Pyranoflavones: A group of small-molecule probes for exploring the active site cavities of cytochrome P450 enzymes 1A1, 1A2, and 1B1

Article abstract of DOI:10.1021/jm4003654

Selective inhibition of P450 enzymes is the key to block the conversion of environmental procarcinogens to their carcinogenic metabolites in both animals and humans. To discover highly potent and selective inhibitors of P450s 1A1, 1A2, and 1B1, as well as to investigate active site cavities of these enzymes, 14 novel flavone derivatives were prepared as chemical probes. Fluorimetric enzyme inhibition assays were used to determine the inhibitory activities of these probes toward P450s 1A1, 1A2, 1B1, 2A6, and 2B1. A highly selective P450 1B1 inhibitor 5-hydroxy-4′-propargyloxyflavone (5H4′FPE) was discovered. Some tested compounds also showed selectivity between P450s 1A1 and 1A2. α-Naphthoflavone-like and 5-hydroxyflavone derivatives preferentially inhibited P450 1A2, while β-naphthoflavone-like flavone derivatives showed selective inhibition of P450 1A1. On the basis of structural analysis, the active site cavity models of P450 enzymes 1A1 and 1A2 were generated, demonstrating a planar long strip cavity and a planar triangular cavity, respectively.

Full text of DOI:10.1021/jm4003654

Article
pubs.acs.org/jmc
Pyranoflavones: A Group of Small-Molecule Probes for Exploring the
Active Site Cavities of Cytochrome P450 Enzymes 1A1, 1A2, and 1B1
Jiawang Liu,† Shannon F. Taylor,† Patrick S. Dupart,† Corey L. Arnold,† Jayalakshmi Sridhar,†
Quan Jiang,† Yuji Wang,‡ Elena V. Skripnikova,§ Ming Zhao,‡ and Maryam Foroozesh*,†
†Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, Louisiana 70125, United States
‡College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, P. R. China
§Cell and Molecular Biology Core, College of Pharmacy, Xavier University of Louisiana, New Orleans, Louisiana 70125, United States
S
* Supporting Information
ABSTRACT: Selective inhibition of P450 enzymes is the key to block the
conversion of environmental procarcinogens to their carcinogenic
metabolites in both animals and humans. To discover highly potent and
selective inhibitors of P450s 1A1, 1A2, and 1B1, as well as to investigate
active site cavities of these enzymes, 14 novel flavone derivatives were
prepared as chemical probes. Fluorimetric enzyme inhibition assays were
used to determine the inhibitory activities of these probes toward P450s 1A1,
1A2, 1B1, 2A6, and 2B1. A highly selective P450 1B1 inhibitor 5-hydroxy-4′-
propargyloxyflavone (5H4′FPE) was discovered. Some tested compounds also showed selectivity between P450s 1A1 and 1A2.
α-Naphthoflavone-like and 5-hydroxyflavone derivatives preferentially inhibited P450 1A2, while β-naphthoflavone-like flavone
derivatives showed selective inhibition of P450 1A1. On the basis of structural analysis, the active site cavity models of P450
enzymes 1A1 and 1A2 were generated, demonstrating a planar long strip cavity and a planar triangular cavity, respectively.
and selective inhibitors of these enzymes have not yet been
discovered.
INTRODUCTION
■
Cytochrome P450s are a ubiquitous enzyme superfamily and
play a predominant role in the metabolism and detoxification of
endogenous and xenobiotic substances.1,2 However, these
versatile enzymes are also involved in the bioactivation of
environmental pollutants leading to certain types of cancers.3
Many procarcinogens (such as polycyclic aromatic hydro-
carbons) are metabolically activated by certain P450 enzymes
into active intermediates that covalently bond to DNA and/or
proteins to form DNA adducts and/or protein adducts,
resulting in DNA mutations and cancer formation.4−7 P450s
1A1, 1A2, and 1B1 are representative family I enzymes that
carry out procarcinogen bioactivation reactions inducing
subsequent mutagenesis and tumorigenesis.3,4 Therefore, the
development of potent and selective P450 enzyme inhibitors
has attracted considerable attention over the years, and
inhibiting family I P450 enzymes specifically has become an
important cancer prevention target.8−16
The project reported here focuses on the development of
selective inhibitors toward P450 family I enzymes, specifically
P450s 1A1, 1A2, and 1B1.15−18 In our previous work, we
obtained considerable information about P450 family I enzyme
inhibitors belonging to various structural cores and found that
the inhibitors with flavone backbone possess very high potency,
leading to follow-up flavone structure modification projects.18,19
Because of the structural similarities of P450s 1A1, 1A2, and
1B1 (P450 1A1 shares 80% amino acid sequence identity with
P450 1A2 and about 38% with P450 1B1),20,21 highly potent
In recent years, several research groups have been working
on developing chemical probes containing a reactive moiety
(such as an acetylenic group) in order to investigate the details
of P450−ligand interactions.22−25 In this study, we aimed to
explore the size and shape of P450 enzyme active site cavities
by using chemical probes with a rigid and inert structure. The
rigidity of the molecule gives fewer conformers for consid-
eration, and the lack of reactive functional groups provides a
clear picture of the pure enzyme−ligand binding interactions.
In brief, our strategy can be described as (1) selecting a
structure core (flavone), (2) incorporating the rigid functional
group(s) (pyranyl and 5-hydroxyl groups) into the core to
obtain the chemical probes, (3) evaluating the inhibitory
activities of the newly designed probes on various enzymes
(P450s 1A1, 1A2, 1B1, 2A6, and 2B1), and (4) constructing the
enzyme active site cavity models with the activity data obtained.
Since the inhibitory activity of a rigid and inert probe positively
correlates with the enzyme−probe affinity, valuable information
about the shape of the active site cavity can be obtained by
studying the most potent probes. As depicted in Figure 1, when
probes A and B show high inhibition (high affinity) of the
target enzyme, the shape of the enzyme’s active site cavity can
be generated based on the structures of these probes.
Received: March 11, 2013
© XXXX American Chemical Society
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inhibitory activity toward P450 1A2.18,26 Since the pyrano-
flavone probes have similar hydrophobic properties and
electronic parameters, only the steric properties (molecular
size and shape) need to be taken into consideration during the
structure−activity analysis. This largely simplified the compar-
ison of probes’ 3D structures and accelerated the generation of
ligand models that best fit in the enzyme active sites.
RESULTS AND DISCUSSION
■
Synthetic Strategy for Pyranoflavones. In order to
extend the flavone core, a propargyl moiety was used to
construct a new six-membered ring adjacent to the flavone core
through an etherification and an annulation reaction (Scheme
1). The first reaction step, the formation of flavonyl propargyl
ether from hydroxyflavone, has been well-documented.26 In
brief, the starting material hydroxyflavone is deprotonated by
sodium hydride (NaH) before reacting with propargyl bromide
to form the corresponding propargyl ether. The purpose of
using a strong base (NaH) is to completely deprotonate the
reactant, even in the case of the hydroxyl group of 5-
hydroxyflavone. The second reaction step is a Claisen
rearrangement followed by a nucleophilic addition.27,28 The
propargyl ether undergoes a Claisen rearrangement to form an
unstable cumulated diene intermediate,28 leading to the ring
closure product pyranoflavone. For each monohydroxy-
substituted flavone, only one major thermal ring closure
product was observed.
Monoetherification of Dihydroxyflavones 5,7dHF,
5,3′dHF, and 5,4′dHF. Since the 5-hydroxyl hydrogen in 5-
hydroxyflavone forms an intramolecular H-bond with the
oxygen of the carbonyl group at position 4, this compound is
less polar and more difficult to deprotonate compared to the
other hydroxyflavones. 5-Hydroxyflavone also selectively
inhibits P450 1A2 over P450 1A1 (about 10 times more
selective).18,26 In order to keep the 5-hydroxyl group intact in
the dihydroxyflavone derivatives, monoetherification was
performed. Because of the intramolecular H-bond of the 5-
Figure 1. Flow diagram for the determination of the enzyme’s active
site cavity shape using small molecule probes. This strategy includes
four steps: (1) determination of an active structure core, (2)
modification of the core with rigid functional group(s), (3) evaluation
of the inhibitory activity of probes on the target enzyme, and (4)
determination of the enzyme’s active site cavity shape based on
structural analysis of the most potent probes.
To investigate the differences among the active site cavities of
P450s 1A1, 1A2, and 1B1, a series of α-naphthoflavone-like, β-
naphthoflavone-like, and flavone C-ring extensional pyrano-
flavone derivatives were designed and synthesized. An addi-
tional 5-hydroxyl functional group was incorporated into some
of the probes in order to verify the recent observation that the
presence of a hydroxyl group in position 5 of flavone increases
a
Scheme 1. Synthesis of Pyranoflavones from Monohydroxyflavones
a
The starting materials in this synthetic route were 5-hydroxyflavone (5HF), 6-hydroxyflavone (6HF), 7-hydroxyflavone (7HF), 2′-hydroxyflavone
(2′HF), 3′-hydroxyflavone (3′HF), and 4′-hydroxyflavone (4′HF). Six flavone propargyl ether (5FPE, 6FPE, 7FPE, 2′FPE, 3′FPE, and 4′FPE)
intermediates and six pyranoflavone products were synthesized through the two facile reactions, respectively. Reagents and conditions: (a) propargyl
bromide, NaH in dimethyl sulfoxide (DMSO) (25 °C); (b) N,N-diethylaniline, reflux.
B
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a
Scheme 2. Synthesis of 5-Hydoxypyranoflavones from Dihydroxyflavones
a
5,7-Dihydroxyflavone, 5,3′-dihydroxyflavone, and 5,4′-dihydroxyflavone were the starting materials used in this synthetic route. To obtain the
monoetherified products (5H7FPE, 5H3′FPE, and 5H4′FPE), a mild basic environment was applied for the etherification reaction. Because of the
introduction of the 5-hydroxyl group, the regioselectivity of cyclization reaction decreased. Reagents and conditions: (a) propargyl bromide,
potassium carbonate in acetone (40 °C); (b) N,N-diethylaniline, reflux.
hydroxyl and 4-position carbonyl groups, using a mild base
(K2CO3) deprotonated the other hydroxyl group on the flavone
without affecting the hydroxyl group at position 5 (Scheme 2,
step a); thus, selective etherification succeeded in high yields
(64−80%).
Synthesis of 5-Hydroxypyranoflavones. As shown in
Scheme 2, through the aforementioned thermal ring closure
reaction, 5-hydroxypropargyloxyflavones (5H7FPE, 5H3′FPE,
and 5H4′FPE) were converted to 5-hydroxypyranoflavones in
acceptable yields. However, the presence of the 5-hydroxyl
group decreased the regioselectivity of Claisen rearrangement.
Thus, multiple annulation products (like 5H76PF and
5H3′4′PF) were produced and used as chemical probes to
investigate the active site cavities of P450 enzymes.
Regioselectivity of Claisen Rearrangement Reaction
on the Flavone Core. As mentioned above, the regioselec-
tivity of Claisen rearrangement in an unsymmetrical system is
an important concern. In recent years, the Claisen rearrange-
ment of allyl aryl ethers has been investigated through synthetic
experiment data, NMR techniques, and computational
calculations.29,30 Generally, an electron-withdrawing environ-
ment, a favored reactant conformation, and low product energy
are beneficial to this reaction. Our work here was focused on
the Claisen rearrangement of aryl propargyl ethers, and
interestingly we have found that the higher chemical shift
value (in the proton NMR spectrum) of the corresponding
hydrogen in a reactant correlates with the product formed. For
instance, 6- and 8-positions are the theoretical reaction sites for
the Claisen rearrangement of 7-flavonyl propargyl ether
(7FPE). However since the chemical shift value of H-8 is
higher than that of H-6, the 7,8-pyranoflavone was observed to
be the predominant product. This general rule was observed for
all of the Claisen rearrangement reactions performed for this
project (Table 1). The chemical shift of a hydrogen reflects the
surrounding chemical environment, and a high chemical shift
correlates to an electron-withdrawing environment. Therefore,
our results support the conclusion that electron-withdrawing
environments favor Claisen rearrangement.29 We plan to
collect more data in relation to the prediction of Claisen
rearrangement products (such as favorable reactant conforma-
tions) and report them in a separate publication.
Determination of 5-Hydroxy-7,8-pyranoflavone and
5-Hydroxy-7,6-pyranoflavone by NOE Experiments. The
decrease of regioselectivity accompanied with the introduction
of the 5-hydroxyl group gives us opportunities to create
miscellaneous small molecule probes; however, some of these
compounds are isomeric and hard to analyze by regular
methods. For example, the Claisen rearrangement of 5-
hydroxy-7-propargyloxyflavone has poor regioselectivity and
produces two ring closure products, 5-hydroxy-7,8-pyrano-
flavone (5H78PF, major product) and 5-hydroxy-7,6-pyrano-
flavone (5H76PF, minor product). Because of the structural
similarity of the two isomers, their normal 1D and 2D NMR
spectra are almost identical (Figure 2). Therefore, 1D NOESY
spectra were used to identify the structures of these two
1
isomers. Figure 2 shows the H NMR spectra of 5H78PF and
5H76PF and the corresponding 1D NOESY spectra by
selectively exciting Hβ and H-2′, respectively. Through analysis
of the steric relationship of Hβ and H-2′, the structures of
5H78PF and 5H76PF were clearly elucidated.
Inhibitory Activities of the Chemical Probes toward
P450s 1A1, 1A2, and 1B1. The inhibitory activities of the 14
chemical probes toward P450s 1A1, 1A2, and 1B1 were
evaluated through the standard fluorimetric enzyme inhibition
assays as described previously.9,16,31 Figure 3 shows the results
observed for the inhibition of P450 1A1-dependent deethyla-
tion of resorufin ethyl ether, P450 1A2-dependent demethyla-
tion of resorufin methyl ether, and P450 1B1-dependent
C
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1
Table 1. H NMR Chemical Shift Values of the Aromatic Hydrogens Next to the Propargyl Group
deethylation of resorufin ethyl ether activities. Chrysin (5,7-
dihydroxyflavone, 57DHF), α-naphthoflavone (αNF), and β-
naphthoflavone (βNF) were used as positive controls.10,12,18
The data are presented here as a volume chart with various
compounds on the horizontal axis and IC50 values on the
vertical axis.
In the study of the inhibitory activities of our chemical
probes toward P450s 1A1 and 1A2, some selectivity was
observed. C-Ring extension did not increase the selectivity
between P450s 1A1 and 1A2, while B-ring extension showed an
obvious trend of selectivity. The data showed that the α-
naphthoflavone-like derivatives, 7,8-pyranoflavone (78PF) and
5-hydroxy-7,8-pyranoflavone (5H78PF), have a high inhibitory
activity for P450 1A2 (IC50 values of 0.059 and 0.014 μM,
respectively) and a relatively low inhibitory activity for P450
1A1 (IC50 values of 0.27 and 0.11 μM, respectively). In
contrast, β-naphthoflavone-like derivatives, 5,6-pyranoflavone
(56PF) and 6,5-pyranoflavone (65PF), showed higher
inhibition of P450 1A1 (IC50 values of 0.32 and 0.15 μM,
respectively) compared to P450 1A2 (IC50 values of 1.13 and
0.76 μM, respectively). Results of further investigation of these
two types of inhibitors are presented in the section entitled
“Ligand Models of P450s 1A1 and 1A2”.
P450s 1A1, 1A2, and 1B1 share a large number of substrates
and inhibitors because of their structural similarities. Finding
selective inhibitors for each of these enzymes is quite
challenging. On the basis of our data, the compounds tested
showed selectivity toward P450 1B1 over P450s 1A1 and 1A2.
Especially, 5H4′FPE showed very high selectivity for P450 1B1
(120 times higher than shown for P450 1A1 and more than 250
times higher than observed for P450 1A2). As the data in
Figure 3 show, 0.1 μM 5H4′FPE inhibited the dealkylation
activity of P450 1B1 by 50% without having any significant
effect on P450s 1A1 and 1A2. This high selectivity makes
5H4′FPE an extremely useful compound in studying the
structure and function of P450 1B1. The effect of 5H4′FPE on
aryl hydrocarbon receptor (AhR) activation in yeast strain
MYA-3637 was determined, and the results are included in the
Supporting Information.
Investigation of the mechanism of inhibition showed that
most of the target compounds are competitive inhibitors of
these P450 enzymes. However, 5-hydroxy-3′-propargyloxyfla-
vone (5H3′FPE) is a mechanism-based inhibitor of P450s 1A1
and 1A2. Kinetic calculations showed that 5H3′FPE reacts
D
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1
Figure 2. H NMR and 1D NOESY spectra of 5H78PF (A) and 5H76PF (B) used for structural determination. The 1D NOESY spectra were
recorded on a Bruker Avance III 400 MHz NMR spectrometer by exciting Hβ and H-2′, respectively. The two-way arrows on the molecular
structures indicate the NOE effects between the corresponding hydrogens.
rapidly with P450 1A1 (limiting kinact = 0.19 min−1, KI = 0.13
μM) before inactivating the enzyme. The reaction rate between
Ligand Models of P450s 1A1 and 1A2. The type I
probes, α-naphthoflavone-like or 5-hydroxyflavone derivatives,
showed selective inhibition toward P450 1A2 (Figure 4). This
group of inhibitors included αNF, 78PF, 5H78PF, 5H7FPE,
57HF, and 5HF (the activity data for 5HF are cited from our
recent paper).18,26 5H78PF showed the highest potency and
notable selectivity for P450 1A2 (about 8 times higher than
observed for P450 1A1). Because of the intramolecular
hydrogen bond, the 5-hydroxyl group of flavone is not as
polar as a regular phenol group, and the whole molecule
exhibits relatively nonpolar and hydrophobic properties similar
to a regular flavone. Therefore, the introduction of 7,8-pyranyl
and 5-hydroxyl groups mainly changes the molecular size and
shape of the flavone molecule without much effect on the
hydrophobic and electronic properties.
5H3′FPE and P450 1A2 was relatively slow (limiting kinact
=
0.022 min−1, KI = 0.071 μM) but still led to irreversible
inhibition of the enzyme. In our previous work, we have shown
that the reactive site of 5H3′FPE with P450 enzymes is the
acetylenic group.
Inhibitory Activities of the Probes toward P450s 2A6
and 2B1. Generally, the tested chemical probes have good
selectivity against P450s 2A6 and 2B1. Most of these
compounds do not possess any activity toward these enzymes.
However, the C-ring modified derivatives (2′,3′-pyranoflavone,
3′,2′-pyranoflaovne, and 4′,3′-pyranoflavone) have relatively
low inhibitory activities toward the P450s 1A1 and 1A2 while
showing a mild inhibition of P450 2B1 (IC50 of 6.29−7.12
μM). This means that modification of flavone C-ring could
increase the inhibitory activities on P450 2B1.
The type II chemical probes, β-naphthoflavone-like deriva-
tives, showed selective inhibition of P450 1A1 (Figure 5). This
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Figure 3. IC50 values of the chemical probes for inhibition of P450s 1A1, 1A2, and 1B1. For convenience, the ordinate axis was plotted on a
logarithmic scale. The IC50 values are represented as the mean SE μM of three independent experiments. The IC50 value of 5H4′FPE for 1A2
inhibition is higher than 20 μM.
Figure 4. α-Naphthoflavone-like and 5-hydroxyflavone derivatives (type I) and their IC50 values for inhibition of P450s 1A1 and 1A2.
Figure 5. β-Naphthoflavone-like derivatives (type II) and their IC50 values for inhibition of P450s 1A1 and 1A2.
group of inhibitors consisted of βNF, 56PF, 65PF, and 4′-
propargyloxy-β-naphthoflavone (βNF4′PE) (data cited from a
recent paper18). These compounds exhibit a stretched
structural characteristic and possess 5−14 times higher
selectivity for P450 1A1 over P450 1A2. Especially, the longest
molecule βNF4′PE showed the highest potency and selectivity
for P450 1A1.
Even though both 5H78PF and βNF4′PE are planar
hydrophobic molecules with a common flavone core, they
show completely opposite selectivity for P450s 1A1 and 1A2.
Thus, investigating the molecular shapes of 5H78PF and
βNF4′PE could disclose the differences between the active site
cavities of P450s 1A1 and 1A2. The molecular surface images
(wire mesh mode) of the two representatives, 5H78PF and
βNF4′PE, were generated in UCSF Chimera 1.6.2. (UCSF San
Francisco, CA) after energy minimization using the conjugate
gradient method with CHARMm force field (Figure 6). P450
1A2-favored ligand (5H78PF) showed a planar heart-shape
structure (with a 9.1 Å long side and a 7.0 Å short side) due to
the incorporation of a 7,8-pyranyl and a 5-hydroxy groups into
the flavone core, while P450 1A1-favored ligand (βNF4′PE)
formed a fish fillet shape conformation with 15.8 Å in length
and 4.6 Å in width. Thus, it can be concluded that P450 1A1
has a narrow and long cavity and can accommodate a planar
long strip molecule (with the maximum width of the strip less
than that of P450 1A2). On the contrary, the P450 1A2 owns a
compact active site cavity, and it can accommodate a triangular
molecule. This active site cavity information will be the
foundation of our future optimization. By employing the
molecular probes, we have obtained critical structural character-
istics of the active site cavities of P450s 1A1 and 1A2 while
discovering selective inhibitors among the probes.
CONCLUSION
■
In order to explore the shape and dimensions of P450 active
site cavities and further discover selective inhibitors of P450
enzymes, we carried out the design, synthesis, and evaluation of
a group of small molecule probes. In this work, we focused on
family I P450 enzymes 1A1, 1A2, and 1B1 and created a group
F
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of Bristol, Bristol, U.K.). Elemental analyses were performed by
Atlantic Microlab, Inc. (Norcross, GA).
General Procedure A: Synthesis of Flavonyl Propargyl
Ethers (FPEs). The synthetic procedure for flavonyl propargyl ethers
has been well-described in our previous work.26 Briefly, 500 mg (2.10
mmol) of the hydroxyflavone starting material was dissolved in 40 mL
of dry dimethyl sulfoxide (DMSO) under nitrogen atmosphere,
followed by the slow addition of 100 mg (2.52 mmol) of 60% sodium
hydride (w/w) in mineral oil. After 5 min, 0.5 mL (4.50 mmol) of 80%
propargyl bromide solution in toluene was added and the reaction
mixture was left to stir under nitrogen atmosphere at room
temperature for 24 h. An equal volume of water was then added,
and the mixture was left to stir for an additional 12 h. The resulting
precipitate was filtered, washed with water, and air-dried on a filter
paper overnight. The crude product was purified by recrystallization
from ethanol to give pure flavonyl propargyl ether as crystals with a
yield of 72−80%. All analytical data for FPEs were reported in the
aforementioned paper.
General Procedure B: Synthesis of Pyranoflavones (PFs). In a
reaction flask, 200 mg (0.72 mmol) of flavonyl propargyl ether was
dissolved in 20 mL of N,N-diethylaniline. The mixture was refluxed
under nitrogen atmosphere until the starting material completely
disappeared on TLC (thin layer chromatography). The reaction
solution was then cooled to room temperature, and 100 mL of
dichloromethane (DCM) was added. The crude was washed
successively with 5% hydrochloric acid (50 mL × 8), brine (50 mL
× 2), saturated sodium bicarbonate (50 mL × 3), and brine (50 mL ×
2). The DCM layer was then dried over anhydrous magnesium sulfate
and concentrated under vacuum. The residue was purified by column
chromatography using silica gel (solvent system varied based on the
products) to provide pure pyranoflavone (yield, 44−66%).
Figure 6. Molecular surface images (wire mesh mode) of 5H78PF (A)
and βNF4′PE (B) generated from UCSF Chimera. The size of the
molecules was measured and labeled around them.
8-Phenylpyrano[2,3-f ]chromen-10(2H)-one (5,6-Pyranofla-
vone, 56PF). Starting with 5-flavonyl propargyl ether, 122 mg
(yield, 62%) of 8-phenylpyrano[2,3-f ]chromen-10(2H)-one (5,6-
pyranoflavone, 56PF) was prepared using general procedure B.
Column chromatography solvent system: DCM/methanol 20:1. Mp
156−158 °C. GC/MS: 276 (M+, 100%), 247 (8), 173 (30). 1H NMR
(CDCl3, 300 MHz) δ = 7.87 (m, 2H), 7.51 (m, 3H), 7.22 (d, J = 8.4
Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.41 (d, J = 9.9 Hz,
1H), 5.80 (dt, J = 9.9 Hz, J = 3.6 Hz, 1H), 5.06 (dd, J = 3.3 Hz, J = 1.8
Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ = 178.12, 161.26, 157.21,
154.08, 131.40, 131.34, 131.21, 128.95, 126.02, 123.65, 120.97, 118.63,
114.07, 109.85, 108.54, 66.18. Anal. Calcd for C18H12O3: C, 78.25; H,
4.38. Found: C, 77.77; H, 4.35.
of novel pyranoflavones as probes based on our previous
studies. These probes showed varied inhibitory activities toward
different P450 enzymes. Most tested compounds showed high
inhibition of family I P450s while showing weak or no
inhibition activities toward P450s 2A6 and 2B1. The
comparison of inhibition activities among P450s 1A1, 1A2,
and 1B1 demonstrated that most of these probes possess higher
inhibitory potential toward P450 1B1 compared to P450s 1A1
and 1A2. Especially, the compound 5H4FPE showed a 100
times higher selectivity toward P450 1B1 over 1A1 and no
inhibition of P450 1A2. Even for the most similar P450
enzymes 1A1 and 1A2, some selectivity was observed with
certain compounds. α-Naphthoflavone-like and 5-hydroxy-
incorporating probes preferentially inhibited P450 1A2, while
the β-naphthoflavone-like probes were selective toward P450
1A1. According to these and previous results, we have found
remarkable differences in the preference for the ligand
structures between the active site cavities of P450s 1A1 and
1A2, which share around 80% amino acid sequence identity. In
summary, employing small molecule probes is a promising
method to gain insight into the internal structure of P450
enzymes.
3-Phenylpyrano[3,2-f ]chromen-1(8H)-one (6,5-Pyranofla-
vone, 65PF). Starting with 6-flavonyl propargyl ether, 120 mg
(yield, 60%) of this product was prepared using general procedure B.
Column chromatography solvent system: DCM. Mp 142−144 °C.
1
GC/MS: 276 (M+, 100%), 247 (35), 174 (8). H NMR (CDCl3, 300
MHz) δ = 8.12 (ddt, J = 10.2 Hz, J = 0.6 Hz, J = 1.8 Hz, 1H), 7.90 (m,
2H), 7.52 (m, 3H), 7.35 (d, J = 9.0 Hz, 1H), 7.16 (dd, J = 9.0 Hz, J =
0.6 Hz, 1H), 6.76 (s, 1H), 5.97 (dt, J = 10.2 Hz, J = 3.3 Hz, 1H), 4.81
(dd, J = 3.9 Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ =
180.61, 161.99, 152.36, 151.28, 131.51, 131.48, 129.01, 126.17, 123.58,
123.04, 122.47, 120.56, 118.68, 118.29, 108.10, 64.85. Anal. Calcd for
C18H12O3: C, 78.25; H, 4.38. Found: C, 77.70; H, 4.41.
2-Phenylpyrano[2,3-f ]chromen-4(8H)-one (7,8-Pyranofla-
vone, 78PF). Starting with 7-flavonyl propargyl ether, 130 mg
(yield, 65%) of this compound was prepared using general procedure
B. Column chromatography solvent system: petroleum ether/ethyl
acetate 3:1. Mp 143−144 °C. GC/MS: 276 (M+, 100%), 247 (13),
173 (40), 146 (18), 102 (18), 89 (20). 1H NMR (CDCl3, 300 MHz) δ
= 7.97 (d, J = 8.7 Hz, 1H), 7.87 (m, 2H), 7.53 (m, 3H), 7.00 (d, J =
10.2 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.78 (s, 1H), 5.90 (m, J = 10.2
Hz, 1H), 4.98 (dd, J = 3.6 Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75
MHz) δ = 177.87, 162.76, 158.53, 152.15, 131.89, 131.56, 129.11,
126.18, 126.12, 121.73, 118.08, 117.51, 114.61, 110.57, 107.29, 66.10.
Anal. Calcd for C18H12O3: C, 78.25; H, 4.38. Found: C, 78.13; H, 4.41.
2′H,4H-[2,8′-Bichromen]-4-one (2′,3′-Pyranoflavone,
2′3′PF). Starting with 2′-flavonyl propargyl ether, 110 mg (yield,
EXPERIMENTAL SECTION
■
Chemistry. The hydroxyflavone starting materials were purchased
from INDOFINE Chemical Company, Inc. (Hillsborough, NJ), and
other chemicals were purchased from Sigma-Aldrich Corporation (St.
Louis, MO) and Fisher Scientific International, Inc. (Hampton, NH).
Mass spectral data were determined using an Agilent 6890 GC
instrument with a 5973 MS instrument. 1H NMR and 13C NMR
spectra were recorded on a Bruker Fourier 300 MHz FT-NMR
spectrometer. 1D NOE experiments were performed on a Bruker
Avance III 400 MHz NMR spectrometer, and the data were processed
with a MestReNova NMR software (School of Chemistry, University
G
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Journal of Medicinal Chemistry
Article
55%) of this product was prepared using general procedure B. Column
4.75 (d, J = 2.1 Hz, 2H), 2.59 (t, J = 2.1 Hz, 1H). 13C NMR (CDCl3,
75 MHz) δ = 183.48, 163.99, 160.71, 157.91, 156.29, 135.43, 132.49,
130.23, 119.59, 118.35, 113.00, 111.44, 110.80, 107.07, 106.23, 77.99,
76.29, 56.02. Anal. Calcd for C18H12O4: C, 73.97; H, 4.14. Found: C,
73.43; H, 4.38.
chromatography solvent system: petroleum ether/DCM 1:2. Mp 132−
1
134 °C. GC/MS: 276 (M+, 100%), 247 (15), 155 (30), 121 (18). H
NMR (CDCl3, 300 MHz) δ = 8.20 (dd, J = 8.1 Hz, J = 1.5 Hz, 1H),
7.71 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.66 (m, 1H), 7.50 (dd, J = 8.4
Hz, J = 0.6 Hz, 1H), 7.38 (m, 1H), 7.14 (s, 1H), 7.06 (dd, J = 7.5 Hz, J
= 1.8 Hz, 1H), 6.96 (t, J = 7.8 Hz, J = 7.5 Hz, 1H), 6.44 (dt, J = 9.9 Hz,
J = 1.8 Hz, 1H), 5.84 (dt, J = 9.9 Hz, J = 3.6 Hz, 1H), 4.93 (dd, J = 3.6
Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ = 178.92, 160.34,
156.40, 152.89, 133.60, 129.50, 128.33, 125.58, 124.95, 124.22, 123.75,
123.24, 122.42, 121.17, 119.57, 118.03, 112.43, 65.99. Anal. Calcd for
C18H12O3: C, 78.25; H, 4.38. Found: C, 78.16; H, 4.28.
5-Hydroxy-2-(4-(propargyloxy)phenyl)-4H-chromen-4-one
(5-Hydroxy-4′-propargyloxyflavone, 5H4′FPE). Starting with
5,4′-dihydroxyflavone (the starting material 5,4′-dihydroxyflavone
purchased from INDOFINE Chemical Company, Inc. was purified
before use), this compound was prepared using general procedure C,
with a yield of 64%. The compound decomposed before melting. GC/
1
MS: 292 (M+, 100%), 155 (25). H NMR (CDCl3, 300 MHz) δ =
12.64 (s, 1H), 7.89 (m, 2H), 7.54 (t, J = 8.4 Hz, 1H), 7.11 (m, 2H),
6.98 (dd, J = 8.4 Hz, J = 0.9 Hz, 1H), 6.81 (dd, J = 8.4 Hz, J = 0.9 Hz,
1H), 6.67 (s, 1H), 4.79 (d, J = 2.4 Hz, 2H), 2.58 (t, J = 2.4 Hz, 1H).
13C NMR (CDCl3, 75 MHz) δ = 183.52, 164.39, 160.82, 160.56,
156.39, 135.26, 128.21, 124.33, 115.46, 111.41, 110.77, 106.98, 104.87,
55.94. Anal. Calcd for C18H12O4: C, 73.97; H, 4.14. Found: C, 73.60;
H, 4.12.
2′H,4H-[2,5′-Bichromen]-4-one (3′,2′-Pyranoflavone,
3′2′PF). Starting with 3′-flavonyl propargyl ether, 132 mg (yield,
66%) of this compound was prepared using general procedure B.
Column chromatography solvent system: DCM. Mp 120−122 °C.
GC/MS: 276 (M+, 100%), 259 (15), 247 (28), 156 (60), 121 (35). 1H
NMR (CDCl3, 300 MHz) δ = 8.25 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H),
7.70 (td, J = 7.2 Hz, J = 1.5, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.44 (t, J =
7.8 Hz, J = 7.2 Hz, 1H), 7.27−7.17 (m, 2H), 6.98 (d, J = 7.8 Hz, 1H),
6.68 (d, J = 10.2 Hz, 1H), 6.49 (s, 1H), 5.92 (dt, J = 10.2 Hz, J = 3.9
Hz, 1H), 4.83 (dd, J = 3.9 Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75
MHz) δ = 178.09, 163.91, 156.54, 154.93, 133.91, 130.07, 129.06,
125.80, 125.40, 123.81, 123.30, 122.17, 121.87, 120.90, 118.68, 118.17,
112.72, 64.74. Anal. Calcd for C18H12O3: C, 78.25; H, 4.38. Found: C,
77.94; H, 4.23.
Preparation of 5-Hydroxy-7,8-pyranoflavone and 5-Hy-
droxy-7,6-pyranoflavone. In a reaction flask, 200 mg (0.68
mmol) of 5-hydroxy-7-propargyloxyflavone was dissolved in 40 mL
of N,N-diethylaniline. The mixture was refluxed under nitrogen
atmosphere until the starting material completely disappeared on
TLC. When the reaction solution was cooled to room temperature,
150 mL of dichloromethane (DCM) was added. The solution was
washed successively with 5% hydrochloric acid (50 mL × 10), brine
(50 mL × 2), saturated sodium bicarbonate (50 mL × 3), and brine
(50 mL × 2). The DCM layer was dried with anhydrous magnesium
sulfate and concentrated under vacuum. The residue was purified by
column chromatography on silica gel (petroleum ether/DCM 1:1) to
provide 73 mg of 5-hydroxy-7,8-pyranoflavone (yield, 37%) and 20 mg
of 5-hydroxy-7,6-pyranoflavone (yield, 10%) as yellow crystals,
respectively.
5-Hydroxy-2-phenylpyrano[2,3-f ]chromen-4(8H)-one (5-Hy-
droxy-7,8-pyranoflavone, 5H78PF). Mp 196−198 °C. GC/MS:
292 (M+, 100%), 264 (15), 189 (25). 1H NMR (CDCl3, 300 MHz) δ
= 12.81 (s, 1H), 7.86 (m, 2H), 7.55 (m, 3H), 6.88 (d, J = 10.2 Hz,
1H), 6.65 (s, 1H), 6.27 (s, 1H), 5.75 (dt, J = 9.9 Hz, J = 3.6 Hz, 1H),
4.93 (dd, J = 3.3 Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ =
182.53, 163.54, 161.82, 160.45, 151.79, 131.89, 131.32, 129.14, 126.16,
118.40, 117.11, 105.82, 105.72, 102.17, 99.89, 66.27. Anal. Calcd for
C18H12O4: C, 73.97; H, 4.14. Found: C, 73.27; H, 4.31.
5-Hydroxy-2-phenylpyrano[3,2-g]chromen-4(8H)-one (5-Hy-
droxy-7,6-pyranoflavone, 5H76PF). Mp 172−174 °C. GC/MS:
292 (M+, 100%), 263 (40), 189 (18). 1H NMR (CDCl3, 300 MHz) δ
= 13.00 (s, 1H), 7.87 (m, 2H), 7.53 (m, 3H), 6.80 (dtd, J = 10.2 Hz, J
= 1.8 Hz, J = 0.6 Hz, 1H), 6.65 (s, 1H), 6.40 (d, J = 0.9 Hz, 1H), 5.74
(dt, J = 10.2 Hz, J = 3.6 Hz, 1H), 4.93 (dd, J = 3.6 Hz, J = 1.8 Hz, 2H).
13C NMR (CDCl3, 75 MHz) δ = 182.52, 163.85, 160.38, 157.14,
156.35, 131.83, 131.26, 129.09, 126.27, 118.96, 117.76, 106.36, 105.94,
105.78, 94.66, 66.39. Anal. Calcd for C18H12O4: C, 73.97; H, 4.14.
Found: C, 73.42; H, 4.34.
Preparation of 5-Hydroxy-3′,2′-pyranoflavone and 5-Hy-
droxy-3′,4′-pyranoflavone. In a reaction flask, 200 mg (0.68
mmol) of 5-hydroxy-3′-propargyloxyflavone was dissolved in 40 mL of
N,N-diethylaniline. The mixture was refluxed under nitrogen
atmosphere until the starting material completely disappeared on
TLC. When the reaction solution was cooled to room temperature,
150 mL of DCM was added. The solution was then washed
successively with 5% hydrochloric acid (50 mL × 10), brine (50 mL
× 2), saturated sodium bicarbonate (50 mL × 3), and brine (50 mL ×
2). The DCM layer was dried with anhydrous magnesium sulfate and
concentrated under vacuum. The residue was purified by column
chromatography on silica gel (petroleum ether/DCM 1:1) to provide
136 mg of 5-hydroxy-3′,2′-pyranoflavone (yield, 68%) and 32 mg of 5-
hydroxy-3′,4′-pyranoflavone (yield, 16%) as yellow crystals, respec-
tively.
2′H,4H-[2,6′-Bichromen]-4-one (4′,3′-Pyranoflavone,
4′3′PF). Starting with 4′-flavonyl propargyl ether, 88 mg (yield,
44%) of this compound was prepared using general procedure B.
Column chromatography solvent system: DCM. Mp 126−127 °C.
1
GC/MS: 275 ([M − 1]+, 100%), 247 (8), 155 (40), 124 (20). H
NMR (CDCl3, 300 MHz) δ = 8.21 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H),
7.71−7.65 (m, 2H), 7.54 (dd, J = 8.4 Hz, J = 0.6 Hz, 1H), 7.48 (d, J =
2.4 Hz, 1H), 7.40 (td, J = 7.2 Hz, J = 1.2 Hz, 1H), 6.85 (d, J = 8.7 Hz,
1H), 6.74 (s, 1H), 6.47 (dt, J = 9.9 Hz, J = 1.8 Hz, 1H), 5.84 (dt, J =
9.9 Hz, J = 3.3 Hz, 1H), 4.94 (dd, J = 3.3 Hz, J = 1.8 Hz, 2H). 13C
NMR (CDCl3, 75 MHz) δ = 178.34, 163.39, 157.26, 156.16, 133.66,
127.64, 125.65, 125.16, 124.66, 124.47, 123.81, 123.70, 122.94, 122.35,
117.97, 116.38, 106.05, 66.23. Anal. Calcd for C18H12O3: C, 78.25; H,
4.38. Found: C, 77.78; H, 4.45.
General Procedure C: Synthesis of 5-Hydroxypropargyloxy-
flavone (5HFPEs). To a solution of 500 mg (1.97 mmol) of 5,7-
dihydroxyflavone, 5,3′-dihydroxyflavone, or 5,4′-dihydroxyflavone in
40 mL of anhydrous acetone, 500 mg (3.62 mmol) of anhydrous
potassium carbonate was added. When the reaction solution turned
yellow, 0.5 mL (4.50 mmol) of 80% propargyl bromide solution in
toluene was added. The reaction solution was heated to 50 °C using a
heating mantle for 2 h. Once the starting material spot disappeared on
TLC, the mixture was cooled to room temperature, passed through a
silica pad, and then concentrated under vacuum to give the crude
product. The pure product was obtained as crystals by recrystallization
from anhydrous ethanol (yield, 64−82%).
5-Hydroxy-2-phenyl-7-(propargyloxy)-4H-chromen-4-one
(5-Hydroxy-7-propargyloxyflavone, 5H7FPE). Starting with 5,7-
dihydroxyflavone, this compound was prepared using general
procedure C (yield, 73%). Mp 154−155 °C. GC/MS: 292 (M+,
100%), 263 (20), 207 (20), 189 (20). 1H NMR (CDCl3, 300 MHz) δ
= 12.64 (br, 1H), 7.80 (m, 2H), 7.49 (m, 3H), 6.59 (s, 1H), 6.50 (d, J
= 2.4 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 2.53
(t, J = 2.4 Hz, 1H). 13C NMR (CDCl3, 75 MHz) δ = 182.50, 164.14,
163.33, 162.17, 157.62, 131.93, 131.18, 129.11, 126.32, 106.18, 105.90,
98.94, 93.54, 77.41, 56.20. Anal. Calcd for C18H12O4: C, 73.97; H,
4.14. Found: C, 73.89; H, 4.00.
5-Hydroxy-2-(3-(propargyloxy)phenyl)-4H-chromen-4-one
(5-Hydroxy-3′-propargyloxyflavone, 5H3′FPE). Starting with
5,3′-dihydroxyflavone, this compound was prepared using general
procedure C, with a yield of 82%. Mp 116−118 °C. GC/MS: 292 (M+,
1
100%), 263 (18), 207 (45), 156 (40), 137 (30). H NMR (CDCl3,
300 MHz) δ = 12.50 (br, 1H), 7.53−7.37 (m, 4H), 7.12 (d, J = 6.9 Hz,
1H), 6.93 (d, J = 8.1 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.65 (s, 1H),
5-Hydroxy-2′H,4H-[2,5′-bichromen]-4-one (5-Hydroxy-3′,2′-
pyranoflavone, 5H3′2′PF). Mp 161−163 °C. GC/MS: 292 (M+,
H
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Article
100%), 263 (15), 156 (50), 137 (30). 1H NMR (CDCl3, 300 MHz) δ
= 12.52 (s, 1H), 7.54 (t, J = 8.4 Hz, 1H), 7.24 (t, J = 7.8 Hz, J = 6.3
Hz, 1H), 7.16 (d, J = 6.6 Hz, 1H), 6.96 (m, 2H), 6.83 (d, J = 8.1 Hz,
1H), 6.65 (d, J = 9.9 Hz, 1H), 6.40 (s, 1H), 5.93 (dt, J = 9.9 Hz, J = 3.9
Hz, 1H), 4.82 (dd, J = 3.6 Hz, J = 1.5 Hz, 2H). 13C NMR (CDCl3, 75
MHz) δ = 183.26, 165.12, 160.87, 156.72, 154.98, 135.53, 129.49,
129.12, 123.55, 121.97, 121.89, 120.93, 119.02, 111.57, 111.15, 110.75,
107.14, 64.74. Anal. Calcd for C18H12O4: C, 73.97; H, 4.14. Found: C,
72.54; H, 4.36.
excitation and 585 nm emission, with a slit width of 2 nm. The
production of 7-hydroxycoumarin was monitored at 338 nm excitation
and 458 nm emission, with a slit width of 2 nm. The reactions were
performed at 37 °C. For each inhibitor, a number of assay runs were
performed using varying inhibitor concentrations (ranging from 0.1 to
100 μM). At least four concentrations of each inhibitor showing 20−
80% inhibition were tested.
Data Analysis.33 IC50 Values. The initial data obtained from the
above assays were a series of reaction progress curves (the time-course
of product formation) in the presence of various inhibitor
concentrations and in the absence of the inhibitor as the control.
The Microsoft Excel program was used to fit these data (fluorescence
intensity vs time) in order to obtain the parameters of the best-fit
second-order curves (y = ax2 + bx + c). The coefficient b in the above
second-order equation represented enzymatic activity (v). Dixon plots
were used (by plotting the reciprocals of the enzymatic activity (1/v)
vs inhibitor concentrations [I]) in order to determine IC50 values (x-
intercepts) for the inhibitors. The results based on the first 6 min of
enzymatic reaction are tabulated in Table S1 of Supporting
5-Hydroxy-2′H,4H-[2,7′-bichromen]-4-one (5-Hydroxy-3′,4′-
pyranoflavone, 5H3′4′PF). Mp 166−167 °C. GC/MS: 292 (M+,
1
100%). H NMR (CDCl3, 300 MHz) δ = 12.55 (s, 1H), 7.50 (t, J =
8.4 Hz, 1H), 7.34 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H), 7.22 (s, 1H), 7.00
(d, J = 7.8 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H),
6.61 (s, 1H), 6.41 (d, J = 9.9 Hz, 1H), 5.87 (dt, J = 9.9 Hz, J = 3.6 Hz,
1H), 4.88 (dd, J = 3.0 Hz, J = 1.8 Hz, 2H). 13C NMR (CDCl3, 75
MHz) δ = 183.49, 163.87, 160.72, 156.29, 154.35, 135.34, 131.48,
126.96, 125.69, 124.72, 123.63, 119.49, 113.41, 111.36, 110.85, 107.03,
105.68, 65.89. Anal. Calcd for C18H12O4: C, 73.97; H, 4.14. Found: C,
72.34; H, 4.15.
Information. The data are represented as the mean
SE μM of
5-Hydroxy-2′H,4H-[2,6′-bichromen]-4-one (5-Hydroxy-4′,3′-
pyranoflavone, 5H4′3′PF). In a reaction flask, 200 mg (0.68 mmol)
of 5-hydroxy-4′-propargyloxyflavone was dissolved in 40 mL of N,N-
diethylaniline. The mixture was heated at 195 °C under nitrogen
atmosphere until the starting material completely disappeared on TLC
(about 48 h). When the reaction solution was cooled to room
temperature, 150 mL of DCM was added. The solution was washed
successively with 5% hydrochloric acid (50 mL × 10), brine (50 mL ×
2), saturated sodium bicarbonate (50 mL × 3), and brine (50 mL ×
2). The DCM layer was dried with anhydrous magnesium sulfate and
concentrated under vacuum. The residue was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 3:1)
followed by recrystallization with acetone to provide 86 mg of 5-
hydroxy-4′,3′-pyranoflavone (yield, 43%) as colorless crystals. The
compound decomposed at higher than 180 °C. GC/MS: 292 (M+,
three independent experiments. Figure 3 shows the IC50 values for the
tested compounds on P450s 1A1, 1A2, and 1B1 as a column chart.
The data for P450s 2A6 and 2B1 are shown in Table 2 (in text).
Table 2. IC50 Values of Tested Chemical Probes for the
a
Inhibition of P450s 2A6 and 2B1
IC50 (μM)
IC50 (μM)
P450
2A6
P450
2A6
P450 2B1
P450 2B1
chrysin
aNF
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
5H7FPE
5H78PF
5H76PF
5H3′FPE
5H3′2′PF
5H3′4′PF
5H4′FPE
5H4′3′PF
>25
>25
>25
>25
>25
>25
>25
>25
>25
13.0 2.6
>25
>25
bNF
>25
56PF
65PF
78PF
2′3′PF
3′2′PF
4′3′PF
>25
4.08 0.48
>25
1
100%), 155 (18). H NMR (CDCl3, 300 MHz) δ = 12.66 (s, 1H),
>25
7.66 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 7.53 (t, J = 8.7 Hz, 1H), 7.48 (d,
J =2.1 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.80
(d, J = 8.1 Hz, 1H), 6.62 (s, 1H), 6.48 (d, J = 9.9 Hz, 1H), 5.85 (dt, J =
9.9 Hz, J = 3.3 Hz, 1H), 4.96 (dd, J = 3.0 Hz, J = 1.8 Hz, 2H). 13C
NMR (CDCl3, 75 MHz) δ = 183.47, 164.43, 160.80, 157.59, 156.35,
135.18, 127.81, 124.76, 123.95, 123.60, 123.05, 122.39, 116.47, 111.36,
110.76, 106.95, 104.56, 66.29. Anal. Calcd for C18H12O4: C, 73.97; H,
4.14. Found: C, 73.87; H, 4.24.
>25
>25
6.29 0.59
7.12 0.62
6.62 0.50
>25
>25
a
The IC50 values are represented as the mean
independent experiments.
SE μM of three
Fluorimetric Enzyme Inhibition Assays of P450s 1A1, 1A2,
1B1, 2A6, and 2B1. The inhibition activities of tested compounds on
P450s 1A1, 1A2, 1B1, 2A6, and 2B1 dependent reactions were tested
through standard methods as previously described.31,32 These studies
included P450 1A1-dependent deethylation of resorufin ethyl ether,
P450 1A2-dependent demethylation of resorufin methyl ether, P450
1B1-dependent deethylation of resorufin ethyl ether, P450 2B1-
dependent depentylation of resorufin pentyl ether, and P450 2A6-
dependent coumarin 7-hydroxylation assays. In brief, potassium
phosphate buffer (1760 μL of a 0.1 M solution, pH 7.6) was placed
in a 1.0 cm quartz cuvette, and 10 μL of a 1.0 M MgCl2 solution, 10 μL
of a 1.0 mM corresponding resorufin or coumarin substrate solution
(final concentration of 5 μM) in dimethyl sulfoxide (DMSO), 10 μL of
the microsomal P450 protein (final concentration of 5 nM), and 10 μL
of an inhibitor solution in DMSO were added. For the controls, 10 μL
of pure DMSO was added in place of the inhibitor solution. The
reaction was initiated by the addition of 200 μL of a NADPH
regenerating solution. The regenerating solution was prepared by
combining 797 μL of a 0.10 M potassium phosphate buffer solution
(pH 7.6), 67 μL of a 15 mM NADP+ solution in buffer, 67 μL of a 67.5
mM glucose 6-phosphate solution in buffer, and 67 μL of a 45 mM
MgCl2 solution and incubating the mixture for 5 min at 37 °C before
the addition of 3 units of glucose 6-phosphate dehydrogenase/mL and
a final 5 min incubation at 37 °C. The final assay volume was 2.0 mL.
The production of resorufin anion was monitored by a spectro-
fluorimeter (OLIS DM 45 spectrofluorimetry system) at 535 nm
KI and Limiting kinact Values: The first-order derivatives (y = 2ax
+ b) of the above second-order curves (y = ax2 + bx + c) represent the
enzymatic activity over time. The semilog plots of the percent relative
activity (Y = log[(y/y0) × 100]) versus time demonstrate the loss of
enzymatic activity with time. The linear portions of the above semilog
plots were used to determine t1/2 (0.693/kinact) values at various
concentrations for the observed time-dependent losses of activity. To
obtain KI and limiting kinact, values of 1/kinact were plotted versus
reciprocals of the inhibitor concentration (1/[I]) (Kitz−Wilson plots).
The limiting kinact values were obtained from the abscissa intercepts of
the plots, and the KI values were calculated from the ordinate
intercepts (−1/KI). The KI and limiting kinact values of the mechanism-
based inhibitor 5H7FPE against P450 1A1and 1A2 are tabulated in
Table S2 of Supporting Information and also described in the text.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra, elemental analysis results, and
activity data of all the tested compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
I
dx.doi.org/10.1021/jm4003654 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(12) He, L.; He, F.; Bi, H.; Li, J.; Zeng, S.; Luo, H. B.; Huang, M.
Isoform-selective inhibition of chrysin towards human cytochrome
P450 1A2. Kinetics analysis, molecular docking, and molecular
dynamics simulations. Bioorg. Med. Chem. Lett. 2010, 20, 6008−6012.
(13) Chang, T. K.; Chen, J.; Yang, G.; Yeung, E. Y. Inhibition of
procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1
enzymes by melatonin. J. Pineal Res. 2010, 48, 55−64.
AUTHOR INFORMATION
Corresponding Author
*Phone: 504-520-5078. Fax: 504-520-7942. E-mail: mforooze@
xula.edu.
■
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by DoD Award W81XWH-11-1-
0105. We thank NIH-MBRS SCORE (Grant S06 GM 08008)
for support of the preliminary work done on this project by the
Foroozesh research group. We also thank the Louisiana Cancer
Research Consortium and the NIH-RCMI Grant
8G12MD007595-04 from the National Institute on Minority
Health and Health Disparities for their support of the Major
Instrumentation and the Cell and Molecular Biology Cores at
Xavier University of Louisiana. The contents are solely the
responsibility of the authors and do not necessarily represent
the official views of the Louisiana Cancer Research Consortium,
the DoD, or the NIH.
ABBREVIATIONS USED
NaH, sodium hydride; DMSO, dimethyl sulfoxide; DCM,
dichloromethane; TLC, thin layer chromatography
■
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