Exploring novel aromatase cytochrome P450-binding ligands: Molecular docking and QSAR study of phorbol esters as aromatase inhibitors

A library comprising thirty-three natural and semi-synthetic phorbol derivatives was constructed and subsequently evaluated for aromatase (CYP19A1) inhibitory activity. The primary goal was to identify novel aromatase inhibitors through selective esterifications at the C-12, C-13 and C-20 positions of the phorbol skeleton. Twenty-two phorbol esters, fifteen of which were previously undescribed (5-9, 12 and 16-24), were obtained via semi-synthetic modifications. Additionally, ten 12-deoxyphorbol esters (25-34), four bearing an extra acyloxy chain at C-16, isolated from Euphorbia resinifera, were tested. Inhibition studies revealed that 12,13-diesters bearing medium-sized ester moieties (such as isobutyrate, tigliate or phenylacetate) exhibited the highest potency (IC₅₀ = 0.9-6.4 μM). Conversely, shorter or longer ester chains significantly reduced activity (IC₅₀ values ranging from 22.4 to 29.8 μM). An enhancement of potency was observed upon reduction of the α,β-unsaturated ketone at C-3 to an allylic alcohol. Molecular docking suggested that the phorbol derivatives bind within the aromatase activator-binding pocket, exerting their inhibitory actions through hydrogen-bond interactions with protein residues, independent of the heme group. The inhibitory potency correlated with the ability to establish these interactions. The activities of the most potent compounds are comparable to nonsteroidal, non-heme-binding aromatase inhibitors, such as stilbene and coumarin derivatives. Moreover, 3D-QSAR models were developed, with the most robust model (R² = 0.908) highlighting the molecular descriptors NQC₁₄ and AP as crucial modulators of the anti-aromatase activity. These findings provide a framework for future optimization of phorbol ester as selective aromatase inhibitors.