Carbohydrate-steroid conjugation by Ugi reaction: One-pot synthesis of triple sugar/pseudo-peptide/spirostane hybrids

Article abstract of DOI:10.1016/j.carres.2012.05.003

The one-pot synthesis of novel molecular chimeras incorporating sugar, pseudo-peptide, and steroidal moieties is described. For this, a new carbohydrate-steroid conjugation approach based on the Ugi four-component reaction was implemented for the ligation of glucose and chacotriose to spirostanic steroids. The approach proved wide substrate scope, as both mono and oligosaccharides functionalized with amino, carboxy, and isocyano groups were conjugated to steroidal substrates in an efficient, multicomponent manner. Two alternative strategies based on the hydrazoic acid variant of the Ugi reaction were employed for the synthesis of tetrazole-based chacotriose-diosgenin conjugates resembling naturally occurring spirostan saponins. This is the first time that triple sugar/pseudo-peptide/steroid hybrids are produced, thus opening up an avenue of opportunities for applications in drug discovery and biological chemistry.

Full text of DOI:10.1016/j.carres.2012.05.003

Carbohydrate Research 359 (2012) 102–110
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Carbohydrate–steroid conjugation by Ugi reaction: one-pot synthesis
of triple sugar/pseudo-peptide/spirostane hybrids
Daniel G. Rivera a,b, , Karell Pérez-Labrada a,c, Liudmila Lambert a, Simon Dörner b,
⇑
Bernhard Westermann b, Ludger A. Wessjohann b
a Center for Natural Products Study, Faculty of Chemistry, University of Havana, Zapata y G, 10400 La Habana, Cuba
b Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle/Saale, Germany
c Institute of Pharmacy and Food, University of Havana, San Lázaro y L, 10400 La Habana, Cuba
a r t i c l e i n f o
a b s t r a c t
Article history:
The one-pot synthesis of novel molecular chimeras incorporating sugar, pseudo-peptide, and steroidal
moieties is described. For this, a new carbohydrate–steroid conjugation approach based on the Ugi
four-component reaction was implemented for the ligation of glucose and chacotriose to spirostanic ste-
roids. The approach proved wide substrate scope, as both mono and oligosaccharides functionalized with
amino, carboxy, and isocyano groups were conjugated to steroidal substrates in an efficient, multicom-
ponent manner. Two alternative strategies based on the hydrazoic acid variant of the Ugi reaction were
employed for the synthesis of tetrazole-based chacotriose–diosgenin conjugates resembling naturally
occurring spirostan saponins. This is the first time that triple sugar/pseudo-peptide/steroid hybrids are
produced, thus opening up an avenue of opportunities for applications in drug discovery and biological
chemistry.
Received 30 March 2012
Received in revised form 3 May 2012
Accepted 3 May 2012
Available online 11 May 2012
Keywords:
Carbohydrates
Steroids
Multicomponent reactions
Conjugation
Hybrids
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the CuI-catalyzed azide–alkyne 1,3-dipolar cycloaddition, thus pro-
ducing amphipathic hybrids with anticancer12c and antimicrobial
The conjugation of steroids to other biomolecules is a common
strategy employed both by nature and chemists to modulate the
biological and chemical behavior of these molecules.1 An impor-
tant family of naturally occurring steroidal conjugates are the
saponins, which possess an oligosaccharide moiety attached to
the steroidal skeleton.2 These steroidal glycosides have found
significant applications in traditional and modern medicine2,3
owing to their cytotoxic,4–6 antifungal,7 antiinflammatory,8 and
antiviral activities.9
The interest on conjugating lipophilic scaffolds like steroids to
oligosaccharides also derives from the recognized capability of
the resulting amphipathic hybrids to interact with phospholipid
membranes and liposomes.10 Very recently, facially amphiphilic
steroid-disaccharide hybrids have proven success on the solubiliza-
tion and stabilization of membrane proteins.11 This has opened up
new perspectives for the extensive manipulation and characteriza-
tion of membrane proteins, as many of the hybrid amphiphiles are
synthetically available in multigram scale. Alternatively, the grow-
ing development of ‘click chemistry’ has had also an impact on the
development of novel sugar/steroids hybrid architectures.12 Differ-
ent types of lipophilic steroids have been ligated to saccharides by
activities.12f Considering the growing importance of sugar/steroid
hybrids in drug discovery and biological chemistry, we were
prompted to pursuit a novel carbohydrate–steroid conjugation
approach alternative to the traditional glycosylation and ‘click’
processes and capable to produce unique types of molecular
chimeras.
Here we report on the use of the Ugi four-component reaction
(Ugi-4CR),13 and its hydrazoic acid variant,14 for the conjugation
of sugars to spirostanic steroids. The Ugi-4CR is one of the most
versatile and explored isocyanide-based multicomponent reac-
tions (MCRs), owing to the great level of molecular diversity and
complexity that generates with low synthetic cost.13,15 This MCR
has been utilized for the assembly of steroidal macrocycles,16 for
the conjugation of carbohydrates to amino acids17 and proteins18
and for the construction of glycoconjugate libraries.19 However,
its utilization in the synthesis of any type of carbohydrate/steroid
hybrid has remained completely unexplored. Accordingly, we de-
scribe the first multicomponent approach for the direct conjuga-
tion of sugars to steroids, thus producing new types of triple
hybrids including a pseudo-peptidic (i.e., peptoid or tetrazole) moi-
ety in-between the sugar and steroidal skeletons.
The simplest version of the Ugi-4CR comprises the condensa-
tion of a primary amine, and aldehyde or ketone, a carboxylic acid
and an isocyanide to afford an N-substituted dipeptide.13 An
⇑
Corresponding author. Tel.: +53 7 8792331.
E-mail address: dgr@fq.uh.cu (D.G. Rivera).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.05.003

D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
103
related and include the formation of an
a
-adduct, which in the
sugar or
steroid
NH2
+
Dipeptide linkage
O
classic Ugi-4CR undergoes the Mumm rearrangement while in
the hydrazoic acid variant performs an electrocyclic ring closure.
Figure 1 depicts the strategy toward carbohydrate/steroid hy-
brids using Ugi-type MCRs, which give rise either to N-substituted
peptide or tetrazole frameworks. Both motifs are considered as
pseudo-peptidic backbones, as they feature structurally modified
peptide bonds or mimics of them. Also, they may allow for
accessing a high level of diversity by varying the combinations of
carbohydrate and steroidal functional groups reacting on the mul-
ticomponent conjugation, that is, 16 combinations for the Ugi-4CR
and 9 combinations for the Ugi-tetrazole reaction (hydrazoic acid
is a fixed component). As shown in Figure 1, it is possible to use
both the sugar and the steroid as each one of the Ugi-components,
though in this work the oxo-component will be fixed to formalde-
hyde in order to avoid the formation of diastereomers of difficult
separation.
sugar or
Ugi-4CR
(CH2O)n
sugar or
steroid
steroid
sugar or
steroid
N
+
C
N
O
O
sugar or
steroid
sugar or
steroid
N
OH
H
Tetrazole linkage
N
Modified
Ugi-4CR
sugar or
steroid
NH2
+
N
N
sugar or
steroid
N
(CH2O)n
HN3
sugar or
steroid
sugar or
steroid
N
C
HN
Figure 1. Strategy for the conjugation of sugars to steroids by the Ugi-4CR and its
hydrazoic acid variant.
2. Results and discussion
β-Chacotrioside
Diosgenin
To assess the scope of the Ugi-4CR-based conjugation approach,
we chose spirostanes as the steroidal Ugi-components owing to
their high incidence in bioactive saponins. Most spirostan sapo-
O
nins4 incorporate an inner
D-glucose unit conjugated to ring A of
O
OH
the spirostanic skeleton and additionally glycosylated with other
glycosyl units. For example, dioscin (1, Fig. 2) is a spirostanyl
glycoside combining high occurrence in the plant kingdom and a
broad spectrum of bioactivity,4,7–9 including potent cytotoxicity
O
O
O
HO
O
O
HO
O
HO
OH
HO
Dioscin (1)
anticancer saponin
against cancer cells. This compound possesses a 4,6-di-O-(
a-L-
OH
HO
rhamnopyranosyl)-b- -glucopyranoside unit, namely chacotriose,
D
linked to C-3 of diosgenin (2). Taking dioscin (1) as model
compound, the aim was posed on addressing the multicomponent
conjugation approach utilizing glucose, chacotriose, and spiros-
tanic derivatives bearing Ugi-reactive functional groups.
Glucose derivatives having amino, carboxylic, and isocyano
groups at C-1 have been previously prepared and utilized in Ugi-
4CRs.17a,b,19b On the other hand, spirostanes functionalized with
amino and carboxylic groups are less common,16c,d while
Figure 2. Structure of the spirostan saponin dioscin, a diosgenyl trisaccharide.
important variation of this approach encompasses the utilization
hydrazoic acid as the acid component, which upon reaction with
an amine, an oxo-compound, and an isonitrile furnishes a 1,5-
disubtituted tetrazole,14 which is commonly considered as an spe-
cial type of peptide bond mimetic. Both processes are chemically
O
O
O
O
ref. 12b
a
b,c
N3
H2N
CN
HO
Diosgenin (2)
5, 83%
3, 77%
4, 91%
O
O
O
O
d, e
f
b, c
N3
H2N
CN
HO
H
H
H
H
OAc
OAc
OAc
OAc
6, ref. 21
7, 87%
8, 96%
9, 80%
ref. 21
O
O
O
O
HO2C
g
HO2C
O
N
O
HO
HO
OAc
12, 95%
O
OAc
11, R = H, ref.23
O
10, ref. 22
Scheme 1. Synthesis of spirostanes functionalized with amino, carboxylic, and isocyano groups. Reagents and conditions: (a) PPh3, THF/H2O; (b) HCO2H/Ac2O, Et3N, THF; (c)
POCl3, Et3N, THF, ꢀ60?0 °C; (d) MsCl, Et3N, CH2Cl2; (e) NaN3, DMPU; (f) H2 (g), 10% Pd/C, EtOAc/AcOH; (g) HClꢁH2NOCH2CO2H, pyridine.

104
D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
spirostanic isocyanides have not been described. Scheme 1 shows
the synthesis of spirostanes functionalized with the Ugi-reactive
groups. Initially, 3b-diosgenyl azide (3) was prepared from
diosgenin (2) as previously described,12b following a protocol
developed for the synthesis of its analogous 3b-cholestanyl azide.20
Subsequent Staudinger azide reduction with PPh3 produced the
3b-diosgenyl amine (4) in 91% yield after column chromatography.
This spirostanic amine was then subjected to formylation by treat-
ment with acetic–formic mixed anhydride followed by phospho-
rylchloride mediated dehydration to afford the 3b-diosgenyl
isocyanide (5) in 83% yield.
yield over two steps. Importantly, configuration of C-3 was not
affected during the synthesis of the two spirostanic isocyanides.
Two different types of spirostanic acids were prepared for the
conjugation to isocyano and aminosugars. The seco-spirostanic
acid 10, which is a naturally occurring sapogenin,22was obtained
from spirostanol 6 according to a reported procedure.21 Alterna-
tively, condensation of ketone 1123with O-(carboxymethyl)-
hydroxylamine gave rise to acid 12, which thus bears the carboxy
functionality attached to C-3 through an oxime linker. Accordingly,
each pair of functionalized spirostanes could be utilized to evaluate
the substrate scope on the Ugi-conjugation approach to glucose
derivatives.
We next turned to the synthesis of 3-amino and 3-isocyanospi-
rostanes having the
a
disposition at C-3, so that the influence of the
Table 1 shows the results of the conjugation of glucose deriva-
tives to a variety of spirostanic steroids by means of the Ugi-4CR
and its hydrazoic acid variant. Based on our experience on Ugi-
4CRs with bulky substrates,16 reaction times were fixed to 24 h
for the classical four-component process and 72 h for the hydrazoic
acid variant.13b Examples were chosen to assess the conjugation
efficiency and to prove the structural diversity of triple sugar/pseu-
do-peptide/steroid hybrids that can be accessed in one-pot
different C-3 stereochemistry on the conjugation efficiency could
be evaluated. For this, the B-ring functionalized 3b-spirostanol
621 was subjected to mesylation and nucleophilic replacement
with azide to furnish the 3a-azidospirostane 7 in 87% yield. This
azide was next subjected to Pd-catalyzed hydrogenation over
24 h to afford amine 8 in 96% yield. Conversion of amine 8 into
the 3a-isocyanospirostane 9 was accomplished as before in 80%
Table 1
Conjugation of glucose derivatives to spirostanic steroids through the Ugi-4CR and its hydrazoic acid varianta
Amine
Acid
Isocyanide
OAc
Sugar-spirostan hybrid
O
OAc
NH2
O
NC
OAc
AcO
AcO
O
O
H
N
O
HO2C
AcO
AcO
N
13
OAc
O
O
O
O
10
O
14, 87%
OAc
NC
O
O
AcO
AcO
NH2
OAc
AcO
AcO
O
OAc
O
O
15
HO2C
O
O
N
AcO
N
N
HO
HO
OAc
O
OAc
12
HN
16, 74%
Cy
OAc
O
O
NC
AcO
AcO
O
OAc
N
OAc
O
HN3
N
N
13
H2N
N
AcO
AcO
N
H
4
OAc
17, 76%
OAc
O
O
MeO2C
NC
O
CO2H
AcO
AcO
O
OAc
O
OAc
18
O
H2N
AcO
AcO
O
H
N
OAc
H
OAc
O
OAc
8
MeO2C
NH
19, 51%
OAc
O
O
NH2
AcO
AcO
O
OAc
15
OAc
O
O
Me
O
AcOH
CN
N
AcO
AcO
H
N
OAc
9
H
H
OAc
OAc
20, 73%
a
All reactions were conducted in MeOH using 1 equiv of each component. Paraformaldehyde was employed as the oxo component in all cases. HN3 was formed in situ from
TMSN3/MeOH.

D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
105
processes. Thus, the spirostanic acids 10 and 12 were conjugated to
glucosyl isocyanide17 13 and glucosyl amine17 15 to furnish
hybrids 14 and 16, respectively, in good yields. As noticed, the
structural differences between both types of conjugates rely not
only in the spirostanic skeleton—that is, 10 is a seco-spirostane
while acid 12 bears the carboxy functionality attached to the ste-
roid through an oxime linker—but on the linear or branched nature
of the pseudo-peptidic backbone connecting the two structures.
Thus, hybrid 14 incorporates the N-glucoside as a secondary
amide, while 16 incorporates the same moiety as a tertiary amide
resulting in a branched peptidic connectivity.
variant of the Ugi-4CR, thus leading to hybrid 23 in 51% yield after
72 h. Indeed, the bulky structure of the trisaccharidic amine 22 is
the main cause of the moderate yield obtained in this procedure,
since other initial experiments with the same isocyanide and smal-
ler amines proved to proceed in up to 90%.
As mentioned above, an important aim of this report is to illus-
trate applications of the multicomponent conjugation approach in
the synthesis of analogs of bioactive sugar/steroids hybrids. To this
end, two different strategies were implemented for the synthesis of
chacotriose/spirostane hybrids resembling the natural spirostan
saponin dioscin (1). As shown in Scheme 2, the b-chacotriosyl azide
2112a was reduced by catalytic hydrogenation to amine 22, which
was then conjugated to diosgenyl isocyanide 5 to afford the triple
chacotriose/tetrazole/diosgenin hybrid 23. This hybrid was sub-
jected to global deprotection to furnish 24 in overall 46% yield.
As noticed from Scheme 2, compound 24 may be considered as a
dioscin analog wherein the traditional glycosidic bond is replaced
by an aminomethyltetrazole linkage, thus encompassing a unique
type of hybrid architecture.
Considering the moderate yield of the direct conjugation of
b-chacotrioside moiety to spirostanic steroids, we turned to
evaluate an alternative route to access analogs of the target
saponin. This second approach comprises the construction of the
2,4-branched trisaccharide moiety starting from the previously
obtained glucose/diosgenin hybrid 17, a route that requires the
We next turned to the conjugation of glucose derivatives to ste-
roidal amines featuring either
a or b stereochemistry. Thus, the
equatorially-disposed 3b-diosgenyl amine (4) was conjugated to
glucosyl isocyanide 13 by the hydrazoic acid variant of the Ugi-
4CR, giving rise to the triple glucose/tetrazole/spirostane hybrid
17 in 76% yield after column chromatography. In this process,
hydrazoic acid is formed in situ by methanolysis of azidotrimeth-
ylsilane, and behaves as acid component upon reaction with the
imine and the isocyanide. Alternatively, the conjugation of the axi-
ally-disposed spirostanyl amine 8 to the carboxymethyl glucoside
18 led to hybrid 19 in only 51% yield after column chromatogra-
phy. This is an expected result considering that axially-disposed
steroidal amines are less reactive than equatorial ones in all types
of transformations.
In contrast, we found that the different stereochemistry of 3-
isocyanosteroids does not have a profound effect of the reaction
yield. Hence, in several initial experiments (not shown) with sim-
ple amino, aldehyde, and carboxylic components, it turned out that
either the equatorial steroidal isocyanide 5 or the axial isocyanide
9 led to Ugi-products in good yields (i.e., 85–90%). Of course, yields
drops significantly when utilizing bulkier substrates as any of the
other components. For example, the axial spirostanic isocyanide
9 was conjugated to glucosyl amine 15 to afford the hybrid 20 in
73% yield after column chromatography (see Table 1). Alterna-
tively, Scheme 2 depicts the conjugation of b-chacotriosyl amine
22 to the 3b-diosgenyl isocyanide 5 by means of the hydrazoic acid
regioselective incorporation of two L-rhamnose units. The se-
quence commenced with acetylation of the aminomethyltetrazole
moiety of 17—to avoid its participation in next steps—followed by
removal of the glucose acetyl groups under typical Zemplen condi-
tions (NaOMe/MeOH). Further selective pivaloylation at OH-3 and
OH-6 according to a reported procedure24 furnished compound 25
in 56% yield over three steps. 2,3,4-Tri-O-acetyl-a-L-rhamnopyr-
anosyl trichloroacetimidate (26)25 was next used as glycosyl donor
for the double glycosylation step following the Schmidt’s inverse
procedure,26 thus leading to the triple chacotriose/tetrazole/dios-
genin hybrid 27 in 77% yield. Final deprotection furnished the
spirostan saponin analog 28, which—apart from the acetamide
OPiv
O
O
O
X
(CH2O)n
+
O
OR1
H
O
O
O
PivO
O
O
AcO
N
b
O
AcO
R1O
O
HN3
OAc
AcO
CN
O
N
N
O
N
R2O
OAc
AcO
5
N
R2O
O
OR2
R2O
23, 51%, R1 = Piv, R2 = Ac
24, 90%, R1 = R2 = H
21, X = N3
22, X = NH2
OR2
a
c
R2O
NH
O
CCl3
O
OAc
O
N
OPiv
N
AcO
N
N
26
N
N
AcO
O
d, e, f
OAc
N
N
AcO
AcO
HO
PivO
N
H
N
Me
O
OAc
OH
g
O
17
25, 56%
O
OR1
N
N
N
O
N
O
R1O
N
O
O
O
R2O
R2O
Me
27, 77%, R1 = Piv, R2 = Ac
28, 88%, R1 = R2 = H
O
OR2
c
R2O
OR2
R2O
Scheme 2. Synthesis of tetrazole-based spirostan saponin analogs. Reagents and conditions: (a) H2 (g), Pd/C, MeOH; (b) MeOH (HN3 is formed in situ from TMSN3); (c) aq
NaOH, THF/MeOH, 50 °C; (d) Ac2O, Et3N, CH2Cl2; (e) NaOMe, MeOH; (f) PivCl, Py, ꢀ15 °C?rt; (g) BF3ꢁEt2O, CH2Cl2, 4 Å MS, ꢀ78 °C?rt.

106
D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
functionality—comprises the same structure of hybrid 24 but with
4.2. (25R)-3b-Isocyano-5-spirostene (5)
the aminomethyltetrazole linkage in a reverse manner.
A mixture of acetic anhydride (2.4 mL, 25 mmol) and formic
acid (1.4 mL, 37 mmol) was stirred at 60 °C for 3 h. The mixture
was cooled to room temperature and added dropwise to a solution
of diosgenyl amine 4 (1.0 g, 2.5 mmol) and Et3N (1.5 mL) in 30 mL
of THF. The reaction mixture was stirred at room temperature
overnight and then diluted with 60 mL of EtOAc. The organic phase
was washed with satd aq NaHCO3 (3 ꢃ 50 mL), aq 10% HCl and
brine (50 mL), dried over anhydrous Na2SO4, and concentrated un-
der reduced pressure to dryness. The resulting formamide was dis-
solved in dry THF (30 mL) and the solution was treated with 4 mL
of Et3N and cooled to ꢀ60 °C. A solution of POCl3 (0.35 mL,
3.75 mmol) in 3 mL of THF was added dropwise under nitrogen
atmosphere and the reaction mixture was stirred at ꢀ60 °C for
3 h and then allowed to reach room temperature. The mixture
was poured into 80 mL of cold water and extracted with Et2O
(2 ꢃ 50 mL). The organic layer was washed with satd aq NaHCO3
(2 ꢃ 50 mL), brine (50 mL), dried over anhyd Na2SO4, and concen-
trated under reduced pressure to dryness. The crude product was
purified by flash column chromatography (n-hexane/EtOAc 5:1)
to afford the diosgenyl isocyanide 5 (878 mg, 83%) as a white solid.
3. Conclusions
We have shown that the Ugi-4CR and its hydrazoic acid variant
are suitable procedures for the conjugation of mono and oligosac-
charides to steroids. The approach proved wide substrate scope,
since both the sugar and the steroidal substrates were utilized with
success as amino, carboxylic, and isocyano components of the Ugi
reactions. These procedures give rise to unique types of conjugates
featuring triple sugar/pseudo-peptide/steroid hybrid architectures.
To our knowledge, this is the first time that multicomponent reac-
tions are employed for the conjugation of carbohydrates to steroi-
dal derivatives. The approach was utilized for the synthesis of
tetrazole-based spirostan saponin analogs, which proved that
applications in the synthesis of natural product analogs are possi-
ble. Considering the remarkable diversity-oriented character of
isocyanide-based MCRs, the present approach shows promise for
the combinatorial production and biological screening of libraries
of saponin analogs.
Mp (MeOH): 135–137 °C. ½a D20
ꢂ
ꢀ10.2 (c 0.95, CHCl3). 1H NMR
4. Experimental
(300 MHz, CDCl3): d = 0.79 (3H, d, J = 6.2 Hz, H-27); 0.80 (3H, s,
H-18); 0.96 (3H, d, J = 6.8 Hz, H-21); 1.06 (3H, s, H-19); 2.57 (1H,
dd, J = 6.8/8.8 Hz); 3.35 (1H, t, J = 10.9 Hz, H-26ax); 3.49 (1H, dd,
Melting points were determined on a Leica DM LS2 apparatus
and are uncorrected. 1H NMR and 13C NMR spectra were recorded
on Varian Mercury spectrometers. Chemical shifts (d) are reported
in ppm relative to the TMS (1H NMR) and to the solvent signal
J = 4.1/10.7 Hz, H-26eq); 3.68 (1H, br m, H-3
a); 4.35–4.40 (1H, m,
H-16a
); 5.33 (1H, m, H-6). 13C NMR (75 MHz, CDCl3): d = 13.2,
(
13C NMR). High resolution ESI mass spectra were obtained from
15.9, 17.1, 18.6 (CH3); 26.6, 28.8 (CH2); 30.2, 30.6 (CH); 31.2,
31.3, 31.4, 32.8 (CH2); 36.9 (C); 37.3, 37.6, 39.8 (CH2); 42.2 (CH);
51.5 (t, J = 5.2 Hz, CH); 51.7, 53.2 (CH); 54.6 (C); 55.6 (CH); 66.9
(CH2); 79.2 (CH); 109.2 (C); 124.2 (CH); 136.0 (C); 155.1 (t,
J = 5.2 Hz, CN). HRMS (ESI-FT-ICR) m/z: 446.3042 [M+Na]+; calcd
for C28H41NO2Na: 446.3035.
a Bruker Apex III Fourier transform ion cyclotron resonance
(FT-ICR) mass spectrometer equipped with an Infinity™ cell, a
7.0 Tesla superconducting magnet, an RF-only hexapole ion guide,
and an external electrospray ion source (Agilent, off axis spray).
ESI-MS spectra were recorded on a Finnigan TSQ 7000, capillary
temperature: 220 °C, ESI positive ion mode: spray voltage
4.5 kV, collision energy ꢀ40 eV, CID pressure 1.8 mT, collision
gas: Ar, ESI negative ion mode: spray voltage 4.0 kV. Unless other-
wise stated, flash column chromatography was carried out using
Merck Silica Gel 60 (0.015–0.040 nm) and analytical thin layer
chromatography (TLC) was performed using Merck Silica Gel 60
F254 aluminium sheets. Solid compounds were recrystallized from
selected solvents for the melting points measurements. All com-
mercially available chemicals were used without further purifica-
tion. Spirostanes 3, 6, and 11 were obtained as described in
Refs.12b,21,23, respectively.
4.3. (25R)-3a-Azido-5a-spirostan-6b-yl acetate (7)
Spirostanol 6 (4.36 g, 7.4 mmol) was dissolved in dry CH2Cl2
(80 mL) and treated with Et3N (4.6 mL, 33.3 mmol) and mesyl
chloride (1.32 mL, 11.1 mmol) at 0 °C. The reaction mixture
was stirred at 0 °C for 1 h, then diluted with 200 mL of CH2Cl2,
and washed with brine (2 ꢃ 100 mL). The organic phase was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to dryness. The resulting crude product was dissolved
in DMPU (50 mL) and the solution was treated with NaN3
(828 mg, 14.8 mmol). The reaction mixture was stirred vigor-
ously under nitrogen atmosphere at 50 °C for 48 h and then
diluted with 300 mL of Et2O. The organic phase was washed
with aq 10% HCl (2 ꢃ 60 mL) and brine (100 mL), dried over an-
hyd Na2SO4 and evaporated under reduced pressure to dryness.
The crude product was purified by flash column chromatogra-
phy (n-hexane/EtOAc 3:1) to give the pure spirostanyl azide 7
4.1. (25R)-3b-Amino-5-spirostene (4)
Azide 3 (1.2 g, 2.9 mmol) was dissolved in 20 mL of THF and
PPh3 (1.13 g, 4.3 mmol) was added. The reaction mixture was stir-
red at room temperature for 8 h, then treated with H2O (180 lL,
10 mmol), and stirred for additional 48 h. The reaction mixture
was concentrated under reduced pressure and the crude product
purified by flash column chromatography (CH2Cl2/Et3N 10:0.1) to
furnish amine 4 (1.09 g, 91%) as a white solid. Mp (MeOH): 166–
(3.21 g, 87%) as a white solid. Mp (MeOH): 152–154 °C. ½a D20
ꢂ
ꢀ124.8 (c 1.30, CHCl3). 1H NMR (400 MHz, CDCl3): d = 0.78
(3H, d, J = 6.4 Hz, H-27); 0.79 (3H, s, H-18); 0.96 (3H, d,
J = 6.8 Hz, H-21); 1.00 (3H, s, H-19); 2.04 (3H, s, CH3CO); 3.37
(1H, t, J = 10.9 Hz, H-26ax); 3.47 (1H, dd, J = 4.0/10.7 Hz, H-
167 °C. ½a 2D0
ꢂ
ꢀ91.4 (c 0.5, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 0.78 (3H, d, J = 6.4 Hz, H-27); 0.79 (3H, s, H-18); 0.97 (3H, d,
26eq); 3.94 (1H, m, H-3b); 4.39 (1H, br m, H-16
a); 4.92 (1H,
J = 6.7 Hz, H-21); 1.09 (3H, s, H-19); 2.70 (1H, m, H-3
(1H, t, J = 10.8 Hz, H-26ax); 3.49 (1H, dd, J = 10.8/4.0 Hz, H-26eq);
4.41 (1H, br m, H-16
); 5.29 (1H, m, H-6). 13C NMR (75 MHz,
CDCl3): d = 13.3, 16.0, 17.1, 18.5 (CH3); 26.8, 28.8 (CH2); 30.1,
30.6 (CH); 31.1, 31.3, 31.5, 32.7 (CH2); 36.8 (C); 37.3, 37.9, 39.9
(CH2); 40.4 (C); 42.0 (CH); 51.6, 53.1, 55.6, 55.9 (CH); 66.9 (CH2);
79.8 (CH); 109.3 (C); 124.2 (CH); 136.0 (C). HRMS (ESI-FT-ICR) m/
z: 414.3369 [M+H]+; calcd for C27H44O2N: 414.3373.
a); 3.36
d, J = 2.7 Hz, H-6
a
). 13C NMR (100 MHz, CDCl3): d = 14.5, 14.7,
16.5, 17.1 (CH3); 20.3 (CH2); 21.4 (CH3); 25.4, 28.7, 29.7
(CH2); 30.3, 30.5 (CH); 31.3, 31.6, 34.3 (CH2); 36.0 (C); 36.4,
39.8 (CH2); 40.5 (C); 41.6, 41.8, 53.7, 55.8, 58.0, 62.0 (CH);
66.8 (CH2); 73.3, 80.6 (CH); 109.2 (C); 170.6 (C@O). HRMS
(ESI-FT-ICR) m/z: 522.3293 [M+Na]+; calcd for C29H45O4N3Na:
522.3302.
a

D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
107
4.4. (25R)-3
a
-Amino-5
a
-spirostan-6b-yl acetate (8)
(3 ꢃ 50 mL), dried over anhyd Na2SO4, and concentrated under re-
duced pressure to afford the spirostanic acid 12 (532 mg, 95%) as a
white solid. Re-crystallization from EtOAc provided an analytical
sample for characterization. Mp (EtOAc): 235–236 °C.
Azide 7 (1.5 g, 3.0 mmol) was dissolved in 40 mL of the solvent
mixture EtOAc/AcOH 9:1 and treated with 10% Pd/C (150 mg). The
reaction mixture was treated successively with hydrogen and vac-
uum and finally stirred under hydrogen atmosphere for 48 h, when
TLC analysis revealed that all of the azide had been consumed. The
catalyst was removed by filtration over a pad of Celite and the
resulting solution was evaporated under reduced pressure to give
the crude amine 8 (1.36 g, 96%). Re-crystallization from MeOH pro-
vided an analytical sample for characterization. Mp (MeOH): 186–
½
a 2D0
ꢂ
ꢀ25.4 (c 0.50, CHCl3). 1H NMR (300 MHz, CDCl3): d = 0.78
(6H, m, H-18 + H-27); 0.95 (3H, d, J = 6.6 Hz, H-21); 1.21 (3H, s,
H-19); 2.07 (3H, s, CH3CO); 3.36 (1H, t, J = 10.9 Hz, H-26ax); 3.46
(1H, m, H-26eq); 4.37 (1H, m, H-16a); 4.52 (1H, m, H-6a); 4.64–
4.78 (2H, m, OCH2). 13C NMR (75 MHz, CDCl3): d = 14.5, 15.6,
16.5, 17.1, 20.7 (CH3); 21.4, 21.5, 27.2, 28.7, 30.1, 30.2, 31.3, 31.6,
32.6 (CH2); 33.9 (CH); 39.7 (C); 39.8, 40.6 (CH); 41.6 (C); 45.0,
55.4, 62.0 (CH); 66.8 (CH2); 74.6 (CH); 76.4 (CH2); 77.2 (C); 80.7
(CH); 109.2 (C); 162.3 (C@N); 170.1, 174.3 (C@O). HRMS (ESI-FT-
ICR) m/z: 584.3196 [M+Na]+; calcd for C31H47NO8Na: 584.3199.
187 °C. ½a 2D0
ꢂ
ꢀ86.7 (c 0.45, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 0.78 (3H, d, J = 6.2 Hz, H-27); 0.79 (3H, s, H-18); 0.96 (3H, d,
J = 6.6 Hz, H-21); 0.99 (3H, s, H-19); 2.05 (3H, s, CH3CO); 3.28
(1H, m, H-3b); 3.37 (1H, t, J = 10.8 Hz, H-26ax); 3.47 (1H, dd,
J = 3.4/10.8 Hz, H-26eq); 4.38 (1H, br m, H-16
a
); 4.90 (1H, d,
4.7. General procedure for the Ugi-4CR-based conjugation
approach
J = 2.3 Hz, H-6
a
). 13C NMR (75 MHz, CDCl3): d = 14.6, 15.6, 16.5,
17.1 (CH3); 20.3 (CH2); 21.4 (CH3); 26.4, 28.7, 30.0 (CH2); 30.2,
30.6 (CH); 31.3, 31.6, 34.1 (CH2); 36.3 (C); 36.5, 39.9 (CH2); 40.6
(C); 41.2, 41.6, 52.1, 53.8, 56.0, 62.0 (CH); 66.8 (CH2); 74.0, 80.7
(CH); 109.2 (C); 170.8 (C@O). HRMS (ESI-FT-ICR) m/z: 474.8535
[M+H]+; calcd for C29H48O4N: 474.8535.
A solution of paraformaldehyde (0.5 mmol) and the amine
(0.5 mmol) in MeOH (30 mL) is stirred for 2 h at room temperature.
The carboxylic or hydrazoic acid (0.5 mmol) and the isonitrile
(0.5 mmol) are then added and the reaction mixture is stirred at
room temperature. The mixture is concentrated under reduced
pressure and the crude product is purified by flash column chro-
matography on silica gel to afford the corresponding sugar-steroid
hybrid.
4.5. (25R)-3a-Isocyano-5a-spirostan-6b-yl acetate (9)
A mixture of acetic anhydride (2.4 mL, 25 mol) and formic acid
(1.43 mL, 37 mmol) was stirred at 60 °C for 3 h. The mixture was
cooled to room temperature and added dropwise to a solution of
amine 8 (1.2 g, 2.5 mmol) and Et3N (1.5 mL) in 30 mL of THF. The
reaction mixture was stirred at room temperature overnight and
then diluted with 80 mL of EtOAc. The organic phase was washed
with satd aq NaHCO3 (3 ꢃ 50 mL), aq 10% HCl and brine (50 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure to dryness. The resulting formamide was dissolved in
dry THF (30 mL) and the solution was treated with 4 mL of Et3N
and cooled to ꢀ60 °C. A solution of POCl3 (0.35 mL, 3.75 mmol)
in 3 mL of THF was added dropwise under nitrogen atmosphere
and the reaction mixture was stirred at ꢀ60 °C for 3 h and then al-
lowed to reach room temperature. The mixture was poured into
80 mL of cold water and extracted with Et2O (2 ꢃ 50 mL). The or-
ganic layer was washed with satd aq NaHCO3 (2 ꢃ 50 mL), brine
(50 mL), dried over anhyd Na2SO4, and concentrated under reduced
pressure to dryness. The crude product was purified by flash col-
umn chromatography (n-hexane/EtOAc 3:1) to afford the spirosta-
nyl isocyanide 9 (960 mg, 80%) as a white solid. Mp (MeOH): 158–
4.8. Glucose/pseudo-peptide/spirostane hybrid 14
Isopropylamine (21 lL, 0.25 mmol), paraformaldehyde (7.5 mg,
0.25 mmol), the spirostanic acid 10 (115 mg, 0.25 mmol), and glu-
cosyl isocyanide 13 (90 mg, 0.25 mmol) were reacted for 24 h
according to the general Ugi-4CR procedure described in Section
4.7. Flash column chromatography purification (n-hexane/EtOAc
2:1) afforded the pure conjugate 14 (193 mg, 87%) as a white solid.
Mp (EtOAc): 207–209 °C. ½a D20
ꢂ
ꢀ54.5 (c 0.75, CHCl3). 1H NMR
(300 MHz, CDCl3): d = 0.75 (3H, s, H-18); 0.77 (3H, d, J = 5.7 Hz,
H-27); 0.95 (3H, d, J = 6.7 Hz, H-21); 1.20 (3H, s, H-19); 1.24 (6H,
d, J = 6.6 Hz, (CH3)2CH); 2.01, 2.02, 2.03, 2.04 (4 ꢃ 3H, 4 ꢃ s,
4 ꢃ CH3CO); 3.19 (1H, m); 3.35 (1H, t, J = 10.9 Hz, H-26ax); 3.46
(1H, dd, J = 4.4/11.0 Hz, H-26eq); 3.65 (2H, m); 3.97 (1H, m);
4.10–4.18 (2H, m); 4.13 (1H, dd, J = 2.2/12.5 Hz); 4.23 (1H, dd,
J = 4.9/12.6 Hz); 4.40 (1H, m, H-16a); 4.63 (1H, m); 4.86 (1H, t,
J = 9.3 Hz); 4.92 (1H, t, J = 9.3 Hz); 5.06 (1H, t, J = 9.4 Hz); 5.26
(1H, m); 7.44 (1H, d, J = 9.2 Hz, NH). 13C NMR (75 MHz, CDCl3):
d = 14.5, 16.3, 16.9, 17.1, 20.2, 20.3, 20.4, 20.7, 20.8, 20.9 (CH3);
28.8 (CH2); 29.0 (CH); 30.2, 30.3, 31.4, 31.6, 35.0 (CH2); 36.8
(CH); 39.6, 40.2 (CH2); 40.3, 41.6 (C); 43.2 (CH); 45.6, 56.5 (CH2);
58.4, 61.5 (CH); 62.0, 66.9 (CH2); 67.4, 68.5, 70.2, 72.9, 73.5, 75.2,
78.1, 80.3 (CH); 109.3 (C); 169.7, 170.2, 170.7, 171.0, 171.7,
172.1, 178.1 (C@O). HRMS (ESI-FT-ICR) m/z: 911.4521 [M+Na]+;
calcd for C46H68N2O15Na: 911.4517.
160 °C. ½a 2D0
ꢂ
ꢀ73.2 (c 0.30, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 0.77 (3H, d, J = 6.4 Hz, H-27); 0.80 (3H, s, H-18); 0.96 (3H, d,
J = 6.8 Hz, H-21); 0.99 (3H, s, H-19); 2.02 (3H, s, CH3CO); 3.36
(1H, t, J = 10.8 Hz, H-26ax); 3.47 (1H, dd, J = 3.7/10.8 Hz, H-26eq);
3.95 (1H, m, H-3b); 4.40 (1H, br m, H-16
a); 4.90 (1H, d,
J = 2.4 Hz, H-6
a
). 13C NMR (75 MHz, CDCl3): d = 14.5, 15.1, 16.4,
17.1 (CH3); 20.4 (CH2); 21.4 (CH3); 25.7, 28.8, 29.8 (CH2); 30.2,
30.6 (CH); 31.3, 31.7, 34.4 (CH2); 36.1 (C); 36.4, 39.7 (CH2); 40.6
(C); 41.7, 41.9 (CH); 51.9 (t, J = 5.1 Hz, CH); 55.8, 56.7, 62.0 (CH);
66.8 (CH2); 73.4, 80.5 (CH); 109.3 (C); 154.3 (t, J = 5.1 Hz, CN),
170.5 (C@O). HRMS (ESI-FT-ICR) m/z: 484.3430 [M+H]+; calcd for
4.9. Glucose/pseudo-peptide/spirostane hybrid 16
Glucosyl amine 15 (88 mg, 0.25 mmol), paraformaldehyde
(7.5 g, 0.25 mmol), spirostanic acid 12 (140 mg, 0.25 mmol), and
C30H46O4N: 484.3427.
cyclohexylisocyanide (22 lL, 0.25 mmol) were reacted for 24 h
4.6. (25R)-6b-Acetoxy-3E-[O-(carboxymethyl)oximino]-5
spirostan-5-ol (12)
a
-
according to the general Ugi-4CR procedure described in Section
4.7. Flash column chromatography purification (n-hexane/EtOAc
3:1) furnished the pure sugar–spirostan conjugate 16 (190 mg,
O-(Carboxymethyl)-hydroxylamine hydrochloride (114 mg,
1.39 mmol) was added to a solution of ketone 11 (488 mg,
1.0 mmol) in 20 mL of dry pyridine. The reaction mixture was stir-
red at room temperature for 10 h and then diluted with 100 mL of
EtOAc. The organic phase was washed with aq 10% HCl
74%) as a white foam. ½a D20
ꢂ
ꢀ82.1 (c 1.20, CHCl3). 1H NMR
(300 MHz, CDCl3): d = 0.74 (3H, d, J = 6.2 Hz, H-27); 0.77 (3H, s,
H-18); 0.91 (3H, d, J = 6.8 Hz, H-21); 1.20 (3H, s, H-19); 1.98,
1.99, 2.00, 2.02, 2.03 (5 ꢃ 3H, 5 ꢃ s, 5 ꢃ CH3CO); 3.26 (1H, t,
J = 10.9 Hz, H-26ax); 3.40–3.44 (2H, m); 3.55 (1H, m); 3.90–4.08

108
D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
(3H, m); 4.15–4.22 (1H, m); 4.36 (1H, m); 4.63 (1H, d, J = 4.5 Hz);
4.70 (1H, m); 4.85 (1H, m); 4.90–5.02 (2H, m); 5.04–5.12 (2H,
m); 5.33–5.45 (2H, m); 6.76 (1H, m, NH). 13C NMR (75 MHz,
CDCl3): d = 15.0, 16.3, 17.1, 17.6, 20.7, 20.8, 21.2, 21.5, 21.8 (CH3);
22.9, 23.8, 25.9, 26.0, 26.1, 26.6, 26.7 (CH2); 29.9, 31.4 (CH); 31.5,
32.4, 32.6, 32.7, 33.7, 40.5, 40.7, 41.1 (CH2); 41.8 (C); 42.9, 46.4,
56.9 (CH); 63.7, 67.8 (CH2); 70.2, 71.4, 72.7, 74.1, 74.2, 75.0, 75.1,
77.3 (CH); 79.5 (C); 82.0 (CH2); 90.9 (CH); 110.4 (C); 166.1, 169.6,
171.3, 171.5, 171.8, 172.1, 172.8, 174.0 (C@O). HRMS (ESI-FT-ICR)
m/z: 1052.5258 [M+Na]+; calcd for C53H79NaO17N3: 1052.5307.
spirostanyl isocyanide 9 (120 mg, 0.25 mmol) were reacted accord-
ing for 24 h to the general Ugi-4CR procedure described in Section
4.7. Flash column chromatography purification (n-hexane/EtOAc
3:1) furnished the pure conjugate 20 (185 mg, 73%) as a white so-
lid. Mp (from MeOH): 224–226 °C. ½a D20
ꢂ
ꢀ63.7 (c 0.50, CHCl3). 1H
NMR (300 MHz, CDCl3): d = 0.77 (3H, d, J = 6.5 Hz, H-27); 0.81
(3H, s, H-18); 0.96 (3H, d, J = 6.8 Hz, H-21); 1.12 (3H, s, H-19);
2.02, 2.04, 2.05, 2.06, 2.08, 2.27 (6 ꢃ 3H, 6 ꢃ s, 6 ꢃ CH3CO); 3.36
(1H, t, J = 10.9 Hz, H-26ax); 3.44 (1H, m, H-26eq); 4.04–4.08 (2H,
m); 4.13 (1H, dd, J = 2.5/12.3 Hz); 4.21 (1H, d, J = 9.0 Hz); 4.23
(1H, dd, J = 4.9/12.3 Hz); 4.27–4.36 (3H, m); 4.38 (1H, m); 4.84
(1H, m); 4.91 (1H, m); 6.72 (1H, m, NH). 13C NMR (75 MHz, CDCl3):
d = 14.7, 15.3, 16.5, 17.2, 20.2, 20.3, 20.5, 20.7, 21.0, 21.4 (CH3);
25.5 (CH2); 28.3 (CH); 28.4, 30.4, 31.4, 31.6 (CH2); 34.7 (CH); 36.3
(CH2); 36.8 (C); 39.9 (CH2); 40.4 (C); 41.5 (CH2); 41.8 (CH); 46.7
(CH2); 53.6, 55.8, 58.1, 61.7 (CH); 62.0, 66.8 (CH2); 69.8, 71.1,
71.3, 72.4, 73.3, 80.6, 80.8, 81.5, 84.7 (CH); 109.2 (C); 169.9,
170.2, 170.5, 170.6, 171.0, 171.5 (C@O). HRMS (ESI-FT-ICR) m/z:
1040.5316 [M+Na]+; calcd for C52H79N3NaO17: 1040.5309.
4.10. Glucose/tetrazole/spirostane hybrid 17
Spirostanyl amine
(15 mg, 0.5 mmol), azidotrimethylsilane (66
4
(206 mg, 0.5 mmol), paraformaldehyde
L, 0.5 mmol), and glu-
l
cosyl isocyanide 13 (180 mg, 0.5 mmol) were reacted for 72 h accord-
ing to the general Ugi-4CR procedure described in Section 4.7. Flash
column chromatography purification (n-hexane/EtOAc 2:1) afforded
the pure conjugate 17 (314 mg, 76%) as a white solid. Mp (EtOAc):
211–212 °C. ½a 2D0
ꢂ
ꢀ60.9 (c 1.10, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 0.77 (3H, d, J = 6.4 Hz, H-27); 0.78 (3H, s, H-18); 0.97 (3H, d,
J = 6.5 Hz, H-21); 0.97 (3H, s, H-19); 2.02, 2.03, 2.05, 2.06 (3 ꢃ 4H,
4 ꢃ s, 4 ꢃ CH3CO); 2.27 (1H, m); 2.40 (1H, ddd, J = 2.1/2.3/15.4 Hz);
3.29 (1H, tt, J = 4.4/11.2 Hz); 3.37 (1H, t, J = 10.9 Hz, H-26ax); 3.44–
3.50 (1H, m); 3.98 (1H, ddd, J = 2.1/5.0/10.1 Hz); 4.13 (1H, dd,
4.13. Chacotriose/tetrazole/spirostane hybrid 23
b-Chacotriosyl azide 21 (230 mg, 0.25 mmol) was dissolved in
25 mL of MeOH and treated with 25 mg of 10% Pd/C. The reaction
mixture was treated successively with hydrogen and vacuum and
finally stirred under hydrogen atmosphere for 48 h, when TLC
analysis revealed that all of the azide had been consumed. The cat-
alyst was removed by filtration over a pad of Celite and the result-
ing solution was evaporated under reduced pressure to furnish b-
chacotriosyl amine (22), which was identified by ESI-MS and used
in the Ugi-4CR conjugation procedure without further purification.
The crude amine 22 (223 mg, 0.25 mmol), paraformaldehyde
J = 2.0/12.5 Hz); 4.28 (1H, dd, J = 5.1/12.7 Hz); 4.40 (1H, m, H-16a);
4.67 (1H, s); 5.23 (1H, m); 5.32–5.38 (1H, m); 5.41 (1H, t,
J = 9.3 Hz); 5.46 (1H, t, J = 9.0 Hz); 5.87 (1H, d, J = 9.2 Hz). 13C NMR
(75 MHz, CDCl3): d = 14.5, 16.2, 17.1, 19.31, 20.2, 20.5, 20.5, 20.7,
20.8 (CH3); 28.2, 28.7 (CH2); 30.2 (CH); 31.3, 31.4 (CH2); 31.8 (CH);
32.0 (CH2); 36.9 (C); 37.1, 38.9, 39.7 (CH2); 40.2 (C); 41.5, 45.0, 50.0,
56.4 (CH); 61.5 (CH2); 62.0 (CH); 66.8 (CH2); 70.1, 72.7, 75.0, 78.9,
80.8, 85.6 (CH); 109.2 (C); 121.5 (CH); 140.6, 159.6 (C); 168.9,
169.3, 169.9, 170.5 (C@O). HRMS (ESI-FT-ICR) m/z: 848.4428
[M+Na]+; calcd for C43H63N5O11Na: 848.4422.
(7.5 mg, 0.25 mmol), azidotrimethylsilane (33 lL, 0.25 mmol),
and the spirostanyl isocyanide 5 (106 mg, 0.25 mmol) were reacted
in MeOH for 72 h according to the general Ugi-4CR procedure de-
scribed in Section 4.7. Flash column chromatography purification
(n-hexane/EtOAc 2:1) furnished the pure conjugate 23 (175 mg,
4.11. Glucose/pseudo-peptide/spirostane hybrid 19
51%) as a white foam. ½a D20
ꢂ
ꢀ70.0 (c 1.25, CHCl3). 1H NMR
The spirostanyl amine 8 (119 mg, 0.25 mmol), paraformalde-
hyde (7.5 mg, 0.25 mmol), acid 18 (102 mg, 0.25 mmol), and methyl
2-isocyano-2-methylpropanoate (32 mg, 0.25 mmol) were reacted
for 24 h according to the general Ugi-4CR procedure described in
Section 4.7. Flash column chromatography purification (n-hexane/
EtOAc 3:1) afforded the pure conjugate 19 (130 mg, 51%) as a white
(400 MHz, CDCl3): d = 0.78 (6H, m, H-18 + H-27); 0.84 (3H, d,
J = 6.2 Hz, CH3 Rha); 0.97 (3H, d, J = 6.9 Hz, H-21); 1.03 (3H, s, H-
´
19); 1.15–1.18 (21H, m, 2 ꢃ (CH3)3C, CH3 Rha); 1.91, 1.95, 1.97,
2.03, 2.04, 2.11 (6 ꢃ 3H, 6 ꢃ s, 6 ꢃ CH3CO); 2.65–2.72 (1H, m, H-5
Rha´); 3.38 (1H, t, J = 10.9 Hz, H-26ax); 3.43–3.48 (1H, m, H-
26eq); 3.88–3.97 (2H, m, H-4 Glc, H-5 Rha); 3.99–4.04 (1H, m, H-
5 Glc); 4.06–4.12 (2H, m, NCH2); 4.29 (1H, dd, J = 4.4/12.2 Hz, H-
solid. Mp (from MeOH): 237–238 °C. ½a D20
ꢂ
ꢀ49.1 (c 0.80, CHCl3). 1H
NMR (300 MHz, CDCl3): d = 0.78 (3H, d, J = 6.3 Hz, H-27); 0.79 (3H,
s, H-18); 0.97 (3H, d, J = 6.6 Hz, H-21); 1.00 (3H, s, H-19); 1.50
(2 ꢃ 3H, 2 ꢃ s, 2 ꢃ CH3); 2.05, 2.03, 2.02, 2.01 (3 ꢃ 5H, 5 ꢃ s,
5 ꢃ CH3CO); 3.37 (1H, t, J = 11.0 Hz, H-26ax); 3.47 (1H, m, H-
26eq); 3.52 (1H, m, H-3b); 3.70 (3H, s, CH3O); 4.04–3.98 (1H, m);
4.14 (1H, m); 4.26–4.22 (2H, m, CH2); 4.29 (1H, dd, J = 4.5/
6a Glc); 4.33–4.43 (3H, m, H-2 Glc, H-16a, H-6b Glc); 4.48–4.52
(1H, m, H-1 Glc); 4.66 (1H, m, H-3
a); 4.77 (1H, d, J = 1.7 Hz, H-1
´
Rha); 4.81 (1H, t, J = 10.0 Hz, H-4 Rha); 4.94 (1H, d, J = 1.8 Hz, H-
´
´
1 Rha); 4.99–5.08 (3H, m, H-2 Rha, H-3 Rha, H-4 Rha,); 5.15 (1H,
dd, J = 1.8/3.2 Hz, H-2 Rha); 5.21 (1H, dd, J = 3.2/10.1 Hz, H-3
Rha); 5.34 (1H, m, H-6 diosgenin); 5.47 (1H, t, J = 7.6 Hz, H-3
Glc). 13C NMR (100 MHz, CDCl3): d = 14.9, 16.4, 17.4, 17.5, 17.8,
19.4, 20.4, 20.5, 20.6 (CH3); 20.8 (CH2); 26.9, 27.1 (CH3); 28.3,
28.7 (CH2); 30.6 (CH); 31.5 (CH2); 31.6 (CH); 31.8, 32.1 (CH2);
36.9 (C); 37.5 (CH2); 38.8, 38.9 (C); 39.0, 39.9 (CH2); 40.4 (C);
41.6, 50.1, 56.5 (CH); 61.7, 61.9 (CH2); 62.3, 68.5, 68.6, 68.7, 69.4,
69.9, 70.7, 75.5, 75.7, 77.4, 79.4, 80.8, 86.7, 97.5, 98.3 (CH); 109.3
(C); 124.3 (CH); 139.6 (C); 169.1, 169.2, 169.4, 169.8, 169.9,
170.1, 177.3, 177.6 (C@O). HRMS (ESI-FT-ICR) m/z: 1392.6938
[M+Na]+; calcd for C69H103N5O23Na: 1392.6942.
12.3 Hz); 4.40–4.36 (2H, m); 4.43 (1H, br m, H-16
a); 4.69 (1H, d,
J = 9.8 Hz); 4.91 (1H, m, H-6 ); 5.26 (1H, t, J = 9.6 Hz); 5.36–5.41
a
(2H, m); 6.95 (1H, br s, NH). 13C NMR (75 MHz, CDCl3): d = 14.6,
15.6, 16.5, 17.0, 20.1, 20.2, 20.5, 20.6, 20.8, 21.2, 21.4 (CH3); 26.5,
28.8 (CH2); 30.0 (CH); 30.2, 30.7, 31.3 (CH2); 34.9 (CH); 36.5 (CH2);
39.8 (C); 40.4 (CH2); 41.3 (C); 41.6, 45.4 (CH); 52.5 (CH3); 53.6,
56.2 (CH); 58.6 (C); 61.6 (CH); 62.0, 62.4, 66.8 (CH2); 70.1, 72.6,
74.2, 75.0, 80.7, 100.4 (CH); 109.2 (C); 168.6, 169.9, 169.7, 169.5,
170.5, 170.8, 171.4, 174.7 (C@O). HRMS (ESI-FT-ICR) m/z:
1041.5150 [M+Na]+; calcd for C52H78N2O18Na: 1041.5146.
4.14. Tetrazole-based dioscin analogue 24
4.12. Glucose/pseudo-peptide/spirostane hybrid 20
An aqueous solution of NaOH (1 M, 1 mL) was added to a solu-
tion of hybrid 23 (140 mg, 0.1 mmol) in THF/MeOH (4 mL, 1:1, v/v)
and the reaction mixture was stirred at 50 °C overnight. The
Glucosyl amine 15 (88 mg, 0.25 mmol), paraformaldehyde
(7.5 mg, 0.25 mmol), acetic acid (15 mg, 0.25 mmol), and the

D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
109
solution was neutralized with acid resin Dowex-50 (H+) and then
4.16. Chacotriose/tetrazole/spirostane hybrid 27
filtered to remove the resin. The filtrates were concentrated under
reduced pressure and the resulting crude product was purified by
flash column chromatography (CHCl3/MeOH 5:1) to afford the
saponin analogue 24 (86 mg, 90%) as a white amorphous solid.
A suspension of hybrid 25 (140 mg, 0.16 mmol) and 4 Å molec-
ular sieves in dry CH2Cl2 (30 mL) was cooled to ꢀ80 °C and treated
with BF3ꢁEt2O (80
lL, 0.64 mmol) under nitrogen atmosphere.
½
a 2D0
ꢂ
ꢀ82.1 (c 0.90, MeOH). 1H NMR (400 MHz, pyridine): d = 0.73
After stirring for 1 h at this temperature, a solution of rhamnopyr-
anosyl trichloroacetimidate 26 (208 mg, 0.48 mmol) in CH2Cl2
(10 mL) was added and the stirring was continued for 5 h at room
temperature. The reaction mixture was diluted with CH2Cl2
(30 mL) and filtered through a pad of Celite. The filtrate was
washed with satd aq NaHCO3 (2 ꢃ 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 chro-
matography (n-hexane/EtOAc 2:1) to afford 27 (173 mg, 77%) as
(3H, d, J = 5.9 Hz, H-27); 0.89 (3H, s, H-18); 1.01 (3H, s, H-19);
1.18 (3H, d, J = 6.9 Hz, H-21); 1.57 (3H, d, J = 6.1 Hz, CH3 Rha);
´
1.65 (3H, d, J = 6.4 Hz, CH3 Rha); 3.08–3.14 (1H, m, H-5 Rha);
3.48–3.56 (1H, m, H-26ax); 3.59–3.63 (1H, m, H-26eq); 3.90–3.94
(1H, m, H-5 Glc); 4.04–4.18 (6H, m, H-6a Glc, H-6b Glc, H-4 Rha,
H-4 Rha´, NCH2); 4.28–4.35 (2H, m, H-3 Glc, H-3 Rha´); 4.40–4.44
(1H, m, H-1 Glc); 4.49–4.58 (4H, m, H-3 Rha, H-4 Glc, H-16
a, H-
3a); 4.69 (1H, m, H-2 Rha); 4.77 (1H, m, H-2 Rha´); 4.83 (1H, t,
J = 9.2 Hz, H-2 Glc); 4.88 (1H, m, H-5 Rha); 5.49 (1H, m, H-6 dios-
a white foam. ½a 2D0
ꢂ
ꢀ48.7 (c 0.25, CHCl3). 1H NMR (400 MHz,
´
genin); 5.88 (1H, d, J = 1.3 Hz, H-1 Rha); 6.12 (1H, d, J = 1.5 Hz, H-
CDCl3): d = 0.78 (6H, m, H-18 + H-27); 0.84 (3H, d, J = 6.2 Hz, CH3
Rha); 0.96 (3H, d, J = 6.9 Hz, H-21); 1.03 (3H, s, H-19); 1.19–1.23
1 Rha). 13C NMR (100 MHz, pyridine): d = 15.6, 16.8, 17.9, 18.8,
´
´
19.5, 19.9 (CH3); 21.6, 29.4, 29.9 (CH2); 31.0, 32.1 (CH); 32.3,
32.5, 32.8 (CH2); 37.6 (C); 37.9, 39.8, 40.3 (CH2); 40.9 (C); 42.4,
50.9, 56.9 (CH); 61.2, 62.6 (CH2); 63.3 (CH); 67.3 (CH2); 70.5,
70.8, 72.5, 72.6, 72.7, 73.1, 74.3, 74.4, 77.9, 78.6, 79.6, 79.7, 80.8,
81.9, 90.4, 101.8, 102.2 (CH); 109.5 (C); 124.1 (CH); 139.4 (C).
HRMS (ESI-FT-ICR) m/z: 972.5152 [M+Na]+; calcd for C47H75N5O15-
Na: 972.5157.
(21H, m, 2 ꢃ (CH3)3C, CH3 Rha); 1.98, 1.99, 2.02, 2.04, 2.06, 2.08,
´
2.12 (7 ꢃ 3H, 7 ꢃ s, 7 ꢃ CH3CO); 2.66–2.73 (1H, m, H-5 Rha); 3.37
(1H, t, J = 10.9 Hz, H-26ax); 3.45–3.49 (1H, m, H-26eq); 3.89–3.96
(2H, m, H-4 Glc, H-5 Rha); 3.98–4.02 (1H, m, H-5 Glc); 4.30 (1H,
dd, J = 4.5/12.3 Hz, H-6a Glc); 4.35–4.45 (3H, m, H-2 Glc, H-16
H-6b Glc); 4.48 (1H, m, H-3 ); 4.78 (1H, d, J = 1.8 Hz, H-1 Rha);
4.81 (1H, t, J = 10.0 Hz, H-4 Rha); 4.92 (1H, d, J = 1.9 Hz, H-1
a,
a
´
´
´
Rha); 4.99–5.08 (5H, m, H-2 Rha, H-3 Rha, H-4 Rha, NCH2); 5.13
(1H, dd, J = 1.9/3.3 Hz, H-2 Rha); 5.18 (1H, dd, J = 3.3/10.1 Hz, H-3
Rha); 5.34 (1H, m, H-6 diosgenin); 5.47 (1H, t, J = 7.6 Hz, H-3
Glc); 5.89 (1H, d, J = 8.6 Hz, H-1 Glc). 13C NMR (100 MHz, CDCl3):
d = 14.5, 16.3, 17.1, 17.2, 17.5, 19.4, 20.5, 20.6, 21.8 (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 (CH); 143.2 (C); 169.1,
169.3, 169.4, 169.8, 169.9, 170.0, 171.4, 176.3, 177.6 (C@O). HRMS
(ESI-FT-ICR) m/z: 1434.7050 [M+Na]+; calcd for C71H105N5O24Na:
1434.7047.
4.15. Glucose/tetrazole/spirostane hybrid 25
To a solution of hybrid 17 (300 mg, 0.36 mmol) in 20 mL of
CH2Cl2 was added Et3N (100
lL, 0.72 mmol) and acetic anhydride
(85 L, 0.9 mmol). The reaction mixture was stirred at room tem-
l
perature for 4 h and then diluted with 50 mL of CH2Cl2. The solu-
tion was washed with aq 10% HCl (2 ꢃ 50 mL), brine (50 mL),
then dried over anhydrous Na2SO4, and concentrated under re-
duced pressure. The crude product was dried in vacuo for 2 h,
then dissolved in MeOH (30 mL, 1:1, v/v) and treated with
NaOMe up to pH 9–10. The mixture was stirred overnight, then
neutralized with acid resin Dowex-50 (H+) and filtered to remove
the resin. The filtrate was concentrated under reduced pressure to
dryness and. The residue was dried in vacuo and then dissolved in
anhydrous pyridine (20 mL). The solution was cooled to ꢀ15 °C
under nitrogen atmosphere and pivaloyl chloride (0.22 mL,
1.8 mmol) was added dropwise. The reaction mixture was stirred
at room temperature until the intermediate disappeared as indi-
cated by TLC. The mixture was then diluted with EtOAc (40 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 chro-
matography (n-hexane/EtOAc 2:1) to afford the dipivaloylated
conjugate 25 (172 mg, 56%) as a white solid. Mp (EtOAc): 208–
4.17. Tetrazole-based dioscin analogue 28
An aqueous solution of NaOH (1 M, 1 mL) was added to a solu-
tion of hybrid 23 (130 mg, 0.09 mmol) in THF/MeOH (4 mL, 1:1, v/
v) and the reaction mixture was stirred at 50 °C overnight. The
solution was neutralized with acid resin Dowex-50 (H+) and then
filtered to remove the resin. The filtrates were concentrated under
reduced pressure and the resulting crude product was purified by
flash column chromatography (CHCl3/MeOH 5:1) to afford the
saponin analogue 28 (80 mg, 88%) as a white amorphous solid.
½
a 2D0
ꢂ
ꢀ61.9 (c 0.85, MeOH). 1H NMR (400 MHz, pyridine-d5):
210 °C. ½a 2D0
ꢂ
ꢀ5.3 (c 1.45, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 0.71 (3H, d, J = 5.8 Hz, H-27); 0.87 (3H, s, H-18); 0.99 (3H, s,
d = 0.78 (3H, d, J = 6.9 Hz, H-27); 0.79 (3H, s, H-18); 0.97 (3H, d,
J = 6.9 Hz, H-21); 1.01 (3H, s, H-19); 1.19, 1.22 (2 ꢃ 9H, 2 ꢃ s,
2 ꢃ (CH3)3C); 2.09 (3H, s, CH3CO); 3.36 (1H, t, J = 10.9 Hz, H-
26ax); 3.41–3.48 (3H, m, H-2 Glc, H-4 Glc, H-26eq); 3.54–3.59
(1H, m, H-5 Glc); 4.22 (1H, dd, J = 6.9/11.7 Hz, H-6a Glc); 4.30–
H-19); 1.16 (3H, d, J = 7.0 Hz, H-21); 1.55 (3H, d, J = 6.1 Hz, CH3
´
Rha); 1.64 (3H, d, J = 6.2 Hz, CH3 Rha); 2.11 (3H, s, CH3CO); 3.09–
3.15 (1H, m, H-5 Rha); 3.47–3.55 (1H, m, H-26ax); 3.59–3.62
(1H, m, H-26eq); 3.89–3.93 (1H, m, H-5 Glc); 4.03–4.21 (4H, m,
´
H-6a Glc, H-6b Glc, H-4 Rha, H-4 Rha); 4.29–4.37 (2H, m, H-3
´
4.34 (1H, m, H-16
a
); 4.40–4.43 (1H, m, H-6b Glc); 4.46 (1H, m,
Glc, H-3 Rha); 4.46 (1H, m, H-3
a); 4.50–4.60 (3H, m, H-3 Rha, H-
´
H-3 ); 4.86 (1H, t, J = 9.1 Hz, H-3 Glc); 4.89–4.95 (2H, m,
a
4 Glc, H-16 ); 4.68 (1H, m, H-2 Rha); 4.77 (1H, m, H-2 Rha);
a
´
NCH2); 5.32 (1H, m, H-6 diosgenin); 5.78 (1H, d, J = 9.1 Hz, H-1
Glc). 13C NMR (75 MHz, CDCl3): d = 11.9, 13.2, 15.9, 17.1, 20.6,
27.0, 27.1 (CH3); 28.3, 28.7, 29.2 (CH2); 30.1 (CH); 31.1, 31.4,
31.5 (CH2); 34.3 (CH); 36.1 (C); 36.4, 37.7 (CH2); 42.2, 44.6, 53.5
(CH); 55.1 (C); 55.4, 55.7 (CH); 63.7, 66.8 (CH2); 70.0, 72.1, 74.2,
77.8, 78.7, 79.1, 101.1 (CH); 109.2 (C); 178.5, 180.1 (C@O). HRMS
(ESI-FT-ICR) m/z: 890.5250 [M+Na]+; calcd for C47H73N5O10Na:
890.5255.
4.84 (1H, t, J = 9.2 Hz, H-2 Glc); 4.88 (1H, m, H-5 Rha); 5.39 (1H,
m, H-6 diosgenin); 5.86 (1H, d, J = 1.3 Hz, H-1 Rha); 6.27 (1H, d,
J = 1.3 Hz, H-1 Rha); 6.31 (1H, d, J = 9.3 Hz, H-1 Glc). 13C NMR
´
(100 MHz, pyridine): d = 15.4, 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,

110
D. G. Rivera et al. / Carbohydrate Research 359 (2012) 102–110
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103.5 (CH); 109.7 (C); 122.1 (CH); 141.4 (C). HRMS (ESI-FT-ICR) m/
z: 1014.5267 [M+Na]+; calcd for C49H77N5O16Na: 1014.5263.
Acknowledgment
We gratefully acknowledge financial support from the Land
Sachsen-Anhalt (HWP).
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