Substituted 2-[(2-Oxo-2H-[1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetamides with Thiazole and Thiadiazole Fragments: Synthesis, Physicochemical Properties, Cytotoxicity, and Anticancer Activity

The series of novel N-R-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamides with thiazole and thiadiazole fragments in a molecule were obtained by alkylation of potassium salts 1.1–1.4 by N-hetaryl-2-chloroacetamides and by aminolysis of activated acids 2.1–2.4 with N,N’-carbonyldiimidazole (CDI). The structures of compounds were determined by IR, 1H NMR, MS, and EI-MS analysis. The results of cytotoxicity evaluated by the bioluminescence inhibition of bacterium Photobacterium leiognathi, Sh1 showed that the compounds have considerable cytotoxicity. The synthesized compounds were tested for anticancer activity in NCI against 60 cell lines. Among the highly active compounds 3.1, 3.2, and 6.5, 2-[(3-methyl-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-N-(1,3-thiazol-2-yl)acetamide (3.1) was found to be the most active anticancer agent against the cell lines of colon cancer (GI50 at 0.41–0.69 μM), melanoma (GI50 0.48–13.50 μM), and ovarian cancer (GI50 0.25–5.01 μM). The structure-activity relationship (SAR-analysis) was discussed.

The identity of the synthesized compounds was confirmed by LC-MS (APCI), elemental analysis, IR, 1 H, 13 C NMR, and MS. The chromato-mass study of the synthesized compounds 3.1-3.6, 4.1-4.11, 5.1-5.9, 6.1-6.5 in a "soft" ionization (chemical ionization at atmospheric pressure) allowed each in each case to register the molecular ion peak [M+1] in high intensity. In addition, the characteristic ion [M+3] was present in the majority of compounds, which characterized the presence of the sulfur isotope in the molecule.
A «classical» chemical shift of carbon atoms of the -S-CH 2 -group in the 13 C NMR spectra of amides 3.1, 3.2, 4.2, 4.5-4.7, 5.2, 5.6, and 5.7 was observed at 35.55-35.30 ppm. It is important that diamides 6.2, 6.3 have two characteristic signals for the carbon atom of the -S-CH 2 -group at 35. 28-35.23 ppm and 38.13-38.12 ppm. This aspect clearly confirms the S-regioselectivity of the reaction. In addition, the low magnetic field deshielded the carbon atom of the -CONH-group and carbons at the 2 and 6 positions of the [1,2,4]triazino [2,3-c] 22-167.25 ppm, 164.77-158.45 ppm, and 161.02-154.59 ppm. Thus, the position and splitting of signals in the spectra of 1 H and 13 C NMR were in accordance with the proposed structures and clearly prove their structure and S-regioselectivity of alkylation.
For thiazolylamide 3.2, low-intensity М +• (3.0%) was observed in the mass spectra, the main directions of fragmentation were caused by the α-and β-break of the bond with the formation of fragmentary ions +• (F 1 ) m/z 320 (

Bioluminescence inhibition test
In the first stage, we researched the cytotoxicity of the synthesized compounds by the bioluminescent test of the luminescent bacteria Photobacterium leiognathi Sh1 (Table 1). Among the synthesized substances, compounds 3.1-3.6, 4.1-4.11, 5.1-5.9, 6.1-6.5 on the model of acute action showed inhibiting properties at the concentration gradient of 0.025-0.25 mg/mL, which were typical for compounds 5.2-5.9 with benzothiazolamide and compounds 6.2-6.5, 4.2, 4.7, 4.9-4.11 with the thiadiazolamide fragment in the molecule.
It's important that among the mentioned compounds, only 4.10, 5.3, and 6.3 have shown high rates of inhibitory activity (inhibition of growth of 94. .70%) at a concentration of 0.25 mg/mL in acute models of action. Meanwhile, in the chronic test, most of the synthesized compounds exhibited cytotoxic effects (Table 1). It should be noted that the compounds 3.1, 3.4, 3.5, 4.2, 4.4, 4.6, 5.1, 5.5, 5.7, and 6.4 in the chronic test showed the effect of hormezis, inhibiting the intensity of bioluminescence in the smallest concentration (0.025 mg/mL). The increasing cytotoxicity of the compounds 3.1, 3.3, 3.5, 3.6, 4.2-4.6, 4.10, 5.1, 5.3-5.6, 5.8, and 6.5 at a concentration of 0.01 and 0.25 mg/mL in the chronic test was expected.

Tab. 1.
Bioluminescence inhibiting assay data, % The SAR study revealed that: [1,2,4]triazino [2,3-c]quinazolin-6-yl)-thio]acetic acids with a thiazolamide fragment exhibited a high cytostatic effect against the luminescent bacteria Photobacterium leiognathi Sh1;  a cytostatic effect of the synthesized compounds was retained with the replacement of thiazolamide by benzothiazole or thiadiazolamide fragments in the molecule,  the replacement of the methyl group in position 3 of the triazino [2,3-c]quinazoline cycle by aryl function in most cases was the reason for the increased cytotoxic action,  the replacement of the methyl group in position 6 in the benzothiazolamide fragment by the methoxygroup or chlorine didn't lead to the loss of cytostatic action, and  the introduction of a butyl substituent in position 5 of the thiadiazolamide fragment resulted in the initiation of growth of the luminescent bacteria Photobacterium leiognathi Sh1.
Thus, high levels of growth inhibition and cytotoxicity of compounds to the bacteria P. leiognathi Sh1 served as a prerequisite for further research on their antitumor activity. [1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetamides with thiazole and thiadiazole fragments in the molecules (3.1-3.6, 4.1-4.11, 5.1-5.9, 6.1-6.5) were submitted and evaluated at the single concentration of 10 μM in the panel of approximately 60 cancer cell lines. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the US NCI protocol, which was described elsewhere (see e.g. http://dtp.nci.nih.gov) [29][30][31]. The results of primary screening are reported as the percent cancer cell line growth (GP%) and are presented in Table 2. The range of growth % shows the lowest and the highest growth % found among the different cancer cell lines.

Anticancer assay for preliminary in vitro testing
The conducted screening studies of compounds at a concentration of 10 μM showed that the compounds 3.1, 3.2, 6.5 turned to be effective against most cancer cell lines (Table 2). [1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetamide (3.1) showed high cytotoxic activity against 57 cell lines (inhibition of growth by 30-100%). Replacement of the methyl substituent at position 3 (compound 3.1) of the triazinoquinazoline cycle by phenyl (3.2) and 4-methoxyphenyl (3.4) substituents led to a decrease in cytotoxicity against most cancer cell lines ( or benzothiazole (5.1, 5.4, 5.7, 5.8) fragments caused a reduction in the spectrum and a decline in the antitumor activity. It is important that these compounds selectively inhibit the growth of the cell lines SSRF-CEM of leukemia. Further modification of the molecule by combining thiadiazole and arylamide fragments (compounds 6.4, 6.5) didn't lead to significantly increased antitumor activity, but retained selective action against the cell line SSRF-CEM of leukemia and expanded the spectrum of compound 6.5 (inhibition of growth of 57 cell lines). Further dose-dependent activity was investigated for the most effective compounds 3.1, 3.2, and 6.5 according to the standard NCI procedure for the 57-59 lines of nine types of cancer at five concentrations at 10-multiply dilution (100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM). In the experimental result, the three dose-dependent parameters were calculated: 1) GI 50 -molar concentration of the compound that inhibits 50% net cell growth; 2) TGI -molar concentration of the compound leading to a total inhibition of cell growth; 3) LC 50 -molar concentration of the compound leading to 50% net cell death. Furthermore, the mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an average activity parameter over all of the cell lines for the tested compounds. For the calculation of the MG_MID, insensitive cell lines were included with the highest concentration tested (Table 3).

Percentage of in vitro
Among the studied substances,  The SAR study revealed that: [1,2,4]triazino [2,3-c]quinazolin-6-yl)-thio]acetic acids with a thiazolamide fragment demonstrated high antitumor activity,  the replacement of thiazolamide by benzothiazole or thiadiazolamide fragments leed to a significant loss in antitumor activity,  the replacement of the methyl group in position 3 of the triazino [2,3-c]quinazoline cycle by the phenyl, 4-methylphenyl, or 4-methoxyphenyl group caused the modest increase in activity against some cancer cell lines,  the replacement of the methyl group at position 6 of the benzothiazolamide fragment by methoxygroup or chlorine didn't lead to a significant increase in activity,  the introduction of the butyl substituent in position 5 of the thiadiazolamide fragment was the reason for a slight increase in activity against some cancer cell lines, and  the combination of one molecule of 2-[(5-amino-1,3,4-thiadiazol-2-yl)thio]-Nphenylacetamide and 2-[(3-R-2-oxo-2H- [1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetamide fragments leed to a high level of antitumor activity and requires further study.

Conclusion
In the present paper, 31 new N-R-2-[(3-R-2-oxo-2H- [1,2,4]triazino [2,3-c]quinazolin-6yl)thio]acetamides with thiazole and thiadiazole fragments in the molecules were described. The results of cytotoxicity evaluated by bioluminescence inhibition of the bacterium Photobacterium leiognathi, Sh1 showed that the compounds revealed significant cytotoxicity. Thirteen of the synthesized compounds were tested and most of them displayed antitumor activity against leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines. Screening of anticancer activity in vitro yielded the most active compounds 3.1, 3.2, and 6.5 in micromolar concentrations at the GI 50 level (GI 50 mean graph midpoint varies from 6.47 up to 19.69).  [1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetic acid by broadening the base of amides is indisputably interesting and is being considered, which will be the premise for the creation of more effective anticancer agents among the barely known heterocyclic system.

Chemistry
Melting points were determined in open capillary tubes and were uncorrected. The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL cube analyzer (USA). Analyses were indicated by the symbols of the elements or functions within ±0.3% of the theoretical values. The IR spectra (4000-600 cm -1 ) were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Bioscience, Germany) using a module for measuring attenuated total reflection (ATR). The 1 H NMR spectra (400 MHz) and 13 [1][2][3]. Other starting materials and solvents were obtained from commercially available sources and used without additional purification.

Method B
N,N'-carbonyldiimidazole (1.95 g, 0.011 mol) was added to a solution of proper acid (2.1-2.4) (0.01 mol) in 10 ml of anhydrous DMF and heated in the water bath at 60-80°С for 1 hour with a calcium chloride tube. The proper 4-R-1,3-thiazolyl-2-аmines (0.01 mol) were added with stirring to the resulting mixture and refluxed for 5-6 hours. The mixture was poured into the water, and neutralized to pH 6-7 by acetic acid. The precipitate was filtered, dried, and recrystallized from the DMF-water (1:1).

Method B
N,N'-carbonyldiimidazole (1.95 g, 0.011 mol) was added to a solution of proper acid (2.1-2.4) (0.01 mol) in 10 ml of anhydrous DMF and heated in the water bath at 60-80°С for 1 hour with a calcium chloride tube. The proper 5-R 1 -1,3,4-thiadiazolyl-2-аmines (0.01 mol) were added with stirring to the resulting mixture and refluxed for 5-6 hours. The mixture was poured into the water, and neutralized to pH 6-7 by acetic acid. The precipitate was filtered, dried, and recrystallized from the DMF-water (1:1).

Cytotoxic activity against malignant human tumor cells
The primary anticancer assay was performed at the human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [27][28][29][30]. Tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with the test agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10 −4 to 10 −8 M. A 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth. Using the seven absorbance measurements [time zero, (T z ), control growth in the absence of the drug (C), and test growth in the presence of the drug at the five concentration levels (T i )], the percentage growth was calculated at each of the drug concentrations levels.
Percentage growth inhibition was calculated as: [(T i − T z )/(C − T z )] × 100 for concentrations for which T i ≥ T z , [(T i − T z )]/T z ] × 100 for concentrations for which T i < T z .
Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(T i − T z )/(C − T z )] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from T i = T z . The LC 50 (concentration of the drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment, was calculated from [(T i − T z )/T z ] ×100 = -50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. The log GI 50 , log TGI, and log LC 50 were then determined, defined as the mean of the logs of the individual GI 50 , TGI, and LC 50 values. The lowest values were obtained with the most sensitive cell lines.