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. [Display omitted] • Pharmacophore-probe reaction guided precision discovery strategy is reported. • Three undescribed withanolides were identified of which the structure of 1 is rare. • Four known withanolides exhibited significant anti-TNBC activity in vitro. • It may be a novel method to determine the absolute configuration of EWs using BTP. [ABSTRACT FROM AUTHOR]