In the present study, four new steroidal saponins, namely vernoniamyoside A–D (1–4), together with the two known steroidal saponins vernoamyoside D (5) and vernonioside B₂ (6) were isolated from the ethanol extract of leaves of the African medicinal plant Vernonia amygdalina Del. (Asteraceae). Their structures were demonstrated by spectral analyses along with 1D and 2D nuclear magnetic resonance (NMR) techniques and mass spectrometry (MS). The cytotoxicity of the compounds was also tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method on the cell lines Hela, MCF-7, BT-549 and MDA-MB-231. Vernoniamyoside A, vernoniamyoside B, and vernonioside B₂ showed cytotoxicity towards BT-549 cell lines. Vernoniamyoside C, vernoniamyoside D and vernoamyoside D showed different levels of cytotoxic activities.

Metabolic heterogeneity resulting from the intra-tumoral heterogeneity mediates massive adverse outcomes of tumor therapy, including chemotherapeutic resistance, but the mechanisms inside remain largely unknown. Here, we find that the de novo pyrimidine synthesis pathway determines the chemosensitivity. Chemotherapeutic drugs promote the degradation of cytosolic Carbamoyl-phosphate synthetase II, Aspartate transcarbamylase, and Dihydroorotase (CAD), an enzyme that is rate-limiting for pyrimidine synthesis, leading to apoptosis. We also find that CAD needs to be cleaved by caspase-3 on its Asp1371 residue, before its degradation. Overexpressing CAD or mutating Asp1371 to block caspase-3 cleavage confers chemoresistance in xenograft and Cldn18-ATK gastric cancer models. Importantly, mutations related to Asp1371 of CAD are found in tumor samples that failed neoadjuvant chemotherapy and pharmacological targeting of CAD-Asp1371 mutations using RMY-186 ameliorates chemotherapy efficacy. Our work reveals the vulnerability of de novo pyrimidine synthesis during chemotherapy, highlighting CAD as a promising therapeutic target and biomarker.