Asymmetric synthesis and biological evaluation of Danshensu derivatives as anti-myocardial ischemia drug candidates

Article abstract of DOI:10.1016/j.bmc.2009.02.065

The synthesis and bioactivities of Danshensu derivatives (R)-methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)propanoate (1a), (R)-methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)propanoate (1b) and their racemates 7 and 10 were reported in this paper. These derivati

Full text of DOI:10.1016/j.bmc.2009.02.065

Bioorganic & Medicinal Chemistry 17 (2009) 3499–3507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Asymmetric synthesis and biological evaluation of Danshensu derivatives
as anti-myocardial ischemia drug candidates
a,
Cunnan Dong a, Yang Wang b, , Yi Zhun Zhu
*
*
a Department of Pharmacology, School of Pharmacy and Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China
b Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and bioactivities of Danshensu derivatives (R)-methyl 2-acetoxy-3-(3,4-diacetoxyphe-
nyl)propanoate (1a), (R)-methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)propanoate (1b) and their
racemates 7 and 10 were reported in this paper. These derivatives were designed to improve their chem-
ical stability and liposolubility by protecting Danshensu’s phenolic hydroxyl groups with acetyl or meth-
ylene which could be readily hydrolyzed to release bioactive Danshensu. The asymmetric synthesis of 1a
and 1b were achieved by catalytic hydrogenation of (Z)-methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)-2-
propenoate (6a) and (Z)-methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)-2-propenoate (6b) in excellent
enantiomeric excesses (92% ee and 98% ee, respectively) and good yields (>89%). An unexpected interme-
diate product, (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl)acrylic acid (4c) was obtained with high chemose-
lectivity in 86% yield by keeping the reaction temperature at 60 °C and its structure was identified by X-
ray single crystal diffraction analysis. 1a, 1b and their racemates 7, 10 as well as 4c exhibited potent pro-
tective activities against hypoxia-induced cellular damage. The in vitro test showed that all these com-
pounds could increase cell viability, and inhibit lipid hyperoxidation. Furthermore, 1a and 4c could
inhibit apoptosis by regulating the expression of apoptosis-related molecule in gene and protein levels,
up-regulating the expression of bcl-2 and down-regulating bax and caspase-3. The in vivo test indicated
that 4c exhibited anti-myocardial ischemic effects featured by reducing infarction size and increasing the
level of the intracellular enzymes detectable in serum. Therefore, these Danshensu derivatives may be
good drug candidates for anti-myocardial ischemia therapy and merit further investigation.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 16 December 2008
Revised 4 February 2009
Accepted 5 February 2009
Available online 21 March 2009
Keywords:
Danshensu derivatives
Asymmetric synthesis
Neonatal rat ventricular myocytes (NRVMs)
Myocardial infarction
Isolation and purification of Danshensu from natural resources
are difficult and time consuming due to the chemical unstability
1. Introduction
of phenolic hydroxyl groups, low content in Danshen and a variety
of structural analogs as impurities. Several synthetic strategies
have been explored to produce ( )-Danshensu,5,6 but asymmetric
synthesis of Danshensu is extremely rare. Findrik developed a
mathematical model for the enzymatic kinetics of the synthesis
of (R)-(+)-3,4-dihydroxyphenyllactic acid, which was catalyzed by
D-lactate dehydrogenase from Lactobacillus leishmannii.7 The prep-
aration of Danshensu using a chemical method based on catalytic
asymmetric hydrogenation has never been reported. Moreover,
being a hydrophilic molecule and thus poorly soluble in lipidic
matrices, Danshensu uneasily enters into cells by crossing the cell
membranes. Therefore, it is highly desirable to search for new
Danshensu derivatives with potent pharmacological activity, good
stability and liposolubility.
Danshensu is an active component of Danshen, the dried root of
salvia miltiorrhiza mostly responsible for many biological activities,
such as dilating coronary arteries, inhibiting platelet aggregation,
myocardial cell apoptosis and anti-inflammatory property.1–3
These activities are at least partially related to its anti-oxidative
activity.4 Accordingly, Danshensu itself and its scaffold are privi-
leged and of great interest as synthetic targets and building blocks
for cardiovascular active drug candidates.
HO
HO
COOH
OH
Danshensu
Danshensu has the structure of phenyllactic acid bearing two
phenolic hydroxyl groups on its phenyl ring which are considered
as the radical scavenging moieties. The presence of different sub-
stituents in the phenol backbone structure may modulate their
antioxidant property. In general, monophenol is a less efficient
* Corresponding authors. Tel.: +86 21 59180012; fax: +86 21 59180008.
E-mail addresses: wangyang@shmu.edu.cn (Y. Wang), zhuyz@fudan.edu.cn (Y.Z.
Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.065

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C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
antioxidant than polyphenol. The introduction of electron donating
groups such as hydroxyl in the ortho or para position increases the
antioxidant activity of phenolic acid.8,9 In addition, the presence of
a carbonyl group, such as aromatic acid, ester, or lactone enhanced
antioxidant activity. Steric hindrance of the phenolic hydroxyls by
a neighboring inert group, such as methoxyl group, enhanced its
antioxidant activity.10 According to such structure-activity rela-
tionship, Danshensu derivatives (1a, 1b) were designed with the
phenolic hydroxyls esterized by acetylation or protected by meth-
ylene. Such structures could enhance the stability and liposolubili-
ty, and is beneficial to the chiral catalysts used in the asymmetric
reduction step. These structural modification of Danshensu were
expected to exhibit similar pharmacological activity with Dansh-
ensu due to the ester and ether linkages are easily hydrolyzed
and readily release bioactive Danshensu.11–15
the starting material, and 3a and 3b were easily accessible by the
condensation of 2a and 2b with N-acetylglycine followed by
hydrolysis in hydrochloric acid to give the key intermediates 4a
and 4b, respectively.16,17
To prepare the enantiomers of Danshensu derivatives, two ap-
proaches were studied. In the first strategy, compound 4b was sub-
jected to hydrogenation in the presence of catalytic amount of Pd–
C and cinchonidine as the chiral ligand, but the enantiomeric ex-
cess of the product was very low (16% ee).18 Recently the excellent
performance of monophosphites, monophosphonites, and mono-
phosphoramidites as ligands provide tremendous interest and po-
tential in asymmetric hydrogenation.19 Thus, to improve the
enantiomeric excess of product, in the second approach, a new
generation of the monodentate phosphoramidites, compound 11
developed by Ding and co-workers19 was employed as the chiral li-
gand and [Rh(cod)2]BF4 as the catalyst precursor in asymmetric
hydrogenation. First, compounds 4a and 4b were protected at the
OH-groups by esterization with acetic anhydride in the presence of
sodium acetate to provide the corresponding products 5a and 5b in
80% and 77% yields, respectively. Followed by the reaction with
diazomethane in ice-cooled dichloromethane, the thoroughly
protected compounds 6a and 6b were obtained in 81% and 85%
yields, respectively. Using 11 as chiral ligand, under the modified
conditions of the literature procedures,19 excellent enantioselectiv-
ities (92–98% ee) and good yields (>89%) of target compounds 1a
and 1b were realized. Racemic compounds 7 and 10 were also syn-
thesized. First, compound 8 was obtained by two methods of
reduction of 4b with KBH4 and catalytic hydrogenation. The con-
densation of compound 8 with acetic anhydride in the presence
of sodium acetate gave compound 9 in 95% yield. Compound 10
was attained by reacting 9 with diazomethane in ice-cooled dichlo-
romethane in 79% yield. Using Pd/C as catalyst, racemic compound
7 was prepared by catalytic hydrogenation of compound 6a in 79%
O
COOCH3
H3C C O
COOCH3
O
O
O
CH3
C
O
O
CH3
C
O
H3C C O
O
1a
1b
Moreover, it is also attractive to study the pharmacological dif-
ference between the enantiomers and racemates. Herein, we report
the efficient asymmetric synthesis of 1a, 1b and an unexpected
intermediate product 4c and their preliminary pharmacological
assay results.
2. Chemistry
The designed compounds were synthesized efficiently accord-
ing to the procedures outlined in Schemes 1 and 2. 3,4-Dihydroxy-
benzaldehyde (2a) and its analog piperonal (2b) were chosen as
HO
HO
CHO
AcO
AcO
COOH
HO
HO
COOH
i
ii
iii
NHCOCH3
OR
2a
3a
4a: R = H
4c: R = Ac
AcO
COOCH3
v
O
O
CH3
AcO
C
O
AcO
AcO
COOH
iv
AcO
AcO
COOCH3
1a
7
O
CH3
C
O
O
CH3
C
O
vi
AcO
AcO
COOCH3
CH3
C
5a
6a
O
H2C
N
H
N
H2C
O
O
P
O
N
H2C
Chiral ligand (11)
Scheme 1. The synthetic route of Danshensu derivatives 1a, 4c and 7. Reaction conditions: (i) N-acetylglycine, acetic anhydride, NaOAc, 100 °C, 2 h, pour into cold water; (ii)
4a: 9% HCl, 100 °C, 6 h; 4c: 9% HCl, 60 °C, 3 h; (iii) acetic anhydride, NaOAc, rt, 5.5 h; (iv) CH2N2, dichloromethane, 0 °C; (v) [Rh(cod)2]BF4, chiral ligand 11, H2, 60 atm, CH2Cl2,
rt, 24 h; (vi) Pd–C, EtOAc, 10 atm, 24 h.

C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
3501
COOH
OH
COOH
CHO
O
O
O
O
O
ii
i
NHCOCH3
O
2b
3b
4b
COOH
COOCH3
COOCH3
O
O
O
O
O
O
iii
iv
v
O
CH3
C
O
CH3
C
O
CH3
C
O
O
O
5b
8
6b
9
1b
COOH
COOCH3
O
O
COOH
vi
O
O
vii
viii
O
CH3
O
CH3
C
O
O
O
C
O
OH
10
Scheme 2. The synthetic route of Danshensu derivatives 1b and 10. Reaction conditions: (i) N-acetylglycine, acetic anhydride, NaOAc, 100 °C, 3 h; add water, 120 °C, 0.5 h; (ii)
9% HCl, 100 °C, 4 h; (iii) acetic anhydride, NaOAc, rt 7 h; (iv) CH2N2, CH2Cl2, 0 °C; (v) [Rh(cod)2]BF4, chiral ligand 11, H2, 60 atm, CH2Cl2, rt, 24 h; (vi) Pd–C, H2, 6 atm, MeOH,
24 h; or KBH4/MeOH, rt, 7 h; (vii) acetic anhydride, HOAc, rt, 24 h; (viii) CH2N2, CH2Cl2, 0 °C.
yield. Interestingly, the hydrolyzation of 3a was found to lead to
different products at different temperatures. 4a was got at 100 °C
for 6 h and at a lower temperature of 60 °C for 3 h compound 4c
was obtained in 86% yield. However, we were unable to identify
the position of acetyl group simply on the basis of its 1H NMR,
13C NMR, and MS spectra data. The single crystals of 4c were ob-
tained by recrystallization from water and analyzed by X-ray single
crystal diffraction (Fig. 1). Unexpectedly, 2-acetamido in 3a was
converted to 2-acetoxy and 30,40-diacetoxy groups hydrolyzed to
30,40-dihydroxyls to form 4c. This result can be rationalized as
follows: 2-acetamido and 30,40-diacetoxy groups was easily hydro-
lyzed to hydroxyls at low temperature, however acetyl recombined
to 2-dihydroxyl to get 2-acetoxy, and such bonding was so tight
that could be hydrolyzed only at high temperature and lasting
time.
3. Pharmacological assay results
3.1. Protective effects of compounds 1a, 1b, 7, 10, 4c on
hypoxia-induced neonatal rat ventricular myocytes (NRVMs)
The protective effects of compounds 1a, 1b, 7, 10 and 4c were
investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide(MTT) assay and LDH leakage assay. Cells were incu-
bated with indicated concentrations of compounds 1a, 1b, 7, 10
and 4c for 12 h before exposure to hypoxia, then cells were sub-
jected to hypoxia for 5 h. Cell viability was assessed by MTT reduc-
tion assay. It showed that compounds 1a, 1b, 7, 10 and 4c at
1 lmol/L significantly increased cell viability compared with hy-
poxia group (Fig. 2). The marker of cell damage LDH leakage was
assessed after hypoxia and it was found that compounds 1a, 1b,
Figure 1. The single crystal structure of compound 4c.

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C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
49
47
45
43
41
39
37
35
5.5
5
∗
∗
∗
4.5
4
*
*
∗
∗
*
3.5
3
2.5
2
1.5
1
control
hypoxia
1a
1b
4c
hypoxia
1a
1b
7
10
4c
Figure 4. Effects of compounds 1a, 1b, 4c at 1 lmol/L on MDA content in hypoxia-
Figure 2. Effects of compounds 1a, 1b, 7, 10 and 4c at 1 lmol/L on cell viability in
induced neonatal rat ventricular myocytes. Values are expressed as means SE
hypoxia-induced neonatal rat ventricular myocytes. Compounds 1a, 1b, 7, 10 and
4c could significantly increase cell viability. Values are expressed as means SE
from six individual examples. *P < 0.05 versus hypoxia.
from six individual examples. *P < 0.05 versus hypoxia.
3.3. Anti-apoptotic activity of compounds 1a and 4c in hypoxia
induced neonatal rat ventricular myocytes
45
40
∗ #
Apoptosis is a programmed cell death that is characterized by
specific structural changes that include cell shrinkage, nuclear con-
densation and DNA fragmentation. To assess the anti-apoptotic
activity of 1a and 4c, Hoechst staining was performed to observe
the morphological changes in hypoxia-induced neonatal rat ven-
tricular myocytes. Administration of compounds 1a and 4c re-
sulted in less nuclei-shrunk and nuclear condensation compared
with the hypoxia group (Fig. 5).
At the molecular level, apoptosis is activated by the aspartate-
specific cysteine protease cascade, including caspase-3 and cas-
pase-12. Caspase-3 is considered to be the most important execu-
tioner caspases and is activated by any of the initiator caspases.20
Besides caspases, members of the bcl-2 family are also critical for
the regulation of apoptosis. The relative concentration of these
proteins in the outer mitochondrial membrane is thought to
determine the survival or death of a cell following an apoptotic
stimulus.21 To further elucidate the mechanism of anti-apoptotic
activity of 1a and 4c, their effects on modulating the expression
of various apoptosis-related biomarkers, including caspase-3,
bax and bcl-2 were investigated. RT-PCR assay showed that the
level of bax and capase-3 mRNA in hypoxia-induced neonatal
rat ventricular myocytes declined significantly after treatment
by compounds 1a and 4c relative to hypoxia group (P < 0.05),
whereas the level of bcl-2 mRNA markedly increased ( Fig. 6).
Western blot showed consistent results with RT-PCR in protein
level (Fig. 7).
35
30
∗
∗
∗
25
20
15
10
5
∗
hypoxia
1a
1b
7
10
4c
Figure 3. Effects of compounds 1a, 1b, 7, 10 and 4c at 1 lmol/L on LDH leakage in
hypoxia-induced neonatal rat ventricular myocytes. Values are expressed as
means SE from six individual examples. *P < 0.05 versus hypoxia, #P < 0.05
between compounds 1a and 7.
7, 10 and 4c at 1
same concentration of 1
higher activity than racemic compounds 7 and 10 in decreasing
cellular damage. Therefore, the antioxidant property of compounds
1a, 1b and 4c was further evaluated.
l
mol/L could decrease LDH leakage (Fig. 3). At the
mol/L, compounds 1a and 1b possessed
l
3.2. Antioxidant activity of compounds 1a, 1b and 4c in
hypoxia-induced neonatal rat ventricular myocytes
To determine the antioxidant activity of compounds 1a, 1b and
4c, malondialdehyde (MDA), a marker of oxidant-mediated lipid
peroxidation in cells was quantified after hypoxia. As illustrated
in Figure 4, when compounds 1a, 1b and 4c were served at
3.4. In vivo protective effects of 4c on acute myocardial
ischemia in adult rat
1 lmol/L, MDA content was decreased compared with the hypoxia
group (P < 0.05), indicating that compounds 1a, 1b and 4c could in-
Considering the readily synthesis and potent pharmacological
hibit the lipid peroxidation of cell membrane.
activity in vitro of 4c, its cardio-protective effects in rats of acute
Figure 5. Effects of compounds 1a, 4c at 1 lmol/l on apoptosis in hypoxia-induced neonatal rat ventricular myocytes. Nuclear staining was achieved using Heochst dye.

C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
3503
Figure 7. Effects of compounds 1a, 4c at 1
lmol/l on the expression of bcl-2, bax,
protein in hypoxia-induced neonatal rat ventricular myocytes. Quantitative anal-
ysis of the protein of bcl-2 (A) and bax (B). The value at each group represents the
normalized mean SE from six individual examples. *P < 0.05 versus hypoxia.
Figure 6. Effects of compounds 1a, 4c at 1 lmol/l on the expression of bcl-2, bax,
caspase-3 gene in hypoxia-induced neonatal rat ventricular myocytes. Quantitative
analysis of the mRNA of bcl-2 (A), bax (B) and caspase-3 (C). The value at each group
represents the normalized mean SE from six individual examples. *P < 0.05 versus
hypoxia.
myocardial infarction were further evaluated. Rats were treated
with 4c at different concentrations (15, 30 and 60 mg/kg). Two
days after myocardial infarction surgery, the hearts were analyzed
by TTC staining to quantify infarct size. As shown in Figure 8, the
infarct areas in compound 4c-treated rats were smaller than in
the model rats, and only the difference between the rats treated
with 4c at 60 mg/kg and model rats was of statistic significance.
The level of clinical markers of cardiac infarction in serum was also
measured. As expected, the levels of serum lactate dehydrogenase
(LDH), creatine kinase (CK) in model rats were dramatically higher
compared with those in the sham-operated rats, which indicated
that myocardial infarction was successfully established. Compared
with model rats, the levels of serum LDH, CK and MDA in rats trea-
ted with 4c were significantly reduced, meanwhile the activity of
SOD in serum was increased in 4c treated rats ( Fig. 9), which
Figure 8. Effects of compound 4c at different concentrations on infarction size in
rat after myocardial infarction. Values are expressed as means SE from six
individual examples. *P < 0.05 versus model.
was consistent with the results of infarction area, indicating that
4c treatment apparently promoted cell integrity and reduced myo-
cardial damage.

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C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
Figure 9. Effects of compound 4c at concentrations of 15, 30, 60 mg/kg on the levels of CK (A), LDH (B), MDA (C) and SOD (D) in serum. Values are expressed as means SE
from six individual examples. *P < 0.05 versus model.
1a and 4c inhibit cell apoptosis was possibly through suppressing
apoptotic signaling. Systemic levels of anti-apoptotic protein bcl-2
were significantly higher in cells treated with compounds 1a and
4c, pro-apoptotic protein bax and apoptosis executive protein
caspase-3 were lower in 1a and 4c treated group compared with
hypoxia group. In addition, the anti-apoptosis activity of com-
pounds 1a and 4c may be partially related to their antioxidant
property.
The results in vivo showed that compound 4c could protect car-
diac myocytes from myocardial infarction injury by reducing in-
farct area and decreasing the level of the intracellular enzymes
detectable in serum. Considering readily synthesis and purification
as well as potent pharmacological activity, compound 4c was
promising for further therapeutic applications.
4. Discussion
Danshensu, the only component of Danshen that has been stud-
ied in human subjects, exhibited well-known pharmacological
activity. However, it is hard to obtain either from natural Danshen
due to the unstability of phenolic hydroxyl or from chemical syn-
thesis due to its chiral structure. In the present study, several novel
Danshensu derivatives were designed and synthesized asymmetri-
cally. The hydroxyl groups in these Danshensu derivatives were
protected by acetyl or methylene, which not only increased their
stability and liposolubility but also was beneficial to asymmetric
synthesis and pharmacological assay. These structural modifica-
tions of Danshensu were expected to exhibit similar pharmacolog-
ical activity with Danshensu due to both the ester and ether
linkages are easily hydrolyzed and readily release bioactive Dansh-
ensu. Using a chemical approach based on asymmetric hydrogena-
tion, (R)-Danshensu derivatives 1a and 1b as well as their
racemates 7 and 10 were successfully obtained in excellent ee
and high yields.
5. Conclusion
Overall, we successfully synthesized new derivatives of Dansh-
ensu, (R)-1a, (R)-1b, ( )-7, ( )-10 and 4c, which are more stable
and liposoluble than Danshensu. Their structures were confirmed
through NMR, MS and X-ray single crystal diffraction analysis.
Pharmacological evaluation showed that these Danshensu deriva-
tives possessed potent cardio-protective activities that occur
through blocking oxidative stress and apoptotic pathways. Further
structural modification, biological screening and the mechanism
study are in progress as these promising pharmacological results
demonstrate that these Danshensu derivatives merit attention as
potential anti-myocardial ischemia drugs.
Compounds 1a, 1b, 7, 10 and 4c significantly increased cell
viability and reduced LDH leakage, which signified that these com-
pounds possessed potent protective effects against hypoxia-in-
duced cellular damage. At the same concentration of 1 lmol/L,
the LDH leakage of (R)-1a treated cells was less than that of cells
treated by 7, indicating that the bioactivity may be related to the
configuration of the chiral carbon and R-configuration was more
favorable. However, the difference of LDH leakage between (R)-
1b and 10 treated cells was not significant, suggesting that differ-
ent substitutions of phenolic hydroxyl groups influence on the
metabolism in vivo and further impact the pharmacological
activity.
6. Experimental
Compounds 1a, 1b and 4c possessed potent antioxidant activity
featured by decreased MDA content, which may be related to the
hydrolysis to expose phenolic hydroxyl groups due to the cleavage
of the labile ester bond. However, there may be another way for
these compounds to act as antioxidant agents, for example increas-
ing the activity of intracellular antioxidant enzyme. Therefore, the
mechanism of antioxidative activity of 1a, 1b and 4c merits further
investigations.
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. Melting points were
determined in open capillary tubes and are uncorrected. Nuclear
magnetic resonance spectra were recorded on a Brucker-DPX
400 MHz spectrometer, Mass spectral data was collected on a
HP5973 N analytical mass spectrometer. HRMS data were deter-
mined on an IonSpec 4.7 Tesla FTMS instrument. X-ray single crys-
tal diffraction was carried out on
diffractometer.
a Bruker Smart CCD
Using Hoechst staining, compounds 1a and 4c showed potent
anti-apoptotic activity. From our results, the mechanism by which

C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
3505
6.1. (Z)-2-Acetamido-3-(3,4-diacetylphenyl)acrylic acid (3a)
(3 ꢀ 30 mL). The organic extract was concentrated and purified
by silica gel chromatography providing 8 as white solid (1.41 g,
70%).
3,4-Dihydroxybenzaldehyde (2a) (10.8 g, 0.078 mol), N-acetyl-
glycine (9.2 g, 0.0789 mol), sodium acetate (6.4 g, 0.078 mol) and
acetic anhydride (30 mL, 32.4 g, 0.319 mol) were mixed together
and stirred at 140 °C for 2 h. After cooling, the dark-coloured
viscous solution was poured into cold water (400 mL). The sticky
yellow gum which separated hardened on keeping overnight. It
was collected and crystallized from EtOAc to give 3a as a yellow
solid (12.1 g, 48%): mp 183–185 °C (lit.16: 183.4 °C). 1H NMR
(400 MHz, CDCl3) d 2.08 (s, 3H, 2-NHCOCH3), 2.28 (s, 6H, 30-
CH3COO, 40-CH3COO), 7.06 (s, 1H, 3-H), 7.14 (m, 3H, 20-H, 50-H,
60-H), 8.01 (s, 1H, 2-NHCOCH3).
6.6. (Z)-2-Acetoxy-3-(3,4-diacetoxyphenyl)acrylic acid (5a)
A mixture of 4a (0.9 g, 4.59 mmol) and sodium acetate (0.5 g,
6.1 mmol) in acetic anhydride (6 mL, 6.48 g, 0.064 mol) was stirred
at rt for 5.5 h. After that, the solution was added water (15 mL), and
was extracted with EtOAc (3 ꢀ 30 mL). The combined organic lay-
ers were dried over anhydrous MgSO4. Concentration of the dried
extracts yielded a residue which was purified by crystallization
from acetic acid to give 5a as yellow solid (1.2 g, 80%): mp 184–
186 °C (lit.22: 189–192 °C). 1H NMR (400 MHz, acetone-d6) d
2.28–2.29 (m, 9H, 2-OCOCH3, 30-OCOCH3, 40-OCOCH3), 7.31 (d,
1H, J = 8.3 Hz, 50-H), 7.34 (s, 1H, 3-H), 7.63 (d, 1H, J = 8.5 Hz, 60-
H), 7.64 (s, 1H, 20-H).
6.2. (Z)-2-Acetamido-3-(3,4-methylenedioxyphenyl)acrylic acid
(3b)
3,4-Methylenedioxyphenyl aldehyde (2b) (5.1 g, 0.034 mol), N-
acetylglycine (5.20 g, 0.044 mol), sodium acetate (4 g, 0.049 mol)
and acetic anhydride (20 mL, 21.6 g, 0.213 mol) were mixed to-
gether and stirred at 100 °C for 3 h. After completion of the reac-
tion, the mixture was allowed to cool to rt. Then water (100 mL)
was added, and stirred at 120 °C for 0.5 h. After cooling, the mix-
ture was filtered and the filtrate was extracted with EtOAc. The or-
ganic extract was evaporated to give 3b as a pale yellow solid
(4.68 g, 55%): mp 215–216 °C (lit.17: 216–216.5 °C).
6.7. (Z)-2-Acetoxy-3-(3,4-methylenedioxyphenyl)acrylic acid
(5b)
A mixture of 4b (1.0 g, 4.8 mmol) and sodium acetate (0.6 g,
7.3 mmol) in acetic anhydride (8 mL, 8.64 g) was stirred at rt for
5.5 h. The formed solid was filtered off, washed with water, air-
dried, and crystallized from EtOAc to give 5b as yellow solid
(0.93 g, 77%): mp 164–166 °C; IR (KBr, cmꢁ1) 2960, 2936, 2853,
2522, 1756, 1691, 1639, 1500, 1444, 1429, 1265, 1245, 1199,
1116, 1039; 1H NMR (400 MHz, acetone-d6) d 2.32 (s, 3H, 2-
OCOCH3), 6.08 (s, 2H, 30,40-OCH2O), 6.92 (d, 1H, J = 7.83 Hz, 3-H),
7.24–7.28 (m, 3H, 20-H, 60-H, 50-H). MS (EI) m/z (%): 250 (M+,
6.44), 208 (39.85), 194 (9.29), 162 (100.00), 150 (6.67), 134
(39.25), 104(15.41), 76(27.58). HRMS calcd mass for C12H10O6
250.0477, found 250.0481.
6.3. (Z)-3-(3,4-Dihydroxyphenyl)-2-hydroxyacrylic acid (4a)
A mixture of 3a (1.0 g, 0.016 mol) in 9% HCl (40 mL) was re-
fluxed at 100 °C for 6 h. Then the resulting mixture was allowed
to cool to rt and filtered. The filtrate was extracted with EtOAc.
The organic extract was evaporated. The crude product was dried
under vacuum and washed with water to get 4a as a slightly yellow
solid (0.53 g, 87%): mp 179–181 °C (lit.17
:
181 °C). 1H NMR
6.8. 2-Acetoxy-3-(3,4-methylenedioxyphenyl)propanoic acid
(9)
(400 MHz, acetone-d6) d 4.5 (br, 3H, OH), 6.44 (s, 1H, 20-H), 6.81
(d, 1H, J = 8.2 Hz, 50-H), 7.12 (d, 1H, J = 8.2 Hz, 60-H), 7.50 (s, 1H,
3-H), 7.61 (br, 1H, COOH).
To a solution of 8 (0.4 g, 2.4 mmol) in acetic anhydride (2 mL,
2.16 mg, 0.021 mmol) was added acetic acid (1 mL) and the mix-
ture was stirred at rt for 24 h. After that, the solution was added
water (5 mL), and was extracted with EtOAc (3 ꢀ 10 mL). The com-
bined organic layers were dried over anhydrous MgSO4. Concentra-
tion of the dried extracts yielded a residue which was purified by
crystallization from acetic acid to give 9 as white solid (0.45 g,
95%): mp 151–153 °C; IR (KBr, cmꢁ1) 2960, 2853, 2567, 1749,
1723, 1710, 1505, 1498, 1450, 1259, 1226, 1070, 1041; 1H NMR
(400 MHz, CDCl3) d 2.09 (s, 3H, 2-OCOCH3), 3.00–3.15 (m, 2H, 3-
H), 5.15–5.18 (m, 1H, 2-H), 5.93 (s, 2H, 30,40-OCH2O), 6.69 (d, 1H,
J = 8.24 Hz, 60-H), 6.73 (s, 1H, 20-H), 6.74 (d, 1H, J = 7.74 Hz, 50-H),
7.86 (br, 1H, COOH). MS (EI) m/z (%): 252 (M+, 0.32), 192
(100.00), 175 (6.00), 135 (99.68), 105(6.57), 77(20.23). HRMS calcd
mass for C12H12O6 252.0634, found 252.0644.
6.4. (Z)-3-(3,4-Methylenedioxyphenyl)-2-hydroxyacrylic acid
(4b)
A mixture of 3b (3 g, 0.012 mol) in 9% HCl (50 mL) was refluxed
at 100 °C for 4 h. After that, the resulting mixture was allowed to
cool to rt and extracted with EtOAc (3 ꢀ 30 mL). The organic ex-
tract was evaporated and the product was dried under vacuum
and washed with water to get 4b as a slightly yellow solid (2 g,
80%): mp 207 °C (lit.17: 206 °C). 1H NMR (400 MHz, acetone-d6) d
5.93 (s, 2H, OCH2O), 6.42 (s, 1H, 20-H), 6.78 (d, 1H, J = 8.22 Hz, 60-
H), 7.11 (d, 1H, J = 8.41 Hz, 50-H), 7.48 (s, 1H, 3-H).
6.5. 3-(3,4-Methylenedioxyphenyl)-2-hydroxypropanoic acid
(8)
6.9. General procedure for the synthesis of 6a, 6b and 10
A mixture of 4b (1.0 g, 4.8 mmol), Pd–C (0.21 g) and anhydrous
MeOH (10 mL) was hydrogenated at 6 atm for 24 h. The mixture
was filtered and concentrated, the residue was purified by silica
gel chromatography, providing 8 as white solid (0.44 g, 43%): mp
96 °C (lit.17: 95–97 °C). 1H NMR (400 MHz, acetone-d6) d 2.77–
3.01 (m, 2H, 3-H), 4.28–4.31 (m, 1H, 2-H), 5.90(s, 2H, OCH2O),
6.69 (s, 2H, 50-H, 60-H), 6.78 (s, 1H, 20-H).
To an ice-cooled solution of 5a or 5b or 9 (0.02 mol) in dry
dichloromethane (20 mL) was added a solution of diazomethane
in ether dropwise with stirring. Drop adding was stopped until
the solution was clear, and the solution was stirred overnight. After
that, the mixture was concentrated and the residue was purified by
silica gel chromatography to give 6a, 6b, 10.
A mixture of 4b (2 g, 9.6 mmol) and 25% methanol (40 mL) was
adjusted pH to 8 by sodium hydroxide then was added KBH4 (1.5 g,
27.8 mmol). The mixture was stirred at room temperature for 7 h
and adjusted pH to 2 by 1 N HCl and then extracted with EtOAc
6.9.1. (Z)-Methyl 2-acetoxy-3-(3,4-diacetylphenyl)acrylate (6a)
White solid, yield 81%, mp 95–96 °C; IR (KBr, cmꢁ1) 3504, 2960,
2936, 2853, 1760, 1722, 1654, 1503, 1380, 1305, 1245, 1212, 1122,

3506
C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
1092; 1H NMR (400 MHz, CDCl3) d 2.30–2.32 (m, 9H, 2-OCOCH3, 30-
OCOCH3, 40-OCOCH3), 3.84 (s, 3H, COOCH3), 7.22 (d, 1H, J = 8.22 Hz,
50-H), 7.25(s, 1H, 3-H), 7.43 (d, 1H, J = 8.61 Hz, 60-H), 7.48 (s, 1H, 20-
H). MS (EI) m/z (%): 336 (M+, 0.60), 294 (25.11), 252 (77.88), 210
(96.24), 150 (100.00), 122 (11.07). HRMS calcd mass for C16H16O8
336.0845, found 336.0849.
105(6.21), 77(18.38). HRMS calcd mass for C13H14O6 226.0790,
found 226.0806.
The enantiomeric excess was determined by HPLC on Chiralpak
AD-H column, hexane:isopropanol = 95:5; flow rate = 0.7 mL/min;
UV detection at k = 214 nm; t = 17.5 min (major), 22.3 min (minor),
respectively.
6.11. Methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)propanoate (7)
6.9.2. (Z)-Methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)-
acrylate (6b)
White solid, yield 85%, mp 84–85 °C; IR (KBr, cmꢁ1) 3061, 2954,
2914, 1773, 1708, 1654, 1615, 1508, 1489, 1338, 1282, 1244, 1195,
1099; 1H NMR (400 MHz, CDCl3) d 2.34 (s, 3H, 2-OCOCH3), 3.83 (s,
3H, COOCH3), 6.01 (s, 2H, 30,40-OCH2O), 6.82 (d, 1H, J =7.82 Hz, 50-
H), 7.06 (d, 1H, J = 8.22 Hz, 60-H), 7.16 (s, 1H, 3-H), 7.23 (s, 1H, 20-
H). MS (EI) m/z (%): 264 (M+, 4.04), 222 (50.29), 162 (100.00),
134 (33.75), 134 (39.25), 104(6.30), 76(16.06). HRMS calcd mass
for C11H9O5 221.0450, found 221.0442.
A mixture of 6a (0.2 g, 0.59 mmol), Pd–C (30 mg) and EtOAc
(4 mL) was hydrogenated at 10 atm for 24 h. The mixture was fil-
tered and concentrated, the residue was purified by silica gel chro-
matography, providing 7 as white solid (160 mg, 79%): mp 64 °C; IR
(KBr, cmꢁ1) 2963, 1771, 1754, 1736, 1503, 1377, 1205, 1178; 1H
NMR (400 MHz, CDCl3) d 2.09 (s, 3H, 2-OCOCH3), 2.28 (s, 6H, 30-
OCOCH3, 40-OCOCH3), 3.05–3.18 (m, 2H, 3-H), 3.72 (s, 3H, COOCH3),
5.17–5.20 (m, 1H, 2-H), 7.06 (s, 1H, 20-H), 7.12 (s, 2H, 50-H, 60-H).
6.12. (Z)-2-Acetoxy-3-(3,4-dihydroxyphenyl)acrylic acid (4c)
6.9.3. Methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)propa-
noate (10)
A mixture of 3a (2 g, 0.012 mol) in 9% HCl (50 mL) was heated at
60 °C for 3 h. The formed solid was filtered off, washed with water,
air-dried, and crystallized from water to give 4c as a colorless slice
(1.27 g, 86%): mp 229–230 °C; IR (KBr, cmꢁ1) 3258, 3037, 1686,
1636, 1617, 1521, 1245, 1225, 1200, 1172, 1118; 1H NMR
White solid, yield 79%, mp 63–65 °C; IR (KBr, cmꢁ1) 2957, 2920,
1760, 1736, 1491, 1448, 1376, 1297, 1253, 1239, 1197, 1038; 1H
NMR (400 MHz, CDCl3) d 2.10(s, 3H, 2-OCOCH3), 2.97–3.10(m,
2H, 3-H), 3.73(s, 3H, COOCH3), 5.14–5.17 (m, 1H, 2-H), 5.94 (s,
2H, 30,40-OCH2O), 6.66 (d, 1H, J = 7.72 Hz, 60-H), 6.71 (s, 1H, 20-H),
6.82 (d, 1H, J = 8.21 Hz, 50-H).
(400 MHz, CD3OD)
d 2.13 (s, 3H, 2-OCOCH3), 6.77 (d, 1H,
J = 8.21 Hz, 50-H), 6.96 (d, 1H, J = 8.41 Hz, 20-H), 7.16 (d, 1H,
J = 1.95 Hz, 60-H), 7.38 (s, 1H, 3-H). 13C NMR (100 MHz, CD3OD) d
22.58, 116.28, 117.55, 123.50, 124.89, 126.60, 137.09, 146.35,
148.81, 168.68, 173.46. MS (ESI) m/z: 238 (M+).
6.10. General procedure for the synthesis of 1a and 1b
[Rh(cod)2]BF4 (2.4 mg, 0.006 mmol), ligand 1119 (7.7 mg,
0.0132 mmol) were dissolved in CH2Cl2 (1 mL) under nitrogen
and the solution was stirred at room temperature for 10 min, the
substrate (200 mg, 0.6 mmol) in CH2Cl2 (1.5 mL) was added to
the above catalyst solution, the mixture was then transferred to
a stainless steel autoclave under nitrogen atmosphere, and then
sealed. After purging with hydrogen for three times, final H2 pres-
sure was adjusted to 60 atm. After stirring at room temperature for
24 h, H2 was released, the mixture was filtered and the filtrate was
concentrated. The residue was purified by silica gel chromatogra-
phy to give compounds 1a and 1b.
Crystal with dimensions of 0.421 mm ꢀ 0.347 mm ꢀ 0.250 mm
for compound 4c was selected and mounted on a Bruker Smart
CCD diffractometer with graphite monochromatized Mo K
a radia-
tion (k = 0.071073 nm). Diffraction data were collected using
x
ꢁ 2h scans at room temperature (293 K). A perspective view of
the structure is depicted in Figure 1.
Empirical formula: C11H10O6. Formula weight: 238.19. Crystal
system: monoclinic. Space group: P2(1)/c. Unit cell dimensions:
a = 1.05986 (13) nm, b = 0.93423 (11) nm, c = 1.19601 (15) nm.
V = 1.0751 (2) nm3. Theta range for data collection is from 2.12°
to 26.50°, Z = 4, Dc = 1.472 g/cm3, F(000) = 496. Refinement meth-
od: full-matrix least-squares on F2. Goodness-of-fit on F2: 1.075. Fi-
6.10.1. (R)-Methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)propa-
nal R indices [I > 2r(I)]: 0.0610, 0.1776. R indices (all data): 0.0700,
0.1858. Largest difference in peak and hole: 0.695 and ꢁ524 e/nm3.
noate (1a)
White solid, yield 89%, ee 92%, mp 81–82 °C, ½a D20
¼ þ3:6 (c = 1.0
ꢂ
in CHCl3); IR (KBr, cmꢁ1) 2963, 1771, 1755, 1735, 1504, 1377, 1205,
1178; 1H NMR (400 MHz, CDCl3) d 2.09 (s, 3H, 2-OCOCH3), 2.28 (s,
6H, 30-OCOCH3, 40-OCOCH3), 3.05–3.18 (m, 2H, 3-H), 3.72 (s, 3H,
COOCH3), 5.17–5.20 (m, 1H, 2-H), 7.07 (s,1H, 20-H), 7.12 (s, 2H,
50-H, 60-H). MS (EI) m/z (%): 338 (M+, 0.66), 278 (3.95), 254
(7.28), 236 (17.92), 194 (100.00), 163 (8.56), 123(22.04). HRMS
calcd mass for C16H18O8 338.1002, found 338.1013.
6.13. Cell culture
Neonatal rat ventricular myocytes (NRVMs) were isolated from
2- or 3-day old Spragur-Dawley rats and cultured as previously de-
scribed.23 Briefly, the hearts were removed and minced in PBS buf-
fer. These tissue fragments were digested by trypsin dissociation.
The dissociated cells were centrifuged for 5 min at 12,000 r/min.
The pellets were re-suspended with DMEM supplemented with
The enantiomeric excess was determined by HPLC on Chiralpak
AS-H column, hexane:isopropanol = 90:10; flow rate = 1.0 mL/min;
UV detection at k = 254 nm; t = 30.6 min (minor), 34.5 min (major),
respectively.
10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml
streptomycin. After incubation for 2 h, the non-adherent cardiac
myocytes were plated at a density of 1 ꢀ 106 cells/ml and seeded
in plastic plates with 100 lM 5-bromo-deoxyuridine (Sigma) and
6.10.2. (R)-Methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)-
incubated in a 5% CO2 humidified incubator at 37 °C. After a period
of 48 h, cells were exposed to different compounds for 12 h, and
then subjected to hypoxia for 5 h.
propanoate (1b)
White solid, yield 89%, ee 98%, mp 70–71 °C, ½a D20
¼ þ6:2 (c = 1.0
ꢂ
in CHCl3); IR (KBr, cmꢁ1) 3014, 2965, 2920, 1753, 1734, 1490, 1239,
1219, 1033; 1H NMR (400 MHz, CDCl3) d 2.10 (s, 3H, 2-OCOCH3),
3.01–3.08 (m, 2H, 3-H), 3.73 (s, 3H, COOCH3), 5.14–5.17 (m, 1H,
2-H), 5.95 (s, 2H, 30,40-OCH2O), 6.66 (d, 1H, J = 7.82 Hz, 60-H), 6.71
(s, 1H, 20-H), 6.74 (d, 1H, J = 7.82 Hz, 50-H). MS (EI) m/z (%): 266
(M+, 12.19), 206 (100.00), 193 (4.15), 175 (41.89), 135 (97.92),
6.14. Assessment of myocardial cells viability and damage
Cell viability was assessed by the measurement of the reduction
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to produce a dark blue formazan product that can be mea-

C. Dong et al. / Bioorg. Med. Chem. 17 (2009) 3499–3507
3507
sured using a microplate reader at 570 nm. The viability of normal
6.18. Measurement of infarct size
cell is presumed as 100%. Cell damage in cardiac myocytes was
quantitatively assessed by the measurement of intracellular lactate
dehydrogenase (LDH), it is presumed that in normal cell group
there is no LDH leakage.
The heart was excised immediately 2 days after the coronary ar-
tery was ligated and the entire ventricular tissue was sliced into
five sections through the transverse axis from the apex to the atrio-
ventricular groove and incubated in 1% triphenyltetrazolium chlo-
ride (TTC) at 37 °C for 15 min. Viable myocardium is stained in red
by TTC, whereas infarction tissue remains unstained. Slices were
imaged and the area of infracted myocardium was defined using
an image analysis software (Scion Image, CA, USA). The size of
the infarction area was estimated by the volume as a percentage
of the left ventricle.
6.15. RNA isolation and RT-PCR
Total RNA was isolated from cardiac myocytes by using Trizol
(Invitrogen, Carlbad, CA) as previously described. Reverse tran-
TM
scription(RT) was conducted by using PrimeScript 1st Strand
cDNA Synthesis Kit according to the manufacture’s procedures.
Primers used for amplification were synthesized as follows: bcl-
2: forward 50-CGGGAGAACAGGGTATGA-30; reverse 50-CAGGCTG
GAAGGA GAAGAT-30. Bax: forward 50-GCAGGGAGGATGGCTGGG
GAGA-30; reverse 50-TCCAGACAAGCAGCCGCTCACG-30. Caspase-3:
forward 50-CTGGACTGCGGTATTGAG-30; reverse 50-GGGTGCGGTAG
AGTAAGC-3. GAPDH: forward 50-TTCAACGGCACAGTCAAGG-30;
reverse 50-CGGCATGTCAGATCCACAA-3. The PCR products were
analyzed by electrophoresis in 1.5% agarose gels. The intensity of
each band was photographed and quantified by using the Fluor
Chem SP system (USA) as a ratio of a target gene over GAPDH.
6.19. Measurements of lactate dehydrogenase (LDH), creatine
kinase (CK), superoxide dismutase (SOD), malondialdehyde (MDA)
The levels of LDH, CK, SOD, MDA in serum were measured
using diagnostic kit (NJBI, China) according to the manufacture’s
protocol.
6.20. Statistic analysis
6.16. Western blot
Data were presented as means SE and analyzed by SPSS soft-
ware. Pictures were processed with Photoshop software. Differ-
ences at P < 0.05 were considered statistically significant.
At the end of hypoxia, cardiac myocytes were scraped off in cell
lysis buffer (Beyotime Biotechnology, Haimen, China) and centrifu-
gated at 10,000 r/min for 10 min. The supernatant was collected,
and protein concentrations were quantified using the enhanced
BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, China).
Thirty micrograms of total proteins were separated on SDS–PAGE
gels as previously described. Separated proteins were transferred
Acknowledgements
This study was supported by Research Fund for the Doctoral
Program of Higher Education of China (No. 20060246054) and
the National Natural Science Foundation of China (No. 20672022).
onto 0.45-lm polyvinylidene difluoride (PVDF) membrane (Milli-
pore Corporation). PVDF membrane was blocked in Tris-buffered
saline (TBS) containing 5% skim milk for 2 h at room temperature
and probed with rabbit anti-rat bcl-2 polyclonal antibody (R&D,
USA) diluted in TBS-T for 1 h at room temperature. Anti-rabbit
IgG diluted in TBS-T was used as secondary antibody. Protein band
intensities were quantified by using a Western blotting detection
system (Alpha Innotech, USA).
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6.17. Model of myocardial infarction
The method was used as described previously.24 Briefly, male
Sprague-Dawley rats weighing 230–250 g, provided by the experi-
mental animal center of fudan university were intubated and arti-
ficially ventilated with a rodent ventilator (DHX-150, China) under
anesthesia with 7% choral hydrate (60 mg/kg ip). The normal elec-
trocardiogram (II) was recorded after electrodes were placed sub-
cutaneously and connected to an electrocardiograph. Then the
chest was opened by left thoracotomy in the 3rd and 4th intercos-
tal space and the pericardium was removed. The left anterior
descending coronary artery was ligated with a 5–0 suture 1–
2 mm below the left atrial appendage. Sham operated rats under-
went an identical surgical procedure except that the left coronary
was not ligation. Rats were randomly divided into five groups: MI
group, sham-operated group, MI and drug-treated groups (15, 30,
60 mg/kg). The drug was injected intraperitoneally 7 days before
surgery and 2 days after surgery once daily. Successful MI model
was confirmed by pallor of the anterior wall of the left ventricle
and ST-segment elevation.
20. Elmore, S. Toxicol. Pathol. 2007, 35, 495.
21. Oltvai, Z. N.; Milliman, C. L.; Korsmeyer, S. J. Cell 1993, 74, 609.
22. Lindén, I. B.; Neuvonen, P. J.; Vapaatalo, H. Acta Pharmacol. Toxicol. 1982, 266.
23. Akao, M.; Ohler, A.; O’Rourke, B.; Marban, E. Circ. Res. 2001, 88, 1267.
24. Sun, H. L.; Jiao, J. D.; Pan, Z. W.; Dong, D. L.; Yang, B. F. Yao Xue Xue Bao 2006, 41,
247.