Effect of C-ring modifications on the cytotoxicity of spirostan saponins and related glycosides

Article abstract of DOI:10.1016/j.bmc.2012.05.018

Twelve C-ring modified spirostanyl glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). With the aim of assessing the influence of the hydrophobic character, the conformational flexibility

Full text of DOI:10.1016/j.bmc.2012.05.018

Bioorganic & Medicinal Chemistry 20 (2012) 4522–4531
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of C-ring modifications on the cytotoxicity of spirostan saponins
and related glycosides
Karell Pérez-Labrada a,b, Ignacio Brouard b, , Sara Estévez c, María Teresa Marrero c, Francisco Estévez c,
⇑
Daniel G. Rivera d,
⇑
a Institute of Pharmacy and Food, University of Havana, San Lázaro y L, 10400 La Habana, Cuba
b Instituto de Productos Naturales y Agrobiología-C.S.I.C., Av. Astrofísico F. Sánchez 3, 38206 La Laguna, Tenerife, Spain
c Department of Biochemistry, Molecular Biology and Physiology, University of Las Palmas de Gran Canaria, Instituto Canario de Investigación del Cáncer,
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:
Received 20 February 2012
Revised 3 May 2012
Accepted 9 May 2012
Available online 17 May 2012
Twelve C-ring modified spirostanyl glycosides were synthesized and evaluated for their cytotoxicity
against the human myeloid leukemia cell line (HL-60). With the aim of assessing the influence of the
hydrophobic character, the conformational flexibility and the stereochemistry of the C-ring functionalities
on the cytotoxic activity, a variety of spirostanic aglycones incorporating methylene, methoxyl,
urated ketone and lactone groups were subjected to a linear glycosylation strategy leading to glycosides
derived from the 3,6-dipivaloylated b- -glucoside and the b-chacotrioside moieties. The 3,6-dipivaloylat-
ed spirostanyl b- -glucosides showed moderate to good cytotoxic activity against HL-60, but no significant
a,b-unsat-
D
Keywords:
Steroids
D
cytotoxicity against benign blood cells. However, the cytotoxicity of spirostanyl b-chacotriosides was
highly dependent on the nature of the C-ring functional groups of the steroidal aglycones. Actually, the
chacotrioside-based saponins either with no functionality or bearing a hydrophobic methylene group at
C-12 were the most cytotoxic ones against both HL-60 and benign blood cells. On the other hand, the
incorporation of very polar functionalities and the opening of the ring C with the consequent loss of rigid-
ity led to a significant drop in the cytotoxicity against HL-60. These results confirm that spirostanyl b-chac-
otriosides including very lipophilic aglycones are the most cytotoxic ones among their congeners.
Ó 2012 Elsevier Ltd. All rights reserved.
Saponins
Glycosides
Cytotoxicity
HL-60 cells
1. Introduction
discovered that an intermediate of the synthetic route, that is,
the dipivaloylated hecogenyl b-glucoside 4,3 exhibits very good
Dioscin (1)1 and filiasparoside A (2)2 are two important members
of the family of spirostan saponins, which show promise as antican-
cer agents owing to their high cytotoxicity against a variety of cancer
cells. In a previous report,3 we have described the synthesis and
cytotoxicity evaluation of a new spirostan saponin incorporating
the trisaccharide portion of dioscin—namely b-chacotriose—and
the spirostanic aglycone of filiasparoside A, that is, hecogenin. As
shown inFigure 1, the new saponin (3) comprises a hybrid structure
integrating structural elements of the two naturally occurring spiro-
stan saponins. Importantly, we found that this hecogenyl b-chaco-
trioside 3 exhibits cytotoxic activity as high as that of the natural
products utilized as models. It was also found that hecogenyl
cytotoxic activity against HL-60 cells, thus providing a new lead
compound for further structural optimization with related pivaloy-
lated spirostanyl glucosides.
Herein we report on the synthesis and cytotoxicity evaluation of
new spirostan saponins having a variety of structural modifications
on ring C of the steroidal skeletons. Accordingly, the focus is posed
on assessing the influence of the hydrophobic character, conforma-
tional flexibility and stereochemistry of the C-ring functionalities
on the cytotoxic activity against HL-60 cells. Additionally, we are
interested on determining whether novel pivaloylated spirostanyl
glucosides—analogous to compound 4 but with such a new type
of functionalities on ring C—show cytotoxicity against such cancer
cells.
saponins including other oligosaccharides—like the 2-O-(
a
-
L
-
-
rhamnopyranosyl)-b-
rhamnopyranosyl)-b-
D
-glucopyranoside and the 4,6-di-O-(
a
-L
D
-glucopyranoside—showed no cytotoxicity
-rhamnosyl residues at-
-glucose. Interestingly, we
2. Results and discussion
2.1. Chemical synthesis
at all, thus proving the importance of the a-L
tached at positions 2 and 4 of the inner
D
⇑
Considering that synthetic hecogenyl glycosides including only
the b-chacotrioside and the 3,6-dipivaloylated b-glucoside moieties
Corresponding authors. Tel.: +53 7 8702102; fax: +53 7 8733502.
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.
http://dx.doi.org/10.1016/j.bmc.2012.05.018

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4523
Hecogenin
O
O
β-Chacotrioside
O
OH
HO
O
O
OH
4
O
O
OH
O
O
HO
2
O
6
HO
O
4
O
O
O
HO
HO
O
O
HO
O
HO
H
HO
HO
OH
OH
HO
OH
Structural Hybridization
Dioscin (1)
Filiasparoside A (2)
O
O
O
O
O
O
OH
OPiv
4
Glycosylation-
Deprotection
O
6
O
O
O
HO
O
H
HO
2
H
PivO
O
3
HO
O
OH
HO
O
HO
HO
3,6-Dipivaloylated Hecogenyl β-glucoside (4)
Intermediate of the route to β-chacotrioside 3
IC50 = 12.0 μM
HO
OH Hecogenyl β-chacotrioside (3)
IC50 = 4.3 μM
Figure 1. New hecogenyl glycosides with cytotoxic activity against HL-60.
have exhibited cytotoxicity,3,4 we implemented a linear glycosyla-
tion strategy capable to ensure the 1,2-trans-glycosidic linkage in
all glycosides along the synthetic route.5 Such a strategy firstly
requires the preparation of the individual spirostanic aglycones
bearing the desired functionalities on ring C, followed by the step-
wise construction of the oligosaccharidic moiety. Importantly, this
type of approach also allows accessing the 3,6-dipivaloylated spiro-
stanyl b-glucosides, which are advanced intermediates in the route
toward the b-chacotriosides.
As depicted in Scheme 1, hecogenin (5) was initially subjected
to silylation of its 3b-OH group according to a reported procedure.6
The resulting TBDMS-hecogenyl derivative 6 was next submitted
to a series of transformations aiming to incorporate more or less
polar functionalities at position 12. Accordingly, Wittig olefination
of 6 with methyltriphenylphosphonium bromide and BuLi as base
followed by TBAF-mediated 3b-OH deprotection afforded the
methylene-spirostane 7 in 77% yield over two steps. Alternatively,
ketone 6 was reduced with NaBH4 in MeOH/CH2Cl2 to yield a mix-
we decided to include in the glycosylation strategies not only the
synthetic spirostanes 7, 8 and 9, but also the natural sapogenins
D
9-hecogenin (10) and tigogenin (11). As shown in Scheme 2, the
five spirostanols were subjected to glycosylation with 2,3,4,6-tet-
ra-O-benzoyl-
a-D
-glucopyranosyl trichloroacetimidate (12)7 in
the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
to furnish the 3-O-b-glucosides 13–17 in 87–96% yield. Global
deprotection of the spirostanyl b-glucosides under typical Zemplen
conditions (NaOMe/MeOH) followed by selective pivaloylation8
gave rise to the 3,6-dipivaloylated b-glucosides 18–22 in 63–68%
yield over two steps. These five glucosides are not only key inter-
mediates of the final saponins, but also important targets for com-
parison of their cytotoxicity with that of the previously reported3
3,6-dipivaloylated hecogenyl glucoside 4.
Finally, glucosides 18–22 were subjected to double glycosyla-
tion with 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl trichloroacetim-
idate (23)9 according to the Schmidt’s inverse procedure,10
followed by deprotection to give the spirostanyl b-chacotriosides
24–28 in 63–69% yield over two steps. As noticed, the overall yield
of this synthetic planning was kept over 35% in all cases, similar to
those reported for other spirostan saponins.3,11
ture of the 12a- and 12b-spirostanols, which was subjected to
methylation without previous separation of their components.
The epimeric mixture was then purified by flash column chroma-
tography and the resulting pure compounds were deprotected
upon treatment with TBAF in THF to afford the 12b- and 12a-
methoxy spirostanes 8 and 9 in 58% and 13% overall yields,
With the five 3,6-dipivaloylated glucosides 13–17 and the five
spirostanyl saponins 24–28 in hands, it is possible to evaluate a set
of compounds endowed either with dissimilar functionalities at
respectively.
position 12 (i.e., alkene, methoxyl and a,b-unsaturated ketone) or
To assess the influence on the cytotoxicity of the more or less
polar nature of the C-ring functionalities of the target glycosides,
with no functionality at all (i.e., saponin 28 derived from tigoge-
nin12). Such a set of glycosides are well suited for comparison of their
cytotoxicity against HL-60 with that of the previously obtained hec-
ogenyl glycosides 3 and 4. Nevertheless, we were also interested in
obtaining spirostanyl glycosides with an opened ring C, thus assess-
ing the influence of such a lack of C-ring rigidity on the cytotoxicity
of the resulting seco-compounds. To this end, we planned a route to-
ward a new chacotrioside-based saponin incorporating a seco-spi-
rostanic aglycone arising from the ring opening of a C-ring lactone
functionality.
As depicted in Scheme 3, lactone 29—produced by the
Baeyer–Villiger oxidation of the 12-oxo function of hecogenin
(5) as previously described6—was subjected to the linear glyco-
sylation strategy developed for the previous spirostanyl glyco-
sides. Thus, glycosylation of 29 with donor 12 leading to the
b-glucoside 30 was followed by deprotection and selective piva-
loylation to furnish the key intermediate 31 in 66% over three
steps. Subsequent glycosylation with donor 23 followed by
simultaneous deprotection and lactone-ring opening led to the
O
H2C
O
O
a, b
HO
RO
H
H
7, 77%
Hecogenin (5), R = H
6, R = TBDMS
Ref. 6
OMe
OMe
c, d, b
HO
+
HO
H
H
8, 58%
9, 13%
Scheme 1. Synthesis of spirostanic aglycones functionalized on the ring C. Reagents
and conditions: (a) Ph3CH3P+Br, BuLi, THF; (b) TBAF, THF, 65 °C; (c) NaBH4, CH2Cl2/
MeOH; (d) MeI, NaH, DMF.

4524
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
OBz
O
R
R
R
O
BzO
BzO
O
BzO
O
CCl3
NH
OBz
O
OPiv
O
b, c
12
BzO
BzO
HO
PivO
HO
O
O
H
H
H
a
7, R = 12-CH2
8, R = 12β-OMe
9, R = 12α-OMe
10, R = Δ9-12-oxo
11, R = H
OBz
13, 96%, R = 12-CH2
14, 89%, R = 12β-OMe
15, 94%, R = 12α-OMe
OH
18, 63%, R =12-CH2
19, 65%, R = 12β-OMe
20, 67%, R = 12α-OMe
21, 68%, R =Δ9-12-oxo
22, 64%, R = H
16, 92%, R = 9-12-oxo
Δ
17, 87%, R = H
O
NH
R
O
CCl3
O
O
OH
O
AcO
AcO
O
OAc
23
O
O
HO
H
HO
O
O
24, 63%, R = 12-CH2
d, e
HO
25, 69%, R = 12β-OMe
26, 67%, R = 12α-OMe
27, 65%, R = Δ9-12-oxo
28, 67%, R = H
HO HO
HO
OH
Scheme 2. Synthesis of C-ring modified spirostanyl b-chacotriosides. 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
O
O
O
O
O
O
O
OBz
O
OPiv
O
12
b, c
BzO
BzO
HO
PivO
HO
O
O
a
H
H
H
OBz
OH
31, 66%
29, ref. 6
30, 95%
O
HO2C
HO
H
O
OH
23
H
O
O
O
O
d, e
HO
H
HO
O
32, 61%
HO
O
HO HO
HO
OH
Scheme 3. Synthesis of a seco-spirostanyl b-chacotrioside. 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, then H+ to pH 5.
Table 1
seco-spirostanyl b-chacotrioside 32 in 61% yield. With this final
saponin in hand, we can next assess the influence of such a
Cytotoxicity of spirostanyl glycosides against HL-60 cellsa
greater conformational flexibility and polarity of ring C on the
cytotoxic activity against HL-60 cells, thus having a more detailed
picture of the structural factors influencing the bioactivity of this
family of compounds.
3,6-Dipivaloylated glucosides
Chacotrioside-based saponins
Compound
IC50
(l
M)
Compound
IC50 (lM)
18
19
20
21
22
31
15.7 1.2
19.2 6.2
21.5 4.7
5.9 0.9
8.9 0.7
9.9 1.8
24
25
26
27
28
32
9.7 2.4
21.8 3.9
31.5 2.9
30.4 10.4
2.6 2.1
>100
2.2. Biological studies
Table 1 shows the results of the in vitro cytotoxic activities of 12
spirostanyl glycosides (i.e., six 3,6-dipivaloylated b-D-glucosides
a
Cells were cultured for 72 h and the IC50 values were calculated as described in
Section 4.
and six b-chacotriosides) 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.
Accordingly, it was shown that human myeloid leukemia HL-60
cells were highly sensitive to the anti-proliferative effect of all
glycosides, with the exception of saponin 32. It seems obvious that
the lower rigidity and lipophilicity of the seco-spirostanic aglycone
of 32 compared to those of the traditional (natural or synthetic)
spirostan saponins leads to the complete loss of cytotoxicity.
Analysis of Table 1 shows that the 3,6-dipivaloylated spirosta-
nyl glucosides exhibited moderate to good cytotoxic activity, being
analogous glucosides herein produced. Interestingly, the spirosta-
nyl glucoside 31 bearing the lactone functionality in ring C resulted
as cytotoxic as glucoside 22, which is derived from the more lipo-
philic tigogenin.
Saponin 28 displayed the greatest cytotoxicity of all spirostanyl
b-chacotriosides, a result that is in agreement with previous
reports on the cytotoxic activity of this compound against other
cancer cell lines.12 It must be noticed that the cytotoxicity of the
hecogenyl b-chacotrioside 3—used as model compound—is in the
same order of magnitude that those of saponins 28 and 24, in
the
D
9-hecogenyl b-glucoside 21 the most potent among all

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4525
which the 12-oxo functionality has been either removed (in 28) or
replaced by a methylene group (in 24). On the other hand, substi-
tution of the 12-oxo group by other functionalities, like 12-meth-
3,6-dipivaloylated glucosides 18, 19, 22 and 31. However, it turned
out that b-chacotriosides 24 and 28 were also cytotoxic against
PBMCs, although the sensitivity of HL-60 cells was higher than that
of PBMCs. Thus, the percentage of cell viability was 23 3.8% and
64.2 10% in HL-60 cells and PBMCs, respectively, upon treatment
oxy (25 and 26) and
D
9-12-oxo (27), led to five to sixfold
decrease in the cytotoxicity against HL-60. In agreement with our
previous report,3 we might conclude that a greater lipophilic char-
acter of the aglycone leads to a more potent cytotoxicity of chaco-
trioside-based spirostan saponins. Of course, this cannot be strictly
applied to the case of the 3,6-dipivaloylated spirostanyl glucosides,
as we have seen that the cytotoxicity depends not only on the
structural features of the aglycone but of the glycoside as a whole.
Actually, analysis of Table 1 demonstrates that the effect of the
structural variations in ring C on the cytotoxicity is much less pro-
nounced in the case of the 3,6-dipivaloylated spirostanyl gluco-
sides 18–22 than in the b-chacotriosides 24–28. This is
supported as well by our previous results,3 in which the 3,6-dipiva-
with 30
ment of both cells with 10
l
M of saponin 24. A similar trend was observed after treat-
M of saponin 28, as the percentage of
l
cell viability was 17 4.8% and 45 10% in HL-60 cells and PBMCs,
respectively.
An important conclusion arises from this study, that is, the 3,
6-dipivaloylated spirostanyl glucosides are much more cytotoxic
for HL-60 cells than for PBMCs, while the selectivity of cytotoxic
spirostanyl b-chacotriosides between blood malign and benign
cells is very low. Whereas this result fully agrees with previous re-
ports stating that spirostanyl monosaccharides are less hemolytic
than trisaccharides, it is known that those spirostanyl monosac-
charides show no cytotoxicity either against cancer cells.12a
Accordingly, this family of 3,6-dipivaloylated glucosides are the
first spirostanyl glycosides exhibiting good cytotoxicity against
cancer cells while no significant cytotoxicity against benign blood
cells. Indeed, this feature show great promise for the development
of cytotoxic agents based on spirostanyl glucoside scaffolds.
loylated glucoside of the less lipophilic 5
turned out to be the most cytotoxic (i.e., IC50 = 2.1 0.5
a
-hydroxy-laxogenin
M) among
l
all tested spirostanyl glycosides. Whereas we have proven that the
cytotoxicity mechanism in HL-60 of the 3,6-dipivaloylated gluco-
sides3 is alike to that of b-chacotrioside saponins, the current re-
sults corroborate that the structural factors influencing the
cytotoxicity of both types of glycosides are not strictly the same.
In order to assess whether the obtained spirostanyl glycosides
were also cytotoxic against non-transformed cell, some of the
active compounds were assayed for their cytotoxicity against hu-
man peripheral blood mononuclear cells (PBMCs). A comparative
study was performed to determine the effect of the 3,6-dipivaloy-
lated glucosides 18, 19, 22 and 31 as well as b-chacotriosides 24
and 28 on the viability of human HL-60 cells and PBMCs. As shown
in Figure 2, PBMCs were more resistant than HL-60 cells for the
3. Conclusions
We have produced a series of 12 C-ring modified spirostanyl
glycosides incorporating either the 3,6-dipivaloylated b-glucoside
or the b-chacotrioside moiety. The six 3,6-dipivaloylated spirosta-
nyl glucosides showed moderate to good cytotoxic activity, regard-
less the more or less lipophilic nature of the C-ring functionalities.
Some of these spirostanyl glucosides exhibited good selectivity be-
HL-60
100
75
50
25
0
3
10 30 60
18
3
10 30 60
19
3
10 30 60
22
3
10 30 60
31
3
10 30 60
24
3
10 30 60
28
μM
PBMC
100
75
50
25
0
3
10 30 60
18
3
10 30 60
19
3
10 30 60
22
3
10 30 60
31
3
10 30 60
24
3
10 30 60
28
μM
Figure 2. Differential effect of selected saponins (18, 19, 22, 31, 24 and 28) on proliferation of normal PBMCs versus HL-60 cells. Proliferation of HL-60 cells and quiescent
PBMCs cultured in presence of the indicated concentrations of the selected saponins for 24 h. Values represent means SEs of two independent experiments each performed
in triplicate.

4526
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
tween malign and benign blood cells, which along with their great
synthetic availability supports our proposal3 of considering this
family of spirostanyl glycosides as a new type of cytotoxic lead
compound. The biological evaluation of the spirostanyl b-chacotri-
osides confirms that those saponins incorporating very lipophilic
aglycones are the most cytotoxic ones among their congeners,
although they were also toxic against benign blood cells. In con-
trast, either the incorporation of more polar functionalities or a
lack of rigidity derived from the opening of the fused-ring system
leads to a significant drop in the cytotoxicity against HL-60. These
results support previous information from our and other groups as
well as provide new insights into the structure–cytotoxicity rela-
tionship of spirostan saponins and related glycosides.
(CH2); 30.3 (CH); 31.2, 31.3, 31.4, 31.8, 31.9 (CH2); 34.9 (CH); 35.7
(C); 36.8, 38.0 (CH2); 41.8, 44.8 (CH); 47.8 (C); 55.6, 56.7, 56.9
(CH); 66.8 (CH2); 71.1, 80.4 (CH); 103.9 (CH2); 109.3, 156.6 (C).
HRMS (ESI-FT-ICR) m/z: 451.3197 [M+Na]+ (calcd for C28H44O3Na:
451.3188).
4.1.2. 12b-Methoxy tigogenin (8) and 12a-methoxy tigogenin
(9)
NaBH4 (503 mg, 13.3 mmol) was added to a solution of com-
pound 6 (4.84 g, 8.89 mmol) in CH2Cl2/MeOH (200 mL, 1:1, v/v)
at ꢀ10 °C. The reaction mixture was stirred at this temperature
for 30 min and then treated with acetic acid (500 lL). The volatiles
were evaporated under reduced pressure to dryness and the result-
ing crude product was dissolved in the solvent mixture of dry DMF/
THF (15 mL, 2:1, v/v). The stirring solution was cooled to 0 °C and
treated with NaH (60% in mineral oil, 355 mg, 8.89 mmol) under
nitrogen atmosphere. The suspension was stirred for 10 min under
nitrogen atmosphere, treated with CH3I (0.7 mL, 10.7 mmol) and
then stirred at room temperature for additional 10 h. The reaction
mixture was poured into 100 mL of cold water and extracted with
CH2Cl2 (3 ꢂ 50 mL). The combined organic phases were washed
with aq 10% HCl (3 ꢂ 40 mL), satd aq NaHCO3 (3 ꢂ 40 mL), brine
(50 mL) and then dried over anhydrous Na2SO4 and concentrated
under reduced pressure to dryness. The crude product was purified
by flash column chromatography (n-hexane/EtOAc 50:1) to furnish
the 12b-OMe isomer (3.09 g, 62% yield after two steps) and the
4. Experimental
4.1. General
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 accomplished
by analysis of the 1H–1H COSY and HSQC data. High resolution ESI
mass spectra were obtained from a Fourier transform ion cyclotron
resonance (FT-ICR) mass spectrometer, an RF-only hexapole ion
guide and an external electrospray ion source. Flash column chro-
matography was carried out using Silica Gel 60 (230–400 mesh)
and analytical thin layer chromatography (TLC) was performed
12a-OMe isomer (698 mg, 14% yield).
4.1.3. 12b-Methoxy tigogenin (8)
D
9-hecogenin
The 12b-OMe isomer (1.55 g, 2.76 mmol) was deprotected with
TBAF (1.44 g, 5.52 mmol) in anhydrous THF (60 mL) in a similar way
as described in Section 4.1.1. Flash column chromatography purifi-
cation (n-hexane/EtOAc 3:1) afforded 8 (1.15 g, 93%) as a white
using silica gel aluminum sheets. Hecogenin (5),
(10) and tigogenin (11) are naturally occurring, commercially avail-
able spirostan sapogenins. Compound 6 was synthesized according
to a reported procedure.6 All commercially available chemicals
were used without further purification.
solid. Rf = 0.22 (n-hexane/EtOAc 3:1). Mp: 193–194 °C.
½ ꢃ
a 2D0
ꢀ115.9 (c 1.5, CHCl3). IR (KBr, cmꢀ1
) m: 3361, 2933, 2877, 1055.
1H NMR (500 MHz, CDCl3): d = 0.73 (s, 3H, CH3); 0.78 (d, 3H,
J = 6.3 Hz, CH3); 0.82 (s, 3H, CH3); 0.99 (d, 3H, J = 6.6 Hz, CH3);
4.1.1. 12-Methylene tigogenin (7)
BuLi (2.2 mL, 2.5 M in n-hexane) was added to a suspension of
the phosphonium salt CH3Ph3P+Br (1.97 g, 5.52 mmol) in THF
(40 mL) under nitrogen atmosphere at 0 °C. The mixture was stirred
for 1 h at this temperature and treated with a solution of compound
6 (1.0 g, 1.84 mmol) in THF (40 mL). The reaction mixture was stir-
red for 3 h and then concentrated under reduced pressure to
dryness. The residue was dissolved in CH2Cl2 (100 mL) and washed
with dilute aq HCl (3 ꢂ 30 mL), satd aq NaHCO3 (3 ꢂ 30 mL) and
brine (50 mL). The organic phase was dried over anhydrous Na2SO4
and evaporated to dryness. The crude product was purified by flash
column chromatography (n-hexane/EtOAc 50:1) to afford corre-
sponding methylene–spirostane as a white solid. This pure com-
pound was dissolved in anhydrous THF (30 mL) and treated with
tetra-n-butyl ammonium fluoride (TBAF, 690 mg, 2.64 mmol). The
reaction mixture was stirred at 65 °C for 8 h and then evaporated
under reduced pressure to dryness. The residue was dissolved in
CH2Cl2 (50 mL) and washed with dilute aq HCl (3 ꢂ 15 mL), satd
aq NaHCO3 (3 ꢂ 15 mL) and brine (30 mL). The organic phase was
dried over anhydrous Na2SO4 and evaporated to dryness. The crude
product was purified by flash column chromatography (n-hexane/
EtOAc 3:1) to afford compound 7 (521 mg, 77%) as a white solid.
2.75 (dd, 1H, J = 11.0/4.4 Hz, H-12
(t, 1H, J = 11.0 Hz, H-26ax); 3.46 (ddd, 1H, J = 11.0/4.4/2.3 Hz, H-
26eq); 3.58 (m, 1H, H-3 ); 4.37 (m, 1H, H-16
). 13C NMR
(125 MHz, CDCl3): d = 11.3, 12.2, 13.4, 17.1 (CH3); 25.3, 28.5, 28.8
(CH2); 30.2 (CH); 31.1, 31.3, 31.4, 31.9 (CH2); 34.2 (CH); 35.7 (C);
36.9, 38.0 (CH2); 42.4, 44.8 (CH); 45.6 (C); 53.1, 54.9 (CH); 56.8
(CH3); 62.0 (CH); 66.8 (CH2); 71.1, 80.6, 89.5 (CH); 109.5 (C). HRMS
(ESI-FT-ICR) m/z: 469.3289 [M+Na]+ (calcd for C28H46O4Na:
469.3294).
a); 3.31 (s, 3H, OCH3); 3.36
a
a
4.1.4. 12
a
-Methoxy tigogenin (9)
-OMe isomer (349 mg, 0.622 mmol) was deprotected
The 12
a
with TBAF (324 mg, 1.24 mmol) in anhydrous THF (15 mL) in a
similar way as described in Section 4.1.1. Flash column chromatog-
raphy purification (n-hexane/EtOAc 3:1) afforded 9 (253 mg, 91%)
as a white solid. Rf = 0.26 (n-hexane/EtOAc 3:1). Mp: 191–193 °C.
½
a 2D0
ꢃ
ꢀ51.8 (c 0.3, CHCl3). IR (KBr, cmꢀ1
) m: 3364, 2933, 2879,
1055. 1H NMR (500 MHz, CDCl3): d = 0.77 (s, 3H, CH3); 0.78 (d,
3H, J = 6.3 Hz, CH3); 0.81 (s, 3H, CH3); 0.96 (d, 3H, J = 7.3 Hz,
CH3); 2.51 (dd, 1H, J = 8.8/6.9 Hz); 3.04 (t, 1H, J = 2.6 Hz, H-12b);
3.30 (s, 3H, OCH3); 3.37 (t, 1H, J = 10.9 Hz, H-26ax); 3.47 (ddd,
Rf = 0.31 (n-hexane/EtOAc 3:1). Mp: 232–234 °C. ½a D20
ꢀ29.3 (c
ꢃ
1.4, CHCl3). IR (KBr, cmꢀ1 max: 3234, 2933, 2885, 1048. 1H NMR
) m
1H, J = 10.9/4.2/2.0 Hz, H-26eq); 3.58 (m, 1H, H-3
a); 4.36 (m, 1H,
H-16a
). 13C NMR (125 MHz, CDCl3): d = 12.2, 14.5, 17.0, 17.1
(400 MHz, CDCl3): d = 0.78 (d, 3H, J = 6.3 Hz, CH3); 0.85 (s, 3H,
CH3); 0.89 (s, 3H, CH3); 1.07 (d, 3H, J = 6.9 Hz, CH3); 2.32 (dd, 1H,
J = 8.8/6.6 Hz); 3.37 (t, 1H, J = 10.9 Hz, H-26ax); 3.47 (ddd, 1H,
(CH3); 23.5, 28.6, 28.8 (CH2); 30.3 (CH); 31.4, 31.5, 31.6, 31.9
(CH2); 35.1 (CH); 35.2 (C); 36.9, 38.2 (CH2); 41.6 (CH); 44.5 (C);
44.7, 47.7, 48.9, 52.9 (CH); 57.4 (CH3); 66.8 (CH2); 71.2, 80.4, 82.2
(CH); 109.2 (C). HRMS (ESI-FT-ICR) m/z: 469.3297 [M+Na]+ (calcd
for C28H46O4Na: 469.3294).
J = 10.9/4.4/2.2 Hz, H-26eq); 3.58 (m, 1H, H-3
16
); 4.55 (s, 1H, C@CH2a); 4.58 (s, 1H, C@CH2b). 13C NMR
(100 MHz, CDCl3): d = 12.1, 14.1, 17.1, 17.4 (CH3); 28.5, 28.8
a); 4.40 (m, 1H, H-
a

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4527
4.1.5. 12-Methylene-tigogenyl 2,3,4,6-tetra-O-benzoyl-b-
glucopyranoside (13)
The glucosyl donor 12 (177 mg, 0.239 mmol) and the spirostanic
acceptor 7 (78.7 mg, 0.184 mmol) were suspended in dry CH2Cl2
(4 mL) in the presence of 4 Å molecular sieves. The reaction mixture
was stirred at room temperature under nitrogen atmosphere for
D
-
in Section 4.1.3. Flash column chromatography purification (n-hex-
ane/EtOAc 3:1) afforded 15 (375.8 mg, 94%) as a white solid.
Rf = 0.37 (n-hexane/EtOAc 3:1). Mp: 176–178 °C. ½a D20
ꢃ
+8.2 (c 1.1,
CHCl3). IR (KBr, cmꢀ1
) m: 2950, 2929, 2870, 2372, 1736, 1316,
1266, 1178, 1096, 1071. 1H NMR (500 MHz, CDCl3): d = 0.69
(s, 3H, CH3); 0.75 (s, 3H, CH3); 0.78 (d, 3H, J = 6.6 Hz, CH3); 0.97
(d, 3H, J = 6.9 Hz, CH3); 2.51 (dd, 1H, J = 8.8/6.9 Hz); 3.03 (t, 1H,
J = 2.5 Hz, H-12b); 3.33 (s, 3H, OCH3); 3.37 (t, 1H, J = 10.9 Hz, H-
15 min and treated with a solution of TMSOTf (1.8 lL, 0.01 mmol)
in CH2Cl2 (0.35 mL). The stirring was continued for 1 h and then
the reaction was quenched by addition of Et3N and filtered. The fil-
trate was evaporated to dryness and the crude product was purified
by flash column chromatography (n-hexane/EtOAc 6:1) to afford 13
(177.9 mg, 96%) as a white solid. Rf = 0.30 (n-hexane/EtOAc 5:1).
26ax); 3.48 (m, 1H, H-26eq); 3.58 (m, 1H, H-3
a); 4.16 (m, 1H, H-5
Glc); 4.36 (m, 1H, H-16 ); 4.53 (dd, 1H, J = 12.0/6.0 Hz, H-6a Glc);
a
4.60 (dd, 1H, J = 11.9/3.4 Hz, H-6b Glc); 4.94 (d, 1H, J = 7.9 Hz, H-1
Glc); 5.49 (dd, 1H, J = 9.8/7.9 Hz, H-2 Glc); 5.63 (t, 1H, J = 9.8 Hz,
H-4 Glc); 5.90 (t, 1H, J = 9.8 Hz, H-3 Glc); 7.25–7.43 (m, 9H, Ar-H);
7.47–7.55 (m, 3H, Ar-H); 7.83, 7.90, 7.95, 8.01 (4 ꢂ m, 4 ꢂ 2H, Ar-
H). 13C NMR (125 MHz, CDCl3): d = 12.0, 14.5, 17.0, 17.1 (CH3);
23.4, 28.5, 28.8, 29.3 (CH2); 30.3 (CH); 31.4, 31.5, 31.7, 34.6 (CH2);
35.0 (CH); 35.2 (C); 36.6 (CH2); 41.6, 44.4 (CH); 44.5 (C); 47.6,
48.8, 53.0 (CH); 57.3 (CH3); 63.4, 66.8 (CH2); 70.2, 72.1, 72.2, 73.1,
79.9, 80.4, 82.2, 100.1 (CH); 109.1 (C); 128.1, 128.2, 128.3, 128.4
(CH); 128.8, 128.9, 129.5 (C); 129.7, 129.8, 132.9, 133.0, 133.1,
133.3 (CH); 165.0, 165.2, 165.8, 166.0 (C@O). HRMS (ESI-FT-ICR)
m/z: 1047.4863 [M+Na]+ (calcd for C62H72O13Na: 1047.4871).
Mp: 182–183 °C. ½a D20
ꢃ
ꢀ6.7 (c 1.4, CHCl3). IR (KBr, cmꢀ1
) m: 2955,
2930, 2874, 1735, 1269, 1096, 1070. 1H NMR (500 MHz, CDCl3):
d = 0.64 (s, 3H, CH3); 0.72 (s, 3H, CH3); 0.81 (d, 3H, J = 6.3 Hz,
CH3); 1.10 (d, 3H, J = 6.8 Hz, CH3); 3.38 (t, 1H, J = 10.9 Hz,
H-26ax); 3.45–3.50 (m, 1H, H-26eq); 3.55–3.64 (m, 1H, H-3
a);
4.11–4.17 (m, 1H, H-5 Glc); 4.38–4.46 (m, 1H, H-16 ); 4.53 (dd,
a
1H, J = 12.1/5.9 Hz, H-6a Glc); 4.60 (dd, 1H, J = 12.1/3.9 Hz, H-6b
Glc); 4.77 (d, 1H, J = 2.0 Hz, C@CH2a); 4.84 (d, 1H, J = 2.1 Hz,
C@CH2b); 4.95 (d, 1H, J = 7.6 Hz, H-1 Glc); 5.49 (dd, 1H, J = 9.8/
7.8 Hz, H-2 Glc); 5.63 (t, 1H, J = 9.8/9.5 Hz, H-4 Glc); 5.89 (t, 1H,
J = 9.6 Hz, H-3 Glc); 7.28–7.56 (m, 12H, Ar-H); 7.83, 7.90, 7.95,
8.01 (4 ꢂ m, 4 ꢂ 2H, Ar-H). 13C NMR (125 MHz, CDCl3): d = 12.1,
14.1, 17.1, 17.4 (CH3); 28.5, 28.8, 29.7 (CH2); 30.3 (CH); 31.2, 31.4,
31.7, 32.2 (CH2); 34.9 (CH); 35.7 (C); 36.8, 38.0 (CH2); 40.6 (C);
41.8, 44.7, 62.2 (CH); 63.5, 66.8 (CH2); 70.6, 71.1, 72.5, 72.6, 80.4,
81.2, 100.1 (CH); 104.3 (CH2); 109.6 (C); 128.6, 128.7, 128.8, 128.9
(CH); 129.2, 129.3, 129.9 (C); 130.1, 130.2, 133.4, 133.5, 133.6,
133.8 (CH); 156.6 (C); 165.5, 165.7, 166.3, 166.5 (C@O). HRMS
(ESI-FT-ICR) m/z: 1029.4763 [M+Na]+ (calcd for C62H70O12Na:
1029.4765).
4.1.8.
glucopyranoside (16)
The glucosyl donor 12 (189.7 mg, 0.256 mmol), the spirostanic
acceptor 10 (84.4 mg, 0.197 mmol) and TMSOTf (1.8 L, 0.01 mmol)
D D-
9-Hecogenyl 2,3,4,6-tetra-O-benzoyl-b-
l
were reacted in dry CH2Cl2 (4 mL) in a similar way as described in
Section 4.1.3. Flash column chromatography purification (n-hex-
ane/EtOAc 3:1) afforded 16 (182.5 mg, 92%) as a white solid.
Rf = 0.27 (n-hexane/EtOAc 3:1). Mp: 195–197 °C. ½a D20
ꢃ
+24.8 (c 1.5,
CHCl3). IR (KBr, cmꢀ1
) m: 2930, 2870, 2371, 1736, 1266, 1096,
1071. 1H NMR (500 MHz, CDCl3): d = 0.79 (d, 3H, J = 6.3 Hz, CH3);
0.90 (s, 3H, CH3); 0.95 (s, 3H, CH3); 1.10 (d, 3H, J = 6.9 Hz, CH3);
2.16 (m, 1H); 2.38 (dd, 1H, J = 8.8/7.3 Hz); 2.42–2.47 (m, 1H); 3.35
(t, 1H, J = 10.9 Hz, H-26ax); 3.47–3.50 (m, 1H, H-26eq); 3.55 (m,
4.1.6. 12b-Methoxy tigogenyl 2,3,4,6-tetra-O-benzoyl-b-D-
glucopyranoside (14)
The glucosyl donor 12 (192 mg, 0.259 mmol), the spirostanic
acceptor 8 (89 mg, 0.199 mmol) and TMSOTf (1.8 L, 0.01 mmol)
l
1H, H-3a); 4.17 (m, 1H, H-5 Glc); 4.38 (m, 1H, H-16a); 4.55 (dd,
were reacted in dry CH2Cl2 (4 mL) in a similar way as described in
Section 4.1.3. Flash column chromatography purification (n-hex-
ane/EtOAc 5:1) afforded 14 (181.6 mg, 89%) as a white solid.
1H, J = 12.0/6.0 Hz, H-6a Glc); 4.59 (dd, 1H, J = 12.0/3.5 Hz, H-6b
Glc); 4.92 (d, 1H, J = 7.6 Hz, H-1 Glc); 5.48 (dd, 1H, J = 9.8/7.9 Hz,
H-2 Glc); 5.61 (m, 2H, H-4 Glc, H-11); 5.89 (t, 1H, J = 9.7 Hz, H-3
Glc); 7.27–7.44 (m, 9H, Ar-H); 7.48–7.58 (m, 3H, Ar-H); 7.82 (dd,
2H, J = 8.5/1.3 Hz, Ar-H); 7.90 (dd, 2H, J = 8.3/1.1 Hz, Ar-H); 7.95
(dd, 2H, J = 8.2/1.3 Hz, Ar-H); 8.01 (dd, 2H, J = 8.5/1.3 Hz, Ar-H).
13C NMR (125 MHz, CDCl3): d = 13.1, 15.0, 17.1, 18.3 (CH3); 27.5,
28.7, 29.1 (CH2); 30.2 (CH); 31.3, 31.4, 32.4, 34.4, 34.5 (CH2); 36.7
(CH); 39.2 (C); 42.3, 42.4 (CH); 51.0 (C); 52.4, 53.7 (CH); 63.3, 66.9
(CH2); 70.0, 72.1, 72.9, 79.4, 79.7, 100.4 (CH); 109.4 (C); 119.9,
128.2, 128.3, 128.4 (CH); 128.7, 128.8, 129.4, 129.5 (C); 129.6,
129.7, 129.8, 133.1, 133.2, 133.3, 133.4 (CH); 165.0, 165.2, 165.8,
166.0 (C@O); 170.8 (C); 204.9 (C@O). HRMS (ESI-FT-ICR) m/z:
1029.4384 [M+Na]+ (calcd for C61H66O13Na: 1029.4401).
Rf = 0.25 (n-hexane/EtOAc 5:1). Mp: 158–160 °C. ½a D20
ꢃ
+24.5 (c 1.1,
CHCl3). IR (KBr, cmꢀ1
) m: 2930, 2877, 2379, 1730, 1271, 1094,
1071. 1H NMR (500 MHz, CDCl3): d = 0.70, 0.71 (2 ꢂ s, 2 ꢂ 3H,
2 ꢂ CH3); 0.78 (d, 3H, J = 6.4 Hz, CH3); 1.00 (d, 3H, J = 6.4 Hz, CH3);
2.74 (dd, 1H, J = 10.8/4.4 Hz, H-12a); 3.33 (s, 3H, OCH3); 3.37 (t,
1H, J = 10.9 Hz, H-26ax); 3.45–3.49 (m, 1H, H-26eq); 3.60 (m, 1H,
H-3 ); 4.13–4.18 (m, 1H, H-5 Glc); 4.38 (m, 1H, H-16 ); 4.53 (dd,
a
a
1H, J = 11.9/5.8 Hz, H-6a Glc); 4.60 (dd, 1H, J = 12.0/3.4 Hz, H-6b
Glc); 4.94 (d, 1H, J = 7.8 Hz, H-1 Glc); 5.48 (dd, 1H, J = 9.7/7.8 Hz,
H-2 Glc); 5.63 (t, 1H, J = 9.7 Hz, H-4 Glc); 5.90 (t, 1H, J = 9.7 Hz, H-
3 Glc); 7.25–7.55 (m, 12H, Ar-H); 7.83, 7.90, 7.96, 8.02 (4 ꢂ m,
4 ꢂ 2H, Ar-H). 13C NMR (125 MHz, CDCl3): d = 11.3, 12.1, 13.4,
17.1 (CH3); 25.2, 28.4, 28.8, 29.3 (CH2); 30.3 (CH); 31.1, 31.4, 31.9
(CH2); 34.2 (CH); 34.4 (CH2); 35.7 (C); 36.8 (CH2); 42.4, 44.7 (CH);
45.6 (C); 53.1, 55.0 (CH); 56.7 (CH3); 62.1 (CH); 63.4, 66.8 (CH2);
70.2, 72.1, 72.2, 73.1, 79.7, 80.5, 89.4, 99.9 (CH); 109.5 (C); 128.2,
128.3, 128.4 (CH); 128.8, 128.9, 129.5 (C); 129.7, 129.8, 132.9,
133.0, 133.1, 133.4 (CH); 165.0, 165.2, 165.8, 166.0 (C@O). HRMS
(ESI-FT-ICR) m/z: 1047.4860 [M+Na]+ (calcd for C62H72O13Na:
1047.4871).
4.1.9. Tigogenyl 2,3,4,6-tetra-O-benzoyl-b-D-glucopyranoside
(17)
The glucosyl donor 12 (1.04 g, 1.40 mmol), the spirostanic accep-
tor 11 (420 mg, 1.01 mmol) and TMSOTf (9.1 L, 0.051 mmol) were
l
reacted in dry CH2Cl2 (20 mL) in a similar way as described in Section
4.1.3. Flash column chromatography purification (n-hexane/EtOAc
4:1?2:1) afforded 17 (874.5 mg, 87%) as a white solid. Rf = 0.29
(n-hexane/EtOAc 4:1). Mp: 143–145 °C. ½a D20
ꢃ
+13.6 (c 1.1, CHCl3).
IR (KBr, cmꢀ1 : 2930, 2874, 1736, 1269, 1095, 1071. 1H NMR
) m
4.1.7. 12
glucopyranoside (15)
The glucsosyl donor 12 (375.7 mg, 0.507 mmol), the spirostanic
acceptor 9 (174 mg, 0.390 mmol) and TMSOTf (3.5 L, 0.02 mmol)
were reacted in dry CH2Cl2 (10 mL) in a similar way as described
a
-Methoxy-tigogenyl 2,3,4,6-tetra-O-benzoyl-b-
D
-
(500 MHz, CDCl3): d = 0.69 (s, 3H, CH3); 0.73 (s, 3H, CH3); 0.78 (d,
3H, J = 6.3 Hz, CH3); 0.95 (d, 3H, J = 6.9 Hz, CH3); 3.37
(t, 1H, J = 10.9 Hz, H-26ax); 3.47 (m, 1H, H-26eq); 3.59 (m, 1H, H-
l
3a); 4.15 (m, 1H, H-5 Glc); 4.38 (m, 1H, H-16
a); 4.52 (dd, 1H,
J = 12.0/6.0 Hz, H-6a Glc); 4.60 (dd, 1H, J = 12.0/3.5 Hz, H-6b Glc);

4528
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4.94 (d, 1H, J = 7.6 Hz, H-1 Glc); 5.48 (dd, 1H, J = 9.6/7.9 Hz, H-2 Glc);
5.62 (t, 1H, J = 9.8 Hz, H-4 Glc); 5.89 (t, 1H, J = 9.6 Hz, H-3 Glc); 7.27–
7.44 (m, 9H, Ar-H); 7.48–7.55 (m, 3H, Ar-H); 7.83, 7.90, 7.95, 8.01
(4 ꢂ m, 4 ꢂ 2H, Ar-H). 13C NMR (125 MHz, CDCl3): d = 12.2, 14.5,
16.4, 17.1 (CH3); 21.0, 28.5, 28.8, 29.3 (CH2); 30.3 (CH); 31.4, 31.7,
32.2, 34.6 (CH2); 35.0 (CH); 35.6 (C); 36.8, 40.1 (CH2); 40.6 (C);
41.6, 44.7, 62.2 (CH); 63.5, 66.8 (CH2); 70.2, 72.1, 72.2, 73.1, 80.0,
80.8, 100.0 (CH); 109.2 (C); 128.1, 128.2, 128.3, 128.4 (CH); 128.8,
128.9, 129.5 (C); 129.7, 129.8, 133.0, 133.1, 133.2, 133.4 (CH);
165.0, 165.3, 165.8, 166.1 (C@O). HRMS (ESI-FT-ICR) m/z:
1017.4747 [M+Na]+ (calcd for C61H70O12Na: 1017.4765).
4.44 (d, 1H, J = 7.6 Hz, H-1 Glc); 4.85 (t, 1H, J = 9.1 Hz, H-3 Glc).
13C NMR (125 MHz, CDCl3): d = 11.3, 12.2, 13.4, 17.1 (CH3); 25.3
(CH2); 27.0, 27.1 (CH3); 28.6, 28.8, 29.3 (CH2); 30.3 (CH); 31.1,
31.3, 31.9 (CH2); 34.2 (CH); 34.3 (CH2); 35.9 (C); 36.9 (CH2); 38.8,
39.0 (C); 42.4, 44.8 (CH); 45.6 (C); 53.1, 54.9 (CH); 56.8 (CH3);
62.0 (CH); 63.5, 66.8 (CH2); 70.1, 72.1, 74.2, 78.0, 78.7, 80.6, 89.4,
100.8 (CH); 109.5 (C); 178.6, 180.2 (C@O). HRMS (ESI-FT-ICR) m/z:
799.4968 [M+Na]+ (calcd for C44H72O11Na: 799.4972).
4.1.12. 12a-Methoxy-tigogenyl 3,6-di-O-pivaloyl-b-D-
glucopyranoside (20)
Glucoside 15 (342.3 mg, 0.334 mmol) was deprotected with
NaOMe in CH2Cl2/MeOH (20 mL, 1:1, v/v) in a similar way as
described in Section 4.1.8. The resulting product was selectively
4.1.10. 12-Methylene-tigogenyl 3,6-di-O-pivaloyl-b-D-
glucopyranoside (18)
NaOMe was added to a solution of compound 13 (157.9 mg,
0.157 mmol) in CH2Cl2/MeOH (10 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
filtered. The filtrate was concentrated under reduced pressure and
the residue was washed with Et2O (5 ꢂ 10 mL) and dried in vacuo
protected with pivaloyl chloride (205 lL, 1.67 mmol) in anhydrous
pyridine (8 mL) according to the procedure described in Section
4.1.8. Flash column chromatography purification (n-hexane/EtOAc
3:1) afforded 20 (173.9 mg, 67%) as a white solid. Rf = 0.32 (n-hex-
ane/EtOAc 3:1). Mp: 167–169 °C. ½a D20
ꢀ26.7 (c 0.5, CHCl3). IR (KBr,
ꢃ
cmꢀ1 : 3454, 2931, 2873, 2379, 1724, 1164, 1083, 1050. 1H NMR
) m
to furnish the corresponding b-
D-glucoside. This product was dis-
(500 MHz, CDCl3): d = 0.77 (s, 3H, CH3); 0.78 (d, 3H, J = 6.3 Hz, CH3);
0.81 (s, 3H, CH3); 0.97 (d, 3H, J = 6.9 Hz, CH3); 1.21, 1.24 (2 ꢂ s,
2 ꢂ 9H, 2 ꢂ (CH3)3C); 2.51 (dd, 1H, J = 8.9/6.7 Hz); 3.04 (t, 1H,
J = 2.6 Hz, H-12b); 3.30 (s, 3H, OCH3); 3.37 (t, 1H, J = 10.9 Hz,
H-26ax); 3.44–3.49 (m, 3H, H-2 Glc, H-4 Glc, H-26eq); 3.55 (m,
solved in anhydrous pyridine (5 mL) and the solution was cooled
to ꢀ15 °C under nitrogen atmosphere. Pivaloyl chloride (97 L,
l
0.785 mmol) was added dropwise and the reaction course was
monitored by TLC. The stirring was continued at room temperature
until the disappearance of the intermediate. The mixture was then
diluted with EtOAc (25 mL) and washed with dilute aq HCl, satd aq
NaHCO3 and brine. The organic phase was dried over anhydrous
Na2SO4 and evaporated to dryness. The crude product was purified
by flash column chromatography (n-hexane/EtOAc 5:1) to afford 18
(75.1 mg, 63%) as a white solid. Rf = 0.26 (n-hexane/EtOAc 5:1). Mp:
1H, H-5 Glc); 3.61 (m, 1H, H-3
a); 4.27 (dd, 1H, J = 12.0/6.6 Hz,
H-6a Glc); 4.36 (m, 1H, H-16 ); 4.40–4.44 (m, 2H, H-6b Glc, H-1
a
Glc); 4.84 (t, 1H, J = 9.1 Hz, H-3 Glc). 13C NMR (125 MHz, CDCl3):
d = 12.2, 14.5, 17.0, 17.1 (CH3); 23.5 (CH2); 27.0, 27.1 (CH3); 28.6,
28.8, 29.3 (CH2); 30.3 (CH); 31.4, 31.5, 31.8, 34.5 (CH2); 35.1
(CH); 35.3 (C); 36.7 (CH2); 38.8, 38.9 (C); 41.6, 44.5 (CH); 44.6
(C); 47.6, 48.9, 52.9 (CH); 57.4 (CH3); 63.5, 66.8 (CH2); 70.1, 72.0,
74.3, 78.1, 78.8, 80.4, 82.2, 100.8 (CH); 109.2 (C); 178.6, 180.3
(C@O). HRMS (ESI-FT-ICR) m/z: 799.4970 [M+Na]+ (calcd for
115–116 °C. ½a 2D0
ꢃ
ꢀ33.1 (c 1.3, CHCl3). IR (KBr, cmꢀ1
) m: 3450, 2955,
2933, 2875, 1721, 1287, 1165, 1074, 1050. 1H NMR (500 MHz,
CDCl3): d = 0.76 (s, 3H, CH3); 0.80 (d, 3H, J = 6.3 Hz, CH3); 1.07 (s,
3H, CH3); 1.10 (d, 3H, J = 6.6 Hz, CH3); 1.21, 1.22 (2 ꢂ s, 2 ꢂ 9H,
2 ꢂ (CH3)3C); 3.37 (t, 1H, J = 11.0 Hz, H-26ax); 3.43–3.52 (m, 3H,
H-2 Glc, H-4 Glc, H-26eq); 3.54–3.59 (m, 1H, H-5 Glc); 3.62–3.67
C44H72O11Na: 799.4972).
4.1.13.
D
9-Hecogenyl 3,6-di-O-pivaloyl-b-
D-glucopyranoside (21)
(m, 1H, H-3
(m, 3H, H-16
4.77 (d, 1H, J = 1.9 Hz, C@CH2b); 4.85 (t, 1H, J = 9.4 Hz, H-3 Glc). 13
a
a
); 4.25 (dd, 1H, J = 11.7/6.6 Hz, H-6a Glc); 4.40–4.46
, H-6b Glc, H-1 Glc); 4.60 (d, 1H, J = 1.6 Hz, C@CH2a);
Glucoside 16 (165.5 mg, 0.164 mmol) was deprotected with
NaOMe in CH2Cl2/MeOH (10 mL, 1:1, v/v) in a similar way as
described in Section 4.1.8. The resulting product was selectively
C
NMR (125 MHz, CDCl3): d = 11.4, 12.1, 12.8, 13.9, 27.0, 27.1 (CH3);
28.6, 28.8, 29.3 (CH2); 30.3, 30.4 (CH); 31.1, 31.4, 31.6 (CH2); 32.2
(CH); 33.8, 36.7, 37.8 (CH2); 38.8, 39.0 (C); 41.8, 44.6 (CH); 46.6
(C); 53.5, 53.8, 56.8, 58.2 (CH); 63.6, 66.8 (CH2); 70.1, 72.0, 74.2,
76.1, 78.0, 79.4, 81.1, 101.1 (CH); 104.9 (CH2); 109.2, 155.7 (C);
178.6, 180.3 (C@O). HRMS (ESI-FT-ICR) m/z: 781.4872 [M+Na]+
(calcd for C44H70O10Na: 781.4867).
protected with pivaloyl chloride (100 lL, 0.82 mmol) in anhydrous
pyridine (5 mL) according to the procedure described in Section
4.1.8. Flash column chromatography purification (n-hexane/EtOAc
2:1) afforded 21 (79.5 mg, 68%) as a white solid. Rf = 0.33 (n-hex-
ane/EtOAc 2:1). Mp: 139–140 °C. ½a D20
ꢀ8.0 (c 1.3, CHCl3). IR (KBr,
ꢃ
cmꢀ1
) m: 3452, 2959, 2933, 2877, 1707, 1285, 1167, 1082, 1052.
1H NMR (500 MHz, CDCl3): d = 0.79 (d, 3H, J = 6.3 Hz, CH3); 0.92
(s, 3H, CH3); 1.07 (s, 3H, CH3); 1.10 (d, 3H, J = 6.9 Hz, CH3); 1.21,
1.24 (2 ꢂ s, 2 ꢂ 9H, 2 ꢂ (CH3)3C); 2.04 (m, 2H); 2.19 (m, 1H);
2.38 (dd, 1H, J = 8.8/6.9 Hz); 2.41 (d, 1H, J = 2.5 Hz); 2.50 (m, 1H);
3.04 (d, 1H, J = 4.7 Hz); 3.35 (t, 1H, J = 10.9 Hz, H-26ax); 3.40–
3.43 (m, 3H, H-2 Glc, H-4 Glc, H-26eq); 3.56 (m, 1H, H-5 Glc);
4.1.11. 12b-Methoxy-tigogenyl 3,6-di-O-pivaloyl-b-D-
glucopyranoside (19)
Glucoside 14 (158.6 mg, 0.155 mmol) was deprotected with
NaOMe in CH2Cl2/MeOH (10 mL, 1:1, v/v) in a similar way as de-
scribed in Section 4.1.8. The resulting product was selectively
3.61 (m, 1H, H-3a); 4.25 (dd, 1H, J = 12.0/6.6 Hz, H-6a Glc); 4.38
protected with pivaloyl chloride (95
lL, 0.775 mmol) in anhydrous
(m, 1H, H-16 ); 4.42 (dd, 1H, J = 12.0/2.2 Hz, H-6b Glc); 4.43 (d,
a
pyridine (5 mL) according to the procedure described in Section
4.1.8. Flash column chromatography purification (n-hexane/EtOAc
4:1) afforded 19 (78.3 mg, 65%) as a white solid. Rf = 0.29 (n-hex-
1H, J = 7.6 Hz, H-1 Glc); 4.85 (t, 1H, J = 9.3 Hz, H-3 Glc), 5.68 (d,
1H, J = 1.9 Hz, H-11). 13C NMR (125 MHz, CDCl3): d = 13.1, 15.0,
17.1, 18.4, 27.0, 27.1 (CH3); 27.7, 28.7, 29.2 (CH2); 30.2 (CH);
31.3, 31.5, 32.4, 34.4, 34.7 (CH2); 36.8 (CH); 38.8, 39.0, 39.3 (C);
42.4, 42.5 (CH); 51.0 (C); 52.4, 53.7 (CH); 63.6, 66.9 (CH2); 70.0,
72.1, 74.2, 77.9, 78.3, 79.7, 101.3 (CH); 109.4 (C); 120.0 (CH);
170.7 (C); 178.6, 180.2, 204.9 (C@O). HRMS (ESI-FT-ICR) m/z:
781.4500 [M+Na]+ (calcd for C43H66O11Na: 781.4503).
ane/EtOAc 4:1). Mp: 142–144 °C. ½a D20
ꢀ73.6 (c 1.4, CHCl3). IR
ꢃ
(KBr, cmꢀ1
) m: 3454, 2932, 2874, 2379, 1722, 1164, 1083, 1049.
1H NMR (500 MHz, CDCl3): d = 0.73 (s, 3H, CH3); 0.78 (d, 3H,
J = 6.6 Hz, CH3); 0.82 (s, 3H, CH3); 1.00 (d, 3H, J = 6.6 Hz, CH3);
1.21, 1.24 (2 ꢂ s, 2 ꢂ 9H, 2 ꢂ (CH3)3C); 2.76 (dd, 1H, J = 11.0/
4.4 Hz, H-12a); 3.32 (s, 3H, OCH3); 3.36 (t, 1H, J = 11.0 Hz, H-
26ax); 3.44–3.47 (m, 3H, H-2 Glc, H-4 Glc, H-26eq); 3.56 (m, 1H,
H-5 Glc); 3.62 (m, 1H, H-3 ); 4.25 (dd, 1H, J = 12.0/6.6 Hz, H-6a
Glc); 4.39 (m, 1H, H-16 ); 4.43 (dd, 1H, J = 11.7/2.5 Hz, H-6b Glc);
4.1.14. Tigogenyl 3,6-di-O-pivaloyl-b-D-glucopyranoside (22)
Glucoside 17 (758.4 mg, 0.762 mmol) was deprotected with
NaOMe in CH2Cl2/MeOH (60 mL, 1:1, v/v) in a similar way as
a
a

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4529
described in Section 4.1.8. The resulting product was selectively
protected with pivaloyl chloride (470 L, 3.81 mmol) in anhydrous
pyridine (15 mL) according to the procedure described in Section
4.1.8. Flash column chromatography purification (n-hexane/EtOAc
3:1) afforded 22 (364.3 mg, 64%) as a white solid. Rf = 0.29 (n-hex-
4.1.16. 12b-Methoxy-tigogenyl 2,4-di-O-(
b- -glucopyranoside (25)
Acceptor 19 (176.9 mg, 0.228 mmol) and donor 23 (307.3 mg,
0.707 mmol) in dry CH2Cl2 (30 mL) were reacted in the presence
L, 1.003 mmol) in a similar way as described in
a
-
L
-rhamnopyranosyl)-
l
D
of BF3ꢁEt2O (127
l
ane/EtOAc 3:1). Mp: 196–197 °C. ½a D20
ꢃ
ꢀ49.5 (c 0.95, CHCl3). IR
Section 4.1.13. The resulting crude product was purified by flash
column chromatography (n-hexane/EtOAc 2:1) to afford the pro-
tected saponin (431 mg), which was deprotected with NaOH
(1.0 M, 5 mL) in THF/MeOH (20 mL, 1:1, v/v) in a similar way as
described in Section 4.1.13. Flash column chromatography purifi-
cation (CHCl3/MeOH 4:1) afforded 25 (141.8 mg, 69%) as a white
(KBr, cmꢀ1
) m: 3451, 2932, 2873, 1721, 1287, 1241, 1164, 1086,
1051. 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); 0.95 (d, 3H, J = 6.9 Hz, CH3); 1.21,
1.24 (2 ꢂ s, 2 ꢂ 9H, 2 ꢂ (CH3)3C); 3.37 (t, 1H, J = 11.0 Hz, H-26ax);
3.44–3.48 (m, 3H, H-2 Glc, H-4 Glc, H-26eq); 3.56 (m, 1H, H-5
Glc); 3.61 (m, 1H, H-3
a); 4.25 (dd, 1H, J = 12.0/6.6 Hz, H-6a Glc);
solid. Rf = 0.35 (CHCl3/MeOH 4:1). Mp: 168–170 °C. ½a D20
ꢀ128.9
ꢃ
4.38 (m, 1H, H-16 ); 4.42 (dd, 1H, J = 12.0/2.5 Hz, H-6b Glc); 4.44
a
(c 1.4, C5H5N). IR (KBr, cmꢀ1
) m: 3396, 2930, 2373, 1625, 1396,
(d, 1H, J = 7.9 Hz, H-1 Glc); 4.85 (t, 1H, J = 9.1 Hz, H-3 Glc). 13C
NMR (125 MHz, CDCl3): d = 12.3, 14.5, 16.5, 17.1 (CH3); 21.0 (CH2);
27.0, 27.1 (CH3); 28.6, 28.8, 29.4 (CH2); 30.3 (CH); 31.4, 31.7, 32.2,
34.5 (CH2); 35.1 (CH); 35.7 (C); 36.9 (CH2); 38.8, 39.0 (C); 40.0
(CH2); 40.5 (C); 41.6, 44.8, 54.3, 56.2, 62.2 (CH); 63.6, 66.8 (CH2);
70.1, 72.0, 74.2, 78.0, 79.0, 80.8, 100.9 (CH); 109.2 (C); 178.6, 180.3
(C@O). HRMS (ESI-FT-ICR) m/z: 769.4874 [M+Na]+ (calcd for
1044. 1H NMR (500 MHz, pyridine-d5): d = 0.71 (d, 3H, J = 5.7 Hz,
CH3); 0.87 (s, 3H, CH3); 0.94 (s, 3H, CH3); 1.27 (d, 3H, J = 6.9 Hz,
CH3); 1.64 (d, 3H, J = 6.3 Hz, CH3 Rha); 1.76 (d, 3H, J = 6.3 Hz, CH3
Rha0); 2.77 (dd, 1H, J = 11.0/4.4 Hz, H-12
a
); 3.38 (s, 3H, OCH3);
3.54 (t, 1H, J = 10.6 Hz, H-26ax); 3.60–3.63 (m, 1H, H-26eq); 3.72
(m, 1H, H-5 Glc); 3.94 (m, 1H, H-3 ); 4.12 (dd, 1H, J = 12.1/
a
3.5 Hz, H-6a Glc); 4.23–4.41 (m, 6H, H-2 Glc, H-3 Glc, H-4 Glc, H-
C43H70O10Na: 769.4867).
6b Glc, H-4 Rha, H-4 Rha0); 4.54–4.63 (m, 3H, H-3 Rha, H-3 Rha0,
H-16a); 4.70 (dd, 1H, J = 3.2/1.4 Hz, H-2 Rha); 4.84 (dd, 1H,
4.1.15. 12-Methylene-tigogenyl 2,4-di-O-(
a
-
L
-rhamnopyranosyl)
J = 3.3/1.7 Hz, H-2 Rha0); 4.90–4.96 (m, 2H, H-5 Rha, H-5 Rha0);
4.98 (d, 1H, J = 7.6 Hz, H-1 Glc); 5.86 (d, 1H, J = 1.7 Hz, H-1 Rha);
6.38 (d, 1H, J = 1.5 Hz, H-1 Rha0). 13C NMR (125 MHz, pyridine-
d5): d = 12.0, 12.7, 14.6, 17.7, 18.9, 19.0 (CH3); 29.3, 29.7, 30.3
(CH2); 31.0 (CH); 31.9, 32.2, 32.5, 34.7 (CH2); 34.9 (CH); 36.4 (C);
37.5 (CH2); 43.4, 45.0 (CH); 46.3 (C); 53.5, 55.5 (CH); 56.8 (CH3);
61.8 (CH2); 63.3 (CH); 67.2 (CH2); 69.9, 70.8, 72.8, 72.9, 73.1,
73.2, 74.3, 74.5, 79.1, 81.4, 89.7, 100.2, 102.5, 103.3 (CH); 109.8
(C). HRMS (ESI-FT-ICR) m/z: 923.4972 [M+Na]+ (calcd for
-b-D
-glucopyranoside (24)
BF3ꢁEt2O (130
lL, 1.034 mmol) was added under nitrogen atmo-
a stirring suspension of acceptor 18 (178.7 mg,
sphere to
0.235 mmol) and 4 Å molecular sieves in dry CH2Cl2 (10 mL) at
ꢀ80 °C. After stirring for 1 h at this temperature, a solution of
rhamnopyranosyl trichloroacetimidate 23 (316.9 mg, 0.729 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 ꢂ 15 mL) and brine (10 mL).
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 the protected
saponin (302 mg, partially unpurified with donor 23). An aqueous
solution of NaOH (1.0 M, 3 mL) was added to a solution of this
crude product in THF/MeOH (15 mL, 1:1, v/v) and the reaction mix-
ture 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 under reduced pressure and
the resulting crude product was purified by flash column chroma-
tography (CHCl3/MeOH 5:1) to afford the pure glycoside 24
(130 mg, 63%) as a white solid. Rf = 0.32 (CHCl3/MeOH 5:1). Mp:
C46H76O17Na: 923.4980).
4.1.17. 12
-b- -glucopyranoside (26)
Acceptor 20 (120.6 mg, 0.155 mmol) and donor 23 (209 mg,
0.481 mmol) in dry CH2Cl2 (20 mL) were reacted in the presence
L, 0.682 mmol) in a similar way as described in
a-Methoxy-tigogenyl 2,4-di-O-(a-L-rhamnopyranosyl)
D
of BF3ꢁEt2O (86
l
Section 4.1.13. The crude product was purified by flash column
chromatography (n-hexane/EtOAc 2:1) to afford the protected
saponin (298 mg). This product was deprotected with NaOH
(1.0 M, 4 mL) in THF/MeOH (16 mL, 1:1, v/v) in a similar way as
described in Section 4.1.13. Flash column chromatography purifi-
cation (CHCl3/MeOH 5:1?4:1) afforded 26 (93.6 mg, 67%) as a
white solid. Rf = 0.42 (CHCl3/MeOH 4:1). Mp: 173–175 °C. ½a D20
ꢃ
159–161 °C. ½a 2D0
ꢃ
ꢀ87.6 (c 1.50, C5H5N). IR (KBr, cmꢀ1
) m: 3390,
ꢀ46.3 (c 0.8, C5H5N). IR (KBr, cmꢀ1
) m: 3401, 2931, 1640, 1403,
2929, 1572, 1050. 1H NMR (500 MHz, pyridine-d5): d = 0.74 (m,
3H, CH3); 0.86 (m, 3H, CH3); 1.04 (m, 3H, CH3); 1.26 (m, 3H,
CH3); 1.64 (d, 3H, J = 6.3 Hz, CH3 Rha); 1.77 (d, 3H, J = 6.3 Hz, CH3
Rha0); 3.50–3.75 (m, 3H, H-26ax, H-26eq, H-5 Glc); 3.89–4.03 (m,
1046. 1H NMR (500 MHz, pyridine-d5): d = 0.70 (d, 3H, J = 5.4 Hz,
CH3); 0.87 (s, 6H, 2 ꢂ CH3); 1.18 (d, 3H, J = 6.9 Hz, CH3); 1.64 (d,
3H, J = 6.3 Hz, CH3 Rha); 1.76 (d, 3H, J = 6.3 Hz, CH3 Rha0); 2.77
(dd, 1H, J = 8.6/6.7 Hz); 3.06 (t, 1H, J = 2.3 Hz, H-12b); 3.34 (s, 3H,
OCH3); 3.51 (t, 1H, J = 10.6 Hz, H-26ax); 3.59 (m, 1H, H-26eq);
1H, H-3a); 4.13 (dd, 1H, J = 12.0/3.9 Hz, H-6a Glc); 4.23–4.41 (m,
6H, H-2 Glc, H-3 Glc, H-4 Glc, H-6b Glc, H-4 Rha, H-4 Rha0); 4.55
3.72 (m, 1H, H-5 Glc); 3.90 (m, 1H, H-3a); 4.13 (dd, 1H, J = 12.1/
(dd, 1H, J = 3.2/9.5 Hz, H-3 Rha); 4.61 (dd, 1H, J = 2.9/9.3 Hz, H-3
Rha0); 4.68–4.72 (m, 2H, H-16 , H-2 Rha); 4.84 (m, 1H, H-2 Rha0);
a
3.5 Hz, 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); 4.54–4.62 (m, 3H, H-3 Rha, H-3 Rha0,
4.92–5.02 (m, 5H, H-5 Rha, H-5 Rha0, H-1 Glc, C@CH2); 5.86 (s, 1H,
H-1 Rha); 6.38 (s, 1H, H-1 Rha0). 13C NMR (125 MHz, pyridine-d5):
d = 12.0, 14.7, 17.7, 17.8, 18.9, 19.1 (CH3); 29.6, 29.8, 30.0, 30.2
(CH2); 31.1, 31.2 (CH); 32.0, 32.1, 32.4, 32.5 (CH2); 32.7, 34.1
(CH); 34.2, 34.9 (CH2); 35.7 (CH); 35.8, 36.9, 37.6, 38.6 (CH2);
42.7, 42.9, 44.9, 45.0, 45.1, 46.3 (CH); 47.4 (C); 48.0, 48.7, 51.6,
54.4, 54.6, 57.6, 57.7, 58.2, 59.5 (CH); 62.0, 67.4, 67.7 (CH2); 69.6,
69.9, 70.9, 72.8, 72.9, 73.2, 73.3, 74.0, 74.4, 74.6, 74.9 (CH); 76.9
(C); 77.4, 77.7, 78.1, 78.4, 78.6, 79.5, 81.2, 81.9, 96.4, 100.3,
100.4, 100.5, 102.6, 103.4 (CH); 105.6, 106.9 (CH2); 109.7, 109.8
(C); 121.6 (CH); 143.9 (C); 156.9 (CH). HRMS (ESI-FT-ICR) m/z:
905.4871 [M+Na]+ (calcd for C46H74O16Na: 905.4875).
H-16a); 4.69 (dd, 1H, J = 3.2/1.6 Hz, H-2 Rha); 4.84 (dd, 1H,
J = 3.5/1.3 Hz, H-2 Rha0); 4.90–4.96 (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 = 1.8 Hz, H-1 Rha0). 13C NMR (125 MHz, pyridine-
d5): d = 12.7, 15.4, 17.6, 17.7, 18.9, 19.0 (CH3); 23.8, 29.3, 29.7,
30.3 (CH2); 31.0 (CH); 32.2, 32.3, 32.7, 34.9 (CH2); 35.7 (CH); 35.9
(C); 37.4 (CH2); 42.4, 45.0 (CH); 45.2 (C); 48.3, 49.7, 54.0 (CH);
57.3 (CH3); 61.8, 67.2 (CH2); 69.9, 70.8, 72.8, 72.9, 73.1, 73.2,
74.3, 74.5, 77.3, 77.6, 78.3, 78.5, 79.1, 81.2, 82.3, 100.3, 102.5,
103.3 (CH); 109.6 (C). HRMS (ESI-FT-ICR) m/z: 923.4980 [M+Na]+
(calcd for C46H76O17Na: 923.4980).

4530
K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4.1.18.
D
9-Hecogenyl 2,4-di-O-(
a
-L
-rhamnopyranosyl)-b-
D
-
4.1.20. (25R)-C-Homo-12a-oxa-3b-hydroxy-5a-spirostan-12-
glucopyranoside (27)
one (29)
Acceptor 21 (216.8 mg, 0.286 mmol) and donor 23 (385.5 mg,
0.887 mmol) in dry CH2Cl2 (40 mL) were reacted in the presence
A solution of m-CPBA (2.2 g, 12.8 mmol) in CH2Cl2 (100 mL) was
added to a stirred solution of hecogenin 5 (2.2 g, 5.12 mmol) in
CH2Cl2 (100 mL) at 0 °C. The reaction was allowed to reach room
temperature and stirred for 5 days in the dark. The mixture was
diluted with CHCl3 (150 mL) and washed with solutions of 10%
Na2SO3 (2 ꢂ 100 mL) and 10% NaHCO3 (2 ꢂ 100 mL). The organic
phase was dried over anhydrous Na2SO4 and evaporated under
reduced pressure. The resulting crude product was purified by
flash column chromatography (n-hexane/EtOAc 1:1) to yield the
lactone 29 (1.58 g, 69%). Rf = 0.38 (n-hexane/EtOAc 2:3). Mp:
of BF3ꢁEt2O (160
lL, 1.258 mmol) in a similar way as described in
Section 4.1.13. The crude product was purified by flash column
chromatography (n-hexane/EtOAc 3:2) to afford the protected
saponin (512 mg). This product was deprotected with NaOH
(1.0 M, 6 mL) in THF/MeOH (24 mL, 1:1, v/v) in a similar way as
described in Section 4.1.13. Flash column chromatography purifica-
tion (CHCl3/MeOH 4:1) afforded 27 (164.2 mg, 65%) as a white solid.
Rf = 0.33 (CHCl3/MeOH 4:1). Mp: 165–167 °C.
½
a 2D0
ꢃ
ꢀ81.6
(c 1.3, C5H5N). IR (KBr, cmꢀ1
)
m
: 3387, 2937, 2877, 2405, 1670,
215–217 °C. ½a 2D0
ꢃ
ꢀ93.0 (c 1.2, CHCl3). IR (KBr, cmꢀ1
) m: 3424,
1380, 1052. 1H NMR (500 MHz, pyridine-d5): d = 0.80 (d,
3H, J = 5.7 Hz, CH3); 1.06 (s, 3H, CH3); 1.11 (s, 3H, CH3); 1.42 (d,
3H, J = 6.9 Hz, CH3); 1.62 (d, 3H, J = 6.3 Hz, CH3 Rha); 1.73 (d, 3H,
J = 6.3 Hz, CH3 Rha0); 2.65 (dd, 1H, J = 8.6/7.1 Hz); 3.54 (t, 1H,
J = 10.7 Hz, H-26ax); 3.64 (m, 1H, H-26eq); 3.69 (m, 1H, H-5 Glc);
2937, 2870, 1700, 1052. 1H NMR (500 MHz, CDCl3): d = 0.77
(s, 3H, CH3); 0.78 (d, 3H, J = 6.3 Hz, CH3); 1.03 (d, 3H, J = 7.3 Hz,
CH3); 1.44 (s, 3H, CH3); 2.01 (m, 1H); 2.31 (m, 1H); 2.47 (dd, 1H,
J = 13.7/12.0 Hz); 2.55 (dd, 1H, J = 9.3/6.4 Hz); 2.74 (d, 1H,
J = 12.9 Hz); 3.31 (t, 1H, J = 11.0 Hz, H-26ax); 3.47 (ddd, 1H,
3.88 (m, 1H, H-3
a
); 4.08 (dd, 1H, J = 12.0/3.5 Hz, H-6a Glc); 4.11–
J = 11.0/4.4/2.2 Hz, H-26eq); 3.60 (m, 1H, H-3
a); 4.36 (m, 1H, H-
4.17 (m, 2H, H-2 Glc, H-3 Glc); 4.22–4.28 (m, 4H, H-4 Glc, H-6b
16a
). 13C NMR (125 MHz, CDCl3): d = 11.6, 13.9, 17.1, 18.5 (CH3);
Glc, H-4 Rha, H-4 Rha0); 4.46 (dd, 1H, J = 9.2/3.3 Hz, H-3 Rha);
28.2, 28.7 (CH2); 30.1 (CH); 31.3, 31.4, 32.2, 34.4, 36.5, 36.6
(CH2); 36.8 (C); 37.9 (CH2); 38.2, 42.6, 43.7, 50.3, 56.7, 62.9 (CH);
66.9 (CH2); 70.7, 75.6 (CH); 86.3, 108.7 (C); 174.7 (C@O). HRMS
(ESI-FT-ICR) m/z: 469.2927 [M+Na]+ (calcd for C27H42O5Na:
469.2930).
4.51 (dd, 1H, J = 9.3/3.2 Hz, H-3 Rha0); 4.57 (m, 1H, H-16
a); 4.61
(dd, 1H, J = 3.2/1.6 Hz, H-2 Rha); 4.74 (dd, 1H, J = 3.2/1.3 Hz, H-2
Rha0); 4.78 (m, 1H, H-5 Rha); 4.82 (m, 1H, H-5 Rha0); 4.90 (d, 1H,
J = 7.3 Hz, H-1 Glc); 5.70 (s, 1H, H-1 Rha); 5.83 (s, 1H, H-11); 6.24
(s, 1H, H-1 Rha0). 13C NMR (125 MHz, pyridine-d5): d = 14.0, 15.6,
17.7, 18.7, 18.8, 18.9 (CH3); 28.2, 29.5, 29.9 (CH2); 30.8 (CH); 32.1,
33.0, 34.5 (CH2); 35.3 (CH); 37.3 (C); 39.3, 42.8 (CH2); 43.2, 51.0,
53.0 (CH); 54.7 (C); 61.7, 67.3 (CH2); 69.6, 70.7, 72.5, 72.6, 72.8,
72.9, 73.9, 74.2, 76.9, 77.1, 78.1, 78.2, 79.5, 80.5, 100.2, 102.2,
103.2 (CH); 109.4 (C); 120.2 (CH); 172.1 (C); 205.6 (C@O). HRMS
(ESI-FT-ICR) m/z: 905.4497 [M+Na]+ (calcd for C45H70O17Na:
905.4497).
4.1.21. (25R)-C-Homo-12a-oxa-12-oxo-5
2,3,4,6-tetra-O-benzoyl-b- -glucopyranoside (30)
The glucosyl donor 12 (216 mg, 0.291 mmol), the spirostanic
acceptor 29 (100 mg, 0.224 mmol) and TMSOTf (2 L,
a-spirostan-3b-yl
D
l
0.0112 mmol) were reacted in dry CH2Cl2 (4.5 mL) in a similar
way as described in Section 4.1.3. Flash column chromatography
purification (n-hexane/EtOAc 2:1) afforded 30 (218.3 mg, 95%) as
a white solid. Rf = 0.26 (n-hexane/EtOAc 2:1). Mp: 211–213 °C.
½
a 2D0
ꢃ
ꢀ79.5 (c 1.1, CHCl3). IR (KBr, cmꢀ1
) m: 2933, 2870, 1734,
1271, 1095, 1071. 1H NMR (500 MHz, CDCl3): d = 0.63 (s, 3H,
CH3); 0.76 (d, 3H, J = 6.3 Hz, CH3); 1.03 (d, 3H, J = 6.9 Hz, CH3);
1.41 (s, 3H, CH3); 2.00 (m, 1H); 2.27 (m, 1H); 2.41 (dd, 1H,
J = 13.6/12.3 Hz); 2.53 (dd, 1H, J = 9.3/6.4 Hz); 2.63 (dd, 1H,
J = 13.6/0.9 Hz); 3.30 (t, 1H, J = 11.0 Hz, H-26ax); 3.46 (ddd, 1H,
4.1.19. Tigogenyl 2,4-di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranoside (28)
Acceptor 22 (100 mg, 0.134 mmol) and donor 23 (180.4 mg,
0.415 mmol) in dry CH2Cl2 (20 mL) were reacted in the presence
of BF3ꢁEt2O (75
lL, 0.590 mmol) in a similar way as described in
Section 4.1.13. The crude product was purified by flash column
chromatography (n-hexane/EtOAc 2:1) to afford the protected
saponin (241 mg). This product was deprotected with NaOH
(1.0 M, 4 mL) in THF/MeOH (16 mL, 1:1, v/v) in a similar way as
described in Section 4.1.13. Flash column chromatography purifi-
cation (CHCl3/MeOH 5:1) afforded 28 (78.2 mg, 67%) as a white
J = 11.0/4.1/2.2 Hz, H-26eq); 3.55 (m, 1H, H-3
a); 4.16 (m, 1H, H-5
Glc); 4.35 (m, 1H, H-16 ); 4.53 (dd, 1H, J = 12.0/6.0 Hz, H-6a
a
Glc); 4.58 (dd, 1H, J = 12.0/3.5 Hz, H-6b Glc); 4.91 (d, 1H,
J = 7.9 Hz, H-1 Glc); 5.46 (dd, 1H, J = 9.8/7.9 Hz, H-2 Glc); 5.59
(t, 1H, J = 9.8/9.5 Hz, H-4 Glc); 5.88 (t, 1H, J = 9.8/9.5 Hz, H-3 Glc);
7.24–7.28 (m, 2H, Ar-H); 7.31–7.42 (m, 7H, Ar-H); 7.46–7.51 (m,
2H, Ar-H); 7.54–7.57 (m, 1H, Ar-H); 7.81 (dd, 2H, J = 8.3/1.1 Hz,
Ar-H); 7.89 (dd, 2H, J = 8.2/1.3 Hz, Ar-H); 7.94 (dd, 2H, J = 8.4/
1.4 Hz, Ar-H); 7.99 (dd, 2H, J = 8.5/1.3 Hz, Ar-H). 13C NMR
(125 MHz, CDCl3): d = 11.4, 13.9, 17.0, 18.5 (CH3); 28.1, 28.7, 29.2
(CH2); 30.1 (CH); 31.4, 32.1, 34.3, 34.5 (CH2); 36.4 (C); 36.5, 36.6
(CH2); 38.0, 42.5, 43.5, 50.2, 56.7, 63.0 (CH); 63.3, 66.9 (CH2);
70.1, 72.0, 72.1, 73.0, 75.6, 79.6 (CH); 86.2 (C); 100.3 (CH); 108.7
(C); 128.2, 128.3, 128.4, 128.5 (CH); 128.7, 128.8, 129.4, 129.5
(C); 129.6, 129.7, 129.8, 129.9 (CH); 164.9, 165.2, 165.8, 166.0,
174.5 (C@O). HRMS (ESI-FT-ICR) m/z: 1047.4508 [M+Na]+ (calcd
for C61H68O14Na: 1047.4507).
solid. Rf = 0.29 (CHCl3/MeOH 5:1). Mp: 164–166 °C. ½a D20
ꢀ105.6
ꢃ
(c 0.9, C5H5N). IR (KBr, cmꢀ1
) m: 3327, 2933, 1622, 1403, 1044,
978. 1H NMR (500 MHz, pyridine-d5): d = 0.71 (d, 3H, J = 5.4 Hz,
CH3); 0.84 (s, 3H, CH3); 0.88 (s, 3H, CH3); 1.15 (d, 3H, J = 6.9 Hz,
CH3); 1.65 (d, 3H, J = 6.3 Hz, CH3 Rha); 1.77 (d, 3H, J = 6.3 Hz, CH3
Rha0); 3.52 (t, 1H, J = 10.7 Hz, H-26ax); 3.61 (m, 1H, H-26eq);
3.73 (m, 1H, H-5 Glc); 3.94 (m, 1H, H-3
12.3 Hz, H-6a Glc); 4.23–4.42 (m, 6H, H-2 Glc, H-3 Glc, H-4 Glc,
H-6b Glc, H-4 Rha, H-4 Rha0); 4.54–4.58 (m, 1H, H-16
); 4.62
a); 4.13 (dd, 1H, J = 3.5/
a
(dd, 1H, J = 9.1/3.5 Hz, H-3 Rha); 4.70 (m, 1H, H-2 Rha); 4.76 (dd,
1H, J = 9.1/3.5 Hz, H-3 Rha0); 4.84 (m, 1H, H-2 Rha0); 4.91–4.97
(m, 3H, H-5 Rha, H-5 Rha0, H-1 Glc); 5.87 (d, 1H, J = 1.6 Hz, H-1
Rha); 6.39 (d, 1H, J = 1.3 Hz, H-1 Rha0). 13C NMR (125 MHz, pyri-
dine-d5): d = 12.9, 15.5, 17.0, 17.8, 18.9, 19.1 (CH3); 21.7, 29.4,
29.7, 30.4 (CH2); 31.0 (CH); 32.3, 32.6, 32.9, 34.9 (CH2); 35.7
(CH); 36.4 (C); 37.7 (CH2); 41.2 (C); 42.4, 45.1, 54.9, 56.9 (CH);
61.9 (CH2); 63.5 (CH); 67.3 (CH2); 69.7, 69.9, 70.9, 72.9, 73.0,
73.2, 73.3, 74.0, 74.4, 74.6, 74.9, 81.6, 100.3, 102.6, 103.4 (CH);
109.7 (C). HRMS (ESI-FT-ICR) m/z: 893.4876 [M+Na]+ (calcd for
4.1.22. (25R)-C-Homo-12a-oxa-12-oxo-5
di-O-pivaloyl-b- -glucopyranoside (31)
a-spirostan-3b-yl 3,6-
D
Glucoside 30 (201.2 mg, 0.196 mmol) was deprotected with
NaOMe in CH2Cl2/MeOH (10 mL, 1:1, v/v) in a similar way as de-
scribed in Section 4.1.8. The resulting product was selectively pro-
tected with pivaloyl chloride (120 lL, 0.98 mmol) in anhydrous
pyridine (6 mL) according to the procedure described in Section
C45H74O16Na: 893.4875).
4.1.8. Flash column chromatography purification (n-hexane/EtOAc

K. Pérez-Labrada et al. / Bioorg. Med. Chem. 20 (2012) 4522–4531
4531
3:2) afforded 31 (100.5 mg, 66%) as a white solid. Rf = 0.23 (n-hex-
medium containing 10% (v/v) heat-inactivated fetal bovine serum,
100 units/mL penicillin and 100 g/mL streptomycin at 37 °C in a
ane/EtOAc 3:2). Mp: 254–256 °C. ½a D20
ꢃ
ꢀ53.3 (c 1.4, CHCl3). IR (KBr,
l
cmꢀ1
)
m
: 3461, 2933, 2874, 2372, 1718, 1160, 1080, 1047. 1H NMR
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. Human peripheral blood mononuclear cells were isolated
from heparin-anticoagulated blood of healthy volunteers by centri-
fugation with Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden),
as previously described.13
(500 MHz, CDCl3): d = 0.77 (s, 3H, CH3); 0.78 (d, 3H, J = 6.3 Hz, CH3);
1.04 (d, 3H, J = 7.3 Hz, CH3); 1.21, 1.23 (2 ꢂ s, 2 ꢂ 9H, 2 ꢂ (CH3)3C);
1.44 (s, 3H, CH3); 2.01 (t, 1H, J = 6.7 Hz); 2.31 (m, 1H); 2.39 (s, 1H);
2.47 (dd, 1H, J = 13.6/12.3 Hz); 2.55 (dd, 1H, J = 9.3/6.5 Hz); 2.73
(d, 1H, J = 13.6 Hz); 3.03 (d, 1H, J = 4.7 Hz); 3.31 (t, 1H, J = 11.0 Hz,
H-26ax); 3.42–3.49 (m, 3H, H-2 Glc, H-4 Glc, H-26eq); 3.56 (m,
1H, H-5 Glc); 3.63 (m, 1H, H-3a); 4.24 (dd, 1H, J = 12.0/6.6 Hz, H-
6a Glc); 4.37 (m, 1H, H-16 ); 4.41 (dd, 1H, J = 12.0/2.3 Hz, H-6b
a
Glc); 4.43 (d, 1H, J = 7.9 Hz, H-1 Glc); 4.85 (t, 1H, J = 9.1 Hz, H-3
Glc). 13C NMR (125 MHz, CDCl3): d = 11.5, 13.9, 17.1, 18.5, 27.0,
27.1 (CH3); 28.3, 28.7, 29.2 (CH2); 30.1 (CH); 31.4, 32.2, 34.4, 36.6,
36.7 (CH2); 38.1 (CH); 38.8, 39.0 (C); 42.6, 43.6, 50.2, 56.6, 63.0
(CH); 63.6, 67.0 (CH2); 70.0, 72.1, 74.2, 75.6, 77.9, 78.4 (CH); 86.2
(C); 101.1 (CH); 108.7 (C); 174.7, 178.6, 180.2 (C@O). HRMS (ESI-
FT-ICR) m/z: 799.4608 [M+Na]+ (calcd for C43H68O12Na: 799.4608).
4.2.2. Cytotoxicity assay
The cytotoxicity of compounds was evaluated by colorimetric
3-(4,5-dimethyl-2-thyazolyl-)-2,5-diphenyltetrazolium bromide
(MTT) assay as previously described.14 Concentrations inducing a
50% inhibition of cell growth (IC50) were determined graphically
using the curve-fitting algorithm of the computer software Prism
4.0 (GraphPad, La Jolla, CA). Values are means SEs from three
independent experiments, each performed in triplicate.
4.1.23. (25R)-3b-O-[2,4-di-O-(
a-
L
-rhamnopyranosyl)-b-
D-
glucopyranosyl)]-12,13-seco-5a-spirostan-12-oic acid (32)
Acceptor 31 (166.2 mg, 0.214 mmol) and donor 23 (288 mg,
Acknowledgements
0.663 mmol) in dry CH2Cl2 (30 mL) were reacted in the presence
of BF3ꢁEt2O (120
l
L, 0.942 mmol) in a similar way as described in
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 FE). K. Pérez-Labrada gratefully acknowledges
to MAEC-AECID for a doctoral scholarship. S.E. and M.T.M. thank
the Spanish Ministry of Education and University of Las Palmas
de Gran Canaria, respectively, for financial support.
Section 4.1.13. The crude product was purified by flash column
chromatography (n-hexane/EtOAc 3:2) to afford the protected
saponin (397 mg). This product was deprotected with NaOH (1 M,
5 mL) in THF/MeOH (20 mL, 1:1, v/v) in a similar way as described
in Section 4.1.13. Flash column chromatography purification
(CHCl3/MeOH 3:1) afforded 32 (120 mg, 61%) as a white solid.
Rf = 0.33 (CHCl3/MeOH 3:1). Mp: 189–191 °C. ½a D20
ꢀ105.6 (c 1.3,
ꢃ
C5H5N). IR (KBr, cmꢀ1
)
m
: 3390, 2932, 2874, 1675, 1399, 1044. 1H
References and notes
NMR (500 MHz, pyridine-d5): d = 0.72 (d, 3H, J = 5.2 Hz, CH3); 0.82
(s, 3H, CH3); 1.24 (d, 3H, J = 5.7 Hz, CH3); 1.40 (s, 3H, CH3); 1.64
(d, 3H, J = 6.3 Hz, CH3 Rha); 1.73 (d, 3H, J = 6.3 Hz, CH3 Rha0); 2.08
(m, 1H); 2.37 (m, 1H); 2.62 (m, 1H); 2.76 (m, 1H); 3.45 (t, 1H,
J = 10.7 Hz, H-26ax); 3.54 (m, 1H, H-26eq); 3.73 (m, 1H, H-5 Glc);
1. (a) Wang, Y.; Che, C.-M.; Chiu, J.-F.; He, Q.-Y. J. Proteome Res. 2007, 6, 4703; (b)
Wang, Y.; Cheung, Y. H.; Yang, Z.; Chiu, J.-F.; Che, C.-M.; He, Q.-Y. Proteomics
2006, 6, 2422; (c) Cheung, J. Y.-N.; Ong, R. C.-Y.; Suen, Y.-K.; Ooi, V.; Wong, H.
N.-C.; Mak, T. C.-W.; Fung, K.-P.; Yu, B.; Kong, S.-K. Cancer Lett. 2005, 217, 203;
(d) Liu, M. J.; Wang, Z.; Ju, Y.; Zhou, J. B.; Wang, Y.; Wong, R. N.-S. Biol. Pharm.
Bull. 2004, 27, 1059; (e) Watanabe, K.; Mimaki, Y.; Sakagami, H.; Sashida, Y. J.
Nat. Prod. 2003, 66, 236; (f) Wang, Z.; Zhou, J.; Ju, Y.; Zhang, H.; Liu, M.; Li, X.
Biol. Pharm. Bull. 2001, 24, 159.
2. Zhou, L.-B.; Chen, T.-H.; Bastow, K. F.; Shibano, M.; Lee, K.-H.; Chen, D.-F. J. Nat.
Prod. 2007, 70, 1263.
3. Pérez-Labrada, K.; Brouard, I.; Estévez, S.; Marrero, M. T.; Estévez, F.; Bermejo,
J.; Rivera, D. G. Bioorg. Med. Chem. 2012, 20, 2690.
4. Pérez-Labrada, K.; Brouard, I.; Morera, C.; Estévez, F.; Bermejo, J.; Rivera, D. G.
Tetrahedron 2011, 67, 7713.
5. Yu, B.; Zhang, Y.; Tang, P. Eur. J. Org. Chem. 2007, 31, 5145.
6. Rivera, D. G.; Pando, O.; Coll, F. Tetrahedron 2006, 62, 8327.
7. Fukase, K.; Winarno, H.; Kusumoto, S. Chem. Express 1993, 8, 409.
8. Jiang, L.; Chan, T.-H. J. Org. Chem. 1998, 63, 6035.
9. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21.
10. Schmidt, R. R.; Toepfer, A. Tetrahedron Lett. 1991, 32, 3353.
11. Yu, B.; Tao, H. J. Org. Chem. 2002, 67, 9099.
12. Whereas saponin 28 has been previously reported, its cytotoxicity against HL-
60 has not been evaluated so far. Accordingly, its synthesis was accomplished
in this work for a comparison of the cytotoxicity with the other C-ring modified
glycosides. For a report of a previous synthesis and cytotoxicity against the A-
549 cell line, see: (a) Wang, Y.; Zhang, Y.; Zhu, Z.; Zhu, S.; Li, Y.; Li, M.; Yu, B.
Bioorg. Med. Chem. 2007, 15, 2528; (b) Li, Y.; Zhang, Y.; Guo, T.; Guan, H.; Hao,
Y.; Yu, B. Synthesis 2006, 775.
3.87 (m, 1H, H-3a); 4.12–4.39 (m, 7H, H-2 Glc, H-3 Glc, H-4 Glc,
H-6a Glc, H-6b Glc, H-4 Rha, H-4 Rha0); 4.55 (dd, 1H, J = 9.1/
3.2 Hz, H-3 Rha); 4.60 (dd, 1H, J = 9.1/3.2 Hz, H-3 Rha0); 4.70 (m,
2H, H-2 Rha, H-16a
); 4.83 (m, 1H, H-2 Rha0); 4.92 (m, 2H, H-5
Rha, H-5 Rha0); 4.96 (d, 1H, J = 7.3 Hz, H-1 Glc); 5.85 (d, 1H,
J = 1.5 Hz, H-1 Rha); 6.36 (d, 1H, J = 1.3 Hz, H-1 Rha0). 13C NMR
(125 MHz, pyridine-d5): d = 12.6, 15.0, 17.7, 18.9, 19.0 (CH3); 29.4,
29.6, 30.2 (CH2); 30.6 (CH3); 31.1 (CH); 31.7, 32.3, 34.9 (CH2);
36.1 (CH); 38.2 (C); 38.3, 44.1, 44.8, 49.6, 52.9 (CH); 62.0 (CH2);
62.2 (CH); 67.5 (CH2); 69.9, 70.9, 72.8, 72.9, 73.1, 73.2, 74.3, 74.6,
77.3, 77.8, 78.4, 78.7, 79.3, 79.5, 100.5, 102.6, 103.4 (CH); 109.1
(C); 175.2 (C@O). HRMS (ESI-FT-ICR) m/z: 941.4708 [M+Na]+ and
963.4525 [M+Na]+ (calcd for C45H74O19Na: 941.4722 and
C45H73O19Na2: 963.4541, respectively).
4.2. Biological activity
4.2.1. Cell culture
HL-60 cells (German Collection of Microorganisms and Cell
Cultures, Braunnschweig, Germany) were cultured in RPMI 1640
13. Rubio, S.; Quintana, J.; Eiroa, J. L.; Triana, J.; Estévez, F. Carcinogenesis 2007, 28,
2105.
14. Burmistrova, O.; Quintana, J.; Díaz, J. G.; Estévez, F. Cancer Lett. 2011, 309, 71.