Compound 15c(Hainan Medical University)

Human cytochrome P450 1B1 (hCYP1B1), a heme-dependent monooxygenase predominantly expressed in extrahepatic tissues, has gained prominence as a critical therapeutic target for overcoming chemotherapy resistance and suppressing tumorigenesis. To explore potential inhibitors of hCYP1B1, three series of azepinoindole derivatives were rationally designed, synthesized, and biologically evaluated in this study. Primary structure-activity relationship (SAR) studies revealed that introducing a para-nitro group (p-NO₂) on the C-ring enhanced anti-hCYP1B1 activity, and 15n (half-maximal inhibitory concentration [IC₅₀] = 1.44 μM) was identified as a second potent hit compound. Further SAR analysis revealed that key structural modifications, such as the introduction of the pyrene group, quinuclidine ring, and p-OMe/F or m-Me substituents on the benzene ring of the C-ring, significantly affected the efficacy of these inhibitors. Ultimately, six optimized derivatives (15c, 15ab, 15ai, 15 am, 15an, and 15ap) were identified as effective hCYP1B1 inhibitors and exhibited submicromolar hCYP1B1 inhibition (IC₅₀ ≤ 0.35 μM). Subsequently, we used the human granulosa cell tumor cell line (KGN, hCYP1B1-positive) and the hepatocellular carcinoma cell line (MHCC-97H, hCYP1B1-negative) to evaluate the cytotoxic effects of the selected derivatives using the CCK-8 assay. The results demonstrated that the cytotoxicity of the compounds was strongly dependent on hCYP1B1 expression. Additionally, RNA sequencing (RNA-seq) analysis of KGN and MHCC-97H cells treated with the derivatives revealed that these compounds exert cytotoxic effects in an hCYP1B1-dependent manner, with selective inhibition of hCYP1B1 while sparing aryl hydrocarbon receptor (AhR) pathway activation. Notably, 15c exhibited favorable inhibition kinetics with a submicromolar inhibitory constant (Ki = 0.053 μM) and minimal residual activity. Molecular docking simulations further demonstrated its stable binding within the catalytic cavity of hCYP1B1, driven by a combination of hydrophobic interactions and hydrogen bonding, suggesting a robust mechanism of action. Finally, computational studies assessing pharmacokinetic properties and toxicity indicated acceptable profiles, including an acute toxicity value of LD₅₀ = 270 mg/kg. To our knowledge, this is the first report on azepinoindole-based inhibitors of hCYP1B1, demonstrating a significant activity enhancement via rational π-π stacking optimization. The promising lead compound 15c exhibits submicromolar potency with AhR-independent anticancer activity, as validated via RNA-seq and cellular assays, thus addressing a critical gap in CYP-targeted therapies. These findings establish a robust chemical framework for developing hCYP1B1-selective antineoplastic agents against hormonal malignancies.