Inhibitory activity of Brazilian green propolis components and their derivatives on the release of cys-leukotrienes

Article abstract of DOI:10.1016/j.bmc.2009.11.007

The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was comparable to that of existing LT synthesis inhibitors. Crown Copyright

Full text of DOI:10.1016/j.bmc.2009.11.007

Bioorganic & Medicinal Chemistry 18 (2010) 151–157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory activity of Brazilian green propolis components and their
derivatives on the release of cys-leukotrienes
Hiroko Tani a, , Keiko Hasumi a,b, Tomoki Tatefuji a, Ken Hashimoto a, Hiroyuki Koshino b,
*
Shunya Takahashi b,
*
a Institute for Bee Products and Health Science, Yamada Apiculture Center, Inc., 194 Ichiba, Kagamino 708-0393, Japan
b Advanced Science Institute, RIKEN, 2-1 Wako 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine
release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key
mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-
LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized
and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major
active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was com-
parable to that of existing LT synthesis inhibitors.
Received 7 October 2009
Revised 3 November 2009
Accepted 4 November 2009
Available online 10 November 2009
Keywords:
Propolis
Baccharis dracunculifolia
Cinnamic acid derivatives
Flavanone
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Allergic rhinitis
Nasal obstruction
Cysteinyl-leukotrienes
1. Introduction
constrictors, cysteinyl-leukotrienes (cys-LTs) synthesis inhibitors
and cys-LTs receptor antagonists.6–10 Leukotrienes are important
Propolis is a resinous substance collected by honeybees from
leaf buds and cracks in the bark of various plants. Crude extracts
of propolis have very complicated compositions, resulting from
variations in geographical and botanical origin. These compounds
can originate from plant exudates collected by bees, bee salivary
gland secretions or substances, which are incorporated into propo-
lis during its processing.1 Brazilian green propolis produced in
Southeast Brazil has been extensively consumed as a dietary sup-
plement, particularly in Japan, and Baccharis dracunculifolia is the
main botanical source.2,3 It contains mainly prenylated derivatives
of cinnamic acid, among which artepillin C (1) is known to be a ma-
jor component.
In a previous study, we reported that the ethanol extract of Bra-
zilian green propolis was able to alleviate symptoms in patients
with seasonal allergic rhinitis by reducing therapeutic drug con-
sumption and delaying the start of symptom onset.4 Treatment
with propolis also significantly relieved nasal obstruction.5
Seasonal allergic rhinitis is usually treated symptomatically
with histamine H1 receptor antagonists, sympathomimetic vaso-
mediators of allergic nasal obstruction, and cys-LTs synthesis
inhibitors and receptor antagonists have been reported to be effec-
tive especially in the treatment of nasal obstruction.9,10
Caffeic acid (2), a representative constituent of Brazilian green
propolis, has been reported to be an inhibitor of 5-lipoxygenase.11
The enzyme 5-lipoxygenase plays a key role in regulating the pro-
duction of leukotrienes. Therefore, other cinnamic acid derivatives
may also suppress leukotriene synthesis and release. Furthermore,
caffeic acid phenethyl ester (9), which is found in European and
Chinese propolis is derived from poplar (Populus) bud is not found
in Brazilian green propolis, and is more potent in several activities
than caffeic acid.12,13 Although 9 has been reported to cause aller-
gic contact dermatitis,14 it has received attention due to its potent
pharmacological activities.12–16
In order to understand the possible mechanism of propolis eth-
anol extract, we investigated the effects of the ethanol extract on
the release of proinflammatory cytokines from peripheral blood
mononuclear cells, as well as histamine and cys-LTs release from
peripheral leukocytes of allergic patients. Furthermore, the synthe-
sis of a series of propolis components (1 and 3–5) and their phen-
ethyl esters (10–13), and their inhibitory effects on the release of
cys-LTs in differentiated HL-60, a human promyelocytic leukemia
cell line are also discussed (Fig. 1).
* Corresponding authors. Fax: +81 86 854 3346 (H.T.); +81 48 462 4627 (S.T.).
E-mail addresses: ht0807@yamada-bee.com (H. Tani), shunyat@riken.jp (S.
Takahashi).
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.007

152
H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
Table 2
HO
HO
Effects of propolis ethanol extract on Cry j1-induced cytokine release from PBMCs of
patients with allergic rhinitis
CO2R
CO2R
HO
Amounta
(lg/ml)
Inhibition (%)
IL-5
IL-13
1: R = H
10: R = (CH2)2Ph
2: R = H
9: R = (CH2)2Ph
3
10
30
8.3 19.5
26.0 15.3
64.4 27.5
10.7 41.9
29.6 29.5
75.2 12.8
CO2R1
CO2R
Each value represented the mean SD of 8 experiments.
a
Propolis extract (solid).
R2O
O
3: R1= H, R2 = H
5: R = H
propolis were prepared as follows: synthesis of artepillin C (1)
was initiated with the Mizoroki–Heck reaction17 of iodide 1418
with t-butyl acrylate in the presence of palladium acetate, tri-o-tol-
ylphosphine and n-tetrabutylammonium chloride in aqueous N,N-
dimethylformamide (DMF),19,20 providing 15 in 81% yield. Treat-
ment of the t-butyl ester with trimethylsilyl trifluoromethanesul-
fonate (TMSOTf) and 2,6-lutidine followed by acidic work-up
gave 1 in 71% yield. Following the same procedure described
above, the known phenol 1621 was transformed into t-butyl ester
18 and then drupanin (3) in 48% overall yield. Acylation of 16 with
phenylpropionyl chloride was the first step in the synthesis of bac-
charin (4), giving 17 in 94% yield. The Mizoroki–Heck reaction of 17
with t-butyl acrylate was accomplished by the action of palladium
acetate–triphenylphosphine in triethylamine and toluene to give
19 in 78% yield. Interestingly, the use of tri-o-tolyphosphine18,21 in-
stead of triphenylphosphine resulted in no reaction. Compound 19
underwent removal of t-butyl group to give baccharin (4) in 90%
yield. Although DDQ oxidation of 1 was reported to produce culif-
olin (5), the yield was quite low (ꢀ25%).22 Therefore, we searched a
more efficient method for the preparation of 5. A Pd-promoted
etherification23–25 of 15 gave a mixture of 20–22 in a variety of ra-
tios (Table 3), while DDQ oxidation of 15 afforded 22 exclusively,
albeit in a low yield. Selenocyclization26 of 15 at À78 °C followed
by H2O2 oxidation provided a mixture of 21 and 22 in 57% yield
(21/22 = 37/63 by 1H NMR). The best results were obtained by
the use of 16 as a starting material. Thus, propargylation27 and
thermal rearrangement28 of 16 gave the 2,2-dimethylpyran deriv-
ative 24 in 70% yield. This reacted smoothly with t-butyl acrylate in
toluene to give 22 in 78% yield. Synthesis of 22 from 18 via the cor-
responding propargyl ether was impractical due to the low yield.
Upon treatment with TMSOTf-2,6-lutidine, 22 afforded culifolin
(5) in 90% yield (Scheme 1).
4: R1= H, R2 = COCH2CH2Ph
11: R1= (CH2)2Ph, R2 = H
13: R = (CH2)2Ph
12: R1= (CH2)2Ph, R2 = COCH2CH2Ph
OR
HO
O
HO
O
OH
OH
O
OH
O
6: R = H
7: R = CH3
8
Figure 1. Structures of 1–13.
2. Results and discussion
To investigate the influence of propolis ethanol extract, which
contained 55% propolis extract as a solid, on cys-LTs, histamine,
and proinflammatory cytokine release, peripheral leukocytes and
peripheral blood mononuclear cells (PBMCs) were prepared from
10 allergic patients. After treatment with the propolis ethanol ex-
tract and Cry j1, which is a major antigen of Japanese cedar pollen,
the amount of cys-LTs and histamine released from peripheral leu-
kocytes, and IL-4, IL-5, and IL-13 released from PBMCs was
measured.
The ethanol extract potently inhibited cys-LTs release in a con-
centration-dependent manner, with an IC50 value of 5.8
ble 1). On the other hand, the release of histamine was inhibited
by the ethanol extract only at high concentration (100 g/ml). In
addition, theenhancedreleaseof IL-5and IL-13seemed tobe slightly
inhibited by the addition of the ethanol extract, however, the values
varied greatly among individual patients and were not statistically
significant (Table 2). IL-4 was not detected even under extreme
lg/ml (Ta-
l
Phenethyl ester 9 has been reported to be more potent in sev-
eral activities than 2.12–16 Such results prompted us to prepare
similar esters of 1 and 3–5 with the expectation of an increase in
the activity. Compounds 1 and 3–5 each reacted with (2-bromo-
ethyl)benzene in the presence of cesium carbonate in DMF to af-
ford the corresponding phenethyl esters 10–13, respectively, in
high yield.
Cry j1 stimulation (10 lg/ml) (data not shown). These results sug-
gested that the inhibition of cys-LTs release was one of the most
important mechanisms of the anti-allergic properties of the ethanol
extract. This was in agreement with our previous clinical observa-
tion, where the ethanol extract prevented nasal obstruction.
To evaluate the effect of propolis components on cys-LTs
release, cinnamic acid derivatives 1 and 3–5 in Brazilian green
Table 3
Cyclization reaction of 15
Table 1
Effects of propolis ethanol extract on Cry j1-induced cys-LTs and histamine release
from peripheral leukocytes of patients with allergic rhinitis
Entry Conditions
Products (%)
20 21 22
Amounta
(lg/ml)
Inhibition (%)
1
2
3
4
5
PdCl2, AcONa, MeOH–H2O, 35 °C
8
9
15 15
26 20
PdCl2, O2, AcONa, MeOH–H2O, 55 °C
PdCl2(CH3CN)2, NaOMe, benzene, 90 °C
DDQ, benzene, rt
cys-LTs
Histamin
7
—
—
—
—
7
6
19
23
3
10
30
28.5 13.4
61.8 12.1
95.9 2.9
96.2 3.6
7.2 8.5
7.5 10.5
10.5 8.1
77.2 17.2
PhSeBr, CH2Cl2, À20?0 °C, then 15% H2O2, pyridine,
0 °C?rt
100
6
7
PhSeCl, CH2Cl2, rt, then 15%H2O2, pyridine, 0 °C?rt
PhSeCl, CH2Cl2, À78 °C, then 15%H2O2, pyridine,
0 °C?rt
—
—
4
24
21 36
Each value represented the mean SD of 7 and 8 experiments.
a
Propolis extract (solid).

H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
153
I
CO2t-Bu
CO2H
CO2 (CH2)2Ph
a
b
c
HO
HO
HO
HO
14
15
1
10
f or g
CO2R
CO2t-Bu
+
CO2t-Bu
CO2t-Bu
b
+
O
O
O
c
O
e
5: R = H
21
22
20
13: R = (CH2)2Ph
I
I
h
i
O
O
23
24
CO2H
I
CO2t-Bu
CO2 (CH2)2Ph
a or e
b
c
RO
RO
d
RO
RO
18: R = H
19: R =CO(CH2)2Ph
3: R = H
4: R =CO(CH2)2Ph
11: R = H
16: R = H
17: R =CO(CH2)2Ph
12: R =CO(CH2)2Ph
Scheme 1. Reagents and conditions: (a) t-butyl acrylate, Pd(OAc)2, (o-Tol)3P, n-Bu4NCl, Et3N, DMF–H2O, 35 °C, 81% for 15, 77% for 18 from 16; (b) TMSOTf, 2,6-lutidine,
dichloromethane, rt, and then dil HCl, rt, 71% for 1, 62% for 3, 90% for 4, 98% for 5; (c) (2-bromoethyl)benzene, Cs2CO3, DMF, rt, 72% for 10, 78% for 11, 83% for 12, 90% for 13;
(d) 3-phenylpropionyl chloride, DMAP, Et3N, dichloromethane, 0 °C, 94%; (e) t-butyl acrylate, Pd(OAc)2, Ph3P, Et3N, toluene, 105 °C, 78% for 19 from 17, 78% for 22 from 24; (f)
PdCl2, O2, AcONa, methanol-H2O, 55 °C, 9% for 20, 26% for 21, 20% for 22; (g) PhSeCl, dichloromethane, À78 °C, and then 15% H2O2, dichloromethane–pyridine, 0 °C, 57% (21/
22 = 37/63; 1H NMR); (h) 3-chloro-3-methyl-1-butyne, DBU, CuCl, acetonitrile, 0 °C, 82% from 16; (i) N,N-diethylaniline, 150 °C, 85%.
All synthesized compounds (1, 3–5, and 10–13) and some
authentic samples (2, 6–8, and 9) were tested for their inhibitory
activity with the ethanol extract on cys-LTs release using differen-
tiated HL-60, a human promyelocytic leukemia cell line.29 Two
types of leukotriene synthesis inhibitors (nordihydroguaiaretic
acid: NDGA and zileuton) were also tested in order to compare
the activities. The ethanol extract and its components (1–8) mod-
erately inhibited the release of cys-LTs (Table 4). The content of 1–
8 in the ethanol extract, which contained 55% propolis extract as a
solid, were approximately 20.7%, 0.1%, 1.9%, 7.5%, 0.3%, 0.2%, 3.6%,
and 3.1% (w/w), respectively, as measured by HPLC analysis. When
considering the contents of the ethanol extract, the main active
components were artepillin C (1), baccharin (4), and kaempferide
(7). It remains to be established whether minor components might
have greater potency than these major components, since the
potency of the ethanol extract was not totally explained by the
potency of these three components.
Among the phenethyl esters, compounds 9–11 showed 667, 74,
and 52 times more potent than each acid showed, and the potency
of 10 was comparable to those of existing leukotriene synthesis
inhibitors (NDGA and zileuton) and also to that of 9, an active com-
ponent of European and Chinese propolis. Structure–activity rela-
tionships of tested compounds indicated that the cys-LTs
inhibitory activity increases with increasing lipophilicity. Further,
the potencies of 12 and 13 were weaker than those of each acids
showed that a phenolic hydroxyl group or an acid was essential
for this activity.
Recently artepillin C was reported to have an inhibitory effect
on prostaglandin E2 and nitric oxide through NF-jB modulation
Table 4
Inhibitory activity of cys-LTs release from differentiated HL-60 by DMSO
using the macrophage cell line RAW 264.7.30 In addition the inhib-
itor of nitric oxide synthase has been reported to prevent nasal
obstruction by inhibiting venodilation of nasal mucosa induced
by antigen challenge.31 These findings suggested that preventing
nasal obstruction using the Brazilian green propolis extract may
be due to the inhibition of several inflammatory pathways.
Further study on the design and synthesis of a new class of cys-
LTs release inhibitors with the cinnamic acid and/or flavanone
scaffold with a favorable safety profile is in progress.
Compounds
IC50 values (
0.08 0.012
0.04 0.0026
4.3 1.5
7.0 0.15
44 18
10.3 2.5
4.4 2.0
1.6 0.39
2.4 0.9
2.0 1.3
6.1 2.1
0.068 0.0055
0.094 0.0027
0.20 0.036
>10
lg/ml)
NDGA
Zileuton
EtOH extracta
Artepillin C (1)
Caffeic acid (2)
Drupanin (3)
Baccharin (4)
Culifolin (5)
Kaempferol (6)
Kaempferide (7)
Pinocembrin (8)
Caffeic acid phenethyl ester (9)
Artepillin C phenethyl ester (10)
Drupanin phenethyl ester (11)
Baccharin phenethyl ester (12)
Culifolin phenethyl ester (13)
3. Experimental section
3.1. General procedures
>10
All reactions were carried out under an argon atmosphere, un-
less otherwise noted. Melting points were determined using a
Yanaco MP-500 apparatus and are uncorrected. IR spectra were
The values represent the mean SD (n = 2).
a
Solid propolis extract.

154
H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
recorded with a JASCO VALOR-III spectrophotometer. 1H and 13C
NMR spectra were obtained using Varian NMR system spectrome-
ter at 500 MHz and 125 MHz, respectively. Chemical shifts were
referenced to a residual signal of CDCl3 (dH 7.26, dC 77.0) and
CD3OD (dH 3.30, dC 49.0). ESIMS were recorded on a Waters Micro-
mass Quattro Premier XE mass spectrometer or JEOL JMS-T100LC
mass spectrometer. Column chromatography was performed on
trated to give a syrup (126 mg), which was dissolved in
tetrahydrofuran (1.5 ml). To this solution was added 0.2 M HCl
(1.5 ml), and the mixture was stirred at rt for 2 h, diluted with
ether, washed successively with water, brine, dried, and concen-
trated to give a brown solid, which was recrystallized from n-hex-
ane–ethyl acetate to give 1 (75 mg, 71%) as a colorless solid; mp
96–97 °C (n-hexane–ethyl acetate); IR (ATR) 3400, 1690, 1620,
1580, 1440, 1430, 1400, 1360, 1320, 1300, 1250, 1210, 1180,
Kanto Silica Gel 60N (spherical, neutral; 40–100 lm). Merck pre-
coated Silica Gel 60 F254 plates, 0.25 mm thickness, was used for
analytical thin-layer chromatography. The solvent extracts were
dried with magnesium sulfate, and the solutions were evaporated
under diminished pressure at 40–42 °C. Compounds on the TLC
plates were detected under UV light (254 nm) and/or by spraying
anisaldehyde–sulfuric acid reagent on the plate and heating it at
110 °C for 20 min. The spray reagent was prepared freshly before
use by adding 1 ml of concentrated H2SO4 to a solution of 0.5 ml
of p-anisaldehyde in 50 ml of acetic acid.
1120, 1020, 970 cmÀ1 1H NMR (500 MHz, CDCl3): d 7.71 (d,
;
J = 16.0 Hz, 1H), 7.21 (s, 2H), 6.29 (d, J = 16.0 Hz, 1H), 5.32 (m,
2H), 3.35 (d, J = 7.5 Hz, 4H), 1.80 (s, 6H), 1.78 (s, 6H); 13C NMR
(125 MHz, CDCl3): d 172.7, 155.4, 147.4, 135.2, 128.4, 127.7,
126.4, 121.3, 114.1, 29.5, 25.8, 17.9; HR MS (ESI) calcd for
C19H24O3Na (M+Na+) 323.1623, found 323.1621.
3.5.3. Artepillin C phenylethyl ester (10)
To a stirred solution of 1 (40.2 mg, 0.13 mmol) and cesium car-
bonate (46 mg, 0.14 mmol) in N,N-dimethylformamide (0.9 ml)
3.2. Chemicals
was added (2-bromoethyl)benzene (30 ll, 0.20 mmol) at rt, and
the mixture was stirred at rt for 18 h, then diluted with water. The
resulting mixture was extracted with ether. The extracts were
washed successively with water, brine, dried, and concentrated.
Theresiduewaschromatographedon silicagel(n-hexane–ethylace-
tate = 30:1) to give 10 (38.0 mg, 72%) as a colorless solid; IR (ATR)
3400, 2950, 2840, 1680, 1620, 1590, 1470, 1450, 1420, 1370, 1340,
The authentic samples of caffeic acid, kaempferol, and kaempfe-
ride were purchased from Wako Co. (Osaka, Japan), and pinocem-
brin and caffeic acid phenethyl ester were from Sigma (St. Louis,
MO).
1260, 1160, 1130, 1080, 1110, 980, 880, 840, 800, 760, 690 cmÀ1
;
3.3. Material
1H NMR (500 MHz, CDCl3):d 7.58 (d, J = 16.0 Hz, 1H), 7.33–7.23 (m,
5H), 7.16 (s, 2H), 6.26 (d, J = 16.0 Hz, 1H), 5.66 (s, 1H), 5.31 (m, 2H),
4.41 (t, J = 7.0 Hz, 2H), 3.34 (d, J = 7.0 Hz, 4H), 3.01 (t, J = 7.0 Hz,
2H), 1.79 (s, 6H), 1.77 (s, 6H); 13C NMR (125 MHz, CDCl3): d 167.4,
155.0, 145.2, 138.0, 135.0, 128.9, 128.5, 128.0, 127.6, 126.6, 126.5,
121.4, 115.0, 64.8, 35.2, 29.5, 25.8, 17.9; HR MS (ESI) calcd for
C27H32O3Na (M+Na+) 427.2249, found 427.2250.
Brazilian green propolis was purchased from Apiários Floresta
(Minas Gerais, Brazil).
3.4. Extraction
Brazilian green propolis (2 g) was pulverized with a homoge-
nizer and extracted in 20 ml of ethanol. After stirring at room tem-
perature for 12 h, the filtration was evaporated until the solid
content reached 55%. The solid content was evaluated after evapo-
ration in vacuo and further drying in an oven at 105 °C for 4 h.
3.5.4. 4-Iodo-2-(3-methylbut-2-enyl)phenyl 3-
phenylpropionate (17)
To a stirred solution of 16 (332 mg 1.15 mmol), N,N-dimethyl-
aminopyridine (10 mg, 0.08 mmol), triethylamine (240 ml,
1.73 mmol) in dichloromethane (3.0 ml) was added dropwise 3-
phenylpropionyl chloride (205 ml, 1.38 mmol) at 0 °C. The mixture
was stitrred at 0 °C?rt for 1 h, diluted with ether, and treated suc-
cessively with aqueous NH4Cl, saturated aqueous NaHCO3. The
resulting mixture was washed with brine, dried, and concentrated.
The residue was chromatographed on silica gel (n-hexane–ethyl
acetate = 100:1?55:1) to give 17 (455 mg, 94%) as a colorless oil;
IR (ATR) 2970, 2910, 1760, 1480, 1375, 1290, 1210, 1170, 1125,
3.5. Synthesis
3.5.1. tert-Butyl (E)-3-[4-hydroxy-3,5-bis(3-methylbut-2-
enyl)phenyl]acrylate (15)
To a stirred solution of 14 (120 mg, 0.34 mmol), t-butyl acrylate
(100
ll, 0.68 mmol), tri-o-tolylphosphine (32 mg, 0.10 mmol), and
n-tetrabutylammonium chloride (29 mg, 0.10 mmol) in N,N-
dimethylformamide–triethylamine–water (9:1:1; 1.2 ml) was
added palladium acetate (15 mg, 0.07 mmol). The mixture was
stirred at 35 °C for 4 h, cooled to rt, diluted with ether, washed suc-
cessively with aqueous NH4Cl, water, brine, dried, and concen-
trated. The residue was chromatographed on silica gel (n-
hexane–ethyl acetate = 80:1?50:1) to give 15 (97.5 mg, 81%) as a
faint yellow solid; IR (ATR) 3420, 2960, 1900, 1700, 1680, 1620,
1090, 865, 660 cmÀ1 1H NMR (500 MHz, CDCl3): d 7.50 (m, 2H),
;
7.33–7.22 (m, 5H), 6.67 (d, J = 8.1 Hz, 1H), 5.14 (m, 1H), 3.09–
3.06 (m, 4H), 2.91–2.88 (m, 2H), 1.74 (s, 3H), 1.65 (s, 3H); 13C
NMR (125 MHz, CDCl3): d 170.9, 148.7, 139.9, 138.8, 136.2, 135.9,
133.9, 128.6, 128.3, 126.5, 124.2, 120.9, 90.6, 35.7, 30.9, 28.3,
25.7, 17.8; HR MS (EI) calcd for C20H21O2I (M+) 420.0586, found
420.0571.
1600, 1470, 1430, 1360, 1340, 1260, 1130, 1080, 980, 840 cmÀ1
;
1H NMR (500 MHz, CDCl3): d 7.49 (d, J = 15.8 Hz, 1H), 7.15 (s,
2H), 6.20 (d, J = 15.8 Hz, 1H), 5.62 (s, 1H), 5.30 (m, 2H), 3.33 (d,
J = 7.3 Hz, 4H), 1.78 (s, 6H), 1.77 (s, 6H), 1.53 (s, 9H);13C NMR
(125 MHz, CDCl3): d 166.8, 154.7, 143.9, 134.8, 127.9, 127.5,
126.9, 121.5, 117.1, 80.1, 29.5, 28.2, 25.8, 17.9; HR MS (EI) calcd
for C23H32O3 (M+) 356.2351, found 356.2349.
3.5.5. tert-Butyl (E)-3-[4-hydroxy-3-(3-methylbut-2-
enyl)phenyl]acrylate (18)
Treatment of 16 (300 mg, 1.04 mmol) as described for prepara-
tion of 15 gave 18 (231 mg, 77%) as a colorless oil; IR (ATR) 3325,
2960, 2900, 1670, 1630, 1600, 1500, 1420, 1360, 1330, 1270, 1240,
1140, 1080, 980, 850, 820 cmÀ1; 1H NMR (500 MHz, CDCl3): d 7.52
(d, J = 16.0 Hz, 1H), 7.27–7.26 (m, 2H), 6.80 (d, J = 9.0 Hz, 1H), 6.23
(d, J = 16.0 Hz, 1H), 5.81 (br s, 1H), 5.31, (m, 1H), 3.35 (d, J = 7.0 Hz,
1H), 1.78 (s, 3H), 1.77 (s, 3H), 1.53 (s, 9H); 13C NMR (125 MHz,
CDCl3): d 167.5, 156.5, 144.2, 134.2, 129.8, 127.5, 126.8, 121.4,
116.6, 115.8, 28.9, 28.2, 25.7, 17.7; HR MS (EI) calcd for C18H24O3
(M+) 288.1725, found 288.1717.
3.5.2. Artepillin C (1)
To a stirred solution of 15 (133 mg, 0.37 mmol), and 2,6-lutidine
(850
ll, 7.3 mmol) in dichloromethane (4.0 ml) was added tri-
methylsilyl trifluoromethanesulfonate (642
ll, 3.7 mmol). The
mixture was stirred at rt for 4.5 h, diluted with ether, washed suc-
cessively with cold aqueous HCl, water, brine, dried, and concen-

H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
155
3.5.6. tert-Butyl (E)-3-[4-(3-phenylpropionyloxy)-3-(3-
methylbut-2-enyl)phenyl]acrylate (19)
(m, 12H), 6.82 (d, J = 8.5 Hz, 1H), 6.35 (d, J = 16.0 Hz, 1H), 5.17
(m, 1H), 4.42 (t, J = 7.0 Hz, 2H), 3.14 (d, J = 7.5 Hz, 2H), 3.08 (t,
J = 7.5 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H), 1.75
(s, 3H), 1.67 (s, 3H); 13C NMR (125 MHz, CDCl3): d 171.0, 166.8,
150.3, 144.1, 139.9, 137.8, 134.2, 133.8, 132.3, 129.9, 129.0,
128.6, 128.5, 128.3, 126.5, 122.7, 120.9, 117.9, 65.0, 35.8, 35.2,
30.9, 28.5, 25.7, 17.8; HR MS (ESI) calcd for C31H32O4Na (M+Na+)
491.2198, found 491.2186.
To a stirred solution of 17 (353 mg, 0.84 mmol), t-butyl acrylate
(630
ll, 4.3 mmol), triphenylphosphine (25 mg, 0.095 mmol), and
triethylamine (240
ll, 1.7 mmol) in toluene (5.7 ml) was added
palladium acetate (10.4 mg, 0.046 mmol). The mixture was stirred
at 105 °C for 6 h, cooled to rt, diluted with ether, washed succes-
sively with aqueous NH4Cl, water, brine, dried, and concentrated.
The residue was chromatographed on silica gel (n-hexane–ethyl
acetate = 30:1?20:1) to give 19 (273 mg, 78%) as a colorless oil;
IR (ATR) 2975, 2915, 1760, 1705, 1640, 1495, 1455, 1370, 1325,
3.5.11. tert-Butyl (E)-3-[2-isopropyl-7-(3-methylbut-2-
enyl)benzofuran-5-yl]acrylate (20), tert-butyl (E)-3-[2,3-
dihydro-7-(3-methylbut-2-enyl)-2-(prop-1-en-2-
yl)benzofuran-5-yl]acrylate (21), and tert-butyl (E)-3-[2,2-
dimethyl-8-(3-methylbut-2-enyl)-2H-chromen-6-yl]acrylate
(22)
1285, 1260, 1225, 1125 cmÀ1 1H NMR (500 MHz, CDCl3): d 7.53
;
(d, J = 16.0 Hz, 1H), 7.36–7.22 (m, 7H), 6.94 (d, J = 7.2 Hz, 1H),
6.29 (d, J = 16.0 Hz, 1H), 5.17 (m, 1H), 3.14 (d, J = 5.6 Hz, 2H),
3.08 (t, J = 6.2 Hz, 2H), 2.91 (t, J = 6.2 Hz, 2H), 1.74 (s, 3H), 1.67 (s,
3H), 1.53 (s, 9H); 13C NMR (125 MHz, CDCl3): d 171.0, 166.2,
150.1, 142.8, 139.9, 134.1, 133.7, 132.6, 129.7, 128.6, 128.3,
126.5, 122.6, 121.0, 120.0, 80.5, 35.8, 30.9, 28.5, 28.2, 25.7, 17.8;
HR MS (ESI) calcd for C27H32O4Na (M+Na+) 443.2198, found
443.2189.
(i) To a stirred solution of 15 (24.0 mg, 0.067 mmol) and sodium
acetate (44 mg, 0.54 mmol) in methanol–water (17:1; 1.2 ml) was
added palladium chloride (6.0 mg, 0.034 mmol). The mixture was
stirred at 55 °C in the air for 1.5 h, diluted with ether, washed with
water, brine, and concentrated. The residue was purified by pre-
parative TLC (n-hexane–ethyl acetate = 12:1, three developments)
to give 20 (2.1 mg, 9%), 21 (6.3 mg, 26%), and 22 (4.8 mg, 20%).
(ii) To a stirred solution of 15 (31.0 mg, 0.087 mmol) in dichlo-
romethane (0.5 ml) was added phenylselenenyl chloride (16 mg,
0.083 mmol) at À78 °C. The mixture was stirred at À78 °C for
40 min, diluted with dichloromethane, washed with brine, concen-
trated to give a syrup (56.0 mg), which was dissolved in dichloro-
methane (0.5 ml) containing a trace amount of pyridine. To this
solution was added 15% hydrogen peroxide (0.1 ml) at 0 °C, and
the mixture was stirred for 45 min, washed with water, brine,
and concentrated. The residue was chromatographed on silica gel
(n-hexane–ethyl acetate = 44:1) to give a mixture of 21 and 22
(17.6 mg, 57%) as a colorless oil (21/22 = 37/63 by 1H NMR
analysis).
3.5.7. Drupanin (3)
Treatment of 18 (100 mg, 0.35 mmol) as described for prepara-
tion of 1 gave 3 (50 mg, 62%) as a colorless solid; mp 143–144 °C
(benzene); IR (ATR) 3300, 3125, 2975, 2925, 2850, 1675, 2525,
1640, 1590, 1510, 1430, 1360, 1310, 1260, 1220, 1150, 1100, 980,
940, 860, 820, 690 cmÀ1 1H NMR (500 MHz, CD3OD): d 7.56 (d,
;
J = 16.0 Hz, 1H), 7.27–7.23 (m, 2H), 6.76 (d, J = 9.0 Hz, 1H), 6.22
(d, J = 16.0 Hz, 1H), 5.30 (m, 1H), 3.28 (d, J = 7.4 Hz, 2H), 1.73 (s,
3H), 1.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d 167.4, 156.5,
144.9, 137.9, 135.5, 130.1, 128.9, 128.5, 127.8, 127.4, 127.2,
126.5, 121.1, 116.1, 115.2, 64.9, 35.2, 29.6, 25.8, 17.9; HR MS
(ESI) calcd for C14H16O3Na (M+Na+) 255.0997, found 255.0997.
3.5.8. Baccharin (4)
(iii) Treatment of 24 (20.0 mg, 0.06 mmol) and t-butyl acrylate
(40 ll, 0.29 mmol) as described for preparation of 19 gave 22
(15.6 mg, 78%).
Treatment of 19 (150 mg, 0.36 mmol) as described for prepara-
tion of 1 gave 4 (118 mg, 90%) as a colorless solid; mp 101–102 °C
(methanol); IR (KBr) 2930, 1755, 1670, 1635, 1495, 1430, 1240,
;
1130, 690, 670 cmÀ1 1H NMR (500 MHz, CDCl3): d 7.75 (d,
Compound 20. IR (ATR) 2960, 2910, 1700, 1630, 1600, 1460,
1360, 1280, 1150, 1120, 1080, 980, 850; 1H NMR (500 MHz,
CDCl3):d 7.65 (d, J = 16.0 Hz, 1H), 7.46 (d, J = 1.5 Hz, 1H), 7.20 (d,
J = 1.5 Hz, 1H), 6.34 (s, 1H), 6.32 (d, J = 16.0 Hz, 1H), 5.39 (m, 1H),
3.57 (d, J = 7.5 Hz, 2H), 3.07 (m, 1H), 1.79 (s, 3H), 1.76 (s, 3H),
1.54 (s, 9H), 1.34 (d, J = 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3): d
166.8, 165.7, 154.3, 144.7, 133.4, 129.4, 129.1, 125.2, 122.8,
121.3, 118.4, 118.1, 100.1, 80.2, 29.7, 28.2, 28.1, 25.8, 20.9, 17.8;
HR MS (EI) calcd for C23H30O3 (M+) 354.2195, found 354.2184.
Compound 21. IR (ATR) 2970, 2900, 1700, 1630, 1600, 1470,
J = 15.9 Hz, 1H), 7.41–7.23 (m, 7H), 6.98 (d, J = 8.8 Hz, 1H), 6.39
(d, J = 15.9 Hz, 1H), 5.19 (m, 1H), 3.16 (d, J = 7.0 Hz, 2H), 3.09 (t,
J = 7.5 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 1.76 (s, 3H), 1.68 (s, 3H);
13C NMR (125 MHz, CDCl3): d 172.1, 170.9, 150.7, 146.4, 139.9,
134.3, 133.9, 131.9, 130.2, 128.6, 128.4, 126.9, 126.5, 122.8,
120.8, 117.1, 35.6, 30.9, 28.4, 25.7, 17.8; HR MS (ESI) calcd for
C23H24O4Na (M+Na+) 387.1572, found 387.1578.
3.5.9. Drupanin phenylethyl ester (11)
1430, 1360, 1250, 1140, 1120, 1080, 980, 890, 850, 820 cmÀ1
;
Treatment of 3 (30 mg, 0.13 mmol) as described for preparation
of 10 gave 11 (34 mg, 78%) as a colorless oil; IR (ATR) 3350, 2900,
1680, 1620, 1600, 1500, 1420, 1380, 1320, 1300, 1260, 1230, 1170,
1160, 1080, 980, 880, 860, 820, 750, 700 cmÀ1; 1H NMR (500 MHz,
CDCl3): d 7.60 (d, J = 16.0 Hz, 1H), 7.33–7.22 (m, 7H), 6.80 (d,
J = 8.0 Hz, 1H), 6.27 (d, J = 16.0 Hz, 1H), 5.44 (s, 1H), 5.31 (m, 1H),
4.41 (t, J = 7.0 Hz, 2H), 3.36 (d, J = 7.0 Hz, 2H), 3.01 (t, J = 7.0 Hz,
2H), 1.79 (br s, 6H); 13C NMR (125 MHz, CDCl3): d 167.4, 156.5,
144.9, 137.9, 135.5, 130.1, 128.9, 128.5, 127.8, 127.4, 127.2,
126.5, 121.1, 116.1, 115.2, 64.9, 35.2, 29.6, 25.8, 17.9; HR MS
(ESI) calcd for C22H24O3Na (M+Na+) 359.1623, found 359.1622.
1H NMR (500 MHz, CDCl3): d 7.51 (d, J = 15.5 Hz, 1H), 7.18 (br
s, 1H), 7.12 (br s, 1H), 6.18 (d, J = 15.5 Hz, 1H), 5.30 (m, 1H),
5.21 (dd, J = 9.5, 8.0 Hz, 1H), 5.07 (br, s, 1H), 4.90 (br s, 1H),
3.36 (dd, J = 15.7, 9.5 Hz, 1H), 3.31 (dd, J = 16.0, 7.5 Hz, 1H),
3.25 (dd, J = 16.0, 7.5 Hz, 1H), 3.02 (dd, J = 15.7, 8.0 Hz, 1H),
1.76 (s, 3H), 1.74 (s, 3H), 1.71 (s, 3H), 1.52 (s, 9H);13C NMR
(125 MHz, CDCl3): d 166.9, 159.7, 144.0, 143.9, 133.2, 128.9,
123.5, 122.0, 121.4, 116.6, 111.8, 85.9, 80.0, 34.6, 28.2, 28.1,
25.8, 17.8, 17.2; HR MS (ESI) calcd for C23H30O3Na (M+Na+)
377.2093, found 377.2106.
Compound 22. IR (ATR) 2960, 2910, 1700, 1630, 1600, 1480,
1410, 1385, 1365, 1360, 1330, 1265, 1140, 1080, 980, 845 cmÀ1
;
3.5.10. Baccharin phenylethyl ester (12)
1H NMR (500 MHz, CDCl3): d 7.48 (d, J = 15.9 Hz, 1H), 7.15 (d,
J = 2.1 Hz, 1H), 6.99 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 9.8 Hz, 1H),
6.19 (d, J = 15.9 Hz, 1H), 5.63 (d, J = 9.8 Hz, 1H), 5.26 (m, 1H),
3.26 (d, J = 7.3 Hz, 2H), 1.74 (s, 3H), 1.73 (s, 3H), 1.55 (s, 6H), 1.54
(s, 9H); 13C NMR (125 MHz, CDCl3): d 166.8, 152.5, 143.7, 132.4,
Treatment of 4 (128 mg, 0.43 mmol) as described for prepara-
tion of 10 gave 12 (167 mg, 83%) as a colorless solid; IR (ATR)
2900, 1760, 1700, 1640, 1600, 1500, 1450, 1410, 1370, 1320,
1270, 1220, 1160, 1150, 1120, 1080, 980, 860, 750, 700 cmÀ1 1H
;
NMR (500 MHz, CDCl3): d 7.62 (d, J = 16.0 Hz, 1H), 7.36–7.24

156
H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
130.9, 129.6, 129.3, 126.8, 124.0, 122.1, 120.9, 117.1, 80.0, 76.8,
28.2, 28.1, 25.8; HR MS (ESI) calcd for C23H30O3Na (M+Na+)
377.2093, found 377.2085.
114.9, 76.9, 64.8, 35.2, 28.2, 28.1, 25.8, 17.9; HR MS (ESI) calcd
for C27H30O3Na (M+Na+) 425.2093, found 425.2084.
3.6. HPLC analysis of extract
3.5.12. 1-(2-Methylbut-3-yn-2-yloxy)-4-iodo-2-(3-methylbut-2-
enyl)benzene (23)
To a stirred solution of 16 (581 mg, 2.02 mmol), 3-chloro-3-
methyl-1-butyne (0.226 ml, 2.02 mmol) and copper chloride
The analysis of the ethanol extract of Brazilian green propolis
were conducted using HPLC and it was carried out with a Cosmosil
5C18-ARII column (Nacalai Tesque, 4.6 Â 250 mm), with the gradi-
ent solvent system of CH3CN (5%, 5 min hold, 50–30%, 5–50 min
linear, and 30–60%, 50–160 min linear) in 0.1% TFA at a flow rate
of 1.0 ml/min and detection at 260 nm..
(0.7 mg, 7 lmol) in acetonitrile (2.0 ml) was added dropwise DBU
(0.33 ml, 2.21 mmol) at 0 °C, and the mixture was stirred at 0 °C
for 1 h. After addition of aqueous NH4Cl, the resulting mixture
was extracted with ether. The extracts washed with water, brine,
dried, and concentrated. The residue was chromatographed on sil-
ica gel (n-hexane–ethyl acetate = 1:0?20:1) to give 23 (584 mg,
82%) as a syrup; IR (ATR) 3290, 2985, 2915, 1580, 1480, 1235,
3.7. Assays
The experiments with human blood were conducted at the
National Sagamihara Hospital in accordance with the principle of
the Declaration of Helsinki. Before withdrawing the blood, each
subject signed an informed written consent form, which had been
approved by the ethical committee of Yamada Apiculture Center,
Inc.
1140, 1090, 880, 860, 815 cmÀ1 1H NMR (500 MHz, CDCl3): d
;
7.41 (br s, 1H), 7.40 (br d, J = 8.1 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H),
5.22 (m, 1H), 3.25 (d, J = 7.4 Hz, 2H), 2.57 (s, 1H), 1.74 (br s, 3H),
1.69 (br s, 3H), 1.66 (s, 6H); 13C NMR (125 MHz, CDCl3): d 153.7,
138.2, 136.3, 134.9, 132.9, 121.9, 120.7, 85.9, 85.5, 74.0, 72.2,
29.6, 28.5, 25.7, 17.8; HR MS (ESI) calcd for C16H19OI (M+)
354.0481, found 354.0480.
3.7.1. Peripheral leukocytes and PBMCs
Thirty milliliters of blood were collected from 10 allergic
patients who had a clinical history of pollinosis with positive
specific IgE to Cry j1/2 antigen (CAP-RAST P class3). Peripheral
blood leukocytes were isolated by dextran sedimentation. Briefly,
heparinized venous blood was incubated with one-tenth volume
of 4.5% dextran at room temperature for 45 min. The blood
suspension was centrifuged and the pellet was washed with phos-
phate-buffered saline (PBS). The pellet is then washed in Tyrode’s
buffer containing human serum albumin (0.1%) and diluted into
same buffer.
3.5.13. 6-Iodo-2,2-dimethyl-8-(3-methylbut-2-enyl)-2H-
chromene (24)
A solution of 23 (233 mg, 0.66 mmol) in N,N-diethylaniline
(10.0 ml) was heated at 150 °C with stirring for 3 h, cooled, diluted
with hexane, and then poured into ice-water. The resulting mix-
ture was extracted with hexane. The extracts were washed succes-
sively with cold aqueous HCl, water, saturated aqueous NaHCO3,
water, brine, dried, and concentrated. The residue was chromato-
graphed on silica gel (n-hexane) to give 24 (198 mg, 85%) as a col-
orless oil; IR (ATR) 2967, 2920, 1735, 1640, 1445, 1380, 1360, 1255,
1200, 1165, 1090, 865, 720 cmÀ1; 1H NMR (500 MHz, CDCl3): d 7.23
(d, J = 2.2 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 6.21 (d, J = 9.8 Hz, 1H),
5.60 (d, J = 9.8 Hz, 1H), 5.23 (m, 1H), 3.20 (d, J = 7.6 Hz, 2H), 1.73
(br s, 3H), 1.71 (br s, 3H), 1.40 (s, 6H); 13C NMR (125 MHz, CDCl3):
d 150.4, 137.6, 132.6, 132.4, 132.0, 131.4, 123.3, 121.9, 121.4, 82.4,
76.3, 31.6, 27.9, 27.8, 25.8, 22.6; HRMS (EI) calcd for C16H19OI (M+)
354.0481, found 354.0485.
Peripheral blood mononuclear cells (PBMC) were obtained by
sodium diatrizoate-Ficoll density gradient centrifugation (MP Bio-
medicals). PBMC were suspended in RPMI 1640 medium (Gibco)
with 10% fetal bovine serum, 2 mM
L-glutamine, penicillin
(100 IU/ml), streptomycin (100
6 days.
lg/ml) at 37 °C under 5% CO2 for
3.7.2. Differentiated HL-60 Cells
HL-60 cells obtained from Health Science Research Resources
Bank (Tokyo, Japan) were grown in RPMI-1640 with 10% FBS in a
humidified atmosphere of 5% CO2 and 95% air at 37 °C. The
density of HL-60 cells was maintained between 2.5 Â 105 and
5 Â 105/ml. The HL-60 cells were cultured in the same medium
containing 1.25% DMSO for 6 days. The differentiation into mye-
loid cells resembling PMN leukocytes were confirmed by NBT
reduction.
3.5.14. Culifolin (5)
Treatment of 22 (238 mg, 0.67 mmol) as described for prepara-
tion of 1 gave 5 (178 mg, 89%) as a colorless solid; mp 130–131 °C
(methanol); IR (ATR) 3000, 2950, 1670, 1620, 1590, 1460, 1440,
1370, 1360, 1330, 1265, 1245, 1200, 1140 cmÀ1
;
1H NMR
(500 MHz, CDCl3): d 7.68 (d, J = 15.5 Hz, 1H), 7.19 (d, J = 1.8 Hz,
1H), 7.05 (d, J = 1.8 Hz, 1H), 6.32 (d, J = 10.0 Hz, 1H), 6.27 (d,
J = 15.5 Hz, 1H), 5.65 (d, J = 10.0 Hz, 1H), 5.27 (m, 1H), 3.27 (d,
J = 7.0 Hz, 2H), 1.75 (s, 3H), 1.74 (s, 3H), 1.44 (s, 6H); 13C NMR
(125 MHz, CDCl3): d 172.8, 153.3, 147.2, 132.7, 131.1, 129.9, 129.8,
126.3, 124.4, 122.0, 121.0, 114.1, 77.0, 28.2, 28.1, 25.8, 17.8; HR MS
(ESI) calcd for C19H22O3Na (M+Na+) 321.1467, found 321.1481.
3.7.3. Analysis of cytokine production
PBMC were seeded in 96-well plates (2.0 Â 105/well) and stim-
ulated by 10 lg/ml Cry j1 in the presence or absence of propolis at
37 °C under 5% CO2 for 6 days. The cytokines were measured by
ELISA (IL-4, IL-5; BioSource, and IL-13; Beckman Coutler).
3.5.15. Culifolin phenylethyl ester (13)
3.7.4. Analysis of histamine and cys-LTs release
Treatment of 5 (22 mg, 0.07 mmol) as described for preparation
of 10 gave 13 (27 mg, 90%) as a colorless solid; IR (ATR) 2950, 2900,
1700, 1620, 1590, 1460, 1420, 1360, 1320, 1290, 1250, 1220, 1200,
Peripheral blood leukocytes were seeded in test tube
(1.5 Â 104 ꢀ 5.1 Â 104/tube) and stimulated by Cry j1 (1 ng/ml) in
the presence or absence of propolis at 37 °C under 5% CO2 for
6 days. Medium from cultures peritoneal lavage fluids were
analyzed quantitatively by histamine autoanalyzer and cys-LTs
ELISA kit (Cayman 520501) according to the instructions.
Differentiated HL-60 cells were seeded in 96-well plates and
stimulated by Ca2+ in the presence or absence of samples at
37 °C under 5% CO2 for 6 days. The concentration of cys-LTs was
determined using ELISA kit (Bühlmann CAST-2000).
1160, 1130, 1070, 970, 940, 870, 840, 710, 690 cmÀ1 1H NMR
;
(500 MHz, CDCl3): d 7.57 (d, J = 16.0 Hz, 1H), 7.33–7.22 (m, 5H),
7.15 (d, J = 2.3 Hz, 1H), 7.01 (d, J = 2.3 Hz, 1H), 6.31 (d, J = 10.0 Hz,
1H), 6.25 (d, J = 16.0 Hz, 1H), 5.64 (d, J = 10.0 Hz, 1H), 5.26 (m,
1H), 4.41 (t, J = 7.3 Hz, 2H), 3.26 (d, J = 7.5 Hz, 2H), 3.01 (t,
J = 7.3 Hz, 2H), 1.74 (s, 3H), 1.73 (s, 3H), 1.43 (s, 6H); 13C NMR
(125 MHz, CDCl3): d 167.3, 152.8, 145.0, 137.9, 132.6, 131.0,
129.7, 129.6, 128.9, 128.5, 126.6, 126.5, 124.1, 122.1, 121.0,

H. Tani et al. / Bioorg. Med. Chem. 18 (2010) 151–157
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Acknowledgments
The authors thank Mr. S. Hikami of Yamada apiculture center,
Inc. for his technical assistance. This work was supported by the
Chemical Genomics Project (RIKEN) and in part by a Grant-in-Aid
for Scientific Research from the Ministry of Education, Science,
Sports, and Culture of Japan.
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