Journal of Medicinal Chemistry
Article
formaldehyde (5 mL). The obtained mixture was allowed to stir at r.t.
for 3 days. The reaction was quenched by adding a cool diluted
hydrochloric acid solution (20 mL). The mixture was extracted by
EtOAc (30 mL × 3), and the combined organic layers were washed
with brine (30 mL × 3) and dried by anhydrous sodium sulfate. The
dry solvent was concentrated under vacuum, and the residue was
purified by flash chromatography (petroleum ether/EtOAc, 10:1 v/v)
Care and Use Committee (IACUC) of Chine Pharmaceutical
University (SYXK (SU) 2016-0011).
Measurement of NO−2 . Analysis of NO−2 contents in vitro was
performed by the Griess assay. Briefly, the tested compounds in
DMSO were diluted into 50 μM in saline, buffer (various molarities of
nucleophiles), or bovine plasma containing 5% DMSO. The samples
were incubated at 37 °C for 24 h. Individual samples (50 μL each)
were mixed in triplicate with the Griess reagent (150 μL) and
incubated at 37 °C for 10 min followed by measurement of the
absorbance at 540 nm. The levels of NO−2 in vivo were determined by
ion chromatography.24 Briefly, individual male SD rats were injected
iv with a single dose of each compound (4a: 10 mg/kg or NaNO2:
2.44 mg/kg, N = 3 per group), and their plasma samples were
collected longitudinally. Individual plasma samples (50 μL) were
immediately mixed with 2.5 ng of oroxylin A (an internal lane
standard, Sigma, St. Louis, USA) in 200 μL of methanol and
centrifuged at 20,000g for 10 min. Their supernatants (10 μL each)
were used for ion chromatography.
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to get 8 as a yellowish oil in a yield of 41%. H NMR (300 MHz,
CDCl3): δ 8.22 (s, 1H), 7.48−7.58 (m, 5H), 4.71 (d, J = 4.2 Hz, 2H),
2.61 (s, 1H); 13C NMR (75 MHz, CDCl3): δ 149.44, 137.67, 131.31,
130.96, 130.19, 129.14, 56.62.
The Synthesis of (E)-(3-Bromo-2-nitroprop-1-en-1-yl)-
benzene (9). To the solution of 8 (107 mg, 0.6 mmol, 1.0 equiv)
in anhydrous CH2Cl2 (15 mL) was added dropwise PBr3 (0.085 mL,
0.9 mmol, 1.5 equiv) at 0 °C. The reaction mixture was allowed to stir
at r.t. for 15 min. The mixture was quenched by adding cool water (40
mL) and extracted by CH2Cl2 (30 mL × 3). The combined organic
solvent was washed by a saturated sodium bicarbonate aqueous
solution (30 mL × 3) and brine (30 mL × 3) and dried by anhydrous
sodium sulfate. The dried solvent was concentrated under vacuum,
and the residue was purified by flash chromatography (petroleum
ether/EtOAc, 20:1 v/v) to give 9 as a yellow solid in a yield of 85%.
1H NMR (300 MHz, CDCl3): δ 8.15 (s, 1H), 7.44−7.54 (m, 5H),
Oxygen−Glucose Deprivation (OGD) and Recovery (R)
Induction. Rat cortical neurons were isolated as described previously
rat embryos at E18−E19 were euthanized by cervical dislocation
under general anesthesia. Their brain cortex tissues were dissected out
and mechanically cut into small pieces. The brain tissues were
enzymatically digested to prepare single cell suspensions. Primary
cortical neurons (1 × 106 cells/mL) were cultured onto poly-lysine-
coated plates in DMEM supplemented with 10% fetal bovine serum
(FBS, Invitrogen, Grand Island, NY) for 4 h at 37 °C in a humidified
atmosphere of 95% air and 5% CO2 to allow cell adhesion. After
removal of the unattached cells, the adhered primary cortical neurons
were pretreated with 4a or NaNO2 (0, 0.9, 11.8, and 3.6 μM) in
primary neuron basal medium supplemented with 2% B27 supple-
ment (Invitrogen) for 24 h. After being washed, the cells were
cultured in glucose- and FBS-free DMEM in 1% O2, 5% CO2, and
94% N2 for 45 min at 37 °C to induce OGD injury. Subsequently, the
cells were cultured in 10% FBS DMEM (5% glucose) for 24 h at 37
°C in a humidified atmosphere of 95% air and 5% CO2. The cell
viability was measured by the MTT assay.
4.54 (s, 2H); 13C NMR (75 MHz, CDCl3): δ 146.85, 137.43, 131.36,
131.20, 130.30, 129.48, 23.26.
General Procedure for the Preparation of 4a−d. To a
solution of respective allyl bromide compounds 3, 6a, 6b, or 9 (1
mmol) in 3 mL of ether was added AgNO2 (183.5 mg, 1.2 mmol).
The obtained mixture was stirred overnight at r.t. protected from
light. The reaction mixture was filtered, and the filtrate was
concentrated under a reduced pressure. The resulting residue was
purified by flash chromatography (petroleum ether/EtOAc, 15:1 v/v)
to afford the target compounds 4a−d.
(E)-2-(Nitromethyl)-1,3-diphenylprop-2-en-1-one (4a). The
title compound was prepared from intermediate 3 and AgNO2 as a
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colorless oil in a yield of 53%. H NMR (300 MHz, CDCl3): δ 7.88
(d, J = 7.3 Hz, 2H), 7.73−7.41 (m, 7H), 7.39−7.28 (m, 2H), 5.60 (s,
2H); 13C NMR (75 MHz, CDCl3): δ 196.18, 148.43, 137.13, 133.49,
132.79, 130.36,130.22, 129.88, 129.88, 129.29, 129.03, 128.65, 71.95;
ESI-MS (m/z): 290.1 [M + Na]+; ESI-HRMS (m/z): calculated for
C16H13NNaO3 [M + Na]+ 290.0788, found 290.0788.
(E)-Methyl 2-(Nitromethyl)-3-phenylacrylate (4b). The title
compound was obtained from 6a as a colorless oil in a yield of 48%.
The NMR data were consistent with a previous report.47 1H NMR
(300 MHz, CDCl3): δ 8.19 (s, 1H), 7.52−7.40 (m, 3H), 7.36−7.29
(m, 2H), 5.36 (s, 2H), 3.87 (s, 3H).
Quantitative Real-Time PCR. Total RNA was extracted from
individual groups of primary cortical neurons or brain tissues using
the Trizol reagent (Invitrogen) according to the manufacturer’s
instructions. After determining the quantity and quality, each RNA
sample was reversely transcribed into cDNA using a Hiscript II
reverse transcriptase kit (Vazyme, Nanjing, China). The relative levels
of target gene mRNA transcripts to control GAPDH were determined
by RT-qPCR using the SYBR-green mix kit (Hiscript II reverse
transcriptase, Vazyme, Nanjing, China) and specific primers
(Supporting information Table S1). The data were normalized to
(Z)-2-(Nitromethyl)-3-phenylacrylonitrile (4c). The title com-
pound was obtained from 6b as a pale-yellow solid in a yield of 25%.
GAPDH and analyzed by 2−ΔΔCt
.
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m.p. 99.2−101.2 °C; H NMR (300 MHz, CDCl3): δ 7.85 (dd, J =
7.6, 1.4 Hz, 2H), 7.57−7.42 (m, 3H), 7.32 (s, 1H), 5.18 (s, 2H); 13C
NMR (75 MHz, CDCl3):δ 152.78, 132.45, 131.82, 129.79, 129.32,
116.73, 99.72, 78.31;ESI-MS (m/z):187.1 [M−H]−; ESI-HRMS (m/
z): calculated forC10H7N2O2[M − H]− 187.0513, found 187.0512.
(E)-(2,3-Dinitroprop-1-en-1-yl)benzene (4d). The title com-
pound was obtained from 927 as a white solid in a yield of 36%. m.p.
107.6−109.4 °C; 1H NMR (300 MHz, CDCl3): δ 8.60 (s, 1H), 7.59−
7.40 (m, 5H), 5.65 (s, 2H); 13C NMR (75 MHz, CDCl3): δ 143.00,
140.01, 132.16, 130.27, 129.78, 129.69, 70.82; ESI-MS (m/z):290.1
[M−NO2]+; ESI-HRMS (m/z): calculated for C9H8NO2[M−NO2]+
162.0550, found 162.0558.
Animals. Male SD rats (12 weeks of age, 200−220 g) were
obtained from the Shanghai Laboratory Animals Center (SLAC,
Shanghai, China) and housed in a specific pathogen-free facility in our
campus. The animals were allowed free access to rat chow and water.
Additional SD embryos at E18−E19 were obtained from the
Experimental Animal Center of Nanjing University, Nanjing, China.
All animal experiments and animal care were conducted in accordance
with the guidelines and laws of the Provision and General
Recommendation of Chinese Experimental Animals in China. The
experimental protocols were approved by the Institutional Animal
Western Blotting. The different groups of primary cortical
neurons were harvested and their cytoplasmic and nuclear proteins
were extracted using the NE-PER nuclear and cytoplasmic extraction
kit (Thermo) according to the manufacturer’s instruction. The
cytoplasmic and nuclear proteins (30 μg/lane) were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) on 8−12% gels and transferred onto nitrocellulose (NC)
membranes. The membranes were blocked with 5% nonfat dry milk in
TBS containing 0.075% Tween-20 (TBST) for 1 h and incubated
with primary antibodies (anti-Nrf2, ab137550, 1:1000 dilution; anti-
lamin A, ab8980, 1:1000 dilution; and anti-β-actin, ab8227, 1:1000
dilution; Abcam, Cambridge, UK) overnight at 4 °C. After being
washed, the bound antibodies were detected with horseradish
peroxidase (HRP)-conjugated secondary antibodies for 1 h at room
temperature and visualized using the chemiluminescence detection kit
(Tanon 6600). The relative levels of target protein expression were
determined by densitometric analysis using Image pro plus 6.0
(Media Cybernetics, Silver Spring, USA).
Rat Blood Pressure Measurements. Male SD rats were injected
iv with 4a (950 μg/kg) or the vehicle control through their tail vein
(N = 3 per group). Their tail artery blood pressures were measured
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J. Med. Chem. 2021, 64, 10919−10933
Wu, Jianbing
