Design, synthesis, biological evaluation, and molecular docking of chalcone derivatives as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2016.12.008

In this study, two series of 35 new chalcone derivatives containing aryl-piperazine or aryl-sulfonyl-piperazine fragment were synthesized and their structures were characterized by¹H,¹³C and ESI-MS. The in vivo and in vitro anti-inflammatory activities of target compounds were evaluated by using classical para-xylene-induced mice ear-swelling model and ELISA assays. Furthermore, docking studies were performed in COX-2 (4PH9). The in vivo anti-inflammatory assays indicated that most of the target compounds showed significant anti-inflammatory activities. Docking results revealed that the anti-inflammatory activities of compounds correlated with their docking results. Especially, compound 6o exhibited the most potent anti-inflammatory activity in vivo with the lowest docking score of ?17.4?kcal/mol and could significantly inhibit the release of LPS-induced IL-6 and TNF-α in a dose-dependent manner in vitro.

Full text of DOI:10.1016/j.bmcl.2016.12.008

Accepted Manuscript
Design, synthesis, biological evaluation, and molecular docking of chalcone
derivatives as anti-inflammatory agents
Jingfen Li, Dong Li, Yiming Xu, Zhenbo Guo, Xu Liu, Hua Yang, Lisheng
Wang, Lichuan Wu
PII:
S0960-894X(16)31275-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.008
BMCL 24497
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 September 2016
12 November 2016
2 December 2016
Please cite this article as: Li, J., Li, D., Xu, Y., Guo, Z., Liu, X., Yang, H., Wang, L., Wu, L., Design, synthesis,
biological evaluation, and molecular docking of chalcone derivatives as anti-inflammatory agents, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, biological evaluation, and molecular docking of
chalcone derivatives as anti-inflammatory agents
Jingfen Li a,†, Dong Li b,†, Yiming Xu b, Zhenbo Guo b, Xu Liu b, Hua Yang b, Lisheng
Wang b,*, Lichuan Wu b,*
a
Department of Life Science, Huzhou Teachers’ College, Huzhou, 313000, People’s
Republic of China
b
School of Chemistry and Chemical Engineering, Guangxi University, Nanning,
530004, People’s Republic of China
† The authors contributed equally to this work
* Corresponding authors: Lisheng Wang, E-mail: w_lsheng@163.com, Daxue East
Road, No.100, Nanning, Guangxi 530004, PR China
Lichuan Wu, E-mail: wulichuan@126.com, Daxue East
Road, No.100, Nanning, Guangxi 530004, PR China
Abstract: In this study, two series of 35 new chalcone derivatives containing
aryl-piperazine or aryl-sulfonyl-piperazine fragment were synthesized and their
1
structures were characterized by H, 13C and ESI-MS. The in vivo and in vitro
anti-inflammatory activities of target compounds were evaluated by using classical
para-xylene-induced mice ear-swelling model and ELISA assays. Furthermore,
docking studies were performed in COX-2 (4PH9). The in vivo anti-inflammatory
assays indicated that most of the target compounds showed significant
anti-inflammatory activities. Docking results revealed that the anti-inflammatory
activities of compounds correlated with their docking results. Especially, compound
6o exhibited the most potent anti-inflammatory activity in vivo with the lowest
docking score of -17.4 Kcal/mol and could significantly inhibit the release of
LPS-induced IL-6 and TNF-α in a dose-dependent manner in vitro.
Keywords: inflammation, chalcone, derivatives, anti-inflammatory activity, COX-2
Inflammation is a complex biological process which seriously threatens human
health. Exaggerated and prolonged inflammation may cause various diseases, such as
arthritis, sepsis, and even cancer.1 At present, the most widely used drugs in treating

inflammation are non-steroidal anti-inflammatory drugs (NSAIDs), which account for
35% of the global market for prescription of pain medications.2-3 Common NSAIDs,
such as aspirin and indomethacin can inhibit both cyclooxygenase-1 (COX-1) and
cyclooxygenase-2 (COX-2)4-5, displaying significant toxicity to gastrointestinal tract
and kidney.6 For that reason, it is of great importance and urgent need to develop new
anti-inflammation drugs.7
Chalcone which belongs to a flavonoid family is widely distributed in plants such
as vegetables, fruits, tea and spices. It has been demonstrated that chalcone posses
many important bioactivities including anti-oxidant8, anti-cancer9, anti-bacterial10,
anti-fungal11, and anti-inflammation12. Recently, chalcone and its derivatives have
been reported to show anti-inflammatory activities in acute lung injury13-14 and could
protect liver from hepatic injury by inhibition of hepatic inflammation and fibrosis.15
Based on its favorable anti-inflammatory activity, chalcone framework has been used
for chemical modification to find novel derivatives with better pharmacological or
pharmacokinetic profiles.16
N-aryl piperazine moieties belong to an important class of organic compounds
which is widely used in medicinal chemistry.17 Recently, N-aryl piperazine derivatives
have been attracting considerable attentions for their versatile properties in
pharmacology18-19 and anti-inflammatory activities in vitro20 and in vivo21. Based on
these data, we introduced N-aryl piperazine moiety into the chalcone skeleton to
synthesize series I of chalcone derivatives. It is well recognized that selective COX-2
inhibitors (the coxibs) exhibit moderate gastrointestinal toxicity comparing with
traditional NSAIDs.22 In view of the structures of coxibs, we discover that the coxibs
bear an arylsulfonyl moiety (Fig. 1), implying that compounds bearing arylsulfonyl
moiety might show selective COX-2 inhibition activity. Thus, to make COX-2
selective compounds, series II was synthesized with an arylsulfonyl moiety insertion
based on the structures of compounds from series I. The anti-inflammatory activities
of these two series were screened in vivo and in vitro. Molecular modeling with
COX-2 was also studied.

Figure 1. Structures of selective COX-2 inhibitors (coxibs).
The synthesis of target compounds is outlined in Scheme 1. Condensation of
4-fluoro acetophenone (1) with substituted benzaldehyde 2a~2c in the presence of
sodium hydroxide solution in enthanol at room temperature afforded the substituted
chalcone 3a~3c.23 For series I containing aryl-piperazine fragment, intermediates
3a~3c reacted with substituted aryl-piperazine in the presence of DMF solution at 120
°C for 12-16 h to give compounds 4a~4s under nitrogen protection. For series II
containing aryl-sulfonyl-piperazine fragment, treatment of 3a~3c with piperazine by
heating in the presence of potassium carbonate under nitrogen protection provided the
intermediate products 5a~5c. Especially, the reaction time of this step must be
controlled under 5 to 6 h otherwise unprecedented complicated products obtained.
Then intermediates 5a~5c reacted with arenlsulfonyl to give compounds 6a~6p as
reported.24 The structures and characterization of target compounds were depicted in
Table 1 and supplementary information, respectively.

Scheme 1. Synthetic routes of chalcone derivatives. Reagents and conditions: (i) 10%
NaOH, EtOH, rt; (ii) aryl piperazine derivatives, K2CO3, DMF, N2, 12~16 h; (iii)
piperazine, K2CO3, 120 °C, N2, 4 h; (IV) (6a~6n): arenelsulfonyl chloride, K2CO3,
acetone, rt, 24h; (6o~6p): arenelsulfonyl chloride, K2CO3, DMF, 50 °C, 12 h.
Table 1. Structures of synthesized chalcone derivatives.
Comp
4a
4b
4c
R1
R2
3,4-Cl
4-NO2
2-F
Yields
58%
56%
53%
56%
63%
51%
58%
52%
55%
51%
57%
60%
57%
61%
56%
54%
58%
57%
Comp
4s
R1
R2
3-OCH3
4-Cl
Yields
57%
78%
77%
81%
68%
71%
79%
80%
81%
75%
76%
78%
69%
66%
73%
76%
72%
-
4’-Cl
-
4’-Cl
6a
6b
6c
-
4’-Cl
4-CF3
-
1-Benzyl
2,4-CH3
3-Cl
-
4-CH3
4d
4e
-
-
4-Cl
6d
6e
-
-
3-(CH=CH)-4
4-Br
4f
-
2,3-Cl
3-OCH3
4-CH3
2-Cl
-
4g
4h
4i
6f
-
-
4-OCH3
2,4,6-CH3
-
6g
6h
6i
-
-
4j
-
-
-
3-CF3
-
-
4-OCF3
2,4,6-isopropyl
4-Butyl
4-CF3
4k
4l
6j
-
-
6k
6l
3’,4’-OCH3
3,4-Cl
-
4m
4n
4o
4p
4q
4r
3’,4’-OCH3 2,4-CH3
-
6m
6n
6o
6p
4’-Cl
4’-Cl
4’-Cl
4’-Cl
2,4-CH3
3-Cl
-
3,4-OCH3
4-Br
3’,4’-OCH3
3’,4’-OCH3
4-OCH3
2-OCH3
4-OCH3
The in vivo anti-inflammatory activities of target compounds were evaluated using
in vivo para-xylene-induced mice ear-swelling model. All the target compounds were
administered at a dose of 40 mg/kg and aspirin (100 mg/kg) and celecoxib (5 mg/kg)
were used as reference drugs. The results showed that for series I, only compounds 4b,

4m, 4n, and 4q exhibited significant anti-inflammatory activities (p < 0.05). While for
series II, almost all the derivatives displayed significant anti-inflammatory activities
except compound 6l (p < 0.05) (Table 2). Compound 6o displayed the most potent
anti-inflammatory activity which was better than that of aspirin and even equal to that
of celecoxib. Preliminary structure activity relationship (SAR) analysis indicated that:
1) In view of the structures of compounds 4i with 6c and 4l with 6i, we found
compounds 6c and 6i contained an extra sulfonyl moiety over compounds 4i and 4l,
respectively. The in vivo anti-inflammatory activities of these compounds were 6c >>
4i, 6i >> 4l. These results implied that sulfonyl group could improve compounds
anti-inflammatory activity. 2) Comparing compounds 4b and 4i, the in vivo
anti-inflammatory activity was 4b >> 4i. Comparing compounds 6d, 6f, 6g, 6j, 6m
and 6c, 6l, the in vivo anti-inflammatory activities were 6d, 6f, 6g, 6j, 6m, 6m > 6c, 6l.
These results implied that substitution at 4 position of aryl-piperazine or
aryl-sulfonyl-piperazine with electron-withdrawing group showed advantage over
electron-donating group in improving compound’s anti-inflammatory activity. 3)
Comparing compounds 4f and 4p, 4h and 4s, the in vivo anti-inflammatory activities
were 4f > 4p, 4h > 4s. These results indicated that substitution at 4’ position of
benzene ring could not benefit from improving compound’s anti-inflammatory
activity.
Table 2. Anti-inflammatory acitivites of chalcone derivatives
Compound
Dose
(mg/kg)
Swelling
degree (mg)
Inhibition
(%)
Docking
Score
p value
(Kcal/mol)
7.0 1.6
1.6 0.7
2.5 0.8
5.5 0.7
6.1 1.5
3.1 1.3
7.0 3.4
control
celecoxib
aspirin
chalcone
4a
5
100
40
40
40
40
77.03
64.91
21.68
13.41
55.35
0.57
-14.53
-9.47
p < 0.001
p < 0.001
p = 0.07
p = 0.36
p < 0.001
p = 0.98
-9.25
-11.12
-13.22
-10.57
4b
4c

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
5.8 1.0
6.0 0.7
6.0 1.4
4.9 2.4
5.9 0.8
6.8 1.3
6.7 2.8
5.3 3.1
6.8 2.0
2.6 0.8
3.6 1.9
5.2 2.6
6.6 2.8
3.2 1.5
6.6 1.2
6.9 2.2
3.0 1.4
2.8 1.0
4.9 0.9
3.7 2.1
4.6 0.9
3.7 0.9
3.8 0.9
3.1 1.1
4.9 0.7
3.7 2.6
2.8 0.7
5.3 3.0
3.7 0.8
17.69
14.98
14.12
30.67
15.98
2.85
-10.37
-11.27
-11.18
-11.12
-10.75
-10.81
-10.20
-12.03
-10.31
-14.99
-13.33
-12.35
-10.90
-13.44
-11.31
-12.08
-13.04
-13.56
-11.50
-11.33
-12.52
-12.64
-12.57
-13.01
-11.20
-12.86
-13.92
-12.68
-12.98
p = 0.15
p = 0.19
p = 0.29
p = 0.10
p = 0.17
p = 0.82
p = 0.80
p = 0.21
p = 0.81
p < 0.001
p = 0.008
p = 0.17
p = 0.77
p < 0.001
p = 0.61
p = 0.88
p < 0.001
p < 0.001
p = 0.01
p = 0.005
p = 0.007
p = 0.02
p = 0.02
p < 0.001
p = 0.01
p = 0.02
p < 0.001
p = 0.25
p < 0.001
4d
4e
4f
4g
4h
4i
4.71
4j
24.96
3.71
4k
4l
63.48
48.50
25.68
5.85
4m
4n
4o
4p
4q
4r
4s
55.06
6.13
2.28
56.63
59.63
30.81
46.79
34.95
47.36
45.22
55.49
29.96
47.36
60.06
23.97
47.93
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m

40
40
40
3.1 1.1
1.7 0.8
2.5 1.0
55.49
75.46
64.05
-12.99
-17.40
-15.98
p < 0.001
p < 0.001
p < 0.001
6n
6o
6p
The statistical significance was calculated between each compound group and control
group by using student’s t-test of unpaired data. P values < 0.05 were considered
statistically significant.
The structure and favorable in vivo anti-inflammatory activities of compounds led
us to study the molecular docking with COX-2 enzyme. The results indicated that the
anti-inflammatory activities of compounds might correlate with their docking results
with COX-2 (Table 2). Compound 6o, 6p, 4m, and 6k which had a lower docking
score than others displayed better anti-inflammatory activities than other compounds.
Especially, compound 6o had the lowest docking score of -17.4 Kcal/mol and the best
anti-inflammatory activity (inhibition rate, 75.46%) which showed two hydrogen
bonds (H-bonds) and two chemical bonds of arene-cation with COX-2 (Fig.2 and Fig.
3). H-bonds were formed between oxygen atom of methoxyl group with amino acid
Tyr386 and Ser531 at distances of 2.46 and 2.78 Å, respectively. The intensities of
H-bonds between compound 6o with COX-2 were 98% and 67%, respectively.
Chemical bonds of arene-cation were formed between benzene ring of compound 6o
and amino acid Arg121 of COX-2. To validate the results of molecular docking,
further enzyme studies on COX-2 are needed.
Figure 2. 2D docking model of compound 6o with COX-2.

Figure 3. 3D docking model of compound 6o with COX-2.
Considering the favorable in vivo anti-inflammatory activity and docking results in
silico, we chose compound 6o for further studies. To reveal the cytotoxity of
compound 6o, MTT assay was conducted in mouse RAW264.7 macrophages. The
results indicated that compound 6o showed no significant cytotoxity with a
concentration less than 50 µM (Fig. 4a). To further detect the effects of compound 6o
on pro-inflammatory factors (TNF-α and IL-6) secretion, RAW264.7 macrophages
were pretreated with different doses (10, 20, 40 µM) of compound 6o for 2 h and then
exposed to LPS (1 µg/ml) for additional 22 h. The levels of TNF-α and IL-6 in the
media were determined. The results displayed that compound 6o significantly
inhibited TNF-α and IL-6 release by RAW264.7 macrophages in a dose-dependent
manner (Fig. 4b).
Figure 4. Cytotoxicity and in vitro anti-inflammatory activity of compound 6o. (a)
MTT assays of compound 6o in mouse RAW264.7 macrophages. (b) Compound 6o
inhibited LPS-induced TNF-α and IL-6 release by RAW264.7 macrophages in a
dose-dependent manner in mouse RAW264.7 macrophages.

In summary, two series of 35 new chalcone derivatives containing aryl-piperazine
or aryl-sulfonyl-piperazine fragment were synthesized. The in vivo anti-inflammatory
results revealed that most of the target compounds exhibited potent anti-inflammatory
activity. Especially, compound 6o exhibited the most potent anti-inflammatory
activity in vivo and could significantly inhibit the release of LPS-induced IL-6 and
TNF-α by RAW264.7 macrophages in a dose-dependent manner in vitro. Furthermore,
docking results indicated that the anti-inflammatory activities of compounds
correlated with their docking results. Our finding might provide information on
developing potentially new and safe anti-inflammatory agents.
Acknowledgements
The work was supported by the Natural Science Foundation of Zhejiang Province of
China（No.Y12H300003）, the high level innovation team and outstanding scholar
project of Guangxi institutions of higher education (gui jiao ren [2014] 49 hao), and
the Postdoctoral Science Foundation of Guangxi Province of China (Y201001928).
References
1. Tilg, H.; Moschen, A. R. Nature reviews. Immunology 2006, 6.
2. Melnikova, I. Nature reviews. Drug discovery 2010, 9.
3. Velazquez, C. A.; Chen, Q. H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J Med
Chem 2008, 51.
4. Dannhardt, G.; Kiefer, W.; Kramer, G.; Maehrlein, S.; Nowe, U.; Fiebich, B. Eur J
Med Chem 2000, 35.
5. Dubost, J. J.; Soubrier, M.; Sauvezie, B. Rev Med Interne 1999, 20.
6. Uchoa Fde, T.; da Silva, T. G.; de Lima Mdo, C.; Galdino, S. L.; Pitta Ida, R.;
Dalla Costa, T. J Pharm Pharmacol 2009, 61.
7. Nagarapu, L.; Mateti, J.; Gaikwad, H. K.; Bantu, R.; Sheeba Rani, M.; Prameela
Subhashini, N. J. Bioorg Med Chem Lett 2011, 21.
8. Awasthi, S. K.; Mishra, N.; Kumar, B.; Sharma, M.; Bhattacharya, A.; Mishra, L.
C.; Bhasin, V. K. Med Chem Res 2009, 18.
9. Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Nakanishi, Y.; Lee, K. H. Bioorganic

& Medicinal Chemistry Letters 2000, 10.
10. Kaur, K.; Kumar, V.; Gupta, G. K. J Fluorine Chem 2015, 178.
11. Lopez, S. N.; Castelli, M. V.; Zacchino, S. A.; Dominguez, J. N.; Lobo, G.;
Charris-Charris, J.; Cortes, J. C. G.; Ribas, J. C.; Devia, C.; Rodriguez, A. M.; Enriz,
R. D. Bioorganic & Medicinal Chemistry 2001, 9.
12. Hsieh, H. K.; Tsao, L. T.; Wang, J. P.; Lin, C. N. J Pharm Pharmacol 2000, 52.
13. Zhang, Y.; Wu, J.; Ying, S.; Chen, G.; Wu, B.; Xu, T.; Liu, Z.; Liu, X.; Huang, L.;
Shan, X.; Dai, Y.; Liang, G. Sci Rep 2016, 6.
14. Chen, W.; Ge, X.; Xu, F.; Zhang, Y.; Liu, Z.; Pan, J.; Song, J.; Dai, Y.; Zhou, J.;
Feng, J.; Liang, G. Bioorg Med Chem Lett 2015, 25.
15. Singh, H.; Sidhu, S.; Chopra, K.; Khan, M. U. Can J Physiol Pharmacol 2016,
94.
16. Chamni, S.; De-Eknamkul, W. Expert Opinion on Therapeutic Patents 2013, 23.
17. Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem Rev 2003, 103.
18. Zhao, H.; Prosser, A. R.; Liotta, D. C.; Wilson, L. J. Bioorg Med Chem Lett 2015,
25.
19. Popovic, M.; Stanojevic, Z.; Tosic, J.; Isakovic, A.; Paunovic, V.; Petricevic, S.;
Martinovic, T.; Ciric, D.; Kravic-Stevovic, T.; Soskic, V.; Kostic-Rajacic, S.; Shakib,
K.; Bumbasirevic, V.; Trajkovic, V. J Neurochem 2015, 135.
20. Gouault, N.; Pinel, B.; Cupif, J. F.; Depince, A.; Martin-Chouly, C. A.; Belleguic,
C.; David, M. J Enzyme Inhib Med Chem 2004, 19.
21. Dundar, Y.; Gokce, M.; Kupeli, E.; Sahin, M. F. Arzneimittel-Forschung 2007, 57.
22. Patrignani, P.; Patrono, C. Biochim Biophys Acta 2015, 1851.
23. Ajay, A.; Singh, V.; Singh, S.; Pandey, S.; Gunjan, S.; Dubey, D.; Sinha, S. K.;
Singh, B. N.; Chaturvedi, V.; Tripathi, R.; Ramchandran, R.; Tripathi, R. P.
Bioorganic & Medicinal Chemistry 2010, 18.
24. Abdel-Aziz, A. A. M.; Asiri, Y. A.; Al-Agamy, M. H. M. European Journal Of
Medicinal Chemistry 2011, 46.