Three new spirocyclic terpenoids from Euphorbia amygdaloides exhibit cytotoxicity against cancerous cell lines through early and late apoptosis

Bioassay-guided fractionation led to the isolation of three new spirocyclic terpenoid compounds from Euphorbia amygdaloides L., named Zagrosin I–III. Their structures were identified by 1D and 2D NMR (1H NMR, 13C NMR, DEPT 135, HMBC, and HSQC-TOCSY) and LC-MS-MS spectrometry. The cytotoxicity of the isolated spirocyclic terpenoids (Zagrosin I-III) was assessed against human breast cancer (MCF-7), human fibrosarcoma (HT1080), and normal human foreskin fibroblast cells with MTT assays (24, 48, and 72 h treatments). The FITC-Annexin V apoptosis flow cytometry assays and cell cycle analysis were performed for Zagrosin I–III.These isolated compounds were identified as: (9)-8a-((benzoyloxy)methyl)-2-methoxy-4,9-dimethyltetrahydro-4H,5H-2,4a-methanobenzo[d] [1,3] dioxine-4-carboxylate (Zagrosin I), ((9)-4-hydroxy-2-methoxy-4,9-dimethyltetrahydro-4H,8aH-2,4a-methanobenzo[d] [1,3] dioxin-8a-yl) methyl benzoate (Zagrosin II), and (9)-2-methoxy-4,9-dimethyl-8a-(phenoxy methyl) tetrahydro-4H,5H-2,4a-methanobenzo[d][1,3]dioxin-4-yl 4-methylpentanoate (Zagrosin III).The IC50 of Zagrosin I on 48-h-treated MCF-7 was calculated as 1.5 μg/mL. Zagrosin II and III exhibited cytotoxicity on 48-h-treated MCF-7 with IC50s of 14.04 and 12.50 μg/mL, respectively. The IC50 of Zagrosin I on human fibrosarcoma (HT1080) was 115.5 μg/mL, Zagrosin III, 16.81 μg/mL (48 h treatment), and Zagrosin II, 142.7 μg/mL (72 h treatment). Zagrosin I-III exhibited significant cytotoxicity against the MCF-7 cell line and human fibrosarcoma (HT1080), with the mechanism of early and late apoptosis affecting cells mostly in G0/G1 fallowed by S and G2 phases. MCF-7 had a higher rate of phosphatidyl serine exposure on the cell membrane than two other studied cells. The cytotoxicity on normal human foreskin fibroblasts was low. Zagrosin I-III can be considered an effective chemical backbone for anticancer drug development.Abbreviations: 2D NMR: Two-Dimensional Nuclear Magnetic Resonance Spectroscopy; API: Atmospheric Pressure Ionization; DEPT: Distortionless Enhancement by Polarization Transfer; ELISA: Enzyme-Linked Immunosorbent assay; ERK: Extracellular signal-regulated kinase; ESI: Electrospray ionization; FBS: Fetal Bovine Serum; FITC: Fluorescein isothiocyante; fr: fraction; HMBC: Heteronuclear Multiple Bond Correlation; HPLC: High-Performance Liquid Chromatography; HSQC: Heteronuclear Single Quantum Coherence; HT1080: Human fibrosarcoma cell line; IC50: Half-maximal inhibitory concentration; MCF-7: Human breast cancer cell line; MHz: Megahertz; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; NMR: Nuclear Magnetic Resonance; PBS: Phosphate Buffered Saline; PI: Propidium Iodide; ppm: Part Per Million; Rf: Retention Factor; TLC: Thin-layer chromatography; TOCSY: Total Correlation Spectroscopy; VLC: Vacuum Liquid Chromatography.