Chemical Research in Toxicology
Article
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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dx.doi.org/10.1021/tx5001865 | Chem. Res. Toxicol. 2014, 27, 1431−1439