Beyond acrylamide: a structure-guided investigation of covalent warheads in the development of CDK7 inhibitors.

To achieve covalent CDK7 inhibitors with superior antiproliferative activity and enhanced selectivity, we designed and synthesized a series of covalent CDK7 inhibitors bearing diverse warheads based on the THZ1 scaffold. Experimental validation confirmed that 2-chloroacrylamide-based covalent inhibitors possess potent CDK7 kinase inhibitory activity. HRMS results revealed that the interaction between these compounds and the cysteine residue of CDK7 extends beyond a simple Michael addition, potentially involving nucleophilic substitution at the covalent warhead. Among them, compound B19 exhibited potent CDK7 kinase inhibition with an IC50 value of 0.370 ± 0.0200 nM, as well as strong anti-proliferative activity against MV-4-11 (IC50 = 0.820 ± 0.0500 nM), Molm-13 (IC50 = 1.14 ± 0.0900 nM) and MDA-MB-231 (IC50 = 20.2 ± 1.34 nM) cells. Furthermore, B19 strongly suppressed CDK7-mediated downstream signaling and induced apoptosis and G0/G1 phase arrest in MV-4-11 cells, supporting its potent antitumor effects. Wash-out assays indicated that compound B19 possesses a prolonged activity profile compared with THZ1. Additionally, B19 displayed favorable selectivity across a panel of other kinases. Collectively, these results identify B19 as a promising lead compound for targeting CDK7.