New insights into the structure-cytotoxicity relationship of spirostan saponins and related glycosides

Article abstract of DOI:10.1016/j.bmc.2012.02.026

A variety of spirostan saponins and related glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). A linear glycosylation strategy allowed for accessing a variety of functionalization pattern

Full text of DOI:10.1016/j.bmc.2012.02.026

Bioorganic & Medicinal Chemistry 20 (2012) 2690–2700
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New insights into the structure–cytotoxicity relationship of spirostan saponins
and related glycosides
Karell Pérez-Labrada a,b, Ignacio Brouard a, , Sara Estévez c, María Teresa Marrero c, Francisco Estévez c,
⇑
Jaime Bermejo a, Daniel G. Rivera d,
⇑
a Instituto de Productos Naturales y Agrobiología-C.S.I.C., Av. Astrofísico Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain
b Institute of Pharmacy and Food, University of Havana, San Lázaro y L, 10400 La Habana, Cuba
c Department of Biochemistry, Molecular Biology and Physiology, University of Las Palmas de Gran Canaria, Unidad Asociada C.S.I.C., Instituto Canario de Investigación del
Cáncer (ICIC), Plaza Dr. Pasteur s/n, 35016, Las Palmas de Gran Canaria, Spain
d Center for Natural Products Study, Faculty of Chemistry, University of Havana, Zapata y G, 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:
A variety of spirostan saponins and related glycosides were synthesized and evaluated for their cytotox-
icity against the human myeloid leukemia cell line (HL-60). A linear glycosylation strategy allowed for
accessing a variety of functionalization patterns at both the spirostanic and the saccharide moieties,
which provides new information regarding the structure–cytotoxicity relationship of this family of ste-
Received 2 December 2011
Revised 3 February 2012
Accepted 8 February 2012
Available online 15 February 2012
roidal glycosides. Intriguing results were achieved with respect to hecogenyl and 5
b-chacotriosides, turning out to be the former very cytotoxic and the latter no cytotoxic at all. Impor-
tantly, the partially pivaloylated b- -glucosides of 5 -hydroxy-laxogenin were the most potent cytotoxic
a-hydroxy-laxogenyl
Keywords:
Steroids
Saponins
Structure–activity relationship
Cytotoxicity
D
a
compounds among all tested glycosides. This comprises the first report on acylated spirostanyl glucosides
displaying significant cytotoxicity, and therefore, it opens up new opportunities toward the development
of saponin analogues as anticancer agents.
Ó 2012 Elsevier Ltd. All rights reserved.
HL-60 cell
1. Introduction
pattern of this steroidal nucleus on the cytotoxicity of the resulting
spirostanyl glycosides. In addition, there are certain gaps on the
Saponins1 are triterpene and steroid glycosides of important
biological and medicinal applications.1,2 Spirostan saponins are
the most abundant steroidal saponins, which bear the oligosaccha-
ride moiety directly attached to the C-3 hydroxyl group. Several of
these saponins have exhibited potent cytotoxic,3 antifungal,4
antiinflammatory,5 and antiviral activities,6 which makes them
important leads for drug development. A very promising property
of spirostan saponins is their anticancer activity, exhibiting dioscin
(1, Fig. 1) the most potent anticancer activity among the members
of this family.3f–h,7
SAR studies concerning either other types of branching or the pres-
ence of acylated functionalities in the saccharide portion.
Herein we report on the synthesis of novel spirostanic saponins
as well as on the new insights in the SAR studies derived from the
cytotoxicity evaluation of these compounds. The synthetic strategy
is directed toward the production of spirostanyl glycosides bearing
varied oxygenated functionalities on the steroidal aglycone and
different sugar moieties, including tri, di and monosaccharides
inspired in the oligosaccharide portions of natural saponins. The
cytotoxic saponins dioscin (1) and filiasparoside A3d (2) were cho-
sen as models for the design of the new analogs. Figure 1 depicts
the structural features of the saponins chosen to be synthesized
and biologically tested for their cytotoxicity against human leuke-
mia cell line (HL-60). It has been proposed that the presence of
Dioscin (1) is a diosgenyl glycoside endowed with a trisaccha-
ride moiety—namely chacotrioside—including two
residues attached at positions 2 and 4 of the inner
a
-
L
-rhamnosyl
D
-glucose. Pre-
viously, structure–activity relationship (SAR) studies based on
dioscin (1) have focused either on the variations of the oligosaccha-
ride moiety,8,6a,3a,b the replacement of the glycosidic linkage by an
heterocyclic ring,9 or the substitution of the spirostanic skeleton by
other steroidal or triterpenic aglycones.6a,10 However, little infor-
mation is available regarding the effect of the functionalization
L
-rhamnose units facilitates the cellular uptake of saponins, prob-
ably due to a better interaction with lectins.3a Accordingly, we
were prompted to accomplish the synthesis of B and C-ring func-
tionalized spirostanyl glycosides not only including the 2,4-
branched trisaccharide moiety of dioscin (i.e., chacotrioside) but
also b-
residues either at position 2 or at positions 4 and 6. The interest
on the -rhamnopyranosyl-(1?2)-b- -glucopyranoside saponins
derives from previous SAR studies, which have shown that the
D-glucopyranosides branched with a-L-rhamnopyranosyl
a
-L
D
⇑
Corresponding authors. Tel.: +53 7 8702102; fax: +53 7 8733502 (D.G.R.).
E-mail addresses: ibrouard@ipna.csic.es (I. Brouard), dgr@fq.uh.cu (D.G. Rivera).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.026

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2691
O
OH
O
O
O
O
HO
O
OH
OH
O
4
O
O
O
HO
4
2
6
O
O
HO
HO
HO
O
O
O
O
Dioscin (1)
HO
Filiasparoside A (2)
H
HO
O
OH
OH
HO
HO
HO
OH
OH
O
OH
OH
R2
OH
OH
4
O
O
O
O
O
HO
HO
2
O
6
HO
2
HO
O
S
O
4
O
O
HO
O
O
HO
HO
HO
SO
O
R1
R1 or R2 = OH, O
O
HO
HO
OH
HO
OH
HO
HO
OH
HO
Figure 1. Natural spirostan saponins and their synthetic analogs.
corresponding diosgenyl disaccharide displays cytotoxic activity
against HL-60 as high as that of dioscin.8b,g On the other hand,
41% and 43% overall yields. Importantly, the overall yields of these
b-chacotriosides were higher than the 32% overall yield reported
for the synthesis of dioscin.18
Additionally, we turned to the synthesis of further saponins
with the same hecogenyl and 5a-hydroxy-laxogenyl aglycones
the interest on the
anosyl-(1?6)]-b- -glucopyranoside scaffold is inspired on filias-
paroside A, which possess the same type of trisaccharide
connectivity but including b- -xylopyranosyl and -arabinopyr-
-rhamnosyl.
a-L-rhamnopyranosyl-(1?4)-[a-L-rhamnopyr-
D
D
a-D
but including structural variations at the oligosaccharide moiety.
Two main structural aspects were chosen for this study: (i) the
anosyl units instead of the desired
2. Results and discussion
2.1. Chemical synthesis
a-L
number and (ii) position of the
inner -glucose. In an important SAR study on diosgenyl saponins,
Mimaki and co-workers reported that while diosgenyl b- -gluco-
side shows no cytotoxic activity against HL-60 cells, the incorpora-
tion of -rhamnosyl at C-2 of the glucosyl unit leads to a
disaccharide saponin with potent cytotoxicity. In contrast, when
-rhamnose is attached to C-3 or C-4 of -glucose, the resulting
diosgenyl saponins do not exhibit significant cytotoxicity.8g In con-
sequence, we decided to obtain spirostan saponins with the
rhamnose joined to C-2 of the b- -glucoside. Alternatively, we
were also interested on the production of novel spirostan saponins
with -rhamnosyl including 4,6-branched trisaccharide moieties,
L-rhamnosyl units attached to the
D
D
a-L
Taking dioscin (1) and filiasparoside A (2) as lead structures, we
first decided to evaluate the influence of the functionalization at
the steroidal aglycone in the cytotoxicity of the resulting spirostan
saponins. Interestingly, despite the importance that has been
attributed to the spirostanic nature of the steroidal aglycone,10a,d
detailed SAR studies focused on assessing the effect of oxygenated
functionalities at different positions of such a skeleton have re-
mained elusive so far. For this, the available spirostan sapogenins
L
D
L-
D
L
which have been neither synthesized nor evaluated for their cyto-
toxic activity.
As shown in Scheme 2, the access to the mentioned saponins
required of more elaborated routes including several protection/
hecogenin (3) and 5a-hydroxy-laxogenin (4) were selected for
the introduction of the b-chacotrioside moiety, thus producing C
and B-ring functionalized analogs of the naturally occurring dios-
cin (1). Hecogenin is the aglycone of the cytotoxic filiasparoside
deprotection procedures. Thus, the b-D-glucosides 8 and 9 were
A and possesses a carbonyl functionality at C-12, whilst 5a-hydro-
capped at positions 4 and 6 as benzylidene acetals and next sub-
jected to selective pivaloylation of the OH-3 groups to afford com-
pounds 19 and 20 in 63% and 58% yields, respectively, over the
two steps. Glycosylation of acceptors 19 and 20 with donor 12
according to the Schmidt’s inverse procedure17 (i.e., the promotor
and the acceptor are first mixed and then added to the trichloro-
xy-laxogenin is a synthetic derivative of diosgenin having a ketol
functionality on ring B.11 Both sapogenins were subjected to a lin-
ear glycosylation strategy in order to produce exclusively the 1,2-
trans-glycosidic linkage between the glucose and the steroid.12
As shown in Scheme 1, glycosylation of sapogenins 3 and 4 with
2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl trichloroacetimidate
(5)13 in the presence of trimethylsilyl trifluoromethanesulfonate
(TMSOTf)14 provided the desired 3-O-b-glucopyranosides 6 and 7
in 91% and 94%, respectively. Remotion of the benzoyl groups un-
der typical Zemplen conditions (NaOMe/MeOH) afforded the
acetimidate donor) led to the fully protected
a-(1?2)-linked spi-
rostan disaccharide only in moderate yields (i.e., about 50%), as a
result of the partial cleavage of the benzylidene acetal. Attempts
to improve the glycosylation efficiency with the use of the
Schmidt’s glycosylation procedure19 (i.e., the promotor is added
to the mixture of the acceptor and the trichloroacetimidate do-
nor) were successful, with increments in the reaction yields up
to about 80%. Finally, a two-step deprotection protocol afforded
saponins 21 and 22 in 75% and 77% yields, respectively, over
the three steps.
Alternatively, synthesis of the 4,6-branched trisaccharide sapo-
nins encompassed acetylation of the 4,6-O-benzylidene-b-D-gluco-
sides 17 and 18, followed by cleavage of the acetal group to afford
acceptors 23 and 24 in good yields. Subsequent glycosylation with
deprotected b-D-glucosides 8 and 9. These intermediates were then
subjected to selective pivaloylation at OH-3 and OH-6 according to
a reported procedure15 to furnish compounds 10 and 11 in 65% and
63%, respectively. The 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl tri-
chloroacetimidate (12)16 was next employed as donor for the dou-
ble glycosylation step following the Schmidt’s inverse procedure,17
which produced the protected spirostanyl trisaccharide 13 and 14
in 72% and 75% yields, respectively. Global deprotection furnished
the hecogenyl and 5a-hydroxy-laxogenyl saponins 15 and 16 in

2692
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
OBz
O
O
R3
R3
O
BzO
BzO
O
O
BzO
O
CCl3
NH
OR4
O
5
R4O
R4O
HO
O
R1
R2
R1R2
a
OR4
Hecogenin, 3, R1 = R2 = H, R3 = O
5α-Hydroxy-laxogenin, 4, R1 = OH, R2 = O, R3 = H
6, R4 = Bz, 91% from 3
7, R4 = Bz, 94% from 4
b
8, R4 = H, 99% from 6
9, R4 = H, 98% from 7
R3
O
NH
CCl3
c
R3
O
O
O
O
OR4
O
AcO
12
OPiv
O
AcO
O
OAc
O
O
R4O
HO
PivO
d
R1R2
O
R5O
R5O
O
R1
O
R2
OH
O
13, R4 = Piv, R5 = Ac, 72% from 10
14, R4 = Piv, R5 = Ac, 75% from 11
15, R4 = R5 = H, 97% from 13
16, R4 = R5 = H, 96% from 14
R5O
5
R O
10, 65% from 8
11, 63% from 9
R5O
OR5
e
Scheme 1. Synthesis of b-chacotrioside-based spirostan saponins functionalized on rings B and C. Reagents and conditions: (a) TMSOTf, 4 Å MS, CH2Cl2, rt; (b) NaOMe, MeOH;
(c) PivCl, Py, ꢀ15 °C?rt; (d) BF3ꢁEt2O, CH2Cl2, 4 Å MS, ꢀ78 °C?rt; (e) aq NaOH, THF/MeOH, 50 °C.
O
R3
O
O
R3
O
R3
O
O
OH
O
a
b
O
O
HO
HO
Ph
Ph
O
O
O
O
O
O
O
R1
R2
R1
HO
PivO
R1
R2
R2
OH
OH
OH
8, R1 = R2 = H, R3 = O
9, R1 = OH, R2 = O, R3 = H
17, 94% from 8
18 ,92% from 9
19, 67% from 17
20, 63% from 18
f), d)
c)12, d), e)
O
O
R3
O
R3
O
OR4
O
R3
OR4
OR4
O
O
OH
O
O
OH
HO
HO
O
O
O
R1
R2
c) 12
O
O
HO
O
R4O
O
R1
AcO
R1
R2
R4O
R4O
R4O
R2
OR4
O
OAc
O
HO
25, R4 = Ac, 69% from 23
26, R4 = Ac, 66% from 24
23, 88% from 17
24, 81% from 18
21, 75% from 19
22, 77% from 20
HO
OH
g
27, R4 = H, 98% from 25
28, R4 = H, 95% from 26
Scheme 2. Synthesis of disaccharide and trisaccharide spirostan saponins including L-rhamnosyl units at different positions. Reagents and conditions: (a) PhCH(OMe)2, p-
TsOH, DMF, 50 °C; (b) PivCl, Py, ꢀ15 °C?rt; (c) BF3ꢁEt2O, CH2Cl2, 4 Å MS, ꢀ78 °C?rt; (d) p-TsOH, THF/MeOH (1:1, v/v); (e) aq NaOH, THF/MeOH, 50 °C; (f) Ac2O, pyridine,
0 °C?rt; (g) NaOMe, MeOH.
donor 12 gave the fully protected spirostan saponins 25 and 26 in
69% and 66% yields, respectively. Global deprotection with NaOMe
in MeOH furnished the final saponins 27 and 28 in 56% and 47%
overall yields, respectively.
As previously reported for diosgenyl b-
ogous hecogenyl b- -glucoside 8 and 5
glucoside showed no significant cytotoxic activity (IC50
>100 M). However, further incorporation of pivaloyl groups at
OH-3 and OH-6 of the -glucose unit of both compounds led to
D
-glucoside,8g their anal-
-hydroxy-laxogenyl b-
D
a
D-
9
l
D
2.2. Biological studies
the appearance of cytotoxicity in glucosides 10 and 11, being this
latter compound the most potent one among all tested glycosides.
Table 1 shows the results of the in vitro cytotoxic activities of
the spirostanyl glycosides against the human myeloid leukemia
HL-60 cell line, as determined by the 3-[4,5-dimethylthiazol-2-
yl-]-2,5-diphenyl tetrazolium bromide (MTT) dye-reduction assay.
Compounds 11, 15 and 20 were the most cytotoxic ones, with IC50
Intriguingly, the partially protected hecogenyl b-
D
-glucoside 19
-gluco-
exhibited low activity whilst the 5 -hydroxy-laxogenyl b-
a
D
side 20 was very cytotoxic against HL-60 cells, displaying the same
behaviour that their congeners 10 and 11, respectively. We may
reason about the effect of such acyl and acetal groups on the bio-
activity of these compounds, since this is the first report in the
values in the order of that of dioscin (1, IC50 3.8 l
M).8g

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2693
Table 1
Cytotoxicity of spirostanyl glycosides against HL-60 cellsa
R1
R2
R3
R4
IC50
(l
M)
R1
R2
R3
R4
IC50
(lM)
8
H
H
H
H
>100
9
H
H
H
H
>100
10
15
19
21
27
H
Rha
H
Rha
H
Piv
H
Piv
H
H
Rha
Piv
H
12 3.5
4.3 1.0
45 8.5
>100
11
16
20
22
28
H
Rha
H
Rha
H
Piv
H
Piv
H
H
Rha
Piv
H
2.1 0.5
>100
3.3 2.2
86.7 10.4
>100
4,6-O-Benzylidene
H
Rha
4,6-O-Benzylidene
H
Rha
H
Rha
H
Rha
H
>100
H
a
Cells were cultured for 72 h and the IC50 values were calculated as described in Section 4.
literature regarding the cytotoxic activity of pivaloylated (or any
other type of acylated) and benzylidene-protected spirostanyl glu-
cosides. Indeed, the lipophilicity of the sugar moiety with some hy-
droxyl groups capped as esters and acetal is significantly increased,
but the more or less hydrophobic nature of the steroidal aglycone
must be also considered. It turns out that there are varied issues
influencing this type of bioactivity: (i) the nature and (ii) position
of the capping functionalities in the inner D-glucose, and (iii) the
functionalization level of the spirostanic skeleton. Such structural
factors might be evaluated in further SAR studies focused exclu-
sively on spirostanyl glucosides with dissimilar protecting groups
at the sugar moiety and varied functionalization patterns in the
spirostanic aglycone.
Analysis of Table 1 also provides important information regard-
ing the effect of the number and position of the L-rhamnose units
as well as the B and C-ring functionalization on the cytotoxicity
of the spirostan saponins. Hecogenyl chacotrioside 15, which is
an hydrid saponin analogue integrating structural elements from
both dioscin (1) and filiasparoside A (2),3d proved to be as cytotoxic
Figure 2. (A) Effect of compound 11 on human HL-60 cell viability. Cells were
cultured in the presence of the indicated concentrations of 11 for 72 h, and
thereafter cell viability was determined by the MTT dye-reduction assay. The
results of a representative experiment are shown. Each point represents the mean
of triplicate determinations. (B) Photomicrographs of representative fields of HL-60
cells in culture using an inverted phase contrast microscope after treatment with a
10 lM solution of compound 11 for 24 h.
as those natural saponins. Contrarily, 5a-hydroxy-laxogenyl chac-
otrioside 16, which is a B-ring functionalized dioscin analogue,
showed no cytotoxicity against HL-60 cells. Although further SAR
studies must be carry out to achieve general conclusions regarding
the effect of further functionalizations on the cytotoxicity of chac-
otrioside saponins, it seems that the incorporation of polar oxygen-
ated groups into ring B leads to a significant reduction of the
cytotoxic activity of spirostanyl chacotriosides. These results are
intriguing when compared with the cytotoxicity of the pivaloylat-
ed glucosides of the same aglycones, which exhibit exactly the
opposite biological behavior, that is, being the B-ring functional-
ized spirostanyl glucoside more cytotoxic than the C-ring function-
alized one.
(i.e., 11) was chosen to explore its mechanism of action and to
assess whether it is similar to that reported for other spirostan
saponins.8b Figure 2 shows the effect of compound 11 on human
HL-60 cell viability, as illustrated by a representative IC50 plot in
HL-60 cell (Fig. 2A). It also reveals that this glucoside induces
important morphological changes and inhibits cellular growth, as
visualized by phase contrast microscopy (Fig. 2B).
In order to evaluate whether compound 11 decreases cell viabil-
ity through activation of apoptosis, morphological changes charac-
teristic of apoptotic cells (condensed and fragmented chromatin)
were analysed by fluorescent microscopy. As shown in Figure 3A,
compound 11 induces the appearance of condensed and frag-
mented chromatin, while control HL-60 cells appeared normal
with the nuclei round and homogeneous. Moreover, the percentage
of hypodiploid cells (i.e., sub-G1 fraction) increased about seven-
fold in compound 11-treated HL-60 compared with control cells
It is also interesting to note that neither the disaccharide sapo-
nins 21 and 22 nor the trisaccharide saponins 27 and 28 showed
significant cytotoxic activity. For the case of the disaccharide sap-
onins, the lack of cytotoxicity against HL-60 cells contrasts with
the considerable cytotoxicity (IC50 2.5
nin having the same disaccharide portion,8g that is,
pyranosyl-(1?2)]-b- -glucopyranoside. As shown before, this
lM) of the diosgenyl sapo-
after 24 h exposure at a concentration of 10 lM (Fig. 3B).
a-L-rhamno-
Apoptosis can occur with or without the activation of caspases,
a family of cysteine proteases which are constitutively expressed
as zymogens. To determine whether caspases were involved in
the response of the cells to compound 11, we examined whether
this compound induces caspases activation. To this end, HL-60 cells
were treated with increasing concentrations of compound 11 and
cell lysates were assayed for cleavage of the tetrapeptide sub-
strates DEVD-pNA, IETD-pNA and LEHD-pNA as specific substrates
for caspase-3/7, caspase-8 and caspase-9, respectively. As shown in
Figure 4, induction of both initiator caspases (caspases-8 and -9)
D
latter information suggests that the cytotoxicity of spirostanyl gly-
cosides depends not only of isolated structural factors but of com-
bination of properties at both the saccharide and the steroidal
moieties. Indeed, much needs to be done to achieve concluding
evidences regarding the structural elements influencing the cyto-
toxic activity of spirostanyl glycosides.
In order to get deeper insights into the cytotoxic effect of the
pivaloylated spirostanyl glycosides, the most active compound

2694
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
Figure 3. (A) Photomicrographs of representative fields of HL-60 cell stained with
Hoechst 33258 to evaluate nuclear chromatin condensation. (B) Cells were
incubated with a 10 lM solution of compound 11 for 24 h and subjected to flow
cytometry using propidium iodide labelling. Hypodiploid cells (apoptotic cells) are
shown in region marked with an arrow.
and the executioner caspase-3/7 activities was significantly detect-
able after 24 h of treatment.
3. Conclusions
We have implemented a linear glycosylation strategy to pro-
duce novel spirostan saponins with structural variations at both
the saccharidic and steroidal moieties. The cytotoxicity evaluation
of the final saponins and the partially protected spirostanyl gluco-
sides provides important information regarding some of the struc-
tural elements required for the cytotoxic activity against HL-60
cells. Such a biological assessment showed that the synthetic hec-
ogenyl saponin including the b-chacotrioside residue was as potent
as the natural ones dioscin and filiasparoside A. However, the b-
Figure 4. Activation of caspase-9, -8 and 3/7 in response to compound 11. HL-60
and U937 cells were treated with the indicated concentrations of compound 11 and
harvested at 24 h. Cell lysates were assayed for caspase-9, -8 and -3/7 activities
using the LEHD-pNA, IETD-pNA and DEVD-pNA colorimetric substrates, respec-
tively. Results are expressed as fold increase in caspase activity relative to control.
Values represent the mean SE; this histogram is representative of two indepen-
dent experiments, each one performed in duplicate.
chacotrioside saponin derived from 5a-hydroxy-laxogenin was
not cytotoxic; suggesting that the more hydrophilic nature of this
aglycone compared that of dioscin (i.e., diosgenin) leads to a
significant drop in the bioactivity. Interestingly, the pivaloylated
lution 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 chromatography was carried out using Silica Gel 60
(230–400 mesh) and analytical thin layer chromatography (TLC)
was performed using silica gel aluminum sheets. All commercially
available chemicals were used without further purification. Hecog-
b-D-glucosides of 5a-hydroxy-laxogenin showed very high cyto-
toxicity, being the first report describing potent cytotoxic activity
for any type of partially or fully acylated spirostanyl glucosides.
Detailed antiproliferative studies with the more active pivaloylated
b-D-glucoside proved that this type of compound exerts cell growth
inhibition effect similar to that of previously reported diosgenyl
saponins. We believe that these results are of outmost importance
for a better understanding of the cytotoxic activity of spirostan
saponins and related glycosides, as they provide new information
on the structure–activity relationship of these amphipathic
molecules.
enin (3) is a commercially available spirostan sapogenin and 5
hydroxy-laxogenin (4) was obtained as described in Ref. 11d
a-
4.1.1. Hecogenyl 2,3,4,6-tetra-O-benzoyl-b-D-glucopyranoside
(6)
The glucosyl donor 5 (1.29 g, 1.75 mmol), hecogenin 3 (579 mg,
1.34 mmol) and 4 Å molecular sieves were suspended in dry
CH2Cl2 (25 mL) and the mixture was stirred at room temperature
under nitrogen atmosphere for 15 min. A solution of TMSOTf
4. Experimental
4.1. General
(12 lL, 0.067 mmol) in CH2Cl2 (2.4 mL) was added and the reaction
mixture was stirred for 1 h. The reaction was quenched by addition
of Et3N and then filtered. The filtrate was evaporated to dryness
and the crude product was purified by flash column chromatogra-
phy (n-hexane/EtOAc 4:1) to afford 6 (1.23 g, 91%) as a white solid.
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 mil-
lion relative to the residual solvent signals, and coupling constants
(J) are reported in hertz. NMR peak assignments were accom-
plished by analysis of the 1H–1H COSY and HSQC data. High reso-
Rf = 0.30 (n-hexane/EtOAc 3:1). Mp: 144–146 °C. ½a D20
ꢂ
+26.1 (c 1.7,
CHCl3). IR (KBr, cmꢀ1
) mmax: 2928, 2871, 2377, 1729, 1266, 1109,
1097, 1069, 1027. 1H NMR (400 MHz, CDCl3): d = 0.77 (s, 3H,

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2695
CH3); 0.78 (d, 3H, J = 7.0 Hz, CH3); 1.02 (s, 3H, CH3); 1.06 (d, 3H,
J = 6.4 Hz, CH3); 2.34 (t, 1H, J = 13.7 Hz); 2.51 (dd, 1H, J = 6.8/
8.5 Hz); 3.34 (t, 1H, J = 11.0 Hz, H-26ax); 3.48 (m, 1H, H-26eq);
umn chromatography (n-hexane/EtOAc 2:1) to afford diol 10
(416 mg, 65%) as a white solid. Rf = 0.30 (n-hexane/EtOAc 2:1).
Mp: 185–187 °C. ½a D20
ꢂ
ꢀ5.0 (c 1.4, CHCl3). IR (KBr, cmꢀ1
) mmax:
3.57 (m, 1H, H-3
a
); 4.13–4.18 (m, 1H, H-5 Glc); 4.30–4.36 (m,
3447, 2958, 2930, 2873, 2373, 2346, 1717, 1458, 1285, 1161,
1076, 1040. 1H NMR (400 MHz, CDCl3): d = 0.77 (d, 3H, J = 6.3 Hz,
CH3); 0.88 (s, 3H, CH3); 1.03 (s, 3H, CH3); 1.04 (d, 3H, J = 6.9 Hz,
CH3); 1.19, 1.22 (2 ꢃ s, 2 ꢃ 9H, 2 ꢃ (CH3)3C); 2.19 (dd, 1H, J = 5.0/
14.2 Hz); 2.38 (t, 1H, J = 13.9 Hz); 2.49 (dd, 1H, J = 6.6/8.8 Hz);
3.33 (t, 1H, J = 10.9 Hz, H-26ax); 3.41–3.46 (m, 3H, H-2 Glc, H-4
1H, H-16 ); 4.54 (dd, 1H, J = 6.1/12.1 Hz, H-6a Glc); 4.59 (dd, 1H,
a
J = 3.5/12.0 Hz, H-6b Glc); 4.92 (d, 1H, J = 7.8 Hz, H-1 Glc); 5.48
(dd, 1H, J = 7.9/9.7 Hz, H-2 Glc); 5.61 (t, 1H, J = 9.7 Hz, H-4 Glc);
5.89 (t, 1H, J = 9.7 Hz, H-3 Glc); 7.27–7.44 (m, 9H, Ar-H); 7.47–
7.57 (m, 3H, Ar-H); 7.83 (dd, 2H, J = 1.2/8.3 Hz, Ar-H); 7.90 (dd,
2H, J = 1.1/8.4 Hz, Ar-H); 7.95 (dd, 2H, J = 1.2/8.4 Hz, Ar-H); 8.00
(dd, 2H, J = 1.2/8.4 Hz, Ar-H). 13C NMR (100 MHz, CDCl3): d = 11.8,
13.2, 15.9, 17.1 (CH3); 28.2, 28.8, 29.1 (CH2); 30.2 (CH); 31.1,
31.4, 31.5 (CH2); 34.3 (CH); 34.4 (CH2); 36.1 (C); 36.3, 37.7 (CH2);
42.2, 44.5, 53.6 (CH); 55.1 (C); 55.5, 55.8 (CH); 63.4, 66.9 (CH2);
70.2, 72.1, 72.2, 73.1, 79.2, 79.8, 100.3 (CH); 109.3 (C); 128.2,
128.3, 128.4, 128.5 (CH); 128.8, 128.9, 129.4, 129.5 (C); 129.6,
129.7, 129.8, 129.9, 133.0, 133.1, 133.4 (CH); 165.0, 165.3, 165.8,
166.0, 213.4 (C@O).HRMS (ESI-FT-ICR) m/z: 1031.4542 [M+Na]+
(calcd for C61H68O13Na: 1031.4558).
Glc, H-26eq); 3.54–3.63 (m, 2H, H-3
a, H-5 Glc); 4.22 (dd, 1H,
J = 6.9/11.7 Hz, H-6a Glc); 4.30–4.34 (m, 1H, H-16
a
); 4.40–4.43
(m, 2H, H-6b Glc, H-1 Glc); 4.86 (t, 1H, J = 9.3 Hz, H-3 Glc). 13C
NMR (100 MHz, CDCl3): d = 11.9, 13.2, 15.9, 17.1, 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); 38.8, 38.9 (C); 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, 213.4
(C@O). HRMS (ESI-FT-ICR) m/z: 783.4655 [M+Na]+ (calcd for
C43H68O11Na: 783.4659).
4.1.2. 5
a
-Hydroxy-laxogenyl 2,3,4,6-tetra-O-benzoyl-b-
D
-
4.1.4. 5a-Hydroxy-laxogenyl 3,6-di-O-pivaloyl-b-D-
glucopyranoside(7)
glucopyranoside (11)
The glucosyl donor 5 (1.29 g, 1.75 mmol), 5
a
-hydroxy-laxoge-
Glucoside 7 (773 mg, 0.75 mmol) was deprotected with NaOMe
in CH2Cl2/MeOH (36 mL, 1:1, v/v) in a similar way as described in
the synthesis of 8 to furnish 9 (435 mg, 98%). This compound was
dissolved in anhydrous pyridine (25 mL) and the solution was
cooled to ꢀ15 °C under nitrogen atmosphere. Pivaloyl chloride
(0.46 mL, 3.75 mmol) was added dropwise and the reaction course
was monitored by TLC. The stirring was continued at room temper-
ature until disappearance of the intermediate. The 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 anhy-
drous Na2SO4 and evaporated to dryness. The crude product was
purified by flash column chromatography (n-hexane/EtOAc 3:2)
to afford diol 11 (367 mg, 63%) as a white solid. Rf = 0.23 (n-hex-
nyl 4 (600 mg, 1.34 mmol) and TMSOTf (12
lL, 0.067 mmol) were
reacted in dry CH2Cl2 (25 mL) in a similar way as described in
the synthesis of 6. Flash column chromatography purification (n-
hexane/EtOAc 5:2) afforded 7 (1.29 g, 94%) as a white solid.
Rf = 0.25 (n-hexane/EtOAc 5:2). Mp: 153–155 °C. ½a D20
ꢀ38 (c 2.0,
ꢂ
CHCl3). IR (KBr, cmꢀ1
) mmax: 3503, 2947, 2871, 2372, 2346, 1725,
1266, 1105, 1093, 1069, 1026. 1H NMR (400 MHz, CDCl3):
d = 0.70 (s, 3H, CH3); 0.73 (s, 3H, CH3); 0.78 (d, 3H, J = 6.4 Hz,
CH3); 0.97 (d, 3H, J = 6.8 Hz, CH3); 2.10 (dd, 1H, J = 4.2/13.1 Hz);
2.65 (t, 1H, J = 12.5 Hz); 3.35 (t, 1H, J = 10.9 Hz, H-26ax); 3.47 (m,
1H, H-26eq); 3.99 (m, 1H, H-3a); 4.10–4.16 (m, 1H, H-5 Glc);
4.36–4.42 (m, 1H, H-16 ); 4.53 (dd, 1H, J = 5.7/12.0 Hz, H-6a
a
Glc); 4.60 (dd, 1H, J = 3.4/12.0 Hz, H-6b Glc); 4.95 (d, 1H,
J = 7.8 Hz, H-1 Glc); 5.49 (dd, 1H, J = 7.8/9.8 Hz, H-2 Glc); 5.63 (t,
1H, J = 9.7 Hz, H-4 Glc); 5.87 (t, 1H, J = 9.6 Hz, H-3 Glc); 7.25–
7.44 (m, 9H, Ar-H); 7.47–7.56 (m, 3H, Ar-H); 7.82 (dd, 2H, J = 1.3/
8.5 Hz, Ar-H); 7.90 (dd, 2H, J = 1.2/8.4 Hz, Ar-H); 7.95 (dd, 2H,
J = 1.3/8.4 Hz, Ar-H); 8.00 (dd, 2H, J = 1.3/8.4 Hz, Ar-H). 13C NMR
(100 MHz, CDCl3): d = 13.9, 14.4, 16.3, 17.1 (CH3); 21.2, 28.3,
28.7, 29.5 (CH2); 30.2 (CH); 31.3, 31.5, 33.1 (CH2); 36.6 (CH); 39.6
(CH2); 41.0 (C); 41.6 (CH); 41.7 (CH2); 42.3 (C); 44.5, 56.1, 62.0
(CH); 63.3, 66.8 (CH2); 70.0, 72.0, 72.1, 73.0, 75.8 (CH); 80.1 (C);
80.4, 99.9 (CH); 109.2 (C); 128.2, 128.4 (CH); 128.7, 128.8, 129.3,
129.6 (C); 129.7, 129.8, 129.9, 133.1, 133.2, 133.4 (CH); 165.1,
165.2, 165.8, 166.1, 211.0 (C@O). HRMS (ESI-FT-ICR) m/z:
1047.4504 [M+Na]+ (calcd for C61H68O14Na: 1047.4507).
ane/EtOAc 3:2). Mp: 199–201 °C. ½a D20
ꢀ82.8 (c 1.8, CHCl3). IR
ꢂ
(KBr, cmꢀ1
) mmax: 3447, 2954, 2932, 2911, 2873, 2371, 2346,
1716, 1458, 1288, 1166, 1076, 1051, 1035. 1H NMR (400 MHz,
CDCl3): d = 0.74 (s, 3H, CH3); 0.78 (d, 3H, J = 6.6 Hz, CH3); 0.79 (s,
3H, CH3); 0.96 (d, 3H, J = 6.9 Hz, CH3); 1.20, 1.23 (2 ꢃ s, 2 ꢃ 9H,
2 ꢃ (CH3)3C); 2.73 (dd, 1H, J = 12.0/12.8 Hz); 3.00, 3.09, 3.24
(3 ꢃ s, 3 ꢃ 1H, 3 ꢃ OH); 3.35 (t, 1H, J = 10.9 Hz, H-26ax); 3.41–
3.48 (m, 3H, H-26eq, H-2 Glc, H-4 Glc); 3.56–3.61 (m, 1H, H-5
Glc); 3.98 (m, 1H, H-3
a); 4.21 (dd, 1H, J = 6.7/11.8 Hz, H-6a Glc);
4.37–4.46 (m, 3H, H-16
a,
H-6b Glc, H-1 Glc); 4.88 (t, 1H,
J = 9.3 Hz, H-3 Glc). 13C NMR (100 MHz, CDCl3): d = 13.9, 14.4,
16.4, 17.1 (CH3); 21.2 (CH2); 27.0, 27.1 (CH3); 28.6, 28.8, 29.6
(CH2); 30.3 (CH); 31.4, 31.5, 33.3 (CH2); 36.7 (CH); 38.9, 39.0 (C);
39.6 (CH2); 41.1 (C); 41.6 (CH); 41.8 (CH2); 42.5 (C); 44.4, 56.1,
62.1 (CH); 63.7, 66.9 (CH2); 70.1, 72.2, 74.1, 76.5, 77.8 (CH); 80.2
(C); 80.5, 102.2 (CH); 109.2 (C); 178.7, 180.2, 212.7 (C@O). HRMS
(ESI-FT-ICR) m/z: 799.4595 [M+Na]+ (calcd for C43H68O12Na:
799.4608).
4.1.3. Hecogenyl 3,6-di-O-pivaloyl-b-D-glucopyranoside(10)
NaOMe was added to a solution of compound 6 (850 mg,
0.84 mmol) in CH2Cl2/MeOH (40 mL, 1:1, v/v) until pH was set to
9–10. The reaction mixture was stirred overnight at room temper-
ature, and then neutralized with acid resin Dowex-50 (H+) and fil-
tered. The filtrate was concentrated under reduced pressure and
the residue was washed with Et2O (5 ꢃ 10 mL) and dried in vacuo
to furnish 8 (506 mg, 99%). This product was dissolved in anhy-
drous pyridine (25 mL) and the solution was cooled to ꢀ15 °C un-
der nitrogen atmosphere. Pivaloyl chloride (0.52 mL, 4.2 mmol)
was added dropwise and the reaction course was monitored by
TLC. The stirring was continued at room temperature until the dis-
appearance of the intermediate. The 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 col-
4.1.5. Hecogenyl 2,4-di-O-(2,3,4-tri-O-acetyl-
rhamnopyranosyl)-3,6-di-O-pivaloyl-b- -glucopyranoside (13)
BF3ꢁEt2O (150 L, 1.195 mmol) was added under nitrogen atmo-
sphere to stirring suspension of acceptor 10 (206.7 mg,
a-L-
D
l
a
0.272 mmol) and 4 Å molecular sieves in dry CH2Cl2 (40 mL) at
ꢀ80 °C. After stirring for 1 h at this temperature, a solution of
rhamnopyranosyl trichloroacetimidate 12 (366 mg, 0.842 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).

2696
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
The organic phase was dried over anhydrous Na2SO4 and evapo-
rated to dryness. The crude product was purified by flash column
chromatography (n-hexane/EtOAc 2:1) to afford 13 (255.7 mg,
72%) as a white solid. Rf = 0.24 (n-hexane/EtOAc 2:1). Mp: 134–
4.1.7. Hecogenyl 2,4-di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranoside(15)
An aqueous solution of NaOH (1 M, 1 mL) was added to a solu-
tion of compound 13 (198 mg, 0.152 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 filtrate was concentrated un-
der reduced pressure and the resulting crude product was purified
by flash column chromatography (CHCl3/MeOH 5:1) to afford 15
(130 mg, 97%) as a white solid. Rf = 0.29 (CHCl3/MeOH 5:1). Mp:
135 °C. ½a 2D0
ꢂ
ꢀ45.0 (c 1.4, CDCl3). IR (KBr, cmꢀ1
) mmax: 2959, 2934,
2874, 2371, 2346, 1749, 1458, 1374, 1369, 1244, 1223, 1147,
1076, 1041. 1H NMR (400 MHz, CDCl3): d = 0.77 (d, 3H, J = 6.3 Hz,
CH3); 0.87 (s, 3H, CH3); 1.03 (s, 3H, CH3); 1.05 (d, 3H, J = 6.9 Hz,
CH3); 1.13 (d, 3H, J = 6.3 Hz, CH3 Rha); 1.15 (s, 9H, (CH3)3C); 1.16
(d, 3H, J = 6.2 Hz, CH3 Rha0); 1.20 (s, 9H, (CH3)3C); 1.93, 1.95, 2.00,
2.02, 2.08, 2.10 (6 ꢃ s, 6 ꢃ 3H, 6 ꢃ CH3CO); 2.21 (dd, 1H, J = 4.9/
278–279 °C. ½a 2D0
ꢂ
ꢀ91.4 (c 1.4, C5H5N). IR (KBr, cmꢀ1
) mmax: 3420,
14.3 Hz, H-11b); 2.37 (t, 1H, J = 14.1 Hz, H-11a); 2.49 (dd, 1H,
2928, 2864, 2371, 2345, 1701, 1654, 1560, 1050. 1H NMR
(500 MHz, pyridine-d5): d = 0.71 (d, 3H, J = 6.0 Hz, CH3); 0.90 (s,
3H, CH3); 1.10 (s, 3H, CH3); 1.37 (d, 3H, J = 6.9 Hz, CH3); 1.64 (d,
3H, J = 6.0 Hz, CH3 Rha); 1.76 (d, 3H, J = 6.3 Hz, CH3 Rha0); 2.13
(m, 1H); 2.28 (dd, 1H, J = 5.0/14.2 Hz); 2.44 (t, 1H, J = 13.7 Hz);
2.78 (dd, 1H, J = 6.7/8.6 Hz); 3.50 (t, 1H, J = 10.7 Hz, H-26ax); 3.60
J = 6.8/8.7 Hz); 3.33 (t, 1H, J = 11.0 Hz, H-26ax); 3.46–3.49 (m, 1H,
H-26eq); 3.59–3.70 (m, 3H, H-2 Glc, H-4 Glc, H-3 ); 3.74–3.78
(m, 1H, H-5 Glc); 3.89 (m, 1H, H-5 Rha); 4.19 (dd, 1H, J = 6.1/
11.8 Hz, H-6a Glc); 4.30–4.38 (m, 2H, H-5 Rha0, H-16
); 4.40 (dd,
a
a
1H, J = 2.2/12.0 Hz, H-6b Glc); 4.56 (d, 1H, J = 7.3 Hz, H-1 Glc);
4.81 (d, 1H, J = 1.6 Hz, H-1 Rha); 4.87 (d, 1H, J = 1.9 Hz, H-1 Rha0);
4.97–5.05 (m, 3H, H-2 Rha0, H-4 Rha, H-4 Rha0); 5.13–5.16 (m,
2H, H-2 Rha, H-3 Rha0); 5.19 (dd, 1H, J = 3.5/9.8 Hz, H-3 Rha);
5.27 (t, 1H, J = 8.7 Hz, H-3 Glc). 13C NMR (125 MHz, CDCl3):
d = 11.8, 13.2, 15.9, 17.0, 17.1, 17.2, 20.61, 20.69, 20.74, 20.76,
26.8, 27.1 (CH3); 28.4, 28.7, 29.1 (CH2); 30.1 (CH); 31.1, 31.4,
31.5, 33.8 (CH2); 34.3 (CH); 36.2 (C); 36.5, 37.7 (CH2); 38.8 (C);
42.2, 44.4, 53.5 (CH); 55.1 (C); 55.4, 55.7 (CH); 63.1 (CH2); 66.4
(CH); 66.9 (CH2); 67.9, 68.7, 68.9, 69.5, 69.9, 70.5, 71.2, 72.3,
75.1, 75.8, 76.9, 77.9, 79.1, 97.0, 97.2, 98.7 (CH); 109.2 (C); 169.5,
169.6, 169.7, 169.8, 169.9, 170.0, 176.5, 177.8, 213.3 (C@O). HRMS
(ESI-FT-ICR) m/z: 1327.6415 [M+Na]+ (calcd for C67H100O25Na:
1327.6451).
(m, 1H, H-26eq); 3.72 (m, 1H, H-5 Glc); 3.89 (m, 1H, H-3
4.11–4.14 (m, 1H, H-6a Glc); 4.20–4.40 (m, 6H, H-2 Glc, H-3 Glc,
H-4 Glc, H-6b Glc, H-4 Rha, H-4 Rha0); 4.47–4.52 (m, 1H, H-16
);
a);
a
4.55 (dd, 1H, J = 3.2/9.1 Hz, H-3 Rha); 4.61 (dd, 1H, J = 3.5/9.5 Hz,
H-3 Rha0); 4.70 (m, 1H, H-2 Rha); 4.84 (m, 1H, H-2 Rha0); 4.89–
4.95 (m, 2H, H-5 Rha, H-5 Rha0); 4.97 (d, 1H, J = 7.3 Hz, H-1 Glc);
5.86 (d, 1H, J = 1.6 Hz, H-1 Rha); 6.38 (d, 1H, J = 1.3 Hz, H-1 Rha0).
13C NMR (125 MHz, pyridine-d5): d = 12.3, 14.3, 16.5, 17.7, 18.9,
19.1 (CH3); 29.1, 29.7, 30.1 (CH2); 31.0 (CH); 31.9, 32.2, 32.3, 34.8
(CH2); 34.9 (CH); 36.9 (C); 37.1, 38.5 (CH2); 43.1, 44.9, 54.8 (CH);
55.8 (C); 56.1, 56.4 (CH); 61.9, 67.4 (CH2); 69.9, 70.9, 72.8, 72.9,
73.2, 73.3, 74.3, 74.6, 76.9, 77.0, 78.2, 78.5, 79.4, 80.1, 100.3,
102.5, 103.4 (CH); 109.8 (C); 213.2 (C@O).HRMS (ESI-FT-ICR) m/z:
907.4676 [M+Na]+ (calcd for C45H72O17Na: 907.4667).
4.1.6. 5
a
-Hydroxy-laxogenyl 2,4-di-O-(2,3,4-tri-O-acetyl-
a-L-
4.1.8. 5
-glucopyranoside (16)
Compound 14 (210 mg, 0.159 mmol) was deprotected with
a-Hydroxy-laxogenyl 2,4-di-O-(a-L-rhamnopyranosyl)-b-
rhamnopyranosyl)-3,6-di-O-pivaloyl-b-
D-glucopyranoside(14)
D
Acceptor 11 (206.5 mg, 0.266 mmol) and donor 12 (358.1 mg,
0.824 mmol) in dry CH2Cl2 (40 mL) were reacted in the presence
NaOH (1 M, 1.5 mL) in THF/MeOH (6 mL, 1:1, v/v) in a similar
was as described in the synthesis of 15. Flash column chromatog-
raphy purification (CHCl3/MeOH 4:1) afforded 16 (138 mg, 96%) as
of BF3ꢁEt2O (148
lL, 1.17 mmol) in a similar way as described in
the synthesis of 13. Flash column chromatography purification
a white solid. Rf = 0.28 (CHCl3/MeOH 4:1). Mp: 274–276 °C. ½a D20
ꢂ
(n-hexane/EtOAc 3:2) afforded 14 (351.5 mg, 75%) as a white solid.
Rf = 0.28 (n-hexane/EtOAc 3:2). Mp: 129–131 °C. ½a D20
ꢀ98.2 (c 0.9,
ꢂ
ꢀ138.6 (c 1.4, C5H5N). IR (KBr, cmꢀ1
) mmax: 3368, 2929, 2871,
CDCl3). IR (KBr, cmꢀ1
) mmax: 3503, 2956, 2871, 2372, 2346, 1749,
2372, 2346, 1702, 1654, 1560, 1131, 1043. 1H NMR (500 MHz, pyr-
idine-d5): d = 0.70 (d, 3H, J = 5.8 Hz, CH3); 0.83 (s, 3H, CH3); 0.98 (s,
3H, CH3); 1.15 (d, 3H, J = 6.9 Hz, CH3); 1.62 (d, 3H, J = 6.3 Hz, CH3
Rha); 1.79 (d, 3H, J = 6.1 Hz, CH3 Rha0); 2.36 (dd, 1H, J = 11.6/
13.4 Hz); 2.79 (dd, 1H, J = 3.8/13.9 Hz); 3.07 (t, 1H, J = 12.6 Hz);
3.40–3.44 (m, 1H, H-5 Glc); 3.49 (t, 1H, J = 10.7 Hz, H-26ax);
3.57–3.60 (m, 1H, H-26eq); 4.02–4.13 (m, 3H, H-6a Glc, H-6b Glc,
H-3 Glc); 4.21 (dd, 1H, J = 7.7/9.1 Hz, H-2 Glc); 4.30–4.37 (m, 3H,
H-4 Glc, H-4 Rha, H-4 Rha0); 4.52 (dd, 1H, J = 3.4/9.3 Hz, H-3
1374, 1246, 1225, 1146, 1053. 1H NMR (400 MHz, CDCl3):
d = 0.73 (s, 3H, CH3); 0.76 (d, 3H, J = 6.6 Hz, CH3); 0.77 (s, 3H,
CH3); 0.94 (d, 3H, J = 6.9 Hz, CH3); 1.13 (d, 3H, J = 6.3 Hz, CH3
Rha); 1.15 (s, 9H, (CH3)3C); 1.17 (d, 3H, J = 6.1 Hz, CH3 Rha0); 1.19
(s, 9H, (CH3)3C); 1.93, 1.94, 2.01, 2.04, 2.08, 2.09 (6 ꢃ s, 6 ꢃ 3H,
6 ꢃ CH3CO); 2.77 (t, 1H, J = 12.6 Hz); 3.33 (t, 1H, J = 10.9 Hz, H-
26ax); 3.43–3.46 (m, 1H, H-26eq); 3.62 (t, 1H, J = 7.3 Hz, H-2
Glc); 3.69 (t, 1H, J = 8.2 Hz, H-4 Glc); 3.83 (m, 1H, H-5 Glc); 3.87–
Rha); 4.56 (m, 1H, H-16
a
); 4.63 (dd, 1H, J = 3.4/9.4 Hz, H-3 Rha0);
); 4.76
3.93 (m, 1H, H-5 Rha); 3.96–4.02 (m, 1H, H-3
J = 6.5/11.8 Hz, H-6a Glc); 4.23–4.29 (m, 1H, H-5 Rha); 4.35–4.40
(m, 2H, H-6b Glc, H-16 ); 4.57 (d, 1H, J = 7.6 Hz, H-1 Glc); 4.86
a); 4.20 (dd, 1H,
4.67 (dd, 1H, J = 1.3/3.3 Hz, H-2 Rha); 4.74 (m, 1H, H-3
a
(dd, 1H, J = 1.4/3.4 Hz, H-2 Rha0); 4.84 (d, 1H, J = 7.7 Hz, H-1 Glc);
4.89–4.93 (m, 2H, H-5 Rha, H-5 Rha0); 5.83 (d, 1H, J = 1.3 Hz, H-1
Rha); 6.36 (d, 1H, J = 1.4 Hz, H-1 Rha0). 13C NMR (125 MHz, pyri-
dine-d5): d = 14.6, 15.4, 16.9, 17.7, 18.9, 19.1 (CH3); 22.0, 29.5,
29.7, 30.7 (CH2); 31.0 (CH); 32.3, 32.4, 33.4 (CH2); 37.4 (CH); 40.4
(CH2); 41.7 (C); 42.4 (CH); 42.6 (CH2); 43.3 (C); 45.2, 56.8 (CH);
61.8 (CH2); 63.4 (CH); 67.3 (CH2); 69.9, 70.9, 72.8, 72.9, 73.1,
74.3, 74.5, 74.6, 77.3, 78.2, 78.4, 79.1 (CH); 80.3 (C); 81.4, 100.1,
102.4, 103.3 (CH); 109.7 (C); 212.7 (C@O). HRMS (ESI-FT-ICR) m/
z: 923.4603 [M+Na]+ (calcd for C45H72O18Na: 923.4616).
a
(m, 2H, H-1 Rha, H-1 Rha0); 4.98 (t, 1H, J = 10.1 Hz, H-4 Rha);
5.01 (t, 1H, J = 9.9 Hz, H-4 Rha0); 5.05 (dd, 1H, J = 1.9/3.2 Hz, H-2
Rha); 5.11 (dd, 1H, J = 1.7/3.3 Hz, H-2 Rha0); 5.14 (dd, 1H, J = 3.2/
10.1 Hz, H-3 Rha); 5.22 (dd, 1H, J = 7.3/7.9 Hz, H-3 Glc); 5.28 (dd,
1H, J = 3.3/10.2 Hz, H-3 Rha0). 13C NMR (125 MHz, CDCl3):
d = 13.9, 14.4, 16.4, 17.0, 17.1, 17.2, 20.63, 20.65, 20.69, 20.71,
20.75 (CH3); 21.2 (CH2); 26.8, 27.1 (CH3); 28.5, 28.7, 29.7 (CH2);
30.2 (CH); 31.3, 31.5, 32.8 (CH2); 36.7 (CH); 38.7, 38.8 (C); 39.6
(CH2); 41.0 (C); 41.6 (CH); 41.8 (CH2); 42.4 (C); 44.3, 56.0, 62.0
(CH); 63.3 (CH2); 66.8 (CH); 66.9 (CH2); 68.0, 68.7, 68.9, 69.8,
69.9, 70.3, 70.4, 71.0, 71.2, 72.8, 74.0, 74.9, 76.8, 77.7 (CH); 80.1
(C); 80.5, 95.7, 97.1, 100.4 (CH); 109.2 (C); 169.6, 169.8, 169.9,
170.1, 170.2, 170.4, 176.7, 177.9, 211.4 (C@O). HRMS (ESI-FT-ICR)
m/z: 1343.6378 [M+Na]+ (calcd for C67H100O26Na: 1343.6401).
4.1.9. Hecogenyl 4,6-O-benzylidene-3-O-pivaloyl-b-D-
glucopyranoside (19)
Benzaldehyde dimethyl acetal (1.8 mL, 11.7 mmol) was added
to a solution of compound 8 (960 mg, 1.623 mmol) in anhydrous

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2697
DMF (30 mL). The pH was adjusted to 3 by addition of p-TsOHꢁH2O
and the mixture was rotated in the rotavapor under reduced pres-
sure at 50 °C for 3 h. The reaction was quenched by addition of
Et3N and the volatiles were removed under reduced pressure. Flash
column chromatography (n-hexane/EtOAc 2:3) afforded com-
pound 17 (1.04 g, 94%). A solution of 17 (519 mg, 0.763 mmol) in
anhydrous pyridine (15 mL) was cooled to ꢀ15 °C and pivaloyl
chloride (0.24 mL, 1.91 mmol) was added dropwise under nitrogen
atmosphere. The reaction course was monitored by TLC and the
stirring was continued at room temperature until disappearance
of the intermediate. The reaction was quenched with MeOH and
concentrated to dryness. The resulting crude product was purified
by flash column chromatography (n-hexane/EtOAc 3:1) to fur-
nish19 (390 mg, 67%) as a white solid. Rf = 0.29 (n-hexane/EtOAc
128.2, 128.9 (CH); 137.0 (C); 178.6, 212.2 (C@O). HRMS (ESI-FT-
ICR) m/z: 803.4299 [M+Na]+ (calcd for C45H64O11Na: 803.4346).
4.1.11. Hecogenyl 2-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranoside (21)
BF3ꢁEt2O (32
lL, 0.254 mmol) was added to a stirring suspen-
sion of 19 (161.8 mg, 0.212 mmol), rhamnopyranosyl trichloroace-
timidate 12 (138.2 mg, 0.318 mmol) and 4 Å molecular sieves in
CH2Cl2 (10 mL) at ꢀ80 °C under nitrogen atmosphere. The reaction
mixture was stirred for 2 h at room temperature and then
quenched with Et3N, filtered and concentrated to dryness. The
resulting crude product was purified by flash column chromatogra-
phy (n-hexane/EtOAc 3:1) to furnish the corresponding glycoside
(184 mg, 87%) as a white amorphous solid. Rf = 0.27(n-hexane/
EtOAc 3:1). HRMS (ESI-FT-ICR) m/z: 1059.5396 [M+Na]+ (calcd
for C57H80O17Na: 1059.5293). This compound (89 mg, 0.089 mmol)
was dissolved in THF/MeOH (2 mL, 1:1, v/v) and p-TsOHꢁH2O
(33.9 mg, 0.178 mmol) was added. The reaction mixture was stir-
red for 3 h, quenched with Et3N and the volatiles were removed
under reduced pressure. An aqueous solution of NaOH (1 mol/L,
0.2 mL) was added to a solution of the crude product in THF/MeOH
(2 mL, 1:1, v/v) and the reaction mixture was stirred at 50 °C over-
night. The solution was neutralized with acid resin Dowex-50 (H+)
and then filtered to remove the resin. The filtrate was concentrated
under reduced pressure and the resulting crude product was puri-
fied by flash column chromatography (CHCl3/MeOH 5:1) to afford
21 (57 mg, 75% from 19) as a white solid. Rf = 0.24(CHCl3/MeOH
3:1). Mp: 171–173 °C. ½a D20
ꢂ
ꢀ29.0 (c 1.0, CHCl3). IR (KBr, cmꢀ1
)
m
max: 3449, 2932, 2874, 2371, 1712, 1178, 1156, 1076, 1047. 1H
NMR (400 MHz, CDCl3): d = 0.78 (d, 3H, J = 6.3 Hz, CH3); 0.90 (s,
3H, CH3); 1.04 (s, 3H, CH3); 1.06 (d, 3H, J = 6.9 Hz, CH3); 1.22 (s,
9H, (CH3)3C); 2.21 (dd, 1H, J = 4.9/14.3 Hz); 2.40 (t, 1H,
J = 13.9 Hz); 2.51 (dd, 1H, J = 6.7/8.6 Hz); 3.34 (t, 1H, J = 11.0 Hz,
H-26ax); 3.47–3.68 (m, 5H, H-26eq, H-2 Glc, H-4 Glc, H-5 Glc, H-
3a); 3.79 (t, 1H, J = 10.2 Hz, H-6a Glc); 4.32–4.38 (m, 2H, H-6b
Glc, H-16 ); 4.56 (d, 1H, J = 7.6 Hz, H-1 Glc); 5.17 (t, 1H,
a
J = 9.5 Hz, H-3 Glc); 5.50 (s, 1H, CHPh); 7.32–7.34 (m, 3H, Ph);
7.41–7.43 (m, 2H, Ph). 13C NMR (100 MHz, CDCl3): d = 11.9, 13.2,
16.0, 17.1, 27.0 (CH3); 28.3, 28.8, 29.1 (CH2); 30.2 (CH); 31.1,
31.4, 31.5 (CH2); 34.3 (CH); 36.2 (C); 36.5, 37.8 (CH2); 38.9 (C);
42.2, 44.4, 53.5 (CH); 55.1 (C); 55.5, 55.7, 66.4 (CH); 66.9, 68.7
(CH2); 73.5, 73.6, 78.4, 78.5, 79.2, 101.1, 101.8 (CH); 109.2 (C);
125.9, 128.2, 128.9 (CH); 137.0 (C); 178.7, 213.5 (C@O). HRMS
(ESI-FT-ICR) m/z: 787.4386 [M+Na]+ (calcd for C45H64O10Na:
787.4397).
5:1). Mp: 199–201 °C. ½a D20
ꢂ
ꢀ77.4 (c 1.2, C5H5N). IR (KBr, cmꢀ1
)
mmax
:
3368, 2937, 2870, 2368, 1709, 1564, 1053. 1H NMR
(500 MHz, pyridine-d5): d = 0.71 (d, 3H, J = 5.7 Hz, CH3); 0.91 (s,
3H, CH3); 1.10 (s, 3H, CH3); 1.37 (d, 3H, J = 6.9 Hz, CH3); 1.77 (d,
3H, J = 6.3 Hz, CH3 Rha); 2.28 (dd, 1H, J = 5.0/13.9 Hz); 2.44 (t, 1H,
J = 13.7 Hz); 2.78 (dd, 1H, J = 6.6/8.5 Hz); 3.50 (t, 1H, J = 10.9 Hz,
H-26ax); 3.60 (dd, 1H, J = 3.3/11.0 Hz, H-26eq); 3.89–3.99 (m, 2H,
4.1.10. 5a-Hydroxy-laxogenyl 4,6-O-benzylidene-3-O-pivaloyl-
b-
D
-glucopyranoside (20)
Compound 9 (490 mg, 0.807 mmol) and benzaldehyde dimethyl
H-3
(m, 4H, H-2 Glc, H-3 Glc, H-6a Glc, H-4 Rha); 4.48–4.54 (m, 1H,
H-16 ); 4.58 (dd, 1H, J = 2.4/11.7 Hz, H-6b Glc); 4.62 (dd, 1H,
a, H-5 Glc); 4.17 (dd, 1H, J = 8.8/9.5 Hz, H-4 Glc); 4.26–4.40
acetal (0.9 mL, 5.81 mmol) in anhydrous DMF (20 mL) were re-
acted in the presence of p-TsOHꢁH2O in a similar was as described
in the synthesis of 17. Flash column chromatography (n-hexane/
EtOAc 1:5) afforded 18 (517 mg, 92%) as a pure compound. A solu-
tion of 18 (93.3 mg, 0.134 mmol) in anhydrous pyridine (5 mL) was
cooled to ꢀ15 °C under nitrogen atmosphere and pivaloyl chloride
a
J = 3.5/9.1 Hz, H-3 Rha); 4.82 (dd, 1H, J = 1.3/3.5 Hz, H-2 Rha);
5.07 (d, 1H, J = 7.6 Hz, H-1 Glc); 6.38 (d, 1H, J = 1.4 Hz, H-1 Rha).
13C NMR (125 MHz, pyridine-d5): d = 12.3, 14.3, 16.5, 17.7, 19.1
(CH3); 29.0, 29.6, 30.1 (CH2); 31.0 (CH); 31.9, 32.1, 32.2, 34.7
(CH2); 34.8 (CH); 36.8 (C); 37.1, 38.4 (CH2); 43.1, 44.8, 54.7(CH);
55.8 (C); 56.0, 56.3 (CH); 63.2, 67.4 (CH2); 69.9, 72.4, 72.9, 73.3,
74.6, 77.0, 78.5, 78.8, 80.1, 100.3, 102.6 (CH); 109.7 (C); 213.3
(C@O). HRMS (ESI-FT-ICR) m/z: 761.4067 [M+Na]+ (calcd for
(41 lL, 0.335 mmol) was added dropwise. The reaction was moni-
tored by TLC and the stirring was continued at room temperature
until disappearance of the intermediate. The reaction was
quenched with MeOH and concentrated to dryness. The resulting
crude product was purified by flash column chromatography (n-
hexane/EtOAc 2:1) to furnish 20 (66 mg, 63%) as a white solid.
C39H62O13Na: 761.4088).
4.1.12. 5a-Hydroxy-laxogenyl 2-O-(a-L-rhamnopyranosyl)-b-D-
Rf = 0.37 (n-hexane/EtOAc 2:1). Mp: 183–185 °C. ½a D20
ꢀ105.2 (c
ꢂ
glucopyranoside(22)
Acceptor 20 (198 mg, 0.254 mmol) and donor 12 (165.6 mg,
0.381 mmol) in dry CH2Cl2 (5 mL) were reacted in the presence
1.0, CHCl3). IR (KBr, cmꢀ1
)
m
max: 3404, 2953, 2877, 1706, 1180,
1162, 1080, 1054, 1036, 984. 1H NMR (400 MHz, CDCl3): d = 0.75
(s, 3H, CH3); 0.78 (d, 3H, J = 6.3 Hz, CH3); 0.81 (s, 3H, CH3); 0.96
(d, 3H, J = 6.9 Hz, CH3); 1.21 (s, 9H, (CH3)3C); 2.11 (dd, 1H, J = 3.9/
12.0 Hz); 2.72 (t, 1H, J = 12.9 Hz); 3.36 (t, 1H, J = 11.0 Hz, H-
26ax); 3.45–3.51 (m, 2H, H-26eq, H-5 Glc); 3.52 (dd, 1H, J = 7.6/
9.1 Hz, H-2 Glc); 3.63 (t, 1H, J = 9.5 Hz, H-4 Glc); 3.77 (t, 1H,
of BF3ꢁEt2O (39
lL, 0.305 mmol) in a similar way as described in
the synthesis of 21. The crude product was purified by flash col-
umn chromatography (n-hexane/EtOAc 2:1) to afford the corre-
sponding glycoside (225 mg, 84%) as a white solid. Rf = 0.26
(EtOAc/n-hexane 3:2). Mp: 194–196 °C. HRMS (ESI-FT-ICR) m/z:
1075.5198 [M+Na]+ (calcd for C57H80O18Na: 1075.5242). This com-
pound (92 mg, 0.087 mmol) was deprotected according to the pro-
cedure described in the synthesis of 21. The resulting crude
product was purified by flash column chromatography (CHCl3/
MeOH 5:1) to afford 22 (60 mg, 77% from 20) as a white solid.
J = 10.2 Hz, H-6a Glc); 4.01 (m, 1H, H-3
10.5 Hz, H-6b Glc); 4.40 (m, 1H, H-16 ); 4.55 (d, 1H, J = 7.6 Hz,
a); 4.33 (dd, 1H, J = 4.8/
a
H-1 Glc); 5.15 (t, 1H, J = 9.5 Hz, H-3 Glc); 5.49 (s, 1H, CHPh);
7.32–7.33 (m, 3H, Ph); 7.40–7.42 (m, 2H, Ph). 13C NMR (100 MHz,
CDCl3): d = 14.0, 14.4, 16.4, 17.1 (CH3); 21.2 (CH2); 27.1 (CH3);
28.5, 28.7, 29.7 (CH2); 30.2 (CH); 31.3, 31.5, 33.2 (CH2); 36.7
(CH); 38.9 (C); 39.6 (CH2); 41.1 (C); 41.6 (CH); 41.8 (CH2); 42.5
(C); 44.4, 56.1, 62.0, 66.3 (CH); 66.9, 68.7 (CH2); 73.4, 73.5, 76.4,
78.6 (CH); 80.4 (C); 80.5, 101.1, 102.8 (CH); 109.3 (C); 125.9,
Rf = 0.31(CHCl3/MeOH 4:1). Mp: 209–211 °C. ½a D20
ꢀ124.2 (c 1.0,
ꢂ
C5H5N). IR (KBr, cmꢀ1
) mmax: 3390, 2937, 2375, 1720, 1564, 1056.
1H NMR (500 MHz, pyridine-d5): d = 0.70 (d, 3H, J = 5.4 Hz, CH3);
0.83 (s, 3H, CH3); 1.01 (s, 3H, CH3); 1.15 (d, 3H, J = 6.9 Hz, CH3);
1.81 (d, 3H, J = 6.0 Hz, CH3 Rha); 2.37 (dd, 1H, J = 11.4/13.3 Hz);

2698
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2.82 (dd, 1H, J = 3.8/12.6 Hz); 3.09 (t, 1H, J = 12.6 Hz); 3.50 (t, 1H,
J = 10.7 Hz, H-26ax); 3.58 (dd, 1H, J = 3.3/10.8 Hz, H-26eq); 3.64
(m, 1H, H-5 Glc); 4.09–4.16 (m, 2H, H-3 Glc, H-4 Glc); 4.26 (dd,
1H, J = 7.9/8.5 Hz, H-2 Glc); 4.30–4.36 (m, 2H, H-6a Glc, H-4
Rha); 4.46 (dd, 1H, J = 2.4/11.8 Hz, H-6b Glc); 4.57 (m, 1H, H-
(CH3); 21.2, 28.1, 28.8, 29.7 (CH2); 30.2 (CH); 31.3, 31.5, 32.8
(CH2); 36.7 (CH); 39.6 (CH2); 41.1 (C); 41.6 (CH); 41.8 (CH2); 42.4
(C); 44.5, 56.1, 62.1 (CH); 62.2, 66.9 (CH2); 69.5, 71.6, 75.1, 75.7,
75.9 (CH); 80.2 (C); 80.5, 99.0 (CH); 109.3 (C); 169.9, 171.8, 212.1
(C@O). HRMS (ESI-FT-ICR) m/z: 715.3671 [M+Na]+ (calcd for
16a); 4.64 (dd, 1H, J = 3.3/9.3 Hz, H-3 Rha); 4.74 (dd, 1H, J = 1.4/
C37H56O12Na: 715.3669).
3.3 Hz, H-2 Rha); 4.82 (m, 1H, H-3
a); 4.91 (d, 1H, J = 7.9 Hz, H-1
Glc); 6.35 (d, 1H, J = 1.5 Hz, H-1 Rha). 13C NMR (125 MHz, pyri-
dine-d5): d = 14.6, 15.4, 16.9, 17.7, 19.1 (CH3); 22.0, 29.5, 29.7,
30.8 (CH2); 31.0 (CH); 32.2, 32.4, 33.2 (CH2); 37.4 (CH); 40.4
(CH2); 41.7 (C); 42.4 (CH); 42.6 (CH2); 43.3 (C); 45.2, 56.8 (CH);
63.1 (CH2); 63.3 (CH); 67.3 (CH2); 69.9, 72.2, 72.9, 73.2, 74.0,
74.6, 78.4, 78.6, 79.9 (CH); 80.2 (C); 81.4, 99.9, 102.5 (CH); 109.7
(C); 212.9 (C@O). HRMS (ESI-FT-ICR) m/z: 777.4040 [M+Na]+ (calcd
for C39H62O14Na: 777.4037).
4.1.15. Hecogenyl 4,6-di-O-(2,3,4-tri-O-acetyl-
a-L-
rhamnopyranosyl)-2,3-di-O-acetyl-b- -glucopyranoside (25)
D
Acceptor 23 (93 mg, 0.137 mmol) and donor 12 (185 mg,
0.425 mmol) in dry CH2Cl2 (10 mL) were reacted in the presence
of BF3ꢁEt2O (76
lL, 0.603 mmol) in a similar way as described in
the synthesis of 13. Flash column chromatography purification
(n-hexane/EtOAc 2:1) afforded 25 (115 mg, 69%) as a white solid.
Rf = 0.45 (n-hexane/EtOAc 1:1). Mp: 137–139 °C. ½a D20
ꢀ59.4 (c
ꢂ
1.6, CDCl3). IR (KBr, cmꢀ1
) mmax: 2933, 2873, 2375, 2346, 1751,
4.1.13. Hecogenyl 2,3-di-O-acetyl-b-
D
-glucopyranoside(23)
1439, 1374, 1369, 1243, 1223, 1138, 1076, 1043. 1H NMR
(400 MHz, CDCl3): d = 0.77 (d, 3H, J = 6.3 Hz, CH3); 0.86 (s, 3H,
CH3); 1.02 (s, 3H, CH3); 1.04 (d, 3H, J = 7.0 Hz, CH3); 1.14 (d, 3H,
J = 6.1 Hz, CH3 Rha); 1.17 (d, 3H, J = 6.3 Hz, CH3 Rha0); 1.95, 1.96,
2.02, 2.03 (4 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3CO); 2.06 (s, 6H, 2 ꢃ CH3CO);
2.09, 2.11 (2 ꢃ s, 2 ꢃ 3H, 2 ꢃ CH3CO); 2.18 (dd, 1H, J = 4.6/
Ac2O (3 mL) was added at room temperature to solution of 17
(346 mg, 0.508 mmol) in anhydrous pyridine (3 mL). The resulting
reaction mixture was stirred 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 THF/
MeOH (10 mL, 1:1, v/v) and treated with p-TsOHꢁH2O (106.3 mg,
0.559 mmol). The reaction mixture was stirred for 3 h, then
quenched with Et3N and concentrated reduced pressure. The crude
product was purified by flash column chromatography (n-hexane/
EtOAc 1:3) to afford diol 23 (302 mg, 88% from 17) as a white solid.
14.1 Hz, H-11b); 2.37 (t, 1H, J = 13.9 Hz, H-11
J = 6.7/8.6 Hz); 3.33 (t, 1H, J = 11.0 Hz, H-26ax); 3.46–3.51 (m, 3H,
H-5 Glc, H-3 , H-26eq); 3.78–3.85 (m, 3H, H-5 Rha, H-4 Glc, H-
6a Glc); 3.94–3.99 (m, 2H, H-5 Rha0, H-6b Glc); 4.29–4.35 (m, 1H,
H-16 ); 4.54 (d, 1H, J = 8.0 Hz, H-1 Glc); 4.79–4.83 (m, 3H, H-2
a); 2.49 (dd, 1H,
a
a
Glc, H-1 Rha, H-1 Rha0); 5.00–5.08 (m, 3H, H-2 Rha, H-3 Rha, H-4
Rha); 5.13 (dd, 1H, J = 3.4/9.9 Hz, H-3 Rha0); 5.16–5.22 (m, 4H, H-
2 Rha0, H-3 Rha0, H-4 Rha0, H-3 Glc). 13C NMR (125 MHz, CDCl3):
d = 12.3, 13.6, 16.4, 17.5, 17.7, 21.0, 21.1, 21.2, 21.5 (CH3); 28.7,
29.2, 29.6 (CH2); 30.6 (CH); 31.5, 31.8, 31.9 (CH2); 34.7 (CH); 34.9
(CH2); 36.5 (C); 36.8, 38.1 (CH2); 42.6, 44.9, 53.9 (CH); 55.5 (C);
55.9, 56.2 (CH); 65.9 (CH2); 66.8 (CH); 67.3 (CH2); 68.2, 69.3,
69.6, 69.9, 70.0, 70.9, 71.3, 72.7, 73.8, 74.4, 77.6, 79.6, 97.8, 100.1,
100.2 (CH); 109.6 (C); 169.9, 170.1, 170.2, 170.3, 170.5, 170.6,
170.7, 170.8, 213.8 (C@O). HRMS (ESI-FT-ICR) m/z: 1243.5492
[M+Na]+ (calcd for C61H88O25Na: 1243.5512).
Rf = 0.33 (EtOAc/n-hexane 3:1). Mp: 228–229 °C. ½a D20
ꢀ18.0 (c 1.5,
ꢂ
CHCl3). IR (KBr, cmꢀ1
) mmax: 3421, 2929, 2873, 2377, 2346, 1748,
1710, 1457, 1374, 1242, 1159, 1076, 1040. 1H NMR (500 MHz,
CDCl3): d = 0.77 (d, 3H, J = 6.3 Hz, CH3); 0.86 (s, 3H, CH3); 1.03 (s,
3H, CH3); 1.04 (d, 3H, J = 7.3 Hz, CH3); 2.03, 2.07 (2 ꢃ s, 2 ꢃ 3H,
2 ꢃ CH3CO); 2.19 (dd, 1H, J = 4.9/14.3 Hz); 2.38 (t, 1H,
J = 14.2 Hz); 2.49 (dd, 1H, J = 6.8/8.7 Hz); 3.18 (m, 1H, OH); 3.33
(t, 1H, J = 11.0 Hz, H-26ax); 3.37–3.41 (m, 1H, H-5 Glc); 3.46–
3.48 (m, 1H, H-26eq); 3.53 (m, 1H, H-3
H-4 Glc); 3.81 (dd, 1H, J = 4.3/11.8 Hz, H-6a Glc); 3.89 (dd, 1H,
J = 3.2/12.0 Hz, H-6b Glc); 4.30–4.36 (m, 1H, H-16 ); 4.58 (d, 1H,
a); 3.73 (t, 1H, J = 9.3 Hz,
a
J = 8.0 Hz, H-1 Glc); 4.84 (dd, 1H, J = 8.0/9.6 Hz, H-2 Glc); 5.01 (t,
1H, J = 9.5 Hz, H-3 Glc). 13C NMR (125 MHz, CDCl3): d = 11.9, 13.2,
15.9, 17.1, 20.7, 20.8 (CH3); 28.3, 28.7, 29.1 (CH2); 30.2 (CH);
31.1, 31.4, 31.5 (CH2); 34.3 (CH); 34.4 (CH2); 36.1 (C); 36.4, 37.7
(CH2); 44.2, 44.5, 53.5 (CH); 55.1 (C); 55.5, 55.7 (CH); 62.2, 66.9
(CH2); 69.4, 71.6, 75.4, 75.9, 79.2, 99.6 (CH); 109.2 (C); 169.5,
171.5, 213.5 (C@O). HRMS (ESI-FT-ICR) m/z: 699.3712 [M+Na]+
(calcd for C37H56O11Na: 699.3720).
4.1.16. 5
a
-Hydroxy-laxogenyl 4,6-di-O-(2,3,4-tri-O-acetyl-
a-L-
rhamnopyranosyl)-2,3-di-O-acetyl-b-
Acceptor 24 (62 mg, 0.090 mmol) and donor 12 (120.6 mg,
0.277 mmol) in dry CH2Cl2 (10 mL) were reacted in the presence
D
-glucopyranoside(26)
of BF3ꢁEt2O (50
lL, 0.394 mmol) in a similar way as described in
the synthesis of 13. Flash column chromatography purification
(n-hexane/EtOAc 1:1) afforded 26 (73 mg, 66%) as a white amor-
phous solid. Rf = 0.28 (n-hexane/EtOAc 1:1). ½a D20
ꢀ112.9 (c 0.9,
ꢂ
CDCl3). IR (KBr, cmꢀ1
) mmax: 3506, 2934, 2872, 1753, 1368, 1245,
4.1.14. 5
a
-Hydroxy-laxogenyl 2,3-di-O-acetyl-b-
D-
1136, 1076, 1043. 1H NMR (400 MHz, CDCl3): d = 0.74 (s, 3H,
CH3); 0.78 (d, 3H, J = 7.0 Hz, CH3); 0.79 (s, 3H, CH3); 0.96 (d, 3H,
J = 6.8 Hz, CH3); 1.14 (d, 3H, J = 6.1 Hz, CH3 Rha); 1.20 (d, 3H,
J = 6.3 Hz, CH3 Rha0); 1.96, 1.97, 2.03, 2.05, 2.06, 2.07, 2.11, 2.12
glucopyranoside (24)
Compound 18 (411 mg, 0.590 mmol) was subjected to acetyla-
tion and benzylidene deprotection in a similar was as described
in the synthesis of 23. Flash column chromatography purification
(n-hexane/EtOAc 1:4) afforded 24 (331 mg, 81% from 18) as a
(8 ꢃ s, 8 ꢃ 3H, 8 ꢃ CH3CO); 2.72 (t, 1H, J = 12.2 Hz, H-7
a); 3.35 (t,
1H, J = 10.9 Hz, H-26ax); 3.45–3.48 (m, 1H, H-26eq); 3.55 (m, 1H,
white solid. Rf = 0.36(EtOAc/n-hexane 4:1). Mp: 233–235 °C. ½a D20
ꢂ
H-5 Glc); 3.73–3.83 (m, 3H, H-4 Glc, H-6a Glc, H-5 Rha); 3.91–
ꢀ116 (c 1.4, CHCl3). IR (KBr, cmꢀ1
)
m
max: 3421, 2951, 2872, 2371,
3.99 (m, 3H, H-6b Glc, H-5 Rha0, H-3
a); 4.39 (m, 1H, H-16a);
2345, 1735, 1724, 1449, 1374, 1242, 1165, 1076, 1052. 1H NMR
(500 MHz, CDCl3): d = 0.75 (s, 3H, CH3); 0.78 (d, 3H, J = 6.6 Hz,
CH3); 0.79 (s, 3H, CH3); 0.96 (d, 3H, J = 6.8 Hz, CH3); 2.07, 2.08
(2 ꢃ s, 2 ꢃ 3H, 2 ꢃ CH3CO); 2.72 (t, 1H, J = 12.1 Hz); 3.10 (s, 1H);
3.35 (t, 1H, J = 10.9 Hz, H-26ax); 3.42–3.50 (m, 2H, H-26eq, H-5
Glc); 3.68 (t, 1H, J = 9.2 Hz, H-4 Glc); 3.79 (dd, 1H, J = 4.9/12.0 Hz,
H-6a Glc); 3.91 (dd, 1H, J = 2.8/11.7 Hz, H-6b Glc); 3.99 (m, 1H,
4.58 (d, 1H, J = 8.0 Hz, H-1 Glc); 4.82 (m, 3H, H-2 Glc, H-1 Rha,
H-1 Rha0); 5.01–5.08 (m, 3H, H-2 Rha, H-3 Rha, H-4 Rha); 5.12–
5.23 (m, 4H, H-3 Glc, H-2 Rha0, H-3 Rha0,H-4 Rha0). 13C NMR
(125 MHz, CDCl3): d = 14.1, 14.4, 16.4, 17.1, 17.2, 17.3, 20.6, 20.7,
20.8, 20.9, 21.0 (CH3); 21.2, 28.3, 28.7, 29.6 (CH2); 30.2 (CH);
31.3, 31.5, 33.0 (CH2); 36.7 (CH); 39.6 (CH2); 41.1 (C); 41.6 (CH);
41.8 (CH2); 42.5 (C); 44.7, 56.1, 62.1 (CH); 65.9 (CH2); 66.6 (CH);
66.9 (CH2); 67.8, 68.8, 69.4, 69.6, 69.7, 70.5, 70.9, 72.2, 73.6, 74.1,
75.1, 77.4, 80.4 (CH); 80.5(C); 97.4, 99.3, 99.6 (CH); 109.3 (C);
169.7, 169.8, 169.9, 170.1, 170.2, 170.3, 170.4, 211.2 (C@O).HRMS
H-3a); 4.40 (m, 1H, H-16a); 4.64 (d, 1H, J = 7.9 Hz, H-1 Glc); 4.83
(dd, 1H, J = 8.0/9.5 Hz, H-2 Glc); 5.04 (t, 1H, J = 9.4 Hz, H-3 Glc).
13C NMR (125 MHz, CDCl3): d = 14.0, 14.4, 16.4, 17.1, 20.8, 20.9

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 2690–2700
2699
(ESI-FT-ICR) m/z: 1259.5439 [M+Na]+ (calcd for C61H88O26Na:
1259.5462).
4.2. Biological activity
4.2.1. Cell culture
4.1.17. Hecogenyl 4,6-di-O-(
glucopyranoside (27)
a
-L
-rhamnopyranosyl)-b-
D
-
Human HL-60 myeloid leukemia cells were grown in RPMI 1640
(Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine
NaOMe was added to a solution of 25 (87 mg, 0.071 mmol) in
THF/MeOH (4 mL, 1:1, v/v) until pH was set to 9–10. The reaction
mixture was stirred at room temperature for 2 h, then neutralized
with acid resin Dowex-50 (H+) and filtered. The filtrate was con-
centrated under reduced pressure to dryness and the resulting
crude product was purified by flash column chromatography
(CHCl3/MeOH 4:1) to afford 27 (62 mg, 98%) as a white amorphous
serum (Sigma) and 100 U/mL penicillin and 100 lg/mL streptomy-
cin at 37 °C in a humidified atmosphere containing 5% CO2. The cell
numbers were counted by a hematocytometer, and the viability
was always greater than 95% in all experiments as assayed by
the 0.025% Trypan blue exclusion method. Stock solutions of
100 mM spirostanyl glycosides were made in dimethylsulfoxide
(DMSO), and aliquots were frozen at ꢀ20 °C.
solid. Rf = 0.31 (CHCl3/MeOH 3:1). ½a D20
ꢂ
+39.2 (c 1.1, C5H5N). IR (KBr,
cmꢀ1
)
m
max: 3367, 2929, 2873, 1701, 1654, 1455, 1375, 1099, 1059,
4.2.2. Assay for growth inhibition and cell viability
1040. 1H NMR (500 MHz, pyridine-d5): d = 0.69 (s, 3H, CH3); 0.71
(d, 3H, J = 6.0 Hz, CH3); 1.09 (s, 3H, CH3); 1.37 (d, 3H, J = 6.9 Hz,
CH3); 1.66 (d, 3H, J = 6.0 Hz, CH3 Rha); 1.73 (d, 3H, J = 6.3 Hz, CH3
Rha0); 2.36 (t, 1H, J = 13.8 Hz); 2.77 (dd, 1H, J = 6.6/8.8 Hz); 3.51
(t, 1H, J = 10.9 Hz, H-26ax); 3.61 (dd, 1H, J = 3.8/11.0 Hz, H-26eq);
The cytotoxicity of spirostanyl glycosides was assessed using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bro-
mide (MTT) assay.20 Briefly, 1 ꢃ 104 exponentially growing cells
were seeded in 96-well microculture plates with various spirosta-
nyl glycoside concentrations (0.3–100 lM) in a volume of 200 lL.
3.84–4.03 (m, 4H, H-2 Glc, H-4 Glc, H-6a Glc, H-3
a
); 4.12 (t, 1H,
DMSO concentration was the same in all the treatments and did
not exceed 0.1% (v/v). After 72 h, surviving cells were detected
based on their ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide (Sigma) into formazan crys-
tals. Optical density was read with an ELISA reader at a wavelength
of 570 nm and was used as a measure of cell viability. The MTT
dye-reduction assay measures mitochondrial respiratory function
and can detect the onset of cell death earlier than dye-exclusion
methods. Cell survival was calculated as the fraction of cells alive
relative to control for each point: cell survival (%) = mean absor-
bance in treated cells/mean absorbance in control wells ꢃ 100.
Concentrations inducing a 50% inhibition of cell growth (IC50) were
determined graphically for each experiment. Parameters describ-
ing the concentration–response curves (IC50) were determined
using the curve fitting routine of the computer software Prism™
(GraphPad) and the equation derived by DeLean et al.21
J = 9.1 Hz, H-3 Glc); 4.21–4.26 (m, 2H, H-5 Glc, H-4 Rha0); 4.33–
4.41 (m, 3H, H-4 Rha, H-5 Rha, H-6b Glc); 4.48–4.52 (m, 2H, H-3
Rha0, H-16
a); 4.56 (dd, 1H, J = 3.2/9.1 Hz, H-3 Rha); 4.60 (m, 1H,
H-2 Rha); 4.68 (m, 1H, H-2 Rha0); 4.92 (d, 1H, J = 7.9 Hz, H-1
Glc); 4.94 (m, 1H, H-5 Rha0); 5.40 (d, 1H, J = 1.6 Hz, H-1 Rha);
5.66 (d, 1H, J = 1.6 Hz, H-1 Rha0). 13C NMR (125 MHz, pyridine-
d5): d = 12.1, 14.3, 16.5, 17.7, 18.9, 19.0 (CH3); 29.1, 29.7, 30.2
(CH2); 31.0 (CH); 31.9, 32.1, 32.2 (CH2); 34.8 (CH); 35.2 (CH2);
36.7 (C); 37.0, 38.4 (CH2); 43.1, 44.8, 54.8 (CH); 55.8 (C); 55.9,
56.3 (CH); 67.4, 67.8 (CH2); 70.2, 71.1, 72.7, 73.0, 73.2, 74.3, 74.4,
75.8, 75.9, 77.2, 77.9, 80.1, 80.2, 102.5, 102.6, 103.3 (CH); 109.7
(C); 213.2 (C@O). HRMS (ESI-FT-ICR) m/z: 907.4668 [M+Na]+ (calcd
for C45H72O17Na: 907.4667).
4.1.18. 5
b- -glucopyranoside(28)
NaOMe was added to a solution of 26 (58 mg, 0.047 mmol) in
a-Hydroxy-laxogenyl 4,6-di-O-(a-L-rhamnopyranosyl)-
D
Acknowledgements
THF/MeOH (4 mL, 1:1, v/v) until pH was set to 9–10. The reaction
mixture was stirred at room temperature for 2 h, then neutralized
with acid resin Dowex-50 (H+) and filtered. The filtrate was con-
centrated under reduced pressure to dryness and the resulting
crude product was purified by flash column chromatography
(CHCl3/MeOH 4:1) to afford 28 (40 mg, 95%)as a white amorphous
This work was partially supported by CSIC (Proyecto Intramural
de Incorporación—2007022), the Ministry of Science and Innova-
tion of Spain and the European Regional Development Fund
(SAF2010–21380 to F.E.). K. Pérez-Labrada gratefully acknowledges
to MAEC-AECID for a doctoral scholarship.
solid. Rf = 0.26 (CHCl3/MeOH 3:1). ½a D20
ꢀ162.5 (c 0.4, C5H5N). IR
ꢂ
(KBr, cmꢀ1
)
m
max: 3368, 2929, 2873, 2371, 2345, 1703, 1654,
References and notes
1457, 1375, 1097, 1059, 1040. 1H NMR (500 MHz, pyridine-d5):
d = 0.69 (d, 3H, J = 6.0 Hz, CH3); 0.78 (s, 3H, CH3); 0.81 (s, 3H,
CH3); 1.14 (d, 3H, J = 6.9 Hz, CH3); 1.66 (d, 3H, J = 6.0 Hz, CH3
Rha); 1.72 (d, 3H, J = 6.3 Hz, CH3 Rha0); 2.66 (m, 1H); 3.00 (t, 1H,
J = 12.6 Hz); 3.49 (t, 1H, J = 10.9 Hz, H-26ax); 3.59 (dd, 1H, J = 3.6/
10.9 Hz, H-26eq); 3.78–3.80 (m, 1H, H-5 Glc); 3.97–4.02 (m, 2H,
H-2 Glc, H-6a Glc); 4.09–4.13 (m, 2H, H-3 Glc, H-4 Glc); 4.23–
4.40 (m, 4H, H-4 Rha, H-4 Rha0, H-5 Rha, H-6b Glc); 4.49 (dd, 1H,
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73.0, 73.1, 74.3, 74.4, 74.9, 75.7, 75.8, 77.1, 79.8 (CH); 80.4 (C);
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