Evaluation of the antiproliferative activity of selected 1,2,3-triazole-4-carboxylic acids — key fragments and precursors of antitumor 1,2,3-triazole-4-carboxamides

Aim. To evaluate in vitro antiproliferative effect of selected 1,2,3-triazole-4-carboxylic acids, which are the key fragments and precursors of antitumor 1,2,3-triazole-4-carboxamides. Methods. Organic synthesis, in vitro cytotoxicity assay, MTT assay, spectrophotometry, sta-tistical analysis. Results. The selected 1,2,3-triazole carboxylic acids and their esters synthesized according to a convenient synthetic procedure were tested for their anticancer activity in NCI60 cell lines within 9 cancer types at the 60 human tumour cell lines panel. These preliminary results allowed identifying the most active compounds and finding the structure-activity rela-tions. The most promising 1,2,3-triazole-4-carboxylic fragments were selected for the design of 1,2,3-triazole-4-carboxamides for screening anticancer activity. Conclusions. The obtained results of antitumour activity of the studied derivatives are interesting for the discovery of selective and active anticancer agents among 1,2,3-triazole-4-carboxamides in terms of a fragment-based drug discovery (FBDD) concept, that proves the necessity of further studies.

Finally, new PROTACs were synthesised via conjugation of corresponding focal adhesion kinase (FAK) or Bruton's tyrosine kinase (BTK) inhibitors with the E3 ligase ligand through 1,2,3-triazole-4-carboxamide containing linkers and showed rapid and reversible degradation with a picomolar DC 50 value in various cell lines in vitro for FAK [28] and BTK [29] respectively. Thus, the compounds with a 1,2,3-triazole-4-carboxamide moiety possess considerable potential for the drug discovery. For this reason, we decided to select and evaluate several 1,2,3-triazole carboxylic acids, which are key fragments and precursors of antitumor 1,2,3-triazole-4-carboxamides for their antiproliferative activity.

Anticancer assay
According to the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, a primary anticancer assay was performed within nine cancer types at approximately 60 human tumour 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 a 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 the growth seen in untreated cells. Growth percent of 0 indicates the 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 [38]. Tumour cells were seeded for 24 h in 96-well 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 h 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 measurement of the reaction results.

Chemistry
The compounds presented in the article were obtained using simple and convenient synthetic protocols based on the Dimroth reaction. Diversity of azides and β-keto esters can be used for substituent variation in all positions of the triazole ring. The reaction is useful for the preparation of the heterocyclic, aromatic and aliphatic 1,2,3-triazole carboxylic acids. The method is well suited for the concept of diversity-oriented synthesis (DOS) and allows obtaining a large number of such acids as convenient structural blocks in the synthesis of carboxamides for drug discovery. The synthesis of acids can be described by the General Scheme 1. Noteworthy, ester formed during the cyclization can be in situ hydrolysed to the corresponding acid or kept as ester depending on the reaction conditions. In particular, the ester is the main product when the reaction is performed in dry DMSO with K 2 CO 3 as a catalyst. Compounds 13 and 17 were obtained in this work for the first time. Their structure was confirmed by NMR, LCMS and elemental analysis.

Evaluation of antiproliferative activity in vitro
The synthesized 24 examples of 1,2,3-triazole carboxylic acids and their esters 1-24 were submitted and evaluated at the single concentration of 10 -5 M towards a panel of approximately sixty cancer cell lines. The human tumour cell lines were derived from nine different cancer types: leukaemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers. Primary anticancer assays were performed according to the US NCI protocol (http://dtp.nci.nih.gov), which was described elsewhere [39][40][41][42]. The results for each compound are reported as growth percentage (GP) in Table 1. The range of growth (%) shows the lowest and highest growth that was found among different cancer cell lines.

Conclusion
In the present article, in vitro anticancer activity of the selected 1,2,3-triazole carboxylic acids and their esters was evaluated. The preliminary results allowed identifying the most active compounds and finding the structureactivity relations. The obtained data on the antitumour activity of such derivatives are interesting for the discovery of selective and active anticancer agents among the fused 1,2,3-triazole-4-carboxamides in terms of a fragment-based drug discovery (FBDD) concept and prove the necessity of further studies.