Targeting pharmacophore with probe-reactivity-guided fractionation to precisely identify electrophilic sesquiterpenes and its activity of anti-TNBC.

Innovative strategy is urgently needed to precisely discover novel natural products as lead compounds for development of new drugs against orphan diseases such as triple-negative breast cancer (TNBC). Herein, we describe a targeting pharmacophore with probe-reactivity-guided strategy for the discovery of electrophilic sesquiterpene (ES), a class of bioactive natural product.This study aimed to identify pharmacophore, based on pharmacophore with probe-reactivity-guided strategy for precisely discovering ESs from ethyl acetate extract of Eupatorium chinense L. (EEEChL) METHODOLOGY: MTT assay combined with ultra-performance liquid chromatography (UPLC) analysis was used to identify pharmacophore. UPLC-mass spectrometry (MS) was applied to carefully compare the intrinsic reactivity characteristics of two chemoselective nucleophilic probes: glutathione (GSH) and 4-bromothiophenol (BTP) reaction with ESs. ESs was isolated and identified from EEEChL by phytochemical methods. Furthermore, stoichiometric ratio and binding site of one typical ES 8β-[4'-hydroxytigloyloxy]-5-desoxy-8-desacyleuparotin (HDDE) reaction with BTP were studied by UPLC-quadrupole time-of-flight (Q-TOF)-MS and two-dimensional nuclear magnetic resonance (NMR).Eleven ESs were identified from EEEChL, MTT assay illustrated that all of the 11 ESs possess fairly good anti-TNBC activity CONCLUSIONS: Electrophilic groups were confirmed as pharmacophore of bioactive compounds contained in EEEChL. An optimised halogenated aromatic probe BTP furnishes ES-BTP conjugates that are highly conspicuous via MS by virtue of a unique isotopic bromine signature, conjugates also have a considerable separation on C18 column. The new probe-reactivity-guided strategy can effectively improve the traditional bioassay-guided approaches, and significantly increase the probability of obtaining designated bioactive compounds.