Design, synthesis, and anti-inflammatory activity of caffeoyl salicylate analogs as NO production inhibitors

Article abstract of DOI:10.1016/j.fitote.2018.05.029

Chlorogenic acid (CGA) has been reported to exhibit potent anti-inflammatory activity. However, the development of anti-inflammatory agent based on CGA has not been investigated. In this paper, a series of caffeoyl salicylate compounds derived from CGA were designed, synthesized, and evaluated by LPS-induced nitric oxide synthase inhibition and QRT-PCR technique. Most compounds showed modest activity to inhibit production of nitric oxide (NO) in RAW 264.7 cells induced by lipopolysaccharides (LPS). Among these compounds, QRT-PCR and western blotting results indicated that compounds 6b, 6c, 6f, 6g and D104 that possess 5-member ring or 6-member ring caused a significant inhibition against expression of the iNOS2 in LPS-induced macrophages. In addition, cytotoxic assay displayed most derivatives have good safety in vitro. This new promising scaffold could be further exploited for the development of anti-inflammatory agent in the future.

Full text of DOI:10.1016/j.fitote.2018.05.029

Fitoterapia129(2018)25–33
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Design, synthesis, and anti-inflammatory activity of caffeoyl salicylate
analogs as NO production inhibitors
Pan Yua,1, Chao-Jie Xiaa,1, Dong-Dong Lia, Jun-Jun Nia, Lin-Guo Zhaoa,⁎, Gang Dingb,
Zhen-Zhong Wangb, Wei Xiaob,⁎
a
College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China
Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang 222001, Jiangsu, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chlorogenic acid (CGA) has been reported to exhibit potent anti-inflammatory activity. However, the devel-
opment of anti-inflammatory agent based on CGA has not been investigated. In this paper, a series of caffeoyl
salicylate compounds derived from CGA were designed, synthesized, and evaluated by LPS-induced nitric oxide
synthase inhibition and QRT-PCR technique. Most compounds showed modest activity to inhibit production of
nitric oxide (NO) in RAW 264.7 cells induced by lipopolysaccharides (LPS). Among these compounds, QRT-PCR
and western blotting results indicated that compounds 6b, 6c, 6f, 6g and D104 that possess 5-member ring or 6-
member ring caused a significant inhibition against expression of the iNOS2 in LPS-induced macrophages. In
addition, cytotoxic assay displayed most derivatives have good safety in vitro. This new promising scaffold could
be further exploited for the development of anti-inflammatory agent in the future.
Chlorogenic acid
Anti-inflammatory
Caffeoyl salicylate analogs
Nitric oxide synthase
1. Introduction
κB. For this reason, we could attempt to design novel caffeoyl salicylate
analogs as anti-inflammatory agent based on these two targets.
Chlorogenic acid (5-O-caffeoylquinic acid, CGA) is a phenolic
compound widely distributed in plants, which has been extensively
studied since it has been reported to display physiological activities
such as anti-inflammatory [1], anti-oxidant [2], anticancer [3], antili-
pidemic [4], and anti-neurodegenerative activities [5]. Moreover,
chlorogenic acid exhibits good activity against a wide range of micro-
organisms, and this property can be used for the food industry as a food
additive. Therefore, chlorogenic acid could be regarded as a very pro-
mising starting point for the drug development.
Chlorogenic acid can be retrieved from the three bioactivity databases
CHEMBL [6], PubChem [7], and BindingDB [8], respectively. In these small
molecule databases, several drug targets such as aldose reductase, HIV type 1
integrase and 1,3-beta-glucan synthase, can be involved with various bio-
logical activities reported by many relevant references. Among these targets,
only two molecular targets peroxisome proliferator-activated receptor
gamma (PPARγ) and nuclear factor kappa-light-chain-enhancer of activated
B cells (NF-κB) are closely related to the anti-inflammatory activity of CGA
[9]. About fifty-seven anti-inflammatory molecules extracted from Selleck
Chemicals website [10] are collected and curated carefully, in which three
compounds are similar structurally with CGA (Fig. 1). It is worth noting that
anti-inflammatory targets of these three compounds are also PPARγ and NF-
In this study, about 1300 kinds of drug-like fragments named
Enamine_golden_fragment_library_sdf are downloaded from the
Enamine website [11]. We would plan to select appropriate moiety in
replacement of quinic acid group, and there are about twenty drug-like
fragments screened by being docked into the PPARγ and NF-κB pro-
teins. Subsequently, two hundred novel small molecules are carefully
designed by the replacement of quinic acid portion of CGA using these
fragments. Among these compounds, one caffeic acid phenethyl ester
(CAPE) analog (compound D104) can be obtained that it was the pre-
cursor to CGA and CAPE, and Glide docking [12] screening (Fig. 2)
reveals that compound D104 would bind more tightly with PPARγ and
NF-κB targets, than CGA or CAPE. Based on this, we sought to discover
novel anti-inflammatory agent by modifying the promising scaffold
using common chemicals in our lab.
2. Results and discussion
2.1. Chemistry
The intermediates (3a-3d) were prepared in the same way ac-
cording to Scheme 1. The 3,4-dihydroxybenzaldehyde 1 was treated
⁎
Corresponding authors.
E-mail addresses: lgzhao@njfu.edu.cn (L.-G. Zhao), xw@kanion.com (W. Xiao).
These authors equally contributed to this work.
1
https://doi.org/10.1016/j.fitote.2018.05.029
Received 4 March 2018; Received in revised form 24 May 2018; Accepted 27 May 2018
Availableonline28May2018
0367-326X/©2018ElsevierB.V.Allrightsreserved.

P. Yu et al.
Fitoterapia129(2018)25–33
OH
O
O
HO
OH
OH
O
(R)
(R)
(R)
HO
(S)
(E)
HO
HO
OH
HO
O
OH
OH
O
Gastrodin
CGA
HO
O
Scheme 1. Synthesis of the cyclized cinnamic acids 3a-3d. Reagents and con-
ditions: (i) K2CO3, dibromoalkane, acetone or DMF, reflux, 4–5 h, yield
50–60%; (ii) malonic acid, pyridine, piperidine, 80 °C, 24 h, yield 70–80%.
O
O
HO
HO
OH
OH
O
HO
OH
Salicin
Caffeic Acid
Ester
(CAPE)
Phenethyl
Fig. 1. Chlorogenic acid analogues in Selleck chemicals.
with the corresponding dibromoalkane and K2CO3 in acetone or N, N-
Dimethylformamide (DMF) to afford the compounds 2a-2d as the major
products. Subsequently, the cyclized cinnamic acids (3a-3d) were
synthesized by reacting 2a-2d with malonic acid in the presence of
pyridine and piperidine. The detailed mechanism of the reaction was
involved with the Knoevenagel condensation reaction. The next step is
that these cinnamic acids were converted to acyl chloride 4a-4d before
reacting with corresponding salicylic aldehyde in Scheme 2. Adding
various salicylic aldehydes into the solution of acyl chloride 4a-4d in
CH2Cl2 or ethyl acetate at ice-bath under an inert (N2) atmosphere
would produce the required esters 5a-5m (Table 1). The aldehyde
group of compounds 5a-5m was subsequently oxidized to acid group
according to the Pinnick oxidation reaction: the designed acids 6a-6m
(Table 1) were obtained by the oxidation of sodium chlorite (NaClO2) in
tert-butyl alcohol and tetrahydrofuran (THF) (Scheme 2). The chemical
structures of all new compounds were characterized by melting point
and 1H NMR spectra. Besides, most of the compounds were
Scheme 2. Synthesis of required acids 6a-6m. Reagents and conditions: (i)
SOCl2, 80 °C, reflux, 2 h, yield 93–95%; (ii) corresponding salicylic aldehyde,
CH2Cl2 or ethyl acetate, pyridine, N2, ice-bath, overnight, yield 40–60%; (iii)
NaClO2, NaH2PO4, 3-Methyl-1-butene, pH 3–4, stirred at rt., 5–6 h, yield
45–60%.
characterized by infra-red spectrum (IR spectrum). What is more,
compound 3b due to intermediate products and two compounds (5a
and 6e) as representative products of aldehydes and acids respectively
were selected to give 13C NMR spectra. In addition, a part of these
compounds was also characterized by general mass spectra (MS) and
high-resolution mass spectrum (HR-MS) (5a, 5f, 6a and 6f).
2.2. Pharmacological screening
2.2.1. Assay for cytotoxicity to RAW264.7
The cytotoxic activities of all these compounds were tested by the
OH
(R)
O
HO
O
HO
(R)
(R)
O
(E)
(S)
HO
HO
OH
O
OH
HO
O
CGA
ester
caffeic acid
phenethyl
(CAPE)
F
O
O
O
F
O
COOH
D104
O
R
O
n
O
O
COOH
new
scaffold
promising
GLIDE Docking (SP) (kcal/mol)
COMPOUND
PPAR
NF- (PDB ID: 4KIK)
D104
CGA
-9.058
-6.720
-7.005
-6.755
-6.970
-7.983
CAPE
Fig. 2. New promising scaffold derived from CGA.
26

P. Yu et al.
Fitoterapia129(2018)25–33
Table 1
Structures of caffeoyl salicylate analogs.
Compounds
n
R1
R2
R3
Compounds
n
R4
R5
R6
5a
5b
5c
5d
5e
5f
5g
5h
5i
1
1
1
1
2
2
2
2
3
3
3
3
4
H
H
Cl
H
H
H
Cl
H
H
Cl
H
H
H
H
H
H
H
6a
6b
6c
6d
6e
6f
6g
6h
6i
1
1
1
1
2
2
2
2
3
3
3
3
4
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
H
H
H
Cl
Cl
H
Cl
Cl
H
N(C2H5)2
H
H
H
N(C2H5)2
H
H
H
N(C2H5)2
H
H
H
N(C2H5)2
H
H
H
H
H
Cl
Cl
H
Cl
Cl
H
H
H
5j
5k
5l
Cl
Cl
H
6j
6k
6l
Cl
Cl
H
N(C2H5)2
H
N(C2H5)2
H
5m
Cl
6m
Cl
MTT method [13] using macrophage cell RAW264.7 cultured in DMEM
medium. The cell monolayer was incubated with caffeoyl salicylate
analogs (5a-5m, 6a-6m, pro-D104 and D104) at the concentration of
50 μM for 72 h, respectively. As shown in Fig. 3, it is obvious that most
of these compounds could not exhibit any toxicity when compared with
control and two positive molecules (CGA and aspirin). Only compounds
5b, 5c, and 5h did significantly affect cell viability. This main reason
could account much for toxicity of aldehyde group.
caffeoyl salicylate analogs are presented in Fig. 4. As shown above,
most of the caffeoyl salicylate analogs exhibited high-potency in-
hibitory activity against the nitrite production in LPS induced RAW
264.7 macrophage. Among these compounds, the inhibitory effects of
compounds 6b, 6c, 6f, 6g, 6k, 5b, 5c, 5d, 5g, 5h and D104 against NO
production were more potent in RAW264.7 than others. The structure-
activity relationships (SARs) for inhibition of nitric oxide synthase
could be demonstrated as follows: (1) compounds that possess 5-
member ring or 6-member ring (compounds 6a-6h) caused a more
significant inhibition of the iNOS2 expression in LPS-induced macro-
phages than compounds with 7-member ring or 8-member ring (com-
pounds 6i-6m); (2) the analogs with electron withdrawing groups such
as chlorine exhibited more effective inhibitory activity than the analogs
with electron donating groups or non-substituted compounds; the
compounds with electron donating groups and the molecular without
substituted exhibited similar potency with non-substituted compounds;
(3) compared to compounds with 5-member ring or 6-member ring,
compounds with one-substituent on benzene ring displayed more ef-
fective inhibitory activity than compounds with double substituents
(compounds 6c and 6g); (4) overall, the anti-inflammatory activity of
caffeoyl salicylaldehyde exhibited better than that of caffeoyl salicylate.
2.2.2. Anti-inflammatory activity
Lipopolysaccharide (LPS), found in the outer membrane of Gram-
negative bacteria, can elicit strong immune responses in animals. In
immune defense mechanism, the production of NO as a free radical
with an unpaired electron is primarily regulated by the inducible nitric
oxide synthase (iNOS), which is a key regulatory factor in immune
response, particularly inflammation, and binds calmodulin at physio-
logically relevant concentrations [14]. Therefore, the inhibition of iNOS
by these small molecules could to some extent reflect anti-inflammatory
activities of these compounds. The chemical structures of caffeoyl sal-
icylate analogs studied are listed in Table 1, and intuitively can be
classified into two main types including caffeoyl salicylaldehyde (5a-
5m) and caffeoyl salicylate (6a-6b) that contribute to follow-up struc-
ture-activity analysis. Meanwhile, the anti-inflammatory activities of
Fig. 4. The effect of inhibiting the production of NO in RAW 264.7 cells in-
duced by LPS in vitro of compounds 5a-5m, 6a-6m, pro-D104 and D104 were
compared with the controls of chlorogenic acid and acetylsalicylic acid. The
inhibitory effects of compounds 6b, 6c, 6f, 6g, 6k, 5b, 5c, 5d, 5g, 5h and D104
against NO production were more potent in RAW264.7 than others. Data was
Fig. 3. Assay for cytotoxicity to RAW264.7 cells in vitro by compounds 5a-5m,
6a-6m, pro-D104 and D104 at the concentration of 50 μM for 72 h. Only
compounds 5b, 5c, and 5h did significantly affect cell viability. Data was re-
presented by the mean
SD of the three independent experiments.
represented by the mean
SD of the three independent experiments.
27

P. Yu et al.
Fitoterapia129(2018)25–33
Based on this, we could surmise: (1) the potent inhibitory activity of
Table 3
Primer used in Real time-PCR assay.
compounds 6b, 6c, 6f, 6g and D104 would be closely related to stable
structure of 5-member ring and 6-member ring; (2) the contribution of
the inductive effect of the halogens to anti-inflammatory activity is
demonstrated by the differences in activity of compounds 6a and 6e
with chlorinated compounds 6b, 6c, 6f and 6g; (3) the N,N-diethyla-
mine group reduces activity due to steric effects and does not take into
consideration electronic factors; (4) the number of electron with-
drawing groups has also a modest effect on activity while comparing
compounds 6b, 6f with 6c, 6g; (5) although compounds 5b, 5c, 5d, 5g
and 5h displayed obvious inhibitory activity, the main reason could
account for toxicity of aldehyde group.
Gene
Primer sequence
Mus-β-actin
Mu-iNOS2
Forward 5′-GGTGTGATGGTGGGAATGGG-3′
Reverse 5′-ACGGTTGGCCTTAGGGTTCAG-3′
Forward 5′-CAGCTGGGCTGTACAAACCTT-3′
Reverse 5′-CATTGGAAGTGAAGCGTTTGG-3′
Seven compounds (6b, 6c, 6f, 6g, 5a, 5e and 5f) were subsequently
selected to investigate the IC50 values [15] of anti-inflammatory ac-
tivity against nitric oxide synthase. IC50 values can be calculated by
formula
through non-linear fitting in origin software. Cexperimental group CLPS and
control are nitric oxide mean concentration of experimental group, only
IC50 = 1 − [(Cexperimental
group – Ccontrol)/(CLPS – Ccontrol)]
C
LPS added group and only cell sap added group, respectively. The re-
sults were determined from replicates of 6 wells from at least three
independent experiments (Table 2). In accordance with the analysis
mentioned above, the most potent compounds are 6f and D104, and it
is obviously noted that the inhibitory activity of acid compounds (6b,
6c, 6f, and 6g) were superior to that of aldehyde compounds (5a, 5e,
and 5f).
2.2.3. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-
time PCR
Fig. 5. The LPS-stimulated macrophages were treated with compounds 6b, 6c,
6f 6g and D104, incubated for 24 h, following which RNA was isolated and
subjected to the RT-PCR analysis for the expression of iNOS2 mRNA.
Compounds 6b, 6c, 6f and 6g and D104 caused a significant reversal of the
iNOS2 inhibition in LPS-induced macrophages with expression values of 0.7297
to 28.43741 in percentage (%) based on a no-additions (NA) control. Data was
In order to explore anti-inflammatory activity at the mRNA mole-
cular level, we carried out mRNA microarray analyses and quantitative
reverse transcription-PCR (QRT-PCR) [16] on iNOS2 in this study. The
PCRs were amplified using β-actin as standard. Sequences of the PCR
primers are listed in Table 3. We chose three concentrations (10 μM,
20 μM and 40 μM) around IC50 value to test iNOS2 expression (Fig. 5).
The obtained results showed that the compounds 6b, 6c, 6f, 6g and
D104 caused a significant reversal of the iNOS2 inhibition in LPS-in-
duced macrophages with expression values of 0.7297 to 28.43741 in
percentage (%) based on a no-additions (NA) control. Among these
compounds, compounds 6f and D104 displayed the most significant
inhibitory activities. The inhibitory activities of 6f and D104 were
approximately 200-fold greater than the LPS group.
represented by the mean
SD of the three independent experiments.
the most potent derivative showing the strongest inhibitory effect on
iNOS protein expression. Different concentrations (10, 30, 100 μM) of
compounds 6f with or without LPS (500 ng/mL) were subsequently
selected to explain 6f work through the iNOS signaling pathway to
downregulate the cytokines. NO regulation of compounds 6b, 6c, 6f
and 6g might be attributed to the iNOS signaling pathway.
2.2.4. Western blotting analysis
3. Conclusions
Based on western blot analysis, we determined whether the NO
inhibitory effects of compounds 6b, 6c, 6f and 6g are related to the
regulation of the protein expression of iNOS. As shown in Fig. 6, pre-
treatment with 100 μM of the four compounds led to a decrease in iNOS
protein level. These results indicate that compounds 6b, 6c, 6f and 6g
exert attenuate LPS-induced pro-inflammatory responses in RAW 264.7
cells through down regulation of NO. Among them, compound 6f was
In summary, our design and synthesis have led to a series of caffeoyl
salicylate analogs derived from CGA. Bioassays showed that compounds
6b, 6c, 6f, 6g and D104 displayed significant activity to inhibit pro-
duction of nitric oxide (NO) in RAW 264.7 cells induced by lipopoly-
saccharides (LPS). Compounds 6b, 6c, 6f, 6g and D104 can therefore be
used as promising treatment candidate compounds for diseases related
with excess NO production. Structure-activity relationships are showed
as follows. (1) The anti NO synthase activity of compounds with 5-
member ring or 6-member ring is higher than that of the seven and
eight elements ring. (2) The electron-withdrawing inductive effect and
steric effect have great effect on anti-inflammatory activity. (3) The
amount and position of substitution groups must attract our attention.
(4) The assay for cytotoxic activity on RAW 264.7 cells demonstrate
that most of the caffeoyl salicylate analogs have good safety in vitro.
Based on this, compounds 6b, 6c, 6f, 6g and D104 will be studied in the
next step and we also give the relevant bioactivity evaluation to these
compounds. Further investigation is needed to determine mechanisms
underlying this effect and the most effective concentrations. What is
more, the compounds with different groups should be synthetized to
intensive study structure-activity relationships of caffeoyl salicylate
analogs derived from chlorogenic acid.
Table 2
Inhibitory effect on NO production of compounds 6b, 6c,
6f, 6g, 5a, 5e, 5f and D104 in LPS-activated RAW264.7
cells.
Compounds
IC50(μM)
Chlorogenic acid
Aspirin
6b
6c
6f
6g
5a
5e
5f
> 100
> 100
23.44
28.84
19.95
31.62
41.78
46.81
65.06
17.59
1.57
1.98
2.47
2.02
0.42
0.90
4.75
1.64
D104
28

P. Yu et al.
Fitoterapia129(2018)25–33
4.1.2. General procedure for the preparation of 2a-2d
mixture of 3,4-dihydroxybenzaldehyde (3.04 g, 22 mM) and
A
acetone or DMF (100 mL) was stirred at room temperature until clear,
and then dibromoalkane (26.4 mM) and K2CO3 (3.64 g, 26.4 mM) were
added. The mixture was heated at reflux about 60 °C for 4–5 h. After
completion of the reaction as indicated by TLC, the solution was eva-
porated under reduced pressure. Then the resulting solid was poured
into H2O (50 mL) and extracted with ethyl acetate for column chro-
matography. Column chromatography was performed using silica gel
(200–300 mesh) eluting with ethyl acetate and petroleum ether to give
2a-2d (Yield: 50–60%).
4.1.2.1. benzo[d][1,3]dioxole-5-carbaldehyde (2a). White solid was
obtained. ESI-MS m/z: 150.03. M.p. 36–38 °C. 1H NMR (400 MHz,
CDCl3) δ 9.79 (s, 1H), 7.39 (dd, J = 7.9, 1.5 Hz, 1H), 7.31 (d,
J = 1.4 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.06 (s, 2H). Purity: 98%.
4.1.2.2. 3,4-dihydro-2H-benzo[b][1,4]dioxepine-7-carbaldehyde
(2c). White solid was obtained. ESI-MS m/z: 178.06. M.p. 49–50 °C. 1H
NMR (400 MHz, CDCl3) δ 9.81 (s, 1H), 7.61–7.34 (m, 2H), 7.17–6.83
(m, 1H), 4.35–4.29 (m, 2H), 4.25 (t, J = 5.9 Hz, 2H), 2.42–2.03 (m,
2H). Purity: 98%.
4.1.2.3. 2,3,4,5-tetrahydrobenzo[b][1,4]dioxocine-8-carbaldehyde
(2d). White solid was obtained. ESI-MS m/z: 192.08. M.p. 77–78 °C. 1H
NMR (400 MHz, CDCl3) δ 9.80 (s, 1H), 7.48 (dq, J = 3.8, 2.0 Hz, 2H),
7.00 (d, J = 8.8 Hz, 1H), 4.61–4.48 (m, 2H), 4.30–4.17 (m, 2H),
2.02–1.92 (m, 2H), 1.81 (td, J = 11.0, 5.8 Hz, 2H). Purity: 98%.
4.1.3. General procedure for the preparation of 3a-3d
The cyclized cinnamic acids were synthesized using Doebner-
Knoevenagel modification. Cinnamic acids were prepared by mixing
cyclized benzaldehydes (4.43 g, 27 mmol), malonic acid (3.37 g,
32.4 mM), pyridine (20 mL) and piperidine (340 μL) together and stir-
ring 80 °C on a magnetic stirrer for 24 h. The reaction mixture was
evaporated under reduced pressure. Precipitates obtained were filtered,
washed with cold water repeatedly, and dried. Finally, the mixtures
were recrystallized with dichloromethane to give these acids. The yields
were between 70% and 80%.
Fig. 6. Effects of compounds 6b, 6c, 6f and 6g on NO regulation. (A) RAW
264.7 cells were treated with compounds 6b, 6c, 6f and 6g (100 μM) for 24 h.
LPS-induced iNOS and GAPDH protein levels were determined by western
blotting. (BeC) Image J software was used to analyze the levels of iNOS with
4.1.3.1. (E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylic acid (3b). Faint
yellow solid was obtained. ESI-MS m/z: 206.06. M.p. 191–193 °C. 1H NMR
(400 MHz, DMSO‑d6) δ 12.23 (s, 1H), 7.47 (d, J = 16.0 Hz, 1H), 7.34–7.05
(m, 2H), 6.87 (d, J = 8.3 Hz, 1H), 6.35 (d, J = 16.0 Hz, 1H), 4.26 (qd,
J = 3.7, 2.1 Hz, 4H)·13C NMR (101 MHz, DMSO- d6) δ 167.72 (s), 145.35
(s), 143.64 (s), 143.51 (s), 127.68 (s), 121.89 (s), 117.40 (s), 117.13 (s),
116.68 (s), 64.31 (s), 63.92 (s). Purity: 97%.
GAPDH as the reference. Data was represented by the mean
independent experiments.
SD of the three
4. Experimental section
4.1. Chemistry
4.1.4. General procedure for the preparation of 5a-5m and pro-D104
The starting materials (compounds 3a-3d) for the synthesis of esters
should be activated in the first procedure: the compounds 3a-3d
(12 mM) and SOCl2 (20 mL) were mixed and stirred at reflux 80 °C for
2 h. The reaction mixture was cooled and evaporated to give reactive
acyl chloride obtained as an oil, which would be dissolved in CH2Cl2 or
ethyl acetate (10–15 mL) in the next step. A solution of acyl chloride
(12 mM) in CH2Cl2 or ethyl acetate was added dropwise to corre-
sponding salicylic aldehyde (10 mM) in CH2Cl2 or ethyl acetate con-
taining pyridine (1.6 mL, 20 mM) under an inert (N2) atmosphere and at
0 °C with constant stirring overnight. The reaction mixture was then
poured in excess of diluted NaOH and extracted with CH2Cl2 or ethyl
acetate. The extraction liquid was purified by a flash chromatography
with ethyl acetate/petroleum ether to give these compounds. The yields
were between 40% and 60%.
4.1.1. Reagents and general methods
1H and 13C NMR spectra were recorded on Bruker AM 101, 400 and
600 MHz spectrometers with tetramethylsilane (TMS) as the internal
standard. Melting points (M.p.) were recorded on SRS OptiMelt-100 full
automatic micro melting point instrument. Electrospray ionization mass
spectra (ESI-MS) and high-resolution mass spectrum (HR-MS) were
recorded by Agilent 6520B Q-TOF. FT-IR spectra were carried out on
Nicolet 380 fourier transformation infrared spectrometer using KBr
pellets in the 400–4000 cm−1 range. Column chromatography (CC):
silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd.; Qingdao;
China). All reactions were monitored using thin layer chromatography
(TLC) on silica gel plates. Reaction reagents were analytical reagent
grade and purchased from Aladdin.
4.1.4.1. (E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acryloyl
chloride
(4b). Faint yellow solid was obtained. ESI-MS m/z: 224.02. 1H NMR
29

P. Yu et al.
Fitoterapia129(2018)25–33
(400 MHz, CDCl3) δ 7.70 (dd, J = 19.1, 15.7 Hz, 1H), 7.13–7.02 (m,
2H), 6.89 (dd, J = 10.0, 8.2 Hz, 1H), 6.38 (dd, J = 76.0, 15.7 Hz, 1H),
4.39–4.21 (m, 4H). Purity: 98%.
4.9 Hz, 4H). Purity: 98%.
4.1.4.9. 4-(diethylamino)-2-formylphenyl
(E)-3-(2,3-dihydrobenzo[b]
[1,4]dioxin-6-yl)acrylate (5h). Faint yellow solid was obtained. ESI-
MS m/z: 381.16. M.p. 122–124 °C. 1H NMR (400 MHz, CDCl3) δ 9.85 (s,
1H), 7.76 (dd, J = 19.7, 12.4 Hz, 2H), 7.20–7.03 (m, 2H), 6.89 (d,
J = 8.3 Hz, 1H), 6.57 (dd, J = 8.9, 2.4 Hz, 1H), 6.51 (d, J = 15.9 Hz,
1H), 6.35 (d, J = 2.4 Hz, 1H), 4.29 (q, J = 5.1 Hz, 4H), 3.42 (q,
J = 7.1 Hz, 4H), 1.21 (t, J = 7.1 Hz, 6H). Purity: 98%.
4.1.4.2. 2-formylphenyl (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylate (5a). Faint
yellow solid was obtained. HR-MS m/z ([M + Na]+): C17H12O5Na,
calculated 319.0577, found 319.0578. M.p. 119–121 °C. IR KBr (cm−1):
3434, 3077, 2914 (CeH), 1735 (CHO), 1709 (C=O), 1628, 1599, 1497,
1450 (C=C). 1H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H), 7.91 (dd, J = 7.7,
1.7 Hz, 1H), 7.81 (d, J = 15.9 Hz, 1H), 7.67–7.57 (m, 1H), 7.38 (t,
J = 7.5 Hz, 1H), 7.24 (s, 1H), 7.10 (d, J = 1.6 Hz, 1H), 7.07 (dd, J = 8.0,
1.6 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H), 6.02 (s,
2H). 13C NMR (101 MHz, CDCl3) δ 188.68 (s), 165.41 (s), 152.49 (s),
150.45 (s), 148.67 (s), 147.64 (s), 135.42 (s), 130.03 (s), 128.50 (s), 128.40
(s), 126.44 (s), 125.43 (s), 123.63 (s), 114.10 (s), 108.83 (s), 106.82 (s),
101.88 (s). Purity: 98%.
4.1.4.10. 2-formylphenyl (E)-3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-
7-yl)acrylate (5i). Faint yellow solid was obtained. ESI-MS m/z:
324.10. M.p. 110–112 °C. 1H NMR (400 MHz, CDCl3) δ 10.22 (s, 1H),
7.95 (dd, J = 7.7, 1.7 Hz, 1H), 7.83 (d, J = 15.9 Hz, 1H), 7.67 (ddd,
J = 8.1, 7.6, 1.8 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.31–7.27 (m, 1H),
7.25 (d, J = 2.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (d,
J = 8.3 Hz, 1H), 6.56 (d, J = 15.9 Hz, 1H), 4.30 (dt, J = 11.5, 5.7 Hz,
4H), 2.32–2.19 (m, 2H). Purity: 98%.
4.1.4.3. 5-chloro-2-formylphenyl (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylate
(5b). Faint yellow solid was obtained. ESI-MS m/z: 330.03. M.p.
181–183 °C. IR KBr (cm−1): 3342, 3074, 2911 (CeH), 1723 (CHO),
1685 (C=O), 1625, 1595, 1501, 1477, 1450 (C=C), 1401 (CeCl). 1H
NMR (400 MHz, CDCl3) δ 10.15 (s, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.83 (d,
J = 15.9 Hz, 1H), 7.60 (dd, J = 8.7, 2.7 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H),
6.86 (d, J = 7.9 Hz, 1H), 6.48 (d, J = 15.8 Hz, 1H), 6.05 (s, 2H). Purity:
98%.
4.1.4.11. 5-chloro-2-formylphenyl(E)-3-(3,4-dihydro-2H-benzo[b][1,4]
dioxepin-7-yl)acrylate (5j). Faint yellow solid was obtained. ESI-MS m/
z: 358.06. M.p. 174–176 °C. 1H NMR (400 MHz, CDCl3) δ 10.14 (s, 1H),
7.89 (d, J = 2.6 Hz, 1H), 7.81 (d, J = 15.9 Hz, 1H), 7.60 (dd, J = 8.7,
2.7 Hz, 1H), 7.25–7.22 (m, 2H), 7.18 (dd, J = 8.3, 2.1 Hz, 1H), 6.99 (d,
J = 8.3 Hz, 1H), 6.52 (d, J = 15.9 Hz, 1H), 4.29 (dt, J = 11.9, 5.7 Hz,
4H), 2.30–2.16 (m, 2H). Purity: 98%.
4.1.4.4. 3,5-dichloro-6-formylphenyl
(E)-3-(benzo[d][1,3]dioxol-5-yl)
acrylate (5c). Faint yellow solid was obtained. ESI-MS m/z: 363.99.
M.p. 152–154 °C. IR KBr (cm−1): 3071, 2908 (CeH), 1729 (CHO), 1691
(C=O), 1622, 1596, 1501, 1494, 1450, 1414 (C=C), 1401 (CeCl). 1H
NMR (400 MHz, CDCl3) δ 10.06 (s, 1H), 7.88 (d, J = 15.8 Hz, 1H), 7.80
(s, 1H), 7.71 (s, 1H), 7.11 (d, J = 11.3 Hz, 2H), 6.86 (d, J = 7.9 Hz,
1H), 6.52 (d, J = 15.8 Hz, 1H), 6.05 (s, 2H). Purity: 98%.
4.1.4.12. 3,5-dichloro-6-formylphenyl(E)-3-(3,4-dihydro-2H-benzo[b]
[1,4]dioxepin-7-yl)acrylate (5k). Faint yellow solid was obtained. ESI-
MS m/z: 392.02. M.p. 165–167 °C. 1H NMR (400 MHz, DMSO-d6) δ
10.00 (s, 1H), 8.18 (d, J = 2.6 Hz, 1H), 7.97 (d, J = 2.6 Hz, 1H), 7.87
(d, J = 16.0 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.45 (dd, J = 8.3,
2.1 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 16.0 Hz, 1H), 4.20
(dt, J = 13.7, 5.6 Hz, 4H), 2.19–2.08 (m, 2H). Purity: 98%.
4.1.4.5. 4-(diethylamino)-2-formylphenyl (E)-3-(benzo[d][1,3]dioxol-5-
yl)acrylate (5d). Faint yellow solid was obtained. ESI-MS m/z:
367.14. M.p. 128–131 °C. 1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H),
7.79 (d, J = 15.9 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.06 (dd, J = 12.1,
4.1 Hz, 2H), 6.82 (d, J = 8.0 Hz, 1H), 6.55 (dd, J = 8.9, 2.3 Hz, 1H),
6.48 (d, J = 15.9 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 6.01 (s, 2H), 3.40
(q, J = 7.1 Hz, 4H), 1.20 (t, J = 7.1 Hz, 6H). Purity: 96%.
4.1.4.13. 4-(diethylamino)-2-formylphenyl(E)-3-(3,4-dihydro-2H-benzo
[b][1,4]dioxepin-7-yl)acrylate (5l). Faint yellow solid was obtained.
ESI-MS m/z: 395.17. M.p. 115–118 °C. IR KBr (cm−1): 3071, 2973,
2922 (CeH), 1735 (CHO), 1664 (C=O), 1608, 1499, 1406 (C=C). 1H
NMR (600 MHz, CDCl3) δ 7.69 (s, 1H), 7.66 (s, 1H), 7.18 (d, J = 2.1 Hz,
2H), 7.13 (d, J = 2.1 Hz, 1H), 7.12 (d, J = 2.1 Hz, 1H), 6.97 (s, 1H),
6.96 (s, 1H), 6.32 (s, 1H), 6.30 (s, 1H), 4.29–4.27 (m, 4H), 4.25 (t,
J = 5.8 Hz, 4H), 2.25–2.20 (m, 4H), 1.25 (s, 2H). Purity: 97%.
4.1.4.6. 2-formylphenyl (E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylate
(5e). Faint yellow solid was obtained. ESI-MS m/z: 310.08. M.p.
117–119 °C. 1H NMR (400 MHz, CDCl3)
J = 7.7 Hz, 1H), 7.79 (d, J = 15.9 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.37
(t, J = 7.5 Hz, 1H), 7.24 (s, 1H), 7.14–7.06 (m, 2H), 6.88 (d, J = 8.2 Hz,
1H), 6.50 (d, J = 15.8 Hz, 1H), 4.28 (d, J = 4.7 Hz, 4H). Purity: 98%.
δ
10.19 (s, 1H), 7.91 (d,
4.1.4.14. 5-chloro-2-formylphenyl (E)-3-(2,3,4,5-tetrahydrobenzo[b][1,4]
dioxocin-8-yl)acrylate (5m). Faint yellow solid was obtained. ESI-MS m/
z: 372.08. M.p. 96–98 °C. 1H NMR (600 MHz, CDCl3) δ 10.14 (s, 1H), 7.88
(d, J = 2.6 Hz, 1H), 7.81 (d, J = 15.9 Hz, 1H), 7.59 (dd, J = 8.7, 2.7 Hz,
1H), 7.24 (dd, J = 5.4, 3.2 Hz, 2H), 7.22 (dd, J = 8.3, 2.2 Hz, 1H), 6.98 (d,
J = 8.3 Hz, 1H), 6.51 (d, J = 15.9 Hz, 1H), 4.51–4.47 (m, 2H), 4.33–4.26
(m, 2H), 1.97 (m, J = 11.4, 5.9 Hz, 2H), 1.92–1.85 (m, 2H). Purity: 98%.
4.1.4.7. 5-chloro-2-formylphenyl (E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)acrylate (5f). Faint yellow solid was obtained. HR-MS m/z
([M + Na]+): C18H13O5ClNa, calculated 367.0344, found 367.0340.
M.p. 182–184 °C. IR KBr (cm− 1): 3309, 2928, 2857 (CeH), 1682
(C=O), 1491, 1439 (C=C), 1411 (CeCl). 1H NMR (400 MHz,
DMSO‑d6) δ 10.05 (s, 1H), 7.94 (d, J = 2.7 Hz, 1H), 7.87–7.77 (m,
2H), 7.46 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.32 (dd,
J = 8.4, 2.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 16.0 Hz,
1H), 4.29 (td, J = 5.2, 3.7 Hz, 4H). Purity: 98%.
4.1.4.15. 3,5-difluoro-2-formylphenyl
(E)-3-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)acrylate (pro-D104). Faint yellow solid was obtained. ESI-
MS m/z: 346.07. M.p. 179–181 °C. IR KBr (cm−1): 3433, 3086, 2891
(CeH), 1751 (CHO), 1699 (C=O), 1641, 1607, 1586, 1508 (C=C),
1438 (CeF). 1H NMR (600 MHz, DMSO‑d6) δ 10.13 (s, 1H), 7.77 (d,
J = 15.9 Hz, 1H), 7.52–7.46 (m, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.33 (d,
J = 2.0 Hz, 1H), 7.32 (d, J = 1.9 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.75
(d, J = 16.0 Hz, 1H), 4.31 (m, 2H), 4.29–4.27 (m, 2H). Purity: 98%.
4.1.4.8. 3,5-dichloro-6-formylphenyl
(E)-3-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)acrylate (5g). Faint yellow solid was obtained. ESI-MS m/
z: 378.01. M.p. 174–176 °C. IR KBr (cm−1): 3028, 2991, 2934 (CeH),
1726 (CHO), 1691 (C=O), 1625, 1599, 1506 (C=C), 1408 (CeCl). 1H
NMR (400 MHz, CDCl3) δ 10.06 (s, 1H), 7.86 (d, J = 15.9 Hz, 1H), 7.80
(d, J = 2.3 Hz, 1H), 7.71 (d, J = 2.3 Hz, 1H), 7.17–7.10 (m, 2H), 6.91
(d, J = 8.3 Hz, 1H), 6.54 (d, J = 15.9 Hz, 1H), 4.31 (dd, J = 10.3,
4.1.5. General procedure for the preparation of 6a-6m and D104
A mixture of 5a-5m and pro-D104 (0.15 g, 0.5 mmol) and tert-butyl
alcohol (8 mL) and THF (6 mL) was stirred at room temperature until
clear, and 3-methyl-1-butene (0.84 mL, 10 mM) what was cooled at 0 °C
was added. Sodium dihydrogen phosphate (0.6 g, 5 mM) and NaClO2
30

P. Yu et al.
Fitoterapia129(2018)25–33
was dissolved in 2 mL aqueous solution and acidified with hydrochloric
acid aqueous solution (3 mM) to pH 3–4. The solution was slowly added
into the prepared aldehyde. Then the reaction mixture was stirred at
room temperature for 5–6 h. The solvent was evaporated away and
purified by a flash chromatography in pleasing yield (Yield: 45–60%).
J = 8.1 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 15.9 Hz, 1H),
5.95 (s, 2H), 2.35 (s, 2H). Purity: 98%.
4.1.5.8. (E)-4-(diethylamino)-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)acryloyl)oxy)benzoic acid (6h). Yellow solid was obtained. ESI-MS
m/z: 397.15. M.p. 107–109 °C. 1H NMR (400 MHz, CDCl3) δ 7.79 (d,
J = 15.9 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.13 (d, J = 1.9 Hz, 1H),
7.10 (dd, J = 8.3, 2.0 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.57 (dd,
J = 8.9, 2.4 Hz, 1H), 6.51 (d, J = 15.9 Hz, 1H), 6.35 (d, J = 2.4 Hz,
1H), 4.29 (q, J = 5.1 Hz, 4H), 3.42 (q, J = 7.1 Hz, 4H), 1.21 (t,
J = 7.1 Hz, 6H). Purity: 98%.
4.1.5.1. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)benzoic acid
(6a). Yellow solid was obtained. HR-MS m/z ([M + Na]+):
C
17H12O6Na, calculated 335.0526, found 335.0527. M.p. 153–155 °C.
IR KBr (cm−1): 3077, 3018, 2917 (CeH), 1735 (COOH), 1697 (C=O),
1625, 1604, 1500 (C=C). 1H NMR (400 MHz, CDCl3) δ 8.09 (d,
J = 7.9 Hz, 1H), 7.78 (d, J = 15.9 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H),
7.34 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 7.10–7.05 (t, 2H),
6.83 (d, J = 7.9 Hz, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.03 (s, 2H). Purity:
98%.
4.1.5.9. (E)-2-((3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)acryloyl)
oxy)benzoic acid (6i). Yellow solid was obtained. ESI-MS m/z: 340.09.
M.p. 163–164 °C. 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 7.8 Hz, 1H),
7.76 (d, J = 15.9 Hz, 1H), 7.64–7.58 (m, 1H), 7.33 (t, J = 7.5 Hz, 1H),
7.21 (d, J = 3.5 Hz, 2H), 7.15 (dd, J = 8.3, 1.8 Hz, 1H), 6.97 (d,
J = 8.3 Hz, 1H), 6.51 (d, J = 15.9 Hz, 1H), 4.28 (dt, J = 11.2, 5.7 Hz,
4H), 2.25–2.21 (m, 2H). Purity: 98%.
4.1.5.2. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-5-
chlorobenzoic acid (6b). Yellow solid was obtained. ESI-MS m/z:
346.02. M.p. 189–191 °C. IR KBr (cm−1): 3080, 2991, 2902 (CeH),
1720 (COOH), 1679 (C=O), 1602, 1503 (C=C), 1452 (CeCl). 1H NMR
(400 MHz, CDCl3) δ 8.06 (d, J = 2.6 Hz, 1H), 7.78 (d, J = 15.6 Hz, 1H),
7.57 (d, J = 11.2 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.09 (s, 1H), 7.06
(d, J = 7.9 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 6.46 (d, J = 15.9 Hz, 1H),
6.04 (s, 2H). Purity: 98%.
4.1.5.10. (E)-5-chloro-2-((3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-
yl)acryloyl)oxy)benzoic acid (6j). Yellow solid was obtained. ESI-MS m/
z: 374.06. M.p. 210–212 °C. 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H),
7.76 (d, J = 15.9 Hz, 1H), 7.57 (dd, J = 8.6, 2.5 Hz, 1H), 7.21 (d,
J = 1.7 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.4 Hz, 1H), 6.49
(d, J = 15.9 Hz, 1H), 4.28 (dt, J = 11.4, 5.8 Hz, 4H), 2.25–2.21 (m,
2H). Purity: 96%.
4.1.5.3. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-3,5-
dichlorobenzoic acid (6c). Yellow solid was obtained. ESI-MS m/z:
379.99. M.p. 172–175 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.08 (d,
J = 2.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.82 (d, J = 15.9 Hz, 1H),
7.56 (d, J = 1.5 Hz, 1H), 7.32 (dd, J = 8.1, 1.5 Hz, 1H), 7.00 (d,
J = 8.0 Hz, 1H), 6.81 (d, J = 15.9 Hz, 1H), 6.11 (s, 2H). Purity: 98%.
4.1.5.11. (E)-3,5-dichloro-2-((3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-
7-yl)acryloyl)oxy)benzoic acid (6k). Yellow solid was obtained. ESI-MS
m/z: 408.02. M.p. 198–199 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (d,
J = 2.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.81 (d, J = 16.0 Hz, 1H),
7.48 (d, J = 2.0 Hz, 1H), 7.43 (dd, J = 8.4, 2.1 Hz, 1H), 7.01 (d,
J = 8.3 Hz, 1H), 6.82 (d, J = 16.0 Hz, 1H), 4.24–4.14 (m, 4H),
2.20–2.07 (m, 2H). Purity: 98%.
4.1.5.4. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)acryloyl)oxy)-4-
(diethylamino)benzoic acid (6d). Yellow solid was obtained. ESI-MS m/
z: 383.14. M.p. 160–162 °C. 1H NMR (600 MHz, CDCl3) δ 7.92 (d,
J = 9.1 Hz, 1H), 7.75 (d, J = 15.9 Hz, 1H), 7.11–7.06 (m, 1H), 7.04
(dd, J = 8.1, 1.6 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.47 (d,
J = 15.9 Hz, 2H), 6.30 (d, J = 2.0 Hz, 1H), 6.02 (s, 2H), 3.38 (q,
J = 7.1 Hz, 4H), 1.18 (t, J = 7.1 Hz, 6H). Purity: 98%.
4.1.5.12. (E)-4-(diethylamino)-2-((3-(3,4-dihydro-2H-benzo[b][1,4]
dioxepin-7-yl)acryloyl)oxy)benzoic acid (6l). Yellow solid was obtained.
ESI-MS m/z: 411.17. M.p. 145–147 °C. 1H NMR (400 MHz, CDCl3) δ
7.92 (d, J = 9.0 Hz, 1H), 7.80–7.68 (m, 1H), 7.21 (d, J = 2.0 Hz, 1H),
7.15 (dd, J = 8.3, 2.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.57–6.46 (m,
2H), 6.31 (d, J = 2.1 Hz, 1H), 4.27 (dt, J = 9.5, 5.7 Hz, 4H), 3.39 (q,
J = 7.0 Hz, 4H), 2.34–2.10 (m, 2H), 1.19 (t, J = 7.1 Hz, 6H). Purity:
98%.
4.1.5.5. (E)-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acryloyl)oxy)
benzoic acid (6e). Yellow solid was obtained. ESI-MS m/z: 326.08. M.p.
102–105 °C. 1H NMR (400 MHz, DMSO‑d6) δ 7.92 (dd, J = 7.8, 1.7 Hz,
1H), 7.71 (d, J = 16.0 Hz, 1H), 7.63 (td, J = 7.8, 1.7 Hz, 1H),
7.41–7.35 (m, 2H), 7.30 (dd, J = 8.4, 2.0 Hz, 1H), 7.24 (dd, J = 8.1,
0.9 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 4.28
(dd, J = 9.9, 5.1 Hz, 4H). 13C NMR (101 MHz, DMSO) δ 165.94 (s),
165.08 (s), 149.98 (s), 145.90 (s), 143.60 (s), 133.40 (s), 131.26 (s),
127.38 (s), 125.94 (s), 125.12 (s), 123.79 (s), 122.48 (s), 117.53 (s),
117.15 (s), 115.31 (s), 64.39 (s), 63.93 (s). Purity: 98%.
4.1.5.13. (E)-5-chloro-2-((3-(2,3,4,5-tetrahydrobenzo[b][1,4]dioxocin-8-
yl)acryloyl)oxy)benzoic acid (6m). Yellow solid was obtained. ESI-MS
m/z: 388.07. M.p. 80–82 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d,
J = 2.7 Hz, 1H), 7.77–7.67 (m, 2H), 7.48–7.40 (m, 2H), 7.31 (d,
J = 8.6 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 16.0 Hz, 1H),
4.40 (t, J = 5.4 Hz, 2H), 4.23 (t, J = 5.3 Hz, 2H), 1.88 (dd, J = 12.5,
6.8 Hz, 2H), 1.80–1.73 (m, 2H). Purity: 98%.
4.1.5.6. (E)-5-chloro-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
acryloyl)oxy)benzoic acid (6f). Yellow solid was obtained. HR-MS m/z
([M + Na]+): C18H13O6ClNa, calculated 383.0293, found 383.0292.
M.p. 157–159 °C. IR KBr (cm−1): 3071, 2997, 2881 (CeH), 1726
(COOH), 1685 (C=O), 1625, 1599, 1506 (C=C), 1436 (CeCl). 1H
4.1.5.14. (E)-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acryloyl)oxy)-
4,6-difluorobenzoic acid (D104). Yellow solid was obtained. ESI-MS m/
z: 362.06. M.p. 154–156 °C. IR KBr (cm−1): 3428, 2959, 2880 (CeH),
1727 (COOH), 1686 (C=O), 1626, 1600, 1573, 1506 (C=C), 1435
(CeF). 1H NMR (600 MHz, DMSO-d6) δ 8.39 (d, J = 2.4 Hz, 1H), 8.12
(d, J = 2.4 Hz, 1H), 7.85 (d, J = 15.9 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H),
7.35 (dd, J = 8.4, 2.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.82 (d,
J = 15.9 Hz, 1H), 4.32–4.30 (m, 2H), 4.30–4.27 (m, 2H). Purity: 98%.
NMR (600 MHz, DMSO‑d6)
δ 7.89 (d, J = 2.7 Hz, 1H), 7.74 (d,
J = 2.8 Hz, 1H), 7.72–7.71 (m, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.34 (d,
J = 8.6 Hz, 1H), 7.31 (dd, J = 8.4, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz,
1H), 6.73 (d, J = 16.0 Hz, 1H), 4.30 (dd, J = 5.6, 2.2 Hz, 2H), 4.27 (dd,
J = 5.4, 2.0 Hz, 2H). Purity: 98%.
4.1.5.7. (E)-3,5-dichloro-2-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
acryloyl)oxy)benzoic acid (6g). Yellow solid was obtained. ESI-MS m/z:
394.00. M.p. 153–155 °C. 1H NMR (400 MHz, DMSO‑d6) δ 7.88 (s, 1H),
7.72 (s, 1H), 7.65 (d, J = 15.9 Hz, 1H), 7.37 (s, 1H), 7.14 (d,
4.2. Biological evaluation
4.2.1. Reagents and cell culture
RAW 264.7 cell line was
a gift from Nanjing University.
31

P. Yu et al.
Fitoterapia129(2018)25–33
Lipopolysaccharides (LPS), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-
nyltetrazolium bromide (MTT), and Griess reagent (1% sulfanilamide,
0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric
acid) were from Sigma. 6-well and 96-well plates were from Beyotime
biotechnology.
RAW 264.7 cell were grown in High glucose Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin and propa-
gated at 37 °C in a humidified atmosphere containing 5% CO2 in air.
Compounds 5a-5m, 6a-6m, pro-D104, D104, chlorogenic acid and
acetylsalicylic acid were dissolved in dimethyl sulfoxide to make stock
solutions, respectively, and kept at −20 °C. The final concentration of
the vehicle in the solution never exceeded 0.1% and had no effects on
NO production and cell viability.
for 5 min at 4 °C, and protein concentrations were measured using the
BCATM protein quantification kit. The iNOS protein level was de-
termined by western blotting analysis of 50 μg of cell extract using the
antibody against iNOS (D6B6S). Briefly, the protein samples were
centrifuged (4 °C, 12000 g, 10 min) and boiled for 10 min, then sub-
jected to a 10% SDS-polyacrylamide gel electrophoresis at a constant
20 mA current for 1 h. The resolved proteins were transferred to a PVDF
membrane by wet rotation at 70 mV and blocked with a blocking buffer
(2% free fat milk, 10 mM Tris-Cl, 50 mM NaCl, 0.1% Tween 20, pH 7.4)
at room temperature for 1 h. The membrane was incubated with pri-
mary antibodies against iNOS and GAPDH overnight on the converter at
low temperature (4 °C). The next day, the membrane was washed using
the washing buffer (10 mM Tris-Cl, 50 mM NaCl, 0.1% Tween 20,
pH 7.4) three times, twice for 5 min and once for 10 min, to remove any
nonspecific primary antibody binding. Then the membrane was in-
cubated with an appropriate dilution of secondary antibody at room
temperature for 2 h. After washing three times with washing buffer, the
membrane was illuminated with ECL reagent according to the manu-
facturer's instructions. A photograph of the gel was taken, and the re-
lative band density was analyzed by optical densitometry using Image
J.
4.2.2. Anti-inflammatory assay
Accumulation of nitrite (NO2−), an indicator of NO synthase ac-
tivity, in culture supernatant fluids was measured based on Griess re-
action [17]. Briefly, Cells (2 × 104) were seeded in 100 μL of DMEM
into 96-well plates and co-incubated with different concentrations
(1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM) of compounds 5a-5m, 6a-
6m, pro-D104 and D104 in the absence or presence of LPS (500 ng/ml)
for 48 h. Meanwhile, chlorogenic acid and acetylsalicylic acid also were
tested as positive controls. Culture supernatant fluids were mixed with
100 μL Griess reagent at room temperature for 5 min. Using NaNO2 to
generate a standard curve, nitrite production was measured by an ab-
sorbance reading at 540 nm.
4.2.6. Assay for cytotoxic activity
Non-toxic concentrations of compounds 5a-5m, 6a-6m, pro-D104
and D104 were determined according to MTT test [20] and con-
centration of 50 μM were chosen to test the effects of caffeoyl salicylate
analogs on nitric oxide production. MTT was dissolved at 4 mg/ml in
PBS and used essentially as previously described. Briefly, cell lines in
logarithmic phase were seeded at a density of 3 × 103 cells/well in
100 μL of DMEM into 96-well microtiter plates. After 24 h, ex-
ponentially growing cells were exposed to the indicated compounds at
various concentrations. After 48 h in final volumes of 200 μL, cell sur-
vival was determined by the addition of an MTT solution (20 μL of
4 mg/mL MTT in PBS) for 4 h. After carefully removing the medium, the
precipitates were dissolved in 200 μL of DMSO, shaken mechanically for
10 min, and then absorbance values at a wavelength of 540 nm were
taken on an LX300 Epson Diagnostic microplate reader. Survival ratios
are expressed in percentages with respect to untreated cells.
4.2.3. Total RNA isolation
The concentration and purity of RNA were detected. The operation
was as follows [18]. Put cells in tripure isolation reagent and incubate
samples at 15–25 °C for 5 min. Then 200 μL chloroform was added. The
mixture were shook vigorously with vortex for 15 s and incubated at
15–25 °C for 2–15 min. After centrifuged at 12000g at 4 °C for 15 min,
aqueous phase was pipetted into a clean screw-cap centrifuge tube
carefully and lower phases were discarded. Aqueous phase was mixed
with an equal volume of isopropyl alcohol and incubated at 15–25 °C
for 5–10 min. Then the mixture was centrifuged at 12000g at 4 °C for
10 min and the precipitate was washed with 1 ml 75% ethanol by
spinning briefly. After centrifuged at 7500g at 4 °C for 5 min, the su-
pernatant was aspirated and precipitate was dried. Then diethyl pyr-
ocarbonate (DEPC/ddH2O) was added and incubated at 55–60 °C for
10–15 min to dissolve precipitate. Total RNA concentration and the
absorbance 260/280 ratios were measured.
Conflict of interest
The authors confirm that this article content has no conflict of in-
terests.
Acknowledgement
4.2.4. QRT-PCR analysis
Quantitative RT-PCR assays [19] to investigate differences in the
expression of the mRNAs of interest were performed on a 7300 Se-
quence Detection System (Applied Biosystems) using EvaGreen Dye
(Biotium, Hayward, CA). Briefly, 2 μL total RNA (1 μg/μL) was reverse
transcribed into cDNA using PrimeScript RTase (Takara Bio, Shiga,
Japan) and a Oligo dT primer (Invitrogen) under the following condi-
tions:30 °C for 10 min, 42 °C for 20 min, and 99 °C for 5 min. The con-
ditions for the PCR were as follows: 95 °C for 5 min, 95 °C for 30 s, and
60 °C for 45 s, for 40 cycles. All reactions, including the no template
controls, were run in triplicate. After the reactions were complete, the
CT values were determined using fixed threshold settings. The mRNA
expression was normalized to β-actin expression in this study. The
amount of mRNA to relative to the internal control β-actin was calcu-
This work was supported by the Jiangsu “333” project of cultivation
of high-level talents (Grant No. BRA2015317), the 11th Six Talents
Peak Project of Jiangsu Province (Grant No. 2014-JY-011), a Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD), and the Doctorate Fellowship
Foundation of Nanjing Forestry University (Grant No. 163030748).
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