Design, synthesis, and characterization of 7-methoxy-4- (aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening

Article abstract of DOI:10.1021/tx980248q

In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4- (aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7- hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 μM with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 μM. K(m) and ν(max) values of MAMC for P450 2D6 were found to be 26.2 ± 2.8 μM and 2.9 ± 0.07 min^-1, respectively. For P450 1A2, MAMC was found to have a K(m) value of 29.7 ± 6.2 μM and a ν(max) of 0.57 ± 0.07 min^-1. Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 μM selective for P450 2D6, and furafylline, at a concentration of 30 μM selective for P450 1A2. In conclusion, O- demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high- throughput screening purposes.

Full text of DOI:10.1021/tx980248q

Chem. Res. Toxicol. 1999, 12, 555-559
555
Communications
Design , Syn th esis, a n d Ch a r a cter iza tion of
7
-Meth oxy-4-(a m in om eth yl)cou m a r in a s a Novel a n d
Selective Cytoch r om e P 450 2D6 Su bstr a te Su ita ble for
High -Th r ou gh p u t Scr een in g
Rob C. A. Onderwater, J ennifer Venhorst, J an N. M. Commandeur, and
Nico P. E. Vermeulen*
Leiden/ Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology,
Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083,
1
081 HV Amsterdam, The Netherlands
Received November 13, 1998
In this study, a selective substrate for cytochrome P450 2D6 was designed using a small
molecule model developed by M. J . De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41-48]. The
substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated
metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective
fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was
characterized using microsomes containing single human P450 isoenzymes and human liver
microsomes. Formation of the metabolic product HAMC was easily assessed in real time with
fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths
differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product
of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was
found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450
isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 µM with only P450
1
2
v
A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19,
E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 µM. K and
max values of MAMC for P450 2D6 were found to be 26.2 ( 2.8 µM and 2.9 ( 0.07 min
value of 29.7 ( 6.2 µM and a vmax
m
-
1
,
respectively. For P450 1A2, MAMC was found to have a K
m
-
1
of 0.57 ( 0.07 min . Formation of HAMC in human liver microsomes could be completely
inhibited by quinidine, at a concentration of 0.5 µM selective for P450 2D6, and furafylline, at
a concentration of 30 µM selective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-
4
-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity
and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for
high-throughput screening purposes.
In tr od u ction
Presently, an increasing need exists in drug discovery
and development for highly selective P450 substrates,
which give rise to easily detectable products, preferably
suited for high-throughput screening. Until recently, only
HPLC and GC methods have been described for P450
Cytochrome P450 2D6 is a member of the P450
superfamily, which is absent in 5-9% of the Caucasian
population as a result of recessive inheritance of gene
mutations (1, 2). First discovered as the debrisoquine/
sparteine polymorphism, this absence results in deficien-
cies in the metabolism of numerous drugs. Deficiencies
in metabolism can lead to a number of problems, such
as undesirably high plasma levels of the drug and
concomitant side effects or lack of activation of a prodrug
2
D6 model substrates such as bufuralol, dextromethor-
phan, debrisoquine, sparteine, and metoprolol. These
assays are all time-consuming and therefore not suitable
for high-throughput screening purposes. Recently, a
fluorescence microplate reader-based method for high-
throughput screening was described by Crespi et al. (4).
This method is based on single expressed P450 isoen-
zymes with high catalytic activity. However, the sub-
strate used in this assay, i.e., 7-ethoxy-3-cyanocoumarin,
was not selective for P450 2D6 and had a low rate of
turnover.
(
3). It is, therefore, important to discover in an early stage
of drug development whether potential drug candidates
might be substrates of P450 2D6.
*
To whom correspondence should be addressed. Telephone:
31204447590. Fax: +31204447610. E-mail: vermeule@chem.vu.nl.
+
1
0.1021/tx980248q CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/18/1999

5
56 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Communications
Several small molecule and homology models have
been developed for P450 2D6 (reviewed in ref 5). One
derived by Koymans et al. (6) and extended and refined
by De Groot et al. (7, 8) suggests that substrates of P450
2
D6 attach with a basic nitrogen moiety to the carboxy-
late group of aspartate 301, that oxidation occurs at 5 or
Å from this basic nitrogen moiety, and that the
7
substrates contain a flat usually aromatic region coplanar
to the oxidation site.
In this study, this small molecule model of P450 2D6
substrates was used to design a new and selective P450
2
D6 substrate which would give rise to a fluorescent
metabolite and be suitable for high-throughput screening.
This computer-predicted substrate was synthesized and
characterized for substrate selectivity in microsomes of
F igu r e 1. Fluorescence spectra of 7-methoxy-4-(aminomethyl)-
coumarin (MAMC) and 7-hydroxy-4-(aminomethyl)coumarin
1
0 different heterologously expressed human P450s and
(
HAMC) at a concentration of 100 µM in 0.1 M potassium
human liver microsomes.
phosphate buffer at pH 7.4 and 37 °C: (A) excitation spectrum
of MAMC (at an emission wavelength of 396 nm), (B) excitation
spectrum of HAMC (at an emission wavelength of 470 nm), (C)
emission spectrum of MAMC (at an excitation wavelength of
Ma ter ia ls a n d Meth od s
3
26 nm), and (D) emission spectrum of HAMC (at an excitation
Ma ter ia ls. 7-Methoxy-4-(bromomethyl)coumarin, resorcinol,
-chloroacetoacetate ethyl ester, and quinidine were obtained
wavelength of 370 nm).
4
from Aldrich (Zwijndrecht, The Netherlands). Furafylline was
obtained from RBI (Natick, MA). NADPH was obtained from
Applichem (Darmstadt, Germany). Microsomes of lymphoblas-
toid cells overexpressing P450 1A2, 2A6, 2B6, 2E1, 2D6, and
Syn t h esis of 7-H yd r oxy-4-(a m in om et h yl)cou m a r in
HAMC). At 0 °C, 50 mL of sulfuric acid (98%) was added to
1.1 g of resorcinol and 18.5 g of 4-chloroacetoacetate ethyl ester
while the mixture was being stirred for 45 min. The mixture
was left stirring for 18 h at 20 °C and subsequently was poured
over ice. The progress of the reaction was followed by silica TLC
with 9/1 acetone/25% ammonium hydroxide as the mobile phase.
(
1
3
A4 were obtained from GENTEST (Woburn, MA). Microsomes
containing P450 2C8, 2C9, 2C18, and 3A5 and human liver
microsomes were a kind gift from P. Beaune (INSERM U75,
Paris, France).
The product (R
f
) 0.5) was detected by UV (256 or 366 nm).
Molecu la r Mod elin g of 7-Meth oxy-4-(a m in om eth yl)-
The product 7-hydroxy-4-(chloromethyl)coumarin was recrystal-
1
cou m a r in (MAMC). Generation of the initial geometry of the
1
lized from methanol, and its identity was established by
NMR: 1H NMR (DMSO-d
) δ 4.90 (s, 2H, CH ), 6.40 (s, 1H, d
CH), 6.70 (m, 2H, Ar), 7.75 (d, 1H, Ar).
H
structure as well as a full conformational analysis was carried
out with the molecular mechanics modeling package Macro-
Model (5.0) (9). The resulting conformations were subsequently
minimized with the BatchMin facility (10) using the AMBER
force field. Further optimization of the lowest-energy conforma-
tion resulting from the conformational search was performed
with the Amsterdam Density Functional (ADF) program version
6
2
7-Hydroxy-4-(chloromethyl)coumarin (1 g) was added under
anaerobic conditions to 50 mL of 25% ammonium hydroxide.
The resultant yellow solution was stirred under nitrogen for 60
min at 50 °C. The progress of the reaction was followed by silica
TLC with 9/1 acetone/25% ammonium hydroxide as the mobile
2
.3 using the double-z basis set (11). The fitting procedure was
essentially as described previously (8) and was carried out with
the molecular modeling program Chem-X 1998 (12). The energy
difference between the fitted and lowest-energy conformation
was determined by means of a single-point calculation with
ADF.
f
phase. The product (R ) 0.3) was detected by UV (256 or 366
nm) and by ninhydrin reactivity. After completion of the
reaction, the mixture was acidified with 6 N hydrochloric acid
and filtered. The filtrate was gently neutralized with 2 N sodium
hydroxide, and the product was filtered off as a beige solid. After
the product (460 mg) had been dried, its identity was established
Syn th esis of MAMC. 7-Methoxy-4-(bromomethyl)coumarin
1
1
by H NMR: H NMR (DMSO-d
6 2
) δ 3.95 (s, 2H, CH ), 6.35 (s,
(500 mg) was added to 50 mL of acetone and 2 mL of 25%
1
H, dCH), 6.85 (m, 2H, Ar), 7.55 (d, 1H, Ar). HAMC exhibited
ammonium hydoxide. The resulting colorless solution was
stirred at room temperature for 60 min. The progress of the
reaction was followed by silica TLC with acetone as the mobile
a fluorescence excitation maximum at 370 nm and a fluorescence
emission maximum at 470 nm (Figure 1).
phase. The product with an R
or 366 nm) and by ninhydrin reactivity. After completion of the
reaction, the yellow reaction mixture was acidified to pH 6 with
f
of 0.6 was detected by UV (256
Micr osom a l MAMC In cu ba tion s. Incubations were per-
formed at 37 °C in a Shimadzu RF-5000 spectrofluorometer with
the excitation wavelength set at 405 nm (5 nm slit) and emission
set at 480 nm (10 nm slit). These wavelength settings deviated
slightly from the excitation and emission maxima of the
metabolic product in order to eliminate background fluorescence
from the parent substrate and NADPH.
6
N hydrochloric acid and the acetone was evaporated. The
product was extracted at pH 10 with ethyl acetate and taken
up in 2-propanol. To this solution 10 drops of concentrated
hydrochloric acid was added, and the product crystallized
pentane as a hydrochloric acid salt (beige plates). The identity
NADPH (10 µL of a 1 mM solution) and 10 µL of the
microsomal fraction were added to 970 µL of 0.1 M potassium
phosphate buffer (pH 7.4) containing 0.4 mM EDTA. After
equilibration at 37 °C, 10 µL of 2.5 or 25 mM MAMC in DMSO
was added and the real-time increase in fluorescence was
recorded. After following the reaction for several minutes, 10
µL of 10 µM HAMC in buffer was added and the reaction rate
was quantified from the resulting increase in fluorescence. The
reaction rate remained constant for at least 20 min. In the
experiments with the inhibitors quinidine and furafylline, 10
µL of solutions with concentrations of 50 µM (in water) and 3
mM (in DMSO) was added, respectively. Furafylline was added
5 min prior to addition of MAMC.
1
of the product (350 mg, >95% yield) was established by H NMR
and with GC/MS after derivatization with acetic acid anhy-
1
dride: H NMR (DMSO-d
6
) δ 3.85 (s, 3H, CH
3
O), 4.40 (s, 2H,
2
CH ), 6.40 (s, 1H, dCH), 7.00 (m, 2H, Ar), 7.70 (d, 1H, Ar); GC/
+
•
+
MS (relative intensity) m/z 247 ([M ], 53%), 205 ([M - Od
+
CdCH
(
2
], 100%), 190 ([M - HNCOCH
2
], 22%), 176 (51%), 162
44%), 161 (53%). MAMC exhibited a fluorescence excitation
maximum at 326 nm and a fluorescence emission maximum at
3
7
96 nm (Figure 1).
1Abbreviations: HAMC, 7-hydroxy-4-(aminomethyl)coumarin; MAMC,
-methoxy-4-(aminomethyl)coumarin.

Communications
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 557
F igu r e 2. Metabolism of MAMC to its fluorescent metabolite
HAMC by P450 2D6.
Incubations were performed in the presence of microsomes
from yeast or lymphoblastoid cells selectively expressing P450
isoenzymes (10 pmol of P450/incubation) and human liver
microsomes.
HP LC An a lysis. MAMC and its O-demethylated metabolite
HAMC were separated on a 4.6 mm × 25 cm Phenomenex
Spherex 5 C18 column with 25% methanol/1% triethylamine (pH
3
) as the mobile phase. The flow rate was set at 0.4 mL/min.
Compounds were detected using a Shimadzu RF 530 fluores-
cence detector with excitation set at 370 nm and emission set
at 470 nm. Under these conditions, HAMC and MAMC were
found to have retention times of 8.4 and 13.5 min, respectively.
Resu lts a n d Discu ssion
F igu r e 3. Overlay of debrisoquine (green), dextromethorphan
This study was aimed at the development of a selective
P450 2D6 substrate which would give rise to a fluorescent
metabolite. Because of its fluorescent properties, the
coumarine skeleton was chosen as a starting point.
(
blue), and 7-methoxy-4-(aminomethyl)coumarin (MAMC, red)
in the small molecule model for P450 2D6 derived by Koymans
et al. (6) and extended and refined by De Groot et al. (7, 8). The
red arrow points to the site of oxidation. The green arrow points
to the interaction site between the basic nitrogen moiety of the
substrate and aspartate 301 of the enzyme.
7
-Hydroxycoumarine has fluorescent properties which
strongly differ from those of 7-alkoxycoumarines; thus,
the site of oxidation of the potential substrate should
preferably be the R-carbon of an alkoxycoumarine. Since
all selective, high-turnover P450 2D6 substrates known
to date contain a basic nitrogen moiety 5-7 Å from their
site of oxidation, an amino-containing substituent was
added to the coumarin skeleton, to fulfill this require-
ment. The small molecule model derived by Koymans et
al. (6) and extended and refined by De Groot et al. (7, 8)
was used to predict where such a moiety would have to
be placed to achieve oxidation at the R-carbon of an
alkoxycoumarin. A methylamine moiety at the 4-position
of the coumarine ring was proposed to direct the site of
oxidation to the R-carbon of alkoxycoumarins (see Figure
2
for the chemical structures of MAMC and its fluorescent
metabolic HAMC).
As can be seen in Figure 3, MAMC fits well in the
refined substrate model according to the criteria previ-
ously defined. The difference between the energy level
of the lowest-energy conformation of MAMC and the
fitted conformation was 7.94 kcal/mol. The fit distances
between the CR and Câ atoms of the aspartic acid residue
coupled to MAMC and the two template molecules were
F igu r e 4. O-Demethylation of MAMC by microsomes of
heterologously expressed human P450s at 25 and 250 µM (n )
3
). P450 1A2, 2A6, 2B6, 2E1, 2D6, and 3A4 were obtained from
0
0
.130 and 0.163 Å for dextromethorphan and 0.224 and
.430 Å for debrisoquine, respectively, which satisfy the
GENTEST. P450 2C8, 2C9, 2C18, and 3A5 were a kind gift from
P. Beaune. nd means not detectable.
fit criteria of the model of 0.5 Å. In addition, MAMC
contains a flat aromatic region coplanar to the oxidation
site.
Incubations were performed at 37 °C in a Shimadzu
RF-5000 spectrofluorometer with a crystal cuvette, and
the increase in fluorescence from incubations was mea-
sured in real time, analogous to the well-known assay of
ethoxyresorufin O-deethylation as a marker for P450
extent 1A2 exhibited activity at a substrate concentration
of 25 µM (Figure 4). The increase in fluorescence in
incubations of P450 2D6-containing microsomes with 250
µM MAMC and 0.1 mM NADPH was found to be linear
for at least 20 min. MAMC was found to have a K
m
value
of 26.2 ( 2.8 µM and a vmax of 2.9 ( 0.07 min- for P450
2D6. MAMC was found to have a K value of 29.7 ( 6.2
1
m
-
1
1
A1/2 activity. MAMC proved to be a relatively selective
P450 2D6 substrate. In a series of microsomes containing
0 different human P450s, only P450 2D6 and to a lesser
µM and a vmax of 0.57 ( 0.07 min for P450 1A2 (Table
1). Activity in microsomes containing P450 2D6 could be
completely inhibited by addition of 0.5 µM quinidine, a
1

5
58 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Communications
Ta ble 1. En zym e Kin etic P a r a m eter s of MAMC
Meta bolism by Releva n t Hu m a n Cytoch r om e P 450
O-demethylation activity of human liver microsomes
exhibit an excellent correlation to their respective vmax
/
a
Isofor m s
K
m
values. At a concentration of 250 µM, other P450
P450 isoform
Km (µM)
vmax (min-1)
isoforms also metabolize MAMC (Figure 4); therefore, we
recommend that MAMC be used in human liver microso-
mal preparations at a concentration near its K value
m
1
2
A2
D6
29.7 ( 6.2
26.2 ( 2.8
0.57 ( 0.07
2.9 ( 0.07
a
for P450 2D6.
n ) 3.
m
HPLC analysis of incubations of 100 µM MAMC (4K )
incubated for 30 min with P450 2D6-containing mi-
crosomes and stopped with 0.3 M trichloroacetic acid
clearly showed the formation of HAMC as the single
metabolite. The formation rate of HAMC (3.5 ( 0.51
-
1
min ) was linear over 30 min and could be completely
inhibited by addition of quinidine (Figure 5). HPLC
analysis of the rate of formation of HAMC indicated that
the entire real-time increase in fluorescence could be
ascribed to the formation of HAMC.
Recently, a high-throughput screening method was
developed for several human P450s (4). In this assay,
7
-ethoxy-3-cyanocoumarin was used as a substrate for
P450 2D6. However, although useful, 7-ethoxy-3-cyano-
coumarin does not appear to be a selective, high-turnover
substrate for P450 2D6 as can be derived from the
substrate model for 2D6 (8). MAMC might easily be
incorporated in this high-throughput method by replacing
7
-ethoxy-3-cyanocoumarin.
In conclusion, MAMC is a highly selective P450 2D6
substrate. Due to its strong fluorescence, 7-hydroxy-4-
aminomethyl)coumarin (HAMC), the O-demethylated
(
metabolite of MAMC, can be quantified directly in the
incubation mixture with negligible interference from
substrate or NADPH fluorescence. In human liver mi-
crosomes, only P450 2D6 and 1A2 contribute to the
O-demethylation of MAMC. After preincubation with the
selective P450 1A2 inhibitor furafylline, all MAMC
O-demethylation activity in human liver microsomes can
be ascribed to P450 2D6. Thus, the O-demethylation of
MAMC may provide a rapid and easy tool for investigat-
ing drug-drug interactions in vitro.
F igu r e 5. Overlay of representative HPLC traces of the
O-demethylation of 100 µM MAMC by P450 2D6 (100 pmol/
mL): (A) HAMC peak of a 0 min incubation, (B) HAMC peak of
a 30 min incubation, and (C) HAMC peak of a 30 min incubation
in the presence of 10 µM quinidine. The positions of the parent
compound MAMC and the inhibitor quinidine are also indicated.
Refer en ces
(
1) Mahgoub, A., Idle, J . R., Dring, L. G., Lancaster, R., and Smith,
R. L. (1977) Polymorphic hydroxylation of Debrisoquine in man.
Lancet II (8038), 584-586.
2) Eichelbaum, M., and Gross, A. S. (1990) The genetic polymor-
phism of debrisoquine/sparteine metabolism: Clinical Aspects.
Pharmacol. Ther. 46, 377-394.
well-known and selective inhibitor of P450 2D6 (13).
Activity in microsomes containing P450 1A2 could be
completely inhibited by preincubation for 5 min with 30
µM furafylline, a well-known and selective inhibitor of
P450 1A2 (13). In human liver microsomes (n ) 3;
obtained from P. Beaune and designated as A1, A8, and
A11), MAMC O-demethylation activity (25 µM MAMC)
was inhibited by 33.9 ( 8.9% by 0.5 µM quinidine and
by 53.4 ( 3.9% by preincubation for 5 min with 30 µM
furafylline. Residual activity after preincubation with 30
µM furafylline could be completely inhibited by addition
of 0.5 µM quinidine. These observations indicate that in
human liver microsomes, MAMC is exclusively metabo-
lized by P450 2D6 and 1A2. Although P450 1A2 has a
much lower vmax value for MAMC O-demethylation than
(
(3) Tucker, G. T. (1994) Clinical implications of genetic polymorphism
in drug metabolism. J . Pharm. Pharmacol. 46, 417-424.
(
4) Crespi, C. L., Miller, V. P., and Penman, B. W. (1997) Microtiter
plate assays for inhibition of human, drug-metabolizing cyto-
chromes P450. Anal. Biochem. 248, 188-190.
(5) De Groot, M. J ., and Vermeulen, N. P. E. (1997) Modeling the
active sites of cytochrome P450s and glutathione S-transferases,
two of the most important biotransformation enzymes. Drug
Metab. Rev. 29, 747-799.
(6) Koymans, L. M. H., Vermeulen, N. P. E., Van Acker, S. A. B. E.,
Te Koppele, J . M., Heykants, J . J . P., Lavrijsen, K., Meuldermans,
W., and Donn e´ -Op den Kelder, G. M. (1992) A predictive model
for substrates of cytochrome P450-debrisoquine (2D6). Chem. Res.
Toxicol. 5, 211-219.
2
D6, due to the higher level of 1A2 (12.7 ( 6.2% of the
(7) De Groot, M. J ., Bijloo, G. J ., Hansen, K. T., and Vermeulen, N.
P. E. (1995) Computer prediction and experimental validation of
cytochrome P4502D6-dependent oxidation of GBR 12909. Drug
Metab. Dispos. 23, 667-669.
(8) De Groot, M. J ., Bijloo, G. J ., Martens, B. J ., Van Acker, F. A. A.,
and Vermeulen, N. P. E. (1997) A refined substrate model for
human cytochrome P450 2D6. Chem. Res Toxicol. 10, 41-48.
total P450) compared to 2D6 (1.5 ( 1.3% of the total
P450) in human liver (14), the contribution of 1A2 to the
overall MAMC O-demethylation in human liver mi-
crosomes is significant. In fact, when their respective
microsomal levels are taken into account, the observed
contributions of P450 1A2 and 2D6 to the overall MAMC
(9) Department of Chemistry, Columbia University (1995) Macro-
model, version 5.0, Columbia University, New York.

Communications
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 559
(
10) Department of Chemistry, Columbia University (1995) BatchMin,
version 4.0, Columbia University, New York.
organic solvents for the measurement of human cytochrome P450
monooxygenase activities. Drug Metab. Dispos. 26, 207-215.
14) Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich,
F. P. (1994) Interindividual variations in human liver cytochrome
P-450 enzymes involved in the oxidation of drugs, carcinogens
and toxic chemicals: studies with liver microsomes of 30 J apanese
and 30 Caucasians. J . Pharmacol. Exp. Ther. 270, 414-423.
(
11) Department of Theoretical Chemistry, Vrije Universiteit Amster-
dam (1997) ADF, version 2.3, Vrije Universiteit Amsterdam,
Amsterdam.
(
(12) Chemical Design Ltd. (1998) ChemX, version J anuary 1998,
Chemical Design, Ltd.
(13) Hickman, D., Wang, J .-P., Wang, Y., and Unadkat, J . D. (1998)
Evaluation of the selectivity of in vitro probes and suitability of
TX980248Q