Design, synthesis, and biological evaluation of novel stachydrine derivatives as potent neuroprotective agents for cerebral ischemic stroke

Article abstract of DOI:10.1007/s00210-020-01868-4

Stachydrine is a natural product with multiple protective biological activities, including those involved in preventing cancer, ischemia, and cardiovascular disease. However, its use has been limited by low bioavailability and unsatisfactory efficacy. To address this problem, a series of stachydrine derivatives (A1/A2/A3/A4/B1/B2/B3/B4) were designed and synthesized, and biological studies were carried out in vitro and in vivo. When compared with stachydrine, Compound B1 exhibited better neuroprotective effects in vitro, and significantly reduced infarction size in the model of the middle cerebral artery occlusion rat model. Therefore, Compound B1 was selected for further research on ischemic stroke. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00210-020-01868-4

Naunyn-Schmiedeberg's Archives of Pharmacology
https://doi.org/10.1007/s00210-020-01868-4
ORIGINAL ARTICLE
Design, synthesis, and biological evaluation of novel stachydrine
derivatives as potent neuroprotective agents for cerebral
ischemic stroke
Liang Zhang1 & Feng Li2,3 & Chenhui Hou1 & Sifeng Zhu1 & Lili Zhong1 & Jianchun Zhao1,4 & Cai Song5 & Wenbao Li1,4,6
Received: 19 December 2019 /Accepted: 2 April 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Stachydrine is a natural product with multiple protective biological activities, including those involved in preventing cancer,
ischemia, and cardiovascular disease. However, its use has been limited by low bioavailability and unsatisfactory efficacy. To
address this problem, a series of stachydrine derivatives (A1/A2/A3/A4/B1/B2/B3/B4) were designed and synthesized, and
biological studies were carried out in vitro and in vivo. When compared with stachydrine, Compound B1 exhibited better
neuroprotective effects in vitro, and significantly reduced infarction size in the model of the middle cerebral artery occlusion
rat model. Therefore, Compound B1 was selected for further research on ischemic stroke.
.
.
.
.
Keywords Stachydrine derivatives Organic synthesis Biological activities Ischemic stroke Neuroprotective agent
Introduction
of all stroke cases (Li et al. 2017). However, the only method
currently approved by the FDA for the treatment of acute
ischemic stroke is intravenous injection of recombinant tissue
plasminogen activator. Unfortunately, the efficacy of throm-
bolytic therapy for ischemic stroke is low because of a narrow
treatment window and potential side effects (Snow 2016;
Denorme et al. 2016; Alberts 2017). Therefore, many initia-
tives to develop new drugs to treat cerebral ischemic stroke are
being pursued (Wang et al. 2018; Ahmed et al. 2014; Aguilera
et al. 2010).
Stroke is a health risk worldwide, and in China alone, the
annual mortality rate is approximately 157 out of 100,000
people (Brainin et al. 2007; Zhou et al. 2014). Ischemic stroke
is the most common type of stroke and accounts for 70–90%
* Feng Li
fengli81@163.com
The pathophysiology of stroke is complex and involves
many complex processes. It is well-known that glutamate is
an important mediator of neuronal degeneration during ische-
mia. It is released from neurons and astrocytes in large quan-
tities, which may lead to intracellular calcium excess. This
calcium excess can lead to necrosis and breakdown of cellular
structures, including DNA, proteins, and membrane phospho-
lipids (Campos et al. 2012).
* Wenbao Li
wbli92128@ouc.edu.cn
1
School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, China
2
Shandong Peninsula Engineering Research Center of
Comprehensive Brine Utilization, Weifang University of Science and
Technology, Weifang 262700, Shandong, People’s Republic of
China
In order to study stroke in vitro, a glutamate-induced neu-
ronal injury model is widely used. Using the experimental
animal model of cerebral ischemia, the process of nerve func-
tion damage and recovery after cerebral ischemia injury was
understood, and the protective effect of drugs was studied
(Wang et al. 2017a; Ghazavi et al. 2017; Kim et al. 2018;
Berger et al. 2008; White et al. 2000). Transient middle cere-
bral artery occlusion (tMCAO) model, which plays an impor-
tant role in the study of temporal artery occlusion and
3
Department of Medicine and Pharmacy, Ocean University of China,
23 HongKong Road, Qingdao 266071, People’s Republic of China
4
Marine Biomedical Research Institute of Qingdao, Qingdao 266071,
China
5
Shenzhen Institute, Guangdong Ocean University, Shenzhen 518116,
China
6
Innovation Center for Marine Drug Screening and Evaluation,
Qingdao National Laboratory for Marine Science and Technology,
Qingdao 266071, China

Naunyn-Schmiedeberg's Arch Pharmacol
subsequent blood flow recovery, has been widely employed in
the development of neuroprotective therapeutic approaches
(Dergunova et al. 2018).
Concrete experimental procedures
N-methyl-L-proline (3) and N-methyl-L-proline hydrochloride
(A2) Formaldehyde (40%, 33.6 mL, 448.8 mmol) and 10%
palladium on carbon (11.75 g) were added to a solution of
L-proline 1 (47 g, 408.7 mmol) in CH3OH (350 mL), under
a hydrogen atmosphere condition. The reaction was stirred at
25 °C for 24 h, filtered, and concentrated to generate
Compound 3 (47.4 g, 90%) as a white solid (Han et al.
2005). Compound 3 (1 g, 7.75 mmol) was then added to
5 mL HCl (1 M), and the reaction mixture was stirred for
30 min at 25 °C. Compound A2 (1.2 g, 94%) formed a white
solid after vacuum concentration. 1H NMR (500 MHz, D2O) δ
4.10–3.98 (m, 1H), 3.63 (ddd, J = 11.8, 7.7, 4.2 Hz, 1H), 3.08
(dt, J = 11.3, 8.3 Hz, 1H), 2.84 (s, 3H), 2.52–2.34 (m, 1H),
2.13–1.98 (m, 2H), 1.96–1.82 (m, 1H). MS (ESI), Calcd for
C6H11NO2 [M + H]+ 130.08, found 130.19.
Stachydrine is extracted from traditional Chinese
medicine motherwort (Fig. 1), which possesses thera-
peutic benefits for the treatment of cardiovascular dis-
eases and cancers (Servillo et al. 2013; Ma et al. 2017).
Recently, there have been several reports of using
stachydrine to treat ischemic stroke (Zhang et al.
2017; Wang et al. 2017b). However, its further applica-
tion has been limited due to low bioavailability and
unsatisfactory efficacy (Feng 2009). Here, we report
the design and synthesis of a series of stachydrine de-
rivatives by adding a lipophilic group, which exhibited
better fat solubility and better stability in Wistar rat
plasma and liver microsomes. The biological activities
of the stachydrine derivatives were evaluated in vitro
and in vivo.
Ethyl N-methyl-L-prolinate (4) and ethyl N-methyl-L-prolinate
hydrochloride (A3) Thionyl chloride (10.2 g, 86 mmol) was
added slowly to the mixture of N-methyl-L-proline 3 (1.5 g,
11.6 mmol) and anhydrous ethanol (20 mL), with vigorous
stirring at 0 °C. The mixture was further stirred for 30 min at
25 °C and refluxed for another 3 h. After cooling down to
room temperature, excess ethanol and thionyl chloride were
removed under reduced pressure, producing ethyl N-methyl-
L-prolinate 4 (1.8 g, 99%) as a white solid (Reddy et al. 2016).
The product was added to 8 mL HCl (2 M) and stirred for
0.5 h, and the reaction mixture was concentrated to give
Material and methods
Chemistry
General experimental procedures
All chemical reagents were purchased from commercial sup-
pliers. Thin-layer chromatography (TLC) was performed on
silica gel GF-254 plates (Yantai Industrial Research Institute,
YanTai, China), and spots were visualized by UV (254 nm).
Column chromatography was performed on silica gel (200–
300 mesh, YanTai, China).
The target Compounds and intermediates were character-
ized by NMR. 1H NMR and 13C NMR spectra were obtained
using a Bruker 500 spectrometer, with tetramethylsilane
(TMS) as an internal standard. Chemical shifts (δ) were re-
ported in ppm: s = singlet; d = doublet; t = triplet; q = quartet;
m = multiplet; br = board singlet. Analyses were carried out
using a 6530 QTOF mass spectrometer (Agilent
Technologies, Santa Clara, CA, USA) equipped with an
electrospray ionization (ESI) interface.
1
Compound A3 (2.1 g, 95%) as oil. H NMR (500 MHz,
D2O), δ 4.13 (q, J = 7.1 Hz, 3H), 3.59 (ddd, J = 11.8, 8.0,
4.1 Hz, 1H), 3.14–3.02 (m, 1H), 2.82 (s, 3H), 2.42 (m, J =
13.4, 7.2 Hz, 1H), 2.10–1.97 (m, 2H), 1.95–1.78 (m, 1H), 1.11
(t, J = 7.2 Hz, 3H). MS (ESI), Calcd for C8H15NO2 [M + H]+
158.11, found 158.19.
Methyl methyl-L-prolinate (Denorme et al. 2016) and (S)-N-
ethyl-1-methylpyrrolidine-2-carboxamide (A4) Thionyl chlo-
ride (4.61 g, 38.76 mmol) was slowly added drop by drop at
0 °C to a solution of N-methyl-L-proline 3 (5 g, 38.76 mmol)
in 50 mL methanol, and the mixture was stirred for 1 h at
25 °C before reflux for another 3 h. After cooling to room
temperature, excess methanol and thionyl chloride were re-
moved under reduced pressure to produce methyl N-methyl-
L-prolinate 5 (5.2 g) (Kasperowiczfrankowska 2014). Without
further purification, methyl N-methyl-L-prolinate (3 g,
23.3 mmol) was dissolved in 70% ethylamine aqueous solu-
tion (15 mL, 233 mmol). After stirring at 25 °C for 12 h, the
reaction mixture was concentrated under reduced pressure.
The residue was then purified by column chromatography
on silica gel (dichloromethane, methanol = 15: 1) to generate
Compound A4 (2.5 g, 75%) as oil. 1H NMR (500 MHz, D2O),
δ 3.23 (q, J = 7.3 Hz, 2H), 3.03 (dd, J = 6.1, 3.2 Hz, 1H),
The purity of Compounds A1, A2, A3, A4, 8a-d, 9e-h, and
Compounds B1, B2, B3, B4 was established to be ≥ 95% by
HPLC (UltiMate 3000 series 250*4.6 mm).
Fig. 1 Structures of stachydrine and B1

Naunyn-Schmiedeberg's Arch Pharmacol
2.92–2.85 (m, 1H), 2.39–2.32 (m, 1H), 2.29 (s, 3H), 2.21–
concentrated. Water (25 mL) was added to the residual oil,
followed by sodium hydroxide (1 M), before the pH was ad-
justed to 13–14. After the mixture was washed with dichloro-
methane, the aqueous layer was adjusted to pH 2 with satu-
rated aq. potassium bisulfate, and then concentrated under
reduced pressure to generate Compound B2 (1.45 g, 45%)
2.09 (m, 1H), 1.87–1.72 (m, 3H), 1.12 (t, J = 7.3 Hz, 3H). 13
C
NMR (125 MHz, D2O), δ 13.57, 22.67, 29.50, 34.13, 39.90,
55.80, 68.77, 175.72 ppm. HRMS (ESI), m/z 157.1338 [M +
H]+ (Calcd for C8H16N2O, 157.1335).
1
(S)-2-(Ethoxycarbonyl)-1, 1-dimethyl-pyrrolidin-1-ium chlo-
ride (A1), and ethyl L-prolinate (Zhou et al. 2014) Thionyl
chloride (10.2 g, 86 mmol) was slowly added drop by drop
to a mixture of L-proline 1 (10 g, 86 mmol) and anhydrous
ethanol (150 mL), with vigorous stirring at 0 C. The mixture
was further stirred for 0.5 h at 25° C and refluxed for another
3 h. After cooling to room temperature, excess ethanol and
thionyl chloride were removed under reduced pressure to ob-
tain ethyl L-prolinate 2 (12 g). Without any further purifica-
tion, potassium carbonate (6.9 g, 0.05 mol) was added to the
mixture of ethyl L-prolinate (7.15 g, 0.05 mol) and acetonitrile
(75 mL). The mixture was stirred for another 1.5 h at 25 C,
and iodomethane (7.75 mL, 0.124 mol) was added dropwise,
before stirring for an additional 24 h at 25 C. After the reac-
tion is completed, the solid is filtered out, and the filtrate is
concentrated by decompression. The crude product was dis-
solved in CH2Cl2, stirred for 30 min, and the filtrate was
concentrated into compound A1 (11.8 g, 79%) as a white solid
(Gao et al. 2008). 1H NMR (500 MHz, D2O), δ 4.40 (t, J =
9.6 Hz, 1H), 4.29–4.16 (m, 2H), 3.70 (ddd, J = 11.9, 7.8,
4.2 Hz, 1H), 3.57 (dd, J = 20.8, 10.0 Hz, 1H), 3.28 (s, 3H),
3.05 (s, 3H), 2.52 (dd, J = 14.3, 5.4 Hz, 1H), 2.44–2.30 (m,
1H), 2.15 (m, J = 10.0, 5.0 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H).
MS (ESI), Calcd for C9H17NO2 [M + H] + 172.23, found
172.27.
as oil. H NMR (500 MHz, CD3OD), δ 3.94 (t, J = 7.9 Hz,
1H), 3.71–3.59 (m, 1H), 3.28 (d, J = 7.1 Hz, 2H), 3.13 (dt, J =
20.8, 10.5 Hz, 1H), 2.88 (s, 3H), 2.59–2.46 (m, 1H), 2.29–
2.22 (m, 2H), 2.22–2.10 (m, 1H), 2.08–1.97 (m, 2H), 1.88–
1.77 (m, 2H). 13C NMR (125 MHz, D2O), δ 22.50, 27.28,
29.02, 31.63, 40.18, 55.22, 56.35, 68.71, and 177.40 ppm.
HRMS (ESI), m/z 214.1551 [M + H]+ (Calcd for
C10H19N3O2, 214.1550).
Methyl-L-prolylglutamine (B3) Formaldehyde (40%, 3 mL,
40 mmol) and 10% palladium on carbon (0.45 g) were added
to Compound 9g (4.5 g, 12.4 mmol) in methanol (50 mL),
under a hydrogen atmosphere. After stirring at 25 °C for 12 h,
the reaction mixture was filtered, and the filtrate was concen-
trated. Water (25 mL) was added to the residual oil, followed
by sodium hydroxide (1 M), and pH was adjusted to 13–14.
After the solution was washed with dichloromethane, the
aqueous layer was adjusted to pH 2 by saturated aq. potassium
bisulfate, and then concentrated under reduced pressure to
produce Compound B3 (1 g, 32%) as oil. 1H NMR
(500 MHz, CD3OD), δ 4.30 (dd, J = 7.4, 5.0 Hz, 1H), 3.79–
3.69 (m, 1H), 3.61–3.49 (m, 1H), 2.96 (dt, J = 18.2, 9.1 Hz,
1H), 2.79 (d, J = 8.7 Hz, 3H), 2.47 (m, J = 12.3, 7.5 Hz, 1H),
2.31–2.23 (m, 2H), 2.23–2.13 (m, 1H), 2.12–2.03 (m, 2H),
1.97 (ddd, J = 20.2, 13.2, 7.2 Hz, 2H). 13C NMR (125 MHz,
D2O), δ 22.47, 24.36, 29.14, 32.28, 39.05, 40.20, 56.33,
68.76, 163.06, 168.26, and 178.79 ppm. HRMS (ESI), m/z
258.1449 [M + H]+ (Calcd for C11H19N3O4, 258.1448).
(Tert-butoxycarbonyl)-L-proline (Alberts 2017) Triethylamine
(2.6 g, 26 mmol) and di-tert-butyl pyrocarbonate (6.1 g,
28 mmol) in dichloromethane (20 mL) were added to L-pro-
line 1 (2.3 g, 0.2 mol) in dichloromethane (30 mL) at 0 °C.
After stirring for 3 h, the reaction mixture was washed with
saturated citric acid. The organic layer was dried by anhydrous
magnesium sulfate, and then filtered and concentrated. The
residue was purified by recrystallization (petroleum ether, eth-
yl acetate = 1:1) to generate Compound 6 (3.6 g, 83.7%) as a
white solid (Loughlin et al. 2013). 1H NMR (500 MHz,
CD3OD), δ 4.28–4.12 (m, 1H), 3.47 (dd, J = 14.0, 8.4 Hz,
1H), 3.40 (dd, J = 16.8, 7.6 Hz, 1H), 2.35–2.20 (m, 1H),
2.05–1.82 (m, 3H), 1.44 (d, J = 18.9 Hz, 9H). MS (ESI),
Calcd for C10H17NO4 [M + H] + 216.12, found 216.25.
Methyl-L-prolylglycine (B4) Formaldehyde (40%, 2.4 mL,
31.8 mmol) and 10% palladium on carbon (0.35 g) were
added to Compound 9h (3.5 g, 12.2 mmol) in methanol
(30 mL), under a hydrogen atmosphere. After stirring at
25 °C for 12 h, the reaction mixture was filtered, and the
filtrate was concentrated. Water (25 mL) was added to the
residual oil, followed by sodium hydroxide (1 M), and pH
was adjusted to 13–14. After the mixture was washed with
dichloromethane, the aqueous layer was adjusted to pH 2 by
saturated aq. potassium bisulfate, and then concentrated under
reduced pressure to generate Compound B4 (2.1 g, 93.7%) as
oil. 1H NMR (500 MHz, CD3OD), δ 4.14 (t, J = 8.3, 1H), 4.00
(s, 2H), 3.71 (ddd, J = 11.4, 7.7, 4.1, 1H), 3.22 (d, J = 11.1,
1H), 2.95 (d, J = 10.5, 3H), 2.58 (dd, J = 12.2, 7.5, 1H), 2.24–
2.02 (m, 3H). 13C NMR (125 MHz, D2O), δ 22.45, 29.04,
40.36, 41.18, 56.45, 68.62, 168.96, and 172.68 ppm. HRMS
(ESI), m/z 187.1079 [M + H]+ (Calcd for C8H14N2O3,
187.1077).
(S)-N-(4-Amino-4-oxobutyl)-1-methylpyrrolidine-2-
carboxamide (B2) Formaldehyde (40%, 2.2 mL, 29.3 mmol)
and 10% palladium on carbon (0.3 g) were added to
Compound 9f (3 g, 9.6 mmol) in methanol (30 mL), under a
hydrogen atmosphere. After stirring at 25 °C for 12 h, the
reaction mixture was filtered, and the filtrate was

Naunyn-Schmiedeberg's Arch Pharmacol
(S)-2-(1-Methylpyrrolidine-2-carboxamido) benzoic acid (B1)
Sodium hydroxide (168 mg, 4.2 mmol) in water (10 mL)
was added to Compound 10i (1 g, 3.8 mmol) in methanol
(35 mL). After stirring at 25 °C for 24 h, the excess methanol
was removed under reduced pressure. The mixture was then
washed with dichloromethane. Hydrogen chloride (2 M) was
added, and pH was adjusted to 2. After concentration under
reduced pressure, Compound B1 (0.77 g, 71%) was produced
triturated and centrifuged at 1000× rpm for 5 min at 4 °C.
The suspension was filtered through a 70-μm nylon cell re-
strainer, and cells were diluted to 1 × 106 cells/mL in a 6-well
plate pretreated with 0.1 g/L polylysine. The cultures were
incubated in a humidified atmosphere under 5% CO2 condi-
tions at 37 °C.
Animals
1
as oil. H NMR (500 MHz, CD3OD), δ 8.45 (d, J = 8.3 Hz,
1H), 8.10 (dd, J = 7.9, 1.3 Hz, 1H), 7.61–7.53 (m, 1H), 7.22 (t,
J = 7.6 Hz, 1H), 4.43 (t, J = 8.4 Hz, 1H), 3.82–3.72 (m, 1H),
3.36–3.22 (m, 1H), 3.02 (s, 3H), 2.77–2.67 (m, 1H), 2.25
(ddd, J = 18.2, 9.2, 3.9 Hz, 2H), 2.17–2.06 (m, 1H). 13C
NMR (125 MHz, D2O), δ 22.56, 28.91, 40.65, 56.56, 69.50,
122.20, 123.11, 125.79, 131.10, 133.65, 136.30, 166.77, and
170.95 ppm. HRMS (ESI): m/z 249.1236 [M + H]+ (Calcd for
C13H16N2O3, 249.1234).
Sprague-Dawley (SD) rats (250–280 g) were purchased from
Qinglong Mountain Animal Breeding Farm in Jiangning
District, Nanjing. All animals were maintained under standard
housing conditions at temperatures between 20 and 23 °C and
a relative humidity of 50% with a 12-h light/dark cycle.
In vitro coagulation assay
Abnormal coagulation is a risk factor for thrombosis and has
been reported to contribute to ischemic stroke (Hanscombe
et al. 2015). To evaluate the effect of synthesized derivatives
on coagulation, we developed a coagulation assay. The assay
was designed with three conditions: a control (saline), synthe-
sized stachydrine derivatives, and stachydrine and
rivaroxaban at three doses (50 nM, 100 nM, 500 nM). All
Compounds were dissolved in physiological saline solution.
Pipes were taken, and 0.2 mL of the corresponding solution
was added. Rabbits were anesthetized with 20% urethane
(Ulatan) at a dose of 5 mL/kg, and the carotid artery was then
intubated before blood was drawn. A total of 1 mL of fresh
blood was added to each tube and mixed in a 37 °C water bath,
at which point a stopwatch was started. Blood coagulation
time was recorded as the time when the tube could be
completely inverted without blood flowing out.
Biological assays
Materials
Cell culture medium and supplement were obtained from
Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT); 2,3, 5-
triphenyltetrazolium chloride (TTC); deoxyribonuclease I
(DNase I); trypsin; dizocilpine (MK-801); rivaroxaban; and
poly-L-lysine were purchased from Sigma-Aldrich (St.
Louis, MO, USA). GlutaMax, fetal bovine serum (FBS), and
glutamate were purchased from Gibco (Carlsbad, CA).
TUNEL Kit, LDH Kit, DCF Kit, SOD Kit, and MDA Kit
were purchased from Beyotime Biotechnology (Nanjing,
China). All procedures were performed according to the US
National Institutes of Health (NIH) Guide for the Care and
Use of Laboratory Animals published by the US National
Academy of Sciences (http://oacu.od.nih.gov/regs/index.
htm), and were approved by the Administration Committee
of Experimental Animals in Shandong Province and the
Ethics Committee of Marine Biomedical Research Institute
of Qingdao.
Primary rat cerebellar granule cell viability assay
in glutamate-induced neuronal injury model
Neuronal death by necrosis and apoptosis are observed during
an ischemic stroke (Kaur et al. 2013). We designed an exper-
iment to evaluate the effect of synthesized derivatives on cell
viability and apoptosis. The conditions tested were as follows:
a control group, model group, MK-801 (dizocilpine) group,
and groups of synthesized derivatives. All Compounds were
dissolved in physiological saline solution, and added on the
sixth day after the isolation and culture of primary rat cerebel-
lar granule nerve cells. Glutamic acid at a concentration of
200 μM was added on the seventh day. Cell viability was
measured with MTT on the eighth day. Control group cells
were not submitted with glutamate (Glu) and added with sa-
line in the same volume of glutamate. Then cultures were
rinsed with 1.2 mM MgCl2-supplemented experimental buffer
and returned to preconditioned media. Cell viability was mea-
sured by MTT assay, and the operation method is as follows:
Cell culture and reagents
Primary rat cerebellar granule cells (CGCs) were isolated from
8-day-old Sprague-Dawley rat pups. Rats were washed with
75% ethanol (v/v) to remove bacteria. Cerebella were collect-
ed and placed in ice-cold Hanks’ balanced salt solution
(HBSS), and then dissolved in the same buffer containing
0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) for
15 min after meninx removal. Trypsin digestion was stopped
by addition of two volumes of neurobasal supplement with
10% B27 (a supplement for neuronal cell culture), 0.5 mM
GlutaMax, and 0.1 mg/mL DNase I. The solution was

Naunyn-Schmiedeberg's Arch Pharmacol
cells were incubated with 0.5 mg/mL MTT for 4 h in a hu-
midified atmosphere of 5% CO2 and 95% air at 37 °C, then the
medium was replaced with DMSO, and absorbance was mea-
sured at 570 nm in a BD plate-reader. Cell viability is directly
proportional to absorbance, and the absorbance of control
group cells is taken as 100%.
We designed an experiment to evaluate the effect of syn-
thesized derivatives on SOD. Assay conditions included a
control group, model group, positive drug (MK-801)
group, and groups of the synthesized Compounds
(20 μM). All Compounds were dissolved in saline solu-
tion on the sixth day after CGCs were isolated and cul-
tured. On the seventh day, glutamic acid (200 μM) was
added to cells. The corresponding doses of Compounds
were introduced 48 h after glutamic acid induction of
cells. CGCs were washed 3 times with cold PBS, and
collected with scrape; an appropriate amount of PBS
was added to homogenize; a part of the sample was used
to determine the protein concentration, then all samples
are adjusted to the same level with PBS, and SOD content
in the samples was detected with an SOD assay kit.
Apoptosis assay
A control group, model group, MK-801 (dizocilpine) group,
and groups of synthesized derivatives were tested in an assay
measuring apoptosis. All Compounds were dissolved in phys-
iological saline solution, and added on the sixth day after the
isolation and culture of primary rat cerebellar granule nerve
cells. Glutamic acid (200 μM) was added on the seventh day.
On the eighth day, cells were stained with TUNEL Kit, and
detected by laser confocal microscopy.
Assay for measuring malondialdehyde content
in a glutamate-induced neuronal injury model
Assay for measuring lactate dehydrogenase activity
in glutamate-induced neuronal injury model cells
Products of lipid peroxidation, such as malondialdehyde
(MDA) and 4-hydroxynonenal, are increased in patients
with thrombotic or cardioembolic ischemic stroke, in
comparison with controls (El Kossi and Zakhary 2000;
Warner et al. 2004). We designed an experiment to
evaluate the effect of synthesized derivatives on MDA.
The conditions tested included a control group, model
group, positive drug (MK-801) groups, and groups of
the synthesized Compounds (20 μM). All Compounds
were dissolved in physiological saline solution on the
sixth day after CGCs were isolated and cultured. On
the seventh day, glutamic acid (200 μM) was added.
The corresponding concentrations of Compounds were
introduced 48 h after glutamic acid induction of cells.
Protein samples were collected, and MDA content in the
sample was detected with an MDA assay kit.
Primary rat cerebellar granule cells (CGCs) were added to
assay Compounds on the sixth day after separation and cul-
ture. On the seventh day, glutamic acid (200 μM) was added.
On the eighth day, the cell supernatant was collected to deter-
mine the content of LDH, and all operations were performed
according to the instructions of LDH kit.
Assay for measuring intracellular reactive oxygen species
in glutamate-induced neuronal injury model
The over-generation of reactive oxygen species (ROS) and
lipid peroxidation contributes to neuronal damage induced
by ischemia-reperfusion (Love 1999). To evaluate the effect
of synthesized derivatives on ROS, we designed several ex-
periments. The conditions tested included a control group,
model group, positive drug (MK-801) group, and synthesized
Compound groups (20 μM). All Compounds were dissolved
in saline solution, and were added on the sixth day after pri-
mary rat cerebellar granule nerve cell isolation and culture.
Glutamic acid (200 μM) was added on the seventh day. On
the eighth day, the probe was loaded using the DCF Kit, and
photographed with a laser confocal microscope. A positive
signal indicated that intracellular reactive oxygen species were
generated.
Testing plasma stability of B1 in vitro
Extraction of fresh rat plasma from the eye socket of
blank rats by heparinized micro -centrifugal tubes, and
immediately centrifuged at 8000 r/min for 10 min.
Compound B1 and stachydrine were incubated together
at 37 °C for a final concentration of 3 μM. Each result
was repeated for three times. After the Compounds were
introduced and rapidly mixed, an aliquot (100 μL) was
taken and precipitated in 150 μL of acetonitrile contain-
ing 20 ng/mL propranolol (internal reference). This
sample was used to determine the 0 min value. Small
samples were collected at 0, 5, 10, 20, 40, 60, 90, 120,
and 180 min of incubation. The samples were centri-
fuged by vortex (10 min, 14,000 rpm, 4 °C), and the
supernatant was transferred into a small bottle and ana-
lyzed by LC-MS/MS.
Assay for measuring superoxide dismutase activity
in a glutamate-induced neuronal injury model
Overexpression of SOD protects against ischemic brain dam-
age by reducing oxidative injury and modifying redox signals,
in addition to alleviating mitochondrial dysfunction and sub-
sequent apoptosis after cerebral ischemia (Chen et al. 2011).

Naunyn-Schmiedeberg's Arch Pharmacol
In vivo pharmacokinetics
Acute toxicity measurement
Pharmacokinetics of Compound B1 was evaluated in male
Wistar rats (about 200 g) that were fasting for one night and
fed 4 h after administration. During the whole experiment, an-
imals were free to take water, and they were conscious and
unconstrained from first to last. Rats were randomly divided
into 4 groups, 5 in each group. Compound B1 (1 mg/mL)
was solubilized in a suspension containing 0.5% of CMC-Na,
and a single intravenous injection study (10 mg/kg) was con-
ducted in the rats. At periodic intervals of 0.0167, 0.167, 0.5, 1,
1.5, and 2 h, blood samples were collected from the eye sockets
in heparinized micro-centrifuge tubes, and immediately centri-
fuged at 8000 r/min for 10 min to obtain plasma. Plasma sam-
ples were precipitated with acetonitrile solution including
100 ng/mL propranolol in double volume (internal reference),
and analyzed using a verified LC-MS/MS method.
Acute toxicity of Compound B1 was evaluated in Kunming
mice. Six mice weighing between 18 and 23 g were placed
into each of five groups, and acclimatized for 7 days. Mice in
group 1 served as the normal control, while the remaining four
groups were treatment groups. The animals were separated in
cages, and the same treatment was given to all mice in the
same cage. Clinical signs were observed every day for 14 days
after treatment, including the presence of tremors, loose bow-
el, abdominal contortions, convulsions, lethargy, salivation,
diarrhea, and dyskinesia. Body weight was recorded, and food
consumption was calculated daily for 14 days.
Results and discussion
Chemistry
Focal cerebral ischemia procedure (tMCAO) and drug
treatment in rats
A total of 8 derivatives were synthesized based on the struc-
ture of stachydrine, through the use of two synthetic routes,
which are outlined in Schemes 1 and 2.
SD rats were randomly divided into a series of groups, and a
minimum of 12 rats were designated per group. We performed
these experiments based on previously reported results (Longa
et al. 1989). Rats were anesthetized with 3.5% isoflurane un-
der the tMCAO model, and anesthesia was maintained with
1.0–2.0% isoflurane in 70% N2O and 30% O2. Body temper-
ature was measured with a rectal probe, and maintained at
37.0 0.5 °C with a heating pad. Cerebral blood flow was
monitored using Doppler flowmetry for 5 min both before
surgery and after ischemia. Ischemia was defined as the point
at which MCA cerebral blood flow levels were reduced by
15% compared with that of before surgery. Rats were anesthe-
tized with isoflurane, and an incision was made in the neck to
access the common carotid artery (CCA), internal carotid ar-
tery (ICA), and external carotid artery (ECA). A monofila-
ment nylon suture (diameter of 0.26 mm) with a proper
amount of silicone on the head was inserted into the ICA
through the ECA stump, and gently advanced to the middle
cerebral artery. After the MCA had been ischemic for 2 h, the
monofilament nylon was gently removed, and blood flow was
measured by Doppler flowmetry. Rats were excluded based
on death or non-satisfactory cerebral blood flow (CBF) during
or 10 min after reperfusion. For pharmacodynamic studies of
Compound B1 in the transient middle cerebral artery occlu-
sion model, SD male rats with weight between 250 and 280 g
were divided into six groups: the sham operation group
(Sham), model group (vehicle), edaravone (Eda, 3 mg/kg)
group, stachydrine (A, 30 mg/kg) group, B1 (30 mg/kg)
group, and B1 (60 mg/kg) group. After arterial occlusion,
the rats were intravenously administered with the
Compounds after 2 h, 4 h, and 6 h, and the amount of cerebral
infarct was detected after 24 h.
Synthesis of Compounds A1–A4 is described in Scheme 1.
The commercial material L-proline 1 was esterified with eth-
anol in the presence of thionyl chloride under refluxing con-
ditions to generate intermediate 2. Without purification, inter-
mediate 2 reacted with methyl iodide in acetonitrile at room
temperature to generate Compound A1. Key intermediate 3
was acquired from the reaction between L-proline 1 and aque-
ous formaldehyde in methanol at 25 °C, under a hydrogen
atmosphere condition. Compound A2 was acquired via treat-
ment of intermediate 3 with diluted hydrochloride at 25 °C.
Intermediate 3 was converted to intermediates 4 and 5 via a
substitution reaction with ethanol or methanol under refluxing
conditions, respectively. Similarly, Compound A3 was obtain-
ed after treatment of intermediate 4 with diluted hydrochloride
in methanol at room temperature. Compound A4 was gener-
ated by an ester exchange reaction between intermediate 5 and
ethylamine in methanol at 25 °C.
Synthesis of Compounds B1–B4 is described in Scheme 2.
L-Proline 1 was also used as the starting material. The amine
group was protected with di-tert-butyl pyrocarbonate to gen-
erate intermediate 6, and intermediate 6 was condensed with
4-nitrophenol to form intermediate 7 at 25 °C. The key inter-
mediates 8a–d were then generated by a reaction of interme-
diate 7 with methylanthranilate, γ-aminobutanamide, gluta-
mine, or glycine in the presence of sodium bicarbonate and
N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline at 25 °C.
After using trifluoroacetic acid to remove the protection group
of intermediates 8a–d at 0 °C, intermediates 9e–h were obtain-
ed. Compounds 10i and B2–B4 were obtained through reac-
tions between intermediates 9e–h and aqueous formaldehyde
in methanol at 25 °C, under hydrogen atmosphere conditions,

Naunyn-Schmiedeberg's Arch Pharmacol
Scheme 1 Synthesis of final Compounds A1–A4. Reagents and
conditions: (a) SOCl2, EtOH, reflux, 3 h; (b) CH3I, K2CO3, CH3CN,
25 °C, 24 h; (c) 40% aq. HCHO, 10% Pd/C, H2, 25 °C, 24 h; (d) HCl
(1 M), MeOH, 25 °C, 30 min; (e) SOCl2, MeOH, reflux, 3 h; (f)
CH3CH2NH2 (70%), MeOH, 25 °C, 12 h. Abbreviations: SOCl2,
thionyl chloride; CH3I, iodomethane; CH3CN, acetonitrile; HCHO,
formaldehyde; Pd/C, palladium on carbon; CH3CH2NH2, ethylamine
Scheme 2 Synthesis of Compounds B1–B4. Reagents and conditions:
(a) Et3N, (Boc)2O, DCM, 0 °C, 3 h; (b) 4-nitrophenol, EDCI, DCM,
25 °C, 12 h; (c) Methylanthranilate or γ-aminobutanamide or glutamine
or glycine, NaHCO3, EEDQ, 1,4-dioxane, H2O, 25 °C, 12 h; (d)
CF3COOH, DCM, 0 °C; (e) HCHO, Et3N, 10% Pd/C, H2, 25 °C, 24 h;
(f) NaOH (0.42 N), MeOH, HCl, 24 h, 25 °C. Abbreviations: Et3N,
triethylamine; (Boc)2O, di-tert-butyl decarbonate; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; DCM, dichlorometh-
ane; NaHCO3, sodium bicarbonate; EEDQ, N-ethoxycarbonyl-2-ethoxy-
1,2-dihydroquinoline; CF3COOH, trifluoroacetic acid; HCHO, formalde-
hyde; Pd/C, palladium on carbon

Naunyn-Schmiedeberg's Arch Pharmacol
Table 1 The effects of the
synthesized derivatives
stachydrine and rivaroxaban on
time to coagulation
Compound
Time to coagulation (s)
Low-dose group (50 nM) Medium-dose group (100 nM) High-dose group (500 nM)
Control (Saline) 1153
NDb
2276
1523
1971
1521
2537
2084
2084
1360
1396
1422
ND
Rivaroxabana
1834
1421
1235
1254
1673
1462
1721
1212
1054
948
2784
1523
1424
1630
2631
2232
2013
1431
1224
1421
Stachydrine
A1
A2
A3
A4
B1
B2
B3
B4
a Rivaroxaban is a drug known to induce coagulation
b ND not detected
respectively. Finally, the hydrolysis reaction of intermediate
10i with sodium hydroxide was performed to generate
Compound B1 at 25 °C.
derivatives on the corresponding pathogenic processes,
we performed assays evaluating blood coagulation, re-
lease of glutamate, and reactive oxygen species (ROS)
formation. Additionally, we determined levels of lactate
dehydrogenase (LDH), malondialdehyde (MDA), and
superoxide dismutase (SOD).
Biology
The pathophysiology of stroke is complex and involves
many processes, including energy loss, ion homeostasis
loss, intracellular calcium excess, excitotoxicity, free
radical activation, cytokine release, blood-brain barrier
destruction, and activation of glial and inflammatory
cells. The series of cascading events is well-coordinated,
and ultimately lead to necrosis and apoptosis (Love
1999). To evaluate the efficacies of several stachydrine
Time to coagulation in vitro
As shown in Table 1, all Compounds exhibited anticoagulant
activity and could prolong the coagulation time when com-
pared with the control. Compounds B1, A3, or A4 were espe-
cially effective at prolonging coagulation at a dose of 100 nM,
with times of 2084 s, 2537 s, or 2084 s, respectively. These
M
T T a s s a y
1 5 0
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1 0 0
5 0
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2
3
4
1
2
2
3
4
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o
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in
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1
A
2
A
A
B
B
2
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i
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1
A
A
A
B
B
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t
3
B
4
r i
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1
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A
A
B
1
B
2
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3
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e l
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t r
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r
0
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c
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y
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K
c
h
c h
a
c
a
a
t
S
S
3 0 µ m o l/ L
1 0 µ m o l/ L
2 0 µ m o l/ L
Fig. 2 Effects of the synthesized derivatives stachydrine and MK-801c on
CGC viability. CGCs were pretreated with various concentrations of MK-
801, stachydrine, A1, A2, A3, A4, B1, B3, and B4 for 24 h, followed by
incubation with 200 μM glutamate (Glu) for an additional 24 h. aData are
presented as mean SD (n = 3). ***P < 0.001 versus the control group
(Saline); ###P < 0.001 versus the model group (Glutamate).
Abbreviations: bND = not detect. cMK-801 is also known as dizocilpine,
a kind of protective drug used in the central nervous system. Notes:
aValues present mean SD from three independent experiments.
***P < 0.001 versus the control group (CN); #P < 0.05, ###P < 0.001 ver-
sus the model group

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 3 Representative images of
TUNEL immunostaining in the
apoptosis assay for Compounds
B1, MK-801, or A3 in a
glutamate-induced neuronal inju-
ry model. CGCs were pretreated
with MK-801, stachydrine, A3,
A4, or B1 for 24 h, followed by
incubation with 200 μM gluta-
mate (Glu) for an additional 24 h,
and apoptotic cells were labeled
with TUNEL staining; (a) control
group (Saline), (b) model group
(Glutamate), (c) B1 group, (d)
MK-801 group, and (e) A3 group
results indicate that Compounds B1, A3, and A4 are more
effective anticoagulant agents than the other derivatives.
release rate of the model group was significantly increased.
In comparison, the LDH release rate of MK-801, A3, or B1
was dramatically decreased. These results indicate that MK-
801, A3, and B1 significantly decrease LDH release rates at a
dose of 20 μmol/L.
Neuroprotective effects of the synthesized derivatives
in a glutamate-induced neural injury model in primary rat
cerebellar granule cells
A3 and B1 reduce intracellular reactive oxygen species
production in a glutamate-induced neuronal injury model
Glutamic acid damage can lead to massive neuronal cell death
after ischemic stroke, which contributes to neuron injury. In
this study, neuronal cell viability was assessed by MTT assay.
As shown in Fig. 2, glutamate (Glu) stimulation significantly
reduced the cell viability of CGCs; however, this decrease
could be prevented by treating with Compounds A3, B1,
B2, or B4.
The intracellular reactive oxygen species (ROS) assay uses
fluorescence as a means to indicate ROS production.
Figure 6 shows fluorescent images of ROS staining, using
fluorescence microscopy. The intensity of the signal was pos-
itively correlated with ROS content of cells. As shown in
A3 and B1 inhibited glutamate-induced apoptosis in CGCs
T U N E L
* * *
Based on the results of Fig. 2, Compounds A3, A4, and B1
were selected for further study in the apoptosis assay. Fig. 3
and Fig. 4 indicate that the percentage of apoptotic neuronal
cells was significantly increased when stimulated with gluta-
mate (Glu). However, the number of apoptotic cells was dra-
matically decreased when cells were treated with A3, B1, or
MK-801, respectively. Figure 3 shows the TUNEL staining in
the apoptosis assay. As seen in Fig. 3b, there were large num-
bers of scattered red fluorescent positive apoptotic cells in the
model group, while in comparison, the number of red fluores-
cent cells was significantly reduced by the Compounds MK-
801, A3, A4, and B1. These results indicate that Compounds
B1 and A3 are able to inhibit neuronal apoptosis and have
neuroprotective effects.
4 0 0
#
3 0 0
# # #
# # #
2 0 0
1 0 0
0
# # #
l
e
3
1
e
o l
n
r
i
A
A
4
B 1
r
8 0
o d
n t
-
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m
c
h
M
K
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t a
S
A3 and B1 reduced lactate dehydrogenase release
after glutamate stimulation
Fig. 4 The effects of MK-801, stachydrine, A3, A4, and B1 on apoptosis
rate in glutamate-induced neuronal injury model. Note: The dose of MK-
801 was 10 μM, while the dose of other assayed Compounds was 20 μM.
The scale bar represents 50 μM. Values present mean SD from three
independent experiments. ***P < 0.001 versus the control group (saline);
#P < 0.05; ###P < 0.001 versus the model group (Glutamate).
After various treatments, cellular cytotoxicity was evaluated
through an LDH release assay. As shown in Fig. 5, LDH

Naunyn-Schmiedeberg's Arch Pharmacol
L D H r e le a s e
Note: The dose of MK-801 was 10 μM, and other
Compounds were dosed at 20 μM. The scale bar represents
50 μM; (a) control group (Saline), (b) model group
(Glutamate), (c) B1 group, (d) MK-801 group, and (e) A3
group. Values present mean SD from three independent
experiments. ***P < 0.001 versus the control group (CN);
###P < 0.001 versus the model group.
4 0 0
3 0 0
2 0 0
1 0 0
0
# # #
* * *
# #
# # #
# # #
The effects of A3 and B1 on superoxide dismutase content
in a glutamate-induced neuronal injury model
To further explore the biological properties of Compounds
A3, A4, and B1, SOD content was measured by an assay
kit. As shown in Fig. 8, in comparison with the model group,
SOD level of cells treated with B1, A3, and MK-801 increased
significantly. These data indicate that Compounds B1 and A3
also had antioxidant effects.
l
l
e
3
1
o
r
0
in
A
A
4
B 1
d e
r
o
n t
- 8
d
o
y
m
K
c
h
M
c
a
S
t
Fig. 5 The effects of MK-801, stachydrine, A3, A4, and B1 on lactate
dehydrogenase (LDH) release in glutamate-induced neuronal injury mod-
el. CGCs were pretreated with MK-801, stachydrine, A3, A4, or B1 for
24 h, and subsequently treated with 200 μM glutamate (Glu) for 24 h..
Note: The dose of MK-801 was 10 μM, and the dose of other assayed
Compounds was 20 μM. Values presents mean SD from three indepen-
dent experiments. ***P < 0.001 versus the control group (saline);
#P < 0.05, ###P < 0.001 versus the model group (Glutamate).
Malondialdehyde content assay in vitro
MDA content of the control, model, MK-801, B1, A3,
A4, and stachydrine groups was measured with an
MDA kit. The results are summarized in Fig. 9. The
relative content of MDA in the model group was sig-
nificantly increased, while in comparison, Compounds
B1 and A3 reduced the relative content of MDA, which
suggests that Compounds B1 and A3 might possess
anti-lipid oxidation activity.
Fig. 6b, a large amount of positive red signal was observed in
the model group, while in comparison, the positive signals for
the other four groups were greatly decreased.
As shown in Fig. 7, compared with the model group,
the content of cellular ROS decreased dramatically after
treatment with Compounds B1 and A3. These results
suggest that Compounds B1 and A3 effectively inhibit
ROS production in a glutamate-induced neuronal injury
model. Therefore, Compounds B1 and A3 might exhibit
neuroprotective effects by suppressing excess production
of intracellular ROS.
In vitro plasma stability experiment with Compound B1
During the in vitro assay, B1 significantly improved survival
rate of neuronal cells, inhibited neuronal apoptosis, and de-
creased levels of LDH, ROS, and MDA, while increasing
SOD activity. Therefore, Compound B1 was selected for fur-
ther stability analysis. Figure 10 shows that B1 maintained
Fig. 6 Typical fluorescent
photomicrographs of ROS
staining using fluorescence
microscopy. CGCs were
pretreated with MK-801,
stachydrine, A3, A4, and B1 for
24 h, followed by stimulation
with 200 μM glutamate (Glu) for
an additional 24 h; (a) control
group (Saline), (b) model group
(Glutamate), (c) B1 group, (d)
MK-801 group, and (e) = A3
group

Naunyn-Schmiedeberg's Arch Pharmacol
R O S
M D A
$ $
2 0 0 0
2 0 0
1 5 0
1 0 0
5 0
* * *
* * *
1 5 0 0
1 0 0 0
5 0 0
# # #
# # #
# # #
# # #
# # #
# # #
0
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a c
A
3
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B 1
8
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-
Fig. 7 The effects of MK-801, stachydrine, A3, A4, and B1 on
intracellular reactive oxygen species (ROS) release rate in glutamate-
induced neuronal injury model
K
m
y d
c o
h
M
c
a
S t
Fig. 9 Effect of A3, A4, B1, stachydrine, and MK-801 on
malondialdehyde (MDA) content. CGCs were pretreated with MK-801,
stachydrine, A3, A4, or B1 for 24 h, followed by stimulation with
200 μM glutamate (Glu) for an additional 24 h. Note: The dose of MK-
801 was 10 μM, and other Compounds were given at a dose of 20 μM.
Data are presented as mean SD (n = 3). ***P < 0.001 versus the control
group (saline); ##P < 0.01, ###P < 0.001 versus the model group
(glutamate).
stability in plasma throughout the 180 min experiment
duration.
Pharmacokinetic studies in vivo
In vivo pharmacokinetic studies were performed after intrave-
nous (10 mg/kg) administration of Compound B1 or
stachydrine in Wistar rats. Blood samples were taken at
0.0167, 0.167, 0.5, 1.0, 1.5, and 2.0 h, and concentration of
Compound was quantified by LC-MS/MS. The Cmax of B1
with intravenous injection was 70,924.9 ng/mL, which was
higher than that for stachydrine at 6396.3 ng/mL. More spe-
cifically, B1 exhibited a 2.63-fold increase in AUC(0-∞), with a
value of 31,611.76 h ng mL−1, in comparison with the
A U C ( 0 - ∞ ) v a l u e f o r s t a c h y d r i n e , w h i c h w a s
12,026.7 h ng mL−1. In addition, the clearance (CL) of B1
was 0.16 L h−1 kg−1, which was lower than that observed
for stachydrine at 0.93 L h−1 kg−1. These pharmacokinetic
profiles of Compound B1 were consistent with experimental
activity in the middle cerebral artery occlusion rat model, as
shown in Fig. 11 and Table 2.
S O D
1 5 0
# # #
1 0 0
# #
# #
* * *
5 0
0
l
l
4
1
1
e
e
0
r o
A 3
A
B
d
in
t
8
r
n
-
m
o
y d
c o
h
M
K
a c
S t
Fig. 8 The effects of A3, A4, B1, stachydrine, and MK-801 on superox-
ide dismutase content. CGCs were pretreated with MK-801, stachydrine,
A3, A4, or B1 for 24 h, followed by stimulation with 200 μM glutamate
(Glu) for an additional 24 h. Note: The dose of MK-801 was 10 μM, and
the dose for other Compounds was 20 μM. Data are presented as mean
SD (n = 3). ***P < 0.001 versus the control group (Saline); ##P < 0.01,
###P < 0.001 versus the model group (Glutamate).
Fig. 10 Percentage of Compound B1 remaining after incubation with
Wistar rat plasma at 37 °C for 180 min. Note: Data are presented as
mean SD (n = 3).
a

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 11 Mean plasma concentration of stachydrine and Compound B1
versus time, after intravenous injection in Wistar rats at a dose of
0.056 mmol/kg and 0.035 mmol/kg
Fig. 12 Compound B1 reduced infarct size and decreased neurological
deficit scores in the model of rat the middle cerebral artery occlusion.
Representative brain infarcts were visualized with 2,3,5-
tetraphenyltetrazolium chloride (TTC) staining in mice treated with dif-
ferent Compounds. The pale regions represent infarct tissues, while the
red regions are non-ischemic areas. Note: Eda = edaravone A =
stachydrine. The six rows represent n = 6 animals.
In vivo studies of biological activity
To further study biological activity in vivo, Compound B1
was administered intravenously in the model of rat the middle
cerebral artery occlusion at doses of 30 mg/kg or 60 mg/kg.
Figure 12 shows the infarct sizes for different groups, with the
results summarized in Table 3. The infarct size of the B1 group
at a dose of 60 mg/kg was 16.13%, which was less than the
vehicle and stachydrine groups at 24.21% and 26.14%, re-
spectively. These results suggest that Compound B1 was more
effective in reducing infarct volume than stachydrine. In ad-
dition, the infarct size of B1 was 16.13%, which is similar to
the control (edaravone) infarct size of 10.0%. Edaravone has
been approved to treat cerebral ischemic stroke in Japan but
requires special labeling due to several side effects, such as
increasing the risk of renal disorders (Hishida 2007; Chen
et al. 2007).
Acute toxicity tests of Compound B1 in vivo
In vivo acute toxicity of Compound B1 and stachydrine was
measured in Kunming mice. No physical signs of toxicity
were observed, such as behavioral change, abnormal breath-
ing, alteration in food intake, or change in body weight in
animals treated over 14 days. This study shows that
Compound B1 and stachydrine are all well-tolerated with
LD50 > 1 g/kg, and also indicates that Compound B1 is rea-
sonably safe in vivo.
Table 2 Pharmacokinetic parameters of B1 and stachydrine after
intravenous injection in Wistar ratsa
Parameter (unit)
max (ng·mL−1
B1
Stachydrine
Table 3 Evaluation of B1 activity in vivo
C
)
70,900
6390
1.83
Compound Molecular Concentration Neurological Infarct size (%
mass
deficit score of brain)b
t
1/2 (h)
AUC(0-t) (h·ng·mL−1
AUC(0-∞) (h·ng·mL−1
Vz (L·kg−1
0.27
)
31,400
31,600
6080
12,000
1.10
Sham
Vehicle
Edaa
A
ND
ND
ND
179.5
284
284
ND
0
0
)
ND
3.13 0.23
1.20 0.20*** 10.00 0.94***
24.21 1.71
)
0.06
3 mg/kg
30 mg/kg
30 mg/kg
60 mg/kg
CL (L·h−1·kg−1
)
0.16
0.48
0.42
2.50 0.19
2.40 0.33
1.50 0.19### 16.13 1.48#
26.14 1.70
23.36 1.27
MRT(0-t) (h)
0.93
B1
a The pharmacokinetic parameters were calculated using the DAS 3.0
pharmacokinetic software (Chinese Pharmacological Association,
Shanghai, China)
B1
a Eda, known as edaravone injection, is a type of positive drug for cerebral
stroke.
b Data are presented as mean SD (n = 8). ***P < 0.001 versus the sham
group; ## P < 0.01, ### P < 0.001 versus the vehicle group. A =
stachydrine
Cmax maximum plasma concentration, t1/2 half-life, AUC(0-t) area under
the curve to termination time, AUC(0-∞) area under the curve extrapolated
to infinity, VZ volume of distribution, CL total clearance, MRT(0-t) mean
residence time

Naunyn-Schmiedeberg's Arch Pharmacol
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Conclusion
In this study, a series of stachydrine derivatives was designed
and synthesized, and biological studies were carried out
in vitro and in vivo. All results indicate that Compound B1
had superior therapeutic activities over stachydrine and other
derivatives. During the in vitro assay, B1 significantly im-
proved survival rate of neuronal cells, inhibited neuronal ap-
optosis, and decreased levels of LDH, ROS, and MDA, while
increasing SOD activity. Moreover, Compound B1 exhibited
better pharmacokinetic parameters than stachydrine. During
the in vivo study, B1 significantly reduced infarct size at a
60 mg/kg dose, and its LD50 was > 1 g/kg. In summary, these
results provide valuable information in the development of
potent neuroprotective agents for treatment of cerebral ische-
mic stroke.
There have been multiple attempts to develop new drugs
for treatment of cerebral ischemic stroke. Among these efforts,
natural products have emerged as a promising source for anti-
stroke drugs, and many reports have suggested stachydrine in
particular for treatment of ischemic stroke. Although
Compound B1 displayed less activity than the approved neu-
roprotective drug edaravone, our work provides guidance for
the development of future drugs. In order to derive
Compounds with better protective activity that can be devel-
oped into anti-ischemic stroke agents, knowledge of how to
design and synthesize new derivatives is crucial.
El Kossi MMH, Zakhary MM (2000) Oxidative stress in the context of
acute cerebrovascular stroke. Stroke. 31(8):1889–1892
Feng XD (2009) Pharmacokinetics of stachydrine in rabbits. China
Pharm 12(12):1696–1697
Author contributions Wenbao Li and Feng Li conceived and designed
research. Liang Zhang, Chenhui Hou, and Sifeng Zhu conducted exper-
iments. Lili Zhong, Jianchun Zhao, and Cai Song made contribution in
ex vivo and in vivo studies. Liang Zhang and Chenhui Hou analyzed data.
Liang Zhang wrote the manuscript. All authors read and approved the
manuscript. We explicitly state that the data were generated in-house, and
we did not use a paper mill.
Gao HS, Hu ZG, Wang JJ, Qiu ZF, Fan FQ (2008) Synthesis and prop-
erties of novel chiral ionic liquids from L-proline. Aust J Chem
61(7):521–525
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S, Ghorbani A, Sadri K, Mahdipour E, Ghasemi F, Forouzanfar F,
Hoseini A, Pasdar AR, Sadeghnia HR, Ghayour-Mobarhan M
(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
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amino acids as chiral ligands in the catalytic asymmetric addition
of phenylacetylene to aldehydes. Eur J Org Chem 36(26):934–938
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PM, Worrall BB, Seshadri S, Sudlow C, the METASTROKE
Consortium, the Wellcome Trust Case Control Consortium 2,
Williams FMK, Markus HS, Lewis CM (2015) Genetic factors
influencing coagulation factor XIII B-subunit contribute to risk of
ischemic stroke. Stroke. 46(8):2069–2074
Funding information This study was funded by the “Zhufeng Scholar
Program” of the Ocean University of China (841412016), the “Major
Projects of Independent Innovation” of Qingdao (15-4-13-zdzx-hy), the
“Outstanding Talents Plan” of Qingdao (15-10-3-15-(34)-zch), and the
Aoshan Talents Training Program of Qingdao National Laboratory for
Marine Science and Technology (No. 2017ASTCP-OS08) to Dr. Wenbao
Li.
Compliance of Ethical Standards
Conflict of interest The authors declare that they have no conflict of
interest.
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post-marketing surveillance. Clin Exp Nephrol 11(4):292–296
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reagents based on esters of N-alkylproline and their application in
the enantioselective incorporation D or L amino acid residue directly
from racemic substrate. Acta Pol Pharm 71(6):994–1003
Kaur H, Prakash A, Medhi B (2013) Drug therapy in stroke: from pre-
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