Anticancer activity evaluation of thieno[3,2-e][1,2,3]triazolo[1,5-a] pyrimidines and thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives

O. Shyyka, N. Pokhodylo, N. Finiuk © 2019 O. Shyyka et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 547.859; 615.27


Introduction
Thieno-fused five-and six-membered nitrogen containing heterocycles [1] play an important role in medical and pharmaceutical chemistry. One of the reasons is that several of fused cores are bioisosters to the natural purine bases. Noteworthy, the implementation of bio-isosteric replacement strategy could be the basis for successful design of drug rational structure [2,3]. In this regard, thienopyrimidines continue to attract considerable attention as the privileged scaffolds and the number of such core compounds, which exhibited various Bioorganic Chemistry ISSN 1993-6842 (on- biological activities, gradually increases [4][5][6].
On the other hand, fused triazolopyrimidines also were found to be biologically active [7][8][9]. Moreover, we previously synthesized several 1,2,3-triazoles which were found to possess significant anticancer activity [10,11]. Recently, we have developed facile synthetic methods for the versatile synthesis of 1,2,3-triazole and thieno-fused compounds, where new efficient and mild procedures from readily available reagents can be employed [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Those protocols could be successfully used for the preparation of fused heterocyclic compounds with both thiophene and 1,2,3-triazole rings. Based on such combination an extended structure-activity investigation focusing on drug-like properties should be performed. The present work is devoted to the evaluation of anticancer activity of substituted thienotriazolopyrimidines synthesized at room temperature in a short time via domino-1,3-dipolar cyclocondensation [16,21,12] that meets the energy-saving and environmentally friendly philosophical concepts of 'green chemistry'. The point of the research was to perform in vitro anticancer activity assay of the fused substituted thienotriazolopyrimidines.

Synthesis:
The thienotriazolopyrimidine derivatives were designed as promising anticancer agents and obtained as we described earlier [16,21,12].

Materials for MTT assay:
The 10 mM stock solution of thienotriazolopyrimidine derivatives was prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA), and additionally dissolved in the culture medium prior to addition to the cell culture. In vitro screening of the anti-proliferative activity of the synthesized compounds and doxorubicin towards tumor cell was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test (99.5 % pure, Sigma-Aldrich, St. Louis, USA). The DMEM cell culture medium was obtained from Sigma-Aldrich, USA; RPMI-1640 -from PPA, Austria. Fetal bovine serum was obtained from Biowest, France. Doxorubicin was obtained from Actavis, Romania.

Cell cultures for MTT assay:
Human acute T-cell leukemia cells of Jurkat line were obtained from a Collection at R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiology (Kyiv, Ukraine). Human myeloid leukemia HL-60 and human ovarian carcinoma cells of Skov3 line were from a Collection of the Institute of Cancer Research at Vienna Medical University (Vienna, Austria). Human hepatocarcinoma cells of HepG2 line and human colon carcinoma cells of HCT116 line were from a Collection at the Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine (Kyiv, Ukraine). Cells were grown in the RPMI-1640 or DMEM culture medium supplemented with 10 % of fetal bovine serum. Cells were cultivated in the CO 2 -thermostate at 37°C in atmosphere of 95 % air and 5 % CO 2 .

Anticancer assay via NCI protocol:
According to the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, a primary anticancer assay was per-formed within nine cancer types at approximately 60 human tumor cell lines panel. The tested compounds were added to the culture at a single concentration (10 -5 M) and left for 48 h incubation. Sulforhodamine B (SRB) was used as protein binding dye for the end-point determinations. The percent of growth of the treated cells when compared to the untreated control cells was taken as a result for each tested compound. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. Growth percent of 100 corresponds to growth seen in untreated cells. Growth percent of 0 indicates absence of net growth over the course of the assay (i.e. equal to the number of cells at time zero). Growth percent of -100 results when all cells are killed.

Cell proliferation (MTT) assay:
In vitro evaluation of anticancer activity of the synthesized compounds and doxorubicin, used as a reference drug control, towards cancer cell lines was carried out by the MTT test [25]. Tumor cells were seeded for 24 hours in 96well microtiter plates at a concentration of 5,000 substrate-dependent cells/well or 10,000 suspension cells/well (100 μL/well), after that cells were incubated for 72 hours with various additions of the synthesized compounds (0-50 μM). MTT, converted to dark violet, water insoluble MTT formazan by the mitochondrial dehydrogenases, was used to determine viable cells according to the Sigma-Aldrich protocol. Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, VT, USA) was used for reaction results measurement.

Statistical analysis:
All data are presented as the mean (M) ± standard deviation (SD), n = 4. Results were analyzed and illustrated with GraphPad Prism (version 6; GraphPad Software, San Diego, CA, USA). Statistical analyses were performed using two-way ANOVA with Bonferroni's multiple comparisons test.

Chemistry
The compounds presented in the article were obtained using simple and convenient synthetic protocols (Scheme 1). The 3-aminothiophenes 1a-c and Gewald thiophenes 1d-g were used as starting materials in the synthesis of isomeric thienotriazolopyrimidine scaffolds 4 and 5.
Starting alkyl amino-thiophene-carboxylates 1a-g were converted into corresponding azides 2a-g, which were found to be reactive in the base catalyzed cycloaddition with activated methylene compounds providing thieno [3,2-

Evaluation of anticancer activity in vitro
The synthesized 24 examples of thienotriazolopyrimidines (4a-i, 5a-o) were submitted and evaluated at the single concentration of 10 -5 M towards a panel of approximately sixty cancer cell lines. The human tumor cell lines were derived from nine different cancer types: leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostatev and breast cancers. Primary anticancer assays were performed according to the US NCI protocol (http://dtp.nci.nih.gov), which was described elsewhere [28][29][30][31]. The results for each compound are reported as the percent growth (GP) ( Table 1) Table 1). The most active compounds are presented in Figure 1.
Moreover, it should be noted that bioisosteric to thieno [2,3- Additionally, in vitro screening of anti-proliferative activity of some compounds (5b, 5d) in different final concentrations (0-50 μM) towards several cancer cell lines (human acute T-cell leukemia cells of Jurkat line, human colon carcinoma HCT116, human liver HepG2 and human ovarian Skov3) was performed by the MTT assay. However, the activity of doxorubicin was higher than the cytotoxicity of synthesized thienotriazolopyrimidines 5b, 5d at the concentration up to 50 μM against chosen cell lines (Figure 3).

Conclusion
In the present article, in vitro anticancer activity of the isomeric thienotriazolopyrimidines was evaluated. These preliminary results allowed identifying the most active compounds: 24 of the synthesized compounds were tested and two of them 4a, 4b displayed moderate antitumor activity against CNS and breast cancers cell lines. Moreover, the compound 5a can be defined as a prospective antitumor agent with the value of GP = -31.50 % on SK-MEL-5 melanoma cell line, the compound has selective influence being active only against a single cell line. The obtained results of antitumor activity of such derivatives are interesting with the hope to get more selective and active anticancer agents among fused pyrimidines and prove the necessity of further investigation.