Synthesis, cytotoxicity and liposome preparation of 28-acetylenic betulin derivatives

Article abstract of DOI:10.1016/j.bmc.2010.08.023

Several novel betulin derivatives were prepared and evaluated for their antitumor activity. 3-O-Acetylbetulinic aldehyde served as an ideal starting material for the synthesis of 28-acetylenic derivatives. These compounds were further transformed into pyrazoles and 1,2,3-triazoles. Also, the synthesis of 3-amino substituted butenolides was carried out. The compounds were screened for their antitumor activity in a panel of 15 human cancer cell lines in a sulforhodamine B (SRB) assay. Several compounds showed a noteworthy antitumor activity. In addition, the possibility of encapsulation into liposomes was examined, thereby resulting in an increased cytotoxicity. The results from a trypan-blue test and from DNA laddering provided evidence for an apoptotic cell death.

Full text of DOI:10.1016/j.bmc.2010.08.023

Bioorganic & Medicinal Chemistry 18 (2010) 7252–7259
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxicity and liposome preparation of 28-acetylenic
betulin derivatives
⇑
René Csuk , Alexander Barthel, Ralph Kluge, Dieter Ströhl
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle, Saale, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel betulin derivatives were prepared and evaluated for their antitumor activity. 3-O-Acetyl-
betulinic aldehyde served as an ideal starting material for the synthesis of 28-acetylenic derivatives.
These compounds were further transformed into pyrazoles and 1,2,3-triazoles. Also, the synthesis of
3-amino substituted butenolides was carried out. The compounds were screened for their antitumor
activity in a panel of 15 human cancer cell lines in a sulforhodamine B (SRB) assay. Several compounds
showed a noteworthy antitumor activity. In addition, the possibility of encapsulation into liposomes was
examined, thereby resulting in an increased cytotoxicity. The results from a trypan-blue test and from
DNA laddering provided evidence for an apoptotic cell death.
Received 16 June 2010
Revised 9 August 2010
Accepted 11 August 2010
Available online 26 August 2010
Keywords:
Betulin
Betulinic acid
SRB assay
Ó 2010 Elsevier Ltd. All rights reserved.
1,3-Dipolar cycloaddition
Liposomes
Antitumor
1. Introduction
potency was examined by a sulforhodamin B assay (SRB) applying
15 human cancer cell lines.
Triterpenes of the lupane-series (Fig. 1) are widely spread in the
plant kingdom; recently, they came focus of scientific interest
because of their promising antitumor and antiviral properties.
Several reports revealed the great potency of betulinic acid (BA)
I and related compounds as potential therapeutics for HIV therapy.
Further studies disclosed their particular action by inhibiting the
virus-cell fusion at the gp41–gp120 interface1,2 or by altering the
process of cell maturation by interfering the CA-SP1 junction in
Gag processing.3,4Also, betulinic acid derivatives have been sug-
gested5 for the therapy of human melanoma tumors; no toxic side
effects were observed in doses up to 500 mg/kg, and their way of
action works by an induction of apoptosis.
2. Chemistry
The synthesis of betulin derivatives bearing an acetylenic side
chain at C-28 was realized starting from 3-O-acetylbetulinic alde-
hyde10,11 (Scheme 1). The reaction of methyl propiolate or of
2-propyn-1-ol in the presence of LDA12 proceeded smoothly. For
phenylacetylene butyllithium13 gave higher yields compared to
the use of LDA. The additions advanced stereoselectively; only
the (28S) isomers could be isolated.
The oxidation of compound 1 (Scheme 2) with Jones reagent14
yielded the 28-keto derivative 4. Furthermore, the acetylenic
Follow-up studies allowed some insights into this process of
apoptosis: Important steps involve the liberation of cytochrome c
and AIF by changing the transmembrane potential of mitochon-
dria6,7 and the generation of reactive oxygen species (ROS).8 A par-
tial contribution of the p38 pathway was suggested, based on the
observation9 of phosphorylated proapoptotic mitogen-activated
protein kinases (MAPKs).
Here, we present a synthetic approach to new betulin-derived
compounds bearing an acetylenic side chain at the C-28 position.
Also, some of these compounds were used for the synthesis of het-
erocyclic compounds by 1,3-dipolar cycloaddition. Their antitumor
⇑
Corresponding author. Tel.: +49 345 55 25660; fax +49 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
Figure 1. Structure of lupane-type triterpenes.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.023

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
7253
Scheme 1. Synthesis of 28-acetylenic betulins. Reagents and conditions: (a) LDA, dry THF ꢀ78 °C, 2 h; (b) n-BuLi, dry THF ꢀ78 °C, 2 h.
Scheme 2. Transformations of 28-acetylenic betulins. Reagents and conditions: (a) CrO3, H2SO4, acetone 0 °C, 2 h; (b) ethyl diazoacetate, toluene, reflux, 48 h; (c)
diazomethane, Et2O, 0 °C; (d) NaN3, DMF, reflux, 72 h; (e) amine, EtOH, 70 °C, 12 h; (f) H2, Pd/BaSO4, quinoline, EtOAc, 30 min.
structure was ideal for the synthesis of the heterocyclic com-
pounds 5–8 by 1,3-dipolar cycloaddition reactions. For the reaction
of 1 with of ethyl diazoacetate15 the two isomers 5 and 6 were ob-
tained in a ratio of ca. 1:1. In general, the formation of compound 6
is favored but steric hindrance of the skeletal structure usually
leads to an increased formation of compound 5.
3. Results and discussion
The compounds were tested for their antitumor potential in a
panel of 15 human cancer cell lines in 96 well plates by using
the colorimetric SRB protocol.20 The calculated IC50 values in Table
1 were obtained from the corresponding dose–response curves.
Compound 1 shows IC50 values that are considerably lower than
those of standard betulinic acid. Changing the carboxymethyl moi-
ety for a hydroxymethyl group led to a decreased activity as ob-
served for compound 2, whereas the presence of a phenyl-
acetylene moiety is unfavorable. Interestingly, oxidizing the hydro-
xyl group of 1 led to a significant loss of activity. The pyrazole
derivatives 5–7 are less active than the parent compound 1, but
compared to betulinic acid for certain cell lines lower IC50 values
have been observed. For the triazole 8 IC50 values between
In contrast, adding diazomethane16 advanced stereoselectively;
the direct reaction with an excess of diazomethane, however, gave
the matching N-alkylated derivative 7. The triazole 8 was obtained
by reaction of 1 with sodium azide in DMF.17 In another approach,
1 was allowed to react with primary and secondary amines.18
Thereby, the Michael addition gave substituted acrylates; intramo-
lecular lactonization afforded the corresponding 3-N-substituted
butenolides 9–11. Hydrogenation of 2 in the presence of Lindlar
catalyst19 yielded the matching alkene 12.

7254
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
Table 1
Cytotoxicity of compounds in a panel of various human cancer cell lines
Cell line
IC50 values in lM for cancer cell lines
BA
1
2
3
4
5
6
7
8
9
10
11
12
518A2
A431
A253
FADU
A549
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
11.9
15.4
11.1
10.4
14.9
11.0
17.5
17.8
13.3
16.1
6.4
5.9
3.4
2.7
5.0
3.1
3.0
3.6
6.0
3.5
5.0
3.6
3.6
2.3
6.0
4.2
11.9
10.5
10.3
11.2
11.9
10.4
NA
11.6
11.3
11.6
6.9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
21.5
7.6
12.5
9.9
14.8
9.1
NA
10.0
9.8
12.0
7.7
8.9
16.4
8.0
16.4
11.7
13.2
13.9
18.2
13.2
NA
13.4
14.0
17.0
11.4
12.0
19.2
11.3
14.0
NA
14.8
16.6
10.3
15.1
17.3
13.4
15.5
13.9
15.6
18.2
14.7
15.4
11.8
14.8
18.2
4.8
3.3
4.9
3.8
6.1
2.6
3.6
1.7
3.2
5.7
2.2
3.1
2.9
6.9
5.3
6.2
2.9
4.2
6.0
6.6
2.5
4.7
1.9
3.2
5.1
8.0
6.0
3.9
4.0
5.8
4.7
2.1
4.6
6.3
7.8
2.2
5.4
0.5
2.1
3.9
5.7
5.3
4.1
3.7
6.0
20.4
12.4
14.8
10.7
12.8
12.6
12.3
10.1
10.5
15.9
17.9
17.8
9.0
12.4
16.0
17.6
24.5
12.7
NA
13.4
20.4
NA
7.0
6.7
8.4
11.5
9.9
10.8
22.8
12.3
14.1
11.6
14.9
9.7
19.4
18.5
11.4
19.9
Values are derived from dose–response curves obtained by measuring the percentage of viable cells relative to untreated controls after 96 h exposure of the cell line to the
test compounds using an SRB-assay for melanoma (518A2), zervic cancer (A431), head and neck tumor (A253, FADU), lung carcinoma (A549), ovarian cancer (A2780), colon
cancer (DLD-1, HCT-8, HCT-116, HT-29, SW-480), anaplastic thyroid cancer (8505c, SW-1736), mamma carcinoma (MCF-7) and Liposarcoma. Values are the average from at
least three independent experiments. Variation was generally 10%. NA = no inhibition of cell growth at the highest concentration (30 lM).
11.8–18.2
l
M were measured. The amino-substituted butenolides,
An apoptose-based mechanism seems reasonable. To gain
insights in the mode of action of our compounds more tests were
performed using 9 and liposomes of compound 1. Thereby, the
floating cells (obtained after treatment with IC90-concentrations
for 24 h) were analyzed by a trypan-blue exclusion test and DNA
gel electrophoresis (Fig. 2). Apoptotic cells have an intact cell
membrane and can exclude the dye whereas necrotic cells are
stained blue. During apoptosis DNA is cleaved into smaller frag-
ments by endonucleases. These fragments are observable in gel
electrophoresis as ladders.23–25
however, showed promising cytotoxicity: Best results were
observed for the dimethylamino derivative 9 with IC50 values be-
tween 2.2 and 6.9 lM. Slightly increased IC50 values were obtained
for the corresponding cyclopropylamine derivative 10 and the pyr-
rolidinyl derivative 11. The allyl alcohol 12 displayed only weak
activities. Our findings parallel previous results21 showing that a
free C-28 carboxylic acid function is important for the cytotoxicity
of these compounds. There are also some exceptions,21 however, to
this general tendency.
Betulinic acid and derivatives often show only poor solubilities
in water hence limited in vivo drug administration. To overcome
this limit we explored the principal possibility of encapsulation into
liposomes. Therefore, compound 1 was introduced into commercial
available liposome formulations. Best results were obtained with
soybean lecithin (Lipoid S75). Subsequent extrusion through a poly-
carbonate membrane22 with a pore size of 100 nm using a Liposo-
4. Conclusion
We have examined betulin derivatives bearing different acety-
lenic side chains at C-28. These derivatives showed promising
cytoxicity and they are also valuable starting materials for the syn-
thesis of betulin-derived pyrazoles, 1,2,3-triazoles and 3-substi-
tuted butenolides. Several of these derivatives revealed a higher
cytotoxicity than betulinic acid. Also we looked into the encapsu-
lation of the title compound 1 into liposomes.
Fast system gave liposomes with
a hydrodynamic diameter
between 70 and 120 nm (Z-average 105.0 nm) as determined by
dynamic light scattering. Noteworthy, encapsulation efficiency
was nearly 100% as determined by HPLC; their physical stability ex-
ceeded 4 weeks. Applicating compound 1 in liposomes in the SRB
assay revealed an extra benefit of encapsulation by observing an in-
creased cytotoxicity for most of the cell lines (Table 2).
5. Experimental section
5.1. General
Table 2
Instrumentation, cell lines and culture conditions, cytotoxicity
assay, dye exclusion test and DNA fragmentation assay as de-
scribed previously.11
IC50 (lM) values of compound 1
Cell line
1 in DMSO
1 Liposome
518A2
A431
A253
FADU
A549
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
5.9
3.4
2.7
5.0
3.1
3.0
3.6
6.0
3.5
5.0
3.6
3.6
2.3
6.0
4.2
3.7
2.4
3.6
4.0
4.2
2.0
3.3
2.8
2.3
4.6
3.7
4.4
4.4
3.4
4.2
5.2. Preparation of liposomes
Unilamellar liposomes of approximately 100 nm diameter were
obtained by the method of Olson22 employing a laboratory extru-
der (LiposoFast, Avestin Inc.). In a typical experiment for preparing
5 ml dispersion of liposomes, 5 mg of the compound were mixed
with an excess (125 mg) of phosphatidylcholin formulation (Lipoid
S75) in chloroform (5 ml) and evaporated to a film. The lipid film
was hydrated with H2O (5 ml) for 24 h at room temperature. The
solution obtained was extruded through a polycarbonate filter of
100 nm pore size. Twenty-one cycles were applied and concentra-
tion of the compound was determined by HPLC (RP18, 4.6 ꢁ 250,
k = 230 nm, methanol, 1.3 ml). Liposomes were characterized by
DLS.
Comparison of the calculated IC50 values derived from dose–response curves of
compound 1 as solution in DMSO and as aqueous liposomal formulation.

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
7255
Figure 2. Trypan-blue exclusion test for ovarian cancer cell line A2780 and DNA laddering for colon cancer cell line SW480 of compound 9 (left) and liposomal compound 1
(right) after treatment with IC90-concentrations for 24 h.
5.3. General procedure for the addition of alkynes to 3-O-
acetylbetulinic aldehyde (GP1)
(OMe), 51.0 (C17, Cquart.), 50.3 (C9, CH), 49.0 (C18, CH), 48.6 (C19,
CH), 43.0 (C14, Cquart.), 40.9 (C8, Cquart.), 38.3 (C1, CH2), 37.8 (C4,
Cquart.), 37.4 (C13, CH), 37.1 (C10, Cquart.), 34.2 (C7, CH2), 34.1
(C16, CH2), 34.0 (C22, CH2), 32.2 (C21, CH2), 27.9 (C23, CH3), 27.8
(C15, CH2), 25.0 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 20.8
(C11, CH2), 18.8 (C29, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3), 16.0
(C25 + C26, 2xCH3), 15.1 (C27, CH3) ppm; MS (i.e., 70 eV): m/z
(%) = 566 (12), 453 (100), 393 (61), 203 (19), 189 (24); Anal. for
To a solution of DIA (416 mg, 4.12 mmol) in dry THF (20 ml),
n-BuLi (2.6 ml, 1.6 M in hexane) was added at ꢀ20 °C. After
15 min, the solution was cooled to ꢀ78 °C and a solution of the cor-
responding alkyne (4.12 mmol) in dry THF (2 ml) was added drop-
wise. Stirring at this temperature was continued for an additional
30 min; then a solution of 3-O-acetylbetulinic aldehyde (0.50 g,
1.03 mmol) in dry THF (2 ml) was added. After 1 h at ꢀ78 °C, the
reaction was quenched by the addition of brine (100 ml). The
phases were separated and the aq layer was extracted with ethyl
acetate (2 ꢁ 50 ml). The combined extracts were dried over Na2SO4
and evaporated to dryness. The residue was subjected to column
chromatography (silica gel, hexane/ethyl acetate, 8:2).
C36H54O5 (566.81): C, 76.28; H, 9.60; found: C, 76.23; H, 9.49.
5.6. (28S) 3-O-Acetyl-28-(3-hydroxyprop-1-ynyl)-lup-20(29)-en-
3,28-diol (2)
Following GP1 but using two equivalents of LDA, compound 2
(0.43 g, 68%) was obtained as a colourless solid from 3-O-acetylbet-
ulinic aldehyde (0.50 g, 1.03 mmol) and 2-propyn-1-ol (0.23 g,
5.4. General procedure for the synthesis of 3-N-substitued
butenolides (GP2)
4.12 mmol); mp 210–213 °C; ½a D20
ꢂ
= +21.6 (c 3.2, CHCl3); Rf = 0.73
= 3423br, 2942s,
(silica gel, hexane/ethyl acetate, 5:5); IR (KBr):
m
2866m, 1733m, 1693s, 1455m, 1369m, 1315w, 1273s, 1117w,
A solution of compound 1 (0.30 g, 0.53 mmol) and the corre-
sponding amine (2.0 mmol) in ethanol (10 ml) was stirred at
70 °C for 12 h. The solution was concentrated and the residue sub-
jected to column chromatography (silica gel, chloroform/diethyl
ether, 9:1).
1038m, 1015m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.91 (s, 1H,
;
CH (28)), 4.68 (br s, 1H, CHa (30)), 4.56 (br s, 1H, CHb (30)), 4.45
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 4.32 (s, 2H, OCH2), 2.89
(ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.09–1.90 (m, 4H, CHa
(16) + CHa (21) + CHa (22) + CH (13)), 2.02 (s, 3H, Ac), 1.66 (s, 3H,
CH3 (29)), 1.76–1.46 (m, 7H, CH (18) + CHa (12) + CHa (1) + CH2
(2) + CHa (15) + CHa (6)), 1.45–1.08 (m, 9H, CH2 (11) + CHb
(6) + CHb (22) + CHb (21) + CH2 (7) + CH (9) + CHb (16) + CHb (12)),
1.04–0.91 (m, 2H, CHb (15) + CHb (1)), 1.02 (s, 3H, CH3 (25)), 0.98
(s, 3H, CH3 (27)), 0.84 (s, 6H, CH3 (26)), 0.83 (s, 6H, CH3 (23)),
0.82 (s, 3H, CH3 (24)), 0.78 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C
NMR (125 MHz, CDCl3): d = 171.0 (C@O), 150.9 (C20, C@CH2),
109.7 (C30, CH2@C), 86.5 (C31, C„C), 84.3 (C32, C„C), 80.9 (C3,
CHOAc), 66.0 (C28, CH), 55.3 (C5, CH), 51.1 (C17, Cquart.), 50.7
(OCH2), 50.2 (C9, CH), 48.9 (C18, CH), 48.7 (C19, CH), 43.0 (C14,
Cquart.), 40.9 (C8, Cquart.), 38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.2
(C13, CH), 37.0 (C10, Cquart.), 34.3 (C7, CH2), 34.1 (C16, CH2), 34.0
(C22, CH2), 32.3 (C21, CH2), 27.9 (C23, CH3), 27.8 (C15, CH2), 25.0
(C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 20.8 (C11, CH2), 18.8
(C29, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3), 16.1 (C26, CH3), 16.0
(C25, CH3), 15.0 (C27, CH3) ppm; MS (ESI, MeOH): m/z (%) = 561.5
(40% [M+Na]+), 1099.1 (100% [2M+Na]+); Anal. for C35H54O4
(538.80): C, 78.02; H, 10.10. Found: C, 77.85; H, 10.33.
5.5. Methyl (28S)-3-[3b-acetoxy-28-hydroxy-lup-20(29)-en-28-
yl]-propiolate (1)
Following GP1, 1 (0.43 g, 73%) was obtained from 3-O-acetyl-
betulinic aldehyde (0.50 g, 1.03 mmol) and methyl propiolate
(0.35 g, 4.12 mmol) as
= +18.5 (c 4.5, CHCl3); Rf = 0.55 (silica gel, hexane/ethyl ace-
tate, 8:2); IR (KBr): = 3475s, 2955s, 2870m, 2228m, 1713s,
1638w, 1455m, 1431m, 1380m, 1250s, 1155w, 1107w, 1066m,
1049m, 1033m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.98 (s, 1H,
a colourless solid; mp 210–212 °C;
½ ꢂ
a 2D0
m
;
CH (28)), 4.67 (br s, 1H, CHa (30)), 4.56 (br s, 1H, CHb (30)), 4.44
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.76 (s, 3H, OCH3), 2.85
(ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.04–1.80 (m, 4H, CHa
(16) + CHa (21) + CHa (22) + CH (13)), 2.02 (s, 3H, Ac), 1.65 (s, 3H,
CH3 (29)), 1.78–1.45 (m, 8H, CH (18) + CHa (12) + CHa (1) + CH2
(2) + CHa (6) + CHa (15) + CHa (11)), 1.45–1.09 (m, 10H, CHb
(6) + CHb (21) + CHb (22) + CHb (16) + CH2 (7) + CH (9) + CHb
(11) + CHb (12) + CHb (15)), 1.04–0.86 (m, 1H, CHb (1)), 1.00 (s,
3H, CH3 (25)), 0.97 (s, 3H, CH3 (27)), 0.83 (s, 3H, CH3 (26)), 0.82
(s, 3H, CH3 (23)), 0.81 (s, 3H, CH3 (24)), 0.77 (d, 1H, J = 9.5 Hz, CH
(5)) ppm; 13C (125 MHz, CDCl3): d = 171.0 (C@O), 153.8 (C@O),
150.6 (C20, C@CH2), 109.8 (C30, CH2@C), 88.3 (C31, C„C), 80.9
(C3, CHOAc), 77.7 (C32, C„C), 66.0 (C28, CH), 55.3 (C5, CH), 52.8
5.7. (28S) 3-O-Acetyl-28-(phenylethynyl)-lup-20(29)-en-3,28-
diol (3)
To a solution of phenylacetylene (0.31 g, 3.00 mmol) in THF
(10 ml) n-butyllithium (1.9 ml, 1.6 M in hexane) was added under

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R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
argon at ꢀ78 C. Stirring at this temperature was continued for an
additional 30 min; then, a solution of 3-O-acetylbetulinic aldehyde
(0.50 g, 1.03 mmol) in dry THF (2 ml) was added. After 1 h at ꢀ78
C, the reaction was quenched by the addition of H2O (15 ml). The
phases were separated and the aq layer was extracted with CHCl3
(2 ꢁ 20 ml). The combined extracts were dried over Na2SO4 and
evaporated to dryness. The residue was subjected to column chro-
matography (silica gel, hexane/ethyl acetate, 8:2) to afford com-
149.8 (C20, C@CH2), 109.9 (C30, CH2@C), 80.3 (C31, C„C), 80.9
(C3, CHOAc), 76.9 (C32, C„C), 62.1 (C17, Cquart.), 55.4 (C5, CH),
53.2 (OMe), 50.4 (C9, CH), 48.5 (C18, CH), 46.3 (C19, CH), 42.4
(C14, Cquart.), 40.7 (C8, Cquart.), 38.4 (C1, CH2), 37.8 (C4, Cquart.),
37.2 (C13, CH), 37.1 (C10, Cquart.), 35.2 (C22, CH2), 34.2 (C7, CH2),
31.2 (C16, CH2), 29.9 (C21, CH2), 29.6 (C15, CH2), 27.9 (C23, CH3),
25.4 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 20.8 (C11, CH2),
19.1 (C29, CH3), 18.1 (C6, CH2), 16.4 (C24, CH3), 16.2 (C26, CH3),
15.9 (C25, CH3), 14.3 (C27, CH3) ppm; MS (ESI, MeOH): m/z (%):
587.3 (40% [M+Na]+), 1154.1 (100% [2M+Na]+); Anal. for C36H52O5
(564.80): C, 76.56; H, 9.28. Found: C, 76.28; H, 8.92.
pound
= +17.3 (c 3.0, CHCl3); Rf = 0.68 (silica gel, hexane/ethyl ace-
tate, 8:2); IR (KBr): = 2943s, 2872m, 2123w, 1734s, 1638w,
1490m, 1456m, 1376m, 1245m, 1071w, 1028m cmꢀ1 1H NMR
3 (0.45 g, 75%) as a white solid; mp 144–146 °C;
½ ꢂ
a 2D0
m
;
(500 MHz, CDCl3): d = 7.44–7.42 (m, 2H, Ph), 7.31–7.29 (m, 3H,
Ph), 5.09 (s, 1H, CH (28)), 4.72 (d, 1H, J = 2.0 Hz, CHa (30)), 4.59
(br s, 1H, CHb (30)), 4.47 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)),
2.95 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.20–2.00 (m, 4H,
CHa (16) + CHa (22) + CHa (21) + CH (13)), 2.03 (s, 3H, Ac), 1.81–
1.58 (m, 6H, CH (18) + CHa (12) + CHa (1) + CHa (15) + CH2 (2)),
1.70 (s, 3H, CH3 (29)), 1.52–1.13 (m, 11H, CH2 (6) + CH2
(11) + CHb (21) + CHb (16) + CH2 (7) + CHb (22) + CH (9) + CHb
(12)), 1.06–0.95 (m, 2H, CHb (15) + CHb (1)), 1.06 (s, 3H, CH3
(25)), 1.01 (s, 3H, CH3 (27)), 0.86 (s, 3H, CH3 (26)), 0.84 (s, 3H,
CH3 (23)), 0.83 (s, 3H, CH3 (24)), 0.80 (d, 1H, J = 9.5 Hz, CH (5));
13C NMR (125 MHz, CDCl3): d = 171.0 (C@O), 151.0 (C20, C@CH2),
131.6 (Ph), 128.3 (Ph), 128.2 (Ph), 122.8 (Ph), 109.6 (C30, CH2@C),
89.9 (C31, C„C), 86.1 (C32, C„C), 80.9 (C3, CHOAc), 66.6 (C28, CH),
55.3 (C5, CH), 51.1 (C17, Cquart.), 50.2 (C9, CH), 49.0 (C18, CH), 48.7
(C19, CH), 43.1 (C14, Cquart.), 40.9 (C8, Cquart.), 38.4 (C1, CH2), 37.8
(C4, Cquart.), 37.3 (C13, CH), 37.1 (C10, Cquart.), 34.5 (C16, CH2),
34.2 (C7, CH2), 34.1 (C22, CH2), 32.5 (C21, CH2), 27.9 (C23, CH3),
27.8 (C15, CH2), 25.1 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3),
20.8 (C11, CH2), 18.8 (C29, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3),
16.2 (C25, CH3), 16.1 (C26, CH3), 15.1 (C27, CH3) ppm; MS (ESI,
MeOH): m/z (%) = 1191.1 (100% [2M+Na]+); Anal. for C40H56O3
(584.87): C, 82.14; H, 9.65. Found: C, 82.00; H, 9.85.
5.9. 28-[3-(Ethylcarboxy)-4-(methylcarboxy)-pyrazol-5-yl]-3,
28-dioxo-28-ethinyllup-20(29)-ene (5) and 28-[3-(ethylcarb-
oxy)-5-(methylcarboxy)-pyrazol-4-yl]-3,28-dioxo-28-ethinyll-
up-20(29)-ene (6)
A solution of compound 1 (0.30 g, 0.53 mmol) and ethyl diazo-
acetate (0.23 g, 2 mmol) in toluene (10 ml) was heated under re-
flux for 48 h. After TLC revealed the absence of starting material,
the reaction mixture was concentrated in vacuo and the residue
subjected to column chromatography (silica gel, hexane/ethyl ace-
tate, 8:2).
Data for 5: Compound 5 (0.14 g, 38%) was obtained as a colour-
less solid; mp 218–210 °C; ½a D20
ꢂ
= +5.75 (c 4.1, CHCl3); Rf = 0.75 (sil-
= 3475s, 2945s,
ica gel, hexane/ethyl acetate, 5:5); IR (KBr):
m
2873m, 1733s, 1701s, 1455m, 1370m, 1249s, 1159m, 1107m,
1060m, 1028m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.53 (s, 1H,
;
CH (28)), 4.71 (d, 1H, J = 2.2 Hz, CHa (30)), 4.58 (br s, 1H, CHb
(30)), 4.46 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 4.36 (q, 2H,
J = 7.0 Hz, OCH2), 3.85 (s, 3H, OCH3), 3.08 (ddd, 1H, J = 11.2, 10.4,
7.0 Hz, CH (19)), 2.23–2.00 (m, 2H, CHa (21) + CH (13)), 2.02 (s,
3H, Ac), 1.87–1.75 (m, 2H, CHa (16) + CH (18)), 1.68 (s, 3H, CH3
(29)), 1.71–1.56 (m, 5H, CHa (12) + CHa (15) + CHa (1) + CH2 (2)),
1.53–1.08 (m, 12H, CH2 (6) + CH2 (11) + CH2 (22) + CH2 (7) + CHb
(21) + CH (9) + CHb (16) + CHb (12)), 1.36 (t, 3H, J = 7.0 Hz, CH3
(Et)), 1.04–0.84 (m, 2H, CHb (1) + CHb (15)), 1.05 (s, 3H, CH3 (26)),
0.98 (s, 3H, CH3 (27)), 0.84 (s, 3H, CH3 (25)), 0.82 (s, 3H, CH3
(23)), 0.81 (s, 3H, CH3 (24)), 0.79 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 171.0 (C@O), 165.3 (C@O), 160.7
(C@O), 151.2 (C20, C@CH2), 150.8 (pyrazole, Cquart.), 113.0 (pyra-
zole, Cquart.), 109.5 (C30, CH2@C), 80.9 (C3, CHOAc), 68.7 (C28,
CH), 61.8 (OCH2), 55.3 (C5, CH), 52.6 (OMe), 50.6 (C18, CH), 50.5
(C17, Cquart.), 50.2 (C9, CH), 48.2 (C19, CH), 43.1 (C14, Cquart.), 40.9
(C8, Cquart.), 38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.0 (C10, Cquart.),
36.8 (C13, CH), 34.5 (C16, CH2), 34.2 (C7, CH2), 33.6 (C22, CH2),
32.6 (C21, CH2), 27.9 (C23, CH3), 27.8 (C15, CH2), 25.3 (C12, CH2),
23.7 (C2, CH2), 21.3 (Ac, CH3), 20.8 (C11, CH2), 19.0 (C29, CH3),
18.2 (C6, CH2), 16.5 (C24, CH3), 16.1 (C25 + C26, 2 ꢁ CH3), 15.1
(C27, CH3), 14.1 (Et, CH3) ppm; MS (ESI, MeOH): m/z (%) = 681.2
(40% [M+H]+), 703.3 (100% [M+Na]+), 1383.1 (40% [2M+Na]+); Anal.
for C40H60N2O7 (680.91): C, 70.56; H, 8.88; N, 4.11. Found: C, 70.48;
H, 8.67; N, 4.02.
5.8. Methyl (28S) 3-[3b-acetoxy-28-oxo-lup-20(29)-en-28-yl]-
propiolate (4)
A solution of chromium(VI) oxid (300 mg) in aq H2SO4 (35%,
1 ml) was slowly added to a stirred solution of compound 1
(1.04 g, 1.84 mmol) in acetone (10 ml) at 0 °C. After TLC revealed
the absence of starting material, the excess chromium(VI) oxid
was destroyed by the addition of isopropanol (2 ml). The solution
was concentrated in vacuo and the residue partitioned between
H2O (30 ml) and CH2Cl2 (30 ml). The layers were separated and
the aqueous layer was extracted with CH2Cl2 (10 ml). The combined
organic extracts were dried over Na2SO4, evaporated to dryness and
purified by column chromatography (silica gel, hexane/ethyl ace-
tate 9:1). Compound 4 (0.40 g, 80%) was obtained as a white solid;
mp 103 °C; ½a 2D0
ꢂ
= +19.2 (c 6.0, CHCl3); Rf = 0.65 (silica gel, hexane/
= 2949s, 2871m, 1728s, 1680m,
1H
ethyl acetate, 8:2); IR (KBr):
m
1643w, 1436m, 1392w, 1368m, 1247s, 1096w, 1024m cmꢀ1
;
(400 MHz, CDCl3): d = 4.70 (br s, 1H, CHa (30)), 4.58 (br s, 1H, CHb
(30)), 4.44 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.82 (s, 3H, OCH3),
2.87 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.38 (ddd, 1H,
J = 13.7, 3.7, 3.3 Hz, CHa (16)), 2.20 (ddd, 1H, J = 12.5, 12.0, 3.7 Hz,
CH (13)), 2.05–1.99 (m, 1H, CHa (22)), 2.01 (s, 3H, Ac), 1.76 (ddd,
1H, J = 10.8, 7.9, 2.5 Hz, CHa (21)), 1.65 (s, 3H, CH3 (29)), 1.70–1.50
(m, 6H, CH (18) + CHa (12) + CHa (1) + CH2 (2) + CHb (16)), 1.49–
1.31 (m, 9H, CH2 (6) + CHb (22) + CHb (21) + CH2 (7) + CHa
(11) + CHa (15)), 1.25–1.17 (m, 3H, CH (9) + CHb (15) + CHb (11)),
1.02–0.90 (m, 2H, CHb (1) + CHb (12)), 0.93 (s, 3H, CH3 (27)), 0.88
(s, 3H, CH3 (25)), 0.82 (s, 6H, CH3 (26)), 0.81 (s, 6H, CH3 (23)), 0.80
(s, 3H, CH3 (24)), 0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d = 190.3 (C@O), 170.9 (C@O), 152.8 (C@O),
Data for 6: Compound 6 (0.16 g, 43%) was obtained as a colour-
less solid; mp 210–212 °C; ½a D20
ꢂ
= +12.9 (c 4.3, CHCl3); Rf = 0.42 (sil-
= 2947s, 2873m,
ica gel, hexane/ethyl acetate, 8:2); IR (KBr):
m
1733s, 1640w, 1550w, 1466m, 1378m, 1247s, 1125w, 1074w,
1020m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.77 (s, 1H, CH (28)),
;
4.74 (d, 1H, J = 2.2 Hz, CHa (30)), 4.54 (br s, 1H, CHb (30)), 4.46–
4.36 (m 3H, CHOAc (3) + OCH2), 3.92 (s, 3H, OCH3), 3.23 (ddd, 1H,
J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.22–2.10 (m, 2H, CH (13) + CHa
(21)), 2.01 (s, 3H, Ac), 1.98–1.70 (m, 2H, CH (18)), 1.68 (s, 3H,
CH3 (29)), 1.71–1.50 (m, 6H, CHa (1) + CH2 (2) + CHa (16) + CHa
(12) + CHa (15)), 1.51–1.08 (m, 11H, CH2 (6) + CH2 (11) + CH2
(7) + CHb (21) + CH2 (22) + CH (9) + CHb (12)), 1.37 (t, 3H,
J = 7.0 Hz, CH3 (Et)), 1.03–0.95 (m, 3H, CHb (1) + CHb (16) + CHb

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
7257
(15)), 1.09 (s, 3H, CH3 (26)), 0.94 (s, 3H, CH3 (27)), 0.84 (s, 3H, CH3
(25)), 0.81 (s, 6H, CH3 (23) + CH3 (24)), 0.77 (d, 1H, J = 9.5 Hz, CH
(5)) ppm; 13C NMR (125 MHz, CDCl3): d = 171.0 (C@O), 167.7
(C@O), 163.7 (C@O), 151.4 (C20, C@CH2), 132.4 (pyrazole, Cquart.),
129.3 (pyrazole, Cquart.), 109.7 (C30, CH2@C), 81.0 (C3, CHOAc),
67.8 (C28, CH), 62.4 (OCH2), 55.3 (C5, CH), 53.0 (OMe), 52.1 (C17,
Cquart.), 51.5 (C18, CH), 50.2 (C9, CH), 47.5 (C19, CH), 43.1 (C14,
Cquart.), 41.1 (C8, Cquart.), 38.4 (C1, CH2), 37.8 (C4, Cquart.), 37.0
(C10, Cquart.), 36.2 (C13, CH), 35.1 (C16, CH2), 34.5 (C22, CH2),
34.2 (C7, CH2), 31.7 (C21, CH2), 27.9 (C23, CH3), 27.8 (C15, CH2),
25.3 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 20.8 (C11, CH2),
19.0 (C29, CH3), 18.2 (C6, CH2), 16.5 (C24, CH3), 16.2 (C25,
2xCH3), 16.1 (C26, 2 ꢁ CH3), 14.9 (C27, CH3), 14.2 (Et, CH3) ppm;
MS (ESI, MeOH): m/z (%) = 681.2 (30% [M+H]+), 703.3 (100%
arated and the aq layer extracted with CHCl3 (20 ml). The com-
bined extracts were dried over Na2SO4 and evaporated to
dryness. The residue was subjected to column chromatography
(silica gel, hexane/ethyl acetate, 8:2). Compound 8 (0.14 g, 43%)
was obtained as a colourless solid; mp 196–200 °C; ½a D20
ꢂ
= +13.2
(c 4.3, CHCl3); Rf = 0.59 (silica gel, hexane/ethyl acetate, 5:5); IR
(KBr):
m
= 3440br, 2947s, 2874m, 1734s, 1708s, 1662m, 1456m,
1377m, 1246s, 1117w, 1029m cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d = 5.64 (d, 1H, J = 9.8, CH (28)), 4.79 (d, 1H, J = 9.8, OH), 4.75 (d,
1H, J = 1.9, CHa (30)), 4.60 (br s, 1H, CHb (30)), 4.47 (dd, 1H,
J = 11.4, 4.8 Hz, CHOAc (3)), 4.00 (s, 3H, OCH3), 3.20 (ddd, 1H,
J = 11.3, 11.3, 6.1 Hz, CH (19)), 2.25–2.15 (m, 2H, CHa (21) + CH
(13)), 2.04 (s, 3H, Ac), 1.95 (ddd, 1H, J = 14.0, 13.8, 3.8 Hz, CHa
(15)), 1.70 (s, 3H, CH3 (29)), 1.86–1.56 (m, 6H, CHa (16) + CH
(18) + CHa (12) + CHa (1) + CH2 (2)), 1.50–1.30 (m, 7H, CH2
(6) + CHa (22) + CHb (21) + CHa (11) + CH2 (7)), 1.29–1.08 (m, 5H,
CH (9) + CHb (11) + CHb (22) + CHb (12) + CHb (16)), 1.00–0.92 (m,
1H, CHb (1) + CHb (15)), 1.10 (s, 3H, CH3 (26)), 1.00 (s, 3H, CH3
(27)), 0.85 (s, 3H, CH3 (25)), 0.83 (s, 3H, CH3 (23)), 0.82 (s, 3H,
CH3 (24)), 0.80 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d = 171.2 (C@O), 163.8 (C@O), 151.3 (C20,
C@CH2), 109.6 (C30, CH2@C), 81.1 (C3, CHOAc), 69.4 (C28, CH),
55.3 (C5, CH), 53.1 (OMe), 51.2 (C17, Cquart.), 50.9 (C18, CH), 50.2
(C9, CH), 48.3 (C19, CH), 43.0 (C14, Cquart.), 41.0 (C8, Cquart.), 38.4
(C1, CH2), 37.8 (C4, Cquart.), 37.0 (C10, Cquart.), 36.9 (C13, CH), 34.2
(C7, CH2), 34.1 (C16, CH2), 34.0 (C22, CH2), 32.5 (C21, CH2), 27.9
(C23, CH3), 27.8 (C15, CH2), 25.2 (C12, CH2), 23.7 (C2, CH2), 21.3
(Ac, CH3), 20.9 (C11, CH2), 18.9 (C29, CH3), 18.2 (C6, CH2), 16.5
(C24, CH3), 16.2 (C25, CH3), 16.1 (C26, CH3), 15.2 (C27, CH3) ppm;
MS (ESI, MeOH): m/z (%) = 610.3 (40% [M+H]+), 632.5 (100%
[M+Na]+); Anal. for C36H55N3O5 (609.83): C, 70.90; H, 9.09; N,
6.89. Found: C, 70.78; H, 9.24; N, 6.76.
[M+Na]+), 1383.1 (50% [2M+Na]+); UV–vis (Methanol): kmax (log
e)
218 nm (4.41); Anal. for C40H60N2O7 (680.91): C, 70.56; H, 8.88; N,
4.11. Found: C, 70.32; H, 8.54; N, 3.89.
5.10. 28-[N-Methyl-3-(methylcarboxy)-pyrazol-4-yl]-3,28-dioxo
-28-ethinyllup-20(29)-ene (7)
An ethereal solution of diazomethane was added to a solution of
compound 1 (0.30 g, 0.53 mmol) in Et2O (10 ml) under cooling
with ice until the reaction mixture remained yellow. After stirring
for 30 min, the excess diazomethane was destroyed with acetic
acid (5%) and the solution was concentrated in vacuo. After column
chromatography compound 7 (0.30 g, 92%) was obtained as a col-
ourless solid; mp 213–214 °C;
Rf = 0.73 (silica gel, hexane/ethyl acetate, 5:5); IR (KBr):
= 2949s, 2873m, 1728s, 1638w, 1455m, 1370m, 1246s, 1196w,
1111m, 1018m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 7.53 (s, 1H,
½
a 2D0
= +16.4 (c 7.1, CHCl3);
ꢂ
m
;
CH (pyrazole)), 5.51 (s, 1H, CH (28)), 4.73 (d, 1H, J = 2.2 Hz, CHa
(30)), 4.58 (br s, 1H, CHb (30)), 4.45 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc
(3)), 4.10 (s, 3H, NCH3), 3.95 (s, 3H, OCH3), 3.08 (ddd, 1H, J = 11.2,
10.4, 7.0 Hz, CH (19)), 2.21–2.07 (m, 3H, CH (13) + CHa (16) + CHa
(21)), 2.03 (s, 3H, Ac), 1.71 (s, 3H, CH3 (29)), 1.83–1.72 (m, 2H,
CH (18) + CHa (12)), 1.71–1.56 (m, 5H, CHa (1) + CHa (15) + CHa
(22) + CH2 (2)), 1.71–1.34 (m, 5H, CH2 (6) + CHa (11) + CHb
(21) + CHb (16)), 1.33–1.16 (m, 6H, CH (9) + CHb (22) + CHb
(11) + CH2 (7) + CHb (12)), 1.06 (s, 3H, CH3 (26)), 1.04–0.94 (m,
2H, CHb (1) + CHb (15)), 1.00 (s, 3H, CH3 (27)), 0.86 (s, 3H, CH3
(25)), 0.83 (s, 3H, CH3 (23)), 0.82 (s, 3H, CH3 (24)), 0.80 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 171.0
(C@O), 161.3 (C@O), 151.5 (C20, C@CH2), 137.7 (pyrazole, CH),
130.2 (pyrazole, Cquart.), 129.0 (pyrazole, Cquart.), 109.5 (C30,
CH2@C), 80.9 (C3, CHOAc), 67.2 (C28, CH), 55.3 (C5, CH), 52.5
(OMe), 50.8 (C18, CH), 50.2 (C9, CH), 50.2 (C17, Cquart.), 48.5 (C19,
CH), 43.0 (C14, Cquart.), 40.9 (C8, Cquart.), 40.7 (NMe), 38.3 (C1,
CH2), 37.8 (C4, Cquart.), 37.1 (C10, Cquart.), 36.9 (C13, CH), 34.2 (C7,
CH2), 34.7 (C22, CH2), 34.1 (C16, CH2), 32.8 (C21, CH2), 27.9 (C23,
CH3), 27.8 (C15, CH2), 25.0 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac,
CH3), 20.9 (C11, CH2), 18.8 (C29, CH3), 18.1 (C6, CH2), 16.5 (C24,
CH3), 16.1 (C25, CH3), 16.0 (C26, CH3), 15.1 (C27, CH3) ppm; MS
(ESI, MeOH): m/z (%) = 645.3 (70% [M+Na]+), 1267.2 (100%
[2M+Na]+); Anal. for C37H56N2O5 (608.85): C, 72.99; H, 9.27; N,
4.60. Found: C, 72.68; H, 9.32; N, 4.52.
5.12. (4R) 3-(Dimethylamino)-4-[3b-hydroxy-28-norlup-20(29)-
en-17b-yl]-2-butenolide (9)
Following GP2, compound 9 (0.21 g, 68%) was obtained from 1
(0.30 g, 0.53 mmol) and dimethylammonium dimethyl carbamate
(0.27 g, 2.00 mmol) as
= +135.0 (c 3.3, CHCl3); Rf = 0.36 (silica gel, chloroform/diethyl
ether, 9:1); IR (KBr): = 3456s, 2942s, 1736s, 1642w, 1606s,
1454m, 1377m, 1301w, 1250s, 1167w, 1133m, 1032m cmꢀ1 1H
a colourless solid; mp 175–178 °C;
½ ꢂ
a 2D0
m
;
NMR (500 MHz, CDCl3): d = 5.26 (s, 1H, CH (32)), 4.88 (s, 1H, CH
(28)), 4.74 (d, 1H, J = 1.9 Hz, CHa (30)), 4.54 (dd, 1H, J = 2.4, 1.4 Hz,
CHb (30)), 4.46 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.04 (ddd, 1H,
J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.82 (s, 3H, NCH3), 2.19 (ddd, 1H,
J = 12.2, 12.2, 3.6 Hz, CH (13)), 2.00–1.95 (m, 1H, CHa (16)), 2.02
(s, 3H, Ac), 1.83–1.71 (m, 3H, CH (18) + CHb (16) + CHa (15)), 1.68–
1.56 (m, 8H, CHa (1) + CHa (12) + CH2 (2) + ), 1.67 (s, 3H, CH3 (29)),
1.53–1.22 (m, 10H, CHa (22) + CH2 (6) + CH2 (7) + CH2 (21) + CH2
(11) + CH (9)), 1.17–0.94 (m, 4H, CHb (15) + CHb (12) + CHb
(22) + CHb (1)), 1.06 (s, 3H, CH3 (26)), 1.04 (s, 3H, CH3 (27)), 0.85
(s, 3H, CH3 (23)), 0.84 (s, 3H, CH3 (25)), 0.83 (s, 3H, CH3 (24)), 0.79
(d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d = 175.6 (C@O), 173.6 (C31, C@C), 171.0 (C@O), 150.8 (C20,
C@CH2), 109.6 (C30, CH2@C), 88.3 (C28, CH), 80.9 (C3, CHOAc),
80.2 (C32, CH), 55.4 (C5, CH), 51.5 (C17, Cquart.), 52.0 (C18, CH),
50.2 (C9, CH), 47.7 (C19, CH), 43.0 (C14, Cquart.), 41.0 (C8, Cquart.),
42.3 (NMe), 38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.1 (C10, Cquart.),
36.5 (C13, CH), 34.2 (C7, CH2), 35.1 (C22, CH2), 32.5 (C16, CH2),
32.4 (C21, CH2), 28.0 (C15, CH2), 27.9 (C23, CH3), 25.4 (C12, CH2),
23.7 (C2, CH2), 21.3 (Ac, CH3), 20.7 (C11, CH2), 19.5 (C29, CH3),
18.1 (C6, CH2), 16.5 (C24, CH3), 16.3 (C25, CH3), 16.1 (C26, CH3),
15.4 (C27, CH3) ppm; MS (ESI, MeOH): m/z (%) = 580.5 (100%
5.11. 28-(4-(Methylcarboxy)-1H-1,2,3-triazol-5-yl)-3,28-dioxo-
28-ethinyllup-20(29)-ene (8)
A solution of compound 1 (0.30 g, 0.53 mmol) and sodium azide
(0.37 g, 5.0 mmol) in dry DMF (10 ml) was heated under reflux for
72 h. The mixture was concentrated in vacuo and the residue was
treated with H2O (10 ml) and CHCl3 (20 ml). The phases were sep-

7258
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 7252–7259
[M+H]+), 602.5 (100% [M+Na]+); Anal. for C37H57NO4 (579.85): C,
76.64; H, 9.91; N, 2.42. Found: C, 76.54; H, 9.74; N, 2.32.
(C15, CH2), 27.9 (C23, CH3), 25.6 (pyrrolid., CH2),25.4 (C12, CH2),
23.7 (C2, CH2), 21.3 (Ac, CH3), 20.7 (C11, CH2), 19.5 (C29, CH3),
18.1 (C6, CH2), 16.5 (C24, CH3), 16.3 (C25, CH3), 16.1 (C26, CH3),
15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z (%) = 606.5 (90%
[M+H]+), 628.5 (100% [M+Na]+); Anal. for C39H59NO4 (605.89): C,
77.31; H, 9.82; N, 2.31. Found: C, 77.01; H, 9.98; N, 2.05.
5.13. (4R) 3-(Cyclopropylamino)-4-[3b-hydroxy-28-norlup-
20(29)-en-17b-yl]-2-butenolide (10)
Following GP2, 10 (0.21 g, 68%) was obtained from 1 (0.30 g,
0.53 mmol) and cyclopropylamine (0.11 g, 2.00 mmol) as an amor-
5.15. (28S) 3-O-Acetyl-28-(3-hydroxyprop-1-enyl)-lup-20(29)-
en-3,28-diol (12)
phous colourless solid. ½a D20
ꢂ
= ꢀ29.8 (c 3.1, CHCl3); Rf = 0.50 (silica
gel, chloroform/diethyl ether, 9:1); IR (KBr):
m
= 2948s, 2873m,
1735s, 1602s, 1508m, 1455m, 1367m, 1300s, 1245s, 1161m,
Hydrogen was bubbled for 30 min through a mixture containing
2 (0.2 g, 0.37 mmol), Pd/BaSO4 (20 mg) and quinoline (200 mg) in
ethyl acetate (10 ml). After completion of the reaction (as observed
by TLC), the reaction was filtered over celite and the solvent re-
moved in vacuo. The residue was subjected to column chromatog-
raphy (silica gel, hexane/ethyl acetate, 8:2). Compound 12 (0.19 g,
1033m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.14 (s, 1H, CH
;
(32)), 5.03 (s, 1H, CH (28)), 4.78 (s, 1H, NH), 4.73 (d, 1H,
J = 1.9 Hz, CHa (30)), 4.58 (dd, 1H, J = 2.4, 1.4 Hz, CHb (30)), 4.46
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.00 (ddd, 1H, J = 11.2, 10.4,
7.0 Hz, CH (19)), 2.54–2.50 (m, 1H, NCH), 2.15 (ddd, 1H, J = 12.2,
12.2, 3.6 Hz, CH (13)), 2.03 (s, 3H, Ac), 1.87–1.46 (m, 11H, CHa
(21) + CH (18) + CHa (16) + CHa (15) + CHa (1) + CHa (12) + CH2
(2) + CHa (22) + CHa (7) + CHa (6)), 1.67 (s, 3H, CH3 (29)), 1.44–
1.20 (m, 7H, CH2 (11) + CHb (7) + CHb (6) + CHb (16) + CHb
(21) + CH (9)), 1.17–1.08 (m, 3H, CHb (22) + CHb (15) + CHb (12)),
1.00–0.93 (m, 1H, CHb (1)), 1.03 (s, 3H, CH3 (26)), 1.01 (s, 3H, CH3
(27)), 0.85 (s, 3H, CH3 (25)), 0.83 (s, 3H, CH3 (24)), 0.82 (s, 3H,
CH3 (23)), 0.82–0.76 (m, 3H, CH2(cycloprop.) + CH (5)), 0.65–0.55
(m, 2H, CH2(cycloprop.)) ppm; 13C NMR (125 MHz, CDCl3):
d = 174.0 (C@O), 171.0 (C@O), 170.9 (C31, C@C), 150.6 (C20,
C@CH2), 109.6 (C30, CH2@C), 86.0 (C28, CH), 80.8 (C3, CHOAc),
79.5 (C32, CH), 55.4 (C5, CH), 49.8 (C17, Cquart.), 51.6 (C18, CH),
50.2 (C9, CH), 47.1 (C19, CH), 43.0 (C14, Cquart.), 41.0 (C8, Cquart.),
38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.0 (C10, Cquart.), 36.6 (C13, CH),
34.3 (C22, CH2), 34.2 (C7, CH2), 34.0 (C16, CH2), 32.4 (C21, CH2),
27.9 (C23, CH3), 27.8 (C15, CH2), 26.6 (NCH), 25.3 (C12, CH2),
23.7 (C2, CH2), 21.3 (Ac, CH3), 20.7 (C11, CH2), 19.6 (C29, CH3),
18.1 (C6, CH2), 16.4 (C24, CH3), 16.3 (C25, CH3), 16.1 (C26, CH3),
15.3 (C27, CH3), 7.2 (cycloprop., CH2), 7.0 (cycloprop., CH2) ppm;
MS (ESI, MeOH): m/z (%) = 592.5 (100% [M+H]+), 614.5 (70%
[M+Na]+); Anal. for C38H57NO4 (591.86): C, 77.11; H, 9.71; N,
2.37. Found: C, 77.08; H, 9.86; N, 2.18.
94%) was obtained as
= +37.4 (c 3.4, CHCl3); Rf = 0.68 (silica gel, hexane/ethyl acetate,
8:2); IR (KBr): = 3520br, 2944s, 1701s, 1637m, 1451m, 1379m,
1283m, 1029m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.75–5.72
a colourless solid; mp 220–222 °C;
½ ꢂ
a 2D0
m
;
(m, 2H, CH (31) + CH (32)), 4.86 (d, 1H, J = 7.3 Hz, CH (28)), 4.65
(d, 1H, J = 1.9 Hz, CHa (30)), 4.51 (br s, 1H, CHb (30)), 4.40 (dd, 1H,
J = 11.4, 4.8 Hz, CHOAc (3)), 4.34 (dd, 1H, J = 12.4, 5.6 Hz, CH (33)),
4.11 (dd, 1H, J = 12.4, 4.0 Hz, CH (33)), 2.85 (ddd, 1H, J = 11.2,
10.4, 7.0 Hz, CH (19)), 2.05 (ddd, 1H, J = 12.2, 12.1, 3.4 Hz, CH
(13)), 1.98–1.85 (m, 2H, CHa (21) + CHa (22)), 1.97 (s, 3H, Ac),
1.70–1.47 (m, 6H, CHa (12) + CH (18) + CHa (1) + CH2 (2) + CHa
(16)), 1.62 (s, 3H, CH3 (29)), 1.44–1.02 (m, 12H, CH2 (6) + CHa
(15) + CH2 (11) + CH2 (7) + CHb (21) + CH (9) + CHb (16) + CHb
(22) + CHb (12)), 1.00 (s, 3H, CH3 (26)), 0.95–0.80 (m, 2H, CHb
(15) + CHb (1)), 0.92 (s, 3H, CH3 (27)), 0.80 (s, 3H, CH3 (26)), 0.77
(s, 3H, CH3 (23)), 0.76 (s, 3H, CH3 (24)), 0.73 (d, 1H, J = 9.5 Hz, CH
(5)) ppm; 13C NMR (125 MHz, CDCl3): d = 171.0 (C@O), 151.2
(C20, C@CH2), 133.3 (C31, CH), 131.0 (C32, CH), 109.6 (C30, CH2@C),
80.9 (C3, CHOAc), 68.0 (C28, CH), 58.7 (OCH2), 55.3 (C5, CH), 50.3
(C9, CH), 49.8 (C18, CH), 49.3 (C17, Cquart.), 48.8 (C19, CH), 42.8
(C14, Cquart.), 40.9 (C8, Cquart.), 38.4 (C1, CH2), 37.8 (C4, Cquart.), 37.0
(C10, Cquart.), 36.9 (C13, CH), 34.2 (C7, CH2), 34.1 (C16, CH2), 32.9
(C22, CH2), 32.6 (C21, CH2), 27.9 (C23, CH3), 27.6 (C15, CH2), 25.1
(C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 21.0 (C11, CH2), 18.8
(C29, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3), 16.2 (C26, CH3), 16.1
(C25, CH3), 15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z (%) = 563.4
(70% [M+Na]+), 1103.2 (100% [2M+Na]+); Anal. for C35H56O4
(540.82): C, 77.73; H, 10.44. Found: C, 77.53; H, 10.66.
5.14. (4R) 3-(Pyrrolidyl)-4-[3b-hydroxy-28-norlup-20(29)-en-
17b-yl]-2-butenolide (11)
Following GP2, 11 (0.12 g, 38%) was obtained from 1 (0.30 g,
0.53 mmol) and pyrrolidine (0.14 g, 2.00 mmol) as a colourless so-
lid; mp 164–166 °C; ½a D20
ꢂ
= +2.7 (c 6.3, CHCl3); Rf = 0.36 (silica gel,
= 2948s, 2874m, 1735s,
chloroform/diethyl ether, 9:1); IR (KBr):
m
Acknowledgments
1641w, 1593s, 1456m, 1392m, 1378m, 1304m, 1246s, 1165m,
1031m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.16 (s, 1H, CH
;
We like to thank Mr. Harish Kommera and Dr. Reinhard Paschke
(Biosolutions GmbH, Halle) for their help with the preparation of
the liposomes and Mr. Ronny Sczepek (Organic Chemistry, Univer-
sität Halle-Wittenberg) for his help with the experimental data.
The cell lines were kindly provided by Dr. Thomas Müller (Dept.
of Haematology/Oncology, Universität Halle-Wittenberg).
(32)), 4.69 (s, 2H, CH (28) + CHa (30)), 4.51 (dd, 1H, J = 2.4, 1.4 Hz,
CHb (30)), 4.41 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.24–3.14 (m,
4H, NCH2), 2.98 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.22
(ddd, 1H, J = 12.2, 12.2, 3.6 Hz, CH (13)), 2.05–1.92 (m, 3H, 2CHa
(pyrrolid.) + CHa (16)), 1.97 (s, 3H, Ac), 1.88–1.71 (m, 5H, 2CHa
(pyrrolid.) + CHa (21) + CHa (15) + CH (18)), 1.68–1.52 (m, 5H, CHa
(1) + CHa (12) + CH2 (2) + CHa (22)), 1.61 (s, 3H, CH3 (29)), 1.47–
1.16 (m, 9H, CH2 (6) + CH2 (7) + CH2 (11) + CHb (16) + CH (9) + CHb
(21)), 1.09–0.88 (m, 3H, CHb (15) + CHb (22) + CHb (12) + CHb (1)),
1.00 (s, 3H, CH3 (26)), 0.97 (s, 3H, CH3 (27)), 0.80 (s, 3H, CH3
(25)), 0.78 (s, 3H, CH3 (23)), 0.77 (s, 3H, CH3 (24)), 0.75 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 173.6
(C@O), 171.6 (C31, C@C), 171.0 (C@O), 150.9 (C20, C@CH2), 109.5
(C30, CH2@C), 86.4 (C28, CH), 81.4 (C32, CH), 80.8 (C3, CHOAc),
55.4 (C5, CH), 52.4 (C18, CH), 51.6 (NCH2), 51.2 (C17, Cquart.), 50.2
(C9, CH), 47.2 (C19, CH), 42.8 (C14, Cquart.), 41.0 (C8, Cquart.), 38.3
(C1, CH2), 37.8 (C4, Cquart.), 37.0 (C10, Cquart.), 36.5 (C13, CH), 35.5
(C22, CH2), 34.3 (C7, CH2), 33.4 (C16, CH2), 32.4 (C21, CH2), 28.0
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