Journal of the American Chemical Society
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12.4 Hz), 6.99 (1H, d, J = 9.2 Hz), 6.03 (1H, d, J = 12.4 Hz), 5.21
(2H, s), 1.40 (9H, s).
continued for 10 min, then the mixture was diluted with AcOEt and
washed with 10% citric acid. The organic layer was evaporated in
vacuo. The residue was taken up in sat. NaHCO3 and extracted with
CHCl3. The organic layer was washed with brine and dried over
Na2SO4. Filtration, evaporation in vacuo, and purification of the
residue by silica gel flash chromatography (n-hexane:AcOEt = 2:1 →
AcOEt only → AcOEt:MeOH = 100:1 → AcOEt:MeOH = 20:1) gave
113 mg (44%) of NOBL-1 as a red solid. This was recrystallized from
n-hexane and CHCl3 to obtain 75 mg (66%) of green crystals: mp
195.3−198.3 °C; 1H NMR (DMSO-d6, 500 MHz, δ; ppm) 12.08 (1H,
brs), 9.82 (1H, s), 7.36 (2H, d, J = 8.2 Hz), 7.32 (2H, d, J = 8.2 Hz),
7.29 (1H, d, J = 2.4 Hz), 7.15 (1H, dd, J = 2.7 Hz, 8.7 Hz), 6.82 (8.5
Hz), 5.38 (2H, s), 2.75 (2H, t, J = 7.8 Hz), 2.63 (6H, s) 2.48 (2H, t, J
= 7.8 Hz), 1.39 (6H, s); 13C NMR (DMSO-d6, 125 MHz, δ; ppm)
173.7, 158.8, 154.9, 149.1, 147.1, 137.1, 132.0, 130.7, 130.7, 129.2,
127.7, 127.5, 123.3, 120.9, 114.9, 113.3, 105.4, 46.8, 33.1, 25.2, 13.5,
13.3. Anal. Calcd for C31H27BF2N6O4·4/3H2O: C, 60.01; H, 4.82; N,
13.55. Found: C, 60.29; H, 4.79; N, 13.28. MS (FAB): m/z 597 [(M +
1)+].
Synthesis of 5. A slurry of 4 (164 mg, 0.462 mmol) and 5% Pd−C
(97 mg) in MeOH (5 mL) was stirred at room temperature under H2
for 18 h. The reaction mixture was filtered on Celite and the filtrate
was evaporated in vacuo. The residue was purified by silica gel flash
chromatography (n-hexane:AcOEt = 1:1) to obtain 86 mg (79%) of 4
as a pale yellow solid: 1H NMR (CDCl3, 500 MHz, δ; ppm) 6.95 (1H,
s), 6.72 (1H, d, J = 8.4 Hz), 6.49 (1H, dd, J = 2.8 Hz, 8.4 Hz),
6.45(1H, d, J = 2.8 Hz), 3.36 (2H, s), 2.77 (2H, t, J = 6.3 Hz), 2.60
(2H, t, J = 6.3 Hz), 1.41 (9H, s).
Synthesis of 7. To a slurry of 6 (5.00 g, 33.3 mmol) in CH2Cl2
(200 mL) was added N-methylmorpholine (3.66 mL, 33.3 mmol, 1.0
equiv), followed by EDC·HCl (7.68 g, 40.1 mmol, 1.2 equiv) and
NHMeOMe·HCl (3.60 g, 36.9 mmol, 1.1 equiv). The mixture was
stirred at room temperature for 4.5 h, and then evaporated in vacuo.
The residue was taken up in Et2O and the mixture was washed with
water and brine. The organic layer was dried over Na2SO4. Filtration
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and evaporation in vacuo gave 4.31 g (67%) of 7 as a yellow oil: H
NMR (CDCl3, 500 MHz, δ; ppm) 10.07 (1H, s), 7.93 (2H, d, J = 8.4
Hz), 7.81 (2H, d, J = 8.2 Hz), 3.54 (3H, s), 3.39 (3H, s).
Synthesis of 13. To a solution of benzaldehyde (100 μL, 0.981
mmol) in CH2Cl2 (10 mL) was added 8 (118 mg, 0.982 mmol, 2.0
equiv), followed by TFA (3 drops). The reaction mixture was stirred at
room temperature for 12 h, and DDQ (223 mg, 0.982 mmol, 2.0
equiv) was added to it. Stirring was continued at room temperature for
20 min, and then DIPEA (854 μL, 4.90 mmol, 10 equiv) was added,
followed by BF3·OEt2 (1.31 mL, 4.91 mmol, 10 equiv). Stirring was
continued at room temperature for 10 min, and the reaction mixture
was quenched with water. The mixture was extracted with CHCl3. The
organic layer was washed with brine and dried over Na2SO4. Filtration,
evaporation in vacuo, and purification of the residue by silica gel flash
chromatography (n-hexane:AcOEt = 8:1) gave 87 mg (48%) of 13 as a
dark green solid. This solid was washed with AcOEt to obtain 58 mg of
Synthesis of 9. To a solution of 7 (1.26 g, 6.51 mmol) and 8 (1.55
g, 12.9 mmol, 2.0 equiv) in CH2Cl2 (50 mL) was added TFA (0.2
mL). The mixture was stirred at room temperature for 19 h, and then
DDQ (2.21 g, 9.71 mmol, 1.5 equiv) was added to it. Stirring was
continued for a further 20 min, then BF3·OEt2 (8.5 mL, 32.4 mmol,
5.0 equiv) was added, followed by DIPEA (6.0 mL, 34.4 mmol, 5.3
equiv) on an ice−water bath. Stirring was continued for a further 10
min on the ice−water bath, then the reaction mixture was quenched
with water and filtered. The organic layer was separated, and the water
layer was extracted with CHCl3. The combined organic layer was
washed with brine and dried over Na2SO4. Filtration, evaporation in
vacuo, and purification of the residue by silica gel flash chromatography
(n-hexane:AcOEt = 1:1 → 1:2) gave 1.92 g (64%) of 9 as a dark red
solid: 1H NMR (CDCl3, 500 MHz, δ; ppm) 7.88 (2H, d, J = 8.2 Hz),
7.35 (2H, d, J = 8.3 Hz), 4.28 (4H, q, J = 7.1 Hz), 3.51 (3H, s), 3.42
(3H, s), 2.84 (6H, s), 1.68 (6H, s), 1.33 (6H, t, J = 7.1 Hz).
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purified 13 as a brick-colored solid: mp 279.6−282.5 °C; H NMR
(CDCl3, 500 MHz, δ; ppm) 7.62−7.61 (3H, m), 7.27 (2H, m), 2.73
(6H, s), 1.58 (6H, s); 13C NMR (CDCl3, 150 MHz, δ; ppm) 159.7,
149.5, 147.1, 132.6, 131.5, 130.6, 130.1, 127.0, 113.5, 106.5, 13.8. Anal.
Calcd for C21H17BF2N4: C, 67.40; H, 4.58; N, 14.97. Found: C, 67.24;
H, 4.75; N, 14.75. MS (EI): m/z 374 [M+].
Synthesis of 10. To a solution of 9 (206 mg, 0.447 mmol) in dry
THF (10 mL) was added Cp2ZrHCl (142 mg, 0.551 mmol, 1.2 equiv).
The mixture was stirred for 5 min, and then quenched with water. The
mixture was extracted with CHCl3. The organic layer was washed with
brine and dried over Na2SO4. Filtration, evaporation in vacuo, and
purification of the residue by silica gel flash chromatography (n-
ESR Analysis Using Iron Dithiocarbamate Complex. A
solution (total volume 200 μL) of FeSO4·6H2O (1.5 mM), N-
methylglucamine dithiocarbamate (6 mM) and NOBL-1 (100 μM) in
PBS (pH 7.0, DMF 1%) was photoirradiated (470−500 nm, 200 mW/
cm2) at room temperature for 15 min as a sample for ESR studies. ESR
spectra were taken on a JES-RE2X spectrometer (JEOL Co. Ltd.,
Tokyo, Japan). The measurement conditions were follows: microwave
power, 10 mW; frequency, 9.4200 GHz; field, 330 mT; sweep width,
7.5 mT; sweep time, 4 min; modulation width, 0.125 mT; time
constant; 0.10 s.
Fluorescence Measurement of DAR-4M. Sample solutions
(total volume 2 mL) containing NOBL-1 (10 μM) and DAR-4M (10
μM) in PBS (pH 7.4, containing 0.3% DMSO) were irradiated or
incubated in the dark. The fluorescence intensity was determined with
an RF5300-PC (Shimadzu) at 575 nm, with excitation at 560 nm. The
slit width was set to 1.5 nm for both excitation and emission.
Detection of NO in HEK293 Cells. HEK293 cells were plated on
3.5 cm glass dishes at 2.0 × 105 cells/dish with 2 mL of Dulbecco’s
Modified Eagle Medium (DMEM). The cells were incubated at 37 °C
in a humidified atmosphere of 5% (v/v) CO2 in air for 2 days. The
medium was replaced with 2 mL of fresh DMEM. The cells were
incubated with 10 μM NOBL-1 (DMSO 0.1%) or DMSO for 1 h
under the above conditions. Then, the medium was replaced with 2
mL of fresh DMEM, and the cells were washed with 2 mL of DMEM
twice and treated with 5 μM DAR-4M AM (DMSO 0.1%) for 30 min
under the above conditions. The medium was replaced with 2 mL of
Dulbecco’s PBS containing Mg2+ and Ca2+ (D-PBS). The cells were
washed with 2 mL of D-PBS and irradiated with a MAX-302 (470−
500 nm, 25 mW/cm2) for 5 min. After irradiation, the cells were
examined under a differential interference contrast microscope
(Olympus, IX71).
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hexane:AcOEt = 2:1) gave 126 mg (70%) of 10 as a red solid: H
NMR (CDCl3, 500 MHz, δ; ppm) 10.37 (1H, s), 8.31 (2H, d, J = 8.3
Hz), 7.74 (2H, d, J = 8.1 Hz), 4.51 (4H, q, J = 7.1 Hz), 3.07 (6H, s),
1.87 (6H, s), 1.55 (6H, t, J = 7.1 Hz).
Synthesis of 11. A solution of 5 (130 mg, 0.549 mmol, 1.0 equiv),
10 (221 mg, 0.550 mmol), and AcOH (0.5 mL) in CH2Cl2 (5 mL)
was stirred at room temperature for 2 h, then NaBH(OAc)3 (353 mg,
1.67 mmol, 3.0 equiv) was added to it and stirring was continued for
10 min. The reaction mixture was quenched with water and extracted
with CHCl3. The organic layer was washed with brine and dried over
Na2SO4. Filtration, evaporation, and purification of the residue by
silica gel flash chromatography (n-hexane:AcOEt =2:1 → 3:2) gave
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283 mg (83%) of 11 as a dark green solid: H NMR (CDCl3, 500
MHz, δ; ppm) 7.61 (2H, d, J = 8.1 Hz), 7.22 (2H, d, J = 8.1 Hz), 6.96
(1H, brs), 6.75 (1H, d, J = 8.6 Hz), 6.43 (1H, dd, J = 2.9 Hz, 8.5 Hz),
6.38 (1H, d, J = 2.8 Hz), 4.43 (2H, s), 2.76 (2H, t, J = 6.2 Hz), 2.72
(6H, s), 2.58 (2H, t, J = 6.2 Hz), 1.59 (6H, s), 1.42 (9H, s).
Synthesis of NOBL-1. A solution of 11 (273 mg, 0.438 mmol) in
4 N HCl AcOEt (7 mL) and AcOEt (3 mL) was stirred at room
temperature for 19.5 h. The reaction mixture was neutralized with 2 N
NaOH and sat. NaHCO3, and extracted with AcOEt. The organic
layer was washed with brine and dried over Na2SO4. Filtration,
evaporation, and purification of the residue by silica gel flash
chromatography (CHCl3:MeOH = 100:1 → 30:1 → 10:1) gave 247
mg (crude) of 12 as a dark green solid. A solution of 12 (crude 247
mg) in AcOH (6 mL) was added to a solution of NaNO2 (34 mg,
0.493 mmol) in water (6 mL) on an ice−water bath. Stirring was
F
dx.doi.org/10.1021/ja5020053 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX