Medicinal foodstuffs. XXIV. Chemical constituents of the processed leaves of Apocynum venetum L.: Absolute stereostructures of apocynosides I and II

Article abstract of DOI:10.1248/cpb.49.845

Two new ionone glucosides, named apocynosides I and II, were isolated from the roasted leaves of Apocynum venetum L. together with nine known compounds. The absolute stereostructures of apocynosides I and II were determined by chemical and physicochemical evidence, which included the application of a modified Mosher's method and the circular dichroism helicity rule.

Full text of DOI:10.1248/cpb.49.845

July 2001
Chem. Pharm. Bull. 49(7) 845—848 (2001)
845
Medicinal Foodstuffs. XXIV.1) Chemical Constituents of the Processed
Leaves of Apocynum venetum L.: Absolute Stereostructures of
Apocynosides I and II
Toshiyuki MURAKAMI,a Akinobu KISHI,a Hisashi MATSUDA,a Masao HATTORI,b and
,a
*
Masayuki YOSHIKAWA
Kyoto Pharmaceutical University,a Misasagi, Yamashina-ku, Kyoto 607–8412, Japan and Institute of Natural Medicine,
Toyama Medical and Pharmaceutical University,b 2630 sugitani, Toyama 930–0194, Japan.
Received January 23, 2001; accepted March 12, 2001
Two new ionone glucosides, named apocynosides I and II, were isolated from the roasted leaves of Apocynum
venetum L. together with nine known compounds. The absolute stereostructures of apocynosides I and II were
determined by chemical and physicochemical evidence, which included the application of a modified Mosher’s
method and the circular dichroism helicity rule.
Key words apocynoside; Apocynum venetum; ionone glucoside; absolute stereostructure; modified Mosher’s method; circular
dichroism helicity rule
The Apocynaceae plant Apocynum (A.) venetum L. (Chi- a fragment ion peak at m/z 223 (MϪC6H11O5)Ϫ, which was
nese name “羅布麻”) is used for the treatment of cardiac dis- derived by cleavage of the glycoside linkage at the 1Ј-posi-
eases, hypertension, and hepatitis. Recently, the roasted tion, was observed in the negative-ion FAB-MS. By acid hy-
leaves of this plant have been used as a health food in Japan. drolysis with 5% aqueous sulfuric acid (H2SO4)–1,4-dioxane,
As chemical constituents of this plant, many phenolic com- 1 liberated D-glucose, which was identified by GLC analysis
pounds have been isolated from the leaves of A. venetum.2)
of the trimethylsilyl thiazolidine derivatives.10) Enzymatic hy-
In the course of our continuing studies on the bioactive drolysis of 1 with b-glucosidase yielded an aglycone called
constituents of medicinal foodstuffs,1,3) we have isolated two apocynol A (3), whose molecular formula, C13H20O3, was de-
new ionone glucosides, called apocynosides I (1) and II (2), termined from the positive-ion FAB-MS [m/z 225 (MϩH)ϩ]
together with nine known compounds from the roasted leaves and by high-resolution MS measurement. The 1H-NMR
of A. venetum L. This paper describes the isolation and struc- (CDCl3) and 13C-NMR (Table 1) spectra of 3, which were as-
tural elucidation of apocynosides I (1) and II (2).
signed by various NMR analytical methods,11) showed the
The roasted leaves of A. venetum L. were extracted with presence of two singlet methyls [d 0.99, 1.04 (both s, 13, 12-
methanol under reflux. The methanol extract was partitioned H3)], a doublet methyl [d 1.28 (d, Jϭ6.4 Hz, 10-H3)], a me-
into an ethyl acetate–water mixture to furnish the ethyl ac- thine and methylene bearing a hydroxyl group [d 4.34 (dd-
etate-soluble portion and water phase. The water phase was like, 9-H), 4.17, 4.23 (both d, Jϭ17.1 Hz, 11-H2)], a disubsti-
further extracted with 1-butanol to give a 1-butanol-soluble tuted olefin [d 5.67 (dd, Jϭ8.5, 15.6 Hz, 7-H), 5.60 (dd,
portion and a water-soluble portion. The ethyl acetate-soluble Jϭ6.5, 15.6 Hz, 8-H)], and an enone function [d 6.18 (s, 4-
portion was subjected to normal- and reversed-phase silica H)] together with a methine [d 2.59 (d, Jϭ8.5 Hz, 6-H)], a
gel column chromatography and HPLC to give quercetin methylene [d 2.13, 2.41 (both d, Jϭ16.8 Hz, 2-H2)], and a
(0.014%), phytol4) (0.0003%), and lupeol5) (0.012%). The 1- quaternary carbon [dC 36.2 (1-C)]. On the other hand, the
butanol-soluble portion was subjected to normal-phase silica 1H-NMR (pyridine-d5) and 13C-NMR (Table 1) spectra of 1
gel column chromatography to give seven fractions. Fraction showed signals due to an apocynol A moiety, together with a
4 was separated by reversed-phase silica gel column chro- b-D-glucopyranosyl part [d 4.84 (d, Jϭ7.6 Hz, 1Ј-H)]. In the
matography and repeated HPLC to give apocynosides I (1, HMBC experiment on 1, long-range correlations were ob-
0.0002%) and II (2, 0.0004%), together with phenethyl alco- served between the following protons and carbons: 2-H2 and
hol xylopyranosyl(1→6)glucopyranoside6) (0.0006%), Z-hex- 1, 3-C, 11-H2 and 4, 5, 6-C, 12, 13-H3 and 1, 2, 6-C, 10-H3
3-en-1-ol xylopyranosyl(1→6)glucopyranoside7) (0.0008%), and 8, 9-C, 8-H and 6, 7, 9-C, 1Ј-H and 11-C (Fig. 1). By
isoquercitrin8) (0.0005%), and kaempferol 3-O-b-D-galac- comparison of the 13C-NMR data for 1 with those for 3, a
topyranoside9) (0.0001%). Fraction 5 was subjected to re-
versed-phase silica gel column chromatography and purified
by HPLC to give benzyl alcohol xylopyranosyl(1→6)glu-
copyranoside6) (0.0007%) and hyperoside9) (0.0002%).
Apocynoside I (1) was isolated as an amorphous powder
with positive optical rotation ([a]D27 ϩ79.2°). The IR spec-
trum of 1 showed absorption bands assignable to hydroxyl
and enone functions at 3432, 1655, 1619, and 1076 cmϪ1. In
the negative- and positive-ion FAB-MS of 1, quasimolecular
ion peaks were observed at m/z 385 (MϪH)Ϫ and m/z 387
(MϩH)ϩ, and the molecular formula C19H30O8 of 1 was de-
termined by high-resolution MS measurement. Furthermore,
Chart 1
To whom correspondence should be addressed. e-mail: shoyaku@mb.kyoto-phu.ac.jp
© 2001 Pharmaceutical Society of Japan

846
Vol. 49, No. 7
Table 1. 13C-NMR Data of Apocynosides I (1) and II (2) and Apocynols A
(3) and B (4)
1a)
2a)
3b)
4a)
1
2
36.1
48.6
42.0
50.3
36.2
48.1
42.0
50.5
3
4
5
6
198.2
124.0
160.9
51.0
198.1
124.1
163.3
78.0
199.3
122.4
163.7
50.8
198.2
122.8
168.5
78.7
7
8
9
141.0
125.4
62.4
129.1
137.6
67.4
138.6
125.4
68.1
129.6
135.8
67.4
Fig. 1. HMBC and 1H–1H COSY Correlations of 1 and 2
10
11
12
13
Glc-1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
24.5
69.3
27.6
27.1
103.8
75.2
78.6
71.7
78.6
24.6
69.3
24.2
23.5
103.9
75.1
78.5
71.7
78.4
13.5
63.9
27.7
27.1
24.6
61.0
24.1
23.6
62.8
62.6
a) 68 MHz, pyridine-d5, b) 125 MHz, CDCl3.
Fig. 2
glycosidation shift was observed around the 11-carbon in 1.
1
These findings and comparisons of the H-NMR and 13C-
NMR spectra for 1 with those for known related ionone glu-
cosides12) led us to formulate the 11-O-b-D-glucopyranosyl-
9,11-dihydroxyionol structure of 1. The relative stereostruc-
1
ture of 1 was characterized by H-NMR nuclear Overhauser
and exchange spectroscopy (NOESY). Namely, NOE corre-
lations were observed between the 12-protons and the 2a and
6-protons and between the 13-protons and the 2b and 7-pro-
tons.
Fig. 3
In order to clarify the absolute stereostructure of the 9-po- while an aglycone, apocynol B (4), was obtained by enzy-
sition in 1, the aglycone (3) was subjected to a modified matic hydrolysis. The 1H-NMR (CDCl3) and 13C-NMR
Mosher’s method.13) Namely, 3 was treated with (R)- and (S)- (Table 1) spectra11) of 4 showed signals assignable to two sin-
a-methoxy-a-trifluoromethylphenyl acetate (MTPA) in the glet methyls [d 1.15, 1.34 (both s, 13, 12-H3)], a doublet
presence of 1-ethyl-3-(3-dimethylaminopropyl)cardoiimide methyl [d 1.39 (d, Jϭ6.4 Hz, 10-H3)], an olefin [d 6.38 (dd,
(EDC·HCl) and 4-dimethylaminopyridine (DMAP) to give Jϭ3.1, 12.8 Hz, 8-H), 6.39 (d, Jϭ12.8 Hz, 7-H)], a methyl-
the 9,11-di-(R)-MTPA ester (3a) and the 9,11-di-(S)-MTPA ene and methine bearing a hydroxyl group [d 4.67 (dd,
ester (3b), respectively. As shown in Fig. 2, signals due to Jϭ3.1, 6.4 Hz, 9-H), 4.81, 5.07 (both d, Jϭ18.6 Hz, 11-H2)],
protons attached to the 7 and 8-positions in 3a were observed and an enone function [d 7.00 (s, 4-H)], together with a
at lower fields (Dd: negative) compared to those of 3b, while methylene [d 2.50, 2.74 (both d, Jϭ16.8 Hz, 2-H2)] and two
the signal due to the 10-position in 3a was observed at a quaternary carbons (dC 42.0, 67.4). On the other hand, the
higher field (Dd: positive) compared to that of 3b. Conse- 1H-NMR (pyridine-d5) and 13C-NMR (Table 1) spectra11) of 2
quently, the absolute configuration at the 9-position was elu- indicated the presence of an apocynol B moiety and a b-D-
cidated as R. Finally, the absolute stereostructure of the 6-po- glucopyranosyl part [d 4.85 (d, Jϭ7.6 Hz, 1Ј-H)]. The proton
sition in 1 was determined by application of the circular and carbon signals in the 1H- and 13C-NMR spectra of 2 and
dichroism (CD) helicity rule.14) That is, since the CD spec- 4 were superimposable on those of 1 and 3, respectively, ex-
trum of 1 showed a positive Cotton effect at 239 nm, the 6- cept for same signals around the 6-position. The connectivi-
position was determined to be an R configuration. On the ties of the quaternary carbons at the 1 and 10-positions and
basis of the above evidence, the absolute stereostructure of the position of the glucosidic linkage were clarified by an
apocynoside I (1) was elucidated.
HMBC experiment with 2. Namely, HMBC correlations
Apocynoside II (2), obtained as an amorphous powder were observed between the following protons and carbons: 2-
with positive optical rotation ([a]D26 ϩ25.1°), showed absorp- H2 and 1, 3-C, 11-H2 and 4, 5, 6-C, 12, 13-H3 and 1, 2, 6-C,
tion bands due to hydroxyl and enone functions in its IR 10-H3 and 8, 9-C, 8-H and 6, 7, 9-C, 1Ј-H and 11-C (Fig. 1).
spectrum. The molecular formula C19H30O9 of 2 was deter- To clarify the absolute configuration of the 6 and 9-positions,
mined from the negative- and positive-ion FAB-MS [m/z 401 2 and 4 were subjected to the application of a modified
(MϪH)Ϫ, m/z 425 (MϩNa)ϩ] and by high-resolution MS Mosher’s method and the CD helicity rule. As shown in Fig.
1
measurement. Acid hydrolysis of 2 liberated D-glucose,11) 3, signals due to the 7 and 8-protons in the H-NMR spec-

July 2001
847
MeOH
max
trum of the (S)-MTPA ester (4b) were observed at higher ϩ10.0 (234) (positive max.), Ϫ0.68 (326) (negative max). UV l
nm
(log e): 325 (3.1), 234 (3.8). IR (KBr): 3432, 1655, 1638, 1076 cmϪ1. H-
NMR (270 MHz, pyridine-d5) d: 1.11 (3H, s, 13-H3), 1.24 (3H, s, 12-H3),
1.42 (3H, d, Jϭ6.3 Hz, 10-H3), 2.41 (1H, d, Jϭ16.5 Hz, 2a-H), 2.68 (1H, d,
Jϭ16.5 Hz, 2b-H), 3.84 (1H, m, 5Ј-H), 4.04 (1H, t-like, 2Ј-H), 4.17 (1H, m,
3Ј-H), 4.19 (1H, m, 4Ј-H), 4.28 (1H, dd, Jϭ5.0, 11.8 Hz), 4.42 (1H, dd,
Jϭ5.0, 11.8 Hz) (6Ј-H2), 4.64 (1H, dd, Jϭ4.7, 6.3 Hz, 9-H), 4.85 (1H, d,
Jϭ7.6 Hz, 1Ј-H), 4.83, 5.20 (1H each, both d, Jϭ16.5 Hz, 11-H2), 6.36 (1H,
dd, Jϭ4.7, 15.5 Hz, 8-H), 6.27 (1H, d, Jϭ15.5 Hz, 7-H), 6.93 (1H, s, 4-H).
13C-NMR (68 MHz, pyridine-d5) dC: given in Table 1. Negative-ion FAB-
MS: m/z 401 (MϪH)Ϫ. Positive-ion FAB-MS: m/z 425 (MϩNa)ϩ.
1
fields compared to those for the (R)-MTPA ester (4a) (Dd:
negative), while the signal due to the 10-protons of the (S)-
MTPA was observed at a lower field than that for the (R)-
MTPA (Dd: positive). Furthermore, the CD spectrum of 2
showed a positive Cotton effect at 237 nm. On the basis of
the above evidence, the 6 and 9-positions were determined to
be 6S and 9R configurations, and the absolute stereostructure
of apocynoside II (2) was characterized as shown.
Acid Hydrolysis of Apocynosides I (1) and II (2) A solution of 1 and
2 (3 mg each) in 5% aq. H2SO4–1,4-dioxane (1 : 1, v/v, 2 ml) was heated
under reflux for 1 h. After cooling, the reaction mixture was neutralized with
Amberlite IRA-400 (OHϪ form), and the residue was removed by filtration.
After removal of the solvent from the filtrate in vacuo, the residue was trans-
ferred to a Sep-Pak C18 cartridge with H2O and MeOH. The H2O eluate was
concentrated and the residue was treated with L-cysteine methyl ester hy-
drochloride (4 mg) in pyridine (0.5 ml) at 60 °C for 1 h. After reaction, the
solution was treated with N,O-bis(trimethylsilyl)trifluoroacetamide (0.2 ml)
at 60 °C for 1 h. The supernatant was then subjected to GLC analysis to
identify the derivatives of D-glucose; GLC conditions: column: Supeluco
STBTM-1, 30 mϫ0.25 mm (i.d.) capillary column, column temperature:
230 °C, He flow rate: 15 ml/min, tR: 24.2 min.
Enzymatic Hydrolysis of Apocynosides I (1) and II (2) Give Apocynols
A (3) and B (4) A solution of 1 or 2 (10 mg each) in 0.2 M acetate buffer
(pH 4.4, 2 ml) was treated with b-glucosidase (Oriental Yeast Co., Ltd.,
10 mg) and stirred at 38 °C for 24 h. After EtOH was added to the reaction
mixture, the solvent was removed in vacuo. The crude product was purified
by normal-phase silica gel column chromatography [1 g, CHCl3–MeOH–
H2O (15 : 3 : 1, lower layer)] to give 3 (5.7 mg, 98%) and 4 (4.6 mg, 74%).
Apocynol A (3): An amorphous powder, [a]D27 ϩ289.9° (cϭ0.2, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C13H21O3 (MϩH)ϩ:
Experimental
The instruments used to obtain physical data and the experimental condi-
tions for chromatography were the same as described in our previous paper.1)
Isolation of Apocynosides I (1) and II (2) and Known Compounds
from the Processed Leaves of A. venetum L. The roasted leaves of A.
venetum L. (17 kg, provided from Toyama Chemical Co., Ltd. and cultivated
and processed in China) were cut and extracted three times with MeOH
under reflux. Evaporation of the solvent under reduced pressure provided an
MeOH extract (2.6 kg, 15.3%), and the extract (2.6 kg) was partitioned into
the AcOEt–H2O (1 : 1) mixture. The H2O phase was further extracted with
n-BuOH. Removal of the solvent under reduced pressure from the AcOEt
and n-BuOH-soluble portion yielded 1.1 kg and 328 g of residue, respec-
tively. The AcOEt-soluble portion (374.3 g) was subjected to normal-phase
silica gel column chromatography [3.6 kg, n-hexane–AcOEt (10 : 1→5 : 1→
2 : 1→1 : 1, v/v)→CHCl3–MeOH (5 : 1→1 : 1, v/v)→MeOH] to give twelve
fractions [Fr. 1 (33.8 g), Fr. 2 (12.5 g), Fr. 3 (10.5 g), Fr. 4 (22.7 g), Fr. 5
(22.4 g), Fr. 6 (44.5 g), Fr. 7 (11.9 g), Fr. 8 (6.7 g), Fr. 9 (12.4 g), Fr. 10
(50.5 g), Fr. 11 (64.4 g), Fr. 12 (81.8 g)]. Fraction 4 (21 g) was further sepa-
rated by reversed-phase silica gel column chromatography [630 g,
MeOH–H2O (80 : 20→90 : 10→95 : 5, v/v)→MeOH] and HPLC [YMC-
Pack ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (90 : 10, v/v)]
to give phytol (47.8 mg, 0.0003%), and lupeol (2010 mg, 0.012%). Fraction
9 (12.4 g) was separated by reversed-phase silica gel column chromatogra-
phy [380 g, MeOH–H2O (50 : 50→60 : 40→70 : 30→95 : 5, v/v)→MeOH]
to give quercetin (2400 mg, 0.014%). The n-BuOH-soluble portion (300 g)
was subjected to normal-phase silica gel column chromatography [BW-200
(Fuji Silysia, Ltd., 3 kg), CHCl3–MeOH–H2O (10 : 3 : 1, lower layer→7 : 3 :
0.5→6 : 4 : 1, v/v)→MeOH] to give seven fractions [Fr. 1 (78.8 g), Fr. 2
(32.7 g), Fr. 3 (36.2 g), Fr. 4 (27.6 g), Fr. 5 (35.8 g), Fr. 6 (82.9 g), Fr. 7
(40.5 g)]. Fraction 4 (25 g) was further separated by reversed-phase silica gel
column chromatography [Chromatorex DM1020T (Fuji Silysia, Ltd., 750 g),
MeOH–H2O (10 : 90→30 : 70→50 : 50→70 : 30, v/v)→MeOH] and re-
peated HPLC [MeOH–H2O (30 : 70, 50 : 50, v/v), CH3CN–H2O (15 : 85,
20 : 80, v/v)] to give apocynosides I (1, 41 mg, 0.0002%) and II (2, 73 mg,
0.0004%), phenethyl alcohol xylopyranosyl(1→6)glucopyranoside (104 mg,
0.0006%), Z-hex-3-en-1-ol xylopyranosyl(1→6)glucopyranoside (133 mg,
0.0008%), isoquercitrin (83 mg, 0.0005%), and kaempferol 3-O-b-D-galac-
topyranoside (10 mg, 0.0001%). Fraction 5 (75 g) was separated by reversed-
phase silica gel column chromatography [750 g, MeOH–H2O (30 : 70→50 :
50→70 : 30, v/v)→MeOH] and purified by HPLC [MeOH–H2O (45 : 55,
v/v), CH3CN–H2O (20 : 80, v/v)] to give benzyl alcohol xylopyranosyl(1→
6)glucopyranoside (116 mg, 0.0007%) and hyperoside (30 mg, 0.0002%).
The known compounds were identified by comparison of their physical data
225.1525. Found: 225.1492. CD (cϭ0.0028, MeOH) De (nm): ϩ20.4 (243)
MeOH
(positive max.), Ϫ1.0 (319) (negative max.). UV l
nm (log e): 236
max
(4.2). IR (KBr): 3401, 1655, 1608, 1055 cmϪ1. H-NMR (500 MHz, CDCl3)
d: 0.99 (3H, s, 13-H3), 1.04 (3H, s, 12-H3), 1.28 (3H, d, Jϭ6.4 Hz, 10-H3),
2.13 (1H, d, Jϭ16.8 Hz, 2a-H), 2.41 (1H, d, Jϭ16.8 Hz, 2b-H), 2.59 (1H, d,
Jϭ8.5 Hz, 6-H), 4.17, 4.23 (1H each, both d, Jϭ17.1 Hz, 11-H2), 4.34 (1H,
dd-like, 9-H), 5.60 (1H, dd, Jϭ6.5, 15.6 Hz, 8-H), 5.67 (1H, dd, Jϭ8.5,
15.6 Hz, 7-H), 6.18 (1H, s, 4-H). 13C-NMR (125 MHz, CDCl3) dC: given in
Table 1. Positive-ion FAB-MS: m/z 225 (MϩH)ϩ.
1
Apocynol B (4): An amorphous powder, [a]D27 ϩ104.7° (cϭ0.8, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C13H20O4Na (MϩNa)ϩ:
263.1294. Found: 263.1272. CD (cϭ0.0018, MeOH) De (nm): ϩ9.2 (232)
MeOH
(positive max.), Ϫ0.4 (322) (negative max.). UV l
nm (log e): 236
max
(3.9). IR (KBr): 3401, 1655, 1611, 1064 cmϪ1 1H-NMR (500 MHz, pyri-
.
dine-d5) d: 1.15 (3H, s, 13-H3), 1.34 (3H, s, 12-H3), 1.39 (3H, d, Jϭ6.4 Hz,
10-H3), 2.50 (1H, d, Jϭ16.8 Hz, 2a-H), 2.74 (1H, d, Jϭ16.8 Hz, 2b-H), 4.67
(1H, dd, Jϭ3.1, 6.4 Hz, 9-H), 4.81, 5.07 (1H each, both d, Jϭ18.6 Hz, 11-
H2), 6.38 (1H, dd, Jϭ3.1, 12.8 Hz, 8-H), 6.39 (1H, d, Jϭ12.8 Hz, 7-H), 7.00
(1H, s, 4-H). 13C-NMR (125 MHz, pyridine-d5) dC: given in Table 1. Posi-
tive-ion FAB-MS: m/z 263 (MϩNa)ϩ.
Preparation of the (R)-(؉)-MTPA Esters (3a, 4a) from Apocynols A
(3) and B (4) A solution of 3 (1.0 mg) in dry CH2Cl2 (1 ml) was treated
with (R)-MTPA (12 mg), EDC HCl (10 mg), and DMAP (10 mg), and the
whole mixture was stirred at room temperature (25 °C) for 30 min. The reac-
tion mixture was poured into brine and the whole was extracted with AcOEt.
The AcOEt extract was washed with 5% aqueous HCl, saturated aqueous
NaHCO3, and brine, then dried over MgSO4. Removal of the solvent from
the extract gave a residue, which was purified by normal-phase silica gel col-
umn chromatography [1 g, n-hexane–AcOEt (2 : 1, v/v)] to furnish 3a
(1.2 mg).
1
([a]D, H-NMR, 13C-NMR) with reported values.4—9) Quercetin was identi-
fied by comparison of an authentic sample.
Apocynoside I (1): An amorphous powder, [a]D27 ϩ79.2° (cϭ1.2, MeOH),
High-resolution positive-ion FAB-MS: Calcd for C19H31O8 (MϩH)ϩ:
387.2019. Found: 387.2036. CD (cϭ0.036, MeOH) De (nm): ϩ20.0 (239)
(positive max.), Ϫ1.4 (314) (negative max). UV lMmeaOx H nm (log e): 236 (4.0).
IR (KBr): 3432, 1655, 1619, 1076 cmϪ1. H-NMR (270 MHz, pyridine-d5)
1
d: 0.87 (3H, s, 13-H3), 0.94 (3H, s, 12-H3), 1.42 (3H, d, Jϭ6.4 Hz, 10-H3),
2.15 (1H, d, Jϭ16.5 Hz, 2a-H), 2.51 (1H, d, Jϭ16.5 Hz, 2b-H), 2.69 (1H, d,
Jϭ8.9 Hz, 6-H), 3.90 (1H, m, 5Ј-H), 4.06 (1H, t-like, 2Ј-H), 4.18 (1H, m, 3Ј-
H), 4.20 (1H, m, 4Ј-H), 4.34 (1H, dd, Jϭ5.2, 11.6 Hz), 4.48 (1H, m) (6Ј-H2),
4.51 (1H, m), 4.67 (1H, d, Jϭ14.9 Hz) (11-H2), 4.53 (1H, m, 9-H), 4.84 (1H,
d, Jϭ7.6 Hz, 1Ј-H), 5.80 (1H, dd, Jϭ8.9, 15.3 Hz, 7-H), 5.91 (1H, dd,
Jϭ5.2, 15.3 Hz, 8-H), 6.70 (1H, s, 4-H). 13C-NMR (68 MHz, pyridine-d5)
dC: given in Table 1. Negative-ion FAB-MS: m/z 385 (MϪH)Ϫ, 223 (MϪ
C6H11O5)Ϫ. Positive-ion FAB-MS: m/z 387 (MϩH)ϩ.
1
(R)-MTPA Ester of Apocynol A (3a): An amorphous powder. H-NMR
(270 MHz, CDCl3) d: 0.91 (3H, s, 13-H3), 0.97 (3H, s, 12-H3), 1.37 (3H, d,
Jϭ6.3 Hz, 10-H3), 2.12 (1H, d, Jϭ16.9 Hz, 2a-H), 2.33 (1H, d, Jϭ16.9 Hz,
2b-H), 2.57 (1H, d, Jϭ7.9 Hz, 6-H), 4.73, 4.82 (1H each, both d, Jϭ18.6 Hz,
11-H2), 5.61 (1H, m, 7-H), 5.54 (1H, m, 9-H), 5.56 (1H, m, 8-H), 6.00 (1H,
s, 4-H).
The (R)-MTPA ester (4a, 1.5 mg) was also prepared form 4 (2.0 mg) by
the procedure described above.
Apocynoside II (2): An amorphous powder, [a]D26 ϩ25.1° (cϭ0.4,
MeOH). High-resolution positive-ion FAB-MS: Calcd for C19H30O9Na
(MϩNa)ϩ: 425.1788. Found: 425.1793. CD (cϭ0.028, MeOH) De (nm):
1
(R)-MTPA Ester of Apocynol B (4a): An amorphous powder. H-NMR
(270 MHz, CDCl3) d: 0.92 (3H, s, 13-H3), 1.01 (3H, s, 12-H3), 1.38 (3H, d,
Jϭ6.3 Hz, 10-H3), 2.27 (1H, d, Jϭ17.1 Hz, 2a-H), 2.37 (1H, d, Jϭ17.1 Hz,

848
Vol. 49, No. 7
Yoshikawa M., Shimoda H., Uemura T., Morikawa T., Kawahara Y.,
Matsuda H., Bioorg. Med. Chem., 8, 2071—2077 (2000); f) Li Y.,
Matsuda H., Wen S., Yamahara J., Yoshikawa M., Eur. J. Pharmacol.,
387, 337—342 (2000); g) Li Y., Matsuda H., Yamahara J., Yoshikawa
M., ibid., 392, 71—77 (2000); h) Murakami T., Emoto A., Matsuda
H., Yoshikawa M., Chem. Pharm. Bull., 49, 54—63 (2001); i) Mu-
rakami T., Kohno K., Matsuda H., Yoshikawa M., ibid., 49, 73—77
(2001).
2b-H), 4.83, 4.97 (1H each, both d, Jϭ15.9 Hz, 11-H2), 5.60 (1H, dq-like, 9-
H), 5.90 (1H, m, 7-H), 5.93 (1H, m, 8-H), 5.93 (1H, s, 4-H).
Preparation of the (S)-(؊)-MTPA Esters (3b, 4b) from Apocynols A
(3) and B (4) A solution of 3 (1.0 mg) in CH2Cl2 (1 ml) was treated with
(S)-MTPA (12 mg), EDC·HCl (10 mg), and DMAP (10 mg), and the whole
mixture was stirred at room temperature (25 °C) for 30 min. It was worked
up as described for the above AcOEt extract, which was purified by normal-
phase silica gel column chromatography [1 g, n-hexane–AcOEt (2 : 1, v/v)]
to furnish 3b (1.4 mg).
4) Gramatica P., Manitto P., Monti D., Speranza G., Tetrahedron, 43,
4481—4486 (1987).
1
(S)-MTPA Ester of Apocynol A (3b): An amorphous powder. H-NMR
5) Sholichin M., Yamasaki K., Kasai R., Tanaka O., Chem. Pharm. Bull.,
28, 1006—1008 (1980).
6) Otsuka H., Takeda Y., Yamasaki K., Phytochemistry, 29, 3681—3683
(1990).
7) Lei Z. H., Yahara S., Tai B. S., Tian R. H., Takiguchi Y., Nohara T.,
Nat. Med., 49, 475—477 (1995).
8) Barbera O., Sanz J. F., Sanchez-Parareda J., Marco J. A., Phytochem-
istry, 25, 2361—2365 (1986).
9) Shigematsu N., Kouno I., Kawano N., Phytochemistry, 21, 2156—
2158 (1982).
10) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843—1845
(1986).
(270 MHz, CDCl3) d: 0.87 (3H, s, 13-H3), 0.93 (3H, s, 12-H3), 1.42 (3H, d,
Jϭ6.3 Hz, 10-H3), 2.09 (1H, d, Jϭ16.8 Hz, 2a-H), 2.25 (1H, d, Jϭ16.8 Hz,
2b-H), 2.50 (1H, d-like, 6-H), 4.63, 4.82 (1H each, both d, Jϭ13.8 Hz, 11-
H2), 5.52 (1H, m, 8-H), 5.53 (1H, m, 7-H), 5.54 (1H, m, 9-H), 5.96 (1H, s,
4-H).
The (S)-MTPA ester (4b, 1.8 mg) was also prepared from 4 (2.0 mg).
1
(S)-MTPA Ester of Apocynol B (4b): An amorphous powder. H-NMR
(270 MHz, CDCl3) d: 0.89 (3H, s, 13-H3), 1.01 (3H, s, 12-H3), 1.43 (3H, d,
Jϭ6.4 Hz, 10-H3), 2.13 (1H, br s, 2a-H), 2.26 (1H, br s, 2b-H), 4.67, 5.05
(1H each, both d, Jϭ15.9 Hz, 11-H2), 5.60 (1H, dq-like, 9-H), 5.67 (1H, m,
7-H), 5.86 (1H, m, 8-H), 5.88 (1H, s, 4-H).
References and Notes
1) Part XXIII: Murakami T., Hirano K., Matsuda H., Yoshikawa M.,
Chem. Pharm. Bull., 2001, submitted.
2) a) Nishibe S., Takemura H., Fujimoto T., Sasahara M., Tanaka T.,
Shoyakugaku Zasshi, 47, 27—33 (1993); b) Fan W., Tezuka Y., Xiong
Q., Hattori M., Namba T., Kadota S., Chem. Pharm. Bull., 47, 1049—
1050 (1999).
11) The 1H- and 13C-NMR spectra of 1 and 2 were assigned on the basis of
homo- and hetero-correlation spectroscopy (1H–1H, 1H–13C COSY),
homo- and heteronuclear Hartmann-Hahn spectroscopy (1H–1H,
1H–13C HOHAHA), and heteronuclear multiple bond correlation
(HMBC) experiments.
12) Yoshikawa M., Shimada H., Saka M., Yoshizumi S., Yamahara J., Mat-
suda H., Chem. Pharm. Bull., 45, 464—469 (1997).
3) a) Matsuda H., Kageura T., Toguchida I., Ueda H., Morikawa T., 13) Ohtani I., Kusumi T., Kashman H., Kakisawa H., J. Am. Chem. Soc.,
Yoshikawa M., Life Sci., 66, 2151—2157 (2000); b) Matsuda H., Li Y.,
113, 4092—4096 (1991).
Yoshikawa M., ibid., 66, PL41—PL46 (2000); c) Idem, ibid., 66, 14) Oritani T., Yamashita K., Tetrahedron Lett., 1972, 2521—2524.
2233—2238 (2000); d) Idem, ibid., 67, 2921—2927 (2000); e)