Inhibitory activity of prostaglandin E2 production by the synthetic 2′-hydroxychalcone analogues: Synthesis and SAR study

Article abstract of DOI:10.1016/j.bmcl.2009.02.001

A series of 2′-hydroxychalcones has been synthesized and screened for their in vitro inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells. Structure-activity relationship study suggested that inhibitory activity against prostaglandin E₂ production was governed to a greater extent by the substituent on B ring of the chalcone, and most of the active compounds have at least two methoxy or benzyloxy groups on B ring. The relationship between chalcone structures and their PGE₂ inhibitory activities was also interpreted by docking study on cyclooxygenase-2.

Full text of DOI:10.1016/j.bmcl.2009.02.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1650–1653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory activity of prostaglandin E2 production by the synthetic
20-hydroxychalcone analogues: Synthesis and SAR study
b
b
c
a,
Thanh-Dao Tran a, , Haeil Park , Hyun Pyo Kim , Gerhard F. Ecker , Khac-Minh Thai
*
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, 41 Dinh Tien Hoang, Dist. 1, Ho Chi Minh City, Viet Nam
b College of Pharmacy, Kangwon National University, Chuncheon 200-701, Republic of Korea
c Emerging Field Pharmacoinformatics, Department of Medicinal Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
A series of 20-hydroxychalcones has been synthesized and screened for their in vitro inhibitory activities
of cyclooxygenase-2 catalyzed prostaglandin production from lipopolysaccharide-treated RAW 264.7
cells. Structure–activity relationship study suggested that inhibitory activity against prostaglandin E2
production was governed to a greater extent by the substituent on B ring of the chalcone, and most of
the active compounds have at least two methoxy or benzyloxy groups on B ring. The relationship
between chalcone structures and their PGE2 inhibitory activities was also interpreted by docking study
on cyclooxygenase-2.
Article history:
Received 16 October 2008
Revised 8 January 2009
Accepted 2 February 2009
Available online 7 February 2009
Keywords:
Chalcone
Anti-inflammation
PGE2
Ó 2009 Elsevier Ltd. All rights reserved.
COX-2
Cytotoxicity
Chalcones, originally isolated from natural sources and consid-
ered as precursors of flavonoids and isoflavonoids, are abundant in
edible plants. Chalcones are open-chain flavonoids in which two
The preparation of the 20-hydroxychalcone derivatives (Table 1)
was carried out via Claisen–Schmidt condensation (Scheme 1).
Thus, an appropriate aryldehyde derivative was reacted with 20-
hydroxyl-acetophenone in MeOH/KOH to give the corresponding
20-hydroxychalcone precipitated as the potassium salt. Subsequent
treatment with HCl yielded the desired product (1–18) with an
average yield of 46–88% (Table 1).14,22 Their structures, established
with 1H NMR spectra, showed that the E-isomers were specifically
generated following the reaction. The physical and analytical data
for the new compounds 7 and 12 are presented in references and
notes.23,24
aromatic rings are joined by a three carbon
a,b-unsaturated car-
bonyl system (1,3-diphenyl-2-propen-1-ones). Being as a minority
subgroup of the flavonoid family, like other members, chalcones
have been reported responsible for a variety of biological activities,
including antiviral,1,2 anticancer,3–7 antimicrobial,8,9 anti-inflam-
matory,3,10–17 antioxidative,6,7 antimalarial,18 anti-leishmania,19
antinociceptive,20 and antiproliferative21 activities. Hence, chal-
cones are considered as a class of important therapeutic potentials.
With respect to anti-inflammatory activity, several studies indi-
cated that chalcone derivatives potently inhibit NO production by
the inducible nitric oxide synthase (iNOS) catalyzed NO production
by the different cellular mechanisms, iNOS down-regulation and/
or iNOS inhibition, depending on their chemical structures.12,15,16
However, chalcone derivatives also inhibited cyclooxygenase-2
(COX-2) catalyzed prostaglandin production14 as well as 5-
lipoxygenase.11
All synthesized 20-hydroxychalcone analogues were screened
for their activities on PGE2 production in the mouse macrophage-
like cell line RAW 264.7, stimulated by lipopolysaccharide (LPS)
at concentration of 10 l
M.14,25,26 The inhibitory activity of prosta-
glandin E2 (PGE2) production are contributed by two enzymes
including COX-2 and PG synthase (down-regulating COX-2 induc-
tion). However in this study, RAW cells were pre-incubated with
LPS for COX-2 induction, after that test compounds and arachi-
donic acid were added to examine the direct inhibitory activity
against COX-2.27 The inhibitory activities of synthetic chalcones
on COX-2 catalyzed PGE2 production from LPS-induced RAW
264.7 cells were estimated and shown in Table 1. Among 18 syn-
thesized chalcones, six compounds including 4-benzyloxy-20-
hydroxychalcone (9), 3,4-dibenzyloxy-20-hydroxychalcone (13),
3-benzyloxy-4-methoxy-20-hydroxychalcone (14), 2,3-dimethoxy-
20-hydroxychalcone (15), 2,4-dimethoxy-20-hydroxychalcone (16),
and 3,4,5-trimethoxy-20-hydroxychalcone (17) showed potential
In this study, various 20-hydroxychalcones with different sub-
stituents in the B ring were synthesized using a classical base cat-
alyzed condensation reaction and tested for anti-inflammatory
activities as well as for cytotoxicity.
* Corresponding authors. Tel.: +84 903 716482; fax: +84 8 3822 5435 (T.-D.
Tran); tel.: +84 909 680385; fax: +84 8 3822 5435 (K.-M. Thai).
E-mail addresses: thanhdaot@yahoo.com (T.-D. Tran), thaikhacminh@gmail.com,
thaikminh@yahoo.com (K.-M. Thai).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.001

T.-D. Tran et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1650–1653
1651
Table 1
Chemical structures, reaction conditions and biological activities of 20-hydroxychalcone analogues
a
Chalcone
B ring substituents
Yield (%)
Reaction conditions
Reaction time (h) Sol. for crystallization
PGE2 inhibition
IC50
(l
M)
Cell viability
@ 10
l
Ma (%)
@ 10 l
Ma (%)
R1
R2
R3
R4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
Br
Cl
Br
OBn
OBn
OCH3
H
H
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
46
85
58
74
65
58
68
63
58
66
88
65
62
59
58
67
55
59
—
16
12
24
16
24
12
12
12
16
12
12
12
10
16
28
12
16
12
—
MeOH
MeOH
MeOH/CH2Cl2
MeOH
MeOH
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH
MeOH/CH2Cl2
MeOH
60
60
24
62
32
62
18
11
102
51
48
55
100
93
101
98
102
< 0
109
103
—
—
—
—
—
—
—
—
4.1
—
—
—
4.6
—
3.8
—
6.2
—
0.05
1.07
119
141
162
152
115
85
87
135
99
CH3
OCH3
SCH3
OCF3
Ph
OBn
H
Cl
OCH3
OBn
OCH3
H
OCH3
OCH3
90
MeOH
155
159
103
102
93
107
65
118
104
111
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH/CH2Cl2
MeOH
17
18
OCH3
OCH3
H
—
—
H
—
—
–O–CH2–O–
NS-398b
Wogoninb
—
—
—
—
—
—
—
—
a
All values represented here were mean of three experiments.
Wogonin (5,7-dihydroxy-8-methoxyflavone) and NS-398 (N-(2-cyclohexyloxy)-4-nitromethanesulfonamide) were used as reference compounds.
b
R2
R2
OH R1
R3
R4
R1
H
R3
OH
CH3
KOH
R4 MeOH. rt
+
O
O
O
Scheme 1. Synthesis of 20-hydroxychalcone derivatives.
inhibitory activity against PGE2 production with inhibition values
larger than 90% (italicized in Table 1) and IC50 in range 3.8–
6.2 lM. In term of structure–activity relationship (SAR), most of
the active compounds possess at least two alkoxy groups (methoxy
and/or benzyloxy) in the B ring, except compound 9 which has an
unique benzyloxy group at 4-position.
The chalcone 9 (IC50 = 4.1 lM) also showed 1.6- to 9.2-fold
stronger of inhibitory activity against COX-2 catalyzed prostaglan-
din production than compounds 1–8 which possess a 4-substituent
of either hydro/chloro/bromo/methyl/methoxy/methylthio/trifluo-
romethy/phenyl group, respectively. Introducing either a strong
electron withdrawal (Cl, Br, CF3) group or a weak electron donating
group (SCH3, CH3, OCH3) to B ring of 20-hydroxychalcone (1) does
not influent PGE2 production inhibition. However, the presence
of a strong electron donating group at 4-position of 20-hydroxy-
chalcone (e.g., chalcone 9 containing benzyloxy moiety) exhibited
a stronger activity than that of other groups. Bioactivity results
indicated that the benzyloxy group at 4-position of 20-hydroxy-
chalcone contributed an important effect on the PGE2 inhibitory
activity. Docked chalcone-9:COX-2 (pdb 1cx2) complexes indi-
cated the importance of oxygen of the benzyloxy moiety at 4-posi-
tion of 20-hydroxychalcone (Fig. 1).28,29 This substituent leads to
formation of both hydrogen bond and/or p-cation interaction with
Arg120 residue, thus plays a critical role on the interaction be-
tween COX-2 and 9. Conversely, no hydrogen bond was observed
for chalcone 8 with the phenyl moiety at the same position versus
compound 9 that explained why chalcone 8 achieved a weak inhib-
itory activity of prostaglandin E2 production (Fig. 1).
Chalcone, containing more than two alkoxy groups on B ring in
which one group substituted at 4-position and the other substi-
tuted at 3- or 5-position of B ring may enhance inhibitory activity
toward PGE2 production. However, the presence of 3,4-dioxymeth-
Figure 1. Relative position of chalcone 9 (magenta carbon) and chalcone 8 (orange
carbon) in the active site of COX-2 generated by MOE docking (A). The hydrogen
bonds (magenta dotted lines) within the binding site are indicated for compound 9.
The benzyloxy moiety at 4-position of 20-hydroxychalcone established a strong
interaction with Arg120 of COX-2 via both H-bond and
p-cation interaction. 2D
interactions between 8 and COX-2 showed in (B) with no hydrogen bond observed.
ylene in B ring (18) destroyed anti-inflammatory activity of chal-
cones. Chalcone 17 (20-hydroxy-3,4,5-trimethoxychalcone) with

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T.-D. Tran et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1650–1653
Figure 2. Docked conformation alignment of chalcone 17 (magenta carbon) and its original scaffold 1 (orange carbon) in COX-2 binding site generated by MOE docking (left
side). In right side, 2D ligand-interactions between these chalcones and COX-2 are also showed. Three methoxy moieties presented in B ring of 17 contributed the additional
hydrogen bonds (magenta dotted lines) with Arg120 and Arg513 of COX-2.
three methoxy groups in B ring formed additional hydrogen bonds
(magenta dotted lines) with Arg120 and Arg513 of COX-2 in com-
parison with its original scaffold 1 (Fig. 2). At concentration of
tion; (ii) the benzyloxy moiety plays an important role on
establishing strong interactions between chalcone and COX-2;
(iii) the inhibition of PGE2 production from RAW 264.7 cells of 2-
hydroxychalcone derivatives is not associated with their cytotoxic-
ity. The SAR information is meaningful to design and develop new
compounds with higher anti-inflammatory activity.
10 lM, chalcone 17 markedly inhibit the PGE2 production
(102.3%). In addition by MTT cell viability assay,29 compound 17
also exhibited a cytotoxic effect that may provide a template to de-
sign new novels with a dual activity of anti-inflammation and anti-
cancer. Except chalcone 17, most synthetic 20-hydroxychalcones
did not show cytotoxicity or less than 10% cell reduction as as-
sessed by MTT assay indicating that they were not significant cyto-
toxicity against RAW 264.7 cells even in the presence or absence of
LPS (Table 1).30 Moreover, the significant increase of cell viability
by synthesized chalcones (1–5, 7, 11, 12, 16, 18) in the MTT test
were observed. Direct cell counting test is currently investigating
for these compounds to figure out which reason, due to an in-
creased cell number or an increased metabolism of the dehydroge-
nase, leads to the increase of cell viability.
Acknowledgments
We thank Ministry of Health & Welfare of Korea (to T.-D. Tran
and H. Park), ASIA UNINET (to T.-D. Tran) for financial support.
The authors wish to thank Dr. Dong Van Quyen, Dr. Chu Thi Thu
Trang, and Pharm. Le Thi Tuong Vy for critical reading and
discussion.
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In the literature, 5,7-diacetylflavone, which is obtained by
means of the subsequent ring closure of the corresponding 20-
hydroxychalcone, strongly inhibited COX-2 (IC50 = 2.7 l
M).31
Molecular modeling showed that 5,7-diacetylflavone fits well into
the binding pocket of COX-2 and their model suggested that a
hydrogen bond exists between the oxygen of the ketone group at
the 7-position and the hydroxy group of Tyr355.31 Docking into
the COX-2 active site, 20-hydroxychalcone analogues showed a
positioning similar to 5,7-diacetylflavone with A ring tend to place
near Tyr355 more than B ring. However, no hydrogen bond with
Tyr355 was formed for 20-hydroxychalcone derivatives. It seems
not surprising that the synthetic compounds do not contain any
substituent in A ring and also no hydrogen bond with Tyr355
was observed for other derivatives of chrysin.31 Conversely, hydro-
gen bonds were formed between those 20-hydroxychalcone com-
pounds and Arg120, Arg513.
In summary, 18 20-hydroxychalcone derivatives were synthe-
sized and evaluated for their PGE2 inhibitory activities and cyto-
toxicity. Among them, six chalcones showed better desired
biological activities than that of parent compound 20-hydroxychal-
cone. The structural requirements for the inhibitory activity of 20-
hydroxychalcone analogues on PGE2 production from RAW 264.7
cells may be drawn as follows: (i) the concomitance of at least
two alkoxy groups on B ring of chalcones in which one group
substituted at 4-position and the other substituted at 3- or 5-posi-
tion of B ring may enhance inhibitory activity toward PGE2 produc-
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26. RAW 264.7 cells obtained from American Type Culture Collection were
cultured with DMEM supplemented with 10% FBS and 1% CO2 at 37 °C and
activated with LPS (Escherichia coli O127:B8). All 20-hydroxychalcone
21. Daskiewicz, J. B.; Depeint, F.; Viornery, L.; Bayet, C.; Comte-Sarrazin, G.; Comte,
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analogues were screened at concentration of 10 lM for their activity on PGE2
production in RAW 264.7 cells stimulated by LPS. Briefly, cells were plated in
96-well plates (2 ꢀ 105 cells/well). Each synthetic chalcone was dissolved in
dimethyl sulfoxide (DMSO) and LPS (1 mg/mL) were added and incubated for
24 h to allow the expression of COX-2 and then, were washed with culture
22. General procedure: 20-hydroxyacetophenone (5 mmol) and benzaldehyde
derivatives (5 mmol) were dissolved in methanol (10 mL) with stirring.
Potassium hydroxide (15 mmol) was added in portions to give a blood-red
solution. Resulting solution was stirred for 8–28 h, during which 20-
hydroxychalcone precipitated as the potassium salt. The solution/suspension
was poured into cold 1 N HCl (10 mL), and further concentrated HCl was added
until the solution was acidic. The resulting yellow solid was filtered, washed
with water (2 ꢀ 20 mL), and recrystallized from corresponding solvent (MeOH
or MeOH/CH2Cl2) to give the product.
medium. Test compounds were added at 10 lM and incubated for 2 h in fresh
culture medium supplemented with arachidonic acid. PGE2 concentration in
the medium was measured using EIA kit for PGE2 according to the
manufacturer’s recommendation. All experiments were carried out at least
three times.
27. Han, K. H.; Son, M. J.; Chang, H. W.; Chi, Y. S.; Park, H.; Kim, H. P. Biol. Pharm.
Bull. 2005, 28, 1366.
28. Molecular Operating Environment MOE, Chemical Computing Group Inc.,
Montreal, H3A 2R7 Canada. http://www.chemcomp.com.
29. Molecular modeling and docking study. Preparation of molecular structures.
The 3D structure of chalcone derivatives were prepared using the build
molecule module in MOE. The structures of molecules are optimized by
23. 20-Hydroxy-4-trifluoromethoxychalcone (7): The product was obtained as yellow
solid, mp 83–85 °C. UV (MeOH, kmax): 203; 221.5 and 309.5 nm. IR (m )
cmꢁ1
1643.2; 1577.7; 1205.4; 1157.2; 754.1 cmꢁ1
.
1H NMR (200 MHz, CDCl3), d:
12.94 (s, 1H, 20-OH), 7.92–7.97 (d, 1H, J = 8.0 Hz, H60); 7.86–7.94 (d, 1H,
J = 15.4 Hz, Hb); 7.68–7.72 (d, 1H, J = 8.6 Hz, H40); 7.67–7.73 (m, 2H, J = 8.8 Hz,
2.8 Hz, H2, H6); 7.52–7.59 (d, 1H, J = 15.2 Hz, H ), 7.48–7.56 (d, 1H, J = 8.4 Hz,
energy minimization until converged to
a
maximum derivative of
a
1.6 Hz, H50); 7.26–7.30 (d, 1H, J = 8.2 Hz, H30); 6.92–7.06 (m, 2H, J = 8.8 Hz,
8.2 Hz, 8.2 Hz, 2.8 Hz, H2 and H6). 13C NMR (50 Hz, CDCl3, ppm), d: 194.2 (C4);
164.4 (C4 and C20); 144.3 (Cb); 137.3 (C40); 133.9 (C60); 130.8 (C1, C2, and C6);
0.001 kcal molꢁ1 Åꢁ1
.
The lowest-energy conformer of each molecule was
selected and stored in mdb database. Preparation of target enzyme structure
and docking. The X-ray crystal structure of COX-2:SC-558 complex (pdb
1cx2) was retrieved from the RCSB Protein Data Bank (www.rcsb.org). The
active site was defined as all the amino acid residues enclosed within 6.5 Å
radius sphere centered by the bound ligand, SC-558 (1-phenylsulfonamied-
3-trifluoromethyl-5-parabromophenyl-pyrazole, a selective COX-2 inhibitor)
and ‘site finder’ in MOE was used to determine the binding site. The docking
and subsequent scoring were performed using the MOE docking programs.
The final of 30 docked conformations per ligand were analyzed and used to
create the illustrative figures.
130.3 (C10) and 121.9 (C ); 121.7 (CF3); 120.6 (C50); 119.6 (C30); 119.4 (C3, C5).
a
Anal. (C16H11F3O2) C, H, O. m/z 309 (M+, 70), 308 (100), 307 (95), 188 (68), 147
(94), 121 (94), 120 (96), 101 (70).
24. 3-Bromo-20-hydroxy-4-methoxychalcone (12): The product was obtained as
yellow solid. Mp 137-139 °C. UV (MeOH, kmax): 204; 249.5; 339.5 and
354 nm. IR
(m )
cmꢁ1 1637.5, 1596.9; 1207.5, 1012.6; 767.6. 1H NMR
(200 MHz, CDCl3), d: 12.85 (s, 1H, 20-OH); 7.95–8.0 (dd, 1H, J = 8.8 Hz,
1.8 Hz, H60); 7.91–7.95 (d, 1H, J = 8.0H, 1.7 Hz, H2); 7.78–7.86 (d, 1H,
J = 15.6 Hz, Hb); 7.49–7.57 (d, 1H, J = 15.6 Hz, H ); 7.46–7.59 (m, 2H, H40,
30. Mossman, T. J. Immunol. Methods 1983, 65, 55.
31. Cho, H.; Yun, C.-W.; Park, W.-K.; Kong, J.-Y.; Kim, K. S.; Park, Y.; Lee, S.; Kim, B.-
K. Pharmacol. Res. 2004, 49, 37.
a
H6); 6.96–7.05 (m, 2H, H30, H50); 6.92–6.96 (d, 1H, J = 8.2 Hz, H5); 3.98 (s, 3H,
OCH3). Anal. (C16H13BrO3) C, H, O.