'Click' synthesis of triazole-based spirostan saponin analogs

Article abstract of DOI:10.1016/j.tet.2011.08.003

Novel analogs of spirostan saponins in which the glycosidic bond has been replaced by a triazole linkage are described. For this, a direct oligosaccharide-steroid conjugation approach based on the Cu^I- catalyzed azide-alkyne 1,3-dipolar cycload

Full text of DOI:10.1016/j.tet.2011.08.003

Tetrahedron 67 (2011) 7713e7727
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‘Click’ synthesis of triazole-based spirostan saponin analogs
a
,
,
d
,
b
d
,
,d
Karell Perez-Labrada c, Ignacio Brouard a , Cercis Morera , Francisco Estevez e, Jaime Bermejo a
,
*
ꢀ
ꢀ
,
Daniel G. Rivera b
*
a
ꢀ
ꢀ
ꢀ
Instituto de Productos Naturales y Agrobiología-C.S.I.C., Instituto Universitario de Bio-Organica, ‘Antonio Gonzalez’, Av. Astrofísico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain
b Center for Natural Products Study and Faculty of Chemistry, University of Havana, Zapata y G, 10400, La Habana, Cuba
c
ꢀ
Institute of Pharmacy and Food, University of Havana, San Lazaro y L, 10400, La Habana, Cuba
d Department of Biochemistry, Molecular Biology and Physiology, University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016, Las Palmas de Gran Canaria, Spain
e
ꢀ
ꢀ
Instituto Canario de Investigaciones del Cancer (ICIC), Av. Astrofísico F. Sanchez 2, 38206 La Laguna Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2011
Received in revised form 1 August 2011
Accepted 2 August 2011
Available online 7 August 2011
Novel analogs of spirostan saponins in which the glycosidic bond has been replaced by a triazole linkage
are described. For this, a direct oligosaccharideesteroid conjugation approach based on the CuI-catalyzed
azideealkyne 1,3-dipolar cycloaddition was implemented, leading to diverse combinations of saponin
analogs with variations in the trisaccharide moiety, the artificial linkage, and the steroid-skeleton
functionalization. This ‘click’ process proved great efficiency for the ligation of two bulky building
blocks (e.g., chacotriose derivatives and spirostanes bearing axial azides), which enabled the rapid cre-
ation of a small library of triazole-based analogs for cytotoxicity evaluation. A molecular modeling study
was performed to understand the conformational and electronic differences between a natural saponin
and its triazole-based analogs.
ꢀ
Dedicated to Professor Margarita Suarez
Navarro on the occasion of her 65th
birthday
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Steroids
Carbohydrates
Saponins
Click chemistry
Cycloaddition reactions
1. Introduction
However, a common feature of saponins is their hemolytic ac-
tivity, a property that has hampered the recognition of these nat-
Saponins1 comprise a family of terpene and steroid glycosides
with a broad variety of biological and medicinal applications.1,2
Steroidal saponins are among the natural products of wider dis-
tribution in the plant kingdom and of greater significance in tra-
ditional medicine. Most abundant steroidal saponins contain
a spirostanic aglycone and the oligosaccharide moiety directly at-
tached to the C-3 hydroxyl group. Several members of this family
have exhibited potent cytotoxic,3 antifungal,4 antiinflammatory,5
and antiviral activities,6 which makes them important targets for
drug development. Indeed, the most promising property of spiro-
ural products as potential anticancer drugs. Fortunately, interesting
SAR studies8 have been accomplished and the general perception
that the cytotoxicity of saponins is correlated to the hemolytic ac-
tivity has been recently dissipated. In a remarkable report,8a Yu and
co-workers evaluated dozens of spirostan saponins and showed,
firstly, that the hemolytic and cytotoxic activities are highly de-
pendent on the structural features of the glycosides, and secondly,
that these activities do not correlate between them. Alternatively,
the same group demonstrated that the cytotoxicity does correlate
with the cellular internalization of the steroidal saponins,9 which
stan saponins is their inhibitory activity against the growth of
provides new evidences for a better understanding of the
3def,7
certain cancer cells,
cancer potency.
showing dioscin (1) the greatest anti-
apoptosis-inducing effect of dioscin (1) and several of its
congeners.3def,7
All these findings have been only possible as a consequence of
extensive synthetic programs directed to access natural spirostan
saponins and analogs for biological evaluation.10 By introducing
modifications on both the aglycone and the oligosaccharide skel-
etons, SAR studies8e10 have showed many of the structural re-
quirements needed at each moiety for these compounds to have
a good cytotoxic activity. However, little attention has been paid to
Abbreviations: DIAD, diisopropylazodicarboxylate; DMAP, N,N-dimethylamino-
pyridine; DMF, dimethylformamide; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-
pyrimidonone; TBAB, tetra-n-butyl ammonium bromide; SAR, Structureeactivity
relationship.
* Corresponding authors. Tel.: þ537 8702102; fax: þ537 8733502; e-mail
addresses: ibrouard@ipna.csic.es (I. Brouard), dgr@fq.uh.cu (D.G. Rivera).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.08.003

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7714
the effect of
a
variation of the traditional glycosidic link-
usefulness of this concept on diverse fields of steroid chemistry.
Whereas spirostaneoligosaccharide conjugates have not been
reported by means of click chemistry, we describe the first ap-
proach to various combinations of spirostan saponin analogs hav-
ing the triazole ring as artificial linkage. These saponin analogs are
then evaluated for their cytotoxicity and studied by molecular
modeling to achieve conclusions regarding the effect of such
a linkage on the biological activity.
agedbetween the oligosaccharide and the spirostan steroiddon
the anticancer potency of the resulting conjugate. Will synthetic
spirostan saponin with an artificial oligosaccharideesteroid linkage
display good cytotoxic activity? This is an important answer to be
addressed, as some alternative conjugation approaches more effi-
cient than glycosylation could be implemented to produce analogs
of steroidal saponins.
Herein we report on the use of the CuI-catalyzed 1,3-dipolar
cycloaddition11 of alkynes and azides for the direct conjugation of
trisaccharides to spirostanic steroids. ‘Click’ chemistry refer to
methods capable to join together organic molecules in high yields
under mild conditions and in the presence of a diverse range of
functional groups.11c The present ‘click’ approach gives rise to novel
spirostan saponin analogs having a triazole ring as linkage of both
building blocks. The triazole linkage comprises several features that
make it a good choice in medicinal and biological chemistry, i.e., it
is hydrolytically and metabolically stable, water soluble, and rigid.
We also considered the easiness on the introduction of alkyne and
azide groups into the steroidal and carbohydrate units, an issue that
would allow for the rapid variation of the functionalization patterns
on both building blocks.
2. Results and discussion
Fig. 1 shows the structure of dioscin (1) and filiasparoside A3b
(2), two naturally occurring cytotoxic saponins that were chosen
as models for the design of the new analogs. Dioscin is probably the
saponin of greatest occurrence in the plant kingdom, and its broad
spectrum of bioactivity3feh,4,5 makes it an interesting lead for drug
development. The trisaccharide moiety of dioscin, namely chaco-
trioside, has been the subject of several SAR studies dealing with
alkylations,10b,d removal or position change of some of its
a-L-
rhamnosyl residues,8,10a,b and conjugation to triterpene or lipidic
aglycones.9,10d In contrast, filiasparoside A (2) possesses a less
common trisaccharide moiety featuring
arabinopyranosyl units linked at positions 4 and 6 of the inner
glucose, respectively. Although using -rhamnosyl as branching
monosaccharide, this type of connectivity will be also employed by
us to construct trisaccharide moieties analogous to chacotrioside
(Fig. 1).
b-D-xylopyranosyl and a-D-
The CuI-catalyzed 1,3-dipolar cycloaddition approach has been
extensively used on glycoconjugation chemistry,12 although only
a few reports have dealt with such a type of ligation between ste-
roids and sugars.13 Very recently, a series of sulfated oligosaccha-
rides have been conjugated to lipophilic steroids through this click
a-L
Fig. 1. Structures of naturally occurring cytotoxic saponins and strategy to produce triazole-based spirostan saponin analogs through CuI-catalyzed azide-alkyne 1,3-dipolar
cycloaddition.
process. This type of artificial ligation produced stable, amphiphilic
conjugates that display potent inhibitory activity against heparin
sulfate-binding proteins, and therefore, inhibition of tumor growth
and metastasis.13a Also, cytotoxic cyclopamine-rhamnose13c con-
jugates and antibacterial cholestan-neomicin B13b conjugates have
been prepared through standard click chemistry, proving the
There are two different strategies for the incorporation of the
oligosaccharide moieties of spirostan saponins to the steroidal
aglycone: (i) the direct conjugation of the oligosaccharide donor to
the steroidal aglycone,10c,d and (ii) the conjugation of the inner
glucose unit to the spirostane followed by protection/deprotection
protocols and further glycosylation with the branching

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7715
monosaccharide donors.10eei Whereas, the first approach is more
straightforward, the glycosylation step usually proceeds with low
stereoselectivity when the inner glucose has position 2 capped
with another sugar moiety. Nevertheless, since our conjugation
process relies on a cycloaddition reaction of sugars previously
functionalized at the anomeric center, the stereoselectivity issue is
not a concern.
Fig. 1 illustrates the strategy to achieve analogs of spirostan
saponins through the direct CuI-catalyzed 1,3-dipolar
cycloaddition-based conjugation of trisaccharides to spirostanes.
Combination A comprises the use of glycosyl azides and spirostanic
steroids bearing a propargyl ether at C-3. Alternatively, the tri-
saccharide moiety can also be functionalized with propargyl alco-
hol and next conjugated to 3-azido-spirostanes, as depicted in
combination B. The versatility of this method toward saponins-li-
brary construction relies both on the efficiency of the direct con-
jugation process and on the wide variety of azido-steroids and
propargyl trisaccharides that can be utilized. As shown in Fig. 1, the
2 and 4 of the glucoside.14 Thus, both the peracetylated glucosyl
azide 415 and propargyl glucoside 816 were fully deprotected and
next subjected to selective pivaloylation of C-3 and C-6, followed by
glycosylation with rhamnopyranosyl trichloroacetimidate 6 to
furnish 7 and 12, respectively, in good overall yields. Alternatively,
propargyl glucoside 8 was completely deacetylated and then cap-
ped at positions 4 and 6 as benzylidene acetal.14 Acetylation of
hydroxyls 2 and 3 followed by removal of the acetal with p-TsOH in
the mixture THFeMeOH afforded acceptor 9, which was subjected
to glycosylation with donor 6 to give the propargyl trisaccharide 11
in 69% overall yield.
For the preparation of the alkyne and azido spirostanic building
blocks, we decided to select natural spirostanic sapogenins with
diverse functionalization patterns on rings B and C, but keeping
their structural resemblance to the aglycones of most cytotoxic
saponins. As shown in Scheme 2, the first option was diosgenin (13,
dioscin’s aglycone), which was propargylated in 74% yield upon
treated with propargyl bromide in a NaH/DMF suspension. Simul-
taneously, the natural sapogenins hecogenin (15) and 5-hydroxy-
laxogenin (16) were chosen for the preparation of the corre-
approach includes not only the use of chacotriose (i.e.,
nopyranosyl-(1/4)-[ -rhamnopyranosyl-(1/2)]- -glucopyr-
anose) but also an analog bearing the two rhamnoses linked at C-4
and C-6 of the glucose (i.e., -rhamnopyranosyl-(1/6)-[
rhamnopyranosyl-(1/4)]- -glucopyranose).
a-L-rham-
a-L
b-D
sponding azides featuring the 3
the model saponins are endowed only with a 3
glycosides, we were prompted to evaluate if either the stereo-
chemistry or of the artificial linkage has some influence on the
bioactivity. Thus, the synthesis of the 3 -azido-spirostanes 17 and
18 required the replacement of the 3 -OH by azide with net re-
a
and 3
b
stereochemistry. Although
a-
L
a
-L-
b
connectivity to the
b-D
Scheme 1 depicts the preparation of the propargyl and azido
trisaccharides that will be employed on the direct conjugation to
spirostanes through click chemistry. Chacotriosyl azide 7 and
propargyl chacotrioside 12 were prepared by an adaptation of
a reported protocol designed for introducing rhamnose at positions
a
b
b
b
tention of the configuration. This was accomplished according to
a reported procedure by using methanesulfonic acid as the acidic/
nucleophilic component of the Mitsunobu reaction, followed by
displacement with NaN3 in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-
pyrimidonone (DMPU) to recover the original C-3 stereochemis-
try.17 Alternatively, the 3
prepared by tosylation of the 3
placement with inversion of the C-3 configuration.
a-azido-spirostanes 19 and 20 were easily
b
-OH and subsequent azide dis-
Scheme 2. Synthesis of 3b-propargyloxy and 3-azido-spirostanes.
As depicted in Scheme 3, the click sugarespirostan conjugation
approach (combination A, Fig. 1) was firstly implemented by uti-
lizing chacotriosyl azide 7 and propargyloxy-diosgenin 14 on the
CuI-catalyzed 1,3-dipolar cycloaddition, thus giving rise to conju-
gate 21 in 89% yield. Typical reaction conditions (i.e., 20% mol of
Scheme 1. Synthesis of trisaccharides functionalized with azido and propargyl groups.

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
Scheme 3. Synthesis of a triazole-based dioscin analog by CuI-catalyzed 1,3-dipolar cycloaddition of chacotriosyl azide (7) and propargyloxy-diosgenin (14).
a CuII salt and 40% mol of sodium ascorbate in a THF/H2O mixture)
Sadly, none of the obtained spirostan saponin analogs showed
significant cytotoxic activity against HL-60 cells (IC50>100 g/mL).
were employed, which proved great success on the ligation of these
bulky building blocks. Complete deprotection of the trisaccharide
moiety was accomplished upon treatment with standard saponi-
fication conditions, thus leading to the triazole-based dioscin an-
alog 22 in 83% yield over the two steps. Unfortunately, dioscin
analog 22 showed no significant cytotoxic activity against HL-60
m
This fact, although disappointing, provides new information re-
garding important structural elements that seem to be required for
the cytotoxicity of spirostan saponins and their synthetic analogs.
Firstly, while it is true that the triazole ring is rigid enough to
provide conformational constrain to the resulting conjugate, it
must be noticed that the methylene group of the propargyl moiety
introduces considerable flexibility to the artificial linkage. As a re-
sult, the saponin analogs may have greater conformational flexi-
bility than the natural ones. Secondly, the distance expanding the
triazole-based artificial linkage is longer that the traditional gly-
cosidic bond, which may vary the overall three-dimensional
structure of the conjugate. Thirdly, the incorporation of a triazole
ring introduces new electronic properties (e.g., the triazole ring is
considered a peptide bond analog) into the conjugate, which might
lead to mechanisms of action and/or transport in the cells com-
pletely different from those of the original spirostanyl glycosides.
To pursuit information about this subject, the conformational
and electronic differences between the natural saponin and two
selected analogs20 were studied. Consequently, two conformational
analyses were accomplished to obtain the most stable conformers
of the natural saponin (1) and its analogs 22 and 24. First, molecular
mechanics with the SYBYL force field (TRIPOS, Inc.) was performed
as implemented in the Spartan’02 windows program.21 Next, the
Multiple Minima Hypersurfaces Methodology (MMH)22 featuring
the semiempirical Hamiltonian AM1 was used to obtain the most
stable conformers. This methodology performs a Monte Carlo
simulation by randomly modifying the specified dihedral angles. In
this study, 100 conformations were created for each molecule and
the selected dihedrals were randomly generated for each confor-
mation. In Table 2 appear the dihedral angles within the MMH
methodology selected for the evaluation of the conformational
space of dioscin (1) and the two saponin analogs. The angles
C2eC3eOeC1 (1), C2eC3eOeCH2 (22), and C2eC3eNeN (24) show
the relative orientation either of the carbohydrate or the carbohy-
drateetriazole hybrid with respect to the steroidal plane. Dihedrals
Csp2eNeC1eO (22) and Csp2eCH2eOeC1 (24) allow for the eval-
uation of the relative orientation of the trisaccharide with respect
to the triazole ring. Single point AM1 energy calculations were
performed to all conformations obtained by molecular mechanics,
which enabled accessing to the whole population distribution of
the three compounds.
cells (IC50>100
m
g/mL) in a preliminary biological evaluation.18
This result made us to turn to combination B (i.e., the use of
propargyl trisaccharides and 3-azido-spirostanes) as a way of cre-
ating greater structural diversity on the resulting conjugates.
It must be mentioned that the preparation of 3a-azido-spiro-
stanes to be used in the click conjugation approach was not ran-
domly decided. Based on conformational studies performed by
molecular dynamic and molecular mechanic calculations,8b Mimaki
et al. found that the preferred conformation of a cytotoxic diosgenyl
diglycoside (i.e.,
side) possess the
a
-
L
-rhamnopyranosyl-(1/2)-
b-D-glucopyrano-
a-L-rhamnosyl unit in a vertical orientation
against the steroidal plane. In contrast, in the conformations of
non-cytotoxic diosgenyl diglycosides having the -rhamnosyl
a-L
unit linked at positions 3 or 4 of the glucoside, the full disaccharide
moiety appear on the same plane of the steroidal aglycone.
Therefore, it was suggested that the perpendicularity between the
branching a-L-rhamnosyl residue and the lipophilic steroid might
be important for the cytotoxicity mechanism of these saponins.
Although it is be difficult to correlate this information to the case of
spirostan trisaccharides,19 we were prompted to prepare artificial
conjugates derived not only from 3
also from the 3 epimers. By doing this we would be populating
a conformational space not accessible to the 3 -derivatives, since in
the 3 epimers the trisaccharideetriazole moiety is directing
completely toward the face as a result of the axial orientation.
b-functionalized spirostanes but
a
b
a
a
Accordingly, a small library of saponin analogs was created by
the direct click conjugation of the propargyl trisaccharides 11 and
12 to the 3-azido-spirostanes 17e20, which possess varied oxy-
genated functions on ring B and C and feature either the
a or
b
orientation of the azide. Table 1 illustrates the results of per-
forming the eight possible combinations between the two above
oligosaccharides and the four spirostanes. As before, the approach
proved great efficiency on producing the triazole-based conjugates,
as most reactions were completed within a few hours with yields of
isolated products higher that 70%. Remarkably, the yields of con-
jugates arising from the sterically demanding 3
a-azido-spirostanes
did not show to be lower than those of the 3
otherwise impossible to achieve by using the traditional glycosyl-
ation of 3-hydroxy-spirostanes. This becomes even more intriguing
b
-epimers, a result
Table 2 also shows the two calculated preferred conformations
for each compound, obtained as explained before. For dioscin (1),
conformation A contributes in 62% to the partition function, whilst
conformation B does it only in 14%. Interestingly, the analysis of
conformation A of dioscin (1) clearly agrees with the results pre-
viously reported by Mimaki et al.8b Therein, the conformation of
for the reaction of the very hindered 3
a-azido group of compound
20, which features a 1,3-diaxial interaction with the 5
a
-OH group
that does not seem to interfere in the reaction outcome. In general,
the CuI-catalyzed azideealkyne 1,3-dipolar cycloaddition proved to
be a very reliable and efficient method for the conjugation of tri-
saccharides to spirostanes, and in addition allowed for a rapid ac-
cess to a small library of saponin analogs for biological screening.
a diosgenyl diglycoside having an a-L-rhamnosyl attached at posi-
tion 2 of glucose possesses that rhamnose unit in a vertical orien-
tation with respect to the steroidal plane, while the glucose unit
was roughly in the same plane of the steroid. This is exactly as

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7717
Table 1
Synthesis of spirontan saponin analogs via CuI-catalyzed 1,3-dipolar cycloaddition of propargyl trisaccharides and 3-azido-spirostanes
Conjugation conditions: 20% mol Cu(AcO)2$H2O, 40% mol sodium ascorbate, THF/H2O.
Deprotection conditions: (A) NaOH (aq), THF/MeOH, 50 ꢀC; (B) NaOMe, MeOH, rt.
appear in dioscin’s preferred conformation A shown in Table 2,
which corroborates the value of such a structural requirement for
a good cytotoxicity. In contrast, most conformations of dioscin
analog 22 exhibit the whole trisaccharideetriazole hybrid moiety
directing toward the a face of the steroid skeleton, and with the
triazole ring almost perpendicular to the steroidal plane. This sys-
tem shows great conformational entropy, which is reflected by the
presence of several conformers with almost the same contribution

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7718
Table 2
Calculated preferred conformations of dioscin (1) and saponin analogs 22 and 24
to the partition function. Thus, the shown conformations A and B
contribute only in 15% and 14%, respectively, and several others
contribute between 8% and 12%. This result confirms our hypothesis
that the methylene group expanding the linkage between the
chacotriosyl triazole and the spirostanol leads to a very high con-
formational flexibility of the entire system. Finally, analysis of an-
alog 24 conveys that conformation A is the most populated one
with 83% of contribution, while the next preferred conformation is
B with only 2%. Indeed, analog 24 has much less conformational
flexibility than its analog 22, presumably owing to the fact that the
rigid triazole is directly attached to steroidal skeleton, thus lying in
the same plane. In both conformations the entire trisaccharide
moiety displays a vertical orientation with respect to the steroidal
plane, also quite different to that of dioscin (1).
addition, it was interesting to assess further information dealing
with the electronic properties of the different conjugates, with
emphasis in a comparison between the triazole-based linkage and
the glycosidic one. For this, the frontier orbitals were calculated for
the most stable conformations of 1, 22, and 24 by means of the
semiempirical AM1 method, and then visualized with the
Fig. 2 shows the geometrical superposition of the most stable
conformer of 1 (black), 22 (red) and 24 (blue), which allows for
a better visualization of the conformational differences between
the three molecules. As noticed from analysis of Table 2, the major
differences are not obviously in the steroidal skeleton but on the
orientation of the trisaccharide moiety with respect to the steroid.
It is expected that such a difference in the three-dimensional
structure of the trisaccharideespirostan conjugate is responsible
for the lack of significant cytotoxicity found for all analogs. In
Fig. 2. Superposition of the molecular geometries of most stable conformers of dioscin
(1, black), and analogs 22 (red) and 24 (blue).

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7719
Spartan’02 package. The location of the frontier orbitals can dis-
close the molecular region involved in a putative electron exchange
processes during interaction with other molecules or molecular
systems.
a triazole ring as surrogate of the traditional glycosidic linkage.
Two different combinations were employed: (i) the conjugation of
chacotriosyl azide to propargyl-spirostanes and (ii) the conjuga-
tion propargyl chacotrioside and a trisaccharidic analog to 3-
Table 3 illustrates the frontier orbitals calculated for the three
molecules. Analysis of the frontier orbitals of dioscin (1) shows the
HOMO and LUMO localized in the D5 double bond of ring B, being
both molecular orbitals mainly formed by the atomic py and pz
orbitals of the olefinic carbon atoms. Saponin analog 22 also has the
azido-spirsotanes featuring either the 3a or 3b configuration.
Both strategies resulted highly efficientdeven for the direct con-
jugation of bulky steroids having sterically hindered axial
azidesdas well as adaptable for the utilization of spirostan sa-
pogenins having diverse functionalization patterns on rings B and
C. Although the obtained saponin analogs showed no significant
cytotoxic activity against leukemia cells (HL-60), the wide spec-
trum of bioactivity exhibited by steroidal saponins shows promise
toward the emergence of biological and medicinal applications for
this new family of natural product analogs. A theoretical study
based on molecular mechanics and semiempirical calculations was
accomplished to provide more information about the conforma-
tional and electronic differences between the obtained saponins
analogs and the natural one. The conformational search disclosed
that there are important differences in the geometry of the pre-
ferred conformations as well as on the location of the frontier
molecular orbitals between the natural product and its analogs.
HOMO localized on the D
5 double bond, but the LUMO is located on
the triazole ring and mainly formed by the atomic px, py, and pz
orbitals of the triazole atoms. Alternatively, analog 24 has both
frontier orbitals localized in the triazole ring, and as in the case of
analog 22, the atomic orbitals of the triazole atoms have the
greatest contribution to the LUMO. As a summary, not only the
preferred conformation of the obtained saponin analogs are unlike
from that of the natural one, but also the distribution and location
of frontier molecular orbitals are quite different. Since the molec-
ular mechanism for the anticancer activity of saponins remains by
far uncertain, the information provided by this theoretical study is
helpful for future SAR studies.
Table 3
Calculated frontier molecular orbitals for the preferred conformations of 1, 22, and 24
Compound
HOMO
LUMO
1
22
24
3. Conclusions
Indeed, this might be a cause for the lack of cytotoxic activity in
the saponin analogs. Overall, this report represents the first ex-
perimental and theoretical work dealing the synthesis and struc-
tural study of spirostan saponin analogs having an artificial linkage
(triazole) between the sugar and the steroid, and remarkably, the
first approach on the use of steroidal azides in ‘click chemistry’
strategies.
We have implemented an oligosaccharideesteroid direct con-
jugation approach based on the CuI-catalyzed azideealkyne 1,3-
dipolar cycloaddition reaction included in the ‘click chemistry’
concept. The approach proved to be versatile and practical for the
production of a series of spirostan saponin analogs bearing

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7720
4. Experimental section
J¼7.9 Hz, H-1 Glc); 4.81 (d, 1H, J¼1.3 Hz, H-1 Rha); 4.85 (d, 1H,
J¼1.9 Hz, H-1 Rha0); 4.98e5.03 (m, 3H, H-2 Rha0, H-4 Rha, H-4
Rha0); 5.13e5.17 (m, 3H, H-2 Rha, H-3 Rha, H-3 Rha0), 5.25 (t, 1H,
4.1. General
J¼7.9 Hz, H-3 Glc). 13C NMR (100 MHz, CDCl3):
¼17.1, 17.2, 20.5,
d
Melting points are uncorrected. 1H NMR and 13C NMR spectra
were recorded at 400 and 500 MHz for 1H, 100 and 125 MHz for 13C,
respectively. Chemical shifts (d) are reported in parts per million
20.6, 20.7, 26.8, 27.0 (CH3); 38.7, 38.8 (C); 61.9 (CH2); 67.4, 68.0, 68.6,
68.9, 69.4, 69.9, 70.4, 70.7, 74.2, 74.8, 75.9, 77.5, 88.0, 97.5, 98.3 (CH);
169.4, 169.6, 169.7, 169.8, 169.9, 176.3, 177.6 (C]O). HRMS (ESI-FT-
ICR) m/z: 940.3546 [MþNa]þ (calculated for C40H59N3O21Na:
940.3539).
relative to the solvent signals, and coupling constants (J) are
reported in hertz. NMR peak assignments were accomplished by
analysis of the 1He1H COSY and HSQC data. High resolution ESI
mass spectra were obtained from a Fourier transform ion cyclotron
resonance (FT-ICR) mass spectrometer, an RF-only hexapole ion
guide and an external electrospray ion source. Flash column chro-
matography was carried out using silica gel 60 (230e400 mesh)
and analytical thin layer chromatography (TLC) was performed
using silica gel aluminum sheets. All commercially available
chemicals were used without further purification.
4.1.3. Propargyl 2,3-di-O-acetyl-b-D-glucopyranoside (9). To a sus-
pension of compound 8 (2.27 g, 5.88 mmol) in MeOH (30 mL) was
added NaOMe until pH 9e10. The mixture was stirred for 1 h, then
neutralized with acid resin Dowex-50 (Hþ), filtered and the filtrate
was concentrated under reduced pressure. The residue was dis-
solved in anhydrous DMF (80 mL) and benzaldehyde dimethyl
acetal (6.4 mL, 42.3 mmol) was added. The pH was adjusted to 3
with p-TsOH and the solution was rotated in the rotavapor under
reduced pressure at 50 ꢀC for 3 h. The reaction was quenched with
Et3N and the volatiles were removed under reduced pressure.
Flash column chromatography (hexane/EtOAc 1:1) afforded
4.1.1. 3,6-Di-O-pivaloyl-b-D-glucopyranosyl azide (5). To a suspen-
sion of compound 4 (2.12 g, 5.7 mmol) in MeOH (30 mL) was
added NaOMe up to pH 9e10. The mixture was stirred for 1 h,
then neutralized with acid resin Dowex-50 (Hþ), filtered and the
filtrate was concentrated under reduced pressure. The residue was
dissolved in anhydrous pyridine (35 mL) and the solution was
cooled to ꢁ15 ꢀC. Pivaloyl chloride (3.1 mL, 28.4 mmol) was added
dropwise under nitrogen atmosphere. The reaction course was
monitored by TLC, and it was stirred at rt until the intermediate
disappeared. The reaction mixture was then diluted with EtOAc
(50 mL) and washed with dilute aq HCl, satd aq NaHCO3, and
brine. The organic phase was dried over anhydrous Na2SO4 and
evaporated to dryness. The crude product was purified by flash
column chromatography (hexane/EtOAc 3:1) to afford diol 5
(1.31 g, 62%) as a white solid. Rf¼0.22 (hexane/EtOAc 3:1). Mp:
propargyl 4,6-O-benzylidene-b-D-glucopyranoside (1.66 g, 92%).
The solution of this product in anhydrous pyridine (10 mL) was
treated with Ac2O (10 mL). The resulting reaction mixture was
stirred at rt overnight and then poured into water and extracted
with EtOAc (30 mL). The organic layer was washed with dilute aq
HCl and brine, then dried over anhydrous Na2SO4 and evaporated
to dryness. The residue was dissolved in 1:1 THFeMeOH (30 mL)
and p-TsOH$H2O (1.13 g, 5.96 mmol) was added. After 3 h the
reaction was quenched with Et3N and the solvents were removed
under reduced pressure. The crude product was purified by flash
column chromatography (hexane/EtOAc 1:4) to afford diol 9
(1.54 g, 94% from 4,6-O-benzylidene-b-D-glucopyranoside and 87%
163e164 ꢀC. ½a 2D0
ꢂ
ꢁ35.3 (c 1.50, CHCl3). IR (KBr, cmꢁ1
)
nmax: 3447,
from 8) as a white foam. Rf¼0.38 (hexane/EtOAc 1:4). ½a D20
ꢂ
ꢁ30.2
2961, 2925, 2853, 2373, 2346, 2118, 1724, 1288, 1160, 1052. 1H NMR
(c 1.20, CHCl3). IR (KBr, cmꢁ1
)
nmax: 3431, 3283, 2935, 2871, 2374,
(400 MHz, CDCl3):
d
¼1.21, 1.23 (2ꢃs, 2ꢃ9H, 2ꢃ (CH3)3C); 3.39 (t,
1743, 1372, 1249, 1077, 1052. 1H NMR (400 MHz, CDCl3):
d
¼2.05,
1H, J¼9.0 Hz, H-2); 3.46 (t, 1H, J¼9.5 Hz, H-4); 3.65e3.70 (m, 1H,
H-5); 4.33 (dd, 1H, J¼5.6/12.2 Hz, H-6a); 4.41 (dd, 1H, J¼2.5/
12.1 Hz, H-6b); 4.65 (d, 1H, J¼8.6 Hz, H-1); 4.93 (t, 1H, J¼9.3 Hz, H-
2.08 (2ꢃs, 2ꢃ3H, 2ꢃ CH3CO); 2.48 (t, 1H, J¼2.3 Hz, C^CH); 2.78
(m, 2H, 2ꢃ OH); 3.42e3.46 (m, 1H, H-5); 3.76 (t, 1H, J¼9.5 Hz, H-
4); 3.84 (dd, 1H, J¼4.3/12.1 Hz, H-6a); 3.92 (dd, 1H, J¼3.2/12.1 Hz,
H-6b); 4.35 (m, 2H, OCH2); 4.76 (d, 1H, J¼8.0 Hz, H-1); 4.90 (dd,
1H, J¼8.0/9.6 Hz, H-2); 5.07 (t, 1H, J¼9.5 Hz, H-3). 13C NMR
3). 13C NMR (100 MHz, CDCl3):
d
¼27.0, 27.1 (CH3); 38.9, 39.0 (C);
62.9 (CH2); 69.1, 72.1, 76.2, 77.6, 90.2 (CH); 179.1, 180.2 (C]O).
HRMS (ESI-FT-ICR) m/z: 396.1749 [MþNa]þ (calculated for
C16H27N3O7Na: 396.1747).
(100 MHz, CDCl3):
d
¼20.7, 20.8 (CH3); 56.2, 61.8 (CH2); 69.0, 71.1
(CH); 75.4 (C); 75.6, 75.7, 98.5 (CH); 169.8, 171.5 (C]O). HRMS
(ESI-FT-ICR) m/z: 325.0897 [MþNa]þ (calculated for C13H18O8Na:
325.0899).
4.1.2. 2,4-Di-O-(2,3,4-tri-O-acetyl-
a-L-rhamnopyranosyl)-3,6-di-O-
pivaloyl- -glucopyranosyl azide (7). A suspension of 5 (661 mg,
b-D
ꢁ
1.77 mmol) and 4 A molecular sieves in CH2Cl2 (5 mL) was cooled to
ꢁ80 ꢀC and BF3$Et2O (0.99 mL, 7.79 mmol) was added under ni-
trogen atmosphere. Stirring was continued for 1 h at this temper-
ature, and then a solution of rhamnopyranosyl trichloroacetimidate
6 (2.39 g, 5.49 mmol) in CH2Cl2 (5 mL) was added. The reaction
mixture was stirred for additional 5 h at rt, then diluted with CH2Cl2
(30 mL) and filtered through a pad of Celite. The filtrate was washed
with satd aq NaHCO3 (15 mL) and brine (10 mL). The organic phase
was dried over anhydrous Na2SO4 and evaporated to dryness. The
crude product was purified by flash column chromatography
(hexane/EtOAc 2:1) to afford trisaccharide 7 (1.27 g, 78%) as a white
4.1.4. Propargyl 3,6-di-O-pivaloyl-b-D-glucopyranoside (10). To
a suspension of compound 8 (1.17 g, 3.03 mmol) in MeOH (15 mL)
was added NaOMe until pH 9e10. The mixture was stirred for 1 h,
then neutralized with acid resin Dowex-50 (Hþ), filtered and the
filtrate was concentrated under reduced pressure. The residue was
dissolved in anhydrous pyridine (20 mL) and the solution was
cooled to ꢁ15 ꢀC. Pivaloyl chloride (1.9 mL, 15.2 mmol) was added
dropwise under nitrogen atmosphere. The reaction was monitored
by TLC, and it was stirring at rt until the intermediate disappeared.
The reaction mixture was then diluted with EtOAc (60 mL) and
washed with a dilute HCl solution, satd aq NaHCO3, and brine. The
organic phase was dried over anhydrous Na2SO4 and evaporated to
dryness. The crude product was purified by flash column chro-
matography (hexane/EtOAc 4:1/3:1) to afford diol 10 (738 mg,
63%) as a white solid. Rf¼0.32 (hexane/EtOAc 2:1). Mp: 96e97 ꢀC.
foam. Rf¼0.25 (hexane/EtOAc 2:1). ½a D20
ꢁ61.5 (c 1.30, CHCl3). IR
ꢂ
(KBr, cmꢁ1
) nmax: 2962, 2926, 2854, 2371, 2346, 2116, 1757, 1246,
1222, 1144, 1043. 1H NMR (400 MHz, CDCl3):
d¼1.13 (d, 3H,
J¼6.4 Hz, CH3 Rha); 1.15 (s, 9H, (CH3)3C); 1.20 (d, 3H, J¼6.4 Hz, CH3
Rha0); 1.21 (s, 9H, (CH3)3C); 1.93, 1.95, 2.01, 2.03, 2.08, 2.09 (6ꢃs,
6ꢃ3H, 6ꢃ CH3CO); 3.50 (t, 1H, J¼7.9 Hz, H-2 Glc); 3.77 (t, 1H,
J¼7.9 Hz, H-4 Glc); 3.80e3.84 (m, 1H, H-5 Glc); 3.86e3.91 (m, 1H,
H-5 Rha); 4.10e4.17 (m, 1H, H-5 Rha0); 4.27 (dd, 1H, J¼4.5/12.2 Hz,
H-6a Glc); 4.49 (dd, 1H, J¼2.2/12.3 Hz, H-6b Glc); 4.64 (d, 1H,
½
a 2D0
ꢂ
ꢁ35.6 (c 1.35, CHCl3). IR (KBr, cmꢁ1
) nmax: 3448, 2973, 2932,
2878, 2373, 1720, 1288, 1159, 1082, 1045. 1H NMR (400 MHz,
CDCl3):
d
¼1.21, 1.24 (2ꢃs, 2ꢃ9H, 2ꢃ (CH3)3C); 2.48 (t, 1H, J¼2.4 Hz,
C^CH); 3.47 (t, 1H, J¼9.6 Hz, H-4); 3.51 (dd, 1H, J¼7.8/9.5 Hz, H-
2); 3.57e3.61 (m, 1H, H-5); 4.30 (dd, 1H, J¼6.0/12.0 Hz, H-6a);

ꢀ
K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7721
4.33e4.44 (m, 3H, OCH2, H-6b); 4.59 (d, 1H, J¼7.7 Hz, H-1); 4.92 (t,
(ESI-FT-ICR) m/z: 953.3623 [MþNa]þ (calculated for C43H62O22Na:
1H, J¼9.3 Hz, H-3). 13C NMR (100 MHz, CDCl3):
d¼27.5 (CH3); 39.3,
953.3630).
39.4 (C); 56.3, 63.6 (CH2); 70.1, 72.3, 74.9 (CH); 75.9 (C); 78.1, 78.7,
100.6 (CH); 179.1, 180.6 (C]O). HRMS (ESI-FT-ICR) m/z: 409.1849
[MþNa]þ (calculated for C19H30O8Na: 409.1838).
4.1.7. (25R)-3b-Propargyloxy-spirost-5-ene (14). NaH (60% in min-
eral oil, 362 mg, 9.05 mmol, washed with hexane) was added to
a stirred solution of diosgenin 13 (2.5 g, 6.03 mmol) in dry DMF
(100 mL) at 0 ꢀC. The suspension was stirred for 10 min under ni-
trogen atmosphere, treated with propargyl bromide (18.1 mL,
9.06 mmol) and then stirred at rt for additional 10 h. The reaction
mixture was poured into 100 mL of cold water and extracted with
CH2Cl2 (3ꢃ50 mL). The combined organic phases were washed with
aq 10% HCl (50 mL), brine (50 mL), and then dried over anhydrous
Na2SO4 and concentrated under reduced pressure to dryness. The
crude product was purified by flash column chromatography (n-
hexane/EtOAc 5:1) to yield compound 14 (2.02 g, 74%) as a pale
4.1.5. Propargyl 4,6-di-O-(2,3,4-tri-O-acetyl-
a-L-rhamnopyranosyl)-
2,3-di-O-acetyl- -glucopyranoside (11). A suspension of
b
-
D
9
ꢁ
(343 mg, 1.14 mmol) and 4 A molecular sieves in CH2Cl2 (3.5 mL)
was cooled to ꢁ80 ꢀC and BF3 $Et2O (0.64 mL, 5.02 mmol) was
added under nitrogen atmosphere. After stirring for 1 h at this
temperature, a solution of rhamnopyranosyl trichloroacetimidate 6
(1.53 g, 3.53 mmol) in CH2Cl2 (3.5 mL) was added and it was stirred
for 5 h at rt. The reaction mixture was diluted with CH2Cl2 (20 mL)
and filtered through a pad of Celite. The filtrate was washed with
satd aq NaHCO3 (15 mL) and brine (10 mL). The organic phase was
dried over anhydrous Na2SO4 and evaporated to dryness. The crude
product was purified by flash column chromatography (hexane/
EtOAc 1:1) to afford trisaccharide 11 (762 mg, 79%) as a white solid.
yellow solid. Rf¼0.34 (n-hexane/EtOAc 5:1). Mp: 215e217 ꢀC. ½a D20
ꢂ
ꢁ20.4 (c 1.40, CHCl3). IR (KBr, cmꢁ1
) nmax: 3567, 2925, 2854, 2371,
1560, 1118. 1H NMR (400 MHz, CDCl3):
d
¼0.78 (d, 3H, J¼6.9 Hz,
CH3); 0.79 (s, 3H, CH3); 0.97 (d, 3H, J¼6.9 Hz, CH3); 1.01 (s, 3H, CH3);
2.40 (t, 1H, J¼2.3 Hz, C^CH); 3.36 (t, 1H, J¼10.9 Hz, H-26ax); 3.39
Rf¼0.34 (hexane/EtOAc 1:1). Mp: 132e134 ꢀC. ½a D20
ꢁ71.5 (c 1.30,
ꢂ
CHCl3). IR (KBr, cmꢁ1
)
nmax: 2982, 2373, 1749, 1373, 1242, 1222,
(m, 1H, H-3
a
); 3.47 (dd, 1H, J¼4.1/11.0 Hz, H-26eq); 4.18 (m, 2H,
1052, 1043. 1H NMR (400 MHz, CDCl3):
d
¼1.13 (d, 3H, J¼6.2 Hz, CH3
OCH2); 4.40 (m, 1H, H-16
CDCl3):
a
); 5.35 (m, 1H, H-6). 13C NMR (100 MHz,
Rha); 1.18 (d, 3H, J¼6.2 Hz, CH3 Rha0); 1.94, 1.95, 2.01, 2.04, 2.05,
2.06, 2.08, 2.10 (8ꢃs, 8ꢃ3H, 8ꢃ CH3CO); 2.46 (t, 1H, J¼2.4 Hz,
C^CH); 3.55e3.59 (m, 1H, H-5 Glc); 3.76e3.89 (m, 3H, H-5 Rha, H-
4 Glc, H-6a Glc); 3.91e3.99 (m, 2H, H-5 Rha0, H-6b Glc); 4.33 (d, 2H,
J¼2.5 Hz, OCH2); 4.74 (d, 1H, J¼7.9 Hz, H-1 Glc); 4.80 (d, 1H,
J¼1.5 Hz, H-1 Rha); 4.81 (d, 1H, J¼1.9 Hz, H-1 Rha0); 4.88 (dd, 1H,
J¼7.9/9.3 Hz, H-2 Glc); 4.99e5.07 (m, 3H, H-4 Rha, H-4 Rha0, H-2
Rha); 5.12 (dd, 1H, J¼3.2/10.3 Hz, H-3 Rha0); 5.16e5.23 (m, 3H, H-2
d
¼14.6, 16.4, 17.2, 19.4 (CH3); 20.9, 28.1, 28.8 (CH2); 30.3,
31.4 (CH); 31.5, 31.9, 32.1, 37.0 (CH2); 37.1 (C); 38.7, 39.8 (CH2); 40.3
(C); 41.6, 50.1 (CH); 55.1 (CH2); 56.5, 62.0 (CH); 66.8 (CH2); 73.9 (C);
78.1, 80.8 (CH); 109.2 (C); 121.5 (CH); 140.4 (C). HRMS (ESI-FT-ICR)
m/z: 475.3153 [MþNa]þ (calculated for C30H44O3Na: 475.3188).
4.1.8. (25R)-3b-Azido-5a-spirostan-12-one (17). Methanesulfonic
acid (0.16 mL, 2.44 mmol) was added to a stirred solution of
hecogenin 15 (500 mg, 1.16 mmol), Ph3P (913 mg, 3.48 mmol), and
DMAP (298 mg, 2.44 mmol) in dry THF (3.5 mL) under nitrogen
atmosphere. DIAD, diisopropylazodicarboxylate (0.69 mL,
3.48 mmol) was added dropwise over a 15 min period. The reaction
mixture was stirred vigorously for 48 h at rt. The solvent was
evaporated under reduced pressure and the resulting residue was
partially purified by flash column chromatography (n-hexane/
Rha0, H-3 Rha, H-3 Glc). 13C NMR (100 MHz, CDCl3):
d¼17.2, 17.3,
20.6, 20.7, 20.8, 20.9, 21.0 (CH3); 55.6, 65.4 (CH2); 66.4, 67.8, 68.8,
69.2, 69.4, 69.6, 70.4, 70.9, 71.7, 73.6, 73.7 (CH); 75.4 (C); 76.9 (CH);
78.1 (C); 97.4, 98.0, 99.8 (CH); 169.6, 169.7, 169.8, 169.9, 170.1, 170.2,
170.3, 170.4 (C]O). HRMS (ESI-FT-ICR) m/z: 869.2684 [MþNa]þ
(calculated for C37H50O22Na: 869.2691).
4.1.6. Propargyl 2,4-di-O-(2,3,4-tri-O-acetyl-
a-
L
-rhamnopyranosyl)-
EtOAc 2:1), yielding impure 3a-methanesulfonate as a white solid
3,6-di-O-pivaloyl- -glucopyranoside (12). A suspension of 10
b
-
D
(610 mg), Rf¼0.68 (n-hexane/EtOAc 1:1). The crude methanesul-
fonate was dissolved in DMPU (10 mL) and NaN3 (189 mg,
2.90 mmol) was added. The resulting mixture was stirred vigor-
ously under nitrogen atmosphere at rt for 48 h and then diluted
with Et2O (100 mL). The organic phase was washed with aq 10% HCl
(2ꢃ30 mL) and brine (30 mL), dried over anhydrous Na2SO4 and
evaporated under reduced pressure to dryness. The crude product
was purified by flash column chromatography (n-hexane/EtOAc
8:1) to give the pure azide 17 (365 mg, 69% from 15) as a white
ꢁ
(620 mg, 1.60 mmol) and 4 A molecular sieves in CH2Cl2 (5 mL) was
cooled to ꢁ80 ꢀC and BF3 $Et2O (0.9 mL, 7.04 mmol) was added
under nitrogen atmosphere. After stirring for 1 h at this tempera-
ture, a solution of rhamnopyranosyl trichloroacetimidate 6 (2.16 g,
4.97 mmol) in CH2Cl2 (5 mL) was added and it was stirred for 5 h at
rt. The reaction mixture was diluted with CH2Cl2 (30 mL) and fil-
tered through a pad of Celite. The filtrate was washed with satd aq
NaHCO3 (15 mL) and brine (10 mL). The organic phase was dried
over anhydrous Na2SO4 and evaporated to dryness. The crude
product was purified by flash column chromatography (hexane/
EtOAc 2:1) to afford trisaccharide 12 (1.28 g, 86%) as a white solid.
solid. Rf¼0.47 (n-hexane/EtOAc 6:1). Mp: 231e232 ꢀC. ½a D20
ꢁ2.1 (c
ꢂ
1.40, CHCl3). IR (KBr, cmꢁ1
) nmax: 3567, 2925, 2854, 2371, 2346,
1702, 1654, 1560, 1118. 1H NMR (400 MHz, CDCl3):
J¼6.4 Hz, CH3); 0.89 (s, 3H, CH3); 1.04 (s, 3H, CH3); 1.06 (d, 3H,
J¼7.0 Hz, CH3); 2.20 (dd, 1H, J¼5.0/14.3 Hz, H-11 ); 2.38 (t, 1H,
J¼14.3 Hz, H-11 ); 3.22e3.29 (m, 1H, H-3 ); 3.33 (t, 1H, J¼10.9 Hz,
H-26ax); 3.45e3.49 (m, 1H, H-26eq); 4.30e4.36 (m, 1H, H-16
). 13
NMR (100 MHz, CDCl3):
¼11.8, 13.2, 16.0, 17.1 (CH3); 27.4, 28.1, 28.8
d
¼0.78 (d, 3H,
Rf¼0.34 (hexane/EtOAc 3:2). Mp: 127e128 ꢀC. ½a D20
ꢁ46.7 (c 1.35,
ꢂ
CHCl3). IR (KBr, cmꢁ1
)
nmax: 2980, 2938, 2870, 2371, 1748, 1369,
b
1244, 1225, 1145, 1080, 1044. 1H NMR (400 MHz, CDCl3):
d¼1.14 (d,
a
a
3H, J¼6.4 Hz, CH3 Rha); 1.15 (s, 9H, (CH3)3C); 1.17 (d, 3H, J¼6.4 Hz,
CH3 Rha0); 1.21 (s, 9H, (CH3)3C); 1.93, 1.95, 2.01, 2.02, 2.08, 2.10 (6ꢃs,
6ꢃ3H, 6ꢃ CH3CO); 2.48 (t, 1H, J¼2.4 Hz, C^CH); 3.57 (t, 1H,
J¼7.3 Hz, H-2 Glc); 3.76 (m, 2H, H-4 Glc, H-5 Glc); 3.89 (m, 1H, H-5
Rha); 4.20 (m, 1H, H-5 Rha0); 4.24 (dd, 1H, J¼4.6/11.9 Hz, H-6a Glc);
4.33 (d, 2H, J¼2.5 Hz, OCH2); 4.47 (dd, 1H, J¼1.6/12.0 Hz, H-6b Glc);
4.72 (d, 1H, J¼7.0 Hz, H-1 Glc); 4.79 (d, 1H, J¼1.6 Hz, H-1 Rha); 4.85
(d, 1H, J¼2.0 Hz, H-1 Rha0); 4.97e5.04 (m, 3H, H-2 Rha0, H-4 Rha, H-
4 Rha0); 5.15e5.28 (m, 4H, H-2 Rha, H-3 Rha, H-3 Rha0, H-3 Glc). 13C
a
C
d
(CH2); 30.2 (CH); 31.1, 31.4, 31.5, 33.8 (CH2); 34.3 (CH); 36.1 (C);
36.6, 37.6 (CH2); 42.2, 45.0, 53.5 (CH); 55.1 (C); 55.4, 55.7, 60.2 (CH);
66.9 (CH2); 79.1 (CH); 109.2 (C); 213.2 (C]O). HRMS (ESI-FT-ICR) m/
z: 478.3023 [MþNa]þ (calculated for C27H41N3O3Na: 478.3046).
4.1.9. (25R)-3b-Azido-5-hydroxy-5a-spirostan-6-one (18). Ketol 16
(2.0 g, 4.48 mmol), methanesulfonic acid (0.61 mL, 9.41 mmol),
Ph3P (3.51 g, 13.4 mmol), DMAP (1.15 g, 9.41 mmol), and diisopro-
pylazodicarboxylate (2.64 mL, 13.4 mmol) in dry THF (15 mL) were
reacted in a similar way as described in the synthesis of 17. Flash
column chromatography purification (hexane/EtOAc 1:1) afforded
NMR (100 MHz, CDCl3):
d
¼17.0, 17.1, 20.6, 20.7, 26.8, 27.1 (CH3);
38.7, 38.8 (C); 55.5, 62.4 (CH2); 66.8, 67.9, 68.7, 69.0, 69.6, 69.9, 70.5,
70.9, 72.3, 75.0, 75.6 (CH); 76.5 (C); 77.7, 78.0, 97.4, 98.1, 98.2 (CH);
169.5, 169.6, 169.7, 169.8, 169.9, 170.0, 176.4, 177.7 (C]O). HRMS

ꢀ
K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7722
the corresponding 3
a
-mesylate as a crude product (2.24 g). This
66.8 (CH2); 79.3, 80.5 (CH); 109.2 (C); 210.6 (C]O). HRMS (ESI-FT-
ICR) m/z: 494.2972 [MþNa]þ (calculated for: C27H41N3O4Na:
494.2995).
compound was subjected to nucleophilic substitution with NaN3
(350 mg, 5.38 mmol) in DMPU (20 mL) in a similar way as described
in the synthesis of 17. The crude product was purified by flash
column chromatography (hexane/EtOAc 5:1) to furnish azide 18
(1.4 g, 66% from 16) as a white solid. Rf¼0.36 (n-hexane/EtOAc 5:1).
4.1.12. Click conjugation procedure: triazole-based trisacchar-
ideespirostan conjugate 21. Alkyne 14 (31 mg, 0.067 mmol) and
azide 7 (51.3 mg, 0.056 mmol) were dissolved in THF (3 mL) and
Mp: 255e257 ꢀC. ½a D20
ꢂ
ꢁ41.7 (c 0.60, CHCl3). IR (KBr, cmꢁ1
) nmax:
2925, 2850, 2373, 2346, 2095, 1701, 1654, 1560, 1465. 1H NMR
treated with a 8 M aqueous solution of Cu(OAc)2$H2O (5.5
0.011 mmol) and a freshly prepared 8 M aqueous solution of so-
dium ascorbate (11 L, 0.022 mmol). The reaction mixture was
mL,
(400 MHz, CDCl3):
0.82 (s, 3H, CH3); 0.97 (d, 3H, J¼6.8 Hz, CH3); 2.16 (dd, 1H, J¼3.5/
13.0 Hz, H-7 ); 3.36 (t, 1H, J¼10.9 Hz,
); 2.73 (t, 1H, J¼13.0 Hz, H-7
H-26ax); 3.46 (dd, 1H, J¼4.3/10.9 Hz, H-26eq); 3.65 (m, 1H, H-3 );
4.41 (m, 1H, H-16
). 13C NMR (100 MHz, CDCl3):
¼14.0, 14.4, 16.4,
d
¼0.76 (s, 3H, CH3); 0.79 (d, 3H, J¼6.2 Hz, CH3);
m
b
a
stirred at rt until completion as indicated by TLC, and then con-
centrated to dryness. The crude product was purified by flash col-
umn chromatography (hexane/EtOAc 3:2) to give conjugate 21
(69 mg, 89%) as a white solid. Rf¼0.26 (hexane/EtOAc 3:2). Mp:
a
a
d
17.1 (CH3); 21.1, 26.3, 28.7, 29.6 (CH2); 30.7 (CH); 31.2, 31.4, 32.8
(CH2); 36.6 (CH); 39.5 (CH2); 41.0 (C); 41.5 (CH); 41.8 (CH2); 42.3,
44.4, 56.0, 56.3, 61.8 (CH); 66.8 (CH2); 79.8 (C); 80.4 (CH); 109.3 (C);
211.6 (C]O). HRMS (ESI-FT-ICR) m/z: 494.2986 [MþNa]þ (calcu-
lated for: C27H41N3O4Na: 494.2995).
179e180 ꢀC. ½a 2D0
ꢂ
ꢁ70.0 (c 1.50, CHCl3). IR (KBr, cmꢁ1
) nmax: 2927,
2854, 2371, 2346, 1750, 1246, 1222, 1143, 1052. 1H NMR (400 MHz,
CDCl3):
d
¼0.78 (m, 6H, 2ꢃ CH3); 0.84 (d, 3H, J¼6.2 Hz, CH3 Rha);
0.96 (d, 3H, J¼6.9 Hz, CH3); 1.01 (s, 3H, CH3); 1.18 (m, 21H, 2ꢃ
(CH3)3C, CH3 Rha0); 1.91, 1.97, 1.98, 2.04, 2.05, 2.12 (6ꢃs, 6ꢃ3H, 6ꢃ
4.1.10. (25R)-3
a-Azido-5
a-spirostan-12-one (19). Hecogenin 15
CH3CO); 2.66e2.73 (m, 1H, H-5 Rha0); 3.31 (m, 1H, H-3
a); 3.37 (t,
(5.0 g, 11.6 mmol) was dissolved in dry pyridine (25 mL) and TsCl
(6.6 g, 34.8 mmol) was added. The reaction mixture was stirred at rt
until TLC (hexane/EtOAc 3:1) analysis revealed that all the heco-
genin was consumed. The reaction mixture was slowly poured into
100 mL of sat aq NaHCO3 and the precipitate formed was separated
by filtration under reduced pressure and washed with water sev-
eral times. The resulting crude product was dried over anhydrous
CaCl2 and recrystallized from EtOH to give the corresponding
tosylate (6.3 g, 93%) as a white solid. This product was dissolved in
DMPU (80 mL) and then NaN3 (2.1 g, 32.3 mmol) was added. The
reaction mixture was stirred vigorously under nitrogen atmosphere
at 60 ꢀC for 48 h and then diluted with Et2O (500 mL). The organic
phase was washed with aq 10% HCl (2ꢃ100 mL) and brine (100 mL),
then dried over anhydrous Na2SO4 and evaporated under reduced
pressure to dryness. The crude product was purified by flash col-
umn chromatography (n-hexane/EtOAc 8:1) to give the azide 19
(4.4 g, 84% from 15) as a white solid. Rf¼0.58 (n-hexane/EtOAc 6:1).
1H, J¼10.9 Hz, H-26ax); 3.45e3.49 (m, 1H, H-26eq); 3.89e3.96 (m,
2H, H-4 Glc, H-5 Rha); 3.98e4.02 (m, 1H, H-5 Glc); 4.30 (dd, 1H,
J¼4.5/12.3 Hz, H-6a Glc); 4.35e4.45 (m, 3H, H-2 Glc, H-16
a, H-6b
Glc); 4.67 (s, 2H, OCH2); 4.78 (d, 1H, J¼1.8 Hz, H-1 Rha); 4.81 (t, 1H,
J¼10.0 Hz, H-4 Rha0); 4.92 (d, 1H, J¼1.9 Hz, H-1 Rha0); 4.99e5.08 (m,
3H, H-2 Rha, H-3 Rha0, H-4 Rha,); 5.13 (dd, 1H, J¼1.9/3.3 Hz, H-2
Rha); 5.18 (dd, 1H, J¼3.3/10.1 Hz, H-3 Rha); 5.34 (m, 1H, H-6 dio-
sgenin); 5.47 (t, 1H, J¼7.6 Hz, H-3 Glc); 5.75 (d, 1H, J¼8.6 Hz, H-1
Glc); 7.71 (s, 1H, H-triazole). 13C NMR (100 MHz, CDCl3):
d
¼14.5,
16.3, 17.1, 17.2, 17.5, 19.4, 20.5, 20.6, 20.7 (CH3); 20.8 (CH2); 26.8, 27.1
(CH3); 28.3, 28.8 (CH2); 30.3 (CH); 31.3 (CH2); 31.4 (CH); 31.8, 32.1
(CH2); 36.9 (C); 37.1 (CH2); 38.8, 38.9 (C); 39.0, 39.8 (CH2); 40.3 (C);
41.6, 50.1, 56.5 (CH); 61.7, 61.9 (CH2); 62.1, 68.3, 68.6, 68.7, 69.4, 69.9,
70.4, 75.2, 75.7, 77.4, 79.1, 80.8, 86.3, 97.4, 98.1 (CH); 109.3 (C); 121.3,
121.6 (CH); 140.6, 146.8 (C); 169.1, 169.3, 169.4, 169.8, 169.9, 170.0,
176.3, 177.6 (C]O). HRMS (ESI-FT-ICR) m/z: 1392.6824 [MþNa]þ
(calculated for C70H103N3O24Na: 1392.6829).
Mp: 222e223 ꢀC. ½a D20
ꢂ
ꢁ92.4 (c 1.10, CHCl3). IR (KBr, cmꢁ1
) nmax:
3567, 2925, 2854, 2371, 2346, 1718, 1654, 1560, 1118. 1H NMR
4.1.13. Deprotection procedure A: saponin analog 22. An aqueous
solution of NaOH (0.5 M, 0.75 mL) was added to a solution of
conjugate 21 (35 mg, 0.026 mmol) in the solvent mixture MeOH/
THF/H2O (1.6 mL, 1:1:1, v/v/v) and the reaction mixture was stirred
at 50 ꢀC overnight. The solution was neutralized with acid resin
Dowex-50 (Hþ) and then filtered to remove the resin. The filtrate
was concentrated under reduced pressure and the resulting crude
product was purified by flash column chromatography (EtOAc/
MeOH 5:1) to furnish 22 (33 mg, 93%) as a white amorphous solid.
(400 MHz, CDCl3):
1.03 (s, 3H, CH3); 1.06 (d, 3H, J¼7.0 Hz, CH3); 2.21 (dd, 1H, J¼5.0/
14.1 Hz, H-11 ); 3.34 (t, 1H, J¼11.1 Hz,
); 2.35 (t, 1H, J¼14.1 Hz, H-11
H-26ax); 3.48 (m, 1H, H-26eq); 3.89 (m, 1H, H-3 ); 4.31e4.37 (m,
1H, H-16
). 13C NMR (100 MHz, CDCl3):
¼11.2,13.2,16.0,17.1 (CH3);
d¼0.78 (d, 3H, J¼6.5 Hz, CH3); 0.87 (s, 3H, CH3);
b
a
b
a
d
25.4, 27.8, 28.7 (CH2); 30.2 (CH); 31.0, 31.4, 32.4 (CH2); 34.2 (CH);
36.4 (C); 37.3 (CH2); 39.8, 42.2, 53.5 (CH); 55.0 (C); 55.2, 55.7, 57.8
(CH); 55.1 (C); 55.2, 55.7, 57.8 (CH); 66.8 (CH2); 79.1 (CH); 109.2 (C);
213.4 (C]O). HRMS (ESI-FT-ICR) m/z: 478.3013 [MþNa]þ (calcu-
lated for C27H41N3O3Na: 478.3046).
Rf¼0.28 (EtOAc/MeOH 5:1). ½a D20
ꢁ82.1 (c 1.45, CHCl3). IR (KBr,
ꢂ
cmꢁ1 nmax: 3567, 2371, 2346, 1654, 1560. 1H NMR (500 MHz, pyr-
)
idine-d5):
d
¼0.71 (d, 3H, J¼5.8 Hz, CH3); 0.87 (s, 3H, CH3); 0.99 (s,
4.1.11. (25R)-3
a
-Azido-5-hydroxy-5
a-spirostan-6-one (20). Ketol 16
3H, CH3); 1.16 (d, 3H, J¼7.0 Hz, CH3); 1.55 (d, 3H, J¼6.1 Hz, CH3 Rha);
(2.0 g, 4.48 mmol) was subjected to tosylation (TsCl, 2.5 g,
13.4 mmol in dry pyridine 15 mL) and followed by nucleophilic
substitution with NaN3 (350 mg, 5.38 mmol) in DMPU (20 mL) in
a similar way as described in the synthesis of 19. The crude product
was purified by flash column chromatography (n-hexane/EtOAc
6:1) to give the azide 20 (1.5 g, 71% from 16) as a white solid.
1.64 (d, 3H, J¼6.2 Hz, CH3 Rha0); 2.40 (m, 1H); 2.63 (m, 1H);
3.09e3.15 (m, 1H, H-5 Rha); 3.47e3.55 (m, 2H, H-26ax, H-3a);
3.59e3.62 (m, 1H, H-26eq); 3.89e3.93 (m, 1H, H-5 Glc); 4.03e4.21
(m, 4H, H-6a Glc, H-6b Glc, H-4 Rha, H-4 Rha0); 4.29e4.37 (m, 2H,
H-3 Glc, H-3 Rha0); 4.50e4.60 (m, 3H, H-3 Rha, H-4 Glc, H-16
a);
4.68 (m, 1H, H-2 Rha); 4.77 (m, 1H, H-2 Rha0); 4.84 (t, 1H, J¼9.2 Hz,
H-2 Glc); 4.88 (m, 1H, H-5 Rha0); 4.95 (d, 1H, J¼11.9 Hz, OCH2); 5.39
(m, 1H, H-6 diosgenin); 5.86 (d, 1H, J¼1.3 Hz, H-1 Rha); 6.27 (d, 1H,
J¼1.3 Hz, H-1 Rha0); 6.31 (d, 1H, J¼9.3 Hz, H-1 Glc); 8.60 (s, 1H, H-
Rf¼0.42 (hexane/EtOAc 6:1). Mp: 243e244 ꢀC. ½a D20
ꢁ129.2 (c 1.20,
ꢂ
CHCl3). IR (KBr, cmꢁ1
) nmax: 3567, 2925, 2854, 2371, 2346, 2115,
1718, 1561, 1458, 1240. 1H NMR (400 MHz, CDCl3):
3ꢃ CH3); 0.96 (d, 3H, J¼6.7 Hz, CH3); 2.73 (t, 1H, J¼12.4 Hz, H-7
3.35 (t, 1H, J¼10.9 Hz, H-26ax); 3.45e3.48 (m, 1H, H-26eq); 3.69 (s,
1H, OH); 4.08 (m, 1H, H-3 ); 4.37e4.43 (m, 1H, H-16
). 13C NMR
(100 MHz, CDCl3):
d
¼0.76 (m, 9H,
a
);
triazole). 13C NMR (125 MHz, pyridine-d5):
d
¼15.4, 16.8, 17.7, 18.9,
19.4, 19.8 (CH3); 21.6, 29.2, 29.7 (CH2); 31.0, 32.1 (CH); 32.2, 32.6,
32.7 (CH2); 37.6 (C); 37.7, 39.8, 40.3 (CH2); 40.9 (C); 42.4, 50.7, 57.1
(CH); 61.2, 62.5 (CH2); 63.3 (CH); 67.3 (CH2); 70.5, 70.9, 72.6, 72.7,
72.8, 73.1, 74.1, 74.2, 77.9, 78.3, 79.5, 79.6, 80.6, 81.5, 88.1, 103.3,
103.5 (CH); 109.7 (C); 122.1, 122.9 (CH); 141.4, 147.2 (C). HRMS (ESI-
b
a
d
¼13.7, 14.4, 16.4, 17.1 (CH3); 20.8, 24.4, 25.5
(CH2); 28.7 (CH); 29.5, 30.2, 31.3, 31.5, 36.9 (CH2); 39.6 (C); 41.0
(CH); 41.5 (CH2); 41.6, 43.3, 44.6 (CH); 56.1 (C); 57.2 (CH); 62.0 (C);

ꢀ
K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7723
FT-ICR) m/z: 972.5042 [MþNa]þ (calculated for C48H75N3O16Na:
a white solid. Rf¼0.36 (hexane/EtOAc 1:2). Mp: 198e200 ꢀC. ½a D20
ꢂ
972.5045).
ꢁ84.4 (c 1.60, CHCl3). IR (KBr, cmꢁ1
) nmax: 2955, 2873, 2372, 1747,
1374, 1245, 1224, 1146, 1075, 1050. 1H NMR (400 MHz, CDCl3):
4.1.14. Triazole-based trisaccharideespirostan conjugate 23. Alkyne
12 (100 mg, 0.107 mmol) and azide 17 (58.3 mg, 0.128 mmol) in THF
(3 mL) were reacted in the presence of Cu(OAc)2$H2O (0.021 mmol)
and sodium ascorbate (0.043 mmol) according to the click conju-
gation procedure. Flash column chromatography purification (hex-
ane/EtOAc 1:1) afforded conjugate 23 (114 mg, 77%) as a white solid.
d
¼0.77 (s, 3H, CH3); 0.78 (d, 3H, J¼6.4 Hz, CH3); 0.91 (s, 3H, CH3);
0.97 (d, 3H, J¼6.7 Hz, CH3); 1.11 (d, 3H, J¼6.2 Hz, CH3 Rha); 1.16 (m,
12H, (CH3)3C, CH3 Rha0); 1.23 (s, 9H, (CH3)3C); 1.86, 1.99, 2.01, 2.08,
2.12, 2.13 (6ꢃs, 6ꢃ3H, 6ꢃ CH3CO); 2.33 (t, 1H, J¼13.3 Hz); 2.78 (t,
1H, J¼12.9 Hz); 3.36 (t,1H, J¼11.1 Hz, H-26ax); 3.45e3.49 (m,1H, H-
26eq); 3.66 (t, 1H, J¼7.3 Hz, H-2 Glc); 3.77 (t, 1H, J¼7.6 Hz, H-4 Glc);
3.81e3.85 (m, 1H, H-5 Glc); 3.88e3.92 (m, 1H, H-5 Rha); 4.00e4.04
(m, 1H, H-5 Rha0); 4.27 (dd, 1H, J¼5.2/11.9 Hz, H-6a Glc); 4.38e4.67
Rf¼0.38 (hexane/EtOAc 1:2). Mp: 187e188 ꢀC. ½a D20
ꢁ48.7 (c 1.50,
ꢂ
CHCl3). IR (KBr, cmꢁ1
) nmax: 2955, 2925, 2857, 2375, 2346,1748,1460,
1369, 1244, 1222, 1042. 1H NMR (400 MHz, CDCl3):
d¼0.78 (d, 3H,
(m, 2H, H-6b Glc, H-16
a
); 4.62 (d,1H, J¼7.7 Hz, H-1 Glc); 4.83 (d,1H,
J¼6.4 Hz, CH3); 1.01 (s, 3H, CH3); 1.06 (s, 3H, CH3); 1.07 (d, 3H,
J¼6.9 Hz, CH3); 1.15e1.17 (m, 15H, (CH3)3C, CH3 Rha, CH3 Rha0); 1.21
(s, 9H, (CH3)3C); 1.93, 1.97, 2.03, 2.05, 2.08, 2.11 (6ꢃs, 6ꢃ3H, 6ꢃ
J¼1.3 Hz, H-1 Rha); 4.86 (d, 1H, J¼1.9 Hz, H-1 Rha0); 4.90e5.06 (m,
6H, H-3 Rha, H-4 Rha, H-4 Rha0, OCH2, H-3
a); 5.08e5.10 (m, 2H, H-2
Rha, H-2 Rha0); 5.13 (dd, 1H, J¼3.1/10.2 Hz, H-3 Rha0); 5.20 (t, 1H,
CH3CO); 2.25 (dd,1H, J¼5.0/14.3 Hz, H-11 ); 2.44 (t,1H, J¼14.0 Hz, H-
b
J¼7.3 Hz, H-3 Glc); 7.76 (s, 1H, H-triazole). 13C NMR (100 MHz,
11
a
); 2.53 (dd, 1H, J¼6.7/8.7 Hz); 3.34 (t, 1H, J¼11.0 Hz, H-26ax);
CDCl3):
d¼13.9, 14.4, 16.4, 16.8, 17.1, 20.6, 20.7, 20.8 (CH3); 21.1
3.46e3.51 (m, 1H, H-26eq); 3.58 (t, 1H, J¼7.5 Hz, H-2 Glc); 3.77 (m,
(CH2); 26.9, 27.2 (CH3); 27.3, 28.7, 29.9 (CH2); 30.2 (CH); 31.3, 31.5,
33.6 (CH2); 36.5 (CH); 38.8, 38.9 (C); 39.5 (CH2); 41.0 (C); 41.6 (CH);
41.8 (CH2); 42.3 (C); 44.2, 56.0, 56.6, 62.1 (CH); 62.2, 62.7 (CH2);
66.8 (CH); 66.9 (CH2); 68.1, 68.8, 69.3, 69.4, 69.9, 70.3, 70.4, 73.8,
74.5, 75.7, 76.0 (CH); 79.1 (C); 80.5, 97.2, 98.9, 99.7 (CH); 109.3 (C);
121.5 (CH); 143.4 (C); 169.5, 169.8, 169.9, 170.1, 170.3, 171.8, 176.7,
178.1, 211.6 (C]O). HRMS (ESI-FT-ICR) m/z: 1424.6703 [MþNa]þ
(calculated for C70H103N3O26Na: 1424.6728).
2H, H-4 Glc, H-5 Glc); 3.87e3.94 (m,1H, H-5 Rha); 4.01e4.08 (m,1H,
H-5 Rha0); 4.27e4.38 (m, 2H, H-16
a, H-6a Glc); 4.46 (m, 1H, H-3a);
4.55 (m, 1H, H-6b Glc); 4.62 (d, 1H, J¼7.5 Hz, H-1 Glc); 4.79 (d, 1H,
J¼1.4 Hz, H-1 Rha); 4.86 (d, 2H, J¼1.9 Hz, H-1 Rha0); 4.92e5.07 (m,
5H, H-2 Rha, H-4 Rha, H-4 Rha0, OCH2); 5.13e5.19 (m, 3H, H-2 Rha, H-
3 Rha, H-3 Rha0); 5.26 (t, 1H, J¼8.0 Hz, H-3 Glc), 7.68 (s, 1H, H-tri-
azole). 13C NMR (100 MHz, CDCl3):
d¼11.9, 13.2, 15.9, 16.9, 17.1, 17.2,
20.6, 20.7, 20.8, 20.9, 26.8, 27.2 (CH3); 28.0, 29.7 (CH2); 30.0 (C); 30.2
(CH); 31.1, 31.4 (CH2); 34.3 (CH); 35.1 (CH2); 36.2 (C); 36.9, 37.1, 37.4,
37.6 (CH2); 38.8, 38.9 (C); 42.2, 45.5, 53.5 (CH); 55.1 (C); 55.3, 55.7
(CH); 62.3 (CH2); 66.7 (CH); 66.9 (CH2); 68.0, 68.8, 68.9, 69.6, 69.9,
70.5, 70.8, 72.5, 75.0, 76.6, 77.6, 79.1, 97.5, 98.0,100.3 (CH); 109.2 (C);
123.2 (CH); 145.1 (C); 169.5, 169.6, 169.8, 169.9, 170.1, 176.4, 177.8,
213.1 (C]O). HRMS (ESI-FT-ICR) m/z: 1408.6794 [MþNa]þ (calcu-
lated for C70H103N3O25Na: 1408.6778).
4.1.17. Saponin analog 26. Compound 25 (42 mg, 0.03 mmol) was
treated with NaOH (0.42 mmol) in H2O/MeOH/THF (1.8 mL,1:1:1, v/
v/v) according to the deprotection procedure A. Flash column
chromatography purification (CHCl3/MeOH 4:1/2:1) afforded
conjugate 26 (27 mg, 91%) as a white amorphous solid. Rf¼0.38
(CHCl3/MeOH 4:1). ½a D20
ꢂ
ꢁ96.0 (c 1.00, MeOH). IR (KBr, cmꢁ1
) nmax:
3368, 2928, 2878, 2373, 1560, 1130, 1052. 1H NMR (500 MHz, pyr-
idine-d5):
d
¼0.70 (d, 3H, J¼6.0 Hz, CH3); 0.84 (s, 3H, CH3); 0.95 (s,
4.1.15. Saponin analog 24. Compound 23 (30 mg, 0.022 mmol) was
treated with NaOH (0.308 mmol) in H2O/MeOH/THF(1.2 mL,1:1:1, v/
v/v) according to the deprotection procedure A. Flash column
chromatography purification (CHCl3/MeOH 4:1) afforded 24 (19 mg,
3H, CH3); 1.16 (d, 3H, J¼6.9 Hz, CH3); 1.52 (d, 3H, J¼6.3 Hz, CH3 Rha);
1.63 (d, 3H, J¼6.0 Hz, CH3 Rha0); 2.62 (m, 1H); 2.82 (t, 1H,
J¼13.0 Hz); 3.10 (t, 1H, J¼12.6 Hz); 3.49 (t, 1H, J¼10.7 Hz, H-26ax);
3.59 (dd, 1H, J¼3.8/10.7 Hz, H-26eq); 3.64 (m, 1H, H-5 Glc); 4.07
(dd, 1H, J¼3.2/12.0 Hz, H-6a Glc); 4.13e4.35 (m, 5H, H-3 Glc, H-6b
Glc, H-2 Glc, H-4 Rha, H-4 Rha0); 4.38 (t, 1H, J¼9.3 Hz, H-4 Glc); 4.53
89%) as a white amorphous solid. Rf¼0.20 (CHCl3/MeOH 4:1). ½a D20
ꢂ
ꢁ51.8 (c 0.85, CHCl3). IR (KBr, cmꢁ1
) nmax: 3587, 3566, 2938, 2373,
2345,1560.1H NMR (500 MHz, pyridine-d5):
d
¼0.72 (d, 3H, J¼6.0 Hz,
(m, 2H, H-3 Rha, H-3 Rha0); 4.55e4.60 (m, 1H, H-16
a); 4.67 (dd, 1H,
CH3); 0.86 (s, 3H, CH3); 1.12 (s, 3H, CH3); 1.39 (d, 3H, J¼6.9 Hz, CH3);
1.58 (d, 3H, J¼6.0 Hz, CH3 Rha); 1.64 (d, 3H, J¼6.3 Hz, CH3 Rha0); 2.24
(dd, 1H, J¼4.9/14.3 Hz); 2.40 (t, 1H, J¼13.9 Hz); 2.79 (dd, 1H, J¼6.7/
8.6 Hz); 3.51 (t, 1H, J¼10.7 Hz, H-26ax); 3.61 (dd, 1H, J¼3.7/10.7 Hz,
H-26eq); 3.66 (m,1H, H-5 Glc); 4.10 (m,1H, H-6a Glc); 4.16e4.36 (m,
5H, H-6b Glc, H-3 Glc, H-2 Glc, H-4 Rha, H-4 Rha0); 4.41 (t, 1H,
J¼1.6/3.2 Hz, H-2 Rha); 4.75 (dd, 1H, J¼1.6/3.5 Hz, H-2 Rha0); 4.78
(m, 1H, H-5 Rha); 4.88 (m, 1H, H-5 Rha0); 4.94 (d, 1H, J¼7.9 Hz, H-1
Glc); 5.04 (d, 1H, J¼12.0 Hz, OCH2a); 5.34 (d, 1H, J¼12.0 Hz, OCH2b);
5.48 (m, 1H, H-3
a
); 5.82 (d, 1H, J¼1.3 Hz, H-1 Rha); 6.34 (d, 1H,
J¼1.3 Hz, H-1 Rha0); 7.69 (s, 1H, H-triazole). 13C NMR (125 MHz,
pyridine-d5):
d
¼14.4, 15.3, 16.9, 17.7, 18.7, 18.8 (CH3); 21.9, 29.1, 29.6
J¼9.3 Hz, H-4 Glc); 4.49e4.60 (m, 4H, H-3 Rha, H-3 Rha0, H-3
a
, H-
(CH2); 30.3 (C); 30.8 (CH2); 30.9 (CH); 32.1, 32.2, 35.3 (CH2); 37.5
(CH); 40.2 (CH2); 41.6 (C); 42.3 (CH); 42.6 (CH2); 43.1 (C); 45.0, 56.7,
57.4 (CH); 61.5 (CH2); 63.3 (CH); 63.8, 67.3 (CH2); 69.7, 70.8, 72.6,
72.9, 73.0, 73.1, 74.2, 74.6, 77.4, 77.8, 78.1, 78.9 (CH); 79.7 (C); 81.3,
102.1, 102.2, 103.3 (CH); 109.6 (C); 123.5 (CH); 145.0 (C); 212.8 (C]
O). HRMS (ESI-FT-ICR) m/z: 1004.4923 [MþNa]þ (calculated for
C48H75N3O18Na: 1004.4943).
16a
); 4.69 (m,1H, H-2 Rha); 4.77 (m,1H, H-2 Rha0); 4.80 (m,1H, H-5
Rha); 4.98 (d, 1H, J¼8.2 Hz, H-1 Glc); 5.08 (d, 1H, J¼11.7 Hz, OCH2a);
5.39 (d, 1H, J¼11.7 Hz, OCH2b); 5.84 (d, 1H, J¼1.3 Hz, H-1 Rha); 6.38
(d, 1H, J¼1.4 Hz, H-1 Rha0); 8.45 (s, 1H, H-triazole). 13C NMR
(125 MHz, pyridine-d5):
d
¼12.1,14.3,16.4,17.7,18.9 (CH3); 28.6, 29.4,
29.6 (CH2); 30.9 (CH); 31.8, 31.9, 32.2 (CH2); 34.6 (CH); 35.9 (CH2);
36.6 (C); 37.2, 38.3 (CH2); 43.0, 45.7, 54.7, 55.6 (CH); 55.8 (C); 56.3,
60.6 (CH); 61.5, 64.0, 67.3 (CH2); 69.7, 70.8, 72.7, 72.9, 73.1, 73.2, 74.3,
74.6, 77.5, 77.8, 78.2, 79.0, 80.0, 102.2, 102.4, 103.3 (CH); 109.7 (C);
123.5 (CH); 144.9 (C); 213.0 (C]O). HRMS (ESI-FT-ICR) m/z:
988.4974 [MþNa]þ (calculated for C48H75N3O17Na: 988.4994).
4.1.18. Triazole-based trisaccharideespirostan conjugate 27. Alkyne
12 (100 mg, 0.107 mmol) and azide 19 (58.3 mg, 0.128 mmol) in THF
(3.2 mL) were reacted in the presence of Cu(OAc)2$H2O
(0.021 mmol) and sodium ascorbate (0.043 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 1:1) afforded 27 (111 mg, 75%) as a white
4.1.16. Triazole-based trisaccharideespirostan conjugate 25. Alkyne
12 (138 mg, 0.148 mmol) and azide 18 (83.6 mg, 0.177 mmol) in THF
(3.2 mL) were reacted in the presence of Cu(OAc)2$H2O (0.03 mmol)
and sodium ascorbate (0.059 mmol) according to the click conju-
gation procedure. Flash column chromatography purification
(hexane/EtOAc 1:1.3) afforded conjugate 25 (154 mg, 74%) as
solid. Rf¼0.22 (hexane/EtOAc 1:1). Mp: 180e182 ꢀC. ½a D20
ꢁ23.8 (c
ꢂ
1.30, CHCl3). IR (KBr, cmꢁ1
) nmax: 2932, 2852, 2373, 2346, 1749,
1242, 1225, 1148, 1052. 1H NMR (400 MHz, CDCl3):
d
¼0.94 (d, 3H,
J¼6.1 Hz, CH3); 0.97 (s, 3H, CH3); 1.03 (s, 3H, CH3); 1.04 (d, 3H,
J¼7.0 Hz, CH3); 1.15 (m,15H, (CH3)3C, CH3 Rha, CH3 Rha0); 1.21 (s, 9H,

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7724
(CH3)3C); 1.93, 1.97, 2.03, 2.05, 2.08, 2.11 (6ꢃs, 6ꢃ3H, 6ꢃ CH3CO);
);
(t, 1H, J¼9.8 Hz, H-4 Rha0); 5.01 (t, 1H, J¼9.9 Hz, H-4 Rha); 5.05 (dd,
1H, J¼2.1/3.1 Hz, H-2 Rha0); 5.10e5.13 (m, 2H, H-2 Rha, H-3 Rha);
5.15 (dd,1H, J¼3.1/10.0 Hz, H-3 Rha0); 5.25 (t,1H, J¼8.2 Hz, H-3 Glc);
2.18 (dd, 1H, J¼5.5/14.4 Hz, H-11
b
); 2.36 (t, 1H, J¼14.2 Hz, H-11
a
2.48 (dd, 1H, J¼6.7/8.8 Hz); 3.33 (t, 1H, J¼11.0 Hz, H-26ax);
3.45e3.49 (m, 1H, H-26eq); 3.58 (t, 1H, J¼7.6 Hz, H-2 Glc);
3.72e3.78 (m, 2H, H-4 Glc, H-5 Glc); 3.87e3.94 (m, 1H, H-5 Rha);
7.76 (s, 1H, H-triazole). 13C NMR (100 MHz, CDCl3):
d¼13.8, 14.4,
16.4, 16.9, 17.0, 17.1, 20.5, 20.6, 20.7, 20.8 (CH3); 20.9, 25.7, 26.4
(CH2); 26.8, 27.1 (CH3); 28.7, 29.4 (CH2); 30.2 (CH); 31.3, 31.5 (CH2);
37.1 (CH); 38.8, 38.9 (C); 39.6 (CH2); 41.1 (C); 41.6 (CH); 43.2 (C);
44.2, 54.6, 56.1, 61.9 (CH); 62.0, 62.8, 66.8 (CH2); 66.9, 68.0, 68.7,
68.9, 69.5, 69.9, 70.4, 70.8, 72.6, 75.0, 76.5 (CH); 76.9 (C); 77.3, 80.5,
97.5, 97.8, 100.4 (CH); 109.2 (C); 123.6 (CH); 144.2 (C); 169.5, 169.7,
169.8, 169.9, 170.0, 176.4, 177.9, 211.6 (C]O). HRMS (ESI-FT-ICR) m/
z: 1424.6715 [MþNa]þ (calculated for C70H103N3O26Na: 1424.6728).
4.03e4.11 (m, 1H, H-5 Rha0); 4.26e4.36 (m, 2H, H-16
a
, H-6a Glc);
4.54 (dd, 1H, J¼1.3/11.8 Hz, H-6b Glc); 4.63 (d, 1H, J¼7.4 Hz, H-1
Glc); 4.66 (m, 1H, H-3
b
); 4.79 (d, 1H, J¼1.6 Hz, H-1 Rha); 4.86 (d, 1H,
J¼1.9 Hz, H-1 Rha0); 4.89e5.07 (m, 5H, H-2 Rha, H-4 Rha, H-4 Rha0,
OCH2); 5.14e5.18 (m, 3H, H-2 Rha0, H-3 Rha, H-3 Rha0); 5.26 (t, 1H,
J¼8.0 Hz, H-3 Glc), 7.70 (s, 1H, H-triazole). 13C NMR (100 MHz,
CDCl3):
d¼11.5, 13.2, 15.9, 16.9, 17.1, 17.2, 20.6, 20.7, 20.8, 20.9 (CH3);
25.5 (CH2); 26.8, 27.2 (CH3); 27.8, 28.8 (CH2); 30.2 (CH); 31.0, 31.1,
31.4, 32.6, 32.8 (CH2); 34.2 (CH); 36.3 (C); 37.2 (CH2); 39.6, 42.2,
53.5, 55.0, 55.6, 56.2 (CH); 62.3, 62.6 (CH2); 66.8 (CH); 66.9 (CH2);
68.0, 68.8, 68.9, 69.6, 69.9, 70.5, 70.9, 72.6, 74.9, 79.1, 97.5, 98.0,
100.3 (CH); 109.2 (C); 122.8 (CH); 144.7 (C); 169.5, 169.6, 169.8,
169.9, 170.0, 176.4, 177.8, 212.9 (C]O). HRMS (ESI-FT-ICR) m/z:
1408.6747 [MþNa]þ (calculated for C70H103N3O25Na: 1408.6778).
4.1.21. Saponin analog 30. Compound 29 (48 mg, 0.035 mmol) was
treated with NaOH (0.49 mmol) in H2O/MeOH/THF (2.1 mL,1:1:1, v/
v/v) according to the deprotection procedure A. Flash column
chromatography purification (CHCl3/MeOH 4:1) afforded 29
(30 mg, 88%) as a white amorphous solid. Rf¼0.28 (CHCl3/MeOH
4:1). ½a 2D0
ꢂ
ꢁ86.9 (c 1.60, CHCl3). IR (KBr, cmꢁ1
)
nmax: 3567, 3033,
¼0.68 (d, 3H,
2374, 2346, 1560. 1H NMR (500 MHz, pyridine-d5):
d
4.1.19. Saponin analog 28. Compound 27 (80 mg, 0.058 mmol) was
treated with NaOH (0.81 mmol) in H2O/MeOH/THF (3.6 mL,1:1:1, v/
v/v) according to the deprotection procedure A. Flash column
chromatography purification (CHCl3/MeOH 4:1) afforded 28
(52 mg, 92%) as a white amorphous solid. Rf¼0.35 (CHCl3/MeOH
J¼6.0 Hz, CH3); 0.84 (s, 3H, CH3); 0.86 (s, 3H, CH3); 1.14 (d, 3H,
J¼6.9 Hz, CH3); 1.58 (d, 3H, J¼6.3 Hz, CH3 Rha); 1.62 (d, 3H, J¼6.3 Hz,
CH3 Rha0); 2.67 (d, 2H, J¼3.5 Hz); 3.19 (t, 1H, J¼12.6 Hz); 3.47 (t, 1H,
J¼10.5 Hz, H-26ax); 3.57 (dd, 1H, J¼3.3/10.7 Hz, H-26eq); 3.63 (m,
1H, H-5 Glc); 4.06e4.15 (m, 2H, H-3 Glc, H-6a Glc); 4.20e4.34 (m,
4H, H-2 Glc, H-6b Glc, H-4 Rha, H-4 Rha0); 4.38 (t, 1H, J¼9.3 Hz, H-4
3:1). ½a 2D0
ꢂ
ꢁ38.8 (c 0.85, CHCl3). IR (KBr, cmꢁ1
) nmax: 3487, 2928,
2878, 2373, 1560, 1130, 1052. 1H NMR (500 MHz, pyridine-d5):
Glc); 4.45e4.53 (m, 3H, H-3 Rha, H-3 Rha0, H-16
a); 4.66 (m, 1H, H-2
d
¼0.70 (d, 3H, J¼6.0 Hz, CH3); 0.81 (s, 3H, CH3); 1.09 (s, 3H, CH3);
Rha); 4.69e4.75 (m, 2H, H-2 Rha0, H-5 Rha); 4.84e4.90 (m, 1H, H-5
Rha0); 4.95 (d, 1H, J¼7.9 Hz, H-1 Glc); 5.01 (d, 1H, J¼12.0 Hz, OCH2);
1.36 (d, 3H, J¼6.9 Hz, CH3); 1.56 (d, 3H, J¼6.3 Hz, CH3 Rha); 1.63 (d,
3H, J¼6.3 Hz, CH3 Rha0); 2.37 (t, 1H, J¼13.6 Hz); 2.48 (m, 1H); 2.78
(dd, 1H, J¼6.7/8.6 Hz); 3.49 (t, 1H, J¼10.7 Hz, H-26ax); 3.60 (dd, 1H,
J¼3.8/10.7 Hz, H-26eq); 3.64 (m, 1H, H-5 Glc); 4.07 (m, 1H, H-6a
Glc); 4.14e4.35 (m, 5H, H-2 Glc, H-3 Glc, H-6b Glc, H-4 Rha, H-4
Rha0); 4.38 (t, 1H, J¼9.2 Hz, H-4 Glc); 4.46e4.54 (m, 3H, H-3 Rha, H-
5.06 (m, 1H, H-3
b
); 5.30 (d, 1H, J¼11.7 Hz, OCH2); 5.81 (d, 1H,
J¼1.1 Hz, H-1 Rha); 6.31 (d, 1H, J¼1.2 Hz, H-1 Rha0); 8.60 (s, 1H, H-
triazole). 13C NMR (125 MHz, pyridine-d5):
d¼14.2, 15.4, 17.0, 17.7,
18.9 (CH3); 21.8, 27.5, 29.6 (CH2); 31.0 (CH); 32.1, 32.2, 32.3 (CH2);
37.8 (CH); 40.4 (CH2); 41.7 (C); 42.4 (CH); 42.5 (CH2); 43.6 (C); 45.1,
54.9, 56.9 (CH); 61.5 (CH2); 63.2 (CH); 63.7, 67.3 (CH2); 69.8, 70.8,
72.8, 72.9, 73.0, 73.1, 74.3, 74.6, 77.5, 77.9 (CH); 78.1 (C); 78.2, 79.0,
81.3, 102.3, 103.3 (CH); 109.7 (C); 124.7 (CH); 144.5 (C); 213.1 (C]
O). HRMS (ESI-FT-ICR) m/z: 1004.4935 [MþNa]þ (calculated for
C48H75N3O18Na: 1004.4943).
3 Rha0, H-16
a); 4.67 (m,1H, H-2 Rha); 4.70 (m,1H, H-3b); 4.75e4.81
(m, 2H, H-2 Rha0, H-5 Rha); 4.85e4.91 (m, 1H, H-5 Rha0); 4.97 (d,
1H, J¼7.9 Hz, H-1 Glc); 5.09 (d, 1H, J¼12.0 Hz, OCH2a); 5.40 (d, 1H,
J¼12.0 Hz, OCH2b); 5.82 (d, 1H, J¼1.6 Hz, H-1 Rha); 6.35 (d, 1H,
J¼1.6 Hz, H-1 Rha0); 8.50 (s, 1H, H-triazole). 13C NMR (125 MHz,
pyridine-d5):
d
¼11.7, 14.3, 16.5, 17.7, 18.9 (CH3); 25.9, 28.3, 29.6
(CH2); 30.9 (CH); 31.7, 32.2, 33.1, 33.3 (CH2); 34.5 (CH); 36.8 (C); 37.9
(CH2); 40.3, 43.0, 54.6, 55.4 (CH); 55.7 (C); 56.1, 56.6 (CH); 61.5, 64.0,
67.3 (CH2); 69.7, 70.8, 72.7, 72.9, 73.1, 73.2, 74.3, 74.6, 77.5, 77.9, 78.2,
79.0, 80.0, 102.2, 102.4, 103.3 (CH); 109.7 (C); 124.4 (CH); 145.0 (C);
213.0 (C]O). HRMS (ESI-FT-ICR) m/z: 988.4992 [MþNa]þ (calcu-
lated for C48H75N3O17Na: 988.4994).
4.1.22. Triazole-based trisaccharideespirostan conjugate 31. Alkyne
11 (80 mg, 0.095 mmol) and azide 17 (51.9 mg, 0.114 mmol) in THF
(3.0 mL) were reacted in the presence of Cu(OAc)2$H2O
(0.019 mmol) and sodium ascorbate (0.038 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 2:3) afforded conjugate 31 (98 mg, 79%) as
a white solid. Rf¼0.19 (hexane/EtOAc 2:3). Mp: 193e195 ꢀC. ½a D20
ꢂ
4.1.20. Triazole-based trisaccharideespirostan conjugate 29. Alkyne
12 (100 mg, 0.107 mmol) and azide 20 (60.3 mg, 0.128 mmol) in THF
(3.0 mL) were reacted in the presence of Cu(OAc)2$H2O
(0.021 mmol) and sodium ascorbate (0.043 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 2:3) afforded conjugate 29 (121.4 mg, 81%)
ꢁ91.1 (c 1.35, CHCl3). IR (KBr, cmꢁ1
) nmax: 2929, 2864, 2375, 1750,
1372, 1238, 1222, 1044. 1H NMR (500 MHz, CDCl3):
d
¼0.79 (d, 3H,
J¼6.3 Hz, CH3); 1.01 (s, 3H, CH3); 1.06 (s, 3H, CH3); 1.07 (d, 3H,
J¼6.0 Hz, CH3); 1.15 (d, 3H, J¼6.3 Hz, CH3 Rha); 1.22 (d, 3H, J¼6.3 Hz,
CH3 Rha0); 1.96, 1.97, 1.99, 2.03, 2.05, 2.06, 2.10, 2.12 (8ꢃs, 8ꢃ3H, 8ꢃ
CH3CO); 2.25 (dd, 1H, J¼5.0/14.5 Hz); 2.44 (t, 1H, J¼13.7 Hz); 2.52
(dd, 1H, J¼6.8/8.7 Hz); 3.35 (t, 1H, J¼10.9 Hz, H-26ax); 3.49 (ddd,
1H, J¼2.2/8.8/11.0 Hz, H-26eq); 3.57e3.60 (m, 1H, H-5 Glc);
3.79e3.84 (m, 1H, H-5 Rha0); 3.85e3.89 (m, 2H, H-6a Glc, H-4 Glc);
as a white solid. Rf¼0.20 (hexane/EtOAc 1:1). Mp: 186e187 ꢀC. ½a D20
ꢂ
ꢁ50.0 (c 1.40, CHCl3). IR (KBr, cmꢁ1
) nmax: 2954, 2935, 2873, 2371,
1750, 1374, 1369, 1244, 1224, 1146, 1074, 1050. 1H NMR (400 MHz,
CDCl3):
d
¼0.75 (s, 3H, CH3); 0.77 (d, 3H, J¼6.4 Hz, CH3); 0.85 (s, 3H,
3.96e4.03 (m, 2H, H-6b Glc, H-5 Rha); 4.33e4.37 (m, 1H, H-16
a
);
CH3); 0.95 (d, 3H, J¼6.8 Hz, CH3); 1.01 (d, 3H, J¼6.2 Hz, CH3 Rha);
1.14 (m, 12H, (CH3)3C, CH3 Rha0); 1.20 (s, 9H, (CH3)3C); 1.92, 1.96,
2.02, 2.04, 2.07, 2.10 (6ꢃs, 6ꢃ3H, 6ꢃ CH3CO); 2.60 (dd, 1H, J¼6.9/
16.6 Hz); 2.87 (t, 1H, J¼12.5 Hz); 3.34 (t, 1H, J¼10.9 Hz, H-26ax);
3.44e3.47 (m, 1H, H-26eq); 3.56 (t, 1H, J¼7.7 Hz, H-2 Glc); 3.73 (m,
2H, H-4 Glc, H-5 Glc); 3.86e3.92 (m, 1H, H-5 Rha); 4.02e4.09 (m,
1H, H-5 Rha0); 4.25 (dd, 1H, J¼3.9/11.9 Hz, H-6a Glc); 4.37e4.42 (m,
4.40e4.46 (m, 1H, H-3
a
); 4.62 (d, 1H, J¼7.9 Hz, H-1 Glc); 4.76 (d, 1H,
J¼12.3 Hz, OCH2a); 4.83 (d, 1H, J¼1.9 Hz, H-1 Rha); 4.84 (d, 1H,
J¼1.6 Hz, H-1 Rha0); 4.89 (dd, 1H, J¼7.9/9.1 Hz, H-2 Glc); 4.93 (d, 1H,
J¼12.4 Hz, OCH2b); 5.02e5.08 (m, 3H, H-4 Rha, H-4 Rha0, H-2 Rha);
5.14 (dd, 1H, J¼3.2/10.1 Hz, H-3 Rha0); 5.17e5.21 (m, 2H, H-3 Glc, H-
2 Rha0); 5.24 (dd, 1H, J¼3.6/10.2 Hz, H-3 Rha); 7.52 (s, 1H, H-tri-
azole). 13C NMR (125 MHz, CDCl3):
d
¼12.0, 13.2, 16.0, 17.1, 17.2, 17.3,
1H, H-16
a
); 4.56e4.60 (m, 2H, H-1 Glc, H-6b Glc); 4.78 (d, 1H,
20.6, 20.7, 20.8, 20.9, 21.0, 21.1 (CH3); 28.0, 28.7, 28.8 (CH2); 30.2
(CH); 31.1, 31.3, 31.4 (CH2); 34.3 (CH); 35.2 (CH2); 36.2 (C); 36.8, 37.6
J¼1.2 Hz, H-1 Rha); 4.84e4.90 (m, 4H, H-1 Rha0, H-3
b, OCH2); 4.95

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7725
(CH2); 42.2, 45.4, 53.6 (CH); 55.1 (C); 55.3, 55.7, 60.1 (CH); 62.4, 65.8
(CH2); 66.5 (CH); 66.9 (CH2); 67.8, 68.8, 69.2, 69.5, 69.6, 70.4, 70.9,
72.0, 73.6, 74.0, 77.2, 79.1, 97.7, 99.4, 99.8 (CH); 109.2 (C); 120.6
(CH); 143.5 (C); 169.6, 169.7, 169.8, 169.9, 170.1, 170.2, 170.3, 213.0
(C]O). HRMS (ESI-FT-ICR) m/z: 1324.5825 [MþNa]þ (calculated for
C64H91N3O25Na: 1324.5839).
deprotection procedure B. Flash column chromatography purifica-
tion (EtOAc/MeOH 5:1) afforded 34 (66 mg, 95%) as a white
amorphous solid. Rf¼0.65 (EtOAc/MeOH 2:1). ½a D20
ꢁ80.0 (c 1.20,
ꢂ
CHCl3). IR (KBr, cmꢁ1 nmax: 3567, 2372, 2346, 1654, 1560. 1H NMR
)
(400 MHz, CD3OD):
d¼0.81 (d, 3H, J¼6.4 Hz, CH3); 0.83 (s, 3H, CH3);
0.95 (s, 3H, CH3); 0.98 (d, 3H, J¼6.9 Hz, CH3); 1.27 (d, 3H, J¼6.1 Hz,
CH3 Rha); 1.29 (d, 3H, J¼6.3 Hz, CH3 Rha0); 2.35 (t, 1H, J¼13.2 Hz);
2.84 (t, 1H, J¼12.6 Hz); 3.26 (dd, 1H, J¼7.8/8.9 Hz, H-2 Glc);
3.35e3.59 (m, 6H, H-26ax, H-26eq, H-4 Rha, H-4 Rha0, H-3 Glc, H-4
Glc); 3.63e3.75 (m, 5H, H-3 Rha, H-3 Rha0, H-5 Rha0, H-5 Glc, H-6a
Glc); 3.84 (dd, 1H, J¼1.8/3.3 Hz, H-2 Rha0); 3.86 (dd, 1H, J¼1.7/
3.3 Hz, H-2 Rha); 3.94e4.02 (m, 2H, H-5 Rha, H-6b Glc); 4.38e4.47
4.1.23. Deprotection procedure B: saponin analog 32. To a solution
of 31 (74 mg, 0.057 mmol) in THF/MeOH (4 mL, 1:1, v/v) was added
NaOMe up to pH 9e10. The reaction mixture was stirred at rt
overnight, then neutralized with acid resin Dowex-50 (Hþ) and
filtered under reduced pressure. The filtrate was concentrated to
dryness and the crude product purified by flash column chroma-
tography (EtOAc/MeOH 6:1) to afford 32 (50 mg, 91%) as a white
(m, 2H, H-1 Glc, H-16
a
); 4.75 (d, 1H, J¼1.3 Hz, H-1 Rha); 4.77 (d, 1H,
J¼12.4 Hz, OCH2); 4.82 (d, 1H, J¼1.6 Hz, H-1 Rha0); 4.84e4.94 (m,
amorphous solid. Rf¼0.70 (EtOAc/MeOH 3:1). ½a D20
ꢂ
ꢁ40.0 (c 1.50,
2H, H-3
CD3OD):
a
, OCH2); 8.11 (s, 1H, H-triazole). 13C NMR (100 MHz,
MeOH). IR (KBr, cmꢁ1
)
nmax: 3384, 2928, 2873, 2371, 1703, 1450,
d
¼14.4,14.9,16.9,17.5,17.9, 18.1 (CH3); 22.3, 29.3, 29.9, 31.2
1376, 1097, 1060. 1H NMR (400 MHz, CD3OD):
d
¼0.83 (d, 3H,
(CH2); 31.5 (CH); 32.5, 34.8 (CH2); 38.4 (CH); 40.9 (CH2); 42.3 (C);
42.9 (CH); 43.0 (CH2); 43.7 (C); 45.9, 57.5, 58.2 (CH); 63.2 (CH2);
63.8 (CH); 67.4, 67.9 (CH2); 69.9, 70.7, 72.3, 72.4, 72.6, 73.8, 74.1,
75.3, 75.7, 76.6, 79.6 (CH); 80.3 (C); 82.0, 102.0, 102.8, 103.5 (CH);
110.6 (C); 123.6 (CH); 145.5 (C); 213.9 (C]O). HRMS (ESI-FT-ICR) m/
z: 1004.4949 [MþNa]þ (calculated for C48H75N3O18Na: 1004.4943).
J¼6.4 Hz, CH3); 1.07 (d, 3H, J¼7.0 Hz, CH3); 1.10 (s, 3H, CH3); 1.13 (s,
3H, CH3); 1.29 (d, 6H, J¼6.1 Hz, CH3 Rha, CH3 Rha0); 2.25 (dd, 1H,
J¼4.7/14.3 Hz, H-11
J¼13.7 Hz, H-11
b
); 2.51 (dd, 1H, J¼6.7/8.8 Hz); 2.58 (t, 1H,
a
); 3.28 (dd, 1H, J¼7.9/8.8 Hz, H-2 Glc); 3.33e3.61
(m, 7H, H-26ax, H-26eq, H-4 Rha, H-4 Rha0, H-3 Glc, H-4 Glc, H-5
Glc); 3.66e3.79 (m, 4H, H-3 Rha, H-3 Rha0, H-5 Rha0, H-6a Glc); 3.86
(dd, 1H, J¼1.7/3.2 Hz, H-2 Rha0); 3.89 (dd, 1H, J¼1.6/3.2 Hz, H-2
Rha); 3.96e4.05 (m, 2H, H-5 Rha, H-6b Glc); 4.34e4.40 (m, 1H, H-
4.1.26. Triazole-based trisaccharideespirostan conjugate 35. Alkyne
11 (80 mg, 0.095 mmol) and azide 19 (51.9 mg, 0.114 mmol) in THF
(3.0 mL) were reacted in the presence of Cu(OAc)2 $H2O
(0.019 mmol) and sodium ascorbate (0.038 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 2:3) afforded conjugate 35 (88.9 mg, 72%) as
16
a
); 4.43 (d, 1H, J¼7.8 Hz, H-1 Glc); 4.57 (m, 1H, H-3
a); 4.77 (d, 1H,
J¼1.3 Hz, H-1 Rha); 4.78 (d, 1H, J¼12.3 Hz, OCH2a); 4.96 (d, 1H,
J¼12.4 Hz, OCH2b); 8.10 (s, 1H, H-triazole). 13C NMR (100 MHz,
CD3OD):
d
¼12.2, 13.7, 16.6, 17.5, 17.9, 18.1 (CH3); 29.3, 29.8, 29.9
(CH2); 31.4 (CH); 32.0, 32.5, 32.6 (CH2); 35.6 (CH); 36.3 (CH2); 37.3
(C); 37.9, 38.7 (CH2); 43.5, 46.6, 55.1 (CH); 56.4 (C); 56.8, 57.2, 61.7
(CH); 63.2, 67.3, 67.9 (CH2); 69.9, 70.7, 72.2 (CH); 72.3 (C); 72.4, 72.5,
73.8, 74.0, 75.2, 75.7, 76.6, 79.6, 80.6, 101.9, 102.8, 103.5 (CH); 110.5
(C); 123.4 (CH); 145.3 (C); 215.7 (C]O). HRMS (ESI-FT-ICR) m/z:
988.4986 [MþNa]þ (calculated for C48H75N3O17Na: 988.4994).
a white solid. Rf¼0.25 (hexane/EtOAc 2:3). Mp: 188 ꢀC. ½a D20
ꢁ65.0
ꢂ
(c 1.40, CHCl3). IR (KBr, cmꢁ1
) nmax: 2933, 2874, 2369, 1751, 1374,
1240, 1224, 1076, 1043. 1H NMR (400 MHz, CDCl3):
d¼0.77 (d, 3H,
J¼6.2 Hz, CH3); 0.97 (s, 3H, CH3); 1.03 (s, 3H, CH3); 1.06 (d, 3H,
J¼7.0 Hz, CH3); 1.14 (d, 3H, J¼6.2 Hz, CH3 Rha); 1.21 (d, 3H, J¼6.2 Hz,
CH3 Rha0); 1.95, 1.96, 1.97, 2.03, 2.04, 2.06, 2.09, 2.11 (8ꢃs, 8ꢃ3H, 8ꢃ
CH3CO); 2.46 (dd, 1H, J¼6.8/8.7 Hz); 3.32 (t, 1H, J¼11.0 Hz, H-26ax);
3.45e3.48 (m, 1H, H-26eq); 3.57e3.61 (m, 1H, H-5 Glc); 3.77e3.82
(m, 1H, H-5 Rha0); 3.84e3.90 (m, 2H, H-6 Glc, H-4 Glc); 3.94e4.03
4.1.24. Triazole-based trisaccharideespirostan conjugate 33. Alkyne
11 (80 mg, 0.095 mmol) and azide 18 (53.5 mg, 0.113 mmol) in THF
(3.0 mL) were reacted in the presence of Cu(OAc)2 $H2O
(0.019 mmol) and sodium ascorbate (0.038 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 2:3) afforded conjugate 33 (100 mg, 81%) as
(m, 2H, H-5 Rha, H-6b Glc); 4.29e4.34 (m, 1H, H-16
H-1 Glc, H-3
a); 4.64 (m, 2H,
b
); 4.76 (d, 1H, J¼12.5 Hz, OCH2); 4.83 (m, 2H, H-1 Rha,
H-1 Rha0); 4.88 (dd, 1H, J¼7.9/9.4 Hz, H-2 Glc); 4.94 (d, 1H,
J¼12.5 Hz, OCH2); 5.00e5.07 (m, 3H, H-4 Rha, H-4 Rha0, H-2 Rha);
5.13 (dd, 1H, J¼3.2/10.3 Hz, H-3 Rha0); 5.16e5.25 (m, 3H, H-2 Rha0,
H-3 Glc, H-3 Rha); 7.58 (s, 1H, H-triazole). 13C NMR (100 MHz,
a white solid. Rf¼0.33 (hexane/EtOAc 1:2). Mp: 201e203 ꢀC. ½a D20
ꢂ
ꢁ83.1 (c 1.30, CHCl3). IR (KBr, cmꢁ1
) nmax: 2951, 2874, 2370, 1751,
1374, 1367, 1242, 1224, 1075, 1051. 1H NMR (400 MHz, CDCl3):
CDCl3):
d
¼11.4, 13.2, 16.0, 17.1, 17.2, 17.3, 20.6, 20.7, 20.8, 20.9, 21.0
d
¼0.77 (s, 3H, CH3); 0.78 (d, 3H, J¼6.4 Hz, CH3); 0.91 (s, 3H, CH3);
(CH3); 25.4, 27.7, 28.8 (CH2); 30.2 (CH); 31.0, 31.1, 31.4, 32.5, 32.7
(CH2); 34.2 (CH); 36.3 (C); 37.2 (CH2); 39.7, 42.2, 53.5, 55.0, 55.5,
56.0 (CH); 62.4, 65.7 (CH2); 66.5 (CH); 66.8 (CH2); 67.8, 68.8, 69.2,
69.5, 69.6, 70.4, 70.9, 72.0, 73.6, 73.9, 77.1, 79.1, 97.6, 99.6, 99.8 (CH);
109.2 (C); 121.9 (CH); 143.5 (C); 169.5, 169.7, 169.8, 169.9, 170.1,
170.2, 170.3, 170.4, 212.9 (C]O). HRMS (ESI-FT-ICR) m/z: 1324.5819
[MþNa]þ (calculated for C64H91N3O25Na: 1324.5839).
0.97 (d, 3H, J¼6.7 Hz, CH3); 1.14 (d, 3H, J¼6.2 Hz, CH3 Rha); 1.22 (d,
3H, J¼6.1 Hz, CH3 Rha0); 1.95, 1.96, 2.02, 2.03, 2.04, 2.05, 2.10, 2.12
(8ꢃs, 8ꢃ3H, 8ꢃ CH3CO); 2.82 (t, 1H, J¼12.1 Hz); 3.36 (t, 1H,
J¼11.0 Hz, H-26ax); 3.45e3.49 (m, 1H, H-26eq); 3.65 (m, 1H, H-5
Glc); 3.77e3.88 (m, 3H, H-4 Glc, H-6a Glc, H-5 Rha0); 3.93e4.04 (m,
2H, H-5 Rha, H-6b Glc); 4.42 (m, 1H, H-16
a
); 4.62 (d, 1H, J¼7.6 Hz,
H-1 Glc); 4.81e4.96 (m, 6H, H-1 Rha, H-1 Rha0, H-2 Glc, H-3
a,
OCH2); 5.01e5.06 (m, 3H, H-2 Rha, H-4 Rha, H-4 Rha0); 5.14 (dd, 1H,
J¼3.2/10.3 Hz, H-3 Rha0); 5.16e5.21 (m, 3H, H-3 Rha, H-2 Rha0, H-3
4.1.27. Saponin analog 36. Compound 35 (55 mg, 0.042 mmol) was
treated with NaOMe in THF/MeOH (4 mL, 1:1, v/v) according to the
deprotection procedure B. Flash column chromatography purifica-
tion (EtOAc/MeOH 5:1) afforded 36 (37 mg, 92%) as a white
Glc); 7.67 (s, 1H, H-triazole). 13C NMR (100 MHz, CDCl3):
d
¼14.1,
14.4, 16.4, 17.1, 17.2, 17.4, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1 (CH3); 27.9,
28.8, 29.9 (CH2); 30.2 (CH); 31.3, 31.5, 34.1 (CH2); 36.7 (CH); 39.5
(CH2); 41.1 (C); 41.6 (CH); 41.9 (CH2); 42.5 (C); 44.4, 56.0, 62.0, 66.6
(CH); 66.9 (CH2); 67.9, 68.8, 69.3, 69.5, 69.7, 70.4, 70.8, 71.9, 73.6,
73.9, 77.4 (CH); 79.4 (C); 80.4, 97.9, 99.7, 99.8 (CH); 109.3 (C); 169.7,
169.9, 170.1, 170.2, 170.3, 170.4, 211.3 (C]O). HRMS (ESI-FT-ICR) m/
z: 1340.5802 [MþNa]þ (calculated for C64H91N3O26Na: 1340.5778).
amorphous solid. Rf¼0.67 (EtOAc/MeOH 3:1). ½a D20
ꢁ40.0 (c 1.15,
ꢂ
CHCl3). IR (KBr, cmꢁ1 nmax: 3567, 2371, 2346, 1654, 1560. 1H NMR
)
(500 MHz, pyridine):
d
¼0.69 (d, 3H, J¼5.8 Hz, CH3); 0.80 (s, 3H,
CH3); 1.09 (s, 3H, CH3); 1.36 (d, 3H, J¼6.9 Hz, CH3); 1.65 (d, 3H,
J¼6.1 Hz, CH3 Rha); 1.70 (d, 3H, J¼6.2 Hz, CH3 Rha0); 2.35 (t, 1H,
J¼13.8 Hz); 2.79 (dd, 1H, J¼6.7/8.6 Hz); 3.49 (t, 1H, J¼10.8 Hz, H-
26ax); 3.58e3.61 (m, 1H, H-26eq); 3.86e3.90 (m, 1H, H-4 Glc);
3.94e4.01 (m, 2H, H-2 Glc, OCH2); 4.08e4.18 (m, 2H, H-3 Glc, H-4
Rha0); 4.24 (t, 1H, J¼9.3 Hz, H-4 Rha); 4.31e4.39 (m, 3H, H-5 Glc,
4.1.25. Saponin analog 34. Compound 33 (93 mg, 0.071 mmol) was
treated with NaOMe in THF/MeOH (6 mL, 1:1, v/v) according to the

ꢀ
K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7726
OCH2, H-5 Rha); 4.47e4.56 (m, 4H, H-3 Rha, H-3 Rha0, H-16
a, H-3
b
);
73.8, 74.0, 75.3, 75.7, 76.6 (CH); 78.7 (C); 79.6, 81.9, 102.0, 102.6,
102.9 (CH); 110.6 (C); 125.0 (CH); 144.6 (C); 214.5 (C]O). HRMS
4.59 (dd, 1H, J¼1.6/3.2 Hz, H-2 Rha); 4.65 (dd, 1H, J¼1.4/3.1 Hz, H-2
Rha0); 4.85e4.93 (m, 1H, H-5 Rha0); 4.97 (d, 1H, J¼7.8 Hz, H-1 Glc);
5.17 (d, 1H, J¼12.2 Hz, H-6a Glc); 5.37 (d, 1H, J¼12.3 Hz, H-6b Glc);
5.40 (d, 1H, J¼1.2 Hz, H-1 Rha); 5.61 (d, 1H, J¼1.2 Hz, H-1 Rha0); 8.18
(ESI-FT-ICR)
m/z:
1004.4950
[MþNa]þ
(calculated
for
C48H75N3O18Na: 1004.4943).
(s, 1H, H-triazole). 13C NMR (125 MHz, pyridine):
d¼11.7, 14.3, 16.5,
Acknowledgements
17.7, 18.9, 19.1 (CH3); 25.9, 28.3, 29.6 (CH2); 30.9 (CH); 31.7, 32.2,
33.1, 33.2 (CH2); 34.6 (CH); 36.8 (C); 37.9 (CH2); 40.3, 43.0, 54.7, 55.4
(CH); 55.7 (C); 56.1, 56.5 (CH); 63.5, 67.3, 67.5 (CH2); 70.3, 71.0, 72.6,
72.9, 73.1, 74.2, 74.4, 75.4, 76.1, 76.9, 79.9, 80.1, 102.5, 103.3, 104.2
(CH); 109.7 (C); 123.7 (CH); 145.2 (C); 213.1 (C]O). HRMS (ESI-FT-
ICR) m/z: 988.4976 [MþNa]þ (calculated for C48H75N3O17Na:
988.4994).
This work was partially supported by CSIC (Proyecto Intramural
ꢀ
de Incorporacion-2007022), the Ministry of Science and Innovation
of Spain and the European Regional Development Fund (SAF2010-
21380 to F.E.). K.P.-L. gratefully acknowledges to MAEC-AECID for
a doctoral scholarship.
References and notes
4.1.28. Triazole-based trisaccharideespirostan conjugate 37. Alkyne
11 (80 mg, 0.095 mmol) and azide 20 (53.5 mg, 0.113 mmol) in THF
(3.0 mL) were reacted in the presence of Cu(OAc)2 $H2O
(0.019 mmol) and sodium ascorbate (0.038 mmol) according to the
click conjugation procedure. Flash column chromatography puri-
fication (hexane/EtOAc 1:2) afforded conjugate 37 (102 mg, 82%) as
1. Hostettmann, K.; Marston, A. Saponins; Cambridge University: Cambridge, UK,
1995.
2. (a) Sparg, S. G.; Light, M. E.; van Staden, J. J. Ethnopharmacol. 2004, 94, 219e243;
(b) Lacaille-Dubois, M. A.; Wagner, H. Stud. Nat. Prod. Chem. 2000, 21, 633e687;
(c) Attele, A. S.; Wu, J. A.; Yuan, C. S. Biochem. Pharmacol. 1999, 58, 1685e1693;
(d) Walt, J. M.; Breyer-Brandwijk, M. G. The Medicinal and Poisonous Plants of
Southern and Eastern Africa; E. and S. Livingstane Ltd.: London, 1962; 69.
3. (a) Yokosuka, A.; Jitsuno, M.; Yui, S.; Yamazaki, M.; Mimaki, Y. J. Nat. Prod. 2009,
72, 1399e1404; (b) Zhou, L.-B.; Chen, T.-H.; Bastow, K. F.; Shibano, M.; Lee, K.-
a white solid. Rf¼0.24 (hexane/EtOAc 1:2). Mp: 196e197 ꢀC. ½a D20
ꢂ
ꢁ98.5 (c 1.30, CHCl3). IR (KBr, cmꢁ1
) nmax: 2951, 2873, 2372, 1751,
1374, 1369, 1242, 1223, 1075, 1050. 1H NMR (400 MHz, CDCl3):
ꢀ
ꢀ
H.; Chen, D.-F. J. Nat. Prod. 2007, 70, 1263e1267; (c) Hernandez, J. C.; Leon, F.;
d
¼0.75 (s, 3H, CH3); 0.77 (d, 3H, J¼6.4 Hz, CH3); 0.85 (s, 3H, CH3);
ꢀ
Estevez, F.; Quintana, J.; Bermejo, J. Bioorg. Med. Chem. 2004, 12, 4423e4429;
(d) Watanabe, K.; Mimaki, Y.; Sakagami, H.; Sashida, Y. J. Nat. Prod. 2003, 66,
0.95 (d, 3H, J¼6.7 Hz, CH3); 1.14 (d, 3H, J¼6.2 Hz, CH3 Rha); 1.21 (d,
3H, J¼6.1 Hz, CH3 Rha0); 1.96 (s, 6H, 2ꢃ CH3CO); 1.99, 2.03, 2.05,
2.06, 2.09, 2.11 (6ꢃs, 6ꢃ3H, 6ꢃ CH3CO); 2.60 (dd, 1H, J¼6.5/
16.3 Hz); 2.86 (t, 1H, J¼12.4 Hz); 3.35 (t, 1H, J¼11.0 Hz, H-26ax);
3.44e3.48 (m, 1H, H-26eq); 3.58 (m, 1H, H-5 Glc); 3.77e3.88 (m,
3H, H-4 Glc, H-6a Glc, H-5 Rha); 3.96e4.02 (m, 2H, H-5 Rha0, H-6b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
236e241; (e) Gonzalez, A. G.; Hernandez, J. C.; Leon, F.; Padron, J. I.; Estevez, F.;
Quintana, J.; Bermejo, J. J. Nat. Prod. 2003, 66, 793e798; (f) Hu, K.; Dong, A. J.;
Yao, X. S.; Kobayashi, H.; Iwasaki, S. Planta Med. 1996, 62, 573e575; (g) Naka-
mura, T.; Komori, C.; Lee, Y.-Y.; Hashimoto, F.; Yahara, S.; Nohara, T.; Ejima, A.
Biol. Pharm. Bull. 1996, 19, 564e566; (h) Chiang, H. C.; Tseng, T. H.; Wang, C. J.;
Chen, C. F.; Kan, W. S. Anticancer Res. 1991, 11, 1911e1917.
4. (a) Takechi, M.; Shimada, S.; Tanaka, Y. Phytochemistry 1991, 30, 3943e3944; (b)
Hufford, C. D.; Liu, S.; Clark, A. M. J. Nat. Prod. 1988, 51, 94e98.
Glc); 4.40 (m, 1H, H-16
a
); 4.63 (d, 1H, J¼7.6 Hz, H-1 Glc); 4.74e4.96
5. (a) Kim, S. Y.; Son, K. H.; Chang, H. W.; Kang, S. S.; Kim, H. P. Arch. Pharmcol. Res.
1999, 22, 313e316; (b) Mimaki, Y.; Nakamura, O.; Sashida, Y.; Nikaido, T.; Oh-
moto, T. Phytochemistry 1995, 38, 1279e1286; (c) Baek, S. H.; Kim, S. H.; Son, K.
H.; Chung, K. C.; Chang, H. W. Arch. Pharm. Res. 1994, 17, 218e222.
6. (a) Song, G.; Yang, S.; Zhang, W.; Cao, Y.; Wang, P.; Ding, N.; Zhang, Z.; Guo, Y.;
Li, Y. J. Med. Chem. 2009, 52, 7368e7371; (b) Ikeda, T.; Ando, J.; Miyazono, A.;
Zhu, X.-H.; Tsumagari, H.; Nohara, T.; Yokomizo, K.; Uyeda, M. Biol. Pharm. Bull.
2000, 23, 363e364.
7. (a) Cheung, JY.-N.; Ong, RC.-Y.; Suen, Y.-K.; Ooi, V.; Wong, HN.-C.; Mak, TC.-W.;
Fung, K.-P.; Yu, B.; Kong, S.-K. Cancer Lett. 2005, 217, 203e211; (b) Liu, M. J.;
Wang, Z.; Ju, Y.; Zhou, J. B.; Wang, Y.; Wong, R. N.-S. Biol. Pharm. Bull. 2004, 27,
1059e1065; (c) Wang, Z.; Zhou, J.; Ju, Y.; Zhang, H.; Liu, M.; Li, X. Biol. Pharm.
Bull. 2001, 24, 159e162.
(m, 6H, H-1 Rha, H-1 Rha0, H-2 Glc, OCH2, H-3
b
); 4.87 (m, 1H, H-2
Glc); 5.01e5.08 (m, 3H, H-2 Rha, H-4 Rha, H-4 Rha0); 5.13 (dd, 1H,
J¼3.2/10.3 Hz, H-3 Rha0); 5.17e5.21 (m, 2H, H-3 Glc, H-2 Rha0); 5.24
(dd, 1H, J¼3.5/10.2 Hz, H-3 Rha); 7.64 (s, 1H, H-triazole). 13C NMR
(100 MHz, CDCl3):
d
¼13.8, 14.4, 16.4, 17.1, 17.2, 17.3, 20.6, 20.7, 20.8,
20.9, 21.0 (CH3); 25.7, 28.8, 29.5 (CH2); 30.2 (CH); 31.3, 31.5 (CH2);
37.0 (CH); 39.6 (CH2); 41.1 (C); 41.5 (CH2); 41.6 (CH); 43.2 (C); 44.2,
54.8, 56.1, 62.0 (CH); 62.2, 65.8 (CH2); 66.6 (CH); 66.8 (CH2); 67.8,
68.8, 69.2, 69.5, 69.6, 70.4, 70.9, 72.1, 73.6, 73.9 (CH); 76.9 (C); 77.1,
80.5, 97.7, 99.4, 99.8 (CH); 109.2 (C); 122.1 (CH); 144.2 (C); 169.7,
169.9, 170.0, 170.1, 170.2, 170.3, 211.5 (C]O). HRMS (ESI-FT-ICR) m/
z: 1340.5773 [MþNa]þ (calculated for C64H91N3O26Na: 1340.5778).
8. (a) Wang, Y.; Zhang, Y.; Shu, Z.; Zhu, S.; Li, Y.; Li, M.; Yu, B. Bioorg. Med. Chem.
2007, 15, 2528e2532; (b) Mimaki, Y.; Yokosuka, A.; Kuroda, M.; Sashida, Y. Biol.
Pharm. Bull. 2001, 24, 1286e1289.
9. Wang, Y.; Zhang, Y.; Yu, B. ChemMedChem 2007, 2, 288e291.
10. For recent examples, see: (a) Hernandez, J. C.; Leon, F.; Brouard, I.; Torres, F.;
ꢀ
ꢀ
4.1.29. Saponin analog 38. Compound 37 (80 mg, 0.061 mmol) was
treated with NaOMe in THF/MeOH (5 mL, 1:1, v/v) according to the
deprotection procedure B. Flash column chromatography purifica-
tion (EtOAc/MeOH 4:1) afforded 38 (57 mg, 96%) as a white
ꢀ
Rubio, S.; Quintana, J.; Estevez, F.; Bermejo, J. Bioorg. Med. Chem. 2008, 16,
2063e2076; (b) Kaskiw, M. J.; Tassotto, M. L.; Th’ng, J.; Jiang, Z.-H. Bioorg. Med.
Chem. 2008, 16, 3209e3217; (c) Miyashita, H.; Kai, Y.; Nohara, T.; Ikeda, T.
Carbohydr. Res. 2008, 343, 1309e1315; (d) Miyashita, H.; Ikeda, T.; Nohara, T.
Carbohydr. Res. 2007, 342, 2182e2191; (e) Li, W.; Qiu, Z.; Wang, Y.; Zhang, Y.; Li,
M.; Yu, J.; Zhang, L.; Zhu, Z.; Yu, B. Carbohydr. Res. 2007, 342, 2705e2715; (f)
Zhu, S.; Zhang, Y.; Li, M.; Yu, J.; Zhang, L.; Li, Y.; Yu, B. Bioorg. Med. Chem. Lett.
2006, 16, 5629e5632; (g) Li, M.; Yu, B. Org. Lett. 2006, 8, 2679e2682; (h) Li, Y.;
Zhang, Y.; Guo, T.; Guan, H.; Hao, Y.; Yu, B. Synthesis 2006, 775e782; (i) Zhang,
Y.; Li, Y.; Guo, T.; Guan, H.; Shi, J.; Yu, Q.; Yu, B. Carbohydr. Res. 2005, 340,
1453e1459; (j) Yu, B.; Hui, Y. Chemical synthesis of bioactive steroidal saponins
In Glycochemistry: Principles, Synthesis, and Application; Wang, P. G., Bertozzi, C.
R., Eds.; Marcel Dekker: New York, NY, 2001; p 163.
amorphous solid. Rf¼0.68 (hexane/EtOAc 2:1). ½a D20
ꢁ96.3 (c 1.35,
ꢂ
MeOH). IR (KBr, cmꢁ1
) nmax: 3366, 2928, 2874, 2374, 1708, 1653,
1449, 1380, 1239, 1174,1060. 1H NMR (400 MHz, CD3OD):
d
¼0.80 (d,
3H, J¼6.6 Hz, CH3); 0.81 (s, 3H, CH3); 0.90 (s, 3H, CH3); 0.98 (d, 3H,
J¼6.9 Hz, CH3); 1.27 (d, 3H, J¼6.1 Hz, CH3 Rha); 1.29 (d, 3H, J¼6.2 Hz,
CH3 Rha0); 2.51 (dd, 1H, J¼5.7/12.1 Hz, H-7
); 2.77 (t, 1H, J¼11.8 Hz,
b
H-7
a
); 3.26 (dd, 1H, J¼7.9/9.1 Hz, H-2 Glc); 3.36e3.59 (m, 7H, H-
11. (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057e3064;
(b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
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26ax, H-26eq, H-4 Rha, H-4 Rha0, H-3 Glc, H-4 Glc, H-5 Glc);
3.63e3.78 (m, 4H, H-3 Rha, H-3 Rha0, H-5 Rha, H-6a Glc); 3.84 (dd,
1H, J¼1.6/3.2 Hz, H-2 Rha0); 3.88 (dd, 1H, J¼1.7/3.4 Hz, H-2 Rha);
3.97e4.02 (m, 2H, H-6b Glc, H-5 Rha0); 4.33 (d, 1H, J¼7.7 Hz, H-1
Glc); 4.43 (m, 1H, H-16
a
); 4.76 (d, 1H, J¼1.5 Hz, H-1 Rha); 4.81 (d,
1H, J¼12.6 Hz, OCH2a); 4.82 (d, 1H, J¼1.3 Hz, H-1 Rha0); 4.91e4.95
(m, 2H, H-3
CD3OD):
b
, OCH2b); 8.13 (s, 1H, H-triazole). 13C NMR (100 MHz,
d
¼14.1, 14.8, 16.9, 17.5, 17.9, 18.1 (CH3); 22.1, 24.7, 27.9, 29.9
(CH2); 31.5 (CH); 32.3, 32.4, 32.5 (CH2); 38.6 (CH); 40.9 (CH2); 42.3
(C); 42.7 (CH2); 42.9 (CH); 44.1 (C); 45.9, 55.7, 57.6 (CH); 62.7 (CH2);
63.6 (CH); 67.3, 67.9 (CH2); 69.9, 70.7, 72.2 (CH); 72.3 (C); 72.4, 72.5,

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K. Perez-Labrada et al. / Tetrahedron 67 (2011) 7713e7727
7727
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20. In order to perform a reliable comparison between molecules with the
greatest structural similarity, only the
combination.
b isomers were selected from each
18. For details of the cytotoxicity assays against HL-60 performed for all saponin
analogs, see Refs. 3c,e, and 10a.
19. Consider, for example, that dioscin (1), filiasparoside A (2), and Xiebai saponin I
(not shown) have different trisaccharide and spirostan moieties and are all
cytotoxic. For reports on Xiebai saponin I, see: (a) Kuroda, M.; Mimaki, Y.;
21. Deppmeier, B. J.; Driessen, A. J.; Hehre, W. J.; Johnson, J. A.; Kluzinger, P. E.;
Watanabe, M.; Yu, J. PC Spartan Pro; Wavefunction: Irvine CA, USA, 2000.
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quez, H.; Acosta, A. J. Am. Chem. Soc. 1998, 120, 12023e12033; (b) Montero, L. A.;
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