Artepillin C isoprenomics: Design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity Yoshihiro Uto, a Shutaro Ae, a Daisuke Koyama, a Mitsutoshi Sakakibara, a Naoki Otomo, a Mamoru Otsuki, a Hideko Nagasawa, a Kenneth L. Kirk b and Hitoshi Hori a, * a Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minamijosanjimacho-2, Tokushima 770-8506, Japan b Laboratory of Bioorganic Chemistry, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA Received 17 March 2006; revised 5 April 2006; accepted 6 April 2006 Available online 11 May 2006 Abstract—We designed and synthesized isoprene analogues of artepillin C, a major component of Brazilian propolis, and investi- gated the inhibitory activity on lipid peroxidation of rat liver mitochondria (RLM) and RLM toxicity based on isoprenomics. We succeeded in the synthesis of artepillin C isoprene analogues using regioselective prenylation within the range from 22% to 53% total yield. Reactivity of artepillin C and its isoprene analogues with ABTS (2,2 0 -Azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical cations showed only a slight difference among the molecules. The isoprene side-chain elongation analogues of artepillin C showed almost the same inhibitory activity against RLM lipid peroxidation as artepillin C. Artepillin C and its isoprene analogues had very weak RLM uncoupling activity. Moreover, artepillin C and its isoprene analogues exhibited a lower inhibitory activity against aden- osine 5 0 -triphosphate (ATP) synthesis by about two orders of magnitude than the effective inhibitory activity against RLM lipid peroxidation. From these results we conclude that artepillin C isoprene analogues could be potent lipid peroxidation inhibitors hav- ing low mitochondrial toxicity. We also conclude that elongation of the isoprene side chain of artepillin C to increase lipophilicity had little influence on the inhibitory activity toward RLM lipid peroxidation. Ó 2006 Elsevier Ltd. All rights reserved. 1. Introduction Mitochondria are indispensable to the maintenance of human life as the site of organization of energy produc- tion. However, it is well known that potentially toxic reactive oxygen species (ROS) are produced by the elec- tron transport chain (ETC) in the mitochondrial inner membrane. 1 In addition, mitochondria are very suscep- tible to lipid peroxidation by ROS because of the pres- ence in the mitochondrion of high concentrations of unsaturated lipids. 2 It has been pointed out that such ROS-mediated peroxidation is related to carcinogenesis and aging. 3,4 Natural anti-oxidants are present to provide protection against this. Thus, ubiquinone (coenzyme Q) is a component of the mitochondrial respiratory chain. 5 A structural feature of ubiquinone is the presence of from 1 to 12 of consecutive prenyl side chains that makes ubiquinone a highly hydrophobic structure with high affinity for membrane. In addition, a semiquinone radical of ubiquinol, as a reduced form of ubiquinone, is relatively stable so it functions as an effective antioxidant in the mitochondria. 6 Paradoxical- ly, coenzyme Q is also involved in superoxide produc- tion by the respiratory chain. 7 The development of additional effective antioxidants targeted to the mito- chondria is an important strategy for the prevention and treatment of mitochondrial dysfunction. a-Tocopherol (vitamin E) is an endogenous antioxidant and potent lipid peroxidation inhibitor, possessing the phenolic hydroxyl group and hydrophobic alkyl side chain present in ubiquinone. Exogenous antioxidants also are known Artepillin C (Fig. 1) is a diprenyl-p- hydroxycinnamic acid derivative first isolated from Bac- 0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2006.04.015 Keywords: Artepillin C isoprene analogue; Isoprenomics; Lipid peroxidation inhibitor; Rat liver mitochondria. * Corresponding author. Tel.: +81 88 656 7514; fax: +81 88 656 9164; e-mail: hori@bio.tokushima-u.ac.jp Bioorganic & Medicinal Chemistry 14 (2006) 5721–5728