Bioactivation of fluorinated 2-aryl-benzothiazole antitumor molecules by human cytochrome P450s 1A1 and 2W1 and deactivation by cytochrome P450 2S1

Article abstract of DOI:10.1021/tx3001994

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 5-fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (GW 610) contain the benzothiazole pharmacophore and possess potent and selective in vitro antitumor properties. Prior studies suggested the involvement of cytochrome P450 (P450) 1A1 and 2W1-mediated bioactivation in the antitumor activities and P450 2S1-mediated deactivation of 5F 203 and GW 610. In the present study, the biotransformation pathways of 5F 203 and GW 610 by P450s 1A1, 2W1, and 2S1 were investigated, and the catalytic parameters of P450 1A1- and 2W1-catalyzed oxidation were determined in steady-state kinetic studies. The oxidations of 5F 203 catalyzed by P450s 1A1 and 2W1 yielded different products, and the formation of a hydroxylamine was observed for the first time in the latter process. Liquid chromatography-mass spectrometry (LC-MS) analysis with the synthetic hydroxylamine and also a P450 2W1/5F 203 incubation mixture indicated the formation of dGuo adduct via a putative nitrenium intermediate. P450 2W1-catalyzed oxidation of GW 610 was 5-fold more efficient than the P450 1A1-catalyzed reaction. GW 610 underwent a two-step oxidation process catalyzed by P450 1A1 or 2W1: a regiospecific O-demethylation and a further hydroxylation. Glutathione (GSH) conjugates of 5F 203 and GW 610, presumably through a quninoneimine and a 1,2-quinone intermediate, respectively, were detected. These results demonstrate that human P450s 1A1 and 2W1 mediate 5F 203 and GW 610 bioactivation to reactive intermediates and lead to GSH conjugates and a dGuo adduct, which may account for the antitumor activities of 5F 203 and GW 610 and also be involved in cell toxicity. P450 2S1 can catalyze the reduction of the hydroxylamine to the amine 5F 203 under anaerobic conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity.

Full text of DOI:10.1021/tx3001994

Article
pubs.acs.org/crt
Bioactivation of Fluorinated 2-Aryl-benzothiazole Antitumor
Molecules by Human Cytochrome P450s 1A1 and 2W1 and
Deactivation by Cytochrome P450 2S1
Kai Wang and F. Peter Guengerich*
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, School of Medicine, 638 Robinson Research
Building, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, United States
S
* Supporting Information
ABSTRACT: Both 2-(4-amino-3-methylphenyl)-5-fluoroben-
zothiazole (5F 203) and 5-fluoro-2-(3,4-dimethoxyphenyl)-
benzothiazole (GW 610) contain the benzothiazole pharma-
cophore and possess potent and selective in vitro antitumor
properties. Prior studies suggested the involvement of
cytochrome P450 (P450) 1A1 and 2W1-mediated bioactiva-
tion in the antitumor activities and P450 2S1-mediated
deactivation of 5F 203 and GW 610. In the present study,
the biotransformation pathways of 5F 203 and GW 610 by
P450s 1A1, 2W1, and 2S1 were investigated, and the catalytic
parameters of P450 1A1- and 2W1-catalyzed oxidation were
determined in steady-state kinetic studies. The oxidations of
5F 203 catalyzed by P450s 1A1 and 2W1 yielded different
products, and the formation of a hydroxylamine was observed
for the first time in the latter process. Liquid chromatography−
mass spectrometry (LC-MS) analysis with the synthetic
hydroxylamine and also a P450 2W1/5F 203 incubation
mixture indicated the formation of dGuo adduct via a putative
nitrenium intermediate. P450 2W1-catalyzed oxidation of GW
610 was 5-fold more efficient than the P450 1A1-catalyzed
reaction. GW 610 underwent a two-step oxidation process
catalyzed by P450 1A1 or 2W1: a regiospecific O-demethylation and a further hydroxylation. Glutathione (GSH) conjugates of
5F 203 and GW 610, presumably through a quninoneimine and a 1,2-quinone intermediate, respectively, were detected. These
results demonstrate that human P450s 1A1 and 2W1 mediate 5F 203 and GW 610 bioactivation to reactive intermediates and
lead to GSH conjugates and a dGuo adduct, which may account for the antitumor activities of 5F 203 and GW 610 and also be
involved in cell toxicity. P450 2S1 can catalyze the reduction of the hydroxylamine to the amine 5F 203 under anaerobic
conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity.
reported that the expression level of P450 2W1 is an
independent prognostic factor in colon cancer, with high
expression levels associated with poor clinical outcome.10 The
functions of these two P450s are still not well understood,
although P450 2W1 has been shown to have activity toward
INTRODUCTION
■
P450 enzymes catalyze the oxidation and reduction of a variety
of drugs, carcinogens, steroids, lipids, and other organic
molecules.1,2 The Human Genome Project has identified 57
P450 genes.3 About one-fourth are still classified as “orphan”
P450s because their functions remain largely unknown.4 P450s
2W1 and 2S1 remain in this category, and a number of
carcinogens were shown to be activated by P450 2W1 but not
P450 2S1 when heterologously expressed in this laboratory.5
The only repeatedly demonstrated reaction of P450 2S1 to date
is the reduction of a di-N-oxide, AQ4N, to its amino derivative
AQ4 [1,4-bis([2-(dimethylamino)ethyl]amino)-5,8-dehydrox-
yanthracene-9,10-dione] under anaerobic conditions.6,7 P450
2W1 has been found to be overexpressed in tumor cells,
especially in colon tumor cells, with only low or undetectable
expression levels in other tissues.8,9 Prior studies have also
some drugs and lysophospholipids.5 33
,
In the screening of 2-arylbenzothiazoles for anticancer
activity, a number of molecules have been discovered with
novel anticancer mechanisms, 2-(4-amino-3-methylphenyl)-5-
fluorobenzothiazole (5F 203) and 5-fluoro-2-(3,4-dimethox-
yphenyl)-benzothiazole (GW 610) being two promising drug
candidates.11 5F 203 showed antiproliferative potency in
sensitive human breast and ovarian cancer cell lines (GI50 < 1
Received: May 1, 2012
Published: June 26, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of GW 610, 5F 203, and Their Putative Metabolites
nM for MCF-7, MDA 468, and IGROV-1 cells) and emerged as
a lead compound, currently in phase 1 clinical trials in the
United Kingdom.12,13 The results of coincubation of 2-4(-
amino-3-methyl-phenyl)benzothiazole (DF 203, an anticancer
agent discovered earlier, yielding inactive metabolites that led
to the discovery of 5F 20312) with α-naphthoflavone, a P450
1A1 inhibitor, showed inhibition of anticancer activity and
revealed a role for P450 1A1 in the anticancer activity of the
benzothiazoles.14 The involvement of P450 1A1 was reported
for 5F 203 bioactivation (to generate reactive intermediates to
form protein and DNA adducts) in the antitumor process
within sensitive cells.13,15 P450 1A1 is expressed in fetal liver
and in some extrahepatic adult human tissues but not at
appreciable levels in adult liver.16,17 5F 203 acts as both a
substrate and an inducer of P450 1A1, the latter process
involving binding to the aryl hydrocarbon receptor.18
Although P450 1A1 has been shown to be involved in the
bioactivation of 5F 203, the products are still uncharacterized.
In a prior study of the metabolism of 5F 203 in human B-
lymphoblastoid cells expressing P450 1A1, 1B1, and 1A2,
respectively,13 P450 1A1 exhibited the highest level of
metabolic capacity. However, no chemical structures of the
metabolites were determined.
reported on the metabolism of GW 610. GW 610,19 like 5F
203,18 can induce P450 1A1 in breast cancer cell lines (MCF-7
and MDA 468). However, P450 1A1 was neither highly
expressed nor could be induced in the sensitive colon cancer
cell lines KM12 and HCC2998. The conclusion was reached
that the antitumor properties of GW 610 were unlikely to
depend upon P450 1A1-mediated bioactivation alone.
Demethylation was proposed as an initial mechanistic step in
the antitumor process,11 although no evidence was provided.
The suggestion has been made that P450 1A1-mediated
metabolism of 5F 203 and GW 610 is involved in their
anticancer activities.20 In addition, P450 2W1-mediated
bioactivation was concluded to be essential for GW 610
activation in colon cancer cells.20 In contrast, 5F 203 and GW
610 showed enhanced growth inhibition effects in P450 2S1
knock-down cancer cells; that is, P450 2S1 deactivated these
two anticancer molecules.20
The biotransformation of GW 610 and 5F 203 by P450 1A1,
2W1, and 2S1 plays a critical role in their potent and selective
antitumor properties, as well as in any toxicity associated with
these molecules. Knowledge of the biotransformation pathways
is important in understanding the antitumor activity and
improvement of drug efficacy. In this article, we report results
on the biotransformation pathways of GW 610 and 5F 203
mediated by P450s 1A1, 2W1, and 2S1, characterization of the
major oxidation products, and evidence for GSH conjugates
and dGuo adduct formation from the reactive products.
More recently, GW 610, a dimethoxyphenyl benzothiazole
derivative, was discovered to exhibit potent (GI50 < 0.1 nM)
and selective in vitro inhibitory activity against colon, lung, and
breast cancer cell lines.19 Even less information has been
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1
with H2O to afford 7 as a pale yellow solid (12 mg, 75%). H NMR
(400 MHz, CDCl3): δ 8.87 (brs, 2H, NH), 8.18 (dd, J = 11.0, 2.8 Hz,
2H, H-3), 8.01 (d, J = 8.4 Hz, 2H, H-5′), 7.73 (s, 2H, H-2′), 7.53 (dd, J
= 8.4, 1.8 Hz, 2H, H-6′), 7.47 (dd, J = 8.6, 6.2 Hz, 2H, H-6), 6.67 (td, J
= 8.1, 2.8 Hz, 2H, H-5), 2.68 (s, 6H, CH3).
EXPERIMENTAL PROCEDURES
■
All commercially obtained solvents and other chemicals were used
directly without further purification. 2-Amino-5-fluorobenzothiazole
and 2-phenylbenzothiazole were purchased from Alfa Aesar (Ward
Hill, MA). 3-Methyl-4-nitrobenzoic acid was purchased from Sigma
Aldrich (St. Louis, MO). One- and two-dimensional NMR spectros-
copy data were obtained with 400 or 600 MHz Bruker NMR
spectrometers, in the solvents noted, in the Vanderbilt facility. UV
spectra were obtained (online) with a Waters Acquity ultra
performance liquid chromatography (UPLC) system equipped with
a photodiode array detector (Waters, Milford, MA).
2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (8, 5F 203).
Compound 8 was synthesized as reported elsewhere.12 To a solution
of concentrated HCl (1 mL), C2H5OH (2 mL), and H2O (0.2 mL)
was added the disulfide 7 (12 mg) and SnCl2·2H2O (80 mg). The
resulting mixture was stirred and heated under reflux for 15 h and
allowed to cool, and then, H2O (7.5 mL) and NaOH (0.40 g) were
added slowly. The mixture was stirred for 1 h, after which the
precipitate was collected and washed with H2O (5 mL). Flash
chromatography (CH2Cl2, 100%) afforded 8 as a pale yellow solid (6.9
Chemical Synthesis (Scheme 1). Bis(aminofluorophenyl)
Disulfide (2). The title compound was prepared using a known
procedure.12 2-Amino-5-fluorobenzothiazole (1) (0.125 g, 0.74 mmol)
was added to a solution of KOH (0.625 g) in H2O (4 mL), and the
resulting mixture was heated to reflux. After 3 h, a clear solution was
formed, and the solution was stirred under reflux for another 2 h. The
solution was cooled to room temperature and acidified to pH 6 by the
addition of CH3CO2H. H2O (1 mL) was added to the mixture, which
was then stirred overnight. The solid precipitate was collected and
purified by flash column chromatography on silica gel
(CH2Cl2:hexanes, 3:1, v/v). Compound 2 was obtained as a pale
1
mg, 68%). UV λmax 347 nm. H NMR (400 MHz, CDCl3): δ 7.83 (s,
1H, H-2′), 7.79−7.72 (m, 2H, H-7, H-6′), 7.69 (dd, J = 9.7, 2.5 Hz,
1H, H-4), 7.09 (td, J = 8.8, 2.5 Hz, 1H, H-6), 6.73 (d, J = 8.3 Hz, 1H,
H-5′), 2.25 (s, 3H, CH3). HRMS m/z calcd for C14H12FN2S (MH+),
259.0700; found, 259.0689.
2-(3-Methyl-4-nitrophenyl)-5-fluorobenzothiazole (9). To a sol-
ution of the disulfide 7 (29 mg, 0.048 mmol) in toluene (3 mL) were
added triphenylphosphine (12.7 mg, 0.048 mmol) and p-toluenesul-
fonic acid monohydrate (1.9 mg, 9.7 μmol). The resulting mixture was
heated under reflux for 24 h. The solvent was removed in vacuo, and
flash column chromatography gave 9 as a pale yellow solid (23 mg,
1
yellow solid (0.077 g, 73% yield). H NMR (400 MHz, CDCl3): δ
7.05 (dd, J = 8.5, 6.5 Hz, 2H, H-6), 6.41 (dd, J = 10.5, 2.6 Hz, 2H, H-
3), 6.29 (td, J = 8.5, 2.6 Hz, 2H, H-5), 4.47 (br s, 4H, NH2).
General Method for the Synthesis of GW 610 and Its
Monodemethylated Analogues, 4a−c. Compounds 4a−c were
synthesized by using a previously reported method.11 Each
disubstituted benzaldehyde (3a−c; 0.20 mmol), p-toluenesulfonic
acid monohydrate (0.040 mmol), and triphenylphosphine (0.20
mmol) was added to a solution of 2 in toluene (5 mL). The resulting
mixture was stirred and heated under reflux for 24 h. After each
mixture was cooled to room temperature, the solvent was removed in
vacuo. Flash chromatography (CH2Cl2, 100%) afforded corresponding
4a−c as white solids in yields of 68−87%.
5-Fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (4a, GW 610).
Compound 4a was prepared from 2 and veratraldehyde (68% yield):
UV λmax 331 nm. 1H NMR (400 MHz, CDCl3): δ 7.80 (dd, J = 8.8, 5.1
Hz, 1H, H-7), 7.73 (dd, J = 9.6, 2.3 Hz, 1H, H-4), 7.72 (d, J = 2.3 Hz,
1H, H-2′), 7.60 (dd, J = 8.4, 2.1 Hz, 1H, H-6′), 7.14 (td, J = 8.8, 2.5
Hz, 1H, H-6), 6.96 (d, J = 8.4 Hz, 1H, H-5′), 4.03 (s, 3H, OCH3-3′),
3.97 (s, 3H, OCH3-4′). HRMS m/z calcd for C15H13FNO2S (MH+),
290.0646; found, 290.0643.
1
83%). UV λmax 323 nm. H NMR (400 MHz, CDCl3): δ 8.10 (d, J =
8.6 Hz, 1H, H-5′), 8.09 (s, 1H, H-2′), 8.02 (dd, J = 8.4, 1.8 Hz, 1H, H-
6′), 7.88 (dd, J = 8.8, 5.0 Hz, 1H, H-7), 7.80 (dd, J = 9.3, 2.5 Hz, 1H,
H-4), 7.22 (td, J = 8.8, 2.5 Hz, 1H, H-6), 2.72 (s, 3H, CH3). HRMS
m/z calcd for C14H10FN2O2S (MH+), 289.0442; found, 289.0429.
2-(4-Hydroxylamino-3-methylphenyl)-5-fluorobenzothiazole (10)
and 2-(3-Methyl-4-nitrosophenyl)-5-fluorobenzothiazole (11). To a
solution of 9 (1.4 mg, 4.9 μmol) in THF (3 mL) was added Pd/C
(10%, catalyst) and hydrazine hydrate (20 μL) under Ar.21 The
reaction mixture was stirred at room temperature. After 2 h, the
reaction was complete. The solvent was removed in vacuo, and the
residue was purified by flash chromatography to afford 10 as a yellow
solid (1.1 mg, 83%) (note: the solubility of 10 in CHCl3 and CH3OH
1
is very poor). UV λmax 339 nm. H NMR (400 MHz, DMSO-d6): δ
8.68 (s, 1H), 8.57 (s, 1H), 8.11 (dd, J = 8.7, 5.4 Hz, 1H, H-7), 7.85 (d,
J = 8.5 Hz, 1H, H-6′), 7.80 (dd, J = 8.8, 2.4 Hz, 1H, H-4), 7.75 (s, 1H,
H-2′), 7.31 (td, J = 8.8, 2.4 Hz, 1H, H-6), 7.20 (d, J = 8.4, 1H, H-5′),
2.19 (s, 3H, H3). HRMS m/z calcd for C14H12FN2OS (MH+),
275.0649; found, 275.0642.
5-Fluoro-2-(3-hydroxy-4-methoxyphenyl)-benzothiazole (4b).
Compound 10 was then dissolved in C2H5OH (100 μL), to which
was added a solution of K3Fe(CN)6 (20 mg, 60 μmol).22 The resulting
mixture was stirred at room temperature for 3 h and then diluted with
H2O (2 mL). The white precipitate (11) was collected by filtration.
UV λmax 362 nm. HRMS m/z calcd for C14H10FN2OS (MH+),
273.0492; found, 273.0496.
Compound 4b was prepared from 2 and 3-hydroxy-4-dimethoxy-
benzaldehyde (87% yield): UV λmax 331 nm. H NMR (400 MHz,
1
CDCl3): δ 7.79 (dd, J = 8.8, 5.1 Hz, 1H, H-7), 7.72 (dd, J = 9.6, 2.5
Hz, 1H, H-4), 7.65 (m, 2H, H-2′, H-6′), 7.13 (td, J = 8.8, 2.5 Hz, 1H,
H-6), 6.95 (d, J = 9.0 Hz, 1H, H-5′), 5.73 (br s, 1H, OH-3′), 3.98 (s,
3H, OCH3-4′). HRMS m/z calcd for C14H11FNO2S (MH+), 276.0489;
found, 276.0487.
5-Fluoro-2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (4c).
Compound 4c was prepared from 2 and vanillin (83% yield): UV
λmax 331 nm. 1H NMR (400 MHz, CDCl3): δ 7.79 (dd, J = 8.8, 5.1 Hz,
1H, H-7), 7.72 (d, J = 2.1 Hz, 1H, H-2′), 7.71 (dd, J = 10.2, 2.1 Hz,
1H, H-4), 7.53 (dd, J = 8.3, 2.0 Hz, 1H, H-6′), 7.13 (td, J = 8.8, 2.5 Hz,
1H, H-6), 7.01 (d, J = 8.3 Hz, 1H, H-5′), 5.95 (br s, 1H, OH-4′), 4.04
(s, 3H, OCH3-3′). HRMS m/z calcd for C14H11FNO2S (MH+),
276.0489; found, 276.0479.
3-Methyl-4-nitrobenzoyl Chloride (6). 3-Methyl-4-nitrobenzoic
acid (0.60 g), thionyl chloride (3 mL), and DMF (0.05 mL) were
heated under reflux for 4 h, cooled, and concentrated in vacuo. The
residue was slurried twice with hexanes and concentrated to yield 6 as
a yellow solid, which was used immediately to the next reaction.
Bis[4-fluoro-2-(3-methyl-4-nitrobenzoyl)aminophenyl] Difulfide
(7). Compound 7 was synthesized as reported previously.12
Compound 6 (25 mg, 0.125 mmol) was added to a solution of 2
(15 mg, 0.053 mmol) in anhydrous pyridine (3 mL). The reaction
mixture was stirred and heated under reflux for 30 min and then
poured into H2O (15 mL). The precipitate was collected and washed
Enzyme Preparations. Human P450s 1A1 and 2W1 were
coexpressed with NADPH-P450 reductase, and P450s 2S1 and 2W1
were expressed in Escherichia coli membranes as previously
described.5,23 Human P450 2S1 and 2W1 were purified as described.5
Human liver microsomes were prepared from 10 randomly chosen
human liver donor samples using previously reported procedures.24
Incubation Conditions. A typical incubation mixture for qualitative
studies contained P450 enzyme (0.3−0.8 μM) in bacterial membranes
or liver microsomes, an NADPH-generating system (10 mM glucose
6-phosphate, 0.5 mM NADP+, and 4 μg mL−1 yeast glucose 6-
phosphate dehydrogenase) and the substrate (100 μM) in 0.10 M
potassium phosphate buffer (pH 7.4).24 When a purified P450 was
used, it was generally reconstituted with a 2-fold excess of NADPH-
P450 reductase and 30 μM L-α-dilauroy-sn-glycero-3-phosphocho-
line.5,7,24,33 The incubations were performed at 37 °C for 30−60 min.
CH2Cl2 (2-fold volume) was added to terminate the reaction, and the
resulting mixture was separated by centrifugation at 2 × 103g for 5 min.
The organic layer was transferred and dried under an N2 stream. The
residue was dissolved in CH3CN for HPLC and liquid chromatog-
raphy−mass spectrometry (LC-MS) analysis.
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Large-scale reactions (total volume of 50−100 mL) were performed
to obtain sufficient amounts of metabolites for NMR analysis. The
incubation conditions were the same as in the qualitative studies
except that catalase (4 μg mL−1, Sigma Aldrich, from bovine
erythrocytes, dialyzed before use to remove thymol) was also present
in the mixture. The reactions were terminated by addition of CH2Cl2
(2-fold volume). The resulting mixtures were centrifuged at 2 × 103g
for 5 min, and the lower layer of each was concentrated using a rotary
evaporator. A minimum amount of CH3CN was added to dissolve the
residue, which was subsequently subjected to preparative HPLC
isolation (vide infra).
Anaerobic Incubations. Anaerobic reduction experiments were
performed in Thunberg tubes, with the samples deaerated with the use
of a manifold, using alternating cycles of argon and vacuum (20
mmHg) (five cycles). A typical 500 μL incubation mixture contained
0.5 μM P450 2S1, 1.0 μM NADPH-P450 reductase, 160 μM L-α-1,2-
dilauoryl-sn-glycero-3-phosphocholine, 100 mM potassium phosphate
buffer (pH 7.4), and 100 μM 10 (from a freshly prepared 10 mM
DMSO stock). After 5 min of preincubation, an NADPH-generating
system was added from the neck of the tube under anaerobic
conditions. The reactions were incubated at 37 °C for 45 min,
terminated by the addition of 1 mL of CH3CN, and analyzed by
HPLC.
Steady-State Kinetic Measurements of P450 Reactions. The
incubation conditions for kinetic studies were similar to those
employed in the qualitative studies. The concentrations of P450 and
the NADPH-generating system remained the same. Reactions with a
series of different substrate concentrations ranging from 0 to 200 μM
were carried out, in duplicate, at 37 °C for 10 and 15 min for GW 610
with P450s 2W1 and 1A1, respectively, and for 15 and 25 min for 5F
203 with P450s 2W1 and 1A1, respectively. After the reactions were
quenched with CH2Cl2 (2-fold volume), a small amount of internal
standard (5-fluoro-2-(3-hydroxy-4-methoxyphenyl)-benzothiazole, 4b,
5 μL, 0.1 mM for GW 610 reactions; 2-phenylbenzothiazole, 12, 5 μL,
0.1 mM for 5F 203 reactions) was added to each reaction mixture. The
mixture was centrifuged at 2 × 103g for 5 min, and the organic layer
was transferred and dried under an N2 stream. The residue was
dissolved in CH3CN for HPLC analysis, from which the P450 enzyme
kinetic data (Km and kcat) were calculated using nonlinear regression
analysis (Prism, GraphPad Software, La Jolla, CA).
GSH Adduct Formation from GW 610 and 5F 203 Oxidation
Products. A typical incubation mixture (total volume of 0.50 mL)
contained P450 enzyme (2W1, 1A1, or microsomes, 0.50 μM) in
bacterial membranes or liver microsomes, an NADPH-generating
system (10 mM glucose 6-phosphate, 0.5 mM NADP+, 4 μg mL−1
yeast glucose 6-phosphate dehydrogenase), catalase (4 μg mL−1), GSH
(5 mM), and the substrate (100 μM) in 0.10 M potassium phosphate
buffer (pH 7.4). The incubations were performed at 37 °C for 12−15
h. CH3CN (2-fold volume of incubation mixture) was added to
terminate the reaction, and the supernatant was analyzed by LC-MS.
Formation of dGuo Adduct with Synthetic 10 and P450
2W1-Catalyzed Oxidation of 5F 203. With Synthetic 10. To a
freshly prepared solution of 10 in CH2Cl2 (10 μM, 1.0 mL), 20 μL of
Ac2O was added. The mixture was swirled for 5 min, the solvent was
removed in vacuo, and 30 μL of CH3CN was added. The CH3CN
solution (5 μL) was added to a solution of 100 mM potassium
phosphate buffer (pH 7.4) containing 0.1 mM EDTA (245 μL),
followed by the addition of aqueous dGuo (250 μL of a 10 mM
solution). The resulting mixture was incubated at 37 °C under argon
for 2 h and analyzed by LC-MS.
resulting mixture was incubated at 37 °C under argon for 2 h and
analyzed by LC-MS.
HPLC-UV Assays. HPLC analysis was performed on a Waters
Acquity UPLC system equipped with two pumps, an autosampler, and
a photodiode array detector (Waters). Samples for qualitative and
kinetic studies were injected onto an octadecylsilane (C18) reversed-
phase column (5 μm, 2.1 mm × 250 mm, Hypersil GOLD, Thermo
Scientific, Odessa, TX). The column was maintained at room
temperature. The eluting solvents used were mobile phase A
(CH3CN:H2O, 5:95, 0.5% HCO2H, v/v) and mobile phase B
(CH3CN:H2O, 95:5, 0.5% HCO2H, v/v). The flow rate was 0.5 mL
min−1. The gradient conditions for all of the qualitative and kinetic
studies (except the kinetic analysis of the transformation of GW 610)
started from 5% B (v/v) during the first 2 min (0−2 min) and was
increased to 100% B over 5 min (2−7 min), maintained at that level
for 4.5 min (7−11.5 min), and then returned to 5% B (v/v) over 0.5
min (11.5−12 min) before equilibration (12−15 min). 5-Fluoro-2-(3-
hydroxy-4-methoxyphenyl)-benzothiazole (4b) was used as the
internal standard in the kinetic analysis of the oxidation of GW 610.
The separation of the products, internal standard, and parent
compound was achieved using gradient conditions as follows: 30% B
(v/v) during the first 2 min (0−2 min), increased to 65% B (v/v) over
10 min (2−12 min), then decreased to 30% B (v/v) over 1 min (12−
13 min) before equilibration (13−15 min). Some products were
identified by comparison with their retention times, UV spectra, and
LC-MS with corresponding reference compounds (vide infra).
Isolation of metabolites was performed using a Hitachi L-7100
HPLC system (Tokyo, Japan) equipped with a single pump and UV
detector (LDC Spectro Monitor III Model 1204D, Thermo). Samples
(typical injection 100−150 μL) were injected onto a semipreparative
octadecylsilane (C18) reversed-phase column (5 μm, 10 mm × 250
mm, Prodigy, Phenomenex, Torrance, CA). The column was
maintained at room temperature. The eluting solvents used were the
same as used for analytical HPLC analysis, mobile phase A
(CH3CN:H2O, 5:95, 0.5% HCO2H, v/v) and mobile phase B
(CH3CN:H2O, 95:5, 0.5% HCO2H, v/v). The flow rate was 4 mL
min−1. Isocratic elution (65% B, v/v) was used for isolation of 4c′. The
gradient for isolation of 8a and 8b started from 60% B (v/v), was
increased to 70% B (v/v) over 10 min (0−10 min), and then returned
to 60% B (v/v) in 2 min (10−12 min) before equilibration (12−20
min). The fractions containing products of interest were collected, and
CH3CN was removed under a stream of N2. The resulting mixture was
extracted with CH2Cl2 three times, and the combined organic layers
were dried over Na2SO4. CH2Cl2 was removed under a stream of N2,
and the residue was dissolved in CDCl3 for NMR analysis.
LC-MS Analysis. LC/MS and LC/MS/MS analyses were performed
on Finnigan LTQ ion trap mass spectrometers (Thermo Fisher
Scientific, San Jose, CA). The samples were analyzed in the
electrospray ionization (ESI) positive ion mode. A Waters Acquity
UPLC system was coupled with the mass spectrometer. An
octadecylsilane (C18) reversed-phase column (5 μm, 2.1 mm × 250
mm, Hypersil GOLD, Thermo Scientific) was used and was
maintained at room temperature in the analyses. The eluting solvents
used were mobile phase A (CH3CN:H2O, 5:95, 0.5% HCO2H, v/v)
and mobile phase B (CH3CN:H2O, 95:5, 0.5% HCO2H, v/v). The
flow rate was 0.5 mL min−1. The gradient conditions were the same as
described above: 5% B (v/v) during the first 2 min (0−2 min), and
increased to 100% B (v/v) in 5 min (2−7 min), maintained at that
level for 4.5 min (7−11.5 min), and then returned to 5% B (v/v) in 0.5
min (11.5−12 min) before equilibration (12−15 min). MS conditions
(N2 gas flow, spray current, voltages, etc.) were tuned to give
maximum sensitivity for the parent compound.
With the P450 2W1-Catalyzed Oxidation of 5F 203. 5F 203 was
incubated with P450 2W1 membranes under the usual incubation
conditions at 37 °C for 45 min (total volume, 0.5 mL), the reaction
was quenched by the addition of 1 mL of CH2Cl2 and mixed with a
vortex device, and the organic layer was transferred. Ac2O (20 μL) was
added to the CH2Cl2 solution, which was swirled for 5 min and dried
under an N2 stream. CH3CN (5 μL) and 100 mM potassium
phosphate buffer (pH 7.4) containing 0.1 mM EDTA (245 μL) were
added to the residue. The contents were mixed with a vortex device
and added to a 10 mM aqueous solution of dGuo (250 μL). The
LC-HRMS was performed on a Waters Acquity UPLC system
coupled with a Waters Synapt hybrid quadropole/OA-TOF mass
spectrometer (Waters). The samples were analyzed in the ESI positive
ion mode. The MS conditions were tuned to give maximum sensitivity
for the parent compound. The LC conditions were the same as in the
LC-MS studies (vide supra).
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the tR 5.55 min peak as a single product (Figure 1F). LC-MS
analysis indicated that the peak had an m/z of 292 (MH+,
Figure S9 in the Supporting Information), 2 Da greater than
GW 610 and 16 Da greater than 4c, suggesting that it might be
a secondary oxidation metabolite. A sufficient amount of 4c′
was obtained from a large-scale incubation of 4c with P450
RESULTS
■
Incubations of 2-Phenylbenzothiazole, GW 610, and
5F 203 with P450 2W1 and 2S1: Preliminary Assays. No
products were observed following the incubation of 2-
phenylbenzothiazole with reconstituted systems containing
recombinant human P450 2W1 or 2S1. In addition, no
products were observed by HPLC-UV following the incubation
of either GW 610 or 5F 203 with P450 2S1. Products were
formed in the incubation of GW 610 or 5F 203 with P450 2W1,
as judged by HPLC-UV assays.
Hydroxylation and Demethylation of GW 610 by
P450s 2W1 and 1A1. An HPLC peak appeared at retention
time (tR) 6.05 min in the incubation of GW 610 (tR 6.55 min)
with P450 2W1 (Figure 1B). Further LC-MS analysis showed
1
2W1 for NMR analysis. In the 1D H NMR spectrum of 4c′
(Figure S2 in the Supporting Information), one high field
proton signal disappeared in the aromatic region and the two
doublet proton signal (in 4c) was replaced with two singlet
proton signals (in 4c′ at 7.27 and 7.34 ppm), suggesting
substitution at 5′ position. The proton coupling pattern on the
fluorine-containing ring was unchanged [2D total correlation
(NMR) spectroscopy (TOCSY) results, Figure 2], supporting
this conclusion. Compound 4c′ was assigned the structure
shown in Scheme 2, based on both the LC-MS and the NMR.
Figure 2. TOCSY spectrum of 4c′ in CDCl3 (600 MHz).
Oxidation of 5F 203 by P450 2W1, 1A1, and Human
Liver Microsomes. Two product peaks appeared at tR 5.45
min (8a, UV λmax 343 nm) and 6.02 min (10, UV λmax 339 nm)
following incubation of 5F 203 (tR 6.31 min, UV λmax 347 nm)
with P450 2W1 (Figure S7A in the Supporting Information).
LC-MS results showed that both peaks had an m/z 275 ion
([MH]+), 16 Da greater than the parent compound 5F 203,
very likely corresponding to oxidation products. Two product
peaks also appeared at tR 5.45 min (8a, UV λmax 343 nm)
(which was present in the P450 2W1 oxidation as well) and tR
5.80 min (8b, UV λmax 356 nm) from the incubation of 5F 203
(tR 6.31 min) with P450 1A1 (Figure S7B in the Supporting
Information). Interestingly, the HPLC results for the
incubation of 5F 203 with human liver microsomes showed
all three major products (8a, 8b, and 10). LC-MS showed that
all three peaks had m/z 275. Arylamines are known to undergo
oxidation to form hydroxylamines and nitroso and nitro
compounds,25 in addition to hydroxylation on the aromatic
rings. The hydroxylamine (10), nitroso (11), and nitro (9)
derivatives of 5F 203 were readily synthesized. The tR values of
10, 11, and 9 were 6.04, 7.43, and 7.11 min, respectively, under
the same conditions. The tR and the UV spectrum of synthetic
10 were identical to those of oxidation product 10 found in the
5F 203 incubation with P450 2W1. A mass signal (m/z 273,
[MH]+) corresponding to the nitroso derivative (11) was
observed, consistent with the tR (7.49 min) in LC-MS.
However, no signal for the nitro derivative (9) was detected.
Figure 1. HPLC chromatograms of GW 610 oxidation products
formed with P450s 1A1 and 2W1. (A) Incubation products of GW
610 formed with P450 1A1 (45 min). (B) Incubation products of GW
610 formed with P450 2W1 (45 min). (C) Chemically synthesized 4c.
(D) Synthetic 4b. (E) Incubation products of GW 610 formed with
P450 2W1 (60 min). (F) Incubation products of 4c formed with P450
2W1 (60 min).
that the peak had m/z 276 (Figure S9 in the Supporting
Information) possibly corresponding to a demethylated
product. To determine the structure of the product, two
chemicals were synthesized as potential products (4b and 4c,
Scheme 1A). Comparison of synthetic 4b and 4c with the
incubation mixture using HPLC showed cochromatography
with 4c (tR 6.05 min, Figure 1C), whereas 4b was eluted
slightly earlier (tR 5.98 min, Figure 1D) under the same
conditions. The tR 6.05 min peak was also the predominant
peak obtained from the incubation of GW 610 with P450 1A1
(Figure 1A).
Using more P450 2W1 and a longer incubation time, a new
peak appeared at tR 5.55 min (Figure 1E). In a separate
experiment, 4c was incubated with P450 2W1 and also yielded
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Scheme 2. P450 2W1, P450 1A1, and Microsomal GW 610 Reactions
Scheme 3. P450 2W1, P450 1A1, and Microsomal 5F 203 Reactions
Compound 10 was also detected following incubation of 5F
203 with P450 1A1, and 8b was detected as well in incubations
of 5F 203 with P450 2W1, respectively, both as minor products
(Figure S10 in the Supporting Information). A large-scale
incubation of 5F 203 with human liver microsomes provided
sufficient amounts of 8a and 8b for NMR spectra [the
instability and poor solubility of 10 in common organic solvents
(e.g., CHCl3, CH2Cl2, and CH3OH) complicated the use of
NMR for comparison]. In the 1D 1H NMR of 8a (Figure S3 in
the Supporting Information), the retention of the two pairs of
doublet signals at δ 7.84 and 6.76 ppm and the disappearance
of a singlet proton signal clearly indicated the presence of a
nonhydrogen substitutent at 2′-position. Additionally, the
homonuclear correlated (NMR) spectroscopy (COSY) NMR
spectrum of 8a (Figure S3 in the Supporting Information)
showed an unambiguous coupling relationship between the two
pairs of doublet signals at δ 7.84 and 6.76 ppm, providing
further evidence for 2′-substitution. Given the LC-MS results,
the structure 8a was proposed, that is, a 2′-hydroxy derivative of
5F 203 (Scheme 3). In the 1H NMR spectrum of 8b (Figure S4
in the Supporting Information), the proton signal splitting
pattern was similar to that of 4c′, where one high field aromatic
proton signal disappeared and two singlet proton signals
replaced the original doublets in the precursors. On the basis of
the LC-MS and NMR data available, the structure 8b was
assigned, that is, the 5′-hydroxy product of 5F 203 (Scheme 3).
Steady-State Kinetic Data for P450 2W1 and 1A1
Oxidation Reactions. Steady-state kinetic results for P450
2W1- and 1A1-catalyzed oxidation reactions with GW 610 and
5F 203 are summarized in Table 1, including the formation of
4c from GW 610 catalyzed by P450 2W1 and 1A1, the
formation of 8a and 10 from 5F 203, and the formation of 8a
Table 1. Steady-State Kinetics of P450 2W1- and 1A1-
Catalyzed Reactions
kcat/Km
substrate P450 product Km (μM)
kcat (min−1
)
(μM−1 min−1
)
2W1
1A1
4c
4c
8a
10
8a
8b
22
10
7
3
3.8 0.4
0.18 0.05
0.036 0.01
0.030 0.01
GW 610
0.37 0.03
0.95 0.1
0.56 0.06
1.7 0.2
31 11
35 12
2W1
1A1
0.016 0.005
0.054 0.02
0.11 0.03
5F 203
32
29
9
8
3.1 0.3
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Figure 3. LC-MS and LC-MS/MS chromatograms and CID spectra showing the presence of a GSH conjugate of GW 610 formed with P450 2W1.
(A) Extracted ion chromatogram of m/z 597. (B) LC-MS spectrum of the peak at tR 4.48 min. (C) Product ion chromatogram of m/z 597. (B) LC-
MS/MS spectrum of the peak at tR 4.45 min.
Figure 4. LC-MS and LC-MS/MS chromatograms and CID spectra showing the presence of a GSH conjugate of 5F 203 formed with P450 2W1.
(A) Extracted ion chromatogram of m/z 580. (B) LC-MS spectrum of the peak at tR 4.62 min. (C) Product ion chromatogram of m/z 580. (B) LC-
MS/MS spectrum of the peak at tR 4.60 min.
and 8b from 5F 203. P450 2W1 was 5-fold more efficient than
P450 1A1 in the formation of 4c from GW 610. In contrast,
P450 1A1 was ∼2-fold more efficient than P450 2W1 in terms
of oxidation of 5F 203 to form 8a. In addition, while P450 2W1
catalyzed 5F 203 to yield the hydroxylamine 10, P450 1A1
oxidized 5F 203 to 8b much more efficiently.
GSH Adduct Formation from GW 610 and 5F 203
Oxidation Products. A peak at tR ∼ 4.5 min appeared in total
ion chromatograms (ESI+) of all LC-MS analyses for incubation
mixtures of GW 610 with P450 2W1 (Figure 3A), P450 1A1, or
human liver microsomes (data not shown) in the presence of
GSH. The MS of that peak (m/z 597, Figure 3B) was
consistent with the structure 4g (the GSH conjugate of 4c′),
and another significant signal in the MS spectrum (m/z 299,
Figure 3B) was determined to be a doubly charged GSH
conjugate ([M + 2H]+), in that its fragmentation pattern was
found to be identical that of the peak (m/z 597) (data not
shown). The LC-MS/MS of the peak at tR ∼ 4.5 min (Figure
3C) gave two major fragment ions: m/z 468 (loss of 129) and
324 (loss of 273), commonly seen for GSH conjugates (Figure
3D).
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Figure 5. LC-MS/MS and LC-MS3 chromatograms and CID spectra showing a dGuo adduct of an oxidation product of 5F 203 formed with P450
2W1. (A) Extracted ion chromatogram of m/z 408. (B) LC-MS/MS spectrum of the peak at tR 4.63 min. (C) Total ion chromatogram of CID of the
m/z 408 product ion. (D) LC-MS3 spectrum of the peak at tR 4.61 min.
Similarly, the LC-MS total ion chromatograms for reactions
of 5F 203 with P450 2W1 and GSH (Figure 4A), as well as
P450 1A1 or human liver microsomes (data not shown) in the
presence of GSH, all showed a peak at tR ∼ 4.6 min under the
same conditions as for GW 610. The peak had m/z 580 (Figure
4B), consistent with 8g (the GSH conjugate of 8b), and the
predominant signal in the MS spectrum (m/z 290.6, Figure 4B)
was also proven to be a doubly charged GSH conjugate ([M +
2H]+) upon comparison of its fragmentation pattern with the
peak (m/z 580) (data not shown). LC-MS/MS analysis of the
peak (m/z 580, Figure 4C) again showed two major fragment
ions: m/z 451 (loss of 129) and 307 (loss of 273), providing
supportive evidence for the formation of a GSH conjugate
(Figure 4D).
Formation of a dGuo Adduct with Synthetic 10 and a
P450 2W1-Catalyzed Oxidation of 5F 203. The analyses of
samples of dGuo incubated with synthetic 10 and the oxidation
product of 5F 203 formed by P450 2W1 (using LC-MS in the
positive ion mode) both showed a peak at tR 4.6 min with m/z
524 (MH+), which is consistent with a dGuo adduct of
compound 10. From the reaction of 5F 203 P450 2W1
incubation products with dGuo, CID of the peak at tR 4.6 min
(Figure 5A, positive ion mode) yielded a characteristic
transition of m/z 524 → 408 (Figure 5B), with a loss of 116,
very typical of dGuo adducts. LC-MS3 of m/z 408 (Figure 5C)
generated a fragment ion with m/z 391 (loss of NH3) as the
major peak (Figure 5D), consistent with the proposed
structure, providing evidence for the presence of a dGuo
adduct. The LC-MS/MS and LC-MS3 analyses of the sample of
synthetic 10 with dGuo showed identical fragmentation results
(Figure S11 in the Supporting Information) with those found
for 5F 203/P450 2W1 incubation products reacting with dGuo.
Anaerobic Reduction of 10 Catalyzed by P450 2S1.
Freshly prepared 10 was incubated with P450 2S1 under
anaerobic conditions (Figure 6A) and normal aerobic
conditions (Figure 6B), in parallel. The peaks appearing at tR
6.02, 6.33, and 7.41 min correspond to 10, 5F 203, and 11,
respectively. When authentic 10 was incubated in 100 mM
potassium phosphate buffer (pH 7.4) containing 0.1 mM
EDTA for 45 min, varying amounts of 11 (Figure 6A-b,B-b)
were formed by oxidation. More 11 was formed under aerobic
conditions. In contrast, 5F 203 was produced from the
reduction of 10 under both anaerobic and aerobic conditions
with P450 2S1 in the presence of NADPH-P450 reductase and
an NADPH-generating system (Figure 6A-c,B-c, respectively).
More 5F 203 was formed under anaerobic conditions. With
only the NADPH-generating system deleted, oxidation of 10 to
11 occurred under both anaerobic (Figure 6A-d) and areobic
conditions (Figure 6B-d), and unidentified products were also
formed, especially under areobic conditions (tR 7.90 min and
others, Figure 6B-d). With 10, NADPH-P450 reductase, and
the NADPH-generating system in buffer (but in the absence of
P450 2S1), oxidation yielded only barely observable amounts of
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Figure 6. Reduction of 10 by P450 2S1. Freshly prepared 10 (5 μL of a 10 mM solution in DMSO) was incubated with P450 2S1 (total volume 0.5
mL) at 37 °C for 45 min under anaerobic (A) and normal aerobic conditions (B). (a) Standard 10. (b) Standard 10 in 100 mM potassium
phosphate buffer (pH 7.4) containing 0.1 mM EDTA, incubated at 37 °C for 45 min. (c) Standard 10 incubated with P450 2S1 in the presence of
NADPH-P450 reductase and an NADPH-generating system. (d) Standard 10 incubated with all typical components of the P450 2S1 system with
the exception of the NADPH-generating system. (e) Standard 10 incubated with all P450 system components with the exception of P450 2S1.
Scheme 4. GSH Conjugates of GW 610 and 5F 203 Oxidation Products
11 under both anaerobic (Figure 6A-e) and normal aerobic
conditions (Figure 6B-e).
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nitrenium ions and formation of dGuo adducts and thus
alleviating the damage caused by 5F 203. These results can
explain the role of P450 2S1 in attenuating the antitumor
activity of 5F 203, a conclusion reached only on the basis of
knock-down experiments.20 It is worth noting that NADPH-
P450 reductase appeared to be able to reduce the nitroso
derivative 11 (to 10) both anaerobically and aerobically,
consistent with similar observations in a previous study of the
nitroso derivative of 2-amino-3-methylimidazo[4,5-f ]-
quinoline.32
In conclusion, the biotransformation pathways of the
antitumor agents GW 610 and 5F 203 involving human
P450s 1A1, 2W1, and 2S1 were studied, and the chemical
structures of major products were determined. The high
catalytic efficiency of P450 2W1 for oxidation of GW 610 may
explain the potent and selective antitumor activity to colon
cancer cells. The hydroxylamine 10 was identified, for the first
time, as a major oxidation product of 5F 203 formed by P450
2W1, which can further react with dGuo to form an adduct
(Figure 5). P450 2S1 can catalyze the reduction of 10 back to
5F 203 under anaerobic conditions and (to a lesser extent)
aerobic conditions, thus resulting in attenuating the anticancer
activity by decreasing the production of nitrenium ions and
thus the level of the dGuo adduct and probably other DNA
adducts. Oxidation products of GW 610 and 5F 203 can form
GSH conjugates, presumably via quinone and quinoneimine
intermediates, which may lead to their pharmacological and
possible toxicological consequences.
DISCUSSION
■
In this study, we characterized the P450-mediated bioactivation
of GW 610 and 5F 203 to form reactive intermediates, which
may lead to their antitumor activity within sensitive tumor
cells.13,20 Our results clearly demonstrate that, in addition to
P450 1A1, P450 2W1 is also involved in the biotransformation
of GW 610. Consistent with a previous hypothesis,11 O-
demethylation is the first step of the process, and it occurred in
a regiospecific manner to give 4c as the single initial product.
Furthermore, steady-state kinetic studies indicated that P450
2W1 has a 5-fold higher catalytic efficiency toward GW 610 as
compared to P450 1A1. This result may explain the observation
in a previous study that the addition of the P450 1A1 inhibitor
resveratrol26 did not affect the potency of GW 610 in sensitive
cell lines,19 because P450 2W1 could also contribute to its
metabolism. The result may also explain the potent selective
antitumor activity of GW 610 in human colon cancer cells,
where P450 2W1 was found to be selectively expressed.8,9
Further oxidation of 4c leads to the formation of a catechol, 4c′,
which can be readily oxidized to an o-quinone and react with
GSH (Scheme 4).
5F 203 can be oxidized by P450 1A1 as well as 2W1, and the
products differ. The two major products of P450 1A1-catalyzed
oxidation of 5F 203 were 8a and 8b, both formed by
hydroxylations on one aromatic ring. While 8a was also one
of the two major products formed by P450 2W1, another
product was the hydroxylamine 10, which had been previously
proposed as a putative 5F 203 bioactivation product but not
characterized.27,28 A compound analogous to 10 (with no F and
methyl groups) was shown to be able to bind to purines and
pyrimidines via a nitrenium ion intermediate28,29 (the
mechanism for the formation of C8-substituted dGuo-arylamine
adduct was reported previously30). To our knowledge, our in
vitro results constitute the first evidence of the formation of 10
in the bioactivation of 5F 203, and indeed, LC-MS results
provide the evidence for the formation of dGuo adduct with
activated 10 (Figure 5). The nitroso derivative (11) was also
detected in LC-MS (Figure S10 in the Supporting Informa-
tion).
Among the 2-aryl-benzothiazoles tested in this study, all of
the substrates of P450 2W1 contain at least one hydrogen-
bonding donor (−NH2, 5F203), acceptor (−OCH3, GW 610),
or both (−OCH3, −OH, 4c). In contrast, the 2-phenyl-
benzothiazole, without a hydrogen-bonding donor or acceptor
as a substituent on the aromatic ring, is not a substrate for P450
2W1.
The formation of GSH conjugates is usually considered as
evidence for chemically reactive intermediates, often associated
with drug toxicity and potential adverse drug reactions.31 The
GW 610 GSH conjugate is presumably formed via a 1,2-
quninone intermediate (Scheme 4A), whereas the 5F 203 GSH
conjugate is presumably formed via a quninoneimine (Scheme
4B). The hydroxylamine generated in the biotransformation of
5F 203 could react with dGuo to produce an adduct via a
nitrenium ion intermediate, in a similar way as shown in studies
using a 5F 203 analogue28,29 or other carcinogenic arylamines.25
Neither GW 610 nor 5F 203 was a substrate for P450 2S1,
that is, no products were observed from the incubations.
However, as shown in the incubation of hydroxylamine 10 with
P450 2S1, reduction of 10 to 5F 203 occurred under both
anaerobic and aerobic conditionsmore so under anaerobic
conditions (Figure 6A-c)decreasing the production of
ASSOCIATED CONTENT
■
S
* Supporting Information
Selective NOE NMR spectra of GW 610, 1H NMR spectrum of
4c′, 1H NMR and HH COSY spectra of 8a, 1H NMR spectrum
of 8b, UV spectra of GW610, 4b, 4c, and 4c′, HPLC
chromatograms of incubations of GW 610 with P450 2W1
using 4b as internal standard, HPLC chromatograms of
incubations of 5F 203 with P450s 2W1 and 1A1, HPLC
chromatograms and UV spectra of synthetic 10, 11, and 9, LC-
MS chromatograms and spectra of 4c and 4c′, LC-MS
chromatograms of 5F 203 oxidation products catalyzed by
P450s 2W1 and 1A1, and LC-MS/MS and LC-MS3 chromato-
grams and CID spectra showing the presence of the dGuo
adduct formed with synthetic 10. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 615-322-2261. Fax: 615-322-4349. E-mail: f.guengerich@
vanderbilt.edu.
Funding
This work was supported in part by the U.S. Public Health
Service (F.P.G., R37 CA090426, P30 ES000267).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
5F 203, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole;
COSY, correlated (NMR) spectroscopy; ESI, electrospray
ionization; GW 610, 5-fluoro-2-(3,4-dimethoxyphenyl)-benzo-
thiazole; LC-MS, liquid chromatography−mass spectrometry;
NOE, nuclear Overhauser effect; TOCSY, total correlation
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