Structure and biogenesis of jolkinin, a highly oxygenated ellagitannin from Euphorbia jolkinii

Article abstract of DOI:10.1021/np0400297

A new ellagitannin, jolkinin (2), was isolated from the fresh whole plant of Euphorbia jolkinii, and its structure, including absolute configuration, was determined on the basis of spectroscopic and chemical evidence. A highly oxygenated acyl group with unique hexacyclic structure is attached to the 2,4-positions of 1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-β-D- glucopyranose. The structural similarity to elaeocarpusin (4) suggests that jolkinin is produced by a condensation reaction between ascorbic acid and geraniin (1), the dominant ellagitannin in this plant, which contains a dehydrohexahydroxydiphenoyl group. A plausible mechanism for jolkinin formation is also proposed.

Full text of DOI:10.1021/np0400297

1
018
J . Nat. Prod. 2004, 67, 1018-1022
Str u ctu r e a n d Biogen esis of J olk in in , a High ly Oxygen a ted Ella gita n n in fr om
Eu ph or bia jolkin ii
†
,‡
§
⊥
Seung-Ho Lee, Takashi Tanaka,* Gen-ichiro Nonaka, and Itsuo Nishioka
Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, J apan, and
Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University,
Bunkyo-Machi 1-14, Nagasaki 852-8521, J apan
Received February 5, 2004
A new ellagitannin, jolkinin (2), was isolated from the fresh whole plant of Euphorbia jolkinii, and its
structure, including absolute configuration, was determined on the basis of spectroscopic and chemical
evidence. A highly oxygenated acyl group with unique hexacyclic structure is attached to the 2,4-positions
of 1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-â-D-glucopyranose. The structural similarity to elaeocarpusin
(4) suggests that jolkinin is produced by a condensation reaction between ascorbic acid and geraniin (1),
the dominant ellagitannin in this plant, which contains a dehydrohexahydroxydiphenoyl group. A plausible
mechanism for jolkinin formation is also proposed.
Recently, various health benefits of polyphenols in plants
have attracted much scientific interest. Among the polyphen-
ols, tannins are a group of compounds that bind to proteins
The known compounds were identified by direct compari-
son of spectroscopic and physical data.
J olkinin was obtained as a tan amorphous powder and
characterized as an ellagitannin by the standard color
reactions with ferric chloride (dark blue) and sodium
1
by hydrophobic, electrostatic, and ionic interactions; there-
fore, tannins inhibit digestive enzymes2 and are believed
,3
4
16
1
13
to act as defensive substances against microorganisms and
nitrite-acetic acid (reddish brown). The H and C NMR
spectra exhibited signals due to a fully acylated hexopy-
ranose along with aromatic and carboxyl carbon signals,
which is consistent with 2 being a hydrolyzable tannin. The
presence of galloyl and HHDP esters was suggested by the
herbivores.5 Ellagitannins are defined as a group of
tannins having hexahydroxydiphenic acid (HHDP) esters
in the molecule, and most ellagitannins have a D-glucose
,6
7
polyalcohol core. When a HHDP ester bridges the C-3 and
1
13
C-6 or the C-1 and C-6 positions of the D-glucopyranose,
H and C NMR signals listed in the Experimental Section,
1
the pyranose ring adopts a C
4
conformation, in which the
and hydrolysis of 2 in hot H O to yield corilagin [1-O-
2
oxygen atoms and the C-6 methylene carbon are fixed in
axial orientations. In such ellagitannins, some oxidized
forms of the HHDP esters are often found at the glucose
galloyl-3,6-(R)-HHDP-â-D-glucopyranose (3), Figure 1] con-
firmed the partial structure. This result showed not only
the location of the galloyl and HHDP esters on the â-D-
glucopyranose ring but also R-atropisomerism of the HHDP
2
1
,4-positions. The most typical one is geraniin (1) (Figure
), which occurs in many Euphorbiaceous plants as a
8
8,17
biphenyl bond.
major phenolic constituent. Euphorbia jolkinii Boiss. (Eu-
phorbiaceae) also contains 1 as a major ellagitannin
The remaining part of the molecule was comprised of an
aromatic ring (C-1′′-C-6′′), three carboxyl (C-7, C-1′, and
C-7′′), seven aliphatic quaternary (C-2, C-4, C-5, C-6, C-2′,
C-3′, and C-4′), and three methine (C-1, C-3, and C-5′)
carbons, and one methylene (C-6′) carbon. Chemical shifts
of the aromatic carbon signals showed that the aromatic
ring is a pyrogallol ring. In the HMBC spectrum, an
aromatic singlet (δ 7.01, H-3′′) of this pyrogallol ring was
correlated to a carboxyl carbon signal at δ 165.4 (C-7′′),
indicating that the C-7′′ carboxyl group is at C-2′′. Three
of the aromatic carbons (C-1′′, C-2′′, and C-6′′) were
correlated to a benzyl methine proton (δ 4.86, H-1), which
was coupled with two acetal carbons (C-5 and C-6), two
quaternary carbons (C-2 and C-2′), and a methine carbon
(
0.098% from fresh whole plant), along with several related
9
compounds. In our continuing chemical studies on tannins
in Euphorbiaceous plants, a new ellagitannin named
jolkinin (2) was isolated from E. jolkinii. This paper deals
with the structural determination of this ellagitannin.
Resu lts a n d Discu ssion
Fresh whole E. jolkinii plants were extracted with 80%
aqueous acetone, and the extract was first fractionated by
Sephadex LH-20 column chromatography. Then each frac-
tion was separated by repeated column chromatography
over Sephadex LH-20, MCI-gel CHP20P, and Bondapak
(C-3). In addition, correlations of the C-3 methine proton
C
18/Porasil B to give jolkinin (2) (0.002%), together with
signal (δ 2.64, H-3) with the C-2, C-5, and another acetal
carbon (C-4) signal suggested that the three acetal carbons
9
9
9
9
1
-O-, 1,6-di-O-, 1,2,3,6-tetra-O-, 1,3,4,6-tetra-O-, 2,3,4,6,-
1
0
tetra-O-, and 1,2,3,4,6-penta-O-galloyl-â-D-glucose, co-
rilagin (3),
carpinusin,9 helioscopinins A and B,
(C-4, C-5, and C-6), two methine carbons (C-1 and C-3),
9
,11
,14
8,9
12
13
geraniin (1), furosin, putranjivain A,
and a quaternary carbon (C-2) formed a cyclohexane ring.
9,15
and jolkianin.9
1
1
This was supported by H- H long-range coupling between
H-1 and H-3 observed in the 1H- H COSY spectrum.
Furthermore, the HMBC correlations of the C-7 carboxyl
carbon with H-1 and H-3 indicated that this group was
attached to C-2. The 1H- H COSY spectrum revealed
linkage between oxygen-bearing methine (C-5′) and meth-
ylene (C-6′) carbons. In the HMBC spectrum, H-6′ cor-
related with C-4′ and C-5′, and H-5′ correlated with C-4′,
1
*
To whom correspondence should be addressed. Tel: 81-95-819-2433.
Fax: 81-95-819-2477. E-mail: t-tanaka@net.nagasaki-u.ac.jp.
†
Kyushu University. Present address: College of Pharmacy, Yeungnam
1
University, Kyongsan, 712-749, Korea.
‡
Kyushu University. Present address: Nagasaki University.
Kyushu University. Present address: Usaien Seiyaku KK, 1-4-6
§
Zaimoku, Saga, 840-0055, J apan.
⊥
Kyushu University.
1
0.1021/np0400297 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/08/2004

Structure and Biogenesis of J olkinin
J ournal of Natural Products, 2004, Vol. 67, No. 6 1019
F igu r e 1. Structures of compounds 1-4.
C-3′, C-2′, C-6′, and C-3. The aforementioned H-3 was
correlated with C-4′, C-3′, and C-2′, and C-2′ was also
coupled with H-1. From these correlations, it was deduced
that C-2′ and C-4′ of the carbon chain C-2′ through C-6′
were connected to the C-2 and C-3 positions of the above-
mentioned cyclohexane ring. Consequently, a cyclopentane
ring was formed by C-2, C-3, C-4′, C-3′, and C-2′, as shown
in Figure 1. The remaining carboxyl carbon C-1′ was
presumed to be attached to C-2′, on the basis of observation
of a small 4J correlation peak with H-3 in the HMBC
spectrum. The ester carbonyl carbons C-7 and C-7′′ of this
acyl group showed HMBC cross-peaks with the glucose H-4
and H-2, respectively, confirming the connection of this acyl
group to the glucopyranose 2,4-positions.
therefore, formation of an ether linkage between C-6 and
C-6′′ was deduced. The remaining two rings were presumed
to be formed by ether linkages between the oxygen-bearing
quaternary carbon C-2′ and the acetal carbon C-5 and
between two acetal carbons C-3′ and C-4.
To obtain further evidence for the structure, methylation
of 2 was attempted. However, under usual conditions using
(CH ) SO or CH N , 2 gave a complex mixture. Therefore,
3
2
4
2
2
the acetonide of 2 was prepared prior to methylation,
because aliphatic vicinal diols were expected to be present
in the acyl group. Methylation of the acetonide successfully
yielded two products, 2a and 2b. The molecular mass of
2b [m/z 1376] was 14 mass units larger than that of 2a
[m/z 1362], which corresponds to the mass of a methyl
The negative ion FABMS of 2 exhibited the [M - H]-
peak at m/z 1143. Taking the above-mentioned chemical
and spectral data into account, the molecular formula of 2
1
group. The H NMR spectra of 2a and 2b (see Experimental
Section) were closely related to each other; however, the
spectrum of 2a showed a sharp singlet signal due to a
2
is C47
H
36
O
34, which was also supported by elemental
hydroxyl proton at δ 5.88, which showed a J HMBC
analysis. The index of unsaturation calculated from the
formula is 30, and 25 degrees of unsaturation were ac-
counted for by three carboxyl groups; the aromatic ring of
the 2,4-acyl group, the corilagin moiety, and a macrocyclic
lactone ring formed between these two parts. The remain-
ing 5 degrees of unsaturation indicated that there are five
ring structures in the aliphatic part of the 2,4-acyl group.
Of the five rings, two were accounted for by the above-
mentioned cyclohexane and cyclopentane rings. Compari-
son of the chemical shifts of the C-6 (δ 106.0) and C-6′′ (δ
correlation with the carbon signal attributable to the C-3′
acetal carbon. An additional methyl proton signal was
observed at δ 3.47 in the spectrum of 2b, and this signal
showed a correlation peak with C-3′ in the HMBC spec-
trum. These spectral differences indicated that 2b was a
methyl ether with an additional methyl group at the C-3′
acetal hydroxyl group of 2a .
The 13C NMR spectrum of 2a exhibited a signal attribut-
able to a ketone carbon (C-4), which was not observed in
the spectrum of 2. The ketone signal showed a cross-peak
with the H-3 of the acyl group in the HMBC spectrum;
therefore, the ketone in 2a was assigned to the C-4 position.
In addition, one of the H-6′ methylene protons of 2a showed
a correlation peak with the C-3′ acetal carbon. Further-
more, the C-6′ carbon signal appeared at a lower field (δ
74.3) than that of 2 (δ 62.7). From these findings, it was
1
49.9) of 2 with those of the two equilibrium forms of the
dehydrohexahydroxydiphenoyl (DHHDP) esters of geraniin
1) indicated that the chemical shifts were similar to those
of the five-membered ring hemiacetal form [1b: δ 108.9
C-6) and 147.3 (C-6′′)] and different from those of the six-
(
(
8
membered ring form [1a : δ 92.5(C-6) and 143.4 (C-6′′)];

1
020 J ournal of Natural Products, 2004, Vol. 67, No. 6
Lee et al.
F igu r e 2. Structures of 1c and 4a and important NOESY correlations for 1c.
Sch em e 1. Acetonide Formation Followed by Methylation
deduced that formation of a new ether linkage between the
acetal C-3′ and the C-6′ hydroxyl group, which was ac-
companied by cleavage of the ether linkage between C-4
and C-3′ in 2, occurred during derivation of 2a from 2
benzyl methine has an R-configuration. Accordingly, the
structure of jolkinin was concluded to be as shown in
formula 2.
Elaeocarpusin (4) and related ellagitannins were previ-
1
5,20
(Scheme 1). The acetonide moieties were presumed to be
ously isolated from various Euphorbiaceous
and Elaeo-
carpaceous plants,19 and it was shown that the elaeocar-
pusinoyl group, the acyl group attached to the glucose 2,4-
positions in 4, was derived by addition of L-ascorbic acid
(5) to the (R)-DHHDP group.20 The structural similarity
to 4 suggested that 2 was also derived from ascorbic acid
and geraniin (1), which is the dominant ellagitannin of E.
jolkinii. However, 4 was not isolated from this plant.
Plausible biogenesis of 2 from 1 is demonstrated in Scheme
2, where 5 reacts from the â side of the DHHDP group,
and the pathway includes an oxidation step. In contrast,
in the biosynthesis of 4 from 1, 5 is attacked from the R
side of the DHHDP group. Hence, jolkinin represents a new
type of ascorbic acid adduct of ellagitannins and could be
important from the viewpoint of ellagitannin metabolism
in the plant kingdom.
attached to the vicinal hydroxyl groups at the C-5′ and C-4′
positions, on the basis of the HMBC correlation between
H-5′ and a quaternary carbon of the acetonide. These
HMBC correlations described for 2a were similarly ob-
served in the spectrum of 2b, and other HMBC correlations
observed for 2a and 2b were consistent with the formula-
tion of the structures of 2a and 2b shown in Scheme 1 and
also supported the carbon skeleton of the acyl group of 2.
The HMBC correlations of the methoxyl groups in 2a
indicated that the C-1′ of 2 was a free carboxyl group and
the C-5 and C-6 were hemiacetal carbons.
The relative stereochemistry of the acyl group attached
to the glucose 2,4-positions was determined by observation
of NOEs for methanolysate 2c (Figure 2), which was
obtained along with methyl 3,4,5-trimethoxybenzoate and
dimethyl (R)-4,4′,5,5′,6,6′-hexamethoxydiphenate.18 One-
dimensional differential NOE experiments showed NOEs
of H-3 with H-1 and H-5′, indicating that these protons are
located on the same side of the molecule. In addition, two-
dimensional NOESY correlations of three acetal methyl
groups, two acetonide methyls, and C-6′ methylene protons
shown in Figure 2 also supported the configuration of 2c.
The absolute configuration of 2c was determined by
comparison of the CD spectrum with that of 4a , which was
derived from elaeocarpusin (4) by methylation followed by
methanolysis.19 The CD spectrum of 2c showed a negative
Cotton effect at 260 nm and a positive Cotton effect at 230
nm, which was similar to that of 4a (negative Cotton at
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with J ASCO DIP-4 and DIP-370 digital
polarimeters. IR spectra were obtained with a J ASCO FT/IR-
4
10 spectrophotometer. CD spectra were measured by using
1
13
1
1
a J ASCO J -600 spectropolarimeter. H, C NMR, H- H
COSY, NOESY, HSQC, and HMBC spectra for 2, 2a , and 2b
were recorded with a Unity plus 500 spectrometer (Varian Inc.)
operating at 500 MHz for 1H and 125 MHz for 13C. NMR
spectra for 2c were measured by using a J EOL GX-270
1
spectrometer operating at 270 MHz for H and 67.5 MHz for
1
3
C. FABMS spectra were recorded by using a J MS DX-303
spectrometer (J EOL Ltd., J apan), using m-nitrobenzyl alcohol
2
63 nm and positive Cotton at 229 nm), where the C-1

Structure and Biogenesis of J olkinin
J ournal of Natural Products, 2004, Vol. 67, No. 6 1021
Sch em e 2. Plausible Biogenesis of 1 from 3 and Ascorbic Acid
or glycerol as the matrix. Elemental analysis was carried out
with a Perkin-Elmer 2400 II analyzer (Perkin-Elmer, Inc.).
Column chromatography was done on MCI-gel CHP 20P
(C-7), 173.2(C-1′), 86.6 (C-2′), 104.8 (C-3′), 86.9 (C-4′), 81.5 (C-
5′), 62.7 (C-6′), 118.5 (C-1′′), 115.3 (C-2′′), 112.9 (C-3′′), 147.3
(C-4′′), 134.8 (C-5′′), 149.9 (C-6′′), 165.4 (C-7′′); HMBC correla-
tions (H/C) glc-1/galloyl-7, glc-2, 3, 5; glc-2/acyl-7′′, glc-1, 3, 4;
glc-3/HHDP-7, glc-1, 2, 4, 5; glc-4/acyl-7, glc-2, 3; glc-5/glc-6;
glc-6/HHDP-7′, glc-4; galloyl-2,6/galloyl-1, 3, 4, 6, 7; HHDP-
(Mitsubishi Chemical Co.), Bondapak C18/Porasil B (37-75 µm,
Waters Associates, Inc.), Sephadex LH-20 (Pharmacia Fine
Chemical Co.), and silica gel 60 (70-230 mesh, Merck). Thin-
layer chromatography (TLC) for tannins was performed on
precoated Kieselgel 60 F254 plates, 0.2 mm thick (Merck), with
benzene-ethyl formate-formic acid (1:7:1, v/v), or cellulose
3
1
2
4
5
/HHDP-1, 2, 4, 5, 7; HHDP-3′/HHDP-1′, 4′, 5′, 7′; 2, 4-acyl-
4
/2, 4-acyl-2, 3, 5, 6, 7, 2′, 1′′, 2′′, 6′′, 7′′( J ); 2, 4-acyl-3/2, 4-acyl-
4
, 4, 5, 7, 1′( J ), 2′, 3′, 4′; 2, 4-acyl-5′/2, 4-acyl-3, 4, 3′, 4′, 6′; 2,
4
-acyl-6/2, 4-acyl-4′, 5′; 2, 4-acyl-3′′/2, 4-acyl-1( J ), 1′′, 2′′, 4′′,
F
254 plates, 0.1 mm thick (Merck), with 2% acetic acid. Spots
4
-
′′, 6′′( J ), 7′′; FABMS m/z 1143 [M - H] ; anal. calcd for
O C, 48.55; H, 3.29; found C, 48.31; H, 3.19.
P a r tia l Hyd r olysis of 2. A solution of 2 in H O (8 mg/mL)
were detected by UV illumination and sprayed with 2%
47 36 34 2
C H O H
ethanolic FeCl
heating.
3
or 10% sulfuric acid reagent followed by
2
was heated at 90 °C for 12 h. The mixture was subjected to
P la n t Ma ter ia l. Whole plants of Euphorbia jolkinii Boiss.
were collected in Yobuko, Saga Prefecture, in April 1989. The
voucher specimen (KPMG 0427-89/4) was deposited at the
Herbarium, Faculty of Pharmaceutical Sciences, Kyushu
University.
MCI-gel CHP20P column chromatography (1 cm i.d. × 7 cm)
2
with H O containing increasing proportions of MeOH, to yield
gallic acid (1.5 mg) and corilagin (1.7 mg). The latter was
1
identified by comparison of the H NMR spectrum with that
of an authentic sample.9
Isola tion . The plant material and extraction procedures
9
P r ep a r a tion of Meth yl Eth er s 2a a n d 2b. A solution of
were described in our previous paper. Fraction 2 (200 g),
2
(300 mg) and p-toluenesulfonic acid (10 mg) in acetone (20
obtained from the aqueous acetone extract of fresh whole
plants (50 kg) by Sephadex LH-20 column chromatography
mL) was heated under reflux for 2 h. After concentration, the
mixture was chromatographed on a Sephadex LH-20 column
(H
2
O-MeOH), was further separated by MCI-gel CHP20P
column chromatography with H O containing increasing
amounts of MeOH, to give fractions 2-1 (15 g), 2-2 (150 g), and
-3 (25 g). Repeated chromatography of fraction 2-1 on
Sephadex LH-20 (60% MeOH), Bondapak C18/Porasil B (H O-
MeOH), and MCI-gel CHP20P (H O-MeOH) yielded carpi-
(
1.5 cm i.d. × 15 cm) with H
tions of MeOH to give an acetonide (250 mg). The acetonide
was methylated by (CH SO (2 mL), with K CO (2 g) in
2
O containing increasing propor-
2
3
)
2
4
2
3
2
acetone (30 mL) under reflux for 2 h. The products were
separated by silica gel column chromatography (hexane-
acetone, 1:1) to give 2a (58.6 mg) and 2b (72.0 mg).
2
2
nusin (1.3 g). Separation of fraction 2-2 by column chroma-
Meth yl Eth er 2a : white amorphous powder; [R]28
(c 0.2, CHCl ), IR νmax (neat) 3450, 2943, 2841, 1750, 1719,
1589, and 1459 cm ; H NMR (500 MHz, CDCl
benzoyl (TMB), δ 7.21 (2H, s, H-2,6), 3.68 (6H, s, C-3, 5-OCH
-102.6°
tography over Sephadex LH-20 (60-100% MeOH) and MCI-
D
gel CHP20P (H
2
O-MeOH) afforded 1 (52 g), 2 (1.4 g), and 3
3
-1 1
(0.9 g). On similar chromatography, fraction 2-3 yielded 1,3,4,6-
3
) trimethoxy-
);
tetra-O-galloyl-D-glucopyranose (18 mg), putranjivain A (40
3
mg), helioscopinin B (2.2 g), and helioscopinin A (3.2 g).
hexamethoxydiphenoyl (HMDP), δ 6.66 (1H, s, H-3), 6.72 (1H,
s, H-3′), 4.00, 3.98, 3.97, 3.95, 3.72, 3.70, 3.25 (each 3H, s,
2
8
J olk in in (2): tan amorphous powder; [R]
D
-55.4° (c 0.7,
acetone); IR νmax (neat) 3448, 1719, 1614, 1526, 1446 1316,
213, and 1036 cm ; H NMR (500 MHz, CDCl
.41 (1H, br s, H-1), 5.27 (1H, br s, H-2), 6.21 (1H, br t, J )
.0 Hz, H-3), 5.18 (1H, br d, J ) 3.0 Hz, H-4), 4.78 (2H, m,
OCH ); glucose, δ 6.62 (1H, br s, H-1), 5.35 (1H, br s, H-2),
3
-
1
1
1
6
3
3
) glucose, δ
6.03 (1H, ddd, J ) 1.4, 2.3, 5.0 Hz, H-3), 5.47 (1H, br d, J ) 5
Hz, H-4), 4.78 (1H, br dd, J ) 8.5, 11.1 Hz, H-5), 5.01 (1H, t,
J ) 11.1 Hz, H-6), 4.42 (1H, dd, J ) 8.5, 11.1 Hz, H-6); 2, 4-acyl
group, δ 5.38 (1H, d, J ) 0.5 Hz, H-1), 3.64 (1H, d, J ) 0.5 Hz,
H-3), 4.42 (1H, d, J ) 4.2 Hz, H-5′), 4.45 (1H, dd, J ) 4.2, 10.1
Hz, H-6′), 4.13 (1H, d, J ) 10.1 Hz, H-6′), 7.47 (1H, s, H-3′′),
1.54 (3H, s, acetonide-H-2), 1.42 (3H, s, acetonide-H-3), 5.88
H-5, H-6), 4.38 (1H, m, H-6); galloyl, δ 7.15 (2H, s, H-2, 6);
HHDP, δ 7.06 (1H, s, H-3), 6.63 (1H, d, H-3′); 2,4-acyl group,
δ 4.86 (1H, s, H-1), 2.64 (1H, s, H-3), 4.31 (1H, dd, J ) 3.9, 4.2
Hz, H-5′), 3.79 (1H, dd, J ) 4.2, 11.6 Hz, H-6′), 3.67 (1H, dd,
1
3
J ) 3.9, 11.6 Hz, H-6′), 7.01 (1H, s, H-3′′); C NMR (500 MHz,
CDCl ) glucose, δ 92.1 (C-1), 70.6 (C-2), 60.8 (C-3), 68.6 (C-4),
2.4 (C-5), 64.0 (C-6); galloyl, δ 120.0 (C-1), 110.5 (C-2, 6), 145.9
(1H, s, C-3′-OH), 3.88 (3H, s, C-5-OCH
OCH ), 4.08 (3H, s, C-1′-OCH ), 3.86 (3H, s, C-4′′-OCH
(3H, s, C-5′′-OCH ); C NMR (125 MHz, CDCl ) ΤΜΒ, δ 122.9
3
), 3.68 (3H, s, C-6-
3
3
3
3
), 3.97
1
3
7
3
3
(
(
(
(
C-3, 5), 139.8 (C-4), 165.3 (C-7); HHDP, δ 117.0 (C-1), 115.0
C-1′), 124.2 (C-2), 125.7 (C-2′), 110.5 (C-3), 107.5 (C-3′), 144.6
C-4), 145.3 (C-4′), 137.7 (C-5), 136.2 (C-5′), 145.1 (C-6), 144.9
C-6′), 166.4 (C-7), 168.7 (C-7′); 2,4-acyl group, δ 53.2 (C-1),
(C-1), 106.9 (C-2, 6), 153.3 (C-3, 5), 143.3 (C-4), 164.1 (C-7);
HMDP, δ 124.5, 120.9 (C-1,1′), 128.1, 125.7 (C-2,2′), 107.5 (C-
3), 104.9 (C-3′), 153.4, 152.6, 152.6, 152.3 (C-4, 4′, 6, 6′), 145.45,
144.02 (C-5, 5′), 164.59 (C-7), 167.50 (C-7′); glucose, δ 91.8 (C-
1), 70.3 (C-2), 59.9 (C-3), 67.0 (C-4), 70.5 (C-5), 63.1 (C-6); 2,
5
4.9 (C-2), 56.5 (C-3), 102.4 (C-4), 96.2 (C-5), 106.0 (C-6), 169.2

1
022 J ournal of Natural Products, 2004, Vol. 67, No. 6
Lee et al.
4
9
1
1
6
2
-acyl group, δ 47.8 (C-1), 66.4 (C-2), 59.5 (C-3), 206.0 (C-4),
6.0 (C-5), 101.9 (C-6), 166.1 (C-7), 170.5 (C-1′), 92.6 (C-2′),
11.1 (C-3′), 94.6 (C-4′), 82.8 (C-5′), 74.3 (C-6′), 110.6 (C-1′′),
24.3 (C-2′′), 113.8 (C-3′′), 152.9 (C-4′′), 142.4 (C-5′′), 147.2 (C-
′′), 165.6 (C-7′′), 114.1 (acetonide-C-1), 26.4 (acetonide-C-2),
acetone, 93:7) to yield methyl trimethoxybenzoate (3 mg),
dimethyl (R)-hexamethoxydiphenate (8 mg), and 2c (10 mg):
white amorphous powder; [R]27
6.2° (c 0.8, acetone); IR νmax
1 1
D
-
(neat) 2951, 2843, 1747, 1720, 1610, and 1510 cm ; H NMR
(270 MHz, CDCl ) δ 7.20 (1H, s, H-3′′), 5.51 (1H, s, H-1), 4.89
(1H, dd, J ) 2, 6 Hz, H-5′), 4.20 (1H, dd, J ) 2, 11 Hz, H-6′),
4.11 (1H, dd, J ) 6, 11 Hz, H-6′), 3.98 (3H, s, 5′′-OCH ), 3.93
(3H, s, 7′′-OCH ), 3.91 (3H, s, 4′′-OCH ), 3.83 (3H, s, 7-OCH ),
3.67 (3H, s, 5-OCH ), 3.51 (3H, s, 1′-OCH ), 3.50 (3H, s, 3′-
OCH ), 3.41 (3H, s, 6-OCH ), 2.97 (1H, s, H-3), 1.46, 1.29 (each
1H, s, acetonide CH ); C NMR (67.5 MHz, CDCl ) δ 198.9
3
6.0 (acetonide-C-3), 52.0 (C-5-OCH
), 56.1 (C-4′′-OCH ); OCH
3
), 56.4 (C-6-OCH
, δ 61.1, 60.9, 60.8, 60.73,
3
), 53.4
(C-1′-OCH
3
3
3
3
6
1
2
0.70, 60.6, 56.4, 56.0, 55.2; HMBC correlations (H/C) 2, 4-acyl-
3
3
3
4
/2, 4-acyl-2, 3, 5, 6, 7, 2′, 4′( J ), 1′′, 2′′, 6′′; 2, 4-acyl-3/2, 4-acyl-
3
3
4
, 4, 5, 7, 1′( J ), 2′, 3′, 4′, 5′; 2, 4-acyl-5′/2, 4-acyl-3, 3′, 6′,
3
3
1
3
acetonide-1; 2, 4-acyl-6′/2, 4-acyl-3′, 4′, 5′; 2, 4-acyl-3′′/2, 4-acyl-
3
3
4
4
4
1
2
4
( J ), 1′′, 2′′, 4′′, 5′′, 6′′( J ), 7′′; 2, 4-acyl-3′-OH/2, 4-acyl-1′( J ),
′, 3′, 4′; 2, 4-acyl-5-OCH /2, 4-acyl-5; 2, 4-acyl-6-OCH /2,
-acyl-6; 2, 4-acyl-1′-OCH /2, 4-acyl-1′, 2′( J ); 2, 4-acyl-4′′-
/2, 4-acyl-4′′; acetonide-2, 3/acetonide-1; FABMS m/z
(C-4), 166.1, 166.0, 165.9 (C-7, 1′, 7′′), 152.3 (C-4′′), 149.9 (C-
6′′), 137.0 (C-5′′), 123.0 (C-2′′), 119.2 (C-1′′), 114.8 (acetonide
C), 112.3 (C-3′), 110.2 (C-6), 107.9 (C-3′′), 99.0 (C-5), 94.4 (C-
4′), 87.1 (C-2′), 79.3 (C-5′), 74.7 (C-6′), 58.1 (C-2), 57.4 (C-3),
3
3
4
3
OCH
3
+
1
5
362 M ; anal. calcd for C64
6.14; H, 4.88.
Meth yl Eth er 2b: white amorphous powder; [R]28
c 0.4, CHCl ); IR νmax (neat) 2945, 2848, 1751, 1718, 1592,
H
66
O
33 C, 56.39; H, 4.88; found C,
60.8 (5′′-OCH
52.1 (×3, 6-OCH
1), 25.9, 25.3 (acetonide CH
(nm), -0.35 (260), 11.5(230); FABMS m/z 681 [M + H] ; anal.
36
calcd for C31H O17 C, 54.70; H, 5.29; found C, 54.89; H, 5.53.
3
), 56.5 (4′′-OCH
3
), 53.2 (5-OCH
3
), 52.5 (7′′-OCH
3
),
3
, 7-OCH , 1′-OCH
3
3
), 51.4 (3′-OCH
3
), 47.6 (C-
-5
D
-28.2°
3
); CD (1.5 × 10 M, EtOH) ∆ꢀ
+
(
3
-
1 1
and 1458 cm ; H NMR (500 MHz, CDCl
s, H-2, 6), 3.69 (6H, s, 3,5-OCH
6
3
) ΤΜΒ, δ 7.21 (2H,
); HMDP, δ 6.68 (1H, s, H-3),
3
.73 (1H, s, H-3′), 3.84 (3H, s, 4′-OCH ), 3.21 (3H, s, 4-OCH );
3
3
Ack n ow led gm en t. The authors are grateful to Mr. Y.
Tanaka, Miss Y. Soeda, and Dr. K. Isobe (Kyushu University)
and Mr. K. Inada and Mr. N. Yamaguchi (Nagasaki Univer-
sity) for NMR and MS measurements.
glucose, δ 6.59 (1H, br s, H-1), 5.29 (1H, br s, H-2), 6.32 (1H,
ddd, J ) 1.1, 2.5, 5.0 Hz, H-3), 5.12 (1H, br d, J ) 5 Hz, H-4),
4
.60 (1H, br dd, J ) 8.2, 10.8 Hz, H-5), 5.03 (1H, t, J ) 10.8
Hz, H-6), 4.48 (1H, dd, J ) 8.2, 10.8 Hz, H-6); 2, 4-acyl group:
δ 5.59 (1H, d, J ) 0.4 Hz, H-1), 3.15 (1H, d, J ) 0.4 Hz, H-3),
Refer en ces a n d Notes
4
.51 (1H, dd, J ) 2.1, 3.9 Hz, H-5′), 4.26 (2H, br s, H-6′), 7.18
(
(
1) Haslam, E. J . Nat. Prod. 1996, 59, 205-215.
(
1H, s, H-7′′), 1.56 (3H, s, acetonide-H-2), 1.36 (3H, s, ac-
etonide-H-3), 3.67 (3H, s, C-5-OCH ), 3.47 (3H, s, C-6-OCH ),
), 3.89 (3H, s, C-4′′-OCH ); OCH : δ 4.00,
.98, 3.973, 3.971, 3.96, 3.75, 3.71 (each 3H, s); 1 C NMR (125
) ΤΜΒ, δ 122.9 (C-1), 106.9 (C-2, 6), 153.3 (C-3,
), 143.3 (C-4), 164.1 (C-7); HMDP, δ 124.2, 121.8 (C-1, 1′),
2) Milic, B. L.; Stojanovic, S.; Vucurevic, N. J . Sci. Food Agric. 1972,
3
3
2
3, 1157-1162.
3
3
.47 (3H, s, C-3′-OCH
3
3
3
(3) Maitra, S.; Ray, A. K. Aquacult. Res. 2003, 34, 93-95.
3
(4) Fukai, K.; Ishigami, T.; Hara, Y. Agric. Biol. Chem. 1991, 55, 1895-
1
897.
MHz, CDCl
3
(
(
5) Zucker, W. V. Am. Nat. 1983, 121, 335-365.
5
1
1
1
6) Clausen, T. P.; Reichardt, P. B.; Bryant, J . P.; Provenza, F. Basic
Life Sci. 1992, 59, 639-651.
28.4, 125.8 (C-2, 2′), 107.7 (C-3), 104.3 (C-3′), 153.8, 152.7,
52.4, 152.3 (C-4, 4′, 6, 6′), 145.4, 144.1 (C-5,5′), 164.6 (C-7),
67.4 (C-7′); glucose, δ 92.6 (C-1), 69.1 (C-2), 60.4 (C-3), 66.9
(
(
7) Haslam, E.; Cai, Y. Nat. Prod. Rep. 1994, 11, 41-66.
8) Okuda, T.; Yoshida, T.; Hatano, T. J . Chem. Soc., Perkin Trans. 1
1
982, 9-14.
(
C-4), 72.2 (C-5), 63.5 (C-6); 2, 4-acyl group, δ 45.3 (C-1), 56.7
(
9) Lee, S.; Tanaka, T.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull.
(C-2), 58.5 (C-3), 198.8 (C-4), 98.4 (C-5), 110.3 (C-6), 165.1 (C-
1991, 39, 630-638.
10) Armitage, R.; Bayliss, G. S.; Gramshaw, J . W.; Haslam, E.; Haworth,
(
7
5
), 165.5 (C-1′), 88.0 (C-2′), 114.1 (C-3′), 92.6 (C-4′), 87.8 (C-
′), 74.0 (C-6′), 116.3 (C-1′′), 120.8 (C-2′′), 109.7 (C-3′′), 152.8
R. D.; J ones, K.; Rogers, H. J .; Searle, T. J . Chem. Soc. 1961, 1842-
1
853.
(
C-4′′), 137.1 (C-5′′), 150.7 (C-6′′), 164.2 (C-7′′), 114.3 (acetonide-
C-1), 25.7 (acetonide-C-2), 24.2 (acetonide-C-3), 54.0 (C-5-
OCH ), 50.3 (C-6-OCH ), 52.3 (C-1′-OCH ), 52.4 (C-3′-OCH ),
6.5 (C-4′′-OCH ); OCH , δ 61.1, 60.83, 60.79, 60.75, 60.70,
0.69, 56.5, 56.0, 55.2; HMBC correlations (H/C) 2, 4-acyl-1/2,
(11) Schmidt, O.; Lademann, R. Liebigs Ann. Chem. 1951, 571, 232-237.
(12) Okuda, T.; Hatano, T.; Yazaki, K. Chem. Pharm. Bull. 1982, 30,
1
113-1116.
13) (a) Ishimatsu, M.; Nonaka, G.; Nishioka, I. Abstracts of papers, The
6th Annual Meeting of the J apanese Society of Pharmacognosy,
3
3
3
3
(
5
6
4
3
3
4
5
2
1
5
3
3
3
Kumamoo, 1989; p 172. (b) Lin, J .; Ishimatsu, M.; Tanaka, T.;
Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1990, 38, 1844-1851.
(c) El-Mekkawy, S.; Meselhy, M.; Kusumoto, I.; Kadota, S.; Hattori,
M.; Namba, T. Chem. Pharm. Bull. 1995, 43, 641-648.
14) Nonaka, G.; Akazawa, M.; Nishioka, I. Heterocycles 1992, 33, 597-
606.
4
4
4
-acyl-2, 3, 5, 6, 7, 2′, 1′( J ), 2′, 4′( J ), 1′′, 2′′, 6′′, 7′′( J ); 2, 4-acyl-
4
/2, 4-acyl-2, 4, 5, 7, 1′( J ), 2′, 3′, 4′, 5′; 2, 4-acyl-5′/2, 4-acyl-3,
′, 6′, acetonide-1; 2, 4-acyl-6′/2, 4-acyl-3′, 4′, 5′; 2, 4-acyl-3′′/2,
(
(
4
4
-acyl-1( J ), 1′′, 2′′, 4′′, 5′′, 6′′( J ), 7′′; 2, 4-acyl-5-OCH
; 2, 4-acyl-6-OCH /2, 4-acyl-6; 2, 4-acyl-1′-OCH /2, 4-acyl-1′,
′( J ); 2, 4-acyl-4′′-OCH /2, 4-acyl-4′′; acetonide-2, 3/acetonide-
3
/2, 4-acyl-
15) Lee, S.; Tanaka, T.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull.
1990, 38, 1518-1523.
3
3
4
3
(
16) Bate-Smith, E. C. Phytochemistry 1972, 11, 1153-1156.
17) Okuda, T.; Yoshida, T.; Nayeshiro, H. Chem. Pharm. Bull. 1977, 25,
1862-1869.
+
; FABMS m/z 1376 M ; anal. calcd for C65
68 33 2
H O H O C,
(
5.96; H, 5.06; found C, 55.78; H, 4.92.
(
18) Ikeya, Y.; Taguchi, H.; Yoshioka, I.; Kobayashi, H. Chem. Pharm.
Bull. 1979, 27, 1383-1394.
19) Tanaka, T.; Nonaka, G.; Nishioka, I.; Miyahara, K.; Kawasaki, T. J .
Chem. Soc., Perkin Trans. 1 1986, 369-376.
Meth a n olysis of 2b. A solution 2b (40 mg) in MeOH (1
mL) and 10% aqueous NaOH (2 mL) was heated at 70 °C for
h. The mixture was acidified with 1 M HCl and extracted
with ether. The ether layer was dried over Na SO and
concentrated. The residue was dissolved in MeOH (2 mL) and
treated with CH ether solution at 0 °C for 2 h. The products
were separated by silica gel column chromatography (toluene-
(
1
(20) (a) Saijo, R.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1989, 37,
2
4
2
063-2070. (b) Lin, J .; Tanaka, T.; Nonaka, G.; Nishioka, I.; Chen,
I. Chem. Pharm. Bull. 1990, 38, 2161-2171.
2
N
2
NP0400297