Reinvestigation of absolute stereostructure of (-)-rosiridol: Structures of monoterpene glycosides, rosiridin, rosiridosides A, B, and C, from Rhodiola sachalinensis

Article abstract of DOI:10.1248/cpb.56.695

Three new (-)-rosiridol glycosides, rosiridosides A, B, and C, were isolated from the roots of Rhodiola sachalinensis together with rosiridin [(-)-rosiridol 1-O-β-D-glucopyranoside]. In the course of the structure elucidation of those new glycosides, the

Full text of DOI:10.1248/cpb.56.695

May 2008
Chem. Pharm. Bull. 56(5) 695—700 (2008)
695
Reinvestigation of Absolute Stereostructure of (ꢀ)-Rosiridol: Structures
of Monoterpene Glycosides, Rosiridin, Rosiridosides A, B, and C, from
Rhodiola sachalinensis1)
Masayuki YOSHIKAWA,* Seikou NAKAMURA, Xuezheng LI, and Hisashi MATSUDA
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received January 28, 2008; accepted February 20, 2008; published online February 29, 2008
Three new (ꢀ)-rosiridol glycosides, rosiridosides A, B, and C, were isolated from the roots of Rhodiola
sachalinensis together with rosiridin [(ꢀ)-rosiridol 1-O-b-D-glucopyranoside]. In the course of the structure elu-
cidation of those new glycosides, the absolute configuration of the 4-position in (ꢀ)-rosiridol was reinvestigated.
On the basis of the application of the modified Mosher’s method for (ꢀ)- and (ꢁ)-rosiridol derivatives, the ab-
solute configuration of the 4-position in (ꢀ)-rosiridol should be revised to be S orientation from the recently as-
signed R form, so that the absolute stereostructures of rosiridosides A, B, and C and rosiridin were determined.
Key words (ꢀ)-rosiridol; rosiridoside; rosiridin; (ꢂ)-rosiridol; Rhodiola sachalinensis; Rhodiolae Radix
In the course of our serial studies on bioactive constituents ture elucidation of three new (ꢀ)-rosiridol glycosides (3, 4,
from Chinese natural medicines,2—11) we have characterized 5).
the structures of aliphatic alcohol and monoterpene oligo-
Isolation of Rosiridin and Rosiridosides A, B, and C
glycosides and cyanogenic glycosides from the roots of The methanolic extract from the roots of R. sachalinensis
Rhodiola (R) quadrifida,12,13) R. sacra,14) and R. crenulata.15) was partitioned into an EtOAc–H2O mixture to furnish an
Among the isolated compounds, several monoterpene oligo- EtOAc-soluble phase (3.5% from the roots) and aqueous
glycosides were found to show histamine release from rat ex- layer, which was further extracted with n-BuOH to give a n-
udate cells induced by an antigen–antibody reaction.14) Fur- BuOH-soluble phase. The EtOAc-soluble fraction was sub-
thermore, from the extracts of R. sachalinensis with a protec- jected to normal-phase and reverse-phase column chro-
tive effect an D-galactosamine-induced cytotoxicity in pri- matographies, and finally HPLC to give rosiridoside C (5,
mary cultured mouse hepatocytes, we isolated monoterpene 0.0013%). The n-BuOH-soluble phase was subjected to Di-
oligoglycosides, flavonol bisdesmosides, and cyanogenic gly- aion HP-20 column chromatography to afford a methanol-
coside named sachalosides I—VIII and reported their struc- eluted fraction (3.5%) as previously reported.1,16) The
tures and hepatoprotective effects.1,16) As a continuing study methanol-eluted fraction was separated by normal- and re-
on R. sachalinensis, we isolated three new (ꢀ)-rosiridol gly- versed-phase column chromatography, and finally HPLC to
cosides termed rosiridosides A (3), B (4), and C (5) together give rosiridosides A (3, 0.00019%) and B (4, 0.00032%) to-
with rosiridin (2) [(ꢀ)-rosiridol 1-O-b-D-glucopyranoside].
The absolute configuration of the 4-position in (ꢀ)-rosiri-
gether with rosiridin (2, 0.49%).
Reinvestigation of the Absolute Configuration of (ꢀ)-
dol (1) was first reported to be S by the total enantioselective Rosiridol Rosiridin (2) was obtained as a colorless viscous
synthesis and chemical transformation to (ꢀ)-eldanolide.17) oil with a negative optical rotation {[a]D25 ꢀ32.1° (cꢃ2.06 in
However, the absolute configuration of the 4-position in (ꢀ)- MeOH), [a]D22 ꢀ31.3° (cꢃ4.65 in acetone)} and the IR spec-
rosiridol and rosiridin was recently revised to be R (1ꢁ, 2ꢁ) by trum of 2 showed absorption bands at 3450, 1638, and
the application of the modified Mosher’s method.18,19) In 1045 cmꢀ1 assignable to hydroxyl, olefin, and ether func-
order to determine the absolute stereostructures of rosirido- tions. The positive-ion first atom bombardment (FAB)-MS of
sides (3—5), we began with a reinvestigation of the absolute 2 exhibited a quasimolecular ion peak at m/z 355 (MꢂNa)ꢂ.
configuration of (ꢀ)-rosiridol. This paper deals with the ap- The high resolution (HR) MS analysis revealed the molecular
plication of the modifiel Mosher’s method for (ꢀ)- and (ꢂ)- formula of 2 to be C16H28O7. Acid hydrolysis of 2 with HCl
rosiridol pivalate (6, 11), which supported the previous syn- 1 M liberated D-glucose, which was identified by HPLC
thetic evidence to be S configuration (1, 2) rather than R form analysis using an optical rotation detector.3,7,16) On the basis
(1ꢁ, 2ꢁ). Furthermore, we described the isolation and struc- of this evidence and by comparison of the physical data with
reported values {[a]D25 ꢀ16.9° (cꢃ0.087 in MeOH),18) [a]D20
ꢀ32.7° (cꢃ1.1 in acetone),20) 1H- and 13C-NMR,18) MS18)}, 2
was identified to be rosiridin, which was isolated from R.
rosea20) and R. sachalinensis, in which the absolute stere-
ostructure of rosiridin was reported to be 2ꢁ.18) Enzymatic
hydrolysis of 2 with b-glucosidase furnished (ꢀ)-rosiridol,
{1, [a]D25 ꢀ7.7° (cꢃ1.98 in acetone), [a]D22 ꢀ14.2° (cꢃ0.42
in CHCl3)}, whose molecular formula C10H18O2 was deter-
mined from a molecular ion peak at m/z 170 (M)ꢂ and by
HR-MS measurement. The 1H-NMR (CD3OD) and 13C-
NMR (Table 1) spectra21) of 1 showed signals assignable to
three methyls [d 1.65, 1.69, 1.74, (3H each, both s, H3-8, 10,
Chart 1
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2008 Pharmaceutical Society of Japan

696
Vol. 56, No. 5
Table 1. 13C-NMR (150 MHz) Data for 1 (CDCl3), 2—5 (CD3OD)
and 11 by treatment with (ꢂ)-MTPA-Cl in pyridine. In the
case of MTPA esterification using S-(ꢀ)- or R-(ꢂ)-MTPA in
the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide (EDC) and 4-dimethylaminopyridine (4-DMAP), 6a
and 6b were also obtained from 6. As shown in Fig. 2, the
signals due to the protons attached to the 5, 6, 8 and 9-posi-
tions in the (S)-MTPA ester (6a) were observed at higher
fields compared with those of the (R)-MTPA ester (6b) [Dd:
negative], while the signals due to the protons on the 1, 2,
and 10-positions in 6a were observed at lower fields com-
pared with those of 6b [Dd: positive]. The same result was
obtained in the application case of the modified Mosher’s
method using synthetic 6. On the other hand, the signals due
to the protons attached to the 1, 2, and 10-positions in the
(S)-MTPA ester (11a) were observed at higher fields com-
pared with those of the (R)-MTPA ester (11b) [Dd: negative],
while the signals due to the protons on the 5, 6, 8, and 9-po-
sitions in 11a were observed at lower fields compared with
those of 11b [Dd: positive]. On the basis of those findings,
the absolute configuration of the 4-position in (ꢀ)-rosiridol
(1) was clarified to be S and also the absolute stereostructure
of (ꢀ)-rosiridol (1) and rosiridin (2) was determined as
shown.
Position
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
6ꢁ
1ꢄ
59.2
124.5
140.4
76.3
66.0
122.8
142.9
78.0
65.9
122.9
142.9
78.1
66.1
122.7
143.3
78.0
34.8
121.6
134.1
18.1
26.1
12.2
102.8
75.0
78.1
72.0
76.8
68.1
110.2
83.3
79.0
85.9
63.1
65.8
122.6
143.5
78.0
34.2
34.8
34.8
34.8
119.7
135.4
18.0
25.9
12.2
121.6
134.0
18.1
26.0
12.1
102.8
75.0
77.9
71.6
78.1
121.6
134.1
18.0
26.0
12.0
103.5
72.5
74.4
69.6
66.9
121.6
134.2
18.1
26.1
12.0
102.5
75.0
78.0
71.7
75.3
62.7
64.9
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢁ-O-Ac
172.8
20.8
Absolute Stereostructures of Rosiridosides A, B, and C
9)], a methylene bearing an oxygen function [d 4.23 (2H, dd, Rosiridoside A (3) was obtained as a colorless viscous oil
Jꢃ3.5, 6.9 Hz H2-1)], a methine bearing an oxygen function with negative optical rotation ([a]D27 ꢀ10.4° in MeOH). The
[d 4.03 (1H, dd, Jꢃ5.5, 7.6 Hz, H-4)], two olefinic protons [d IR spectrum of 3 showed absorption bands at 3545, 1653,
5.12 (1H, t-like, Jꢃca. 7.0 Hz, H-6), 5.67 (1H, t-like, Jꢃca. and 1040 cmꢀ1 assignable to hydroxyl, olefin, and ether func-
7 Hz, H-2)], and a methylene [d 2.27 (2H, m, H2-5)]. These tions. The positive-ion FAB-MS of 3 showed a pseudomolec-
results supported that the planner structure of (ꢀ)-rosiridol ular ion peak at m/z 325 (MꢂNa)ꢂ and the HR-MS analysis
(1) was the same as the reported structure.17,19) On the other revealed the molecular formula of 3 to be C15H26O6. Acid hy-
hand, although all the optical rotations of reported (ꢀ)-rosiri- drolysis of 3 with HCl 1 M liberated L-arabinose, which was
dol showed negative values {[a]D20 ꢀ7.7° (cꢃ1.3 in identified by HPLC analysis using an optical rotation detec-
acetone),20) [a]D25 ꢀ7.1° (cꢃ0.4 in acetone),17) [a]D24 ꢀ7.1° tor.3,7,16) Enzymatic hydrolysis of 3 with naringinase yielded
(cꢃ0.1 in acetone),19) [a]D24 ꢀ21.1° (cꢃ0.1 in CHCl3)19)}, (ꢀ)-rosiridol (1). The H- (CD3OD) and 13C-NMR (Table 1)
1
the absolute configuration at the 4-position was first reported spectra21) of 3 exhibited the presence of a (ꢀ)-rosiridol moi-
to be S17 and then changed to R form.18,19) In order to confirm ety [d 1.62, 1.65, 1.68 (3H each, both s, H3-8, 10, 9), 2.24
the absolute configuration of (ꢀ)-rosiridol, the application of (2H, t like, Jꢃca. 7 Hz, H2-5), 4.23, 4.30 (1H each, both dd,
the modified Mosher’s method for (ꢀ)- and (ꢂ)-rosiridol Jꢃ6.8, 12.4 Hz, H2-1), 3.96 (1H, t, Jꢃ6.9 Hz, H-4), 5.10
was carried out. (ꢀ)- And (ꢂ)-rosiridol 1-pivalates (6, 11) (1H, t, Jꢃ6.8 Hz, H-6), 5.56 (1H, t, Jꢃ6.8 Hz, H-2)] and an
having the different configulation at the 4-position were pre- a-L-arabinopyranosyl part [4.23 (1H, d, Jꢃ6.8 Hz, H-1ꢁ)].
pared from (ꢀ)-rosiridol (1) in 5-steps and applied the modi- As shown in Fig. 1, the double quantum filter correlation
fied Mosher’s method. Namely, selective acylation of 1 with spectroscopy (DQF COSY) experiment on 3 indicated the
pivaloyl chloride yielded the 1-pivalate (6), which was presence of partial structures written in bold lines, and in the
treated with CrO3 in pyridine to afford the 4-ketone (7). Re- heteronuclear multiple-bond correlations (HMBC) experi-
duction of 7 with NaBH4 in the presence of CeCl3 furnished ment, long-range correlation was observed between the 1ꢁ-
an enantiomeric mixture (8), which was treated with (5S)- proton and 1-carbon. Furthermore, comparison of the 13C-
allyl-2-oxabicyclo[3,3,0]oct-8-ene (ALBO)22) followed by NMR data of 3 with those of 1 indicated the presence of a
separation to give 9 (21%) and 10 (39%). Acid treatment of 9 glycosylation shift around the 1-position. Consequently, the
and 10 with p-TsOH furnished 6 and 11, respectively. Syn- absolute stereostructure of rosiridoside A (3) was determined
thetic 6 was identified by comparison with authentic (ꢀ)- as shown.
rosiridol 1-pivalate (6) {[a]D25 ꢀ5.9° (cꢃ0.45 in acetone), IR,
Rosiridoside B (4) was also isolated as a colorless viscous
1H- and 13C-NMR, MS}. Deacylation of 11 with 1% NaOMe oil with negative optical rotation ([a]D27 ꢀ73.7° in MeOH)
1
yielded (ꢂ)-rosiridol (12). The physical data (IR, H- and and its IR spectrum showed absorption bands due to hy-
13C-NMR, positive FAB-MS, HR-MS) of 11 and 12 were in droxyl, olefin, and ether functions. In the positive and nega-
accord with those of 6 and 1, respectively, except for the op- tive-ion FAB-MS of 4, quasimolecular ion peaks were ob-
tical rotation {11: [a]D25 ꢂ4.4° (cꢃ0.45 in acetone); 12: [a]D25 served at m/z 487 (MꢂNa)ꢂ and m/z 463 (MꢀH)ꢀ and the
ꢂ6.1° (cꢃ0.32 in acetone), [a]D22 ꢂ14.1° (cꢃ0.32 in molecular formula C21H36O11 was determined based on the
CHCl3)}. Treatment of 6 and 11 with (ꢀ)-MTPA-Cl in pyri- results of HR-FAB-MS measurement. The acid hydrolysis of
dine yielded the (S)-MTPA ester (6a, 11a), respectively. 4 liberated D-glucose and L-arabinose, whereas the enzymatic
1
Whereas, the (R)-MTPA ester (6b, 11b) were derived from 6 hydrolysis of 4 afforded (ꢀ)-rosiridol (1). The H- (CD3OD)

May 2008
697
Fig. 1. Synthesis of (ꢀ)- and (ꢂ)-Rosiridol
cose,3,7,16) while alkaline hydrolysis of 5 furnished rosiridin
(2) and acetic acid, which was identified by HPLC analysis of
its p-nitrobenzyl derivative.23,24) The proton and carbon sig-
nals in the 1H- and 13C-NMR spectra21) of 5 was very similar
to those of 2, except for the signal due to the acetyl group. In
the HMBC experiment of 5, a long-range correlation was ob-
served between the 6ꢁ-proton and acetyl carbonyl carbon.
Furthermore, comparison of the 13C-NMR data of 5 with
those of 2 indicated an acetylation shift around the 6ꢁ-posi-
tion of 5. Those findings led us to formulate the stereostruc-
ture of rosiridoside C (5) as shown.
Experimental
General Experimental Procedures The following instruments were
used to obtain physical data: specific rotations, Horiba SEPA-300 digital po-
larimeter (lꢃ5 cm); IR spectra, Shimadzu FTIR-8100 spectrometer; EI-MS
and HR-EI-MS, JEOL JMS-GCMATE mass spectrometer; FAB-MS and
HR-FAB-MS, JEOL JMS-SX 102A mass spectrometer; 1H-NMR spectra,
JEOL EX-270 (270 MHz), JNM-LA500 (500 MHz), and JEOL ECA-600K
(600 MHz) spectrometers; 13C-NMR spectra, JEOL EX-270 (68 MHz) JNM-
LA500 (125 MHz), and JEOL ECA-600K (150 MHz) spectrometers with
tetramethylsilane as an internal standard; HPLC detector, Shimadzu RID-6A
refractive index detector; and HPLC column, YMC-Pack ODS-A (YMC,
Inc., 250ꢅ4.6 mm i.d.) and (250ꢅ20 mm i.d.) columns were used for analyt-
ical and preparative purposes, respectively.
The following experimental materials were used for chromatography: nor-
mal-phase silica gel column chromatography, Silica gel BW-200 (Fuji
Silysia Chemical, Ltd., 150—350 mesh); reversed-phase silica gel column
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd.,
100—200 mesh); Diaion HP-20 column chromatography (Nippon Rensui);
TLC, precoated TLC plates with Silica gel 60F254 (Merck, 0.25 mm) (ordi-
nary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (reversed phase);
reversed-phase HPTLC, precoated TLC plates with Silica gel RP-18 WF254S
(Merck, 0.25 mm); and detection was achieved by spraying with 1%
Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Plant Material The dried roots of R. sachalinensis were collected at
Jilin and Heilongjiang provinces of China in 2005 and identified by one of
authors (X. Li). A voucher of the plant is on file in our laboratory (2006.
China-06).
Extraction and Isolation The methanolic extract (1457 g, 14.6%) was
obtained from dried roots of R. sachalinensis (10.0 kg) as reported previ-
ously.16) The aliquot (1395 g) from the extract was partitioned into an
EtOAc–H2O (1 : 1, v/v) mixture to furnish an EtOAc-soluble fraction (336 g,
3.5%) and an aqueous phase. The aqueous phase was further extracted with
Fig. 2
and 13C-NMR (Table 1) spectra21) of 4 showed signals due to
a (ꢀ)-rosiridol moiety together with b-D-glucopyranosyl [d
4.29 (1H, d, Jꢃ7.6 Hz, H-1ꢁ)] and a-L-arabinofuranosyl [d
4.96 (1H, br s, H-1ꢄ)] parts. The proton and carbon signals in
the H- and 13C-NMR data of 4 were superimposable on
1
those of 3, except for the signals due to the a-L-arabinopyra-
nosyl part of 3. The HMBC experiment on 4 showed long-
range correlations between the 1ꢁ-proton and 1-carbon and
between the 1ꢄ-proton and 6-carbon. On the basis of this evi-
dence and detail comparison of 13C-NMR data of 4 with
those of 1 and 2, the absolute stereostructure of rosiridoside
B (4) was characterized as shown.
Rosiridoside C (5), a colorless viscous oil, [a]D25 ꢀ16.7° in
MeOH, showed absorption bands at 3566, 1734, 1647 and
1037 cmꢀ1 assignable to hydroxyl, carbonyl, olefin, and ether
functions in the IR spectrum. The molecular formula
C18H30O8 was determined based on the results of the posi-
tive-ion FAB-MS at m/z 397 (MꢂNa)ꢂ and HR-FAB-MS
measurement. The acid hydrolysis of 5 liberated D-glu- n-BuOH to give an n-BuOH-soluble fraction (418 g, 4.4%) and an H2O-sol-

698
Vol. 56, No. 5
Fig. 3. Significant DQF COSY and HMBC Correlations of New Constituents 3—5
uble fraction (620 g, 6.5%). The n-BuOH-soluble fraction (366 g) was sub- (cꢃ0.94, MeOH); IR (Film) nmax 3566, 1734, 1647, 1037 cmꢀ1
jected to Diaion HP-20 column chromatography (4.0 kg, H2O→MeOH) to (CD3OD, 600 MHz) d: 1.62, 1.67, 1.69, 2.06 (3H each, both s, H3-8, 10, 9,
give H2O- and MeOH-eluted fractions 65.9 g, 0.8% and 290.7 g, 3.5%), re- Ac), 2.24 (2H, t like, Jꢃca. 7 Hz, H2-5), 3.97 (1H, t, Jꢃ6.9 Hz, H-4), 4.30,
;
1H-NMR
spectively. The EtOAc fraction (139 g) was subjected to ordinary-phase sil- 4.32 (1H each, both m, H2-1), 4.30 (1H, d, Jꢃ7.6 Hz, H-1ꢁ), 5.10 (1H, t,
ica gel column chromatography [3.0 kg, CHCl3→CHCl3–MeOH (20 : 1)→ Jꢃ6.9 Hz, H-6), 5.55 (1H, t like, Jꢃca. 7 Hz, H-2); 13C-NMR data see Table
CHCl3–MeOH–H2O (15 : 3 : 1, lower layer→10 : 3 : 1, lower layer→7 : 3 : 1, 1; positive-ion FAB-MS m/z: 397 [MꢂNa]ꢂ; HR-FAB-MS m/z: 397.1841
lower layer)→MeOH] to give six fractions [Fr. 1 (18.0 g), Fr. 2 (38.7 g), Fr. 3 (Calcd for C18H30O8Na [MꢂNa]ꢂ, 397.1838).
(19.8 g), Fr. 4 (13.1 g), Fr. 5 (15.1 g), Fr. 6 (17.4 g)]. Fraction 4 (13.0 g) was
Acid Hydrolysis of 2—5 A solution of 2—5 (each 1.0 mg) in HCl 1 M
subjected to reversed-phase silica gel column chromatography [450 g, (2.0 ml) were each heated under reflux for 3 h. After cooling, each reaction
MeOH–H2O (30 : 70→40 : 60→50 : 50→60 : 40→70 : 30→90 : 10, v/v)→ mixture was neutralized with Amberlite IRA-400 (OHꢀ form) and filtrated,
MeOH] to give twelve fractions [Fr. 4-1 (1157 mg), Fr. 4-2 (241 mg), Fr. 4-3 and the solution was partitioned with EtOAc to give two layers. The aqueous
(689 mg), Fr. 4-4 (538 mg), Fr. 4-5 (752 mg), Fr. 4-6 (2058 mg), Fr. 4-7 layer was evaporated and then subjected to HPLC analysis using Kaseisorb
(540 mg), Fr. 4-8 (479 mg), Fr. 4-9 (1701 mg), Fr. 4-10 (976 mg), Fr. 4-11 LC NH2-60-5 column (4.6 mmꢅ250 mm i.d., Tokyo Kasei Co., Ltd., Tokyo,
(248 mg), Fr. 4-12 (2391 mg)]. Fr. 4-9 (1771 mg) was further purified by Japan) and an optical rotation detector (Shodex OR-2, Showa Denko Co.,
HPLC [MeOH–H2O (55 : 45, v/v)] to give rosiridoside C (5, 38.5 mg). The
MeOH-eluted fraction (188.8 g) was subjected to ordinary-phase silica gel
column chromatography [3.0 kg, CHCl3–MeOH–H2O (15 : 3 : 1, lower
layer→10 : 3 : 1, lower layer→7 : 3 : 1, lower layer→6 : 4 : 1, v/v/v)→MeOH]
Ltd., Tokyo, Japan) with CH3CN–H2O [(75 : 25, v/v), 0.5 ml/min]. D-glucose
and L-arabinose were confirmed by comparison of the retention times with
the authentic samples (Wako Pure Chemicals Ltd., Osaka, Japan); tR:
13.8 min (D-(ꢂ)-glucose, positive optical rotation); tR: 12.3 min (L-(ꢂ)-ara-
to give seven fractions [Fr. 1 (428 mg), Fr. 2 (1.8 g), Fr. 3 (60.5 g), Fr. 4 binose, positive optical rotation).
(13.4 g), Fr. 5 (16.6 g), Fr. 6 (12.6 g), Fr. 7 (64.1 g)]. Fraction 3 (60.5 g) was
Alkaline Hydrolysis of 5 A solution of 5 (7.0 mg) in 5% aqueous KOH
subjected to reversed-phase silica gel column chromatography [1.5 kg, (1.0 ml) was stirred at 37 °C for 5 h. The reaction mixture was neutralized
MeOH–H2O (10 : 90→20 : 80→30 : 70→40 : 60→50 : 50→60 : 40→70 : 30,
with Amberlite HCR-W2 (Hꢂ form) and filtrated. The evaporated reaction
v/v)→MeOH] to give 11 fractions [Fr. 3-1 (12.1 g), Fr. 3-2 (3.4 g), Fr. 3-3 mixture was subjected to SiO2 column chromatography [CHCl3–MeOH–
(2.3 g), Fr. 3-4 (822 mg), Fr. 3-5 (572 mg), Fr. 3-6 (26.5 g), Fr. 3-7 (956 mg), H2O (20 : 3 : 1, lower layer→15 : 3 : 1, lower layer)] to give 2 (4.6 mg, 74.2%)
Fr. 3-8 (221 mg), Fr. 3-9 (347 mg), Fr. 3-10 (626 mg), Fr. 3-11 (356 mg). Fr. and the organic acid fraction. The obtained compound 2 was identified by
3-6 (26.5044 g) was identified as rosiridin (2, 26.5044 g). Fr. 3-7 (956.3 mg) comparison of their physical data ([a]D, 1H-NMR, 13C-NMR, MS) with iso-
was separated by HPLC [MeOH–H2O (45 : 55, v/v)] to give rosiridoside A lated compound 2. The organic acid fraction was dissolved in (CH2)2Cl2
(3, 10.5 mg). Fraction 5 (15.1 g) was subjected to reversed-phase silica gel (2.0 ml) and the solution was treated with p-nitrobenzyl-N,Nꢁ-diisopyopy-
column chromatography [450 g, MeOH–H2O (20 : 80→30 : 70→50 : 50→
lisourea (10 mg), then stirred at 80 °C for 1 h. The reaction solution was sub-
70 : 30→90 : 10, v/v)→MeOH] to afford nine fractions [Fr. 5-1 (142 mg), Fr. jected to HPLC analysis [column: YMC-pack ODS-A, 250ꢅ4.6 mm i.d.;
5-2 (5021 mg), Fr. 5-3 (860 mg), Fr. 5-4 (761 mg), Fr. 5-5 (6560 mg), Fr. 5-6 mobile phase: CH3CN–H2O (45 : 55, v/v); detection: UV (254 nm); flow
(212 mg), Fr. 5-7 (381 mg), Fr. 5-8 (386 mg), Fr. 5-9 (143 mg)]. Fraction 5-5 rate: 1.0 ml/min] to identify the p-nitrobenzyl ester of acetic acid (tR
(446 mg) was separated by HPLC [MeOH–H2O (35 : 65, v/v)] to give rosiri- 28.5 min).
doside B (4, 17.6 mg).
Rosiridin (2): Obtained as colorless viscous oil; [a]D25 ꢀ32.1° (cꢃ2.06, (35.2 mg) in H2O (2 ml) was treated with b-glucosidase (from Almond, Ori-
MeOH); [a]D22 ꢀ31.3° (cꢃ4.65, acetone); IR (Film) nmax 3450, 1638,
ental Yeast Co., Ltd., Japan, 25 mg) and the solution was stirred at 37 °C for
1045 cmꢀ1; 1H-NMR (CD3OD, 600 MHz) d: 1.62, 1.66, 1.68, (3H each, both 12 h. After EtOH was added to the reaction mixture, the solvent was re-
Enzymatic Hydrolysis of 2 with b-Glucosidase A solution of 2
s, H3-8, 9, 10), 2.23 (2H, t, Jꢃ6.8 Hz, H2-5), 3.97 (1H, t, Jꢃ6.8 Hz, H-4),
4.28 (1H, dd, Jꢃ6.3, 11.7 Hz, H-1a), 4.32 (1H, m, H-1b), 4.30 (1H, d,
Jꢃ7.7 Hz, H-1ꢁ), 5.12 (1H, t, Jꢃ6.8 Hz, H-6), 5.56 (1H, t like, Jꢃca. 7 Hz,
moved under reduced pressure and the residue was purified by HPLC
[MeOH–H2O (45 : 55, v/v)] to furnish (ꢀ)-rosiridol (1, 14.7 mg, 82%).
(ꢀ)-Rosiridol (1): Obtained as colorless viscous oil; [a]D25 ꢀ7.7°
H-2); 13C-NMR data see Table 1; positive-ion FAB-MS m/z: 355 [MꢂNa]ꢂ; (cꢃ1.98, acetone); [a]D22 ꢀ14.2° (cꢃ0.42, CHCl3); IR (Film) nmax 3500,
HR-FAB-MS m/z: 355.1742 (Calcd for C16H28O7Na [MꢂNa]ꢂ, 355.1733).
2971, 1655 cmꢀ1; 1H-NMR (CDCl3, 600 MHz) d: 1.65, 1.69, 1.74, (3H each,
Rosiridoside A (3): Obtained as colorless viscous oil; [a]D27 ꢀ10.4° both s, H3-8, 10, 9), 2.27 (2H, m, H2-5), 4.03 (1H, dd, Jꢃ5.5, 7.6 Hz, H-4),
(cꢃ0.52, MeOH); IR (Film) nmax 3545, 1653, 1040 cmꢀ1
;
1H-NMR 4.23 (2H, dd, Jꢃ3.5, 6.9 Hz H2-1), 5.12 (1H, t-like, Jꢃca. 7 Hz, H-6), 5.67
(CD3OD, 600 MHz) d: 1.62, 1.65, 1.68 (3H each, both s, H3-8, 10, 9), 2.24 (1H, t-like, Jꢃca. 7 Hz, H-2); 13C-NMR data see Table 1; EI-MS m/z: 170
(2H, t like, Jꢃca. 7 Hz, H2-5), 4.23, 4.30 (1H each, both dd, Jꢃ6.8, 12.4 Hz, [M]ꢂ, HR-EI-MS m/z: 170.1300 (Calcd for C10H18O2 [M]ꢂ, 170.1307).
H2-1), 3.96 (1H, t, Jꢃ6.9 Hz, H-4), 4.23 (1H, d, Jꢃ6.8 Hz, H-1ꢁ), 5.10 (1H,
t, Jꢃ6.8 Hz, H-6), 5.56 (1H, t, Jꢃ6.8 Hz, H-2); 13C-NMR data see Table 1;
Enzymatic Hydrolysis of 3 and 4 with Naringinase A solution of 3
(4.9 mg) in 0.1 M acetate buffer (pH 3.8, 2.0 ml) was treated with naringinase
positive-ion FAB-MS m/z: 325 [MꢂNa]ꢂ; HR-FAB-MS m/z: 325.1633 (Sigma Chemical Co., 10 mg), and the solution was stirred at 40 °C for 24 h.
(Calcd for C15H26O6Na [MꢂNa]ꢂ, 325.1627).
Rosiridoside B (4): Obtained as colorless viscous oil; [a]D27 ꢀ73.7° under reduced pressure and the residue was purified by HPLC [MeOH–H2O
(cꢃ0.57, MeOH); IR (Film) nmax 3565, 1634, 1041 cmꢀ1 1H-NMR (45 : 55, v/v)] to furnish (ꢀ)-rosiridol (1, 1.5 mg, 54%). Through a similar
After EtOH was added to the reaction mixture, the solvent was removed
;
(CD3OD, 600 MHz) d: 1.62, 1.67, 1.69 (3H each, both s, H3-8, 10, 9), 2.24 procedure, enzymatic hydrolysis of 4 (7.8 mg) was carried out to afford (ꢀ)-
(2H, t like, Jꢃca. 7 Hz, H2-5), 4.30, 4.32 (1H each, both m, H2-1), 3.96 (1H,
m, H-4), 4.29 (1H, d, Jꢃ7.6 Hz, H-1ꢁ), 4.96 (1H, br s, H-1ꢄ), 5.10 (1H, t,
Jꢃ7.0 Hz,, H-6), 5.56 (1H, t like, Jꢃca. 7 Hz, H-2); 13C-NMR data see
rosiridol (1, 1.8 mg, 63%).
Pivaloylation of (ꢀ)-Rosiridol (1)
A Solution of 1 (337 mg,
1.98 mmol) in pyridine (2.0 ml) was treated with pivaloyl chloride (0.23 ml,
Table 1; positive-ion FAB-MS m/z: 487 [MꢂNa]ꢂ; negative-ion FAB-MS 1.98 mmol) and the mixture was stirred at 0 °C for 2 h. The reaction mixture
m/z: 463 [MꢀH]ꢀ; HR-FAB-MS m/z: 487.2162 (Calcd for C21H36O11Na was poured into water (2.0 ml) and the whole was extracted with EtOAc
[MꢂNa]ꢂ, 487.2155).
(10 ml). The EtOAc extract was successively washed with H2O and brine,
Rosiridoside C (5): Obtained as colorless viscous oil; [a]D25 ꢀ16.7° then dried over Na2SO4 powder and filtered. Removal of the solvent from the

May 2008
699
filtrate under reduced pressure furnished a residue, which was purified by 1.59, 1.69, 1.69, (3H each, both s, H3-8, 10, 9), 1.66 (1H, m, OCH2CHaHb),
normal-phase silica gel column chromatography [n-hexane–EtOAc (2 : 1, 1.90 (1H, m, OCH2CHaHb), 2.10 (1H, dd, Jꢃ7.6, 13.7 Hz, CH2ꢃCH–
v/v)] to give 6 (395 mg, 78%).
CHaHb), 2.10 (2H, m, H-5a), 2.22 (1H, dd, Jꢃ6.9, 14.5 Hz, H-5b), 2.27 (1H,
The 1-Pivalate (6): Obtained as colorless viscous oil; [a]D25 ꢀ7.1° dd, Jꢃ7.6, 13.7 Hz, CH2ꢃCH–CHaHb), 3.65 (1H, dd, Jꢃ7.6, 15.1 Hz,
(cꢃ0.24, acetone); IR (Film) nmax 3500, 2972, 1732, 1655, 1049 cmꢀ1; H-
OCHaHbCH2), 3.74 (1H, m, OCHaHbCH2), 3.98 (1H, t, Jꢃ6.9 Hz, H-4), 4.59
1
NMR (CDCl3, 600 MHz) d: 1.20 (9H, s, (CH3)3CCOO), 1.64, 1.71, 1.72, (2H, m, H2-1), 5.05 (2H, m, CH2ꢃCHCH2), 5.05 (1H, m, H-6), 5.49 (1H, t,
(3H each, both s, H3-8, 10, 9), 2.27 (2H, m, H2-5), 4.04 (1H, dd-like, H-4), Jꢃ6.9 Hz, H-2), 5.85 (1H, m, CH2ꢃCHCH2); 13C-NMR (CDCl3, 150 MHz)
4.62 (2H, d, Jꢃ6.1 Hz, H2-1), 5.09 (1H, t, Jꢃ7.3 Hz, H-6), 5.58 (1H, t,
Jꢃ6.1 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) d: 12.4 (C-10), 18.0 (C-8),
25.9 (C-9), 27.2 [(CH3)3CCOO], 34.1 (C-5), 38.7 [(CH3)3CCOO], 61.0 (C-
1), 76.1 (C-4), 119.5 (C-2), 119.8 (C-6), 135.4 (C-7), 142.3 (C-3), 178.6
d: 12.4 (C-10), 17.9 (C-8), 25.8 (C-9), 27.2 [(CH3)3CCOO], 33.7 (C-5),
36.3, 34.7, 21.4 (CCH2CH2CH2), 38.3 (OCH2CH2), 38.7 [(CH3)3CCOO],
40.4 (CH2ꢃCHCH2), 54.7 (OCH2CH2C), 61.0 (C-1), 66.1 (OCH2CH2), 78.7
(C-4), 116.6 (CH2ꢃCH), 117.9 [OCO(C)(CH2)], 118.5 (C-2), 120.8 (C-6),
[(CH3)3CCOO]; positive-ion FAB-MS m/z: 277 [MꢂNa]ꢂ; HR-FAB-MS 132.8 (C-7), 136.9 (CH2ꢃCH), 143.8 (C-3), 178.5 [(CH3)3CCOO]; EI-MS
m/z: 277.1786 (Calcd for C15H26O3Na [MꢂNa]ꢂ, 277.1780).
m/z: 404 [M]ꢂ; HR-EI-MS m/z: 404.2917 (Calcd for C25H40O4 [M]ꢂ,
Oxidation of 6 A solution of 6 (395 mg, 1.56 mmol) in pyridine (3 ml) 404.2926).
was treated with CrO3 (315 mg, 3.15 mmol) and the mixture was stirred at rt
Treatment of 9 and 10 with p-TsOH A solution of 9 (31.0 mg,
for 2 h. The reaction mixture was poured into water (10 ml) and the whole 0.077 mmol) in MeOH (1 ml) was treated with p-TsOH (1 mg), and the mix-
was extracted with EtOAc (10 ml). The EtOAc extract was washed with H2O ture was stirred at rt for 2 h. The reaction mixture was poured into saturated
and brine, then dried over Na2SO4 powder and filtered. Removal of the aqueous solution of sodium hydrogen carbonate and extracted with EtOAc
solvent from the filtrate under reduced pressure furnished a residue, which (10 ml). The EtOAc extract was washed with H2O and brine, then dried over
was purified by normal-phase silica gel column chromatography [n- Na2SO4 powder and filtered. Removal of the solvent under reduced pressure
hexane–EtOAc (50 : 1→30 : 1, v/v)] to give 7 (117 mg, 30%).
gave a residue, which was purified by normal-phase silica gel column chro-
matography [n-hexane–EtOAc (2 : 1, v/v)] to give synthetic 6 (18.8 mg,
The 4-Ketone (7): Obtained as colorless viscous oil; IR (Film) nmax 1734,
1684, 1653, 1076 cmꢀ1 1H-NMR (CDCl3, 600 MHz) d: 1.24 (9H, s, 97%). Through a similar procedure, 11 (20.4 mg, 87%) was obtained from
;
(CH3)3CCOO), 1.59, 1.75, 1.83 (3H each, both s, H3-8, 10, 9), 3.40 (2H, d, 10 (37.2 mg, 0.092 mmol).
Jꢃ7.4 Hz, H2-5), 4.79 (2H, d, Jꢃ6.1 Hz, H2-1), 5.29 (1H, t, Jꢃ7.4 Hz, H-6),
Synthetic 6: Obtained as colorless viscous oil; IR (Film) nmax 3502, 2972,
6.57 (1H, t, Jꢃ6.1 Hz, H-2); EI-MS m/z: 252 [M]ꢂ; HR-EI-MS m/z: 1730, 1655, 1049 cmꢀ1; [a]D25 ꢀ5.9° (cꢃ0.45, acetone); The proton and car-
252.1731 (Calcd for C15H24O3 [M]ꢂ, 252.1725).
bon signals of H- and 13C-NMR were completely the same values as those
of 6; positive-ion FAB-MS m/z: 277 [MꢂNa]ꢂ; HR-FAB-MS m/z: 277.1784
(Calcd for C15H26O3Na [MꢂNa]ꢂ, 277.1780).
1
Reduction of 7 A solution of 7 (117 mg, 0.46 mmol) in methanol (2 ml)
was treated with NaBH4 (17.4 mg, 0.46 mmol) and CeCl3 7H2O (17.3 mg,
0.05 mmol), and the mixture was stirred at rt for 1 h. The reaction mixture
Compound 11: Obtained as colorless viscous oil; [a]D25 ꢂ4.4° (cꢃ0.45,
was quenched in acetone, and then removal of the solvent under reduced acetone); IR (Film) nmax 3503, 2973, 1730, 1655, 1049 cmꢀ1 1H-NMR
;
pressure gave a residue, which was purified by normal-phase silica gel col- (CDCl3, 600 MHz) d: 1.20 (9H, s, (CH3)3CCOO), 1.65, 1.72, 1.73, (3H
umn chromatography [n-hexane–EtOAc (30 : 1, v/v)] to give 8 (112 mg,
96%).
each, both s, H3-8, 10, 9), 2.27 (2H, m, H2-5), 4.04 (1H, dd, Jꢃ6.4, 6.8 Hz,
H-4), 4.62 (2H, d, Jꢃ6.4 Hz, H2-1), 5.09 (1H, t, Jꢃ7.3 Hz, H-6), 5.59 (1H, t,
Jꢃ6.4 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) d: 12.4 (C-10), 18.0 (C-8),
The 4-Enantiomeric mixture (8). Obtained as colorless viscous oil; [a]D24
ꢂ0.14° (cꢃ1.47, acetone); IR (Film) nmax 3500, 2971, 1655, 1048 cmꢀ1; 1H- 25.9 (C-9), 27.2 [(CH3)3CCOO], 34.1 (C-5), 38.7 [(CH3)3CCOO], 60.9 (C-
NMR (CDCl3, 600 MHz) d: 1.20 (9H, s, (CH3)3CCOO), 1.64, 1.71, 1.72 1), 76.1 (C-4), 119.5 (C-2), 119.8 (C-6), 135.5 (C-7), 142.3 (C-3), 178.5
(3H each, both s, H3-8, 10, 9), 2.27 (2H, dd like, H2-5), 4.04 (1H, t,
Jꢃ6.4 Hz, H-4), 4.62 (2H, d, Jꢃ6.6 Hz, H2-1), 5.09 (1H, t-like, Jꢃca. 7 Hz,
H-6), 5.58 (1H, t, Jꢃ6.6 Hz, H-2); positive-ion FAB-MS m/z: 277
[(CH3)3CCOO]; positive-ion FAB-MS m/z: 277 [MꢂNa]ꢂ; HR-FAB-MS
m/z: 277.1776 (Calcd for C15H26O3Na [MꢂNa]ꢂ, 277.1780).
Deacylation of 11 A solution of 11 (15.0 mg, 0.059 mmol) in 1%
[MꢂNa]ꢂ; HR-FAB-MS m/z: 277.1776 (Calcd for C15H26O3Na [MꢂNa]ꢂ, NaOMe–MeOH (1.0 ml) was stirred at rt for 12 h. The reaction mixture was
277.1780).
neutralized with Dowex HCR-W2 (Hꢂ form) and the resin was removed by
filtration. Evaporation of the solvent from the filtrate under reduced pressure
Isolations of 9 and 10 by Chiral Resolution A solution of 8 (69.0 mg,
0.27 mmol) in CH2Cl2 (2.0 ml) was treated with (5S)-allyl-2-oxabicy- gave a residue, which was purified by normal-phase silica gel column chro-
clo[3.3.0]oct-8-ene (ALBO, 0.04 ml, 0.326 mmol) and p-TsOH (1 mg), and matography [CHCl3→CHCl3–MeOH (10 : 1, v/v)] to give (ꢂ)-rosiridol (12,
the mixture was stirred slowly from 0 °C to rt for 1 h. The reaction mixture 3.2 mg, 32%).
was poured into saturated aqueous solution of sodium hydrogen carbonate
(ꢂ)-Rosiridol (12): Obtained as colorless viscous oil; [a]D25 ꢂ6.1°
and extracted with EtOAc (10 mlꢅ3). The EtOAc extract was successively (cꢃ0.32, acetone); [a]2D2 ꢂ14.1° (cꢃ0.32, CHCl3); IR (Film) nmax 3500,
washed with H2O and brine, then dried over Na2SO4 powder and filtered. 2975, 1655 cmꢀ1; 1H-NMR (CDCl3, 600 MHz) d: 1.65, 1.69, 1.74 (3H each,
Removal of the solvent under reduced pressure gave a residue, which was
both s, H3-8, 10, 9), 2.27 (2H, m, H2-5), 4.03 (1H, dd, Jꢃ5.5, 7.6 Hz, H-4),
purified by normal-phase silica gel column chromatography [n- 4.23 (2H, dd, Jꢃ3.4, 6.9 Hz, H2-1), 5.12 (1H, t-like, Jꢃca. 7 Hz, H-6), 5.67
hexane–EtOAc (100 : 1→90 : 1→70 : 1, v/v)] to give 9 (42.8 mg, 21%) and
10 (42.6 mg, 39%).
(1H, t-like, Jꢃca. 7 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) d: 12.2 (C-10),
18.0 (C-8), 25.9 (C-9), 34.2 (C-5), 59.2 (C-1), 76.3 (C-4), 124.5 (C-2), 119.7
Compound 9: Obtained as colorless viscous oil; [a]D24 ꢀ25.9° (cꢃ1.28, (C-6), 135.4 (C-7), 140.4 (C-3); EI-MS m/z: 170 [M]ꢂ, HR-EI-MS m/z:
acetone); IR (Film) nmax 1732, 1653, 1047 cmꢀ1
;
1H-NMR (CDCl3, 170.1300 (Calcd for C10H18O2 [M]ꢂ, 170.1307).
Preparation of the (S)- and (R)-MTPA Esters (6a, 6b) with MTPA-Cl
600 MHz) d: 1.18 (9H, s, (CH3)3CCOO), 1.43—1.56 (6H, m, CH2CH2CH2),
1.61, 1.68, 1.68, (3H each, both s, H3-8, 10, 9), 1.68 (1H, m, OCH2CHaHb), A Solution of 6 and synthetic 6 (each 2.0 mg, 0.008 mmol) in pyridine (1 ml)
1.93 (1H, m, OCH2CHaHb), 2.07 (1H, dd, Jꢃ7.6, 13.7 Hz, CH2ꢃCH– was each treated with (ꢀ)-MTPA-Cl (both 0.01 ml, 0.066 mmol), and the
CHaHb), 2.09 (2H, m, H-5a), 2.24 (1H, dd, Jꢃ7.6, 14.5 Hz, H-5b), 2.30 (1H,
mixture was stirred at rt for 6 h. The reaction mixture was poured into water
dd, Jꢃ6.8, 13.7 Hz, CH2ꢃCH–CHaHb), 3.76 (1H, dd like, Jꢃ8.2, 15.8 Hz, (1 ml) and the whole was extracted with EtOAc (6 ml). The EtOAc extract
OCHaHbCH2), 3.81 (1H, ddd, Jꢃ4.1, 7.6, 15.8 Hz, OCHaHbCH2), 4.14 (1H, was washed with H2O and brine, then dried over Na2SO4 powder and fil-
dd, Jꢃ6.2, 7.7 Hz, H-4), 4.55 (1H, dd, Jꢃ6.8, 12.4 Hz, H-1a), 4.61 (1H, dd,
Jꢃ7.6, 12.4 Hz, H-1b), 5.03 (2H, m, CH2ꢃCHCH2), 5.08 (1H, dd-like, H-
tered. Removal of the solvent from the filtrate under reduced pressure fur-
nished a residue, which was purified by normal-phase silica gel column
6), 5.49 (1H, dd, J ꢃ6.8, 7.6 Hz, H-2), 5.85 (1H, m, CH2ꢃCHCH2); 13C- chromatography [n-hexane–EtOAc (30 : 1, v/v)] to give (S)-MTPA ester de-
NMR (CDCl3, 150 MHz) d: 12.0 (C-10), 17.9 (C-8), 21.6, 34.8, 36.4 rivative (6a, 4.1 mg, 79% from 6; 2.4 mg, 65% from synthetic 6). Through a
(CCH2CH2CH2), 25.8 (C-9), 27.2 [(CH3)3CCOO], 34.1 (C-5), 38.2 similar procedure, the (R)-MTPA ester derivative (6b, 3.1 mg, 69% from 6;
(OCH2CH2), 38.7 [(CH3)3CCOO], 40.4 (CH2ꢃCHCH2), 54.5 (OCH2CH2C), 2.7 mg, 73% from synthetic 6) was obtained from 6 (2.5 mg, 0.010 mmol)
60.8 (C-1), 65.8 (OCH2CH2), 77.9 (C-4), 116.7 (CH2ꢃCH), 117.6 and synthetic 6 (2.1 mg, 0.008 mmol), respectively, using (ꢂ)-MTPA-Cl.
[OCO(C)(CH2)], 120.0 (C-2), 121.1 (C-6), 132.6 (C-7), 136.9 (CH2ꢃCH),
143.6 (C-3), 178.4 [(CH3)3CCOO]; EI-MS m/z: 404 [M]ꢂ; HR-EI-MS m/z: (3H, s, H3-8), 1.63 (3H, s, H3-9), 1.72 (3H, s, H3-10), 2.28 (1H, m, H-5a),
404.2932 (Calcd for C25H40O4 [M]ꢂ, 404.2926).
2.42 (1H, m, H-5b), 4.61 (2H, dd-like, H2-1), 4.92 (1H, m, H-6), 5.38 (1H,
Compound 10: Obtained as colorless viscous oil; [a]D25 ꢂ15.0° (cꢃ0.07, dd-like, H-4), 5.67 (1H, t-like, H-2); 13C-NMR (CDCl3, 150 MHz) d: 12.6
acetone); IR (Film) nmax 1732, 1653, 1048 cmꢀ1 1H-NMR (CDCl3, (C-10), 17.8 (C-8), 25.7 (C-9), 31.4 (C-5), 60.4 (C-1), 80.7 (C-4), 118.1 (C-
600 MHz) d: 1.19 (9H, s, (CH3)3CCOO), 1.47—2.06 (6H, m, CH2CH2CH2), 6), 123.8 (C-2), 135.2 (C-7), 137.4 (C-3).
(S)-MTPA Ester Derivative (6a): 1H-NMR (CDCl3, 600 MHz) d: 1.54
;

700
(R)-MTPA Ester Derivative (6b): 1H-NMR (CDCl3, 600 MHz) d: 1.59
(3H, s, H3-8), 1.60 (3H, s, H3-10), 1.70 (3H, s, H3-9), 2.32 (1H, m, H-5a),
2.49 (1H, m, H-5b), 4.58 (2H, dd-like, H2-1),5.04 (1H, t-like, H-6), 5.34
(1H, dd-like, H-4), 5.60 (1H, t-like, H-2); 13C-NMR (CDCl3, 150 MHz) d:
12.3 (C-10), 17.9 (C-8), 25.8 (C-9), 31.5 (C-5), 60.4 (C-1), 80.9 (C-4), 118.5
(C-6), 123.5 (C-2), 135.3 (C-7), 137.3 (C-3).
Vol. 56, No. 5
3) Zhang Y., Morikawa T., Nakamura S., Ninomiya K., Matsuda H.,
Muraoka O., Yoshikawa M., Heterocycles, 71 1565—1576 (2007).
4) Morikawa T., Zhang Y., Nakamura S., Matsuda H., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 55, 435—441 (2007).
5) Ninomiya K., Morikawa T., Zhang Y., Nakamura S., Matsuda H., Mu-
raoka O., Yoshikawa M., Chem. Pharm. Bull., 55, 1185—1191 (2007).
6) Yoshikawa M., Wang T., Morikawa T., Xie H., Matsuda H., Chem.
Pharm. Bull., 55, 1308—1315 (2007).
Preparation of the (S)- and (R)-MTPA Esters (6a, 6b) with MTPA
A
Solution of 6 (3.0 mg, 0.012 mmol) in CH2Cl2 (1 ml) was added (S)-MTPA
(5.5 mg, 0.025 mmol), EDC (9.0 mg, 0.047 mmol), and 4-DMAP (3.5 mg,
0.028 mmol). The mixture was stirred at rt for 12 h. The reaction mixture
was poured into water (1 ml) and the whole was extracted with EtOAc
(6 ml). The EtOAc extract was successively washed with H2O and brine,
then dried over Na2SO4 powder and filtered. Removal of the solvent from the
filtrate under reduced pressure furnished a resudue, which was purified by
normal-phase silica gel column chromatography [n-hexane–EtOAc (30 : 1,
v/v)] to give (S)-MTPA ester derivative 6a (0.9 mg, 16%). Through a similar
procedure, the (R)-MTPA ester derivative (6b, 1.0 mg, 18%) was obtained
from 6 (3.0 mg, 0.012 mmol) using (R)-MTPA.
7) Yoshikawa M., Morikawa T., Zhang Y., Nakamura S., Muraoka O.,
Matsuda H., J. Nat. Prod., 70, 575—583 (2007).
8) Yoshikawa M., Matsuda H., Morikawa T., Xie H., Nakamura S.,
Muraoka O., Bioorg. Med. Chem., 14, 7468—7475 (2006).
9) Xie H., Morikawa T., Matsuda H., Nakamura S., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 54, 669—675 (2006).
10) Morikawa T., Xie H., Matsuda H., Yoshikawa M., J. Nat. Prod., 69,
881—886 (2006).
11) Morikawa T., Xie H., Matsuda H., Wang T., Yoshikawa M., Chem.
Pharm. Bull., 54, 506—513 (2006).
12) Yoshikawa M., Shimada H., Shimoda H., Murakami N., Yamahara J.,
Matsuda H., Chem. Pharm. Bull., 44, 2086—2091 (1996).
13) Yoshikawa M., Shimada H., Shimoda H., Matsuda H., Yamahara J.,
Murakami N., Chem. Pharm. Bull., 43, 1245—1247 (1995).
14) Yoshikawa M., Shimada H., Horikawa S., Murakami T., Shimoda H.,
Yamahara J., Matsuda H., Chem. Pharm. Bull., 45, 1498—1503
(1997).
Preparation of the (S)- and (R)-MTPA Esters (11a, 11b) with MTPA-
Cl (S)- and (R)-MTPA esters, 11a (2.8 mg, 54%) and 11b (3.9 mg, 72%),
were each obtained from 11 (2.5 mg, 0.010 mmol for 11a, 2.9 mg,
0.011 mmol for 11b) by using the similar procedure when 6a and 6b were
given.
(S)-MTPA Ester Derivative (11a): 1H-NMR (CDCl3, 600 MHz) d: 1.60
(3H, s, H3-8), 1.62 (3H, s, H3-10), 1.70 (3H, s, H3-9), 2.32 (1H, m, H-5a),
2.49 (1H, m, H-5b), 4.57 (2H, dd, Jꢃ6.9, 9.7 Hz, H2-1), 5.04 (1H, t-like, H-
6), 5.33 (1H, dd, Jꢃ5.5, 8.3 Hz, H-4), 5.60 (1H, t, Jꢃ6.9 Hz, H-2); 13C-
NMR (CDCl3, 150 MHz) d: 12.3 (C-10), 17.9 (C-8), 25.8 (C-9), 31.5 (C-5),
60.4 (C-1), 80.9 (C-4), 118.5 (C-6), 123.5 (C-2), 135.3 (C-7), 137.4 (C-3).
(R)-MTPA Ester Derivative (11b): 1H-NMR (CDCl3, 600 MHz) d: 1.54
(3H, s, H3-8), 1.63 (3H, s, H3-9), 1.72 (3H, s, H3-10), 2.28 (1H, m, H-5a),
2.42 (1H, m, H-5b), 4.61 (2H, dd-like, H2-1), 4.92 (1H, t-like, H-6), 5.38
(1H, dd, Jꢃ6.2, 7.6 Hz, H-4), 5.67 (1H, t-like, H-2); 13C-NMR (CDCl3,
150 MHz) d: 12.6 (C-10), 17.8 (C-8), 25.7 (C-9), 31.4 (C-5), 60.4 (C-1),
80.7 (C-4), 118.1 (C-6), 123.8 (C-2), 135.2 (C-7), 137.4 (C-3).
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1
21) The H- and 13C-NMR spectra of 1—5 were assigned with the aid of
Acknowledgments This research was supported by the 21st COE Pro-
gram, Academic Frontier Project, and a Grant-in Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
distortionless enhancement by polarization transfer (DEPT), double
quantum filter correlation spectroscopy (DQF COSY), heteronuclear
multiple quantum coherence (HMQC), and heteronuclear multiple
bond connectivity (HMBC) experiments.
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References and Notes
1) This paper is number 30 in the series “Bioactive Constituents from
Chinese Natural Medicines”. For paper number 29, see: Li X., Naka-
mura S., Matsuda H., Yoshikawa M., Chem. Pharm. Bull., 56, 612—
615 (2008).
24) Murakami T., Nakamura J., Kageura T., Matsuda H., Yoshikawa M.,
Chem. Pharm. Bull., 48, 1720—1725 (2000).
2) Morikawa T., Xie H., Wang T., Matsuda H., Yoshikawa M., Helv.
Chim. Acta, in press (2008).