Alkylidene branched lupane derivatives: Synthesis and antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2012.04.023

Several novel alkylidene branched lupane derivatives have been prepared. Many of these compounds showed a significant cytotoxicity. The most active compound, 2-methylene-betulonic acid, showed IC₅₀ values between 0.2 and 0.6 μM for 15 different human cancer cell lines. Cytotoxicity can be improved by encapsulation in liposomes. These compounds act by triggering apoptotic cell death as shown by DNA-laddering experiments and acridine orange/ethidium bromide staining.

Full text of DOI:10.1016/j.ejmech.2012.04.023

European Journal of Medicinal Chemistry 53 (2012) 337e345
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Alkylidene branched lupane derivatives: Synthesis and antitumor activity
*
René Csuk , Sebastian Stark, Christoph Nitsche, Alexander Barthel, Bianka Siewert
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 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 alkylidene branched lupane derivatives have been prepared. Many of these compounds
Received 22 February 2012
Received in revised form
16 April 2012
Accepted 17 April 2012
Available online 25 April 2012
showed a significant cytotoxicity. The most active compound, 2-methylene-betulonic acid, showed IC50
values between 0.2 and 0.6 mM for 15 different human cancer cell lines. Cytotoxicity can be improved by
encapsulation in liposomes. These compounds act by triggering apoptotic cell death as shown by DNA-
laddering experiments and acridine orange/ethidium bromide staining.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Betulonic acid
Methylene compounds
Antitumor activity
Apoptosis
1. Introduction
decided to incorporate the a,b-unsaturated functional group within
the triterpenoid skeleton. A compound meeting these require-
ments can be obtained by an oxidation of the secondary hydroxyl
group at carbon C(3) in betulin followed by a subsequent meth-
ylenation reaction at the adjacent carbon C(2).
The natural occurringlupane-typetriterpene betulin(1, Scheme 1)
is widely spread inplants. Betulin and derivatives show antiviral [1,2],
antiplasmodial [3] and anti-inflammatory [1] activity. Many of its
derivatives [4e7] inhibit the growth of human tumor cell lines by
triggering apoptosis [8e10].
2. Results
Several structureeactivity relationships have been established
for betulin derivatives. Thus, the presence of a carboxylic or
carbonylic group at carbon C28 seems mandatory for high cyto-
toxicity as well as the oxidation of hydroxyl group at carbon C3
afforded derivatives showing higher cytotoxicity. The same is true
for introducing an unsaturation between carbons C20/C29. Acyla-
tion and carbon C3 and/or esterification at C28 diminish cytotox-
icity. Oxidation of betulin either at position C3 and/or at C30 affects
its binding to topoisomerases I/II, and hence to derivatives
providing higher cytotoxicity [11].
2.1. Chemistry
Commercially available betulin (1) was oxidized to betulonal (2)
using Swern oxidation conditions (Scheme 1), and 2 was obtained
in >90% yield. Betulonic acid (3) was obtained from 1 by Jones
oxidation. Jones oxidation of 1 followed by an esterification yielded
methyl betulonate (4) [12,13].
Previously, methylenation in a-position of a cyclohexanone has
been performed by several different approaches, among them
Previously we could show that betulin derivatives holding an
cascade syntheses [14], the use of silyl enolethers [15e17], frag-
extra a,b-unsaturated functional group display stronger cytotox-
icity toward human tumor cell lines than their parent compounds
[12,13]. It is assumed that this improved cytotoxic effect is con-
mentation of
zirconocene [19] or palladium catalysts [20,21]. One of the oldest
approaches to -alkylidene cycloalkanones, however, has been
g-oxosulfonium methylides [18] and the use of several
a
nected to the reaction of the a,b-unsaturated functional group in
described as early as 1920 by Carl Mannich [22,23]. Recently, an
interesting variation has been described using ionic liquids [24].
There are no precedences for Mannich-type reactions using the
CeH acidic protons at C(2) of a triterpenoid skeleton. Treatment of
2 with dimethylammonium hydrochloride, aq. formalin in tBuOH at
60 ꢀC for 5 days did not result in forming the corresponding
Mannich base. Formation of this product was invariably followed
Michael addition reactions with suitable intracellular targets.
Further, to improve the solubility of betulin (which is a noto-
rious problem in screening and administration of betulin) we
* Corresponding author. Tel.: þ49 (0) 345 55 25660; fax: þ 49 (0) 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
by a
b-elimination reaction, and 2-methylene compound 5 was
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.023

338
R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
Scheme 1. a) (COCl)2, DCM, DMSO, NEt3, 90%; b) CrO3, H2SO4, 1 h, 25 ꢀC, 52%; c) K2CO3, acetone, MeI, 24 h, 25 ꢀC, 94%, d) NHMe2.HCl, aq. HCHO, t.BuOH, 60 ꢀC, 5 days, 75%; e)
NHMe2.HCl, aq. HCHO, t.BuOH, 60 ꢀC, 3 days, 60%; f) K2CO3, paraformaldehyde, DMF, 90 ꢀC, 1 h, 59%; g) NHMe2.HCl, aq. HCHO, t.BuOH, 60 ꢀC, 6 days, 54%; h) paraformaldehyde,
K2CO3, DMF, 90 ꢀC, 1 h, 71%.
obtained. Similar treatment of acid 3 or ester 4 invariably gave the
C-2 methylene substituted compounds 6 and 7, respectively.
Treatment of 4 with dimethylammonium hydrochloride and para-
formaldehyde in glacial acetic acid, however, allowed to isolate the
Mannich base 8. On standing in water or phosphate buffer,
From these experiments we assumed the aldol condensation
might be a useful method for obtaining C-2 alkylidene branched
derivatives. Previously, it has been shown that for the reaction of 4
with benzaldehyde or furfural [25] the products of an aldol
condensation could be obtained. These products, however, did not
compound 8 decomposes rapidly, and 7 is formed by a
b-elimina-
show any significant cytotoxicity (IC50 > 30 mmol). Treating 3 with
tion reaction. The absolute configuration at carbon C(2) in
compound 8 was determined to be (S) with an axially oriented
hydrogen substituent at C(2) by extensive 1H NMR NOESY experi-
ments (Fig. 1). In the 1H NOESY spectra HeC(2) shows three long-
range couplings with the proton HeC(1) and protons HaxeC(23)
and HaxeC(25).
paraformaldehyde in the presence of finely grounded potassium
carbonate gave the methylene compound 6. Similarly, from 4
compound 7 was obtained. Treatment of 4 with acetaldehyde/LDA
gave (2E)-2-ethylidene 9 (Scheme 2) in 67% yield; from 4 and
acrolein/LDA (2Z)-2-prop-2-enylidene 10 was obtained. The
assignment of the absolute configuration of the alkenic bond was
performed again using 1H NMR NOESY spectra. Thus, in 9 HeqeC(1)
shows a long-range coupling with the methyl protons of the eth-
ylidene moiety, whereas in 10 no long-range couplings for
H
eqeC(1) can be found e therefore paralleling previous assign-
ments made for the respective benzylidene or furfurylidene
derivatives [25].
Treatment of 6 with oxalyl chloride followed by the reaction
with thiomorpholine yielded the ester 11 (Scheme 3). Michael
reaction of 7 with 2-mercaptoethanol in the presence of DBU gave
the 2-hydroxyethylmethylthio compound 12 whose oxidation with
hydrogen peroxide in glacial acetic acid yielded the sulfonyl
compound 13. Similarly, by a Michael reaction of 7 with nitro-
methane/DBU compound 14 was obtained.
Fig. 1. Assignment of the absolute configuration at C(2) from 1H NOESY NMR
experiments.
The absolute configuration of the newly created stereogenic
center C(2) in compounds 11e13 was again deduced from the 1H

R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
339
Fig. 2. Left: Results from the AO/EB test: treatment of A549 lung carcinoma cells with 6 (0.8 mM); right: DNA-laddering (colon cancer cell line SW480; after treatment with IC90
concentration of 6 for 24 h).
NOESY NMR spectra. A [2 þ 3] cycloaddition reaction of 7 with
diazomethane gave spiro-compound 15.
an excellent cytotoxicity with the ester 7 being weakest in activity
and the acid 5 showing IC50 values between 0.2 and 0.6 mol for the
m
different tumor cell lines. Cytotoxicity drops for the alkylidene
compounds 9 and 10 significantly.
2.2. Biology
The weaker activity of 9 and 10 compared to 7 might be
explained by steric hindrance, the higher activity of acid 6
compared to ester 7 parallels previous findings showing that
betulinic esters show a reduced cytotoxicity compared to the free
acid.
The compounds 5e15 were tested for their cytotoxicity against
15 human cancer cell lines and mouse fibroblasts NiH3T3 using the
colorimetric SRB protocol [26e28]. The summarized IC50 values in
Table 1 were obtained from the doseeresponse curves.
Compound 14 showed no sufficient solubility in the test,
compound 8 was too unstable, and 15 was regarded inactive
(IC50 > 30 mmol). All the C(2)-methylene derivatives 5e7 showed
Betulin and derivatives often show only poor solubility in water;
hence, their bioavailability is reduced. To overcome this limit we
encapsulated ester 7 using a commercially available liposome
Scheme 2. a) LDA, MeCHO, THF, ꢁ78 / 25 ꢀC, 67%; b) NHMe2.HCl, paraformaldehyde, HOAc, 80 ꢀC, 30 min, 47%; c) LDA, acrolein, THF, ꢁ78 / 25 ꢀC, 63%; d) H2O, 40 ꢀC, 6 h, quant.

340
R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
Scheme 3. a) (COCl)2, toluene, NEt3, DMF, 30 min, 25 ꢀC, then thiomorpholine, NEt3, 100 ꢀC, 3 h, 55%; b) HSeCH2eCH2eOH, DBU, DCM, 25 ꢀC, 48 h, 60%; c) MeNO2, DBU, DCM, 25 ꢀC,
5 days, 66%; d) CH2N2, Et2O, 25 ꢀC, 3 days, 32%; e) H2O2, AcOH, 25 ꢀC, 30 min, 83%.
formulation [29]. Best results were obtained with soybean lecithin
(Lipoid S75). Subsequent extrusion through polycarbonate
membrane [30] with a pore size of 100 nm using a LiposoFast
system gave liposomes with a hydrodynamic diameter between 70
and 120 nm (Z-average 105 nm) as determined by dynamic light
scattering. Encapsulation was >96% as determined by HPLC. Their
physical stability exceeds 4 weeks. Applicating compound 7 in
liposomes in the SRB assay revealed an extra benefit from encap-
sulation by observing an increased cytotoxicity for all the cell lines.
For the products resulting from the Michael addition reactions
only compound 12 showed a good cytotoxicity; this compound is
roughly three times more active than its parent compound 7.
Cytoxicity becomes weaker for oxidized 13. Hence, the presence of
a
Table 1
Cytotoxicity in a panel of various human cancer cell lines.
an a,b-unsaturated system in ring A is not a prerequisite for high
cytotoxicity, However, it cannot ruled out that 12 is transformed in
the cells back into compound 7, and the higher cytotoxicity asso-
ciated with compound 12 is indicative for a higher intracellular
concentration of 12. In consequence, compound 12 would be able
to act as a pro-drug for ester 7 or acid 6.
Compounds 6, 7 and 12 were investigated regarding their
potential to induce apoptosis. All of these compounds showed
DNA-laddering (using the colon cancer cell line SW480), hence
giving a good indication for apoptosis [31]. These compounds were
also tested in an acridine orange/ethidium bromide (AO/EB) assay;
all compounds showed green fluorescence in the AO/EB tests,
which indicate apoptosis (Fig. 2).
cell line
IC50 values in mM for cancer cell lines
5
6
7
7L
9
10
11
12
13
518A2
A431
A253
FADU
A549
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
3.8
3.0
1.0
5.1
1.8
1.3
3.7
7.1
3.2
7.1
2.7
1.3
2.1
2.1
2.7
4.7
0.5
0.2
0.4
0.5
0.6
0.6
0.4
0.2
0.2
0.4
0.4
0.6
0.4
0.3
0.6
0.8
4.2
4.9
4.0
7.7
5.1
5.1
6.4
9.0
4.5
5.9
4.5
5.0
4.9
4.4
4.6
4.9
1.6
1.6
1.5
2.6
2.3
2.5
2.6
2.1
2.2
2.2
1.5
2.3
1.9
1.8
1.9
2.3
>30
26.4
11.5
13.0
10.4
17.0
13.0
11.2
14.2
14.0
14.6
10.9
10.5
11.3
15.0
11.2
11.6
13.1
1.5
1.4
1.4
1.6
1.5
1.0
1.5
1.5
1.3
1.6
1.5
1.5
1.5
1.4
1.5
1.9
1.5
1.7
1.5
2.5
2.1
1.6
2.5
2.5
1.9
1.4
1.5
2.3
2.1
1.5
1.8
2.1
2.6
2.8
2.5
3.8
3.3
2.6
4.4
4.0
2.8
2.5
2.6
3.4
3.3
2.6
2.7
3.2
21.6
22.4
>30
16.1
>30
17.2
24.0
>30
16.0
26.4
21.9
27.2
27.8
29.1
3. Conclusions
NiH3T3
We have examinated betulonates bearing an exo-alkylidene
moiety at C(2). These compounds are easily synthesized either by
Mannich reactions followed by b-elimination or by aldol conden-
sation reactions. These compounds showed promising cytotoxicity
for 15 human tumor cell lines; they are also valuable starting
materials for following Michael addition reactions. Encapsulation of
one of these compounds increased cytotoxicity by a factor of three.
Values are derived from doseeresponse curves obtained by measuring the
percentage of viable cells relative to untreated controls after 96 h exposure of the
test compounds to the cell line 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, SW480), anaplastic
thyroid cancer (8505c, SW1736), mamma carcinoma (MCF-7), liposarcoma (LIPO)
and mouse fibroblasts (NiH3T3). Values are the average from at least three inde-
pendent experiments. Variation was generally ꢂ 10%; 7L corresponds to compound
7 liposome encapsulated.
The presence of an
a,b-unsaturated system in ring A is not

R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
341
a prerequisite for an excellent cytotoxicity. Increased cytotoxicity
4.4. 2-Methylene-3-oxolup-20(29)en-28-oic acid (6)
was observed also for a 2-hydroxyethylthio-derivative; the activity
of this compound to induce cell death might result from a higher
intracellular concentration of the compound. The results from
DNA-laddering experiments and an AO/EB assay provided evidence
for an apoptotic cell death.
4.4.1. Mannich reaction
A solution of 3 (1.35 g, 2.90 mmol), dimethylammonium
hydrochloride (550 mg, 6.75 mmol) and aq. formalin (37%, 0.7 ml,
8.6 mmol) in t-butanol (15 ml) was stirred at 60 ꢀC for 3 days. The
solvents were removed under reduced pressure, and the residue
was subjected to chromatography (silica gel, n-hexane/ethyl
acetate, 9:1) to yield 6 (810 mg, 60%) as colorless solid.
4. Experimental
4.1. General
4.4.2. Aldol condensation
A mixture of finely grounded K2CO3 (7.0 g, 54.6 mmol), para-
formaldehyde (7.0 g, 0.23 mol) and 3 (3.3 g, 7.26 mmol) was sus-
pended in dry DMF (80 ml) and heated for 1 h at 90 ꢀC. After cooling
to 25 ꢀC, the mixture was poured into cold water (200 ml) and
extracted with ethyl acetate (3 ꢃ 100 ml). The extracts were washed
with brine (30 ml), dried (Na2SO4), and the solvents were removed
under diminished pressure. The residue was subjected to chroma-
tography (silica gel, n-hexane/ethyl acetate, 4:1) to yield 6 (2 g,
59.0%) as a colorless solid.
Instrumentation, cell lines and culture conditions, SRB cyto-
toxicity assay, AO/EB staining and DNA fragmentation assay as
described previously [26e28,31].
4.2. Preparation of the liposomes
Unilamellar liposomes of approximately 100 nm diameter were
obtained by the method of Olson [30] employing a laboratory
extruder (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 phosphatidylcholine
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 poly-
carbonate filter of 100 nm pore size. Twenty-one cycles were
applied and concentration of the compound was determined by
4.4.3. Data for 6
M.p. 242e245 ꢀC; RF ¼ 0.47 (n-hexane/ethyl acetate, 8:2);
[a
]
D
¼ þ39.3ꢀ (c ¼ 5.40, CHCl3); IR (KBr):
¼ 3069 m, 2943 s, 2872 s,
n
1690 s, 1639 w, 1602 m, 1459 m, 1398 w, 1380 m, 1358 w, 1316 w,
1290 w, 1229 m, 1196 w, 1152 w, 1111 w, 1084 w, 1060 m cmꢁ1
;
UVevis (methanol): lmax (log ε) ¼ 218 (4.15), 252 (3.85) nm; 1H
NMR (500 MHz, CDCl3):
d
¼ 10.76 (br s, 1 H, COOH (28)), 5.95 (br m,
HPLC (RP18, 4.6 ꢃ 250,
l
¼ 230 nm, methanol, 1.3 ml). Liposomes
1 H, CHa (31)), 5.11 (br m, 1 H, CHb (31)), 4.73 (br m, 1 H, CHa (29)),
4.60 (br s, 1 H, CHb (29)), 3.00 (ddd, 1 H, J ¼ 10.6, 10.6, 4.5 Hz, CH
(19)), 2.68 (d, 1 H, J ¼ 15.3 Hz, CHa (1)), 2.31e2.18 (m, 2 H, CH
13 þ CHa (16)), 2.05 (d, 1 H, J ¼ 15.3 Hz, CHb (1)), 2.04e1.91 (m, 2 H,
CHa (21) þ CHb (22)), 1.77e1.66 (m, 1 H, CHa (12)), 1.68 (s, 3 H, CH3
(30)), 1.63 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.57e1.15 (m, 12 H, CH
(5) þ CH (9) þ CHa (15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2
(6) þ CH2 (7) þ CH2 (11)), 1.14e1.02 (m, 2 H, CHb (12) þ CHb (15)),
1.09 (s, 3 H, CH3 (23)), 1.01 (s, 3 H, CH3 (24)), 0.98 (s, 3 H, CH3 (27)),
0.97 (s, 3 H, CH3 (26)), 0.82 (s, 3 H, CH3 (25)) ppm; 13C NMR
were characterized by DLS.
4.3. 2-Methylene-3-oxolup-20(29)en-28-al (5)
A solution of 2 (097 g, 2.21 mmol), dimethylammonium
hydrochloride (200 mg, 2.45 mmol) and aq. formalin (37%, 0.3 ml,
3.76 mmol) in t-butanol (15 ml) was stirred at 60 ꢀC for 5 days. The
solvents were removed under reduced pressure, and the residue
was subjected to chromatography (silica gel, n-hexane/ethyl
acetate, 9:1) to yield 5 (975 mg, 75%) as a colorless foam; m.p.
(125 MHz, CDCl3):
d
¼ 207.6 (C3, C]O), 182.5 (C28, COOH), 150.3
205 ꢀC (decomp.); RF
¼
0.78 (n-hexane/ethyl acetate, 4:1);
(C20, C]CH2), 142.2 (C2, C]CH2), 123.7 (C31, CH2), 109.8 (C29,
CH2), 56.4 (C17, Cquart.), 54.0 (C5, CH), 49.2 (C9, CH), 48.4 (C18, CH),
47.1 (C1, CH2), 46.9 (C19, CH), 45.8 (C4, Cquart.), 42.5 (C14, Cquart.),
40.5 (C8, Cquart.), 38.5 (C13, CH), 37.0 (C10, Cquart.), 36.9 (C22, CH2),
33.4 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.7 (C15, CH2), 28.1
(C23, CH3), 25.5 (C12, CH2), 22.3 (C24, CH3), 21.4 (C11, CH2), 20.1 (C6,
CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.5 (C25, CH3), 14.6 (C27,
CH3) ppm; MS (ESI, MeOH): m/z ¼ 956.1 (40% [2M þ Na]þ), 521.3
(75% [M þ Na þ MeOH]þ); 1399.5 (15% [3MeH]ꢁ), 954.3 (65%
[
a]
¼ þ56.6ꢀ (c ¼ 5.70, CHCl3); IR (KBr):
n
¼ 3432 m, 2945 s,
D
2868 m, 1718 m, 1687 m, 1642 m, 1454 m, 1377 m, 1246 m, 1141 w,
1056 m, 967 w, 884 m cmꢁ1; UVevis (MeOH): lmax (nm)
(log ε) ¼ 232 (0.44); 1H NMR (500 MHz, CDCl3):
d
¼ 9.67 (d, 1 H,
J ¼ 1.5 Hz, CH (28)), 5.96 (m, 1 H, CHa (31)), 5.12 (m, 1 H, CHb (31)),
4.76 (m, 1 H, CHa (29)), 4.64 (m, 1 H, CHb (29)), 2.88 (ddd, 1 H,
J ¼ 11.4, 11.2, 5.8 Hz, CH (19)), 2.69 (d, 1 H, J ¼ 15.1 Hz, CHa (1)),
2.11e2.03 (m, 3 H, CHb (1) þ CH (13) þ CHa (21)), 1.93e1.18 (m, 17 H,
CHa (16) þ CHa (15) þ CHa (12) þ CHb (16) þ CH (18) þ CH (9) þ CHa
(22) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (21) þ CHb (15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6) þ CH (5)), 1.70 (s, 3 H, CH3 (30)),
1.12e1.03 (m, 1 H, CHb (12)), 1.10 (s, 3 H, CH3 (24)), 1.04 (s, 3 H, CH3
(23)), 1.00 (s, 3 H, CH3 (27)), 0.96 (s, 3 H, CH3 (25)), 0.83 (s, 3 H, CH3
[2M
þ
Nae2H]ꢁ), 931.7 (75% [2MeH]ꢁ), 497.8 (20%
[M þ MeOHeH]ꢁ), 465.6 (100% [M-H]ꢁ); analysis for C31H46O3
(466.70): C, 79.78; H, 9.93; found: C, 79.54; H, 10.11.
4.5. Methyl 2-methylene-3-oxolup-20(29)en-28-oate (7)
(26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 207.5 (C3, CO), 206.4
(C28, CHO), 149.6 (C20, C]CH2), 142.2 (C2, C]CH2), 123.6 (C31, C]
CH2), 110.2 (C29, C]CH2), 59.3 (C17, Cquart.), 54.0 (C5, CH), 48.3 (C9,
CH), 47.9 (C18, CH), 47.4 (C19, CH), 47.1 (C1, CH2), 45.8 (C4, Cquart.),
42.6 (C14, Cquart.), 40.7 (C8, Cquart.), 38.7 (C13, CH), 36.9 (C10, Cquart.),
33.4 (C7, CH2), 33.1 (C16, CH2), 29.8 (C15, CH2), 29.1 (C21, CH2), 28.8
(C22, CH2), 28.1 (C24, CH3), 25.5 (C12, CH2), 22.4 (C23, CH3), 21.3
(C11, CH2), 20.1 (C6, CH2), 19.0 (C30, CH3), 15.6 (C25, CH3), 15.5 (C26,
CH3), 14.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 923.4 (86%
4.5.1. Mannich reaction
A solution of 4 (938 mg, 2.06 mmol), dimethylammonium
hydrochloride (150 mg, 1.84 mmol) and aq. formalin (37%, 0.4 ml,
4.9 mmol) in t-butanol (15 ml) was stirred at 60 ꢀC for 6 days. The
solvents were removed under reduced pressure, and the residue
was subjected to chromatography (silica gel, n-hexane/ethyl
acetate, 9:1) to yield 7 (540 mg, 54%) as a colorless foam.
[2M
þ
Na]þ), 901.5 (12% [2M
þ
H]þ), 505.1 (77%
4.5.2. Aldol condensation
A mixture of finely grounded K2CO3 (2.5 g, 18.1 mmol), para-
formaldehyde (2.5 g, 83.3 mmol) and 4 (2.0 g, 4.3 mmol) was
[M þ Na þ MeOH]þ), 451.4 (18% [M þ H]þ); analysis for C31H46O2
(450.70): C, 82.61; H, 10.29; found: C, 82.52; H, 10.37.

342
R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
suspended in dry DMF (30 ml) and heated for 1 h at 90 ꢀC. After
cooling to 25 ꢀC, the mixture was poured into cold water (200 ml)
and extracted with ethyl acetate (3 ꢃ 100 ml). The extracts were
washed with brine (30 ml), dried (Na2SO4), and the solvents were
removed under diminished pressure. The residue was subjected to
chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 7
(1.45 g, 70.7%) as a colorless solid.
NMR (125 MHz, CDCl3):
d
¼ 217.3 (C3, CO), 176.6 (C28, CO), 150.4
(C20, C]CH2), 109.7 (C29, C]CH2), 59.9 (C32, CH2), 57.3 (C5, CH),
56.4 (C17, Cquart.), 51.2 (C31, CH3), 50.0 (C9, CH), 49.4 (C18, CH), 48.4
(C4, Cquart.), 47.0 (C19, CH), 46.2 (C1, CH2), 45.8 (C33 þ C34, 2 ꢃ CH3),
42.5 (C14, Cquart.), 40.7(C8, Cquart.), 40.3 (C2, CH), 38.3 (C13, CH), 37.3
(C10, Cquart.), 36.9 (C22, CH2), 34.1 (C7, CH2), 32.1 (C16, CH2), 30.5
(C21, CH2), 29.6 (C15, CH2), 25.4 (C12, CH2), 25.3 (C24, CH3), 21.6
(C23, CH3), 21.2 (C11, CH2), 19.3 (C6, CH2), 19.2 (C30, CH3), 16.1 (C25,
CH3), 16.0 (C26, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/
z ¼ 526.4 (100% [M þ H]þ); analysis for C34H55NO3 (525.81): C,
77.66; H, 10.54; N, 2.66; found: C, 77.57; H, 10.76; N, 2.42.
4.5.3. Data for 7
M.p. 152e155 ꢀC (decomp.); RF ¼ 0.83 (n-hexane/ethyl acetate,
4:1); [
a
]
¼ þ69.5ꢀ (c ¼ 2.6, CHCl3); IR (KBr):
n
¼ 3435 m, 2949 s,
D
2869 m, 1729 s, 1690 m, 1643 m, 1453 m, 1377 m, 1318 m, 1266 w,
1188 m, 1155 s, 1135 s, 1061 m, 1010 m, 985 m, 883 m cmꢁ1; UVevis
(methanol): lmax (nm) (log ε) ¼ 238 (0.40), 205 (1.90); 1H NMR
4.7. Methyl (2 E) 2-ethyliden-3-oxolup-20(29)en-28-oate (9)
(500 MHz, CDCl3):
d
¼ 5.95 (m,1 H, CHa (32)), 5.10 (m,1 H, CHb (32)),
To a freshly prepared solution of LDA [from diisopropylamine
(200 mg, 2.0 mmol) and n-butyllithium (1.0 ml, 1.6 mmol, 1.6 M in
n-hexane) in dry THF (10 ml)] at ꢁ78 ꢀC a solution of 4 (300 mg,
0.64 mmol) in THF (5 ml) was added, and stirring was continued for
another hour. A solution of acetaldehyde (150 mg, 3.41 mmol) in
THF (5 ml) was slowly added, and the mixture was allowed to warm
to 25 ꢀC. The reaction was quenched by the careful addition of aq.
hydrochloric acid (150 ml, 1.0 M) and extracted with ethyl acetate
(3 ꢃ 100 ml). The organic layers were washed with brine (100 ml),
dried (Na2SO4), and the solvent was removed. The residue was
subjected to chromatography (silica gel, n-hexane/ethyl acetate,
9:1) to yield 9 (210 mg, 66.6%) as a colorless solid; m.p. 140e142 ꢀC;
4.72 (m,1 H, CHa (29)), 4.58 (m,1 H, CHb (29)), 3.66 (s, 3 H, CH3 (31)),
2.99 (m, 1 H, CH (19)), 2.67 (d, 1 H, J ¼ 15.3 Hz, CHa (1)), 2.29e2.19
(m, 2 H, CHa (16) þ CH (13)), 2.05 (d, 1 H, J ¼ 15.3 Hz, CHb (1)),
1.93e1.81 (m, 2 H, CHa (21) þ CHa (22)), 1.76e1.64 (m, 1 H, CHa (12)),
1.68 (s, 3 H, CH3 (30)), 1.60 (dd, 1 H, J ¼ 11.5, 11.5 Hz, CH (18)),
1.53e1.14 (m, 13 H, CH (9) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb
(16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb
(6) þ CHb (15) þ CH (5)), 1.12e1.01 (m, 1 H, CHb (12)), 1.08 (s, 3 H,
CH3 (24)), 1.03 (s, 3 H, CH3 (23)), 0.97 (s, 3 H, CH3 (27)), 0.95 (s, 3 H,
CH3 (25)), 0.83 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 207.5 (C3, CO), 176.6 (C28, CO), 150.3 (C20, C]CH2), 142.3 (C2,
C]CH2),123.6 (C31, C]CH2),109.8 (C29, C]CH2), 56.5 (C17, Cquart.),
53.9 (C5, CH), 51.2 (C31, CH3), 49.4 (C18, CH), 48.4 (C9, CH), 47.1 (C1,
CH2), 46.9 (C19, CH), 45.8 (C4, Cquart.), 42.4 (C14, Cquart.), 40.6 (C8,
Cquart.), 38.3 (C13, CH), 36.9 (C10, CH2), 36.9 (C22, Cquart.), 33.5 (C7,
CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.0 (C24,
CH3), 25.5 (C12, CH2), 22.4 (C23, CH3), 21.4 (C11, CH2), 20.0 (C6,
CH2), 19.3 (C30, CH3), 15.6 (C25, CH3), 15.4 (C26, CH3), 14.6 (C27,
CH3); MS (ESI, MeOH): m/z ¼ 983.2 (72% [2M þ Na]þ), 535.3 (60%
[M þ H þ MeOH]þ), 481.4 (100% [M þ H]þ); analysis for C32H48O3
(480.72): C, 79.95; H, 10.06; found: C, 79.73; H, 10.16.
RF ¼ 0.75 (n-hexane/ethyl acetate, 8:2); [
a]
D
¼ þ 57.4ꢀ (c ¼ 4.65,
CHCl3); IR (KBr):
n
¼ 3432 s, 2949 s, 2869 m, 1728 s, 1683 m, 1618 m,
1457 m, 1380 m, 1188 m, 1153 m, 1133 m cmꢁ1; UVevis (methanol):
lmax (log ε) ¼ 218 (3.96), 263 (3.78) nm; 1H NMR (500 MHz, CDCl3):
d
¼ 6.65 (ddq, 1 H, J ¼ 7.5, 2.5, 1.7 Hz, CH (32)), 4.74 (d, 1 H, J ¼ 1.7 Hz,
CHa (29)), 4.60 (br s, 1 H, CHb (29)), 3.66 (s, 3 H, CH3 (31)), 3.00 (ddd,
1 H, J ¼ 10.8, 10.8, 4.3 Hz, CH (19)), 2.65 (d, 1 H, J ¼ 16.1 Hz, CHa (1)),
2.32e2.19 (m, 2 H, CH (13) þ CHa (16)), 1.94e1.84 (m, 2 H, CHa
(21) þ CHa (22)), 1.82 (d, 1 H, J ¼ 16.1 Hz, CHb (1)), 1.78e1.73 (m, 1 H,
CHa (12)), 1.68 (s, 3 H, CH3 (30)), 1.66 (d, 3 H, J ¼ 7.5 Hz, CH3 (33)),
1.62 (dd, 1 H, J ¼ 11.5, 11.5 Hz, CH (18)), 1.58e1.21 (m, 12 H, CH
(5) þ CH (9) þ CHa (15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2
(6) þ CH2 (7) þ CH2 (11)), 1.20e1.01 (m, 2 H, CHb (12) þ CHb (15)),
1.06 (s, 3 H, CH3 (24)), 1.03 (s, 3 H, CH3 (23)), 0.99 (s, 3 H, CH3 (27)),
0.95 (s, 3 H, CH3 (25)), 0.76 (s, 3 H, CH3 (26)) ppm; 13C NMR
4.6. Methyl (2a)-2-[(dimethylamino)methyl]-3-oxolup-20(29)en-
28-oate (8)
A solution of 4 (1.01 g, 2.15 mmol), dimethylammonium
hydrochloride (800 mg, 9.81 mmol) and paraformaldehyde
(300 mg, 10.0 mmol) in glacial acetic acid (5 ml) was stirred at 80 ꢀC
for 30 min. After cooling to 0 ꢀC, an aq. solution of Na2CO3 (satd.,
50 ml) was added dropwise, and the mixture was extracted with
ethyl acetate (3 ꢃ 75 ml). The solvent was stripped off under
reduced pressure, and the remaining yellowish product dissolved
in hot pentane (15 ml), insoluble material was filtered off, and the
solvent was removed. Compound 8 (1.06 g, 94%) was obtained as
a colorless, amorphous solid; m.p: 76e79 ꢀC; RF ¼ 0.07 (n-hexane/
(125 MHz, CDCl3):
d
¼ 207.6 (C3, C]O), 176.6 (C28, COOCH3), 150.5
(C20, C]CH2), 135.8 (C32, CH), 135.2 (C2, C]CH), 109.6 (C29, CH2),
56.5 (C17, Cquart.), 53.0 (C5, CH), 51.2 (C31, CH3), 49.4 (C18, CH), 48.5
(C9, CH), 46.9 (C19, CH), 44.9 (C4, Cquart.), 42.4 (C1, CH2), 42.4 (C14,
Cquart.), 40.4 (C8, Cquart.), 38.4 (C13, CH), 36.9 (C22, CH2), 36.0 (C10,
Cquart.), 33.2 (C7, CH2), 32.0(C16, CH2), 30.5 (C21, CH2), 29.6 (C15,
CH2), 29.1 (C23, CH3), 25.6 (C12, CH2), 22.1 (C24, CH3), 21.6 (C11,
CH2), 20.3 (C6, CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.4 (C25,
CH3), 14.6 (C27, CH3), 13.6 (C33, CH3) ppm; MS (ESI, MeOH): m/
z ¼ 549.0 (79% [M þ Na þ MeOH]þ), 495.4 (100% [M þ H]þ);
analysis for C33H50O3 (494.75): C, 80.11; H,10.19; found: C, 79.95; H,
10.26.
ethyl acetate, 4:1); [
a
]
¼ ꢁ5.8ꢀ (c ¼ 4.10, CHCl3); IR (KBr):
D
n
¼ 3432 m, 2947 s, 2869 m, 2764 m,1729 s,1703 m,1641 w,1458 m,
1378 m, 1158 m, 963 w cmꢁ1; UVevis (methanol): lmax (nm)
(log ε) ¼ 300 (0.05), 258 (0.30), 225 (1.65); 1H NMR (500 MHz,
CDCl3):
d
¼ 4.66 (m, 1 H, CHa (29)), 4.52 (m, 1 H, CHb (29)), 3.60 (s, 3
4.8. (2 Z) methyl 3-oxo-2-prop-2-en-1-ylid-lup-20(29)en-28-oate
(10)
H, CH3 (31)), 2.93 (ddd, 1 H, J ¼ 10.8, 10.8, 4.3 Hz, CH (19)), 2.73 (m, 1
H, CH (2)), 2.58 (dd, 1 H, J ¼ 12.4, 5.0 Hz, CHa (32)), 2.21e2.09 (m, 4
H, CH (13) þ CHa (16) þ CHb (32) þ CHa (1)), 2.10 (s, 6 H, 2 ꢃ CH3
(33) þ (34)), 1.86e1.77 (m, 2 H, CHa (21) þ CHa (22)), 1.69e1.15 (m,
12 H, CH (18) þ CHa (12) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb
(16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb
(6)), 1.60 (s, 3 H, CH3 (30)), 1.50 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (9)),
1.10e1.01 (m, 3 H, CH (5) þ CHb (15) þ CHb (12)), 0.89e0.80 (m, 1 H,
CHb (1)), 1.02 (s, 3 H, CH3 (25)), 0.99 (s, 3 H, CH3 (23)), 0.97 (s, 3 H,
CH3 (24)), 0.91 (s, 3 H, CH3 (26)), 0.87 (s, 3 H, CH3 (27)) ppm; 13C
To a freshly prepared solution of LDA [from diisopropylamine
(200 mg, 2.0 mmol) and n-butyllithium (1.0 ml, 1.6 mmol, 1.6 M in
n-hexane) in dry THF (10 ml)] at ꢁ78 ꢀC a solution of 4 (300 mg,
0.64 mmol) in THF (5 ml) was added, and stirring was continued for
another hour. A solution of acrolein (150 mg, 2.7 mmol) in THF
(5 ml) was slowly added, and the mixture was allowed to warm to
25 ꢀC. The reaction was quenched by the careful addition of aq.
hydrochloric acid (150 ml, 1.0 M) and extracted with ethyl acetate

R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
343
(3 ꢃ 100 ml). The organic layers were washed with brine (100 ml),
dried (Na2SO4), and the solvent was removed. The residue was
subjected to chromatography (silica gel, n-hexane/ethyl acetate,
9:1) to yield 10 (320 mg, 63.2%) as a colorless solid; m.p.
(C20, C]CH2), 142.4 (C2, C]CH2), 123.5 (C31, CH2), 109.2 (C29,
CH2), 54.8 (C17, Cquart.), 54.1 (C5, CH), 52.8 (C18, CH), 48.8 (C9, CH),
48.0 (br, C32, CH2), 47.1 (C1, CH2), 45.9 (C4, Cquart.), 45.6 (C19, CH),
42.0 (C14, Cquart.), 40.5 (C8, Cquart.), 37.0 (C13, CH), 36.9 (C10, Cquart.),
35.9 (C22, CH2), 33.4 (C7, CH2), 32.4 (C16, CH2), 31.3 (C21, CH2), 29.9
(C15, CH2), 28.0 (C23, CH3), 27.7 (C33, CH2), 25.6 (C12, CH2), 22.4
(C24, CH3), 21.7 (C11, CH2), 20.1 (C6, CH2), 19.7 (C30, CH3), 15.7 (C26,
CH3), 15.5 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/
z ¼ 606.0 (46% [M þ Na þ MeOH]þ), 552.4 (100% [M þ H]þ);
analysis for C35H53NO2S (551.87): C, 76.17; H, 9.68; N, 2.54; S, 5.81
found: C, 76.00; H, 9.79; N, 2.41; S, 5.69.
175e180 ꢀC; RF ¼ 0.79 (n-hexane/ethyl acetate, 8:2); [
a]
¼ þ77.5ꢀ
D
(c ¼ 5.11, CHCl3); IR (KBr):
n
¼ 3435 m, 3072 w, 2950 s, 2869 s,
1728 s, 1684 m, 1642 w, 1456 m, 1380 m, 1318 w, 1188 s, 1156 s,
1133 s, 1082 m, 987 m cmꢁ1; UVevis (methanol): lmax (log ε) ¼ 218
(4.10), 259 (3.98) nm; 1H NMR (500 MHz, CDCl3):
d
¼ 6.92 (d, 1 H,
J ¼ 11.5 Hz, CH (32)), 6.45 (ddd, 1 H, J ¼ 16.8, 11.5, 10.2 Hz, CH (33)),
5.52 (br d, 1 H, J ¼ 16.8 Hz, CHa (34)), 5.41 (br d, 1 H, J ¼ 10.2 Hz, CHb
(34)), 4.69 (s, 1 H, CHa (29)), 4.55 (s, 1 H, CHb (29)), 3.61 (s, 3 H, CH3
(31)), 2.95 (ddd, 1 H, J ¼ 10.8, 10.8, 4.4 Hz, CH (19)), 2.81 (d, 1 H,
J ¼ 16.4 Hz, CHa (1)), 2.25e2.14 (m, 2 H, CH (13) þ CHa (16)), 1.91 (d,
1 H, J ¼ 16.4 Hz, CHb (1)), 1.88e1.78 (m, 2 H, CHa (21) þ CHa (22)),
1.75e1.67 (m, 1 H, CHa (12)), 1.63 (s, 3 H, CH3 (30)), 1.56 (dd, 1 H,
J ¼ 11.3, 11.3 Hz, CH (18)), 1.53e1.16 (m, 12 H, CH (5) þ CH (9) þ CHa
(15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2
(11)), 1.16e0.96 (m, 2 H, CHb (12) þ CHb (15)), 1.03 (s, 3 H, CH3 (24)),
1.00 (s, 3 H, CH3 (23)), 0.93 (s, 3 H, CH3 (27)), 0.90 (s, 3 H, CH3 (25)),
4.10. Methyl (2a) 2-{[(2-hydroxyethyl)thio]methyl}-3-oxolup-
20(29)en-28-oate (12)
To a solution of 7 (480 mg, 1.0 mmol) and 2-mercaptoethanol
(0.9 ml, 1.0 g, 12.8 mmol) in dichloromethane (10 ml), DBU
(0.49 ml, 0.50 g, 3.3 mmol) was added, and the mixture was
stirred for 48 h at 25 ꢀC. After dilution with water (200 ml) and
extraction with ethyl acetate (3 ꢃ 100 ml), the organic phase was
washed with aq. hydrochloric acid (100 ml, 0.5 M), aq. NaHCO3-
Lösung (100 ml) and brine (100 ml). The extract was dried
(Na2SO4), the solvents were removed, and the residue subjected to
chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to yield 12
(336 mg, 60.1%) as a colorless foam; RF ¼ 0.50 (n-hexane/ethyl
0.72 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 207.8
(C3, C]O), 176.6 (C28, COOCH3), 150.5 (C20, C]CH2), 136.7 (C32,
CH), 133.8 (C2, Cquart.), 131.6 (C33, CH), 125.2 (C34, CH2), 109.6 (C29,
CH2), 56.5 (C17, Cquart.), 53.0 (C5, CH), 51.3 (C31, CH3), 49.4 (C18, CH),
48.4 (C9, CH), 46.9 (C19, CH), 45.1 (C4, Cquart.), 42.6 (C14, Cquart.),
42.4 (C1, CH2), 40.5 (C8, Cquart.), 38.4 (C13, CH), 36.9 (C22, CH2), 36.1
(C10, Cquart.) 33.2 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.6
(C15, CH2), 29.2 (C23, CH3), 25.6 (C12, CH2), 22.4 (C24, CH3), 21.6
(C11, CH2), 20.3 (C6, CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.4 (C25,
CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 507.5 (100%
[M þ H]þ); analysis for C34H50O3 (506.76): C, 80.58; H, 9.94; found:
C, 80.41; H, 10.07.
acetate, 8:2); [
a
]
¼ þ71.7ꢀ (c ¼ 5.00, CHCl3); IR (KBr):
¼ 3447 m,
n
D
2949 s, 2868 s, 1727 s, 1642 w, 1458 m, 1386 m, 1160 m,
1064 w cmꢁ1; UVevis (methanol): lmax (log ε) ¼ 216 nm (4.01); 1H
NMR (500 MHz, CDCl3):
d
¼ 4.71 (s, 1 H, CHa (29)), 4.58 (s, 1 H, CHa
(29)), 3.72 (t, 2 H, J ¼ 5.8 Hz, CH2 (34)), 3.64 (s, 3 H, CH3 (31)),
3.00e2.84 (m, 3 H, CH (2) þ CH (19) þ CHa (32)), 2.70 (t, 2 H,
J ¼ 5.8 Hz, CH2 (33)), 2.35 (dd, 1 H, J ¼ 12.2, 6.0 Hz, CHb (32)),
2.25e2.17 (m, 2 H, CH (13) þ CHa (16)), 1.98 (dd, 1 H, J ¼ 13.1,
10.8 Hz, CHa (1)), 1.91e1.83 (m, 2 H, CHa (21) þ CHa (22)), 1.75e1.64
(m, 2 H, CH (5) þ CHa (12)), 1.67 (s, 3 H, CH3 (30)), 1.60 (dd, 1 H,
J ¼ 11.4 Hz, 11.4 Hz, CH (18)), 1.50e1.20 (m, 12 H, CH (9) þ CHa
(15) þ CHb (1) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2
(7) þ CH2 (11)), 1.19e0.93 (m, 2 H, CHb (12) þ CHb (15)), 1.06 (s, 3
H, CH3 (23)), 1.01 (s, 3 H, CH3 (24)), 0.99 (s, 3 H, CH3 (27)), 0.88 (s, 3
H, CH3 (25)), 0.66 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz,
4.9. 2-Methylene-28-oxo-28-thiomorpholin-4-yl-lup-20(29)en-3-
one (11)
A solution of 6 (107 mg, 0.23 mmol) and oxalyl chloride (0.2 ml,
0.30 g, 2.36 mmol) in dry toluene (5 ml) containing triethylamine
(0.1 ml, 73 mg, 0.72 mmol) and DMF (0.1 ml, 95 mg, 1.30 mmol) was
stirred at 25 ꢀC for 30 min. The solvents were removed under
reduced pressure, and the residue was suspended in dry toluene
(5 ml), and thiomorpholine (0.5 ml, 513 mg, 4.97 mmol) and trie-
thylamine (0.2 ml, 146 mg, 1.44 mmol) were added. After stirring at
100 ꢀC for an additional 30 min, the mixture was cooled to 25 ꢀC,
diluted with water (200 ml) and extracted with ethyl acetate
(3 ꢃ 100 ml). The extracts were washed with brine (100 ml), dried
(Na2SO4), and the solvents were removed. The residue was sub-
jected to chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to
yield 11 (69 mg, 54.5%) as a colorless solid; m.p. 250e255 ꢀC;
CDCl3):
d
¼ 218.8 (C3, C]O), 176.6 (C28, COOCH3), 150.4 (C20, C]
CH2), 109.6 (C29, CH2), 60.5 (C34, CH2OH), 56.5 (C14, Cquart.), 52.3
(C5, CH), 51.2 (C31, CH3), 50.0 (C9, CH), 49.3 (C18, CH), 48.8 (C1,
CH2), 46.9 (C19, CH), 46.7 (C4, Cquart.), 42.4 (C14, Cquart.), 42.1 (C2,
CH), 40.6 (C8, Cquart.), 38.5 (C13, CH), 36.9 (C10, Cquart.), 36.9 (C22,
CH2), 36.2 (C33, CH2), 32.9 (C7, CH2), 32.2 (C16, CH2), 32.0 (C32,
CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.9 (C23, CH3), 25.6 (C12,
CH2), 22.0 (C11, CH2), 20.0 (C6, CH2), 19.3 (C24, CH3), 19.3 (C30,
CH3), 18.5 (C26, CH3), 15.2 (C25, CH3), 14.6 (C27, CH3) ppm; MS
RF ¼ 0.65 (n-hexane/ethyl acetate, 8:2); [
a
]
¼ þ28.5ꢀ (c ¼ 5.25,
(ESI, MeOH): m/z
C34H54SO4 (558.86): C, 73.07; H, 9.74; S, 5.74 found: C, 72.86; H,
9.99; S, 5.53.
¼
581.4 (100% [M
þ
Na]þ); analysis for
D
CHCl3); IR (KBr):
n
¼ 3442 m, 2948 s, 2867 m, 1687 m, 1636 s,
1457 w, 1403 m, 1288 w, 1247 w, 1167 m, 1056 w cmꢁ1; UVevis
(methanol): lmax (log ε) ¼ 218 nm (4.28); 1H NMR (500 MHz,
CDCl3):
d
¼ 5.94 (s, 1 H, CHa (31)), 5.11 (s, 1 H, CHb (31)), 4.72 (s, 1 H,
4.11. Methyl (2a) 2-{[(2-hydroxyethyl)sulfonyl]methyl}-3-oxolup-
20(29)en-28-oate (13)
CHa (29)), 4.58 (s,1 H, CHb (29)), 4.00e3.73 (br d, 4 H, CH2 (32)), 2.97
(ddd, 1 H, J ¼ 11.0, 10.9, 3.5 Hz, CH (19)), 2.90 (ddd, 1 H, J ¼ 13.0, 13.0,
3.4 Hz, CH (13)), 2.69 (d,1 H, J ¼ 15.3 Hz, CHa (1)), 2.64e2.53 (m, 4 H,
CH2 (33)), 2.11e2.00 (m, 2 H, CHa (16) þ CHb (1)), 1.96e1.90 (m, 1 H,
CHa (22)), 1.88e1.80 (m, 1 H, CHa (21)), 1.77e1.72 (m, 1 H, CHa (12)),
1.67 (s, 3 H, CH3 (30)), 1.62e1.14 (m, 13 H, CH (5) þ CH (9) þ CHb
(16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11) þ CH2
(15)), 1.57 (dd, 1 H, J ¼ 11.3, 11.3 Hz, CH (18)), 1.09 (s, 3 H, CH3 (23)),
1.03 (s, 3 H, CH3 (24)), 1.01e0.88 (m, 1 H, CHb (12)), 0.97 (s, 3 H, CH3
(26)), 0.97 (s, 3 H, CH3 (27)), 0.83 (s, 3 H, CH3 (25)) ppm; 13C NMR
(125 MHz, CDCl3): d ¼ 207.6 (C3, C]O), 173.6 (C28, CON), 151.1
To a solution of 12 (200 mg, 0.36 mmol) in glacial acetic acid
(15 ml) hydrogen peroxide (10 ml, 0.10 mol, 35% in water) was
added, and the mixture was stirred at 25 ꢀC for 30 min. The
mixture was diluted with water (200 ml), extracted with ethyl
acetate (3 ꢃ 100 ml), the extracts were washed with aq. NaHCO3
(satd., 100 ml), an aq. solution of (NH4)2Fe(SO4)2 [5.0 g,
12.75 mmol in water (100 ml)] and brine (100 ml). After drying
(Na2SO4), the solvents were removed, and the residue was sub-
jected to chromatography (silica gel, ethyl acetate/methanol, 95:5)

344
R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345
to yield 13 (177 mg, 83.3%) as a colorless solid; m.p. 140e145 ꢀC;
4.13. (2 R)-Spiro[pyrazol-30,2-28-methoxy-3-oxolup-20(29)-en-28-
one] (15)
RF ¼ 0.74 (ethyl acetate/methanol, 9:1); [
a
]
¼ þ51.5ꢀ (c ¼ 5.85,
D
CHCl3); IR (KBr):
n
¼ 3429 m, 3072 w, 2948 s, 2868 s, 1725 s,
1642 w, 1455 m, 1384 m, 1318 w, 1287 w, 1188 m, 1156 s, 1133 m,
To a solution of 7 (515 mg, 1.07 mmol) in dry THF (10 ml),
a solution of diazomethane (15 ml, 15 mmol, 1 M in ether) was
added, and the mixture was stirred for 3 days at 25 ꢀC. A drop of
acetic acid was added, the mixture diluted with water (100 ml) and
extracted with ether (3 ꢃ 100 ml). The organic phases were dried
(Na2SO4), and the solvent was removed. The residue was subjected
to chromatography (silica gel, n-hexane/ethyl acetate, 9:1), and 15
(180 mg, 32.2%) was obtained as a colorless solid; m.p. 97e100 ꢀC
1062 m, 1012 w cmꢁ1; UVevis (methanol): lmax (log ε) ¼ 214 nm
(4.13); 1H NMR (500 MHz, CDCl3):
d
¼ 4.70 (s, 1 H, CHa (29)), 4.57
(s, 1 H, CHb (29)), 4.16e4.05 (m, 2 H, CH2 (34)), 3.63 (s, 3 H, CH3
(31)), 3.49e3.38 (m, 1 H, CH (2)), 3.25e3.17 (m, 1 H, CHa (32)),
3.09e2.81 (m, 3 H, CH (19) þ CH2 (33)), 2.48 (dd, 1 H, J ¼ 12.9,
3.1 Hz, CHb (32)), 2.25e2.14 (m, 2 H, CH (13) þ CHa (16)), 2.40 (dd,
1 H, J ¼ 12.9, 11.2 Hz, CHa (1)), 1.91e1.81 (m, 2 H, CHa (21) þ CHa
(22)), 1.80e1.74 (m, 1 H, CH (5)), 1.72e1.62 (m, 1 H, CHa (12)), 1.65
(s, 3 H, CH3 (30)), 1.58 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.53e1.10
(m, 13 H, CH (9) þ CHb (1) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2
(6) þ CH2 (7) þ CH2 (11) þ CH2 (15)), 1.09 (s, 3 H, CH3 (23)),
1.05e0.96 (m, 1 H, CHb (12)), 1.01 (s, 3 H, CH3 (24)), 0.97 (s, 3 H,
CH3 (27)), 0.87 (s, 3 H, CH3 (25)), 0.66 (s, 3 H, CH3 (26)) ppm; 13C
(decomp.); RF
¼
0.79 (n-hexane/ethyl acetate, 8:2);
[a]
¼ þ128.4ꢀ (c ¼ 2.34, CHCl3); IR (KBr):
n
¼ 3435 w, 2949 s,
D
2869 s, 1728 s, 1703 s, 1642 w, 1549 w, 1452 w, 1383 m, 1318 w,
1279 w, 1187 m, 1155 s, 1134 s, 1083 w, 1059 w, 1032 w cmꢁ1
;
UVevis (methanol): lmax (log ε) ¼ 215 nm (4.14); 1H NMR
(500 MHz, CDCl3):
d
¼ 4.70 (br s, 1 H, CHa (29)), 4.59 (ddd, 1 H,
NMR (125 MHz, CDCl3):
d
¼ 218.0 (C3, C]O), 176.6 (COOCH3),
J ¼ 17.4, 9.4, 3.8 Hz, CHa (50)), 4.56 (br s,1 H, CHb (29)), 4.24 (ddd,1 H,
J ¼ 17.4, 9.0, 8.6 Hz, CHb (50)), 3.65 (s, 3 H, CH3 (31)), 2.97 (ddd, 1 H,
J ¼ 11.1, 11.0, 4.5 Hz, CH (19)), 2.60 (ddd, 1 H, J ¼ 12.6, 9.0, 3.8 Hz, CHa
(40)), 2.46 (dd, 1 H, J ¼ 10.3, 3.8 Hz, CH (5)), 2.28e2.18 (m, 2 H, CH
(13) þ CHa (16)), 2.05 (d, 1 H, J ¼ 14.4 Hz, CHa (1)), 1.92e1.83 (m, 2 H,
CHa (21) þ CHa (22)), 1.79 (d, 1 H, J ¼ 14.4 Hz, CHb (1)), 1.74e1.15 (m,
14 H, CH (9) þ CH (18) þ CHb (22) þ CH2 (7) þ CHb (16) þ CHb
(21) þ CH2 (15) þ CHa (12) þ CH2 (11) þ CH2 (6)), 1.65 (s, 3 H, CH3
(30)), 1.10 (br s, 6 H, CH3 (23) þ CH3 (24)), 1.07e0.90 (m, 2 H, CHb
(40), CHb (12)), 0.99 (s, 3 H, CH3 (27)), 0.94 (s, 3 H, CH3 (25)), 0.80 (s,
150.3 (C20, C]CH2), 109.7 (C29, CH2), 56.8 (C34, CH2), 56.5 (C17,
Cquart.), 54.5 (C33, CH2), 53.8 (C32, CH2), 52.2 (C5, CH), 51.2 (C31,
CH3), 49.9 (C9, CH), 49.3 (C18, CH), 49.1 (C1, CH2), 46.8 (C19, CH),
46.8 (C4, Cquart.), 42.4 (C14, Cquart.), 40.6 (C8, Cquart.), 38.5 (C13, CH),
37.3 (C10, Cquart.), 36.9 (C22, CH2), 36.6 (C2, CH), 32.8 (C7, CH2),
32.0 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.7 (C23, CH3),
25.5 (C12, CH2), 22.0 (C11, CH2), 19.9 (C6, CH2), 19.3 (C30, CH3), 19.2
(C24, CH3), 18.8 (C26, CH3), 15.2 (C25, CH3), 14.5 (C27, CH3) ppm;
MS (ESI, MeOH): m/z ¼ 613.3 (56% [M þ Na]þ), 591.3 (74%
[M þ H]þ); analysis for C34H54SO6 (590.85): C, 69.11; H, 9.21; S,
5.43; found: C, 68.89; H, 9.07; S, 5.35.
3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 211.5 (C3, C]
O),176.6 (C28, COOCH3),150.4 (C20, C]CH2),109.6 (C29, CH2),101.7
(C2/C30, Cspiro), 76.7 (C50, CH2), 56.5 (C17, Cquart.), 54.1 (C1, CH2), 51.2
(C31, CH3), 50.9 (C5, CH), 49.3 (C18, CH), 49.2 (C9, CH), 46.9 (C19,
CH), 46.2 (C4, Cquart.), 42.5 (C14, Cquart.), 40.5 (C8, Cquart.), 38.4 (C13,
CH), 36.9 (C22, CH2), 36.8 (C10, Cquart.), 32.8 (C7, CH2), 32.0 (C16,
CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 29.3 (C40, CH2), 27.5 (C23,
CH3), 25.5 (C12, CH2), 22.1 (C24, CH3), 21.9 (C11, CH2), 20.1 (C6, CH2),
19.3 (C30, CH3), 18.4 (C26, CH3), 15.2 (C25, CH3), 14.6 (C27, CH3)
ppm; MS (ESI, MeOH): m/z ¼ 545.3 (60% [M þ Na]þ), 495.4 (100%
[M þ H-N2]þ); analysis for C33H50N2O3 (522.76): C, 75.82; H, 9.64;
N, 5.36; found: C, 75.61; H, 9.88; N, 5.17.
4.12. Methyl (2a) 2-(2-nitroethyl)-3-oxolup-20(29)en-28-oate (14)
To a solution of 7 (365 mg, 0.76 mmol) in nitromethane (10 ml)
and dichloromethane (5 ml) DBU (2.0 ml, 2.04 g, 13.40 mmol) was
added, and the mixture was stirred for 5 days at 25 ꢀC. After diluting
with water (200 ml) and extraction with ethyl acetate (3 ꢃ 100 ml),
the extracts were washed with brine (100 ml) and dried (Na2SO4).
The solvents were removed, and the residue was subjected to
chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to yield 14
(273 mg, 66.3%) as a colorless solid; m.p. 165e168 ꢀC; RF ¼ 0.59 (n-
hexane/ethyl acetate, 8:2); [
a
]
¼ þ1.7ꢀ (c ¼ 5.34, CHCl3); IR (KBr):
Acknowledgments
D
n
¼ 3444 w, 2955 s, 2877 s, 1730 s, 1698 s, 1550 m, 1380 w, 1320 m,
1277 w, 1190 m, 1144 s, 1130 m, 1029 w, 980 w cmꢁ1; UVevis
We like to thank Dr. D. Ströhl for the NMR experiments, Dr. R.
Kluge for the ESI-MS spectra and Mrs. F. Flemming (Organic
Chemistry, Universität Halle-Wittenberg) for her help with some
experiments. The cell lines were kindly provided by Dr. Thomas
Müller (Dept. of Haematology/Oncology, Universität Halle-
Wittenberg). Support by “Gründerwerkstatt e Biowissenschaften”
is gratefully acknowledged.
(methanol): lmax (log ε) ¼ 218 nm (4.00); 1H NMR (500 MHz,
CDCl3):
d
¼ 4.67 (s, 1 H, CHa (29)), 4.53 (br s, 1 H, CHb (29)),
4.48e4.28 (m, 2 H, CH2 (33)), 3.60 (s, 3 H, CH3 (31)), 2.92 (ddd, 1 H,
J ¼ 10.9, 10.7, 4.5 Hz, CH (19)), 2.83e2.72 (m, 1 H, CH (2)), 2.30e2.11
(m, 3 H, CH (13) þ CHa (16) þ CHa (32)), 1.99e1.75 (m, 4 H, CHa
(1) þ CHa (21) þ CHa (22) þ CHb (32)), 1.70e1.58 (m, 2 H, CH
(5) þ CHa (12)), 1.62 (s, 3 H, CH3 (30)), 1.55 (dd, 1 H, J ¼ 11.4, 11.4 Hz,
CH (18)), 1.42e0.93 (m, 14 H, CH (9) þ CHb (1) þ CHb (12) þ CHb
(16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11) þ CH2
(15)), 0.99 (s, 3 H, CH3 (23)), 0.96 (s, 3 H, CH3 (24)), 0.94 (s, 3 H, CH3
(27)), 0.83 (s, 3 H, CH3 (25)), 0.61 (s, 3 H, CH3 (26)) ppm; 13C NMR
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