Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cytochrome P450-catalyzed N-dealkylation of amines occurs through processes that involve formation of carbinolamine intermediates with ultimate decomposition of these intermediates to a carbonyl compound and an amine, e.g., from propranolol yielding acetone and desisopropylpropranolol (Fig. 1). The carbinolamine is generally thought to arise via oxygen rebound to a carbon-centered radical intermediate. This mechanism is supported by studies with ¹⁸O₂ and/or H₂¹⁸O (McMahon et al., 1969; Shea et al., 1982; Kedderis et al., 1983) showing that the oxygen in the carbinolamine comes from O₂. The step(s) to the formation of the carbon-centered radical remains a subject of conjecture. The carbinolamine may arise from an earlier intermediate nitrogen-centered cation radical via proton transfer and electron reorganization (Miwa et al., 1983; Guengerich et al., 1996), or perhaps directly by hydrogen atom abstraction (Karki et al., 1995; Karki and Dinnocenzo, 1995; Manchester et al., 1997). Different interpretations of very similar metabolic data and of data from model systems (Baciocchi et al., 1998; Goto et al., 1998) have generated conflicting views on their origin. Regardless of how they arise mechanistically, carbinolamines are usually unstable, and they are isolated or shown to exist only under very specific circumstances.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Pathway of P450-catalyzed N-dealkylation of propranolol (1). Ar = 1-naphthyl.

Carbinolamines have been isolated as metabolites of well chosen relatively nonbasic N-alkyl-substituted compounds, such as N-alkyltriazines (Jackson et al., 1991; Lang et al., 1996, 1997), arylamides (Fujimaki et al., 1995), and highly conjugated arylamines (Schwartz and Kolis, 1972; Gorrod and Temple, 1976; Shea et al., 1982; Kedderis et al., 1983). N-Hydroxymethylcarbazole from the microsomal metabolic oxidation of the arylamine N-methylcarbazole in the presence of ¹⁸O₂ or H₂¹⁸O did not show the incorporation of oxygen from H₂O (Shea et al., 1982; Kedderis et al., 1983), indicating not only that the source of the oxygen is O₂ but also that this carbinolamine does not exchange hydroxide ion with the aqueous medium.

For most carbinolamines, the reversible loss of water or hydroxide ion to form imine or iminium ion intermediates competes with the loss of aldehyde or ketone from the carbinolamine (Fig. 1). Rehydration of the imine or iminium ion results in replacement of the oxygen atom arising via the enzyme-catalyzed activation of O₂ with an oxygen atom from solvent water, e.g., from the tertiary amine sparteine (Ebner et al., 1991a,b). In spite of the dehydration-rehydration of these carbinolamine intermediates, ¹⁸O-labeled benzaldehyde formed as a result of microsomal N-dealkylation in the presence of ¹⁸O₂ was successfully reduced to an ¹⁸O-alcohol without complete loss of the label (McMahon et al., 1969).

Although iminium ions formed in the metabolism of tertiary amines have been trapped by reaction with cyanide, e.g., nicotine (Murphy, 1973), trapping of imines with cyanide has not been successful. This is probably due to the instability of the -aminonitrile products of this reaction (Taillades and Commeyras, 1974; Shetty and Nelson, 1985).

Many years ago, formation of acetone and desisopropropylpropranolol in incubations of propranolol (1) in rat liver microsomes was demonstrated (Bakke et al., 1973). In the case of propranolol, cyclization of the imine to form an oxazolidine can occur (Fig. 2). Attempts have been made in this laboratory to demonstrate the existence of the carbinolamine, imine, and oxazolidine intermediates expected in the cytochrome P450-catalyzed N-dealkylation of propranolol (1). Although the imine (3) and oxazolidine (4) species were readily observed by NMR spectroscopy in organic solution, neither was observed in extracts from in vitro metabolic experiments, probably due to the rapid decomposition of the carbinolamine (2) to primary amine 5 and acetone (Shetty and Nelson, 1985).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Carbinolamine, imine, and oxazolidine intermediates in the P450-catalyzed N-dealkylation of propranolol (1) and trifluoropropranolol (6).

Carbinolamines can be stabilized by electron-withdrawing groups attached to the tetrahedral carbon (Rosenberg et al., 1974; Sayer and Jencks, 1977), similar to the stabilization of hydrates of aldehydes or ketones by adjacent electron-withdrawing substituents (Bover and Zuman, 1973; Greenzaid, 1973; Lamaty et al., 1986a,b). Electron-withdrawing groups also stabilize related species, such as hemiacetals (Crampton, 1975), oxazolidines (Alva Astudillo et al., 1985), and cyanohydrins (Ching and Kallen, 1978). Addition of fluorine atoms to the carbon atom  to the carbonyl stabilizes the hydrates and carbinolamines of carbonyl compounds significantly (Szinai et al., 1970a,b; Guthrie, 1975; Buschmann et al., 1980, 1982; Mispelaere and Roques, 1999). The aldehyde-hydrate and aldehyde-hemiacetal equilibria from trifluoroacetaldehyde favor hydrate and hemiacetal formation to a greater extent than the related equilibria from trifluoroacetone (Guthrie, 1975).

It seemed possible that trifluoropropranolol (6)-related carbinolamines, imines, or oxazolidines (7-9; Fig. 2) would be stable enough to observe as metabolites. Here we present ¹⁹F NMR and mass spectral data for trifluoromethyl carbinolamines, imines, and oxazolidines and our assessment of their stability in organic and aqueous solution and under mass spectral conditions. By ¹⁹F NMR we observed the formation of carbinolamines, imines, and in some cases oxazolidines from the reaction of primary amines 5, 10, and 11 with trifluoroacetone or trifluoroacetaldehyde in organic solution (Fig. 3). We also present evidence for the formation of relatively stable imine metabolites of trifluoromethyl-substituted propranolol analogs (Fig. 4) in incubations with rat liver microsomes and human recombinant CYP1A2.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Carbinolamines, imines, and oxazolidines from propranolol-related primary amines and trifluoroacetone or trifluoroacetaldehyde.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Substrates for metabolic experiments.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Synthesis of Amines. Trifluoropropranolol (6) was prepared by the reductive amination of trifluoroacetone using desispropropylpropranol (5) (Upthagrove et al., 1999b). Deshydroxydesisopropylpropranolol (11) was prepared by reported methodology (Glennon et al., 1989). The O-methyl ether of desisopropylpropranolol (10) was prepared by conversion of desisopropylpropranolol (5) to its N-t-butyl carbamate, O-methylation, followed by acid-catalyzed hydrolysis of the carbamate ester. Reductive amination of trifluoroacetone and trifluoroacetaldehyde using 10 provided needed O-methyl ethers 23 and 24, respectively. Imines and oxazolidines (Fig. 3) used for characterization of the intermediates were obtained from the condensations of desisopropylpropranolol (5), 10 and 11 with trifluoroacetaldehyde methyl hemiacetal, and trifluoroacetone, respectively.

Desisopropylpropranolol O-methyl ether (10). Desisopropylpropranolol (5) (1.02 g, 4.71 mmol) was dissolved in CH₂Cl₂ (20 ml). Di-t-butyl dicarbonate (1.13 g, 5.2 mmol) and triethylamine (2.60 g, 18.8 mmol) were added. The mixture was heated at reflux for 1.5 h, and the solvent and triethylamine was evaporated. Methylene chloride (30 ml) was added, and the mixture was washed with 2 × 20 ml of aqueous 0.5 N HCl and then with 20 ml of H₂O. The CH₂Cl₂ layer was dried over Na₂SO₄, and evaporation afforded 1.50 g (~100% yield) of the t-butyl carbamate ester of 10. This carbamate ester (1.50 g, 4.71 mmol) was dissolved in 50 ml of tetrahydrofuran, and 1.36 g (23.5 mmol) of powdered KOH was added. Methyl iodide (3.0 ml, 47 mmol) was then added, the flask was sealed, and the mixture was stirred at room temperature. After 2 h, when thin-layer chromatography monitoring showed the reaction was complete, the mixture was partitioned between 100 ml each of H₂O and CH₂Cl₂. The CH₂Cl₂ was washed with 50 ml of H₂O, dried over Na₂SO₄, and the solvent evaporated to yield 1.51 g (97%) of the intermediate O-methyl ether carbamate ester. The oil was purified by flash column chromatography eluting with CH₂Cl₂. The O-methyl ether carbamate ester (1.51 g, 4.58 mmol) was dissolved in a mixture of 1.0 ml of concentrated HCl and 4.0 ml of EtOAc¹ and stirred at room temperature for 30 min. The acid solution was made alkaline by the addition of aqueous 5 N NaOH, the EtOAc layer removed, washed with water, dried over Na₂SO₄, and the solvent removed to yield the O-methyl ether of desisopropylpropranolol (10) as a yellow waxy solid, which was used without further purification. ¹H NMR (CDCl₃):  8.24 (1H, m, H-8'), 7.79 (1H, m, H-5'), 7.50-7.40 (3H, m, H-7',-6',-4'), 7.36 (1H, dd, H-3'), 6.81 (1H, d, H-2'), 4.20 (2H, m, H-3), 3.71 (1H, m, H-2), 3.58 (3H, s, OCH₃), 3.01 (2H, m, H-1). ESI-MS/MS [MH]⁺ 232  [C₁₃H₁₁O]⁺ 183, [C₄H₁₀NO]⁺ 88.

Trifluoroethylpropranolol O-methyl ether (23). Desisopropylpropranolol O-methyl ether (10) (200 mg, 0.9 mmol), trifluoroacetaldehyde methyl hemiacetal (518 mg, 4.0 mmol), and p-toluenesulfonic acid (2 mg, 0.02 mmol) were dissolved in benzene (30 ml). The mixture was refluxed, and water collected in a Dean-Stark trap. After 16 h, the benzene was evaporated, and the residue partitioned between CH₂Cl₂ (20 ml) and aqueous 2 N NaOH (10 ml). The aqueous layer was extracted with an additional 10 ml of CH₂Cl₂. The combined organic extracts were washed with H₂O (5 ml), dried over Na₂SO₄, and evaporated. The residual oil and sodium cyanoborohydride (280 mg, 4.5 mmol) were dissolved in CH₃OH (10 ml). Acetic acid (~50 µl) was added, and the reaction mixture was allowed to stir at room temperature for 8 h. At the end of this time, CH₂Cl₂ (20 ml) and aqueous 1 N NaOH (10 ml) were added to the reaction mixture, and the product extracted into CH₂Cl₂. The aqueous phase was extracted with additional CH₂Cl₂ (10 ml). The combined CH₂Cl₂ extracts were washed with H₂O (10 ml), dried over Na₂SO₄, and the solvent was evaporated to leave a tan solid. This solid was purified by flash column chromatography on silica gel with a stepwise gradient of CH₂Cl₂ to 20% EtOAc in CH₂Cl₂, affording 79 mg (29%) of 23. ¹H NMR (CDCl₃):  8.23 (1H, m, H-8'), 7.80 (1H, m, H-5'), 7.51-7.43 (3H, m, H-7',-6',-4'), 7.37 (1H, dd, H-3'), 6.82 (1H, d, H-2'), 4.22 (2H, m, H-3), 3.85 (1H, m, H-2), 3.58 (3H, s, OCH₃), 3.26 (2H, qm, H-2"), 3.07 (2H, m, H-1). ESI-MS/MS [MH]⁺ 314  [C₁₃H₁₁O]⁺ 183, [C₆H₁₁NOF₃]⁺ 170.

Trifluoropropranolol O-methyl ether (24). Desisopropylpropranolol O-methyl ether (10) (200 mg, 0.9 mmol) and trifluoroacetone (~200 µl, 2.2 mmol) were dissolved in CH₂Cl₂ (5 ml) and heated in a sealed vial to 70°C for 24 h. After cooling, the reaction mixture was transferred into CH₃OH (5 ml). Sodium cyanoborohydride (259 mg, 4.1 mmol) and acetic acid (~50 µl) were added, and the reaction mixture was allowed to stir for 4 h. The mixture was partitioned between CH₂Cl₂ (20 ml) and aqueous 1 N NaOH (20 ml). The organic layer was washed with H₂O (5 ml), dried over Na₂SO₄, and the solvent was evaporated. The crude product was purified by flash column chromatography on silica gel using CH₂Cl₂, affording 128 mg (45%) of 24. ¹H NMR (CDCl₃):  8.23 (1H, m, H-8'), 7.80 (1H, m, H-5'), 7.52-7.43 (3H, m, H-7',-6',-4'), 7.37 (1H, dd, H-3'), 6.83 (1H, d, H-2'), 4.23 (2H, m, H-3), 3.84 (1H, m, H-2), 3.58 (3H, s, OCH₃), 3.20 (1H, m, H-2"), 3.13, 3.00 (2H, 2 m, H-1), 1.29, 1.26 (3H, 2d, 2"-CH₃). ESI-MS/MS [MH]⁺ 328  [C₇H₁₃NOF₃]⁺ 184, [C₁₃H₁₁O]⁺ 183.

Trifluoroethyl imine and oxazolidine of desisopropylpropranolol (17 and 22). Desisopropylpropranolol (5) (200 mg, 0.92 mmol) was dissolved in 10 ml of C₆H₆, and 0.50 ml of trifluoroacetaldehyde methyl hemiacetal (0.62 g, 5.6 mmol) was added. The reaction vessel was sealed and heated at 60°C for approximately 24 h. ¹⁹F NMR (C₆H₆):  71.46. In samples examined at earlier time points, diastereomeric carbinolamine 12 was present as indicated by ¹⁹F NMR (C₆H₆):  81.44, 81.46. Oxazolidine 22 was also observed in these samples. The reaction mixture for preparation of the imine (17) was heated at 40°C for 6 to 10 days resulted in more complete conversion to the oxazolidine (22). ¹⁹F NMR (C₆H₆):  79.65 and 79.67.

Trifluoropropyl imine of desisopropylpropranolol (8). Desisopropylpropranolol (5) (200 mg, 0.92 mmol) was dissolved in 10 ml of CH₂Cl₂, and 0.50 ml of trifluoroacetone (0.62 g, 5.6 mmol) was added. The reaction vessel was sealed and heated to 40°C for approximately 24 h. Nearly complete conversion to the imine was indicated by proton NMR in CDCl₃. The imine (8) was isolated by flash column chromatography on silica gel using CH₂Cl₂ as eluent. ¹H NMR (CDCl₃):  8.22 (1H, m, H-8'); 7.80 (1H, m, H-5'); 7.53-7.44 (3H, m, H-7', -6', -4'); 7.37 (1H, dd, H-3'); 6.84 (1H, d, H-2'); 4.49 (1H, m, H-2); 4.25 (2H, m, H-3); 3.72 (2H, m, H-1); 2.05 (3H, s, 2"-CH₃). ¹⁹F NMR (C₆D₆):  73.71; (CDCl₃):  75.26. ESI-MS/MS [MH]⁺ 312  [C₁₃H₁₁O]⁺ 183, [C₆H₉NOF₃]⁺ 168. In samples maintained at 25°C, significant amounts of the intermediate carbinolamine diastereoisomers 7 were present. ¹⁹F NMR (C₆D₆):  82.58, 82.88.

Trifluoropropyl oxazolidine of desisopropylpropranolol (9). Desisopropylpropranolol (5) (200 mg, 0.92 mmol) was dissolved in 10 ml of CH₂Cl₂, and 0.50 ml of trifluoroacetone (0.62 g, 5.6 mmol) was added. The reaction vessel was sealed and heated to 40°C for several days, resulting in nearly complete conversion to diastereomeric oxazolidines 9. Isolation was accomplished by column chromatography on silica gel using CH₂Cl₂ as eluent. ¹H NMR (CDCl₃):  8.23, 8.19 (1H, 2 m, H-8'); 7.81 (1H, m, H-5'); 7.53-7.44 (3H, m, H-7', -6', -4'); 7.38, 7.36 (1H, 2dd, H-3'); 6.85, 6.83 (1H, 2d, H-2'); 4.68, 4.50 (1H, 2 m, H-2); 4.40, 4.19 (2H, 2 m, H-3); 3.57, 3.16 (2H, 2 m, H-1); 1.61, 1.59 (3H, 2s, 2"-CH₃). ¹⁹F NMR (C₆D₆):  81.13, 81.44; (CDCl₃):  82.79, 83.00. ESI-MS/MS [MH]⁺ 312  [C₁₃H₁₁O]⁺ 183, [C₆H₉NOF₃]⁺ 168.

Trifluoroethyl imine of desisopropylpropranolol O-methyl ether (18). Desisopropylpropranolol O-methyl ether (10) (500 mg, 2.17 mmol) was dissolved in 10 ml of CH₂Cl₂ or CDCl₃. Trifluoroacetaldehyde methyl hemiacetal (0.50 ml) (0.62 g, 5.6 mmol) was added, the reaction vessel sealed, and heated at 40°C for approximately 24 h. ¹⁹F NMR (C₆D₆): 71.56. Shorter reaction times showed the presence of the diastereomeric carbinolamine 13. ¹⁹F NMR (C₆D₆): 81.51 (d, ³J_HF = 5 Hz), 81.57 (³J_HF = 5 Hz).

Trifluoropropyl imine of desisopropylpropranolol O-methyl ether (20). Desisopropylpropranolol O-methyl ether (10) (500 mg, 2.17 mmol) was dissolved in 10 ml of CH₂Cl₂ or CDCl₃. Trifluoroacetone (0.50 ml) (0.62 g, 5.6 mmol) was added, and the reaction vessel was sealed and heated at 40°C for approximately 24 h. The imine was isolated by column chromatography on silica gel eluting with CH₂Cl₂. ¹H NMR (CDCl₃):  8.25 (1H, m, H-8'); 7.80 (1H, m, H-5'); 7.53-7.44 (3H, m, H-7', -6', -4'); 7.37 (1H, dd, H-3'); 6.84 (1H, d, H-2'); 4.29 (2H, m, H-3); 4.11 (1H, m, H-2); 3.79 (2H, m, H-1); 3.58 (3H, s, OCH₃); 2.04 (3H, s, 2"-CH₃). ¹⁹F NMR (C₆D₆):  73.19; (CDCl₃):  75.29. ESI-MS/MS [MH]⁺ 326  [C₁₃H₁₁O]⁺ 183, [C₇H₁₁NOF₃]⁺ 182.

Trifluoroethyl imine of 3-(1-naphthoxy)propylamine (19). 3-(1-Naphthoxy)propylamine (11) (400 mg, 2.0 mmol) and trifluoroacetaldehyde methyl hemiacetal (500 mg, 3.85 mmol) were dissolved in 5 ml of benzene, and the mixture flushed with dry argon and stirred at room temperature for 5 to 8 days. At the end of this time, the benzene was evaporated. ¹⁹F NMR (C₆D₆):  70.76 (d, ³J_HF = 2 Hz). Shorter reaction times showed the presence of carbinolamine 14. ¹⁹F NMR (C₆D₆):  81.15 (d, ³J_HF = 5 Hz).

Trifluoropropyl imine of 3-(1-naphthoxy)propylamine (21). 3-(1-Naphthoxy)propylamine (11) (400 mg, 2.0 mmol) and trifluoroacetone (500 mg, 4.5 mmol) were dissolved in 5 ml of benzene, and the mixture flushed with dry argon and stirred at room temperature for 7 days. At the end of this time, the benzene was evaporated. ¹H NMR (CDCl₃):  8.32 (1H, m, H-8'); 7.86 (1H, m, H-5'); 7.58-7.37 (4H, m, H-7', -6', -4', -3'); 6.85 (1H, d, H-2'); 4.26 (2H, t, H-3); 3.77 (2H, t, H-1); 2.38 (2H, m, H-2); 2.03 (3H, s, 2"-CH₃). ¹⁹F NMR (C₆D₆):  73.69. Shorter reaction times showed the presence of the intermediate carbinolamine 16. ¹⁹F NMR (C₆D₆):  83.24.

NMR Spectroscopy. NMR spectra were acquired on a Varian VXR 300 spectrometer (Varian Inc., Palo Alto, CA) or a Bruker AF-300 spectrometer (Bruker Instruments Inc., Billerica, MA). ¹⁹F NMR spectra were calibrated using neat trifluoroethanol (Aldrich, Milwaukee, WI) as an external reference,  = 77.8 ppm relative to CFCl₃ (Everett, 1995). Reactions of 4 to 10 mg (20-50 µmol) of primary amine and one to two equivalents of trifluoroacetone (Aldrich) or trifluoroacetaldehyde methyl hemiacetal (PCR, Gainesville, FL) were carried out in deuterated solvents (total volume approximately 1 ml) in 5-mm NMR tubes.

Mass Spectrometry. ESI mass spectra were obtained using a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan/Thermoquest, San Jose, CA) equipped with a capacitive electrospray ion source modification (Wang and Hackett, 1998) of the Finnigan API interface. Collision-induced dissociation was carried out in the mass analyzer on an ion selected from the mass spectrum, using the helium gas present in the trap. The samples, either synthetic standards or metabolic product mixtures in methanol, were infused directly via a syringe pump at a flow rate of 300 nl/min. The heated capillary was maintained at 160°C and the source voltage at 1.7 kV.

Metabolism.

Incubations with rat liver microsomes Substrates were incubated at 37°C for 20 to 60 min in the presence of 3-methylcholanthrene-induced rat liver microsomes. The typical incubation volume was 250 µl, substrate concentration 50 µM, microsomal protein concentration 1 mg/ml, NADPH concentration 1.0 mM, in 100 mM sodium phosphate buffer, pH 7.4. After incubation, the samples were made alkaline by the addition of aqueous-saturated sodium carbonate (50 µl). Samples were extracted into EtOAc (500 µl). The EtOAc solution was dried over sodium sulfate. This EtOAc solution was injected into the liquid chromatography/MS system.

Incubations with recombinant human CYP1A2. The substrate was incubated at 37°C for 20 min in the presence of insect cell-expressed recombinant human CYP1A2 (GENTEST Supersomes, Woburn, MA). The incubation volume was 1.2 ml, substrate concentration 50 µM, P450 concentration 10 pmol/ml, NADPH concentration 1.0 mM, in 100 mM sodium phosphate buffer, pH 7.4. After incubation, the sample was made alkaline by the addition of aqueous saturated sodium carbonate (100 µl). Samples were extracted into EtOAc (5 ml). The EtOAc solution was dried over sodium sulfate, then the solvent was evaporated. The residue was dissolved in 200 µl of CH₃OH. This CH₃OH solution was infused into the mass spectrometer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of the synthesized carbinolamines, imines, and oxazolidines and detection of them as metabolites required development of analytical methods. ¹⁹F NMR proved to be very useful in monitoring the formation and stability of carbinolamines, imines, and other species produced in the reactions of primary amines with fluorinated carbonyl compounds. Because we were unable to achieve sufficient sensitivity by NMR analysis to demonstrate the presence of carbinolamine, imine, or oxazolidine metabolites from incubations of trifluoromethyl substrates with P450s, other analytical methods were sought.

Attempts to adapt standard gas chromatography/MS methods used for propranolol metabolite assays that involve derivatizations, e.g., trialkylsilyl ether formation or trifluoroacetylation (Shetty and Nelson, 1985), were not successful due to low sensitivity of the assay and instability of the carbinolamine species. Although imine 8 generated an N-trifluoroacetyl derivative via isomerization of the imine to an enamine, we could not attain complete conversion due to significant decomposition.

Attempts to develop a liquid chromatography/MS method to separate the metabolites were unsuccessful. Under reverse phase conditions, elution of the trifluoropropranolol-related substrates from the column required low pH. The intermediates were not stable to these acidic conditions. Under normal phase high-performance liquid chromatography conditions, separation of imine 8, oxazolidine 9, and trifluoropropranolol (6) on a CN column was successful. However, elution of the N-dealkylation product 5 from the column required a steep hexane/ethanol gradient with a relatively high concentration of triethylamine (4% by volume), conditions that suppressed ionization of these compounds in the mass spectrometer.

APCI ionization is amenable to nonaqueous conditions. Small volumes of sample in EtOAc or CH₃OH solution were infused into the mass spectrometer with hexane flowing at a rate of 100 to 200 µl/min. Under these unusual conditions, mass spectra of fluorinated imines and oxazolidines were obtained; however, nonfluorinated components of compound mixtures, e.g., N-dealkylation products 5 and 10, ionized very poorly under these conditions.

Mass spectra of the fluorinated imines and oxazolidines were successfully obtained using a capacitive ESI source and ion trap mass spectrometer. Methanol served as the proton source because in the presence of water only the primary amine decomposition product was observed. Ionization did not occur in ethyl acetate and acetonitrile solutions of samples. The capacitive ESI source had the advantage over APCI that both fluorinated and nonfluorinated compounds ionized and produced satisfactory mass spectra under the same conditions.

NMR Spectra. By ¹⁹F NMR spectroscopy, formation of carbinolamines, imines, and in some cases oxazolidines from the reaction of primary amines 5, 10, and 11 with trifluoroacetone or trifluoroacetaldehyde in organic solution was observed (Fig. 3). ¹⁹F NMR chemical shifts of the trifluoromethyl groups from the carbinolamine and imines from the three amines and the two fluorinated carbonyl compounds obtained in solution are given in Table 1. Besides carbinolamines and imines, resonances of the trifluoromethyl groups of other species were observed, e.g., oxazolidines and aminals. Where possible, the compound identities were confirmed by ¹H and ¹³C NMR spectra and/or mass spectra (under Materials and Methods; Table 2). ¹⁹F Resonances for the trifluoromethyl groups of the imines are the farthest downfield and consistently downfield from those of the carbinolamines by ca. 10 ppm (Figs. 5-7; Table 1). Carbinolamine trifluoromethyl group resonances are downfield from those of the hydrates of the carbonyl compounds by about 3 ppm.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Reaction of desisopropylpropranolol (5) with trifluoroacetone in benzene at 60°C monitored by 19F NMR. a, formation of diastereomeric carbinolamine 7 ( 82.2 ppm) at 3 h, 60°C. b, 24 h, 60°C. Imine 8 ( 73.2 ppm). c, after 6 days, nearly complete conversion to 8. d, mixture of oxazolidine diastereoisomer standards (9). e, imine 8 in acetonitrile/water (4:1), 19 h. f, oxazolidine 9 in acetonitrile/water (4:1), 19 h. g, hydrolysis of 8 occurs after addition of one equivalent of HCl.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Reaction of O-methyl desisopropylpropranolol (10) with trifluoroacetone in benzene monitored by 19F NMR. a, initial formation of carbinolamine 15 at 2 h (room temperature). Imine 20 ( 73.1 ppm) is beginning to form. b, after heating, 20 is the major product. c, trifluoroacetone and its hydrate in benzene. d, imine 20 in the presence of acetonitrile/water (4:1), after 20 h. e, addition of approximately one equivalent of HCl to 20 in acetonitrile/water (4:1) affords trifluoroacetone hydrate. f, trifluoroacetone in acetonitrile/water (4:1) affords trifluoroacetone hydrate.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Reaction of 10 with trifluoroacetaldehyde methyl hemiacetal in benzene was monitored by 19F NMR spectroscopy. a, diastereomeric carbinolamine (13,  81.5 ppm), hemiacetal, and the hydrate of trifluoroacetaldehyde. b, heating the mixture catalyzed formation of imine 18. c, control spectrum of trifluoroacetaldehyde methyl hemiacetal in benzene. d, rehydration of 18 in a mixture of acetonitrile and water. Carbinolamine 13 is stable for many hours under these conditions. e, addition of approximately one equivalent of HCl to 13 in acetonitrile/water. f, trifluoroacetaldehyde methyl hemiacetal in acetonitrile/water with approximately one equivalent of acid.

Mass Spectra. Imine and oxazolidine synthetic standards in methanol were examined by mass spectrometry to aid in the assignment of their structures as possible metabolites. The compounds appearing in Table 2 were examined under ESI-ion trap conditions. Because these samples were analyzed in methanol solution, methanol adducts were commonly observed (Table 2). In some cases, NMR samples containing predominantly imine but also other species, e.g., primary amines and aminals, were used to acquire mass spectra. Molecular ions of desisopropylpropranolol (5) were observed in the mass spectra of purified imine 8 and oxazolidine 9, however, suggesting that some hydrolysis of the imines and oxazolidines to the primary amine occurs under the mass spectral conditions. There were differences in the behavior of imine 8 and oxazolidine 9 under ESI-ion trap conditions. The relative amount of hydrolysis of imine 8 to primary amine 5 was large, whereas oxazolidine 9 produced only a small amount of 5 (Table 2). Ions for methanol adducts to imine 8 or oxazolidine 9 were not observed.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Mass spectra of trifluoropropranolol (6) and metabolites in extracts from incubation with human CYP1A2. a, MS/MS spectrum of m/z 314, the molecular ion for trifluoropropranolol. b, MS/MS spectrum of m/z 330. Fragment ions at m/z 199 and 170 indicate that the m/z 330 ion is due to ring hydroxylation product(s). c, MS/MS of m/z 312. Fragment ions at m/z 168 and 183 indicate the loss of 2 amu from the aliphatic side chain, consistent with imine or oxazolidine. d and e, MS/MS spectra of imine 8 and oxazolidine 9 standards.

Metabolic Experiments. Three fluorinated propranolol analogs were incubated with 3-methylcholanthrene-induced rat liver microsomes and analyzed for the formation of carbinolamines and imines: trifluoropropranolol (6), trifluoropropranolol O-methyl ether (24), and trifluoroethylpropranolol O-methyl ether (23). These samples were analyzed under the APCI-MS conditions described above. Trifluoropropranolol (6) was also incubated with recombinant human CYP1A2 and analyzed using the ESI-MS method, giving very similar results to those obtained from incubations with rat liver microsomes. In addition to the N-dealkylation products, imine and ring-hydroxylated metabolites were formed from the three substrates 6, 23, and 24. Mass spectral evidence for these metabolites is shown in Figs. 8 and 9.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Mass spectra of trifluoroethylpropranolol O-methyl ether (23) and metabolites in extracts from incubation with rat liver microsomes. a, MS/MS spectrum of m/z 314, the molecular ion for the substrate. b, MS/MS spectrum of m/z 330. Fragment ions at m/z 199 and 170 indicate that the m/z 330 ion is due to ring hydroxylation product(s). c, MS/MS of m/z 312. Fragment ions at m/z 168 and 183 indicate the loss of 2 amu from the aliphatic side chain, consistent with imine 18. d, MS/MS spectrum of imine 18 synthetic standard.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The ¹⁹F NMR experiments clearly demonstrate the existence of carbinolamine and imine species from these primary amines and fluorinated carbonyl compounds in solution. No systematic effort was made to determine the equilibrium ratios of imine to carbinolamine because the focus of the work was on determining whether they were produced metabolically. Because the equilibrium for hydration of acetaldehyde favors hydration to a much greater extent than for acetone (Buschmann et al., 1980) and for trifluoroacetaldehyde versus trifluoroacetone (Guthrie, 1975), it seemed that this might be true for their respective imines, i.e., more carbinolamine versus imine from the trifluoroacetaldehyde-derived imines. However, the situation is much more complex because equilibrium conditions were not assured, and the conditions were not carefully controlled for pH, solvent composition, or temperature, important factors that affect the composition of imine-oxazolidine-carbinolamine mixtures (Kurono et al., 1994). In addition, the process of hydration of the ketone or aldehyde competes under these conditions. These studies do provide evidence that the carbinolamines are produced and slowly dehydrate to form imines in organic solutions. The carbinolamines derived from trifluoroacetaldehyde formed from rehydration of their corresponding imines, suggesting that at equilibrium in neutral aqueous solution, these carbinolamines would be present.

Our attempts to use ¹⁹F NMR to demonstrate the presence of carbinolamine, imine, or oxazolidine metabolites from incubations of trifluoromethyl substrates with rat liver microsomes or recombinant human CYP1A2 expressed in insect cells were unsuccessful. We believe this was due to our inability to optimize the NMR instrument to detect small concentrations of the metabolites in situ, and the instability of the carbinolamines to methods for concentrating the samples. A ¹⁹F NMR method to detect and quantitate low micromolar concentrations of more stable metabolites from P450 incubations has been reported (Vervoort et al., 1990), but these investigators used an instrument dedicated to ¹⁹F NMR on biological samples.

The mass spectral experiments under APCI and ESI-ion trap conditions clearly demonstrate imine and/or oxazolidine metabolite(s) and metabolic products of N-dealkylation and aromatic hydroxylation from fluorinated propranolol analogs in the presence of rat liver microsomes and CYP1A2. The structure of the metabolic product shown in Fig. 8c from trifluoropropranolol (6) could be imine 8 and/or oxazolidine 9. Since the NMR experiments indicated that imine 8 was relatively stable in a neutral aqueous environment, it is the more likely structure, although the oxazolidine 9 cannot be ruled out completely. The MS/MS spectra unambiguously showed that the imines 20 and 21 were formed as metabolites of substrates 23 and 24, respectively.

In spite of mass spectral evidence for the presence of imines in the metabolism of some of these trifluorinated propranolol analogs, we were unable to detect their expected carbinolamine precursors. These carbinolamines do not appear sufficiently stable to determine under the mass spectral conditions used. In the APCI experiments, even though nonaqueous solvents like hexane can be used for the sample infusion, they were not detected. In the less energetic ESI process, hydroxylic solvents are needed as a proton source for ionization. Methanol used in these experiments may be detrimental to the stability of the carbinolamines, as shown in the decomposition of imines under the ESI-ion trap conditions.

In conclusion, the addition of fluorines on the -carbon in these aliphatic amines, stabilized imines, and carbinolamines to the extent that they could be detected by ¹⁹F NMR in solution. Direct determination of the carbinolamines' mass spectrally was unsuccessful. Their presence in microsomal incubations is inferred from the demonstration that the imine or oxazolidine is a metabolite from trifluoropropranolol (6) and imine metabolites of the other related secondary amines with -trifluoromethyl groups are formed.