Toxicities and beneficial protection of H2S donors based on nonsteroidal anti-inflammatory drugs

Article abstract of DOI:10.1039/c8md00611c

On the basis of our previous study, the H₂S donors based on nonsteroidal anti-inflammatory drugs (NSAIDs) were further evaluated with regard to the following aspects: animal blood and urine analyses, liver and kidney toxicities, gastrointestinal protection, anti-hypertension, and myocardial protection. The test results showed that after the successive administration of the compound for 14 days, the number of white blood cells in the blood of rats reduced, and protein and leukocytes appeared in urine; further, α-lipoic-acid-acetaminophen ester (1) and ibuprofen-ADTOH ester (2) had a certain effect on the physiological tissue and function of rat liver, and their side-effects on the kidneys were obvious. However, when compared with NSAIDs as the precursors, the tested compounds displayed much fewer side-effects; particularly, for the gastrointestinal mucosa of rats, there were hardly any side-effects. Moreover, all the three compounds decreased blood pressures in spontaneously hypertensive rats in a concentration-dependent manner, even though this antihypertensive effect was weaker than those induced by nifedipine and captopril. In addition, three compounds protected H9c2 cells from injury via an antioxidation pathway; and they improved myocardial injury in spontaneously hypertensive rats. Compound 2 is the derivative of ibuprofen and has lower toxicity to rat cardiomyocytes than ibuprofen. Therefore, it may become a better substitute of ibuprofen in patients due to its lower cardiotoxicity.

Full text of DOI:10.1039/c8md00611c

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Toxicities and beneficial protection of H2S donors basDeOdI: 1o0.1n03V9i/eCw8AMrtDicl0e0O6n1l1inCe
non-steroidal anti-inflammatory drugs
Jinlong Zhang1, Qiuping Zhang1, Yanni Wang1, Jili Li1, Zhongjie Bai1, Quanyi Zhao *1,
Zhen Wang1, Dian He*1, Jingke Zhang2, Yonglin Chen3
(1 Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University; 2 GLP Lab
Centre, School of Basic Medicine, Lanzhou University; 3 The First Affiliated Hospital of Lanzhou
University, Lanzhou, 730000, China)
Abstract: On the basis of our previous work, the H2S donors based on non-steroidal
anti-inflammatory drugs (NSAIDs) were further evaluated in following aspects,
animal blood and urine analysis, liver and kidney toxicities, gastrointestinal protection,
anti-hypertension and myocardial protection. The testing results showed: after
successive administration of the compound for 14 days, the numbers of white blood
cells in the blood of rats reduced, and protein and leukocytes appeared in urine; and α-
lipoic acid-acetaminophen ester (1) and ibuprofen-ADTOH ester (2) had a certain
effect on the physiological tissue and function of rat liver, and their side-effects on
kidney was obvious. However, compared with the NSAIDs as precursors, the tested
compounds displayed much lower side effects, especially, for the gastrointestinal
mucosa of rats, there has hardly side-effect; moreover, all three compounds decreased
blood pressures in spontaneously hypertensive rats in concentration dependent
manner even though this antihypertensive effect was weaker than those of nifedipine
and captopril. In addition, three compounds protected H9c2 cells from injure through
anti-oxidation pathway; and they improved myocardial injury in spontaneously
hypertensive rats. Compound 2 is the derivative of ibuprofen and has lower toxicity
than ibuprofen to rat cardiomyocytes. So possibly, it will be a better substitute of
ibuprofen for patients due to lower cardiotoxicity.
Keywords: NSAIDs; H2S donors; gastrointestinal protection; anti-hypertension;
myocardial protection
Introduction
Endogenous H2S is the third endogenous gas signal molecule after nitric oxide
(NO) and carbon monoxide (CO), and plays an important physiological role in the
body.[1-4] H2S significantly reduced lipopolysaccharide-induced accumulation of
Corresponding author, Email: zhaoqy@lzu.edu.cn; hed@lzu.edu.cn
1

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neutrophils in tissues and organs (liver and lung), reduced tumor necrosisDfOaI:c1t0o.1r0-3α9/C8MD00611C
(TNF-α) and plasma interleukin-1β (IL-1β) Level, and increased plasma IL-10 levels,
thereby inhibiting inflammatory response.[5] In the cardiovascular system, H2S caused
vasodilation and lowered blood pressure by promoting KATP channels open;[6]
meanwhile, it produced myocardial protection through activating protein kinase C
(PKC) and myocardium KATP channels. [7]
Since H2S is toxic gas and its dosage is not easy to control, it cannot be directly
applied to the clinic. Therefore, using H2S donors is simple and convenient way to
replace H2S. The earliest H2S donors were sulfide salts, such as Na2S, CaS, they
released H2S too fast under physiological conditions, which was not beneficial for its
function. GYY4137 was found as a novel water-soluble H2S releaser, and it displayed
anti-shock, anti-tumor, antithrombotic and other functions. [8-11] After that, many H2S
donors were discovered and synthesized, such as desipramine (ADTOH) derivatives
[12], 4-hydroxythiobenzamide (TBZ) derivatives[13], alpha-lipoic acid derivatives, and
so on.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat pain,
inflammation and fever in clinic. [14] However, NSAIDs have many side effects, such
as gastrointestinal bleeding, ulcers, high blood pressure, and increased risk of
myocardial infarction. [15-17] Recently, H2S donor on the basic of naproxen (ATB-346)
was reported to be a good inhibitor of COX-2, moreover, it significantly reduced the
gastrointestinal damage compared with naproxen.[18] Later, our group designed and
synthesized a series of H2S donors modified by traditional NSAIDs. The testing
results showed all the compounds had good anti-inflammatory activity, lower
cytotoxicity and reproductive toxicity, which suggests these compounds may be a
kind of more potential anti-inflammatory drugs with lower toxicity. However, as we
know, traditional NSAIDs cause myocardiac injure and blood pressure rising after
administrated over a long period time. H2S has anti-hypertension effect and
myocardial protection, the NSAIDs modified by H2S donors possibly counter or
compensate for these side effects caused by NSAIDs.
[19-20]
Based on our previous work,
in this paper, we further evaluate these
compounds from gastrointestinal protection, antihypertensive and myocardial
protection. By which, we hope to obtain useful data and information to
comprehensively evaluate NSAIDs-based H2S donors whether they are more
beneficial in clinic.
Results and discussion
1. Synthesis and characterization of compounds
All three compounds were prepared according to the method we used before. [19]
Compound 1 was synthesized by the reaction of α-lipoic acid and acetaminophen, and
compounds 2 and 3 were prepared by TBZ and ADTOH reacting with ibuprofen,
respectively (Scheme 1). The reaction conditions are mild, the yields are high. All
2

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DOI: 10.1039/C8MD00611C
three target compounds were obtained by separation and purification using column
chromatography.
The spectra of three compounds were consistent with the expected. In IR spectra,
there are strong absorption peaks in the range of 1728-1766 cm-1, which are
characteristic absorptions of C=O in the ester group. For compound 1, the absorption
for the C-S bond appears at 604 cm-1; for compound 2, the strong absorption at 1152
cm-1 belongs to C=S; whereas the characteristic absorption for C=S for compound 3
appears at 1136 cm-1. In the 1H-NMR spectrum of compound 1, the chemical shift of
hydrogen atoms in CH3CO is at 2.14 ppm; for compound 2, the signals of two
hydrogen atoms in CONH2 occur in the range of 9.52-9.58 and 9.90-9.95 ppm. As for
compound 3, the signal of the hydrogen atom in trisulfide ring appears at around 7.40
ppm.
S
OH
O
O
S
S
O
HO
EDCI/DMAP
EDCI/DMAP
S
O
H3C
N
H3C
N
O
H
H
1
S
NH2
S
CH3
O
CH3
O
OH
O
NH2
CH3
CH3
H3C
H3C
HO
2
S
S
S
S
CH3
S
OH
CH3
O
S
EDCI/DMAP
CH3
O
O
CH3
H3C
HO
H3C
3
Scheme 1 Synthetic route and structures of compounds 1−3
2. H2S release of the compounds
The H2S release ability of the compounds was examined using the methylene
blue (MB+) method. The MB+ method is according to the reaction between zinc
acetate and H2S to form zinc sulfide in the presence of acid and ferric ion, and then
reacting with N, N-dimethyl-1,4-phenylenediamine sulfate to form methylene blue.
The absorbance at 670 nm was measured to quantify H2S. Because the compounds
were slower H2S releasers, we used the methylene blue method to detect the ability of
all compounds to release H2S in the presence of TECP or cysteine.
The test results are shown in Fig.1. The release half-lives of the three compounds
are all less than 30 min, and the release rates are roughly consistent with the first-
order kinetics. This suggests they are all rapid H2S releasers. The release half-life of
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DOI: 10.1039/C8MD00611C
compound 1 is 14.1 min and its maximum release amount is 46.0 μM. For compound
2, its H2S release profile is shown in Fig.1 (b); its half-life is 20.1 min and maximum
release amount is 20.9 μM. Significantly, the H2S release ability of compound 2 is
weaker than that of compound 1. Fig.1 (c) is the H2S release curve of compound 3, its
half-life is 1.4 min, the maximum release is 13.8μM. In contrast, its H2S release
ability is the weakest among the three compounds, but its rate is the fastest.
Fig. 1 H2S release curves of the compounds, a for 1; b for 2; and c for 3
3. Toxicity of the compounds after successive administration
Since the three compounds did not show toxicity to the mice as model in the
acute toxicity test, we selected rats to further evaluate their toxicity after successive
administration. Each compound was administered repeatedly by intraperitoneal
injection at a dose of 0.01 mmol•kg-1 for 14 days. The urine was collected and the rats
were sacrificed 14 days later. Blood and liver, kidney and other major organs were
collected and further examined. The blood test results are shown in Table 1. Seen
from Table 1, some blood indexes are abnormality: the numbers of leukocyte
significantly decreased compared with the blank control; the platelet numbers
decreased too much. The platelets are only 194, 161, 114×109/L after rats treated with
compound 1, 2 and 3, respectively, while the control is 670×109/L. However, the
numbers of red blood cell and hemoglobin are within the normal range. This shows
that the compounds have a certain impact on the blood system.
Table 1 Results of blood test after rat successive administration for 14 days
Blank
Test items
Compd 1
Compd 2
Compd 3
Control
6.88
54.7
42.1
0.5
leukocyte(109/L)
2.89
31.7
64.2
1.3
1.72
44.4
52.8
1.8
3.27
35.9
60.4
0.9
Neutrophil ratio(%)
Lymphocyte ratio(%)
Monocyte ratio(%)
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Eosinophil ratio(%)
Basophilic ratio(%)
Red blood cell (1012/L)
2.6
1
2.6
0.2
5,9
1.8DOI: 10.1039/C8MD00611C
0.2
0
2.2
6.08
5.5
6.83
135
194
119
161
136
114
138
670
Hemoglobin（g/L）
Platelet (109/L)
The urine test results of rats are shown in Table 2. Some indexes are abnormality.
Compared with the control group, the urine pH value decreased, and both
urobilinogen and bilirubin levels increased. Among three compounds, compound 3
led to urobilinogen and bilirubin in rat urine increasing beyond 50-fold as high as the
level of the control. Protein was detected in the urine of the three test rats, and the
content was over 3 μmol•L-1. Moreover, leukocytes were found in urine of compound
2 and 3 test groups. This suggests three compounds possibly injured glomeruli of
kidney in some degree, further have an effect on rat kidney function.
Table 2 Results of urine test after rat successive administration after 14 days
Blank
Control
Test items
Compd 1
Compd 2
Compd 3
3.3
12
16
66
1
Urobilinogen（μmol/L）
Bilirubin（μmol/L）
Ketone body（mmol/L）
Occult blood(cacells/μL)
protein(g/L)
17
50
10
4
4
Neg
Neg
3.0
0.5
Neg
3.0
+
80
10
0
3.2
Neg
Neg
Neg
1.005
Nitrite
Neg
70
9
leukocyte(cacells/μL)
glucose
500
Neg
1.005
10
0
Neg
1.005
proportion
1.01
PH
VC
7.5
7.5
7.5
8.4
10
Neg
Neg
Neg
In order to make out the degree of damage of the test compounds to the rat liver
and kidney, we do a liver and kidney biopsy through HE staining. Under microscopy,
for the control group, the hepatic lobule was structurally intact, the hepatocytes were
arranged neatly, and no obvious gap was observed between the cells; the renal tissue
was stained deep and the glomerular structure was intact. After administration of 10
mg•kg-1 of compound in rats by intraperitoneal injection for 14 days, compound 2
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group: hepatic lobule structure was intact, central venous congestion, hepaDtiOcI: 1s0i.n10u3V9si/eCw8AMrtDicl0e0O6n1l1inCe
congestion was obvious, hepatocytes were swollen and visible gaps were observed,
cytoplasm was lightly stained; for kidney section, the small ball was intact, the cells
of the proximal convoluted tubule were swollen and visible gaps were visible, the
cytoplasm was loose, lightly stained, and some renal tubular epithelial cells were
necrotic. As for compound 3 group: the liver tissue damage was similar to that of
compound 2 group, the renal tissue was congested and the cells were swollen, the
cytoplasm was loose, and the light staining was significantly lighter. This indicates
that compounds 2 and 3 have a certain degree of damage to the physiological structure
of rat liver and kidney tissue. In contrast, the damage caused by compound 2 was
severer to rat kidney than that of compound 3.
Fig.2 Optical microscopy morphological images of rat liver (A and B) and kidney tissues (C and
D) after successive administration compound for 14d. Kidney section: A for compound 2 and B
for compound 3; kidney section: C for compound 2 and D for 3. Black arrows indicate tissue
congestion.
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Fig.3 TEM morphological images of rat liver after successive administration compound for 14d.
A, B, and C were the control group; a, b, and c for compound 2 group (6000, 10000, 20000-fold,
respectively). The arrows in Fig.3a and 3b indicate an increase in lipid droplets.
Fig. 4 TEM morphological images of rat kidney after successive administration compound for 14d.
A, B, and C were the control group; a, b, and c for compound 2 group (6000, 10000, 20000-fold,
respectively). The arrow in Fig.4a indicates swelling of glomerular endothelial cells; the arrow in
Fig.4b indicates swelling of glomerular endothelial cells, and stromal hyperplasia; the arrow in
Fig.4c indicates a large amount of exudation (precipitate) in the glomerulus.
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In order to further understand the reasons of liver and kidney dysfunction, we
observed the liver and kidney tissues of the test rats by transmission electron
microscopy. The results are shown in Fig.3 and 4. After treated with compound 2, the
rat liver cells had abnormal nuclear morphology, increased heterochromatin,
chromatin edge collection, and increased lipid droplets in the cytoplasm. The
glomerular endothelial cells were swollen, the stromal hyperplasia, and a large
amount of exudation in the glomerulus. This indicates that compound 2 gave rise to
the liver and kidney cells of the rat greatly damaged, which further affect their
biological functions.
4. Gastrointestinal protection of the compounds
In order to figure out whether the compound as H2S donor has protective effect
on the gastrointestinal tract, the rats were given a continuous dose of 10 mg•kg-1
compound 2 for 14 days, the compound precursor ibuprofen as a positive control
(ibuprofen: daily dose of 6.1mg•kg-1; Note: clinical adult dose does not exceed
5.0mg•kg-1). Gastrointestinal tissues were collected and further examined under
microscopy. The results are shown in Fig.5. After HE staining, the gastrointestinal
tissues were observed by optical microscopy. The control group: the gastric mucosal
cells were arranged neatly, and the gastric mucosa showed no obvious hyperemia
(Fig.5 control stomach); intestinal mucosa showed no hyperemia, inflammatory cell
infiltration and intestinal mucosal edema (Fig.5 control intestinal).
Compared with the blank group, the ibuprofen group (positive control) showed
hyperemia and inflammatory cell infiltration in the gastric mucosa, and gastric
mucosal cells swelled (Fig.5A); intestinal mucosa became hyperemia, inflammatory
cell infiltration and intestinal mucosal edema (Fig.5C). This indicates ibuprofen has
serious side effects to gastrointestinal, especially for the patients needing for long-
term medication. In contrast, in compound 2 group, gastric congestion symptoms
were significantly reduced compared with ibuprofen group, and no gastric cell
swelling occurred (Fig.5B); intestinal mucosal congestion symptoms were also
significantly reduced (Fig.5D). This indicates that compound 2 has a certain
improvement effect on gastrointestinal side effects caused by NSAIDs.
It is to be noted that all the side effects of ibuprofen on the gastrointestinal tract
are caused by the free carboxyl group in its molecule, and in compound 2, the
carboxyl group was transformed into ester, which reducing the irritation to the
gastrointestinal tract. Compound 2 is a pro-drug that acts individually through esterase
hydrolysis in the blood. At the same time, compound 2 not only improved the
gastrointestinal side effects of ibuprofen, but also changed the absorption, distribution
and metabolism of ibuprofen in vivo.
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Fig. 5 Optical microscopy morphological images of rat stomach (A and B) and intestinal tissues
(C and D) after successive administration compound for 14d. Stomach sections: A for ibuprofen
group; B for compound 2 group; intestinal sections: C for ibuprofen group; D for compound 2
group. Black arrows indicate tissue congestion and red arrows indicate swelling of gastric cells.
5. The anti-hypertension of the compound
In order to confirm the anti-hypertension of the compounds as H2S donors, we
selected spontaneously hypertensive rats (SHRs) as a model of hypertension, and the
test compound was administered by intraperitoneal injection; anifedipine and
captopril were used as positive control. Blood pressure was measured by a rat blood
pressure monitor.
The test results showed compound 1 displayed a good antihypertensive effect,
and it was concentration dependent. When compound 1 was 10, 20, 40 mg•kg-1, the
mean minimum systolic blood pressure of the SHR was 139.1, 127.6, and 115.3
mmHg, respectively (Fig. 6a). The statistical difference was significant between
different concentrations, p<0.01; the duration of blood pressure was longer and about
60 min, this is because compound 1 has a strong H2S release capacity and a longer
release half-life. Compound 2 has a similar antihypertensive effect as compound 1,
but it has a shorter duration (Fig. 6b). Compound 3 is a norepine trisulfide compound.
Under the same condition, the antihypertensive effect of compound 3 was weaker
than those of the two compounds. The average minimum systolic blood pressure at
three concentrations was 142.9, 136.7, and 135.1 mmHg, respectively (Fig. 6-c). This
may be related to less H2S released from compound 3.
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Fig. 6 Effect of compound concentration on systolic blood pressure in SHR rats. a for
compound 1; b for compound 2, and c for compound 3. Each column represents the mean ± SD
of four independent experiments.
Fig.7 Effect of compound on systolic blood pressure in SHR rats. a for compound 1; b for
compound 2; c for compound 3, and each column represents the mean ± SD of four
independent experiments.
The effect of the compounds as H2S donors on the diastolic blood pressure of
SHRs was similar to that of systolic blood pressure. Compound 2 had the strongest
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effect on reducing diastolic blood pressure among three compounds (Fig. 8b), and it
also had a significant concentration-dependent, but the duration of diastolic blood
pressure reduction was shorter. The effect of compound 1 on reducing diastolic blood
pressure was also concentration-dependent and had the longest duration (Fig. 8a),
which may be related to the strong H2S release ability and longer release half-life of
compound 1. When compound 1 was at 10, 20 and 40 mg•kg-1, the minimum diastolic
blood pressures of SHRs were 113.8, 110.9 and 99.4 mmHg, respectively. Compound
3 had the weakest effect on reducing diastolic blood pressure, and the concentration
dependence was not obvious. When compound 3 was 10, 20 and 40 mg•kg-1, the
minimum diastolic blood pressure of SHRs was 116.3, 115.1, 112.4 mmHg,
respectively (Fig.8c).
However, at the same concentration (10 mg•kg-1), regardless of systolic or
diastolic blood pressure, the anti-hypertension of the compound was weaker than the
same concentration of nifedipine and captopril, and the duration of blood pressure
was shorter than that of nifedipine and captopril (Fig. 9); the compounds with higher
concentration lowered the blood pressure of SHRs to the blood pressure level of
normal WKY rats. During the hypotensive process, the antihypertensive effect of the
compound is related to the release ability of H2S. Compound 1 with strong H2S
releasing ability displayed a stronger antihypertensive effect, which indicates that the
antihypertensive effect of compound 1 was possibly mediated by H2S.
Fig. 8 Effect of compound concentration on diastolic blood pressure in SHR rats: a for compound
1; b for compound 2; c for compound 3. Each column represents the mean ± SD of four
independent experiments.
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Fig. 9 Effect of compound on diastolic blood pressure in SHR rats: a for compound 1; b for
compound 2; c for compound 3. Each column represents the mean ± SD of four independent
experiments.
It can be seen from the above results that the compounds as H2S donors can
reduce the systolic blood pressure and diastolic blood pressure of SHRs in a
concentration-dependent manner. In the process of modulating blood pressure, the
effects of compounds on systolic blood pressure and diastolic blood pressure are
related to H2S release ability. Compound 1 with strong H2S release ability showed a
stronger antihypertensive effect, indicating that the antihypertensive effect of the
compound may be mediated by H2S.
6. Protective effect of the compounds as H2S donors on myocardium
6.1 Protection of the compounds against cardiomyocyte H9c2
To confirm the effect of the compounds as H2S donors on myocardial protection,
we used H9c2 cell lines as the test model. The cell viability of the H9c2 cells was
measured under test conditions and then determined the myocardial protection of the
compound. The cell tests were divided into blank group, H2O2 group, control group
(acetaminophen or ibuprofen), compound-H2O2 group. The damage models of light,
medium and heavy were obtained only through the cardiomyocyte treated with H2O2
for 1h, 4h and 8h respectively.
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Fig. 10 H9c2 cell viability after treated with compound for 24 h
Seen from Fig. 10, in the range of 10-80 μM, the tested compounds had no effect
on cardiomyocyte survival rate compared with the blank control. This indicates the
three compounds were less toxic and the results of myocardial protection against the
cells in this concentration range will be meaningful.
Fig. 11 Effect of compound concentration on the survival rate of H9c2 cells. Each column
represents the mean ± SD of three independent experiments, *P <0.05, **P <0.01
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In following tests, the compounds displayed protective effects on cell DdOaI:m10a.1g03e9./C8MD00611C
Seen from Fig. 11 (A, B and C), after treatment with H2O2 of 400 μM for 1h, the
survival rate of H9c2 cells was 82.3%; in the presence of compound 1 or 2 or 3, the
cell survival rates increased in dose-dependent manner; and when they all were 80 μM,
the survival rates of the cells increased by 13.3%, 11.9% and 9.4%, respectively. We
found the compound displayed a different activity with the cell injure degree. When
the H9c2 cells treated for more time, the survival rates of the cells increased little
even in the presence of the compounds at the same concentration. When H9c2 cells
treated with H2O2 for 8 h, compound 1 made cell viability increase by 8.6%, while
compounds 2 and 3 less than 5.0%. Obviously, when the myocardial cells injured
slightly, the protective effect of compound on cardiomyocyte is significant; and the
cell damaged seriously, the activity of the compound is low, this is because the cell
damaged too severe to repair. In comparison, compound 1 has a stronger activity,
which possibly resulting from the larger maximum release of H2S.
To understand the mechanism of myocardial protection of compounds as H2S
donors, we measured the SOD and MDA levels in H9c2 cells.
Fig. 12 Effect of compound on SOD and MDA content in H9c2 cells, each column representing
the mean ± SD of four independent experiments; *P < 0.05, **P < 0.01.
As shown in Fig. 12, when the H9c2 cardiomyocytes were treated with only
H2O2 for 24h, the level of SOD in cells was significantly decreased, but after the cells
incubated with the tested compounds, the levels of SOD were significantly increased.
Accordingly, MDA levels decreased in concentration-dependent manner. Therefore,
we speculate the myocardial protection of the compound possibly results from the
antioxidant of the H2S donors. The positive control acetaminophen and ibuprofen
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displayed weak antioxidant activity, which it suggests the antioxidant effect of the
compound is mediated through H2S released from the compound.
In the process of test, the cells were stained by giemsa and observe the H9c2 cell
morphology under microscope. In the blank group, the cells were long spindle-shaped,
and adherent firm cell; but in H2O2 group, the cells showed an irregular shape, and
some cells contracted and became round. With the prolongation of H2O2 intervention
time, the number of deformed cells increased (Fig. 13-d). But in compound-H2O2
group, the number of cells reduced significantly; moreover, with the concentration of
compound 1, the repair effect was more pronounced (Fig. 13-l). Meanwhile, we see
acetaminophen as a control also showed anti-oxidation activity due to phenolic
hydroxyl group (Fig. 13g-h). In contrast, under the same concentration, compound 1
displayed better activity than acetaminophen did, just because H2S released from
compound 1 also has anti-oxidation activity. This is consistent with the previous
conclusions.
Fig. 13 Morphology of H9c2 cells under all kinds conditions: a, e, and i are blank groups; b, c, and
d for H2O2 treated for 1h, 4h, and 8h, respectively; f, g, and h for acetaminophen treated for 1h,
4h, and 8h, respectively; j, k, and l for compound 1 treated for 1h, 4h, and 8h, respectively.
6.2 Myocardial protective effect of target molecules on hypertensive rats
The compound as H2S releasing molecule has displayed H9c2 cardiomyocytes
protection in some degree, but we want to know whether it also can reverse the
myocardial remodeling and protect the cardiac function for animal. For this reason,
hypertensive rats were administrated the tested compounds at the dose of 10 mg•kg-1
per day by intraperitoneal injection for 14 consecutive days, ibuprofen as the positive
control. Then the myocardial tissues were removed, and observed after HE staining
under light microscopy. As seen in Fig. 14. The positive control group: the
myocardial fibers of the rats were neatly arranged and coarser, the muscle bundle gaps
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were increased to varying degrees compared with the blank control, and some
myocardial interstitial collagen fibers were visible. But compound 1 group: the
myocardial fibers of the rats were arranged neatly, and the muscle bundle gap was
slightly reduced compared with the ibuprofen group, and the collagen fibers were not
significantly stained in the interstitium. Myocardial fibers are significantly thinner,
which indicating compound 1 has lower toxicity than the positive control. As for
compound 2 group: myocardial fibers were arranged neatly, and the muscle bundle
gap was significantly reduced, and the thickening of the myocardial fibers have a
certain improvement effect compared with the ibuprofen group, indicating that
compound 2 as non-steroidal anti-inflammatory drug modified H2S donor has lower
side-effect than the original compound ibuprofen.
Fig. 14 Optical microscopy morphological images of rat myocardial tissues. The control group:
ibuprofen; A for compound 1; B for compound 2; C for normal control group. The black arrow
indicates the muscle bundle gap and the red arrow indicates the myocardial fiber.
In order to make the injury of myocardial cells more visible, myocardial tissues
were observed under TEM. As shown in Fig. 15, the positive control group, the rat
myocardial interstitial vascular swelled, myocardial fibers loosed, with myocardial
fibrous submucosal swelling and myocardial fiber melting; meanwhile, myocardial
contraction band formed, and myocardial disk dissociated and mitochondrial swelled.
Mitochondria were damaged, causing energy production, release and transmission
disorders, which led to ventricular pumping dysfunction. But for compound 2 group,
the myofibrils of the rats were arranged substantially neatly compared with the control
group, no obvious muscle fiber dissolution, no significant swelling of the
mitochondria, and no myocardial disk dissociation.
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Fig. 15 TEM images of myocardial tissues of rats after administration of the tested compound for
14 days: A, B, and C for the positive control group; a, b, and c for compound 2 group (6000,
10000, 20000-fold); The arrow in Fig. 15a indicates swelling of cardiomyocytes; the arrow in
Fig.15B indicates dissolution of cardiomyocytes; the arrow in Fig.15C indicates swelling of
mitochondria.
This result indicates that ibuprofen has more toxicity to rat cardiomyocytes than
compound 2. Compound 2 is the derivative of ibuprofen, and has good anti-
inflammatory activity like ibuprofen. Maybe, compound 2 will be a better substitute
of ibuprofen for patients due to lower cardiotoxicity.
Conclusion
In the presence of cysteine or TECP, all three compounds were H2S releasers
rapidly. After successive administration for14 days, the test compound reduced the
white blood cell number of rats, and the protein and white blood cells appeared in
urine. Seen under the microscope, the structure of the kidney tissues were abnormal in
some degree. This indicates that the compounds rat still have side-effect to kidney for
the patients with long-term medication. However, compared with ibuprofen, their side
effects were less. Secondly, compared with ibuprofen, the side effects of
gastrointestinal tract significantly alleviated; and there were no symptoms such as
gastric mucosa and intestinal mucosal hyperemia. Thirdly, the test compounds as H2S
donors have a good concentration-dependent anti-hypertension on SHRs even though
its antihypertensive effect is weaker than nifedipine and captopril. Fourthly, the tested
compound displayed protective effect on H9c2 cardiomyocytes, and its effect was
achieved by reducing intracellular SOD and MDA levels, and the compounds have
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protective effect on the myocardium tissues of rat. Among three compounds,
compound 2 has lower toxicity than ibuprofen to rat cardiomyocytes. Compound 2 is
the derivative of ibuprofen and will be a better substitute of ibuprofen for patients due
to lower cardiotoxicity.
Experimental part
1. Experimental instruments and materials
AVANCE III 400M NMR instrument; maXis4G TOF mass spectrometer
(Brucker, Germany); Nicolet NEXUS 360 FT-IR infrared spectrometer (Nieolet,
USA); Lambda25 UV-visible spectrometer (PerkinElmer, USA); CO2 incubator;
microplate reader (ThermoVarioskan Flash 3000) Beckman-Coulter LX-20 fully
automated biochemical analyzer; JEM-1230 transmission electron microscope.
BP-300A automatic large mouse non-invasive blood pressure detector (Chengdu
Taimeng Software Co., Ltd.); Captopril, purity 98%, supplied by Beijing Bailingwei
Technology Co., Ltd., product number: 237181; nifedipine, purity 98%, supplied by
Beijing Bailingwei Technology Co., Ltd., product number: 307429; other chemical
reagents used in the experiment are analytically pure The reagents used were all dried.
The experimental animals were purchased at the GLP Laboratory of
Experimental Animal Center of Lanzhou University, and the experimental animal
certificate: SCXK (Gan) 2009-0004; WKY (Wistar-Kyoto) rat; Clean male
spontaneously hypertensive rat (SHR), Zhou Ling, 8 weeks; weight is 300 ± 25g. Rats
were housed in cages, one per cage, kept at room temperature (23 ± 2) °C, relative
humidity of 60-65%, free access to drinking water, and all animals were kept for 7
days before the experiment. All animal procedures were performed in accordance
with the Guidelines for Care and Use of Laboratory Animals of LanZhou University
and Experiments were approved by the Animal Ethics Committee of agree.
2. Synthesis of H2S donors
The synthesis of compound 1 is described in [22]. 206 mg (1 mmol) of α-lipoic
acid was dissolved in 10 mL of dried dichloromethane, and 227 mg (1.1 mmol) of
DCC was added, and stirred at room temperature for 30 min. Then 150 mg (1 mmol)
of acetaminophen and 18 mg (0.15 mmol) of DMAP were added and stirred at room
temperature for 3h. The precipitate was removed by filtration, and the filtrate was
washed three times with water, 5% acetic acid solution, and then dried over
anhydrous sodium sulfate overnight. The solvent was evaporated under reduced
pressure and the crude material was applied to column chromatography (CH2Cl2-
CH3OH = 30:1) to yield 103 mg of pale yellow solid. IR (KBr, cm-1): 1751 (v, C=O),
1663 (v, C=O), 604 (w, C-S). 1H NMR (DMSO-d6, ppm): δ 1.55 (m, 2H , Dithiolane-
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C-CH2), 1.76 (m, 4H, Dithiolane-CH2, O=CC-CH2), 1.93 (m, 1H, 5H-DitDhOioI:l1a0n.10e3)9,/C8MD00611C
2.14 (s, 3H, CH3), 2.47 (m, 1H ,3H-Dithiolane), 2.50 (t, J=6.0Hz, 2H, 4H-Dithiolane),
3.09 (m, 2H, O=C-CH2), 3.60 (m, 1H, 5H-Dithiolane), 7.00 (d, J = 8.8 Hz, 2H, Ar-H),
7.47 (d, J = 8.8 Hz, 2H, Ar-H), 7.53 (s, 1H, NH). 13C NMR (DMSO-d6): δ 24 .4, 24.6,
28.5, 33.8, 34.5, 38.6, 39.7, 40.4, 56.5, 120.3, 122.3, 137.4, 146.1, 168.7, 172.3.
HRMS (m/z): calcd. For C16H21NO3S2Na [M+Na]+ : 362.0861; found 362.0857.
The synthesis of compound 2 is described in [23]. Ibuprofen (206 mg, 1 mmol)
was dissolved in 8 mL of dried acetonitrile, EDCI (210 mg, 1.1 mmol) was added,
and stirred at room temperature for 30 min. 4-Hydroxythiobenzamide (153 mg, 1
mmol) and DMAP (18 mg, 0.15 mmol) were added to the reaction system, and the
mixture was reacted at room temperature for 1 h. After the reaction was completed,
the mixture was filtered, and the filtrate was evaporated under reduced pressure. The
crude product was separated by column chromatography (CH2Cl2-CH3OH = 30:1) to
give the product as a pale yellow solid, 145.8 mg, yield 43%. IR (KBr, cm-1): 3374 (w,
1
NH2), 3281 (w, NH2), 1741 (v, COO), 1152 (v, C=S). H NMR (DMSO-d6, ppm) : δ
0.86 (d, J = 6.4 Hz, 6H, C(CH3)2), 1.50 (d, J = 6.8 Hz, 3H, Ar-C-CH3), 1.83 (m, 1H,
Bn-CH), 2.44 (d, J = 7.2 Hz, 2H, Ar-CH2), 4.07 (q, J = 7.2 Hz, 1H, Ar-CH), 7.07 (d, J
= 8.4 Hz, 2H, Ar-H), 7.17 (d , J = 7.6 Hz, 2H, Ar-H), 7.21 (d, J = 8.0 Hz, 2H, Ar-H),
7.91 (d, J = 8.8 Hz, 2H, Ar-H), 9.54 (s, 1H , H of NH2), 9.92 (s, 1H, H of NH2). 13C
NMR (DMSO-d6, ppm): δ 19.2, 22.9, 30.3, 44.9, 121.6, 127.9, 129.5, 130.0, 137.8,
138.0, 140.8, 153.3, 173.3, 199.6. HRMS (m/z): calcd. for C20H23NO2SNa [M+Na]+:
364.1347; found 364.1337.
The synthesis of compound 3 was similar to that of compound 2, and 226 mg (1
mmol) of ADTOH was used in place of the above 4-hydroxythiobenzamide; the
mobile phase in the crude product column chromatography was adjusted to:
chloroform-methanol = 50:1 . Brownish yellow oily liquid, 180.0 mg. Yield: 44.1%.
IR (KBr, cm-1): 1753 (s, COO), 1136 (v, C=S). 1H NMR (DMSO-d6, ppm): δ 0.94 (d,
J = 6.0 Hz, 6H, C (CH3)2), 1.65 (d, J = 8.0 Hz, 3H, Ar-C-CH3), 1.90 (m, 1H, Ar-C-
CH), 2.50 (d, 2H, Ar-CH2), 3.98 ( q, J = 8.0 Hz, 1H, Ar-CH), 7.18 (m, 4H, Ar-H),
7.31 (m, 2H, Ar-H), 7.40 (s, 1H, 4H-1, 2-dithiole- 3-thione), 7.67 (d, J = 8.0 Hz, 2H,
Ar-H). 13C NMR (DMSO-d6, ppm): δ18.9, 22.7, 30.1, 44.7, 123.3, 127.7, 129.2, 129.3,
129.8, 136.3, 137.7, 140.6, 172.9, 173.0, 215.9. HR-MS (m/z): calcd. for
C22H22O2S3Na [M+Na]+: 437.0680; found 437.0701.
3. H2S release detection
3.1 Na2S standard curve plotting
A buffer solution of 20 mM pH 7.4 was first prepared for 1 L. Then the 120.2mg
Na2S•9H2O was dissolved in the 100mL volumetric bottle, and the 5 mM standard
solution of Na2S was prepared with the ph=7.4 phosphate buffer solution. 50, 100,
200, 400, 600, 800, 1000 μL of Na2S standard solution of 5 mM were moved into
50mL volumetric bottle respectively, and added phosphate buffer solution up to the
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scale line. The standard solution of Na2S with a concentration gradient of 5, 1D0O,I2: 100.,104309/,C8MD00611C
60, 80 and 150 mM were obtained. The gradient standard solution of 1mL was added
to methylene blue (MB) mixture. The blank control was a mixture of methylene blue
with phosphoric acid buffer solution of pH=7.4 of 1mL. 20 min later, the absorbance
of the solution was measured at 670nm by ultraviolet visible spectrophotometer
(lambda 25), and the standard curve between absorbance and Na2S concentration was
plotted.
3.2 H2S release detection of the compounds
75 μL of 40 mM compound solution was added to the volumetric flask, and the
promoter TECP (1.0 mM) or L-cysteine (1.0 mM) was added. Then 2.0 mL of the
above mixed solution was moved into a cuvette, and methylene blue (MB+) mixture
was added. 20min later, the absorbance was measured at a wavelength of 670 nm, and
the H2S concentration was calculated using a Na2S calibration curve. Finally, the
concentration versus time H2S release curve was plotted.
4. Toxicity of compounds after repeated administration
In the dose range set, the mice did not show significant toxicity in a single dose.
Therefore, we selected rats and studied the toxicity of the target molecule by multiple
administrations. The target compound was dissolved in 10% 1,3-propanediol to
prepare a standard solution of the compound, and then normal WKY rats were
administered by intraperitoneal injection according to the dose of 0.01 mol•kg-1, and
the control group was given by intraperitoneal injection. An equal volume of 10%
1,3-propanediol in blank (6 rats per group) was administered for 14 consecutive days
during which rat urine was collected by a rat metabolic cage. After the end of the
administration, blood was taken through the eyeball, and the liver, kidney and other
tissues of the rat were collected, and the tissue was immediately fixed with 4%
glutaraldehyde solution. After sectioning, the tissue was irradiated by transmission
electron microscopy, and the liver and kidney tissues of the rat were observed by HE
staining. The physiological condition determines whether the target compound has an
effect on the structure and function of the liver and kidney of the rat, and analyzes the
urine and blood of the rat by biochemical instruments to further evaluate the influence
of the target compound on the physiological condition of the rat.
5. Gastrointestinal protection
WKY rats were divided into two groups (3 rats each group): control group,
compound group. Rats were administrated the tested compound for 14 days, each
dose was 10 mg•kg-1, and the control was ibuprofen. The dose was 6.1 mg•kg-1. After
anesthesia with ether, the rats were sacrificed by cervical dislocation and supine. The
rats were fixed on the surgical board, with 70% alcohol disinfection of the abdominal
skin, and taken the midline incision of the abdomen, fully exposing the organs of the
abdominal cavity. The stomach was removed. A section of the complete small
intestine about 10cm was taken at the proximal end of the ileocecal. The contents of
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the stomach and small intestine were removed by refrigerating saline. The pDroOcI:e10d.1u0r3e9/C8MD00611C
is as follows: 1. the fresh materials were cut into small piece of 1.0 cm and
immediately put it into the fixing agent for fixing. 2. The fixed material was removed
from the water in the tissue by ethanol, and after being transparent to xylene, it was
immersed in the melted paraffin to be soaked and embedded. 3. A 5-10 μm slice was
cut with a microtome, attached to a glass slide, dewaxed, stained, hematoxylin and
eosin stained (HE stained). 4. Observed under an optical microscope.
6. Measurement of blood pressure in hypertensive rats
Male SHRs were randomly divided into 4 groups (6 rats in each group):
hypertension control group, captopril group (10 mg•kg-1), nifedipine group (10
mg•kg-1), compound group (25, 50, 100 mg•kg-1), male WKY rats were the normal
blood pressure control group. All compounds were administered by intraperitoneal
injection. The solvent was 0.5% (w/v) mixed solution of sodium carboxymethyl
cellulose and DMSO 3/1(v/v), and the SHRs were administrated at the dosage per day
was 2 mL•kg-1 at 8 am every day. The control group and the WKY group were
administrated equal amount of blank solvent. Blood pressures and heart rates of the
rats were measured before administration, and were measured every 30 minutes after
administration for 6 hours. Arterial systolic blood pressure (SBP), arterial diastolic
blood pressure (DBP) and heart rate were recorded before and after rat administration.
Measurement method: When the rat was quiet and awake, fixed it on the special
holder, and passed the rat tail through the pressure cuff to the root. Immediately after
the cuff, bound the pulse transducer to the rat tail, and the ventral tibial artery was in
good contact. When the rat pulse wave was stable, the measurement was started. The
inflation pressure was applied, the blocking pressure was 250 mmHg (the pulse wave
disappeared when the blocking pressure reached the systolic pressure level), and the
pressure was slowly decompressed after the blocking pressure was maintained for 6 s.
When the pulse wave appeared, the corresponding blood pressure at that time was the
arterial systolic pressure. The peak of the corresponding curve was the arterial
diastolic pressure, and the system automatically displayed the heart rate. Each group
was continuously measured 6 times, and the average value was taken.
7. Myocardial protective effect of SHRs
After the end of blood pressure measurement, the compound 1 group, the
compound 2 group and the hypertensive control group were sacrificed after anesthesia
with ether. The rat supine position was fixed on the surgical plate, 70% alcohol was
used to disinfect the abdominal skin, the midline incision was taken, the organs in the
abdominal cavity were fully exposed, the heart was removed and the heart was
removed, and the tissue was immediately fixed with 4% glutaraldehyde solution.
Then, the sections were irradiated by transmission electron microscopy, and the
physiological condition of the rat myocardium was observed by HE staining.
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8. Cardiomyocyte H9c2 protection
The rat cardiac H9c2 cells were purchased from the Cell Bank of the Chinese
Academy of Sciences (Shanghai,China) and cultured in DMEM supplemented with
10% FBS,100 U•mL-1 of penicillin, and 100 µgmL-1 of streptomycin at 37°C in a
humidified atmosphere of 5% CO2 .
The cardiomyocytes grown to log phase were collected and adjusted for cell
concentration. The cells were seeded at a cell density of 5 × 104 cells/mL in a 96-well
plate at 100 μL per well, and cultured at 37 ° C in a 5% CO2 incubator for 8 h to
adhere the cells. The cytotoxicity of each tested compound was firstly determined
before the compounds were evaluated. The tests were divided into blank group,
control group (acetaminophen, ibuprofen) and compound group (10μM, 40μM,
80μM). The blank group only added cells without drug or compound. The cell
survival rate was measured after the cells were incubated with the drug or compound
for 24 h. By which cardioprotective effects of tested compound was evaluated.
Conflict of Interest
The authors confirm that this article content has no conflict of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundations of
China (no. 21171079) and the Recruitment Program of Global Experts (1000 Talents Plan).
References
[1] Wang R. Two's company, three's a crowd: Can H2S be the third endogenous gaseous
transmitter [J]. FASEB J, 2002, 6: 1792-1798.
[2] Lu M, Liu YH, Goh HS, et al. Hydrogen sulfide inhibits plasma renin activity [J]. J Am Soc
Nephrol, 2010, 21: 993-1002.
[3] Huang C W, Feng W, Peh M T, et al. A novel slow-releasing hydrogen sulfide donor, FW1256,
exerts anti-inflammatory effects in mouse macrophages and in vivo [J]. Pharmacological research,
2016, 113: 533-546.
[4] Huang C, Wei F, Dymock B W, et al. 85: Identification of novel hydrogen sulfide (H2S)
donors with controlled release rates that elicit anti-inflammatory effects in RAW264.7 cells [J].
Cytokine, 2014, 70(1): 48.
[5] Li L, Rossoni G, Sparatore A, et al. Anti-inflammatory and gastrointestinal effects of a novel
diclofenac derivative[J]. Free Radical Biology and Medicine, 2007, 42(5): 706-719.
[6] Zhao W, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous
KATP channel opener [J]. The EMBO journal, 2001, 20(21): 6008-6016.
[7] Du J, Hui Y, Cheung Y, et al. The possible role of hydrogen sulfide as a smooth muscle cell
proliferation inhibitor in rat cultured cells [J]. Heart and vessels, 2004, 19(2): 75-80.
22

Page 23 of 24
MedChemComm
View Article Online
[8] Li L, Whiteman M, Guan Y Y, et al. Characterization of a Novel, Water-Soluble DHOyI:d1r0o.1g0e3n9/C8MD00611C
Sulfide–Releasing Molecule (GYY4137) [J]. Circulation, 2008, 117(18): 2351-2360.
[9] Li L, Salto-Tellez M, Tan C H, et al. GYY4137, a novel hydrogen sulfide-releasing molecule,
protects against endotoxic shock in the rat [J]. Free Radical Biology and Medicine, 2009, 47(1):
103-113.
[10] Lee Z W, Zhou J, Chen C S, et al. The slow-releasing hydrogen sulfide donor, GYY4137,
exhibits novel anti-cancer effects in vitro and in vivo [J]. PloS one, 2011, 6(6): e21077.
[11] Grambow E, Mueller-Graf F, Delyagina E, et al. Effect of the hydrogen sulfide donor
GYY4137 on platelet activation and microvascular thrombus formation in mice [J]. Platelets, 2014,
25(3): 166-174.
[12] Wallace J L, Caliendo G, Santagada V, et al. Gastrointestinal safety and anti-inflammatory
effects of a hydrogen sulfide–releasing diclofenac derivative in the rat [J]. Gastroenterology, 2007,
132(1): 261-271.
[13] Zhao Y, Wang H, Xian M. Cysteine-activated hydrogen sulfide (H2S) donors [J]. Journal of
the American Chemical Society, 2010, 133(1): 15-17.
[14] Laneuville O, Breuer D K, Dewitt D L, et al. Differential inhibition of human prostaglandin
endoperoxide H synthases-1 and-2 by nonsteroidal anti-inflammatory drugs [J]. Journal of
Pharmacology and Experimental Therapeutics, 1994, 271(2): 927-934.
[15] I. Tegeder, NSAIDs, Adverse Effects.” Encyclopedia of Pain, Springer, Berlin Heidelberg,
2007, 1463-1465.
[16] Sostres C, Gargallo C J, Arroyo M T, et al. Adverse effects of non-steroidal anti-
inflammatory drugs (NSAIDs, aspirin and coxibs) on upper gastrointestinal tract [J]. Best practice
& research Clinical gastroenterology, 2010, 24(2): 121-132.
[17] Fosslien E. Adverse effects of nonsteroidal anti-inflammatory drugs on the gastrointestinal
system [J]. Annals of Clinical & Laboratory Science, 1998, 28(2): 67-81.
[18] Wallace J L, Caliendo G, Santagada V, et al. Markedly reduced toxicity of a hydrogen
sulphide‐releasing derivative of naproxen (ATB‐346) [J]. British journal of pharmacology,
2010, 159(6): 1236-1246.
[19] Li M, Li J, Zhang T, et al. Syntheses, toxicities and anti-inflammation of H2S-donors based
on non-steroidal anti-inflammatory drugs[J]. European Journal of Medicinal Chemistry, 2017.
138,51-65.
[20] Bai Z, Zhang J, et al. Synthesis, toxicities and bio-activities of manganese complexes with
CO and H2S dual donors[J]. European Journal of Medicinal Chemistry,2018, 159,339-356.
[21] Hasegawa U, van der Vlies A J. Design and synthesis of polymeric hydrogen sulfide donors
[J]. Bioconjugate chemistry, 2014, 25(7): 1290-1300.
[22] Cao Z, Mendoza J, DeJesus A, et al. Synthesis and antitumor activity of aromatic
camptothecin esters[J]. International journal of molecular medicine, 2008, 21(4): 477-487.
[23] Dhaon M K, Olsen R K, Ramasamy K. Esterification of N-protected. alpha.-amino acids with
alcohol/carbodiimide/4-(dimethylamino) pyridine. Racemization of aspartic and glutamic acid
derivatives[J]. The Journal of Organic Chemistry, 1982, 47(10): 1962-1965.
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79x39mm (300 x 300 DPI)