Anticancer properties of some triazolo[3,4-b

In the present work, we presented an efficient synthesis and anticancer activity evaluation of some new 3-R-6-(5-arylfuran-2-yl-[1,2,4]triazolo[3,4-b ][1,3,4]thiadiazoles. We have shown that the proposed synthetic protocols provided the possibility to design triazolothiadiazoles diversity with a considerable chemical novelty. The structures of target substances were confirmed by using 1 H NMR spectroscopy, mass spectrometry and elemental analysis. The synthesized compounds were selected by the National Cancer Institute Developmental Therapeutic Program for the in vitro cell line screening. Among all the substances tested, three compounds exhibited significant cytotoxic activity.


Introduction
Cancer disorders are one of the most dangerous medical, biological and social problems today and are the cause of up to 12% of all deaths and inferior to this indicator only to cardiovascular diseases. 1 Despite the rapid development of modern organic, pharmaceutical and medicinal chemistry, the effectiveness of antitumor drugs remains low. This is largely due to the nonspecificity of their action, the resistance of tumors, insufficient study of the mechanisms of disease pathogenesis. In addition, the treatment of serious diseases is expensive, for example, the therapy of stage III cancer costs 25-30 thousand dollars, and oncohematology -50 thousand dollars. Nevertheless, even with full funding, the effectiveness of treatment is no more than 50%. Most of the obtained compounds are not clinically used in consequence to their high toxicity, poor solubility in water, indiscriminate action and a number of other side effects. 2 Therefore, the problem of investigating new, more effective drugs remains relevant. To overcome these limitations, the search for new effective and safe anticancer drugs continues around the world.
One of the promising methods to solve this problem is the screening of potential antitumor agents among new synthesized compounds. Nitrogen-based heterocyclic analogues are an extremely important class of organic substances that are widely used in medicinal chemistry, as more than 60% of drugs and more than 85% of biologically active substances described in the literature contain a nitrogen-containing heterocycle. 3 In recent years, there has been an increasing interest in condensed nitrogen-containing heterocyclic systems, as many of them exhibit different types of pharmacological activity. [4][5][6] [1,2,4]Triazolo [3,4-b] [1,3,4]thiadiazoles are one of the little-studied and hard-to-reach representatives of this class of 814 compounds. 7 The presence of considerable material on the chemistry and biological properties of this class of compounds allows us to consider them as one of the promising classes of biologically active compounds with a wide range of action. Among the triazolothiadiazole derivatives, there have been identified compounds that possess antitumor, [8][9][10] antimicrobial, [11][12][13] antidipressant, 13 anticonvulsant 14 and other activities. They are inhibitors of viral helicase, 15 cholinesterases, 16 carbonic anhydrases, 17 c-Met kinases. 18 Considering mentioned above, the search for new antitumor agents among this class of compounds is an interesting and relevant direction.
Methods of quantitative elemental analysis and 1 H NMR spectroscopy were used to confirm the structure and individuality of the synthesized substances. Interpretation of the spectra revealed that the signals for protons of all structural units were observed in their characteristic ranges.

Anticancer activity
The anticancer activity of the synthesized compounds was evaluated within the framework of an international cooperation program with the National Cancer Institute (Bethesda, Maryland, USA) DTP NCI (Developmental Therapeutic Program). The primary anticancer assay was performed at approximately sixty human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda. [30][31][32][33] The quantitative criterion of the compounds activity was the percentage of the cancer cell lines growth (GP,%) in comparison with the control. The results of the anticancer activity study are presented in Table 1. It was found that the synthesized compounds showed anticancer activity at different levels. The most active were compounds 3d and 3e with values of average mitotic activity of 48.95% and 49.39%. A feature of their structure is the presence of a benzyl radical in position 3 of the [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazoles cycle. It should also be noted the high cytotoxic effect of compound 3e relative to COLO 205 Colon Cancer -21.37%, MALME-3M Melanoma -9.92%, SK-MEL-5 Melanoma -7.64% and compound 3j relative to TK-10 Renal Cancer -21.22% and ACHN Renal Cancer -11.46%. According to the standard NCI procedure Compounds 3d, 3e and 3j were selected for the secondary phase of the study, which consisted of testing them on 60 cancer cell lines at five concentrations at 10-fold dilution (100μM, 10μM, 1μM, 0.1μM and 0.01μM. Based on experimental data of thorough in vitro screening of compounds, three dose-dependent parameters were calculated: GI50 -concentration that causes inhibition of 50% of cell lines growth; TGI -the concentration of the compound that leads to a complete inhibition of growth; LC50 is the concentration of a substance that leads to 50% cell death. GI50 is interpreted as an effective level of inhibition, TGI as a cytostatic effect, and LC50 is a lethal concentration that characterizes the cytotoxic effect. If the studied parameters values (GI50, TGI and LC50) are less than 100µM, the compounds are considered to be active (relative to this line).
The obtained results of thorough in vitro screening of compounds are shown in table 2. The most active compound was 3d for which MG-MID GI50 was 3.410. In contrast, the activity of compounds 3e and 3j was an order of magnitude lower.  To objectively interpret the data of the anticancer activity study, the selectivity index (SI) of the effect of compounds at the level of effective inhibition was calculated, which is the ratio of the mean MID GI 50 for all cancer cell lines to the mean for a particular disease. The value of the selectivity index in the range of 3-6 is interpreted as moderate selectivity, the value of SI> 6 indicates a high selectivity of the anticancer effect. The activity parameters of the test compounds are shown in table 3. In the case of compound 3d, high selectivity was observed for leukemia SI = 50.15, epithelial bowel cancer SI = 15.787 and prostate cancer SI = 10.36. Instead, compound 3e selectively acted on the line of epithelial bowel cancer SI = 7.036 and melanoma SI = 7.559. Compound 3j did not possess any selectivity. The next step in the investigation of anticancer activity was to compare the results of compounds 3d, 3e and 3j with known drugs -5-Fluorouracil (5-FU), Cisplatinum, as well as a natural anticancer substance -Curcumin. As can be seen in table 4, the anticancer activity of compound 3d at the GI level is significantly higher than 5-FU and is comparable to Cisplatinum and Curcumin. At the same time, the activity of compounds 3e and 3j was commensurate with 5-Fluorouracil and significantly lower than the activity of Cisplatinum and Curcumin. Thus, the studied 3-R-6-(5-arylfuran-2-yl- [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazoles possess a pronounced selective anticancer activity, which gives grounds to consider this condensed systems as a promising molecular framework for the design of potential anticancer agents.

Conclusions
In summary, we presented efficient synthetic approaches to a number of 3-R-6-(5-arylfuran-2-yl- [1,2,4]triazolo [3,4b] [1,3,4]thiadiazoles for their anticancer activity evaluation. We have shown that the proposed synthetic protocols provided the possibility to design triazolothiadiazoles diversity with a considerable chemical novelty. The obtained results of the performed biological activity evaluation have shown that synthesized compounds have expressive anticancer properties. Further optimization of the structure to improve biological activity is currently in progress.

Chemistry
All chemicals were of analytical grade and commercially available. When performing the synthetic part of the work, the reagents of the company Merck (Germany) and Sigma-Aldrich (USA) were used. All reagents and solvents were used without further purification and drying. All the melting points were determined in an open capillary and are uncorrected. 1 H-NMR spectra were recorded on a Varian Mercury 400 (Agilent Technologies, San Francisco, USA) instrument with TMS or deuterated solvent as an internal reference. Mass spectra were run using Agilent 1100 series LC/MSD (Agilent Technologies, San Francisco, USA) with an API-ES/APCI ionization mode. Elemental analysis was performed on an Elementar Vario L cube instrument (Elementar Analysen systeme GmbH, Hanau, Germany). Satisfactory elemental analyses were obtained for new compounds (C±0.17, H±0.21, N±0.19).

Anticancer activity
During the first step (primary screening), test compounds were added at a concentration of 10 -5 M to cell cultures and incubated for 48 hours. The end point was estimated using the dye-sulforodamine B. Results for each compound were expressed as Growth percent (GP%) of cells related to the control cells growth without test samples. During the secondary screening the study of compounds in 5 concentrations was executed. Human tumor cells from the screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mm L-glutamine. A 0.4% solution of sulforodamine B (SRB) (100 μl) in 1% acetic acid was added to each hole, and the plates were incubated for 10 min at room temperature. After staining, the unbound dye was removed, and absorption was detected on an automated reader at a wavelength of 515 nm. The percentage of cell growth for each concentration was calculated based on 7 absorption measurements: zero time (Tz), growth in the control sample (C) and growth in the presence of the test substances at 5 concentrations (Ti). The percentage of growth inhibition was evaluated by the formulas: [(Ti-Tz) / (C-Tz)] × 100 for concentrations for which Ti> / = Tz [(Ti-Tz) / Tz] x 100 for concentrations for which Ti < As a result, 3 dose-dependent parameters were calculated for each test substance: 1) the concentration of the substance that causes growth inhibition of 50% of cells -GI50 (growth inhibition); 2) TGI (total growth inhibition) -the concentration of a substance that completely inhibits cell growth; 3) LC50 (lethal concentration) -the concentration that causes the death of 50% of tumor cells (http://dtp.nci.nih.gov).