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.

Twenty-one styryl and phenethyl aryl ureas have been synthetized and biologically evaluated as multitarget inhibitors of Vascular endothelial growth factor receptor-2 VEGFR-2 and programmed death-ligand-1 (PD-L1) proteins in order to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, MCF-7, HeLa and A549), on the endothelial cell line human microvascular endothelial cells (HMEC)-1 and on the non-tumor cell line human embryonic kidney cells (HEK)-293 has been determined. Some derivatives were evaluated for their antiangiogenic properties such as their ability to inhibit microvessel formation using HMEC-1 or their effect on VEGFR-2 in both cancer and endothelial cell lines. In addition, the immunomodulator action of a number of selected compounds was also studied on PD-L1 and c-Myc proteins. Compounds 16 and 23 (Z) and (E)-styryl p-bromophenyl urea, respectively, showed better results than sorafenib in down-regulation of VEGFR-2 and also improved the effect of the anti-PD-L1 compound BMS-8 on both targets, PD-L1 and c-Myc proteins.

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3′-aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound.

Aims: In search of new promising anti-cancer agents. Background: Six N-acyl derivatives of aminocombretastatin A-4 have been synthesized and evaluated as regards their interaction with tubulin and as c-Myc downregulators. Objective: This study is focused on the synthesis and the biological evaluation of N-acyl derivatives of aminocombretastatin A-4 (CA-4). Docking studies were carried out in order to find out whether the synthetic derivatives could bind to tubulin at the colchicine site in a conformation similar to that of CA-4. The effect of the synthetic derivatives on the proliferation of several cancer cells and on non-cancer cells has been measured. In addition, their effect on tubulin polymerization, on cell cycle distribution, on the microtubule network and on c-Myc expression have also been evaluated. Method: A set of six N-acyl derivatives were achieved by means of a peptide-type coupling of aminocombretastatin A-4 and the corresponding carboxylic acid. The ability of the synthetic compounds to inhibit cell proliferation was measured by MTT assay against three human carcinoma cell lines (colorectal HT-29, lung A549 and breast adenocarcinoma MCF-7) and one non-tumor cell line (HEK-293). Inhibition of tubulin polymerization was evaluated by turbidimetry time-course measurements. The action of the synthetic derivatives on cell cycle distribution was measured by flow cytometry and their effects on the microtubule network was determined by immunofluorescence microscopy. Finally, the downregulation of the synthetic derivatives on c-Myc protein was quantified by ELISA assay while the effect on cMyc gene was measured by RT-qPCR analysis. Results: Derivatives bearing pentanoyl (compound 2), hexanoyl (compound 3), and heptanoyl (compound 4) side chains show antiproliferative activities on the HT-29 line in the low nanomolar range, with values similar to that exhibited by AmCA-4 but far exceeding those of CA-4. Compounds 1 (butanoyl side chain) and 2-3 inhibit tubulin polymerization in vitro in a manner similar to that of CA-4 and AmCA-4 whereas compounds 4, 5 (octanoyl side chain) and 6 (dodecanoyl side chain) may be considered as partial inhibitors of tubulin polymerization. While all derivatives are able to accumulate cells in G2/M phase, compounds with the longest acyl chains (5 and 6) are the least active ones in this particular action. Moreover, compounds 2-3 were the most active ones as c-Myc downregulators. Conclusion: Our studies show that the most active compounds in the disruption of the microtubule network are also the most potent ones in the downregulation of c-Myc expression. Other: Compounds 2 and 3 are good candidates for in vivo studies as they combine the best anti-mitotic and c-Myc downregulation activities at low doses.

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