Amurensiosides A-K, 11 new pregnane glycosides from the roots of Adonis amurensis

Article abstract of DOI:10.1016/j.steroids.2009.10.008

Five new pregnane tetraglycosides, amurensiosides A-E (1-5), two new pregnane hexaglycosides, amurensiosides F (6) and I (9), two new 18-norpregnane hexaglycosides, amurensiosides G (7) and H (8), and two new pregnane octaglycosides, amurensiosides J (10) and K (11), were isolated from the MeOH extract of the roots of Adonis amurensis. The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including two-dimensional (2D) NMR data, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activity against HSC-2 human oral squamous cell carcinoma cells.

Full text of DOI:10.1016/j.steroids.2009.10.008

Steroids 75 (2010) 83–94
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Amurensiosides A–K, 11 new pregnane glycosides from the roots of
Adonis amurensis
Minpei Kurodaa,∗, Satoshi Kuboa, Shingo Uchidaa, Hiroshi Sakagamib, Yoshihiro Mimakia,∗
a School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
b Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, 1-1 Keyaki-dai, Sakado, Saitama 350-0283, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 August 2009
Received in revised form 21 October 2009
Accepted 21 October 2009
Available online 31 October 2009
Five new pregnane tetraglycosides, amurensiosides A–E (1–5), two new pregnane hexaglycosides,
amurensiosides F (6) and I (9), two new 18-norpregnane hexaglycosides, amurensiosides G (7) and H (8),
and two new pregnane octaglycosides, amurensiosides J (10) and K (11), were isolated from the MeOH
extract of the roots of Adonis amurensis. The structures of the new compounds were determined on the
basis of extensive spectroscopic analysis, including two-dimensional (2D) NMR data, and the results of
hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activity against HSC-2
human oral squamous cell carcinoma cells.
Keywords:
Adonis amurensis
Ranunculaceae
Pregnane glycosides
Amurensiosides A–K
Cytotoxic activity
HSC-2 cells
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Experimental
Plants of the family Ranunculaceae can be found worldwide,
but is common in the temperate and cold areas of the north-
ern hemisphere, and consists of 58 genera totaling about 2500
species [1]. We have already made phytochemical screenings of
three Ranunculaceae plants, Helleborus orientalis [2–5], Cimicifuga
racemosa [6,7] and Eranthis cilicica [8,9], and isolated a variety
of new steroidal, bufadienolide and tritrepene glycosides, and
chromone derivatives. Adonis amurensis Regel et Radde is mainly
found in Japan, Russia, and China. The chemical constituents
of the roots of A. amurensis have been investigated, and more
than 20 pregnanes and cardenolides [10–15] have been isolated
and identified. As part of our continuous study on biologically
active secondary metabolites from Ranunculaceae plants, we have
now examined the fresh roots of A. amurensis, resulting in the
isolation of 11 new pregnane glycosides, named amurensiosides
A–K (1–11). This paper deals with the structure elucidation of the
new glycosides on the basis of extensive spectroscopic analysis,
including two-dimensional (2D) NMR data, and the results of
hydrolytic cleavage. The cytotoxic activity of the glycosides against
HSC-2 human oral squamous cell carcinoma cells is also described.
2.1. General methods
Optical rotations were measured using a JASCO P-1030 (Tokyo,
Japan) automatic digital polarimeter. IR spectra were recorded
on a JASCO FT-IR 620 spectrophotometer and UV spectra on
a JASCO V-520 spectrophotometer. NMR spectra were recorded
on a Bruker DRX-500 spectrometer (500 MHz for 1H NMR, Karl-
sruhe, Germany) using standard Bruker pulse programs. Chemical
shifts are given as ı-value with reference to tetramethylsilane
as an internal standard. ESI-TOF-MS data were obtained on a
Waters-Micromass LCT (Manchester, U.K.) mass spectrometer.
Porous-polymer polystyrene resin (Diaion HP-20, Mitsubishi-
Chemical, Tokyo, Japan), silica gel (300 mesh, Fuji-Silysia Chemical,
Aichi, Japan), and octadecylsilanized (ODS) silica gel (75 ␮m,
Nacalai-Tesque, Kyoto, Japan) were used for column chromatogra-
phy. TLC was carried out on precoated Silica gel 60 F254 (0.25 mm,
Merck, Darmstadt, Germany) and RP-18 F254S (0.25 mm thick,
Merck) plates, and spots were visualized by spraying with 10%
H2SO4 solution followed by heating. HPLC was performed by using
a system comprised of a CCPM pump (Tosoh, Tokyo, Japan), a CCP
PX-8010 controller (Tosoh), an RI-8010 refractive index detector
(Tosoh) or a Shodex OR-2 optical rotation detector (Showa-Denko,
Tokyo, Japan), and a Rheodyne injection port. A Capcell Pak
C18 UG120 column (10 mm i.d. × 250 mm, 5 ␮m, Shiseido, Tokyo,
Japan) was employed for preparative HPLC. The following reagents
were obtained from the indicated companies: Dulbecco’s modi-
∗
Corresponding authors. Tel.: +81 42 676 4575; fax: +81 42 676 4579.
E-mail addresses: kurodam@toyaku.ac.jp (M. Kuroda),
mimakiy@toyaku.ac.jp (Y. Mimaki).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.10.008

84
M. Kuroda et al. / Steroids 75 (2010) 83–94
fied Eagle medium (DMEM) (Gibco, Grand Island, NY, U.S.A.); fetal
bovine serum (FBS) (JRH Biosciences, Lenexa, KS, U.S.A.); penicillin
G and streptomycin sulfate (Meiji-Seika, Tokyo, Japan); and 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) (Sigma, St. Louis, MO, U.S.A.). All other chemicals used were
of biochemical reagent grade.
cooling, the reaction mixture was neutralized by passage through
an Amberlite IRA-96SB column and then chromatographed on ODS
silica gel eluted with MeCN–H2O (1:1) to give an aglycone (12-
O-benzoylisolineolon, 5.8 mg) and a sugar fraction (3.5 mg). The
sugar fraction was analyzed by HPLC under the following condi-
tions: column, Shodex Sugar SC1011 (8.0 mm i.d. × 300 mm, 5 ␮m,
Showa-Denko); solvent, H2O; flow rate, 1.0 mL/min; column tem-
perature, 80 ◦C; detection, RI and OR. Identification of d-glucose,
d-cymarose, and d-diginose was carried out by comparison of
their retention times and optical rotations with those of authen-
tic samples; tR (min) d-glucose (7.61, positive optical rotation),
d-cymarose (9.14, positive optical rotation), and d-diginose (9.55,
positive optical rotation).
2.2. Plant material
A. amurensis was purchased from a nursery in Heiwaen, Japan,
in April 2002 and was identified by Dr. Yutaka Sashida, emeri-
tus professor of Medicinal Pharmacognosy at Tokyo University
of Pharmacy and Life Sciences. A voucher specimen has been
deposited in our laboratory (Voucher No. 02-4-20-AM, Laboratory
of Medicinal Pharmacognosy).
2.7. Amurensioside B (2)
2.3. Extraction and isolation
An amorphous solid; [␣]D −13.4◦ (c 0.10, MeOH); UV ꢀmax
25
nm (log ε): 230 (4.11); IR ꢁmax (film) cm−1: 3426 (OH), 2931 (CH),
1712 (C O), 1608 and 1450 (aromatic ring); HRESITOFMS m/z:
1085.5277 [M+Na]+ (calcd for C55H82O20Na, 1085.5230); 1H and
13C NMR (C5D5N): see Tables 1, 3 and 4.
The plant material (fresh weight, 16.2 kg) was extracted with hot
MeOH (28 L). The MeOH extract was concentrated under reduced
pressure, and the viscous concentrate (670 g) was passed through
a Diaion HP-20 column eluted with 20% MeOH in H2O, EtOH, and
EtOAc. The EtOH eluate (fraction II, 110 g) was chromatographed
on silica gel eluted with CHCl3–MeOH gradients (19:1; 9:1; 4:1;
2:1), and finally with MeOH alone, to give six fractions (II-I to II-vi).
Fraction II-iii was subjected to column chromatography on ODS
silica gel eluted with MeOH–H2O (2:3; 1:1; 3:2; 7:4; 7:3), silica gel
with CHCl3–MeOH (9:1), and to preparative HPLC using MeCN–H2O
(2:1; 1:1; 1:3) and MeOH–H2O (9:2; 7:3) to yield 1 (58.9 mg), 2
(26.8 mg), 4 (41.8 mg), 5 (19.2 mg), 6 (9.5 mg), 9 (45.7 mg), and 11
(16.3 mg). Column chromatography of the EtOAc eluate (fraction
III, 20 g) on silica gel eluted with CHCl3–MeOH (19:1) followed
by MeOH alone to gave fractions (III-i and III-ii). Fraction III-i was
chromatographed on ODS silica gel eluted with MeOH–H2O (3:2)
to yield 10 (68.7 mg). Fraction III-ii was subjected to ODS silica gel
column chromatography eluted with MeOH–H2O (6:5) followed
by MeCN–H2O (1:1; 3:2), and preparative HPLC using MeCN–H2O
(1:1) to give 3 (23.9 mg), 7 (75.6 mg), and 8 (18.0 mg).
2.8. Acid hydrolysis of 2
Compound 2 (12.5 mg) was subjected to acid hydrolysis as
described for 1 to give an aglycone (12-O-benzoyllineolon, 3.8 mg)
and a sugar fraction (3.5 mg). HPLC analysis of the sugar frac-
tion under the same conditions as for 1 showed the presence of
d-glucose, d-cymarose, and d-diginose; tR (min) d-glucose (7.66,
positive optical rotation), d-cymarose (9.17, positive optical rota-
tion), and d-diginose (9.69, positive optical rotation).
2.9. Amurensioside C (3)
An amorphous solid; [␣]D25 +14.0◦ (c 0.10, MeOH); UV ꢀmax nm
(log ε): 261 (3.46), 221 (3.89); IR ꢁmax (film) cm−1: 3399 (OH), 2933
(CH), 1720 (C O), 1592 and 1457 (aromatic ring); HRESITOFMS
m/z: 1064.5435 [M+H]+ (calcd for C54H82NO20, 1064.5429); 1H and
13C NMR (C5D5N): see Tables 1, 3 and 4.
2.4. Preparation of 2,6-dideoxy sugars for comparison
A solution of the crude glycoside fraction (1.3 g) in 0.025 M HCl
(dioxane–H2O, 1:1, 200 mL) was heated at 95 ◦C for 5 h under an Ar
atmosphere. After cooling, the reaction mixture was neutralized
by passage through an Amberlite IRA-96SB (Organo, Tokyo, Japan)
column and then chromatographed on ODS silica gel eluted
with MeCN–H2O (1:2) to give five fractions (I–V). Fraction II was
subjected to column chromatography on ODS silica gel eluted
with MeCN–H2O (1:3), and silica gel eluted with hexane–EtOAc
(1:9), EtOAc and CHCl3–MeOH (19:1; 14:1) to yield d-cymarose
(21.9 mg), d-diginose (2.9 mg), and d-oleandrose (23.0 mg). d-
2.10. Acid hydrolysis of 3
Compound 3 (10.3 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the agly-
cone (2.2 mg) and a sugar fraction (2.4 mg). HPLC analysis of the
sugar fraction under the same conditions as for 1 showed the pres-
ence of d-glucose, d-cymarose, and d-diginose; tR (min) d-glucose
(7.69, positive optical rotation), d-cymarose (9.22, positive optical
rotation), and d-diginose (9.59, positive optical rotation).
Cymarose: [␣]D +46.5◦ (c 0.10, H2O); d-diginose: [␣]D +16.4◦
27
27
2.11. Amurensioside D (4)
(c 0.05, H2O); d-oleandrose: [␣]D −15.3◦ (c 0.10, H2O) [16,17].
27
+27.6◦ (c 0.10, MeOH); IR ꢁmax
25
An amorphous solid; [␣]D
2.5. Amurensioside A (1)
(film) cm−1: 3411 (OH), 1685 (C O); HRESITOFMS m/z: 981.5075
[M+Na]+ (calcd for C48H78O19Na, 981.5034); 1H and 13C NMR
(C5D5N): see Tables 1, 3 and 4.
An amorphous solid; [␣]D +10.2◦ (c 0.10, MeOH); UV ꢀmax
25
nm (log ε): 231 (3.70); IR ꢁmax (film) cm−1: 3409 (OH), 2933 (CH),
1712 (C O), 1610 and 1450 (aromatic ring); HRESITOFMS m/z:
1085.5225 [M+Na]+ (calcd for C55H82O20Na, 1085.5230). 1H and
13C NMR (C5D5N): see Tables 1, 3 and 4.
2.12. Acid hydrolysis of 4
Compound 4 (20.0 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the agly-
cone (3.1 mg) and a sugar fraction (3.3 mg). HPLC analysis of the
sugar fraction under the same conditions as for 1 showed the pres-
ence of d-glucose, d-cymarose, and d-diginose; tR (min) d-glucose
2.6. Acid hydrolysis of 1
A solution of 1 (20.0 mg) in 0.025 M HCl (dioxane–H2O, 1:1,
5 mL) was heated at 95 ◦C for 90 min under an Ar atmosphere. After

M. Kuroda et al. / Steroids 75 (2010) 83–94
85
Table 1
1H NMR data (500 MHz, C5D5N) for compounds 1–5.
Positions
1
2
3
4
5
ı
J (Hz)
ı
J (Hz)
ı
J (Hz)
ı
J (Hz)
ı
H
J (Hz)
H
H
H
H
Aglycone
Aglycone
Aglycone
Aglycone
Aglycone
1
2
ax
eq
ax
eq
1.11
1.81
1.83
2.07
3.87 m
2.46
2.57
–
5.35 br d
2.54
2.51
–
1.11
1.77
1.80
2.07
3.86 m
2.43
2.56
–
5.29 br s
2.53
2.35
–
1.12
1.83
1.82
2.10
3.87 m
2.59
2.47
–
5.35 br d
2.55
2.35
–
1.11 br dd
1.84
1.77
2.05
3.86 m
2.47
2.56 dd
–
5.36 br s
2.51
2.30
–
11.7, 3.5
3.30
1.41
2.01
2.16
3.93 m
2.63 (2H)
3
4
W
= 22.3
W
= 21.8
W
= 19.8
W
= 20.7
W
= 18.7
1/2
1/2
1/2
1/2
1/2
ax
eq
12.6, 3.4
5
6
7
–
4.1
4.3
5.39 br d
2.57
2.28
–
5.2
a
b
8
9
10
11
1.74 dd
–
2.40
2.05
5.25 dd
–
12.9, 2.8
12.0, 3.8
1.76
–
2.32
2.13
5.41 dd
–
1.74 dd
–
2.40
2.10
5.20
–
12.9, 3.8
12.0, 3.8
1.60 dd
–
2.38 q-like
1.90
3.75 dd
–
–
–
2.30
2.01
2.17
12.9, 2.9
12.1
1.78 d
–
4.77 ddd
–
1.89
2.11
–
11.1
ax
eq
11.1, 11.1, 4.1
12
11.8, 4.0
11.9, 3.6
13
14
15
–
–
–
–
–
–
–
a
b
a
b
2.00
2.10
1.95
2.26
3.26 dd
1.65 s
1.37 s
–
2.10
1.94
2.54
1.84
3.54
2.04 s
1.33 s
–
2.12
2.02
2.31
1.94
3.26 dd
1.63 s
1.38 s
–
2.26 (2H)
–
16
1.90
2.02
2.89 dd
1.43 s
1.80 s
–
1.95
17
18
19
20
21
9.3, 5.7
9.2, 5.8
3.86 m
1.53 s
1.37 s
–
9.5, 4.8
2.20 s
2.11 s
2.23 s
2.28 s
2.20 s
Bz
Bz
Nic
1
2
3
4
5
6
7
–
–
–
–
–
–
8.37 br d
7.53 br dd
7.60 br dd
7.53 br dd
8.37 br d
7.8
8.27 br d
7.47 br dd
7.53 br dd
7.47 br dd
8.27 br d
8.0
8.92 dd
–
8.54 ddd
7.43 dd
9.65 d
4.8, 1.8
7.8, 7.8
7.8, 7.8
7.8, 7.8
7.8
8.0, 8.0
8.0, 8.0
8.0, 8.0
8.0
8.0, 1.8, 1.8
8.0, 4.8
1.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Cym
Cym
Cym
Cym
Cym
ꢀ
ꢀ
1
2
5.29 dd
1.92 ddd
2.34 br d
4.09 q-like
3.52 dd
4.23 m
9.5, 1.6
12.0, 9.5, 2.9
12.0
2.7
9.5, 2.7
5.28 dd
1.92 ddd
2.31 br d
4.09 q-like
3.51 dd
4.23 m
9.4, 1.3
12.0, 9.4, 2.9
12.0
2.7
9.4, 2.7
5.29 br d
1.91 ddd
2.35 br d
4.09 q-like
3.48 dd
4.23 m
9.4
5.27 br d
1.87 ddd
2.30 br d
4.06 q-like
3.49 dd
4.21 m
9.5
5.31 dd
1.92 ddd
2.31 br d
4.07 q-like
3.48 dd
4.20 m
9.5, 1.4
12.4, 9.5, 2.9
12.4
ax
eq
12.0, 9.4, 2.9
12.0
2.6
12.0, 9.5, 3.0
12.0
2.7
ꢀ
ꢀ
ꢀ
ꢀ
3
4
5
6
2.5
9.7, 2.5
9.2, 2.6
9.7, 2.5
1.40 d
6.3
1.40 d
6.2
1.37 d
6.4
1.36 d
6.3
1.36 d
6.2
OMe
3.61 s
3.61 s
3.41 s
3.59 s
3.59 s
ꢀꢀ
Cym
ꢀꢀ
Cym
ꢀꢀ
Cym
ꢀꢀ
Cym
ꢀꢀ
Cym
ꢀꢀ
ꢀꢀ
1
2
5.11 dd
1.78 ddd
2.33 br d
4.00 q-like
3.48 dd
4.17 m
9.5, 1.3
12.4, 9.5, 2.7
12.4
2.7
9.6, 2.7
5.10 br dd
1.78 ddd
2.31 br d
4.00 q
3.47 dd
4.16 m
1.38 d
9.4, 1.1
12.4, 9.4, 2.9
12.4
2.7
9.3, 2.7
5.11 br d
1.79 ddd
2.31 br d
4.00 q-like
3.52 dd
4.20 m
9.4
5.08 br d
1.76 ddd
2.28 br d
3.98 q-like
3.46 dd
4.13 m
9.5
5.09 dd
1.78 ddd
2.30 br d
3.98 q-like
3.46 dd
4.15 m
9.6, 1.4
12.4, 9.6, 2.9
12.4
ax
eq
12.4, 9.4, 2.9
12.4
2.6
12.4, 9.5, 2.9
12.4
2.6
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
3
4
5
6
2.4
9.6, 2.4
9.2, 2.6
9.5, 2.6
1.37 d
6.1
6.2
1.40 d
6.4
1.36 d
6.3
1.36 d
6.2
OMe
3.41 s
3.41 s
3.61 s
3.39 s
3.39 s
ꢀꢀꢀ
Dgn
ꢀꢀꢀ
Dgn
ꢀꢀꢀ
Dgn
ꢀꢀꢀ
Dgn
ꢀꢀꢀ
Dgn
ꢀꢀꢀ
ꢀꢀꢀ
1
2
4.65 dd
2.30 q-like
2.18 br dd
3.46 ddd
4.17 br s
3.56 m
9.7, 1.7
11.0
12.2, 5.0
12.2, 4.3, 2.9
4.65 dd
2.30 q-like
2.18 br dd
3.46 ddd
4.17 br s
3.55 m
9.6, 1.7
11.2
12.8, 5.1
12.0, 4.0, 2.9
4.65 br d
2.34 q-like
2.19 br dd
3.45 ddd
4.18 br s
3.57 m
9.4
11.0
12.5, 5.0
12.5, 4.5, 2.9
4.65 dd
2.27 q-like
2.17 br dd
3.47 ddd
4.18 br s
3.56 m
9.6, 1.2
10.5
13.1, 5.0
12.9, 4.5, 3.0
4.65 dd
2.28 q-like
2.16 br dd
3.46 ddd
4.17 br s
3.56
9.6, 1.8
11.0
ax
eq
13.1, 5.0
12.3, 4.7, 2.6
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
3
4
5
6
1.55 d
6.4
1.55 d
6.3
1.64 d
6.2
1.54 d
6.0
1.54 d
6.3
OMe
3.39 s
3.39 s
3.39 s
3.39 s
3.39 s
ꢀꢀꢀꢀ
Glc
5.13 d
ꢀꢀꢀꢀ
Glc
5.12 d
ꢀꢀꢀꢀ
Glc
5.13 d
ꢀꢀꢀꢀ
Glc
5.09 d
ꢀꢀꢀꢀ
Glc
5.11 d
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
1
2
3
4
5
6
7.7
7.6
7.6
7.7
7.8
3.95 dd
4.22 dd
4.16 dd
3.94 m
4.57 dd
4.36 dd
8.9, 7.7
8.9, 8.9
8.9, 8.9
3.96 dd
4.22 dd
4.17 dd
3.94 m
4.58 dd
4.36 dd
8.9, 7.6
8.9, 8.9
8.9, 8.9
3.96 dd
4.23 dd
4.18 dd
3.95 m
4.58 dd
4.36 dd
8.9, 7.6
8.9, 8.9
8.9, 8.9
3.92 dd
4.21 dd
4.15 dd
3.92 m
4.53 dd
4.34 dd
8.8, 7.7
8.8, 8.8
8.8, 8.8
3.94 dd
4.22 dd
4.15 dd
3.94 m
4.55 dd
4.35 dd
9.0, 7.8
9.0, 9.0
9.0, 9.0
a
b
11.7, 2.5
11.7, 2.5
11.6, 2.4
11.6, 5.8
11.6, 1.5
11.6, 5.8
11.7, 2.2
11.7, 5.7
11.6, 2.3
11.6, 5.7
Bz: benzoyl; Nic: nicotinoyl; Cym: ␤-d-cymaropyranosyl; Dgn: ␤-d-diginopyranosyl; Glc: ␤-d-glucopyranosyl.

Table 2
1H NMR data (500 MHz, C5D5N) for compounds 6, 6a, and 7–11.
Positions
6
6a
7
8
9
10
11
ıH
J (Hz)
ıH
J (Hz)
ıH
J (Hz)
ıH
J (Hz)
ıH
J (Hz)
ıH
J (Hz)
ıH
J (Hz)
Aglycone
Aglycone
Aglycone
Aglycone
Aglycone
Aglycone
Aglycone
1
2
ax 1.07
1.15
1.84
1.75
2.07
1.63
2.30
1.71
2.05
1.06
1.74
1.71
2.10
3.78 m
2.40
2.62
–
1.14
1.82
1.71
2.05
3.80 m
2.46
2.62
–
1.27
1.87
1.37
2.10
3.87 m
2.62
2.53
–
5.44 br d
2.53
2.33
–
1.79
–
2.41
2.07
5.26
–
1.15
1.87
1.85
2.12
3.91 m
2.63
2.53
–
eq 1.77
ax 1.67
eq 2.07
3.80
ax 2.52
eq 2.34
–
3
4
W1/2 = 22.6 3.83 m
W1/2 = 21.5 3.76 m
W1/2 = 23.9
W1/2 = 24.1
W1/2 = 22.5
W1/2 = 23.2
W1/2 = 24.1
2.55
2.56
–
2.34
2.57
–
5.41 dd
2.17
1.71
2.48
1.71
–
5
6
7
5.36 t-like
2.20
2.5
5.32 t-like
2.6
5.0
5.49 t-like 2.6
5.57 t-like 2.4
4.4
5.45 br d 4.1
a
2.20
1.89
1.99
1.29
–
2.26
1.65
1.30
2.11
–
1.99
2.64
2.82
1.80
–
2.65
2.51
–
2.74
1.94
1.99
2.05
–
1.91 (2H)
–
4.88 br d
–
2.53
2.31
–
1.49
–
1.99
1.44
1.37
1.49
–
b
1.89
1.95
1.27
–
8
9
10
11
ax 2.35
eq 1.60
ax 2.07
eq 1.31
–
2.32
2.42
–
12
2.6
13
14
15
–
–
–
–
–
–
–
–
–
–
–
a
b
a
b
1.84
1.77
1.59 (2H)
1.86
1.77
1.58 (2H)
1.47 (2H)
6.40 d
4.0
4.0
1.78
1.50
1.29
2.07
3.21
1.04 s
1.05 s
3.97
1.35 d
2.08
2.04
2.30
1.96
3.27 dd
1.64 s
1.37 s
–
2.12
1.87
1.99
1.90
2.82 dd
1.33 s
1.35 s
–
16
2.05
1.98
4.05
–
0.90 s
–
7.41 d
17
18
19
20
21
2.06 brs
–
0.95 s
–
2.61 t-like
–
1.01 s
–
1.57 s
2.4
9.5, 4.9
–
–
9.3, 5.7
9.5, 4.9
1.00 s
–
2.48 s
1.57 s
2.28 s
5.9
2.19 s
2.22 s
Bz
1
–
2
–
3, 7
4, 6
5
8.38 br d
7.53 br dd 7.8
7.59 br dd 7.8
7.8
Oleꢀ
4.80 br d
ax 1.83 q-like 10.8
eq 2.43 br d
3.53
Oleꢀ
Oleꢀ
4.81 dd
11.8, 11.8, 9.6 1.81 ddd
Oleꢀ
4.81 dd
11.8, 11.8, 9.7 1.81 ddd
Oleꢀ
Oleꢀ
4.85 dd
1ꢀ
2ꢀ
9.6
4.80 dd
1.78 ddd
2.44 br d
3.56
3.53 dd
3.52 m
1.41 d
3.51 s
9.6, 1.6
9.7, 1.6
9.6, 1.6
11.3, 11.3, 9.6
9.9
4.83 dd
1.77 ddd
2.49 br d
3.55
9.6, 1.3
9.7, 1.7
11.8, 11.8, 9.6 1.80 ddd 11.8, 11.8, 9.7
10.4
11.8
2.45 br d
3.56
3.51 dd
3.55 m
1.45 d
3.52 s
11.8
2.45 br d
3.56
3.51 dd
3.53 m
1.45 d
3.52 s
11.8
2.51 br d 11.8
3.56
3.52
3.53 m
1.46 d
3.51 s
3ꢀ
4ꢀ
3.53 dd
3.53 m
1.46 d
9.5, 9.5
6.0
9.5, 9.5
6.3
9.5, 9.5
6.2
9.5, 9.5
6.3
3.52
5ꢀ
3.53 m
1.46 d
3.51 s
6ꢀ
6.1
9.7
6.1
OMe
3.51 s
Oleꢀꢀ
4.91 br d
ax 1.73 q-like 10.9
eq 2.45 br d
3.55
Oleꢀꢀ
Oleꢀꢀ
4.91 dd
11.1, 11.1, 9.7 1.77 ddd
Oleꢀꢀ
4.91 dd
11.1, 11.1, 9.7 1.75 ddd
Oleꢀꢀ
Oleꢀꢀ
4.91 dd
1ꢀꢀ
2ꢀꢀ
9.7
4.90 dd
1.74 ddd
2.49 br d
3.56
9.7, 1.7
9.7, 1.7
9.8, 1.5
11.1, 11.1, 9.8
9.9
4.91 br d
1.73 ddd
2.45 br d
3.55
9.7, 1.7
11.1, 11.1, 9.7 1.73 ddd 11.1, 11.1, 9.7
10.6
11.1
2.50 br d
3.54
3.53 dd
11.1
2.47 br d
3.56
3.51 dd
11.1
2.51 br d 11.1
3.56
3.52
3ꢀꢀ
4ꢀꢀ
3.52 dd
9.6, 9.6
3.53 dd
9.6, 9.6
9.6, 9.6
9.6, 9.6
3.52

5ꢀꢀ
3.53 m
1.42 d
3.54 s
3.52 m
1.44 d
3.56 s
3.51 m
1.45 d
3.56 s
3.53 m
1.44 d
3.56 s
3.53 m
1.42 d
3.54 s
3.53 m
1.42 d
3.53 s
6ꢀꢀ
6.1
6.2
6.4
6.4
6.1
6.2
OMe
Cymꢀꢀꢀ
Cymꢀꢀꢀ
Cymꢀꢀꢀ
Cymꢀꢀꢀ
Oleꢀꢀꢀ
Oleꢀꢀꢀ
1ꢀꢀꢀ
2ꢀꢀꢀ
5.28 br d
1.84 ddd
2.34 br d
4.07 q-like
3.47 br d
4.19 m
9.7
5.28 dd
1.83 ddd
2.34 br d
4.07 q-like
3.46 dd
4.20 m
1.36 d
9.6, 1.6
12.1, 9.6, 3.1
12.1
2.8
9.4, 2.8
5.29 dd
1.83 ddd
2.35 br d
4.08 q-like
3.46 dd
4.19 m
1.36 d
9.7, 1.6
12.1, 9.7, 3.1
12.1
2.9
9.4, 2.9
5.29 dd
1.84 ddd
2.35 br d
4.07 q-like
3.46 dd
4.21 m
1.35 d
9.7, 1.5
12.0, 9.7, 2.9
12.0
2.8
9.5, 2.8
4.89 br d
1.73 ddd
2.49 br d
3.55
9.7
4.89 dd
1.73 ddd
2.51 br d
3.56
9.7, 1.7
11.1, 11.1, 9.7
11.1
ax
eq
12.2, 9.7, 2.9
13.5
2.7
11.1, 11.1, 9.7
11.1
3ꢀꢀꢀ
4ꢀꢀꢀ
10.7
3.52
3.52
5ꢀꢀꢀ
3.53 m
1.42 d
3.50 s
3.53 m
1.42 d
3.56 s
6ꢀꢀꢀ
1.36 d
3.60 s
6.3
6.2
6.2
6.3
6.0
6.0
OMe
3.60 s
3.60 s
3.60 s
Cymꢀꢀꢀꢀ
Cymꢀꢀꢀꢀ
Cymꢀꢀꢀꢀ
Cymꢀꢀꢀꢀ
Cymꢀꢀꢀꢀ
Cymꢀꢀꢀꢀ
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
5.09 br d
1.79 ddd
2.30 br d
4.00 q-like
3.48 br d
4.19 m
9.4
5.08 dd
1.76 ddd
2.32 br d
3.99 q-like
3.48 dd
4.19 m
9.6, 1.5
12.3, 9.6, 3.1
12.3
2.8
9.6, 2.8
5.09 dd
1.77 ddd
2.29 br d
4.00 q-like
3.48 dd
4.17 m
9.6, 1.5
12.3, 9.6, 3.1
12.3
2.7
9.6, 2.7
5.09 dd
1.77 ddd
2.29 br d
4.00 q-like
3.48 dd
4.16 m
9.6, 1.4
12.4, 9.6, 2.7
12.4
2.8
9.5, 2.8
5.29 br d
1.85 ddd
2.36 br d
4.07 q-like
3.47 dd
4.17 m
9.5
5.29 dd
1.84 ddd
2.35 br d
4.07 q-like
3.46 dd
4.21 m
9.7, 1.5
12.0, 9.7, 2.9
12.0
2.8
9.5, 2.8
ax
eq
12.3, 9.4, 2.9
13.1
2.4
12.0, 9.5, 2.9
12.0
2.6
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
10.9
9.5, 2.6
5ꢀꢀꢀꢀ
6ꢀꢀꢀꢀ
1.39 d
6.2
1.38 d
6.2
1.38 d
6.2
1.37 d
6.5
1.33 d
6.8
1.35 d
6.3
OMe
3.41 s
3.41 s
3.41 s
3.41 s
3.60 s
3.60 s
Dgnꢀꢀꢀꢀꢀ
Dgnꢀꢀꢀꢀꢀ
Dgnꢀꢀꢀꢀꢀ
Dgnꢀꢀꢀꢀꢀ
Cymꢀꢀꢀꢀꢀ
Cymꢀꢀꢀꢀꢀ
1ꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀ
4.65 br d
2.32 q-like
2.20 br dd
3.47 ddd
4.17 br s
3.56 m
9.5
10.5
12.6, 4.9
12.6, 4.4, 3.2
4.65 br d
2.30 q-like
2.18 br dd
3.47 ddd
4.17 br s
3.56 m
9.6
11.0
13.0, 5.1
13.0, 4.4, 2.3
4.65 dd
2.31 q-like
2.18 br dd
3.46 ddd
4.17 br s
3.55 m
9.7, 1.7
11.0
12.4, 4.1
12.4, 4.3, 2.8
4.65 dd
2.31 q-like
2.20 br dd
3.46 ddd
4.17 br s
3.56 m
9.7, 1.8
11.0
12.2, 5.0
12.2, 4.3, 2.9
5.10 br d
1.79 ddd
2.29 br d
4.00 q-like
3.48 dd
4.14 m
9.5
5.09 dd
1.77 ddd
2.29 br d
4.00 q-like
3.48 dd
4.16 m
9.6, 1.4
12.4, 9.6, 2.7
12.4
2.8
9.5, 2.8
ax
eq
12.4, 9.5, 2.7
12.4
2.6
3ꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀ
OMe
9.5, 2.6
1.55 d
3.39 s
6.5
1.55 d
3.39 s
6.3
1.56 d
3.39 s
6.4
1.55 d
3.39 s
6.3
1.35 d
3.41 s
6.5
1.37 d
3.41 s
6.5
Glcꢀꢀꢀꢀꢀꢀ
5.12 d
Glcꢀꢀꢀꢀꢀꢀ
5.12 d
Glcꢀꢀꢀꢀꢀꢀ
5.13 d
Glcꢀꢀꢀꢀꢀꢀ
5.13 d
Dgnꢀꢀꢀꢀꢀꢀ
Dgnꢀꢀꢀꢀꢀꢀ
1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀ
7.8
7.8
7.8
7.8
4.66 br d
2.32 q-like
2.21 br dd
3.45 ddd
4.17 br s
3.54 m
8.9
11.0
12.2, 5.0
12.2, 4.3, 2.9
4.65 dd
2.31 q-like
2.20 br dd
3.46 ddd
4.17 br s
3.56 m
9.7, 1.8
11.0
12.2, 5.0
12.2, 4.3, 2.9
3.95 dd
4.22 dd
4.15 dd
3.95 m
4.57 dd
4.36 dd
9.0, 7.8
9.0, 9.0
9.0, 9.0
3.95 dd
4.22 dd
4.15 dd
3.95 m
4.56 br d
4.36 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
3.96 dd
4.22 dd
4.15 dd
3.95 m
4.57 dd
4.35 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
3.96 dd
4.21 dd
4.15 dd
3.95 m
4.57 br d
4.35 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
a
b
11.6, 2.1
11.6, 5.7
11.5, 2.2
11.5, 5.7
11.7, 2.4
11.7, 5.7
10.8
10.8, 4.9
1.53 d
6.3
1.55 d
6.3
3.39 s
3.39 s
Glcꢀꢀꢀꢀꢀꢀꢀ
Glcꢀꢀꢀꢀꢀꢀꢀ
5.13 d
7.8
5.13 d
7.8
3.95 dd
4.21 dd
4.15 dd
3.94 m
4.57 dd
4.35 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
3.96 dd
4.21 dd
4.15 dd
3.95 m
4.57 br d
4.35 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
11.6, 2.3
11.6, 5.8
10.8
10.8, 4.9
Bz: benzoyl; Ole: ␤-d-oleandropyranosyl; Cym: ␤-d-cymaropyranosyl; Dgn: ␤-d-diginopyranosyl; Glc: ␤-d-glucopyranosyl.

88
M. Kuroda et al. / Steroids 75 (2010) 83–94
(7.63, positive optical rotation), d-cymarose (9.18, positive optical
rotation), and d-diginose (9.65, positive optical rotation).
tion), d-cymarose (9.22, positive optical rotation), and d-diginose
(9.59, positive optical rotation).
2.17. Adonilide (6a)
2.13. Amurensioside E (5)
An amorphous solid; [␣]D
+5.62◦ (c 0.10, MeOH); IR ꢁmax
25
25
An amorphous solid; [␣]D
+15.7◦ (c 0.10, MeOH); IR ꢁmax
(film) cm−1: 3150 (OH), 2955 (CH), 1775 (C O); HRESITOFMS m/z:
367.1887 [M+Na]+ (calcd for C21H28O4Na, 367.1885); 1H and 13C
NMR: see Tables 2 and 3.
(film) cm−1: 3401 (OH), 2933 (CH), 1689 (C O); HRESITOFMS m/z:
981.4995 [M+Na]+ (calcd for C48H78O19Na, 981.5034); 1H and 13
NMR (C5D5N): see Tables 1, 3 and 4.
C
2.18. Amurensioside G (7)
2.14. Acid hydrolysis of 5
An amorphous solid; [␣]D −45.6◦ (c 0.10, MeOH); UV ꢀmax
25
Compound 5 (10.0 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the agly-
cone (1.4 mg) and a sugar fraction (1.3 mg). HPLC analysis of the
sugar fraction under the same conditions as for 1 showed the pres-
ence of d-glucose, d-cymarose, and d-diginose; tR (min) d-glucose
(7.69, positive optical rotation), d-cymarose (9.22, positive optical
rotation), and d-diginose (9.59, positive optical rotation).
nm (log ε): 250 (3.91); IR ꢁmax (film) cm−1: 3093 (OH), 2965
(CH), 1710 (C O); HRESITOFMS m/z: 1219.6228 [M+Na]+ (calcd for
C61H96O23Na, 1219.6234); 1H and 13C NMR: see Tables 2–4.
2.19. Acid hydrolysis of 7
Compound 7 (24.8 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the
aglycone (5.1 mg) and a sugar fraction (9.9 mg). HPLC analysis of
the sugar fraction under the same conditions as for 1 showed the
presence of d-glucose, d-cymarose, d-oleandrose, and d-diginose;
tR (min) d-glucose (7.41, positive optical rotation), d-oleandrose
(9.16, negative optical rotation), d-cymarose (9.19, positive optical
rotation), and d-diginose (9.60, positive optical rotation).
2.15. Amurensioside F (6)
+0.49◦ (c 0.11, MeOH); IR ꢁmax
25
An amorphous solid; [␣]D
(film) cm−1: 3131 (OH), 1779 (C O); HRESITOFMS m/z: 1249.6392
[M+Na]+ (calcd for C62H98O24Na, 1249.6345); 1H and 13C NMR: see
Tables 2–4.
2.16. Acid hydrolysis of 6
2.20. Amurensioside H (8)
Compound 6 (8.3 mg) was subjected to acid hydrolysis as
described for 1 to give an aglycone (6a) (adonilide, 1.5 mg) and a
sugar fraction (4.1 mg). HPLC analysis of the sugar fraction under
the same conditions as for 1 showed the presence of d-glucose, d-
cymarose, d-oleandrose, and d-diginose; tR (min) d-glucose (7.69,
positive optical rotation), d-oleandrose (9.20, negative optical rota-
An amorphous solid; [␣]D −22.6◦ (c 0.10, MeOH); UV ꢀmax
25
nm (log ε): 385 (3.81), 329 (3.90), 245 (3.99); IR ꢁmax (film) cm−1
3378 (OH), 2923 (CH), 1729 (C O); HRESITOFMS m/z: 1233.6001
[M+Na]+ (calcd for C61H94O24Na, 1233.6032); 1H and 13C NMR: see
Tables 2–4.
:
Table 3
13C NMR data (125 MHz, C5D5N) for the aglycone moieties of compounds 1–6, 6a, and 7–11.
Positions
1
2
3
4
5
6
6a
38.0
7
8
9
10
11
1
2
3
4
5
6
7
8
9
38.9
29.9
77.7
39.2
139.1
119.4
35.8
74.3
45.1
37.6
24.7
78.6
55.0
86.5
36.6
24.6
59.3
12.7
18.6
214.0
31.6
38.9
29.8
77.6
39.2
139.4
119.2
35.1
74.5
44.7
37.5
25.0
73.7
56.1
87.5
34.2
22.1
60.3
15.9
18.2
209.5
32.2
38.9
29.9
77.7
39.2
139.1
119.4
35.7
74.3
45.1
37.6
24.6
79.3
55.0
86.5
36.5
24.6
56.2
12.7
18.4
213.9
31.4
39.0
29.9
77.7
39.2
41.0
30.5
78.4
40.1
37.6
30.2
77.3
39.2
36.6
29.8
77.4
39.2
36.8
29.9
77.4
39.2
140.7
121.4
31.3
31.6
50.1
37.1
34.4
189.8
120.8
158.8
119.5
137.1
122.0
–
19.4
184.2
21.9
37.7
30.4
77.7
39.5
38.9
29.8
77.9
39.2
139.1
119.5
35.9
74.3
45.1
37.6
24.7
78.5
55.0
86.6
36.6
24.6
59.3
12.7
18.3
214.1
31.6
39.1
29.9
77.9
39.3
139.1
119.9
36.0
74.6
47.4
37.7
19.1
39.1
49.8
85.9
36.9
24.6
63.8
17.1
18.4
216.7
32.1
32.5
71.0
43.4
141.3
120.1
27.4
35.6
45.7
37.2
20.8
21.9
59.1
91.5
28.8
18.5
58.4
175.9
19.5
113.5
15.6
139.1
119.6
36.1
74.4
45.4
37.5
28.3
73.8
56.5
86.6
37.1
24.6
58.2
11.6
18.4
216.8
32.2
140.8
118.9
36.1
76.9
53.2
39.6
66.1
50.2
50.5
85.7
37.2
24.8
64.1
18.9
17.8
216.3
32.0
140.3
120.8
27.4
35.5
45.6
37.2
20.7
21.8
59.1
91.4
28.8
18.5
58.4
175.9
19.4
113.5
15.6
141.2
121.0
29.9
33.7
49.5
37.1
34.0
196.4
136.7
169.4
34.0
26.9
55.5
–
140.1
123.0
26.1
35.5
39.9
36.9
29.2
74.8
49.1
85.2
33.5
25.2
47.5
16.8
19.6
69.7
24.4
10
11
12
13
14
15
16
17
18
19
20
21
19.0
209.3
29.4
Bz
Bz
Nic
Bz
1
2
3
4
5
6
7
166.5
131.2
130.0
129.1
133.6
129.1
130.0
165.5
131.3
129.9
128.9
133.2
128.9
129.9
165.5
–
166.6
131.2
130.0
129.1
133.6
129.1
130.0
154.1
126.9
137.2
124.0
151.2
Bz: benzoyl; Nic: nicotinoyl.

M. Kuroda et al. / Steroids 75 (2010) 83–94
89
Table 4
13C NMR data (C5D5N, 125 MHz) for the sugar moieties of compounds 1–11.
Positions
1
2
3
4
5
6
7
8
9
10
11
Cymꢀ
96.4
37.2
78.0
83.4
69.0
18.6
58.8
Cymꢀ
96.4
37.2
78.0
83.4
69.0
18.6
58.8
Cymꢀ
96.4
37.1
78.0
83.4
69.0
18.6
58.8
Cymꢀ
96.3
37.0
77.9
83.3
69.0
18.5
58.7
Cymꢀ
96.5
37.3
78.2
83.6
69.2
18.8
58.9
Oleꢀ
Oleꢀ
Oleꢀ
Oleꢀ
Oleꢀ
Oleꢀ
1ꢀ
97.9
37.9
79.3
83.3
71.5
18.8
57.3
98.0
37.9
79.3
83.1
71.5
18.7
57.3
98.1
37.9
79.3
83.2
71.6
18.8
57.5
98.0
37.9
79.4
83.1
71.5
18.8
57.3
98.0
37.8
79.3
83.3
71.6
18.8
57.5
98.0
37.8
79.3
83.3
71.6
18.8
57.2
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
OMe
Cymꢀꢀ
100.4
36.5
77.5
83.1
68.9
18.6
58.3
Cymꢀꢀ
100.4
36.5
77.5
83.1
68.9
18.6
58.3
Cymꢀꢀ
100.4
36.5
77.5
83.1
68.9
18.6
59.4
Cymꢀꢀ
100.3
36.3
77.4
83.0
68.9
18.5
58.2
Cymꢀꢀ
100.6
36.6
77.6
83.3
69.1
18.7
58.5
Oleꢀꢀ
100.2
37.9
79.2
83.0
71.8
18.7
57.5
Oleꢀꢀ
100.2
37.9
79.1
83.0
71.8
18.7
57.5
Oleꢀꢀ
100.2
37.9
79.1
83.0
71.8
18.7
57.3
Oleꢀꢀ
100.2
37.9
79.2
83.0
71.8
18.7
57.5
Oleꢀꢀ
100.2
37.9
79.3
83.0
71.7
18.7
57.4
Oleꢀꢀ
100.2
37.9
79.3
83.1
71.7
18.7
57.4
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6ꢀꢀ
OMe
Dgnꢀꢀꢀ
102.7
32.8
79.8
73.8
71.0
17.9
56.2
Dgnꢀꢀꢀ
102.6
32.8
79.8
73.7
71.0
17.9
56.2
Dgnꢀꢀꢀ
102.7
32.8
80.0
73.7
71.0
17.9
58.3
Dgnꢀꢀꢀ
102.5
32.8
79.7
73.7
70.9
17.8
56.1
Dgnꢀꢀꢀ
102.8
33.0
80.0
73.9
71.2
18.0
56.4
Cymꢀꢀꢀ
98.5
36.9
78.0
83.2
69.2
18.4
58.7
Cymꢀꢀꢀ
98.5
36.9
78.0
83.2
69.2
18.4
58.7
Cymꢀꢀꢀ
98.5
36.9
78.0
83.2
69.2
18.4
58.7
Cymꢀꢀꢀ
98.5
36.9
78.1
83.2
69.2
18.4
58.8
Oleꢀꢀꢀ
100.2
37.9
79.1
82.9
71.8
18.7
57.3
Oleꢀꢀꢀ
100.2
37.9
79.1
82.9
71.8
18.7
57.3
1ꢀꢀꢀ
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
5ꢀꢀꢀ
6ꢀꢀꢀ
OMe
Glcꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
78.5
63.1
Glcꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
78.5
63.1
Glcꢀꢀꢀꢀ
104.9
75.9
78.4
71.9
78.6
63.1
Glcꢀꢀꢀꢀ
104.8
75.8
78.2
71.8
78.5
62.9
Glcꢀꢀꢀꢀ
105.1
76.1
78.5
72.0
78.7
63.3
Cymꢀꢀꢀꢀ
100.4
36.4
77.5
83.1
69.0
18.6
58.3
Cymꢀꢀꢀꢀ
100.4
36.4
77.4
83.1
69.0
18.6
58.3
Cymꢀꢀꢀꢀ
100.4
36.4
77.5
83.1
69.0
18.6
58.3
Cymꢀꢀꢀꢀ
100.4
36.5
77.5
83.2
69.0
18.6
58.3
Cymꢀꢀꢀꢀ
98.5
36.9
78.1
83.2
69.2
18.4
58.8
Cymꢀꢀꢀꢀ
98.5
37.0
78.1
83.3
69.2
18.4
58.8
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
6ꢀꢀꢀꢀ
OMe
Dgnꢀꢀꢀꢀꢀ
102.7
32.8
79.8
73.8
71.0
17.9
56.2
Dgnꢀꢀꢀꢀꢀ
102.6
32.8
79.8
73.7
71.8
17.9
56.2
Dgnꢀꢀꢀꢀꢀ
102.7
32.8
79.8
73.7
71.0
17.9
56.2
Dgnꢀꢀꢀꢀꢀ
102.7
32.8
79.8
73.8
71.0
17.9
56.2
Cymꢀꢀꢀꢀꢀ
100.4
36.5
77.5
83.1
69.0
18.6
58.3
Cymꢀꢀꢀꢀꢀ
100.4
36.5
77.5
83.0
69.0
18.6
58.3
1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀ
OMe
Glcꢀꢀꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
78.5
63.1
Glcꢀꢀꢀꢀꢀꢀ
104.4
75.9
78.4
71.9
78.5
63.1
Glcꢀꢀꢀꢀꢀꢀ
104.9
75.9
78.4
71.9
78.5
63.2
Glcꢀꢀꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
78.5
63.2
Dgnꢀꢀꢀꢀꢀꢀ
102.7
32.8
79.8
73.8
71.0
Dgnꢀꢀꢀꢀꢀꢀ
102.7
32.8
79.8
73.8
71.0
1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀ
OMe
17.9
56.2
17.9
56.2
Glcꢀꢀꢀꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
Glcꢀꢀꢀꢀꢀꢀꢀ
105.0
75.9
78.4
71.9
1ꢀꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀꢀ
78.5
63.1
78.6
63.2
Cym: ␤-d-cymaropyranosyl; Dgn: ␤-d-diginopyranosyl; Ole: ␤-d-oleandropyranosyl; Glc: ␤-d-glucopyranosyl.
2.21. Acid hydrolysis of 8
2.22. Amurensioside I (9)
25
Compound 8 (9.6 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the
aglycone (1.3 mg) and a sugar fraction (1.5 mg). HPLC analy-
sis of the sugar fraction under the same conditions as for 1
showed the presence of d-glucose, d-cymarose, d-oleandrose, and
d-diginose; tR (min) d-glucose (7.69, positive optical rotation),
d-oleandrose (9.20, negative optical rotation), d-cymarose (9.22,
positive optical rotation), and d-diginose (9.59, positive optical
rotation).
An amorphous solid; [␣]D −14.0◦ (c 0.10, MeOH); IR ꢁmax
(film) cm−1: 3397 (OH), 2933 (CH), 1637 (C O); HRESITOFMS m/z:
1233.7043 [M+H]+ (calcd for C62H105O24, 1233.6995); 1H and 13C
NMR: see Tables 2–4.
2.23. Acid hydrolysis of 9
Compound 9 (20.9 mg) was subjected to acid hydrolysis as
described for 1 to give several decomposed compounds of the

90
M. Kuroda et al. / Steroids 75 (2010) 83–94
aglycone (4.3 mg) and a sugar fraction (5.5 mg). HPLC analy-
sis of the sugar fraction under the same conditions as for 1
showed the presence of d-glucose, d-cymarose, d-oleandrose, and
d-diginose; tR (min) d-glucose (7.69, positive optical rotation),
d-oleandrose (9.20, negative optical rotation), d-cymarose (9.22,
positive optical rotation), and d-diginose (9.59, positive optical
rotation).
2.26. Amurensioside K (11)
An amorphous solid; [␣]D
+4.30◦ (c 0.10, MeOH); IR ꢁmax
25
(film) cm−1: 3399 (OH), 2931 (CH), 1687 (C O); HRESITOFMS m/z:
1397.7471 [M+Na]+ (calcd for C69H114O27Na, 1397.7397); 1H and
13C NMR (C5D5N): see Tables 2–4.
2.27. Acid hydrolysis of 11
2.24. Amurensioside J (10)
Compound 11 (12.1 mg) was subjected to acid hydrolysis as
described for 1 to give an aglycone (fukujusone, 2.1 mg) and a
sugar fraction (2.6 mg). HPLC analysis of the sugar fraction under
the same conditions as for 1 showed the presence of d-glucose, d-
cymarose, d-oleandrose, and d-diginose; tR (min) d-glucose (7.69,
positive optical rotation), d-oleandrose (9.20, negative optical rota-
tion), d-cymarose (9.22, positive optical rotation), and d-diginose
(9.59, positive optical rotation).
An amorphous solid; [␣]D +2.79◦ (c 0.10, MeOH); UV ꢀmax
25
nm (log ε): 231 (4.01); IR ꢁmax (film) cm−1: 3392 (OH), 2931 (CH),
1714 (C O), 1601 and 1450 (aromatic ring); HRESITOFMS m/z:
1517.7706 [M+Na]+ (calcd for C76H118O29Na, 1517.7655); 1H and
13C NMR: see Tables 2–4.
2.25. Acid hydrolysis of 10
Compound 10 (14.2 mg) was subjected to acid hydrolysis as
described for 1 to give an aglycone (12-O-benzoylisolineolon,
2.28. HSC-2 cell culture assay
3.1 mg) and
a
sugar fraction (5.3 mg). HPLC analysis of the
showed
HSC-2 cells were maintained as monolayer cultures at 37 ◦C
in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL
penicillin G, and 100 ␮g/mL streptomycin sulfate in a humidi-
fied 5% CO2 atmosphere. Cells were trypsinized and inoculated at
6 × 103 to 1.2 × 104 per each 96-microwell plate (Falcon, flat bot-
tom, treated polystyrene, Becton Dickinson, San Jose, CA, U.S.A.)
and incubated for 24 h. After washing once with PBS, they were
sugar fraction under the same conditions as for
1
the presence of d-glucose, d-cymarose, d-oleandrose, and d-
diginose; tR (min) d-glucose (7.69, positive optical rotation),
d-oleandrose (9.20, negative optical rotation), d-cymarose (9.22,
positive optical rotation), and d-diginose (9.59, positive optical
rotation).
Fig. 1. Structures of compunds 1–11.

M. Kuroda et al. / Steroids 75 (2010) 83–94
91
treated for 24 h without or with increasing concentrations of test
compounds. They were then washed once with PBS and incubated
for 4 h with 0.2 mg/mL MTT in DMEM supplemented with 10%
FBS. After the medium was removed, the cells were lysed with
0.1 mL DMSO and the relative viable cell number was determined
by measuring the absorbance at 540 nm of the cell lysate, using Lab-
systems Multiskan (Biochromatic, Helsinki, Finland) connected to a
Star/DOTMatrix printerJL-10 [18,19]. TheIC50 value, which reduces
the viable cell number by 50%, was determined from dose–response
curve.
patterns and coupling constants (Table 1) indicated the presence of
two ␤-d-cymaropyranosyl (4C1) units, a ␤-d-diginopyranosyl (4C1)
unit, and a ␤-d-glucopyranosyl (4C1) unit. The proton resonances
correlated with those of the one-bond coupled carbons using the
HMQC spectrum. The existence of a terminal ␤-d-glucopyranosyl
unit (Glcꢀꢀꢀꢀ), a C-4 substituted ␤-d-diginopyranosyl unit (Dgnꢀꢀꢀ),
and two C-4 substituted ␤-d-cymaropyranosyl units (Cymꢀ, Cymꢀꢀ)
was confirmed by comparison of their carbon chemical shifts
with those of the sugar moieties of reported steroidal glycosides
[16,17,21]. The ␤-orientations of the anomeric centers of the Cymꢀ,
Cymꢀꢀ, Dgnꢀꢀꢀ, and Glcꢀꢀꢀꢀ units were ascertained by the relatively
3
large JH-1, H-2(ax) values of their anomeric protons (7.7–9.7 Hz).
3. Results and discussion
In the HMBC spectrum of 1 (Fig. 2), long-range correlations were
observed between ı 5.13 (H-1 of Glcꢀꢀꢀꢀ) and ı 73.8 (C-4 of Dgnꢀꢀꢀ),
ı 4.65 (H-1 of Dgnꢀꢀꢀ) and ı 83.1 (C-4 of Cymꢀꢀ), ı 5.11 (H-1 of
Cymꢀꢀ) and ı 83.4 (C-4 of Cymꢀ), and between ı 5.29 (H-1 of Cymꢀ)
and ı 77.7 (C-3 of aglycone), establishing the sequence of the
sugar moieties. From the above spectroscopic and chemical data,
the structure of 1 was formulated as 12-O-benzoylisolineolon
3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-
O-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
The fresh roots of A. amurensis were extracted with MeOH, and
the MeOH extract was passed through a Diaion HP-20 column
eluted with 20% MeOH in H2O, EtOH, and EtOAc. Through multi-
ple chromatographic steps over silica gel and ODS silica gel, as well
as preparative HPLC, amurensiosides A (1; 58.9 mg), B (2; 26.8 mg),
D (4; 41.8 mg), E (5; 19.2 mg), F (6; 9.5 mg), I (9; 45.7 mg), and K (11;
16.3 mg) were isolated from the EtOH eluate, and amurensiosides
C (3; 23.9 mg), G (7; 75.6 mg), H (8; 18.0 mg), and J (10; 68.7 mg)
were isolated from the EtOAc eluate (Fig. 1).
Amurensioside B (2) was deduced to be C55H82O20 on the basis
of the HRESITOFMS data (m/z 1085.5277 [M+Na]+). The 1H and
13C NMR spectra of 2 were almost identical to those of 1, except
for the signals due to the H-17 and Me-18 protons and C-12 and
C-20 carbons, suggesting that 2 is an epimer of 1 with regard to
the configuration at C-17, that is, 17␣. This was confirmed by
the presence of a NOESY correlation between ı 3.54 (H-17) and
ı 2.04 (Me-18). Furthermore, acid hydrolysis of 2 with 0.025 M
HCl furnished 12-O-benzoyllineolon [22] as the aglycone, and
d-cymarose, d-diginose, and d-glucose as the sugar residues.
Thus, the structure of 2 was formulated as 12-O-benzoyllineolon
3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-
O-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Amurensioside A (1) was obtained as an amorphous solid and
was shown to have a molecular formula of C55H82O20 on the
basis of the HRESITOFMS data, exhibiting an [M+Na]+ peak at m/z
1085.5225. The IR spectrum of 1 showed absorption bands for
hydroxy groups at 3409 cm−1 and a carbonyl group at 1712 cm−1
.
The UV absorption at 231 nm (log ε 3.70) is consistent with the
presence of an aromatic ring in the structure of 1. The 1H NMR
spectrum of 1 in C5D5N contained three three-proton singlet
signals at ı 2.20, 1.65, and 1.37, and one olefinic proton signal at ı
5.35 (br d, J = 4.1 Hz), which were characteristic of the pregn-5-en-
20-one skeleton, as well as signals for four anomeric protons at ı
5.29 (dd, J = 9.5, 1.6 Hz), 5.13 (d, J = 7.7 Hz), 5.11 (dd, J = 9.5, 1.3 Hz),
and 4.65 (dd, J = 9.7, 1.7 Hz). The methyl carbon signals at ı 18.6
(2× CH3) and 17.9, and proton signals at ı 1.55 (d, J = 6.4 Hz), 1.40
(d, J = 6.3 Hz), and 1.37 (d, J = 6.1 Hz), respectively, were indicative
of 1 possessing three deoxy sugars. In addition, the presence of
a benzoyl group was shown by the following 1H and 13C NMR
signals; ıH 8.37 (2H, br d, J = 7.8 Hz), 7.60 (1H, br dd, J = 7.8, 7.8 Hz),
7.53 (2H, br dd, J = 7.8, 7.8 Hz)/ıC 166.5 (C O), 133.6 (C), 131.2 (C),
130.0 (CH ×2), and 129.1 (CH ×2). Acid hydrolysis of 1 with 0.025 M
HCl gave a pregnane aglycone with a benzoyl group, identified as
12␤-(benzoyloxy)-3␤,8␤,14␤-trihydroxypreg-5-en-20-one (12-
O-benzoylisolineolon, 1a) by comparison with literature data [20],
and d-cymarose, d-diginose, and d-glucose as the carbohydrate
components. Identification of the monosaccharides, including their
absolute configurations, was carried out by direct HPLC analysis
of the hydrolysate, using a combination of refractive index (RI)
and optical rotation (OR) detectors. The 1H–1H COSY and TOCSY
experiments with 1 allowed for the sequential assignments from
H-1 to H2-6 or Me-6 of each monosaccharide. Their signal multiplet
Amurensioside C (3) was isolated as an amorphous solid
with a molecular formula, C54H81NO20, as determined by the HRE-
SITOFMS data (m/z 1064.5435 [M+H]+). The spectral properties of 3
were essentially analogous with those of 1, except for the aromatic
region signals for the benzoyl moiety. The aromatic acid linked to
the aglycone moiety of 3 was suggested to be nicotinic acid by the
1H NMR [ı 9.65 (d, J = 1.8 Hz), 8.92 (dd, J = 4.8, 1.8 Hz), 8.54 (ddd,
J = 8.0, 1.8, 1.8 Hz), and 7.43 (dd, J = 8.0, 4.8 Hz)] and 13C NMR [ı
165.5 (C O), 154.1 (CH), 151.2 (CH), 137.2 (CH), 126.9 (C), and 124.0
(CH)] spectra. The linkage position of the nicotinoyl group to C-12
of the aglycone moiety was ascertained by a long-range correlation
from the H-12 proton at ı 5.20 (dd, J = 12.0, 3.8 Hz) to the carbonyl
carbon resonance of the nicotinoyl moiety at ı 165.5 in the HMBC
spectrum of 3. Thus, 3 was identified as 12-O-nicotinoylisolineolon
3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-
O-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Amurensioside D (4) was obtained as an amorphous solid. On
the basis of the HRESITOFMS (m/z 981.5075 [M+Na]+) data, its
Fig. 2. HMBC correlations of the sugar moiety of 1.

92
M. Kuroda et al. / Steroids 75 (2010) 83–94
molecular formula was determined to be C48H78O19, which was
less than that of 1 by C6H4O, corresponding to a benzoyl unit. When
the 1H and 13C NMR spectra of 4 were compared with those of 1, the
signals assigned to the benzoyl group could not be detected, and
the carbon resonance of C-12 of the aglycone moiety was shifted
upfield by 4.8 ppm and observed at ı 73.8 in 4. All other 1H and
13C NMR signals, including those assignable to the tetraglycoside
moiety, appeared at almost the same positions between the two
compounds. Thus, the structure of 4 was established as isolineolon
3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-
O-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Amurensioside E (5) was isolated as an amorphous solid with a
molecular formula, C48H78O19, which was determined on the basis
of the HRESITOFMS data (m/z 981.4995 [M+Na]+). Acid hydrolysis
of 5 with 0.025 M HCl gave d-cymarose, d-diginose, and d-glucose,
together with several unidentified degradation products; no agly-
cone could be obtained. The molecular formula of 5 was the same
as that of 4, and the 1H and 13C NMR spectra of 5 were essentially
analogous to those of 4 with the exceptions of the signals assignable
to the ring C part of the aglycone. In the HMBC spectrum of 5, the
Me-18 angular methyl protons at ı 1.43 exhibited correlations with
not only two quaternary carbons at ı 50.5 (C-13) and 85.7 (C-14)
but also a methylene carbon at ı 50.2 (C-12), which was associated
with methylene protons at ı 2.11 and 1.89 (H2-12) by analysis of the
HMQC spectrum. In the 1H–1H COSY spectrum, the methylene pro-
ton signals at ı 2.11 and 1.89 exhibited correlations with an oxyme-
thine proton at ı 4.77 (H-11). The oxymethine proton, in turn, dis-
played a correlation with a methine proton at ı 1.78 (d, J = 11.1 Hz,
H-9). These data are consistent with the presence of a hydroxy
group at C-11 of the aglycone moiety of 5. NOE correlations between
ı 4.77 (H-11) and ı 1.43 (Me-18)/ı 1.80 (Me-19) in the PHNOESY
spectrum, and the proton spin-coupling constants between H-
9 and H-11 (3JH-9, H-11 = 11.1 Hz) and between H-11 and H-12
(3JH-11, H-12ax = 11.1 Hz, 3JH-11, H-12eq = 4.1 Hz) indicate that the con-
figuration of the C-11 hydroxy group is ␣. Thus, 5 was defined as 3␤-
[(O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-
O-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranosyl)oxy]-
8␤,11␣,14␤-trihydroxypregn-5-en-20-one.
Amurensioside F (6) was obtained as an amorphous solid,
and its molecular formula was determined to be C62H98O24 by
the HRESITOFMS data (m/z 1249.6392 [M+Na]+). The 1H and 13C
NMR spectral properties of 6 were suggestive of a pregnane
hexaglycoside. Acid hydrolysis of 6 with 0.025 M HCl gave a six-
membered pregnane derivative (6a; C21H28O4) as the aglycone,
and d-cymarose, d-diginose, d-oleandrose and d-glucose as the
carbohydrate components. The pregnane (6a) appeared to have
a structure identical to adonilide, which was isolated by Shimizu
et al. in 1967 [10]. However, no spectroscopic data for adonilide
are available. The structure of 6a, 14␤,20-epoxy-3␤-hydroxypregn-
5-ene-13-carboxylic acid-20-lactone, was identified by combined
analysis of the 1H–1H COSY, HMQC, HMBC, and NOESY spectra
(Figs. 3 and 4).
Fig. 3. Key HMBC correlations of 6a.
six monosaccharide units, including identification of their signal
multiplet patterns and coupling constants. The proton resonances
correlated with those of the corresponding one-bond coupled
carbons using the HMQC and HSQC–TOCSY spectra, leading to
unambiguous assignments of the carbon shifts. Comparison of
the carbon chemical shifts thus assigned with those reported in
the literature [16,17,21], taking into account the known effects
of the O-glycosylation shift, indicate that 6 contains two 4-
substituted ␤-d-oleandropyranosyl (4C1) units (Oleꢀ and Oleꢀꢀ),
two 4-substituted ␤-d-cymaropyranosyl (4C1) units (Cymꢀꢀꢀ and
Cymꢀꢀꢀꢀ), a 4-substituted ␤-d-diginopyranosyl (4C1) unit (Dgnꢀꢀꢀꢀꢀ),
and a terminal ␤-d-glucopyranosyl (4C1) unit (Glcꢀꢀꢀꢀꢀꢀ). The ␤-
orientations of the anomeric centers of these monosaccharides
3
were supported by the relatively large JH-1, H-2ax values of their
anomeric protons (7.8–9.7 Hz). In the HMBC spectrum of 6 (Fig. 5),
correlation peaks were observed between ı 5.12 (H-1 of Glcꢀꢀꢀꢀꢀꢀ
)
and ı 73.8 (C-4 of Dgnꢀꢀꢀꢀꢀ), ı 4.65 (H-1 of Dgnꢀꢀꢀꢀꢀ) and ı 83.1 (C-4
of Cymꢀꢀꢀꢀ), ı 5.09 (H-1 of Cymꢀꢀꢀꢀ) and ı 83.2 (C-4 of Cymꢀꢀꢀ), ı
5.28 (H-1 of Cymꢀꢀꢀ) and ı 83.0 (C-4 of Oleꢀꢀ), ı 4.91(H-1 of Oleꢀꢀ)
and ı 83.3 (C-4 of Oleꢀ), and between ı 4.80 (H-1 of Oleꢀ) and ı
77.3 (C-3 of aglycone). All of these data for 6 are consistent with
the structure adonilide 3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-
diginopyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-O-␤-d-
cymaropyranosyl-(1 → 4)-O-␤-d-oleandropyranosyl-(1 → 4)-␤-d-
oleandropyranoside.
Amurensioside G (7), isolated as an amorphous solid, showed
an [M+Na]+ ion at m/z 1219.6228 in HRESITOFMS, corresponding
to the empirical molecular formula C61H96O23. Comparison of
the 1H and 13C NMR spectral data of 7 with those of 6 revealed
that the structures of the ring A and B portions of the aglycone
and the hexaglycoside moiety attached to C-3 of the aglycone
are identical to that of 6. Analysis of the 1H–1H COSY, 2D TOCSY,
The severe overlapping of the proton signals for the sugar
moieties of 6 excluded the possibility of a complete assignment
in a straightforward way using conventional 2D NMR methods,
such as the 1H–1H COSY, 2D TOCSY, and HMQC spectra. Analysis
of the 1D TOCSY spectra followed by interpretation of the 1H–1H
COSY, HSQC–TOCSY, and HMQC spectra allowed for the exact
sugar sequences of the sugars to be determined. The 1H NMR
subspectra of the individual monosaccharide units were obtained
by using selective irradiation of easily identifiable anomeric
proton signals at ı 5.28, 5.12, 5.09, 4.91, 4.80, and 4.65, as well as
irradiation of other non-overlapping proton signals at ı 1.55, 1.46,
1.42, 1.39 and 1.36 in a series of 1D TOCSY experiments [23,24].
Subsequent analysis of the 1H–1H COSY spectrum resulted in the
sequential assignments of all the proton resonances due to the
Fig. 4. Key NOE correlations of 6a.

M. Kuroda et al. / Steroids 75 (2010) 83–94
93
Fig. 5. HMBC correlations of the sugar moiety of 6.
HMQC, HMBC, and NOESY spectra of 7 allowed for the structure
of the aglycone moiety of 7 to be assigned as 3␤-hydroxy-
Amurensioside I (9) was shown to have a molecular formula of
C62H104O24 on the basis of HRESITOFMS (m/z 1233.7043 [M+H]+).
The 1H and 13C NMR signals of the aglycone part of 9 were similar to
those of pregn-5-ene-3␤,14␤,20-triol (calogenin) 3-O-glycosides
[25]. However, the signals for the C-12 methylene group at ıH 1.17
and 1.74, and ıC 37.6 in the calogenin glycosides were displaced by
those assigned to a hydroxymethine group at ıH 4.88 and ıC 74.8 in
9. An HMBC correlation between ı 1.84 (Me-18) and ı 74.8 (C-12)
is consistent with the presence of a hydroxy group at C-12. The
proton spin-coupling constant between H-12 and H2-11 (2.6 Hz),
along with an NOE correlation between H-12 and Me-18, indicate
that the C-12 hydroxy group is an ␣-axial-orientated configuration.
Unfortunately, no information concerning the configurations at
C-17 and C-20 of the aglycone of 9 could be obtained from the
NOE spectral data. The configurations at C-17 and C-20 remain to
be determined. As for the sugar sequence of 9, the hexaglycoside
attached to C-3 of the aglycone was shown to be identical with
that of 6–8 on the basis of the 1H and 13C NMR, and HMBC spectral
data, and the results of acid hydrolysis. Thus, the structure of
18-norpregna-5,13(14)-diene-12,20-dione
(fukujusonorone),
which was reported by Shimizu et al. in 1969 [12]. Although
fukujusonorone could not be obtained by acid hydrolysis of 7,
the structure was unequivocally identified by analysis of the
1H–1H COSY, 2D TOCSY, HMQC, HMBC, and NOESY spectra. The
structure of 7 was characterized as fukujusonorone 3-O-␤-d-
glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-O-␤-d-
cymaropyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-O-␤-
d-oleandropyranosyl-(1 → 4)-␤-d-oleandropyranoside.
Amurensioside H (8) had a molecular formula of C61H94O24 on
the basis of HRESITOFMS (m/z 1233.6001 [M+Na]+). The 1H and 13
C
NMR spectral features of 8 showed a close similarity to those of 7.
On comparison of the 1H and 13C NMR spectra of 8 with those of
7, the methine proton and carbon signals assigned to H-17/C-17 at
ıH 4.05 and ıC 55.5, were missing from the spectrum of 8. Instead,
a quaternary carbon signal bearing a hydroxy group was observed
at ı 122.0, accompanied by significant upfield shifts of C-13 (by
15.9 ppm) and C-20 (by 25.1 ppm). Furthermore, the coupled
methylene proton signals at H2-15 and H2-16 at ı 2.05 and 1.98
were replaced by a pair of olefinic proton signals at ı 6.40 and 7.41,
which were associated with the corresponding olefinic carbon sig-
nals at ı 119.5 and 137.1, respectively. Thus, 8 was assigned as the
C-15/C-16 dehydro and C-17 hydroxy derivative of 7. The presence
of a double bond between C-15 and C-16, and a hydroxy group at
C-17 was supported by HMBC correlations between ı 2.48 (Me-21)
and ı 122.0 (C-17)/ı 184.2 (C-20), ı 6.40 (H-15) and ı 120.8 (C-13)/ı
158.8 (C-14)/C-17, and between ı 7.41 (H-16) and C-13/C-14/C-17.
The hexaglycoside attached to C-3 of the aglycone was confirmed
to be the same as that of 6 and 7 by analysis of the HMBC spectrum
of 8, and the results of acid hydrolysis. Thus, the structure of 8
was formulated as 3␤-[(O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-
diginopyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-O-␤-
d-cymaropyranosyl-(1 → 4)-O-␤-d-oleandropyranosyl-(1 → 4)-
␤-d-oleandropyranosyl)oxy]-17-hydroxy-18-norpregna-5,13,15-
triene-12,20-dione. The configuration at C-17 remains to be
determined.
9
was established as 12␣,14␤,20-trihydroxypregn-5-en-3␤-yl
O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-diginopyranosyl-(1 → 4)-O-
␤-d-cymaropyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-
O-␤-d-oleandropyranosyl-(1 → 4)-␤-d-oleandropyranoside.
Amurensioside J (10) was obtained as an amorphous solid,
and its molecular formula was determined to be C76H118O29
on the basis of HRESITOFMS (m/z 1517.7706 [M+Na]+). The 1H
NMR spectrum of 10 showed signals for three tertiary methyl
groups at ı 2.19, 1.64, and 1.37, one olefinic proton at ı 5.44 (br
d, J = 4.4 Hz), and five aromatic protons of a benzene ring at ı 8.38
(2H, br d, J = 7.8 Hz), 7.59 (1H, br dd, J = 7.8 Hz) and 7.53 (2H, br dd,
J = 7.8 Hz), as well as signals for seven anomeric protons at ı 5.29
(br d, J = 9.7 Hz), 5.13 (d, J = 7.8 Hz), 5.10 (br d, J = 9.5 Hz), 4.91 (br d,
J = 9.7 Hz), 4.89 (br d, J = 9.7 Hz), 4.83 (dd, J = 9.6, 1.3 Hz) and 4.66 (br
d, J = 8.9 Hz), six methoxy protons at ı 3.60, 3.54, 3.51, 3.50, 3.41
and 3.39 (each s), and six secondary methyl protons at ı 1.53 (d,
J = 6.3 Hz), 1.46 (d, J = 6.1 Hz), 1.42 (d, J = 6.0 Hz), 1.42 (d, J = 6.1 Hz),
1.35 (d, J = 6.5 Hz) and 1.33 (d, J = 6.8 Hz). Acid hydrolysis of 10 with
0.025 M HCl gave 12-O-benzoylisolineolon (1a) as the aglycone,
Fig. 6. HMBC correlations of the sugar moiety of 10.

94
M. Kuroda et al. / Steroids 75 (2010) 83–94
Table 5
natural product chemistry. The aglycones of 5, 8, and 9 are new
Cytotoxic activity of compounds 1–11 against HSC-2 cells.
polyoxygenated pregnane derivatives. Although the aglycones
of 6 and 7, adonilide and fukujusonorone, respectively, were
reported in the 1960s, their glycosides such as 6 and 7 have not
been previously reported. The oligoglycosides attached to C-3 of
the aglycone of 1–11 are novel tetra-, hexa-, and heptaglycosides
containing deoxysugars characteristic of plant pregnane and
cardiotonic glycosides.
Compounds
IC50 (␮g/mL)
Compounds
IC50 (␮g/mL)
1
2
3
4
5
6
66
26
>120
47
58
>120
7
8
9
10
>120
>120
>120
>120
>120
13
11
Melpharan
HSC-2 (human oral squamous cell carcinoma).
References
[1] Tsukamoto Y. The grand dictionary of horticulture, vol. 3. Tokyo: Shogakukan;
1988. pp. 89–96.
and d-cymarose, d-diginose, d-oleandrose, and d-glucose as the
carbohydrate moieties. These data suggest that 10 is a 12-O-
benzoylisolineolon heptaglycoside. In the 13C NMR spectrum of 10,
the signal due to C-3 of the aglycone residue was observed at ı 77.9,
indicating that the sugar is linked to C-3, as in 1. Using the same
procedures as described for 6, all the 1H and 13C NMR signals arising
from the sugar moieties of 10 were assigned to three 4-substituted
␤-d-oleandropyranosyl (4C1) units (Oleꢀ, Oleꢀꢀ, and Oleꢀꢀꢀ), two 4-
substituted ␤-d-cymaropyranosyl (4C1) unit (Cymꢀꢀꢀꢀ and Cymꢀꢀꢀꢀꢀ),
a 4-substituted ␤-d-diginopyranosyl (4C1) unit (Dgnꢀꢀꢀꢀꢀꢀ), and a
terminal ␤-d-glucopyranosyl (4C1) unit (Glcꢀꢀꢀꢀꢀꢀꢀ). In the HMBC
spectrum of 10 (Fig. 6), long-range correlations were observed
between ı 5.13 (H-1 of Glcꢀꢀꢀꢀꢀꢀꢀ) and ı 73.8 (C-4 of Dgnꢀꢀꢀꢀꢀꢀ), ı 4.66
[2] Watanabe K, Mimaki Y, Sakuma C, Sashida Y.
A novel polyoxygenated
spirostanol trisdesmoside from the rhizomes of Helleborus orientalis. Chem Lett
2002;8:772–3.
[3] Watanabe K, Mimaki Y, Sakagami H, Sashida Y. Bufadienolide and
spirostanol glycosides from the rhizomes of Helleborus orientalis. J Nat Prod
2003;66:236–41.
[4] Mimaki Y, Watanabe K, Sakuma C, Sakagami H, Sashida Y. Novel polyoxy-
genated spirostanol glycosides from the rhizomes of Helleborus orientalis. Helv
Chim Acta 2003;86:398–407.
[5] Watanabe K, Sakagami H, Mimaki Y. Four new steroidal saponins from the
rhizomes of Helleborus orientalis. Heterocycles 2005;65:775–85.
[6] Watanabe K, Mimaki Y, Sakagami H, Sashida Y. Cycloartane glycosides from the
rhizomes of Cimicifuga racemosa and their cytotoxic activities. Chem Pharm Bull
2002;50:121–5.
[7] Mimaki Y, Nadaoka I, Yasue M, Ohtake Y, Ikeda M, Watanabe K, et al. Neocimi-
cigenosides A and B, cycloartane glycosides from the rhizomes of Cimicifuga
racemosa and their effects on CRF-stimulated ACTH secretion from AtT-20 cells.
J Nat Prod 2006;69:829–32.
[8] Watanabe K, Mimaki Y, Sakuma C, Sashida Y. Eranthisaponins A and B, two new
bisdesmosidic triterpene saponins from the tubers of Eranthis cilicica. J Nat Prod
2003;66:879–82.
(H-1 of Dgnꢀꢀꢀꢀꢀꢀ) and ı 83.1 (C-4 of Cymꢀꢀꢀꢀꢀ), ı 5.10 (H-1 of Cymꢀꢀꢀꢀꢀ
)
and ı 83.2 (C-4 of Cymꢀꢀꢀꢀ), ı 5.29 (H-1 of Cymꢀꢀꢀꢀ) and ı 82.9 (C-4 of
Oleꢀꢀꢀ), ı 4.89 (H-1 of Oleꢀꢀꢀ) and ı 83.0 (C-4 of Oleꢀꢀ), ı 4.91 (H-1 of
Oleꢀꢀ) and ı 83.2 (C-4 of Oleꢀ), and between ı 4.83 (H-1 of Oleꢀ) and ı
77.9 (C-3 of aglycone). Thus, the structure of 10 was characterized
as 12-O-benzoyllineolon 3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-
diginopyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-O-␤-d-
cymaropyranosyl-(1 → 4)-O-␤-d-oleandropyranosyl-(1 → 4)-O-␤-
d-oleandropyranosyl-(1 → 4)-␤-d-oleandropyranoside.
[9] Kuroda M, Uchida S, Watanabe K, Mimaki Y. Chromones from the tubers of
Eranthis
cilicica
and
their
antioxidant
activity.
Phytochemistry
2009;70:288–93.
[10] Shimizu Y, Sato Y, Mitsuhashi H. Isolation and structure of adonilide. Chem
Pharm Bull 1967;15:2005–6.
[11] Shimizu Y, Sato Y, Mitsuhashi H. Isolation and structures of new pregnane
derivatives from Adonis amurensis. Chem Pharm Bull 1969;17:2391–4.
[12] Shimizu Y, Sato Y, Mitsuhashi H. Isolation and characterization of fukuju-
sonorone, an 18-norpregnane derivative from Adonis amurensis. Experientia
1969;25:1129–30.
[13] Ponomarenko AA, Komissarenko NF, Stukkei KL. Cardenolides from Adonis
amurensis. Khim Prir Soedin 1971;7:848–9.
[14] Shimizu Y, Sato Y, Mitsuhashi H. A study of the chemical constituents of Adonis
amurensis. Lloydia 1978;41:1–16.
[15] Ponomarenko AA, Komissarenko NF, Stukkei KL. Coumarins from Adonis
amurensis. Khim Prir Soedin 1971;5:661–2.
[16] Liu Y, Tang W, Yu S, Qu J, Liu J, Liu Y. Eight new C-21 steroidal glycosides from
Dregea sinensis var. corrugata. Steroids 2007;72:514–23.
[17] Bai H, Li W, Koike K, Satou T, Chen Y, Nikaido T. Cynanosides A–J, ten novel preg-
nane glycosides from Cynanchum atratum. Tetrahedron 2005;61:5797–811.
[18] Furuya S, Takayama F, Mimaki Y, Sashida Y, Satoh K, Sakagami H. Cytotoxic
activity of steroidal saponins against human oral tumor cell lines. Anticancer
Res 2000;20:4189–94.
[19] Furuya S, Takayama F, Mimaki Y, Sashida Y, Satoh K, Sakagami H. Cytotoxic
activity of saponins from Camassia leichtlinii against human oral tumor cell
lines. Anticancer Res 2001;21:959–64.
Amurensioside K (11) was determined to be C69H114O27 on
the basis of HRESITOFMS (m/z 1387.7471 [M+Na]+). The deduced
molecular formula of 11 was lower than that of 10 by C7H5O2. When
the 1H and 13C NMR spectra of 11 were compared with those of 10,
the signals assignable to the benzoyloxy moiety attached to C-12 of
the aglycone were absent from the spectrum of 11. Acid hydrolysis
of 11 with 0.025 M HCl gave 3␤,8␤,14␤-trihydroxypregnan-5-
en-20-one (fukujusone) [14] as the aglycone, and d-cymarose,
d-diginose, d-oleandrose, and d-glucose as the carbohydrate
moieties. Analysis of the HMBC spectrum of 11 and the results of
acid hydrolysis indicate that the heptaglycoside attached to C-3 of
the aglycone is the same as that of 10. Thus, the structure of 11 was
established as fukujusone 3-O-␤-d-glucopyranosyl-(1 → 4)-O-␤-
d-diginopyranosyl-(1 → 4)-O-␤-d-cymaropyranosyl-(1 → 4)-O-␤-
d-cymaropyranosyl-(1 → 4)-O-␤-d-oleandropyranosyl-(1 → 4)-O-
␤-d-oleandropyranosyl-(1 → 4)-␤-d-oleandropyranoside.
[20] Warashina T, Noro T. Cardenolide and oxypregnane glycosides from the root of
Asclepias incarnata L. Chem Pharm Bull 2000;48:516–24.
Amurensiosides A–K (1–11) were evaluated for their cyto-
toxic activity against HSC-2 cells, using a modified MTT assay
method (Table 5). Although 3 and 6–11 did not show any apparent
cytotoxicity at a sample concentration of 120 ␮g/mL, 1, 2, 4, and
5 were moderately cytotoxic to HSC-2 cells with IC50 values of 66,
26, 47, and 58 ␮g/mL, respectively, while that of melphalan used
as a positive control was 13 ␮g/mL.
The present phytochemical study of the roots of A. amurensis
resulted in the isolation and characterization of 11 new pregnane
glycosides named amurensiosides A–K. The structures of these
new compounds are notable for the following viewpoints of
[21] Abe F, Yamauchi T. Digitoxigenin oleandroside and 5␣-adymerin in the leaves
of Nerium odorum (Nerium 9). Chem Pharm Bull 1978;26:3023–7.
[22] Kitagawa I, Zhang RS, Park JD, Baek NI, Takeda Y, Yoshikawa M, et al. Indone-
sian medicinal plants. I. Chemical structures of calotroposides A and B, two
new oxypregnane-oligoglycosides from the root of Calotropis gigantea (Ascle-
piadaceae). Chem Pharm Bull 1992;40:2007–13.
[23] Yokosuka A, Mimaki Y, Sakuma C, Sashida Y. New glycosides of the campesterol
derivative from the rhizomes of Tacca chantrieri. Steroids 2005;70:257–65.
[24] Yokosuka A, Kawakami S, Haraguchi M, Mimaki Y. Stryphnosides A–F, six new
triterpene glycosides from the pericarps of Stryphnodendron fissuratum. Tetra-
hedron 2008;64:1474–81.
[25] Pawar RS, Shukla YJ, Khan IA. New calogenin glycosides from Hoodia gordonii.
Steroids 2007;72:881–91.