The synthesis and the study of the antitumor activity of 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]- thiadiazine hydrobromides

Aim. To synthesize and study the antitumor activity of 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine derivatives. Results and discussion. To determine the antitumor activity of 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine hydrobromides, the in vitro study was conducted on 60 lines of cancer cells (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) according to the standard procedure of the mitotic activity assessment of new potential bioactive compounds by the fluorescent coloring method (sulforhodamine B as a dye). It was performed in the US National Сancer Institute within the Development Therapeutic Program. It has been found that derivatives of 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine exhibit the antineoplastic activity against a wide range of cancer cells lines and are promising core structures for creating new effective anticancer agents. Experimental part. 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine hydrobromides were synthesized by the interaction of 4-amino-5-R-4H-1,2,4-triazole-3-thiols with 4-methoxyphenacyl bromide in ethyl acetate. The 1Н NMR spectra were recorded on a Bruker VXR-300 spectrometer (Germany) with the working frequency of 299.945 MHz. Conclusions. A series of 3-R-6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine hydrobromides has been synthesized. The anticancer activity of the compounds obtained has been studied in the National Cancer Institute on 60 lines of tumor cells. Compounds that exhibit high levels of the antitumor activity have been found. It has been shown that the replacement of 3-H in compound 3a with ethyl or pentyl radicals leads to increase in the antitumor activity against MDA-MB-468 breast cancer cells.

ISSN 2518-1548 (Online) ISSN 2308-8303 (Print) Cancer is the second most important cause of mortality in the world. Thus, in 2018, 9.6 million people died from the disease according to the WHO. About one third of deaths from cancer are due to five major sources of risk -a high body mass index, low levels of fruit and vegetable consumption, the lack of physical activity, smoking and excessive alcohol use. Smoking is the most significant risk factor for cancer development, accounting for almost 22 % of the world deaths from cancer [1]. Up to 25 % of cases of cancer in low-and middle-income countries are due to infections, such as hepatitis and human papillomavirus [2]. The prostate and lungs in men and the mammary gland in women are the most commonly affected by cancer.
Nowadays, cyclophosphamide, methotrexate, vincristine, adriablastin are widely used to treat tumor diseases. These drugs exhibit the necessary healing properties, but have significant side effects on the hematopoietic system (leukopenia, anemia, thrombocytopenia), central nervous system (feeling tired, dizziness, headache, aphasia, drowsiness, seizures), reproductive system (disorder of oogenesis and spermatogenesis, oligospermia, menstrual irregularity, decreased libido, impotence), urinary system (hematuria, cystitis, severe renal dysfunction), allergic and dermatological reactions, etc. In this way, the search for new highly effective antitumor drugs remains a pressing issue today.
Previously, triazole derivatives have been proven to have the antitumor activity. The known drug anastrozole [3 -7] (a derivative of 1,2,4-triazole) is active against estrogen dependent tumors of the breast in women. It is a selective non-steroidal enzyme antagonist of aromatase, which leads to a decrease in estradiol levels in peripheral tissues.
To determine the antineoplastic activity of 3-R-6-(4-methoxyphenyl)-7H-[1, 2,4]triazolo [3,4-b][1,3,4]thiadiazine hydrobromides 3 the in vitro study was conducted on 60 lines of cancer cells (leukemia, nonsmall cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) according to the standard procedure of the mitotic activity assessment of new potential bioactive compounds by the fluorescent coloring method (sulforhodamine B as a dye) performed in the US National Cancer Institute within the Development Therapeutic Program [14]. The substances were used in the concentration of 10 -5 mol/L, cancer cells were incubated with the compounds for 48 hours.
The results of the studies conducted were expressed as a percentage of the cancer cell growth compared to the reference drug 5-fluorouracil (Table 1). In vitro experiments revealed high levels of the anti-tumor activity of the test compounds against almost all lines of cancer cells.
According to the Table 1 compounds 3c -g possessed higher levels of the antineoplastic activity against cells of leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer compared to those of the reference drug 5-fluorouracil. Compounds 3a,b showed the activity at the level of the reference drug. Compound 3a exceeded the activity of the reference drug for 23 lines of cancer cells, 3bfor 21 lines, 3c -for 51 lines, 3d -for 52 lines, 3efor 59 lines, 3f -for 54 lines, and 3g -for 57 lines among 60 lines studied.
It should be noted that compound 3c stopped the growth and division of MDA-MB-468 cells of breast cancer, and destroyed them at the level of -0.91 %.  Compound 3f demonstrated the activity against NCI-H522 cells of non-small cell lung cancer at the level of -0.85 %, SK-MEL-5 melanoma -0.14 %, OVCAR-3 ovarian cancer -(-11.98 %), A498 renal cancer -(-1.24 %) and MDA-MB-468 breast cancer -19.00 %.   At the second stage of the study the most active compounds 3c, 3f and 3g were tested in five concentrations in 10-fold dilution series (100 μm, 10 μm, 1 μm, 0.1 μm and 0.01 μm) on the lines of human can-cer cells listed above. As a result of the experiment, three dose-dependent parameters were calculated: the GI 50 value (the growth inhibitory activity, effective inhibition level) corresponded to the concentration of the compound causing 50 % decrease in the net cell growth, the TGI value (cytostatic activity) -the concentration of the compound resulting in the total growth inhibition (TGI), and the LC 50 value (cytotoxic activity)the concentration of the compound causing a net 50 % loss of initial cells at the end of the incubation period.
If logarithmic values of the parameters studied (lgGI 50 , lgTGI and lgLC 50 ) were less than -4.00, the compound was considered to be active [14 -16].
According to the screening results (Table 2), the compounds demonstrated the considerable level of the anti- Table 2 The results of the in-depth in vitro screening of compounds 3c,f,g in the concentration gradient of 10 -4 -10 -8 М
The melting points were measured on a smallsized heating table with a RNMK 05 observation device (VEB Analytik, Dresden).

Biology Part
The methodology of the NCI procedure for the primary anticancer assay is detailed in Anticancer Drug Development Guide [14]. Briefly, the protocol was performed on 60 human tumor cell lines derived from different nine neoplastic diseases. NCI-60 testing was performed as a single concentration, which was tested on all 60 cell lines in a single dose of 10 −5 M. All of the assays were in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, USA. If the results obtained met selection criteria, then the compound was tested again on all 60 cell lines in 5 × 10-fold dilutions with the top dose being 10 −4 M.
Conflict of interests: the authors have no conflict of interests to declare.