Synthesis and cytotoxicity of bidesmosidic betulin and betulinic acid saponins

Article abstract of DOI:10.1021/np800579x

The naturally occurring cytotoxic saponin 28-O-β-D- glucopyranosylbetulinic acid 3β-O-α-L-arabinopyranoside (3) was easily synthesized along with seven bidesmosidic saponins starting from the lupane-type triterpenoids betulin (1) and betulinic acid (2). A

Full text of DOI:10.1021/np800579x

72
J. Nat. Prod. 2009, 72, 72–81
Synthesis and Cytotoxicity of Bidesmosidic Betulin and Betulinic Acid Saponins
Charles Gauthier, Jean Legault, Serge Lavoie, Simon Rondeau, Samuel Tremblay, and Andre´ Pichette*
Laboratoire d’Analyse et de Se´paration des Essences Ve´ge´tales (LASEVE), De´partement des Sciences Fondamentales, UniVersite´ du Que´bec a`
Chicoutimi, 555 BouleVard de l’UniVersite´, Chicoutimi, Que´bec, Canada, G7H 2B1
ReceiVed September 14, 2008
The naturally occurring cytotoxic saponin 28-O-ꢀ-D-glucopyranosylbetulinic acid 3ꢀ-O-R-L-arabinopyranoside (3) was
easily synthesized along with seven bidesmosidic saponins starting from the lupane-type triterpenoids betulin (1) and
betulinic acid (2). As highlighted by the preliminary cytotoxicity evaluation against A549, DLD-1, MCF7, and PC-3
human cancer cell lines, the bidesmosidic betulin saponin 22a, bearing R-L-rhamnopyranoside moieties at both C-3 and
C-28 positions, was determined to be a potent cytotoxic agent (IC50 1.8-1.9 µM).
Bidesmosidic saponins are naturally occurring compounds that
consist of a triterpenoid or steroid aglycone bearing two sugar
moieties usually at the C-3 and C-28 positions.1 Biological activities
exhibited by saponins are quite diversified (cytotoxic, antitumor,
anti-inflammatory, molluscicidal) and have been reviewed exten-
sively.2 However, clinical development of saponins as pharmaco-
logical agents is strongly hampered because of their hemolytic
activity, inducing toxicity in most animals when delivered intra-
venously.3 Interestingly, it has been reported that bidesmosidic
saponins are considerably less hemolytic compared to monodes-
mosides4 and thus represent attractive chemical targets for
structure-activity relationship (SAR) studies.
The first synthesis of a bidesmosidic saponin was achieved by
the group of Biao Yu5 in 1999. Since this accomplishment, several
syntheses of bidesmosides have been published, although most of
them are solely related to diosgenin6-8 or oleanolic acid5,9-12 as
aglycones. Betulin (1) and betulinic acid (2) are cytotoxic lupane-
type triterpenoids widely distributed in nature.13,14 Synthesis of
monodesmosidic lupane-type saponins has been reported by us15,16
and by other groups.17-22 However, to our knowledge, the only
example of the synthesis of betulinic acid bidesmosides is the
preparation of the 3,28-bis-ꢀ-D-glucopyranoside derivative.20 Natu-
ral bidesmosidic saponins of the lupane-type are scarce and have
been isolated principally from plant species of the Schefflera23-25
and Pulsatilla26-28 genera. Braca and co-workers25 isolated the
3ꢀ-O-(R-L-arabinopyranosyl)lup-20(29)-ene-28-O-ꢀ-D-glucopyra-
nosyl ester (3) from the aerial parts of S. rotundifolia, a plant used
as a folk remedy in Asian countries. Bidesmosidic saponin 3
exhibited noticeable cytotoxic activity against J774.A1, HEK-293,
and WEHI-164 cell lines and was found, in this study, to be more
active than glycosides having oleanolic acid or hederagenin as
aglycones.
dazole and 4-dimethylaminopyridine (DMAP) in refluxing tetrahy-
drofuran (THF) to give 4 (90%) protected at the C-28 primary
hydroxyl position.7 The latter was glycosylated with the known
2,3,4-tri-O-benzoyl-ꢀ-L-arabinopyranosyl trichloroacetimidate (5)5
or 2,3,4-tri-O-R-L-rhamnopyranosyl trichloroacetimidate (6)29 under
the promotion of the Lewis acid trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) in dry CH2Cl2 at room temperature to afford
protected monodesmosides 7a and 7b in yields of 71% and 76%,
respectively. Desilylation of 7a and 7b under standard conditions,7
i.e., tetrabutylammonium bromide (TBAF) and acetic acid (HOAc)
in refluxing THF, readily furnished benzoylated betulin saponins
8a (75%) and 8b (87%). Since the next step consisted in the
glucosylation at the C-28 position, we tried to couple 2,3,4,6-tetra-
O-benzoyl-R-D-glucopyranosyl trichloroacetimidate (9)30 with 8a
using the above-mentioned glycosylation conditions. However, the
reaction afforded the rearrangement product allobetulin 3ꢀ-O-2,3,4-
tri-O-benzoyl-R-L-arabinopyranoside in 42% yield with no trace
of the desired bidesmosidic glycoside 10a. Similar treatment of
the acceptor 8b with the donor 9 led to the exclusive formation of
We now report the synthesis of the natural bidesmosidic betulinic
acid saponin 3 along with seven other bidesmosides (16a, 16b, 19,
21a, 21b, 22a, and 22b) containing D-glucose, L-rhamnose, and
L-arabinose moieties starting from the parent triterpenoids betulin
(1) and betulinic acid (2). The in Vitro cytotoxic activity of the
synthesized saponins was evaluated against human cancer cell lines
(A549, DLD-1, MCF7, and PC-3).
Results and Discussion
In order to synthesize bidesmosidic betulin saponins, we first
planned to introduce arabinopyranosyl or rhamnopyranosyl moieties
at the C-3 position of 1 prior to glucosylating the C-28 position.
As depicted in Scheme 1, betulin (1)15 was treated with tert-
butyldiphenylsilyl chloride (TBDPSCl) in conjunction with imi-
* Corresponding author. Tel: +1 418 545-5011. Fax: +1 418 545-5012.
E-mail: andre_pichette@uqac.ca.
10.1021/np800579x CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/30/2008

Bidesmosidic Betulin and Betulinic Acid Saponins
Journal of Natural Products, 2009, Vol. 72, No. 1 73
Scheme 1. Attempts to Synthesize Bidesmosidic Betulin Saponins (10a and 10b)a
a A: donor 9 (1.5 equiv), TMSOTf, CH2Cl2, 4 Å molecular sieves, rt, 16 h; B: inverse procedure, donor 9 (1.5 equiv), TMSOTf, CH2Cl2, 4 Å molecular sieves, -10
°C to rt, 2.5 h; C: donor 12 (1.5 equiv), AgOTf, CH2Cl2, 4 Å molecular sieves, -78 to 0 °C, 2 h; D: donor 11 (1.5 equiv), BF3 · OEt2, CH2Cl2, 4 Å molecular sieves,
-78 to 0 °C, 24 h; E: donor 12 (1.3 equiv), K2CO3, Bu4NBr, CH2Cl2/H2O 1:1, reflux, 5 h.
the trans-esterification product 28-O-benzoylbetulin 3ꢀ-O-2,3,4-
tri-O-benzoyl-R-L-rhamnopyranoside in 42% yield. As shown in
Scheme 1, further modifications of the glycosylation conditions were
considered using the acceptor 8b in conjunction with various
glucosyl donors (9, 11, and 12) and promoters such as boron
trifluoride diethyl etherate (BF3 ·OEt2) and silver trifluoromethane-
sulfonate (AgOTf). Both Schmidt’s inverse procedure31 and phase-
transfer conditions32were also tried in order to glucosylate the C-28
position of 8b. Unfortunately, all these attempts failed to yield the
target bidesmoside 10b. Instead, rapid decomposition of sugar donor
(9, 11, and 12) was generally observed on the basis of TLC analysis.
It is worth noting that 8b was nearly quantitatively transformed into
allobetulin 3ꢀ-O-2,3,4-tri-O-benzoyl-R-L-rhamnopyranoside when the
Lewis acid AgOTf was used as promoter of the glycosylation reaction.
The yields of the rearrangement were comparable to those reported
by Li and co-workers for the preparation of allobetulin from betulin
(1) catalyzed by solid acids.33
Therefore, we turned to another approach for the synthesis of
bidesmosidic betulin saponins. According to Scheme 2, the known
betulin 3-acetate (13)16 was prepared in good yield (86%, two steps)
from 1 following a reported procedure. Once again, attempts to
glucosylate the acceptor 13 with 9 under the catalytic action of
TMSOTf (0.1 equiv) in dry CH2Cl2 20 mL mmol-1 afforded
rearrangement products (allobetulin 3-acetate, 30% yield) and trans-
esterification (28-O-benzoylbetulin 3-acetate, 17% yield) instead
of the desired glycoside 14. However, condensation of 13 and 9
proceeded smoothly to furnish 14 (60% yield) when only 0.05 equiv
of TMSOTf was used in 40 mL mmol-1 of dry CH2Cl2. Thereafter,
deacetylation of the C-3 position was achieved by treatment of 14
with acetyl chloride (AcCl)34 in dry CH2Cl2/MeOH (1:2) to afford
15 in good yield (75%). The latter acceptor was coupled with the
donor 5 or 6 using TMSOTf as the promoter to give the fully
benzoylated bidesmosides 10a (62%) and 10b (72%), which were
deprotected using standard conditions (NaOH, MeOH/THF/H2O,
1:2:1) to provide the target bidesmosidic betulin saponins 16a and
16b in excellent yields (86% and 80%, respectively). The overall
yields for the syntheses were 24% for 16a and 26% for 16b over
four linear steps starting from betulin 3-acetate (13).
Synthesis of the natural bidesmosidic betulinic acid saponin 3
along with the non-natural saponin 19 was achieved in a straight-
forward manner. As depicted in Scheme 3, the lupane-type
triterpenoid betulinic acid (2) was condensed with the known donor
2,3,4,6-tetra-O-benzoyl-R-D-glucopyranosyl bromide (12)35 under
phase-transfer conditions32 using K2CO3 and TBAF in a refluxing
solution of CH2Cl2/H2O (1:1) to furnish 17 in excellent yield (90%).
The latter was coupled with the donor 5 or 6 under the promotion
of TMSOTf to afford 18a (63%) and 18b (86%). Subsequent
removal of the benzoyl groups by treatment with NaOH in MeOH/
THF/H2O provided the target bidesmosidic saponins 3 (75%) and
19 (81%). The overall yields for the syntheses were 43% for 3 and
63% for 19 over three linear steps starting from 1. Unexpectedly,
it was found that the physical and analytical data (1H NMR, 13C
NMR, and [R]D) of saponin 3 were not in agreement with those
reported for the natural product isolated from S. rotundifolia.25
Indeed, under the same NMR experimental conditions (300 K,
MeOD), the chemical shifts and coupling constants of the sugar
moieties of saponin 3 were different from those of the isolated
compound (Table 1). Recently, we also found such differences in
NMR spectral data during the synthesis of a structurally similar
betulinic acid saponin isolated by Braca and co-workers.36 High-
resolution electrospray ionization mass spectra (HRESIMS) and
extensive 1D and 2D NMR analyses (1H, 13C, DEPT-135, COSY,
TOCSY, HSQC, and HMBC) further proved that the structure of
the synthetic saponin 3 was correct.

74 Journal of Natural Products, 2009, Vol. 72, No. 1
Gauthier et al.
Scheme 2. Completion of the Synthesis of Bidesmosidic Betulin Saponins (16a and 16b)
Scheme 3. Synthesis of Bidesmosidic Betulinic Acid Saponins (3 and 19)
Surprisingly, glucosylation at the C-3 position of 28-O-2,3,4,6-
tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulinic acid (17) proved to be
very difficult. In fact, as shown in Scheme 4, all attempts to
condense the acceptor 17 with either the trichloroacetimidate sugar
donor 9 under Schmidt’s normal37 and inverse procedure31 or the
bromide sugar donor 12 in conjunction with silver oxide (Ag2O)38
and AgOTf39 (modified Koenigs-Knorr methods) failed to yield
the fully protected bidesmosidic betulinic acid saponin 20. Ac-
cording to TLC and NMR analysis, no coupling product was
observed in any assays and the acceptor 17 was nearly fully
recovered. Thus, we chose to adopt another strategy in which the
unprotected betulin (1) and betulinic acid (2) are glycosylated at
both C-3 and C-28 positions via Schmidt’s inverse procedure31
(Scheme 5). Using this methodology, the acceptors (1 or 2) and
the promoter (TMSOTf) were premixed before the dropwise
addition of the sugar donors (6 or 9, 3 equiv) at low temperature
(-10 °C). Deprotection of the crude product (NaOH, MeOH/THF/
H2O) and purification by C-18 inversed phase flash chromatography
afforded the target saponins (21a, 21b, 22a, and 22b) in yields
ranging from 37% to 84% over two steps. As expected, the 1,2-
trans-glycosidic linkage (R-L-rhamnoside and ꢀ-D-glucoside) of
1
saponins was clearly proved by H NMR analysis (δ 4.98, d, J1,2
7.8 Hz and δ 4.30, d, J1,2 7.6 Hz, H-1′ for 21a and 21b; δ 4.76, br
s and δ 4.72, d, J1,2 1.3 Hz, H-1′ for 22a and 22b).40

Bidesmosidic Betulin and Betulinic Acid Saponins
Journal of Natural Products, 2009, Vol. 72, No. 1 75
Table 1. Comparison of 1H and 13C NMR Spectral Data (300
residues, was more cytotoxic than betulinic acid (2) against MCF7
and PC-3 cell lines (IC50 9.5 and 5.3 µM, respectively).
K, MeOD, sugar moieties) and [R]25 between Synthetic and
D
Isolated Saponin 3
In this SAR study, the most active saponins were generally those
bearing R-L-rhamnopyranoside moieties. Indeed, bidesmosides 16b,
19, 22a, and 22b inhibited the growth of human cancer cell lines
with IC50 values ranging from 1.7 to 23 µM. Saponins 22a and
22b, containing an R-L-rhamnopyranoside moiety at both C-3 and
C-28 positions, were highly cytotoxic against all tested cancer cell
lines (IC50 1.7-1.9 and 6.0-7.2 µM, respectively) and significantly
more active than their parent triterpenes (P < 0.05). Notably,
bidesmosidic betulin saponin 22a was the most potent of all tested
compounds to inhibit the growth of human cancer cell lines. The
increase in cytoxicity correlated with the presence of rhamnose
moieties was also reported in the literature for solasodine steroidal
glycosides.46,47 It was suggested that certain types of cancer cells
may have protein receptors, such as lectins,48-50 that recognize
rhamnose moieties and facilitate movement of the drug into the
cellular cytoplasm.46 Thus, these rhamnose receptors could serve
to deliver the anticancer agent directly to the tumor.51-53
In summary, eight bidesmosidic saponins (3, 16a, 16b, 19, 21a,
21b, 22a, and 22b) were synthesized in moderate to good overall
yields starting from betulin (1) and betulinic acid (2). The syntheses
were achieved by a combination of Schmidt’s procedures and phase-
transfer conditions using fully benzoylated trichloroacetimidate and
sugar bromide donors. This SAR study suggests that the relative
cytotoxicities of bidesmosidic betulin and betulinic acid saponins
are strongly influenced by the nature of both the aglycone and the
sugar moieties. Bidesmosides 22a and 22b bearing R-L-rhamnopy-
ranosyl moieties at both C-3 and C-28 positions were highly
cytotoxic. Therefore, these preliminary results indicate that bides-
mosidic saponins having betulin (1) or betulinic acid (2) as the
aglycone may have clinical potential as anticancer agents. The
relatively high polarity of these compounds should facilitate
the preparation of nontoxic injectable formulations for further in
ViVo studies on animal models. Work on the evaluation of the
hemolytic activity and the mechanism of action of these new “lead”
compounds (22a and 22b) is currently in progress in our laboratory,
and results will be reported in due course.
synthetic saponina
δH δC
(position) (ppm) J (Hz) (ppm) δH (ppm)
isolated saponinb
sugar
J
δC
(Hz)
(ppm)
Ara (1)
Ara (2)
Ara (3)
Ara (4)
Ara (5a)
Ara (5b)
Glc (1)
Glc (2)
Glc (3)
Glc (4)
Glc (5)
Glc (6a)
Glc (6b)
4.26 d (6.6) 107.1
4.50
3.50
3.66
4.02
3.90
3.60
5.40
3.42
3.49
3.39
3.41
3.87
3.61
d (6.8)
dd (8.5,6.8)
dd (8.5, 3.0)
m
dd (12.0, 2.0)
dd (12.0, 3.0)
d (7.5)
dd (9.0, 7.5)
t (9.0)
t (9.0)
105.2
72.4
75.2
70.5
66.0
3.54
3.50
3.79
3.81
3.51
m
m
m
m
m
72.8
74.3
69.5
66.3
5.49 d (8.1)
95.2
74.1
78.4
71.1
78.8
62.4
95.6
74.2
77.9
71.0
78.1
62.2
3.32
3.37
3.37
3.37
3.84
3.70
m
m
m
m
m
m
m
dd (12.0, 3.0)
dd (12.0, 5.0)
+93 (c 0.1, MeOH)
[R]25
+12.2 (c 0.1, MeOH)
D
a Synthetic product of the present work (NMR 400 MHz). b Spectral
data of ref 25 (NMR 600 MHz).
Scheme 4. Attempts to Synthesize Benzoylated Bidesmosidic
Betulinic Saponins (20)a
a A: donor 9 (1.5 equiv), TMSOTf, CH2Cl2, 4 Å molecular sieves, rt, 16 h;
B: inverse procedure, donor 9 (3 equiv), TMSOTf, CH2Cl2, 4 Å molecular sieves,
-10 °C to rt, 3.5 h; C: donor 12 (1.5 equiv), Ag2O, CH3CN/CH2Cl2, 4 Å
molecular sieves, rt, 4 days; D: donor 12 (1.5 equiv), AgOTf, CH2Cl2, 4 Å
molecular sieves, 0 to 16 °C, 2 h.
In Vitro cytotoxic activity of bidesmosidic saponins was evaluated
against four human cancer cell lines including lung carcinoma
(A549) and colorectal (DLD-1), breast (MCF7), and prostate (PC-
3) adenocarcinomas.41 The parent triterpenoids betulin (1)15 and
betulinic acid (2)42 and the clinically used etoposide were used as
positive controls. The cytotoxicity of 28-O-ꢀ-D-glucopyranosides
of betulin15 and betulinic acid18 was also investigated. Moreover,
cytotoxic activity was assessed against human normal skin fibro-
blasts (WS1), but no selectivity was observed for the new series of
bidesmosidic saponins.
Experimental Section
General Experimental Procedures. Chemical reagents were pur-
chased from Sigma-Aldrich Co. Canada or Alfa Aesar Co. and were
used as received. Solvents were obtained from VWR International Co.
and were used as received. Air- and water-sensitive reactions were
performed in flame-dried glassware under argon. Moisture-sensitive
reagents were introduced via a dry syringe. Dichloromethane and
acetone were distilled from anhydrous CaH2 under argon. Tetrahydro-
furan (THF) was distilled from sodium/benzophenone ketyl under argon.
MeOH was distilled from Mg and I2 under argon. Analytical thin-layer
chromatography was performed with silica gel 60 F254, 0.25 mm
precoated TLC plates (Silicycle, Que´bec, Canada). Compounds were
visualized using UV254 and cerium molybdate (2 g Ce(SO4)4(NH4)4,
5 g MoO4(NH4)2, 200 mL H2O, 20 mL H2SO4) with charring. Flash
column chromatography was carried out using 230-400 mesh silica
gel (Silicycle, Que´bec, Canada). All chemical yields represent the
highest result obtained for at least three independent experiments. NMR
spectra were recorded on a Bruker Avance spectrometer at 400 MHz
(1H) and 100 MHz (13C), equipped with a 5 mm QNP probe.
Elucidations of chemical structures were based on 1H, 13C, COSY,
TOCSY, HMBC, HSQC, and DEPT-135 experiments. Chemical shifts
are reported in ppm (δ) relative to tetramethylsilane (TMS). Optical
rotations were obtained at the sodium D line at ambient temperature
on a Rudolph Research Analytical Autopol IV automatic polarimeter.
High-resolution electrospray ionization mass spectra (HRESIMS) were
obtained at the Department of Chemistry, Universite´ de Montre´al,
Que´bec, Canada. Compound 1154 was synthesized from D-glucose.
Betulin (1) was extracted from the outer bark of Betula papyrifera
March. and recrystallized with an azeotropic mixture of 2-butanol/H2O
(37:13) to afford crude 1 with purity >95% according to GC-MS.
Betulinic acid (2) was purchased from Indofine Chemical Company
It had been shown in previous structure-activity relationship
(SAR) studies that the free C-28 carboxylic acid function is
important to preserve the cytotoxicity of betulinic acid (2).18,43-45
As revealed in Table 2, in our SAR study, this assertion was verified
for the two monodesmosidic betulin and betulinic acid saponins
bearing a single glucopyranoside moiety at C-28 (IC50 >100 µM).
On the other hand, the cytotoxicity profile of most of the synthesized
bidesmosidic saponins bearing an additional sugar moiety at C-3
was generally similar or higher than betulinic acid (2) against tested
cancer cell lines. Bidesmosidic saponins 21a and 21b were the sole
exceptions to this general tendency since the presence of ꢀ-D-
glucopyranoside moieties at both C-3 and C-28 positions seems to
have a detrimental effect on cytotoxicity. Nevertheless, saponins
21a and 21b were preferentially cytotoxic and significantly (P <
0.05) more active than betulinic acid (2) against breast adenocar-
cinoma (MCF7) cells (IC50 14.5 and 20 µM, respectively).
It is noteworthy that the natural bidesmosidic betulinic acid
saponin 3, which features an R-L-arabinopyranoside moiety at C-3,
was only moderately cytotoxic against the cancer lines (IC50 23-76
µM), whereas the betulin analogue 16a, bearing the same sugar

76 Journal of Natural Products, 2009, Vol. 72, No. 1
Gauthier et al.
Scheme 5. Synthesis of Bidesmosidic Saponins (21a, 21b, 22a, and 22b) by Schmidt’s “Inverse Procedure”
Table 2. Cytotoxicity (IC50) of Bidesmosidic Saponins against Cancer Cell Linesa
IC50 (µmol·L-1)
compd
R1
R2
CH2OH
A549
DLD-1
MCF7
PC-3
WS1
1
2
-
-
21a
21b
16a
3
16b
19
22a
22b
H
H
H
H
3.8 ( 0.1
10.3 ( 0.4
>100
6.6 ( 0.3
15.0 ( 0.3
>100
23.3 ( 0.5
41 ( 1
>100
>100
14.5 ( 0.9
20 ( 2
9.5 ( 0.8
23 ( 1
9.0 ( 0.7
5.7 ( 0.6
1.7 ( 0.2
6.0 ( 0.6
0.7 ( 0.1
17.9 ( 0.9
40 ( 2
3.6 ( 0.1
12 ( 1
>100
COOH
CH2O-ꢀ-D-Glc
COO-ꢀ-D-Glc
CH2O-ꢀ-D-Glc
COO-ꢀ-D-Glc
CH2O-ꢀ-D-Glc
COO-ꢀ-D-Glc
CH2O-ꢀ-D-Glc
COO-ꢀ-D-Glc
CH2O-R-L-Rha
COO-R-L-Rha
>100
>100
>100
>100
>100
ꢀ-D-Glc
ꢀ-D-Glc
R-L-Ara
R-L-Ara
R-L-Rha
R-L-Rha
R-L-Rha
R-L-Rha
>100
27 ( 2
>100
20 ( 2
35 ( 3
4.5 ( 0.3
50 ( 7
5.3 ( 0.4
9 ( 1
>100
>100
66 ( 3
>100
19 ( 2
5.3 ( 0.6
68 ( 7
76 ( 4
60 ( 5
16.8 ( 0.9
23 ( 1
10.6 ( 0.9
11.0 ( 0.5
1.9 ( 0.1
7.3 ( 0.3
27 ( 5
6.9 ( 0.4
11.2 ( 0.8
1.8 ( 0.1
7.2 ( 0.5
1.7 ( 0.2
1.9 ( 0.1
7.2 ( 0.5
1.2 ( 0.1
1.3 ( 0.1
4.9 ( 0.7
34 ( 4
Etoposide
a Glc, glucopyranose; Rha, rhamnopyranose; Ara, arabinopyranose.
Inc. 28-O-ꢀ-D-Glucopyranosylbetulin,15 28-O-ꢀ-D-glucopyranosylbetu-
linic acid,18 28-O-tert-butyldiphenylsilylbetulin (4),36 and betulin
3-acetate (13)16 were synthesized according to reported procedures.
28-O-tert-Butyldiphenylsilylbetulin 3ꢀ-O-2,3,4-tri-O-benzoyl-r-
L-arabinopyranoside (7a). The acceptor 4 (750 mg, 1.10 mmol) and
the donor 5 (1.00 g, 1.65 mmol) were stirred at room temperature in
anhydrous CH2Cl2 (16.5 mL, 15 mL ·mmol-1) with 4 Å molecular
sieves under argon during 60 min. Then, the promoter TMSOTf (12
µL, 0.055 mmol) was injected in the medium via a dry syringe while
keeping rigorous anhydrous conditions. The mixture was stirred 2.5 h
at room temperature and quenched by addition of Et3N (0.61 mL, 4.4
mmol). The solvents were evaporated under reduced pressure, then the
resulting oily residue was purified by flash chromatography (hexanes/
28-O-tert-Butyldiphenylsilylbetulin 3ꢀ-O-2,3,4-tri-O-benzoyl-r-
L-rhamnopyranoside (7b). This compound was prepared from the
acceptor 4 (500 mg, 0.734 mmol) and the donor 6 (684 mg, 1.10 mmol)
in the same manner as that described for compound 7a. Purification
by flash chromatography (isocratic hexanes/Et2O, 9:1) gave 7b (634
mg, 76%) as a white, crystalline powder: [R]25D +46.6 (c 0.5, CHCl3);
1H NMR (CDCl3, 400 MHz) δ 8.13-7.21 (25H, H-Ar), 5.84 (1H, dd,
J ) 10.1, 3.3 Hz, H-3′), 5.68 (1H, m, H-4′), 5.65 (1H, m, H-2′), 5.08
(1H, d, J ) 1.1 Hz, H-1′), 4.60 (1H, d, J ) 1.8 Hz, H-29), 4.53 (1H,
br s, H-29), 4.30 (1H, m, H-5′), 3.70 (1H, d, J ) 9.9 Hz, H-28), 3.34
(1H, d, J ) 9.9 Hz, H-28), 3.20 (1H, t, J ) 8.3 Hz, H-3), 2.27 (1H, td,
J ) 10.8, 5.6 Hz, H-19), 1.65 (3H, s, H-30), 1.33 (3H, d, J ) 6.2 Hz,
H-6′), 1.07 (9H, s, C(CH3)3), 1.06 (3H, s, H-23), 0.94 (3H, s, H-24),
0.94 (3H, s, H-27), 0.83 (3H, s, H-25), 0.72 (3H, s, H-26); 13C NMR
(CDCl3, 100 MHz) δ 165.8-165.6 (3 × CO), 150.8 (C-20), 135.7-127.6
(C-Ar), 109.4 (C-29), 99.7 (C-1′), 90.0 (C-3), 72.0 (C-4′), 71.2 (C-2′),
70.2 (C-3′), 66.8 (C-5′), 61.1 (C-28), 55.4 (C-5), 50.3 (C-9), 48.4 (C-
18), 48.4 (C-17), 47.8 (C-19), 42.6 (C-14), 40.8 (C-8), 39.1 (C-4), 38.6
(C-1), 37.2 (C-13), 36.9 (C-10), 34.5 (C-22), 34.1 (C-7), 29.9 (C-21),
29.5 (C-16), 28.3 (C-23), 27.0 (C-15), 26.9 (C(CH3)3), 25.6 (C-2), 25.1
(C-12), 20.8 (C-11), 19.4 (C(CH3)3), 19.1 (C-30), 18.3 (C-6), 17.6 (C-
6′), 16.4 (C-24), 16.1 (C-25), 15.7 (C-27), 14.7 (C-26); HRESIMS m/z
1161.6262 [M + Na]+ (calcd for C73H90O9SiNa, 1161.6246).
Et2O, 9:1 to 17:3) to afford 7a (874 mg, 71%) as a white, crystalline
1
powder: [R]25 +71.0 (c 1.0, CHCl3); H NMR (CDCl3, 400 MHz) δ
D
8.08-7.27 (25H, H-Ar), 5.78 (1H, dd, J ) 8.7, 6.5 Hz, H-2′), 5.68
(1H, m, H-4′), 5.60 (1H, dd, J ) 8.9, 3.5 Hz, H-3′), 4.78 (1H, d, J )
6.4 Hz, H-1′), 4.59 (1H, br s, H-29), 4.52 (1H, br s, H-29), 4.32 (1H,
dd, J ) 13.0, 3.8 Hz, H-5′), 3.86 (1H, dd, J ) 12.9, 1.8 Hz, H-5′),
3.68 (1H, d, J ) 9.9 Hz, H-28), 3.32 (1H, d, J ) 10.0 Hz, H-28), 3.13
(1H, dd, J ) 11.4, 4.8 Hz, H-3), 2.26 (1H, td, J ) 11.0, 5.6 Hz, H-19),
1.64 (3H, s, H-30), 1.06 (9H, s, C(CH3)3), 0.91 (3H, s, H-27), 0.77
(3H, s, H-23), 0.75 (3H, s, H-25), 0.68 (3H, s, H-26), 0.64 (3H, s,
H-24); 13C NMR (CDCl3, 100 MHz) δ 165.8-165.2 (3 × CO), 150.7
(C-20), 135.7-127.6 (C-Ar), 109.4 (C-29), 103.0 (C-1′), 90.1 (C-3),
70.8 (C-3′), 70.2 (C-2′), 68.7 (C-4′), 62.6 (C-5′), 61.0 (C-28), 55.5 (C-
5), 50.3 (C-9), 48.4 (C-18), 48.4 (C-17), 47.8 (C-19), 42.6 (C-14), 40.7
(C-8), 39.0 (C-4), 38.6 (C-1), 37.2 (C-13), 36.8 (C-10), 34.5 (C-22),
34.1 (C-7), 29.8 (C-21), 29.5 (C-16), 27.7 (C-23), 27.0 (C-15), 26.9
(C(CH3)3), 26.1 (C-2), 25.1 (C-12), 20.7 (C-11), 19.4 (C(CH3)3), 19.1
(C-30), 18.1 (C-6), 16.0 (C-24), 16.0 (C-25), 15.7 (C-26), 14.6 (C-
27); HRESIMS m/z 1147.6111 [M + Na]+ (calcd for C72H88O9SiNa,
1147.6090).
Betulin 3ꢀ-O-2,3,4-tri-O-benzoyl-r-L-arabinopyranoside (8a). To
a solution of 7a (200 mg, 0.178 mmol) in anhydrous THF (1.94 mL)
were added HOAc (224 µL, 3.91 mmol) and 1 M TBAF in THF (3.88
mL) at room temperature under argon. The mixture was refluxed
overnight or until TLC showed no remaining 7a. The mixture was
diluted with EtOAc, washed with H2O, dried over anhydrous MgSO4,
and filtered, and the solvents were evaporated under reduced pressure.
The residue was purified by flash chromatography (hexanes/Et2O, 9:1
to 3:2) to furnish 8a (117 mg, 75%): white, amorphous solid; [R]25
D
+103.6 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.09-7.27 (15H,

Bidesmosidic Betulin and Betulinic Acid Saponins
Journal of Natural Products, 2009, Vol. 72, No. 1 77
H-Ar), 5.77 (1H, dd, J ) 8.9, 6.5 Hz, H-2′), 5.67 (1H, m, H-4′), 5.60
(1H, dd, J ) 8.9, 3.5 Hz, H-3′), 4.78 (1H, d, J ) 6.5 Hz, H-1′), 4.68
(1H, d, J ) 1.8 Hz, H-29), 4.57 (1H, br s, H-29), 4.33 (1H, dd, J )
13.0, 3.8 Hz, H-5′), 3.88 (1H, dd, J ) 12.9, 1.9 Hz, H-5′), 3.78 (1H,
d, J ) 10.7 Hz, H-28), 3.32 (1H, d, J ) 10.7 Hz, H-28), 3.14 (1H, dd,
J ) 11.3, 4.8 Hz, H-3), 2.38 (1H, td, J ) 10.7, 5.6 Hz, H-19), 1.68
(3H, s, H-30), 0.98 (3H, s, H-26), 0.95 (3H, s, H-27), 0.80 (3H, s,
H-25), 0.76 (3H, s, H-23), 0.64 (3H, s, H-24); 13C NMR (CDCl3, 100
MHz) δ 165.8-165.2 (3 × CO), 150.4 (C-20), 133.3-128.3 (C-Ar),
109.7 (C-29), 103.0 (C-1′), 90.1 (C-3), 70.7 (C-3′), 70.2 (C-2′), 68.7
(C-4′), 62.6 (C-5′), 60.4 (C-28), 55.5 (C-5), 50.3 (C-9), 48.7 (C-18),
47.7 (C-17), 47.7 (C-19), 42.6 (C-14), 40.9 (C-8), 39.0 (C-4), 38.7
(C-1), 37.2 (C-13), 36.8 (C-10), 34.1 (C-7), 33.9 (C-22), 29.7 (C-21),
29.1 (C-16), 27.7 (C-23), 27.0 (C-15), 26.1 (C-2), 25.2 (C-12), 20.8
(C-11), 19.1 (C-30), 18.1 (C-6), 16.0 (C-25), 16.0 (C-24), 15.9 (C-
26), 14.7 (C-27).; HRESIMS m/z 909.4957 [M + Na]+ (calcd for
C56H70O9Na, 909.4912).
(1H, t, J ) 9.7 Hz, H-3′′), 5.67 (1H, t, J ) 9.7 Hz, H-4′′), 5.56 (1H,
dd, J ) 9.7, 7.8 Hz, H-2′′), 4.79 (1H, d, J ) 8.0 Hz, H-1′′), 4.64 (1H,
m, H-6′′), 4.63 (1H, m, H-29), 4.54 (1H, m, H-29), 4.53 (1H, m, H-6′′),
4.17 (1H, m, H-5′′), 3.66 (1H, d, J ) 8.9 Hz, H-28), 3.58 (1H, d, J )
8.9 Hz, H-28), 3.17 (1H, dd, J ) 11.0, 4.6 Hz, H-3), 2.27 (1H, m,
H-19), 1.63 (3H, s, H-30), 0.96 (3H, s, H-23), 0.83 (3H, s, H-26), 0.83
(3H, s, H-27), 0.77 (3H, s, H-25), 0.76 (3H, s, H-24); 13C NMR (CDCl3,
100 MHz) δ 166.1-165.0 (4 × CO), 150.4 (C-20), 133.4-128.3 (C-
Ar), 109.6 (C-29), 102.0 (C-1′′), 78.9 (C-3), 72.8 (C-3′′), 72.2 (C-5′′),
71.7 (C-2′′), 70.0 (C-4′′), 68.8 (C-28), 63.3 (C-6′′), 55.2 (C-5), 50.3
(C-9), 48.6 (C-18), 48.0 (C-19), 46.9 (C-17), 42.5 (C-14), 40.7 (C-8),
38.8 (C-4), 38.6 (C-1), 37.6 (C-13), 37.1 (C-10), 34.7 (C-22), 33.8
(C-7), 29.6 (C-21), 29.2 (C-16), 28.0 (C-23), 27.3 (C-2), 27.0 (C-15),
25.0 (C-12), 20.8 (C-11), 19.0 (C-30), 18.1 (C-6), 16.1 (C-25), 15.7
(C-26), 15.4 (C-24), 14.8 (C-27); HRESIMS m/z 1043.5295 [M + Na]+
(calcd for C64H76O11Na, 1043.5280).
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulin 3ꢀ-O-
2,3,4-tri-O-benzoyl-r-L-arabinopyranoside (10a). The acceptor 15
(150 mg, 0.147 mmol) and the donor 5 (134 mg, 0.220 mmol) were
stirred at room temperature in anhydrous CH2Cl2 (2.9 mL) with 4 Å
molecular sieves under argon during 60 min. The temperature was
lowered to 0 °C with an ice/water bath, then a solution of TMSOTf in
CH2Cl2 (100 µL, 150 mM) was injected in the medium via a dry syringe
while keeping rigorous anhydrous conditions. The mixture was stirred
3 h at room temperature and quenched by addition of Et3N (82 µL,
0.59 mmol). The solvents were evaporated under reduced pressure. The
residue was purified by flash chromatography (hexanes/EtOAc, 9:1 to
Betulin 3ꢀ-O-2,3,4-tri-O-benzoyl-r-L-rhamnopyranoside (10b).
This compound was prepared from 7b (200 mg, 0.176 mmol) in the
same manner as that described for compound 8a. Purification by flash
chromatography (hexanes/EtOAc, 9:1 to 3:2) gave 10b (138 mg, 87%):
white, crystalline powder; [R]25 +76.6 (c 1.0, CHCl3); 1H NMR
D
(CDCl3, 400 MHz) δ 8.13-7.23 (15H, H-Ar), 5.82 (1H, dd, J ) 10.2,
3.3 Hz, H-3′), 5.67 (1H, t, J ) 10.0 Hz, H-4′), 5.64 (1H, dd, J ) 3.3,
1.8 Hz, H-2′), 5.08 (1H, d, J ) 1.4 Hz, H-1′), 4.69 (1H, d, J ) 2.1 Hz,
H-29), 4.58 (1H, br s, H-29), 4.30 (1H, ddt, J ) 9.7, 6.2, 6.2 Hz, H-5′),
3.81 (1H, d, J ) 10.8 Hz, H-28), 3.34 (1H, d, J ) 10.8 Hz, H-28),
3.20 (1H, dd, J ) 8.7, 7.5 Hz, H-3), 2.39 (1H, td, J ) 10.5, 5.6 Hz,
H-19), 1.68 (3H, s, H-30), 1.33 (3H, d, J ) 6.2 Hz, H-6′), 1.05 (3H,
s, H-23), 1.04 (3H, s, H-26), 0.98 (3H, s, H-27), 0.93 (3H, s, H-24),
0.89 (3H, s, H-25); 13C NMR (CDCl3, 100 MHz) δ 165.9-165.6 (3 ×
CO), 150.5 (C-20), 133.4-128.3 (C-Ar), 109.7 (C-29), 99.7 (C-1′),
90.0 (C-3), 72.0 (C-4′), 71.2 (C-2′), 70.2 (C-3′), 66.8 (C-5′), 60.6 (C-
28), 55.5 (C-5), 50.4 (C-9), 48.8 (C-18), 47.8 (C-19), 47.8 (C-17), 42.7
(C-14), 41.0 (C-8), 39.2 (C-4), 38.7 (C-1), 37.3 (C-13), 36.9 (C-10),
34.2 (C-7), 34.0 (C-22), 29.8 (C-21), 29.2 (C-16), 28.3 (C-23), 27.0
(C-15), 25.7 (C-2), 25.2 (C-12), 20.9 (C-11), 19.1 (C-30), 18.3 (C-6),
17.6 (C-6′), 16.4 (C-24), 16.2 (C-25), 16.0 (C-26), 14.8 (C-27);
HRESIMS m/z 923.5111 [M + Na]+ (calcd for C57H72O9Na, 923.5069).
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulin 3-ace-
tate (14). This compound was prepared from the acceptor 13 (700 mg,
1.44 mmol) and the donor 9 (1.61 g, 2.17 mmol) in the same manner
as that described for compound 7a except for the molar volume of
CH2Cl2 (40 mL ·mmol-1). Purification by flash chromatography (hex-
3:2) to afford 10a (132 mg, 62%) as a white foam: [R]25 +80.3 (c
D
1
0.2, CHCl3); H NMR (CDCl3, 400 MHz) δ 8.10-7.25 (35H, H-Ar),
5.94 (1H, t, J ) 9.7 Hz, H-3′′), 5.78 (1H, dd, J ) 8.9, 6.7 Hz, H-2′),
5.68 (1H, m, H-4′), 5.67 (1H, m, H-4′′), 5.60 (1H, dd, J ) 9.1, 3.5 Hz,
H-3′), 5.56 (1H, dd, J ) 9.9, 8.0 Hz, H-2′′), 4.79 (1H, m, H-1′′), 4.78
(1H, m, H-1′), 4.65 (1H, m, H-6′′), 4.63 (1H, br s, H-29), 4.55 (1H, br
s, H-29), 4.53 (1H, m, H-6′′), 4.33 (1H, dd, J ) 13.0, 3.5 Hz, H-5′),
4.17 (1H, ddd, J ) 9.5, 5.4, 3.3 Hz, H-5′′), 3.87 (1H, dd, J ) 13.0, 1.9
Hz, H-5′), 3.66 (1H, d, J ) 8.8 Hz, H-28), 3.57 (1H, d, J ) 8.8 Hz,
H-28), 3.12 (1H, dd, J ) 11.3, 4.6 Hz, H-3), 2.28 (1H, m, H-19), 1.62
(3H, s, H-30), 0.81 (3H, s, H-27), 0.79 (3H, s, H-26), 0.76 (3H, s,
H-23), 0.76 (3H, s, H-25), 0.65 (3H, s, H-24), 0.58 (1H, d, J ) 10.8
Hz, H-5), 0.50 (1H, br d, J ) 13.5 Hz, H-15); 13C NMR (CDCl3, 100
MHz) δ 166.1-164.9 (7 × CO), 150.3 (C-20), 133.4-128.3 (C-Ar),
109.6 (C-29), 103.0 (C-1′), 102.0 (C-1′′), 90.0 (C-3), 72.8 (C-3′′), 72.1
(C-5′′), 71.7 (C-2′′), 70.7 (C-3′), 70.2 (C-2′), 70.0 (C-4′′), 68.9 (C-28),
68.7 (C-4′), 63.2 (C-6′′), 62.6 (C-5′), 55.4 (C-5), 50.2 (C-9), 48.5 (C-
18), 47.9 (C-19), 46.9 (C-17), 42.4 (C-14), 40.6 (C-8), 38.9 (C-4), 38.6
(C-1), 37.6 (C-13), 36.7 (C-10), 34.6 (C-22), 33.7 (C-7), 29.6 (C-21),
29.1 (C-16), 27.6 (C-23), 26.9 (C-15), 26.0 (C-2), 25.0 (C-12), 20.7
(C-11), 19.0 (C-30), 17.9 (C-6), 16.0 (C-24), 16.0 (C-25), 15.7 (C-
26), 14.7 (C-27); HRESIMS m/z 1487.6499 [M + Na]+ (calcd for
C90H96O18Na, 1487.6489).
anes/EtOAc, 4:1 to 7:3) gave 14 (903 mg, 60%): white foam; [R]25
D
+24.7 (c 0.2, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.06-7.26 (20H,
H-Ar), 5.93 (1H, t, J ) 9.7 Hz, H-3′′), 5.67 (1H, t, J ) 9.7 Hz, H-4′′),
5.56 (1H, dd, J ) 9.8, 8.0 Hz, H-2′′), 4.79 (1H, d, J ) 8.0 Hz, H-1′′),
4.65 (1H, m, H-6′′), 4.63 (1H, m, H-29), 4.55 (1H, m, H-29), 4.53
(1H, m, H-6′′), 4.45 (1H, m, H-3), 4.17 (1H, ddd, J ) 9.4, 5.6, 3.3 Hz,
H-5′′), 3.67 (1H, d, J ) 8.9 Hz, H-28), 3.58 (1H, d, J ) 8.9 Hz, H-28),
2.28 (1H, m, H-19), 2.05 (3H, s, CH3CO), 1.63 (3H, s, H-30), 0.84
(3H, s, H-23), 0.84 (3H, s, H-24), 0.83 (3H, s, H-26), 0.82 (3H, s,
H-27), 0.80 (3H, s, H-25); 13C NMR (CDCl3, 100 MHz) δ 171.1
(CH3CO), 166.2-165.3 (4 × CO), 150.4 (C-20), 133.5-128.3 (C-Ar),
109.7 (C-29), 102.1 (C-1′′), 80.9 (C-3), 72.9 (C-3′′), 72.2 (C-5′′), 71.8
(C-2′′), 70.1 (C-4′′), 68.9 (C-28), 63.3 (C-6′′), 55.3 (C-5), 50.2 (C-9),
48.6 (C-18), 48.0 (C-19), 47.0 (C-17), 42.5 (C-14), 40.7 (C-8), 38.3
(C-1), 37.8 (C-4), 37.6 (C-13), 37.0 (C-10), 34.7 (C-22), 33.8 (C-7),
29.6 (C-21), 29.2 (C-16), 28.0 (C-23), 27.0 (C-15), 25.0 (C-12), 23.7
(C-2), 21.4 (CH3CO), 20.8 (C-11), 19.0 (C-30), 18.1 (C-6), 16.5 (C-
24), 16.2 (C-25), 15.8 (C-26), 14.7 (C-27); HRESIMS m/z 1085.5384
[M + Na]+ (calcd for C66H78O12Na, 1085.5386).
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulin 3ꢀ-O-
2,3,4-tri-O-benzoyl-r-L-rhamnopyranoside (10b). This compound
was prepared from the acceptor 15 (17 mg, 0.017 mmol) and the donor
6 (16 mg, 0.025 mmol) in the same manner as that described for
compound 10a except for the concentration of the solution of TMSOTf
in CH2Cl2 (20 mM). Purification by flash chromatography (hexanes/
EtOAc, 9:1 to 3:1) gave 10b (18 mg, 72%) as a white foam: [R]25
D
+57.1 (c 0.2, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.15-7.24 (35H,
H-Ar), 5.95 (1H, t, J ) 9.7 Hz, H-3′′), 5.83 (1H, dd, J ) 10.2, 3.3 Hz,
H-3′), 5.68 (1H, m, H-4′′), 5.68 (1H, m, H-4′), 5.65 (1H, m, H-2′),
5.56 (1H, dd, J ) 9.9, 8.0 Hz, H-2′′), 5.07 (1H, d, J ) 1.3 Hz, H-1′),
4.80 (1H, d, J ) 8.1 Hz, H-1′′), 4.66 (1H, m, H-6′′), 4.63 (1H, m,
H-29), 4.55 (1H, m, H-29), 4.54 (1H, m, H-6′′), 4.32 (1H, dd, J ) 9.7,
6.0 Hz, H-5′), 4.18 (1H, ddd, J ) 9.4, 5.4, 3.3 Hz, H-5′′), 3.67 (1H, d,
J ) 9.1 Hz, H-28), 3.59 (1H, d, J ) 9.1 Hz, H-28), 3.18 (1H, t, J )
8.1 Hz, H-3), 2.29 (1H, m, H-19), 1.63 (3H, s, H-30), 1.33 (3H, d, J
) 6.2 Hz, H-6′), 1.04 (3H, s, H-23), 0.94 (3H, s, H-24), 0.85 (3H, s,
H-26), 0.84 (3H, s, H-25), 0.83 (3H, s, H-27); 13C NMR (CDCl3, 100
MHz) δ 166.2-165.0 (7 × CO), 150.4 (C-20), 133.4-128.3 (C-Ar),
109.6 (C-29), 102.1 (C-1′′), 99.7 (C-1′), 90.0 (C-3), 72.8 (C-3′′), 72.2
(C-5′′), 72.0 (C-4′′), 71.7 (C-2′′), 71.2 (C-2′), 70.2 (C-3′), 70.0 (C-4′),
68.9 (C-28), 66.8 (C-5′), 63.3 (C-6′′), 55.4 (C-5), 50.2 (C-9), 48.6 (C-
18), 48.0 (C-19), 46.9 (C-17), 42.5 (C-14), 40.7 (C-8), 39.1 (C-4), 38.6
(C-1), 37.6 (C-13), 36.8 (C-10), 34.7 (C-22), 33.8 (C-7), 29.6 (C-21),
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulin (15). To
a solution of 14 (840 mg, 0.790 mmol) in anhydrous CH2Cl2/MeOH
(1:2) (60 mL) was added AcCl (1.19 mL, 16.8 mmol) at 0 °C (ice/
water bath). The mixture was stirred overnight at room temprature or
until TLC (hexanes/EtOAc, 7:3) showed no remaining 14. Then, the
reaction was quenched with Et3N (4.68 mL, 33.6 mmol) and the solvents
were evaporated under reduced pressure. The residue was purified by
flash chromatography (hexanes/EtOAc, 4:1 to 3:2) to afford 15 (523
mg, 75%, corrected yield) as a white crystalline powder along with 14
(87 mg, 10%, recovery yield) as a white foam: [R]25 +27.0 (c 0.5,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.05-7.25 (20H, H-Ar), 5.93

78 Journal of Natural Products, 2009, Vol. 72, No. 1
Gauthier et al.
Table 3. 13C NMR Data of Bidesmosidic Saponins 3, 16a, 16b, 19, 21a, 21b, 22a, and 22ba
position
3b
16ab
16bb
19c
21ad
21bc
22ab
22bc
1
2
3
4
5
6
7
8
39.5 (t)
26.7 (t)
90.3 (d)
39.8 (s)
56.5 (d)
18.8 (t)
35.0 (t)
41.5 (s)
51.4 (d)
37.6 (s)
21.6 (t)
26.3 (t)
38.8 (d)
43.1 (s)
30.2 (t)
32.4 (t)
57.4 (s)
50.1 (d)
47.7 (d)
151.1 (s)
31.0 (t)
37.1 (t)
28.2 (q)
16.5 (q)
16.6 (q)
16.3 (q)
15.1 (q)
175.9 (s)
110.1 (t)
19.5 (q)
106.2 (d)
72.1 (d)
73.6 (d)
68.4 (d)
65.4 (t)
39.2 (t)
26.4 (t)
90.2 (d)
39.6 (s)
56.1 (d)
18.6 (t)
34.6 (t)
41.4 (s)
50.8 (d)
37.3 (s)
21.3 (t)
25.7 (t)
38.0 (d)
43.1 (s)
27.5 (t)
29.9 (t)
47.6 (s)
49.3 (d)
48.3 (d)
151.0 (s)
30.1 (t)
35.1 (t)
28.2 (q)
16.5 (q)
16.5 (q)
16.4 (q)
15.1 (q)
68.9 (t)
110.0 (t)
19.4 (q)
105.5 (d)
71.7 (d)
73.1 (d)
67.8 (d)
64.9 (t)
39.1 (t)
26.0 (t)
89.7 (d)
39.5 (s)
55.9 (d)
18.7 (t)
34.6 (t)
41.4 (s)
50.9 (d)
37.3 (s)
21.3 (t)
25.7 (t)
38.0 (d)
43.1 (s)
27.5 (t)
29.9 (t)
47.6 (s)
49.3 (d)
48.3 (d)
151.0 (s)
30.1 (t)
35.1 (t)
28.3 (q)
16.4 (q)
16.4 (q)
16.3 (q)
15.1 (q)
68.8 (t)
109.9 (t)
19.3 (q)
103.3 (d)
71.5 (d)
71.9 (d)
73.4 (d)
68.8 (d)
17.5 (q)
104.4 (d)
74.2 (d)
77.0 (d)
70.8 (d)
76.5 (d)
62.1 (t)
39.9 (t)
26.8 (t)
90.4 (d)
40.2 (s)
56.9 (d)
19.4 (t)
35.5 (t)
42.0 (s)
52.0 (d)
38.1 (s)
22.1 (t)
26.9 (t)
39.4 (d)
43.6 (s)
30.8 (t)
32.8 (t)
57.9 (s)
50.6 (d)
48.4 (d)
151.8 (s)
31.5 (t)
37.5 (t)
28.7 (q)
16.8 (q)
16.8 (q)
16.7 (q)
15.2 (q)
176.1 (s)
110.3 (t)
19.5 (q)
104.4 (d)
72.5 (d)
72.5 (d)
74.1 (d)
69.9 (d)
17.9 (q)
95.2 (d)
74.1 (d)
78.4 (d)
71.1 (d)
78.8 (d)
62.4 (t)
39.4 (t)
27.1 (t)
89.2 (d)
40.0 (s)
56.2 (d)
18.8 (t)
34.8 (t)
41.5 (s)
51.0 (d)
37.4 (s)
21.3 (t)
26.0 (t)
38.0 (d)
43.3 (s)
28.0 (t)
30.4 (t)
48.1 (s)
49.5 (d)
48.4 (d)
151.3 (s)
30.5 (t)
35.6 (t)
28.5 (q)
16.4 (q)
17.2 (q)
16.7 (q)
15.3 (q)
68.9 (t)
110.4 (t)
19.6 (q)
107.3 (d)
76.2 (d)
79.2 (d)
72.2 (d)
78.7 (d)
63.4 (t)
106.4 (d)
75.8 (d)
79.0 (d)
72.2 (d)
79.0 (d)
63.3 (t)
40.1 (t)
27.2 (t)
90.9 (d)
40.3 (s)
57.2 (d)
19.3 (t)
35.5 (t)
42.1 (s)
52.0 (d)
38.1 (s)
22.1 (t)
26.9 (t)
39.4 (d)
43.6 (s)
30.9 (t)
32.8 (t)
58.0 (s)
50.6 (d)
48.4 (d)
151.9 (s)
31.5 (t)
37.5 (t)
28.4 (q)
16.8 (q)
16.8 (q)
16.7 (q)
15.2 (q)
176.2 (s)
110.3 (t)
19.5 (q)
106.8 (d)
75.7 (d)
78.3 (d)
71.7 (d)
77.7 (d)
62.8 (t)
95.2 (d)
74.1 (d)
78.4 (d)
71.1 (d)
78.8 (d)
62.4 (t)
39.0 (t)
25.9 (t)
89.7 (d)
39.4 (s)
55.8 (d)
18.6 (t)
34.6 (t)
41.3 (s)
50.8 (d)
37.2 (s)
21.2 (t)
25.6 (t)
38.0 (d)
43.1 (s)
27.5 (t)
30.1 (t)
47.3 (s)
49.2 (d)
48.3 (d)
150.8 (s)
30.3 (t)
35.3 (t)
28.3 (q)
16.4 (q)
16.4 (q)
16.2 (q)
15.0 (q)
66.4 (t)
110.0 (t)
19.3 (q)
103.1 (d)
71.4 (d)
71.9 (d)
73.3 (d)
68.8 (d)
17.7 (q)
101.1 (d)
71.3 (d)
71.8 (d)
73.1 (d)
68.7 (d)
17.5 (q)
39.9 (t)
26.8 (t)
90.4 (d)
40.2 (s)
56.9 (d)
19.4 (t)
35.6 (t)
42.0 (s)
51.9 (d)
38.1 (s)
22.2 (t)
26.9 (t)
40.0 (d)
43.7 (s)
30.8 (t)
33.1 (t)
58.3 (s)
50.5 (d)
48.8 (d)
151.5 (s)
31.8 (t)
38.0 (t)
28.7 (q)
16.8 (q)
16.8 (q)
16.8 (q)
15.2 (q)
175.6 (s)
110.6 (t)
19.6 (q)
104.4 (d)
72.5 (d)
72.6 (d)
74.1 (d)
69.9 (d)
17.9 (q)
95.1 (d)
71.4 (d)
72.8 (d)
73.4 (d)
69.9 (d)
18.2 (q)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1‘
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
94.6 (d)
73.4 (d)
77.7 (d)
70.6 (d)
78.0 (d)
62.0 (t)
104.3 (d)
74.2 (d)
76.9 (d)
70.8 (d)
76.3 (d)
62.3 (t)
a Spectra recorded at 100 MHz. The multiplicities were deduced from DEPT experiments. b CDCl3/CD3OD. c CD3OD. d Pyridine-d5.
29.2 (C-16), 28.2 (C-23), 26.9 (C-15), 25.6 (C-2), 25.0 (C-12), 20.8
(C-11), 19.0 (C-30), 18.1 (C-6), 17.6 (C-6′), 16.4 (C-24), 16.1 (C-25),
15.7 (C-26), 14.8 (C-27); HRESIMS m/z 1501.6648 [M + Na]+ (calcd
for C91H98O18Na, 1501.6645).
[R]25D -42.8 (c 0.2, MeOH); 1H NMR (CDCl3/CD3OD, 1:1, 400 MHz)
δ 4.76 (1H, br s, H-1′), 4.68 (1H, br s, H-29), 4.58 (1H, br s, H-29),
4.25 (1H, d, J ) 7.8 Hz, H-1′′), 3.90 (1H, m, H-6′′), 3.89 (1H, m,
H-2′), 3.78 (1H, m, H-6′′), 3.75 (1H, m, H-5′), 3.70 (1H, m, H-28),
3.69 (1H, m, H-3′), 3.62 (1H, d, J ) 9.2 Hz, H-28), 3.43 (1H, m, H-4′′),
3.42 (1H, m, H-3′′), 3.39 (1H, m, H-4′), 3.31 (1H, m, H-5′′), 3.27 (1H,
m, H-2′′), 3.08 (1H, dd, J ) 11.4, 4.6 Hz, H-3), 2.43 (1H, m, H-19),
2.09 (1H, br d, J ) 12.1 Hz, H-16), 1.69 (3H, s, H-30), 1.27 (3H, d,
J ) 6.0 Hz, H-6′), 1.05 (3H, s, H-26), 0.99 (3H, s, H-27), 0.93 (3H, s,
H-23), 0.85 (3H, s, H-25), 0.76 (3H, s, H-24); 13C NMR (CDCl3/
CD3OD, 1:1, 100 MHz), see Table 3; HRESIMS m/z 773.4794 [M +
Na]+ (calcd for C42H70O11Na, 773.4810).
28-O-ꢀ-D-Glucopyranosylbetulin 3ꢀ-O-r-L-arabinopyranoside
(16a). To a solution of 10a (94 mg, 0.064 mmol) in MeOH/THF/H2O
(1:2:1) (4.4 mL) was added NaOH (52 mg, 1.3 mmol). The reaction
mixture was stirred 5 h at room temperature or until TLC (CH2Cl2/
MeOH, 9:1) showed no remaining 10a and then acidified to pH ≈ 4
with aqueous HCl 10%. The solvents were evaporated under reduced
pressure. The residue was purified by C-18 reversed-phase flash
chromatography (MeOH/H2O, 4:1 to 9:1) to furnish 16a (40 mg, 86%)
as a white, amorphous powder: [R]25D -15.6 (c 0.1, MeOH); 1H NMR
(CDCl3/CD3OD, 1:1, 400 MHz) δ 4.68 (1H, d, J ) 1.6 Hz, H-29),
4.58 (1H, br s, H-29), 4.34 (1H, d, J ) 5.9 Hz, H-1′), 4.25 (1H, d, J
) 7.8 Hz, H-1′′), 3.89 (1H, m, H-6′′), 3.88 (1H, m, H-4′), 3.88 (1H,
m, H-5′), 3.79 (1H, dd, J ) 11.9, 4.6 Hz, H-6′′), 3.68 (1H, m, H-28),
3.65 (1H, m, H-2′), 3.61 (1H, m, H-3′), 3.61 (1H, m, H-28), 3.53 (1H,
dd, J ) 13.8, 3.8 Hz, H-5′), 3.45 (1H, m, H-4′′), 3.44 (1H, m, H-3′′),
3.31 (1H, m, H-5′′), 3.27 (1H, m, H-2′′), 3.13 (1H, dd, J ) 11.3, 4.3
Hz, H-3), 2.43 (1H, td, J ) 10.3, 5.7 Hz, H-19), 1.69 (3H, s, H-30),
1.04 (3H, s, H-26), 1.01 (3H, s, H-23), 0.98 (3H, s, H-27), 0.84 (3H,
s, H-25), 0.82 (3H, s, H-24), 0.73 (1H, d, J ) 10.3 Hz, H-5); 13C NMR,
see Table 3; HRESIMS m/z 759.4635 [M + Na]+ (calcd for
C41H68O11Na, 759.4654).
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulinic Acid
(17). To a solution of the acceptor 2 (500 mg, 1.10 mmol) and the
donor 12 (939 mg, 1.42 mmol) in CH2Cl2 (12.7 mL) were added H2O
(12.7 mL), K2CO3 (378 mg, 2.74 mmol), and Bu4NBr (141 mg, 0.438
mmol). The resulting mixture was vigorously stirred and refluxed for
6 h. Then, the mixture was diluted with CH2Cl2 and washed with H2O
and brine. The solvents of the dried (MgSO4) organic solution were
evaporated under reduced pressure. The brown residue was purified
by flash chromatography (100% CH2Cl2 to CH2Cl2/MeOH, 49:1) to
afford 17 (1.015 g, 90%): white, crystalline powder; [R]25 +38.0 (c
D
1
0.5, CHCl3); H NMR (CDCl3, 400 MHz) δ 8.07-7.25 (20H, H-Ar),
6.03 (1H, d, J ) 8.4 Hz, H-1′′), 6.02 (1H, t, J ) 9.5 Hz, H-3′′), 5.76
(1H, dd, J ) 9.9, 8.4 Hz, H-2′′), 5.73 (1H, t, J ) 9.8 Hz, H-4′′), 4.71
(1H, br s, H-29), 4.59 (1H, m, H-6′′), 4.58 (1H, m, H-29), 4.48 (1H,
dd, J ) 12.2, 5.6 Hz, H-6′′), 4.29 (1H, ddd, J ) 9.5, 5.3, 2.9 Hz, H-5′′),
3.13 (1H, dd, J ) 11.0, 4.6 Hz, H-3), 2.93 (1H, td, J ) 11.1, 4.8 Hz,
H-19), 2.17 (1H, br d, J ) 13.2 Hz, H-16), 2.03 (1H, td, J ) 12.2, 3.2
Hz, H-13), 1.91 (1H, dd, J ) 12.7, 8.0 Hz, H-22), 1.63 (3H, s, H-30),
28-O-ꢀ-D-Glucopyranosylbetulin 3ꢀ-O-r-L-rhamnopyranoside
(16b). This compound was prepared from 10b (84 mg, 0.057 mmol)
in the same manner as that described for compound 16a. Purification
by C-18 reversed-phase flash chromatography (MeOH/H2O, 4:1, to
100% MeOH) gave 16b (33 mg, 80%): white, amorphous powder;

Bidesmosidic Betulin and Betulinic Acid Saponins
Journal of Natural Products, 2009, Vol. 72, No. 1 79
0.93 (3H, s, H-23), 0.79 (3H, s, H-27), 0.73 (3H, s, H-24), 0.68 (3H,
s, H-25), 0.60 (1H, br d, J ) 14.3 Hz, H-15), 0.54 (1H, br d, J ) 10.5
Hz, H-5), 0.47 (3H, s, H-26), 0.38 (1H, br d, J ) 11.0 Hz, H-7); 13C
NMR (CDCl3, 100 MHz) δ 174.0 (C-28), 166.1-164.7 (4 × CO), 150.3
(C-20), 133.5-128.3 (C-Ar), 109.5 (C-29), 91.4 (C-1′′), 78.9 (C-3),
73.0 (C-5′′), 72.8 (C-3′′), 70.3 (C-2′′), 69.4 (C-4′′), 62.7 (C-6′′), 56.8
(C-17), 55.2 (C-5), 50.4 (C-9), 49.1 (C-18), 46.6 (C-19), 42.2 (C-14),
40.2 (C-8), 38.8 (C-4), 38.6 (C-1), 38.0 (C-13), 37.0 (C-10), 36.3 (C-
22), 33.4 (C-7), 31.5 (C-16), 30.2 (C-21), 29.9 (C-15), 28.0 (C-23),
27.4 (C-2), 25.4 (C-12), 20.7 (C-11), 19.5 (C-30), 18.0 (C-6), 16.0
(C-25), 15.4 (C-26), 15.4 (C-24), 14.5 (C-27); HRESIMS m/z 1057.5114
[M + Na]+ (calcd for C64H74O12Na, 1057.5073).
H-3′), 3.52 (1H, d, J ) 10.3 Hz, H-5′), 3.46 (1H, m, H-3′′), 3.42 (1H,
m, H-4′′), 3.41 (1H, m, H-5′′), 3.37 (1H, m, H-2′′), 3.13 (1H, dd, J )
11.1, 4.0 Hz, H-3), 3.00 (1H, td, J ) 11.0, 4.6 Hz, H-19), 1.69 (3H, s,
H-30), 1.01 (3H, s, H-23), 0.99 (3H, s, H-27), 0.95 (3H, s, H-26), 0.85
(3H, s, H-25), 0.81 (3H, s, H-24), 0.73 (1H, d, J ) 9.5 Hz, H-5); 13C
NMR, see Table 3; HRESIMS m/z 773.4444 [M + Na]+ (calcd for
C41H66O12Na, 773.4447).
28-O-ꢀ-D-Glucopyranosylbetulinic acid 3ꢀ-O-r-L-rhamnopyra-
noside (19). This compound was prepared from 18b (147 mg, 0.0986
mmol) in the same manner as that described for compound 16a.
Purification by C-18 reversed-phase flash chromatography (MeOH/H2O,
3:2 to 9:1) gave 19 (61 mg, 81%): white, amorphous powder; [R]25
D
-32.4 (c 0.1, MeOH); 1H NMR (CD3OD, 400 MHz) δ 5.49 (1H, d, J
) 8.1 Hz, H-1′′), 4.71 (1H, m, H-1′), 4.71 (1H, m, H-29), 4.59 (1H,
m, H-29), 3.84 (1H, m, H-6′′), 3.82 (1H, m, H-2′), 3.70 (1H, m, H-5′),
3.70 (1H, m, H-6′′), 3.63 (1H, dd, J ) 9.5, 3.3 Hz, H-3′), 3.43 (1H, m,
H-3′′), 3.38 (1H, m, H-4′′), 3.37 (1H, m, H-5′′), 3.36 (1H, m, H-4′),
3.31 (1H, m, H-2′′), 3.06 (1H, dd, J ) 11.6, 4.8 Hz, H-3), 3.00 (1H,
td, J ) 10.8, 6.2 Hz, H-19), 1.69 (3H, s, H-30), 1.22 (3H, d, J ) 6.2
Hz, H-6′), 1.00 (3H, s, H-27), 0.95 (3H, s, H-26), 0.93 (3H, s, H-23),
0.86 (3H, s, H-25), 0.77 (3H, s, H-24); 13C NMR, see Table 3;
HRESIMS m/z 787.4607 [M + Na]+ (calcd for C42H68O12Na, 787.4603).
28-O-ꢀ-D-Glucopyranosylbetulin 3ꢀ-O-ꢀ-D-glucopyranoside (21a).
A solution of the acceptor 1 (250 mg, 0.565 mmol) in anhydrous CH2Cl2
(11.3 mL) was stirred for 60 min with 4 Å molecular sieves at -10 °C
(ice water/acetone bath). TMSOTf (20 µL, 0.113 mmol) was added
under argon while keeping rigorous anhydrous conditions. Then, a
solution of the donor 9 (1.26 g, 1.70 mmol) in anhydrous CH2Cl2 (8.5
mL) was added dropwise over 5 min with continuous stirring. The
reaction was allowed to warm to room temperature over 4 h and
quenched by addition of Et3N (0.31 mL, 2.3 mmol), and the solvents
were evaporated under reduced pressure. The residue was immediately
dissolved in a solution of MeOH/THF/H2O, 1:2:1 (37 mL), to which
was added NaOH (438 mg, 11.0 mmol). The reaction mixture was
stirred overnight at room temperature and then acidified to pH ≈ 4
with aqueous HCl 10%. The solvents were evaporated under reduced
pressure. The solid residue was purified by C-18 reversed-phase flash
chromatography (MeOH/H2O, 7:3 to 9:1) to afford 21a (363 mg, 84%,
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulinic acid
3ꢀ-O-2,3,4-tri-O-benzoyl-r-L-arabinopyranoside (18a). This com-
pound was prepared from the acceptor 17 (250 mg, 0.241 mmol) and
the donor 5 (220 mg, 0.362 mmol) in the same manner as that described
for compound 7a except for the molar volume of CH2Cl2 (20
mL·mmol-1). Purification by flash chromatography (hexanes/EtOAc,
9:1 to 7:3) gave 18a (224 mg, 63%): white, crystalline powder; [R]25
D
+88.9 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.09-7.23 (35H,
H-Ar), 6.04 (1H, m, H-1′′), 6.03 (1H, m, H-3′′), 5.78 (1H, m, H-2′),
5.76 (1H, m, H-2′′), 5.75 (1H, m, H-4′′), 5.68 (1H, m, H-4′), 5.61 (1H,
m, H-3′), 4.77 (1H, d, H-1′), 4.71 (1H, m, H-29), 4.60 (1H, m, H-6′′),
4.58 (1H, m, H-29), 4.50 (1H, m, H-6′′), 4.32 (1H, m, H-5′), 4.30 (1H,
m, H-5′′), 3.87 (1H, m, H-5′), 3.09 (1H, m, H-3), 2.94 (1H, m, H-19),
1.64 (3H, s, H-30), 0.77 (3H, s, H-27), 0.74 (3H, s, H-23), 0.67 (3H,
s, H-25), 0.62 (3H, s, H-24), 0.44 (3H, s, H-26); 13C NMR (CDCl3,
100 MHz) δ 174.0 (C-28), 166.0-164.7 (7 × CO), 150.2 (C-20),
133.5-128.3 (C-Ar), 109.6 (C-29), 103.0 (C-1′), 91.4 (C-1′′), 90.1 (C-
3), 73.0 (C-5′′), 72.8 (C-3′′), 70.7 (C-3′), 70.2 (C-2′′), 70.2 (C-2′), 69.4
(C-4′′), 68.7 (C-4′), 62.7 (C-6′′), 62.7 (C-5′), 56.8 (C-17), 55.4 (C-5),
50.3 (C-9), 49.1 (C-18), 46.6 (C-19), 42.1 (C-14), 40.2 (C-8), 38.9
(C-4), 38.6 (C-1), 38.0 (C-13), 36.7 (C-10), 36.3 (C-22), 33.3 (C-7),
31.5 (C-16), 30.2 (C-21), 29.8 (C-15), 27.7 (C-23), 26.0 (C-2), 25.4
(C-12), 20.7 (C-11), 19.5 (C-30), 17.8 (C-6), 16.1 (C-24), 15.9 (C-
25), 15.3 (C-26), 14.4 (C-27); HRESIMS m/z 1501.6347 [M + Na]+
(calcd for C90H94O19Na, 1501.6282).
28-O-2,3,4,6-Tetra-O-benzoyl-ꢀ-D-glucopyranosylbetulinic acid
3ꢀ-O-2,3,4-tri-O-benzoyl-r-L-rhamnopyranoside (18b). This com-
pound was prepared from the acceptor 17 (250 mg, 0.241 mmol) and
the donor 6 (225 mg, 0.362 mmol) in the same manner as that described
for compound 7a except for the molar volume of CH2Cl2 (20
mL·mmol-1). Purification by flash chromatography (hexanes/EtOAc,
2 steps) as a white, amorphous powder: [R]25 +1.2 (c 0.5, MeOH);
D
1H NMR (Pyr-d5, 400 MHz) δ 5.05 (1H, d, J ) 7.6 Hz, H-1′′), 4.98
(1H, d, J ) 7.8 Hz, H-1′), 4.83 (1H, d, J ) 2.1 Hz, H-29), 4.71 (1H,
br s, H-29), 4.67 (1H, m, H-6′′), 4.63 (1H, m, H-6′), 4.49 (1H, dd, J )
12.1, 5.1 Hz, H-6′′), 4.45 (1H, dd, J ) 11.6, 5.3 Hz, H-6′), 4.34 (1H,
m, H-3′′), 4.34 (1H, m, H-4′′), 4.28 (1H, m, H-3′), 4.27 (1H, m, H-4′),
4.14 (1H, m, H-2′′), 4.13 (1H, m, H-5′′), 4.10 (1H, m, H-28), 4.08
(1H, m, H-2′), 4.03 (1H, m, H-5′), 3.95 (1H, d, J ) 9.7 Hz, H-28),
3.43 (1H, dd, J ) 11.4, 4.3 Hz, H-3), 1.72 (3H, s, H-30), 1.33 (3H, s,
H-23), 1.03 (3H, s, H-27), 1.01 (3H, s, H-24), 0.94 (3H, s, H-26), 0.80
(3H, s, H-25), 0.74 (1H, br d, J ) 8.9 Hz, H-5); 13C NMR, see Table
3; HRESIMS m/z 789.4747 [M + Na]+ (calcd for C42H70O12Na,
789.4760).
9:1 to 4:1) gave 18b (311 mg, 86%): white, amorphous powder; [R]25
D
+72.5 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.10-7.21 (35H,
H-Ar), 6.08 (1H, m, H-1′′), 6.07 (1H, m, H-3′′), 5.83 (1H, m, H-3′),
5.82 (1H, m, H-2′′), 5.77 (1H, m, H-4′′), 5.69 (1H, m, H-4′), 5.67 (1H,
m, H-2′), 5.08 (1H, br s, H-1′), 4.72 (1H, br s, H-29), 4.62 (1H, dd, J
) 12.3, 2.9 Hz, H-6′′), 4.59 (1H, br s, H-29), 4.52 (1H, dd, J ) 12.3,
5.4 Hz, H-6′′), 4.34 (1H, m, H-5′′), 4.33 (1H, m, H-5′), 3.17 (1H, t, J
) 8.1 Hz, H-3), 2.96 (1H, td, J ) 10.8, 4.6 Hz, H-19), 2.20 (1H, br d,
J ) 12.7 Hz, H-16), 1.64 (3H, s, H-30), 1.34 (3H, d, J ) 6.2 Hz,
H-6′), 1.03 (3H, s, H-23), 0.92 (3H, s, H-24), 0.81 (3H, s, H-27), 0.77
(3H, s, H-25), 0.51 (3H, s, H-26), 0.44 (1H, br d, J ) 11.4 Hz, H-7);
13C NMR (CDCl3, 100 MHz) δ 174.0 (C-28), 166.0-163.5 (7 × CO),
150.1 (C-20), 133.6-128.2 (C-Ar), 109.5 (C-29), 99.7 (C-1′), 91.4 (C-
1′′), 90.0 (C-3), 72.9 (C-5′′), 72.8 (C-3′′), 71.9 (C-4′), 71.1 (C-2′), 70.2
(C-3′), 70.2 (C-2′′), 69.3 (C-4′′), 66.7 (C-5′), 62.7 (C-6′′), 56.7 (C-17),
55.3 (C-5), 50.3 (C-9), 49.0 (C-18), 46.6 (C-19), 42.1 (C-14), 40.2
(C-8), 39.0 (C-4), 38.5 (C-1), 37.9 (C-13), 36.7 (C-10), 36.3 (C-22),
33.3 (C-7), 31.4 (C-16), 30.2 (C-21), 29.8 (C-15), 28.2 (C-23), 25.5
(C-2), 25.3 (C-12), 20.7 (C-11), 19.4 (C-30), 17.9 (C-6), 17.5 (C-6′),
16.3 (C-24), 16.0 (C-25), 15.3 (C-26), 14.4 (C-27); HRESIMS m/z
1515.6419 [M + Na]+ (calcd for C91H96O19Na, 1515.6438).
28-O-ꢀ-D-Glucopyranosylbetulinic acid 3ꢀ-O-ꢀ-D-glucopyrano-
side (21b). This compound was prepared from the acceptor 2 (50 mg,
0.109 mmol) and the donor 9 (243 mg, 0.328 mmol) in the same manner
as that described for compound 21a. Purification by C-18 reversed-
phase flash chromatography (MeOH/H2O, 7:3 to 17:3) gave 21b (49
mg, 58%, 2 steps): white, amorphous powder; [R]25 -6.8 (c 0.1,
D
1
MeOH); H NMR (CD3OD, 400 MHz) δ 5.49 (1H, d, J ) 8.1 Hz,
H-1′′), 4.71 (1H, br s, H-29), 4.60 (1H, br s, H-29), 4.30 (1H, d, J )
7.6 Hz, H-1′), 3.84 (1H, m, H-6′′), 3.83 (1H, m, H-6′), 3.70 (1H, dd,
J ) 11.9, 3.0 Hz, H-6′′), 3.65 (1H, dd, J ) 11.9, 5.3 Hz, H-6′), 3.42
(1H, m, H-3′′), 3.39 (1H, m, H-4′′), 3.38 (1H, m, H-5′′), 3.33 (1H, m,
H-3′), 3.31 (1H, m, H-2′′), 3.28 (1H, m, H-4′), 3.24 (1H, m, H-5′),
3.18 (1H, m, H-2′), 3.15 (1H, m, H-3), 3.01 (1H, td, J ) 10.8, 4.5 Hz,
H-19), 1.69 (3H, s, H-30), 1.03 (3H, s, H-23), 0.99 (3H, s, H-27), 0.95
(3H, s, H-26), 0.86 (3H, s, H-25), 0.82 (3H, s, H-24); 13C NMR, see
Table 3; HRESIMS m/z 803.4537 [M + Na]+ (calcd for C42H68O13Na,
803.4552).
28-O-ꢀ-D-Glucopyranosylbetulinic acid 3ꢀ-O-r-L-arabinopyra-
noside (3). This compound was prepared from 18a (100 mg, 0.068
mmol) in the same manner as that described for compound 16a.
Purification by C-18 reversed-phase flash chromatography (MeOH/H2O,
3:2 to 9:1) gave 3 (38 mg, 75%): white, amorphous powder; [R]25
28-O-r-L-Rhamnopyranosylbetulin 3ꢀ-O-r-L-rhamnopyranoside
(22a). This compound was prepared from the acceptor 1 (100 mg, 0.226
mmol) and the donor 6 (421 mg, 0.678 mmol) in the same manner as
that described for compound 21a. Purification by C-18 reversed-phase
flash chromatography (MeOH/H2O, 3:2 to 9:1) gave 22a (53 mg, 32%,
2 steps): white, amorphous powder; [R]25D -58.4 (c 0.1, CHCl3/MeOH,
D
1
+12.2 (c 0.1, MeOH); H NMR (CDCl3/CD3OD, 1:2, 400 MHz) δ
5.51 (1H, d, J ) 8.1 Hz, H-1′′), 4.72 (1H, br s, H-29), 4.60 (1H, br s,
H-29), 4.31 (1H, d, J ) 6.3 Hz, H-1′), 3.86 (1H, m, H-5′), 3.86 (1H,
m, H-6′′), 3.84 (1H, m, H-4′), 3.74 (1H, dd, J ) 12.1, 4.0 Hz, H-6′′),
3.61 (1H, dd, J ) 8.4, 6.4 Hz, H-2′), 3.55 (1H, dd, J ) 8.4, 3.0 Hz,

80 Journal of Natural Products, 2009, Vol. 72, No. 1
Gauthier et al.
1:1); 1H NMR (CDCl3/CD3OD, 1:1, 400 MHz) δ 4.76 (1H, br s, H-1′),
4.70 (1H, m, H-1′′), 4.70 (1H, m, H-29), 4.59 (1H, m, H-29), 3.88
(1H, m, H-2′), 3.88 (1H, m, H-2′′), 3.75 (1H, dd, J ) 9.4, 6.2 Hz,
H-5′′), 3.69 (1H, m, H-3′), 3.69 (1H, m, H-3′′), 3.60 (1H, dd, J ) 9.4,
6.4 Hz, H-5′), 3.51 (1H, d, J ) 9.2 Hz, H-28), 3.43 (1H, m, H-28),
3.40 (1H, m, H-4′), 3.38 (1H, m, H-4′′), 3.07 (1H, dd, J ) 11.6, 4.8
Hz, H-3), 2.47 (1H, m, H-19), 1.69 (3H, s, H-30), 1.33 (3H, d, J ) 6.2
Hz, H-6′), 1.26 (3H, d, J ) 6.4 Hz, H-6′′), 1.03 (3H, s, H-26), 0.99
(3H, s, H-27), 0.92 (3H, s, H-23), 0.84 (3H, s, H-25), 0.76 (3H, s,
H-24), 0.72 (1H, br d, J ) 10.0 Hz, H-5); 13C NMR (CDCl3/CD3OD,
1:1, 100 MHz), see Table 3; HRESIMS m/z 757.4843 [M + Na]+ (calcd
for C42H70O10Na, 757.4861).
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28-O-r-L-Rhamnopyranosylbetulinic acid 3ꢀ-O-r-L-rhamnopy-
ranoside (22b). This compound was prepared from the acceptor 2 (100
mg, 0.219 mmol) and the donor 6 (408 mg, 0.657 mmol) in the same
manner as that described for compound 21a. Purification by C-18
reversed-phase flash chromatography (MeOH, 7:3 to 17:3) gave 22b
(60 mg, 37%, 2 steps): white, amorphous powder; [R]25D -47.0 (c 0.5,
1
MeOH); H NMR (CD3OD, 400 MHz) δ 6.00 (1H, d, J ) 1.6 Hz,
H-1′′), 4.75 (1H, d, J ) 1.3 Hz, H-29), 4.72 (1H, d, J ) 1.3 Hz, H-1′),
4.62 (1H, br s, H-29), 3.82 (1H, dd, J ) 3.2, 1.6 Hz, H-2′), 3.79 (1H,
dd, J ) 3.3, 1.9 Hz, H-2′′), 3.70 (1H, m, H-5′), 3.67 (1H, m, H-3′′),
3.67 (1H, m, H-5′′), 3.63 (1H, m, H-3′), 3.46 (1H, t, J ) 9.4 Hz, H-4′′),
3.36 (1H, t, J ) 9.4 Hz, H-4′), 3.07 (1H, dd, J ) 11.6, 4.9 Hz, H-3),
3.02 (1H, td, J ) 10.7, 4.6 Hz, H-19), 1.71 (3H, s, H-30), 1.27 (3H, d,
J ) 6.2 Hz, H-6′′), 1.22 (3H, d, J ) 6.2 Hz, H-6′), 1.02 (3H, s, H-27),
0.94 (3H, s, H-26), 0.93 (3H, s, H-23), 0.87 (3H, s, H-25), 0.77 (3H,
s, H-24); 13C NMR, see Table 3; HRESIMS m/z 771.4639 [M + Na]+
(calcd for C42H68O11Na, 771.4654).
Cell Lines and Culture Conditions. Human lung carcinoma (A549),
human colorectal adenocarcinoma (DLD-1), human breast adenocar-
cinoma (MCF7), human prostate adenocarcinoma (PC-3), and human
normal skin fibroblasts (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% fetal bovine serum
(Hyclone), vitamins (1×), penicillin (100 IU/mL), streptomycin (100
µg/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.
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 µL of culture medium and were allowed to adhere for
16 h before treatment. Increasing concentrations of each compound in
biotech DMSO (Sigma-Aldrich) 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 assessed using
resazurin55 on an automated 96-well Fluoroskan Ascent F1 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 percentage 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.
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Acknowledgment. We thank Professor Franc¸ois-Xavier Garneau for
reviewing the manuscript. The financial support of Fonds Que´be´cois
de la Recherche sur la Nature et les Technologies (FQRNT, fonds
forestiers 02) is gratefully acknowledged. C.G. thanks Programme
d′Aide Institutionnel a` la Recherche de l′Universite´ du Que´bec a`
Chicoutimi (PAIR-UQAC), Fondation de l′UQAC, Association Fran-
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