7-ethynylcoumarins: Selective inhibitors of human cytochrome P450s 1A1 and 1A2

Article abstract of DOI:10.1021/tx300023p

To discover new selective mechanism-based P450 inhibitors, eight 7-ethynylcoumarin derivatives were prepared through a facile two-step synthetic route. Cytochrome P450 activity assays indicated that introduction of functional groups in the backbone of coumarin could enhance the inhibition activities toward P450s 1A1 and 1A2, providing good selectivity against P450s 2A6 and 2B1. The most potent product 7-ethynyl-3,4,8-trimethylcoumarin (7ETMC) showed IC ₅₀ values of 0.46 μM and 0.50 μM for P450s 1A1 and 1A2 in the first six minutes, respectively, and did not show any inhibition activity for P450s 2A6 and 2B1 even at the dose of 50 μM. All of the inhibitors except 7-ethynyl-3-methyl-4-phenylcoumarin (7E3M4PC) showed mechanism-based inhibition of P450s 1A1 and 1A2. In order to explain this mechanistic difference in inhibitory activities, X-ray crystallography data were used to study the difference in conformation between 7E3M4PC and the other compounds studied. Docking simulations indicated that the binding orientations and affinities resulted in different behaviors of the inhibitors on P450 1A2. Specifically, 7E3M4PC with its two-plane structure fits into the P450 1A2's active site cavity with an orientation leading to no reactive binding, causing it to act as a competitive inhibitor.

Full text of DOI:10.1021/tx300023p

Article
pubs.acs.org/crt
7-Ethynylcoumarins: Selective Inhibitors of Human Cytochrome
P450s 1A1 and 1A2
Jiawang Liu,† Thong T. Nguyen,† Patrick S. Dupart,† Jayalakshmi Sridhar,† Xiaoyi Zhang,‡ Naijue Zhu,†
Cheryl L. Klein Stevens,†,§ and Maryam Foroozesh*,†
†Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana 70125, United States
‡College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
S
* Supporting Information
ABSTRACT: To discover new selective mechanism-based
P450 inhibitors, eight 7-ethynylcoumarin derivatives were
prepared through a facile two-step synthetic route. Cyto-
chrome P450 activity assays indicated that introduction of
functional groups in the backbone of coumarin could enhance
the inhibition activities toward P450s 1A1 and 1A2, providing
good selectivity against P450s 2A6 and 2B1. The most potent
product 7-ethynyl-3,4,8-trimethylcoumarin (7ETMC) showed
IC50 values of 0.46 μM and 0.50 μM for P450s 1A1 and 1A2 in
the first six minutes, respectively, and did not show any
inhibition activity for P450s 2A6 and 2B1 even at the dose of
50 μM. All of the inhibitors except 7-ethynyl-3-methyl-4-
phenylcoumarin (7E3M4PC) showed mechanism-based in-
hibition of P450s 1A1 and 1A2. In order to explain this mechanistic difference in inhibitory activities, X-ray crystallography data
were used to study the difference in conformation between 7E3M4PC and the other compounds studied. Docking simulations
indicated that the binding orientations and affinities resulted in different behaviors of the inhibitors on P450 1A2. Specifically,
7E3M4PC with its two-plane structure fits into the P450 1A2’s active site cavity with an orientation leading to no reactive
binding, causing it to act as a competitive inhibitor.
hydrocarbon procarcinogens due to their occupation or high-
level environmental pollution.14−16
INTRODUCTION
■
Cytochrome P450 enzymes are a large superfamily of
hemoprotein monooxygenases involved in the metabolism,
detoxification, and bioactivation of endogenous and xenobiotic
chemicals and also associated with the formation and
development of certain cancers.1,2 Therefore, developing
selective P450 enzyme inhibitors has attracted considerable
attention over the years.3,4 P450s 1A1 and 1A2 play important
roles in the bioactivation of a variety of procarcinogenic
polycyclic aromatic hydrocarbons.5,6 As a classic example,
benzo[a]pyrene could be metabolized by P450s 1A1 and 1A2
into benzo[a]pyrene-7,8-diol-9,10-epoxide, the ultimate muta-
gen, which could form DNA and protein adducts, leading to
tumor formation and organ toxicity.6,7 Another example is that
of the bioactivation of aflatoxin B1 mainly by P450s 1A2, 2B1,
and 3A4 to form a reactive epoxide intermediate, acting as a
DNA intercalating and alkylating agent and resulting in an
increased risk of cancer formation.8−10 In addition, recent
epidemiological evidence suggests that genetic polymorphisms
of human CYP1A1 and 1A2 genes show significant correlation
with the susceptibilities to lung and breast cancers.11−13
Therefore, it is expected that inhibitors of P450s 1A1 and
1A2 could be developed to serve as cancer chemo-preventive
agents, especially for individuals exposed to polycyclic aromatic
A number of small molecules including polycyclic aromatic
hydrocarbons, coumarins, flavones, and anthraquinones have
been developed in our laboratory and evaluated for their
inhibition of various P450 enzymes.17−21 Among these planar
molecules, coumarins are known substrates for a number of
P450 enzymes (such as P450s 1A1, 1A2, 3A4, 2A6, and P450s
from the 2B subfamily). P450 2A6 metabolizes coumarin into
7-hydroxycoumarin through a 7-hydroxylation reaction, which
accounts for more than 70% of the coumarin metabolism in
humans. Both 7-ethoxycoumarin and 7-ethoxy-4-
(trifluoromethyl)coumarin are known substrates for the P450
2B enzymes, and their major metabolites are also 7-
hydroxycoumarins.22,23 Coumarin could also be metabolized
by P450s 1A1, 1A2, and 3A4 into coumarin-3,4-epoxide and 3-
hydroxycoumarin through minor pathways.24 Thus, coumarin
derivatives are expected to be potential substrates and/or
inhibitors for P450 enzymes.
In this study, in order to develop a group of coumarin
derivatives which selectively inhibit P450s 1A1 and 1A2 but are
not metabolized by P450 2A6 and those from the 2B subfamily,
Received: January 17, 2012
Published: March 23, 2012
© 2012 American Chemical Society
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C18 110A). Mass spectral data were determined by Agilent 6890 GC
the key metabolic site (7-position) on coumarin was modified
(Figure 1). Since a number of aromatic acetylenic molecules
1
with a 5973 MS. H NMR and 13C NMR spectra were recorded on a
Varian 300 MHz NMR spectrometer. Elemental analysis was
performed by Atlantic Microlab, Inc. (Norcross, GA). X-ray crystal
diffraction patterns of 7E3M4PC were recorded on Bruker AXS
SMART X2S.
Preparation of 7-Ethynylcoumarin (7EC) (Method A, Scheme
1). To a solution of 500 mg (3.1 mmol) of 7-hydroxycoumarin in 10
mL of anhydrous pyridine, 1.0 mL (5.9 mmol) of triflic anhydride was
added while cooling in an ice bath and under nitrogen atmosphere.
The reaction solution was stirred on ice for 2 h before moving into a
heating mantle. To this reaction solution, 400 mg (0.57 mmol) of
bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh3)2Cl2), 60
mg (0.32 mmol) of CuI, and 50 mL of diisopropylamine (DIPA) were
added. After 10 min of stirring, 800 μL (5.7 mmol) of
trimethylsilylacetylene was also added, and the reaction mixture was
refluxed for 2 h. It was then allowed to cool to room temperature
before quenching with 100 mL of diethyl ether. A black precipitate
formed. After filtration, the filtrate was washed with 5% KHSO4 (50
mL × 7) followed by saturated NaCl (20 mL × 2), dried over
anhydrous sodium sulfate, and concentrated under vacuum. The crude
7-trimethylsilylethynylcoumarin intermediate (compound 1, Scheme
1) was purified through a column chromatography with petroleum
ether/ethyl acetate 20:1 as solvent to give 480 mg (Yield, 64%) of a
pure gray powder. GC/MS: 242 (M+, 20%), 277 ([M-CH3]+, 100%).
1H NMR (CDCl3, 300 HMz): δ = 7.588 (d, J = 9.6 Hz, 1H), 7.306−
7.109 (m, 3H), 6.334 (d, J = 9.6 Hz, 1H), 0.200 (s, 9H). To a solution
of 300 mg (1.2 mmol) of this 7-trimethylsilylethynylcoumarin
intermediate in 50 mL of methanol, 1.0 mL (1 M) of
tetrabutylammonium fluoride was added. The reaction mixture was
stirred at 70 °C for 0.5 h and quenched with 50 mL of deionized water
to form a white precipitate. After filtration, 180 mg (Yield 88%) of the
final product was obtained in gray powder form. mp 126−128 °C.
GC/MS: 170 (M+, 100%), 142 (100), 114 (30), 88 (25), 63 (30). 1H
NMR(CDCl3, 300 HMz): δ = 7.684 (d, J = 9.6 Hz, 1H), 7.447−7.421
(m, 2H), 7.368 (dd, J = 6.8 Hz, J = 1.5 Hz, 1H), 6.433 (d, J = 9.6 Hz,
1H), 3.269 (s, 1H). 13C NMR(CDCl3, 75 HMz): 142.787, 128.257,
127.923, 120.513, 117.522, 80.808. Anal. Calcd for C11H6O2: C, 77.64;
H, 3.55; O, 18.80. Found: C, 76.93; H, 3.52; O, 18.21.
Figure 1. Design of 7-ethynylcoumarins as selective mechanism-based
inhibitors of P450s from the 1A subfamily. The rationale for the
inhibition mechanism was based on the previous investigation of
aromatic acetylenes.25−27
.
have been shown to inactivate P450s in a mechanism-based
manner, and their metabolic site has been shown to be the
acetylene group, the acetylene functional group was chosen to
modify the 7-position of coumarin (Figure 1).25−28 The design
of these 7-ethynylcoumarins was thus expected to yield
selective mechanism-based inhibitors of P450s 1A1 and 1A2.
Starting from substituted 7-hydroxycoumarins, eight 7-
ethynylcoumarins were synthesized through a facile two-step
reaction route. The products were evaluated as potential
inhibitors of P450s 1A1, 1A2, 2A6, and 2B1 in order to identify
the extent of the inhibition activity, dynamic behavior, and
selectivity. The KI and limiting Kinact values of the mechanism-
based inhibitors were determined. Structure−inhibition activity
relationships of the 7-ethynylcoumarin derivatives on P450 1A2
were investigated based on the 3D structures of the inhibitors
and docking simulations.
MATERIALS AND METHODS
7-Ethynyl-6-methoxycoumarin (7E6MOC). Starting from 7-
hydroxy-6-methoxycoumarin, the target compound was prepared by
method A. The colorless crystals were recrystallized from methanol.
Yield, 70%. mp 138−141 °C. GC/MS: 200 (M+, 100%), 185 (35), 171
(15), 157 (20), 129 (20), 101 (22), 75 (15), 51 (20). 1H NMR
■
Chemistry. All of the chemicals were purchased from Sigma-
Aldrich Corporation (St. Louis, MO) and Fisher Scientific Interna-
tional, Inc. (Hampton, NH). RP-HPLC was performed on an hp
Hewlett-Packard Series 1050 (Column: Phenomenex Gemini-NX 5u
a
Scheme 1. Synthetic Route of 7-Ethynylcoumarins
a
Reagents and conditions: (a) triflic anhydride, pyridine, 0 °C, 2 h; (b) Pd(PPh3)2Cl2, copper(I) iodide, trimethylsilylacetylene, diisopropylamine,
reflux, 2 h; (c) tetrabutylammonium fluoride, methanol, 70 °C, 0.5 h.
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Figure 2. (A) Production of resorufin by P450 1A1 in the absence (control) and presence of 0.125, 0.25, 0.5, and 1.0 μM of 7ETMC (final
concentrations). The reactions were performed at 37 °C, and the formation of resorufin anion was monitored continuously over 6-min as described
in the Materials and Methods section. Compound 7ETMC inhibits the production of resorufin in a concentration-dependent manner, and the
second-order product formation curves (y = ax2 + bx + c) were obtained employing the Trendline tool in Microsoft Excel. The differential curves (y
= 2ax + b) of the product formation curves represent the instantaneous enzymatic activity values. (B) Time- and concentration-dependent inhibition
of P450 1A1-dependent EROD activity by compound 7ETMC. The final concentrations of 7ETMC were 0.125, 0.25, 0.5, and 1.0 μM. The
enzymatic activity values were calculated through the first-order derivatives of the product formation curves at different times, and the percentages of
activity remaining was obtained according to the formula (Ainhibitor/Acontrol)%. The activity loss with time indicates the time-dependent inhibition of
P450 1A1 by compound 7ETMC. (C) Kitz-Wilson plots (1/kinact vs 1/[I]). Kinact values were obtained using the curves in graph B as described in the
Materials and Methods section, and [I]s were the final concentrations of the inhibitor. In this case, the final concentrations of the inhibitor
(7ETMC) were 0.125, 0.25, 0.5, and 1.0 μM. Using the linear equation shown in this graph, KI and limiting Kinact values were determined. (D) The
production of resorufin by P450 1A1 in the presence of 7ETMC (0.125 μM). The reaction solution underwent a 5-min preincubation process in the
presence and absence of NADPH. The sharp decrease of product formation in the test with NADPH preincubation indicated that compound
7ETMC inhibited the O-deethylation activity of P450 1A1 in an NADPH-dependent manner.
(CDCl3, 300 MHz) δ = 7.635 (d, J = 9.5 Hz, 1H), 7.411 (s, 1H), 6.875
(s, 1H), 6.427 (d, J = 9.5 Hz, 1H), 3.933 (s, 3H), 3.468 (s, 1H). 13C
NMR (CDCl3, 75 MHz) δ = 142.666, 122.229, 117.917, 108.230,
84.756, 56.667. Anal. Calcd for C12H8O3: C, 72.00; H, 4.03; O, 23.98.
Found: C, 71.67; H, 4.12; O, 23.59.
(CDCl3, 75 MHz) δ = 128.029, 124.749, 120.619, 115.897, 80.657,
18.723. Anal. Calcd for C12H8O2: C, 78.25; H, 4.38; O, 17.37. Found:
C, 77.42; H, 4.47; O, 17.22.
7-Ethynyl-3,4,8-trimethylcoumarin (7ETMC). Starting from 7-
hydroxy-3,4,8-trimethylcoumarin, the target compound was prepared
by method A. The colorless crystals were recrystallized from methanol.
Yield, 68%. mp 196−198 °C. GC/MS: 212 (M+, 100%), 184 (65), 169
(55), 115 (20). 1H NMR (CDCl3, 300 HMz) δ = 7.357 (s, 2H), 3.409
(s, 1H), 2.552 (s, 3H), 2.377 (s, 3H), 2.218 (s, 3H). 13C NMR
(CDCl3, 75 MHz) δ = 127.801, 121.591, 83.481, 81.780, 15.353,
13.743. Anal. Calcd for C14H12O2: C, 79.22; H, 5.70. Found: C, 79.29;
H, 5.67.
Preparation of 7-Ethynyl-4-methylcoumarin (7E4MC) (Meth-
od B, Scheme 1). To a solution of 500 mg (3.1 mmol) of 7-
hydroxycoumarin in 10 mL of anhydrous pyridine, 1.0 mL (5.9 mmol)
of triflic anhydride was added while cooling in an ice bath and under
nitrogen atmosphere. The reaction mixture was stirred on ice for 2 h
before placing in a heating mantle. To this reaction solution, 400 mg
(0.57 mmol) of Pd(PPh3)2Cl2, 60 mg (0.32 mmol) of CuI, and 50 mL
of diisopropylamine (DIPA) were added. After 10 min of stirring, 800
μL (5.7 mmol) of trimethylsilylacetylene was also added, and the
reaction mixture was refluxed for 2 h. The reaction mixture was then
concentrated under vacuum to 10 mL before the addition of 1.0 mL
(1.0 M) of tetrabutylammonium fluoride in 50 mL of methanol. The
reaction mixture was stirred at 70 °C for 0.5 h and then concentrated
under vacuum. The residue was purified through a column
chromatography with petroleum ether/ethyl acetate 4:1 as solvent to
give 260 mg (Yield 50%) of the final product as a gray powder. mp
133−135 °C. GC/MS: 184 (M+, 100%), 156 (95), 127 (20), 102 (10),
7-Ethynyl-4-(trifluoromethyl)coumarin (7E4TFC). Starting
from 7-hydroxy-4-(trifluoromethyl)coumarin, the target compound
was prepared by method B. Yield, 45%. mp 147−149 °C. GC/MS: 238
(M+, 100%), 210 (98), 191 (10), 162 (8), 132 (12), 113 (9), 87 (6),
1
63 (11). H NMR(CDCl3, 300 MHz) δ = 7.666 (dd, J = 8.1 Hz, J =
1.8 Hz, 1H), 7.483 (d, J = 1.5 Hz, 1H), 7.433 (dd, J = 8.1 Hz, J = 1.5
Hz, 1H), 6.797 (s, 1H), 3.338 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ
= 128.773, 127.376, 125.493, 123.368, 121.090, 119.708, 116.717,
116.626, 82.160, 81.674. Anal. Calcd for C12H5F3O2: C, 60.52; H,
2.12; O, 13.44. Found: C, 59.93; H, 2.45; O, 13.17.
1
77 (8), 63 (12), 51 (10). H NMR (CDCl3, 300 HMz) δ = 7.526−
7-Ethynyl-3-phenylcoumarin (7E3PC). Starting from 7-hydroxy-
3-phenylcoumarin, the target compound was prepared by method A.
7.195 (m, 3H), 6.222 (s, 1H), 3.186 (s, 1H), 2.361 (s, 3H). 13C NMR
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a
Table 1. Inhibition Activities of 7ECs on P450s 1A1, 1A2, 2A6, and 2B1 Represented as IC50 Values
a
Calculations are based on the first 6 min of the enzymatic reaction.
Yield, 65%. mp 153−155 °C. GC/MS: 246 (M+, 100%), 218 (60), 189
effects were applied. Full-matrix least-squares refinement was on F2
against all reflections. Final R = 0.0396, Rw = 0.1078, and S = 1.022 for
F2 > 2σ(F2). A crystallographic information file (cif) is provided in
Supporting Information.
Assays. Human P450 enzymes 1A1, 1A2, and 2A6 (human
CYP1A1, 1A2, and 2A6 + reductase, BD Supersomes), and rat P450
2B1 enzyme (Rat CYP2B1 + reductase + cytochrome b5, BD
Supersomes) were purchased from B.D. Biosciences Corporation
(Woburn, MA). The P450 1A1, 1A2, and 2B1-dependent activities
were assayed in resorufin alkyl ether dealkylation assays using resorufin
ethyl ether, resorufin methyl ether, and resorufin pentyl ether
fluorescent substrates, respectively.29 P450 2A6-dependent 7-hydrox-
ylation of coumarin was used in a similar assay with minor differences
as described below for measuring 2A6 activity.23
1
(50), 109 (10), 94 (15). H NMR (CDCl3, 300 HMz) δ = 7.768 (s,
1H), 7.704−7.675 (m, 2H), 7.489−7.363 (m, 6H), 3.283 (s,1H). 13C
NMR (CDCl3, 75 MHz) δ = 139.204, 129.335, 128.758, 128.393,
128.029, 125.326, 120.255, 120.012, 82.555, 80.900. Anal. Calcd for
C17H10O2: C, 82.91; H, 4.09. Found: C, 82.36; H, 4.03.
7-Ethynyl-4-methyl-3-phenylcoumarin (7E4M3PC). Starting
from 7-hydroxy-4-methyl-3-phenylcoumarin, the title compound was
prepared by method B. Yield, 35%. mp 127−129 °C. GC/MS: 260
(M+, 100%), 231 (50), 202 (30), 155 (10), 101 (8). 1H NMR (CDCl3,
300 HMz) δ = 7.600−7.147 (m, 8H), 3.197 (s, 1H), 2.224 (s, 3H).
13C NMR (CDCl3, 75 MHz) δ = 130.170, 128.697, 128.621, 128.150,
125.357, 120.392, 80.641, 16.719. Anal. Calcd for C18H12O2: C, 83.06;
H, 4.65. Found: C, 82.18; H, 4.48.
7-Ethynyl-3-methyl-4-phenylcoumarin (7E3M4PC). Starting
from 7-hydroxy-3-methyl-4-phenylcoumarin, the target compound
was prepared by method A. The light yellow crystals were
recrystallized from methanol/acetone. Yield, 57%. mp 159−161 °C.
GC/MS: 259 ([M-1]+, 100%), 231 (30), 202 (29), 155 (10), 101 (9).
1H NMR (CDCl3, 300 HMz) δ = 7.593−7.473 (m, 4H), 7.297−7.190
(m, 3H), 6.965 (d, J = 8.2, 1H), 3.239 (s, 1H), 2.024 (s, 3H). 13C
NMR (CDCl3, 75 MHz) δ = 129.198, 129.046, 128.530, 127.832,
127.103, 120.164, 80.171, 15.019. Anal. Calcd for C18H12O2: C, 83.06;
H, 4.65; O, 12.29. Found: C, 83.03; H, 4.56; O, 12.32.
X-ray Crystallography (7E3M4PC). Single crystals were grown
by slow evaporation from an acetone/methanol solution. Data were
collected at 173(2) K on a Bruker SMART X2S diffractometer with a
microfocus sealed Mo target X-ray tube. The structure was solved and
refined using the SHELX-97 software package. All hydrogen atoms
were calculated and treated as riding on the parent carbon atom.
Corrections for absorption, extinction, and Lorentz and polarization
Ethoxyresorufin O-Deethylation (EROD), Methoxyresorufin
O-Demethylation (MROD), Pentoxyresorufin O-Depentylation
(PROD), and Coumarin 7-Hydroxylation Assays. 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
(f.c. Five μM) in dimethyl sulfoxide (DMSO), 10 μL of the
microsomal P450 protein (f.c. Five 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 of
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incubation at 37 °C. The final assay volume was 2.0 mL. The
production of resorufin anion was monitored by a spectrofluorimeter
(OLIS DM 45 Spectrofluorimetry System) at 530 nm 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.
fluorescence loss vs log of Inhibitor concentration) employing the
equation log(F0/F − 1) = log Ka + n log [I] in the Microsoft Excel
Program. The fluorescence quenching assays were performed at 37 °C.
Docking Simulations of the 7-Ethynylcoumarins in Human
P450 1A2. The in-silico studies for 7ECs were carried out using the
LigandFit module in Accelrys Discovery Studio (Accelrys, San Diego,
CA). The crystal structure of human cytochrome P450 1A2 in
complex with α-naphthoflavone (PDB ID: 2HI4) is available from the
Protein Data Bank (PDB).33 The active site of P450 1A2 was defined
as a sphere of radius 5.37 Å surrounding the α-naphthoflavone
molecule. The water molecules were removed, and hydrogen atoms
were added to the P450 template under the CHARMm force field.
The structures of the eight ethynylcoumarin molecules used in this
study are presented in Table 1. The 3D structures of 7ECs were built
using a 3D-sketcher module in Accelrys Discovery Studio. Partial
atomic charges were assigned to each atom with the Gasteiger Charge
method, and energy minimization of each molecule was performed
using the conjugate gradient method with CHARMm force field. The
minimization was terminated when the energy gradient convergence
criterion of 0.001 kcal/mol·Å was reached. The 3D structure of
compound 7E3M4PC was identified using X-ray crystallography since
the single crystal of 7E3M4PC was prepared in order to investigate the
binding postures and orientations.
To explore the binding modes of the 3D-structures of the 7-
ethynylcoumarins to P450 1A2, the docking program LigandFit of
Accelrys DS was used to automatically dock the ligands into the active
site cavity of the enzyme. In the LigandFit program, the standard
flexible docking protocol was performed. As described by
Venkatachalam et al,34 this method employs a cavity detection
algorithm for detecting invaginations in the protein as candidate active
site regions. A shape comparison filter is combined with a Monte
Carlo conformational search for generating ligand poses consistent
with the active site shape. Candidate poses are minimized in the
context of the active site using a grid-based method for evaluating
protein−ligand interaction energies.34 In this case, 20 conformers of
each ligand were automatically formed, and docked into the defined
active site cavity. The prediction of the ranking of ligands
corresponding to their activity was done by a Ligand scoring protocol
(including LigScore1&2, PLP1&2, PMF, Jain, and Ludi scoring
functions).34−36 rms deviation was used to evaluate the accuracy of the
docking performance, and the value for the native ligand α-
naphthoflavone was 0.26 Å, suggesting that the accuracy of this
docking performance in LigandFit was highly satisfactory.37−39
Data Analysis.28 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 (Figure 2A). 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 enzymatic reaction are tabulated in Table 1.
KI and Limiting Kinact Values. The first-order derivatives (y = 2ax +
b) of the above second-order curves (y = ax2 + bx + c) represented the
enzymatic activity over time. The semilog plots of the percent relative
activity versus time as shown in Figure 2B demonstrated the loss of
enzymatic activity. The linear portions of the semilog plots (Figure
2B) 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).
NADPH-Dependency Assay. To confirm mechanism-based
inhibition, preincubation assays were performed as follows. All assay
solution components had the same concentrations as in the previously
described assays. For preincubation assays in the presence of NADPH,
potassium phosphate buffer (1560 μL of a 0.1 M solution, pH 7.6) was
placed in a 1.0 cm quartz cuvette followed by 10 μL of a 1.0 M MgCl2
solution, 10 μL of the microsomal P450 protein, 10 μL of an inhibitor
solution in DMSO (f.c., the concentration leading to 20% enzymatic
activity inhibition), and 200 μL of a NADPH regenerating solution.
The assay mixture was incubated for five minutes at 37 °C before
reaction initiation by the addition of 200 μL of buffer and 10 μL of the
corresponding substrate solution. For the preincubation assays in the
absence of NADPH, potassium phosphate buffer (1760 μL of a 0.1 M
solution, pH 7.6) was placed in a 1.0 cm quartz cuvette followed by 10
μL of a 1.0 M MgCl2 solution, 10 μL of the microsomal P450 protein,
and 10 μL of an inhibitor solution in DMSO (f.c., the concentration
leading to 20% enzymatic activity inhibition). The assay mixture was
incubated for five minutes at 37 °C, before reaction initiation by the
addition of 200 μL of the NADPH regenerating solution and 10 μL of
the corresponding substrate solution. The final assay volume for both
assays was 2.0 mL. The production of P450-dependent reaction
products were monitored as described above. The reactions were
performed at 37 °C.
RESULTS
■
Synthesis of 7-Ethynylcoumarins. The synthetic route
and conditions for the synthesis of the 7-ethynylcoumarin
derivatives are described in Scheme 1. The 7-position of the
starting materials is activated through the formation of triflate
intermediates followed by substitution with the trimethylsilyl-
protected acetylene. After removing the protecting group
(trimethylsilyl group), the final 7-ethynylcoumarin products are
obtained. This synthesis is based on a route previously
reported.25 Since all the reactions involved can occur in basic
environment, in this project, a successful improvement was
made by the combination of the first two steps (method A) or
all the steps in one pot (method B). The target compounds
7EC, 7E6MOC, 7ETMC, 7E3PC, and 7E3M4PC were
produced by method A with yields of 56−70%, while the
other target molecules were synthesized through method B
with yields of 30−50%. On the basis of these results, method A
proved to be a facile pathway to prepare these coumarin
derivatives.
Quenching of P450 1A1 Fluorescence with the 7-
Ethynylcoumarins. The Fluorescence quenching spectra of P450
1A1 with the 7-ethynylcoumarins and their binding constants (Ka)
were obtained using a method previously reported.30−32 P450 1A1 was
diluted in 0.1 M potassium phosphate buffer (pH 7.6) to form 2.0 mL
of a 1.0 μM solution (f.c. Five μM), and the fluorescence intensity was
measured between 300 and 400 nm (excitation wavelength at 295
nm). Binding of the 7-ethynylcoumarins by P450 1A1 was monitored
by detecting decreases in fluorescence intensity after the addition of
different concentrations of the 7-ethynylcoumarins (ranging from
0.25−10 μM). The binding constant (Ka) of each compound was
estimated by linear regression analysis (plotting log of relative
Inhibition Activities of 7ECs on P450s 1A1, 1A2, 2A6,
and 2B1. The inhibition activities of 7ECs on P450s 1A1, 1A2,
2A6, and 2B1-dependent reactions were tested through
standard methods as previously described.23,29 Table 1 contains
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the results observed for the inhibition of P450 1A1-dependent
deethylation of resorufin ethyl ether, P450 1A2-dependent
demethylation of resorufin methyl ether, P450 2B1-dependent
depentylation of resorufin pentyl ether, and P450 2A6-
dependent coumarin 7-hydroxylation activities.
Table 3. KI, Limiting Kinact, and Consensus Scores of the
Inhibitors on P450 1A2
a
compd
7EC
KI (μM)
limiting Kinact (min−1
)
consensus score
5.83
78.90
2.65
0.87
3.15
0.65
0.83
ND
0.09
0.35
0.39
0.44
0.31
0.03
0.10
ND
0
2
3
3
3
5
4
2
7E6MOC
7E4MC
The obtained data show a high selectivity for the 7-
ethynylcoumarin derivatives toward P450s 1A1 and 1A2
compared to P450s 2A6 and 2B1. Almost all of the studied
compounds show inhibition of P450s 1A1 and 1A2 and not
P450 2A6, suggesting that the selectivity of these coumarin
derivatives for P450s 1A1 and 1A2 are achieved as expected by
the design of 7-position acetylene-modified coumarins. Only
two of the compounds (7E4TFC and 7E3M4PC) inactivate
P450 2B1, suggesting that most of the target compounds
possess selectivity for P450s 1A1 and 1A2 compared to 2B1.
Considering the inhibition activities of the coumarin
derivatives toward P450s 1A1 and 1A2, the most potent
compound on both of these enzymes is compound 7ETMC,
with IC50 values of 0.50 μM and 0.46 μM, respectively. Further
observation indicates that the compounds with a 4-position
monosubstitution (7E4MC and 7E4TFC) more effectively
inhibit P450 1A2 than P450 1A1 (around 5-fold difference in
the IC50 values). However, the 3-phenyl substituted compound,
7E3PC, is about five times more potent toward P450 1A1 than
P450 1A2. This observation indicates that although P450s 1A1
and 1A2 have a very high degree of similarity (about 80%
amino acid sequence similarity), it is possible to develop
selective inhibitors for each of them through investigating
structure characteristics of their active site cavities.
Identification of the Inhibition Behavior of 7ECs on
1A1, 1A2, and 2B1. To further study the mode of activity of
the inhibitors toward P450s 1A1, 1A2, and 2B1, apparent
dissociation constants of enzyme−inhibitor complexes (KI) and
apparent rate constants for time-dependent loss of P450-
dependent activity (limiting Kinact) were determined based on
the same data obtained from the above enzyme activity assays.
The IC50 values could well represent the inhibition activity for
the inhibitors with competitive behavior (inhibition degree only
depending on the inhibitor’s concentration). However, the
mechanism-based inhibitors show a time-, concentration-, and
NADPH-dependent loss of enzymatic activity (Figures 2A, B,
and D). Therefore, the KI and limiting Kinact (dynamic
parameter) values of the inhibitors were calculated employing
Kitz−Wilson plots (Figure 2C) as shown in Tables 2 and 3.
The inhibitors of P450 1A1 studied here show mechanism-
based inhibition behavior. In terms of the inhibition activities
toward P450 1A1, the KI values vary greatly from 0.57 μM to
104.03 μM (about 200-fold), while the limiting Kinact values
7ETMC
7E4TFC
7E3PC
7E4M3PC
7E3M4PC
a
ND: not determined.
show a relatively small variance from 0.21 to 0.37 min−1. This
indicates that the activities of 7ECs on P450 1A1 mainly
depend on the binding affinity, and 7ECs possess similar
reaction rates with the enzyme. Since this affinity seems to be
critical for the inhibition of P450 1A1 by these compounds,
another affinity constant, the binding constant (Ka), is
determined through the fluorescence quenching assay of
P450 1A1 by the inhibitors as shown in Table 2.
The inhibition behaviors of 7ECs toward P450 1A2 are not
the same as those toward P450 1A1. The compound ranking in
the second place (based on the IC50 value of 1.68 μM),
7E3M4PC, does not show a time-dependent loss of enzymatic
activity based on the obtained data. Thus, this compound is a
competitive inhibitor. As to the inhibitors showing mechanism-
based inhibition behavior, the limiting Kinact values greatly vary,
ranging from 0.03 min−1 to 0.44 min−1 (>10 fold). This implies
that they have different reaction rates with P450 1A2, resulting
from their different reaction abilities and/or probabilities. The
following docking simulations of 7ECs in P450 1A2 were done
as an effort to explain the differences of the inhibition behaviors
based on the binding style and orientation.
For P450 2B1, two inhibitors, 7E4TFC and 7E3M4PC, were
analyzed. The results show that 7E4TFC is a competitive
inhibitor, while 7E3M4PC acts as a mechanism-based inhibitor.
The KI and limiting Kinact values of 7E3M4PC are 18.45 μM
and 0.29 min−1, respectively.
Single Crystal Structure of 7E3M4PC. During the
preparation of the 3D-structures of 7ECs, it was found that
the 3D-structure of 7E3M4PC was not similar to the others.
Due to the steric H−H and H−CH3 interactions, the 4-phenyl
ring of 7E3M4PC could not rotate freely and favored a torsion
angle predicted by Accelrys Discovery Studio to be 64.17°
between the phenyl ring plane and the coumarin core plane.
The X-ray crystal structure of 7E3M4PC was completed in
order to clarify the actual torsion angle between the two planes.
The 3D-structure of 7E3M4PC is shown in Figure 3A. The
torsion angle was determined to be 68.8(2)° for C13−C12−
C3−C4. There are no unusual inter- or intramolecular bonds or
angles. There is only a slight difference (rmsd = 0.27A)
between the crystal structure conformation and the computer-
generated conformation, suggesting a good performance of
conformation prediction (Figure 3B). Among the eight
molecules, 7E3M4PC is the sole competitive inhibitor of
P450 1A2 and the sole mechanism-based inhibitor of P450 2B1,
suggesting that the two-plane structure is critical for the
inhibition abilities and behaviors observed toward these
enzymes. Therefore, docking studies were performed based
on the crystallographic coordinates of 7E3M4PC and the
computer-generated 3D structures of other 7ECs.
Table 2. KI, Limiting Kinact, and Ka Values of the Inhibitors
a
on P450 1A1
compd
7EC
KI (μM)
limiting Kinact (min−1
)
Ka (μM)
ND
ND
0.34
0.21
0.34
0.23
0.25
0.31
0.37
ND
0.11
0.25
1.23
0.29
0.93
0.33
0.38
7E6MOC
7E4MC
104.03
38.60
0.57
7ETMC
7E4TFC
7E3PC
16.85
0.38
7E4M3PC
7E3M4PC
5.16
3.58
a
ND: not determined.
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Figure 3. (A) Single crystal structure of 7E3M4PC with thermal ellipsoids plotted at the 50% probability level. Due to hindrance on both sides, the
3-phenyl ring of 7E3M4PC forms a 68.8(2)° torsion angle with the plane of the coumarin core. (B) The alignment of the single crystal conformation
(gray) and the computer generated minimum-energy conformation (blue) of 7E3M4PC.
Docking Simulations of 7ECs Based on the Crystal
Complex of P450 1A2 with α-Naphthoflavone. Docking
studies were performed with LigandFit in the Accelrys DS
studio according to the protocol described in the Materials and
Methods Section. The standard ligand, α-naphthoflavone,
underwent the same process as the test compounds 7ECs (in
short, structure sketching and energy minimization), and was
then redocked into the active site cavity of P450 1A2 in order
to evaluate the accuracy of the docking performance. Figure 4A
shows the overlapped structures of X-ray conformation of α-
naphthoflavone and the docked pose. The root-mean-square
deviation (rmsd) value is 0.26 Å, suggesting a good docking
performance.
The prediction of the ranking of ligands was done by ligand
scoring protocol (including LigScore1&2, PLP1&2, PMF, Jain,
and Ludi scoring functions) after the docking process. The
comprehensive scores of the docking patterns are represented
as consensus values (shown in Table 3). A high consensus value
indicates a high affinity ligand. The results show that the
docking poses of 7E3PC rank in the first place (score: 5; the
most favorable pose displayed in Figure 4B), and the binding
poses of 7E4M3PC rank in the second place (score: 4; the most
favorable pose displayed in Figure 4C). Both of these
compounds possess a 3-phenyl functional group and own the
lowest KI values (0.65 and 0.83 μM, respectively) in the P450
1A2 inhibition assays. Specifically, 7E3PC and 7E4M3PC share
the same docking orientation with the standard ligand α-
naphthoflavone in the active site cavity of P450 1A2, in which
the 3-phenyl group faces the enzymatic active center heme,
while the coumarin rings are embedded in the narrow cavity
formed by Phe226, Phe125, and α-helix I of the enzyme
(Figures 4B and C). These observations suggest that the double
fitness in both the phenyl pocket and the narrow cavity makes
them suitable for the binding site of P450 1A2.
7E4M3PC, and the standard ligand (with the phenyl group
next to the heme, Figures 4G and H).
DISCUSSION
■
Using a facile synthetic method, eight 7-ethynylcoumarin
derivatives were prepared. Method A (two-step method) gave
higher yields than both the one-pot method (method B) and
the original three-step method previously described.25
The inhibition activities of these 7-ethynylcoumarins on
P450s 1A1, 1A2, 2A6, and 2B1-dependent reactions were
studied through enzyme activity assays. As expected, most of
the compounds selectively inhibit P450s 1A1 and 1A2 rather
than P450s 2A6 and 2B1. 7ECs act as inhibitors toward P450
1A1 in a mechanism-based manner, and the similar dynamic
parameter (limiting Kinact) values indicate that the inhibitors
probably share the same inhibition mechanism and have the
same enzyme binding pattern.
For P450 1A2, the inhibition behaviors of compounds differ.
Compound 7E3M4PC, possessing a phenyl group in position 4,
does not show time- and NADPH-dependent inhibition, acting
as a competitive inhibitor. The obviously low limiting Kinact
values of compounds 7E3PC and 7E4M3PC, which possess a
phenyl group in position 3, suggest weak reactive abilities of
7E3PC and 7E4M3PC with P450 1A2. Interestingly, the
unsubstituted compound 7EC also shows a weak reactive ability
with P450 1A2. This indicates that the midsize 7-
ethynylcoumarins (7E6MOC, 7E4MC, 7ETMC, and
7E4TFC) are prone to react with P450 1A2 compared to
their smaller or bigger counterparts.
To investigate why the high-affinity ligands 7E3PC and
7E4M3PC (KI values of 0.65 and 0.83 μM, respectively) show
low reactivity with P450 1A2, docking simulations were
performed with the LigandFit protocol. The active site cavity
of P450 1A2 is made of two tightly connected sections. A
narrow water inaccessible cavity (part A) contains Phe-226 on
α-helix F, Phe-125 in the B′-C loop region with π−π
interactions (on one side), and the peptide backbone of α-
helix I with hydrophobic interactions (on the other side)
(Figure 5A). The π−π interactions, noncovalent interactions
between aromatic rings, are important forces in molecular
recognition and play an important role in controlling the
binding locations and orientations of ligands.40,41 The geometry
preferences and energetic forces of these interactions have been
However, the midsize 7-ethynylcoumarins (7E6MOC,
7E4MC, 7ETMC, and 7E4TFC) got relatively lower scores
(from 2 to 3) in docking energy evaluation. It was also observed
that they could be docked into the enzyme binding site with
different poses. Figures 4D−F show three representative poses
of 7ETMC in the cavity. Unpredicted, the unsubstituted 7-
ethynylcoumarin (7EC) got the poorest score of 0. The crystal
conformation of 7E3M4PC could also be docked into the active
site cavity with a similar orientation as seen for 7E3PC,
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Figure 4. Results and stereoviews of the docking studies. (A) Redocking of α-naphthoflavone into the active site cavity of P450 1A2, yielding an
rmsd value of 0.26 Å between the starting X-ray conformation of α-naphthoflavone (pink) and the docked pose (yellow) suggesting a good
performance of docking simulation. (B−H) The docking images of 7E3PC (B), 7E4M3PC (C), 7ETMC (D−F), 7E3M4PC (G), and the
overlapped structures of 7E3M4PC and α-naphthoflavone (yellow) (H). The heme residue is located on the bottom, and some amino acid residues
(Phe-226, Phe-125, Thr-124, Leu-386, Ile-382, etc.) are shown in the docking images.
widely studied in recent years. Our earlier studies have
demonstrated the importance of aromatic interactions in
determining the inhibition potency toward P450 1A2.19,42 In
the case of α-naphthoflavone, the π−π interactions between the
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Figure 5. (A) Diagram of the active site cavity of P450 1A2: a narrow, but extended channel (part A), and a spherical hydrophobic pocket (part B).
(B) The binding patterns of compound 7ETMC with P450 1A2, which suggests that the midsize 7-ethynylcoumarins could freely rotate in the
narrow cavity (part A) if only it is parallel with Phe-226 and the peptide bond in the α-helix I in order to form π−π interactions. (C) The possible
binding patterns of compound 7E3PC with P450 1A2. Phenyl-facing-heme orientation (left) takes an obvious priority over the acetylene-facing-
heme orientation (right). (D) The possible binding pattern (left) and impossible binding pattern (right) of compound 7E3M4PC with P450 1A2,
suggesting that there is no reactive pose for the docking of 7E3M4PC with this enzyme.
flavone ring and the phenyl side chains of Phe-226 (parallel
displaced) and Phe-125 (T-Shaped) contribute to the binding
affinity. The distance between the two sides of the cavity is
about 7.1−7.3 Å, resulting in the parallel conformation of the
ligand’s rigid plane with the phenyl plane of Phe-226 to keep
good π−π interactions and van der Waals interactions. A
polycyclic aromatic molecule could fit into this cavity. The next
section of the cavity (part B) is a hydrophobic semispherical
pocket, consisting of the heme residue, α-helix I, Phe-125, Leu-
386, Ile-382, Thr-124, Leu-497, and Thr-498 with hydrophobic
interactions. Its maximum diameter is about 10.5 Å, and the
minimum diameter is about 9.5 Å (detected according to the
crystal structure 2HI4).33 Thus, part B can accommodate a
group with the size of one phenyl ring at the most, but this
group once inside could rotate since there is no direction-
oriented residue(s) around this pocket (Figure 5A).
In terms of the poses of 7ETMC (the representative of the
midsize 7-ethynylcoumarins) docked into P450 1A2, several
energetically favorable binding poses exist (Figure 5B). This
means that 7ETMC could make free rotation with similar
binding energies if only it is kept parallel with the part A’s plane
forming the π−π interactions. Thus, 7ETMC always has the
probability that its acetylene group can face the heme residue
(Figure 5B, top-left) and react with the enzyme’s active center.
The other midsize compounds 7E6MOC, 7E4MC, and
probabilities of 7E3PC and 7E4M3PC with the enzyme are
greatly lower than that of midsize coumarins, due to the lower
probabilities of the reactive conformation (acetylene group
facing the heme). In general, the higher the affinity of the
phenyl-facing-heme pose with the enzyme, the lower the
probability of a reaction between the acetylene group and the
enzyme. This observation explains why compound 7E3PC
possesses the highest affinity with P450 1A2 (KI 0.65 μM and
docking score 5), while possessing the lowest reactivity with
that enzyme (limiting Kinact, 0.03 min−1). Therefore, the
inhibition activity of compound 7E3PC toward P450 1A2
mainly results from competitive inhibition and not from
mechanism-based inhibition.
On the basis of docking simulations, the 4-phenyl derivative
7E3M4PC fits into the enzyme’s active site in one orientation
only (the phenyl group facing the heme, Figure 5D, left). This
is due to the fact that if the 4-phenyl ring is clamped in part A
of the enzyme’s active site, the coumarin bicyclic ring could not
be contained in part B of the enzyme’s active site (Figure 5D
right). Therefore, compound 7E3M4PC does not show a
reactive pose (the acetylene group facing the heme), and its
inhibition activity toward P450 1A2 is completely competitive.
All of the inhibitors studied here inactivate P450 1A1 in a
mechanism-based manner, suggesting that P450 1A1 accom-
modates a relatively large range of inhibitors of various sizes
compared to P450 1A2. Part B of the P450 1A1 active site
cavity is expected to be larger than that of P450 1A2. At
present, the crystal structure of P450 1A1 is still unavailable,
and docking studies with P450 1A1 were based on the
homology models obtained using modeling programs (such as
Composer, Modeler, SWISS-MODEL, etc.).20,31,43−46 Among
these homology modeling studies, Yamazaki et al. reported that
7E4TFC, which show similar reaction rates (limiting Kinact
)
and similar docking scores, probably share a similar reaction
probability with 7ETMC.
For the 3-phenyl derivatives, 7E3PC and 7E4M3PC, the
docking orientation where the 3-phenyl group faces the heme
(Figure 5C left) takes an obvious priority over the acetylene-
facing-heme orientation (Figure 5C right). Thus, the reaction
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(5) Ma, Q., and Lu, A. Y. (2007) CYP1A induction and human risk
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the volume of the active site of P450 1A1 was larger than that
of P450 1A2,44,47 which is consistent with our observations
based on the size of their ligands, while the other studies either
reported the opposite19,48 or did not report this parameter.20,43
Since the homology modeling studies were performed using
different modeling programs and there are no criteria for
evaluating homology modeling results obtained from various
programs, this debate will continue until the 3D crystal
structure of P450 1A1 is reported.
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W. (2009) Insights into the structure, function, and regulation of
human cytochrome P450 1A2. Curr. Drug Metab. 10, 713−729.
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(1995) Oxidation of aflatoxin B1 by bacterial recombinant human
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Crystallographic information file (cif); raw 13C NMR
spectra, raw elemental analysis reports, and HPLC purity
reports. This material is available free of charge via the Internet
at http://pubs.acs.org.
́
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AUTHOR INFORMATION
■
Corresponding Author
*Department of Chemistry, Xavier University of Louisiana, 1
Drexel Dr., New Orleans, LA 70125. Tel: 504-520-5078. Fax:
504-520-7942. E-mail: mforooze@xula.edu.
Present Address
§Ogden College of Science and Engineering, Western Kentucky
University, 1906 College Heights Boulevard #11075, Bowling
Green, Kentucky 42101-1075, United States.
Funding
We thank NIH-MBRS SCORE (grant number 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 for purchase of software licenses, NIH-
RCMI (grant number G12RR026260) for purchase of the
SMART X2S diffractometer and support of the Molecular
Structure and Modeling Core, and NIH-SCORE (grant
number SC1GM084722) for research support for C.L.S. and
salary support for J.S.
(13) Khvostova, E. P., Pustylnyak, V. O., and Gulyaeva, L. F. (2012)
Genetic polymorphism of estrogen metabolizing enzymes in Siberian
women with breast cancer. Genet. Test. Mol. Biomarkers 16, 167−173.
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Notes
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and Guengerich, F. P. (1997) Aryl acetylenes as mechanism-based
inhibitors of cytochrome P450-dependent monooxygenase enzymes.
Chem. Res. Toxicol. 10, 91−102.
The authors declare no competing financial interest.
ABBREVIATIONS
■
7EC, 7-ethynylcoumarin; 7E6MOC, 7-ethynyl-6-methoxycou-
marin; 7E4MC, 7-ethynyl-4-methylcoumarin; 7ETMC, 7-
ethynyl-3,4,8-trimethylcoumarin; 7E4TFC, 7-ethynyl-4-
(trifluoromethyl)coumarin; 7E3PC, 7-ethynyl-3-phenylcoumar-
in; 7E4M3PC, 7-ethynyl-4-methyl-3-phenylcoumarin;
7E3M4PC, 7-ethynyl-3-methyl-4-phenylcoumarin; rmsd, root-
mean-square deviation; DIPA, diisopropylamine; Pd-
(PPh3)2Cl2, bis(triphenylphosphine)palladium(II) dichloride
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