Ethynylflavones, highly potent, and selective inhibitors of cytochrome P450 1A1

Article abstract of DOI:10.1021/tx5001865

The flavone backbone is a well-known pharmacophore present in a number of substrates and inhibitors of various P450 enzymes. In order to find highly potent and novel P450 family I enzyme inhibitors, an acetylene group was incorporated into six different p

Full text of DOI:10.1021/tx5001865

Article
pubs.acs.org/crt
Open Access on 07/17/2015
Ethynylflavones, Highly Potent, and Selective Inhibitors of
Cytochrome P450 1A1
Navneet Goyal,† Jiawang Liu,† La’Nese Lovings,† Patrick Dupart,† Shannon Taylor,† Sydni Bellow,†
Lydia Mensah,† Erika McClain,† Brandan Dotson,† Jayalakshmi Sridhar,† Xiaoyi Zhang,‡ Ming Zhao,‡
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: The flavone backbone is a well-known
pharmacophore present in a number of substrates and
inhibitors of various P450 enzymes. In order to find highly
potent and novel P450 family I enzyme inhibitors, an acetylene
group was incorporated into six different positions of flavone.
The introduction of an acetylene group at certain locations of
the flavone backbone lead to time-dependent inhibitors of
P450 1A1. 3′-Ethynylflavone, 4′-ethynylflavone, 6-ethynylflavone, and 7-ethynylflavone (KI values of 0.035−0.056 μM) show
strong time-dependent inhibition of P450 1A1, while 5-ethynylflavone (KI value of 0.51 μM) is a moderate time-dependent
inhibitor of this enzyme. Meanwhile, 4′-ethynylflavone and 6-ethynylflavone are highly selective inhibitors toward this enzyme.
Especially, 6-ethynylflavone possesses a Ki value of 0.035 μM for P450 1A1 177- and 15-fold lower than those for P450s 1A2 and
1B1, respectively. The docking postures observed in the computational simulations show that the orientation of the acetylene
group determines its capability to react with P450s 1A1 and 1A2. Meanwhile, conformational analysis indicates that the shape of
an inhibitor determines its inhibitory selectivity toward these enzymes.
flavonoids, and alkaloids have been identified as effective
inhibitors of P450 family I enzymes.12−19 In our recent study
on 7-ethynylcoumarins, we have reported a highly potent
mechanism-based inhibitor (MBI), 7-ethynyl-3-phenylcoumar-
in (7E3PC), possessing selectivity toward P450 1A1 in
comparison with P450s 1A2, 2A6, and 2B1 (Figure 1).17 In
this molecule, the 3-phenylcoumarin residue is the major
pharmacophore, fitting into the narrow active site cavity of
P450 1A1 with multiple π−π interactions, while the acetylene
group could react with the enzyme, leading to its deactivation.
According to these results, we incorporated the acetylene group
into the flavone backbone (an analogue of 3-phenylcoumarin
but much more powerful than that compound)20 to produce a
series of flavone derivatives, ethynylflavones. The major points
of this design could be summarized as (i) introducing the
flavone backbone to enhance the inhibitory activity and (ii)
modifying flavones in various positions with the ethynyl group
to regulate the selectivity while keeping the mechanism-based
inhibition capability.
INTRODUCTION
■
Cytochrome P450s 1A1, 1A2, and 1B1 are major P450 family I
enzymes implicated in the detoxification and metabolic
activation of drugs, environmental chemicals, and other
xenobiotics. P450 1A2 largely metabolizes aromatic amines,
whereas P450s 1A1 and 1B1 deal with the polycyclic aromatic
hydrocarbons (PAHs) and polyhalogenated aromatic hydro-
carbons (PHAHs).1−4 PAHs are widely distributed environ-
mental pollutants that ubiquitously occur as the byproducts of
the petroleum industry, diesel-engine exhaust, cigarette smoke,
and charcoal-grilled food.5 As toxicants, they are of concern
because some of these compounds have been identified as
carcinogenic, mutagenic, and teratogenic agents. In vitro and in
vivo experiments have shown that P450-mediated metabolism
(like P450s 1A1 and 1B1) converts certain PAHs into reactive
intermediates which can covalently bond to DNA and proteins,
forming DNA- and protein-adducts.6,7 These adducts lead to
DNA or protein damage and often induce organ toxicity and
possible tumorigenicity. However, P450 1A2 is notable for its
capacity to bioactivate arylamines such as 2-amino-3-
methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP) into potent mutagenic or
carcinogenic components.8 Therefore, selective inhibition of
P450 family I enzymes has been selected as a molecular target
for potential cancer chemoprevention.9−11
MATERIALS AND METHODS
■
Chemistry. The hydroxyflavone starting materials were purchased
from INDOFINE Chemical Company, Inc. (Hillsborough, NJ), and all
other chemicals were purchased from Sigma-Aldrich Corporation (St.
Louis, MO) and Fisher Scientific International, Inc. (Hampton, NH).
Over the years, a large number of naturally occurring and
synthetic PAHs, anthraquinones, stilbenoids, coumarins,
Received: May 11, 2014
Published: July 17, 2014
© 2014 American Chemical Society
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Figure 1. Structural optimization from ethynylcoumarins to ethynylflavones.
Mass spectral data were collected using an Agilent 6890 GC with a
5973 MS. 1H NMR and 13C NMR spectra were recorded on a Bruker
Fourier 300 MHz FT-NMR spectrometer and an Agilent 400 MHz
NMR spectrometer. The data was processed using MestReNova NMR
software (School of Chemistry, University of Bristol, Bristol, UK).
Elemental analyses were performed by Atlantic Microlab, Inc.
(Norcross, GA).
NMR (CDCl3, 75 HMz): 177.77, 163.72, 155.78, 131.86, 131.45,
129.13, 128.76, 127.73, 126.33, 125.82, 123.83, 121.76, 107.78, 81.97,
81.03. Anal. Calcd for C17H10O2: C, 82.91; H, 4.09; O, 12.99. Found:
C, 81.91; H, 4.22.
Preparation of 2′-Ethynylflavone (2′EF). To a solution of 500
mg (2.1 mmol) of 2′-hydroxyflavone in 15 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring on ice for 1 h, the
reaction mixture was transferred to a heating mantle. To this solution,
800 mg (1.14 mmol) of Pd(PPh3)2Cl2, 60 mg (0.32 mmol) of CuI,
and 40 mL of DIPA were added. After 10 min of stirring, 1.2 mL (8.43
mmol) of trimethylsilylacetylene was also added, and the reaction
mixture was refluxed for 2 h. After cooling down to room temperature,
the reaction mixture was concentrated by vacuum to a black residue
which was dissolved in a mixture of 10 mL of methanol and 10 mL of
diethyl ether. To start the final step, 1.0 mL (1 M in methanol, 1.0
mmol) of tetrabutylammonium fluoride was added. The reaction
mixture was stirred at 70 °C for 1.0 h and concentrated under vacuum.
The residue was purified using column chromatography with
petroleum ether/ethyl acetate 4:1 as the eluent to give 85 mg (yield,
16%) of 2′-ethynylflavone as yellow crystals. mp 106−108 °C. GC/
MS: 246 (M+, 100%), 218 (96), 189 (92), 92 (90). 1H NMR (CDCl3,
300 HMz): δ = 8.25 (dd, J = 8.1 Hz, J = 1.8 Hz, 1 H), 7.76−7.65 (m,
3H), 7.53−7.39 (m, 4H), 6.97 (s, 1H), 3.39 (s, 1H). 13C NMR
(CDCl3, 75 HMz): 177.41, 163.48, 155.94, 136.92, 131.79, 131.48,
129.84, 129.09, 126.30, 123.85, 119.50, 118.43, 107.94, 81.89, 78.37.
Anal. Calcd for C17H10O2: C, 82.91; H, 4.09; O, 12.99. Found: C,
81.67; H, 4.21.
Preparation of 3′-Ethynylflavone (3′EF). To a solution of 500
mg (2.1 mmol) of 3′-hydroxyflavone in 15 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring on ice for 1 h, the
reaction mixture was transferred to a heating mantle. To this solution,
800 mg (1.14 mmol) of Pd(PPh3)2Cl2, 60 mg (0.32 mmol) of CuI,
and 40 mL of DIPA were added. After 10 min of stirring, 1.2 mL (8.43
mmol) of trimethylsilylacetylene was also added, and the reaction
mixture was refluxed for 2 h. After cooling down to room temperature,
the reaction mixture was concentrated under vacuum to a black
residue which was dissolved in a mixture of 10 mL of methanol and 10
mL of diethyl ether. To start the final step, 1.0 mL (1 M in methanol,
1.0 mmol) of tetrabutylammonium fluoride was added. The reaction
mixture was stirred at 70 °C for 1.0 h and concentrated under vacuum.
The residue was purified using column chromatography with
petroleum ether/ethyl acetate 4:1 as the eluent to give 94 mg (yield,
18%) of 3′-ethynylflavone as a brown powder. mp 129−130 °C. GC/
MS: 246 (M+, 100%), 218 (55), 120 (50), 92 (60). 1H NMR (CDCl3,
400 HMz): δ = 8.24 (dd, J = 8.1 Hz, J = 1.8 Hz, 1 H), 8.12 (d, J = 8.6
Hz, 1H), 7.92 (d, J = 8.0 Hz1H), 7.72 (d, J = 8.6 Hz, 2H), 7.58 (dd, J
= 8.4 Hz, J = 0.8 Hz, 1H), 7.74 (dd, J = 8.1 Hz, 1.2 Hz, 1H), 6.82 (s,
1H), 3.24 (s, 1H). 13C NMR (CDCl3, 100 HMz): 177.35, 162.20,
156.76, 132.63, 132.48, 131.66, 131.29, 129.04, 126.20, 124.60, 121.00,
Preparation of 7-Ethynylflavone (7EF). To a solution of 500 mg
(2.1 mmol) of 7-hydroxyflavone in 10 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring on ice for 2 h, the
reaction mixture was quenched with 100 mL of ethyl acetate. The
reaction mixture was washed with 5% KHSO4 (50 mL × 8) and
saturated NaCl (50 mL × 2), dried over anhydrous MgSO4, and
concentrated under vacuum to give the crude product which was
recrystallized from 30 mL of anhydrous ethanol to give 545 mg (yield,
70%) of pure flavon-7-triflate as colorless crystals. GC/MS: 370 (M+,
1
95%), 209 ([M-CF3SO2]+, 100). H NMR (CDCl3, 300 HMz): δ =
8.35 (d, J = 8.7 Hz, 1H), 7.93 (m, 2H), 7.60−7.54 (m, 4H), 7.35 (dd, J
= 8.7 Hz, J = 2.1 Hz, 1H), 6.87 (s, 1H). 13C NMR (CDCl3, 75 HMz):
176.89, 164.25, 165.44, 132.15, 131.00, 129.22, 128.44, 126.38, 123.71,
120.82, 118.62, 116.57, 111.59, 107.95. Flavon-7-triflate (500 mg, 1.35
mmol) was dissolved in a mixed solution of 5 mL of anhydrous
pyridine and 40 mL of diisopropylamine (DIPA). To this solution, 800
mg (1.14 mmol) of bis(triphenylphosphine)palladium(II) dichloride
(Pd(PPh3)2Cl2) and 60 mg (0.32 mmol) of CuI were added. After 10
min of stirring, 1.2 mL (8.43 mmol) of trimethylsilylacetylene was also
added, and the reaction mixture was refluxed for 2 h. After cooling
down to room temperature, the reaction mixture was concentrated to a
black residue to which 100 mL of diethyl ether was added. A black
precipitate formed. After filtration, the filtrate was washed with 5%
KHSO4 (50 mL × 3) followed by saturated NaCl (20 mL × 2), dried
over anhydrous MgSO4, and concentrated under vacuum. The crude
7-trimethylsilylethynylflavone was purified using column chromatog-
raphy with petroleum ether/ethyl acetate 10:1 as the eluent to give
290 mg (yield, 68%) of silvery crystals. GC/MS: 318 (M+, 30%), 303
1
([M-CH3]+, 100). H NMR (CDCl3, 300 HMz): δ = 8.13 (d, J = 8.4
Hz, 1H), 7.88 (m, 2H), 7.66 (d, J = 1.2 Hz, 1H), 7.50 (m, 3H), 7.45
(dd, J = 8.4 Hz, J = 1.8 Hz, 1H), 6.79 (s, 1H), 0.28 (s, 9H). 13C NMR
(CDCl3, 75 HMz): 177.80, 163.52, 155.78, 131.75, 131.53, 129.09,
128.74, 128.62, 126.26, 125.62, 123.54, 121.37, 107.75, 103.12, 98.94,
−0.21. To a solution of 200 mg (0.63 mmol) of 7-trimethylsilylethy-
nylflavone in 10 mL of methanol and 10 mL of diethyl ether, 1.0 mL
(1 M in methanol, 1.0 mmol) of tetrabutylammonium fluoride was
added. The reaction mixture was stirred at 70 °C for 0.5 h and
concentrated under vacuum. The crude product was purified using
column chromatography with petroleum ether/ethyl acetate 3:1 as the
eluent to produce 112 mg (yield, 72%) of 7-ethynylflavone as a
yellowish powder. mp 170−173 °C. GC/MS: 246 (M+, 100%), 218
(45), 144 (30), 116 (28). 1H NMR (CDCl3, 300 HMz): δ = 8.16 (d, J
= 8.1 Hz, 1H), 7.91 (m, 2H), 7.69 (d, J = 1.2 Hz, 1H), 7.53 (m, 3H),
7.49 (dd, J = 8.1 Hz, J = 1.2 Hz, 1H), 6.83 (s, 1H), 3.31 (s, 1H). 13C
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118.92, 108.41, 83.35, 82.21. Anal. Calcd for C17H10O2: C, 82.91; H,
4.09; O, 12.99. Found: C, 81.33; H, 4.09.
60 mg (0.32 mmol) of CuI were added. After 10 min of stirring, 1.2
mL (8.43 mmol) of trimethylsilylacetylene was also added, and the
reaction mixture was refluxed for 2 h. After cooling down to room
temperature, the reaction mixture was concentrated by vacuum to a
black residue which was dissolved in a mixture of 10 mL of methanol
and 10 mL of diethyl ether. To start the final step, 1.0 mL (1 M in
methanol, 1.0 mmol) of tetrabutylammonium fluoride was added. The
reaction mixture was stirred at 70 °C for 0.5 h and concentrated under
vacuum. The residue was purified using column chromatography with
petroleum ether/ethyl acetate 3:1 as the eluent to give 120 mg
(37.38%) of the product. mp 157−160 °C. GC/MS: 246 (M+, 100%),
218 (40), 189 (30), 144 (30), 116 (50). 1H NMR (CDCl3, 400 HMz):
δ = 8.24 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 7.41−7.64 (m, 6H), 6.72 (s,
1H), 3.42 (s, 1H). 13C NMR (CDCl3, 100 HMz): 177.35, 162.20,
156.76, 132.63, 132.48, 131.66, 131.29, 129.04, 126.20, 124.60, 121.00,
118.92, 108.41, 83.31, 82.34. Anal. Calcd for C17H10O2: C, 82.91; H,
4.09; O, 12.99. Found: C, 82.04; H, 4.20.
Materials in Bioassays. Gentest human CYP1A1, CYP1A2,
CYP1B1, and CYP2A6 supersomes and rat CYP2B1 supersomes were
purchased from BD Biosciences (Franklin Lakes, NJ). D-Glucose-6-
phosphate sodium salt, β-nicotinamide adenine dinucleotide phos-
phate sodium salt (NADP+), and glucose-6-phosphate dehydrogenase
were purchased from Sigma-Aldrich Corporation. Other reagents in
bioassays were purchased from Fisher Scientific International, Inc.
Figures were plotted with Prism 6 (GraphPad Software, Inc., La Jolla,
CA).
Fluorimetric Enzyme Inhibition Assays of P450s 1A1, 1A2,
1B1, 2A6, and 2B1. The inhibition activities of the target compounds
toward P450s 1A1-, 1A2-, 1B1-, 2A6-, and 2B1-dependent reactions
were tested through standard methods as previously described.21,22
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 1.6 nM for P450
1A1 and 5 nM for P450s 1A2, 1B1, 2A6, and 2B1), 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
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 535 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 gradually diluted inhibitor solutions. At least four concentrations
of each inhibitor showing 20−80% inhibition were tested.
Preparation of 4′-Ethynylflavone (4′EF). To a solution of 500
mg (2.1 mmol) of 4-hydroxyflavone in 10 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring on ice for 2 h, the
reaction mixture was transferred to a heating mantle. To this solution,
800 mg (1.14 mmol) of Pd(PPh3)2Cl2, 60 mg (0.32 mmol) of CuI,
and 40 mL of DIPA were added. After 10 min of stirring, 1.2 mL (8.43
mmol) of trimethylsilylacetylene was also added, and the reaction
mixture was refluxed for 2 h. After cooling down to room temperature,
the reaction mixture was concentrated under vacuum to a black
residue which was dissolved in a mixture of 10 mL of methanol and 10
mL of diethyl ether. To start the final step, 1.0 mL (1 M in methanol,
1.0 mmol) of tetrabutylammonium fluoride was added. The reaction
mixture was stirred at 70 °C for 0.5 h, and concentrated under
vacuum. The residue was purified using column chromatography with
petroleum ether/ethyl acetate 3:1 as the eluent to give 80 mg (yield,
15%) of 4-ethynylflavone as a brown powder. mp 164 °C decomposed.
GC/MS: 246 (M+, 100%), 218 (35), 120 (35). 1H NMR (CDCl3, 300
HMz): δ = 8.24 (dd, J = 8.1 Hz, J = 1.2 Hz, 1H), 7.91 (d, J = 8.7 Hz,
2H), 7.73 (ddd, J = 8.4 Hz, J = 8.4 Hz, J = 1.5 Hz, 1H), 7.65 (d, J = 8.7
Hz, 2H), 7.58 (dd, J = 8.4 Hz, J = 0.6 Hz, 1H), 7.73 (ddd, J = 8.1 Hz, J
= 8.1 Hz, J = 1.2 Hz, 1H), 6.85 (s, 1H), 3.27 (s, 1H). 13C NMR
(CDCl3, 75 HMz): 178.35, 162.45, 156.21, 134.00, 133.32, 132.72,
131.82, 126.16, 125.75, 125.43, 123.89, 118.10, 107.96, 82.72, 80.16.
Anal. Calcd for C17H10O2: C, 82.91; H, 4.09; O, 12.99. Found: C,
82.50; H, 4.26.
Preparation of 6-Ethynylflavone (6EF). To a solution of 500 mg
(2.1 mmol) of 6-hydroxyflavone in 10 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring on ice for 1 h, the
reaction mixture was transferred to a heating mantle. To this solution,
800 mg (1.14 mmol) of Pd(PPh3)2Cl2, 60 mg (0.32 mmol) of CuI,
and 40 mL of DIPA were added. After 10 min of stirring, 1.2 mL (8.43
mmol) of trimethylsilylacetylene was also added, and the reaction
mixture was refluxed for 2 h. After cooling down to room temperature,
the reaction mixture was concentrated under vacuum to a black
residue which was dissolved in a mixture of 10 mL of methanol and 10
mL of diethyl ether. To start the final step, 1.0 mL (1 M in methanol,
1.0 mmol) of tetrabutylammonium fluoride was added. The reaction
mixture was stirred at 70 °C for 1.0 h and concentrated under vacuum.
The residue was purified using column chromatography with
petroleum ether/ethyl acetate 4:1 as the eluent to give 72 mg (yield,
14%) of 6-ethynylflavone as a blue powder. mp 154−156 °C. GC/MS:
246 (M+, 100%), 218 (15), 144 (95), 116 (60). 1H NMR (CDCl3, 300
HMz): δ = 8.34 (d, J = 1.8 Hz, 1 H), 7.93−7.89 (m, 1H), 7.78 (m,
1H), 7.55−7.51 (m, 4H), 6.82 (s, 1H), 3.14 (s, 1H). 13C NMR
(CDCl3, 100 HMz): 177.41, 163.48, 155.94, 136.92, 131.79, 131.48,
129.84, 129.09, 126.30, 123.85, 119.50, 118.43, 107.94, 81.89, 78.37.
Anal. Calcd for C17H10O2: C, 82.91; H, 4.09; O, 12.99. Found: C,
80.86; H, 4.53.
Preparation of 5-Ethynylflavone (5EF). To a solution of 500 mg
(2.1 mmol) of 5-hydroxyflavone in 15 mL of anhydrous pyridine
under nitrogen atmosphere and cooling in an ice bath, 1.0 mL (5.9
mmol) of triflic anhydride was added. After stirring at room
temperature for 3 days, the reaction mixture was quenched with 100
mL of ethyl acetate. The reaction mixture was washed with 5% KHSO4
(50 mL × 8) and saturated NaCl (50 mL × 2), dried over anhydrous
MgSO4, and concentrated under vacuum to give the crude product
which was recrystallized from 30 mL of anhydrous ethanol to give 560
mg (yield, 72%) of pure flavon-5-triflate as colorless crystals. GC/MS:
Data Analysis. Ki 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 Ki values (x-intercepts)
for the inhibitors. The results based on the first 6 min of the enzymatic
1
370 (M+, 95%), 209 ([M-CF3SO2]+, 100). H NMR (CDCl3, 300
HMz): δ 7.91−7.87 (m, 2H), 7.75−7.63 (m, 2H), 7.58−7.51 (m, 3H),
7.24 (d, J = 1.2 Hz, 1H), 6.79 (s, 1H). 13C NMR (CDCl3, 75 HMz):
176.00, 162.83, 157.21, 146.84, 133.35, 132.10, 130.77, 129.19, 126.32,
120.96, 119.11, 118.87, 117.84, 116.71, 108.74. Flavon-5-triflate (500
mg, 1.35 mmol) was dissolved in a mixture of 10 mL of anhydrous
pyridine and 40 mL of DIPA. To this solution, 800 mg (1.14 mmol) of
bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh3)2Cl2) and
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Figure 2. Ki values of αNF, 7E3PC, and ethynylflavones for the inhibition of P450s 1A1, 1A2, and 1B1. For convenience, the ordinate axis was
plotted on a logarithmic scale. Ki values are represented as the mean SE micromolar of three independent experiments. The exact Ki values are
listed in Supporting Information, Table S1.
a
Table 1. KI and Limiting kinact Values of Ethynylflavones for the Inhibition of P450 1A1
b
2′EF
3′EF
4′EF
5EF
6EF
7EF
KI (μM)
limiting kinact (min−1
0.053 0.007
0.46 0.02
0.056 0.014
0.33 0.08
0.51 0.09
0.043 0.008
0.33 0.03
0.035 0.012
0.42 0.04
)
0.066 0.023
a
b
The KI and limiting kinact values are represented as the mean SE μM of three independent experiments. 2′EF is not a time-dependent inhibitor of
P450 1A1.
a
Scheme 1. Synthesis and Structures of Ethynylflavones
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.
reactions are tabulated in Table S1 of Supporting Information. The
data are represented as the mean SE micromolar of three
independent experiments. Figure 2 plots the Ki values of the tested
compounds for the inhibition of P450s 1A1, 1A2, and 1B1 as a column
chart.
versus reciprocals of the inhibitor concentration (1/[I]) (Kitz−Wilson
plots). The limiting kinact values were 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 time-dependent
inhibitors for P450 1A1 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) represent the
enzymatic activity over time. The semilog plots of the percent relative
activity (Y = log[(y/y0) × 100]) versus time demonstrate the
centesimal loss of enzymatic activity with time. The linear portions of
the above semilog plots were used to determine t1/2 values (the time of
enzymes losing half of their activities, which equals 0.693/kinact) at
various concentrations for the observed time-dependent losses of
activity. To obtain KI and limiting kinact values, 1/kinact were plotted
NADPH-Dependency Assay. All assay solution components had
the same concentrations as in the above fluorimetric enzyme inhibition
assays. For preincubation assays in the presence of NADPH,
potassium phosphate buffer (1560 μL, 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 (the concentration leading to approximately 20% enzymatic
activity inhibition), and 200 μL of a NADPH regenerating solution.
The assay mixture was incubated for 5 min at 37 °C before reaction
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Figure 3. Effects of NADPH preincubation on the production of resorufin by P450 1A1 in the presence of ethynylflavones. Five-minute
preincubation was applied in the presence (red triangles) and absence (blue squares) of NADPH for each test. The concentrations used for 2′EF,
3′EF, 4′EF, 5EF, 6EF, and 7EF were 0.1 μM, 0.00625 μM, 0.00625 μM, 0.1 μM, 0.00625 μM, and 0.00625 μM, respectively. For 2′EF, preincubation
with NADPH exhibited a slight influence on enzymatic activity of P450 1A1. For 5EF, preincubation with NADPH caused a considerable inhibition
of P450 1A1. Whereas for 3′EF, 4′EF, 6EF, and 7EF, 5 min preincubation with NADPH (0.00625 μM of inhibitor) lead to a complete inhibition of
P450 1A1.
initiation by the addition of 200 μL of buffer and 10 μL of the
RESULTS
■
corresponding substrate solution. For the preincubation assays in the
Synthesis of Ethynylflavones. Ethynylflavones were
absence of NADPH, potassium phosphate buffer (1760 μL, pH 7.6)
synthesized from hydroxyflavones through a similar three-step
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
procedure described previously17 (Scheme 1). In the synthesis
of 7EF, each intermediate was purified and described in detail
above, as an example. In the syntheses of 2′EF, 3′EF, 4′EF, and
6EF, intermediates were not purified and rather directly used in
the next steps. Thus, a simplified method was applied in the
syntheses of these compounds. In the synthesis of 5EF, the first
reaction is extremely slow due to the influence of an
intramolecular hydrogen bond. Thus, the synthesis of 5EF
was also described separately in detail. All spectra of target
compounds were supplied in the Supporting Information.
Ethynylflavones’ Inhibition of P450s 1A1, 1A2, and
1B1. Effects of the target ethynylflavones on P450 1A1-
mediated ethoxyresorufin O-deethylation (EROD), 1A2-
mediated methoxyresorufin O-demethylation (MROD), and
1B1-mediated EROD were determined through fluorescence
enzyme assays, and the data are shown in Figure 2. 7-Ethynyl-3-
phenylcoumarin (7E3PC) and α-naphthoflavone (αNF,
Scheme 1) serve as positive controls for the convenient
comparison with the previous data.17,19
As is shown in Figure 2, ethynylflavones 3′EF, 4′EF, 6EF,
and 7EF possess much stronger inhibitory activities toward
P450 1A1 (Ki values ranging from 0.024 to 0.041 μM) than 7-
ethynyl-3-phenylcoumarin (Ki value of 0.74 μM). However,
2′EF and 5EF exhibit a relatively weak inhibition of P450 1A1
(Ki, 1.64 and 1.23 μM, respectively). Time-course curves show
that 3′EF, 4′EF, 5EF, 6EF, and 7EF time- and concentration-
dependently inhibit P450 1A1, suggesting that they are time-
dependent inhibitors (TDIs). Of these five TDIs, 3′EF and 7EF
possess the highest kinact values which will be discussed below.
an inhibitor solution in DMSO (the concentration leading to
approximately 20% enzymatic activity inhibition). The assay mixture
was incubated for 5 min 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.
Computational Chemistry. Docking simulations of the ethynyl-
flavones with human P450s 1A1 and 1A2 were performed using the
LigandFit module in Accelrys Discovery Studio 3.5 (Accelrys, San
Diego, CA). The crystal structures of human cytochrome P450s 1A1
and 1A2 in complex with α-naphthoflavone (PDB ID: 4I8V and 2HI4)
are available from the Protein Data Bank (PDB).23,24 For the crystal
structure of P450 1A1, subunit A was used for docking simulations.
Water molecules in the crystal structures were removed, and hydrogen
atoms were added to the P450 templates under the CHARMm force
field. The 3D structures of ethynylflavones 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. To explore the binding
modes of the 3D-structures of ethynylflavones to P450s 1A1 and 1A2,
the docking program LigandFit was used to automatically dock the
ligands into the active site cavities of the enzymes. In the docking
process, the standard flexible docking protocol was performed. Ten
conformers of each molecule were automatically formed, which are the
best fit into the defined active site cavities.
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Figure 4. Stereoviews of compounds αNF (A), 2′EF (B), 3′EF (C), 5EF (D), and 6EF (E) with P450 1A1. The protein is shown as ribbons (α-helix
I in pink for emphasis), while the heme and ligands are shown as sticks. Selected amino acid residues around the active site cavity are labeled and
exhibited as sticks. αNF is the standard ligand in the crystal structure of P450 1A1 (PDB ID: 4I8V). To compare the relative positions between αNF
and the other compounds, the surface representation of αNF is shown in each image. The acetylene groups of 3′EF, 5EF, and 6EF point toward the
heme, while that of 2′EF is pointing away from the heme.
4′EF and 6EF have relatively weak inhibition toward P450
1A2, with the Ki values of 3.26 and 6.20 μM, respectively. While
2′EF, 3′EF, 5EF, and 7EF exhibit moderately strong inhibition
of P450 1A2 (Ki values ranging from 0.23 to 0.81 μM). 2′EF is
the most potent inhibitor toward P450 1A2 among the six
tested compounds, with a Ki value of 0.23 μM (Figure 2).
As for P450 1B1, the ethynylflavones show small variations in
their inhibition activities, with Ki values ranging from 0.13 to
0.61 μM. Interestingly, none of the compounds showed time-
dependent inhibition toward P450s 1A2 and 1B1.
Effects of Ethynylflavones on P450s 2A6 and 2B1. To
determine the inhibitory activities of the ethynylflavones toward
P450s 2A6 and 2B1, coumarin 7-hydroxylation and pentoxy-
resorufin O-depentylation (PROD) assays were performed,
respectively. 2′EF showed a weak inhibition toward P450 2B1
with a Ki value of 15.7 μM, while no inhibition was observed for
the other compounds with concentrations under 25 μM,
suggesting selectivity of the ethynylflavones toward family I
over family II P450 enzymes.
3′EF, 4′EF, 5EF, 6EF, and 7EF Time- and NADPH-
Dependent Inhibition of P450 1A1. The time-course curves
of the tested compounds in NADPH-dependency assays are
summarized in Figure 3. Among the six compounds, 3′EF,
4′EF, 5EF, 6EF, and 7EF exhibit NADPH-dependency in the
inhibition toward P450 1A1, suggesting that 3′EF, 4′EF, 5EF,
6EF, and 7EF are potential mechanism-based inhibitors (MBIs)
of P450 1A1. However, 2′EF does not NADPH-dependently
inhibit P450 1A1. This type of assay was also performed for
P450s 1A2, 1B1, 2A6, and 2B1. No time-dependent inhibition
was observed for these enzymes. The kinetic parameters of
TDIs 3′EF, 4′EF, 5EF, 6EF, and 7EF in the inhibition of P450
1A1 were determined through a 6 min continuous observation
with various concentrations of the inhibitors. The data are listed
in Table 1. 3′EF and 7EF possess the highest limiting kinact
values, 0.46
0.02 and 0.42
0.04 min−1, respectively,
suggesting that 3′EF and 7EF have very high affinity toward
P450 1A1 and that their ethynyl groups tend to react with the
active site of this enzyme. 5EF exhibits a relatively poor affinity
toward and reaction rate with P450 1A1, with a KI value of 0.51
μM and a kinact value of 0.066 min−1.
Computational Simulation of Ethynylflavones Bind-
ing to P450s 1A1 and 1A2. To investigate the interaction
between target compounds and P450 enzymes 1A1 and 1A2,
the docking simulations were performed with the LigandFit
protocol in the Accelrys Discovery Studio described above.
After 3D structure sketching and energy minimization, the
standard ligand, α-naphthoflavone, as well as test compounds
were docked into the active site cavities of P450s 1A1 and 1A2
in order to obtain the binding patterns. Figure 4A−E shows the
docking postures of α-naphthoflavone, 2′EF, 3′EF, 5EF, and
6EF with the crystal structure of P450 1A1 (PDB ID: 4I8V).
These images demonstrate that certain ethynylflavones such as
3′EF, 5EF, and 6EF dock into P450 1A1 with the acetylene
group oriented toward the reactive center, heme. On the
contrary, the acetylene group of 2′EF is oriented away from the
heme when docked with P450 1A1. These observations explain
why 3′EF, 4′EF, 5EF, 6EF, and 7EF are TDIs of P450 1A1,
while 2′EF is only a competitive inhibitor of this enzyme since
close interaction between the acetylene group and the heme is
essential for the metabolism of these compounds by the
enzyme into active intermediates which bind the protein and
lead to the inactivation of the enzyme.
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Figure 5. Stereoviews of compounds αNF (A), 2′EF (B), 3′EF (C), 5EF (D), and 6EF (E) with P450 1A2. The protein is shown as ribbons (α-helix
I in pink for emphasis), while the heme and ligands are shown as sticks. Selected amino acid residues around the active site cavity are labeled and
exhibited as sticks. αNF is the standard ligand in the crystal structure of P450 1A2 (PDB ID: 2HI4). To compare the relative positions between αNF
and the other compounds, the surface representation of αNF is shown in each image. The acetylene groups of all docked compounds are pointing
away from the heme.
Figure 5A−E shows the docking postures of α-naphtho-
flavone, 2′EF, 3′EF, 5EF, and 6EF with the crystal structure of
P450 1A2 (PDB ID: 2HI4). As seen in these docking images,
the acetylene group of all tested compounds are away from the
heme, indicating that they are not TDIs of this enzyme. These
docking simulation studies show the importance of relative
position between the heme and the acetylene group of a TDI.
In our projects focused on the discovery of new P450
inhibitors, the active site cavities of P450 enzymes were
thoroughly investigated. In our recent studies, evidence shows
that P450 1A1 has a planar long-strip active site cavity and that
P450 1A2 owns a planar triangle cavity.19,20 As to the long-strip
molecules 4′EF and 6EF in this study, they exhibit 85-fold and
177-fold higher selectivity toward P450 1A1 over P450 1A2,
respectively (Figure 6). However, the triangle molecule 2′EF
shows a 7.2-fold higher selectivity toward P450 1A2 over 1A1.
These observations are consistent with the results of our
previous studies.19
DISCUSSION
■
Considering that P450 family I enzymes play an important role
in carcinogenesis, discovering selective inhibitors of these
enzymes is of great value. Superior to competitive inhibitors,
mechanism-based inhibitors exhibit higher potency by covalent
binding to the target enzymes and completely and irreversibly
inactivating them. In this study, we identified five TDIs (3′EF,
4′EF, 5EF, 6EF, and 7EF) of P450 1A1. They are probably
MBIs since they show time-, concentration-, and NADPH-
dependent inhibition of P450 1A1, and the ethynyl functional
group is a typical residue of an MBI. Some of these compounds
exhibit compatible IC50 values to well-known P450 1A1
inhibitors, αNF and pyrene (both Kis around 0.04 μM).19,25
Especially, compounds 3′EF and 7EF possessing kinact values of
0.46 0.02 and 0.42 0.04 min−1, respectively, irreversibly
inactivate half of the P450 1A1 enzymes in less than 2 min
(their limiting t1/2 values are 1.52 and 1.65 min, respectively).
This dramatically strong inactivation can be directly observed in
the NADPH-dependency assays (Figure 3). With preincubation
in the presence of NADPH for 5 min, 0.00625 μM of 3′EF or
7EF completely subvert the enzymatic activity of P450 1A1 in
EROD assays.
Figure 6. Inhibitory selectivity of ethynylflavones for P450 1A1 over
P450 1A2 shows dependence on the molecular shape. (A)
Superimposed 4′EF and 6EF; (B) superimposed 3′EF, 5EF, and
7EF; (C) compound 2′EF. Ki(1A2)/Ki(1A1) is the ratio of the Ki values of
a compound against P450 1A2 to that of P450 1A1.
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In general, the introduction of an acetylene group decreases
the inhibitory activity of the flavone derivatives toward P450
1B1, compared with αNF and other flavone derivatives
previously reported.19 Thus, for the compounds reported
here, considerable selectivity toward P450 1A1 over P450 1B1
was observed. For example, 6EF and 7EF exhibit 15- and 18-
fold higher selectivity toward P450 1A1 over P450 1B1.
The introduction of an acetylene group seems to increase the
potential of the compounds for acting as inhibitors of P450s
1A1 and 1A2. At the same time, these compounds also provide
us with a tool to investigate the interactions between the small
molecules and the enzymes. Docking simulations in this study
show that TDIs of P450 1A1 bind with the enzyme’s active site
cavity with the acetylene group facing toward the heme,
whereas for all the tested molecules docked into the active site
cavity of P450 1A2, the acetylene group is oriented away from
the heme leading to the compounds acting as competitive
inhibitors of this enzyme. Because of the orientation of a TDI,
its binding pattern(s) could be easily determined compared
with that of other types of inhibitors. Thus, as expected, TDIs
are useful tools for the investigation of enzyme−ligand
interactions.
flavone; PROD, pentoxyresorufin O-depentylation; MBIs,
mechanism-based inhibitors; TDIs, time-dependent inhibitors
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra, elemental analysis results, and
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AUTHOR INFORMATION
Corresponding Author
*Phone: 504-520-5078. Fax: 504-520-7942. E-mail: mforooze@
xula.edu.
■
Funding
This work was supported by DoD Award W81XWH-11-1-
0105. We thank NIH-MBRS SCORE (grant number S06 GM
008008) 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-NIMHD
grant 8G12MD007595 for their support of the Major
Instrumentation at Xavier University of Louisiana. We also
acknowledge the NIH-NIGMS supported RISE and MARC
Programs at Xavier University of Louisiana (grant numbers
2R25GM060926-09 and 2T34GM007716) for the support of
our undergraduate students working on this project.
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not necessarily represent the official views of the Louisiana
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ABBREVIATIONS
■
PAHs, polycyclic aromatic hydrocarbons; PHAH, polyhalo-
genated aromatic hydrocarbons (PHAHs); IQ, 2-amino-3-
methylimidazo[4,5-f ]quinolone; PhIP, 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine; 7EC, 7-ethynylcoumarin;
7E3PC, 7-ethynyl-3-phenylcoumarin; EF, ethynylflavone;
DIPA, d iisopropylamine; Pd (PPh3 )2 C l 2 , b i s-
(triphenylphosphine)palladium(II) dichloride; NADP+, β-nic-
otinamide adenine dinucleotide phosphate sodium salt; DMSO,
dimethyl sulfoxide; EROD, ethoxyresorufin O-deethylation;
MROD, methoxyresorufin O-demethylation; αNF, α-naphtho-
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