Synthesis and structure-activity relationship study of cytotoxic germanicane- and lupane-type 3β-O-monodesmosidic saponins starting from betulin

Article abstract of DOI:10.1016/j.bmc.2007.06.033

Germanicane-type triterpenes allobetulin (3) and 28-oxoallobetulin (4) can be obtained by the Wagner-Meerwein rearrangement of the more available lupane-type triterpenes betulin (1) and betulinic acid (2), respectively. The medical uses of betulinic acid (2) and its derivatives are limited because of their poor hydrosolubility and pharmacokinetics properties. In order to overcome this major problem, we synthesized and studied the in vitro anticancer activity of a series of 3β-O-monodesmosidic saponins derived from betulin (14-16), betulinic acid (20-22), allobetulin (23-28) and 28-oxoallobetulin (29-34) based on six different natural sugar residues (d-glucose, l-rhamnose, d-arabinose, d-galactose, d-mannose and d-xylose). This structure-activity relationship study confirmed that betulinic acid saponins are generally better in vitro anticancer agents than those derived from betulin with the exception of betulin 3β-O-α-d-mannopyranoside (15) which exerted a potent cytotoxic activity against lung carcinoma (A-549) and colorectal adenocarcinoma (DLD-1) human cell lines with IC₅₀ ranging from 7.3 to 10.1 μmol/L. Furthermore, although the synthesis of novel germanicane-type saponins was carried out with success, the bioactivity measured for these glycosides was not as high as we anticipated since only the 3β-O-β-d-glucopyranoside and 3β-O-β-d-galactopyranoside of allobetulin (23, 24) showed moderate anticancer activity (IC₅₀ 30-40 μmol/L).

Full text of DOI:10.1016/j.bmc.2007.06.033

Bioorganic & Medicinal Chemistry 15 (2007) 6144–6157
Synthesis and structure–activity relationship study of cytotoxic
germanicane- and lupane-type 3b-O-monodesmosidic
saponins starting from betulinq
Dominic Thibeault,ꢀ Charles Gauthier,ꢀ Jean Legault, Jimmy Bouchard,
*
´
Philippe Dufour and Andre Pichette
Laboratoire d’Analyse et de Se´paration des Essences Ve´ge´tales, De´partement des Sciences Fondamentales,
´
`
´
Universite´ du Que´bec a Chicoutimi, 555 boul. de l’Universite, Chicoutimi, Quebec, Canada G7H 2B1
Received 9 March 2007; revised 11 June 2007; accepted 13 June 2007
Available online 20 June 2007
Abstract—Germanicane-type triterpenes allobetulin (3) and 28-oxoallobetulin (4) can be obtained by the Wagner-Meerwein rear-
rangement of the more available lupane-type triterpenes betulin (1) and betulinic acid (2), respectively. The medical uses of betulinic
acid (2) and its derivatives are limited because of their poor hydrosolubility and pharmacokinetics properties. In order to overcome
this major problem, we synthesized and studied the in vitro anticancer activity of a series of 3b-O-monodesmosidic saponins derived
from betulin (14–16), betulinic acid (20–22), allobetulin (23–28) and 28-oxoallobetulin (29–34) based on six different natural sugar
residues (D-glucose, L-rhamnose, D-arabinose, D-galactose, D-mannose and D-xylose). This structure–activity relationship study con-
firmed that betulinic acid saponins are generally better in vitro anticancer agents than those derived from betulin with the exception
of betulin 3b-O-a-D-mannopyranoside (15) which exerted a potent cytotoxic activity against lung carcinoma (A-549) and colorectal
adenocarcinoma (DLD-1) human cell lines with IC50 ranging from 7.3 to 10.1 lmol/L. Furthermore, although the synthesis of novel
germanicane-type saponins was carried out with success, the bioactivity measured for these glycosides was not as high as we antic-
ipated since only the 3b-O-b-D-glucopyranoside and 3b-O-b-D-galactopyranoside of allobetulin (23,24) showed moderate anticancer
activity (IC50 30–40 lmol/L).
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
which are present in the inner and the outer birch bark
residues.10 Because of its selective anti-melanoma activ-
ity and its favourable therapeutic index,3 betulinic acid
(2) is currently undergoing clinical trials at the National
Cancer Institute (NCI). Nevertheless, the medical uses
of betulinic acid (2) and its derivatives in the pharma-
ceutical industry are limited because of their poor
hydrosolubility and pharmacokinetic properties
(absorption, distribution, metabolism and elimina-
tion).11 To overcome this major problem, some labora-
tories have undertaken the synthesis of more water-
soluble betulinic acid (2) derivatives. For example, it
was reported that the introduction of polar moieties at
the C-3 and C-28 positions such as amino acids12 and
phthalates13 enhances, in certain cases, the hydrosolu-
bility and the anticancer activity of betulin (1) and bet-
ulinic acid (2).
Lupane-type derivatives have attracted increased atten-
tion in the past decade1 since they exhibit a broad range
of biological and medicinal properties such as antican-
cer,2 antitumoral,3,4 anti-inflammatory,5 anti-HIV,6
anti-malarial7 and antifeedant8 activities. Among these
lupanes, betulin (1) and betulinic acid (2) (Fig. 1) are
natural pentacyclic triterpenes that can be found in the
external bark of various Betula species such as B. papy-
rifera.9 These compounds and more especially betulin
(1) are regarded by the scientific community as accessi-
ble, abundant and valuable bioactive natural products
Keywords: Germanicane; Lupane; Glycoside; Anticancer.
q
This article is dedicated to the memory of Linda Grenon, secretary
at the UQAC Fundamental Sciences Department, who passed away
from lung cancer while the preparation of this article was in progress.
Allobetulin (3) and 28-oxoallobetulin (4) (Fig. 1) are
a
*
Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545
5012; e-mail: andre_pichette@uqac.ca
These authors contributed equally to this work.
members of the germanicane-type triterpene family,14
rare class of natural compounds. In contrast to the well
ꢀ
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.033

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6145
evaluation of germanicane-25,31–33 and lupane-type34–38
saponins. To the best of our knowledge and with respect
to the germanicane-type triterpenes, only 3b-O-2-deoxy-
a-glycopyranosides25,31 and 3b-O-b-D-glucopyrano-
side32,33 of allobetulin (3) were synthesized and no
screening of their bioactivities was reported. Moreover,
natural saponins based on betulin (1), betulinic acid (2),
allobetulin (3) and 28-oxoallobetulin (4) aglycones are
especially rare.39,40 Braca et al.40 reported the isolation
of bidesmosidic saponins from the aerial parts of Sche-
fflera rotundifolia where the most active constituents
have, interestingly, betulinic acid (2) as a residue.
Figure 1. Structures of lupane (1,2) and germanicane-type (3,4)
triterpenes.
Recently, we focused our attention on the synthesis of
3b-O-monodesmosidic lupane-type saponins.41 Our re-
sults showed that adding a sugar moiety at the C-3 posi-
tion can give stronger in vitro anticancer agents than
betulinic acid (2). Moreover, glycosides of betulinic acid
(2) showed a selectivity on the cancerous cells up to 12-
fold higher than on healthy cells. These promising
results prompted us to extend our study to the
germanicane-type allobetulin (3) and 28-oxoallobetulin
(4). In order to establish meaningful structure–activity
relationship (SAR) and to modulate the pharmacoki-
netic properties of the triterpenes (1–4), we wanted to
synthesize glycosides incorporating other natural sugar
moieties. Hence, we report here the synthesis of several
3b-O-monodesmosidic saponins derived from lupane-
(14–16, 20–22) and germanicane-type (23–34) triterpenes
based on six different natural sugar residues (D-glucose,
L-rhamnose, D-arabinose, D-galactose, D-mannose and
D-xylose). Cytotoxicity of triterpenes and glycosides
was assessed against lung carcinoma (A-549), colorectal
adenocarcinoma (DLD-1) and normal skin fibroblasts
(WS1) human cell lines. Also, in silico calculations of
pharmacokinetic properties were carried out using the
ADME prediction program QikProp42,43 version 2.5 in
order to evaluate important pharmaceutical parameters
such as lipophilicity (logP) and aqueous solubility
(logS).
distributed oleanane-type triterpenes in the plant king-
dom,15 the stereochemistry of the hydrogen atom at C-
18 is in the a-configuration for the germanicanes rather
than the b-configuration. Triterpenes 3 and 4 can be ob-
tained by the Wagner-Meerwein rearrangement of the
more abundant betulin (1) and betulinic acid (2), respec-
tively, in the presence of Lewis or solid acids.16,17
Although the syntheses of allobetulin (3) and oxyallo-
betulin (4) and analogues were already reported in the
literature,17–24 few studies were reported regarding the
development of their derivatives for potential therapeu-
tic applications.25,26 This is probably due to the hydro-
phobic character of these compounds which
complicates the solubilization and the formulation in
several biological assays.
Triterpenoid glycosides, saponins, are widely distributed
throughout the plant kingdom.27 They are considered
responsible for the bioactivity, including cytotoxic and
anticancer activities, of several medicinal plants.28,29
The synthesis of glycosides is an interesting approach
to enhance the hydrosolubility and, consequently, the
pharmacological and pharmacokinetic properties of
lead compounds.30 Nonetheless, few studies exist in
the literature regarding the synthesis and the biological
Scheme 1. Synthesis of 3-acetylbetulinic acid (8). Reagents and conditions: (a) Ac2O (2.5 equiv), DMAP (0.1 equiv), pyridine, rt, quantitative; (b)
Al(i-OPr)3 (5 equiv), i-PrOH, reflux 1.5 h, 86%; (c) PCC (2 equiv), CH2Cl2, rt, 2 h, 96%; (d) NaClO2/NaH2PO4, t-BuOH/THF/2-methyl-2-butene,
0 ꢁC to rt, 60 min, 81%.

6146
D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3-acetylbetulinic acid (8). The main
source of betulin (1) used in this study was the available
Betula papyrifera March. widely distributed in the bor-
eal forest of North America. Finely powdered outer
bark was heated under reflux in chloroform or methy-
lene chloride to provide crude betulin (1). This extract
was purified following the Eckerman and Ekman44 pro-
cedure. Recrystallized betulin (1) was the starting mate-
rial used directly for the synthesis of allobetulin (3) and
3-acetylbetulinic acid (8) while the latter was the precur-
sor of allyl betulinate (9) and 28-oxoallobetulin (4). The
synthetic pathway employed for the synthesis of 3-acet-
ylbetulinic acid (8) (Scheme 1) was inspired by the pro-
cedure developed by Krasutsky et al. with slight
modifications.45 Initially, betulin (1) was acetylated at
the C-3 and C-28 positions with acetic anhydride
(Ac2O) and 4-dimethylaminopyridine (DMAP) in anhy-
drous pyridine to afford betulin 3,28-diacetate (5) in
quantitative yield without further purification. Thereaf-
ter, compound 5 was deacetylated at the C-28 position
in 86% isolated yield in the presence of five equivalents
of aluminium isopropoxide. Third, the alcohol 6 was
successively converted into aldehyde 7 using pyridinium
chlorochromate (PCC) as oxidizing agent in 96% yield.46
Finally, the crude aldehyde 7 was directly oxidized to
3-acetylbetulinic acid (8) by the combined action of so-
dium chlorite (NaClO2) and sodium dihydrogenophos-
phate monohydrate (NaH2PO4ÆH2O) in a t-BuOH/
THF/2-methyl-2-butene mixture. This procedure was
developed by Clive et al.47 and adapted for substrate 7
to afford compound 8 in only 60–70 min at room
temperature in 81% isolated yield. To the best of our
knowledge, this synthetic pathway (Scheme 1) is the
most efficient methodology for an easy access to 3-acet-
ylbetulinic acid (8) and its derivatives.48
Scheme 2. Synthesis of betulin (11–16) and allobetulin (23–28)
saponins. Reagents and conditions: (a) Ac2O (1.05 equiv), pyridine,
DMAP (0.1 equiv), rt, 2 h, 73%; (b) Fe(NO3)3/SiO2, DCM, reflux,
45 min, 72%; (c) trichloroacetimidate (1.5 equiv), TMSOTf (0.05
2.1.2. Synthesis of saponins. The first series of glycosides
(14–16, 23–28) were synthesized according to the
reaction sequence shown in Scheme 2. In order to avoid
multiple glycosylation reactions, betulin (1) was first
acetylated at the C-28 position with Ac2O/DMAP in
pyridine to give betulin 28-acetate (10). Allobetulin (3)
was obtained from the Wagner-Meerwein rearrange-
ment of 1 by the action of Fe(NO3)3/SiO2 (1:4) in
refluxed CH2Cl2. Aglycone acceptors 10 and 3 were
coupled with the appropriate trichloroacetimidate sugar
donors49 (Fig. 2) under the catalytic promotion of the
Lewis acid trimethylsilyl trifluoromethanesulfonate
(TMSOTf).50 Thereafter, deprotection of the acetyl
and benzoyl groups (NaOH, MeOH/THF/H2O 1:2:1)
affording betulin (14–16) and allobetulin (23–28) sapo-
nins in isolated yields ranging from 19% to 49% and
67% to 89%, respectively (two steps). The second series
of glycosides (20–22, 29–34) came from 3-acetylbetulinic
acid (8) and were synthesized following to the reaction
sequence shown in Scheme 3. In order to avoid the
formation of by-products, the C-28 allyl ester 9 was first
prepared as already reported by our group.41 28-Oxoal-
lobetulin (4) was obtained from the Wagner-Meerwein
˚
equiv), 4 A MS, DCM, rt, 1.5 h; (d) NaOH 0.25 M, MeOH/THF/H2O,
rt, 2 h.
rearrangement by the action of FeCl3/SiO2 (1:4) on the
substrate 8 in refluxed CH2Cl2. Thereafter, the synthesis
of glycosides with aglycone acceptors 9 and 4 was
achieved using the same glycosylation protocol men-
tioned above. Deprotection of the benzoyl groups affor-
ded 28-oxoallobetulin saponins (29–34) in isolated yields
ranging from 20% to 70% (two steps). Betulinic acid sap-
onins (20–22) were obtained after the supplementary
deprotection of the allylic function under the catalytic
action of tetrakistriphenylphosphine palladium(0) in
isolated yields ranging from 23% to 58% (three steps).
The synthesis of other betulin (11–13) and betulinic acid
(17–19) glycosides was previously reported by our
group.41 As we expected, the presence of benzoyl pro-
tecting groups in position 2 of the sugar trichloroacetim-
idates directed the anomeric selectivity of the
glycosidation reaction51 to give exclusively 1,2-trans-
monodesmosides (b-D-glucopyranosides, a-L-rhamno-
pyranosides, a-D-arabinopyranosides, b-D-galactopyr-

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6147
Figure 2. Trichloroacetimidate (TCA) sugar donors used for the glycosidation.
anosides, b-D-xylopyranosides and a-D-mannopyrano-
sides). This was confirmed by the chemical shifts and
the vicinal coupling constants of the anomeric protons
control in this experimentation (IC50 10.3–15 lmol/L).
Compounds with IC50 values >75 lmol/L were consid-
ered as inactive.
1
in H NMR experiments.52 The structures of all newly
synthesized saponins (14–16, 20–22, 23–34) were con-
firmed and elucidated by extended 1D and 2D NMR
experiments (1H, 13C, DEPT135, COSY, HSQC,
HMBC), IR spectra and HRMS. Saponins 1639d and
1839c are naturally occurring products whose synthesis
had not been reported in the literature until now.
Our previous SAR investigations showed that adding a
sugar moiety at the C-3 position of betulin (1) signifi-
cantly decreased cytotoxicity compared to its carboxylic
derivative 2.41 This extended study revealed that there
are some exceptions to this general tendency. In fact,
betulin 3b-O-a-D-mannopyranoside (15) was found to
exhibit more potent cytotoxicity than betulinic acid (2)
with IC50 values ranging from 7.3 to 10.1 lmol/L
against cancer cell lines. In contrast to other sugars
linked to the C-3 position of betulin (1) which suppress
the anticancer activity, the specific stereochemistry of D-
mannose seems to have a beneficial effect on the cytotox-
icity of betulin (1). With regard to betulinic acid (2), we
have previously shown that glycosidation at the C-3 po-
sition significantly increased both the activity and the
selectivity towards cancer cell lines for D-glucose, L-
rhamnose and D-arabinose monosaccharides.41 In this
study, betulinic acid 3b-O-a-D-mannopyranoside (21)
and betulinic acid 3b-O-b-D-xylopyranoside (22) exerted
moderate activity against A-549 (21: IC50 34 lmol/L; 22:
IC50 15 lmol/L) and DLD-1 (21: IC50 15 lmol/L; 22:
IC50 18 lmol/L) with cytotoxicity profiles slightly infe-
rior to aglycone 2, while the 3b-O-b-D-galactopyrano-
side 20 was inactive (IC50 > 75 lmol/L). Moreover, no
significant increase in the selectivity was measured for
these new betulinic acid (2) saponins. These results sug-
gest that both the anticancer activity and the differential
selectivity of betulinic acid (2) are strongly influenced by
the nature of the carbohydrate moiety at the C-3 posi-
2.2. Cytotoxic activity
The in vitro antiproliferative activity of these triterpenes
(1–4, 8) and saponins (11–34) was investigated against
human lung carcinoma (A-549) and human colorectal
adenocarcinoma (DLD-1) cancer cell lines. We chose
these types of cancer because they have a great incidence
in the human population. Assessments were also carried
out on normal human skin fibroblasts (WS1) to evaluate
the differential selectivity of the target compounds. Cell
lines cultured in essential medium were plated on 96-well
microplates (5.0 · 103 cells per well) and allowed to ad-
here for 16 h before treatment at 37 ꢁC in a humidified
environment containing 5% CO2. Thereafter, increasing
concentrations of each compound in DMSO were
added. The cell viability was assessed through the resa-
zurin reduction test as previously described in the litera-
ture.53 Measurements of fluorescence were carried out
after 48 continuous hours of contact between com-
pounds and cells. Results presented in Table 1 are ex-
pressed as the concentration inhibiting 50% of the cell
growth (IC50). Betulinic acid (2) was used as a positive

6148
D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
thought that glycosidation could have a significant im-
pact on the in vitro anticancer activity of these rare
members of the germanicane-type triterpenoid family.
Interestingly, 3b-O-b-D-glucopyranoside and 3b-O-b-D-
galactopyranoside of allobetulin (23,26) exhibited mod-
erate cytotoxicity towards cancer cell lines with IC50 val-
ues ranging from 30 to 40 lmol/L. It should be noted
that, contrary to betulinic acid (2), the introduction of
these sugar moieties at the C-3 position of 3 resulted
in an enhancement of the activity. Furthermore, all
3b-O-glycosides of 28-oxoallobetulin (29–34) were inef-
fective to inhibit the growth of the cancerous cell lines
(IC50 > 75 lmol/L). In terms of SAR, these in vitro cyto-
toxic results suggest that lupane-type triterpenoid sapo-
nins are generally stronger anticancer agents than
germanicane-type triterpenoid saponins and particularly
those with betulinic acid (2) as aglycone.
2.3. Predicted lipophilicity and aqueous solubility
In order to pass through biological membranes (gastro-
intestinal membrane, blood–brain barrier, cellular mem-
brane, etc.), a therapeutic agent must be relatively solu-
ble in water. In medicinal chemistry, the aqueous
solubility and lipophilicity of a drug have proven to be
important molecular parameters determining the
absorption, the bioavailability and, sometimes, the bio-
activity.55 The aqueous solubility (logS) reflects the con-
centration S of the compound in mol/L for a saturated
aqueous solution in equilibrium with the crystalline
material, while the lipophilicity is indicated by the loga-
rithm of a partition coefficient (logP) which reflects the
concentration ratio of the drug at equilibrium partition-
ing between octanol and water phases.42 In this study,
we have used the ADME prediction program QikProp
version 2.5 to evaluate in silico these parameters (Table
1). As we expected, adding a sugar moiety enhances the
aqueous solubility (>logS) and decreases the lipophilic-
ity (<logP) of lupane- and germanicane-type triterpe-
noids (1–4). These theoretical results are in good
agreement with our experimental results since glycosides
were shown to be more soluble in polar solvents
(DMSO, MeOH) used for in vitro bioassays than their
corresponding aglycones (data not shown). Interest-
ingly, all synthesized glycosides (11–34) have logP val-
ues smaller than 5. Thus, concerning the partition
coefficient, these compounds satisfy one of the ‘Lipinski
Rule-of-5’ which is important to ensure a good intestinal
absorption and permeation of the therapeutic agent
when orally delivered.56 Moreover, if we compare logP
values of the most active betulinic acid (2) glycosides, we
observe that the order of anticancer activity potential
(18 > 19 > 22 > 21) generally follows the order of lipo-
philicity (18 > 19 > 22 > 21). Nevertheless, it is obvious
that lipophilicity and aqueous solubility alone cannot
be invoked to explain the difference in cytotoxicity of lu-
pane- and germanicane-type triterpenoid saponins.
Scheme 3. Synthesis of betulinic acid (17–22) and 28-oxoallobetulin
(29–34) saponins. Reagents and conditions: (a) allyl bromide (2 equiv),
K2CO3 (3 equiv), DMF, 55 ꢁC, 7 h; (b) NaOH 0.25 M, MeOH/THF/
H2O, rt, 2 h, 93% (two steps); (c) FeCl3/SiO2, DCM, reflux, 3 h; (d)
NaOH 0.25 M, MeOH/THF/H2O, reflux, overnight, 91% (two steps);
˚
(e) trichloroacetimidate (1.5 equiv), TMSOTf (0.05 equiv), 4 A MS,
DCM, rt, 1.5 h; (f) NaOH 0.25 M, MeOH/THF/H2O, rt, 2 h; (g)
Pd(PPh3)4 (0.3 equiv), PPh3 (0.6 equiv), pyrrolidine (2 equiv), THF, rt,
1–3 d.
tion. It is interesting to note that the 3b-O-acetyl deriv-
ative of betulinic acid (8) was significantly less cytotoxic
against human normal fibroblasts than against cancer-
ous cell lines.
It appeared that modifications in the parent structure of
lupane-type triterpenoids such as the loss of the isoprop-
ylene group at the C-20 position and the expansion of
ring E to six carbons seem to have a deleterious effect
on the cytotoxicity profile. Indeed, no significant dimi-
nution of the cancerous cells’ growth was observed at
the maximum tested concentration for both allobetulin
(3) and 28-oxoallobetulin (4) (IC50 > 75 lmol/L). These
results are in good agreement with those of Kim
et al.54 since they conclude that the C-20 isopropylene
group of betulinic acid (2) is not a favourable position
to derive in order to enhance the cytotoxicity. We
3. Conclusion
In summary, starting from betulin (1) isolated from the
outer bark of B. papyrifera, we successfully synthesized

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6149
Table 1. In vitro cytotoxicity of lupane and germanicane triterpenes (1–4, 8) and saponins (11–34)
O
R3
OH
H
H
R2
R1O
R1O
R1
R2
R3
Cell line IC50 (lmol/L SD)
logPe
logSf
a
Compound
A-549b
DLD-1c
WS1d
1
2
H
H2
O
—
—
H2
O
3.80 0.09g
10.3 0.4g
>75
6.6 0.3g
15.0 0.3g
>75
3.58 0.07g
12 1g
>75
6.06
6.31
6.24
5.55
7.16
3.80
4.57
4.43
3.86
4.08
4.44
4.03
4.81
4.76
4.15
4.08
4.66
3.95
4.71
4.73
4.17
3.99
4.67
3.32
4.09
4.00
3.40
3.34
3.92
ꢀ6.70
ꢀ6.91
ꢀ7.44
ꢀ6.83
ꢀ8.30
ꢀ6.09
ꢀ6.32
ꢀ5.94
ꢀ6.04
ꢀ6.27
ꢀ6.64
ꢀ6.33
ꢀ6.62
ꢀ6.80
ꢀ6.13
ꢀ6.27
ꢀ6.75
ꢀ6.61
ꢀ6.94
ꢀ6.97
ꢀ6.30
ꢀ6.67
ꢀ6.96
ꢀ6.31
ꢀ6.56
ꢀ6.64
ꢀ6.29
ꢀ6.16
ꢀ6.57
H
3
H
—
—
O
4
H
Ac
>75
18 2h
>75g
22 3g
41 3g
>75
>75
20 2h
>75e
50 10g
63 8g
>75
>75
57
8
—
—
—
—
—
—
—
—
—
—
—
—
—
H2
H2
H2
H2
H2
H2
O
6
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Glc
Rha
Ara
Gal
Man
Xyl
Glc
Rha
Ara
Gal
Man
Xyl
Glc
Rha
Ara
Gal
Man
Xyl
Glc
Rha
Ara
Gal
Man
Xyl
H2
H2
H2
H2
H2
H2
O
> 75e
33 5g
59 5g
>75
7.3 0.4
>75
>75e
2.6 0.6g,h
10 2g,h
>75
10.1 0.5
>75
32 9g,h
3.9 0.4g,h
17 3g,h
>75
5.1 0.6
>75
> 75g
31 3g
47 5g
>75
O
O
O
O
34
15
4
2
3
15
18
1
2
13
20
3
1
4
O
—
—
—
—
—
—
—
—
—
—
—
—
31
40 10
>75
>75
40
>75
>75
>75
>75
30 10
>75
>75
>75
>75
>75
>75
>75
>75
40 10
>75
>75
30 10
>75
>75
>75
>75
>75
>75
>75
>75
>75
>75
O
O
>75
>75
O
O
>75
>75
O
Glc, b-D-glucopyranose; Rha, a-L-rhamnopyranose; Ara, a-D-arabinopyranose; Gal, b-D-galactopyranose; Man, a-D-mannopyranose; Xyl, b-D-
xylopyranose; Ac, acetate.
a Data represent mean values standard deviation for three independent experiments made in triplicate.
b Human lung carcinoma.
c Human colorectal adenocarcinoma.
d Human normal skin fibroblasts.
e Predicted octanol/water partition coefficient.
f Predicted aqueous solubility.
g Results previously reported.41
h Significantly more sensitive than fibroblasts; p < 0.05, Student’s t test.
a library of 3b-O-monodesmosidic saponins (11–34)
having betulin (1), betulinic acid (2), allobetulin (3)
and 28-oxoallobetulin (4) as aglycones, and b-D-gluco-
pyranose, a-L-rhamnopyranose, a-D-arabinopyranose,
b-D-galactopyranose, a-D-mannopyranose and b-D-
xylopyranose as sugar moieties, in order to enhance
the cytotoxicity and the hydrosolubility of germanicane-
and lupane-type triterpenes. Saponins 16 and 18 are
naturally occurring products whose synthesis had not
been reported in the literature until now. This extended
SAR study confirmed that saponins of betulinic acid
(17–22) are generally better in vitro anticancer agents
than those derived from betulin (11–16) with the excep-
tion of betulin 3b-O-a-D-mannopyranoside (15) which
exhibited a strongly potent cytotoxic activity against
cancerous cell lines. Furthermore, although the synthe-
sis of novel germanicane-type saponins was carried out
with success, the bioactivity measured for these glyco-
sides was not as high as we anticipated since only 3b-
O-b-D-glucopyranoside and 3b-O-b-D-galactopyrano-
side of allobetulin (23,26) showed moderate anticancer
activity.

6150
D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
4. Experimental
respectively, following a procedure previously reported by
our laboratory.41 2,3,4,5-Tetra-O-benzoyl-a,b-D-galacto-
pyranose trichloroacetimidate,58 2,3,4,6-tetra-O-benzoyl-
a,b-D-mannopyranose trichloroacetimidate59 and 2,3,4-
tri-O-benzoyl-a,b-D-xylopyranose trichloroacetimidate49
were prepared from D-galactose, D-mannose and D-xylose,
respectively, as previously reported. Crude betulin (1) was
extracted from the outer bark of B. papyrifera March. and
recrystallized with an azeotropic mixture of 2-butanol/
water (74:26) to afford 1 with an acceptable purity
(>95%) according to GC–MS. The synthesis of lupane-
type saponins (11–13 and 17–19) was already reported by
our laboratory.41
4.1. Chemicals
Chemical reagents were purchased from Sigma–Aldrich
Co. Canada or Alfa Aesar Co. and were used as
received. The usual solvents were obtained from VWR
International Co. and were used as received. Dichloro-
methane (DCM) was distilled from anhydrous CaH2
under an argon atmosphere. Tetrahydrofuran (THF)
was distilled from sodium/benzophenone ketyl under
an argon atmosphere. All anhydrous and air sensitive
reactions were performed in oven-dried glassware under
positive argon pressure. Tetrakistriphenylphosphine
palladium(0) was prepared as mentioned in the litera-
ture57 and stored under argon. Analytical thin-layer
4.2. Betulin 3,28-diacetate (5)
chromatography was performed with silica gel 60 F254
,
To a solution of betulin (1) (2.33 g, 5.26 mmol) dissolved
in 20 mL of pyridine at room temperature was added
1.33 mL of Ac2O (14.10 mmol) followed by 64 mg of
DMAP (0.53 mmol) and stirred for 1 h. Fifty millilitres
of CH2Cl2 and 50 mL of distilled water were then
poured into the solution, the mixture was acidified with
H2SO4 6.2 M (25 mL) at 0 ꢁC and then extracted three
times with CH2Cl2 (25 mL). The combined organic
phases were washed with saturated NaHCO3 until pH
ꢁ7. The organic phase was dried over Na2SO4, filtered
and evaporated under reduced pressure. Simple filtra-
tion over silica gel using EtOAc/hexanes (2.5:97.5) as
eluent afforded 2.77 g of compound 5 as a white solid
´
0.25 mm pre-coated TLC plates (Silicycle, Quebec,
Canada). Aliphatic compounds were visualised using
aqueous sulfuric acid solution of ammonium heptamo-
lybdate tetrahydrate (10 g/100 mL H2SO4 + 900 mL
H2O) and aromatic compounds were visualised using
254 nm and/or 365 nm UV wavelength. Flash column
chromatographies were performed on 230–400 mesh
´
silica gel R12030B (Silicycle, Quebec, Canada). Infrared
spectra (IR) were recorded with Perkin-Elmer
a
Spectrum One FT-IR spectrophotometer (Shelton,
Connecticut, USA) on a NaCl window from a thin film
of the analyzed compounds and only significant
absorption bands were reported in cmꢀ1. Nuclear
magnetic resonance (NMR) spectra were recorded on
a Bruker Avance spectrometer at 400 MHz (1H) and
100 MHz (13C), equipped with a 5 mm QNP probe.
in a quantitative yield: mp 216–218 ꢁC, lit.60 mp 223–
20
224 ꢁC; ½aꢂ +19.7 (c 1.67, CHCl3); IR: 2945, 2872,
D
1738 (C@O), 1641, 1455, 1365, 1243 (C–O ester), 1030,
979, 883, 738. 1H and 13C NMR spectral data of 5 were
in agreement with those published in the literature.61
1
Elucidations of chemical structures were based on H,
13C, COSY, HMBC, HSQC and DEPT-135 experi-
ments. Signals are reported as m (multiplet), s (singlet),
d (doublet), dd (doublet of doublet), ddt (doublet of
doublet of triplet), br s (broad singlet) and coupling
constants are reported in hertz (Hz). The chemical shifts
are reported in ppm (d) relative to residual solvent peak.
The labile OH NMR signals appearing sometimes were
not listed. Optical rotations were obtained in a solution
of 10% MeOH in CHCl3 (v/v) unless otherwise specified
using sodium D line at ambient temperature on a Jasco
DIP360 digital polarimeter. High-resolution mass
spectra (HRMS) were obtained at the Department of
Chemistry, Queen’s University, Ontario, Canada.
HPLC preparative methods for the purification of sapo-
nins 20, 21 and 22 in DMSO (10 mg/mL) were carried
out with an Agilent 1100 system equipped with a frac-
tion collector using Zorbax ODS C18 column
(21.2 · 250 mm; 7 lmol/L), at a flow rate of 16.0 mL/
min with a column temperature of 25 ꢁC. Compounds
were detected by UV absorption at 254 nm following
this elution gradient: 70–100% B where B = MeOH
and A = H2O + 0.1% HCOOH in 30 min (increasing
concentration of B by 1%/min).
4.3. Betulin 3-acetate (6)
Compound 5 (250 mg, 0.470 mmol) and Al(i-OPr)3
(490 mg, 2.35 mmol) were stirred under reflux in 8 mL
of i-PrOH for 2 h. The crude mixture was concentrated
under reduced pressure, diluted with CH2Cl2 (25 mL)
and water (25 mL). The mixture was then extracted with
CH2Cl2 (3· 25 mL). The combined organic layers were
dried over Na2SO4, filtered and evaporated under re-
duced pressure. The crude product was purified by flash
chromatography on silica gel using EtOAc/hexanes
(10:90 to 20:80) as eluent to afford 0.20 g (86% yield)
of compound 6 as a white solid: mp 258–260 ꢁC, lit.60
20
mp 256–258 ꢁC; ½aꢂ +25.7 (c 0.92, CHCl3); IR: 3438
D
(broad, OH), 2944, 2872, 1732 (C@O), 1454, 1374,
1245 (C–O ester), 1105, 1028 (C–OH), 979, 882, 738.
1H and 13C NMR spectral data of 6 were in agreement
with those published in the literature.62
4.4. 3-Acetylbetulinal (7)
Compound 6 (9.77 g, 20.2 mmol) and PCC (8.73 g,
40.3 mmol) were added to 980 mL of CH2Cl2 and stirred
at room temperature for 2 h. Silica gel (30–50 g) was
poured into the mixture and the solvent was evaporated
under reduced pressure to dryness. The residual dry-
pack was directly used for purification by flash chroma-
tography on silica gel using EtOAc/hexanes (5:95) as
Sugar donors 2,3,4,6-tetra-O-benzoyl-a,b-D-glucopyra-
nose trichloroacetimidate, 2,3,4-tri-O-benzoyl-a,b-L-
rhamnopyranose trichloroacetimidate and 2,3,4-tri-O-
benzoyl-a,b-D-arabinopyranose trichloroacetimidate were
synthesizedfrom D-glucose, L-rhamnose, and D-arabinose,

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6151
eluent to afford 9.34 g (96% yield) of aldehyde 7 as a
+31.7ꢁ (c 0.71, CHC13), lit.60
dried over Na2S2O4, filtered and evaporated under re-
duced pressure. Crude product was purified by flash
chromatography on silica gel using EtOAc/hexanes
(5:95 to 17.5:82.5) as eluent to afford 922 mg (93% yield
20
D
white solid: ½aꢂ
20
D
½aꢂ +30.3 (c 2.0, CHCl3); IR: 2943, 1731 (C@O), 1641,
1
1451, 1375, 1244 (C–O ester), 1027, 979, 883, 738. H
NMR (CDCl3): 9.66 (s, 1H), 4.75 (br s, 1H, H-29),
4.62 (br s, 1H, H-29), 4.45 (dd, 1H, J = 9.9 Hz,
J = 5.8 Hz, H-3), 2.86 (m, 1H), 2.20–0.70 (24H), 2.03
(s, 3H), 1.69 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H), 0.83 (s,
6H), 0.82 (s, 3H); 13C NMR (CDCl3): 206.85, 171.19,
149.85, 110.34, 81.04, 59.47, 55.52, 50.51, 48.17, 47.69,
42.69, 40.97, 38.81, 38.54, 37.92, 37.22, 34.38, 33.35,
29.98, 29.36, 38.93, 28.07, 25.62, 23.82, 21.46, 20.89,
19.11, 18.28, 16.62, 16.34, 16.03, 14.36; HR-EI-MS m/z
482.3779 [M]+ (calcd for C32H50O3, 482.3760).
for two steps) of compound 9 as a white solid: mp 152–
20
D
154 ꢁC; ½aꢂ +3.9 (c 1.00, CHCl3); IR: 3421, 2945, 2869,
1725, 1642, 1452, 1376, 1270, 1152, 1131, 1106, 1043,
982, 884, 738. 1H and 13C NMR spectral data of 9 were
in agreement with those previously reported by our
laboratory.41
4.7. Betulin 28-acetate (10)
Compound 1 (1.00 g, 2.07 mmol) and DMAP (28 mg,
0.21 mmol) were dissolved in 15 mL of pyridine and stir-
red at room temperature. Then, 0.21 mL of Ac2O
(2.07 mmol) was added dropwise and stirred for 1 h.
After, the solution was diluted with 50 mL of CH2Cl2
and poured into a separatory funnel filled with ice and
40 mL of H2SO4 6.2 M was slowly added. The aqueous
phase was extracted three times with CH2Cl2 (50 mL)
and combined organic phases were dried over Na2SO4,
filtered and evaporated under reduced pressure. The
crude product was purified by flash chromatography
on silica gel using EtOAc/hexanes (5:95 to 20:80) as elu-
ent. Betulin 28-acetate (10) (0.80 g, 73% yield) was ob-
tained with betulin 3,28-diacetate (5) (0.13 g, 11%
4.5. 3-Acetylbetulinic acid (8)
Compound 7 (1.00 g, 2.27 mmol) was dissolved in
50 mL of t-BuOH, 10 mL of distilled THF and 15 mL
of 2-methyl-2-butene. The solution was stirred and
cooled in an ice-bath. Then, 30 mL of freshly prepared
solution of aqueous NaH2PO4/NaClO2 (2.50 g/2.50 g
in 30 mL of distilled water) was slowly added to the
solution and the mixture was stirred for 15 min. The
mixture was then raised to rt and stirred for 1 h. Finally,
the mixture was poured into 50 mL of a saturated solu-
tion of NH4Cl and extracted three times with CH2Cl2.
The combined organic layers were dried over Na2SO4,
filtered and evaporated under reduced pressure. Purifi-
cation of the crude product by flash chromatography
using isocratic 7% ethyl acetate in hexanes as eluent
yield) and starting betulin (1) (0.13 g, 13% yield), all as
20
D
white solids: mp 210–212 ꢁC; ½aꢂ +8.5 (c 1.58, CHCl3);
IR: 3468 (broad, OH), 2942, 2870, 1739 (C@O), 1641,
1454, 1388, 1364, 1237 (C–O ester), 1033 (C–OH), 981,
1
afforded 772 mg (81% yield) of carboxylic acid 8 as a
883, 738. H and 13C NMR spectral data of 10 were in
20
D
white solid: ½aꢂ +26.4ꢁ (c 0.54, CHCl3); IR: 2945,
agreement with those published in the literature.61
1735 (C@O), 1696 (C@O), 1452, 1369, 1244 (C–O ester),
1027, 979. 1H NMR (CDCl3): 4.74 (br s, 1H, H-29), 4.61
(br s, 1H, H-29), 4.47 (dd, 1H, J = 10.4 Hz, J = 5.6 Hz,
H-3), 3.00 (m, 1H), 2.30–0.70 (25H), 2.04 (s, 3H), 1.69
(s, 3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.85 (s, 3H), 0.84 (s,
3H), 0.83 (s, 3H); 13C NMR (CDCl3): 182.19, 171.21,
150.51, 109.90, 81.09, 56.54, 55.55, 50.53, 49.40, 47.09,
42.56, 40.83, 38.56, 38.52, 37.95, 37.27, 37.19, 34.37,
32.30, 30.71, 29.84, 28.10, 25.58, 23.84, 21.48, 20.99,
19.50, 18.31, 16.62, 16.33, 16.19, 14.81; HR-EI-MS m/z
521.3629 [M+Na]+ (calcd for C32H50O4Na, 521.3607).
4.8. Allobetulin (3)
This compound was prepared as previously reported16
following this procedure: 5.00 g of betulin (1)
(11.29 mmol), dissolved in 500 mL of CH2Cl2 with a mix-
ture of Fe(NO3)3/SiO2 (1:4) grounded on a mortar (9.13 g/
36.50 g, 22.58 mmol of Fe(NO3)3) were refluxed for
45 min. The solution was then filtered and washed with
CH2Cl2 and evaporated under reduced pressure. The
crude product was purified by flash chromatography on
silica gel using EtOAc/hexanes (10:90 to 20:80) as eluent
4.6. Allyl betulinate (9)
to afford 3.60 g (72% yield) of allobetulin (3) as a white so-
20
D
lid: ½aꢂ +44.5ꢁ (c 0.65, CHCl3); IR: 3452, 2926, 2863,
Compound 8 (1.00 g, 2.01 mmol) was solubilized with
14 mL of anhydrous dimethylformamide (DMF) and
the solution was stirred at 55 ꢁC. Then, 831 mg of anhy-
drous K2CO3 (6.03 mmol) and 339 lL of allyl bromide
(4.02 mmol) where added to the mixture and stirred at
this temperature for 7 h. After cooling, CH2Cl2 was
added and the organic layer was washed twice with
HCl 10% and once with saturated NaHCO3. The organ-
ic layer was dried over Na2SO4, filtered and evaporated
under reduced pressure. The crude 3-acetoxy-28-allyl
betulinate was dissolved in a 1:2:1 MeOH/THF/H2O
(100 mL) solution containing NaOH (ꢁ0.25 N) and stir-
red for 3 h (TLC monitored). After, 50 mL of CH2Cl2
was added and the solution was neutralized with HCl
10% until pH 4–5 and extracted with CH2Cl2 three times
with portions of 50 mL. Combined organic layers were
1450, 1386, 1264, 1180, 1138, 1088, 1042, 1005, 987,
971, 887, 810, 768, 737. 1H NMR (CDCl3): 3.76 (d, 1H,
J = 7.6 Hz, H-28), 3.52 (s, 1H, H-19), 3.43 (d, 1H,
J = 7.8 Hz, H-28), 3.19 (m, 1H, H-3), 2.00–1.00 (24H),
0.96 (s, 6H), 0.92 (s, 3H), 0.90 (s, 3H), 0.83 (s, 3H), 0.79
(s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3): 88.06, 79.08,
71.39, 55.60, 51.20, 46.95, 41.60, 40.83, 40.73, 39.04,
39.01, 37.38, 36.87, 36.39, 34.26, 34.03, 32.83, 28.94,
28.11, 27.54, 26.58, 26.57, 26.39, 24.68, 21.11, 18.38,
16.62, 15.84, 15.52, 13.64; HR-EI-MS m/z 443.3905
[M + Na]+ (calcd for C30H50O2Na, 443.3889).
4.9. 28-Oxoallobetulin (4)
Carboxylic acid 8 (500 mg, 1.00 mmol), was stirred un-
der reflux in 25 mL of CH2Cl2 with a mixture of

6152
D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
FeCl3/SiO2 (1:4) grounded on a mortar (0.50 g/1.95 g,
3.00 mmol of FeCl3) for 3 h. The mixture was then fil-
tered on celite and washed with CH2Cl2, evaporated
and dissolved in a 1:2:1 MeOH/THF/H2O (50 mL) solu-
tion and was refluxed with 1.00 g of NaOH (25 mmol)
overnight. Then, 25 mL of CH2Cl2 was added and the
solution was neutralized with HCl 10% until pH 4–5
and extracted with CH2Cl2 three times with portions
of 50 mL. Combined organic layers were dried over
Na2S2O4, filtered and evaporated, affording crude prod-
uct which was purified by flash chromatography on sil-
ica gel with MeOH/CH2Cl2 (1:99 to 3:97) as eluent to
The organic phase was dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was dis-
solved in a solution of triphenylphosphine (PPh3) (0.6
equiv) and pyrrolidine (2 equiv) in dry THF (0.22 M),
then Pd0 (PPh3)4 (0.3 equiv) was added and the resulting
mixture was stirred for 1–3 days at room temperature
under argon atmosphere. After evaporation of the sol-
vent under reduced pressure, the residue was purified
by flash chromatography using 2–20% MeOH in
CH2Cl2 as eluent.
4.12. Betulin 3b-O-b-D-galactopyranoside (14)
give 417 mg (91% yield over two steps) of compound 4
20
D
2941, 1760, 1446, 1388, 1153, 1119, 1045, 966, 922,
as a white solid: ½aꢂ +51.7ꢁ (c 0.49, CHCl3); IR: 3377,
1
733. H NMR (CDCl3): 3.93 (s, 1H, H-19), 3.20 (dd,
This compound was prepared from 10 (250 mg,
0.52 mmol) to afford 60 mg of 14 (19% yield) as a white
20
solid: ½aꢂ +9.5ꢁ (c 0.60); IR: 3373, 2920, 2853, 1457,
D
1H, J = 11.2 Hz, J = 4.9 Hz, H-3), 2.00–0.50 (24H),
1.02 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.90 (s, 3H),
0.86 (s, 3H), 0.83 (s, 3H), 0.75 (s, 3H); 13C NMR
(CDCl3): 179.86, 85.99, 78.89, 55.49, 51.23, 46.70,
46.09, 40.55, 39.91, 38.93, 38.87, 37.25, 36.00, 33.71,
33.54, 32.31, 31.93, 28.74, 27.94, 27.88, 27.35, 26.51,
25.54, 23.95, 20.87, 18.14, 16.53, 15.51, 15.34, 13.65;
HR-EI-MS m/z 457.3696 [M+Na]+ (calcd for
C30H48O3Na, 457.3682).
1353, 1246, 1145, 1029, 973, 876. 1H NMR (Pyr-d5):
4.90 (m, 2H, H-10, H-29), 4.75 (s, 1H, H-29), 4.62 (s,
1H, H-40), 4.51 (m, 3H, H-60 (2x), H-20), 4.20 (m, 1H,
H-30), 4.16 (m, 1H, H-50), 4.12 (m, 1H, H-28), 3.68
(m, 1H, H-28), 3.43 (m, 1H, H-3) 2.70–0.60 (25H),
1.78 (s, 3H), 1.33 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H),
0.98 (s, 3H), 0.80 (s, 3H); 13C NMR (Pyr-d5): 151.64,
110.33, 107.98, 89.14, 77.25, 75.91, 73.60, 70.72, 62.89,
59.82, 56.24, 51.02, 49.51, 48.94, 48.73, 43.37, 41.57,
40.05, 39.45, 37.95, 37.46, 35.26, 34.99, 30.78, 30.39,
28.52, 27.94, 27.27, 26.11, 21.45, 19.66, 18.87, 17.20,
16.75, 16.50, 15.33; HR-ESI-MS m/z 627.4214
[M+Na]+ (calcd for C36H60O7Na, 627.4237).
4.10. General procedure for the synthesis of glycosides
11–22 and 29–34
One equivalent of the appropriate acceptor and 1.5
equivalents of the appropriate donor were dissolved in
˚
dry CH2Cl2 (30 mM) with 4 A molecular sieves and stir-
4.13. Betulin 3b-O-a-D-mannopyranoside (15)
red at room temperature. TMSOTf (0.05 equiv) was
then added dropwise under an argon atmosphere while
keeping rigorous anhydrous conditions. The reaction
was usually performed in approximately 1.5 h and then
neutralized by adding triethylamine (8 M). The crude
mixture was filtered and evaporated under reduced pres-
sure. The residue was dissolved in a NaOH 0.25 N solu-
tion of MeOH/THF/H2O (0.01 M) and stirred at rt for
2 h. The mixture was diluted in CH2Cl2 and washed with
HCl 10% and brine. The organic phase was dried over
Na2SO4, filtered and evaporated under reduced pres-
sure. The resulting residue was purified by flash chroma-
tography on silica gel using 2–10% MeOH in CH2Cl2 as
eluent.
This compound was prepared from 10 (261 mg,
0.54 mmol) to afford 159 mg of 15 (49% yield) as a white
20
powder: ½aꢂ +57.5ꢁ (c 0.58); IR: 3303, 2933, 2866, 1451,
D
1374, 1058, 1056, 978, 880, 679. 1H NMR (Pyr-d5): 5.61
(br s, 1H, H-10), 4.90 (d, 1H, J = 2.2 Hz, H-29), 4.76 (s,
1H, H-29), 4.73 (m, 1H, H-40), 4.64 (m, 1H, H-30), 4.62
(m, 1H, H-60), 4.57 (m, 1H, H-20), 4.51 (m, 1H, H-50),
4.45 (m, 1H, H-60), 4.09 (d, 1H, J = 11.2 Hz, H-28),
3.67 (d, 1H, J = 10.7 Hz, H-28), 3.52 (dd, 1H,
J = 11.5 Hz, J = 4.2 Hz, H-3), 2.70–0.60 (25H), 1.78 (s,
3H), 1.16 (s, 3H), 1.02 (s, 3H), 0.96 (s, 3H), 0.84 (s,
3H), 0.78 (s, 3H); 13C NMR (Pyr-d5): 151.65, 110.33,
98.12, 81.99, 76.39, 73.63, 73.40, 69.61, 63.80, 59.82,
56.17, 50.94, 49.49, 48.92, 48.72, 43.34, 41.54, 39.10,
38.81, 37.93, 37.62, 35.25, 34.90, 30.77, 30.40, 29.27,
27.92, 26.05, 22.60, 21.42, 19.66, 18.88, 17.15, 16.67,
16.50, 15.33; HR-ESI-MS m/z 627.4243 [M+Na]+ (calcd
for C36H60O7Na, 627.4237).
4.11. General procedure for the synthesis of glycosides
23–28
One equivalent of the appropriate acceptor and 1.5
equivalents of the appropriate donor were solubilized
˚
in dry CH2Cl2 (30 mM) with 4 A molecular sieves and
4.14. Betulin 3b-O-b-D-xylopyranoside (16)
stirred at room temperature. TMSOTf (0.05 equiv)
was then added dropwise under an argon atmosphere
while keeping rigorous anhydrous conditions. The reac-
tion was usually performed in approximately 1.5 h and
then neutralized by adding triethylamine (8 M). The
crude mixture was filtered and evaporated under re-
duced pressure. The residue was dissolved in a NaOH
0.25 N solution of MeOH/THF/H2O (0.01 M) and stir-
red at room temperature for 2 h. The mixture was di-
luted in CH2Cl2 and washed with HCl 10% and brine.
This compound was prepared from 10 (251 mg,
0.52 mmol) to afford 81 mg of 16 (27% yield) as a white
20
D
solid: ½aꢂ +2.4 ꢁ (c 0.38); IR: 3343, 2937, 2866, 1450,
1
1374, 1242, 1161, 1039, 974, 880, 635. H NMR (Pyr-
d5): 4.90 (d, 1H, J = 2.1 Hz, H-29), 4.88 (d, 1H,
J = 7.6 Hz, H-10), 4.75 (s, 1H, H-29), 4.40 (m, 1H, H-
50), 4.26 (m, 1H, H-40), 4.19 (m, 1H, H-30), 4.11 (d,
1H, J = 10.6 Hz, H-28), 4.06 (m, 1H, H-20), 3.80 (m,
1H, H-50), 3.68 (d, 1H, J = 10.4 Hz, H-28), 3.41 (dd,
1H, J = 11.7 Hz, J = 4.4 Hz, H-3), 2.70–0.70 (25H),

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6153
1.77 (s, 3H), 1.33 (s, 3H), 1.09 (s, 3H), 1.02 (s, 3H), 0.99
(s, 3H), 0.83 (s, 3H); 13C NMR (Pyr-d5): 151.06, 110.35,
108.08, 89.07, 79.04, 75.97, 71.64, 67.54, 59.76, 56.24,
51.03, 49.50, 48.93, 48.72, 43.35, 41.58, 40.10, 39.41,
37.94, 37.51, 35.26, 34.96, 30.76, 30.41, 28.49, 27.94,
27.35, 26.06, 21.43, 19.64, 18.86, 17.20, 16.76, 16.51,
15.29; HR-ESI-MS m/z 597.4146 [M+Na]+ (calcd for
C35H58O6Na, 597.4131).
0.88 (s, 6H), 0.85 (s, 3H), 0.77 (s, 3H); 13C NMR (Pyr-
d5): 103.39, 88.21, 85.19, 75.21, 72.96, 71.63, 70.03,
67.49, 56.74, 51.61, 47.51, 42.00, 41.30, 41.19, 39.26,
39.01, 37.69, 37.30, 36.90, 34.85, 34.58, 33.54, 29.57,
28.90, 27.12, 27.09, 26.90, 24.95, 24.16, 21.68, 18.96,
17.29, 16.95, 16.16, 14.00; HR-ESI-MS m/z 597.4130
[M+Na]+ (calcd for C35H58O6Na, 597.4131).
4.18. Allobetulin 3b-O-b-D-galactopyranoside (26)
4.15. Allobetulin 3b-O-b-D-glucopyranoside (23)
This compound was prepared from 3 (100 mg,
0.23 mmol) to afford 91 mg of 26 (67% yield) as a white
This compound was prepared from
0.18 mmol) to afford 82 mg of 23 (75% yield) as a white
solid: ½aꢂ +20.3ꢁ (c 0.57); IR: 3350, 2923, 2865, 1448,
3
(80 mg,
20
solid: ½aꢂ +21.6ꢁ (c 0.41); IR: 3407, 2941, 2868, 1641,
D
20
D
1387, 1374, 1358, 1304, 1162, 1072, 1035, 1022, 893,
1449, 1386, 1140, 1056, 978, 667. 1H NMR (Pyr-d5):
4.92, (d, 1H, J = 7.8 Hz, H-10), 4.63 (d, 1H,
J = 3.0 Hz, H-40), 4.53 (m, 2H, H-60), 4.50 (m, 1H, H-
20), 4.22 (m, 1H, H-30), 4.17 (m, 1H, H-50), 3.87 (d,
1H, J = 8.0 Hz, H-28), 3.68 (s, 1H, H-19), 3.51 (d, 1H,
J = 7.8 Hz, H-28), 3.41 (m, 1H, H-3), 2.32 (m, 1H, H-
2), 1.92 (m, 1H, H-2), 1.70–0.70 (22H), 1.33 (s, 3H),
1.09 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H),
0.85 (s, 3H), 0.80 (s, 3H); 13C NMR (Pyr-d5): 107.57,
88.67, 87.82, 76.87, 75.48, 73.18, 71.22, 70.30, 62.49,
55.97, 51.23, 47.11, 41.60, 40.89, 40.77, 39.65, 39.12,
37.10, 36.90, 36.50, 34.47, 34.19, 33.14, 29.17, 28.09,
26.86, 26.72, 26.70, 26.50, 24.54, 21.25, 18.36, 16.79,
16.66, 15.76, 13.63; HR-ESI-MS m/z 627.4215
[M+Na]+ (calcd for C36H60O7Na, 627.4237).
1
766. H NMR (Pyr-d5): 4.98 (d, 1H, J = 7.8 Hz, H-10),
4.64 (m, 1H, H-60), 4.45 (m, 1H, H-60), 4.26 (m, 2H,
H-30 and H-40), 4.07 (m, 1H, H-20), 4.04 (m, 1H, H-
50), 3.87 (d, 1H, J = 7.8 Hz, H-28), 3.68 (s, 1H, H-19),
3.51 (d, 1H, J = 7.6 Hz, H-28), 3.41 (m, 1H, H-3), 2,28
(m, 1H, H-2), 1.87 (m, 1H, H-2), 1.70–0.70 (22H), 1.34
(s, 3H), 1.07 (s, 3H), 1.03 (s, 3H), 0.97 (s, 3H), 0.88 (s,
3H), 0.84 (s, 3H), 0.79 (s, 3H); 13C NMR (Pyr-d5):
107.36, 89.21, 88.23, 79.18, 78.77, 76.21, 72.27, 71.63,
63.48, 56.37, 51.63, 47.52, 42.02, 41.31, 41.18, 40.04,
39.49, 37.51, 37.32, 36.92, 34.89, 34.61, 33.55, 29.60,
28.51, 27.18, 27.15, 27.11, 26.93, 24.97, 21.67, 18.79,
17.25, 17.07, 16.18, 14.05; HR-ESI-MS m/z 627.4220
[M+Na]+ (calcd for C36H60O7Na, 627.4237).
4.19. Allobetulin 3b-O-a-D-mannopyranoside (27)
4.16. Allobetulin 3b-O-a-L-rhamnopyranoside (24)
This compound was prepared from 3 (100 mg,
0.23 mmol) to afford 121 mg of 27 (89% yield) as a white
This compound was prepared from
3
(100 mg,
0.23 mmol) to afford 110 mg of 24 (83% yield) as a white
20
solid: ½aꢂ +79.8ꢁ (c 0.54); IR: 3364, 2924, 2868, 1443,
D
20
D
solid: ½aꢂ ꢀ3.3ꢁ (c 0.27); IR: 3408, 2926, 1448, 1386,
1386, 1123, 1069, 1033, 811, 713. 1H NMR (Pyr-d5):
5.62 (d, 1H, J = 1.2 Hz, H-10), 4.76 (m, 1H, H-40), 4.65
(m, 1H, H-30), 4.63 (m, 1H, H-60), 4.59 (m, 1H, H-20),
4.50 (m, 1H, H-50), 4.48 (m, 1H, H-60), 3.87 (d, 1H,
J = 7.8 Hz, H-28), 3.68 (s, 1H, H-19), 3.51 (d, 1H,
J = 7.6 Hz, H-28), 3.51 (m, 1H, H-3), 1.84 (m, 1H, H-
2), 1.70–0.70 (23H), 1.18, (s, 3H), 1.08 (s, 3H), 0.91 (s,
3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.77 (s,
3H); 13C NMR (Pyr-d5): 98.09, 88.21, 81.85, 76.43,
73.67, 73.42, 71.62, 69.60, 63.81, 56.33, 51.55, 47.51,
42.02, 41.29, 41.16, 39.13, 38.88, 37.68, 37.30, 36.91,
34.85, 34.51, 33.54, 29.58, 29.26, 27.13, 27.08, 26.89,
24.95, 22.55, 21.65, 18.82, 17.17, 17.00, 16.16, 14.05;
HR-ESI-MS m/z 627.4221 [M+Na]+ (calcd for
C36H60O7Na, 627.4237).
1
1130, 1106, 1051, 974, 811. H NMR (Pyr-d5): 5.36 (d,
1H, J = 1.2 Hz, H-10), 4.61 (m, 1H, H-20), 4.50 (m,
1H, H-30), 4.36 (m, 1H, H-50), 4.34 (m, 1H, H-40), 3.87
(d, 1H, J = 7.4 Hz, H-28), 3.68 (s, 1H, H-19), 3.51 (d,
1H, J = 7.8 Hz, H-28), 2.00 (m, 1H, H-2), 1.90–0.60
(23H), 1.71 (d, 3H, J = 5.7 Hz, H-60), 1.09 (s, 3H),
0.94 (s, 3H), 0.94 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H),
0.83 (s, 3H), 0.81 (s, 3H); 13C NMR (Pyr-d5): 104.89,
88.87, 88.22, 74.52, 73.33, 72.90, 71.62, 70.25, 56.11,
51.60, 47.50, 42.00, 41.29, 41.15, 39.70, 39.32, 37.49,
37.30, 36.91, 34.88, 34.53, 33.54, 29.58, 28.50, 27.13,
27.09, 26.90, 26.44, 24.96, 21.67, 18.92, 18.87, 17.01,
16.94, 16.16, 14.01; HR-ESI-MS m/z 611.4267
[M+Na]+ (calcd for C36H60O6Na, 611.4288).
4.17. Allobetulin 3b-O-a-D-arabinopyranoside (25)
4.20. Allobetulin 3b-O-b-D-xylopyranoside (28)
This compound was prepared from
3
(100 mg,
0.23 mmol) to afford 103 mg of 25 (79% yield) as a white
This compound was prepared from 3 (100 mg,
0.23 mmol) to afford 110 mg of 28 (85% yield) as a white
20
D
20
solid: ½aꢂ +51.9ꢁ (c 0.47); IR: 3343, 2939, 2926, 2871,
solid: ½aꢂ +17.7ꢁ (c 0.57); IR: 3250, 2923, 1441, 1385,
D
2855, 1450, 1386, 1337, 1290, 1252, 1069, 1033, 1001,
939, 767, 714. 1H NMR (Pyr-d5): 4.76 (d, 1H,
J = 7.1 Hz, H-10), 4.46 (m, 1H, H-20), 4.41 (m, 1H, H-
50), 4.38 (m, 1H, H-40), 4.23 (m, 1H, H-30), 3.88 (m,
1H, H-50), 3.85 (d, 1H, J = 6.8 Hz, H-28), 3.69 (s, 1H,
H-19), 3.51 (d, 1H, J = 7.7 Hz, H-28), 3.46 (dd, 1H,
J = 12.4 Hz, J = 4.6 Hz, H-3), 2.03 (m, 1H, H-2), 1.80–
0.60 (24H), 1.22 (s, 3H), 1.08 (s, 3H), 0.96 (s, 3H),
1165, 1086, 1032, 969, 892, 767. 1H NMR (Pyr-d5):
4.88 (d, 1H, J = 7.6 Hz, H-10), 4.43, (m, 1H, H-50),
4.29 (m, 1H, H-40), 4.22 (m, 1H, H-30), 4.07 (m, 1H,
H-20), 3.87 (d, 1H, J = 8.2 Hz, H-28), 3.82 (m, 1H, H-
50), 3.68 (s, 1H, H-19), 3.52 (d, 1H, J = 8.0 Hz, H-28),
3.38 (m, 1H, H-3), 2.24 (m, 1H, H-2), 1.95 (m, 1H, H-
2), 1.70–0.70 (22H), 1.33 (s, 3H), 1.09 (s, 3H), 1.02 (s,
3H), 0.96 (s, 3H), 0.88 (s, 3H), 0.85 (s, 3H), 0.79 (s,

6154
D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
3H); 13C NMR (Pyr-d5): 108.13, 89.01, 88.21, 79.06,
75.98, 71.65, 71.63, 67.56, 56.40, 51.66, 47.51, 42.00,
41.30, 41.18, 40.12, 39.54, 37.56, 37.30, 36.91, 34.86,
34.60, 33.54, 29.58, 28.45, 27.35, 27.13, 27.09, 26.91,
24.96, 21.67, 18.78, 17.19, 17.09, 16.18, 14.01; HR-
ESI-MS m/z 597.4144 [M+Na]+ (calcd for C35H58O6,
597.4131).
16.75 (2x), 15.22; HR-ESI-MS m/z 587.3961 [MꢀH]ꢀ
(calcd for C35H55O7, 587.3953).
4.24. 28-Oxoallobetulin 3b-O-b-D-glucopyranoside (29)
This compound was prepared from
0.18 mmol) to afford 56 mg of 29 (50% yield) as a white
4
(80 mg,
20
D
1447, 1388, 1375, 1304, 1154, 1072, 1016, 969, 923,
solid: ½aꢂ +23.1ꢁ (c 0.41); IR: 3388, 2943, 2869, 1766,
4.21. Betulinic acid 3b-O-b-D-galactopyranoside (20)
1
532. H NMR (Pyr-d5): 4.98 (d, 1H, J = 7.8 Hz, H-10),
This compound was prepared from
0.42 mmol) to afford 111 mg of 20 (43% yield) as a white
9
(207 mg,
4.64 (m, 1H, H-60), 4.45 (m, 1H, H-60), 4.26 (m, 2H,
H-30, H-40), 4.08 (m, 1H, H-20), 4.06 (s, 1H, H-19),
4.04 (m, 1H, H-50), 3.40 (m, 1H, H-3), 2.28 (m, 1H,
H-2), 2.00 (m, 1H, H-16), 1.86 (m, 2H, H-2, H-18),
1.70–0.70 (20H), 1.32 (s, 3H), 1.04 (s, 3H), 1.00 (s,
3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.78 (s, 3H), 0.75 (s,
3H); 13C NMR (Pyr-d5): 179.92, 107.36, 89.16, 86.25,
79.16, 78.77, 76.19, 72.25, 63.48, 56.33, 51.72, 47.18,
46.60, 41.10, 40.55, 40.00, 39.47, 37.46, 36.88, 34.40,
34.11, 33.12, 32.44, 29.19, 28.65, 28.47, 27.14, 26.97,
26.38, 24.05, 21.51, 18.66, 17.19, 17.07, 15.86, 14.12;
HR-ESI-MS m/z 641.4038 [M+Na]+ (calcd for
C36H58O8Na, 641.4029).
20
D
solid: ½aꢂ ꢀ3.9ꢁ (c 0.20); IR: 3325, 2936, 2864, 1687,
1
1449, 1375, 1214, 1152, 1056, 976, 879. H NMR (Pyr-
d5): 4.96 (s, 1H, H-29), 4.90 (d, 1H, J = 7.6 Hz, H-10),
4.77 (s, 1H, H-29), 4.63 (m, 1H, H-40), 4.50 (m, 3H,
H-60 (2x), H-20), 4.21 (m, 1H, H-30), 4.15 (m, 1H, H-
50), 3.56 (m, 1H, H-19), 3.42 (m, 1H, H-3) 2.80–0.60
(24H), 1.80 (s, 3H), 1.32 (s, 3H), 1.12 (s, 3H), 1.03 (s,
3H), 0.96 (s, 3H), 0.76 (s, 3H); 13C NMR (Pyr-d5):
179.32, 151.69, 110.35, 107.95, 89.11, 77.25, 75.91,
73.62, 70.66, 62.84, 57.02, 56.31, 51.20, 50.13, 48.16,
43.22, 41.45, 40.04, 39.44, 38.96, 37.98, 37.50, 35.13,
33.27, 31.59, 30.65, 28.51, 27.26, 26.46, 21.56, 19.84,
18.83, 17.18, 16.76, 16.74, 15.25; HR-ESI-MS m/z
641.4005 [M+Na]+ (calcd for C36H58O8Na, 641.4029).
4.25. 28-Oxoallobetulin 3b-O-a-L-rhamnopyranoside (30)
This compound was prepared from 4 (100 mg,
0.22 mmol) to afford 92 mg of 30 (70% yield) as a white
4.22. Betulinic acid 3b-O-a-D-mannopyranoside (21)
20
D
solid: ½aꢂ ꢀ6.8ꢁ (c 0.27); IR: 3310, 2935, 1757, 1443,
1
This compound was prepared from
0.40 mmol) to afford 58 mg of 21 (23% yield) as a white
9
(201 mg,
1387, 1146, 1117, 1053, 965, 921, 810. H NMR (Pyr-
d5): 5.36 (d, 1H, J = 1.2 Hz, H-10), 4.62 (m, 1H, H-20),
4.53 (m, 1H, H-30), 4.37 (m, 1H, H-50), 4.35 (m, 1H,
H-40), 4.07 (s, 1H, H-19), 3.17 (m, 1H, H-3), 2.00 (m,
1H, H-2), 2.00 (m, 1H, H-16), 1.87 (m, 1H, H-18),
1.80–0.60 (21H), 1.72 (d, 3H, J = 5.7 Hz, H-60), 1.04
(s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.87 (s, 3H), 0.80 (s,
3H), 0.79 (s, 3H), 0.76 (s, 3H); 13C NMR (Pyr-d5):
179.94, 104.94, 88.84, 86.26, 74.53, 73.35, 72.92, 70.28,
56.10, 51.72, 47.19, 46.59, 41.08, 40.56, 39.69, 39.31,
37.47, 36.88, 34.34, 34.12, 33.13, 32.45, 29.20, 28.66,
28.48, 26.97, 26.43, 26.39, 24.06, 21.55, 18.95, 18.77,
17.05, 16.91, 15.87, 14.10; HR-ESI-MS m/z 625.4055
[M+Na]+ (calcd for C36H58O7Na, 625.4080).
20
D
solid: ½aꢂ +71.6ꢁ (c 0.08); IR: 3382, 2944, 1686, 1440,
1
1376, 1241, 1106, 1058, 1028, 975, 881, 814. H NMR
(Pyr-d5): 5.60 (br s, 1H, H-10), 4.96 (br s, 1H, H-29),
4.78 (br s, 1H, H-29), 4.75 (m, 2H, H-40), 4.63 (m, 2H,
H-30, H-60), 4.57 (br s, 1H, H-20), 4.49 (m, 2H, H-50,
H-60), 3.55 (m, 1H, H-19), 3.53 (m, 1H, H-3), 3.00–
0.50 (24H), 1.80 (s, 3H), 1.16 (s, 3H), 1.04 (s, 3H),
1.02 (s, 3H), 0.81 (s, 3H), 0.74 (s, 3H); 13C NMR (Pyr-
d5): 179.29, 151.73, 110.35, 98.10, 81.95, 76.41, 73.67,
73.41, 69.63, 63.82, 57.01, 56.24, 51.12, 50.11, 50.06,
43.19, 41.43, 39.09, 38.92, 38.81, 37.98, 37.66, 35.04,
33.24, 31.57, 30.62, 29.26, 26.42, 22.56, 21.52, 19.82,
18.87, 17.13, 16.74, 16.65, 15.25; HR-ESI-MS m/z
641.4017 [M+Na]+ (calcd for C36H58O8Na, 641.4029).
4.26. 28-Oxoallobetulin 3b-O-a-D-arabinopyranoside (31)
4.23. Betulinic acid 3b-O-b-D-xylopyranoside (22)
This compound was prepared from 4 (250 mg,
0.10 mmol) to afford 26 mg of 31 (20% yield) as a
white solid: ½aꢂ +50.9ꢁ (c 0.25); IR: 3280, 2941,
20
D
2921, 1757, 1442, 1386, 1360, 1137, 1068, 1002, 965,
This compound was prepared from
0.40 mmol) to afford 138 mg of 22 (58% yield) as a
9
(200 mg,
20
D
1
white solid: ½aꢂ +11.9ꢁ (c 0.15); IR: 3376, 2931,
945, 921. H NMR (Pyr-d5): 4.75 (d, 1H, J = 7.1 Hz,
2865, 1687, 1638, 1453, 1375, 1161, 1046, 974, 882.
1H NMR (Pyr-d5): 4.96 (s, 1H, H-29), 4.87 (d, 1H,
J = 7.0 Hz, H-10), 4.78 (s, 1H, H-29), 4.39 (m, 1H,
H-50), 4.26 (m, 1H, H-40), 4.20 (m, 1H, H-30), 4.05
(m, 1H, H-20), 3.80 (m, 1H, H-50), 3.56 (m, 1H, H-
19), 3.40 (m, 1H, H-3), 2.80–0.70 (24H), 1.79 (s,
3H), 1.32 (s, 3H), 1.11 (s, 3H), 1.04 (s, 3H), 0.99 (s,
3H), 0.78 (s, 3H); 13C NMR (Pyr-d5): 179.27,
151.67, 110.39, 108.09, 89.06, 79.05, 75.98, 71.64,
67.54, 57.01, 56.31, 51.20, 50.12, 48.16, 43.20, 41.46,
40.09, 39.45, 38.94, 37.97, 37.56, 35.11, 33.24, 31.57,
30.64, 28.48, 27.35, 26.44, 21.56, 19.81, 18.85, 17.18,
H-10), 4.45 (m, 1H, H-20), 4.41 (m, 1H, H-50), 4.38
(m, 1H, H-40), 4.23 (m, 1H, H-30), 4.07 (s, 1H, H-
19), 3.85 (d, 1H, J = 12.6 Hz, H-50), 3.43 (m, 1H,
H-3), 2.20–0.70 (24H), 1.24 (s, 3H), 1.03 (s, 3H),
0.93 (s, 3H), 0.88 (s, 3H), 0.86 (s, 3H), 0.78 (s, 3H),
0.73 (s, 3H); 13C NMR (Pyr-d5): 179.95, 103.33,
86.23, 85.07, 75.20, 72.94, 70.01, 67.47, 56.71, 51.71,
47.18, 46.57, 41.10, 40.55, 39.24, 38.98, 37.65, 36.84,
34.38, 34.09, 33.10, 32.42, 29.16, 28.86, 28.63, 26.96,
26.37, 24.10, 24.03, 21.54, 18.84, 17.23, 16.96, 15.85,
14.06; HR-ESI-MS m/z 611.3935 [M+Na]+ (calcd for
C35H56O7Na, 611.3924).

D. Thibeault et al. / Bioorg. Med. Chem. 15 (2007) 6144–6157
6155
4.27. 28-Oxoallobetulin 3b-O-b-D-galactopyranoside (32)
This compound was prepared from (100 mg,
0.22 mmol) to afford 83 mg of 32 (61% yield) as a white so-
(WS1) cell lines were obtained from the American Type
Culture Collection (ATCC). All cell lines were cultured
in minimum essential medium containing Earle’s salts
and L-glutamine (Mediatech Cellgro, VA), to which
were added 10% foetal bovine serum (Hyclone), vita-
mins (1·), penicillin (100 IU/mL) and streptomycin
(100 lg/mL), essential amino acids (1·) and sodium
pyruvate (1·) (Mediatech Cellgro, VA). Cells were kept
at 37 ꢁC in a humidified environment containing 5%
CO2.
4
lid: ½aꢂ20 +28.0ꢁ (c 0.47); IR: 3378, 2935, 1758, 1446, 1389,
D
1153, 1055, 966, 922, 756. 1H NMR (Pyr-d5): 4.91, (d, 1H,
J = 7.7 Hz, H-10), 4.63 (d, 1H, J = 3.0 Hz, H-40), 4.53 (m,
2H, H-60), 4.51 (m, 1H, H-20), 4.22 (m, 1H, H-30), 4.17 (m,
1H, H-50), 4.07 (s, 1H, H-19), 3.40 (m, 1H, H-3), 2.32 (m,
1H, H-2), 2.01 (m, 1H, H-16), 1.90 (m, 1H, H-2), 1.88 (m,
1H, H-18), 1.70–0.70 (20H), 1.32 (s, 3H), 1.04 (s, 3H), 0.97
(s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H);
13C NMR (Pyr-d5): 179.94, 107.99, 89.02, 86.25, 77.29,
75.88, 73.58, 70.72, 62.91, 56.36, 51.75, 47.18, 46.59,
41.10, 40.56, 40.04, 39.52, 37.49, 36.88, 34.41, 34.12,
33.13, 32.44, 29.19, 28.66, 28.47, 27.24, 26.99, 26.39,
24.05, 21.53, 18.67, 17.16, 17.10, 15.87, 14.12; HR-ESI-
MS m/z 641.4037 [M+Na]+ (calcd for C36H58O8Na,
641.4029).
4.31. Cytotoxicity assay
Exponentially growing cells were plated in 96-well
microplates (Costar, Corning Inc.) at a density of
5 · 103 cells per well in 100 lL of culture medium and
were allowed to adhere for 16 h before treatment.
Increasing concentrations of each compound in DMSO
(Sigma–Aldrich) were then added (100 lL per well) and
the cells were incubated for 48 h. The final concentration
of DMSO in the culture medium was maintained at
0.5% (v/v) to avoid solvent toxicity. Cytotoxicity was as-
sessed using resazurin53 on an automated 96-well Fluo-
roskan Ascent F1TM plate reader (Labsystems) using
excitation and emission wavelengths of 530 and
590 nm, respectively. Fluorescence was proportional to
the cellular metabolic activity in each well. Survival per-
centage was defined as the fluorescence in experimental
wells compared to that in control wells after subtraction
of blank values. Each experiment was carried out three
times in triplicate. IC50 results were expressed as
means standard deviation.
4.28. 28-Oxoallobetulin 3b-O-a-D-mannopyranoside (33)
This compound was prepared from 4 (100 mg,
0.22 mmol) to afford 62 mg of 33 (46% yield) as a white
20
D
solid: ½aꢂ +83.1ꢁ (c 0.30); IR: 3330, 2940, 1757, 1443,
1
1388, 1119, 1067, 965, 921. H NMR (Pyr-d5): 5.62 (d,
1H, J = 1.1 Hz, H-10), 4.76 (m, 1H, H-40), 4.65 (m,
1H, H-30), 4.63 (m, 1H, H-60), 4.59 (m, 1H, H-20), 4.50
(m, 1H, H-50), 4.48 (m, 1H, H-60), 4.06 (s, 1H, H-19),
3.51 (m, 1H, H-3), 1.99 (m, 1H, H-16), 1.86 (m, 1H,
H-18), 1.84 (m, 1H, H-2), 1.70–0.70 (21H), 1.16 (s,
3H), 1.02 (s, 3H), 0.92 (s, 3H), 0.84 (s, 3H), 0.83 (s,
3H), 0.77 (s, 3H), 0.73 (s, 3H); 13C NMR (Pyr-d5):
179.99, 98.05, 86.23, 81.72, 76.44, 73.67, 73.42, 69.62,
63.84, 56.29, 51.66, 47.17, 46.59, 41.07, 40.54, 39.15,
38.87, 37.65, 36.84, 34.32, 34.11, 33.12, 32.43, 29.23,
29.18, 28.65, 26.95, 26.36, 24.04, 22.50, 21.51, 18.71,
17.12, 17.02, 15.85, 14.13; HR-ESI-MS m/z 641.4043
[M+Na]+ (calcd for C36H58O8Na, 641.4029).
Acknowledgments
This work was supported by grants from le Fonds
´ ´
Quebecois de la Recherche sur la Nature et les Technol-
ogies (FQRNT) and la Scierie Thomas-Louis-Tremblay
´
de Sainte-Monique-de-Honfleur, Quebec, Canada. We
thank Sebastien Vanier for his contribution in organic
4.29. 28-Oxoallobetulin 3b-O-b-D-xylopyranoside (34)
´
synthesis.
This compound was prepared from 4 (100 mg,
0.22 mmol) to afford 28 mg of 34 (22% yield) as a white so-
lid: ½aꢂ20 +37.3ꢁ (c 0.07); IR: 3230, 2922, 2853, 1757, 1443,
D
References and notes
1386, 1260, 1166, 1044, 966, 921, 712. 1H NMR (Pyr-d5):
4.88 (d, 1H, J = 7.4 Hz, H-10), 4.43 (m, 1H, H-50), 4.28 (m,
1H, H-40), 4.22 (m, 1H, H-30), 4.07 (m, 1H, H-20), 4.06 (s,
1H, H-19), 3.82 (m, 1H, H-50), 3.37 (m, 1H, H-3), 2.24 (m,
1H, H-2), 1.95 (m, 1H, H-2), 1.80–0.70 (24 H), 1.32 (s,
3H), 1.03 (s, 3H), 1.00 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H),
0.79 (s, 3H), 0.78 (s, 3H); 13C NMR (Pyr-d5): 179.95,
108.16, 88.94, 86.24, 79.08, 75.98, 71.65, 67.57, 56.37,
51.76, 47.18, 46.58, 41.10, 40.55, 40.10, 39.52, 37.53,
36.85, 34.40, 34.10, 33.11, 32.44, 29.18, 28.65, 28.41,
27.32, 26.96, 26.38, 24.05, 21.53, 18.67, 17.13, 17.11,
15.86, 14.08; HR-ESI-MS m/z 611.3914 [M+Na]+ (calcd
for C35H56O7Na, 611.3924).
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