Phase 2 Metabolites of N-Hydroxylated Amidines (Amidoximes

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis. Paloma Riquelme , Judith ...
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Chem. Res. Toxicol. 2001, 14, 319-326

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Phase 2 Metabolites of N-Hydroxylated Amidines (Amidoximes): Synthesis, in Vitro Formation by Pig Hepatocytes, and Mutagenicity Testing Bernd Clement,* Kai Christiansen, and Ulrich Girreser Pharmazeutisches Institut, Christian-Albrechts-Universita¨ t Kiel, Gutenbergstrasse 76, D-24118 Kiel, Germany Received May 10, 2000

A pig hepatocyte culture system was used for phase 2 biotransformation studies in vitro. The viability of the cultured hepatocytes was characterized daily during cultivation by lactate dehydrogenase release into the supernatant and albumin synthesis of the cells. The metabolic activity of the hepatocyte cultures was measured by 7-ethoxycoumarin (ECOD) and 7-ethoxyresorufin O-deethylation (EROD). The viability and metabolic activity of these pig hepatocytes were preserved for several days by culturing the cells in a monolayer culture system. Besides the known reduction of N-hydroxylated benzamidine (benzamidoxime) (2) to benzamidine (1), glucuronidation and, to a much smaller extent, sulfation of 2 to benzamidoxime O-glucuronide (3) and benzamidoxime O-sulfate (4) by cultured pig hepatocytes were found. The analyses were performed using HPLC and LC/MS studies. For unequivocal assignment, the hitherto unknown metabolites 3 and 4 were synthesized and characterized by spectroscopic techniques. Examination of benzamidoxime O-glucuronide and benzamidoxime O-sulfate for mutagenicity by means of the Ames test revealed that both phase 2 conjugates have no mutagenic effects in the TA98 and TA100 strains. So the phase 2 conjugation of benzamidoxime is significant in terms of detoxification.

Introduction Biotransformation of xenobiotics by liver enzymes can result in inactivation or detoxification but also in formation of pharmacological active metabolites. Such effects should be revealed as soon as possible during preclinical development of new active compounds. Several in vitro systems have been developed for short-term biotransformation studies, i.e., liver perfusion, liver slices, cultured hepatocytes, liver homogenates, microsomes, and purified enzymes (1, 2). Among these systems, cultured hepatocytes represent a useful tool in biotransformation studies, due to the ability to produce a wide range of metabolites normally produced in vivo, including phase 2 conjugates (2). Pig liver becomes more and more important as a model for the biotransformation in humans (3) and is considered as a xenotransplant (4). In this study, cultured pig hepatocytes were characterized for their viability and metabolic activities and used to explore phase 2 conjugation of N-hydroxylated amidines (amidoximes). Amidoxime functional groups are components of numerous drug candidates in particular developed as prodrugs of amidines (5-8). Our investigations into the biotransformation of this functional group have demonstrated that reduction of this functional group is the main phase 1 reaction, as shown in Scheme 1 (5, 9, 10). The unsubstituted benzamidoxime induces DNA singlestrand breaks in rat hepatocytes and DNA amplification in SV-40 transformed hamster cells, whereas benzami* To whom correspondence should be addressed. Phone: (431) 8801126. Fax: (431) 880-1352.

Scheme 1. N-Hydroxylation of Benzamidine 1, Reduction of Benzamidoxime 2, and Phase 2 Conjugation of Benzamidoxime

dine itself exhibits only a low mutagenicity in the TA98 strain in the presence of rabbit liver S91 fractions (11). Other N-hydroxylated amidines used as prodrugs of amidines exhibit no mutagenic effects, and some seem to have even lower toxicity than the amidines (8). These amidoximes are easily reduced to the active principles, the amidines (5-8). 1 Abbreviations: ECOD, 7-ethoxycoumarin O-deethylation; ELISA, enzyme-linked immunosorbent assay; EROD, 7-ethoxyresorufin Odeethylation; KRB, Krebs Ringer buffer; LDH, lactate dehydrogenase; S9, 9000g supernatant.

10.1021/tx000105f CCC: $20.00 © 2001 American Chemical Society Published on Web 02/20/2001

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In previous studies aimed at the detection of conjugates of benzamidoxime, only indirect evidence was obtained for the possible formation of a glucuronide and a sulfate (10, 11), and the comparative synthesis of both metabolites has not yet been reported. However, it was postulated (11) that conjugates of N-hydroxylated amidines may be unstable under physiological conditions and yield electrophilic nitrenium ions which react with cellular macromolecules in a manner similar to the activation of several aromatic amines and certain aromatic amides (12). So in following up this hypothesis, we tested the mutagenicity of the Oglucuronide and O-sulfate of benzamidoxime in the Salmonella typhimurium assay of Ames et al. (13, 14) and its formation by cultured pig hepatocytes incubated with benzamidoxime. The aim of our investigations was also the independent synthesis of 3 and 4. Here, we describe the methods for the isolation, cultivation, and characterization of pig hepatocytes, the preparation of benzamidoxime O-glucuronide and benzamidoxime O-sulfate, and a LC/MS method for the direct elucidation of these conjugates in cultured hepatocytes. Whereas nonbasic nitrogen-containing funtional groups have been investigated in numerous studies with respect to their metabolic activation, only a few studies exist dealing with N-hydroxylated derivatives of strongly basic functional groups such as amidines.

Experimental Procedures Caution: Cadmium salts are cancerogenic. The positive controls used in the Ames test are known to be highly potent carcinogens. If not stated otherwise, all chemicals were obtained from E. Merck (Darmstadt, Germany) and were analytical grade. If necessary, solvents were dried and purified by distillation using standard procedures. Livers. Livers from German landrace pigs with a body weight of 70-100 kg were used from the local slaughterhouse. The animals were electrocuted and killed by exsanguination. The organs were transferred to calcium-free Krebs Ringer buffer (KRB) at pH 7.4 and 4 °C and placed into a perfusion apparatus. The perfusion was initiated 50 min after exsanguination. Hepatocyte Isolation and Culture. The portal and hepatic veins of a liver piece were cannulated using the method described by Reese and Byard (15) with slight modifications. The specimen was sequentially perfused with a calciumfree Krebs Ringer buffer (KRB) for 15 min, followed by KRB containing 0.6 mg/mL collagenase (type CLS IV, Biochrom, Berlin, Germany) and 5 mM CaCl2, for an additional 15 min. After perfusion, the cells were dispersed, filtered, and washed twice with KRB. The viability of the isolated hepatocytes ranged between 79 and 93% as assessed by trypan blue exclusion. The cells were seeded onto 60 mm Petri dishes (Sarstedt, Nu¨mbrecht, Germany), which had been coated with collagen (Boehringer Ingelheim Bioproducts, Heidelberg, Germany) using the method of Suzuki et al. (16), at a density of 2 × 106 cells. The medium used was Williams E (Biochrom), which was supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Biochrom), 9.6 µg/mL prednisolone, 0.014 µg/mL glucagon, and 0.16 unit/mL insulin (all from Sigma, Deisenhofen, Germany); 200 units/mL penicillin and 200 µg/mL streptomycin (Biochrom) were added. After 2 h at 37 °C in a humidified 95% air/5% CO2 atmosphere to allow attachment, the culture medium (2 mL) was removed along with nonadherent cells. The medium was changed every 24 h. Viability Studies. The hepatocytes were cultured for up to 9 days. Cell damage was assessed by the release of lactate dehydrogenase (LDH) into the medium, using the Cytotoxicity

Clement et al. Detection Kit-LDH (Boehringer, Mannheim, Germany). For this study, the supernatant of the cultures was collected every 24 h, and the change in LDH release into the medium was quantitated. A 200 µL aliquot from the medium was diluted 1:10 in 0.15 M sodium chloride solution. This diluted solution (100 µL) was added to 100 µL of LDH reagent in a 96-well plate (Sarstedt, Nu¨mbrecht, Germany). Fresh culture medium not being applied onto culture dishes was treated in the same way and was used as a blank. The change in absorbance at 492 nm was measured with a Titertek Plus MK II Malvern Instruments Plate Reader (Titertek, Huntsville, AL). Albumin ELISA. Culture supernatants were stored at -10 °C prior to analysis by enzyme-linked immunosorbent assay (ELISA), described in detail by Dunn et al. (17). Pig albumin was purchased from Sigma, and horseradish peroxidaseconjugated antibody to pig albumin was purchased from Universal Biologicals Ltd. (Gloucestershire, England). 7-Ethoxycoumarin O-Deethylation (ECOD) Assay. 7-Hydroxycoumarin (Sigma) and its conjugation with D-glucuronic acid and sulfate were assessed as described by Ulrich and Weber (18) and Fry and Bridges (19) using a substrate concentration of 100 µM 7-ethoxycoumarin (Sigma) in Williams E medium without supplements. The cells were washed twice with 2 mL of a warm 0.15 M sodium chloride solution. Then, 1500 µL of the incubation medium was added to the culture dishes, and the cells were incubated for 1 h by 37 °C in 5% CO2 in a humidified air condition. The incubations were stopped by aspirating the supernatant from the culture dishes. A 250 µL aliquot was diluted 1:8 in 0.15 M sodium chloride solution and measured fluorimetrically with 362 nm as the excitation wavelength and 450 nm as the emission wavelength (free hydroxycoumarin). For measuring the total amount of hydroxycoumarin, 500 µL of the supernatant was deconjugated by adding 200 µL of acetate buffer at pH 4.5 (Sigma) containing 1 mg of glucuronidase/sulfatase (from Helix promatia, Sigma) and incubation for 2 h at 37 °C in a shaking water bath. The deconjugated solution (250 µL) was diluted 1:8 in a 0.15 M sodium chloride solution and assessed as described above (total hydroxycoumarin). 7-Ethoxyresorufin O-Deethylase (EROD) Assay. Resorufin (Sigma) and its conjugation with D-glucuronic acid and sulfate were assessed by a modification of assays developed by Burke and Mayer (20) and Klotz et al. (21) using a substrate concentration of 5 µM 7-ethoxyresorufin (Sigma) in Williams E medium without supplements. The cells were washed twice with 2 mL of a warm 0.15 M sodium chloride solution. Then 2000 µL of the incubation medium was added to the culture dishes, and the cells were incubated for 1 h at 37 °C in 5% CO2 in a humidified air condition. The incubations were stopped by aspirating the supernatant from the culture dishes. A 800 µL aliquot was mixed with 1200 µL of a 0.15 M sodium chloride solution and assessed fluorimetrically with 560 nm as the excitation wavelength and 585 nm as the emission wavelength (free resorufin). For measuring the total amount of resorufin, 800 µL of the supernatant was deconjugated by adding 200 µL of acetate buffer (pH 4.5) containing 1 mg of glucuronidase/ sulfatase (from H. promatia, Sigma) and incubation for 2 h at 37 °C in a shaking water bath. The deconjugated solution (800 µL) was mixed with 1200 µL of a 0.15 M sodium chloride solution and assessed as described above (total resorufin). Benzamidoxime Metabolism. The cells were incubated at day 1, 2, and 3 of culturing with 200 µM benzamidoxime in KRB (pH 7.4) for 60 min at 37 °C in a humidified 95% air/5% CO2 atmosphere. At the end of the incubation period, the cell culture supernatant was aspirated from the Petri dishes and immediately frozen at -20 °C. HPLC Analysis. The frozen supernatant was freeze-dried and resolved in 200 µL of bidistilled water, mixed, centrifugated for 5 min at 10000g, and taken for HPLC analysis. The resulting clear supernatant was analyzed using an HP 1090 Series II high-performance liquid chromatograph (HewlettPackard, Heilbronn, Germany) equipped with a variable-

Phase 2 Metabolites of N-Hydroxylated Amidines wavelength UV detector (HP VWD 1050) set to 250 nm. Separation was performed at room temperature on two prepacked, reversed phase columns (2 × 125 mm × 4 mm i.d., particle size of 5 µm; Lichrospher RP Select B, E. Merck), with 4 mm × 4 mm guard columns of the same material. The following gradient was used: 100% 25 mM ammonium acetate buffer (pH 4.8) from 0 to 15 min, linearly changing to 95% 25 mM ammonium acetate buffer (pH 4.8) and 5% acetonitrile from 15 to 20 min and linearly changing to 70% 25 mM ammonium acetate buffer (pH 4.8) and 30% acetonitrile from 35 to 40 min. The mobile phase was passed through the columns at a rate of 0.5 mL/min. The injected sample volume was 200 µL. Solvents used in the analysis were filtered through a Sartolen 0.45 µm membrane filter (Sartorius AG, Go¨ttingen, Germany) and degassed by bubbling with helium or sonication. Calibration was performed with independently synthesized reference substances. The retention times were 29.6 ( 0.3 min for 2, 27.5 ( 0.3 min for 1, 18.6 ( 0.2 min for 3, and 13.8 ( 0.2 min for 4.

Chem. Res. Toxicol., Vol. 14, No. 3, 2001 321 Scheme 2. Synthesis of Benzamidoxime O-Glucuronide 3

Mass Spectra of Benzamidoxime O-Glucuronide and Bezamidoxime O-Sulfate by LC/MS Coupling. For mass spectroscopic analysis, the same incubations were performed as described for HPLC analysis. A 200 µL aliquot of the clear supernatant was taken for HPLC/MS analysis, using the conditions given above for HPLC analysis. Mass spectra were recorded using an HP 5989 A mass spectrometer (HewlettPackard). The interface to the mass spectrometer was via a thermospray LC/MS interface (Hewlett-Packard). The running temperatures were adjusted as follows: ion source, 226 °C; quadrupole, 120 °C; and stem, 106 to 102 °C during the run. Thermospray mass spectra of synthetic metabolites 3 and 4 were analyzed in the concentration range expected for hepatocyte incubation using positive ion detection and either filament or discharge ionization and the HPLC eluent mentioned above. The discharge mode proved to be most effective; however, the quasimolecular ions were not detected. For the glucuronide, only ions at 180, 139, and 121 amu were characteristic. For the sulfate, only the ion at 121 amu was found to be characteristic. These ions and their relative ratio were employed in the confirmation of the metabolite using selected ion monitoring (SIM). Furthermore, the hepatocyte incubations were assessed in the full scan mode in the range of 80-250 and 150-400 amu to detect any other phase 2 metabolites of benzamidoxime.

Synthesis of Benzamidoxime (2). Benzamidoxime (2) was synthesized using the method of Tiemann and Kru¨ger (24).

Mutagenicity in S. typhimurium. S. typhimurium tester strains TA98 and TA100 were kindly supplied by H. Marquardt (Department of Toxicology, University of Hamburg, Hamburg, Germany). S9 samples from rats pretreated with Aroclor 1254 were obtained from Organon Teknika (Eppelheim, Germany). Purified agar Code L28 and nutrient broth no. 2 were from Oxoid (Basingstoke, England). 2-Nitrofluorene was purchased from Aldrich (Steinheim, Germany) and 2-aminoanthracene from Sigma. Tests for reversion of histidine auxotrophic S. typhimurium strains to prototrophy were essentially performed using the protocol of Maron and Ames (22) with the relevant modification of preincubation (23). The inoculates were grown in 20 mL of nutrient broth no. 2 at 37 °C for 7 h in the dark. Metabolic activation was achieved by the use of S9 from rats induced with Aroclor 1254. A 0.1 mL aliqout of an S. typhimurium culture (strains TA98 and TA100), benzamidoxime Oglucuronide, or benzamidoxime O-sulfate in 20 µL of Me2SO and 0.1 mL of phosphate buffer (pH 7.4) (without metabolic activation), or 0.1 mL of S9 mix, containing 10% S9 (with metabolic activation), were preincubated at 37 °C for 30 min. Then 2 mL of top agar was added, mixed, and poured immediately onto minimal glucose agar plates. A 100 mL aliqout of top agar contains 0.6 g of NaCl and 0.6 g of purified agar. To this was added 10 mL of 0.45 mM L-histidine and 0.5 mM biotine. Benzamidoxime O-glucuronide was tested at concentrations of 0.213, 0.43, 0.85, 2.13, 4.25, and 8.5 µmol/plate. Benzamidoxime O-glucuronide (3) cannot be obtained as a stable compound on a preparative scale; thus, 7 was dissolved in 210.6 µL of Me2SO, and 50 µL of 4 M NaOH was added and mixed for 10 min to set free the unprotected benzamidoxime O-glucuronide (3).

Synthesis of Benzamidoxime O-Glucuronide (3). To a suspension of 681 mg (5.00 mmol) of benzamidoxime (2) and 1.72 g (10.0 mmol) of cadmium carbonate in 75 mL of refluxing toluene was added slowly a solution of 3.97 g (10.0 mmol) of 5 (25) (see Scheme 2) in 100 mL of toluene under nitrogen, while distilling off the same amount of solvent. Then a further aliquot of 50 mL of toluene was added and distilled off. The reaction mixture was allowed to cool to room temperature overnight and filtered through a short column of silica gel (silica gel 60, Merck), and the solvent was evaporated in vacuo. The remaining oil was purified by column chromatography on 40 g of silica gel using an ethyl acetate/dichloromethane mixture (10/90, v/v) to afford 550 mg (1.22 mmol, 24%) of methyl {2,3,4-tri-O-acetyl1-O-[(2′-amino-2′-phenylmethylene)imino]-β-D-glucopyranosyl}uronate (6) as a white powder after recrystallization from hot toluene (maximum temperature of 60 °C): mp 95 °C; Rf ) 0.70 (silica gel, solvent mixture given above), Rf ) 0.0 [RP 18-silica gel (Merck), 25/75 (v/v) methanol/water mixture]; 1H NMR (300 MHz, DMSO-d6) δ 1.99/1.99/2.00 (3 × s, 3 × 3 H, CH3), 3.63 (s, 3 H, OCH3), 4.57 (d, 1 H, J ) 9.9 Hz, H-5), 4.99 (t, 1 H, J ) 9.7 Hz, H-4), 5.09 (dd, 1 H, J ) 9.6, 8.3 Hz, H-2), 5.42 (d, 1 H, J ) 8.3 Hz, H-1), 5.46 (t, 1 H, J ) 9.6 Hz, H-3), 6.14 (br s, 2 H, NH2), 7.43 (m, 3 H, Harom-3′/4′/5′), 7.64 (m, 2 H, Harom-2′/6′); 13C NMR (75 MHz, DMSO-d6) δ 20.2/20.3/20.5 (Me), 52.4 (OMe), 69.3 (C-5), 69.8 (C-4), 71.0 (C-3), 71.6 (C-2), 100.3 (C-1), 126.1 (Carom-3′/5′), 128.2 (Carom-2′/6′), 129.9 (Carom-4′), 131.7 (Carom-1′), 153.6 (CdN), 167.4/169.1/169.2/169.5 (CdO); 15N NMR (35.5 MHz, CDCl3, INEPT pulse sequence, CH3NO2 at 0.0 ppm) δ -78.2 (J ) 2.4 Hz, NO), -314.3 (t, J ) 89.5 Hz, NH2); MS (70 eV, EI, relative intensity) m/z 452 (M+, 1), 317 (10), 257 (21),

Fifty microliters of 99.8% acetic acid was added to reach pH 7.4. Benzamidoxime O-sulfate was tested at concentrations of 0.01, 0.1, 0.5, 1.0, 5.0, and 8.0 µmol/plate. Both substances were tested as freshly prepared solutions. After incubation for 2 days at 37 °C, the colonies on the plates were manually counted. If the Ames test was considered positive, the his+ character of the colonies has been verified by testing the growth on agar containing no histidine.

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197 (18), 155 (78), 136 (14), 127 (42), 119 (43), 104 (13), 77 (25), 43 (100); MS (thermospray, 80% methanol/20% aqueous 0.1 M ammonium acetate mixture, pH 6.3) m/z 573 (M + 2CH3CO2H + H+, 2), 513 (M + CH3CO2H + H+, 1), 454 (24), 453 (M + H+, 100); IR (KBr pellet) ν˜ 3466/3358, 3030, 2950, 1752, 1592, 1438, 1226 cm-1. Anal. Calcd for C20H24O10N2 (452.42 g/mol): C, 53.10; H, 5.35; N, 6.19. Found: C, 52.91; H, 5.27; N, 5.82. To a solution of 200 mg (0.44 mmol) of 6 in 10 mL of methanol was added 84 mg (1.54 mmol) of sodium methylate (Merck) at 4 °C, and the mixture was stirred for 1 h. The solvent was evaporated in vacuo, dissolved in a small portion of methanol, and filtered through a small bed of silica gel to afford 160 mg of a slightly brownish oil. This oil was subjected to column chromatography on ∼30 g of silica gel using an ethyl acetate/methanol gradient; the solvents were evaporated, and 6 was precipitated with toluene to afford 66.4 mg (0.20 mmol, 47%) of methyl {1-O-[(2′amino-2′-phenylmethylene)imino]-β-D-glucopyranosyl}uronate (7): Rf ) 0.15 (silica gel, ethyl acetate), Rf ) 0.40 [RP 18-silica gel, 25/75 (v/v) methanol/water mixture]; 1H NMR (300 MHz, DMSO-d6) δ 3.20-3.40 (m, 3 H, H-2/3/4), 3.66 (s, 3 H, OMe), 3.80 (d, J ) 9.5 Hz, H-5), 4.08 (br s, 1 H, OH), 4.72 (d, J ) 7.9 Hz, 1 H, H-1), 5.25 (br s, 1 H, OH), 5.37 (br s, 1 H, OH), 6.25 (br s, 2 H, NH2), 7.40 (m, 3 H, Harom-3′/4′/5′), 7.65 (m, 2 H, Harom2′/6′); 13C NMR (75 MHz, DMSO-d6) δ 51.8 (OMe), 71.6 (C-5), 72.1 (C-4), 75.4/75.5 (C-2/3), 104.2 (C-1), 126.0 (Carom-3′/5′), 128.2 (Carom-2′/6′), 129.6 (Carom-4′), 132.2 (Carom-1′), 153.2 (CdN), 169.4 (CdO); MS (thermospray, 80% methanol/20% aqueous 0.1 M ammonium acetate mixture, pH 6.3, relative intensity) m/z 327 (M + H+, 68), 309 (14), 295 (3), 153 (16), 139 (7), 137 (10), 122 (13), 121 (100), 114 (10), 100 (27); IR (KBr pellet) ν˜ 3374 (br), 1734, 1636 cm-1. Sodium {1-O-[(2′-amino-2′-phenylmethylene)imino]-β-D-glucopyranosyl}uronate (3) was obtained by treatment of 2-3 mg of 7 with an excess of an aqueous sodium hydroxide solution at room temperature, and HPLC analysis of this solution showed complete cleavage of the methyl ester group. Attempts to isolate the acid by acidification failed. After evaporation of the solvent, the sodium salt was characterized as follows: Rf ) 0.83 [RP 18-silica gel, 25/75 (v/v) methanol/ water mixture]; 1H NMR (300 MHz, DMSO-d6) δ 3.20-3.40 (m, 3 H, H-2/3/4), 3.66 (s, 3 H, OMe), 3.80 (d, J ) 9.5 Hz, H-5), 4.08 (br s, 1 H, OH), 4.72 (d, J ) 7.9 Hz, 1 H, H-1), 5.25 (br s, 1 H, OH), 5.37 (br s, 1 H, OH), 6.25 (br s, 2 H, NH2), 7.40 (m, 3 H, Harom-3′/4′/5′), 7.65 (m, 2 H, Harom-2′/6′); 13C NMR (75 MHz, DMSO-d6) δ 72.1 (C-5), 72.3 (C-4), 73.3/75.8 (C-2/3), 103.6 (C1), 126.3 (Carom-3′/5′), 128.4 (Carom-2′/6′), 130.0 (Carom-4′), 131.9 (Carom-1′), 155.2 (CdN), 173.3 (CdO); MS (thermospray, 80% methanol/20% aqueous 0.1 M ammonium acetate mixture, pH 6.3, relative intensity) m/z 313 (M + H+, 2), 241 (2), 175 (4), 169 (3), 153 (11), 149 (16), 139 (13), 137 (28), 136 (13), 122 (10), 121 (100). Synthesis of Benzamidoxime O-Sulfate (4). To a solution of 1.0 g (7.4 mmol) of 2 in 50 mL of dry DMF was added 4.1 g (25.7 mmol, 3.5 equiv) of the pyridine-SO3 complex. The solution was stirred for 1 day at room temperature. Then another aliquot of 2 (1.0 g, 7.4 mmol) was added and the mixture stirred for an additional 3 d. The solvent was evaporated in vacuo. Benzamidoxime-O-sulfonic acid was obtained after chromatography of the remainder on ∼15 g of silica gel using an ethyl acetate/methanol mixture (80/20, v/v). Concentrating the fractions containing the product to ∼5 mL and addition of 1.1 mL of a 1 M KOH solution in methanol led to the formation of 4 (0.63 g, 2.5 mmol, 16%) of a white solid (mp 142 °C) which was dried over P4O10. 4 could be further purified by chromatography on Sephadex LH-20, but only with great loss. Attempts to recrystallize 4 with a suitable solvent system let to decomposition and formation of K2SO4 according to IR spectroscopy. 4 was characterized as follows: Rf ) 0.42 [silica gel, 80/20 (v/v) ethyl acetate/methanol mixture], Rf ) 0.81 (RP-18 silica gel, water); 1H NMR (300 MHz, DMSO-d6) δ 6.06 (br s, 2 H, NH2), 7.39 (m, 3 H, Harom-3/4/5), 7.69 (m, 2 H, Harom-2/6); 13C NMR (75 MHz, DMSO-d6) δ 126.2 (Carom-3/5), 128.1 (Carom-2/6),

Clement et al.

Figure 1. Albumin synthesis and LDH release (high absorption rates indicating a high level of LDH release) in pig hepatocyte cultures. Data represent the means ( SD of three experiments. 129.6 (Carom-4), 132.6 (Carom-1), 152.8 (CdN); 15N NMR (35.5 MHz, DMSO-d6, INEPT pulse sequence, CH3NO2 at 0.0 ppm) δ -310.1 (t, J ) 90.4 Hz, NH2); inverse gated decoupling δ -86.1 (NO), -310.1 (NH2); IR (KBr pellet) ν˜ 3474/3312, 1632, 1276/ 1256, 1064 cm-1. Anal. Calcd for C7H7O4N2SK: C, 33.06; H, 2.77; N, 11.02; O, 25.17; S, 12.61. Found: C, 33.10; H, 2.81; N, 11.07; O, 25.07; S, 12.55.

Results and Discussion Hepatocyte Isolation, Cultivation, and Characterization. Because of their ability to produce a wide range of phase 1 and phase 2 metabolites, hepatocytes were used in the study of the metabolic fate of amidoximes. Pig hepatocytes were chosen as model for the biotransformation in humans (3). Pig hepatocytes were isolated and then cultivated for 7-9 days. Assessing the albumin synthesis and the release of lactate dehydrogenase is a sensitive marker of viability and cellular damage (26). After the viability increased in the first 3 days of culturing, the viability decreases until day 7-9 (Figure 1). The cells are damaged by the isolation process, but after the repair mechanism, the hepatocytes are acclimated to this new environment. The level of LDH release into the medium decreases, and the hepatocytes recover from the isolation process in the first days of culturing. Cytochrome P450 activities were measured by O-deethylation of ethoxycoumarin (Figure 2) and ethoxyresorufin (Figure 3).The capacity of forming conjugates of the created phenols was evaluated as well. Ethoxycoumarin is a substrate of several P450 isoforms, whereas ethoxyresorufin is exclusively metabolized by cytochrome P450 1A (27, 28). Metabolic activities for all reactions decreased from a maximum on day 1 during the next days of culturing. Previous studies also demonstrated that in particular the level of formation of glucuronides increases in the first days of culturing (29, 30). Thus, benzamidoxime was incubated during the first 3 days of culturing. In summary, tests on metabolic activity and metabolic competence demonstrated the suitability of our pig hepatocyte culture for metabolism studies.

Phase 2 Metabolites of N-Hydroxylated Amidines

Chem. Res. Toxicol., Vol. 14, No. 3, 2001 323 Scheme 3. Synthesis of Benzamidoxime O-Sulfate 4

Figure 2. Ethoxycoumarin O-deethylase activity in cultured pig hepatocytes. The metabolite 7-hydroxycoumarin was measured before and after deconjugation as described in Experimental Procedures. Data represent the means ( SD of three experiments.

Figure 3. Ethoxyresorufin O-deethylase activity in cultured pig hepatocytes. The metabolite resorufin was assessed before and after deconjugation as described in Experimental Procedures. Data represent the means ( SD of three experiments.

Synthesis of Benzamidoxime O-Glucuronide. The O-glucuronide of benzamidoxime was synthesized by coupling of methyl(tri-O-acetyl-R-D-glucopyranosylbromide)uronate (5) with benzamidoxime (2) (Scheme 2). This so-called Meystre-Miescher modification of the Koenigs-Knorr reaction (31) uses cadmium carbonate according to the procedure developed by Conrow and Bernstein (32). The bromide 5 was obtained using the procedure reported by Bollenback et al. (25). Protecting groups were split off subsequently with sodium methylate and aqueous sodium hydroxide (32, 33). Due to the instability of the intermediate 7, it proved to be advisable to perform the whole synthetic sequence without delay;

7, for example, decomposed completely within a couple of weeks when stored at 0 °C. The nature of these decomposition products was not investigated. The formation of an O-glucuronide existing in the oxime type tautomeric form, and not of an N-glucuronide, is derived from the 1H and 15N NMR data. For the amino group, a broad signal corresponding to two protons is observed in the 1H NMR spectra of 3, 6, and 7. In the case of an N-glucuronide, two signals would be expected. Furthermore, the amino group is detected as a “triplet” with a -1, 0, +1 intensity pattern using the INEPT pulse sequence with a coupling of about 90 Hz in the 15N NMR spectrum of 6. The quaternary nitrogen of the NdO linkage only exhibits a splitting corresponding to a long-range coupling on the order of 2 Hz, presumably with H-1. Due to the instability of 7 upon storage, the glucuronide sodium {1-O-[(2′-amino2′-phenylmethylene)imino]-β-D-glucopyranosyl}uronate (3) was formed in situ by treatment with sodium hydroxide and proved to be sufficiently stable for characterization. Attempts to isolate the pure compound on a preparative scale were not successful. The large coupling observed for H-1 with H-2 in the range of 8 Hz for 3, 6, and 7 (33) shows that benzamidoxime is linked to the sugar moiety in the β-anomeric form. No signs of E,Z-isomers of the CdN double-bond could be observed in the spectra, indicating that probably only one isomer has been formed. Like other N,N-unsubstituted benzamidoximes, which exist exclusively in the Z-form (34), it is believed that 3, 6, and 7 also prefer this configuration as a hydrogen bond between one hydrogen atom of the NH2 group and the oxime oxygen atom is still possible. This is the reason for the preferred Z-isomer in N,Nunsubstituted and N-monosubstituted benzamidoximes (35). Synthesis of Benzamidoxime O-Sulfate. Benzamidoxime O-sulfate (4) was synthesized using a modification of the methods of Walsh et al. (36) and Scha¨nzer et al. (37) by reaction of 2 with the pyridine-SO3 complex at room temperature (Scheme 3). This type of sulfonic acid is known to be unstable or difficult to purify (36, 37); thus, the potassium salt was formed and isolated. Again, as in the case of 6, two absorption bands in the IR spectrum at ∼3500/3300 cm-1 and the “triplet” observed by 15N NMR spectroscopy show the presence of an amino group, and the sulfation by chemical means

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Figure 4. Representative HPLC chromatograms (section) of the incubation of benzamidoxime (2) with cultured pig hepatocytes. The incubations were performed as described in Experimental Procedures: (A) complete system and (B) without hepatocytes. 1 is benzamidine (27.5 ( 0.3 min), 2 benzamidoxime (29.6 ( 0.3 min), 3 benzamidoxime Oglucuronide (18.6 ( 0.2 min), and 4 benzamidoxime O-sulfate (13.8 ( 0.2 min).

takes place regioselectively at the hydroxyl group of 2. All the spectroscopic data obtained for 3 and its synthetic precursors as well as for 4 do not give any evidence of the presence of other tautomeric forms or the E-isomers. O-Glucuronidation and O-Sulfation of Benzamidoxime. In previous studies, only indirect evidence was obtained for the possible formation of phase 2 metabolites by measuring the benzamidoxime concentration before and after deconjugation with glucuronidase (10, 11). In this study, direct proof is presented for the formation of the O-glucuronide and the O-sulfate of the benzamidoxime in vitro by cultured pig hepatocytes. A representative HPLC chromatogram recorded after the incubation of benzamidoxime with pig hepatocyte cultures is given in Figure 4A. The use of cultured hepatocytes leads to several signals in the HPLC chromatogram arising from contents of the medium. Indeed, the complex culture medium and the numerous resulting peaks shown in the HPLC chromatogram (see traces A and B of Figure 4) make it more difficult to identify one metabolite, especially when the whole medium is analyzed in one chromatographic run without any laborious extraction of single metabolites. These extraction procedures used in previous studies (11) can be a potential source of error in the case of labile metabolites, which, for example, decompose during extraction. However, none of the other signals belongs to further benzamidoxime metabolites, as the characteristic fragment ion, the sensitive probe for benzamidine and benzamidoxime derivatives at 121 amu (see below), is missing. The retention time of the first metabolite (4) (13.8 min) agrees with that of independently synthesized benzamidoxime O-sulfate, and the retention time of the second metabolite (3) (18.6 min) agrees with that of independently synthesized benzamidoxime O-glucuronide.

Clement et al.

Figure 5. Ion chromatograms for the signal at 121 amu as a characteristic fragment of benzamidoxime O-sulfate with a retention time of 13.8 ( 0.2 min (A) for the produced metabolite benzamidoxime O-sulfate (4) and (B) for the blank. See Experimental Procedures for details of the incubation procedure, sampling, and analysis.

The chromatogram of the incubation mixture in the absence of hepatocytes (Figure 4B) did not reveal peaks for 3 and 4. The ion chromatogram of the produced metabolite 4 shown in Figure 5A exhibits the characteristic signal at 121 amu at 13.8 min. The synthetic benzamidoxime O-sulfate exhibits a comparable ion chromatogram for the signal at 121 amu obtained under identical conditions. Signals at 139, 154, and 180 amu are observed only at higher concentrations of benzamidoxime O-sulfate so that only the signal at 121 amu is characteristic for benzamidoxime O-sulfate at low concentrations, as formed by incubation. In the blank run, the characteristic signal at 121 amu is not found (Figure 5B). At low concentrations of the synthetic metabolite 3, which is in the same concentration range as that formed by incubation, the quasi-molecular ion with 313 amu is not observed. Rather, the signals at 121, 139, and 180 amu are characteristic for the glucuronide 3. The ion chromatogram of the produced metabolite 3 shown in Figure 6A shows the characteristic signals at 121, 139, and 180 amu at 18.6 min. The peaks at 4-10 min are produced by the large amount of proteins in the incubations, generating ions over the whole mass range (see also panels A and C of Figure 6). The synthetic benzamidoxime O-glucuronide exhibits a comparable ion chromatogram for the signals at 121, 139, and 180 amu (Figure 6B) obtained under identical conditions. The signals at 121, 139, and 180 amu correspond to fragments arising from the benzamidinium ion [PhC(NH2)2]+ (121 amu), and this ion is found as a major fragment in the thermospray mass spectra of benzamidine, benzamidoxime, benzamidoxime O-sulfate, and benzamidoxime O-glucuronide which were run for comparison using the solvent system described in Experimental Procedures. Other characteristic ions arise by cluster formation of the benzamidinium ion, namely, [PhC(NH2)2 + H2O]+ at 139 amu and [PhC(NH2)2 + CH3-

Phase 2 Metabolites of N-Hydroxylated Amidines

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cultured pig hepatocytes by direct methods. This was made possible by the synthesis of the two metabolites as reference compounds. The main metabolite formed in benzamidoxime incubations is the amidine (benzamidine). The nontoxicity of several amidoximes developed as prodrugs of amidines (5-8) can partly be seen in their facile reduction (5, 9, 10, 38) to amidines (see Scheme 1) which is the predominant reaction in vivo, although phase 2 reactions also occur. In phase 2, the O-glucuronide and O-sulfate of the N-hydroxylated amidine are formed by cultured pig hepatocytes. Both compounds exhibit no mutagenicity in the Ames test in TA98 and TA100 strains so that phase 2 conjugation of benzamidoxime also leads to nonmutagenic compounds and can be considered as a detoxification reaction in the metabolism of benzamidoxime. Further studies will be directed toward the formation and mutagenicity of the O-acetyl derivative of benzamidoxime.

Acknowledgment. We are grateful to Prof. Dr. H. Marquardt, Department of Toxicology, University of Hamburg, for allowing us to to carry out the mutagenicity tests. We also thank M. Ko¨nig and M. Wollny for their technical assistance. This work was supported by grants from the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (Grant 0311245), the Fonds der Chemischen Industrie, and the Deutsche Forschungsgemeinschaft (Grant DFG Cl 56/6-1).

References Figure 6. Ion chromatograms for the signals at 121, 139, and 180 amu as characteristic fragments of benzamidoxime O-glucuronide (A) for the produced metabolite benzamidoxime O-glucuronide (3), (B) for the synthesized benzamidoxime O-glucuronide, and (C) for the blank. See Experimental Procedures for the details of the incubation procedure, sampling, and analysis.

CO2]+ at 180 amu. For benzamidoxime, the following ions were observed: [M + CH3CO2H]+ at 196 amu, [M + H]+ at 137 amu, and [PhC(NH2)2]+ at 121 amu. Furthermore, [PhC(NH2)2 + CH3CN]+ at 162 amu is observed for 1 and 2. The HPLC chromatogram in Figure 4 shows that the predominant reaction of benzamidoxime in vitro is the reduction to the amidine, and that conjugation occurs only as a side reaction. Mutagenicity Studies. In previous studies, it was shown that benzamidoxime was mutagenic in the TA98 strain in the presence of untreated rabbit liver S9 fractions (11). To study wether conjugation with glucuronic acid or active sulfate leads to toxification or detoxification, benzamidoxime O-sulfate and benzamidoxime O-glucuronide were tested in the Ames test. Both phase 2 metabolites, found after incubation of benzamidoxime with cultured pig hepatocytes, exhibit no mutagenicity in TA98 and TA100 strains.

Conclusions In previous studies, only indirect evidence for the formation of phase 2 metabolites of benzamidoxime was obtained. This study demonstrates the metabolic formation of benzamidoxime O-sulfate and benzamidoxime O-glucuronide after incubation of benzamidoxime with

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