Exploring Pyrazoline-Thiophene Hybrids as CDK2 Inhibitors: Synthesis, Mechanistic, Biological Studies, and Computational Insights

Abstract

Pyrazoline-linked thiophene (4a-p), a novel family of compounds, was designed and synthesized, and its anticancer potential was assessed. The most effective analogues of bis-thiophene (4n and 4o) and tris-thiophene (4p) were discovered to have sub-micromolar cytotoxicity against MCF-7 breast cancer (IC₅₀ = 0.17 µM) and A549 lung cancer (IC₅₀ = 0.78 µM) cells. Using flow cytometry, compound 4p triggered apoptosis and caused an G1/S-phase arrest (36.74% total apoptotic cells, compared to 0.99% in the control). Intrinsic apoptotic pathway was activated, as evidenced by downregulation of Bcl-2 and upregulation of P53, Bax, PUMA, caspases-3, and -9, as validated by RT-PCR analysis. The potency of compound 4p (IC₅₀ = 148 nM) against CDK2 was much higher than that of roscovitine (IC₅₀ = 700 nM). Molecular docking supported these results by demonstrating stable hydrogen-bond interactions with Leu83 and Lys89, as well as significant hydrophobic interactions in the ATP-binding region. In vivo, 4p significantly reduced tumor burden in the Ehrlich carcinoma model (TIR% = 65%), recovered hematological parameters, and demonstrated negligible systemic toxicity. ADMET projections further highlighted positive drug-like qualities. Taking together, compound 4p is a promising anticancer candidate that targets CDK2 and exhibits strong in vivo efficacy, with supporting molecular evidence.