{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T17:13:10Z","timestamp":1784826790209,"version":"3.55.0"},"reference-count":29,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T00:00:00Z","timestamp":1768435200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["NRF-2022R1A2C1008642"],"award-info":[{"award-number":["NRF-2022R1A2C1008642"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001459","name":"National Research Facilities and Equipment Center","doi-asserted-by":"publisher","award":["2019R1A6C1010001"],"award-info":[{"award-number":["2019R1A6C1010001"]}],"id":[{"id":"10.13039\/501100001459","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Jeju RISE Center","award":["2025-RISE-17-001"],"award-info":[{"award-number":["2025-RISE-17-001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: Flavonoids are widely used as lead structures in drug discovery, and their pharmacological and metabolic properties are strongly influenced by structural features such as positional isomerism. This study aimed to compare the metabolic profiles and underlying mechanisms of two isoflavone-based positional isomers, ACF-02 (2-(4-hydroxy-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one) and ACF-03 (2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one). Methods: The metabolic pathways of synthetically prepared ACF-02 and ACF-03 were investigated using an in vitro incubation system with human liver microsomes (HLMs) supplemented with an NADPH-regenerating system, followed by liquid chromatography\u2013high-resolution tandem mass spectrometry (LC\u2013HRMS\/MS) analysis. Metabolites were identified based on LC\u2013HRMS\/MS data and molecular networking-based node connectivity with the parent compounds. Major metabolites were further characterized by CYP phenotyping using recombinant CYP450 isoforms, and the potential for drug\u2013drug interactions of ACF-03 was evaluated using a CYP probe substrate cocktail approach. Results: HLM incubation of ACF-02 and ACF-03 produced both hydroxylated and O-demethylated metabolites, with O-demethylation as the predominant pathway; notably, the most abundant O-demethylated metabolite differed in an isomer-dependent manner, occurring at the B2 ring for ACF-02 and at the A ring for ACF-03, with distinct CYP isoform involvement. Molecular networking supported the relationships between the parent compounds and their metabolites, and both compounds exhibited relatively high metabolic stability with limited CYP inhibition. Conclusions: Despite differing only in the position of a single methyl substituent, ACF-02 and ACF-03 exhibited distinct isomer-dependent metabolic profiles. These findings demonstrate that even subtle positional isomerism can significantly influence metabolic behavior and should be carefully considered during lead optimization and drug design.<\/jats:p>","DOI":"10.3390\/pharmaceutics18010114","type":"journal-article","created":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T16:21:13Z","timestamp":1768494073000},"page":"114","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Metabolism of the Isoflavone Derivative Structural Isomers ACF-02 and ACF-03 in Human Liver Microsomes"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1681-829X","authenticated-orcid":false,"given":"Zhuoning","family":"Liang","sequence":"first","affiliation":[{"name":"BK21 FOUR KNU Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eui-Hyeon","family":"Kim","sequence":"additional","affiliation":[{"name":"Clinical Omics Institute, School of Medicine, Kyungpook National University, Daegu 41405, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ga-Young","family":"Kim","sequence":"additional","affiliation":[{"name":"BK21 FOUR KNU Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2374-2314","authenticated-orcid":false,"given":"Jin-Hyuk","family":"Choi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, School of Medicine, Jeju National University, Jeju 63243, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0489-2486","authenticated-orcid":false,"given":"Hyung-Ju","family":"Seo","sequence":"additional","affiliation":[{"name":"BK21 FOUR KNU Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3285-5594","authenticated-orcid":false,"given":"Kwang-Hyeon","family":"Liu","sequence":"additional","affiliation":[{"name":"BK21 FOUR KNU Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea"},{"name":"Mass Spectrometry Based Convergence Research Institute, Kyungpook National University, Daegu 41566, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4799-7584","authenticated-orcid":false,"given":"Moonjae","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, School of Medicine, Jeju National University, Jeju 63243, Republic of Korea"},{"name":"ACHEMBIO Inc., Jeju 63169, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1332","DOI":"10.2741\/229","article-title":"Dietary flavonoids as cancer-preventive and therapeutic biofactors","volume":"3","author":"Nishiumi","year":"2011","journal-title":"Front. 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