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Vol. 50, No. 10
starting material in the synthetic work, 2-methoxy-6-methylbenzoic acid
ethyl ester, from Tokyo Kasei Kogyo Co., Ltd., respectively. The authentic
samples of (R)- and (S)-1-phenylethanols were also available as (R)-(ϩ)-
and (S)-(Ϫ)-1-phenylethyl alcohols from Tokyo Kasei Kogyo Co., Ltd.
Plant Material Leaves of J. occidentalis were collected in Harney
to yield the desired 1-phenylethanol derivative (7) in a
racemic form, which was identified with each of the agly-
1
cones 1a and 2a by comparison of H- and 13C-NMR (400,
100.5 MHz, respectively; in pyridine-d5) and EI-MS spectra.
The absolute structures for the two aglycones were deter- county, Oregon, U.S.A. in 1992. A voucher specimen (Murata J. et al., No.
363) was deposited in the Herbarium of the Botanical Gardens, Faculty of
Science, University of Tokyo.
Extraction and Isolation The dried and cut leaves (584.3 g) were
soaked in acetone (2 l), and the washed plant materials were extracted twice
mined as follows. Aglycones 1a ([a]D Ϫ42.5°, MeOH) and
2a ([a]D ϩ43.8°, MeOH) showed optical rotations of the
same magnitude in an opposite sense, indicating that they are
an enantiomeric pair. The signs in the optical rotations of 1a
and 2a were compared with those of a model enantiomeric
pair, i.e., commercially available authentic (1R)- and (1S)-1-
phenylethanols [the (1R)-isomer: [a]D ϩ44.65° (cϭ0.98,
MeOH) and the (1S)-isomer: [a]D Ϫ44.12° (cϭ0.98,
MeOH)] and it was concluded that 1a corresponds to the
(1S)-isomer and hence, 2a is the (1R)-isomer, the antipode of
1a.
with EtOH (each 3 l) at room temperature for 1 week. The resulting EtOH
extract (16.0 g) was chromatographed on silica gel, and eluted successively
with CHCl3–MeOH (50 : 1®20 : 1), the lower phase of CHCl3–MeOH–H2O
(9 : 3 : 1), and a mixture solvent of CHCl3–MeOH–H2O (6 : 4 : 1) to give 13
fractions (Nos. 1 to 13). Fraction No. 7 (830 mg) was rechromatographed
over silica gel [eluting with CHCl3–MeOH (10 : 1)], followed by preparative
HPLC [70% aqueous (ϭaq.) MeOH] to afford 3b-hydroxy sandaracopimaric
acid (15.4 mg). Fraction No. 8 (530 mg) was subjected to reversed-phase
(ODS) column chromatography (eluting with 70% aq. MeOH), followed by
preparative HPLC separation (JAIGEL-GS column; eluting with MeOH) to
yield cupressuflavone (39.0 mg) and amentoflavone (37.8 mg) in that eluting
order. Fraction No. 9 (350 mg) was further column-chromatographed over
silica gel [eluting with CHCl3–MeOH (7 : 1)] and ODS (eluting with 50%
aq. MeOH), successively, followed by preparative HPLC separation (eluting
with 50% aq. MeOH) to give 1 (73.9 mg) and 2 (21.9 mg).
As the final step in the structural elucidation of both glu-
cosides, the location of the b-D-glucopyranosyl residue on
each aglycone is decided as follows. Each glucoside showed
the NOESY cross peak between the anomeric H and the aro-
matic 3Ј-H, as well as the HMBC correlation between the
anomeric H and 2Ј-C bearing the phenolic O-function (Fig.
2), proving that the b-D-glucopyranosyl moiety is linked at
Juniperoside I (1): Colorless needles from MeOH, mp 89—91 °C, [a]D
Ϫ26.3° (cϭ1.0, pyridine). FAB- and HR-FAB-MS (negative mode) m/z:
313.1281 (Calcd for C15H21O7, [MϪH]Ϫ: 313.1288), 151 [MϪHϪGlc]Ϫ.
the phenolic (2Ј-) OH of the respective aglycones in 1 and 2. 1H- and 13C-NMR: Given in Tables 1 and 2, respectively.
Juniperoside II (2): Colorless needles from MeOH, mp 97—99 °C, [a]D
In conclusion, the entire structures of 1 and 2 are defined as
(1S)- and (1R)-1-(2Ј-hydroxy-6Ј-methylphenyl)ethanol 2Ј-O-
b-D-glucopyranosides, respectively.
Ϫ8.4° (cϭ1.0, pyridine). FAB- and HR-FAB-MS (negative mode) m/z:
313.1281 (Calcd for C15H21O7, [MϪH]Ϫ: 313.1288), 151 [MϪHϪGlc]Ϫ.
1H- and 13C-NMR: Given in Tables 1 and 2, respectively.
To our knowledge, the aglycones in 1 and 2 must belong to
structurally novel and unique phenolic compounds which
have not yet been found in nature. Furthermore, glucosides 1
and 2 must also be novel and rare types of naturally occur-
ring components.
Enzymatic Hydrolysis of 1 A solution of 1 (20.0 mg) in acetate buffer
(pH 5.0; 2 ml) was incubated with almond emulsin (20.0 mg) at 37 °C for
2 h. The reaction mixture was extracted with ether and the solvent was evap-
orated to dryness under reduced pressure. The residue was purified by silica
gel column chromatography (n-hexane : etherϭ3 : 1 as the eluent) to yield 1a
(4.5 mg), colorless needles of mp 78—79 °C, [a]D Ϫ42.5° (cϭ0.80,
MeOH). EI- and HR-EI-MS m/z (%): 152.0841 (Calcd for C9H12O2, Mϩ:
152.0837, 30), 134 ([MϪH2O]ϩ, 100). 1H- and 13C-NMR: Given in Tables 1
and 2.
Enzymatic Hydrolysis of 2 A solution of 2 (10.0 mg) in acetate buffer
(pH 5.0; 2 ml) was incubated with almond emulsin (10.0 mg) at 37 °C for
2 h. Work-up and purification procedure of the reaction mixture as described
in the hydrolysis of 1 furnished 2a (2.2 mg), colorless needles, mp 80—
81 °C, [a]D ϩ43.8° (cϭ0.22, MeOH). EI- and HR-EI-MS m/z (%):
152.0840 (Calcd for C9H12O2, Mϩ: 152.0837, 30), 134 ([MϪH2O]ϩ, 100).
1H- and 13C-NMR: Given in Tables 1 and 2.
In this study, two known biflavones, cupressuflavone and
amentoflavone and a known diterpene, 3b-hydroxy sandara-
copimaric acid were also isolated from the same plant mater-
ial for the first time. Identities of cupressuflavone2,3) and
amentoflavone3,4) were established by comparison of 1H- and
13C-NMR data with those reported for the respective authen-
tic specimens. The physicochemical and spectral data {mp,
1
[a]D, H- and 13C-NMR (600, 150 MHz, respectively), EI-
MS} of the isolated diterpene were in agreement with the re-
ported values for authentic 3b-hydroxy sandaracopimaric
acid.5)
Synthesis of the Racemic Aglycone (7) The preparation of the syn-
thetic aglycone (7) from the commercially available starting compound (3)
via 4 reaction processes (Chart 1) is briefly described below.
2-Methoxy-6-methylbenzyl Alcohol (4) A 1 M solution of DIBAL-H in
n-hexane (10 ml, 1 mmol) was added to a stirred solution of 3 (970.0 mg,
0.5 mmol) in dry ether (10 ml) at Ϫ78 °C under nitrogen. The mixture was
stirred at Ϫ78 °C for a further 10 min under nitrogen, acidified with 5% hy-
drochloric acid, and extracted into ether. The organic layer was washed with
brine until neutral, dried (anhydrous Na2SO4), and evaporated in vacuo to af-
ford 4 (734.0 mg; in a 97% yield) as a pale yellow oil.
Although none of juniperosides I and II, cupressuflavone,
amentoflavone, and 3b-hydroxy sandaracopimaric acid
showed significant anti-HIV-1 activity, further component
analysis of the aerial part of the plant is continuing with the
intention of isolating the anti-HIV-1 active compound.
2-Methoxy-6-methylbenzaldehyde (5) A mixture of 4 (734.0 mg, 0.48
mmol) and active MnO2 (3.67 g) in benzene (10 ml) was refluxed with stir-
ring for 5 h. The mixture was filtered through a short bed of Celite and the
filtrate was evaporated in vacuo to give 5 (619.0 mg; in a 86% yield) as a
pale yellow oil.
Experimental
General Remarks All melting points were recorded on a Yanagimoto
melting point apparatus without correction. H- and 13C-NMR spectra were
1
measured on a GE-Omega 600 (1H at 600 and 13C at 150 MHz) and a JEOL
JNM-GX 400 (1H at 400 and 13C at 100.5 MHz) spectrometers with pyri-
dine-d5 as a solvent. Chemical shifts are given in d (ppm) values relative to
internal tetramethylsilane (TMS). FAB- and HR-FAB-MS spectra in a nega-
tive mode (matrix, triethanolamine), along with EI- and HR-EI-MS spectra,
were obtained with a JEOL JMS-700T spectrometer. Optical rotations were
determined on a JASCO DIP-140 polarimeter. GLC was carried out on a
Shimadzu GC-7AG instrument. For column chromatography, Kieselgel 60
(Merck) and Chromatorex ODS DM1020T (Fuji Silysia) were used. Prepar-
ative HPLC was performed on a JAI LC-908 instrument and unless other-
wise noted, a JAIGEL-ODS column was used. The almond emulsin used in
this work was commercially obtained from Sigma-Aldrich Japan Co. and the
2-Hydroxy-6-methylbenzaldehyde (6)
A mixture of 5 (300.0 mg,
0.2 mmol) and LiCl (254 mg, 0.6 mmol) in N,N-dimethylformamide (DMF)
(5 ml) was refluxed with stirring for 15 h. The cooled reaction mixture was
diluted with 10% aq. NaOH and washed with ether. The aq. solution was
acidified with 10% hydrochloric acid and extracted with ether. The ether
layer was washed with brine and dried (anhydrous Na2SO4). After evapora-
tion of the solvent, the residue was purified by silica gel column chromatog-
raphy (elution with n-hexane : etherϭ3 : 1) to give 6 (111.3 mg; in a 41%
yield) as colorless needles.
1-(2
-Hydroxy-6
-methylphenyl)ethanol (7) A 1.09 M solution of MeLi