Spirostanols obtained by cyclization of pseudosaponin derivatives and comparison of anti-platelet agglutination activities of spirostanol glycosides

Article abstract of DOI:10.1016/S0223-5234(00)00151-3

Naturally occurring saponins 3 and 4 have a normal type F ring and α- arranged CH₃-21 group. Treatments of pseudosaponin peracetates 18 and 19 derived from 3 and 4, respectively, with alcoholic KOH, followed by acidification with acetic acid, gave spirostanols 20 and 22 having iso type F rings as major products. Structural analyses of sapogenins and saponins derived from pseudo derivatives 11, 12, 18 and 19 were performed by comparisons of their ¹H-NMR spectral data and the X-ray analytical data of 3-O-p-bromobenzoyl sarsasapogenin 7, 3-O-acetyl diosgenin 13 and saponin 20. The mechanisms of ring-closure reaction of the side chain at C-22 of pseudosapogenins and pseudosaponins were deduced using stereomodels of the spirostanols derived from 11 under various reaction conditions. Inhibitory activities of saponin diglycosides 3, 4, 20, 21 and 25 on human platelet agglutinations induced by ADP and ristocetin were compared. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00151-3

Eur. J. Med. Chem. 35 (2000) 511−527
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001513/FLA
511
Original article
Spirostanols obtained by cyclization of pseudosaponin derivatives and
comparison of anti-platelet agglutination activities of spirostanol glycosides
Akihiko Tobaria, Mutsumi Teshimaa, Junich Koyanagia, Masami Kawasea, Hiroshi Miyamaeb,
Kenji Yozac, Akihiko Takasakid, Yoich Nagamurad, Setsuo Saitoa*
aFaculty of Pharmaceutical Sciences, Josai University, Keyakidai 1-1, Sakado, Saitama 350-0295, Japan
bDepartment of Chemistry, Josai University, Keyakidai 1-1, Sakado, Saitama 350-0295, Japan
cBruker Japan AXS group, 3-21-5 Ninomiya Tsukuba, Ibaraki, Japan
dSchool of Hygiene, Fujita Health University, Kusukake, Toyoake, Japan
Received 28 June 1999; revised 6 December 1999; accepted 9 December 1999
Abstract – Naturally occurring saponins 3 and 4 have a normal type F ring and α-arranged CH3-21 group. Treatments of pseudosaponin
peracetates 18 and 19 derived from 3 and 4, respectively, with alcoholic KOH, followed by acidification with acetic acid, gave spirostanols
20 and 22 having iso type F rings as major products. Structural analyses of sapogenins and saponins derived from pseudo derivatives 11, 12,
18 and 19 were performed by comparisons of their 1H-NMR spectral data and the X-ray analytical data of 3-O-p-bromobenzoyl
sarsasapogenin 7, 3-O-acetyl diosgenin 13 and saponin 20. The mechanisms of ring-closure reaction of the side chain at C-22 of
pseudosapogenins and pseudosaponins were deduced using stereomodels of the spirostanols derived from 11 under various reaction
conditions. Inhibitory activities of saponin diglycosides 3, 4, 20, 21 and 25 on human platelet agglutinations induced by ADP and ristocetin
were compared. © 2000 Éditions scientifiques et médicales Elsevier SAS
saponin / isospirostanol / pseudosapogenin / pseudosaponin / anti-platelet agglutination
1. Introduction
deloides BUNGE (Liliaceae) [10–14]. The rhizome has
been known to have anti-platelet agglutination activity
[10], anti-diabetic activity [15] and anti-diuretic activity
[16].
There are two different structures, normal- (I) and
iso-type (II), with respect to the F rings in spirostanols
(figure 1). So far only the normal-type derivatives have
been found in the naturally occurring spirostanols [1].
Marker et al. [2] and Wall et al. [3] reported that iso
structures were formed during the acid hydrolysis of the
plant steroidal saponins and were in a more stable form in
strong acid medium. Furthermore several investigators
[2–8] revealed that the isomerization of normal structures
to the iso structures proceeded via pseudo type interme-
diates such as (III) formed during acid hydrolyses. One of
the examples of the isomerization is the formation of
smilagenin 1 from sarsasapogenin 2. This procedure was
reported by Wall and Walens [8], and Marker and
Rhormann [9]; sarsasapogenin 2 was refluxed in alco-
holic hydrogen chloride solution to give smilagenin 1.
Sarsasapogenin 2 is the aglycon of timosaponin A-III 3,
which is isolated from rhizomes of Anemarrhena aspho-
In this paper we report the formation of iso type
spirostanols from sarsasapogenin 2, timosaponin A-III 3
and markogenin diglycoside 4, the latter of which was
isolated together with 3 as a minor constituent from the
same plant, in controlled reaction conditions. The struc-
1
tures of the products are established by H-NMR and
X-ray crystallography. The anti-platelet agglutination ac-
tivities of saponin diglycosides 3, 4, 20, 21 and 25 are
further compared.
2. Chemical results and discussion
In order to obtain smilagenin 1 from timosaponin A-III
3 according to the procedure of Marker et al. [2], 3 was
refluxed with 2 N HCl in dioxane/H2O (1:1) to quantita-
tively afford a crystalline compound which was acety-
lated to give acetate 5. In the 1H-NMR spectra, signals of
* Correspondence and reprints: saitoset@josai.ac.jp

512
structure (R-configuration at C-22), and CH3-21 and
CH3-27 groups arrange in α and ꢀ, respectively. These
results indicated that the hydrolysis of naturally occurring
timosaponin A-III 3 in 2 N HCl caused no isomerization
at the 22 position, and consequently gave quantitatively
only normal type sapogenin 2. These results suggested
that the crystalline hydrolysate from 3 was sarsasapoge-
nin 2 but not smilagenin 1, a different result from that of
Marker et al. [2]
The preparation of smilagenin 1 from sarsasapogenin 2
was further re-investigated in the same reaction condi-
tions reported by Marker and Rohrmann [9]; sarsasapo-
genin 2 was refluxed in a solution of 5% HCl in MeOH
for 4 days, then post-treated according to the described
method to obtain a crystalline residue. However, the
residue showed only one peak, whose retention time
agreed with that of 2 on high performance liquid chro-
matography, and also the acetate of the residue gave the
same 1H-NMR spectrum as that of 5. Thus, our results of
the treatments of timosaponin A-III 3 and sarsasapogenin
2 with strong mineral acid were different from those
reported by Marker and Rohmann.
Figure 1. Structures of F rings of spirostanols.
5 were superimposable on the corresponding signals of
the aglycon moiety of timosaponin A-III acetate 6 except
for the signal of H-3 (see Experimental section). The
structure of the hydrolysate was unequivocally confirmed
by X-ray diffraction study [17, 18] using single crystals
of 3-O-p-bromobenzoate 7 derived from the hydrolysate
(figure 2). In the crystal, the F ring of 7 has a normal type
Next, ring-closure reactions of pseudosapogenin and
pseudosaponin derivatives were investigated. Pseudosar-
Figure 2. Structures of 1–7 and 17.

513
sasapogenin 11 and pseudodiosgenin 12 were obtained
via the corresponding diacetates 8 and 10 derived from
sarsasapogenin 2 and diosgenin 9, respectively, according
to the methods of Scheer et al. [5]. Treatments of 11 and
12 with 5% HCl in MeOH for 12 h at room temperature
gave only sarsasapogenin 2 and diosgenin 9, respectively.
The absolute structure of 9 was confirmed by the X-ray
analysis using its monoacetate 13 (ORTEP not repre-
sented) [17, 18]. In the crystal of 13, the F ring has a
normal type structure (R-configuration at C-22) as well as
7, and CH3-21 arranged in α (S-configuration at C-20),
and CH3-27 groups are equatorial (R-configuration at
C-25). However, when the ring-closure reaction of 11 was
carried out in acetic acid instead of 5% HCl in MeOH,
products 2, 14 and 15 were obtained in the yields of 2.7,
13.2 and 57.4%, respectively (figure 3). These products
showed the same quasimolecular ion peak [M + Na]+ at
m/z 439 in the fast-atom-bombardment mass spectra
(FABMS). In order to elucidate the structures of the
suggests that 9 and 16 have the same normal type F-rings.
As the CH3-21 group of 9 has α-configuration, the
CH3-21 group of 16 is therefore deduced to arrange in ꢀ.
Thus, it became apparent that iso type spirostanol 15
was obtained via the pseudosapogenin 11 in acetic acid,
though pseudodiosgenin 12 gave only normal type
spirostanols 9 and 16. Our next investigation was focused
on the preparations of iso type spirostanol glycosides.
Pseudosaponin peracetates 18 and 19 were prepared from
saponin acetates 6 and 17, respectively, in the same
manner as pseudosarsasapogenin acetate 8 mentioned
above. Treatment of 18 with 5% KOH in EtOH/H2O
(1:1), followed by acidification with acetic acid, gave
products 3, 20 and 21 in the yields of 2.8, 46.5 and
20.1%, respectively (figure 6). The products 3, 20 and 21
showed the same quasimolecular ion peak [M + Na]+ at
m/z 763 in the FABMS spectra. Compound 3 was
identified with authentic timosaponin-AIII by HPLC and
1H- and 13C-NMR spectra. Fortunately compound 20 was
obtained as good crystals for the X-ray analysis. From the
crystalline structure (figure 7), it was confirmed that the F
ring of 20 was an iso type structure and the CH3-21 and
CH3-27 groups of 20 arranged in ꢀ and α, respectively.
The signals due to the protons at the 26 positions of 20
and 21 appeared at the similar fields at δ 3.6–3.68,
suggesting that 21 has the same iso type F ring as 20. As
the CH3-21 group of 20 was ꢀ, it was deduced that
compound 21 was an isospirostanol having an α-arranged
CH3-21 group.
1
products, H-NMR spectra of compounds 2, 14 and 15
were compared (figure 4 and table I). Figure 4 showed
the spectra of 2, 14 and 15 in the regions of δ 3.2–4.6, in
which the proton signals at positions 3, 16 and 26 were
observed. The chemical shifts and coupling constants of
H-26a (δ 3.30, d, J = 11.0 Hz) and H-26b (δ 3.95, dd,
J = 11.0, 2.8 Hz) on the F ring of 2 were similar to those
(δ 3.25, d, J = 11.3 Hz for H-26a; δ 4.03, dd, J = 11.3,
3.1 Hz for H-26b) on the F ring of 14. From the spectral
data and the X-ray evidence, it is suggested that 14 has
the same normal type F ring as well as 2. The difference
of the structures between 2 and 14 consequently seemed
to be due to that of the configurations of CH3-21 groups.
As the configuration of the CH3-21 group of 2 was α it
was speculated that the CH3-21 group of 14 had
ꢀ-configuration. The signals of both protons at the 26
position of 15 closely appeared at δ 3.46–3.52, different
from those of 2 and 14, which indicates that compound 15
has a different F-ring structure, namely iso type structure
C or D (figure 5), from 2 and 14. The actual structure of
15 was, however, obscure at this moment, although the
structure assignment of 15 was described later.
In order to elucidate the configuration of the CH3-21
group of 15, the chemical shifts of the proton at the 20
position of 15 was compared with those of sapogenins 2,
1
9, 14 and 16 and saponins 3, 20 and 21 in the H-NMR
spectra (table I). The H-20 signals of 14, 16 and 21 were
observed as quintets at δ 2.47–2.70, while those of 2, 3,
9 and 20 were shifted to a higher field than δ 2.2. The
orientations of the protons at the 20 positions and the
oxygen atoms on the F rings of 14, 16 and 21 are cis,
whereas those of 2, 3, 9 and 20 are trans. Thus, when an
H-20 signal was observed at lower field than about δ
2.47, the orientation relationship between the proton at
the 20 position and the oxygen atom on the F ring in one
spirostanol structure was thought to be cis. On the other
hand, when an H-20 signal shifted at higher field than δ
2.2, the orientation relationship was thought to be trans.
The H-20 signal of 15 appeared at higher field than δ 2.2
as well as those of 2, 3, 9 and 20, which suggests that the
orientation of the H-20 and the oxygen atom on the F-ring
of 15 is trans. Therefore it was deduced that the structure
of 15 was C in figure 5, in which the CH3-21 group
arranges in ꢀ.
The ring-closure reaction of pseudodiosgenin 12 in
acetic acid gave two products, 9 as a minor product
(12.6% yield) and 16 as a major product (68.3% yield).
Compounds 9 and 16 showed the same quasimolecular
ion peak [M + Na]+ at m/z 437 in the FABMS spectra. As
it was already confirmed that compound 9 had a normal
type F ring and that the CH3-21 group had α-configuration
[19, 20], the 1H-NMR spectrum of 16 was compared with
that of 9 to elucidate the structure. In the 1H-NMR spectra
(table I), the chemical shifts and multiplicities of the
H-26a and H-26b of 9 and 16 were similar, which

514
Figure 3. Structures of 8–16.
Treatment of pseudosaponin peracetate 19 with 5%
KOH, followed by acidification with acetic acid, four
spirostanol diglycosides 4, 22, 23 and 24 were obtained in
the yields of 3.8, 41.5, 11.3 and 2.0%, respectively.
Although X-ray analyses were not performed because
they were all obtained as amorphous powders, the struc-
tures of the products were analysed by comparison of the
1H-NMR spectra of 4 and 22–24 with those of 3, 20 and
21. Compounds 4, 22 and 24 gave similar 1H-NMR
spectra to those of 3, 20 and 21, respectively, except for
the signals due to the 2 positions (table I and Experimen-
tal section), which suggests that the structures of the F
rings and the configurations at the CH3-21 groups of 4, 22
and 24 were the same as those of 3, 20 and 21,
respectively. As compound 4 was one of two obtainable
normal type spirostanol diglycosides and both 22 and 24
were iso type spirostanol diglycosides, it was thought that
compound 23 was an epimer of 4 with respect to the 20
Figure 4. 1H-NMR (500 MHz) spectra (pyridine-d5) (δ 3.2–4.6)
of compounds 2, 14 and 15.
1
position. In the H-NMR spectrum of 23, the signals of

515
Table I. 1H-NMR (pyridine-d5) signals used for comparisons in structural analyses of spirostanol derivatives, types of F-rings and
configurations of CH3-21 and CH3-27 groups.
Compound CH3-18
CH3-19
CH3-21
CH3-27
H-20
H-26a
H-26b
Type of configuration of
F-ring
CH3-21 CH3-27
2
0.76 (s)a 0.98 (s)
0.99 (d)
(6.7)b
1.17 (d)
(7.0)
1.11 (d)
(7.3)
0.98 (d)
(7.0)
1.15 (d)
(7.9)
1.08 (d)
(7.0)
1.24 (d)
(7.3)
1.14 (d)
(7.9)
1.08 (d)
(7.3)
1.22 (d)
(7.3)
1.15 (d)
(7.9)
1.12 (d)
(7.9)
1.08 (d)
(7.0)
1.06 (d)
(7.3)
0.78 (d)
(6.4)
0.79 (d)
(6.1)
0.79 (d)
(6.4)
1.15 (d)
(7.0)
0.71 (d)
(5.5)
0.72 (d)
(5.8)
1.15 (d)
(7.0)
0.71 (d)
(5.5)
1.08 (d)
(7.3)
0.71 (d)
(5.8)
2.2<
3.30 (d)
(11.0)
3.95 (dd)
(11.0, 2.8)
4.03 (dd)
(11.3, 3.1)
normal
normal
iso
α
ꢀ
ꢀ
α
ꢀ
α
ꢀ
α
α
ꢀ
ꢀ
α
ꢀ (axial)
14
15
9
0.93 (s)
1.02 (s)
0.79 (s)
0.97 (s)
0.82 (s)
1.13 (s)
0.96 (s)
0.80 (s)
1.11 (s)
0.94 (s)
0.94 (s)
0.97 (s)
0.98 (s)
1.03 (s)
1.03 (s)
0.97 (s)
0.99 (s)
0.96 (s)
0.95 (s)
0.98 (s)
0.96 (s)
0.96 (s)
2.48 (quin.)c 3.25 (d)
(8.5)
2.2<
ꢀ (axial)
(11.3)
3.46–3.52*
α (equatorial)
α (equatorial)
α (equatorial)
ꢀ (axial)
2.2<
3.37–3.46
(m)
normal
normal
normal
iso
16
3
2.47 (quin.)c 3.48
(8.5)
2.2<
(m)
3.37 (d)
(11.0)
3.60–3.68
(m)
4.07 (dd)
(11.0, 2.4)
20
21
4
2.2<
α (equatorial)
α (equatorial)
ꢀ (axial)
2.70 (quin.)c 3.60–3.65
(8.5)
2.2<
iso
(m)
3.37 (d)
(11.0)
3.31–3.36
(m)
3.83 (dd)
(11.0, 3.1)
normal
iso
22
23
24
2.2<
α (equatorial)
ꢀ (axial)
2.68 (quin.)c 3.36 (d)
(8.5)
2.68 (quin.)c 3.60–3.70
(8.6) (m)
3.82 (dd)
(11.0, 3.1)
normal
iso
(11.0)
α (equatorial)
a Multiplicities were indicated as s (singlet), d (doublet), dd (doublet of doublets), quin. (quintet) and m (multiplet).. b Coupling constants
(J in Hz) are given in parentheses.. c These protons shifted at lower fields in the cases of cis relationships between 20-Hs and F-ring oxygens
than those of trans relationships.. * These proton signals observed closely at δ 3.46 (dd, J = 11.0, 4.6 Hz, H-26a) and δ 3.52 (dd, J = 11.0,
11.0 Hz, H-26b) as shown in figure 4.
H-26a and H-26b were observed separately from each
other at δ 3.36 (d, J = 11.0 Hz) and 3.82 (dd, J = 11.0 and
3.1 Hz), respectively, as well as 4. The H-20 signal of 23
was observed at the same position of δ 2.68 as a quintet
(J = 8.5 Hz) as that of 24. From these spectral data it is
indicated that compound 23 is a normal type spirostanol
diglycoside having ꢀ-arranged CH3-21 group.
The mechanisms of the ring-closure reactions of the
side chains at the 22 positions of pseudo derivatives were
investigated using pseudosarsasapogenin 11. As men-
tioned above, hydrolysis of saponin 3 in 2 N HCl gave
only normal type sapogenin 2. Treatment of pseudosapo-
genin 11 in 5% HCl in MeOH also quantitatively gave 2.
On the other hand, treatment of 11 with acetic acid gave
three products: 2, 14 and 15. These results indicated that
under strong mineral acid conditions, the normal type
sapogenin 2 was the most stable structure, while in a
weak acid, such as acetic acid, other cyclized products 14,
which was an epimer of 2, with respect to C-20 and 15
which was an iso type spirostanol having a ꢀ-arranged
CH3-21 group, were obtained as major products accom-
panied by a minor product 2. Hereupon, the ring-closure
reaction of 11 was performed under control conditions.
After 5 min of dissolving 11 in a methanolic hydrochloric
acid solution adjusted to pH 4.0, the reaction mixture was
poured into 5% KOH solution, then extracted with
AcOEt. The extract showed only two peaks due to
compounds 14 and 15 as minor and major products,
respectively, on the HPLC. From these results together
with the fact that 11 was rather stable in neutral and
alkaline solutions, the mechanisms of the ring-closure
reaction of 11 inferred as follows (figure 8): the trigger of
the ring-closure reaction of 11 was thought to be proto-
nation at C-20. When the protonation at C-20 occurs from
the ꢀ-site, the attacking of the proton to C-20 may be
greatly sterically hindered by the bulky CH3-18 group,

516
Figure 5. Stereomodels A–D (MM2-based, Chem 3-D plus) of compounds 2, 14, 15 and 1, respectively.
whereas it is thought that the protonation at C-20 from
α-site does not suffer from such a steric hindrance.
Consequently in the protonation step, pseudostructure 11
seemed to give an intermediate [I], having a ꢀ-arranged
CH3-21 group in larger amounts than an intermediate [II]
having an α-arranged CH3-21 group. In the cyclizations
of the side chain at the 22 position of the intermediate [I]
to form F rings, when the resulting F ring obtained from
[I] is a normal type (structure b in figure 8), the 1,3-
diaxial interactions between the axial CH3-27 group and
a lone pair orbital of the oxygen on the F ring are caused.
On the other hand, an equatorial CH3-27 group on the iso
type F ring (structure a) does not suffer from such an
interaction. Therefore, it was thought that in the early
steps of the ring-closure reaction, 11 was protonated at
C-20 from mainly the α-site to give an intermediate [I]
whose CH3-21 group arranged in ꢀ, from which iso type
spirostanol 15 was obtained in much higher yield than 14.

517
Figure 6. Structures of 18–25.
Another intermediate [II] having an α-arranged CH3-21
group seems to be produced in small amounts so that 2
was obtained as a minor product in the ring-closure
reaction of 11.
among three substituents of CH3-18, ꢀ-CH3-21 and
CH2-23 in B (14), among three substituents of CH3-18,
ꢀ-CH3-21 and the F ring oxygen in C (15), and between
CH3-18 and the lone pair orbital on the F-ring oxygen in
D (1) might have occurred, while such interactions are
not considered in structure A (2). Therefore compound 2
is thought to be most thermodynamically stable con-
former of four spirostanols 1, 2, 14 and 15.
However, treatments of sapogenins 14 and 15 and
saponins 20 and 21 as well as that of 3 with strong
mineral acid gave only one sapogenin 2, which suggests
that sapogenin 2 has a normal type F ring and an
α-arranged CH3-21 group is the most stable spirostanol
structure, and 2 is the thermodynamically favoured prod-
uct under strong acid condition. This suggestion was
revealed by comparisons of the stereomodels A–D which
were obtained by molecular dynamic calculations (MM2)
(Chem 3-D plus, Chembridge Scientific Company, Inc.)
(figure 5). In the structures B–D, some steric interactions,
3. Pharmacological results
Anti-platelet agglutination activities of saponins 3, 4,
20 and 21 and 26-O-acetyl pseudotimosaponin A-III 25
derived from 18 were compared. In this study adenosine-

518
Figure 7. ORTEP representation of 20 at the 20% probability level. Hydrogen atoms are drawn as spheres of arbitrary radius.
5′-diphosphate (ADP) and ristocetin sulfate (ristocetin)
were used as agglutination inducers of platelets prepared
from healthy adult blood. First, the inhibitory activities of
spirostanol diglycosides 3 and 4 were investigated on
human platelet agglutinations induced by ADP. The
agglutination inhibitions of 3 were 0, 25.4, 79.8 and
100%, and those of 4 were 0, 13.2, 60.0 and 100%, with
dose concentrations of 50, 100, 200 and 400 µg/mL,
respectively. Thus, it appeared that the inhibitory efficien-
cies of 3 and 4 on ADP-induced platelet agglutinations
were dose-dependent and the results agreed with those
reported by Niwa et al. [11].
Next, the inhibitory activities of compounds 20, 21 and
25 against both ADP- and ristocetin-induced platelet
agglutinations were compared with those of 3 (figure 9) at
the drug concentration of 400 µg/mL that showed 100%
inhibition in the assay of 3 on the ADP-induced platelet
agglutinations. On the ADP-induced platelet agglutina-
tions, spirostanol diglycosides 3 and 21 possessed potent
inhibitory activities, and isospirostnol 20 and pseudosa-
ponin 25 showed 60–70% inhibitory activities. On the
contrary, in the inhibitions of the ristocetin-induced
platelet agglutinations, 20 and 25 exhibited high activities
(100%), although 3 exhibited no activity and 21 poor
activity (29%). These results indicate that thermodynami-
cally favoured spirostanols 3, 4 and 21 have potent
activities against the ADP-induced platelet agglutina-
tions. On the other hand, thermodynamically unfavoured
saponin 20 and pseudosaponin 25 have potent activity
against ristocetin-induced platelet agglutinations.
Although these differences of inhibitory activities on both
ADP- and ristocetin-induced platelet agglutinations have
not yet been elucidated, they might come from the
stability of the saponins. Saponins 3 and 21 are stable
spirostanols which exhibited potent inhibitory activities
on the ADP-induced platelet agglutinations. On the other
hand, isospirostanol 20 is thermodynamically unfavoured
so that it may produce a pseudosaponin during the assay,
and the resulting pseudostructure may appear to be the
potent activity against ristocetin-induced platelet aggluti-
nations, because pseudosponin 25 showed potent activity
on the agglutinations.
4. Conclusions
It was reported that iso type spirostanol sapogenins
were obtained by epimerization at C-22 of the corre-
sponding normal type spirostanol sapogenins and were
more stable structures than normal ones in the strong
mineral acid solutions [2]. However, in our study, treat-
ments of sarsasapogenin 2 and timosaponin A-III 3 with
hot strong mineral acid solutions gave only 2. Further-
more, treatments of pseudosapogenins 11 and 12 with

519
Figure 8. Stereomodel (MM2-based, Chem 3-D plus) of pseudosarsasapogenin 11 and proposed mechanisms for ring-closure reaction
of 11.
strong mineral acid solutions also gave the corresponding
normal type spirostanols 2 and 9, respectively. These
results were different from those reported by Marker et al.
[2]. On the other hand, treatment of 11 with acetic acid
gave iso type spirostanol 15 accompanied by minor
normal type spirostanols 2 and 14, and treatments of
pseudosaponin peracetates 18 and 19 with 5% KOH in
methanol, followed by acidification with acetic acid
produced iso type spirostanol derivatives accompanied by
minor normal type ones; 20 and 21 accompanied by 3
from 18, and 22 and 24 accompanied by 4 and 23 from
19. It became apparent that the signal assignments of
protons at C-26 and C-20 of the spirostanols in the
1H-NMR spectra were a very useful means of determin-
ing the conformations of the F rings and the configura-
tions at C-20: when the signals of protons at the 26
position of spirostanols were separately observed at δ
3.25–3.37 (Hs-26a) and 3.81–4.07 (Hs-26b) as shown in
figure 4, the spirostanols had a normal type F ring. On the
other hand, when both protons at the 26 position were
closely shifted, the spirostanols had iso type F ring. When
the protons at the 20 positions of the spirostanols ob-
served at lower fields than δ 2.47, the orientation rela-
tionships between the protons at the 20 positions and the
oxygen atoms on the F rings were cis as shown in the
cases of 14, 16, 21, 23 and 24. Therefore, the CH3-21
groups of 14, 16 and 23 which had normal type F rings
arranged in ꢀ, and those of 21 and 24 which had iso type
F ring arranged in α. On the other hand, when the protons
at the 20 position shifted at higher field than δ 2.2, the
orientation relationships were trans, as shown in the
cases of 2, 3, 4, 9, 15, 20 and 22, which describes that the
CH3-21 groups of 2, 3, 4 and 9 which had normal type F
rings arranged in α, and those of 15, 20 and 22 which had
iso type F rings arranged in ꢀ. In addition, the configu-
rations of CH3-27 groups of spirostanols were deduced
from the following results: the signals of ꢀ-arranged
(axial) CH3-27 groups were observed at lower fields than
δ 1.0, whereas those of α-arranged (equatorial) CH3-27
groups were at higher fields than δ 0.8.
The mechanisms of the ring closure reactions of the
side chains of the pseudo derivatives are explained using
11 as follows (figure 8): the attack of a proton at the 20
position of 11 from the ꢀ-site exerts a steric hindrance on
the CH3-18 group, while that from the α-site does not.
Consequently, intermediate [I] might be produced in
much larger amounts than [II]. In the F-ring formation
from the intermediate [I], a spirostanol having an iso type

520
Figure 9. Inhibitory activities on human platelet agglutinations induced by ADP (shaded bars) and ristocetin sulfate (closed bars) at
the concentration of 400 µg/mL of compounds 3, 20, 21 and 25. C: control. Materials and assay methods were described in the
Experimental section. All data indicate the average of three experiments.
F ring (a in figure 8) is obtained as a major product,
because in another spirostanol with a normal F ring (b in
figure 8), a steric interaction between the axial CH3-27
and a lone pair orbital of oxygen atoms on the F ring is
generated, whereas such interaction is not considered in
the iso type F ring in structure a. In the four structures
A–D in figure 5, steric interactions among the CH3-18,
ꢀ-CH3-21 and CH2-23 in B, among the CH3-18, ꢀ-CH3-21
and the F-ring oxygen in C, and between the CH3-18 and
the lone pair orbital on the F-ring oxygen in D might
occur, while such interactions might not in A. Therefore,
it was reasonable that the treatment of 2 with strong
mineral acid did not give smilagenin 1 and the hydrolyses
of saponin 3, 20 and 21 in 2 N HCl gave only the most
stable sapogenin 2.
from the lone pair orbital of the F-ring oxygen. On the
other hand, the hindrance is not considered in structures
E and G which have a normal type of F ring. Further-
more, an intermediate such as [I] in figure 8 might be
more stable than another intermediate such as [II] in the
first step of the reaction. Therefore, the mechanisms for
the formations of spirostanols mentioned above also
agreed in this case; the ring-closure reaction of 12 in
acetic acid gave 16 as a major product and 9 as a minor
product.
Although Callow and James [21], Yu et al. [22] and
Asano et al. [23] reported that epimerizations at C-25 on
normal F rings of spirostanols occurred without any
conversion of the F rings in strong mineral acid, however,
such epimerizations were not observed in our study.
Inhibitory activities on human platelet agglutinations
induced by ADP and restocetin of steroidal saponins were
investigated. The inhibitory activities of compounds 20,
21 and 25 on ADP- and ristocetin-induced platelet agglu-
tinations were compared with those of 3 at the drug
In the ring-closure reaction of pseudodiosgenin 12 in
which the configuration at C-25 was R, the structures
E–H (figure 10) were considered for the products. In
those structures, the axial CH3-27 groups of structures F
and H which have iso type F rings suffer steric hindrance

521
Figure 10. Possible structures E–H formed in the ring-closure reaction of 12.
concentration of 400 µg/mL (figure 9). Stable spirostanols
3 and 21 exhibited potent inhibitory activities, whereas
unstable saponin 20 and pseudosaponin 25 showed
60–70% inhibition at the concentration of 400 µg/mL
against ADP-induced platelet agglutinations. On the con-
trary, 25 and 20 showed 100% inhibition on ristocetin-
induced platelet agglutinations, although 3 and 21 showed
poor inhibitory activities (0% in 3 and 20% in 21). These
results indicate that thermodynamically favoured
spirostanols exhibit highly inhibitory activities against
ADP-induced platelet agglutination. On the other hand,
thermodynamically unfavored saponins showed highly
inhibitory activities on ristocetin-induced platelet agglu-
tinations.
chased from SIGMA Chemical Co., USA. Other chemi-
cals and solvents were of reagent grade and were ob-
tained from commercial sources. Melting points were
determined on a Yanagimoto micromelting point appara-
tus and are uncorrected. The thin-layer chromatography
(TLC) utilized Kieselgel 60 F254 (E. Merck), and spots
were detected by spraying the plates with 1:9 Ce(SO4)2/
10% H2SO4 reagent, followed by heating at 100 °C for
l0 min. Column chromatography was carried out on a
Wakogel C-200, and the eluates were monitored by TLC.
An SSC-6300 HPLC instrument (Senshu Scientific Co.
Ltd.) was employed for analytical HPLC using a DOCO-
SIL (10 × 250 mm; flow rate, 1.0 mL/min, column temp,
40 °C) column, and was further equipped with an SSC
autoinjector 6310 and an SSC fraction collector 6320 for
preparative HPLC using a DOCOSIL (10 × 250 mm; flow
rate, 1.0 mL/min, column temp, 40 °C) preparative col-
5. Experimental protocols
1
umn. H- and 13C-NMR at 500 and 125 MHz, respec-
5.1. General procedures
1
1
tively, as well as H-1H and H-13C COSY, DEPT and
HMBC spectra, were obtained with a JEOL JNM-A500
FT NMR spectrometer. Tetramethylsilane was used as an
internal standard, and chemical shifts are given in ppm.
Multiplicities of 1H-NMR signals are indicated as s
Timosaponin A-III was isolated from rhizomes of
Anemarrhena asphodeloides BUNGE (Liliaceae) accord-
ing to the method by Saito et al. [24]. Diosgenin,
adenosine-5′-diphosphate and ristocetin sulfate were pur-

522
Table II. 13C-NMR spectral data of compounds 2, 9 and 14–16a.
(singlet), d (doublet), dd (doublet of doublets), quin.
(quintet) and m (multiplet). Fast-atom-bombardment mass
spectra (FABMS) were recorded on a JEOL JMS-DX 300
mass spectrometer.
2
14
15
9
16
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
29.9b
27.7
67.0
33.5
36.4
26.5
26.5
35.2
39.8
35.2
20.8
40.2
40.6
56.4
31.7
81.0
62.0
16.4
23.8
42.0
14.3
109.7
25.7
25.9
27.0
65.1
16.0
29.9
27.8
67.0
33.5
36.5
26.5
26.2
35.0
39.7
35.1
20.4
40.2
42.2
57.5
32.0
81.1
60.8
16.2
23.9
46.6
11.5
108.8
25.9
25.7
27.4
66.8
16.5
29.9
27.9
67.1
34.0
36.6
26.6
26.5
34.5
39.9
35.3
20.6
41.1
42.0
57.0
33.6
83.2
60.1
15.9
23.9
45.9
9.9
37.2
31.6
71.7
42.2
140.8
121.4
32.0
31.4
50.0
36.6
20.8
39.7
40.2
56.5
31.8
80.8
62.0
16.2
19.4
41.6
14.5
109.3
31.3
28.7
30.2
66.8
17.1
37.2
31.5
71.6
42.2
140.8
121.3
31.7
30.5
49.9
36.5
20.4
39.7
41.7
57.5
32.1
81.0
60.4
16.1
19.4
46.4
11.3
108.3
30.6
28.5
31.2
68.1
17.1
5.1.1. Chemistry
5.1.1.1. Acid hydrolysis of 3
After refluxing a solution of 3 (3.5 g, 4.72 mmol) in
2 N HCl in dioxane/H2O (1:1, 20 mL) for 24 h, the
mixture was diluted with distilled H2O (50 mL), ex-
tracted with AcOEt (50 mL × 3). The combined extracts
were washed with H2O, dried over Na2SO4, then filtered.
The filtrate was evaporated to give a residue. The residue
was subjected to column chromatography (a gradient of
0–5% MeOH in CH2Cl2) to give sapogenin 2 (1.6 g,
81.3%) as fine needles: m.p. more than 300 °C after
recrystallization from acetone/H2O (lit. [11] 317–322 °C);
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
C-22
C-23
C-24
C-25
C-26
C-27
1
FABMS: m/z 439 [M + Na]+; H-NMR (d5-pyridine) δ:
4.10 (1H, br. s, H-3), 4.40 (1H, ddd, J = 7.0, 7.0, 3.7 Hz,
H-16). Other assignable signals in the 1H-NMR spectrum
were listed in table I; 13C-NMR spectral data was listed
in table II.
106.8
29.1
34.9
30.0
67.5
17.1
5.1.1.2. Acetylations of 2 and 3
Acetylations of 2 and 3 were performed in Ac2O/
pyridine (1:1). After-treatments of the reaction mixtures
in the usual way quantitatively gave acetates 5 and 6,
respectively. FABMS of 5: m/z 481 [M + Na]+. FABMS
a Spectra were obtained in CDCl3.. b Chemical shifts are in ppm
from internal (CH3)4Si. Signal assignments were based on DEPT,
1H-1H, 1H-13C COSY and 1H-13C-long-range COSY spectral data.
1
of 6: m/z 1057 [M + Na]+. The H-NMR spectra of 5
(CDCl3) (only assignable signals were listed) δ 5.06 (1H,
br. s, H-3), 4.41 (1H, ddd, J = 7.6, 7.6, 6.4 Hz, H-16),
3.95 (1H, dd, J = 11.0, 2.8 Hz, H-26a), 3.30 (1H, d, J =
11.0 Hz, H-26b), 2.04 (3H, s, Ac), 1.08 (3H, d, J = 7.0 Hz,
CH3-27), 0.99 (3H, d, J = 6.9 Hz, CH3-21), 0.98 (3H, s,
CH3-19) and 0.76 (3H, s, CH3-18): The 1H-NMR spectra
of 6 (CDCl3) (only assignable signals were listed) δ 5.31
(1H, d, J = 3.4 Hz, H-4′), 5.14 (1H, dd, J = 9.5, 9.5 Hz,
H-3′′), 5.05 (1H, dd, J = 9.5, 9.5 Hz, H-4′′), 4.96 (1H, dd,
J = 10.1, 7.9 Hz, H-2′), 4.89 (1H, dd, J = 9.5, 8.2 Hz,
H-2′′), 4.78 (1H, d, J = 8.2 Hz, H-1′′), 4.33 (1H, d, J = 7.9
Hz, H-1′), 4.41 (1H, ddd, J = 7.6, 7.6, 6.4 Hz, H-16), 4.33
(1H, dd, J = 12.5, 4.6 Hz, H-6′′a), 4.14 (1H, dd, J = 11.3,
6.7 Hz, H-6′a), 4.06–4.10 (2H, H-6′b and 6′′b), 4.00 (1H,
br. s, H-3), 3.96 (1H, dd, J = 11.0, 2.7 Hz, H-26a), 3.88
(1H, dd, J = 10.1, 7.9 Hz, H-2′), 3.83 (1H, dd, J = 7.2, 7.2
Hz, H-5′), 3.71 (1H, ddd, J = 9.8, 4.6, 2.8 Hz, H-5′′), 3.30
(1H, d, J = 11.3 Hz, H-26b) and 2.14, 2.07, 2.06, 2.02,
2.01, 2.00 and 1.99 (each 3H, s, Ac).
and allowed to stand overnight. The reaction mixture was
poured into ice water (100 mL) and extracted with
CH2Cl2 (50 mL × 3). The combined extracts were suc-
cessively washed with 5% HCl, NaHCO3-saturated wa-
ter, and water, then dried over anhydrous Na2SO4, and
filtered. The filtrate was evaporated to obtain a residue
that was subjected to column chromatography (a gradient
of 0–10% AcOEt in benzene) to give compound 7 (1.2 g,
83.4%): m.p. 207–209 °C (after recrystallization from
ether/n-hexane); FABMS: m/z 621 [M + Na]+ and 623 [M
1
+ Na + 2]+ (1:1 as the peak-height ratios); H-NMR
(CDCl3) (only assignable signals were listed) δ 7.90 and
7.57 (each 2H, AB quartet, aromatic protons), 5.32 (1H,
br. s, 3-H), 4.42 (1H, ddd, J = 7.6, 7.6, 6.4 Hz, H-16),
3.96 (1H, dd, J = 11.2, 2.6 Hz, H-26a), 3.30 (1H, d, J =
11.2 Hz, H-26b), 1.08 (3H, d, J = 6.9 Hz, 27-CH3), 1.03
(3H, s, 19- CH3), 1.00 (3H, d, J = 6.6 Hz, 21- CH3), 0.77
(3H, s, 18- CH3); 13C-NMR (CDCl3) δ: 165.2 (CO),
131.7 (C-2′ and 6′), 131.0 (C-3′ and 5′), 130.0 (C-4′),
127.7 (C-1′), 109.7 (C-22), 80.7 (C-16), 71.8 (C-3), 65.1
5.1.1.3. Sarsasapogenin p-bromobenzoate 7
To a solution of 2 (1.0 g, 2.4 mmol) in pyridine (5 mL),
p-bromobenzoyl chloride (1.0 g, 4.56 mmol) was added

523
(C-26), 62.1 (C-17), 56.4 (C-14), 42.1 (C-20), 40.7
(C-13), 40.2 (C-12), 40.0 (C-9), 37.7 (C-5), 35.3 (C-8),
35.1 (C-10), 31.8 (C-15), 31.1 (C-1), 30.7 (C-4), 27.1
(C-25), 26.5 (C-6), 26.5 (C-7), 25.9 (C-24), 25.8 (C-23),
25.1 (C-2), 24.1 (C-19), 20.9 (C-11), 16.5 (C-18), 16.1
(C-27) and 14.4 (C-21); Anal. calcd. for C34H47O4Br: C,
68.10; H, 7.90. Found: C, 67.75; H, 7.69.
(C-1), 38.1 (C-4), 37.0 (C-12), 36.7 (C-10), 34.1 (C-15),
32.1 (C-7), 32.1 (C-8), 31.2 (C-25), 30.8 (C-24), 27.7
(C-2), 23.2 (C-11), 21.4 (COCH3), 21.0 (COCH3), 21.0
(C-23), 19.3 (C-19), 16.7 (C-27), 13.9 (C-18) and 11.6
(C-21); Anal. calcd. for C31H46O5: C, 74.66; H, 9.30.
Found: C, 74.49; H, 9.33.
5.1.1.6. Pseudosarsasapogenin 11
5.1.1.4. Pseudosarsasapogenin diacetate 8
A solution of 8 (3.0 g, 6.0 mmol) in 5% methanolic
potassium hydroxide (20 mL) was allowed to stand
overnight at room temperature. The solution was diluted
with ice water (100 mL) and the mixture was extracted
with CH2Cl2 (100 mL × 2). The combined extracts were
washed with water, then dried over anhydrous Na2SO4,
and filtered. The filtrate was evaporated to obtain a
residue that was subjected to column chromatography (a
gradient of 0–5% AcOEt in benzene) to give compound
11 (2.1 g, 84.1%, m.p. 169–170 °C after recrystallization
from ether) (lit. [2] m.p. 171–173 °C): FABMS: m/z 439
[M + Na]+; 1H-NMR (d5-pyridine) (only assignable
signals were listed) δ 4.87 (1H, ddd, J = 10.1, 7.9, 5.8 Hz,
H-16), 4.38 (1H, br. s, H-3), 3.77 (1H, dd, J = 10.4, 5.8
Hz, H-26a), 3.69 (1H, dd, J = 10.4, 6.4 Hz, H-26b), 2.54
(1H, d, J = 10.1 Hz, H-17), 1.67 (3H, s, CH3-21), 1.11
(3H, d, J = 6.7 Hz, CH3-27), 1.03 (3H, s, CH3-19) and
0.75 (3H, s, CH3-18); 13C-NMR (pyridine-d5) δ 152.6
(C-22), 103.5 (C-20), 84.6 (C-16), 67.5 (C-26), 66.1
(C-3), 64.7 (C-17), 54.9 (C-14), 43.9 (C-13), 40.2 (C-12),
40.0 (C-9), 37.0 (C-5), 36.1 (C-25), 35.6 (C-8), 35.3
(C-10), 34.5 (C-15), 34.4 (C-4), 31.3 (C-1), 30.6 (C-24),
28.6 (C-7), 27.2 (C-6), 27.0 (C-2), 24.3 (C-19), 23.8
(C-11), 21.4 (C-23), 17.1 (C-27), 14.5 (C-18) and 11.9
(C-21); Anal. calcd. for C27H44O3: C, 77.84; H, 10.64.
Found: C, 77.49; H, 10.28.
A solution of 2 (5.0 g, 12.0 mmol) in acetic anhydride
(50 mL) was heated in a bomb tube at 200 °C for 12 h.
The reaction mixture was co-evaporated with toluene
(50 mL × 5) to give a residue that was subjected to
column chromatography (a gradient of 0–10% AcOEt in
benzene), followed by application of preparative HPLC
(1:9 H2O/acetone), to give compound 8 as white foam
1
(3.8 g, 63.2%): FABMS: m/z 523 [M + Na]+; H-NMR
(CDCl3) (only assignable signals were listed) δ 5.06 (1H,
br. s, H-3), 4.72 (ddd, J = 10.4, 7.9, 5.8 Hz, H-16), 3.94
(1H, dd, J = 10.7, 6.7 Hz, H-26a), 3.87 (1H, dd, J = 10.7,
5.8 Hz, H-26b), 2.47 (1H, d, J = 10.4 Hz, H-17), 2.05
(3H, s, Ac), 2.04 (3H, s, Ac), 1.58 (3H, s, CH3-21), 0.98
(3H, s, CH3-19), 0.94 (3H, d, J = 6.7 Hz, CH3-27) and
0.66 (3H, s, CH3-18); 13C-NMR (CDCl3) δ 171.1 (CO),
170.5 (CO), 151.3 (C-22), 103.7 (C-20), 84.2 (C-16),
70.5 (C-3), 69.1 (C-26), 64.2 (C-17), 54.8 (C-14), 43.6
(C-13), 39.8 (C-9), 39.8 (C-12), 37.2 (C-5), 35.0 (C-8),
34.9 (C-10), 33.9 (C-15), 32.0 (C-25), 30.6 (C-24), 30.6
(C-1), 30.5 (C-4), 26.4 (C-7), 26.3 (C-6), 24.9 (C-2), 23.7
(C-19), 23.1 (C-11), 21.4 (COCH3), 20.9 (C-23), 20.8
(COCH3), 16.5 (C-27), 14.0 (C-18) and 11.5 (C-21);
Anal. calcd. for C31H48O5: C, 71.36; H, 9.66. Found: C,
71.17; H, 9.68.
5.1.1.5. Pseudodiosgenin diacetate 10
The general procedure was employed with diosgenin 9
(5.0 g, 12.1 mmol) in acetic anhydride (50 mL) to give a
residue that was subjected to column chromatography (a
gradient of 0–10% AcOEt in benzene), followed by
application of preparative HPLC (1:9 H2O/acetone), to
give compound 10 as a white foam (3.4 g, 56.3%):
5.1.1.7. Pseudodiosgenin 12
Compound 10 (2.0 g, 4.0 mmol) was deacetylated in
the same manner as 8 to obtain 12 (1.3 g, 78.5%):
FABMS of 12: m/z 437 [M + Na]+; H-NMR (CDCl3)
1
(only assignable signals were listed) δ: 5.34 (1H, d, J =
5.2 Hz, H-6), 4.73 (1H, ddd, J = 10.1, 7.9, 5.8 Hz, H-16),
3.50 (1H, m, H-3), 3.49 (1H, dd, J = 10.6, 5.5 Hz, H-26a),
3.43 (1H, dd, J = 10.6, 6.0 Hz, H-26b), 2.47 (1H, d, J =
10.1 Hz, H-17), 1.59 (3H, s, CH3-21), 1.02 (3H, s,
CH3-19), 0.93 (3H, d, J = 6.7 Hz, CH3-27) and 0.69 (3H,
s, CH3-18); 13C-NMR (CDCl3) δ: 151.5 (C-22), 140.8
(C-5), 121.2 (C-6), 103.7 (C-20), 84.2 (C-16), 71.5 (C-3),
67.7 (C-26), 64.1 (C-17), 55.0 (C-14), 50.0 (C-9), 43.2
(C-13), 42.2 (C-4), 39.4 (C-1), 37.2 (C-12), 36.5 (C-10),
35.3 (C-25), 34.0 (C-15), 32.1 (C-7), 31.5 (C-24), 31.2
(C-8), 30.4 (C-2), 23.1 (C-11), 20.9 (C-23), 19.4 (C-19),
1
FABMS: m/z 521 [M + Na]+; H-NMR (CDCl3) (only
assignable signals were listed) δ 5.38 (1H, d, J = 4.3 Hz,
H-6), 4.74 (1H, ddd, J = 10.2, 7.9, 5.6 Hz, H-16), 4.60
(1H, m, H-3), 3.95 (1H, dd, J = 10.9, 5.9 Hz, H-26a), 3.87
(1H, dd, J = 10.9, 6.6 Hz, H-26b), 2.47 (1H, d, J = 10.2
Hz, H-17), 2.05 (3H, s, Ac), 2.03 (3H, s, Ac), 1.59 (3H,
s, CH3-21), 1.04 (3H, s, CH3-19), 0.94 (3H, d, J = 6.7 Hz,
CH3-27) and 0.69 (3H, s, CH3-18); 13C-NMR (CDCl3) δ
171.2 (CO), 170.5 (CO), 151.4 (C-22), 139.7 (C-5), 122.3
(C-6), 103.8 (C-20), 84.3 (C-16), 73.8 (C-3), 69.1 (C-26),
64.2 (C-17), 54.9 (C-14), 50.0 (C-9), 43.2 (C-13), 39.5

524
16.5 (C-27), 13.9 (C-18) and 11.6 (C-21); Anal. calcd. for
C27H42O3: C, 78.21; H, 10.21. Found: C, 78.01; H, 10.25.
5.1.1.11. Pseudotimosaponin A-III octaacetate 18
The general procedure was employed with timosapo-
nin A-III heptaacetate 6 (10.0 g, 9.7 mmol) which was
heated in acetic anhydride (50 mL) to give a residue that
was subjected to column chromatography (a gradient of
0–10% AcOEt in benzene), followed by application of
preparative HPLC (1:9 H2O/acetone), to give compound
18 (white foam, 5.8 g, 55.8%). FABMS: m/z 1099 [M +
5.1.1.8. Ring-closure reactions of 11 and 12 in 5% HCl
in methanol
Solutions of 11 and 12 in 5% HCl in methanol were
allowed to stand for 12 h at room temperature. These
reactions quantitatively gave 2 and 9, respectively. Com-
pounds 2 and 9 were identified as sarsasapogenin and
1
Na]+; H-NMR (CDCl3) (only assignable signals were
1
diosgenin, respectively, with HPLC and H-NMR spec-
listed) (protons on the aglycon) δ 4.72 (1H, ddd, J = 10.1,
7.9, 5.8 Hz, H-16), 4.00 (1H, br. s, H-3), 3.94 (1H, dd, J
= 11.0, 6.1 Hz, H-26a), 3.88 (1H, d, J = 11.0 Hz, H-26b),
2.47 (1H, d, J = 10.1 Hz, H-17), 1.58 (3H, s, CH3-21),
1.00 (3H, s, CH3-19), 0.93 (3H, d, J = 7.0 Hz, CH3-27)
and 0.66 (3H, s, CH3-18); (protons on galactopyranose) δ
5.30 (1H, d, J = 3.4 Hz, H-4′), 4.96 (1H, dd, J = 10.1, 3.4
Hz, H-3′), 4.45 (1H, d, J = 7.6 Hz, H-1′), 4.13 (1H, dd, J
= 11.3, 6.4 Hz, H-6′a), 4.06–4.10 (H-6′b,), 3.86 (1H, dd,
J = 10.1, 7.6 Hz, H-2′) and 3.83 (1H, dd, J = 6.4, 6.4 Hz,
H-5′); (protons on glucopyranose) δ 5.14 (1H, dd, J = 9.5,
9.5 Hz, H-3′′), 5.05 (1H, dd, J = 9.5, 7.3 Hz, H-4′′), 4.83
(1H, dd, J = 9.5, 8.2 Hz, H-2′′), 4.78 (1H, d, J = 8.2 Hz,
H-1′′), 4.33 (1H, dd, J = 12.5, 4.6 Hz, H-6′′), 4.06–4.10
(6′′a and 6′′b) and 3.70 (1H, ddd, J = 7.3, 4.9, 2.8 Hz,
H-5′′), and 2.14, 2.07, 2.06, 2.05, 2.03, 2.01, 2.00 and
2.00 (each 3H, Ac); 13C-NMR (CDCl3) δ 171.1, 170.5,
170.3, 170.2, 170.1, 169.8, 169.2 and 169.1 (each CO),
151.3 (C-22), 103.7 (C-20), 100.4 (C-1′′), 100.1 (C-1′),
84.3 (C-16), 75.6 (C-3), 74.9 (C-2′), 73.0 (C-3′), 72.8
(C-3′′), 71.8 (C-2′′), 71.8 (C-5′′), 70.3 (C-5′), 69.1
(C-26), 68.4 (C-4′′), 67.3 (C-4′), 64.3 (C-17), 62.2
(C-6′′), 61.1 (C-6′), 54.8 (C-14), 43.6 (C-13), 40.0 (C-9),
39.9 (C-12), 36.2 (C-5), 35.0 (C-8), 34.9 (C-10), 33.9
(C-15), 32.0 (C-25), 30.6 (C-24), 30.0 (C-1), 30.0 (C-4),
26.6 (C-7), 26.4 (C-6), 26.2 (C-2), 23.8 (C-19), 23.1
(C-23), 20.9 (C-11), 20.9 (COCH3), 20.7 (COCH3), 20.6
(COCH3), 20.6 (COCH3), 20.5 (COCH3), 20.5 (COCH3),
20.4 (COCH3), 20.4 (COCH3), 16.5 (C-27), 14.0 (C-18)
and 11.5 (C-21); Anal. calcd. for C55H80O21: C, 61.32; H,
7.49. Found: C, 61.05; H, 7.56.
tra.
5.1.1.9. Ring-closure reaction of 11 in acetic acid
Compound 11 (1.0 g, 2.4 mmol) was dissolved in
acetic acid (5 mL) and the mixture was allowed to stand
at room temperature for 12 h. The reaction mixture was
poured into ice water (50 mL) and extracted with CH2Cl2
(30 mL × 3). The combined extracts were successively
washed with water, NaHCO3-saturated water and water,
then dried over anhydrous MgSO4, and filtered. The
filtrate was evaporated to give a residue that was sub-
jected to preparative HPLC (2:8 H2O/MeOH) to give
compounds 2, 14 and 15 in 2.7, 13.2 and 57.4% yields,
respectively. Compounds 2, 14 and 15 showed the same
quasimolecular ion peak at m/z 439 [M + Na]+.
Signals of CH3-18, CH3-19, CH3-21, CH3-27, H-20,
H-26a and H-26b in the H-NMR spectra (CDCl3) of 14
1
and 15 were listed in table I. Other assignable signals of
14 and 15 were as follows; δ 4.45 (1H, ddd, J = 7.0, 7.0,
3.7 Hz, H-16) and 4.10 (1H, br. s, H-3) for 14, and δ 4.42
(1H, ddd, J = 7.6, 7.6, 5.5 Hz, H-16) and 4.10 (1H, br. s,
H-3) for 15. 13C-NMR (CDCl3) spectra of 14 and 15 were
listed in table II. Anal. calcd. for C27H44O3 of 14: C,
77.84; H, 10.64. Found: C, 78.53; H, 10.35. Anal. calcd.
for C27H44O3 of 15: C, 77.84; H, 10.64. Found: C, 78.67;
H, 10.50.
5.1.1.10. Ring-closure reaction of 12 in acetic acid
Treatment of compound 12 (1.0 g, 2.4 mmol) with
acetic acid gave a residue that was subjected to prepara-
tive HPLC (2:8 H2O/acetone) to give compounds 9
(white powder, 126 mg, 12.6%) and 16 (white powder,
683 mg, 68.3%). Compound 9 was identified as diosgenin
5.1.1.12. Pseudomarcogenin diglycoside nonaacetate 19
The general procedure was employed with compound
17 (1.0 g, 0.91 mmol) which was heated in acetic anhy-
dride (10 mL) to give a residue that was subjected to
column chromatography (a gradient of 0–10% AcOEt in
benzene), followed by application of preparative HPLC
(1:9 H2O/acetone), to give compound 19 (white foam,
640 mg, 61.7%): FABMS: m/z 1157 [M + Na]+; 1H-NMR
(CDCl3) (only assignable signals were listed) (protons on
the aglycon) δ 4.73 (2H, H-3 and H-16), 4.10 (1H, m,
H-2), 3.94 (1H, dd, J = 10.7, 6.4 Hz, H-26a), 3.88 (1H,
1
with HPLC and H-NMR spectrum. FABMS of 16: m/z
437 [M + Na]+. Signals of CH3-18, CH3-19, CH3-21,
CH3-27, H-20, H-26a and H-26b in the 1H-NMR spectra
(CDCl3) of 16 were listed in table I. Other assignable
signals of 16 were as follows: δ 5.34 (1H, d, J = 5.27 Hz,
H-6), 4.44 (1H, ddd, J = 7.0, 7.0, 4.0 Hz, H-16) and 3.50
(1H, m, H-3); Anal. calcd. for C27H42O3: C, 78.21; H,
10.21. Found: C, 78.10; H, 10.24. 13C-NMR (CDCl3)
spectra of 9 and 16 were listed in table II.

525
dd, J = 10.7, 6.1 Hz, H-26b), 2.47 (1H, d, J = 10.1 Hz,
H-17), 1.57 (3H, s, CH3-21), 1.02 (3H, s, CH3-19), 0.94
(3H, d, J = 6.7 Hz, CH3-27) and 0.65 (3H, s, CH3-18);
(protons on galactopyranose) δ 5.29 (1H, d, J = 3.7 Hz,
H-4′), 4.93 (1H, dd, J = 10.1, 3.7 Hz, H-3′), 4.32 (1H, d,
J = 7.6 Hz, H-1′), 3.98 (1H, dd, J = 10.1, 7.6 Hz, H-2′),
3.82 (1H, dd, J = 7.3, 7.3 Hz, H-5′) and 4.05–4.15 (H-6′a
and H-6′b); (protons on glucopyranose) δ 5.13 (1H, dd, J
= 9.8, 9.5 Hz, H-3′′), 4.98 (1H, dd, J = 9.8, 9.8 Hz, H-4′′),
4.83 (1H, dd, J = 9.5, 8.2 Hz, H-2′′), 4.73 (1H, d, J = 8.2
Hz, H-1′′) and 4.05–4.15 (H-6′′a and H-6′′b), 3.73 (1H,
ddd, J = 9.8, 5.2, 3.7 Hz, H-5′′) and 2.15, 2.09, 2.08, 2.06,
2.05, 2.04, 2.01, 1.99 and 1.98 (each 3H, Ac); 13C-NMR
(CDCl3) δ 171.1, 170.5, 170.5, 170.3, 170.1, 170.1,
169.8, 169.3 and 169.2 (each CO), 151.3 (C-22), 103.7
(C-20), 100.8 (C-1′), 100.4 (C-1′′), 84.2 (C-16), 77.0
(C-2), 74.0 (C-2′), 73.7 (C-3′), 72.7 (C-3′′), 71.7 (C-5′′),
71.4 (C-2′′), 70.5 (C-5′), 70.0 (C-3), 69.1 (C-26), 68.7
(C-4′′), 67.3 (C-4′), 64.2 (C-17), 62.5 (C-6′′), 61.2 (C-6′),
54.6 (C-14), 45.3 (C-13), 41.0 (C-9), 39.6 (C-12), 36.7
(C-10), 35.1 (C-8), 35.0 (C-5), 33.9 (C-15), 30.6 (C-1),
30.6 (C-24), 30.1 (C-4), 26.6 (C-6), 25.6 (C-7), 23.5
(C-19), 23.1 (C-23), 21.0 (C-11), 21.2 (C-25), 20.9–20.3
(COCH3 × 9), 16.6 (C-27), 14.0 (C-18) and 11.5 (C-21);
Anal. calcd. for C57H82O23: C, 60.31; H, 7.28. Found: C,
60.01; H, 7.30.
J = 8.9, 7.6 Hz, H-2′′),.4.01 (1H, dd, J = 6.4, 6.1 Hz,
H-5′) and 3.85 (1H, ddd, J = 9.5, 6.1, 3.4 Hz, H-5′′);
13C-NMR spectrum of 20 was listed in table III; Anal.
calcd. for C39H64O13.H2O: C, 61.72; H, 8.77. Found; C,
61.66; H, 8.81. FABMS of 21 m/z 763 [M + Na]+; signals
of CH3-18, CH3-19, CH3-21, CH3-27, H-20, H-26a and
H-26b in the 1H-NMR spectra (pyridine-d5) were listed in
table I; other assignable signals were as follows: δ 5.23
(1H, d, J = 7.6 Hz, H-1′′), 4.89 (1H, d, J = 7.6 Hz, H-1′),
4.62 (1H, dd, J = 9.5, 7.6 Hz, H-2′), 4.61 (1H, ddd, J =
7.3, 7.3, 3.4 Hz, H-16), 4.58 (1H, d, J = 4.0 Hz, H-4′),
4.53 (1H, m, H-16), 4.31 (1H, br. s, H-3), 4.35–4.47 (4H,
H-6′a, 6′b, 6′′a and 6′′b), 4.27 (1H, dd, J = 9.5, 9.2 Hz,
H-4′′), 4.24 (1H, dd, J = 9.5, 4.0 Hz, H-3′), 4.14 (1H, dd,
J = 9.2, 8.9 Hz, H-3′′), 4.03 (1H, dd, J = 8.9, 7.6 Hz,
H-2′′), 4.00 (1H, dd, J = 6.1, 6.1 Hz, H-5′) and 3.81 (1H,
ddd, J = 9.5, 5.5, 4.0 Hz, H-5′′); 13C-NMR spectrum of
21 was listed in table III;Anal. calcd. for C39H64O13.H2O:
C, 61.72; H, 8.77. Found: C, 61.33; H, 8.65.
5.1.1.14. Acid hydrolysis of 20 and 21
Solutions of 20 and 21 in 2 N HCl in dioxane/H2O
were heated at 80 °C for 16 h. After-treatment of both
solutions in the same manner as hydrolysis of 3, 2 was
obtained quantitatively.
5.1.1.15. Ring-closure reaction of 19
5.1.1.13. Ring-closure reaction of 18
A solution of 19 (0.6 g, 0.53 mmol) in 5% KOH in
EtOH/H2O (1:1, 5 mL) was allowed to stand overnight at
room temperature. After-treatment of the solution in the
same manner as the ring-closure reaction of 18 gave
compounds 4 (15 mg, 3.8%), 22 (166 mg, 41.5%), 23
(45 mg, 11.3%) and 24 (8 mg, 2.0%). Compound 4 was
identified with marcogenin diglycoside by comparison of
HPLC and 1H-NMR spectrum. FABMS of 22 m/z 779 [M
+ Na]+; signals of CH3-18, CH3-19, CH3-21, CH3-27,
H-20, H-26a and H-26b in the 1H-NMR specrum
(pyridine-d5) were listed in table I. Other assignable
signals were as follows: δ 5.27 (1H, d, J = 7.6 Hz, H-1′′),
4.99 (1H, d, J = 7.6 Hz, H-1′), 4.71 (1H, dd, J = 8.9, 7.6
Hz, H-2′), 4.51 (1H, d, J = 3.1 Hz, H-4′), 4.53–4.43 (5H,
H-16, 6′a, 6′b, 6′′a and 6′′b), 4.38 (1H, br. s, H-3), 4.27
(2H, H-3′ and 4′′), 4.19 (1H, dd, J = 8.9, 8.9 Hz, H-3′′),
4.11 (1H, dd, J = 8.9, 7.6 Hz, H-2′′), 4.09 (1H, m, H-5′)
and 3.85 (1H, m, H-5′′); 13C-NMR spectrum were listed
in table III; Anal. calcd. for C39H64O14.H2O: C, 60.45; H,
8.58. Found: C, 60.19; H, 8.47. FABMS of 23 m/z 779 [M
+ Na]+; signals of CH3-18, CH3-19, CH3-21, CH3-27,
H-20, H-26a and H-26b in the 1H-NMR specrum
(pyridine-d5) were listed in table I; other assignable
signals of 23 were as follows; δ 5.29 (1H, d, J = 7.6 Hz,
H-1′′), 5.03 (1H, d, J = 7.6 Hz, H-1′), 4.73 (1H, dd, J =
A solution of 18 (5 g, 4.64 mmol) in 5% KOH in
EtOH/H2O (1:1, 20 mL) was allowed to stand overnight
at room temperature. The reaction mixture was acidified
with acetic acid (pH 4.0), then neutralized with pyridine.
The mixture was evaporated to give a residue that was
subjected to column chromatography (CHCl3/MeOH/
H2O, 65:35:10, lower layer), followed by application of
preparative HPLC (3:7 H2O/MeOH), to give compound 3
(fine needles, m.p. more than 300 °C after recrystalliza-
tion from acetone/ethanol, 95 mg, 2.8%), 20 (white
needles, m.p. more than 300 °C after recrystallization
from acetone/ethanol, 1.6 g, 46.5%), and 21 (white foam,
690 mg, 20.1%). Compound 3 was identified as timosa-
ponin A-III by HPLC and 1H-NMR spectrum. FABMS of
20 m/z 763 [M + Na]+; signals of CH3-18, CH3-19,
CH3-21, CH3-27, H-20, H-26a and H-26b in the 1H-NMR
spectra (pyridine-d5) were listed in table I. Other assign-
able signals were as follows: δ 5.25 (1H, d, J = 7.6 Hz,
H-1′′), 4.90 (1H, d, J = 7.6 Hz, H-1′), 4.64 (1H, dd, J =
9.5, 7.6 Hz, H-2′), 4.54 (1H, d, J = 3.1 Hz, H-4′), 4.53
(1H, ddd, J = 7.3, 7.3, 5.5 Hz, H-16), 4.35–4.55 (4H,
H-6′a, 6′b, 6′′a and 6′′b), 4.32 (1H, br. s, H-3), 4.29 (1H,
dd, J = 9.5, 9.2 Hz, H-4′′), 4.26 (1H, dd, J = 9.5, 3.1 Hz,
H-3′), 4.17 (1H, dd, J = 9.2, 8.9 Hz, H-3′′), 4.06 (1H, dd,

526
Table III. 13C-NMR spectral data of compounds 3, 4 and 20–24a.
8.61. FABMS of 24 m/z 779 [M + Na]+; signals of
CH3-18, CH3-19, CH3-21, CH3-27, H-20, H-26a and
H-26b in the 1H-NMR specrum (pyridine-d5) were listed
in table I; other assignable signals of 24 were as follows:
δ 5.29 (1H, d, J = 7.6 Hz, H-1′′), 5.00 (1H, d, J = 7.6 Hz,
H-1′), 4.74 (1H, dd, J = 8.0, 7.6 Hz, H-2′), 4.60 (1H, dd,
J = 10.1, 7.6 Hz, H-6′′a), 4.58 (1H, d, J = 4.0 Hz, H-4′),
4.53–4.42 (4H, H-16, 6′a, 6′b and 6′′b), 4.39 (1H, br. s,
H-3), 4.32 (1H, dd, J = 9.5, 9.2 Hz, H-4′′), 4.30 (1H, dd,
J = 7.9, 4.0 Hz, H-3′), 4.21 (1H, dd, J = 9.2, 8.9 Hz,
H-3′′), 4.12 (2H, H-5′ and 2′′) and 3.87 (1H, ddd, J = 9.5,
7.6, 4.3 Hz, H-5′′); 13C-NMR spectrum were listed in
table III; Anal. calcd. for C39H64O14.H2O: C, 60.45; H,
8.58. Found: C, 60.22; H, 8.46.
3
20
21
4
22
23
24
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
30.9b 30.9
31.0
26.9
75.5
30.9
36.9
26.5
26.8
35.1
40.1
35.1
20.7
40.0
42.3
57.4
32.3
81.4
60.0
16.4
24.0
46.6
11.5
40.5
67.2
81.7
31.8
36.5
26.7
26.3
35.5
41.3
37.0
21.3
40.2
40.8
56.3
32.1
81.3
62.9
16.5
23.8
42.4
14.9
40.5
67.2
81.7
31.8
36.6
26.7
26.3
34.7
41.4
37.0
21.0
41.1
42.1
56.7
34.2
83.3
60.3
16.2
23.9
46.1
10.2
40.6
67.2
81.8
31.8
36.6
26.5
26.3
35.2
41.3
37.0
20.9
40.3
42.3
57.3
32.3
81.4
61.0
16.4
23.9
47.0
11.6
40.3
67.2
81.8
31.9
36.6
26.5
26.3
35.2
41.3
37.0
20.9
40.6
42.3
57.3
32.3
81.4
61.0
16.4
23.9
46.7
11.5
27.0
75.4
30.9
36.8
26.8
26.8
35.5
40.2
35.2
21.1
40.3
40.9
56.4
35.2
81.3
62.9
16.6
24.0
40.3
14.9
27.0
75.6
30.8
36.9
26.6
26.8
34.7
40.2
35.2
20.8
41.2
42.2
56.9
34.2
83.3
60.3
16.2
24.0
46.1
10.2
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
C-22
C-23
C-24
C-25
C-26
C-27
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
C-6′′
5.1.1.16. 26-O-Acetyl pseudotimosaponin A-III 25
A solution of 18 (1 g, 0.93 mmol) in 5% NH3 in MeOH
(20 mL) was allowed to stand overnight at room tempera-
ture. The mixture was sucked for 10 min and evaporated
to give a residue that was subjected to column chroma-
tography (0–10% AcOEt in benzene) to give 25 (510 mg,
70.2%) as white powder: FABMS m/z 805 [M + Na]+;
1H-NMR (pyridine-d5) (only assignable signals were
listed) δ 5.24 (1H, d, J = 7.6 Hz, H-1′′), 4.90 (1H, d, J =
7.6 Hz, H-1′), 4.86 (2H, H-16, overlapped with H-1′),
4.62 (1H, dd, J = 9.2, 8.2 Hz, H-2′), 4.54 (1H, br.s, H-4′),
4.35–4.50 (5H, m, H-26a, 6′a, 6′b, 6′′a and 6′′b), 4.33
(1H, br. s, H-3), 4.20–4.30 (2H, m, H-3′ and 4′′), 4.16
(1H, dd, J = 9.2, 8.9 Hz, H-3′′), 4.00–4.10 (3H, m, H-26b,
5′ and 2′′), 3.81 (1H, m, H-5′′), 2.50 (1H, d, J = 7.0 Hz,
H-17), 2.01 (3H, s, Ac), 1.64 (3H, s, CH3-21), 0.98 (3H,
s, CH3-19), 0.95 (3H, d, J = 6.4 Hz, CH3-27) and 0.72
(3H, s, CH3-18); 13C-NMR (pyridine-d5) δ 170.8 (CO),
151.9 (C-22), 107.5 (C-1′′), 103.9 (C-20), 102.5 (C-1′),
84.6 (C-16), 81.7 (C-2′), 78.3 (C-5′′), 77.9 (C-3′′), 76.8
(C-2′′), 76.5 (C-5′), 75.4 (C-3), 75.1 (C-3′), 71.6 (C-4′′),
69.7 (C-4′), 69.1 (C-26), 64.6 (C-17), 62.7 (C-6′′), 62.1
(C-6′), 54.7 (C-14), 43.8 (C-13), 40.1 (C-9), 40.1 (C-12),
36.9 (C-5), 35.2 (C-8), 35.2 (C-10), 34.4 (C-15), 32.3
(C-25), 31.1 (C-1), 30.9 (C-4), 30.9 (C-7), 26.9 (C-2),
26.8 (C-6), 26.8 (C-24), 24.0 (C-19), 23.4 (C-11), 21.3
(C-23), 20.8 (COCH3), 16.7 (C-27), 14.4 (C-18) and 11.8
(C-21); Anal. calcd. for C41H66O14.H2O: C, 61.48; H,
8.56. Found: C, 61.19; H, 8.67.
109.7 106.8 106.4 109.6 106.8 109.0 108.4
26.4
26.2
27.5
65.1
16.3
35.2
29.5
30.2
67.7
17.3
30.9
29.9
30.9
68.1
17.4
26.2
26.2
27.5
65.1
16.3
35.2
29.5
30.2
67.7
17.3
26.2* 31.0*
25.9* 29.0*
27.8
66.6
16.6
30.9
68.1
17.4
102.5 102.5 102.5 103.3 103.3 103.4 103.5
81.7
75.1
69.8
76.5
62.1
81.8
75.1
69.7
76.5
62.1
81.7
75.1
69.7
76.5
62.1
81.9
76.9
69.7
76.9
61.9
82.0
76.9
69.7
76.9
61.9
82.1
77.0
69.7
77.0
62.0
82.1
77.0
69.7
77.0
62.0
106.0 106.0 106.0 106.1 106.2 106.2 106.2
76.8
77.9
71.6
78.3
62.7
76.8
77.9
71.6
78.3
62.7
76.8
77.9
71.6
78.3
62.7
75.1
78.5
71.6
77.9
62.7
75.1
78.5
71.6
77.9
62.7
75.1
78.5
71.7
78.0
62.8
75.2
78.6
71.7
78.0
62.8
a Spectra were obtained in pyridine-d5.. b Chemical shifts are in
ppm from internal (CH3)4Si. Signal assignments were based on
DEPT, 1H-1H and 1H-13C COSY and 1H-13C long-range COSY
spectral data.. * These values may be interchangeable in each
column.
8.0, 7.6 Hz, H-2′), 4.58 (1H, dd, J = 10.7, 7.3 Hz, H-6′′a),
4.53 (1H, d, J = 3.1 Hz, H-4′), 4.50–4.39 (4H, H-16, 6′a,
6′b and 6′′b), 4.39 (1H, br. s, H-3), 4.31 (1H, dd, J = 9.9,
9.5 Hz, H-4′′), 4.29 (1H, dd, J = 8.0, 3.1 Hz, H-3′), 4.21
(1H, dd, J = 9.9, 8.2 Hz, H-3′′), 4.11 (2H, H-5′ and 2′′)
and 3.87 (1H, ddd, J = 9.5, 7.3, 4.3 Hz, H-5′′); 13C-NMR
spectrum were listed in table III; Anal. calcd. for
C39H64O14.H2O: C, 60.45; H, 8.58. Found: C, 60.33; H,
5.1.1.17. X-ray diffraction
The measurements of 7, 13 and 20 were made on a
Bruker SMART diffractometer with graphite monochro-
mated Mo Kα radiation (λ = 0.71073 Å), a Mac Science
MXC 3KHF diffractometer with graphite monochro-
mated Mo Kα radiation (λ = 0.71073 Å) and a Rigaku

527
AFC5 diffractometer with graphite monochromated Cu
Ka radiation (λ = 1.5418 Å), respectively.
considered as 100% agglutination. Suppression rate of
agglutination was calculated by the following equation:
suppression efficiency of agglutination = 100 × (agglu-
tination intensity with DMSO – agglutination intensity
with sample)/agglutination intensity with DMSO.
5.1.1.18. Crystal data for 7
C34H47BrO4, M = 599.63, prismatic crystal (colour-
less, 0.63 × 0.15 × 0.15 mm), monoclinic, space group
P21, a = 10.339 (1), b = 6.450 (1), c = 23.533 (2) Å, ꢀ =
101.697 (2)°, V = 3725.0 (6) Å3, Z = 2, DX = 1.296 g/cm3,
final R = 0.042, WR = 0.075, GOF = 0.833.
References
[1] Devon T.K., Scott A.I., Handbook of Naturally Occurring Com-
pounds, Vol. II, Terpenes, Academic Press, NY, 1972, pp. 404–411.
[2] Marker R.E., Wagner R.B., Ulshafer P.R., Wittbecker E.L., Gold-
5.1.1.19. Crystal data for 13
smith D.P.J., Ruof C.H., J. Am. Chem. Soc. 69 (1947) 2167–2230.
C29H44O4, M = 456.64, prismatic crystal (colourless,
0.25 × 0.20 × 0.15 mm), monoclinic, space group P21, a
= 14.553 (2), b = 6.211 (1), c = 14.795 (2) Å, ꢀ = 100.036
(7)°, V = 1316.8 (3) Å3, Z = 2, DX = 1.150 g/cm3, final R
= 0.049, WR = 0.050, GOF = 0.563.
[3] Wall M.E., Eddy C.R., Serota S., J. Am. Chem. Soc. 76 (1954) 2849.
[4] Wall M.E., Serota S., J. Am. Chem. Soc. 76 (1954) 2850–2852.
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641–646.
[6] Ziegler J.B., Rosen W.E., Shabica A.C., J. Am. Chem. Soc. 77
(1955) 1223–1229.
5.1.1.20. Crystal data for 20
[7] Wall M.E., Serota S., Eddy C.R., J. Am. Chem. Soc. 77 (1955)
1230–1237.
C39H64O13, M = 730.93, prismatic crystal (colourless,
0.45 × 0.30 × 0.20 mm), orthoronbic, space group P212121,
a = 11.579 (2), b = 43.263 (3), c = 7.967 (2) Å, V = 3990
(3) Å3, Z = 4, DX = 1.233 g/cm3, final R = 0.109, WR =
0.092, GOF = 5.678.
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[9] Marker R.E., Rohrmann E., J. Am. Chem. Soc. 61 (1939) 846–851.
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Kohda H. et al., Yakugaku Zasshi 108 (1988) 555–561.
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892–896.
5.1.2. Pharmacology
[12] Kawasaki T., Yamauchi T., Chem. Pharm. Bull. 11 (1963) 1221–1224.
[13] Morita N., Shimizu M., Fukuda M., Yakugaku Zasshi 85 (1965)
5.1.2.1. Materials
374–375.
Platelet rich plasma (PRP) was prepared with freshly
collected blood from healthy adults, and its concentration
was adjusted to 3.0 × 105 cells/µL by dilution with
platelet poor plasma (PPP) [25, 26]. The PRP was used
within 3 h of preparation. Spirostanol diglycosides were
dissolved in DMSO at a concentration of 40 mg/mL as
stock solutions.
[14] Nagumo S., Kishi S., Inoue T., Nagai M., Yakugaku Zassh 111
(1991) 306–310.
[15] Takahashi M., Konno C., Hikino H., Planta Med. 51 (1985)
100–102.
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Pharm. Sci. 61 (1971) 1838–1840.
[17] Hall S.R., Stewart J.M., Xtal 3.2, Univs. of Western Australia,
Australia and Maryland, USA.
[18] Full X-ray crystal data are deposited in the Cambridge Crystallo-
graphic Data Centre (CCDC).
5.1.2.2. Assay of platelet agglutination activities
[19] Marker R.E., Tsukamoto T., Turner D.L., J. Am. Chem. Soc. 62
The platelet agglutination was monitored with an
aggregometer using PPP as a reference [27]. Two hundred
and ninety-four microlitres of PRP in a glass cell appa-
ratus were pre-incubated with 3 µL of samples for 5 min
at 37 °C in a glass chamber. Final concentration of each
samples was adjusted to 400, 200, 100 and 50 µg/mL.
After 5 min of pre-incubation, assays were started by
addition of agglutination inducers; 3 µL of 1 mM ADP or
100 mg/mL ristocetin, and the change of light transmit-
tance induced by platelet agglutination was monitored
with an Platelet Aggregometer (Chrono-Log Co. Tokyo).
Agglutination activity and effect of each sample on it
were evaluated by agglutination intensity. The agglutina-
tion activity with DMSO instead of a specimen was
(1940) 2525–2532.
[20] Agrawal P.K., Jain D.C., Gupta P.K., Thakur R.S., Phytochemistry
24 (1985) 2479–2496.
[21] Callow R.K., James V.H.T., J. Chem. Soc. (1955) 1671–1674.
[22] Yu B.Y., Hirai Y., Shoji J., Xu G.J., Chem. Pharm. Bull. 38 (1990)
1931–1935.
[23] Asano T., Murayama T., Hirai Y., Shoji J., Chem. Pharm. Bull. 41
(1993) 566–570.
[24] Saito S., Nagase S., Ichinose K., Chem. Pharm. Bull. 42 (1994)
2342–2345.
[25] Garder A., Jonsen J., Laland S., Hellem A., Owren P.A., Nature 192
(1961) 531–532.
[26] MacFarlane D.E., Stibbe J., Kirby E.P., Zucker M.B., Grant R.A.,
McPherson J., Thromb. Diath. Haemorrh. 34 (1975) 306–308.
[27] Born G.V.R., Nature 194 (1962) 927–929.