Design, synthesis, and biological evaluation of pyrazole-based combretastatin A-4 analogues as potential cytotoxic agents

The design of pyrazole-based combretastatin A-4 analogues 4a-g depended on preservation of the cis olefinic configuration of combretastatin A-4 (CA-4) by replacing it with a pyrazole ring, aiming to enhance the cytotoxic activity. Furthermore, the pyrazoline moiety of 4a-g was included to add a polar center for binding interactions and to contribute to Apoptosis induction. Synthesis of pyrazoline derivatives 4a-g was achieved through a new chalcone analogues 3a-g and were confirmed by spectral data and high-resolution mass spectroscopy. The cytotoxic activity was the intended biological target of the new compounds, and upon preliminary in vitro investigation against the NCI-60 panel at one dose, and it was found that the pyrazoline derivative 4a showed remarkable growth inhibition against fifteen Cancer cell lines. Also, the cytotoxic activity of compounds 4a-g was screened in vitro against non-small cell lung Cancer cell line A549 and ovarian Cancer cell line OVCAR-4, along with the normal lung cell line WI-38, and their half-maximal inhibitory concentration (IC₅₀) values were calculated. Compound 4a was found to be the most potent cytotoxic agent (IC₅₀ = 3.46 and 5.93 μM against OVCAR-4 and A549 cells, respectively) and the most selective derivative towards A549 cells with a selectivity index equal to 7.2. The inhibition of tubulin polymerization by the most active compound, 4a, was comparable to CA-4 and was visualized by means of immunofluorescent staining of A549 cells. Prediction of Reactive Oxygen Species (ROS) production by pyrazole-pyrazoline hybrid 4a was confirmed as a rise of ROS level by compound 4a was observed to be almost 243 %- in respect to control- and ROS scavenger enzyme catalase inhibited apoptotic cell death induced by 4a by 10.5 %. Moreover, compound 4a caused a decline of the mitochondrial membrane potential by approximately 56 % less than the control, which confirms the presence of ROS. The apoptotic cell death by 4a was further studied, and it showed a 3.7-folds elevation of Caspase-3 expression compared to the control. Additionally, a molecular docking study of compound 4a into the colchicine binding site of tubulin confirmed the anti-tubulin activity. Indeed, pyrazoline derivative 4a is a promising new druggable molecule that acts as a tubulin polymerization inhibitor and ROS-mediated apoptosis-inducing agent.

2-pyrazoline; Apoptosis; CA-4; CBSIs; Cytotoxic activity; Pyrazole; ROS; Tubulin.