Pharmacophore-probe reaction guided purification to precisely identify electrophilic withanolides from Tubocapsicum anomalum Makino and their anti-TNBC activity

Pharmacophore-probe reaction guided purification strategy can reduce the workload of natural product chemistry and raise the probability of obtaining undescribed and high-bioactive target compounds. In this study, a probe of N-acetyl cysteine (NAC) was used to confirm the pharmacophore of Tubocapsicum anomalum (Franch. et Sav.) Makino. Furthermore, a thiol probe named 4-bromothiophenol (BTP) guided the discovery of three undescribed (1-3) and nine known (4-12) electrophilic withanolides (EWs) featured potential anti-triple negative breast cancer (TNBC) pharmacophore. Notably, co-incubation with BTP made the single crystals of EW conjugates much more accessible, which facilitated the absolute configuration determination of EWs. Electrophilic natural products with the potential of thio-alkylation modification and covalent inhibition key proteins in tumor cell signal transduction pathways may display significant antitumor activity. The MTT results indicated that most EWs showed anti-TNBC activity and were expected to develop anti-TNBC candidate drugs with high selectivity and novel mechanism.