Novel cajaninstilbene acid derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2015.06.008

Discovery of novel antibacterial agents with new structural scaffolds that combat drug-resistant pathogens is an urgent task. Cajaninstilbene acid, which is isolated from pigeonpea leaves, has shown antibacterial activity. In this study, a series of cajaninstilbene acid derivatives were designed and synthesized. The antibacterial activities of these compounds against gram-negative and gram-positive bacteria, as well as nine strains of methicillin-resistant staphylococcus aureus (MRSA) bacteria are evaluated, and the related structure-activity relationships are discussed. Assays suggest that some of the synthetic cajaninstilbene acid derivatives exhibit potent antibacterial activity against gram-positive bacterial strains and MRSA. Among these compounds, 5b, 5c, 5j and 5k show better antibacterial activity than the positive control compounds. The results of MTT assays illustrate the low cytotoxicity of the active compounds.

Full text of DOI:10.1016/j.ejmech.2015.06.008

European Journal of Medicinal Chemistry 100 (2015) 235e245
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel cajaninstilbene acid derivatives as antibacterial agents
,
,
,
Zhi-Zhong Geng a 1, Jian-Jun Zhang a 1, Jing Lin a *, Mei-Yan Huang a, Lin-Kun An b,
Hong-Bin Zhang c, Ping-Hua Sun a, Wen-Cai Ye a, Wei-Min Chen a
,
*
a College of Pharmacy, Jinan University, Guangzhou 510632, China
b School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
c Department of Clinical Laboratory, Guangzhou Liuhuaqiao Hospital, Guangzhou 510010, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Discovery of novel antibacterial agents with new structural scaffolds that combat drug-resistant path-
ogens is an urgent task. Cajaninstilbene acid, which is isolated from pigeonpea leaves, has shown anti-
bacterial activity. In this study, a series of cajaninstilbene acid derivatives were designed and synthesized.
The antibacterial activities of these compounds against gram-negative and gram-positive bacteria, as
well as nine strains of methicillin-resistant staphylococcus aureus (MRSA) bacteria are evaluated，and
the related structure-activity relationships are discussed. Assays suggest that some of the synthetic
cajaninstilbene acid derivatives exhibit potent antibacterial activity against gram-positive bacterial
strains and MRSA. Among these compounds, 5b, 5c, 5j and 5k show better antibacterial activity than the
positive control compounds. The results of MTT assays illustrate the low cytotoxicity of the active
compounds.
Received 17 April 2015
Received in revised form
27 May 2015
Accepted 3 June 2015
Available online 10 June 2015
Keywords:
Cajaninstilbene acid
Synthesis
Antibacterial
© 2015 Elsevier Masson SAS. All rights reserved.
MRSA
Structure-activity relationship
1. Introduction
the erythromycin positive group, through it is lower than that of
penicillin or chloramphenicol [4]. As a natural active compound
With the expansion of the use and abuse of antibiotics,
increasing numbers of drug-resistant strains have emerged in the
clinic [1]. One of the prototypical “superbugs” caused by antibiotic
use and overuse is methicillin-resistant Staphylococcus aureus
(MRSA) [2], against which almost all antibiotics are ineffective. As a
consequence, novel antibacterial agents with new structural scaf-
folds are urgently needed to overcome the growing problem of
drug resistance. Natural products are an important source of new
drugs, and searching for novel antibacterial agents from natural
products and their derivatives is an important approach to the
discovery of antibacterial agents that can overcome drug resistance.
It has been reported that cajaninstilbene acid (CSA, Fig. 1), iso-
lated from pigeonpea leaves [3], shows antibacterial activity against
gram-positive bacteria. The minimal inhibitory concentrations
(MIC) of CSA for Staphylococcus aureus, Staphylococcus epidermidis,
extracted from edible beans, CSA is only mildly toxic [5], so it and its
derivatives may have good potential as drugs. CSA may have
promising activity, but there is still a large gap between its anti-
bacterial effect and that of several existing drugs such as penicillin,
norfloxacin and linezolid. In this study, a series of CSA derivatives
were designed and synthesized in a search for compounds with
improved antibacterial activity, and their antibacterial activity
against gram-negative, gram-positive, and MRSA bacteria were
evaluated, and the structure-activity relationships (SAR) were
elucidated and discussed. Among these compounds, several new
compounds with low toxicity and promising antibacterial activity
were identified.
2. Chemistry
and Bacillus subtilis are 13, 25, and 25 mg/mL respectively. Its anti-
bacterial activity against Staphylococcus aureus is equal to that of
In an effort to develop novel antibacterial agents, a variety of
CSA derivatives have been designed and synthesized. As shown in
Fig. 1, the drug design strategies include: (a) removal of the iso-
prenyl group of CSA, to obtain compounds 3ae3k, 6a, 6c and 6f; (b)
esterification of the carboxyl group, to synthesize 4ae4k; (c)
cleavage of the cinnamenyl group, giving compound 10; and (d)
introduction of substituents into the phenyl ring B, to obtain CSA
derivatives 5be5k.
* Corresponding authors.
E-mail addresses: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn
(W.-M. Chen).
1
These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.ejmech.2015.06.008
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

236
Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
Fig. 1. Design of novel cajaninstilbene acid (CSA) derivatives.
The synthetic routes to the compounds in Fig. 1 are depicted in
acid and its derivatives 5ae5k were obtained by hydrolysis of the
ester group of 4ae4k (Scheme 1). Another series (6a, 6c and 6f) was
conveniently prepared by hydrolysis of 3a, 3c and 3f (Scheme 2). In
addition, as shown in Scheme 3, intermediate 8 was prepared from
7 by oxidative aromatization using iodine in methanol [10]. Com-
pound 10 was synthesized by coupling the isoprenyl group and
ester hydrolysis.
Schemes 1e3. Intermediate 1 was prepared according to a pub-
lished method [6]. Compounds 2ae2k were synthesized by react-
ing 1 with different benzaldehydes under the conditions of the
HornereWadswortheEmmons reaction [7]. During this reaction,
the E-configuration products can be isolated in high yield and high
selectivity when a catalytic amount of 15-crown-5 (the cyclic
pentamer of ethylene oxide) is used [8]. Compounds 3ae3k were
obtained from selective demethylation of the 2-OMe group of
2ae2k. Compounds 3ae3k were then treated with prenyl bromide
and NaH to give 4ae4k. The yield of 4ae4k depends on the amount
of NaH and prenyl bromide and the optimum ratio (1.2 equiv) was
determined in a number of experiments [9]. Finally, cajaninstilbene
3. Results and discussion
3.1. Evaluation of antibacterial activity
The antibacterial activities of all synthesized compounds were
Scheme 1. General synthetic route of cajaninstilbene acid derivatives 3ae3k, 4ae4k and 5ae5k. Reagents and conditions: (i) aromatic aldehyde, 15-crown-5, NaH, THF, ꢀ2 ꢁC, 2 h;
(ii) BCl3, CH2Cl2, ꢀ30 ꢁC, 2 h; (iii) prenyl bromide, NaH, 78 ꢁC, 2 h; (iv) KOH, EtOH, H2O, 70 ꢁC, 2 h.

Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
237
Scheme 2. Synthetic route of compounds 6a, 6c and 6f.
Scheme 3. Synthetic route of compound 10. Reagents and conditions: (i) I2, CH3OH, reflux, 4 h; (ii) prenyl bromide, 78 ꢁC, 2 h; (iii) KOH, EtOH, H2O, 70 ꢁC, 2 h.
evaluated in vitro by a serial dilution method to obtain their min-
imum inhibitory concentration (MIC) values against a variety of
different strains, including gram-positive (Staphylococcus aureus,
Staphylococcus epidermidis, Bacillus subtilis), gram-negative
(Eschemeerichia coli, Proteus vulgaris, Pesudomonas aeruginosa)
and nine strains of methicillin-resistant Staphylococcus aureus
bacteria. The MIC is defined as the lowest concentration of anti-
bacterial agent at which no growth of the strain can be observed
[11]. Penicillin, norfloxacin were used as positive controls in the
assays. The MIC values are summarized in Tables 1e3.
values were mostly between 8 and 32 mg/mL for the tested bacterial
strains, the antibacterial activities of ester derivatives were signif-
icantly decreased, indicating that the free carboxyl group of CSA is
also necessary for its antibacterial activity.
3.1.3. Cajaninstilbene acid derivatives with no cinnamenyl group
(10)
A new compound (10), which does not contain the cinnamenyl
group, was synthesized to determine whether the cinnamenyl
group is essential for the antibacterial activity of CSA. Assays
(Table 2) revealed that the antibacterial activity of 10 (MIC:
3.1.1. Cajaninstilbene acid derivatives lacking the isoprenyl group
(3ae3k, 6a, 6c and 6f)
32e64 mg/mL) is much less than that of CSA (MIC: 8e32 mg/mL).
This result indicates that the cinnamenyl group is also necessary for
the antibacterial activity. Combination of all the above results, leads
to the conclusion that the isopentenyl, the free carboxyl, as well as
the cinnamenyl group of CSA are all required for the antibacterial
activity of CSA. Therefore, the subsequent design was focused on
derivatization of the stilbene acid.
The isoprenyl group is considered to be an active essential group
of many natural products [12], such as prenylated stilbene, which is
an effective agent for the treatment of tapeworm infection [13]. In
order to investigate whether the isoprenyl group of CSA is also
necessary for its antibacterial activity, we synthesized a series of
compounds lacking an isoprenyl group (3ae3k). A preliminary test
of bioactivity showed that 3ae3k exhibited no antibacterial activ-
ity; their minimal inhibitory concentration (MIC) values were
3.1.4. Cajaninstilbene acid derivatives with different substituents on
phenyl ring B (5be5k)
greater than 128 mg/mL against all tested strains (Table 1). We then
In order to explore the impact of the substituents on the phenyl
ring B on the antibacterial activity and to develop improved anti-
bacterial agents, a variety of cajaninstilbene acid derivatives with
different substituents on benzene ring B (5be5k) were synthe-
sized. Since trifluoromethyl and fluorine are well known to improve
the efficiency of many drugs [15e17] trifluoromethyl (5j) and
fluorine (5b, 5c, 5d and 5k) were introduced into the benzene ring
B. The MIC results (Table 3) shown that all the compounds with a
fluorine atom (5be5d, 5j and 5k) exhibit strong inhibitory activities
towards all the three wild gram-negative bacterial strains (S.a, S.e,
B.u) and nine methicillin resistant staphylococcus aureus bacteria
synthesized 6a, 6c and 6f, which lacks an isoprenyl group but, like
CSA has a free carboxyl group, to determine if the loss of antibac-
terial activity is caused by the loss of the isoprenyl group. The MIC
results showed that 6a, 6c and 6f also have no antibacterial activity.
(Table 1, MIC: ꢂ 64
mg/mL), indicating that the isoprenyl group is
necessary for the antibacterial activity of CSA.
3.1.2. Cajaninstilbene acid ester derivatives (4ae4k)
Carboxylic acid esters can be absorbed in vivo quickly and
completely, and have been widely used to improve the bioavail-
ability of drugs, such as cefuroxime ester [14]. Thus a series of
cajaninstilbene acid ester derivatives were synthesized in this
study, in which the carboxyl group of cajaninstilbene acid (CSA) on
the A ring was esterified by methanol (4ae4k) to investigate
whether the esterification of CSA will improve its activity or
druggability. The results demonstrated that cajaninstilbene acid
esters have no significant antibacterial property, as shown in
Table 2. The MIC values of the compounds against the tested bac-
with MIC values in the range of 0.5e8
significantly superior to CSA (8e32
m
g/mL, and were therefore
mg/mL). For example, the
inhibitory activity of compound 5j was 2e64 times greater than
that of CSA. Since both trifluoromethyl and fluorine are electron
withdrawing groups, we further investigated whether other elec-
tron withdrawing substituent groups on the benzene ring B, such as
chlorine (5e) and cyano (5i), can also enhance the antibacterial
activity. The results show that the minimal inhibitory concentra-
terial strains were all greater than 64
m
g/mL, and most of them were
tions of 5e and 5i against S.a, S.e, B.s and MRSA were 0.5e8
mg/mL,
even greater than 128 g/mL. Compared with the CSA, whose MIC
m
and thus 5e and 5i are also superior to CSA. On the other hand, we

238
Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
Table 1
In vitro antibacterial activity of compounds 3ae3k, 6a, 6c and 6f.
Compound
MICa
(
m
g/mL)
S.ab
S.ec
B.sd
128
E.ce
P.vf
P.ag
128
128
128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>64
43300h
523512i
425055j
513045k
62202l
51033m
52056n
515992o
510019p
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
6a
6c
6f
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
128
128
128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
16
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
64
>128
>128
128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
128
>128
128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
128
128
64
>128
128
>128
>128
>128
16
>128
>128
>128
>128
>128
32
>128
>128
>128
16
>128
>128
>128
16
>128
64
32
>128
>128
8
>128
>128
16
>128
16
>128
32
32
CSA(5a)
Penicillin
Norfloxacin
2
2
2
>16
16
32
32
32
32
32
64
32
32
2
2
2
32
16
16
4
8
4
4
8
4
8
16
8
a
MIC is defined as the minimum concentration of a compound that inhibits growth by 99%. Identical values were obtained for each compound in three replicates by visual
investigation as stated in the experimental section.
b
Staphylococcus aureus ATCC25923.
Staphylococcus epidermidis ATCC12228.
Bacillus subtilis ATCC6633.
ESchemeerichia coli ATCC25922.
Proteus vulgaris ATCC49101.
Pesudomonas aeruginosa ATCC2785.
MRSA ATCC43300.
MRSA ATCC 523512.
MRSA ATCC425055.
MRSA ATCC513045.
MRSA ATCC62202.
MRSA ATCC51033.
MRSA ATCC52056.
MRSA ATCC515992.
MRSA ATCC510019.
c
d
e
f
g
h
i
j
k
l
m
n
o
p
also investigated the effects of introducing of electron donating
group into the phenyl ring B. The results (Table 3) showed that both
a methoxy group (5f, 5h) and a methyl group (5g) reduce the
antibacterial candidates for the treatment of multidrug-resistant
pathogens.
antibacterial activity (MIC: 16e64 mg/mL). Our results clearly show
3.3. Cytotoxicity assay
therefore that introduction of electron withdrawing group into the
benzene ring B can increase the antibacterial activity while intro-
duction of electron donating groups reduces the antibacterial ac-
tivity. Additionally, it can be noted that there is no significant
difference among the activities of 5b, 5c and 5k (MIC 0.5e8 mg/mL),
which indicates that the number of substituents has no influence
The in vitro cytotoxicity of compounds 5b, 5c, 5j and 5k were
further examined in mouse macrophage cell lines (RAW 264.7) in
terms of a maximum tolerated test (MTT) to determine their
selectivity between bacterial and normal cells [18]. As shown in
Table 4, Compounds 5b, 5c, 5j and 5k all had no obvious cytotox-
on the activity.
icity in the RAW 264.7 cells at concentration of 8 mg/mL (the largest
of the MIC values), and the relative cell viabilities of the treated cells
were all more than 90%. The cytotoxic concentrations for normal
cells, indicated by the IC50 were much higher than their MIC values,
and their selectivity index (SI) was good (Table 4). The low cyto-
toxicity for normal cells and good selective index values of these
compounds indicate their potential usefulness in the development
of drugs for infectious diseases.
3.2. Antibacterial activity summary
Antibacterial assay results showed that an overwhelming ma-
jority of compounds have no activity against gram-negative bac-
teria, while some compounds exhibited strong antibacterial activity
on gram-positive bacteria, as well as methicillin-resistant Staphy-
lococcus aureus (MRSA). In particular, the antibacterial activities of
compounds 5b, 5c, 5j and 5k (MIC: 0.5e8
than those of the natural product cajaninstilbene acid (MIC:
8e32 g/mL). These results illustrated that the modification of CSA
in this study has greatly improved its antibacterial activity. It should
be noted that compounds 5b, 5c, 5j and 5k also have good anti-
bacterial activity against methicillin-resistant Staphylococcus
aureus (MRSA); the MIC values of these compounds for certain
m
g/mL), were much better
3.4. Structure-activity relationships summary
m
Based on our results, the following structure-activity relation-
ships (SARs) of the CSA derivatives can be summarized (Fig. 2). First,
the activities of compounds 5ae5k were significantly better than
those of compounds 4ae4k, which indicates that the free carboxyl
on the benzene ring A was necessary for the activity. Second,
compounds3ae3k, 6a, 6c and 6f had no antibacterial activity,
indicating that the loss of the isoprenyl group leads to the signifi-
cant decrease of activity. Third, the activity of 10 was significantly
lower than that of compounds 5ae5k, which indicates that
MRSA strains are 0.5
8e32 g/mL), 64e128-fold superior to penicillin (MIC: 32e64
mL), and 8e32-fold than norfloxacin (MIC: 4e16 g/mL). These
results indicate that compounds 5b, 5c, 5j and 5k are promising
mg/mL, 16-fold superior to that of CSA (MIC:
m
mg/
m

Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
239
Table 2
In vitro antibacterial activity of compounds 4ae4k and 10.
Compound
MICa
(
m
g/mL)
S.ab
S.ec
B.sd
E.ce
P.vf
P.ag
43300h
523512i
425055j
513045k
62202l
51033m
52056n
515992o
510019p
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
10
>128
>128
64
>128
>128
128
>128
>128
>128
64
>128
>128
>128
>128
128
>128
64
64
128
64
>128
>128
128
>128
>128
>128
128
>128
>128
128
128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>64
128
128
>128
>128
>128
128
128
>128
>128
>128
>128
32
>128
>128
64
>128
>128
128
>128
>128
>128
64
128
128
>128
>128
>128
128
128
>128
>128
64
128
32
16
32
4
128
128
128
128
>128
128
>128
>128
>128
128
>128
32
128
128
128
>128
128
>128
>128
>128
128
128
64
>128
>128
128
>128
>128
>128
128
128
>128
>128
128
32
>128
>128
>128
128
>128
128
>128
>128
>128
128
128
64
>128
>128
>128
128
>128
>128
>128
64
>128
>128
>128
32
>128
>128
128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
32
16
64
64
16
2
64
32
32
32
32
8
32
8
CSA(5a)
Penicillin
Norfloxacin
32
2
2
16
32
4
16
32
4
16
32
4
16
64
8
32
32
16
32
32
8
2
2
>16
16
16
16
2
32
8
a
MIC is defined as the minimum concentration of a compound that inhibits growth by 99%. Identical values were obtained for each compound in three replicates by visual
investigation as stated in the experimental section.
b
Staphylococcus aureus ATCC25923.
Staphylococcus epidermidis ATCC12228.
Bacillus subtilis ATCC6633.
ESchemeerichia coli ATCC25922.
Proteus vulgaris ATCC49101.
Pesudomonas aeruginosa ATCC2785.
MRSA ATCC43300.
MRSA ATCC 523512.
MRSA ATCC425055.
MRSA ATCC513045.
MRSA ATCC62202.
MRSA ATCC51033.
MRSA ATCC52056.
MRSA ATCC515992.
MRSA ATCC510019.
c
d
e
f
g
h
i
j
k
l
m
n
o
p
Table 3
In vitro antibacterial activity of compounds 5be5k.
Compound
MICa
(
mg/mL)
S.ab
S.ec
B.sd
E.ce
64
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
64
P.vf
P.ag
43300h
523512i
425055j
513045k
62202l
51033m
52056n
515992o
510019p
5b
5c
5d
5e
5f
5g
5h
5i
4
4
8
1
2
4
0.5
0.5
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>16
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
1
1
4
2
2
4
0.5
2
2
4
8
16
16
1
0.5
0.5
16
32
4
2
2
8
4
16
8
16
4
8
0.5
0.5
1
1
1
1
0.5
32
16
64
2
1
1
16
32
4
2
0.5
1
2
1
8
2
1
1
8
4
2
2
4
2
2
4
1
32
16
64
4
2
1
16
2
2
32
32
32
2
16
16
16
16
1
2
32
2
16
32
16
8
2
2
16
32
4
16
32
16
2
0.5
2
32
32
8
16
32
32
0.5
0.5
1
8
32
8
16
16
32
4
4
2
16
64
8
16
32
16
2
0.5
1
32
32
16
16
32
16
2
2
2
32
32
8
5j
5k
2
0.5
16
2
2
CSA(5a)
Penicillin
Norfloxacin
16
32
4
2
2
32
16
a
MIC is defined as the minimum concentration of a compound that inhibits growth by 99%. Identical values were obtained for each compound in three replicates by visual
investigation as stated in the experimental section.
b
Staphylococcus aureus ATCC25923.
Staphylococcus epidermidis ATCC12228.
Bacillus subtilis ATCC6633.
ESchemeerichia coli ATCC25922.
Proteus vulgaris ATCC49101.
Pesudomonas aeruginosa ATCC2785.
MRSA ATCC43300.
MRSA ATCC 523512.
MRSA ATCC425055.
MRSA ATCC513045.
MRSA ATCC62202.
MRSA ATCC51033.
MRSA ATCC52056.
MRSA ATCC515992.
MRSA ATCC510019.
c
d
e
f
g
h
i
j
k
l
m
n
o
p

240
Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
Table 4
IC50
(
m
g/mL) and selectivity index (SI) values of active compounds (5b, 5c, 5j and 5k) against mouse macrophage cell lines (RAW 264.7a).
MICb
(m
g/mL)
% Cell viability at 8
m
g/mL
IC50
(
m
g/mL)
SI valuec,d
c
Compound
5b
5c
5j
0.5e4
0.5e4
0.5e2
0.5e2
92.68 1.24
91.72 0.78
90.56 1.28
94.36 0.45
24.69 2.75
20.17 3.60
25.71 0.87
46.54 2.82
6.17e41.38
5.04e40.34
12.86e51.42
23.27e93.08
5k
a
RAW264.7 monolayers were used as an in vitro model of cervicovaginal epithelium for testing the cytotoxicity of the new compounds.
b
Minimum inhibitory concentration against S.a, S.e, B.s, MRSA43300, MRSA523512, MRSA425055, MRSA513045, MRSA62202l, MRSA51033, MRSA52056, MRSA515992,
MRSA510019.
c
IC50 is defined as the concentration at which 50% growth is inhibited.
Selectivity index (in vitro): IC50 in RAW 264.7 cells/MIC.
d
cinnamenyl group is very important for the activity. Fourth, the
introduction of electron withdrawing groups such as tri-
fluoromethyl, fluorine, and chlorine into the phenyl ring B can in-
crease the activity, especially in the case of fluorine. The number
and position of the fluorine atoms have no obvious effect on the
activity since the activity of 5k is similar to that of 5b and 5c. In
contrast, when the benzene ring B was substituted with electron
donating group, a decrease in activity is observed.
oil) were dissolved in dry THF (20 mL) followed by the addition of
the solution of compound 1 (1.38 g, 2 mmol) and aromatic alde-
hydes (0.025 mol) at ꢀ2 ꢁC. After 20 min, the solution was warmed
to room temperature and reacted for 2 h, after which most of the
THF was evaporated, ice H2O was added and the solution was
extracted with ether. The combined ether layer was dried over
anhydrous Na2SO4, filtered and concentrated to give a residue,
which was purified over silica gel (petroleum ether: EtOAc ¼ 8:1) to
afford compound 2ae2k as a colorless solid.
4. Conclusion
5.1.1.1. (E)-Methyl 2,4-dimethoxy-6-styrylbenzoate (2a). Yield 88%;
1H NMR (300 MHz, CDCl3)
d
7.50 (d, J ¼ 7.2 Hz, 2H), 7.38 (m, 3H),
A series of cajaninstilbene acid derivatives were designed and
synthesized for the first time. Some of the derivatives were found to
have better antibacterial activities than positive controls and caja-
ninstilbene acid against Staphylococcus aureus, Staphylococcus epi-
dermidis, Bacillus subtilis, especially MRSA. The MIC values of
7.28 (d, J ¼ 16.2, 2H), 6.79 (d, J ¼ 2.4 Hz,1H), 6.78 (d, J ¼ 16.2 Hz,1H),
6.42 (d, J ¼ 2.4 Hz, 1H), 3.94 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H). 13
C
NMR (75 MHz, CDCl3) d 168.5, 161.5, 158.3, 137.6, 136.9, 131.8, 128.7,
128.1, 126.8, 125.5, 116.0, 101.5, 98.1, 56.0, 55.5, 52.3.
compounds 5b, 5c, 5j and 5k against MRSA are up to 0.5 mg/mL,
8e32-fold superior to that of norfloxacin. The cytoxic assay exhibits
a good selective index between bacterial and normal cells for the
active compounds 5b, 5c, 5j and 5k. These findings indicate the
potential usefulness of 5b, 5c, 5j and 5k in drug development.
Moreover, some structure-activity relationships of CSA derivatives
were determined and will be useful in the future to guide the
design and modification of new candidate CSA analogues as anti-
bacterial agents.
5.1.1.2. (E)-Methyl
ꢀ2.4-dimethoxy-6-
styrylbenzoate
7.55 (td, J ¼ 7.7, 1.7 Hz, 1H),
(2b).
Yield 92%; 1H NMR (300 MHz, CDCl3)
d
7.25 (dt, J ¼ 4.5, 3.9 Hz, 1H), 7.20 (d, J ¼ 1.7 Hz, 2H), 7.17e7.02 (m,
2H), 6.80 (d, J ¼ 2.1 Hz, 1H), 6.43 (d, J ¼ 2.1 Hz, 1H), 3.94 (s, 3H), 3.88
(s, 3H), 3.83 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 168.4, 161.6, 158.3,
137.5,129.4,129.3,127.7, 127.7, 127.4, 127.3, 124.8, 124.7, 124.3, 124.3,
124.0, 123.9, 116.0, 116.0, 115.7, 101.5, 98.3, 56.0, 55.5, 52.4; ESI-
LRMS m/z: 317.1 [MþH]þ. ESI-HRMS m/z: 317.1188 [MþH]þ, calcd
for C18H17FO4 317.1184.
5. Experimental protocols
5.1.1.3. (E)-Methyl-2-(3-fluorostyryl)-4,6-dimethoxybenzoate (2c).
Yield 88%; 1H NMR (300 MHz, CDCl3)
d
7.30 (td, J ¼ 7.9, 5.9 Hz, 1H),
5.1. Chemistry
7.22 (d, J ¼ 7.8 Hz, 1H), 7.19e7.13 (m, 1H), 7.12e6.91 (m, 3H), 6.74
(d, J ¼ 2.1 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H),
Intermediate 1 was synthesized according to the literature [6].
Compound 2 and the major chemical reagents were purchased
from Alfa Aesar or Sigma Aldrich Co. The synthetic routes to the
cajaninstilbene acid derivatives 2ae2k, 3ae3k, 4ae4k, 5ae5k, 6a,
6c, 6f and 10 are depicted in Schemes 1e3. All the compounds were
fully analyzed and characterized by 1H and 13C-nuclear magnetic
resonance (NMR), mass spectrometry (MS) and high resolution
mass spectrometry (ESI-HRMS) before biological screening.
3.82 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 168.4, 158.3, 139.3, 139.2,
137.1, 130.6, 130.5, 130.2, 130.1, 126.8, 122.7, 122.7, 116.0, 115.0,
114.7, 113.3, 113.0, 101.6, 98.3, 56.0, 55.5, 52.4; ESI-LRMS m/z: 317.2
[MþH]þ. ESI-HRMS m/z: 317.1184 [MþH]þ, calcd for C18H17FO4
317.1184.
5.1.1.4. (E)-Methyl 2-(4-fluorostyryl)-4,6-dimethoxybenzoate (2d).
Yield 89%; 1H NMR (300 MHz, CDCl3)
d 7.52e7.34 (m, 2H),
5.1.1. General procedure for the synthesis of compounds 2ae2k
7.13e6.83 (m, 4H), 6.74 (d, J ¼ 2.1 Hz, 1H), 6.40 (d, J ¼ 2.1 Hz, 1H),
15-crown-5 (40 mg) and NaH (100 mg, 2.5 mmol, 60% in mineral
3.93 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H). 13C NMR (75 MHz, CDCl3)
Fig. 2. Structure-activity relationships of cajaninstilbene acid (CSA) derivatives.

Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
241
d
168.5, 161.6, 158.3, 137.4, 133.1, 133.1, 130.5, 128.4, 128.3, 125.2,
(75 MHz, CDCl3) d 168.4,161.3,158.0,137.4, 128.2,127.3, 122.9,122.7,
125.1, 115.9, 115.7, 115.5, 101.5, 98.0, 55.9, 55.4, 52.3; ESI-LRMS m/z:
317.1 [MþH]þ. ESI-HRMS m/z: 317.1188 [MþH]þ, calcd for C18H17FO4
317.1184.
115.9, 111.8, 111.8, 111.5, 104.5, 104.1, 101.4, 98.3, 56.0, 55.2, 52.0; ESI-
LRMS m/z: 335.3 [MþH]þ. ESI-HRMS m/z: 335.1100 [MþH]þ, calcd
for C18H16F2O4 335.1089.
5.1.1.5. (E)-Methyl 2-(4-chlorostyryl)-4,6-dimethoxybenzoate (2e).
5.1.2. General procedure for the synthesis of compounds 3a-3k
Compound 2a-2k (0.02 mol) was dissolved in dry CH2Cl2
(100 mL) and BCl3 (2 mL,1 mol/L) was added at ꢀ30 ꢁC. The reaction
mixture was stirred for 2 h at ꢀ20 ꢁC after which it was quenched
by the addition of iced H2O. The organic layer was separated and
dried over anhydrous Na2SO4, filtered and concentrated to give a
residue, which was purified over silica gel (petroleum ether:
Et2O ¼ 20:1) to give compound 3a-3k as a white solid.
Yield 93%; 1H NMR (300 MHz, CDCl3)
d 7.44e7.36 (m,1H), 7.35e7.28
(m, 1H), 7.07 (d, J ¼ 16.1 Hz, 1H), 6.97 (d, J ¼ 16.1 Hz, 1H), 6.74 (d,
J ¼ 2.1 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 1H), 3.92 (s, 1H), 3.87 (s, 1H), 3.83
(s, 1H). 13C NMR (75 MHz, CDCl3)
d 168.4, 161.6, 158.3, 137.3, 135.4,
133.7, 130.4, 128.9, 128.0, 126.0, 115.9, 101.6, 98.2, 56.0, 55.5, 52.4;
ESI-LRMS m/z: 333.3 [MþH]þ. ESI-HRMS m/z: 333.0889 [MþH]þ,
calcd for C18H18ClO4 333.0888.
5.1.1.6. (E)-Methyl 2,4-dimethoxy-6-(4-methoxystyryl)benzoate (2f).
5.1.2.1. (E)-Methyl 2-hydroxy-4-methoxy-6-styrylbenzoate (3a).
Yield 85%; 1H NMR (300 MHz, CDCl3)
d
7.50e7.38 (m, 2H), 7.03 (d,
1H NMR (300 MHz, CDCl3)
d
11.70 (s, 1H), 7.73 (d, J ¼ 16.2 Hz, 1H),
J ¼ 16.1 Hz, 1H), 6.96 (d, J ¼ 16.2 Hz, 1H), 6.93e6.86 (m, 2H), 6.77 (d,
7.50e7.38 (m, 2H), 7.43 e 7.34 (m, 2H), 7.31 (d, J ¼ 16.2 Hz，1H)，
6.83 (d, J ¼ 16.2 Hz, 1H)，6.78 (d, J ¼ 2.1 Hz，1H)，6.45 (d,
J ¼ 2.1 Hz, 1H)，3.95 (s, 3H), 3.85 (s, 3H). 13C NMR (75 MHz, CDCl3)
J ¼ 2.1 Hz, 1H), 6.41 (d, J ¼ 2.1 Hz, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.84
(s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 168.6, 161.5, 159.7,
158.2, 137.9, 131.3, 129.7, 128.1, 123.2, 115.8, 114.1, 101.3, 97.7, 56.0,
55.5, 55.3, 52.3; ESI-LRMS m/z: 329.4 [MþH]þ. ESI-LRMS m/z: 333.3
[MþH]þ. ESI-HRMS m/z: 329.1385 [MþH]þ, calcd for C19H20O5
329.1384.
d 171.7, 165.2, 164.2, 142.9, 137.4, 130.9, 129.9, 128.8, 127.9, 126.7,
108.0, 104.0, 100.2, 55.5, 52.3.
5.1.2.2. (E)-Methyl 2-(2-fluorostyryl)-6-hydroxy-4-methoxybenzoate
(3b). Yield 90%; 1H NMR (300 MHz, CDCl3)
d 11.72 (s, 1H), 7.80 (d,
5.1.1.7. (E)-Methyl 2,4-dimethoxy-6-(4-methylstyryl)benzoate (2g).
J ¼ 16.1 Hz, 1H), 7.57 (td, J ¼ 7.6, 1.7 Hz, 1H), 7.32e7.22 (m, 1H), 7.13
(m, 2H), 6.95 (d, J ¼ 16.1 Hz, 1H), 6.66 (d, J ¼ 2.6 Hz, 1H), 6.47 (d,
J ¼ 2.6 Hz, 1H), 3.97 (s, 3H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3)
Yield 72%; 1H NMR (300 MHz, CDCl3)
d
7.37 (d, J ¼ 8.1 Hz, 2H), 7.16
(d, J ¼ 8.0 Hz, 2H), 7.11e6.96 (t, 2H), 6.76 (d, J ¼ 2.1 Hz, 1H), 6.40 (d,
J ¼ 2.1 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H), 2.36 (s, 3H). 13
C
d 171.6, 165.2, 164.2, 142.8, 132.4, 132.4, 129.1, 129.0, 127.6, 127.6,
NMR (75 MHz, CDCl3)
d
168.6, 161.5, 158.2, 138.1, 137.7, 134.1, 131.7,
125.3,125.1,124.3,124.2,123.3,123.3,116.1,115.8,108.1,104.0,100.4,
55.5, 52.3; ESI-LRMS m/z: 303.3 [MþH]þ. ESI-HRMS m/z: 303.1028
[MþH]þ, calcd for C17H15FO4 303.1027.
129.4, 126.7, 124.4, 115.9, 101.4, 97.9, 56.0, 55.5, 52.3, 21.3; ESI-LRMS
m/z: 313.3 [MþH]þ. ESI-HRMS m/z: 313.1434 [MþH]þ, calcd for
C
19H20O4 313.1434.
5.1.2.3. (E)-Methyl 2-(3-fluorostyryl)-6-hydroxy-4-methoxybenzoate
5.1.1.8. (E)-Methyl 2,4-dimethoxy-6-(3,4,5-trimethoxystyryl)benzo-
ate (2h). Yield 80%; 1H NMR (300 MHz, CDCl3)
(3c). Yield 90%; 1H NMR (300 MHz, CDCl3)
d 11.72 (s, 1H), 7.80 (d,
d
6.97 (d, J ¼ 16.1 Hz,
J ¼ 16.1 Hz, 1H), 7.57 (td, J ¼ 7.6, 1.7 Hz, 1H), 7.32e7.22 (m, 1H),
7.16e7.25 (m, 2H), 6.95 (d, J ¼ 16.1 Hz, 1H), 6.66 (d, J ¼ 2.6 Hz, 1H),
6.47 (d, J ¼ 2.6 Hz, 1H), 3.97 (s, 3H), 3.87 (s, 3H). 13C NMR (75 MHz,
1H), 6.91 (d, J ¼ 16.1 Hz, 1H), 6.70 (d, J ¼ 2.1 Hz, 1H), 6.66 (s, 2H),
6.36 (d, J ¼ 2.1 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 6H), 3.83 (s, 3H), 3.82 (s,
3H), 3.77 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
168.5, 161.6, 158.3,
CDCl3) d 171.5, 165.2, 164.2, 142.3, 139.8, 139.7, 131.2, 130.3, 130.1,
153.4, 138.3, 137.5, 132.7, 131.7, 125.0, 115.7, 103.9, 101.6, 98.0, 60.9,
56.1, 56.0, 55.4, 52.2; ESI-LRMS m/z: 411.7 [MþNa]þ. ESI-HRMS m/z:
389.1597 [MþH]þ, calcd for C21H24O7 389.1595.
129.6,129.6,122.7,122.7,114.8,114.5,113.0,112.7,108.2,103.9,100.4,
55.5, 52.4; ESI-LRMS m/z: 303.3 [MþNa]þ. ESI-HRMS m/z: 303.1028
[MþH]þ, calcd for C17H15FO4 303.1027.
5.1.1.9. (E)-Methyl 2-(3-cyanostyryl)-4,6-dimethoxybenzoate (2i).
5.1.2.4. (E)-Methyl 2-(4-fluorostyryl)-6-hydroxy-4-methoxybenzoate
Yield 87%; 1H NMR (300 MHz, CDCl3)
d
7.69 (dd, J ¼ 7.4, 6.2 Hz, 2H),
(3d). Yield 92%; 1H NMR (300 MHz, CDCl3)
d 11.67 (s, 1H), 7.60 (d,
7.54 (dt, J ¼ 7.6,1.3 Hz,1H), 7.45 (t, J ¼ 7.7 Hz,1H), 7.15 (d, J ¼ 16.2 Hz,
1H), 6.98 (d, J ¼ 16.1 Hz, 1H), 6.73 (d, J ¼ 2.1 Hz, 1H), 6.44 (d,
J ¼ 2.1 Hz, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H). 13C NMR
J ¼ 15.9 Hz, 1H), 7.51e7.39 (m, 2H), 7.06 (t, J ¼ 8.7 Hz, 2H), 6.76 (d,
J ¼ 15.9 Hz, 1H), 6.60 (d, J ¼ 2.6 Hz, 1H), 6.44 (d, J ¼ 2.6 Hz, 1H), 3.94
(s, 3H), 3.85 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.6, 165.2, 164.2,
(75 MHz, CDCl3)
d
168.3, 161.7, 158.5, 138.1, 136.8, 131.2, 130.7, 130.3,
142.7, 133.6, 129.7, 128.2, 128.1, 115.8, 115.6, 108.0, 103.9, 100.2, 55.5,
52.3; ESI-LRMS m/z: 303.3 [MþH]þ. ESI-HRMS m/z: 303.1028
[MþH]þ, calcd for C17H15FO4 303.1027.
129.5, 129.3, 128.2, 118.4, 116.1, 113.0, 101.8, 98.7, 56.1, 55.6, 52.5;
ESI-LRMS m/z: 324.3 [MþH]þ. ESI-HRMS m/z: 324.1233 [MþH]þ,
calcd for C19H17NO4 324.123.
5.1.2.5. (E)-Methyl 2-(4-chlorostyryl)-6-hydroxy-4-methoxybenzoate
5.1.1.10. (E)-Methyl 2,4-dimethoxy-6-(4-(trifluoromethyl)styryl)ben-
(3e). Yield 86%; 1H NMR (300 MHz, CDCl3)
d 11.66 (s, 1H), 7.65 (d,
zoate (2j). Yield 92%; 1H NMR (300 MHz, CDCl3)
d
7.56 (q, J ¼ 8.6 Hz,
J ¼ 16.0 Hz, 1H), 7.46e7.38 (m, 2H), 7.37e7.29 (m, 2H), 6.74 (d,
4H), 7.18 (d, J ¼ 16.1 Hz, 1H), 7.03 (d, J ¼ 16.1 Hz, 1H), 6.75 (d,
J ¼ 15.9 Hz, 1H), 6.60 (d, J ¼ 2.6 Hz, 1H), 6.44 (d, J ¼ 2.6 Hz, 1H), 3.94
J ¼ 2.1 Hz, 1H), 6.43 (d, J ¼ 2.1 Hz, 1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.82
(s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.5, 165.2, 164.2,
(s, 3H). 13C NMR (75 MHz, CDCl3)
d
168.3, 161.7, 158.4, 140.4, 137.0,
142.5,135.9, 133.4, 130.5, 129.5,128.9,127.8, 108.1, 103.9,100.3, 55.5,
52.3; ESI-LRMS m/z: 319.3 [MþH]þ. ESI-HRMS m/z: 319.0732
[MþH]þ, calcd for C17H16ClO4 319.0732.
130.2, 128.0, 126.9, 125.7, 125.6, 125.6, 125.5, 116.1, 101.8, 98.5, 56.0,
55.5, 52.4; ESI-LRMS m/z: 367.1 [MþH]þ. ESI-HRMS m/z: 367.1154
[MþH]þ, calcd for C19H17F3O4 367.1152.
5.1.2.6. (E)-Methyl 2-hydroxy-4-methoxy-6-(4-methoxystyryl)ben-
5.1.1.11. (E)-Methyl 2-(2,4-difluorostyryl)-4,6-dimethoxybenzoate
zoate (3f). Yield 92%; 1H NMR (300 MHz, CDCl3)
d 11.69 (s, 1H), 7.57
(2k). Yield 92%; 1H NMR (300 MHz, CDCl3)
d
7.56e7.43 (m, 1H),
(d, J ¼ 15.9 Hz, 1H), 7.43 (d, J ¼ 8.7 Hz, 2H), 6.91 (d, J ¼ 8.7 Hz, 2H),
6.77 (d, J ¼ 15.9 Hz, 1H), 6.62 (d, J ¼ 2.6 Hz, 1H), 6.42 (d, J ¼ 2.6 Hz,
1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3)
7.10 (s, 2H), 6.92e6.77 (m, 2H), 6.75 (d, J ¼ 2.0 Hz, 1H), 6.42 (d,
J ¼ 2.0 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). 13C NMR

242
Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
d
171.8, 165.1, 164.1, 159.5, 143.2, 130.5, 130.2, 127.9, 127.7, 114.2,
5.1.4. General procedure for the synthesis of compounds 4ae4k and
9
107.7, 104.0, 100.0, 55.5, 55.4, 52.2; ESI-LRMS m/z: 337.2 [MþNa]þ.
ESI-HRMS m/z: 315.1226 [MþH]þ, calcd for C17H16O5 315.1227.
Compound 3ae3k or 8 (7.0 mmol) was dissolved in dry toluene
(50 mL) and NaH (8.4 mmol, 60% in mineral oil) was added at 45 ꢁC.
The reaction mixture was stirred at 45 ꢁC for 0.5 h after which the
prenyl bromide (8.4 mmol) was added at room temperature. The
mixture was stirred for 2 h at 78 ꢁC, then quenched by the addition
of saturated NaCl and the organic layer was separated and dried
over anhydrous Na2SO4. It was then filtered and concentrated to
give a residue, which was purified over silica gel (petroleum ether:
Et2O ¼ 100:1) to give compound 4ae4k or 9 as a white solid
separately.
5.1.2.7. (E)-Methyl 2-hydroxy-4-methoxy-6-(4-methylstyryl)benzo-
ate (3g). Yield 95%; 1H NMR (300 MHz, CDCl3)
d 11.71 (s, 1H), 7.65
(d, J ¼ 15.9 Hz, 1H), 7.40 (d, J ¼ 8.1 Hz, 2H), 7.19 (d, J ¼ 8.0 Hz, 2H),
6.79 (d, J ¼ 15.9 Hz, 1H), 6.64 (d, J ¼ 2.5 Hz, 1H), 6.44 (d, J ¼ 2.6 Hz,
1H), 3.94 (s, 3H), 3.85 (s, 3H), 2.38 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
171.8, 165.2, 164.2, 143.1, 137.8, 134.7, 130.8, 129.5, 128.9, 126.6,
107.8, 104.0, 100.1, 55.5, 52.2, 21.3; ESI-LRMS m/z: 299.1 [MþH]þ.
ESI-HRMS m/z: 299.1278 [MþH]þ, calcd for C18H18O4 299.1278.
5.1.4.1. (E)-Methyl 2-hydroxy-4-methoxy-3-(3-methylbut-2-en-1-
5.1.2.8. (E)-Methyl 2-hydroxy-4-methoxy-6-(3,4,5-trimethoxystyryl)
yl)-6-styrylbenzoate (4a). Yield 41%; 1H NMR (300 MHz, CDCl3)
benzoate (3h). Yield 90%; 1H NMR (300 MHz, CDCl3)
d
11.65 (s, 1H),
d
11.74 (s,1H), 7.55 (m,3H), 7.39 (t, J ¼ 7.2 Hz, 2H), 7.28 (t, J ¼ 7.2 Hz,
7.56 (d, J ¼ 15.9 Hz, 1H), 6.70 (s, 2H), 6.68 (d, J ¼ 15.9 Hz, 1H) 6.58 (d,
1H), 7.08 (d, J ¼ 16.2 Hz, 1H), 6.85 (s,1H), 5.27 (t, J ¼ 7.2 Hz, 1H), 3.96
(s, 3H), 3.95 (s, 3H), 3.43 (d, J ¼ 7.2 Hz, 2H), 1.84 (s, 3H), 1.73 (s, 3H).
J ¼ 2.5 Hz, 1H), 6.40 (d, J ¼ 2.6 Hz, 1H), 3.92 (s, 3H), 3.89 (s, 6H), 3.86
(s, 3H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
171.6, 165.2, 164.2,
13C NMR (75 MHz, CDCl3)
d 172.0, 161.5, 161.4, 140.3, 137.5, 131.9,
153.4, 142.7, 138.1, 133.2, 130.7, 129.4, 107.8, 103.9, 103.8, 100.2, 61.0,
56.1, 55.4, 52.2; ESI-LRMS m/z: 375.3 [MþH]þ. ESI-HRMS m/z:
375.1439 [MþH]þ, calcd for C20H22O7 375.1438.
130.7, 130.1, 128.8, 127.7, 126.6, 122.1, 116.7, 104.6, 102.9, 55.7, 52.2,
25.8, 22.1, 17.8.
5.1.4.2. (E)-Methyl-2-hydroxy-3-(3-methylbut-2-enyl)-4-dimethoxy-
6-styrylbenzoate (4b). Yield 44%; 1H NMR (300 MHz, CDCl3)
d 7.80
5.1.2.9. (E)-Methyl 2-(3-cyanostyryl)-6-hydroxy-4-methoxybenzoate
(d, J ¼ 16.2 Hz, 1H), 7.57 (td, J ¼ 7.6, 1.6 Hz, 1H), 7.25 (m, 1H),
7.20e7.04 (m, 2H), 6.92 (d, J ¼ 16.1 Hz, 1H), 6.62 (s, 1H), 5.29e5.13
(m, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.39 (d, J ¼ 7.0 Hz, 2H),1.80 (s, 3H),
(3i). Yield 92%; 1H NMR (300 MHz, CDCl3)
d 11.64 (s, 1H),
7.80e7.63 (m, 3H), 7.54 (dt, J ¼ 7.6, 1.3 Hz, 1H), 7.47 (d, J ¼ 7.7 Hz,
1H), 6.73 (d, J ¼ 16.0 Hz, 1H), 6.58 (d, J ¼ 2.4 Hz, 1H), 6.45 (d,
J ¼ 2.6 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3)
1.69 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.9, 162.1, 161.5, 161.4,
140.2,133.1,133.1,131.9,129.0,128.9,127.5,127.4,125.4,125.3,124.3,
124.3, 122.4, 122.0, 116.9, 116.1, 115.8, 104.5, 102.9, 55.7, 52.2, 25.9,
22.2, 17.8; ESI-LRMS m/z: 371.2 [MþH]þ; ESI-HRMS m/z: 371.1655
[MþH]þ, calcd for C22H24FO4 371.1653.
d
171.3, 165.2, 164.2, 141.8, 138.7, 132.5, 130.9, 130.8, 130.0, 129.6,
128.4, 118.8, 113.0, 108.4, 103.9, 100.7, 55.6, 52.4; ESI-LRMS m/z:
310.3 [MþH]þ. ESI-HRMS m/z: 310.1074 [MþH]þ, calcd for
C
18H15NO4 310.1074.
5.1.4.3. (E)-Methyl-6-(3-fluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)- benzoate (4c). Yield 40%; 1H NMR (300 MHz,
5.1.2.10. (E)-Methyl-2-hydroxy-4-methoxy-6-(4-(trifluoromethyl)
styryl)benzoate (3j). Yield 92%; 1H NMR (300 MHz, CDCl3)
11.71 (s,
d
CDCl3)
d
11.72 (s, 1H), 7.74 (d, J ¼ 15.9 Hz, 1H), 7.44e7.15 (m, 3H),
1H), 7.63 (d, J ¼ 15.9 Hz, 1H), 7.47 (m, 1H), 7.08 (t, J ¼ 8.7 Hz, 2H),
7.06e6.92 (m, 1H), 6.75 (d, J ¼ 15.9 Hz, 1H), 6.61 (s, 1H), 3.96 (s, 3H),
6.78 (d, J ¼ 15.9 Hz, 1H), 6.63 (d, J ¼ 2.4 Hz, 1H), 6.46 (d, J ¼ 2.6 Hz,
3.95 (s, 3H), 3.41 (d, J ¼ 7.1 Hz, 2H), 1.82 (s, 3H), 1.71 (s, 3H). 13C NMR
1H), 3.97 (s, 3H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
171.6,
(75 MHz, CDCl3) d 171.8, 161.5,161.4, 139.9,139.9,139.8,132.0, 132.0,
165.2, 164.2, 160.8, 142.7, 133.6, 129.7, 128.2, 128.1, 115.9, 115.6,
108.0, 103.9, 100.2, 55.5, 52.3; ESI-LRMS m/z: 351.3 [MꢀH]þ. ESI-
HRMS m/z: 353.0993 [MþH]þ, calcd for C18H15F3O4 353.0995.
130.3, 130.1, 128.8, 122.6, 122.5, 122.0, 117.0, 114.7, 114.4, 113.0, 112.7,
104.5, 102.9, 55.7, 52.4, 25.9, 22.2, 17.8; ESI-LRMS m/z: 371.4
[MþH]þ; ESI-HRMS m/z: 371.1643 [MþH]þ, calcd for C22H24FO4
371.1653.
5.1.2.11. (E)-Methyl-2-(2,4-difluorostyryl)-6-hydroxy-4-
5.1.4.4. (E)-Methyl-6-(4-fluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoate (4d). Yield 38%; 1H NMR (300 MHz,
methoxybenzoate (3k). Yield 95%; 1H NMR (300 MHz, CDCl3)
d 11.70
(s, 1H), 7.72 (d, J ¼ 16.1 Hz, 1H), 7.53 (td, J ¼ 8.6, 6.5 Hz, 1H), 6.97 (d,
J ¼ 16.1 Hz, 1H), 6.96e6.77 (m, 2H), 6.63 (d, J ¼ 2.5 Hz, 1H), 6.47 (d,
J ¼ 2.6 Hz, 1H), 3.96 (s, 3H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3)
CDCl3)
d
11.67 (s, 1H), 7.63 (d, J ¼ 15.9 Hz, 1H), 7.53e7.41 (m, 2H),
7.07 (t, J ¼ 8.7 Hz, 2H), 6.74 (d, J ¼ 15.9 Hz, 1H), 6.59 (s, 1H), 5.22 (m,
1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.38 (d, J ¼ 7.0 Hz, 2H), 1.80 (s, 3H),
d
171.5, 165.1, 164.1, 142.5, 131.9, 128.5, 128.3, 122.3, 111.5, 108.2,
1.69 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.9, 161.5, 161.4, 140.1,
104.5, 104.2, 103.8, 100.4, 100.3, 55.4, 52.1; ESI-LRMS m/z: 319.3 [M-
H]þ. ESI-HRMS m/z: 321.0934 [MþH]þ, calcd for C17H14F2O4
321.0933.
133.8, 131.9, 130.5, 130.5, 128.9, 128.1, 128.0, 122.1, 116.8, 115.8, 115.6,
104.5, 102.8, 55.7, 52.3, 25.8, 22.2, 17.8; ESI-LRMS m/z: 371.4
[MþH]þ. ESI-HRMS m/z: 371.1643 [MþH]þ, calcd for C22H24FO4
371.1653.
5.1.3. Procedure for the synthesis of compounds 8
To a solution of compound 7 (10 g, 54 mmol) in MeOH (100 mL)
was added I2 (27.6 g, 108 mmol). The mixture was heated at reflux
for 4 h. Excess MeOH was evaporated, then the mixture was
extracted with EtOAc (300 mL ꢃ 3), and washed with H2O
(50 mL ꢃ 2) and brine (50 mL), dried with Na2SO4, and filtered. The
filtrate was concentrated to give compound 8 (5.7 g) as a white
5.1.4.5. (E)-Methy6-(4-chlorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoate (4e). Yield 43.4%; 1H NMR (300 MHz,
CDCl3)
d
11.68 (s, 1H), 7.68 (d, J ¼ 15.9 Hz, 1H), 7.43 (d, J ¼ 8.5 Hz,
2H), 7.34 (d, J ¼ 8.5 Hz, 2H), 6.72 (d, J ¼ 15.9 Hz, 1H), 6.58 (s, 1H),
5.22 (t, J ¼ 7.0 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.38 (d, J ¼ 7.0 Hz,
2H),1.80 (s, 3H), 1.69 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.8,161.5,
solid, yield 54%.1H NMR (300 MHz, CDCl3)
d
11.77 (s, 1H), 6.30 (d,
161.4, 139.9, 136.0, 133.3, 132.0, 131.3, 129.0, 128.8, 127.8, 122.0,
116.9, 104.5, 102.9, 55.7, 52.3, 25.9, 22.2, 17.8; ESI-LRMS m/z: 409.3
[MþNa]þ. ESI-HRMS m/z: 387.1358 [MþH]þ, calcd for C22H24FO4
387.1358.
J ¼ 2.6 Hz, 1H), 6.25 (d, J ¼ 2.6 Hz, 1H), 3.90 (s, 3H), 3.77 (s, 3H), 2.46
(s, 3H). 13C NMR (75 MHz, CDCl3)
105.2, 98.7, 55.2, 51.8, 24.3.
d 172.2, 165.5, 163.9, 143.1, 111.1,

Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
243
5.1.4.6. (E)-Methyl-2-hydroxy-4-methoxy-6-(4-methoxystyryl)-3-(3-
5.1.4.12. Methyl-2-hydroxy-4-methoxy-6-methyl-3-(3-methylbut-2-
methylstyryl)benzoate (4f). Yield 36%; 1H NMR (300 MHz, CDCl3)
en-1-yl)benzoate (9). Yield 42.3%; 1H NMR (300 MHz, CDCl3)
d
11.70 (s, 1H), 7.59 (d, J ¼ 16.0 Hz, 1H), 7.45 (d, J ¼ 8.7 Hz, 2H), 6.92
d 11.80 (s, 1H), 6.28 (s, 1H), 5.43e4.90 (m, 1H), 3.92 (s, 3H), 3.85 (s,
(d, J ¼ 8.8 Hz, 2H), 6.75 (d, J ¼ 15.9 Hz, 1H), 6.60 (s, 1H), 5.22 (m, 1H),
3H), 3.33 (d, J ¼ 7.0 Hz, 2H), 2.52 (s, 3H), 1.78 (s, 3H), 1.67 (s, 3H). 13
C
3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.38 (d, J ¼ 7.0 Hz, 2H), 1.80 (s,
NMR (75 MHz, CDCl3)
d 172.6, 161.9, 161.1, 140.6, 131.6, 122.5, 114.7,
3H), 1.69 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
172.1, 161.4, 161.3,
106.1, 105.7, 55.5, 51.8, 25.8, 24.7, 21.9, 17.8; ESI-LRMS m/z: 265.3
[MþH]þ. ESI-HRMS m/z: 265.1441 [MþH]þ, calcd for C17H15FO4
265.1434.
159.4, 140.6, 131.8, 130.3, 129.6, 128.5, 127.8, 122.2, 116.3, 114.2,
104.5, 102.6, 55.7, 55.4, 52.2, 25.9, 22.2, 17.9; ESI-LRMS m/z: 383.2
[MþH]þ; ESI-HRMS m/z: 383.1834 [MþH]þ, calcd for C23H27O5
383.1853.
5.1.5. General procedure for the synthesis of compounds 5a (CSA)e
5k, 10, 6a, 6c and 6f
The compound 4ae4k (1.42 mmol) was dissolved in EtOH, H2O
(10 mL: 2 mL) and KOH (4.26 mmol) were added. The mixture was
heated to 90 ꢁC for 2 h. Then the pH of the mixture was adjusted to
2 with 10% HCl followed by extraction with EtOAc (100 mL). The
organic layer was dried over anhydrous Na2SO4, filtered and
concentrated to give a residue, which was purified by column
chromatography (petroleum ether:Me2CO ¼ 10:1) to give 5a
(CSA)e5k as white solid respectively. In addition, 10, 6a, 6c and 6f
were synthesized from compounds 9, 3a, 3c and 3e by this method.
5.1.4.7. (E)-Methyl-2-hydroxy-4-methoxy-3-(3-methylbut-2-en-1-
yl)-6-(4-methylstyryl)- benzoate (4g). Yield 35%; 1H NMR
(300 MHz, CDCl3)
d
11.71 (s, 1H), 7.68 (d, J ¼ 15.9 Hz, 2H), 7.41 (d,
J ¼ 8.1 Hz, 1H), 7.19 (d, J ¼ 8.0 Hz, 1H), 6.77 (d, J ¼ 15.9 Hz, 2H), 6.61
(s, 1H), 5.32e5.14 (m, 1H), 3.93 (s, 3H), 3.93 (s, 3H), 3.39 (d,
J ¼ 7.1 Hz, 2H), 2.38 (s, 3H), 1.81 (s, 3H), 1.70 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 172.1, 161.5, 161.3, 140.5, 137.7, 134.8, 131.9, 130.0,
129.7, 129.5, 126.5, 122.1, 116.5, 104.5, 102.8, 55.7, 52.3, 25.9, 22.2,
21.3, 17.9; ESI-LRMS m/z: 389.4 [MþNa]þ. ESI-HRMS m/z: 367.1818
[MþH]þ, calcd for C23H27O4 367.1904.
5.1.5.1. (E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-
styrylbenzoic acid (CSA or 5a). 1H NMR (300 MHz, CDCl3)
d 11.53 (s,
5.1.4.8. (E)-Methyl-2-hydroxy-4-methoxy-3-(3-methylbut-2-en-1-
1H), 7.86 (d, J ¼ 16.2 Hz, 1H), 7.55 (d, J ¼ 7.2 Hz, 2H), 7.39 (t,
J ¼ 7.2 Hz, 2H), 7.34 (t, J ¼ 7.2 Hz, 1H), 6.82 (d, J ¼ 16.2 Hz, 1H), 6.69
(s, 1H), 5.25 (t, J ¼ 6.8 Hz, 1H), 3.98 (s,3H), 3.40 (d, J ¼ 6.8 Hz, 2H),
yl)-6-(3,4,5-trimethoxystyryl)benzoate (4h). Yield 35%; 1H NMR
(300 MHz, CDCl3)
d
11.32 (s, 1H), 7.17 (d, J ¼ 16.4 Hz, 1H), 6.66 (s,
2H), 6.45 (s,1H), 6.22 (d, J ¼ 16.4 Hz,1H), 5.08 (t, J ¼ 6.6 Hz,1H), 3.90
1.83 (s, 3H), 1.72 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.7, 165.2,
(s, 6H), 3.88 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.32 (d, J ¼ 6.4 Hz, 2H),
164.2, 142.9, 137.3, 132.0, 130.9, 129.9, 128.7, 127.9, 126.7, 121.9,
116.8, 108.0, 103.9, 55.5, 52.3.
1.68 (s, 3H), 1.61 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.9, 162.8,
162.6,153.4,140.8, 137.7, 133.5,131.6, 130.7, 127.7, 123.8, 121.6, 104.6,
103.2, 98.3, 61.0, 56.1, 55.7, 52.1, 26.9, 26.1, 25.8, 18.1; ESI-LRMS m/z:
443.3 [MþH]þ；ESI-HRMS m/z: 443.2064 [MþH]þ, calcd for
5.1.5.2. (E)-6-(2-Fluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoic acid (5b). Yield 92.5%; 1H NMR
C
25H31O7 443.2064.
(300 MHz, Me2CO-d6):
d
8.10 (d, J ¼ 16.2 Hz, 1H), 7.73 (td, J ¼ 7.7,
1.7 Hz, 1H), 7.40e7.30 (m, 1H), 7.28e7.13 (m, 2H), 7.08 (d,
J ¼ 16.2 Hz, 1H), 6.86 (s, 1H), 5.38e5.07 (m, 1H), 4.01 (s, 3H), 3.37 (d,
J ¼ 7.2 Hz, 2H), 1.78 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, Me2CO-
5.1.4.9. (E)-Methyl-6-(3-cyanostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)- benzoate (4i). Yield 36%; 1H NMR (300 MHz,
CDCl3)
d
11.66 (s, 1H), 7.72 (d, J ¼ 16.0 Hz, 1H), 7.60 (m, 2H), 7.55 (dd,
d6):
d 173.2, 162.1, 161.6, 140.7, 133.1, 133.1, 130.7, 129.3, 129.2, 127.7,
J ¼ 6.4, 1.3 Hz, 1H), 7.48 (t, J ¼ 7.7 Hz, 1H), 6.73 (d, J ¼ 16.0 Hz, 1H),
6.57 (s, 1H), 5.27e5.15 (m, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.38 (d,
J ¼ 7.1 Hz, 2H), 1.79 (s, 3H), 1.68 (s, 3H). 13C NMR (75 MHz, CDCl3)
127.6, 125.4, 125.3, 124.6, 124.5, 122.3, 122.2, 122.1, 116.3, 115.8,
115.5, 104.0, 102.7, 55.3, 25.0, 21.8, 17.0; ESI-LRMS m/z: 357.3
[MþH]þ. ESI-HRMS m/z: 357.1494 [MþH]þ, calcd for C21H22FO4
357.1497.
d
171.6, 161.5, 161.4, 139.3, 138.8, 133.4, 132.1, 130.9, 130.6, 130.0,
129.6, 127.7, 121.8, 118.8, 117.4, 113.0, 104.6, 103.0, 55.7, 52.5, 25.9,
22.2, 17.8; ESI-LRMS m/z: 378.0 [MþH]þ. ESI-HRMS m/z: 378.1691
[MþH]þ, calcd for C23H24NO4 378.1700.
5.1.5.3. (E)-6-(3-Fluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl) benzoic acid (5c). Yield 94%; 1H NMR
(300 MHz, Me2CO-d6):
d
8.08 (d, J ¼ 16.1 Hz, 1H), 7.54e7.26 (m, 3H),
5.1.4.10. (E)-Methyl-6-(3-cyanostyryl)-2-hydroxy-4-methoxy-3-(3-
7.16e6.95 (m, 2H), 6.88 (s, 1H), 5.30e5.13 (m, 1H), 3.99 (s, 3H), 3.36
methylbut-2-en-1-yl)- benzoate (4j). Yield 34%; 1H NMR (300 MHz,
(d, J ¼ 7.3 Hz, 2H), 1.78 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz,
CDCl3)
d
11.67 (s, 1H), 7.80 (d, J ¼ 15.9 Hz, 1H), 7.67e7.55 (m, 4H),
Me2CO-d6):
d 173.2, 162.0, 161.5, 140.6, 140.4, 131.9, 130.7, 130.5,
6.78 (d, J ¼ 16.0 Hz, 1H), 6.60 (s, 1H), 5.22 (m, 1H), 3.93 (s, 6H), 3.39
130.4, 128.9, 122.8, 122.8, 122.3, 116.2, 114.3, 114.0, 112.8, 112.5,
104.2,102.6, 55.3, 25.0, 21.8,17.0; ESI-LRMS m/z: 357.3 [MþH]þ; ESI-
HRMS m/z: 357.1499 [MþH]þ, calcd for C21H22FO4 357.1497.
(d, J ¼ 7.1 Hz, 2H), 1.80 (s, 3H), 1.69 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
171.7, 161.5, 161.4, 139.6, 133.2, 132.0, 128.5, 126.7, 125.7, 125.7,
121.9, 117.3, 104.6, 103.0, 55.7, 52.3, 25.9, 22.2, 17.8; ESI-LRMS m/z:
421.4 [MþH]þ. ESI-HRMS m/z: 421.1610 [MþH]þ, calcd for
5.1.5.4. (E)-6-(4-Fluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl) benzoic acid (5d). Yield 95%; 1H NMR
C
23H24F3O4 421.1621.
(300 MHz, Me2CO-d6)
d
7.96 (d, J ¼ 16.1 Hz, 1H), 7.81e7.54 (m, 2H),
5.1.4.11. (E)-Methyl 6-(2,4-difluorostyryl)-2-hydroxy-4-methoxy-3-
(3-methylbut-2-en-1-yl)benzoate (4k). Yield 34%; 1H NMR
7.16 (t, J ¼ 8.8 Hz, 2H), 7.01 (d, J ¼ 16.1 Hz,1H), 6.86 (s,1H), 5.31e5.10
(m, 1H), 3.99 (s, 3H), 3.36 (d, J ¼ 7.2 Hz, 2H), 1.78 (s, 3H), 1.65 (s, 3H).
(300 MHz, CDCl3)
d
11.67 (s, 1H), 7.72 (d, J ¼ 16.1 Hz, 1H), 7.56e7.49
13C NMR (75 MHz, Me2CO-d6)
d 173.3, 162.1, 161.5, 140.8, 134.3,
(m, 1H), 6.98e6.8 (m, 2H), 6.83 (d, J ¼ 16.1 Hz, 1H), 6.59 (s,
1H),5.24e5.20 (m, 1H), 3.93 (s, 6H), 3.38 (d, J ¼ 7.1 Hz, 2H), 1.80 (s,
134.3, 130.7, 130.3, 130.3, 129.0, 128.5, 128.4, 122.3, 116.0, 115.6,
115.3, 104.0, 102.5, 55.3, 25.0, 21.8, 17.0; ESI-LRMS m/z: 357.2
[MþH]þ. ESI-HRMS m/z: 357.1504 [MþH]þ, calcd for C21H22FO4
357.1497.
3H),1.68 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 171.8, 161.5,161.4,140.1,
132.8, 128.3, 128.1, 122.0, 121.5, 118.2, 111.8, 111.5, 104.6, 104.5, 104.2,
103.9, 102.9, 55.7, 52.3, 25.9, 22.2, 17.8; ESI-LRMS m/z: 389.5
[MþH]þ. ESI-HRMS m/z: 389.1558 [MþH]þ, calcd for C22H22F2O4
389.1559.
5.1.5.5. (E)-6-(4-Chlorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoic acid (5e). Yield 92%; 1H NMR

244
Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
(300 MHz, Me2CO-d6):
d
12.29 (s, 1H), 8.01 (d, J ¼ 16.1 Hz, 1H), 7.59
133.0,130.7,130.4,129.0,128.4,122.0,121.9,116.0,112.0,111.3,104.0,
102.7, 55.1, 25.0, 21.8, 17.0; ESI-LRMS m/z: 473.4 [MþH]þ. ESI-HRMS
m/z: 375.1401 [MþH]þ, calcd for C21H20F2O4 375.1402.
(d, J ¼ 8.5 Hz, 2H), 7.41 (d, J ¼ 8.5 Hz, 2H), 7.01 (d, J ¼ 16.0 Hz, 1H),
6.86 (s, 1H), 5.22 (t, J ¼ 7.1 Hz, 1H), 3.98 (s, 3H), 3.36 (d, J ¼ 7.1 Hz,
2H), 1.78 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d 173.2,
162.1, 161.5, 140.6, 136.7, 132.7, 131.2, 130.7, 128.9, 128.7, 128.1, 122.3,
116.2, 103.9, 102.6, 55.3, 25.0, 21.8, 17.0; ESI-LRMS m/z: 395.3
[MþNa]þ; ESI-HRMS m/z: 373.1174 [MþH]þ, calcd for C21H22ClO4
373.1201.
5.1.5.12. 2-Hydroxy-4-Methoxy-6-methyl-3-(3-methylbut-2-en-1-yl)
benzoic acid (10). Yield 95%; 1H NMR (300 MHz, Me2CO-d6)
d 6.49
(s, 1H), 5.33e5.07 (m, 1H), 3.90 (s, 3H), 3.30 (d, J ¼ 7.2 Hz, 2H), 2.59
(s, 3H), 1.75 (s, 3H), 1.63 (s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d
173.8, 162.5, 161.3, 141.3, 130.3, 122.7, 114.0, 106.1, 105.1, 55.1, 25.0,
5.1.5.6. (E)-2-Hydroxy-4-methoxy-6-(4-methoxystyryl)-3-(3-
23.8, 21.6, 16.9; ESI-LRMS m/z: 251.3 [MþH] þ; ESI-HRMS m/z:
methylbut-2-en-1-yl)benzoic acid (5f). Yield 92%; 1H NMR
251.1271 [MþH]þ, calcd for C14H19O4 251.1278.
(300 MHz, Me2CO-d6)
d
7.89 (d, J ¼ 16.0 Hz, 1H), 7.52 (d, J ¼ 8.7 Hz,
2H), 6.97 (m, 2H), 6.86 (s, 1H), 5.22 (m, 1H), 3.99 (s, 3H), 3.83 (s, 3H),
5.1.5.13. (E)-2-Hydroxy-4-methoxy-6-styrylbenzoic
acid
(6a).
3.35 (d, J ¼ 7.2 Hz, 2H), 1.78 (s, 3H), 1.65 (s, 3H). 13C NMR (75 MHz,
Yield 95%; 1H NMR (300 MHz, Me2CO-d6)
d
7.88 (d, J ¼ 16.0 Hz, 1H),
Me2CO-d6)
d
173.5, 162.0, 161.5, 159.6, 141.3, 130.6, 130.4, 130.0,
7.54e7.48 (m, 2H), 7.23e7.38 (m, 2H), 7.30e7.22 (m, 1H), 6.87 (d,
J ¼ 16.0 Hz, 1H), 6.68 (d, J ¼ 2.4 Hz, 1H), 6.42 (d, J ¼ 2.5 Hz, 1H), 3.86
128.0, 127.9, 122.4, 115.6, 114.1, 103.9, 102.2, 55.3, 54.7, 25.0, 21.8,
17.0; ESI-LRMS m/z: 369.4 [MþH]þ. ESI-HRMS m/z: 369.1672
[MþH]þ, calcd for C22H25O5 369.1697.
(s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d 173.0, 164.9, 164.0, 143.0,
137.6, 130.3, 129.5, 128.3, 127.3, 126.3, 106.3, 104.3, 99.8, 54.6; ESI-
LRMS m/z: 271.4.3 [MþH]þ. ESI-HRMS m/z: 271.0964 [MþH]þ,
calcd for C16H15O4 271.0965.
5.1.5.7. (E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-(4-
methylstyryl)benzoic acid (5g). Yield 93%; 1H NMR (300 MHz,
Me2CO-d6)
d
7.98 (d, J ¼ 16.0 Hz, 1H), 7.47 (d, J ¼ 8.1 Hz, 2H), 7.20 (d,
5.1.5.14. (E)-2-(3-Fluorostyryl)-6-hydroxy-4-methoxybenzoic
acid
J ¼ 8.0 Hz, 2H), 6.99 (d, J ¼ 16.0 Hz,1H), 6.86 (s, 1H), 5.22 (dd, J ¼ 7.9,
(6c). Yield 93%; 1H NMR (300 MHz, Me2CO-d6)
d
12.15 (s, 1H), 8.02
6.6 Hz, 1H), 3.99 (s, 3H), 3.36 (d, J ¼ 7.2 Hz, 2H), 2.34 (s, 3H), 1.78 (s,
(d, J ¼ 16.0 Hz, 1H), 7.43 (m,2H), 7.40e7.29 (m, 1H), 7.11e7.03 (m,
3H), 1.65 (s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d 173.4, 162.1, 161.5,
1H), 6.99 (d, J ¼ 16.0 Hz, 1H), 6.75 (d, J ¼ 2.5 Hz, 1H), 6.48 (d,
141.1, 137.4, 135.1, 130.6, 130.3,129.3,126.6,122.4,115.8,103.9,102.4,
55.3, 25.0, 21.8, 20.3,17.0; ESI-LRMS m/z: 375.3 [MþNa]þ. ESI-HRMS
m/z: 353.1762 [MþH]þ, calcd for C22H25O4 353.1747.
J ¼ 2.6 Hz, 1H), 3.91 (s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d 172.7,
165.9, 164.8, 164.5, 161.6, 142.8, 140.3, 140.2, 131.3, 130.5, 130.4,
129.4, 122.9, 122.8, 114.4, 114.1, 112.9, 112.6, 107.2, 103.6, 100.4, 55.1;
ESI-LRMS m/z: 289.3 [MþH]þ. ESI-HRMS m/z: 289.0873 [MþH]þ,
calcd for C17H15FO4 289.0871.
5.1.5.8. (E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-
(3,4,5-trimethoxystyryl)benzoic acid (5h). Yield 95%; 1H NMR
(300 MHz, Me2CO-d6)
d
12.32 (s, 1H), 7.94 (d, J ¼ 16.0 Hz, 1H), 6.96
5.1.5.15. (E)-2-Hydroxy-4-methoxy-6-(4-methoxystyryl)benzoic acid
(d, J ¼ 16.1 Hz, 1H), 6.89 (s, 2H), 6.84 (s, 1H), 5.22 (t, J ¼ 7.2 Hz, 1H),
(6f). Yield 95%; 1H NMR (300 MHz, Me2CO-d6)
d 12.01 (s, 1H), 7.85
3.99 (s, 3H), 3.87 (s, 6H), 3.75 (s, 3H), 3.36 (d, J ¼ 7.3 Hz, 2H), 1.78 (s,
(d, J ¼ 16.1 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H), 6.96 (m, 3H), 6.74 (d,
3H), 1.65 (s, 3H). 13C NMR (75 MHz, Me2CO-d6)
d
173.4, 162.0, 161.5,
J ¼ 2.5 Hz, 1H), 6.43 (d, J ¼ 2.6 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H). 13
C
153.7, 141.0, 138.4, 133.5, 130.7, 130.4, 129.8, 122.3, 115.8, 104.2,
102.3, 59.7, 55.5, 55.3, 25.0, 21.8,17.0; ESI-LRMS m/z: 429.4 [MþH]þ;
ESI-HRMS m/z: 429.1879 [MþH]þ, calcd for C24H29O7 429.1908.
NMR (75 MHz, Me2CO-d6) d 173.0, 165.8, 164.4, 159.7, 143.7, 130.5,
130.3,128.0,127.3,114.1,106.6,103.5, 99.9, 55.1, 54.7; ESI-LRMS m/z:
301.3 [MþH]þ. ESI-HRMS m/z: 301.1072 [MþH]þ, calcd for C17H16O5
301.1071.
5.1.5.9. (E)-6-(3-Cyanostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoic acid (5i). Yield 91%; 1H NMR
5.2. Biological assays
(300 MHz, Me2CO-d6)
d
8.16 (d, J ¼ 16.1 Hz, 1H), 8.05e7.85 (m, 3H),
7.65 (m, 2H), 7.06 (d, J ¼ 16.1 Hz, 1H), 6.88 (s, 1H), 5.22 (m, 1H), 3.99
5.2.1. In vitro antibacterial activity evaluation by MIC assay
The MIC values were determined using a broth dilution method
[19]. The starting concentration of the compounds tested was
(s, 3H), 3.36 (d, J ¼ 7.2 Hz, 3H), 1.78 (s, 3H), 1.65 (s, 3H). 13C NMR
(75 MHz, Me2CO-d6)
d 173.2, 162.0, 161.5, 140.2, 139.2, 133.0, 130.8,
130.7, 130.0, 129.9, 127.9, 122.2, 118.4, 116.5, 112.8, 104.3, 102.7, 55.3,
25.0, 21.8, 17.0; ESI-LRMS m/z: 364.3 [MþH]þ. ESI-HRMS m/z:
364.1539 [MþH]þ, calcd for C22H22NO4 364.1543.
256
added to 285
mg/mL. The solution of the compound in DMSO (15 mL) was
m
L of bacterial culture (5 ꢃ 105 cells/mL) at the first
well of a flat-bottomed 96-well tissue culture plate. The solution
was then double diluted. Bacterial culture solution containing
5.1.5.10. (E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-(4-
appropriate compound (150
mL) was discarded at the last well in
(trifluoromethyl)styryl)benzoic acid (5j). Yield 93%; 1H NMR
order to ensure 150 L volume of bacterial culture in every well. The
m
(300 MHz, CDCl3)
d
11.51 (s, 1H), 7.92 (d, J ¼ 16.0 Hz, 1H), 7.74e7.56
plates were incubated at 37 ꢁC for 24 h in electro-heating standing-
temperature cultivator and were read visually. The minimum
concentration of the sample showing no turbidity was recorded as
the MIC.
(m, 4H), 6.83 (d, J ¼ 15.9 Hz, 1H), 6.64 (s, 1H), 5.20 (t, J ¼ 7.1 Hz, 1H),
3.96 (s, 3H), 3.38 (d, J ¼ 7.1 Hz, 2H),1.79 (s, 3H),1.69 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 175.0, 162.5, 162.3, 141.1, 132.9, 132.1, 129.2, 126.9,
125.7, 121.7, 117.4, 103.5, 103.0, 55.8, 25.8, 22.1, 17.8; ESI-LRMS m/z:
429.3 [MþNa]þ. ESI-HRMS m/z: 407.1442 [MþH]þ, calcd for
5.2.2. Cytotoxicity assay
C
22H22F3O4 407.1465.
The active compounds (5b, 5c, 5j and 5k) were further exam-
ined for cytotoxicity in mouse macrophage cell lines (RAW 264.7) at
concentration gradients [20]. The cells were cultured in DMEM
5.1.5.11. (E)-6-(2,4-Difluorostyryl)-2-hydroxy-4-methoxy-3-(3-
methylbut-2-en-1-yl)benzoic acid (5k). Yield 92%; 1H NMR
medium supplemented with 10% fetal bovine serum and 2 mM L-
(300 MHz, Me2CO-d6)
d
8.05 (d, J ¼ 16.2 Hz, 1H), 7.85e7.71 (m, 1H),
glutamine and 100 units/mL penicillin/streptomycin. Cell cultures
were maintained in a humidified atmosphere of 5% CO2 at 37 ꢁC.
Cells were passaged at preconfluent densities using a solution
containing 0.25% trypsin and 0.5 mM EDTA. Cells were seeded at a
7.13e7.06 (m, 2H), 7.02 (d, J ¼ 16.1 Hz, 1H), 6.85 (s, 1H), 5.41e5.08
(m, 1H), 4.00 (s, 3H), 3.36 (d, J ¼ 7.2 Hz, 2H), 1.78 (s, 3H), 1.65 (s, 3H).
13C NMR (75 MHz, Me2CO-d6)
d 205.3, 173.2, 162.1, 161.6, 140.3,

Z.-Z. Geng et al. / European Journal of Medicinal Chemistry 100 (2015) 235e245
245
Cajaninstilbene acid (CSA) exerts cytoprotective effects against oxidative
stress through the Nrf 2-dependent antioxidant pathway, Toxicol. Lett. 219
(2013) 254e261.
density of 10,000 cell/well in a 96 well plate for 12 h with 10% FBS,
1% penicillin and streptomycin, and 1% -glutamine resulting in 80%
L
confluency. Each dose was prepared in 1% FBS medium by
1000 ꢃ dilution of the drug which was prepared in DMSO solution
to ensure DMSO concentration less than 0.1%. Control experiments
showed that 0.1% DMSO had no effect on cytotoxicity. After 72 h of
exposure, viability was assessed on the basis of cellular conversion
of MTT into a formazan product with DMSO for 45 min. Absorbance
was detected by a Gen5 Reader at 562 nm. The experiment was
performed in 4 replicate wells for each compound or concentration
with at least three experimental runs (N ¼ 12).
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We gratefully acknowledge support for this research from the
Natural
Science
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of
Guangdong
Province
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