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

Article abstract of DOI:10.1016/S0223-5234(03)00064-3

Sophoricoside isolated from Sophora japonica is a glycoside of isoflavonone as an inhibitor of interleukin (IL)-5. To identify structural requirements of this isoflavonone for its inhibitory activity against IL-5, isoflavonones, isoflavanones, and their glycosides were prepared and their inhibitory activity was tested against IL-5. Among them, 5-benzyloxy-3-(4-hydroxyphenyl)chromen-4-one (4b, 87.9% inhibition at 50 μM, IC₅₀=15.3 μM) shows the most potent activity, comparable with that of sophoricoside. The important structural requirements of these isoflavonone analogs exhibiting the inhibitory activity against IL-5 were recognized as (1) planarity of chromen-4-one ring, (2) existence of phenolic hydroxyl at 4-position of B ring, and (3) introduction of benzyloxy at 5-position, which may act as a bulky group for occupying hydrophobic pocket in putative binding site. However the glucopyranosyl moiety of sophoricoside is not an essential motif for the activity.

Full text of DOI:10.1016/S0223-5234(03)00064-3

European Journal of Medicinal Chemistry 38 (2003) 537ꢁ545
/
www.elsevier.com/locate/ejmech
Preliminary communication
Structural requirement of isoflavonones for the inhibitory activity
of interleukin-5
Sang-Hun Jung a,*, Soo-Hyun Cho a, The Hung Dang a, Jee-Hyun Lee a, Jung-Hun Ju b,
Mi-Kyung Kim c, Seung-Ho Lee d, Jae-Chun Ryu e, Youngsoo Kim b
a College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea
b College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea
c College of Medicine, Chungbuk National University, Cheongju 361-763, South Korea
d College of Pharmacy, Yeungnam University, Kyungsan, South Korea
e Toxicology Laboratory, Korea Institute of Science and Technology, Seoul 130-650, South Korea
Received 30 July 2002; received in revised form 20 December 2002; accepted 23 December 2002
Abstract
Sophoricoside isolated from Sophora japonica is a glycoside of isoflavonone as an inhibitor of interleukin (IL)-5. To identify
structural requirements of this isoflavonone for its inhibitory activity against IL-5, isoflavonones, isoflavanones, and their glycosides
were prepared and their inhibitory activity was tested against IL-5. Among them, 5-benzyloxy-3-(4-hydroxyphenyl)chromen-4-one
(4b, 87.9% inhibition at 50 mM, IC50
ꢀ15.3 mM) shows the most potent activity, comparable with that of sophoricoside. The
/
important structural requirements of these isoflavonone analogs exhibiting the inhibitory activity against IL-5 were recognized as (1)
planarity of chromen-4-one ring, (2) existence of phenolic hydroxyl at 4-position of B ring, and (3) introduction of benzyloxy at 5-
position, which may act as a bulky group for occupying hydrophobic pocket in putative binding site. However the glucopyranosyl
moiety of sophoricoside is not an essential motif for the activity.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Sophoricoside; Isoflavonones; Inhibitory activity of interleukin-5
1. Introduction
affinity [8]. Even though IL-5Rb alone does not bind to
IL-5, it is required for high affinity binding in combina-
tion 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
cytokines display several overlapping biological effects
[10,11]. Rather than coordinating T-cell effector devel-
opment or antibody isotype secretion by B-cells, biolo-
gical effects of IL-5 are confined primarily to growth,
differentiation, survival, chemotaxis and activation of
Eosinophilic inflammation is the main histological
correlate of airway hyper-responsiveness 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) cytokines 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 proinflammatory 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 IL-5Ra by itself binds to IL-5 with low
eosinophils [12ꢁ14]. The contribution of IL-5 to allergic
/
disease has been greatly facilitated by the generation of
mice deficient in the IL-5 gene. In contrast to normal
mice, allergic IL-5-deficient mice do not generate
eosinophilia in the bone marrow, blood or lung in
response to allergen provocation [15]. Airway instilla-
tion of recombinant IL-5 to allergic IL-5-deficient mice
completely restores allergen-induced eosinophilia to
levels normally observed in allergic wild-type mice [16].
* Corresponding author.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00064-3

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S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ545
/
Thus, IL-5 is critically involved in eosinophilia-asso-
ciated allergic inflammation.
h and then heating at 50 8C after addition of hydro-
chloric acid in one pot afforded the corresponding
isoflavonones 4 in good yield. This conversion has
been known as a key step for this classical preparation
of isoflavonone [20,21]. To remove the benzyl-protecting
group on A ring of 4, two different hydrogenolysis
conditions were applied in the presence of 10% pal-
Interfering with the action of IL-5 represents one of
the new immunomodulatory therapeutic strategies in the
treatment of allergic diseases including bronchial
asthma. Compared to established immunosuppressive
agents like corticosteroids, a major advantage of this
strategy is the specificity of reducing eosinophilic
inflammation, thus possibly acting nearly without side
effects. However small organic compounds to inhibit IL-
5 activity have been rarely found. The isothiazolones
were identified as the IL-5 antagonists through mod-
ification of Cys66 in IL-5Ra [17]. Sophoricoside and its
analogs (Fig. 1) were isolated from Sophora japonica, a
plant of Leguminosae family, as inhibitors of IL-5
bioactivity [18], and showed differential inhibition on
IL-3 and GM-CSF bioactivities [19]. These isoflavo-
noids are unrelated to the structural unit of IL-5 and are
the first natural products to show the inhibitory activity
against IL-5 bioactivity. Therefore it is necessary to
determine the structure activity relationship of sophor-
icoside analogs for the design of potent derivatives.
Thus a number of isoflavonone compounds have been
prepared and their inhibitory effects on IL-5 bioactivity
have been evaluated.
ladiumꢁcarbon in methanol. Hydrogen pressure less
/
than 20 psi only removed benzyl group to form
isoflavonones 5. However increasing hydrogen pressure
to 30 psi resulted in both removal of the benzyl and
reduction of 2,3-double bond to give isoflavanone 7.
Controlling hydrogen pressure in this reaction makes
this synthetic scheme a versatile method for the pre-
paration of isoflavonone and isoflavanone derivatives.
Treatment of 5b with 1 equiv. of benzyl bromide in the
presence of potassium carbonate selectively gave 6b, in
which hydroxyl at 4-position of B ring is benzylated.
This selectivity might be originated from the steric
hinderence, thus the alkylation might occur at the less
hindered hydroxyl function. Such selectivity had been
demonstrated in the glycosidation of genistein to
acetylated sophoricoside [22]. Glycosidation of 4 to
glucopyranosylisoflavonones 8 was successfully per-
formed by the 2-day reaction at room temperature
with 2-bromoglucopyranosyl acetate and potassium
carbonate in the presence of benzyl triethyl ammonium
chloride as a phase transfer catalyst [23,24]. Under
2. Chemistry
catalytic hydrogenolysis condition (10% PdÃ
/
C, 30 psi
hydrogen), removal of the benzyl protecting group and
reduction of 2,3-double bond of 4?-glycosylisoflavonone
8b was accomplished to give isoflavanone 9b. Mean-
while catalytic hydrogenolysis under reduced pressure of
hydrogen gas (20 psi) of 8b gave compound 10b.
Treatment of the acetates 8a and 8b with sodium
methoxide in methanol gave the corresponding glyco-
sides 11a and 11b. Benzyl group of 11b was selectively
removed by the low-pressure hydrogenolysis condition
used for the preparation of 5 to produce 12b. Sophorico-
side isolated from Sophora japonica [17] was reduced to
isoflavanone 14 by catalytic hydrogenation at 30-psi
The synthetic pathways of sophoricoside analogs are
outlined in Fig. 2. Sophoricoside (13) is a glycoside of
isoflavonone. Thus its analogs were prepared using
conventional isoflavonone synthesis [20,21], which is
the most convenient method considering the availability
of starting material. To obtain the selectively substituted
sophoricoside analogs, benzyl protected 2-hydroxyace-
tophenones 1 were used as starting material in initial
step. 2-Hydroxyphenylpropenones 3 were readily ob-
tained by aldol condensation between 2-hydroxyaceto-
phenones 1 and 4-methoxymethoxybenzaldehyde (2) in
the presence of sodium hydroxide in 90% aqueous
ethanol. Sequential treatments of compounds 3 with
thallium nitrate in methanol at room temperature for 24
hydrogen in the presence of 10% palladiumꢁcarbon as
/
shown in Fig. 3.
3. Pharmacology
The measurement of inhibitory activity of IL-5 of
these analogs was performed with the comparison of cell
proliferation of Y16 cell with/without the samples [18].
An Y16 cell line originated from murine early B cell is
proliferated in the presence of IL-5, which was used as
the parameter of IL-5 bioactivity. Non-radioactive
procedures that measure cell metabolism as an index
of proliferation have now become popular. 2-(4-Iodo-
phenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-
Fig. 1. Naturally occurring isoflavonoids.

S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ
/
545
539
Fig. 2. Preparation of sophoricoside analogs. Substituent for compounds 3, 5, 8, and 11 are the same as those of compound 1. (a) 9% NaOH in 90%
aqueous ethanol; (b) Tl(NO3)2×3H2O for 1 day and then HCl in methanol; (c) 20 psi H2, 10% PdÃC in methanol; (d) NaOH, benzyl bromide; (e) 30
psi H2, 10% PdÃC in methanol; (f) K2CO3, 2-bromoglucopyranosyl acetate; (g) NaOCH3 in methanol.
/
/
/
tetrazolium sodium salt (WST-1) was used to form
soluble formazan product on exposure to the dehydro-
genase activity in metabolising cells. Proliferation of
Y16 cells in the presence of IL-5 was specifically blocked
by treatment of polyclonal antibody against IL-5 or
monoclonal antibody against IL-5Ra. Sample and IL-5
were simultaneously added to the Y16 cells, and the
bioassay system can identify the IL-5 antagonist and its
signal inhibitor.
4. Results and discussion
The results of biological screening of sophoricoside
analogs against IL-5 are listed in Table 1 as % inhibition
at 50 mM and IC50 values. Sophoricoside, genistin,
genistein, and orobol isolated from fruit of Sophora
japonica L. (Leguminosae) as the active principles
against cytokine IL-5 [18,19]. Among them sophorico-
side shows the most potent activity, which is much
Fig. 3. Reduction of sophoricoside.

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S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ545
/
Table 1
Inhibitory activity of sophoricoside analogs against IL-5
Entry no.
Compound no.
Molecular formula
% Inhibition at 50 (mM)
IC50 (mM)
1
2
4a
4b
C
15H10O3
C22H16O4
22H16O4
C15H10O4
15H10O4
C22H16O4
15H12O4
C29H28O12
36H34O13
16.29
87.89
57.79
57.39
69.69
41.09
17.29
8.09
39.49
33.69
55.09
5.09
91.39
95.39
93.09
17.69
70.39
/
0.3
6.4
1.0
3.4
2.8
4.3
3.2
9.3
3.9
3.6
3.1
2.7
2.4
0.4
0.8
2.9
2.1
ꢀ
/
50
/
15.3
36.3
45.1
30.6
50
3
4c
C
/
4
5b
/
5
5c
C
/
6
6b
7b
/
ꢀ
/
7
C
/
ꢀ
/
50
8
8a
8b
/
ꢀ
/
50
9
C
/
ꢀ
/
50
10
11
12
13
14
15
16
17
9b
10b
C29H30O13
C29H28O13
C21H20O8
/
ꢀ/
50
/
35.3
50
11a
11b
ꢂ/
/
ꢀ/
C
28H26O9
/
23.4
26.1
10.3
50
12b
13
C21H20O9
C21H20O10
/
/
14
Budesonide
C
21H22O10
/
ꢀ/
ꢁ/
/
26.8
stronger than oxyphenylbutazone [18]. Although re-
moval of glucopyranosyl moiety of sophoricoside de-
creases the activity 34 times based on IC50 value
comparison [18], the structure activity relationships of
these isoflavonones are essentially unknown. To find out
the primary pharmacophoric motif of these analogs,
hydroxyl on A ring shows very weak activity. Alkylation
of hydroxyl group at 4-position of B ring of isoflavo-
none as in 6b reduces the activity dramatically. There-
fore hydroxyl function at this position is considered to
be essential for the activity. Although these hydroxyl
compounds 5b, 5c, 10b, and 12b do not have hydro-
phobic binding moiety on A ring like benzyloxy group,
increased hydrophilicity of these compounds by hydrox-
yl function on A ring might be a factor for the
enhancement of the activity.
Since sophoricoside has stronger activity than its
aglycon [18] and IL-5 is heavily glycosylated, it was
assumed that the glucopyranosyl moiety has its unique
role for the activity. To confirm the role of glucopyr-
anosyl moiety at 4-hydroxyl group of B ring of
isoflavonones, glucopyranosyl analogs 11b and 12b
were prepared and their activity were compared to those
of their corresponding aglycons 4b and 5b. Compound
compounds 4aꢁc, 5b [25], 5c [25], 6b, 7b, 8a, 8b, 9b, 10b,
/
11a, 11b, 12b, 13 (sophoricoside), 14 were tested.
Introduction of benzyloxy moiety at 5- or 7-positions
on A ring of isoflavonones increases the activity.
Isoflavonones 4b (87.9% inhibition at 50 mM, IC50ꢀ
/
15.3 mM) and 4c (57.7% inhibition at 50 mM, IC50ꢀ
/
36.3 mM) containing benzyloxy at 5- or 7-position show
relatively strong activity, which are comparable with
sophoricoside activity (90.0% inhibition at 50 mM,
IC50ꢀ
mM, IC50ꢀ
/
10.3 mM). However 4a (16.2% inhibition at 50
50 mM) without benzyloxy moiety on A ring
/
exhibits very weak activity. This trend continues in the
activity of compounds 8 and 11 series. Compounds 8a
and 11a do not have benzyloxy group on A ring and
show very weak activity. Whereas compounds 8b and
11b possessing benzyloxy on A ring show much more
potent activity. Compound 4b with benzyloxy moiety at
5-position appears more active than 4c at the 7-position.
However compound 6b (17.2% inhibition at 50 mM,
11b (91.3% inhibition at 50 mM, IC50ꢀ
same level of activity with 4b. Compound 12b (95.3%
inhibition at 50 mM, IC50ꢀ26.1 mM) is slightly more
active than compound 5b (57.3% inhibition at 50 mM,
IC50ꢀ45.1 mM). Introduction of glucopyranosyl moiety
/
23.4 mM) has the
/
/
at this position dose not much affect the activity. Rapid
metabolic cleavage of glycosidic linkage of isoflavonoid
glycoside has been known in physiological condition
[26]. This might reflect that weak glycosidic bond should
be cleaved metabolically or chemically under physiolo-
gical condition before binding to the receptor. Thus the
activity of 11b, 12b, and sophoricoside (13) are essen-
tially those of the corresponding aglycons 4b, 5b, and
genistein. Therefore the glucopyranosyl moiety may not
be an essential motif for the intrinsic activity of these
analogs.
IC50ꢀ50 mM) with benzyloxy group at 4-position of B
/
ring shows very weak activity. These results should
suggest that benzyloxy moiety on A ring of isoflavonone
capable to bind with hydrophobic pocket in putative
binding site increases the activity.
Compounds (5b, 5c, 10b, 12b) possessing hydroxyl
groups on both A and B rings shows compatible level of
activity compared to the corresponding compounds (4b,
4c, 8b, 11b) with benzyloxy group on A ring and
hydroxyl group on B ring. Compound 4a without

S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ
/
545
541
To identify the importance of planar chromen-4-one
moiety, isoflavanones 7b, 9b and 14 containing chro-
man-4-one were prepared. These compounds show
reduced activity compared to the corresponding isofla-
vonones 5b, 10b, and 13. This result indicates that the
planarity of this region should be critical for the activity.
We reported that sophoricoside (13) showed potent
inhibitory effect on IL-5 involved in eosinophilic in-
flammation but did not inhibit proinflammatory cyto-
kines such as TNF and IL-1 involved in broad spectra of
inflammatory responses [19]. Among isoflavonones
prepared and tested in this study, compound 4b showed
5.1.1. General procedure [21,28] for the preparation of
compounds 1
2,6-Dihydroxyacetophenone (40 mmol) or 2,4-dihy-
droxyacetophenone was dissolved in dry acetonitrile (80
mL) and potassium carbonate (6.624 g, 48 mmol)) was
added. The resulting mixture was refluxed for 1 h and
then benzyl bromide (8.208 g, 48 mmol) in acetonitrile
(10 mL) was dropped for 15 min. The reaction mixture
was refluxed for one and half hours. After cooling to
room temperature, dichloromethane (320 mL) was
added and washed with 5% aqueous sodium hydroxide
and water three times. The organic layer was dried with
anhydrous sodium sulphate and concentrated under
vacuum. The crude product was recrystallized from
the strongest inhibitory effect (IC50ꢀ
Inhibitory potency of 4b on IL-5 was significantly
stronger than budesonide (IC50ꢀ26.8 mM) used as an
/
15.3 mM) on IL-5.
/
acetoneꢁhexane (2:1).
/
inhaled corticosteroid for asthma therapy (Table 1).
Like mode of action shown by sophoricoside, com-
pound 4b did not show inhibitory effect at concentration
of 50 mM on TNF and IL-1 bioactivities. A study to
address selectivity and mechanism of compound 4b on
IL-5 inhibition is in progress.
From this study, we could summarize the structural
requirement of sophoricoside analogs possessing the
inhibitory activity against IL-5 as (1) planarity of
chromen-4-one ring, (2) existence of phenolic hydroxyl
at 4-position of B ring, and (3) introduction of
benzyloxy at 5-position, which may act as a bulky
group for binding hydrophobic pocket in putative
receptor. However the glucopyranosyl moiety of so-
phoricoside is not an essential motif for the activity.
5.1.1.1. 6-Benzyloxy-2-hydroxyacetophenone 1b. Yellow
solid; Rfꢀ0.53 (hexanes:ethyl acetateꢀ5:1); yield
71.4%; m.p. 104.5ꢁ105.3 8C; IR (KBr) 3030, 2850,
1600, 1450 cmꢂ1 1H-NMR (CDCl3, 89.45 MHz) d
2.62 (s, 3H), 5.13 (s, 2H), 6.47 (d, Jꢀ8.1 Hz, 1H), 6.59
(d, Jꢀ8.1 Hz, 1H), 7.34ꢁ7.49 (m, 6H), 13.24 (s, 1H);
/
/
/
;
/
/
/
Anal. Calc. for C15H14O3: C, 74.36; H; 5.82. Found: C,
74.25; H, 5.90.
5.1.1.2. 4-Benzyloxy-2-hydroxyacetophenone 1c. White
solid; Rfꢀ
92.9%; m.p. 116.8ꢁ
/
0.40 (hexanes:ethyl acetateꢀ
117.7 8C; IR (KBr) 3030, 2850,
1620 cmꢂ1; H-NMR (CDCl3, 89.45 MHz) d 2.53 (s,
3H), 5.09 (s, 2H), 6.51 (m, 2H), 7.28ꢁ7.69 (m, 6H), 12.73
/
5:1); yield
/
1
/
(s, 1H); Anal. Calc. for C15H14O3: C, 74.36; H, 5.82.
Found: C, 74.27; H, 5.92.
5. Experimental protocols
5.1.2. Preparation [29] of 4-
methoxymethoxybenzaldehyde 2
4-Hydroxybenzaldehyde (6.1 g, 50.0 mmol) was dis-
solved in dry acetonitrile (80 mL) and potassium tert-
butoxide (6.2 g, 52.5 mmol) was added. The resulting
mixture was stirred for 30 min at room temperature.
After addition of 18-crown-6 (1.322 g, 5 mmol),
chloromethyl methyl ether (5.636 g, 70 mmol) was
added. The reaction mixture was stirred for one and a
half hours and filtered. The filtrate was concentrated
under vacuum and dissolved in ether. After washed with
5% aqueous sodium hydroxide and water three times,
the organic layer was dried with anhydrous sodium
sulphate and evaporated under vacuum. The product
was very pure based on TLC and NMR spectroscopic
analyses. Thus the product was used in the next step
without further purification.
5.1. Chemistry
Melting points (m.p.) were determined on Electro-
thermal 1A 9100 MK2 apparatus and are uncorrected.
All commercial chemicals were used as obtained and all
solvents were purified by the standard procedures prior
to use [27]. 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 column chromatography was performed
with E Merck silica gel (230ꢁ400 mesh). IR spectra were
/
recorded with Jasco IR-Report-100 IR spectrometer in
cmꢂ1 and corrected against peak at 1601 cmꢂ1 of
polystyrene. NMR spectra were measured against the
peak of tetramethylsilane by JEOL JNM-EX90 NMR
(89.45 MHz) and Varian Unity Inova 400 NMR (400
MHz) spectrometers. Elemental analysis was performed
with EA1110 elemental analyser (CE Instrument).
Brown oil; Rfꢀ
/
0.39 (hexanes:ethyl acetateꢀ
/
3:1);
1H-
yield 98.1%; IR (KBr) 1700, 1600, 1500 cmꢂ1
;
NMR (CDCl3, 89.45 MHz) d 3.48 (s, 3H), 5.26 (s, 2H),
7.14 (d, Jꢀ8.8 Hz, 2H), 7.84 (d, Jꢀ8.8 Hz, 2H), 9.90
(s, 1H).
/
/

542
S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ545
/
5.1.3. General procedure for the preparation of
compounds 3
The corresponding 2-hydroxyacetophenone
vacuum. The crude product was purified by flash
column chromatography.
1 (1
equiv.) was added to 90% aqueous ethanol solution of
2 equiv. of sodium hydroxide (8.7% w/w). Aldehyde 2
(1.02 equiv.) was then added and the resulting solution
was stirred for 2 h at 50 8C. After removal of ethanol
under vacuum, the crude mixture was dissolved in water
and neutralized with hydrochloric acid. The resulting
mixture was extracted with dichloromethane three
times. The combined organic layers were dried with
anhydrous sodium sulphate and evaporated under
vacuum. The crude product was purified by flash
column chromatography.
5.1.4.1. 3-(4-Hydroxyphenyl)chromen-4-one 4a. Yellow
solid; Rfꢀ
51.3%; m.p. 178.6ꢁ
1620, 1540, 1460 cmꢂ1
MHz) d 7.23ꢁ7.74 (m, 4H), 7.78ꢁ
(s, 1H), 8.09ꢁ8.19 (m, 2H), 10.66 (s, 1H); Anal. Calc. for
/
0.39 (hexanes:ethyl acetateꢀ
179.3 8C; IR (KBr) 3300, 1640,
1H-NMR (CDCl3, 89.45
7.95 (m, 2H), 8.06
/
3:1); yield
/
;
/
/
/
C15H10O3: C, 75.62; H, 4.23. Found: C, 75.38; H, 4.35.
5.1.4.2. 5-Benzyloxy-3-(4-hydroxyphenyl)chromen-4-
one 4b. Yellow solid; Rfꢀ
1:1); yield 69.3%; m.p. 161.7ꢁ
3100, 1620, 1460 cmꢂ1; 1H-NMR (CDCl3, 400 MHz) d
5.29 (s, 2H), 6.08 (broad s, 1H), 6.84ꢁ7.04 (m, 4H),
7.28ꢁ7.60 (m, 8H), 7.84 (s, 1H); Anal. Calc. for
/
0.44 (hexanes:ethyl acetateꢀ
/
/
162.5 8C; IR (KBr) 3400ꢁ
/
5.1.3.1. 1-(2-Hydroxyphenyl)-3-[4-
(methoxymethoxy)phenyl]propenone 3a. Yellow solid;
/
/
Rfꢀ
m.p. 71.3ꢁ
/
0.63 (hexanes:ethyl acetateꢀ
72.8 8C; IR (KBr) 1640, 1580ꢁ
cmꢂ1; H-NMR (CDCl3, 89.45 MHz) d 3.49 (s, 3H),
5.23 (s, 2H), 6.84ꢁ7.16 (m, 4H), 7.40ꢁ7.67 (m, 4H),
7.83ꢁ8.00 (m, 2H), 12.91 (s, 1H); Anal. Calc. for
/
3:1); yield 78.0%;
C22H16O4: C, 76.73; H, 4.68. Found: C, 76.55; H, 4.81.
/
/
1560, 1505
1
5.1.4.3. 7-Benzyloxy-3-(4-hydroxyphenyl)chromen-4-
/
/
one 4c. Yellow solid; Rfꢀ
1:1); yield 80.1%; m.p. 228.4ꢁ
3000, 2850, 1620, 1510, 1440 cmꢂ1; H-NMR (acetone-
d6, 89.45 MHz) d 5.31 (s, 2H), 6.72ꢁ7.02 (m, 2H), 7.08ꢁ
7.21 (m, 2H), 7.34ꢁ8.13 (m, 8H), 8.18 (s, 1H), 8.43
/
0.47 (hexanes:ethyl acetateꢀ
/
/
/
229.7 8C; IR (KBr) 3400ꢁ
/
C17H16O4: C, 71.82; H, 5.67. Found: C, 71.54; H, 5.71.
1
/
/
5.1.3.2. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-[4-
(methoxymethoxy)phenyl]propenone 3b. Yellow solid;
/
(broad s, 1H); Anal. Calc. for C22H16O4: C, 76.73; H,
4.68. Found: C, 76.57; H, 4.79.
Rfꢀ
/
0.39 (hexanes:ethyl acetateꢀ5:1); yield 65.8%;
/
m.p. 87.6ꢁ
/
88.5 8C; IR (KBr) 1640, 1580ꢁ1560, 1510,
/
1450 cmꢂ1; H-NMR (CDCl3, 89.45 MHz) d 3.50 (s,
3H), 5.13 (s, 2H), 5.20 (s, 2H), 6.59 (m, 2H), 6.97 (m,
1
5.1.5. General procedure for the preparation of
compounds 5
Compound 4 (0.3 mmol) was dissolved in methanol
4H), 7.53ꢁ7.75 (m, 8H), 13.61 (s, 1H); Anal. Calc. for
/
C24H22O5: C, 73.83; H, 5.68. Found: C, 73.67; H, 5.77.
(25 mL) and 10% PdÃ/C (15 mg) was added. The
resulting mixture was stirred under hydrogen gas (20
psi) at room temperature for 2 h. After removal of
catalyst by filtration with aid of celite pad, the solvent
was removed under vacuum. The crude product was
purified by flash column chromatography.
5.1.3.3. 1-(4-Benzyloxy-2-hydroxyphenyl)-3-[4-
(methoxymethoxy)phenyl]propenone 3c. Yellow solid;
Rfꢀ
m.p. 96.4ꢁ
/
0.23 (hexanes:ethyl acetateꢀ
97.3 8C; IR (KBr) 3250, 1620, 1580, 1500
cmꢂ1; H-NMR (CDCl3, 89.45 MHz) d 3.48 (s, 3H),
5.09 (s, 2H), 5.28 (s, 2H), 6.48ꢁ6.62 (m, 2H), 7.08 (d,
Jꢀ11.5 Hz, 2H), 7.25ꢁ7.68 (m, 6H), 7.82ꢁ7.95 (m, 4H),
/
5:1); yield 61.5%;
/
1
/
5.1.5.1. 5-Hydroxy-3-(4-hydroxyphenyl)chromen-4-one
/
/
/
5b [25]. Yellow solid; Rfꢀ
ateꢀ3:1); yield 63.7%; m.p. 151.5ꢁ
3400, 2925, 1650, 1610, 1580, 1520, 1470 cmꢂ1
NMR (CDCl3, 400 MHz) d 5.03 (broad s, 1H), 6.83 (d,
Jꢀ4.0 Hz, 1H), 7.10 (m, 4H), 7.42 (dd, Jꢀ2.0, 6.4 Hz,
2H), 7.55 (t, Jꢀ8.4 Hz, 1H), 7.96 (s, 1H); Anal. Calc.
/
0.24 (hexanes:ethyl acet-
153.0 8C; IR (KBr)
1H-
13.50 (s, 1H); Anal. Calc. for C24H22O5: C, 73.83; H,
5.68. Found: C, 73.72; H, 5.75.
/
/
;
5.1.4. General procedure [20,21] for the preparation of
compounds 4
/
/
/
To the solution of 3 (3.6 mmol) in methanol (400 mL),
thallium nitrate trihydrate (7.2 mmol) was added. The
resulting solution was stirred at room temperature for 1
day. After concentration to about 200 mL, hydrochloric
acid (2 N, 25 mL) was added and the resulting mixture
was stirred at 50 8C for 5 h. After removal of insoluble
material by filtration, the filtrate was concentrated at
room temperature under vacuum. The crude product
was dissolved in chloroform (50 mL) and washed with
water three times. The organic layer was dried with
anhydrous sodium sulphate and concentrated under
for C15H10O4: C, 70.89; H, 3.96. Found: C, 70.69; H,
4.14.
5.1.5.2. 7-Hydroxy-3-(4-hydroxyphenyl)chromen-4-one
5c [25]. White solid; Rfꢀ
1:1); yield 84.0%; m.p. 344.1ꢁ
3000, 1630, 1520, 1460 cmꢂ1; 1H-NMR (CD3OD, 89.45
MHz) d 6.79ꢁ7.00 (m, 4H), 7.37 (m, 2H), 8.06 (s, 1H),
/
0.36 (hexanes:ethyl acetateꢀ
/
/
345.6 8C; IR (KBr) 3400ꢁ
/
/
8.13 (s, 1H); Anal. Calc. for C15H10O4: C, 70.89; H,
3.96. Found: C, 70.71; H, 4.08.

S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ
/
545
543
5.1.6. Preparation of 3-(4-benzyloxyphenyl)-5-
hydroxychromen-4-one 6b
(m, 2H), 5.15ꢁ
7.54 (m, 4H), 7.72ꢁ
Hz, 1H), 8.02 (d, Jꢀ
Jꢀ1.4, 7.8 Hz, 1H); Anal. Calc. for C29H28O12: C,
/
5.22 (m, 2H), 5.31ꢁ
7.76 (m, 1H), 7.82 (dd, Jꢀ
2.0 Hz, 1H), 8.09 (s, 1H), 8.31 (dd,
/
5.34 (m, 2H), 7.42ꢁ
/
/
/2.4, 8.8
Compound 5b (160 mg, 0.63 mmol) was dissolved in
dry acetonitrile (40 mL) and potassium carbonate (95
mg, 0.69 mmol) was added. The resulting mixture was
stirred at 60 8C for 1 h and benzyl bromide (108 mg, 0.63
mmol) was added. After 2 h stirring at 60 8C, the solvent
was removed under vacuum. Chloroform (50 mL) was
added to the residue and washed with water three times.
The organic layer was dried with anhydrous sodium
sulphate and concentrated under vacuum. The crude
product was purified by flash column chromatography.
/
/
61.27; H, 4.96. Found: C, 61.01; H, 5.13.
5.1.8.2. 5-Benzyloxy-3-[4-(2?,3?,4?,6?-tetra-O-acetyl-b-
D
-glucopyranosyloxy)phenyl]chromen-4-one 8b. White
solid; Rfꢀ0.31 (hexanes:ethyl acetateꢀ1:1); yield
82.9%; m.p. 159.0ꢁ 2850,
/
/
/
160.1 8C; IR (KBr) 3100ꢁ
/
1750, 1640, 1600, 1500, 1450 cmꢂ1; H-NMR (CDCl3,
400 MHz) d 2.04 (s, 3H), 2.06 (s, 3H), 2.08 (s, 3H), 2.09
(s, 3H), 3.87 (m, 1H), 4.18 (m, 1H), 4.30 (m, 1H), 5.10
1
Yellow solid; Rfꢀ
yield 68.1%; m.p. 145.2ꢁ
1640, 1605, 1580, 1500, 1470 cmꢂ1; H-NMR (CDCl3,
89.45 MHz) d 5.11 (s, 2H), 6.77ꢁ7.10 (m, 4H), 7.18ꢁ
/
0.45 (hexanes:ethyl acetateꢀ
/
3:1);
/
146.0 8C; IR (KBr) 3600ꢁ3200,
/
1
(m, 1H), 5.17 (m, 1H), 5.28 (s, 2H), 5.31 (m, 2H), 6.86ꢁ
/
/
/
7.07 (m, 4H), 7.26ꢁ7.61 (m, 8H), 7.83 (s, 1H); Anal.
/
7.61 (m, 8H), 7.96 (s, 1H), 12.68 (s, OH); Anal. Calc. for
C22H16O4: C, 76.73; H, 4.68. Found: C, 76.62; H, 4.75.
Calc. for C36H34O13: C, 64.09; H, 5.08. Found: C, 63.87;
H, 5.17.
5.1.7. Preparation [30] of 5-hydroxy-3-(4-
hydroxyphenyl)chroman-4ꢁ
Compound 6b (90 mg, 0.2614 mmol) was dissolved in
methanol (50 mL) and 10% PdÃC (50 mg) was added.
The resulting mixture was stirred under hydrogen (30
psi) for 2 h at room temperature. After removal of
catalyst by filtration with aid of celite pad, the solvent
was removed under vacuum. The crude product was
purified by flash column chromatography.
/
one 7b
5.1.9. Preparation of 5-benzyloxy-3-[4-(2?,3?,4?,6?-tetra-
O-acetyl-b-D-glucopyranosyloxy)phenyl]chroman-4-one
9b
Procedure used for the preparation of 7b was utilized
for the preparation of 9b starting with 8b (80 mg, 0.12
mmol).
/
Yellow solid; Rfꢀ
/
0.50 (hexanes:ethyl acetateꢀ1:1);
/
yield 85.9%; m.p. 67.8ꢁ68.9 8C; IR (KBr) 3300, 2950,
/
White solid; Rfꢀ
yield 68.7%; m.p. 97.5ꢁ
2900, 1650, 1520, 1460 cmꢂ1; H-NMR(CDCl3, 89.45
MHz) d 3.96 (t, Jꢀ6.9 Hz, 1H), 4.63 (d, Jꢀ6.9 Hz,
2H), 6.42ꢁ6.59 (m, 2H), 6.76ꢁ7.11 (m, 2H), 7.20ꢁ7.55
/
0.23 (hexanes:ethyl acetateꢀ
/5:1);
1750 cmꢂ1; 1H-NMR (CDCl3, 400 MHz) d 2.04 (s, 3H),
2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 3.85 (m, 1H), 4.00
/
98.7 8C; IR (KBr) 3600ꢁ3100,
/
1
(dd, Jꢀ
(dd, Jꢀ
(d, Jꢀ7.2 Hz, 1H), 5.16 (t, Jꢀ
1H), 6.47 (d, Jꢀ8.0 Hz, 1H), 6.54 (d, Jꢀ
7.00 (d, Jꢀ8.8 Hz, 2H), 7.23 (d, Jꢀ8.4 Hz, 2H), 7.39 (t,
Jꢀ8.2 Hz, 1H), 11.70 (d, Jꢀ2.0 Hz, 1H); Anal. Calc.
/
5.8, 8.6 Hz, 1H), 4.16 (d, Jꢀ
4.4, 13.2 Hz, 1H), 4.61 (t, Jꢀ
9.6 Hz, 1H), 5.29 (m,
8.8 Hz, 1H),
/
12.4 Hz, 1H), 4.28
/
/
/
/6.8 Hz, 2H), 5.07
/
/
/
/
/
(m, 4H), 11.81 (s, 1H); Anal. Calc. for C15H12O4: C,
70.31; H, 4.27. Found: C, 70.15; H, 4.32.
/
/
/
/
/
/
5.1.8. General procedure [23,24] for the preparation of
compounds 8
for C29H30O13: C, 59.38; H, 5.16. Found: C, 59.18; H,
5.25.
Compound 5a or 5b (0.25 mmol) and 2-bromogluco-
pyranosyl acetate (3 equiv.) were dissolved in chloro-
form (6 mL). To the resulting solution, benzyl triethyl
ammonium chloride (0.2 equiv.), potassium carbonate
(6 equiv.) and water (5% w/w of potassium carbonate)
were added. The resulting mixture was stirred for 2 days.
After addition of chloroform (40 mL), the organic layer
was washed with water three times, dried with anhy-
drous sodium sulphate, and concentrated under va-
cuum. The crude product was purified by flash column
chromatography.
5.1.10. Preparation of 5-hydroxy-3-[4-(2?,3?,4?,6?-tetra-
O-acetyl-b-D-glucopyranosyloxy)phenyl]chromen-4-one
10b
Procedure used for the preparation of 5b was utilized
for the preparation of 10b starting with 8b (80 mg, 0.12
mmol).
Yellow solid; Rfꢀ
yield 78.8%; m.p. 98.2ꢁ
1750, 1650ꢁ
1680, 1510 cmꢂ1; H-NMR (CDCl3, 400
MHz) d 2.05 (s, 3H), 2.06 (s, 3H), 2.08 (s, 3H), 2.09 (s,
3H), 3.75 (m, 1H), 4.19 (dd, Jꢀ2.4, 12.4 Hz, 1H), 4.31
(m, 1H), 5.12 (m, 1H), 5.18 (m, 1H), 5.30 (m, 2H), 6.83
(d, Jꢀ8.0 Hz, 1H), 6.93 (d, Jꢀ8.0 Hz, 1H), 7.09 (d,
Jꢀ4.4 Hz, 2H), 7.48 (d, Jꢀ4.4 Hz, 2H), 7.56 (t, Jꢀ8.4
/
0.36 (hexanes:ethyl acetateꢀ
/
1:1);
/
99.5 8C; IR (KBr) 3500ꢁ2950,
/
1
/
5.1.8.1. 3-[4-(2?,3?,4?,6?-Tetra-O-acetyl-b-
glucopyranosyloxy)phenyl]chromen-4-one 8a. Yellow so-
lid; Rfꢀ0.27 (hexanes:ethyl acetateꢀ1:1); yield 65.0%;
m.p. 173.2ꢁ174.4 8C; IR (KBr) 3100ꢁ
2900, 1750 cmꢂ1
1H-NMR(CDCl3, 400 MHz) d 2.05 (s, 3H). 2.06 (s, 3H),
2.11 (s, 3H), 2.14 (s, 3H), 3.87ꢁ3.92 (m, 1H), 4.25ꢁ4.28
D-
/
/
/
/
/
/
/
;
/
/
/
Hz, 1H), 7.97 (s, 1H), 12.60 (s, 1H); Anal. Calc. for
C29H28O13: C, 59.59; H, 4.82. Found: C, 59.45; H, 4.94.
/
/

544
S.-H. Jung et al. / European Journal of Medicinal Chemistry 38 (2003) 537ꢁ545
/
1
5.1.11. General procedure for the preparation of
compounds 11
1640, 1500 cmꢂ1; H-NMR (CDCl3, 400 MHz) d 3.15
(m, 2H), 3.24 (m, 3H), 3.32 (m, 2H), 3.46 (m, 2H), 3.67
Acetate 8a or 8b (0.54 mmol) was dissolved in
methanol (10 mL) and sodium methoxide (5 equiv.)
was then added. The resulting solution was stirred for 1
h at room temperature. After removal of solvent under
vacuum, the crude product was purified by flash column
chromatography.
(m, 1H), 3.74 (s, 1H), 4.06 (t, Jꢀ
Jꢀ5.2 Hz, 2H), 4.84 (d, Jꢀ7.6 Hz, 1H), 5.82 (d, Jꢀ
Hz, 2H), 7.00 (d, Jꢀ8.8 Hz, 2H), 7.20 (d, Jꢀ8.8 Hz,
2H), 12.19 (s, 1H); Anal. Calc. for C21H22O10: C, 58.06;
H, 5.10. Found: C, 57.93; H, 5.24.
/
6.6 Hz, 1H), 4.54 (d,
/
/
/4.8
/
/
5.2. Inhibitory activity against interleukin-5
5.1.11.1. 3-[4-(b-
Glucopyranosyloxy)phenyl]chromen-4-one 11a. white so-
lid; Rfꢀ0.54 (chloroform:methanolꢀ3:1); yield 57.9%;
3000, 1640, 1540
D-
The Y16 cell line was grown in RPMI (10.4 mg mLꢂ1
RPMI-1640, 24 mM NaHCO3, 100 units mLꢂ1 benzyl-
penicillin potassium, 100 mg mLꢂ1 streptomycin sul-
phate, pH 7.1) containing 8% fetal bovine serum (FBS)
and 5 units mLꢂ1 IL-5 at 37 8C with 5% CO2. The
grown Y16 cells were harvested by centrifugation at
/
/
m.p. 103.5ꢁ
/
104.5 8C; IR (KBr) 3600ꢁ
/
cmꢂ1; H-NMR (dimethylsulphoxide-d6, 400 MHz) d
1
3.30 (m, 1H), 3.70 (m, 1H), 4.58 (m, 3H), 5.14 (m, 3H),
5.32 (d, Jꢀ
(m, 1H), 7.40 (d, Jꢀ
3H), 8.00 (d, Jꢀ8.0 Hz, 1H), 8.17 (m, 1H), 8.71 (s, 1H);
/
4.4 Hz, 1H), 5.38 (d, Jꢀ/4.4 Hz, 1H), 7.00
/8.8 Hz, 1H), 7.54 (m, 2H), 7.80 (m,
250ꢃg for 10 min at 4 8C, washed twice with Hanks’
/
solution (9.8 mg mLꢂ1 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
/
Anal. Calc. for C21H20O8: C, 63.00; H, 5.03. Found: C,
62.85; H, 5.14.
1ꢃ
Viability of the cells was more than 95% in all
preparations. One hundred mL of 1ꢃ
104 cells were
/
105 cells mLꢂ1 with RPMI containing 8% FBS.
5.1.11.2. 5-Benzyoxy-3-[4-(b-
glucopyranosyloxy)phenyl]chromen-4-one 11b. Yellow
solid; Rfꢀ0.45 (chloroform:methanolꢀ5:1); yield
82.0%; m.p. 93.5ꢁ94.6 8C; IR (KBr) 3500ꢁ3000, 1600
cmꢂ1; H-NMR (DMSO, 400 MHz) d 3.26ꢁ
3.50 (m,
3H), 3.71(d, Jꢀ11.2 Hz, 2H), 4.12 (broad s, 1H), 4.91
(d, Jꢀ7.6 Hz, 1H), 4.99 (d, Jꢀ7.2 Hz, 1H), 5.26 (s,
2H), 5.22ꢁ5.33 (m, 3H), 7.07ꢁ7.19 (m, 3H), 7.32ꢁ7.52
(m, 5H), 7.62 (d, Jꢀ7.6 Hz, 2H), 7.70 (t, Jꢀ8.4 Hz,
D-
/
/
/
/
/
dispensed to each well of a 96-well microplate (Nunc,
Denmark), and 50 mL of 4 units mLꢂ1 IL-5 and 50 mL
of the sample were added. Control group was treated
with RPMI containing 8% FBS instead of sample, and
blank group with RPMI containing 8% FBS instead of
IL-5. After incubation at 37 8C with 5% CO2 for 48 h,
Y16 cells in each well were treated with 20 mL of WST-1
solution (3.3 mg WST-1 and 0.7 mg 1-methoxy-5-
methyl-phenazinium methylsulphate per mL of phos-
phate buffered saline), and incubated 37 8C with 5%
1
/
/
/
/
/
/
/
/
/
2H), 8.31 (s, 1H); Anal. Calc. for C28H26O9: C, 66.40; H,
5.17. Found: C, 66.23; H, 5.32.
5.1.12. Preparation of 5-hydroxy-3-[4-(b-D-
glucopyranosyloxy)phenyl]chromen-4-one 12b
Procedure used for the preparation of 5b was utilized
for the preparation of 12b starting with 11b (70 mg, 0.14
mmol).
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 mM concentration was expressed as %
/
White solid; Rfꢀ
yield 79.9%; m.p. 84.3ꢁ
1630, 1500, 1460 cmꢂ1; 1H-NMR (CDCl3, 400 MHz) d
3.19ꢁ3.47 (m, 3H), 3.71 (m, 2H), 4.60 (broad s, 1H),
4.84 (m, 2H), 4.93 (d, Jꢀ6.8 Hz, 1H), 5.06 (broad s,
1H), 5.36 (broad s, 1H), 6.85 (d, Jꢀ8.4 Hz, 1H), 7.12
(m, 3H), 7.54 (d, Jꢀ8.8 Hz, 2H), 7.69 (t, Jꢀ8.4 Hz,
/
0.59 (chloroform:methanolꢀ
/3:1);
inhibition, [1-(sample A450
ꢂ
/
blank A450)/(control A450
ꢂ
/
85.4 8C; IR (KBr) 3600ꢁ3000,
/
blank A450)]ꢃ100. Data were collected as mean9
/
/
S.E.M. of three independent tests. Samples showing
more than 50% inhibition at 50-mM concentration were
serially diluted, and then subjected to the IL-5 bioassay
to determine IC50 values.
/
/
/
/
/
1H), 8.57 (s, 1H), 12.65 (broad s, 1H); Anal. Calc. for
C21H20O9: C, 60.58; H, 4.84. Found: C, 60.45; H, 4.96.
Acknowledgements
5.1.13. Preparation of 5,7-dihydroxy-3-[4-(b-
glucopyranosyloxy)phenyl]chroman-4-one 14
Procedure used for the preparation of 7b was utilized
for the preparation of 14 starting with sophoricoside
(12, 100 mg, 0.23 mmol) isolated from Sophora japonica
[18]. The reaction was run for 15 h.
D-
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.
References
White solid; Rfꢀ
/
0.41 (chloroform:methanolꢀ
/3:1);
yield 94.5%; m.p. 82.3ꢁ
/
83.4 8C; IR (KBr) 3600ꢁ3000,
/
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