3 L. Constantino, E. Rosa and J. Iley, Biochem. Pharmacol., 1992, 44,
at timed intervals and added to ethanol (200 µl) to terminate the
reaction. 1 M Methanolic KOH (200 µl) was added the solu-
tions were left for 10 min then acidifed with 1 M methanolic
HCl (200 µl). The samples were evaporated to dryness and
resuspended in acetonitrile for GC analysis or methanol–water
(1 : 1) for HPLC analysis (see below).
651.
4 J. Iley and R. Tolando, J. Chem. Soc., Perkin Trans. 2, 2000,
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9 For the nomenclature of the P450 family, see e.g. F. J. Gonzalez,
Pharmacol. Ther., 1990, 45, 1.
10 J. A. Fruetel, Y.-T. Chang, J. Collins, G. Loew and P. R. Ortiz de
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11 J. H. Capdevila, S. Wei, C. Helvig, J. R. Falck, Y. Belosludtsev,
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1996, 271, 22663.
12 S. Graham-Lorence, G. Truan, J. A. Peterson, J. R. Falck, S. Wei,
C. Helvig and J. H. Capdevila, J. Biol. Chem., 1997, 272,
1127.
Microsomal incubations
Liver microsomes from phenobarbital-induced rats were
obtained from the Institute of Cancer Research, Sutton, Surrey,
UK. The P450 content of these microsomes was 1.95 nmol
mgϪ1 microsomal protein. Incubations with these microsomes
were performed in 0.1 M pH 7.4 phosphate buffer using
microsomal concentrations of 1.5 mg protein cmϪ3 in the pres-
ence of an NADPH generating system comprising NADPϩ
(1.25 mmol cmϪ3), MgCl2 (6 mmol cmϪ3), glucose-6-phosphate
(6.25 nmol cmϪ3) and of glucose-6-phosphate dehydrogenase
(2.5 units cmϪ3). The incubations were maintained at 37 ЊC and
reactions were initiated by addition of substrate (to give a sub-
strate concentration in the range 0.5–10 mM). Aliquots (100 µl)
were withdrawn at timed intervals and added to a 10% (w/v)
solution of trichloroacetic acid (200 µl) to precipitate protein.
After centrifugation (4500 rpm, 5 min) the supernatant was
evaporated to dryness and analysed for products as described
below. Only products of N-dealkylation or ring oxidation
adjacent to the nitrogen atom were detected. No evidence for
the competing formation of phenols (from oxidation of the
aromatic groups) or of ring oxidation at positions other than
next to the amide nitrogen atom was obtained. For the kinetic
reactions the supernatant was treated with 1 M sodium
hydroxide (200 µl) for 10 min, acidified with 1 M HCl (200 µl)
for 10 min, evaporated to dryness then analysed as described
below.
13 J. P. Jones, M. Shou and K. R. Korzekwa, Biochemistry, 1995, 34,
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14 M. Shou, F. J. Gonzalez and H. V. Gelboin, Biochemistry, 1996, 35,
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15 R. E. White, J. P. Miller, L. V. Favreau and A. B. Bhattacharyya,
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18 C. J. Sinal, M. Hirst, C. D. Webb and J. R. Bend, Drug Metab.
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20 J. R. Lindsay Smith and D. N. Mortimer, J. Chem. Soc., Perkin
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21 J. I. Manchester, J. P. Dinnocenzo, L. A. Higgins and J. P. Jones,
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22 L. R. Hall and J. P. Hanzlik, Xenobiotica, 1991, 21, 1127.
23 R.-S. Wang and T. Nakajima, Arch. Toxicol., 1991, 65, 39.
24 L. Constantino, PhD Thesis, University of Lisbon, 1994.
25 S. Yoshifuji and Y. Arakawa, Chem. Pharm. Bull., 1989, 37, 3380.
26 V. V. Dunina, Zh. Org. Khim., 1978, 14, 2064.
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Analytical methods
The GC analysis of 6–8 and 22, 25 and 28 employed direct
injection of the sample under isothermal conditions at 90 ЊC.
Analysis of 23, 24, 26, 27, 29 and 30 required derivatisation of
the sample as follows. The dried sample was dissolved in
acetonitrile (100 µl); a portion (50 µl) of this solution was added
to N-tert-butyldimethylsilyl-N-methyltrifluoracetamide (50 µl),
the mixture sonicated for 5 min then heated at 100 ЊC for 2 h.
After cooling, the sample was analysed using a thermal gradi-
ent: 2 min at 90 ЊC, 90 to 140 ЊC at 5 ЊC minϪ1, 140 ЊC for 1 min.
Reactions involving compound 5, 9 and 10 were analysed by
HPLC. A gradient comprising two eluants, A and B, was
employed: eluant A, 5% acetonitrile in 0.05 M pH 2 phosphate
buffer; eluant B, 60% acetonitrile in 0.05 M pH 2 phosphate
buffer. The gradient was as follows: 0–15 min, 60% B to 100%
B; 15–18 min, 100% B; 18–19 min, 100% B to 60% B. The flow
rate was 1 cm3 minϪ1; detection wavelength 254 nm.
37 N. H. Cromwell and K. E. Cook, J. Am. Chem. Soc., 1958, 80, 4573.
38 T. Hase, Synthesis, 1980, 1, 36.
39 C.-Y. Cheng, H.-B. Tsai and M.-S. Lin, J. Heterocycl. Chem., 1995,
32, 73.
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