A new [ 2 H]-labelled α-trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates Georg Heinkele, Mirjam C. K. Geditz, Boian Ganchev, Reinhold Kerb, Ute Hofmann, and Thomas E. Mürdter * A new reaction pathway for the synthesis of a [ 2 H]-labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope-labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis. Keywords: 2,3,4-[ 2 H 3 ]-6-Methyl-2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-D-glucopyranuronate; glucuronidation; stable isotope- labelled internal standard; 2′,3′,4′-[ 2 H 3 ]-Dihydroartemisinin-12α,1′β-glucuronide; 2′,3′,4′-[ 2 H 3 ]-Clomiphene-4-O-1′β-glucuronide Introduction Glucuronidation is the major phase 2 pathway involved in xenobiotic metabolism in humans. The conjugation reaction is catalysed by the superfamily of membrane-bound uridine-5′- diphospho-glucuronosyltransferases (UGTs) comprised of 22 isoforms in four families. UGT 1A, 2A and 2B are mainly responsible for the transfer of glucuronic acid from uridine-5′- diphosphoglucuronic acid to hydroxyl, sulfhydryl, amino or acyl groups of various endogenous and xenobiotic substrates. 1 The increased water solubility of the resulting glucuronides enhances urinary elimination. 2 Glucuronidation is thought to be a detoxification pathway, which is generally true for the majority of glucuronides such as O-alkyl- and O-aryl-glucuronides. 3 However, some glucuronides (mostly O-acyl-glucuronides such as diclofenac-glucuronide) are held responsible for adverse drug reactions. 4 On the other hand, some glucuronides, for example, morphine-6-glucuronide or ezetimibe-glucuronide, exhibit biological effects that significantly contribute to the efficacy of the parent drugs. 5,6 Together with the fact that glucuronidation can play a role in drug–drug interactions with clinical relevance, these findings led to an increased interest in pharmacokinetic data of drug glucuronides. 7 State-of-the-art liquid chromatography–mass spectrometric analysis requires both reference compounds and stable isotope-labelled internal standards for accurate and fast quantification. 8–11 Usually, the isotope label is located in the aglycone moiety of the respective glucuronide. 12–14 However, stable isotope- labelled phase 1 metabolites as precursors are not always available in sufficient amounts to conduct glucuronide syntheses, which are often burdened with low yields. An approach to overcome this is to include the isotope label into the glucuronic acid moiety as proposed by Latli et al. 15 Starting with [ 13 C 6 ]-glucose a five-step synthesis afforded allyl-[ 13 C 6 ]- glucuronate, an excellent precursor for the synthesis of stable isotope-labelled acyl glucuronides. In contrast, the synthesis of O-alkyl and O-aryl glucuronides is often achieved by the approach of Koenigs–Knorr or Schmidt. 16 Here, we describe a seven-step reaction sequence for the synthesis of the deuterium-labelled trichloroacetimidate 2,3,4-[ 2 H 3 ]-6-methyl-2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl- α-D-glucopyranuronate (1) (Figure 1). The usability of (1) was proven via synthesis of 2′,3′,4′-[ 2 H 3 ]-dihydroartemisinin-12α,1′β- glucuronide and 2′,3′,4′-[ 2 H 3 ]-clomiphene-4-O-1′β-glucuronide. Results and discussion The synthesis of the stable isotope-labelled trichloroacetimidate precursor was achieved by a seven-step synthesis pathway starting with 1-methyl-α-D-glucopyranoside (2) as shown in Scheme 1. The hydrogen/deuterium (H/D) exchange of 1-methyl-α-D- glucopyranoside (2) was performed according to Koch et al. with 2 H 2 O on Raney nickel catalyst. 17 The progress of the H/D exchange was monitored following derivatization with N- methyl-bis-trifluoroacetamide by gas chromatography–mass spectrometry (MS). According to the specific fragmentation pattern of the tetrakis(trifluoroacetyl)-α-D-methylglucoside described by Ando et al., the predominant integration of five deuterium atoms was observed. 18 The absolute isotope Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Auerbachstrasse 112, 70376 Stuttgart, Germany *Correspondence to: Thomas E. Mürdter, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376, Stuttgart, Germany. E-mail: Thomas.muerdter@ikp-stuttgart.de Copyright © 2014 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2014 Research Article Received 11 July 2014, Revised 15 August 2014, Accepted 26 September 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jlcr.3243