1. New N,C-Diaryl-1,2,4-triazol-3-ones: Synthesis and Evaluation as Anticancer Agents
Miguel Carda, Celia Martín-Beltrán, Eva Falomir, Rosa M Claramunt, José Elguero, Dolores Santa María Med Chem . 2019;15(4):360-372. doi: 10.2174/1573406414666180821103604.
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.
2. Neonicotinoid nitroguanidine insecticide metabolites: synthesis and nicotinic receptor potency of guanidines, aminoguanidines, and their derivatives
David B Kanne, Ryan A Dick, Motohiro Tomizawa, John E Casida Chem Res Toxicol . 2005 Sep;18(9):1479-84. doi: 10.1021/tx050160u.
Four neonicotinoid nitroguanidine insecticides (imidacloprid, thiamethoxam, clothianidin, and dinotefuran) acting as nicotinic agonists account for 10-15% of worldwide insecticide sales. General methods are needed for synthesis of their guanidine and aminoguanidine metabolites so they may be used as analytical standards and for evaluation of nicotinic receptor potency. The guanidines are obtained by treating the parent nitroguanidines with Fe powder in aqueous C2H5OH containing NH4Cl and isolated by silica chromatography. The aminoguanidines are prepared as mixtures with the guanidines on reaction of the parent nitroguanidines and Zn powder in glacial acetic acid. The imidacloprid aminoguanidine is isolated as the acetone imine or trifluoroacetamide and the clothianidin and dinotefuran aminoguanidines as the acetone imines using silica chromatography. Deprotection under acidic conditions then leads to the aminoguanidine.HCl salts. Because of stability considerations, a pH partitioning method is used to separate thiamethoxam aminoguanidine and guanidine. An alternate procedure to the aminoguanidine of imidacloprid (but not thiamethoxam, clothianidin, or dinotefuran) is reaction with hydrazine hydrate and NH4Cl in anhydrous C2H5OH. Ambiguities in further biological reactions are clarified by synthesizing authentic standards of three purported metabolites formed via the imidacloprid aminoguanidine: the 1,2,4-triazol-3-one derivative with ethyl chloroformate or ethyl pyrocarbonate, the acetaldehyde imine with acetaldehyde, and the 3-methyl-1,2,4-triazin-4-one derivative with ethyl pyruvate in refluxing toluene. The purported triazolone metabolite is reassigned as the aminoguanidine acetaldehyde imine probably formed as an artifact from acetaldehyde present in the ethyl acetate used for metabolite extraction. Potency at the Drosophila nicotinic receptor is greatly decreased on converting a nitroguanidine to a guanidine or aminoguanidine. In sharp contrast, potency at the vertebrate alpha4beta2 nicotinic receptor is generally increased on conversion from the nitroguanidine to aminoguanidine and particularly guanidine derivatives.