3-Arylcoumarins: Synthesis and potent anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmcl.2014.10.033

Chronic inflammation is the persistent and excessive immune response and can lead to a variety of diseases. Aiming to discover new compounds with anti-inflammatory activity, we report herein the synthesis and biological evaluation of 3-arylcoumarins. Thirty five 3-arylcoumarins were prepared through Perkin condensation and further acid-promoted hydrolysis if necessary. In lipopolysaccharide-activated mouse macrophage RAW264.7 cells, 6,8-dichloro-3-(2-methoxyphenyl)coumarin (16) and 6-bromo-8-methoxy-3-(3-methoxyphenyl)coumarin (25) exhibited nitric oxide production inhibitory activity with the IC₅₀ values of 8.5 μM and 6.9 μM, respectively, providing a pharmacological potential as anti-inflammatory agents.

Full text of DOI:10.1016/j.bmcl.2014.10.033

Accepted Manuscript
3-Arylcoumarins: Synthesis and potent anti-inflammatory activity
Wenchen Pu, Yuan Lin, Jianshuo Zhang, Fei Wang, Chun Wang, Guolin Zhang
PII:
S0960-894X(14)01093-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.10.033
BMCL 22088
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 December 2013
19 February 2014
9 October 2014
Please cite this article as: Pu, W., Lin, Y., Zhang, J., Wang, F., Wang, C., Zhang, G., 3-Arylcoumarins: Synthesis
and potent anti-inflammatory activity, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/
10.1016/j.bmcl.2014.10.033
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1
Bioorganic & Medicinal Chemistry Letters
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3-Arylcoumarins: Synthesis and potent anti-inflammatory activity
Wenchen Pu a,b, Yuan Lin a,b,c, Jianshuo Zhang c, Fei Wang a, Chun Wang a, Guolin Zhang a,∗
a Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China
b University of Chinese Academy of Sciences, Beijing 100049, PR China
c College of Life Sciences, Sichuan University, Chengdu 610064, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
Chronic inflammation is the persistent and excessive immune response and can lead to a variety
of diseases. Aiming to discover new compounds with anti-inflammatory activity, we report
herein the synthesis and biological evaluation of 3-arylcoumarins. Thirty five 3-arylcoumarins
were prepared through Perkin condensation and further acid-promoted hydrolysis if necessary.
In lipopolysaccharide-activated mouse macrophage RAW264.7 cells, 6,8-dichloro-3-(2-
methoxyphenyl)coumarin (16) and 6-bromo-8-methoxy-3-(3-methoxyphenyl)coumarin (25)
exhibited nitric oxide production inhibitory activity with the IC50 values of 8.5 µM and 6.9 µM,
respectively, providing a pharmacological potential as anti-inflammatory agents.
Revised
Accepted
Available online
Keywords:
anti-inflammatory activity
nitric oxide production
coumarin
2009 Elsevier Ltd. All rights reserved.
Perkin reaction
Inflammation is one of the protective immune responses which
occurs as a result of chemical, physical, immunological, and/or
biological stimuli to human tissue.1 Persistent and excessive
immune response can promote tissue damage, resulting in
chronic inflammation and generating a vicious cycle between
inflammation and the accompanying pathological state, such as
arteriosclerosis, periodontitis, allergic rhinitis, inflammatory
bowel disease, rheumatoid arthritis, Alzheimer’s disease and
cancer.2 The most widely explored targets for controlling
inflammation are inflammatory mediators.3 Nitric oxide (NO),
one of the important inflammatory mediators, was secreted by
activated immune cells (such as macrophages). High levels of
NO in chronic inflammation state can result in various
pathological conditions.4 Therefore, the development of new anti-
inflammatory agents for the control of NO production in immune
cells is the subject of interest in recent years.
Coumarins and their natural and/or synthetic derivatives are
biologically interesting compounds because of their biological
activities and pharmacological potentials. They have been
reported with anticancer, antioxidant, anti-inflammatory,
antimicrobial, antiviral and enzyme-inhibitory activities.5
Hyuganin A-D, anomalin and isoteryxin from the roots of
Angelica furcijuga KITAGAWA,6 fukanefuromarin D from the
roots of Ferula fukanensis7 and a series of coumarins from the
flowers of Mammea siamensis (Calophyllaceae)8 demonstrated
potent inhibitory activity on lipopolysaccharide (LPS)-induced
NO production in mouse macrophages (Figure 1). Recently,
Figure 1. Coumarins with potent inhibitory activity on LPS-induced NO
production in mouse macrophages
———
∗ Corresponding author. E-mail address: zhanggl@cib.ac.cn (G.-L. Zhang)

2
Table 1. Synthesis of 3-arylcoumarin via Perkin reactiona
Compound
R1
H
R2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
Ar (Phenyl)c
3,4-dimethoxyl
4-methoxyl
Yield (%)b
65
Compound
R1
H
R2
H
Ar (Phenyl)c
3,4-dimethoxyl
4-methoxyl
Yield (%)b
59
1
2
18
19
20
21
22
23
24
25
26
27
28
29
30
32
34
35
H
58
H
H
60
3
H
3-methoxyl
73
H
H
3-methoxyl
64
d
4
H
3,4,5-trimethoxyl
47
H
H
-
63
d
5
H
-
63
H
H
4-fluoro
3,4-dimenthoxyl
4-methoxyl
56
6
H
4-chloro
4-fluoro
60
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
66
7
H
56
64
8
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3,4-dimethoxyl
4-methoxyl
2-methoxyl
3,4,5-trimethoxyl
4-chloro
72
3-methoxyl
59
9
H
54
2-methoxyl
52
10
11
12
13
14
15
16
17
H
47
3,4,5-trimethoxyl
58
d
H
51
-
62
H
59
4-fluoro
3-acetoxyl
53
Cl
3,4-dimethoxyl
4-methoxyl
3-methoxyl
2-methoxyl
73
72
Cl
70
4-acetoxyl
74
Cl
67
2,5-dimethoxyl
pyridin-3-yl
69
Cl
46
55
d
Cl
-
54
aSee supplementary data for the general procedure of 3-arylcoumarin preparation. bIsolated yield. c3-Aryl of compound 35 is pyridine-3-yl. dNo substituents.
prenylated coumarins, columbianadin9 and glycycoumarin10,
showed moderate inhibitory activity and suggested the potential
to be developed into anti-inflammatory agents. However,
considering the pharmaceutical applications, these inhibitors
were not favorable as a result of limited natural abundance,
complicated structure and/or poor activity. Therefore, efforts are
still needed to develop new coumarin derivatives with both high
activity and production feasibility.
With considerable efforts to develop synthetic strategies,
coumarins can be prepared by Perkin, Pechmann, Knoevenagel,
Wittig, Kostanecki-Robinson or Reformatsky reactions.5 The
most wildly used method was Perkin reaction staring from
salicylaldehyde in the presence of base and acid anhydride.11 In
this work, we present the synthesis of manifold 3-aryl substituted
coumarins and their inhibitory activity on LPS-induced NO
production in RAW 264.7 cells.
The preparation of 3-arylcoumarin was performed via Perkin
reaction using salicylaldehydes and phenylacetic acids as starting
materials. In the presence of acetic anhydride and triethylamine,
the reaction mixture was stirred at 120℃ overnight and quenched
with ice water. After filtration, 3-arylcoumarins were obtained
OH
OAc
Br
Br
acetone, HCl aq.
80oC
O
O
O
O
OCH3
OCH3
30
31
: 3'-hydroxyl (93%)
33: 4'-hydroxyl (96%)
: 3'-acetoxyl
32: 4'-acetoxyl
Scheme 1. Acid-promoted hydrolysis of compounds 30 and 32
Figure 2. Inhibitory Effect of Compounds 16 and 25 on NO Production
Stimulated by LPS13

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Table 2. Inhibitory effects on LPS-Induced NO production and cytotoxity of compounds 1-35 in RAW264.7 cells at 5 mg/L13
Inhibition on LPS-
induced NO
production (%)
Inhibition on LPS-
induced NO
production (%)
Compounds
Cell viability (%)
Compounds
Cell viability (%)
Curcumina
77.04 2.22
32.33 5.41
44.58 10.28
91.36 2.78
52.01 9.96
78.4 3.09
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
47.36 8.26
59.64 5.72
49.23 1.24
69.45 2.18
9.98 4.06
53.52 2.6
44.25 14.66
72.28 2.08
47.65 13.86
47.28 12.69
111.38 0.2
33.88 9.61
84.13 2.7
115.72 0.4
77.78 1.22
91.33 2.06
45.86 9.08
58.65 11
47.03 6.17
93.44 5.1
1
2
87.71 0.25
81.29 1.92
34.19 10.37
54.4 3.04
48.44 2.47
103.88 2.87
110.45 6.93
98.03 0.06
99.88 2.36
116.42 6.7
95.72 6.06
115.19 2.09
29.62 2.16
102.58 1.06
66.48 1.71
28.46 0.45
61.72 5.68
24.45 5.14
93.53 3.01
84.87 1.62
3
4
5
64.46 5.95
93.54 0.52
126.61 1.1
105.34 3.8
101.85 14.14
115.38 9.99
78.41 13.06
113.58 0.79
117.83 7.63
99.31 0.63
105.26 24.2
119.17 1.67
103.52 1.07
6
61.74 5.39
47.61 11.18
31.25 2.55
47.53 0.85
65.73 8.18
57.62 2.77
12.11 20.16
27.27 15.12
20.79 10.12
35.99 4.71
76.9 1.52
7
8
9
10
11
12
13
14
15
16
17
53.38 8.19
The values are mean SEM (n = 3). aCurcumin was used as a positive control.
after purification with the yields from 46% to 74% (Table 1). 3-
Arylcoumarins 31 and 33 were further synthesized via acid-
promoted hydrolysis of 30 and 32, respectively (Scheme 1).
evaluated for their inhibitory effects on LPS-Induced NO
production and cytotoxic effects on RAW264.7 cells.
Compounds 16 and 25 showed potent inhibitory activity with the
IC50 values of 8.5 µM and 6.9 µM, respectively. Our results
suggested that it is possible to develop synthetic coumarins as
anti-inflammatory agents for related diseases of chronic
inflammation.
Compounds 1-35 were subsequently evaluated for their
inhibitory activity on LPS-induced NO production in RAW 264.7
cells. As shown in Table 2, compounds 3, 5, 16, 21, 25, 28 and
30-33 caused low percentage of NO production at 5 mg/L.
Simultaneously, cytotoxic effects of compounds 1-35 were
examined in mouse macrophage RAW264.7 cells. The results
revealed that, compounds 1, 2, 6-10, 12-17, 19, 21-27, 29, 34 and
35 had no cytotoxic activity in these cells at 5 mg/L, while
compounds 3, 18, 20, 28, 31 and 33 showed strong cytotoxicity.
With regard to these proofs, we believed that the low percentage
of NO production in the presence of compounds 3, 5, 28 and 30-
33 may be as a result of low cell viability of RAW 264.7 cells.
As shown in Figure 2, IC50 values of compounds 16 and 25 were
2.7 mg/L (8.5 µM) and 2.5 mg/L (6.9 µM), respectively, which
were comparable with the value of curcumin (6.2 µM, positive
control12). Considering reported anti-inflammatory coumarins
(Figure 1), these results demonstrated that anti-inflammatory
activity of coumarins could be maintained by introducing
aromatic groups into 3-position and the replacement of long
and/or asymmetric alkyl side chains with simple functional
groups. Low cell viability of compounds 31 and 33 (28.46% and
24.45%) indicated that the presence of hydroxyl on 3-aryl group
could cause intense cytotoxicity. 4′-Halogen substituted 3-
arylcoumarins resulted in poor inhibitory activities, as can be
seen from 12, 22 and 29 (12.11%, 9.98% and 33.88%,
respectively). The inhibitory activities of compounds 16 and 25
were 76.9% and 72.28%. Our results indicated that the
introduction of halogen into 6-position, substituents (other than
hydrogen) at 8-position as well as Ar group together could favor
the activity, which is in accord with Bansal’s speculation.3
Acknowledgments
This work was financially supported by National Natural
Science Foundation of China (Grants 20932007 and 21372213).
Supplementary Material
General synthetic procedures and spectral data (1H-NMR, 13C-
NMR and HRMS) associated with this article can be found in the
online version.
References and notes
1. Ferrero-Miliani, L.; Nielsen, O. H.; Anderson, P. S.;
Girardin, S. E. Clin. Exp. Immunol. 2007, 147, 227.
2. Khan, S.; Shin, E. M.; Choi, R. J.; Jung, Y. H.; Kim, J.
W.; Tosun, A.; Kim, Y. S. J. Cell. Biochem. 2011, 112,
2179.
3. Bansal, Y.; Sethi, P.; Bansal, G. Med. Chem. Res. 2013,
22, 3049.
4. Motai, T.; Kitanaka, S. Chem. Pharm. Bull. 2004, 52,
1215.
5. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.;
Uriarte, E. Curr. Med. Chem. 2005, 12, 887.
6. Matsuda, H.; Murakami, T.; Kageura, T.; Ninomiya, K.;
Toguchida, I.; Nishida, N.; Yoshikawa, M. Bioorg. Med.
Chem. Lett. 1998, 8, 2191.
In conclusion, a series of 3-arylcoumarins were prepared via
low-cost Perkin reaction (and further hydrolysis if necessary) and
7. Motai, T.; Daikonya, A.; Kitanaka, S. J. Nat. Prod.

4
2004, 67, 432.
compounds for 24 h. After AlamarBlue reagent was
added to each well, and fluorescence intensity was
measured with excitation at 544 nm and excitation at
590 nm using Thermo Scientific Varioskan Flash
Multimode Reader. Cytotoxicity was defined as the
ratio of the fluorescence intensity in test wells
8. Morikawa, T.; Sueyoshi, M.; Chaipech, S.; Matsuda, H.;
Nomura, Y.; Yabe, M.; Matsumoto, T.; Ninomiya, K.;
Yoshikawa, M.; Pongpiriyadacha, Y.; Hayakawa, T.;
Muraoka, O. Bioorg. Med. Chem. 2012, 20, 4968.
9. Zhang, Y. B.; Li, W.; Yang, X. W. Phytochemistry
2012, 81, 109.
compared to solvent control wells (0.1% DMSO). The
10. Fu, Y.; Chen, J.; Li, Y. J.; Zheng, Y. F.; Li, P. Food
Chemistry 2013, 141, 1063.
11. Shen, W.; Mao, J. F.; Sun, J.; Sun, M. J.; Zhang, C.
Med. Chem. Res. 2013, 22, 1630.
assay was conducted 3 times in triplicate. Measurement
of nitric oxide content: The RAW264.7 macrophages
were seeded in 96-well plates at 1 × 104 cells/well, and
pretreated with various concentrations of compounds
for 2 h, followed by 1 µg/mL LPS for an additional 24 h.
After that the cell culture medium was used for NO
measurements using a commercial available kit
(Beyotime, Haimen, China). Nitrite production was
measured at OD550. The assay was conducted 3 times in
triplicate. Percentage inhibition was calculated using the
following equation:
12. Lin, Y.; Wang, F.; Yang, L. J.; Chun, Z.; Bao, J. K.;
Zhang, G. L. Phytochemistry 2013, 95, 242.
13. Cell culture: RAW264.7 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM)
(Invitrogen, Carlsbad, CA, USA) supplemented with
1% penicillin/streptomycin and 10% fetal calf serum
(Gibco-Invitrogen). For experiments of nitric oxide
content, RAW264.7 cells were maintained in medium
devoid of Phenol Red (Invitrogen). Cell proliferation
assay: Cell proliferation was assayed as described
previously.14 In brief, 1 x 104 RAW264.7 cells was
seeded in 96-well plates, and treated with the
%Inhibition = (A – B)/(A – C) × 100
A: LPS (+), sample (-); B: LPS (+), sample (+); C: LPS
(-), sample (-)
14. Yang, L. J.; Chen, Q. F.; Wang, F.; Zhang, G. L. J.
Ethnopharmacol. 2011, 135, 553.

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3-Arylcoumarins: Synthesis and potent anti-
inflammatory activity
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Wenchen Pu, Yuan Lin, Jianshuo Zhang, Fei Wang, Chun Wang, Guolin Zhang
R1
CHO
OH
R1
Ar
O
Ac2O
+
HOOC
Ar
NEt3
O
R2
R2
35 examples
R1
Cl
R2
Cl
Inhibition on NO production(IC50
)
Cell viability (%)
119.17 1.67
8.5 μM
6.9 μM
±
Br OCH3
116.42 6.7
±
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