Synthesis of N -acetyl-S -(3-coumarinyl)-cysteine methyl ester and HPLC analysis of urinary coumarin metabolites Gerhard Eisenbrand *, Michael Otteneder, Weici Tang Division of Food Chemistry and Environmental Toxicology, Department of Chemistry, University of Kaiserslautern, D-67663 Kaiserslautern, Germany Received 5 March 2003; received in revised form 6 May 2003; accepted 6 May 2003 Abstract N -Acetyl-S -(3-coumarinyl)cysteine, a metabolite of coumarin in rodents, has been synthesized as methyl ester. A new synthetic route to prepare N -acetyl-S -(3-coumarinyl)-D,L-cysteine methyl ester (1) comprises reaction of 3- mercaptocoumarin (3) with N -acetyl-3-chloro-D,L-alanine methyl ester (4). N -acetyl-S -(4-coumarinyl)-L-cysteine (10) was obtained by reaction of 3-bromocoumarin (12) and N -acetyl-L-cysteine (13). A method for the determination of N - acetyl-S -(3-coumarinyl)cysteine as its methyl ester in urine by HPLC has been developed. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Coumarin; N -Acetyl-S -(3-coumarinyl)cysteine; Metabolism; Synthesis; HPLC-determination 1. Introduction Coumarin (2H-1-benzopyran-2-one (11)), is a widely occurring secondary plant metabolite that has been used as a fragrance in food and cosmetic products. After rapid absorption from the gastro- intestinal tract coumarin is distributed throughout the body (Cohen, 1979; Fentem and Fry, 1993). Coumarin has also been found to be well absorbed through the skin (Beckley-Kartey et al., 1997; Yourick and Bronaugh, 1997). The daily intake of coumarin has been estimated to reach 0.02 mg kg 1 body weight via food and 0.04 mg kg 1 body weight from cosmetic products (Lake, 1999). Coumarin has been reported to be used clinically for treatment of lymphedemas, mainly in breast cancer patients (Burgos et al., 1999; Loprinzi et al., 1999); for treatment of renal cell carcinoma (Sagaster et al., 1995; Marshall et al., 1994); and for prophylaxis of thromboembolism in gynecolo- gic oncology (Graf et al., 1998). The biotransformation of coumarin is known to be species-dependent (Pearce et al., 1992; Bogan et al., 1996). It appears that 7-hydroxylation of coumarin is the major phase I pathway in the majority of human subjects (Rautio et al., 1992; Bogan et al., 1995), catalyzed predominantly by cytochrome P450 isoenzyme CYP2A6 (Li et al., 1997). In phase II biotransformation, 7-hydroxy- coumarin is converted into glucuronide or sulfate * Corresponding author. Tel.:  /49-631-205-2974; fax:  /49- 631-205-3085. E-mail address: eisenbra@rhrk.uni-kl.de (G. Eisenbrand). Toxicology 190 (2003) 249  /258 www.elsevier.com/locate/toxicol 0300-483X/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00204-X