Design, synthesis and biological evaluation of hydrogen sulfide releasing derivatives of 3-n-butylphthalide as potential antiplatelet and antithrombotic agents

Article abstract of DOI:10.1039/c4ob00830h

In the present study, a series of hydrogen sulfide (H₂S) releasing derivatives (8a-g and 9a-f) of 3-n-butylphthalide (NBP) were designed, synthesized and biologically evaluated. The most promising compound 8e significantly inhibited the adenosi

Full text of DOI:10.1039/c4ob00830h

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Biomolecular Chemistry
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Design, synthesis and biological evaluation
of hydrogen sulfide releasing derivatives of
3-n-butylphthalide as potential antiplatelet and
antithrombotic agents†
Cite this: Org. Biomol. Chem., 2014,
12, 5995
Xiaoli Wang,‡a,b,c Linna Wang,‡b,d Xiao Sheng,‡b,e Zhangjian Huang,b,e Tingting Li,b,d
Ming Zhang,a,b Jinyi Xu,b,e Hui Ji,*b,d Jian Yin*a,b,c and Yihua Zhang*b,e
In the present study, a series of hydrogen sulfide (H2S) releasing derivatives (8a–g and 9a–f) of 3-n-
butylphthalide (NBP) were designed, synthesized and biologically evaluated. The most promising com-
pound 8e significantly inhibited the adenosine diphosphate (ADP) and arachidonic acid (AA)-induced
platelet aggregation in vitro, superior to NBP, ticlopidine hydrochloride and aspirin. Furthermore, 8e could
slowly produce moderate levels of H2S in vitro, which could be beneficial for improving cardiovascular
and cerebral circulation. Most importantly, 8e protected against the collagen and adrenaline induced
thrombosis in mice, and exhibited greater antithrombotic activity than NBP and aspirin in rats. Overall, 8e
could warrant further investigation for the treatment of thrombosis-related ischemic stroke.
Received 22nd April 2014,
Accepted 19th June 2014
DOI: 10.1039/c4ob00830h
www.rsc.org/obc
need for new drugs with multiple actions to combat ischemic
stroke.
1. Introduction
Ischemic stroke, a leading cause of death and long-term dis-
The racemic 3-n-butylphthalide (NBP) was approved by the
ability worldwide, is a major socioeconomic burden due to the State Food and Drug Administration (SFDA) of China in 2002
lack of a suitable therapy.1 Ischemic stroke is characterized by as a new anti-ischemic stroke drug, which has beneficial
thrombosis initiated by abnormal platelet function, which effects on stroke through multiple actions,6–8 including
results in insufficient cerebral blood flow, and further leads to inhibiting platelet aggregation and thrombosis, improving
ischemic brain damage.2–5 Current drug therapies for preven- microcirculation, reducing the area of cerebral infarct and
tion and treatment of ischemic stroke are primarily dependent decreasing oxidative damage, etc. Despite these advantages,
on high affinity and single-target drugs. However, a drug the clinical application of NBP has been limited due to its
directed against a single molecular target may not be effective moderate potency.9,10 To achieve the ideal anti-ischemic stroke
in treatment of ischemic stroke because of its multi-factorial drugs, a wide range of studies of structural modification and
pathology. Therefore, there is a significantly unmet medical related research on NBP have been carried out. Previous
studies have demonstrated that the potassium salt of 2-(1-
hydroxypentyl)-benzoate (HPBA), a ring-opening derivative of
NBP, has an improved aqueous solubility and comparable
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of
potency to NBP.11 Recently, we also reported several ring-
Education, School of Biotechnology, Jiangnan University, Lihu Avenue 1800, Wuxi
opening derivatives of NBP as potent anti-ischemic stroke
214122, P. R. China. E-mail: jianyin@jiangnan.edu.cn; Fax: +86-0510-85328229;
agents.12–14
Tel: +86-0510-85328229
bState Key Laboratory of Natural Medicines, China Pharmaceutical University,
Hydrogen sulfide (H2S) is considered as a new member of
gasotransmitter family, following nitric oxide (NO) and carbon
monoxide (CO). Recent studies have revealed that H2S has a
positive effect on cerebral ischemia reperfusion injury. Indeed,
augmentation of H2S production can induce vasodilatation,
inhibit platelet aggregation, improve CBF, promote angio-
genesis, and protect neurons against oxidative stress and free
radicals, benefiting patients with ischemic stroke.15–18And a
number of H2S-releasing compounds have been developed and
are already undergoing intensive investigation.19–21
Nanjing 210009, P. R. China. E-mail: zyhtgd@163.com, huijicpu@163.com;
Fax: +86-025-83271015, +86-25-86635503; Tel: +86-25-83271015, +86-25-86021369
cSynergetic Innovation Center of Food Safety and Nutrition, Jiangnan University,
Lihu Avenue 1800, Wuxi 214122, P. R. China
dDepartment of Pharmacology, China Pharmaceutical University, Nanjing 210009,
P. R. China
eCenter of Drug Discovery, China Pharmaceutical University, Nanjing 210009,
P. R. China
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ob00830h
‡These authors contributed equally to this work.
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In this study, we hypothesized that H2S-releasing derivatives H2S would synergistically inhibit platelet aggregation and
of NBP could display synergistic effects exerted by both thrombosis.
NBP and H2S on platelet aggregation and thrombosis. Thereby,
a series of hybrids from HPBA and H2S were designed,
synthesized and evaluated as potential antiplatelet and anti-
thrombotic agents in vitro and in vivo.
2.2. Chemistry
The synthesis of the target compounds 8a–g and 9a–f are illus-
trated in Scheme 1. The H2S-donor ADTOH (compound 2) was
prepared as described previously26 and converted to the corres-
ponding bromo-substituted ethers 3a–g using K2CO3 in
acetone. NBP was subjected to saponification and sequential
acidification to form the ring-opening acid 4. Treatment of 4
with acetyl chloride or chloroacetyl chloride gave the acylates 5
or 6. Compound 5 was condensed with 3a–g to generate the
target compounds 8a–g. Treatment of 6 with diethylamine or
N-methyl piperazine led to ester 7a or 7b, which was then
reacted with 3c–d to provide the target compounds 9a–f. All of
the new compounds were purified by column chromatography
2. Results
2.1. Strategy for the design of H2S–NBP
It has been demonstrated that conjugation of an H2S-donor
moiety with a “native” molecule enhances therapeutic effect
and/or reduces the adverse effect of the native molecule.22–24
In view of the fact that ADTOH, 5-(4-hydroxyphenyl)-3H-1,2-
dithiole-3-thione, a known H2S-donor, is able to release H2S
in vitro and in vivo,25 we designed a novel class of compounds
through connecting ADTOH to HPBA with different linkers. In
addition, amines, such as diethylamine or N-methyl piper-
azine, were introduced to the side chain of HPBA as a substi-
tuted acetate linkage, respectively. We expected that the ester
bonds of H2S–NBP would be cleaved in vivo by esterases to
and characterized by ESI-MS, H NMR, 13C NMR, and HRMS.
The individual compounds with a chemical purity of >95%
(determined by HPLC analysis) were used for subsequent
experiments.
1
2.2. Antiplatelet aggregation effects in vitro
release HPBA and H2S, the former would subsequently The individual compounds were evaluated for inhibition of
undergo ring closing to generate NBP; and that both NBP and platelet aggregation in rabbit platelet rich plasma (PRP) in
Scheme 1 Synthesis of 8a–g and 9a–f. Reagents and conditions: (a) pyridine hydrochloride, 215 °C, 0.5 h; (b) Br(CH2)nBr, K2CO3, acetone, reflux,
6–8 h; (c) (i) 2 M NaOH, CH3OH–H2O, 50 °C, 0.5 h; (ii) 5% HCl, −10 to 0 °C; (d) CH3COCl, Et3N, DMAP, CH2Cl2, −10 °C, 5 h; (e) ClCH2COCl, Et3N,
DMAP, CH2Cl2, −10 °C, 5 h; (f) 3a–g, K2CO3, acetone, reflux, 6–10 h; (g) diethylamine or N-methyl piperazine, K2CO3, acetone, reflux, 6–8 h; (h)
3c–e, K2CO3, acetone, reflux, 4–8 h.
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Table 1 The IC50 values of 8a–g and 9a–f against platelet aggregation
in vitro
IC50 (mM)
Compd
ADP (10 μM)
AA (1.0 mM)
Control
Ticlida
Aspirinb
NBP
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
—
—
—
0.36 0.04
—
0.14 0.02
0.58 0.06
0.15 0.05
0.14 0.06
0.14 0.08
0.15 0.07
0.09 0.01
0.16 0.05
0.20 0.06
0.18 0.03
0.17 0.04
0.15 0.06
0.37 0.10
0.23 0.09
0.23 0.08
0.74 0.20
0.23 0.09
0.20 0.11
0.18 0.10
0.17 0.13
0.14 0.06
0.19 0.14
0.24 0.12
0.23 0.14
0.21 0.13
0.19 0.11
0.71 0.19
0.52 0.17
0.49 0.14
Fig. 1 Inhibition of 8e, 2, 3e and 5 on the ADP-induced platelet aggre-
gation in vitro. Rabbit platelet suspensions were preincubated with the
testing compound (200 μM) at 37 °C for 5 min and exposed to 10 μM of
ADP, followed by continually monitoring. Rabbit platelet suspensions
that had been treated with the vehicle and exposed to ADP were used as
positive controls. Data are expressed as mean SD of each group (n =
6) for two separate experiments. ***P < 0.001 by one-way ANOVA fol-
lowed by post hoc Tukey’s test.
a Ticlid is an inhibitor of the ADP-induced platelet aggregation.
b Aspirin is an inhibitor of the AA-induced platelet aggregation. IC50
values (a dose achieved 50% inhibition of platelet aggregation) are
expressed as mean SD (n = 6) and analyzed by one-way analysis of
variance (ANOVA) followed by post hoc Tukey’s test.
response to the adenosine diphosphate (ADP) and arachidonic
acid (AA) using the Born’s turbidimetric method.27 Ticlopidine
hydrochloride (Ticlid) and aspirin, as inhibitors of the ADP-
and AA-induced platelet aggregation,28,29 respectively, were
employed as positive controls, and NBP as a reference com-
pound. The inhibitory activity of individual compounds at five
different concentrations against the ADP (10 μM) and AA
(1.0 mM) induced platelet aggregation. Data were calculated
and expressed as the IC50 values.
As shown in Table 1, all the target compounds presented
better inhibitory effects than NBP both against the ADP- and
AA-induced platelet aggregation. The compounds with
6-carbon alkanes (8e, 9c) displayed the best inhibitory activity.
Furthermore, the compounds containing a hydrogen atom
(8c–e) exhibited stronger inhibitory activity than that bearing
diethylamino and N-methyl piperazino moieties in the side
chain (9a–f). Notably, 8e (R = H, n = 6) was the most potent,
whose IC50 value (0.14 mM) on the ADP-induced platelet aggre-
gation was significantly less than that of NBP (0.74 mM)
and Ticlid (0.36 mM), and the IC50 value (0.09 mM) on the
AA-induced platelet aggregation was also less than that of NBP
(0.58 mM) and aspirin (0.14 mM). These data indicated that
8e was a potent inhibitor of both the ADP- and AA-induced
platelet aggregation. Therefore, 8e was chosen for the follow-
ing investigations.
Fig. 2 Inhibition of 8e and combinations of its moieties on the ADP-
induced platelet aggregation in vitro. Data are expressed as mean SD
of each group (n = 6) for two separate experiments. *P < 0.05 by one-
way ANOVA followed by post hoc Tukey’s test.
of them was substantially less effective than 8e (62.90%),
suggesting that these moieties may have synergistic effects
on the inhibition of 8e against the ADP-induced platelet
aggregation.
To confirm the synergistic effects between these moieties,
we then tested the moieties of 8e in combination. Obviously 8e
had a better antiplatelet activity than NBP together with 2
(*P < 0.05, Fig. 2) which supports our hypothesis that 8e affect
the platelet aggregation greater than the combination of its
mother compound-NBP and H2S releasing moiety-ADTOH
(compound 2). Although the combination of 3e and 5 showed
weaker inhibition on platelet aggregation than 8e, no statisti-
cal significance was found between them.
In order to examine whether each moiety (2, 3e and 5) of 8e
contributes to the inhibitory activity of this compound, we
further investigated their corresponding effects on the ADP-
induced platelet aggregation in vitro. As shown in Fig. 1, com-
pounds 2, 3e and 5 inhibited the ADP-induced platelet aggre-
gation by 28.04%, 30.05% and 26.80%, respectively, but each
2.3. Antithrombotic activity of 8e in mice
We then evaluated the antithrombotic activity of 8e on a
mouse model of thromboembolism, that is, an acute vascular
occlusion initiated by intravascular platelet induced aggrega-
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thrombosis.31 SD rats were randomized and treated intragastri-
cally (i.g.) with normal saline (sham group), NBP (80 mg kg−1),
aspirin (80 mg kg−1), and 8e (80 and 235 mg kg−1). After a
7-day oral administration of individual compounds, the wet
and dry thrombus weights were measured (Fig. 4). All four
treatment groups showed significant difference from the sham
group on wet weight of thrombus. Treatment with 8e at a dose
of 80 or 235 mg kg−1, which is equal mass to or equal molar to
that of NBP, reduced wet weight by 27.87% and 37.16%,
respectively, more effective than treatment with NBP (15.02%),
and the potency of the higher dose group was similar to that
of aspirin (36.74%). Similarly, four treatment groups shared
Fig. 3 The survival of mice after injection of 0.21 mg kg−1 collagen and
44.5 μg kg−1 adrenaline and subsequent oral administration of 8e, NBP
and aspirin, respectively, at the indicated doses and time points, and the significant difference from the sham group on dry weight
of thrombus. 8e at 235 mg kg−1 reduced the dry weight of
results were expressed as the percentage of mice alive (n = 15) as a
function of time.
thrombus by 32.58%, which was more potent than both NBP
(18.72%) and aspirin (30.60%). Treatment with 8e at 80 mg kg−1
also effectively reduced the thrombus dry weight by 26.34%,
which was higher than NBP but lower than aspirin. These
results were consistent with our previous research, and clearly
indicated that 8e was a potent thrombosis inhibitor in vivo.
tion through infusion of a mixture of collagen and adrenaline
into the jugular vein.30 Male ICR mice were randomized and
treated orally with vehicle, NBP (160 mg kg−1), aspirin (160 mg
kg−1), or 8e (160 mg kg−1 and 470 mg kg−1, which are equal
mass to and equal molar to that of NBP, respectively) for seven
days. Two hours after the last administration, individual
mice were injected intravenously with a mixture of collagen
(0.21 mg kg−1) and adrenaline (44.5 μg kg−1) to induce acute
systemic vascular thromboembolism. As shown in Fig. 3, treat-
ment with 8e (470 mg kg−1) significantly reduced the onset of
hemiplegia and death in mice, and the percentage of survivors
(66.7%) was greater than that of aspirin (60.0%) and NBP
(53.3%), while the percentage (60.0%) by treatment with 8e
(160 mg kg−1) was slightly greater than that of NBP and com-
parable to that of aspirin in this model at the same dosage.
These results clearly demonstrated that 8e efficiently inhibited
acute systemic intravascular thrombosis in mice leading to
protective effects.
2.5. Assay of hydrogen sulfide release in vitro
The target compounds were designed to be NBP derivatives,
which could release H2S. In order to show that this was indeed
the case, the levels of H2S produced by 8e were measured in
phosphate buffer and rat liver homogenate,32 respectively. As
shown in Fig. 5, little but still detectable H2S release was
observed when 8e was incubated with phosphate buffer.
However, in liver homogenate, 8e could produce moderate
levels of H2S peaking at 60 min which remained slightly elev-
ated for a further 30 min, suggesting that 8e could slowly
release H2S in liver homogenate, which would be expected to
increase the duration of intracellular H2S exposure leading to
the enhanced potency of 8e relative to NBP. Based on the
chemical structure of 8e, we speculated that it could be firstly
metabolized by esterases to release HPBA, which would
2.4. Antithrombotic activity of 8e in rats
Parallelly, we investigated the antithrombotic activity of 8e in a subsequently undergo ring closure to generate NBP, and H2S
rat arterio-venous (A-V) shunt model mimicking arterial donor ADTOH. Incubation of 8e in buffer released much
Fig. 4 Effects of 8e, NBP and aspirin on thrombus wet weights (A) and dry weights (B) in rats. SD rats were treated with the indicated doses of each
drug by gavage daily for seven days. Two hours after the last treatment, the rats were subjected to the A-V shunt using a silk thread for 15 min. The
wet and dry weights of the threads were measured. Data are expressed as mean SD of each group (n = 12) and analyzed by ANOVA and post hoc
Tukey test. ***P < 0.001, **P < 0.01 vs. the control group, ###P < 0.001, #P < 0.05 vs. the NBP group.
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apparatus. H and 13C NMR spectral data were obtained from
a Bruker Avance 300 MHz spectrometer at 300 K using TMS as
an internal standard. MS spectra were recorded on a Mariner
Mass Spectrum (ESI). Analytical and preparative TLC were per-
formed on silica gel GF/UV 254, and the chromatograms were
conducted on silica gel (200–300 mesh) and visualized under
UV light at 254 and 365 nm. The purities of the compounds
were characterized by HPLC analysis (LC-10A HPLC system
consisting of LC-10ATvp pumps and a SPD-10Avp UV detector)
and HRMS (Agilent technologies LC/MSD TOF). The target
compounds 8a–g and 9a–f with a chemical purity of >95%
(see the ESI†).
All experiments were approved and performed in accord-
ance with National Institutes of Health Guide for Care and Use
of Laboratory Animals (publication no. 85-23, revised 1985),
and all animal experimental protocols were approved by the
Animal Research Protection Committee of China Pharma-
ceutical University, Nanjing, China.
1
Fig. 5 Release of H2S from 8e in vitro. 8e (100 μM) was incubated
(37 °C) for 90 min with fresh rat liver homogenate or buffer in vitro. H2S
concentration was determined. Data are expressed as accumulate %
conversion of 8e to H2S at each time point, n = 6.
smaller quantities of H2S which also implied that the gas
released from the parent molecule is mainly as a result of
metabolic events.
5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione
(2). Pyridine
hydrochloride (68.0 g, 590 mmol) was added to 1 (25.0 g,
100 mmol) in a dry flask, mixed and then heated to melt at
215 °C under argon protection for 0.5 h. After being cooled to
100 °C, warm water (200 mL) was added and hot-filtered. The
cake was placed in a beaker, and 10% NaOH (300 mL) was
added. This was stirred for 4 h, filtered, the cake was dissolved
in water (3 L), and then adjusted to pH 2 with concentrated
hydrochloric acid. The red precipitate was filtered and washed
to neutral pH using water, and then dried in a vacuum desiccator
to yield a yellow solid (18.0 g, 76.5%), mp 172–174 °C (found:
C, 47.78; H, 2.67; O, 7.08; S, 42.53. Calcd for C9H6OS3: C,
47.76; H, 2.67; O, 7.07; S, 42.50%); 1H NMR (CDCl3, 300 Hz, δ):
10.47 (brs, 1H, OH), 7.84 (d, 2H, J = 8.8 Hz, ArH), 7.70 (s, 1H,
vCH), 6.88 (d, 2H, J = 8.8 Hz, ArH).
General procedure for the preparation of compounds 3a–g.
To a mixture of 2 (0.226 g, 1.0 mmol), appropriate amounts of
dibromoalkanes (3.0 mmol), K2CO3 (0.690 g, 5.0 mmol), and
acetone (20 mL) were added under constant stirring and argon
protection. It was then heated to 65 °C and refluxed for 6–8 h.
After completion of the reaction (monitored by TLC), the reac-
tion mixture was filtered, and the filtrate was concentrated
under reduced pressure. The resulting residue was purified by
flash chromatography (PE–acetone 10 : 1 v/v) to give the title
compounds (70–83%).
3. Discussion and conclusions
As far as structure–activity relationships are concerned, in
general, extension of the carbon chain (CH2)n connecting to
the H2S releasing moiety (n = 2–6), the platelet aggregation
inhibitory activity of the compounds was enhanced, and the
activity was declined while n = 8. Furthermore, the compounds
containing a hydrogen atom linking to the acetyl group in the
side-chain exhibited stronger inhibitory activity than that
bearing diethylamino and N-methylpiperazino moieties in the
side chain. As a matter of fact, 8e exhibited the strongest
inhibitory activity among all compounds. Interestingly, all of
the structural moieties of 8e substantially inhibited the ADP-
induced platelet aggregation but much less than 8e, suggesting
that they may act synergistically. Moreover, administration of 8e
alone had better antiplatelet aggregation activity than combi-
nations from its corresponding moieties. However, the precise
SAR of the target compounds remains to be further investigated.
In summary, we designed and synthesized a series of novel
H2S releasing derivatives of NBP, and found that compound 8e
was the most potent in inhibiting the ADP- and AA-induced
platelet aggregation in vitro, superior to NBP, Ticlid and
aspirin. Furthermore, 8e could produce moderate levels of H2S
in vitro, which could be beneficial for improving cardiovascular
and cerebral circulation. Most importantly, 8e protected
against the collagen and adrenaline induced thrombosis in
mice, and exhibited greater antithrombotic activity in rats.
Therefore, our findings may aid in the design of new thera-
peutic reagents for the treatment of thrombosis-related
ischemic stroke and other vascular diseases.
5-(4-(2-Bromoethoxy)phenyl)-3H-1,2-dithiole-3-thione (3a). The
title compound was obtained as a reddish brown solid (80%),
mp 134–136 °C. IR (cm−1, KBr): νmax 595, 819, 1184, 1400,
1
1486, 1599. H NMR (300 Hz, CDCl3): δ 3.68 (t, 2H, J = 6.0 Hz,
CH2Br), 4.36 (t, 2H, J = 6.0 Hz, ArOCH2), 7.00 (d, 2H, J =
8.8 Hz, ArH), 7.40 (s, 1H, vCH), 7.63 (d, 2H, J = 8.8 Hz, ArH).
5-(4-(3-Bromopropoxy)phenyl)-3H-1,2-dithiole-3-thione
(3b).
The title compound was obtained as a reddish brown solid
(78%), mp 131–133 °C. IR (cm−1, KBr): νmax 591, 815, 1185,
1400, 1485, 1600. 1H NMR (300 Hz, CDCl3): δ 2.32–2.40 (m,
2H, CH2CH2Br), 3.62 (t, 2H, J = 6.3 Hz, CH2Br), 4.12 (t, 2H, J =
4. Experimental section
4.1. Chemistry
General. Melting points are uncorrected and were measured 5.8 Hz, ArOCH2), 7.00 (d, 2H, J = 8.7 Hz, ArH), 7.40 (s, 1H,
in open capillary tubes using a Gallenkamp melting point vCH), 7.63 (d, 2H, J = 8.7 Hz, ArH).
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5-(4-(4-Bromobutoxy)phenyl)-3H-1,2-dithiole-3-thione (3c). The washed with water, dried, filtered, and evaporated to dryness.
title compound was obtained as a reddish brown solid (81%), The residue was recrystallized from n-hexane to obtain 5 as a
mp 129–131 °C. IR (cm−1, KBr): νmax 590, 823, 1189, 1400, white crystal (0.85 g, 68%, over two steps). mp 65–66 °C. MS
1489, 1600. 1H NMR (300 Hz, CDCl3): δ 1.95–2.43 (m, 4H, (ESI): m/z 249.1 [M − H]−. IR (cm−1, KBr): νmax 1412, 1691,
1
CH2CH2CH2Br), 3.60 (t, 2H, J = 6.4 Hz, CH2Br), 4.07 (t, 2H, J = 1734, 2958, 3450. H NMR (300 Hz, CDCl3): δ 0.93 (t, 3H, CH3,
6.0 Hz, ArOCH2), 6.97 (d, 2H, J = 8.8 Hz, ArH), 7.40 (s, 1H, J = 8.5 Hz), 1.37–1.42 (m, 4H, 2 × CH2), 1.88–1.91 (m, 2H,
vCH), 7.62 (d, 2H, J = 8.8 Hz, ArH).
CH2), 2.13–2.33 (m, 3H, COCH3), 6.61–6.72 (m, 1H, OCHCH2),
7.37–7.40 (m, 1H, ArH), 7.56–7.62 (m, 2H, ArH), 8.05 (d, 1H,
ArH, J = 8.1 Hz), 10.98 (brs, 1H, COOH). 13C NMR (75 Hz,
CDCl3): δ 172.0, 166.5, 140.8, 133.1, 130.3, 130.0, 127.1, 125.7,
74.8, 41.0, 36.3, 27.8, 22.4, 13.8.
( )-2-[1-(2-Chloroacetoxy)pentyl]benzoic acid (6). Compound 6
was synthesized in the same manner as compound 5. Starting
from compound 4 (1.04 g, 5.00 mmol) and 2-chloroacetyl
chloride (2.08 mL, 15.00 mmol), compound 6 was obtained as
a white crystal (0.93 g, 65%, over two steps). mp 67–68 °C. MS
(ESI): m/z 283 [M − H]−. IR (cm−1, KBr): νmax 1412, 1691, 1734,
2958, 3450. 1H NMR (300 Hz, CDCl3): δ 0.93 (t, 3H, CH3, J =
4.2 Hz), 1.37–1.42 (m, 4H, 2 × CH2), 1.88–1.91 (m, 2H, CH2),
4.11–4.32 (m, 2H, COCH2Cl), 6.71–6.78 (m, 1H, OCHCH2),
7.36–7.42 (m, 1H, ArH), 7.56–7.62 (m, 2H, ArH), 8.08 (d, 1H,
ArH, J = 8.1 Hz), 10.89 (brs, 1H, COOH). 13C NMR (75 Hz,
CDCl3): δ 172.0, 166.5, 140.8, 133.1, 130.3, 130.0, 127.1, 125.7,
5-(4-((5-Bromopentyl)oxy)phenyl)-3H-1,2-dithiole-3-thione (3d).
The title compound was obtained as a reddish brown solid
(75%), mp 125–127 °C. IR (cm−1, KBr): νmax 592, 826, 1184,
1400, 1482, 1600. 1H NMR (300 Hz, CDCl3): δ 1.60–1.70 (m,
2H, CH2CH2CH2Br), 1.81–2.45 (m, 4H, CH2CH2CH2CH2Br),
3.46 (t, 2H, J = 6.6 Hz, CH2Br), 4.04 (t, 2H, J = 6.2 Hz, ArOCH2),
6.96 (d, 2H, J = 8.8 Hz, ArH), 7.40 (s, 1H, vCH), 7.61 (d, 2H, J =
8.8 Hz, ArH).
5-(4-((6-Bromohexyl)oxy)phenyl)-3H-1,2-dithiole-3-thione (3e).
The title compound was obtained as a reddish brown solid
(83%), mp 122–124 °C. IR (cm−1, KBr): νmax 596, 827, 1189,
1400, 1482, 1599. 1H NMR (300 Hz, CDCl3): δ 1.25–1.33 (m,
2H, CH2(CH2)2Br), 1.60–1.70 (m, 2H, ArO(CH2)2CH2), 1.81–2.45
(m, 4H, ArOCH2CH2, CH2CH2Br), 3.44 (t, 2H, J = 6.7 Hz,
CH2Br), 4.02 (t, 2H, J = 6.3 Hz, ArOCH2), 6.96 (d, 2H, J =
8.6 Hz, ArH), 7.40 (s, 1H, vCH), 7.60 (d, 2H, J = 8.6 Hz, ArH).
5-(4-((8-Bromooctyl)oxy)phenyl)-3H-1,2-dithiole-3-thione (3f). 74.8, 41.0, 36.3, 27.8, 22.4, 13.8.
( )-2-(1-(2-(Diethylamino)acetoxy)pentyl)benzoic acid (7a). To
The title compound was obtained as a reddish brown solid
(76%), mp 118–120 °C. IR (cm−1, KBr): νmax 596, 833, 1184,
1400, 1483, 1598. 1H NMR (300 Hz, CDCl3): δ 1.26–1.38 (m,
4H, CH2CH2(CH2)3Br), 1.56–1.72 (m, 4H, ArO(CH2)2CH2,
CH2(CH2)2Br), 1.81–2.45 (m, 4H, ArOCH2CH2, CH2CH2Br), 3.42
(t, 2H, J = 6.7 Hz, CH2Br), 4.02 (t, 2H, J = 6.4 Hz, ArOCH2), 6.96
(d, 2H, J = 8.6 Hz, ArH), 7.40 (s, 1H, vCH), 7.60 (d, 2H, J =
8.6 Hz, ArH).
a solution of 6 (284 mg, 1.0 mmol) and K2CO3 (276 mg,
2.0 mmol) in acetone (20 mL) was added diethylamine
(0.2 mL, 2.0 mmol), and the solution was heated to 65 °C and
refluxed for 4 h. The solution was then filtered, and the filtrate
was reconstituted in EtOAc (20 mL) and the solvent was
removed under reduced pressure. The crude was purified by
flash chromatography (CH2Cl2–MeOH = 100/1–50/1, v/v), to
5-(4-((12-Bromododecyl)oxy)phenyl)-3H-1,2-dithiole-3-thione give 7a as a light yellow oil (231 mg, 72%). MS (ESI): m/z 322
1
[M + H]+. H NMR (300 Hz, CDCl3): δ 0.83 (t, 3H, CH3, J = 7.5
(3g). The title compound was obtained as a reddish brown
solid (75%), mp 113–115 °C. IR (cm−1, KBr): νmax 597, 833,
Hz), 1.02 (t, 6H, 2 × NCH2CH3, J = 7.0 Hz), 1.29–1.37 (m, 4H,
1
1186, 1401, 1483, 1598. H NMR (300 Hz, CDCl3): δ 1.26–1.38
(m, 12H, 6 × CH2(CH2)3Br), 1.43–1.62 (m, 4H, ArO(CH2)2CH2,
CH2(CH2)2Br), 1.78–2.09 (m, 4H, ArOCH2CH2, CH2CH2Br), 3.41
(t, 2H, J = 6.8 Hz, CH2Br), 4.02 (t, 2H, J = 6.5 Hz, ArOCH2), 6.96
(d, 2H, J = 8.7 Hz, ArH), 7.40 (s, 1H, vCH), 7.60 (d, 2H, J =
8.7 Hz, ArH).
2 × CH2CH3), 1.87–1.88 (m, 2H, CHCH2), 2.94 (q, 4H, 2 × CH2,
J = 7.0 Hz), 3.62–3.76 (m, 2H, COCH2N), 6.69 (m, 1H, CH),
7.21–7.26 (m, 1H, ArH), 7.34–7.43 (m, 2H, ArH), 7.80 (d, 1H,
ArH, J = 7.5 Hz), 10.67 (brs, 1H, COOH).
( )-2-(1-(2-(4-Methylpiperazin-1-yl)acetoxy)pentyl)benzoic acid
(7b). Compound 7b was synthesized in the same manner as
( )-2-(1-Hydroxypentyl)benzoic acid (4). To a stirred solution compound 7a. Starting from compound 6 (284 mg, 1.0 mmol)
of NBP (1.90 g, 10.00 mmol) in CH3OH–H2O (20 mL, 1 : 1 v/v) and N-methyl piperazine (0.22 mL, 2.0 mmol), compound 7b
was added NaOH (0.80 g, 20.00 mmol). The reaction mixture was obtained as a light yellow oil (271 mg, 78%). MS (ESI): m/z
1
was heated at 50 °C for 0.5 h. The solvent was removed under 349 [M + H]+. H NMR (300 Hz, CDCl3): δ 0.83 (t, 3H, CH3, J =
reduced pressure and dissolved in water (20 mL), followed 7.5 Hz), 1.29–1.37 (m, 4H, 2 × CH2CH3), 1.87–1.88 (m, 2H,
by acidification with 5% HCl to pH 3–4 at −10 to 0 °C. The CHCH2), 2.26 (t, 3H, NCH3, J = 7.0 Hz), 2.45 (m, 8H, 4 ×
mixture was extracted with cold Et2O (10 mL × 3) and quickly NCH2), 3.62–3.76 (m, 2H, COCH2N), 6.69 (m, 1H, CH),
used for the next step without any purification.
7.21–7.26 (m, 1H, ArH), 7.34–7.43 (m, 2H, ArH), 7.80 (d, 1H,
ArH, J = 7.5 Hz), 10.67 (brs, 1H, COOH).
( )-2-(1-Acetoxypentyl)benzoic acid (5). A solution of acetyl
chloride (1.06 mL, 15.00 mmol) in dry CH2Cl2 (10 mL) was
added dropwise to a mixture of 4 (1.04 g, 5.00 mmol), Et3N
(2.09 mL, 15.00 mmol) and DMAP (0.50 mmol) in CH2Cl2
(80 mL) at −10 °C and the solution was left stirring at −10 °C
for 5 h. The mixture was acidified with 1 M HCl to pH 2 and
stirred for 1 h at room temperature. The organic layer was
General procedure for the preparation of the target com-
pounds 8a–g. To a solution of 5 (50 mg, 0.2 mmol) and K2CO3
(55 mg, 0.4 mmol) in acetone (10 mL) were added 3a–g
(0.2 mmol), and the solution was heated to 65 °C and refluxed
for 6–8 h. The solution was then filtered, and the filtrate was
reconstituted in EtOAc (20 mL) and the solvent was removed
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under reduced pressure. The crude compounds were purified 7.28–7.33 (m, 1H, ArH), 7.39 (s, 1H, CvCH), 7.51–7.52 (m, 2H,
by flash chromatography (PE–EtOAc = 10/1–1 : 1, v/v), to give ArH), 7.59 (d, 2H, ArH, J = 8.7 Hz), 7.88 (d, 1H, ArH, J =
the title compounds (67–78%).
7.8 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2,
125.8, 124.1, 115.5, 92.6, 72.9, 68.0, 64.9, 36.6, 29.7, 28.4, 25.8,
25.4, 22.5, 21.2, 14.0. HRMS (ESI): m/z calcd for C28H32O5S3,
[M + H]+ 545.1412; found 545.2204.
( )-2-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl 2-(1-acetoxy-
pentyl)benzoate (8a). The title compound was obtained as a
reddish brown solid (75%), mp 86–88 °C. MS (ESI): m/z 503.1
1
[M + H]+. H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.9
Hz), 1.26–1.48 (m, 4H, CH2CH2CH3), 1.74–1.83 (m, 2H,
( )-6-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)hexyl 2-(1-acet-
CHCH2), 2.07 (s, 3H, OOCCH3), 4.10 (t, 2H, ArOCH2, J = oxypentyl)benzoate (8e). The title compound was obtained as a
6.2 Hz), 4.41 (t, 2H, ArCOOCH2, J = 6.2 Hz), 6.53 (dd, 1H, CH, reddish brown oil (78%), MS (ESI): m/z 559.4 [M + H]+. 1H
J = 4.7 Hz, 8.0 Hz), 6.96 (d, 2H, ArH, J = 8.6 Hz), 7.27–7.32 NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.8 Hz), 1.26–1.45
(m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.51–7.52 (m, 2H, ArH), (m, 4H, CH2CH2CH3), 1.47–1.56 (m, 4H, ArCOO(CH2)2CH2,
7.58 (d, 2H, ArH, J = 8.6 Hz), 7.87 (d, 1H, ArH, J = 7.8 Hz). ArO(CH2)2CH2), 1.70–1.73 (m, 2H, CHCH2), 1.78–1.85 (m, 4H,
13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3, 143.7, ArCOOCH2CH2, ArOCH2CH2), 2.07 (s, 3H, OOCCH3), 4.03 (t,
134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2, 125.8, 2H, ArOCH2, J = 6.3 Hz), 4.34 (t, 2H, ArCOOCH2, J = 6.6 Hz),
124.1, 115.5, 92.6, 72.9, 67.8, 64.6, 36.3, 28.1, 25.4, 22.5, 21.2, 6.54 (dd, 1H, CH, J = 4.8 Hz, 8.1 Hz), 6.96 (d, 2H, ArH, J =
14.0. HRMS (ESI): m/z calcd for C25H26O5S3, [M + H]+ 503.0942; 8.7 Hz), 7.28–7.33 (m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.51–7.53
found 503.1018.
(m, 2H, ArH), 7.58 (d, 2H, ArH, J = 8.7 Hz), 7.88 (d, 1H, ArH, J =
7.6 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2,
125.8, 124.1, 115.5, 92.6, 72.9, 68.3, 65.1, 36.7, 28.9, 28.6, 28.1,
25.8, 25.7, 22.5, 21.2, 14.0. HRMS (ESI): m/z calcd for
C29H34O5S3, [M + H]+ 559.1568; found 559.1626.
( )-3-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)propyl 2-(1-acet-
oxypentyl)benzoate (8b). The title compound was obtained as a
reddish brown solid (78%), mp 83–85 °C. MS (ESI): m/z 517.1
1
[M + H]+. H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.9
Hz), 1.26–1.48 (m, 4H, CH2CH2CH3), 1.74–1.83 (m, 2H,
CHCH2), 1.98–2.04 (m, 2H, ArCOOCH2CH2), 2.07 (s, 3H,
( )-8-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)octyl 2-(1-acetoxy-
OOCCH3), 4.10 (t, 2H, ArOCH2, J = 6.2 Hz), 4.41 (t, 2H, pentyl)benzoate (8f). The title compound was obtained as a
ArCOOCH2, J = 6.2 Hz), 6.53 (dd, 1H, CH, J = 4.7 Hz, 8.0 Hz), reddish brown oil (70%), MS (ESI): m/z 587.2 [M + H]+. 1H
6.96 (d, 2H, ArH, J = 8.6 Hz), 7.27–7.32 (m, 1H, ArH), 7.38 (s, NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.8 Hz), 1.26–1.42
1H, CvCH), 7.51–7.52 (m, 2H, ArH), 7.58 (d, 2H, ArH, J = (m, 4H, CH2CH2CH3), 1.47–1.68 (m, 12H, ArCOOCH2(CH)6),
8.6 Hz), 7.87 (d, 1H, ArH, J = 7.8 Hz). 13C NMR (75 Hz, CDCl3): 1.70–1.73 (m, 2H, CHCH2), 2.06 (s, 3H, OOCCH3), 4.02 (t, 2H,
δ 173.1, 170.3, 166.9, 162.3, 143.7, 134.8, 134.6, 132.3, 131.3, ArOCH2, J = 6.4 Hz), 4.32 (t, 2H, ArCOOCH2, J = 6.7 Hz), 6.54
130.1, 128.6, 127.1, 126.2, 125.8, 124.1, 115.5, 92.6, 72.9, 67.8, (dd, 1H, CH, J = 4.7 Hz, 8.0 Hz), 6.96 (d, 2H, ArH, J = 8.7 Hz),
64.6, 36.3, 28.1, 25.4, 22.5, 21.2, 14.0. HRMS (ESI): m/z calcd 7.28–7.33 (m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.51–7.52 (m, 2H,
for C26H28O5S3, [M + H]+ 517.1099; found 517.1297.
ArH), 7.59 (d, 2H, ArH, J = 8.8 Hz), 7.88 (d, 1H, ArH, J =
7.7 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2,
125.8, 124.1, 115.5, 92.6, 72.9, 68.3, 65.1, 36.7, 29.6, 29.3, 29.1,
25.9, 25.8, 22.5, 21.2, 14.0. HRMS (ESI): m/z calcd for
C29H34O5S3, [M + H]+ 587.2246; found 587.2324.
( )-4-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)butyl 2-(1-acetoxy-
pentyl) benzoate (8c). The title compound was obtained as a
reddish brown oil (72%), MS (ESI): m/z 531.2 [M + H]+. 1H
NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.9 Hz), 1.26–1.48
(m, 4H, CH2CH2CH3), 1.74–1.83 (m, 2H, CHCH2), 2.00–2.06
(m, 4H, ArCOOCH2(CH2)2), 2.07 (s, 3H, OOCCH3), 4.10 (t, 2H,
( )-12-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)dodecyl
2-(1-
ArOCH2, J = 6.2 Hz), 4.41 (t, 2H, ArCOOCH2, J = 6.2 Hz), 6.53 acetoxypentyl)benzoate (8g). The title compound was obtained
(dd, 1H, CH, J = 4.7 Hz, 8.0 Hz), 6.96 (d, 2H, ArH, J = 8.6 Hz), as a reddish brown oil (67%), MS (ESI): m/z 665.2 [M + Na]+.
7.27–7.32 (m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.51–7.52 (m, 2H, 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.8 Hz),
ArH), 7.58 (d, 2H, ArH, J = 8.6 Hz), 7.87 (d, 1H, ArH, J = 1.26–1.42 (m, 4H, CH2CH2CH3), 1.45–1.66 (m, 20H,
7.8 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3, ArCOOCH2(CH)10), 1.70–1.73 (m, 2H, CHCH2), 2.06 (s, 3H,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2, OOCCH3), 4.02 (t, 2H, ArOCH2, J = 6.4 Hz), 4.32 (t, 2H,
125.8, 124.1, 115.5, 92.6, 72.9, 67.8, 64.6, 36.3, 28.1, 25.8, 25.4, ArCOOCH2, J = 6.7 Hz), 6.54 (dd, 1H, CH, J = 4.7 Hz, 8.0 Hz),
22.5, 21.2, 14.0. HRMS (ESI): m/z calcd for C27H30O5S3, 6.96 (d, 2H, ArH, J = 8.7 Hz), 7.28–7.33 (m, 1H, ArH), 7.38
[M + H]+ 531.1255; found 531.1375.
( )-5-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)pentyl 2-(1-acet-
(s, 1H, CvCH), 7.51–7.52 (m, 2H, ArH), 7.59 (d, 2H, ArH, J =
8.8 Hz), 7.88 (d, 1H, ArH, J = 7.7 Hz). 13C NMR (75 Hz, CDCl3):
oxypentyl) benzoate (8d). The title compound was obtained as δ 173.1, 170.3, 166.9, 162.3, 143.7, 134.8, 134.6, 132.3, 131.3,
a reddish brown oil (74%), MS (ESI): m/z 545.3 [M + H]+. 1H 130.1, 128.6, 127.1, 126.2, 125.8, 124.1, 115.5, 92.6, 72.9, 68.3,
NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.9 Hz), 1.24–1.47 65.1, 36.7, 29.8, 29.7, 29.6, 29.3, 29.1, 25.9, 25.8, 22.5, 21.2,
(m, 4H, CH2CH2CH3), 1.67–1.73 (m, 2H, CHCH2), 1.78–2.05 14.0. HRMS (ESI): m/z calcd for C35H46O5S3, [M + H]+ 643.2507;
(m, 6H, ArCOOCH2(CH2)3), 2.07 (s, 3H, OOCCH3), 4.06 (t, 2H, found 643.2601.
ArOCH2, J = 6.3 Hz), 4.36 (t, 2H, ArCOOCH2, J = 6.2 Hz), 6.54
General procedure for the preparation of the target com-
(dd, 1H, CH, J = 4.8 Hz, 8.1 Hz), 6.96 (d, 2H, ArH, J = 8.8 Hz), pounds 9a–f. To a solution of 7a or 7b (0.2 mmol) and K2CO3
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(55 mg, 0.4 mmol) in acetone (10 mL) were added 3c–e 64.6, 47.6, 36.7, 29.7, 28.9, 28.6, 28.1, 25.8, 25.6, 22.4, 14.0,
(0.2 mmol), and the solution was heated to 65 °C and refluxed 12.2. HRMS (ESI): m/z calcd for C33H43NO5S3, [M + H]+
for 6–8 h. The solution was then filtered, and the filtrate was 630.2303; found 630.2387.
( )-4-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)butyl 2-(1-(2-(4-
methylpiperazin-1-yl)acetoxy)pentyl)benzoate (9d). The title
compound was obtained as a reddish brown oil (71%), MS
reconstituted in EtOAc (20 mL) and the solvent was removed
under reduced pressure. The crude compounds were purified
by flash chromatography (PE–EtOAc = 5/1–1 : 1, v/v), to give the
title compounds (65–71%).
1
(ESI): m/z 629.2 [M + H]+. H NMR (300 Hz, CDCl3): δ 0.89 (t,
3H, CH3, J = 6.9 Hz), 1.25–1.42 (m, 4H, CH2CH2CH3), 1.54–1.62
( )-4-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)butyl
2-(1-(2-
(diethylamino) acetoxy)pentyl)benzoate (9a). The title com- (m, 2H, CHCH2), 1.80–1.86 (m, 4H, ArCOOCH2(CH2)2), 2.28 (s,
pound was obtained as a reddish brown oil (66%), MS (ESI): 3H, NCH3), 2.48–2.58 (m, 8H, 4 × NCH2), 3.24 (s, 2H,
m/z 602.2 [M + H]+. 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, OCOCH2N), 4.10 (t, 2H, ArOCH2, J = 6.2 Hz), 4.40 (t, 2H,
CH3, J = 6.9 Hz), 1.06 (t, 6H, 2 × NCH2CH3, J = 7.2 Hz), ArCOOCH2, J = 6.4 Hz), 6.62 (dd, 1H, CH, J = 5.1 Hz, 7.7 Hz),
1.26–1.45 (m, 4H, CH2CH2CH3), 1.76–1.88 (m, 2H, CHCH2), 6.96 (d, 2H, ArH, J = 8.8 Hz), 7.26–7.31 (m, 1H, ArH), 7.39 (s,
2.00–2.04 (m, 4H, ArCOOCH2(CH2)2), 2.68 (q, 4H,
2
×
1H, CvCH), 7.50–7.51 (m, 2H, ArH), 7.59 (d, 2H, ArH, J =
NCH2CH3, J = 7.1 Hz), 3.42 (s, 2H, OCOCH2N), 4.10 (t, 2H, 8.8 Hz), 7.86 (d, 1H, ArH, J = 8.0 Hz). 13C NMR (75 Hz, CDCl3):
ArOCH2, J = 6.2 Hz), 4.41 (t, 2H, ArCOOCH2, J = 6.2 Hz), 6.62 δ 173.1, 170.3, 166.9, 162.3, 143.7, 134.8, 134.6, 132.3, 131.3,
(dd, 1H, CH, J = 5.1 Hz, 7.8 Hz), 6.96 (d, 2H, ArH, J = 8.8 Hz), 130.1, 128.6, 127.1, 126.2, 125.8, 124.1, 115.5, 92.6, 73.1, 67.8,
7.27–7.32 (m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.52–7.55 (m, 2H, 64.6, 59.4, 54.8, 52.9, 45.9, 36.6, 29.7, 28.0, 25.4, 22.6, 14.0.
ArH), 7.58 (d, 2H, ArH, J = 8.8 Hz), 7.87 (d, 1H, ArH, HRMS (ESI): m/z calcd for C32H40N2O5S3, [M + H]+ 629.2099;
J = 7.6 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, found 629.2183.
( )-5-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)pentyl 2-(1-(2-(4-
methylpiperazin-1-yl)acetoxy)pentyl)benzoate (9e). The title com-
pound was obtained as a reddish brown oil (70%), MS (ESI):
m/z 643.2 [M + H]+. 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H,
CH3, J = 6.9 Hz), 1.24–1.46 (m, 4H, CH2CH2CH3), 1.52–1.56 (m,
2H, CHCH2), 1.84–1.92 (m, 6H, ArCOOCH2(CH2)3), 2.27 (s, 3H,
NCH3), 2.47–2.57 (m, 8H, 4 × NCH2), 3.23 (s, 2H, OCOCH2N),
4.06 (t, 2H, ArOCH2, J = 6.2 Hz), 4.36 (t, 2H, ArCOOCH2, J = 6.4
Hz), 6.62 (dd, 1H, CH, J = 5.0 Hz, 7.8 Hz), 6.96 (d, 2H, ArH, J =
8.8 Hz), 7.27–7.30 (m, 1H, ArH), 7.39 (s, 1H, CvCH), 7.50–7.51
(m, 2H, ArH), 7.59 (d, 2H, ArH, J = 8.8 Hz), 7.87 (d, 1H, ArH, J =
7.8 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2,
125.8, 124.1, 115.5, 92.6, 72.2, 67.3, 64.1, 58.5, 53.9, 52.0, 45.0,
35.7, 28.7, 28.0, 27.6, 22.6, 21.6, 14.0. HRMS (ESI): m/z calcd
for C33H42N2O5S3, [M + H]+ 643.2256; found 643.2339.
( )-6-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)hexyl 2-(1-(2-(4-
methylpiperazin-1-yl)acetoxy)pentyl)benzoate (9f). The title com-
pound was obtained as a reddish brown oil (65%), MS (ESI):
m/z 657.0 [M + H]+. 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H,
CH3, J = 6.8 Hz), 1.25–1.47 (m, 4H, CH2CH2CH3), 1.50–1.56 (m,
2H, CHCH2), 1.80–1.84 (m, 8H, ArCOOCH2(CH2)4), 2.28 (s, 3H,
NCH3), 2.48–2.58 (m, 8H, 4 × NCH2), 3.24 (s, 2H, OCOCH2N),
4.03 (t, 2H, ArOCH2, J = 6.4 Hz), 4.33 (t, 2H, ArCOOCH2, J = 6.6
Hz), 6.62 (dd, 1H, CH, J = 5.0 Hz, 7.8 Hz), 6.96 (d, 2H, ArH, J =
8.8 Hz), 7.28–7.32 (m, 1H, ArH), 7.39 (s, 1H, CvCH), 7.49–7.50
(m, 2H, ArH), 7.59 (d, 2H, ArH, J = 8.8 Hz), 7.87 (d, 1H, ArH, J =
7.6 Hz). 13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3,
143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2,
125.8, 124.1, 115.5, 92.6, 72.2, 67.3, 64.1, 58.5, 53.9, 52.0, 45.0,
35.7, 28.7, 28.0, 27.6, 24.8, 22.8, 22.4, 14.0. HRMS (ESI): m/z
calcd for C34H44N2O5S3, [M + H]+ 657.2412; found 657.2497.
162.3, 143.7, 134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1,
126.2, 125.8, 124.1, 115.5, 92.6, 72.9, 67.8, 64.6, 47.6,
36.3, 28.1, 25.8, 25.4, 22.5, 21.2, 14.0, 12.2. HRMS (ESI):
m/z calcd for C31H39NO5S3, [M
+
H]+ 602.1990; found
602.2074.
( )-5-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)pentyl
2-(1-(2-
(diethylamino)acetoxy)pentyl)benzoate (9b). The title compound
was obtained as a reddish brown oil (67%), MS (ESI): m/z 616.2
[M + H]+. 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3,
J = 6.8 Hz), 1.06 (t, 6H, 2 × NCH2CH3, J = 7.1 Hz), 1.26–1.45
(m, 4H, CH2CH2CH3), 1.60–1.67 (m, 2H, CHCH2), 1.75–2.04
(m, 6H, ArCOOCH2(CH2)3), 2.68 (q, 4H, 2 × NCH2CH3,
J = 7.1 Hz), 3.42 (s, 2H, OCOCH2N), 4.05 (t, 2H, ArOCH2,
J = 6.2 Hz), 4.36 (t, 2H, ArCOOCH2, J = 6.4 Hz), 6.62 (dd, 1H,
CH, J = 4.9 Hz, 8.0 Hz), 6.96 (d, 2H, ArH, J = 8.8 Hz), 7.28–7.32
(m, 1H, ArH), 7.38 (s, 1H, CvCH), 7.51–7.54 (m, 2H, ArH),
7.58 (d, 2H, ArH, J = 8.8 Hz), 7.87 (d, 1H, ArH, J = 7.6 Hz).
13C NMR (75 Hz, CDCl3): δ 173.1, 170.3, 166.9, 162.3, 143.7,
134.8, 134.6, 132.3, 131.3, 130.1, 128.6, 127.1, 126.2, 125.8,
124.1, 115.5, 92.6, 72.9, 67.8, 64.6, 47.6, 36.7, 29.7, 28.7, 28.4,
22.6, 22.5, 21.2, 14.0, 12.2. HRMS (ESI): m/z calcd for
C32H41NO5S3, [M + H]+ 616.2147; found 616.2230.
( )-6-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)hexyl
2-(1-(2-
(diethylamino)acetoxy)pentyl)benzoate (9c). The title compound
was obtained as a reddish brown oil (70%), MS (ESI): m/z 630.2
[M + H]+. 1H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.8 Hz),
1.07 (t, 6H, 2 × NCH2CH3, J = 7.1 Hz), 1.26–1.47 (m, 4H,
CH2CH2CH3), 1.60–1.66 (m, 2H, CHCH2), 1.82–1.84 (m, 8H,
ArCOOCH2(CH2)4), 2.70 (q, 4H, 2 × NCH2CH3, J = 7.0 Hz), 3.44
(s, 2H, OCOCH2N), 4.03 (t, 2H, ArOCH2, J = 6.3 Hz), 4.34 (t, 2H,
ArCOOCH2, J = 6.6 Hz), 6.62 (dd, 1H, CH, J = 5.0 Hz, 7.8 Hz),
6.96 (d, 2H, ArH, J = 8.8 Hz), 7.28–7.32 (m, 1H, ArH), 7.38
(s, 1H, CvCH), 7.51–7.54 (m, 2H, ArH), 7.58 (d, 2H, ArH, J =
8.8 Hz), 7.87 (d, 1H, ArH, J = 7.6 Hz). 13C NMR (75 Hz, CDCl3):
4.2. Antiplatelet aggregation effect in vitro
δ 173.1, 170.3, 166.9, 162.3, 143.7, 134.8, 134.6, 132.3, 131.3, Blood samples were withdrawn from rabbit carotid artery and
130.1, 128.6, 127.1, 126.2, 125.8, 124.1, 115.5, 92.6, 72.9, 67.8, mixed with 3.8% sodium citrate solution (9 : 1, v/v), followed
6002 | Org. Biomol. Chem., 2014, 12, 5995–6004
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by centrifuging at 500 rpm for 10 min at room temperature. right carotid artery with the left jugular vein of rats. The poly-
After the resulting platelet-rich plasma (PRP) supernatant was ethylene tube (14 cm, containing 6 cm long 4 braided silk
collected, the residue was centrifuged at 3000 rpm for another threads) was filled with saline before installation. After 15 min
10 min at room temperature to obtain platelet-poor plasma circulation of blood through the shunt tube, both ends of the
(PPP). The PRP was adjusted with PPP in order to obtain plate- tubing were pinched, and the cotton thread was removed from
let counts of 400–450 × 109 Pl L−1. Platelet aggregation was the shunt tube. The wet weight of the cotton thread was
determined by Born’s turbidimetric method using a four- measured immediately and the cotton thread was dried at the
channel aggregometer (LG-PABER-I Platelet-Aggregometer, room temperature for 6 h, followed by measuring the dry
Beijing, China) within 3 h after blood collection. Briefly, PRP weight. The wet and dry weights of thrombus formed on the
(240 µL) was pre-incubated with vehicle, positive control or cotton threads were determined by subtracting the pre-experi-
different concentrations (0.1, 0.2, 0.4, 0.8 and 1.6 mM) of indi- ment weight of the dry 6 cm long threads.
vidual compounds (30 µL) for 5 min at 37 °C, followed by the
addition of 10 µM of the adenosine 5′-diphosphate sodium
salt (ADP, Sigma-Aldrich, USA) or 0.33 mM of the arachidonic
acid (AA, Cayman Chemical, USA) respectively to induce the
platelet aggregation. The maximum aggregation rate (MAR)
4.5. Assay of hydrogen sulfide release in vitro
Compound 8e (100 μM) was incubated (37° C) for timed inter-
vals (0–90 min) with phosphate buffer or fresh rat liver homo-
genate (430 μL) and the concentration of released H2S was
was recorded within 5 min at 37 °C. The inhibition rate of the
measured as described previously.32 After incubation, zinc
tested compounds on platelet aggregation was calculated with
acetate (1% w/v, 250 μL) was injected followed by trichloroacetic
acid (10% w/v, 250 μL) to stop the reaction. Subsequently,
N,N-dimethyl-p-phenylenediamine sulfate (20 μM; 133 μL) in
7.2 M HCl was added followed by FeCl3 (30 μM, 133 μL) in
the following formula: Inhibition rate (%) = (100% − MAR of
tested compound/MAR of vehicle). The IC50 value of each com-
pound was calculated accordingly.
1.2 M HCl. The resulting solution was determined 10 min
thereafter using a microplate reader at 670 nm. The H2S con-
centration of 8e was calculated against a standard curve of
NaHS (3.125, 6.25, 12.5, 25, 50, 100 μM). Results are expressed
as total % conversion of 8e to H2S at each time point.
4.3. Inhibitory effect on the thrombosis in mice
Male ICR mice (30–35 g) were purchased from B&K Universal
Group Limited, Shanghai, China. Animals were kept under
standard laboratory conditions and maintained in a 12 h
light–dark cycle with free access to food and water. All pro-
cedures were performed following institutional approval in
accordance with the NIH Guide for the Care and Use of Lab-
oratory Animals. A model of acute systemic vascular thrombo-
embolism induced by infusion of a mixture of collagen and
adrenaline was performed as described previously.30 Briefly,
the mice were divided into groups randomly and treated orally
with vehicle dimethyl sulfoxide (DSMO), aspirin (160 mg kg−1),
NBP (160 mg kg−1), or 8e (160 or 470 mg kg−1) by gavage daily
for seven days. Two hours after the last administration, the
mice were injected intravenously with a mixture of 0.21 mg
kg−1 collagen and 44.5 μg kg−1 adrenaline. Mice were observed
for thrombosis-related death within 15 min. The results are
expressed as the percentage of mice alive as a function of time.
The protective rates against vehicle are also calculated.
Acknowledgements
This study was financially supported by grants from the Major
National Science and Technology Program of China for Innova-
tive Drug during the Eleventh Five-Year Plan Period (no.
2009ZX09103-095), the Project Program of State Key Laboratory
of Natural Medicines, China Pharmaceutical University (no.
ZJ11176), 2014 Independent Research Projects of Youth Fund,
Jiangnan University (no. 1012050205142380) and 2013 Inno-
vation Program of National College Students, Jiangnan Univer-
sity (no. 1012050205135820).
4.4. Antithrombotic activity assay in rats
Notes and references
Male Sprague-Dawley (SD) rats (250–280 g) were randomized
and divided into six groups (n = 12 per group): (1) control
group (0.5% CMC-Na), (2) vehicle group (A-V + 0.5% CMC-Na),
(3) A-V + NBP (80 mg kg−1) group, (4) A-V + aspirin (80 mg
kg−1) group, (5) A-V + 8e (80 mg kg−1) group, and (6) A-V + 8e
(235 mg kg−1) group. The 3–6 groups of rats were treated by
gavage with the indicated dose of the compound in saline
daily for consecutive 7 days. Subsequently, the rats were
anesthetized by intraperitoneal (i.p.) injection of pentobarbital
(50 mg kg−1). The control group of rats received a sham
surgery while the remaining rats were subjected to the arterio-
venous (A-V) shunt procedure.31 Briefly, individual rats were
inserted with an A-V shunt tube that directly connected the
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