Structural requirement of chalcones for the inhibitory activity of interleukin-5

Article abstract of DOI:10.1016/j.bmc.2006.10.007

Novel chalcones were found as potent inhibitors of interleukin (IL)-5. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (2b, 78.8% inhibition at 50 μM, IC₅₀ = 25.3 μM) was initially identified as a potent inhibitor of IL-5. Th

Full text of DOI:10.1016/j.bmc.2006.10.007

Bioorganic & Medicinal Chemistry 15 (2007) 104–111
Structural requirement of chalcones for the inhibitory
activity of interleukin-5
Hyun-Mo Yang,a Hye-Rim Shin,a Soo-Hyun Cho,a Seong-Cheol Bang,a Gyu-Yong Song,a
Jung-Hun Ju,b Mi-Kyeong Kim,c Seung-Ho Lee,d Jae-Chun Ryu,e
Youngsoo Kimb and Sang-Hun Junga,*
aCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
bCollege of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University,
Cheongju 361-763, Republic of Korea
cCollege of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea
dCollege of Pharmacy, Yeungnam University, Kyungsan, Republic of Korea
eToxicology Laboratory, Korea Institute of Science and Technology, Seoul 130-650, Republic of Korea
Received 14 August 2006; revised 3 October 2006; accepted 6 October 2006
Available online 10 October 2006
Abstract—Novel chalcones were found as potent inhibitors of interleukin (IL)-5. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxy-
phenyl)-2-propen-1-one (2b, 78.8% inhibition at 50 lM, IC50 = 25.3 lM) was initially identified as a potent inhibitor of IL-5. This
shows the compatible activity with budesonide or sophoricoside. To identify structural requirements, 26 chalcones were prepared
and their inhibitory activities were tested against IL-5. Among them, compound 4-[(E)-3-(2-cyclohexylmethoxy-6-hydroxyphe-
nyl)-3-oxoprop-1-enyl]benzenesulfonamide (2w, 99.5% inhibition at 50 lM, IC50 = 1.8 lM) shows the most potent activity. The
important structural requirements of these chalcone analogs exhibiting the inhibitory activity against IL-5 were recognized as the
following. (1) The hydrophobic group such as benzyloxy or cyclohexylmethoxy at 6-position of A ring is necessary. (2) The existence
of phenolic hydroxyl at 6-position of A ring is critical. (3) Propenone unit as a,b-unsaturated ketone is essential. (4) Electron with-
drawing groups with hydrogen acceptor property at 4-position of B ring enhance the activity and quantitative structure–activity
relationship of 2 regarding these substituents was determined.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
IL-5Ra by itself binds to IL-5 with low affinity.8 Even
though IL-5Rb alone does not bind to IL-5, it is re-
quired for high affinity binding in combination with
IL-5Ra and is essential for signal transduction.9
IL-5Rb is shared by IL-3 and GM-CSF receptors as
the common signal transducer, and thereby these cyto-
kines display several overlapping biological effects.10,11
Rather than coordinating T-cell effector development
or antibody isotype secretion by B-cells, biological ef-
fects of IL-5 are confined primarily to growth, differen-
tiation, survival, chemotaxis, and activation of
eosinophils.12–14. The contribution of IL-5 to allergic
disease has been greatly demonstrated by the generation
of mice deficient in the IL-5 gene. In contrast to normal
mice, allergic IL-5-deficient mice do not generate eosin-
ophilia in the bone marrow, blood or lung in response to
allergen provocation.15 Airway instillation of recombi-
nant IL-5 to allergic IL-5-deficient mice completely re-
stores allergen-induced eosinophilia to levels normally
Eosinophilic inflammation the main histological corre-
late of airway hyperresponsiveness and tissue injury in
the pathogenesis of bronchial asthma.1,2 There is strong
evidence for a central role of T helper type 2 (Th2) cyto-
kines that contributes importantly to diseases such as
asthma, and therapeutic strategies that target the Th2
cytokines are of potential benefit in allergic disease.3,4
Interleukin (IL)-5 appears to be one of the main proin-
flammatory mediators among a growing number of
cytokines and chemokines that induce eosinophilic
inflammation.5,6 IL-5 displays its cellular response by
binding to a specific receptor (IL-5R), composed
of two distinct polypeptides of a and b chains.7 The
Keywords: Chalcones; Inhibitor; SAR; Interleukin-5.
*
Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823
6566; e-mail: jungshh@cnu.ac.kr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.007

H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
105
R1
O
R3
R1
O
A
B
R3
R2
OH
R2
O
1a: Sophoricoside R1, R2 = OH, R3= OGlu
1b: R1 = benzyloxy, R2 = H, R3 = OH
1c: R1 = H, R2 = benzyloxy, R3 = OH
2: substituents listed in
Table 1.
O
O
O
O
OH
OH
OH
O
O
4
3
Figure 1. Isoflavonoids (1) and chalcone analogs.
observed in allergic wild-type mice.16 Thus, IL-5 is crit-
ically involved in eosinophilia-associated allergic
inflammation.
mation, thus possibly acting nearly without side effects.
However small organic compounds to inhibit IL-5 activ-
ity have been rarely found. The isothiazolones were
identified as the IL-5 antagonists through modification
of Cys66 in IL-5Ra.17 Sophoricoside (1a) and its ana-
logs (Fig. 1) were isolated from Sophora japonica, a
plant of Leguminosae family, as inhibitors of interleukin
(IL)-5 bioactivity,18 and showed differential inhibition
on IL-3 and GM-CSF bioactivities.19 During the study
of structure–activity relationship of sophoricoside
Interfering with the action of IL-5 represents one of the
new immunomodulatory therapeutic strategies in the
treatment of allergic diseases including bronchial asth-
ma. Compared to established immunosuppressive
agents like corticosteroids, a major advantage of this
strategy is the specificity of reducing eosinophilic inflam-
Table 1. Inhibitory activity of chalcone analogs against IL-5
% inhibition at 50 lMa
IC50 (lM)
a
Entry
Compound
Substituents
R1
R2
R3 (rp)
1
2a
2b
H
H
H
H
4-OH
4-OH
50.0 2.2
78.8 1.4
47.8 4.6
77.6 1.4
99.0 0.5
13.0 2.7
59.7 1.8
100.0 0.0
100.0 0.0
100.0 0.0
98.6 0.1
100.0 0.0
98.1 1.1
ꢀ18.9 0.1
ꢀ18.7 0.1
99.6 1.7
98.8 0.0
16.4 11.8
99.2 0.1
82.3 0.6
99.3 0.0
99.8 0.1
99.5 0.0
99.5 0.0
15.0 1.0
15.6 0.1
70.3 2.1
48.2
25.3
>50
28.8
12.6
>50
21.4
10.2
14.2
9.4
2
BNOb
BNO
3
2c20
4-OCH2OCH3
4-OH
4
2d
2e
H
BNO
H
5
CHMOc
CHMO
H
4-OH (ꢀ0.37)
4-OCH2OCH3
4-OH
6
2f
2g
H
CHMO
7
8
2h
2i
BNO
H
H
4-COOH
9
BNO
H
4-COOH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
29
2j
2k
CHMO
CHMO
H
4-COOH (0)f
4-COOCH3 (0.45)
4-COOH
H
3.9
2l
2m
CHMO
H
10.8
14.1
>50
>50
14.3
7.1
CHMO
CHMO
H
3-COOH
2-COOH
2n
2o
H
H
4-COOH
H (0)
2p
2q
CHMO
CHMO
CHMO
CHMO
CHMO
CHMO
CHMO
CHMO
BNO
H
H
Cl (0.06)
4-NH2
2r
H
>50
6.4
2s
H
4-NHCOCH3 (0)
4-CH2CH3 (ꢀ0.15)
4-CH2OH (0)
4-CHO (0.42)
4-SO2NH2 (0.57)
4-SO2NH2
4-COOH
2t
2u
H
27.9
6.4
H
2v
2w
H
4.5
1.8
H
2x
3d
H
7.0
BNO
CHMO
H
>50
>50
26.8
4e
Budesonide
H
4-COOH
a Data are means SD obtained from triplicate experiments.
b BNO, benzyloxy.
c CHMO, cyclohexylmethoxy.
d Compound 3; 4-[(E)-3-(2-benzyloxyphenyl)-3-oxoprop-1-enyl]benzoic acid.
e Compound 4; 4-[3-(2-cyclohexylmethoxyphenyl)-3-oxoprpyl]benzoic acid.
f rp value (0) for carboxylate anion was used for finding equation 1 as a real form of carboxylic acid under the bioassay condition.

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H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
analogs,20 chalcones 2a–2d had been prepared as inter-
mediates for the preparation of the corresponding isofl-
avonones and their inhibitory effects on IL-5 bioactivity
have been evaluated as shown in Table 1. The inhibitory
was prepared by the catalytic hydrogenation of 2j
(Scheme 2).
activity of 2b (78.8% inhibition at 50 lM, IC50
25.3 lM) and 2d (77.6% inhibition at 50 lM, IC50
=
=
3. Pharmacology
28.8 lM) are compatible with the most potent 5-benzyl-
oxy-3-(4-hydroxyphenyl)chromen-4-one (1b, 87.9% inhi-
bition at 50 lM, IC50 = 15.3 lM).20 Although a number
of chalcones had been investigated for the various bio-
logical properties such as anticancer,21–23 enzyme inhib-
itors,24,25 inhibitor of IL-1 biosynthesis,26 and
antimalarials,27 chalcones have never been investigated
as an inhibitor of IL-5. Thus a number of chalcone
derivatives 2, 3 and its saturated analog 4 were prepared
and their inhibitory activities were tested against IL-5
bioactivity for establishing the structure–activity
relationship.
The measurement of inhibitory activity of IL-5 of these
analogs was performed with the comparison of cell pro-
liferation of Y16 cell with/without the samples.18 An
Y16 cell line originated from murine early B cell is pro-
liferated in the presence of IL-5, which was used as the
parameter of IL-5 bioactivity. Non-radioactive proce-
dures that measure cell metabolism as an index of
proliferation have now become popular. 2-(4-Iodophe-
nyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetra-
zolium sodium salt (WST-1) was used to form soluble
formazan product on exposure to the dehydrogenase
activity in metabolizing cells. Proliferation of Y16 cells
in the presence of IL-5 was specifically blocked by treat-
ment of polyclonal antibody against IL-5 or monoclonal
antibody against IL-5Ra. Sample and IL-5 were simul-
taneously added to the Y16 cells, and the bioassay sys-
tem can identify the IL-5 antagonist and its signal
inhibitor. The results of biological screening of chalcone
analogs against IL-5 are listed in Table 1 as % inhibition
at 50 lM and IC50 values.
2. Chemistry
Preparation of 2 was accomplished by the aldol con-
densation of substituted 2-hydroxyacetophenones 520
and appropriate benzaldehydes 6 in the presence of
sodium hydroxide or potassium hydroxide in ethanol
at 50 ꢁC for 2 h as illustrated in Scheme 1.20 The sub-
stituents of 2 are listed in Table 1. Trans stereochem-
istry of propenone moiety of 2 was confirmed by the
coupling constants of vinyl hydrogens (15–16 Hz).
Acetophenones 5 used in the preparation of 2 were
prepared by the partial alkylation of 2,6 (or 4)-dihydr-
oxyacetophenone.20 Most benzaldehydes 6 used in
preparation of 2 are commercially available. 4-Form-
ylbenzenesulfonamide used for the preparation of 2w
and 2x was synthesized by van Es’s procedure.28 Com-
pound 2k was prepared by esterification of 2j. Com-
pound 2s was converted to 2r by the hydrolysis.
Chalcone 3 was obtained by the reaction of 2-benzyl-
oxyacetophenone and 4-formylbenzoic acid using the
same procedure for the preparation 2. Compound 4
4. Results and discussion
Considering the activity of budesonide (70.3% inhibition
at 50 lM, IC50 = 26.8 lM) being used for the treatment
of chronic asthma, the activities of 2b (78.8% inhibition
at 50 lM, IC50 = 25.3 lM) and 2d (77.6% inhibition at
50 lM, IC50 = 28.8 lM) appear to be very potent. To
explore structure–activity relationship of 2, benzyloxy
group was initially varied. Omission of benzyloxy group
as shown in 2a and 2o reduces the activity. Cyclohexyl-
methoxy analog 2e (99.0% inhibition at 50 lM,
IC50 = 12.6 lM) shows two times more potency than
2-benzyloxy analog 2b. The same trend appears in
R1
O
R1
O
KOH
O
+
Methanol
R3
R3
H
R2
OH
R2
OH
6
2
5
Scheme 1. Preparation of chalcone analogs.
O
O
O
O
H2/Pd-C
OH
OH
OH
OH
O
O
4
2j
Scheme 2. Reduction of 2j.

H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
107
comparison of 4-cyclohexylmethoxy analog 2g and 4-
benzyoxy analog 2d. Regardless of their positions on
A ring, the hydrophobic groups on A ring obviously
produce favorable effect for the enhancement of activity
of these series. Compounds 2b and 2e containing 2-alk-
oxy group have slightly better activity than 2d and 2e
possessing 4-alkoxy group. Thus 2-alkoxy-6-hydroxy
analogs 2 were mostly prepared and tested.
To identify the role of hydroxyl group on A ring of 2,
compound 3 without hydroxyl group on A ring was pre-
pared. This compound shows very weak activity (15.6%
inhibition at 50 lM, IC50 > 50 lM). Therefore hydroxyl
function on A ring of 2 is essential for the activity.
To determine the role of propenone system for the activ-
ity of 2, compound 2j was converted to its propanone
analog 4 by the catalytic hydrogenation. Inhibitory
activity of 4 (15.0% inhibition at 50 lM, IC50 > 50 lM)
against IL-5 was abolished. Thus the a,b-unsaturated
ketone of 2 is critical for the activity.
Substituents at 4-position of B ring greatly influence the
change of activity of 2. Comparison of the activities of 2j
(4-carboxylic acid analog), 2m (3-carboxylic acid ana-
log), and 2n (2-carboxylic acid analog) indicates that car-
boxylic acid at 4-position of B ring most enhances the
activity. Therefore, 4-substituted derivatives of 2 were
mainly investigated. Replacement of hydroxyl group of
2b or 2e with electron withdrawing group capable to be
hydrogen bonding acceptor such as carboxylic acid (2h
and 2j), carboxylic ester (2k), aldehyde (2v), chloro
(2q), and sulfonamide (2w, 2x) increases the activity.
The activities of compounds 2e, 2j, 2k, 2q, 2s, 2u, 2v,
and 2w are well correlated with Hammet parameter
(rp)29 as shown in Eq. 1. In case of 2j, rp value (0) for car-
boxylate anion was used for finding equation 1 as a real
form of carboxylic acid under the bioassay condition.
5. Conclusion
Novel chalcones were prepared and identified as potent
inhibitors of IL-5 bioactivity. Among them, compound
2w (4-[(E)-3-(2-(cyclohexylmethoxy)-6-hydroxyphenyl)-
3-oxoprop-1-enyl]benzenesulfonamide) is the most ac-
tive derivative (99.5% inhibition at 50 lM,
IC50 = 1.8 lM). The structural requirement of chalcone
analogs possessing the potent inhibitory activity against
IL-5 could be summarized as the following. (1) The
hydrophobic group such as benzyloxy or cyclohexyl-
methoxy at 2-position of A ring is necessary. (2) The
existence of phenolic hydroxyl at 6-position of A ring
is critical. (3) Propenone motif as a,b-unsaturated ke-
tone is essential. (4) Electron withdrawing groups with
hydrogen acceptor property at 4-position of B ring as
small as three-atom size enhance the activity. The quan-
titative structure–activity relationship of 2 regarding
these substituents was well determined.
log 1=IC50 ¼ 0:763rp ꢀ 0:86ðn ¼ 8; R2 ¼ 0:86Þ
ð1Þ
Despite rp values (0) of H in 2o and ethyl in 2t being the
same as those for COOꢀ in 2h and NHCOCH3 in 2s, the
activities of 2o (99.6% inhibition at 50 lM,
IC50 = 14.3 lM) and 2t (82.3% inhibition at 50 lM,
IC50 = 27.9 lM) are much weaker than those of 2j and
2s. These differences imply that substituents at 4-position
of B ring of 2 should have strong hydrogen acceptor prop-
erty in hydrogen bonding in addition to large electron
withdrawing property for the potent inhibitory activity
against IL-5 induced proliferation of eosinophil. The
activity of 2k with methyl ester was much more enhanced
compared to that of the corresponding carboxylic acid
analog 2j. Methyl ester has larger rp compared to
caboxylate anion and hydrogen acceptor property with-
out ability of hydrogen donor. Thus this also supports
that these electron withdrawing and hydrogen acceptor
properties of substituents are important for the activity
of 2.
6. Experimental
6.1. Chemistry
Melting points (mp) were determined on Electrothermal
1A 9100 MK2 apparatus and are uncorrected. All com-
mercial chemicals were used as obtained and all solvents
were purified by the standard procedures prior to use.30
Thin-layer chromatography was performed on E Merck
silica gel GF-254 precoated plates and the identification
was done with UV light and colorization with spray 10%
phosphomolybdic acid followed by heating. Flash col-
umn chromatography was performed with E. Merck sil-
ica gel (230-400mesh). IR spectra were recorded with
Jasco IR-Report-100 IR spectrometer in cmꢀ1 and cor-
rected against peak at 1601 cmꢀ1 of polystyrene. 1H
NMR spectra were measured against the peak of tetra-
methylsilane by Varian Unity Inova 400 NMR
(400 MHz) spectrometers. Elemental analysis was per-
formed with EA1110 elemental analyzer (CE
Instrument).
The size of substituents at 4-position of B ring should be
important factor for the activity of 2. Comparison of
the activities of hydroxyl analogs 2b and 2e and their
methoxmethoxy analogs 2c (47.8% inhibition at 50 lM,
IC50 > 50 lM) and 2f (13.0% inhibition at 50 lM,
IC50 > 50 lM) reveals that methoxymethoxy group dra-
matically reduces the activity. Weaker activities of 2c
and 2f than those of 2b and 2e might indicate that meth-
oxymethoxy group may be too large to fit with putative
binding pocket regardless of its hydrogen acceptor prop-
erty. In spite of its incapability in hydrogen bonding, ethyl
analog 2t (82.3% inhibition at 50 lM, IC50 = 27.9 lM)
has fairly strong activity. Hydroxymethyl analog 2u
(99.3% inhibition at 50 lM, IC50 = 6.4 lM) with hydro-
gen bonding capability possesses the more potent activity.
Therefore the substituents at 4-position of B ring should
be as small as three atom size.
6.2. General procedure20 for the preparation of com-
pounds (5)
2,6-Dihydroxyacetophenone or 2,4-dihydroxyacetophe-
none (40 mmol) was dissolved in dry acetonitrile
(80 mL) and potassium carbonate (6.624 g, 48 mmol)

108
H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
was added. The resulting mixture was refluxed for 1 h
and then cyclohexylmethyl methanesulfonate (9.216 g,
48 mmol) in acetonitrile (10 mL) was dropped for fifteen
min. The reaction mixture was refluxed for 1.5 h. After
cooling to room temperature, dichloromethane
(320 mL) was added and washed with 5% aqueous sodi-
um hydroxide and water three times. The organic layer
was dried with anhydrous sodium sulfate and concen-
trated under vacuum. The crude product was purified
by flash column chromatography.
6.3.1. (E)-3-(4-Hydroxyphenyl)-1-(2-hydroxyphenyl)-2-pro-
pen-1-one (2a). Yield 93.8%, orange solid; mp 133.5–
134.7 ꢁC; Rf 0.20 (hexanes/ethyl acetate = 3:1). IR
1
(KBr) 3300, 3100, 2950, 1640 cmꢀ1. H NMR (CDCl3)
d 6.90 (d, J = 8.4 Hz, 1H), 6.98 (m, 2H), 7.50 (t,
J = 8.4 Hz, 1H), 7.54 (d, J = 15.6 Hz, 1H), 7.60 (d,
J = 8.4 Hz, 2H), 7.89 (d, J = 15.6 Hz, 1H), 7.92 (d,
J = 6.4 Hz, 2 H), 12.92 (s, 1H); Anal. Calcd for
C15H12O3: C, 74.99; H, 5.03. Found: C, 74.73; H, 4.97.
6.3.2. (E)-1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxy-
phenyl)-2-propen-1-one (2b). Yield 86.4%, orange solid;
mp 143.2–144.3 ꢁC; Rf 0.26 (hexanes/ethyl ace-
6.2.1. 2-Cyclohexylmethoxy-6-hydroxyacetophenone (5a).
Yield 71.0%; yellow solid; mp 71.3–72.4 ꢁC; Rf 0.30
(hexanes/ethyl acetate = 20:1). IR (KBr) 3420, 3050,
1
tate = 3:1). IR (KBr) 3400, 3100, 2950, 1640 cmꢀ1. H
2900, 1620 cmꢀ1
.
1H NMR (CDCl3) d 1.07–1.33 (m,
NMR (CDCl3) d 5.19 (s, 2H), 6.69–6.89 (m, 3H), 7.25
(d, J = 8.6 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.50 (m,
6H), 7.76 (d, J = 15.6 Hz, 2H), 13.64 (s, 1H); Anal.
Calcd for C22H18O4: C, 76.29; H, 5.24. Found: C,
76.18; H, 5.15.
5H), 1.68–2.02 (m, 6H), 2.71 (s, 3H), 3.83 (d, J = 5.6,
2H), 6.36 (d, J = 8.4 Hz, 1 H), 6.54 (d, J = 8.4 Hz,
1H), 7.31 (t, J = 8.4 Hz, 1H), 7.68 (s, 1H); Anal. Calcd
for C15H20O3: C, 72.55; H, 8.12. Found: C, 72.36; H,
8.09.
6.3.3. (E)-1-(4-Benzyloxy-2-hydroxyphenyl)-3-(4-hydroxy-
phenyl)-2-propen-1-one (2d). Yield 81.2%; brown solid;
mp 164.1–165.0 ꢁC; Rf 0.14 (hexanes/ethyl ace-
6.2.2. 2-Cyclohexylmethoxy-6-hydroxyacetophenone (5b).
Yield 67.9%; yellow solid; mp 73.0–74.2 ꢁC; Rf 0.32
(hexanes/ethyl acetate = 20:1). IR (KBr) 3400, 3030,
1
tate = 5:1). IR (KBr) 3400, 3080, 2850, 1630 cmꢀ1. H
2850, 1620 cmꢀ1
.
1H NMR (CDCl3) d 1.19–1.29 (m,
NMR (CDCl3) d 5.11 (s, 2H), 6.55 (d, J = 8.4 Hz, 1H),
6.59 (s, 1H), 6.93 (m, 3H), 7.35 (d, J = 8.8 Hz, 2H),
7.40 (m, 5H), 7.43 (d, J = 15.2 Hz, 1H), 7.86 (d,
J = 15.2 Hz, 1H), 13.54 (s, 1H); Anal. Calcd for
C22H18O4: C, 76.29; H, 5.24. Found: C, 75.99; H, 5.14.
5H), 1.69–1.87 (m, 6H), 2.72 (s, 3H), 3.81 (d, J = 6.0,
2H), 6.40 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H),
7.29 (t, J = 8.4 Hz, 1H), 7.57 (s, 1H); Anal. Calcd for
C15H20O3: C, 72.55; H, 8.12. Found: C, 72.40; H, 8.07.
6.2.3. 2-Benzyloxyacetophenone (5c). Using the method
for the preparation of 3a, 3c was prepared from
2-hydroxybenzaldehyde and benzyl bromide.
6.3.4. (E)-1-(2-Cyclohexylmethoxy-6-hydroxyphenyl)-3-
(4-hydroxyphenyl)-2-propen-1-one (2e). Yield 52.9%; yel-
low solid; mp 173.7–174.4 ꢁC; Rf 0.31 (hexanes/ethyl
acetate = 3:1). IR (KBr) 3400, 3100, 2850, 1620 cmꢀ1
.
Yield 63.2%; yellow solid; mp 92.2–93.4 ꢁC; Rf 0.32
(hexanes/ethyl acetate = 10:1). IR (KBr) 3030,
1H NMR (CDCl3) d 1.07–1.26 (m, 6H), 1.64–1.72 (m,
5H), 3.86 (d, J = 6.4, 2H), 6.40 (d, J = 8.4 Hz, 1H),
6.87 (d, J = 8.4 Hz, 2H), 7.32 (t, J = 8.4 Hz,1H), 7.52
(d, J = 8.4 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H), 7.84 (d,
J = 15.6 Hz, 1H), 13.27(s, 1H); Anal. Calcd for
C22H24O4: C, 74.98; H, 6.86. Found: C, 74.79; H, 6.77.
2930, 1620 cmꢀ1 1H NMR (CDCl3) d 2.62 (s, 3H),
.
5.13 (s, 2H), 6.47 (d, J = 8.4 Hz, 1H), 6.59(d,
J = 8.4 Hz, 1H), 7.33(t, J = 8.4 Hz, 1H), 7.41 (m, 5H);
Anal. Calcd for C15H14O2: C, 79.62; H, 6.24. Found:
C, 79.42; H, 6.17.
6.3.5. (E)-1-(2-Cyclohexylmethoxy-6-hydroxyphenyl)-3-
(4-methoxymethoxyphenyl)-2-propen-1-one (2f). Yield
71.1%, yellow solid; mp 90.4–91.7 ꢁC; Rf 0.36 (hex-
anes/ethyl acetate = 7:1). IR (KBr) 3350, 3100, 2950,
6.3. General procedure for the preparation of
compounds 2
The corresponding 2-hydroxyacetophenone 5 (1 equiv)
was added to 90% aqueous ethanol solution of two
equivalents of sodium hydroxide or potassium hydrox-
ide (8.7% w/w). The substituted benzaldehyde 6 (1.02
equiv) was then added and the resulting solution was
stirred for 2 h at 50 ꢁC. After removal of ethanol under
vacuum, the crude mixture was dissolved in water and
neutralized with hydrochloric acid. The resulting mix-
ture was extracted with dichloromethane three times.
The combined organic layers were dried with anhydrous
sodium sulfate and evaporated under vacuum. The
crude product was purified by flash column chromatog-
raphy to give 2. In case of preparation of 2a, 2b, 2d, 2e,
and 2g, the corresponding intermediates 1-(2-hydroxy-
phenyl)-3-[4-(methoxymethoxy)phenyl]propenones such
as 2c or 2f were hydrolyzed in hydrochloric acid in aque-
ous methanol (90%) by reflux for 2 h to convert 1-(2-
hydroxyphenyl)-3-(4-hydroxyphenyl)propenones 2.
1630 cmꢀ1 1H NMR (CDCl3) d 1.06–1.25 (m, 5H),
.
1.63–1.71 (m, 3H), 1.87–1.90 (m, 3H), 3.49 (s, 3H),
3.86 (d, J = 5.6 Hz, 2H,), 5.22 (s, 2H), 6.40 (d,
J = 7.6 Hz, 1H), 6.59 (d, J = 7.6 Hz, 1H), 7.06 (d,
J = 8.8 Hz, 2H), 7.32 (t, J = 8.4 Hz, 1H), 7.56 (d,
J = 8.8 Hz, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.85 (d,
J = 15.6 Hz, 1H), 12.97 (s, 1H); Anal. Calcd for
C24H28O5: C, 72.70; H, 7.12. Found: C, 72.56; H,
6.98.
6.3.6. (E)-1-(4-Cyclohexylmethoxy-2-hydroxyphenyl)-3-
(4-hydroxyphenyl)-2-propen-1-one (2g). Yield 50.9%, yel-
low solid; mp 171.8–172.4 ꢁC; Rf 0.26 (hexanes/ethyl
acetate = 3:1). IR (KBr) 3400, 3100, 2900, 1640 cmꢀ1
.
1H NMR (CDCl3) d 1.19–1.29 (m, 5H), 1.69–1.87 (m,
6H), 3.81 (d, J = 6.0 Hz, 2H), 6.46 (m, 2H), 6.88 (d,
J = 8.8 Hz, 2 H), 7.45 (d, J = 15.2 Hz, 1H), 7.57 (d,
J = 8.8 Hz, 2H), 7.79 (s 1H), 7.84 (d, J = 15.2 Hz, 1H),

H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
109
13.07(s, 1H); Anal. Calcd for C22H24O4: C, 74.98; H,
6.86. Found: C, 74.83; H, 6.75.
yellow solid; mp 176.3–177.2 ꢁC; Rf 0.37 (hexanes/ethyl
acetate = 1:1). IR (KBr) 3410, 3100, 2930, 2850, 1700,
1640 cmꢀ1 1H NMR (CDCl3) d 1.00–1.14 (m, 6H),
.
6.3.7. 4-[(E)-3-(2-Benzyloxy-6-hydroxyphenyl)-3-oxopro-
pen-1-yl]benzoic acid (2h). Yield 68.7%, yellow solid; mp
187.5–188.1 ꢁC; Rf 0.33 (hexanes/ethyl acetate = 1:2). IR
1.63–1.71 (m, 5H), 3.80 (s, 2H), 6.09 (t, J = 8.0 Hz,
1H), 6.51 (d, J = 8.4 Hz, 2H), 7.29 (t, J = 8.4 Hz, 1H),
7.60 (t, J = 8.4 Hz, 1H), 7.69 (d, J = 15.6 Hz, 1H), 7.77
(m, 2H), 7.84 (d, J = 15.6 Hz, 1H), 13.02 (s, 1H); Anal.
Calcd for C23H24O5: C, 72.61; H, 6.36. Found: C, 72.58;
H, 6.27.
(KBr) 3400, 3100, 2950, 1690, 1630 cmꢀ1 1H NMR
.
(CDCl3) d 5.12 (s, 2H), 6.51 (d, J = 8.4 Hz, 1H), 6.69
(d, J = 8.4 Hz, 1H), 7.07 (t, J = 8.4 Hz, 1H), 7.16 (d,
J = 8.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.44 (m,
6H), 7.75 (d, J = 15.2 Hz, 1H), 7.85 (d, J = 15.2 Hz,
1H), 10.02 (s, 1H), 13.03(s, 1H); Anal. Calcd for
C23H18O5: C, 73.79; H, 4.85. Found: C, 73.65; H, 4.73.
6.3.13. (E)-4-[3-(2-Hydroxyphenyl)-3-oxopropen-1-yl]ben-
zoic acid (2o). Yield 90.0%; yellow solid; mp 248.5–
252.0 ꢁC; Rf 0.21 (hexanes/ethyl acetate = 1:2). IR
(KBr) 3400, 3100, 2950, 1690, 1630 cmꢀ1 1H NMR
.
6.3.8. 4-[(E)-3-(4-Benzyloxy-2-hydroxyphenyl)-3-oxopro-
pen-1-yl]benzoic acid (2i). Yield 37.9%; yellow solid; mp
236.1–237.2 ꢁC; Rf 0.46 (hexanes/ethyl acetate = 1:2). IR
(DMSO-d6) d 7.01 (m, 2H), 7.58 (t, J = 7.6 Hz, 1H),
7.85 (d, J = 15.6 Hz, 1H), 8.01 (m, 4H), 8.12 (d,
J = 15.6, Hz, 1H), 8.23 (d, J = 15.6 Hz, 1H), 8.23 (d, J
=8.0 Hz, 1H), 12.30 (s, 1H), 13.04 (s, 1H); Anal. Calcd
for C16H12O4: C ,71.64; H, 4.51. Found: C, 71.45; H,
4.46.
(KBr) 3400, 3100, 2930, 1700, 1650 cmꢀ1 1H NMR
.
(DMSO-d6) d 5.23 (s, 2H), 6.62 (s, 1H), 6.66 (d,
J = 7.1, 1H), 7.42 (d, J =8.8 Hz, 2H), 7.69 (d,
J = 15.2 Hz, 1H), 7.92 (d, J = 7.1 Hz, 1H), 7.99 (d,
J = 15.2 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 10.60 (s,
1H), 13.17(s, 1H); Anal. Calcd for C23H18O5: C, 73.79;
H, 4.85. Found: C, 73.64; H, 4.79.
6.3.14. (E)-1-[2-(Cyclohexylmethoxy)-6-hydroxyphenyl]-
3-phenylprop-2-en-1-one (2p). Yield 58.3%; orange solid;
mp 94.2–96.4 ꢁC; Rf 0.21 (hexanes/ethyl acetate = 10:1).
IR (KBr) 3400, 3100, 2950, 1640 cmꢀ1 1H NMR
.
6.3.9. 4-[(E)-3-(2-Cyclohexylmethoxy-6-hydroxyphenyl)-
3-oxopropen-1-yl]benzoic acid (2j). Yield 49.2%; yellow
solid; mp 213.8–214.9 ꢁC; Rf 0.35 (hexanes/ethyl ace-
tate = 1:1). IR (KBr) 3400, 3100, 2950, 1680,
(DMSO-d6) d 1.01–1.23 (m, 5H), 1.59–1.89 (m, 6H),
3.84 (d, J = 6.0 Hz, 2H), 6.39 (d, J = 8.0 Hz, 1H), 6.59
(d, J = 8.4 Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.40 (m,
4H), 7.60 (m, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.94 (d,
J = 15.6 Hz, 1H), 12.84 (s, 1H); Anal. Calcd for
C22H24O3: C, 78.54; H, 7.19. Found: C, 78.45; H, 7.07.
1
1630 cmꢀ1. H NMR (DMSO-d6) d 1.01 (m, 5H), 1.54
(m, 5H), 2.51 (m, 1H), 3.80 (d, J = 5.6 Hz, 1H), 6.54
(d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 1H), 7.38 (d,
J = 8.2 Hz, 2H), 7.75 (d, J = 15.2 Hz, 1H), 7.84 (d,
J = 8.2 Hz, 2H), 8.05 (d, J = 15.2 Hz, 1H), 10.80 (s,
1H), 13.07 (s, 1H); Anal. Calcd for C23H24O5: C,
72.61; H, 6.36. Found: C, 72.49; H, 6.24.
6.3.15. (E)-3-(4-Chlorophenyl)-1-[2-(cyclohexylmethoxy)-
6-hydroxyphenyl]-2-propen-1-one (2q). Yield 53%,
orange solid; mp 120.7–122.6 ꢁC; Rf 0.29 (hexanes/ethyl
acetate = 10:1). IR (KBr) 3400, 3100, 2950, 1640 cmꢀ1
.
1H NMR (CDCl3) d 1.01–1.21 (m, 5H), 1.59–1.88 (m,
6H), 3.85 (d, J = 6.0 Hz, 2H), 6.39 (d, J = 8.4 Hz, 1H),
6.59 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 8.4 Hz, 1H), 7.37
(d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.72 (d,
J = 15.6 Hz, 1H), 7.90 (d, J = 15.6 Hz, 1H), 13.04 (s,
1H); Anal. Calcd for C22H23ClO3: C, 71.25; H, 6.25.
Found: C, 71.12; H, 6.17.
6.3.10.
4-[(E)-3-(4-Cyclohexylmethoxy-2-hydroxyphe-
nyl)-3-oxopropen-1-yl]benzoic acid (2l). Yield 55.9%; yel-
low solid; mp 249.4–250.2 ꢁC; Rf 0.46 (hexanes/ethyl
acetate = 1:1). IR (KBr) 3400, 3100, 2950, 2850, 1680,
1640 cmꢀ1. H NMR (DMSO-d6) d 1.15 (m, 5H), 1.71
1
(m, 6H), 3.87 (d, J = 5.7 Hz, 2H), 6.51 (d, J = 8.2 Hz,
2H), 7.61 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H),
7.91 (d, J = 15.2 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H),
8.23 (d, J = 15.2 Hz, 1H), 10.84 (s, 1H), 13.31 (s, 1H);
Anal. Calcd for C23H24O5: C, 72.61; H, 6.36. Found:
C, 72.47; H, 6.25.
6.3.16. (E)-N-4-{3-[2-(Cyclohexylmethoxy)-6-hydroxy-
phenyl]-3-oxoprop-1-enyl}phenyl acetamide (2s). Yield
64.2%; yellow solid; mp 203.0–204.0 ꢁC; Rf 0.21 (hex-
anes/ethyl acetate = 6:1). IR (KBr) 3300, 3100, 2950,
1680, 1640 cmꢀ1 1H NMR (CDCl3) d 1.05–1.25 (m,
.
6.3.11. 3-[(E)-3-(2-(Cyclohexylmethoxy)-6-hydroxyphe-
nyl)-3-oxopropen-1-yl]benzoic acid (2m). Yield 60.0%;
yellow solid; mp 188.2–189.1 ꢁC; Rf 0.46 (hexanes/ethyl
acetate = 2:1). IR (KBr) 3370, 3100, 2900, 2850, 1690,
5H), 1.63–1.70 (m, 3H), 1.87–1.89 (m, 3H), 2.21 (s,
3H), 3.85 (d, J = 6.0 Hz, 2H), 6.39 (d, J = 8.4 Hz, 1H),
6.58 (d, J = 8.4 Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.44
(s, 1H), 7.56 (s, 4H), 7.75 (d, J = 15.6 Hz, 1H), 7.88 (d,
J = 15.6 Hz, 1H), 13.02 (s, 1H); Anal. Calcd for
C24H27O4: C, 73.26; H, 6.92. Found: C, 73.14; H, 6.84.
1
1640 cmꢀ1. H NMR (DMSO-d6) d 1.01–1.08 (m, 5H),
1.48–1.68 (m, 6H), 3.80 (d, J = 6.0 Hz, 2H), 6.52 (d,
J = 8.8 Hz, 1H), 6.55 (d, J = 8.8, 1H), 7.28 (t,
J = 8.8 Hz, 1H), 7.41 (d, J = 16.0 Hz, 1H), 7.51 (d, J
=8.2 Hz, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.95 (m, 2H),
8.18 (s, 1H), 13.15 (s, 1H); Anal. Calcd for C23H24O5:
C, 72.61; H, 6.36. Found: C, 72.51; H, 6.31.
6.3.17. (E)-1-(2-(Cyclohexylmethoxy)-6-hydroxyphenyl)-
3-(4-ethylphenyl)-2-propen-1-one (2t). Yield 69.3%;
orange solid; mp 81.0–82.1 ꢁC; Rf 0.29 (hexanes/ethyl
acetate = 10:1). IR (KBr) 3400, 3100, 2950, 1640 cmꢀ1
.
NMR (CDCl3)
d
1.03–1.27 (m, 8H), 1.66 (t,
6.3.12. 2-((E)-3-(2-(Cyclohexylmethoxy)-6-hydroxyphe-
nyl)-3-oxopropen-1-yl)benzoic acid (2n). Yield 67.7%;
J = 14.4 Hz, 3H), 1.88 (m, 3H), 2.68 (q, J = 7.6 Hz,
2H), 3.85 (d, J = 5.6 Hz, 2H), 6.39 (d, J = 8.4 Hz, 1H),

110
H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
6.58 (d, J = 8.4 Hz, 1H), 7.22 (d, J =8.0 Hz, 2H), 7.31 (t,
J = 8.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.79 (d,
J = 15.6 Hz, 1H), 7.91 (d, J = 15.6 Hz, 1H), 12.95 (s,
1H); Anal. Calcd for C24H28O3: C, 79.09; H, 7.74.
Found: C, 78.86; H, 7.65.
water three times and 10% aqueous sodium bicarbonate.
The organic layer was dehydrated with anhydrous sodi-
um sulfate and concentrated under vacuum. The crude
product was purified by flash column chromatography
to give 2k.
6.3.18. (E)-1-(2-(Cyclohexylmethoxy)-6-hydroxyphenyl)-
3-(4-(hydroxymethyl)phenyl)-2-propen-1-one (2u). Yield
66.2%; orange solid; mp 93.3–94.5 ꢁC; Rf 0.45 (hex-
anes/ethyl acetate = 2:1). IR (KBr) 3300, 2930, 2850,
Yield 40%, yellow solid; mp 118.7–119.5 ꢁC; Rf 0.22
(hexanes/ethyl acetate = 8:1). IR (KBr) 3450, 3100
1
2950, 1720, 1630 cmꢀ1. H NMR (CDCl3) d 1.04–1.19
(m, 5H), 1.65–1.69 (m, 3H), 1.85–1.88 (m, 3H), 3.86
(d, J = 5.6 Hz, 2H) 3.95 (s, 3H), 6.40 (d, J = 8.4 Hz,
1H), 6.60 (d, J = 6.4 Hz, 1H), 7.34 (t, J = 8.4 Hz, 1H),
7.65 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.37
(d, J = 15.6 Hz, 1H), 7.98 (d, J = 15.6 Hz, 1H), 8.07
(d, J = 8.4 Hz, 2H), 13.07 (s, 1H); Anal. Calcd for
C24H26O5: C, 73.08; H, 6.64. Found: C, 72.88; H, 6.53.
1640 cmꢀ1 1H NMR (CDCl3) d 1.03–1.25 (m, 6H),
.
1.67–1.90 (m, 5H), 3.87 (s, 3H), 4.75 (s, 2H), 6.39 (d,
J = 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 7.33 (t,
J = 8.4 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.59 (d,
J = 8.0 Hz, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.95 (d,
J = 15.6 Hz, 1H), 13.20 (s, 1H); Anal. Calcd for
C23H26O4: C, 75.38; H, 7.15. Found: C, 75.25; H, 7.07.
6.5. Preparation of (E)-3-(4-aminophenyl)-1-[2-(cyclo-
hexylmethoxy)-6-hydroxyphenyl]-2-propen-1-one (2r)
6.3.19. (E)-4-{3-[2-(Cyclohexylmethoxy)-6-hydroxyphe-
nyl]-3-oxoprop-1-enyl}benzalde hyde (2v). Yield 60.5%;
orange solid; mp 116.0–117.2 ꢁC; Rf 0.19 (hexanes/ethyl
acetate = 8:1). IR (KBr) 3420, 3100, 2950, 1700,
Hydrochloric acid (2 N, 10 mL) was added to the solu-
tion of 2s (0.27 g, 0.69 mmol) in ethanol (50 mL). The
resulting mixture was refluxed for 3 h and concentrated
under vacuum. The crude mixture was dispersed in
dichloromethane (50 mL) and basified with saturated
aqueous sodium bicarbonate solution. The organic layer
was separated, dehydrated with anhydrous sodium sul-
fate, and concentrated under vacuum. The crude prod-
uct was purified by flash column chromatography.
1640 cmꢀ1 1H NMR (CDCl3) d 1.00–1.18 (m, 5H),
.
1.59–1.87 (m, 6H), 3.86 (d, J = 6.0 Hz, 2H), 6.40 (d,
J = 8.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 7.36 (t,
J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2 H), 7.77 (d,
J = 15.6 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 8.01 (d,
J = 15.6 Hz, 1H), 10.10 (s, 1H), 13.02 (s, 1H); Anal.
Calcd for C23H24O4: C, 75.80; H, 6.64. Found: C,
75.67; H, 6.48.
Yield 75.2%; brown solid; mp 122.7–128.5 ꢁC; Rf 0.20
(hexanes/ethyl acetate = 3:1). IR (KBr) 3450, 3200,
6.3.20. 4-((E)-3-(2-(Cyclohexylmethoxy)-6-hydroxyphe-
nyl)-3-oxoprop-1-enyl)benznesulfonamide (2w). Yield
56.2%; yellow solid; mp 130.1–131.3 ꢁC; Rf 0.33 (hex-
anes/ethyl acetate = 2:1). IR (KBr) 3420, 3400, 3100,
2900, 1620 cmꢀ1 1H NMR (CDCl3) d 1.00–1.19 (m,
.
5H), 1.58–1.67 (m, 3H), 1.83–1.97 (m, 3H), 3.77 (d,
J = 6.0 Hz, 2H), 3.94 (s, 1H), 6.31 (d, J = 8.4 Hz, 1H),
6.50 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 2H), 7.22
(t, J = 8.4 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.73 (s,
2H), 12.94 (s, 1H); Anal. Calcd for C22H25O3: C,
75.19; H, 7.17. Found: C, 75.03; H, 7.02.
1
1640 cmꢀ1. H NMR (DMSO-d6) d 1.01–1.10 (m, 5H),
1.52–1.99 (m, 6H), 3.79 (d, J = 5.6 Hz, 2H), 6.53 (d,
J = 80 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.26 (t,
J = 8.4 Hz, 1H), 7.39 (d, J = 15.6 Hz, 2H), 7.45 (s,
2H), 7.82 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H),
10.63 (s, 1H); Anal. Calcd for C22H25NO5S: C, 63.59;
H, 6.06. Found: C, 63.42; H, 5.97.
6.6. Preparation of 4-[(E)-3-(2-benzyloxyphenyl)-3-oxo-
propen-1-yl]benzoic acid (3)
6.3.21. 4-((E)-3-(2-(Benzyloxy)-6-hydroxyphenyl)-3-oxo-
prop-1-enyl)benzenesulfonamide (2x). Yield 52.4%; yel-
low solid; mp 182.0–183.2 ꢁC; Rf 0.33 (hexanes/ethyl
Using the method for the preparation of 2a, compound
3 was synthesized from 1-(2-(benzyloxy))acetophenone.
acetate = 2:1). IR (KBr) 3420, 3400, 3100, 1640 cmꢀ1
.
Yield 57.3%; yellow solid; mp 140.1–141.5 ꢁC; Rf 0.48
(hexanes/ethyl acetate = 2:1). IR (KBr) 2930, 2850,
1H NMR (DMSO-d6)
d
5.13 (s, 2H), 6.57 (d,
1
J = 8.4 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 7.26 (t,
J = 8.4 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.44–7.72
(m, 7H), 7.78 (d, J = 15.6 Hz, 1H), 7.82 (d,
J = 15.6 Hz, 1H), 10.65 (s, 1H); Anal. Calcd for
C22H19NO5S: C, 64.53; H, 4.68. Found: C, 64.39; H,
4.57.
1640 cmꢀ1 . H NMR (CDCl3) d 5.16 (s, 2H), 7.06–
7.11 (m, 2H), 7.31–7.44 (m, 8H), 7.34 (d, J = 15.6 Hz,
1H), 7.40 (d, J = 15.6 Hz, 1H), 7.51 (t, J = 8.4 Hz,
1H), 8.01 (d, J = 8.4 Hz, 2H), 12.10 (s, 1H); Anal. Calcd
for C23H24O4: C, 75.80; H, 6.64. Found: C, 75.62; H,
6.48.
6.4. Preparation of (E)-methyl 4-{3-[2-(cyclohexylmeth-
oxy)-6-hydroxyphenyl]-3-oxoprop-1-enyl}benzoate (2k)
6.7. Preparation of 4-{3-[2-(cyclohexylmethoxy)-6-
hydroxyphenyl]-3-oxopropyl} benzoic acid (4)
Compound 2j (0.5 g, 1.3 mmol) and sulfuric acid 1 mL
were dissolved in methanol (50 mL) and then the reac-
tion mixture was refluxed for 2 h. After cooling, the mix-
ture was concentrated under vacuum. The residue was
dispersed in dichloromethane and washed with cold
Compound 2j (200 mg, mmol) was dissolved in metha-
nol (50 mL) and 10% Pd/C (20 mg) was added. The
resulting mixture was stirred under hydrogen (20 psi)
for two hours at room temperature. After removal of
catalyst by filtration with aid of Celite pad, the solvent

H.-M. Yang et al. / Bioorg. Med. Chem. 15 (2007) 104–111
111
3. Gelfand, E. W. Allergy Asthma Proc. 1998, 19, 365.
4. Riffo-Vasquez, Y.; Spina, D. Pharmacol. Ther. 2002, 94,
185.
was removed under vacuum. The crude product was
purified by flash column chromatography.
5. Hamelmann, E.; Gelfand, E. W. Int. Arch. Allergy
Immunol. 1999, 120, 8.
6. Allakhverdi, Z.; Allam, M.; Renzi, P. M. Am. J. Respir.
Crit. Care Med. 2002, 165, 1015.
Yield 77%, white solid; mp 198.2–199.4 ꢁC; Rf 0.45 (hex-
anes/ethyl acetate = 4:1). IR (KBr) 3300, 2930, 2850,
1720, 1690 cmꢀ1 1H NMR (CDCl3) d 0.96–1.12 (m,
.
5H), 1.64–1.77 (m, 6H), 3.11 (t, J = 8.0 Hz, 2H), 3.51
(t, J = 8.0 Hz, 2H), 3.81 (d, J = 5.6 Hz, 2H), 6.36 (d,
J = 8.4 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.31 (t,
J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 8.04 (d,
J = 8.4 Hz, 2H), 9.88 (s, 1H), 12.01 (s, 1H); Anal. Calcd
for C23H26O5: C, 72.23; H, 6.85. Found: C, 72.18; H,
6.80.
7. Takaki, S.; Tominaga, A.; Hitoshi, Y.; Mita, S.; Sonoda,
E.; Yamaguchi, N.; Takatsu, K. EMBO J. 1990, 9, 4367.
8. Murata, Y.; Takaki, S.; Migita, M.; Kikuchi, Y.; Tomi-
naga, A.; Takatsu, K. J. Exp. Med. 1992, 175, 341.
9. Mita, S.; Takaki, S.; Tominaga, A.; Takatsu, K.
J. Immunol. 1993, 151, 6924.
10. Tomaki, M.; Zhao, L. L.; Sjostrand, M.; Linden, A.;
Ichinose, M. J. Lotvall. Pulm. Pharmacol. Ther. 2002, 15,
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6.8. Inhibitory activity against interleukin-5
The Y16 cell line was grown in RPMI (10.4 mg/mL
RPMI-1640, 24 mM NaHCO3, 100 U/mL benzylpenicil-
lin potassium, and 100 lg/mL streptomycin sulfate, pH
7.1) containing 8% fetal bovine serum (FBS) and
5 U/mL IL-5 at 37 ꢁC with 5% CO2. The grown Y16
cells were harvested by centrifugation at 250g for
10 min at 4 ꢁC, washed twice with Hanks’ solution
(9.8 mg/mL Hanks’ balanced salts, 24 mM NaHCO3,
pH 7.1), and resuspended in a small volume of RPMI
containing 8% FBS. Number of the cells were counted
after trypan blue exclusion and then diluted to 1 · 105
cells/mL with RPMI containing 8% FBS. Viability of
the cells was more than 95% in all preparations. One
hundred microliters of 1 · 104 cells was dispensed to
each well of a 96-well microplate (Nunc, Denmark),
and 50 lL of 4 U/mL IL-5 and 50 lL of the sample were
added. Control group was treated with RPMI contain-
ing 8% FBS instead of sample, and blank group with
RPMI containing 8% FBS instead of IL-5. After incuba-
tion at 37 ꢁC with 5 CO2 for 48 h, Y16 cells in each well
were treated with 20 lL WST-1 solution (3.3 mg WST-1
and 0.7 mg 1-methoxy-5-methyl-phenazinium methyl-
sulfate per mL of phosphate-buffered saline) and incu-
bated 37 ꢁC with 5% CO2 for 4 h. Absorbance at
wavelength 450 nm (A450) was measured by using a
microplate reader (Molecular Device, USA). Inhibitory
effect on the IL-5 bioassay by sample at 50 lM concen-
tration was expressed as % inhibition [1-(sample
A450 ꢀ blank A450)/(control A450 ꢀ blank A450)] · 100.
Data were collected as means SEM of three indepen-
dent tests. Samples showing more than 50% inhibition
at 50 lM concentration were serially diluted and then
subjected to the IL-5 bioassay to determine IC50 values.
27. Li, R.; Kenyon, G. L.; Cohen, F. E.; Chen, X.; Gong, B.;
Dominguez, J. N.; Davidson, E.; Kurzban, G.; Miller, R.
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pp. 631-633.
Acknowledgments
This work was supported by the Grant (00-PJ2-PG1-
CD02-0003) of the Good Health R&D Project, Ministry
of Health & Welfare, R.O.K.
29. Bowen, K.. In Comphrehensive Medicinal Chemistry;
Hansch, C., Ed.; Pergamon Press, 1990; Vol. 4, pp 234–
235.
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