The synthesis and biological evaluation of novel Danshensu-cysteine analog conjugates as cardiovascular-protective agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.016

A series of novel amide and thioester conjugates between Danshensu and cysteine derivatives have been designed and synthesized based on the strategy of "medicinal chemical hybridization". Pharmacological evaluation indicated that the amide conjugates 3a/4a/17a and thioester conjugates 6a-d exhibited obvious protective effects on H₂O₂-induced human umbilical vein endothelial cells (HUVECs). Pretreated with these conjugates could increase glutathione (GSH) activity and decrease malondialdehyde (MDA) level. Further study on mechanism of compound 4a revealed that it was related to its mitochondrial-protective effect and regulation of apoptosis-related proteins expression (Bax, p53, PARP, caspase-3, caspase-9 and Bcl-2). These results indicate that these Danshensu-cysteine analog conjugates possess significant cardiovascular-protective effects and merit further investigation.

Full text of DOI:10.1016/j.ejmech.2012.07.016

European Journal of Medicinal Chemistry 55 (2012) 176e187
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis and biological evaluation of novel Danshensuecysteine analog
conjugates as cardiovascular-protective agents
,
,
,
*
Yaoling Jia a 1, Xiaoyi Dong b 1, Pengfei Zhou a, Xinhua Liu b, Lilong Pan b, Hong Xin b, Yi Zhun Zhu b
,
Yang Wang a
,
*
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Department of Pharmacology, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel amide and thioester conjugates between Danshensu and cysteine derivatives have been
designed and synthesized based on the strategy of “medicinal chemical hybridization”. Pharmacological
evaluation indicated that the amide conjugates 3a/4a/17a and thioester conjugates 6aed exhibited
obvious protective effects on H2O2-induced human umbilical vein endothelial cells (HUVECs). Pretreated
with these conjugates could increase glutathione (GSH) activity and decrease malondialdehyde (MDA)
level. Further study on mechanism of compound 4a revealed that it was related to its mitochondrial-
protective effect and regulation of apoptosis-related proteins expression (Bax, p53, PARP, caspase-3,
caspase-9 and Bcl-2). These results indicate that these Danshensu-cysteine analog conjugates possess
significant cardiovascular-protective effects and merit further investigation.
Received 28 March 2012
Received in revised form
18 June 2012
Accepted 9 July 2012
Available online 20 July 2012
Keywords:
Danshensu
Cysteine
Conjugate
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cardiovascular-protective
1. Introduction
indicated that the key intermediate, 2-acetoxy caffeic acid (2) exhibi-
ted anti-myocardial ischemic effects featured by reducing infarction
Danshensu, (R)-(þ)-3-(3,4-dihydroxyphenyl)-2-hydroxypropan-
oic acid, is one of the main hydrosoluble active ingredients of Radix
Salviae Miltiorrhizae (Danshen) which has been applied in clinical
practice for centuries in Asia (Scheme 1). It has been widely reported
on its pharmacological effects such as potent anti-oxidative, inhibiting
platelet aggregation, anti-coagulant, anti-inflammatory, improving
heart function effects and anti-tumor activities [1e3]. Moreover,
Danshensu is one of the basic building blocks in many active compo-
nents of culinary plants (sage, rosemary, mint, melissa, thyme,
prunella, origan, sweet basil, etc.) mostly responsible for antiinfective,
antiinflammatory, and antioxidative activity of these herbs, and
therefore its scaffold is easily accepted by the human body and privi-
leged for further exploring novel drug candidates among these activ-
ities [4,5]. In our previous work [4], a series of optically active
Danshensu derivatives (1) were synthesized by asymmetric hydro-
genation and the preliminary pharmacological studies showed that
they exhibited significant activity for cardiovascular protection by
blocking oxidative stress and apoptosis pathway. The in vivo test
size and increasing the level of the intracellular enzymes detectable in
serum. The phenolic esterified or etherified protection of Danshensu
were not inclined to be oxidized, with high liposoluble and low toxic
property (The median lethal dose of these Danshensu derivatives is
5 g/kg, assayed by the National Shanghai Center for New Drug Safety
Evaluation and Research). These results prompted us to further design
new chemical entities by combining Danshensu with other biologi-
cally active molecules under the guidance of “medicinal chemical
hybridization” (MCH) which has been widely used to design poly-
valent drug [6e13]. It can be carried out by joining two drugs through
an appropriate linker or bond which is likely to be cleaved metaboli-
cally. The most obvious advantages are potentially increased affinity
and improved predictable pharmacokinetic profile, because single
product is capable of working at two separate pharmacological sites of
action directly or following metabolism. Such examples abound in
recent years [6e13].
Recently, ample publications have probed hydrogen sulfide
(H2S) which has been proposed as the third gasotransmitter besides
NO and CO [14e19]. It has been confirmed that endogenous H2S
possesses many important physiological functions in the regulation
of vasodilation and vascular reconstruction in recent years [20e23].
Its cardiovascular bioactivities include relaxing vascular smooth
muscle, inhibiting the proliferation of vascular smooth muscle cells
* Corresponding authors. Tel./fax: þ86 21 51980115.
E-mail addresses: zhuyz@fudan.edu.cn (Y.Z. Zhu), wangyang@shmu.edu.cn
(Y. Wang).
1
Both authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.016

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
177
Scheme 1. The structures of Danshensu, L-cysteine and their conjugated derivatives.
and preventing the generation of transient hypertension. Endoge-
nous H2S can also improve the ischemia-reperfusion injury by
increasing the activity of antioxidant enzymes and direct removing
part of the radical [21,22]. Endogenous H2S is produced in vivo from
cysteine, methionine and other organosulfur compounds in
acetylglycine followed by hydrolysis in hydrochloric acid [4]. To
prepare the racemic Danshensu derivatives 15aeb, compounds
14aeb were subjected to hydrogenation in the presence of catalytic
amount of 10% PdeC under the pressure of 15 atm and 1 atm
respectively. Besides the normal products 15aeb isolated in 68%
and 30% yields respectively, the overreductive products 16aeb
were simultaneously obtained in yields of 15% and 59%, respec-
tively. To investigate the effects of 2,3-double bond, 2-acetoxy
group and phenolic protective groups on the bioactivity, six
Allium species catalyzed by cystathionine-
g-lyase (CSE) and cys-
tathionine- -synthetase (CBS), two key enzymes of the trans-sul-
b
furation pathway in the CNS and cardiovascular system, which
leads to inactivation of HMG-CoA reductase and accordingly
inhibits the biosynthesis of cholesterol [24].
carboxylic acids 14e16 were condensed with -cysteine derivative 8
L
Based on the MCH strategy, a series of novel amide conjugates of
Danshensu-cysteine derivatives (3aeb) and 2-acetoxy caffeic acid-
cysteine derivatives (4aeb) as well as the thioester conjugates
(5aed, 6aed) were designed via amidation and thioesterification to
expect to enhance bioactivity through synergic effect (Scheme 1). In
the present study, we reported the synthesis of these target conju-
gates, their pharmacological activities and the possible mechanism.
to afford amide conjugates 4aeb, 3aeb and 17aeb in good yields
respectively. Similarly, the thioesterification between 14e16 and 10
were carried out in the presence of bis(2-oxo-3-oxazolidinyl)
phosphonic chloride (BOP-Cl) and triethylamine (TEA) to afford
thioester conjugates 6aeb, 5aeb and 18aeb in 79e92% yields
respectively. In order to get rid of the effect of the bulky Boc and
release the amino groups, the deprotection were subsequently
performed in the presence of TFA at 10 ꢀC to give the corresponding
conjugates 6ced, 5ced and 18ced in excellent yields [25].
2. Chemistry
L
-cysteine derivatives 8 and 10 with amino and mercapto group
exposed respectively were prepared according to the general proce-
dures outlined in Scheme 2. -cysteine reacted with benzyl bromide
3. Pharmacological assay results
L
3.1. Protective effects of Danshensu-cysteine analog conjugates on
H2O2-induced HUVECs
in the presence of NaOH/EtOH solution to provided compound 7,
which was esterified by MeOH to obtain product 8 in 78% total yield.
Compound 10 was attained via esterification of carboxyl group fol-
lowed by protection of amino-group in 9 by tert-butoxycarboxylic
anhydride in relatively low yield of 45% for two steps due to the
unavoidable formation of its bisulfide even under N2 atmosphere.
The designed conjugates were synthesized as outlined in
Scheme 3 via the key intermediates 14aeb, which could be facilely
attained by the condensation of aldehydes 11aeb with N-
Effects of these synthesized conjugates on the viability of H2O2-
induced HUVECs were investigated by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) analysis [26]. N-
acetyl-
experiments. Cells were incubated with indicated concentrations of
compounds for 4 h before exposure to 200 mol/L H2O2 for 12 h. As
shown in Fig. 1, 200 M H2O2 treatment decreased cell viability to
L-cysteine (NAC) was served as positive control in the
m
m
around 45%, but pre-treatment with 3a/4a/17a, 6aed, 5d and
positive control NAC (5 mmol/L) prevented cell death, restoring cell
viability (*P < 0.05 vs. vehicle group).
As the activity of Boc deprotected conjugates are basically
consistent with their precursors, compounds 3a/4a/17a and 6aeb
were chosen for further investigation. The protective effects of
these chosen Danshensu-cysteine analog conjugates were further
evaluated by lactate dehydrogenase (LDH) assay, another indicator
of cell injury [26]. Compounds 3a/4a/17a, 6aeb at the same
concentration of 50 mmol/L and positive control NAC (5 mmol/L)
were all effective in attenuating H2O2-induced increase in LDH
release (Fig. 2A). As shown in Fig. 2B, compound 4a could
Scheme 2. The synthesis of L-cysteine derivatives 8 and 10. Reaction conditions: (i)
BnBr, NaOH/EtOH, rt, 1 h, 92%; (ii) MeOH, SOCl2, rt, 6 h, 85% (8). (iii) Boc2O, TEA, CH2Cl2
under N2 atmosphere, 6 h, 45% yield for two steps.

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Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
Scheme 3. The synthesis of target conjugates. Reaction conditions: (i) N-acetylglycine, Ac2O/NaOAc, 120 ꢀC, 2e3 h, then poured into H2O, yield: 75% (12a), 65% (12b); (ii) 9% HCl,
100 ꢀC, 3e4 h, yield: 87% (13a), 96% (13b); (iii) Ac2O/NaOAc, rt, 6 h, yield: 90% (14a), 92% (14b); (iv) H2 (15 atm for a and 1 atm for b), 10% PdeC, rt, 24 h, yield: 68% (15a), 15% (16a),
30% (15b), 59% (16b); (v) 8 (1.1 eq.), EDC, HOBt, DIPEA, rt, 2 h, yield: 78% (3a), 72% (3b), 80% (4a), 82% (4b), 75% (17a), 76% (17b); (vi) 10 (1.2 eq.), BOP-Cl, TEA, rt, 2e12 h, yield: 92%
(5a), 87% (5b), 86% (6a), 89% (6b), 82% (18a), 79% (18b); (vii) TFA (2 eq.), CH2Cl2, 10 ꢀC, 6 h, yield: 85% (5c), 89% (5d), 92% (6c), 94% (6d), 91% (18c), 88% (18d).
significantly decrease LDH leakage in a dose-dependent manner.
The results were consistent with that determined by MTT assay.
Therefore, the protective effect of these compounds on HUVECs
against H2O2-induced injury was further evaluated.
dependently restored the mitochondrial potential that decreased
due to H2O2 exposure which was shown as decreased red fluores-
cence, indicating that compound 4a possesses mitochondrial-
protective effect on H2O2-induced HUVECs.
3.4. Compound 4a attenuated H2O2-induced apoptosis in HUVECs
3.2. Effects of compounds 3a/4a/17a and 6aeb on MDA and GSH in
H2O2-induced HUVECs
Apoptosis is a programmed cell death which is characterized by
specific structural changes that include cell shrinkage, nuclear
condensation and DNA fragmentation [27]. In order to evaluate the
anti-apoptotic activity of 4a, Hoechst staining was carried out to
observe the morphological changes in H2O2-induced HUVECs. As
shown in Fig. 5A, administration of compound 4a (5, 50, 100 mmol/
L) resulted in less nuclei-shrunk and nuclear condensation dose-
dependently compared with the H2O2-induced group.
To determine the antioxidant activity of compounds 3a/4a/17a
and 6aeb, malondialdehyde (MDA), a marker of oxidant-mediated
lipid peroxidation in cells and glutathione (GSH), an important
antioxidant in cells, were quantified after H2O2-incubation. Treat-
ment of HUVECs with 200
increase in the level of MDA and the decrease in the activity of GSH.
mmol/L of H2O2 for 12 h caused the
However, pre-incubation with compounds 3a/4a/17a, 6aeb at
To gain insight into anti-apoptotic effects of compound 4a in
50
mmol/L and positive control NAC (5 mmol/L) significantly
H2O2-induced HUVECs quantitatively,
Annexin V-FITC fluorescence was measured by flow cytometric
analysis. Pre-incubation with compound 4a (5, 50, 100 mol/L) for
a display of PI versus
inhibited the increase of MDA level and the loss of GSH activity
(Fig. 3A and B) and compound 4a manifested this antioxidant
activity in a dose-dependent manner as indicated in Fig. 3C and D.
m
4 h prior to H2O2 exposure inhibited both late and total apoptosis
dose-dependently and the values of total apoptosis were decreased
to 20.1 ꢁ 1.9%, 9.0 ꢁ 0.7% and 5.4 ꢁ 1.8%, respectively (Fig. 5B).
3.3. Compound 4a abolished H2O2-induced mitochondrial membrane
potential reduction in HUVECs
Mitochondrial membrane potential (6jm), tested by JC-1 assay,
is an important parameter reflecting the function of mitochondria.
JC-1 exhibits potential-dependent accumulation in mitochondria
which is manifested by a fluorescence emission shift from green to
red. As illustrated in Fig. 4, pre-treatment with compound 4a dose-
3.5. Effects ofcompound 4a on the levels ofapoptosis-related proteins
To further elucidate the mechanism of anti-apoptotic activity of
compound 4a, expression levels of apoptosis-related proteins were
examined at protein level.

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
179
Fig. 1. (A) Effects of conjugates 3aeb/4aeb/17aeb on cell viability in H2O2-induced HUVECs. (B) Effects of conjugates 5aeb/6aeb/18aeb on cell viability in H2O2-induced HUVECs.
(C) Effects of conjugates 5ced/6ced/18ced on cell viability in H2O2-induced HUVECs. The viability of control cell is presumed to be 100%. HUVECs were pre-incubated with different
concentrations of danshensu-cysteine conjugates (5, 50, 100 mmol/L) and NAC (5 mmol/L) for 4 h and then exposed to H2O2 for 12 h. NAC denotes N-acetyl-L-cysteine. Values are
expressed as means ꢁ S.D. from six individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group.
Western blot analysis revealed that the levels of pro-apoptotic
proteins Bax, p53 and poly (ADP-ribose) polymerase (PARP) in
H2O2-induced HUVECs declined significantly, whereas the level of
anti-apoptotic protein Bcl-2 markedly increased, all in a dose-
dependent manner, after treatment by compound 4a compared
with H2O2-induced group (P < 0.05) (Fig. 6). As shown in Fig. 7, the
amount of pro-caspase-3 and pro-caspase-9 in H2O2-induced
HUVECs were markedly decreased, which is a sign of caspase
activation that lead to apoptosis. However, compound 4a
pretreatment attenuated the H2O2-induced caspase-3 and caspase-
9 activation dose-dependently (P < 0.05). All of these results
indicate that compound 4a inhibits apoptosis markedly in H2O2-
induced HUVECs.
cardiovascular diseases. Human umbilical vein endothelial cells
(HUVECs) are commonly accepted as a tool in the study of the
mechanisms involved in the pathogenesis of cardiovascular
diseases, and hydrogen peroxide (H2O2) is extensively referred to as
one of the major oxidative stimuli in antioxidative studies [28e30].
In the present study, a series of Danshensu-cysteine analog
conjugates were synthesized and their pharmacological activities
as well as possible protective mechanism were investigated on
H2O2-induced HUVECs.
MTT results showed that the amide conjugates 3a/4a/17a with
phenolic acetyl protective groups increased cell viability signifi-
cantly after H2O2 treatment, indicating that both Danshensu and
caffeic acid were effective scaffolds in this type of conjugates.
However, the amidates 3b/4b/17b with the methylene protective
groups were inert, which may be attributed to the fact that their
methylene phenolic protective groups were hard to cleave to
release hydroxyls compared with acetyl in 3a/4a/17a. All thioester
conjugates 6aed with caffeic acid scaffold were demonstrated
4. Discussion
Enhanced oxidative damage after various stimuli has been
confirmed to be an initial event in the development of
Fig. 2. (A) Effects of 3a/4a/17a and 6aeb on LDH release in H2O2-induced HUVECs. HUVECs were pre-incubated with 50 mmol/L 3a/4a/17a, 6aeb and 5 mmol/L NAC for 4 h and then
exposed to H2O2 for 12 h. (B) Effect of 4a on LDH release in H2O2-induced HUVECs. HUVECs were pre-incubated with 5, 50, 100 mmol/L 4a and 5 mmol/L NAC for 4 h and then
exposed to H2O2 for 12 h. NAC denotes N-acetyl-L-cysteine. Values are expressed as means ꢁ S.D. from three individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus
control group.

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Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
Fig. 3. (A) Effects of 3a/4a/17a and 6aeb on MDA level in H2O2-induced HUVECs. (B) Effects of 3a/4a/17a and 6aeb on total GSH level in H2O2-induced HUVECs. HUVECs were pre-
incubated with 50 mol/L 3a/4a/17a, 6aeb and 5 mmol/L NAC for 4 h and then exposed to H2O2 for 12 h. (C) Effect of 4a on MDA level in H2O2-induced HUVECs. (D) Effect of 4a on
m
total GSH level in H2O2-induced HUVECs. HUVECs were pre-incubated with 5, 50, 100 mmol/L 4a and 5 mmol/L NAC for 4 h and then exposed to H2O2 for 12 h. NAC denotes N-acetyl-
L-cysteine. Values are expressed as means ꢁ S.D. from three individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group; $ P < 0.05 versus 6a, 6b, 17aetreated
groups, respectively.
bioactive albeit most thioesters (5aec/18aed) were inactive, sug-
gesting that the carbonecarbon double bond was the crucial
pharmacophore in the thioester conjugates.
reduction of mitochondrial membrane potential, indicating that
anti-apoptotic activity of 4a was related to its protection of
mitochondria.
Compounds 3a/4a/17a and 6aeb all decreased LDH leakage
after H2O2 treatment and manifested potent antioxidant activity
featured by decreased formation of MDA and increased GSH
activity in H2O2-induced HUVECs. At the same concentration of
The members of Bcl-2 family, a series of anti- or pro-apoptotic
regulators, are critical for the regulation of apoptosis. Bcl-2
protein is known to promote cell survival as well as to suppress
cell death, whereas Bax is a pro-apoptotic protein that promotes or
accelerates cell death [32]. P53 is a tumor suppressor protein which
can lead to apoptosis. The results showed that 4a up-regulated the
level of Bcl-2 markedly and down-regulated the levels of Bax and
p53. The caspase family is another key regulator of apoptotic
signaling pathway, which is categorized into initiator (caspase-8,
-9) and executioner (caspase-3, -6, -7) [33]. Damaged mitochondria
will activate upstream inactive proteases (caspase-9) to trigger
a cascade reaction which finally turns downstream executive
proteins such as caspase-3 into active forms. Active executive
protein caspase-3 can further cleave downstream substrates
involved in apoptotic process, such as PARP, resulting in cell
apoptosis [28,34,35]. In the current study, H2O2-induced activation
of caspase-9 and caspase-3, increased p53 and Bax expression, as
well as the cleavage of PARP were all inhibited by pretreated 4a
significantly. Therefore, the mechanism by which 4a suppressed
cell apoptosis in H2O2-induced HUVECs was possibly through the
regulation in the intrinsic mitochondria-mediated pathway such as
Bcl-2 and caspase family-related signals.
50 mmol/L, the GSH activity of cells treated by the amide conjugates
3a/4a was higher than that of cells treated by the thioester conju-
gates 6aeb, illuminating that the bioactivity of amide conjugates is
more potent than those conjugated by thioester bond. Besides, the
fact that 3a/4a exhibited higher activities than 17a in GSH assay
showed that 2-acetoxy substitution was favorable to anti-oxidant
activity. However, no significant difference of GSH activity was
exhibited between 4a and 3a treated cells, indicating that the
double bond was not the crucial pharmacophore in the amide
conjugates.
Morphological analysis via Hoechst staining showed that 4a
lessened the strong staining and nuclear shrunk after H2O2 expo-
sure. Meanwhile, the flow cytometric evaluation gave similar
results that 4a dose-dependently inhibited both late and total
apoptosis in H2O2-induced HUVECs. These results suggested that 4a
possessed potent anti-apoptotic activity.
Mitochondria are complex cell organelles which are essential in
the maintenance of cell survival. Mitochondrial dysfunction is
a prominent feature of cell apoptosis, which is marked by a loss of
mitochondrial membrane potential. It has been revealed that the
intrinsic program of apoptosis was mainly triggered by the mito-
chondrial pathway [31]. Therefore, to further elucidate the
protective mechanism of 4a, its inhibitory effect on the loss of
mitochondrial membrane potential was determined. As expected,
pretreatment of 4a greatly prevented HUVECs from H2O2-induced
5. Conclusion
The synthesis and pharmacological evaluation of a series of
novel amide and thioester conjugates between Danshensu and
cysteine derivatives have been reported and 3a/4a/17a/6aed
demonstrated significant protective effect on H2O2-induced

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
181
Fig. 4. Effect of 4a on loss of mitochondrial membrane potential in H2O2-induced HUVECs. NAC denotes N-acetyl-L-cysteine. Values are expressed as means ꢁ S.D. from three
individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group.
HUVECs. Pharmacological evaluation has shown that these conju-
gates exhibit well-ordered structureeactivity relationship. For
amide conjugates 3a/4a/17a, the phenolic acetyl protective groups
and 2-acetoxy substitution are favorable to their activity. For thio-
ester conjugates 6aed, the scaffold of caffeic acid was more privi-
leged than saturated analogs. The protective mechanism of these
conjugates may be related to their anti-oxidative and anti-
apoptotic properties. Further study are in progress as these prom-
ising results demonstrate that these Danshensu-cysteine analog
conjugates merit further investigation as potential cardiovascular-
protective agents.
b-actin were purchased from Proteintech Group, Inc. (IL, USA); anti-
p53 and anti-caspase-3 were obtained from Cell Signaling Tech-
nology, Inc. (MA, USA); anti-(ADP-ribose)polymerase (PARP) was
purchased from Epitomics, Inc. (CA, USA). Dulbecco’s modified
Eagle’s medium (DMEM) and fetal bovine serum (FBS) were ob-
tained from Gibco (Gibco-BRL, Paisley, UK). The kits for lactate
dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH)
were purchased from Jiancheng Bioengineering Institute (Nanjing,
China). The kits for JC-1 and Hoechst 33258 staining were obtained
from Beyotime Bioengineering Institute (Lianyungang, China). The
kit for AnnexinV-FITC/PI was purchased from BD Biosciences (CA,
USA). All compounds (except for positive control NAC) solution in
dimethyl sulfoxide (DMSO) was freshly prepared. Final DMSO
concentration in media did not exceed 0.05%.
6. Experimental
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. Melting points were
determined in open capillary tubes on X-4 apparatus and were
uncorrected. Nuclear magnetic resonance spectra were recorded on
a Brucker-DPX 400 MHz spectrometer. Mass spectral data was
collected on Shimadzu LCMS-2010EV, Agilent 5973N MSD and
Agilent LC/MSD analytical mass spectrometer. HRMS data were
determined on Kratos Concept 1H and Bruker Daltonics or IonSpec
6.1. (R)-Methyl 2-amino-3-(benzylthio)propanoate hydrochlorid
(8)
To a rapidly stirring solution of 2N sodium hydroxide (15 mL)
and ethanol (35 mL) was added -cysteine hydrochloride (1.3 g,
L
8.2 mmol) and benzyl bromide (0.98 mL, 8.2 mmol). The reaction
mixture was neutralized after 1 h to pH 6e7 by careful addition of
concentrated hydrochloric acid. The precipitate was filtered and
washed successively with water, ethanol and ether to give S-ben-
zylcysteine (7) as a white powder (1.87 g, 92%), mp 210e212 ꢀC (lit
[36].: 215e216 ꢀC). To a solution of compound 7 (1 g, 5 mmol) in
4.7 T FTMS instrument. H2O2, N-acetyl-
L-cysteine (NAC), 3-(4, 5-
dimetrylthiazol)-2, 5-diphenyltetrazolium
bromide (MTT),
dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical
(St. Louis, MO, USA). Anti-Bax, anti-Bcl-2, anti-caspase-9 and anti-

182
Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
Fig. 5. (A) Effect of 4a on morphological features in H2O2-induced HUVECs. Fluorescence photomicrographs of cells stained with Hoechst 33258 (400 ꢂ ). Each photograph is
a representative of three independent observations. (B) Anti-apoptotic effect of 4a in H2O2-induced HUVECs determined by AnnexinV-FITC/PI. NAC denotes N-acetyl-L-cysteine.
Values are expressed as means ꢁ S.D. from three individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group.

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
183
Fig. 6. (A) Effect of 4a on the protein expression of Bcl-2 in H2O2-induced HUVECs. (B) Effect of 4a on the protein expression of Bax in H2O2-induced HUVECs. (C) Effect of 4a on the
protein expression of p53 in H2O2-induced HUVECs. (D) Effect of 4a on the protein expression of PARP in H2O2-induced HUVECs. NAC denotes N-acetyl-L-cysteine. Values are
expressed as means ꢁ S.D. from three individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group.
methanol (30 mL) at 0 ꢀC was dropped SOCl2 (1.5 mL, 7 mmol) and
then the mixture was stirred at room temperature for 6 h. Methanol
was evaporated to give 8 as a white solid (1.3 g, 85%), mp
148e150 ꢀC (lit [37].: 160e162 ꢀC; lit. [38].: 151e152 ꢀC). 1H NMR
6.2. (R)-Methyl 2-(tert-butoxycarbonylamino)-3-mercaptopropanoate
(10)
To a solution of -cysteine hydrochloride (2 g, 12.6 mmol) in
L
(400 MHz, CDCl3)
d
7.23e7.32 (m, 5H, AreH), 3.73 (s, 2H, eCH2Ph),
methanol (40 mL) at 0 ꢀC was dropped SOCl2 (1.5 mL, 7 mmol) and
then the mixture was reacted at room temperature for 24 h.
Methanol was evaporated to give 9 as a colorless oil which was
3.72 (s, 3H, eCOOCH3), 3.58e3.61 (m, 1H, H-2), 2.80e2.85 (m, 1H,
H-3), 2.64e2.69 (m, 1H, H-3).
Fig. 7. (A) Effect of 4a on the protein expression of pro-caspase-3 in H2O2-induced HUVECs. (B) Effect of 4a on the protein expression of pro-caspase-9 in H2O2-induced HUVECs.
NAC denotes N-acetyl-L-cysteine. Values are expressed as means ꢁ S.D. from three individual samples. *P < 0.05 versus vehicle group; #P < 0.05 versus control group.

184
Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
directly used in the next process. To a solution of 9 and triethyl-
amine (2.1 mL, 15 mmol) in CH2Cl2 (30 mL) was added Boc2O and
reacted under N2 atmosphere for 6 h. The solid was filtered out and
the filtrate was purified by silica gel column chromatography
(eluent: PE-EtOAc, 10:1e3:1) providing 10 as a colorless oil (1.33 g,
J ¼ 7.9 Hz, 1H, eNHCHe), 5.36e5.40 (m, 1H, ArCH2CHe), 4.71e4.75
(m, 1H, eNHCHe), 3.74 (s, 3H, eCOOCH3), 3.65 (s, 2H, eSCH2Ph),
3.11e3.21 (m, 2H, ArCH2CH-), 2.78e2.87 (m, 2H, eCH2SBn),
2.25e2.27 (m, 6H, 2 ꢂ AreOCOCH3), 2.11, 2.13 (2s, 3H, CH3COO-
2). 13C NMR (100 MHz, CDCl3)
d 170.8, 169.5, 168.8, 168.2, 168.1,
45% for two steps). 1H NMR (400 MHz, CDCl3)
d
5.41 (br s, 1H,
141.8,141.0,137.5,134.5,129.0,128.9,128.6,127.6,127.3,124.7,123.3,
73.7, 52.7, 51.2, 36.9, 36.4, 33.3, 33.0, 20.8, 20.6. ESI-MS m/z (%):
554.8 (M þ Naþ, 100). HRMS calcd mass for C26H30NO9S [M þ Hþ]
532.1636, found 532.1633.
eNHe), 4.58e4.60 (m, 1H, H-2), 3.76 (s, 3H, eCOOCH3), 2.93e2.96
(m, 2H, H-3), 1.78 (s, 1H, eSH), 1.46 (s, 9H, eOC(CH3)3). The 1H NMR
data are consistent with those reported in literature [39].
6.3. 2-Acetoxy-3-(3,4-diacetoxyphenyl)propanoic acid (15a) and 3-
6.5.2. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-
(3,4-diacetoxyphenyl)propanoic acid (16a)
3-(3,4-methylenedioxyphenyl)propanamide (3b)
Eluent: PE-EtOAc, 2:1. White solid, yield 72%, mp 84e85 ꢀC.
25
(Z)-2-Acetoxy-3-(3,4-diacetoxyphenyl)acrylic acid (14a) and (Z)-
2-acetoxy-3-(3,4-methylenedioxyphenyl)acrylic acid (14b) were
prepared by using aldehydes 11aeb as the starting materials
according to the literature method [4].
[a
]
ꢃ27.5 (c 0.5, acetone). UV lmax (nm) (log
3 ) (MeOH): 206
D
(4.49), 286 (3.56). IR (neat), nmax (cmꢃ1): 3361, 2925, 1742, 1674,
1491, 1443, 1370, 1245, 1038, 931, 808, 704. 1H NMR (400 MHz,
CDCl3)
d 7.24e7.35 (m, 5H, ePh), 6.58e6.79 (m, 4H, eNHCHe,
A mixture of 14a (1 g, 3.1 mmol), 10% PdeC (200 mg) and MeOH
(9 mL) was hydrogenated at 15 atm for 24 h. The mixture was
filtered and concentrated, the residue was purified by silica gel
column chromatography (eluent: PE-EtOAc, 20:1e5:1), providing
15a (0.68 g, 68%) and 16a (148 mg, 15%) as an oil and a white solid
AreH), 5.87e5.92 (m, 2H, eOCH2Oe), 5.32e5.37 (m, 1H,
ArCH2CHe), 4.73e4.79 (m, 1H, eNHCHe), 3.74 (s, 3H, eCOOCH3),
3.64, 3.66 (s, s, 2H, eSCH2Ph), 3.00e3.17 (m, 2H, ArCH2CHe),
2.80e2.88 (m, 2H, eCH2SBn), 2.11, 2.15 (2s, 3H, CH3COO-2). 13C
NMR (100 MHz, CDCl3)
d 170.8, 169.4, 168.8, 147.6, 146.6, 137.4,
respectively. 15a: 1H NMR (400 MHz, CDCl3)
d
7.09e7.12 (m, 3H,
128.9, 128.6, 127.3, 122.7, 122.6, 109.9, 109.8, 108.2, 74.3, 52.7, 51.3,
37.3, 36.5, 36.4, 20.9. ESI-MS m/z (%): 460.2 (M þ Hþ, 100). HRMS
(MALDI-DHB) calcd mass for C23H25NO7SNa [M þ Naþ] 482.1244,
found 482.1244.
AreH), 5.21 (br s, 1H, eCOOH), 3.12e3.18 (m, 2H, H-3), 2.27 (s, 6H,
AreOCOCH3), 2.17 (s, 3H, CH3COO-2). 16a: mp 75e78 ꢀC (lit [40].:
79e80 ꢀC), 1H NMR (CDCl3)
d 7.10 (s, 2H, AreH), 7.04 (s, 1H, AreH),
2.95 (t, J ¼ 7.6 Hz, 2H, H-2), 2.68 (t, J ¼ 7.6 Hz, 2H, H-3), 2.28 (s, 6H,
eOCOCH3).
6.5.3. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-
3-(3,4-diacetoxyphenyl)propenamide (4a)
6.4. 2-Acetoxy-3-(3,4-methylenedioxyphenyl)propanoic acid (15b)
and 3-(3,4-methylenedioxyphenyl)propanoic acid (16b)
Eluent: PE-EtOAc, 1:1. White needles, yield 80%: mp 111e112 ꢀC.
25
[
a
]
ꢃ29 (c 1.0, CHCl3). UV lmax (nm) (log
3
) (MeOH): 202 (4.56),
D
289 (4.13), 320 (4.20). IR (KBr), nmax (cmꢃ1): 1745, 1671, 1640, 1504,
A mixture of 14b (525 mg, 3.1 mmol), 10% PdeC (103 mg) and
MeOH (9 mL) was hydrogenated at atmospheric pressure for 24 h.
The mixture was filtered and concentrated, the residue was purified
by silica gel column chromatography (eluent: CH2Cl2eCH3OH, 8:1),
providing 15b (130 mg, 30%) and 16b (230 mg, 59%) as pale yellow
solids respectively. 15b was purified by crystallization from acetic
acid to give 15b as white solid: mp 150e152 ꢀC (lit [4].: 151e153 ꢀC).
1449, 1244, 1184, 1308. 1H NMR (400 MHz, CDCl3)
d
7.18e7.44 (m,
9H, AreH, -CH]), 6.84 (d, J ¼ 7.3 Hz, 1H, eNHCHe), 4.84e4.86 (m,
1H, eNHCHe), 3.77 (s, 3H, eCOOCH3), 3.70 (s, 2H, eSCH2Ph),
2.91e3.03 (m, 2H, eSCH2e), 2.34 (s, 3H, eOCOCH3), 2.30 (s, 6H,
AreOCOCH3). 13C NMR (100 MHz, CDCl3)
d 171.0, 167.9, 167.8, 162.0,
142.5, 142,1, 139.8, 137.5, 130.8, 128.9, 128.6, 127.9, 127.3, 123.7, 52.8,
51.9, 36.6, 33.2, 20.7. ESI-MS m/z (%): 530.0 (M þ Hþ, 100). HRMS
calcd mass for C26H28NO9S [M þ Hþ] 530.1479, found 530.1467.
1H NMR (400 MHz, CDCl3)
d 6.68e6.75 (m, 3H, AreH), 5.94 (s, 2H,
eOCH2Oe), 5.17e5.21 (m, 1H, H-2), 3.01e3.16 (m, 2H, H-3), 2.12 (s,
3H, eOCOCH3). 16b: mp 80e82 ꢀC (lit [41].: 87e88 ꢀC), 1H NMR
6.5.4. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-
3-(3,4-methylenedioxyphenyl)propenamide (4b)
(CDCl3)
d 6.66e6.75 (m, 3H, AreH), 5.93 (s, 2H, eOCH2Oe), 2.89 (t,
J ¼ 7.2 Hz, 2H, H-3), 2.64 (t, J ¼ 7.2 Hz, 2H, H-2).
Eluent: PE-EtOAc,1:1. White needles, yield 82%: mp 100e102 ꢀC.
[a
]
25 ꢃ30.0 (c 1.0, CHCl3). UV lmax (nm) (log
3
) (MeOH): 205 (4.38),
D
6.5. General procedure for the amide conjugates 3aeb, 4aeb and
17aeb
289 (3.85), 320 (3.91). IR (neat), nmax (cmꢃ1): 2958, 2925, 2855,
1766, 1638, 1493, 1187, 1036, 967. 1H NMR (400 MHz, CDCl3)
d
7.24e7.31 (m, 5H, ePh), 7.16 (s, 1H, AreH), 7.08 (s, 1H, eCH]Ce),
To a solution of carboxylic acid 15aeb, 14aeb or 16aeb
(0.5 mmol) in CH2Cl2 (8 mL) was added 1-hydroxybenzotriazole
(HOBt, 82 mg, 0.6 mmol) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI, 116 mg, 0.6 mmol). The
mixture was stirred at room temperature for 15 min, then methyl S-
7.02 (d, J ¼ 8.3 Hz, 1H, AreH), 6.77e6.84 (m, 2H, eNHCHe, AreH),
6.00 (s, 2H, eOCH2Oe), 4.84e4.86 (m, 1H, eNHCHe), 3.77 (s, 3H,
eCOOCH3), 3.70 (s, 2H, eSCH2Ph), 2.91e3.03 (m, 2H, eSCH2e), 2.36
(s, 3H, eOCOCH3). 13C NMR (100 MHz, CDCl3)
d 171.1, 167.9, 162.4,
148.7, 148.0, 137.6, 137.5, 128.9, 128.6, 127.3, 126.2, 125.5, 124.3,
108.8, 108.6, 101.5, 52.8, 51.9, 36.7, 33.3, 20.9. ESI-MS m/z (%): 458.0
(M þ Hþ, 100). HRMS calcd mass for C23H24NO7S [M þ Hþ]
458.1273, found 458.1286.
benzyl- -cysteine hydrochloride (8) (244 mg, 0.55 mmol) followed
L
by diisopropylethylamine (0.1 mL, 0.6 mmol) were added. The
solution was kept at room temperature for 2 h and then purified by
silica-gel column chromatography to give the corresponding amide
conjugates 3aeb, 4aeb and 17aeb.
6.5.5. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-3-(3,4-
diacetoxyphenyl)propanamide (17a)
6.5.1. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-
Eluent: PE-EtOAc, 1:1. White solid, yield 75%: mp 86e88 ꢀC.
25
3-(3,4-diacetoxyphenyl)propanamide (3a)
[
a
]
þ4.8 (c 1.0, CHCl3). UV lmax (nm) (log
3
) (MeOH): 202 (4.56).
D
25
D
Eluent: PE-EtOAc, 1:1. Sticky oil, yield 78%. [
CHCl3). UV lmax (nm) (log
(cmꢃ1): 1771, 1748, 1679, 1507, 1437, 1372, 1213. 1H NMR (400 MHz,
CDCl3) 7.24e7.33 (m, 5H, ePh), 7.05e7.09 (m, 3H, AreH), 6.75 (t,
a
]
þ0.2 (c 1.0,
IR (neat), nmax (cmꢃ1): 3370, 2924, 1745, 1652, 1504, 1436, 1370,
3
) (MeOH): 203 (4.61). IR (neat), nmax
1207, 1181, 1109, 1013, 897, 703. 1H NMR (400 MHz, CDCl3)
d
7.24e7.33 (m, 5H, ePh), 7.06e7.08 (m, 2H, AreH), 7.04 (s, 1H,
AreH), 6.08 (d, J ¼ 7.4 Hz, 1H, eNHe), 4.77e4.78 (m, 1H, eNHCHe),
d

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
185
3.73 (s, 3H, eCOOCH3), 3.67 (s, 2H, eSCH2Ph), 2.95 (t, J ¼ 7.5 Hz, 2H,
ArCH2CHe), 2.84e2.87 (m, 2H, eCH2SBn), 2.46e2.51 (m, 2H,
ArCH2CHe), 2.27 (s, 6H, eOCOCH3). 13C NMR (100 MHz, CDCl3)
3H, eCOOCH3), 3.30e3.42 (m, 2H, eSCH2e), 2.92e3.00 (m, 2H,
ArCH2e), 2.11 (s, 3H, eOCOCH3), 1.45 (s, 9H, eOC(CH3)3). 13C NMR
(100 MHz, CDCl3)
d 197.8, 170.8, 169.7, 169.6, 147.7, 146.7, 122.6,
d
171.4, 171.2, 168.3, 168.2, 142.0, 140.5, 139.5, 137.8, 128.9, 128.7,
109.7, 108.3, 101.0, 80.3, 78. 8, 52.8, 52.7, 37.9, 30.6, 28.3, 20.7. MS
(ESI) m/z: 492.3 [M þ Naþ]. HRMS calcd mass for C21H27NO9SNa
[M þ Naþ] 492.1299, found 492.1298.
127.3, 126.5, 123.3, 52.6, 21.6, 37.5, 36.7, 33.5, 30.6, 20.6. ESI-MS m/z
(%): 496.12 (M þ Naþ, 100). HRMS calcd mass for C24H28NO7S
[M þ Hþ] 474.1581, found 474.1574.
6.6.3. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
6.5.6. N-((R)-3-Benzylthio-1-methoxy-1-oxo-2-propanyl)-3-(3,4-
2-acetoxy-3-(3,4-diacetoxyphenyl)propenethioate (6a)
methylenedioxy phenyl)propanamide (17b)
Eluent: PE-EtOAc, 3:1. White solid, mp 121e123 ꢀC, yield 86%.
25
Eluent: PE-EtOAc, 2:1. White needles, yield 76%: mp 76e78 ꢀC.
[a
]
þ32 (c 1.0, CHCl3). UV lmax (nm) (log
3
) (MeOH): 203 (3.83),
D
25
[
a]
þ7.6 (c 1.0, CHCl3). UV lmax (nm) (log
3
) (MeOH): 204 (4.63),
248 (4.27), 300 (3.80). IR (neat), nmax (cmꢃ1): 1772, 1714, 1503, 1369,
D
286 (3.62). IR (neat), nmax (cmꢃ1): 1744,1654,1491,1443,1246,1039,
1261, 1160, 1113, 1014, 757. 1H NMR (400 MHz, CDCl3)
d
7.49 (s, 1H,
704. 1HNMR (400 MHz, CDCl3)
d
7.22e7.34 (m, 5H, ePh), 6.64e6.72
AreH), 7.43 (d, J ¼ 8.6 Hz, 1H, AreH), 7.22e7.24 (m, 2H, AreH and
eCH]), 5.23 (br d,1H, eNHe), 4.58 (br s,1H, eCH(COOCH3)e), 3.77
(s, 3H, eCOOCH3), 3.49e3.52 (m, 2H, eSCH2e), 2.34 (s, 3H,
(m, 3H, AreH), 6.06 (d, J ¼ 6.3 Hz, 1H, eNHe), 5.89 (s, 2H,
eOCH2Oe), 4.77e4.81 (m, 1H, eNHCHe), 3.73 (s, 3H, eCOOCH3),
3.65 (s, 2H, eSCH2Ph), 2.81e2.89 (m, 4H, ArCH2CHe, eCH2SBn),
eOCOCH3), 2.31 (s, 6H, 2 ꢂ ArOCOCH3), 1.45 (s, 9H, eOC(CH3)3). 13
C
2.43e2.48 (m, 2H, ArCH2CHe). 13C NMR (100 MHz, CDCl3)
d
171.7,
NMR (100 MHz, CDCl3) d 186.6, 170.8, 167.9, 155.1, 143.2, 142.6,
171.3, 147.7, 146.0, 137.7, 134.4, 128.9, 128.6, 127.3, 121.2, 108.8, 108.3,
100.8, 77.2, 52.6, 51.5, 38.3, 36.7, 33.5, 31.1. ESI-MS m/z (%): 402.1
(M þ Hþ, 100). HRMS calcd mass for C21H23NO5SNa [M þ Naþ]
424.1195, found 424.1192.
142.3, 130.2, 128.5, 125.0, 124.1, 123.9, 80.3, 53.0, 52.8, 31.4, 30.2,
29.7, 28.3, 20.7. ESI-MS m/z: 562.3 [M þ Naþ]. HRMS calcd mass for
C24H29NO11SNa [M þ Naþ] 562.1354, found 562.1360.
6.6.4. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
6.6. General procedure for the thioester conjugates 5aeb, 6aeb
and 18aeb
2-acetoxy-3-(3,4-methylenedioxyphenyl)propenethioate (6b)
25
Eluent: PE-EtOAc, 5:1. Yellow oil, yield 89%. [
CHCl3). UV lmax (nm) (log
(4.15). IR (neat), nmax (cmꢃ1): 1715, 1504, 1261, 1160, 1038. 1H NMR
(400 MHz, CDCl3)
a
]
þ40 (c 0.5,
D
3
) (MeOH): 202 (4.26), 250 (3.88), 342
To a solution of carboxylic acid 15aeb, 14aeb or 16aeb
(1 mmol) in CH2Cl2 (10 mL) was added methyl N-(tert-butox-
d
7.21 (s, 1H, ArCH]), 7.12 (d, J ¼ 1.5 Hz, 1H,
ycarbonyl)-
L
-cysteine (10) (282 mg, 1.2 mmol). The mixture was
AreH), 7.06 (dd, J ¼ 8.2, 1.5 Hz, 1H, AreH), 6.83 (d, J ¼ 8.2 Hz, 1H,
AreH), 6.01 (s, 2H, eOCH2Oe), 5.30 (d, J ¼ 7.8 Hz, 1H, eNHe),
4.53e4.58 (m, 1H, eCH(COOCH3)-), 3.76 (s, 3H, eCOOCH3),
3.41e3.52 (m, 2H, eSCH2e), 2.36 (s, 3H, eOCOCH3), 1.44 (s, 9H,
stirred at 0 ꢀC for 15 min, then triethylamine (0.5 mL, 4 mmol)
followed by bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP-
Cl) (381 mg, 1.5 mmol) were added. The solution was kept at room
temperature for 3 h. After the removal of the solvent under the
reduced pressure, the residue was added EtOAc (30 mL) and
washed with water (3 ꢂ 2 mL), brine (10 mL) and dried over
anhydrous Na2SO4. After the solvent was removed under the
reduced pressure, the residue was purified by silica-gel column
chromatography to give the corresponding thioester conjugates
5aeb, 6aeb or 18aeb.
eOC(CH3)3). 13C NMR (100 MHz, CDCl3)
d 186.5, 170.9, 168.0, 155.1,
149.5, 148.2, 140.6, 126.8, 126.1, 125.7, 109.2, 108.8, 101.7, 80.3, 53.1,
52.8, 31.2, 20.8. ESI-MS m/z: 490.2 [M þ Naþ]. HRMS calcd mass for
C21H25NO9SNa [M þ Naþ] 490.1142, found 490.1156.
6.6.5. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
3-(3,4-diacetoxyphenyl)propanethioate (18a)
25
D
Eluent: PE-EtOAc, 5:1. Colorless oil, yield 82%. [
CHCl3). UV lmax (nm) (log
(cmꢃ1): 1773, 1714, 1506, 1369, 1213, 1182. 1H NMR (400 MHz,
CDCl3)
a
]
þ26 (c 1.0,
6.6.1. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
3 ) (MeOH): 202 (4.46). IR (neat), nmax
2-acetoxy-3-(3,4-diacetoxyphenyl)propanethioate (5a)
25
D
Eluent: PE-EtOAc, 5:1. Colorless oil, yield 92%. [
CHCl3). UV lmax (nm) (log
(cmꢃ1): 1751, 1506, 1370, 1211, 1114. 1H NMR (400 MHz, CDCl3)
a
]
þ66 (c 1.0,
d
7.10 (d, J ¼ 8.3 Hz, 1H, AreH), 7.05 (dd, J ¼ 8.3, 2.0 Hz, 1H,
3 ) (MeOH): 201 (4.49). IR (neat), nmax
AreH), 7.00 (d, J ¼ 2.0 Hz, 1H, AreH), 5.24 (br d, 1H, eNHe),
4.51e4.55 (m, 1H, eCH(COOCH3)e), 3.73 (s, 3H, eCOOCH3),
3.30e3.39 (m, 2H, eSCH2e), 2.85e2.98 (m, 4H, eCH2CH2Ar), 2.28
(s, 6H, 2 ꢂ ArOCOCH3), 1.44 (s, 9H, eOC(CH3)3). 13C NMR (100 MHz,
d
7.07e7.14 (m, 2H, AreH), 7.05 (d, J ¼ 1.7 Hz, 1H, AreH), 5.38 (dd,
J ¼ 8.9, 3.9 Hz, 1H, eCH(OCOCH3)e), 5.23 (br s, 1H, eNHe),
4.49e4.53 (m, 1H, eCH(COOCH3)e), 3.74 (2s, 3H, eCOOCH3),
3.31e3.42 (m, 2H, eSCH2e), 3.14e3.18 (m, 1H, ArCH2e),
3.03e3.07 (m, 1H, ArCH2e), 2.28 (2s, 6H, ArOCOCH3), 2.10 (s, 3H,
eOCOCH3), 1.44 (s, 9H, eOC(CH3)3). 13C NMR (100 MHz, CDCl3)
CDCl3)
d 197.1, 170.9, 168.4, 168.3, 155.1, 142.0, 140.6, 138.6, 126.5,
123.4, 123.3, 80.2, 77.3, 53.0, 52.7, 44.9, 31.2, 30.6, 28.3, 20.6. MS
(ESI) m/z: 506.5 [M þ Naþ]. HRMS calcd mass for C22H29NO9SNa
[M þ Naþ] 506.1455, found 506.1449.
d
197.6, 170.7, 169.8, 169.7, 168.2, 168.2, 141.9, 141.1, 134.2, 127.5,
124.6, 123.4, 80.3, 78.1, 52.8, 37.3, 30.7, 29.7, 28.3, 20.7, 20.6. MS
(ESI) m/z: 564.5 [M þ Naþ]. HRMS calcd mass for C24H31NO11SNa
[M þ Naþ] 564.1510, found 564.1502.
6.6.6. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
3-(3,4-methylenedioxyphenyl)propanethioate (18b)
25
D
Eluent: PE-EtOAc, 5:1. Colorless oil, yield 79%. [
CHCl3). UV lmax (nm) (log
(3.79). IR (neat), nmax (cmꢃ1): 1712, 1491, 1442, 1366, 1245, 1164,
1038. 1H NMR (400 MHz, CDCl3)
a
]
þ54 (c 1.0,
3 ) (MeOH): 204 (4.66), 233 (4.14), 287
6.6.2. S-((R)-3-methoxy-3-oxo-2-(tert-butoxycarbonylamino)propyl)
2-acetoxy-3-(3,4-methylenedioxyphenyl)propanethioate (5b)
25
d
6.72 (d, J ¼ 7.9 Hz,1H, AreH), 6.66
Eluent: PE-EtOAc, 5:1. Yellow oil, yield 87%. [
CHCl3). UV lmax (nm) (log
(4.36). IR (neat), nmax (cmꢃ1): 2927, 1748, 1697, 1490, 1443, 1367,
1216, 1162, 1037, 929, 756. 1H NMR (400 MHz, CDCl3)
6.74 (d,
a
]
þ135 (c 1.0,
D
(s, 1H, AreH), 6.62 (d, J ¼ 7.9 Hz, 1H, AreH), 5.92 (s, 2H, eOCH2Oe),
5.22 (d, J ¼ 7.8 Hz, 1H, eNHe), 4.49e4.54 (m, 1H, -CH(COOCH3)-),
3.74 (s, 3H, eCOOCH3), 3.27e3.40 (m, 2H, eSCH2e), 2.80e2.90 (m,
4H, eCH2CH2Ar), 1.44 (s, 9H, eOC(CH3)3). 13C NMR (100 MHz,
3
) (MeOH): 207 (4.90), 235 (4.66), 287
d
J ¼ 7.7 Hz, 1H, AreH), 6.69 (s, 1H, AreH), 6.65 (d, J ¼ 7.7 Hz, 1H,
AreH), 5.95 (s, 2H, eOCH2Oe), 5.34e5.37 (m, 1H, eCH(OCOCH3)e),
5.21 (br s, 1H, eNHe), 4.52e4.55 (m, 1H, eCH(COOCH3)e), 3.75 (s,
CDCl3)
d 197.4, 170.9, 155.1, 147.7, 146.1, 133.5, 121.2, 108.8, 108.3,
100.9, 80.2, 77.3, 53.0, 52.7, 45.7, 31.2, 31.1, 28.3, 28.3. MS (ESI) m/z:

186
Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
434.2 [M þ Naþ]. HRMS calcd mass for C19H25NO7SNa [M þ Naþ]
(3.86). IR (neat), nmax (cmꢃ1): 3346, 2925, 1744, 1641, 1504, 1448,
1247, 1037. 1H NMR (400 MHz, CDCl3)
1H, AreH), 7.03 (d, J ¼ 8.3 Hz, 1H, AreH), 6.90 (d, J ¼ 7.0 Hz, 2H,
eNH2), 6.82 (d, J ¼ 8.3 Hz, 1H, AreH), 6.00 (s, 2H, eOCH2Oe),
4.92e4.94 (m, 1H, eCH(COOCH3)e), 3.82 (s, 3H, eCOOCH3),
3.05e3.14 (m, 2H, eSCH2e), 2.38 (s, 3H, eOCOCH3). 13C NMR
434.1244, found 434.1244.
d
7.17 (s, 1H, ¼CHAr), 7.09 (s,
6.7. General procedure for the thioester conjugates 5ced, 6ced and
18ced
To a solution of thioester conjugates 5aeb, 6aeb or 18aeb
(0.2 mmol) in CH2Cl2 (8 mL) was added trifluoroacetic acid
(46 mg, 0.4 mmol) at 0 ꢀC. The mixture was stirred at 10 ꢀC for 6 h.
The solution was kept at 0 ꢀC and neutralized with saturated
saturated NaHCO3 (aq.) to pH 8e9, then extracted with CH2Cl2
(3 ꢂ 6 mL). The combined organic layers were washed with water
(10 mL), brine (10 mL) and dried over anhydrous Na2SO4. After the
removal of the solvent under the reduced pressure, the residue was
submitted to silica-gel column chromatography separation to
afford the corresponding product 5ced, 6ced or 18ced.
(100 MHz, CDCl3) d 170.4, 168.1, 162.2, 159.1, 148.1, 140.9, 129.8,
125.6, 124.4, 110.0, 108.9, 101.5, 53.7, 53.0, 31.9, 22.7. MS (ESI) m/z:
368.2 [M þ Hþ]. HRMS calcd mass for C16H17NO7SNa [M þ Naþ]
390.0618, found 390.0613.
6.7.5. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 3-(3,4-diacetoxyphe-
nyl)propanethioate (18c)
25
Eluent: PE-EtOAc, 2:1. Colorless oil, yield 91%. [
CHCl3). UV lmax (nm) (log
(neat), nmax (cmꢃ1): 1770, 1658, 1507, 1372, 1214. 1H NMR (400 MHz,
CDCl3)
a]
þ15 (c 0.5,
D
3
) (MeOH): 200 (4.52), 202 (3. 65). IR
d
7.10 (s, 2H, AreH), 7.05 (s, 1H, AreH), 6.31 (d, J ¼ 7.5 Hz, 2H,
6.7.1. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 2-acetoxy-3-(3,4-
eNH2), 4.85e4.87 (m, 1H, eCH(COOCH3)e), 3.77 (s, 3H, eCOOCH3),
2.92e3.00 (m, 4H, ArCH2- and eSCH2e), 2.54e2.61 (m, 2H,
eCH2COSe), 2.25, 2.27 (2s, 6H, 2 ꢂ ArOCOCH3). 13C NMR (100 MHz,
diacetoxyphenyl)propanethioate (5c)
25
D
Eluent: PE-EtOAc, 2:1. Colorless oil, yield 85%. [
CHCl3). UV lmax (nm) (log
(neat), nmax (cmꢃ1): 2926,1744,1672,1528,1443,1373,1228, 763. 1H
NMR (400 MHz, CDCl3) 7.03e7.11 (m, 3H, AreH), 6.87 (d,
a
]
þ5.5 (c 0.5,
3 ) (MeOH): 202 (4.44), 276 (3.12). IR
CDCl3) d 197.6, 171.5, 170.6, 168.4, 142.0, 140.4, 139.4, 126.6, 123.4,
123.3, 52.8, 52.1, 37.5, 30.8, 30.5, 20.7. MS (ESI) m/z: 406.3
[M þ Naþ]. HRMS calcd mass for C17H21NO7SNa [M þ Naþ]
406.1058, found 406.1056.
d
J ¼ 7.0 Hz, 2H, eNH2), 5.33e5.42 (m, 1H, eCH(OCOCH3)e),
4.76e4.79 (m, 1H, eCH(COOCH3)-), 3.74, 3.76 (2s, 3H, eCOOCH3),
3.08e3.24 (m, 2H, eSCH2e), 2.84e2.97 (m, 2H, ArCH2e), 2.26 (s,
6H, 2 ꢂ ArOCOCH3), 2.13, 2.17 (2s, 3H, eOCOCH3). 13C NMR
6.7.6. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 3-(3,4-methylenedio-
xyphenyl)propanethioate (18d)
25
(100 MHz, CDCl3)
d
196.6, 170.2, 169.8, 169.7, 169.3, 147.1, 134.8,
Eluent: PE-EtOAc, 5:1. Colorless oil, yield 88%. [
CHCl3). UV lmax (nm) (log
(neat), nmax (cmꢃ1): 1743, 1652, 1490, 1442, 1246, 1039. 1H NMR
(400 MHz, CDCl3) 6.64e6.74 (m, 3H, AreH), 6.26 (br d, 2H, eNH2),
a
]
þ28 (c 0.5,
D
128.0, 122.3, 121.9, 118.6, 73.6, 53.0, 52.8, 37.0, 30.4, 21.0, 20.9, 20.8.
MS (ESI) m/z: 459.4 [M þ NHþ4 ]. HRMS calcd mass for C19H23NO9SNa
[M þ Naþ] 464.1030, found 464.1032.
3
) (MeOH): 203 (4.21), 285 (3.25). IR
d
5.92 (s, 2H, eOCH2Oe), 4.86e4.88 (m, 1H, eCH(COOCH3)e), 3.78 (s,
3H, eCOOCH3), 2.88e2.97 (m, 4H, eSCH2e and ArCH2e), 2.47e2.56
6.7.2. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 2-acetoxy-3-(3,4-
methylenedioxyphenyl)propanethioate (5d)
(m, 2H, ArCH2CH2e). 13C NMR (100 MHz, CDCl3)
d 171.7, 170.5, 147.7,
25
D
Eluent: PE-EtOAc, 2:1. Colorless oil, yield 89%. [
acetone). UV lmax (nm) (log
(3.55). IR (neat), nmax (cmꢃ1): 3339, 2926, 1741, 1675, 1490, 1443,
1245, 1038, 930. 1H NMR (400 MHz, CDCl3)
6.80 (br s, 2H, eNH2),
a
]
ꢃ1.8 (c 0.5,
146.1, 134.2, 121.2, 108.9, 108.4, 100.9, 53.4, 52.8, 38.5, 31.3, 26.9. MS
(ESI) m/z: 312.1 [M þ Hþ]. HRMS calcd mass for C14H17NO5SNa
[M þ Naþ] 334.0720, found 334.0722.
3 ) (MeOH): 203 (4.56), 257 (2.85), 285
d
6.61e6.74 (m, 3H, AreH), 5.93 (s, 2H, eOCH2Oe), 5.29e5.39 (m, 1H,
eCH(OCOCH3)e), 4.79e4.83 (m, 1H, eCH(COOCH3)e), 3.76, 3.78
(2s, 3H, eCOOCH3), 2.88e3.12 (m, 4H, ArCH2- and eSCH2e), 2.13,
6.8. Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs) were obtained
from ATCC (Manassas, VA). Cells were cultured in DMEM supple-
mented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and
2.18 (2s, 3H, eOCOCH3). 13C NMR (100 MHz, CDCl3)
d 170.1, 169.3,
168.5, 147.7, 146.8, 129.0, 122.8, 110.0, 108.3, 101.0, 77.2, 74.1, 53.3,
52.9, 37.2, 21.0. MS (ESI) m/z: 370.0 [M þ Hþ]. HRMS calcd mass for
C16H19NO7SNa [M þ Naþ] 392.0774, found 392.0783.
100
m
g/mL streptomycin at 37 ꢀC in a humidified atmosphere
comprised of 95% air and 5% CO2. Passage 3e8 was used for
experiments. Cells were exposed to different Danshensu-cysteine
6.7.3. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 2-acetoxy-3-(3,4-
analog conjugates (5, 50, 100
mmol/L) or NAC (5 mmol/L) for 4 h,
diacetoxyphenyl)propenethioate (6c)
and then subjected to 200 mol/L H2O2 for 12 h.
m
Eluent: PE-EtOAc, 1:1. Colorless oil, yield 92%. [
CHCl3). UV lmax (nm) (log
a
]
25 þ20.2 (c 1.0,
D
3
) (MeOH): 202 (4.27). IR (neat), nmax
(cmꢃ1): 2925, 1745, 1505, 1434, 1371, 1206, 1181, 1112, 1014, 901. 1H
NMR (400 MHz, CDCl3)
6.9. Determination of cell viability
d
7.45 (d, J ¼ 1.8 Hz, 1H, AreH), 7.39 (dd,
J ¼ 8.7, 1.8 Hz, 1H, AreH), 7.21 (d, J ¼ 8.7 Hz, 1H, AreH), 7.20 (s, 1H,
]CHAr), 6.95 (d, J ¼ 6.5 Hz, 2H, eNH2), 4.92e4.97 (m, 1H,
eCH(COOCH3)e), 3.83 (s, 3H, eCOOCH3), 3.05e3.14 (m, 2H,
Cell viability analysis was performed based on the capacity of
mitochondrial enzymes to transform 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) to formazan. HUVECs
were seeded in 96-well plate (150 mL DMEM with 10% FBS per well)
and incubated overnight. After growing to sub-confluence, cells
eSCH2e), 2.36 (s, 3H, eOCOCH3), 2.30 (s, 6H, 2 ꢂ ArOCOCH3). 13
C
NMR (100 MHz, CDCl3)
d
174.2,170.8,169.7,168.3,168.2,141.8,141.0,
134.6, 127.6, 124.8, 123.2, 78.2, 52.9, 52.8, 38.8, 34.6, 20.7. MS (ESI)
m/z: 462.4 [M þ Naþ]. HRMS calcd mass for C19H21NO9SNa
[M þ Naþ] 462.0974, found 462.0978.
were pretreated with various concentrations of Danshensu-
cysteine analog conjugates (5, 50, 100
FBS for 4 h. After that, H2O2 with a final 200
m
mol/L) in DMEM without
mol/L concentration
L of MTT
m
was added to the culture medium for 12 h. Next, 100
m
6.7.4. S-((R)-3-methoxy-3-oxo-2-aminopropyl) 2-acetoxy-3-(3,4-
(5 mg/mL) was added to each well and cells were incubated at 37 ꢀC
for 4 h. Then, the culture medium with MTT was abandoned and the
methylenedioxyphenyl)propenethioate (6d)
25
D
Eluent: PE-EtOAc, 1:1. Colorless oil, yield 94%. [
CHCl3). UV lmax (nm) (log
a
]
ꢃ0.8 (c 0.2,
colored formazan was dissolved in 100
mL of dimethyl sulfoxide
3 ) (MeOH): 203 (4.43), 288 (3.87), 320
(DMSO). The absorption values were measured at 570 nm using

Y. Jia et al. / European Journal of Medicinal Chemistry 55 (2012) 176e187
187
a microplate reader (Tecan infinite 200). The viability of HUVECs in
6.15. Statistic analysis
each well was presented as percentage of control cells.
Data were presented as means ꢁ S.D. and analyzed by SPSS
software. Two-tailed Student’s t-test was carried out to determine
statistical significance. Differences were considered significant at
P < 0.05.
6.10. Preparation of cell lysates
The cells were grown to confluence in 24-well plates. After drug
treatment the culture supernatant was collected for analysis of
LDH. The cells were scraped from the plates into ice-cold RIPA lysis
buffer (Beyotime, China) and protein concentration was deter-
mined by bicinchoninic acid protein assay kit (Biocolor Bioscience &
technology company, Shanghai, China). Aliquots were stored
at ꢃ80 ꢀC until detection for MDA and GSH level.
Acknowledgements
Financial support from the National Natural Science Foundation
of China (No. 21172044) and National Drug Innovative Program
(Grant No. 2009ZX09301-011) is gratefully acknowledged.
6.11. LDH, MDA, GSH measurement
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measurements were performed according to the manufacturer’s
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phoresis and transferred to PVDF membranes (Millipore Corpora-
tion). The membranes were blocked with 5% nonfat dried milk in
0.1% Tween 20 in Tris-buffered saline (TBST) for 1 h, and incubated
with primary antibodies (Bax, Blc-2, caspase-3, p53, PARP, caspase-
9 and
b
-actin) overnight at 4 ꢀC. Antibodies were detected by
means of HRP-conjugated secondary antibody for 1 h at room
temperature. Immunoreactive bands were visualized using
enhanced chemiluminescence reagents (Beyotime, China) and
densitometric analysis was performed with the use of Alpha Image
(Alpha Innotech, USA).