Structural optimization of caffeoyl salicylate scaffold as NO production inhibitors

Article abstract of DOI:10.1248/cpb.c19-00366

Chlorogenic acid (CGA) has been considered as one of important active components in a number of medicinal herbs. Recently our group demonstrated that caffeoyl salicylate scaffold derived from CGA can be employed for the development of novel anti-inflammatory agents. The most active compound D104 can be a very promising starting point for the further structural optimization. A series of novel caffeoyl salicylate analogs were designed, synthesized, and evaluated by preliminary biological evaluation. The obtained results showed that the two compounds B12 and B13 can not only inhibit production of nitric oxide (NO) in RAW264.7 cells induced by lipopolysaccharides (LPS) effectively, but also have high safety in in vitro cytotoxic test, which could be comparable with D104. Molecular docking study on the peroxisome proliferator-activated receptor γ (PPARγ) protein revealed that compounds B12 and B13 can follow the same binding mode with D104, and the carboxyl group of caffeoyl salicylate scaffold might play a key role in the interaction with protein target, which implied the carboxyl group should be retained in the further optimization.

Full text of DOI:10.1248/cpb.c19-00366

1
006
Chem. Pharm. Bull. 67, 1006–1014 (2019)
Vol. 67, No. 9
Regular Article
Structural Optimization of Caffeoyl Salicylate Scaffold as NO
Production Inhibitors
a,b,#
a,b,#
,a,b
c
c
Pan Yu,
Chao-Jie Xia,
Dong-Dong Li,* Zhenzhong Wang, Wei Xiao, and
a,b
Lin-Guo Zhao
a
ꢀ
JiangsuꢀCo-InnovationꢀCenterꢀofꢀEfficientꢀProcessingꢀandꢀUtilizationꢀofꢀForestꢀResources,ꢀNanjingꢀForestryꢀ
b
University;ꢀNanjingꢀ210037,ꢀPeople’sꢀRepublicꢀofꢀChina:ꢀ ꢀCollegeꢀofꢀChemicalꢀEngineering,ꢀNanjingꢀForestryꢀ
University;ꢀ159ꢀLongꢀPanꢀRoad,ꢀNanjingꢀ210037,ꢀPeople’sꢀRepublicꢀofꢀChina:ꢀandꢀ Jiangsu Kanion Pharmaceutical
c
Co.,ꢀLtd.;ꢀ58ꢀHaichangꢀSouthꢀRoad,ꢀLianyungangꢀ222001,ꢀJiangsuꢀProvince,ꢀPeople’sꢀRepublicꢀofꢀChina.
Received May 6, 2019; accepted June 17, 2019
Chlorogenic acid (CGA) has been considered as one of important active components in a number of
medicinal herbs. Recently our group demonstrated that caffeoyl salicylate scaffold derived from CGA can
be employed for the development of novel anti-inflammatory agents. The most active compound D104 can
be a very promising starting point for the further structural optimization. A series of novel caffeoyl salicy-
late analogs were designed, synthesized, and evaluated by preliminary biological evaluation. The obtained
results showed that the two compounds B12 and B13 can not only inhibit production of nitric oxide (NO) in
RAW264.7 cells induced by lipopolysaccharides (LPS) effectively, but also have high safety in in vitro cyto-
toxic test, which could be comparable with D104. Molecular docking study on the peroxisome proliferator-
activated receptor γ (PPARγ) protein revealed that compounds B12 and B13 can follow the same binding
mode with D104, and the carboxyl group of caffeoyl salicylate scaffold might play a key role in the interac-
tion with protein target, which implied the carboxyl group should be retained in the further optimization.
Key words chlorogenic acid; caffeoyl salicylate scaffold; anti-inflammatory
Introduction
stituents including 3,5-, 5-, 3-bromine-5-chlorine, 3-ethoxy,
Chlorogenic acid (CGA, Fig. 1) is one of the most available and 4-methoxy, were directly introduced into the B ring of
active substances widely distributed in not only lots of Chi- the scaffold. Given the appropriate size of oxygen-containing
nese herbal medicine but also in various fruits and vegetables. ring adjacent to the A ring part, the 5-, 6-, and 7-member
It has been extensively demonstrated for wide-ranging physi- oxygen-containing ring structures were better to be retained
1)
2)
ological activities such as anti-obesity, anti-diabetic, anti- in the new molecules. As presented in Table 1, thirteen novel
3)
4)
hypertension, antimicrobial, and anti-inflammatory activi- caffeoyl salicylate analogs in all were designed, synthesized,
5
–7)
ties.
Analyzing from the structure, CGA is the simple ester and evaluated by the corresponding biological assay. Accord-
of caffeic acid with quinic acid, two common natural metabo- ingly, the SARs of the scaffold would be further discussed on
lites, which suggests that it has strong potential for chemical analysis of these compounds, as well as compounds reported
8)
modification. CGA could be regarded as a very promising in the previous research.
privileged structure for drug discovery.
As shown in Fig. 1, we described that the most anti-inflam- Results and Discussion
matory activity compound D104 was screened by a series of
Chemistry According to Chart 1, 3,4-dihydroxybenzal-
caffeoyl salicylate analogs designed by scaffold hopping meth- dehyde 1 was mixed with the corresponding dibromoalkane
od based on the chemical structure of CGA and caffeic acid and K CO in acetone or N,N-dimethylformamide (DMF) to
2
3
8)
phenethyl ester (CAPE) in the previous study. The structure– obtain compounds 2a–2c as the major intermediates. Subse-
activity relationships (SARs) of these compounds presents quently, the cyclized cinnamic acids (3a–3c) were prepared by
that: A) compounds with 5- or 6-member oxygen-containing the Knoevenagel condensation reaction, in which compounds
ring can exhibit superior potency to compounds with 7- or 2a–2c can react with malonic acid in the presence of pyridine
8
-member oxygen-containing ring; B) electron withdrawing and piperidine, respectively. After the three cinnamic acids
groups of B ring contributed more to anti-inflammatory activi- were converted to the corresponding acyl chlorides 4a–4c,
ty than electron donating groups or no substituent; C) caffeoyl the solution of acyl chloride 4a–4c in CH Cl or ethyl acetate
2
2
salicylaldehydes generally have potent cytotoxicity, compared (EtOAc) at ice-bath under an inert atmosphere (N ) were
2
to caffeoyl salicylates. Nevertheless, the kinds, quantity and added into selected salicylic aldehydes for the synthesis of
position of substituents seem to be kind of limited. The im- the required caffeoyl salicylaldehydes. The aldehyde group of
provement for anti-inflammatory activity based on the scaffold compounds A1–A13 can be oxidized to the acid group by the
still has great potential.
Pinnick oxidation reaction: the caffeoyl salicylates B1–B13
In order to further investigate the anti-inflammatory activity (Table 1) were prepared by the oxidation of compounds
of the caffeoyl salicylate scaffold, five different types of sub- A1–A13 under sodium chlorite (NaClO ) in tert-butyl alcohol
2
or tetrahydrofuran (THF). The chemical structures of all new
caffeoyl salicylates were characterized by H-NMR spectra
#
1
These authors contributed equally to this work.
To whom correspondence should be addressed. e-mail: lidon@njfu.edu.cn
*
©
2019 The Pharmaceutical Society of Japan

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
1007
Fig. 1. The Design Workflow of New Caffeoyl Salicylate Analogs
Table 1. Chemical Structure of Caffeoyl Salicylate Analogs
1
2
3
1
2
3
Cmpd
n
R
R
R
Cmpd
n
R
R
R
A1
A2
1
2
3
1
2
3
1
2
3
1
2
1
2
Br
Br
Br
H
H
Br
Br
Br
Br
Br
Br
H
B1
B2
1
2
3
1
2
3
1
2
3
1
2
1
2
Br
Br
Br
H
H
Br
Br
Br
Br
Br
Br
H
H
H
A3
H
B3
H
A4
H
B4
H
A5
H
H
H
B5
H
H
H
A6
H
B6
H
A7
H
–OCH3
–OCH3
–OCH3
H
B7
H
–OCH3
–OCH3
–OCH3
H
A8
H
H
B8
H
H
A9
H
H
B9
H
H
A10
A11
A12
A13
Br
Br
Cl
Cl
H
B10
B11
B12
B13
Br
Br
Cl
Cl
H
H
H
–OC H
H
–OC H
H
2
5
2
5
–OC H
H
H
–OC H
H
H
2
5
2
5
and general mass spectra (electrospray ionization (ESI)-MS). ability, given that the concentration of 50µM was very high
Besides, all the intermediates such as 2a–2c and 3a–3c had for toxicity test. As expected, the caffeoyl salicylaldehydes
8
)
been characterized in the previous study.
(A1–A13) were proved to have potent toxicity against A549
cells, probably due to the high membrane permeability of
aldehyde group of these compounds mentioned in the previ-
Biological Activity
8)
Cytotoxicity to A549 Cells and RAW264.7 Cells The ous study. Hence, these caffeoyl salicylates as well as D104
cytotoxic activities of caffeoyl salicylaldehydes (A1–A13) were selected for the further cytotoxicity evaluation against
and caffeoyl salicylates (B1–B13) were evaluated by the RAW264.7 cells at the corresponding concentration of 25 and
3
-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro- 50µM, respectively. Figure 3 demonstrated most caffeoyl sa-
9)
mide (MTT) method using adenocarcinomic human alveolar licylates exhibited no toxicity against RAW264.7 cells, except
basal epithelial cells A549 and macrophage cells RAW264.7, B7 and B10 that possess low cytotoxicities at high concentra-
respectively. As shown in Fig. 2, most of caffeoyl salicylates tion of 50µM. It is noteworthy that compound B3 has no tox-
exhibited no toxicity against A549 cells when comparing with icity against RAW264.7 cells but can inhibit A549 cells, which
blank control and two positive molecules (D104 and aspirin). implied that B3 might have potential antitumor activity.
Only compounds B3, B7, and B10 had a bit impact on cell vi-
Anti-inflammatory Activity Generally, the inhibition of

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Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
Reagents and conditions: (i) K CO , dibromoalkane, acetone or DMF, reflux, 4–5h; (ii) malonic acid, pyridine, piperidine, 80°C, 24h; (iii) SOCl , 80°C, reflux, 2h; (iv)
2
3
2
corresponding salicylic aldehyde, CH Cl2 or ethyl acetate, pyridine, N , ice-bath, overnight; (v) NaClO , NaH PO , 3-methyl-1-butene, pH 3–4, stirred at r.t., 5–6h.
2
2
2
2
4
Chart 1. Synthesis of the Caffeoyl Salicylates B1–B13
Fig. 2. Assay for Cytotoxicity to A549 Cells (Adenocarcinomic Human Alveolar Basal Epithelial Cells) in Vitro by Compounds A1–A13, B1–B13,
Aspirin, and D104 at the Concentration of 50µM for 72h
Most caffeoyl salicylaldehyde compounds (A1–A13) did significantly affect cell viability. Data was represented by the mean ± standard error (S.E.) of the three indepen-
dent experiments.
inducible nitric oxide synthase (iNOS) induced by lipopoly- ing compounds B2, B5, B8, B11 and B13 with others; Gener-
saccharide (LPS) can be measured using nitric oxide (NO) ally, the inhibition rate of 6-membered-ring derivatives was
release, which to some extent reflects anti-inflammatory ac- less than 5- and 7-membered-ring derivatives. (2) compounds
10)
tivities of these compounds. The anti-inflammatory activi- with one-substituent of B ring actually displayed more potent
ties of caffeoyl salicylaldehydes (A1–A13) and caffeoyl salicy- inhibitory activity than compounds with double substituents;
lates (B1–B13) at the concentration of 50µM can be clearly (3) When introducing one-substituent on B ring and compar-
presented in Fig. 4. In accordance with expectation, all the ing B4–B6 with B7–B9, B12, and B13, we could observe that
designed caffeoyl analogs exhibited high-potency inhibitory as follows: a) in terms of substituents positions, introducing
1
2
activity against NO production in LPS-induced RAW264.7 groups at the R and R position exhibited stronger activities
3
macrophage. However, with regard to caffeoyl salicylalde- than that at the R position; b) in consideration of the size of
hydes, the inhibition of NO release should attribute to the high functional groups, bigger substituents (methoxyl and ethyoxyl)
toxicity of caffeoyl salicylaldehydes with high membrane per- might be more beneficial than smaller substituents (bromine
meability of aldehyde group.
and chlorine); c) besides, electron property of substituents
As to caffeoyl salicylates (B1–B13), the SARs for inhibition could be hardly determined for inhibitory activity of caffeoyl
of NO production could be concluded into three points in the salicylates, and electron withdrawing groups such as chlorine
previous manuscript: the size of oxygen-containing ring, elec- was not necessarily the best.
tron property of substituents, and the number of substituents
In addition, ten caffeoyl salicylates except B5, B7, and
at the B ring (Fig. 1). Consequently, from analysis of the data B10 were investigated for their inhibitory activities of NO
presented in Fig. 4, it could be obtained as follows: (1) the size production under a decreasing concentration gradient of
of oxygen-containing ring adjacent to the A ring took obvious 100, 50, 25, 12.5, and 6.25µM. As shown in Fig. 5, most of
effect on inhibitory activity of NO production when compar- these compounds can exhibit significant inhibitory activities

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1009
Fig. 3. Assay for Cytotoxicity to RAW264.7 Cells in Vitro by Compounds B1–B13 and D104 at the Concentration of 25 and 50µM for 72h, Respec-
tively
Most caffeoyl salicylate compounds exhibit almost no toxicity except B7 and B10. Data was represented by the mean± S.D. of the three independent experiments.
Fig. 4. The Inhibition Rate for NO Production in RAW264.7 Cells Induced by LPS in Vitro of Compounds A1–A13 and B1–B13 at the Concentra-
tion of 50µM, as Well as the Three Positive Controls of Aspirin, Chlorogenic Acid (CGA), and D104
The inhibitory activities of most compounds against NO production were more potent in RAW264.7 when compared to Aspirin and CGA. Data was represented by the
mean± S.D. of the three independent experiments.
at high concentrations. Among these compounds, the in- salicylate scaffold.
hibitory effects of two compounds B12 and B13 against NO
Binding Mode Analysis Previous study demonstrated
production were more potent in RAW264.7 than the positive that the caffeoyl salicylate scaffold was obtained by vir-
11)
control D104, even if under low concentrations such as 12.5 tual screening using the Glide docking study on the basis
or 6.25µM. Interestingly, the other positive controls Aspirin of the peroxisome proliferator-activated receptor γ (PPARγ)
12)
and CGA can also reach a modest inhibition of NO produc- protein. Accordingly, docking study of the two most ac-
tion at low concentrations, albeit the inhibitory effect under tive compounds B12 and B13, together with D104 and CGA,
high concentrations seemed to not be obvious. In brief, Fig. were performed to identify the possible binding mode at the
5
presented that all the caffeoyl salicylates can inhibit NO active pocket of PPARγ protein (PDB ID: 3U9Q). The dock-
production with a dose-dependent manner, and the two most ing results can be provided in Fig. 6, which displayed that the
effective compounds were B12 and B13, respectively, both of three caffeoyl salicylates (D104, B12, and B13) can follow the
which have an ethoxyl at the 3-position of B ring of caffeoyl same binding mode in the pocket, although CGA adopted the

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Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
Conclusion
Recently our group demonstrated that caffeoyl salicylate
scaffold derived from CGA can be regarded as one promising
starting template for the development of novel anti-inflamma-
tory agents. Accordingly, on the basis of the primary SARs of
the scaffold, a series of novel caffeoyl salicylate analogs were
designed, synthesized, and evaluated by preliminary biological
evaluation. The obtained results showed that the two com-
pounds B12 and B13 can not only inhibit production of NO
in RAW264.7 cells induced by LPS effectively, but also have
high safety in in vitro cytotoxic test, which can be better than
D104. Molecular docking study on the PPARγ protein revealed
that compounds B12 and B13 can follow the same binding
mode with D104, and the carboxyl group of caffeoyl salicylate
scaffold might play a key role in the interaction with protein
target, which implied the carboxyl group should be retained in
the further optimization.
Experimental 1
Chemistry H spectra were recorded on Bruker AM
6
00MHz spectrometers with tetramethylsilane (TMS) as the
internal standard. ESI-MS and high-resolution (HR)-MS were
recorded by Agilent 6520B Q-TOF. Melting points (mp) were
recorded on SRS OptiMelt-100 full automatic micro melting
point instrument. Analytical HPLC was performed on a Agi-
lent 1260 HPLC system using a Chromolith SpeedROD RP-18
column (4.6×50mm). A linear gradient elution was performed
with eluent A (H O/TFA, 100:0.01) containing 0% of solvent
2
B (CH CN/H O/TFA, 90:10:0.01) rising to 100% of B during
3
2
2
0min with a flow rate of 1.0mL/min. Column chromatogra-
phy (CC): silica gel (200–300mesh; Qingdao Makall Group
Co., Ltd.; Qingdao; China). All reactions were monitored
using TLC on silica gel plates. Reaction reagents were analyti-
cal reagent grade and purchased from Aladdin.
Fig. 5. The Inhibition Rate for NO Production in RAW264.7 Cells
Induced by LPS in Vitro of Selected Caffeoyl Salicylates at the Concen-
tration Gradient of 100, 50, 25, 12.5, and 6.25µM, in Comparison with
Asprin, Chlorogenic Acid (CGA), and D104 as the Positive Controls
General Procedures for the Synthesis of Compounds
2
2
a–2c All the procedures and spectral data of compounds
8)
a–2c can refer to the previous paper.
General Procedures for the Synthesis of Compounds
A) Compounds B1–B4 as well as the tree controls above. B) Compounds B6, B8,
B9, B11–B13. Data was represented by the mean± S.D. of the three independent
experiments.
3
3
a–3c All the procedures and spectral data of compounds
8)
a–3c can refer to the previous paper.
General Procedures for the Synthesis of Compounds
reverse orientation completely. Comparing with each other 4a–4c All the procedures of compounds 4a–4c can refer to
8)
from Fig. 6A to D, the four molecules can insert into the cleft the previous paper.
located at the active pocket of PPARγ protein in a linear man- General Procedures for the Synthesis of Compounds
ner. Besides, the docking scores of B12 and B13 were both A1–A13 A solution of acyl chloride (12mM) in CH Cl or
2
2
better than that of D104.
ethyl acetate was added dropwise to corresponding salicylic
More importantly, the local pocket around the B ring of aldehyde (10mM) in CH Cl or ethyl acetate containing pyri-
2
2
caffeoyl salicylates seemed enough for providing the further dine (1.6mL, 20mM) under an inert (N ) atmosphere and at
2
optimization space of this scaffold. This is the reason why 0°C with constant stirring overnight. The reaction mixture
compounds with various substituents still exhibited potent in- was then poured in excess of diluted NaOH and extracted with
hibitory activity against NO production, unless the volume of CH Cl or ethyl acetate. The extraction liquid was purified
2
2
substituent was too large such as the N,N-diethylamine group by a flash chromatography with ethyl acetate/petroleum ether
8)
of compounds 6d, 6h, and 6l in the previous paper, which to give these compounds. The yields were between 40% and
may lead to the decrease of inhibitory activity. In addition, 60%.
the carboxyl group of B ring can form three hydrogen bonds
2,4-Dibromo-6-formylphenyl(E)-3-(benzo[d][1,3]dioxol-5-
with HIS323, TYR473 of the binding site, and ester group of yl)acrylate (A1)
−
compounds itself, respectively. Therefore, we can infer that
the carboxyl group of caffeoyl salicylate scaffold would play
Mp 240–242°C. MS (ESI): 450.89 (C H Br O , [M−H] ).
17 10 2 5
1
H-NMR (600MHz, dimethyl sulfoxide-d6 (DMSO-d )) δ:
6
a key role in the interaction with protein target, and should be 9.95 (s, 1H), 8.39 (d, J=2.4Hz, 1H), 8.12 (dd, J=3.5, 2.4Hz,
retained in the further optimization.
2H), 7.92 (d, J=2.5Hz, 1H), 7.88 (d, J=15.9Hz, 1H), 7.57 (d,
J=1.7Hz, 1H), 7.01 (d, J=8.0Hz, 1H), 6.85 (d, J=15.9Hz,

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
1011
Fig. 6. The Binding Poses of Small Molecules at the Active Pocket of the PPARγ Protein (PDB ID: 3U9Q)
A) Chlorogenic acid (CGA); B) compound D104; C) compound B12; D) compound B13.
1
1H), 6.12 (s, 2H). Purity by anal. HPLC: 96% (254nm).
H-NMR (600MHz, DMSO-d ) δ: 10.03 (s, 1H), 8.07 (d,
6
2
,4-Dibromo-6-formylphenyl(E)-3-(2,3-dihydrobenzo[b]- J=2.5Hz, 1H), 7.96 (dd, J=8.6, 2.6Hz, 1H), 7.80 (d,
1,4]dioxin-6-yl)acrylate (A2) J=15.9Hz, 1H), 7.41–7.38 (m, 2H), 7.33 (dd, J=8.4, 2.1Hz,
Mp 248–250°C. MS (ESI): 464.91 (C H Br O , [M−H] ). 1H), 6.94 (d, J=8.3Hz, 1H), 6.77 (d, J=16.0Hz, 1H), 4.31
[
−
18
12
2
5
1
H-NMR (600MHz, DMSO-d ) δ: 9.95 (s, 1H), 8.39 (d, (dd, J=5.5, 2.2Hz, 2H), 4.28 (dd, J=5.5, 2.2Hz, 2H). Purity
6
J=2.4Hz, 1H), 8.12 (d, J=2.4Hz, 1H), 7.85 (d, J=15.9Hz, by anal. HPLC: 97% (254nm).
1
H), 7.43 (d, J=2.1Hz, 1H), 7.35 (dd, J=8.4, 2.1Hz, 1H),
.94 (d, J=8.3Hz, 1H), 6.82 (d, J=15.9Hz, 1H), 4.32–4.30 [1,4]dioxepin-7-yl)acrylate (A6)
4-Bromo-2-formylphenyl(E)-3-(3,4-dihydro-2H-benzo[b]-
6
−
(m, 2H), 4.30–4.27 (m, 2H). Purity by anal. HPLC: 95%
Mp 199–201°C. MS (ESI): 401.01 (C H BrO , [M−H] ).
19
15
5
1
(254 nm).
H-NMR (600MHz, DMSO-d ) δ: 10.03 (s, 1H), 8.07 (d,
6
2
,4-Dibromo-6-formylphenyl(E)-3-(3,4-dihydro-2H- J=2.6Hz, 1H), 7.96 (dd, J=8.6, 2.6Hz, 1H), 7.82 (d,
benzo[b][1,4]dioxepin-7-yl)acrylate (A3)
J=16.0Hz, 1H), 7.47 (d, J=2.2Hz, 1H), 7.42 (dd, J=8.3,
−
Mp 256–258°C. MS (ESI): 478.92 (C H Br O , [M−H] ). 2.2Hz, 1H), 7.40 (d, J=8.6Hz, 1H), 7.02 (d, J=8.3Hz,
H-NMR (600MHz, DMSO-d ) δ: 9.95 (s, 1H), 8.38 (d, 1H), 6.80 (d, J=16.0Hz, 1H), 4.21 (t, J=5.6Hz, 2H), 4.18
J=2.4Hz, 1H), 8.12 (d, J=2.4Hz, 1H), 7.87 (d, J=16.0Hz, (t, J=5.6Hz, 2H), 2.14 (p, J=5.6Hz, 2H). Purity by anal.
19
14
2
5
1
6
1
7
H), 7.49 (d, J=2.2Hz, 1H), 7.45 (dd, J=8.4, 2.2Hz, 1H), HPLC: 97% (254nm).
.02 (d, J=8.3Hz, 1H), 6.85 (d, J=16.0Hz, 1H), 4.22 (t, 2-Formyl-5-methoxyphenyl(E)-3-(benzo[d][1,3]dioxol-5-yl)-
J=5.6Hz, 2H), 4.18 (t, J=5.6Hz, 2H), 2.14 (p, J=5.6Hz, acrylate (A7)
H). Purity by anal. HPLC: 95% (254nm).
-Bromo-2-formylphenyl(E)-3-(benzo[d][1,3]dioxol-5-yl)-
acrylate (A4)
−
2
Mp 156–158°C. MS (ESI): 325.08 (C H O , [M−H] ).
18 14 6
1
4
H-NMR (600MHz, DMSO-d ) δ: 9.92 (s, 1H), 7.88 (d,
6
J=8.7Hz, 1H), 7.81 (d, J=15.9Hz, 1H), 7.53 (d, J=1.7Hz,
−
Mp 182–184°C. MS (ESI): 372.98 (C H BrO , [M−H] ). 1H), 7.31 (dd, J=8.0, 1.7Hz, 1H), 7.06 (dd, J=8.7, 2.4Hz,
H-NMR (600MHz, DMSO-d ) δ: 10.03 (s, 1H), 8.07 (d, 1H), 7.00 (d, J=8.0Hz, 1H), 6.96 (d, J=2.4Hz, 1H), 6.79
17
11
5
1
6
J=2.5Hz, 1H), 7.96 (dd, J=8.6, 2.6Hz, 1H), 7.83 (d, (d, J=15.9Hz, 1H), 3.88 (s, 3H), 3.29 (s, 2H). Purity by anal.
J=15.9Hz, 1H), 7.54 (d, J=1.7Hz, 1H), 7.40 (d, J=8.6Hz, HPLC: 95% (254nm).
1
6
9
H), 7.32 (dd, J=8.1, 1.7Hz, 1H), 7.01 (d, J=8.0Hz, 1H),
.80 (d, J=15.9Hz, 1H), 6.12 (s, 2H). Purity by anal. HPLC: dioxin-6-yl)acrylate (A8)
7% (254nm).
-Bromo-2-formylphenyl(E)-3-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)acrylate (A5)
2-Formyl-5-methoxyphenyl(E)-3-(2,3-dihydrobenzo[b][1,4]-
−
Mp 164–166°C. MS (ESI): 339.09 (C H O , [M−H] ).
19
16
6
1
4
H-NMR (600MHz, DMSO-d ) δ: 9.92 (s, 1H), 7.87 (d,
6
J=8.6Hz, 1H), 7.78 (d, J=15.9Hz, 1H), 7.38 (s, 1H), 7.31 (d,
−
Mp 190–192°C. MS (ESI): 386.99 (C H BrO , [M−H] ). J=8.3Hz, 1H), 7.05 (d, J=8.6Hz, 1H), 6.98 (m, 2H), 6.75 (d,
18
13
5

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Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
J=16.0Hz, 1H),4.30 (t, 2H), 4.28 (t, J=5.4Hz, 2H), 3.88 (s, 1H), 7.36 (d, J=8.3Hz, 1H), 7.15 (d, J=16.0Hz, 1H), 4.57 (t,
H). Purity by anal. HPLC: 95% (254nm). J=5.5Hz, 2H), 4.54 (t, J=5.6Hz, 2H), 2.86 (s, 2H). Purity
-Formyl-5-methoxyphenyl(E)-3-(3,4-dihydro-2H-benzo[b]- by anal. HPLC: 96% (254nm).
1,4]dioxepin-7-yl)acrylate (A9) (E)-2-((3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-5-
3
2
[
−
Mp 171–173°C. MS (ESI): 353.11 (C H O , [M−H] ). bromobenzoic Acid (B4)
2
0
18
6
1
−
H-NMR (600MHz, DMSO-d ) δ: 9.92 (s, 1H), 7.87 (d,
Mp 220–222°C. MS (ESI): 388.97 (C H BrO , [M−H] ).
H-NMR (600MHz, DMSO-d ) δ: 8.02 (d, J=2.6Hz, 1H),
6
17 11
6
1
J=8.7Hz, 1H), 7.80 (d, J=16.0Hz, 1H), 7.45 (d, J=2.1Hz,
6
1
H), 7.42 (d, J=8.3Hz, 1H), 7.06 (dd, J=8.7, 2.4Hz, 1H), 7.85 (dd, J=8.5, 2.6Hz, 1H), 7.75 (d, J=15.9Hz, 1H), 7.53
.02 (d, J=8.3Hz, 1H), 6.96 (d, J=2.4Hz, 1H), 6.78 (d, (d, J=1.7Hz, 1H), 7.29 (dd, J=8.1, 1.7Hz, 1H), 7.27 (d,
J=16.0Hz, 1H), 4.23–4.20 (m, 2H), 4.19 (t, J=5.6Hz, 2H), J=8.6Hz, 1H), 6.99 (d, J=8.0Hz, 1H), 6.76 (d, J=15.9Hz,
.88 (s, 1H), 2.15 (t, J=5.6Hz, 2H). Purity by anal. HPLC: 1H), 6.11 (s, 2H). Purity by anal. HPLC: 96% (254nm).
7% (254nm). (E)-5-Bromo-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
-Bromo-4-chloro-6-formylphenyl(E)-3-(benzo[d][1,3]- acryloyl)oxy)benzoic Acid (B5)
7
3
9
2
−
dioxol-5-yl)acrylate (A10)
Mp 211–213°C. MS (ESI): 406.94 (C H BrClO , [M−H] ).
Mp 227–229°C. MS (ESI): 402.99 (C H BrO , [M−H] ).
1
8
13
6
−
1
H-NMR (600MHz, DMSO-d ) δ: 8.00 (s, 1H), 7.79 (d,
17
10
5
6
1
H-NMR (600MHz, DMSO-d ) δ: 9.97 (s, 1H), 8.28 (d, J=8.6Hz, 1H), 7.71 (d, J=15.9Hz, 1H), 7.35 (s, 1H), 7.29 (d,
6
J=2.5Hz, 1H), 8.00 (d, J=2.6Hz, 1H), 7.88 (d, J=15.9Hz, J=8.4Hz, 1H), 7.22 (d, J=8.7Hz, 1H), 6.91 (d, J=8.4Hz,
1
7
H), 7.56 (d, J=1.7Hz, 1H), 7.34 (dd, J=8.1, 1.7Hz, 1H), 1H), 6.69 (d, J=15.9Hz, 1H), 4.29 (d, J=13.9Hz, 4H). Purity
.01 (d, J=8.0Hz, 1H), 6.84 (d, J=15.9Hz, 1H), 6.12 (s, 2H). by anal. HPLC: 95% (254nm).
Purity by anal. HPLC: 95% (254nm). (E)-5-Bromo-2-((3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-
-Bromo-4-chloro-6-formylphenyl(E)-3-(2,3-dihydrobenzo- yl)acryloyl)oxy)benzoic Acid (B6)
2
−
[b][1,4]dioxin-6-yl)acrylate (A11)
Mp 235–237°C. MS (ESI): 417.01 (C H BrO , [M−H] ).
1
9
15
6
−
1
Mp 218–220°C. MS (ESI): 420.96 (C H BrClO , [M−H] ).
H-NMR (600MHz, DMSO-d ) δ: 8.38 (s, 1H), 8.21 (d,
18
12
5
6
1
H-NMR (600MHz, DMSO-d ) δ: 9.97 (s, 1H), 8.28 (d, J=7.8Hz, 1H), 8.10 (d, J=16.0Hz, 1H), 7.79 (s, 1H), 7.76 (d,
6
J=2.6Hz, 1H), 8.00 (d, J=2.6Hz, 1H), 7.85 (d, J=16.0Hz, J=8.4Hz, 1H), 7.62 (d, J=8.6Hz, 1H), 7.36 (d, J=8.3Hz,
1
6
4
H), 7.42 (d, J=2.1Hz, 1H), 7.35 (dd, J=8.4, 2.1Hz, 1H), 1H), 7.10 (d, J=16.0Hz, 1H), 4.56 (t, J=6.0Hz, 2H), 4.54
.94 (d, J=8.4Hz, 1H), 6.81 (d, J=15.9Hz, 1H), 4.32 (t, 2H), (t, J=5.3Hz, 2H), 2.86 (s, 2H). Purity by anal. HPLC: 95%
.29 (t, 2H). Purity by anal. HPLC: 95% (254nm).
General Procedures for the Synthesis of Compounds B1–
(254 nm).
(E)-2-((3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-4-
B13 A mixture of A1–A13 (0.15g, 0.5mmol) and tert-butyl methoxybenzoic Acid (B7)
−
alcohol (8mL) and THF (6mL) was stirred at room tempera-
ture until clear, and 3-methyl-1-butene (0.84mL, 10mM) that
Mp 193–195°C. MS (ESI): 341.07 (C H O , [M−H] ).
18 14 7
1
H-NMR (600MHz, DMSO-d ) δ: 7.89 (d, J=8.6Hz, 1H),
6
was cooled at 0°C was added. Sodium dihydrogen phosphate 7.72 (d, J=15.9Hz, 1H), 7.53 (d, J=1.7Hz, 1H), 7.51 (d,
0.6g, 5mM) and NaClO2 was dissolved in 2mL aqueous J=1.7Hz, 1H), 7.31 (dd, J=8.1, 1.7Hz, 2H), 7.06 (dd, J=8.7,
(
solution and acidified with hydrochloric acid aqueous solu- 2.5Hz, 1H), 7.00 (d, J=8.0Hz, 1H), 6.96 (d, J=2.5Hz,
tion (3mM) to pH 3–4. The solution was slowly added into 1H), 6.11 (s, 3H), 3.88 (s, 4H). Purity by anal. HPLC: 97%
the prepared aldehyde. Then the reaction mixture was stirred (254 nm).
at room temperature for 5–6h. The solvent was evaporated
away and purified by a flash chromatography in pleasing yield oxy)-4-methoxybenzoic Acid (B8)
Yield: 45–60%).
E)-2-((3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-3,5-
dibromobenzoic Acid (B1)
(E)-2-((3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acryloyl)-
−
(
Mp 201–203°C. MS (ESI): 355.09 (C H O , [M−H] ).
19 16 7
1
(
H-NMR (600MHz, DMSO-d ) δ: 12.56 (s, 1H), 7.91 (d,
6
J=8.8Hz, 1H), 7.69 (d, J=16.0Hz, 1H), 7.36 (d, J=2.1Hz,
−
Mp 226–228°C. MS (ESI): 466.88 (C H Br O , [M−H] ). 1H), 7.29 (dd, J=8.4, 2.1Hz, 1H), 6.96–6.90 (m, 2H), 6.83 (d,
17
10
2
6
1
H-NMR (600MHz, DMSO-d ) δ: 8.27 (d, J=2.4Hz, 1H), J=2.6Hz, 1H), 6.70 (d, J=16.0Hz, 1H), 4.31–4.29 (m, 2H),
6
8
(
.03 (d, J=2.4Hz, 1H), 7.78 (d, J=15.9Hz, 1H), 7.41 4.29–4.26 (m, 2H), 3.84 (s, 3H). Purity by anal. HPLC: 97%
d, J=2.1Hz, 1H), 7.33 (dd, J=8.4, 2.1Hz, 1H), 6.92 (d, (254 nm).
J=8.3Hz, 1H), 6.78 (d, J=15.9Hz, 1H), 4.30 (t, J=3.7Hz, (E)-2-((3-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)-
H). Purity by anal. HPLC: 97% (254nm). acryloyl)oxy)-4-methoxybenzoic Acid (B9)
E)-3,5-Dibromo-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6- Mp 209–211°C. MS (ESI): 369.11 (C H O , [M−H] ).
yl)acryloyl)oxy)benzoic Acid (B2)
Mp 234–236°C. MS (ESI): 480.90 (C H Br O , [M−H] ). J=8.8Hz, 1H), 7.71 (d, J=16.0Hz, 1H), 7.43 (d, J=2.2Hz,
2
−
(
2
0
18
7
1
H-NMR (600MHz, DMSO-d ) δ: 12.48 (s, 1H), 7.91 (d,
6
−
18
12
2
6
1
H-NMR (600MHz, DMSO-d ) δ: 8.25 (d, J=2.5Hz, 1H), 1H), 7.39 (dd, J=8.3, 2.2Hz, 1H), 7.00 (d, J=8.3Hz, 1H),
6
8
(
.03 (d, J=2.4Hz, 1H), 7.78 (d, J=16.0Hz, 1H), 7.40 6.95 (dd, J=8.8, 2.6Hz, 1H), 6.83 (d, J=2.5Hz, 1H), 6.73 (d,
d, J=2.1Hz, 1H), 7.33 (dd, J=8.4, 2.1Hz, 1H), 6.92 (d, J=16.0Hz, 1H), 4.19 (dt, J=15.0, 5.5Hz, 4H), 3.84 (s, 3H),
J=8.4Hz, 1H), 6.76 (d, J=15.9Hz, 1H), 4.34 –4.30 (m, 2H), 2.17–2.10 (m, 2H). Purity by anal. HPLC: 97% (254nm).
4.28–4.26 (m, 2H). Purity by anal. HPLC: 97% (254nm).
(E)-2-((3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-3-
(
E)-3,5-Dibromo-2-((3-(3,4-dihydro-2H-benzo[b][1,4]- bromo-5-chlorobenzoic Acid (B10)
−
dioxepin-7-yl)acryloyl)oxy)benzoic Acid (B3)
Mp 242–244°C. MS (ESI): 494.92 (C H Br O , [M−H] ).
H-NMR (600MHz, DMSO-d ) δ: 8.58 (s, 1H), 8.38 (s, 1H), J=2.6Hz, 1H), 7.82 (d, J=15.8Hz, 1H), 7.54 (s, 1H), 7.32 (d,
Mp 196–198°C. MS (ESI): 422.93 (C H BrClO , [M−H] ).
17
10
6
−
1
H-NMR (600MHz, DMSO-d ) δ: 8.15 (s, 1H), 7.91 (d,
19
14
2
6
6
1
6
8
.15 (d, J=16.0Hz, 1H), 7.82 (s, 1H), 7.78 (d, J=8.4Hz, J=8.0Hz, 1H), 6.99 (d, J=8.0Hz, 1H), 6.80 (d, J=15.9Hz,

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
1013
1
H), 6.11 (s, 2H). Purity by anal. HPLC: 96% (254nm).
4h. After carefully removing the medium, the precipitates
10min, and then absorbance values at a wavelength of 540nm
(
E)-3-Bromo-5-chloro-2-((3-(2,3-dihydrobenzo[b][1,4]- were dissolved in 200µL of DMSO, shaken mechanically for
dioxin-6-yl)acryloyl)oxy)benzoic Acid (B11)
−
Mp 204–206°C. MS (ESI): 436.95 (C H BrClO , [M−H] ). were taken on a SpectraMax 190 microplate reader (Molecular
18
12
6
1
H-NMR (600MHz, DMSO-d ) δ: 8.15 (s, 1H), 7.91 (d, Devices, U.S.A.). Survival ratios are expressed in percentages
6
J=2.6Hz, 1H), 7.78 (d, J=16.0Hz, 1H), 7.40 (d, J=2.0Hz, with respect to untreated cells.
1
6
2
H), 7.33 (dd, J=8.4, 2.1Hz, 1H), 6.92 (d, J=8.4Hz, 1H),
.77 (d, J=15.9Hz, 1H), 4.31–4.30 (m, 2H), 4.28–4.27 (m, (NO ), an indicator of NO synthase activity, in culture su-
Anti-inflammatory Assay Accumulation of nitrite
−
2
13)
H). Purity by anal. HPLC: 96% (254nm).
E)-2-((3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-3- Briefly, Cells (2×10 ) were seeded in 100µL of DMEM into
ethoxybenzoic Acid (B12) 96-well plates and co-incubated with different concentra-
Mp 205–207°C. MS (ESI): 355.09 (C H O , [M−H] ). tions (6.25, 12.5, 25, 50, 100µM) of compounds B1–B4, B6,
pernatant fluids was measured based on Griess reaction.
4
(
−
19
16
7
1
H-NMR (600MHz, DMSO-d ) δ: 12.88 (s, 1H), 7.73 B8, B9, B11–B13, D104, CGA, and Aspirin in the absence
6
(
dd, J=15.9, 2.0Hz, 1H), 7.52 (d, J=1.7Hz, 1H), 7.44 or presence of LPS (500ng/mL) for 48h. Meanwhile, chloro-
(d, J=7.7Hz, 1H), 7.35 (dd, J=8.3, 1.7Hz, 1H), 7.31 (d, genic acid and acetylsalicylic acid also were tested as positive
J=7.9Hz, 1H), 7.28 (dd, J=7.9, 1.8Hz, 1H), 6.98 (d, controls. Culture supernatant fluids were mixed with 100µL
J=8.0Hz, 1H), 6.76 (dd, J=15.9, 1.5Hz, 1H), 6.10 (s, 2H), Griess reagent at room temperature for 5min. Using NaNO to
2
4
.07 (q, J=7.0Hz, 2H), 1.26 (t, J=7.0Hz, 3H). Purity by generate a standard curve, nitrite production was measured by
anal. HPLC: 97% (254nm).
E)-2-((3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acryloyl)-
oxy)-3-ethoxybenzoic Acid (B13)
Mp 212–214°C. MS (ESI): 369.11 (C H O , [M−H] ).
an absorbance reading at 540nm.
(
Molecular Docking Study
Compounds B12, B13, D104, and CGA were imported
−
2
0
18
7
1
H-NMR (600MHz, DMSO-d ) δ: 12.90 (s, 1H), 7.70 (d, into the LigPreb module of the Schrodinger 2015 suite. The
6
J=16.0Hz, 1H), 7.44 (dd, J=7.7, 1.6Hz, 1H), 7.37 (d, PPARγ protein crystal complex (PDB ID: 3U9Q) had been
14)
J=2.1Hz, 1H), 7.35 (d, J=6.7Hz, 1H), 7.32–7.29 (m, 2H), downloaded from the PDB website, and prepared in Protein
.92 (d, J=8.3Hz, 1H), 6.73 (d, J=16.0Hz, 1H), 4.30–4.29 Preparation Wizard. Subsequently, four prepared small mol-
6
(
m, 2H), 4.28–4.27 (m, 2H), 1.25 (t, J=6.9Hz, 3H). Purity by ecules, together with PPARγ protein, were imported into the
anal. HPLC: 97% (254nm).
GLIDE module integrated in the Schrodinger 2015 suite. The
docking study was performed at the GLIDE standard precision
(SP) mode. All the docking parameter was set default values.
Biological Evaluation
Reagents and Cell Culture RAW264.7 cells and A549
cells were obtained from State Key Laboratory of Medicine,
Acknowledgments This work was supported by the
Nanjing University. LPS, MTT, and Griess reagent (1% sulfa- Natural Science Foundation of Jiangsu Higher Education
nilamide, 0.1% naphthylethylenediamine dihydrochloride, and Institutions (Grant number: 18KJB350004), the Project for
2
9
% phosphoric acid) were purchased from Sigma. The 6- and Young Teachers of Nanjing Forestry University (Grant num-
6-well plates were purchased from Beyotime Biotechnology. ber: CX2017005), and the Doctorate Fellowship Foundation of
RAW264.7 cells and A549 cells were grown in High glu- Nanjing Forestry University (Grant number: 163030748).
cose Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), 100U/mL peni-
Conflict of Interest The authors declare no conflict of
cillin, and 100µg/mL streptomycin and propagated at 37°C in interest.
a humidified atmosphere containing 5% CO in air.
2
Compounds A1–A13, B1–B13, D104, CGA, and acetyl-
Supplementary Materials The online version of this ar-
salicylic acid (Aspirin) were dissolved in dimethyl sulfoxide ticle contains supplementary materials.
to make stock solutions, respectively, and kept at −20°C.
The final concentration of the vehicle in the solution never References
exceeded 0.1% and had no effects on NO production and cell
viability.
1) Santana-Gálvez J., Cisneros-Zevallos L., Jacobo-Velázquez D. A.,
Molecules, 22, 358 (2017).
2)
Tajik N., Tajik M., Mack I., Enck P., Eur.ꢀ J.ꢀ Nutr., 56, 2215–2244
Assay for Cytotoxic Activity Compounds A1–A13,
B1–B13, and D104 were assayed according to the MTT
(2017).
9)
3) Naveed M., Hejazi V., Abbas M., Kamboh A. A., Khan G. J.,
Shumzaid M., Ahmad F., Babazadeh D., FangFang X., Modarresi-
Ghazani F., WenHua L., XiaoHui Z., Biomed.ꢀ Pharmacother., 97,
test under the concentration of 50µM against A549 cells.
Moreover, compounds B1–B13 and D104 were also chosen
to test the cytotoxic effects against RAW264.7 cells under
the two concentrations (25 and 50µM). MTT was dissolved
at 4mg/mL in phosphate buffered saline (PBS) and used es-
sentially as previously described. Briefly, cell lines in loga-
67–74 (2018).
4)
Ren S., Wu M., Guo J., Zhang W., Liu X., Sun L., Holyst R., Hou
S., Fang Y., Feng X., Sci.ꢀRep.ꢀUK, 5, 10464 (2015).
5) Shin H. S., Satsu H., Bae M.-J., Zhao Z., Ogiwara H., Totsuka M.,
3
rithmic phase were seeded at a density of 3×10 cells/well in
Shimizu M., FoodꢀChem., 168, 167–175 (2015).
6
7
)
)
Tsang M. S. M., Jiao D., Chan B. C. L., Hon K.-L., Leung P. C.,
Lau C. B. S., Wong E. C. W., Cheng L., Chan C. K. M., Lam C. W.
K., Wong C. K., Molecules, 21, 519 (2016).
Hwang S. J., Jun S. H., Park Y., Cha S.-H., Yoon M., Cho S., Lee
H.-J., Park Y., NBM, 11, 1677–1688 (2015).
100 µL of DMEM into 96-well microtiter plates. After 24h,
exponentially growing cells were exposed to the indicated
compounds at various concentrations. After 48h in final vol-
umes of 200µL, cell survival was determined by the addition
of an MTT solution (20µL of 4mg/mL MTT in PBS) for

1
014
Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
8
)
Yu P., Xia C.-J., Li D.-D., Ni J.-J., Zhao L.-G., Ding G., Wang Z.-Z.,
Xiao W., Fitoterapia, 129, 25–33 (2018).
Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D., Mitchell
J. B., CancerꢀRes., 47, 943–946 (1987).
(2004).
12) Rosen E. D., Sarraf P., Troy A. E., Bradwin G., Moore K., Milstone
D. S., Spiegelman B. M., Mortensen R. M., Mol. Cell, 4, 611–617
(1999).
9)
1
0) Gurkan A. S., Karabay A. Z., Buyukbingol Z., Buyukbingol E., Eur.
13) Han H.-E., Kim T.-K., Son H.-J., Park W. J., Han P.-L., Biomol.
Ther., 21, 21–28 (2013).
J.ꢀMed.ꢀChem., 46, 468–479 (2011).
1
1) Friesner R. A., Banks J. L., Murphy R. B., Halgren T. A., Klicic J.
J., Mainz D. T., Repasky M. P., Knoll E. H., Shelley M., Perry J. K.,
Shaw D. E., Francis P., Shenkin P. S., J.ꢀMed.ꢀChem., 47, 1739–1749
14) Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F. Jr., Brice
M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M., J.
Mol. Biol., 112, 535–542 (1977).