Synthesis of lupane-type saponins bearing mannosyl and 3,6-branched trimannosyl residues and their evaluation as anticancer agents

Article abstract of DOI:10.1016/j.carres.2008.02.011

The saponins modified with mono- or trimannosyl residues can provide a convenient means of delivering drugs to certain human cells via interactions with mannose receptors. In the study reported therein, we developed a convenient approach for the synthesis of 3-O-mannoside and branched trimannoside derivatives of the saponin lupeol and of C-28 acyl esters of 3-O-acetyl-betulinic acid bearing the same mannosyl entities. Lupeol and 3-O-acetyl-betulinic acid were mannosylated with tetra-O-benzoyl- or tetra-O-acetyl-α-d-mannopyranosyl trichloroacetimidates. De-esterification followed by regioselective dimannosylation of the unprotected monosaccharide derivatives with 2 equiv of tetra-O-benzoyl-α-d-mannopyranosyl trichloroacetimidate selectively yielded O-3,O-6-linked trimannosides. The cytotoxic activity of selected lupane-type saponins (derivatives of lupeol, betulinic acid, and betulin) toward normal human fibroblasts and various cancer cell lines was also compared.

Full text of DOI:10.1016/j.carres.2008.02.011

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 995–1003
Synthesis of lupane-type saponins bearing mannosyl
and 3,6-branched trimannosyl residues and their
evaluation as anticancer agents
b
b
Piotr Cmoch,a Zbigniew Pakulski,a, Jana Swaczynova and Miroslav Strnad
*
´
aInstitute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, PL-01-224 Warszawa, Poland
bLaboratory of Growth Regulators, Palacky University & Institute of Experimental Botany ASCR,
Slechtitelu 11, CZ-783 71 Olomouc, Czech Republic
Received 10 October 2007; received in revised form 11 February 2008; accepted 12 February 2008
Available online 20 February 2008
Abstract—The saponins modified with mono- or trimannosyl residues can provide a convenient means of delivering drugs to certain
human cells via interactions with mannose receptors. In the study reported therein, we developed a convenient approach for the
synthesis of 3-O-mannoside and branched trimannoside derivatives of the saponin lupeol and of C-28 acyl esters of 3-O-acetyl-
betulinic acid bearing the same mannosyl entities. Lupeol and 3-O-acetyl-betulinic acid were mannosylated with tetra-O-benzoyl-
or tetra-O-acetyl-a-D-mannopyranosyl trichloroacetimidates. De-esterification followed by regioselective dimannosylation of the
unprotected monosaccharide derivatives with 2 equiv of tetra-O-benzoyl-a-D-mannopyranosyl trichloroacetimidate selectively
yielded O-3,O-6-linked trimannosides. The cytotoxic activity of selected lupane-type saponins (derivatives of lupeol, betulinic acid,
and betulin) toward normal human fibroblasts and various cancer cell lines was also compared.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Saponin; Betulin; Lupeol; Betulinic acid; Glycosylation
1. Introduction
Saponins are a large family of steroid or triterpenoid
glycosides, widely distributed in plants and in some mar-
ine organisms,5 in which hydrophilic mono- or oligosac-
charides are attached to a hydrophobic sapogenin
backbone. They have received considerable attention
because of their diverse, promising biological and phar-
maceutical properties, including antitumor,6 antiviral,7
antifungal,8 antiinflamatory,9 and other10 activities.
They have also been shown recently to have significant
effects on plant growth.11
Lupeol [1, 3-b-hydroxy-lup-20(29)-ene, Chart 1] is
found in many plant species,10,12 betulin (3-b-28-dihydr-
oxylup-20(29)-ene) is a highly abundant component of
birch bark, and betulinic acid [2, 3b-hydroxyl-
up-20(29)-ene-28-oic acid], which has very interesting and
promising biological properties,13 can also be isolated
from various plants,12c,e,13d,f or easily prepared from
betulin.14 Natural saponins based on lupeol15 and
betulinic acid16 are rarely found in nature, and reports
Terminal a-D-mannopyranosyl units that are recog-
nized by the receptors of macrophages1 and dendritic
cells2 commonly occur in glycoproteins of pathogenic
bacteria, yeasts, viruses, and various parasites. The
branched trisaccharide 3,6-di-O-(a-D-mannopyranos-
yl)-a-D-mannopyranosyl ligand binds with especially
high affinity to these mannose receptors,3 providing a
possible way to transport antigens or drugs into human
dendritic cells or macrophages. The interaction of car-
bohydrates with proteins responsible for such effects
is intensively studied, and information regarding trim-
annoside binding has been published in several recent
studies.4
*
Corresponding author. E-mail: pakul@icho.edu.pl
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.011

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P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
2. Results and discussion
H
RO
RO
RO
RO
2.1. Synthesis of lupeol mannosides
O
H
R'
H
OCCCl3
NH
For the synthesis of saponins having mannosyl or tri-
mannosyl residues connected with lupeol and betulinic
acid, the synthetic strategy previously applied by us
for the preparation of betulin derivatives was used.18
Reacting lupeol (1) with 2,3,4,6-tetra-O-benzoyl-a-D-
mannopyranosyl trichloroacetimidate (5) in the presence
of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
gave the expected glycoside 7 in good yield (95%).
Debenzoylation with potassium carbonate in methanol
afforded the free mannoside 8 (79%), which was further
mannosylated with 2 equiv of 5 to yield the (1?3, 1?6)-
linked trisaccharide 9 purified by chromatography as its
diacetate 10 (27% after two steps). According to NMR
spectra, this product was contaminated by small
amounts (approx. 15%) of inseparable 1-O-acetyl-
2,3,4,6-tetra-O-benzoyl-D-mannopyranose.
No other regioisomer was detected in the above reac-
tion, in accordance with our previous observations that
O-benzoyl-protected donors afforded (1?3, 1?6)-
linked mannosides regioselectively.18,23h Typical depro-
tection with potassium carbonate in methanol afforded
the final saponin 11 (88%), slightly contaminated by
small amounts of free mannose (Scheme 1).
RO
H
1: R = H, R' = CH3 (lupeol)
2: R = H, R' = CO2H (betulinic acid)
3: R = Ac, R' = CH3
5: R = Bz
6: R = Ac
4: R = Ac, R' = CO2H
Chart 1.
on their synthesis are also sporadic.17 We have discussed
various aspects of the chemistry and biological activity
of betulin and its derivatives in a previous paper.18
Promisingly, for their potential clinical use, these tri-
terpenoids have shown no hemolytic activity at high
concentrations (100 mmol/L), and very weak hemolytic
activity even at extremely high concentrations
(500 mmol/L).10a For instance, for some derivatives of
betulin and betulinic acid, which showed high anti-
HIV activity, EC50 (concentration which produces 50%
of the maximum response) values <6.6 ꢀ 10ꢁ4 lM and
remarkably high therapeutic index (TI), exceeding
20,000, have been obtained, compared to 1.5 lM and
12,000, respectively, for azidothymidine (AZT).19 Some
derivatives, for which IC50 (concentrations leading to
50% inhibition of viability) values were approx. 20–
80 lM, have also shown significant cytotoxicity and
anti-tumor properties.20 Although betulin and lupeol
themselves are usually inactive, betulinic acid was found
to be selectively cytotoxic against several cancer cell
lines.13a
Recently, we were interested in the preparation of sap-
onins from several lupane-type triterpenes (lupeol, betul-
inic acid, and betulin). Attaching trimannoside moieties
to sapogenins as a hydrophilic transport-facilitating
functional group could both improve their ability to
enter target cells via interactions with mannose receptors
and increase their solubility, thus providing a convenient
drug delivery strategy. Additionally, due to the presence
of the sugar fragment and potential affinity with the
dendritic cells, these derivatives may be considered as
components of carbohydrate-based cancer vaccines.21
Several formal routes leading to 3,6-di-O-(a-D-man-
nopyranosyl)-a-D-mannopyranose have been reported,
including glycosylation of partially protected mannopy-
ranoside derivatives.22 A direct approach for synthesiz-
ing this mannotriose derivative has also been reported
via the selective O-3,O-6 mannosylation of unprotected
mannosides.18,23 In this paper, as a continuation of
our studies on regioselective glycosylation,18,23d,h we
report a convenient method for synthesizing lupeol
trimannoside and trimannosyl betulinate, which are
potential antitumor and antiviral agents.
2.2. Synthesis of betulinic acid mannosyl esters
Very similar transformations were used to prepare sapo-
nins 14 and 17. Glycosylation of 3-O-acetyl betulinic
acid (4) with 5 in the presence of TMSOTf gave betulinic
acid derivative 12 (91%). However, its debenzoylation
with potassium carbonate gave a mixture of the
expected product 14 together with the compound
deacetylated at the triterpene O-3 position. Repetition
of mannosylation of 4 with 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranosyl trichloroacetimidate (6) in the presence
of TMSOTf afforded derivative 13 (93%). Its deprotec-
tion with sodium methoxide in methanol gave 14
(90%), without affecting the acetyl group at the triter-
pene O-3 position.
Treatment of 14 with 2 equiv of 5 as described above,
followed by acetylation of crude 15, purification of di-
acetate 16 (41% after two steps), and removal of benzoyl
protecting groups gave the saponin glycosyl-ester 17
(quant., Scheme 2).
Observed reactivity of the acetyl group at the betulinic
acid’s O-3 position is rather unexpected. It was not
affected under Zemplen conditions (NaOMe/MeOH, in
the case of 13) but partially deprotected when treated
with potassium carbonate in methanol (in the case of
12). Previously, we showed that acetyl groups at the
O-3 and O-28 positions of betulin derivatives were resis-

P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
997
H
H
a
c
RO
H
H
RO
RO
RO
O
H
H
HO
O
H
H
1
7: R = Bz
8: R = H
b
BzO
HO
BzO
HO
HO
HO
O
O
H
H
BzO
BzO
b
RO
HO
H
H
O
O
O
O
RO
HO
H
H
O
O
O
O
BzO
O
HO
O
H
H
BzO
HO
OBz
OH
BzO
HO
9: R = H
10: R = Ac
11
d
Scheme 1. Reagents and conditions: (a) 5 (1 equiv), CH2Cl2, TMSOTf, ꢁ40 °C; (b) K2CO3, MeOH; (c) 5 (2 equiv), MeCN–CH2Cl2, TMSOTf,
ꢁ40 °C; (d) Ac2O, pyridine.
OR
RO
OR
H
H
OR
O
O
a
c
H
CO2H
H
O
H
H
b
AcO
AcO
H
H
4
12: R = Bz
13: R = Ac
14: R = H
OBz
OH
BzO
HO
BzO
HO
OBz
OH
BzO
HO
HO
HO
O
O
BzO
BzO
OR
OH
OBz
OH
O
O
O
O
O
O
e
H
H
H
OR
OH
O
O
O
O
H
O
O
H
H
AcO
AcO
H
H
15: R = H
16: R = Ac
17
d
Scheme 2. Reagents and conditions: (a) 5 or 6 (1 equiv), CH2Cl2, TMSOTf, ꢁ40 °C; (b) NaOMe, MeOH; (c) 5 (2 equiv), acetonitrile–CH2Cl2,
TMSOTf, ꢁ40 °C; (d) Ac2O, pyridine; (e) K2CO3, MeOH.
tant to alkaline hydrolysis (potassium carbonate in
methanol).18
indicated characteristic deshielding effects of the C-3
and C-6 resonances.
We decided to leave the O-3 acetyl group at the sapo-
genin portion to improve the solubility of the final com-
pound 17. This acetate group should be removed by the
action of esterases, thereby releasing the free betulinic
acid derivative inside the target cells.
Moreover, results of HMBC analyses of 10 showed
that H signals of H-3 at 4.39 ppm and both protons
1
H-6 at 3.70 and 4.02 ppm were correlated with the 13C
signals at 99.0 (C-10) and 97.3 ppm (C-100), respectively.
These observations clearly proved that the glycosidic
linkages were at O-3 and O-6 of the central mannose
ring. In addition, 1J(1H–13C) coupling constants of
171.3, 173.5, and 173.5 Hz observed for the anomeric
carbon atoms (C-1, C-10, and C-100, respectively) indicate
the presence of a-linkages.24
2.3. Configurational assignments
1
The H NMR spectra of diacetates 10 and 16 showed
the H-2 and H-4 resonances of the central mannose res-
idues being deshielded by ca. 0.9–1.2 ppm with respect
to those of H-3. The 13C NMR spectra of 10 and 16
Similarly, HMBC analysis of 16 showed correlations
1
between H signals of H-3 (4.38 ppm) and H-6 protons

998
P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
13
(4.02 and 3.76 ppm) with C signals of C-10 (99.4 ppm)
MCF-7, lung carcinoma A-549, multiple myeloma
RPMI 8226, cervical carcinoma HeLa, and malignant
melanoma G361 lines. Cells of all of these lines were
exposed to six serial 4-fold dilutions of each drug for
72 h, the proportions of surviving cells were then esti-
mated and IC50 values were calculated. The results ob-
tained from Calcein AM assays are presented in Table 1.
The most potent compounds were monosaccharide
derivatives (8, 14, 22, 24) that showed cytotoxic activity
against all of the tumor cell lines. However, these com-
pounds also had significant toxicity toward normal cells
(BJ fibroblasts). Compound 4 was also tested using the
panel of cancer cells. The cytotoxicity values of 4 varied
between 6.7 and 43 lM and were comparable with those
of the monosaccharide analogues. A striking observa-
tion from these data was that much lower lupane-medi-
ated loss of viability was observed in the BJ fibroblasts,
suggesting that the lupane derivative induces different
responses in cancer and normal cells. At present, only
a few natural agents are known to possess the potential
ability for selective/preferential elimination of cancer
cells without affecting the growth of normal cells.25 Tri-
saccharides (11, 17, 23, 25) showed no cytotoxicity
1
and C-100 (98.0 ppm), respectively. Observed J(1H–13C)
coupling constants for C-1 (179.3 Hz), C-10 (174.2 Hz),
and C-100 (175.5 Hz) proved the anomeric configuration
to be a.24
2.4. Biological activity
Anticancer activity was tested for parent triterpenoids
[lupeol (1) and its acetate (3), betulinic acid (2) and its
acetate (4), betulin (18) and its acetylated derivatives
(19–21)], mono- and trisaccharide derivatives of lupeol
(8, 11), and betulinic acid (14, 17) prepared above as well
as mono- and trimannoside of betulin (22–25), which
were obtained during our previous studies (Chart 2).18
Several model normal and cancer cell lines were cul-
tured and used in experiments to examine the struc-
ture–activity relationships of lupane-type saponins
with respect to their activities against human cancers.
We compared the in vitro cytotoxic activity of selected
analogues against human BJ-H-tert fibroblasts and can-
cer cell lines of various histopathological origins, includ-
ing T-lymphoblastic leukemia CEM, breast carcinoma
OH
RO
OR
H
H
H
OH
O
OR'
OAc
O
H
HO
H
H
RO
HO
RO
O
H
H
H
RO
O
AcO
H
H
H
18: R = R' = H (betulin)
19: R = R' = Ac
20: R = Ac, R' = H
21: R = H, R' = Ac
22: R = H
HO
24: R = H
HO
HO
HO
O
O
HO
HO
23: R =
25: R =
HO
HO
Chart 2.
Table 1. IC50 (lM) values obtained from the Calcein AM assays with the tested cancer and normal cell lines; means SD obtained from three
independent experiments performed in triplicate
Compound
Cell lines (IC50, lM)
CEM
MCF 7
A-549
HeLa
BJ-H-tert
RPMI 8226
G 361
1
2
27.6 1.4
40 2.8
>50
10 0.2
33.3 4.7
>50
>50
>50
>50
21.8 5.5
34.1 0.1
>50
>50
>50
>50
43 0.6
45.1 1.4
>50
>50
47.6 1.9
>50
14.5 4.3
32.1 0.4
>50
>50
>50
>50
>50
33.5 5.3
>50
37.5 5.6
34.6 1.5
44.1 2.2
6.7 0.4
26.2 1.5
>50
>50
>50
>50
32.3 0.6
31.8 1.7
>50
3
4
8
11
14
17
18
19
20
21
22
23
24
25
10.4 0.7
>50
22.7 2.2
>50
>50
>50
>50
46.7 0.9
35.5 1.9
>50
39.2 5.7
>50
43.3 0.3
>50
>50
>50
>50
>50
44.6 0.2
>50
44.6 0.2
>50
34.7 2.5
>50
>50
38.4 4.6
>50
37.9 0.1
42.8 1.3
>50
38.7 0.5
>50
48.6 0.1
>50
>50
>50
43.1 0.5
>50
35.6 7.9
>50
19.4 0.5
>50
22.7 1.4
>50
13.4 0.8
>50
42.1 1.9
35.9 0.1
27.9 0.1
>50
22.3 3.9
>50
21.2 3.4
34.8 0.2
30.4 2.2
30.2 0.7
12.9 1.1
>50
12.9 1.6
28.1 0.5
19.9 2.3
22.4 1.7
18.5 0.5
>50
27.7 5.3
>50
45.7 3.5
>50
29.2 3.8
>50
Betulinic acid (2) was used as a positive control.

P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
999
toward the cancer and normal cell lines used, even when
tested at concentrations up to 50 lM. Parent com-
pounds (1, 18–21) were more active against leukemia cell
lines CEM and RPMI 8226 (IC50 12.9–34.8 lM) than
the other types of cancer cells. The results also show that
nonmalignant cells (BJ-H-tert fibroblasts) tolerated
higher doses of most of the tested compounds than
tumor cells, demonstrating that they have preferential
cytotoxicity for malignant cells.26
fied by acetylation in a refluxed mixture of Ac2O
(120 mL), acetic acid (50 mL), and pyridine (1 mL) for
2 h. Solvents were evaporated to dryness and the residue
was purified by column chromatography (40:1?5:1
hexane–diethyl ether) as described earlier.18 The fastest
moving fraction during chromatographic separation of
individual components contained lupeol acetate (3). It
was further purified by column chromatography (50:1
1
hexane–EtOAc) to afford pure 3 (4.0 g). H NMR spec-
tral data matched those reported.27 13C NMR (CDCl3)
d: 170.9 (C@O), 150.9 (C-20), 109.3 (C-29), 81.0 (C-3),
55.4, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.9 (C), 40.0
(CH2), 38.4 (CH2), 38.1, 37.8 (C), 37.1 (C), 35.6
(CH2), 34.2 (CH2), 29.8 (CH2), 27.9, 27.4 (CH2), 25.1
(CH2), 23.7 (CH2), 21.3, 20.9 (CH2), 19.3, 18.2 (CH2),
18.0, 16.5, 16.2, 16.0, 14.5.
3. Conclusions
In summary, a rapid, efficient method for synthesizing
novel lupeol a-D-mannopyranosides and 3-O-acetyl-
betulinic acid a-D-mannopyranosyl esters has been
developed. Structure–activity analysis showed that
lupane-type saponins can inhibit the growth of various
cancer cell lines at micromolar concentrations, despite
having limited effects on normal human fibroblasts.
Monosaccharide derivatives of lupeol (8), betulinic acid
(14), and betulin (22, 24) were strongly cytotoxic,
whereas trisaccharide analogues were inactive.
A soln of lupeol acetate (3, 3.93 g, 8.4 mmol) and
potassium hydroxide (1.25 g, 22 mmol) in EtOH
(30 mL) was refluxed for 5 h, cooled to room tempera-
ture, and concentrated. The oily residue was suspended
in CH2Cl2 and filtered through a short silica pad (5:1
hexane–EtOAc). The filtrate was concentrated and the
residue was purified by column chromatography (9:1
hexane–EtOAc) to yield lupeol (1, 3.06 g, 85%): mp
20
209–211 °C; lit.28 mp 215–216 °C; ½aꢂD +25.0 (c 1.15,
20
1
4. Experimental
CHCl3); lit.28 ½aꢂD +26.4 (CHCl3). H and 13C NMR
spectral data matched those reported.12a,27
4.1. General methods
4.3. 3b-O-(2,3,4,6-Tetra-O-benzoyl-a-D-mannopyran-
osyl)-lup-20(29)-ene (7)
Silica gel HF-254 and Silica Gel 230–400 mesh (E.
Merck) were used for TLC and column chromatography,
respectively. 1H and 13C NMR spectra were recorded at
303 K (500 and 125 MHz, respectively) with a Bruker
Avance DRX-500 spectrometer. An internal TMS was
A soln of 5 (572 mg, 0.77 mmol) and lupeol (1, 320 mg,
0.75 mmol) in CH2Cl2 (15 mL) was stirred for 30 min at
˚
room temperature over molecular sieves (4 A, 700 mg,
1
used as the H and 13C NMR chemical shift standard.
finely ground), then cooled to ꢁ40 °C and TMSOTf
(50 lL) was added. After 20 min, the reaction was
quenched with Et3N (0.2 mL), and the solvents were
evaporated under diminished pressure. Column chroma-
tography (9:1?5:1 hexane–EtOAc) of the residue gave 7
Signals of the aromatic groups observed for the typical
values were omitted for simplicity. High resolution mass
spectra (HRMS) were acquired with a MARINER mass
spectrometer. Optical rotations were measured with a
JASCO P-1020 automatic polarimeter. Configurational
assignments were based on NMR measurements includ-
ing DEPT and two-dimensional techniques, including
gradient-selected COSY, 1H–13C gradient selected HSQC
(g-Heteronuclear Single Quantum Correlation; C, H cor-
relation via double INEPT transfer in the phase sensitive
20
1
(716 mg, 95%) as a foam: ½aꢂD ꢁ2.4 (c 0.5, CHCl3). H
NMR (CDCl3) d: 6.08 (t, 1H, J4,3 = J4,5 = 10.1 Hz,
H-4), 5.92 (dd, 1H, J3,2 3.2 Hz, H-3), 5.62 (dd, 1H, J2,1
1.8 Hz, H-2), 5.28 (d, 1H, H-1), 4.70 (d, 1H, J 2.0 Hz,
0
lupene-H-29), 4.67 (dd, 1H, J6,5 2.3, J6;6 11:9 Hz,
H-6), 4.56 (m, 2H, H-5, lupene-H-29), 4.48 (dd, 1H,
J6,5 5.1 Hz, H-6), 3.35 (dd, 1H, J 4.3, 11.7 Hz, lupene-
H-3), 2.38 (td, 1H, J 5.7, 11.0 Hz, lupene-H-19), 1.78–
1.98 (m, 2H), 0.66–1.75 (m), 1.70 (s, 3H, CH3), 1.11 (s,
3H, CH3), 1.05 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.94
(s, 3H, CH3), 0.89 (s, 3H, CH3), 0.80 (s, 3H, CH3).
13C NMR (CDCl3) d: 166.1 (C@O), 165.6 (C@O),
165.6 (C@O), 165.5 (C@O), 150.9 (lupene-C-20), 109.4
(lupene-C-29), 94.4 (C-1), 84.3 (lupene-C-3), 71.6, 70.3,
69.5, 67.1, 63.1 (CH2), 55.7, 50.5, 48.3, 48.0, 43.0 (C),
42.9 (C), 40.9 (C), 40.0 (CH2), 38.6 (C), 38.3 (CH2),
38.1, 37.1 (C), 35.6 (CH2), 34.3 (CH2), 29.9 (CH2),
1
mode), H–13C gradient selected HMBC (g-Heteronu-
clear Multiple Bond Correlation; long-range correl-
ation), as well as TOCSY experiments.
4.2. Lupeol [3b-hydroxy-lup-20(29)-ene] (1)
A sample (300 g) of the outer bark of a white birch tree
collected in Poraj (South Poland) was cut into small
pieces, air dried for 7 days and extracted with MeOH
in a Soxhlet apparatus for 8 h. The extract was concen-
trated to afford 58.0 g of crude betulin, which was puri-

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P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
28.8, 27.5 (CH2), 25.2 (CH2), 22.2 (CH2), 21.0 (CH2),
19.3, 18.3 (CH2), 18.0, 16.5, 16.2, 16.0, 14.6. HRESIMS:
calcd for C64H76NaO10 [M+Na]+, 1027.5331. Found:
m/z 1027.5388.
(251 mg, 27% after two steps) as an amorphous glass.
1H NMR (CDCl3) d: 6.23 (t, 1H, J4,3 = J4,5 = 10.0 Hz,
H-40), 6.14 (t, 1H, J4,3 = J4,5 = 10.1 Hz, H-400), 5.91 (dd,
1H, J3,2 3.4 Hz, H-300), 5.84 (dd, 1H, J3,2 3.2 Hz, H-30),
5.78 (dd, 1H, J2,1 1.8 Hz, H-200), 5.56 (dd, 1H, J2,1
2.0 Hz, H-20), 5.49 (t, 1H, J4,3 = J4,5 = 10.1 Hz, H-4),
5.40 (d, 1H, H-10), 5.34 (dd, 1H, J2,3 3.3, J2,1 1.7 Hz,
H-2), 5.14 (d, 1H, H-100), 5.08 (d, 1H, H-1), 4.48–4.75
(m, 8H, H-50, 500, 60, 600, lupene-H-29), 4.39 (dd, 1H,
H-3), 4.23 (m, 1H, H-5), 4.02 (dd, 1H, J6,5 6.4,
4.4. 3b-O-(a-D-Mannopyranosyl)-lup-20(29)-ene (8)
To a soln of 7 (910 mg, 0.91 mmol) in MeOH (20 mL),
K2CO3 (200 mg) was added, the mixture was stirred
overnight, and concentrated to dryness. Column
chromatography (5:1 hexane–EtOAc then 5:3:1?1:1:1
hexane–EtOAc–MeOH) of the residue gave 8 (420 mg,
0
0
J6;6 10:7 Hz, H-6), 3.70 (dd, 1H, J6 ;5 2:5 Hz, H-6), 3.33
(dd, 1H, J 4.3, 11.7 Hz, lupene-H-3), 2.31 (s, 3H, CH3),
2.30 (s, 3H, CH3), 0.75–1.63 (m, other protons). 13C
NMR (CDCl3) d: 170.8 (C@O), 170.3 (C@O), 166.1
(C@O), 166.0 (C@O), 165.4 (C@O), 165.3 (C@O),
165.2 (C@O), 165.2 (C@O), 165.1 (C@O), 150.8 (lupen-
e-C-20), 109.3 (lupene-C-29), 99.0 (1JC–H 173.5 Hz,
C-10), 97.3 (1JC–H 173.5 Hz, C-100), 94.3 (1JC–H
171.3 Hz, C-1), 84.4 (lupene-C-3), 75.2 (C-3), 72.0
(C-2), 70.7 (C-20), 70.3, 70.2 and 70.2 (C-200,300,5), 69.6
(C-50), 69.4 (C-30), 68.7 (C-500), 68.4 (C-4), 66.9 (C-6),
66.8 (C-400), 66.3 (C-40), 62.9 (C-600), 62.4 (C-60), 55.3,
50.1, 48.2, 47.9, 42.9 (C), 42.5 (C), 40.7 (C), 39.9 (CH2),
38.6 (C), 38.3 (CH2), 37.9, 37.0 (C), 35.4 (CH2), 34.1
(CH2), 29.8 (CH2), 28.9, 27.2 (CH2), 24.9 (CH2), 22.5
(CH2), 21.1 (CH3), 20.9 (CH3), 18.2 (CH2), 17.9, 16.5,
16.0, 15.9, 14.0. HRESIMS: calcd for C108H116NaO26
[M+Na]+, 1851.7647. Found: m/z 1851.7730.
20
79%) as white crystals: mp 244–245 °C; ½aꢂD +77.5 (c
1
0.5, CHCl3–MeOH). H NMR (1:1 CDCl3–CD3OD) d:
4.94 (br s, 1H), 4.70 (d, 1H, J 2.0 Hz, lupene-H-29),
4.57 (m, 1H), 3.72–3.84 (m, 5H), 3.68 (m. 1H), 3.35 (m,
1H), 3.23 (dd, 1 H, J 4.3, 11.7 Hz, lupene-H-3), 2.40
(td, 1H, J 5.8, 11.1 Hz, lupene-H-19), 1.92 (m, 2H),
0.66–1.80 (m), 1.69 (s, 3H, CH3), 1.06 (s, 3H, CH3),
0.99 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.86 (s, 3H, CH3),
0.81 (s, 3H, CH3), 0.78 (s, 3H, CH3). 13C NMR
(CDCl3–CD3OD 1:1) d: 151.3 (lupene-C-20), 109.7
(lupene-C-29), 97.0 (C-1), 83.1 (lupene-C-3), 73.6, 72.3,
72.1, 67.6, 62.0 (CH2), 56.2, 51.0, 48.9, 48.6, 43.5 (C),
43.3 (C), 41.4 (C), 40.5 (CH2), 38.9 (C), 38.8 (CH2),
38.6, 37.6 (C), 36.1 (CH2), 34.8 (CH2), 30.3 (CH2),
28.9, 27.9 (CH2), 25.7 (CH2), 22.4 (CH2), 21.5 (CH2),
19.5, 18.8 (CH2), 18.3, 16.6, 16.5, 16.4, 14.9. HRESIMS:
calcd for C36H60NaO6 [M+Na]+, 611.4282. Found: m/z
611.4311. Anal. Calcd for C36H60O6ꢃH2O: C, 71.25; H,
10.30. Found: C, 71.02; H, 10.38.
4.7. 3b-O-(a-D-Mannopyranosyl)-(1?3)-[(a-D-manno-
pyranosyl)-(1?6)]-a-D-manno-pyranosyl-lup-20(29)-
ene (11)
4.5. 3b-O-(2,3,4,6-Tetra-O-benzoyl-a-D-mannopyran-
osyl)-(1?3)-[(2,3,4,6-tetra-O-benzoyl-a-D-mannopyran-
osyl)-(1?6)]-a-D-mannopyranosyl-lup-20(29)-ene (9)
A suspension of 10 (190 mg, 0.1 mM) and K2CO3
(40 mg) in MeOH (5 mL) was stirred for 2 h, then neu-
tralized with Amberlyst 15 resin (H+ form), filtered
through a short silica pad (MeOH as eluent), and the
filtrate was evaporated to dryness. The residual methyl
benzoate was removed by adding water (3 mL) and was
freeze dried to afford 11 (84 mg, 88%) as a white powder.
13C NMR (pyridine-d5) d: 151.1 (lupene-C-20), 109.9
(lupene-C-29), 103.8 (C-1), 101.8 (C-1), 98.6 (C-1), 82.9,
81.1, 75.5, 75.1, 74.5, 73.2, 73.1, 72.4, 72.1, 72.1, 69.5,
69.3, 67.2 (CH2), 67.1, 63.2 (CH2), 63.0 (CH2), 55.8,
52.0, 50.5, 48.6, 48.3, 43.2 (C), 43.1 (C), 41.1 (C), 40.3
(CH2), 38.7 (C), 38.5 (CH2), 38.3, 37.3 (C), 35.8 (CH2),
34.7 (CH2), 30.2 (CH2), 27.8 (CH2), 25.6 (CH2), 22.7
(CH2), 21.1 (CH2), 19.5, 18.6 (CH2), 18.2, 16.7, 16.3,
16.2, 14.7. HRESIMS: calcd for C48H80NaO16
[M+Na]+, 935.5339. Found: m/z 935.5343.
A soln of 8 (295 mg, 0.5 mmol) in 1:1 CH2Cl2–MeCN
(20 mL) was stirred at room temperature over molecular
˚
sieves (4 A, 500 mg, finely ground) for 30 min, then
cooled to ꢁ40 °C and TMSOTf (55 lL) was added
followed by 5 (815 mg, 1.1 mmol) in CH2Cl2 (10 mL)
dropwise over 15 min. The soln was stirred for a further
30 min, neutralized with Et3N (0.2 mL), and concen-
trated to dryness. Column chromatography (5:1?7:3
hexane–EtOAc, then 5:3:1 hexane–EtOAc–MeOH)
afforded 430 mg of crude 9, which was used in the next
step without further purification.
4.6. 3b-O-(2,3,4,6-Tetra-O-benzoyl-a-D-mannopyran-
osyl)-(1?3)-[(2,3,4,6-tetra-O-benzoyl-a-D-mannopyran-
osyl)-(1?6)]-2,4-di-O-acetyl-a-D-mannopyranosyl-
lup-20(29)-ene (10)
4.8. 1-O-[3-b-Acetoxy-lup-20(29)-ene-28-oyl)-2,3,4,6-
tetra-O-benzoyl-a-D-mannopyranosyl (12)
The above crude product (9) was acetylated under
standard conditions (Ac2O, Py) and purified by column
chromatography (5:1?7:3 hexane–EtOAc) to yield 10
Betulinic acid acetate18 (4, 500 mg, 1.0 mM) was
converted into the glycosyl ester 12 using 5 and the

P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
1001
procedure described for 7 to yield 12 (985 mg, 91%) as a
eluent), and the filtrate was evaporated to dryness. Col-
umn chromatography (7:3 hexane–EtOAc, then 5:3:1?
1:1:1 hexane–EtOAc–MeOH) of the residue gave 14
25
1
foam. ½aꢂD ꢁ18.6 (c 0.6, CHCl3). H NMR (CDCl3) d:
6.44 (d, 1H, J1,2 2.0 Hz, H-1), 6.19 (t, 1H, J4,3
=
20
J4,5 = 10.1 Hz, H-4), 5.87 (dd, 1H, J3,2 3.3 Hz, H-3),
5.72 (dd, 1H, H-2), 4.80 (br s, 1H, lupene-H-29), 4.66
(m, 2H), 4.42–4.52 (m, 3H), 3.05 (ddd, 1H, J 5.0,
11.1 Hz), 2.42 (m, 1H), 2.31 (m, 1H), 2.12 (m, 1H),
2.04 (s, 3H, CH3). 13C NMR (CDCl3) d: 173.1 (C@O),
171.0 (C@O), 166.1 (C@O), 165.5 (C@O), 165.3
(C@O), 165.1 (C@O), 150.0 (lupene C-20), 110.0 (lupene
C-29), 90.3 (C-1), 80.9 (lupene C-3), 71.5, 70.0, 69.3,
66.3, 62.6 (C-6), 57.1 (C), 55.5, 50.5, 49.4, 46.9, 42.5
(C), 40.7 (C), 38.4 (CH2), 38.2, 37.8 (C), 37.1 (C), 37.0
(CH2), 34.2 (CH2), 32.4 (CH2), 30.5 (CH2), 29.6
(CH2), 27.9, 25.5 (CH2), 23.7 (CH2), 21.3 (CH3), 20.8
(CH2), 19.4, 18.2 (CH2), 16.4 (CH3), 16.1 (CH3), 16.0
(CH3), 14.7 (CH3). HRESIMS: calcd for C66H76NaO13
[M+Na]+, 1099.5178. Found: m/z 1099.5142.
(910 mg, 90%) as a foam; ½aꢂD +48.4 (c 0.6, 1:1
CHCl3–MeOH). 1H NMR (1:1 CDCl3–CD3OD) d:
6.04 (d, 1H, J1,2 1.8 Hz, H-1), 2.01 (s, 3H, CH3), 1.66
(s, 3H, CH3), 0.96 (s, 3H, CH3), 0.90 (s, 3H, CH3),
0.83 (s, 3H, CH3), 0.81 (s, 6H, 2 ꢀ CH3). 13C NMR
(1: 1 CDCl3–CD3OD,) d: 173.7 (C@O), 171.3 (C@O),
149.5 (lupene C-20), 109.1 (lupene C-29), 92.7 (C-1),
80.9 (lupene C-3), 75.0, 70.8, 69.3, 65.7, 60.6 (C-6),
56.3 (C), 54.9, 50.0, 48.7, 46.4, 41.9 (C), 40.1 (C), 37.8
(CH2), 37.7, 37.1 (C), 36.5 (C), 36.1, 33.7 (CH2), 31.4
(CH2), 29.7 (CH2), 29.0 (CH2), 27.1 (CH2), 24.9, 23.0
(CH2), 20.3 (CH2), 20.2, 18.3, 17.5 (CH2), 15.6, 15.3,
15.2, 13.9. HRESIMS: calcd for C38H60NaO9
[M+Na]+, 683.4130. Found: 683.4161. Anal. Calcd for
C38H60O9ꢃ1.5H2O: C, 66.35; H, 9.23. Found: C, 66.35;
H, 9.14.
4.9. 1-O-[3-b-Acetoxy-lup-20(29)-ene-28-oyl]-2,3,4,6-
tetra-O-acetyl-a-D-mannopyranosyl (13)
4.11. 1-O-[3-b-Acetoxy-lup-20(29)-ene-28-oyl]-(2,3,4,6-
tetra-O-benzoyl-a-D-mannopyranosyl)-(1?3)-[(2,3,4,6-
tetra-O-benzoyl-a-D-mannopyranosyl)-(1?6)]-a-D-
mannopyranosyl (15)
Betulinic acid acetate (4, 500 mg, 1.0 mM) and 6 were
converted into the glycosyl ester 13 using the procedure
20
described for 7 to yield 13 (772 mg, 93%) as a foam: ½aꢂD
1
+32.4 (c 0.6, CHCl3). H NMR (CDCl3) d: 6.13 (d, 1H,
Mannoside 14 (330 mg, 0.5 mM) and 5 (815 mg,
1.1 mM) were converted into trimannoside 15 using
the procedure described for 9. The product was used
in the next reaction without further purification.
J1,2 2.0 Hz, H-1), 5.35 (t, 1H, J4,3 = J4,5 = 10.0 Hz, H-4),
5.29 (dd, 1H, J3,2 3.3 Hz, H-3), 5.24 (dd, 1H, H-2), 4.74
(d, 1H, J 1.8 Hz, lupene H-29), 4.61 (br s, lupene H-29),
4.46 (dd, 1H, J 8.0 and 10.3 Hz, lupene H-17), 4.29 (dd,
0
1H, J6,5 4.9, J6;6 12:4 Hz, H-6), 4.06 (dd, 1H,
0
4.12. 1-O-[3-b-Acetoxy-lup-20(29)-ene-28-oyl]-(2,3,4,6-
tetra-O-benzoyl-a-D-mannopyranosyl)-(1?3)-[(2,3,4,6-
tetra-O-benzoyl-a-D-mannopyranosyl)-(1?6)]-2,4-di-
O-acetyl-a-D-mannopyranosyl (16)
J6 ;5 2:5 Hz, H-60), 3.98 (m, 1H, H-5), 2.94 (m, 1H,
lupene H-19), 2.18 (s, 3H, CH3), 2.08 (s, 3H, CH3),
2.05 (s, 3H, CH3), 2.03 (s, 3H, CH3), 1.99 (s, 3H,
CH3), 1.68 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.90 (s,
3H, CH3), 0.83 (s, 6H, 2 ꢀ CH3), 0.81 (s, 3H, CH3).
13C NMR (CDCl3) d: 172.9 (C@O), 171.0 (C@O),
170.6 (C@O), 169.9 (C@O), 169.7 (C@O), 169.5
(C@O), 149.8 (lupene C-20), 110.0 (lupene C-29), 90.1
(C-1), 80.9 (lupene C-3), 71.1, 69.0, 68.3, 65.4, 62.2
(C-6), 56.9 (C), 55.4, 50.5, 49.3, 46.8, 42.4 (C), 40.7
(C), 38.4 (CH2), 38.1, 37.8 (C), 37.1 (C), 36.8 (CH2),
34.2 (CH2), 32.2 (CH2), 30.3 (CH2), 29.5 (CH2), 27.9,
25.4 (CH2), 23.7 (CH2), 21.3, 20.8 (CH2), 20.7, 20.7,
20.6, 20.5, 19.3, 18.1 (CH2), 16.4, 16.1, 16.0, 14.6. HRE-
SIMS: calcd for C46H68NaO13 [M+Na]+, 851.4552.
Found: m/z 851.4578.
The crude product 15 was acetylated under standard
conditions (Ac2O, Py) and purified by column chroma-
tography (7:3 hexane–EtOAc then 5:3:0.2?5:3:0.5
hexane–EtOAc–MeOH) to yield 16 (391 mg, 41% after
20
two steps) as an amorphous glass; ½aꢂD ꢁ13.9
(c 0.5, CHCl3). 1H NMR (CDCl3) d: 6.27 (t, 1H,
J4,3 = J4,5 = 10.1 Hz, H-400), 6.25 (d, 1H, J1,2 1.9 Hz,
H-1), 6.17 (m, 1H, J 9.6, 10.9 Hz, H-40), 5.83 (m, 3H,
H-20, 30, 300), 5.63 (t, 1H, J4,3 = J4,5 = 9.9 Hz, H-4),
5.56 (dd, 1H, J2,1 2.0, J2,3 3.1 Hz, H-200), 5.45 (dd, 1H,
J2,3 3.3 Hz, H-2), 5.39 (d, 1H, H-100), 5.14 (br s, 1H,
H-10), 4.75 (m, 2H, H-60, lupene H-29), 4.44–4.65 (m,
6H, H-50, 500, 60, 600, 600, lupene H-29), 4.38 (m, 2H,
H-3, lupene H-3), 4.02 (m, 2H, H-5, 6), 3.76 (dd, 1H,
4.10. 1-O-[3-b-Acetoxy-lup-20(29)-ene-28-oyl]-a-D-
mannopyranosyl (14)
0
J6,5 2.3, J6;6 10:6 Hz, H-6), 2.98 (m, 1H, J 4.7,
11.1 Hz), 2.38 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.01 (s,
3H, CH3), 1.69 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.87
(s, 3H, CH3), 0.66 (s, 3H, CH3), 0.61 (s, 3H, CH3),
0.57 (s, 3H, CH3). 13C NMR (CDCl3) d: 173.3 (C@O),
171.0 (C@O), 170.4 (C@O), 170.1 (C@O), 166.2
(C@O), 166.0 (C@O), 165.5 (C@O), 165.4 (C@O),
To a soln of 13 (1.27 g, 1.53 mM) in MeOH (15 mL), a
soln of freshly prepared sodium methoxide in MeOH
(0.24 M, 0.2 mL) was added and stirred for 90 min.
The soln was neutralized with Amberlyst 15 resin (H+
form), filtered through a short silica pad (MeOH as

1002
P. Cmoch et al. / Carbohydrate Research 343 (2008) 995–1003
165.4 (C@O), 165.3 (2 ꢀ C@O), 165.2 (C@O), 149.8
(lupene C-20), 110.2 (lupene C-29), 99.4 (1JC–H
174.2 Hz, C-10), 98.0 (1JC–H 175.5 Hz, C-100), 90.2
(1JC–H 179.3 Hz, C-1), 80.9 (lupene C-3), 75.5 (C-3),
72.3 (C-5), 70.7 (C-200), 70.4 (C-30 or C-300), 70.1 (C-20),
69.8 (C-500), 69.8 (C-2), 69.4 (C-30 or C-300), 68.9 (C-50),
67.6 (C-4), 67.2 (C-6), 66.6 (C-40), 66.4 (C-400), 62.8
(C-60), 62.4 (C-600), 57.1 (C), 55.4, 50.4, 49.3, 47.1, 42.5
(C), 40.7 (C), 38.4, 38.2 (CH2), 37.6 (C), 37.1 (CH2),
37.1 (C), 36.9 (C), 34.3 (CH2), 32.4 (CH2), 30.5 (CH2),
29.5 (CH2), 27.8, 25.5 (CH2), 23.6 (CH2), 21.3, 21.0,
20.8, 20.8 (CH2), 19.3, 18.0, 16.3, 16.1, 16.0, 14.6. HRE-
SIMS: calcd for C110H116NaO29 [M+Na]+, 1923.7495.
Found: m/z 1924.7516. Anal. Calcd for C110H116O29ꢃ
2H2O: C, 68.17; H, 6.24. Found: C, 68.05; H, 6.29.
Control cultures were treated with Me2SO alone. The
final concentration of Me2SO in the reaction mixtures
never exceeded 0.6%. Fourfold dilutions of the intended
test concentration were added at time zero in 20 lL
aliquots to the microtiter plate wells. Usually, each test
compound was evaluated at six 4-fold dilutions and in
routine testing, the highest well concentration was
50 lM, although this varied in a few cases, depending
on the test compound. After 72 h of culture, the cells
were incubated with Calcein AM solution (Molecular
Probes) for 1 h. The fluorescence of viable cells was
quantified using
(Microsystems). The percentage of surviving cells in
each well was calculated from the equation IC50
(ODdrug
a Fluoroscan Ascent instrument
=
well/mean ODcontrol wells) ꢀ 100%. The
exposed
IC50 value was calculated from the obtained dose-
response curves.
4.13. 1-O-[3-b-Acetoxy-lup-20(29)-en-28-oyl]-(a-D-
mannopyranosyl)-(1?3)-[(a-D-mannopyranosyl)-(1?6)]-
a-D-mannopyranosyl (17)
Acknowledgments
A suspension of 16 (160 mg, 0.084 mM) and K2CO3
(40 mg) in MeOH (5 mL) was stirred for 2 h, neutralized
with Amberlyst 15 resin (H+ form), filtered through a
short silica pad (MeOH as eluent), and the filtrate was
evaporated to dryness. The residual methyl benzoate
was removed by adding water (3 mL) and was freeze
dried to afford 17 (82 mg, quant.) as a white powder.
13C NMR (pyridine-d5) d: 174.3 (C@O), 170.7 (C@O),
150.7 (lupene C-20), 110.2 (lupene C-29), 104.1 (C-1),
102.2 (C-1), 95.1 (C-1), 80.8, 80.5, 76.8, 75.5, 75.2,
73.1, 73.1, 72.3, 72.0, 70.3, 69.5, 69.2, 66.9 (CH2), 66.5,
63.1 (CH2), 63.0 (CH2), 57.2 (C), 55.7, 52.0, 50.8, 49.7,
47.6, 42.8 (C), 41.1 (C), 38.6 (CH2), 38.0 (C), 37.3 (C),
37.2 (CH2), 34.6 (CH2), 32.5 (CH2), 30.9 (CH2), 29.9
(CH2), 28.1, 26.0 (CH2), 24.1 (CH2), 21.2 (CH, CH2),
19.4, 18.5 (CH2), 16.8, 16.3, 14.9. HR-MS (ESI) calcd
for C50H80NaO19 [M+Na]+, 1007.5186. Found:
1007.5177. Anal. Calcd for C50H80O19ꢃH2O: C, 59.86;
H, 8.24. Found: C, 59.77; H, 8.21.
This work was supported by grant 3 TO9A 110 29 from
the Ministry of Science and Higher Education, Poland,
and Grant MSM 6198959216 from the Ministry of Edu-
cation of the Czech Republic.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.02.011.
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