Neoflavonoids and related constituents from nepalese propolis and their nitric oxide production inhibitory activity

Article abstract of DOI:10.1021/np050009k

A methanolic extract of Nepalese propolis yielded 10 new open-chain neoflavonoids (1-10), a new chalcone (11), and eight previously reported compounds (12-19). Their structures were determined on the basis of spectroscopic data and chemical conversion. Th

Full text of DOI:10.1021/np050009k

858
J. Nat. Prod. 2005, 68, 858-864
Neoflavonoids and Related Constituents from Nepalese Propolis and Their
Nitric Oxide Production Inhibitory Activity
Suresh Awale, Suraj Prakash Shrestha, Yasuhiro Tezuka, Jun-ya Ueda, Katsumichi Matsushige, and
Shigetoshi Kadota*
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
Received January 11, 2005
A methanolic extract of Nepalese propolis yielded 10 new open-chain neoflavonoids (1-10), a new chalcone
(11), and eight previously reported compounds (12-19). Their structures were determined on the basis
of spectroscopic data and chemical conversion. The isolated compounds other than 5, 8, and 16 showed
dose-dependent inhibitory activities on nitric oxide production in lipopolysaccharide-activated macrophage-
like J774.1 cells and were more active than a positive control, NG-monomethyl-L-arginine (IC50, 27.1 µM).
The most potent activities, of 6 and 7 (IC50, 0.5 µM), were greater than another positive control, caffeic
acid phenethyl ester (IC50, 4.8 µM).
Propolis, a resinous substance collected by bees from
various plants, has a pleasant aromatic odor and yellow-
green to dark brown color depending on source and age. It
is used by bees to seal holes in their hive, smooth out the
internal walls, and protect the entrance against intruders.1
The plant source of propolis depends on the specific flora
at the site of collection. Thus, the constituents of propolis
of different places may be different.2 Propolis is a tradi-
tional remedy in Eastern Europe and is well known for its
valuable biological activities such as antibacterial, anti-
fungal, immunostimulating, antitumor, antiinflammatory,
and antioxidant.1,3,4 Because of the broad spectrum of
biological activities, propolis has become a subject of
increasing interest concerning its constituents, and more
than 300 compounds have been reported so far from
propolis.2,3 In our continued work on propolis of various
origin5-11 we recently found that methanol and water
extracts of propolis collected from Nepal possessed potent
nitric oxide (NO) production inhibitory activity (IC50 < 2
µg/mL) in lipopolysaccharide (LPS)-activated macrophage-
like J774.1 cells. Since no previous research has been
reported on Nepalese propolis, we carried out a detailed
chemical investigation. The work led to the isolation of 10
new open-chain neoflavonoids (1-10) and a new chalcone
(11), together with eight known compounds. In this paper,
we report the structure elucidation of the new compounds
and their NO production inhibitory activity.
molecular formula C16H20O3. The IR spectrum of 1 showed
Results and Discussion
absorptions of hydroxyl (3730 cm-1), carbonyl (1720 cm-1),
and phenyl (1605, 1455 cm-1) groups. The 1H NMR
spectrum of 1 exhibited signals due to a phenyl ring (δΗ
7.32-7.14, 5Η), a vinyl (δΗ 6.07, 5.06, 5.00), two oxygen-
substituted methines (δΗ 4.30, 3.42), two aliphatic methines
(δΗ 3.70, 3.25), two methylenes (δH 2.77, 2.62; δH 1.94, 1.23),
The MeOH extract was subjected to a series of chro-
matographic separations resulting in the isolation of 10
new open-chain neoflavonoids (1-10) and a new chalcone
(11) together with eight known compounds: (S)-4-meth-
oxydalbergione(12),12 cearoin(13),13 9-hydroxy-6,7-dimethoxy-
dalbergiquinol (14),14 obtusaquinol (15),15 2′,4,4′-trihy-
droxychalcone (16),16 medicarpin (17),17 (+)-vesticarpan
(18),18 and 4-hydroxymedicarpin (19).19 The structures of
compounds 12-19 are included in the Supporting Informa-
tion.
and a methoxyl group (δΗ 3.35). On the other hand, its 13
C
NMR spectrum showed the signals of 16 carbons, including
those for a ketone carbonyl (δC 208.2), two oxygen-
substituted sp3 carbons, and two olefinic carbons (Table 1).
The partial connectivities C2-C3-C4-C5-C6 and C2-C7-
C8-C9 were deduced by the analysis of the COSY and
HMQC spectra. In the HMBC spectrum, the correla-
tions of the ketone carbonyl carbon (δC 208.2) with H-2,
H2-3, H-5, and H2-6 suggested the connectivity of C-2 and
C-6 via the ketone carbonyl carbon (C-1), while the cor-
relations between H-2′,6′ and C-7 indicated the phenyl
Compound 1 was obtained as a yellow oily substance
with [R]D24 -178° (CHCl3). It showed the molecular ion at
m/z 260.1406 (M+) in HREIMS, which corresponds to the
* To whom correspondence should be addressed. Tel: 81-76-434-7625.
Fax: 81-76-434-5059. E-mail: kadota@ms.toyama-mpu.ac.jp.
10.1021/np050009k CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/21/2005

Neoflavonoids from Nepalese Propolis
Journal of Natural Products, 2005, Vol. 68, No. 6 859
Figure 1. Chemical conversion of 1 to S5. Reagents and conditions: (i) NaBH4, THF, CH3OH, 0 °C, 6 h; (ii) PhSeCl, K2CO3, CH2Cl2, 4 h; (iii)
Bu3SnH, AIBN, toluene, reflux, 5 h; (iv) Jones reagent, acetone, 0 °C.
Table 1. 13C NMR (100 MHz) Data (δ) for Compounds 1-10 in
CDCl3
posi-
tion
1
2
3
4
5
6
7
8
9
10
1
208.2 208.9 207.3 207.4 67.1 199.9 197.9 199.1 130.5 130.4
47.7 41.9 51.7 51.6 32.7 49.9 50.7 47.5 147.2 116.4
32.2 83.1 32.8 32.8 76.5 34.9 34.2 31.9 116.4 149.5
65.6 67.6 69.9 69.9 65.5 63.7 67.9 65.0 149.4 147.4
81.2 32.5 81.1 42.2 28.6 149.6 152.2 174.6 114.4 114.4
42.8 51.3 42.2 81.1 38.2 128.9 129.4 101.8 117.3 117.3
47.7 49.6 48.8 48.9 52.7 49.5 46.9 48.6 48.9 49.1
139.9 139.6 138.3 138.2 141.2 139.6 135.9 139.7 139.2 139.1
115.2 115.3 116.9 116.9 144.9 115.6 118.5 115.2 117.0 117.1
115.2 141.0 143.1 143.1 142.9 141.2 142.5 141.3 141.4 141.2
2
3
4
5
6
7
8
9
1′
2′,6′ 141.3 128.7 128.5 128.4 128.6 128.9 128.5 128.5 128.6 128.6
3′,5′ 128.2 128.2 127.9 127.8 127.4 127.9 128.1 128.4 128.6 128.6
4′
126.5 126.7 126.3 126.3 126.3 126.9 126.5 126.6 126.7 126.8
OMe 56.4 56.7 56.8 56.7 55.9
56.0
Table 2. Inhibitory Effects of Constituents of Nepalese
Propolis on NO Production in LPS-Activated J774.1 Cells
compound
IC50 (µM)a
1
13.2
9.6
Figure 2. (a) Key NOE (arrow) observed in the difference NOE spectra
and (b) CD spectrum and octant projection of S5.
2
3
9.6
4
2.3
5
100
0.5
correlations H-2/H-6ax and H-3ax/H-5 suggested the chair
conformation of the cyclohexanone ring and equatorial
orientation of the methoxyl group. Similarly, the ROESY
correlations of H-4 with H2-3 and H-5 indicated the
equatorial orientation of H-4, i.e., the hydroxyl group to
be axial. Then, to determine the absolute stereochemistry,
1 was converted to the ketone S5 via NaBH4 reduction,
selenoetherification, Bu3SnH reduction, and Jones oxida-
tion (Figure 1). In the difference NOE experiment on S5,
NOE enhancements from H-1 to H-2, H2-6, H-8, and H-2′,6′,
from H-2 to H-6ax, and from H-3ax to H-5 and H-7
indicated the conformation depicted in Figure 2a. Finally,
the absolute stereochemistry of S5 was deduced to be
1S,2S,5S,7R,8R by applying the octant rule20 to the nega-
tive minimum ([θ]300 -1426) in its CD spectrum (Figure
2b). Thus, the absolute configuration of 1 was concluded
to be 2S,4R,5S,7R.
6
7
0.5
8
48.5
5.5
9
10
8.4
11
19.2
10.2
15.4
12.8
6.3
12
13
14
15
16
29.3
10.1
3.4
17
18
19
5.4
CAPE
L-NMMA
4.8
27.1
a IC50 values were calculated from the mean of data of four
determinations.
group to be at C-7. Likewise, a correlation was observed
between the methoxyl protons and C-5, suggesting the
methoxyl group to be at C-5. Thus, 1 was determined to
be an open-chain neoflavanoid with a cyclohexanone ring.
The relative configuration of 1 was determined from the
ROESY analysis and coupling constant data. The trans-
diaxial coupling constant for H-2 and H-3ax (J ) 12.5 Hz)
and for H-5 and H-6ax (J ) 10.6 Hz) and the ROESY
HREIMS of compounds 2 and 3 showed the same
1
molecular formula (C16H20O3) as that of 1. Their H (see
Experimental Section) and 13C NMR (Table 1) spectra were
also similar. Analysis of the COSY, HMQC, and HMBC
spectra indicated 2 and 3 to be stereoisomers of 1. The
coupling constants and the ROESY correlations indicated
that 2 had the same stereochemistry in the cyclohexanone

860 Journal of Natural Products, 2005, Vol. 68, No. 6
Awale et al.
Figure 3. LC-UV profile chromatogram (a) and LC-MS profile chromatogram (b) of the MeOH extract of Nepalese propolis.
ring as 1, and the CD spectrum revealed the absolute
stereochemistry of the cyclohexanone ring to be the same
as 1. Thus, 2 should be a stereoisomer of 1 at C-7. As for
compound 3, the ROESY correlations H-2/H-4, H-2/H-6ax,
H-2/H-3eq, and H-3eq/H-4 and the trans-diaxial coupling
constant (J ) 12.9 Hz) between H-2 and H-3ax indicated
a chair conformation of the cyclohexanone ring, â-equato-
rial orientation of the 4-OH group, and â-axial orientation
of the 5-OMe group. These observations and the positive
maximum ([θ]291 +4600) in the CD spectrum indicated the
2R,4R,5S absolute configuration, but the absolute config-
uration at C-7 could not be determined due to the meager
amount of compound isolated.
The 1H and 13C NMR spectra of 4 closely resembled those
of 3, but analysis of the COSY and HMQC spectra revealed
C-5 to be an aliphatic methylene and C-6 to be an oxygen-
substituted methine. The HMBC analysis indicated the
methoxyl group to be at C-6 instead at C-5 as in 3. ROESY
correlations H-2/H-4, H-2/H-5ax, H-5ax/H-6, and H-5eq/
H-6 and NOE enhancements from H-4 to both H-5ax and
H-5eq indicated a boat conformation of the cyclohexanone
ring and â-equatorial orientation of the 4-OH group. The
small coupling constants between H-5R and H-6 (J ) 3.2
Hz) and between H-5â and H-6 (J ) 4.4 Hz) indicated
â-axial orientation of the methoxyl group. A positive
maximum ([θ]286 +1700) due to nfπ* transition in the CD
spectrum indicated R configuration at C-2, i.e., 4 to have
a 2R,4R,6R configuration. But, due to the meager amount
of the compound isolated, the absolute configuration at C-7
could not be determined.
The molecular formulas of compounds 6 and 7 (C15H16O2)
were determined by HREIMS. Their IR spectra showed the
presence of hydroxyl and R,â-unsaturated ketone function-
1
alities. Their H NMR spectra displayed signals due to a
cis-olefin, a vinyl group, an oxygen-substituted methine, a
methylene, two methines, and a phenyl group. Their 13C
NMR spectra displayed 15 signals corresponding to the
above groups and an R,â-unsaturated ketone (Table 1). The
partial structures C(6)HdC(5)H-C(4)H(O)-C(3)H2-C(2)H-
C(7)H-C(8)HdC(9)H2 were deduced by the COSY and
HMQC spectra, while the HMBC correlations of the ketone
carbonyl carbon with H-2, H-3, H-5, H-6, and H-7 suggested
the carbonyl carbon to be C-1, and the correlation between
C-7 and H-1′,6′ and between C-1′ and H-7 indicated the
location of the phenyl group at C-7. In 6, on the other hand,
small coupling constants for H-2 and H2-3 (J ) 4.9 Hz) and
ROESY correlations H-2/H-3R and H-2/H-3â suggested the
1-phenyl-2-propenyl group to be R-axial, and the large
coupling constant between H-3â and H-4 (J ) 10.7 Hz)
suggested the â-equatorial orientation of the 4-OH group.
In addition, by applying an inverse octant rule,21,22 6 was
determined to have an S configuration at C-2 ([θ]322 +748),
i.e., 2S,4R. Similarly, the configuration of 7 was determined
to be 2R,4R, on the basis of the coupling constants of H-3ax
with H-2 (J ) 12.4 Hz) and H-4 (J ) 10.7 Hz), the ROESY
correlation between H-2 and H-4, and the negative mini-
mum ([θ]339 -3664) in the CD spectrum. The absolute
configuration at C-7 of both 6 and 7 could not be deter-
mined due to meager amounts obtained.
The 1H and 13C NMR spectra of 8 closely resembled those
of 6, but were characterized by the absence of a signal due
to an olefinic proton and the presence of a methoxyl signal.
The methoxyl group was deduced to be at C-5 on the basis
of HMBC correlations between the methoxyl protons and
C-5. ROESY correlations H-2/H-3R, H-2/H-3â, H-4/H-3R,
and H-4/H-3â and their coupling constants indicated an
The HREIMS of 5 showed the molecular ion at m/z
262.1572 (M+), consistent with the molecular formula
C16H22O3. The 1H and 13C NMR spectra and the [R]D value
(+26.4°, CHCl3) were the same as those of synthesized
compound S2 (Figure 1). Thus, 5 was determined to be the
alcohol S2.

Neoflavonoids from Nepalese Propolis
Journal of Natural Products, 2005, Vol. 68, No. 6 861
R-axial orientation of both the 4-hydroxyl and 1-phenyl-2-
propenyl groups. The CD data ([θ]307 +30704) indicated C-2
to have an S configuration, i.e., 2S,4S, but the absolute
configuration at C-7 could not be determined due to the
meager amount of available sample.
As a part of our ongoing research on propolis, we
previously established an LC-MS technique for the analysis
of propolis samples from locations in Brazil, Peru, China,
and The Netherlands.8 To compare Nepalese propolis with
those of other origin, further work on LC-MS was carried
out. Both LC-UV and LC-MS fingerprint profiles were
obtained using the LC-UV handling mode in a JEOL JMS-
700T four-sector mass spectrometer, in the negative atmo-
spheric pressure chemical ionization (APCI) mode (Figure
3). Peak identification in the profile was carried out on the
basis of the retention time and pseudomolecular ion (M -
H)- of all the standard compounds.
The isolated compounds were assayed against NO pro-
duction by LPS-activated macrophage-like J774.1 cells. All
of the compounds displayed significant concentration-
dependent inhibition of NO production in macrophage-like
J774.1 cells. The activities of 1-3, 9-15, 17, and 19 were
greater than a positive control, NG-monomethyl-L-arginine
(L-NMMA; IC50, 27.1 µM), a nonselective nitric oxide
synthase (NOS) inhibitor,34 while the activities of 4, 6, 7,
and 18 were more potent than another positive control,
caffeic acid phenethyl ester (CAPE; IC50, 4.8 µM), an
inhibitor of nuclear factor κB activation35 (Table 2). CAPE
is an active principle of Europian propolis, reported to
inhibit inducible nitric oxide synthase of Raw 264.7 cells36
and to show an antiinflammatory effect in a rat model of
carrageenin-induced subcutaneous inflammation by in-
creasing leukocyte apoptosis and decreasing leukocyte
concentration in exudate.37
In the present study, structure-activity relationships
were deduced as follows. Among the open-chained neofla-
vanoids, those having a ketone carbonyl at C-1 in ring A
exhibited stronger activity than those without (1, 2, 3, 4,
6, 7, 8, 12 > 5). Compounds without a substituent at C-5
showed stronger activity than those having a methoxyl
substituent (4 > 1, 2, 3; 6, 7 . 8). Among the compounds
possessing the phenyl ring A system, an increase in the
number of hydroxyl groups improved activity (9, 10, 15 >
14). These phenomena were also observed in pterocarpans
(17, 18 > 16). Conversely, an increase in the number of
methoxyl groups in ring A decreased the activity (6, 7 > 8,
and 15 > 14). The absence of a vinyl group at C-7 also
showed a decrease in activity (15 > 13).
Compounds 9 and 10 both had the molecular formula
C15H14O2. Their 1H and 13C NMR spectra resembled those
of 14 and 15, but they indicated the presence of a 1,2,4-
trisubstituted benzene ring instead of the 1,2,4,5-tetrasub-
stituted benzene ring in 14 and 15. HMBC correlations
between H-7 and the carbons at δC 130.5 (C-1), 147.2 (C-
2), and 117.3 (C-6) and the coupling patterns of H-3 (d, J
) 2.6 Hz), H-5 (dd, J ) 8.6, 2.6 Hz), and H-6 (d, J ) 8.6
Hz) in 9 suggested the two hydroxyl groups to be located
at C-2 and C-4. Similarly, HMBC correlations between H-7
and the carbons at δC 130.4 (C-1), 116.4 (C-2), and 117.3
(C-6) and the coupling patterns of H-2 (d, J ) 2.9 Hz), H-5
(d, J ) 8.5 Hz), and H-6 (dd, J ) 8.5, 2.9 Hz) in 10
suggested the hydroxyl groups to be located at C-3 and C-4.
Their absolute stereochemistry was determined to be 7S
by comparing their [R]D values and CD data with those of
related compounds.23,24 In addition, the absolute stereo-
chemistry of the known compound 14, which has not been
determined in the literature,14 was determined to be 7R
by the same method.
Compound 11 had the molecular formula C16H18O3. Its
IR spectrum showed the presence of hydroxyl and R,â-
unsaturated ketone groups. The 1H NMR spectrum showed
signals due to a monosubstituted benzene ring, three
methylenes, three olefinic protons, and a methoxyl group.
The 13C NMR spectrum displayed signals due to 16 carbons
including those of an R,â-unsaturated carbonyl group (δC
198.3), an oxygen-substituted sp2 carbon (δC 177.6), and
an oxygen-substituted quaternary carbon (δC 72.0). The
partial structures C(7)H2-C(8)HdC(9)H and C(5)H2-C(6)-
H2 were obtained from the COSY and HMQC spectra. The
planar structure was deduced from the HMBC correlations
of H-2, H-6, and H-5 with C-1 and C-4, of the methoxyl
protons with C-3, of the methylene protons at δH 2.74 and
2.62 (H2-7) with C-4, and of the olefinic proton at δH 6.50
(H-9) with the carbons of the phenyl ring.
Thus, we report 11 new flavonoids (1-11) together with
eight known compounds (12-19, structures in the Sup-
porting Information). Among the isolated compounds, 1,
12, and 15 were the major constituents of Nepalese
propolis. Compounds 12 and 15 were earlier reported as
constituents of Dalbergia and Machaerium woods,15,24-28
while neoflavonoids 13 and 14 were reported only from
Dalbergia species.13,26-30 Moreover, “Chitwan”, where the
propolis used in this study was collected, has many plants
of various Dalbergia species such as D. sissoo. Thus, the
source of Nepalese propolis might be plants belonging to
the genus Dalbergia. Neoflavonoids such as obtusaquinone,
R-3,4-dimethoxydalbergione, R-4-methoxydalbergione, and
other quinones isolated from Dalbergia species have been
reported as allergens.31-33 They are also reported to inhibit
the growth of microorganisms, such as the marine borer
Teredo navalis, and are reported to be useful in protective
coatings on ships.31 As bees prepare propolis to protect their
hive from foreign intruders, including microorganisms,
these neoflavanoids may have a defensive role in protecting
the hive. However, the use of propolis as a health food that
contains such allergens should be a matter of further
research. To the best of our knowledge, this is the first
report on the isolation of open-chain neoflavonoids from
propolis.
Cell viability in the present experiment was determined
by the MTT method to find out whether inhibition of NO
production was due to the cytotoxicity of tested compounds
(data not shown). Compounds 6 and 7 exhibited cytotoxicity
above 50 µM concentration. However, the IC50 values of
all the compounds lie within the nontoxic concentration
range, suggesting that the inhibition of nitrite accumula-
tion was not due to its cytytoxic properties but was due to
its inhibitory activity on NO production. NO is produced
by inducible NOS (iNOS) and acts as a host defense by
damaging pathogenic DNA and as a regulatory molecule
with homeostatic activities.38 However, excessive produc-
tion has detrimental effects on many organ systems of the
body, leading to tissue damage and even leading to a fatal
development (septic shock). Therefore, the effective inhibi-
tion of NO accumulation by inflammatory stimuli may be
beneficial for thereapy.39,40
Experimental Section
General Experimental Procedures. Optical rotations
were measured on a JASCO DIP-140 digital polarimeter. IR
spectra were measured with a Shimadzu IR-408 spectropho-
tometer in CHCl3 solution. NMR spectra were taken on a
JEOL JNM-LA400 spectrometer with tetramethylsilane (TMS)
as an internal standard, and chemical shifts are expressed in

862 Journal of Natural Products, 2005, Vol. 68, No. 6
Awale et al.
δ values. EIMS and HREIMS measurements were performed
on a JEOL JMS-700T spectrometer using a direct inlet system
at the ionization voltage of 70 eV. CD spectra were measured
in a JASCO J-805 spectropolarimeter. Column chromatogra-
phy was performed with BW-820MH silica gel (Fuji Silysia,
Aichi, Japan) and LiChroprep RP-18 (Merck, Darmstadt,
Germany). Analytical and preparative TLC was carried out
on precoated silica gel 60F254 or RP-18F254S plates (Merck, 0.25
or 0.50 mm thickness).
Biological Material. Nepalese propolis was collected at
Chitwan, Nepal, in 2001. A voucher specimen (TMPW24045)
is preserved in the Museum of Materia Medica, Research
Center for Ethnomedicines, Institute of Natural Medicine,
Toyama Medical and Pharmaceutical University, Toyama,
Japan.
Extraction and Isolation. Nepalese propolis (500 g) was
successively extracted with H2O and MeOH under reflux to
give H2O (45 g) and MeOH (55 g) extracts, respectively. Part
of the MeOH extract (20 g) was chromatographed on silica gel
(5 × 46 cm) with a MeOH-CHCl3 solvent system to give five
fractions [1: MeOH-CHCl3 (1:99) eluate, 2.64 g; 2: MeOH-
CHCl3 (2:98) eluate, 2.06 g; 3: MeOH-CHCl3 (3:97) eluate,
1.38 g; 4: MeOH-CHCl3 (5:95) eluate, 5.97 g; 5: MeOH-
CHCl3 (7:93) eluate, 1.88 g].
Fraction 2 (2.06 g) was rechromatographed on silica gel (3.5
× 36 cm) with MeOH-CHCl3 (1% f 5% MeOH) to afford four
subfractions (2-1, 504 mg; 2-2, 374 mg; 2-3, 201 mg; 2-4, 326
mg). Subfraction 2-1 gave 12 (24.0 mg). Subfractions 2-2 to
2-4 were rechromatographed on silica gel (2 × 34 cm) with
2% MeOH-CHCl3, followed by normal-phase preparative TLC
with CH3CN-C6H6 (1:10), to give 13 (70.1 mg), 14 (25.2 mg),
and 16 (60.4 mg), respectively.
(1H, ddd, J ) 13.4, 7.6, 5.4 Hz, H-3); 13C NMR, see Table 1;
HREIMS m/z 260.1382 [calcd for C16H20O3, 260.1412].
24
Compound 3: white waxy substance; [R]D +180.1° (c
0.045, CHCl3); IR νmax 3570, 1715, 1490, 1455, 1330, 1220, 995
cm-1; CD (c 3.841 × 10-4 M, EtOH) [θ]292 +4600; 1H NMR (CD3-
OD) δ 7.30 - 7.15 (5H, m, Ph), 5.89 (1H, ddd, J ) 17.3, 10.4,
7.8 Hz, H-8), 5.11 (1H, dd, J ) 17.3, 1.0 Hz, H-9), 5.10 (1H,
dd, J ) 10.4, 1.0 Hz, H-9), 4.10 (1H, ddd, J ) 10.7, 4.4, 3.2
Hz, H-4), 3.91 (1H, t, J ) 7.8 Hz, H-7), 3.81 (3H, s, OMe), 3.76
(1H, dt, J ) 4.4, 3.2 Hz, H-5), 2.83 (1H, ddd, J ) 12.9, 7.8 Hz,
5.1, H-2), 2.74 (1H, dd, J ) 14.4, 4.4 Hz, H-6), 2.39 (1H, dd, J
) 14.4, 3.2, H-6), 2.16 (1H, ddd, J ) 11.7, 4.4, 5.1 Hz, H-3),
1.98 (1H, ddd, J ) 12.9, 11.7 Hz, 10.7, H-3); 13C NMR, see
Table 1; HREIMS m/z 260.1445 [calcd for C16H20O3, 260.1412].
Compound 4: white waxy substance; [R]D24 +210.5° (c 0.06,
CHCl3); IR νmax 3560, 1715, 1495, 1455, 995 cm-1; CD (c 3.841
× 10-4 M, EtOH) [θ]286 +1700; 1H NMR (CD3OD) δ 7.30-7.15
(5H, m, Ph), 5.86 (1H, ddd, J ) 16.8, 10.0, 8.2 Hz, H-8), 5.11
(1H, dd, J ) 16.8, 0.72 Hz, H-9), 5.09 (1H,dd, J ) 10.0, 0.72
Hz, H-9), 4.08 (1H, br s, H-4), 3.90 (1H, t, J ) 8.2 Hz, H-7),
3.75 (1H, dd, J ) 4.4, 3.2 Hz, H-6), 3.38 (1H, d, J ) 2.6 Hz,
OMe), 2.76 (1H,ddd, J ) 12.2, 8.2, 5.4 Hz, H-2), 2.70 (1H, dd,
J ) 14.4, 4.4 Hz, H-5), 2.38 (1H, dd, J ) 14.4, 3.2 Hz, H-5),
2.15 (1H, ddd, J ) 12.9, 5.4, 4.4 Hz, H-3), 1.95 (1H, ddd, J )
12.9, 12.2, 10.5 Hz, H-3); 13C NMR, see Table 1; HREIMS m/z
260.1400 [calcd for C16H20O3, 260.1412].
22
Compound 5: colorless oil; [R]D +26.4° (c 0.055, CHCl3);
IR νmax 3590, 3350, 1490, 1455, 1350, 1025 cm-1; 1H NMR (CD3-
OD) δ 7.30-7.15 (5H, m, Ph), 5.97 (1H, dt, J ) 16.9, 10.0 Hz,
H-8), 5.12 (1H, dd, J ) 16.9, 1.2 Hz, H-9), 5.01 (1H, dd, J )
10.0, 1.2 Hz, H-9), 4.23 (1H, br d, J ) 2.4 Hz, H-1), 4.02 (1H,
br d, J ) 2.4 Hz, H-4), 3.53 (1H, ddd, J ) 11.7, 4.6, 2.4 Hz,
H-5), 3.28 (1H, s, OMe), 3.19 (1H, t, J ) 10.0 Hz, H-7), 2.18
(1H, m, H-2), 2.18 (1H, m, H-6), 1.81 (1H, ddd, J ) 13.9, 11.7,
2.2 Hz, H-6), 1.45 (2H, m, H-3); 13C NMR, see Table 1;
HREIMS m/z 262.1572 [calcd for C16H22O3, 262.1568].
Fraction 3 (1.38 g) was rechromatographed on silica gel (3
× 35 cm) with 2.5% MeOH-CHCl3 to give 15 (357 mg).
Fraction 4 (5.97 g) was rechromatographed on ODS with
CH3CN-acetone-H2O (1:1:5 f 1:1:1) to afford four subfrac-
tions (4-1, 534 mg; 4-2, 1.45 g; 4-3, 930 mg; 4-4, 811 mg).
Subfraction 4-1 (534 mg) was rechromatographed on silica gel
(2 × 37 cm) with 10% CH3CN-C6H6, followed by normal-phase
preparative TLC with CH3CN-C6H6 (1:10), to give 2 (80.6 mg),
3 (12.2 mg), 4 (8.1 mg), and 8 (10.8 mg). Subfraction 4-2 (1.45
g) was rechromatographed on ODS with CH3CN-acetone-
H2O (1:1:5 f 1:1:1), followed by normal-phase preparative TLC
with 2% MeOH-CHCl3 or reversed-phase preparative TLC
with CH3CN-acetone-H2O (1:1:3), to give 5 (182 mg), 6 (9.3
mg), 7 (6.7 mg), 9 (60.6 mg), and 10 (35.0 mg). Subfraction
4-3 (930 mg) gave 1. Subfraction 4-4 (811 mg) was rechro-
matographed on ODS with CH3CN-acetone-H2O (1:1:5 f 1:1:
1), followed by normal-phase preparative TLC with 10%
CH3CN-C6H6, to give 11 (22.2 mg), 17 (20.0 mg), 18 (33.9 mg),
and 19 (57.3 mg). Structures of 12-19 are shown in the
Supporting Information.
Compound 6: brown oil; [R]D22 +173.7° (c 0.05, CHCl3); IR
νmax 3600, 1675, 1600, 1450 cm-1; CD (c 4.380 × 10-4 M, EtOH)
1
[θ]322 +748; H NMR (CD3OD) δ 7.36-7.20 (5H, m, Ph), 6.83
(1H, dd, J ) 10.4, 3.4 Hz, H-5), 6.02 (1H, ddd, J ) 16.8, 10.2,
8.7 Hz, H-8), 5.94 (1H, d, J ) 10.4 Hz, H-5), 5.02 (1H, dd, J )
10.2, 1.0 Hz, H-9), 4.93 (1H, dd, J ) 16.8, 1.0 Hz, H-9), 4.57
(1H, br d, J ) 8.7 Hz, H-4), 3.64 (1H, t, J ) 8.7 Hz, H-7), 2.97
(1H, dt, J ) 8.7, 4.9 Hz, H-2), 1.97 (1H, ddd, J ) 13.1, 8.7, 4.9
Hz, H-3), 1.95 (1H, ddd, J ) 13.1, 4.9, 5.2 Hz, H-3); 13C NMR,
see Table 1; HREIMS m/z 228.1108 [calcd for C15H16O2,
228.1150].
Compound 7: brown oil; [R]D22 +214.0° (c 0.09, CHCl3); IR
νmax 3595, 1680, 1600, 1450, 1380 cm-1; CD (c 4.380 × 10-4
1
M, EtOH) [θ]339 -3664; H NMR (CD3OD) δ 7.32-7.10 (5H,
m, Ph), 6.86 (1H, dd, J ) 8.2, 1.9 Hz, H-5), 6.02 (1H, ddd, J )
17.6, 9.7, 8.6 Hz, H-8), 5.98 (1H, d, J ) 8.2 Hz, H-5), 5.18 (1H,
dd, J ) 9.7, 1.2 Hz, H-9), 5.17 (1H, dd, J ) 17.6, 1.2 Hz, H-9),
4.50 (1H, br s, H-4), 4.32 (1H, dd, J ) 8.6, 4.1 Hz, H-7), 2.64
(1H, dt, J ) 12.4, 4.1 Hz, H-2), 2.34 (1H, ddd, J ) 13.9, 4.8,
Compound 1: yellow oil; [R]D24 -178° (c 0.075, CHCl3); IR
νmax 3650, 1720, 1605, 1510, 1470, 1455, 1370, 1340, 1120 cm-1
;
CD (c 3.841 × 10-4 M, EtOH) [θ]291 -5263; 1H NMR (CD3OD)
δ 7.32-7.14 (5H, m, Ph), 6.07 (1H, ddd, J ) 17.3, 10.3, 7.8
Hz, H-8), 5.06 (1H, dd, J ) 10.3. 1.2 Hz, H-9), 5.00 (1H, dd, J
) 17.3, 1.2 Hz, H-9), 4.30 (1H, br s, H-4), 3.70 (1H, dd, J )
8.4, 7.8 Hz,H-7), 3.42 (1H, ddd, J ) 10.6, 5.1, 2.9 Hz, H-5),
3.35 (1H, s, OMe), 3.25 (1H, ddd, J ) 12.5, 8.4, 5.5 Hz, H-2),
2.77 (1H, dd, J ) 12.6, 5.1 Hz, H-6), 2.62 (1H, dd, J ) 12.6,
10.6 Hz, H-6), 1.94 (1H, ddd, J ) 14.3, 5.5, 2.6 Hz, H-3), 1.23
(1H, ddd, J ) 14.3, 12.5, 2.6 Hz, H-3); 13C NMR, see Table 1;
HREIMS m/z 260.1406 [calcd for C16H20O3, 260.1412].
4.1 Hz, H-3), 1.90 (1H, ddd, J ) 13.9, 12.4, 10.7 Hz, H-3); 13
C
NMR, see Table 1; HREIMS m/z 228.1135 [calcd for C15H16O2,
228.1150].
Compound 8: dark brown oil; [R]D22 +26.7° (c 0.11, CHCl3);
IR νmax 3400 br, 1655, 1615 cm-1; CD (c 3.871 × 10-4 M, EtOH)
[θ]307 +30704; 1H NMR (CD3OD) δ 7.32-7.18 (5H, m, Ph), 6.10
(1H, dd, J ) 17.6, 10.4, 7.6 Hz, H-8), 5.29 (1H, s, H-5), 5.07
(1H, dd, J ) 10.4, 1.2 Hz, H-9), 5.02 (1H, dd, J ) 17.6, 1.2 Hz,
H-9), 4.42 (1H, dd, J ) 5.4, 4.8 Hz, H-4), 3.85 (1H, t, J ) 7.6
Hz, H-7), 3.71 (1H, s, OMe), 3.02 (1H, ddd, J ) 7.6, 4.9, 4.3
Hz, H-2), 1.99 (1H, ddd, J ) 13.7, 5.4, 4.9 Hz, H-3), 1.84 (1H,
ddd, J ) 13.7, 4.8, 4.3 Hz, H-3); 13C NMR, see Table 1;
HREIMS m/z 258.1243 [calcd for C16H18O3, 258.1256].
Compound 9: dark brown greasy substance; [R]D22 +34.7°
(c 0.06, CHCl3); IR νmax 3600, 1675, 1600, 1450 cm-1; CD (c
4.419 × 10-4 M, EtOH) [θ]286 +1700; 1H NMR (CD3OD) δ 7.31-
7.15 (5H, m, Ph), 6.64 (1H, d, J ) 8.6 Hz, H-5), 6.56 (1H, dd,
J ) 8.6, 2.6 Hz, H-5), 6.53 (1H, d, J ) 2.6 Hz, H-3), 6.25 (1H,
ddd, J ) 16.5, 10.5, 6.6 Hz, H-8), 5.98 (1H, d, J ) 16.5 Hz,
H-9), 5.22 (1H, d, J ) 10.5 Hz, H-9), 4.90 (1H, d, J ) 6.6 Hz,
Compound 2: yellow waxy substance; [R]D24 -57.0° (c 0.05,
CHCl3); IR νmax 3350, 1690, 1490, 1455, 1370, 1340, 1220, 1000
cm-1; CD (c 3.841 × 10-4 M, EtOH) [θ]290 -3466; 1H NMR (CD3-
OD) δ 7.35-7.15 (5H, m, Ph), 6.00 (1H, ddd, J ) 17.1, 10.0,
8.0, H-8), 5.02 (1H, d, J ) 8.0 Hz, H-9), 5.00 (1H, d, J ) 17.1
Hz, H-9), 4.03 (1H, ddd, J ) 7.6, 5.4, 3.6 Hz, H-4), 3.63 (1H,
dd, J ) 10.0, 8.8 Hz, H-7), 3.50 (1H, dt, J ) 7.4, 3.6 Hz, H-5),
3.36 (3H, s, OMe), 3.02 (1H, ddd, J ) 8.8, 5.8, 5.4 Hz, H-2),
2.87 (1H, dd, J ) 13.4, 3.6 Hz, H-6), 2.50 (1H, dd, J ) 13.4,
7.4 Hz, H-6), 1.76 (1H, ddd, J ) 13.4, 5.8, 3.6 Hz, H-3), 1.55

Neoflavonoids from Nepalese Propolis
Journal of Natural Products, 2005, Vol. 68, No. 6 863
H-7); 13C NMR, see Table 1; HREIMS m/z 226.0989 [calcd for
H-3), 1.70 (1H, m, H-3), 1.64 (1H, dt, J ) 13.9, 3.4 Hz, H-6),
1.26 (1H, dt, J ) 13.9, 7.2, Hz, H-6), 1.56 (1H, d, J ) 6.3 Hz,
H-9).
C15H14O2, 226.0994].
Compound 10: dark brown greasy substance; [R]D22 +36.5°
(c 0.05, CHCl3); IR νmax 3600, 1600, 1490 cm-1; CD (c 4.419 ×
Oxidation of S4. To the stirred solution of S4 (6.1 mg) in
CH2Cl2 (1.5 mL) was Jones reagent drop by drop at 0 °C. After
the reaction was completed, the solution was extracted with
CH2Cl2. The organic layer was dried and chromatographed on
silica gel with 3% MeOH-CHCl3 to give ketone S5 (4.0 mg):
1H NMR (CD3OD) δ 7.32 (2H, m, H-2′,6′), 7.27 (1H, m, H-4′),
7.18 (2H, m, H-3′,5′), 4.57 (1H, dt, J ) 9.3, 4.2 Hz, H-1), 4.00
(1H, dq, J ) 9.0, 5.8 Hz, H-8), 3.90 (1H, dd, J ) 10.5, 4.8 Hz,
H-5), 3.50 (3H, s, OMe), 2.81 (1H, dddd, J ) 9.3, 6.8, 6.8, 4.2
Hz, H-2), 2.63 (1H, dd, J ) 15.5, 6.8 Hz, H-3), 2.46 (1H, dt, J
) 14.4, 4.2 Hz, H-6), 2.40 (1H, dd, J ) 9.0, 6.8 Hz, H-7), 2.35
(1H, dd, J ) 15.6, 4.2 Hz, H-3), 2.10 (1H, ddd, J ) 14.4, 10.5,
4.2 Hz, H-6), 1.27 (3H, d, J ) 5.8 Hz, H-9); 13C NMR (CD3OD)
δ 209.5 (C-4), 139.6 (C-1′), 128.9 (C-2′,6′), 127.7 (C-3′,5′), 127.2
(C-4′), 81.6 (C-8), 79.1 (C-5), 74.8 (C-1), 60.7 (C-7), 58.3 (OMe),
47.2 (C-2), 40.5 (C-3), 34.1 (C-6), 19.1 (C-9); CD (c 3.844 ×
10-4M, EtOH) [θ]300 -1426; HREIMS m/z 260.1389 [calcd for
C15H14O2, 260.1412].
1
10-4 M, EtOH) [θ]304 -2807; H NMR (CD3OD) δ 7.35 - 7.2
(5H, m, Ph), 6.71 (1H, d, J ) 8.5 Hz, H-5), 6.64 (1H, d, J )
8.5, 2.9 Hz, H-5), 6.54 (1H, d, J ) 2.9 Hz, H-2), 5.86 (1H, m,
H-8), 5.30 (1H, d, J ) 10.3 Hz, H-9), 5.01 (1H, d, J ) 17.1 Hz,
H-9), 4.90 (1H, d, J ) 6.6 Hz, H-7); 13C NMR, see Table 1;
HREIMS m/z 226.0980 [calcd for C15H14O2, 226.0994].
22
Compound 11: brown oil; [R]D -14.2° (c 0.055, CHCl3);
IR νmax 3530, 1720, 1620, 1600, 1500 cm-1; 1H NMR (CD3OD)
δ 7.40-7.21 (5H, m, Ph), 6.50 (1H, d, J ) 15.9 Hz, H-9), 6.22
(1H, ddd, J ) 15.9, 8.3, 6.8 Hz, H-8), 5.35 (1H, s, H-2), 3.76
(3H, s, OMe), 2.74 (1H, dd, J ) 14.2, 6.8 Hz, H-7), 2.62 (1H,
dd, J ) 14.2, 8.3 Hz, H-7), 2.56 (1H, dt, J ) 17.3, 5.3 Hz, H-6),
2.42 (1H, ddd, J ) 17.3, 11.0, 5.1 Hz, H-6), 2.22 (1H, dt, J )
13.6, 5.1 Hz, H-5), 2.10 (1H, ddd, J ) 13.6, 11.0, 5.3 Hz, H-5);
13C NMR (CD3OD) δ 198.3 (C-1), 177.6 (C-3), 136.9 (C-1′), 134.5
(C-9), 128.6 (C-2′,6′), 128.3 (C-4′), 127.6 (C-3′,5′), 123.4 (C-8),
102.0 (C-2, C-7), 72.0 (C-4), 56.2 (OMe), 33.9 (C-6), 33.0 (C-5);
HREIMS: m/z 258.1249 [calcd for C16H18O3, 258.1256].
LC-MS Analysis. The LC-MS analyses were performed
using a JEOL JMS-700T four-sector mass spectrometer,
coupled to a Hewlett-Packard (Waldbronn, Germany) model
HP1100 HPLC system, operated in the negative atmospheric
pressure chemical ionization (APCI) mode [capillary temper-
ature, 400 °C; ion injection time, 0.1 s; column, Shim-pack
(Shimadzu, Kyoto, Japan) CLC-ODS (150 × 6.0 mm i.d.);
column temperature, 40 °C; mobile phase, linear gradient of
0.5% acetic acid:MeOH from 70:30 (v/v) to 20:80 in 30 min;
flow-rate, 1 mL/min]. Samples of the standards 1-19 were
dissolved in MeOH (5 mg/mL), and 10 µL aliquots were
injected for LC-MS analysis. The Nepalese propolis extract was
dissolved in HPLC grade MeOH (5 mg/mL), filtered using a
Millipore (Millipore, Bedford, MA) SJLG 250 filter, and
aliquots of the filtrate (10 µL) were injected for analysis.
Nitric Oxide Inhibitory Assay. The J774.1 cell line was
propagated in 75 cm2 plastic culture flasks (Falcone, Becton
Dickinson, NJ), containing RPMI 1640 medium supplemented
with penicillin G (100 U/mL), streptomycin (100 U/mL), and
10% FCS. The cells were harvested with trypsin and diluted
to a suspension in fresh medium. The cells were seeded in 96-
well plastic plates with 1 × 105 cells/well and allowed to adhere
for 2 h at 37 °C in a humidified atmosphere containing 5%
CO2. Then, the medium was replaced with fresh medium,
containing LPS (10 µg/mL) and test compounds at indicated
concentrations, and the cells were incubated for 24 h. NO
production was determined by measuring the accumulation
of nitrite in the culture supernatant using Griess reagent.42
Briefly, 50 µL of the supernatant from incubates was mixed
with equal volume of Griess reagent (0.05% sulfanilamide and
0.05% naphthylenediamide dihydrochloride in 2.5% H3PO4)
and was allowed to stand for 10 min at room temperature.
Absorbance at 550 nm was measured using an HTS 7000
microplate reader (Perkin-Elmer, CT). The blank correction
was carried out by subtracting the absorbance due to medium
only from the absorbance reading of each well. The percentage
inhibition was calculated as follows: % inhibition ) [(Ac - As)/
Ac] × 100, where Ac and As are absorbance of control group
treated with LPS alone and absorbance of the sample, respec-
tively.
Reduction of Compound 1. Compound 1 (96.2 mg) in 5
mL of THF was treated at 0 °C with NaBH4 (55.9 mg) in MeOH
(5 mL). After 3 h, an excess of acetone was added to the
reaction mixture, which was then partitioned with water and
EtOAc. The EtOAc layer was evaporated to dryness and then
subjected to normal-phase preparative TLC with 30% CH3CN-
C6H6 to give S1 (17.9 mg) and S2 (22.6 mg). S1: 1H NMR (CD3-
OD) δ 7.35-7.20 (5H, m, Ph), 6.17 (1H, ddd, J ) 17.1, 10.2,
8.6 Hz, H-8), 5.90 (1H, br d, J ) 10.2 Hz, H-9), 5.17 (1H, br d,
J ) 17.1 Hz, H-9), 3.93 (1H, br d, J ) 2.7 Hz, H-4), 3.57 (3H,
s, OMe), 2.27 (1H, m, H-7), 3.45 (1H, dt, J ) 9.3, 3.9 Hz, H-1),
3.19 (1H, ddd, J ) 9.3, 3.9, 2.7 Hz, H-5), 2.27 (1H, m, H-2),
1.97 (1H, dt, J ) 12.7, 3.9 Hz, H-6), 1.85 (1H, dt, J ) 12.7, 9.3
Hz, H-6), 1.83 (1H, dt, J ) 13.6, 2.7 Hz, H-3), 0.97 (1H, ddd,
J ) 13.6, 11.2, 2.7 Hz, H-3). S2: [R]D22 +26.4° (c 0.055, CHCl3);
1H NMR (CD3OD) δ 7.30-7.15(5H, m, Ph), 5.97 (1H, dt, J )
16.9, 10.0 Hz, H-8), 5.12 (1H, dd, J ) 16.9, 1.2 Hz, H-9), 5.01
(1H, dd, J ) 10.0, 1.2 Hz, H-9), 4.23 (1H, br d, J ) 2.2 Hz,
H-1), 4.02 (1H, br d, J ) 2.4 Hz, H-4), 3.53 (1H, ddd, J ) 11.7,
4.6, 2.4 Hz, H-5), 3.28 (3H, s, OMe), 3.19 (1H, t, J ) 10 Hz,
H-1), 2.18 (1H, m, H-6), 2.18 (1H, m, H-2), 1.81 (1H, ddd, J )
13.9, 11.7, 2.2 Hz, H-6), 1.45 (2H, m, H-3).
Selenoetherification of S2. To a magnetically stirred
solution of S2 (13.5 mg) in dry CH2Cl2 (1 mL) were added
PhSeCl (10.5 mg) and K2CO3 (7.6 mg), and the mixture was
stirred at room temperature until the red-orange solid of
PhSeCl was dissolved and TLC indicated completion of the
reaction.41 The product mixture was partitioned with H2O and
CH2Cl2, and the organic layer was separated, dried, and
subjected to normal-phase preparative TLC with 30% CH3CN-
C6H6 to afford selenoether S3 (17.2 mg): 1H NMR (CD3OD) δ
6.82-6.58 (5H, m, Ph), 4.60 (1H, dt, J ) 9.3, 4.3 Hz, H-1),
4.40 (1H, dt, J ) 12.4, 5.6 Hz, H-8), 3.60 (1H, br d, J ) 2.6
Hz, H-4), 3.4 (3H, s, OMe), 3.31 (1H, ddd, J ) 11.4, 4.4, 2.6
Hz, H-5), 3.15 (2H, d, J ) 2.6 Hz, H-9), 3.12 (1H, dd, J ) 12.4,
5.6 Hz, H-7), 2.84 (1H, ddd, J ) 12.9, 8.5, 2.6 Hz, H-3), 2.40
(1H, ddd, J ) 12.9, 4.6, 3.6 Hz, H-3), 2.34 (1H, dt, J ) 11.2,
4.3 Hz, H-6), 2.28 (1H, dddd, J ) 12.4, 8.5, 9.3, 4.6 Hz, H-2),
1.81 (1H, ddd, J ) 11.4, 11.2, 4.3 Hz, H-6).
Reduction of Selenoether S3. To a solution of selenoether
S3 (15.1 mg) in freshly distilled toluene (1 mL) were added
n-Bu3SnH (15.9 mg) and azobisisobutyronitrile (AIBN, 0.02
M toluene solution, 1 mL). The mixture was degassed with a
stream of argon for 15 min and heated to 110 °C for 6 h under
argon.41 The mixture was partitioned with H2O and CHCl3,
and the organic layer was dried and subjected to normal-phase
preparative TLC with 30% CH3CN-C6H6 to afforded S4 (7.5
mg): 1H NMR (CD3OD) δ 7.32-7.18 (5H, m, Ph), 4.44 (1H,
ddd, J ) 9.8, 7.2, 3.4 Hz, H-1), 4.35 (1H, dq, J ) 8.7, 6.3 Hz,
H-8), 3.50 (1H, dt, J ) 13.9, 3.4 Hz, H-5), 3.40 (1H, br d, J )
3.4 Hz, H-4), 3.28 (3H, s, OMe), 2.51 (1H, dd, J ) 11.4, 8.7
Hz, H-7), 2.30 (1H, m, H-2), 2.14 (1H, dt, J ) 10.7, 3.4 Hz,
Acknowledgment. A part of this work was supported by
a Grant-in-Aid for International Scientific Research (No.
16406002) from The Ministry of Education, Culture, Sports,
Science and Technology, Japan, and The Tokyo Biochemical
Research Foundation, Japan.
Supporting Information Available: Figure S1, showing the
structures of the known compounds (12-19) isolated form Nepalese
propolis, and Figure S2, showing the key HMBC and selected ROESY
correlations observed for 1, 4, 5, and 6, are available free of charge
via the Internet at http://pubs.acs.org.

864 Journal of Natural Products, 2005, Vol. 68, No. 6
Awale et al.
(21) Snatzke, G. Tetrahedron 1965, 21, 413-419.
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