Design, synthesis, and bioevaluation of pyrazole-containing tubulin polymerisation inhibitors based on conformational constraint strategy

Based on conformational preference of SMART analogues and conformational constraint strategy, two series of new tubulin polymerisation inhibitors (<b>4a </b>-<b> 4k</b> and <b>5a </b>-<b> 5h</b>/<b>6a </b>-<b> 6h</b>) were designed <i>via</i> hydrogen bonding, steric effect (for <b>4a </b>-<b> 4k</b>) and ring-closing approach by fused five- and seven-membered ring (for <b>5a </b>-<b> 5h</b>/<b>6a </b>-<b> 6h</b>) which was first adopted in the design of new SMART analogues. Among these compounds, <b>4k</b> and <b>5a</b> showed potent activities with IC₅₀ values of 15 nM and 6 nM against PC-3 cell line. Mechanism studies indicated that <b>4k</b> and <b>5a</b> could inhibit tubulin polymerisation, arrest cell cycle at G₂/M phase, induce cell apoptosis, and inhibit cell migration and colony formation. Molecular docking suggested that compounds <b>4k</b> and <b>5a</b> could bind into the colchicine binding site at the pose similar to DAMA-colchicine. Western blot assays revealed that <b>4k</b> and <b>5a</b> regulated the expression of cell cycle and apoptosis-related proteins. Prediction of physicochemical properties indicated good drug-likeness of <b>4k</b> and <b>5a</b>.