Synthesis and biological evaluation of antitumour-active betulin derivatives

Article abstract of DOI:10.1016/j.bmc.2009.12.024

The reaction of betulinic aldehydes with various carbon nucleophiles gave a series of new betulin derivatives, among them epoxides, glycidic derivatives and β-hydroxy carbonyl compounds. Subsequent transformations of the β-hydroxy carbonyls lead to 1,3-diketo- and α,β-unsaturated betulin derivatives. These compounds were assayed for cytotoxicity using 15 human cancer cell lines and a colorimetric SRB-assay. Several compounds revealed significant antitumour activity.

Full text of DOI:10.1016/j.bmc.2009.12.024

Bioorganic & Medicinal Chemistry 18 (2010) 1344–1355
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of antitumour-active betulin derivatives
a
a
a
b
b
René Csuk a, , Alexander Barthel , Ralph Kluge , Dieter Ströhl , Harish Kommera , Reinhard Paschke
*
a Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle, Saale, Germany
b BioSolutions 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 reaction of betulinic aldehydes with various carbon nucleophiles gave a series of new betulin deriv-
Received 28 September 2009
Revised 1 December 2009
Accepted 8 December 2009
Available online 23 December 2009
atives, among them epoxides, glycidic derivatives and b-hydroxy carbonyl compounds. Subsequent trans-
formations of the b-hydroxy carbonyls lead to 1,3-diketo- and a,b-unsaturated betulin derivatives. These
compounds were assayed for cytotoxicity using 15 human cancer cell lines and a colorimetric SRB-assay.
Several compounds revealed significant antitumour activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Betulin
Betulinic acid
Apoptosis
Antitumour activity
1. Introduction
aldehydes as starting materials for further transformations. We
examined carbanionic reactions for the synthesis of epoxides, gly-
Betulin I and betulinic acid (BA) II are triterpenes; they are
widely spread in plant kingdom and possess a lupane-skeleton
(see Fig. 1).
cidic derivatives and b-hydroxy esters. Their antitumour potency
was examined by a sulforhodamine-B (SRB) assay for 15 cancer cell
lines.
They aroused interest by their versatile pharmacological prop-
erties. Firstly recognized were BA and several derivatives for inhib-
iting HIV-1 propagation in lymphocyte cells.1 Extending studies
revealed two fundamental mechanisms depending on the deriva-
tized position. Thus, BA compounds varied at the hydroxy group
interrupts CA-SP1 junction in Gag processing which alters the cell
maturation.2,3 In contrast, amide compounds of BA, for example,
RPR103611 affect the interface between gp120 and gp41 resulting
in the inhibition of virus–cell fusion.4,5 Also, betulin derivatives6
revealed their great potential for cancer therapy because of their
high selectivity for cancer cells. They trigger apoptosis7 by causing
a loss of transmembrane potential in mitochondria, and they re-
lease soluble apoptotic factors8, for example, cytochrome c and
AIF to the cytoplasm. Other studies revealed also the activation
of caspases and the formation of reactive oxygen species (ROS) as
essential steps of the cell death.9 Also a partial involvement of
the p38 pathway in apoptosis was suggested due to the observa-
tion10 of phosphorylated proapoptotic mitogen-activated protein
kinases (MAPKs).
2. Chemistry
Several11,12 of betulinic aldehydes were allowed to react with
different carbon nucleophiles (Scheme 1).
The synthesis of epoxide compounds was achieved either by the
application of sulfonium ylids or Darzens reaction.13 However, the
use of the trimethylsulfoxonium iodide/sodium hydride14 leads to
the formation of the two diastereomeric epoxides 1 and 2 in a ratio
of 3:1. Noticeable amounts of the pure isomers could only be
Because of the high selectivity for cancer cells; the synthesis of
betulin derivatives has become an appealing research field for can-
cer therapy. So far only little attention has been paid to betulinic
* Corresponding author. Tel.: +49 345 55 25660; fax: +49 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
Figure 1. Structure of betulin and betulinic acid.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.024

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
1345
Scheme 1. Addition of C-nucleophiles to betulinic aldehydes. Reagents and conditions: (a) trimethylsulfoxonium iodide, NaH, DMSO; (b) DIA, n-BuLi, BF3OEt2, dry THF,
ꢀ78 °C?25 °C, 1 h; (c) DIA, n-BuLi, dry THF, ꢀ78 °C?25 °C, 1 h; (d) silyl enol ether, BF3OEt2, dry THF, ꢀ78 °C?25 °C, 2 h.
earned by laborious preparative HPLC. Therefore we refrained from
the synthesis of further derivatives and turned towards Darzens
reaction15 to produce glycidic derivatives 3–6. This reaction was
3. Results and discussion
The compounds 1–19 were tested for their cytotoxicity against
15 human cancer cell lines by using the SRB-protocol.22 The sum-
marized IC50-values in Table 1 were obtained from the correspond-
ing dose–response curves. The epoxide compounds 1 and 2 showed
no cytotoxicity for several cell lines at the highest tested concen-
tration, but IC50-values below 20 lM were observed for colon can-
cer cell lines HT-29 and SW480. Glycidic derivative 4 and 6
revealed a lack of activity.
Betulinic acid has three positions (C-3, C-20 and C-28) where
modifications can be performed to yield derivatives for struc-
ture–activity relationship studies. In this work, compound 5
showed a high antitumour potency with an averaged IC50-value
performed using the corresponding a-halogen carbonyl compound
and LDA. A high stereoselectivity was observed yielding only the
shown trans-isomer. Structure elucidation was performed by com-
parison of the observed NOE-correlations with the calculated
structure (Fig. 2).
The addition of ester enolates16 proceeds smoothly under Lewis
acid catalysis. Alternatively the reaction could be carried out with
the corresponding silyl enol ethers.17 The conversion with ethyl
acetate gave besides the b-hydroxy esters 7 and 9 also b-keto es-
ters 8 and 10. The b-hydroxy-c-butyrolactones 11 and 12 were ob-
tained by addition of the corresponding silyl enol ethers. Thereby
only the anti-product was formed. However, lithium enolates or si-
lyl enol ethers of ketones or aldehydes gave no reaction at all. Fur-
ther derivatizations were carried out with b-hydroxy esters 7 and
9. However, their reactivity was limited. The oxidation under
Swern18 or Jones19 conditions gave b-keto-esters 13 and 14. The
of 5.5 lM. In general, the b-hydroxy esters 7 and 9 are less active
then the matching b-keto esters 8 and 10. Interestingly, oxidizing
the b-hydroxy group of compounds 7 and 9 increases the activity,
whereas the elimination products 15 and 16 are not cytotoxic
even at the highest tested concentration. Similar results were ob-
synthesis of
mesylation and subsequently elimination.20 Similar to the open
chained derivatives the -butyrolactone compound 11 was trans-
formed to the b-keto-derivative 17 by Jones oxidation and to the
-methylene- -butyrolactone 18. Treating the b-hydroxy com-
pounds 7 and 8 with trifluoroacetic acid resulted in a rearrange-
ment21 of the isopropylidene moiety thus resulting in the
formation of the allobetulin derivatives 19 and 20, respectively
(see Scheme 2).
a,b-unsaturated esters 15 and 16 was achieved by
served for analogue
derivative 17 showed an averaged IC50-value of 8.2
the -methylene- -butyrolactone 18 possesses no cytotoxicity in
many cell lines at the highest tested concentration of 30 M.
These results support assumptions from other groups, the keto-
group at C-28 position is important for antitumour potency.23,24
Rearranging compound 8 to the allobetulin derivative 20 leads
c
-butyrolactone compounds. The b-keto-
l
M whereas
c
a
c
l
a
c
to an higher activity with an averaged IC50-value of 8.5
lM. Our
investigations demonstrate that simple modifications of the

1346
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
Figure 2. Calculated structures (PM3) and observed characteristic NOE-correlations of 3 (upper left), 6 (upper right), 11 (lower left) and 12 (lower right).
parent structure of betulinic acid can provide potentially active
derivatives.
in comparison to betulinic acid. The best compounds in this
screening are glycidic amide 5 with an average IC50-value of
To control whether the compounds induce cell death by trigger-
ing apoptosis further investigations were performed for selected
compounds 5, 13 and 19 (Fig. 3). The trypan-blue dye exclusion
test is a qualitative test to distinguish between apoptotic cells with
an intact cell membrane and necrotic cells that have a lysed cell
membrane. The intact cells can exclude the blue dye whereas ne-
crotic cells are stained blue. A characteristically appearance in
apoptosis is the fragmentation of the DNA into smaller pieces of
ca. 180 bp which can be noted in gel electrophoresis as ladders.25,26
5.5
l
M and b-keto-esters 13 and 14 with intermediate IC50-value
M, respectively. However, a greater number of
of 3.4 and 5.7
l
derivatives is needed for establishing a reliable structure–activity
relationship study to design a more active betulinic acid derived
antitumour agent.
5. Experimental
5.1. Chemistry
4. Conclusion
5.1.1. General
Melting points are uncorrected (Leica hot stage microscope),
NMR spectra were recorded using the Varian spectrometers Gem-
ini 200, Gemini 2000 or Unity 500 (d given in ppm, J in Hz, internal
Me4Si), optical rotations were obtained using a Perkin–Elmer 341
polarimeter (1 cm microcell), IR spectra (film or KBr pellet) on a
Perkin–Elmer FT-IR spectrometer Spectrum 1000, MS spectra were
The synthesis of new betulin derivatives with potent pharmaco-
logical properties can be achieved starting from substituted betuli-
nic aldehydes by reaction with carbon nucleophiles.
These additions proceed stereoselectively for lithium enolates
and silyl enol ethers. Several compounds hold a higher cytotoxicity

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
1347
Scheme 2. Reactions of b-hydroxy esters and b-hydroxy ketones. Reagents and conditions: (a) CrO3, H2SO4, acetone, 0 °C, 30 min; (b) (CO)2Cl2, DMSO, TEA, dry CH2Cl2,
ꢀ78 °C?25 °C, 1 h; (c) i—methanesulfonyl chloride, TEA, dry CH2Cl2, 0 °C?25 °C, 3 h; ii—K2CO3, DMF, 60 °C, 24 h; (d) TFA, CH2Cl2, 24 h.
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) instrument. TLC was performed on silica gel (Merck
5554). Spots were detected by spraying a solution of ammonium
molybdate and cerium(IV) sulfate in sulfuric acid, followed by
gently heating. The solvents were dried according to usual
procedures.
5.1.2. General procedure for Darzens reactions (GP1)
To a solution of diisopropylamine (417 mg, 4.12 mmol) in dry
THF under argon was added at 0 °C butyllithium (1.6 M in hexane,
2.6 ml, 4.12 mmol) and then cooled to ꢀ78 °C. Subsequently, a
solution of the corresponding 2-halo ester (4.12 mmol) was added
and stirring was continued for 20 min at ꢀ78 °C. Then a solution of
3-acetylbetulinic aldehyde (0.50 g, 1.03 mmol) and BF3OEt2

1348
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
Table 1
Cytoxicity of compounds in a panel of various human cancer cell lines
Cell line
IC50-values in lM for cancer cell lines
BA
1
2
3
4
5
6
7
8
9
10
11
12
13
2.5
14
15
16
17
9.5
18
19
20
518A2
A431
A253
FADU
A549
A2780
DLD-1
HCT-8
HCT-116
HT-29
SW480
8505C
SW1736
MCF-7
Lipo
11.9
15.4
11.1
10.4
14.9
11.0
17.5
17.8
13.3
16.1
6.4
26.8
20.1
18.8
NA
NA
28.2
NA
20.6
19.9
15.4
16.2
22.3
NA
NA
NA
NA
NA
NA
NA
12.3
NA
NA
18.1
NA
NA
NA
17.6
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
7.0
4.3
3.5
5.9
5.6
4.7
7.9
2.7
2.3
9.6
6.0
8.3
2.7
4.6
7.7
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
18.7
NA
11.6
16.1
10.0
10.5
16.8
9.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
19.4
20.7
17.0
16.2
14.6
12.1
14.0
12.5
14.6
16.8
15.3
17.6
13.8
20.7
17.8
17.9
11.7
16.8
17.5
21.1
12.4
17.4
8.3
16.5
8.9
15.7
12.3
17.0
8.8
16.3
14.7
8.5
29.9
12.8
17.0
14.7
12.5
15.9
6.3
3.9
7.4
6.5
5.3
4.3
7.9
4.1
3.6
4.1
5.9
7.9
7.2
4.7
6.4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
19.4
NA
NA
NA
NA
NA
NA
13.9
NA
NA
NA
18.8
NA
NA
NA
10.4
9.8
10.9
7.0
5.2
4.7
7.2
6.1
7.0
13.2
5.8
23.5
20.1
NA
NA
NA
2.6
2.2
3.0
2.2
1.8
3.9
3.0
2.4
12.0
2.2
2.3
2.3
6.8
2.2
7.3
7.0
5.8
5.5
6.4
9.5
7.8
6.2
14.8
9.6
9.2
6.2
7.7
10.0
22.6
25.1
24.7
21.9
11.9
NA
12.0
9.3
16.7
17.3
NA
NA
28.2
21.0
NA
16.5
13.0
21.8
NA
NA
9.3
24.6
20.3
18.2
16.8
24.3
NA
11.3
13.0
18.5
13.0
9.6
10.6
13.9
9.0
10.7
NA
16.4
22.2
18.5
11.7
17.4
6.7
NA
8.9
11.3
9.2
11.6
14.9
9.7
23.5
27.3
NA
25.9
27.1
NA
23.6
22.4
28.6
24.7
10.5
Values are derived from dose–response curves obtained by measuring the percentage of viable cells relative to untreated controls after 96 h exposure of the cell line to the
test compounds using an SRB-assay for melanoma (518A2), zervic cancer (A431), head and neck tumour (A253, FADU), lung carcinoma (A549), ovarian cancer (A2780), colon
cancer (DLD-1, HCT-8, HCT-116, HT-29, SW480), anaplastic thyroid cancer (8505c, SW-1736), mamma carcinoma (MCF-7) and liposarcoma. Values are the average from at
least three independent experiments. Variation was 10%. NA = no inhibition of cell growth at the highest concentration (30
lM).
Figure 3. (from left to right): Trypan-blue exclusion test of compounds 5 for colon cancer cell line HCT-116, 13 for head and neck tumour A253 and 19 for lung carcinoma
A549 and DNA laddering of compounds 5 for colon cancer cell line SW480, 13 for colon cancer cell line HCT-8 and 19 for colon cancer cell line SW480 after treatment with
IC90-concentrations for 24 h.
(0.1 ml) was added. The reaction mixture was warmed to room
temperature over 2 h and stirred over night. The solution was di-
luted with brine (20 ml), the phases were separated and the aque-
ous layer was extracted with diethyl ether (2 ꢁ 20 ml). The
combined organic extracts were dried over Na2SO4, concentrated
in vacuo and the residue purified by column chromatography (sil-
ica gel, hexane/ethyl acetate 8:2).
The reaction mixture was warmed to room temperature and stir-
red over night. Subsequently, the solution was diluted with brine
(20 ml), the phases were separated and the aqueous layer ex-
tracted with diethyl ether (2 ꢁ 20 ml). The combined organic ex-
tracts were dried over Na2SO4, concentrated in vacuo and the
residue purified by column chromatography (silica gel, hexane/
ethyl acetate 8:2).
5.1.3. General procedure for the addition of lithium enolates
(GP2)
5.1.5. General procedure for the Swern oxidation (GP4)
To a solution of oxalyl chloride (0.6 g, 4.7 mmol) in dry dichlo-
romethane (CH2Cl2) (10 ml) at ꢀ78 °C a solution of dry DMSO
(0.78 ml) in dry dichloromethane (CH2Cl2) (10 ml) was slowly
added and stirring at ꢀ78 °C was continued for another 30 min.
Maintaining this temperature a solution of the b-hydroxy-carbonyl
compound (4.26 mmol) in dry CH2Cl2 (5 ml) was slowly added and
stirring at this temperature was continued for another 2 h. Then
dry TEA (1.4 ml) was added and then the reaction mixture was al-
lowed to warm to room temperature. Diluted aq HCl (10%, 100 ml)
was added under vigorous stirring, the phases were separated, the
organic layer was washed with aq Na2CO3 (2 ꢁ 10 ml), water
(2 ꢁ 10 ml), and brine (2 ꢁ 10 ml), the solvents were removed,
and the residue was subjected to chromatography (silica gel, hex-
ane/ethyl acetate 9:1).
To a solution of diisopropylamine (417 mg, 4.12 mmol) in dry
THF under argon was added at 0 °C butyllithium (1.6 M in hexane,
2.6 ml, 4.12 mmol) and then cooled to ꢀ78 °C. Subsequently, a
solution of the corresponding ester (4.12 mmol) was added and
stirring was continued for 20 min at ꢀ78 °C. Then a solution of
3-O-acetylbetulinic aldehyde (0.50 g, 1.03 mmol) and BF3OEt2
(0.1 ml) was added. The reaction mixture was warmed to room
temperature over 2 h and stirred over night. The solution was
diluted with brine (20 ml), the phases were separated and the
aqueous layer extracted with diethyl ether (2 ꢁ 20 ml). The com-
bined organic extracts were dried over Na2SO4, concentrated in
vacuo and the residue purified by column chromatography (silica
gel, hexane/ethyl acetate 8:2).
5.1.4. General procedure for the addition of silyl enol ether
(GP3)
A mixture of 3-O-acetylbetulinic aldehyde (0.50 g, 1.03 mmol)
and the corresponding silyl enol ether (3 mmol) in THF (20 ml)
under argon was cooled to ꢀ78 C, then BF3OEt2 (0.1 ml) was added.
5.1.6. General procedure for the Jones oxidation (GP5)
Chromium(VI) oxide (300 mg) was dissolved in aq H2SO4 (35%,
1 ml) and added dropwise to a stirred solution of the b-hydroxy-
carbonyl compound (1.84 mmol) in acetone (10 ml). After TLC re-
vealed the absence of starting material, the excess chromium(VI)

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
1349
oxide was destroyed by the addition of isopropanol (2 ml). The
solution was concentrated in vacuo and the residue partitioned be-
tween H2O (30 ml) and CH2Cl2 (30 ml). The layers were separated
and the aqueous layer was extracted with CH2Cl2 (10 ml). The com-
bined organic extracts were dried over Na2SO4, evaporated to dry-
ness and purified by column chromatography (silica gel, hexane/
ethyl acetate 9:1).
37.3 (C13, CH), 37.1 (C10, Cquart.), 34.2 (C7, CH2), 33.0 (C22, CH2),
32.0 (C16, CH2), 30.8 (C21, CH2), 28.1 (C15, CH2), 27.9 (C23, CH3),
27.4 (C2, CH2), 24.9 (C12, CH2), 20.9 (C11, CH2), 18.7 (C29, CH),
18.2 (C6, CH2), 16.1 (C26, CH3), 15.9 (C25, CH3), 15.3 (C24, CH3),
14.5 (C27, CH3) ppm; MS (e.i., 70 eV): m/z (%) 454 (34), 423 (29),
411 (51), 246 (27), 217 (63), 207 (88), 201 (57), 189 (92), 175
(45), 159 (57), 145 (64), 135 (79), 119 (82), 107 (95), 95 (82), 81
(100); Anal. Calcd for C31H50O2 (454.73): C, 81.88; H, 11.08. Found:
C, 81.53; H, 10.80.
5.1.7. General procedure for the eliminations (GP6)
To a solution of b-hydroxy-carbonyl compound (0.35 mmol),
DMAP (24 mg, 0.2 mmol) and TEA (0.35 g, 3.5 mmol) in dry dichlo-
romethane (CH2Cl2) (10 ml) was added methanesulfonyl chloride
(0.4 g, 3.5 mmol) and stirring over night. The reaction was
quenched by the addition of saturated Na2CO3 solution (20 ml).
The phases were separated and the aq layer was extracted with
CH2Cl2 (2 ꢁ 10 ml). The combined extracts were dried over Na2SO4
and concentrated in vacuo. The crude mesylate and K2CO3 (0.48 g,
3.5 mmol) were dissolved in DMF (10 ml) and heated to 70 °C for
5 h. Then the mixture was concentrated in vacuo and treated with
water (20 ml) and CH2Cl2 (20 ml). The phases were separated and
the aq layer was extracted with CH2Cl2 (2 ꢁ 10 ml). The combined
organic layers were dried over Na2SO4, evaporated to dryness and
purified by column chromatography (silica gel, hexane/ethyl ace-
tate 9:1).
Data for 2. Mp 217 °C; ½a2D0ꢂ 23.6 (c 5.0, CHCl3); RF 0.48 (silica gel,
hexane/ethyl acetate, 8:2); IR (KBr):
m 2941s, 2870m, 1642w,
1453w, 1376w, 1293w, 1181w, 1105w, 1082w, 1045w cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 4.60 (d, 1H, J = 2.3 Hz, CHa (30)), 4.49–
4.46 (m, 1H, CHb (30)), 3.07 (dd, 1H, J = 11.5, 4.7 Hz, CHOH (3)),
3.02 (dd, 1H, J = 4.2, 2.9 Hz, CH (28)), 2.63 (dd, 1H, J = 4.6, 4.2 Hz,
CHa (31)), 2.41 (dd, 1H, J = 4.6, 2.9 Hz, CHb (31)), 2.36 (ddd, 1H,
J = 11.5, 11.0, 6.8 Hz, CH (19)), 1.90 (ddd, 1H, J = 12.2, 12.2,
3.8 Hz, CH (13)), 1.82 (ddd, 1H, J = 13.4, 13.9, 4.4 Hz, CHa (15)),
1.68–1.56 (m, 2H, CHa (16)+CHa (12)), 1.56 (s, 3H, CH3 (29)),
1.55–1.36 (m, 6H, CHa (1)+CH2 (2)+CH (18)+CHa (21)+CHa (6)),
1.34–1.17 (m, 9H, CH2 (11)+CHb (6)+CHb (16)+CH2 (7)+CH
(9)+CHa (22)+CHb (21)), 1.06–0.92 (m, 3H, CHb (22)+CHb (15)+CHb
(12)), 0.93 (s, 3H, CH3 (27)), 0.88 (s, 3H, CH3 (25)), 0.85 (s, 3H,
CH3 (23)), 0.80 (ddd, 1H, J = 13.3, 13.3, 3.7 Hz, CHb (1)), 0.71 (s,
3H, CH3 (26)), 0.64 (s, 3H, CH3 (24)), 0.57 (d, 1H, J = 9.5 Hz, CH
(5)) ppm; 13C NMR (125 MHz, CDCl3): d 149.8 (C20, C@CH2),
110.3 (C30, CH2@C), 78.9 (C3, CHOH), 55.3 (C5, CH), 52.8 (C28,
CH), 50.4 (C9, CH), 48.7 (C19, CH), 48.6 (C18, CH), 46.1 (C31,
CH2), 45.5 (C17, Cquart.), 42.6 (C14, Cquart.), 41.0 (C8, Cquart.), 38.8
(C4, Cquart.), 38.7 (C1, CH2), 37.5 (C13, CH), 37.1 (C10, Cquart.), 34.2
(C7, CH2), 32.7 (C22, CH2), 31.2 (C16, CH2), 30.5 (C21, CH2), 27.9
(C23, CH3), 27.7 (C15, CH2), 27.4 (C2, CH2), 25.0 (C12, CH2), 20.8
(C11, CH2), 18.6 (C29, CH), 18.3 (C6, CH2), 16.1 (C26, CH3), 15.9
(C25, CH3), 15.3 (C24, CH3), 14.7 (C27, CH3) ppm; MS (e.i., 70 eV):
m/z (%) 454 (34), 423 (18), 246 (20), 215 (47), 207 (68), 201 (57),
189 (100), 173 (44), 159 (53), 145 (63), 135 (74), 119 (68), 107
(86), 93 (82), 81 (78); Anal. Calcd for C31H50O2 (454.73): C,
81.88; H, 11.08. Found: C, 81.50; H, 10.75.
5.1.8. General procedure for allobetulin rearrangement (GP7)
To a solution of the corresponding b-hydroxy carbonyl com-
pound (0.35 mmol) in dichloromethane (CH2Cl2) (20 ml) was
added TFA (1 ml) and stirred over night. The solution was concen-
trated in vacuo and the residue purified by column chromatogra-
phy (silica gel, hexane/ethyl acetate 8:2).
5.1.9. 3b-Hydroxy-17b-[(2R)-2-oxiranyl]-28-norlup-20(29)-en
(1) and 3b-hydroxy-17b-[(2S)-2-oxiranyl]-28-norlup-20(29)-en
(2)
To a mixture of trimethylsulfoxonium iodide (2.7 mmol) in
DMSO (10 ml), NaH (60% suspension in mineral oil, 0.11 g,
2.7 mmol) was added and stirred for 15 min. Then betulinic alde-
hyde (0.6 g, 1.35 mmol) was added and stirring was continued
for 24 h. The reaction mixture was diluted with H2O (100 ml)
and extracted with ethyl acetate (2 ꢁ 100 ml). The combined or-
ganic extracts were dried over Na2SO4, concentrated in vacuo
and the residue purified by column chromatography (silica gel,
hexane/ethyl acetate 8:2). The obtained mixture of the diastereo-
meric epoxides (0.58 g, 95%, 28R/28S ratio: 3:1) was separated by
semi-preparative HPLC (RP 18, 25 ꢁ 250 mm, 10 ml/min, CH3CN/
H2O, 86:14, k = 210 nm).
5.1.10. (3S,2R)-3-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-
oxirane-2-carboxylic acid methyl ester (3)
Compound 3 (0.25 g, 45%) was obtained from 3-O-acetylbetuli-
nic aldehyde (0.50 g, 1.03 mmol) and methyl 2-chloroacetate
(0.46 g, 4.12 mmol) following GP1 as a colourless solid; mp
189 °C; ½a2D0ꢂ 28.0 (c 4.7, CHCl3); RF 0.35 (silica gel, hexane/ethyl
acetate, 9:1); IR (KBr):
m 3424br, 2952s, 2869m, 1743s, 1721s,
1641m, 1455m, 1392m, 1378m, 1246s, 1159w, 1131w, 1105w,
Data for 1.Mp 236 °C; ½a2D0ꢂ 26.5 (c 4.3, CHCl3); RF 0.48 (silica gel,
1081w, 1016m cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.73 (br s, 1H,
;
hexane/ethyl acetate, 8:2); IR (KBr):
m
2942s, 2870m, 1639w,
CHa (30)), 4.59 (br s, 1H, CHb (30)), 4.44 (dd, 1H, J = 11.4, 4.8 Hz,
CHOAc (3)), 3.78 (s, 3H, OCH3), 3.33–3.27 (m, 2H, CH (28)+CH
(31)), 2.58 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)), 2.02 (s, 3H,
Ac), 1.99–1.85 (m, 2H, CH (13)+CHa (21)), 1.83–1.74 (m, 1H, CHa
(12)), 1.66 (s, 3H, CH3 (29)), 1.70–1.51 (m, 7H, CH (18)+CHa
(1)+CHa (16)+CH2 (2)+CHa (6)+CHa (15)), 1.50–1.13 (m, 9H, CHa
(22)+CH2 (11)+CHb (6)+CHb (16)+CH2 (7)+CH (9)+CHb (21)), 1.12–
0.86 (m, 4H, CHb (22)+CHb (15)+CHb (12)+CHb (1)), 0.96 (s, 3H,
CH3 (25)), 0.94 (s, 3H, CH3 (27)), 0.83 (s, 6H, CH3 (23)+CH3 (26)),
0.81 (s, 3H, CH3 (24)), 0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C
NMR (125 MHz, CDCl3): d 170.9 (C@O), 170.2 (C@O), 149.6 (C20,
C@CH2), 110.3 (C30, CH2@C), 80.6 (C3, CHOAc), 60.0 (C28, CH),
55.3 (C5, CH), 52.4 (OCH3), 50.6 (C31, CH), 50.3 (C9, CH), 48.8
(C18, CH), 48.3 (C19, CH), 46.0 (C17, Cquart.), 42.8 (C14, Cquart.),
40.9 (C8, Cquart.), 38.4 (C1, CH2), 37.8 (C4, Cquart.), 37.5 (C13, CH),
37.0 (C10, Cquart.), 34.1 (C7, CH2), 32.8 (C22, CH2), 32.5 (C16, CH2),
30.3 (C21, CH2), 28.0 (C15, CH2), 27.9 (C23, CH3), 24.9 (C12, CH2),
23.6 (C2, CH2), 21.2 (Ac, CH3), 20.8 (C11, CH2), 18.8 (C29, CH3),
1561w, 1454w, 1376w, 1275w, 1191w, 1106w, 1043w cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 4.64 (d, 1H, J = 2.6 Hz, CHa (30)), 4.48
(s, 1H, CHb (30)), 3.07 (dd, 1H, J = 11.5, 4.7 Hz, CHOH (3)), 3.05
(dd, 1H, J = 4.4, 2.9 Hz, CH (28)), 2.60 (dd, 1H, J = 4.8, 4.4 Hz, CHa
(31)), 2.46 (ddd, 1H, J = 11.5, 11.0, 6.8 Hz, CH (19)), 2.41 (dd, 1H,
J = 4.8, 2.9 Hz, CHb (31)), 1.96–1.88 (m, 1H, CH (13)), 1.82–1.76
(m, 1H, CHa (21)), 1.74–1.68 (m, 1H, CHa (12)), 1.56 (s, 3H, CH3
(29)), 1.55–1.42 (m, 6H, CHa (1)+CH2 (2)+CH (18)+CHa (22)+CHa
(6)), 1.35–1.15 (m, 9H, CH2 (11)+CHb (6)+CHb (22)+CH2 (7)+CHa
(16)+CH (9)+CHb (21)), 1.05–0.90 (m, 3H, CH2 (15)+CHb (12)),
0.89 (s, 3H, CH3 (27)), 0.88 (s, 3H, CH3 (25)), 0.85 (s, 3H, CH3
(23)), 0.87–0.74 (m, 2H, CHb (16)+CHb (1)), 0.71 (s, 3H, CH3 (26)),
0.64 (s, 3H, CH3 (24)), 0.56 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C
NMR (125 MHz, CDCl3): d 150.1 (C20, C@CH2), 110.0 (C30, CH2@C),
78.9 (C3, CHOH), 55.3 (C5, CH), 52.9 (C28, CH), 50.4 (C9, CH), 48.9
(C19, CH), 48.8 (C18, CH), 45.6 (C17, Cquart.), 43.6 (C31, CH2), 42.4
(C14, Cquart.), 40.9 (C8, Cquart.), 38.8 (C4, Cquart.), 38.7 (C1, CH2),

1350
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
18.1 (C6, CH2), 16.4 (C24, CH3), 16.2 (C25, CH3), 15.7 (C26, CH3),
14.4 (C27, CH3) ppm; MS (e.i., 70 eV): m/z (%) 554 (16), 494 (16),
465 (21), 451 (46), 215 (40), 201 (47), 189 (100) 159 (39), 145
(52), 135 (95), 121 (81), 107 (76), 95 (68), 81 (60); Anal. Calcd
for C35H54O5 (554.80): C, 75.77; H, 9.81. Found: C, 75.60; H, 9.69.
(C15, CH2), 27.9 (C23, CH3), 24.9 (C12, CH2), 23.7 (C2, CH2), 21.3
(Ac, CH3), 20.9 (C11, CH2), 18.7 (C29, CH3), 18.1 (C6, CH2), 16.4
(C24, CH3), 16.2 (C25, CH3), 15.9 (C26, CH3), 14.8 (NCH2CH3), 14.5
(C27, CH3), 12.8 (NCH2CH3) ppm; MS (ESI, MeOH): m/z 596.4
(100%, [M+H]+), 618.5 (80%, [M+Na]+); Anal. Calcd for C38H61NO4
(595.90): C, 76.59; H, 10.32; N, 2.35. Found: C, 76.10; H, 9.88; N,
2.32.
5.1.11. (3S, 2R)-3-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-
oxirane-2-carbonitril (4)
Compound 4 (0.6 g, 89%) was obtained from 3-O-acetylbetulinic
aldehyde (0.50 g, 1.03 mmol) and 2-chloroacetonitrile (0.62 g,
5.1.13. (2S, 3S)-2-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-1,5-
dioxa-spiro[2.4]heptan-4-one (5)
Compound 6 (0.6 g, 89%) was obtained from 3-O-acetylbetulinic
aldehyde (0.50 g, 1.03 mmol) and 2-bromo-c-butyrolactone
4.12 mmol) following GP1 as a colourless solid; mp 243 °C; ½aD20
ꢂ
39.3 (c 4.2, CHCl3); RF 0.63 (silica gel, hexane/ethyl acetate, 8:2);
IR (KBr):
m
2950s, 2869m, 2242w, 1718s, 1642m, 1465m, 1392m,
(0.68 g, 4.12 mmol) following GP1 as a colourless solid; mp
1377s, 1320w, 1257s, 1198m, 1148w, 1105w, 1079w, 1027m cm
249 °C; ½a2D0ꢂ 41.0 (c 4.1, CHCl3); RF 0.16 (silica gel, hexane/ethyl
ꢀ1
;
1H NMR (500 MHz, CDCl3): d 4.74 (d, 1H, J = 2.1 Hz, CHa (30)),
acetate, 9:1); IR (KBr): m 2944s, 2875m, 1798s, 1724s, 1640w,
4.58 (dd, 1H, J = 2.1, 1.2 Hz, CHb (30)), 4.45 (dd, 1H, J = 10.4,
5.8 Hz, CHOAc (3)), 3.53 (d, 1H, J = 2.1 Hz, CH (28)), 3.20 (d, 1H,
J = 2.1 Hz, CH (31)), 2.48 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz, CH (19)),
2.02 (s, 3H, Ac), 1.98 (ddd, 1H, J = 12.2, 12.2, 3.8 Hz, CH (13)),
1.86–1.78 (m, 2H, CHa (21)+CHa (12)), 1.66 (s, 3H, CH3 (29)),
1.66–1.40 (m, 8H, CH (18)+CHa (1)+CHa (15)+CH2 (2)+CHa
(16)+CHa (6)+CHa(11)), 1.40–1.08 (m, 10H, CHb (6)+CH2 (7)+CHb
(21)+CH (9)+CHa (22)+CHb(11)+CHb (16)+CHb (15)+CHb (12)),
1.03–0.94 (m, 2H, CHb (22)+CHb (1)), 1.03 (s, 3H, CH3 (25)), 0.98
(s, 3H, CH3 (27)), 0.85 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (23)),
0.82 (s, 3H, CH3 (24)), 0.79 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C
NMR (125 MHz, CDCl3): d 170.9 (C@O), 149.0 (C20, C@CH2),
117.0 (C„N), 110.7 (C30, CH2@C), 80.8 (C3, CHOAc), 60.8 (C28,
CH), 55.4 (C5, CH), 50.3 (C9, CH), 48.7 (C18, CH), 48.5 (C19, CH),
46.0 (C17, Cquart.), 42.4 (C14, Cquart.), 40.9 (C8, Cquart.), 38.7 (C31,
CH), 38.4 (C1, CH2), 37.8 (C4, Cquart.), 37.2 (C13, CH), 37.0 (C10,
Cquart.), 34.1 (C7, CH2), 32.6 (C16, CH2), 32.0 (C22, CH2), 30.2 (C21,
CH2), 28.0 (C15, CH2), 27.9 (C23, CH3), 24.8 (C12, CH2), 23.7 (C2,
CH2), 21.3 (Ac, CH3), 20.8 (C11, CH2), 18.6 (C29, CH3), 18.1 (C6,
CH2), 16.5 (C24, CH3), 16.2 (C25, CH3), 16.0 (C26, CH3), 14.5 (C27,
CH3) ppm; MS (ESI, MeOH): m/z 447.3 (100%, [M+H]+), 469.4
(80%, [M+Na]+); Anal. Calcd for C34H51NO3 (521.77): C, 78.26; H,
9.85; N, 2.68. Found: C, 78.00; H, 9.55; N, 2.31.
1447m, 1392m, 1376m, 1244s, 1115m, 1084w, 1024m cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 4.72 (d, 1H, J = 2.1 Hz, CHa (30)), 4.58
(br s, 1H, CHb (30)), 4.52 (ddd, 1H, J = 9.5, 9.5, 2.1 Hz, CHa (33)),
4.44 (dd, 1H, J = 10.8, 5.4 Hz, CHOAc (3)), 4.37 (ddd, 1H, J = 9.9,
9.5, 6.6 Hz, CHb (33)), 3.41 (s, 1H, CH (28)), 2.61 (ddd, 1H,
J = 13.3, 9.9, 9.5 Hz, CHa (32)), 2.43 (ddd, 1H, J = 11.2, 10.4, 7.0 Hz,
CH (19)), 2.34–2.30 (m, 1H, CHa (16)), 2.23 (ddd, 1H, J = 13.3, 6.6,
2.1 Hz, CHb (32)), 2.02 (s, 3H, Ac), 1.98–1.82 (m, 3H, CH (13)+CHa
(21)+CHa (12)), 1.67 (s, 3H, CH3 (29)), 1.69–1.54 (m, 6H, CHa
(1)+CHa (22)+CH (18)+CH2 (2)+CHa (6)), 1.50–1.08 (m, 11H, CHb
(6)+CH2 (11)+CH2 (15)+CHb (22)+CHb (16)+CH2 (7)+CH (9)+CHb
(21)), 1.05–0.88 (m, 2H, CHb (12)+CHb (1)), 0.98 (s, 3H, CH3 (27)),
0.97 (s, 3H, CH3 (24)), 0.84 (s, 6H, CH3 (25)), 0.83 (s, 6H, CH3
(23)), 0.81 (s, 3H, CH3 (26)), 0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm;
13C NMR (125 MHz, CDCl3): d 172.4 (C@O), 170.9 (C@O), 149.7
(C20, C@CH2), 110.1 (C30, CH2@C), 80.7 (C3, CHOAc), 69.0 (C28,
CH), 64.4 (C33, CH2), 59.0 (C31, Cquart.), 55.3 (C5, CH), 50.4 (C9,
CH), 49.9 (C18, CH), 48.6 (C19, CH), 45.6 (C17, Cquart.), 42.0 (C14,
Cquart.), 40.8 (C8, Cquart.), 38.3 (C1, CH2), 37.7 (C4, Cquart.), 37.6
(C13, CH), 37.0 (C10, Cquart.), 34.1 (C7, CH2), 33.6 (C22, CH2), 32.5
(C16, CH2), 30.3 (C21, CH2), 29.6 (C32, CH2), 28.4 (C15, CH2), 27.8
(C23, CH3), 24.7 (C12, CH2), 23.6 (C2, CH2), 21.2 (Ac, CH3), 20.9
(C11, CH2), 18.6 (C29, CH3), 18.1 (C6, CH2), 16.4 (C24, CH3), 16.2
(C25, CH3), 15.8 (C26, CH3), 14.5 (C27, CH3) ppm; MS (ESI, MeOH):
m/z 567.1 (40%, [M+H]+), 589.4 (100%, [M+Na]+); Anal. Calcd for
C36H54O5 (566.81): C, 76.28; H, 9.60. Found: C, 76.10; H, 9.88.
5.1.12. (3S, 2R)-3-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-
oxirane-2-carboxylic acid diethylamide (5)
Compound 5 (0.6 g, 89%) was obtained from 3-O-acetylbetulinic
aldehyde (0.50 g, 1.03 mmol) and chloro-N,N-diethylacetamide
(0.62 g, 4.12 mmol) following GP1 as a colourless solid; mp
106 °C; ½a2D0ꢂ 27.8 (c 5.0, CHCl3); RF 0.30 (silica gel, hexane/ethyl
5.1.14. Ethyl-(3R)-3-[3b-acetoxy-28-norlup-20(29)-en-17b-yl]-
3-hydroxy-propionate (7)
Compound 7 (0.43 g, 73%) was obtained as major product from
3-O-acetylbetulinic aldehyde (0.50 g, 1.03 mmol) and ethyl acetate
(0.68 g, 4.12 mmol) following GP2 or by reaction with ketene ethyl
trimethylsilyl acetal (0.66 g, 4.12 mmol) following GP3 in 65% yield
as a colourless solid; mp 226 °C; ½a2D0ꢂ 28.0 (c 9.2, CHCl3); RF 0.66
acetate, 8:2); IR (KBr):
m
2944s, 2873m, 1733s, 1654s, 1464m,
1H NMR (500 MHz,
1378m, 1246s, 1149w, 1106w, 1028w cmꢀ1
;
CDCl3): d 4.74 (br s, 1H, CHa (30)), 4.58 (br s, 1H, CHb (30)), 4.44
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 3.49–3.37 (m, 4H, CH
(28)+NCH2), 3.33 (d, 1H, J = 2.1 Hz, CH (31)), 2.62 (ddd, 1H,
J = 11.2, 10.4, 7.0 Hz, CH (192.08 (ddd, 1H, J = 12.2, 12.2, 3.8 Hz,
CH (13)))), 2.02 (s, 3H, Ac), 1.97–1.88 (m, 1H, CHa (21)), 1.84–
1.74 (m, 1H, CHa (12)), 1.66 (s, 3H, CH3 (29)), 1.66–1.50 (m, 7H,
CH (18)+CHa (1)+CHa (16)+CH2 (2)+CHa (6)+CHa (15)), 1.50–1.19
(m, 12H, CHa (22)+CH2 (11)+CHb (6)+CHb (16)+CH2 (7)+CH
(9)+CHb (21), 1.24 (t, 3H, J = 7.1 Hz, CH2CH3), 1.18 (t, 3H,
J = 7.1 Hz, CH2CH3), 1.13–0.92 (m, 4H, CHb (22)+CHb (15)+CHb
(12)+CHb (1)), 0.98 (s, 3H, CH3 (25)), 0.97 (s, 3H, CH3 (27)), 0.82
(s, 6H, CH3 (23)+CH3 (26)), 0.81 (s, 3H, CH3 (24)), 0.77 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d 170.9
(C@O), 167.0 (C@O), 149.6 (C20, C@CH2), 110.3 (C30, CH2@C),
80.9 (C3, CHOAc), 59.5 (C28, CH), 55.4 (C5, CH), 50.4 (C31, CH),
50.3 (C9, CH), 48.9 (C18, CH), 48.5 (C19, CH), 45.8 (C17, Cquart.),
42.5 (C14, Cquart.), 41.2 (NCH2), 40.9 (C8, Cquart.), 40.5 (NCH2), 38.4
(C1, CH2), 37.8 (C4, Cquart.), 37.2 (C13, CH), 37.1 (C10, Cquart.), 34.1
(C7, CH2), 33.6 (C22, CH2), 32.4 (C16, CH2), 30.6 (C21, CH2), 28.0
(silica gel, hexane/ethyl acetate, 8:2); IR (KBr):
m 2939s, 2864m,
1737s, 1707s, 1643w, 1446m, 1373m, 1339w, 1267s, 1176m,
1107w, 1079w, 1027s cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.69
;
(d, 1H, J = 2.5 Hz, CHa (30)), 4.56 (dd, 1H, J = 1.2, 2.5 Hz, CHb (30)),
4.52 (d, 1H, J = 10.0, Hz, CH (28)), 4.47 (dd, 1H, J = 10.4, 5.8 Hz,
CHOAc (3)), 4.18 (q, 2H, J = 7.0 Hz, OCH2), 2.94 (ddd, 1H, J = 11.2,
11.0, 5.4 Hz, CH (19)), 2.49 (dd, 1H, J = 16.2, 2.1 Hz, CHa (31)),
2.40 (dd, 1H, J = 16.2, 10.0 Hz, CHb (31)), 2.09 (ddd, 1H, J = 12.2,
12.2, 3.7 Hz, CH (13)), 2.05–1.97 (m, 1H, CHa (21)), 2.02 (s, 3H,
Ac), 1.80 (ddd, 1H, J = 12.8, 9.2, 1.5 Hz, CHa (16)), 1.71–1.49 (m,
8H, CHa (12)+CH (18)+CHa (1)+CH2 (2)+CHa (22)+CHa (15)+CHa
(6)), 1.66 (s, 3H, CH3 (29)), 1.48–1.20 (m, 8H, CH2 (11)+CH2
(7)+CHb (6)+CHb (22)+CHb (21)+CH (9)), 1.27 (t, 3H, J = 7.0 Hz,
CH2CH3), 1.19–1.08 (m, 2H, CHb (12)+CHb (16)), 1.02–0.91 (m, 2H,
CHb (1)+CHb (15)), 1.05 (s, 3H, CH3 (25)), 0.99 (s, 3H, CH3 (27)),
0.85 (s, 3H, CH3 (24)), 0.83 (s, 3H, CH3 (26)), 0.82 (s, 3H, CH3
(23)), 0.79 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
1351
CDCl3): d 173.9 (C@O), 170.9 (C@O), 151.3 (C20, C@CH2), 109.3
(C30, CH2@C), 80.9 (C3, CHOAc), 68.8 (C28, CH), 60.7 (OCH2), 55.4
(C5, CH), 50.3 (C18, CH), 50.2 (C9, CH), 49.2 (C17, Cquart.), 48.7
(C19, CH), 42.9 (C14, Cquart.), 40.9 (C8, Cquart.), 38.3 (C1, CH2), 37.8
(C4, Cquart.), 37.4 (C31, CH2), 37.0 (C10, Cquart.), 36.7 (C13, CH),
34.1 (C7, CH2), 34.0 (C22, CH2), 33.2 (C16, CH2), 32.6 (C21, CH2),
27.9 (C23, CH3), 27.7 (C15, CH2), 25.1 (C12, CH2), 23.7 (C2, CH2),
21.3 (Ac, CH3), 20.9 (C11, CH2), 19.0 (C29, CH3), 18.1 (C6, CH2),
16.5 (C24, CH3), 16.1 (C25+C26, 2 ꢁ CH3), 15.1 (C27, CH3), 14.7
(CH2CH3) ppm; MS (ESI, MeOH): m/z 571.3 (20%, [M+H]+), 593.4
(100%, [M+Na]+); Anal. Calcd for C36H58O5 (570.84): C, 75.75; H,
10.24. Found: C, 75.40; H, 9.97.
(16)+CHa (1)+CH (18)+CHa (12)), 1.66 (s, 3H, CH3 (29)), 1.61–1.22
(m, 12H, CH2 (22)+CH2 (11)+CH2 (7)+CH2 (6)+CHa (15)+CHb
(2)+CH (9)+CHb (21)), 1.27 (t, 3H, J = 7.0 Hz, CH2CH3), 1.20–1.04
(m, 2H, CHb (12)+CHb (16)), 1.05 (s, 3H, CH3 (25)), 1.01–0.91 (m,
1H, CHb (15)), 0.99 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (23)), 0.86–
0.78 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3 (24)), 0.73 (s, 3H, CH3
(26)), 0.65 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d 173.9 (C@O), 151.4 (C20, C@CH2), 109.3 (C30, CH2@C),
88.6 (C3, CHOMe), 68.8 (C28, CH), 60.7 (OCH2), 57.4 (OMe), 55.9
(C5, CH), 50.4 (C18+C9, CH), 49.2 (C17, Cquart.), 48.7 (C19, CH),
42.9 (C14, Cquart.), 41.0 (C8, Cquart.), 38.8 (C1, CH2), 38.6 (C4, Cquart.),
37.4 (C31, CH2), 37.2 (C10, Cquart.), 36.8 (C13, CH), 34.2 (C7, CH2),
34.1 (C22, CH2), 33.3 (C16, CH2), 32.7 (C21, CH2), 28.0 (C23, CH3),
27.8 (C15, CH2), 25.2 (C12, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2),
19.1 (C29, CH3), 18.1 (C6, CH2), 16.2 (C24+C25, 2 ꢁ CH3), 16.1
(C26, CH3), 14.8 (C27, CH3), 14.2 (CH2CH3) ppm; MS (ESI, MeOH):
m/z 543.3 (20%, [M+H]+), 565.5 (100%, [M+Na]+); Anal. Calcd for
C35H58O4 (542.83): C, 77.44; H, 10.77. Found: C, 75.45; H, 10.68.
5.1.15. Ethyl-(5R)-5-[3b-acetoxy-28-norlup-20(29)-en-17b-yl]-
5-hydroxy-3-oxo-valerate (8)
Compound 8 (0.13 g, 20%) was obtained as minor product from
3-O-acetylbetulinic aldehyde (0.50 g, 1.03 mmol) and ethyl acetate
(0.68 g, 4.12 mmol) following GP2 or by reaction with ketene ethyl
trimethylsilyl acetal (0.66 g, 4.12 mmol) following GP3 in 17% yield
as a colourless solid; mp 134 °C; ½a2D0ꢂ 28.7 (c 5.2, CHCl3); RF 0.14
5.1.17. Ethyl-(5R)-5-[3b-methoxy-28-norlup-20(29)-en-17b-yl]-
5-hydroxy-3-oxo-valerate (10)
(silica gel, hexane/ethyl acetate, 9:1); IR (KBr):
m 2941s, 2868m,
1739s, 1707s, 1643w, 1446m, 1378m, 1326m, 1269s, 1157w,
Compound 10 (0.09 g, 15%) was obtained as minor product from
3-O-methylbetulinic aldehyde (0.50 g, 1.03 mmol) and ethyl ace-
tate (0.68 g, 4.12 mmol) following GP2 or by reaction with ketene
ethyl trimethylsilyl acetal (0.66 g, 4.12 mmol) following GP3 in
18% yield as a colourless foam; ½a2D0ꢂ 27.9 (c 4.0, CHCl3); RF 0.16 (sil-
1083w, 1039m cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.62 (d, 1H,
;
J = 2.5 Hz, CHa (30)), 4.56 (d, 1H, J = 7.6, 4.3 Hz, CH (28)), 4.50 (dd,
1H, J = 1.2, 2.5 Hz, CHb (30)), 4.40 (dd, 1H, J = 10.3, 6.6 Hz, CHOAc
(3)), 4.14 (q, 2H, J = 7.0 Hz, OCH2), 3.43 (s, 2H, CH2 (33)), 2.86
(ddd, 1H, J = 11.1, 11.1, 5.9 Hz, CH (19)), 2.65–2.63 (m, 2H, CH2
(31)), 2.01 (ddd, 1H, J = 12.2, 12.2, 3.7 Hz, CH (13)), 1.98–1.91 (m,
1H, CHa (21)), 1.97 (s, 3H, Ac), 1.71 (ddd, 1H, J = 12.3, 9.0, 1.5 Hz,
CHa (16)), 1.71–1.48 (m, 5H, CHa (12)+CH (18)+CHa (1)+CH2 (2)),
1.61 (s, 3H, CH3 (29)), 1.48–1.17 (m, 8H, CH2 (22)+CHa (15)+CHa
(6)+CH2 (11)+CH2 (7)+CHb (6)+CHb (21)+CH (9)), 1.22 (t, 3H,
J = 7.0 Hz, CH2CH3), 1.15–1.01 (m, 2H, CHb (12)+CHb (16)), 0.94–
0.85 (m, 2H, CHb (1)+CHb (15)), 0.99 (s, 3H, CH3 (25)), 0.93 (s, 3H,
CH3 (27)), 0.79 (s, 3H, CH3 (26)), 0.78 (s, 3H, CH3 (24)), 0.77 (s,
6H, CH3 (23)), 0.72 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d 204.4 (C@O), 170.9 (C@O), 167.7 (C@O),
151.3 (C20, C@CH2), 109.3 (C30, CH2@C), 80.9 (C3, CHOAc), 68.2
(C28, CH), 61.5 (OCH2), 55.4 (C5, CH), 50.3 (C18, CH), 50.2 (C9,
CH), 50.0 (C33, CH2), 49.1 (C17, Cquart.), 48.7 (C19, CH), 45.8 (C31,
CH2), 42.9 (C14, Cquart.), 40.9 (C8, Cquart.), 38.3 (C1, CH2), 37.8 (C4,
Cquart.), 37.0 (C10, Cquart.), 36.7 (C13, CH), 34.1 (C7, CH2), 34.0
(C22, CH2), 33.5 (C16, CH2), 32.6 (C21, CH2), 27.9 (C23, CH3), 27.7
(C15, CH2), 25.1 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac, CH3), 20.9
(C11, CH2), 19.0 (C29, CH3), 18.1 (C6, CH2), 16.4 (C24, CH3), 16.1
(C25, CH3), 16.1 (C26, CH3), 15.1 (C27, CH3), 14.1 (CH2CH3) ppm;
MS (ESI, MeOH): m/z 613.3 (20%, [M+H]+), 635.4 (100%,
[M+Na]+); Anal. Calcd for C38H60O6 (612.87): C, 74.47; H, 9.87.
Found: C, 74.40; H, 9.65.
ica gel, hexane/ethyl acetate, 9:1); IR (KBr):
m 2943s, 1730m,
1706m, 1646w, 1465w, 1370w, 1318w, 1249w, 1103w, 1025w
cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.68 (d, 1H, J = 2.5 Hz, CHa
;
(30)), 4.61 (d, 1H, J = 7.9, 3.5 Hz, CH (28)), 4.55 (dd, 1H, J = 1.2,
2.5 Hz, CHb (30)), 4.14 (q, 2H, J = 7.0 Hz, OCH2), 3.48 (s, 2H, CH2
(33)), 3.33 (s, 3H, OMe), 2.90 (ddd, 1H, J = 11.1, 11.1, 5.9 Hz, CH
(19)), 2.71–2.66 (m, 2H, CH2 (31)), 2.61 (dd, 1H, J = 11.7, 4.3 Hz,
CHOMe (3)), 2.03 (ddd, 1H, J = 12.2, 12.2, 3.7 Hz, CH (13)), 1.94–
1.83 (m, 1H, CHa (21)), 1.80–1.42 (m, 8H, CHa (2)+CHa (16)+CHa
(12)+CHa (1)+CH (18)+CHa (22)+CHa (15)+CHa (6)), 1.66 (s, 3H,
CH3 (29)), 1.41–1.17 (m, 9H, CH2 (11)+CHb (2)+CHb (6)+CHb
(22)+CH2 (7)+CHb (21)+CH (9)), 1.27 (t, 3H, J = 7.0 Hz, CH2CH3),
1.16–1.03 (m, 2H, CHb (12)+CHb (16)), 1.01–0.93 (m, 1H, CHb
(15)), 1.03 (s, 3H, CH3 (25)), 0.98 (s, 3H, CH3 (27)), 0.93 (s, 3H,
CH3 (23)), 0.82 (s, 3H, CH3 (24)), 0.89–0.77 (m, 1H, CHb (1)), 0.73
(s, 3H, CH3 (26)), 0.66 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d 204.4 (C@O), 167.0 (C@O), 151.4 (C20, C@CH2),
109.2 (C30, CH2=@C), 88.7 (C3, CHOMe), 68.2 (C28, CH), 61.5
(OCH2), 57.5 (OMe), 55.9 (C5, CH), 50.4 (C9, CH), 50.3 (C18, CH),
50.0 (C33, CH2), 49.1 (C17, Cquart.), 48.6 (C19, CH), 45.8 (C31,
CH2), 42.9 (C14, Cquart.), 41.0 (C8, Cquart.), 38.8 (C1, CH2), 38.5 (C4,
Cquart.), 37.2 (C10, Cquart.), 36.8 (C13, CH), 34.2 (C7, CH2), 34.1
(C22, CH2), 33.5 (C16, CH2), 32.6 (C21, CH2), 27.9 (C23, CH3), 27.7
(C15, CH2), 25.2 (C12, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2), 19.0
(C29, CH3), 18.1 (C6, CH2), 16.2 (C24, CH3), 16.1 (C25, CH3), 16.0
(C26, CH3), 15.1 (C27, CH3), 14.1 (CH2CH3) ppm; MS (ESI, MeOH):
m/z 607.4 (20%, [M+Na]+), 1191.3 (100%, [2M+Na]+); Anal. Calcd
for C37H60O5 (584.87): C, 75.98; H, 10.34. Found: C, 75.88; H, 9.98.
5.1.16. Ethyl-(3R)-3-[3b-methoxy-28-norlup-20(29)-en-17b-yl]-
3-hydroxy-propionate (9)
Compound 9 (0.42 g, 75%) was obtained as major product from
3-O-methylbetulinic aldehyde (0.470 g, 1.03 mmol) and ethyl ace-
tate (0.68 g, 4.12 mmol) following GP2 or by reaction with ketene
ethyl trimethylsilyl acetal (0.66 g, 4.12 mmol) following GP3 in
65% yield as a colourless solid; mp 198–200 °C; ½a2D0ꢂ 29.3 (c 4.0,
5.1.18. (R)-2-((R)-[3b-Acetoxy-28-norlup-20(29)-en-17b-
yl]hydroxymethyl)-c-butyro-lactone (11)
CHCl3); RF 0.38 (silica gel, hexane/ethyl acetate, 9:1); IR (KBr):
m
Compound 11 (0.6 g, 89%) was obtained from 3-O-acetylbetuli-
nic aldehyde (0.50 g, 1.03 mmol) and (4,5-dihydrofuran-2-
yloxy)trimethylsilane (0.65 g, 4.12 mmol) following GP3 as a col-
ourless solid; mp 232 °C; ½aD20ꢂ 15.9 (c 5.8, CHCl3); RF 0.40 (silica
2936s, 2840m, 1718s, 1642w, 1453w, 1392w, 1374m, 1192m,
1100m, 1023w cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.69 (d, 1H,
;
J = 2.5 Hz, CHa (30)), 4.56 (dd, 1H, J = 1.2, 2.5 Hz, CHb (30)), 4.51
(d, 1H, J = 10.0, Hz, CH (28)), 4.17 (q, 2H, J = 7.0 Hz, OCH2), 3.33
(s, 3H, OMe), 2.93 (ddd, 1H, J = 11.2, 11.0, 5.4 Hz, CH (19)), 2.62
(dd, 1H, J = 11.6, 4.1 Hz, CHOMe (3)), 2.49 (dd, 1H, J = 16.1,
2.4 Hz, CHa (31)), 2.40 (dd, 1H, J = 16.1, 10.0 Hz, CHb (31)), 2.13–
1.95 (m, 2H, CH (13)+CHa (21)), 1.80–1.63 (m, 5H, CHa (2)+CHa
gel, hexane/ethyl acetate, 8:2); IR (KBr):
m 2947s, 2873m, 1735s,
1640w, 1453m, 1375s, 1316w, 1247s, 1217m, 1169m, 1109w,
1027s cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.78 (d, 1H, J = 4.1 Hz,
;
CH (28)), 4.67 (d, 1H, J = 2.5 Hz, CHa (30)), 4.56 (dd, 1H, J = 1.2,
2.5 Hz, CHb (30)), 4.44 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 4.36

1352
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
(ddd, 1H, J = 9.1, 8.7, 1.7 Hz, CHa (33)), 4.19–4.10 (m, 1H, CHb (33)),
2.78 (ddd, 1H, J = 11.2, 10.4, 6.2 Hz, CH (19)), 2.75–2.69 (m, 1H, CH
(31)), 2.66–2.56 (m, 1H, CHa (32)), 2.26–2.11 (m, 1H, CHb (32)),
2.11–2.02 (m, 2H, CHa (21)+CH (13)), 2.02 (s, 3H, Ac), 1.80 (ddd,
1H, J = 11.2, 8.7, 1.7 Hz, CHa (16)), 1.75–1.45 (m, 8H, CHa (12)+CH
(18)+CHa (1)+CHa (22)+CH2 (2)+CHa (15)+CHa (6)), 1.66 (s, 3H,
CH3 (29)), 1.45–1.20 (m, 8H, CH2 (11)+CHb (22)+CH2 (7)+CHb
(6)+CHb (21)+CH (9)), 1.19–1.08 (m, 2H, CHb (12)+CHb (16)),
1.02–0.91 (m, 2H, CHb (1)+CHb (15)), 1.08 (s, 3H, CH3 (27)), 1.00
(s, 3H, CH3 (25)), 0.85 (s, 3H, CH3 (24)), 0.82 (s, 6H, CH3 (23)+CH3
(26)), 0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d 179.7 (C@O), 170.9 (C@O), 151.0 (C20, C@CH2), 109.6
(C30, CH2@C), 80.9 (C3, CHOAc), 69.2 (C28, CH), 66.9 (C33, CH2),
55.3 (C5, CH), 50.8 (C18, CH), 50.3 (C9, CH), 49.1 (C17, Cquart.),
49.1 (C19, CH), 43.2 (C31, CH), 42.9 (C14, Cquart.), 40.9 (C8, Cquart.),
38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.1 (C13, CH), 37.0 (C10, Cquart.),
34.7 (C22, CH2), 34.1 (C7, CH2), 33.8 (C16, CH2), 32.6 (C21, CH2),
27.9 (C23, CH3), 27.8 (C15, CH2), 25.0 (C12, CH2), 23.7 (C2, CH2),
22.8 (C32, CH2), 21.3 (Ac, CH3), 20.9 (C11, CH2), 18.8 (C29, CH3),
18.1 (C6, CH2), 16.5 (C24, CH3), 16.1 (C25+C26, 2 ꢁ CH3), 15.1
(C27, CH3) ppm; MS (ESI, MeOH): m/z 568.3 (100%, [M+H]+),
591.3 (100%, [M+Na]+); Anal. Calcd for C36H56O5 (568.82): C,
76.01; H, 9.92. Found: C, 75.77; H, 9.60.
CHa (30)), 4.56 (dd, 1H, J = 2.4, 1.5 Hz, CHb (30)), 4.45 (dd, 1H,
J = 10.4, 5.8 Hz, CHOAc (3)), 4.20–4.15 (m, 2H, OCH2), 3.50 (d, 1H,
J = 15.1 Hz, CHa (31)), 3.44 (dd, 1H, J = 15.1 Hz, CHb (31)), 2.94
(ddd, 1H, J = 11.2, 11.2, 4.4 Hz, CH (19)), 2.32 (ddd, 1H, J = 12.7,
12.2, 3.7 Hz, CH (13)), 1.97 (ddd, 1H, J = 14.2, 3.4, 2.9 Hz, CHa
(16)), 2.03 (s, 3H, Ac), 1.85 (ddd, 1H, J = 12.7, 8.3 Hz, CHa (22)),
1.78–1.52 (m, 7H, CHa (21)+CHa (12)+CHa (1)+CH (18)+CH2
(2)+CHb (16)), 1.66 (s, 3H, CH3 (29)), 1.50–1.16 (m, 11H, CHb
(22)+CH2 (7)+CH2 (6)+CHb (21)+CH2 (15)+CH2 (11)+CH (9)), 1.26
(t, 3H, J = 7.0 Hz, CH2CH3), 1.02–0.92 (m, 2H, CHb (12)+CHb (1)),
0.94 (s, 3H, CH3 (27)), 0.89 (s, 3H, CH3 (25)), 0.83 (s, 3H, CH3
(24)), 0.82 (s, 3H, CH3 (26)), 0.81 (s, 6H, CH3 (23)), 0.77 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d 206.4
(C@O), 170.9 (C@O), 167.7 (C@O), 150.3 (C20, C@CH2), 109.6
(C30, CH2@C), 80.9 (C3, CHOAc), 62.2 (OCH2), 61.1 (C17, Cquart.),
55.4 (C5, CH), 50.5 (C9, CH), 49.4 (C18, CH), 45.9 (C19, CH), 44.7
(C31, CH2), 42.5 (C14, Cquart.), 40.7 (C8, Cquart.), 38.4 (C1, CH2),
37.8 (C4, Cquart.), 37.1 (C10, Cquart.), 37.1 (C13, CH), 35.0 (C22,
CH2), 34.2 (C7, CH2), 31.4 (C21, CH2), 30.2 (C16, CH2), 29.6 (C15,
CH2), 27.9 (C23, CH3), 25.5 (C12, CH2), 23.7 (C2, CH2), 21.3 (Ac,
CH3), 20.9 (C11, CH2), 19.3 (C29, CH3), 18.1 (C6, CH2), 16.4 (C24,
CH3), 16.2 (C26, CH3), 16.0 (C25, CH3), 14.4 (C27, CH3), 14.1
(CH2CH3) ppm; MS (ESI, MeOH): m/z 569.7 (10%, [M+H]+), 591.6
(20%, [M+Na]+), 1159.4 (100%, [2M+Na]+); Anal. Calcd for
C36H56O5 (568.83): C, 76.01; H, 9.92. Found: C, 75.77; H, 9.63.
5.1.19. (R)-4-((S)-[3b-Acetoxy-28-norlup-20(29)-en-17b-
yl]hydroxymethyl)-2-buten-4-olide (12)
Compound 12 (0.43 g, 73%) was obtained from 3-O-acetylbetu-
linic aldehyde (0.50 g, 1.03 mmol) and 2-trimethylsilyloxyfuran
(0.48 g, 3.00 mmol) following GP3 as a colourless solid; mp
251 °C; ½a2D0ꢂ 51.6 (c 5.0, CHCl3); RF 0.20 (silica gel, hexane/ethyl
5.1.21. Ethyl-3-[3b-methoxy-28-norlup-20(29)-en-17b-yl]-3-
oxo-propionate (14)
Compound 14 (0.49 g, 97%) was obtained from compound 9
(0.50 g, 0.92 mmol) following GP4 or GP5 as a colourless solid;
mp 151 °C; ½a2D0ꢂ 14.4 (c 4.1, CHCl3); RF 0.55 (silica gel, hexane/ethyl
acetate, 8:2); IR (KBr):
m 3502m, 3066w, 2942s, 2875m, 1761s,
1715s, 1637w, 1453w, 1375w, 1335w, 1265s, 1165w, 1100w,
acetate, 9:1); IR (KBr): m 3448m, 3076w, 2941s, 2873m, 2837m,
1039w cmꢀ1 1H NMR (500 MHz, CDCl3): d 7.58 (dd, 1H, J = 5.8,
;
1754m, 1709m, 1643w, 1465m, 1391m, 1376w, 1364m, 1322w,
1.2 Hz, CH (32)), 6.17 (dd, 1H, J = 5.8, 2.1 Hz, CH (33)), 5.20 (ddd,
1H, J = 3.3, 2.1, 1.2 Hz, CH (31)), 4.67 (d, 1H, J = 2.5 Hz, CHa (30)),
4.56 (dd, 1H, J = 1.2, 2.5 Hz, CHb (30)), 4.53 (dd, 1H, J = 3.3, 3.8 Hz,
CH (28)), 4.45 (dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 2.79 (ddd, 1H,
J = 11.2, 10.4, 6.2 Hz, CH (19)), 2.02 (s, 3H, Ac), 2.01–1.86 (m, 2H,
CHa (21)+CH (13)), 1.82–1.52 (m, 9H, CHa (22)+CHa (16)+CHa
(12)+CH (18)+CHa (1)+CH2 (2)+CHa (15)+CHa (6)), 1.66 (s, 3H, CH3
(29)), 1.51–1.14 (m, 10H, CHb (22)+CH2 (11)+CH2 (7)+CHb (6)+CH
(9)+CHb (21)+CHb (16)+CHb (12)), 1.09–1.04 (m, 1H, CHb (15)),
1.00–0.91 (m, 1H, CHb (1)), 1.00 (s, 6H, CH3 (25)+CH3 (27)), 0.84
(s, 3H, CH3 (23)), 0.82 (s, 3H, CH3 (26)), 0.81 (s, 3H, CH3 (24)),
0.77 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d
172.7 (C@O), 170.9 (C@O), 153.5 (C33, CH@), 150.5 (C20, C@CH2),
122.8 (C32, CH@), 109.9 (C30, CH2@C), 84.6 (C31, CH), 80.8 (C3,
CHOH), 71.6 (C28, CH), 55.3 (C5, CH), 51.0 (C18, CH), 50.2 (C9,
CH), 48.6 (C19, CH), 48.5 (C17, Cquart.), 42.7 (C14, Cquart.), 40.9 (C8,
Cquart.), 38.3 (C1, CH2), 37.8 (C4, Cquart.), 37.2 (C13, CH), 37.0 (C10,
Cquart.), 34.1 (C7, CH2), 34.0 (C16, CH2), 33.5 (C22, CH2), 32.4 (C21,
CH2), 27.9 (C23, CH3), 28.1 (C15, CH2), 24.8 (C12, CH2), 23.6 (C2,
CH2), 21.3 (Ac, CH3), 20.9 (C11, CH2), 18.7 (C29, CH3), 18.1 (C6,
CH2), 16.4 (C24, CH3), 16.1 (C25+C26, 2 ꢁ CH3), 15.2 (C27, CH3)
ppm; MS (ESI, MeOH): m/z 568.3 (80%, [M+H]+), 591.3 (100%,
[M+Na]+); Anal. Calcd for C36H54O5 (566.81): C, 76.28; H, 9.60.
Found: C, 75.77; H, 9.60.
1300m, 1250m, 1183m, 1156w, 1104m, 1033m cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d = 4.69 (d, 1H, J = 2.4 Hz, CHa (30)), 4.56 (dd,
1H, J = 2.4, 1.5 Hz, CHb (30)), 4.20–4.15 (m, 2H, OCH2), 3.54 (d,
1H, J = 15.1 Hz, CHa (31)), 3.41 (dd, 1H, J = 15.1 Hz, CHb (31)), 3.32
(s, 3H, OMe), 2.93 (ddd, 1H, J = 11.2, 11.2, 4.4 Hz, CH (19)), 2.53
(dd, 1H, J = 11.7, 4.2 Hz, CHOMe (3)), 2.32 (ddd, 1H, J = 12.7, 12.2,
3.7 Hz, CH (13)), 2.08 (ddd, 1H, J = 14.2, 3.4, 2.9 Hz, CHa (16)),
1.84 (ddd, 1H, J = 12.7, 8.3 Hz, CHa (22)), 1.77–1.50 (m, 4H, CHa
(21)+CHa (2)+CHa (12)+CHa (1)), 1.66 (s, 3H, CH3 (29)), 1.50–1.13
(m, 14H, CH (18)+CHb (16)+CH2 (6)+CH2 (11)+CHb (22)+CHb
(2)+CHb (21)+CH2 (7)+CH2 (15)+CH (9)), 1.24 (t, 3H, J = 7.0 Hz,
CH2CH3), 1.02–0.93 (m, 2H, CHb (12)), 0.93 (s, 3H, CH3 (27)), 0.92
(s, 3H, CH3 (23)), 0.88 (s, 3H, CH3 (24)), 0.79 (s, 3H, CH3 (26)),
0.81–0.72 (m, 1H, CHb (1)), 0.71 (s, 3H, CH3 (25)), 0.64 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d 206.3
(C@O), 167.8 (C@O), 150.4 (C20, C@CH2), 109.5 (C30, CH2@C),
88.6 (C3, CHOMe), 62.1 (C17, Cquart.), 61.2 (OCH2), 57.4 (OMe),
55.9 (C5, CH), 50.6 (C9, CH), 49.4 (C18, CH), 45.9 (C19, CH), 44.7
(C31, CH2), 42.5 (C14, Cquart.), 40.8 (C8, Cquart.), 38.7 (C4, Cquart.),
38.6 (C1, CH2), 37.2 (C10, Cquart.), 37.1 (C13, CH), 35.0 (C22, CH2),
34.3 (C7, CH2), 31.4 (C16, CH2), 30.2 (C21, CH2), 29.6 (C15, CH2),
28.0 (C23, CH3), 25.6 (C12, CH2), 22.2 (C2, CH2), 20.9 (C11, CH2),
19.3 (C29, CH3), 18.1 (C6, CH2), 16.1 (C24+C26, 2 ꢁ CH3), 16.0
(C25, CH3), 14.4 (C27, CH3), 14.1 (CH2CH3) ppm; MS (ESI, MeOH):
m/z 541.3 (15%, [M+H]+), 563.4 (20%, [M+Na]+), 1103.4 (100%,
[2M+Na]+); Anal. Calcd for C35H56O4 (540.81): C, 77.73; H, 10.44.
Found: C, 77.45; H, 10.29.
5.1.20. Ethyl-3-[3b-acetoxy-28-norlup-20(29)-en-17b-yl]-3-oxo-
propionate (13)
Compound 13 (0.49 g, 97%) was obtained from compound 7
(0.50 g, 0.88 mmol) following GP4 or GP5 as a colourless solid;
mp 170 °C; ½a2D0ꢂ 15.0 (c 4.6, CHCl3); RF 0.28 (silica gel, hexane/ethyl
5.1.22. Ethyl-3-[3b-acetoxy-28-norlup-20(29)-en-17b-yl]-
acrylate (15)
Compound 15 (0.31 g, 65%) was obtained from compound 7
(0.50 g, 0.88 mmol) and following GP6 as a colourless solid; mp
207–209 °C; ½a2D0ꢂ ꢀ7.7 (c 4.0, CHCl3); RF 0.73 (silica gel, hexane/
acetate, 9:1); IR (KBr):
m
2940s, 2870m, 1748s, 1726s, 1706m,
1645w, 1448m, 1392m, 1365m, 1305m, 1286m, 1246s, 1165w,
1029m cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.70 (d, 1H, J = 2.4 Hz,
;

R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
1353
ethyl acetate, 9:1); IR (KBr):
m
2938s, 2873m, 1728s, 1643w,
1H
ing NMR data represent chemical shifts of the major isomer; mp
1468m, 1450m, 1392m, 1366s, 1249s, 1178m, 1032m cmꢀ1
;
256–259 °C; ½a2D0ꢂ 16.3 (c 6.8, CHCl3); RF 0.33 (silica gel, hexane/
NMR (500 MHz, CDCl3): d 7.23 (d, 1H, J = 16.1 Hz, CH (28)), 5.87
(d, 1H, J = 16.1 Hz, CH (31)), 4.70 (d, 1H, J = 2.5 Hz, CHa (30)), 4.58
(dd, 1H, J = 1.2, 2.5 Hz, CHb (30)), 4.44 (dd, 1H, J = 10.4, 5.8 Hz,
CHOAc (3)), 4.18 (dq, 2H, J = 7.0, 1.0 Hz, OCH2), 2.49 (ddd, 1H,
J = 11.2, 11.0, 5.4 Hz, CH (19)), 2.01 (s, 3H, Ac), 1.91–1.81 (m, 2H,
CHa (21)+CHa (16)), 1.72 (ddd, 1H, J = 12.7, 12.2, 3.7 Hz, CH (13)),
1.67 (s, 3H, CH3 (29)), 1.67–1.51 (m, 8H, CHa (12)+CHa (1)+CHa
(22)+CHa (15)+CH (18)+CH2 (2)+CHb (21)), 1.50–1.43 (m, 1H, CHa
(6)), 1.41–1.17 (m, 7H, CHa (11)+CHb (22)+CHb (16)+CH2 (7)+CHb
(6)+CH (9)), 1.29 (t, 3H, J = 7.0 Hz, CH2CH3), 1.22–1.14 (m, 1H,
CHb (11)), 1.11–1.02 (m, 1H, CHb (12)), 1.03–0.89 (m, 2H, CHb
(15)+CHb (1)), 0.95 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (25)), 0.82
(s, 6H, CH3 (23)+CH3 (24)), 0.81 (s, 3H, CH3 (26)), 0.75 (d, 1H,
J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d 170.9
(C@O), 167.0 (C@O), 153.6 (C28, CH@), 149.7 (C20, C@CH2), 120.3
(C31, CH@), 110.0 (C30, CH2@C), 80.9 (C3, CHOAc), 60.3 (OCH2),
55.4 (C5, CH), 50.3 (C9, CH), 49.9 (C17, Cquart.), 49.7 (C18, CH),
47.7 (C19, CH), 42.7 (C14, Cquart.), 40.8(C8, Cquart.), 38.7 (C13, CH),
38.6 (C22, CH2), 38.3 (C1, CH2), 37.7 (C4, Cquart.), 37.0 (C10, Cquart.),
34.2 (C7, CH2), 33.4 (C21, CH2), 29.8 (C16, CH2), 27.9 (C23, CH3),
27.7 (C15, CH2), 25.2 (C12, CH2), 23.6 (C2, CH2), 21.3 (Ac, CH3),
20.7 (C11, CH2), 19.2 (C29, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3),
16.1 (C25, CH3), 16.0 (C26, CH3), 14.6 (C27, CH3), 14.3 (CH2CH3)
ppm; MS (ESI, MeOH): m/z 553.1 (10%, [M+H]+), 575.3 (20%,
[M+Na]+), 1127.2 (100%, [2M+Na]+); Anal. Calcd for C36H56O4
(552.83): C, 78.21; H, 10.21. Found: C, 78.00; H, 10.18.
ethyl acetate, 8:2); IR (KBr): m 2943s, 2874m, 1781s, 1728s,
1705m, 1642w, 1452m, 1392m, 1372m, 1247s, 1219m, 1155m,
1024m cmꢀ1 1H NMR (500 MHz, CDCl3): d 4.69 (br s, 1H, CHa
;
(30)), 4.57 (br s, 1H, CHb (30)), 4.55–4.45 (m, 1H, CHa (33)), 4.44
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 4.30 (ddd, 1H, J = 8.7, 7.1 Hz,
CHb (33)), 4.04 (dd, 1H, J = 8.2, 8.0 Hz, CH (31)), 2.87 (ddd, 1H,
J = 11.2, 10.4, 6.2 Hz, CH (19)), 2.46–2.23 (m, 4H, CH2 (32)+CH
(13)+CHa (22)), 2.10 (ddd, 1H, J = 14.1, 3.0, 3.0 Hz, CHa (21)), 2.01
(s, 3H, Ac), 1.87 (ddd, 1H, J = 11.2, 8.7, 1.7 Hz, CHa (16)), 1.71–
1.51 (m, 6H, CHa (12)+CH (18)+CHa (1)+CHb (21)+CH2 (2)), 1.65
(s, 3H, CH3 (29)), 1.50–1.14 (m, 11H, CH2 (6)+CH2 (11)+CHb
(16)+CHb (22)+CH2 (7)+CH2 (15)+CH (9)), 1.00–0.88 (m, 2H, CHb
(12)+CHb (1)), 0.94 (s, 3H, CH3 (27)), 0.86 (s, 3H, CH3 (25)), 0.82
(s, 3H, CH3 (24)), 0.81 (s, 3H, CH3 (26)), 0.80 (s, 3H, CH3 (23)),
0.75 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d
209.4 (C@O), 174.2 (C@O), 170.9 (C@O), 150.3 (C20, C@CH2),
109.5 (C30, CH2@C), 80.8 (C3, CHOAc), 67.5 (C33, CH2), 62.8 (C17,
Cquart.), 55.4 (C5, CH), 50.5 (C9, CH), 49.3 (C18, CH), 47.3 (C31,
CH), 46.0 (C19, CH), 42.6 (C14, Cquart.), 40.7 (C8, Cquart.), 38.4 (C1,
CH2), 37.8 (C4, Cquart.), 37.1 (C10, Cquart.), 37.0 (C13, CH), 34.3 (C7,
CH2), 33.6 (C22, CH2), 31.2 (C21, CH2), 30.2 (C16, CH2), 29.4 (C15,
CH2), 28.8 (C32, CH2), 27.9 (C23, CH3), 25.5 (C12, CH2), 23.7 (C2,
CH2), 21.3 (Ac, CH3), 20.9 (C11, CH2), 19.4 (C29, CH3), 18.1 (C6,
CH2), 16.5 (C24, CH3), 16.2 (C25, CH3), 16.0 (C26, CH3), 14.4 (C27,
CH3) ppm; MS (ESI, MeOH): m/z 565.7 (100%, [MꢀH]ꢀ); Anal. Calcd
for C36H54O4 (550.81): C, 76.28; H, 9.60. Found: C, 76.15; H, 9.35.
5.1.23. Ethyl-3-[3b-methoxy-28-norlup-20(29)-en-17b-yl]-
acrylate (16)
5.1.25. 2-((R)-[3b-Acetoxy-28-norlup-20(29)-en-17b-
yl]hydroxymethyl)-c-butyrolactone (18)
Compound 16 (0.34 g, 71%) was obtained from compound 9
(0.50 g, 0.92 mmol) and following GP6 as a colourless solid; mp
185–190 °C; ½a2D0ꢂ ꢀ6.3 (c 3.9, CHCl3); RF 0.50 (silica gel, hexane/
Compound 18 (50 mg, 11%) was obtained as a colourless solid
from compound 11 (0.50 g, 0.88 mmol) following GP6 with an ex-
tended reaction time of 120 h for the mesylation reaction; mp
295–299 °C; ½a2D0ꢂ ꢀ28.4 (c 3.2, CHCl3); RF 0.48 (silica gel, hexane/
ethyl acetate, 9:1); IR (KBr):
m 2938s, 2837m, 1723s, 1646m,
1451m, 1392m, 1375m, 1364m, 1313s, 1248m, 1180s, 1100s,
ethyl acetate, 8:2); IR (KBr):
m
2945s, 1753s, 1736s, 1662w,
1034m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 7.22 (d, 1H,
;
1639w, 1448w, 1383m, 1244s, 1189m, 1156w, 1030m cmꢀ1 1H
;
J = 16.1 Hz, CH (28)), 5.87 (d, 1H, J = 16.1 Hz, CH (31)), 4.72 (d,
1H, J = 2.5 Hz, CHa (30)), 4.58 (dd, 1H, J = 1.2, 2.5 Hz, CHb (30)),
4.20 (q, 2H, J = 7.0 Hz, OCH2), 3.33 (s, 3H, OMe), 2.61 (dd, 1H,
J = 11.7, 4.3 Hz, CHOMe (3)), 2.50 (ddd, 1H, J = 11.2, 11.0, 5.1 Hz,
CH (19)), 1.92–1.84 (m, 2H, CHa (21)+CHa (16)), 1.77–1.53 (m,
7H, CH (13)+CHa (2)+CH2 (22)+CH (18)+CHa (12)+CHa (15)), 1.68
(s, 3H, CH3 (29)), 1.53–1.17 (m, 11H, CHb (21)+CH2 (6)+CH2
(11)+CHb (2)+CHb (16)+CHa (1)+CH2 (7)+CH (9)), 1.30 (t, 3H,
J = 7.0 Hz, CH2CH3), 1.13–1.05 (m, 2H, CHb (12)+CHb (15)), 0.96 (s,
3H, CH3 (25)), 0.94 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (23)), 0.86–
0.78 (m, 1H, CHb (1)), 0.81 (s, 3H, CH3 (24)), 0.73 (s, 3H, CH3
(26)), 0.66 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d 167.1 (C@O), 153.6 (C28, CH@), 151.4 (C20, C@CH2),
120.3 (C31, CH@), 110.0 (C30, CH2@C), 88.6 (C3, CHOMe), 60.2
(OCH2), 57.4 (OMe), 55.8 (C5, CH), 50.4 (C9, CH), 50.0 (C17, Cquart.),
49.7 (C18, CH), 47.6 (C19, CH), 42.8 (C14, Cquart.), 40.8 (C8, Cquart.),
38.8 (C13, CH), 38.7 (C1, CH2), 38.7 (C22, CH2), 38.6 (C4, Cquart.),
37.2 (C10, Cquart.), 34.3 (C7, CH2), 33.4 (C21, CH2), 29.7 (C16, CH2),
28.0 (C23, CH3), 27.7 (C15, CH2), 25.2 (C12, CH2), 22.2 (C2, CH2),
20.8 (C11, CH2), 19.2 (C29, CH3), 18.1 (C6, CH2), 16.1 (C24, CH3),
16.0 (C25, CH3), 15.9 (C26, CH3), 14.7 (C27, CH3), 14.3 (CH2CH3)
ppm; MS (ESI, MeOH): m/z 525.1 (20%, [M+H]+), 547.4 (20%,
[M+Na]+), 1071.1 (100%, [2M+Na]+); Anal. Calcd for C36H56O4
(552.83): C, 80.10; H, 10.76. Found: C, 80.05; H, 10.8.
NMR (500 MHz, CDCl3): d 6.95 (s, 1H, CH (28)), 4.68 (d, 1H,
J = 2.5 Hz, CHa (30)), 4.55 (dd, 1H, J = 1.2, 2.5 Hz, CHb (30)), 4.39
(dd, 1H, J = 11.4, 4.8 Hz, CHOAc (3)), 4.35–4.23 (m, 2H, CH2 (33)),
2.95–2.80 (m, 2H, CH2 (32)), 2.52 (ddd, 1H, J = 11.0, 10.8, 4.6 Hz,
CH (19)), 2.02 (s, 3H, Ac), 1.97 (ddd, 1H, J = 10.2, 3.6, 2.8 Hz, CHa
(16)), 1.77 (ddd, 1H, J = 12.5, 12.5, 3.3 Hz, CH (13)), 1.69–1.46 (m,
7H, CHa (21)+CHa (22)+CHa (12)+CHa (1)+CH2 (2)+CH (18)), 1.63
(s, 3H, CH3 (29)), 1.45–1.11 (m, 8H, CH2 (6)+CHb (16)+CH2
(11)+CHb (21)+CHa (15)+CHb (22)+CH2 (7)+CH (9)), 1.07–0.90 (m,
3H, CHb (12)+CHb (15)+CHb (1)), 0.99 (s, 3H, CH3 (27)), 0.83 (s,
3H, CH3 (25)), 0.77 (s, 3H, CH3 (24)), 0.77 (s, 6H, CH3 (26)), 0.76
(s, 6H, CH3 (23)), 0.71 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3): d 172.2 (C@O), 170.9 (C@O), 149.5 (C20, C@CH2),
146.1 (C28, CH@C), 124.1 (C31, C@CH), 110.1 (C30, CH2@C), 80.9
(C3, CHOAc), 65.1 (C33, CH2), 55.4 (C5, CH), 50.4 (C9, CH), 50.1
(C18, CH), 50.0 (C17, Cquart.), 47.3 (C19, CH), 42.2 (C14, Cquart.),
40.7 (C8, Cquart.), 38.9 (C13, CH), 38.4 (C1, CH2), 37.8 (C4, Cquart.),
37.1 (C10, Cquart.), 37.0 (C22, CH2), 34.2 (C7, CH2), 31.3 (C16, CH2),
30.1 (C21, CH2), 27.9 (C23, CH3), 28.8 (C15, CH2), 25.9 (C32, CH2),
25.3 (C12, CH2), 23.7 (C2, CH2), 21.2 (Ac, CH3), 20.8 (C11, CH2),
19.3 (C29, CH3), 18.1 (C6, CH2), 16.4 (C24, CH3), 16.1 (C25, CH3),
16.0 (C26, CH3), 14.4 (C27, CH3) ppm; MS (ESI, MeOH): m/z 550.3
(30%, [M+H]+), 1123 (100%, [2M+Na]+); Anal. Calcd for C36H54O4
(550.81): C, 78.50; H, 9.88. Found: C, 78.37; H, 9.60.
5.1.24. 2-((RS)-[3b-Acetoxy-28-norlup-20(29)-en-17b-yl]-3-
oxomethyl)-c-butyrolactone (17)
5.1.26. (28 R)-3-Acetyl-28-(2-ethoxy-2-oxoethyl)allobetulin
(19)
Compound 17 (50 mg, 11%) was obtained as a colourless solid
from compound 11 (0.50 g, 0.88 mmol) following GP5. The follow-
Compound 19 (0.20 g, 97%) was obtained from compound 7
(0.20 g, 0.35 mmol) following GP7 as a colourless solid; mp

1354
R. Csuk et al. / Bioorg. Med. Chem. 18 (2010) 1344–1355
210 °C; ½a2D0ꢂ 46.4 (c 4.9, CHCl3); RF 0.57 (silica gel, hexane/ethyl
added to each well and absorbance was measured at 570 nm (using
a 96-well plate reader, Tecan Spectra, Crailsheim, Germany). The
IC50 was estimated from the semi-logarithmic dose–response
curves.
acetate, 8:2); IR (KBr):
m 2944s, 2861m, 1727s, 1643w, 1450w,
1368m, 1250s, 1178w, 1140w, 1073w, 1026m cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d 4.51 (dd, 1H, J = 8.7, 4.1 Hz, CH (28)), 4.46
(dd, 1H, J = 11.5, 4.7 Hz, CHOAc (3)), 4.20–4.10 (m, 2H, OCH2),
3.51 (s, 1H, CH (19)), 2.63 (dd, 1H, J = 14.9, 8.7 Hz, CHa (31)), 2.35
(dd, 1H, J = 14.9, 4.1 Hz, CHb (31)), 2.03 (s, 3H, Ac), 1.73–1.42 (m,
3H, CHa (1)+CHa (21)+CHa (12)+CH (18)+CH2 (2)+CHa (6)), 1.44–
1.17 (m, 7H, CH2 (22)+CHa (15)+CHa (11)+CHb (21)+CH (13)+CH2
(7)+CH2 (16)+CHb (6)+CH (9)), 1.26 (t, 3H, J = 7.0 Hz, CH3), 1.08–
0.91 (m, 4H, CHb (11)+CHb (15)+CHb (1)+CHb (12)), 0.98 (s, 3H,
CH3 (25)), 0.89 (s, 6H, CH3 (27)+CH3 (30)), 0.86 (s, 3H, CH3 (24)),
0.83 (s, 3H, CH3 (26)), 0.82 (s, 6H, CH3 (23)), 0.80 (s, 3H, CH3
(29)), 0.80 (d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d 172.4 (C@O), 171.0 (C@O), 86.5 (C19, CH), 80.9 (C3,
CHOAc), 75.4 (C28, CH), 62.4 (OCH2), 55.5(C5, CH), 50.9 (C9, CH),
47.6 (C18, CH), 42.3 (C17, Cquart.), 41.1 (C14, Cquart.), 40.5 (C8,
Cquart.), 38.6 (C1, CH2), 37.8 (C4, Cquart.), 37.2 (C10, Cquart.), 35.7
(C20, Cquart.), 34.3 (C31, CH2), 33.7 (C7, CH2), 33.6 (C21, CH2), 33.3
(C13, CH), 32.9 (C16, CH2), 28.7 (C30, CH3), 27.9 (C23, CH3), 26.3
(C22, CH2), 26.2 (C12, CH2), 26.1 (C15, CH2), 23.6 (C2, CH2), 25.3
(C29, CH3), 21.3 (Ac), 20.8 (C11, CH2), 18.1 (C6, CH2), 16.4
(C24+C26, 2 ꢁ CH3), 15.7 (C25, CH3), 14.2 (CH2CH3), 13.5 (C27,
CH3) ppm; MS (ESI, MeOH): m/z 571.4 (70%, [M+H]+), 1163.3
(100%, [2M+Na]+); Anal. Calcd for C34H56O4 (528.80): C, 77.22; H,
10.67. Found: C, 77.10; H, 10.33.
5.2.3. Apoptosis test—dye exclusion test
Apoptotic cell death was analysed by trypan-blue dye (Sigma–
Aldrich, Germany) on A431 and A2780 cell lines. The cell culture
flasks with 70% to 80% confluence were treated with IC90 does of
the compounds for 24 h. The supernatant medium with floating
cells was collected after treatment and centrifuged to collect dead
and apoptotic cells. This pellet was re-suspended in serum free
media. Equal amounts of cell suspension and trypan-blue were
mixed and analysed under a microscope. Viable cells exclude the
dye and appear colourless whereas cells whose cell membrane is
destroyed are stained in blue colour. If there are more colourless
cells than stained cells, then death of the cells can be characterised
as apoptotic.
5.2.4. Apoptosis test—DNA fragmentation assay
Determination of apoptotic cell death was performed by DNA
gel electrophoresis. Briefly, cell lines A431 and A2780 were treated
with respective IC90 doses of the compounds for 24 h. Floating
cells—as induced by drug exposure—were collected, re-suspended
in HBSS (1 ml) and transferred to 70% ethanol (10 ml). The cells
were collected and treated with PCB (40
PO4 and 4 parts of 0.1 M citric acid (pH 7.8)) for 1 h at RT. The
supernatant was collected and treated with RNAse A (3 l, 1 mg/
ml) and Nonide NP40 (3 l 0.25% in H2O) at 37 °C for 30 min. Then
proteinase K (3 l, 1 mg/ml) was added and incubated for 30 min
ll, 96 parts of 0.2 M Na2H-
5.1.27. (28R)-3-acetyl-28-(4-ethoxy-2,4-dioxobut-1-
yl)allobetulin (20)
Compound 20 (0.20 g, 95%) was obtained from compound 8
(0.21 g, 0.35 mmol) following GP7 as a colourless solid; mp
184 °C; ½a2D0ꢂ 66.2 (c 3.0, CHCl3); RF 0.33 (silica gel, hexane/ethyl
acetate, 9:1); IR (KBr):
l
l
l
at 37 °C. DNA laddering was observed by running the samples on
2% agarose gel followed by ethidium bromide (Sigma–Aldrich)
staining.
Acknowledgments
5.2. Biology
The authors are grateful to Stefan Schwarz for assistance during
the screening of the compounds. All cell lines were kindly provided
by Dr. Thomas Müller, Department of Hematology/Oncology, Mar-
tin-Luther-Universität Halle-Wittenberg, Halle, Saale, Germany.
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 foetal bovine serum (Biochrom
AG, Berlin, Germany) and penicillin/streptomycin (PAA Laborato-
ries) at 37 °C in a humidified atmosphere of 5% CO2/95% air.
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The cytotoxicity of the compounds was evaluated using the sul-
forhodamine-B (SRB) (Sigma–Aldrich) microculture colorimetric
assay. In short, exponentially growing cells were seeded into 96-
well plates on day 0 at the appropriate cell densities to prevent
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(0–30 lM) for 96 h. The final concentration of DMSO or DMF sol-
vent never exceeded 0.5%, which was non-toxic to the cells. The
percentages of surviving cells relative to untreated controls were
determined 96 h after the beginning of drug exposure. After a
96 h treatment, the supernatant medium from the 96-well plates
was thrown away and the cells were fixed with 10% TCA. For a
thorough fixation, the plates were allowed to rest at 4 °C. After fix-
ation, the cells were washed in a strip washer. The washing was
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