrial protein which interacts and antagonises inhibitors of apoptosis proteins (IAPs) thus allowing the activation of caspases and apoptosis 62 . BAX accelerates programmed cell death by binding to mitochondrial membrane (MOMP) releasing cytochrome c, promoting activation of caspase-3 and triggering apoptosis 63 . It seems that compounds 5 and 8 induce BAX to form a channel in MOMP and release cytochrome c to activate caspases 9 and 3 (promoted also by SMAC/Diablo) thus initiating apoptosis through the intrinsic mitochondrial pathway. However, further studies are required to confirm the precise mechanism of this anticancer action.

Conclusion
We report here efficient synthesis of 10H-1,9-diazaphenothiazine in the sulphurisation reaction of diphenylamine with elemental sulphur and its transformation into new 10-substituted derivatives, containing the alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenotiazine ring system was identified with advanced 1 H and 13 C NMR techniques and confirmed by single-crystal X-ray crystallography of the methyl derivative. X-ray diffraction analysis revealed nonplanar tricyclic ring system with the substituent at the thiazine nitrogen atom in an equatorial location. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. The expression of H3, TP53, CDKN1A, BCL-2 and BAX genes for those two compounds was detected by the RT-QPCR method. The analysis of the gene expression revealed that both compounds inhibited the proliferation in all cells (H3) and activated of mitochondrial events of apoptosis (BAX/BCL-2) in two cancer cell lines (SNB-19 and MDA-MB-231). The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza-and 3,6diazaphenothiazine).

Disclosure statement
No potential conflict of interest was reported by the authors.