Synthesis and structure-activity relationship study of cytotoxic lupane-type 3β-O-monodesmosidic saponins with an extended C-28 side chain

Article abstract of DOI:10.1016/j.tet.2014.03.006

A concise synthesis of lupane triterpenes with an elongated carbon chain at the C-28 position, as well as saponins containing d-mannose, l-arabinose, and l-rhamnose moieties at the C-3 position is described. The overall synthesis of the new triterpenes involved seven linear steps starting from natural betulin: selective protection of a hydroxyl group, oxidation, elongation of the carbon chain by Grignard reaction, and deoxygenation. O-Glycosides were obtained by glycosylation of triterpenes with classical Schmidt's donors. Additionally, all new compounds were evaluated in vitro for their cytotoxic activities. Several triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against human cancer cell lines. The therapeutical index of active triterpenes is very high, since almost none of them were cytotoxic for normal BJ fibroblasts. These results open the way to the synthesis of various lupane-type saponin derivatives as potentially bioactive compounds.

Full text of DOI:10.1016/j.tet.2014.03.006

Tetrahedron 70 (2014) 2717e2730
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and structureeactivity relationship study of cytotoxic
lupane-type 3b-O-monodesmosidic saponins with an extended C-28
side chain
a
a
b,
*
ꢀ b
*
ꢁ
ꢀ b
ꢁ
ꢀ
Piotr Cmoch , Anna Korda , Lucie Rarova , Jana Oklestkova , Miroslav Strnad
,
Roman Luboradzki c d, Zbigniew Pakulski a
a Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland
,
,
b
ꢀ
ꢀ
Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University & Institute of
ꢁ
ꢂ
Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc, Czech Republic
c Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland
d Cardinal Stefan Wyszynski University, Dewajtis 5, 01-815 Warszawa, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A concise synthesis of lupane triterpenes with an elongated carbon chain at the C-28 position, as well as
Received 26 December 2013
Received in revised form 14 February 2014
Accepted 3 March 2014
saponins containing D-mannose, L-arabinose, and L-rhamnose moieties at the C-3 position is described.
The overall synthesis of the new triterpenes involved seven linear steps starting from natural betulin:
selective protection of a hydroxyl group, oxidation, elongation of the carbon chain by Grignard reaction,
and deoxygenation. O-Glycosides were obtained by glycosylation of triterpenes with classical Schmidt’s
donors. Additionally, all new compounds were evaluated in vitro for their cytotoxic activities. Several
triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against
human cancer cell lines. The therapeutical index of active triterpenes is very high, since almost none of
them were cytotoxic for normal BJ fibroblasts. These results open the way to the synthesis of various
lupane-type saponin derivatives as potentially bioactive compounds.
Available online 11 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
branching at the C-28 position. Herein we report on the synthesis of
novel lupane-type triterpenes and saponins, and studies derived
Saponins are steroid or triterpenoid glycosides widely distrib-
uted in plants and in some marine organisms.1,2 They possess in-
teresting biological properties including antitumor, antiviral,
antifungal, and antiinflammatory activities, which have been ex-
tensively studied and reviewed in the last years.3e10 Their bi-
ological effects were correlated to both sugar residues and
aglycones.3,11e13 Usually, mono-, di-, tri- or tetrasaccharides are
attached to the sapogenin backbone, they constitutes a hydrophilic
part, while sapogenin is a hydrophobic fragment.
from the cytotoxicity evaluation of these compounds. The synthetic
strategy is directed toward an elongation of the carbon chain at the
lupeol C-28 position and introduction of different sugar moieties at
the O-3 position. Fig.1 depicts the structural features of the lupanes
1e3 to be synthesized. Those lupanes were further transformed
into saponins whose cytotoxicity against a series of cell lines have
been tested.
Natural saponins based on the betulin scaffold occur less fre-
quently than those having other triterpene-type aglycones, al-
though a growing interest in their synthesis is noticeable.14e23
However, little information is available regarding the effect of the
non-sugar substitution at the lupane C-28 position and cytotoxicity
of the resulting saponins bearing sugar moiety at the C-3 posi-
tion.24e28 In addition, there are certain gaps on the structur-
eeactivity relationships (SAR) studies concerning other types of
* Corresponding authors. E-mail addresses: miroslav.strnad@upol.cz (M. Strnad),
zbigniew.pakulski@icho.edu.pl (Z. Pakulski).
Fig. 1. Target chain-elongated derivatives of lupeol.
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.03.006

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P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2. Results and discussion
Due to problems with the purification of 12, we decided to use
3-O-acetylbetulinal13 (13) instead of 3-O-allylbetulinal (7) as
starting material for the synthesis of the target triterpenes. Its re-
action with methylmagnesium bromide gave elongated alcohols 14
in 82% yield. Reaction with ethylmagnesium bromide afforded al-
cohols 15 in 86% yield. In this case, partial deacetylation was also
observed and small amounts of the corresponding diols 16 were
isolated as dibenzoates 17 in 2e11% yield (as inseparable mixture),
depending on batch. The composition of the main products was
additionally confirmed by acetylation of the analytical samples of
pure diastereoisomers 15a and 15b (to 18). Similarly, reaction with
propylmagnesium chloride gave elongated alcohols 19 in 73% yield.
A small sample of a 15ab mixture was further acylated with benzoyl
chloride providing benzoate 20 used for studying the influence of
substitution on cytotoxicity. Then, alcohols 14, 15, and 19 were
acylated with phenyl chlorothionoformate yielding the expected
thiocarbonates 21e23. In all cases, minute percentages of the in-
separable corresponding carbonates 24e26 were also detected.
Composition of the carbonates 24e26 was confirmed by acylation
of analytical samples of the starting alcohols with phenyl chlor-
2.1. Chemistry
2.1.1. Synthesis of lupanes with carbon chain elongated at the C-28
position. For the elongation of the carbon chain at the C-28 position
of lupeol, we chose the Grignard reaction of betulinal derivatives
followed by removal of the newly formed hydroxyl group. First we
used 3-O-allylbetulinal (7), because of the flexibility of the allyl
group, which may be selectively removed under very mild condi-
tions, or used for linker construction. Compound 7 was easily
available by acidic allylation of 28-O-acetylbetulin29 (4) with allyl
trichloroacetimidate30 to yield derivative 5, followed by subsequent
hydrolysis of the acetyl group and oxidation of the hydroxyl group
at C-28eOH with PCC. The reaction of aldehyde 7 with methyl- or
ethylmagnesium bromide afforded corresponding alcohols 8 and 9.
In both cases a mixture of diastereoisomers (partially separated by
column chromatography) was obtained. Full separation was not
necessary due to the subsequent deoxygenation, which destroys
this newly created stereogenic center (Scheme 1).
Scheme 1. Synthesis of 3-O-allyl triterpenes and their elongation at C-28 position. Reagents and conditions: (a) CH2]CHCH2OC(NH)CCl3, TMSOTf, DCM, 0 ꢀC, 1 h, 75%; (b) KOH,
EtOH, reflux, 2 h, 82%; (c) PCC, DCM, 0 ꢀC, 90 min, 75%; (d) MeMgBr, THF, ꢁ40 ꢀC, 94%; (e) EtMgBr, THF, ꢁ40 ꢀC, 61%; (f) PhOC(S)Cl, DCM, Py, rt, 24 h; (g) (Me3Si)3SiH, AIBN, toluene,
80 ꢀC, 5 h.
We expected that the RobinseWilson31 modification of the
BartoneMcCombie32 deoxygenation would provide efficient and
convenient ways to the target triterpenes. The reaction of 9a with
phenyl chlorothionoformate yielded expected the thiocarbonate
10 in high yield. However, this product contained a significant
amount of a byproduct in which the H-28 protons appeared at
higher field (5.34 ppm) than for thiocarbonate (6.00 ppm). In the
13C NMR spectrum the thiocarbonyl group of 10 was observed at
oformate. The obtained derivatives had identical NMR spectra as
the above additives. In the next step the thiocarbonate group was
removed by reduction with tris(trimethylsilyl)silane in the pres-
ence of AIBN to afford acetates 27e29. At this stage, unreacted
carbonates were readily removed by column chromatography.
Structures of the acetylated target triterpenes 27e29 were un-
equivocally confirmed by X-ray analysis (Fig. 2). Superimposition of
X-ray structures shows only minimal differences in the space ar-
rangement around the aliphatic chain at C-28 position (Fig. 3).
Acetyl groups present in betulin derivatives are known to be
hydrolytically stable, and classical methods for their deprotection
(basic treatment) usually do not work.34,35 Therefore, deacetylation
at the O-3 position was performed by reduction with LiAlH4. All
target compounds (1e3) were obtained in 96% yield (Scheme 2).
It is clearly visible that changing the 3-O-protective group from
allyl to acetyl was crucial for the successful synthesis of required
triterpenes. A similar influence of a substituent distant to the
reacting center of lupane-type triterpenes was observed earlier by
d
w195 ppm, whereas the carbonyl carbon of the contamination
appeared at 154 ppm. The molecular mass of the contaminant
d
was 16 units lower than for thiocarbonate. Such data suggests that
thiocarbonate 10 was contaminated with the corresponding car-
bonate 11. Its origin is unclear,33 but this hypothesis was con-
firmed during further studies by independent synthesis of similar
compounds. Free-radical deoxygenation of 10 with tris(-
trimethylsilyl)silane in the presence of AIBN afforded the expected
product 12, although badly contaminated with several inseparable
byproducts.

P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2719
Fig. 2. Crystal structures of compounds 27 (a), 28 (b), and 29 (c). View along the C17eC18 bond.
elemental analysis and HRMS (Tables 1 and 2, Experimental
section).
2.2. In vitro results
Cytotoxic activity of the parent triterpenoid lupanes with dif-
ferent carbon chains at the C-28 position, as well as their 3b-O-
monodesmosidic saponin derivatives was tested. Several normal
and cancer cell lines were cultured and used in experiments to
examine the structureeactivity relationships of this lupane-type
derivative, with respect to their activities against human cancers.
We compared the in vitro cytotoxic activity of selected analogues
against human BJ-H-tert fibroblasts and cancer cell lines of various
histopathological origins, including T-lymphoblastic leukemia
CEM, breast carcinoma MCF-7, and cervical carcinoma HeLa lines.
Cells of all of these lines were exposed to six serial four-fold di-
lutions of each drug for 72 h, the proportions of surviving cells were
then estimated and IC50 values (50% inhibitory concentrations)
were calculated. The results obtained from Calcein AM assays are
presented in Table 3.
The most potent triterpene derivative was compound 1, which
showed cytotoxic activity against all of the tumor cell lines. How-
ever, this compound had also significant toxicity towards normal
cells (BJ fibroblasts). Triterpenoid derivatives (1, 6, 9a, 13, 15a, 19a,
19b) were more active against cervical carcinoma HeLa line (IC50
12.3e43.4 mM) than the other types of cancer cells.
The therapeutical index of these type of compounds is very high,
as none of the terpenoids (except 1) were cytotoxic for normal BJ
Fig. 3. Overlaid molecules of 27 ( , only one of two symmetrically independent
molecules was used), 28 ( ), and 29 ( ).
Pichette.15 Its origin is unclear, but it must be considered as an
important factor affecting synthesis of the particular lupane de-
rivatives, and as an explanation for some unsuccessful trials.
2.1.2. Synthesis of saponins. Glycosylation of triterpenes 1e3 was
performed by treatment of glycosyl acceptors with perbenzoylated
donors 30,36 31,37 and 3237 in the presence of TMSOTf under
standard conditions (Scheme 3).35 Protected saponins were ob-
tained in good to high yields (61e90%). As expected, the presence of
benzoyl protecting groups in position 2 of the sugar donors directed
the anomeric selectivity of the glycosidation reaction.38 In all cases
fibroblasts. Furthermore, compounds 1 (IC50 30.8
mM), 10a (IC50
27.8 M), 13 (IC50 38.3 M), and 15a (IC50 41.9 M) were also active
m
m
m
1,2-trans-monodesmosidic saponins (
a
-
D
-mannopyranosides,
a
-
L
-
against T-lymphoblastic leukemia CEM cells. A striking observation
from this data was that much lower lupane-mediated loss of via-
bility was observed in the BJ fibroblasts, suggesting that the lupane
derivative induces different responses in cancer and normal cells.
At present, only a few natural agents are known to possess the
potential ability for selective/preferential elimination of cancer
cells without affecting the growth of normal cells.39e41 This was
also observed in our previous study.35 There were two unique
compounds 14ab and 19ab exhibiting selective cytotoxic activity
arabinopyranosides, and -rhamnopyranosides) were obtained
a-L
exclusively, which was confirmed by the chemical shifts and the
vicinal coupling constants of the anomeric protons. Final depro-
tection of the hydroxyl groups was performed by treatment of
benzoates with potassium carbonate in methanol. With one ex-
ception (60% for 33d) all saponins were obtained in very good
yields (87e98%). The structures of all synthesized saponins were
confirmed by extended 1D and 2D NMR experiments, as well as

2720
P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
Scheme 2. Synthesis of triterpenes elongated at the C-28 position. Reagents and conditions: (a) 13/14, MeMgBr, THF, ꢁ40 ꢀC, 82%; (b) 13/15, EtMgBr, THF, ꢁ40 ꢀC, 86%þ16
(2e11%); (c) 13/19, PrMgCl, THF, ꢁ40 ꢀC, 73%; (d) Ac2O, Py; (e) PhOC(S)Cl, DCM, Py, rt, 24 h, 14/21, 15/22, 19/23; (f) PhOC(O)Cl, DCM, Py, rt, 24 h, 14/24, 15/25, 19/26; (g)
(Me3Si)3SiH, AIBN, toluene, 80 ꢀC, 5 h; (h) LiAlH4, THF, rt, 30 min.
ꢂ
Scheme 3. Synthesis of saponins from triterpenes elongated at the C-28 position. Reagents and conditions: (a) 30, 31, or 32, TMSOTf, DCM, molecular sieves 4 A; (b) K2CO3, MeOH.
for breast carcinoma MCF-7. On the other hand, several of these
derivatives (1, 15b, 20, 24a, and 25a) were cytotoxic to normal fi-
broblasts, thus demonstrating unusable the substitutions of the
lupane scaffold for cancer treatment.
The majority of the lupane-type 3b-O-monodesmosidic sapo-
nins (33f, 34f, 35b,d,f) showed zero cytotoxicity towards the
cancer and normal cell lines used, even when tested at concen-
trations up to 50
mM. The potent compounds were saponin

Table 1
1H NMR spectroscopic data of compounds 1e3, 6, 18b, and 33e35bdf
Atom no.
1a
2a
3a
6a,c
18bd
33ba
34ba
Triterpene unit
1
2
3
0.90, 1.66
1.57
3.18
0.90, 1.66
1.57
3.18
0.90, 1.66
1.58
3.18
0.82, 1.68
1.47, 1.71
2.80
0.98, 1.66
1.60
4.46
0.81, 1.67
1.39, 1.70
3.14
0.81, 1.66
1.39, 1.70
3.14
5
0.68
0.68
0.68
0.68
0.78
0.67
0.67
6
7
1.39, 1.52
1.38
1.40, 1.52
1.38
1.39, 1.52
1.38
1.39, 1.51
1.39
1.40, 1.49
1.37, 1.49
1.37
1.37, 1.49
1.37
9
1.28
1.27
1.27
1.25
1.27
1.25
1.24
11
12
13
1.22, 1.42
1.06, 1.64
1.77
1.23, 1.42
1.06, 1.64
1.79
1.23, 1.42
1.06, 1.64
1.79
1.21, 1.41
1.04, 1.63
1.64
1.25, 1.41
1.11, 1.61
1.86
1.21, 1.40
1.06, 1.64
1.77
1.22, 1.40
1.06, 1.65
1.80
15
16
18
0.95, 1.55
1.12, 1.72
1.46
0.95, 1.57
1.14, 1.71
1.42
0.95, 1.57
1.13, 1.72
1.43
1.05, 1.70
1.21, 1.92
1.59
0.94, 1.93
0.95, 1.54
1.12, 1.72
1.46
0.95, 1.57
1.14, 1.72
1.42
1.72
19
2.40
2.40
2.40
2.38
2.62
2.39
2.43
21
22
23
1.31, 1.85
0.92, 1.63
0.76
1.32, 1.87
0.95, 1.63
0.76
1.31, 1.86
0.95, 1.63
0.76
1.41, 1.96
1.04, 1.86
0.78
1.42, 1.90
1.10, 1.90
0.83
1.31, 1.85
0.93, 1.64
0.74
1.32, 1.87
0.96, 1.64
0.74
24
0.97
0.97
0.97
0.97
0.84
0.92
0.92
25
0.83
0.83
0.83
0.83
0.85
0.82
0.82
26
1.02
1.03
1.03
1.02
1.09
1.02
1.03
27
0.96
0.96
0.96
0.97
0.97
0.95
0.95
28
0.98, 1.49
0.77
d
0.90, 1.42
1.19
0.91
d
0.90, 1.46
1.30
1.15
3.33, 3.80
d
5.28
0.99, 1.49
0.77
d
0.90, 1.42
1.17, 1.22
0.92
28ae
1.54, 1.72
0.87
d
28be
d
28ce
d
0.92
d
d
d
29
30
4.56, 4.67
1.68
4.55, 4.67
1.68
4.56, 4.68
1.68
4.58, 4.68
1.68
4.69, 4.59
1.68
4.57, 4.68
1.68
4.58, 4.69
1.68
Monosaccharide unit
1
2
3
4
5
6
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
4.96
3.82
3.80
3.94
3.63
3.72, 3.94
4.96
3.81
3.82
3.94
3.63
3.72, 3.94
Atom no.
35ba
33da
34db
35da
33fb
34fa
35fb
Triterpene unit
1
2
3
0.81, 1.66
1.39, 1.70
3.14
0.89, 1.66
1.69, 1.83
3.13
0.90, 1.66
1.68, 1.82
3.13
0.88, 1.65
1.68, 1.81
3.11
0.92, 1.68
1.66, 1.76
3.07
0.88, 1.65
1.64, 1.74
3.05
0.90, 1.66
1.66, 1.76
3.06
5
0.67
0.70
0.71
0.69
0.72
0.68
0.71
6
7
1.37, 1.49
1.37
1.38, 1.50
1.38
1.39, 1.51
1.38
1.37, 1.49
1.37
1.40, 1.51
1.40
1.37, 1.48
1.37
1.39, 1.50
1.38
9
1.25
1.25
1.27
1.25
1.30
1.25
1.28
11
12
13
1.22, 1.40
1.06, 1.65
1.80
1.22, 1.41
1.05, 1.64
1.77
1.24, 1.42
1.06, 1.65
1.81
1.22, 1.41
1.05, 164
1.80
1.24, 1.43
1.07, 1.66
1.80
1.22, 1.41
1.06, 1.64
1.79
1.24, 1.42
1.07, 1.65
1.80
15
16
18
0.95, 1.56
1.14, 1.72
1.43
0.95, 1.54
1.12, 1.72
1.46
0.96, 1.58
1.14, 1.72
1.44
0.95, 1.56
1.13, 1.71
1.43
0.97, 1.57
1.14, 1.74
1.48
0.95, 1.56
1.14, 1.71
1.42
0.96, 1.58
1.14, 1.72
1.44
19
2.42
2.40
2.42
2.41
2.41
2.43
2.42
21
22
23
1.32, 1.87
0.96, 1.64
0.74
1.31, 1.85
0.89, 1.66
0.80
1.32, 1.87
0.96, 1.64
0.81
1.31, 1.86
0.95, 1.63
0.79
1.32, 1.86
0.94, 1.65
0.76
1.31, 1.87
0.96, 1.63
0.74
1.32, 1.87
0.96, 1.64
0.75
24
0.91
0.97
1.00
0.97
0.92
0.89
0.91
25
0.82
0.83
0.84
0.82
0.85
0.83
0.84
26
1.02
1.02
1.04
1.02
1.04
1.02
1.04
27
0.95
0.95
0.96
0.94
0.98
0.95
0.96
28
0.90, 1.46
1.31
1.15
0.98, 1.48
0.77
d
0.91, 1.44
1.17, 1.22
0.93
0.90, 1.46
1.31
1.15
1.01, 1.51
0.78
d
0.90, 1.42
1.16, 1.22
0.92
0.91, 1.47
1.32
1.16
28ae
28be
28ce
0.92
d
d
0.92
d
d
0.93
29
30
4.58, 4.69
1.69
4.56, 4.68
1.68
4.56, 4.68
1.69
4.57, 4.68
1.68
4.56, 4.68
1.69
4.57, 4.68
1.68
4.56, 4.68
1.69
Monosaccharide unit
1
2
3
4
5
6
4.96
3.81
3.82
3.94
3.63
3.72, 3.94
4.39
3.77
3.71
3.92
3.56, 3.90
d
4.33
3.65
3.60
3.87
3.52, 3.88
d
4.32
3.75
3.67
3.92
3.52, 3.90
d
4.76
3.88
3.69
3.38
3.75
1.26
4.80
3.93
3.75
3.44
3.79
1.27
4.76
3.88
3.70
3.38
3.75
1.26
a
Recorded in CDCl3 at 600 MHz,
Recorded in CDCl3/CD3OD mixture (3:1) at 600 MHz,
d
in ppm.
b
c
d
in ppm.
3-O-Allyl group: OCH2 3.89e3.85 (m) and 4.12e4.08 (m) ppm; ]CH 5.95e5.88 (m) ppm; ]CH2 5.12e5.09 (m) and 5.27e5.22 (m) ppm.
3-O-Acetyl group: 2.03 (s) ppm; 28-O-acetyl group: 2.07 (s) ppm.
d
e
Atom numbering according to IUPAC rules (Fig. 4).42,43

2722
P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
Table 2
13C NMR spectroscopic data of compounds 1e3, 6, 18b, and 33e35bdf
Atom no.
1a
2a
3a
6a,c
18bd
33ba
34ba
35ba
33da
34db
35da
33fb
34fa
35fb
Triterpene unit
1
2
3
4
5
6
7
8
38.7
27.4
79.0
38.9
55.3
18.3
34.2
40.8
50.5
37.2
21.0
25.1
37.0
42.5
27.1
30.3
45.8
49.7
47.4
151.1
30.0
35.0
15.3
28.0
16.1
16.0
14.8
19.3
8.1
38.7
27.4
79.0
38.9
55.3
18.3
34.2
40.9
50.5
37.2
21.0
25.1
37.0
42.5
27.2
31.2
45.7
49.8
47.4
151.2
30.1
35.8
15.3
28.0
16.1
16.0
14.9
30.0
17.0
15.4
d
38.7
27.4
79.0
38.9
55.3
18.3
34.2
40.9
50.5
37.2
21.0
25.1
37.0
42.5
27.2
31.2
45.6
49.8
47.4
151.2
30.1
35.7
15.3
28.0
16.1
16.0
14.9
26.9
23.8
26.0
14.2
109.2
19.3
38.6
23.1
86.3
38.9
55.9
18.3
34.3
41.0
50.4
37.1
20.9
25.3
37.3
42.7
27.0
29.2
38.3
23.7
80.9
37.8
55.4
18.1
33.9
41.0
50.2
37.0
20.8
25.2
36.9
43.0
28.1
33.9
50.2
49.9
48.2
150.4
31.6
34.2
16.5
27.9
16.0
15.9
14.9
76.5
24.7
11.5
d
38.3
22.1
82.9
38.4
55.6
18.3
34.2
40.9
50.4
37.1
21.0
25.1
37.0
42.5
27.1
30.3
45.8
49.7
47.4
151.1
30.0
35.0
16.4
28.6
16.1
16.0
14.9
19.3
8.1
38.3
22.1
82.9
38.4
55.6
18.3
34.2
40.9
50.4
37.1
21.0
25.1
37.0
42.5
27.2
31.2
45.7
49.8
47.4
151.1
30.1
35.8
16.4
28.6
16.1
16.0
14.9
30.0
17.0
15.4
d
38.3
22.1
82.9
38.4
55.6
18.2
34.2
40.9
50.4
37.1
21.0
25.1
37.0
42.5
27.2
31.1
45.6
49.8
47.4
151.1
30.1
35.7
16.4
28.6
16.1
16.0
14.9
27.0
23.9
26.0
14.2
109.3
19.4
38.7
26.0
90.0
39.1
55.6
18.2
34.2
40.9
50.5
36.9
21.0
25.2
37.0
42.5
27.1
30.4
45.8
49.7
47.4
151.2
30.0
35.0
16.4
28.2
16.1
16.0
14.8
19.3
8.1
38.9
26.2
90.0
39.3
55.8
18.4
34.4
41.0
50.6
37.1
21.2
25.4
37.1
42.7
27.4
31.4
45.8
50.2
47.6
151.3
30.3
36.0
16.3
28.0
16.2
16.1
15.0
30.1
17.2
15.5
d
38.7
26.0
89.8
39.1
55.6
18.2
34.2
40.9
50.4
36.9
21.0
25.1
37.0
42.5
27.2
31.1
45.6
49.8
47.4
151.1
30.1
35.7
16.4
28.0
16.1
16.0
14.8
26.9
23.8
26.0
14.2
109.2
19.4
38.7
25.5
89.2
39.0
55.5
18.3
34.2
40.9
50.5
36.9
21.0
25.2
37.0
42.5
27.1
20.3
45.8
49.8
47.5
151.0
30.0
35.0
16.0
27.9
16.0
15.9
14.7
19.3
7.8
38.6
25.5
89.6
39.0
55.4
18.3
34.2
40.9
50.4
36.9
21.0
25.1
37.0
42.5
27.2
31.2
45.7
49.8
47.4
151.1
30.1
35.8
16.2
28.2
16.1
16.0
14.9
30.0
17.0
15.4
d
38.9
25.7
89.5
39.2
55.7
18.5
34.4
41.1
50.7
37.1
21.2
25.4
37.2
42.7
27.4
31.3
45.8
50.0
47.6
151.4
30.3
35.9
16.3
28.2
16.3
16.2
15.0
27.2
24.0
26.2
14.3
109.4
19.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
28ae
28be
28ce
29
30
48.8
47.8
150.5
29.8
34.0
16.3
28.1
16.1
16.0
14.8
60.6
d
d
d
d
d
d
d
d
d
d
d
109.2
19.3
109.2
19.3
109.7
19.1
110.1
18.7
109.2
19.3
109.3
19.3
109.2
19.3
109.3
19.4
109.1
19.1
109.2
19.3
Monosaccharide unit
1
2
3
4
5
6
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
96.5
71.9
71.8
66.1
72.7
60.9
96.5
71.7
71.9
66.1
72.8
60.9
96.5
71.7
71.9
66.1
72.8
60.9
104.3
71.6
72.3
66.9
64.0
d
105.2
71.4
72.8
67.6
64.6
d
104.8
71.5
72.6
67.5
64.6
d
102.8
71.1
71.5
72.9
68.3
17.0
102.2
71.3
72.0
73.6
67.8
17.3
102.8
71.3
71.7
73.2
68.4
17.4
a
b
c
Recorded in CDCl3 at 150 MHz,
Recorded in CDCl3/CD3OD mixture (3:1) at 150 MHz,
d
in ppm.
d
in ppm.
3-O-Allyl group: OCH2 70.7 ppm; ]CH 135.9 ppm; ]CH2 115.9 ppm.
3-O- and 28-O-Acetyl groups: 171.0 and 170.9 ppm.
d
e
Atom numbering according to IUPAC rules (Fig. 4).42,43
2.2.1. Cell cycle and apoptosis. We have tested whether the two
most active compounds on HeLa cells, 13 and 15a, could influence
their cell cycle. Flow cytometric analysis was used to quantify the
distribution of HeLa cells in cell cycle phases, and the subG1 fraction
as a marker of the proportion of apoptotic cells, following in-
cubation with 13 and 15a. The results show that treatment with
50 mM 13 increased the proportions of S-phase and G2/M cells, with
concomitant reductions in the proportions of G0/G1 cells. The
proportion of cells with subG1 amounts of DNA (apoptotic cells)
increased after two treatments with 13, from 3.7% in control cells to
Fig. 4. Atom numbering.
5.8% in cells treated with 50 mM 13. Compound 15a had no effect on
the cell cycle of HeLa cells (Fig. 5).
derivatives 33b and 33d that showed cytotoxic activity against
almost all of the tumor cell lines, but also much stronger toxicity
towards normal BJ-H-tert fibroblasts. Monosaccharide analogue
3. Conclusions
34d containing
L
-arabinose fragment (IC50 36.1
m
M) was slightly
In conclusion, a series of the lupane-type triterpenes elongated
potent to CEM leukemia with comparable cytotoxicity to the sa-
ponin analogue 34b. The results show several new unique terpe-
noid lupane structures exhibiting cytotoxic activity in low
micromolar range with higher tolerated doses for nonmalignant
cells (BJ-H-tert fibroblasts), demonstrating their potential for fur-
ther development as anticancer drugs.
at the C-28 position, and saponins bearing the
D-mannose, L-arab-
inose, and -rhamnose moieties at the C-3 position obtained from
L
the above triterpenes were synthesized and evaluated for their
cytotoxic activities. Several triterpenes, as well as the correspond-
ing saponins show an interesting cytotoxic activity profile against
human cancer cell lines.

P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2723
Table 3
IC50 M) values obtained from the Calcein AM assays with the tested cancer and
NMR spectra were recorded at 298 K with a Varian NMR-
VNMRS600 or VNMRS500 spectrometers. Standard experimental
conditions and standard Varian programs (ChemPack 4.1) were
used. Configurational assignments were based on the NMR mea-
surements including two-dimensional techniques like COSY,
and 1He13C gradient selected HSQC (g-HSQC), as well as 1He13C
gradient selected HMBC (g-HMBC) experiments, which were
(
m
normal cell lines; meansꢃSD obtained from three independent experiments per-
formed in triplicate. Betulinic acid was used as a positive control
Compound
Cell lines (IC50
CEM
m
M)
MCF7
HeLa
BJ
Triterpenes
employed in several cases. Internal TMS was used as the 1H and 13
C
Betulinic acid
1
2
3
5
6
8
9a
10a
13
14a
14ab
15a
15b
19a
19b
19ab
20ab
21a
22a
22b
23a
23b
24a
25a
26a
27
40ꢃ2.8
30.8ꢃ0.6
>50
>50
>50
>50
>50
>50
>50
46.0ꢃ5.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
32.1ꢃ3.3
>50
>50
>50
>50
36.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
47.6ꢃ1.9
28.5ꢃ5.0
>50
>50
>50
>50
NMR chemical shift standard. J values are given in Hertz. High
resolution mass spectra (HRMS ESI) were acquired with a MARINER
and MaldiSYNAPT G2-S HDMS (Waters) mass spectrometers. Op-
tical rotations were measured with a JASCO P-2000 automatic po-
larimeter. IR spectra were recorded on Jasco 6200 FT-IR
spectrophotometer.
45.0ꢃ1.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
44.7
>50
>50
>50
29.0ꢃ3.2
>50
43.4ꢃ1.0
>50
27.8ꢃ4.3
38.3ꢃ15.6
>50
Diffraction data for 27e29 were collected at 100 K using an
12.3ꢃ0.8
Agilent SuperNova diffractometer with Cu Ka radiation. All struc-
>50
tures were solved by direct methods (SHELXS-97), and refined on F2
by full-matrix least-squares method (SHELXL-97). In all cases
crystals suitable for X-ray analysis were obtained by cooling of a hot
methanol solution.
>50
>50
41.9ꢃ11.5
16.3ꢃ8.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
19.8ꢃ11.6
31.3ꢃ5.9
20.5ꢃ11.3
>50
>50
>50
>50
>50
>50
>50
Data for 27: a¼15.3096(6), b¼10.3795(5), c¼18.0492(7),
b
¼97.943(4), monoclinic, P21, R1¼0.0499, wR2¼0.1325, S¼1.037.
28.3ꢃ6.9
Structure contains two symmetrically independent molecules of 27
in the unit cell.
>50
>50
>50
>50
>50
Data for 28: a¼12.4455(2), b¼14.5119(2), c¼16.1425(3), ortho-
rhombic, P212121, R1¼0.0322, wR2¼0.0845, S¼1.055.
Data for 29: a¼12.5606(3), b¼14.1350(3), c¼16.7391(4), ortho-
rhombic, P212121, R1¼0.0373, wR2¼0.0989, S¼1.061.
Crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the Cambridge
Crystallographic Data Centre as Supplementary data (deposition
numbers: CCDC 927960e927962). Copies of the data can be ob-
tained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: þ44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
The absolute configuration of the diastereoisomeric alcohols
obtained during Grignard reaction and their esters was not de-
termined. For analytical reasons, individual diastereoisomers
were described as a and b in order of appearance during column
chromatography, provided that at least partial separation took
place.
45.9ꢃ5.8
>50
>50
>50
>50
47.5
>50
>50
>50
>50
28
29
>50
Saponins
33b
33d
33f
34b
34d
34f
35b
35d
35f
33.3ꢃ1.6
32.0ꢃ1.3
>50
44.4ꢃ0.6
>50
>50
>50
>50
>50
>50
>50
>50
39.7ꢃ2.1
33.0ꢃ0.6
>50
14.8ꢃ1.3
14.9ꢃ2.3
>50
43.3ꢃ2.7
44.0ꢃ1.2
>50
>50
>50
>50
>50
47.1ꢃ3.3
36.1ꢃ1.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
4.2. Chemistry
4.2.1. 28-O-Acetyl-3-O-allylbetulin (5). To a solution of 4 (2.46 g,
5.08 mmol) in DCM (50 mL) cooled in an ice-bath, allyl tri-
chloroacetimidate (2.91 g, 15.27 mmol) and TMSOTf (1 mL) were
added. The reaction mixture was stirred at 0 ꢀC for 1 h, then water
(50 mL) was added, the organic phase was washed with aq satd
NaHCO3, water, and dried with Na2SO4. Column chromatography
(hexane/ethyl acetate, 50:1/30:1) of the residue gave 2.00 g (75%)
of the title compound as colorless crystals, mp: 180e182 ꢀC; ½a D20
ꢂ
27.8 (c 0.3, CHCl3); nmax (film) 2943, 2870, 1740, 1643, 1233,
757 cmꢁ1
; dH (400 MHz, CDCl3) 5.97e5.89 (m, 1H, CHeallyl),
5.28e5.24 (m, 1H, CHeallyl), 5.13e5.11 (m, 1H, CHeallyl), 4.69 (br d,
1H, J 2.3 Hz, H-29), 4.59e4.58 (m, 1H, H-29), 4.24 (dd, 1H, J 1.6 and
11.0 Hz, H-28), 4.10e4.14 (m,1H, CHeallyl), 3.90e3.85 (m, 2H, H-28,
CHeallyl), 2.79 (dd, 1H, J 4.4, 11.8 Hz, H-3), 2.48e2.41 (m, 1H, H-19),
2.07 (s, 3H, OAc), 1.69 (s, 3H, CH3),1.03 (s, 3H, CH3), 0.97 (s, 3H, CH3),
0.95 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.78 (s, 3H, CH3), 2.00e0.66 (m,
26H, lupane protons); dC (125 MHz, CDCl3) 171.6 (C), 150.2 (C),
135.9, 115.9 (CH2), 109.8 (CH2), 86.3, 70.6 (CH2), 62.8 (CH2), 55.8,
50.4, 48.8, 47.7, 46.3 (C), 42.7 (C), 40.9 (C), 38.9 (C), 38.6 (CH2), 37.6,
37.1, 34.6 (CH2), 34.2 (CH2), 29.8 (CH2), 29.6 (CH2), 28.1, 27.1 (CH2),
25.2 (CH2), 23.1 (CH2), 21.0, 20.8 (CH2), 19.1, 18.2 (CH2), 16.3, 16.1,
Fig. 5. Cell cycle and apoptosis.
4. Experimental section
4.1. General notes
Silica gel HF254 and Silica gel 230e400 mesh (E. Merck) were
used for TLC and column chromatography, respectively. 1H and 13
C

2724
P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
16.0, 14.7; HRMS (ESI): MNaþ, found 547.4122. C35H56NaO3 requires
547.4127.
4.2.4.2. 3-O-Allyl-28-C-methylbetulin (8). Starting from 3-O-
allylbetulinal (7, 1.20 g, 2.5 mmol) and 3 M methylmagnesium
bromide in Et2O (4.2 mL, 12.6 mmol), 8a (745 mg, 60%), and 8ab
(420 mg, 34%, a:bz3:1) were obtained as white foams (column
chromatography, hexane/ethyl acetate, 20:1). Data for major isomer
(8a): [found: C, 82.08; H, 11.23. C34H56O2 requires C, 82.20; H,
4.2.2. 3-O-Allylbetulin (6). A solution of 5 (500 mg, 0.95 mmol) and
KOH (110 mg, 2.0 mmol) in ethanol (15 mL) was refluxed for 2 h, the
solvent was evaporated under reduced pressure, and the solid
residue dissolved in ethyl acetate (50 mL) and water (30 mL). The
organic phase was dried with Na2SO4. Column chromatography
(hexane/ethyl acetate, 20:1/10:1) of the residue gave 378 mg
(82%) of the title compound as colorless crystals, mp: 190e192 ꢀC;
[found: C, 82.03; H, 11.22. C33H54O2 requires C, 82.10; H, 11.27%];
11.36%]; ½a 2D0
ꢂ
36.2 (c 0.3, CHCl3); nmax (film) 3474 (br), 2942, 2871,
dH (500 MHz, CDCl3)
1455, 1375, 1091, 1068, 919, 883, 758 cmꢁ1
;
5.97e5.89 (m, 1H, CHeallyl), 5.29e5.24 (m, 1H, CHeallyl),
5.14e5.10 (m, 1H, CHeallyl), 4.71 (d, 1H, J 2.7 Hz, H-29), 4.58e4.57
(m, 1H, H-29), 4.33 (q, 1H, J 6.4 Hz, H-28), 4.15e4.10 (m, 1H,
CHeallyl), 3.91e3.86 (m, 1H, CHeallyl), 2.90e2.84 (m, 1H), 2.80 (dd,
1H, J 4.2, 11.5 Hz, H-3), 1.69 (s, 3H, CH3), 1.17 (d, 3H, J 6.4 Hz, H-28a),
1.03 (s, 3H, CH3),1.00 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.84 (s, 3H, CH3),
0.78 (s, 3H, CH3), 2.10e0.65 (m, 25H, lupane protons); dC (125 MHz,
CDCl3) 151.4 (C), 135.9, 115.9 (CH2), 109.4 (CH2), 86.3, 70.6 (CH2),
68.3, 55.9, 50.4, 50.2, 50.1 (C), 49.0, 43.0 (C), 41.0 (C), 38.9 (C), 38.6
(CH2), 37.1 (C), 36.9, 34.6 (CH2), 34.3 (CH2), 32.8 (CH2), 32.3 (CH2),
28.1, 27.7 (CH2), 25.2 (CH2), 23.1 (CH2), 21.0 (CH2), 19.8, 18.9, 18.2
(CH2), 16.3, 16.1, 16.1, 15.2.
½
a 2D0
758 cmꢁ1
ꢂ
38.8 (c 0.3, CHCl3); nmax (film) 2942, 2869, 1642, 1454, 1026,
;
1H NMR data, see Table 1; 13C NMR data, see Table 2.
4.2.3. 3-O-Allylbetulinal (7). To a solution of 6 (4.71 g, 9.77 mmol)
in DCM (100 mL) cooled in an ice-bath PCC (12.71 g, 59.0 mmol)
was added in one portion and stirred at rt for 1.5 h. The solution was
decanted from the gummy residue, which was washed with DCM
twice, and the collected organic layers were evaporated to dryness.
Column chromatography (hexane/ethyl acetate, 50:1) of the resi-
due gave 3.50 g (75%) of the title compound as colorless crystals,
mp: 150e152 ꢀC; [found: C, 82.03; H, 10.97. C33H52O2 requires C,
4.2.4.3. 3-O-Acetyl-28-C-ethylbetulin (15). Starting from 3-O-
acetylbetulinal (13, 560 mg, 1.16 mmol) and 3 M ethylmagnesium
bromide in Et2O (1.9 mL, 5.7 mmol),15a (351 mg, 59%),15ab (77 mg,
13%), and 15b (86 mg, 14%) were obtained as white foams. In some
experiments deacetylation on O-3 position was observed. The
corresponding diols 16 were isolated as the dibenzoates 17 (white
foam) after acylation (pyridine, benzoyl chloride) of slowest mov-
ing fraction in 2e11% yield calculated on starting betulinal. Data for
15a: [found: C, 79.17; H, 11.12. C34H56O3 requires C, 79.63; H,
82.44; H, 10.90%]; ½a D20
ꢂ
43.1 (c 0.35, CHCl3); nmax (film) 2943, 2866,
dH (600 MHz,
1726, 1643, 1453, 1376, 1084, 920, 885, 739 cmꢁ1
;
CDCl3) 9.68 (d, 1H, J 1.4 Hz, CHO), 5.96e5.89 (m, 1H, CHeallyl),
5.28e5.24 (m, 1H, CHeallyl), 5.13e5.10 (m, 1H, CHeallyl), 4.76 (br d,
1H, J 1.6 Hz, H-29), 4.63e4.62 (m, 1H, H-29), 4.14e4.10 (m, 1H,
CHeallyl), 3.90e3.86 (m,1H, CHeallyl), 2.89e2.83 (m,1H), 2.79 (dd,
1H, J 4.3,11.8 Hz, H-3),1.70 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.95 (s, 3H,
CH3), 0.91 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.77 (s, 3H, CH3), 2.10e0.65
(m, 24H, lupane protons); dC (150 MHz, CDCl3) 206.7 (CHO), 149.7
(C), 135.9, 115.9 (CH2), 110.1 (CH2), 86.3, 70.6 (CH2), 59.3 (C), 55.8,
50.5, 48.1, 47.5, 42.5 (C), 40.9 (C), 38.7, 38.6 (CH2), 36.6 (C), 37.1 (C),
34.3 (CH2), 33.2 (CH2), 29.8 (CH2), 29.3 (CH2), 28.8 (CH2), 28.1, 25.5
(CH2), 23.1 (CH2), 20.8 (CH2), 19.0, 18.2 (CH2), 16.3, 16.1, 15.9, 14.2.
11.01%]; ½a 2D0
ꢂ
27.2 (c 0.3, CHCl3); dH (500 MHz, CDCl3) 4.71 (d, 1H, J
2.3 Hz, H-29), 4.57e4.56 (m, 1H, H-29), 4.47 (dd, 1H, J 5.8, 10.7 Hz,
H-3), 3.96e3.91 (m, 1H, H-28), 2.95e2.89 (m, 1H), 2.04 (s, 3H, OAc),
1.69 (s, 3H, CH3), 1.04 (s, 3H, CH3), 1.02 (t, 3H, J 7.4 Hz, CH3), 1.00 (s,
3H, CH3), 0.86 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.84 (s, 3H, CH3),
2.10e0.76 (m, 27H, lupane protons); dC (125 MHz, CDCl3) 171.0 (C),
151.5 (C), 109.4 (CH2), 80.9, 74.4, 55.4, 50.5, 50.3, 50.1 (C), 49.0, 43.0
(C), 41.0 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 36.9, 34.3 (CH2), 34.1
(CH2), 33.2 (CH2), 32.9 (CH2), 27.9, 27.8 (CH2), 25.2 (CH2), 23.7 (CH2),
21.3, 20.9 (CH2), 18.9, 18.1, 16.5, 16.1, 15.2, 11.4. Data for 15b: [found:
4.2.4. Grignard reaction. General procedure
4.2.4.1. 3-O-Acetyl-28-C-methylbetulin (14). To a solution of 3-
O-acetylbetulinal (13, 2.900 g, 6.00 mmol) in THF (60 mL) cooled to
ꢁ40 ꢀC, a 3 M solution of methylmagnesium bromide in Et2O
(10 mL, 30.0 mmol) was slowly added. Stirring was continued for
30 min at ꢁ40 ꢀC. The reaction was quenched by the addition of
satd aq NH4Cl (2 mL). Silica gel (10e15 g) was poured into the
mixture and the solvent was evaporated under reduced pressure to
dryness. The residue was used for purification by flash chroma-
tography (hexane/ethyl acetate, 40:1/10:1). Products were par-
tially separated and 14a (1.272 g, 42%) and 14ab (1.195 g, 40%,
a:bz4:1) were obtained as white foams. Data for 14a: [found: C,
C, 79.30; H, 10.75. C34H56O3 requires C, 79.63; H, 11.01%]; ½a D20
ꢂ
24.1
(c 0.25, CHCl3); dH (600 MHz, CDCl3) 4.69 (d,1H, J 2.3 Hz, H-29), 4.58
(s, 1H, H-29), 4.45 (dd, 1H, J 5.5, 11.0 Hz, H-3), 3.70 (br d, 1H, J 9.7 Hz,
H-28), 2.66e2.61 (m, 1H), 2.02 (s, 3H, CH3CO), 1.68 (s, 3H, CH3), 1.02
(t, 3H, J 7.1 Hz, CH3), 1.00 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.83 (s, 6H,
2ꢄ CH3), 0.82 (s, 3H, CH3), 2.10e0.75 (m, 27H, lupane protons); dC
(150 MHz, CDCl3) 171.0 (C), 150.7 (C), 109.9 (CH2), 80.9, 74.5, 55.4,
50.4 (C), 50.3, 50.1, 48.2, 42.9 (C), 41.0 (C), 38.3 (CH2), 37.8 (C), 37.0
(C), 36.8, 34.2 (CH2), 34.0 (CH2), 33.2 (CH2), 31.4 (CH2), 28.2 (CH2),
27.9, 26.5 (CH2), 25.3 (CH2), 23.7 (CH2), 21.3, 20.9 (CH2), 18.7, 18.2
(CH2), 16.5, 16.1, 15.9, 14.9, 12.1.
79.50; H, 11.07. C33H54O3 requires C, 79.46; H, 10.91%]; ½a D20
ꢂ
28.1 (c
0.4, CHCl3); nmax (film) 2945, 2873, 1718, 1374, 1028, 979, 882,
758 cmꢁ1
; dH (600 MHz, CDCl3) 4.71 (d, 1H, J 2.4 Hz, H-29),
4.58e4.57 (m, 1H, H-29), 4.47 (dd, 1H, J 5.6, 10.9 Hz, H-3), 4.32 (q,
1H, J 6.3 Hz, H-28), 2.89e2.84 (m, 1H), 2.04 (s, 3H, CH3CO), 1.69 (s,
3H, CH3), 1.17 (d, 3H, J 6.3 Hz, CH3), 1.03 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 0.85 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.83 (s, 3H, CH3), 2.10e0.76
(m, 25H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C), 151.3 (C),
109.5 (CH2), 80.9, 68.3, 55.4, 50.3, 50.1, 50.1 (C), 49.0, 42.9 (C), 40.9
(C), 38.3 (CH2), 37.8 (C), 37.0 (C), 36.8, 34.6 (CH2), 34.1 (CH2), 32.8
(CH2), 32.2 (CH2), 27.9, 27.7 (CH2), 25.1 (CH2), 23.7 (CH2), 21.3, 20.9
(CH2), 19.8, 18.8, 18.1 (CH2), 16.5, 16.1, 16.1, 15.2. Data for 14b: dH
(600 MHz, CDCl3)dselected signals 4.60e4.59 (m, 1H, H-29), 4.11
(q, 1H, J 6.2 Hz), 2.65e2.62 (m, 1H), 2.04 (s, 3H, OAc); dC (150 MHz,
CDCl3)dselected signals 150.6 (C), 110.0 (CH2), 80.9, 67.7, 50.3 (C),
49.9, 48.2, 41.0 (C), 36.8, 34.2 (CH2), 33.2 (CH2), 33.1 (CH2), 28.1
(CH2), 25.2 (CH2), 20.9, 20.8 (CH2), 16.0, 14.9.
4.2.4.4. 3,28-Di-O-benzoyl-28-C-ethylbetulin (17). White foam.
[Found: C, 81.41; H, 9.25. C46H62O4 requires C, 81.37; H, 9.20%]; dH
(500 MHz, CDCl3)dmajor diastereoisomer 8.08e8.04 (m, 4H, Ar),
7.57e7.42 (m, 6H, Ar), 5.79 (dd,1H, J 2.4,10.1 Hz, H-28), 4.72 (dd,1H,
J 5.1, 11.0 Hz, H-3), 4.44e4.43 (m, 1H, H-29), 4.31e4.30 (m, 1H, H-
29), 2.77e2.72 (m,1H),1.60 (s, 3H, CH3),1.23 (s, 3H, CH3),1.03 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.93 (s, 6H, 2ꢄ CH3), 0.91 (s, 3H, CH3),
2.40e0.85 (m, 26H, lupane protons); dC (125 MHz, CDCl3) 166.6 (C),
166.3 (C), 150.7 (C), 132.8, 132.7, 131.0, 130.9, 129.5, 129.4, 128.4,
128.3, 128.3, 109.4 (CH2), 81.6, 76.2, 55.5, 50.4, 50.3, 50.2 (C), 48.4,
43.0 (C), 41.1 (C), 38.4 (CH2), 38.2 (C), 37.1 (C), 36.5, 34.7 (CH2), 34.3
(CH2), 33.6 (CH2), 32.3 (CH2), 28.1, 27.9 (CH2), 25.1 (CH2), 24.0 (CH2),
23.8 (CH2), 21.0 (CH2), 18.6, 18.2 (CH2), 16.8, 16.2, 16.2, 15.2, 10.9.

P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2725
4.2.4.5. 3,28-Di-O-acetyl-28-C-ethylbetulin
(18). Analytical
150.7 (C), 109.9 (CH2), 80.9, 72.1, 55.4, 50.3, 50.2, 50.2 (C), 48.2, 42.9
(C), 41.0 (C), 38.3 (CH2), 37.8 (C), 37.0 (C), 36.8, 35.8 (CH2), 34.2
(CH2), 34.1 (CH2), 33.1 (CH2), 31.3 (CH2), 28.3 (CH2), 27.9, 25.3 (CH2),
23.7 (CH2), 21.3, 20.9 (CH2), 20.3 (CH2), 18.7, 18.2 (CH2), 16.5, 16.1,
15.9, 14.9, 14.0.
samples of the corresponding diacetates were obtained as white
foams by acylation of 15a and 15b with acetic anhydride in pyridine
under standard conditions. Data for 18a: [found: C, 77.43; H, 10.66.
C
36H58O4 requires C, 77.93; H, 10.54%]; ½a D
ꢂ
20 21.2 (c 0.3, CHCl3); nmax
dH (600 MHz,
(film) 2938, 2872, 1733, 1367, 1244, 1017, 760 cmꢁ1
;
4.2.4.8. 3-O-Acetyl-28-O-benzoyl-28-C-propylbetulin
(20). Analytical sample of the title compound was obtained as
white foam by acylation of 19ab with benzoyl chloride in pyridine.
[Found: C, 79.92; H, 9.98. C42H62O4 requires C, 79.95; H, 9.90%]; dH
(600 MHz, CDCl3)dmajor diastereoisomer 8.07e7.26 (m, 5H, Ar),
5.84 (dd, 1H, J 1.8, 9.9 Hz, H-28), 4.49e4.46 (m, 1H, H-3), 4.43e4.41
(m, 1H, H-29), 4.30 (br d, 1H, J 1.9 Hz, H-29), 2.04 (s, 3H, CH3CO),
1.58 (s, 3H, CH3), 1.21 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.92 (t, 3H, J
7.4 Hz, CH3), 0.88 (s, 3H, CH3), 0.85 (s, 6H, 2ꢄ CH3), 2.40e0.76 (m,
29H, lupane protons); dC (150 MHz, CDCl3)dmajor diastereoisomer
(selected peaks) 171.0 (C),166.4 (C),150.8 (C),109.4 (CH2), 81.0, 74.5.
CDCl3) 5.46 (dd, 1H, J 2.1, 10.2 Hz, H-28), 4.60 (d, 1H, J 1.5 Hz, H-29),
4.56 (br s, 1H, H-29), 4.46 (dd, 1H, J 6.6, 9.9 Hz, H-3), 2.33e2.28 (m,
1H), 2.07 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO), 1.66 (s, 3H, CH3), 1.11
(s, 3H, CH3), 0.97 (s, 3H, CH3), 0.86 (t, 3H, J 7.4 Hz, CH3), 0.85 (s, 3H,
CH3), 0.83 (s, 3H, CH3), 0.83 (s, 3H, CH3), 2.12e0.76 (m, 26H, lupane
protons); dC (150 MHz, CDCl3) 171.0 (C), 170.8 (C), 150.9 (C), 109.4
(CH2), 80.9, 75.4, 55.4, 50.4, 50.2, 50.1 (C), 48.9, 42.8 (C), 41.0 (C),
38.4 (CH2), 37.8 (C), 37.1 (C), 36.5, 34.6 (CH2), 34.3 (CH2), 33.7 (CH2),
32.4 (CH2), 27.9, 27.8 (CH2), 25.1 (CH2), 23.7 (CH2), 23.5 (CH2), 21.7,
21.3, 21.0 (CH2), 18.7, 18.1 (CH2), 16.5, 16.1, 16.1, 15.1, 10.7. Data for
18b: [found: C, 77.45; H, 10.69. C36H58O4 requires C, 77.93; H,
10.54%]; ½a 2D0
ꢂ
23.1 (c 0.4, CHCl3); nmax (film) 2938, 2870, 1728, 1366,
1247, 1017, 760 cmꢁ1; 1H NMR data, see Table 1; 13C NMR data, see
Table 2.
4.2.5. Acylation with phenyl chlorothionoformate. General procedure
4.2.5.1. Phenyl (3-O-acetyl-28-C-methyl)betulinyl thiocarbonate
(21). To a solution of 14 (1.206 g, 2.42 mmol) in DCM (20 mL),
pyridine (1.22 mL, 6.2 mmol), and phenyl chlorothionoformate
(0.86 mL, 6.2 mmol) were added, and the reaction stirred at rt for
24 h. Solvents were evaporated, column chromatography (hexane/
ethyl acetate, 40:1) of the residue gave 1.510 g of 21 as a pale yellow
foam contaminated by a small amount of phenyl (3-O-acetyl-28-C-
methyl)betulinyl carbonate (24). Data for 21: [found: C, 75.47; H,
9.05; S, 5.03. C40H58O4S requires C, 75.66; H, 9.21; S, 5.05%]; dH
(600 MHz, CDCl3)dmajor diastereoisomer 7.42e7.08 (m, 5H, Ar),
5.96 (q, 1H, J 6.3 Hz, H-28), 4.68 (br d, 1H, H-29), 4.59e4.58 (m, 1H,
H-29), 4.46 (dd, 1H, J 5.8, 10.6 Hz, H-3), 2.50e2.44 (m, 1H), 2.03 (s,
3H, OAc), 1.68 (s, 3H, CH3), 1.34 (d, 3H, J 6.3 Hz, CH3), 1.07 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.82 (s, 6H, CH3), 0.82 (s,
3H, CH3), 2.10e0.75 (m, 24H, lupane protons); dC (150 MHz, CDCl3)
194.0 (C]S), 171.0 (C]O), 153.3 (C), 150.8 (C), 129.5, 126.4, 122.0,
109.7 (CH2), 83.3, 80.9, 55.4, 50.4, 50.3 (C), 49.9, 48.9, 43.0 (C), 41.0
(C), 38.4 (CH2), 37.8 (C), 37.2, 37.1 (C), 34.3 (CH2), 34.2 (CH2), 33.0
(CH2), 31.9 (CH2), 27.9, 27.7 (CH2), 25.1 (CH2), 23.7 (CH2), 21.3, 21.0
(CH2), 18.1 (CH2), 16.5, 16.5, 16.1, 15.2, 15.1.
4.2.4.6. 3-O-Allyl-28-C-ethylbetulin (9). Starting from 3-O-allyl-
betulinal (7, 1.086 g, 2.26 mmol) and 3 M ethylmagnesium bromide
in Et2O (3.8 mL,11.4 mmol) 9a (437 mg, 38%), and 9ab (270 mg, 23%,
a:bz3:2), were obtained as white foams (column chromatography,
hexane/ethyl acetate, 20:1). Data for major isomer (9a): [found: C,
82.23; H, 11.38. C35H58O2 requires C, 82.29; H, 11.44%]; ½a D20
ꢂ
38.2 (c
0.3, CHCl3); nmax (film) 2943, 2871, 1458, 1375, 1216, 1091, 1070, 975,
921, 882, 758 cmꢁ1
; dH (600 MHz, CDCl3) 5.95e5.88 (m, 1H,
CHeallyl), 5.27e5.23 (m, 1H, CHeallyl), 5.12e5.08 (m, 1H,
CHeallyl), 4.71 (br d, 1H, J 2.3 Hz, H-29), 4.57e4.56 (m, 1H, H-29),
4.13e4.09 (m, 1H, CHeallyl), 3.92 (br d, 1H, H-28), 3.89e3.85 (m,
1H, CHeallyl), 2.93e2.88 (m, 1H, H-19), 2.79 (dd, 1H, J 4.4, 11.9 Hz,
H-3), 1.67 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.00 (t, 3H, J 7.3 Hz, CH3),
0.99 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.77 (s, 3H,
CH3), 2.10e0.65 (m, 27H, lupane protons); dC (150 MHz, CDCl3)
151.5 (C), 135.9, 115.9 (CH2), 109.3 (CH2), 86.3, 74.4, 70.6 (CH2), 55.8,
50.5, 50.4, 50.1 (C), 49.0, 43.0 (C), 41.0 (C), 38.8 (C), 38.6 (CH2), 37.1
(C), 36.9, 34.3 (CH2), 34.2 (CH2), 32.2 (CH2), 32.9 (CH2), 28.1, 27.8
(CH2), 25.3 (CH2), 25.2 (CH2), 23.1 (CH2), 20.9 (CH2), 19.0, 18.2 (CH2),
16.3, 16.1, 16.0, 15.2, 11.4.
4.2.5.2. Phenyl (3-O-acetyl-28-C-ethyl)betulinyl thiocarbonate
(22). Starting from 15 (1.314 g, 2.56 mmol) 1.495 g of 22 contam-
inated by small amount of phenyl (3-O-acetyl-28-C-ethyl)betulinyl
carbonate (25) was obtained as white foam. Data for major isomer
(22a)d[found: C, 75.96; H, 9.48; S, 4.74. C41H60O4S requires C,
75.88; H, 9.32; S, 4.94%]; dH (600 MHz, CDCl3) 7.42e7.08 (m, 5H, Ar),
5.99 (dd, 1H, J 3.0, 9.7 Hz, H-28), 4.70 (br d, 1H, J 1.8 Hz, H-29), 4.60
(br s, 1H, H-29), 4.46 (dd, 1H, J 5.7, 10.6 Hz, H-3), 2.50e2.44 (m, 1H),
2.03 (s, 3H, CH3CO), 1.69 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.06 (t, 3H, J
7.4 Hz, CH3), 1.00 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.81 (s, 3H, CH3),
0.81 (s, 3H, CH3), 2.40e0.75 (m, 26H, lupane protons); dC (150 MHz,
CDCl3) 195.0 (C), 171.0 (C), 153.3 (C), 150.8 (C), 129.5, 126.4, 122.0,
109.6 (CH2), 87.6, 80.9, 55.4, 51.1 (C), 50.4, 50.4, 48.6, 42.9 (C), 41.1
(C), 38.4 (CH2), 37.8 (C), 37.1, 37.0 (C), 34.6 (CH2), 34.3 (CH2), 33.7
(CH2), 32.2 (CH2), 27.9, 27.8 (CH2), 25.1 (CH2), 24.2 (CH2), 23.7 (CH2),
21.3, 21.0 (CH2), 18.1 (CH2), 16.6, 16.5, 16.1, 15.3, 10.7; HRMS (ESI):
2MNaþ, found 1319.8317. C82H120NaO2S2 requires 1319.8322. Data
for minor isomer (22b)d[found: C, 75.86; H, 9.38; S, 4.85.
4.2.4.7. 3-O-Acetyl-28-C-propylbetulin (19). Starting from 3-O-
acetylbetulinal (13, 560 mg, 1.16 mmol) and 2 M propylmagnesium
chloride in Et2O (2.9 mL, 5.8 mmol), 19a (355 mg, 58%), 19ab
(38 mg, 6%, a:bz8:1), and 19b (53 mg, 9%) were obtained as white
foams. Data for 19a: [found: C, 80.02; H, 11.18. C35H58O3 requires C,
79.79; H, 11.10%]; ½a D20
ꢂ
34.3 (c 0.3, CHCl3); nmax (film) 2953, 2872,
dH (500 MHz, CDCl3) 4.70 (d, 1H, J
1733, 1718, 1639, 1249, 757 cmꢁ1
;
2.4 Hz, H-29), 4.58e4.57 (m, 1H, H-29), 4.47 (dd, 1H, J 5.7, 10.7 Hz,
H-3), 4.06 (br d, 1H, J 8.2 Hz, H-28), 2.94e2.88 (m, 1H), 2.04 (s, 3H,
CH3CO), 1.69 (s, 3H, CH3), 1.04 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.96 (t,
3H, J 7.2 Hz, CH3), 0.85 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.84 (s, 3H,
CH3), 2.10e0.76 (m, 29H, lupane protons); dC (125 MHz, CDCl3)
171.0 (C), 151.5 (C), 109.4 (CH2), 80.9, 72.0, 55.4, 50.5, 50.3, 50.0 (C),
49.0, 43.0 (C), 41.0 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 36.9, 34.6 (CH2),
34.3 (CH2), 34.1 (CH2), 33.2 (CH2), 32.7 (CH2), 27.9, 27.8 (CH2), 25.2
(CH2), 23.7 (CH2), 21.3, 20.9 (CH2), 19.6 (CH2), 18.9, 18.1 (CH2), 16.5,
16.1, 16.0, 15.2, 14.0. Data for 19b: [found: C, 79.86; H, 10.99.
C41H60O4S requires C, 75.88; H, 9.32; S, 4.94%]; dH (600 MHz, CDCl3)
C
35H58O3 requires C, 79.79; H, 11.10%]; ½a D20
ꢂ
18.6 (c 0.2, CHCl3); nmax
dH (600 MHz,
7.40e7.07 (m, 5H, Ar), 5.74 (dd, 1H, J 1.7, 10.4 Hz, H-28), 4.70 (br d,
1H, H-29), 4.60 (s, 1H, H-29), 4.46 (dd, 1H, J 5.4, 11.0 Hz, H-3),
2.81e2.76 (m, 1H), 2.03 (s, 3H, CH3CO), 1.69 (s, 3H, CH3), 1.10 (s, 3H,
CH3), 1.07 (t, 3H, J 7.3 Hz, CH3), 1.00 (s, 3H, CH3), 0.83 (s, 6H, 2ꢄ CH3),
0.81 (s, 3H, CH3), 2.18e0.75 (m, 26H, lupane protons); dC (150 MHz,
CDCl3) 195.8 (C), 171.0 (C), 153.5 (C), 150.2 (C), 129.4, 126.3, 122.0,
110.3 (CH2), 89.3, 80.9, 55.4, 50.9 (C), 50.3, 50.2, 47.9, 43.1 (C), 41.1
(film) 2951, 2870, 1735, 1719, 1640, 1249, 758 cmꢁ1
;
CDCl3) 4.72 (d, 1H, J 2.0 Hz, H-29), 4.60 (s, 1H, H-29), 4.47 (dd, 1H, J
5.5, 11.0 Hz, H-3), 3.85 (br d, 1H, J 10.3 Hz, H-28), 2.68e2.63 (m, 1H),
2.04 (s, 3H, CH3CO), 1.70 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 0.99 (s, 3H, CH3), 0.85 (s, 6H, 2ꢄ CH3), 0.83 (s, 3H, CH3),
2.15e0.76 (m, 29H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C),

2726
P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
(C), 38.4 (CH2), 37.8 (C), 37.0, 37.0 (C), 34.9 (CH2), 34.1 (CH2), 33.2
(CH2), 31.3 (CH2), 28.8 (CH2), 27.9, 25.6 (CH2), 25.3 (CH2), 23.7 (CH2),
21.3, 20.9 (CH2), 18.1 (CH2), 16.5, 16.1, 15.0, 11.7.
(CH2), 16.3, 16.1, 16.1, 15.2, 10.5; m/z (ESI) 653.6 (100, MNaþ), 1284.0
(37, 2MNaþ).
4.2.6. Phenyl
(3-O-acetyl-28-C-alkyl)betulinyl
carbonate
4.2.5.3. Phenyl (3-O-acetyl-28-C-propyl)betulinyl thiocarbonate
(23). Starting from 19 (1.496 g, 2.84 mmol) 1.614 g of 23 contam-
inated by a small amount of phenyl (3-O-acetyl-28-C-propyl)
betulinyl carbonate (26) was obtained as a white foam. Data for
major isomer (23a). [Found: C, 75.98; H, 9.33; S, 4.81. C42H62O4S
requires C, 76.09; H, 9.43; S, 4.84%]; dH (600 MHz, CDCl3) 7.43e7.09
(m, 5H, Ar), 6.03 (dd, 1H, J 2.5, 9.7 Hz, H-28), 4.71 (br s, 1H, H-29),
4.61 (br s, 1H, H-29), 4.47 (dd, 1H, J 5.7, 10.6 Hz, H-3), 2.04 (s, 3H,
CH3CO), 1.71 (s, 3H, CH3), 1.10 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.00 (t,
3H, J 7.3 Hz, CH3), 0.84 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.82 (s, 3H,
CH3), 2.52e0.75 (m, 29H, lupane protons); dC (150 MHz, CDCl3)
194.9 (C), 171.0 (C), 153.5 (C), 150.9 (C), 129.5, 126.4, 122.2, 109.6
(CH2), 86.3, 81.0, 55.4, 51.1 (C), 50.5, 50.4, 48.6, 43.0 (C), 41.1 (C),
38.4 (CH2), 37.8 (C), 37.1, 37.1 (C), 34.7 (CH2), 34.4 (CH2), 33.8 (CH2),
33.4 (CH2), 32.3 (CH2), 28.0, 27.8 (CH2), 25.1 (CH2), 23.7 (CH2), 21.3,
21.1 (CH2), 19.1 (CH2), 18.1 (CH2), 16.6, 16.5, 16.2, 15.3, 14.5. Data for
minor isomer (23b)d[found: C, 76.04; H, 9.62; S, 4.70. C42H62O4S
requires C, 76.09; H, 9.43; S, 4.84%]; dH (600 MHz, CDCl3) 7.43e7.07
(m, 5H, Ar), 5.78 (br d, 1H, J 9.9 Hz, H-28), 4.70 (br s, 1H, H-29), 4.61
(br s, 1H, H-29), 4.47 (dd, 1H, J 5.4, 11.0 Hz, H-3), 2.84e2.79 (m, 1H),
2.04 (s, 3H, CH3CO), 1.71 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.01 (m, 6H,
2ꢄ CH3), 0.84 (s, 6H, 2ꢄ CH3), 0.82 (s, 3H, CH3), 2.18e0.75 (m, 28H,
lupane protons); dC (150 MHz, CDCl3) 195.7 (C), 171.0 (C), 153.5 (C),
150.2 (C), 129.5, 126.3, 122.0, 110.3 (CH2), 87.7, 80.9, 55.4, 50.8 (C),
50.3, 50.3, 47.8, 43.1 (C), 41.1 (C), 38.4 (CH2), 37.8 (C), 37.0, 37.0 (C),
35.2 (CH2), 34.7 (CH2), 34.1 (CH2), 32.9 (CH2), 31.2 (CH2), 28.8 (CH2),
27.9, 25.3 (CH2), 23.7 (CH2), 21.3, 20.9 (CH2), 19.8 (CH2), 18.7, 18.1
(CH2), 16.5, 16.1, 14.9, 14.4.
(24e26). Analytical samples of the corresponding carbonates were
obtained by acylation of 14a, 15a, and 19a with phenyl chlor-
oformate under standard conditions (DCM, pyridine).
4.2.6.1. Phenyl
(3-O-acetyl-28-C-methyl)betulinyl
23.1 (c 0.3, CHCl3); nmax (film) 2946, 2873, 1782, 1755,
dH
carbonate
(24). ½a 2D0
ꢂ
1732, 1593, 1493, 1247, 1181, 1160, 1022, 790, 768, 752, 688 cmꢁ1
;
(600 MHz, CDCl3) 7.42e7.26 (m, 5H, Ar), 5.45 (q, 1H, J 6.4 Hz, H-28),
4.69 (br d,1H, J 2.1 Hz, H-29), 4.60e4.59 (m,1H, H-29), 4.47 (dd,1H, J
5.6, 10.8 Hz, H-3), 2.54e2.49 (m, 1H), 2.04 (s, 3H, CH3CO), 1.70 (s, 3H,
CH3), 1.31 (d, 3H, J 6.3 Hz, CH3), 1.06 (s, 3H, CH3), 1.00 (s, 3H, CH3),
0.84 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.83 (s, 3H, CH3), 2.06e0.75 (m,
24H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C), 153.2 (C), 151.2
(C), 151.0 (C), 150.8 (C), 129.6, 126.3, 120.9, 109.7 (CH2), 80.9, 76.6,
55.4, 50.3, 49.8, 48.9, 42.9 (C), 40.9 (C), 38.4 (CH2), 37.8 (C), 37.1 (C),
36.9, 34.2 (CH2), 32.8 (CH2), 32.0 (CH2), 27.9, 27.7 (CH2), 25.1 (CH2),
23.7 (CH2), 21.3, 20.9 (CH2),18.8 (CH2),18.1 (CH2),16.5,16.2,16.1,15.2;
HRMS (ESI): MNaþ, found 641.4172. C40H58NaO5 requires 641.4182.
4.2.6.2. Phenyl
(3-O-acetyl-28-C-ethyl)betulinyl
carbonate
(25). Compound (25): [found: C, 77.89; H, 9.70. C41H60O5 requires
C, 77.80; H, 9.56%]; ½a D20
ꢂ
32.4 (c 0.3, CHCl3); nmax (film) 2946, 2874,
dH (600 MHz, CDCl3)
1756, 1733, 1640, 1251, 1211, 758 cmꢁ1
;
7.40e7.15 (m, 5H, Ar), 5.33 (dd, 1H, J 2.5, 10.4 Hz, H-28), 4.69 (br d,
1H, J 2.0 Hz, H-29), 4.60 (br s, 1H, H-29), 4.46 (dd, 1H, J 5.7, 10.7 Hz,
H-3), 2.51e2.45 (m, 1H), 2.02 (s, 3H, CH3CO), 1.69 (s, 3H, CH3), 1.07
(s, 3H, CH3), 1.02 (t, 3H, J 7.3 Hz, CH3), 1.01 (s, 3H, CH3), 0.83 (s, 3H,
CH3), 0.81 (s, 6H, 2ꢄ CH3), 2.18e0.75 (m, 26H, lupane protons); dC
(150 MHz, CDCl3) 171.0 (C), 154.0 (C), 151.3 (C), 150.8 (C), 129.5,
125.9, 121.2, 109.6 (CH2), 81.7, 80.9, 55.4, 50.4, 50.2, 48.9, 42.9 (C),
41.0 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 36.7, 34.4 (CH2), 34.3 (CH2),
33.5 (CH2), 32.3 (CH2), 27.9, 27.8 (CH2), 25.2 (CH2), 23.7 (CH2), 23.5
(CH2), 21.3, 21.0 (CH2), 18.8, 18.1 (CH2), 16.5, 16.1, 15.2, 10.5; HRMS
(ESI): MNaþ, found 655.4340. C41H60NaO5 requires 655.4333.
4.2.5.4. Phenyl (3-O-allyl-28-C-ethyl)betulinyl thiocarbonate (10)
and phenyl (3-O-allyl-28-C-ethyl)betulinyl carbonate (11). Compounds
(10) and (11) were prepared from stereoisomer 9a. Column chro-
matography (hexane/ethyl acetate, 40:1) of the residue gave 10
(390 mg) as white foam contaminated by approx. 8% of 11. An ana-
lytical sample was separated on preparative TLC to afford pure
samples of the title compounds. Data for 10: [found: C, 77.85; H, 9.72.
4.2.6.3. Phenyl
(3-O-acetyl-28-C-propyl)betulinyl
25.5 (c 0.3, CHCl3); nmax (film) 2951, 2873, 1782, 1755,
1732, 1593, 1493, 1246, 1181, 1160, 752, 688 cmꢁ1
; dH (500 MHz,
carbonate
C
42H62O3S requires C, 77.97; H, 9.66%]; ½a D20
ꢂ
62.5 (c 0.25, CHCl3); dH
(26). ½a 2D0
ꢂ
(600 MHz, CDCl3) 7.47e7.21 (m, 5H, Ar), 6.00 (dd, 1H, J 3.1, 9.7 Hz, H-
28), 5.96e5.89 (m, 1H, CHeallyl), 5.27e5.23 (m, 1H, CHeallyl),
5.13e5.10 (m, 1H, CHeallyl), 4.71 (br d, 1H, J 1.9 Hz, H-29), 4.61e4.60
(m, 1H, H-29), 4.13e4.09 (m, 1H, CHeallyl), 3.88e3.85 (m, 1H,
CHeallyl), 2.79 (dd, 1H, J 4.3, 11.8 Hz, H-3), 1.70 (s, 3H, CH3), 1.09 (s,
3H, CH3), 1.07 (t, 3H, J 7.4 Hz, CH3), 1.00 (s, 3H, CH3), 0.95 (s, 3H, CH3),
0.80 (s, 3H, CH3), 0.77 (s, 3H, CH3), 2.50e0.60 (m, 27H, lupane pro-
tons); dC (150 MHz, CDCl3)dselected signals 194.8 (C]S), 153.6 (C),
115.9 (CH2), 87.6, 86.3, 70.6 (CH2), 55.9, 50.6, 50.4, 38.7 (CH2), 37.1,
34.6 (CH2), 34.5 (CH2), 33.7 (CH2), 28.1, 27.8 (CH2), 24.2 (CH2), 23.1
(CH2), 21.0 (CH2), 18.2 (CH2), 16.6, 16.3, 16.1, 15.3, 10.7; m/z (ESI) 669.4
(100, MNaþ), 1317.1 (36, 2MNaþ). Data for 11: [found: C, 80.01; H,
CDCl3) 7.36e7.09 (m, 5H, Ar), 5.36 (br d, 1H, J 9.5 Hz, H-28), 4.62 (br
d, 1H, J 2.0 Hz, H-29), 4.54e4.52 (m, 1H, H-29), 4.40 (dd, 1H, J 5.9,
10.5 Hz, H-3), 2.46e2.40 (m, 1H), 1.96 (s, 3H, CH3CO), 1.63 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.92 (t, 3H, J 7.1 Hz, CH3),
0.77 (s, 3H, CH3), 0.75 (s, 6H, 2ꢄ CH3), 2.18e0.75 (m, 28H, lupane
protons); dC (125 MHz, CDCl3) 171.0 (C),153.9 (C),151.3 (C),151.0 (C),
129.6,126.3,120.9,109.6 (CH2), 80.9, 79.9, 55.4, 50.4, 50.2, 48.9, 42.9
(C), 41.0 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 36.7, 34.5 (CH2), 34.3 (CH2),
33.6 (CH2), 32.7 (CH2), 32.3 (CH2), 27.9, 27.7 (CH2), 25.2 (CH2), 23.7
(CH2), 21.0 (CH2), 19.0 (CH2), 18.1 (CH2), 16.5, 16.1, 15.2, 14.0; HRMS
(ESI): MNaþ, found 669.4495. C42H62NaO5 requires 669.4495.
10.11. C42H62O4 requires C, 79.95; H, 9.90%]; ½a D20
ꢂ
53.6 (c 0.6, CHCl3);
dH (600 MHz, CDCl3) 7.42e7.24 (m, 5H, Ar), 5.96e5.89 (m, 1H,
CHeallyl), 5.34 (dd, 1H, J 2.5, 10.3 Hz, H-28), 5.27e5.23 (m, 1H,
CHeallyl), 5.12e5.10 (m, 1H, CHeallyl), 4.70 (br d, 1H, J 2.1 Hz, H-29),
4.60e4.59 (m, 1H, H-29), 4.13e4.09 (m, 1H, CHeallyl), 3.89e3.85 (m,
1H, CHeallyl), 2.78 (dd, 1H, J 4.3, 11.8 Hz, H-3), 2.51e2.46 (m, 1H, H-
19), 1.70 (s, 3H, CH3),1.07 (s, 3H, CH3),1.03 (t, 3H, J 7.3 Hz, CH3),1.00 (s,
3H, CH3), 0.94 (s, 3H, CH3), 0.80 (s, 3H, CH3), 0.77 (s, 3H, CH3),
2.20e0.65 (m, 26H, lupane protons); dC (150 MHz, CDCl3)dselected
signals 154.0 (C]O), 151.3 (C), 135.9, 115.8 (CH2), 109.6 (CH2), 86.3,
81.6, 70.6 (CH2), 55.9, 50.5, 50.3, 48.9, 42.9 (C), 41.0 (C), 38.8 (C), 38.6
(CH2), 37.1 (C), 36.7, 34.5 (CH2), 34.4 (CH2), 33.5 (CH2), 32.3 (CH2),
28.1, 27.8 (CH2), 25.2 (CH2), 23.5 (CH2), 23.1 (CH2), 21.0, 18.9, 18.2
4.2.7. Reduction of thiocarbonates. General procedure. A solution of
thiocarbonate (21, 22, or 23; 0.59 mmol) in toluene was degassed,
then AIBN (20 mg) and tris(trimethylsilyl)silane (0.97 mmol) were
added and the mixture was heated under an argon atmosphere at
80 ꢀC for 5 h. Silica gel (5e10 g) was poured into the mixture and
the solvent was evaporated to dryness. The residue was used for
purification by flash chromatography (hexane/ethyl acetate, 40:1).
Crude product was crystallized from methanol yielded expected
product as colorless crystals.
4.2.7.1. 3-O-Acetyl-28-C-methyl-lup-20(29)-en-3-ol (27). Yield
88%; mp 198e200 ꢀC; [found: C, 82.11; H, 11.26. C33H54O2 requires

P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2727
C, 82.10; H, 11.27%]; ½a D20
ꢂ
29.6 (c 0.2, CHCl3); nmax (film) 2946, 2872,
dH
4.2.9. Synthesis of saponins (glycosylation). General procedure. A
solution of trichloroacetimidate (30, 31 or 32, 0.25 mmol) and
a suitable acceptor (1, 2 or 3, 0.20 mmol) in DCM (7 mL) was stirred
1732, 1641, 1456, 1376, 1246, 1038, 979, 882, 758 cmꢁ1
;
(600 MHz, CDCl3) 4.66 (br d, 1H, J 2.3 Hz, H-29), 4.55e4.54 (m, 1H,
H-29), 4.46 (dd, 1H, J 5.5, 10.9 Hz, H-3), 2.38 (m, 1H), 2.03 (s, 3H,
OAc),1.68 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.84 (s, 3H,
CH3), 0.83 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.76 (t, 3H, J 7.4 Hz),
1.88e0.75 (m, 26H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C),
151.1 (C), 109.2 (CH2), 80.9, 55.4, 50.4, 49.7, 47.4, 45.7 (C), 42.5 (C),
40.9 (C), 38.3 (CH2), 37.8 (C), 37.1 (C), 36.9, 35.0 (CH2), 34.1 (CH2),
30.3 (CH2), 30.0 (CH2), 27.9, 27.1 (CH2), 25.1 (CH2), 23.7 (CH2), 21.3,
21.0 (CH2), 19.3 (CH2), 18.2 (CH2), 16.5, 16.1, 16.0, 14.8, 8.1.
ꢂ
for 20e30 min at rt over molecular sieves (4 A, 300 mg, finely
ground), then cooled to ꢁ40 ꢀC and TMSOTf (20
mL, 0.1 mmol) was
added. After 20 min, the reaction was quenched with Et3N (0.2 mL),
and the solvents were evaporated under diminished pressure.
Column chromatography (hexane/ethyl acetate, 10:1) gave the
protected saponins as foams.
4.2.9.1. 28-C-Methyllupeol
3b-O-(2,3,4,6-tetra-O-benzoyl-a-D-
mannopyranoside) (33a). Yield 90%; ½a D20
ꢂ
ꢁ5.8 (c 0.3, CHCl3); nmax
4.2.7.2. 3-O-Acetyl-28-C-ethyl-lup-20(29)-en-3-ol
(28). Yield
(film) 2946, 2871, 1730, 1451, 1266, 1109, 1068, 757, 710 cmꢁ1
; dH
80%; mp 247e249 ꢀC; [found: C, 80.76; H, 11.23. 2ꢄ C34H56O2ꢄ-
(600 MHz, CDCl3) 8.11e7.25 (m, 20H, Ar), 6.09 (t, 1H, J 10.1 Hz, H-40),
5.91 (dd,1H, J 3.2,10.1 Hz, H-30), 5.62 (dd,1H, J 1.7, 3.2 Hz, H-20), 5.29
(br d, 1H, J 1.7 Hz, H-10), 4.71e4.66 (m, 2H, H-29, H-60), 4.58e4.53
(m, 2H, H-29, H-50), 4.48 (dd, 1H, J 5.1, 12.0 Hz, H-60), 3.35 (dd, 1H, J
4.4, 11.8 Hz, H-3), 2.44e2.38 (m, 1H), 1.70 (s, 3H, CH3), 1.11 (s, 3H,
CH3), 1.04 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.88 (s,
3H, CH3), 0.78 (t, 3H, J 7.4 Hz, CH3), 1.90e0.68 (m, 26H, lupane
protons); dC (150 MHz, CDCl3) 166.1 (C), 165.6 (C), 165.5 (C), 151.1
(C), 133.4, 133.1, 133.0, 129.8e128.3 (aromatic carbons), 109.3 (CH2),
94.3 (C-10), 84.2, 71.6, 70.3, 69.4, 67.0, 63.1 (CH2), 55.7, 50.5, 49.7,
47.4, 45.8 (C), 42.5 (C), 40.9 (C), 38.6 (C), 38.2 (CH2), 37.1 (C), 37.0,
35.1 (CH2), 34.2 (CH2), 30.4 (CH2), 30.0 (CH2), 28.8, 27.1 (CH2), 25.1
(CH2), 22.2 (CH2), 21.0 (CH2), 19.3 (CH2), 16.5 (CH2), 16.1, 16.0, 14.9,
14.2, 8.1; HRMS (ESI): MNaþ, found 1041.5486. C65H78NaO10 re-
quires 1041.5493.
CH3OH requires C, 80.80; H, 11.40%]; ½a D20
ꢂ
14.4 (c 0.3, CHCl3); nmax
(film) 2949, 2870, 1733, 1640, 1245, 758 cmꢁ1
; dH (600 MHz, CDCl3)
4.72 (d, 1H, J 2.4 Hz, H-29), 4.60e4.59 (m, 1H, H-29), 4.51 (dd, 1H, J
5.6, 10.9 Hz, H-3), 2.48e2.43 (m, 1H), 2.08 (s, 3H, CH3CO), 1.73 (s,
3H, CH3), 1.07 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.96 (t, 3H, J 7.3 Hz,
CH3), 0.90 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.88 (s, 3H, CH3),
1.95e0.80 (m, 28H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C),
151.1 (C), 109.2 (CH2), 80.9, 55.4, 50.4, 49.8, 47.4, 45.7 (C), 42.5 (C),
40.9 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 37.0, 35.8 (CH2), 34.1 (CH2),
31.2 (CH2), 30.1 (CH2), 30.0 (CH2), 27.9, 27.2 (CH2), 25.1 (CH2), 23.7
(CH2), 21.3, 21.0 (CH2), 19.3, 18.2 (CH2), 17.0 (CH2), 16.5, 16.1, 16.0,
15.4, 14.8; HRMS (ESI): Mþ, found 496.4274. C34H56O2 requires
496.4280.
4.2.7.3. 3-O-Acetyl-28-C-propyl-lup-20(29)-en-3-ol (29). Yield
71%; mp 213e214 ꢀC; [found: C, 82.08; H, 11.66. C35H58O2 requires
4.2.9.2. 28-C-Methyllupeol
3b-O-(2,3,4-tri-O-benzoyl-a-L-arabi-
C, 82.29; H, 11.44%]; ½a D
ꢂ
20 21.6 (c 0.2, CHCl3); nmax (film) 2948, 2870,
nopyranoside) (33c). Yield 86%; [found: C, 76.00; H, 8.02.
1733, 1640, 1455, 1375, 1246, 1027, 979, 758 cmꢁ1
;
dH (600 MHz,
C
57H72O8ꢄH2O requires C, 75.80; H, 8.26%]; ½a D20
ꢂ
114.2 (c 0.3,
CDCl3) 4.68 (d, 1H, J 2.2 Hz, H-29), 4.57e4.56 (m, 1H, H-29), 4.47
(dd, 1H, J 5.5, 10.9 Hz), 2.44e2.39 (m, 1H), 2.04 (s, 3H, CH3CO), 1.68
(s, 3H, CH3), 1.03 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.92 (t, 3H, J 7.3 Hz,
CH3), 0.85 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.84 (s, 3H, CH3),
1.90e0.75 (m, 30H, lupane protons); dC (150 MHz, CDCl3) 171.0 (C),
151.1 (C), 109.2 (CH2), 80.9, 55.4, 50.4, 49.8, 47.4, 45.6 (C), 42.5 (C),
40.9 (C), 38.4 (CH2), 37.8 (C), 37.1 (C), 37.0, 35.7 (CH2), 34.1 (CH2),
31.1 (CH2), 30.1 (CH2), 27.9, 27.2 (CH2), 26.9 (CH2), 26.0 (CH2), 25.1
(CH2), 23.8, 23.7 (CH2), 21.3, 21.0 (CH2), 19.3, 18.2 (CH2), 16.5, 16.1,
15.9, 14.8, 14.2.
CHCl3); nmax (film) 2945, 2870, 1729, 1452, 1282, 1263, 1093, 1069,
1028, 758, 710 cmꢁ1
; dH (600 MHz, CDCl3) 8.07e7.26 (m, 15H, Ar),
5.76 (dd, 1H, J 6.4, 8.8 Hz, H-20), 5.69e5.66 (m, 1H, H-40), 5.59 (dd,
1H, J 3.6, 8.8 Hz, H-30), 4.78 (d, 1H, J 6.4 Hz, H-10), 4.67 (br d, 1H, J
2.1 Hz, H-29), 4.57e4.55 (m, 1H, H-29), 4.32 (dd, 1H, J 3.9, 12.9 Hz,
H-50), 3.87 (dd, 1H, J 2.1, 12.9 Hz, H-50), 3.13 (dd, 1H, J 4.7, 11.7 Hz,
H-3), 2.42e2.36 (m, 1H), 1.68 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.94 (s,
3H, CH3), 0.81 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.76 (t, 3H, J 7.5 Hz,
CH3), 0.64 (s, 3H, CH3), 1.90e0.60 (m, 26H, lupane protons); dC
(150 MHz, CDCl3) 165.8 (C), 165.6 (C), 165.2 (C), 151.1 (C), 133.3,
133.2, 133.1, 129.9e128.3 (aromatic carbons), 109.2 (CH2), 103.0 (C-
10), 90.1, 70.7, 70.3, 68.7, 62.5 (CH2), 55.6, 50.4, 49.7, 47.4, 45.7 (C),
42.5 (C), 40.8 (C), 39.0 (C), 38.8 (CH2), 38.7 (C), 36.9, 36.9 (CH2),
35.0 (CH2), 34.2 (CH2), 30.3 (CH2), 30.0, 27.7, 27.1 (CH2), 26.1 (CH2),
25.1 (CH2), 21.0 (CH2), 19.3 (CH2), 18.1 (CH2), 16.1, 16.0, 14.8, 8.1;
HRMS (ESI): MNaþ, found 907.5120. C57H72NaO8 requires
907.5125.
4.2.8. Reduction of acetyl group. General procedure. To a suspension
of LiAlH4 (575 mg, 15.1 mmol) in THF (20 mL) the corresponding
acetate (27, 28, or 29; 3.64 mmol) was slowly added. Stirring was
continued at rt for 30 min, then ethyl acetate (5 mL) was slowly
added and the whole mixture was filtered through a short silica
column (hexane/ethyl acetate, 1:1). Crude product was purified by
column chromatography (hexane/ethyl acetate, 40:1/5:1) to af-
ford product as foam.
4.2.9.3. 28-C-Methyllupeol
3b-O-(2,3,4-tri-O-benzoyl-a-L-rham-
nopyranoside) (33e). Yield 89%; [found: C, 77.61; H, 8.33. C58H74O8
4.2.8.1. 28-C-Methyl-lup-20(29)-en-3-ol (1). Yield 96%; [found:
requires C, 77.47; H, 8.30%]; ½a D20
ꢂ
82.0 (c 0.3, CHCl3); nmax (film)
C, 84.63; H, 12.02. C31H52O requires C, 84.48; H, 11.89%]; ½a D
ꢂ
20 15.3 (c
2944, 2871, 1731, 1451, 1278, 1263, 1108, 1069, 1028, 758, 711 cmꢁ1
;
0.25, CHCl3); nmax (film) 2943, 2871, 1457, 1041, 881, 766 cmꢁ1
;
1H
dH (600 MHz, CDCl3) 8.11e7.26 (m, 15H, Ar), 5.83 (dd, 1H, J 3.3,
10.1 Hz, H-30), 5.67 (t, 1H, J 9.9, 10.1 Hz, H-40), 5.64 (dd, 1H, J 1.8,
3.3 Hz, H-20), 5.08 (br d, 1H, H-10), 4.68 (br d, 1H, J 2.1 Hz, H-29),
4.57e4.56 (m, 1H, H-29), 4.30 (dq, 1H, J 9.9, 6.2 Hz, H-50), 3.21 (dd,
1H, J 7.4, 9.1 Hz, H-3), 2.43e2.38 (m, 1H), 1.68 (s, 3H, CH3), 1.33 (d,
3H, J 6.2 Hz, H-60), 1.05 (s, 3H, CH3), 1.04 (s, 3H, CH3), 0.97 (s, 3H,
CH3), 0.94 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.78 (t, 3H, J 7.4 Hz, CH3),
1.90e0.70 (m, 26H, lupane protons); dC (150 MHz, CDCl3) 165.8 (C),
165.7 (C), 165.6 (C), 151.2 (C), 133.4, 133.2, 133.0, 129.9e128.2 (ar-
omatic carbons), 109.2 (CH2), 99.7 (C-10), 90.1, 72.0, 71.2, 70.2, 66.7,
55.5, 50.4, 49.7, 47.4, 45.8 (C), 42.5 (C), 40.9 (C), 39.1 (C), 38.6 (CH2),
37.0, 36.9 (C), 35.0 (CH2), 34.2 (CH2), 30.3 (CH2), 30.0 (CH2), 28.2,
NMR data, see Table 1; 13C NMR data, see Table 2.
4.2.8.2. 28-C-Ethyl-lup-20(29)-en-3-ol (2). Yield 96%; [found: C,
84.56; H, 12.11. C32H54O requires C, 84.51; H, 11.97%]; ½a D
20 6.1 (c 0.4,
CHCl3); nmax (film) 2951, 2869, 1640, 1455, 1215, 1042, 882,
759 cmꢁ1 1H NMR data, see Table 1; 13C NMR data, see Table 2.
ꢂ
;
4.2.8.3. 28-C-Propyl-lup-20(29)-en-3-ol (3). Yield 96%; [found:
C, 84.50; H, 12.25. C33H56O requires C, 84.55; H, 12.04%]; ½a D20
ꢂ
6.3 (c
0.2, CHCl3); nmax (film) 2942, 2869, 1455, 1376, 1042, 882, 759 cmꢁ1
;
1H NMR data, see Table 1; 13C NMR data, see Table 2.

2728
P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
27.1 (CH2), 25.7 (CH2), 25.1 (CH2), 21.0 (CH2), 19.3 (CH2), 18.3 (CH2),
17.6, 16.4, 16.2, 16.0, 14.8, 8.1.
5.29 (br s, 1H, H-10), 4.71e4.66 (m, 2H, H-29, H-60), 4.58e4.57 (m,
1H, H-29), 4.57e4.53 (m, 1H, H-50), 4.48 (dd, 1H, J 5.1, 11.9 Hz, H-60),
3.35 (dd, 1H, J 4.3, 11.8 Hz, H-3), 2.45e2.40 (m, 1H), 1.70 (s, 3H, CH3),
1.11 (s, 3H, CH3), 1.05 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.94 (s, 3H, CH3),
0.93 (t, 3H, J 7.4 Hz, CH3), 0.88 (s, 3H, CH3), 1.90e0.65 (m, 30H,
lupane protons); dC (150 MHz, CDCl3) 166.1 (C), 165.6 (C), 165.5 (C),
151.1 (C), 133.4, 133.1, 133.0, 129.9e128.3 (aromatic carbons), 109.3
(CH2), 94.3 (C-10), 84.2, 71.6, 70.2, 69.4, 67.0, 63.1 (CH2), 55.7, 50.5,
49.9, 47.4, 45.6 (C), 42.5 (C), 40.9 (C), 38.6 (C), 38.2 (CH2), 37.1 (C),
37.0, 35.8 (CH2), 34.2 (CH2), 31.2 (CH2), 30.1 (CH2), 28.8, 27.3 (CH2),
27.0 (CH2), 26.0 (CH2), 25.1 (CH2), 23.9 (CH2), 22.2 (CH2), 21.0 (CH2),
19.3, 18.3 (CH2), 16.5, 16.1, 16.0, 14.9, 14.2; HRMS (ESI): MNaþ, found
1069.5801. C67H82NaO10 requires 1069.5806.
4.2.9.4. 28-C-Ethyllupeol
3b-O-(2,3,4,6-tetra-O-benzoyl-a-D-
mannopyranoside) (34a). Yield 65%; ½a D20
ꢁ9.2 (c 0.3, CHCl3); nmax
ꢂ
(film) 2952, 2869, 1730, 1451, 1266, 1109, 1068, 1027, 758, 710 cmꢁ1
;
dH (600 MHz, CDCl3) 8.11e7.25 (m, 20H, Ar), 6.10 (t, 1H, J 10.1 Hz, H-
40), 5.91 (dd, 1H, J 3.2, 10.1 Hz, H-30), 5.62 (dd, 1H, J 1.7, 3.2 Hz, H-20),
5.29 (br d, 1H, J 1.7 Hz, H-10), 4.71e4.66 (m, 2H, H-29, H-60),
4.59e4.57 (m, 1H, H-29), 4.57e4.54 (m, 1H, H-50), 4.48 (dd, 1H, J 5.1,
12.0 Hz, H-60), 3.35 (dd, 1H, J 4.3, 11.7 Hz, H-3), 2.46e2.40 (m, 1H),
1.70 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.06 (s, 3H, CH3), 0.97 (s, 3H, CH3),
0.95 (s, 3H, CH3), 0.93 (t, 3H, J 7.3 Hz, CH3), 0.89 (s, 3H, CH3),
1.90e0.65 (m, 28H, lupane protons); dC (150 MHz, CDCl3) 166.1 (C),
165.6 (C), 165.5 (C), 151.1 (C), 133.4, 133.1, 133.0, 129.8e128.3 (aro-
matic carbons), 109.3 (CH2), 94.3 (C-10), 84.2, 71.6, 70.3, 69.4, 67.0,
63.1 (CH2), 55.7, 50.5, 49.9, 47.4, 45.7 (C), 42.5 (C), 40.9 (C), 38.6 (C),
38.2 (CH2), 37.1 (C), 37.0, 35.8 (CH2), 34.2 (CH2), 31.2 (CH2), 30.1
(CH2), 30.0 (CH2), 28.8, 27.3 (CH2), 25.1 (CH2), 22.2 (CH2), 21.0 (CH2),
19.3, 18.3 (CH2), 17.0 (CH2), 16.5, 16.1, 16.0, 15.4, 14.9; HRMS (ESI):
MNaþ, found 1055.5645. C66H80NaO10 requires 1055.5649.
4.2.9.8. 28-C-Propyllupeol
3b-O-(2,3,4-tri-O-benzoyl-a-L-arabi-
nopyranoside) (35c). Yield 79%; [found: C, 77.89; H, 8.09. C59H76O8
requires C, 77.60; H, 8.39%]; ½a D20
ꢂ
68.9 (c 0.3, CHCl3); nmax (film)
dH
2944, 2869, 1730, 1451, 1282, 1262, 1104, 1093, 758, 710 cmꢁ1
;
(600 MHz, CDCl3) 8.07e7.25 (m, 15H, Ar), 5.76 (dd, 1H, J 6.4, 8.8 Hz,
H-20), 5.67 (m, 1H, H-40), 5.59 (dd,1H, J 3.6, 8.8 Hz, H-30), 4.78 (d, 1H,
J 6.4 Hz, H-10), 4.68 (br d, 1H, J 2.2 Hz, H-29), 4.56 (s, 1H, H-29), 4.32
(dd, 1H, J 3.9, 13.0 Hz, H-50), 3.87 (dd, 1H, J 2.0, 13.0 Hz, H-50), 3.13
(dd, 1H, J 4.7, 11.6 Hz, H-3), 2.44e2.38 (m, 1H), 1.68 (s, 3H, CH3), 1.00
(s, 3H, CH3), 0.93 (s, 3H, CH3), 0.91 (t, 3H, J 7.3 Hz, CH3), 0.81 (s, 3H,
CH3), 0.77 (s, 3H, CH3), 0.65 (s, 3H, CH3), 1.90e0.60 (m, 30H, lupane
protons); dC (150 MHz, CDCl3) 165.8 (C), 165.6 (C), 165.2 (C), 151.1
(C), 133.3, 133.2, 133.1, 129.9e128.3 (aromatic carbons), 109.2 (CH2),
103.0 (C-10), 90.1, 70.7, 70.3, 68.7, 62.6 (CH2), 55.6, 50.4, 49.8, 47.4,
45.6 (C), 42.5 (C), 40.9 (C), 39.0 (C), 38.7 (CH2), 37.0, 36.9 (C), 35.7
(CH2), 34.2 (CH2), 31.1 (CH2), 30.1 (CH2), 27.7, 27.2 (CH2), 26.9 (CH2),
26.1 (CH2), 26.0 (CH2), 25.1 (CH2), 23.8 (CH2), 21.0 (CH2), 19.3, 18.1
(CH2), 16.0, 15.9, 14.8, 14.2.
4.2.9.5. 28-C-Ethyllupeol 3b-O-(2,3,4-tri-O-benzoyl-a-L-arabino-
pyranoside) (34c). Yield 87%; [found: C, 76.46; H, 8.05.
C
58H74O8ꢄ½H2O requires C, 76.70; H, 8.32%]; ½a D20
ꢂ
110.3 (c 0.25,
dH
CHCl3); nmax (film) 2950, 2869, 1730, 1281, 1262, 1093, 709 cmꢁ1
;
(600 MHz, CDCl3) 8.07e7.26 (m, 15H, Ar), 5.76 (dd, 1H, J 6.3, 8.8 Hz,
H-20), 5.69e5.66 (m, 1H, H-40), 5.59 (dd, 1H, J 3.6, 8.8 Hz, H-30), 4.78
(d, 1H, J 6.3 Hz, H-10), 4.68 (br d, 1H, J 2.2 Hz, H-29), 4.55e4.57 (m,
1H, H-29), 4.32 (dd, 1H, J 3.9, 12.9 Hz, H-50), 3.88 (dd, 1H, J 2.1,
12.9 Hz, H-50), 3.13 (dd, 1H, J 4.7, 11.6 Hz, H-3), 2.44e2.38 (m, 1H),
1.68 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.91 (t, 3H, J
7.3 Hz, CH3), 0.81 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.65 (s, 3H, CH3),
1.90e0.60 (m, 28H, lupane protons); dC (150 MHz, CDCl3) 165.8 (C),
165.6 (C), 165.2 (C), 151.1 (C), 133.3, 133.2, 133.1, 129.9e128.3 (aro-
matic carbons), 109.3 (CH2), 103.0 (C-10), 90.1, 70.7, 70.3, 68.7, 62.6
(CH2), 55.6, 50.4, 49.8, 47.4, 45.7 (C), 42.5 (C), 40.9 (C), 39.0 (C), 38.7
(CH2), 37.0, 36.9 (C), 35.8 (CH2), 34.2 (CH2), 31.2 (CH2), 30.1 (CH2),
30.0 (CH2), 27.7, 27.2 (CH2), 26.1 (CH2), 25.1 (CH2), 21.0 (CH2), 19.3,
18.1 (CH2), 17.0 (CH2), 16.1, 16.0, 15.4, 14.8; HRMS (ESI): MNaþ, found
921.5277. C58H74NaO8 requires 921.5281.
4.2.9.9. 28-C-Propyllupeol
3b-O-(2,3,4-tri-O-benzoyl-a-L-rham-
nopyranoside) (35e). Yield 78%; [found: C, 77.59; H, 8.55. C60H78O8
requires C, 77.72; H, 8.48%]; ½a D20
ꢂ
75.6 (c 0.3, CHCl3); nmax (film)
dH (600 MHz,
2954, 2865, 1731, 1450, 1265, 1107, 757, 710 cmꢁ1
;
CDCl3) 8.10e7.23 (m, 15H, Ar), 5.82 (dd, 1H, J 3.3, 10.1 Hz, H-30), 5.67
(dd, 1H, J 9.9, 10.1 Hz, H-40), 5.63 (dd, 1H, J 1.8, 3.3 Hz, H-20), 5.07 (br
s, 1H, H-10), 4.67 (br d, 1H, J 2.2 Hz, H-29), 4.55 (s, 1H, H-29), 4.30
(dq, 1H, J 9.9, 6.2 Hz, H-50), 3.19 (dd, 1H, J 7.6, 8.7 Hz, H-3),
2.44e2.38 (m, 1H), 1.67 (s, 3H, CH3), 1.32 (d, 3H, J 6.2 Hz, H-60), 1.04
(s, 6H, 2ꢄ CH3), 0.95 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.92 (t, 3H, J
7.4 Hz), 0.89 (s, 3H, CH3), 1.90e0.70 (m, 30H, lupane protons); dC
(150 MHz, CDCl3) 165.8 (C), 165.7 (C), 165.5 (C), 151.2 (C), 133.4,
133.2, 133.0, 129.9e128.2 (aromatic carbons), 109.2 (CH2), 99.7 (C-
10), 90.1, 72.0, 71.2, 70.2, 66.7, 55.5, 50.4, 49.8, 47.4, 45.6 (C), 42.5
(C), 40.9 (C), 39.1 (C), 38.6 (CH2), 37.0, 36.9 (C), 35.7 (CH2), 34.2
(CH2), 31.1 (CH2), 30.1 (CH2), 28.2, 27.2 (CH2), 27.0 (CH2), 26.0 (CH2),
25.7 (CH2), 25.1 (CH2), 23.9 (CH2), 19.3, 18.3 (CH2), 17.6, 16.4, 16.1,
16.0, 14.9, 14.2.
4.2.9.6. 28-C-Ethyllupeol 3b-O-(2,3,4-tri-O-benzoyl-a-L-rhamno-
pyranoside) (34e). Yield 68%; [found: C, 77.39; H, 8.53. C59H76O8
requires C, 77.60; H, 8.39%]; ½a D20
ꢂ
75.9 (c 0.3, CHCl3); nmax (film)
dH (600 MHz,
2944, 2868, 1730, 1451, 1263, 1110, 758, 712 cmꢁ1
;
CDCl3) 8.10e7.23 (m, 15H, Ar), 5.82 (dd, 1H, J 3.3, 10.1 Hz, H-30), 5.67
(dd, 1H, J 9.9, 10.1 Hz, H-40), 5.64 (dd, 1H, J 1.8, 3.3 Hz, H-20), 5.07 (br
d, 1H, H-10), 4.67 (br d, 1H, J 2.1 Hz, H-29), 4.56e4.55 (m, 1H, H-29),
4.30 (dq, 1H, J 9.9, 6.2 Hz, H-50), 3.20 (dd, 1H, J 7.3, 9.1 Hz, H-3),
2.44e2.38 (m, 1H), 1.67 (s, 3H, CH3), 1.32 (d, 3H, J 6.2 Hz, H-60), 1.04
(s, 6H, 2ꢄ CH3), 0.95 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.91 (t, 3H, J
7.3 Hz), 0.89 (s, 3H, CH3), 1.90e0.70 (m, 28H, lupane protons); dC
(150 MHz, CDCl3) 165.8 (C), 165.7 (C), 165.6 (C), 151.2 (C), 133.4,
133.2, 133 0, 129.9e128.2 (aromatic carbons), 109.2 (CH2), 99.7 (C-
10), 90.1, 72.0, 71.2, 70.2, 66.7, 55.5, 50.4, 49.8, 47.4, 45.7 (C), 42.5 (C),
40.9 (C), 39.2 (C), 38.6 (CH2), 37.0, 36.9 (C), 35.8 (CH2), 34.2 (CH2),
31.2 (CH2), 30.1 (CH2), 30.0 (CH2), 28.2, 27.2 (CH2), 25.7 (CH2), 25.1
(CH2), 21.0 (CH2), 19.3, 18.3 (CH2), 17.6, 17.0 (CH2), 16.4, 16.1, 16.0,
15.4, 14.9.
4.2.10. General procedure for the debenzoylation reaction. A sus-
pension of protected saponin (0.10 mM) and K2CO3 (20 mg) in
MeOH (3 mL) was stirred overnight, then neutralized with
Amberlyst 15 resin (Hþ form) and filtered through a PTFE syringe
filter (MeOH as eluent), and the filtrate was evaporated to dryness.
The residual methyl benzoate was removed by adding water (3 mL)
and freeze-drying to afford the free saponin as a white powder.
4.2.10.1. 28-C-Methyllupeol
3b-O-a-D-mannopyranoside
4.2.9.7. 28-C-Propyllupeol
3
b
-O-(2,3,4,6-tetra-O-benzoyl-
a
-
D
-
(33b). Yield 96%; [found: C, 72.25; H, 10.51. C37H62O6ꢄ½H2O re-
mannopyranoside) (35a). Yield 61%; ½a D20
ꢂ
ꢁ8.8 (c 0.3, CHCl3); nmax
quires C, 72.63; H, 10.38%]; ½a D20
ꢂ
60.0 (c, 0.3, CHCl3); nmax (film)
(film) 2950, 2869, 1731, 1451, 1266, 1109, 1068, 757, 710 cmꢁ1
;
dH
3380 (br), 2942, 2871, 1455, 1376, 1104, 1059, 1027, 758 cmꢁ1 1H
;
(600 MHz, CDCl3) 8.11e7.25 (m, 20H, Ar), 6.09 (t,1H, J 10.1 Hz, H-40),
5.91 (dd, 1H, J 3.2, 10.1 Hz, H-30), 5.62 (dd, 1H, J 1.8, 3.2 Hz, H-20),
NMR data, see Table 1; 13C NMR data, see Table 2; HRMS (ESI):
MNaþ, found 625.4445. C37H62NaO6 requires 625.4444.

P. Cmoch et al. / Tetrahedron 70 (2014) 2717e2730
2729
4.2.10.2. 28-C-Methyllupeol
3
b
-O-
a
-
L
-arabinopyranoside
purchased from Sigma (MO, USA). Calcein AM was obtained from
Molecular Probes (Invitrogen Corporation, CA, USA).
(33d). Yield 60%; [found: C, 74.02; H, 10.85. C36H60O5ꢄ½H2O re-
quires C, 74.31; H, 10.57%]; ½a D20
ꢂ
3.5 (c, 0.25, CHCl3); nmax (film)
The screening cell lines (T-lymphoblastic 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 medium (Sigma, MO, USA), supplemented with 10% heat-
3411 (br), 2944, 2871, 1454, 1376, 1087, 1070, 1000, 759 cmꢁ1 1H
;
NMR data, see Table 1; 13C NMR data, see Table 2; HRMS (ESI):
MNaþ, found 595.4340. C36H60NaO5 requires 595.4338.
4.2.10.3. 28-C-Methyllupeol
3
b
-O-
a
-
L
-rhamnopyranoside
inactivated fetal bovine serum, 2 mmol/L L-glutamine, 10,000 U
(33f). Yield 94%; [found: C, 74.37; H, 10.86. C37H62O5ꢄ½H2O re-
penicillin and 10 mg/mL streptomycin. The cell lines were main-
tained under standard cell culture conditions at 37 ꢀC and 5% CO2 in
a humid environment. Cells were subcultured twice (fibroblasts) or
three times a week using the standard trypsinization procedure,
and used from up to thirtieth passage.
quires C, 74.58; H, 10.66%]; ½a D20
ꢁ19.0 (c 0.3, CHCl3þ10% metha-
ꢂ
nol); nmax (film) 3398 (br), 2937, 2867, 1452, 1131, 1105, 1069, 1052,
982, 757 cmꢁ1; 1H NMR data, see Table 1; 13C NMR data, see Table
2; HRMS (ESI): MNaþ, found 609.4493. C37H62NaO5 requires
609.4495.
4.3.2. Calcein AM assay. Suspensions of tested cell lines (ca.
1.0ꢄ105 cells/mL) were placed in 96-well microtiter plates and after
24 h of stabilization (time zero) the tested compounds were added
4.2.10.4. 28-C-Ethyllupeol 3b-O-a-D-mannopyranoside (34b). Yield
97%; [found: C, 73.14; H, 10.87. C38H64O6ꢄ½H2O requires C, 72.92; H,
10.47%]; ½a 2D0
ꢂ
52.5 (c, 0.3, CHCl3); nmax (film) 3386 (br), 2943, 2870,
(in four 20 mL aliquots) in serially diluted concentrations in dime-
1454, 1102, 1060, 1026, 977, 882, 759 cmꢁ1; 1H NMR data, see Table 1;
thylsulfoxide (DMSO). Control cultures were treated with DMSO
alone, and the final concentration of DMSO in the incubation
mixtures never exceeded 0.6%. The tested compounds were typi-
cally evaluated at six three-fold dilutions and the highest final
13C NMR data, see Table 2; HRMS (ESI): MNaþ, found 639.4598.
C
38H64NaO6 requires 639.4601.
4.2.10.5. 28-C-Ethyllupeol3b-O-a-L
-arabinopyranoside (34d). Yield
concentration was generally 50 mM. After 72 h incubation, 100 ml
96%; [found: C, 73.81; H, 10.84. C37H62O5ꢄH2O requires C, 73.47; H,
Calcein AM solution (Molecular Probes, Invitrogen, CA, USA) was
added, and incubation was continued for a further hour. The fluo-
rescence of viable cells was then quantified using a Fluoroskan
Ascent instrument (Labsystems, Finland). The percentage of sur-
viving 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 ob-
tained for selected compounds are shown in Table 1.
10.66%]; ½a 2D0
ꢁ3.8 (c 0.3, CHCl3); nmax (film) 3416 (br), 2951, 2870,
ꢂ
1454, 1375, 1141, 1087, 1070, 999, 882, 759 cmꢁ1 1H NMR data, see
;
Table 1; 13C NMR data, see Table 2; HRMS (ESI): MNaþ, found
609.4498. C37H62NaO5 requires 609.4495.
4.2.10.6. 28-C-Ethyllupeol 3b-O-a-L-rhamnopyranoside (34f). Yield
87%; [found: C, 74.86; H, 10.89. C38H64O5ꢄ½H2O requires C, 74.83; H,
10.74%]; ½a 2D0
ꢁ27.3 (c 0.3, CHCl3þ10% methanol); nmax (film) 3401
ꢂ
(br), 2942, 2870, 1453, 1376, 1127, 1053, 982, 881, 758 cmꢁ1; 1H NMR
data, see Table 1; 13C NMR data, see Table 2.
4.2.10.7. 28-C-Propyllupeol
3
b
-O-
a
-
D
-mannopyranoside
4.3.3. Flow cytometric analysis of the cell cycle and apoptosis. HeLa
cervical carcinoma cells were trypsinized, seeded in 100-mm cul-
ture dishes, and immediately incubated with the test compounds.
After 24 h, the cells were again detached with trypsin, washed,
fixed and stained in 0.1% [v/v] Triton X-100, 0.2 mg/mL RNase A and
10 mg/mL propidium iodide in PBS. Their DNA contents were then
assessed with a flow cytometer (Cell Lab Quanta SCdMPL, Beck-
man Coulter, CA USA), and the distribution of cells in subG1 (‘apo-
(35b). Yield 93%; [found: C, 72.61; H, 10.63. C39H66O6ꢄH2O re-
quires C, 72.18; H, 10.56%]; ½a D20
ꢂ
53.8 (c, 0.3, CHCl3); nmax (film)
3387 (br), 2941, 2870, 1455, 1376, 1103, 1060, 1026, 979, 882,
759 cmꢁ1 1H NMR data, see Table 1; 13C NMR data, see Table 2;
;
HRMS (ESI): MNaþ, found 653.4753. C39H66NaO6 requires
653.4757.
4.2.10.8. 28-C-Propyllupeol
3
b
-O-
a
-
L
-arabinopyranoside
ptotic cells’), G0/G1, S and G2/M peaks were quantified, by
(35d). Yield 98%; [found: C, 73.55; H, 10.89. C38H64O5ꢄH2O re-
histogram analysis, using MultiCycle AV software (Phoenix Flow
Systems, CA, USA).
quires C, 73.74; H, 10.75%]; ½a D20
ꢁ1.0 (c 0.25, CHCl3); nmax (film)
ꢂ
3408 (br), 2943, 2870, 1454, 1376, 1141, 1087, 1070, 1000, 882,
759 cmꢁ1 1H NMR data, see Table 1; 13C NMR data, see Table 2;
;
Acknowledgements
HRMS (ESI): MNaþ, found 623.4664. C38H64NaO5 requires
623.4651.
The support from Grant No. 2012/07/B/ST5/00823 from the
National Science Centre (ZP), and from Grant No. POIG.01.01.02-14-
102/09 part-financed by the European Union within the European
Regional Development Fund (RL), Poland, is acknowledged. This
research was also supported (LR, JO, MS) by IGA grant No.
IGA_PrF_2014_024 and by Czech Ministry of Education grant from
the National Program for Sustainability I (LO1204).
4.2.10.9. 28-C-Propyllupeol
3b-O-a-L-rhamnopyranoside
(35f). Yield 92%; [found: C, 74.00; H, 10.98. C39H66O5ꢄH2O re-
quires C, 74.00; H, 10.83%]; ½a D20
ꢁ26.5 (c 0.2, CHCl3þ10% meth-
ꢂ
anol); nmax (film) 3388 (br), 2937, 2868, 1452, 1377, 1277, 1131,
1069, 1051, 984 cmꢁ1 1H NMR data, see Table 1; 13C NMR data,
;
see Table 2; HRMS (ESI): MNaþ, found 637.4813. C39H66NaO5 re-
quires 637.4808.
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