Design, Synthesis, and Biological Evaluation of Organic Nitrite (NO 2-) Donors as Potential Anticerebral Ischemia Agents

Article abstract of DOI:10.1021/acs.jmedchem.1c00282

The treatment of ischemic stroke (IS) remains a big challenge in clinics, and it is urgently needed to develop novel, safe, and effective medicines against IS. Here, we report the design, synthesis, and biological evaluation of organic NO2- donors as potential agents for the treatment of IS. The representative compound 4a was able to slowly generate low concentrations of NO2- by reaction with a thiol-containing nucleophile, and the NO2- was selectively converted into NO under ischemic/hypoxia conditions to protect primary rat neurons from oxygen-glucose deprivation and recovery (OGD/R)-induced cytotoxicity by enhancing the Nrf2 signaling and activating the NO/cGMP/PKG pathway. Treatment with 4a at 2 h before or after ischemia mitigated the ischemia/reperfusion-induced brain injury in middle cerebral artery occlusion (MCAO) rats by producing NO and enhancing Nrf2 signaling. Furthermore, 4a significantly promoted endothelial cell proliferation and angiogenesis within the ischemic penumbra. Our findings suggest that this type of NO2- donors, like 4a, may be valuable to fight IS and other ischemic diseases.

Full text of DOI:10.1021/acs.jmedchem.1c00282

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Article
Design, Synthesis, and Biological Evaluation of Organic Nitrite (NO−2)
Donors as Potential Anticerebral Ischemia Agents
Jianbing Wu,# Wei Yin,# Zhangjian Huang,* Yinqiu Zhang, Jian Jia, Huimin Cheng, Fenghua Kang,
Kai Huang, Tao Sun, Jide Tian, Xiaojun Xu,* and Yihua Zhang*
Cite This: J. Med. Chem. 2021, 64, 10919−10933
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ABSTRACT: The treatment of ischemic stroke (IS) remains a big challenge in clinics, and it is urgently needed to develop novel,
safe, and effective medicines against IS. Here, we report the design, synthesis, and biological evaluation of organic NO−2 donors as
potential agents for the treatment of IS. The representative compound 4a was able to slowly generate low concentrations of NO−2 by
reaction with a thiol-containing nucleophile, and the NO−2 was selectively converted into NO under ischemic/hypoxia conditions to
protect primary rat neurons from oxygen−glucose deprivation and recovery (OGD/R)-induced cytotoxicity by enhancing the Nrf2
signaling and activating the NO/cGMP/PKG pathway. Treatment with 4a at 2 h before or after ischemia mitigated the ischemia/
reperfusion-induced brain injury in middle cerebral artery occlusion (MCAO) rats by producing NO and enhancing Nrf2 signaling.
Furthermore, 4a significantly promoted endothelial cell proliferation and angiogenesis within the ischemic penumbra. Our findings
suggest that this type of NO−2 donors, like 4a, may be valuable to fight IS and other ischemic diseases.
density is crucial for the survival of patients,5 the enhancement
of therapeutic angiogenesis has become an attractive strategy
for development of anti-IS drugs.
INTRODUCTION
■
Ischemic stroke (IS) is a disease with a high mortality and
morbidity, leading to a heavy socioeconomic burden globally.
Nitric oxide (NO) is one of the most appealing gaseous
Although the intervention of IS has been advanced in the past
molecules in the field of biomedical sciences. It functions as a
decades, there is a lack of safe and effective treatments for this
key mediator in a wide range of biological events, including
fatal disease.1 Endovascular thrombectomy has shown great
improving blood flow by inducing vessel dilation and
value for the treatment of acute IS (AIS). However, few stroke
promoting endothelial cells to form microvasculature. More-
patients actually have received this treatment, and less than half
of those with the treatment will benefit permanently.2 Tissue
plasminogen activator (tPA) is the only anti-AIS specific
medicine approved by the Food and Drug Administration
over, NO can protect tissues against ischemic damage by
reducing the process of cellular respiration.6 However, vascular
NO bioavailability is reduced in ischemic tissues.7−9
Fortunately, the exogenous administration of NO donors can
(FDA). Unfortunately, the very narrow time window and the
enhance angiogenesis in ischemic tissues and protect from
potential risk of intracerebral hemorrhage of tPA greatly limit
its wider clinical applications.3 Therefore, it is urgently needed
Received: February 14, 2021
Published: July 22, 2021
to develop novel, safer, and more effective drugs against IS.
It is well established that surrounding the core of cerebral
infarction in an AIS patient, there is a hypoxic area, named
″ischemic penumbra″, which is sensitive to secondary injury
and can be rescued by improving the blood flow to protect
neurons from ischemic damage.4 Given that microvessel
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a
Scheme 1. Synthetic Route of Target Compounds 4a−d
a
Reagents and conditions: (a) benzaldehyde, NaOH, ethanol, 70 °C, 12 h; (b) NBS, AIBN, CCl4, reflux, 10 h; (c) AgNO2, diethyl ether, r.t.,
overnight; (d) methyl acrylate or acrylonitrile, DABCO, r.t., 7−10 days; (e) PBr3, CH2Cl2, 0 °Cr.t., 15 min; (f) AgNO2, diethyl ether, r.t.,
overnight; (g) nitromethane, ammonium acetate, 100 °C, 8 h; (h) formaldehyde, imidazole, THF, r.t., 72 h; (i) PBr3, CH2Cl2, 0 °Cr.t., 15 min;
and (j) AgNO2, diethyl ether, r.t., overnight.
ischemia injury.10,11 Nevertheless, the therapeutic effects of
NO donors are both dose- and microenvironment context-
dependent. It is well known that high concentrations of NO
may cause cytotoxicity, impeding the utilities of NO donor-
based therapy for ischemic diseases.12 We hypothesize that it
may be a safe and effective strategy to develop a NO donor
that generates a relatively low amount of NO specifically in the
ischemic region for the treatment of ischemic injury.
Interestingly, nitrite can be converted into NO through an
enzyme-dependent or -independent manner in the ischemic
region.13−15 In the ischemic regions, due to the lower pH and
NO bioavailability, nitrite anion (NO−2 ) can be reduced to NO
by deoxygenated hemoglobin (deoxy-Hb), xanthine oxidor-
eductase (XOR), aldehyde oxidase, and others to promote
vessel dilation and blood supply.16 In contrast, NO2− is usually
oxidized into nitrate (NO−3 ) in normoxic tissues and excreted
in the urine.17 Thus, NO2− acts as a prodrug, which can be
selectively converted into NO in the ischemic tissues to exert
its beneficial effects.
Several sodium nitrite (NaNO2)-based therapies have
exhibited promising efficacy for the treatment of ischemic
disorders in rodents.18−21 For example, the early intravenous
administration of NaNO2 reduced infarction volumes and
improved cerebral blood flow and functional recovery to
mitigate brain injury in a rat model of ischemia/reperfusion (I/
R) injury.22 In the murine hindlimb ischemia model, a low
dose of NaNO2 significantly restored ischemic hindlimb blood
flow, increased ischemic limb vascular density, and stimulated
endothelial cell proliferation in a time-dependent manner.18 In
addition, NaNO2 at lower doses also exerts excellent
cytoprotective effects in mouse models of I/R heart, liver, or
kidney injury.20,21 Importantly, treatment with NaNO2 benefits
AIS patients and displays relatively high safety.19
controlled. Additionally, high doses of NaNO2 failed to
generate the desirable protective effects in vivo.18,22 Probably,
the high levels of NO−2 produce a massive amount of NO that
subsequently generates high levels of the stronger oxidant
peroxynitrite, leading to protein nitration, DNA damage, and
energy failure.12 Hence, the discovery of organic NO−2 donors
that release NO−2 in a controlled and sustained manner for the
treatment of ischemic diseases will be of great interest.
Currently, there are few reported NO−2 donors except for
allylic nitro compounds, exemplified by 2-(nitromethyl)-
cyclohex-2-en-1-one (1, Scheme 1), which was synthesized
by King’s group in 2006.23 However, the pharmacological
effects of these compounds were not disclosed, and actually,
this type of donors released NO−2 too fast to be controlled (see
our data below).
Chalcone or (E)-1,3-diphenyl-2-propene-1-one is a common
simple scaffold found in many naturally occurring com-
pounds.24 Chalcone derivatives and analogues have shown
many interesting biological activities, including the antioxidant
activity by activation of the nuclear factor (erythroid-derived
2)-like 2 (Nrf2) pathway, which is critical for mitigating the I/
R-induced tissue damage in experimental ischemic stroke.25,26
Since the NO−2 release of compound 1 is too fast, which may
be due to its high rigidity in structure resulting in the
insufficient stability, in this study, we designed and synthesized
compound 4a by replacing the cyclohexenone structure with
chain-like acrylketone, which was incorporated with a chalcone
scaffold. Subsequently, we designed and synthesized a series of
analogues of 4a using other common unsaturated structures
(carboxylate, cyano, and nitro groups, respectively) to replace
the carbonyl in 4a, and each of them linked to one nitromethyl
group, like compound 1 as a NO2− donating moiety.
Furthermore, we investigated their stability and NO−2 releasing
as well as evaluated their anti-ischemic effects in vitro and in
vivo. Compound 4a was one representative compound shown
in Figure 1.
However, as an inorganic salt, NaNO2 is easily absorbed and
rapidly metabolized after oral or intravenous administration,
making the in vivo NO−2 levels difficult to be accurately
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Figure 1. The rational design of NO−2 donor (4a) and its potential NO-dependent action.
Figure 2. NO−2 releasing behavior and neuronal protecting effects of NO2− donors. The levels of NO−2 released (indicated as % of NO−2 released
from tested compounds, 200 μM) from 1 and 4a−d (A) in saline and McIlvaine buffer; (B) in the presence of cysteine, glutathione, or proline; and
(C) in bovine plasma. Data are expressed as the mean SD of each group from three separate experiments. (D) The effect of indicated compounds
on cell viability of primarily cultured rat neurons following OGD/R damage from three separate experiments. ***P < 0.001 vs the control group;
##P < 0.01, ###P < 0.001 vs the OGD/R group; ^P < 0.05, ^^^P < 0.001 vs the NaNO2 group.
presence of a catalytic amount of ammonium acetate,
benzaldehyde reacted with nitromethane to form the
mononitro compound 7. Treatment of 7 with formaldehyde
catalyzed by imidazole provided nitro allyl alcohol 8, which
was brominated by phosphorus tribromide to give bromide
intermediate 9. The target compound 4d was finally obtained
by the reaction of 9 with silver nitrite in the dark.27
RESULTS
■
Chemistry. The synthetic route of the target compounds
4a−d and their structures are depicted in Scheme 1. Starting
from the aldol condensation of propiophenone with
benzaldehyde, the resulting intermediate 2 was brominated
using NBS and AIBN to give allyl bromide compound 3. The
subsequent nitration of 3 using silver nitrite offered 4a. The
treatment of benzaldehyde with methyl acrylate or acrylonitrile
in the presence of 1,4-diaza[2.2.2]bicyclooctane (DABCO)
produced allyl alcohol compounds 5a and 5b, respectively.
Bromination of 5a and 5b with phosphorus tribromide
generated allyl bromide compounds 6a and 6b. The nitration
of 6a and 6b with silver nitrite away from light furnished target
compounds 4b and 4c, respectively. On the other hand, in the
4a−d Release NO−2 under Various Conditions. Given
that an ideal organic NO−2 donor compound should slowly
release NO−2 by a given trigger, we next examined and
compared the levels of NO−2 released from NO2− donor 1 and
4a−d under various conditions (Figure 2). The relationship
between the structures and NO−2 releasing behaviors of these
target compounds revealed that after incubation in physio-
logical saline or McIlvaine buffer, 1 and 4d with two nitro
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groups in their structure released relatively high levels of NO−2 ,
while 4a−c each with one nitro group failed to release or
released very low levels of NO−2 (Figure 2A), suggesting that
the target compounds 4a−c may be more stable than both 1
and 4d in physiological conditions. To examine whether the
target compounds could be activated by exogenous nucleo-
philes to release NO−2 , we incubated each compound with
cysteine (Cys), glutathione (GSH), or proline (Pro) for 1 h
and measured the levels of NO−2 (Figure 2B). Compounds 1
and 4d released approximately 99% of their theoretical levels of
NO−2 (except for 1 in the presence of Pro). In contrast, 4c
released very low levels of NO−2 in the presence of these three
nucleophiles. Interestingly, 4a and 4b released moderate levels
of NO−2 in the presence of GSH or Cys in a concentration-
dependent manner but did not release detectable NO−2 in the
presence of Pro, indicating that 4a and 4b were only activated
by thiol-containing nucleophiles. In addition, we observed that
1 and 4d rapidly released NO−2 , while 4a−c slowly released
lower levels of NO−2 in bovine plasma (Figure 2C).
Briefly, male Sprague−Dawley (SD) rats were injected iv with
an equimolar 4a (10 mg/kg) or NaNO2 (2.44 mg/kg). The
blood concentrations of 4a and NO−2 were quantified at 2, 5,
15, 30, 60, 90, and 120 min post injection by LC−MS/MS and
ion chromatography,24 respectively. The results were shown in
Figure 3, Figure S3 (Supporting Information), and Table S1
Figure 3. In vivo determination of NO−2 released by 4a. The
concentrations of orbital blood NO−2 in rats were tested at 2, 5, 15, 30,
60, 90, and 120 min post iv administration of 4a (10 mg/kg) or
NaNO2 (2.44 mg/kg), respectively. Data are presented as means
SD of each group (n = 3 per group) of rats. *P < 0.05, **P < 0.01 vs
the NaNO2 group at the indicated time points.
4a−d Protect from the OGD/R-Induced Neuronal
Injury. Next, we examined the activity of all target compounds
on their neuroprotective effects. Briefly, primary rat neurons
were treated with NaNO2 and 4a−d (1.8 μM each) for 24 h
and subjected to OGD for 2 h followed by culture in normoxic
conditions for 24 h. The cell viability of each group of cells was
tested (Figure 2D). The relationship between the structures
and neuroprotective effects of the target compounds indicated
that 4a with both the NO−2 releasing group and the chalcone
scaffold remarkably reduced the OGD/R-induced damage in
primarily cultured neurons, superior to NaNO2 and all other
target compounds, probably because 4a slowly released
moderate levels of NO−2 . The compounds 4b and 4c had
only one NO−2 releasing group as well as one carboxylate or
cyano group in their structures and displayed neuroprotective
activities weaker than 4a, which may stem from the relatively
lower levels of NO−2 released by 4b and 4c than that by 4a.
And 4b had a slightly stronger neuroprotective activity than 4c,
which may also be due to 4b releasing somewhat larger
amounts of NO−2 than 4c. Additionally, 4d with two nitro
groups in its structure displayed moderate levels of neuro-
protective activity, which may be due to its high NO−2 releasing
rate, leading to the generation of a massive amount of NO
toxic to neurons. Based on these results, we chose 4a as the
representative compound for the following experiments.
Examination of 4a Stability. The stability of 4a in the
presence of light, in PBS solutions at different pHs, and in rat
plasma was examined (Figure S1, Supporting Information).
We found that 4a had similar stability in the presence or
absence of light with almost no change during a 24 h
observation period, and 4a was relatively stable at pH 7.4 in
saline and in rat plasma with a half-life period (t1/2) of 38.5 and
5.3 h, respectively, but relatively unstable at pH 6 (t1/2 = 1.1
h). Additionally, the reaction rate of 4a with GSH was
positively correlated with the levels of GSH (Figure S2,
Supporting Information), and the reaction rate constant K for
1, 4, 8, and 32 equiv of GSH was calculated as 0.0214, 0.0299,
0.0363, and 0.0560 h−1, respectively.
(Supporting Information). We found that the administration of
NaNO2 induced a rapid increase in the blood concentrations
of NO−2 with a peak at 5−15 min and a gradual decrease to the
basal level at 90 min. In contrast, treatment with 4a
significantly reduced the peak blood NO−2 concentrations but
maintained lower levels of blood NO−2 until 120 min post
injection (the longest time point for analysis). These results
indicated that 4a produced moderate levels of NO−2 in vivo for
a longer period relative to NaNO2, which may be beneficial for
the treatment of ischemia.
1
Mechanism Study on NO−2 Release from 4a. The H
NMR technique was used for the first time to study the
possible mechanism of NO−2 release from 4a, which was used
alone or mixed with 1.5 equiv of N-acetyl Cys for 5, 30, 60,
120, and 600 min in anhydrous d6-DMSO at 37 °C (Figure 4).
As shown in Figure 4A−D, following the addition of N-acetyl
Cys to the 4a solution, mono-SR intermediate 10 was formed
by a rapid thio-addition and nitro-elimination sequence with
the characteristic signals for Ha (δ = 5.3, J = 6 Hz) and for Hb
and Hc (δ = 6.2 and 5.8, J = 9 Hz) as well as for Hd (δ = 3.9,
the proton adjacent to the NHAc group in N-acetyl Cys). The
double bond of 10 was then gradually converted to a single
bond, and the signals for Hb and Hc were shifted to high field
and overlapped in the signal peaks for N-acetyl Cys (Figure
4E,F), suggesting that another molecule of N-acetyl Cys may
attack the double bond at the β-position of 10 to form an
intermediate, which is rapidly converted to di-SR compound
12.
Another alternative route of NO−2 release might result from a
direct SN2 nucleophilic substitution of the nitro group in 4a by
N-acetyl Cys, which produced mono-SR intermediate 11 with
a signal of Hd’ (δ = 3.8). However, 11 might further undergo
thio-addition reaction to generate di-SR compound 12.
Because the polarity of each intermediate was substantially
increased after reaction with thiols, it was very difficult to
separate or purify these unstable intermediates. Fortunately,
however, we identified the molecular weights of these
intermediates using LC-HRMS (see Supporting Information).
4a Releases NO−2 In Vivo. Before the in vitro and in vivo
biological evaluation, the physicochemical property of 4a was
examined. We found that 4a had a water solubility of 33.21
3.49 mg/L (124.38 13.06 μmol/L, 37 °C) and a lipo-hydro
partition coefficient logP of 0.54 0.025. We then tested the
ability of 4a to release NO−2 and the PK behavior of 4a in vivo.
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Figure 4. 1H NMR spectra of 4a (40 mM) in anhydrous d6-DMSO at 37 °C. (A) 4a was used alone. (B−F) 4a was mixed with N-acetyl Cys (60
mM) for 5, 30, 60, 120, and 600 min, respectively.
The presence of intermediates formed by the reaction of
compound 4a with one and/or two GSH/N-acetyl Cys could
be preliminarily identified by HRMS from their molecular
weights of 527/383 and 834/546, respectively. These data may
support our above mechanistic study on NO−2 release from
chalcone 4a.
TBHQ, remarkably reduced the OGD/R-mediated cytotox-
icity in the cultured primary rat neurons, which was mitigated
by the treatment with a Nrf2 inhibitor, ML385 (5 μM)29,30
(Figure S4, Supporting Information). These results indicated
that 4a had potent neuroprotective activity against the OGD/
R-induced neuronal injury, at least partially, by enhancing the
Nrf2 signaling.
4a Protects from the OGD/R-Induced Neuronal
Injury. 4a Enhances the Nrf2 Signaling. Given that 4a−d
bearing a chalcone scaffold can serve as a potential Michael
acceptor, it may biologically form a covalent bond with the
sulfhydryl of cysteine in proteins, displaying pharmacological
activities. It is notable that chalcones can activate the Kelch-
like ECH-associated protein 1 (Keap1)/Nrf2/ARE pathway
through the covalent modification of the cysteines of Keap1 to
release Nrf2, inducing the expression of phase II enzymes and
antioxidant enzymes that protect from the oxidative-stress-
induced I/R injury.28 Accordingly, the neuroprotective effect
of the 4a was first examined and compared with NaNO2, and a
Nrf2 activator, tert-butylhydroquinone (TBHQ). Primary rat
cortical neurons were isolated and pretreated with 4a, NaNO2,
or TBHQ at the optimized concentration of 1.8 μM for 24 h
and subjected to OGD for 2 h followed by culture in
normoglycemic and normoxic conditions for 24 h. The
apoptosis rate, cell viability, and lactate dehydrogenase
(LDH) activity in the supernatants of cultured cells were
tested. In comparison with the control, the OGD/R
significantly increased the percentages of apoptotic cells
(Figure 5A,B) and LDH activities (Figure 5C) but significantly
reduced the cell viability (Figure 5D) in the cultured primary
rat neurons in vitro (P < 0.001 for all). Treatment with NaNO2
mitigated the OGD/R-increased LDH activity (P < 0.01) but
did not significantly alter cell apoptosis and viability in the
neurons following OGD/R. In contrast, treatment with 4a, like
Next, to determine whether 4a may activate the Keap1/Nrf2
signaling, rat primary cortical neurons were treated with or
without 4a (1.8 μM), NaNO2 (1.8 μM), or TBHQ (1.8 μM)
for 24 h and subjected to OGD/R. The relative levels of Nrf2,
HO-1, and NQO1 mRNA transcripts were determined by RT-
qPCR, and cytoplasmic and nuclear Nrf2 protein levels were
evaluated by Western blot analysis. In comparison with the
control, the OGD/R or pretreatment with any of the
compounds did not significantly alter the relative levels of
Nrf2 mRNA transcripts (Figure 5E). Furthermore, while the
OGD/R significantly reduced the relative ratios of nuclear to
cytoplasmic Nrf2 protein (Figure 5F−H), pretreatment with
4a, and compound 2 (with the chalcone backbone of 4a but
without the nitro group) or TBHQ, but not NaNO2,
significantly mitigated the effect of OGD/R by increasing the
relative ratios of nuclear to cytoplasmic Nrf2 protein (P <
0.001), indicating that 4a enhanced the Nrf2 signaling by
promoting its nuclear translocation in the primary rat neurons
following OGD/R. Consequently, pretreatment with 4a or
TBHQ significantly mitigated the OGD/R-decreased HO-1
and NQO1 (target genes of Nrf2) mRNA transcription in
primary rat neurons (Figure 5I,J). These data further
evidenced that 4a protected the primary rat nerurons from
the OGD/R-induced neuronal injury by enhancing the Nrf2
signaling in vitro.
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Figure 5. 4a protects from the OGD/R-induced neuronal injury by enhancing the Nrf2 signaling. (A and B) Flow cytometry analysis of the
frequency of apoptotic cells. (C) LDH activity. (D) MTT analysis of cell viability. (E) RT-qPCR analysis of the relative levels of Nrf2 mRNA
transcripts. (F−H) Western blot analysis of nuclear and cytoplasmic Nrf2 protein. (I and J) RT-qPCR analysis of the relative levels of NQO-1 and
HO-1 mRNA transcripts. Data are representative images or expressed as the mean
SD of each group of cells from three independent
experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the control group; ##P < 0.01, ###P < 0.001 vs the OGD/R group; ^^^P < 0.001 vs the NaNO2
group.
4a Activates the NO/cGMP/PKG Pathway. Additionally,
we examined whether 4a functioned via the NO−2 /NO/
cGMP/PKG pathway in primary rat neurons. The results
showed that 4a protected neurons from the OGD/R-induced
injury and increased the levels of cGMP and PKG2 expression
in the primarily cultured neurons under OGD/R as compared
to those in the untreated control. Importantly, most of the
effects of 4a were abrogated by treatment with a known sGC
inhibitor, NS2028 (Figure S5, Supporting Information).
Pretreatment with 4a Protects from the I/R-Induced
Brain Injury in MCAO Rats. Next, we tested whether
pretreatment with 4a could protect from the I/R-induced brain
injury in rats. The different groups of rats were injected iv with
the indicated compounds through their tail vein. Two hours
later, they were subjected to middle cerebral artery occlusion
(MCAO) for 2 h followed by reperfusion. One day post the I/
R procedure, their neurologic deficits were scored and their
brain tissue ischemic volumes and cerebral edema degrees were
evaluated in a blinded manner. As shown in Figure 6,
treatment with different doses of NaNO2 did not significantly
alter the brain infarct volumes, cerebral edema degrees, or
neurologic deficit scores in rats relative to those in the vehicle-
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Figure 6. Pretreatment with 4a protects rats from cerebral I/R injury. Rats were treated iv with the vehicle saline, NaNO2, 4a, or 4a + PTIO, and 2
h later, the rats were subjected to the MCAO procedure. At 24 h post I/R procedure, the rats were scored for their neurological deficits.
Subsequently, the rats were euthanized and their brain tissues were stained by TTC. (A) Infarction volumes (%) were calculated as infarct area/
whole area for each group. (B) Cerebral edema (%) was calculated by the formula illustrated in the method section. (C) The neurological deficit
scores were analyzed by the Kruskal−Wallis test followed by the Mann−Whitney U test for multiple comparisons. (D) The representative images
of brain sections in each group after TTC staining. Data are presented as mean SD or median IQR of each group (n = 8). *P < 0.05, **P <
0.01.
Figure 7. Treatment with 4a after MCAO reduces the cerebral I/R injury in rats. Two hours after MCAO, the rats were randomized and treated iv
with the vehicle (as the MCAO group), NaNO2, 4a, or 4a + PTIO immediately after perfusion. At 24 h post I/R, their neurological deficits were
scored and their brain sections were stained by TTC. (A) Infarction volumes (%) were calculated as infarct area/whole area for each rat. (B)
Cerebral edema (%) was calculated by the formula illustrated in the method section. (C) The neurological deficit scores were analyzed by the
Kruskal−Wallis test followed by the Mann−Whitney U test for multiple comparisons. (D) The representative images of brain sections in each
group after TTC staining. Data are presented as mean SD or median IQR of each group (n = 8). *P < 0.05, **P < 0.01.
treated MCAO group. Treatment with different doses of 4a
substantially reduced the brain infarct volumes, cerebral edema
degrees, and neurologic deficit scores in rats, and their
protective effects appeared to be dose-dependent. Importantly,
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Figure 8. 4a promotes angiogenesis in rat cerebral ischemic penumbra. Following treatment with saline, the indicated doses of NaNO2, or 4a for a
week, the CD31 expression and Ki67+ and Aurora B+ cells were determined by IHC and immunofluorescent assays. (A) Representative images of
anti-Ki67 (green), anti-Aurora B (green), anti-CD31 (red), and nuclear DAPI (blue) staining in the brain sections, respectively. (B and C)
Quantitative analysis of the percentages of CD31+/DAPI+ and Ki67+/DAPI+ endothelial cells. (D and E) Quantitative analysis of the percentages of
CD31+/DAPI+ and Aurora B+/DAPI+ endothelial cells. Scale bar, 50 μm. Data were presented as mean SD of each group (N = 5). *P < 0.05,
**P < 0.01.
treatment with 4a, together with a NO scavenger, PTIO (2-
phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide, 1 mg/
kg), almost abolished the therapeutic effect of 4a in MCAO
rats. Together, these results indicated that pretreatment with
4a significantly prevented the I/R-mediated brain injury in rats
predominantly by producing NO.
a 24 h observation period (Figure S6, Supporting Informa-
tion). This may be because 4a, as an organic NO−2 donor,
would not release high levels of NO directly in the blood
circulation. Instead, 4a sustainably released moderate levels of
NO−2 that was converted into low levels of NO under the
hypoxic conditions in ischemic regions. Therefore, NO2−
donating compounds, like 4a, may not significantly alter
systemic blood pressure.
In addition, we observed that iv injection with 4a (950 μg/
kg) displayed little effect on the systemic blood pressure during
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Treatment with 4a after I/R Reduces the Cerebral I/R
Brain Injury in MCAO Rats. To investigate the therapeutic
effects of 4a on the I/R-induced brain damage in rats, male SD
rats were subjected to MCAO for 2 h followed by blood
reperfusion. The rats were randomized and injected iv with the
vehicle, 4a, NaNO2, or 4a + PTIO at the indicated doses
immediately after reperfusion. A group of rats received a sham
procedure (the sham group) and was injected iv with the
vehicle. The brain infarct volumes, cerebral edema degrees, and
neurologic deficit scores in all rats were evaluated in a blinded
manner at 24 h post I/R procedure (Figure 7). The MCAO
group of rats displayed severe brain tissue infarction, higher
degrees of brain edema, and higher neurologic deficit scores.
Treatment with NaNO2 at 24.5 or 245.4 μg/kg significantly
reduced the brain infarct volumes, cerebral edema degrees, and
neurologic deficit scores with a trend of dose-dependence (P <
0.05 or P < 0.01 for all). However, treatment with a higher
dose of NaNO2 only generated a minor reduction in the brain
infarct volumes. In contrast, treatment with low and medium
doses of 4a significantly decreased the brain infarct volumes,
cerebral edema degrees, and neurologic deficit scores (P < 0.01
and P < 0.05). The therapeutic effect of 4a at the high dose
was similar to that of its medium dose. Importantly, PTIO (1
mg/kg) almost abrogated the therapeutic effects of 4a on the
MCAO-mediated brain injury in rats. These data indicated that
4a treatment reduced the MCAO-induced brain damages in
rats predominantly by producing NO.
4a Enhances the Keap1/Nrf2/ARE Signaling in Rat
Cerebral Ischemic Penumbra. Given that 4a effectively
enhanced the Nrf2 signaling to induce its target gene
expression in the cultured primary rat neurons following
OGD/R, we tested whether treatment with 4a could also
enhance the Nrf2 signaling by increasing its phosphorylation
and target gene expression in rat ischemic brains by
immunohistochemistry. First, phosphorylated Nrf2 signals
were detected predominantly in the nuclei, while NQO-1
and HO-1 signals were observed mainly in the cytoplasm of
cells (Figure S7A, Supporting Information). Quantitative
analysis indicated that the I/R process significantly enhanced
the Nrf2 phosphorylation and NQO-1 and HO-1 expression in
rat cerebral ischemic penumbra (Figure S7B, Supporting
Information). Treatment with 4a, but not NaNO2, significantly
further increased the relative levels of Nrf2 phosphorylation
and NQO1 and HO-1 expression in rat ischemic brain regions,
suggesting that 4a treatment may enhance the Nrf2 activation
in the ischemic penumbra of rat brains.
chalcone scaffold in 4a may synergistically enhance tube
formation in HUVECs.
Furthermore, we tested whether continual treatment with 4a
for 7 days could enhance angiogenesis in rat cerebral ischemic
penumbra by immunofluorescence. As shown in Figure 8, the
I/R procedure significantly increased the CD31 expression and
Ki67+ cell frequency relative to those of the sham group,
reflecting compensative responses after I/R process. Treatment
with a low or medium dose of NaNO2, but not a high dose of
NaNO2, significantly increased the CD31 expression and the
percentages of Ki67+ cells or Aurora B+ cells, particularly for
those Ki67+ or Aurora B+ endothelial cells (double staining of
anti-CD31 and anti-Ki67 or double staining of anti-CD31 and
anti-Aurora B). More importantly, treatment with either dose
of 4a, like the low or moderate dose of NaNO2, significantly
enhanced the CD31 expression and increased the frequency of
Ki67+ or Aurora B+ endothelial cells, which were almost
abrogated by co-treatment with PTIO. These results suggest
that treatment with 4a after I/R may significantly enhance the
I/R-related angiogenesis, mainly dependent on its NO
production in rat cerebral ischemic penumbra.
DISCUSSION
■
NO can induce vasodilation and ischemic tolerance.35
However, in an acute hypoxic status, NO levels are reduced
due to decreased levels of NOS expression.17 Instead, a variety
of molecules, including hemoglobin-associated globulin,
molybdenum-containing enzymes, cytochrome P450, and
others, recycle NO from NO2− in a hypoxic condition,
providing an alternative pathway for NO production.15,36,37
A number of NaNO2-based therapies have been reported to be
promising for the treatment of IS in both animals and humans.
Unfortunately, the enteral administration with NaNO2 may
have the potential issue of bioavailability,17 while the
intravenous administration with NaNO2 can cause its rapid
metabolism. Accordingly, the in vivo process of NO−2 is
difficultly controlled, limiting its wider application.
In this study, we developed a group of chalcone derivative 4a
and its analogues 4b−d, which acted as organic NO−2 donors.
We observed that 4a and 4b released moderate levels of NO2−
in bovine plasma and in the presence of cysteine or glutathione
but not proline. 4a generated lower maximum concentrations
of NO−2 for a longer duration than NaNO2 in rats. We
1
employed the H NMR technique for the first time to study
the mechanism underlying thiol-induced NO−2 release from 4a
and found that 4a liberated NO−2 by sequential thio-addition
and nitro-elimination reaction mechanisms.
4a Promotes Angiogenesis In Vitro and in Rat
Cerebral Ischemic Penumbra. Enhancement of angio-
genesis is crucial for the restoration of blood flow in the
ischemic penumbra and closely correlated with the prognosis
of cerebral stroke.31 Given that the activation of the Nrf2
signaling and its target HO-1 expression promote angiogenesis
following the I/R induction,32−34 we evaluated the effects of
4a, NaNO2, compound 2, and the combination of NaNO2 and
2 on angiogenesis by using a commercial angiogenesis tube
formation assay kit. As shown in Figure S8 (Supporting
Information), treatment with 4a (1.8 μM), with the same dose
as NaNO2, remarkably promoted tube formation in HUVECs,
and the effect of 4a on tube formation was significantly
diminished by treatment with a known sGC inhibitor, NS2028.
Compound 2 exerted a weaker effect; however, the
combination of NaNO2 and 2 increased tube formation in
HUVECs, suggesting that both NO−2 releasing group and
Additionally, in OGD/R primary cortical neurons and
MCAO rats, 4a enhanced the Nrf2 signaling to protect the
cells and rat brains from I/R injury, which was superior to
NaNO2. The results suggest that the chalcone scaffold in 4a
may enhance the Nrf2 signaling partly through a Michael
addition reaction of the sulfhydryl in Keap1 to release Nrf2 and
subsequent phosphorylation in the neurons in rat ischemic
lesions.26 Meanwhile, the NO−2 released from 4a activated the
NO/cGMP/PKG pathway. Thus, both Nrf2 enhancement and
cGMP/PKG activation synergistically contributed to the
neuroprotection effects of 4a. More importantly, the NO
scavenger PTIO almost abrogated the therapeutic effects of 4a
on the MCAO-mediated brain injury in rats, and a known sGC
inhibitor, NS2028, eliminated most of the protection of 4a
from the OGD/R-induced injury, suggesting that 4a may exert
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(427.2 mg, 2.4 mmol) in dry CCl4 (10 mL) was added AIBN (32.8
mg, 0.2 mmol). The mixture was refluxed for 10 h under a nitrogen
atmosphere. The resulting reaction mixture was cooled to room
temperature and filtered, and the filtrate was concentrated under a
reduced pressure. The resulting residue was extracted with ethyl
acetate (10 mL) three times. The organic layer was separated and
dried using anhydrous sodium sulfate, concentrated, and purified by
flash chromatography (petroleum ether/EtOAc, 15:1 v/v) to afford 3
(492 mg, 82%) as a colorless oil. The NMR data were consistent with
a previous report.46
its anti-ischemic activity mainly by activating the NO/cGMP/
PKG pathway.
One of the promising therapeutic strategies for the
treatment of I/R-related injury is to promote angiogenesis
and ameliorate penumbra damage after I/R. Indeed, we did
find that 4a promoted angiogenesis in vitro, treatment of
MCAO rats with 4a promoted endothelial cell proliferation
and stimulated angiogenesis within the ischemic penumbra of
rats, and most of the effects of 4a were abrogated by a NO
scavenger, PTIO. These observations suggest that 4a may
slowly release moderate levels of NO−2 that was subsequently
converted into NO in hypoxic penumbra to protect and rescue
neurons in ischemic lesions. More importantly, the preventive
and therapeutic effects of 4a in vivo suggest that 4a-like NO2−
donors may have a broad therapeutic window for intervention
of I/R-related tissue injury in clinical applications. In addition,
unlike NaNO2, treatment with 4a avoids the uptake of Na+
into the blood, which may reduce the risk of Na+-induced
cardiovascular events.38
General Procedure for the Preparation of 5a−b. To a
solution of benzaldehyde (1.06 g, 10.0 mmol, 1.0 equiv) and DABCO
(1.12 g, 10.0 mmol, 1.0 equiv) in methanol (20 mL) was added the
respective substituted ethylene (50 mmol, 5.0 equiv). The reaction
mixture was allowed to stir at r.t. for 1−4 days. The mixture was
diluted by EtOAc (100 mL), washed by water (50 mL × 3) and brine
(50 mL × 3), dried by anhydrous sodium sulfate, concentrated under
vacuum, and purified by flash chromatography (petroleum ether/
EtOAc, 1:1 v/v) to get 5a−b.
Methyl 2-(Hydroxy(phenyl)methyl)acrylate (5a). The title
compound was prepared from the reaction of benzaldehyde with
1
methyl acrylate as a colorless oil in a yield of 35%. H NMR (300
MHz, CDCl3): δ 7.15−7.64 (m, 5H), 6.32 (s, 1H), 5.81 (s, 1H), 5.54
CONCLUSIONS
■
(s, 1H), 3.70 (s, 3H), 2.83 (br., OH).
In summary, 4a was the first organic NO−2 donor bearing a
chalcone scaffold with significant preventive and therapeutic
effects on protecting against I/R brain injury by producing
NO, enhancing the Nrf2 signaling, activating the NO/cGMP/
PKG pathway, and promoting angiogenesis in the ischemic
penumbra of rats. Therefore, 4a may be a valuable lead
compound for the development of new therapeutic agents for
the treatment of ischemic stroke. This type of organic NO2−
donors may be beneficial and helpful to promote the NO−2 -
based therapies against ischemic diseases, including ischemic
stroke.
2-(Hydroxy(phenyl)methyl)acrylonitrile (5b). The title com-
pound was prepared from the reaction of benzaldehyde with
acrylonitrile as a yellowish oil in a yield of 41%. 1H NMR (300
MHz, CDCl3): δ 7.43−7.37 (m, 5H), 6.08 (s, 1H), 6.0 (s, 1H), 5.25
(d, J = 4 Hz, 1H), 3.14 (d, J = 4 Hz, 1H).
General Procedure for the Preparation of 6a−b. To the
solution of 5a−b (10 mmol, 1.0 equiv) in anhydrous CH2Cl2 (15
mL) was added dropwise PBr3 (1.42 mL, 15 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
solvent was concentrated under vacuum and purified by flash
chromatography (petroleum ether/EtOAc, 100:1 v/v) to give 6a−b.
(Z)-Methyl 2-(Bromomethyl)-3-phenylacrylate (6a). The title
compound was prepared from the reaction of 5a with PBr3 in a yield
of 63% as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.79 (s, 1H),
7.54 (m, 2H), 7.40 (m, 3H), 4.36 (s, 2H), 3.83 (s, 3H); 13C NMR
(75 MHz, CDCl3): δ 166.37, 142.75, 134.24, 129.58, 128.84, 128.80,
52.27, 26.66.
EXPERIMENTAL SECTION
■
General. 1H NMR and 13C spectra were recorded with a
BrukerAvance 300 MHz spectrometer at 303 K using TMS as an
internal standard. MS spectra were recorded on a Mariner Mass
Spectrum (ESI) and high-resolution mass spectrometry (HRMS) on
an Agilent Technologies LC/MSD TOF. Analytical and preparative
TLC was performed on silica gel (200−300 mesh) GF/UV 254
plates, and the chromatograms were visualized under UV light at 254
and 365 nm. The purity of all target compounds was determined by
HPLC (Shimadzu LC-20A HPLC system consisting of LC-20AT
pumps and an SPD-20AV UV detector), and the compounds with a
purity of >95% were used for the following experiments (see HPLC
traces in Supporting Information). All solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Solutions after reactions and extractions were concentrated using a
rotary evaporator operating at a reduced pressure of ca. 20 Torr.
Compounds 4a−4c,39−41 5a−5c,39−416,42 7,43 and 844 were
synthesized as previously described.
(E/Z)-2-(Bromomethyl)-3-phenylacrylonitrile (6b). The title
compound was prepared from the reaction of 5b with PBr3 in a yield
of 68% as a white solid. 5b was composed of E and Z isomers, and the
1
ratio was E/Z = 1:3. H NMR (300 MHz, CDCl3): δ 7.40−7.50 &
7.76−7.84 (m, 5H, aromatic), 7.21 & 7.35 (2s, 1H, olefinic HCC),
4.19 & 4.22 (2s, 2H, CH2); 13C NMR (75 MHz, CDCl3): δ 146.52 &
147.20 (HCC), 132.69 (aromatic), 132.45, 131.36, 130.46, 129.22,
129.05, 118.65 (HCC & CN), 117.05, 112.05, 108.10, 26.62 &
32.70 (CH2).
The Synthesis of (E)-(2-Nitrovinyl)benzene (7). To a solution
of benzaldehyde (1.06 g, 10.0 mmol, 1.0 equiv) in nitromethane (15
mL) was added ammonium acetate (77 mg, 1.0 mmol, 0.1 equiv).
The obtained mixture was heated to 100 °C and stirred for 8 h. 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 solvent was concentrated under
vacuum and purified by flash chromatography (petroleum ether/
The Synthesis of (E)-2-Methyl-1,3-diphenylprop-2-en-1-one
(2). To a stirred solution of propiophenone (10 mmol, 1.34 g) and
benzaldehyde (0.224 g, 12 mmol, 1.27 g) in EtOH (20 mL) was
added NaOH (0.8 g, 20 mmol). The mixture was heated to 70 °C and
stirred for 12 h. The reaction mixture was acidified to pH 2 using aq 2
N HCl and extracted with ethyl acetate (20 mL × 3). The combined
organic layers were dried using anhydrous sodium sulfate,
concentrated, and purified by flash chromatography (petroleum
ether/EtOAc, 20:1 v/v) to afford 2 (1.44 g, 65%) as a colorless oil.45
1H NMR (300 MHz, CDCl3): δ 7.72−7.78 (m, 2H), 7.51−7.58 (m,
1H), 7.31−7.50 (m, 7H), 7.18 (d, 1H, J = 1.4 Hz), 2.28 (d, 3H, J =
1.4 Hz); 13C NMR (75 MHz, CDCl3): δ 199.2, 142.0, 138.4, 136.7,
135.6, 131.5, 129.6, 129.3, 128.5, 128.3, 128.1, 14.3.
The Synthesis of (Z)-2-(Bromomethyl)-1,3-diphenylprop-2-
en-1-one (3). To a solution of 2 (444.2 mg, 2 mmol) and NBS
1
EtOAc, 10:1 v/v) to give 6 as a yellowish solid in a yield of 85%. H
NMR (300 MHz, CDCl3): δ 8.00 (dd, J = 13.7, 1.8 Hz, 1H), 7. 60
(dd, J = 13.7, 1.1 Hz, 1H), 7.52−7.56 (m, 2H), 7.48−7.52 (m, 1H),
7.41−7.48 (m, 2H); 13C NMR (75 MHz, CDCl3): δ 139.23, 137.22,
132.30, 130.17, 129.53, 129.29.
The Synthesis of (E)-2-Nitro-3-phenylprop-2-en-1-ol (8). 7
(119 mg, 0.8 mmol, 1.0 equiv) was dissolved in dry THF (5 mL) and
mixed with imidazole (54.4 mg, 0.8 mmol, 1.0 equiv) and
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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.
1
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
(https://www.nature.com/articles/s41598-017-05342-9). In brief, SD
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
1
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
.
1
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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longitudinally at 0, 1, 2, 3, 4, 5, 6, and 24 h post drug injection using a
thermostat noninvasive blood pressure monitor (XH200; Beijing
Zhongshi Dichuang Science & Technology Development, Beijing,
China).
anti-mouse IgG (green) followed by nuclear staining with DAPI. The
fluorescent signals were captured and photo imaged under a
fluorescent microscope. Vascular density was calculated by CD31-
positive cells divided by DAPI-positive cells. Proliferating vascular
cells were measured as the percentages of CD31+/Ki67+ cells divided
by CD31+ cells.
Statistical Analysis. Data are expressed as the mean SD. The
difference between groups was analyzed by Student’s t test. The
difference in neurologic deficit scores among groups was calculated
using the Kruskal−Wallis test and Mann−Whitney U test. All the
other data were compared using one-way ANOVA and post hoc
Tukey’s test. All statistical analyses were performed using the SPSS
version 20 for Windows. A P value of <0.05 was considered
statistically significant.
Drug Treatment and In Vivo Anti-IS Evaluation. Male SD rats
were randomized and injected iv with the vehicle, sodium nitrite
(24.5, 245.4, and 2454 μg/kg), or 4a (95, 950, and 9500 μg/kg) 2 h
before or after ischemia (N = 8 per group). One group of rats received
sham surgery without MCAO, and another group of rats was injected
iv with PTIO (1 mg/kg) 30 min before receiving 4a treatment.
Subsequently, these rats were injected iv with chloral hydrate (350
mg/kg) and subjected to a procedure of middle cerebral artery
occlusion (MCAO).48,49 In brief, the MCA of individual rats was
exposed and inserted with a 0.30−0.35 mm nylon intraluminal suture
to induce MCAO. At 2 h after ischemia, the nylon suture was
removed to reperfuse the brain. At 24 h after reperfusion, the
neurologic deficits in individual rats were examined by Longa’s
methods.48 The rats were euthanized, and their whole brains were
dissected and weighed (wet weight). Their brain sections (2 mm)
were stained with triphenyltetrazolium chloride (TTC, Sigma).50 The
whole brains from some rats in each group were dried in a 105 °C
baker for 24 h and weighed (dry weight). The infarction volumes (%)
were calculated as infarct area/whole area for each rat. The degrees of
brain edema were quantified by the following formula: (wet weight −
dry weight)/(wet weight) × 100 (%).51
Immunohistochemistry. The levels of NQO-1 and HO-1
expression and Nrf2 phosphorylation in the brain tissues of different
groups of rats were analyzed by immunohistochemistry. Briefly, the
paraformaldehyde-fixed paraffin-embedded brain tissue sections (3
μm) were deparaffinized, rehydrated, and subjected to antigen
retrieval. After being blocked with 10% normal goat sera in PBS,
the sections were incubated with rabbit anti-NQO-1 (ab34173, 1:200
dilution), anti-HO-1 (ab13243, 1:200 dilution; Abcam, Cambridge,
UK), and anti-phos-Nrf2 (bs-2013R, 1:100 dilution; Beijing Biosyn-
thesis Biotechnology, Beijing, China) at 4 °C overnight, and rabbit
IgG from healthy animals served as the negative control. After being
washed, the bound antibodies were detected with HRP-conjugated
goat anti-rabbit IgG followed by visualizing with diaminobenzidine
(DAB; Sigma, St. Louis, MO) and counterstaining with hematoxylin.
The staining intensity and the frequency of positively stained cells
were scored by the average optical density (AOD) of the positive cells
in five fields/sample with the Image-Pro Plus 6.0 software in a blinded
manner.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00282.
The stability of 4a in different conditions; reaction
kinetics of 4a and GSH; the pharmacokinetic behavior
of 4a (including concentration−time curve and calcu-
lated pharmacokinetic parameters); the effects of 4a on
enhancing Nrf2 signaling and activating the sGC/
cGMP/PKG pathway in OGD/R rat primary cortical
neurons, on blood pressure in rats, on enhancing the
Nrf2 pathway in MCAO rats, and on the angiogenesis in
vitro; the primers used in the real-time quantitative PCR;
the HPLC analytical parameters of plasma NO−2 levels
and a representative chromatogram; HRMS spectra of
the intermediates formed by the reaction of 4a with
1
thiols; the H NMR, 13C NMR, and HRMS spectra for
compounds 4a, 4c, and 4d; and the HPLC traces for the
purity of compounds 4a−d (PDF)
Molecular formula strings for compounds 4a−d (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Zhangjian Huang − State Key Laboratory of Natural
Medicines, Jiangsu Key Laboratory of Drug Discovery for
Metabolic Diseases, Jiangsu Key Laboratory of Drug
Screening, China Pharmaceutical University, Nanjing
210009, P.R. China; orcid.org/0000-0001-6409-8535;
Phone: +86-25-83271072; Email: zhangjianhuang@
cpu.edu.cn; Fax: +86-25-83271072
Xiaojun Xu − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China;
Phone: +86 18066069538; Email: xiaojunxu2000@
163.com
Yihua Zhang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China;
orcid.org/0000-0003-2378-7064; Phone: +86-25-
83271015; Email: zyhtgd@163.com; Fax: +86-25-
83271015
In Vitro Tube Formation Assay for the Evaluation of
Angiogenesis. The tube formation experiment was conducted
according to a previous report with minor modifications.52 Briefly,
HUVECs at between two and six generations were cultured in the
DMEM with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate,
100 U/mL penicillin, and 100 μg/mL streptomycin. Matrigel (Cat.
No. 356234; Corning, NY, USA) was added to prechilled 96-well
plates, and the plates were left in the incubator for 30 min. Then, the
HUVEC suspension (at final density of 8 × 104 per well) was added
to each well of plates. These HUVECs were incubated with the
vehicle or tested compounds, cultured for 8 h at 37 °C and 5% CO2
atmosphere. The tube formation was observed and counted under an
inverted microscope (Ts2R; Nikon, Tokyo, Japan).
In Vivo Determination of Angiogenesis. The different groups
of rats (N = 5 per group) were treated with the indicated dose of each
drug immediately after reperfusion and then daily for 7 consecutive
days. The rats were euthanized, and their brain tissues were dissected
and fixed. The paraffin-embedded brain tissue sections (3 μm) were
subjected to immunohistochemistry using mouse anti-CD31
(ab264089, 1:100; Abcam, Cambridge, UK), as described above.
Furthermore, some tissue sections were analyzed by immunofluor-
escence.53,18 Briefly, the brain tissue sections were incubated with
anti-CD31 (ab264089, 1:100; Abcam, Cambridge, UK), rabbit anti-
Ki67 (ab197547, 1:100; Abcam, Cambridge, UK), or rabbit anti-
Aurora (ab45145, 1:100; Abcam, Cambridge, UK) at 4 °C overnight,
and after being washed, the bound antibodies were reacted with Cy5-
conjugated anti-rabbit IgG (red) and Alexa Fluor 488-conjugated
Authors
Jianbing Wu − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
10930
https://doi.org/10.1021/acs.jmedchem.1c00282
J. Med. Chem. 2021, 64, 10919−10933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Pharmaceutical University, Nanjing 210009, P.R. China;
orcid.org/0000-0003-2725-9859
Wei Yin − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases,
Jiangsu Key Laboratory of Drug Screening, China
monophosphate; Cys, cysteine; DAB, diaminobenzidine;
DABCO, 1,4-diaza[2.2.2]bicyclooctane; deoxy-Hb, deoxygen-
ated hemoglobin; ESI, electrospray ionization; FBS, fetal
bovine serum; FDA, Food and Drug Administration; GAPDH,
reduced glyceraldehyde-phosphate dehydrogenase; GSH,
glutathione; HO, hemeoxygenase-1; HPLC, high-performance
liquid chromatography; HRMS, high-resolution mass spec-
trometry; HRP, horseradish peroxidase; HUVECs, human
umbilical vein endothelial cells; IHC, immunohistochemistry;
IS, ischemic stroke; I/R, ischemia/reperfusion; IQR, inter-
quartile range; Keap1, Kelch-like ECH-associated protein 1;
LDH, lactate dehydrogenase; MCA, middle cerebral artery;
MCAO, middle cerebral artery occlusion; MTT, methyl
thiazolyl tetrazolium; NaNO2, sodium nitrite; NC, nitro-
cellulose; NO, nitric oxide; NO−2 , nitrite; NO3−, nitrate; NQO-
1, NADPH quinineoxidoreductase-1; Nrf2, nuclear factor
(erythroid-derived 2)-like 2; OGD/R, oxygen−glucose depri-
vation/recovery; PKG, protein kinase G; Pro, proline; PTIO,
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; RT-
qPCR, reverse transcription-quantitative polymerase chain
reaction; sGC, soluble guanylate cyclase; SD, Sprague−
Dawley; SDS-PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis; TBHQ, tert-butylhydroquinone; TBST,
TBS containing 0.075% Tween-20; TTC, triphenyltetrazolium
chloride; tPA, tissue plasminogen activator; t1/2, half-life
period; XOR, xanthine oxidoreductase
Pharmaceutical University, Nanjing 210009, P.R. China
Yinqiu Zhang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China
Jian Jia − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases,
Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China
Huimin Cheng − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China
Fenghua Kang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China
Kai Huang − The Department of Cardiology, Institute of
Cardiovascular Disease, Union Hospital, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430074, P.R. China
Tao Sun − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases,
Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P.R. China
Jide Tian − Department of Molecular and Medical
Pharmacology, University of California, Los Angeles,
California 90095, United States
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Natural Science
Foundation of China (Nos. 81773573, 21372261, 81673305,
and 81273378); Jiangsu Province Funds for Distinguished
Young Scientists (BK20160033) and the 111 Project
(B16046); the Open Project Program of State Key Laboratory
of Natural Medicines, China Pharmaceutical University
(SKLNMZZCX201820); and the ″Double First-Class″ Uni-
versity Project (CPU2018GF04) for the financial support.
ABBREVIATIONS
AIS, acute ischemic stroke; AOD, average optical density;
ARE, antioxidant response element; cGMP, cyclic guanosine
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