2’-Hydroxychalcone Analogues: Synthesis and Structure-PGE2 Inhibitory Activity Relationship

Hydroxychalcone Analogues: Synthesis and
Structure-PGE2 Inhibitory Activity Relationship


Chalcones, originally isolated from natural plant sources, considered as precursors of flavonoids and
isoflavonoids are abundant in edible plants. Chemically, chalcones are open-chain flavonoids in which
two aromatic rings are joined by a three carbon , -unsaturated carbonyl system (1,3-diphenyl-2-
propen-1-ones). Being a majority subgroup of the flavonoid family, chalcones have been reported to
have a variety of biological activities, including antiviral and anticancer,1-3 anti-microbial,4, 5 antiinflammatory,
3, 6, 7 anti-ulcer and spasmolytic8, 9 and antiproliferative10 activities. Hence, chalcones is
considered as a class with important therapeutic potentials.
In this study, various 2’-hydroxychalcones having different substituents in B-ring were synthesized
using a classical base catalyzed condensation reaction and tested for anti-inflammatory activity via the
inhibition of PGE2 production from RAW 264.7 cells as well as for cytotoxicity at concentration of 10
μM.
RESULTS AND DISCUSSION
Chemistry
The preparation of the 2’-hydroxychalcone derivatives (Table 1) was carried out via Claisen-Schmidt
condensation (Scheme 1). Thus, an appropriate aryldehyde derivaties was reacted with 2’-
hydroxyacetophenone in MeOH/KOH to give the corresponding 4’,6’-diprotected-2’-
hydroxychalcones precipitated as the potassium salt. Subsequent treatment with an HCl yielded the
desired product (CH.01-CH.20) with an average yield of 45-80% (Table 1). Their structures
established with 1H-NMR spectra showed that the E-isomers were specifically generated via this
reaction. The spectral data of the synthesized compounds as well as detailed procedure for the
synthesis are presented in experimental section.
OH
O
R1
R2
R3
R4
OH
O
CH3
R1
R2
R3
R4
O
H
+
KOH
MeOH. rt
Scheme 1. Synthesis of 2’-hydroxychalcone derivatives
 Chemical structures, yields and reaction conditions of 2’-hydroxychalcone analogues
Product
B-ring substituents Yield
(%)
Reaction Conditions
R1 R2 R3 R4 Reaction time (h) Sol. for crystalization
CH.01 H H Cl H 85 12 MeOH
CH.02 H Cl Cl H 88 12 MeOH
CH.03 H H H H 46 16 MeOH
CH.04 H H OCH3 H 65 24 MeOH
CH.05 H H CH3 H 74 16 MeOH
CH.06 H H Br H 58 24 MeOH and CH2Cl2
CH.07 H OCH3 OBn H 59 24 MeOH and CH2Cl2
CH.08 H OBn OBn H 62 10 MeOH and CH2Cl2
CH.09 H H OBn H 58 16 MeOH and CH2Cl2
CH.10 H OBn OCH3 H 59 16 MeOH and CH2Cl2
CH.11 H OCH3 OCH3 OCH3 55 16 MeOH and CH2Cl2
CH.12 OCH3 OCH3 H H 58 28 MeOH and CH2Cl2
CH.13 H OCH3 OCH3 H 56 28 MeOH and CH2Cl2
CH.14 OCH3 H OCH3 H 67 12 MeOH and CH2Cl2
CH.15 H H SCH3 H 58 12 MeOH and CH2Cl2
CH.16 H H OCF3 H 68 12 MeOH and CH2Cl2
CH.17 H Br OCH3 H 65 12 MeOH and CH2Cl2
CH.18 H -O-CH2-O- H 59 12 MeOH
CH.19 H H Ph H 63 12 MeOH
CH.20 H Br H H 66 12 MeOH
Biological activities
All synthesized 2’-hydroxychalcone analogues were screened for their activity on PGE2 production in
RAW 264.7 cells stimulated by lipopolysaccharide (LPS) at concentration of 10 M. The inhibitory
activities of synthetic chalcones on cyclooxygenase-2 (COX2) catalyzed PGE2 production from LPSinduced
RAW 264.7 cells were estimated and shown in Table 2. In addition, MTT cell viability assay
also performed and all tested compounds showed no or less than 10% reduction indicating that those
compounds were not significantly cytotoxicity to RAW 264.7 cells in the presence or absence of LPS
(Table 2).
Table 2. Biological activities of chalcone analogues
Chalcone
Biological activities (10 μM)
Chalcone
Biological activities (10 μM)
PGE2 inhibition (%)a Cell viability (%)a PGE2 inhibition (%)a Cell viability (%)a
CH.01 59.68 140.5 CH.11 102.3 64.90
CH.02 48.05 154.6 CH.12 100.9 92.80
CH.03 60.30 119.1 CH.13 26.59 88.20
CH.04 32.22 115.3 CH.14 98.30 107.3
CH.05 62.07 152.2 CH.15 62.12 85.00
CH.06 23.56 161.5 CH.16 18.10 86.70
CH.07 < 0 102.0 CH.17 54.62 158.8
CH.08 99.56 102.7 CH.18 < 0 117.6
CH.09 101.7 98.90 CH.19 10.86 135.3
CH.10 92.9 102.4 CH.20 51.23 90.20
NS-398b 109.1 103.6 Wogoninb 102.6 111.1
aAll values represented here were arithmetic of duplicate.
bWogonin (5,7-dihydroxy-8-methoxyflavone) and NS-398 (N-(2-cyclohexyloxy)-4-nitromethanesulfonamide) were used
as reference compounds.
These compounds inhibited PGE2 production at 10 M concentration with values in percentage (%)
range. In those 20 synthesized chalcones, 6 compounds including CH.08-12 and CH.14 were
indicated as the most potential inhibited PGE2 production (inhibition values larger than 90% were
underlined in Table 2). In term of structure-activity relationship (SAR), most of active compounds
possess more than two alkoxy groups (methoxy and/or benzyloxy) in the B ring, excepting the CH.09
having an unique benzyloxy group at 4-position. The chalcone CH.09 also showed three-fold stronger
than that observed for CH.04 (possessing a methoxy group at the same position).
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The 2’-hydroxychalcone analogues possessing the unique substituent at 4-position of B-ring such as
CH.01, CH.04-06, CH.15, CH.17, CH.19 (having a substituent either chloro/
methoxy/methyl/bromo/methylthio/trifluoromethyl/phenyl group, respectively) showed the same or
less inhibitory activity than non-substituted 2’-hydroxychalcone (CH.03). Introducing either a strong
electron withdrawal (Cl, Br, CF3) group or a weak electron donating group (SCH3, CH3, OCH3) to
ring B of 2’-hydroxychalcone (CH.03) does not indicate the effect on PGE2 inhibition. However, a
strong electron donating group at 4-position of 2’-hydroxychalcone (for example chalcone CH.09
containing benzyloxy moiety) exhibited a stronger activity than that of other 4-substituted-2’-
hydroxychalcone analogues. Bioactivity results indicated that the benzyloxy group at 4-position of 2’-
hydroxychalcone was contributed an important effect on the PGE2 inhibitory activity. Docked
CH.09:COX2 complexes indicated the important of oxygen of the benzyloxy moiety at 4-position of
2’-hydroxychalcone (Figure 1). This substituent formed both hydrogen bond and -cation interaction
with Arg120 thus plays the major role on the interaction between COX2 and CH.09. Conversely, no
hydrogen bond was observed for compound CH.19 with phenyl moiety at the same position versus
CH.09 that explained a weak anti-inflammatory activity (Figure 1).
Figure 1. Relative position of CH.09 (magenta carbon) and CH.19 (orange carbon) in the active site of COX2 generated by
MOE docking (left side). 2D interactions between CH.19 and COX2 is showed in right side with no H-bond observed. The
hydrogen bonds (magenta dotted lines) within the binding site are indicated for compound CH.09. The benzyloxy moiety
at 4-position of 2’-hydroxychalcone established a strong interaction with Arg120 of COX2 via both H-bond and -cation
interaction.
Compound CH.11 (2’-hydroxy-3,4,5-trimethoxychalcone) with 3 methoxyl groups in ring B formed
additional hydrogen bonds (magenta dotted lines) with Arg120 and Arg513 of COX2 in comparison
with its original scaffold CH.03 (Figure 2). At concentration of 10μM, CH.11 proved strong effect to
inhibit the PGE2 production (102.3%). However, CH.11 also indicated the effect on cell viability that
provided a template to design new novels with a dual activity of anti-inflammation and anticancer.
Except CH.11, most synthetic 2’-hydroxychalcones did not showed cytotoxicity which no or less than
10% cell reduction during MTT assay. Hence, the inhibition of PGE2 production by chalcone
derivatives might be not associated with their cytotoxicity at 10 μM.
In summary, twenty 2’-hydroxychalcone derivatives were synthesized and evaluated for their PGE2
inhibitory activity and cytotoxicity. Among them, six chalcones showed better biological activities
than that of 2’-hydroxychalcone. The structural requirements for the inhibitory activity of 2’-
hydroxychalcone analogues on PGE2 production from RAW 264.7 cells are drawn as follow: (i) the
concomitance of more than two alkoxy groups on B ring in which one group sustituted at 3-position
and the other substituted at 2- or 4-position of B-ring may enhance inhibitory activity of PGE2
production; (ii) the benzyloxy plays a role as good legand, that established strongly interactions
between compound and drug target; (iii) the inhibition of PGE2 from RAW 264.7 cells is not
associated with their cytotoxicity.
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Figure 2. Docked conformation alignment of CH.11 (magenta carbon) and its original scaffold CH.03 (orange carbon) in
COX2 binding site generated by MOE docking (left side). In right side, 2D ligand-interactions between these chalcones
and COX2 are also showed. Three methoxyl moieties presented in ring B of CH.11 contributed the additional hydrogen
bonds (magenta dotted lines) with Arg120 and Arg513 of COX2.
EXPERIMENTAL
Chemistry
All chemicals were obtained from commercial suppliers, and used without further purification. All
solvents used for reaction were freshly distilled from proper dehydrating agent under nitrogen gas. All
solvents used for chromatography were purchased and directly applied without further purification.
1H-NMR spectra were recorded on a Varian Gemini 2000 instrument (200 MHz) spectrometer.
Chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an
internal standard. Peak splitting patterns are abbreviated as m (multiplet), s (singlet), bs (broad
singlet), d (doublet), bd (broad doublet), t (triplet) and dd (doublet of doublets). Analytical thin-layer
chromatography was performed using commercial glass plate with silica gel 60F254 purchased from
Merck.
General procedure: 2'-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 8-12 hours, during which 2'-
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 x 20 ml), and recrystallized from corresponding
solvent (MeOH or MeOH/CH2Cl2) to give the product (Table 1).
CH.01: 4-chloro-2’-hydroxychalcone. Yellow crystals, m.p. 139 oC. UV (methanol, max): 203.5; 225 and 319 nm. IR
(KBr, cm-1): 1639.4; 1564, 752.8 cm-1. 1H-NMR (200 MHz, CDCl3), δ: 12.76 (s, 1H, 2’-OH), 7.84-7.92 (d, 1H, J = 15.6
Hz, Hβ,); 7.63-7.67 (d, 2H, J = 8 Hz, H3 and H5); 7.44-7.51 (d, 1H, J = 15.6 Hz, Hα); 7.48-7.67 (m, 3H, H2, H6 and H6’);
7.40-7.44 (m, 3H, H3’, H4’ and H5’); 6.92-7.06 (m, 2H, H3 and H5). Anal. C,H,O.
CH.02: 3,4-dichloro-2’-hydroxychalcone. Yellow crystals, m.p. 142 oC. UV (methanol, max): 204, 249 and 351 nm. IR
(KBr, cm-1): 1645.2, 1589, 750.3 cm-1. 1H-NMR (200 MHz, CDCl3,), δ: 12.68 (s, 1H, 2’-OH ), 7.85-7.94 (t, 2H, J = 8.4
Hz, 16.2 Hz, Hβ and H6’), 7.78 (s, 1H, H2), 7.60-7.67 (d, 1H, J = 15.6 Hz, Hα), 7.46-7.59 (m, 3H, H4’, H5 and H6), 6.94-
7.04 (m, 2H, H3’and H5’). Anal. C,H,O.
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CH.03: 2’-hydroxychalcone. Yellow crystals, m.p. 78 oC. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.78 (s, 1H, 2’-OH),
7.91-7.96 (d, 1H, J = 7.6 Hz, and 1.8 Hz, H6’); 7.91-7.96 (dd, 1H, J = 14.4 Hz, Hβ), 7.35-7.70 (m, 7H, Hα, H4’, H2, H3,
H4, H5, H6); 7.02-7.09 (m, 2H, H3’, and H5’). Anal. C,H,O.
CH.04: 4-methoxy-2’-hydroxychalcone. Yellow crystals, m.p. 94 oC; UV (methanol, max): 205; 240; 339,5 and 365
nm. IR (KBr, cm-1): 1641.5, 1608.9; 1211.5, 827,4; 763 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.95 (s, 1H, 2’-OH),
7.88-7.96 (d, 1H, J = 15.2 Hz, Hβ), 7.87-7.91 (d, 1H, J = 7.4 Hz, H6’); 7.59-7.66 (d, 1H, J = 15.2 Hz, Hα); 7.62-7.66 (d,
2H, J = 8.8 Hz, H2 and H6); 7.45-7.49 (t, 1H, J = 8.8 Hz, and 7.6 Hz, H4’); 6.90-7.05 (m, 4H, H3, H5, H3’ and h5’); 3.87
(s, 3H, OCH3). Anal. C,H,O.
CH.05: 4-methyl-2’-hydroxychalcone. Yellow solid, m.p.120 oC. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.88 (s, 1H, 2’-
OH), 7.87 – 9.95 (d, 1H, J = 15.2 Hz, Hβ); 7.91-7.95 (d, 1H, J = 9.2 Hz, H6’); 7.58-7.66 (d, 1H, J = 15.2 Hz, Hα); 7.46-
7.59 (m, 3H, J = 7.8 Hz, 8.0 Hz, 1.2 Hz, H2, H4’and H6); 7.22-7.26 (d, 2H, J = 8.0 Hz, H3 and H5); 6.90-7.01 (m, 2H, J =
8.0 Hz, 1.0 Hz, H3’and H5’); 2.40 (s, 3H, CH3). Anal. C,H,O.
CH.06: 4-bromo-2’-hydroxychalcone. Yellow solid, m.p. 136 oC. UV (methanol, max): 203; 223.5; and 320 nm. IR
(KBr, cm-1): 1639; 1575; 1205.5; 758 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.87 (s, 1H, 2’-OH), 7.92 – 8.0 (d, 1H, J
= 15.6 Hz, Hβ); 7.92-7.96 (d, 1H, J = 7.6 Hz, H6’); 7.68-7.76 (d, 1H, J = 15.6 Hz, Hα); 7.55-7.65 (m, 3H, J = 7.8 Hz, 8.0
Hz, H2, H4’ and H6); 7.92-7.96 (m, 2H, J = 8.2 Hz, H3 and H5); 6.90-7.05 (m, 2H, J = 8.0 Hz, H3’ and H5’). Anal.
C,H,O.
CH.07: 4-benzyloxy-3-methoxy-2’-hydroxychalcone. Yellow solid. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.87 (s, 1H,
2’-OH), 8.17-8.25 (d, 1H, J = 15.6 Hz, Hβ); 7.90-7.94 (d, 1H, J = 8.0 Hz, H6’); 7.50-7.72 (d, 1H, J = 15.6 Hz, Hα); 7.27-
7.31 (t, 1H, J = 6.2 Hz, 7.8 Hz, 1.8 Hz, H4’), 7.20-7.43 (m, 7H, H4’, H5’, Aryl); 6.90-7.05 (m, 3H, H2, H5 and H6), 5.25
(s, 2H, Ar-CH2-); 3.95 (s, 3H, OCH3). Anal. C,H,O.
CH.08: 3,4-dibenzyloxy-2’-hydroxychalcone. Yellow solid. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.91 (s, 1H, 2’-OH),
7.85-7.89 (d, 1H, J = 7.2 Hz, H6’); 7.78-7.89 (d, 1H, J = 15.2 Hz, Hβ); 7.43-7.47 (d, 1H, J = 7.2 Hz, H4’); 7.39-7.47 (d,
1H, J = 15.2 Hz, Hα), 7.20-7.36 (m, 12H, H3’, H5’, 2xAryl); 6.95-7.05 (m, 3H, H2, H5 and H6), 5.24 (s, 4H, 2xAr-CH2-).
Anal. C,H,O.
CH.09: 4-benzyloxy-2’-hydroxychalcone.Yellow solid, m.p. 103-105 oC; UV (methanol, max): 204; 241; 379 nm. IR
(KBr, cm-1): 1637.5, 1562.2; 1174.6; 821.6; 765.7 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.94 (s, 1H, 2’-OH), 7.91-
7.95 (d, 1H, J = 8.6 Hz, H6’); 7.87-7.95 (d, 1H, J = 14.8 Hz, Hβ); 7.62-7.66 (d, 1H, J = 8.2 Hz, H4’); 7.59-7.66 (d, 1H, J =
14.8 Hz, Hα), 7.74-7.51 (m, 7H, H3’, H5’, Aryl); 6.93-7.05 (m, 4H, J = 8.8 Hz, 8.2 Hz, 8.2 Hz, 2.8 Hz, H2, H3, H5 and
H6), 5.14 (s, 2H, Ar-CH2-). Anal. C,H,O.
CH.10: 3-benzyloxy-4-methoxy-2’-hydroxychalcone. Yellow solid, m.p. 105 oC. 1H-NMR (200 MHz, CDCl3), δ ppm:
12.93 (s, 1H, 2’-OH), 7.91-7.95 (d, 1H, J = 7.4 Hz, H6’); 7.82-7.92 (d, 1H, J = 15.2 Hz, Hβ); 7.47-7.55 (d, 1H, J = 15.2
Hz, Hα), 7.20-7.43 (m, 7H, H4’, H5’, Aryl); 6.90-7.05 (m, 3H, H2, H5 and H6), 5.25 (s, 2H, Ar-CH2-); 3.95 (s, 3H,
OCH3). Anal. C,H,O.
CH.11: 3,4,5-trimethoxy-2’-hydroxychalcone. Yellow solid, m.p. 157 oC; UV (methanol, max): 215.5; 252; 339.5 and
363.5 nm. IR (KBr, cm-1): 1637.5, 1566.7; 1296.1; 1026.1; 835.1; 769.5 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.84
(s, 1H, 2’-OH), 7.92-7.96 (d, 1H, J = 7.8 Hz, H6’); 7.83-7.90 (d, 1H, J = 14.4 Hz, Hβ); 7.50-7.58 (d, 1H, J = 14.4 Hz, Hα),
7.48-7.53 (d, 1H, H4’); 6.93-7.06 (m, 2H, H3’ and H5’); 6.90 (s, 2H, H2’ and H6’); 3.95 (s, 9H, 3xOCH3); 3.92 (s, 3H,
OCH3). Anal. C,H,O.
CH.12: 2,3-dimethoxy-2’-hydroxychalcone. Yellow solid, m.p. 107 oC. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.89 (s,
1H, 2’-OH), 8.18-8.26 (d, 1H, J = 15.6 Hz, Hβ); 7.91-7.95 (d, 1H, J = 8.2 Hz, H6); 7.72-7.80 (d, 1H, J = 15.6 Hz, Hα),
7.47-7.55 (t, 1H, H4’); 7.28-7.32 (d, 1H, H3’); 6.92-7.16 (m, 4H, H4, H5. H6 and H6’); 3.92 (s, 6H, 2xOCH3). Anal.
C,H,O.
CH.13: 3,4-dimethoxy-2’-hydroxychalcone. Yellow solid, m.p. 115 oC. IR (KBr, cm-1): 1629.7; 1598.9; 1028; 744.5. 1HNMR
(200 MHz, CDCl3,, δ ppm: 12.88 (s, 1H, 2’-OH), 8.18-8.26 (d, 1H, J = 15.6 Hz, Hβ); 7.91-7.95 (d, 1H, J = 8.2 Hz,
H6’); 7.49-7.57 (d, 1H, J = 15.4 Hz, Hα); 7.46-7.54 (m, 1H, J = 8.6 Hz, J = 1.6 Hz, H4’); 7.18-7.30 (m, 2H, J = 8.2 Hz, 1.8
Hz, H3’ and H5’); 6.90-7.05 (m, 3H, H2, H4, H5); 3.97, 3.95 (s, 6H, 2x OCH3). Anal. C,H,O.
CH.14: 2,4-dimethoxy-2’-hydroxychalcone. Yellow solid, m.p. 111 oC; UV (methanol, max): 208; 258.5 and 383.5 nm.
IR (KBr, cm-1) 1629.7; 1560.3; 1203.5; 1024.1; 769.5 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 13.10 (s, 1H, 2’-OH),
8.14-8.22 (d, 1H, J = 15.4 Hz, Hβ); 7.90-7.94 (d, 1H, J = 8.2 Hz, H6’); 7.64-7.75 (d, 1H, J = 15.4 Hz, Hα); 7.58-7.62 (d,
1H, J = 8.6 Hz, H6); 7.44-7.52 (m, 1H, J = 8.0 Hz, H4’); 6.99-7.03 (d, 1H, J = 8.6 Hz, H5’); 6.93-6.97 (d, 1H, J = 8.4 Hz,
H3’); 6.55-6.59 (d, 1H, J = 7.6 Hz, H5), 6.50 (ds, 1H, J = 1.4 Hz, H3); 3.93, 3.87 (s, 6H, 2x OCH3). Anal. C,H,O.
CH.15: 4-methylthio-2’-hydroxychalcone. Yellow solid, m.p. 85 oC; UV (methanol, max): 203; 253 and 375.5 nm. IR
(KBr, cm-1) 1637.5, 1566.7; 1201.5; 812.0; 758 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.87 (s, 1H, 2’-OH); 7.90-
7.95 (dd, 1H, J = 7.8 Hz, 1.8 Hz, H6’); 7.86-7.94 (d, 1H, J = 15.6 Hz, Hβ); 7.58-7.65 (d, 1H, J = 15.6 Hz, Hα); 7.46-7.65
(m, 3H, H2, H4’, H6); 7.25-7.29 (d, 2H, J = 8.6 Hz, 2.0 Hz, H3, H5); 6.91-7.05 (m, 2H, H3’ and H5’); 2.53 (s, 3H, SCH3).
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Anal. C,H,O.
CH.16: 4-trifluoromethoxy-2’-hydroxychalcone. Yellow solid, m.p. 81 oC; UV (methanol, max): 203; 221.5 and 309.5
nm. IR (KBr, cm-1): 1643.2; 1577.7; 1205.4; 1157.2; 754.1 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.94 (s, 1H, 2’-
OH), 7.92-7.97 (d, 1H, J = 8.0 Hz, H6’); 7.86-7.94 (d, 1H, J = 15.4 Hz, Hβ); 7.68-7.72 (d, 1H, J = 8.6 Hz, H4’); 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, Hα), 7.48-7.56 (d, 1H, J = 8.4 Hz, 1.6 Hz, H5’); 7.26-
7.30 (d, 1H, J = 8.2 Hz, H3’); 6.92-7.06 (m, 2H, J = 8.8 Hz, 8.2 Hz, 8.2 Hz, 2.8 Hz, H2 and H6). Anal. C,H,O.
CH.17: 3-bromo-4-methoxy-2’-hydroxychalcone. Yellow solid, m.p. 137-139 oC; UV (methanol, max): 204; 249.5;
339.5 and 354 nm. IR (KBr, cm-1) 1637.5, 1596.9; 1207.5, 1012.6; 767.6. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.85 (s,
1H, 2’-OH); 7.95-8.0 (dd, 1H, J = 8.8 Hz, 1.8 Hz, H6’); 7.91-7.95 (d, 1H, J = 8.0H, 1.7 Hz, H2); 7.78-7.86 (d, 1H, J =
15.6 Hz, Hβ); 7.49-7.57 (d, 1H, J = 15.6 Hz, Hα); 7.46-7.59 (m, 2H, H4’, H6); 6.96-7.05 (m, 2H, H3’, H5’); 6.92-6.96 (d,
1H, J = 8.2 Hz, H5); 3.98 (s, 3H, OCH3). Anal. C,H,O.
CH.18: 3,4-dioxymethylene-2’-hydroxychalcone. Yellow solid, m.p. 108 oC; UV (methanol, max): 208; 266 and 373
nm. IR (KBr, cm-1) 1641.3, 1566; 1242.1; 1037.6; 759.9 cm-1. 1H-NMR (200 MHz, CDCl3), δ ppm: 12.90 (s, 1H, 2’-OH);
7.92-7.96 (dd, 1H, J = 8.2 Hz, 1.4 Hz, H6’); 7.82-7.89 (d, 1H, J = 15.2 Hz, Hβ); 7.68-7.72 (m, 1H, H4’); 7.46-7.54 (d, 1H,
J = 15.2 Hz, Hα), 6.84-7.15 (m, 5H, H2, H3’, H5, H5’, H6); 6.0 (s, 2H,-O-CH2-O-). Anal. C,H,O.
CH.19: 4-phenyl-2’-hydroxychalcone. Yellow solid, m.p. 147 oC; UV (methanol, max): 204; 249 and 351 nm. IR (KBr,
cm-1) 1637.5, 1571.9; 1203.5, 989,4; 756 cm-1. 1H-NMR (200 MHz, CDCl3, ppm), δ ppm: 12.71 (s, 1H, 2’-OH); 7.88-7.95
(d, 1H, J = 14.0 Hz, Hβ); 7.87-7.91 (d, 1H, J = 7.6 Hz, H6’); 7.68 (s, 1H, H2); 6.93-7.60 (m, 7H, Hα
, H4, H5, H3’, H4’,
H5’, H6). Anal. C,H,O.
CH.20: 3-bromo-2’-hydroxychalcone. Yellow solid, m.p. 139 oC; UV (methanol, max): 206 and 308 nm. IR (KBr, cm-
1): 1641.3; 1577.7; 1205.4; 1022.2; 761 cm-1. 1H-NMR (200 MHz, CDCl3, ppm), δ ppm: 12.88 (s, 1H, 2’-OH); 7.93-8.0
(dd, 1H, J = 15.6 Hz, Hβ); 7.92-7.96 (d, 1H, J = 8.0 Hz, H6’); 7.69-7.76 (d, 1H, J = 15.6 Hz, Hα), 7.38-7.62 (m, 9H, H2,
H3, H5, H6, Aryl); 6.99 -7.05 (m, 1H, J = 8.2 Hz, H5’); 6.92-6.96 (d, 1H, J = 8.0 Hz, H3’). Anal. C,H,O.
Biological Assays
PGE2 inhibitory assay. The PGE2 inhibitory assay was performed according to the previous published
procedure.11 RAW 264.7 cells obtained from American Type Culture Collection were cultured with
DMEM supplemented with 10% FBS and 1% CO2 at 37 oC and activated with LPS (Escherichia coli
O127:B8). All 2’-hydroxychalcone analogues were screened at concentration of 10 M for their
activity on PGE2 production in RAW 264.7 cells stimulated by LPS. Briefly, cells were plated in 96-
well plates (2x105 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 COX2 and then,
were washed with culture medium. Test compounds were added at 10 M 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 twice and they gave similar results.
Cytotoxicity. Cell viability was assessed with MTT assay based on the experimental procedures
described previously.12
Molecular modeling and docking study
Preparation of molecular structures. The 3D structure of 20 chalcones were prepared using the build
molecule module in MOE.13 The structures of molecules are optimized by energy minimization until
converged to a maximum derivative of 0.001 kcal mol-1 Å-1. 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 COX2:L-758048
complex (pdb 2cx2) was retrieved from the RCSB Protein Data Bank.14 The active site was defined as
all the amino acid residues enclosed within 6.5Å radius sphere centered by the bound ligand, L-
758048 (a benzyl-indole COX2 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.13 The
final of 30 docked conformations per ligand were analyzed and used to create the illustrative figures.
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ACKNOWLEDGEMENTS
We thank Ministry of Health & Welfare of Korea (to TDT and HP) and ASIA UNINET (to TDT)
for financial support. We also thank Prof. Huyn Pyo Kim at Kangwon National University for the use
of bioassay facilities.