Bioassay-guided isolation of a new cytotoxic compound targeting carbonic anhydrase-II: in-vitro structure-activity relationships and dynamics studies.

Bioassay-guided isolation of Anogeissus dhofarica A.J. Scott afforded one new natural product (lupeol butyl ether, 1), along with sixteen known metabolites (2-17) reported from this source for the first time. Structural elucidation of the isolates was performed by NMR and mass spectrometry. An in vitro carbonic anhydrase-II (CA-II) inhibition assay was performed on the crude extract, the fractions, and the resulting pure constituents. The activity against CA-II of the crude extract and fractions was in the range of IC₅₀ 45.10-102.56 µg/mL. Among the isolates, 14 was the most active with an IC₅₀ of 7.19 ± 0.20 µM followed by 10 (IC₅₀ = 13.61 ± 0.30 µM), 12 (IC₅₀ = 17.30 ± 0.58 µM) and 1 (21.63 ± 0.48 µM), with the remaining compounds having moderate to low inhibition. Fourteen compounds were evaluated against breast cancer (MDA-MB-231) and normal cell (3T3-L1) lines in an MTT assay, with most natural products exhibiting moderate activity against MDA-MB-231 cells (1, IC₅₀ = 34.5 ± 0.8 μM/mL), and less active to the normal cell line. Additionally, the molecular binding of the active hits was predicted through an in-silico approach, specifically docking molecular dynamic (MD) simulations, which revealed that acetate and carboxyl moieties play an important role in ligand binding with the Zn²⁺ ion of the CA-II active site. The MD simulation of the structure dynamics revealed that the most active inhibitors (10 and 14) had strong affinity with the CA-II active site and brought structural conformational changes to the protein.