Synthesis and biological activity of new homolupanes and homolupane saponins

Article abstract of DOI:10.1016/j.tet.2015.02.008

A concise synthesis of 28a-homolupane triterpenes and the corresponding saponins containing d-mannose, d-idose, d-arabinose, and l-rhamnose moieties was elaborated. The overall synthesis of the new triterpenes involved three linear steps starting from readily available 3-O-acetyl-betulinal: elongation of the carbon chain by Wittig reaction followed by enol ether hydrolysis and reduction (or oxidation) of the elongated aldehyde. Saponins were obtained by glycosylation of triterpenes with classical Schmidt donors. Cytotoxic activities of new lupane and homolupane compounds were evaluated in vitro. Several triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against human cancer cell lines. Influence of the side-chain structure and substituents on the cytotoxicity of betulin and homobetulin derivatives was investigated. These results open the way to the synthesis of various lupane-type triterpene and saponin derivatives as potential anticancer compounds.

Full text of DOI:10.1016/j.tet.2015.02.008

Accepted Manuscript
Synthesis and biological activity of new homolupanes and homolupane saponins
Katarzyna Sidoryk, Anna Korda, Lucie Rárová, Jana Oklešťková, Miroslav Strnad,
Piotr Cmoch, Zbigniew Pakulski, Katarzyna Gwardiak, Romuald Karczewski, Roman
Luboradzki
PII:
S0040-4020(15)00142-8
10.1016/j.tet.2015.02.008
TET 26396
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 October 2014
Revised Date: 22 January 2015
Accepted Date: 2 February 2015
Please cite this article as: Sidoryk K, Korda A, Rárová L, Oklešťková J, Strnad M, Cmoch P, Pakulski
Z, Gwardiak K, Karczewski R, Luboradzki R, Synthesis and biological activity of new homolupanes and
homolupane saponins, Tetrahedron (2015), doi: 10.1016/j.tet.2015.02.008.
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ACCEPTED MANUSCRIPT
Graphical abstract
1
/ 19

ACCEPTED MANUSCRIPT
Synthesis and biological activity of new homolupanes and homolupane saponins.
a,b
a
c
c
,c
a
Katarzyna Sidoryk, Anna Korda, Lucie Rárová, Jana Oklešťková, Miroslav Strnad,* Piotr Cmoch,
,
a
a
a
d
Zbigniew Pakulski,* Katarzyna Gwardiak, Romuald Karczewski, and Roman Luboradzki,
a
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
b
Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland
c
Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the Region Haná for
Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacký University, Šlechtitelů
1, 783 71 Olomouc, Czech Republic
Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
1
d
*
Authors for correspondence:
zbigniew.pakulski@icho.edu.pl; http://www.icho.edu.pl/eng/
miroslav.strnad@upol.cz; http://www.ueb.cas.cz/en/
Abstract: A concise synthesis of 28a-homolupane triterpenes and the corresponding saponins
containing D-mannose, D-idose, D-arabinose and L-rhamnose moieties was elaborated. The overall synthesis
of the new triterpenes involved three linear steps starting from readily available 3-O-acetyl-betulinal:
elongation of the carbon chain by Wittig reaction followed by enol ether hydrolysis and reduction (or
oxidation) of the elongated aldehyde. Saponins were obtained by glycosylation of triterpenes with classical
Schmidt donors.
Cytotoxic activities of new lupane and homolupane compounds were evaluated in vitro. Several
triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against human
cancer cell lines. Influence of the side-chain structure and substituents on the cytotoxicity of betulin and
homobetulin derivatives was investigated. These results open the way to the synthesis of various lupane-type
triterpene and saponin derivatives as potential anticancer compounds.
Keywords: Homobetulin; Homobetulinic acid; Glycosylation; Lupane saponins; Homolupane saponins;
SAR study.
1
. Introduction
Betulin (1) and betulinic acid (2) are natural compounds with proven anticancer, antibacterial,
1
-6
antimalarial, antiviral and antiinflammatory activities. These triterpene class compounds have attracted
7
-10
high interest due to their strong biological activities and low toxicity.
Recent investigations have
demonstrated that even a simple modification of betulin or betulinic acid can profoundly influence their
1
1-13
anticancer and anti-HIV activities.
The best example is Bevirimat (3), betulinic acid modified at the C-3
2
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ACCEPTED MANUSCRIPT
hydroxyl group, a potent anti-HIV agent that blocks HIV-1 replication, which is currently in phase IIb
14-16
clinical trials (Figure 1).
H
H
R
H
R'O
H
1
2
3
: R = CH OH, R' = H
2
: R = CO H, R' = H
O
2
: R = CO H, R' =
2
O
CO H
2
Figure 1. Structures of betulin (1), betulinic acid (2), and Bevirimat (3).
We are particularly interested in the chemistry and biological roles of lupane saponins. Saponins are
steroid or triterpenoid glycosides, widely distributed in plants and some marine organisms, which have
17,18
interesting biological properties.
those with other triterpene-type aglycones, but interest in their synthesis is growing.
strenuous efforts, no lupane derivatives that could be used as anticancer drugs have been found to date,
Natural saponins based on the betulin scaffold occur less frequently than
1
9-23
However, despite
2
4,25
generally because they have too low cytotoxicity.
Therefore, identifying new betulin derivatives with
high cytotoxic activity and potential as drug candidates is still a major challenge.
Early investigations demonstrated that even simple modifications at C-3 and/or C-28 positions of
2
5-30
betulin and betulinic acid significantly change the cytotoxic activity of the resulting compounds.
However, little information is available on the effects of substitution at the lupane C-28 carbon and
2
-4,11,27
cytotoxicity of the resulting saponins.
In addition, there are gaps in the knowledge of the structure-
activity relationships (SAR) of variations in the branching at the C-28 position.
Herein we report the synthesis of novel homolupane triterpenes via elongation of the side-chain at the
lupeol C-17 position by Wittig-type reaction and transformation of the products into saponins, and tests of
their cytotoxicity against a series of cell lines. We also discuss effects of the substituents and elongation of
the side-chain on cell cytotoxicity.
2
2
. Results and discussion
.1. Synthesis of homolupanes
The synthesis of homobetulin (ichopanol, 8) and homobetulinic acid (ichopanic acid, 10) is presented in
27
Scheme 1. The Wittig reaction of easily available aldehyde 4 with a (methoxymethyl)triphenylphosphorane
obtained from (methoxymethyl)triphenylphosphonium chloride by treatment with n-butyllithium] gave enol
[
ether 5 as a mixture of (E)- and (Z)-isomers in 66% yield. Enol ether 5 was hydrolytically stable and its
transformation into aldehyde 6 by treatment with PPTS in the acetone/water mixture required a long reaction
time (48 h) and elevated temperature (60 °C). The expected elongated aldehyde 6 was obtained in 68% yield.
3
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Further reduction of 6 with NaBH gave homobetulin acetate 7 in 84% yield. Deacetylation by treatment
4
with refluxing ethanolic KOH solution afforded ichopanol (8) quantitatively. Oxidation of aldehyde 6 by
treatment with Jones reagent at 0 °C in acetone provided the corresponding acid 9 in 84% yield.
Deacetylation of 9, as described for alcohol 8, gave the free ichopanic acid (10) in 77% yield.
Scheme 1. Reagents and conditions: (a) (methoxymethyl)triphenylphosphonium chloride (5 equiv.), n-butyllithium (4
equiv.), 0 °C; (b) PPTS, acetone/water, 60 °C; (c) NaBH , CH OH; (d) KOH (2.5 equiv.), ethanol, reflux; (e) Jones
4
3
reagent, acetone.
Scheme 2. Reagents and conditions: (a) PdCl , MeOH, DCM.
2
Isomeric homobetulin 12, in which the hydroxyl group is connected to the C-28 atom was obtained in
2
7
7
1% yield by palladium(II) chloride-induced deallylation of alcohol 11 (Scheme 2). Betulinal (14) was
2
7
prepared in 80% yield by similar deallylation of aldehyde 13 (Scheme 2).
4
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2
.2. Synthesis of saponins
Scheme 3. Reagents and conditions: (a) 15–18, CH Cl , TMSOTf, –40 °C; (b) K CO , methanol; (c) 19, CH Cl ,
2
2
2
3
2
2
TMSOTf, –40 °C.
3
1
28
Ichopanol acetate (7) was glycosylated by treatment with perbenzoylated glycosyl donors 15, 16,
2
8
32
26
1
7, and 18 in the presence of TMSOTf under standard conditions (Scheme 3). The glycosides 20-23
were obtained in excellent yields ranging from 89% to 99%. As expected, the presence of benzoyl protecting
3
3
groups in position 2 of the sugar donors directed anomeric selectivity of the glycosidation reaction. In all
cases 1,2-trans-monodesmosidic saponins (α- -mannopyranosides, α- -arabinopyranosides, α-
rhamnopyranosides, and α- -idopyranosides) were obtained exclusively, as confirmed by the chemical shifts
D
D
L-
D
and vicinal coupling constants of the anomeric protons. Final deprotection of the hydroxyl groups was
performed by treating protected saponins with potassium carbonate in methanol. Under the applied reaction
conditions, deacetylation at the O-3 position of the lupane core was also observed. This contrasts with our
26,34
earlier observations that acetyl groups in lupanes are hydrolytically stable.
obtained in good yields (56-94%).
Free saponins 26-29 were
By comparison, attempt to glycosylation of ichopanol acetate (7) with 2,3,4-tri-O-acetyl-D-
3
5
xylopyranosyl trichloroacetimidate (19) under standard conditions completely failed, and diacetate 24 was
obtained as the only product in high yield (83%).
5
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Scheme 4. Reagents and conditions: (a) 15, TMSOTf, CH Cl , –40 °C; (b) K CO , CH OH.
2
2
2
3
3
Glycosylation of ichopanic acid acetate (9) with 2,3,4,6-tetra-O-benzoyl-α-
D
-mannopyranosyl
trichloroacetimidate (15) gave the expected glycosyl ester 25 in 97% yield. Its treatment with potassium
carbonate in methanol gave methyl ester 30 as the only product in good yield (63%). When sodium
methoxide in methanol was used, decomposition of the starting material occurred (Scheme 4).
The structures of all new compounds were confirmed by extended 1D and 2D NMR experiments
(
(
Tables 1 and 2), as well as elemental analysis and HRMS. In addition, the crystal structure of homobetulin
8) was unequivocally confirmed by X-ray analysis (Figure 2).
Figure 2. X-Ray structure of 8.
2.3. Results of in vitro anticancer activity assays
Anticancer activities of the studied homolupane triterpenes and saponins with a modified side-chain at
the C-17 position were tested in vitro and compared with activities of the structurally similar compounds we
2
6,27
have previously described.
Several normal and cancer cell lines were cultured and used to examine
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
lines. Cells of all of these lines were exposed to six serial 3-fold dilutions 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.
6
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1
Table 1. H NMR spectroscopic data for compounds 7-10, 20-23, and 25-29.
Atom
No.
a,b
a
a,b
a
a,b
a,b
a,b
7
8
9
10
20
21
22
triterpene unit:
1.00, 1.67 0.91, 1.67 0.98, 1.66 0.90, 1.66 1.00, 1.68 0.96, 1.64 1.0, 1.68
1
2
3
5
6
7
9
1.62
4.48
0.79
1.38, 1.61
3.19
1.61
4.47
0.79
1.58
3.19
0.69
1.63
4.48
0.80
1.59
4.46
0.75
1.59, 1.62
4.48
0.68
0.85
1.40, 1.57 1.40, 1.52 1.40, 1.51 1.40, 1.53 1.40, 1.51 1.32, 1.46 1.39, 1.51
1.40
1.31
1.38
1.28
1.40
1.30
1.40
1.28
1.41
1.32
1.29
1.23, 1.24
1.42
1.33
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
5
6
8
9
1
2
3
4
5
6
7
8
1.23, 1.43 1.23, 1.43 1.22, 1.42 1.23, 1.43 1.27, 1.44 1.15, 1.38 1.28, 1.45
1.07, 1.66 1.06, 1.65 1.08, 1.64 1.07, 1.64 1.06, 1.69 1.01, 1.58 1.10, 1.70
1.78
1.78
1.79
1.61
1.83
1.65
1.84
1.01, 1.65 1.01, 1.66 1.06, 1.75 1.06, 1.76 1.04, 1.69 0.82, 1.50 1.08, 1.69
1.22, 1.64 1.22, 1.64 1.32, 1.64 1.32, 2.00 1.25, 1.66 1.11, 1.57 1.44, 1.92
1.42
2.42
1.42
2.42
1.54
2.35
1.54
2.35
1.50
2.48
1.36
2.28
1.48
2.48
1.36, 1.93 1.36, 1.93 1.41, 2.00 1.40, 2.00 1.39, 1.96 1.30, 1.85 1.41, 1.99
1.05, 1.65 1.04, 1.64 1.16, 1.95 1.16, 1.94 1.25, 1.64 0.97, 1.59 1.28, 1.70
0.85
0.83
0.86
1.04
0.96
0.76
0.97
0.83
1.05
0.96
0.84
0.85
0.86
1.04
0.97
0.76
0.96
0.83
1.04
0.97
0.84
0.85
0.87
1.10
0.97
0.84
0.85
0.81
0.83
0.87
0.83
0.84
0.87
1.11
0.99
1.28, 1.81 1.28, 1.82 0.90, 2.04 2.04, 2.53 1.41, 1.97 1.28, 1.80 1.44, 1.92
3.70-3.63 3.70, 3.64 3.58, 3.85 3.54, 3.94 3.57, 3.84
4.58, 4.69 4.58, 4.69 4.59, 4.69 4.58, 4.68 4.61, 4.74 4.55, 4.63 4.56, 4.68
2
8a
–
–
2
3
9
0
1.68
1.68
1.69
1.69
1.71
1.64
1.71
monosaccharide unit:
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.09
5.69
5.93
6.07
4.42
4.74
5.73
5.59
5.01
5.64
5.84
5.67
4.19
1.37
2
3
4
5
6
–
–
–
–
–
5.69
3.91, 4.34
–
4.51, 4.69
a
b
c
Recorded in CDCl at 600 MHz, δ in ppm. 3-O-Acetyl group: OCH 2.04 (s) ppm. Recorded in CDCl / CD OD
3
3
3
3
mixture (1:1) at 600 MHz, δ in ppm.
7
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Table 1 contd. H NMR spectroscopic data for compounds 7-10, 20-23, and 25-29.
1
Atom
No.
a,b
a,b
c
c
c
c
2
3
25
26
27
28
29
triterpene unit:
0.97, 1.66 1.01, 1.69 0.92, 1.68 0.90, 1.67 0.91, 1.67 0.92, 1.68
1
2
3
5
6
7
9
1.59, 1.62
4.46
1.62
4.53-4.43
0.82
1.56, 1.61
3.12
1.57
3.17
0.69
1.58
3.17
0.68
1.59
3.16
0.69
0.77
0.70
1.34, 1.48 1.43, 1.51 1.43, 1.54 1.39, 1.52 1.40, 1.52 1.40, 1.53
1.32
1.27
1.46
1.34
1.42
1.34
1.38
1.28
1.39, 1.66
1.28
1.40
1.30
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
5
6
8
9
1
2
3
4
5
6
7
8
1.18, 1.41 1.28, 1.45 1.26, 1.44 1.22, 1.43 1.23, 1.43 1.24, 1.44
1.05, 1.64 1.12, 1.70 1.10, 1.69 1.06, 1.65 1.06, 1.66 1.08, 1.67
1.70
1.69
1.82
1.77
1.76
1.77
0.91, 1.53 1.17, 1.85 1.07, 1.72 1.01, 1.62 0.96, 1.62 0.97, 1.63
1.16, 1.54 1.38, 2.03 1.25, 1.73 1.22, 1.62 1.14, 1.72 1.16, 1.54
1.40
2.38
1.58
2.44
1.48
2.42
1.44
2.40
1.44
2.40
1.46
2.40
1.28, 1.89 1.47, 2.06 1.35, 1.94 1.35, 1.91 1.35, 1.91 1.37, 1.91
0.94, 1.58 1.22, 1.94 1.05, 1.73 1.04, 1.64 1.03, 1.64 1.03, 1.64
0.83
0.84
0.82
0.88
0.92
0.85
0.86
0.89
1.13
0.99
0.75
0.94
0.86
1.08
0.99
0.76
0.96
0.83
1.03
0.95
0.76
0.96
0.83
1.04
0.97
0.76
0.96
0.85
1.05
0.97
1.33, 1.88 2.23, 2.71 1.32, 1.86 1.32, 1.88 1.29, 1.79 1.30, 1.88
3.50, 3.86 1.16, 1.22 3.46, 3.78 3.55, 3.85 3.40, 3.74 3.81
4.60, 4.70 4.64, 4.76 4.57, 4.69 4.57, 4.68 4.58, 4.69 4.58, 4.69
2
8a
2
3
9
0
1.68
1.72
1.68
1.68
1.68
1.69
monosaccharide unit:
1
5.13
5.23
5.62
5.38
4.88
6.40
5.72
5.86
4.73
3.77
3.68
3.60
3.52
4.25
3.62
3.60
4.72
3.86
3.70
3.39
3.63
4.89
3.66
3.86
3.80
4.06
2
3
4
5
6.17-6.08
4.53-4.43
3.89
3.54, 3.95
4
.70,
6
4.57, 4.70
3.70, 3.82
–
1.32
3.89
4
.53-4.43
a
b
c
Recorded in CDCl at 600 MHz, δ in ppm. 3-O-Acetyl group: OCH 2.04 (s) ppm. Recorded in CDCl / CD OD
3
3
3
3
mixture (1:1) at 600 MHz, δ in ppm.
8
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Table 2. C NMR spectroscopic data for compounds 7-10, 20-23, and 25-29.
1
3
Atom
No.
a,b
a
a,b
a
a,b,c
a,b,c
a,b,c
7
8
9
10
20
21
22
triterpene unit:
38.3
23.6
80.9
37.7
55.3
18.1
34.1
40.8
50.3
37.0
20.9
25.0
37.1
42.5
27.3
31.5
44.7
50.1
47.3
150.6
29.9
36.1
27.9
16.4
16.1
16.0
14.8
30.5
60.0
109.5
19.2
1
2
3
4
5
6
7
8
9
38.7
27.3
78.98
38.8
55.3
18.3
34.2
40.8
50.4
37.1
20.9
25.1
37.12
42.5
27.3
31.5
44.7
50.1
47.3
150.6
29.9
36.1
27.9
15.3
16.1
16.1
14.8
30.5
60.1
109.5
19.3
38.3
23.6
80.9
37.7
55.3
18.1
34.0
40.7
50.2
37.0
20.8
24.9
37.4
42.5
27.2
31.5
46.1
49.8
47.3
150.1
29.6
36.2
27.9
16.4
16.1
15.9
14.8
33.4
178.9
109.8
19.3
38.7
27.3
79.0
38.8
55.3
18.2
34.1
40.8
50.3
37.1
20.8
25.1
37.5
42.6
27.2
31.5
46.1
49.9
47.3
150.5
29.7
36.2
27.9
15.3
16.1
16.0
14.9
33.4
178.7
109.7
19.3
38.4
38.3
38.4
23.7
80.9
37.7
55.4
18.2
34.1
40.9
50.4
37.0
20.9
25.1
37.2
42.5
27.3
31.4
44.8
50.1
47.3
150.5
29.9
36.1
27.9
16.4
16.1
16.2
14.8
26.9
65.9
109.7
19.3
23.6
80.9
37.7
55.3
18.1
33.9
40.7
50.2
37.0
20.8
24.9
37.0
42.3
27.1
31.3
44.6
49.9
47.2
150.5
29.8
35.9
27.9
16.4
16.1
16.0
14.7
27.0
67.4
109.5
19.4
23.7
80.9
37.8
55.4
18.2
34.1
40.9
50.4
37.1
21.0
25.0
37.2
42.5
27.3
27.0
44.7
50.1
47.3
150.5
29.9
35.8
27.9
16.4
16.2
16.2
14.9
27.1
65.5
109.6
19.3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
8a
2
3
9
0
monosaccharide unit:
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
97.6
70.6
70.0
67.1
68.9
63.1
101.1
69.9
70.7
68.5
62.7
–
97.5
71.0
70.0
71.9
66.8
17.7
2
3
4
5
6
–
–
–
–
–
a
b
c
Recorded in CDCl at 150 MHz, δ in ppm. 3-O-Acetyl group: COCH = 171.0 ppm, COCH = 21.3 ppm. C=O
groups: 165-166 ppm, aromatic carbons: 128-133 ppm. Recorded in CDCl / CD OD mixture (1:1) at 150 MHz, δ in
3
3
3
d
3
3
ppm.
9
/ 19

ACCEPTED MANUSCRIPT
Table 2 contd. C NMR spectroscopic data for compounds 7-10, 20-23, and 25-29.
1
3
Atom
No.
a,b,c
a,b,c
d
d
d
d
2
3
25
26
27
28
29
triterpene unit:
38.3
23.7
80.9
37.8
55.3
18.1
34.1
40.8
50.3
37.0
20.9
25.0
37.1
42.4
27.2
31.3
44.6
49.9
47.4
150.1
30.0
36.1
27.9
16.4
16.1
16.0
14.8
27.1
65.5
109.2
19.2
1
2
3
4
5
6
7
8
9
38.3
40.0
28.0
79.6
39.4
56.8
19.4
35.4
42.1
51.8
38.3
22.1
26.4
38.6
43.6
28.5
32.6
46.1
51.3
48.8
151.8
31.1
37.2
28.6
16.1
16.7
16.6
15.4
28.04
65.8
110.2
19.6
38.6
26.9
78.7
38.7
55.6
18.1
34.1
40.7
50.3
37.0
20.8
24.9
37.1
42.4
27.2
31.3
44.5
49.9
47.2
150.5
29.8
35.9
27.8
15.2
15.97
16.0
14.7
26.7
66.7
109.4
19.1
38.5
26.8
78.6
38.6
55.1
18.1
34.0
40.6
50.2
36.9
20.7
24.9
37.0
42.3
27.1
31.3
44.5
49.8
47.2
150.4
29.7
35.8
27.6
15.1
15.85
15.81
14.6
26.7
64.6
109.3
19.0
38.4
26.5
78.3
38.5
55.0
17.9
33.9
40.5
50.1
36.7
20.5
24.7
36.8
42.1
26.9
31.1
44.4
49.7
47.0
151.1
29.5
35.6
27.4
14.9
15.66
15.64
14.4
26.5
65.3
109.2
18.8
23.7
80.9
37.8
55.4
18.2
34.1
40.8
50.2
37.0
20.9
25.0
37.6
42.6
27.2
31.6
46.6
49.8
47.3
149.8
29.7
36.3
27.9
16.4
16.2
16.1
14.9
33.7
170.2
110.1
19.2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
8a
2
3
9
0
monosaccharide unit:
1
100.7
67.2
66.94
66.91
64.5
90.5
101.6
72.3
72.7
68.6
74.7
62.9
102.6
71.1
72.6
67.5
65.0
–
99.7
70.6
71.3
72.7
67.9
17.2
102.2
67.9
68.8
69.6
66.5
62.1
2
3
4
5
6
69.2
69.8
66.3
71.3
62.7
63.7
a
b
c
Recorded in CDCl at 150 MHz, δ in ppm. 3-O-Acetyl group: COCH = 171.0 ppm, COCH = 21.3 ppm. C=O
groups: 165-166 ppm, aromatic carbons: 128-133 ppm. Recorded in CDCl / CD OD mixture (1:1) at 150 MHz, δ in
3
3
3
d
3
3
ppm.
Ichopanol (8, entry 1) was highly selective against CEM cell lines (IC 15.3 µM), whereas the parent
5
0
betulin (1, entry 2) was less active. 28a-Homolupeol (31, entry 3) was less active than the above compounds,
less selective and slightly toxic against normal cells. Such data may suggest that both hydroxyl groups (at C-
3
and C-28 / C-28a) are involved in the interactions between triterpene and cell receptors. This speculation
may be confirmed by testing of the acetylated derivatives (entries 4-6). 3-O-Acetyl-ichopanol (7) was less
active and less selective than the parent diol 8, as well as 3-O-acetyl-betulin (32). Acetylation of both
hydroxyls (to afford diacetate 24) cancelled the activity. All acetates were nontoxic against normal cells.
1
0 / 19

ACCEPTED MANUSCRIPT
Table 3. IC (ꢀM) values obtained from Calcein AM assays with the tested cancer and normal cell lines; means
50
±
SD obtained from three independent experiments performed in triplicate. Betulinic acid (2) was used as a positive
control.
IC [µM]
50
Entry
R
H
H
R'
No
8
CEM
15.3±1.4
21.2±3.4
30.8±0.6
19.8±1.9
30.4±2.2
>50
33.7±5.4
>50
>50
38.3±15.6
5.4±1.1
19.4±0.7
40.0±2.8
9.6±0.6
10.0±0.2
>50
MCF7
>50
>50
46.0±5.7
38.4±0.7
>50
>50
44.9±6.4
>50
>50
>50
37.4±7.2
>50
>50
31.0±1.2
21.8±5.5
>50
>50
>50
>50
>50
HeLa
>50
>50
28.5±5.0
27.9±5.6
>50
>50
37.5±0.6
>50
BJ
>50
48.6±0.1
45.0±1.3
>50
>50
>50
42.1±0.3
>50
>50
>50
12.6±3.0
>50
>50
24.7±0.1
>50
>50
>50
>50
43.9±2.4
>50
1
2
3
4
5
6
7
8
9
–CH CH OH
2
2
26
–CH OH
1
31
2
27
H
–C H
2
5
Ac
Ac
Ac
H
Ac
Ac
Ac
H
H
H
Ac
Ac
Ac
H
–CH CH OH
7
2
2
2
6
–CH OH
32
24
12
2
–CH CH OAc
–CH(OH)CH3
–CH(OH)CH3
2
2
27
33
–CH CHO
6
4
>50
2
2
7
1
0
1
2
3
4
5
6
7
8
9
0
–CHO
–CHO
12.3±0.8
7.1±0.6
42.9±5.5
47.6±1.9
13.5±0.5
14.5±4.3
>50
>50
>50
23.6±0.1
>50
1
1
1
1
1
1
1
1
1
2
14
10
–CH CO H
2
2
26
–CO H
2
2
–CH CO H
9
34
2
2
26
–CO H
2
–CH CO Me
30
2
2
–CH CH O-α-
–CH CH O-α-
–CH CH O-α-
D
-
-
-
-
Manp
Arap
Rhap
Idop
26
27
28
29
>50
>50
34.0±3.6
>50
2
2
H
H
H
D
2
2
L
2
2
–CH CH O-α-
D
2
2
Homobetulin with a secondary hydroxyl at the C-28 position (12, entry 7) was less active and less
selective than isomeric ichopanol (8). Acetylation of the O-3 hydroxyl (33, entry 8) removed the activity. It
is likely that sterically hindered hydroxyl group at the C-28 position is unable to bind to receptors, and the
only possible interactions are between the C-3 hydroxyl group and receptors.
Homologated aldehyde 6 (entry 9) was inactive, whereas betulinal (14) was the most active against both
CEM and HeLa cell lines (IC50 5.4 and 7.1 µM, respectively), although it was also most toxic to normal
cells. Ichopanic acid (10) was more active than the parent betulinic acid (2, entries 12 and 13, respectively).
In these cases, acetylation at the O-3 position increased activity (9 and 34, respectively; entries 14 and 15) in
comparison to the parent acids. Esterification of the carboxyl group in 9 leading to ester 30 (entry 16)
completely suppressed the activity. Cytotoxicity to normal human BJ fibroblasts was usually very low in this
group.
Surprisingly, saponins based on the ichopanol core (26-29) were inactive except
L-rhamnoside 28,
which was moderately cytotoxic (IC 23.6-34.0 µM) and slightly toxic to normal cells (IC 43.9 µM). Thus,
5
0
50
adding a sugar moiety to the O-28a position of homobetulin decreased cytotoxic activity. No such effect has
2
6
been observed in previously studied betulin derivatives.
1
1 / 19

ACCEPTED MANUSCRIPT
These results clearly demonstrate that the C-28 or C-28a primary hydroxyl group is important for the
cytotoxicity of (homo)lupane triterpenes. The free hydroxyl at the C-3 position significantly increases
cytotoxicity. Unexpectedly, coupling of homolupanes with a sugar moiety at these positions can cause a loss
of activity. 28a-Homo-28a-hydroxylupane triterpenes may be considered as interesting starting materials for
the preparation of highly cytotoxic derivatives. In comparison to the parent triterpenes they are more
cytotoxic and usually nontoxic against normal cells.
3
. Conclusion
In conclusion, a series of 28a-homo-28a-hydroxylupane triterpenes and saponins bearing
idose, -arabinose, and -rhamnose moieties were synthesized and evaluated for their cytotoxic activities
D-mannose, D-
L
L
towards normal and cancer cell lines. Comparison with a series of known lupane triterpenes and saponins
revealed that oxygen substituents at the C-28a (or C-28) position affect the cytotoxic properties of these
lupanes. Several triterpenes, as well as selected saponins, showed interesting cytotoxic activity profiles
against human cancer cell lines.
4
. Experimental section
.1. General
Silica gel HF254 and Silica gel 230–400 mesh (E. Merck) were used for TLC and column
4
1
13
chromatography, respectively. H and C NMR spectra were recorded at 298 K with a Varian NMR-
vnmrs600 or vnmrs500 spectrometer, using standard experimental conditions and Varian software
(
ChemPack 4.1). Configurational assignments were based on the NMR measurements, generated using two-
1
13
1
13
dimensional techniques like COSY and H- C gradient selected HSQC (g-HSQC), as well as H- C
1
13
gradient selected HMBC (g-HMBC) in several cases. Internal TMS was used as the H and C NMR
chemical shift standard. J values are given in Hertz. High-resolution mass spectra (HRMS ESI) were
acquired with MARINER and MaldiSYNAPT G2-S HDMS (Waters) mass spectrometers. Optical rotations
were measured with a JASCO P-2000 automatic polarimeter. IR spectra were recorded on Jasco 6200 FT-IR
spectrophotometer. Vario EL III (Elementar) was used for determination of C and H contents.
Single crystal X-ray diffraction measurements were acquired using an Agilent Supernova
diffractometer, at 100 K with graphite monochromated Cu Ka radiation (1.54184 Å). The data were reduced
3
6
2
using CrysAlisPRO software then structures were solved by direct methods and refined on F by full-matrix
3
7
least-squares using SHELXS97 and SHELXL97. All non-hydrogen atoms were refined as anisotropic
while hydrogen atoms were placed in calculated positions, and refined in riding mode. Structures contained
the solvent (methanol) in 1:1stoichiometry. Both CH (C-30) and CH (C-29) groups connected to the C-20
3
2
carbon atom are disordered, with 0.54 and 0.46 occupancy, respectively. Data for 8: orthorombic, P22 2 ,
1
1
3
-1
-1
a=6.9273(3) b=12.2412(4) c=33.8983(13) Å, V=2874.52(19) Å , Z=4, D =1.129g cm , m=0.532 mm ,
calc
R1=0.04320 for 4260Fo, [ Fo > 4s(Fo)] and 0.0493 for all data, wR2=0.1092, S=1.038. Crystallographic data
(
excluding structure factors) for the structure described in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary material (deposition number: CCDC 996102).
1
2 / 19

ACCEPTED MANUSCRIPT
4
4
.2. Synthesis
.2.1. 3 -O-Acetyl-17-(2-methoxyethenyl)-28-norlup-20(29)-en-3-ol (5). To an ice cooled suspension of
β
(
methoxymethyl)triphenylphosphonium chloride (1.51 g, 4.40 mmol) in anhydrous THF (15 mL), n-
butyllithium (1.6 M in hexane, 1.41 mL, 3.52 mmol) was added dropwise and stirred for 30 min. Then, a
solution of 3-O-acetylbetulinal (4, 429 mg, 0.88 mmol) in anhydrous THF (5 mL) was added, the whole
mixture was stirred at 5 °C for an additional 15 min, and for 1 h at r.t. The reaction was quenched with a
saturated solution of NH Cl (0.5 mL), a small portion of silica gel was added and the solvents were
4
evaporated under diminished pressure. Column chromatography of the residue (hexane – ethyl acetate, 40:1
→
10:1) yielded the title compound as a mixture of (E)- and (Z)-isomers (white solid, 550 mg, 66%). An
2
0
analytical sample of pure (E)-isomer was isolated and had the following physicochemical properties: [α]D
-1 1
1
7.2 (c 0.2, CHCl ); νmax (film): 2952, 2871, 1731, 1715, 1451, 1375, 1247, 1024, 980, 896, 757 cm . H
3
NMR (500 MHz, CDCl ) δ: 5.78 (d, 1 H, J 6.9 Hz, -CH=CHOCH ), 4.68 (br s, 1 H, H-29), 4.56 (br s, 1 H,
3
3
H-29), 4.46 (dd, 1 H, J 10.8, 5.5 Hz, H-3), 4.28 (d, 1 H, J 7.1 Hz, -CH=CHOCH ), 3.55 (s, 3 H, -
3
CH=CHOCH ), 2.37-2.39 (m, 1 H, H-19), 2.20-2.22 (m, 1 H), 2.04 (s, 3 H, CH ), 1.68 (s, 3 H, CH ), 1.61-
3
3
3
1
.64 (m, 5 H), 1.28-1.48 (m, 8 H), 1.07-1.20 (m, 10 H), 0.97 (s, 3 H, CH ), 0.93 (s, 3 H, CH ), 0.85 (s, 3 H,
3 3
1
3
CH ), 0.84 (s, 3 H, CH ), 0.83 (s, 3 H, CH ); C NMR (500 MHz, CDCl ) δ: 170.9 (OCOCH ), 151.0 (C-
3
3
3
3
3
2
0), 149.8, 145.8, 109.6 (C-29), 104.0, 80.8 (C-3), 59.5, 55.4, 50.3, 48.1, 45.6, 44.5, 42.5, 41.7 (CH ), 40.7,
2
3
8.4, 37.9 (CH ), 37.1, 36.5, 35.8 (CH ), 33.6 (CH ), 30.0 (CH ), 29.1, 27.4, 26.4 (CH ), 24.4 (CH ), 23.2
2
2
2
2
2
2
(
CH ), 20.3 (CH ), 17.6 (CH ), 16.0, 15.7, 15.5, 14.4. HR MS-EI calcd. for C H O : 510.4073. Found
2
2
2
34 54
3
5
10.4067.
4.2.2. 3β-O-Acetyl-28a-homobetulinal (6). PPTS (4.0 g) was added to a solution of 5 (3.00 g, 5.87
mmol) in acetone (15 mL) and water (1.5 mL). The solution was heated at 60 °C for 48 h, then the solvents
were evaporated. Column chromatography (hexane – ethyl acetate, 40:1 → 20:1) of the residue gave 6 as a
2
0
white foam (1.99 g, 68%). [α]D 13.7 (c 0.2, CHCl ). ν (film): 2947, 2871, 1723, 1453, 1374, 1247, 1028,
3
max
-1 1
9
80, 886, 757 cm . H NMR (500 MHz, CDCl ) δ: 9.83-9.85 (m, 1 H, CHO), 4.69 (1 H, H-29), 4.60 (br s, 1
3
H, H-29), 4.47 (dd, 1 H, J 10.7, 5.5 Hz, H-3), 2.53-2.56 (m, 1 H, H-28), 2.33-2.36 (m, 1 H, H-19), 2.09-2.10
(
m, 1 H), 2.04 (s, 3 H, CH ), 1.83-1.85 (m, 2 H), 1.69 (s, 3 H, CH ), 1.60-1.66 (m, 6 H), 1.39-1.50 (m, 9 H),
3 3
1
.24-1.29 (m, 3 H), 1.09-1.10 (m, 2 H), 1.04 (s, 3 H, CH ), 0.97-0.98 (m, 2 H), 0.97 (s, 3 H, CH ), 0.85 (s, 3
3 3
1
3
H, CH ), 0.84 (s, 3 H, CH ), 0.83 (s, 3 H, CH ). C NMR (500 MHz, CDCl ) δ: 204.1 (CHO), 171.0 (C=O),
3
3
3
3
1
49.8 (C-20), 110.1 (C-29), 80.8 (C-3), 55.3, 50.2, 50.1, 47.5, 45.6 (C), 42.4 (C), 42.2 (CH ), 40.8 (C), 38.4
2
(
(
CH ), 37.7 (C), 37.5, 37.05 (C), 36.3 (CH ), 34.1 (CH ), 32.1 (CH ), 29.5 (CH ), 27.9, 26.9 (CH ), 24.9
2 2 2 2 2 2
CH ), 23.6 (CH ), 21.2, 20.8 (CH ), 19.3 (C), 18.1 (CH ), 16.4, 16.1, 16.0, 14.8. HR MS-EI calcd. for
2
2
2
2
C H O : 496.3916. Found 496.3912.
33
52
3
1
3 / 19

ACCEPTED MANUSCRIPT
4
.2.3. 3β-O-Acetyl-28a-homobetulin (7). To a solution of 6 (950 mg, 1.91 mmol) in MeOH (10 mL),
NaBH (217 mg, 5.74 mmol) was added. The solution was stirred at r.t. for 30 min. and concentrated to
4
dryness. Column chromatography (hexane – ethyl acetate, 20:1 → 5:1) of the residue gave 7 (803 mg, 84%)
2
0
as a white solid; m.p. 191-192 °C; [α]D 20.4 (c 0.2, CHCl ). ν (film): 3418, 2946, 2871, 1732, 1454,
3
max
-1 1
13
1
375, 1247, 1027, 980, 882, 739 cm . H NMR data, see Table 1; C NMR data, see Table 2. Anal. Calcd.
for C H O (498.78): C, 79.46; H, 10.91. Found: C 79.42; H 10.76.
33
54
3
4.2.4. 28a-Homobetulin (8). To a solution of 7 (80 mg, 0.16 mmol) in EtOH (2 mL), potassium
hydroxide (23 mg, 0.40 mmol) was added, and the mixture was refluxed for 1 h, then the reaction mixture
was cooled to room temperature, the solvent was evaporated to dryness, and the residue was purified by
column chromatography (hexane – ethyl acetate, 20:1 → 1:1) to give 8 as a white solid (73 mg, quant.); m.p.
2
0
-1 1
2
04-206 °C; [α]D 15.9 (c 0.2, CHCl ). ν (film): 3342, 2942, 2869, 1454, 1376, 1045, 883, 758 cm . H
3 max
1
3
NMR data, see Table 1; C NMR data, see Table 2. Anal. Calcd. for C H O × CH OH (488.78): C, 78.63;
3
1
52
2
3
H, 11.55. Found: C, 78.87; H, 11.48; HR MS-EI calcd. for C H O : 456.3980. Found 456.3967.
3
1
52
2
4.2.5. 3β-O-Acetyl-28a-homobetulinic acid (9). To an iced cooled solution of 6 (100 mg, 0.2 mmol) in
acetone (2 mL) and THF (2 mL) Jones reagent (3 mL) was added, the mixture was stirred at r.t. for 5 h. The
reaction was quenched with isopropyl alcohol (1.5 mL) solvents were decanted from the gummy residue and
evaporated under diminished pressure. Column chromatography (hexane – ethyl acetate, 20:1 → 7:3) of the
2
0
residue gave the title compound as a foam (87 mg, 84%). [α]D 22.7 (c 0.3, CHCl ). ν (film): 2946, 2872,
3
max
-
1
1
13
1
732, 1703, 1454, 1376, 1247, 1029, 980, 884, 758 cm . H NMR data, see Table 1; C NMR data, see
Table 2. Anal. Calcd. for C H O × H O (530.76): C, 74.67; H, 10.25 Found: C, 74.69; H, 10.19.
3
3
52
4
2
4.2.6. 28a-Homobetulinic acid (10). To a solution of 9 (56 mg, 0.11 mmol) in EtOH (2.5 mL),
potassium hydroxide (15 mg, 0.27 mmol) was added, and the mixture was refluxed for 3 h, then cooled to r.t.
and the solvents were evaporated to dryness. Column chromatography (hexane – ethyl acetate, 10:1 → 1:1)
2
0
of the residue gave 10 as a white solid (40 mg, 77%); m.p. 199-202 °C; [α]D 12.9 (c 0.2, CHCl ). ν
3
max
-1
1
13
(
film): 2943, 2871, 1704, 1453, 1377, 758 cm . H NMR data, see Table 1; C NMR data, see Table 2. HR
-
MS-ESI calcd. for C H O [M-H] : 469.3687. Found 469.3682.
3
1
49
3
2
7
4
.2.7. 28-C-Methyl-betulin (12). To a solution of 3-O-allyl-28-C-methyl-betulin (11, main isomer from
the Grignard reaction, 550 mg, 1.11 mmol) in methanol (10 mL) and DCM (5 mL), palladium(II) chloride
50 mg) was added and the mixture was stirred at r.t. overnight. The solvents were evaporated to dryness.
Column chromatography of the residue (hexane – ethyl acetate, 10:1 → 1:1) afforded 12 (359 mg, 71%) as
(
2
0
-1 1
white foam. [α]D 11.1 (c 1.0, CHCl ). ν (film): 3409, 2941, 2870, 1452, 1375, 1042, 883, 757 cm . H
3
max
NMR (500 MHz, CDCl ) δ: 4.71 (d, 1 H, J 2.2 Hz, H-29), 4.57 (dd, 1 H, J 2.3, 1.3 Hz, H-29), 4.33 (q, 1 H, J
3
6
.3 Hz, H-28), 3.19 (dd, 1 H, J 4.9, 11.4 Hz, H-3), 2.84-2.90 (m, 1 H), 2.05-2.10 (m, 1 H), 1.90-1.97 (m, 1
1
4 / 19

ACCEPTED MANUSCRIPT
H), 1.82-1.87 (m, 1 H), 1.69 (s, 3 H, CH ), 1.18 (d, 3 H, J 6.3 Hz, H-28a), 1.04 (s, 3 H, CH ), 1.01 (s, 3 H,
3
3
1
3
CH ), 0.97 (s, 3 H, CH ), 0.83 (s, 3 H, CH ), 0.76 (s, 3 H, CH ), 0.68-1.73 (m, 22 H, lupane protons). C
3
3
3
3
NMR (500 MHz, CDCl ) δ: 151.4 (C-20), 109.4 (C-29), 79.0, 68.3, 55.3, 50.4, 50.2, 50.1 (C), 49.0, 42.9 (C),
3
4
0.9 (C), 38.8 (C), 38.7 (CH ), 37.1 (C), 36.9, 34.6 (CH ), 34.2 (CH ), 32.8 (CH ), 32.2 (CH ), 28.0, 27.7
2 2 2 2 2
(
CH ), 27.4 (CH ), 25.2 (CH ), 20.9 (CH ), 19.8, 18.9 (C), 18.3 (CH ), 16.1, 16.1, 15.3, 15.2. HR-MS (ESI)
2 2 2 2 2
+
calc. for C H O (M) : 456.3967. Found: 456.3970. Anal. Calcd for C H O × H O (474.77): C, 78.43; H,
3
1
52
2
31 52
2
2
1
1.46. Found: C, 78.42; H, 11.37.
4.2.8. Betulinal (14). To a solution of 3-O-allyl-betulinal (13, 100 mg, 0.20 mmol) in methanol (8 mL)
and DCM (4 mL), palladium(II) chloride (20 mg) was added and the mixture was stirred at r.t. overnight.
The solvents were evaporated to dryness. Column chromatography of the residue (hexane – ethyl acetate,
2
1
0:1) afforded the title compound (64 mg, 80%) as white foam. νmax (film): 3409, 2941, 2869, 1691, 1452,
-1
38
388, 1370, 1041, 1030, 756 cm . NMR spectroscopic data of 14 matches the literature.
4.3. General method for glycosylation
A solution of glycosyl donor (0.24 mmol) and the corresponding triterpene (0.20 mmol) in CH Cl (10
2
2
mL) was stirred for 20 min. at r.t. over molecular sieves (4 Å, 200 mg, finely ground), then cooled to –40 °C
and TMSOTf (20 µL, 0.1 mmol) was added. After 25 min the reaction was quenched with Et N (1 mL), and
3
the solvents were evaporated under diminished pressure. Column chromatography (hexane – ethyl acetate,
4
0:1 → 5:1) of the residue gave the protected saponins as white foams.
4.3.1.
3β-O-Acetyl-28a-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-28a-homolup-20(29)-ene
(20). Yield 89% (191 mg). νmax (film): 2947, 2871, 1730, 1451, 1265, 1108, 1096, 1069, 1028, 978, 757, 711
-
1
20
1
13
cm . [α] –28.2 (c 0.3, CHCl ). H NMR data, see Table 1; C NMR data, see Table 2. Anal. Calcd. for
D
3
C H O (1077.34): C, 74.69; H, 7.53 Found: C, 74.55; H, 7.53.
67
80 12
4.3.2. 3β-O-Acetyl-28a-O-(2,3,4-tri-O-benzoyl-α-D-arabinopyranosyl)-28a-homolup-20(29)-ene (21).
2
0
Yield 92% (173 mg). [α]D –62.1 (c 0.3, CHCl ). ν (film): 2946, 2871, 1730, 1452, 1282, 1262, 1251,
3
max
-1
1
13
1
093, 1063, 1028, 757, 711 cm . H NMR data, see Table 1; C NMR data, see Table 2. Anal. Calcd. for
C H O (943.21): C, 75.13; H, 7.91 Found: C, 75.21; H, 8.08.
59
74 10
4.3.3. 3β-O-Acetyl-28a-O-(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-28a-homolup-20(29)-ene (22).
2
0
Yield 99% (190 mg). [α]D 71.4 (c 0.3, CHCl ). ν (film): 2946, 2872, 1731, 1451, 1278, 1263, 1248,
3
max
-1
1
13
1
108, 1069, 1028, 979, 757, 711 cm . H NMR data, see Table 1; C NMR data, see Table 2. HR MS-ESI
+
calcd. for C H NO [M+NH ] : 974.5782. Found 974.5782.
6
0
80
10
4
1
5 / 19

ACCEPTED MANUSCRIPT
4
.3.4. 3
β-O-Acetyl-28a-O-(2,3,4,6-tetra-O-benzoyl-α-D-idopyranosyl)-28a-homolup-20(29)-ene (23).
2
0
Yield 97% (209 mg). [α]D 40.9 (c 0.2, CHCl ). ν (film): 2947, 2871, 1727, 1263, 1248, 1108, 1069,
3
max
13
-1 1
1
027, 756, 710 cm . H NMR data, see Table 1; C NMR data, see Table 2. Anal. Calcd. for C H O
6
7
80 12
(
1077.34): C, 74.69; H, 7.48 Found: C, 74.70; H, 7.68.
4.3.5. 3β,28a-Di-O-acetyl-28a-homobetulin (24). Yield 83% (90 mg), white solid; m.p. 224-226 °C.
20
-1 1
[
α]D 21.0 (c 0.2, CHCl ). ν (film): 2946, 2871, 1737, 1455, 1366, 1246, 1029, 980, 883, 757 cm . H
3
max
NMR (500 MHz, CDCl ) δ: 4.67-4.68 (m, 1 H, H-29), 4.57-4.58 (m, 1 H, H-29), 4.46 (dd, 1 H, J 10.8, J 5.5
3
Hz, H-3), 4.12 (ddd, 1 H, J 6.1, J 10.2, J 10.2 Hz, H-28a), 4.02 (ddd, 1 H, J 5.5, J 10.2, J 10.2 Hz, H-28a),
2
.37-2.41 (m, 1 H, H-19), 2.05 (s, 3 H, CH ), 2.04 (s, 3 H, CH ), 1.85-1.95 (m, 3 H), 1.78-1.73 (m, 2 H), 1.68
3 3
(
s, 3 H, CH ), 1.58-1.66 (m, 6 H), 1.35-1.51 (m, 5 H), 1.21-1.31 (m, 5 H), 1.08-1.10 (m, 2 H), 1.04 (s, 3 H,
3
CH ), 1.01 (m, 2 H), 0.95 (s, 3 H, CH ), 0.85 (s, 3 H, CH ), 0.84 (s, 3 H, CH ), 0.83 (s, 3 H, CH ), 0.79 (m, 1
3
3
3
3
3
1
3
H). C NMR (500 MHz, CDCl ) δ: 171.2 (C=O), 170.9 (C=O), 150.4 (C-20), 109.6 (C-29), 80.9 (C-3), 62.2,
3
5
5.3 (CH ), 50.3 (CH ), 50.0 (CH ), 47.3 (CH ), 44.7 (C), 42.5 (C), 40.8 (C), 38.3, 37.7 (C), 37.1 (CH ), 37.0
2 2 2 2 2
(
C), 35.9, 34.1, 31.3, 29.8, 27.9 (CH ), 27.2, 26.0, 25.0, 23.6, 21.3 (CH ), 21.1 (CH ), 20.9, 19.2, 18.1, 16.4
2 2 2
(
CH ), 16.1 (CH ), 16.0 (CH ), 14.8. Anal. Calcd. for C H O (540.82): C, 77.73; H, 10.44. Found: C,
2
2
2
35 56
4
7
7.69; H, 10.30.
4.3.6.
1-O-[3β-O-Acetyl-28a-homolup-20(29)-ene-28a-oyl)-2,3,4,6-tetra-O-benzoyl-α-D-
2
0
mannopyranosyl (25). Yield 97% (212 mg). [α]D –20.9 (c 0.3, CHCl ). ν (film): 2947, 2871, 1731, 1452,
3
max
-1 1
13
1
263, 1107, 1095, 1027, 978, 757, 710 cm . H NMR data, see Table 1; C NMR data, see Table 2. Anal.
Calcd. for C H O (1091.33): C, 73.74; H, 7.20. Found: C, 73.61; H, 7.39.
67
78 13
4.5. General procedure for the debenzoylation reaction
A suspension of protected saponin (0.10 mmol) and K CO (20 mg) in MeOH (3 mL) was stirred for 48
2
3
+
h, then neutralized with Amberlyst 15 resin (H form) and filtered through a PTFE syringe filter (MeOH as
eluent), and the filtrate was evaporated to dryness. The residue was purified by column chromatography to
afford unprotected saponin as white foam.
4.5.1. 28a-O-(α-D-Mannopyranosyl)-28a-homobetulin (26). Column chromatography (hexane – ethyl
acetate, 10:1 → 1:1, and hexane – ethyl acetate – methanol, 5:3:1). νmax (film): 3348, 2941, 2870, 1563,
-
1
20
1
454, 1380, 1132, 1082, 1046, 1032, 981, 880, 681 cm . Yield 94% (78 mg). [α] 29.8 (c 0.2, CHCl3-
D
1
13
CH OH). H NMR data, see Table 1; C NMR data, see Table 2. HR MS-ESI calcd. for C H NaO
7
3
37 62
+
[
M+Na] : 641.4392. Found 641.4393.
4.5.2. 28a-O-(α-D-Arabinopyranosyl)-28a-homobetulin (27). Column chromatography (hexane – ethyl
2
0
acetate, 7:3 → 1:1, and hexane – ethyl acetate – methanol, 5:3:1). Yield 78% (66 mg). [α]D 6.2 (c 0.3,
1
6 / 19

ACCEPTED MANUSCRIPT
-
1 1
CHCl -CH OH). ν (film): 3387, 2942, 2869, 1451, 1375, 1070, 1040, 996, 878, 755 cm . H NMR data,
3
3
max
13
+
see Table 1; C NMR data, see Table 2. HR MS-ESI calcd. for C H NaO [M+Na] : 611.4287. Found
3
6
60
6
6
11.4288.
4.5.3. 28a-O-(α-L-Rhamnopyranosyl)-28a-homobetulin (28). Column chromatography (hexane – ethyl
2
0
acetate, 40:1 → 5:1, and hexane – ethyl acetate – methanol, 5:3:1). Yield 56% (55 mg). [α]D –20.5 (c 0.3,
-1 1
CHCl -CH OH). ν (film): 3374, 2943, 2870, 1466, 1454, 1389, 1376, 1134, 1062, 1044, 983, 880 cm . H
3
3
max
1
3
+
NMR data, see Table 1; C NMR data, see Table 2. HR MS-ESI calcd. for C H NaO [M+Na] : 625.4442.
37
62
6
Found 625.4444.
4.5.4. 28a-O-(α-D-Idopyranosyl)-28a-homobetulin (29). Column chromatography (hexane – ethyl
2
0
acetate, 10:1 → 1:1, and hexane – ethyl acetate – methanol, 5:3:1). Yield 86% (84 mg). [α]D 32.2 (c 0.3,
-1 1
CHCl -CH OH). ν (film): 3388, 2941, 2869, 1453, 1389, 1377, 1093, 1026, 879, 718 cm . H NMR data,
3
3
max
13
+
see Table 1; C NMR data, see Table 2. HR MS-ESI calcd. for C H NaO [M+Na] : 641.4393. Found
3
7
62
7
6
41.4411.
4.5.5. 3β-O-Acetyl-28a-homobetulinic acid methyl ester (30). A suspension of 25 (80 mg, 0.07 mmol)
and K CO (40 mg) in MeOH (5 mL) was stirred at r.t. for 1 h, then the solvents were evaporated under
2
3
diminished pressure. Column chromatography (hexane – ethyl acetate, 40:1 → 10:1) of the residue gave 30
2
0
as a white foam (24 mg, 63%). [α]D 25.5 (c 0.2, CHCl ). ν (film): 2947, 2871, 1734, 1640, 1454, 1377,
3
max
-
1 1
1
2
245, 1019, 980, 884, 758 cm . H NMR (500 MHz, CDCl ) δ: 4.68 (br s, 1 H, H-29), 4.58-4.61 (m, 1 H, H-
3
9), 4.47 (dd, 1 H, J 10.8, J 5.5 Hz, H-3), 3.65 (s, 3 H, CH ), 2.49-2.52 (m, 1 H, H-28), 2.32-2.36 (m, 1 H,
3
H-19), 2.04 (s, 3 H, CH ), 1.80-1.95 (3 H), 1.75-1.78 (m, 1 H), 1.69 (s, 3 H, CH ), 1.50-1.65 (7 H), 1.40 (5
3
3
H), 1.20-1.33 (4 H), 1.10-1.16 (3 H), 1.04 (s, 3 H, CH ), 1.02 (1 H), 0.96 (s, 3 H, CH ), 0.85 (s, 3 H, CH ),
3
3
3
1
3
0
.84 (s, 3 H, CH ), 0.83 (s, 3 H, CH ), 0.79-0.80 (m, 1 H). C NMR (500 MHz, CDCl ) δ: 173.6 (C=O),
3 3 3
1
70.9 (C=O), 150.2 (C-20), 109.7 (C-29), 80.9 (C-3), 55.3 (CH ), 51.2 (CH ), 50.2 (CH ), 49.8 (CH ), 47.3
2
2
2
2
(
(
CH ), 46.1 (C), 42.5 (C), 40.8 (C), 38.3, 37.7 (C), 37.4 (CH ), 37.0 (C), 36.3, 34.0, 33.5, 31.6, 29.7, 27.9
2 2
CH ), 27.2, 25.0, 23.6, 21.3 (CH ), 20.8, 19.3 (C), 18.1, 16.4 (CH ), 16.1 (CH ), 15.9 (CH ), 14.8 (CH ).
2
2
2
2
2
2
Anal. Calcd. for C H O (526.79): C, 77.52; H, 10.33. Found: C, 77.63; H, 10.33.
3
4
54
4
4
4
.6. Biological evaluation
.6.1. Cell culture
Stock solutions (10 mmol/L) of the tested compounds were prepared by dissolving an appropriate quantity of
each substance in dimethylsulfoxide (DMSO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), L-glutamine, penicillin and streptomycin were purchased from Sigma (MO, USA). Calcein
AM was obtained from Molecular Probes (Invitrogen Corporation, CA, USA). The screening cell lines (T-
lymphoblastic leukemia cell line CEM, breast carcinoma cell line MCF7, cervical carcinoma cell line HeLa,
1
7 / 19

ACCEPTED MANUSCRIPT
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% fetal bovine
serum, L-glutamine (2 mmol/L), penicillin (10 000 U) and streptomycin (10 mg/mL). The cell lines were
maintained under standard cell culture conditions at 37 ºC and 5% CO in a humid environment. Cells were
2
subcultured two or three times a week using the standard trypsinization procedure.
4.6.2. Calcein AM assay
5
Suspensions of the tested cell lines (ca. 1.0 × 10 cells/mL) were placed in 96-well microtiter plates and after
2
4 h of stabilization (time zero), the tested compounds were added (in three 20 µL aliquots) in serially
diluted concentrations in dimethylsulfoxide (DMSO). Control cultures were treated with DMSO alone, and
the final concentration of DMSO in the incubation mixtures never exceeded 0.6%. The test compounds were
typically evaluated at six 3-fold dilutions and the highest final concentration was generally 50 µM. After 72
h incubation, Calcein AM solution (100 µL, Molecular Probes, Invitrogen, CA, USA) was added, and
incubation was continued for a further hour. The fluorescence of viable cells was then quantified using a
Fluoroskan Ascent instrument (Labsystems, 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 IC values, the concentrations of the respective compounds that were lethal to 50% of the
5
0
tumor cells, were calculated.
5. Acknowledgements
This work was financed by grants from the National Science Centre, Poland (No.
012/07/B/ST5/00823) and Czech Ministry of Education National Program for Sustainability I (No.
2
LO1204). We thank Olga Hustáková for excellent technical assistance.
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[
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1
9 / 19

9
.834
ACCEPTED MANUSCRIPT
7.265
4
.703
.699
.608
.605
4
4
4
2.041
1.695
1.646
1.635
1.624
1.617
1.614
1.605
1.481
1.430
1.422
1.404
1.398
1.290
1.048
0.974
0.857
0.847
0.839
-0.000

ACCEPTED MANUSCRIPT
204.105
171.003
149.825
110.051
80.881
77.257
77.004
76.747
55.367
50.267
50.060
47.503
45.696
42.495
42.214
40.862
38.370
37.781
37.492
37.059
36.328
34.116
32.074
29.531
27.922
26.933
24.946
23.668
2
1.299
0.826
8.149
6.466
6.162
6.015
2
1
1
1
1
14.893

ACCEPTED MANUSCRIPT
7.267
4
.688
.684
.579
.578
4
4
4
2.041
1.685
1.659
1.650
1.639
1.630
1.625
1.420
1.401
1.394
1.386
1.045
0.954
0.863
0.857
0.852
0.845
0.837

ACCEPTED MANUSCRIPT
171.025
150.620
109.563
80.941
77.213
77.002
76.791
60.057
55.362
50.331
50.082
47.365
44.763
42.506
40.866
38.359
37.769
37.099
37.057
36.133
34.156
31.550
30.496
29.975
27.917
27.319
25.035
23.675
21.291
2
0.924
8.168
1
1
6.463
6.156
6.091
4.838
1
1
1

ACCEPTED MANUSCRIPT
7.264
4
.689
.685
.577
.576
4
4
4
3.488
1.681
1.659
1.651
1.627
1.420
1.385
1.320
1.273
1.269
1.044
0.968
0.959
0.832
0.763

ACCEPTED MANUSCRIPT
150.646
109.528
78.979
77.205
76.994
76.780
60.087
55.289
50.434
50.105
47.349
44.774
42.517
40.854
38.842
38.685
37.148
37.118
36.141
34.232
31.565
30.508
29.990
27.963
27.377
27.335
25.085
20.916
18.287
16.099
15.340
14.880

ACCEPTED MANUSCRIPT
7.262
5.300
4
.695
.692
.597
4.595
4
4
2.052
2.042
2.030
1.693
1.651
1.629
1.623
1.611
1.536
1.416
1.397
1.317
1.295
1.043
0.967
0.854
0.846
0.838
0.000

ACCEPTED MANUSCRIPT
178.927
171.052
150.075
109.824
80.926
77.201
76.990
76.776
55.355
50.239
49.863
47.330
46.173
42.575
40.793
38.348
37.773
37.474
37.045
36.240
34.064
33.416
31.504
29.657
27.913
27.243
24.986
2
3.663
1.299
0.828
8.149
6.459
6.137
5.953
4.880
2
2
1
1
1
1
1

ACCEPTED MANUSCRIPT
7.263
4
.695
.692
.593
.591
.588
4
4
4
4
3.489
3.188
2
.046
.024
.010
.987
.961
2
2
1
1
1.689
1.653
1.630
1.615
1.608
1.552
1.533
1.409
1.394
1.273
1.269
1.042
0.972
0.967
0.828
0.762
0.000

ACCEPTED MANUSCRIPT
178.666
150.114
109.774
79.002
77.205
76.990
76.780
55.266
50.335
49.882
47.311
46.150
42.579
40.777
38.831
38.666
37.482
37.129
36.256
34.133
33.412
31.519
29.680
27.948
2
7.319
7.258
5.035
0.816
8.264
6.072
5.957
5.336
2
2
2
1
1
1
1
14.923