New N,C-Diaryl-1,2,4-triazol-3-ones: Synthesis and Evaluation as Anticancer Agents
Authors: María Dolores Santa , Claramunt Rosa M. , Elguero José , Carda Miguel , Falomir Eva , Martín-Beltrán Celia 
Published: August 2018

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.