Synthesis, Anticancer Activity, and Apoptosis Induction of Novel 3,6-Diazaphenothiazines †

New 10-substituted derivatives of 3,6-diazaphenothiazine, containing the triple bond linker terminated with tertiary cyclic and acyclic amine groups, were synthesized and screened for their anticancer action. The compounds exhibited varied anticancer activities against human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cell lines, depending on the nature of the substituents. The most active 3,6-diazaphenothiazine, 4, was the derivative with the N,N-diethylamino-2-butynyl substituent against glioblastoma SNB-19, and was ten times more potent than cisplatin. For this compound, the expression of H3, TP53, CDKN1A, BCL-2, and BAX genes was detected by the RT-qPCR method. The gene expression ratio BAX/BCL-2 indicated the induction of mitochondrial apoptosis in cancer cell lines. The transformation of the propynyl substituent into amino-2-butynyl can be a method applicable to the search for more anticancer-active azaphenothiazines.

Molecules 2019, 24, x 3 of 9 in good yields (68-84%). The structures of the new compounds were characterized with the use of spectroscopic data: 1 H-NMR, 13 C-NMR, FAB-MS, and HR-MS.

No
Anticancer Activity IC50 (μg/mL) SNB The derivatives 3 and 4, with the N-methyl-N-ethylamino-2-butynyl and N,N-diethylamino-2butynyl groups, were found to be the most active against the SNB-19 cell line (IC50 = 0.11 μg/mL). Strong activity was observed also for derivative 4 against the C-32 cell line. In those cases, the activity was even higher than for cisplatin. Significant activity (IC50 = 0.45-0.74 μg/mL) was also demonstrated by the derivative 9 with the N-phenylpiperazinyl-2-butynyl fragments against cell lines 1 and 3. The compounds 5-7 with the aminobutynyl substituents, containing the pyrrolidine, piperidine, and morpholine moieties, showed very weak activity in the range of tested concentrations. Derivative 2 (possessing only the propynyl group), being the starting material in the synthesis of the tested Scheme 1. Synthesis of 3,6-diazaphenothiazines with dialkylaminoalkynyl substituents.

Anticancer Activity
The activity of target compounds (3)(4)(5)(6)(7)(8)(9) was investigated in vitro using cultured human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cell lines and cisplatin as a reference. The tested compounds exhibited varied degrees of activity depending on the type of tested cell lines and the nature of the substituent at the thiazine nitrogen atom (Table 1). The derivatives 3 and 4, with the N-methyl-N-ethylamino-2-butynyl and N,N-diethylamino-2-butynyl groups, were found to be the most active against the SNB-19 cell line (IC 50 = 0.11 µg/mL). Strong activity was observed also for derivative 4 against the C-32 cell line. In those cases, the activity was even higher than for cisplatin. Significant activity (IC 50 = 0.45-0.74 µg/mL) was also demonstrated by the derivative 9 with the N-phenylpiperazinyl-2-butynyl fragments against cell lines 1 and 3. The compounds 5-7 with the aminobutynyl substituents, containing the pyrrolidine, piperidine, and morpholine moieties, showed very weak activity in the range of tested concentrations. Derivative 2 (possessing only the propynyl group), being the starting material in the synthesis of the tested compounds, showed low anticancer activity (IC 50 = 32.9-45.1 µg/mL), as described earlier [33]. This means that the transformation of the propynyl substituent into amino-2-butynyl can increase the anticancer activity. The finally obtained derivatives were compared with the isomeric derivatives of the 1,6-, 1,8-and 2,7-diazaphenothiazines previously described. Compound 4 is the most potent derivative in the group of dipyridothiazines described up to now. The activity is at least 300 times more potent than its isomers [28,32]. Based on the conducted research, it can be concluded that the places of additional nitrogen atoms in the phenothiazine moiety have an influence on antiproliferative activity.

Apoptosis Assay
The most active derivative 4 was selected for studies on the mechanism of antitumor activity through the use of gene expression analysis: proliferation marker (H3), cell cycle regulators (TP53, CDKN1A), and markers of the apoptosis pathway (BCL-2 and BAX). Antiproliferative activity of the tested compound for C-32 cells was confirmed in the analysis of the expression of a gene encoding the histone H3, which is an accepted marker of proliferation in molecular studies. The product of TP53 gene is the P53 protein, which acts as a genome guardian and is also involved in the regulation of many cell processes. One of this protein's major tasks is stopping the cell cycle when DNA damage occurs that is not repaired, and the P53 protein can initiate apoptosis. In cells, apoptosis can be initiated by intrinsic mitochondrial pathway with BAX and BCL-2 involved (pro-and antiapoptotic proteins family). A new target in anticancer therapies is to restore the P53 activity in tumor cells, which could lead to the precise degradation of cancer cells. This protein product also regulates the P21 protein gene (CDKN1A) expression. This protein selectively binds to cyclin-dependent kinase complexes with cyclins and regulates the cell cycle. This means that an inadequate number of the P21 protein, or its mutations in cells, can induce an oncogenic transformation [35][36][37][38].
The results of the analysis of H3, TP53, CDKN1A, BCL-2, and BAX genes in SNB-19, C-32, MDA-MB231 cells after 24 h of treatment are collected in Table 2. Compound 4 reduced, significantly, the number of mRNA copies of H3 in all cell lines, suggesting an alteration in chromatin structure. There is also a reduction in the number of copies of the gene TP53, significantly in the C-32 cells, but slightly in others. An increase in the number of CDKN1A copies in the SNB-19 and MDA-MB231 cells points to the possibility of the induction of cell cycle arrest and apoptosis.
The P53 protein can also stimulate the cell to changes in the gene expression of proapoptopic BAX and antiapoptopic BCL-2 involved in mitochondrial pathway apoptosis. BAX protein's role in cells is the increase of the mitochondrial membrane permeability by the formation of pores, while BCL-2 protein is responsible for the release of cytochrome C into the cytosol [39]. As a result of the described study, an increased BAX/BCL-2 ratio indicates the activation of mitochondrial apoptosis in the SNB-19 cells. Transcriptional activity of these genes in MDA-MB231 and C-32 cells suggests a different way of cell death and, possibility, of a protective activation.

Cell Proliferation and Viability
Antiproliferative effect of the obtained compounds on cancer cells was determined using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). This assay is based on the viable cell's ability to cleave the bright red-colored tetrazolium salt [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] to a dark red soluble formazan by cellular enzymes. 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 by measuring 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 µg/mL and 100 µg/mL (prepared initially at a concentration of 1 mg/mL in DMSO). The cells were incubated with WST-1 (10 µL) for 1 h and the absorbance of the samples against a background control was measured at 450 nm using a microplate reader with a reference wavelength of 600 nm. The results are expressed as 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 (concentration of the compound that is required for 50% inhibition) were calculated from the dose-response relationship with respect to control.

RT-qPCR Method
Gene transcriptional activity (H3, TP53, CDKN1A, BCL-2, BAX) was evaluated by real time RT-qPCR method with OPTICON TM DNA Engine (MJ Research, Watertown, NY, USA) and QuantTect ® SYBR ® Green RT-PCR Kit (Qiagen, Valencia, spain). Cells were exposed to compound 4 at 0.5 µg/mL concentration for 24 h. The RNA extraction was made by using Quick-RNA™ Kit MiniPrep (ZYMO RESEARCH, Irvine, CA, USA). Total RNA integrity was analyzed using 1.2% agarose electrophoresis with added ethidium bromide. 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, USA). All values were expressed as means ± SE.

Conclusions
We report here synthesis of new 10-substituted derivatives of 3,6-diazaphenothiazine with more rigid substituents than dialkylaminoalkyl, containing the triple bond linker ended with tertiary cyclic and acyclic amine groups. These compounds exhibited varied anticancer activities against human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cancer lines, depending on the nature of the substituents. Three compounds (3, 4, and 9) were more active than the parent molecule, 10H-3,6-diazaphenothiazine. The most active compound was 3,6-diazaphenothiazine 4 with the N,N-diethylamino-2-butynyl substituent against the glioblastoma SNB-19, ten times more potent than cisplatin. This compound was more potent than its 1,6-, 1,8-, and 2,7-analogs. The location of the azine nitrogen atoms in the dipyridothiazine system seems to be crucial for anticancer activity. For this compound, the expressions of H3, TP53, CDKN1A, BCL-2, and BAX genes were detected by the RT-qPCR method. The gene expression ratio of BAX/BCL-2 indicated the induction of mitochondrial apoptosis in the SNB-19 cells. The transformation of the propynyl substituent into dialkylamino-2-butynyl via the Mannich reaction can be a method for searching for more anticancer active azaphenothiazines.

Conflicts of Interest:
The authors have declared no conflict of interest.