P450-Catalyzed N-Dealkylation Reactions
A R T I C L E S
µ silica gel plates with detection by UV light and I2. For flash
chromatography, Selecto Scientific 63-200 mesh silica gel was em-
ployed. 1H NMR spectra were obtained at either 400 or 500 MHz with
phatidyl choline (32 nmol in 20 µL of buffer) were combined and
vortexed occasionally over 40 min at room temperature. After this
preincubation, 434 µL of phosphate buffer (0.1 M, pH 7.4) and 500
nmol of substrate (in 2.5 µL of MeCN) were added, and the mixture
was incubated at 37 °C in a shaking water bath for 3 min. NADPH
(1.0 µmol in 20 µL of buffer) was added to initiate the reaction and
again after 30 min; after 60 min at 37 °C, the reaction was quenched
with aqueous ZnSO4 solution (15% w/v, 100 µL) and extracted with
Et2O (300 µL) for GC/EIMS analysis.
1
Bruker DRX 400 and DRX 500 spectrometers, while H-decoupled
13C NMR spectra were obtained at 100.6 MHz on a Bruker 400
spectrometer or 125.8 MHz on a Bruker 500 spectrometer. Proton
chemical shifts are reported in ppm (δ) relative to tetramethylsilane as
inferred from shifts of residual protons in the deuterated solvents (i.e.,
7.26 ppm for CDCl3, 4.80 ppm for D2O, and 2.05 ppm for d6-acetone);
13C chemical shifts are in ppm relative to internal CDCl3 (77.23 ppm).
Metabolite Analysis by HPLC and GC/EIMS. Procedures for the
direct analysis of metabolites and their derivatives by HPLC and GC/
EIMS have been described in detail previously.28 The preparation and
analysis of standard solutions of potential carbonyl metabolites, and
the characterization of authentic DNP-carbonyl standards for formal-
dehyde, acetone, acrolein (9a), and 3-hydroxypropanal (10a), are also
described elsewhere;28 this same procedure was employed to synthesize
the DNP-adducts of the potential acyclic carbonyl metabolites of 2.
Thus, methyl vinyl ketone (9b, 420 µL, 5.1 mmol) was added dropwise
with stirring to 0.15 M DNPH trapping solution (35 mL, 5.3 mmol); a
bright orange precipitate formed instantaneously. After 30 min, the
orange crystals were isolated by suction filtration, washed with H2O
(100 mL), and purified by flash chromatography (10% EtOAc in
hexanes) to afford a cyclic DNP derivative (2-(2,4-dinitrophenyl)-(2-
methyl)2,3-dihydropyrazole, 204 mg, 16%) as bright orange crystals.
Microsome Preparation. Male Sprague-Dawley rats (188-205 g),
pretreated with sodium phenobarbital (80 mg/kg, ip for 3 days), were
anesthetized with carbon dioxide and euthanized by decapitation. Their
livers were immersed in ice-cold KH2PO4 buffer (50 mM, pH 7.4)
containing 150 mM potassium chloride and 5 mM Na2EDTA. The
unperfused livers were individually sliced with scissors, and the mince
was homogenized with a Teflon/glass homogenizer in ice-cold 50 mM
phosphate buffer (pH 7.4) containing 150 mM potassium chloride and
5 mM Na2EDTA using 3 mL of buffer/g liver. The homogenate was
first centrifuged at 1000g for 10 min, and that supernatant was
centrifuged again at 12 000g for 20 min. The supernatant was again
decanted and centrifuged at 100 000g for 60 min, and the supernatant
was discarded. The microsomes were removed from the glycogen pellet
by swirling gently with cold phosphate buffer. After being rehomog-
enized in buffer, the microsomes were resedimented at 100 000g for
60 min. The resulting microsomal pellet was resuspended in ice-cold
100 mM KH2PO4 (pH 7.4) buffer containing 1 mM Na2EDTA and
glycerol (20%, v/v) and frozen at -70 °C. The protein concentration
(80.1 mg/mL) was determined by the method of Bradford60 using bovine
serum albumin as a standard. The cytochrome P450 and b5 contents
(2.66 nmol/mg protein and 0.87 nmol/mg protein, respectively) were
determined by difference spectra according to Omura and Sato.61 Just
prior to incubation, thawed microsomes were diluted 50% with 100
mM KH2PO4 (pH 7.4) buffer.
1
mp 148-149 °C. TLC (hexanes:EtOAc, 10:1) Rf ) 0.32. H NMR
(400 MHz, d6-acetone): δ 2.25 (s, 3H), 5.63 (d, J ) 11.0 Hz, 1H),
5.90 (d, J ) 17.8 Hz, 1H), 6.65 (m, J ) 9.6 Hz, 1H), 8.08 (d, J ) 9.6
Hz, 1H), 8.44 (dd, J ) 8.3, 1.7 Hz, 1H), 9.02 (d, J ) 2.6 Hz, 1H),
11.25 (s, 1H). GC tR ) 25.0 min. EIMS: [M]+ ) 250. Anal. Calcd for
C10H10N4O4: C, 48.00; H, 4.03; N, 22.39. Found: C, 48.30; H, 4.20;
N, 22.14. Similarly, 4-hydroxy-2-butanone (10b, 500 µL, 5.8 mmol)
was used as described above and afforded the DNP derivative of methyl
vinyl ketone (150 mg, 10%).
NMR Analysis of Metabolites. Microsomal incubations were
conducted as described above and contained 5 µmol of substrate 2a in
a final volume of 10 mL. Incubations using both [1′-14C]-2a and [1′-
13C]-2a were also conducted similarly and contained 2.5 µmol of each
isotopically labeled probe. After 90 min (>80% oxidation of 2a as
determined by HPLC), the incubation was quenched with 15% ZnSO4
(2 mL), and the microsomal protein was sedimented by centrifugation.
The supernatant (∼10 mL) was transferred by Pasteur pipet to a separate
vial in which it was concentrated in vacuo to ca. 500 µL. The
concentrate was added to a 5 mm NMR tube containing 50 µL of D2O,
Microsomal Incubations. Incubations (10 mL) were performed in
a capped 25 mL Erlenmeyer flask under air at 37 °C in a shaking water
bath. Each incubation contained 500 µL of microsomes (2 mg of
protein/mL 0.1 M KH2PO4 buffer (pH 7.4)), an NADPH regeneration
system composed of 100 µmol of glucose 6-phosphate and 13.3 units
of glucose 6-phosphate dehydrogenase, 50 µL of 100 mM aniline
substrate dissolved in acetonitrile (yielding an initial substrate concen-
tration of 0.5 mM), and 5 µmol of NADPH. Sample aliquots (1 mL)
were removed by micropipet at 0, 2, 5, 10, 15, 20, 30, 45, 60, and 90
min after the addition of NADPH and quenched with 15% ZnSO4 (200
µL) to precipitate microsomal protein. The protein was sedimented by
centrifugation, and the supernatant (1 mL) immediately underwent three
analytical procedures: 20 µL was analyzed by HPLC (A254 and A320);
400 µL was diluted with H2O (100 µL), extracted with Et2O (300 µL),
and a 1 µL aliquot of the organic layer was analyzed by GC/EIMS;
and 500 µL was treated with 15 µL of 0.15 M DNPH trapping solution
for 30 min at room temperature, the solution was extracted with EtOAc
(2 × 250 µL), and a 1 µL aliquot of the organic layer was analyzed by
GC/EIMS. For the determination of 14C within microsomal protein,
the precipitated pellet was treated with 2 N KOH (200 µL) at room
temperature for 48 h following supernatant removal. An aliquot (100
µL) of the basic solution was mixed with LSC-cocktail (6 mL) and
analyzed by LSC.
1
and the H-decoupled 13C NMR spectrum was obtained by a Bruker
500 MHz NMR at a spectral frequency of 125.8 MHz (NS ) 64 250).
Spectra of authentic standards of 11 and 12 were obtained similarly.
11 13C NMR (125.8 MHz, D2O): δ 13.54, 79.21. 12 13C NMR (125.8
MHz, D2O): δ 8.16, 27.00, 179.94.
N-Methylacetanilide. Acetyl chloride (450 µL, 6.3 mmol) was added
dropwise to an ice-cooled stirred solution of Kugelrohr-distilled
N-methylaniline (850 µL, 7.8 mmol) and triethylamine (1.1 mL, 7.9
mmol) in CH2Cl2 (10 mL). After 10 min at 0 °C, the solution was
stirred at room temperature for 3 h. The reaction was quenched with 1
N HCl (20 mL), extracted with CH2Cl2 (5 × 5 mL), dried (MgSO4),
and concentrated in vacuo. The crude solid was recrystallized from
pentane to afford the title compound (938.8 mg, 99%) as white crystals.
mp 98-100 °C. TLC (EtOAc:hexanes, 1:1) Rf ) 0.32. 1H NMR (400
MHz, CDCl3): δ 1.87 (s, 3H), 3.27 (s, 3H), 7.19 (d, J ) 7.4, 2H),
7.34 (t, J ) 7.4, 1H), 7.42 (t, J ) 7.5, 2H). 13C NMR (100.6 MHz,
CDCl3): δ 22.59, 37.33, 121.71, 127.26, 127.88, 129.90, 170.74. GC
tR ) 10.7 min. EIMS: [M]+ ) 149.
Reconstituted CYP2B1 Incubation Procedure. CYP2B162 and
NADPH-cytochrome P450 reductase63 were isolated as described. For
reconstitution of monooxygenase activity, P450 (0.75 nmol in 10 µL
of buffer), reductase (1.5 nmol in 13 µL of buffer), and dilauroylphos-
N-(1′-Methyl)cyclopropyl-N-methylaniline (2b).64 Ethylmagnesium
(60) Bradford, M. Anal. Biochem. 1976, 76, 248-254.
bromide (3.0 M in Et2O, 3.4 mL, 10 mmol) was added dropwise to a
(61) Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370-2378.
(62) Alterman, M. A.; Chaurasia, C. S.; Lu, P.; Hanzlik, R. P. Biochem. Biophys.
Res. Commun. 1995, 214, 1089-1094.
(64) Chaplinski, V.; de Meijere, A. Angew. Chem., Int. Ed. Engl. 1996, 35,
413-414.
(63) Yasukochi, Y.; Masters, B. S. S. J. Biol. Chem. 1976, 251, 5337-5344.
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