Synthesis and biological evaluation of antitumor-active γ-butyrolactone substituted betulin derivatives

Article abstract of DOI:10.1016/j.bmc.2010.02.042

The plant triterpenes betulin and betulinic acid (BA) are triterpenes featuring interesting pharmacological properties. Starting from substituted betulinic aldehydes, we used them as lead structures for the synthesis of several γ-butyrolactones and butenolides. Their antitumor activity was examined for 15 cancer cell lines using a SRB-assay and their apoptotic action was documented by trypan-blue test and DNA laddering. Several compounds revealed a higher activity than betulinic acid.

Full text of DOI:10.1016/j.bmc.2010.02.042

Bioorganic & Medicinal Chemistry 18 (2010) 2549–2558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of antitumor-active
substituted betulin derivatives
c
-butyrolactone
a
a
b
b
René Csuk a, , Alexander Barthel , Stefan Schwarz , Harish Kommera , Reinhard Paschke
*
a Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
b BioSolution GmbH, Weinbergweg 22, 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:
The plant triterpenes betulin and betulinic acid (BA) are triterpenes featuring interesting pharmacological
properties. Starting from substituted betulinic aldehydes, we used them as lead structures for the synthe-
sis of several c-butyrolactones and butenolides. Their antitumor activity was examined for 15 cancer cell
lines using a SRB-assay and their apoptotic action was documented by trypan-blue test and DNA ladder-
ing. Several compounds revealed a higher activity than betulinic acid.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 17 September 2009
Revised 13 January 2010
Accepted 21 February 2010
Available online 2 March 2010
Keywords:
Betulin
Betulinic acid
c
-Butyrolactone
Apoptosis
Antitumor activity
1. Introduction
of action. In addition, it decreases the permeability of the mito-
chondrian transition pore complex,8 it releases cytochrome c and
Betulin I and betulinic acid II (Scheme 1) are naturally occurring
lupan-type triterpenes displaying interesting pharmacological
properties.1 They are widespread in the plant kingdom and readily
available from different plants, for example from the bark of white
birch (Betula alba) (Fig. 1).
Initially, for betulinic acid and derivatives an anti-HIV activity
in infected lymphocytic cells2 was reported. However, in contrast
to many other compounds, these compounds do not substantially
interfere with HIV-1 proteases, reverse transcriptase or integrase.
They rather have a unique way of action depending on the position
of substituents. For a betulinic acid derivative holding an extra
30,30-dimethylsuccinyl-group at the 3-position, a change of the cell
maturation was reported (by affecting the CA-SP1 junction in Gag
processing); this formed intact virus cells with defective cores.3,4
However, derivatives showing variations at the carboxylic group,
for example RPR103611, were reported5,6 to inhibit the virus-cell
fusion at the interface between gp120 and gp41.
apoptosis inducing factor (AIF) into the cytosol.9 Adding perme-
ability stabilizing agents, for example bongkrekic acid, impeded
the release of cell proteins and apoptosis. Also, caspases were acti-
vated and the generation of the reactive oxygen species was dem-
onstrated.10 The phosphorylation of the proapoptotic mitogen-
activated protein kinases (MAPKs) p38 and SAP/JNK was also ob-
served, therefore suggesting at least a partial contribution of the
p38 apoptosis pathway.11 Betulinic acid is also an inhibitor of topo-
isomerase I; however, it only prevents the formation of DNA ad-
ducts and does not form apoptotic topoisomerase I cleavable
complexes.12 The unique action of betulin and betulinic acid deriv-
atives for potential treatment of HIV infections and their high
selectivity for cancer cells at nontoxic doses makes them interest-
ing as targets for the synthesis of derivatives. Here, we present the
synthesis of betulin derivatives bearing an extra c-lactone moiety
at C-28. We examined their antitumor potency for 15 different can-
cer cell lines by an SRB-assay.
In athymic mice betulin derivatives acted7 as potent melanoma
specific cytotoxic agents leading to a tumor regression up to 80% at
a dose of 50 mg/kg; interestingly enough, no toxic side effects were
noted even at high concentrations up to 500 mg/kg. It was as-
sumed that cell death is provoked by inducting apoptosis. Thus,
betulinic acid is a versatile compound capable of several modes
2. Chemistry
For the synthesis of compounds holding a c-lactone at C-28, the
corresponding aldehydes are suitable precursors. Thus, the efficient
synthesis of substituted betulinic aldehydes (Scheme 2) became a
first objective. The betulinic aldehyde 1 was easily accessible by
Swern oxidation from O,O0-bis-(trimethylsilyl)-betulin.13 This
procedure is preferred to procedures using toxic chromium(VI)-
* Corresponding author. Tel.: +49 345 55 25660; fax: +49 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.042

2550
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
Scheme 1. Synthesis of C3 substituted betulin aldehydes. Reagents and conditions: (a) TMSCl, imidazole, CH2Cl2, 0 °C; (b) oxalyl chloride, DMSO, TEA, CH2Cl2, ꢀ78 °C; (c)
Boc2O, Mg(ClO4)2, CH2Cl2; (d) Ac2O, TEA, DMAP, CH2Cl2, 0 °C; (e) MeI, NaH, DMF; (f) NaOMe, MeOH, THF, reflux; (g) CaH, MeOH, THF.
3-O-methyl derivative 5. Deacetylation with sodium methoxide in
methanol followed by oxidation yielded the corresponding 3-O-
methylbetulinic aldehyde 7.
Treating the 3,28-O,O0-bis-acetylbetulin
4 with catalytic
amounts of calcium hydride in a MeOH–THF mixture enabled the
selective deprotection of the primary hydroxy group thus yielding
3-O-acetylbetulin 8. Subsequent Swern oxidation gave the 3-O-
acetylbetulinic aldehyde 9.
The aldehydes were transformed into various saturated and
unsaturated
c-lactones (Scheme 3). We focused on the synthesis
of -methylene-
a
c
-lactones. These were obtained in good yields
from Dreiding–Schmidt reactions using methyl 2-(bromomethyl)-
acrylate and zinc.17 Although we expected the formation of two
diastereoisomers, the (28R)-diastereomer was exclusively formed
Figure 1. Pharmacological important triterpenes betulin (I) and betulinic acid, BA
(II).
during the reaction. The
c-butyrolactones 14–16 were obtained
compounds.14 Aldehydes substituted at C-3 were prepared either
after suitable protection or by direct functionalisation of aldehyde
1. This direct approach was suitable for the synthesis of tert-butyl-
betulinic aldehyde (2) being obtained by the reaction of 1 with di-
tert-butyldicarbonate in the presenceof perchlorate.15 The synthesis
of the 3-O-acetyl- and 3-O-methyl derivatives was achieved by
selective acetylation–deacetylation procedures.16 Thus, treating
betulin with a slightly excess of acetic anhydride gave a mixture of
the compounds 3 and 4 in 69% and 25% yield, respectively. The use
of an excess of acetic anhydride allowed the exclusive preparation
of bisacetylbetulin 4. The 28-O-acetylbetulin 3 afforded on reaction
with methyl iodide and sodium hydride in DMF the corresponding
by a SmI2 induced reductive coupling using methyl acrylate.18
Again, the (28R)-isomer was exclusively obtained and a small
amount of the corresponding betulin. From 2-substituted acrylates,
mixtures of two isomeric compounds were obtained, whereas 3-
substituted acrylates gave no reaction at all. For the synthesis of
the butenolides 20–22, a two-step-procedure was applied (Scheme
3). Therefore, the aldehydes were allowed to react with methyl
propiolate followed by hydrogenation in the presence of Lindlar
catalyst.19 Michael addition of nitromethane with the butenolide
21 proceeded stereoselectively yielding the trans-isomer 2320 only.
Thus, butenolides are suitable starting materials for further deriv-
atization; this is currently under investigation in our laboratory.

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
2551
Scheme 2. Synthesis of the c-butyrolactones. Reagents and conditions: (a) methyl 2-(bromomethyl)acrylate, zinc, THF, ultrasound; (b) (1) samarium, 1,2-diiodoethane, THF,
ultra sonic, (2) methyl acrylate, tBuOH, THF, 0 °C.
Scheme 3. Synthesis of betulin derivatives carrying a butenolide. Reagents and conditions: (a) LDA, methyl propiolate, THF, ꢀ78 °C; (b) H2, Lindlar catalyst, quinoline, 1 atm;
(c) DBU, nitromethane.
compound in this study was the propiolate 18 having an acetyl-
group at C-3 with an average IC50-value of 4.0 M (Table 2).
3. Results and discussion
l
Based on the promising cytotoxicity of the compounds, we se-
lected compounds 10 and 21 to be examined whether the cell death
was mediated by apoptosis. Therefore, the floating cells (obtained
after treatment with IC90-concentrations for 24 h) were analyzed
by a trypan-blue exclusion test and DNA gel electrophoresis24,25
(Figure 2). In this test, apoptotic cells are characterized by an intact
cell membrane and therefore they exclude the blue dye. The pro-
gramed cell death is characterized by fragmentation of DNA into
smaller parts of 180 bp; these can be observed as DNA laddering
using gel electrophoresis.
The compounds 10–23 were assayed for their cytotoxic activity
against 15 human cancer cell lines using a colorimetric21 SRB-as-
say; a summary of the IC50-values is shown in Table 1. The activity
of the compounds depends on the modification at C-28 but also
from substituents at the C-3. The
having an OH-group at C-3 shows an average IC50-value of
10.3 M. Its matching 3-O-methyl derivative 12, however, shows
a slightly decreased IC50-value of 9.1 M whereas the presence of
a-methylene-c-butyrolactone
l
l
a 3-O-tert-butyl group (as in 11) results in a dramatically loss of
activity. This finding is in perfect agreement with results from
Salvador and co-workers showing that there is a size limitation
at position C-3.22 The low IC50 values for the
a
-methylene-c-buty-
4. Conclusion
rolactone 13 and for the
c-butyrolactone 16 reveal the valuable ef-
fect of a 3-O-acetyl moiety. This result is consistent with the
reported data23 for the antitumor activity of acetyl derivatives of
betulin. Interestingly, the butenolides 20–22 showed—compared
with their precursors 17–19—a decreased activity. Our most active
We synthesized a series of novel betulin derived c-butyrolac-
tones and butenolides. Their antitumor activity was examined for
15 cancer cell lines using an SRB-assay and their apoptotic action

2552
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
Table 1
Cytotoxicity (IC50 values,
5. Experimental
lM) in a panel of various cancer cell lines: the values 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 were obtained by an SRB-assay after 96 h of treatment and are
the average from at least two independent experiments
5.1. Chemistry
Melting points are uncorrected (Leica hot stage microscope),
optical rotations were obtained using a Perkin–Elmer 341 polar-
imeter (1 cm micro cell, 20 °C), NMR spectra were recorded
using the Varian spectrometers Gemini 200, Gemini 2000 or
Unity 500 (d given in ppm, J in hertz, internal Me4Si), IR spectra
Cell line
BA
10
11
12
13
14
15
16
8.2
518A2
A431
11.9
15.4
11.1
10.4
14.9
11.0
17.5
17.8
13.3
16.1
6.4
11.3
9.6
26.6
29.5
NA
12.6
8.1
8.1
6.2
5.7
8.5
4.1
3.6
9.5
7.1
6.3
5.3
5.6
3.7
5.8
7.5
5.7
21.8
22.2
20.7
25.5
25.0
20.2
26.8
23.4
17.8
15.2
22.4
22.4
24.8
20.1
24.5
NA
NA
4.6
3.9
9.0
4.7
4.9
10.4
4.6
7.9
10.6
5.1
2.6
6.0
5.7
8.6
A253
FADU
A549
11.0
12.2
11.9
10.4
11.7
9.0
8.3
8.8
9.7
11.6
7.9
9.0
13.4
8.6
8.6
8.8
9.3
7.0
9.8
7.0
18.7
22.6
18.9
24.6
NA
26.4
18.8
NA
21.3
19.5
22.7
NA
(film or KBr pellet) on
a Perkin–Elmer FT-IR spectrometer
NA
NA
17.4
14.4
NA
NA
NA
NA
Spectrum 1000, MS spectra were taken on a Intectra GmbH
AMD 402 (electron impact, 70 eV) or on a Finnigan MAT TSQ
7000 (electrospray, voltage 4.5 kV, sheath gas nitrogen) instru-
ment; for elemental analysis a Foss-Heraeus Vario EL instrument
was used; TLC was performed on silica gel (Merck 5554, detec-
tion by UV absorption or by treatment with a solution of 10%
sulfuric acid, ammonium molybdate and cerium(IV) sulfate) fol-
lowed by gentle heating. The solvents were dried according to
usual procedures.
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
6.7
27.5
19.0
NA
6.4
11.6
14.9
9.7
10.5
10.6
7.4
9.4
12.1
NA
NA
5.1.1. General procedure for the Swern oxidation (GP1)
To a solution of oxalyl chloride (6.0 g, 47.1 mmol) in dry dichlo-
romethane (100 ml) at ꢀ78 °C a solution of dry DMSO (7.8 ml) in
dry (100 ml) was slowly added and stirring at ꢀ78 °C was contin-
ued for another 30 min. At this temperature a solution of the cor-
responding betulinol (25.0 g, 42.6 mmol) in dry dichloromethane
(50 ml) was slowly added and stirring at this temperature was con-
tinued for another 2 h. Then, dry TEA (14 ml) was added and the
reaction mixture was allowed to warm to room temperature. Di-
luted aq HCl (10%, 100 ml) was added under vigorous stirring,
the phases were separated, the organic layer was washed with
aq Na2CO3 (2 ꢁ 50 ml), water (2 ꢁ 50 ml), and brine (2 ꢁ 50 ml),
the solvents were removed, and the residue was subjected to chro-
matography (silica gel, hexane–ethyl acetate 9:1) to afford the cor-
responding betulinic aldehyde.
Variation was 10%; NA: no activity <30 lM.
Table 2
Cytotoxicity (IC50 values,
Table 1
lM) in a panel of various cancer cell lines: cf. comments to
Cell line
17
18
19
20
21
3.6
22
23
518A2
A431
A253
FADU
A549
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
6.0
4.0
5.3
6.2
5.9
6.9
6.0
2.6
4.8
4.9
5.9
5.9
5.2
5.8
5.9
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
7.5
5.0
4.8
7.2
4.4
4.3
5.7
8.2
3.5
4.7
5.5
3.7
5.0
7.3
5.5
6.9
6.1
5.9
6.4
6.3
7.1
8.4
7.7
3.5
5.9
6.1
7.1
7.7
6.4
6.9
18.4
10.9
11.1
NA
12.7
16.9
17.6
15.1
3.5
16.7
13.8
18.0
15.9
NA
22.4
11.2
4.7
5.3
3.9
8.5
4.2
3.7
6.1
4.0
3.5
12.7
5.3
5.7
3.4
4.0
6.4
6.2
22.1
15.3
NA
11.3
10.1
17.4
9.8
28.7
7.0
25.8
15.3
5.1.2. General procedure for the synthesis of
butyrolactones (GP2)
a-methylene-c-
A suspension of zinc (0.54 g, 8.20 mmol) in THF was activated26
with a few crystals iodine under sonification. After the consump-
tion of the iodine, the corresponding aldehyde (1.35 mmol), methyl
2-bromomethylacrylate (0.54 g, 3.00 mmol) and hydroquinone
(10 mg) were added. The suspension was sonificated until TLC re-
vealed the absence of starting material. The precipitate was filtered
off, washed with THF and the solution was concentrated in vacuo.
Purification was achieved by column chromatography (silica gel,
hexane–ethyl acetate, 8:2).
NA
was documented by trypan-blue test and DNA laddering. These
studies reveal the favorable effect of a 3-O-acetyl moiety. Our most
active compound was a 3-O-acetylated propiolate 18 showing an
average IC50-value of 4.0
lM. Several of these novel compounds re-
vealed a higher activity than betulinic acid.
Figure 2. Trypan-blue exclusion test and DNA laddering of compound 10 for adenocarcinoma cell line HCT-8 (left) and compound 21 for ovarian cancer cell line A2780 (right)
after treatment with IC90-concentrations for 24 h.

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
2553
5.1.3. General procedure for the synthesis of
(GP3)
c
-butyrolactones
CH), 42.5 (C14, Cquart.), 40.8 (C8, Cquart.), 39.1 (C1, CH2), 39.0 (C4,
Cquart.), 38.7 (C13, CH), 37.1 (C10, Cquart.), 34.5 (C7, CH2), 33.2
(C22, CH2), 29.9 (C16, CH2), 29.3 (C21, CH2), 29.2 (t-Bu, CH3), 28.8
(C15, CH2), 28.7 (C23, CH3), 27.2 (C2, CH2), 25.6 (C12, CH2), 20.8
(C11, CH2), 19.0 (C29, CH3), 18.5 (C6, CH2), 16.4 (C26, CH3), 16.3
(C25, CH3), 15.9 (C24, CH3), 14.2 (C27, CH3) ppm. MS (i.e., 70 eV):
m/z (%) = 496 (6), 383 (100), 355 (31), 245 (50), 189 (47), 165
(71). Anal. Calcd for C34H56O2 (496.81): C, 82.20; H, 11.36. Found:
C, 81.96; H, 11.49.
A mixture of samarium (0.6 g, 4.0 mmol) and 1,2-diiodoethane
(1.1 g, 4.0 mmol) in dry THF (20 ml) was sonificated under argon
until a homogeneous deep blue solution was formed. After cooling
to 0 °C, a solution of the corresponding aldehyde (1.36 mmol),
methyl acrylate (0.12 g, 1.36 mmol) and t-BuOH (0.10 g,
1.36 mmol) in dry THF (5 ml) was added. The mixture was stirred
over night and then treated with brine (50 ml). The phases were
separated and the aq layer was extracted with ethyl acetate
(2 ꢁ 50 ml). The combined extracts were concentrated in vacuo
and the residue was subjected to column chromatography (silica
gel, hexane–ethyl acetate, 8:2).
5.1.7. 28-O-Acetylbetulin (3) and O,O0-diacetylbetulin (4)
To a suspension of betulin (15 g, 34 mmol), DMAP (0.1 g) in dry
CH2Cl2 (150 ml) and TEA (4.2 g, 40.8 mmol) was added dropwise a
solution of Ac2O (4.2 g, 40.8 mmol) in dry CH2Cl2 (30 ml) at 0 °C.
Stirring was continued for 1 h at room temperature. The precipi-
tate was filtered off and washed with CH2Cl2 (2 ꢁ 50 ml). The fil-
trate was washed with saturated Na2CO3 solution (100 ml) and
brine (100 ml), dried over Na2SO4 and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, hex-
ane–ethyl acetate, 9:1) and afforded compound 3 (11.3 g, 69%)
and 4 (4.5 g, 25%) as white solids. Data for 3: mp 209–212 °C
5.1.4. General procedure for the synthesis of 3-substituted
methyl propiolates (GP4)
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 methyl propi-
olate (0.34 g, 4.12 mmol) in dry THF (2 ml) was added dropwise.
Stirring at this temperature was continued for additional 30 min;
then a solution of the corresponding aldehyde (1.03 mmol) was
added. After 1 h at ꢀ78 °C, the reaction was quenched by the addi-
tion of brine (100 ml). The phases were separated and the aq layer
was extracted with ethyl acetate (2 ꢁ 50 ml). The combined ex-
tracts were dried over Na2SO4 and evaporated to dryness. The res-
idue was subjected to column chromatography (silica gel, hexane–
ethyl acetate, 8:2).
(lit.: 217–219 °C;27 210–212 °C16); [
D +8.5 (c 1.58 CHCl3),16 D +11.0 (c 1.0 CHCl3)28); Rf 0.47 (silica
gel, hexane–ethyl acetate, 8:2). Data for 4: mp 217–218 °C (lit.:
a]
+7.4 (c 3.4, CHCl3) (lit.:
D
[a
]
[a]
220 °C;29 218–219 °C30); [
a] +18.5 (c 3.7, CHCl3) (lit.: [a] +21 (c
D D
0.8 CHCl3),29
acetate, 9:1).
[a]
+22 (CHCl3)30); Rf 0.62 (silica gel, hexane–ethyl
D
5.1.8. 28-O-Acetyl-3-O-methylbetulin (5)
5.1.5. General procedure for the synthesis of butenolides (GP5)
Hydrogen was bubbled for 30 min through a mixture containing
the corresponding methyl propiolates (0.35 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 removed in vacuo. The residue
was subjected to column chromatography (silica gel, hexane–ethyl
acetate, 8:2).
To a solution of 28-O-acetyl-betulin (10.0 g, 20.6 mmol) in dry
DMF (100 ml) NaH (60% in mineral oil, 1.0 g, 24.7 mmol) was added
in several portions. After gas evolution has ceased methyl iodide
(3.5 g, 24.7 mmol) was added and stirred for 5 h at room tempera-
ture. The solvent was removed in vacuo and the residue treated with
CH2Cl2 (200 ml) and H2O (200 ml). After separation of the phases,
the organic layer was dried over Na2SO4 and concentrated in vacuo
to afford 5 (10.0 g, 97%) as a white solid. An analytical sample was
obtained by column chromatography (silica gel, hexane–ethyl ace-
5.1.6. 3-O-tert-Butylbetulinic aldehyde (2)
tate, 8:2); mp 163–165 °C; [
gel, hexane–ethyl acetate, 9:1); IR (KBr):
2868m, 1723m, 1646m, 1452m, 1389m, 1367m, 1346m, 1260s,
1191w, 1127w, 1107m, 1087w, 1044m cmꢀ1 1H NMR (400 MHz,
a] +8.7 (c 4.1, CHCl3); Rf 0.71 (silica
D
Prior to use MgClO4, was dried in vacuo at 130 °C (oil bath tem-
m = 3562s, 3086w, 2941s,
perature) for 2 h. To
a solution of betulin aldehyde (1.0 g,
2.27 mmol) and MgClO4 (1.1 g, 5.0 mmol), di-tert-butyldicarbonate
(5.0 g, 23 mmol) was added and the mixture was stirred for 1 h at
room temperature. When TLC revealed the absence of starting
material, the solution was washed with diluted HCl otherwise an-
other portion of di-tert-butyldicarbonate (5.0 g, 23 mmol) was
added. The organic layer was dried over Na2SO4 and concentrated
in vacuo. After purification by column chromatography (silica gel,
hexane–ethyl acetate, 95:5) 2 (1.0 g, 90%) was obtained as a col-
;
CDCl3): d = 4.67 (d, 1H, J = 2.1 Hz, CHa (30)), 4.57 (dd, 1H, J = 1.7,
1.2 Hz, CHb (30)), 4.22 (d, 1H, J = 11.2 Hz, CHa (28)), 3.84 (d, 1H,
J = 11.2 Hz, CHb (28)), 3.32 (s, 1H, OCH3), 2.62 (dd, 1H, J = 11.6,
4.2 Hz, CHOCH3 (3)), 2.43 (ddd, 1H, J = 11.2, 10.8, 5.8 Hz, CH (19)),
2.05 (s, 3H, Ac), 1.98–1.90 (m, 1H, CHa (21)), 1.85–1.86 (m, 1H, CHa
(16)), 1.78–1.50 (m, 7H, CHa (2)+CHa (22)+CHa (1)+CHa (15)+CH
(13)+CHa (12)+CH (18)), 1.66 (s, 3H, CH3 (29)), 1.44–1.32 (m, 7H,
CH2 (6)+CHa (11)+CHb (2)+CH2 (7)+CHb (21)), 1.27–1.10 (m, 3H,
CHb (16)+CH (9)+CHb (11)), 1.07–0.98 (m, 1H, CHb (22)+CHb
(15)+CHb (12)), 1.01 (s, 3H, CH3 (25)), 0.95 (s, 3H, CH3 (27)), 0.93 (s,
3H, CH3 (23)), 0.81 (s, 3H, CH3 (26)), 0.78–0.74 (m, 1H, CHb (1)),
0.72 (s, 3H, CH3 (24)), 0.65 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d = 171.6 (C@O), 150.2 (C20, C@CH2), 109.8
(C30, CH2@C), 88.6 (C3, CHOCH3), 62.8 (C28, CH2), 57.5 (OCH3),
55.9 (C5, CH), 50.4 (C9, CH), 49.8 (C18, CH), 47.7 (C19, CH), 46.3
(C17, Cquart.), 42.7 (C14, Cquart.), 41.9 (C8, Cquart.), 38.8 (C1, CH2),
38.6 (C4, Cquart.), 37.6 (C13, CH), 37.2 (C10, Cquart.), 34.6 (C7, CH2),
34.2 (C22, CH2), 29.8 (C16, CH2), 29.6 (C21, CH2), 28.0 (C23, CH3),
27.1 (C15, CH2), 25.2 (C12, CH2), 22.2 (C2, CH2), 21.0 (Ac), 20.8
(C11, CH2), 19.1 (C29, CH3), 18.2 (C6, CH2), 16.1 (C24, CH3), 16.0
(C26, CH3), 16.0 (C25, CH3), 14.7 (C27, CH3) ppm; MS (i.e., 70 eV):
m/z (%) = 498 (67), 466 (44), 425 (41), 221 (48), 203 (46), 189
(100), 135 (52). Anal. Calcd for C33H54O3 (498.78): C, 79.47; H,
10.91. Found: C, 79.38; H, 11.05.
ourless solid; mp 184 °C; [
gel, hexane–ethyl acetate, 8:2); IR (KBr):
1727 m, 1642w, 1453w, 1364w, 1252w, 1191w, 1056w, 1021w
cmꢀ1 1H NMR (400 MHz, CDCl3): d = 9.66 (d, 1H, J = 1.5 Hz, CHO),
a] +24.1 (c 4.4, CHCl3); Rf 0.87 (silica
D
m
= 2948s, 2875 m,
;
4.74 (d, 1H, J = 2.0 Hz, CHa (30)), 4.61 (dd, 1H, J = 2.0, 1.5 Hz, CHb
(30)), 2.96 (dd, 1H, J = 7.5, 5.9 Hz, CHOtBu (3)), 2.84 (ddd, 1H,
J = 11.0, 11.1, 5.8 Hz, CH (19)), 2.08–1.85 (m, 3H, CH (13)+CHa
(21)+CHa (16)), 1.78–1.52 (m, 7H, CHa (22)+CHa (12)+CH
(18)+CHa (1)+CH2 (2)+CHa (6)), 1.68 (s, 3H, CH3 (29)), 1.52–1.12
(m, 11H, CHb (21)+CHa (15)+CH2 (11)+CHb (6)+CH2 (7)+CHb
(22)+CHb (12)+CHb (16)+CH (9)), 1.16 (s, 9H, t-Bu), 1.08–0.98 (m,
1H, CHb (15)), 0.96 (s, 3H, CH3 (27)), 0.90 (s, 3H, CH3 (25)), 0.87
(s, 3H, CH3 (23)), 0.89–0.82 (m, 1H, CHb (1)), 0.81 (s, 3H, CH3
(26)), 0.72 (s, 3H, CH3 (24)), 0.68 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 206.7 (C@O), 149.7 (C20, C@CH2),
110.1 (C30, CH2@C), 78.5 (C3, CHOtBu), 72.8 (t-Bu, C), 59.3 (C17,
Cquart.), 56.1 (C5, CH), 50.6 (C9, CH), 48.1 (C18, CH), 47.5 (C19,

2554
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
5.1.9. 3-O-Methylbetulin (6)
5.1.11. (R)-4-[3b-Hydroxy-28-norlup-20(29)-en-17b-yl]-2-methy-
lene- -butyrolactone (10)
Following GP2, 10 (0.45 g, 65%) was obtained from betulinic
aldehyde (0.60 g, 1.35 mmol) as a colourless solid; mp 253–
A solution of 5 (10.0 g, 20.0 mmol) and sodium methoxide
(1.08 g, 20 mmol) in THF (200 ml) and MeOH (100 ml) was heated
under reflux until the absence of starting material was confirmed
by TLC (normally within 24 h). The solvents were removed in va-
cuo, the residue was suspended in 2 N HCl (100 ml) and extracted
with ethyl acetate (2 ꢁ 200 ml). The combined organic layers were
dried over Na2SO4 and concentrated in vacuo to yield 6 (8.7 g, 95%)
as a white solid. The crude product was used without further puri-
fication; an analytical sample was obtained by column chromatog-
c
256 °C; [
acetate, 8:2); IR (KBr):
1453w, 1371w, 1284w, 1250w, 1132w, 1108w, 1033w cmꢀ1
a
]
ꢀ8.0 (c 4.8, CHCl3); Rf 0.25 (silica gel, hexane–ethyl
D
m
= 3423s, 2941s, 2872m, 1756s, 1640w,
1H
;
NMR (500 MHz, CDCl3): d = 6.18 (dd, 1H, J = 2.9, 2.6 Hz, CHa (34)),
5.58 (dd, 1H, J = 2.5, 2.3 Hz, CHb (34)), 4.89 (dd, 1H, J = 7.6, 7.6 Hz,
CH (28)), 4.70 (d, 1H, J = 2.1 Hz, CHa (30)), 4.54–4.53 (m, 1H, CHb
(30)), 3.12 (dd, 1H, J = 11.4, 4.8 Hz, CHOH (3)), 2.88–2.79 (m, 2H,
CHa (31)+CH (19)), 2.70–2.63 (m, 1H, CHb (31)), 2.00 (ddd, 1H,
J = 12.2, 12.2, 3.8 Hz, CH (13)), 1.92–1.82 (m, 1H, CHa (21)), 1.78–
1.65 (m, 2H, CH (18)+CHa (12)), 1.62 (s, 3H, CH3 (29)), 1.65–1.42
(m, 7H, CHa (1)+CHa (22)+CHa (16)+CH2 (2)+CHa (6)+CHa (15)),
1.40–1.00 (m, 11H, CH2 (11)+CHb (6)+CHb (16)+CH2 (7)+CH
(9)+CHb (22)+CHb (21)+CHb (12)+CHb (15)), 0.96 (s, 3H, CH3 (27)),
0.95 (s, 3H, CH3 (25)), 0.90 (s, 3H, CH3 (23)), 0.92–0.88 (m, 1H,
CHb (1)), 0.77 (s, 3H, CH3 (26)), 0.70 (s, 3H, CH3 (24)), 0.62 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 170.5
(C@O), 150.3 (C20, C@CH2), 134.6 (C32, C@CH2), 121.6 (C34,
CH2@C), 110.0 (C30, CH2@C), 78.9 (C3, CHOH), 78.1 (C28, CH),
55.3 (C5, CH), 50.3 (C9, CH), 49.4 (C18, CH), 47.7 (C19, CH), 45.5
(C17, Cquart.), 42.8 (C14, Cquart.), 40.9 (C8, Cquart.), 38.8 (C4, Cquart.),
38.7 (C1, CH2), 37.1 (C10, Cquart.), 36.9 (C13, CH), 34.2 (C7, CH2),
32.9 (C22, CH2), 31.5 (C16, CH2), 31.2 (C21, CH2), 30.4 (C31, CH2),
28.0 (C23, CH3), 27.8 (C15, CH2), 27.3 (C2, CH2), 24.9 (C12, CH2),
20.8 (C11, CH2), 18.8 (C29, CH3), 18.3 (C6, CH2), 16.1 (C26, CH3),
15.9 (C25, CH3), 15.3 (C24, CH3), 15.2 (C27, CH3) ppm; MS (ESI,
MeOH): m/z = 508.4 (30% [M+H]+), 1039.4 (100% [2M+Na]+). Anal.
Calcd for C34H52O3 (508.78): C, 80.26; H, 10.30. Found: C, 80.00;
H, 10.45.
raphy (silica gel, hexane–ethyl acetate, 8:2); mp 230–233 °C; [
ꢀ7.3 (c 2.7, CHCl3); Rf 0.45 (silica gel, hexane–ethyl acetate, 8:2);
IR (KBr): = 3442m, 3070m, 2939s, 2867m, 1734w, 1643w,
1558w, 1453m, 1390w, 1373m, 1246w, 1181m, 1103m, 1013m
cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.66 (d, 1H, J = 2.1 Hz, CHa
a]
D
m
;
(30)), 4.55 (dd, 1H, J = 2.1, 1.2 Hz, CHb (30)), 3.77 (dd, 1H, J = 10.8,
1.7 Hz, CHa (28)), 3.32 (s, 3H, OCH3), 3.31 (d, 1H, J = 10.8 Hz, CHb
(28)), 2.60 (dd, 1H, J = 12.0, 4.6 Hz, CHOCH3 (3)), 2.36 (ddd, 1H,
J = 10.9, 10.9, 5.8 Hz, CH (19)), 1.97–1.80 (m, 2H, CHa (21)+CHa
(16)), 1.78–1.55 (m, 6H, CHa (2)+CHa (15)+CHa (1)+CH (13)+CHa
(12)+CH (18)), 1.66 (s, 3H, CH3 (29)), 1.52–1.32 (m, 6H, CH2
(6)+CHa (11)+CHa (22)+CHb (2)+CH2 (7)), 1.27–1.14 (m, 5H, CH
(9)+CHb (22) CHb (11)+CHb (21)+CHb (12)), 1.06–1.00 (m, 1H, CHb
(15)+CHb (16)), 0.99 (s, 3H, CH3 (25)), 0.95 (s, 3H, CH3 (27)), 0.93
(s, 3H, CH3 (23)), 0.86–0.81 (m, 1H, CHb (1)), 0.80 (s, 3H, CH3
(26)), 0.72 (s, 3H, CH3 (24)), 0.65 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 150.5 (C20, C@CH2), 109.6 (C30,
CH2@C), 88.6 (C3, CHOCH3), 60.5 (C28, CH), 57.5 (OCH3), 55.8 (C5,
CH), 50.4 (C9, CH), 48.8 (C18, CH), 47.8 (C17, Cquart.), 47.7 (C19,
CH), 42.7 (C14, Cquart.), 40.9 (C8, Cquart.), 38.8 (C1, CH2), 38.6 (C4,
Cquart.), 37.3 (C13, CH), 37.2 (C10, Cquart.), 34.2 (C7, CH2), 34.0
(C16, CH2), 29.6 (C22, CH2), 29.2 (C21, CH2), 28.0 (C23, CH3), 27.0
(C15, CH2), 25.2 (C12, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2), 19.1
(C29, CH3), 18.2 (C6, CH2), 16.1 (C24, CH3), 16.1 (C25, CH3), 16.0
(C26, CH3), 14.7 (C27, CH3) ppm; MS (i.e., 70 eV): m/z (%) = 456
(43), 425 (52), 234 (57), 221 (61), 203 (64), 189 (100), 135 (41).
Anal. Calcd for C31H52O2 (456.74): C, 81.52; H, 11.48. Found: C,
81.47; H, 11.61.
5.1.12. (R)-4-[3b-tert-Butyloxy-28-norlup-20(29)-en-17b-yl]-2-
methylene-
Compound 11 (0.50 g, 89%) was obtained as a colourless solid
from 2 (0.50 g, 1.00 mmol) following GP2; mp 243–245 °C; [
ꢀ3.0 (c 5.6, CHCl3); Rf 0.57 (silica gel, hexane–ethyl acetate, 8:2);
IR (KBr): = 2943s, 2873m, 1765s, 1718m, 1639m, 1465m,
1387m, 1365m, 1283m, 1248m, 1195m, 1132m, 1108m, 1083m,
1020m cmꢀ1 1H NMR (400 MHz, CDCl3): d = 6.22 (dd, 1H, J = 2.9,
c-butyrolactone (11)
a
]
D
m
5.1.10. 3-O-Methylbetulinic aldehyde (7)
Following GP1, 7 (6.8 g, 97%) was obtained from 6 (7.0 g,
;
15.3 mmol) as a colourless solid; mp 192–194 °C; [
CHCl3); Rf 0.80 (silica gel, hexane–ethyl acetate, 9:1); IR (KBr):
= 3441m, 3072w, 2944s, 2869m, 1709m, 1646w, 1449m, 1389w,
1373w, 1355w, 1248w, 1181w, 1102m, 1000w cmꢀ1 1H NMR
a
]
D +30.3 (c 5.3,
2.9 Hz, CHa (34)), 5.62 (dd, 1H, J = 2.5, 2.1 Hz, CHb (34)), 4.94 (dd,
1H, J = 7.9, 7.9 Hz, CH (28)), 4.75 (d, 1H, J = 2.1 Hz, CHa (30)),
4.58–4.54 (m, 1H, CHb (30)), 2.94 (dd, 1H, J = 10.8, 5.4 Hz, CHOtBu
(3)), 2.92–2.80 (m, 2H, CHa (31)+CH (19)), 2.75–2.67 (m, 1H, CHb
(31)), 2.06 (ddd, 1H, J = 12.2, 12.2, 3.8 Hz, CH (13)), 1.98–1.85 (m,
1H, CHa (21)), 1.79–1.53 (m, 8H, CHa (12)+CH (18)+CHa (16)+CHa
(22)+CHa (1)+CH2 (2)+CHa (6)), 1.66 (s, 3H, CH3 (29)), 1.52–1.10
(m, 11H, CHa (15)+CHb (22)+CH2 (11)+CHb (6)+CH2 (7)+CHb
(12)+CHb (16)+CH (9)+CHb (21)), 1.16 (s, 9H, t-Bu), 1.06–0.99 (m,
1H, CHb (15)), 1.00 (s, 3H, CH3 (27)), 0.99 (s, 3H, CH3 (25)), 0.87
(s, 3H, CH3 (23)), 0.89–0.82 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3
(26)), 0.72 (s, 3H, CH3 (24)), 0.68 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 170.5 (C@O), 150.3 (C20, C@CH2),
134.6 (C32, C@CH2), 121.5 (C34, CH2@C), 110.1 (C30, CH2@C),
78.5 (C3, CHOtBu), 78.1 (C28, CH), 72.8 (t-Bu, C), 56.0 (C5, CH),
50.5 (C9, CH), 49.5 (C17, Cquart.), 49.4 (C18, CH), 47.7 (C19, CH),
42.8 (C14, Cquart.), 40.9 (C8, Cquart.), 39.0 (C4, Cquart.), 38.9 (C1,
CH2), 37.0 (C10, Cquart.), 36.9 (C13, CH), 34.4 (C7, CH2), 33.0 (C22,
CH2), 31.9 (C16, CH2), 31.7 (C21, CH2), 30.4 (C31, CH2), 29.2 (t-Bu,
CH3), 28.7 (C23, CH3), 27.8 (C15, CH2), 27.2 (C2, CH2), 25.0 (C12,
CH2), 20.8 (C11, CH2), 19.0 (C29, CH3), 18.5 (C6, CH2), 16.4 (C26,
CH3), 16.2 (C25, CH3), 16.1 (C24, CH3), 15.2 (C27, CH3) ppm; MS
(ESI, MeOH): m/z = 587.5 (10% [M+Na]+), 1151.5 (100% [2M+Na]+),
1167.3 (70% [2M+K]+). Anal. Calcd for C38H60O3 (564.88): C,
80.80; H, 10.71. Found: C, 80.67; H, 10.92.
m
;
(400 MHz, CDCl3): d = 9.65 (d, 1H, J = 1.7 Hz, CHO), 4.74 (d, 1H,
J = 1.7 Hz, CHa (30)), 4.61 (dd, 1H, J = 2.1, 1.7 Hz, CHb (30)), 3.32 (s,
1H, OCH3), 2.84 (ddd, 1H, J = 11.2, 11.2, 5.8 Hz, CH (19)), 2.60 (dd,
1H, J = 11.6, 4.6 Hz, CHOCH3 (3)), 2.08–1.96 (m, 2H, CHa (16)+CH
(13)), 1,89–1,82 (m, 1H, CHa (21)), 1.78–1.65 (m, 5H, CHa (2)+CHa
(22)+CHa (12)+CH (18)+CHa (1)), 1.68 (s, 3H, CH3 (29)), 1.49–1.12
(m, 13H, CH2 (6)+CHb (16)+CHb (21)+CH2 (15)+CH2 (11)+CHb
(2)+CH2 (7)+CHb (22)+CH (9)), 1.05–0.99 (m, 1H, CHb (12)), 0.95 (s,
3H, CH3 (27)), 0.92 (s, 3H, CH3 (23)), 0.89 (s, 3H, CH3 (25)), 0.86–
0.82 (m, 1H, CHb (1)), 0.80 (s, 6H, 2CH3 (24+26)), 0.64 (d, 1H,
J = 9.1 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 206.6
(C@O), 149.7 (C20, C@CH2), 110.1 (C30, CH2@C), 88.6 (C3, CHOCH3),
59.3 (C17, Cquart.), 57.4 (C31, CH3), 55.9 (C5, CH), 50.5 (C9, CH), 48.1
(C18, CH), 47.5 (C19, CH), 42.5 (C14, Cquart.), 40.8 (C8, Cquart.), 38.8
(C1, CH2), 38.7 (C13, CH), 38.6 (C4, Cquart.), 37.2 (C10, Cquart.), 34.3
(C7, CH2), 33.2 (C22, CH2), 29.8 (C21, CH2), 29.2 (C16, CH2), 28.8
(C15, CH2), 28.0 (C23, CH3), 25.5 (C12, CH2), 22.2 (C2, CH2), 20.8
(C11, CH2), 19.0 (C29, CH3), 18.1 (C6, CH2), 16.1 (C24+C26, 2CH3),
15.9 (C25, CH3), 14.2 (C27, CH3) ppm; MS (i.e., 70 eV): m/z
(%) = 454 (74), 425 (31), 221 (63), 189 (100), 135 (43). Anal. Calcd
for C31H50O2 (454.73): C, 81.88; H, 11.08. Found: C, 81.66; H, 11.18.

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
2555
5.1.13. (R)-4-[3b-Methoxy-28-norlup-20(29)-en-17b-yl]-2-methy-
lene- -butyrolactone (12)
Following GP1, 12 (0.50 g, 87%) was obtained from aldehyde 7
(0.50 g, 1.00 mmol) as a colourless solid; mp 219–220 °C; [ 1.7
(c 4.28, CHCl3); Rf 0.69 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3441m, 3070w, 2954s, 2947s, 1757s, 1638w, 1456m,
1388w, 1358w, 1282m, 1250m, 1185m, 1127m, 1105m, 1088m,
1011m cmꢀ1 1H NMR (400 MHz, CDCl3): d = 6.22 (dd, 1H,
1123.4 (100% [2M+Na]+). Anal. Calcd for C36H54O4 (550.81):
C, 78.50; H, 9.88. Found: C, 78.41; H, 10.11.
c
a
]
D
5.1.15. (R)-4-[3b-Hydroxy-28-norlup-20(29)-en-17b-yl]-c-butyro-
lactone (14)
m
Following GP3, 14 (0.32 g, 47%) was obtained from aldehyde 1
(0.60 g, 1.36 mmol) as a colourless solid; mp 190–192 °C; [
ꢀ9.6 (c 4.2, CHCl3); Rf 0.17 (silica gel, hexane–ethyl acetate,
8:2); IR (KBr): = 3432s, 2942s, 2872m, 2221w, 1767s, 1641w,
1454w, 1376w, 1352m, 1224m, 1191s, 1142m, 1109w, 1033w
cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.90 (dd, 1H, J = 6.6,
a
]
D
;
J = 2.9, 2.5 Hz, CHa (33)), 5.58 (dd, 1H, J = 2.5, 2.5 Hz, CHb (33)),
4.94 (dd, 1H, J = 7.4, 7.9 Hz, CH (28)), 4.75 (d, 1H, J = 2.1 Hz, CHa
(30)), 4.58 (dd, 1H, J = 2.1, 1.7 Hz, CHb (30)), 3.33 (s, 1H, OCH3),
2.94–2.83 (m, 2H, CHa (31)+CH (19)), 2.75–2.67 (m, 1H, CHb
(31)), 2.61 (dd, 1H, J = 11.6, 4.6 Hz, CHOCH3 (3)), 2.06 (ddd, 1H,
J = 12.0, 12.0, 3.3 Hz, CH (13)), 1.97–1.86 (m, 1H, CHa (21)),
1.80–1.59 (m, 6H, CHa (2)+CH (18)+CHa (12) +CHa (1) +CHa
(22)+CHa (16)), 1.67 (s, 3H, CH3 (29)), 1.50–1.08 (m, 13H, CH2
(6)+CH2 (11)+CHb (22)+CHa (15)+CHb (2)+CH2 (7)+CHb (21)+CHb
(12)+CHb (16)+CH (9)), 1.05–1.02 (m, 1H, CHb (15)), 1.00 (s, 3H,
CH3 (27)), 0.99 (s, 3H, CH3 (25)), 0.93 (s, 3H, CH3 (23)), 0.85–
0.77 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3 (26)), 0.72 (s, 3H, CH3
(24)), 0.66 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d = 170.5 (C@O), 150.3 (C20, C@CH2), 134.6 (C32, C@CH2),
121.6 (C33, C@CH2), 109.8 (C30, CH2@C), 88.5 (C3, CHOCH3), 78.1
(C28, CH), 57.5 (OCH3), 55.8 (C5, CH), 50.3 (C9, CH), 49.5 (C17,
Cquart.), 49.4 (C18, CH), 47.7 (C19, CH), 42.8 (C14, Cquart.), 41.0
(C8, Cquart.), 38.8 (C1, CH2), 38.5 (C4, Cquart.), 37.2 (C10, Cquart.),
36.9 (C13, CH), 34.2 (C7, CH2), 33.0 (C22, CH2), 31.8 (C16, CH2),
31.6 (C21, CH2), 30.4 (C31, CH2), 28.0 (C23, CH3), 27.8 (C15,
CH2), 25.0 (C12, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2), 19.0 (C29,
CH3), 18.1 (C6, CH2), 16.2 (C24, CH3), 16.1 (C26, CH3), 16.0 (C25,
CH3), 15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 523.3 (10%
[M+H]+), 540.3 (47% [M+Na]+), 576.8 (75% [M+Na+MeOH]+),
1067.2 (100% [2M+Na]+). Anal. Calcd for C35H54O3 (522.80): C,
80.41; H, 10.41. Found: C, 80.31; H, 10.62.
m
;
10.0 Hz, CH (28)), 4.73 (d, 1H, J = 2.1 Hz, CHa (30)), 4.54 (dd, 1H,
J = 2.1, 1.6 Hz, CHb (30)), 3.16 (dd, 1H, J = 11.4, 4.8 Hz, CHOH
(3)), 2.79 (ddd, 1H, J = 12.0, 10.4, 6.6 Hz, CH (19)), 2.53–2.46 (m,
2H, CH2 (32)), 2.21–2.11 (m, 1H, CHa (31)), 2.03 (ddd, 1H,
J = 12.2, 12.2, 3.8 Hz, CH (13)), 2.00–1.88 (m, 2H, CHb (31)+CHa
(21)), 1.76–1.62 (m, 5H, CH (18)+CHa (12)+CHa (16)+CHa (1)+CHa
(22)), 1.66 (s, 3H, CH3 (29)), 1.61–1.36 (m, 9H, CH2 (2)+CH2
(6)+CHa (11)+CHa (15)+CHb (22)+CHb (16)+CHa (7)), 1.36–1.09
(m, 5H, CHb (7)+CHb (21)+CHb (11)+CH (9)+CHb (12)), 1.06–1.01
(m, 1H, CHb (15)), 1.01 (s, 3H, CH3 (27)), 1.00 (s, 3H, CH3 (25)),
0.95 (s, 3H, CH3 (23)), 0.91–0.85 (m, 1H, CHb (1)), 0.82 (s, 3H,
CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.66 (d, 1H, J = 9.5 Hz, CH (5))
ppm; 13C NMR (125 MHz, CDCl3): d = 177.5 (C@O), 150.2 (C20,
C@CH2), 110.0 (C30, CH2@C), 81.3 (C28, CH), 78.9 (C3, CHOH),
55.3 (C5, CH), 50.3 (C9, CH), 49.4 (C18, CH), 49.2 (C17, Cquart.),
47.8 (C19, CH), 42.8 (C14, Cquart.), 40.9 (C8, Cquart.), 38.8 (C4,
Cquart.), 38.7 (C1, CH2), 37.1 (C10, Cquart.), 36.9 (C13, CH), 34.2
(C7, CH2), 32.7 (C22, CH2), 32.1 (C16, CH2), 31.6 (C21, CH2), 28.6
(C32, CH2), 28.0 (C23, CH3), 27.8 (C15, CH2), 27.3 (C2, CH2), 24.9
(C12, CH2), 24.5 (C31, CH2), 20.8 (C11, CH2), 18.8 (C29, CH3),
18.2 (C6, CH2), 16.2 (C26, CH3), 16.1 (C25, CH3), 15.3 (C24, CH3),
15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 497.4 (20%
[M+H]+), 1015.4 (100% [2M+Na]+). Anal. Calcd for C33H52O3
(496.76): C, 79.79; H, 10.55. Found: C, 79.62; H, 10.73.
5.1.14. (R)-4-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-2-methy-
lene-c-butyrolactone (13)
5.1.16. (R)-4-[3b-Methoxy-28-norlup-20(29)-en-17b-yl]-c-butyro-
Following GP1, 13 (0.60 g, 80%) was obtained from 3-O-acetyl-
lactone (15)
betulinaldehyde 9 (0.65 g, 1.35 mmol) as a colourless solid; mp
Following GP3, 15 (0.38 g, 56%) was obtained from 7 (0.60 g,
1.32 mmol) as a colourless solid; mp 231–234 °C; [ ꢀ0.6 (c
3.84, CHCl3); Rf 0.55 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3441m, 2943s, 1777m, 1639w, 1456m, 1454m, 1385m,
1356m, 1183m, 1140w, 1100m, 1020w cmꢀ1 1H NMR (400 MHz,
245–247 °C; [
ethyl acetate, 8:2); IR (KBr):
1466m, 1374m, 1335w, 1284m, 1247s, 1195m, 1132m, 1108m,
1028m cmꢀ1 1H NMR (400 MHz, CDCl3): d = 6.23 (dd, 1H,
a]
D
ꢀ5.1 (c 3.3, CHCl3); Rf 0.50 (silica gel, hexane–
a]
D
m
= 2941s, 2873m, 1762s, 1730s,
m
;
;
J = 2.9, 2.9 Hz, CHa (34)), 5.62 (dd, 1H, J = 2.5, 2.1 Hz, CHb (34)),
4.94 (dd, 1H, J = 7.9, 7.9 Hz, CH (28)), 4.75 (d, 1H, J = 2.1 Hz, CHa
(30)), 4.58–4.54 (m, 1H, CHb (30)), 4.45 (dd, 1H, J = 10.8, 5.4 Hz,
CHOAc (3)), 2.94–2.83 (m, 2H, CHa (31)+CH (19)), 2.76–2.67 (m,
1H, CHb (31)), 2.02 (s, 3H, Ac), 2.06 (ddd, 1H, J = 12.2, 12.2,
3.8 Hz, CH (13)), 1.96–1.86 (m, 1H, CHa (21)), 1.79–1.55 (m, 8H,
CHa (12)+CH (18)+CHa (1)+CHa (16)+CHa (22)+CH2 (2)+CHa (6)),
1.53 (s, 3H, CH3 (29)), 1.53–1.10 (m, 11H, CHa (15)+CHb
(22)+CH2 (11)+CHb (6)+CH2 (7)+CHb (12)+CHb (16)+CH (9)+CHb
(21)) 1.08–0.92 (m, 1H, CHb (15), 1.00 (s, 6H, CH3 (27)+CH3
(25)), 0.84 (s, 3H, CH3 (23)), 0.92–0.88 (m, 1H, CHb (1)), 0.83 (s,
3H, CH3 (26)), 0.82 (s, 3H, CH3 (24)), 0.78 (d, 1H, J = 9.5 Hz, CH
(5)) ppm; 13C NMR (125 MHz, CDCl3): d = 170.8 (C@O), 170.4
(C@O), 150.2 (C20, C@CH2), 134.7 (C32, C@CH2), 121.5 (C34,
CH2@C), 110.1 (C30, CH2@C), 80.9 (C3, CHOAc), 78.1 (C28, CH),
55.3 (C5, CH), 50.3 (C9, CH), 49.7 (C17, Cquart.), 49.6 (C18, CH),
47.8 (C19, CH), 43.0 (C14, Cquart.), 41.1 (C8, Cquart.), 38.4 (C1,
CH2), 37.8 (C4, Cquart.), 37.1 (C10, Cquart.), 37.0 (C13, CH), 34.3
(C7, CH2), 33.1 (C22, CH2), 31.9 (C16, CH2), 31.7 (C21, CH2), 30.5
(C31, CH2), 28.0 (C23, CH3), 27.9 (C15, CH2), 25.1 (C12, CH2),
23.8 (C2, CH2), 20.9 (C11, CH2), 21.3 (Ac), 18.8 (C29, CH3), 18.2
(C6, CH2), 16.5 (C26, CH3), 16.3 (C25, CH3), 16.2 (C24, CH3), 15.3
(C27, CH3) ppm; MS (ESI, MeOH): m/z = 551.2 (30% [M+H]+),
CDCl3): d = 4.90 (dd, 1H, J = 6.2, 10.0 Hz, CH (28)), 4.74 (d, 1H,
J = 2.1 Hz, CHa (30)), 4.57 (dd, 1H, J = 1.7, 1.2 Hz, CHb (30)), 3.33
(s, 1H, OCH3), 2.83–2.74 (m, 1H, CH (19)), 2.61 (dd, 1H, J = 11.6,
4.2 Hz, CHOCH3 (3)), 2.53–2.46 (m, 2H, CH2 (32)), 2.20–2.11 (m,
1H, CHa (31)), 2.04 (ddd, 1H, J = 12.0, 12.0, 3.3 Hz, CH (13)), 2.00–
1.88 (m, 2H, CHb (31)+CHa (21)), 1.80–1.60 (m, 6H, CHa (2)+CHa
(12) +CH (18)+ CHa (1)+CHa (16)+CHa (22)), 1.67 (s, 3H, CH3 (29)),
1.51–1.20 (m, 12H, CH2 (6)+CH2 (11)+CHb (22)+CHa (15)+CHb
(2)+CH2 (7)+CHb (21)+CHb (12)+ CH (9)), 1.19–1.12 (m, 1H, CHb
(16)), 1.05–0.99 (m, 1H, CHb (15)), 1.00 (s, 6H, 2 ꢁ CH3 (25+27)),
0.93 (s, 3H, CH3 (23)), 0.86–0.77 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3
(26)), 0.73 (s, 3H, CH3 (24)), 0.66 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 177.5 (C@O), 150.4 (C20, C@CH2),
110.1 (C30, CH2@C), 88.6 (C3, CHOCH3), 81.3 (C28, CH), 57.5
(OCH3), 55.8 (C5, CH), 50.4 (C9, CH), 49.4 (C18, CH), 49.2 (C17,
Cquart.), 47.9 (C19, CH), 42.8 (C14, Cquart.), 41.0 (C8, Cquart.), 38.8
(C1, CH2), 38.6 (C4, Cquart.), 37.2 (C10, Cquart.), 37.0 (C13, CH), 34.3
(C7, CH2), 32.7 (C22, CH2), 32.1 (C16, CH2), 31.6 (C21, CH2), 28.6
(C32, CH2), 28.0 (C23, CH3), 27.8 (C15, CH2), 25.0 (C12, CH2), 24.5
(C31, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2), 19.0 (C29, CH3), 18.1
(C6, CH2), 16.2 (C24, CH3), 16.1 (C26, CH3), 16.1 (C25, CH3), 15.2
(C27, CH3) ppm; MS (ESI, MeOH): m/z = 511.3 (5% [M+H]+), 528.4
(47% [M+Na]+), 565.0 (75% [M+Na+MeOH]+), 1043.4 (100%

2556
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
[2M+Na]+). Anal. Calcd for C34H54O3 (510.79): C, 79.95; H, 10.66.
Found: C, 79.77; H, 10.81.
Calcd for C34H52O4 (524.77): C, 77.82; H, 9.99. Found: C, 77.67; H,
10.17.
5.1.17. (R)-4-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-
butyrolactone (16)
c
-
5.1.19. Methyl (28S)-3-[3b-methoxy-28-hydroxy-lup-20(29)-en-
28-yl]-propiolate (18)
Following GP3, 16 (0.35 g, 52%) was obtained from 9 (0.60 g,
1.24 mmol) as a colourless solid; mp 242–246 °C; [ ꢀ4.9 (c
5.4, CHCl3); Rf 0.39 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 2944s, 2872m, 1766s, 1735s, 1641m, 1459m, 1393m,
1369m, 1250s, 1187m, 1142w, 1073w, 1019m cmꢀ1 1H NMR
Following GP4, 18 (0.40 g, 72%) was obtained from 7 (0.47 g,
a
]
1.03 mmol) as a colourless solid; mp 235–237 °C; [
3.52, CHCl3); Rf 0.67 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3518 m, 3078w, 2955s, 2842m, 2228m, 1693s, 1649m,
1454m, 1433m, 1391m, 1376m, 1357w, 1244m, 1180m, 1098m,
1064m, 1041m, 1017w cmꢀ1 1H NMR (400 MHz, CDCl3):
a]
+21.7 (c
D
D
m
m
;
(500 MHz, CDCl3): d = 4.90 (dd, 1H, J = 6.6, 10.0 Hz, CH (28)),
4.73 (d, 1H, J = 2.1 Hz, CHa (30)), 4.57 (dd, 1H, J = 2.1, 1.6 Hz,
CHb (30)), 4.45 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 2.77 (ddd,
1H, J = 12.0, 10.4, 6.6 Hz, CH (19)), 2.51–2.45 (m, 2H, CH2 (32)),
2.19–2.11 (m, 1H, CHa (31)), 2.01 (s, 3H, Ac), 2.08–1.86 (m, 3H,
CH (13)+CHb (31)+CHa (21)), 1.76–1.52 (m, 7H, CH (18)+CHa
(12)+CHa (16)+CHa (1)+CHa (22)+CH2 (2)), 1.66 (s, 3H, CH3 (29)),
1.51–1.09 (m, 12H, CH2 (6)+CH2 (11)+CHb (22)+CHa (15)+CH2
(7)+CHb (21)+CH (9)+CHb (12)+CHb (16)), 1.02–0.95 (m, 1H, CHb
(15)), 1.00 (s, 3H, CH3 (25)), 0.99 (s, 3H, CH3 (27)), 0.84 (s, 3H,
CH3 (23)), 0.91–0.80 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3 (26)),
0.81 (s, 3H, CH3 (24)), 0.78 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C
NMR (125 MHz, CDCl3): d = 177.4 (C@O), 171.0 (C@O), 150.3
(C20, C@CH2), 110.1 (C30, CH2@C), 81.3 (C28, CH), 80.8 (C3,
CHOAc), 55.3 (C5, CH), 50.2 (C9, CH), 49.4 (C18, CH), 49.2 (C17,
Cquart.), 47.8 (C19, CH), 42.8 (C14, Cquart.), 40.9 (C8, Cquart.), 38.3
(C1, CH2), 37.8 (C4, Cquart.), 37.0 (C10, Cquart.), 36.9 (C13, CH),
34.1 (C7, CH2), 32.7 (C22, CH2), 32.1 (C16, CH2), 31.6 (C21,
CH2), 28.5 (C32, CH2), 27.9 (C23, CH3), 27.8 (C15, CH2), 24.8
(C12, CH2), 24.5 (C31, CH2), 23.6 (C2, CH2), 21.2 (Ac, CH3), 20.8
(C11, CH2), 18.8 (C29, CH3), 18.2 (C6, CH2), 16.4 (C24, CH3),
16.2 (C25, CH3), 16.1 (C26, CH3), 15.1 (C27, CH3) ppm; MS (ESI,
MeOH): m/z = 538.4 (30% [M+H]+), 557.4 (100% [M+Na]+). Anal.
Calcd for C35H54O4 (538.80): C, 78.02; H, 10.10. Found: C,
77.83; H, 10.21.
;
d = 4.99 (s, 1H, CH (28)), 4.68 (d, 1H, J = 2.1 Hz, CHa (30)), 4.57
(dd, 1H, J = 2.1, 1.7 Hz, CHb (30)), 3.77 (s, 1H, OCH3), 3.33 (s, 1H,
OCH3), 2.86 (ddd, 1H, J = 11.2, 10.8, 5.8 Hz, CH (19)), 2.61 (dd,
1H, J = 11.6, 4.2 Hz, CHOCH3 (3)), 2.06–1.84 (m, 4H, CHa
(16)+CHa (21)+CHa (22)+CH (13)), 1.79–1.67 (m, 4H, CHa (2)+CH
(18)+CHa (12) +CHa (1)), 1.66 (s, 3H, CH3 (29)), 1.57–1.23 (m,
12H, CHa (15)+CH2 (6)+CH2 (11)+CHb (22)+ CHb (2)+CH2 (7)+CHb
(21)+ CHb (16)+CH (9)), 1.19–1.11 (m, 1H, CHb (12)), 1.05–1.02
(m, 1H, CHb (15)), 1.01 (s, 3H, CH3 (25)), 0.98 (s, 3H, CH3 (27)),
0.94 (s, 3H, CH3 (23)), 0.85–0.77 (m, 1H, CHb (1)), 0.82 (s, 3H,
CH3 (26)), 0.73 (s, 3H, CH3 (24)), 0.66 (d, 1H, J = 9.1 Hz, CH (5))
ppm; 13C NMR (125 MHz, CDCl3): d = 153.8 (C@O), 150.7 (C20,
C@CH2), 109.8 (C30, CH2@C), 88.6 (C3, CHOCH3), 88.3 (C31,
C„C), 77.8 (C32, C„C), 66.0 (C28, CH), 57.5 (OCH3), 55.8 (C5,
CH), 52.7 (OCH3), 51.0 (C17, Cquart.), 50.3 (C9, CH), 49.0 (C18,
CH), 48.5 (C19, CH), 43.0 (C14, Cquart.), 40.9 (C8, Cquart.), 38.8 (C1,
CH2), 38.6 (C4, Cquart.), 37.4 (C13, CH), 37.1 (C10, Cquart.), 34.2
(C7+C16, 2 ꢁ CH2), 34.0 (C22, CH2), 32.3 (C21, CH2), 28.0 (C23,
CH3), 27.8 (C15, CH2), 25.1 (C12, CH2), 22.2 (C2, CH2), 20.8 (C11,
CH2), 18.8 (C29, CH3), 18.1 (C6, CH2), 16.2 (C24, CH3), 16.1 (C26,
CH3), 16.0 (C25, CH3), 15.1 (C27, CH3) ppm; MS (i.e., 70 eV): m/z
(%) = 538 (12), 425 (94), 393 (62), 221 (38), 203 (57), 189 (100),
135 (46). Anal. Calcd for C35H54O4 (538.80): C, 78.02; H, 10.10.
Found: C, 77.86; H, 10.24.
5.1.18. Methyl (28S)-3-[3b,28-dihydroxy-lup-20(29)-en-28-yl]-
propiolate (17)
5.1.20. Methyl (28S)-3-[3b-acetoxy-28-hydroxy-lup-20(29)-en-
28-yl]-propiolate (19)
Following GP4, 17 (0.36 g, 67%) was obtained from betulinic
aldehyde 1 (0.47 g, 1.03 mmol) as a colourless solid; mp 139–
Following GP4, 19 (0.43 g, 73%) was obtained from 9 (0.50 g,
1.03 mmol) as a colourless solid; mp 210–212 °C; [
4.5, CHCl3); Rf 0.55 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3475s, 2955s, 2870 m, 2228m, 1713s, 1638w, 1455m,
1431m, 1380m, 1250s, 1155w, 1107w, 1066m, 1049m, 1033m
cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.98 (s, 1H, CH (28)), 4.67
a]
+18.5 (c
D
142 °C; [
acetate, 8:2); IR (KBr):
1717s, 1640w, 1455m, 1435m, 1377m, 1252s, 1139w, 1108w,
1070w, 1033m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.00 (s,
a] +5.8 (c 4.3, CHCl3); Rf 0.28 (silica gel, hexane–ethyl
D
m
= 3423br, 2943s, 2870m, 2231m,
m
;
;
1H, CH (28)), 4.67 (br s, 1H, CHa (30)), 4.56 (br s, 1H, CHb (30),
3.76 (s, 3H, OCH3), 3.18 (dd, 1H, J = 11.4, 4.8 Hz, CHOH (3)),
2.86 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.07–1.85 (m, 4H,
CHa (16)+CHa (21)+CHa (22)+CH (13)), 1.78–1.45 (m, 7H, CH
(18)+CHa (12)+CHa (1)+CH2 (2)+CHa (6)+CHa (15)), 1.66 (s, 3H,
CH3 (29)), 1.45–1.09 (m, 10H, CHb (6)+CHb (21)+CHb (22)+CHb
(16)+CH2 (7)+CH (9)+CH2 (11)+CHb (12)), 1.05–0.98 (m, 1H, CHb
(15)), 1.01 (s, 3H, CH3 (27)), 0.99 (s, 3H, CH3 (25)), 0.95 (s, 3H,
CH3 (23)), 0.81 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.68 (d,
1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d = 153.8 (C@O), 150.6 (C20, C@CH2), 109.7 (C30, CH2@C), 88.3
(C31, C„C), 78.9 (C3, CHOH), 77.8 (C32, C„C), 66.0 (C28, CH),
55.3 (C5, CH), 52.7 (OMe), 51.0 (C17, Cquart.), 50.2 (C9, CH), 49.0
(C18, CH), 48.5 (C19, CH), 43.0 (C14, Cquart.), 40.9 (C8, Cquart.),
38.8 (C1, CH2), 38.7 (C4, Cquart.), 37.4 (C13, CH), 37.1 (C10, Cquart.),
34.2 (C7+C16, 2 ꢁ CH2), 34.0 (C22, CH2), 32.2 (C21, CH2), 28.0
(C23, CH3), 27.8 (C15, CH2), 27.4 (C2, CH2), 25.0 (C12, CH2), 20.8
(C11, CH2), 18.8 (C29, CH3), 18.2 (C6, CH2), 16.1 (C26, CH3),
16.0 (C25, CH3), 15.3 (C24, CH3), 15.1 (C27, CH3) ppm; MS (ESI,
MeOH): m/z = 524.4 (10% [M+H]+), 1071.4 (100% [2M+Na]+). Anal.
(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, 6H, CH3 (26)), 0.82 (s, 6H, CH3 (23)), 0.81
(s, 3H, CH3 (24)), 0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(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 (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):

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
2557
15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 563.4 (100%
[M+Na+MeOH]+), 1071.2 (80% [2M+Na+MeOH]+). Anal. Calcd for
C34H52O3 (508.78): C, 80.26; H, 10.30. Found: C, 80.06; H, 10.45.
m/z (%) = 566 (12), 453 (100), 393 (61), 203 (19), 189 (24). Anal.
Calcd for C36H54O5 (566.81): C, 76.28; H, 9.60. Found: C, 76.17; H,
9.75.
5.1.23. (R)-4-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-2-
butenolide (22)
5.1.21. (R)-4-[3b-Hydroxy-28-norlup-20(29)-en-17b-yl]-2-
butenolide (20)
Following GP5, 22 (0.17 g, 90%) was obtained from 19 (0.20 g,
Following GP5, 20 (0.17 g, 90%) was obtained from 17 (0.20 g,
0.35 mmol) as a colourless solid; mp 273–275 °C; [
CHCl3); Rf 0.42 (silica gel, hexane–ethyl acetate, 8:2); IR (KBr):
= 3063w, 2952s, 2875 m, 1735s, 1639w, 1596w, 1466m,
1392m, 1370m, 1250s, 1190w, 1166m, 1095m, 1032m cmꢀ1 1H
a] +0.4 (c 4.6,
D
0.38 mmol) as a colourless solid; mp 106–109 °C; [
2.5, CHCl3); Rf 0.36 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3446m, 2942m, 2870m, 1753m, 1706m, 1640m,
1454m, 1377m, 1210m, 1098m, 1035m cmꢀ1 1H NMR
a]
+13.5 (c
D
m
m
;
;
NMR (500 MHz, CDCl3): d = 7.53 (dd, 1H, J = 5.6, 1.5 Hz, CH (31)),
6.18 (dd, 1H, J = 5.6, 2.2 Hz, CH (32)), 5.50 (s, 1H, CH (28)), 4.75
(br s, 1H, CHa (30)), 4.60 (br s, 1H, CHb (30)), 4.46 (dd, 1H,
J = 11.4, 4.8 Hz, CHOAc (3)), 2.90 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz,
CH (19)), 2.12 (ddd, 1H, J = 12.3, 11.9, 3.7 Hz, CH (13)), 1.86–1.72
(m, 4H, CHa (16)+CHa (21)+CH (18)+CHa (12)), 2.02 (s, 3H, Ac),
1.67 (s, 3H, CH3 (29)), 1.70–1.48 (m, 6H, CHa (1)+CH2 (2)+CHa
(6)+CHa (15)+CHa (11)), 1.47–1.09 (m, 10H, CHb (6)+CH2 (7)+CHb
(21)+CH2 (22)+CHb (16)+CH (9)+CHb (11)+CHb (12)+CHb (15)),
1.04–0.95 (m, 1H, CHb (1)), 1.03 (s, 3H, CH3 (25)), 1.02 (s, 3H, CH3
(27)), 0.85 (s, 3H, CH3 (26)), 0.84 (s, 3H, CH3 (23)), 0.83 (s, 3H,
CH3 (24)), 0.79 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d = 173.2 (C@O), 170.9 (C@O), 155.3 (C31,
CH@CH), 150.0 (C20, C@CH2), 122.2 (C32, CH@CH), 110.2 (C30,
CH2@C), 84.7 (C28, CH), 80.7 (C3, CHOAc), 55.3 (C5, CH), 50.2 (C9,
CH), 49.8 (C17, Cquart.), 49.7 (C18, CH), 47.7 (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.), 34.2 (C7, CH2), 33.5 (C16, CH2), 32.3
(C22, CH2), 31.8 (C21, CH2), 27.9 (C23, CH3), 27.8 (C15, CH2), 24.9
(C12, CH2), 23.7 (C2, CH2), 21.2 (Ac, CH3), 20.8 (C11, CH2), 19.1
(C29, CH3), 18.1 (C6, CH2), 16.4 (C24, CH3), 16.2 (C25, CH3), 16.1
(C26, CH3), 15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 537.3
(30% [M+Na]+), 1095.3 (100% [2M+Na]+). Anal. Calcd for C35H52O4
(536.79): C, 78.31; H, 9.76. Found: C, 78.20; H, 9.97.
(500 MHz, CDCl3): d = 7.53 (dd, 1H, J = 5.8, 1.1 Hz, CH (31)), 6.19
(dd, 1H, J = 5.8, 2.1 Hz, CH (32)), 5.51 (br s, 1H, CH (28)), 4.76 (d,
1H, J = 1.5 Hz, CHa (30)), 4.59 (dd, 1H, J = 2.2, 1.5 Hz, CHb (30)),
3.18 (dd, 1H, J = 11.5, 4.8 Hz, CHOH (3)), 2.90 (ddd, 1H, J = 11.6,
10.7, 6.1 Hz, CH (19)), 2.12 (ddd, 1H, J = 12.1, 11.8, 3.4 Hz, CH
(13)), 1.89–1.64 (m, 6H, CHa (21)+CHa (16)+CHa (12)+CH
(18)+CHa (1)+CHa (15)), 1.67 (s, 3H, CH3 (29)), 1.61–1.05 (m, 15H,
CH2 (2)+CH2 (6)+CHb (16)+CH2 (11)+CH2 (7)+CHb (21)+CH2
(22)+CH (9)+CHb (12)+CHb (15)), 1.03 (s, 6H, 2 ꢁ CH3 (25+27)),
0.96 (s, 3H, CH3 (23)), 0.95–0.90 (m, 1H, CHb (1)), 0.83 (s, 3H, CH3
(26)), 0.75 (s, 3H, CH3 (24)), 0.68 (dd, 1H, J = 11.6, 2.1 Hz, CH (5))
ppm; 13C NMR (125 MHz, CDCl3): d = 173.2 (C@O), 155.4 (C31,
CH@CH), 150.0 (C20, C@CH2), 122.2 (C32, CH@CH), 110.2 (C30,
CH2@C), 84.7 (C28, CH), 78.9 (C3, CHOH), 55.3 (C5, CH), 50.3 (C9,
CH), 49.8 (C17, Cquart.), 49.7 (C18, CH), 47.7 (C19, CH), 43.0 (C14,
Cquart.), 40.9 (C8, Cquart.), 38.8 (C1, CH2), 38.7 (C4, Cquart.), 37.3
(C13, CH), 37.1 (C10, Cquart.), 34.3 (C7, CH2), 33.5 (C16, CH2), 32.3
(C22, CH2), 31.9 (C21, CH2), 28.0 (C23, CH3), 27.8 (C15, CH2), 27.4
(C2, CH2), 25.0 (C12, CH2), 20.8 (C11, CH2), 19.1 (C29, CH3), 18.2
(C6, CH2), 16.2 (C26, CH3), 16.1 (C25, CH3), 15.3 (C24, CH3), 15.2
(C27, CH3) ppm; MS (ESI, MeOH): m/z = 549.3 (50% [M+Na+Me-
OH]+), 1011.4 (100% [2M+Na]+), 1043.4 (50% [2M+Na+MeOH]+).
Anal. Calcd for C33H50O3 (494.75): C, 80.11; H, 10.19. Found:
80.02; H, 10.23.
5.1.24. (3S,4R)-4-[3b-Hydroxy-28-norlup-20(29)-en-17b-yl]-3-
nitromethyl-c-butyrolactone (23)
5.1.22. (R)-4-[3b-Methoxy-28-norlup-20(29)-en-17b-yl]-2-
butenolide (21)
To a solution of 22 (0.1 g, 0.2 mmol) in nitromethane (3 ml)
DBU (6 mg, 0.04 mmol) was added and stirred at room tempera-
ture. When TLC revealed the absence of starting material, the solu-
tion was concentrated in vacuo and the residue purified by column
chromatography (silica gel, hexane–ethyl acetate, 8:2). Compound
23 (0.1 g, 88%) was obtained as a colourless solid; mp 306–309 °C;
Following GP5, 21 (0.17 g, 92%) was obtained from 18 (0.20 g,
0.37 mmol) as a colourless solid; mp 259–261 °C; [
2.3, CHCl3); Rf 0.73 (silica gel, hexane–ethyl acetate, 8:2); IR
(KBr): = 3482 m, 3074w, 2943s, 1703m, 1641w, 1439m, 1408w,
1391m, 1375m, 1231m, 1179m, 1100m, 1035w, 1019m cmꢀ1 1H
a
]
D
+39.2 (c
m
;
NMR (500 MHz, CDCl3): d = 7.53 (dd, 1H, J = 5.8, 1.3 Hz, CH (31)),
6.19 (dd, 1H, J = 5.8, 2.1 Hz, CH (32)), 5.51 (br s, 1H, CH (28)),
4.76 (d, 1H, J = 2.0 Hz, CHa (30)), 4.60 (dd, 1H, J = 2.2, 1.3 Hz, CHb
(30)), 3.34 (s, 1H, CHOCH3 (3)), 2.91 (ddd, 1H, J = 11.2, 10.4,
7.0 Hz, CH (19)), 2.12 (ddd, 1H, J = 12.2, 11.7, 3.6 Hz, CH (13)),
1.90–1.63 (m, 6H, CHa (21)+CHa (16)+CH (18)+CHa (2)+CHa
(12)+CHa (1)), 1.67 (s, 3H, CH3 (29)), 1.57–1.05 (m, 15H, CH2
(6)+CH2 (11)+CHb (2)+CH2 (7)+CH2 (22)+CHb (21)+CH (9)+CHb
(12)+CH2 (15)+CHb (16)), 1.03 (s, 6H, 2 ꢁ CH3 (25+27)), 0.94 (s,
3H, CH3 (26)), 0.85–0.80 (m, 1H, CHb (1)), 0.83 (s, 3H, CH3 (23)),
0.74 (s, 3H, CH3 (24)), 0.67 (dd, 1H, J = 11.6, 2.2 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d = 173.2 (C@O), 155.4 (C31, CH@CH),
150.0 (C20, C@CH2), 122.2 (C32, CH@CH), 110.2 (C30, CH2@C),
88.5 (C3, CHOCH3), 84.7 (C28, CH), 57.5 (OCH3), 55.8 (C5, CH),
50.3 (C9, CH), 49.8 (C17, Cquart.), 49.7 (C18, CH), 47.7 (C19, CH),
43.0 (C14, Cquart.), 41.0 (C8, Cquart.), 38.8 (C1, CH2), 38.5 (C4, Cquart.),
37.3 (C13, CH), 37.2 (C10, Cquart.), 34.3 (C7, CH2), 33.5 (C16, CH2),
32.3 (C22, CH2), 31.9 (C21, CH2), 28.0 (C23, CH3), 27.8 (C15, CH2),
25.0 (C12, CH2), 22.2 (C2, CH2), 20.8 (C11, CH2), 19.1 (C29, CH3),
18.1 (C6, CH2), 16.2 (C24, CH3), 16.1 (C25, CH3), 16.0 (C26, CH3),
[
a
]
ꢀ0.5 (c 4.1, CHCl3); Rf 0.17 (silica gel, hexane–ethyl acetate,
D
8:2); IR (KBr):
m
= 2942s, 2870m, 1772s, 1725s, 1641w, 1555s,
1H NMR (500 MHz,
1467w, 1378m, 1250s, 1178m, 1031m cmꢀ1
;
CDCl3): d = 4.71 (d, 1H, J = 3.9 Hz, CH (28)), 4.68 (d, 1H, J = 1.9 Hz,
CHa (30)), 4.58 (dd, 1H, J = 1.9, 1.5 Hz, CHb (30)), 4.47 (dd, 1H,
J = 12.9, 5.6 Hz, NO2CHa), 4.47–4.41 (m, 2H, CHOAc (3)+NO2CHb),
3.14–3.06 (m, 1H, CH (31)), 2.84 (dd, 1H, J = 18.8, 10.5 Hz, CHa
(32)), 2.80 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.43 (dd, 1H,
J = 18.8, 4.6 Hz, CHb (32)), 1.97 (s, 3H, Ac), 1.96 (ddd, 1H, J = 12.3,
11.9, 3.7 Hz, CH (13)), 1.82–1.70 (m, 2H, CHa (21)+CH (18)), 1.61
(s, 3H, CH3 (29)), 1.65–1.51 (m, 7H, CHa (22)+CHa (12)+CHa
(1)+CHa (16)+CH2 (2)+CHa (15)), 1.47–1.07 (m, 11H, CHb (22)+CH2
(6)+CH2 (11)+CH2 (7)+CHb (21)+CHb (16)+CH (9)+CHb (12)), 1.05–
1.00 (ddd, 1H, J = 14.4, 4.0, 2.5 Hz, CHb (15)), 0.95–0.87 (m, 1H,
CHb (1)), 0.96 (s, 3H, CH3 (25)), 0.95 (s, 3H, CH3 (27)), 0.80 (s, 3H,
CH3 (26)), 0.78 (s, 3H, CH3 (23)), 0.77 (s, 3H, CH3 (24)), 0.72 (d,
1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 174.1
(C@O), 170.9 (C@O), 149.9 (C20, C@CH2), 110.3 (C30, CH2@C),
82.2 (C28, CH), 80.7 (C3, CHOAc), 78.1 (C16, CH2NO2), 55.3 (C5,
CH), 50.4 (C17, Cquart.), 50.1 (C9, CH), 49.8 (C18, CH), 47.1 (C19,

2558
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 2549–2558
CH), 43.0 (C14, Cquart.), 41.0 (C8, Cquart.), 38.3 (C1, CH2), 37.8 (C4,
Cquart.), 37.0 (C10, Cquart.), 36.7 (C13, CH), 35.8 (C31, CH), 34.1 (C7,
CH2), 33.4 (C16, CH2), 32.6 (C32, CH2), 32.5 (C22, CH2), 31.4 (C21,
CH2), 27.9 (C23, CH3), 27.7 (C15, CH2), 25.0 (C12, CH2), 23.6 (C2,
CH2), 21.3 (Ac, CH3), 20.7 (C11, CH2), 19.1 (C29, CH3), 18.0 (C6,
CH2), 16.4 (C24, CH3), 16.1 (C25+C26, 2 ꢁ CH3), 15.1 (C27, CH3)
ppm; MS (ESI, MeOH): m/z = 537.3 (30% [M+Na]+), 1095.3 (100%
[2M+Na]+). Anal. Calcd for C36H55NO6 (597.83): C, 72.33; H, 9.27;
N, 2.34. Found: C, 72.21; H, 9.41; N, 2.29.
duced by drug exposure) were collected, washed with PBS and
lysed with lysis buffer (100 mM Tris–HCL pH 8.0; 20 mM EDTA;
0.8% SDS) (all from Sigma Aldrich). Then they are treated with
RNAse A at 37 °C for 2 h and proteinase K at 50 °C (both from Roche
Diagnostics chemical company, Mannheim, Germany). DNA lad-
dering was observed by running the samples on 2% agarose gel fol-
lowed by ethidium bromide (Sigma–Aldrich) staining.
Acknowledgments
5.2. Biology
We like to thank Dr. Ralph Kluge for numerous ESI-MS mea-
surements and Dr. Dieter Ströhl for the NMR measurements. The
cell lines were kindly provided by Dr. Thomas Müller (Dept. of Hae-
matology/Oncology, Univ. Halle).
5.2.1. Cell lines and culture conditions
The cell lines 518A2, 8505C, A253, A2780, A431, A549, DLD-1,
FaDu, HCT-116, HCT-8, HT-29, LIPO, MCF-7, SW1736 and SW480
were included in this study. Cultures were maintained as mono-
layer in RPMI 1640 (PAA Laboratories, Pasching, Germany) supple-
mented with 10% heat inactivated fetal bovine serum (Biochrom
AG, Berlin, Germany) and penicillin/streptomycin (PAA Laborato-
ries) at 37 °C in a humidified atmosphere of 5% CO2/95% air.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.042.
References and notes
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The cytotoxic activities of our compounds were evaluated using
the sulforhodamine-B (SRB) (Sigma–Aldrich) microculture colori-
metric assay. In short, exponentially growing cells were seeded
into 96-well plates on day 0 at the appropriate cell densities to pre-
vent confluence of the cells during the period of experiment. After
24 h, the cells were treated with serial dilutions of the compounds
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to the cells. The percentages of surviving cells compared to un-
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the 96 well plates was discarded and the cells were fixed with
10% TCA. For a thorough fixation, the plates were allowed to stand
at 4 °C. After fixation, the cells were washed in a strip washer for
four times with water using alternate dispensing and aspiration
procedures. The plates were dyed with 100 ll of 0.4% SRB (sulfo-
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with floating cells was collected and centrifuged to collect the dead
and apoptotic cells. The cell pellet was suspended in serum free
media. Equal amounts of cell suspension and trypan-blue were
mixed and analyzed by microscope. The viable cells exclude the
dye and are colorless and cells whose cell membrane was de-
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respective IC 90 doses of the compounds for 24 h. Floating cells (in-
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