Discovery of a ring-opened derivative of 3-n-butylphthalide bearing NO/H2S-donating moieties as a potential anti-ischemic stroke agent

Article abstract of DOI:10.1016/j.ejmech.2016.03.044

To search for novel anti-ischemic stroke agents with higher potency than a known drug 3-n-butylphthalide (NBP), a series of ring-opened derivatives of NBP bearing both nitric oxide (NO) and hydrogen sulfide (H₂S)-donating moieties (NO/H₂S-NBP) (8a-8o) were designed, synthesized, and biologically evaluated. The most active compound 8d was more potent than NBP and the corresponding H₂S-NBP 10 or NO-NBP 13 in inhibition of the ADP-induced platelet aggregation in vitro. In addition, 8d produced moderate levels of NO and H₂S, which could be beneficial for improving cardiovascular and cerebral circulation. More importantly, in a rat model of transient focal cerebral ischemia, oral treatment with 8d improved neurobehavioral function, reduced the infarct brain size and brain-water content, and enhanced the levels of brain antioxidant SOD, GSH and GSH-Px but diminished the level of oxidant MDA. These protective effects of 8d against the ischemia/reperfusion (I/R)-related brain damage were greater than that of NBP, suggesting that 8d may be a promising agent for further investigation.

Full text of DOI:10.1016/j.ejmech.2016.03.044

European Journal of Medicinal Chemistry 115 (2016) 369e380
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of a ring-opened derivative of 3-n-butylphthalide bearing
NO/H2S-donating moieties as a potential anti-ischemic stroke agent
,
b
,
,
,
b
,
,
Wei Yin a 1, Li Lan a 1, Zhangjian Huang a 1, Jing Ji a, Jiangen Fang a b, Xiaoli Wang c,
Hui Ji a ***, Sixun Peng a b, Jinyi Xu a **, Yihua Zhang a
,
,
,
, b, *
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, PR China
c The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Avenue 1800,
Wuxi 214122, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To search for novel anti-ischemic stroke agents with higher potency than a known drug 3-n-butylph-
thalide (NBP), a series of ring-opened derivatives of NBP bearing both nitric oxide (NO) and hydrogen
sulfide (H2S)-donating moieties (NO/H2S-NBP) (8a-8o) were designed, synthesized, and biologically
evaluated. The most active compound 8d was more potent than NBP and the corresponding H2S-NBP 10
or NO-NBP 13 in inhibition of the ADP-induced platelet aggregation in vitro. In addition, 8d produced
moderate levels of NO and H2S, which could be beneficial for improving cardiovascular and cerebral
circulation. More importantly, in a rat model of transient focal cerebral ischemia, oral treatment with 8d
improved neurobehavioral function, reduced the infarct brain size and brain-water content, and
enhanced the levels of brain antioxidant SOD, GSH and GSH-Px but diminished the level of oxidant MDA.
These protective effects of 8d against the ischemia/reperfusion (I/R)-related brain damage were greater
than that of NBP, suggesting that 8d may be a promising agent for further investigation.
Received 24 December 2015
Received in revised form
16 March 2016
Accepted 17 March 2016
Available online 19 March 2016
Keywords:
Nitric oxide
Hydrogen sulfide
Anti-platelet aggregation
Ischemic stroke
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
The 3-n-butylphthalide (NBP, Fig. 1) was approved by the State
Food and Drug Administration (SFDA) of China as a new drug for
Ischemic stroke accounts for more than 80% of all strokes, and is
a leading cause of death and long-term disability globally [1,2]. In
the past years, remarkable progresses have been made in the un-
derstanding of the pathogenesis of ischemic stroke, including ge-
netic high risk, thrombosis, and chronic inflammatory diseases
such as hypertension, diabetes, and others [3e5]. To date, many
drugs are available for the intervention of ischemic stroke, such as
anti-platelet aggregation drugs, anticoagulant drugs, neuro-
protective drugs, and thrombolytic drugs [6e9]. However, the ef-
ficacy of the current drugs is not satisfactory. Therefore, the
development of novel and more effective drugs for the treatment of
ischemic stroke will be of great significance.
the treatment of ischemic stroke in 2002. Previous studies have
shown that NBP possesses beneficial effects on stroke through
multiple actions that affect different pathophysiological processes
[10,11], including inhibiting platelet aggregation, reducing
thrombus formation, improving microcirculation, regulating en-
ergy metabolism, decreasing brain infarct volume and oxidative
damage. However, the efficacy of NBP is moderate. To improve its
therapeutic effects, it is often administered by combination with
other anti-platelet drug(s) [12]. In addition, a range of structural
modification and related studies on NBP have been carried out. It is
shown that the potassium salt of 2-(1-hydroxypentyl)-benzoate
(HPBA, Fig. 1), a ring-opened NBP, has comparable potency to NBP
and that a lot of HPBA derivatives have also been investigated
[13e15]. However, the potency of these derivatives is still not
satisfactory. Therefore, new derivatives of NBP with more potency
are needed for the treatment of ischemic stroke.
* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, Nanjing 210009, PR China.
** Corresponding author.
Nitric oxide (NO), a free radical gas, is a messenger molecule
with a variety of physiological roles, such as relaxing vascular
smooth muscle, inhibiting platelet aggregation, reducing
*** Corresponding author.
E-mail address: zyhtgd@163.com (Y. Zhang).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.03.044
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
10 and 13 for comparison in biological evaluation where 10 was
used as H2S-NBP while 13 was used as NO-NBP.
2.2. Anti-platelet aggregation activity in vitro
The inhibitory effects of the target compounds on the adenosine
diphosphate (ADP)-induced in vitro platelet aggregation in rabbit
platelet rich plasma (PRP) were preliminarily evaluated using
Born's turbidimetric method. NBP was used as a positive control. As
shown in Fig. 2, all the target compounds displayed better inhibi-
tory effects (19.74%e43.04%) than NBP (12.95%) at the same con-
centration (0.1 mM), among which 8d (43.04%) was the most active.
Subsequently, the eight most potent compounds (8d, 8e, 8g, 8h,
8j, 8k, 8m and 8o) were further assayed for their anti-platelet ac-
tivity against the ADP induced platelet aggregation. Data were
calculated and expressed as the IC50 values. As shown in Table 1, all
of the tested compounds displayed higher inhibitory activity than
NBP. Among them, 8d was the most active inhibitor
(IC50 ¼ 0.14 mM), which was 5.2-fold more potent than NBP
(IC50 ¼ 0.74 mM). Therefore, 8d was selected for the following
investigations.
Given that 8d was composed of two moieties (3d and 7a), we
further investigated their corresponding effects on the ADP-
induced platelet aggregation in vitro. As shown in Fig. 3, 3d and
7a at 0.1 mM inhibited the ADP-induced platelet aggregation by
16.75% and 18.00%, respectively, which were lower than that of 8d
(40.95%), suggesting that these moieties may synergistically inhibit
the ADP-induced platelet aggregation in vitro. To confirm that, 7a
and 3d in combination were then tested for the inhibitory activity.
As shown in Fig. 3, 8d had a stronger anti-platelet aggregation ac-
tivity than combination of 7a with 3d, which further support that
both 3d and 7a displayed synergistic effects on anti-platelet ag-
gregation of 8d. Importantly, as shown in Fig. 3, the inhibitory ac-
tivity of either 10 (27.81%) or 13 (19.75%) was significantly less than
8d (40.95%), indicating that the NO/H2S-NBP is more potent than
the NO-NBP or H2S-NBP alone in inhibition of the ADP-induced
platelet aggregation. Furthermore, the effects of NO and H2S on
the antiplatelet aggregation activity of 8d were investigated. The
Fig. 1. Chemical structures of NBP and HPBA.
neutrophil adherence and activation, mitigating cell death, and
promoting cell proliferation [16e21]. Thanks to these advantages,
conjugation of NO donor with a known drug has been recognized as
a means to improve activity of the anti-ischemic stroke drugs [22].
Recently, structurally diverse NO-donating derivatives of NBP have
been reported by our group, and several derivatives exhibit greater
anti-platelet and anti-thrombotic activities than NBP [22e24].
Hydrogen sulfide (H2S) is known as the third gasotransmitter,
following NO and carbon monoxide (CO). In central nervous system
(CNS), H2S is produced through the enzymes cystathionine
(CSE), cystathionine -synthase (CBS), and 3-mercaptopyruvate
sulfurtransferase (3-MST) [25]. H2S can regulate neurotransmis-
sion, acting as neuroprotectant via its antioxidant, anti-
g-lyase
b
a
inflammatory, and anti-apoptotic effects. Indeed, evidence accu-
mulated over the past decades shows that H2S has a potential
therapeutic value in the treatment of several CNS diseases,
including ischemic stroke, Alzheimer's disease, Parkinson's disease,
etc [25e29]. More recently, our group has reported H2S-based
derivatives of NBP, some of which show better anti-platelet and
anti-thrombotic activities than NBP [30].
Based on the above investigation, we hypothesized that NO/
H2S-donating derivatives of NBP (NO/H2S-NBP) could display syn-
ergistic effects on ant-ischemic stroke with more potency than NBP,
and corresponding NO-NBP or H2S-NBP. To verify this hypothesis, a
novel class of NO/H2S-NBP were synthesized by using HPBA as a
scaffold to which NO and H2S donors were coupled. Nitrate (-(CH2)
mONO2) was employed as an NO-donating moiety attached to
HPBA through ferulic acid as a linker, which has anti-oxidant and
anti-platelet aggregation activities [31]. In addition, 5-(4-
antiplatelet effect of 8d was attenuated by treatment with 20 mM of
carboxy-PTIO (a known NO scavenger [34]) or bismuth (Ⅲ) subsa-
licylate (BSS, a reported H2S scavenger [35]), from 40.95% to 29.15%
and 40.95%e20.86%, respectively. These results further support the
important roles of NO and H2S-releasing groups for antiplatelet
effect of 8d.
hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH),
a
known
H2S-donor, in which 1,2-dithiole-3-thione is a H2S-donating moiety
[32], was introduced to the side chain of HPBA via a substituted
acetate linkage. Herein, we report synthesis of these NO/H2S-NBP,
and their in vitro anti-platelet aggregative and in vivo anti-ischemic
activities.
2.3. Assay of NO and H2S release in vitro
2. Results and discussion
In order to examine whether the target compounds could
release both NO and H2S, we first determined the levels of NO
produced by 8d, 8e, 8g, 8h, 8j, 8k, 8m and 8o by Griess assay [36].
As shown in Fig. 4, the active compounds 8d, 8o and 8k released
2.1. Chemistry
The synthesis of compounds 8a-8o was depicted in Scheme 1.
Compounds 1a-1e, 6, 11 and 12 were synthesized as previously
described [22,30,33]. Treatment of 1a-1e with N-boc-piperazine
gave protected intermediates 2a-2e. The subsequent removal of the
t-Boc protecting group by anhydrous HCl in ethyl acetate furnished
H2S donors 3a-3e. The ferulic acid was converted into the corre-
sponding bromo-substituted esters 4a-4c using Et3N in acetone.
The SN2 reaction of 4a-4c with silver nitrate in CH3CN afforded
corresponding NO donors 5a-5c. Compound 6 was coupled with
5a-5c and methyl ferulate, respectively, in the presence of DCC/
DMAP to yield corresponding esters 7a-7c and 9. Finally, com-
pounds 3a-3e were condensed with 7a-7c to provide the target
compounds 8a-8o, respectively. In addition, compound 9 and 7a
were condensed with 3d and 12 in the presence of Et3N leading to
moderate amount of NO (10.57, 10.22 and 9.57 mM, respectively). In
contrast, the less active compounds 8h, 8e, 8j, 8m and 8g under the
same conditions released lower levels of NO (8.37, 7.51, 5.56, 5.38
and 3.32 mM, respectively).
Next, the levels of H2S produced by 8d, 8e, 8g, 8h, 8j, 8k, 8m and
8o were measured using the method as previously reported [37]. As
shown in Fig. 5, the active compounds 8d, 8k and 8o released
moderate levels of H2S (4.4%, 3.82% and 3.32%, respectively) while
the less active compounds 8h, 8e, 8j, 8m and 8g released lower
levels of H2S (2.72%, 2.41%, 1.82%, 1.35% and 1.23%, respectively).
2.4. Anti-cerebral ischemic activity in rats
We further investigated the anti-cerebral ischemic activity of 8d

W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
371
Scheme 1. Synthesis of 8a-8o, 10 and 13. Reagents and conditions: (a) N-boc-piperazine, DMF, 70 ꢀC, 8 h; (b) HCl/EtOAc, r.t.; (c) Br(CH2)mBr, Et3N, acetone, reflux, 1e2 d; (d) AgNO3,
CH3CN, reflux, 6 h; (e) 5a-5c, DCC, DMAP, CH2Cl2, 0 ꢀC to r.t.; (f) 3a-3e, Et3N, CH2Cl2, r.t., 12 h; (g) Methyl ferulate, DCC, DMAP, CH2Cl2, 0 ꢀC to r.t.; (h) 3d, Et3N, CH2Cl2, r.t., 12 h; (i)
Br(CH2)5Br, K2CO3, DMF, r.t., 24 h; (j) Piperazine, K2CO3, EtOH, 70 ꢀC, 8 h; (k) 7a, Et3N, CH2Cl2, r.t., 12 h.
in a rat model of transient focal cerebral ischemia by intraluminal
occlusion of the middle cerebral artery (MCAO) for 2 h followed by
recirculation, which has been widely used to evaluate the protec-
tive effects of anti-stroke agents [38]. Male SpragueeDawley rats
were randomly and orally treated with 0.5% sodium carboxyl
methyl cellulose (CMC-Na) (sham group and model group), NBP
(80 mg/kg), or 8d (80 mg/kg and 380 mg/kg, which were equal dose
and molar to NBP, respectively) for seven days, and the rats were
subjected to sham surgery or the MCA occlusion and reperfusion.
The neurological functions of individual rats were assessed using
Longa's method 24 h after reperfusion. As shown in Fig. 6, rats in
the sham-operated group had no obvious neuronal abnormality,
while neurological deficit scores in ischemia/reperfusion (I/R)
model group were significantly higher (P < 0.001), and no signifi-
cant difference in neurological deficit was found between the 8d-
treated group (80 mg/kg) and the model group. However, the

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W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
Fig. 3. Inhibition of ADP-induced rabbit platelet aggregation by selected compounds
in vitro. Rabbit platelet suspensions were pre-incubated with tested compounds
(0.1 mM) at 37 ꢀC for 5 min followed by addition of ADP (10
M). 20 M of Carboxy-
m
m
PTIO or BSS was added and incubated with the drug-platelet suspension in the indi-
cated group. Data are expressed as the means (%) SD of each compound from three
independent experiments. **P < 0.01, ***P < 0.001 vs. 8d.
Fig. 2. The effects of compounds on the ADP (10 mM)-induced platelet aggregation
in vitro. Rabbit platelet suspensions were preincubated with 0.1 mM of each compound
tested at 37 ꢀC for 5 min followed by the addition of ADP (10
M). Data are expressed
as the means (%) SD of each compound from three independent experiments.
with both doses of 8d (80 mg/kg and 380 mg/kg) remarkably
decreased MDA level (from 1.46 to 1.07 nmol/mg protein and from
1.46 to 0.86 nmol/mg protein, respectively). Meanwhile, the GSH
level in 8d-treated group (380 mg/kg) was enhanced to 28.05 nmol/
mg protein, which was somewhat greater than that in the NBP-
m
*P < 0.05, **P < 0.01, ***P < 0.001 vs. the NBP.
neurological deficit scores in the 8d-treated I/R rats (380 mg/kg),
but not in the NBP-treated group (80 mg/kg), were significantly
reduced as compared with the model group (P < 0.01). These re-
sults suggested that 8d displayed a better protective effect at an
equal molar concentration of NBP on the surgical MCAO injury.
Similarly, the infarct size of individual rats were evaluated by
2,3,5-triphenyltetrazolium chloride (TTC) assay. In comparison
with the model group, the cerebral infarct size in the 8d-treated I/R
rats was significantly reduced, particularly in rats treated with
higher dose of 8d (P < 0.001) (Fig. 7A). Additionally, administration
of 8d (380 mg/kg) markedly reduced the water content in I/R rat
brain (Fig. 7B).
2.5. Antioxidative effects
In order to explore the mechanism underlying the anti-cerebral
ischemic effects of 8d, we evaluated the levels of malondialdehyde
(MDA), glutathione (GSH), superoxide dismutase (SOD) and gluta-
thione peroxidase (GSH-Px) in the brain tissues. In comparison
with that in the sham group of rats, there was a significant elevation
in MDA level (from 0.87 to 1.46 nmol/mg protein, Fig. 8A) and
decline in GSH level (from 29.21 to 23.47 nmol/mg protein, Fig. 8B)
in the model group. In contrast to the model group, pretreatment
Fig. 4. NO-releasing assay of selected compounds in vitro. The levels of NO produced
by selected compounds (0.1 mM) and presented as nitrite were determined by Griess
assay. Individual compounds were incubated for the indicated periods and reacted
with Griess reagent, followed by measuring at 540 nm. The levels of NO produced by
individual compounds were calculated, according to the standard curve of nitrite. Data
are expressed as the means of individual compounds tested at each time point and
intra-group variations were less than 10%.
Table 1
The IC50 values of compounds against platelet ag-
gregation in vitro.
Compd.
IC50 (mM)
ADP (10 mM)
Control
NBP
8d
8e
8g
8h
8j
8k
8m
8o
d
0.74 0.20
0.14 0.08
0.43 0.12
0.43 0.04
0.36 0.02
0.39 0.03
0.21 0.02
0.32 0.02
0.19 0.03
IC50 values (a dose achieved 50% inhibition of platelet
aggregation) are expressed as mean SD (n ¼ 6) and
analyzed by one-way analysis of variance (ANOVA)
followed by post hoc Tukey's test.
Fig. 5. Release of H2S from selected compounds in vitro. Selected compounds (0.1 mM)
was incubated (37 ꢀC) for 90 min with fresh rat liver homogenate in vitro. H2S con-
version was determined. Data are expressed as accumulate % conversion of selected
compounds to H2S at each time point, n ¼ 6.

W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
373
were obviously decreased in model group by 26.64% (from 119.10 to
87.37 U/mg protein, Fig. 8C) and 45.34% (from 28.21 to 15.42 U/mg
protein, Fig. 8D), respectively, compared with that in the sham
group. In contrast, pretreatment with 8d at 80 and 380 mg/kg
significantly enhanced the SOD activity by 23.07% (from 87.37 to
107.53 U/mg protein) and 32.02% (from 87.37 to 115.35 U/mg pro-
tein), respectively, compared with that in the model group. More-
over, the GSH-Px levels of 8d-treated group (80 and 380 mg/kg)
were elevated by 29.57% (from 15.42 to 19.98 U/mg protein) and
69.26% (from 15.42 to 26.10 U/mg protein), respectively. Notably,
SOD and GSH-Px activities in the 8d-treated group (380 mg/kg)
were somewhat greater than those in the NBP group (24.36%,
108.65 U/mg protein; 63.55%, 25.22 U/mg protein, respectively).
3. Conclusion
In summary, a series of NO/H2S-donating derivatives of NBP
were designed, synthesized and biologically evaluated. The most
active compound 8d displayed the more potent inhibitory activity
on the ADP-induced platelet aggregation in vitro than NBP, and the
corresponding NO-NBP or H2S-NBP. Furthermore, 8d produced
moderate levels of NO and H2S in vitro, which could be beneficial for
improving cardiovascular and cerebral circulation. Most impor-
tantly, treatment with 8d improved neurobehavioral function,
reduced the infarct brain size and brain-water content, thus
Fig. 6. Effects of 8d and NBP on neurological deficit score in rats subjected to I/R
(n ¼ 12). Data are assessed 24 h after reperfusion and analyzed by KruskaleWallis test,
followed by the ManneWhitney U test. ***P < 0.001 vs. the sham group, #P < 0.05,
##P < 0.01 vs. the model group.
treated group (25.22 nmol/mg protein) and in vehicle group
(23.47 nmol/mg protein).
Further analysis revealed that the activities of SOD and GSH-Px
Fig. 7. (A) Effects of NBP and 8d on infarction after cerebral I/R in rats. (a) TTC staining analysis of the infarcted brain regions. Data shown are representative examples from each
treatment group of rats. (b) Quantitative analysis of the infarcted brain regions. The ratios of infarct area to whole brain areas were calculated. Data are expressed as the mean SD
(n ¼ 6) and analyzed by ANOVA followed by post hoc Tukey test. **P < 0.01, ***P < 0.001 vs. the model group, #P < 0.05 vs. the NBP group. (B) Effects of NBP and 8d on brain edema
after cerebral I/R in rats. Data are expressed as the mean SD (n ¼ 6) and analyzed by ANOVA followed by post hoc Tukey test. ***P < 0.001 vs. the sham group. ##P < 0.01,
###P < 0.001 vs. the model group. &P < 0.05 vs. the NBP group.

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W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
Fig. 8. Effect of 8d on the levels of brain MDA (A), GSH (B), SOD activity (C) and GSH-Px activity (D) in rats after I/R in ischemic cerebral cortex. Data are expressed as the mean SD
of individual groups of rats (n ¼ 6) and were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Turkey test. ***P < 0.01 vs. the sham group. #P < 0.05,
##P < 0.01, ###P < 0.001 vs. the vehicle group. &P < 0.05 vs. the NBP group.
diminished cerebral damage in a rat model of MCAO. These pro-
tective effects of 8d on the I/R-related brain damage were greater
than that of NBP. In addition, treatment with 8d significantly
attenuated the I/R related oxidative stress in rat brains by
increasing the levels of brain antioxidant SOD, GSH and GSH-Px but
reducing the level of brain MDA, an index of reduced lipid peroxi-
dation in the brains. Apparently, 8d inhibited oxidative stress,
which at least partially contributed to its anti-I/R brain injury in
rats. Collectively, our findings suggest that 8d may be a promising
agent for further investigation.
necessary, were purified and dried by standard methods. Solutions
after reactions and extractions were concentrated using a rotary
evaporator operating at a reduced pressure of ca. 20 Torr. Com-
pounds 1a-1e, bromo-substituted esters 4a-4c, corresponding ni-
trate 5a-5c, 6, 11 and 12 were synthesized as previously described
[22,30,33,39,40].
4.1.1. General procedure for the preparation of 2a-2e
To a stirred solution of 1a-1e (1.0 mmol) and N-boc-piperazine
(0.224 g, 1.2 mmol) in DMF (20 mL) was added K2CO3 (0.415 g,
3.0 mmol). The mixture was then heated to 70 ꢀC and stirred for 8 h.
The reaction mixture was filtered, and the filtrate was concentrated
under reduced pressure. The resulting residue was extracted with
dichloromethane (20 mL) three times. The organic layer was
separated and dried using anhydrous sodium sulfate, concentrated,
purified by flash chromatography (petroleum ether/EtOAc, 1:1 v/v)
to afford the title compounds.
4. Experimental protocols
4.1. Chemical analysis
Melting points of individual compounds were determined on a
Mel-TEMP II melting point apparatus and uncorrected. 1H NMR and
13C spectra were recorded with a Bruker Avance 300 MHz spec-
trometer at 303 K, using TMS as an internal standard. All com-
pounds were routinely checked by TLC and 1H NMR. TLC were
performed on silica gel GF/UV 254, and the chromatograms were
conducted on silica gel (200e300 mesh) and visualized under UV
light at 254 and 365 nm. All solvents were reagent grade and, when
4.1.1.1. tert-Butyl
4-(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
ethyl)piperazine-1- carboxylate (2a). The title compound was ob-
tained as a reddish brown oil, 65% yield. Analytical data for 2a: 1H
NMR (300 MHz, CDCl3)
J ¼ 8.8 Hz, 2H), 4.16 (t, J ¼ 5.6 Hz, 2H), 3.45 (t, J ¼ 6.0 Hz, 4H), 2.85 (t,
d
7.59 (d, J ¼ 8.8 Hz, 2H), 7.37 (s, 1H), 6.97 (d,

W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
375
J ¼ 5.6 Hz, 2H), 2.53 (t, J ¼ 6.0 Hz, 4H), 1.45 (s, 9H); 13C NMR
4.1.2.2. 5-(4-(3-(Piperazin-1-yl)propoxy)phenyl)-3H-1,2-dithiole-3-
thione (3b). The title compound was obtained as a reddish brown
oil, 45% yield. Analytical data for 3b: 1H NMR (300 MHz, DMSO)
(75 MHz, CDCl3)
d 215.17, 173.05, 162.09, 154.77, 134.75, 128.69,
124.41, 115.62, 79.86, 66.37, 57.07, 53.50, 28.52; ESI-MS (m/z): 439.2
[MþH]þ.
d
7.85 (d, J ¼ 8.8 Hz, 2H), 7.74 (s, 1H), 7.05 (d, J ¼ 8.8 Hz, 2H), 4.09 (t,
J ¼ 6.1 Hz, 2H), 3.13e3.03 (m, 4H), 2.64e2.53 (m, 4H), 2.51e2.43 (m,
2H), 1.91e1.86 (m, 2H); 13C NMR (75 MHz, DMSO)
214.82, 173.92,
d
4.1.1.2. tert-Butyl
4-(3-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
162.18, 134.18, 129.09, 123.66, 115.55, 66.25, 53.98, 49.46, 25.86;
propyl)piperazine-1- carboxylate (2b). The title compound was
obtained as a reddish brown oil, 68% yield. Analytical data for 2b:
ESI-MS (m/z): 353.1 [MþH]þ.
1H NMR (300 MHz, CDCl3)
d
7.60 (d, J ¼ 8.8 Hz, 2H), 7.39 (s, 1H), 6.96
4.1.2.3. 5-(4-(4-(Piperazin-1-yl)butoxy)phenyl)-3H-1,2-dithiole-3-
thione (3c). The title compound was obtained as a reddish brown
oil, 47% yield. Analytical data for 3c: 1H NMR (300 MHz, DMSO)
(d, J ¼ 8.8 Hz, 2H), 4.09 (t, J ¼ 6.2 Hz, 2H), 3.50e3.34 (m, 4H), 2.54 (t,
J ¼ 6.2 Hz, 2H), 2.45e2.35 (m, 4H), 2.07e1.93 (m, 2H), 1.45 (s, 9H);
13C NMR (75 MHz, CDCl3)
d 215.22, 173.21, 162.50, 154.85, 134.72,
d
7.85 (d, J ¼ 8.8 Hz, 2H), 7.75 (s, 1H), 7.06 (d, J ¼ 8.8 Hz, 2H), 4.07 (t,
128.71, 124.20, 115.56, 79.83, 66.61, 55.02, 53.15, 28.55, 26.55; ESI-
MS (m/z): 453.1 [MþH]þ.
J ¼ 6.2 Hz, 2H), 3.16e2.99 (m, 4H), 2.59 (s, 4H), 2.47e2.34 (m, 2H),
1.84e1.65 (m, 2H), 1.62e1.44 (m, 2H); 13C NMR (75 MHz, DMSO)
d
214.78, 173.90, 162.16, 134.13, 129.05, 123.57, 115.51, 67.83, 56.81,
4.1.1.3. tert-Butyl
4-(4-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
49.27, 26.24, 22.29; ESI-MS (m/z): 367.1 [MþH]þ.
butyl)piperazine-1- carboxylate (2c). The title compound was ob-
tained as a reddish brown oil, 73% yield. Analytical data for 2c: 1H
4.1.2.4. 5-(4-((5-(Piperazin-1-yl)pentyl)oxy)phenyl)-3H-1,2-dithiole-
3-thione (3d). The title compound was obtained as a reddish brown
oil, 40% yield. Analytical data for 3d: 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
7.58 (d, J ¼ 8.7 Hz, 2H), 7.37 (s,1H), 6.93 (d,
J ¼ 8.7 Hz, 2H), 4.03 (t, J ¼ 6.1 Hz, 2H), 3.51e3.35 (m, 4H), 2.50e2.38
(m, 6H), 1.90e1.76 (m, 2H), 1.73e1.63 (m, 2H), 1.44 (s, 9H); 13C NMR
d
7.54 (d, J ¼ 8.8 Hz, 2H), 7.32 (s, 1H), 6.90 (d, J ¼ 8.8 Hz, 2H), 3.97 (t,
(75 MHz, CDCl3)
d 215.11, 173.21, 162.48, 154.78, 134.61, 128.67,
J ¼ 6.2 Hz, 2H), 3.26e3.06 (m, 4H), 2.64 (s, 4H), 2.50e2.30 (m, 2H),
124.08,115.49, 79.80, 68.14, 58.07, 52.94, 28.50, 27.06, 23.14; ESI-MS
1.86e1.70 (m, 2H), 1.60e1.38 (m, 4H); 13C NMR (75 MHz, CDCl3)
(m/z): 467.1 [MþH]þ.
d
214.84, 173.29, 162.46, 134.37, 128.57, 123.86, 115.39, 68.14, 57.81,
49.82, 28.81, 26.13, 23.67; ESI-MS (m/z): 381.1 [MþH]þ.
4.1.1.4. tert-Butyl
4-(5-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
pentyl)piperazine-1- carboxylate (2d). The title compound was ob-
4.1.2.5. 5-(4-((6-(Piperazin-1-yl)hexyl)oxy)phenyl)-3H-1,2-dithiole-
3-thione (3e). The title compound was obtained as a reddish brown
oil, 41% yield. Analytical data for 3e: 1H NMR (300 MHz, DMSO)
tained as a reddish brown oil, 69% yield. Analytical data for 2d: 1H
NMR (300 MHz, CDCl3)
d
7.58 (d, J ¼ 8.8 Hz, 2H), 7.37 (s,1H), 6.93 (d,
J ¼ 8.8 Hz, 2H), 4.00 (t, J ¼ 6.3 Hz, 2H), 3.51e3.37 (m, 4H), 2.51e2.33
d
7.86 (d, J ¼ 8.8 Hz, 2H), 7.76 (s, 1H), 7.06 (d, J ¼ 8.8 Hz, 2H), 4.05 (t,
(m, 6H), 1.92e1.73 (m, 2H), 1.64e1.33 (m, 13H); 13C NMR (75 MHz,
J ¼ 6.3 Hz, 2H), 3.13e2.94 (m, 4H), 2.61e2.53 (m, 4H), 2.40e2.28 (m,
CDCl3)
d 215.13, 173.24, 162.55, 154.78, 134.61, 128.67, 124.05,
2H), 1.79e1.65 (m, 2H), 1.50e1.13 (m, 6H); 13C NMR (75 MHz,
115.49, 79.81, 68.26, 58.44, 52.97, 29.00, 28.51, 26.30, 24.00; ESI-MS
DMSO) d 214.78, 173.91, 162.20, 134.12, 129.07, 123.54, 115.50, 68.02,
(m/z): 481.1 [MþH]þ.
57.35, 49.33, 28.50, 26.51, 25.86, 25.35; ESI-MS (m/z): 395.1
[MþH]þ.
4.1.1.5. tert-Butyl
4-(6-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
hexyl)piperazine-1- carboxylate (2e). The title compound was ob-
4.1.3. General procedure for the preparation of 7a-7c
tained as a reddish brown oil, 63% yield. Analytical data for 2e: 1H
To a solution of compound 6 (2.1 mmol) and the corresponding
nitrates 5a-5c (2.0 mmol) in anhydrous CH2Cl2 (25 mL) was added
DMAP (0.1 mmol) and the solution was left stirring at room tem-
perature for 10 min. DCC (2.1 mmol) was then added and the so-
lution was solution was left stirring at room temperature for 6 h.
The solution was then filtered, and the filtrate was concentrated
under reduced pressure. The resulting residue was purified by flash
chromatography (petroleum ether/EtOAc, 5:1 v/v) to afford the title
compounds.
NMR (300 MHz, CDCl3)
d
7.57 (d, J ¼ 8.6 Hz, 2H), 7.36 (s,1H), 6.93 (d,
J ¼ 8.6 Hz, 2H), 3.99 (t, J ¼ 6.2 Hz, 2H), 3.57e3.34 (m, 4H), 2.63e2.21
(m, 6H), 1.95e1.72 (m, 2H), 1.69e1.11 (m, 15H); 13C NMR (75 MHz,
CDCl3)
d 215.07, 173.25, 162.59, 154.75, 134.56, 128.64, 123.98,
115.49, 79.79, 68.36, 58.51, 52.91, 29.04, 28.49, 27.26, 26.42, 25.95;
ESI-MS (m/z): 495.2 [MþH]þ.
4.1.2. General procedure for the preparation of 3a-3e
Compounds 2a-2e (1.0 mmol) were dissolved in ethyl acetate
(20 mL) and the solution was saturated with dry hydrogen chloride
at 0 ꢀC. The mixture was stirred for 3 h to complete the depro-
tection, and the solvent was evaporated. The resulting residue was
extracted with dichloromethane (3 ꢁ 20 mL) and washed with
saturated NaHCO3 solution (10 mL). The organic layer was sepa-
rated and dried using anhydrous sodium sulfate, concentrated,
purified by flash chromatography (dichloromethane/methanol,
10:1 v/v) to afford the title compounds.
4.1.3.1. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-chloroacetoxy)pentyl)benzoate (7a). The ti-
tle compound was obtained as a colorless oil, 85% yield. Analytical
data for 7a: 1H NMR (300 MHz, CDCl3): 8.17 (d, J ¼ 7.7 Hz, 1H), 7.71
(d, J ¼ 16.0 Hz, 1H), 7.61e7.59 (m, 2H), 7.49e7.33 (m, 1H), 7.25e7.15
(m, 3H), 6.71e6.67 (m, 1H), 6.42 (d, J ¼ 16.0 Hz, 1H), 4.76e4.73 (m,
2H), 4.56e4.44 (m, 2H), 4.10 (s, 2H), 3.89 (s, 3H), 1.95e1.85 (m, 2H),
1.53e1.15 (m, 4H), 0.85 (t, J ¼ 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d
166.56,166.37,164.62,151.68,145.46,143.58,141.87,133.26,131.10,
4.1.2.1. 5-(4-(2-(Piperazin-1-yl)ethoxy)phenyl)-3H-1,2-dithiole-3-
thione (3a). The title compound was obtained as a reddish brown
oil, 52% yield. Analytical data for 3a: 1H NMR (300 MHz, DMSO)
127.75, 127.28, 126.33, 123.60, 121.61, 117.24, 111.47, 75.02, 70.71,
60.39, 56.04, 41.15, 36.61, 27.97, 22.50, 14.03; ESI-MS (m/z): 572.1
[MþNa]þ; ESI-HRMS (m/z): calculated for C26H28ClNO10Na
[MþNa]þ 572.1294, found 572.1306.
d
7.87 (d, J ¼ 8.8 Hz, 2H), 7.77 (s, 1H), 7.08 (d, J ¼ 8.9 Hz, 2H), 4.18 (t,
J ¼ 5.2 Hz, 2H), 3.15e3.02 (m, 4H), 2.78 (t, J ¼ 5.1 Hz, 2H), 2.75e2.65
(m, 4H); 13C NMR (75 MHz, DMSO)
214.84, 173.85, 161.88, 134.23,
d
4.1.3.2. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-chloroacetoxy)pentyl)benzoate (7b). The title
compound was obtained as a colorless oil, 88% yield. Analytical data
129.07, 123.78, 115.62, 65.94, 56.01, 49.60; ESI-MS (m/z): 339.1
[MþH]þ.

376
W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
for 7b: 1H NMR (300 MHz, CDCl3):
d
8.14 (d, J ¼ 7.6 Hz, 1H), 7.64 (d,
144.41, 141.99, 134.67, 133.26, 133.05, 131.05, 128.67, 127.48, 127.36,
J ¼ 15.9 Hz, 1H), 7.59e7.52 (m, 2H), 7.39e7.34 (m, 1H), 7.21e7.06 (m,
3H), 6.71e6.67 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.45 (t, J ¼ 5.9 Hz,
2H), 4.19 (t, J ¼ 5.5 Hz, 2H), 4.08 (s, 2H), 3.84 (s, 3H), 1.95e1.71 (m,
6H), 1.46e1.19 (m, 4H), 0.82 (t, J ¼ 7.1 Hz, 3H); 13C NMR (75 MHz,
126.39, 124.16, 123.68, 121.61, 117.24, 115.59, 111.55, 73.27, 70.70,
66.74, 60.42, 59.53, 56.10, 54.89, 53.08, 52.99, 36.72, 28.12, 26.58,
22.53, 14.07; ESI-MS (m/z): 866.2 [MþH]þ, 888.2 [MþNa]þ; ESI-
HRMS (m/z): calculated for C42H48N3O11S3 [MþH]þ 866.2445,
found 866.2468.
CDCl3):
d 166.43, 166.31, 164.36, 151.38, 144.07, 143.29, 141.38,
133.26, 133.01, 130.78, 127.49, 127.01, 126.04, 123.23, 121.11, 117.91,
111.19, 74.64, 72.63, 63.45, 55.71, 40.91, 36.33, 27.69, 24.85, 23.44,
22.19, 13.76; ESI-MS (m/z): 600.2 [MþNa]þ; ESI-HRMS (m/z):
calculated for C28H32ClNO10Na [MþNa]þ 600.1607, found 600.1625.
4.1.4.3. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(4-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)butyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8c). The title
compound was obtained starting from 3c and 7a. Reddish brown
oil, 15% yield. Analytical data for 8c: 1H NMR (300 MHz, CDCl3):
4.1.3.3. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl
(7c). The title compound was obtained as a colorless oil, 80% yield.
Analytical data for 7c: 1H NMR (300 MHz, CDCl3):
8.16 (d,
J ¼ 7.8 Hz,1H), 7.67 (d, J ¼ 16.0 Hz,1H), 7.62e7.57 (m, 2H), 7.50e7.33
(m, 1H), 7.26e7.11 (m, 3H), 6.71e6.67 (m, 1H), 6.42 (d, J ¼ 16.0 Hz,
1H), 4.47e4.42 (m, 2H), 4.23e4.19 (m, 2H), 4.10 (s, 2H), 3.88 (s, 3H),
1.92e1.88 (m, 2H), 1.85e1.73 (m, 4H), 1.47e1.39 (m, 4H), 1.39e1.18
2-(1-(2-chloroacetoxy)pentyl)benzoate
d
8.15 (d, J ¼ 7.6 Hz, 1H), 7.71 (d, J ¼ 16.0 Hz, 1H), 7.61e7.57 (m, 4H),
7.45e7.37 (m, 2H), 7.24e7.16 (m, 3H), 6.95 (d, J ¼ 8.7 Hz, 2H),
6.66e6.63 (m, 1H), 6.42 (d, J ¼ 16.0 Hz, 1H), 4.80e4.71 (m, 2H),
4.58e4.47 (m, 2H), 4.05e4.02 (m, 2H), 3.89 (s, 3H), 3.33e3.19 (m,
2H), 2.60e2.40 (m, 10H), 1.85e1.81 (m, 4H), 1.69e1.67 (m, 2H),
1.46e1.27 (m, 4H), 0.84 (t, J ¼ 6.4 Hz, 3H); 13C NMR (75 MHz,
d
CDCl3):
d 215.23, 173.23, 169.60, 166.39, 164.68, 162.58, 151.78,
(m, 4H), 0.84 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d
166.89,
145.53, 144.43, 142.01, 134.69, 133.27, 133.07, 131.07, 128.70, 127.50,
127.38, 126.42, 124.15, 123.71, 121.63, 117.25, 115.57, 111.57, 73.28,
70.71, 68.28, 60.44, 59.58, 58.10, 56.12, 53.06, 36.75, 28.14, 27.16,
23.88, 22.55, 14.08; ESI-MS (m/z): 880.2 [MþH]þ, 902.2 [MþNa]þ;
ESI-HRMS (m/z): calculated for C43H50N3O11S3 [MþH]þ 880.2602,
found 880.2618.
166.48, 164.58, 151.56, 144.10, 143.49, 141.53, 133.55, 133.18, 131.01,
127.68, 127.26, 126.25, 123.44, 121.33, 118.37, 111.32, 74.94, 73.26,
64.40, 55.95, 41.09, 36.56, 28.53, 27.90, 26.69, 25.61, 25.40, 22.43,
13.97; ESI-MS (m/z): 628.2 [MþNa]þ; ESI-HRMS (m/z): calculated
for C30H36ClNO10Na [MþNa]þ 628.1920, found 628.1937.
4.1.4. General procedure for the preparation of 8a-8o
4.1.4.4. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(5-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)pentyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8d). The title
compound was obtained starting from 3d and 7a. Reddish brown
oil, 21% yield. Analytical data for 8d: 1H NMR (300 MHz, CDCl3):
To a solution of compounds 7a-7c (1.0 mmol) and Et3N (0.122 g,
1.2 mmol) in anhydrous dichloromethane (10 mL) was added 3a-3e
(1.0 mmol), and the mixture was stirred at room temperature for
12 h. The dichloromethane was removed in vacuo, and the resulting
residue was purified by flash chromatography (petroleum ether/
EtOAc, 1:1 v/v) to afford the title compounds.
d
8.14 (d, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 16.0 Hz, 1H), 7.62e7.54 (m, 4H),
7.43e7.35 (m, 2H), 7.25e7.14 (m, 3H), 6.97e6.92 (m, 2H), 6.66e6.63
(m, 1H), 6.42 (d, J ¼ 16.0 Hz, 1H), 4.77e4.74 (m, 2H), 4.52e4.49 (m,
2H), 4.03e4.01 (m, 2H), 3.89 (s, 3H), 3.33e3.19 (m, 2H), 2.60e2.35
(m, 10H), 1.90e1.76 (m, 4H), 1.65e1.50 (m, 4H), 1.38e1.22 (m, 4H),
4.1.4.1. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)ethyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8a). The title
compound was obtained starting from 3a and 7a. Reddish brown
oil, 21% yield. Analytical data for 8a: 1H NMR (300 MHz, CDCl3):
0.84 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 215.06, 173.18,
169.54, 166.29, 164.60, 162.56, 151.69, 145.41, 144.35, 141.92, 134.53,
133.18, 133.00, 130.97, 128.62, 127.41, 127.28, 126.35, 123.99, 123.61,
121.54, 117.18, 115.47, 111.49, 73.17, 70.66, 68.30, 60.35, 59.54, 58.53,
56.05, 53.11, 36.66, 28.99, 28.49, 28.06, 26.54, 22.47, 14.01; ESI-MS
(m/z): 894.3 [MþH]þ, 916.3 [MþNa]þ; ESI-HRMS (m/z): calculated
for C44H52N3O11S3 [MþH]þ 894.2758, found 894.2771.
d
8.14 (d, J ¼ 7.9 Hz, 1H), 7.71 (d, J ¼ 16.0 Hz, 1H), 7.64e7.54 (m, 4H),
7.44e7.36 (m, 2H), 7.26e7.14 (m, 3H), 6.96 (d, J ¼ 8.8 Hz, 2H),
6.65e6.61 (m, 1H), 6.41 (d, J ¼ 16.0 Hz, 1H), 4.77e4.74 (m, 2H),
4.52e4.49 (m, 2H), 4.17e4.10 (m, 2H), 3.88 (s, 3H), 3.34e3.19 (m,
2H), 2.85 (t, J ¼ 5.6 Hz, 2H), 2.64 (s, 8H), 1.91e1.79 (m, 2H),
1.39e1.27 (m, 4H), 0.84 (t, J ¼ 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3):
4.1.4.5. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(6-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)hexyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8e). The title
compound was obtained starting from 3e and 7a. Reddish brown
oil, 24% yield. Analytical data for 8e: 1H NMR (300 MHz, CDCl3):
d
215.25, 173.08, 169.54, 166.39, 164.68, 162.16, 151.75, 145.52,
144.39, 141.98, 134.79, 133.27, 133.09, 131.08, 128.70, 127.51, 127.34,
126.38, 124.43, 123.69, 121.63, 117.24, 115.66, 111.54, 73.34, 70.71,
66.30, 60.43, 59.47, 56.92, 56.11, 53.43, 52.88, 36.73, 28.14, 22.55,
14.08; ESI-MS (m/z): 852.2 [MþH]þ, 874.2 [MþNa]þ; ESI-HRMS (m/
d
8.13 (d, J ¼ 7.8 Hz, 1H), 7.69 (d, J ¼ 16.0 Hz, 1H), 7.63e7.49 (m, 4H),
z): calculated for
852.2305.
C
41H46N3O11S3 [MþH]þ 852.2289, found
7.42e7.30 (m, 2H), 7.23e7.10 (m, 3H), 6.93 (d, J ¼ 8.8 Hz, 2H),
6.64e6.60 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.75e4.72 (m, 2H),
4.50e4.47 (m, 2H), 3.99 (t, J ¼ 6.4 Hz, 2H), 3.87 (s, 3H), 3.32e3.18
(m, 2H), 2.63e2.58 (m, 8H), 2.45e2.34 (m, 2H), 1.92e1.74 (m, 4H),
1.59e1.28 (m, 10H), 0.82 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz,
4.1.4.2. ( )-(E)-2-Methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(3-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)propyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8b). The title
compound was obtained starting from 3b and 7a. Reddish brown
oil, 23% yield. Analytical data for 8b: 1H NMR (300 MHz, CDCl3):
CDCl3):
d 215.08, 173.24, 169.51, 166.34, 164.64, 162.61, 151.72,
145.46, 144.34, 141.95, 134.59, 133.23, 133.07, 131.02, 128.66, 127.47,
127.31, 126.37, 124.03, 123.65, 121.58, 117.21, 115.53, 111.52, 73.29,
70.68, 68.37, 60.39, 59.41, 58.44, 56.08, 52.92, 52.59, 36.69, 29.03,
28.09, 27.24, 26.39, 25.93, 22.50, 14.04; ESI-MS (m/z): 908.3
[MþH]þ, 930.3 [MþNa]þ; ESI-HRMS (m/z): calculated for
d
8.14 (d, J ¼ 7.8 Hz, 1H), 7.70 (d, J ¼ 16.0 Hz, 1H), 7.63e7.51 (m, 4H),
7.44e7.34 (m, 2H), 7.23e7.13 (m, 3H), 6.95 (d, J ¼ 8.8 Hz, 2H),
6.65e6.61 (m, 1H), 6.41 (d, J ¼ 16.0 Hz, 1H), 4.76e4.73 (m, 2H),
4.51e4.48 (m, 2H), 4.10e4.04 (m, 2H), 3.88 (s, 3H), 3.33e3.19 (m,
2H), 2.60e2.50 (m, 10H), 2.03e1.96 (m, 2H), 1.88e1.83 (m, 2H),
1.42e1.31 (m, 4H), 0.83 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
C
45H54N3O11S3 [MþH]þ 908.2915, found 908.2935.
4.1.4.6. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)
d
215.22, 173.20, 169.58, 166.37, 164.67, 162.54, 151.76, 145.49,

W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
377
phenoxy)ethyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8f). The title
compound was obtained starting from 3a and 7b. Reddish brown
oil, 19% yield. Analytical data for 8f: 1H NMR (300 MHz, CDCl3):
1.60e1.43 (m, 4H), 1.31e1.27 (m, 4H), 0.83 (t, J ¼ 7.0 Hz, 3H); 13C
NMR (75 MHz, CDCl3):
d 215.15, 173.23, 169.56, 166.80, 164.68,
162.58, 151.69, 144.52, 144.39, 141.75, 134.61, 133.44, 133.04, 131.03,
128.66, 127.45, 127.33, 126.37, 124.05, 123.61, 121.41, 118.07, 115.51,
111.47, 73.25, 72.76, 68.31, 63.71, 59.55, 58.44, 56.07, 53.03, 52.98,
36.72, 29.02, 28.11, 26.50, 25.19, 24.04, 23.83, 22.52, 14.06; ESI-MS
(m/z): 922.3 [MþH]þ, 944.3 [MþNa]þ; ESI-HRMS (m/z): calculated
for C46H56N3O11S3 [MþH]þ 922.3071, found 922.3082.
d
8.15 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 16.0 Hz, 1H), 7.63e7.51 (m, 4H),
7.44e7.34 (m, 2H), 7.24e7.16 (m, 3H), 6.97 (d, J ¼ 8.8 Hz, 2H),
6.66e6.62 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.53 (t, J ¼ 5.9 Hz, 2H),
4.26e4.18 (m, 2H), 4.16 (t, J ¼ 5.6 Hz, 2H), 3.89 (s, 3H), 3.34e3.20
(m, 2H), 2.85 (t, J ¼ 5.5 Hz, 2H), 2.64 (s, 8H), 1.87 (s, 6H), 1.39e1.28
(m, 4H), 0.84 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 215.16,
173.03, 169.51, 166.77, 164.66, 162.16, 151.69, 144.49, 144.34, 141.75,
134.70, 133.45, 133.02, 131.02, 128.65, 127.46, 127.35, 126.35, 124.35,
123.60, 121.39, 118.08, 115.63, 111.49, 73.26, 72.74, 66.36, 63.70,
59.47, 56.91, 56.07, 53.43, 52.90, 36.70, 28.09, 25.18, 23.83, 22.50,
14.04; ESI-MS (m/z): 880.3 [MþH]þ, 902.2 [MþNa]þ; ESI-HRMS (m/
4.1.4.10. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(6-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)hexyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8j). The title
compound was obtained starting from 3e and 7b. Reddish brown
oil, 29% yield. Analytical data for 8j: 1H NMR (300 MHz, CDCl3):
z): calculated for
880.2619.
C
43H50N3O11S3 [MþH]þ 880.2602, found
d
8.13 (d, J ¼ 7.8 Hz, 1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.60e7.56 (m, 4H),
7.45e7.32 (m, 2H), 7.23e7.15 (m, 3H), 6.94 (d, J ¼ 8.8 Hz, 2H),
6.65e6.61 (m, 1H), 6.39 (d, J ¼ 16.0 Hz, 1H), 4.52 (t, J ¼ 5.9 Hz, 2H),
4.25 (t, J ¼ 5.6 Hz, 2H), 4.00 (t, J ¼ 6.4 Hz, 2H), 3.88 (s, 3H), 3.32e3.18
(m, 2H), 2.59e2.42 (m, 8H), 2.38e2.30 (m, 2H), 1.94e1.73 (m, 8H),
1.57e1.26 (m, 10H), 0.83 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz,
4.1.4.7. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(3-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)propyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8g). The title
compound was obtained starting from 3b and 7b. Reddish brown
oil, 24% yield. Analytical data for 8g: 1H NMR (300 MHz, CDCl3):
CDCl3):
d 215.19, 173.24, 169.58, 166.81, 164.69, 162.65, 151.72,
144.54, 144.40, 141.79, 134.63, 133.46, 133.04, 131.04, 128.67, 127.46,
127.37, 126.40, 124.06, 123.63, 121.42, 118.09, 115.55, 111.51, 73.27,
72.76, 68.43, 63.72, 59.58, 58.58, 56.09, 53.05, 52.99, 36.73, 29.08,
28.12, 27.34, 26.72, 26.00, 25.22, 23.86, 22.53, 14.06; ESI-MS (m/z):
936.3 [MþH]þ, 958.3 [MþNa]þ; ESI-HRMS (m/z): calculated for
d
8.15 (d, J ¼ 7.8 Hz, 1H), 7.67 (d, J ¼ 16.0 Hz, 1H), 7.63e7.51 (m, 4H),
7.44e7.34 (m, 2H), 7.23e7.16 (m, 3H), 6.95 (d, J ¼ 8.7 Hz, 2H),
6.66e6.62 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.53 (t, J ¼ 5.9 Hz, 2H),
4.26 (t, J ¼ 5.5 Hz, 2H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.88 (s, 3H), 3.36e3.21
(m, 2H), 2.66 (s, 10H), 2.09e2.04 (m, 2H), 1.93e1.78 (m, 6H),
1.38e1.28 (s, 4H), 0.83 (d, J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3):
C
47H58N3O11S3 [MþH]þ 936.3228, found 936.3251.
d
214.98, 173.17, 169.50, 166.72, 164.60, 162.43, 151.58, 144.42,
4.1.4.11. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl 2-(1-(2-(4-(2-(4-(3-thioxo-3H-1,2-dithiol-
144.29, 141.64, 134.49, 133.36, 133.00, 130.96, 128.58, 127.40, 127.20,
126.28, 124.00, 123.52, 121.33, 117.99, 115.46, 111.37, 73.18, 72.72,
66.62, 63.65, 59.40, 55.99, 54.80, 52.94, 52.86, 36.63, 28.03, 26.44,
25.10, 23.73, 22.44, 14.00; ESI-MS (m/z): 894.3 [MþH]þ, 916.3
[MþNa]þ; ESI-HRMS (m/z): calculated for C44H52N3O11S3 [MþH]þ
894.2758, found 894.2782.
5-yl)phenoxy)ethyl)piperazin-1-yl)acetoxy)pentyl)benzoate
(8k).
The title compound was obtained starting from 3a and 7c. Reddish
brown oil, 15% yield. Analytical data for 8k: 1H NMR (300 MHz,
CDCl3):
d
8.14 (d, J ¼ 7.8 Hz, 1H), 7.67 (d, J ¼ 16.0 Hz, 1H), 7.61e7.56
(m, 4H), 7.44e7.35 (m, 2H), 7.24e7.14 (m, 3H), 6.96 (d, J ¼ 8.8 Hz,
2H), 6.66e6.61 (m, 1H), 6.41 (d, J ¼ 16.0 Hz, 1H), 4.46 (t, J ¼ 6.6 Hz,
2H), 4.22 (t, J ¼ 6.5 Hz, 2H), 4.15 (t, J ¼ 5.6 Hz, 2H), 3.88 (s, 3H),
3.34e3.19 (m, 2H), 2.85 (t, J ¼ 5.6 Hz, 2H), 2.64 (s, 8H), 1.91e1.70 (m,
6H), 1.53e1.28 (m, 8H), 0.84 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz,
4.1.4.8. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(4-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)butyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8h). The title
compound was obtained starting from 3c and 7b. Reddish brown
oil, 28% yield. Analytical data for 8h: 1H NMR (300 MHz, CDCl3):
CDCl3):
d 215.27, 173.03, 169.54, 166.99, 164.72, 162.22, 151.74,
144.36, 144.23, 141.72, 134.79, 133.63, 133.02, 131.07, 128.69, 127.49,
126.41, 124.46, 123.62, 121.42, 118.47, 115.69, 111.52, 73.33, 73.30,
66.41, 64.49, 59.52, 56.95, 56.11, 53.46, 52.94, 36.75, 28.66, 28.13,
26.85, 25.73, 25.52, 22.54,14.06; ESI-MS (m/z): 930.3 [MþNa]þ; ESI-
HRMS (m/z): calculated for C45H53N3O11S3Na [MþNa]þ 930.2734,
found 930.2758.
d
8.15 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 16.0 Hz, 1H), 7.63e7.52 (m, 4H),
7.48e7.34 (m, 2H), 7.24e7.16 (m, 3H), 6.95 (d, J ¼ 8.8 Hz, 2H),
6.66e6.62 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.53 (t, J ¼ 6.0 Hz, 2H),
4.27 (t, J ¼ 5.7 Hz, 2H), 4.04 (t, J ¼ 6.1 Hz, 2H), 3.89 (s, 3H), 3.34e3.20
(m, 2H), 2.63e2.49 (m, 10H), 1.97e1.81 (m, 8H), 1.73e1.71 (m, 2H),
1.40e1.28 (m, 4H), 0.84 (t, J ¼ 7.0 Hz, 3H); 13C NMR (CDCl3,
300 MHz):
d 215.09, 173.25, 169.49, 166.81, 164.68, 162.49, 151.68,
144.53, 144.32, 141.73, 134.58, 133.46, 133.10, 131.05, 128.68, 127.51,
127.30, 126.36, 124.09, 123.60, 121.4, 118.09, 115.52, 111.47, 73.33,
72.79, 68.12, 63.73, 59.31, 57.82, 56.07, 52.70, 52.46, 36.70, 29.78,
28.11, 27.03, 25.19, 23.82, 22.51, 14.07; ESI-MS (m/z): 908.3 [MþH]þ;
ESI-HRMS (m/z): calculated for C45H54N3O11S3 [MþH]þ 908.2915,
found 908.2941.
4.1.4.12. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl2-(1-(2-(4-(3-(4-(3-thioxo-3H-1,2-dithiol-
5-yl)phenoxy)propyl)piperazin-1-yl)acetoxy)pentyl)benzoate
(8l).
The title compound was obtained starting from 3b and 7c. Reddish
brown oil, 12% yield. Analytical data for 8l: 1H NMR (300 MHz,
CDCl3):
d
8.15 (d, J ¼ 7.7 Hz, 1H), 7.67 (d, J ¼ 16.0 Hz, 1H), 7.61e7.57
(m, 4H), 7.45e7.37 (m, 2H), 7.23e7.16 (m, 3H), 6.96 (d, J ¼ 8.8 Hz,
2H), 6.66e6.62 (m, 1H), 6.41 (d, J ¼ 15.9 Hz, 1H), 4.47 (t, J ¼ 6.6 Hz,
2H), 4.22 (t, J ¼ 6.5 Hz, 2H), 4.08 (t, J ¼ 6.2 Hz, 2H), 3.88 (s, 3H),
3.34e3.20 (m, 2H), 2.61e2.53 (m, 10H), 2.02e1.93 (m, 2H),
1.89e1.75 (m, 6H), 1.57e1.43 (m, 4H), 1.39e1.22 (m, 4H), 0.84 (t,
4.1.4.9. ( )-(E)-2-Methoxy-4-(3-(4-(nitrooxy)butoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1-(2-(4-(5-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe-
noxy)pentyl)piperazin-1-yl)acetoxy)pentyl)benzoate (8i). The title
compound was obtained starting from 3d and 7b. Reddish brown
oil, 28% yield. Analytical data for 8i: 1H NMR (300 MHz, CDCl3):
J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 215.10, 172.88, 166.93,
d
8.13 (d, J ¼ 7.8 Hz, 1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.60e7.56 (m, 4H),
164.68, 161.47, 151.60, 144.05, 141.54, 134.76, 133.63, 133.21, 131.07,
128.69, 127.72, 127.32, 126.23, 124.74, 123.45, 121.34, 118.50, 115.41,
111.46, 73.29, 64.47, 56.02, 36.48, 28.55, 27.97, 26.73, 25.63, 25.41,
23.79, 22.40, 13.94; ESI-MS (m/z): 922.3 [MþH]þ; ESI-HRMS (m/z):
calculated for C46H56N3O11S3 [MþH]þ 922.3071, found 922.3089.
7.43e7.32 (m, 2H), 7.23e7.15 (m, 3H), 6.93 (d, J ¼ 8.8 Hz, 2H),
6.65e6.61 (m, 1H), 6.39 (d, J ¼ 16.0 Hz, 1H), 4.52 (t, J ¼ 6.0 Hz, 2H),
4.25 (t, J ¼ 5.7 Hz, 2H), 4.00 (t, J ¼ 6.4 Hz, 2H), 3.88 (s, 3H), 3.32e3.18
(m, 2H), 2.60e2.53 (m, 8H), 2.41e2.33 (m, 2H), 1.94e1.74 (m, 8H),

378
W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
4.1.4.13. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl 2-(1-(2-(4-(4-(4-(3-thioxo-3H-1,2-dithiol-
purified by flash chromatography (petroleum ether/EtOAc, 5:1 v/v)
to afford 9 as a colorless oil, 85% yield. Analytical data for 9: 1H NMR
5-yl)phenoxy)butyl)piperazin-1-yl)acetoxy)pentyl)benzoate
(8m).
(300 MHz, CDCl3):
d
8.16 (d, J ¼ 7.7 Hz, 1H), 7.67 (d, J ¼ 16.0 Hz, 1H),
The title compound was obtained starting from 3c and 7c. Reddish
7.59 (d, J ¼ 3.4 Hz, 2H), 7.42e7.36 (m, 1H), 7.22e7.11 (m, 3H),
6.72e6.68 (m, 1H), 6.41 (d, J ¼ 16.0 Hz, 1H), 4.09 (s, 2H), 3.86 (s, 3H),
3.79 (s, 3H), 1.95e1.85 (m, 2H), 1.46e1.26 (m, 4H), 0.84 (t, J ¼ 7.0 Hz,
brown oil, 17% yield. Analytical data for 8m: 1H NMR (300 MHz,
CDCl3):
d
8.14 (d, J ¼ 7.8 Hz, 1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.62e7.50
(m, 4H), 7.43e7.33 (m, 2H), 7.23e7.15 (m, 3H), 6.94 (d, J ¼ 8.8 Hz,
2H), 6.66e6.61 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.46 (t, J ¼ 6.6 Hz,
2H), 4.22 (t, J ¼ 6.5 Hz, 2H), 4.03 (t, J ¼ 6.2 Hz, 2H), 3.88 (s, 3H),
3.33e3.24 (m, 2H), 2.76e2.27 (m, 10H), 1.83e1.60 (m, 10H),
1.48e1.28 (m, 8H), 0.83 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz,
3H); 13C NMR (75 MHz, CDCl3):
d 167.20, 166.43, 164.52, 151.50,
144.15, 143.45, 141.48, 133.48, 133.12, 130.96, 127.63, 127.20, 126.19,
123.39, 121.24, 118.03, 111.32, 74.87, 55.88, 51.70, 41.03, 36.50, 27.86,
22.37, 13.92; ESI-MS (m/z): 497.1 [MþNa]þ; ESI-HRMS (m/z):
calculated for C25H27ClO7Na [MþNa]þ 497.1338, found 497.1343.
CDCl3):
d 215.20, 173.24, 169.57, 167.01, 164.71, 162.55, 151.69,
144.40, 144.24, 141.69, 134.67, 133.59, 133.05, 131.06, 128.69, 127.48,
127.36, 126.39, 124.12, 123.60, 121.42, 118.42, 115.55, 111.44, 73.31,
68.24, 64.49, 59.53, 58.06, 56.08, 52.99, 36.75, 28.64, 28.13, 27.13,
26.81, 25.72, 25.51, 23.30, 22.55, 14.08; ESI-MS (m/z): 936.3
[MþH]þ; ESI-HRMS (m/z): calculated for C47H58N3O11S3 [MþH]þ
936.3228, found 936.3231.
4.1.6. ( )-(E)-2-Methoxy-4-(3-methoxy-3-oxoprop-1-en-1-yl)
phenyl 2-(1-(2-(4-(5-(4-(5-Thioxo-2,5-dihydrothiophen-3-yl)
phenoxy)pentyl)piperazin-1-yl)acetoxy)pentyl)benzoate (10)
To a solution of compound 9 (1.0 mmol) and Et3N (0.122 g,
1.2 mmol) in anhydrous dichloromethane (10 mL) was added 3d
(1.0 mmol), and the mixture was stirred at room temperature for
12 h. The dichloromethane was removed in vacuo, and the resulting
residue was purified by flash chromatography (petroleum ether/
EtOAc, 1:1 v/v) to afford 10 as a reddish brown oil, 35% yield.
4.1.4.14. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl2-(1-(2-(4-(5-(4-(3-thioxo-3H-1,2-dithiol-
5-yl)phenoxy)pentyl)piperazin-1-yl)acetoxy)pentyl)benzoate
(8n).
Analytical data for 10: 1H NMR (300 MHz, CDCl3):
d 8.13 (d,
The title compound was obtained starting from 3d and 7c. Reddish
J ¼ 7.6 Hz,1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.60e7.52 (m, 4H), 7.41e7.32
(m, 2H), 7.22e7.10 (m, 3H), 6.93 (dd, J ¼ 6.9, 4.9 Hz, 2H), 6.64e6.60
(m, 1H), 6.39 (d, J ¼ 16.0 Hz, 1H), 3.98 (t, J ¼ 6.3 Hz, 2H), 3.86 (s, 3H),
3.79 (s, 3H), 3.34e3.19 (m, 2H), 2.68 (s, 8H), 2.54e2.47 (m, 2H),
1.90e1.73 (m, 4H), 1.69e1.54 (m, 2H), 1.49e1.42 (m, 2H), 1.39e1.23
brown oil, 18% yield. Analytical data for 8n: 1H NMR (300 MHz,
CDCl3):
d
8.13 (d, J ¼ 7.8 Hz, 1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.59e7.56
(m, 4H), 7.43e7.32 (m, 2H), 7.23e7.13 (m, 3H), 6.93 (d, J ¼ 8.8 Hz,
2H), 6.68e6.55 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.45 (t, J ¼ 6.6 Hz,
2H), 4.21 (t, J ¼ 6.5 Hz, 2H), 4.00 (t, J ¼ 6.4 Hz, 2H), 3.87 (s, 3H),
3.31e3.17 (m, 2H), 2.68e2.42 (m, 8H), 2.38e2.33 (m, 2H), 1.89e1.70
(m, 8H), 1.62e1.45 (m, 8H), 1.39e1.25 (m, 4H), 0.83 (t, J ¼ 7.0 Hz,
(m, 4H), 0.82 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 215.04,
173.20, 169.36, 167.31, 164.62, 162.47, 151.58, 144.24, 141.58, 134.53,
133.49, 133.06, 131.00, 128.62, 127.46, 127.24, 126.27, 124.01, 123.51,
121.31, 118.07, 115.45, 111.38, 73.36, 68.10, 59.11, 57.97, 56.00, 52.54,
51.96, 51.82, 36.63, 28.83, 28.06, 25.57, 23.83, 22.46, 14.02; ESI-MS
(m/z): 819.3 [MþH]þ, 841.3 [MþNa]þ; ESI-HRMS (m/z): calculated
for C43H50N2O8S3Na [MþNa]þ 841.2621, found 841.2611.
3H); 13C NMR (75 MHz, CDCl3):
d 215.13, 173.20, 169.56, 166.94,
164.67, 162.58, 151.67, 144.37, 144.19, 141.68, 134.59, 133.55, 133.00,
131.00, 128.65, 127.43, 127.36, 126.37, 124.04, 123.57, 121.37, 118.40,
115.51, 111.43, 73.27, 73.23, 68.32, 64.44, 59.57, 58.46, 56.05, 53.05,
36.71, 29.02, 28.61, 28.09, 26.78, 26.57, 25.68, 25.47, 24.04, 22.50,
14.04; ESI-MS (m/z): 950.3 [MþH]þ, 972.3 [MþNa]þ; ESI-HRMS (m/
4.1.7. ( ) (E)-2-methoxy-4-(3-(2-(nitrooxy)ethoxy)-3-oxoprop-1-
en-1-yl)phenyl 2-(1- (2-(4-(5-phenoxypentyl)piperazin-1-yl)
acetoxy)pentyl)benzoate (13)
z): calculated for
950.3363.
C
48H60N3O11S3 [MþH]þ 950.3384, found
To a solution of compound 7a (1.0 mmol) and Et3N (0.122 g,
1.2 mmol) in anhydrous dichloromethane (10 mL) was added 12
(1.0 mmol), and the mixture was stirred at room temperature for
12 h. The dichloromethane was removed in vacuo, and the resulting
residue was purified by flash chromatography (petroleum ether/
EtOAc, 1:1 v/v) to afford 13 as a colorless oil, 73% yield. Analytical
4.1.4.15. ( )-(E)-2-Methoxy-4-(3-((6-(nitrooxy)hexyl)oxy)-3-
oxoprop-1-en-1-yl)phenyl 2-(1-(2-(4-(6-(4-(3-thioxo-3H-1,2-dithiol-
5-yl)phenoxy)hexyl)piperazin-1-yl)acetoxy)pentyl)benzoate
(8o).
The title compound was obtained starting from 3e and 7c. Reddish
brown oil, 27% yield. Analytical data for 8o: 1H NMR (300 MHz,
CDCl3):
d
8.13 (d, J ¼ 7.7 Hz, 1H), 7.66 (d, J ¼ 16.0 Hz, 1H), 7.61e7.51
data for 13: 1H NMR (300 MHz, CDCl3)
d
8.15 (d, J ¼ 7.8 Hz, 1H), 7.71
(m, 4H), 7.41e7.31 (m, 2H), 7.21e7.10 (m, 3H), 6.93 (d, J ¼ 8.7 Hz,
2H), 6.68e6.55 (m, 1H), 6.40 (d, J ¼ 16.0 Hz, 1H), 4.45 (t, J ¼ 6.5 Hz,
2H), 4.20 (t, J ¼ 6.5 Hz, 2H), 3.99 (t, J ¼ 6.1 Hz, 2H), 3.87 (s, 3H),
3.32e3.18 (m, 2H), 2.64e2.32 (m, 10H), 1.93e1.61 (m, 8H), 1.49e1.18
(d, J ¼ 16.0 Hz, 1H), 7.58 (d, J ¼ 3.9 Hz, 2H), 7.42e7.36 (m, 1H),
7.30e7.14 (m, 5H), 6.94e6.86 (m, 3H), 6.66e6.62 (m, 1H), 6.42 (d,
J ¼ 16.0 Hz, 1H), 4.75e4.72 (m, 2H), 4.51e4.48 (m, 2H), 3.94 (t,
J ¼ 6.4 Hz, 2H), 3.88 (s, 3H), 3.33e3.19 (m, 2H), 2.60e2.50 (m, 8H),
2.44e2.33 (m, 2H), 1.95e1.74 (m, 4H), 1.59e1.27 (m, 8H), 0.84 (t,
(m, 14H), 0.83 (t, J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 215.15,
173.21, 169.55, 166.95, 164.68, 162.59, 151.68, 144.37, 144.20, 141.68,
134.61, 133.56, 133.01, 131.02, 128.66, 127.44, 127.37, 126.37, 124.06,
123.58, 121.38, 118.41, 115.52, 111.44, 73.28, 73.26, 68.32, 64.45,
59.55, 58.44, 56.07, 53.04, 52.97, 36.71, 29.02, 28.62, 28.52, 28.10,
26.79, 26.51, 25.69, 25.47, 24.04, 22.51, 14.05; ESI-MS (m/z): 964.3
[MþH]þ, 986.3 [MþNa]þ; ESI-HRMS (m/z): calculated for
J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 169.56, 166.33, 164.61,
159.06, 151.66, 145.46, 144.42, 141.88, 133.16, 133.05, 130.99, 129.46,
127.41, 127.20, 126.33, 123.62, 121.57, 120.55, 117.13, 114.48, 111.40,
73.19, 70.66, 67.64, 60.33, 59.55, 58.52, 56.02, 53.00, 36.69, 29.22,
28.09, 26.57, 24.11, 22.49, 14.05; ESI-MS (m/z): 762.4 [MþH]þ, 784.4
[MþNa]þ; ESI-HRMS (m/z): calculated for C41H52N3O11 [MþH]þ
762.3596, found 762.3612.
C
49H62N3O11S3 [MþH]þ 964.3541, found 964.3544.
4.1.5. ( )-(E)-2-Methoxy-4-(3-methoxy-3-oxoprop-1-en-1-yl)
phenyl 2-(1-(2-chloroacetoxy)pentyl)benzoate (9)
4.2. Biological assays
To a solution of compound 6 (2.1 mmol) and methyl ferulate
(2.0 mmol) in anhydrous CH2Cl2 (25 mL) was added DMAP
(0.1 mmol) and the solution was left stirring at room temperature
for 6 h. The solution was then filtered, and the filtrate was
concentrated under reduced pressure. The resulting residue was
4.2.1. Anti-platelet aggregation in vitro
Blood was drawn from rabbit carotid artery and mixed with 3.8%
sodium citrate (9:1, v/v). After centrifugation at 500g for 10 min at
room temperature to obtain platelet-rich plasma (PRP), the
remaining blood was further centrifuged at 3000 g for another

W. Yin et al. / European Journal of Medicinal Chemistry 115 (2016) 369e380
379
10 min to obtain platelet-poor plasma (PPP). Platelet aggregation
completely recovered from anesthesia.
was measured by Born's turbidimetric method in a platelet
aggregometer (LG-PABER-I platelet aggregometer, Beijing) at 37 ꢀC
within 3 h after blood collection. Briefly, PRP (240 mL) was pre-
4.2.4.1. Neurological deficit scores. Neurological deficits of individ-
ual rats were evaluated at 24 h post reperfusion by the Longa's
method in a blinded manner. Tests were always performed be-
tween 10 and 11 a.m. to exclude the possible interference of
circadian rhythm-related behavioral changes.
incubated in duplicate for 5 min at 37 ꢀC with vehicle, the indi-
vidual compounds, or reference drugs (ASP, NBP, and Ticlid) at the
same concentrations (0.1, 0.2, 0.4, 0.8, and 1.6 mM) and the platelet
aggregation in individual PPR samples was induced by ADP (final
concentration of 10
mM), followed by recording of light trans-
4.2.4.2. Measurement of infarct size. At 24 h post reperfusion, some
rats (n ¼ 6) from each group were sacrificed and their brains were
rapidly removed onto ice and coronally sectioned (2 mm). The
sections were immediately incubated in 2% TTC for 30 min at 37 ꢀC
and fixed in10% formalin overnight. The infarct areas (white) in
individual sections were measured and analyzed using Image-Pro
Plus (Version 6.0). The infarct size was expressed as the percent-
age size in the total area of whole brain.
mission at maximal aggregation within 5 min. The inhibition rate of
the tested individual compounds on the platelet aggregation was
calculated as the following formula: inhibition rate
(%) ¼ 100 ꢁ [(1 ꢂ (the platelet aggregation in samples with the
tested compound)/(the platelet aggregation in control samples)]. In
addition, the concentrations of selected compounds that inhibited
the platelet aggregation to 50% (IC50) were calculated.
4.2.2. In vitro nitric oxide release assay
4.2.4.3. Measurement of brain-water content. At 24 h post reper-
fusion, some rats (n ¼ 6) form each group were sacrificed and their
brains were rapidly removed on to ice, followed by isolating the
ischemic hemispheres. The tissue samples were immediately
weighed to obtain the wet weight. The brains were then dried at
100 ꢀC for 24 h and determined for its dry weight. The percentage of
brain water content was calculated as (wet weight ꢂ dry weight)/
wet weight ꢁ 100%.
In vitro NO formation of individual compounds was determined
by Griess assay. Briefly, 0.1 mM of each compound in phosphate
buffer solution (PBS) containing 2% dimethyl sulfoxide and 5.0 mM
L-cysteine at pH 7.4 was incubated at 37 ꢀC for 15e300 min and
were sampled every 15 min for 120 min and then every 30 min for
the remaining time. The collected samples (2 mL) were mixed with
0.5 ml of Griess reagent and incubated at 37 ꢀC for 10 min, followed
by measuring at 540 nm. The different concentrations of nitrite
were used as standards to calculate the concentrations of NO
formed by individual compounds.
4.2.5. In vivo antioxidant activity assay
At 24 h after reperfusion, the rats were sacrificed and their
brains were rapidly removed, rinsed with 0.9% cold saline, and
blotted with filter paper before being kept at ꢂ70 ꢀC. After quan-
tification of protein concentrations, the levels of GSH, SOD and
GSH-Px activities and the levels of MDA content in brain tissues
were determined using commercial analysis kits (Jiancheng Insti-
tute of Biotechnology, Nanjing, China) according to the manufac-
turer's protocol.
4.2.3. In vitro hydrogen sulfide release assay
Individual compounds (100
timed intervals (0e90 min) with fresh rat liver homogenate
(430 L) and the concentration of released H2S was measured as
described previously [37]. After incubation, zinc acetate (1% w/v,
m
M) were incubated (37 ꢀC) for
m
250
250
m
m
L) was injected followed by trichloroacetic acid (10% w/v,
L) to stop the reaction. Subsequently, N,N-dimethyl-p-
M; 133 L) in 7.2 M HCl was
M, 133 L) in 1.2 M HCl. The resulting
phenylenediamine sulfate (20
added followed by FeCl3 (30
m
m
Acknowledgments
m
m
solution was determined 10 min thereafter using a microplate
reader at 670 nm. The H2S concentration of individual compounds
was calculated against a standard curve of NaHS (3.125, 6.25, 12.5,
25, 50, 100 mM). Results are expressed as total % conversion of in-
dividual compounds to H2S at each time point.
This study was financially supported by grants from the National
Natural Science Foundation of China (No. 81373419 and 21502071)
and the Ph.D. Program Foundation of the Ministry of Education of
China, China Pharmaceutical University (No. 20130096110005).
Appendix A. Supplementary data
4.2.4. The cerebral ischemia/reperfusion (I/R) model in rats
Male SD rats (280e320 g) were randomly divided into five
groups (n ¼ 12 per group): (1) sham (0.5% CMC-Na), (2) model (I/
R þ 0.5% CMC-Na), (3) I/R þ NBP (80 mg/kg), (4) I/R þ 8d (380 mg/
kg), (5) I/R þ 8d (80 mg/kg). The rats were treated with the indi-
cated compound at the indicated dose by gavage daily for seven
days. Two hours after the last treatment, the rats were anesthetized
with chloral hydrate (300 mg/kg, ip) and subjected to a middle
cerebral artery occlusion (MCAO), as described previously with
some modification. Briefly, the right common carotid artery (CCA),
internal carotid artery (ICA), and external carotid artery (ECA) were
exposed. A 40 monofilament nylon suture (Beijing Sunbio Biotech,
Beijing, China) was carefully inserted from the ECA stump into the
ICA and was gently advanced to occlude the origin of the right
middle cerebral artery (MCA) until a light resistance was felt
(18e20 mm from CCA bifurcation). Two hours later, the suture was
withdrawn to restore blood flow (reperfusion). The sham group of
rats received a sham operation by inserting the filament into the
ICA and immediately withdrawing it. The core body temperature
was monitored and maintained at 37 0.5 ꢀC with a heating pad
and warm light throughout surgical procedures until the rats
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.044.
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