Design, synthesis and evaluation of 3-substituted coumarin derivatives as anti-inflammatory agents

Article abstract of DOI:10.1248/CPB.C19-01085

Coumarin moiety has garnered momentous attention especially in the design of compounds with significant biological activities. In this work, a series of 3-substituted coumarin derivatives 6a-6l were synthesized and fully characterized. Most of the compoun

Full text of DOI:10.1248/CPB.C19-01085

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c19-01085
Advance Publication
March 13, 2020
Chem. Pharm. Bull.
Regular Article
Design, Synthesis and Evaluation of 3-Substituted Coumarin Derivatives as
Anti-Inflammatory Agents
a,b
b
b
b
b
b
,b
Tao Wang, Tao Peng, Xiaoxue Wen, Gang Wang, Shuchen Liu, Yunbo Sun, Shouguo Zhang,* and
*
,a,b
Lin Wang,
a
College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100124, P. R. China;
Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China.
b
*
To whom correspondence should be addressed. e-mail: zhangshouguo1409@sina.com; wanglin@bmi.ac.cn
Ⓒ
2020 The Pharmaceutical Society of Japan

Coumarin moiety has garnered momentous attention especially in the design of compounds with significant
biological activities. In this work, a series of 3-substituted coumarin derivatives 6a–6l were synthesized and fully
characterized. Most of the compounds could obviously inhibit the activity of COX-1 at the concentration of 10 μM.
Besides, 6h and 6l exhibited highest inhibitory effects against COX-2 with inhibition rates of 33.48% and 35.71%,
respectively. Detailed structure-activity relationships (SARs) were also discussed. In vivo studies, 6b, 6i and 6l could
remarkably repress the xylene-induced ear swelling in mice at the dose of 20 mg/kg. Especially, 6l seemed to be the
most effective compound at the dose of 10 mg/kg, displaying favorable anti-inflammatory activity comparable to
indomethacin. All of these findings suggested that 6l might be utilized as a candidate for the treatment of
inflammatory diseases.
Key words 3-substituted coumarin; anti-inflammatory; tumor necrosis factor; cyclooxygenase; xylene-induced ear
swelling;
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Inflammation is a primary defensive response of living tissue to various damage factors, such as
1
,2)
biological pathogens, toxic chemicals, irritants and other harmful stimuli. As a complex biological and
physiological process, inflammation is characterized by five main symptoms, including swelling, redness,
3
)
heat, pain and local dysfunction. Inflammation is a protective immune response and is usually beneficial.
However, persistent and exaggerated inflammation will promote tissue damage and lead to diseases, for
4
-6)
instance, arthritis, sepsis, atherosclerosis, and even cancer.
Based on the structure and therapeutic mechanism, anti-inflammatory drugs can be divided into two
types, of which nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely administered drugs
7
)
for the treatment of inflammation. NSAIDs, such as indomethacin and ibuprofen, act their antipyretic,
analgesic and anti-inflammatory effects by inhibiting cyclooxygenase-1 and cyclooxygenase-2 (COX-1
8
,9)
and COX-2), which are key enzymes involved in the pathway that produces prostaglandins (PGs).
However, NSAIDs may cause some unexpected side effects, such as peptic ulcer, liver damage and
1
0)
anaphylaxis. Thus, it is still quite necessary for us to develop and explore anti-inflammatory drugs with
better therapeutic effects.
The coumarin skeleton, also known as benzo-α-pyrone, has attracted voluminous attention for its
1
1)
ability to form non-covalent interaction with the active sites of the target protein. Given its favorable
pharmacological activity, benzo-α-pyrone has been used as an indispensible structural subunit for the
1
2)
discovery of drugs with improved pharmacological profiles. In recent years, coumarins and related
1
3)
14)
derivatives have displayed their diverse biological activities, such as anti-cancer, antibacterial,
1
5)
16)
antioxidant and anti-inflammatory . Furthermore, some coumarins with different pharmacophores at
1
7-20)
C-3 position have been evaluated for anti-inflammatory activities.
Sulfone and sulfoxide derivatives containing heterocyclic moieties belong to an important class of
2
1,22)
active compounds possessing various biological activities.
It has been reported that the combination
of distinct pharmacophores in the same structure is very likely to obtain compounds with significant
2
3)
activity. Thus, in order to develop novel anti-inflammatory agents, benzyl sulfone/sulfoxide moieties
were introduced to the C-3 position of coumarin skeleton and the target compounds, 3-substituted
coumarin derivatives were designed and synthesized (Fig. 1 and Chart 1). Anti-inflammatory activity of
compounds 6a–6l was preliminarily evaluated in mouse RAW 264.7 macrophages. Most of the
compounds could markedly restrain the release of lipopolysaccharide (LPS)-induced tumor necrosis
factor (TNF)-α at non-cytotoxic concentrations. Besides, all compounds were evaluated for
cyclooxygenase inhibitory activity in cellular level by the enzyme-linked immuno-sorbent assay (ELISA)
in vitro. In addition, 6b, 6c, 6d, 6h, 6i, 6l were selected for further anti-inflammatory study in vivo by the
xylene-induced ear swelling method.
Fig. 1. The design of 3-substituted coumarin derivatives
Chemical and Pharmaceutical Bulletin Advance Publication

Results and Discussion
Chemistry
The target compounds 6a–6l were synthesized via a three-step synthetic route from substituted
benzylchloride/bromide (1a–1j) as outlined in Chart 1. The starting material 1a–1c were treated with
mercaptoacetic acid at the presence of sodium hydroxide to give benzylmercaptoacetic acids 2a–2c in 63–
2
4)
87% yields.
Treatment of 2a–2c with 30% hydrogen peroxide at room temperature gave
2
4)
benzylsulfinylacetic acids 3a–3c or at heating condition gave benzylsulfonylacetic acids 4a–4c with
2
5)
26)
satisfactory yields. Finally, the target compounds 6a–6l were synthesized via knoevenagel reaction
between 3a–3c or 4a–4c and substituted salicylaldehydes (5a–5d). The expected compounds 6a–6h were
prepared from 3a, 3b, 3c or 4c with 5a–5d at the presence of EDCI in 26–58% yields. And 6i–6l were
obtained from 4a, 4b with 5a, 5c or 5d in acetic anhydride with different yields ranging from 29% to
6
2%. All the target compounds were purified by recrystallization or flash chromatography and their
1
13
structures were confirmed by H-NMR, C-NMR and HRMS spectra analysis.
Cell Viability Assay
The cytotoxicity of coumarin derivatives 6a–6l (2.5 μM, 5 μM, 10 μM and 20 μM ) on RAW 264.7
2
7)
macrophages was evaluated by CCK-8 (Cell Counting Kit-8, WST-8) assay after 24 h of treatment. As
observed from the cell viability data in Fig. 2(A), at the concentration of up to 10 μM, all compounds
generated no cytotoxicity to RAW264.7 with cell viability higher than 85%. At the concentration of 20
μM, the viability of 6h-treated cells was just 81.79%. Thus, the concentration of 10 μM was selected to
evaluate coumarin derivatives in the following TNF-α detection.
Evaluation of TNF-α Production Induced by LPS
Macrophages are well known to play an important role in the initiation and development of
2
8)
inflammation. Activated macrophages induced by LPS produce cytokines such as TNF-α, interleukin,
2
9)
and pre-inflammatory mediators, including nitric oxide (NO) and PGs. Studies have proved that over
3
0)
release of cytokines and pro-inflammatory mediators will lead to inflammatory diseases. In order to
evaluate the anti-inflammatory activity of all the target compounds 6a–6l in vitro, ELISA was used to
3
1)
screen the production of TNF-α induced by LPS in RAW 264.7 macrophages. As showed in Fig. 2(B),
at the concentration of 10 μM, most of the tested compounds could significantly inhibit the secretion of
TNF-α compared with the LPS group. Especially, 6c, 6d, 6h and 6i most strongly restrained the secretion
of TNF-α.
Chemical and Pharmaceutical Bulletin Advance Publication

OH
OH
S
R1
O
O
ii
OH
3
a-3c
X
i
S
R1
R1
O
O
S
iii
R1
1a-1c
2a-2c
O
O
R2
R1
4a-4c
S
O
n
O
O
vi
O
a-6h
CHO
OH
OH
S
6
R2
R1
n
+
R2
O
O
O
v
3
a-3c, 4a-4c
R1
5a-5d
S
O
O
6i-6l
1
1
2
1
1
1
2
2
2
5
5
5
5
a: R = 3-OCH
3
, X = Cl
, 3-NO
3 3 2
6a: R = 3-OCH , R = 5,7-(OCH ) , n = 1
1
1
2
b: R = 4-OCH
3
2
, X = Br 6b: R = 4-Br, R = 6-OCH
3
, n = 2
, n = 1
6d: R = 4-Br, R = 5,7-(OCH , n = 2
1
1
2
c: R = 4-Br, X = Br
6c: R = 4-Br, R = 6-OCH
3
1
1
2
a, 3a, 4a: R = 3-OCH
3
3 2
)
1
1
2
b, 3b, 4b: R = 4-OCH
3
, 3-NO
2
3 2
6e: R = 4-Br, R = 5,7-(OCH ) , n = 1
1
1
2
c, 3c, 4c: R = 4-Br
6f: R = 4-OCH
3
, 3-NO
2
3 2
, R = 5,7-(OCH ) , n = 1
2
1
2
a: R = 4,6-(OCH
3
)
2
6g: R = 4-OCH
3
, 3-NO
2
, R = 7-OCH
3
, n = 1
2
1
2
b: R = 5-OCH
3
6h: R = 3-OCH
3
, R = 6-Br, n = 1
2
1
2
c: R = 4-OCH
3
6i: R = 3-OCH
3
, R = 5,7-(OCH
3
)
2
, n = 2
2
1
2
d: R = 5-Br
6j: R = 4-OCH
3
, 3-NO
2
, R = 5,7-(OCH
3
)
2
1
2
6
6
k: R = 4-OCH
3
, 3-NO
2
, R = 7-OCH
3
1
2
l: R = 3-OCH
3
, R = 6-Br
Chart 1. Reagents and conditions: (i) HSCH
NaOH, H O, rt, 4 h, 61% 88%; (iii) H , CH
rt, 1 h, 26% COONa, (CH CO)
2
COOH, NaOH, CH
3
OH, rt, 1-2 h, 63%
–
2 2
87%; (ii) H O ,
o
2
–
2
O
2
3
COOH, 55 C, 5 h, 63%
–77%; (iv) EDCI, DMAP, CH
3
CN,
o
–
58%; (v) CH
3
3
2
O, 110 C, 0.5 h, 29%
–62%.
1
1
1
1
30
20
10
00
20 μM
10 μM
5 μM
2.5 μM
110
100
90
*
*
*
*
*
*
80
*
*
*
**
** **
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
70
6
5
4
3
2
1
0
0
0
0
0
0
0
**
**
A
B
6
a
6b 6c 6d 6e
6f
6g 6h
6i
6j
6k
6l
Fig. 2. (A) In vitro viability rate of RAW264.7 cells treated with compounds 6a
concentrations of 2.5 μM
–
6l at different
–
20 μM. The results were presented as the percent of LPS control; (B) Effects of
coumarin derivatives on LPS-induced TNF-α production in RAW264.7 cells.
Data were expressed as the mean ± SD of three independent experiments. Compared with the LPS group, *P<0.01,
*
*P<0.001. DEX: dexamethasone; IND: indomethacin.
Chemical and Pharmaceutical Bulletin Advance Publication

In vitro Cyclooxygenase Inhibition and SAR Study
The 12 newly synthesized compounds 6a‒6l were first evaluated for cyclooxygenase inhibitory activity
7
)
in cellular level by ELISA assay, using indomethacin as a comparison. Based on the results displayed in
Table 1, all compounds exhibited favorable inhibitory activity against COX-1 at the concentration of 10
μM, except for 6j. Compounds 6a, 6e and 6g exhibited excellent inhibitory potency with inhibition rates
of 46.76%, 46.24% and 45.57%, respectively, which were comparable to that of indomethacin (51.11%).
Compounds 6b, 6c, 6d, 6h, 6i, 6k and 6l also exhibited moderate inhibitory effects against COX-2 with
inhibition rates above 25% at the concentration of 10 μM. Besides, 6h and 6l exhibited the highest
potency with inhibition rates of 33.48% and 35.71%, respectively, and could be potent COX-2 inhibitors.
Table 1. The COX-1 and COX-2 inhibitory activities of compounds 6a‒6l
Inhibition Rates (%)
Compound
COX-1
COX-2
5 μM
10 μM
5 μM
10 μM
IND
35.14 ± 5.88
21.66 ± 3.97
21.00 ± 2.97
29.99 ± 2.81
23.25 ± 5.28
28.67 ± 3.90
23.65 ± 3.60
25.63 ± 6.44
30.38 ± 2.78
19.82 ± 2.39
12.15 ± 4.59
11.62 ± 4.80
31.04 ± 5.24
51.11 ± 6.59
46.76 ± 4.56
34.74 ± 6.44
43.59 ± 2.87
41.61 ± 4.24
46.24 ± 4.64
36.20 ± 6.00
45.57 ± 1.99
31.70 ± 5.96
41.22 ± 4.38
19.02 ± 4.20
40.29 ± 7.05
38.84 ± 5.17
19.02 ± 2.17
13.01 ± 3.54
16.80 ± 5.56
16.24 ± 3.93
13.79 ± 4.79
8.90 ± 2.00
10.01 ± 2.37
10.12 ± 1.90
16.13 ± 2.59
20.58 ± 5.24
11.90 ± 3.18
9.45 ± 2.22
18.91 ± 2.73
35.26 ± 5.34
21.58 ± 4.59
25.47 ± 4.89
21.02 ± 2.37
24.36 ± 3.36
12.46 ± 3.91
13.46 ± 3.21
15.24 ± 2.41
33.48 ± 5.02
32.15 ± 3.72
19.35 ± 4.54
22.91 ± 2.52
35.71 ± 4.01
6
a
6b
6
c
6d
6
e
6
f
6
g
6h
6
i
6
j
6k
6
l
From the data of COX-1 inhibitory activities, some structure–activity relationships (SARs) can be
observed: (i) the bioactivity of sulfoxides was higher than the corresponding sulfones (6a > 6i, 6c > 6b);
1
(
ii) the type, number and position of R seemed to play important roles for the activity: 7-OCH
3
>
5
3 2
,7-(OCH ) > 6-Br (6g > 6f, 6a > 6h). When it came to COX-2 inhibitory activity, some interesting
SARs were illustrated: (i) the bioactivity of sulfones was higher than the corresponding sulfoxides (6i >
1
6
a, 6b > 6c); (ii) the type, number and position of R also played important roles for the activity: 6-Br
(
6h)> 5,7-(OCH
3 2 3 3 2
) (6a), 7-OCH (6g) > 5,7-(OCH ) (6f).
NSAIDs exert their anti-inflammatory effects mainly by inhibiting COX-2, while the inhibition of
3
2)
COX-1 may contribute to their unwanted side effects, such as gastric and renal damage. Compounds
with higher inhibitory potency on COX-2 but lower inhibitory activity against COX-1 were thought to be
idea anti-inflammatory agents. Then 6b, 6c, 6d, 6h, 6i, 6l were selected for further study in vivo.
Anti-inflammatory Activity Evaluation in vivo
The process of inflammation is related to the increase of blood flow, capillary permeability and
migration of macrophages and neutrophils from capillaries to interstitial spaces. As more fluid continues
3
3)
to accumulate in the interstitial space, the damaged tissue begins to swell. Thus, swelling becomes one
Chemical and Pharmaceutical Bulletin Advance Publication

3
4)
of the main symptoms of inflammation. The anti-inflammatory activities in vivo were screened in mice
model of xylene-induced ear swelling, with dexamethasone and indomethacin as reference drugs.
According to the results in Table 2, the target compounds (6b, 6c, 6d, 6h, 6i and 6l) exhibited different
degrees of anti-inflammatory activities under the experimental conditions. At the dose of 20 mg/kg, these
compounds could obviously repress ear swelling with inhibition rates from 27.76% to 41.43%. At the
dose of 10 mg/kg, 6b, 6i and 6l suppressed the swelling with inhibition rates above 30%. Remarkably,
compound 6l exhibited the best anti-inflammatory activity, with inhibition rate of 34.56%, which was
comparable to indomethacin.
Table 2. Inhibitory effects of 6a‒6l on xylene-induced ear swelling
Swelling degree (mg)
0 mg/kg 10 mg/kg
16.01 ± 3.81
7.02 ± 1.94
Inhibition rate (%)
20 mg/kg 10 mg/kg
Group
2
Control
DEX
–
*
*
*
*
N.T.
N.T.
N.T.
N.T.
56.14
38.03
31.58
19.78
25.47
28.80
30.46
34.56
IND
9.92 ± 2.14
10.96 ± 2.03
12.84 ± 2.84
*
*
**
6b
9.43 ± 2.19
41.08
27.76
32.34
35.04
39.28
41.43
*
6
c
11.57 ± 2.99
*
*
*
6
d
h
10.83 ± 2.40
10.40 ± 3.07
11.93 ± 2.99
11.40 ± 3.05
*
*
*
6
*
*
*
**
6
i
l
9.72 ± 1.68
9.38 ± 2.83
11.57 ± 2.67
10.48 ± 3.12
*
**
6
Data were expressed as the mean ± SD, n = 9. Compared with the LPS group, *P < 0.05, **P < 0.01.
NT”: not test.
“
Conclusion
In summary, to obtain effective lead compounds that can serve as anti-inflammatory agents, we have
designed and synthesized a total of twelve coumarin derivatives linked substituted benzyl
sulfone/sulfoxide moieties at C-3 position. The anti-inflammatory effects of these compounds were
evaluated in vitro and in vivo, including the inhibition of TNF-α production induced by LPS in RAW
2
64.7 macrophages, cyclooxygenase inhibition study and xylene-induced ear swelling in mice. Results of
the in vitro study provided evidence that most of the compounds could repress the release of TNF-α and
exhibited favorable inhibitory activity against COX-1 at the concentration of 10 μM. Moreover, 6h and 6l
exhibited the highest inhibitory potency on COX-2. In addition, at the dose of 20 mg/kg, the active
compounds 6b, 6c, 6d, 6h, 6i and 6l could obviously repress ear swelling in vivo. Especially, 6l displayed
satisfactory inhibitory activity similar to indomethacin at the dose of 10 mg/kg. All of these results reveal
that 6l may be a lead compound working on cyclooxygenase in inflammation therapy and is worthy of
further study and optimization.
Experimental Supplementary General chemistry methods, synthesis procedures, spectral data, and
bioassay methods are given in Supplemental information.
Chemical and Pharmaceutical Bulletin Advance Publication

Acknowledgments This research was supported by National Natural Science Foundation of China
Grant No. 81273431).
(
Conflict of Interest The authors declare no conflict of interest.
Supplementary Materials The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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