Novel Stachydrine-Leonurine Conjugate SL06 as a Potent Neuroprotective Agent for Cerebral Ischemic Stroke

Article abstract of DOI:10.1021/acschemneuro.1c00200

As major active ingredients of the traditional Chinese medicine motherwort, stachydrine and leonurine were found to have protective effects against cerebral ischemia. However, their bioavailability in vivo was low, and their efficacy was unsatisfactory, which limited their further application. To solve these problems, the conjugates based on the structures of stachydrine and leonurine were designed and synthesized. SL06 was found to have neuronal cell survival improvement, neuronal apoptosis restraining, activation of superoxide dismutase (SOD) activity, and inhibition of lactic dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA) in vitro. In vivo, the infarction size was significantly reduced by SL06 in the middle cerebral artery occlusion rat model. SL06 could also activate protein kinase B (AKT)/glycogen synthase kinase 3β (GSK-3β) activity and promoted the expression of antiapoptoticprotein Bcl-2. On the other hand, the expression of the apoptosis-associated protein cleaved caspase-3 would be inhibited as well. Thus, SL06 as the neuroprotective agent has potential for the treatment of cerebral ischemic stroke.

Full text of DOI:10.1021/acschemneuro.1c00200

pubs.acs.org/chemneuro
Research Article
Novel Stachydrine−Leonurine Conjugate SL06 as a Potent
Neuroprotective Agent for Cerebral Ischemic Stroke
Feng Li,* Sifeng Zhu, Qihui Jiang, Chenhui Hou, Tao Pang, Liang Zhang, and Wenbao Li*
Cite This: ACS Chem. Neurosci. 2021, 12, 2478−2490
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: As major active ingredients of the traditional Chinese medicine motherwort, stachydrine and leonurine were found to
have protective effects against cerebral ischemia. However, their bioavailability in vivo was low, and their efficacy was unsatisfactory,
which limited their further application. To solve these problems, the conjugates based on the structures of stachydrine and leonurine
were designed and synthesized. SL06 was found to have neuronal cell survival improvement, neuronal apoptosis restraining,
activation of superoxide dismutase (SOD) activity, and inhibition of lactic dehydrogenase (LDH), reactive oxygen species (ROS),
malondialdehyde (MDA) in vitro. In vivo, the infarction size was significantly reduced by SL06 in the middle cerebral artery
occlusion rat model. SL06 could also activate protein kinase B (AKT)/glycogen synthase kinase 3β (GSK-3β) activity and promoted
the expression of antiapoptoticprotein Bcl-2. On the other hand, the expression of the apoptosis-associated protein cleaved caspase-3
would be inhibited as well. Thus, SL06 as the neuroprotective agent has potential for the treatment of cerebral ischemic stroke.
KEYWORDS: Stachydrine−leonurine conjugate, ischemic stroke, biological activities, neuroprotective agent
1. INTRODUCTION
Cerebral stroke, including ischemic stroke and hemorrhagic
stroke, is a devastating disease of the world.1 Approximately
1.57‰ of people died of cerebral stroke on average, and the
ischemic stroke accounted for 70−90% of all stroke cases.2−4
Thrombolytic therapy, which conducts with standard-dose
recombinant tissue plasminogen activator infusion, is the only
approved treatment for acute ischemic stroke by the FDA.
However, thrombolytic exhibited low therapy efficacy due to
its serious safety-related restrictions and adverse effects.5−7
Thus, many attempts have been made to develop potent
neuroprotective agents against cerebral ischemia.8−10
Figure 1. Structures of stachydrine, leonurine, and SL06.
Natural products are very promising to be developed into
new drugs for the treatment of cerebral ischemic stroke in
these attempts. Stachydrine and leonurine (Figure 1) are the
major active ingredients of motherwort, one traditional
Received: April 1, 2021
Accepted: June 17, 2021
Published: June 28, 2021
Chinese medicine (TCM). It was reported that stachydrine
was effective for treatment of cardiovascular diseases and
cancers.11−14 However, low bioavailability and unsatisfactory
efficacy limited its further application.15 To address these
problems, we report the design and synthesis of these novel
https://doi.org/10.1021/acschemneuro.1c00200
© 2021 American Chemical Society
ACS Chem. Neurosci. 2021, 12, 2478−2490
2478

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
a
Scheme 1. Synthesis of Stachydrine−Leonurine Conjugates SL01−SL05
a
Reagents and conditions: (a) acetic anhydride, 140 °C, 2 h, 83%; (b) (Boc)2O, NaOH, H2O/1, 4-dioxane, rt, 12 h; (c) 4-amino-1-butanol, DMF,
rt, 12 h, 83% for two steps; (d) DPTS, DIC, DCM, rt, 5 h, 67%; (e) MeONa, DCM, 4 °C, 5 h, 96%; (f) DPTS, DIC, DCM, 30 °C, 3 h, 39−89%;
(g) TFA, DCM, 0−25 °C, 8 h, 98%; (h) N-methyl-L-proline, HATU, DIPEA, rt, 1 h, 66−99%.
a
Scheme 2. Synthesis of Stachydrine−Leonurine Conjugates SL06−SL09
a
Reagents and conditions: (a) NaOH, CuSO4, NH4OH, 0−135 °C, 12 h, 45%; (b) 5, DPTS, DIC, DCM, 30 °C, 3 h, 28%; (c) Boc-glycine,
HATU, DIPEA, DMF, rt, 2 h, 56.5%; (d) TFA, DCM, 0−25 °C, 8 h, 98%; (e) (1) N-methyl-L-proline, HATU, DIPEA, DMF, rt, 1 h, 43−72%; (2)
HCl/MeOH, MeOH, rt, 10 min, 99%.
derivatives based on the structure of stachydrine and the
evaluation of their biological activities in vitro and in vivo.
Among these derivatives, the biological activity of Compound
B1 was higher than those of stachydrine and other novel
derivatives, and it was slightly lower than that of edaravone, a
clinically used drug for neuroprotection.16 On the other hand,
2479
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 2. Effects of stachydrine−leonurine conjugates and MK-801 on neuronal cell viability of CGC. First, pretreatment of CGCs was carried out
for 24 h with different concentrations of MK-801, SL01, SL02, SL03, SL04, SL05, SL06, SL07, SL08, and SL09 and then incubation for 24 h with
200 μM glutamate (Glu). Data are expressed as mean SEM (n = 3). ***P < 0.001 versus the control group (Saline); ###P < 0.001 versus the
model group (Glutamate); $, P < 0.05; $$, P < 0.01. Abbreviations: ND = not detected. MK-801 is also called dizocilpine, a clinically used
neuroprotective drug.
the prevention of leonurine on myocardial and cerebral
ischemia was discovered.17−22
leonurine derivative 6 was obtained in 67% yield by the
condensation of 2 with isothiourea 5 in the presence of N,N′-
diisopropylcarbodiimide (DIC) and 4-(dimethylamino)-pyr-
idinium-4-toluene sulfonate (DPTS). Compound 5 was
synthesized from methyl-isothiourea 3 with (Boc)2O, followed
by condensation of the resulting 4 with 4-hydroxyl butylamine
in 81% overall yield (over two steps). Subsequently, the acetyl
group of 6 was hydrolyzed in the presence of a catalytic
amount of sodium methoxide to give the key intermediate 7 in
96% yields. Then, 8a−8e were obtained through condensation
with different linkers in the presence of DIC and DPTS. After
removal of the protective groups of 8a−8e by trifluoroacetic
acid, the desired 9a−9e were afford in almost quantitative
yields. Finally, stachydrine−leonurine conjugates SL01−SL05
were obtained through condensation of 9a−9e with N-methyl-
L-proline in the presence of 2-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) and N,N-diisopropylethylamine (DIPEA) followed
by an equivalent hydrogen chloride in methanol.
Incorporation of two mutually complementary biological
active entities by conjugation has been widely used in drug
design. Antimalarial agents, such as trioxaquine, showed good
results in drug efficacy improvement.23−29 Inspired by these
paradigms, in this study, stachydrine and leonurine were
combined via various linkers to form new conjugates. After it
was screened, SL06 was found to have a high therapeutic
efficacy in vitro and in vivo (Figure 1), and it also exhibited low
toxicity.
2. RESULTS AND DISCUSSION
2.1. Chemistry. The stachydrine−leonurine conjugates
SL01−SL05 and SL06−SL09 were synthesized by a sequence
linear step from syringic acid 1 and 4-bromo-3,5-dimethox-
ybenzoic acid 10, respectively (Schemes 1 and 2). The
chemical structures were characterized by nuclear magnetic
resonance (NMR) and a high-resolution mass spectrometer
(HRMS).
2.1.1. Synthesis of Stachydrine−Leonurine Conjugates
SL01−SL05. The synthetic route to SL01−SL05 was depicted
as Scheme 1. First, syringic acid 1 was acetylated with acetic
anhydride at 148 °C for 2 h to afford 2 in 82% yield. Then,
2.1.2. Synthesis of Stachydrine−Leonurine Conjugates
SL06−SL09. Stachydrine−leonurine conjugates SL06−SL09
were synthesized from the starting material 4-bromo-3,5-
dimethoxybenzoic acid (10). First, transformation of the
bromo group of 10 into the amino group was accomplished
2480
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Table 1. Effects of Compound SL06 and MK-801 on CGC Viability (n = 3)
compound cell viability % of control (0 μM) cell viability % of control (10 μM) cell viability % of control (20 μM) cell viability % of control (30 μM)
control
model
MK-801
SL06
100.00 2.03
56.94 0.57
104.15 0.59
71.73 0.42
80.90 3.75
84.09 1.46
with ammonia in the presence of sodium hydroxide and copper
sulfate in 45% yield. Then, condensation of the resulting 11
with isothiourea 5 afforded the desired leonurine derivative 12
in the presence of DIC and DPTS in 28% yield. The key
intermediate 14 was obtained via a sequence of condensation
of leonurine derivative 12 with N-tert-butoxycarbonylglycine in
the presence of a catalytic amount of HATU and DIPEA in
56% yield and removal of the protective group of the resulting
13 by trifluoroacetic acid in quantitative yield. Finally, the
stachydrine−leonurine conjugates SL06−SL09 were obtained
via the key intermediate 14 reacted with different substituted
L-proline followed by an equivalent hydrogen chloride in
methanol.
Table 2. Effects of Compound SL06 on Time to the Rabbit
Blood Coagulation (n = 3)
a
time of coagulation (s)
parameter (unit)
50 nM
100 nM
500 nM
control
stachydrine
SL06
1009 39.01
1421 22.45
1419 105.10
1523 17.35
1703 77.17
1523 11.20
1994 74.64
a
Values represent means SD (n = 3). The time of the rabbit blood
coagulation was measured by stopwatch.
2.2. Biological Evaluation. Various complex processes
were involved in the pathophysiology of stroke. As is well-
known, the neuronal degeneration was mediated by glutamate
during ischemia. Neurons and astrocytes produced large
amounts of glutamate, which could cause an excess of
intracellular calcium. Necrosis and decomposition could be
observed in the cellular structures of proteins, DNA, and
membrane phospholipids due to overload of calcium.30 The
neuronal injury model induced by glutamate was commonly
used in stroke study. The effect of neuroprotective agents was
tested with the animal model of cerebral ischemia.31−35 Also,
the model of ischemia induced by transient occlusion of the
middle cerebral artery (tMCAO) is widely used to study the
neuroprotective effect of various treatment approaches,
because the model is very important for studying occlusion
of the temporal artery and subsequent restoration of blood
flow.36
Figure 3. Time of coagulation in each group after administration.
***P < 0.001 between the control group and the drug-treated groups.
higher activity than those of other drug-treated groups (P <
0.001).
2.2.1. Neuroprotective Effects in Primary Rat Cerebellar
Granule Neuronal Cells (CGCs). The death of a large number
of neuronal cells could be induced by glutamate damage; then,
neuron injury was observed. The effect of neuronal cell was
tested by MTT assay in this experiment. The neuronal cell
viability of CGCs was significantly reduced by glutamate (Glu)
stimulation (Figure 2, Table 1). Gratifyingly, SL06 significantly
increased cell viability at different doses (P < 0.001) in
comparison with that of the model group, while poor efficacy
in protecting rat cerebellar granule cells was observed in other
groups. Compared with other stachydrine−leonurine con-
jugates at the same concentration, SL06 expressed a higher
neuroprotection activity. Thus, SL06 was selected for further
study.
2.2.3. Effects of SL06 on ROS, SOD, LDH, and MDA. It was
reported that oxidative stress (OS) contributes to acute brain
tissue damage, which might be a potential contributor to the
pathogenesis of ischemic stroke.37 After such brain damage by
ischemic stroke, the production of reactive oxygen species
(ROS) and lactic dehydrogenase (LDH) may increase,
sometimes drastically leading to brain tissue damage or
neuronal death via several different cellular molecular path-
ways. Additionally, the reduction of superoxide dismutase
(SOD) expressions and the rise of the malondialdehyde
(MDA) level in neuronal cells during cerebral ischemia were
involved in subcellular damage, which could lead to brain
edema formation after ischemia reperfusion.38,39
In this study, in comparison with the model group (control),
a decrease in ROS was observed in the SL06-treated group and
in LDH production (Figure 4A). It was indicated that SL06
was able to attenuate brain tissue damage or neuronal death by
oxidative stress. Also, in comparison with the model group, an
increase in SOD activity and the decrease in MDA were
observed in the SL06-treated group (Figure 4B). It is apparent
that SL06 had effects on improving ischemic neuronal injury
not only by decreasing ROS, LDH, and MDA levels but also by
markedly promoting the expression of SOD. As expected, the
results indicated that SL06 exhibited a more potent neuro-
2.2.2. Effects of SL06 on Rabbit Blood Coagulation. It was
reported that blood clots, a kind of deep vein thrombosis, was
involved in ischemic stroke. Thus, the anticoagulant activity to
rabbit blood was studied with Compound SL06. Average time
of the rabbit blood coagulation was 1009
39.01 s in the
normal group (Table 2, Figure 3). In comparison with the
control group, the time of the rabbit blood coagulation was
increased after it was treated with SL06. The average time of
the rabbit blood coagulation in a high dose of the SL06-treated
group was increased to 1994 74.64 s, which also exhibited a
2481
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Table 3. Evaluation of Compound SL06 in Rat Model of
Middle Cerebral Artery Occlusion (tMCAO)
neurological deficit
volume of cerebral infarction
a
group
score
(%)
sham
0
0
vehicle
Eda (3 mg/kg)
SL06 (5 mg/kg)
3.45 0.52
1.13 0.64
0.55 0.53
33.63 3.61
7.34 5.67
6.11 3.05
b
a
Values represent means
SEM from eight independent experi-
b
ments. Eda is short for edaravone, a clinically used drug for
treatment of cerebral stroke.
deficit score at 3.45 0.52 was caused in the middle cerebral
artery occlusion (Table 3, Figure 5C). In contrast, the
neurological deficit score of the SL06-treated group was 0.55
0.53, which was lower than that of the model group
(control) at 3.45
0.52 and that of the edaravone-treated
group at 1.13 0.64. Thus, SL06 had a beneficial effect on
cerebral infarction and neurologic deficit improvement.
2.2.5. Preliminary Toxicology Study In Vivo. In vivo, the
lethal toxicology of SL06 in mice was tested. Sixty mice (18−
23 g) were divided into cages and raised for 7 days in five
groups, including one normal group and four treatment groups.
After the administration, clinical signs, body weight, and food
consumption were recorded every day for a total of 14 days.
During the record of 14 days, no signs of toxicity were
Figure 4. (A) Effects of SL06 on LDH and MDA level on glutamate-
induced rat cerebellar granule cells. (B) Effects of SL06 on SOD and
ROS activity in glutamate-induced rat cerebellar granule cells. MK-
801 denotes positive control group. Values are expressed as means
SEM from three individual samples. ##P < 0.01, ###P < 0.001 versus
model group; ***P < 0.001 versus control group.
observed. It was showed that SL06 was tolerated with LD50
>
85 mg/kg, which indicated that Compound SL06 has a high
safety window in vivo.
protection than that of the control group with the same
concentration.
2.2.6. Regulatory Effects of Compound SL06. PTEN
(phosphatase and tensin homologue deleted on chromosome
10) is a dual specificity phosphatase and acts as a crucial
mediator for neuronal death in neural cells exposed to hypoxic-
ischemic.40 Protein kinase B (AKT) and glycogen synthase
kinase 3β (GSK-3β) are two downstream signal proteins for
PTEN, which play key roles in neuronal death in ischemic
stroke. Activation of the AKT and GSK-3β pathway would
inhibit neuronal apoptosis and ischemic brain injury, which
was a reliable strategy for neuroprotection in ischemic
stroke.41,42 Thus, the effects of SL06 on the expression of
the AKT and GSK-3β pathway were investigated in ischemic
stroke rats. The protein expression rate of p-AKT/p-GSK-3β
was significantly increased within 5 h (Figure 6A). Western
blotting showed that SL06 highly increased p-AKT/p-GSK-3β
2.2.4. Evaluation of Compound SL06. In vivo, the activity
of SL06 was evaluated in the middle cerebral artery occlusion
of the model rat. After they were treated with different drugs,
the typical infarcts were stained with 2,3,5-triphenyte-trazolium
chloride (TTC). The infarct parts were shown in pale colors,
and the normal parts were shown in red (Figure 5). As shown
in Table 3 and Figure 5A, a severe cerebral infarction was
caused in the tMCAO rat model, which accounted for 33.63
3.61% of cerebral volume. In contrast, a light cerebral
infarction was showed in the SL06-treated group at 6.11
3.05% of cerebral volume, while the positive control of the
edaravone-treated group showed a cerebral infarction at 7.34
5.67% of cerebral volume. Additionally, a high neurological
Figure 5. Compound SL06 decreased infarct volume and improved neurological deficit in a model of tMCAO. (A) Gross views of the brains in the
tMCAO rat model treated by SL06 or Eda. (B) The volume of cerebral infarction in the tMCAO rat model in each group at end of administration.
Sham denotes the control group. Vehicle denotes model group. Eda denotes positive control group. (C) Neurological deficit score in the tMCAO
rat model treated by SL06 or Eda. Values are expressed as means SEM from eight individual samples. **P < 0.01, ***P < 0.001 versus sham
group; ##P < 0.01, ###P < 0.001 versus model group; $$P < 0.01 versus positive control group.
2482
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 6. Western blotting showed that SL06 increased protein expressions. (A) Effects of SL06 on the protein expression of p-AKT and p-GSK-
3β in glutamate-induced rat cerebellar granule cells at different times. (B) Quantitative analysis of p-AKT and p-GSK-3β protein expression levels at
different times after SL06 treatment. Representative images and quantitative data are shown. The initial expression rate is 100% (all n = 3 per
group). Data were expressed as mean values standard deviation (SEM). *P < 0.05, **P < 0.01, ***P < 0.001 versus initial time.
Figure 7. Effect of Compound SL06 on apoptosis in model of neuronal injury induced by glutamate. (A) Effect of SL06 on apoptosis in model of
neuronal injury induced by glutamate. (B) Representative images of TUNEL immunostaining in the apoptosis assay (confocal laser microscope
image) for Compound SL06 or MK-801 in model of neuronal injury induced by glutamate. CN denotes the control group. Glu denotes the model
group (control). MK-801 denotes the positive control group. Values are expressed as means SEM from three individual samples. ###P < 0.001
versus the control group (Saline); ***P < 0.001 versus the model group (control) (Glutamate). First, pretreatment of CGCs was carried out for 24
h with SL06 or MK-801; then, they were incubated for 24 h with 200 μM glutamate (Glu). Finally, apoptotic cells were marked with TUNEL
staining. Red fluorescent denotes the positive apoptotic cells.
protein expressions (Figure 6B). Therefore, we infer that SL06
exhibited anti-ischemic stroke efficacy by activating the signal
pathway of AKT/GSK-3β.
group (control) (Figure 7B), while its quantity could be
markedly decreased by MK-801 or SL06 treatment. These
results indicated that Compound SL06 was able to inhibit
neuronal apoptosis and exhibited a higher neuroprotective
efficacy.
2.2.8. Effects of Compound SL06 on Genes and Protein
Expression. Although the previous data indicated that SL06
could prevent neuronal apoptosis and exhibited a higher
protective efficacy for neuronal cells, its precise mechanism has
not been elucidated yet. As is well-known, Bcl-2 and caspase
families once were considered as a major regulator of the
2.2.7. Effects of Compound SL06 on Apoptosis. Effect of
Compound SL06 on apoptosis was studied in the neuronal
injury model induced by glutamate. As shown in Figure 7A,
glutamate caused a significant increase in the proportion of
apoptotic neuronal cells, while the proportion of apoptotic
neuronal cells was significantly reduced in the SL06-treated
group. As shown, many scattered positive apoptotic cells
stained with red fluorescence were observed in the model
2483
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
apoptotic pathway.43,44 In the present study, glutamate
significantly decreased the expression of Bcl-2, which is
consistent with the suggested roles of these genes in the
regulation of apoptotic cell death. As shown in Figure 8A,B,
potential applicability of SL06 as a novel and powerful
neuroprotective treatment against ischemic stroke.
4. EXPERIMENT
4.1. General Information. Reagent and solvents were
purchased from Sinopharm Chemical Reagent Co., Ltd., Qing-
Dao Chemical Company, Beijing InnoChem Science and
Technology Company Acros, and other suppliers. NMR data
were recorded with an Agilent 500 spectrometer. Chemical
shifts (δ) values were expressed in parts per million (ppm)
with tetra-methylsilane (TMS) as an internal standard. Mass
spectra (ESI) were measured on a VG Autospec 3000 mass
spectrometer. HPLC analysis was carried out with a linear
gradient of acetonitrile containing 0.2% (v/v) formic acid at a
flow rate of 300 μL min−1. The MG-III column (50 mm × 2.0
mm, 5 μm, Shiseido Co., Ltd., Kyoto, Japan) and UV detection
at 254 nm on a API-4000 (Applied Biosystems Corpo-ration,
Ltd., Carlsbad, America) were selected. The microTOF-2
focus (Bruker Dalton-ics) mass spectrometer was used to
record ESI-TOF-MS.
4.2. Synthesis. 4.2.1. Synthesis of 4-Acetoxy-3,5-dime-
thoxy-benzoic Acid (2). A suspension of syringic acid (1) (1g,
5.04 mmol) in acetic anhydride (7 mL) was heated to 140 °C
under nitrogen atmosphere for 2 h. After it was cooled to 50
°C, the mixture was poured into ice water (20 mL). Then, after
it was stirred at 0 °C for 1 h, the mixture was filtered and
washed with chilled water three times. Residue was dried to
1
afford 2 (amorphous white powder, 1 g, yield 83%). H NMR
Figure 8. Effects of SL06 on the gene expression of Bcl-2 and caspase-
3 in glutamate-induced rat cerebellar granule cells at different doses
(A). Quantitative analysis of the gene expression of Bcl-2 or caspase-3
(B). SL06 was pretreated for 24 h before glutamate addition. CN
denotes the control group. Glu denotes the model group (control).
(500 MHz, DMSO-d6): δ 7.28 (s, 2H), 3.81 (s, 6H), 2.27 (s,
3H). MS (ESI, m/z): 262.79 [M + Na]+.
4.2.2. Synthesis of N,N′-Boc-methyl-isothiourea (4).
Methyl-isothiourea 3 (14 g, 50.29 mmol) was dissolved in
dichloromethane/water = 1/1 (150 mL). Saturated sodium
hydroxide solution (100 mL) and ditert-butyl decarbonate
(14.26 g, 63.37 mmol) were successively added. The mixture
was stirred at room temperature with nitrogen atmosphere for
12 h. After it was filtered, the residue was stirred in water (300
mL) at 50 °C for 2 h. Then, the mixture was filtered and dried
Data were expressed as mean values
SEM from three individual
samples. ###P < 0.001 versus control group; *P < 0.05, **P < 0.01
versus model group (control).
1
glutamate severely inhibited Bcl-2 expression (0.44-fold) and
promoted cleaved caspase-3 expression (1.88-fold). In
contrast, SL06 (50 μM) remarkably reversed the effects of
glutamate on Bcl-2 (0.85-fold) and increased the caspase-3
ratio to 1.16 (P < 0.01). Obviously, it was demonstrated that
SL06 plays the antiapoptotic role by regulating apoptosis-
associated protein expression at the molecular level.
to afford 4 (amorphous white solid, 13.4 g, yield 92%). H
NMR (500 MHz, DMSO-d6): δ 11.05 (s, 1H), 2.27 (s, 3H),
1.43 (d, J = 7.7 Hz, 18H). MS (ESI, m/z): 291.13 [M + H]+.
4.2.3. Synthesis of N,N′-Boc-N″-(4-hydroxy-butyl)-guani-
dine (5). Compound 4 (1.45 g, 5 mmol) and 4-amino-1-
butanol (450 mg, 5 mmol) were added in N,N-dimethylfor-
mamide (10 mL) at room temperature. After it was stirred for
9 h, the mixture was diluted with dichloromethane (20 mL)
and water (20 mL) (1:1). Then, the organic phase was
separated and washed with brine (10 mL × 3), dried with
anhydrous sodium sulfate, and evaporated to dryness in vacuo,
and it was purified with silica gel (petroleum ether/ethyl
acetate, 2:1) to afford 5 (amorphous white solid, 1.48 g, yield
90%). 1H NMR (500 MHz, DMSO-d6): δ 11.49 (s, 1H), 8.28
(t, J = 5.0 Hz, 1H), 4.41 (s, 1H), 3.40 (d, J = 5.8 Hz, 2H), 3.28
(d, J = 6.3 Hz, 2H), 1.52 (dd, J = 14.4, 7.2 Hz, 2H), 1.47 (s,
9H), 1.45−1.41 (m, 2H), 1.39 (s, 9H). MS (ESI, m/z): 354.01
[M + Na]+.
4.2.4. Synthesis of 3,5-Dimethoxy-4-acetoxy-benzoic Acid
4-(N,N′-tert-butyl Carbonyl-guanidino)-butyl Ester (6). To a
suspension of 5 (5.52 g, 16.66 mmol) in dichloromethane (100
mL), 2 (6 g, 24.98 mmol), 4-(dimethylamino) pyridinium
tosylate (DPTS) (9.8 g, 33.32 mmol), and N,N′-diisopropyl-
carbodiimide (DIC) (4.2 g, 33.32 mmol) were added
successively. After stirring at 25 °C for 5 h under nitrogen
3. CONCLUSION
In summary, the designed novel conjugates based on the
structure of stachydrine and leonurine were synthesized and
evaluated for biological activities. Compound SL06 showed a
higher efficacy than those of other conjugates in the screening
results. SL06 could improve neuronal cell viability and SOD
activity and decrease the percentage of apoptotic neuronal cells
and LDH, ROS, and MDA levels in vitro. Moreover, in vivo, the
infarction size was significantly reduced by SL06 in the
tMCAO rat model. Furthermore, SL06 showed a high safety
treatment window in mice. Furthermore, the study of the
mechanism showed that SL06 was related to the increase in
SOD and AKT/GSK-3β activity, as well as the decrease in
LDH and ROS levels. Also, it could not only promote the
expression of antiapoptoticprotein Bcl-2 but also inhibit the
expression of apoptosis-associated protein cleaved caspase-3.
In conclusion, this experimental evidence suggests the
2484
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
atmosphere, the suspension was filtered and washed with water
three times and then extracted with dichloromethane (20 mL
× 3). The organic phase was washed with brine (20 mL × 3),
dried with anhydrous sodium sulfate, evaporated to dryness in
vacuo, and purified with silica gel (petroleum ether/ethyl
acetate, 4:1) to afford 6 (amorphous pale solid, 6.17 g, yield
67%). 1H NMR (500 MHz, DMSO-d6): δ 11.48 (s, 1H), 8.33
(t, J = 5.5 Hz, 1H), 7.28 (s, 2H), 4.31 (t, J = 6.3 Hz, 2H), 3.82
(s, 6H), 3.36 (q, J = 6.6 Hz, 2H), 2.28 (s, 3H), 1.73 (dd, J =
14.0, 6.6 Hz, 2H), 1.66−1.61 (m, 2H), 1.46 (s, 9H), 1.37 (s,
9H). MS (ESI, m/z): 576.08 [M + Na]+.
3,5-dimethoxybenzoate (8e). To a solution of 7 (0.54 g, 1.05
mmol in N,N-dimethylformamide (20 mL), sodium hydride
was added at 0 °C. After it was stirred for 15 min, tert-butyl N-
(2-bromoethyl)carbamate (0.5 g, 2.1 mmol) was added and
stirred for 1 h at 80 °C under nitrogen atmosphere and then
diluted with water (40 mL) and extracted with dichloro-
methane (20 mL × 3). The dichloromethane layer was washed
with brine (20 mL × 3), dried with anhydrous sodium sulfate,
and concentrated to afford 8e (colorless oil, 614 mg, yield
89%). MS (ESI, m/z): 677.29 [M + H]+.
4.2.11. Synthesis of 4-Guanidinobutyl 4-(2-Aminoace-
toxy)-3,5-dimethoxybenzoate (9a). Compound 8a (100 mg,
0.15 mmol) and trifluoroacetic acid (0.5 mL) were added in
dichloromethane (10 mL) at 0 °C and then stirred for 8 h at
room temperature under nitrogen atmosphere. After the
reaction, the solvent was evaporated in vacuo, and the mixture
was diluted with water (20 mL) and extracted with
dichloromethane (20 mL × 3). The dichloromethane layer
was washed with brine (20 mL × 3), dried with anhydrous
sodium sulfate, and concentrated in vacuo to afford 9a (white
foamy solid, 54 mg, yield 98%). 1H NMR (500 MHz,
methanol-d4): δ 7.36 (s, 2H), 5.36 (dd, J = 5.6, 4.2 Hz, 1H),
4.39 (t, J = 6.5 Hz, 2H), 3.91 (s, 6H), 3.66−3.54 (m, 4H), 1.88
(dt, J = 14.0, 6.5 Hz, 2H), 1.77 (q, J = 7.5 Hz, 2H). MS (ESI,
m/z): 391.17 [M + Na]+.
4.2.12. Synthesis of 4-Guanidinobutyl 4-((2-
Aminopropanoyl)oxy)-3,5-dimethoxybenzoate (9b). Com-
pound 8b (500 mg, 0.66 mmol), trifluoroacetic acid (2 mL),
dichloromethane (10 mL); 295 mg white foamed solid with a
yield 97%. MS (ESI, m/z): 383.08 [M + H]+.
4.2.13. Synthesis of 4-Guanidinobutyl 4-((3-
Aminopropanoyl)oxy)-3,5-dimethoxybenzoate (9c). Com-
pound 8c (500 mg, 0.73 mmol), trifluoroacetic acid (2.1
mL), dichloromethane (20 mL); 276 mg white foamed solid
with a yield 99%. MS (ESI, m/z): 383.31 [M + H]+.
4.2.5. Synthesis of 3,5-Dimethoxy-4-hyroxy-benzoic Acid
4-(N,N′-tert-butyl Carbonyl-guanidino)-butyl Ester (7). To a
solution of 6 (10.8 g, 19.50 mmol, 1 equiv) in dichloro-
methane (100 mL), 7.2 mL of sodium methoxide in methanol
(5.4 mol/L) was added. After it was stirred for 5 h at 4 °C
under nitrogen atmosphere, the mixture was diluted with water
(100 mL), adjusted to pH 7 with dilute hydrochloric acid (6
mol/L), and extracted with dichloromethane (20 mL × 3)
successively. Next, the organic phase was washed with brine
(20 mL × 3), dried with anhydrous sodium sulfate, and
evaporated to afford 7 (white foamed solid, 9.56 g, yield 96%).
1H NMR (500 MHz, DMSO-d6): δ 11.48 (s, 1H), 9.31 (s,
1H), 8.33 (s, 1H), 7.20 (s, 2H), 4.24−4.20 (m, 1H), 4.02 (q, J
= 7.1 Hz, 1H), 3.80 (s, 6H), 1.98 (s, 1H), 1.75−1.58 (m, 5H),
1.46 (s, 9H), 1.37 (s, 9H). MS (ESI, m/z): 512.20 [M + H]+.
4.2.6. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-(2-((tert-Butoxycarbonyl)amino)-
acetoxy)-3,5-dimethoxybenzoate (8a). Compound 7 (1 g,
1.95 mmol) was dissolved in dichloromethane (50 mL), and
Boc-glycine (0.38 g, 2.17 mmol), DPTS (1.14 g, 3.88 mmol),
and DIC (0.5 g, 3.96 mmol) were added successively. The
mixture was stirred at 30 °C under nitrogen atmosphere for 3
h. After the reaction, the mixture was diluted with water (50
mL) and extracted with dichloromethane (20 mL × 3). The
organic phase was washed with brine (20 mL × 3), dried with
anhydrous sodium sulfate, and evaporated in vacuo. Then, it
was purified with silica gel (petroleum ether/ethyl acetate, 4:1)
to get 8a (foamed solid, 9.78 g, yield 75%). MS (ESI, m/z):
775.37 [M + H]+.
4.2.14. Synthesis of (S)-4-Guanidinobutyl 4-((2-Amino-3-
(prop-2-yn-1-yl-thio)propanoyl)oxy)-3,5-dimethoxyben-
zoate (9d). Compound 8d (500 mg, 0.73 mmol), trifluoro-
acetic acid (2.1 mL), dichloromethane (20 mL); 276 mg white
1
foamed solid with a yield 98%. H NMR (500 MHz, CDCl3):
δ 7.32 (s, 2H), 7.27 (s, 2H), 4.35 (t, 4H), 3.95 (s, 6H), 3.32−
3.13 (m, 2H), 2.81 (t, 1H), 2.34−2.26 (m, 4H), 1.62 (s, 2H),
1.49 (d, J = 6.3 Hz, 4H). MS (ESI, m/z): 453.23 [M + H]+.
4.2.15. Synthesis of 4-Guanidinobutyl 4-(2-Aminoethoxy)-
3,5-dimethoxybenzoate (9e). Compound 8e (614 mg, 0.93
mmol), trifluoroacetic acid (3 mL), dichloromethane (20 mL);
4.2.7. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-((2-((tert-Butoxycarbonyl)amino)-
propanoyl)oxy)-3,5-dimethoxybenzoate (8b). Compound 7
(500 mg, 0.98 mmol), Boc-L-alanine (204 mg, 1.08 mmol),
DPTS (574 mg, 1.96 mmol), DIC (247 mg, 1.96 mmol),
dichloromethane (20 mL); 388 mg foamed solid with a yield
58%. MS (ESI, m/z): 705.35 [M + Na]+.
1
310 mg white foamed solid with a yield 94%. H NMR (500
MHz, DMSO-d6): δ 7.90 (t, J = 5.1 Hz, 1H), 7.26 (s, 2H), 4.29
(t, J = 6.3 Hz, 2H), 4.10 (t, J = 4.9 Hz, 2H), 3.86 (s, 6H),
3.20−3.14 (m, 2H), 3.09 (t, J = 4.6 Hz, 2H), 1.79−1.70 (m,
2H), 1.65−1.55 (m, 2H). MS (ESI, m/z): 355.08 [M + H]+.
4.2.16. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-(2-(1-methylpyrrolidine-2-carboxamido)acetoxy)benzoate
Hydrochloride (SL01). To a suspension of 9a (120 mg, 0.22
mmol) in N,N-dimethylformamide (20 mL), N-methyl-L-
proline (43 mg, 0.33 mmol), 2-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) (250 mg, 0.66 mmol) and N,N-diisopropylethyl-
amine (DIPEA) (84 mg, 0.66 mmol) were added successively.
The mixture was stirred at room temperature under nitrogen
atmosphere for 1 h. After the reaction, the solvent was
evaporated in vacuo, and the residue was purified with silica gel
(dichloromethane/methyl alcohol, 7:1). Then, it reacted with
4.2.8. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-((3-((tert-Butoxycarbonyl)amino)-
propanoyl)oxy)-3,5-dimethoxybenzoate (8c). Compound 7
(1 g, 1.96 mmol), Boc-β-alanine (408 mg, 2.16 mmol), DPTS
(1.15 g, 3.92 mmol), DIC (494 mg, 3.92 mmol), dichloro-
methane (40 mL); 740 mg foamed solid with a yield 55%. MS
(ESI, m/z): 705.35 [M + Na]+.
4.2.9. Synthesis of (S)-4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-((2-((tert-Butoxycarbonyl)amino)-3-
(prop-2-yn-1-ylthio)propanoyl)oxy)-3,5-dimethoxybenzoate
(8d). Compound 7 (2.8 g, 5.47 mmol), 4-Boc-L-(S-propynyl)-
cysteine (1.1 g, 6.9 mmol), DPTS (3.2 g, 10.9 mmol), DIC
(1.38 g, 10.9 mmol), dichloromethane (120 mL); 1.6 g foamed
solid with a yield 38.7%. MS (ESI, m/z): 775.37 [M + Na]+.
4.2.10. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-(2-((tert-Butoxycarbonyl)amino)ethoxy)-
2485
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
hydrochloric acid in methanol (2 mL) at room temperature for
10 min, and the solvent was evaporated in vacuo to afford SL01
(light yellow solid, 320 mg, yield 99%). [α]25D −69.09° (c 0.50,
HRMS (ESI) m/z calcd for C26H38ClN5O7S [1/2M + H]+
282.6343; found, 282.6285.
4.2.20. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-(2-(1-methylpyrrolidine-2-carboxamido)ethoxy)benzoate
Hydrochloride (SL05). Compound 9e (1.3 g, 3.67 mmol), N-
methyl-L-proline (479 mg, 3.71 mmol), HATU (2.79 g, 7.34
mmol), DIPEA (948 mg, 7.34 mmol), DMF (20 mL),
hydrochloric acid in methanol (2 mL); 940 mg white solid
with a yield 55%. [α]25D −71.81° (c 0.11, CH3OH); 1H NMR
(500 MHz, methanol-d4): δ 8.54 (s, 1H), 7.35 (s, 2H), 4.37 (t,
J = 6.5 Hz, 2H), 4.11−4.08 (m, 2H), 3.90 (s, 6H), 3.61−
3.58(m, 2H), 3.49−3.48 (m, 2H), 3.27 (t, J = 7.1 Hz, 2H),
2.41 (s, 3H), 2.26−2.19 (m, 1H), 1.93−1.80 (m, 4H), 1.77−
1.73 (m, 4H). 13C NMR (125 MHz, methanol-d4): δ 166.2
(2C), 157.3, 153.1 (2C), 140.8, 125.5, 106.5 (2C), 71.1, 68.8,
64.4, 56.1, 55.5 (2C), 40.7, 39.8, 39.5, 29.6, 25.7, 25.3, 22.7.
HRMS (ESI) m/z calcd for C22H36ClN5O6 [1/2M + H]+
234.6367; found, 234.6370.
4.2.21. Synthesis of 4-Amino-3,5-dimethoxybenzoic Acid
(11). To a solution of benzoic acid 10 (10 g, 38.3 mmol) in an
ammonia solution (35 mL) at 0 °C, sodium hydroxide (1.55 g,
38.7 mmol) and copper sulfate (1.67 g, 10.4 mmol) were
added successively. After it was stirred at 135 °C for 12 h, the
mixture was acidified with hydrochloric acid (12 mol/L) for
the adjustment of the pH to 3, and it was filtered. The filter
cake was stirred in methanol (100 mL) for 2 h; then, the
mixture was filtered. The filtrate was evaporated in vacuo; then,
the residue was stirred and filtered in ethyl acetate (150 mL)
and water (80 mL) for 2 h, respectively, to afford 11 (light red
powder, 3.4 g, yield 45%). 1H NMR (500 MHz, DMSO-d6): δ
7.14 (s, 2H), 4.94 (s, 2H), 3.80 (s, 6H). 13C NMR (125 MHz,
DMSO-d6): δ 167.6, 145.0 (2C), 131.3, 116.5, 105.8 (2C),
55.7 (2C). MS (ESI, m/z): 198.23 [M + H]+.
4.2.22. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl4-amino-3,5-dimethoxybenzoate (12). To
a suspension of 5 (2 g, 6.03 mmol) in dichloromethane (50
mL), Compound 11 (1.43 g, 7.25 mmol), DPTS (3.53 g, 12.06
mmol), and DIC (1.52 g, 12.06 mmol) were added
successively, and the mixture was stirred at 30 °C under
nitrogen atmosphere for 3 h. The mixture was then diluted
with water (100 mL) and extracted with dichloromethane (3 ×
20 mL). The organic phase was separated, washed with brine
(20 mL × 3), and dried with anhydrous sodium sulfate. The
solvent was removed under reduced pressure. The residue was
purified with silica gel (petroleum ether/ethyl acetate, 7:1) to
afford 12 (amorphous pale solid, 0.86 g, yield 28%). 1H NMR
(500 MHz, DMSO-d6): δ 11.49 (s, 1H), 8.33 (s, 1H), 7.15 (s,
2H), 5.05 (s, 2H), 4.22 (t, J = 6.1 Hz, 2H), 3.81 (s, 6H), 3.35
(d, J = 6.1 Hz, 2H), 1.73−1.67 (m, 2H), 1.65−1.59 (m, 2H),
1.46 (s, 9H), 1.38 (s, 9H). 13C NMR (125 MHz, DMSO-d6): δ
165.9, 163.1, 155.2, 152.0, 145.0 (2C), 131.9 (2C), 115.3
(2C), 105.7 (2C), 82.8, 78.0 (2C), 63.6, 55.7 (2C), 27.9 (2C),
27.5 (2C), 25.8, 25.3. MS (ESI, m/z): 511.32 [M + H]+.
4.2.23. Synthesis of 4-(2,3-Bis(tert-butoxycarbonyl)-
guanidino)butyl 4-(2-((tert-Butoxycarbonyl)amino)-
acetamido)-3,5-dimethoxybenzoate (13). To a suspension
of 12 (1 g, 1.95 mmol) in N,N-dimethylformamide (25 mL),
Boc-glycine (375 mg, 2.2 mmol), HATU (1.48 g, 3.9 mmol),
and DIPEA (505 mg, 3.9 mmol) were added successively and
stirred for 2 h at 30 °C. After dilution with water (50 mL), the
mixture was extracted with dichloromethane (3 × 20 mL). The
organic phase was separated, washed with brine (20 mL × 3),
and dried with anhydrous sodium sulfate. The solvent was
1
CH3OH); H NMR (500 MHz, Methanol-d4): δ 7.75−7.73
(m, 2H), 7.39 (d, J = 1.3 Hz, 2H), 7.27−7.25 (m, 2H), 4.41 (t,
J = 6.8 Hz, 2H), 4.34 (d, J = 6.1 Hz, 2H), 3.90 (d, J = 1.3 Hz,
6H), 3.30 (t, J = 7.0 Hz, 2H), 3.25 (dd, J = 9.0, 4.8 Hz, 1H),
3.13−3.07 (m, 1H), 2.40 (s, 3H), 2.29 (td, J = 9.6, 5.3 Hz,
1H), 1.96−1.73 (m, 8H). 13C NMR (125 MHz, methanol-d4):
δ 152.2 (2C), 140.4, 128.4 (2C), 125.6 (2C), 105.8 (2C),
68.7, 64.6, 56.1, 55.5 (2C), 40.7, 40.1, 39.9, 30.2, 25.6, 25.2,
23.3, 19.9. HRMS (ESI) m/z calcd for C22H34ClN5O7 [M +
H]+ 480.2453; found, 480.2456.
4.2.17. Synthesis of 4-Guanidinobutyl 3,5-Dimethoxy-4-
((2-((S)-1-methylpyrrolidine-2-carboxamido)propanoyl)-
oxy)benzoate Hydrochloride (SL02). Compound 9b (100 mg,
0.26 mmol), N-methyl-L-proline (34 mg, 0.27 mmol), HATU
(198 mg, 0.52 mmol), DIPEA (68 mg, 0.52 mmol), DMF (10
mL), hydrochloric acid in methanol (2 mL); 53 mg white solid
with a yield 99%. [α]20 −64.67° (c 0.51, CH3OH); mp =
D
63.2−74.1 °C; 1H NMR (500 MHz, Methanol-d4): δ 7.69 (d, J
= 7.9 Hz, 2H), 7.35 (d, J = 7.7 Hz, 2H), 7.22 (d, J = 7.6 Hz,
2H), 4.35 (t, J = 6.2 Hz, 2H), 3.83 (s, 6H), 3.69 (d, J = 15.2
Hz, 1H), 3.59 (d, J = 6.9 Hz, 1H), 3.28 (t, J = 22.7 Hz, 2H),
2.96 (dd, J = 38.8, 12.3 Hz, 1H), 2.35 (s, 3H), 1.84 (s, 1H),
1.73−1.61 (t, 8H), 1.29 (s, 3H). 13C NMR (125 MHz,
methanol-d4): δ 166.6, 154.8 (2C), 128.8, 117.9 (2C), 113.3,
109.3, 105.1 (2C), 70.2, 65.0, 58.1, 55.5 (2C), 40.8 (2C), 37.8,
30.1, 27.1, 26.1 (2C), 20.1, 12.1. HRMS (ESI) m/z calcd for
C23H36ClN5O7 [M + H]+ 494.2609; found, 494.2611.
4.2.18. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-((3-(1-methylpyrrolidine-2-carboxamido)propanoyl)oxy)-
benzoate Hydrochloride (SL03). Compound 9c (200 mg, 0.52
mmol), N-methyl-L-proline (74 mg, 0.57 mmol), HATU (395
mg, 1.04 mmol), DIPEA (134 mg, 1.04 mmol), DMF (20
mL), hydrochloric acid in methanol (2 mL); 159 mg white
1
solid with a yield 99%. [α]20 −80.06° (c 0.52, H2O); H
D
NMR (500 MHz, methanol-d4): δ 7.74 (d, J = 6.4 Hz, 2H),
7.39 (s, 1H), 7.34 (s, 1H), 7.27−7.25 (m, 2H), 4.42−4.32 (m,
4H), 3.91 (s, 6H), 3.34−3.30 (m, 2H), 3.28 (s, 1H), 3.09 (m,
1H), 2.85 (t, J = 5.5 Hz, 2H), 2.39 (s, 3H), 2.3−2.21 (m, 1H),
1.92−1.86 (m, 8H). 13C NMR (125 MHz, methanol-d4): δ
153.5 (2C), 141.7, 129.8 (2C), 126.9 (2C), 107.2 (2C), 82.8
(2C), 70.0, 65.9, 57.5, 56.9 (2C), 41.2, 42.0, 31.5, 27.0, 26.6,
24.6, 21.6. HRMS (ESI) m/z calcd for C23H36ClN5O7 [M +
H]+ 494.2609; found, 494.2616.
4.2.19. Synthesis of 4-Guanidinobutyl 3,5-Dimethoxy-4-
(((R)-2-((S)-1-methylpyrrolidine-2-carboxamido)-3-(prop-2-
yn-1-ylthio)propanoyl)oxy)benzoate Hydrochloride (SL04).
Compound 9d (250 mg, 0.55 mmol), N-methyl-L-proline (71
mg, 0.55 mmol), HATU (418 mg, 1.1 mmol), DIPEA (142
mg, 1.1 mmol), DMF (20 mL), hydrochloric acid in methanol
(2 mL); 105 mg white solid with a yield 99%. [α]25D −23.64°
1
(c 0.50, CH3OH); H NMR (500 MHz, methanol-d4): δ 7.38
(s, 2H), 5.07 (dd, J = 9.5, 4.3 Hz, 1H), 4.39 (t, J = 6.4 Hz,
2H), 4.16−4.07 (m, 2H), 3.87 (s, 6H), 3.73 (s, 1H), 3.58−
3.48 (m, 2H), 3.41 (d, J = 16.8 Hz, 1H), 3.26 (t, J = 7.0 Hz,
3H), 3.10 (dd, J = 14.5, 9.4 Hz, 1H), 2.96 (s, 3H), 2.70 (s,
1H), 2.62 (dd, J = 12.9, 8.0 Hz, 1H), 2.28−2.02 (m, 4H), 2.01
(s, 1H), 1.87 (p, J = 6.6 Hz, 2H), 1.75 (q, J = 7.3 Hz, 2H). 13
C
NMR (125 MHz, methanol-d4): δ 167.7, 167.5, 165.7, 157.2,
152.0, 132.0, 128.6, 106.7, 105.7, 78.8, 71.8, 68.6, 64.5, 56.0,
55.5, 51.8, 40.6, 39.5, 32.5, 28.9, 25.5, 25.1, 22.3, 18.5, 13.0.
2486
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
removed under reduced pressure, and the residue was purified
with silica gel (petroleum ether/ethyl acetate, 5:1) to afford 13
(white solid, 735 mg, yield 56.5%). MS (ESI, m/z): 668.73 [M
+ H]+.
2-oxoethyl)carbamoyl) Pyrrolidine-1- carboxylate Hydro-
chloride (SL08). Compound 14 (200 mg, 0.55 mmol), (S)-
1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (120 mg,
0.56 mmol), HATU (420 mg, 1.1 mmol), DIPEA (142 mg, 1.1
mmol), N,N-dimethylformamide (15 mL), hydrochloric acid
in methanol (2 mL); 212 mg foamed solid with a yield 68.5%.
4.2.24. Synthesis of 4-Guanidinobutyl 4-(2-Aminoaceta-
mido)-3,5-dimethoxybenzoate (14). Compound 13 (500 mg,
0.75 mmol) and trifluoroacetic acid (2 mL) were added into
dichloromethane (20 mL) at 0 °C and then stirred for 2 h at
room temperature under nitrogen atmosphere. After the
solvent was evaporated in vacuo, the residue was diluted with
water (20 mL). The mixture was extracted with dichloro-
methane (20 mL × 3) and washed with brine (20 mL × 3) and
then dried with anhydrous sodium sulfate and concentrated in
[α]20 −65.10° (c 0.51, CH3OH); 1H NMR (500 MHz,
D
methanol-d4): δ 7.32 (d, J = 4.5 Hz, 2H), 4.38 (t, J = 6.4 Hz,
2H), 4.26−4.20 (m, 1H), 4.16−3.98 (m, 2H), 3.86 (d, J = 7.7
Hz, 6H), 3.51 (s, 1H), 3.43 (d, J = 6.5 Hz, 1H), 3.27 (t, J = 7.1
Hz, 2H), 2.24 (d, J = 7.4 Hz, 1H), 2.08−1.81 (m, 6H), 1.79−
1.72 (m, 2H), 1.41 (s, 4H), 1.35 (s, 5H). 13C NMR (125
MHz, methanol-d4): δ 165.9, 155.6, 105.4, 104.9 (2C), 80.0,
64.3, 60.4, 55.1, 55.1, 54.9, 40.6, 39.9, 30.9, 29.7, 27.1 (2C),
26.7, 26.7, 25.5, 25.1, 24.0, 23.1. HRMS (ESI) m/z calcd for
C26H41ClN6O8 [M + H]+ 565.2980; found, 565.2992.
4.2.28. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-(2-(pyrrolidine-2-carboxamido)acetamido)benzoate Hy-
drochloride (SL09). Compound 14 (200 mg, 0.55 mmol), L-
proline (65 mg, 0.56 mmol), HATU (420 mg, 1.1 mmol),
DIPEA (142 mg, 1.1 mmol), N,N-dimethylformamide (15
1
vacuo to afford 14 (light yellow oil, 270 mg, yield 98%). H
NMR (500 MHz, DMSO-d6): δ 9.72 (s, 1H), 8.16 (s, 3H),
7.90 (s, 1H), 7.26 (s, 4H), 4.32 (s, 2H), 3.82 (s, 8H), 3.18 (d, J
= 6.1 Hz, 2H), 1.75 (s, 2H), 1.62 (d, J = 6.1 Hz, 2H). 13C
NMR (126 MHz, DMSO-d6): δ 165.2, 156.9 (2C), 155.2
(2C), 109.5, 105.1 (2C), 64.5 (2C), 55.9 (2C), 25.4 (2C),
25.1 (2C). MS (ESI, m/z): 368.14 [M + H]+.
4.2.25. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-(2-(1-methylpyrrolidine-2-carboxamido)acetamido)-
benzoate Hydrochloride (SL06). To a solution of 14 (715 mg,
1.95 mmol) in N,N-dimethylformamide (20 mL), N-methyl-L-
proline (273 mg, 2.1 mmol), HATU (1.5 g, 3.9 mmol), and
DIPEA (503 mg, 3.9 mmol) were added successively and
stirred for 3 h at 30 °C under nitrogen atmosphere. After the
reaction, the mixture was diluted with water (50 mL) and
extracted with dichloromethane (20 mL × 3). The organic
phase was washed with brine (20 mL × 3), dried with
anhydrous sodium sulfate, and evaporated in vacuo. Then, it
was purified with silica gel (petroleum ether/ethyl acetate, 4:1)
mL), hydrochloric acid in methanol (2 mL); 183 mg white
1
solid with a yield 72%. [α]25 −69.54° (c 0.51, CH3OH); H
D
NMR (500 MHz, DMSO-d6): δ 10.47 (s, 1H), 9.33 (s, 1H),
8.96 (s, 1H), 8.53 (s, 1H), 8.15 (s, 1H), 7.52 (d, J = 51.0 Hz,
2H), 7.23 (s, 2H), 4.30 (t, J = 6.2 Hz, 2H), 4.23 (s, 1H), 3.97
(s, 2H), 3.80 (s, 6H), 3.24−3.16 (m, 4H), 2.30 (d, J = 7.2 Hz,
1H), 1.87 (s, 3H), 1.80−1.74 (m, 2H), 1.63−1.56 (m, 2H).
13C NMR (125 MHz, DMSO-d6): δ 168.5, 165.3, 157.3, 155.3
(2C), 128.8, 119.0, 105.0 (2C), 64.5, 58.6, 56.0 (2C), 45.4
(2C), 40.2, 29.7, 25.4, 25.2, 23.5 (2C). HRMS (ESI) m/z
calcd for C21H33ClN6O6 [M + H]+ 465.2456; found, 465.2460.
4.3. Biological Evaluation. 4.3.1. Cell Culture and
Reagents. CGCs were obtained through Sprague−Dawley
(SD) 7-day-old rats from Comparative Medicine Centre
(Yangzhou University, China). After washing with ethanol
(75%), cerebella were removed and stored in Hanks’ balanced
salt solution (HBSS) which was cooled with ice; then, the
meninx was removed and placed for 15 min in HBSS
containing 0.05% trypsin. Next, two volumes of neurobasal
solution containing neuronal cell culture supplement B27
(10%), GlutaMax (0.5 mM), gentamycin (10 μg/mL), and
KCl (20 mM) were added to terminate trypsin digestion. The
resulting solution was triturated and centrifuged (1000× rpm,
5 min, 4 °C) and filtered using a nylon cell restrainer (70 μm).
After dilution (1 × 106 cells/mL), the cells were incubated in a
polylysine (0.1 g/L) treated 6-well plate under a humidified
atmosphere containing 5% CO2 at 37 °C.
to afford SL06 (light brown solid, 751 mg, yield 75%). [α]20
−65.77° (c 0.51, CH3OH); H NMR (500 MHz, Methanol-
D
1
d4): δ 7.33 (s, 2H), 4.39 (t, J = 6.4 Hz, 2H), 4.13 (s, 2H), 3.88
(s, 6H), 3.35 (s, 1H), 3.28 (t, J = 7.0 Hz, 2H), 3.22 (q, J = 7.3
Hz, 1H), 2.71 (s, 1H), 2.61 (s, 3H), 2.36 (s, 1H), 2.00−1.83
(m, 5H), 1.77 (dd, J = 15.0, 7.3 Hz, 2H), 1.33 (t, J = 7.3 Hz,
2H). 13C NMR (125 MHz, methanol-d4): δ 166.0, 155.4,
118.2, 105.0, 68.7, 64.4, 56.1, 55.2, 46.4, 40.6, 40.0, 29.8, 25.6,
25.2, 23.0, 7.8. HRMS (ESI) m/z calcd for C22H35ClN6O6 [1/
2M + H]+ 240.1343; found, 240.1349.
4.2.26. Synthesis of (S)-4-Guanidinobutyl 3,5-Dimethoxy-
4-(2-(2-(1-methylpyrrolidine-2-carboxamido)acetamido)-
acetamido)benzoate Hydrochloride (SL07). Compound 14
(500 mg, 0.88 mmol), (S)-2-(1-methylpyrrolidine-2-
carboxamido)acetic acid (163 mg, 0.88 mmol), HATU (672
mg, 1.76 mmol), DIPEA (227 mg, 1.76 mmol), N,N-
dimethylformamide (10 mL), hydrochloric acid in methanol
4.3.2. Animals. SD rats weighing about 250−280 g were
obtained from Qinglong Mountain Animal Breeding Farm,
District Jiangning, Nanjing, Jiangsu Province, China. All rats
were raised under standard conditions at temperatures 20−23
°C, a relative humidity 50%, and a 12 h light/dark cycle.
4.3.3. Analysis of Cell Viability and Western Blotting.
Excitotoxicity was induced by exposing CGCs to 200 μM
glutamate for 24 h. To determine the neuroprotective effects of
all compounds, the cells were pretreated with different
concentrations of the compound for 24 h. Cell viability was
measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium (MTT) assay (Beyotime, China). Briefly, cells were
incubated with 0.5 mg/mL MTT in a humidified atmosphere
of 5% CO2 and 95% air at 37 °C for 4 h. Then, cells were lysed
in dimethyl sulfoxide (DMSO), and the amount of MTT
(2 mL); 210 mg off-white solid with a yield 42%. [α]20
D
238.24° (c 0.51, CH3OH); 1H NMR (500 MHz, methanol-d4):
δ 8.33 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 4.3 Hz, 1H), 7.32
(s, 2H), 4.38 (t, J = 6.4 Hz, 2H), 4.18−3.93 (m, 6H), 3.86 (s,
6H), 3.69 (ddd, J = 11.3, 7.5, 4.3 Hz, 1H), 3.27 (t, J = 7.1 Hz,
4H), 2.94 (s, 3H), 2.61−2.55 (m, 1H), 2.24−2.00 (m, 4H),
1.87 (p, J = 6.6 Hz, 2H), 1.78−1.71 (m, 2H). 13C NMR (125
MHz, methanol-d4): δ 169.8, 168.8, 165.9, 157.2, 155.5, 129.6,
118.2, 109.9, 104.9 (2C), 68.5, 64.3, 56.0, 55.1 (2C), 42.0,
42.0, 40.6, 39.7, 29.0, 29.0, 25.5, 25.1, 22.4. HRMS (ESI) m/z
calcd for C24H38ClN7O7 [1/2M + H]+ 268.6450; found,
268.6451.
4.2.27. Synthesis of (S)-tert-Butyl 2-((2-((4-((4-
Guanidinobutoxy)carbonyl)-2,6-dimethoxyphenyl)amino)-
2487
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
formazan was quantified by absorbance at 570 nm in a
microplate reader. The absorbance (control group’s absorb-
ance set as 100%) was positively correlated with cell viability.
For Western blotting, after SL-06 treatment at different
times or with glutamate incubation, cells were collected by 1×
Tris-Glycine SDS lysis buffer, and the lysates were boiled for
10 min. The extracted whole cell proteins were separated by
electrophoresis on SDS-PAGE gels to determine the levels of
phospho-GSK-3β (p-GSK-3β), phosphor-Akt (p-Akt), Bcl-2,
and cleaved caspase-3 protein. Proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes. The mem-
branes were blocked for 1 h with 5% bovine serum albumin
(BSA) in Tris-buffered saline plus Tween20 (TBST, pH 7.4)
and incubated with primary antibodies (rabbit anti-p-GSK-3β
= 1:1000; rabbit anti-p-Akt = 1:1000; rabbit anti-Bcl-2 =
1:1000; rabbit anticleaved caspase-3 = 1:1000; mouse anti-β-
actin = 1:2000, all purchased from Cell Signaling Technology,
Danvers, MA, USA). After they were washed with TBST, the
membranes were exposed to the corresponding secondary
antibodies for 1 h at room temperature. Following four washes
with TBST, the blots were visualized using chemiluminescence
and Bio-Rad ChemiDoc XRS. The resulting bands were
quantified using densitometric analysis and were normalized to
the levels of β-actin protein.
weight, and food consumption were recorded every day for a
total of 14 days. The clinical signs include the presence of
tremors, loose bowel, abdominal contortions, convulsions,
lethargy, salivation, diarrhea, and dyskinesia.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00200.
■
sı
Chemistry synthesis and 1H NMR, 13C NMR, and
HRMS spectra for all final compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Feng Li − Shandong Peninsula Engineering Research Center of
Comprehensive Brine Utilization, Weifang University of
Science and Technology, Weifang 262700, China;
orcid.org/0000-0003-2795-1693; Phone: (+86) 536-
5101992; Email: fengli81@163.com
Wenbao Li − Shandong Peninsula Engineering Research
Center of Comprehensive Brine Utilization, Weifang
University of Science and Technology, Weifang 262700,
China; School of Medicine and Pharmacy, Ocean University
of China, Qingdao 266003, China; Marine Biomedical
Research Institute of Qingdao, Qingdao 266071, China;
Phone: (+86) 536-5101992; Email: wbli92128@163.com
4.3.4. ROS, LDH, MDA, SOD Activity, and Apoptosis. LDH
and MDA were detected with assay kits of LDH (Beyotime,
China) and MDA (Beyotime, China). The reactive oxygen
species (ROS) was detected with DCFH-DA Kit (Beyotime,
China). The activity of superoxide dismutase (SOD) was
detected with the SOD assay kit (Beyotime, China). The
apoptosis rate of cells was measured with TUNEL kit (Vazyme
Biotech, China). All tests were done according to the
manufacturer’s protocol.
Authors
Sifeng Zhu − School of Medicine and Pharmacy, Ocean
University of China, Qingdao 266003, China
Qihui Jiang − New Drug Screening Center, Institute of
Pharmaceutical Sciences, China Pharmaceutical University,
Nanjing 210009, China
4.3.5. Focal Cerebral Ischemia Procedure (tMCAO) and
Drug Treatment. SD rats were grouped randomly. Each group
was designated no fewer than 12 rats. Methods were
performed based on previously reported literature.45 Isoflurane
(3.5%) was used to anesthetize rats, and 1.0−2.0% isoflurane
in 70% N2 and 29% O2 was used to maintain anesthesia. The
body temperature was monitored with a rectal probe and kept
within 37.0 0.5 °C with a heating pad. The cerebral blood
flow at 5 min before the operation and 5 min after ischemia
was measured by Doppler flowmetry. A 15% decrease of blood
flow levels of point MCA cerebral was considered as ischemia.
After the rats were anesthetized with isoflurane, the common
carotid artery (CCA), internal carotid artery (ICA), and
external carotid artery (ECA) could be accessed through an
incision made in the neck of rats. With a proper amount of
silicone on the head, a monofilament nylon suture (d = 0.26
mm) was threaded into ECA, ICA, and MCA successively and
then removed gently after ischemia for 2 h. After reperfusion
for 10 min, rats with dead or nonsatisfactory cerebral blood
flow (CBF) monitoring with Doppler flowmetry were
excluded. Rats were administered intravenously 2, 4, and 6 h
after arterial occlusion according to the sham group (Sham),
control group (Vehicle), edaravone (3 mg/kg) group, and
SL06 (5 mg/kg) group. The volume of cerebral infarction in
each group was measured 24 h after the administration.
4.3.6. Acute Toxicity Measurement. Kunming mice were
used to test the acute toxicity of SL06 in vivo. Sixty mice (18−
23 g) were separated in cages and raised for 7 days in five
groups, including one normal group and four treatment groups.
After the same treatment in the same cage, clinical signs, body
Chenhui Hou − School of Medicine and Pharmacy, Ocean
University of China, Qingdao 266003, China
Tao Pang − New Drug Screening Center, Institute of
Pharmaceutical Sciences, China Pharmaceutical University,
Nanjing 210009, China
Liang Zhang − School of Medicine and Pharmacy, Ocean
University of China, Qingdao 266003, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00200
Author Contributions
F.L., S.Z., and Q.J. contributed equally to this work. F.L. and
W.L. conceived the research funding, supervised the projects,
interpreted the results, and drafted the manuscript. S.Z., C.H.,
and L.Z. conducted chemical synthesis and structural
characterization under the supervision of F.L. In vitro and in
vivo assays were performed by Q.J. under the supervision of
T.P. All authors contributed to manuscript preparation and
revision.
Funding
This work was supported by the “Zhufeng Scholar Program” of
Ocean University of China (841412016).
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Zhang, K., Tu, M. J., Gao, W., Cai, X., Song, F., Chen, Z., Zhang,
Q., Wang, J., Jin, C., Shi, J., et al. (2019) Hollow Prussian blue
nanozymes drive neuroprotection against ischemic stroke via
2488
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
attenuating oxidative stress, counteracting inflammation, and
suppressing cell apoptosis. Nano Lett. 19, 2812−2823.
(2) Brainin, M., Teuschl, Y., and Kalra, L. (2007) Acute treatment
and long-term management of stroke in developing countries. Lancet
Neurol. 6, 553−561.
(3) Zhou, B. S., Bu, G. Y., Li, M., Chang, B. G., and Zhou, Y. P.
(2014) Tagging SNPs in the MTHFR gene and risk of ischemic
stroke in a Chinese population. Int. J. Mol. Sci. 15, 8931−8940.
(4) Li, M., Cui, F., Yang, F., and Huang, X. (2017) Original article
association between fiber intake and ischemic stroke risk: a meta-
analysis of prospective studies. Int. J. Clin. Exp. Med. 10, 4659−4668.
(5) Snow, S. J. (2016) Stroke and t-PA-triggering new paradigms of
care. N. Engl. J. Med. 374, 809−811.
(6) Denorme, F., Langhauser, F., Desender, L., Vandenbulcke, A.,
Rottensteiner, H., Plaimauer, B., Francois, O., Andersson, T.,
Deckmyn, H., Scheiflinger, F., et al. (2016) ADAMTS13-mediated
thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.
Blood 127, 2337−2345.
(7) Alberts, M. J. (2017) Stroke treatment with intravenous tissue-
type plasminogen activator. Circulation 135, 140−142.
(8) Wang, Y. J., Huang, Y., Xu, Y., Ruan, W., Wang, H., Zhang, Y.,
Saavedra, J. M., Zhang, L., Huang, Z., and Pang, T. (2018) A dual
AMPK/Nrf2 activator reduces brain inflammation after stroke by
enhancing microglia M2 polarization. Antioxid. Redox Signaling 28,
141−163.
(9) Wang, H., Xu, X., Guan, X., Shen, S., Huang, X., Kai, G., Zhao,
S., Ruan, W., Zhang, L., Pang, T., et al. (2020) Liposomal 9-
aminoacridine for treatment of ischemic stroke: From drug discovery
to drug delivery. Nano Lett. 20, 1542−1551.
(10) Gao, C., Hou, G., Liu, J., Ru, T., and Li, X. (2020) Synthesis
and target identification of benzoxepane derivatives as potential anti-
neuroinflammatory agents for ischemic stroke. Angew. Chem., Int. Ed.
Engl. 59, 2429−2439.
(11) Servillo, L., D’Onofrio, N., Longobardi, L., Sirangelo, I.,
Giovane, A., Cautela, D., Castaldo, D., Giordano, A., and Balestrieri,
M. L. (2013) Stachydrine ameliorates high-glucose induced
endothelial cell senescence and SIRT1 downregulation. J. Cell.
Biochem. 114, 2522−2530.
(12) Ma, N., Wei, F., and Guo, J. (2017) Anti-tumor activity of
stachydrine by inhibiting histone diacetylase enzyme in gastric cancer.
Biomed. Res. 28, 802−806.
mitochondria dysfunction in H9c2 Cells. Curr. Pharm. Biotechnol.
11, 895−905.
(21) Liu, X., Pan, L., Gong, Q., and Zhu, Y. (2010) Leonurine
(SCM-198) improves cardiac recovery in rat during chronic
infarction. Eur. J. Pharmacol. 649, 236−241.
(22) Loh, K., Qi, J., Tan, B., Liu, X., Wei, B., and Zhu, Y. (2010)
Leonurine protects middle cerebral artery occluded rats through
antioxidant effect and regulation of mitochondrial function. Stroke 41,
2661−2668.
(23) Liu, C., Gu, X., and Zhu, Y. (2010) Synthesis and biological
evaluation of novel leonurine−SPRC conjugate as cardioprotective
agents. Bioorg. Med. Chem. Lett. 20, 6942−6946.
(24) Koch-Weser, J. (1974) Vasodilator drugs in the treatment of
hypertension. Arch. Intern. Med. 133, 1017−1027.
(25) Nair, M. G., Chen, S. Y., Kisliuk, R. L., Gaumont, Y., and
Strumpf, D. (1979) Folate analogs altered in the C9-N10 bridge
region: 11-thiohomofolic acid. J. Med. Chem. 22, 850−855.
(26) Singh, C., Malik, H., and Puri, S. K. (2004) Synthesis and
antimalarial activity of a new series of trioxaquines. Bioorg. Med. Chem.
12, 1177−1182.
(27) Singh, C., Gupta, N., and Puri, S. K. (2002) Photo-Oxygenation
of Geraniol: Synthesis of a novel series of hydroxy-functionalized anti-
malarial 1,2,4-Trioxanes. Bioorg. Med. Chem. Lett. 33, 1913−1916.
(28) Posner, G. H., Jeon, H. B., Ploypradith, P., Paik, I. H., Borstnik,
K., Xie, S., and Shapiro, T. A. (2002) Orally active, water-soluble
antimalarial 3-aryltrioxanes: short synthesis and preclinical efficacy
testing in rodents. J. Med. Chem. 45, 3824−3828.
(29) Dechy-Cabaret, O., Benoit-Vical, F., Robert, A., and Meunier,
B. (2000) Preparation and antimalarial activities of ″trioxaquines″,
new modular molecules with a trioxane skeleton linked to a 4-
aminoquinoline. ChemBioChem 1, 281−283.
(30) Campos, F., Sobrino, T., Ramos-Cabrer, P., and Castillo, J.
(2012) Oxaloacetate: A novel neuroprotective for acute ischemic
stroke. Int. J. Biochem. Cell Biol. 44, 262−265.
(31) Wang, L., Liu, H., Zhang, L., Wang, G., Zhang, M., and Yu, Y.
(2017) Neuroprotection of dexmedetomidine against cerebral
ischemia-reperfusion injury in rats: Involved in inhibition of NF-κB
and inflammation response. Biomol. Ther. 25, 383−389.
(32) Ghazavi, H., Hoseini, S. J., Ebrahimzadeh-Bideskan, A.,
Mashkani, B., Mehri, S., Ghorbani, A., Sadri, K., Mahdipour, E.,
Ghasemi, F., Forouzanfar, F., Hoseini, A., Pasdar, A. R., Sadeghnia, H.
R., Ghayour-Mobarhan, M., et al. (2017) Fibroblast growth factor
type 1 (FGF1)-overexpressed adipose-derived mesenchaymal stem
cells (AD-MSCFGF1) induce neuroprotection and functional
recovery in a rat stroke model. Stem. Cell Rev. Rep. 13, 670−685.
(33) Kim, Y., Kim, Y. S., Kim, H. Y., Noh, M. Y., Kim, J. Y., Lee, Y. J.,
Kim, J., Park, J., and Kim, S. H. (2018) Early treatment with
poly(ADP-ribose) polymerase-1 inhibitor (JPI-289) reduces infarct
volume and improves long-term behavior in an animal model of
ischemic stroke. Mol. Neurobiol. 55, 7153−7163.
(34) Berger, C., Stauder, A., Xia, F., Sommer, C., and Schwab, S.
(2008) Neuroprotection and glutamate attenuation by acetylsalicylic
acid in temporary but not in permanent cerebral ischemia. Exp.
Neurol. 210, 543−548.
(35) White, B. C, Sullivan, J. M, DeGracia, D. J, O’Neil, B. J,
Neumar, R. W, Grossman, L. I, Rafols, J. A, and Krause, G. S (2000)
Brain ischemia and reperfusion: molecular mechanisms of neuronal
injury. J. Neurol. Sci. 179, 1−33.
(13) Zhang, L., Zheng, N., Ma, C., and Duan, R. (2017) Stachydrine
ameliorates the cerebral ischemia by inhibiting the activity of histone
deacetylase in neonatal rats. Biomed. Res. 28, 973−977.
(14) Miao, M., Wang, T., Lou, X., Bai, M., Xi, P., Liu, B., and Chang,
B. (2017) The influence of stachydrine hydrochloride on the
reperfusion model of mice with repetitive cerebral ischemia. Saudi J.
Biol. Sci. 24, 658−663.
(15) Feng, X. (2009) Pharmacokinetics of stachydrine in rabbits.
China Pharmacist 12, 1696−1697.
(16) Zhang, L., Li, F., Hou, C., Zhu, S., Zhong, L., Zhao, J., Song, C.,
and Li, W. (2020) Design, synthesis, and biological evaluation of
novel stachydrine derivatives as potent neuroprotective agents for
cerebral ischemic stroke. Naunyn-Schmiedeberg's Arch. Pharmacol. 393,
2529−2542.
(17) Liu, X., Chen, P., Pan, L., Silva, R., and Zhu, Y. (2009) 4-
Guanidino-n-butyl syringate (Leonurine, SCM 198) protects H9c2
rat ventricular cells from hypoxia-induced apoptosis. J. Cardiovasc.
Pharmacol. 54, 437−444.
(36) Dergunova, L. V., Filippenkov, I. B., Stavchansky, V. V.,
Denisova, A. E., Yuzhakov, V. V., Mozerov, S. A., Gubsky, L. V., and
Limborska, S. A. (2018) Genome-wide transcriptome analysis using
RNA-Seq reveals a large number of differentially expressed genes in a
transient MCAO rat model. BMC Genomics 19, 655−671.
(37) Schaller, B. (2005) Prospects for the future: the role of free
radicals in the treatment of stroke. Free Radical Biol. Med. 38, 411−
425.
(18) Liu, X., Xin, H., Hou, A., and Zhu, Y. (2009) Protective effects
of Leonurine in neonatal rat hypoxic cardiomyocytes and rat infarcted
heart. Clin. Exp. Pharmacol. Physiol. 36, 696−703.
(19) Liu, X., Pan, L., Chen, P., and Zhu, Y. (2010) Leonurine
improves ischemiainduced myocardial injury through antioxidative
activity. Phytomedicine 17, 753−759.
(20) Hua Liu, X., Long Pan, L., Hai Gong, Q., and Zhun Zhu, Y.
(2010) Antiapoptotic effect of novel compound from Herba Leonuri-
Leonurine (SCM-198): a mechanism through inhibition of
(38) Liu, P. (2003) Ischemia−reperfusion-related repair deficit after
oxidative stress: implications of faulty transcripts in neuronal
sensitivity after brain injury. J. Biomed. Sci. 10, 4−13.
2489
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(39) Love, S. (1999) Oxidative stress in brain ischemia. Brain Pathol.
9, 119−131.
(40) Zhu, Y., Hoell, P., Ahlemeyer, B., Sure, U., Bertalanffy, H., and
Krieglstein, J. (2007) Implication of PTEN in production of reactive
oxygen species and neuronal death in in vitro models of stroke and
Parkinson’s disease. Neurochem. Int. 50, 507−516.
(41) Li, X., Zhang, J., Chai, S., and Wang, X. (2014) Progesterone
alleviates hypoxic-ischemic brain injury via the Akt/GSK-3 signaling
pathway. Exp. Ther. Med. 8, 1241−1246.
(42) Hanumanthappa, P., Densi, A., and Krishnamurthy, R. G.
(2014) Glycogen synthase kinase-3 in ischemic neuronal death. Curr.
Neurovasc. Res. 11, 271−278.
(43) Zhao, Z. Q., Nakamura, M., Wang, N. P., Wilcox, J. N., Shearer,
S., Ronson, R. S., Guyton, R. A., and Vinten-Johansen, J. (2000)
Reperfusion induces myocardial apoptotic cell death. Cardiovasc. Res.
45, 651−660.
(44) Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G.,
Thornberry, N. A., Wong, W. W., and Yuan, J. (1996) Human ICE/
CED-3 protease nomenclature. Cell 87, 171.
(45) Longa, E. Z., Weinstein, P. R., Carlson, S., and Cummins, R.
(1989) Reversible middle cerebral artery occlusion without
craniectomy in rats. Stroke 20, 84−91.
2490
https://doi.org/10.1021/acschemneuro.1c00200
ACS Chem. Neurosci. 2021, 12, 2478−2490