Glycosidation of lupane-type triterpenoids as potent in vitro cytotoxic agents

Article abstract of DOI:10.1016/j.bmc.2006.05.075

The weak hydrosolubility of betulinic acid (3) hampers the clinical development of this natural anticancer agent. In order to circumvent this problem and to enhance the pharmacological properties of betulinic acid (3) and the lupane-type triterpenes lupeol (1), betulin (2), and methyl betulinate (7), glycosides (β-d-glucosides, α-l-rhamnosides, and α-d-arabinosides) were synthesized and in vitro tested for cytotoxicity against three cancerous (A-549, DLD-1, and B16-F1) and one healthy (WS1) cell lines. The addition of a sugar moiety at the C-3 or C-28 position of betulin (2) resulted in a loss of cytotoxicity. In contrast, the 3-O-β-d-glucosidation of lupeol (1) improved the activity by 7- to 12-fold (IC₅₀ 14-15.0 μM). Moreover, the results showed that cancer cell lines are 8- to 12-fold more sensitive to the 3-O-α-l-rhamnopyranoside derivative of betulinic acid (IC₅₀ 2.6-3.9 μM, 22) than the healthy cells (IC₅₀ 31 μM). Thus, this study indicates that 3-O-glycosides of lupane-type triterpenoids represent an interesting class of potent in vitro cytotoxic agents.

Full text of DOI:10.1016/j.bmc.2006.05.075

Bioorganic & Medicinal Chemistry 14 (2006) 6713–6725
Glycosidation of lupane-type triterpenoids as potent in vitro
cytotoxic agents
*
´
Charles Gauthier, Jean Legault, Maxime Lebrun, Philippe Dufour and Andre Pichette
Laboratoire LASEVE, De´partement des Sciences Fondamentales, UQAC, Chicoutimi, Que´bec, Canada G7H 2B1
Received 1 May 2006; revised 26 May 2006; accepted 30 May 2006
Available online 19 June 2006
Abstract—The weak hydrosolubility of betulinic acid (3) hampers the clinical development of this natural anticancer agent. In order
to circumvent this problem and to enhance the pharmacological properties of betulinic acid (3) and the lupane-type triterpenes
lupeol (1), betulin (2), and methyl betulinate (7), glycosides (b-D-glucosides, a-L-rhamnosides, and a-D-arabinosides) were synthe-
sized and in vitro tested for cytotoxicity against three cancerous (A-549, DLD-1, and B16-F1) and one healthy (WS1) cell lines. The
addition of a sugar moiety at the C-3 or C-28 position of betulin (2) resulted in a loss of cytotoxicity. In contrast, the 3-O-b-D-glu-
cosidation of lupeol (1) improved the activity by 7- to 12-fold (IC50 14–15.0 lM). Moreover, the results showed that cancer cell lines
are 8- to 12-fold more sensitive to the 3-O-a-L-rhamnopyranoside derivative of betulinic acid (IC50 2.6–3.9 lM, 22) than the healthy
cells (IC50 31 lM). Thus, this study indicates that 3-O-glycosides of lupane-type triterpenoids represent an interesting class of potent
in vitro cytotoxic agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In the boreal forest of North America, lupeol (1), betulin
(2), and betulinic acid (3) (Fig. 1) are found in the exter-
nal bark of yellow (Betula alleghaniensis) and white (Bet-
ula papyrifera) birches.1,2 These pentacyclic triterpenes
of the lupane-type have recently been investigated by
the scientific community for their various pharmacolog-
ical and medicinal properties.3–6 Lupeol (1) known for
its in vivo anti-inflammatory activity7 exhibited in vitro
cytotoxicity against human hepatocellular carcinoma
(Hep-G2) and human epidermoid carcinoma (A-431),8
while it did not affect the growth of tumor cell lines such
as human melanoma (MEL-2), human lung carcinoma
(A-549), and murine melanoma (B16-F10).9 Moreover,
lupeol (1) was found to exhibit a significant antiangio-
Figure 1. Structures of lupane-type triterpenoids.
genic activity on in vitro tube formation of human
umbilical vein endothelial cells (HUVEC).9 In addition,
induction of apoptosis (programmed cell death) by lu-
peol (1) was observed in human leukemia HL-60 cells.10
lin (2) in the bark of white birches. In addition to
various medicinal properties including anti-inflammato-
ry,13 anti-malarial,14 and anti-HIV15 activities, betulinic
acid (3) possesses a strong in vitro cytotoxicity against a
broad panel of human cancers like melanoma,16 neuro-
ectodermal,17 and malignant brain18 tumor cell lines.
The in vivo activity was first confirmed by experiments
on human melanoma-bearing mice. Indeed, after subcu-
taneous injection of human MEL-2 cells, betulinic acid
Betulinic acid (3) is easily synthesized in a two-step pro-
cess11,12 by taking advantage of the abundance of betu-
Keywords: Triterpenes; Betulinic acid; Glycosides; Cytotoxicity.
*
Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545
5012; e-mail: andre_pichette@uqac.ca
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.075

6714
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
(3) demonstrated highly effective tumor growth inhibi-
tion without inducing any side effects such as weight loss
or mortality.16 Moreover, betulinic acid (3) seems to in-
duce apoptosis in cancer cells via the activation of casp-
ases independently of the p53 gene status.17 Due to the
apparent lack of toxicity on normal cells19 and the
favorable therapeutic index, betulinic acid (3) is a very
promising anticancer agent.3–6
tigations were also carried out on human normal skin
fibroblasts (WS1) to evaluate the differential cytotoxicity
with respect to the healthy cells.
2. Results and discussion
2.1. Chemistry
However, a major inconvenience for the future clinical
development of betulinic acid (3) and analogues resides
in their poor solubility in aqueous media like blood ser-
um and polar solvents used for bioassays.3 To circum-
vent this problem of hydrosolubility and to enhance
pharmacological properties, many derivatives were syn-
2.1.1. Extraction and synthesis of triterpenes. First of all,
the external bark of yellow and white birches was refluxed
in CHCl3. Purification of the extracts on silica gel fol-
lowed by treatment with activated charcoal gave, respec-
tively, the natural triterpenes 1 (1.2%) and 2 (17%). To
perform the glycosidation at the C-3 and C-28 positions
of 2, we needed to prepare the corresponding acetates.
As the reactivity of the C-28 hydroxyl group of 2 is much
higher than the one at C-3, 28-acetoxybetulin (5) was ob-
tained in moderate yield (73%) by using an excess of acetic
anhydride (Ac2O) in CH2Cl2 during a 24 h period at room
temperature. As shown in Scheme 1, diacetylation of 2
withAc2O, pyridine, and acatalytic amount of dimethyla-
minopyridine (DMAP) in CH2Cl2 afforded 3,28-diacet-
oxybetulin (4) in excellent yield (95%).35 Subsequent
selective deprotection of the C-28 alcohol using
Mg(OCH3)2 in dry CH3OH and THF furnished the 3-
acetoxybetulin (6) in good yield (85%) as previously
reported.36 However, it is important to note that, in the
same experimental conditions, contrary to the results of
Xu and co-workers, the reaction was complete after 4 h
instead of 3 days. As shown in Scheme 2, the methyl ester
7 of the commercially available 3 was synthesized in mod-
erate yield (71%) by treatment with iodomethane in the
presence of DBU.37 Methods used to regenerate the car-
boxylic acid (NaOH 1 N refluxed in DMF or dioxane
and Ba(OH)2Æ8H2O in CH3OH) from methyl betulinate
glycosides (18–20) failed to yield the corresponding betu-
linic acid glycosides (21–23). Therefore, another more
versatile protection group for the C-28 acid function
was considered. To this end, the synthesis of allyl betuli-
nate (8) was carried out in good yield (84%) by reaction
of 3 using allyl bromide in DMF in the presence of
K2CO3.38
thesized and evaluated for cytotoxic activity.20–25
A
study showed that C-20 modifications involve the loss
of cytotoxicity.20 Another study demonstrated the
importance of the presence of the COOH group since
compounds substituted at this position like lupeol (1)
and methyl betulinate (7) were less active on human mel-
anoma than betulinic acid (3).21 Moreover, some C-28
amino acid22 and C-3 phthalate23 derivatives of 3 exhib-
ited higher cytotoxic activity against cancer cell lines
with improved selective toxicity and water solubility.
On the other hand, little work has been carried out on
the synthesis and the structure–activity relationships of
triterpenoid lupane-type saponins.26–30 Chatterjee and
co-workers28 obtained the 28-O-b-D-glucopyranoside
of 3 by microbial transformation with Cunninghamella
species, while Baglin and co-workers29 obtained it by
organic synthesis. This glucoside did not exhibit any sig-
nificant in vitro activity on human melanoma (MEL-2)28
and human colorectal adenocarcinoma (HT-29)29 cell
lines which confirms the importance of the carboxylic
acid function to preserve the cytotoxicity.
Our research group is interested in the synthesis of tri-
terpenoid saponins of the lupane-type. Saponins have
long been well known for their biological activities
including antitumoral and cytotoxic activities.31,32
Many natural products including triterpenes are present
as glycoconjugates. Sugar moieties are known to influ-
ence the pharmacokinetic properties of the respective
compounds such as absorption, distribution, metabo-
lism, and elimination.33 Since it is generally accepted
that glycosides are more water-soluble than the respec-
tive aglycones, glycosidation of triterpenes should
increase hydrosolubility and ameliorate pharmacologi-
cal properties. Furthermore, it is known that cancerous
cells need a more significant sugar contribution than
normal cells.34 Consequently, this difference could be
exploited to support the absorption of the therapeutic
agent by the tumoral site.
2.1.2. Synthesis of activated sugars. Protection of sugar
alcohols (Scheme 3) was achieved by using benzoyl
chloride in pyridine with DMAP as catalyst to
afford 1,2,3,4,6-penta-O-benzoyl-a,b-D-glucopyranose
(24, 92%), 1,2,3,4-tetra-O-benzoyl-a,b-L-rhamnopyra-
nose (27, 82%), and 1,2,3,4-tetra-O-benzoyl-a,b-D-arabi-
nopyranose (29, 89%).39 Thereafter, bromination
(HBr/HOAc 33%) of the benzoylated sugars followed
by basic hydrolysis with silver carbonate (Ag2CO3) in
acetone/H2O 20:1 allowed the selective deprotection of
the anomeric position in good yield for 2,3,4,6-tetra-O-
benzoyl-a,b-D-glucopyranose (25, 86%) and in a quanti-
tative way for L-rhamnose and D-arabinose deriva-
tives.40 Finally, trichloroacetimidate derivatives 2641
(85%), 2842 (72%, 2 steps), and 30 (78%, 2 steps) were
synthesized from the corresponding 1-OH sugars
according to Schmidt’s procedure43 using trichloro-
acetonitrile (CCl3CN) and a catalytic amount of cesium
carbonate (Cs2CO3) in CH2Cl2.44
In this study, we report the synthesis of several mono-
desmosides (b-D-glucoside, a-L-rhamnoside, and a-D-
arabinoside) (9–23) of triterpenoid saponins at the C-3
position for lupeol (1), betulinic acid (3), and its methyl
ester 7, and at the C-3 and C-28 positions for betulin (2).
Cytotoxicity of compounds was evaluated on human
lung carcinoma (A-549), human colorectal adenocarci-
noma (DLD-1), and mouse melanoma (B16-F1). Inves-

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
6715
Scheme 1. Reagents and conditions: (a) Ac2O, Py, DMAP, 0 ꢁC to rt, 5 h; (b) Mg(OCH3)2, CH3OH–THF, rt, 4 h; (c) Ac2O, CH2Cl2, rt, 24 h; (d) i—
trichloroacetimidate, TMSOTf, 4 A MS, CH2Cl2, rt, 30 min; ii—CH3OH/THF/H2O 1:2:1, NaOH 0.25 N, rt, 3–24 h.
˚
2.1.3. Synthesis of glycosides. Glycosidations of the
lupane-type triterpenoids were achieved by the reaction
of acceptors (1, 5–8) with donors (26, 28, and 30) at
room temperature in CH2Cl2 under the catalytic promo-
tion of the Lewis acid trimethylsilyl trifluoromethane-
sulfonate (TMSOTf).40 Subsequent removal of the
protecting groups (benzoyl and acetate) by using NaOH
0.25 N in CH3OH/THF/H2O 1:2:1 gave glycosides 9–20
(52–87%, 2 steps). Betulinic acid glycosides 21–23 (41–
50%, 3 steps) were only obtained after the regeneration
of the C-28 acid function in the presence of a catalytic
amount of tetrakistriphenylphosphine palladium
Pd0(PPh3)4 and pyrrolidine in dry THF.38 Since the gly-
cosyl donors contained benzoyl participating neighbor-
ing groups, exclusively 1,2-trans-glycosides were
synthesized as confirmed by 1H NMR experiments.
Indeed, the chemical shifts and the vicinal coupling
constants of the anomeric protons were characteristic
for b-D-glucosides (d = 4.22–4.95 ppm, J = 7.7–8.7 Hz),
a-L-rhamnosides (d = 4.72–5.39 ppm, broad singlets),
and a-D-arabinosides (d = 4.06–4.70 ppm, J = 5.6–
7.1 Hz).45 The glycosides showed a greater solubility
than corresponding triterpenes in the polar solvents
(DMSO and CH3OH) used for bioassays (data not
shown).
2.2. Cytotoxic activity
The in vitro cytotoxicity of triterpenes (1–8) and corre-
sponding glycosides (9–23) was assessed toward human
cancer (A-549, DLD-1), mouse melanoma (B16-F1),
and human normal skin fibroblast (WS1) cell lines using
the resazurin reduction test (RTT test) as previously
described in the literature.46 Measurements of fluores-
cence were carried out after 48 continuous hours of
contact between compounds and cells. Results presented
in Table 1 are expressed as the concentration inhibiting
50% of the cell growth (IC50). Known for its activity
against A-549, betulinic acid (3) was used as a positive
control in this experimentation. Based on the IC50 val-
ues, we considered that compounds with IC50 <20 lM
were strongly active, those with IC50 ranging from
ꢀ20 to 75 lM were moderately active and those with
IC50 ranging from ꢀ75 to 165 lM were weakly active.
Otherwise, the compounds were considered as inactive.
2.2.1. Triterpenes. Lupeol (1) was found to be weakly
cytotoxic against cancer cell lines with IC50 ranging
from 104 to 165 lM. These results are in good agree-
ment with the group of Moriarity and co-workers.8
Surprisingly, results demonstrated that, among all test-

6716
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
˚
Scheme 2. Reagents and conditions: (a) DBU, CH3I, THF, 0 ꢁC to rt, 24 h; (b) i—trichloroacetimidate, TMSOTf, 4 A MS, CH2Cl2, rt, 30 min; ii—
CH3OH/THF/H2O 1:2:1, NaOH 0.25 N, rt, 3 h; (c) AllBr, K2CO3, 55 ꢁC, 7 h; (d) Pd0(PPh3)4, PPh3, pyrrolidine, THF, 24 h.
Scheme 3. Reagents and conditions: (a) BzCl, Py, DMAP, 0 ꢁC to rt, 24 h; (b) i—HBr/HOAc 33%, CH2Cl2, rt, 2 h; ii—Ag2CO3, acetone/H2O 20:1,
rt, 1 h; (c) CCl3CN, Cs2CO3, CH2Cl2, rt, 4 h.
ed triterpenes, betulin (2) exhibited the strongest cyto-
toxicity toward cancer lines (IC50 3.80–13.8 lM).47 In
comparison to other well-known compounds like betu-
linic acid (IC50 10.3 lM, 3) and methyl betulinate (IC50
19 lM, 7), betulin (2) was 3- to 5-fold more potent
against A-549 (IC50 3.80 lM). With regard to acetate
derivatives of betulin (2), only the 28-acetoxy derivative
expected, the allyl ester 8 was a totally inactive com-
pound (IC50 >225 lM). It is significant to note that
tested triterpenes including betulinic acid (3) are not
selective toward cancer cell lines and inhibit also
healthy cells’ growth.
2.2.2. Lupeol and betulin glycosides. The 3-O-b-D-glucos-
idation of 1 to give compound 9 substantially enhanced
the cytotoxicity about 7- to 12-fold with IC50 values
5
exerted
a
moderate cytotoxic activity (IC50
43–75 lM) against cancer lines. Moreover, as we

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
Table 1. In vitro cytotoxicity of lupane-type triterpenoids and glycosides
6717
R2
R1O
Compound
R1
R2
Cell line IC50 (lM SD)a
DLD-1c B16-F1d
125 104
A-549b
165
WS1e
63
1
2
H
CH3
CH2OH
COOH
8
6
6
3
H
3.80 0.09
10.3 0.4
>95
6.6 0.3
15.0 0.3
>95
13.8 0.5
16.1 0.5
>95
3.58 0.07
3
H
12
>95
44
1
2
2
4
Ac
H
CH2OAc
CH2OAc
CH2OH
COOCH3
COOAll
CH3
5
75
>253
19
7
3
1
2
56
>253
25
4
4
1
6
43
>253
26
2
6
Ac
H
>253
19
>225
7
1
8
H
>225
14
>225
14
>225
15.0 0.7
>178
9
Glc
Rha
Ara
Glc
Rha
Ara
H
13.3 0.5
>178
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CH3
CH3
>178
28
>178
50
>200
27
>200
18
2
15.8 0.8
>200
CH2OH
CH2OH
CH2OH
CH2O-Glc
CH2O-Rha
CH2O-Ara
COOCH3
COOCH3
COOCH3
COOH
>200
22
41
3
3
50 10
63
1
3
33
59
5
5
8
38
>248
>228
>175
8.4
>248
>228
>175
>248
>228
>175
>248
>228
>175
H
H
Glc
Rha
Ara
Glc
Rha
Ara
3
3
3.93 0.09
>183
7.1 0.3
55
9.3 0.2
53
59
2
2
13.5 0.6
>178
2.6 0.6
18
32
1
9
13.3 0.4
49 13
3.9 0.4
12.5 0.4
> 178
COOH
COOH
3.9 0.4
17
31
47
3
5
10
2
3
11
1
Glc, b-D-glucopyranose; Rha, a-L-rhamnopyranose; Ara, a-D-arabinopyranose; Ac, acetate; All, allyl.
a Data represent mean values ( SD) for three independent experiments.
b Human lung carcinoma.
c Human colorectal adenocarcinoma.
d Mouse melanoma.
e Human normal skin fibroblasts.
ranging from 14 to 15.0 lM. In contrast, 3-O-a-L-
rhamnopyranoside of lupeol (10) was inactive (IC50
>178 lM), while the D-arabinoside derivative 11 dis-
played a moderate cytotoxicity (IC50 27–50 lM). As
for lupeol (1), we anticipated that adding a glucose sec-
tion at the C-3 position of betulin (2) should ameliorate
its biological activity. In fact, the cytotoxicity of 2 was
lost against all tested cell lines (IC50 >200 lM) when it
was converted to the 3-O-b-D-glucopyranoside 12. How-
ever, the replacement of D-glucose by L-rhamnose or
D-arabinose at the same position gave compounds exert-
ing moderate cytotoxicity (IC50 18–50 lM, 13;
38–63 lM, 14). In the same way that the 28-O-b-D-glu-
coside of betulinic acid (3) did not exhibit any significant
activity,28,29 glycosidation on the C-28 primary alcohol
of betulin (2) completely suppressed the activity (IC50
>248 lM, 15; >228 lM, 16; >175 lM, 17). These results
suggest that the C-28 alcohol function of betulin (2) is
important for the cytotoxicity and, therefore, coupling
a sugar moiety is not a desirable modification to carry
out at this position.
2.2.3. Betulinic acid and methyl betulinate glycosides. In
this study, the most interesting monodesmosides synthe-
sized and evaluated were betulinic acid (3) and methyl
betulinate (7) glycosides (18–23). Indeed, it appears that
3-O-b-D-glucopyranoside of methyl betulinate (18)
exhibited strong cytotoxicity toward DLD-1 and B16-
F1 (IC50 3.93 and 7.1 lM, respectively). The D-arabino-
side derivative 20 also displayed an effective activity
similar to 3 and 9 (IC50 13.3–18 lM) while the L-rham-
noside derivative 19 exerted moderate activity only
against A-549 and B16-F1 (IC50 59 and 55 lM, respec-
tively). Moreover, strong cytotoxicities were obtained
for 3-O-a-L-rhamnopyranoside (22) and 3-O-a-D-arabi-
nopyranoside (23) of betulinic acid (IC50 2.6–3.9 and
10–17 lM, respectively). In addition to their higher
solubility in polar solvents (data not shown), betulinic
acid glycosides (21–23) are the only compounds in this
study displaying differential cytotoxicity toward cancer
cell lines in comparison to healthy cell lines. Indeed,
22 and 23 exhibit only a moderate activity against
WS1 (IC50 31 and 47 lM, respectively), while the 3-O-

6718
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
b-D-glucopyranoside derivative 21 was inactive toward
healthy cells (IC50 >178 lM). These results show that
the glycosidation of betulinic acid (3) at the C-3 position
gives potent and selective cytotoxic agents contrary to
the previously reported inactive 28-O-b-D-glucoside
derivative.28,29 It appeared that, for all synthesized gly-
cosides, the acid function at the C-28 position seems
to be important for the activity and the selectivity
against tested cancer cell lines. Indeed, by adding an
a-L-rhamnopyranose moiety at the C-3 position of bet-
ulinic acid (3), we obtained the strongest in vitro anti-
cancer agent (22) of all tested triterpenes and
glycosides, while the differential cytotoxicity with re-
spect to the healthy cells increased up to 12-fold.
recorded at 400 MHz and 13C NMR were recorded at
100 MHz on an Avance 400 Bruker spectrometer
equipped with a 5 mm QNP probe. Elucidations of
1
chemical structures were based on H, 13C, DEPT135,
COSY, HSQC, and HMBC NMR experiments. Chemi-
cal shifts are reported in parts per million (ppm) relative
to residual solvent peaks. Signals are reported as m
(multiplet), s (singlet), d (doublet), t (triplet), q (quin-
quet), c (complex), br s (broad singlet), and coupling
constants are reported in hertz (Hz). Melting points
were determined in capillaries and are uncorrected.
Optical rotations were obtained using sodium D line
at ambient temperature on a Jasco DIP-360 digital
polarimeter. Mass spectral data (HRMS) were obtained
at the Department of Chemistry, Queen’s University
Ontario, Canada.
3. Conclusion
4.2. Isolation of lupeol (1)
From our in vitro cytotoxic results and in terms of struc-
ture–activity relationships, we can conclude that: (i) the
glycosides 18 and 22 exert a stronger activity than betu-
linic acid (3) against tested cancerous cell lines; (ii) 3-O-
and 28-O-glycosidation of betulin (2) decreases the
activity; (iii) 3-O-b-D-glucosidation of lupeol (1) and
methyl betulinate (7) enhances the activity; (iv) 3-O-a-
L-rhamnosidation decreases the activity except for betu-
linic acid (3); (v) 3-O-a-D-arabinosidation enhances the
activity except for betulin (2); (vi) 3-O-glycosidation of
betulinic acid (3) increases the selectivity against tested
cancerous cell lines and enhances the activity in the case
of L-rhamnose and D-arabinose. Thus, our study indi-
cates that 3-O-glycosides of lupane-type triterpenoids
represent an interesting class of compounds for further
pharmacological studies and preclinical developments.
In vivo assessments on tumor-bearing mice are now in
progress in our laboratory to evaluate the toxicity and
the anticancer activity of the promising 3-O-a-L-
rhamnopyranoside of betulinic acid (22).
The finely ground external bark (150 g) of the yellow
birch (B. alleghaniensis Britton), collected in Saguenay,
Quebec, Canada, was extracted in CHCl3 (1 L) with a
soxhlet apparatus, refluxed for 1 day, and purified by
flash chromatography (CH2Cl2 to CH2Cl2/CH3OH
99:1) to give 1 as a white powder (1.77 g, 1.2%): Rf
0.63 (CH2Cl2); mp 213–215 ꢁC, lit.49 mp 215–216 ꢁC;
20
½aꢁ +19.6 (c 1.2, CHCl3), lit.49 [a]D +26.4 (CHCl3).
D
1H and 13C NMR spectral data of 1 were in agreement
with those published in the literature.6 HR-EI-MS m/z
426.3854 [M]+ (calcd for C30H50O, 426.3862).
4.3. Isolation of betulin (2)
The finely ground external bark (150 g) of the white
birch (B. papyrifera Marsh.), collected in Saguenay,
Quebec, Canada, was soaked in CH2Cl2 (1 L), refluxed
for 1 day, and purified by flash chromatography
(CH2Cl2 to CH2Cl2/CH3OH 49:1) to give 2 as a white
powder (25 g, 17%): Rf 0.17 (CH2Cl2); mp 250–252 ꢁC,
20
lit.49 mp 251–252 ꢁC; ½aꢁ +19.1 (c 0.67, C5H5N), lit.49
D
15
D
1
4. Experimental
4.1. Chemicals
½aꢁ +20.0 (C5H5N). H and 13C NMR spectral data
of 2 were in agreement with those published in the liter-
ature.50 HR-EI-MS m/z 442.3804 [M]+ (calcd for
C30H50O2, 442.3811).
Air and water sensitive reactions were performed in
flame-dried glassware under a nitrogen or argon atmo-
sphere. Moisture sensitive reagents were introduced via
a dry syringe. Dichloromethane was distilled from
CaH2. THF was distilled from sodium with benzophe-
none as indicator of moisture. Betulinic acid (3) was
purchased from Indofine Chemical Company. Tetraki-
striphenylphosphine palladium(0) was prepared as men-
tioned in the literature48 and stored under nitrogen. All
other chemicals and materials were purchased from Sig-
ma–Aldrich and were used as received. Flash chroma-
tography was carried out using 60–230 mesh silica gel.
Analytical thin-layer chromatography was performed
with silica gel 60 F254, 0.25 mm pre-coated TLC plates
and visualized using UV254 and cerium molybdate (2 g
Ce(SO4)4(NH4)4, 5 g MoO4(NH4)2, 200 mL H2O, and
20 mL H2SO4) with charring. All of the chemical yields
are not optimized and generally represent the result of
4.4. 3,28-Diacetoxybetulin (4)
Acetic anhydride (4.8 mL, 50 mmol) was added to a
cooled solution (ice-water bath) of 2 (7.50 g, 17 mmol)
in pyridine (182 mL) with DMAP (100 mg, 0.82 mmol)
as catalyst. After stirring at room temperature for 5 h,
the mixture was diluted with CH2Cl2, then, washed with
cold H2SO4 3 N, saturated NaHCO3 solution, and
brine. The solvents of the dried solution (MgSO4) were
evaporated under reduced pressure and the residue
was purified by flash chromatography (hexanes to hex-
anes/EtOAc 97:3) to give 4 as a white crystalline powder
(8.48 g, 95%): Rf 0.74 (CH2Cl2); mp 216–218 ꢁC, lit.49
20
20
D
mp 223–224 ꢁC; ½aꢁ +19.7 (c 1.67, CHCl3), lit.49 ½aꢁ
D
+22. 1H and 13C NMR spectral data of 4 were in
agreement with those published in the literature.35
HR-ESI-MS m/z 549.3925 [M+Na]+ (calcd for
C34H54O4Na, 549.3920).
1
the mean of two experiments. H NMR spectra were

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
6719
4.5. 28-Acetoxybetulin (5)
aqueous layer was extracted with EtOAc (3·) and the
combined organic layers were washed with saturated
NaHCO3, and brine. After the solution was dried
(MgSO4), the solvents were evaporated under reduced
pressure. The resulting residue was purified by flash
chromatography (CH2Cl2) to give 8 as a white crystal-
line powder (458 mg, 84%): Rf 0.58 (CH2Cl2/CH3OH
Acetic anhydride (300 mL, 3.1 mol) was added to a solu-
tion of 2 (11.6 g, 26.2 mmol) in CH2Cl2 (750 mL). After
stirring overnight at room temperature, the mixture was
washed exhaustively with saturated NaHCO3 solution
and brine. The solvents of the dried solution (MgSO4)
were evaporated under reduced pressure and the residue
was purified by flash chromatography (CH2Cl2 to
20
1
99:1); mp 152–154 ꢁC; ½aꢁ +3.9 (c 1.00, CHCl3). H
D
NMR (CDCl3) d: 0.77, 0.83, 0.92 (all s, each 3H, H-
24, H-25, H-26), 0.97 (s, 6H, H-23, H-27), 1.69 (s, 3H,
H-30), 3.02 (m, 1H, H-19), 3.19 (dd, 1H, J = 11.0 Hz,
J = 5.1 Hz, H-3), 4.58 (m, 2H, CH2CH@CH2), 4.61 (br
s, 1H, H-29a), 4.74 (br s, 1H, H-29b), 5.24 (d, 1H,
J = 10.5 Hz, CH2CH@CH2, Ha), 5.35 (d, 1H,
J = 17.1 Hz, CH2CH@CH2, Hb), 5.94 (ddt, 1H,
J = 17.1 Hz, J = 10.5 Hz, J = 5.7 Hz, CH2CH@CH2),
0.69–2.28 (all m, remaining protons). 13C NMR (CDCl3)
d: 14.75, 15.44, 16.00, 16.19, 18.33, 19.44, 20.92, 25.56,
27.43, 28.04, 29.68, 30.61, 32.15, 34.36, 37.03, 37.22,
38.24, 38.77, 38.89, 40.77, 42.42, 46.94, 49.48, 50.59,
55.39, 56.59, 64.61 (CH2CH@CH2), 78.91 (C-3), 109.64
(C-29), 118.15 (CH2CH@CH2), 132.56 (CH2CH@CH2),
150.53 (C-20), 175.72 (C-28). HR-ESI-MS m/z 497.3985
[M+H]+ (calcd for C33H53O3, 497.3995).
CH2Cl2/CH3OH 49:1) to give 5 as a white powder
20
D
(9.28 g, 73%): Rf 0.31 (CH2Cl2); mp 210–212 ꢁC; ½aꢁ
1
+8.5 (c 1.58, CHCl3). H and 13C NMR spectral data
of 5 were in agreement with those published in the liter-
ature.26,35 HR-EI-MS m/z 484.3903 [M]+ (calcd for
C32H52O3, 484.3916).
4.6. 3-Acetoxybetulin (6)
A solution of Mg(OCH3)2 in CH3OH (224 mL, 8%) was
added under N2 to a solution of 4 (6.14 g, 11.7 mmol) in
dry THF (181 mL) and dry CH3OH (542 mL). After
stirring for 4 h at room temperature, the mixture was
acidified with HCl 10% and extracted with CH2Cl2
(3·). Then, the organic layer was washed with saturated
NaHCO3 solution and brine. The solvents of the dried
solution (MgSO4) were evaporated under reduced pres-
sure and the residue was purified by flash chromatogra-
phy (hexanes to hexanes/EtOAc 9:1) to give 6 as a white
solid (4.80 g, 85%): Rf 0.49 (CH2Cl2); mp 258–260 ꢁC,
4.9. 1,2,3,4,6-Penta-O-benzoyl-a,b-D-glucopyranose (24)
BzCl (77 mL, 666 mmol) was slowly added to a cooled
solution (ice-water bath) of D-glucose (20.0 g,
111 mmol) in anhydrous pyridine (280 mL) with DMAP
(136 mg, 1.1 mmol) as catalyst. The reaction was per-
formed overnight at room temperature with constant
stirring and then quenched with CH3OH (31 mL). The
mixture was diluted with CH2Cl2 and washed with cold
H2SO4 3 N, saturated NaHCO3 solution and brine. The
solvents of the dried solution (MgSO4) were evaporated
under reduced pressure and the residue was purified by
flash chromatography (CH2Cl2) to give 24 as a white
solid (71.6 g, 92%): Rf 0.68 (CH2Cl2); mp 172–174 ꢁC;
20
1
lit.36 mp 256–258 ꢁC; ½aꢁ +25.7 (c 0.92, CHCl3). H
D
and 13C NMR spectral data of 6 were in agreement with
those published in the literature.36 HR-EI-MS m/z
484.3904 [M]+ (calcd for C32H52O3, 484.3916).
4.7. Methyl betulinate (7)
DBU (0.17 mL, 1.1 mmol) and CH3I (0.21 mL,
3.3 mmol) were slowly added under N2 to a cooled solu-
tion (ice-water bath) of 3 (502 mg, 1.09 mmol) in dry
THF (10 mL). The reaction mixture was stirred over-
night at room temperature, then filtered off and washed
with dry THF. The filtrate and the combined washings
were concentrated to give a yellow solid. This residue
was acidified (HCl 6 N) and extracted with CH2Cl2
(3·). After that, the organic layer was washed with
H2O, dried (MgSO4) and then the solvents were evapo-
rated under reduced pressure. The resulting residue was
purified by flash chromatography (CH2Cl2) to give 7 as
20
D
1
½aꢁ +104.9 (c 1.25, CHCl3). H and 13C NMR spectral
data of 24 were in agreement with those published in the
literature.39,55 HR-ESI-MS m/z 723.1818 [M+Na]+
(calcd for C41H32O11Na, 723.1842).
4.10. 2,3,4,6-Tetra-O-benzoyl-a,b-D-glucopyranose (25)
HBr/HOAc (10 mL, 33%) was added under N2 to a solu-
tion of 24 (10.0 g, 14.3 mmol) in dry CH2Cl2 (42 mL).
The reaction mixture was stirred at room temperature
for 4 h, then, the solution was washed with saturated
NaHCO3 solution and brine. The organic layer was
dried (MgSO4), filtered and the solvents were evaporat-
ed under reduced pressure. After the residue was dis-
solved in acetone (75 mL) and water (3 mL), Ag2CO3
(6.50 g, 23.6 mmol) was added portionwise. The hydro-
lysis was performed in 1 h at room temperature with
constant stirring, then, the mixture was filtered through
a bed of Celite. The filtrate was concentrated under re-
duced pressure and the residue was purified by flash
chromatography (CH2Cl2/CH3OH 99:1 to 49:1) to give
25 as a white foam (7.32 g, 86%): Rf 0.28 (CH2Cl2/
CH3OH 99:1); mp 116–118 ꢁC, lit.56 mp 118–120 ꢁC;
a white powder (367 mg, 71%): Rf 0.54 (CH2Cl2); mp
20
D
218–220 ꢁC, lit.51 217-220 ꢁC; ½aꢁ +1.3 (c 0.58, CHCl3),
25
26
lit.51 ½aꢁ +5 (c 0.17, CHCl3), lit.52 ½aꢁ +4.0 (c 0.5,
D
D
1
CHCl3). H and 13C NMR spectral data of 7 were in
agreement with those published in the literature.52–54
HR-EI-MS m/z 470.3744 [M]+ (calcd for C31H50O3,
470.3760).
4.8. Allyl betulinate (8)
Allyl bromide (0.19 mL, 2.2 mmol) and K2CO3 (454 mg,
3.28 mmol) were added to a solution of 3 (501 mg,
1.10 mmol) in DMF (7 mL). The reaction mixture was
stirred for 7 h at 55 ꢁC. After cooling, EtOAc was added
and the organic layer was washed with 1 N HCl. The
20
D
22
D
½aꢁ +70.1 (c 1.42, CHCl3), lit.56 ½aꢁ +72.2 (c 0.5,

6720
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
1
CHCl3). H and 13C NMR spectral data of 25 were in
agreement with those published in the literature.41,56
HR-ESI-MS m/z 619.1567 [M+Na]+ (calcd for
C34H28O10Na, 619.1580).
crystalline powder (1.78 g, 72%, 2 steps): Rf 0.74
20 20
(CH2Cl2); ½aꢁ +83.6 (c 1.33, CHCl3), lit.42 ½aꢁ +97.5
D
D
1
(c 1.0, CHCl3). H and 13C NMR spectral data of 28
were in agreement with those published in the litera-
ture.42 HR-ESI-MS m/z 658.0189 [M+K]+ (calcd for
C29H24NO8Cl3K, 658.0204).
4.11. 2,3,4,6-Tetra-O-benzoyl-a-D-glucopyranose trichlo-
roacetimidate (26)
4.14. 1,2,3,4-Tetra-O-benzoyl-a,b-D-arabinopyranose
(29)
CCl3CN (6 mL, 59.8 mmol) was added to a solution of
25 (5.81 g, 9.74 mmol) and Cs2CO3 (315 mg, 0.97 mmol)
in CH2Cl2 (100 mL). The reaction mixture was stirred
for 4 h at room temperature and then filtered off. The
solvents of the filtrate were evaporated under reduced
pressure and the residue was purified by flash chroma-
This compound was prepared from D-arabinose (4.92 g,
32.8 mmol) in the same manner as that described for com-
pound 24. Purification by flash chromatography
(CH2Cl2) afforded 29 as a white crystalline powder
20
tography (CH2Cl2) to give 26 as a white crystalline pow-
(16.5 g, 89%): Rf 0.59 (CH2Cl2); ½aꢁ ꢂ274.2 (c 1.00,
D
20
D
der (6.13 g, 85%): Rf 0.64 (CH2Cl2/CH3OH 99:1); ½aꢁ
CHCl3). 1H NMR (CDCl3) d: 4.21 (dd, 1H, J = 13.4 Hz,
J = 1.8 Hz, H-5a), 4.44 (d, 1H, J = 13.0 Hz, H-5b), 5.93
(s, 1H, H-4), 6.10 (br s, 2H, H-2, H-3), 6.90 (br s, 1H,
H-1), 7.26–7.34 (m, 4H, H-Ar), 7.42–7.56 (m, 6H, H-
Ar), 7.61–7.68 (m, 2H, H-Ar), 7.88–7.93 (m, 4H, H-Ar),
8.13–8.18 (m, 4H, H-Ar). 13C NMR (CDCl3) d: 63.07
(C-5), 67.82 (C-2), 68.23 (C-3), 69.53 (C-4), 91.12 (C-1),
128.44–133.89 (C-Ar), 164.73, 165.62, 165.76, 165.79
(4· CO). HR-ESI-MS m/z 589.1457 [M+Na]+ (calcd for
C33H26O9Na, 589.1474).
+76.5 (c 1.67, CHCl3). H and 13C NMR spectral data
of 26 were in agreement with those published in the lit-
erature.41 HR-ESI-MS m/z 778.0410 [M+K]+ (calcd for
C36H28NO10Cl3K, 778.0415).
1
4.12. 1,2,3,4-Tetra-O-benzoyl-a,b-L-rhamnopyranose (27)
This compound was prepared from L-rhamnose (2.05 g,
12.5 mmol) in the same manner as that described for
compound 24. Purification by flash chromatography
(CH2Cl2) afforded 27 as a white crystalline powder
4.15. 2,3,4-Tri-O-benzoyl-a-D-arabinopyranose trichloro-
acetimidate (30)
20
D
(5.95 g, 82%): Rf 0.65 (CH2Cl2); ½aꢁ +33.6 (c 0.25,
1
CHCl3). H NMR (CDCl3) d: 1.52 (d, 3H, J = 6.2 Hz,
H-6), 4.20 (m, 1H, H-5), 5.85 (t, 1H, J = 9.6 Hz, H-4),
5.91 (dd, 1H, J = 10.0 Hz, J = 3.2 Hz, H-3), 6.24 (d,
1H, J = 3.0 Hz, H-2), 6.54 (br s, 1H, H-1), 7.20–7.25
(m, 2H, H-Ar), 7.28–7.41 (m, 5H, H-Ar), 7.44–7.54
(m, 4H, H-Ar), 7.58–7.64 (m, 1H, H-Ar), 7.88–7.92
(m, 2H, H-Ar), 7.97–8.05 (m, 4H, H-Ar), 8.23–8.27
(m, 2H, H-Ar). 13C NMR (CDCl3) d: 17.84 (C-6),
69.88 (C-5), 71.44 (C-2), 71.62 (C-3), 71.75 (C-4),
91.38 (C-1), 128.39–133.75 (C-Ar), 164.27, 165.51,
165.74, 165.85 (4· CO). HR-ESI-MS m/z 603.1613
[M+Na]+ (calcd for C34H28O9Na, 603.1631).
This compound was prepared from 29 (5.70 g,
10.1 mmol) in the same manner as that described for
compound 28. Purification by flash chromatography
(CH2Cl2) afforded 30 as a white foam (4.76 g, 78%, 2
20
steps): Rf 0.55 (CH2Cl2); ½aꢁ ꢂ182.8 (c 1.00, CHCl3).
D
1H NMR (CDCl3) d: 4.19 (dd, 1H, J = 13.3 Hz,
J = 2.0 Hz, H-5a), 4.43 (d, 1H, J = 12.8 Hz, H-5b),
5.88 (m, 1H, H-4), 6.02 (ddd, 2H, J = 16.7 Hz,
J = 10.7 Hz, J = 3.0 Hz, H-2, H-3), 6.83 (d, 1H,
J = 3.0 Hz, H-1), 7.26–7.33 (m, 2H, H-Ar), 7.34–7.40
(m, 2H, H-Ar), 7.44–7.55 (m, 4H, H-Ar), 7.60–7.66
(m, 1H, H-Ar), 7.84–7.88 (m, 2H, H-Ar), 7.96–8.00
(m, 2H, H-Ar), 8.09–8.15 (m, 2H, H-Ar), 8.64 (br s,
1H, NH). 13C NMR (CDCl3) d: 63.18 (C-5), 68.00 (d,
C-2, C-3), 69.45 (C-4), 90.89 (CCl3), 94.35 (C-1),
128.38–133.57 (C-Ar), 160.80 (C@NH), 165.59, 165.66,
165.69 (3· CO). HR-ESI-MS m/z 644.0076 [M+K]+
(calcd for C28H22NO8Cl3K, 644.0048).
4.13. 2,3,4-Tri-O-benzoyl-a-L-rhamnopyranose trichloro-
acetimidate (28)
HBr/HOAc (2.3 mL, 33%) was added at room tempera-
ture under N2 to a solution of 27 (2.31 g, 3.98 mmol) in
dry CH2Cl2 (10 mL). The reaction mixture was stirred at
room temperature for 2 h, then, the solution was washed
with saturated NaHCO3 solution and brine. The organic
layer was dried over MgSO4, filtered and the solvents
were evaporated under reduced pressure. After the resi-
due was dissolved in acetone (19 mL) and water
(0.8 mL), Ag2CO3 (1.50 g, 5.44 mmol) was added por-
tionwise. The hydrolysis was performed in 1 h at room
temperature with constant stirring, then, the mixture
was filtered through a bed of Celite. The filtrate was con-
centrated under reduced pressure and dissolved in
CH2Cl2 (50 mL). Cs2CO3 (130 mg, 0.40 mmol) was add-
ed, followed by CCl3CN (2.4 mL, 23.9 mmol), and the
reaction mixture was stirred for 4 h at room tempera-
ture. The mixture was then filtered off, concentrated un-
der reduced pressure and the residue was purified by
flash chromatography (CH2Cl2) to give 28 as a white
4.16. 3-O-b-D-Glucopyranoside of lupeol (9)
The acceptor 1 (1.01 g, 2.34 mmol) and the donor 26
(2.60 g, 3.52 mmol) were stirred in dry CH2Cl2
˚
(80 mL) for 1 h with 4 A MS. At this time, TMSOTf
(24 lL, 0.13 mmol) was added under Ar while keeping
rigorous anhydrous conditions. The reaction was usu-
ally performed in 30 min, then quenched by addition
of Et3N (0.3 mL). The solvents were evaporated under
reduced pressure and the resulting residue was immedi-
ately dissolved in a NaOH 0.25 N solution of CH3OH/
THF/H2O 1:2:1 (240 mL). The reaction mixture was
stirred at room temperature for 2 h, dissolved in
CH2Cl2, and washed with HCl 10% and brine. Once
the solution was dried (MgSO4), the solvents were

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
6721
evaporated under reduced pressure and the residue was
purified by flash chromatography (CH2Cl2/CH3OH,
49:1 to 47:3) to give 9 as a white powder (1.38 g,
90%, 2 steps): Rf 0.24 (CH2Cl2/CH3OH 9:1); mp 176–
1H, J = 11.4 Hz, H0-5a), 3.65 (m, 1H, H0-3), 3.68 (m,
1H, H0-2), 3.93 (br s, 1H, H0-4), 3.94 (d, 1H,
J = 11.4 Hz, H0-5b), 4.34 (d, 1H, J = 5.9 Hz, H0-1),
4.57 (br s, 1H, H-29a), 4.68 (br s, 1H, H-29b), 0.70–
1.92 (all m, remaining protons). 13C NMR (CDCl3) d:
14.47, 15.98, 16.10, 16.39, 18.00, 18.30, 19.32, 20.96,
23.01, 25.13, 27.41, 28.20, 29.84, 34.26, 35.56, 37.03,
38.02, 38.22, 38.39, 40.00, 40.88, 42.82, 43.02, 47.98,
48.30, 50.39, 55.84, 64.83 (C0-5), 67.49 (C0-4), 71.62
(C0-3), 72.68 (C0-2), 84.59 (C-3), 99.53 (C0-1), 109.33
(C-29), 151.01 (C-20). HR-ESI-MS m/z 581.4163
[M+Na]+ (calcd for C35H58O5Na, 581.4181).
20
1
178 ꢁC; ½aꢁ +7.9 (c 0.50, CHCl3). H NMR (CDCl3)
D
d: 0.79, 0.80, 0.83, 0.93, 0.99, 1.02 (all s, each 3H, H-
23, H-24, H-25, H-26, H-27, H-28), 1.68 (s, 3H, H-
30), 2.37 (m, 1H, H-19), 2.63 (br s, 4H, 4· OH), 3.13
(dd, 1H, J = 11.2 Hz, J = 4.8 Hz, H-3), 3.36 (m, 1H,
H0-5), 3.42 (t, 1H, J = 8.3 Hz, H0-2), 3.58 (q, 2H,
J = 8.7 Hz, H0-3, H0-4), 3.80 (dd, 1H, J = 11.8 Hz,
J = 4.2 Hz, H0-6a), 3.86 (dd, 1H, J = 12.0 Hz,
J = 3.1 Hz, H0-6b), 4.36 (d, 1H, J = 7.7 Hz, H0-1),
4.57 (br s, 1H, H-29a), 4.69 (br s, 1H, H-29b), 0.67–
1.92 (all m, remaining protons). 13C NMR (CDCl3)
d: 14.70, 16.15, 16.38, 16.74, 18.16, 18.35, 19.50,
21.00, 25.26, 26.48, 27.60, 28.09, 30.02, 34.46, 35.74,
37.02, 38.20, 38.93, 39.35, 40.15, 40.99, 42.95, 43.17,
48.15, 48.45, 50.57, 55.77, 61.94 (C0-6), 69.69 (C0-4),
73.98 (C0-2), 75.29 (C0-5), 76.51 (C0-3), 90.29 (C-3),
105.32 (C0-1), 109.54 (C-29), 151.08 (C-20). HR-ESI-
MS m/z 611.4267 [M+Na]+ (calcd for C36H60O6Na,
611.4287).
4.19. 3-O-b-D-Glucopyranoside of betulin (12)
This compound was prepared from the acceptor 5
(500 mg, 1.03 mmol) and the donor 26 (1.15 g,
1.55 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 12 as a white
crystalline powder (406 mg, 65%, 2 steps): Rf 0.21
20
D
(CH2Cl2/CH3OH 9:1); mp 192–194 ꢁC; ½aꢁ +2.7 (c
1
0.58, CH3OH). H NMR (CD3OD) d: 0.84, 0.88, 1.02,
1.05, 1.08, 1.69 (all s, each 3H, H-23, H-24, H-25, H-
26, H-27, H-30), 2.42 (m, 1H, H-19), 3.16 (dd, 1H,
J = 11.2 Hz, J = 5.0 Hz, H-3), 3.18 (t, 1H, J = 9.8 Hz,
H0-2), 3.25 (m, 1H, H0-5), 3.28 (t, 1H, J = 11.7 Hz, H0-
4), 3.28 (d, 1H, J = 11.7 Hz, H-28a), 3.28 (dd, 1H,
J = 11.9 Hz, J = 5.1 Hz, H0-6a), 3.33 (t, 1H,
J = 9.8 Hz, H0-3), 3.74 (d, 1H, J = 11.7 Hz, H-28b),
3.84 (dd, 1H, J = 11.9 Hz, J = 1.9 Hz, H0-6b), 4.31 (d,
1H, J = 7.8 Hz, H0-1), 4.58 (br s, 1H, H-29a), 4.69 (br
s, 1H, H-29b), 0.74–1.98 (all m, remaining protons).
13C NMR (CD3OD) d: 15.22, 16.54, 16.77, 16.82,
19.28, 19.38, 21.99, 26.62, 27.19, 28.17, 28.41, 30.37,
30.84, 35.10, 35.47, 38.02, 38.70, 40.00, 40.28, 42.16,
43.81, 48.53, 49.25, 50.03, 51.83, 57.10, 60.35 (C-28),
62.79 (C0-6), 71.64 (C0-4), 75.66 (C0-2), 77.68 (C0-5),
78.27 (C0-3), 90.79 (C-3), 106.74 (C0-1), 110.26 (C-29),
151.87 (C-20). HR-ESI-MS m/z 627.4218 [M+Na]+
(calcd for C36H60O7Na, 627.4236).
4.17. 3-O-a-L-Rhamnopyranoside of lupeol (10)
This compound was prepared from the acceptor 1
(502 mg, 1.18 mmol) and the donor 28 (1.09 g,
1.76 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 10 as a white
powder (485 mg, 72%, 2 steps): Rf 0.33 (CH2Cl2/CH3OH
20
1
9:1); mp 214–216 ꢁC; ½aꢁ ꢂ17.9 (c 0.50, CHCl3). H
D
NMR (CDCl3) d: 0.75, 0.79, 0.83, 0.90, 0.94, 1.02 (all
s, each 3H, H-23, H-24, H-25, H-26, H-27, H-28), 1.28
(d, 3H, J = 6.1 Hz, H0-6), 1.69 (s, 3H, H-30), 2.38 (m,
1H, H-19), 3.07 (dd, 1H, J = 11.3 Hz, J = 4.8 Hz, H-3),
3.43 (t, 1H, J = 9.2 Hz, H0-4), 3.77 (t, 1H, J = 5.2 Hz,
H0-3), 3.81 (dd, 1H, J = 9.0 Hz, J = 6.1 Hz, H0-5), 3.95
(br s, 1H, H0-2), 4.57 (br s, 1H, H-29a), 4.69 (br s,
1H, H-29b), 4.82 (br s, 1H, H0-1), 0.68–1.93 (all m,
remaining protons). 13C NMR (CDCl3) d: 14.55,
15.98, 16.15, 16.25, 17.35 (C0-6), 18.01, 18.30, 19.33,
20.95, 25.14, 25.52, 27.44, 28.19, 29.86, 34.25, 35.59,
36.89, 38.05, 38.64, 39.06, 40.01, 40.85, 42.83, 43.02,
48.00, 48.31, 50.40, 55.45, 67.65 (C0-5), 71.26 (C0-2),
71.98 (C0-3), 74.00 (C0-4), 89.71 (C-3), 101.67 (C0-1),
109.33 (C-29), 151.01 (C-20). HR-ESI-MS m/z
595.4335 [M+Na]+ (calcd for C36H60O5Na, 595.4338).
4.20. 3-O-a-L-Rhamnopyranoside of betulin (13)
This compound was prepared from the acceptor 5
(252 mg, 0.52 mmol) and the donor 28 (484 mg,
0.78 mmol) in the same manner as that described for com-
pound 9. Purification by flash chromatography (CH2Cl2/
CH3OH, 49:1 to 47:3) afforded 13 as a white crystalline
powder (159 mg, 52%, 2 steps): Rf 0.29 (CH2Cl2/CH3OH
20
D
4.18. 3-O-a-D-Arabinopyranoside of lupeol (11)
9:1); mp >200 ꢁC; ½aꢁ ꢂ20.3 (c 0.50, CH3OH). 1H NMR
(CD3OD) d: 0.79, 0.88, 0.94, 1.02, 1.08 (all s, each 3H, H-
23, H-24, H-25, H-26, H-27), 1.22 (d, 3H, J = 6.3 Hz, H0-
6), 1.69 (s, 3H, H-30), 2.42 (m, 1H, H-19), 3.07 (dd, 1H,
J = 11.3 Hz, J = 4.6 Hz, H-3), 3.28 (d, 1H, J = 10.9 Hz,
H-28a), 3.36 (t, 1H, J = 9.5 Hz, H0-4), 3.63 (dd, 1H,
J = 9.5 Hz, J = 3.2 Hz, H0-3), 3.70 (m, 1H, H0-5), 3.74
(d, 1H, J = 10.9 Hz, H-28b), 3.82 (br s, 1H, H0-2), 4.57
(br s, 1H, H-29a), 4.68 (br s, 1H, H-29b), 4.72 (br s, 1H,
H0-1), 0.76–1.95 (all m, remaining protons). 13C NMR
(CD3OD) d: 15.20, 16.51, 16.72, 16.77, 17.83 (C0-6),
19.34, 19.38, 21.98, 26.58, 26.76, 28.14, 28.61, 30.34,
30.82, 35.09, 35.40, 38.06, 38.68, 39.82, 40.15, 42.15,
This compound was prepared from the acceptor 1
(251 mg, 0.59 mmol) and the donor 30 (531 mg,
0.88 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 11 as a white
solid (286 mg, 87%, 2 steps): Rf 0.33 (CH2Cl2/CH3OH
20
1
9:1); mp 212–214 ꢁC; ½aꢁ +26.8 (c 1.25, CHCl3). H
D
NMR (CDCl3) d: 0.77, 0.79, 0.84, 0.92, 1.00, 1.02, 1.68
(all s, each 3H, H-23, H-24, H-25, H-26, H-27, H-28,
H-30), 2.38 (m, 1H, H-19), 2.64 (br s, 3H, 3· OH),
3.26 (dd, 1H, J = 11.9 Hz, J = 3.8 Hz, H-3), 3.54 (d,

6722
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
43.82, 48.53, 49.24, 50.00, 51.77, 56.79, 60.33 (C-28),
69.88 (C0-5), 72.48 (C0-2), 72.50 (C0-3), 74.07 (C0-4),
90.36 (C-3), 104.43 (C0-1), 110.25 (C-29), 151.86 (C-20).
HR-ESI-MS m/z 611.4266 [M+Na]+ (calcd for
C36H60O6Na, 611.4287).
4.23. 28-O-a-L-Rhamnopyranoside of betulin (16)
This compound was prepared from the acceptor 6
(250 mg, 0.52 mmol) and the donor 28 (480 mg,
0.77 mol) in the same manner as that described for com-
pound 9 except for the basic hydrolysis reaction time
(overnight). Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 16 as a white
4.21. 3-O-a-D-Arabinopyranoside of betulin (14)
This compound was prepared from the acceptor 5
(250 mg, 0.52 mmol) and the donor 30 (442 mg,
0.78 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 14 as a white
powder (203 mg, 67%, 2 steps): Rf 0.31 (CH2Cl2/CH3OH
20
D
9:1); mp >200 ꢁC; ½aꢁ ꢂ42.9 (c 0.83, CH3OH). 1H NMR
(C5D5N) d: 0.87, 0.95, 0.98, 1.03, 1.22, 1.73 (all s, each
3H, H-23, H-24, H-25, H-26, H-27, H-30), 1.73 (d, 3H,
J = 6.3 Hz, H0-6), 2.60 (m, 1H, H-19), 3.45 (m, 1H, H-
3), 3.61 (d, 1H, J = 9.4 Hz, H-28a), 3.83 (d, 1H,
J = 9.4 Hz, H-28b), 4.22 (c, 1H, H0-5), 4.33 (t, 1H,
J = 9.2 Hz, H0-4), 4.51 (dd, 1H, J = 9.1 Hz, J = 2.9 Hz,
H0-3), 4.63 (br s, 1H, H0-2), 4.73 (br s, 1H, H-29a), 4.88
(br s, 1H, H-29b), 5.39 (br s, 1H, H0-1), 0.79-2.12 (all
m, remaining protons). 13C NMR (C5D5N) d: 14.89,
16.12, 16.37, 16.43, 18.74 (C0-6), 19.32, 21.00, 25.64,
27.55, 27.55, 28.31, 28.66, 30.33, 30.48, 34.59, 35.39,
37.46, 37.68, 39.27, 39.53, 41.15, 42.93, 47.31, 48.07,
49.07, 50.71, 55.83, 66.18 (C-28), 70.06 (C0-5), 72.45
(C0-2), 73.14 (C0-3), 73.94 (C0-4), 78.08 (C-3), 102.30
(C0-1), 110.11 (C-29), 150.89 (C-20). HR-ESI-MS m/z
611.4268 [M+Na]+ (calcd for C36H60O6Na, 611.4287).
powder (196 mg, 66%, 2 steps): Rf 0.29 (CH2Cl2/CH3OH
20
D
9:1); mp >200 ꢁC; ½aꢁ +17.4 (c 0.25, CH3OH). 1H
NMR (C5D5N) d: 0.75, 0.84, 0.95, 1.05, 1.22, 1.75 (all
s, each 3H, H-23, H-24, H-25, H-26, H-27, H-30), 2.61
(m, 1H, H-19), 3.42 (dd, 1H, J = 11.4 Hz, J = 4.2 Hz,
H-3), 3.64 (d, 1H, J = 10.1 Hz, H-28a), 3.80 (d, 1H,
J = 11.0 Hz, H0-5), 4.07 (d, 1H, J = 10.1 Hz, H-28b),
4.18 (dd, 1H, J = 8.7 Hz, J = 2.8 Hz, H0-3), 4.32 (br s,
1H, H0-4), 4.34 (d, 1H, J = 11.0 Hz, H0-5), 4.39 (t, 1H,
J = 7.9 Hz, H0-2), 4.70 (d, 1H, J = 7.1 Hz, H0-1), 4.74
(br s, 1H, H-29a), 4.88 (br s, 1H, H-29b), 4.99 (br s,
3H, 3· OH), 0.72–2.42 (all m, remaining protons). 13C
NMR (C5D5N) d: 14.90, 16.12, 16.25, 16.91, 18.65,
19.26, 21.06, 23.86, 25.70, 27.54, 28.55, 29.98, 29.99,
30.02, 34.58, 34.87, 37.56, 38.80, 41.08, 41.21, 42.98,
48.35, 48.53, 49.13, 50.61, 56.20, 59.41 (C-28), 67.05
(C0-5), 69.61 (C0-4), 72.55 (C0-2), 74.79 (C0-3), 84.93
(C-3), 102.98 (C0-1), 109.93 (C-29), 151.25 (C-20). HR-
ESI-MS m/z 587.4143 [M+Na]+ (calcd for C35H58O6Na,
597.4131).
4.24. 28-O-a-D-Arabinopyranoside of betulin (17)
This compound was prepared from the acceptor 6
(250 mg, 0.52 mmol) and the donor 30 (469 mg,
0.77 mmol) in the same manner as that described for
compound 9 except for the basic hydrolysis reaction
time (overnight). Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 17 as a white
4.22. 28-O-b-D-Glucopyranoside of betulin (15)
crystalline powder (178 mg, 60%, 2 steps): Rf 0.43
20
D
This compound was prepared from the acceptor 6
(501 mg, 1.03 mmol) and the donor 26 (1.15 g,
1.55 mmol) in the same manner as that described for
compound 9 except for the basic hydrolysis reaction
time (overnight). Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 15 as a white
(CH2Cl2/CH3OH 9:1); mp 204–206 ꢁC; ½aꢁ +4.6 (c
0.25, CH3OH). 1H NMR (DMSO-d6) d: 0.65, 0.76,
0.87, 0.93, 0.97, 1.63 (all s, each 3H, H-23, H-24, H-
25, H-26, H-27, H-30), 2.40 (m, 1H, H-19), 2.96 (m,
1H, H-3), 2.99 (d, 1H, J = 9.3 Hz, H-28a), 3.32 (m,
1H, H0-3), 3.33 (m, 1H, H0-2), 3.35 (d, 1H,
J = 11.8 Hz, H0-5a), 3.61 (m, 1H, H0-4), 3.66 (dd, 1H,
J = 11.8 Hz, J = 3.4 Hz, H0-5b), 3.89 (d, 1H,
J = 9.3 Hz, H-28b), 4.06 (d, 1H, J = 5.6 Hz, H0-1), 4.54
(br s, 1H, H-29a), 4.67 (br s, 1H, H-29b), 0.62-1.94
(all m, remaining protons). 13C NMR (DMSO-d6) d:
14.58, 15.67, 15.82, 15.90, 17.97, 18.76, 20.35, 24.74,
26.67, 27.18, 28.11, 29.29, 29.46, 33.76, 34.03, 36.68,
37.00, 38.25, 38.51, 40.45, 42.19, 46.60, 47.33, 48.33,
49.83, 54.86, 64.80 (C0-5), 66.33 (C-28), 67.40 (C0-4),
70.59 (C0-2), 72.60 (C0-3), 76.80 (C-3), 103.81 (C0-1),
109.77 (C-29), 150.17 (C-20). HR-ESI-MS m/z
597.4156 [M+Na]+ (calcd for C35H58O6Na, 597.4131).
powder (338 mg, 54%, 2 steps): Rf 0.21 (CH2Cl2/CH3OH
20
D
9:1); mp >200 ꢁC; ½aꢁ ꢂ12.8 (c 0.25, CH3OH). 1H
NMR (CD3OD) d: 0.76, 0.87, 0.96, 1.01, 1.09, 1.69 (all
s, each 3H, H-23, H-24, H-25, H-26, H-27, H-30), 2.46
(m, 1H, H-19), 3.13 (dd, 1H, J = 11.1 Hz, J = 4.9 Hz,
H-3), 3.19 (t, 1H, J = 8.4 Hz, H0-2), 3.28 (d, 1H,
J = 4.7 Hz, H0-5), 3.28 (d, 1H, J = 6.0 Hz, H0-4), 3.36
(t, 1H, J = 8.9 Hz, H0-3), 3.61 (d, 1H, J = 9.5 Hz, H-
28a), 3.68 (dd, 1H, J = 11.8 Hz, J = 5.0 Hz, H0-6a),
3.73 (d, 1H, J = 9.5 Hz, H-28b), 3.89 (d, 1H,
J = 11.6 Hz, H0-6b), 4.22 (d, 1H, J = 7.7 Hz, H0-1),
4.57 (br s, 1H, H-29a), 4.68 (br s, 1H, H-29b), 0.71–
2.14 (all m, remaining protons). 13C NMR (CD3OD)
d: 15.33, 16.18, 16.67, 16.75, 19.46, 19.50, 22.03, 26.66,
28.08, 28.40, 28.66, 30.69, 30.89, 35.51, 35.87, 38.32,
38.97, 40.00, 40.09, 42.18, 43.86, 46.96, 49.31, 50.17,
51.89, 56.85, 62.87 (C0-6), 68.91 (C-28), 71.77 (C0-4),
75.29 (C0-2), 77.96 (C0-5), 78.21 (C0-3), 79.70 (C-3),
105.35 (C0-1), 110.23 (C-29), 152.00 (C-20). HR-ESI-
MS m/z 627.4229 [M+Na]+ (calcd for C36H60O7Na,
627.4236).
4.25. 3-O-b-D-Glucopyranoside of methyl betulinate (18)
This compound was prepared from the acceptor 7
(251 mg, 0.53 mmol) and the donor 26 (593 mg,
0.80 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 18 as a white
crystalline powder (189 mg, 56%, 2 steps): Rf 0.24

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
6723
(CH2Cl2/CH3OH 9:1); mp 196–198 ꢁC, lit.27 mp 197–
3.70 (m, 1H, H0-2), 3.93 (m, 1H, H0-4), 3.95 (d, 1H,
J = 9.4 Hz, H0-5b), 4.31 (d, 1H, J = 6.1 Hz, H0-1), 4.59
(br s, 1H, H-29a), 4.73 (br s, 1H, H-29b), 0.68-2.22
(all m, remaining protons). 13C NMR (CDCl3) d:
14.76, 16.09, 16.23, 16.54, 18.42, 19.51, 21.04, 23.15,
25.63, 28.32, 29.78, 30.73, 32.29, 34.44, 37.11, 37.18,
38.34, 38.37, 38.54, 40.85, 42.51, 47.10, 49.59, 50.63,
51.44 (COOCH3), 56.02, 56.69, 65.10 (C0-5), 67.80 (C0-
4), 71.69 (C0-3), 72.85 (C0-2), 84.81 (C-3), 99.79 (C0-1),
109.72 (C-29), 150.74 (C-20), 176.81 (C-28). HR-ESI-
MS m/z 625.4073 [M+Na]+ (calcd for C36H58O7Na,
625.4080).
20
200 ꢁC; ½aꢁ ꢂ6.6 (c 0.50, CHCl3), lit.27 [a]D ꢂ3 (c
D
1
0.38, CH3OH). H NMR (C5D5N) d: 0.75, 0.94, 0.98,
1.02, 1.30, 1.72 (s, 3H, H-23, H-24, H-25, H-26, H-27,
H-30), 3.30 (m, 1H, H-19), 3.40 (dd, 1H, J = 11.7 Hz,
J = 4.3 Hz, H-3), 3.70 (s, 3H, COOCH3), 4.01 (m, 1H,
H0-5), 4.05 (t, 1H, J = 8.3 Hz, H0-2), 4.23 (t, 1H,
J = 8.8 Hz, H0-4), 4.26 (t, 1H, J = 8.5 Hz, H0-3), 4.41
(dd, 1H, J = 11.6 Hz, J = 5.4 Hz, H0-6a), 4.59 (dd, 1H,
J = 11.6 Hz, J = 2.2 Hz, H0-6b), 4.72 (br s, 1H, H-
29a), 4.88 (br s, 1H, H-29b), 4.95 (d, 1H, J = 7.7Hz,
H0-1), 0.73–2.45 (all m, remaining protons). 13C NMR
(C5D5N) d: 14.80, 16.16, 16.32, 16.84, 18.42, 19.37,
21.05, 25.90, 26.76, 28.13, 30.04, 30.91, 32.31, 34.64,
37.08, 37.08, 38.49, 38.99, 39.63, 40.98, 42.67, 47.54,
49.75, 50.69, 51.33 (COOCH3), 55.87, 56.77, 63.04 (C0-
6), 71.84 (C0-4), 75.82 (C0-2), 78.35 (C0-5), 78.79 (C0-3),
88.81 (C-3), 106.92 (C0-1), 110.12 (C-29), 150.82 (C-
20), 176.45 (C-28). HR-ESI-MS m/z 655.4164
[M+Na]+ (calcd for C37H60O8Na, 655.4186).
4.28. 3-O-b-D-Glucopyranoside of betulinic acid (21)
The acceptor 8 (107 mg, 0.22 mmol) and the donor 26
(239 mg, 0.32 mmol) were stirred in dry CH2Cl2 (10 mL)
˚
for 1 h with 4 A MS. At this time, TMSOTf (3 lL,
0.01 mmol) was added under Ar while keeping rigorous
anhydrous conditions. The reaction was usually per-
formed in 30 min, then quenched by addition of Et3N
(50 lL). The solvents were evaporated under reduced
pressure and the resulting residue was immediately dis-
solved in a NaOH 0.25 N solution of CH3OH/THF/
H2O 1:2:1 (30 mL). The reaction mixture was stirred at
room temperature for 2 h, dissolved in CH2Cl2, and
washed with HCl 10% and brine. Once the solution was
dried (MgSO4), the solvents were evaporated under re-
duced pressure to give an oily residue. It was dissolved
in a solution of PPh3 (32 mg, 0.121 mmol) and pyrrolidine
(34 lL, 0.403 mmol) in dry THF (1 mL), then Pd0(PPh3)4
(70 mg, 0.060 mmol) was added and the reaction mixture
was stirred overnight at room temperature. After evapo-
ration of the solvent under reduced pressure, the residue
was purified by flash chromatography (CH2Cl2/CH3OH,
49:1 to 4:1) to give 21 as a white powder (63 mg, 47%, 3
steps): Rf 0.38 (CH2Cl2/CH3OH 4:1); mp 234–236 ꢁC;
4.26. 3-O-a-L-Rhamnopyranoside of methyl betulinate
(19)
This compound was prepared from the acceptor 7
(201 mg, 0.43 mmol) and the donor 28 (398 mg,
0.64 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 19 as a white
powder (176 mg, 67%, 2 steps): Rf 0.24 (CH2Cl2/CH3
20
1
OH 9:1); mp >200 ꢁC; ½aꢁ ꢂ17.1 (c 0.42, CHCl3). H
D
NMR (C5D5N) d: 0.77 (s, 6H, H-25, H-26), 0.89, 0.96,
1.00 (all s, each 3H, H-23, H-24, H-27), 1.65 (d, 3H,
J = 5.4 Hz, H0-6), 1.72 (s, 3H, H-30), 3.14 (dd, 1H,
J = 11.7 Hz, J = 4.3 Hz, H-3), 3.30 (m, 1 H, H-19),
3.70 (s, 3H, COOCH3), 4.29 (m, 1H, H0-4), 4.32 (m,
1H, H0-5), 4.49 (m, 1H, H0-3), 4.72 (br s, 1H, H0-2),
4.72 (br s, 1H, H-29a), 4.88 (br s, 1H, H-29b), 5.32 (br
s, 1H, H0-1), 0.66–2.45 (all m, remaining protons). 13C
NMR (C5D5N) d: 14.77, 16.14, 16.27, 16.54, 18.52 (C0-
6), 19.35, 21.05, 21.13, 25.88, 26.05, 28.13, 30.02,
30.90, 32.29, 33.71, 34.56, 37.07, 38.46, 38.80, 39.28,
40.96, 42.65, 47.53, 49.73, 50.66, 51.34 (COOCH3),
55.61, 56.77, 69.87 (C0-5), 72.51 (C0-2), 72.91 (C0-3),
74.12 (C0-4), 88.51 (C-3), 104.42 (C0-1), 110.13 (C-29),
150.80 (C-20), 176.44 (C-28). HR-ESI-MS m/z
639.4223 [M+Na]+ (calcd for C37H60O7Na, 639.4237).
20
1
½aꢁ +1.3 (c 0.33, CH3OH). H NMR (C5D5N) d: 0.73,
D
0.97, 1.01, 1.09, 1.30, 1.77 (all s, each 3H, H-23, H-24,
H-25, H-26, H-27, H-30), 3.41 (dd, 1H, J = 11.6 Hz,
J = 4.0 Hz, H-3), 3.54 (m, 1H, H-19), 4.02 (m, 1H, H0-
5), 4.05 (t, 1H, J = 11.1 Hz, H0-2), 4.24 (m, 1H, H0-4),
4.26 (m, 1H, H0-3), 4.42 (dd, 1H, J = 11.6 Hz,
J = 5.2 Hz, H0-6a), 4.60 (d, 1H, J = 11.1 Hz, H0-6b),
4.75 (br s, 1H, H-29a), 4.93 (br s, 1H, H-29b), 4.95 (d,
1H, J = 7.8 Hz, H0-1), 0.73–2.69 (all m, remaining pro-
tons). 13C NMR (C5D5N) d: 14.84, 16.31, 16.35, 16.82,
18.44, 19.43, 21.15, 26.05, 26.76, 28.19, 30.25, 31.18,
32.85, 34.72, 37.11, 37.57, 38.56, 39.00, 39.63, 41.07,
42.83, 47.76, 49.71, 50.77, 55.88, 56.62, 63.03 (C0-6),
71.84 (C0-4), 75.82 (C0-2), 78.34 (C0-5), 78.78 (C0-3),
88.82 (C-3), 106.92 (C0-1), 109.95 (C-29), 151.29 (C-20),
178.87 (C-28). HR-ESI-MS m/z 641.4019 [M+Na]+ (calcd
for C36H58O8Na, 641.4029).
4.27. 3-O-a-D-Arabinopyranoside of methyl betulinate
(20)
This compound was prepared from the acceptor 7
(200 mg, 0.42 mmol) and the donor 30 (387 mg,
0.64 mmol) in the same manner as that described for
compound 9. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 47:3) afforded 20 as a white
4.29. 3-O-a-L-Rhamnopyranoside of betulinic acid (22)
powder (169 mg, 66%, 2 steps): Rf 0.24 (CH2Cl2/CH3OH
20
D
9:1); mp >200 ꢁC; ½aꢁ +22.7 (c 0.42, CHCl3). 1H NMR
This compound was prepared from the acceptor 8
(100 mg, 0.20 mmol) and the donor 28 (187 mg,
0.30 mmol) in the same manner as that described for
compound 21. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 4:1) afforded 22 as a white sol-
id (50 mg, 41%, 3 steps): Rf 0.18 (CH2Cl2/CH3OH 9:1);
(CDCl3) d: 0.75, 0.81, 0.90, 0.93, 0.98, 1.68 (all s, each
3H, H-23, H-24, H-25, H-26, H-27, H-30), 3.00 (m,
1H, H-19), 3.02 (br s, 3H, 3· OH), 3.23 (dd, 1H,
J = 11.8 Hz, J = 3.8 Hz, H-3), 3.52 (d, 1H, J = 11.4 Hz,
H0-5a), 3.66 (s, 3H, COOCH3), 3.66 (m, 1H, H0-3),

6724
C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
20
D
1
mp >200 ꢁC; ½aꢁ ꢂ22.8 (c 0.42, CH3OH). H NMR
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 resazurin46 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 triplicata. IC50 results were expressed as
means standard deviation.
(C5D5N) d: 0.75, 0.76, 0.89, 1.02, 1.07 (all s, each 3H,
H-23, H-24, H-25, H-26, H-27), 1.66 (d, 3H,
J = 5.0 Hz, H0-6), 1.77 (s, 3H, H-30), 3.16 (dd, 1H,
J = 11.5 Hz, J = 4.0 Hz, H-3), 3.53 (m, 1H, H-19), 4.29
(m, 1H, H0-4), 4.31 (m, 1H, H0-5), 4.48 (m, 1H, H0-3),
4.58 (br s, 1H, H0-2), 4.75 (br s, 1H, H-29a), 4.93 (br
s, 1H, H-29b), 5.33 (br s, 1H, H0-1), 0.67–2.71 (all m,
remaining protons). 13C NMR (C5D5N) d: 14.83,
16.28, 16.36, 16.54, 18.49, 18.53 (C0-6), 19.44, 21.18,
25.80, 26.06, 28.15, 30.26, 31.20, 32.86, 34.68, 37.13,
37.58, 38.56, 38.84, 39.30, 41.07, 42.84, 47.77, 49.73,
50.77, 55.65, 56.64, 69.88 (C0-5), 72.52 (C0-2), 72.93
(C0-3), 74.15 (C0-4), 88.53 (C-3), 104.42 (C0-1), 109.97
(C-29), 151.29 (C-20), 178.88 (C-28). HR-ESI-MS m/z
625.4057 [M+Na]+ (calcd for C36H58O7Na, 625.4080).
4.30. 3-O-a-D-Arabinopyranoside of betulinic acid (23)
Acknowledgments
This compound was prepared from the acceptor 8
(102 mg, 0.21 mmol) and the donor 30 (187 mg,
0.31 mmol) in the same manner as that described for
compound 21. Purification by flash chromatography
(CH2Cl2/CH3OH, 49:1 to 4:1) afforded 23 as a white
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.
powder (60 mg, 50%, 3 steps): Rf 0.19 (CH2Cl2/CH3OH
Charles Gauthier thanks Fondation Gaston-L. Tremb-
lay de l’UQAC for graduate scholarships and Jimmy
Bouchard for his help in organic synthesis.
20
D
9:1); mp >200 ꢁC; ½aꢁ +14.0 (c 1.00, CH3OH). 1H
NMR (C5D5N) d: 0.71, 0.81, 1.01, 1.07, 1.21, 1.78 (all
s, each 3H, H-23, H-24, H-25, H-26, H-27, H-30), 3.42
(dd, 1H, J = 11.6 Hz, J = 4.0 Hz, H-3), 3.53 (m, 1H,
H-19), 3.80 (d, 1H, J = 11.0 Hz, H0-5a), 4.18 (dd, 1H,
J = 8.7 Hz, J = 2.7 Hz, H0-3), 4.33 (br s, 1H, H0-4),
4.34 (d, 1H, J = 11.0 Hz, H0-5b), 4.39 (t, 1H,
J = 7.9 Hz, H0-2), 4.67 (d, 1H, J = 7.0 Hz, H0-1), 4.77
(br s, 1H, H-29a), 4.94 (br s, 1H, H-29b), 0.73-2.72
(all m, remaining protons). 13C NMR (C5D5N) d:
14.80, 16.20, 16.33, 16.86, 18.62, 19.40, 21.16, 23.84,
26.04, 28.53, 30.22, 31.15, 32.83, 34.71, 37.29, 37.56,
38.53, 38.78, 38.81, 41.08, 42.81, 47.75, 49.72, 50.76,
56.25, 56.60, 67.02 (C0-5), 69.58 (C0-4), 72.51 (C0-2),
74.75 (C0-3), 84.93 (C-3), 102.97 (C0-1), 109.96 (C-29),
151.30 (C-20), 178.82 (C-28). HR-ESI-MS m/z
611.3908 [M+Na]+ (calcd for C35H56O7Na, 611.3924).
References and notes
1. O’Connel, M. M.; Bently, M. D.; Campbell, C. S.; Cole, B.
J. W. Phytochemistry 1988, 27, 2175–2176.
2. Habiyaremye, I.; Stevanovic-Janezic, T.; Riedl, B.; Gar-
neau, F.-X.; Jean, F.-I. J. Wood Chem. Technol. 2002, 22,
83–91.
3. Cichewicz, R. H.; Kouzi, S. A. Med. Res. Rev. 2004, 24,
90–114.
4. Eiznhamer, D. A.; Xu, Z.-Q. IDrugs 2004, 7, 359–373.
´
5. Baglin, I.; Mitaine-Offer, A.-C.; Nour, M.; Tan, K.; Cave,
C.; Lacaille-Dubois, M.-A. Mini Rev. Med. Chem. 2003, 3,
159–165.
6. Setzer, W. N.; Setzer, M. C. Mini Rev. Med. Chem. 2003,
3, 540–556.
7. Geetha, T.; Varalakshmi, P. J. Ethnopharmacol. 2001, 76,
77–80.
4.31. Cell lines and culture conditions
8. Moriarity, D. M.; Huang, J.; Yancey, C. A.; Zhang, P.;
Setzer, W. N.; Lawton, R. O.; Bates, R. B.; Caldera, S.
Planta Med. 1998, 64, 370–372.
9. You, Y.-J.; Nam, N.-H.; Kim, Y.; Bae, K.-H.; Ahn, B.-Z.
Phytother. Res. 2003, 17, 341–344.
10. Aratanechemuge, Y.; Hibasami, H.; Sanpin, K.; Katsu-
zaki, H.; Imai, K.; Komiya, T. Oncol. Rep. 2004, 11, 289–
292.
11. Kim, D. S. H. L.; Chen, Z.; Nguyen, V. T.; Pezzuto, J. M.;
Qiu, S.; Lu, Z.-Z. Synth. Commun. 1997, 27, 1607–1612.
12. Pichette, A.; Liu, H.; Roy, C.; Tanguay, S.; Simard, F.;
Lavoie, S. Synth. Commun. 2004, 34, 3925–3937.
13. Safayhi, H.; Sailer, E.-R. Planta Med. 1997, 63, 487–493.
14. Steele, J. C. P.; Warhurst, D. C.; Kirby, G. C.; Simmonds,
M. S. J. Phytother. Res. 1999, 13, 115–119.
Human lung carcinoma (A-549), human colon adeno-
carcinoma (DLD-1), human normal fibroblasts (WS1),
and mice melanoma (B16-F1) cell lines were obtained
from the American Type Culture Collection (ATCC).
All cell lines were cultured in minimum essential medi-
um containing Earle’s salts and L-glutamine (Mediatech
Cellgro, VA), to which were added 10% foetal bovine
serum (Hyclone), vitamins (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 environ-
ment containing 5% CO2.
4.32. Cytotoxicity assay
15. Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino,
L. M.; Ballas, L. M.; Jiang, J. B.; Janzen, W. P.; Chen, I.-
S.; Lee, K. H. J. Nat. Prod. 1994, 57, 243–247.
16. Pisha, E.; Chai, H.; Lee, I.-S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
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

C. Gauthier et al. / Bioorg. Med. Chem. 14 (2006) 6713–6725
34. Smith, T. A. Br. J. Biomed. Sci. 1999, 56, 285–292.
6725
Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani,
M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.;
Pezzuto, J. M. Nat. Med. 1995, 1, 1046–1051.
17. Fulda, S.; Friesen, C.; Los, M.; Scaffidi, C.; Mier, W.;
Benedict, M.; Nunez, G.; Krammer, P. H.; Peter, M. E.;
Debatin, K. M. Cancer Res. 1997, 57, 4956–4964.
18. Fulda, S.; Jeremias, I.; Steiner, H. H.; Pietsch, T.; Debatin,
K.-M. Int. J. Cancer 1999, 82, 435–441.
35. Hiroya, K.; Takahashi, T.; Miura, N.; Naganuma, A.;
Sakamoto, T. Bioorg. Med. Chem. 2002, 10, 3229–3236.
36. Xu, Y.-C.; Bizuneh, A.; Walker, C. J. Org. Chem. 1996,
61, 9086–9089.
37. Mal, D. Synth. Commun. 1986, 16, 331–335.
´
38. Ple, K.; Chwalek, M.; Voutquenne-Nazabadioko, L. Eur.
J. Org. Chem. 2004, 1588–1603.
´
39. Trujillo, M.; Morales, E. Q.; Vazquez, J. T. J. Org. Chem.
1994, 59, 6637–6642.
40. Deng, S.; Yu, B.; Xie, J.; Hui, Y. J. Org. Chem. 1999, 64,
7265–7266.
41. Fukase, K.; Winarno, H.; Kusumoto, S. Chem. Express
1993, 8, 409–412.
42. Ziegler, T.; Bien, F.; Jurisch, C. Tetrahedron: Asymmetry
1998, 9, 765–780.
43. Schmidt, R. R. Adv. Carbohydr. Chem. Biochem. 1994, 50,
21–123.
44. Urban, F. J.; Moore, B. S.; Breitenbach, R. Tetrahedron
Lett. 1990, 31, 4421–4424.
45. Agrawal, P. K. Phytochemistry 1992, 31, 3307–3330.
46. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J.
Biochem. 2000, 267, 5421–5426.
19. Zuco, V.; Supino, R.; Righetti, S. C.; Cleris, L.; Marchesi,
E.; Gambacorti-Passerini, C.; Formelli, F. Cancer Lett.
2002, 175, 17–25.
20. Kim, J. Y.; Koo, H.-M.; Kim, D. S. H. L. Bioorg. Med.
Chem. Lett. 2001, 11, 2405–2408.
21. Kim, D. S. H. L.; Pezzuto, J. M.; Pisha, E. Bioorg. Med.
Chem. Lett. 1998, 8, 1707–1712.
22. Jeong, H.-J.; Chai, H.-B.; Park, S.-Y.; Kim, D. S. H. L.
Bioorg. Med. Chem. Lett. 1999, 9, 1201–1204.
23. Kvasnica, M.; Sarek, J.; Klinitova, E.; Dzubak, P.;
Hadjduch, M. Bioorg. Med. Chem. 2005, 13, 3447–3454.
24. Mukherjee, R.; Jaggi, M.; Siddiqui, M. J. A.; Srivastava,
S. K.; Rajendran, P.; Vardhan, A.; Burman, A. C. Bioorg.
Med. Chem. Lett. 2004, 14, 4087–4091.
25. Symon, A. V.; Veselova, N. N.; Kaplun, A. P.; Vlasenk-
ova, N. K.; Federova, G. A.; Lyutik, A. I.; Gerasimova,
G. K.; Shvets, V. I. Russ. J. Bioorg. Chem. 2005, 31, 320–
325.
47. Unpublished results.
48. Coulson, D. R. Inorg. Synth. 1972, 13, 121–124.
49. Connolly, J. D.; Hill, R. A.. In Dictionary of Triterpenoids.
Di- and Higher Terpenoids; Chapman and Hall: Cam-
bridge, 1991; Vol. 2, p 1460.
26. Ohara, S.; Hishiyama, S. Mokuzai Gakkaishi 1994, 40,
444–451.
´
ˇ ˇ
´
27. Klinotova, E.; Krecek, V.; Klinot, J.; Endova, M.;
Eisenreichova, J.; Budesınsky, M.; Stıcha, M. Collect.
50. Tinto, W. F.; Blair, L. C.; Alli, A. J. Nat. Prod. 1992, 55,
395–398.
´
ˇ
´
´
ˇˇ
´
Czech. Chem. C 1997, 62, 1776–1798.
51. Ziegler, H. L.; Franzyk, H.; Sairafianpour, M.; Tabatabai,
M.; Tehrani, M. D.; Bagherzadeh, K.; Ha¨gerstrand, H.;
Staerk, D.; Jaroszewski, J. W. Bioorg. Med. Chem. 2004,
12, 119–127.
28. Chatterjee, P.; Pezzuto, J. M.; Kouzi, S. A. J. Nat. Prod.
1999, 62, 761–763.
29. Baglin, I.; Poumaroux, A.; Nour, M.; Tan, K.; Mitaine-
´
Offer, A. C.; Lacaille-Dubois, M. A.; Chauffert, B.; Cave,
52. Kojima, H.; Tominaga, H.; Sato, S.; Ogura, H. Phyto-
chemistry 1987, 26, 1107–1111.
C. J. Enzyme Inhib. Med. Chem. 2003, 18, 111–117.
30. Samoshina, N. F.; Denisenko, M. V.; Denisenko, V. A.;
Uvarova, N. I. Chem. Nat. Compd. 2003, 39, 575–582.
31. Konoshima, T. In Saponins Used in Traditional and
Modern Medicine; Plenum Press: New York, 1996; pp
87–100.
53. Takeoka, G.; Dao, L.; Teranishi, R.; Wong, R.; Flessa, S.;
Harden, L.; Edwards, R. J. Agric. Food Chem. 2000, 48,
3437–3439.
54. Yagi, A.; Okamura, N.; Haraguchi, Y.; Noda, K.;
Nishioka, I. Chem. Pharm. Bull. 1978, 26, 1798–1802.
55. D’Accorso, N. B.; Thiel, I. M. E. Carbohydr. Res. 1983,
124, 177–184.
32. Sparg, S. G.; Light, M. E.; van Staden, J. J. Ethnophar-
macol. 2004, 94, 219–243.
ˇ
´ ´
33. Kren, V.; Martınkova, L. Curr. Med. Chem. 2001, 8,
56. Salinas, A. E.; Sproviero, J. F.; Deulofeu, V. Carbohydr.
Res. 1987, 170, 71–99.
1313–1338.