New N,C-Diaryl-1,2,4-triazol-3-ones: Synthesis and Evaluation as Anticancer Agents

Abstract

Background: A set of 2,5-diaryl-1,2,4-triazol-3-ones was synthesized in two steps and evaluated as regards their activity in some relevant biological targets related to cancer. Objective: This study is focused on the synthesis and the biological evaluation of 2,5-diaryl-1,2,4- triazol-3-ones. In this sense, the effect of the synthetic triazolones on the proliferation of HT-29 and A549 cancer cells and on HEK non-cancer cells has been measured. In addition, the effects of triazolones on the expression of hTERT, c-Myc and PD-L1 genes and on the production of c-Myc and PD-L1 proteins have also been evaluated. Method: A set of 2,5-diaryl-1,2,4-triazol-3-ones was synthesized in two steps. Firstly, N- (aminocarbonyl)-3-methoxybenzamide was prepared by coupling 3-methoxybenzoic acid and cyanamide followed by aqueous HCl hydrolysis. Then, the 2,5-diaryl-1,2,4-triazol-3-ones were obtained upon reaction of N-(aminocarbonyl)-3-methoxybenzamide with arylhydrazines in decaline at 170ºC. The ability of the triazolones to inhibit cell proliferation was measured against two human carcinoma cell lines (colorectal HT-29 and lung A549), and one non-tumor cell line (HEK- 293) by MTT assay. The downregulation of the synthetic triazolones on the expression of the hTERT, c-Myc and PD-L1 genes was measured by an RT-qPCR analysis. Their ability to regulate the expression of the c-Myc and PD-L1 proteins, as well as their direct interaction with c-Myc protein, was determined by the ELISA method. Finally, the direct interaction of triazolones with PD-L1 protein was assessed by the thermal shift assay. Results: Ten 2,5-diaryl-1,2,4-triazol-3-ones were synthesized and characterized by spectroscopic methods. A thorough study by 1H, 13C, 15N and 19F NMR spectroscopy showed that all the synthetic compounds exist as 4H-triazolones and not as hydroxytriazoles or 1H-triazolones. Some triazolones showed relatively high activities together with very poor toxicity in non-tumor cell line HEK-293. 2-(2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (4) was particularly active in downregulating c-Myc and PD-L1 gene expression although 2-(4- chloro-2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (8) is the one that combines the best downregulatory activities in the three genes studied. Considering protein expression, the most active compounds are 2-(4-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro- 3H-1,2,4-triazol-3-one (5) and 2-(2,4,6-trifluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H- 1,2,4-triazol-3-one (10) (c-Myc expression) and 2-(2,3,5,6-tetrafluorophenyl)-5-(3-methoxyphenyl)- 2,4-dihydro-3H-1,2,4-triazol-3-one (11) and (8) (PD-L1 expression). Conclusion: Some of the triazolones studied have shown relevant activities in the inhibition of the hTERT, c-Myc and PD-L1 genes, and in the inhibition of c-Myc and PD-L1 protein secretion, the 2-(4-chloro-2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (8) was found to be a particularly promising lead compound.

A structural analysis of 2,5‐diaryl‐4H‐2,4‐dihydro‐3H‐1,2,4‐triazol‐3‐ones: NMR in the solid state, X‐ray crystallography, and GIPAW calculations

“…As we have already pointed out, [1] Compounds 1-10 exist as 4H-triazolones and not as hydroxytriazoles or 1Htriazolones tautomers, [22,23] which is consistent with the presence of a N4-H group that appears in the 1 H NMR spectra as a broad signal between 12.40 (1) and 12.88 ppm (10) and in 15 N NMR at around −242 ppm. The N2-aryl signals were observed in the range of −200.9 (3) (note that in 1 appears at −202.5 being the second smaller) to −225.1 ppm (10) and the N1 signal between −112.6 (1) and −116.1 ppm (10).…”

“…Due to its biological properties, many other triazol-3-ones, not necessarily having a 2,5-diaryl pattern, have previously been described; they are Ca 2+ conductance openers activated potassium channels, [2] antimicrobials, [3] antifungals, [4,5] TNF-α inhibitors, [6] selective CB1 receptor antagonists, [7] angiotensin II AT1 receptor antagonists, [8,9] antioxidants, [10] and AChE inhibitors. [11] A thorough understanding of biological properties requires information about the structure of molecules, among them tautomerism [1] and conformational isomerism. Conformational data are usually obtained in solution by nuclear magnetic resonance (NMR) [12,13] and quantum chemical calculations [14,15] and in the solidstate by NMR, [16,17] crystallography, [18,19] and again by quantum calculations.…”

“…Conformational data are usually obtained in solution by nuclear magnetic resonance (NMR) [12,13] and quantum chemical calculations [14,15] and in the solidstate by NMR, [16,17] crystallography, [18,19] and again by quantum calculations. [20,21] In our previous paper, [1] the NMR data of these compounds were determined in solution and reported in the experimental section without comments. We elected to pursue a more detailed discussion of compounds previously reported [1] with the following differences:…”

“…[20,21] In our previous paper, [1] the NMR data of these compounds were determined in solution and reported in the experimental section without comments. We elected to pursue a more detailed discussion of compounds previously reported [1] with the following differences:…”

Conformational analysis of 2,5‐diaryl‐4‐methyl‐2,4‐dihydro‐3H‐1,2,4‐triazol‐3‐ones: Multinuclear NMR and DFT calculations

“…Coupling constants J (C-F) were directly determined from the proton decoupled 13 C NMR spectra and in some cases J(F-N) coupling constants could also be measured. All data are consistent with those obtained for the NH precursors described previously by us [1,2], and the most significant are:…”