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Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (Y.K., C.S., U.M.K., P.F.H.); Department of Medicinal Chemistry & Laboratory for Applied Drug Design and Synthesis, University of Mississippi, University, Mississippi (S.G.P., J.M.R.); and Department of Anesthesiology, University of Michigan and Veteran Affairs Health Service, Ann Arbor, Michigan (H.Z., L.W.)
(Received June 12, 2006; Accepted September 20, 2006)
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Abstract |
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; Zhou et al., 2005), or due to idiosyncratic drug reactions caused by reactive metabolites that escaped from endogenous scavengers such as glutathione and reacted with various cellular macromolecules (Seguin and Uetrecht, 2003; Uetrecht, 2003a; Evans et al., 2004; Nassar and Lopez-Anaya, 2004). Thus, precise structure-activity relationships (SARs) are required, in the early stages of contemporary drug discovery, to understand the biochemical mechanisms of metabolism and the generation of reactive intermediates that may act as mechanism-based inactivators (MBIs), in the effort to provide safer drugs to the clinical stage.
Recent efforts to understand the SARs involved in mechanism-based inactivation of P450s have seen an intensified interest in elucidating the structural basis of P450 function, thus aiding in the discovery and design process of drugs metabolized by P450 enzymes. Several studies have reported a variety of different functional groups likely to cause MBIs. These functional groups are easily transformed to radical or carbene species that can react with the apoprotein or the heme of the P450s (Correia and Ortiz de Montellano, 2005). In some cases, the ability of a series of compounds to act as MBIs varies depending on the structures of the analogs, even though the compounds contain the same reactive functional group that theoretically could lead to the formation of a reactive intermediate. For instance, tienilic acid is a potent MBI of P450 2C9 (Lopez Garcia et al., 1993, 1994), and the inactivation is due to the metabolism of tienilic acid at its thiophenyl structure (Koenigs et al., 1999). However, ticlopidine and clopidogrel, which also contain the thiophenyl substructure, show much weaker inhibition of P450 2C9, but inhibit P450s 2C19 and 2B6 in a mechanism-based manner (Ha-Duong et al., 2001; Richter et al., 2004). Similarly, the antidiabetic drugs troglitazone, pioglitazone, and rosiglitazone are all thiazolidinedione derivatives causing mechanism-based inactivation of P450 3A4. However, the potencies for inactivation by the three compounds varied 6-fold (Lim et al., 2005). Troglitazone, the most potent MBI, contained other substructures that could be responsible for production of reactive intermediates (Yamamoto et al., 2002; He et al., 2004; Reddy et al., 2005). This suggests that a single potential reactive functional group may not be the only contributing factor for inactivation and that other functional substructures within the compounds may also contribute to mechanism-based inactivation. Thus, in the drug discovery process, it becomes necessary to investigate more precisely the "determinant factor(s)" in the structure of a compound which may be responsible for the mechanism-based inactivation rather than to investigate only the potential reactive group. It has been reported previously that several (4-alkoxyphenyl-)diaziridines inactivated P450 2B6 in a mechanism-based manner, whereas (4-(methylthio)phenyl)diaziridine had no effect on P450 2B6 (Sridar et al., 2006). These results are of interest because all of the alkoxy and the thiophenyl compounds contained a diaziridine moiety. The purpose of the current study was to investigate the SARs for mechanism-based inactivation by a series of these diaziridines in conjunction with additional aryl diaziridine analogs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). 3-(3-Methoxyphenyl)-3-(trifluoromethyl)diaziridine (7) and 3-(trifluoromethyl)-3-phenyl-diaziridine (9) were also synthesized as described previously (Brunner et al., 1980; Hatanaka et al., 1994), and the analytical and spectroscopic analyses were in accord with the published data. 4-Bromphenol-2,3,5,6-d4 (CAS 152404-44-9) was purchased from C/D/N Isotopes (Pointe Claire, QC, Canada) and methylated according to the reported procedure for the synthesis of 12b (Cho et al., 1993). All remaining chemicals and reagents for the synthesis of compounds used in this study were purchased from Sigma-Aldrich (St. Louis, MO). Thin-layer chromatography was performed on Merck silica gel 60 F254 plates. Column chromatography was performed using standard grade Sorbent Technologies (Atlanta, GA) silica gel with a particle size of 32 to 63 mM. Mass spectral data were acquired using a Waters ZQ LC/MS system (ESI+ mode), and HRMS data were acquired using a Micromass QTOF (Waters, Milford, MA). Elemental analysis was performed using a PerkinElmer Series II 2400 CHNS/O Elemental Analyzer (PerkinElmer Life and Analytical Sciences, Boston, MA). 1H NMR spectra were recorded at 400 MHz or 500 MHz, and 13C NMR spectra were recorded at 100 MHz or 125 MHz using Bruker Avance DXR systems (Bruker, Newark, DE). NADPH, bovine serum albumin, glutathione ethyl ester (GSHEE), and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was obtained from Invitrogen (Carlsbad, CA). Bergamottin was purchased from Indofine Chemical Co., Inc. (Hillsborough, NJ).
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Synthesis. Synthesis of 2,2,2-Trifluoroacetophenones (13a and 13b). Mg turnings (1.2 g, 0.05 mol), substituted bromobenzenes, 12a or 12b (0.05 mol), and anhydrous tetrahydrofuran (40 ml) were placed in a round-bottom flask. The mixture was slowly heated to reflux and maintained until all the magnesium was dissolved. The mixture was cooled in an ice bath, and a solution of N-trifluoroacetylpiperidine (0.05 mol) in anhydrous tetrahydrofuran (15 ml) was added slowly to the Grignard reagent over a period of 0.5 h with stirring at 0°C. The reaction mixture was stirred for 2 h at ambient temperature, and the reaction was quenched by the addition of saturated aqueous ammonium chloride (5 ml). The precipitated solids were filtered, the filtrate was dried over Na2SO4 and evaporated in vacuo, and the residual oil was purified by silica gel column chromatography eluting with hexanes/CH2Cl2 (95:5) to give the product ketones as pale yellow to colorless oils.
1-(4-Chlorophenyl)-2,2,2-trifluoroethanone (13a). Yield, 6.6 g (65%); 1H NMR (CDCl3): 
7.50 (d, 2H, J = 8.0 Hz), 8.07 (d, 2H, J = 8.0 Hz) (Kesavan et al., 2000).
2,2,2-Trifluoro-1-(4-methoxy-(2,3,5,6-2H4)phenyl)ethanone (13b). Yield, 6.3 g (62%); 1H NMR (CDCl3): 3.91 (s, 3H). 13C NMR (CDCl3): 55.80, 114.03 (t, J = 25 Hz, C-D), 116.87 (q, J = 288 Hz, CF3), 122.50, 132.19 (t, J = 25 Hz, C-D), 165.14, 178.57 (q, J = 34 Hz, O=C-CF3).
Synthesis of Oximes (14a and 14b). Hydroxylamine hydrochloride (0.0625 mol) was added to a solution of ketone 13a or 13b (0.025 mol) in absolute ethanol (15 ml) and dry pyridine (20 ml) and heated at 60°C for 8.0 h. The solvent was then removed in vacuo and the remaining residue was dissolved in diethyl ether (40 ml) and washed with 1 N HCl to remove residual pyridine. The organic layer was then washed successively with water (50 x 2 ml), brine, and dried over Na2SO4. After evaporation of the solvent, the crude oxime was purified by silica gel column chromatography eluting with ethyl acetate/dichloromethane (5:95) to yield product oximes as white solids.
1-(4-Chlorophenyl)-2,2,2-trifluoroethanone oxime (14a). Yield, 4.3 g (78%); mp 65–67°C; 1H NMR (CDCl3): 7.50 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 9.36 (bs, 1H). 13C NMR (CDCl3): 120.37 (q, J = 272 Hz, CF3), 123.90, 128.89, 130.06, 136.94, 146.51 (q, J = 32 Hz, N=C-CF3). HRMS: m/z [MH]+ 224.0091 (C8H6ClF3NO requires 224.0090).
2,2,2-Trifluoro-1-(4-methoxy-(2,3,5,6-2H4)phenyl)ethanone oxime (14b). Yield, 3.7 g (68%); mp 100–103°C; 1H NMR (CDCl3): 3.85 (s, 3H), 8.95 (bs, 1H). 13C NMR (CDCl3): 55.71(OCH3), 113.57 (t, J = 24 Hz, 2C-D), 117.70, 118.66 (q, J = 241 Hz, CF3), 130.05 (t, J = 24 Hz, 2C-D), 146.90 (q, J = 66 Hz, N = C-CF3), 160.87. HRMS: m/z [MH]+ 224.0830 (C9H5D4F3NO2 requires 224.0836).
Synthesis of p-Toluenesulfonyl Oximes (15a and 15b). p-Toluenesulfonyl chloride (12.0 mmol) was added to a stirred solution of oxime 14a or 14b (5.0 mmol) in triethylamine (9.0 mmol) and 4-dimethylaminopyridine (1.0 mmol) in methylene chloride (10 ml) at 0°C in lot-wise manner under N2. The reaction was allowed to stir for 2.0 h at room temperature. The reaction mixture was quenched with water and the organic phase was separated, washed successively with water and brine, and dried over Na2SO4. The solvent was evaporated in vacuo, and the crude product was purified by silica gel column chromatography eluting with hexanes/CH2Cl2 (80:20) yielding product tosyl oximes as white solids.
1-(4-Chlorophenyl)-2,2,2-trifluoro-1-ethanone-O-[(4-methylphenyl)sulfonyl] oxime (15a). Yield, 1.35 g (72%); mp 130–131°C; 1H NMR (CDCl3): 2.50 (s, 3H), 7.38 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.48 (d, 2H, J = 8.5 Hz), 7.90 (d, 2H, J = 8.0 Hz). 13C NMR (CDCl3): 22.16, 119.42 (q, J = 275 Hz, CF3), 122.80, 129.20 (4C), 129.80 (2C), 129.84 (2C), 130.93, 138.06, 146.15, 152.76 (q, J = 33 Hz, N = C-CF3). Anal. Calcd for C15H11ClF3NO3S: C, 47.69; H, 2.93; N, 3.71. Found: C, 47.58; H, 2.81; N, 3.64. HRMS: m/z [MH]+ 378.0176 (C15H12ClF3NO3S: requires 378.0179).
2,2,2-Trifluoro-1-(4-methoxy-(2,3,5,6-2H4)phenyl)-1-ethanone-O-[(4-methylphenyl)sulfonyl] oxime (15b). Yield, 1.22 g (65%); mp 114–115°C; 1H NMR (CDCl3): 2.50 (s, 3H) 3.88 (s, 3H), 7.40 (d, 2H, J = 8.0 Hz), 7.91 (d, 2H, J = 8.0 Hz). 13C NMR (CDCl3): 22.14, 55.66, 113.8 (t, J = 25 Hz, 2C-D), 116.22, 120.50 (q, J = 235 Hz, CF3), 129.18 (2C), 129.73 (2C), 130.27 (t, J = 25 Hz, 2C-D), 131.21, 145.85, 152.90 (q, J = 60 Hz, N=C-CF3), 161.81. (Anal. Calcd for C16H10D4F3NO4S: C, 50.92; (H + D) as H, 3.78; N, 3.71. Found: C, 50.90; H, 3.54; N, 3.64. HRMS: m/z [MH]+ 378.0929 (C16H11D4F3NO4S: requires 378.0925).
General Procedure for the Preparation of Diaziridines (8 and 11). Tosyl oximes (15a or 15b, 1.0 mmol) and anhydrous diethyl ether (10 ml) were placed in a three-necked round-bottom flask equipped with a dry ice condenser and a gas inlet. The solution was cooled to –78°C, and approximately 5 ml of anhydrous NH3 was condensed into the flask. The solution was stirred for 1.0 h at –78°C. The cooling bath was removed and the gas inlet was replaced with a drying tube. The solution was stirred at ambient temperature while NH3 refluxed for 2.0 h. The condenser was removed and the ammonia was allowed to evaporate. The remaining residue was dissolved in ethyl ether, washed with water and brine, dried over Na2SO4, and concentrated to afford the crude diaziridines, which were subsequently purified by column chromatography (Si gel), 1 to 5% ethyl acetate/99–95% CHCl3) to yield product diaziridines as white solids.
3-(4-Chlorophenyl)-3-(trifluoromethyl)diaziridine (8). Yield, 0.16 g (75%); amorphous solid, 1H NMR (CDCl3): 2.22 (bs, 1H), 2.83 (bs, 1H), 7.43 (d, 2H, J = 8.5 Hz), 7.58 (d, 2H, J = 8.5 Hz). Anal. Calcd for C8H6ClF3N2:C, 43.17; H, 2.72; N, 12.58. Found: C, 43.25; H, 2.78; N, 12.49. HRMS: m/z [MH]+ 223.0258 (C8H7ClF3N2 requires 223.0250).
3-(4-Methoxy-(2,3,5,6-2H4)phenyl-3-(trifluoromethyl)diaziridine (11). Yield, 0.14 g (65%); mp 74°C; 1H NMR (CDCl3): 2.19 (bs, 1H), 2.78 (bs, 1H), 3.85 (s, 3H). Anal. Calcd for C9H5D4F3N2O: C, 48.65; (H + D) as H, 4.16; N, 12.61. Found: C, 48.43; H, 3.89; N, 12.36. HRMS: m/z [MH]+ 223.0987. (C9H6D4F3N2O requires 223.0996).
Purification of Enzymes. P450 NADPH-reductase was expressed in Escherichia coli Topp3 cells and the purification was carried out as described previously (Hanna et al., 1998). P450 2B6 was expressed in E. coli Topp3 cells and purified as described previously (Hanna et al., 2000).
Time-Dependent Inactivation of P450 2B6 7-EFC Activity. P450 2B6 was reconstituted with reductase at 4°C for 45 min as described previously (Sridar et al., 2006). The primary reaction mixture contained 0.05 mM P450 2B6, 0.1 mM NADPH-reductase, 50 units of catalase, and 50 mM potassium phosphate buffer, pH 7.4, in a total volume of 0.1 ml. The phenyl diaziridine analogs or the ketone analog of compound 1 in DMSO were added to each sample. The final concentrations were 20 µM for compounds 1, 7, 8, and 9, and 100 µM for compounds 6 and 10. Control samples received only DMSO. The final concentration of DMSO in each sample was 1% (v/v). After the reaction mixtures were allowed to equilibrate at 30°C for 15 min, the reactions were initiated by the addition of 1.2 mM NADPH (primary mixture). Aliquots (8 µl, 0.4 pmol of P450 2B6) were removed at 0, 2, 4, 10, and 16 min and added to 988 µl of a secondary reaction mixture containing 1 mM NADPH, 100 µM 7-EFC, and 40 µg/ml bovine serum albumin in 50 mM potassium phosphate buffer, pH 7.4. The secondary reaction was allowed to proceed at 30°C for 10 min and was then stopped with 334 µl of ice-cold acetonitrile. The amount of 7-(hydroxy-4-trifluoromethyl)coumarin that was formed was determined spectrofluorometrically on a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD) with excitation at 410 nm and emission at 510 nm.
Metabolism. Reaction mixtures contained 0.08 nmol of purified P450 2B6, 0.16 nmol of reductase, and 50 units of catalase in a total volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.4. The compounds (1 and 6-10 dissolved in DMSO) were added to each sample. The final concentration of the compounds was 200 µM and the final concentration of the DMSO was 1% (v/v). The reactions were initiated by the addition of 1.2 mM NADPH to the 50-µl incubation mixtures. The control sample received the same amount of water instead of NADPH. The samples were incubated at 30°C for 90 min and the reactions were terminated by the addition of 1 ml of acetonitrile. Bergamottin (10 µM) was added as an internal standard. The mixtures were centrifuged at 13,200g at room temperature for 30 min, and 100 µl of the supernatants was injected onto a 4.6- x 150-mm Waters Symmetry C18 reverse phase column. HPLC was performed using a Waters 600E HPLC system with Waters 501 series pumps, a Waters 996 photodiode array detector, and a Waters 717 autosampler. The initial conditions were 98% solvent A (0.1% v/v acetic acid in water) and 2% solvent B (0.1% v/v acetic acid in acetonitrile) for 2 min at a flow rate of 0.8 ml/min. The percentage of B was increased by a linear gradient to 70% B from 2 to 15 min, and 70% B was maintained for 5 min. The percentage of B was then increased linearly to 95% B from 20 to 25 min and maintained from 25 to 35 min. The percentage of B was then decreased to the initial conditions over the course of 5 min, and the column was equilibrated at 2% B for 10 min before a new injection. The peak areas of the compounds were integrated at their maximum absorbance wavelength between 210 and 300 nm, i.e., 270 nm for compound 1, 260 nm for compound 6, 274 nm for compound 7, 220 nm for compound 8, 260 nm for compound 9, and 297 nm for compound 10. The internal standard bergamottin was integrated at 308 nm. The amount of the compound metabolized in each sample was estimated from the ratio of the peak area of the compound of interest compared with the area of the internal standard. The metabolic ratio of each compound after incubation with P450 was estimated from the ratio of the amount of the compound in each sample compared with the amount in the control sample.
Metabolite Identification Using GC-MS. The reaction mixtures containing compounds 1 or 6 were prepared as above except that methanol was used as the solvent in the stock solutions for these compounds. The reaction mixtures were quenched with 1 ml of ethyl acetate and extracted twice with ethyl acetate. After extraction, the organic phases were evaporated under a stream of nitrogen to approximately 20 µl, and 2 µl of each sample was injected onto the GC-MS system. Metabolites were analyzed on a HP6890/MSD5973 gas chromatography-mass spectrometer (Agilent Technologies, Palo Alto, CA). The metabolites were separated on a DB-210 capillary column (20 m x 0.18 mm x 0.3 µm; Agilent Technologies) with helium as the carrier gas. Aliquots (2-µl) of the ethyl acetate extracts were injected by pulsed splitless injection. The injector temperature was 250°C. The pulse pressure was set at 40 psi for 1 min. The initial oven temperature was 50°C and it was increased to 200°C at 20°C/min after injection. After each run, the oven temperature was increased to the isothermal temperature of the DB-210 column (240°C) and held for 5 min to clean the column. The mass spectra for the compounds eluting from the DB-210 column were obtained by scanning the MS detector in the range of 35 to 550 amu.
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) in a total volume of 0.125 ml of 50 mM potassium phosphate buffer, pH 7.4. Each compound tested was added from a stock solution prepared in DMSO. The final concentration of each compound was 400 µM. The metabolic reaction was initiated by the addition of 1.2 mM NADPH. Control samples received the same amount of water instead of NADPH. The samples were incubated at 30°C for 80 min, and the reactions were terminated by the addition of 1 ml of acetonitrile. The mixtures were centrifuged at 13,200g at room temperature for 30 min. The supernatants were transferred to new tubes and dried down under a stream of nitrogen. The residues were dissolved in 150 ml of 50% (v/v) acetonitrile in water and injected (70 µl) onto a Phenomenex Luna C18 reverse phase column (4.6 x 100 mm; Phenomenex, Torrance, CA). ESI-LC-MS/MS was carried out using a ThermoQuest LCQ ion trap mass spectrometer (Thermo Electron Corporation, Waltham, MA) interfaced with a Hewlett Packard 1100 series HPLC system (Hewlett Packard, Palo Alto, CA). The sheath gas was set at 80 (arbitrary units) and the auxiliary gas was set at 15 (arbitrary units). The spray voltage was 5 kV and the capillary temperature was 200°C. The flow rate was 0.3 ml/min. Initial conditions were 95% of 0.1% (v/v) acetic acid in water (solvent A) and 5% of 0.1% (v/v) acetic acid in acetonitrile (solvent B). The percentage of B was maintained at 5% for 5 min followed by a linear gradient to 30% B from 5 to 15 min, to 80% B from 15 to 35 min, and to 90% B from 35 to 40 min. The column was washed with 90% B for 15 min before returning to the initial conditions and equilibrating the column for 10 min at the initial conditions before the next injection. |
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). After 16 min of incubation, only 33% of the activity remained (Fig. 2). It has been reported previously that compounds 2 to 5 also cause mechanism-based inactivation of P450 2B6 (Sridar et al., 2006).
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Glutathione Ethyl Ester Conjugates of the Metabolites of Phenyldiaziridines. The electrophilic reactive metabolites that were generated by incubating compounds 1 to 5 with recombinant 2B6 in the reconstituted system in the presence of NADPH were trapped using the nucleophilic trapping reagent, GSHEE, and analyzed by LC/MS (Soglia et al., 2004). GSHEE has been reported to be a more useful trapping reagent than glutathione for monitoring trapped adducts of electrophilic intermediates by LC/MS analysis. Figure 5 shows a representative extracted ion chromatogram obtained from a reaction mixture in which P450 2B6 was incubated with compound 1 and NADPH in the presence of GSHEE. The peak eluting at approximately 25 min with m/z = 510 (Fig. 5a) was observed only in samples incubated in the presence of NADPH. Figure 5a also shows the MS/MS fragmentation pattern for the ion with m/z 510. The MS/MS spectrum of the adduct exhibits prominent ions at m/z 381 and 407, which are characteristic of a loss of 129 and 103 mass units corresponding to loss of the pyroglutamate and glycylether ester of the GSHEE moiety, respectively. These results indicate that the peak corresponds to a GSHEE adduct generated by the attack of an electrophilic intermediate produced during metabolism of 1 by P450 2B6 on GSHEE. The fragmentation pattern of the GSHEE adduct (Fig. 5a) also suggests that the diaziridine substructure of compound 1 was metabolized to form the reactive intermediate. Interestingly, the MS/MS results also indicate that the methoxy group of compound 1 has been converted to a hydroxyl group, presumably by O-demethylation. The exact position where the GSHEE was adducted to the reactive diaziridine intermediate could not be determined from the MS spectrum.
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Similar adducts were observed for compounds 2 to 5 when they were incubated with recombinant 2B6 in a reconstituted system. Their MS/MS spectra were similar to that observed with compound 1 (data not shown). The structures of the GSHEE adducts of compounds 1 to 5 are summarized in Fig. 6. All of the adduct structures indicate that the reactive intermediates are generated by the metabolism-dependent loss of both the diaziridine moiety and the alkoxy group from the parent compounds 1 to 5.
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Discussion |
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), which demonstrated that a series of diaziridine compounds inactivated P450 2B6 in a time-, concentration-, and NADPH-dependent fashion in the reconstituted system. The five 3-trifluoromethyl-3-(4-alkoxyphenyl)diaziridines labeled 1 to 5 in Fig. 1 all inhibited P450 2B6 in a mechanism-based manner. Kinetic parameters for the inactivation of P450 2B6 by these five compounds were determined. Analysis of the inactivated samples by determination of changes in the reduced-CO spectrum or HPLC of the heme suggested that the primary loss in activity was not due to heme destruction. Replacement of the methoxy moiety on the diaziridine compounds with a methylthio group abolished the mechanism-based inactivation of P450 2B6 as measured using the 7-EFC assay.
In this study, a series of analogs of the diaziridines have been used to understand the structural determinants involved in the mechanism of inactivation. The presence of a trifluoromethyl group in each of the diaziridine analogs served to enhance the reactivity of the carbene, if formed via the P450-catalyzed reaction. Carbene formation from trifluoromethylaryl diazirine photolysis is well established, and leads to O-H, N-H, and C-H insertion with no intramolecular rearrangements (Hatanaka et al., 1996). In addition, GSHEE was used as a nucleophilic trapping agent to identify the pathway leading to the formation of the reactive intermediate. An analog of compound 1 containing a ketone moiety instead of the diaziridine substructure, 3-trifluoromethyl-3-(4-methoxyphenyl)ethanone (10), was synthesized (Fig. 1). It is interesting to note that no inactivation of 7-EFC activity was seen when compound 10 was incubated with P450 2B6 in the presence of NADPH. Although compound 10 was presumably produced from compound 1 as a result of the metabolism by 2B6 (Fig. 4), compound 10 itself is not the reactive metabolite responsible for inactivating the enzyme. This suggests that the diaziridine structure is essential but not sufficient for mechanism-based inactivation of the P450s. 3-Trifluoromethyl-3-phenyl diaziridine (9), an analog that does not contain another substituent on the phenyl ring, also did not inactivate P450 2B6, indicating the need for a substituted phenyl ring for the inactivation. 3-Trifluoromethyl-3-(3-methoxyphenyl)diaziridine (7), an analog in which the methoxy group was placed at the 3-position of the phenyl ring, also did not inactivate P450 2B6. Another analog of compound 1, 3-trifluoromethyl-3-(4-chlorophenyl-)diaziridine (8), in which a chlorine- was substituted for the methoxy- at the 4-position also lacked the ability to inactivate P450 2B6. Taken together, these data clearly demonstrate the combined importance of the 4-alkoxy substructure in addition to the diaziridine substructure for the mechanism-based inactivation of P450 2B6 by these compounds.
If those compounds that did not lead to mechanism-based inactivation were not metabolized by the P450, SAR determinant factors affecting metabolism alone would be easy to consider. However, as shown by the results in Figs. 3 and 4, the reason for the inability of these compounds to inactivate was not because they were not metabolized by P450 2B6. These results indicate that these MBI-negative compounds were, in fact, metabolized by 2B6 but did not form a reactive intermediate capable of inactivating the P450, whereas the MBI-positive 4-alkoxyphenyl diaziridines, even though they were metabolized to a lesser extent (Fig. 3), generated sufficient levels of reactive intermediates to inactivate the enzyme.
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). GSHEE adducts were observed using LC/MS for all five of the MBI-positive diaziridines 1 to 5, which indicated that an electrophilic reactive intermediate was indeed generated during the metabolism of these compounds by P450 2B6. Interestingly, MS/MS analysis of all of the GSHEE conjugates revealed that not only was the diaziridine substructure lost during metabolism but that the alkoxy group was also removed from the respective parent compounds (Figs. 5 and 6). MS/MS analysis of the GSHEE adduct formed from a deuterated analog (11) of compound 1 demonstrated that the GSHEE moiety was not adducted to the phenyl ring (Fig. 5) and suggested that this was, in all probability, also the case for all the other MBI-positive diaziridines 2 to 5. Thus, it appears that the most probable structures for the GSHEE adducts are related to structure 18 in Scheme 1.
The most plausible sequence of reactions that takes into consideration all of these results is depicted in Scheme 1. The structure of the reactive intermediate that is ultimately responsible for inactivation and adduct formation must be a p-quinone methide (quinomethane), namely, the 4-ethylidene-cyclohexa-2,5-dienone (17) (Scheme 1a). The sequence of steps leading to its formation involves the initial abstraction of a hydrogen radical from the parent compound (1-5) by a one-electron oxidation by 2B6. When the second oxidation step occurs at the 3-carbon of the diaziridine, the final metabolite will be a ketone 10, which does not inactivate the enzyme. However, when the second oxidation occurs at the 4-carbon of the phenyl ring, the alkoxy group is lost and the quinone methide 17 is formed. The quinone methide, trapped as a GSHEE-adduct, 18, is the most likely candidate to be the intermediate that results in the inactivation of P450 2B6. It is well established that quinone-related compounds can form potent reactive intermediates (Fan and Bolton, 2001; Yan et al., 2001, Monks and Jones, 2002; Uetrecht, 2003a,b).
Because the diaziridines 6 to 9 do not have a 4-alkoxy group, they are much less likely to be transformed into quinone methide reactive intermediates during metabolism by P450s than are compounds 1 to 5. A suggested pathway for the metabolism of diaziridine 6 to give the ketone metabolite, compound 16, is shown in Scheme 1b. The inability of compounds 6 to 9 to act as MBIs is further explained by the inability to trap a reactive intermediate with GSHEE. Therefore, the possibility of metabolic transformation of compounds 6 to 9 into quinone methides such as compound 17 should be considered to be a critical determinant that decides whether or not a given compound in the series of aryl diaziridines is capable of being an MBI.
Diaziridine substructures tend to be easily metabolized to form radical or carbene intermediates by a one-electron oxidation. This was indeed the initial reason why a series of these phenyldiaziridine compounds was designed to serve as molecular probes that were expected to attack and modify the P450 heme. It was therefore surprising that these diaziridines did not modify the heme of P450 2B6 but, rather, the apoprotein (Sridar et al., 2006) and that the reactive intermediate responsible for MBI was not a carbene or a radical but rather an electrophile. However, at this point, it is not possible to rule out completely that the other reactive species such as carbenes or radicals may be involved in the inactivation but that they could not be trapped by GSHEE. However, if carbenes or radicals were responsible for mechanism-based inactivation, then the diaziridines 6 to 9 should also have been MBIs of P450 2B6. An alternative mechanism that accounts for both inactivation and the glutathione trapping experiments implicates an initial O-dealkylation, with the direct formation of 4-hydroxy diaziridine 19, and its subsequent oxidation to the electrophilic p-quinone methide 20 (Scheme 2). However, our attempts at synthesizing 19 to test its role in inactivation highlighted the instability of this compound. Reaction of a suitably protected precursor (p-O-tert-butyldiphenylsilyl diaziridine) with tetrabutylammonium fluoride (well established protocols for deprotection of O-silyl phenols) led to extensive decomposition (unpublished data).
In conclusion, these results suggest that the determinant factor for mechanism-based inactivation by the series of aryl diaziridines investigated here is the formation of the reactive p-quinone methide intermediate, namely, 4-ethylidene-cyclohexa-2,5-dienone, which can be generated during the metabolism of 4-alkoxyphenyldiaziridines by P450 2B6. It is also important to note that it was shown here that more than one substructure may be required for a compound to be an MBI of P450 2B6. For the phenyldiaziridine compounds studied in this report, the expected diaziridine substructure was not the only essential component, but a 4-alkoxy group on the phenyl ring was also necessary to cause inactivation.
The importance of drug design that avoids the formation of potential reactive intermediates during metabolism has recently been emphasized during various stages of drug discovery to prevent potentially serious drug side effects. The studies discussed here strongly suggest that these drug design studies must be done with extreme care because the SARs for the formation of reactive intermediates may be much more complicated than expected from the apparent chemical structures. These studies also demonstrate that to better understand the structure-activity relationships for mechanism-based inactivators an entire series of analog structures may need to be taken into consideration.
This report also demonstrates that the elucidation of the structures of reactive intermediates by mass spectrometry is an effective method of investigating SAR of mechanism-based inactivators. The synthesis of various analogs and the structural elucidation of the reactive intermediates should complement each other to clarify the more complicated SAR of inactivators. To create safer drugs, a close collaborative effort between the fields of synthetic chemistry and metabolism appears to be increasingly more essential during the early stages of drug discovery.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: SAR, structure-activity relationship; P450, cytochrome P450; reductase, NAPDH-cytochrome P450 reductase; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; MBI, mechanism-based inactivator; LC, liquid chromatography; GC, gas chromatography; HR, high resolution; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ESI, electrospray ionization; GSHEE, glutathione ethyl ester; DMSO, dimethyl sulfoxide; amu, atomic mass unit(s); HPLC, high-performance liquid chromatography; r.t., retention time.
Address correspondence to: Dr. Paul F. Hollenberg, Department of Pharmacology, The University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: phollen{at}umich.edu
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