Synthesis of Novel 3,7-Substituted-2-(3′,4′-dihydroxyphenyl)flavones with Improved Antioxidant Activity Fre ´de ´rique A. A. van Acker,* ,†,‡ Jos A. Hageman, ‡ Guido R. M. M. Haenen, # Wim J. F. van der Vijgh, † Aalt Bast, # and Wiro M. P. B. Menge ‡ Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands, LACDR, Department of Pharmacochemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, and Department of Pharmacology and Toxicology, University Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands Received March 24, 2000 A series of 3,7-disubstituted-2-(3′,4′-dihydroxyphenyl)flavones was synthesized as potential cardioprotective agents in doxorubicin antitumor therapy. The influence of substituents on the 3 and 7 positions of the flavone nucleus on radical scavenging and antioxidant properties was explored to improve the antioxidant activity of our lead compound monoHER. In the TEAC assay most compounds had a similar potency (3.5-5 times as potent as trolox), but in the LPO assay IC 50 values ranged from 0.2 to 37 µM. In general, the 3-substituted flavones (9a-j) were the most potent compounds in the LPO assay. The number of hydroxyl groups is not the only prerequisite for antioxidant activity. Substitution in ring A of the flavonoid is not necessary for high activity, but the presence of a 7-OH group significantly modifies the antioxidant activity. The compounds are good antioxidants, which makes it interesting to evaluate them as cardioprotective agents. Introduction Flavonoids form a class of benzo-γ-pyrone derivatives which are ubiquitous in plants. The immediate family members of flavonoids include flavones, flavanes, fla- vonols, anthocyanidins, and catechins. They possess a wide spectrum of biological activities. Some flavonoids have been found to possess anticancer, 1,2 antiischemic, 3 antiallergic, antiinflammatory, 4-6 and several other activities. Recent interest in these substances has been stimulated by the potential health benefits arising from the antioxidant activity of these polyphenolic com- pounds. These are the result of their high propensity to transfer electrons, to chelate ferrous ions, and to scavenge reactive oxygen species. 7 Because of these properties, flavonoids have been considered as potential protectors against chronic cardiotoxicity caused by the cytostatic drug doxorubicin. Doxorubicin is a very effective antitumor agent, but its clinical use is limited by the occurrence of a cumula- tive dose-related cardiotoxicity, resulting in, for ex- ample, congestive heart failure. Although the mecha- nism causing this chronic cardiotoxicity has not been fully elucidated, it is generally believed that the forma- tion of oxygen free radicals plays a crucial role (for reviews, see refs 8-12). In the presence of traces of iron, doxorubicin facilitates the formation of radicals. More- over, the iron-catalyzed Haber-Weiss reaction (eq 1) or the Fenton reaction (eq 2) is induced, leading to the formation of the extremely reactive hydroxyl radical. Recently, our group has demonstrated that 7-monohy- droxyethylrutoside (monoHER; Chart 1), a semisyn- thetic flavonoid belonging to a class of hydroxyethylru- tosides, provides dose-dependent protection against the doxorubicin-induced cardiotoxicity. 13 In the present study monoHER has been used as a lead structure for the development of new flavones with improved antioxidant activity. The general structure of the new compounds consists of a flavone backbone with a C2-C3 double bond and a catechol moiety on ring B. According to previously published structure-activity relationships (SARs) of commercially available fla- vonoids, incorporation of these structural elements leads to potent antioxidants. 14-18 All new compounds were tested for their antioxidant activity in two different assays. First by the trolox equivalent antioxidant capa- city assay (TEAC) which is a chemical assay that is a one-phase system containing 2,2′-azinobis(3-ethylben- zthiazoline-6-sulfonic acid) radicals (ABTS). This assay allows us to study the radical scavenging activity directly without interference by other factors. In addi- tion to this chemical assay, the compounds were tested in a lipid peroxidation assay using rat liver microsomes. In this two-phase system with a lipid and an aqueous phase, not only radical scavenging but, for example, also iron chelation and lipophilicity of the compounds play a role. By comparing the results of these two assays, the influence of the iron chelation and lipophilicity on the antioxidant activity can be estimated. Previously SAR studies have been performed using either commercially available flavonoids or flavonoids * Corresponding author: F. A. A. van Acker, Dept. of Medical Oncology, BR-232, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Tel: +31204442773. Fax: +31204443844. E-mail: f.vanacker@azvu.nl. † University Hospital Vrije Universiteit. ‡ Vrije Universiteit. # University Maastricht. H 2 O 2 + O 2 •- + Fe 2+ f HO • + HO - + O 2 + Fe 2+ (1) H 2 O 2 + Fe 2+ f HO • + HO - + Fe 3+ (2) 3752 J. Med. Chem. 2000, 43, 3752-3760 10.1021/jm000951n CCC: $19.00 © 2000 American Chemical Society Published on Web 09/15/2000