Abstract 5736: Design of CYP1-activated 2,4-diaryl-substituted pyridine analogues with significant anti-proliferative effects in breast cancer cells <i>in vitro</i>
Authors: Federico Brucoli, Ketan Ruparelia
Journal: Cancer Research (2025)

Abstract Cytochromes P450-1A1 and 1B1 (CYP1A1 and CYP1B1) are over-expressed in several human malignancies and represent an excellent target for enzyme prodrug therapy of cancer. The extrahepatic CYP1A1 isoform is constitutively expressed in a number of human breast cancers. CYP1B1 has been consistently detected in both pre-malignant tissue and metastatic cancer and its expression is associated with hormone-dependent tumours (e.g., breast and ovary). We have developed lead-prodrugs that selectively targeted CYP1 family enzymes. Here, we report the extensive chemistry work, which included the synthesis of more than 150 analogues, cytotoxicity screening results and CYP1 metabolisation data that led to the identification of a series of 2, 4-diaryl-substituted pyridine lead-compounds. In our in vitro breast cancer model, the CYP1-activated pro-drugs were highly active against triple negative breast cancer MDA-MB-468 cells (IC50=0.08µM), which constitutively express CYP1B1. The pro-drugs also had significant antiproliferative activity against MCF7 cells, which were induced to express CYP1A1 upon treatment with aryl hydrocarbon agonist 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The hit-compounds exhibited no toxicity in "normal" human breast MCF10A cells (IC50&amp;gt;100µM), which have no basal CYP1 expression. Cytotoxicity assays conducted in the presence of known CYP1 inhibitor α-naphthoflavone resulted in a 300-fold increase in the pro-drugs IC50 values from 0.01 to 3µM in MDA-MB-468 cells. Dose-response studies using the NCI 60 human tumour cells panel showed that selected prodrugs were 4-fold more selective towards breast cancer cells (GI50=41nM) compared to the other cell lines tested. In vitro metabolism of selected lead-compounds was assessed by separate incubation with human CYP1A1, CYP1A2, CYP1B1 and CYP3A4 enzymes using HPLC methods. Both CYP1A1 and A2 were the most efficient catalysts of the pro-drugs metabolism, followed by CYP1B1. The prodrugs were not metabolised by CYP3A4. Tandem mass spectrometry (MS/MS) fragmentation and HPLC co-elution analysis allowed for the identification of the structure of the main metabolite of a benzodioxole-containing lead-compound that was produced by CYP1A1, CYP1A2 and CYP1B1 isozymes. The CYP1-generated metabolite was synthesised and cytotoxicity screening revealed that the compound exhibited antiproliferative activity with an IC50 &amp;lt;0.07µM against all the cell lines used in this study (e.g., MDA-MB-468, MCF10A and MCF7) irrespective of their levels of CYP1 expression. Further investigations on the metabolite mode of action are underway. In summary, we have identified cancer pro-drug chemotherapeutic agents that exhibit significant anti-proliferative activity in a CYP1-enzyme expressing breast cancer in vitro model and are suitable candidates for pre-clinical development. Citation Format: Federico Brucoli, Ketan Ruparelia. Design of CYP1-activated 2, 4-diaryl-substituted pyridine analogues with significant anti-proliferative effects in breast cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5736.