Synthesis of 4,4'-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ols) and evaluation of their antioxidant and anticancer activities

Abstract

Background: Pyrazoles have attracted particular attention due to the diverse biological activities associated with this heterocyclic system, and some have been shown to be cytotoxic to several human cell lines. Several drugs currently on the market have this heterocycle as the key structural motif, and some have been approved for the treatment of different types of cancer.

Results: 4,4'-(Arylmethylene)bis(1H-pyrazol-5-ols) derivatives 3a-q were synthetized by a three components reaction of 3-methyl-1-phenyl-5-pyrazolone (1) with various benzaldehydes 2 catalyzed by sodium acetate at room temperature. The structures of all synthesized compounds were characterized by physicochemical properties and spectral means (IR and NMR) and were evaluated for their radical scavenging activity by DPPH assay and tested in vitro on colorectal RKO carcinoma cells in order to determine their cytotoxic properties. All 4,4'-(arylmethylene)bis(1H-pyrazol-5-ols) derivatives 3a-q were synthetized in high to excellent yield, and pure products were isolated by simple filtration. All compounds have good radical scavenging activity, and half of them are more active than ascorbic acid used as standard.

Conclusion: Several derivatives proved to be cytotoxic in the RKO cell line. In particular, compound 3i proved to be a very potent scavenger with an IC₅₀ of 6.2 ± 0.6 µM and exhibited an IC₅₀ of 9.9 ± 1.1 μM against RKO cell. Autophagy proteins were activated as a survival mechanism, whereas the predominant pathway of death was p53-mediated apoptosis.

Keywords: 4,4ʹ-(arylmethylene)bis(1H-pyrazol-5-ols); Antioxidant; Apoptosis; Autophagy.

Fig. 1

Approved anti-cancer pyrazole drugs

Scheme 1

Synthesis of 4,4ʹ-(arylmethylene)bis(1H-pyrazol-5-ols) 3a–q

Fig. 2

Cytotoxic effect of compound 3i. RKO cell line were exposed to 10–70 µM of 3i for 24 and 48 h and evaluated through Trypan Blue dye exclusion assay. A cell population, and B percentages of cell viability. The tests for significance were limited to ANOVA-Dunnett post-test, *p < 0.01, **p < 0.001, ***p < 0.0001 vs. control. C Cell morphology after exposure at 30, 40, and 50 µM; Horizontal bar in bottom figure = 50 µm

Fig. 3

RKO cell line was exposed to different doses of compound 3i for 24 h. Expression of protein: A p53 and p21 protein. B Pro-autophagic LC3 II and p62. C Pro-apoptotic proteins: Bax, Bcl-2, cleaved caspase-3, and PARP-1. Actin was used as control. D Quantification expression protein. The tests for significance were limited to ANOVA-Dunnet post-test: ∗ p < 0.01, ∗  ∗ p < 0.001, ∗  ∗  ∗ p < 0.0001 vs. control. Full-length gels are presented in Additional file 1: Fig. S1