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Chem. Res. Toxicol. 1996, 9, 1414-1424
NMR Spectroscopic and Theoretical Chemistry Studies on the Internal Acyl Migration Reactions of the 1-O-Acyl-β-D-glucopyranuronate Conjugates of 2-, 3-, and 4-(Trifluoromethyl)benzoic Acids Andrew W. Nicholls,† Kazuki Akira,†,‡ John C. Lindon,† R. Duncan Farrant,§ Ian D. Wilson,| John Harding,| David A. Killick,| and Jeremy K. Nicholson*,† Department of Chemistry, Birkbeck College, University of London, Gordon House, 29, Gordon Square, London WC1H 0PP, U.K., Physical Sciences Research Unit, Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, U.K., and Department of Safety of Medicines, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, SK10 4TG, U.K. Received March 13, 1996X
High resolution 19F NMR spectroscopy has been used to investigate the kinetics of internal acyl migration and hydrolysis of the synthetic β-1-O-acyl-D-glucopyranuronates of 2-, 3-, and 4-(trifluoromethyl)benzoic acids (TFMBAs) in phosphate buffer solutions at 30 °C as models of drug ester glucuronides. Apparent first-order degradation of the 1-O-acyl glucuronide and the sequential appearance of 2-, 3-, and 4-O-acyl isomers as both R- and β-anomeric forms were observed for each TFMBA isomer. The overall degradation rate constants of the 2-, 3-, and 4-TFMBA 1-O-acyl isomers were 0.065 h-1, 0.25 h-1, and 0.52 h-1. In order to probe the reasons for these differences in reactivity, theoretical structural and electronic parameters for the β-anomers of the 1-O-acyl glucuronides, their β-2-O-acyl isomers, and both structures of the postulated ortho-acid ester intermediate were computed using semiempirical molecular orbital (AM1 and PM3) methods. The distinction between the slowly reacting 2-TFMBA glucuronide and the much faster reacting 3- and 4-TFMBA glucuronides could be observed by calculation of the relative bond order of the C-O bonds in the ortho-acid ester intermediates. The slow internal acyl migration rate of the 2-TFMBA isomer was also partly attributed to the high degree of steric hindrance of the trifluoromethyl group obstructing attack by the glucuronic acid 2-hydroxy group on the carbonyl carbon to form the ortho-acid ester intermediate. Some calculated molecular orbital properties, namely, dipole moment, energy of the lowest unoccupied molecular orbital (LUMO), LUMO density, and nucleophilic frontier density on the carbonyl carbon, were also shown to be related to the measured half-lives. This work gives insight into the molecular physicochemical properties that influence the acyl migration kinetics of simple model drug glucuronides and is of potential importance in understanding more complex drug glucuronide acyl migration reactions of toxicological interest.
Introduction Conjugation of xenobiotic carboxylic acids with β-Dglucuronic acid to yield β-1-O-acyl-D-glucopyranuronates (β-1-O-acyl glucuronides)1 is a major in vivo metabolic pathway for many drugs including a range of hypolipidemic and nonsteroidal anti-inflammatory drugs (NSAIDs) (1). Ester glucuronides are unstable in aqueous solutions at pH 7.4 due to the susceptibility of the acyl groups to nucleophilic attack. These compounds undergo both spontaneous (buffer solution) and enzymatic (biofluid) hydrolysis along with hydroxide ion catalyzed intra* Correspondence to be addressed to: Professor Jeremy K. Nicholson, Department of Chemistry, Birkbeck College, University of London, Gordon House, 29, Gordon Sq., London WC1H 0PP, U.K. Tel:+44 171 380 7468; Fax:+44 171 380 7464; E-mail:
[email protected]. † University of London. ‡ Present address: Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. § Glaxo Wellcome Medicines Research Centre. | Zeneca Pharmaceuticals. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: TFMBA, (trifluoromethyl)benzoic acid; β-1-O-acyl glucuronides, 1-O-acyl-β-D-glucopyranuronates; MO, molecular orbital; FIDs, free induction decays; SCF, self-consistent field; SAS, solvent accessible surface area; LUMO, lowest unoccupied molecular orbital; HOMO, highest occupied molecular orbital; DFT, density functional theory; TFA, trifluoroacetic acid.
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molecular acyl migration (buffer solution (2, 3), biofluids (4-6)) to isomers of the 1-O-acyl glucuronide. The mechanism of internal acyl migration is well established (7-9) and involves the transfer of the acyl group to the C-2, C-3, or C-4 position of the glucuronic acid ring (3). Acyl groups migrate away from C-1 toward C-4 via orthoacid ester intermediates (10) which are formed by nucleophilic attack of neighboring glucuronic acid hydroxyl groups on acyl carbonyl groups as shown in Scheme 1. These glucuronide isomers readily ring-open and mutarotate giving R- and β-anomers which can then undergo further acyl migration reactions since all reactions appear to be reversible except the initial β-1-O-acyl to β-2-O-acyl migration reaction (11) (Scheme 2). The rates of degradation by acyl migration and hydrolysis have been reported for various drug 1-O-acyl glucuronides (3), and the rates of acyl migration of the isomers of fluorinated benzoic acid glucuronides have recently been reported by us (12, 13). In vitro, acyl migration has been observed to predominate with respect to hydrolysis (9, 14-16), but the exact extent of acyl migration and hydrolysis in vivo is still unknown (6) due to the complication caused by enzymatic hydrolysis. The acyl glucuronide positional isomers produced by acyl migration and mutarotation have been shown to © 1996 American Chemical Society
Acyl Migration of Model Drug Glucuronides Scheme 1. The Reaction Products Arising from Internal Acyl Migration of a Drug 1-O-Acyl Glucuronidea
a An ortho-acid ester intermediate is postulated in all steps. Mutarotation is possible in the 2-, 3-, and 4-O-acyl isomers.
react with proteins to form covalent adducts in vitro for fenoprofen (17) and diflunisal (18). In vivo studies have shown the existence of covalent binding between plasma proteins and the parent drugs zomepirac (19), tolmetin (4, 20), probenecid (21), and diflunisal (21, 22). However, in vivo covalent binding of the drug acyl glucuronide or the positional isomers with plasma proteins has yet to be observed. Since covalent binding may be a general phenomenon for labile acyl glucuronides, such protein binding would be of toxicological concern since these chemically modified proteins may be immunogenic in vivo (23). Recent in vivo studies using tolmetin glucuronideprotein conjugates in mice (20) and using diflunisal glucuronide-protein conjugates in rats (22) have shown the formation of antibodies. Moreover, it was shown that the immunoglobulins produced were minimally crossreactive with regard to other NSAIDs, but did show crossreactivity to other drug glucuronides of similar structure (20). These observations have added support to the hypothesis that acyl glucuronide-protein conjugates may be responsible for observations of drug hypersensitivity, but the general applicability to all glucuronides remains to be shown.
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Two mechanisms for covalent protein binding have been proposed. The first mechanism, proposed by van Breeman and Fenselau (24), involves direct nucleophilic displacement of the glucuronic acid moiety of a 1-O-acyl glucuronide by an -SH, -NH2, or -OH group of a protein to give the corresponding thioester, amide, or ester derivative (Scheme 2A). This mechanism has been demonstrated in the covalent binding of oxaprozin glucuronide to human serum albumin (25) in vitro, but the covalent adduct has not been found in vivo. Smith et al. (26) proposed an alternative mechanism (the “imine” mechanism) where the covalent binding follows acyl migration, by the subsequent formation of a Schiff base linkage between the free aldehyde of the open-chain acyl migrated glucuronide and a nucleophilic amine group normally from a lysyl residue of a protein (Scheme 2B). The validity of this hypothesis has been demonstrated by the discovery of covalent protein binding with human serum albumin of zomepirac glucuronide (26) and etodolac glucuronide (14) with the covalent adduct of the zomepirac glucuronide also being found in vivo (26). Both mechanisms result in the formation of protein adducts, but the nucleophilic displacement reaction by protein residues may account for only a small proportion of the adducts in those drug acyl glucuronides which readily undergo intramolecular acyl migration. For xenobiotic glucuronides which undergo rapid acyl migration to the glucuronide isomers, the major mechanism of covalent binding would appear to be the “imine” mechanism. However, the general applicability of this observation is as yet unknown. The metabolism of acyl glucuronides may involve excretion into the bile with subsequent β-D-glucuronidase hydrolysis in the small intestine to the unconjugated drug and reabsorption into the systemic circulation (27). 1-Oacyl glucuronides are also hydrolyzed by tissue and serum esterases, and the resulting aglycones again become available for further biotransformation (28). The 2-, 3-, and 4- positional isomers formed by acyl migration have been shown to be resistant to β-D-glucuronidase (7), but can be hydrolyzed back to the aglycone by esterases (6). Hence under physiological conditions, the enzymatic hydrolysis of the acyl isomers may result in a reduction of the potential drug immunogenicity.
Scheme 2. The Proposed Mechanisms of Protein Binding for Acyl Glucuronidesa
a(A) Direct nucleophilic displacement of the acid moiety. (B) Formation of a Schiff base linkage between the glucuronide and an amine group on the protein.
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It has been postulated that the extent of formation of the immunotoxic species may be related to the rate of the initial 1-O-acyl to 2-O-acyl migration, although this is highly dependent on the pH, temperature, and the physical nature of the solution (e.g., it may vary between buffer solution, blood plasma, urine, and bile). The 1-Oacyl glucuronide degradation rates are influenced by the chemical structures and molecular physicochemical properties of the aglycone (16). Indeed, Benet et al. have shown a strong relationship between the substitution pattern on the carbon R to the carbonyl carbon and the rate of acyl migration (29). However, no detailed systematic or quantitative studies have been undertaken on the relationships between the physicochemical properties of drugs and acyl migration. Derivation of relationships between the drug structure and the propensity for acyl migration would potentially be useful in drug design studies and aid the avoidance of potential toxicological difficulties related to glucuronide reactivity. We have previously studied the metabolism of (trifluoromethyl)benzoic acids (TFMBAs) in the rat using high-field NMR spectroscopy for quantitative identification of metabolites and have used computational chemistry to provide physicochemical molecular descriptors for predicting the metabolic fate (30, 31). Using 19F NMR, 2- and 4-TFMBAs were found to be mainly biotransformed to acyl glucuronides, while 3-TFMBA was converted to a glycine conjugate in vivo with a minor glucuronide component (32, 33). Thus, the use of 19F NMR and the TFMBA glucuronides as model compounds was considered to be useful for the studies on structuremetabolism and structure-acyl migration relationships. In the present study, we have investigated the kinetics of internal acyl migration of synthetic 2-, 3-, and 4TFMBA glucuronides in phosphate buffer solution at pH 7.25 using 19F NMR spectroscopy. Semiempirical molecular orbital (MO) methods have also been employed to derive theoretical structural and electronic parameters in order to evaluate whether such parameters are useful as predictors of the variation of the glucuronide acyl migration rates and hence the toxicological risk factors associated with reactive glucuronide formation.
Experimental Section All reagents were of analytical grade and purchased from Aldrich Chemical Co. Ltd. (Gillingham, Dorset, U.K.). The structures of synthesized compounds were confirmed by 1H NMR spectroscopy (see below). The structures are given in Figure 1. Synthesis of the β-1-O-Acyl Glucuronides of 2-, 3-, and 4-(Trifluoromethyl)benzoic Acid (V-VII). Benzyl 2,3,4-triO-benzyl-1-O-(trichloroacetimidoyl)-R-D-glucopyranuronate (I) was synthesized according to the literature (34). A mixture of 2-, 3-, or 4-(trifluoromethyl)benzoic acid (TFMBA) (0.5 mmol) (I) (1 mmol), boron trifluoride etherate (22 mL), and crushed molecular sieves (2-3 mg) in dry dichloromethane (15 mL) was stirred under argon at -20 °C for 3 h. The reaction mixture was allowed to reach room temperature and stirred for a further hour. The reaction mixture was then successively washed with 10% aqueous sodium bicarbonate and water. The dichloromethane layer was dried and evaporated to dryness under reduced pressure to give a colorless oil in each case. The products containing 2-TFMBA and 3-TFMBA moieties were purified by “flash chromatography” using petroleum ether/ ethyl acetate (8:2 v/v). The 4-TFMBA analogue was purified by “flash chromatography” using two solvent systems, viz., petroleum ether/ethyl acetate (8:2 v/v) and then hexane/ethyl acetate (9:1 v/v). The overall yields of II, III, and IV were 82%,
Nicholls et al.
Figure 1. The TFMBA glucuronides and the synthetic precursors. Bn ) benzyl. I ) benzyl 2,3,4-tri-O-benzyl-1-O-(trichloroacetimidoyl)-R-D-glucopyranuronate; II ) benzyl 2,3,4-tri-Obenzyl-1-O-(2-(trifluoromethyl)benzoyl)-β- D -glucopyranuronate; III ) benzyl 2,3,4-tri-O-benzyl-1-O-(3-(trifluoromethyl)benzoyl)-β-D-glucopyranuronate; IV ) benzyl 2,3,4-tri-O-benzyl1-O-(4-(trifluoromethyl)benzoyl)-β-D-glucopyranuronate; V ) 1-O-(2-(trifluoromethyl)benzoyl)-D-glucopyranuronic acid; VI ) 1-O-(3-(trifluoromethyl)benzoyl)-D-glucopyranuronic acid; VII ) 1-O-(4-(trifluoromethyl)benzoyl)-D-glucopyranuronic acid. 74%, and 28%, respectively. The structures of the products were confirmed to be benzyl 2,3,4-tri-O-benzyl-1-O-(2-trifluoromethylbenzoyl)-β-D-glucopyranuronate (II), benzyl 2,3,4-tri-O-benzyl1-O-(3-trifluoromethylbenzoyl)-β-D-glucopyranuronate (III), and benzyl 2,3,4-tri-O-benzyl-1-O-(4-trifluoromethylbenzoyl)-β-D-glucopyranuronate (IV), respectively, by 1H NMR spectroscopy (see below). 1H NMR (DMSO-d ): Benzyl 2,3,4-tri-O-benzyl-1-O-(2-(tri6 fluoromethyl)benzoyl)-β-D-glucopyranuronate: δ 7.90-7.98 (m, 4H, aromatic), 7.39-7.04 (m, 20H, benzyl), 6.62 (d, 1H, H1R), 6.09 (d, 1H, H1β), 5.18 (s, 2H, BnCH2OCO), 4.85-4.34 (m, 7H, 3BnCH2O, H5), 4.02-3.70 (m, 3H, H2, H3, H4). 1H NMR (DMSO-d ): Benzyl 2,3,4-tri-O-benzyl-1-O-(3-(tri6 fluoromethyl)benzoyl)-β-D-glucopyranuronate: δ 8.29 (s, 1H, aromatic), 8.23 (d, 1H, aromatic), 8.10 (d, 1H, aromatic), 7.81 (t, 1H, aromatic), 7.40-7.05 (m, 20H, benzyl), 6.61 (d, 1H, H1R), 6.10 (d, 1H, H1β), 5.15 (s, 2H, BnCH2OCO), 4.90-4.40 (m, 7H, 3BnCH2O, H5), 4.15-3.70 (m, 3H, H2, H3, H4). 1H NMR (DMSO-d ): Benzyl 2,3,4-tri-O-benzyl-1-O-(4-(tri6 fluoromethyl)benzoyl)-β-D-glucopyranuronate: δ 8.29 (d, 2H, R aromatic), 8.18 (d, 2H, β aromatic), 7.90 (d, 2H, aromatic), 7.387.08 (m, 20H, benzyl), 6.64 (d, 1H, H1R), 6.09 (d, 1H, H1β), 5.15 (s, 2H, BnCH2OCO), 4.85-4.36 (m, 7H, 3BnCH2O, H5), 4.113.77 (m, 3H, H2, H3, H4). A mixture of II, III, or IV purified above and 10% Pd/C (w/w equivalent) in tetrahydrofuran (20 mL) was stirred under hydrogen at room temperature for 18 h. The reaction mixture was filtered through Celite and the filtrate evaporated to dryness under reduced pressure. The product was dissolved in water, filtered through a Millipore filter, and freeze-dried. V, VI, and VII (see Scheme 2) were obtained in essentially quantitative yield. The structures of the products derived from 2-, 3-, and 4-CF3 analogues were confirmed to be 1-O-(2(trifluoromethyl)benzoyl)-D-glucopyranuronic acid (V), 1-O-(3(trifluoromethyl)benzoyl)-D-glucopyranuronic acid (VI), and 1-O(4-(trifluoromethyl)benzoyl)-D-glucopyranuronic acid (VII), respectively, by 1H NMR spectroscopy and FAB-mass spectrometry. The 1H NMR spectra of V, VI, and VII were as follows: 1-O(2-(Trifluoromethyl)benzoyl)-D-glucopyranuronic acid (V): δ 8.03 (d, 1H, aromatic), 7.93 (d, 1H, aromatic), 7.80 (m, 2H, aromatic), 6.43 (d, 1H, H1R), 5.82 (d, 1H, H1β), 3.94 (d, 1H, H5), 3.72-3.58 (m, 3H, H2, H3, H4). 1-O-(3-(Trifluoromethyl)benzoyl)-D-glucopyranuronic acid (VI): δ 8.48 (s, 1H, aromatic), 8.39 (d, 1H,
Acyl Migration of Model Drug Glucuronides aromatic), 8.07 (d, 1H, aromatic), 7.79 (t, 1H, aromatic), 6.48 (d, 1H, H1R), 5.88 (d, 1H, H1β), 3.98 (d, 1H, H5), 3.79-3.63 (m, 3H, H2, H3, H4). 1-O-(4-(Trifluoromethyl)benzoyl)-D-glucopyranuronic acid (VII): δ 8.35 (d, 2H, aromatic), 7.97 (d, 2H, aromatic), 6.53 (d, 1H, H1R), 5.93 (d, 1H, H1β), 4.02(d, 1H, H5), 3.85-3.67 (m, 3H, H2, H3, H4). FAB-Mass Spectrometry Results. 1-O-(2-(Trifluoromethyl)benzoyl)-D-glucopyranuronic acid (V): m/z 365 (M - H)glycerol matrix; 1-O-(3-(trifluoromethyl)benzoyl)-D-glucopyranuronic acid (VI): m/z 365 (M - H)- glycerol/methanol matrix; acid 1-O-(4-(trifluoromethyl)benzoyl)-D-glucopyranuronic (VII): m/z 365 (M - H)- glycerol/ethanol matrix. 1H NMR spectroscopy confirmed that the ratios of anomers β:R were approximately 7:1, 3:1, and 3:1 for V, VI, and VII, respectively. Glucuronide Solution Preparations. A solution of each TFMBA glucuronide in 475 µL of 0.1 M disodium phosphate buffer (pH 7.4) and 25 µL of 2H2O (added to provide a 2H signal for field frequency lock) was prepared. The concentrations were 8.2, 13.1, and 20.8 mM for V, VI, and VII, respectively. Each solution was transferred to an NMR tube following measurement of the pH (7.25 ( 0.05), and the subsequent reaction was then monitored as quickly as possible (
