Synthesis of lupane-type saponins containing an unusual α-D-idopyranoside fragment as potent cytotoxic agents

Article abstract of DOI:10.1002/ejoc.201402187

A practical method for the preparation of benzoyl protected allyl and benzyl α-D-idopyranosides, and D-idopyranosyl trichloroacetimidate, from 1,2,3,4,6-penta-O-acetyl-α-D-idopyranose, is described. All derivatives can be prepared on a multigram scale and require only simple chromatographic purification. A concise synthesis of lupane triterpenes bearing an unusual D-idopyranoside fragment is described. All new compounds were evaluated in vitro for their cytotoxic activities. Novel saponins exhibited interesting cytotoxic activity in the micromolar range against human cancer cell lines including T-lymphoblastic leukemia CEM, breast adenocarcinoma MCF7, and cervical carcinoma HeLa, but also against normal human fibroblasts BJ. A concise synthesis of 2,3,4,6-tetra-O-benzoyl-D-idopyranosyl trichloroacetimidate and lupane triterpenes bearing an unusual D-idopyranoside fragment is described. All new compounds were evaluated in vitro for their cytotoxic activities, and all exhibited activity in the micromolar range against human cancer cell lines. Copyright

Full text of DOI:10.1002/ejoc.201402187

Job/Unit: O42187
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Date: 06-05-14 17:56:27
Pages: 11
FULL PAPER
DOI: 10.1002/ejoc.201402187
Synthesis of Lupane-Type Saponins Containing an Unusual α-
D
-Idopyranoside
Fragment as Potent Cytotoxic Agents
[a]
[a]
[b]
[b]
[b]
ˇ
Piotr Cmoch, Anna Korda, Lucie Rárová, Jana Oklest’ková, Miroslav Strnad,*
Katarzyna Gwardiak,[a] Romuald Karczewski,[a] and Zbigniew Pakulski*[a]
Dedicated to Professor Mieczysław Ma˛kosza on the occasion of his 80th birthday
Keywords: Carbohydrates / Glycoconjugates / Glycosylation / Steroids / Saponins / Drug discovery
A practical method for the preparation of benzoyl protected
allyl and benzyl α- -idopyranosides, and -idopyranosyl tri-
chloroacetimidate, from 1,2,3,4,6-penta-O-acetyl-α-
new compounds were evaluated in vitro for their cytotoxic
activities. Novel saponins exhibited interesting cytotoxic ac-
-idopyr- tivity in the micromolar range against human cancer cell
D
D
D
anose, is described. All derivatives can be prepared on a
multigram scale and require only simple chromatographic
purification. A concise synthesis of lupane triterpenes bear-
lines including T-lymphoblastic leukemia CEM, breast ade-
nocarcinoma MCF7, and cervical carcinoma HeLa, but also
against normal human fibroblasts BJ.
ing an unusual
D-idopyranoside fragment is described. All
1. Introduction
The chemistry of l-idose and l-idopyranuronic acid is
well known, and numerous syntheses of these carbohydrates
have been published because of the significant role played
by these sugars as components of some glycosaminoglycans
(e.g., heparin).[27,28] In comparison, d-idose has not been
isolated from natural sources, although it is present in the
capsular polysaccharide (CPS) structure of Campylobacter
jejuni in the form of 6-deoxy-d-ido-heptose,[29,32] and was
extracted from the unusual resurrection plant Craterostigma
plantagineum as d-glycero-d-ido-2-octulose.[30,31] Chemical
syntheses of d-idose and its derivatives, however, are ex-
tremely limited, and only a few examples have been re-
ported. Paulsen reported a rearrangement of tetra-O-acetyl-
d-glucopyranosyl chloride into α-d-idopyranose peracetate
in the presence of antimony pentachloride.[33,34] Another
example involved isomerization of the d-galactose part of
lactose into d-idose by its selective sulfonylation, epoxide
formation, followed by an opening of the epoxide ring,
which finally afforded 4-O-β-d-idopyranosyl-d-glucopyr-
anose.[35,36] Free d-idose was also prepared by an elong-
ation of the d-xylose chain by the Nef reaction.[37] Recently,
a stereoselective conversion of d-galactose into orthogo-
nally protected β-d-idopyranosides was reported.[38] During
the realization of this project, the syntheses of new deriva-
tives of peracetylated d-idopyranoses and d-idopyranosides
Saponins are steroid or triterpenoid glycosides that are
widely distributed in plants and in some marine organ-
isms.[1,2] They possess interesting biological properties in-
cluding antitumor, antiviral, antifungal, and antiinflamma-
tory activities, which have been extensively studied and re-
viewed.[3–10] Their biological effects have been correlated to
both sugar residues and aglycones.[3,11–13] Usually, mono-,
di-, tri-, or tetrasaccharides are attached to the sapogenin
backbone, where they constitutes a hydrophilic part,
whereas sapogenin forms a hydrophobic fragment. Natural
saponins based on a betulin scaffold occur less frequently
than those having other triterpene-type aglycones, although
growing interest in their synthesis is noticeable.[14–26] Fur-
ther investigations are required to address questions regard-
ing structure–activity relationships (SAR) and the role of
the saccharide part. The high importance of l-idose deriva-
tives in biological systems[27,28] and growing interest in d-
idose derivatives, which occur very rarely in nature,[29–31]
prompted us to prepare a series of lupane saponins contain-
ing a d-idopyranoside ring.
[a] Institute of Organic Chemistry, Polish Academy of Sciences,
Kasprzaka 44/52, 01-224 Warszawa, Poland
E-mail: zbigniew.pakulski@icho.edu.pl
http://www.icho.edu.pl/eng/
[b] Laboratory of Growth Regulators, Centre of the Region Haná were published by Thiem.[39] However, the synthesis of sim-
for Biotechnological and Agricultural Research, Palacký
ple benzoylated d-idopyranosyl trichloroacetimidate (used
as glycosyl donor), or the corresponding d-idopyranosides,
remains challenging.
Glycosylation of lupane triterpenes with acetylated
glycosyl donors usually causes serious problems and, in
University & Institute of Experimental Botany ASCR,
ˇ
Slechtitelu˚ 11, 78371 Olomouc, Czech Republic
E-mail: miroslav.strnad@upol.cz
http://www.ueb.cas.cz/en/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402187.
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M. Strnad, Z. Pakulski et al.
FULL PAPER
many cases, the desired products cannot be obtained. dard methods of hydrolysis of the anomeric acetate [hydraz-
Acetyl migration from glycosyl donor to acceptor is usually ine acetate[40] or ammonium carbonate in N,N-dimethyl-
observed as the main, or even as the only process. This formamide (DMF)[41]] were unsuccessful and caused de-
problem was fully solved by application of benzoylated composition of the starting material. Therefore, the anom-
glycosyl donors in which migration of the benzoyl group eric acetate was removed by using a two-step procedure
does not occur.
(Scheme 1).[42] First, treatment of 9 with HBr in acetic acid
Herein, we report on the synthesis of perbenzoylated d- gave glycosyl bromide 10, which, in the second step, was
idose derivatives, as well as novel lupane-type saponins converted into 2,3,4,6-tetra-O-acetyl-d-idopyranose (11) by
bearing a d-idopyranoside moiety, and their anticancer hydrolysis induced by silver carbonate. The overall yield
properties determined by a cytotoxicity evaluation of these (86% after two steps) was slightly better than that obtained
compounds. This is a continuation of our research on the by using Thiem’s method (treatment of 9 with BnNH2, 80%
synthesis and utility of saponins obtained from triterpenes yield).[39] Compound 11 was transformed in 69% yield into
isolated from white birch bark.[24–26] The synthetic strategy the required acetylated trichloroacetimidate 7 under the
is directed toward glycosylation of 3-O-acetylbetulinic acid standard conditions (Scheme 1).
1, betulin derivatives 2–4, lupeol (5), and 28-O-acetyl-epi-
betulin (6) with d-idopyranosyl trichloroacetimidates 7[39]
and 8 (Figure 1). Subsequent saponification afforded free
saponins. The cytotoxicities of these saponins were tested
against a series of normal and cancer cell lines.
Scheme 1. Synthesis of acetylated d-idose derivatives. Reagents and
conditions: (i) HBr in acetic acid; (ii) Ag2CO3, acetone/water;
(iii) Cl3CCN, K2CO3.
Reaction of 7 with benzyl alcohol in the presence of tri-
methylsilyl triflate (TMSOTf) gave benzyl α-d-idopyrano-
side 12 in 96% yield. Benzyl (12; 83%) and allyl α-d-idopyr-
anosides (16; 72%) may be also prepared directly from per-
acetate 9 by treatment with benzyl or allyl alcohol, respec-
tively, in the presence of BF3·Et2O complex.
Similar rearrangement of d-glucose perbenzoate into α-
d-idopyranose perbenzoate is possible, but the product is
formed in very low yield (12%).[43] Therefore, we decided
to use idopyranosides 12 and 16 as starting materials for
the preparation of the required benzoyl-protected d-idose
derivatives. Deacetylation of 12 and 16 by methanolic so-
dium methoxide (to give 13 and 17) followed by acylation
with benzoyl chloride in pyridine afforded tetrabenzoates
14 and 18 in excellent yield (89% and 96%, respectively,
after two steps).
Figure 1. Parent triterpenes and glycosyl donors.
2. Results and Discussion
2.1. Synthesis of D-Idose Derivatives
Hydrogenolysis of benzyl idopyranoside 14 gave 2,3,4,6-
The synthesis was initiated from α-d-idopyranose per- tetra-O-benzoyl-d-idopyranose (15) in high yield (90%).
acetate (9), which was easily prepared on a multigram scale The latter sugar was also prepared by hydrolysis (with HgO/
according to the method reported by Paulsen.[33,34] Stan- HgCl2;[44] 96%) of 1-propenyl α-d-idoside 19, which was
Scheme 2. Synthesis of benzoylated d-idose derivatives. Reagents and conditions: (i) BnOH, BF3·Et2O (from 9); (ii) BnOH, TMSOTf (from
7); (iii) MeOH, MeONa; (iv) BzCl, pyridine; (v) H2, 10%Pd/C; (vi) Cl3CCN, K2CO3, DBU; (vii) AllOH, BF3·Et2O; (viii) [Ir(COD)-
(MePPh2)2]PF6; (ix) HgO/HgCl2, acetone, water.
2
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Lupane-Type Saponins Containing an α-d-Idopyranoside Fragment
obtained in 98% yield by isomerization of allyl glycoside 18 tion of 28-O-acetyl-epi-betulin (6)[47] with 8 gave the re-
with iridium complex [Ir(COD)(MePh2P)2]PF6.[45] Trans- quired product 34 in only 52% yield.
formation of 15 into the required donor 8 was achieved
Final deprotection of the hydroxy groups was performed
in 80% yield by treatment with trichloroacetonitrile in the by treatment of benzoates with potassium carbonate in
presence of potassium carbonate and 1,8-diazabicyclo- methanol, except for acetate protected 20, which was
[5.4.0]undec-7-ene (DBU); the use of this mixture of both treated with sodium methoxide in methanol. All saponins
bases was crucial (Scheme 2).
were prepared in good yields (60–83%) with the exception
of bidesmosidic saponin 33, which was isolated in 40%
yield, probably due to the very poor solubility and high
affinity to silica of the latter, which caused unusual loss of
product (Figure 2). The structures of all synthesized sapo-
nins were confirmed by extensive 1D and 2D NMR analy-
sis, as well as by elemental and HRMS analysis.
2.2. Synthesis of Saponins
Glycosylation of 3-O-acetylbetulinic acid 1[24] was per-
formed by treatment with peracetylated (7) or perbenzoyl-
ated (8) d-idopyranosyl trichloroacetimidate in the presence
of TMSOTf under standard conditions.[25] Protected esters
were obtained in high yields (89% for 20 and 86% for 21).
Similar reaction of peracetylated donor 7 with lupeol (5)[25]
2.3. Discussion
It is widely accepted that 1,2,3,4,6-penta-O-acetyl-α-d-
and 28-O-acetylbetulin (3)[24] afforded acetylated triterp- idopyranose (9) exists mainly in 4C1 or 0S2 conforma-
enes as the only products, as a result of acetyl migration tions.[49,50] In these forms, the H-1, H-2, H-3, and H-4 ring
from glycosyl donor to acceptor. Reactions of perbenzoyl- protons are in equatorial positions, which results in small
ated donor 8 with lupeol (5) and 3-O-acetylbetulin (2)[24] coupling constants (2–3 Hz). In fact, all coupling constants
afforded the expected α-d-idosides 23 (97%), and 25 (87%). observed in the 1H NMR spectra of the studied compounds
When 28-O-acetylbetulin (3) was used as starting material, were small. This confirmed the d-ido configuration and the
glycoside 27 was obtained in 81% yield. Reaction of tert- α-position of substituents at the anomeric center. Full
1
butyldiphenylsilyl protected betulin derivative 4[46] with do- analysis of the H NMR spectra was, however, not trivial.
nor 8 gave α-d-idopyranoside 29 in 87% yield. Desilylation Signals were poorly resolved and often occurred as complex
5
of 29 with acetic acid buffered tetrabutylammonium fluo- multiplets caused by long-range couplings (4J1,3, J1,4, and
ride solution gave saponin 30 (78%). Because bidesmosidic 4J2,4), usually observed for idose derivatives.[49]
saponins bearing sugar moieties at both C-3 and C-28 posi-
The coupling constants for the olefinic protons of the 1-
tions were considered to be less haemolytic than monodes- propenyl part of 19 (12.3 Hz) confirmed the (E) configura-
mosides,[10,48] we investigated the synthesis of saponin 32. tion across the double bond. The (Z) isomer of 19 was not
Glycosylation of 30 with donor 8 afforded bidesmosidic sa- identified in the reaction mixture, although it may be pres-
ponin 32 in excellent yield (97%). Unexpectedly for us, reac- ent in a small amount (Ͻ 5%).
Figure 2. Synthesis of lupane saponins. Reagents and conditions: (i) K2CO3, MeOH; (ii) NaOMe, MeOH; (iii) Bu4NF, AcOH, THF.
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As expected, the presence of the acetyl and benzoyl pro- series of lupane saponins containing a d-idopyranose ring
tecting groups in the donor molecules directed the anomeric is thus not very high because of their high cytotoxicity for
selectivity of the glycosidation reaction,[54] and in all cases normal human fibroblasts. The novel lupane-type saponins
studied in this report, 1,2-trans-idopyranosides were formed were, except for 24, 33, and 36, also more potent for HeLa
exclusively. This observation was strongly supported by cell line, whereas bidesmosidic saponin 33 was the most
1JC1,H1 coupling constants of 174 (in the case of 20) and active against CEM but less active than the parent com-
170 Hz (in the case of 23), which clearly indicated an equa- pound 26. The results show that several unique saponins
torial proton at C-1.[51–53]
bearing the d-idopyranose ring exhibit anticancer activities
on several human cancer cell lines in the low micromolar
range.
2.4. In Vitro Results
Anticancer activities of the lupane triterpenes bearing the
unusual d-idopyranoside fragment were tested in vitro. Sev-
eral normal and cancer cell lines were cultured and used to
examine the structure–activity relationships of these lupane
triterpenes. We compared the in vitro cytotoxic activity of
selected analogues against human BJ fibroblasts and cancer
cell lines of various histopathological origins, including T-
lymphoblastic leukemia CEM, breast adenocarcinoma
MCF7, and cervical carcinoma HeLa. Cells of all of these
lines were exposed to six serial threefold dilutions of each
drug for 72 h, the proportions of surviving cells were then
estimated and IC50 values (50% inhibitory concentrations)
were calculated by using betulinic acid (36; Figure 3) as a
positive control. The results obtained from Calcein AM as-
says are presented in Table 1.
3. Conclusions
We have developed a practical method with which to ob-
tain a series of d-idopyranose derivatives. Rearrangement
of glucose peracetate, according to Paulsen and co-workers,
provides rare monosaccharide d-idose. Simple manipula-
tion with the protecting groups afforded series of acetyl and
benzoyl protected derivatives, including allyl and benzyl
glycosides, and the corresponding trichloroacetimidates,
which are valuable glycosyl donors. This constitutes the first
convenient method for the preparation of benzoyl protected
d-idopyranose derivatives, which are especially useful in the
synthesis of lupane-type saponins.
A series of the lupane-type saponins bearing the d-ido-
pyranose ring were also synthesized and evaluated for their
cytotoxic activities. Many of the studied compounds
showed interesting cytotoxic activities in the low micromo-
lar range against human cancer cell lines CEM, MCF7, and
HeLa, but also comparable cytotoxicity on normal human
fibroblasts (BJ).
4. Experimental Section
Figure 3. Betulinic acid (36).
4.1. General Notes: Silica gel HF254 and silica gel 230–400 mesh (E.
Merck) were used for TLC and column chromatography, respec-
tively. 1H and 13C NMR spectra were recorded at 298 K with a
Varian NMR-vnmrs600 or vnmrs500 spectrometers. Standard ex-
perimental conditions and standard Varian programs (ChemPack
4.1) were used with TMS as internal standard. Configurational as-
signments were based on the NMR measurements including two-
dimensional techniques. High-resolution mass spectra (HRMS
ESI) were acquired with MARINER and MaldiSYNAPT G2-S
HDMS (Waters) mass spectrometers. Optical rotations were mea-
sured with a JASCO P-2000 automatic polarimeter. IR spectra
were recorded with a Jasco 6200 FTIR spectrophotometer.
Table 1. IC50 [μm] values obtained from the Calcein AM assays
with the tested cancer and normal cells; meanϮSD values obtained
from three independent experiments performed in triplicate. Betul-
inic acid (36; Figure 3) was used as a positive control.
CEM
MCF7
HeLa
BJ
22
24
26
28
31
33
35
36
25.5Ϯ1.8
38.6Ϯ3.3
10.9Ϯ1.6
29.4Ϯ1.6
32.9Ϯ3.4
14.7Ϯ1.0
31.6Ϯ1.6
40.0Ϯ2.8
33.0Ϯ4.9
Ͼ 50
19.4Ϯ2.2
34.2Ϯ4.0
9.3Ϯ1.0
17.3Ϯ2.5
27.7Ϯ4.1
21.1Ϯ7.0
16.2Ϯ1.5
47.6Ϯ1.9
20.1Ϯ2.5
16.1Ϯ0.8
7.3Ϯ1.2
13.9Ϯ0.1
42.1Ϯ0.6
21.2Ϯ2.9
9.9Ϯ1.6
Ͼ 50
17.6Ϯ3.5
37.6Ϯ3.0
47.2Ϯ2.4
30.1Ϯ0.1
33.7Ϯ12.2
Ͼ 50
4.2. Syntheses
4.2.1. 2,3,4,6-Tetra-O-acetyl-α,β-D-idopyranose (11): 2,3,4,6-Tetra-
O-acetyl-α-d-idopyranosyl bromide (10) was obtained by a modi-
fied literature method.[55] To a solution of 1,2,3,4,6-penta-O-acetyl-
α-d-idopyranose[33,34] (9; 11.50 g, 29.5 mmol) in 1,2-dichloroethane
(100 mL), 33% HBr in acetic acid (30 mL) was added and the mix-
ture was stirred at room temp. for 24 h. The mixture was diluted
with 1,2-dichloroethane (100 mL) and the organic solution was
washed with ice water (2ϫ 100 mL), satd. NaHCO3 (50 mL), cold
water, dried with anhydrous sodium sulfate, and concentrated to
afford crude 2,3,4,6-tetra-O-acetyl-α-d-idopyranosyl bromide (10;
All tested lupane-type saponins bearing the d-idopyran-
oside moiety showed cytotoxic activity towards the cancer
and normal cells used (except for 24 and 36 on MCF7
cells). Potent compounds were saponins 26, 28, and 35,
which showed cytotoxic activity against all of the tumor cell
lines at low micromolar range (IC50 9.3–37.6 μm) but usu-
ally also much stronger toxicity towards normal BJ fibro-
blasts (IC50 7.3–20.1 μm). The therapeutic index of this 11.00 g, 91%). Bromide 10 was dissolved in acetone (100 mL) and
4
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Lupane-Type Saponins Containing an α-d-Idopyranoside Fragment
water (3 mL), silver carbonate (8.50 g, 30.8 mmol) was added and acetate 9 (781 mg, 2.00 mmol), allyl alcohol (0.70 mL, 10.0 mmol),
the resulting suspension was stirred in the dark until disappearance
of the starting material was confirmed by TLC (ca. 3 h). The mix-
ture was filtered through a Celite pad, concentrated, and the resi-
due was purified by column chromatography (hexane/ethyl acetate,
7:3Ǟ1:1) to afford the title compound (8.81 g, 86% after two
steps) with physicochemical properties identical to those re-
ported.[39]
and molecular sieves (4 Å, 500 mg, finely ground), in dichlorometh-
ane (20 mL) were treated with BF3·Et2O (1.3 mL, 10.0 mmol), and
purified by column chromatography to afford the title compound
(556 mg, 72%) as a thick oil; the physicochemical properties were
identical to those reported.[39]
4.2.5. Benzyl 2,3,4,6-Tetra-O-benzoyl-α-D-idopyranoside (14): To a
solution of 12 (2.92 g, 6.66 mmol) in methanol (25 mL), sodium
methoxide in methanol (0.45 m, 0.20 mL) was added and the mix-
ture was stirred for 1 h. The mixture was concentrated and the
residue (crude 13) was dissolved in pyridine (40 mL). Benzoyl
chloride (4.6 mL, 40.0 mmol) was slowly added and the mixture
was stirred overnight. The excess of benzoyl chloride was decom-
posed by addition of methanol (10 mL), then solvents were coevo-
parated with toluene, and the residue was purified by column
chromatography (hexane/ethyl acetate, 40:1Ǟ1:1) to afford the title
compound (4.07 g, 89% after two steps) as a foam. [α]2D0 = 23.0 (c
4.2.2. 2,3,4,6-Tetra-O-acetyl-α,β-D-idopyranosyl Trichloroacetimid-
ate (7): To a solution of 2,3,4,6-tetra-O-acetyl-d-idopyranose (11;
4.00 g, 11.5 mmol) and trichloroacetonitrile (4.3 mL, 43.0 mmol) in
dichloromethane (100 mL), potassium carbonate (3.90 g,
28.2 mmol) was added and the mixture was stirred at room temp.
for 24 h. The mixture was then concentrated and the residue was
purified by column chromatography (hexane/ethyl acetate,
20:1Ǟ1:1) to afford the title compound (α,β-mixture, 3.93 g, 69%)
1
as a foam. H NMR (400 MHz, CDCl3): δ = 8.75 (s, 1 H, NHα),
8.66 (s, 1 H, NHβ), 6.39 (d, J1,2 = 2.9 Hz, 1 H, 1-Hβ), 6.28 (br. s,
1 H, 1-Hα), 5.48 (dd, J3,2 = J3,4 = 7.0 Hz, 1 H, 3-Hβ), 5.17 (dd,
J2,1 = 2.9, J2,3 = 7.2 Hz, 1 H, 2-Hβ), 5.09–5.05 (m, 2 H, 3-Hα, 4-
Hβ), 5.02–5.01 (m, 1 H, 2-Hα), 4.94–4.91 (m, 1 H, 4-Hα), 4.65–
4.60 (m, 1 H, 5-Hα), 4.53–4.48 (m, 1 H, 5-Hβ), 4.38–4.35 (m, 2 H,
6-,6Ј-Hβ), 4.25 (dd, J5,6 = 5.3, J6,6Ј = 11.6 Hz, 1 H, 6-Hα), 4.17 (dd,
J5,6Ј = 7.3, J6,6Ј = 11.6 Hz, 1 H, 6Ј-Hα) ppm. 13C NMR (125 MHz,
CDCl3): δ = 170.4 (α-C=O), 170.3 (β-C=O), 169.7 (α-C=O), 169.6
(β-C=O), 169.5 (β-C=O), 169.0 (β-C=O), 168.9 (α-C=O), 168.8 (α-
C=O), 160.8 (β-C=NH), 159.9 (α-C=NH), 94.0 (C-1β), 93.9 (C-
1α), 71.9 (β), 67.6 (β), 67.6 (β), 66.7 (β), 66.3 (α), 65.7 (α), 65.6 (α),
65.2 (α),62.8 (C-6β), 62.1 (C-6α) ppm. HRMS (ESI): m/z calcd. for
= 0.3, chloroform). IR (film): νmax = 1723, 1451, 1266, 1109, 1027,
˜
709 cm–1. 1H NMR (600 MHz, CDCl3): δ = 8.15–7.18 (m, 25 H,
ArH), 5.66–5.64 (m, 1 H, 3-H), 5.39–5.37 (m, 1 H, 4-H), 5.28–5.25
(m, 2 H, 1,2-H), 4.98–4.95 (m, 1 H, 5-H), 4.91 and 4.62 (ABq, J =
11.2 Hz, 2 H, PhCH2), 4.72 (dd, J6,5 = 8.0, J6,6Ј = 11.6 Hz, 1 H, 6-
H), 4.55 (dd, J6Ј,5 = 4.5, J6,6Ј = 11.6 Hz, 1 H, 6Ј-H) ppm. 13C NMR
(125 MHz, CDCl3): δ = 166.1 (C=O), 165.3 (C=O), 165.1 (C=O),
164.5 (C=O), 133.5, 133.4, 133.3, 133.2, 130.2–127.9 (aromatic car-
bons), 97.0 (C-1), 69.7 (PhCH2), 67.4, 66.6 (2ϫ CH), 64.6, 63.6
(C-6) ppm. C41H34O10 (686.72): calcd. C 71.71, H 4.99; found C
71.74, H 5.10.
C16H20Cl3NNaO10 [M
+
Na]+ 514.0045; found 514.0032.
4.2.6. Allyl 2,3,4,6-Tetra-O-benzoyl-α-D-idopyranoside (18): Ob-
C16H20Cl3NO10 (492.70): calcd. C 39.00, H 4.09, N 2.84, Cl 21.59;
found C 38.83, H 4.17, N 2.86, Cl 21.40.
tained as described for the synthesis of 14. Allyl 2,3,4,6-tetra-O-
acetyl-α-d-idopyranoside (16; 528 mg, 1.36 mmol) was treated with
sodium methoxide to give crude 17, followed by benzoyl chloride
(875 μL, 7.5 mmol) to obtain the title compound (835 mg, 96%
4.2.3. Benzyl 2,3,4,6-Tetra-O-acetyl-α-D-idopyranoside (12): Method
A: A solution of trichloroacetimidate 7 (3.00 g, 6.1 mmol) and
benzyl alcohol (0.76 mL, 7.34 mmol) in dichloromethane (60 mL)
was stirred for 20–30 min at room temperature over molecular
sieves (4 Å, 1.8 g, finely ground), then cooled to 0 °C and TMSOTf
(360 μL, 2.00 mmol) was added and the mixture was stirred at 0 °C
for 20 min. The reaction was quenched with Et3N (2.0 mL), then
the mixture was concentrated and the residue was purified by col-
umn chromatography (hexane/ethyl acetate, 10:1) to give the title
compound (2.58 g, 96%) as a thick oil.
1
after two steps) as a foam. [α]2D0 = 27.0 (c = 0.5, chloroform). H
NMR (500 MHz, CDCl3): δ = 8.14–7.17 (m, 20 H, ArH), 6.00–
5.91 (m, 1 H, allyl CH=), 5.65–5.63 (m, 1 H, 3-H), 5.40–5.38 (m,
1 H, 4-H), 5.27–5.32 (m, 1 H, allyl =CH2), 5.24–5.22 (m, 1 H, 2-
H), 5.18–5.22 (m, 2 H, 1-H, allyl =CH2), 4.93–4.89 (m, 1 H, 5-H),
4.71 (dd, J6,5 = 7.9, J6,6Ј = 11.6 Hz, 1 H, 6-H), 4.54 (dd, J6Ј,5 = 4.7,
J6,6Ј = 11.6 Hz, 1 H, 6Ј-H), 4.34–4.39 (m, 1 H, allyl OCH), 4.11–
4.16 (m, 1 H, allyl OCH) ppm. 13C NMR (125 MHz, CDCl3): δ =
166.1 (C=O), 165.3 (C=O), 165.1 (C=O), 164.6 (C=O), 133.5,
133.5, 133.4, 133.3, 130.1–128.2 (aromatic protons), 117.8 (allyl
=CH2), 96.8 (C-1), 68.7 (allyl OCH2), 67.4, 66.8, 66.7, 64.5, 63.4
(C-6) ppm. C37H32O10 (636.66): calcd. C 69.80, H 5.07; found C
69.87, H 5.07.
Method B: A solution of peracetate 9 (3.13 g, 8.00 mmol) and
benzyl alcohol (3.10 mL, 30.0 mmol) in dichloromethane (60 mL)
was stirred for 20–30 min at room temperature over molecular si-
eves (4 Å, 1.0 g, finely ground), then BF3·Et2O (3.8 mL, 30.0 mmol)
was added and the mixture was stirred at room temperature for
4 h. The reaction was quenched with Et3N (7.0 mL), then the mix-
ture was concentrated and purified by column chromatography
(hexane/ethyl acetate, 40:1Ǟ2:1) to yield the title compound
(2.92 g, 83%) as a thick oil. [α]2D0 = 75.3 (c = 0.3, chloroform). IR
4.2.7. (E)-1-Propenyl 2,3,4,6-Tetra-O-benzoyl-α-D-idopyranoside
(19): To degassed THF (10 mL), [Ir(COD)(MePPh2)2]PF6 (7 mg,
0.008 mmol) was added under an Ar atmosphere. The Ar in the
system was replaced with H2 and the suspension was stirred at
room temperature for 15 min. Insoluble red crystals slowly dis-
solved and the clear solution became colorless. This solution was
transferred into a solution of allyl α-d-idopyranoside 18 (632 mg,
1.00 mmol) in THF (20 mL), stirred at room temperature for 1 h,
and then concentrated. The residue was purified by column
chromatography (hexane/ethyl acetate, 40:1Ǟ5:1) to give the title
compound (630 mg, 98%) as a foam. [α]2D0 = 17.8 (c = 0.2, chloro-
(film): ν
= 1746, 1371, 1225, 1052 cm–1. 1H NMR (500 MHz,
˜
max
CDCl3): δ = 7.36–7.30 (m, 5 H, ArH), 5.00–5.02 (m, 1 H, 3-H),
4.92–4.90 (m, 3 H, 1,2,4-H), 4.77 and 4.54 (ABq, J = 11.9 Hz, 2
H, PhCH2), 4.50–4.46 (m, 1 H, 5-H), 4.20 (d, 2 H, 6,6Ј-H), 2.12 (s,
3 H, CH3), 2.08 (s, 3 H, CH3), 2.06 (s, 3 H, CH3), 2.05 (s, 3 H,
CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.4 (C=O), 169.7
(C=O), 169.2 (C=O), 169.1 (C=O), 136.9, 128.4, 127.9, 127.5, 96.7
(C-1), 69.2 (PhCH2), 67.2, 67.1, 66.8, 64.6, 62.4 (C-6), 20.8 (CH3),
20.7 (CH3), 20.7 (CH3), 20.6 (CH3) ppm. C21H26O10 (438.44):
calcd. C 57.53, H 5.98; found C 57.81, H 6.19.
form). IR (film): ν
= 1725, 1602, 1452, 1316, 1268, 1172, 1112,
˜
max
1070, 1028, 757, 710 cm–1. 1H NMR (500 MHz, CDCl3): δ = 8.17–
7.17 (m, 20 H, ArH), 6.29 (dq, 4J = 1.6, J = 12.3 Hz, 1 H, OCH=),
5.66–5.64 (m, 1 H, 3-H), 5.39 (br. s, 1 H, 4-H), 5.37 (br. s, 1 H, 1-
H), 5.26–5.25 (m, 1 H, 2-H), 5.25–5.19 (dq, J = 12.3, 6.8 Hz, 1 H,
4.2.4. Allyl 2,3,4,6-Tetra-O-acetyl-α-
D-idopyranoside (16): Obtained
as described for the synthesis of compound 12 (Method B). Per-
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5

Job/Unit: O42187
/KAP1
Date: 06-05-14 17:56:27
Pages: 11
M. Strnad, Z. Pakulski et al.
C36H28Cl3NO10 (740.99): calcd. C 58.35, H 3.81, N 1.89, Cl 14.35;
FULL PAPER
=CH-), 4.92–4.88 (m, 1 H, 5-H), 4.67 (dd, J5,6 = 8.2, J6,6Ј
=
11.6 Hz, 1 H, 6-H), 4.53 (dd, J6Ј,5 = 4.3, J6,6Ј = 11.6 Hz, 1 H, 6Ј- found C 58.13, H 3.88, N 1.87, Cl 14.43.
H), 1.49 (dd, 4J = 1.6, J = 6.8 Hz, 3 H, -CH3) ppm. 13C NMR
4.2.10. Synthesis of Saponins (Glycosylation); General Procedure: A
(125 MHz, CDCl3): δ = 166.1 (C=O), 165.3 (C=O), 165.1 (C=O),
164.5 (C=O), 142.4 (=CH), 133.6, 133.4, 133.2, 130.1–128.2 (aro-
matic protons), 105.1 (=CH), 96.6 (C-1), 66.6, 66.6, 66.4, 64.9, 63.4
(C-6), 12.3 (CH3) ppm. C37H32O10 (636.66): calcd. C 69.80, H 5.07;
found C 69.74, H 5.15.
solution of d-idopyranosyl trichloroacetimidate (7 or 8, 0.28 mmol)
and a suitable triterpene acceptor (0.25 mmol) in dichloromethane
(15 mL) was stirred for 20–30 min at room temperature over mo-
lecular sieves (4 Å, 300 mg, finely ground), then cooled to –40 °C
and TMSOTf (30 μL, 0.15 mmol) was added. The mixture was
stirred for 30 min, and the reaction was quenched with Et3N
(0.5 mL). The mixture was concentrated, and the residue was puri-
fied by column chromatography (hexane/ethyl acetate, 10:1Ǟ7:3)
to give the protected saponin as a foam.
4.2.8. 2,3,4,6-Tetra-O-benzoyl-α,β-D-idopyranose (15): Method A:
To a solution of benzyl α-d-idopyranoside 14 (3.70 g, 5.39 mmol)
in either ethyl acetate (75 mL) or THF (30 mL), 10% palladium on
charcoal (300 mg) was added and the mixture was stirred under
hydrogen atmosphere for 16 h. The mixture was filtered through a
short silica pad, and purified by column chromatography (hexane/
ethyl acetate, 10:1Ǟ1:1) to give recovered starting material (0.18 g,
5%), and the title compound (2.89 g, 90%) as a foam.
4.2.10.1.
acetyl-α-
1-O-[3-β-Acetoxylup-20(29)-ene-28-oyl]-2,3,4,6-tetra-O-
D-idopyranosyl (20): Yield 89% starting from 3-O-acetyl-
betulinic acid (1) and 2,3,4,6-tetra-O-acetyl-d-idopyranosyl tri-
chloroacetimidate (7). [α]2D0 = 33.7 (c = 0.3, chloroform). H NMR
1
(500 MHz, CDCl3): δ = 6.11 (br. d, J1,2 = 1.3 Hz, 1 H, 1Ј-H), 5.11–
5.08 (m, 1 H, 3Ј-H), 4.95–4.93 (m, 1 H, 4Ј-H), 4.85–4.83 (m, 1 H,
2Ј-H), 4.73 (br. s, 1 H, 29-H), 4.61 (br. s, 1 H, 29-H), 4.47 (dd, J =
6.1, 10.2 Hz, 1 H, 3-H), 4.42 (ddd, J = 2.3, 6.4 Hz, 1 H, 5Ј-H),
4.20–4.17 (m, 2 H, 6Ј-H, 6Ј-H), 3.00–2.93 (m, 1 H, 19-H), 2.33–
2.21 (m, 3 H), 2.13 (s, 3 H, CH3), 2.11 (s, 3 H, CH3), 2.10 (s, 3 H,
CH3), 2.06 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 1.92–1.85 (m, 2 H),
1.68 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.94 (s, 3 H, CH3), 0.84 (s,
6 H, 2ϫ CH3), 0.83 (s, 3 H, CH3), 1.63–1.20 (m, 19 H, lupane
protons) ppm. 13C NMR (125 MHz, CDCl3): δ = 173.2 (C=O),
171.0 (C=O), 170.3 (C=O), 169.6 (C=O), 169.0 (C=O), 168.7
(C=O), 150.0 (C-20), 109.9 (C-29), 89.8 (1JC1,H1 = 174 Hz, C-1Ј),
80.9 (C-3), 66.6, 66.4, 66.3, 66.0, 61.8 (C-6Ј), 56.7 (C), 55.5, 50.5,
49.2, 46.6, 42.4 (C), 40.7 (C), 38.4 (CH2), 38.0, 37.8 (C), 37.1 (C),
36.6 (CH2), 34.3 (CH2), 32.0 (CH2), 30.3 (CH2), 29.5 (CH2), 27.9,
25.4 (CH2), 23.7 (CH2), 21.3, 20.9 (CH2), 20.7, 20.6, 20.6, 19.3,
18.1 (CH2), 16.5, 16.2, 16.0, 14.6 ppm. HRMS (ESI): m/z calcd. for
C46H68NaO13 [M + Na]+ 851.4552; found 851.4545.
Method B: To a solution of 19 (600 mg, 0.94 mmol) in a mixture
of acetone/water (10:1, 10 mL), yellow mercury oxide (270 mg,
1.25 mmol) followed by a solution of mercury(II) chloride (300 mg,
1.10 mmol) in a mixture of acetone/water (10:1, 2 mL) were added
and the mixture was stirred at room temperature for 4 h. The mix-
ture was concentrated, and the residue was purified by column
chromatography (hexane/ethyl acetate, 20:1Ǟ3:1) to give the title
compound (540 mg, 96%) as a foam. IR (film): νmax = 1716, 1450,
˜
1
1262, 1109, 1090, 1069, 707 cm–1. H NMR (500 MHz, CDCl3): δ
= 8.12–7.19 (m, 20 H, ArH), 5.87 (t, J3,2 = J3,4 = 3.9 Hz, 1 H, 3-
Hβ), 5.74 (ddd, J = 1.0, 3.7 Hz, 1 H, 3-Hα), 5.54 (br. d, J = 3.7 Hz,
1 H, 1-Hα), 5.49 (dd, J1,2 = 8.2, J1,3 = 1.8 Hz, 1 H, 1-Hβ), 5.47–
5.45 (m, 1 H, 4-Hα), 5.43–5.40 (m, 1 H, 4-Hβ), 5.34–5.32 (m, 1 H,
2-Hβ), 5.27–5.25 (m, 1 H, 2-Hα), 5.09–5.05 (m, 1 H, 5-Hα), 4.79
(dd, J6,5 = 7.0, J6,6Ј = 11.3 Hz, 1 H, 6-Hβ), 4.73 (dd, J6,5 = 7.1,
J6,6Ј = 11.6 Hz, 1 H, 6-Hα), 4.71–4.68 (m, 1 H, 5-Hβ), 4.61 (dd,
J6Ј,5 = 5.4, J6,6Ј = 11.3 Hz, 1 H, 6Ј-Hβ), 4.55 (dd, J6Ј,5 = 5.3, J6,6Ј
= 11.6 Hz, 1 H, 6Ј-Hα) ppm. 13C NMR (125 MHz, CDCl3): δ =
166.2 (C=O α), 166.2 (C=O β), 165.9 (C=O β), 165.4 (C=O α),
165.3 (C=O α), 165.2 (C=O β), 164.6 (C=O α), 164.1 (C=O β),
133.8, 133.6, 133.6, 133.5, 133.5, 133.4, 133.2, 133.2, 130.1–128.2
(aromatic protons), 92.6 (C-1α), 92.5 (C-1β), 72.3 (β), 68.8 (β), 68.3
(α),68.0 (β), 67.2 (α), 67.0 (α),66.6 (β), 64.9 (α),63.3 (C-6β), 63.1
(C-6α) ppm. C34H28O10 (596.60): calcd. C 68.45, H 4.73; found C
68.40, H 4.77.
4.2.10.2.
benzoyl-α-
1-O-[3-β-Acetoxylup-20(29)-ene-28-oyl]-2,3,4,6-tetra-O-
D-idopyranosyl (21): Yield 86% starting from 3-O-acetyl-
betulinic acid (1) and 2,3,4,6-tetra-O-benzoyl-d-idopyranosyl tri-
chloroacetimidate (8). [α]2D0 = 34.1 (c = 0.25, chloroform). IR (film):
ν
= 2948, 2871, 1728, 1451, 1263, 1250, 1107, 1069, 1027,
˜
max
711 cm–1. 1H NMR (500 MHz, CDCl3): δ = 8.12–7.19 (m, 20 H,
ArH), 6.48 (br. s, 1 H, 1Ј-H), 5.71–5.69 (m, 1 H, 3Ј-H), 5.52–5.50
(m, 1 H, 4Ј-H), 5.28–5.26 (m, 1 H, 2Ј-H), 4.90 (ddd, J = 1.9, 6.6 Hz,
4.2.9. 2,3,4,6-Tetra-O-benzoyl-α,β-D-idopyranosyl Trichloroacet-
1 H, 5Ј-H), 4.71 (br. s, 1 H, 29-H), 4.68 (dd, J6,5 = 6.6, J6,6Ј
=
imidate (8): Obtained as described for the synthesis of 7. d-Idopyr-
anose 15 (540 mg, 0.91 mmol) was treated with trichloroacetonitrile
(0.40 mL, 4.00 mmol) in the presence of potassium carbonate
(100 mg, 0.72 mmol) and DBU (3 drops) in dichloromethane
11.6 Hz, 1 H, 6Ј-H), 4.58 (br. s, 1 H, 29-H), 4.55 (dd, J6Ј,5 = 6.6,
J6,6Ј = 11.6 Hz, 1 H, 6Ј-H), 4.46 (dd, J = 5.8, 10.3 Hz, 1 H, 3-H),
2.98–2.92 (m, 1 H, 19-H), 2.34–2.29 (m, 2 H), 2.04 (s, 3 H, CH3),
1.84–1.76 (m, 2 H), 1.64 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.84 (s,
3 H, CH3), 0.83 (s, 3 H, CH3), 0.82 (s, 3 H, CH3), 0.78 (s, 3 H,
CH3), 1.62–1.10 (m, 20 H, lupane protons) ppm. 13C NMR
(125 MHz, CDCl3): δ = 173.2 (C=O), 171.0 (C=O), 166.0 (C=O),
165.2 (C=O), 164.9 (C=O), 164.5 (C=O), 150.1 (C-20), 133.8,
133.6, 133.5, 133.2, 109.8 (C-29), 89.9 (C-1Ј), 80.9 (C-3), 66.8, 66.7,
66.5, 66.2, 62.5 (C-6Ј), 56.7 (C), 55.4, 50.5, 49.4, 46.4, 42.4 (C),
40.6 (C), 38.4 (CH2), 37.8, 37.8 (C), 37.1 (C), 36.4 (CH2), 34.1
(CH2), 32.1 (CH2), 30.3 (CH2), 29.4 (CH2), 27.9, 25.4 (CH2), 23.7
(CH2), 21.3, 20.8 (CH2), 19.3, 18.2 (CH2), 16.5, 16.2, 15.9,
14.6 ppm. HRMS (ESI): m/z calcd. for C66H76NaO13 [M + Na]+
1099.5184; found 1099.5179.
1
(20 mL) to give the title compound (533 mg, 80%) as a foam. H
NMR (500 MHz, CDCl3): δ = 8.80 (s, 1 H, NH-α), 8.61 (s, 1 H,
NH-β), 8.22–7.16 (m, 20 H, ArH), 6.74 (d, J1,2 = 3.0 Hz, 1 H, 1-
Hβ), 6.64 (br. s, 1 H, 1-Hα), 6.23 (t, J = 7.7 Hz, 1 H, 3-Hβ), 5.73–
5.71 (m, 1 H, 3-Hα), 5.64–5.68 (m, 2 H, 4-,2-Hβ), 5.47–5.45 (m, 1
H, 4-Hα), 5.45–5.43 (m, 1 H, 2-Hα), 5.08 (ddd, J5,4 = 1.1, J5,6
8.1, J5,6Ј = 4.4 Hz, 1 H, 5-Hα), 5.00–4.96 (m, 1 H, 5-Hβ), 4.87 (dd,
J6,5 = 6.6, J6,6Ј = 11.8 Hz, 1 H, 6-Hβ), 4.76 (dd, J6Ј,5 = 6.4, J6,6Ј
=
=
11.8 Hz, 1 H, 6Ј-Hβ), 4.67 (dd, J6,5 = 8.1, J6,6Ј = 11.7 Hz, 1 H, 6-
Hα), 4.58 (dd, J6Ј,5 = 4.4, J6,6Ј = 11.7 Hz, 1 H, 6Ј-Hα) ppm. 13C
NMR (125 MHz, CDCl3): δ = 166.0 (α-C=O), 165.9 (β-C=O),
165.4 (β-C=O), 165.3 (β-C=O), 165.1 (α-C=O), 165.0 (β-C=O),
164.8 (α-C=O), 164.4 (α-C=O), 160.7 (β-C=N), 160.1 (α-C=N), 4.2.10.3.
3β-O-(2,3,4,6-Tetra-O-benzoyl-α-
20(29)-ene (23): Yield 97% starting from lupeol (5) and 2,3,4,6-
tetra-O-benzoyl-d-idopyranosyl trichloroacetimidate (8). [α]2D0
58.9 (c = 0.3, chloroform). 1H NMR (600 MHz, CDCl3): δ = 8.18–
D-idopyranosyl)lup-
133.8, 133.7, 133.2, 130.4–128.2 (aromatic protons), 94.5 (C-1β),
94.2 (C-1α), 90.8 (C-Cl), 72.4 (β), 68.9 (β), 68.7 (β), 67.0 (β), 66.8
(α), 66.3 (α), 66.1 (α), 65.9 (α), 63.9 (C-6β), 63.3 (C-6α) ppm.
=
6
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O42187
/KAP1
Date: 06-05-14 17:56:27
Pages: 11
Lupane-Type Saponins Containing an α-d-Idopyranoside Fragment
7.15 (m, 20 H, ArH), 5.59–5.58 (m, 1 H, 3Ј-H), 5.40 (br. s, 1 H, 4Ј-
(CH2), 21.0, 20.8 (CH2), 19.2, 18.2 (CH2), 16.2, 16.1, 16.0,
H), 5.32 (br. s, 1 H, 2Ј-H), 5.20–5.19 (m, 1 H, 1Ј-H), 5.00–4.96 (m, 14.9 ppm. HRMS (ESI): m/z calcd. for C66H78NaO12 [M + Na]+
1 H, 5Ј-H), 4.69 (d, J = 1.9 Hz, 1 H, 29-H), 4.66 (dd, J6Ј,5 = 8.0,
J6,6Ј = 11.8 Hz, 1 H, 6Ј-H), 4.59 (dd, J6Ј,5 = 4.2, J6,6Ј = 11.8 Hz, 1
H, 6Ј-H), 4.57 (br. s, 1 H, 29-H), 3.35 (dd, J = 4.2, 11.8 Hz, 1 H,
3-H), 2.41–2.35 (m, 1 H), 1.96–1.89 (m, 1 H), 1.86–1.81 (m, 1 H),
1.69 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.95 (s,
3 H, CH3), 0.79 (s, 6 H, 2ϫ CH3), 0.69 (s, 3 H, CH3), 0.57–0.54
(m, 1 H), 1.68–0.75 (m, 21 H, lupane protons) ppm. 13C NMR
(150 MHz, CDCl3): δ = 166.1 (C=O), 165.3 (C=O), 165.2 (C=O),
164.7 (C=O), 150.9 (C-20), 133.5, 133.5, 133.2, 133.2, 109.4 (C-29),
93.8 (1JC1,H1 = 170 Hz, C-1Ј), 83.0 (C-3), 68.2, 67.2, 66.8, 64.6, 63.7
(CH2), 55.8, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.8 (C), 40.0
(CH2), 38.4 (C), 38.3 (CH2), 38.0, 37.0 (C), 35.6 (CH2), 34.2 (CH2),
29.8 (CH2), 28.6, 27.4 (CH2), 25.1 (CH2), 21.5 (CH2), 20.9 (CH2),
19.3, 18.2 (CH2),18.0, 16.2, 16.1, 15.9, 14.7 ppm. HRMS (ESI): m/z
calcd. for C64H76NaO10 [M + Na]+ 1027.5336; found 1027.5341.
C64H76O10·3/2H2O (591.88): calcd. C 74.46, H 7.71; found C 74.63,
H 7.73.
1085.5391; found 1085.5397. C66H78O12·H2O (1081.37): calcd. C
73.31, H 7.46; found C 73.22, H 7.66.
4.2.10.6. 28-O-tert-Butyldiphenylsilyl-3β-O-(2,3,4,6-tetra-O-benzo-
yl-α-D-idopyranosyl)lup-20(29)-ene (29): Yield 87% starting from
28-O-tert-butyldiphenylsilylbetulin (4) and 2,3,4,6-tetra-O-benzoyl-
d-idopyranosyl trichloroacetimidate (8). [α]D20 = 35.4 (c = 0.3, chloro-
form). IR (film): ν
= 2942, 2860, 1727, 1452, 1265, 1108, 1027,
˜
max
709 cm–1. 1H NMR (500 MHz, CDCl3): δ = 8.16–7.12 (m, 30 H,
ArH), 5.59–5.57 (m, 1 H, 3Ј-H), 5.40 (br. s, 1 H, 4Ј-H), 5.31 (s, 1
H, 1Ј-H), 5.19–5.18 (m, 1 H, 2Ј-H), 4.99–4.95 (m, 1 H, 5Ј-H), 4.66
(dd, J6,5 = 8.2, J6,6Ј = 11.6 Hz, 1 H, 6Ј-H), 4.56–4.60 (m, 2 H, 6Ј-
H, 29-H), 4.53 (br. s, 1 H, 29-H), 3.70–3.66 (m, 1 H, 28-H), 3.31–
3.35 (m, 2 H, 3-H, 28-H), 2.29–2.23 (m, 1 H), 2.16–2.10 (m, 1 H),
1.87–1.78 (m, 2 H), 1.54 (s, 3 H, CH3), 1.06 (s, 9 H, tert-butyl),
1.01 (s, 3 H, CH3), 0.93 (s, 3 H, CH3), 0.72 (s, 3 H, CH3), 0.68 (s,
3 H, CH3), 0.67 (s, 3 H, CH3), 1.53–0.78 (m, 18 H, lupane pro-
tons) ppm. 13C NMR (125 MHz, CDCl3): δ = 166.1 (C=O), 165.3
(C=O), 165.2 (C=O), 164.7 (C=O), 150.8 (C-20), 135.6, 133.8,
133.5, 133.2, 130.2, 109.4 (C-29), 93.8 (C-1Ј), 83.0 (C-3), 77.2, 68.2,
67.3, 66.8, 64.6, 63.7 (CH2), 61.1 (CH2), 55.8, 50.3, 48.5, 48.4 (C),
47.8, 42.6 (C), 40.8 (C), 38.4 (C), 38.2 (CH2), 37.2, 37.0 (C), 34.5
(CH2), 34.1 (CH2), 29.9 (CH2), 29.5 (CH2), 28.6, 27.1 (CH2), 26.9
(3ϫ CH3), 25.1 (CH2), 21.5 (CH2), 20.7 (CH2), 19.4 (C), 19.1, 18.2
(CH2), 16.2, 16.0, 15.7, 14.9 ppm. C80H91O11Si (1259.72): calcd. C
76.28, H 7.52; found C 76.41, H 7.40.
4.2.10.4. 3β-O-Acetyl-28-O-(2,3,4,6-tetra-O-benzoyl-α-D-idopyran-
osyl)lup-20(29)-ene (25): Yield 87% starting from 3-O-acetylbetulin
(2) and 2,3,4,6-tetra-O-benzoyl-d-idopyranosyl trichloroacetimid-
ate (8). [α]2D0 = 45.9 (c = 0.3, chloroform). IR (film): ν
= 2947,
˜
max
2871, 1727, 1451, 1264, 1249, 1108, 1028, 710 cm–1. 1H NMR
(500 MHz, CDCl3): δ = 8.16–7.17 (m, 20 H, ArH), 5.63–5.62 (m,
1 H, 3Ј-H), 5.40 (br. s, 1 H, 4Ј-H), 5.27–5.25 (m, 1 H, 2Ј-H), 5.01
(s, 1 H, 1Ј-H), 4.87–4.83 (m, 1 H, 5Ј-H), 4.64–4.68 (br. s, 1 H, 29-
H), 4.67–4.60 (m, 1 H, 6Ј-H), 4.62 (dd, J6Ј,5 = 6.3, J6,6Ј = 11.6 Hz,
1 H, 6Ј-H), 4.58–4.57 (m, 1 H, 29-H), 4.45 (dd, J = 4.3, 11.0 Hz, 1
H, 3-H), 4.08–4.05 (m, 1 H, 28-H), 3.25 (d, J = 9.8 Hz, 1 H, 28-
H), 2.39–2.30 (m, 1 H), 2.04 (s, 3 H, CH3), 1.67 (s, 3 H, CH3), 0.94
(s, 3 H, CH3), 0.87 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.82 (s, 3 H,
CH3), 0.74 (s, 3 H, CH3), 1.90–1.15 (m, 24 H, lupane protons) ppm.
13C NMR (125 MHz, CDCl3): δ = 171.0 (C=O), 166.0 (C=O),
165.3 (C=O), 165.1 (C=O), 164.5 (C=O), 150.3 (C-20), 133.5,
133.3, 133.2, 109.8 (C-29), 98.6 (C-1Ј), 80.9 (C-3), 67.4, 66.8, 66.5,
64.4, 62.9 (C-6), 55.3, 50.2, 48.8, 48.1, 47.0 (C), 42.6 (C), 40.8 (C),
38.3 (CH2), 37.8 (C), 37.5, 37.0 (C), 34.5 (CH2), 34.0 (CH2), 29.8
(CH2), 27.9 (CH, CH2), 27.2 (CH2), 25.0 (CH2), 23.6 (CH2), 21.3,
20.6 (CH2), 19.0, 18.1 (CH2), 16.4, 16.1, 15.9, 14.7 ppm. HRMS
(ESI): m/z calcd. for C66H78NaO12 [M + Na]+ 1085.5391; found
1085.5386. C66H78O12·0.5H2O (1072.36): calcd. C 73.92, H 7.43;
found C 73.76, H 7.34.
4.2.10.7.
3β-O-(2,3,4,6-Tetra-O-benzoyl-α-D-idopyranosyl)lup-
20(29)-en-28-ol (30): To a solution of 29 (440 mg, 0.35 mmol) in
THF (12 mL), tetrabutylammonium fluoride (1 m in THF,
1.75 mL, 1.75 mmol) was added followed by acetic acid (100 μL,
1.75 mmol) and the mixture was stirred in a sealed tube at 60 °C
for 17 h. The mixture was then concentrated and the residue was
purified by column chromatography (hexane/ethyl acetate,
7:1Ǟ3:1) to give the title compound (279 mg, 78%) as a foam.
[α]2D0 = 48.9 (c = 0.4, chloroform). IR (film): ν
= 2942, 2871,
˜
max
1726, 1451, 1265, 1107, 1027, 710 cm–1. 1H NMR (500 MHz,
CDCl3): δ = 8.16–7.14 (m, 20 H, ArH), 5.59–5.57 (m, 1 H, 3Ј-H),
5.41 (br. s, 1 H, 4Ј-H), 5.32 (s, 1 H, 1Ј-H), 5.20–5.18 (m, 1 H, 2Ј-
H), 5.00–4.96 (m, 1 H, 5Ј-H), 4.65–4.70 (m, 2 H, 6Ј-H, 29-H), 4.56–
4.60 (m, 2 H, 6Ј-H, 29-H), 3.79 (d, J = 10.2 Hz, 1 H, 28-H), 3.32–
3.36 (m, 2 H, 3-H, 28-H), 2.42–2.35 (m, 1 H), 1.96–1.82 (m, 2 H),
1.70 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.98 (s,
3 H, CH3), 0.78 (s, 3 H, CH3), 0.69 (s, 3 H, CH3), 1.72–0.95 (m,
23 H, lupane protons) ppm. 13C NMR (125 MHz, CDCl3): δ =
166.1 (C=O), 165.3 (C=O), 165.2 (C=O), 164.7 (C=O), 150.4 (C-
20), 133.5, 133.2, 109.7 (C-29), 93.8 (C-1Ј), 83.0 (C-3), 77.2, 68.2,
67.2, 66.8, 64.6, 63.7 (CH2), 60.5 (CH2), 55.8, 50.3, 48.8, 47.8 (C,
CH), 42.7 (C), 40.9 (C), 38.4 (C), 38.3 (CH2), 37.3, 37.0 (C), 34.2
(CH2), 34.0 (CH2), 29.7 (CH2), 29.2 (CH2), 28.6, 27.0 (CH2), 25.2
(CH2), 21.5 (CH2), 20.8 (CH2), 19.1, 18.2 (CH2), 16.2, 16.1, 15.9,
14.9 ppm. HRMS (ESI): m/z calcd. for C64H76NaO11 [M + Na]+
1043.5285; found 1043.5289.
4.2.10.5. 28-O-Acetyl-3β-O-(2,3,4,6-tetra-O-benzoyl-α-D-idopyran-
osyl)lup-20(29)-ene (27): Yield 81% starting from 28-O-acetylbetu-
lin (3) and 2,3,4,6-tetra-O-benzoyl-d-idopyranosyl trichloroacetim-
idate (8). [α]2D0 = 54.9 (c = 0.2, chloroform). IR (film): νmax = 2945,
˜
2871, 1726, 1452, 1265, 1248, 1107, 1028, 711 cm–1. 1H NMR
(500 MHz, CDCl3): δ = 8.16–7.15 (m, 20 H, ArH), 5.59–5.57 (m,
1 H, 3Ј-H), 5.41 (br. s, 1 H, 4Ј-H), 5.32 (s, 1 H, 1Ј-H), 5.20–5.18
(m, 1 H, 2Ј-H), 4.98 (ddd, J = 1.5, 4.2, 7.2 Hz, 1 H, 5Ј-H), 4.69–
4.67 (m, 1 H, 29-H), 4.67 (dd, J6,5 = 8.2, J6,6Ј = 11.6 Hz, 1 H, 6Ј-
H), 4.56–4.60 (m, 2 H, 6Ј-H, 29-H), 4.27–4.24 (m, 1 H, 28-H), 3.85
(d, J = 11.1 Hz, 1 H, 28-H), 3.34 (dd, J = 4.4, 11.8 Hz, 1 H, 3-H),
2.48–2.41 (m, 1 H), 2.07 (s, 3 H, CH3), 2.00–1.92 (m, 1 H), 1.70 (s, 4.2.10.8. 3β,28-Di-O-(2,3,4,6-tetra-O-benzoyl-α-
3 H, CH3), 1.02 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.98 (s, 3 H, 20(29)-ene (32): Yield 97% starting from 30 and 2,3,4,6-tetra-O-
CH3), 0.78 (s, 3 H, CH3), 0.69 (s, 3 H, CH3), 1.85–0.55 (m, 23 benzoyl-d-idopyranosyl trichloroacetimidate (8). [α]2D0 = 51.0 (c =
H, lupane protons) ppm. 13C NMR (125 MHz, CDCl3): δ = 171.6
0.3, chloroform). IR (film): ν = 2946, 2871, 1726, 1452, 1266,
(C=O), 166.1 (C=O), 165.3 (C=O), 165.2 (C=O), 164.7 (C=O), 1108, 1028, 757, 710 cm–1. H NMR (600 MHz, CDCl3): δ [sugar
D-idopyranosyl)lup-
˜
max
1
150.1 (C-20), 109.9 (C-29), 93.9 (C-1Ј), 83.1 (C-3), 68.2, 67.2, 66.8,
64.6, 63.7 (CH2), 62.8 (CH2), 55.8, 50.3, 48.8, 47.7, 46.3 (C), 42.7
(C), 40.9 (C), 38.4 (CH2), 38.3 (C), 37.5, 37.0 (C), 34.6 (CH2), 34.2
(CH2), 29.8 (CH2), 29.6 (CH2), 28.6, 27.1 (CH2), 25.2 (CH2), 21.6
protons with prime (Ј) mark belong to idose ring at position 3 of
lupane, with double prime (ЈЈ) to idose ring at position 28 of lup-
ane] = 8.16–6.95 (m, 40 H, ArH), 5.64–5.62 (m, 1 H, 3ЈЈ-H), 5.60–
5.58 (m, 1 H, 3Ј-H), 5.41 (br. s, 2 H, 4Ј,4ЈЈ-H), 5.31 (s, 1 H, 1Ј-H),
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O42187
/KAP1
Date: 06-05-14 17:56:27
Pages: 11
M. Strnad, Z. Pakulski et al.
3-H), 3.98–4.13 (m, 5 H), 3.64 (br. s, 1 H), 3.04–2.93 (m, 1 H),
FULL PAPER
5.27–5.26 (m, 1 H, 2ЈЈ-H), 5.20–5.19 (m, 1 H, 2Ј-H), 5.10 (s, 1 H,
1ЈЈ-H), 5.00–4.96 (m, 1 H, 5Ј-H), 4.87–4.84 (m, 1 H, 5ЈЈ-H), 4.56– 2.34–2.18 (m, 3 H), 2.05 (s, 3 H, CH3), 1.68 (s, 3 H, CH3), 0.96 (s,
4.69 (m, 5 H, 6Ј,6Ј,6ЈЈ,6ЈЈ,29-H), 4.05 (br. d, J = 9.3 Hz, 1 H, 28- 3 H, CH3), 0.92 (s, 3 H, CH3), 0.85 (s, 3 H, CH3), 0.84 (s, 3 H,
H), 3.34 (dd, J = 4.3, 11.7 Hz, 1 H, 3-H), 3.26 (d, J = 9.7 Hz, 1 H,
CH3), 0.83 (s, 3 H, CH3), 1.93–1.18 (m, 25 H, lupane protons) ppm.
28-H), 2.38–2.33 (m, 1 H), 1.68 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 13C NMR (125 MHz, CDCl3): δ = 173.9 (C=O), 171.1 (C=O),
0.95 (s, 3 H, CH3), 0.85 (s, 3 H, CH3), 0.68 (s, 3 H, CH3), 0.67 (s, 150.0 (C-20), 109.9 (C-29), 93.9 (C-1Ј), 80.9 (C-3), 72.0, 68.4, 68.3,
3 H, CH3), 2.00–0.85 (m, 25 H, lupane protons) ppm. 13C NMR
67.0, 64.9 (C-6Ј), 56.9 (C), 55.4, 50.5, 49.3, 46.6, 42.5 (C), 40.7 (C),
(125 MHz, CDCl3): δ = 166.1 (C=O), 166.0 (C=O), 165.3 (C=O),
38.4 (CH2), 38.1, 37.8 (C), 37.1 (C), 36.8 (CH2), 34.3 (CH2), 32.3
165.3 (C=O), 165.2 (C=O), 165.2 (C=O), 164.7 (C=O), 164.5 (CH2), 30.3 (CH2), 29.6 (CH2), 27.9, 25.4 (CH2), 23.7 (CH2), 21.3,
(C=O), 150.2 (lupane C-20), 133.5, 133.3, 133.2, 109.8 (lupane C- 20.9 (CH2), 19.3, 18.2 (CH2), 16.5, 16.2, 14.7 ppm. HRMS (ESI):
29), 98.7 (C-1), 93.9 (C-1), 83.1 (lupane C-3), 77.2, 68.2, 67.4, 67.2,
67.2 (CH2), 66.9, 66.8, 66.5, 64.6, 64.5, 63.6 (CH2), 62.9 (CH2), C38H60O9·1.5H2O (687.92): calcd. C 66.35, H 9.23; found C 66.71,
m/z calcd. for C38H60NaO9 [M + Na]+ 683.4135; found 683.4139.
55.8, 50.3, 48.8, 48.0, 47.1 (C), 42.7 (C), 40.8 (C), 38.4 (C), 38.2
(CH2), 37.5, 37.0 (C), 34.5 (CH2), 34.1 (CH2), 29.8 (CH2), 28.6,
27.2 (CH2), 25.0 (CH2), 21.5 (CH2), 21.3, 20.6 (CH2), 19.0, 18.2
(CH2), 16.2, 16.0, 15.9, 14.9 ppm. HRMS (ESI): m/z calcd. for
C98H102NaO20 [M + Na]+ 1621.6862; found 1621.6917.
H 9.41.
4.2.11.2. 3β-O-(α-D-Idopyranosyl)lup-20(29)-ene (24): Yield 81%
starting from 23. [α]2D0 = 73.4 (c = 0.3, chloroform). IR (film): ν
˜
max
= 3402 (br), 2943, 2871, 1055, 1016, 882, 759 cm–1. 1H NMR
(500 MHz, CDCl3): δ = 5.13 (s, 1 H), 4.69 (br. d, J = 2.2 Hz, 1 H),
4.58–4.56 (m, 1 H), 4.16 (br. s, 1 H), 4.07–3.99 (m, 3 H), 3.95–3.90
(m, 1 H), 3.65 (br. s, 1 H), 3.53 (d, J = 9.1 Hz, 1 H, 29-H), 3.30
(dd, J = 4.2, 11.8 Hz, 1 H, 3-H), 2.41–2.34 (m, 1 H), 1.97–1.88 (m,
1 H), 1.68 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 0.96 (s, 3 H, CH3),
0.94 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.79 (s, 3 H, CH3), 0.76
(s, 3 H, CH3), 0.74–0.70 (m, 1 H), 1.80–0.75 (m, 25 H, lupane
4.2.10.9. 28-O-Acetyl-3α-O-(2,3,4,6-tetra-O-benzoyl-α-D-idopyran-
osyl)lup-20(29)-ene (34): Yield 52% starting from 28-O-acetyl-epi-
betulin (6) and 2,3,4,6-tetra-O-benzoyl-d-idopyranosyl trichloro-
acetimidate (8). [α]2D0 = 23.0 (c = 0.25, chloroform). IR (film): ν
˜
max
= 2945, 2871, 1727, 1451, 1265, 1247, 1107, 1028, 710 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 8.15–7.12 (m, 20 H, ArH), 5.62–
5.60 (m, 1 H, 3Ј-H), 5.39 (br. s, 1 H, 4Ј-H), 5.35 (br. s, 1 H, 2Ј-H), protons) ppm. 13C NMR (125 MHz, CDCl3): δ = 150.9 (C-20),
5.20 (s, 1 H, 1Ј-H), 5.04–5.00 (m, 1 H, 5Ј-H), 4.74 (br. d, J = 3.5 Hz,
1 H, 29-H), 4.67–4.72 (m, 2 H, 6Ј,29-H), 4.59 (dd, J6Ј,5 = 4.8, J6,6Ј
= 11.7 Hz, 1 H, 6Ј-H), 4.21 (d, J = 11.0 Hz, 1 H, 28-H), 3.85 (d, J
109.4 (C-29), 98.4 (C-1Ј), 84.9 (C-3), 72.7, 68.5, 68.2, 65.3 (CH2),
65.1, 55.5, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.8 (C), 40.0 (CH2),
38.3 (C), 38.2 (CH2), 38.0, 37.1 (C), 35.6 (CH2), 34.2 (CH2), 29.8
= 11.0 Hz, 1 H, 28-H), 3.44 (br. s, 1 H, 3-H), 2.47–2.41 (m, 1 H), (CH2), 28.7, 27.4 (CH2), 25.1 (CH2), 22.3 (CH2), 20.9 (CH2), 19.3,
2.06 (s, 3 H, CH3), 2.02–1.93 (m, 1 H), 1.77 (s, 3 H, CH3), 0.96 (s, 18.3 (CH2),18.0, 16.5, 16.0, 16.0, 14.5 ppm. HRMS (ESI): m/z
3 H, CH3), 0.90 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.76 (s, 3 H,
CH3), 0.67 (s, 3 H, CH3), 1.85–0.85 (m, 23 H, lupane protons) ppm.
13C NMR (125 MHz, CDCl3): δ = 171.6 (C=O), 166.2 (C=O),
165.4 (C=O), 165.1 (C=O), 164.6 (C=O), 150.3 (C-20), 133.6,
133.5, 109.8 (C-29), 99.7 (C-1Ј), 85.0 (C-3), 67.6, 67.5, 67.4, 65.0,
63.5 (CH2), 62.8 (CH2), 49.8, 49.6, 48.9, 47.7, 46.3 (C), 42.6 (C),
40.9 (C), 38.3 (C), 37.4 (CH2), 37.1 (C), 34.5 (CH2), 34.1 (CH2),
33.7 (CH2), 29.7 (CH2), 29.7 (CH2), 28.1, 27.0 (CH2), 25.2 (CH2),
24.4 (CH2), 22.2, 21.0, 20.4 (CH2), 19.4, 17.8 (CH2), 16.0, 15.9,
14.7 ppm. HRMS (ESI): m/z calcd. for C66H78NaO12 [M + Na]+
1085.5391; found 1085.5419. C66H78O12·H2O (1081.37): calcd. C
73.31, H 7.46; found C 73.54, H 7.48.
calcd. for C36H60NaO6 [M + Na]+ 611.4288; found 611.4296.
4.2.11.3. 3β-O-Acetyl-28-O-(α-
Yield 76% starting from 25. [α]2D0 = 46.6 (c = 0.3, chloroform). IR
(film): ν = 3434 (br), 2945, 2872, 1727, 1455, 1375, 1250, 1090,
D-idopyranosyl)lup-20(29)-ene (26):
˜
max
1
1060, 1028, 980, 757 cm–1. H NMR (500 MHz, CDCl3): δ = 4.94
(s, 1 H, 1Ј-H), 4.70 (br. d, J = 1.9 Hz, 1 H), 4.62–4.60 (m, 1 H),
4.48 (dd, J = 5.3, 10.9 Hz, 1 H, 3-H), 4.01–4.13 (m, 4 H), 4.00–
3.98 (m, 1 H), 3.93 (br. s, 1 H), 3.73–3.71 (m, 1 H), 3.09 (d, J =
9.3 Hz, 1 H, 28-H), 2.42–2.36 (m, 1 H), 2.04 (s, 3 H, CH3), 1.98–
1.83 (m, 2 H), 1.68 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.97 (s, 3 H,
CH3), 0.85 (s, 3 H, CH3), 0.85 (s, 3 H, CH3), 0.84 (s, 3 H, CH3),
1.75–1.15 (m, 26 H, lupane protons) ppm. 13C NMR (125 MHz,
CDCl3): δ = 171.1 (C=O), 149.9 (C-20), 110.0 (C-29), 102.4 (C-1Ј),
80.9 (C-3), 72.5, 68.2, 67.7, 67.3 (CH2), 65.4 (CH2), 64.8, 55.3, 50.2,
48.8, 47.8, 46.7 (C), 42.7 (C), 40.9 (C), 38.3 (CH2), 37.8 (C), 37.6,
37.1 (C), 34.4 (CH2), 34.2 (CH2), 30.2 (CH2), 29.6 (CH2), 27.9, 27.1
(CH2), 25.1 (CH2), 23.6 (CH2), 21.3, 20.8 (CH2), 19.1, 18.1 (CH2),
16.5, 16.1, 16.1, 14.8 ppm. HRMS (ESI): m/z calcd. for
C38H62NaO8 [M + Na]+ 669.4342; found 669.4344.
4.2.11. Debenzoylation of Glycosides; General Procedure (Method
A): A suspension of protected saponin (0.20 mm) and K2CO3
(50 mg) in MeOH (10 mL) was stirred for 1 h, then neutralized with
Amberlyst 15 resin (H+ form), filtered through a PTFE syringe fil-
ter, and concentrated. The residue was purified by column
chromatography (hexane/ethyl acetate, 3:1Ǟethyl acetate) to give
unprotected saponin as an amorphous powder.
4.2.11.1. 1-O-[3-β-Acetoxylup-20(29)-ene-28-oyl]-α-
D-idopyranosyl 4.2.11.4. 28-O-Acetyl-3β-O-(α-D-idopyranosyl)lup-20(29)-ene (28):
(22): Yield 69% starting from 21. This compound was also ob- Yield 82% starting from 27. [α]2D0 = 54.1 (c = 0.3, chloroform). IR
tained from acetate 20 by treatment with sodium methoxide
(Method B): To a suspension of 20 (166 mg, 0.20 mm) in MeOH
(10 mL), sodium methoxide (0.95 m in methanol, 50 μL) was added
and the mixture was stirred at room temperature for 2 h. The mix-
ture was neutralized with Amberlyst 15 resin (H+ form), filtered
through a PTFE syringe filter, concentrated, and the residue was
purified by column chromatography (hexane/ethyl acetate,
3:1Ǟethyl acetate) to give the title compound (110 mg, 83%) as
(film): ν
= 3407 (br), 2943, 2871, 1738, 1455, 1389, 1364, 1240,
˜
max
1030, 757 cm–1. 1H NMR (500 MHz, CDCl3): δ = 5.13 (br. s, 1 H),
4.70–4.68 (m, 1 H), 4.59 (br. s, 1 H), 4.16–4.26 (m, 3 H), 3.99–4.07
(m, 3 H), 3.95–3.92 (m, 1 H), 3.85 (d, J = 11.2 Hz, 1 H, 28-H),
3.65 (br. s, 1 H), 3.52–3.49 (m, 1 H), 3.29 (dd, J = 4.2, 11.7 Hz, 1
H, 3-H), 2.47–2.41 (m, 1 H), 2.07 (s, 3 H, CH3), 2.01–1.91 (m, 1
H), 1.68 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.96
(s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 1.85–0.72 (m,
25 H, lupane protons) ppm. 13C NMR (125 MHz, CDCl3): δ =
171.7 (C=O), 150.1 (C-20), 109.9 (C-29), 98.4 (C-1Ј), 84.9 (C-3),
72.6, 68.5, 68.3, 65.3 (CH2), 65.1, 62.8 (CH2), 55.5, 50.3, 48.8, 47.7,
46.3 (C), 42.7 (C), 40.9 (C), 38.3 (C), 38.2 (CH2), 37.5, 37.0 (C),
an amorphous powder. [α]2D0 = 43.9 (chloroform). IR (film): ν
=
˜
max
3418 (br), 2947, 2872, 1726, 1376, 1072, 1031, 978, 757 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 6.19 (br. s, 1 H, 1Ј-H), 4.73 (br. s, 1
H, 29-H), 4.61 (br. s, 1 H, 29-H), 4.46 (dd, J = 5.9, 10.4 Hz, 1 H,
8
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O42187
/KAP1
Date: 06-05-14 17:56:27
Pages: 11
Lupane-Type Saponins Containing an α-d-Idopyranoside Fragment
34.5 (CH2), 34.1 (CH2), 29.7 (CH2), 29.6 (CH2), 28.7, 27.0 (CH2),
25.1 (CH2), 22.3 (CH2), 21.0, 20.8 (CH2), 19.1, 18.2 (CH2), 16.5,
16.0, 16.0, 14.7 ppm. HRMS (ESI): m/z calcd. for C38H62NaO8 [M
+ Na]+ 669.4342; found 669.4346. C38H62O8·2H2O (682.95): calcd.
C 66.83, H 9.74; found C 66.81, H 9.70.
4.3.1. Cell culture: Stock solutions (10 mmol/L) of tested com-
pounds were prepared by dissolving an appropriate quantity of
each substance in dimethyl sulfoxide (DMSO). Dulbecco’s modi-
fied Eagle’s medium (DMEM), fetal bovine serum (FBS), l-glut-
amine, penicillin and streptomycin were purchased from Sigma
(MO, USA). Calcein AM was obtained from Molecular Probes (In-
vitrogen Corporation, CA, USA). The screening cell lines (T-lym-
phoblastic leukemia cell line CEM, breast carcinoma cell line
MCF7, cervical carcinoma cell line HeLa, and human fibroblasts
BJ) were obtained from the American Type Culture Collection
(Manassas, VA, USA). All cell lines were cultured in DMEM me-
dium (Sigma, MO, USA), supplemented with 10% fetal bovine se-
rum, l-glutamine (2 mmol/L), penicillin (10000 U) and streptomy-
cin (10 mg/mL). The cell lines were maintained under standard cell
culture conditions at 37 °C and 5% CO2 in a humid environment.
Cells were subcultured two or three times a week by using the stan-
dard trypsinization procedure.
4.2.11.5. 3β-O-(α-
60% starting from 30. [α]2D0 = 67.3 (c = 0.25, chloroform). IR (film):
max = 3420 (br), 2940, 2869, 1672, 1641, 1454, 1018, 880 cm–1; 1H
D-Idopyranosyl)lup-20(29)-en-28-ol (31): Yield
ν
˜
NMR (500 MHz, CDCl3 + 10% CD3OD): δ = 5.01 (br. s, 1 H),
4.68 (br. d, J = 1.7 Hz, 1 H), 4.58 (br. s, 1 H), 4.18–4.15 (m, 1 H),
3.89–3.93 (m, 3 H), 3.77 (d, J = 10.7 Hz, 1 H, 28-H), 3.63 (br. s, 1
H), 3.28–3.33 (m, 1 H), 1.84–2.00 (m, 3 H), 1.68 (s, 3 H, CH3),
1.02 (s, 3 H, CH3), 0.98 (s, 6 H, 2ϫ CH3), 0.83 (s, 3 H, CH3), 0.76
(s, 3 H, CH3), 1.75–0.75 (m, 29 H, lupane protons) ppm. 13C NMR
(125 MHz, CDCl3 + 10% CD3OD): δ = 150.4 (C-20), 109.6 (C-
29), 97.9 (C-1Ј), 84.6 (C-3), 71.2, 68.3, 65.8, 63.8 (CH2), 60.1 (CH2),
55.4, 50.3, 48.7, 47.7, 47.6 (C), 42.6 (C), 40.8 (C), 38.2 (C), 38.1
(CH2), 37.2, 37.0 (C), 34.1 (CH2), 33.9 (CH2), 29.6 (CH2), 29.0
(CH2), 28.6, 26.9 (CH2), 25.1 (CH2), 22.1 (CH2), 20.8 (CH2), 19.0,
18.2 (CH2), 16.4, 15.9, 15.9, 14.6 ppm. HRMS (ESI): m/z calcd. for
C36H60NaO7 [M + Na]+ 627.4237; found 627.4223.
4.3.2. Calcein AM assay: Suspensions of tested cell lines (ca.
1.0ϫ105 cells/mL) were placed in 96-well microtiter plates and, af-
ter 24 h stabilization (time zero), the tested compounds were added
(in three 20 μL aliquots) in serially diluted concentrations in
DMSO. Control cultures were treated with DMSO alone, and the
final concentration of DMSO in the incubation mixtures never ex-
ceeded 0.6%. The test compounds were typically evaluated at six
threefold dilutions and the highest final concentration was gen-
erally 50 μm. After 72 h incubation, Calcein AM solution (100 μL,
Molecular Probes, Invitrogen, CA, USA) was added, and incu-
bation was continued for 1 h. The fluorescence of viable cells was
then quantified by using a Fluoroskan Ascent instrument (Labsys-
tems, Finland). The percentage of surviving cells in each well was
calculated by dividing the intensity of the fluorescence signals from
the exposed wells by the intensity of signals from control wells and
multiplying by 100. These ratios were then used to construct dose-
response curves from which IC50 values, the concentrations of the
respective compounds that were lethal to 50% of the tumor cells,
were calculated. The results obtained for selected compounds are
shown in Table 1.
4.2.11.6. 3β,28-Di-O-(α-
40% starting from 32. [α]2D0 = 61.9 (c = 0.25, chloroform). IR (film):
= 3333 (br), 2938, 2871, 1725, 1642, 1456, 1056, 1018, 979,
D-idopyranosyl)lup-20(29)-ene (33): Yield
ν
˜
max
1
880 cm–1; H NMR (500 MHz, CDCl3 + 10% CD3OD): δ = 5.01
(s, 1 H), 4.91 (s, 1 H), 4.69 (br. s, 1 H), 4.60 (br. s, 1 H), 4.18–4.15
(m, 1 H), 3.89–4.03 (m, 8 H), 3.72–3.70 (m, 1 H), 3.65–3.63 (m, 1
H), 3.31 (dd, J = 4.2, 11.6 Hz, 1 H, 3-H), 3.09 (d, J = 9.5 Hz, 1 H,
28-H), 1.68 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 0.98 (s, 6 H, 2ϫ
CH3), 0.84 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 1.98–0.70 (m, 26 H,
lupane protons) ppm. 13C NMR (125 MHz, CDCl3
+ 10%
CD3OD): δ = 150.0 (lupane C-20), 109.9 (lupane C-29), 101.9 (C-
1), 97.9 (C-1), 84.5 (lupane C-3), 71.2, 71.2, 68.4, 68.4, 68.3, 67.6,
67.1 (CH2), 65.8, 65.5, 63.9 (CH2), 63.8 (CH2), 55.4, 50.2, 48.7,
47.7, 46.6 (C), 42.7 (C), 40.8 (C), 38.2 (C), 38.1 (CH2), 37.4, 37.0
(C), 34.3 (CH2), 34.1 (CH2), 30.1 (CH2), 29.6 (CH2), 29.5 (CH2),
28.6, 26.9 (CH2), 25.0 (CH2), 22.1 (CH2), 20.8 (CH2), 19.0, 18.1
(CH2), 16.4, 15.9, 14.7 ppm. HRMS (ESI): m/z calcd. for
Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of the H and 13C NMR spectra are provided.
C42H70NaO12 [M
+
Na]+ 789.4765; found 789.4754.
C42H70O12·H2O (785.04): calcd. C 64.26, H 9.24; found C 64.35, H
9.26.
Acknowledgments
This work was financed by the National Science Centre, Poland
(grant number 2012/07/B/ST5/00823), by the Czech Ministry of
Education (National Program for Sustainability I, grant number
LO1204) and by the Grant Agency of the Czech Republic (grant
number 14-19590S). The authors thank Olga Hustáková for excel-
lent technical assistance.
4.2.11.7. 28-O-Acetyl-3α-O-(α-
Yield 81% starting from 34. [α]2D0 = 16.2 (c = 0.3, chloroform). IR
(film): ν = 3434 (br), 2942, 2871, 1454, 1242, 1086, 1059, 1028,
D-idopyranosyl)lup-20(29)-ene (35):
˜
max
757 cm–1. 1H NMR (600 MHz, CDCl3): δ = 5.04 (br. s, 1 H), 4.68–
4.67 (m, 1 H), 4.58 (br. s, 1 H), 4.24 (d, J = 11.0 Hz, 1 H, 28-H),
4.04–4.11 (m, 4 H), 3.96 (br. s, 1 H), 3.84 (d, J = 11 Hz, 1 H, 28-
H), 3.80–3.79 (m, 1 H), 3.13 (br. s, 1 H, 3-H), 2.46–2.41 (m, 1 H),
2.07 (s, 3 H, CH3), 1.75–2.00 (m, 4 H), 1.67 (s, 3 H, CH3), 1.02 (s,
3 H, CH3), 0.96 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.85 (s, 3 H,
CH3), 0.84 (s, 3 H, CH3), 1.65–0.85 (m, 24 H, lupane protons) ppm.
13C NMR (150 MHz, CDCl3): δ = 171.7 (C=O), 150.2 (C-20),
109.8 (C-29), 104.7 (C-1Ј), 88.1 (C-3), 72.8, 68.3, 67.6, 65.4 (CH2),
65.0, 62.9 (CH2), 50.6, 50.5, 48.8, 47.8 (C), 47.7, 46.3 (C), 42.7 (C),
41.0 (C), 38.2 (C), 37.5, 37.1 (C), 34.9 (CH2), 34.5 (CH2), 34.0
(CH2), 29.7 (CH2), 29.6 (CH2), 28.5, 27.0 (CH2), 25.1 (CH2), 24.2
(CH2), 21.9, 21.1, 20.7 (CH2), 19.1, 18.0 (CH2), 16.0, 16.0,
14.9 ppm. HRMS (ESI): m/z calcd. for C38H62NaO8 [M + Na]+
669.4342; found 669.4348. C38H62O8·2H2O (682.95): calcd. C
66.83, H 9.74; found C 66.38, H 9.75.
[1] S. B. Mahato, A. N. Ganguly, N. P. Sahu, Phytochemistry 1982,
21, 959.
[2] K. Hostettmann, A. Marston, in: Saponins, Cambridge Uni-
versity Press, Cambridge, UK, 1995.
[3] C. Gauthier, J. Legault, M. Piochon-Gauthier, A. Pichette,
Phytochem. Rev. 2011, 10, 521–544.
[4] R.-Y. Kuo, K. Qian, S. L. Morris-Natschke, K.-H. Lee, Nat.
Prod. Rep. 2009, 26, 1321–1344.
[5] C. Bachran, S. Bachran, M. Sutherland, D. Bachran, H. Fuchs,
Mini-Rev. Med. Chem. 2008, 8, 575–584.
[6] D. Yu, S. L. Morris-Natschke, K.-H. Lee, Med. Res. Rev. 2007,
27, 108–132.
[7] H. Sun, W.-S. Fang, Bot. Stud. 2006, 47, 339–368.
[8] S. G. Sparg, M. E. Light, J. van Staden, J. Ethnopharmacol.
2004, 94, 219–243.
4.3. Biological Evaluation
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
9

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M. Strnad, Z. Pakulski et al.
FULL PAPER
[9]
[31] O. W. Howarth, N. Pozzi, G. Vlahov, D. Bartels, Carbohydr.
Res. 1996, 289, 137–142.
A. V. Rao, D. M. Gurfinkel, Drug Metab. Drug Interact. 2000,
17, 211–235.
[32] P. Guerry, F. Poly, M. Riddle, A. C. Maue, Y.-H. Chen, M. A.
Monteiro, Front. Cell. Infect. Microbiol. 2012, 2, Article 7,
DOI: 10.3389/fcimb.2012.00007.
[33] H. Paulsen, W.-P. Trautwein, F. Garrido Espinosa, K. Heyns,
Chem. Ber. 1967, 100, 2822–2836.
[34] H. Paulsen, Methods Carbohydr. Chem. 1972, 6, 142–149.
[35] T. Chiba, S. Tejima, Chem. Pharm. Bull. 1977, 25, 1049–1054.
[36] T. Chiba, S. Tejima, Chem. Pharm. Bull. 1978, 26, 3426–3432.
[37] M. Dromowicz, P. Köll, Carbohydr. Res. 1998, 308, 169–171.
[38] R. Hevey, A. Morland, C.-C. Ling, J. Org. Chem. 2012, 77,
6760–6772.
[39] S. Kopitzki, J. Thiem, Eur. J. Org. Chem. 2013, 4008–4016.
[40] R. Blattner, R. H. Furneaux, Z. Pakulski, Carbohydr. Res.
2006, 341, 2115–2125.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
K. Oda, H. Matsuda, T. Murakami, S. Katayama, T. Ohgitani,
M. Yoshikawa, Biol. Chem. 2000, 381, 67–74.
S. Man, W. Gao, Y. Zhang, L. Huang, C. Liu, Fitoterapia 2010,
81, 703–714.
Y. Wang, Y. Zhang, Z. Zhu, S. Zhu, Y. Li, M. Li, B. Yu, Bioorg.
Med. Chem. 2007, 15, 2528–2532.
D. Thibeault, C. Gauthier, J. Legault, J. Bouchard, P. Dufour,
A. Pichette, Bioorg. Med. Chem. 2007, 15, 6144–6157.
J. Gao, X. Li, G. Gu, B. Sun, M. Cui, M. Ji, H.-X. Lou, Bioorg.
Med. Chem. Lett. 2011, 21, 622–627.
C. Gauthier, J. Legault, M. Piochon, S. Lavoie, S. Tremblay, A.
Pichette, Bioorg. Med. Chem. Lett. 2009, 19, 2310–2314.
C. Gauthier, J. Legault, A. Pichette, Mini-Rev. Org. Chem.
2009, 6, 321–344.
C. Gauthier, J. Legault, S. Rondeau, A. Pichette, Tetrahedron
Lett. 2009, 50, 988–991.
O. B. Flekhter, L. A. Baltina, G. A. Tolstikov, J. Nat. Prod.
2000, 63, 992–994.
[41] R. Bukowski, L. M. Morris, R. I. Woods, T. Weimar, Eur. J.
Org. Chem. 2001, 2697–2705.
[42] I. A. Ivanova, A. J. Ross, M. A. J. Ferguson, A. V. Nikolaev, J.
Chem. Soc. Perkin Trans. 1 1999, 1743–1753.
[43] H. Paulsen, C.-P. Herold, Chem. Ber. 1970, 103, 2450–2462.
[44] R. Gigg, C. D. Warren, J. Chem. Soc. C 1968, 1903–1911.
[45] J. J. Oltvoort, C. A. A. van Boeckel, J. H. de Koning, J. H.
van Boom, Synthesis 1981, 305–308.
[46] C. Gauthier, J. Legault, S. Lavoie, S. Rondeau, S. Tremblay, A.
Pichette, Tetrahedron 2008, 64, 7386–7399.
[47] I. C. Sun, H.-K. Wang, Y. Kashiwada, J.-K. Shen, L. M. Co-
sentino, C.-H. Chen, L.-M. Yang, K.-H. Lee, J. Med. Chem.
1998, 41, 4648–4657.
L. E. Obinokova, M. W. Denisenko, W. A. Denisenko, N. I.
Uvarova, Khim. Prir. Soedin. 1988, 212–217.
[20] L. E. Obinokova, G. I. Oshitok, W. A. Denisenko, W. F. Anuf-
riev, A. M. Tolkatz, N. I. Uvarowa, Khim. Prir. Soedin. 1984,
182–187.
[21] N. I. Uvarova, L. N. Atopkina, G. B. Elyakov, Carbohydr. Res.
1980, 83, 33–42.
[22] N. I. Uvarova, G. I. Oshitok, G. B. Elyakov, Carbohydr. Res.
1973, 27, 79–87.
[23] N. I. Uvarova, G. I. Oshitok, A. K. Dzizenko, V. V. Isakov,
G. B. Elyakov, Dokl. Akad. Nauk SSSR 1972, 202, 368–370.
[24] Z. Pakulski, Pol. J. Chem. 2005, 79, 361–367.
[25] P. Cmoch, Z. Pakulski, J. Swaczynová, M. Strnad, Carbohydr.
Res. 2008, 343, 995–1003.
[26] P. Cmoch, A. Korda, L. Rárová, J. Oklesˇt’ková, M. Strnad, R.
Luboradzki, Z. Pakulski, Tetrahedron 2014, 70, 2717–2730.
[27] I. Capila, R. J. Linhardt, Angew. Chem. Int. Ed. 2002, 41, 390–
412; Angew. Chem. 2002, 114, 426.
[28] R. Lever, C. P. Page, Nature Rev. Drug Discov. 2002, 1, 140–
148.
[29] Y.-H. Chen, F. Poly, Z. Pakulski, P. Guerry, M. A. Monteiro,
Carbohydr. Res. 2008, 343, 1034–1040.
[30] G. Bianchi, A. Gamba, C. Murelli, F. Salamini, D. Bartels,
Plant J. 1991, 1, 355–359.
[48] L. Voutquenne, C. Lavaud, G. Massiot, L. Le Men-Olivier,
Pharm. Biol. 2002, 40, 253–262.
[49] N. S. Bhacca, D. Horton, H. Paulsen, J. Org. Chem. 1968, 33,
2484–2487.
[50] D. A. Horita, P. J. Hajduk, L. E. Lerner, Glycoconjugate J.
1997, 14, 691–696.
[51] K. Bock, I. Lundt, C. Pedersen, Tetrahedron Lett. 1973, 14,
1037–1040.
[52] K. Bock, C. Pedersen, J. Chem. Soc. Perkin Trans. 2 1974, 293–
297.
[53] K. Bock, C. Pedersen, Acta Chem. Scand., Ser. B 1975, 29,
258–264.
[54] P. Fügedi, in: The Organic Chemistry of Sugars (Eds.: D. E.
Levy, P. Fügedi), CRC Press, Boca Raton, 2006, chapter 4.
[55] R. J. Ferrier, P. C. Tyler, J. Chem. Soc. Perkin Trans. 1 1980,
1528–1534.
Received: March 4, 2014
Published Online: ᭿
10
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O42187
/KAP1
Date: 06-05-14 17:56:27
Pages: 11
Lupane-Type Saponins Containing an α-d-Idopyranoside Fragment
Glycoconjugates
P. Cmoch, A. Korda, L. Rárová,
ˇ
J. Oklesˇtková, M. Strnad,* K. Gwardiak,
R. Karczewski, Z. Pakulski* ........... 1–11
Synthesis of Lupane-Type Saponins Con-
taining an Unusual α-d-Idopyranoside
Fragment as Potent Cytotoxic Agents
A concise synthesis of 2,3,4,6-tetra-O-benz-
oyl-d-idopyranosyl trichloroacetimidate
and lupane triterpenes bearing an unusual
new compounds were evaluated in vitro for
their cytotoxic activities, and all exhibited
activity in the micromolar range against
human cancer cell lines.
Keywords: Carbohydrates
/ Glycoconju-
gates / Glycosylation / Steroids / Saponins /
Drug discovery
d-idopyranoside fragment is described. All
Eur. J. Org. Chem. 0000, 0–0
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11