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Bioconjugate Chem. 1999, 10, 150−154
Enzyme-Assisted Synthesis and Structural Characterization of Nitrocatechol Glucuronides Leena Luukkanen,† Ilkka Kilpela¨inen,‡ Heli Kangas,† Pekka Ottoila,§ Eivor Elovaara,| and Jyrki Taskinen*,† Department of Pharmacy, Division of Pharmaceutical Chemistry, POB 56, FIN-00014, University of Helsinki, Finland, Institute of Biotechnology, NMR Laboratory, FIN-00014, University of Helsinki, Finland, Orion Corporation, Orion Pharma, POB 65, FIN-02101 Espoo, Finland, and Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland. Received June 8, 1998; Revised Manuscript Received November 7, 1998
Enzyme-assisted synthesis and characterization are described for 3-O-β-D-glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (nitecapone) 3a and 4′methyl-3,4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-glucuronides 5b and 6b of the aglycon 1,2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucuronidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1-O-β-D- and 2-O-β-D-glucuronides. Purification of the crude products by C18 solid-phase extraction and/or flash chromatography gave compounds 1b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were characterized on the basis of UV and IR spectra and confirmed with FAB-MS and NMR spectroscopy.
INTRODUCTION
Nitrocatechol derivatives entacapone (E-isomer) 1a, its Z-isomer (Z-entacapone) 2a, nitecapone 3a, and tolcapone 4a (Figure 1) are potent inhibitors of catechol-O-methyltransferase (EC 2.1.1.6, COMT), with Ki values in the nanomolar range (1). Entacapone and tolcapone are used clinically as adjuncts in the L-DOPA therapy of Parkinson’s disease (2). The main biotransformation reaction for these compounds is glucuronidation (3-5), catalyzed by UDP-glucuronosyltransferase, UGT, enzyme family (EC 2.4.1.17). Products of the enzymatic reactions represent the β-anomer. Futile cycling, including the glucuronidation and subsequent hydrolysis of the conjugates by β-glucuronidase, may affect the net glucuronidation in vivo (6, 7) since microsomal β-glucuronidase (EC 3.2.1.31) egasyn complex is located within the lumen of the endoplasmic reticulum (8), as is the active site of UGTs (9). The hydrolysis of nitrocatechol glucuronides by β-glucuronidase from Escherichia coli in the intestine may also be of importance in vivo as suggested by the biliary excretion of nitrocatechol glucuronides (10). Well-characterized reference glucuronides are needed for pharmacokinetic and toxicokinetic studies as well as for enzyme kinetic studies of both glucuronidation by UGTs and glucuronide hydrolysis by β-glucuronidase. A variety of methods are available for the synthesis of β-glucuronide conjugates (11). Most synthetic methods yield mixtures of R- and β-anomers and side-products such as ortho ester. * To whom correspondence should be addressed. Phone: 358 9 70859191. Fax: 358 9 70859556. E-mail: Jyrki.Taskinen@ helsinki.fi. † Department of Pharmacy. ‡ Institute of Biotechnology. § Orion Corporation. | Finnish Institute of Occupational Health.
Figure 1. Structures of the nitrocatechol derivatives and their glucuronides.
Enzyme-assisted synthesis offers an alternative way of producing glucuronides. The advantage of biocatalyzed reactions is that the formation of solely the biologically relevant stereo- and regioisomeric products can be expected. Glucuronides have been prepared in laboratory scale by batch synthesis with microsomal enzyme sus-
10.1021/bc980064n CCC: $18.00 © 1999 American Chemical Society Published on Web 12/31/1998
Enzyme Assisted Synthesis of Nitrocatechol Glucuronides Table 1. Yields and Purities of the Glucuronides 1b-6b yield
impurities, %
product
%
mg
Aglycon
UDP, UDPGA, SL
1b 2b 3b 4b 5b 6b
76 94 46 75 38a 38a
73 84 51 60 29 21
0.5 0.1 0.8 0.5 nd nd
