Multi-targeted pyrazole scaffolds: synthesis, mechanistic insights, and therapeutic potential in anticancer, antioxidant, and anti-inflammatory applications

Abstract

Pyrazole derivatives are a class of heterocyclic compounds known to have broad pharmacological effects. This study aimed to synthesize and characterize a new series of pyrazole derivatives and investigate their effects on a series of in vitro assays. Five synthesized compounds were confirmed structurally using standard spectroscopic techniques, and their cytotoxic activity was evaluated against HeLa cells using MTT assay and morphological examination of viability. DNA damage and fragmentation were analyzed using a comet assay, agarose gel electrophoresis, and diphenylamine reaction. Antioxidant properties were evaluated with radical scavenging assays, and anti-inflammatory potential was assessed with in vitro inhibition assays (COX-1/2). Pyrazolethione 5 showed antiproliferative effects at all concentrations, with preserved cell confluence at ≤62.5 µg/mL. It also demonstrates DPPH scavenging activity similar to that of ascorbic acid in a dose-dependent manner, reaching a maximum activity of 85.9% with an IC₅₀ of 48.77. In addition, it demonstrated antioxidant activity, although significantly lower than that of gallic acid, with a maximum scavenging percentage of 83.9% at 1000 μg/mL and an IC50 of 54.97 μg/mL. In the comet assay, the treatment significantly increased DNA fragmentation to 26.2% ± 0.88% from 12.8% ± 0.77% in the control cells (p < 0.01), representing a two-fold induction of apoptosis. The test compound inhibited COX-1 in a dose-dependent manner (86.29% inhibition at 1000 µg/mL), producing an IC50 value of 29.14 ± 0.97 µg/mL, while it produced moderate inhibition of COX-2, 82.03% inhibition at 1000 µg/mL, and had an IC50 value of 51.49 ± 2.13 µg/mL. According to flow cytometry analysis, untreated HeLa cells were mainly in G1 (54.56%) and G0 (34.86%), with fewer cells in S (9.63%), and G2-M (0.94%). In pyrazolethione-treated HeLa cells, G0 accumulation (73.57%) and fewer proliferative phases (S 3.10% and G2-M 2.78%) indicated substantial alteration to the cell cycle. Pyrazolethione 5 demonstrates a favorable safety profile, with minimal cytotoxicity at lower concentrations, and impressive antioxidant and anti-inflammatory activities. The DPPH and gallic acid-like radical scavenging activities, moderate COX-1 and COX-2 inhibition, and DNA fragmentation suggest multiple bioactivities. Furthermore, the compound significantly altered cell cycle progression in HeLa cells, leading to G0/G1 arrest and reduced proliferation. Taken together, the data indicate pyrazolethione 5 is a potential lead compound for further development/investigation as an anticancer and anti-inflammatory agent with low toxicity.