Synthesis of Novel Pyrido[4,3-e][1,2,4]triazino[3,2-c][1,2,4]thiadiazine 6,6-dioxide Derivatives with Potential Anticancer Activity

A series of novel 3-/2,3-substituted pyrido[4,3-e][1,2,4]triazino[3,2-c][1,2,4]thiadiazine 6,6-dioxides 4–28 have been synthesized by the reaction of 3-amino-2-(4-thioxo-1,4-dihydropyridin-3-yl-sulfonyl)guanidine with either 2-oxoalkanoic acids and its esters, or phenylglyoxylic hydrates in glacial acetic acid. Some of them exhibited reasonable or moderate anticancer activity toward human cancer cell lines, HCT-116, MCF-7 and HeLa. The structure of this novel heterocyclic ring system was confirmed by 1D-NMR and 2D-NMR spectroscopic data including COSY, ROESY and HMBC, elemental analyses and MS spectrometry.


Introduction
Cancer is the second most life threatening non-communicable disease after cardiovascular disease, according to the World Health Organization [1].Despite the availability of numerous anticancer drugs, cancer is still hard to cure, especially without exhibiting any side effects.Due to the serious toxicity of conventional cytotoxic medicines contemporary medicinal chemistry is focused on the development of potent and selective anticancer drugs.Heterocyclic scaffolds play an important role for the design of novel drugs by enhancing their biological effects when fused with other ring systems.Among the many heteroaromatic rings, pyridothiadiazines constitutes an important framework of anticancer and antibacterial agents (I-III, Figure 1) [2] as well as anticancer and tuberculostatic compounds (IV, Figure 1) [3].At the same time, there are also many reports indicating the significant anticancer properties of the 1,2,4-triazine fragment (V-X, Figure 1) [4][5][6][7][8][9][10].It is known that 2-alkylthio-5-chloro-N-(1,2,4-triazin-yl)benzenesulfonamides V exhibit anticancer activity against colon, CNS, melanoma, ovarian, breast, renal, and leukemia cell lines [4].The 6-azauridine VI, an inhibitor of de novo pyrimidine biosynthesis, has been described as an effective agent for inducing remission of acute myelocytic leukemia in children [5].Other analogs of 6-azauridine, the S-alkyl derivatives of 1,2,4-triazinone VII, present cytotoxic activities against human breast cancer (MCF-7), colon carcinoma (HCT-116) and hepatocellular carcinoma (Hep-G2) cell lines [6].Tirapazamine VIII is a known bioreductive hypoxia-selective cytotoxin, currently undergoing a variety of phase I, II and III clinical trials for the treatment of various human cancers, including non-small cell lung, cervical, head and neck, and ovarian cancers.Tirapazamine derives its medicinal activity by inducing DNA damage in poorly oxygenated tumor cells [7].The heterobicyclic derivative bearing the 1,2,4-triazine moiety IXa have demonstrated remarkable inhibitory effects against uterus cancer (SiHa) cells and human colon adenocarcinoma (LS 180) cells while simultaneously inducing DNA cleavage [8].Moreover, its close analog IXb was identified as capable of inducing significantly higher levels of necrotic cells in human breast cancer (T47D) cell lines and human cervical epithelial carcinoma (HeLa) cells [9].Some of 1,2,4-triazine-5-ones X have distinct antiptoliferative activities against the chronic myeloid leukemia (K-562) cell line combined with a low cytotoxicity [10].IXa have demonstrated remarkable inhibitory effects against uterus cancer (SiHa) cells and human colon adenocarcinoma (LS 180) cells while simultaneously inducing DNA cleavage [8].Moreover, its close analog IXb was identified as capable of inducing significantly higher levels of necrotic cells in human breast cancer (T47D) cell lines and human cervical epithelial carcinoma (HeLa) cells [9].Some of 1,2,4-triazine-5-ones X have distinct antiptoliferative activities against the chronic myeloid leukemia (K-562) cell line combined with a low cytotoxicity [10].It has been known that arylsulfonamides exhibit a variety of biological activities including anticancer properties [11,12].Our previous reports underlined the anticancer activity of arylsulfonamide analogues containing various heterocyclic ring systems attached to the benzenesulfonamide core [13][14][15][16][17][18].Recently we have developed of a new method for the synthesis of 2,3-diaryl-9,9-dioxo-1H-9-thia-1,4,4a,7,10-pentaazaphenanthrene-2-ols [19].This original class of compounds prompted us to investigate anticancer activity of arylsulfonamides modified by pyridothiadiazine and 1,2,4-triazine cores.In our research for novel various substituted pyrido [4,3-e] [1,2,4]triazino-[3,2-c][1,2,4]thiadiazine 6,6-dioxides (XI,XII, Figure 1) with potential anticancer activity, we developed synthetic pathways for their preparation and evaluated their cytotoxic activity toward human cancer cell lines HCT-116, HeLa and MCF-7.

Chemistry
The synthesis of the key starting material, i.e., 3-amino-2-(4-thioxo-1,4-dihydropyridin-3-yl-sulfonyl)guanidine (3) was achieved in a three-step reaction sequence starting from 3-methylthio-1,1-dioxopyrido[4,3-e]-1,4,2-dithiazine (1) according to the previously described procedure [19] (Scheme 1).An attempt was made to apply 3-amino-2-(4-thioxo-1,4-dihydropyridin-3-ylsulfonyl)guanidine (3) to the reaction with phenylglyoxal hydrates due to the well-known fact that in acidic conditions the electrophlicity of its formyl group is superior to that of a carbonyl group in the reaction with An attempt was made to apply 3-amino-2-(4-thioxo-1,4-dihydropyridin-3-ylsulfonyl)guanidine (3) to the reaction with phenylglyoxal hydrates due to the well-known fact that in acidic conditions the electrophlicity of its formyl group is superior to that of a carbonyl group in the reaction with substituted aminoguanidines [20].Such an approach may open a convenient way to obtain the similar pyrido [4,3-e] The structures of 4-28 were confirmed by IR, NMR and MS data and elemental analyses.In the IR spectra of compounds 4-20 the characteristic OH group appeared as overlapping broad absorption bands in the 3543-3417 cm −1 .In turn, in the 1 H-NMR spectra of the compounds 4-28 the chemical shifts of protons H-7, H-9 and H-10 attributable to pyridine ring of the tricyclic fused ring system were found in the region of 9.06-9.25,9.03-9.80and 7.79-8.18ppm, respectively (Figures S1-S28, see Supplementary Materials).

Anticancer Activity
Compounds 4-28 were evaluated in vitro for their effects on the viability of three human cancer cell lines: HCT-116 (colon cancer), HeLa (cervical cancer) and MCF-7 (breast cancer).The concentration required for 50% inhibition of cell viability IC50 was calculated and compared with the reference drug cisplatin, and the results are given in Table 1.The compounds 5, 15, 17, 21-23 and 25-28 exhibited either reasonable, moderate or weak anticancer activity against HCT-116 cell line, while five of them i.e. 22, 23, 25, 26 and 28 were also potent against MCF-7 and HeLa human cancer cell lines, whereas remaining compounds 4, 6-14, 16, 18-20 and 24 were essentially inactive in tested concentration range.The structures of 4-28 were confirmed by IR, NMR and MS data and elemental analyses.In the IR spectra of compounds 4-20 the characteristic OH group appeared as overlapping broad absorption bands in the 3543-3417 cm ´1.In turn, in the 1 H-NMR spectra of the compounds 4-28 the chemical shifts of protons H-7, H-9 and H-10 attributable to pyridine ring of the tricyclic fused ring system were found in the region of 9.06-9.25,9.03-9.80and 7.79-8.18ppm, respectively (Figures S1-S28, see Supplementary Materials).

Anticancer Activity
Compounds 4-28 were evaluated in vitro for their effects on the viability of three human cancer cell lines: HCT-116 (colon cancer), HeLa (cervical cancer) and MCF-7 (breast cancer).The concentration required for 50% inhibition of cell viability IC 50 was calculated and compared with the reference drug cisplatin, and the results are given in Table 1.The compounds 5, 15, 17, 21-23 and 25-28 exhibited either reasonable, moderate or weak anticancer activity against HCT-116 cell line, while five of them i.e., 22, 23, 25, 26 and 28 were also potent against MCF-7 and HeLa human cancer cell lines, whereas remaining compounds 4, 6-14, 16, 18-20 and 24 were essentially inactive in tested concentration range.
To summarize the biological test results, the replacement of an aryl (Ar) substituent by a hydroxy (OH) functionality at position 3 with simultaneously shifting of the substituent R 2 = alkyl, benzyl, substituted aryl to the position 2 caused the loss of activity towards the HeLa and MCF-7 cell lines in compounds 7-20.Among a series of 2,3-substituted derivatives only weak activity against HCT-116 cell lines for 5 (R 2 = Bn), 15 (R 2 = 4-i-PrC 6 H 4 ) and 17 (R 2 = 4-t-BuC 6 H 4 ) was observed.
The rational design of more effective and safe compounds may be supported by in silico approaches that predict the human in vivo metabolism and reactivity of small molecules.Therefore in the present studies we applied an on-line accessible tool for accurate prediction of xenobiotic metabolism sites, called XenoSite Cytochrome P450 Prediction Models [21] and estimated the stability of the most active compounds 22, 23, 25 and 28 in the presence of human liver microsomes.In silico results illustrated in Figure 2 show which atoms on a molecule are likely to be oxidized by human liver microsomes.Thus, it was found that the prominent compound 23 demonstrated not only the minimal IC 50 values against tested cell lines, but also the high metabolic stability with regard to oxidative metabolic processes.In turn, drug toxicity, frequently described by the quantitative strength of a molecule's reactivity with glutathione, was also predicted by use of the XenoSite Reactivity Model [22].The results of the in silico analysis did not indicate any toxicity of compounds 22, 23, 25 and 28.  1).
To summarize the biological test results, the replacement of an aryl (Ar) substituent by a hydroxy (OH) functionality at position 3 with simultaneously shifting of the substituent R 2 = alkyl, benzyl, substituted aryl to the position 2 caused the loss of activity towards the HeLa and MCF-7 cell lines in compounds 7-20.Among a series of 2,3-substituted derivatives only weak activity against HCT-116 cell lines for 5 (R 2 = Bn), 15 (R 2 = 4-i-PrC6H4) and 17 (R 2 = 4-t-BuC6H4) was observed.
The rational design of more effective and safe compounds may be supported by in silico approaches that predict the human in vivo metabolism and reactivity of small molecules.Therefore in the present studies we applied an on-line accessible tool for accurate prediction of xenobiotic metabolism sites, called XenoSite Cytochrome P450 Prediction Models [21] and estimated the stability of the most active compounds 22, 23, 25 and 28 in the presence of human liver microsomes.In silico results illustrated in Figure 2 show which atoms on a molecule are likely to be oxidized by human liver microsomes.Thus, it was found that the prominent compound 23 demonstrated not only the minimal IC50 values against tested cell lines, but also the high metabolic stability with regard to oxidative metabolic processes.In turn, drug toxicity, frequently described by the quantitative strength of a molecule's reactivity with glutathione, was also predicted by use of the XenoSite Reactivity Model [22].The results of the in silico analysis did not indicate any toxicity of compounds 22, 23, 25 and 28.     4) by proton and carbon assignments, which are given in Tables 2-5.The resonance signal of proton H7 in compound 7 was identified as the only one singlet showing correlations to four sp 2 carbons C7a, C9, C10 and C10a in HMBC spectrum.The COSY spectrum of 7 allowed the connectivities within two structural blocks, namely C9-C10 and C14-C16 to be traced.The C7, C9-C10 and C14-C16 spin systems were distinguished by the presence of H7/H9 ROE and upon the C2/H14 heteronuclear correlation in HMBC spectrum.Finally, the assignments of quaternary sp 2 carbon atoms C2, C7a, C10a and C13 were made upon several heteronuclear correlations observed in the HMBC spectrum, listed in Tables 2 and 3.     4) by proton and carbon assignments, which are given in Tables 2-5.The resonance signal of proton H7 in compound 7 was identified as the only one singlet showing correlations to four sp 2 carbons C7a, C9, C10 and C10a in HMBC spectrum.The COSY spectrum of 7 allowed the connectivities within two structural blocks, namely C9-C10 and C14-C16 to be traced.The C7, C9-C10 and C14-C16 spin systems were distinguished by the presence of H7/H9 ROE and upon the C2/H14 heteronuclear correlation in HMBC spectrum.Finally, the assignments of quaternary sp 2 carbon atoms C2, C7a, C10a and C13 were made upon several heteronuclear correlations observed in the HMBC spectrum, listed in Tables 2 and 3.

NMR Studies
The  4) by proton and carbon assignments, which are given in Tables 2-5.The resonance signal of proton H7 in compound 7 was identified as the only one singlet showing correlations to four sp 2 carbons C7a, C9, C10 and C10a in HMBC spectrum.The COSY spectrum of 7 allowed the connectivities within two structural blocks, namely C9-C10 and C14-C16 to be traced.The C7, C9-C10 and C14-C16 spin systems were distinguished by the presence of H7/H9 ROE and upon the C2/H14 heteronuclear correlation in HMBC spectrum.Finally, the assignments of quaternary sp 2 carbon atoms C2, C7a, C10a and C13 were made upon several heteronuclear correlations observed in the HMBC spectrum, listed in Tables 2 and 3.

Biological Methods
Cell Culture and Cell Viability Assay All chemicals, if not stated otherwise, were obtained from Sigma-Aldrich (St. Louis, MO, USA).The MCF-7 cell line was purchased from Cell Lines Services (Eppelheim, Germany), the HeLa and HCT-116 cell lines were obtained from the Department of Microbiology, Tumor and Cell Biology, Karolinska Institute (Stockholm, Sweden).Cells were cultured in in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin.Cultures were maintained in a humidified atmosphere with 5% CO 2 at 37 ˝C in an incubator (Hera Cell, Heraeus, Langenselbold, Germany).Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) assay.Cells were seeded in 96-well plates at a density of 5 ˆ10 3 cells/well and treated for 72 h with the examined compounds in the concentration range 1-100 µM.Cisplatin was used as a control compound and was examined in the concentration range 0.01-10 µM.Following treatment, MTT (0.5 mg/mL) was added to the medium and cells were further incubated for 2 h at 37 ˝C.Cells were lysed with DMSO and the absorbance of the formazan solution was measured at 550 nm with a plate reader (1420 multilabel counter, Victor, Jügesheim, Germany).The optical density of the formazan solution was measured at 550 nm with a plate reader (Victor 1420 multilabel counter).The experiment was performed in triplicate.Results are expressed as IC 50 values.Values are expressed as the mean ˘SD of at least three independent experiments.

Figure 2 .
Figure 2. Sites of metabolism predicted for 22, 23, 25 and 28 by the XenoSite software[21].The green color indicates more vulnerability to biotransformation than blue.Some significant differences are additionally pointed out.

Figure 2 .
Figure 2. Sites of metabolism predicted for 22, 23, 25 and 28 by the XenoSite software[21].The green color indicates more vulnerability to biotransformation than blue.Some significant differences are additionally pointed out.

Molecules 2016, 21 , 41 7 of 15 Figure 2 .
Figure 2. Sites of metabolism predicted for 22, 23, 25 and 28 by the XenoSite software[21].The green color indicates more vulnerability to biotransformation than blue.Some significant differences are additionally pointed out.

Table 3 .
13C-NMR data for 7 with long-range C/H couplings observed in HMBC spectrum.

Table 5 .
13C-NMR data for 25 with long-range C/H couplings observed in HMBC spectrum.