Methoxyflavone inhibitors of cytochrome P450

Article abstract of DOI:10.1007/s10870-007-9310-x

Cytochrome P450 enzymes are a superfamily of enzymes involved in the metabolism of endogenous compounds as well as xenobiotics. Due to the large number of reactions catalyzed by these enzymes and their importance in drug metabolism and carcinogenesis, they have been the focus of many studies over the years. Based on the knowledge that flavones are natural substrates of certain P450 enzymes (such as P450 1A2) involved in carcinogenesis, we have synthesized and studied a number of flavonoids as potential inhibitors of these enzymes. These compounds are structurally very similar to the natural flavone substrates of these enzymes but have methoxy substituents at various positions. Here we are reporting the synthesis, structural analysis, X-ray crystal structures, and preliminary inhibition studies of four methoxyflavones from this series. Crystallographic data: 2′-methoxyflavone, P-1, a = 7.2994(8) A, b = 8.3322(7) A, c = 10.8240(10) A, α = 97.905(8)°, β = 92.779(10)°, γ = 111.105(8)°, V = 604.9(1) A³; 3′-methoxyflavone, P2₁/n, a = 15.1313(16) A, b = 3.9699(4) A, c = 19.9454(16) A, β = 91.673(8)°, V = 1197.6(2) A³; 4′-methoxyflavone, P2₁/n, a = 16.451(12) A, b = 3.881(1) A, c = 19.529(16) A, β = 106.65(1)°, V = 1195.1(4) A³; 3′,4′- dimethoxyflavone, C2/c, a = 30.819(5) A, b = 4.0857(7) A, c = 26.100(3) A, β = 124.21(1)°, V = 2717.6(7) A³. Methoxyflavone Inhibitors of Cytochrome P450 Michael McKendall, Tasha Smith, Kien Anh, Jamie Ellis, Terri McGee, Maryam Foroozesh, Naijue Zhu and Cheryl L. Klein Stevens *This paper is a report of the synthesis, structural analysis, X-ray crystal structures, and preliminary inhibition studies of 2′-methoxyflavone, 3′-methoxyflavone, 4′-methoxyflavone, and 3′,4′-dimethoxyflavone. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10870-007-9310-x

J Chem Crystallogr (2008) 38:231–237
DOI 10.1007/s10870-007-9310-x
ORIGINAL PAPER
Methoxyflavone Inhibitors of Cytochrome P450
Michael McKendall Æ Tasha Smith Æ Kien Anh Æ Jamie Ellis Æ Terri McGee Æ
Maryam Foroozesh Æ Naijue Zhu Æ Cheryl L. Klein Stevens
Received: 28 August 2007 / Accepted: 19 November 2007 / Published online: 5 December 2007
Ó Springer Science+Business Media, LLC 2007
Abstract Cytochrome P450 enzymes are a superfamily of
enzymes involved in the metabolism of endogenous com-
pounds as well as xenobiotics. Due to the large number of
reactions catalyzed by these enzymes and their importance in
drug metabolism and carcinogenesis, they have been the
focus of many studies over the years. Based on the knowl-
edge that flavones are natural substrates of certain P450
enzymes (such as P450 1A2) involved in carcinogenesis, we
have synthesized and studied a number of flavonoids as
potential inhibitors of these enzymes. These compounds are
structurally very similar to the natural flavone substrates of
these enzymes but have methoxy substituents at various
positions. Here we are reporting the synthesis, structural
analysis, X-ray crystal structures, and preliminary inhibition
studies of four methoxyflavones from this series. Crystallo-
Introduction
Cytochrome P450 enzymes are a superfamily of hemo-
proteins involved in the metabolism of endogenous
compounds such as steroids, bile acids, and fatty acids, as
well as xenobiotics such as natural products, environmental
pollutants, and drugs [1–5]. The primary purpose of these
enzymes is to detoxify non-polar compounds through
oxidation into more polar and excretable ones [6]. The
metabolism process, however, may lead to the activation of
certain procarcinogenic compounds into their ultimate
carcinogenic forms capable of binding DNA and initiating
cancer [7, 8]. Cytochrome P450s 1A1, 1A2, 2A6, and 2B1
are important enzymes involved in the metabolism of a
number of important drugs and environmental chemicals
such as caffeine, acetaminophen, cigarette smoke compo-
nents, and polycyclic aromatic hydrocarbons. These
enzymes are also very important in the carcinogenesis
process [1, 9–15]. Flavonoids, which are phytochemicals
abundant in nature and human diet, often interact with
P450 enzymes, especially P450 1A2, as substrates or
reversible inhibitors [16, 17].
0
˚
graphic data: 2 -methoxyflavone, P-1, a = 7.2994(8) A,
˚
˚
b = 8.3322(7) A,c = 10.8240(10) A,a = 97.905(8)°,b =
3
0
˚
9
2.779(10)°, c = 111.105(8)°, V = 604.9(1) A ; 3 -meth-
˚
oxyflavone, P2 /n, a = 15.1313(16) A, b = 3.9699(4) A,
˚
1
3
˚
˚
c = 19.9454(16) A, b = 91.673(8)°, V = 1197.6(2) A ;
0
˚
-methoxyflavone, P2 /n, a = 16.451(12) A, b = 3.881
1
4
˚
˚
(
(
1) A, c = 19.529(16) A, b = 106.65(1)°, V = 1195.1
3
4) A ; 3 ,4 -dimethoxyflavone, C2/c, a = 30.819(5) A,
˚
0
0
˚
In this project we have synthesized a family of flav-
ones very similar to the natural flavone substrates of these
enzymes with additional structural features predicted to
make them into suicide (mechanism-based time depen-
dent) inhibitors. Some of the compounds synthesized are
isomeric and the differences in inhibitory activities will
be useful in the determination of structural requirements
for optimum selective irreversible inhibition. A well-
designed suicide inhibitor will be physiologically inert
until metabolized by the target enzyme, so these com-
pounds are potentially useful reagents for in vivo studies.
Due to their inherent properties, suicide inhibitors can be
used as anti-initiation agents as well as to disrupt gene
˚
˚
b = 4.0857(7) A, c = 26.100(3) A, b = 124.21(1)°, V =
3
717.6(7) A .
˚
2
Keywords Flavone ꢀ P450 inhibitors ꢀ X-ray crystal
structures
M. McKendall ꢀ T. Smith ꢀ K. Anh ꢀ J. Ellis ꢀ T. McGee ꢀ
M. Foroozesh ꢀ N. Zhu ꢀ C. L. K. Stevens (&)
Department of Chemistry, Xavier University of Louisiana,
New Orleans, LA 70125, USA
e-mail: cklein@xula.edu
123

2
32
J Chem Crystallogr (2008) 38:231–237
transcription and modify stages of promotion and pro-
gression [18–20].
mixture was refluxed for 2.5 h, poured over 75 mL of cru-
shed ice, stirred vigorously for 45 min, and left in the
refrigerator to crystallize overnight before filtration. The
products were purified by column chromatography using a
gradient of elution, recrystallized from ethanol and water,
Here we are reporting the synthesis, purification, struc-
tural analysis, and X-ray crystal structures of three isomeric
0
0
0
0
0
flavonoids, 2 -, 3 -, 4 -methoxyflavones, and the 3 ,4 -di-
methoxyflavone derivative as well as preliminary assay
results for the in vitro inhibition of human P450s 1A1,
1
13
and characterized by GC/MS, H, and C NMR.
1
A2, 2A6, and rat P450 2B1-dependent reactions.
0
2
-Methoxyflavone
O
O
The yield after purification was 30%. GC/MS showed
1
00% purity; m/z (%): 252 (75), 121 (100). H NMR
OCH
3
1
OCH
3
(DMSO-d6): d 3.92 (s, 3H), 6.92 (s, 1H), 7.13 (t, 1H), 7.22
O
O
(
d, 1H), 7.48 (t, 1H), 7.54 (t, 1H), 7.70 (d, 1H), 7.80 (t, 1H),
2
'-methoxyflavone
3'-methoxyflavone
1
3
7
5
1
1
.90 (d, 1H), and 8.04 (d, 1H). C NMR (DMSO-d6): d
5.89, 111.56, 112.43, 118.39, 119.83, 120.67, 123.06,
24.68, 125.26, 129.08, 132.82, 134.11, 155.85, 157.59,
60.55, and 177.07.
O
O
O
OCH
3
3
O
0
3
-Methoxyflavone
OCH
4
'-methoxyflavone
OCH
3
3',4'-dimethoxyflavone
The yield after purification was 51%. GC/MS showed 100%
1
purity; m/z (%): 252 (100). H NMR (DMSO-d6/CD Cl ): d
Experimental
2
2
3.87 (s, 3H), 6.95 (s, 1H), 7.12 (d, 1H), 7.45 (t, 2H), 7.54
Synthesis
(s, 1H), 7.60 (d, 1H), 7.69 (d, 1H), 7.75 (t, 1H), and 8.07
1
3
(
d, 1H). C NMR (DMSO-d6/CD Cl ): d 55.16, 107.05,
2
2
A variation of the method published by Ares et al. [21] was
used for the synthesis of our target compounds: 1.5 eq. of
potassium tert-butoxide (KOtBu) was dissolved in 50 mL of
dry tetrahydrofuran (THF) and cooled to 0–5 °C in an ice
bath. The starting material, 2-hydroxyacetophenone dis-
solved in THF was added dropwise, and the reaction mixture
was left to warm up to room temperature. After 30 min of
stirring at room temperature, it was cooled back to 0–5 °C
before the dropwise addition of 1.5 eq. of o-, m-, p-methoxy
111.30, 117.11, 118.12, 118.28, 123.30, 124.64, 125.02,
129.86, 132.46, 133.76, 155.58, 159.55, 162.26, and 177.05.
0
4 -Methoxyflavone
The yield after purification was 86%. GC/MS showed
1
[95% purity; m/z (%): 252 (100). H NMR (DMSO-d6/
CD Cl ): d 3.86 (s, 3H), 6.86 (s, 1H), 7.07 (d, 2H), 7.45
2
2
0
0
anisoyl chloride, or 3 ,4 -dimethoxy anisoyl chloride in order
0
(t, 1H), 7.63 (t, 1H), 7.67 (d, 1H), 7.96 (d, 2H), and 8.14
13
0
0
to synthesize the four target flavone derivatives, 2 -, 3 -, 4 -
(d, 1H). C NMR (DMSO-d6/CD Cl ): d 55.23, 105.32,
2
2
0
0
methoxy flavones, or 3 ,4 -dimethoxy flavone, respectively.
The reaction mixture was allowed to warm to room tem-
perature again and left to stir for 2 h before the addition of
another 1.5 eq. of KOtBu. It was then left to stir at room
temperature overnight. On day two, the reaction mixture was
refluxed for 12 h at 60–67 °C and left to stir at room tem-
perature overnight. On day 3, 3 N HCl was added dropwise
until pH of the solution was lowered to three. The solvent was
evaporated under vacuum, and the residue was dissolved in
dichloromethane. Extractions were performed using a 5%
sodium bicarbonate solution, followed by deionized water.
114.24, 118.00, 123.19, 124.63, 124.91, 127.84, 129.10,
129.46, 131.03, 131.38, 155.54, 161.99, 163.04, and
176.86.
0
0
3
,4 -Dimethoxyflavone
The yield after purification was 54%. GC/MS showed
1
95% purity; m/z (%): 282 (100). H NMR (DMSO-d6/
[
CD Cl ): d 3.86 (s, 3H), 3.90 (s, 3H), 6.89 (s, 1H), 7.03
2
2
(
(
d, 1H), 7.42 (t, 1H), 7.50 (s, 1H), 7.62 (d, 1H), 7.64
1
d, 1H), 7.75 (t, 1H), and 8.05 (d, 1H). C NMR (DMSO-
3
The organic layer was dried over anhydrous MgSO . The
4
d6/CD Cl ): d 55.42, 55.56, 105.55, 108.97, 111.18,
solvent was evaporated and the intermediate isolated. The
intermediate was dissolved in 40 mL of glacial acetic acid
before the addition of 0.37 eq. of sulfuric acid. This reaction
2
2
117.94, 119.52, 123.28, 124.57, 124.79, 133.44, 148.85,
151.75, 155.51, 162.52, and 176.90.
1
23

J Chem Crystallogr (2008) 38:231–237
233
Table 1 Crystal and refinement data for the methoxyflavone cytochrome P450 inhibitors
0
0
0
0
0
Compound
2 -Methoxyflavone
3 -Methoxyflavone
4 -Methoxyflavone
3 ,4 -Dimethoxyflavone
Dimensions/color (mm)
0.95 9 0.35 9 0.23
Yellow
0.65 9 0.12 9 0.06
Yellow
0.65 9 0.28 9 0.13
Yellow
1.00 9 0.18 9 0.08
Yellow
Solvent system
Chemical formula
Crystal system
Ethanol/acetone (1:2)
Ethyl acetate/acetone (1:1)
Methanol/acetone (1:3)
2-Propanol/acetone (1:2)
C
16 12
H O
3
C
16 12
H O
3
C
16 12
H O
3
17 14 4
C H O
Triclinic
P-1
Monoclinic
P2 /n
Monoclinic
P2 /n
Monoclinic
C2/c
Space group
1
1
Unit cell dimensions
˚
a (A)
7.2994(8)
8.3322(7)
10.8240(10)
97.905(8)
92.779(10)
111.105(8)
604.9(1)
2
15.1313(16)
3.9699(4)
19.9454(16)
90.
16.451(2)
3.881(1)
19.529(3)
90.
30.819(5)
4.0857(7)
26.100(3)
90.
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
91.673(8)
90.
106.65(1)
90.
124.21(1)
90.
3
˚
Volume (A )
1197.6(2)
4
1195.1(4)
4
2717.6(7)
8
Z
3
Density (calculated) (mg/m )
1.385
1.399
1.402
1.380
-1
)
Absorption coefficient (cm
0.95
0.96
0.96
0.98
h range for data collection (°)
Reflections measured
0.1 B h B 30
3,679
0.1 B h B 25
2,613
0.1 B h B 25
4,495
0.1 B h B 25
5,665
Independent reflections
3,509
2,172
3,852
2,383
Number of observed reflections
(
2,559
1,200
2,370
1,248
I [ 3r(I))
Rmerge
R factor
R
0.026
0.041
0.095
0.039
0.049
0.099
0.68
0.034
0.064
0.954
647764
0.054
0.123
1.51
0.042
0.119
1.98
2
w
R (on F )
Goodness of fit
CCDC deposit number
647765
647763
647762
3
˚
X-ray Crystallography
and sp geometry and a C–H bond length of 0.95 A. Full-
matrix least-squares refinements were on F with non-
hydrogen atoms refined anisotropically. Well behaved
Samples were recrystallized by slow evaporation of solvent
mixtures listed in Table 1. The structures were determined
by X-ray crystallographic techniques described below. All
data were measured at room temperature.
0
0
0
hydrogen atom positions in 2 -, 3 -, and 4 -methoxyflavone
derivatives were refined with isotropic temperature factors
fixed. Hydrogen atom positions and isotropic temperature
0
0
1
A single crystal of each compound was mounted sepa-
rately on an Enraf-Nonius CAD4 diffractometer with a
normal focus molybdenum target X-ray tube, a 1.3 mm
collimator, and a graphite crystal monochromator. All three-
dimensional intensity data were collected in the x:2h scan
mode. Reflections with I [ 3r(I) were considered observed
and were included in the refinements. The data were cor-
rected for Lorentz and polarization effects. Absorption as a
function of w was minimal and not corrected. Three standard
reflections measured every 2 h showed no significant change
in intensity. Symmetry equivalent reflections were averaged.
Structures were solved using SIR [22]. The hydrogen atom
factors in 3 ,4 -dimethoxyflavone were fixed.
Inhibition Assays
Purified human and rat P450 enzymes were purchased from
BD Biosciences (San Jose, CA).
P
P
1
Conventional reliability indices: R = ||F | - |F
o
c
||/ |F |, and
o
are observed
P
P
2
|) / w|F
2 ½
| ) , where F
R
w
= ( (|F
o
| - |F
c
o
o
and F
c
0
0
0
positions for the 2 -, 3 -, and 4 -methoxyflavone derivatives
2
and for 3 ,4 -dimethoxyflavone were calculated based on sp
2
and calculated structure factors, w = 1/r(F) , and where r(F) is the
0
0
estimated standard deviation in F
o
.
123

2
34
J Chem Crystallogr (2008) 38:231–237
Assay of 7-Alkoxyresorufin (7-Alkoxyphenoxazone)
Dealkylation in Rat, and Human Liver Microsomes
Data Analysis
The data obtained from these assays were analyzed by a
computer analysis method of the reaction progress curve in
the presence of various inhibitor concentrations and in the
absence of the inhibitor as the control run [26]. An equa-
tion describing product formation with respect to reaction
time in seconds was obtained for each inhibitor concen-
tration and the control by transferring the original data
from the spectrofluorimeter to a computer. Microsoft Excel
Program was used to curve fit the data obtaining the
parameters of the best-fit curves. Using these equations, the
percent remaining activity values were calculated by
comparing the fluorescent intensity in the presence of the
inhibitor to that of the control at 120 and 240 s.
The P450 1A1, 1A2, and 2B1 dependent activities were
assayed using ethoxyresorufin, methoxyresorufin, and ben-
zyloxyresorufin fluorescent substrates, respectively [23].
7
-Ethoxyresorufin O-deethylation (EROD), 7-methoxyres-
orufin O-demethylation (MROD), and 7-benzyloxyresorufin
O-debenzylation assays were performed and given below.
Potassium phosphate buffer (1,750 lL, 0.1 M, pH 7.6)
was placed in a 1.0 cm quartz cuvette, and 10 lL of
1
.0 mM MgCl , 15 lL of 1.0 mM 7-alkoxyresorufin
2
solution in Me SO (DMSO), 10 lL of corresponding P450
2
protein (&12 lg), and 15 lL of an inhibitor solution in
DMSO or pure DMSO (for the control) were added. The
reaction was initiated by the addition of 200 lL of NADPH
regeneration solution (see below). The final assay volume
was 2.0 mL. The production of 7-hydroxyresorufin anion
was monitored by a spectrofluorimeter at 530 nm excita-
tion and 585 nm emission, with a slit width of 2 nm. The
reaction was performed at 37 °C. A number of assays were
performed for each flavone at varying concentrations.
NADPH regenerating solution was prepared as follows:
Results and Discussion
Cytochrome P450 enzymes oxidatively metabolize a wide
variety of xenobiotic compounds such as drugs, natural
products, pesticides, carcinogens, and other environmental
chemicals [1–3, 6, 27], as well as endogenous compounds
such as prostaglandins [10], fatty acids [13], and steroids
[1, 27]. The various P450 enzymes exhibit substrate
specificity that depends on the identity and orientation of
active site amino acids [1]. Human P450 1A2 is involved in
the metabolism of a number of important drugs and envi-
ronmental chemicals such as caffeine, acetaminophen,
cigarette smoke components, and estradiol [1, 9, 11,
28–31]. The goal of this project was to synthesize and
assay a number of new potentially selective mechanism-
based inactivators of P450 1A2. In our previous studies, we
have established that a number of aromatic acetylenes are
selective suicide inhibitors of cytochrome P450-dependent
monooxygenases. It is possible to design isozyme selective
substrates, which after metabolism by the enzyme into
reactive species, regiospecifically bind to certain amino
acid residues of the active site leading to irreversible
modification and loss of the catalytic activity. Since to be
effective, the suicide inhibitors have to be metabolized by
the enzyme, they can be designed based on structural
similarities to known selective substrates of the target
isozyme. These mechanism-based irreversible inhibitors
are especially potent and lead to long lasting results. Sev-
eral flavones have been shown to inhibit the P450 1A2-
dependent demethylation of methoxyresorufin, in an in
vitro assay of this specific enzyme’s activity [16, 17]. In
this project we synthesized a new family of flavones,
determined their crystal structures, and studied their
in vitro inhibitory effects on a number of P450 enzymes
including P450 1A2. These compounds are very similar to
the natural flavone substrates of this enzyme but are
7
97 lL of 0.1 M potassium phosphate buffer, pH 7.6, was
+
combined with 67 lL of 0.015 M NADP solution in
phosphate buffer, 67 lL of 0.0675 glucose 6-phosphate
solution in phosphate buffer, 67 lL of 0.045 M MgCl2
solution in water. The solution was incubated at 37 °C for
5
min before the addition of 2 lL (3 units) of glucose
6
-phosphate dehydrogenase type XXIII. The solution was
then incubated again for an additional 5 min at 37 °C, and
placed in ice until use.
Assay of Coumarin 7-Hydroxylation in Human Liver
Microsomes
Human P450 2A6 dependent coumarin 7-hydroxylation
activity was assayed [24, 25] and given below.
Potassium phosphate buffer (1,900 lL, 0.1 M, pH 7.4)
was placed in a 1.0 cm quartz cuvette, and 10 lL of a
0
.25–100 lM solution of the substrate in Me SO (DMSO),
2
4
0 lL of microsomal P450 protein (final concentration of
about 30 lg/mL) in the same buffer, and 10 lL of an
inhibitor solution in DMSO or pure DMSO (for the control)
were added. The reaction was then initiated by the addition
of 40 lL of NADPH regenerating solution. The final assay
volume was 2.0 mL. The production of 7-hydroxycouma-
rin was monitored by a spectrofluorimeter at 338 nm
excitation and 458 nm emission, with a slit width of
2
nm. The reaction was performed at 37 °C. A number
of assays were performed for each flavone at varying
concentrations.
1
23

J Chem Crystallogr (2008) 38:231–237
235
expected to possess the capability of acting as suicide
inhibitors. Three of the compounds synthesized are iso-
meric and the differences in their inhibitory activities help
us in the determination of structural features needed for
optimum irreversible inhibition.
X-ray Crystallography Results
0
The numbering systems may be found in Fig. 1 for 2 -meth-
0
oxyflavone, Fig. 2 for 3 -methoxyflavone, Fig. 3 for
0
0 0
4
-methoxyflavone, and Fig. 4 for 3 ,4 -dimethoxyflavone.
Stereographic projections have been drawn at the 50% prob-
ability level. Fractional coordinates, isotropic thermal
parameters, bond lengths, and bond angles have been depos-
ited. There are no hydrogen bonding interactions or unusual
close intermolecular contacts in the crystal structures of these
compounds. It is interesting to note that the unit cell dimen-
0
Fig. 2 Stereographic projection of 3 -methoxyflavone
0
0
sionsof3 -methoxyflavone and 4 -methoxyflavone aresimilar
resulting in a lattice packing that is also similar.
All four compounds crystallized in a conformation that is
nearly planar and are chemically reasonable. The torsion
0
0
0
0
angles (C2–C1–C1 –C2 and O2–C1–C1 –C6 ) that describe
the rotation of the methoxy substituted phenyl ring indicate
0
that the molecules are essentially planar [2 -methoxyflavone:
0
-
2.8(3)° and -2.0(3)°; 3 -methoxyflavone: 2.5(3)° and
0
0
0
2
.4(3)°; 4 -methoxyflavone: -1.4(2)° and 178.8(4)°; 3 ,4 -
dimethoxyflavone: -2.68(12)° and -2.75(10)°]. Preliminary
molecular modeling studies (SYBYL [32] MMFF94 force
field) indicate that the lowest energy conformations are nearly
planar but that higher energy conformations do exist for
structures with much larger torsion angles. This indicates that
other conformations may exist in solution. However, the
aromatic character of these flavone molecules stabilizes the
planar conformation found in the X-ray crystal structures.
0
Fig. 3 Stereographic projection of 4 -methoxyflavone
0
0
Fig. 4 Stereographic projection of 3 ,4 -dimethoxyflavone
Inhibition Studies
Tables 2 and 3 contain the data obtained for the inhibition
assays at 100 lM and 10 lM final assay concentrations of
the flavonoids, respectively. At 100 lM, all four compounds
are very effective inhibitors of reactions catalyzed by human
P450s 1A1, 1A2, and 2A6 causing near-complete loss of
0
Fig. 1 Stereographic projection of 2 -methoxyflavone with ellipsoids
drawn at the 50% probability level
123

2
36
J Chem Crystallogr (2008) 38:231–237
Table 2 Preliminary results for the P450 inhibition assays are presented here as percent activity remaining in the presence of each compound
100 lM final assay concentration) compared to control after 120 and 240 s
(
Name of compound/ P450 enzyme
1A1
1A2
2A6
2B1
Assay time points
120 s
240 s
120 s
240 s
120 s
240 s
120 s
240 s
0
2
3
4
3
-Methoxyflavone
-Methoxyflavone
-Methoxyflavone
0
0.92
3.86
0.56
0.24
0
0
0
0
0
0
0
0
0
0
28.96
60.32
14.04
36.88
27.55
40.20
15.58
20.16
0
0
0
2.85
0.52
0
0
0
3.55
0
1.75
7.32
0
,4 -Dimethoxyflavone
Table 3 Preliminary results for the P450 inhibition assays are presented here as percent activity remaining in the presence of each compound
10 lM final assay concentration) compared to control after 120 and 240 s
(
Name of compound/ P450 enzyme
1A1
1A2
2A6
2B1
Assay time points
120 s
240 s
120 s
240 s
120 s
240 s
120 s
240 s
0
2
3
4
3
-Methoxyflavone
-Methoxyflavone
-Methoxyflavone
22.24
1.82
24.94
5.20
0
6.33
0
0
0
82.80
62.83
66.25
45.50
75.06
63.33
64.46
39.80
0
0
0
0
76.90
42.65
25.65
82.04
54.16
20.88
20.32
4.40
24.13
6.63
0
2.54
30.78
0
,4 -Dimethoxyflavone
24.17
activity. There is also some inhibition of P450 2B1-depen-
0
It is interesting that these compounds show some degree of
selectivity as inhibitors of P450s 1A1, 1A2, and 2A6 but
not toward P450 2B1. Since all four molecules are essen-
tially planar, this selectivity cannot be explained by the
variations in the crystallographic features of the molecules.
As long as the active site can accommodate the molecule,
the inhibitory activity will depend on the steric and elec-
tronic characteristics of the substituents as they approach
and bind to the active site. Future studies involving the
docking of these molecules into the active sites of the P450
enzymes are planned in order to examine the differences in
selectivity.
dent activities but at a much lower level. 4 -Methoxyflavone
seems to be the best inhibitor for this rat enzyme (percent
remaining activity of 14.04 at 120 s and 15.58 at 240 s). At
the lower 10 lM concentration, we can better observe the
inhibition selectivity. At this concentration, for P450 1A1
0
0
0
3
-methoxyflavone is the best inhibitor followed by 3 ,4 -
0
dimethoxyflavone, followed by 4 -methoxyflavone with a
0
relatively large difference, followed by 2 -methoxyflavone.
0
0
0
For P450 1A2, 2 -, 3 -, and 4 -methoxyflavones all cause a
complete (0% remaining activity) or near-complete inhibi-
0
0
tion. 3 ,4 -Dimethoxyflavone is not nearly as strong (percent
remaining activity of 24.17 at 120 s and 30.78 at 240 s). The
results for P450 2A6 inhibition are also very interesting in
0
that only 2 -methoxyflavone causes a complete loss of
Supplementary Material
activity. The other three compounds act as inhibitors but are
0
0
not nearly as potent. 3 -Methoxyflavone acts as the weakest
The supplementary crystallographic data for (a) 2 -meth-
0
inhibitor for this enzyme while it was the strongest for both
P450s 1A1, and 1A2. For the rat P450 2B1, all four com-
pounds cause some loss of enzymatic activity at 10 lM, but
oxyflavone, (CCDC 647765), (b) 3 -methoxyflavone
0
(CCDC 647764), (c) 4 -methoxyflavone (CCDC 647763),
0
0
and (d) 3 -4 -dimethoxyflavone (CCDC 647762) have been
deposited. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via http://
www.ccdc.cam.ac.uk/data_request/cif.
0
0
only 3 ,4 -dimethoxyflavone causes more than 50%
inhibition.
We conclude that, as predicted, the flavones studied are
strong inhibitors of human P450s 1A1, 1A2, and 2A6 with
0
Acknowledgements Support of this research from the NIH/MBRS-
SCORE Program (S06 GM08008) is gratefully acknowledged.
some degree of selectivity. 2 -Methoxyflavone shows
0
selectivity towards human P450s 1A2, and 2A6; 3 -meth-
oxyflavone shows selectivity towards P450s 1A1, and 1A2;
0
4
-methoxyflavone shows selectivity towards P450 1A2;
0
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