Three new polyoxypregnane glycosides from Marsdenia tenacissima

Article abstract of DOI:10.1002/hlca.200590208

Three new polyoxypregnane glycosides, marsdenosides I-K (1-3), were isolated from the stem of Marsdenia tenacissima. The structures were elucidated on the basis of in-depth spectroscopic analyses, and by means of chemical evidence. Marsdenoside I (=(3β,5α

Full text of DOI:10.1002/hlca.200590208

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Three New Polyoxypregnane Glycosides from Marsdenia tenacissima
by Jun Deng, Zhixin Liao, and Daofeng Chen*
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 200032, P. R. China
(phone: +86-21-54237453; fax: +86-21-64170921; e-mail: dfchen@shmu.edu.cn)
Three new polyoxypregnane glycosides, marsdenosides I–K (1–3), were isolated from the stem of Mars-
denia tenacissima. The structures were elucidated on the basis of in-depth spectroscopic analyses, and by
means of chemical evidence. Marsdenoside I (=(3b,5a,11a,12b,14b,17a,20R)-3-[(2,6-dideoxy-4-O-(6-deoxy-3-
O-methyl-b-D-allopyranosyl)-3-O-methyl-b-D-arabino-hexopyranosyl)oxy]-8,20 :11,20-diepoxypregnane-12,14-
diol; 1) is the first C21 steroidal glycoside with a rigid cage. Also, the isolation of 1 demonstrated that tenacigenin
A (1a) is a true natural product as well, rather than an artifact.
Introduction. – Marsdenia tenacissima (ROXB.) WIGHT. et ARN. (Asclepiadaceae) is a
perennial climber widespread from tropical to subtropical Asia. Its stem, known as
’tong-guang-san’, has long been used for the treatment of asthma, cancer, trachitis, ton-
sillitis, pharyngitis, cystitis, and pneumonia in Chinese folk medicine [1]. The crude
CHCl3 extract from the stem of M. tenacissima was reported to exhibit mild anti-asth-
matic properties [2]. The phytochemical investigations of M. tenacissima have focused
on polyoxypregnane constituents because of their antitumor activities and high con-
tents in the plant [2–10]. We have previously reported some polyoxypregnane glyco-
sides from M. tenacissima [11][12]. In this paper, we report the isolation and character-
© 2005 Verlag Helvetica Chimica Acta AG, Zürich

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ization of three new such polyoxypregnane glycosides, marsdenosides I–K (1–3), and
of two known glycosides.
Results and Discussion. – Compounds 1–3 gave rise to positive Liebermann–Bur-
chard, Keller–Kiliani, and xanthydrol reactions, indicating that they were all steroidal
glycosides containing 2-deoxy sugar moieties [13–15].
Marsdenoside I (1) had the molecular formula C35H56O12 based on the quasi-molec-
ular ion at m/z 707.3414 ([M+K]+) in its HR-ESI mass spectrum. The IR spectrum of 1
displayed absorption bands for OH (3480 and 3440 cmꢀ1) and CꢀO moieties (1028,
1058 cmꢀ1), but not for C=O groups.
The 1H-NMR spectrum of 1 (see Table 1 in the Exper. Part) displayed two anomeric
signals at d(H)4.80 (d, J=8.3, 1 H) and 4.60 (dd, J=9.7, 1.7 Hz, 1 H), with correspond-
ing 13C-NMR signals at d(C) 98.9 and 96.9, respectively, suggesting that 1 was a disac-
charide glycoside. Both glycosidic linkages were b-oriented, as deduced from the cou-
pling constants (8.3 and 9.7 Hz) of the two anomeric signals. The 13C-NMR spectro-
scopic data ascribed to the sugar moiety of 1 (see Table 2 in the Exper. Part) were
the same as those reported for pachybiose1) [16], which was further confirmed by
examination of the corresponding TOCSY, NOESY, HMQC, and HMBC spectra
(Figs. 1 and 2). Mild acid hydrolysis of 1 gave only one sugar fragment, which was iden-
tified as pachybiose by paper chromatography and TLC comparison with an authentic
sample.
Fig. 1. Key HMBC correlations for 1
For the aglycone moiety of 1, 21 C-atoms were left, suggesting a C21 steroid. This
was supported by the three Me singlets at d(H) 1.14, 1.18, and 1.22 in the 1H-NMR spec-
trum. The 1H- and 13C-NMR data of the aglycone were in good agreement with those of
tenacigenin A (1a) [2][3][17], except C(2), C(3), and C(4), which were shifted by Dd
ꢀ2.2, +5.0, and ꢀ2.9 ppm, respectively, due to glycosylation. The resonance at d(H)
1.18 (s, Me(21)) indicated that C(20) was quaternary. The HMBC correlations of
d(C) 99.3 with both d(H) 1.18 (Me(21)) and 1.86 (HꢀC(17)) indicated that this 13C-
NMR resonance was due to C(20). Consequently, C(20) was linked to two O-atoms
1
[17]. In the H,1H-COSY spectrum, the signal at d(H) 4.28 (br. d, J=2.6 Hz, 1 H)
was coupled with those at 3.98 (d, J=2.2 Hz, 1 H) and 2.43 (br. s, HꢀC(9)), while
the signal at d(H) 3.98 was correlated only with that at d(H) 4.28, which substantiated
1
)
Pachybiose=2,6-dideoxy-4-O-(6-deoxy-3-O-methyl-b-D-allopyranosyl)-3-O-methyl-b-D-arabino-hexopyr-
anose.

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Fig. 2. Key NOESY correlations for 1
that the signal at d(H)4.28 was due to HꢀC(11), and that the one at d(H) 3.98 was due
to HꢀC(12).
The HMBC correlations between d(H) 4.28 (HꢀC(11)) and d(C) 99.3 (C(20)),
between d(H) 4.28 and d(C) 24.6 (C(21)), and between d(H) 1.18 (Me(21)) and d(C)
71.3 (C(11)) indicated that there was an O-atom between C(20) and C(11). From the
HMBC correlations between d(H) 1.86 (HꢀC(17)) and d(C) 82.0, between d(H) 1.22
(Me(18)) and d(C) 82.0, and between d(H) 3.98 (HꢀC(12)) and d(C) 82.0, the reso-
nance at d(C) 82.0 was ascribed to C(14). Similarly, from the correlations between
d(H) 4.28 (HꢀC(11)) and d(C) 77.9, and between d(H) 2.43 (HꢀC(9)) and d(C) 77.9,
the resonance at d(C) 77.9 was assigned to C(8). The HMBC correlation between
d(H) 1.18 (Me(21)) and d(C) 77.9 (C(8)) supported that C(20) was also linked to
C(8) via an oxy bridge (Fig. 1). Finally, the NOESY correlation between d(H) 1.22
(Me(18)) and 1.86 (HꢀC(17)) confirmed that C(17) was a-configured (Fig. 2). From
these data, the aglycone of 1 was identified as tenacigenin A (1a).
Mild acid hydrolysis of 1 yielded an aglycone, whose molecular weight and 1H- and
13C-NMR spectroscopic data were identical with those reported for tenacigenin A (1a)
[17]. The observed shifts at C(2), C(3), and C(4) (see above) indicated that the oligo-
saccharide chain was linked to the 3-OH group of the aglycone [18], which was sup-
ported by HMBC correlations between d(H) 4.60 (HꢀC(1)Ole2) and d(C) 77.1 (C(3)),
and between d(H) 3.66–3.71 (HꢀC(3)) and d(C) 96.9 (C(1)Ole) (see Fig. 1).
From the above data, the structure of marsdenoside I (1) was established as 3-O-
pachybiosyl-tenacigenin A3). Compound 1 is the first reported glycoside of tenacigenin
A (1a). The isolation of 1 demonstrates that tenacigenin A is a true natural product, and
not, as proposed earlier [4], an artifact formed under acidic or basic conditions.
The HR-ESI mass spectrum of marsdenoside J (2) showed a quasi-molecular ion
peak at m/z 883.3889 ([M+K]+), in accord with the molecular formula C45H64O15.
The 1H- and 13C-NMR spectroscopic data of the sugar moiety of 2 were in good agree-
ment with those of 1 (see Tables 1 and 2 in the Exper. Part), so it also contained a pachy-
biosyl residue. The 13C-NMR data of the aglycone of 2 (Table 2) resembled those of the
1
11,12-diester of tenacigenin B previously reported [5]. The H-NMR signals at d(H)
5.31 (t, J=10.1, HbꢀC(11)) and 4.95 (d, J=10.1, HaꢀC(12)) indicated two ester-bearing
2
)
)
Sugar abbreviations: Allo=allose, Ole=oleandrose, Glc=glucose.
For systematic names, see the Exper. Part.
3

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HELVETICA CHIMICA ACTA – Vol. 88 (2005)
methine groups. The signals at d(H)1.64 (s, 3 H) and d(C) 20.2 suggested the presence
of an acetyl (Ac) group. The signals at d(H) 6.75 and 7.09 (2d, J=8.5 Hz each, 2×1 H)
and the 13C-NMR singlet at d(C) 155.1 indicated that 2 contained a 4-hydroxyphenyl
group. In the HMBC spectrum, the aromatic resonances at d(C) 125.4 and 130.7
were correlated with the signal at d(H) 3.39 (s, 2 H), and d(H) 7.09 correlated with
the d(C) 41.4 (Fig. 3). Also, the HMQC spectrum displayed a cross-peak between
d(H) 3.39 and d(C) 41.4, and the HMBC spectrum showed a correlation between
d(H) 3.39 and d(C) 168.4. Therefore, a CH2 group (d(H) 3.39, d(C) 41.4) was located
between the 4-hydroxyphenyl moiety and the C=O group (d(C) 168.4). Hence, 2 con-
tained a (4-hydroxyphenyl)acetyl (HPA) group.
Fig. 3. Key HMBC and NOESY correlations for 2
As shown in Fig.3, the HMBC correlations between d(H)5.31 (HbꢀC(11)) and d(C)
168.4 (C(1’)HPA) and between d(H)4.95 (HaꢀC(12)) and d(C)170.7 (C(1’’)Ac) supported
that the HPA and Ac groups were at C(11) and C(12), respectively. Coupling constants
of 10.1 Hz for HꢀC(9), HꢀC(11) and HꢀC(12) indicated that these H-atoms were all
in an axial positions. The NOESY correlations between d(H) 1.05 (Me(18)) and 2.90
(HꢀC(17)), and between d(H) 2.19 (Me(21)) and 4.95 (HaꢀC(12)) confirmed that
the C(17) side chain was in an a-orientation (Fig. 3). The glycosyl-induced shifts at
C(2), C(3), and C(4) of the aglycone indicated that the oligosaccharide chain was
attached at the 3-OH group [18], which was confirmed by the HMBC correlation
between d(H)4.58 (HꢀC(1)ole) and d(C)76.2 (C(3)). From the above data, the structure
of 2 was determined as 12-O-acetyl-11-O-[(4-hydroxyphenyl)acetyl]-3-O-pachybiosyl-
tenacigenin B3).
Marsdenoside K (3) had the molecular formula C50H72O19 on the basis of its quasi-
molecular ion at m/z 1015.4309 ([M+K]+) in the HR-ESI mass spectrum. Its 1H-NMR
spectrum exhibited three anomeric signals at d(H)4.36 (d, J=6.7, 1 H), 4.53 (dd, J=9.8,
1.6, 1 H), and 4.78 (d, J=6.7 Hz, 1 H), which were correlated with d(C)104.3, 97.1, and
100.1, respectively, in the HMQC spectrum, thus, indicating three sugar units. The unit
with an anomeric double doublet was a 2-deoxypyranose. The two Me signals at d(H)

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1.26 (d, J=5.8 Hz, 3 H) and 1.31 (d, J=5.6 Hz, 3 H), and the two MeO groups at d(H)
3.35 and 3.57 (s, 3 H each) suggested that two of the three sugar units were 6-deoxy-3-
O-methyl-pyranoses [19]. The signals in the 1H-NMR, 13C-NMR, 1H,1H-COSY,
HMQC, HMBC, and NOESY spectra of 3 due to these two 6-deoxy-3-O-methyl-pyra-
nose units were identical with those in the spectra of 1, except for the chemical shifts of
C(3), C(4), and C(5) of the 6-deoxy-3-O-methyl-allose, with Dd values of ꢀ0.6, +7.8,
and ꢀ2.2 ppm, respectively. The other sugar unit was identified as a glucopyranose
on the basis of its characteristic coupling pattern of the all-axial ring H-atoms and
CH2(6) (d(H) 3.61–3.74 and 3.86–3.93, both ABM spin systems) [20].
When 3 was exposed to b-glucosidase, enzymolysis yielded only one sugar fragment
of glucose, in accord with the above experiments. The b-linkages of each of the sugars
1
were evident from the H-NMR coupling constants of the anomeric signals. The
sequence of the sugar units was deduced as b-
methyl-b-D-allopyranosyl-(1 ! 4)-b-D-oleandropyranosyl from the correlations
between the following pairs: HꢀC(1)Glc/C(4)Allo, HꢀC(1)Allo/C(4)Ole, and HꢀC(1)Ole
D
-glucopyranosyl-(1 ! 4)-6-deoxy-3-O-
/
C(3)Steroid in the HMBC spectrum. Thus, the sugar moiety of 3 was identified as neocon-
durangotriose4), as further supported by the fact that the 13C-NMR data of the glycone
of 3 (in (D5)pyridine; Table 2) coincided well with those reported in the literature [21].
The 1H- and 13C-NMR spectroscopic data of the aglycone of 3 matched those of 2,
except that the signals of the HPA group of 2 were replaced with those of a benzoyl
(Bz) group in 3. The Bz group was identified from d(H) 7.42 (t, J=7.7, HꢀC(4’,6’)),
7.55 (t, J=7.4, HꢀC(5’)), and 7.95 (d, J=7.2 Hz, HꢀC(3’,7’)). HMBC Correlations
between d(H) 5.60 (t, J=10.1, HbꢀC(11)) and d(C) 166.0 (C(1’)Bz), and between
d(H) 5.12 (d, J=10.1 Hz, HaꢀC(12)) and d(C) 170.7 (C(1’’)Ac) indicated that the Bz
and Ac groups were at C(11) and C(12), respectively. The HMBC correlation between
d(H) 4.53 (HꢀC(1)ole) and d(C) 76.5 (C(3)) suggested that the oligosaccharide chain
was attached to the 3-OH group of the steroid. Thus, the structure of 3 was established
as 12-O-acetyl-3-O-neocondurangotriosyl-tenacigenin B3). The selective enzymatic
degradation of the neocondurangotriosyl moiety of 3 with b-glucosidase furnished
the corresponding pachybiosyl conjugate 3a, known as tenacissoside I (3a) [8], which
further corroborated the structure of 3.
Finally, the two known compounds were identified as tenacissoside F and tenacisso-
side D by comparing their ORD, UV, IR, 1H-NMR, 13C-NMR, and MS data with those
reported in the literature [8][5].
The authors are grateful to Prof. Jinlan Yuan, Department of Pharmaceutical Chemistry, School of Phar-
macy, Tongji Medical College, Huazhong University of Science and Technology, P. R. China, for kindly provid-
ing an authentic sample of pachybiose.
Experimental Part
General. Anal. TLC was performed on precoated silica gel F254 plates (0.15 mm; Yantai Institute of Chemical
Technology) and RP-18 F254 S plates (Merck). For prep. TLC, precoated silica gel F254 plates (0.5 mm; Yantai
Institute of Chemical Technology) were used. Spots were visualized by spraying with 0.5% vanillin in conc.
4
)
Systematic name: b-
D
-glucopyranosyl-(1 ! 4)-6-deoxy-3-O-methyl-b- -allopyranosyl-(1 ! 4)-2,6-dide-
D
oxy-3-O-methyl-b- -arabino-hexopyranose
D

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HELVETICA CHIMICA ACTA – Vol. 88 (2005)
Table 1. 1H-NMR Data of the Sugar Moieties of 1–3. At 500 MHz, in CDCl3; d in ppm, J in Hz. Abbreviations:
Allo, allosyl; Ole, oleandrosyl; Glc, glucosyl.
1
2
3
Ole:
HꢀC(1)
4.60 (dd, J=9.5, 1.6)
1.42–1.51 (m),
2.33 (dd, J=11.6, 3.7)
3.38–3.43 (m)
3.36 (t, J=7.4)
3.32–3.38 (m)
1.37 (d, J=5.0)
3.38 (s)
4.58 (dd, J=8.2, 2.9)
1.46–1.56 (m),
2.31 (ddd, J=12.1, 4.8, 1.7)
3.38–3.44 (m)
3.37 (t, J=7.3)
3.34–3.38 (m)
4.53 (dd, J=9.8, 1.6)
1.38–1.50 (m),
2.25 (br. d, J=8.7)
3.27–3.35 (m)
3.26 (t, J=8.4)
3.23–3.31 (m)
1.31 (d, J=5.6)
3.35 (s)
CH2(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
3-MeO
Allo:
1.40 (d, J=6.2)
3.39 (s)
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
4.80 (d, J=8.3)
3.49 (br. d, J=8.2)
3.80 (t, J=2.7)
3.19 (br. d, J=8.7)
3.52–3.59 (m)
1.26 (d, J=6.2)
3.67 (s)
4.80 (d, J=8.3)
3.49 (dd, J=8.2, 2.8)
3.80 (t, J=2.9)
3.20 (br. d, J=7.0)
3.53–3.60 (m)
1.27 (d, J=6.2)
3.67 (s)
4.78 (d, J=6.7)
3.44 (br. d, J=7.7)
3.88 (br. s)
3.25 (br. d, J=8.4)
3.84–3.88 (m)
1.26 (d, J=5.8)
3.57 (s)
3-MeO
Glu:
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
4.36 (d, J=6.7)
3.33 (t, J =7.2)
3.52 (t, J=7.2)
3.59 (t, J=7.3)
3.25–3.33 (m)
3.61–3.74 (m),
3.86–3.93 (m)
H2SO4, followed by heating at 1008. For paper chromatography (PC), Xinhua filter paper (middle rate) was used,
and spots were visualized with vanillin/perchloric acid. Column chromatography (CC): silica gel (200–300 or
300–400 mesh; Qingdao Marine Chemical Factory) and Cosmosil 140 C18-OPN (Nacalai Tesque). UV Spectra:
Shimadzu UV-260 spectrophotometer, in anh. MeOH; lmax (log e) in nm. Optical rotations (ORD): JASCO P-
1020 spectropolarimeter. IR Spectra: Avatar 360 ESP-TM FT-IR spectrophotometer (Thermo Nicolet), as KBr
pellets; in cmꢀ1. 1H- and 13C-NMR Spectra: Bruker AV-500 spectrometer, in CDCl3; d in ppm rel. to Me4Si (=0
ppm), J in Hz. ESI- and HR-ESI-MS: Q-TOF Micro mass spectrometer; in m/z.
Plant Material. The stems of Marsdenia tenacissima were collected in August 2001 in Yunnan Province, P. R.
China, and identified by Dr. Daofeng Chen. A voucher specimen (DFC-TGS0108) was deposited at the Herba-
rium of Materia Medica, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, P. R.
China.
Extraction and Isolation. The dried and ground stems of M. tenacissima (5 kg) were repeatedly percolated
with 95% EtOH at r.t. The solvent was evaporated in vacuo to give a dark-brown extract (436 g), which was
partitioned between petroleum ether (PE) and H2O, followed by CHCl3 and H2O. A sample (100 g) of the
CHCl3-soluble part (314 g) was subjected to CC (SiO2 ; CHCl3/MeOH 100 :0 ! 85 :15) to give Fr. 1 (13 g),
Fr. 2 (22 g), Fr. 3 (17 g), Fr. 4 (21 g), and Fr. 5 (19 g). Fr. 4 was applied to repeated CC (SiO2 ; 1. CHCl3/
MeOH 90 :10, 2. PE/acetone 60 :40) to afford 1 (13 mg) and fraction Fr. 4.1 (2.3 g). The latter was subjected
to prep. TLC (SiO2; CHCl3/acetone 80 :20) to yield tenacissoside F (14 mg) and Fr. 4.2. The latter was further
purified by prep. HPLC (MeOH/H2O 60 :40) to afford 2 (1 mg). Fr. 5 was applied to CC (1. SiO2, CHCl3/MeOH
85 :15; 2. Cosmosil, MeOH/H2O 60 :40) to afford tenacissoside D (8 mg) and Fr. 5.1. The latter was subjected to
prep. HPLC (MeOH/H2O 60 :40) to afford 3 (30 mg).
Marsdenoside I (=(3b,5a,11a,12b,14b,17a,20R)-3-[(2,6-Dideoxy-4-O-(6-deoxy-3-O-methyl-b-D-allopyra-
nosyl)-3-O-methyl-b-D-arabino-hexopyranosyl)oxy]-8,20 :11,20-diepoxypregnane-12,14-diol; 1). Colorless
amorphous powder. [a]2D2 =ꢀ15.3 (c=1.2, MeOH). IR: 3455, 2929, 1455, 1382, 1301, 1292, 1244, 1206, 1175,
1130, 1070, 1026, 1008. 1H-NMR (500 MHz, CDCl3 ; aglycone): 1.14 (s, Me(19)); 1.18 (s, Me(21)); 1.22 (s,

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Table 2. 13C-NMR Data of 1–3 and 1a. In CDCl3 (unless noted otherwise); d in ppm. Abbreviations: Ac, acetyl;
Bz, benzoyl; HPA, (4-hydroxyphenyl)acetyl; Allo, allosyl; Ole, oleandrosyl; Glu, glucosyl.
Position
Aglycone
Position
Sugar moieties
1
1a
2
3
1
2
3
3a)
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
38.4
28.7
77.1
34.5
46.0
28.0
33.8
77.9
58.0
35.9
71.3
71.2
44.7
82.0
34.1
23.7
53.7
17.4
16.0
99.3
24.6
38.3
30.9
72.1
37.4
46.2
27.9
34.2
77.9
58.1
35.8
71.3
71.2
44.8
82.1
34.5
23.7
53.8
17.4
16.1
99.3
24.6
37.4
29.1
76.2
34.8
44.0
26.6
31.7
66.7
51.1
39.0
69.0
74.9
45.8
71.4
26.8
24.9
60.1
16.7
12.7
37.4
29.1
76.5
34.8
44.0
26.8
31.8
66.9
51.3
39.1
69.7
75.2
45.9
71.2
26.6
25.0
59.9
16.6
12.7
Ole:
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
3-MeO
Allo:
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
3-MeO
Glc:
97.1
36.1
78.8
79.1
71.3
18.6
55.6
97.1
36.1
78.9
79.4
71.4
18.6
55.9
97.1
36.2
79.1
82.5
71.4
18.5
55.9
97.3
38.5
79.8
83.3
72.0
18.9
57.5
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
HPA or Bz:
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
Ac:
99.1
71.8
81.0
72.8
71.4
17.9
61.9
99.3
72.0
81.0
72.9
71.3
17.9
62.7
100.1
70.9
80.4
80.6
69.2
18.0
61.2
102.0
72.9
83.4
83.5
69.9
19.7
62.0
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
104.3
73.8
76.1
70.1
75.5
62.1
106.6
75.7
78.5
72.2
78.6
63.2
210.7
29.9
210.8
30.1
168.4
41.4
125.4
130.7
115.6
155.1
166.0
130.1
129.6
128.5
133.2
–
MeCO
MeCO
170.7
20.2
170.7
20.4
a) In C5D5N
Me(18)); 1.86 (br. s, HbꢀC(17)); 2.43 (br. s, HꢀC(9)); 3.66–3.71 (m, HꢀC(3)); 3.98 (dd, J=8.5, 4.0, HaꢀC(12));
4.28 (br. d, J=2.6, HbꢀC(11)); for sugar resonances, see Table 1. 13C-NMR: see Table 2. HR-ESI-MS: 707.3414
([M+K]+, C35H56KO1þ2 ; calc. 707.3409).
Marsdenoside J (=(3b,5a,11a,12b,14b,17a)-12-Acetoxy-3-[(2,6-dideoxy-4-O-(6-deoxy-3-O-methyl-b-
D-allo-
pyranosyl)-3-O-methyl-b- -arabino-hexopyranosyl)oxy]-20-oxo-8,14-epoxypregnan-11-yl (4-Hydroxyphenyl)-
D
acetate; 2). Colorless amorphous powder. [a]2D2 =+10.2 (c=0.15, MeOH). UV (MeOH): 212 (3.52), 276
(3.27). IR: 3443, 2928, 1739, 1723, 1630, 1517, 1469, 1372, 1250, 1162, 1131, 1098, 1069, 1034, 1005, 996, 952,
934. 1H-NMR (500 MHz, CDCl3 ; aglycone): 1.00 (s, Me(19)); 1.05 (s, Me(18)); 1.64 (s, Me(2’’)); 1.96 (d,
J=10.0, HꢀC(9)); 2.19 (s, Me(21)); 2.90 (br. d, J=7.2, HbꢀC(17)); 3.53–3.61 (m, HꢀC(3)); 4.95 (d, J=10.1,
HaꢀC(12)); 5.31 (t, J=10.1, HbꢀC(11)); 6.75 (d, J=8.5, HꢀC(5’,7’)); 7.09 (d, J=8.5, HꢀC(4’,8’)); for sugar res-
onances, see Table 1. 13C-NMR: see Table 2. HR-ESI-MS: 883.3889 ([M+K]+, C45H70KO1þ4 ; calc. 883.3882).
Marsdenoside
methyl-b-
K
(=(3b,5a,11a,12b,14b,17a)-12-Acetoxy-3-[(b-
D
-glucopyranosyl-(1 ! 4)-6-deoxy-3-O-
D
-allopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-
D-arabino-hexopyranosyl)oxy]-20-oxo-8,14-epoxy-
pregnan-11-yl Benzoate; 3). Colorless amorphous powder. [a]2D2 =ꢀ5.0 (c=2.2, MeOH). UV (MeOH): 280
(2.89), 275 (3.04), 230 (4.08). IR: 3439, 2934, 1725, 1646, 1600, 1446, 1372, 1314, 1276, 1214, 1163, 1070, 992,
930, 923, 714. 1H-NMR (500 MHz, CDCl3 ; aglycone): 1.12 (s, Me(19)); 1.14 (s, Me(18)); 1.60 (s, Me(2’’));

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HELVETICA CHIMICA ACTA – Vol. 88 (2005)
2.17 (d, J=10.3, HꢀC(9)); 2.20 (s, Me(21)); 2.94 (br. d, J=7.2, HbꢀC(17)); 3.52–3.62 (m, HꢀC(3)); 5.12 (d,
J=10.1, HaꢀC(12)); 5.60 (t, J=10.1, HbꢀC(11)); 7.42 (t, J=7.7, HꢀC(4’,6’)); 7.55 (t, J=7.4, HꢀC(5’)); 7.95
(d, J=7.2, HꢀC(3’,7’)); for sugar resonances, see Table 1. 13C-NMR: see Table 2. HR-ESI-MS: 1015.4309
([M+K]+, C47H68KO1þ4 ; calc. 1015.4305).
Acid Hydrolysis of 1. A soln. of 1 (5 mg) in MeOH (3 ml) and 0.1M H2SO4 (1 ml) was kept at 608 for 30 min.
Then, H2O (3 ml) was added, the mixture was concentrated to a volume of ca. 4 ml, and kept at 608 for another
30 min, before cooled to r.t. The soln. was extracted with Et2O (3×5 ml), the org. layer was washed with H2O
(4×5 ml), dried (Na2SO4), and evaporated to dryness. The resulting residue was recrystallized from PE/
AcOEt. The aq. acidic layer of the hydrolysate was neutralized with 5% aq. Ba(OH)2 soln. The precipitate
was filtered, and the filtrate was evaporated. The residue, pachybiose, was compared with an authentic sample
by paper chromatography (upper layer of BuOH/AcOH/H2O 4 :1 :5) and TLC (CHCl3/MeOH 9 :1).
Data of Tenacigenin A (1a). Colorless amorphous powder. 1H-NMR (500 MHz, CDCl3): 1.15 (s, Me(19));
1.18 (s, Me(21)); 1.22 (s, Me(18)); 1.86 (br. s, HꢀC(17)); 2.43 (br. s, HꢀC(9)); 3.61–3.67 (m, HꢀC(3)); 3.98 (d,
J=2.4, HaꢀC(12)); 4.30 (br. d, J=2.6, HbꢀC(11)). 13C-NMR: see Table 2. ESI-MS: 387.4 ([M+Na]+, C21H32-
NaOþ5 ).
Enzymatic Hydrolysis of 3. A soln. of 0.1M AcOH/AcONa (pH 4.6) buffer (1 ml) and b-glucosidase (30 ml;
250 U/ml; Sigma) was added to 3 (2 mg). The mixture was incubated at 608 for 30 min, and then extracted with
CHCl3 (3×5 ml). The CHCl3 layer was evaporated under reduced pressure, and the residue was identified as
tenacissoside I [8] by TLC comparison with an authentic sample (visualization with CHCl3/MeOH 9 :1). The
aq. layer contained only one monosaccharide identified as glucose by paper chromatography (upper layer of
BuOH/AcOH/H2O 4 :1:5).
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Received April 27, 2005