Synthesis of 1,2,3-Triazole Derivatives and Evaluation of their Anticancer Activity

Anticancer screening of several 1,2,3-triazoles with heterocyclic fragments has been performed. The 1,2,3-triazole derivatives were synthesized from available starting materials according to convenient synthetic procedures. The antitumor activity of the synthesized compounds was tested in vitro by the National Cancer Institute in NCI60 cell lines. It was observed that some compounds showed slight anticancer activity. One of them possessed a moderate activity against melanoma, colon, and breast cancer. Standard COMPARE analysis was performed at the GI50 level.


Introduction
Triazoles and their derivatives are of great importance in medicinal chemistry and can be used for the synthesis of numerous heterocyclic compounds with different biological activities such as antiviral, antibacterial, antifungal, antituberculosis, anticonvulsant, antidepressant, anti-inflammatory, anticancer activities, etc [1]. They have been reported to be inhibitors of glycogen synthase kinase-3 [2], antagonists of GABA receptors [3,4], agonists of muscarine receptors [5], be neuroleptic [6], and these compounds also show anti-HIV-1 [7], cytotoxic [8], antihistaminic [9], and antiproliferative activities [10]. Thus, the design and synthesis of novel triazole derivatives are the prospective direction of medicinal chemistry for the scientists working in this field.
Herein we describe the synthesis and anticancer activity of 1,2,3-triazoles containing some heterocyclic cores. The structures shown in the article were preselected from a number of substructure molecules by computer simulation and the most active of their representatives were tested.
The reactions started at approximately 60°C and were complete in 30 min to yield compounds 6a,b. Initial tetrazoles 5a,b were obtained by 1,3-dipolar cycloaddition of sodium azide to nitriles in the presence of ammonium chloride as a phase-transfer catalysis.
Compounds containing triazole and flavonoid fragments in one molecule were prepared by a three-step synthetic route. First, by the acylation of 1-(2-hydroxy-5-methylphenyl) ethanone 7 with the 1-phenyl-5-methyl-1H-1,2,3-triazole-4-carboxylic acid chloride 4a. The resulting ester 8 in the presence of base underwent the Baker-Venkataraman rearrangement to yield 1,3-diketone 9. The presence of the 1,3-diketone group and OH group in ortho-position in the arene ring allowed the acid catalysis heterocyclization to form compound 10 with a 56% general yield.
Moreover, the new activated ketomethylenic compounds β-nitrilesulfones 17 [14] and 1-(5-(R-amino)-1,2,4-thiadiazol-3-yl)propan-2-ones 18 [15] were used for 1,2,3-triazole synthesis in the Dimroth cyclization. The triazoles 19 and 20 were formed as mentioned earlier by the reaction of arylazides in sodium methoxide in methanol solution. It was found out that the corresponding 1,2,3-triazoles 19 and 20 were formed immediately after mixing the reagents at room temperature and precipitated in good yields from the reaction medium. Finally, compounds 21 were obtained by the reaction of azides with 1-(triphenylphosphoranylidene)acetone 16. Acid chlorides 22 were used for the synthesis of amides 25 and oxadiazoles 26 from amine 23 and tetrazole 24, respectively. Tetrazole 24 was readily prepared from the 3-cyanopyridines reaction with sodium azide in DMF in the presence of ammonium chloride [12].

Biological activity
The main focus of the biological activity studies was on the search for compounds with antitumor activity. The newly synthesized compounds were selected by the National Cancer Institute (NCI) within the Developmental Therapeutic Program (www.dtp.nci.nih.gov) for in vitro cell line screening. Anticancer assays were performed according to the US NCI protocol, which was described elsewhere [16][17][18][19][20]. The compounds were first evaluated at one dose of the primary anticancer assay towards approximately 60 cell lines (concentration 10 −5 M). The human tumor cell lines represent all forms of cancer (such as non-small-cell lung cancer, colon cancer, breast cancer, ovarian cancer, leukemia, renal cancer, melanoma, prostate cancer). In the screening protocol, each cell line was inoculated and pre-incubated for 24-48 h on a microtiter plate. Test agents were then added at a single concentration and the culture was incubated for an additional 48 h. The endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). The results for each test agent were reported as the percent growth of the treated cells compared to the untreated control cells. The preliminary screening results are shown in Table 1. The results for each compound are reported as the percent growth (GP). Range of growth (%) shows the lowest and the highest growth that was found among different cancer cell lines.
The synthesized 1,2,3 triazoles displayed slight 15a, 6b, 25 or low activity in the in vitro screen on the tested cell lines. However, there was a selective influence observed in some of the compounds on several cancer cell lines ( Table 1) Finally, compound 25 was selected for in vitro testing against a full panel of about 60 tumor cell lines at 10-fold dilutions of five concentrations (100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM). Based on the cytotoxicity assays, three antitumor activity dose-response parameters were calculated for each experimental agent against each cell line: GI 50molar concentration of the compound that inhibits 50% net cell growth; TGI -molar concentration of the compound leading to total inhibition; and LC 50 -molar concentration of the compound leading to 50% net cell death. Values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value was expressed as greater or less than the maximum or minimum concentration tested. Mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an averaged activity parameter over all cell lines for each compound. For the calculation of the MG_MID, insensitive cell lines were included with the highest concentration tested.

Tab. 1.
Anticancer activity screening at one dose assay ( The tested compound showed a broad spectrum of growth inhibition activity against human tumor cells, as well as some distinctive patterns of selectivity. In general, compound 25 selectively inhibited the growth of the colon cancer cell lines. We found that  Table 3. It was found that 1,2,3-triazoles with the thiazole ring are quite active against tumor cell lines. It should be noted that compounds with the thiazole fragment directly bound to the 1,2,3-triazole core were not selected for the second stage of investigation in the NCI. On the contrary, 1,2,3-triazole amides with the thiazole moiety possessed moderate activity, among which compound 25 was the most active. Nowadays, new examples of such compounds are being synthesized and tested in the NCI. The analysis of the activity of the triazole derivatives 6a, 6b allowed us to conclude that the presence of the methoxy group in the 1,3,4-oxadiazole fragment increased the anticancer activity on SR cell line (leukemia) up to 20%.
The combination of both 1,2,3-triazole and quinoline rings in one molecule resulted in interesting antitumor activity. However, compounds with the quinoline ring, bound directly to the 1,2,3-triazole core, were not selected for further investigation at the NCI at all or possessed low anticancer activity. On the contrary, compounds 15a, 15b were more active. In the case of compound 15a, removal of the carboxyl group in the 1,2,3-triazole fragment led to the increase in the antitumor activity against the UO-31 cell line (renal cancer) up to 20%.

COMPARE analysis
NCI's COMPARE algorithm [21][22][23][24] allows the supposition of the biochemical mechanisms of action of novel compounds on the basis of their in vitro activity profiles when comparing with those of standard agents. Similarity of pattern to that of the seed is expressed quantitatively as a Pearson correlation coefficient (PCC). The results obtained with the COMPARE algorithm indicate that compounds high in this ranking may possess a mechanism of action similar to that of the seed compound. We used an accessible online tool -NCI COMPARE analysis to discover the similarity of compound 25 to the seed one (Table 4). Correlations with a PCC > 0,6 were selected as significant. Standard COMPARE analysis was performed at the GI 50 level. Compound 25 did not yield any significant activity correlation with any standard agents. The obtained correlation coefficients didn't allow a distinction between cytotoxicity mechanisms of the tested compounds with a high probability. Nevertheless, the compound showed moderate correlation with 4-ipomeanol (NSC: S349438). This may indicate that it has a unique mode of anticancer action.
Acid chloride 4 or 22, 15 mmol, was added to the solution of 15 mmol of tetrazole 5a,b or 24 in 15 mL of pyridine. The mixture was heated until nitrogen no longer evolved, then heated for 30 min under reflux, cooled, and diluted with 50 mL of water. The precipitate was filtered off, washed on a filter with water (up to 50 mL), dried in air, and purified by recrystallization with ethanol.

Synthesis of 6-Methyl-2-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)-4H-chromen-4-one (10)
1-(2-Hydroxy-5-methylphenyl)ethanone (7) 1.5 g (0.01 mole) was dissolved in 5 mL of pyridine and triazole acid chloride 4a 2.21 g (0.01 mole) was added, heated to 100 º C and left to cool for 30 min at room temperature. Then the reaction mixture was poured into a mixture of 10 g of ice and 20 mL of 1M hydrochloric acid. The precipitate was filtered and crystallized from alcohol. Yield 76%. m.p. = 107-108 °C. Ester 8 2.37 g (0.007 mole) was dissolved in 3 mL of pyridine at 50 °C. The mixture was added to a solution of potassium hydroxide 0.55 g (0.01 mole) and maintained for 1 h at 50 °C under stirring until the mixture became a homogeneous dense mass. The reaction mixture was poured into cool ice water and 10% solution of acetic acid. The precipitate was filtered and crystallized from alcohol. Yield 70%. m.p. = 59-60 ° C. To a suspension of diketone 9 0.27 g (0.0008 mole) in 1 mL of glacial acetic acid the concentrated sulfuric acid 0.08 g was added and heated under reflux for 1 h. After cooling to room temperature the reaction mixture was poured into 15 g of ice and left for 30 min. Precipitate was filtered and crystallized with alcohol. Yield: 67%, mp 203-203°C (Ethanol). 1

Cytotoxic activity against malignant human tumor cells
A primary anticancer assay was performed on a panel of approximately 60 human tumor cell lines derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda. The tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent growth of the treated cells when compared to the untreated control cells. The percent 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. The 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 drug, (C), and test growth in the presence of drug at the five concentration levels (T i )], the percent growth was calculated at each of the drug concentrations levels. Percent 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 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 more or less than the maximum or minimum concentration tested. The logGI 50 , logTGI, logLC 50 were then determined, defined as the mean of the logs of the individual GI 50 , TGI, LC 50 values. The lowest values were obtained with the most sensitive cell lines. Compounds having these values ≤ 4 were declared to be active.