Syntheses, toxicities and anti-inflammation of H2S-donors based on non-steroidal anti-inflammatory drugs

Article abstract of DOI:10.1016/j.ejmech.2017.06.012

Three series of H₂S donors based on NSAIDs were synthesized and characterized by ¹H-NMR, IR and ESI-HRMS. The H₂S-release abilities of all compounds were evaluated in the presence of TECP or cysteine. The results show all

Full text of DOI:10.1016/j.ejmech.2017.06.012

Accepted Manuscript
Syntheses, toxicities and anti-inflammation of H S-donors based on non-steroidal
2
anti-inflammatory drugs
Meng Li, Jili Li, Taofeng Zhang, Quanyi Zhao, Jie Cheng, Bin Liu, Zhen Wang, Libo
Zhao, Chenwei Wang
PII:
S0223-5234(17)30458-0
10.1016/j.ejmech.2017.06.012
EJMECH 9507
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 March 2017
Revised Date: 4 June 2017
Accepted Date: 6 June 2017
Please cite this article as: M. Li, J. Li, T. Zhang, Q. Zhao, J. Cheng, B. Liu, Z. Wang, L. Zhao, C. Wang,
Syntheses, toxicities and anti-inflammation of H S-donors based on non-steroidal anti-inflammatory
2
drugs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.012.
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ACCEPTED MANUSCRIPT
Syntheses, toxicities and anti-inflammation of H2S-donors
based on non-steroidal anti-inflammatory drugs
Meng Li, 1 Jili Li, 1 Taofeng Zhang,1 Quanyi Zhao,1* Jie Cheng, 2 Bin Liu, 3 Zhen Wang,1 Libo
Zhao,3 Chenwei Wang,3
(1 Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University; 2 GLP Lab
centre, School of Basic medicine of Lanzhou University；3 School of Stomatology of Lanzhou
University, Lanzhou 730000, China)
Abstract
Three series of CORMs H2S-donors based on NSAIDs were evaluated from
toxicities and anti-inflammatory activities, and the synergistic effect of H2S and
NSAIDs was preliminary explored.

ACCEPTED MANUSCRIPT
Syntheses, toxicities and anti-inflammation of H2S-donors
based on non-steroidal anti-inflammatory drugs
Meng Li, 1 Jili Li, 1 Taofeng Zhang,1 Quanyi Zhao,1 Jie Cheng, 2 Bin Liu, 3 Zhen Wang,1 Libo
Zhao,3 Chenwei Wang,3
(1 Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University; 2 GLP Lab
centre, School of Basic medicine of Lanzhou University；3 School of Stomatology of Lanzhou
University, Lanzhou 730000, China)
Abstract Three series of H2S donors based on NSAIDs were synthesized and
1
characterized by H-NMR, IR and ESI-HRMS. The H2S-release abilities of all
compounds were evaluated in the presence of TECP or cysteine. The results show all
compounds were fast H2S-releasers, and their half-lives were in range of 0–20 min.
Under the same condition, H2S released from compound 9 was more than any other
compounds. In cytotoxicity aspect, all compounds but 1 and 2 displayed much lower
toxicities to both LO2 and HepG2 cell lines, and the IC50 values of most compounds
were over 800µM. Compounds 1 and 2 had a stronger anti-proliferative activity to
both cell lines, but they displayed lower toxicities to LO2 than to HepG2. Based on
the cytotoxicity, the developmental toxicities of the compounds were assessed using
zebrafish embryos. The results show all tested compounds 2, 9 and 15 had effects on
the mortality, hatching rate and spontaneous movements of zebrafish embryos, and
caused embryos teratogenesis; and the compounds had dose-dependent toxicities to
both embryonic and larval zebrafish. In addition, all compounds had a better
anti-inflammatory activity. In the test of anti-inflammatory activities, the tested
compounds all reduced the levels of intracellular nitrite and pro-inflammatory
cytokines (TNF-α，COX-2)，increased the levels of anti- inflammatory cytokines
(IL-10, HO-1). All these suggest these H2S donors based on NSAIDs have a potential
to be a candidate medicine.
Keywords: H2S-donors; NSAIDs; toxicity; anti-inflammatory
1. Introduction
Corresponding author. Tel./fax: +86 9318915686
E-mail address: zhaoqy@lzu.edu.cn (Q. Zhao).
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Hydrogen sulfide (H2S) has been recognized as a new endogenous gaseous
transmitter like nitric oxide NO and CO [1-4]. Many tests showed H2S affected
several organ systems and biological processes in the human body, for example,
inhibiting the inflammatory response[5-7], significantly reducing the accumulation of
lipopolysaccharide induced neutrophils in the tissues and organs (liver and lung),
reducing tumor necrosis factor-α (TNF-α) and plasma interleukin-1 (IL-1β) levels,
and increasing plasma IL-10 levels [8]. In vascular, H2S is an important endogenous
vascular dilation factor as a gaseous opener of KATP channels [9], and inhibits the
proliferation of vascular smooth muscle through reducing the activity of mitogen
activated protein kinase [10]. H2S also has an effect of nerve regulation by regulating
the dynamic balance of neuronal Ca2+ and intracellular pH homeostasis [11, 12].
H2S is not used directly in clinic because of uncontrollable dose and high toxicity,
H2S donors is a potential substitute to provide suitable H2S. NaSH can release H2S,
and it was reported it inhibited aspirin-induced leukocyte adherence in rat mesenteric
venules and reduced leukocyte infiltration in a carrageenan-induced air pouch model
of inflammation [13]. However, NaSH relases H2S too fast to be used. Lawesson's
reagent is the first to use as H2S donor, which it spontaneously released H2S in
aqueous solution, but because its water solubility is poor to limit its further application
[14]. In 2008, based on the structure of Lawesson's reagent, Moore synthesized a novel
water-soluble H2S releaser GYY4137 [15], and studies showed GYY4137 has the
function of anti-shock, anti-tumor, anti-thrombosis and other effects [16-18]. After
that, more and more novel H2S donors appeared, such as dithiothione derivatives
ADTOH [19], thiobenzamide derivatives [20], S-acetylthosulfide compounds [21],
α-thioctic acid, thioamino acids compounds [22]. All these H2S donors can release H2S
under special condition.
Non-steroidal anti-inflammatory drugs (NSAIDs) have a very wide range of
applications all over the world, which are basically used for the treatment of pain,
inflammation and fever, the mechanism of action is the inhibition of the
cyclooxygenase enzymes [23]. However, current available NSAIDs have a lot of side
effects, like gastrointestinal hemorrhage, ulceration and kidney failure [24-26].
Considering H2S anti-inflammatory activity and the mechanism of action which is
completely different from NSAIDs, the researchers combined H2S donors and
NSAIDs together to get a kind of new molecules. In 2007, Wallace blended H2S donor
(ADTOH) with diclofenac to obtain a new compound ATB-337, and the
pharmacological tests showed ATB-337 not only decreased the adhesion of leukocyte,
but increased the activity of anti-inflammatory and reduced the side effects of
diclofenac [27]. In 2010, ATB-346 was synthesized by combination H2S donor with
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naproxen, the tested results showed ATB-346 inhibited COX-2 activity more
effectively than naproxen, and distinctly reduced the gastrointestinal injury [28].
As a strategy for drug design [29-31], we want to know other anti-inflammatory
drugs combined with H2S donors whether also improve their activities and decrease
their side effects. In addition, several special compounds which H2S donors modified
NSAIDs showed better activities and lower side effects, but we still do not know
whether they can be used in clinic [32]. After all, they are new active compounds, and
there are not enough data about preclinical studies. Especially, the toxicity is crucial
element to determinate them to investigate further or give up.
Therefore, in this paper, we synthesized 15 compounds based on three kinds of
exogenous H2S release donors: 4-hydroxythiobenzamide, ADTOH, α-thioctic acid.
And we assessed their cytotoxicities using both LO2 cells and HepG2 cells, and
genetic toxicities using zebrafish embryos. Meanwhile, their anti-inflammatory
activities were evaluated by measuring nitrite content and four cytokines in
macrophage RAW264.7. By which, we hope to obtain some information about real
H2S-releasing molecules fit for clinical application.
2. Results and discussion
2.1 Syntheses characterization and properties
Compounds 1-15 were synthesized by the reaction of three kinds of parent
molecules and aromatic organic acids A1-15 [33]. These acids have been used as
anti-inflammatory drugs in clinic. The structures and synthetic routes are shown in
schemes 1, 2 and 3. The reaction conditions are warm, and the yields are high. The
testing samples were obtained by the separation and purification using column
chromatography. Among three parent compounds, ADTOH was obtained by
treatment of 5-(4-methoxyphenyl)-3H-1, 2-dithiole-3-thione (ADT-OCH3) with
pyridine hydrochloride [34].
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Scheme 1 Synthetic route for the target compounds 1-8
The spectra of the compounds correspond with the expected. In IR spectra of all
the compounds, there has a strong absorption peak at in range of 1728-1766 cm-1,
which is the characteristic absorption of ester C=O. For compounds 1-8, a strong peak
appeared in 1138-1181cm-1 is the absorption bands for C=S; As for compounds 9-10,
the signals of C-S bond appeared 604-624cm-1; whereas the characteristic absorptions
of C=S in compounds 11-15 appeared in the range of 1120-1160cm-1. In the 1H-NMR
spectra of compounds 1-8, the signals of two protons in CONH2 were observed at
about 9.56 and 9.93ppm as two singlet peaks. For compound 9, the signal appeared at
2.14ppm as singlet belongs to the protons of CH3CO. Compound 10 has α, β –
unsaturated lactone structure, the signals of α-H and β-H appeared at 6.40ppm and
7.70ppm as doublet, respectively. For every compound among 11-15, a singlet at
about 7.40ppm was the signal of the proton in trithionum ring.
Scheme 2 Synthetic route for the target compounds 9-10
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Scheme 3 Synthetic route for the target compounds 10-15
Compounds 1-10 are pale yellow solid, compounds 11-15 are red solid or oil. All
the compounds are insoluble in water, and easily dissolve in organic solvents, like
DMSO, methanol, THF, and so on. All the compounds are stable in air at room
temperature, and do not change even for several weeks. In their solution, pH values
affect their stability; for compounds 11-15, they hydrolyze easily with pH increasing.
2.2 H2S releasing measurement
H2S-release capability of the donors was measured using the methylene blue
(MB+) method [35]. The MB+ method is based on the reaction of zinc acetate with
H2S to form zinc sulfide, which reacting with N, N-dimethyl-1,4-phenylenediamine
sulfate to generate methylene blue in the presence of ferric ions under acid medium
condition. The H2S is quantified by measuring the absorbance of the solution at
670nm.
Not like allyldisulfide, allyltrisulfide, thiobenzamides and GYY 4137 [36-38],
ADTOH and its derivatives were much slower H2S-releasing molecules, and even
some of them were not release H2S at all under the same conditions. But recently,
Hasegawa reported ADTOH released H2S in the presence of TCEP (a water-soluble
phosphine reducing agent) [39]. We found TECP also promoted compounds 1-8 to
release H2S. For compounds 9-10, TECP did not active α-thioctic acid to release H2S,
we chose cysteine as activator to measure H2S release abilities of them, which have
been reported in several papers [20]. Therefore, the ability of H2S release was
evaluated in a MB+-based aqueous assay in the presence of TECP or cysteine.
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Table 1 H2S-releasing half-lives and Cmax of the compounds at 40µM
Compound
t1/2(min
）
0.9
H2S Cmax
(µM)
106.1
23.5
20.9
Compound
t1/2 (min)
H2S Cmax
(µM)
Thiobenzamide a
α-thioctic b
118.1
14.1
4.6
28.4
46.0
37.9
13.4
20.1
1
2
9
10
ADTOH a
11
3.1
3.3
24.0
23.5
3.9
3.1
14.1
11.3
3
4
4.4
14.4
7.1
17.7
21.9
18.2
16.3
1.4
3.4
1.9
1.8
13.8
14.2
9.5
5
6
7
8
12
13
14
15
9.8
14.0
a, t1/2 and H2S concentration of their derivatives and themselves were measured in the
presence of TECP; b, t1/2 and H2S concentration of its derivatives and itself were measured in
the presence of cys.
In the process of measurement, a solution of the MB+ cocktail was prepared and
subsequently treated with a solution of TECP with the compounds at 37.5 °C. To
evaluate both the total amount of H2S released and the rate of H2S release, H2S
amount released from the compound was measured every 5 min, then described the
curve about H2S releasing amount vs time.
The concentrations of H2S which released from the tested compounds were
calculated according to the Na2S standard curve (SI-Fig.1). Fig. 1 is the representative
release curves about the H2S concentration to time. The testing results demonstrate all
compounds released H2S under the promotion of appropriate activator. They were all
fast H2S releasing agents, and the half-lives of most compounds were all less than 20
min (Table 1). However, interestingly, each releasing characteristics was not
distinctly different from each other. Fig. 1-a and Fig. 1-d are the release curves of
4-hydroxythiobenzamide derivatives, compounds 1 and 2, their release rates of H2S
accorded with the first-order kinetics. The H2S max amount released from compounds
1 and 2 were only 23.5µM, 20.9µM respectively, while the H2S amount from
4-hydroxythiobenzamide arrived at 106.1µM. Compared with the parent compound,
the derivatives released H2S amount decreasing sharply, and their half lives of H2S
release were longer than that of parent 4-hydroxythiobenzamide. Possibly, this is
caused by A1-8 structural fragments, because the big steric hindrance disturbs H2S
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release, and the larger the structure of the fragment, the stronger the inhibition of
H2S-releasing.
Compounds 9 and 10 are derivatives of α-thioctic acid. Seen from Fig.1-b,
compound 9 was also fast H2S releaser in the presence of L-cysteine, and it released
H2S up to 46.0µM while α-thioctic acid released 28.4µM under the same condition.
We inferred A9 fragment (acetaminophen) promoted H2S release of H2S in some
degree. In the curve of α-thioctic acid (seen SI-Fig. 2-b), there were two rapid release
periods, we thought the two sulfur atoms in the α-thioctic acid were transformed H2S
stepwise. The max amount of H2S-release from compound 10 was 37.9µM.
Compared with the parent compound α-thioctic acid, fragments A9 and A10
promoted H2S-release in some degree, the half-releasing time of H2S was shorter than
that of α-thioctic acid.
Compounds 11-15 belong to the derivatives of ADTOH. Fig. 1-f shows compound
15 was fast H2S releasing agents under the promotion of TECP, and its release rate of
H2S accorded with the first-order kinetics; the H2S max amount was 14.0µM, in
contrast, it was almost the same as that from ADTOH 14.1µM. Similarly, the max
amount of H2S from compounds 12 and 13 were 13.8µM and 14.2µM，respectively,
which shows the fragments A11-15 did not affect H2S release. Possibly, like ADTOH
parent compound, ADTOH derivatives do not release H2S until after lactones ring
hydrolysis.
Fig.1 H2S-releasing curve of compounds, a for compound 1, b for compound 9, c for compound
12, d for compound 2, e for compound 10, f for compound 15. Each bar represents the mean ± SD
of four independent experiments.
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2.3 Cytotoxicities
Compounds 1-15 were assessed for toxicities against LO2 cell lines (normal liver
cell) and HepG2 cell lines (liver tumor cell) using MTT assay. Stock solutions of the
tested compounds in DMSO (8000 µM) were prepared freshly immediately prior to
testing. Initially, cells were seeded in a 96 well plate about 105cells/well, then let
adhere overnight. The cells were then dosed with 12.5 µM, 25µM, 50µM, 100µM,
200µM, 400µM and 800µM tested compound respectively, and cell survival relative
to control was assessed after 24 h.
The results are shown in Table 2. Compounds 1-8 are the derivatives of
4-Hydroxythiobenzamide; and their IC50 values for HepG2 cells were over 1000µM
except those of compounds 1, 2 and 5; among eight compounds, compound 2 showed
the strongest toxicity to HepG2, and its IC50 value was 89.3µM; as for LO2 cell lines,
all eight compounds but 2 showed lower toxicities, and their IC50 values were over
800µM. In contrast, the toxicities of the compounds to HepG2 were slightly stronger
than those of LO2. As a whole, except for compound 2, all seven compounds showed
almost no toxicities to both LO2 and HepG2 cell lines.
For compounds 9-10 which are the derivatives of α-thioctic acid, their IC50 values
were above 900µM against both LO2 and HepG2 cell lines. Similarly, two
compounds displayed slightly stronger toxicities to HepG2 cell lines than to LO2 cell
lines. As we expected, compounds 11-15 also showed low toxicities, their IC50 values
to HepG2 were beyond 800µM and to LO2 over 300µM; and just like compounds 9
and 10, they displayed a bit stronger toxicities to LO2 cell lines than to HepG2 cell
lines also.
Table 2 IC50 values of all the compounds
Compound
IC50(µM)
Compound
IC50(µM)
HepG2
227.6
LO2
HepG2
965.8
LO2
1792.1
1
2
3
4
5
6
7
8
3614.9
9
89.3
221.0
1480.7
>800
10
11
12
13
14
15
916.8
2541.4
1042.7
889.3
543.8
536.5
337.2
1080.2
1683.1
410.0
1118.6
922.7
>800
1277.0
1076.7
3294.4
1058.1
1058.1
1073.4
>800
1091.1
1177.3
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In summary, the toxicities of the series compounds are low to both normal liver
cells LO2 and tumor cells HepG2, so that these compounds do not harm liver severely
in the range of tested dose.
2.4. Toxicity of zebrafish embryos
2.4.1 Compound-induced mortality and hatchability of zebrafish
To further evaluate the toxicities of the compounds, we chose zebrafish embryos
as test subjects; and in order to get the information as comprehensive as about the
toxicities on the development, the hatchability and the embryos alive were measured at
24, 48, 72, 96 and 120 hpf in the presence of tested compounds, respectively. As
shown in Fig. 2, DMF as solvent showed toxicity to zebrafish embryo in some degree.
For every tested compound, the rate of embryo alive had slight changes in the period
of 120hpf, and the percents of embryos alive decreased gradually with the
concentrations increasing; clearly it was dose-dependent. For compound 2, there was
almost no change in the mortality after treated embryo with 5µM and 10µM, and the
percents of embryos alive were about 60%. Similarly, compound 9 also decreased the
percent of embryos alive at the same concentration compared to the control group; the
percents were 51% and 41% after 120 hpf when its concentrations were 10 and 20 µM
respectively. In contrast, compound 9 has slightly stronger toxicity to zebrafish
embryos than compound 2. As for compound 15, its harmful effect on embryos was in
the middle of compound 2 and compound 9 (SI-Fig. 4-a) under the same condition.
The normal zebrafish embryos had a hatching period from 48 to 72 hpf. In the
process of hatching, some compounds may harm severely embryo development
because they inhibit key enzymes which promote embryo to develop. The hatching
rate of embryos is inversely proportional to the toxicity of compound. Seen from Fig.
3, for compounds 2 and 9, the hatching rates of embryos were in accord with the alive
embryos percents; at the low concentration (0.5 and 1.0µM), the hatching rate was no
significant change compared with that of the control (DMF) group, while in 5.0, 10
and 20 µM-treated groups, their hatchability of the embryos decreased steeply with
the concentration increasing. But for compound 15, when the concentration was not
up to 5µM until it showed inhibitory effects on the embryos to hatch (SI-Fig. 4-b).
This demonstrates these compounds have toxicities to zebrafish embryos on the
development in other aspect.
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Fig. 2 Effects of the compounds on zebrafish embryos alive. A: for compound 2. B: for compound
9. *P＜0.05 and **P＜0.01 compared to control (DMF). Data shown represents three independent
experiments. Values represent the mean ± SD of three replicates. DMF was used as a solvent
control and had no effect on experiment of zebrafish in the dilution used (1:1000) compared with
control.
Fig. 3 Hatch rate of compounds at 48 hpf (n=100). A: for compound 2. B: for compound 9. *P＜
0.05 and **P＜0.01 compared to control (DMF). Data shown represents three independent
experiments. Values represent the mean ± SD of three replicates. DMF was used as a solvent
control and had no effect on experiment of zebrafish in the dilution used (1:1000) compared with
control.
2.4.2 Effect of compound on zebrafish embryonic spontaneous movement
To evaluate the toxicities of the compounds on the nerves of zebrafish, the
embryonic spontaneous movement frequency (1min) was measured at 24 hpf. As
shown in Fig. 4, as a whole, at low concentration (0.5µM), the tested compounds did
not show significantly toxicity compared to the control (DMF); but at higher
concentration, the frequencies reduced with the compound concentration increasing.
For compound 2, the spontaneous movement frequencies of 1.0, 5.0, 10 and 20 µM
–treated group were about 11.0 times and lower than that of the control group
obviously (15.0 times). As for compound 9, it did not show toxicity till to 1.0 µM;
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however, the spontaneous movement frequencies of 5.0, 10 and 20 µM-treated groups
were lower than that of the control group (15.9 times), and they were 14.1, 13.5 and
12.2 times, respectively. This provided the evidence to prove the high toxicity of
higher doses of compound 9. Therefore, we draw a conclusion that the compounds
have embryonic toxicity in another aspect.
Fig. 4 Effects of the compounds on zebrafish spontaneous movement at 24 hpf (n=20);*P＜0.05
compared with control (DMF). Data shown represents three independent experiments. Values
represent the mean ± SD of three replicates. DMF was used as a solvent control and had no effect
on experiment of zebrafish in the dilution used (1:1000) compared with control.
2.4.3 Compound-induced malformation of embryos
The zebrafish were exposed to 0.5 µM tested compound from 24 to 96 hpf and the
malformation were observed at 96 hpf. We found at low concentration (0.5µM), the
tested compounds did not make the embryos malformed compared to the control
(DMF); but when their concentrations rose up, every compound displayed different
toxicity to embryos. Compound 2 induced remarkably zebrafish embryos
malformation. The malformation rate increased with the concentration rising, which
shows that the malformation rate of zebrafish is dependent on concentration. When its
concentration was up to 20µM, the malformation was approximately 5% (Fig. 5A).
For compounds 9 and 15, they also led to malformation of zebrafish, but their rates of
malformation only 3 and 2% at most in the range of 0.5-20 µM (Fig. 5B, SI-Fig. 4-c).
Several malformation patterns (including pericardial edema, vitelline cyst and bent
spine) were observed (Fig. 6). These observations showed that pericardial edema and
vitelline cyst were the typical malformations in the embryos induced by compound 2.
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Fig. 5 Malformation rate of zebrafish embryos exposed to compounds 2 and 9 (n=100). A: for
compound 2. B: for compound 9.*P＜0.05 and **P＜0.01 compared to control (DMF). Data
shown represents three independent experiments. Values represent the mean ± SD of three
replicates. Control (DMF): DMF was used as a solvent control and had no effect on experiment
of zebrafish in the dilution used (1:1000) compared with control.
Fig. 6 Malformation of zebrafish embryos exposed to 0.5 µM compound 2 (A) Normal larvae
and (B-D) abnormal larvae. Malformations are indicated by red arrows. PE, pericardial edema;
VC, vitelline cyst; BS, bent spine
2.5 Effects of compounds on zebrafish larval locomotor activity
The larval locomotor activities of the zebrafish during a 5 min period were
recorded at 144 hpf, to determine whether complexes 2 and 9 exposure had a
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persistent effect on larval behavior. In the visible light test, the total swimming
distances decreased concentration-dependently. Seen from Fig. 7, compared to the
controls (DMF), complex 2 and complex 9 at 10 µM treatments caused a significant
decrease in the total swimming distance, while at the low concentration (0.5 and
1.0µM), the total distance have no significant differences with the control group. In
contrast, compound 9 has stronger effect to the larval locomotor activities of the
zebrafish than compound 2. This trend is similar to embryo of the zebrafish for both
compounds.
Fig. 7 Effects of the compounds on zebrafish total distance of movement. A: for compound 2.
B: for compound 9.*P＜0.05 ** P＜0.01 compared with control (DMF). Data shown
represents three independent experiments. Values represent the mean ± SD of three replicates.
Control (DMF): DMF was used as a solvent control and had no effect on experiment of
zebrafish in the dilution used (1:1000) compared with control.
Therefore, on the basis of testing data, we draw a conclusion that the compounds
have toxicities to the zebrafish embryo and the larval, especially their concentrations
are higher. The compounds will not be used at higher concentration, and not fit for the
female in pregnancy even at low concentration.
2.6. Anti-inflammatory activities
2.6.1 Cell viability
In the process of anti-inflammatory test, we chose to use macrophages RAW264.7.
In order to obtain more accurate results of anti-inflammatory experiments, eliminate
the effects of cell death which the tested compounds give rise to on the results as can
as possible, macrophages were exposed for 24 h to the compounds (10, 50, and
100µM) in the presence or absence of LPS (1µg mL−1), then the cell viability was
measured using MTT method. The results demonstrate compound 9 and compound 15
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had much lower toxicities to macrophages and did not kill them at all (Fig. 8), and for
the other tested compounds (compounds 1, 2 and 12), macrophages viability rates was
up to 70–110%, this suggests the compounds at the range of 10-100µM are acceptable
and effective in anti-inflammatory tests.
Fig. 8 Effects of the compounds on cell viability. Measurement in the media of murine RAW264.7
macrophages exposed for 24 h to 10, 50 and 100 µM of each compound in the presence or absence
of 1 µg mL−1 LPS. a for compound 1, b for 2, c for 9,d for 12,e for 15. Each bar represents the
mean ± SD of four independent experiments.
2.6.2 Effect of the compounds on LPS-mediated nitrite production
NO is one of the inflammatory mediators. Excessive NO up-regulates inducible
NO synthase (iNOS), which is a critical step in the initiation and propagation of the
inflammatory response [40, 41]. NO is oxidated easily to nitrite by superoxide ions in
vivo. H2S not only can quench peroxynitrite but also interacts chemically with NO to
yield a novel nitrosothiol [42, 43]. The anti-inflammatory activity of H2S donors can
be assessed by measurement of the amount of nitrite.
LPS can activate iNOS to increase NO level. In order to make the effect more
pronounced, LPS-activated macrophages were used to evaluate the anti-inflammatory
activities of the compounds. The testing results show tested compounds all decreased
nitrite levels in the presence of LPS. As shown in Fig. 9, they attenuated LPS-induced
nitrite production in a concentration-dependent manner. Among five compounds,
compound 9 displayed the strongest activity to inhibit iNOS; compared with the
control, it inhibited approximately 60% nitrite produce at 50µM. From the H2S
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releasing curves, we can see that compound 9 released H2S most among five
compounds. Maybe, this is one of the reasons why compound 9 showed better
anti-inflammatory activity than the others.
Fig.9 Effect of the compounds on nitrite. Measurement in the media of murine RAW 264.7
macrophages exposed to 10, 50 and 100 µM of each compound for 24 h. a for compound1, b for 2,
c for 9, d for 12, e for 15. Each bar represents the mean ± SD of four independent experiments. # p
< 0.05, ##p < 0.01 vs. untreated cells. *p < 0.05, **p < 0.01, vs. LPS-treated cells
2.6.3 Effect of the compounds on LPS-mediated TNF-α and IL-10
TNF-α is a tumor necrosis factor, and it promotes T cells to produce a variety of
inflammatory factors, thereby promoting the occurrence of inflammatory response.
Studies have found H2S significantly can reduce LPS-induced neutrophils in the
tissues and organs (liver and lung), decrease the level of TNF-α and increase IL-10
levels in plasma, thereby inhibiting inflammatory mediators [8]. Therefore, the level
of TNF-α in cell was used as an important index to assess anti-inflammatory activity
of H2S donors.
For this reason, we measured the levels of TNF-α in LPS-induced macrophages
RAW264.7. As shown in Fig.10, all tested compounds significantly reduced levels of
TNF-α. Among them, compounds 12 and 15 displayed stronger activities than
compound 9, the levels of TNF-α decreased to 5-20% of the control when the cells
treated with both compounds at 100µM; in contrast, compound 9 only reached
approximately 50% under the same condition. We know compound 9 released more
H2S than compounds 12 and 15 at 40µM, why its activity is lower than the two
compounds? In fact, there are many factors to impair the effects of action for the
compounds. Compound 9 has longer H2S-releasing half-life than compounds 12 and
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15, which means compound 9 takes effects more slowly than both of them; possibly,
the concentrations were up to 100µM, the effects were more distinct. In addition,
TNF-α possibly has a different sensibility to all kinds H2S donors. As a whole, the
compounds reduce TNF-α level in dose-dependent manner. This further shows H2S
plays an important role in anti-inflammatory process.
Fig.10 Effect of the compounds on TNF-α. Measurement in the media of murine RAW 264.7
macrophages exposed to 10, 50 and 100 µM of each compound for 24 h. a for compound 1, b for
2, c for 9, d for 12, e for 15. Each bar represents the mean ± SD of four independent experiments.
# p < 0.05, ##p < 0.01 vs. untreated cells; *p < 0.05, **p < 0.01, vs. LPS-treated cells
Fig.11 Effect of the compounds on interleukin-10 (IL-10). Measurement in the media of murine
RAW 264.7 macrophages exposed to 10, 50 and 100 µM of each compound for 24 h. a for
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compound 1, b for 2, c for 9, d for 12,e for 15. Each bar represents the mean ± SD of four
independent experiments. # p < 0.05, ## p < 0.01 vs. untreated cells. *p < 0.05, **p < 0.01, vs.
LPS-treated cells
Like TNF-α, IL-10 also regulates the inflammatory response; however, it is an
anti-inflammatory factor. The level of IL-10 in cell can reflect the anti-inflammatory
activity of compound. We found the tested compounds all significantly increased the
level of IL-10 in both the presence and absence of LPS, as shown in Fig. 11. Among
the compounds, compound 9 displayed stronger activity to increase IL-10 level than
the others. The levels of IL-10 in cells treated with compound 9 at 100µM are as 3
times as the controls of blank and with LPS. We infer that more H2S released from
compound 9 maybe is one of the reasons why it showed better activity to increase the
level of IL-10.
2.6.4 Effects of the compounds on Heme-oxygenase-1(HO-1)
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the heme degradation
processes, and it catalyzes heme to transform into biliverdin, iron ion and CO. CO and
biliverdin were shown to mediate many beneficial effects, including modulation of
inflammation. And several studies have demonstrated HO-1 has anti-inflammatory
property in itself in vivo [44, 45]. The level of HO-1 expression reflects
anti-inflammatory ability of the cell in some degree. Recently, the scientists found
that H2S donor, like NaHS, enhanced hypoxia-induced HO-1 expression in smooth
muscle cells of small pulmonary arteries [46].
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Fig.12 Effect of the compounds on Heme-oxygenase-1(HO-1). Measurement in the media of
murine RAW 264.7 macrophages exposed for 24 h to 10, 50 and 100 µM of each compound after
1 µg mL−1 LPS administration for 4h. a for compound 1, b for 2, c for 9, d for 12, e for 15. Each
bar represents the mean ± SD of four independent experiments. # p < 0.05, ##p < 0.01, vs.
untreated cells; *p < 0.05, **p < 0.01, vs. LPS-treated cells.
In order to evaluate the effects of the compounds on HO-1, we treated RAW264.7
macrophages with the compounds at set concentrations, and examined HO-1
expression level. As shown in Fig.12, the tested compounds all increased HO-1 levels
in a concentration dependent manner. When the tested compounds arrived at 100µM,
the level of HO-1 in cells rose up to 2 times as high as the controls; for compounds 2
and 12, the expression levels were beyond 3 times as much as the controls. This
suggests the more H2S released from the compound, the more HO-1 expression.
2.6.5 Effects of the compounds on COX-2
The inducible cyclooxygenase enzymes COX-2 is key enzyme which catalyzing
the biotransformation of arachidonic acid to the prostaglandin E, the latter is one of
inflammatory mediators. Traditional NSAIDs produce anti-inflammatory effect by
inhibiting the activity of COX-2. Because the compounds contain anti-inflammatory
agent structural fragments, and link them with ester bond, these compounds must be
had anti-COX-2 activities. In order to test these compounds whether have better
activities against COX-2, we examined the effects of the compounds on COX-2
expression in RAW264.7 macrophages. As shown in Fig.13, all tested compounds
displayed strong activities to COX-2. Among the tested compounds, compound 12
displayed stronger activity to decrease COX-2 level than the others; when it was 100
µM, COX-2 level decreased to approximately 40% of the controls.
2.7 Conclusion
To avoid the unnecessary loss, early failing and early eliminating is the best way
to seek a new drug. In this paper, to establish the drug-like properties of all the H2S
donors, we evaluated all compounds from several aspects including H2S-releasing
ability, toxicity and anti-inflammatory activities.
As a result, all the compounds were fast H2S releasing agents, their half-lives
were in range of 0–20 min. Among them, compounds 1 and 2 had higher toxicity to
HepG2 cells (IC50 89.3 µM for compound 2), the others displayed much lower
toxicities to both LO2 cells and HepG2 cells, their IC50 values were above 800µM and
300µM respectively. After zebrafish embryos were exposed to the tested compounds,
the survival rate, hatchability and spontaneous movement all decreased with the
concentration increasing; the survival rate and hatchability decreased in dose
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dependent manner. Moreover, tested compounds caused a dose-dependent
malformation of zebrafish. On the other hand, the tested compounds had strong
anti-inflammatory activities, compound 9 displayed better anti-inflammatory activity
than the others. Under the same condition, compound 9 released more H2S than
compounds 2 and 15, which is corresponding with their activities.
Fig.13 Effect of the compounds on cyclooxygenase-2 (COX-2). Measurement in the media of
murine RAW 264.7 macrophages exposed for 24 h to 10, 50 and 100 µM of each compound after
1 µg mL−1 LPS administration for 4h. a for compound 1, b for 2, c for 9, d for 12, e for 15. Each
bar represents the mean ± SD of four independent experiments. # p < 0.05, ## p < 0.01, vs.
untreated cells. * p < 0.05, **p < 0.01, vs. LPS-treated cells.
The compounds not only decreased PGE level (inflammatory mediator) by
inhibiting COX-2 activity, but decreased NO level (another inflammatory mediator)
through inhibiting iNOS. Meanwhile compound 9 also took effects by TNF-α and
IL-10 pathway which regulated by H2S. This demonstrates that the anti-inflammatory
action of compound is a result of synergistic effect involving in several pathways.
Furthermore, the toxicities of these compounds are very low, and H2S released
from the compounds played a role during the anti-inflammation, such as inhibiting
iNOS, decreasing TNF-α levels and increasing HO-1 and IL-10 express levels, and so
on. All suggest the compounds maybe are potential anti-inflammatory drugs for
substitution for classical NSAIDs.
3. Experimental
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3.1 Reagents and instruments
All reactions were carried under nitrogen atmosphere. Solvents for reactions were
degassed and distilled from the proper drying agents. Column chromatography was
carried out using 200–300 mesh silica gel; RAW264.7macrophages lines, HepG2 cell
lines and LO2 cell lines from cell resources Center for Shanghai Life Science Institute
of Chinese Academy of Sciences (China), zebrafish from Center for Shanghai Life
Science Institute of Chinese Academy of Sciences (China).
IR spectra were recorded on a Nicolet NEXUS 360 spectrophotometer, and NMR
spectra on a BrukerAM-400 MHz spectrometer. A Lambda 25 UV–Visible
spectrophotometer and a Maxis-4G TOF Mass spectrometer (ESI) were used.
3.2 Experimental protocol
3.2.1 H2S Measurement
A 5mM solution of Na2S in sodium phosphate buffer (20 mM, pH 7.4) was
prepared (Na2S. 9H2O, 120.20 mg in 100 mL volumetric flask) and used as the stock
solution. Aliquots of 50, 100, 200, 400, 600, 800, 1000, 1500 µL of the Na2S stock
solution were added into a 50 mL volumetric flask and dissolved in sodium phosphate
buffer to obtain the standard solutions in 5, 10, 20, 40, 60, 80, 100, 150 µM,
respectively. 1 ml aliquot of the respective solution was reacted with the methylene
blue (MB+) cocktail: 30mM FeCl3 (200 µL) in 1.2 M HCl, 20 mM of N,
N-dimethyl-1, 4-phenylenediamine sulfate (200 µL) in 7.2 M HCl, 1%w/v of
Zn(OAc)2 (100 µL) in H2O at room temperature for at least 15 min (each reaction was
performed in triplicate). The absorbance of methylene blue was measured at λmax=670
nm in UV-Vis spectrophotometer (Lambda 25). The Na2S calibration curve was
obtained.
The reaction was initiated by adding 75 µL of stock solution of the donor (40 mM,
in THF) into pH 7.4 phosphate buffer (30 mL) containing accelerator for TECP (1.0
mM) or L-Cysteine (1.0 mM). Then 2.0 mL of reaction aliquots were periodically
taken and transferred to colorimetric cuvette containing zinc acetate (1% w/v, 200 µL)
and N,N-dimethyl-1,4-phenylenediamine sulfate (20 mM, 400 µL) in 7.2 M HCl and
ferric chloride (30 mM, 400 µL) in 1.2 M HCl. The absorbance (670 nm) of the
resulted solution was determined 15 min thereafter using an UV−Vis spectrometer
(Lambda 25). The H2S concentration of each sample was calculated against a
calibration curve of Na2S. The H2S releasing curve was obtained by plotting H2S
concentration versus time.
3.2.2 Cytotoxic assay
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The LO2 cells and HepG2 cells were cultivated at 37 , 10% CO2, 100%
humidity in RPMI1640 medium, enriched with glucose and supplemented with 10%
fetal bovine serum, non-essential amino acids, antibiotics (penicillin/streptomycin)
and antifungals. Typically, 0.08mmol of the compound was dissolved in 0.5 mL of
DMSO, which after homogenization was injected over 10mL of culture medium with
vigorous stirring at 55 °C. Growth inhibitory effect toward LO2 (or HepG2) cells line
was determined by means of MTT colorimetric assay. The cells were seeded into
96-well plates at 6×103 cells/well and a certain volume of the above solution was
added. Culture medium was added until completion of 0.2 mL and the final
concentration of the compound in each well was 12.5–800 µM. Triplicate cultures
were established for all compounds and for the control. The mixture was incubated at
37 °C in a humid atmosphere containing 10% CO2 for 24h. Cell viability was
quantified after 24h by the MTT method. Descriptive data were expressed as mean±
standard deviation and values IC50 were carried out using SPSS 20.0 software.
3.2.3 Zebrafish toxicity test
Adult zebrafish of the wild-type strain (AB) were raised and maintained at 28±
1 °C with a 14h light/10h dark photoperiod (lights on at 8:00) in a recirculation
system. The fish water supplied to the system was filtered by reverse osmosis
(pH6.5–7.5), and instant ocean salt was added to the water, to raise the conductivity to
450–500 µs/cm. The zebrafish were fed twice daily with decapsulated, freshly
hatched brine shrimps (Brine Shrimp Direct, USA) according to the description of
Zhou [47]. The zebrafish embryos were obtained from spawning adults in tanks
overnight with a sex ratio of 1:1. The embryos were collected within 1h after the light
was switched on and washed using standard zebrafish E3culture medium. The
zebrafish use and handling protocol conformed to the Guide for the Care and Use of
Laboratory Animals and was approved by the Institutional Animal Use and Care
Committee (IAUCC) of the Lanzhou University. At 4 hour post-fertilization (hpf), the
embryos were examined under a dissecting light microscope, and the specimens that
had developed normally were selected for the further experiments according to the
description of Kimmel [48].
The zebrafish embryos were exposed to compound for 24, 48, 72, 96, 120, 144
and 168 hpf and then assessed for toxicity. The toxicological end points were
determined based on previous reports in the literature [49]. Each group consisted of
100 embryos randomly divided into three replicate groups. The embryos were kept in
sterile 96-well plates with one embryo per well containing 200 µL of the solution. The
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plates were covered with sealing film to prevent evaporation and the mortality of the
zebrafish was recorded at 24, 48, 72, 96, 120, 144 and 168 hpf.
Normal embryos were exposed to control vehicle and complex from 4 to 24 hpf.
Ten embryos were selected randomly from control and experimental groups. The
zebrafish embryonic spontaneous movement (5min) was recorded using a
stereoscopic dissecting microscope and Media Cruiser recording software (Canopus
Corporation, Kobe, Japan). Data were analyzed using the Etho Vision XT10.0
software (Noldus Information Technology, Wageningen, Netherlands). When normal
embryos were exposed to the control vehicle and compound from 4 to 72 hpf, the
hatch rate was measured at 48 hpf. The normal embryos (4 hpf) were exposed to the
control vehicle compound for 96 hpf, and then the malformation of the zebrafish were
observed using a stereoscopic dissecting microscope.
3.2.4 Anti‑inflammatory actions assays
Murine RAW264.7 monocyte macrophages were cultured in Dulbecco's modified
Eagle's medium (DMEM) with high-glucose from HyClone which is supplemented
with 2mM glutamine, antibiotics (100 U/ml penicillin A and 100 U/ml streptomycin),
and 10% heat-inactivated fetal bovine serum and maintained in a 37 °C humidified
incubator containing 5% CO2.
Macrophages were exposed for 24 h to LPS (1µg mL−1) in the presence or absence
of compound 1, 2, 9, 12 and 15 (10, 50 and 100 µM), cell viability, nitrite levels and
TNF-α, HO-1, IL-10, and COX-2 protein expression were determined at the end of
the incubation. In each experiment, compound 1, 2, 9, 12 and 15 (10, 50 and 100 µM)
were added to cells 24 h after incubation with LPS for 4 h.
3.2.5 Cell viability
Cell viability was determined using MTT method and it was previously reported
[50].The assay is based on the cleavage of the yellow tetrazolium salt, MTT, to form a
soluble blue formazan product by mitochondrial enzymes, and the amount of
formazan produced is directly proportional to the number of living, not dead cells, it
has become a very popular technique for quantification of viable cells in culture.
Briefly, after the cells were exposed for 24h in 96-well plate, MTT was added to each
well (20µL per well) for 4h, after that discard the culture, and then DMSO was added
to each well (100µL per well), the plate was shaken for 10 min to make sure all of
blue formazan were dissolved in DMSO, which is calculated as the absorbance in
570nm on a microplate reader and expressed as a percentage of control.
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3.2.6 Assay for nitrite levels
Nitrite levels were determined using the Griess method as previously described
[51]. The measurement of this parameter is widely accepted as indicative of NO
production. Briefly, the medium from treated cells was removed and placed into a
96-well plate (50µL per well). The Griess reagent Ⅰ and Ⅱ were added to each
well to begin the reaction, the plate was shaken for 10 min and the absorbance read at
570 nm on a microplate reader. The nitrite level in each sample was calculated from a
standard curve generated with sodium nitrite (0–100 µM in cell culture medium).
3.2.7 Bicinchoninic acid (BCA) Protein Assay
In order to exclude the influence of the total amount of protein on the
determination of the following cytokine levels, protein quantification of each protein
was carried out using BCA protein Assay. Briefly, samples were removed and placed
into a 96-well plate (20 µL per well). BCA reagent A and reagent B were formulated
as working solutions, which was added to each well (200 µL per well) and left to react
for 0.5 h in 37 ℃. And then the plate was shaken for 10 min and the absorbance read
at 562 nm on a microplate reader. The concentration of protein in each sample was
calculated from a standard curve generated with standard solution.
3.2.8 Determination of four cytokines (TNF-α, HO-1, IL-10, and COX-2) levels
The level of four cytokines present in each sample was determined using a
commercially available kit from Shanghai MLBIO Biotechnology Co.Ltd. The assay
was performed according to the manufacturers’ instructions. Briefly, cell were
collected from 6-well plate immediately after lysed by RIPA Lysis Buffer and spun at
12,000×g for 10 min to remove any particulates. Each protein solution was measured
by BCA Protein Assay Kit. The protein solution for concentration of 0.1mg ml-1 was
added to a 96-well plate precoated with affinity-purified polyclonal antibodies
specific for the mouse of our cytokines. An enzyme-linked polyclonal antibody
specific for the mouse of our cytokines was added to the wells and left to react for 0.5
h followed by a final wash to remove any unbound antibody-enzyme reagent. The
intensity of the color detected at 450 nm was measured after addition of chromogen
solution A, B and stop solution and was proportional to the amount of four cytokines
produced. The four cytokines levels which in each sample was calculated from a
standard curve generated with standard solution.
3.3 Syntheses
3.3.1 Preparation of compounds 1-8
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Compound 1: To the solution of (naproxen, 230mg 1.0mmol) in 8mL of
acetonitrile, DMAP (18mg, 0.15mmol) and EDCI (190mg, 1.0mmol) were added with
stirring at room temperature for 0.5h. To the reaction mixture
4-hydroxythiobenzamide (153 mg, 1.0 mmol) was added and stirred for 4 h at room
temperature. After filtration, the filtrate was evaporated under reduced pressure to
remove the solvent. The oily residue thus obtained was dissolved in trichloromethane;
the organic layer was washed with brine, with NaCl 5%, and then dried on anhydrous
Na2SO4, filtered and the solvent evaporated. The crude product was chromatographed
on a silica gel (CHCl3/CH3OH 30:1), and 148.8 mg pale yellow solid was obtained.
Yield: 42.0%. IR (KBr, cm−1): 3398 (w, NH2), 3298 (w, NH2), 1755 (v, COO), 1214
(s, COC), 1170 (v, C=S). 1H NMR (DMSO-d6, TMS，ppm): δ 1.59 (d, J=7.2Hz, 3H,
Ar-C-CH3), 3.88 (s, 3H, O-CH3), 4.25 (q, J=7.2Hz, 1H, Ar-CH), 7.09 (d, J=8.8Hz, 2H,
Ar-H), 7.18 (dd, J=8.8Hz, 2.4Hz, 1H, Ar-H), 7.33 (s, 1H, Ar-H), 7.52 (d, J=8.4Hz, 1H,
Ar-H), 7.85 (d, J=4.4Hz, 2H, Ar-H), 7.87 (s, 1H, Ar-H), 7.90 (d, J=8.4Hz, 2H, Ar-H),
13
9.53 (s, 1H, NH), 9.91 (s, 1H, NH). C NMR (DMSO-d6, TMS, ppm): δ18.9, 45.0,
55.7, 106.3, 119.4, 121.5, 126.4, 126.7, 127.8, 129.0, 129.3, 129.7, 133.9, 135.6,
153.2, 157.8, 173.1. ESI-HRMS (m/z): calcd. for C21H19NO3SNa [M+Na]+: 388.0983;
found 388.0979.
The preparation and workup of compounds 2-8 were similar to compound 1.
Compound 2: a pale yellow solid 145.8 mg. Yield: 43.0%. IR (KBr, cm−1): 3374 (w,
1
NH2), 3281 (w, NH2), 1741 (v, COO), 1152 (v, C=S). H NMR (DMSO-d6, TMS,
ppm): δ 0.86 (d, J=6.4Hz, 6H, C(CH3)2), 1.50 (d, J=6.8Hz, 3H, Ar-C-CH3), 1.83 (m,
1H, Bn-CH), 2.44 (d, J=7.2Hz, 2H, Ar-CH2), 4.07 (q, J=7.2Hz, 1H, Ar-CH), 7.07 (d,
J=8.4Hz, 2H, Ar-H), 7.17 (d, J=7.6Hz, 2H, Ar-H), 7.21 (d, J=8.0Hz, 2H, Ar-H), 7.91
13
(d, J=8.8Hz, 2H, Ar-H), 9.54 (s, 1H, NH), 9.92 (s, 1H, NH). C NMR (DMSO-d6,
TMS, ppm): δ19.2, 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. ESI-HRMS (m/z): calcd. for C20H23NO2SNa [M+Na]+: 364.1347;
found 364.1337.
Compound 3: pale yellow solid 307.1 mg. Yield: 62.5%. IR (KBr, cm−1): 3371 (w,
1
NH2), 3267 (w, NH2), 1740 (v, COO), 1328 (m, S=O), 1168 (v, C=S). H NMR
(DMSO-d6, TMS, ppm): δ 2.22 (d, J=24.0Hz, 3H, Cp-CH3), 2.82 (d, J=12.0Hz, 3H,
O=S-CH3), 4.03 (s, 2H, COO-CH2), 6.76 (m, 1H, C=C-H), 7.21 (m, 4H, Ar-H), 7.45
(s, 1H, Ar-H), 7.79 (m, 4H, Ar-H), 7.93 (m, 2H, Ar-H), 9.52 (s, 1H, NH), 9.89 (s, 1H,
NH). 13C NMR (DMSO-d6, TMS, ppm): δ10.9, 31.2, 43.6, 106.6, 106.8, 111.0, 121.6,
123.7, 123.8, 124.4, 129.3, 129.8, 130.4, 130.8, 131.6, 137.7, 138.9, 140.6, 146.9,
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153.1, 169.2, 199.5. ESI-HRMS (m/z): calcd. for C27H23FNO3S2 [M+H]+: 492.1103;
found 492.1365.
Compound 4: pale yellow solid 130.9 mg. Yield: 34.5%. IR (KBr, cm−1): 3379 (w,
1
NH2), 3281 (w, NH2), 1748 (v, COO), 1276 (s, C-F), 1163 (v, C=S). H NMR
(CDCl3, TMS, ppm): δ 1.58 (d, J=8.0Hz, 3H, CH-CH3), 4.23 (q, J=8.0Hz, 1H,
Ar-CH), 7.15 (d, J=8.0Hz, 2H, Ar-H), 7.37 (m, 3H, Ar-H), 7.48 (m, 2H, Ar-H), 7.55
(m, 3H, Ar-H), 7.93 (d, 2H, J=8.0Hz, Ar-H), 9.53 (s, 1H, NH), 9.90 (s, 1H, NH). 13C
NMR (DMSO-d6, TMS, ppm): δ19.1, 44.7, 115.9, 116.2, 121.7, 124.8, 128.6, 129.3,
129.4, 129.5, 131.7, 135.8, 137.7, 142.6, 153.7, 158.3, 163.5, 173.1, 199.6.
ESI-HRMS (m/z): calcd. for C23H20NO3S [M+H]+: 380.1042; found 380.1131.
Compound 5: pale yellow solid 51.5 mg. Yield: 18.2%. IR (KBr, cm−1): 3359 (w,
1
NH2), 3279 (w, NH2), 1729 (v, COO), 1630 (v, C=C), 1139 (v, C=S). H NMR
(DMSO-d6, TMS, ppm): δ 6.91 (d, J=16Hz, 1H, C=O-CH), 7.27 (d, J=8.8Hz, 2H,
Ar-H), 7.48 (d, J=4.0Hz, 3H, Ar-H), 7.82 (d, J=4,0Hz, 2H, Ar-H), 7.89 (d, J=7.2Hz,
13
1H, Ar-CH), 7.96 (d, J=8.4Hz, 2H, Ar-H), 9.54 (s, 1H, NH), 9.90 (s, 1H, NH). C
NMR (DMSO-d6, TMS, ppm): δ117.4, 121.8, 129.2, 129.3, 129.5, 131.5, 134.3,
137.5, 147.3, 153.2, 165.2, 199.5. ESI-HRMS (m/z): calcd. for C16H13NO2SNa
[M+Na]+: 306.0565; found 306.0556.
Compound 6: pale yellow solid 73.4 mg. Yield: 25.2%. IR (KBr, cm−1): 3321 (w,
1
NH2), 3275 (w, NH2), 1767 (v, COO), 1171 (v, C=S). H NMR (DMSO-d6, TMS,
ppm): δ 7.36 (d, J=8.0Hz, 2H, Ar-H), 7.70 (d, J=8.0Hz, 2H, Ar-H), 7.99 (d, J=8.0Hz,
2H, Ar-H), 8.15 (d, J=8Hz, 2H, Ar-H), 9.57 (s, 1H, NH), 9.94 (s, 1H, NH). 13C NMR
(DMSO-d6, TMS, ppm): δ121.0, 126.7, 127.2, 129.9, 131.7, 132.0, 133.8, 135.1,
147.1, 153.5, 167.2, 199.6. ESI-HRMS (m/z): calcd. for C14H10ClNO2SNa [M+Na]+:
314.0018; found 314.0044.
Compound 7: pale yellow solid 103.4 mg. Yield: 35.4%. IR (KBr, cm−1): 3387 (w,
1
NH2), 3281 (w, NH2), 1740 (v, COO), 1168 (v, C=S). H NMR (DMSO-d6, TMS,
ppm): δ 7.38 (d, J=8.0Hz, 2H, Ar-H), 7.57 (m, 1H, Ar-H), 7.70 (d, J=8.0Hz, 2H,
Ar-H), 8.00 (d, J=8Hz, 2H, Ar-H), 8.12 (d, J=8Hz, 1H, Ar-H), 9.58 (s, 1H, NH), 9.95
13
(s, 1H, NH). C NMR (DMSO-d6, TMS, ppm): δ120.8, 127.0, 128.3, 128.5, 132.1,
139.3, 146.9, 153.4, 165.7, 199.5. ESI-HRMS (m/z): calcd. for C14H10ClNO2SNa
[M+Na]+: 314.0018; found 314.0046.
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Compound 8: pale yellow oil 137.2 mg. Yield: 36.2%. IR (KBr, cm−1): 3388 (w, NH2),
3284 (w, NH2), 1751 (v, COO), 1660 (s, CO), 1181 (v, C=S). 1H NMR (CDCl3, TMS,
ppm): δ 1.57 (d, J=8.0Hz, 3H, CH-CH3), 4.30 (q, J=8.0Hz, 1H, Ar-CH), 7.12 (d,
J=8.0Hz, 2H, Ar-H), 7.57 (m, 3H, Ar-H), 7.69 (m, 2H, Ar-H), 7.77 (m, 4H, Ar-H),
7.94 (d, 2H, J=12.0Hz, Ar-H), 9.54 (s, 1H, NH), 9.92 (s, 1H, NH). 13C NMR
(DMSO-d6, TMS, ppm): δ19.2, 44.9, 121.6, 129.3, 129.5, 129.8, 130.3, 132.6, 133.5,
137.6, 137.9, 138.1, 141.2, 153.3, 154.5, 156.1, 163.8, 172.9, 199.6. ESI-HRMS
(m/z): calcd. for C23H19NO3SNa [M+Na]+: 412.0983; found 412.1045.
3.3.2 Preparation of compounds 9-10
Compound 9: A solution of α-thioctic acid (1mmol), DCC (1.1mmol) and
paracetamol (150 mg, 1.0mmol) (1mmol) in dichloromethane (10 ml) with catalytic
amount of DMAP was stirred mechanically at room temperature until esterification
was complete. The N, N-dicyclohexylurea was filtered off and the filtrate was washed
with water (3×50 ml), 5% acetic acid (3×50 ml) again with water (3×50 ml) and then
dried over anhydrous sodium sulphate. The solvent was removed under reduced
pressure to give the ester which was chromatographed over a column of silica gel
using (trichloromethane /methyl alcohol 30:1) as eluent gave 103.0 mg pale yellow
solid. Yield: 30.4%. IR (KBr, cm−1): 1751 (v, COO), 1663 (v, C=O), 604 (w, C-S). 1H
NMR (DMSO-d6, TMS, ppm): δ 1.55 (m, 2H, Dithiolane-C-CH2), 1.76 (m, 4H,
Dithiolane-CH2, O=C-C-CH2), 1.93 (m, 1H, 5H-Dithiolane), 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=8.8Hz, 2H, Ar-H), 7.47 (d,
J=8.8Hz, 2H, Ar-H), 7.53 (s, 1H, NH). 13C NMR (DMSO-d6, TMS, ppm): δ24.4,
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.
ESI-HRMS (m/z): calcd. for C16H21NO3S2Na [M+Na]+: 362.0861; found 362.0857.
Compound 10: pale yellow solid 127.4 mg. Yield: 36.4%. IR (KBr, cm−1): 1748 (v,
1
COO), 1705 (v, C=O), 625 (w, C-S). H NMR (CDCl3, TMS, ppm): δ 1.53 (m, 2H,
Dithiolane-C-CH2), 1.78 (m, 4H, Dithiolane-CH2, O=C-C-CH2), 1.92 (m, 1H,
5H-Dithiolane), 2.47 (m, 1H, 3H-Dithiolane), 2.63 (t, J=7.6Hz, 2H, 4H-Dithiolane),
3.12 (m, 2H, O=C-CH2), 3.60 (m, 1H, 5H-Dithiolane), 6.40 (d, J=9.6Hz, 1H,
3H-Coumarin), 7.05 (dd, J=8.8Hz 2.0Hz, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.50 (d,
J=8.4Hz, 1H, Ar-H), 7.70 (d, J=9.6Hz, 1H, 4H-Coumarin). ESI-HRMS (m/z): calcd.
for C17H18O4S2Na [M+Na]+: 373.0544; found 373.0587.
3.3.3 Preparation of compounds 11-15
26

ACCEPTED MANUSCRIPT
Compound 11-15 were prepared using a procedure similar to that for compound 1
except ADTOH (226mg, 1.0mmol) were used instead of 4-hydroxythiobenzamide.
Chromatography on silica gel with (CHCl3/CH3OH 50:1) as eluent gave the product.
Compound 11, brownish red solid 98.4 mg. Yield: 22.5%. IR (KBr, cm−1): 1744 (s,
COO), 1217 (s, Ar-O-C), 1165 (v, C=S). 1H NMR (DMSO-d6, TMS, ppm): δ 1.73 (d,
J=8.0Hz, 3H, Ar-C-CH3), 3.95 (s, 3H, Ar-O-CH3), 4.15 (d, J=8.0Hz, 1H, Ar-CH),
7.17 (m, 4H), 7.39 (s, 1H, 4H-1,2-dithiole-3-thione), 7.51 (d, J=8.0Hz, 1H, Ar-H),
7.64 (d, J=8.0Hz, 2H, Ar-H), 7.80 (m, 3H, Ar-H). 13C NMR (DMSO-d6, TMS, ppm):
δ 18.8, 31.2, 45.0, 55.7, 106.3, 119.4, 123.3, 126.4, 126.7, 127.8, 129.0, 129.2, 129.3,
129.7, 134.0, 135.5, 136.3, 153.9, 157.8, 172.9, 173.0, 216.0. ESI-HRMS (m/z):
calcd. for C23H18NO3S3 [M–H]–: 437.0418; found 437.1935.
Compound 12: brownish red oil180.0 mg. Yield: 44.1%. IR (KBr, cm−1): 1753 (s,
1
COO), 1136 (v, C=S). H NMR (DMSO-d6, TMS, ppm): δ 0.94 (d, J=6.0Hz, 6H,
C(CH3)2), 1.65 (d, J=8.0Hz, 3H, Ar-C-CH3), 1.90 (m, 1H, Ar-C-CH), 2.50 (d, 2H,
Ar-CH2), 3.98 (q, J=8.0Hz ,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=8.0Hz, 2H, Ar-H). 13C NMR
(DMSO-d6, TMS, ppm): δ 18.9, 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. ESI-HRMS (m/z): calcd. for C22H22O2S3Na
[M+Na]+: 437.0680; found 437.0701.
Compound 13: brownish red oil124.7 mg. Yield: 27.0%. IR (KBr, cm−1): 1751 (s,
1
COO), 1660 (s, CO), 1146 (v, C=S). H NMR (DMSO-d6, TMS, ppm): δ 1.59 (d,
J=8.0Hz, 3H, CH3), 4.00 (q, J=8.0Hz, 1H, Ar-CH), 7.07 (m, 2H, Ar-H), 7.28 (s, 1H,
4H-1,2-dithiole-3-thione), 7.41 (m, 3H, Ar-H), 7.53 (m, 4H, Ar-H), 7.64 (m, 1H,
13
Ar-H), 7.73 (m, 2H, Ar-H), 7.78 (s, 1H, Ar-H). C NMR (DMSO-d6, TMS, ppm):
δ17.9, 42.7, 121.5, 123.3, 128.5, 128.8, 129.1, 130.3, 131.2, 132.4, 135.1, 136.2,
137.6, 140.6, 172.9, 173.1, 215.9. ESI-HRMS (m/z): calcd. for C25H18NO3S3 [M+H]+:
463.0496; found 463.0487.
Compound 14: brownish red solid 109.0 mg. Yield: 30.5%. IR (KBr, cm−1): 1739 (s,
1
COO), 1630 (s, C=C), 1130 (v, C=S). H NMR (DMSO-d6, TMS, ppm): δ 6.66 (d,
J=16.0Hz, 1H, -CHCO), 7.36 (m, 2H, Ar-H), 7.45 (s, 1H, 4H-1,2-dithiole-3-thione),
7.47 (m, 3H, Ar-H), 7.63 (m, 2H, Ar-H), 7.74 (m, 2H, Ar-H), 7.93 (d, J=16.0Hz, 1H,
Ar-CH). 13C NMR (DMSO-d6, TMS, ppm): δ117.3, 123.7, 129.2, 129.3, 129.5, 131.5,
134.3, 136.3, 147.6, 154.0, 165.0, 173.2, 216.0. ESI-HRMS (m/z): calcd. for
C18H13O2S3 [M+H]+: 357.0078; found 357.0134.
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ACCEPTED MANUSCRIPT
Compound 15: brownish red solid 197.0 mg. Yield: 34.9%. IR (KBr, cm−1): 1757(m,
1
COO), 1681 (s, CO), 1320 (s, Ar-O-C), 1121 (v, C=S). H NMR (DMSO-d6, TMS,
ppm): δ 2.41 (s, 3H, Cp-CH3), 3.77 (s, 3H, Ar-OCH3), 3.87 (s, 2H, COO-CH2), 6.64
(dd, J=4.0Hz, J=8.0Hz, 1H), 6.81 (d, J=8.0Hz, 1H, Ar-H), 6.97 (d, J=2.0Hz, 1H,
Ar-H), 7.15 (m, 3H, Ar-H), 7.32 (s, 1H, 4H-1,2-dithiole-3-thione), 7.42 (m, 2H,
Ar-H), 7.60 (m, 4H, Ar-H). 13C NMR (DMSO-d6, TMS, ppm): δ13.8, 29.8, 56.0,
102.2, 112.0, 112.5, 115.2, 123.5, 129.2, 129.4, 129.6, 130.9, 131.7, 134.5, 136.3,
136.4, 138.2, 154.0, 156.2, 169.6, 173.1, 216.0. ESI-HRMS (m/z): calcd. for
C28H21ClNO4S3 [M+H]+: 566.0321; found 566.0407.
Acknowledgements
This work was financially supported by the National Natural Science Foundations
of China (no. 21171079) and Science Development Project of Lanzhou (No.
2014-2-34).
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