New Lupane Derived Compounds with Pro-Apoptotic Activity in Cancer Cells: Synthesis and Structure-Activity Relationships

Article abstract of DOI:10.1021/jm020854p

Cellular screening of various synthetic triterpenoid compounds formally derived from lupane has identified a number of analogues as potential anticancer drug candidates. Here we describe the synthesis and structure-activity relationships of betulin and betulinic acid derivatives containing an E-ring modified with different oxygen functions. Thus compounds containing the lup-18-en-21-one, lup-18-ene-21,22-dione, 18,19-secolupane, and the highly oxygenated 18,19-secolupane systems, as well as des-E-lupane derivatives, were prepared from the readily available natural pentacyclic triterpene betulin using oxidative procedures. These compounds were named betulinines. We demonstrate that only selected compounds, particularly those containing a lupane E-ring-derived unsaturated ketone or diketone function, possessed in vitro cytotoxic activity against tumor cell lines, suggesting a structure-activity relationship.

Full text of DOI:10.1021/jm020854p

5402
J . Med. Chem. 2003, 46, 5402-5415
New Lu p a n e Der ived Com p ou n d s w ith P r o-Ap op totic Activity in Ca n cer Cells:
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s
J an Sˇarek,† J irˇ´ı Klinot,† Petr Dzˇuba´k,‡ Eva Klinotova´,† Veˇra Noskova´,† Va´clav Krˇecˇek,† Gabriela Korˇ´ınkova´,‡
J ean Oliver Thomson,‡ Anna J anosˇt’a´kova´,‡ Shudong Wang,| Simon Parsons,§ Peter M. Fischer,|
Nikolai Z. Zhelev,| and Maria´n Hajdu´ch*,‡
Department of Organic and Nuclear Chemistry, Charles University in Prague, Hlavova 8, 128 43 Prague 2, Czech Republic,
Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacky´ University and
Faculty Hospital in Olomouc, Puskinova 6, 775 20 Olomouc, Czech Republic, Department of Chemistry,
The University of Edinburgh, J oseph Black Chemistry Building, The King’s Buildings, West Mains Road,
Edinburgh, EH9 3J J , United Kingdom, and Cyclacel Limited, J ames Lindsay Place, Dundee DD1 5J J , United Kingdom
Received February 25, 2002
Cellular screening of various synthetic triterpenoid compounds formally derived from lupane
has identified a number of analogues as potential anticancer drug candidates. Here we describe
the synthesis and structure-activity relationships of betulin and betulinic acid derivatives
containing an E-ring modified with different oxygen functions. Thus compounds containing
the lup-18-en-21-one, lup-18-ene-21,22-dione, 18,19-secolupane, and the highly oxygenated
18,19-secolupane systems, as well as des-E-lupane derivatives, were prepared from the readily
available natural pentacyclic triterpene betulin using oxidative procedures. These compounds
were named betulinines. We demonstrate that only selected compounds, particularly those
containing a lupane E-ring-derived unsaturated ketone or diketone function, possessed in vitro
cytotoxic activity against tumor cell lines, suggesting a structure-activity relationship.
Ch a r t 1
In tr od u ction
The antitumor properties of lupane (1, Chart 1)
derived triterpenoid compounds were first discovered
over 20 years ago when extracts from the stem bark of
various plants was tested for cytostatic activity using
different in vivo cancer model systems.1-5 This led to
the isolation of the pentacyclic triterpene betulinic acid
(BA; 2b), the best-known representative of the lupane-
derived compounds with antiproliferative properties.6
Early data about both the in vitro and in vivo antitumor
activity of BA remain controversial, however. For ex-
ample, original observations2 regarding the efficacy of
BA in the Walker 256 murine carcinosarcoma model
could not be reproduced using a similar system.3 Anti-
tumor activity of BA against P388D1 murine leukaemia
tumors was also suggested.4
More recent studies have shown selective cytotoxicity
of BA against human melanoblastoma cells.7 Cell cycle
analysis of melanoma cells exposed to BA revealed a
G0/G1 block after 32 h exposure, and induction of
apoptosis was observed after 56-72 h. In addition to
this antimelanoma specificity, other studies demon-
strated an antitumor effect against neuroectodermal
tumors. Furthermore, there is also evidence that BA
induces apoptosis via the activation of caspases and that
the cytotoxic activity is independent of cellular p53 gene
status and CD95 activation.8 Interestingly, apoptosis is
mediated via direct effects on mitochondria, since induc-
tion of mitochondrial permeability transition by BA
alone was sufficient to trigger a full apoptosis progres-
sion.9 It is also worth mentioning that BA itself, as well
as some derivatives, have been described as potent anti-
HIV agents.10,11
The screening of various lupane-derived triterpenoid
compounds synthesized in our laboratories has now
identified a number of novel potentially active anti-
cancer drugs. In this report, we describe the synthesis
and structure-activity relationships of derivatives of
BA, in which the E-ring is modified with different
oxygen functions, e.g., 18-lupen-21-one, 18-lup-21,22-
dione, 18,19-secolupane, the highly oxygenated 18,19-
secolupane, and des-E-lupane. We have named these
compounds betulinines. It is known12 from the literature
that the cytotoxic activity of isoprenoid carboxylic acid
derivatives is often related to the presence of a free
carboxyl group in the molecule. The activity of the
corresponding alkyl esters is often much less.12 This fact
is often explained by the comparatively poor hydrolytic
lability of alkyl esters under physiological conditions.
Pivaloyloxymethyl (Pom) and acetoxymethyl (Acm) es-
ters, on the other hand, which are based on dihy-
droxymethane, are hydrolyzed readily by various non-
specific enzymes.13 These esters can be used as suitable
* To whom correspondence should be addressed. Telephone: +420-
58-5854421. Fax: +420-58-58525227. E-mail: hajduchm@atlas.cz.
† Charles University.
‡ Palacky´ University and Faculty Hospital in Olomouc.
§ The University of Edinburgh.
| Cyclacel Limited.
10.1021/jm020854p CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/12/2003

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5403
hydroxy ketone 3b,16 whose carbinol function was
oxidized to the carboxylic acid 3c in high yield with
RuO4. Methyl ester 3d , which has been described
previously,17 was also characterized. 1,8-Diazabicyclo-
[5.4.0]undec-7-ene (DBU)-mediated reactions of keto
acid 3c with chloromethyl pivalate13 or with bromo-
methyl acetate in dichloromethane-acetonitrile mix-
tures afforded the Pom and Acm esters 3e and 3f,
respectively. Selenium dioxide oxidation of 21-ketones
3a and 3d -f in dioxane/acetic acid cleanly yielded the
corresponding 21,22-diketones 4a -d . Using 1H NMR
analysis it was found that R-diketones 4b-d reacted
very easily with simple alcohols, producing complicated
mixtures of hemiketals and ketals; this finding ruled
out the use of alcohols as recrystallization solvents.
Sch em e 1
The 21-ketones 3a and 3d -f, as well as 21,22-
diketone 4a , were used for the synthesis of the 18,19-
secolupane derivatives 5 and 6. For fission of the 18,19-
double bond in the 21-oxo and 21,22-dioxo derivatives,
ruthenium(IV) oxide was used in a catalytic mode (15
mol %). Ruthenium(VIII) oxide was generated in situ
from ruthenium(IV) oxide with sodium periodate in a
two-phase system of ethyl acetate and water. A similar
approach was used by Russian authors18 for the fission
of the double bond in unsubstituted 18-lupenes. Regio-
selectivity and reaction yields were excellent with this
system, although reaction times were often long (1-6
days). The resulting triketones 5a -d and tetraketone
6 are stable crystalline compounds, existing, according
to 13C NMR, in exclusively the nonhydrated oxo-form.
However, attempts to cleave the diketone functions in
these compounds with lead tetraacetate or sodium
periodate did not lead to nor-derivatives of tetranoracids
of type 7. Instead, the latter compounds were obtained
through Baeyer-Villiger rearrangement with the aid
of peroxyacetic acid and they were characterized as the
free acids 7a and 7d , or in the form of methyl esters 7b
and 7e-g. Tetranoracid 7a was then lactonized easily
to the previously known spirolactone 14.19 Through
retro-aldol reaction of 7a , followed by methylation and
acetylation, pentanorlupane derivative 7c was obtained.
This compound had previously been prepared by a
different approach.19
prodrug groups for carboxylic acids. Because of their
lipophilic character, they are membrane permeable and
their purification is much easier than that of free
carboxylic acids.
In this study, 3â-acetoxylupane derivatives with
oxidative modifications of the E-ring and variously
bearing acetoxymethyl, carboxyl, methoxycarbonyl,
(pivaloyloxy)methoxycarbonyl, and acetoxymethoxycar-
bonyl groups at C17 were prepared and tested. The
cytotoxic activity of this series of derivatives was as-
sessed comparatively using human tumor cell lines.
Tetraketone 6 was obtained by ruthenium-catalyzed
oxidative degradation of diketone 4a . Yields of 6 were
improved by addition of acetonitrile to the reaction
mixture20 and an increase in the catalytic amount of
ruthenium(IV) oxide to 17 mol %. Carboxylic acid 8a
was prepared by cleavage of tetraketone 6 with peroxy-
acetic acid as reported.21 Compound 8a could also be
obtained by oxidative fission of the diene 10, which in
turn was obtained by a single-step reaction22 from lup-
18-ene-3â,28-diyl diacetate. However, despite the shorter
synthetic route from betulin, the latter method is not
as suitable for larger scale preparations of key com-
pound 8a as the former route via tetraketone 6, or
indeed an alternative route via anhydride 9a we have
reported on previously.23 Methyl ester 8b was obtained
in the usual manner, whereas attempts to prepare Pom-
ester 8c were not successful. Similarly, application of
the DBU-mediated alkylation method referred to above
did not permit preparation of Acm-ester 8d and only
decomposition products were obtained. Nevertheless, 8d
Ch em istr y
The starting material for the preparation of the
compounds described here was 21-oxolup-18-ene-3â,28-
diyl diacetate 3a (Scheme 1). This unsaturated ketone
is readily obtained from the natural triterpene betulin
2a through successive acetylation, acid-catalyzed isomer-
ization of the 20(29)-double bond,14 and allylic oxidation
of the resulting 18,19-unsaturated product.15 Selective
saponification of the 28-acetate ester then furnished

5404 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
Sch em e 2
could be prepared by alkylation of the silver carboxylate
salt of acid 8a in dimethylformamide.
explanations of the origin of the ethoxy derivative 13a
is Michael addition of ethanol to methylene ketone 12
under the basic conditions applied. If dioxane instead
of ethanol is used as the solvent, high yields of pure
heptanor ketone 11a could be isolated.
Oxidative fission of the 17(28)-double bond of meth-
ylene ketone 12 produced heptanor-17,18-seco diacid
15a .25 This acid can also be prepared by fission of the
double bond in hydroxy ketone 3b or acid 3c. Dimethyl
ester 15b25 was prepared from the diacid 15a . The new
diPom-ester 15c was also prepared, but it was impos-
sible to obtain it in crystalline form. The structures of
all the compounds were confirmed by spectral analysis.
Furthermore, the structure and absolute configuration
of key compound 8a was confirmed through a single-
crystal X-ray structure (see Figure 1; refer also to the
Supporting Information).
The relative stability of keto acid 8a , which on
theoretical grounds one would expect to decarboxylate
spontaneously, is probably due to steric congestion
induced by the 17â-acetoxymethyl group, thus hindering
formation of the requisite six-membered ring intermedi-
ates necessary in the cyclic mechanism of decarboxyla-
tion between the 18-oxo and 17R-carboxylate groups. It
is known,18 however, that if the carboxylic function is
in position 17â, the corresponding 18-keto derivatives
decarboxylate spontaneously.
â-Keto acid 8a was used for the preparation of other
highly degraded lupane and des-E lupane derivatives
(Scheme 2). Heating of â-keto acid 8a in boiling diglyme
led to formation of a complicated product mixture
through thermal decomposition. Two of the three main
components could be identified, the known heptanor-
ketoacetate 11b24 and methylene ketone 12, an unstable
compound that was isolated by lyophilization from
benzene; the third compound was very unstable and
could not be identified.
Very unusual is the reaction of â-keto acid 8a with
potassium hydroxide. If this reaction is performed in
the presence of ethanol, the ether 13a appears in the
product mixture along with the normal product of the
retroaldol reaction, which then undergoes decarboxy-
lation to the heptanorketone 11a . One of the possible
F igu r e 1. ORTEP-style plot of one of the two crystallographi-
cally independent molecules of 8a in the single-crystal struc-
ture. Ellipsoids are drawn at the 30% level.

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5405
F igu r e 2. Screening of cytotoxic activity of synthesized lupane derivatives on the chemosensitive CEM T-lymphoblastic leukaemia
cell line.
Synthesis of the compound 8a , described here, was
successfully scaled-up to 100 g, with the same yields
and purity. Betulin, the starting material for the six-
step synthesis of 8a , is a waste product from the
industrial production of paper from birch wood. Esti-
mated production of betulin is thousands of tons per
annum. Therefore, a large-scale availability of the
compound 8a is both cost-effective and ecological. Ecol-
ogy of production is further supported by the fact that
the six-step process was optimized with no industrial
waste.
and species origin (Table 1). It can be seen that
nonmalignant cells, e.g. NIH3T3 fibroblasts and normal
human lymphocytes, tolerated substantially higher
doses of betulinines than the tumor cells, thus demon-
strating a preferential cytotoxicity for malignant cells
and a favorable therapeutic index.
We show here that only selected derivatives of BA
(2b) were active against CEM cells under in vitro
conditions, suggesting a structure-activity relationship.
From the results of the in vitro MTT tests it follows that
the cytotoxic activity of the betulinines is often due to
the presence in the triterpenic molecule of a â-dicarbonyl
group, e.g. as in compounds 4b, 4d , 5d , 7a , and 8a , or
of an R-unsaturated ketone, e.g. in compounds 3a and
16.
The cell cycle and apoptosis analyses clearly demon-
strate that the most active BA (2b) derivatives (4a , 8a ,
16) induce apoptosis in CEM cells, which was demon-
strated by both the flow-cytometry and scanning elec-
tron microscopy techniques (Figures 4 and 5). However,
the compounds differed mostly in time dependency of
apoptosis induction. The most active was compound 8a ,
where the first sub-G1 cells appeared as soon as 3 h
after the treatment, although no specific cell cycle
alterations were observed. On the other hand, in the
CEM cells treated with compounds 4b and 16 the first
signs of apoptosis were between 24 and 48 h after
treatment. Interestingly, compound 16 also induced an
accumulation of cells in the G2/M and S regions. We
hypothesize that the increase in the S phase fraction
could be due to the induction of apoptosis at the G2/M
Resu lts a n d Discu ssion
Since betulinines are complicated natural compounds
with lipophilic and rigid carbon skeleton, they are
soluble in chlorinated solvents (e.g. chloroform) or ethers
(e.g. THF, dioxane) but poorly soluble in water and
water-based solvents. However, some of these com-
pounds, particularly those with a free carboxylic group,
can be dissolved in a water-ethanol system using
pharmaceutically acceptable vehicles.
To evaluate the anticancer potency of our betulinines,
their cytotoxic activity against the drug-sensitive CEM
cell line was initially examined in the screening assay
(Figure 2). It was followed by expanded activity analysis
against typical representatives of epithelial, neuro-
ectodermal, and mesodermal tumor cell lines (refer to
the Supporting Information). Potent compounds with
TCS50 (concentration with 50% tumor cell survival) in
the low micromolar concentration range were further
tested on a panel of cell lines of different histogenetic

5406 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
Ta ble 1. Cytotoxicity of Selected Lupane Derivatives (2b, 4b, 8a , 16) in a Panel of Cell Lines of Different Histogenetic Origin and
Having Various Phenotype Alterations (mean ( SD)
cytotoxicity (TCS50, µM)
cell line
descriptiona
2b
4b
8a
16
B16
B16F
SW620
U87MG
HepG2
A549
MCF-7
U2OS
Saos2
BT549
MDA-MB-2 38
LNCaP
DU145
HT-29
OVCAR-3
Caco-2
MEL-3
lymphocytes
NIH3T3
K562
K562-CdA
K562-GEM
K562-ARA-C
K562-FLU D
CEM
mouse melanoma
mouse melanoma, metastatic
human colon cancer, metastasis
human glioblastoma
30.5 ( 3.72 35.4 ( 3.47
4.6 ( 0.38 67.9 ( 5.14
0.43 ( 0.25
4.66 ( 0.52
1.22 ( 0.11
1.85 ( 0.53
1.67 ( 0.09
9.32 ( 1.05
12.4 ( 0.92
5.86 ( 0.61
98.3 ( 3.95
7.12 ( 0.82
>250
5.89 ( 0.47
>228 ( 24.7 23.8 ( 2.43
3.61 ( 0.28 5.14 ( 0.55
human hepatocellular carcinoma
human lung adenocarcinoma
79.3 ( 6.3
6.7 ( 0.73
2.8 ( 0.34 43.27 ( 3.78
human breast cancer, estrogen dependent, p53+/+, RB+/+
194 ( 15.8 12.9 ( 1.17
2.3 ( 0.20
1.53 ( 0.15
1.78 ( 0.22
2.03 ( 0.17
1.35 ( 0.1
1.12 ( 0.15
0.84 ( 0.11
4.3 ( 0.52
3.94 ( 0.43
4.8 ( 0.51
6.28 ( 0.49
2.37 ( 0.18
3.55 ( 0.36
2.75 ( 0.30
human osteosarcoma, p53+/-, RB
>250
>250
>250
4.28 ( 0.44
7.9 ( 0.83
15.3 ( 1.31
+/-
human rhabdomyosarcoma, p53-/-, RB-/-
human breast cancer, p53mut/mut
human breast cancer, estrogen independent, p53mut/mut
human prostate cancer, androgen dependent
human prostate cancer, androgen independent, RB-/-
human colon cancer
human ovarian cancer
human colon cancer
human melanoma
human normal lymphocytes
mouse immortalized fibroblasts
human promyelocytic leukemia
human promyelocytic leukemia, cladribin resistant
human promyelocytic leukemia, gemcitabin resistant
human promyelocytic leukemia, cytarabin resistant
human promyelocytic leukemia, fludarabin resistant
human T-lymphoblastic leukemia
195 ( 15.3 3.28 ( 0.39
244 ( 11.7 3.26 ( 0.29
241 ( 22.9 1.98 ( 0.25
>250
5.23 ( 0.44
164 ( 15.8 4.65 ( 0.38
19.6 ( 2.04 8.13 ( 0.79
2.65 ( 0.35 5.86 ( 0.63
3.57 ( 0.24 12.31 ( 1.68
0.9 ( 0.10
2.97 ( 0.28
1.28 ( 0.21
12.5 ( 1.13
2.59 ( 0.19
7.72 ( 0.86
3.68 ( 0.37
24.9 ( 2.11
15.7 ( 1.84
>250
85.3 ( 9.15
>250
96.8 ( 10.37 7.21 ( 0.83
53.9 ( 5.79 1.85 ( 0.22
0.19 ( 0.20 15.12 ( 1.93
>250
5.17 ( 0.62
0.28 ( 0.03
0.94 ( 0.10
0.63 ( 0.07
0.35 ( 0.41
1.0 ( 0.14
0.61 ( 0.58
1.05 ( 0.09
3.27 ( 0.39
2.93 ( 0.33
2.51 ( 3.07
2.13 ( 0.24
2.36 ( 0.25
1.59 ( 0.18
1.27 ( 0.13
4.0 ( 0.32
1.58 ( 0.17
3.26 ( 0.41
5.94 ( 0.47
7.82 ( 0.86
6.95 ( 0.58
101 ( 9.77 5.04 ( 0.61
>250
>250
>250
6.42 ( 0.49
4.85 ( 0.54
4.4 ( 0.23
10.3 ( 0.11
5.18 ( 0.49
CEM-DNR 1/C2 human T-lymphoblastic leukemia, daunorubicin resistant >250
CEM-DNR bulk human T-lymphoblastic leukemia, daunorubicin resistant >250
CEM-VCR 1/F3 human T-lymphoblastic leukemia, vincristin resistant
CEM-VCR 3/D5 human T-lymphoblastic leukemia, vincristin resistant
CEM-VCR bulk human T-lymphoblastic leukemia, vincristin resistant
19.1 ( 2.04 38.3 ( 4.15
24.1 ( 2.63 12.5 ( 1.08
68.5 ( 7.19 28.2 ( 2.95
a
The majority of genetic alterations referred to here are described at http://www.atcc.org/.
transition and the appearance of the sub-G2 cells, i.e.,
the population analogous to sub-G1 cells but having a
tetraploid DNA content. Although this explanation is
further supported by the persistence of the S phase/sub-
G2 cells for as long as 18 h after the disappearance of
the G0/1 populations among the paclitaxel-treated CEM
lymphoblasts, further studies should precisely specify
the site and mechanism of action of these molecules.
Although BA has been reported to be preferentially
effective against neuroectodermal tumors,7-9 its deriva-
tives described in this study have lost their specificity
and demonstrated a broad anticancer potency (Table 1).
Thus, the lack of significant activity correlation of BA
with active betulinines of this study (Figure 3) is
consistent with this observation. On the other hand, the
effectiveness of most of the active betulinines described
in Table 1 is significantly correlated (Figure 3), sug-
gesting a common mechanism of action, although the
different kinetics of apoptosis induction of 8a versus 4b/
16 compounds would suggest independent targets (Fig-
ure 4). However, we propose that this difference in
timing of apoptosis is rather due to the dissimilar
physical and/or pharmaceutical properties of these
molecules, e.g., solubility, intracellular penetration,
stability, metabolic activation.
the hormonal status of the cancer cells, and therefore,
compounds should be equally effective in the treatment
of hormone-dependent and -independent cancers (Table
1). Finally, the betulinines were effective on both the
drug-resistant cell lines and the drug-sensitive coun-
terparts. These data demonstrate that the classical
mechanisms of multidrug resistance will apparently not
apply to this novel generation of compounds, and thus,
they could provide significant therapeutic benefit to
chemotherapy-resistant cancer patients.
Exp er im en ta l Section
Gen er a l. Melting points were determined using a Kofler
block and are uncorrected. Optical rotations were measured
using CHCl3 solutions (unless otherwise stated) on an Autopol
III (Rudolph Research, Flanders, NJ ) polarimeter, with an
accuracy of (2°. NMR spectra were recorded on a Varian
UNITY INOVA 400 instrument (1H NMR spectra at 399.95
MHz) using CDCl3 solutions (unless otherwise stated), with
SiMe4 as an internal standard. EIMS spectra were recorded
on an INCOS 50 (Finigan MAT) spectrometer at 70 eV and
an ion source temperature of 150 °C. The samples were
introduced from a direct exposure probe at a heating rate of
10 mA/s. Relative abundances stated are related to the most
abundant ion in the region of m/z > 50. TLC was carried out
using silica gel 60 F254, and detection was by spraying with
10% aqueous H2SO4 and heating to 150-200 °C. Column
chromatography was performed using silica gel 60 (Merck
7734). For preparative HPLC, a normal phase silica gel column
(250 × 25 mm, Biospher 7 µm), pump Gilson, and differential-
refractometer detector (Laboratornı´ prˇ´ıstroje, Praha, Czech
Republic) were used.
Notably, a similar effectiveness of the betulinines was
also found on cell lines bearing various mutations or
deletions in the cell-cycle-associated proteins (Table 1).
This fact indicates that betulinines such as compound
8a should be equally effective on tumors with various
alterations of tumor suppressor genes, such as p53 and
the retinoblastoma protein pRb. We also note that the
cytotoxic activity of the betulinines is independent of
Acetate esters were prepared by overnight esterification at
room temperature with a 1:1 mixture of pyridine and Ac2O.
Workup refers to pouring of the reaction mixture into H2O;
extraction of the product with Et2O; and washing of the organic

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5407
F igu r e 3. Spearmann correlation analysis of in vitro cytotoxic activity of betulinic acid (2b) and the most effective derivatives
16, 4b, and 8a (r, correlation coefficient; p, statistical signicance).
layer successively with H2O, dilute aqueous HCl, H2O, satu-
rated aqueous NaHCO3, and again H2O, followed by drying
over MgSO4, filtration, and evaporation of the filtrate under
the reduced pressure. Analytical samples were dried over P2O5
under diminished pressure. 1,8-Diazabicyclo[5.4.0]undec-7-ene
(DBU), bromomethyl acetate (Acm-Br), chloromethyl pivalate
(Pom-Cl), silver carbonate, and ruthenium(IV) oxide hydrate
were purchased from Aldrich. Ketone diacetate 3a ,15 diene
10,22 and aldehyde 1626 were prepared according to literature
procedures. Betulinic acid 2b was obtained by extraction of
the bark of plane trees (Platanus x hybrida) with MeOH and
several crystallizations from the same solvent.
Hz, 1H, C28Hâ), 4.49 (dd, J ) 11.0 Hz, J ′ ) 5.5 Hz, 1H, C3HR).
Ref.16 mp 292-294 °C, [R]D -69° (c 0.57).
3â-Acetoxy-21-oxolu p -18-en -28-oic Acid (3c). A suspen-
sion of hydroxy ketone 3b (1.59 g, 3.2 mmol) in EtOAc (135
mL) was added to a mixture of Ru(IV)O2‚H2O (40 mg, 0.3
mmol), NaIO4 (4 g, 19 mmol), H2O (120 mL), and CF3COOH
(2 mL). The biphasic mixture was stirred vigorously for 6 h.
EtOH was then added and the mixture was filtered. The
organic phase was separated and filtered through a short
column of SiO2. The column was washed with EtOAc and the
filtrate was evaporated under reduced pressure. The residue
was washed with Et2O and was crystallized from butanone to
afford 3c (1.06 g, 66%, mp 222-239 °C): [R]D -40° (c 0.42
dioxane); 1H NMR δ 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.92
(s, 3H, CH3), 0.94 (s, 3H, CH3), 1.06 (s, 3H, CH3), 1.21 (d, 3H
(J ) 7.0 Hz, CH3), 1.22 (d, J ) 7.0 Hz, CH3, 3H), 2.03 (s, 3H,
OCOCH3), 2.20 (br. d, J ) 18.6 Hz, 1H, C22HR), 2.47 (m, 1H,
C16Hâ), 2.59 (d, J ) 18.6 Hz, 1H, C22Hâ), 2.76 (dd, J ) 12.7
Hz, J ′ ) 3.2 Hz, 1H, C13Hâ), 3.21 (sept., J ) 7.0 Hz, 1H, C20H),
4.49 (dd, J ) 11.0 Hz, J ′ ) 5.5 Hz, 1H, C3HR); EIMS m/z 512
(M+, 13), 468 (16), 452 (8), 437 (4), 426 (1), 409 (28), 393 (7),
365 (8), 317 (3), 264 (8), 249 (78), 229 (11), 218 (11), 205 (100),
189 (94). Anal. (C32H48O5) C, H.
28-Hydr oxy-21-oxolu p-18-en -3â-yl Acetate (3b). A slightly
modified literature procedure16 was used: a solution of usatu-
rated ketone diacetate 3a (10 g, 18.5 mmol) and KOH (1.25 g,
22.5 mmol) in PhMe/EtOH (1:1, 0.6 L) was stirred vigorously
for 6 h, when TLC (hexane/EtOAc 1:1) indicated complete
reaction. After workup a white crystalline residue was ob-
tained, which was recrystallized from EtOAc to afford 3b (8.2
1
g, 90%, mp 310-313 °C dec): [R]D -67° (c 0.48); H NMR δ
0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.95 (s,
3H, CH3), 1.13 (s, 3H, CH3), 1.19 (d, J ) 6.9 Hz, 3H, CH3),
1.21 (d, J ) 6.9 Hz, 3H, CH3), 1.92 (d, J ) 18.6 Hz, 1H, C22HR),
2.05 (s, 3H, OCOCH3), 2.44 (d, J ) 18.6 Hz, C22Hâ), 2.78 (dd,
J ) 12.5 Hz, J ′ ) 3.4 Hz, 1H, C13Hâ), 3.19 (sept., J ) 6.9 Hz,
1H, C20H), 3.67 (d, J ) 10.7 Hz, 1H, C28HR), 3.72 (d, J ) 10.7
Meth yl 3â-Acetoxy-21-oxolu p -18-en -28-oa te (3d ). This
compound was obtained from keto acid 3c (250 mg, 0.49 mmol)
by esterification with diazomethane. Crystallization from

5408 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
F igu r e 4. Apoptosis and cell cycle analysis of CEM cells untreated (control) or treated with betulinic acid (2b) and the most
effective derivatives 16, 4b, and 8a (10 µM). Paclitaxel-treated cells (1 µM) were used as a positive control for both apoptosis and
specific cell cycle block on G2/M transition (typical example of several independnet experiments).
MeOH afforded 3d (213 mg, 83%, mp 220-222 °C): [R]D -33°
C22HR), 2.46 (d, J ) 18.7 Hz, 1H, C22Hâ), 2.48 (ddd, J ) 13.5
Hz, J ′ ) 3.9 Hz, J ′′ ) 2.7 Hz, 1H, C16Hâ), 2.64 (dd, J ) 12.7
Hz, J ′ ) 3.2 Hz, 1H, C13Hâ), 3.20 (sept., J ) 7.0 Hz, 1H, C20H),
3.70 (s, 3H, (OCH3), 4.49 (dd, J ) 11.0 Hz, J ′ ) 5.3 Hz, 1H,
1
(c 0.39). H NMR δ 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91
(s, 3H, CH3), 0.93 (s, 3H, CH3), 1.03 (s, 3H, CH3), 1.21 (d, J )
7.0 Hz, 6H), 2.05 (s, 3H, OCOCH3), 2.13 (d, J ) 18.7 Hz, 1H,

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5409
F igu r e 5. Scanning electron microscopy study of apoptosis induction in CEM cells untreated (control) or treated with betulinic
acid (2b) and the most effective derivatives 16, 4b, and 8a (10 µM). (A) Typical morphology of cells after 24-hour treatment.
Arrows indicate morphological markers of apoptosis: cytoplasm membrane blabbing, cellular fragmentation, and formation of
apoptotic bodies (magnification 3600×). (B) Dynamics of the process is expressed as percentages of apoptotic cells (x ( SD) at
different time points.
C3HR); EIMS m/z 526 (M+, 4), 483 (1), 466 (2), 441 (1), 435 (1),
423 (5), 369 (1), 331 (1), 315 (1), 287 (1), 276 (3), 263 (56), 203
(13), 189 (22). Ref.17 mp 238-241 °C (CH2Cl2/MeOH), [R]D -29°
(CHCl3). Anal. (C33H50O5), C, H.
(4), 536 (5), 512 (4), 467 (22), 452 (12), 437 (2), 423 (2), 409
(20), 391 (4), 363 (17), 333 (76), 249 (48), 216 (7), 203 (100),
189 (38). Anal. (C38H58O7) C, H.
Acetoxym eth yl 3â-Acetoxy-21-oxolu p-18-en -28-oate (3f).
Keto acid 3c (500 mg, 0.98 mmol) was treated with Acm-Br
and DBU in CH2Cl2 (2.5 mL) and MeCN (1 mL) in the same
manner as in the preparation of Pom-ester 3e. Crystallization
from butanone produced the Acm-ester 1f as colorless needles
(P iva loyloxy)m et h yl 3â-Acet oxy-21-oxolu p -18-en -28-
oa te (3e). DBU (165 µL, 1.1 mmol) and Pom-Cl (160 µL; 1.1
mmol) were added to a solution of the keto acid 3c (500 mg,
0.98 mmol) in CH2Cl2 (2.5 mL) and MeCN (1 mL). The mixture
was stirred at room temperature for 3 h. It was then diluted
with ice-cold H2O and extracted with CHCl3. The extract was
washed with cold brine, dried, and evaporated. The residual
viscous pale yellow oil (750 mg) was chromatographed on SiO2
(PhMe). Crystallization from MeOH afforded the Pom-ester
3e as colorless needles (453 mg, 72%, mp 179-180 °C): [R]D
1
(433 mg, 76%, mp 233-235 °C): [R]D -26° (c 0.4); H NMR δ
0.84 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.94 (s,
3H, CH3), 1.05 (s, 3H, CH3), 1.20 (d, J ) 7.0 Hz, 3H CH3), 1.21
(d, J ) 7.0 Hz, 3H CH3), 2.05 (s, 3H, OCOCH3), 2.10 (s, 3H,
OCOCH3), 2.15 (dd, J ) 18.6 Hz, 1H, C22HR), 2.47 (d, J ) 18.6
Hz, 1H, C22Hâ), 2.48 (ddd, J ) 13.4 Hz, J ′ ) 4.1 Hz, J ′′ ) 2.7
Hz, 1H, C16Hâ), 2.67 (dd, J ) 12.6 Hz, J ′ ) 3.3 Hz, 1H, C13Hâ),
3.19 (sept., J ) 7.0 Hz, 1H, C20H), 4.49 (dd, J ) 11.0 Hz, J ′ )
5.3 Hz, 1H, C3HR), 5.70 (d, J ) 5.5 Hz, 1H, OCH2O), 5.79 (d,
J ) 5.5 Hz, 1H, OCH2O). EIMS m/z 584 (M+, 30), 554 (11),
541 (23), 512 (44), 481 (48), 467 (26), 452 (49), 409 (77), 391
(11), 321 (100), 249 (59), 203 (33), 189 (25). Anal. (C35H52O7)
C, H.
1
-18° (c 0.36); H NMR δ 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3),
0.91 (s, 3H, CH3), 0.93 (s, 3H, CH3), 1.06 (s, 3H, CH3), 1.19 (d,
J ) 7.0 Hz, 3H, CH3), 1.21 (d, J ) 7.0 Hz, 3H, CH3), 1.20 (s,
9H, C(CH3)3), 2.05 (s, 3H OCOCH3), 2.14 (dd, J ) 18.7 Hz, J ′
) 0.8 Hz, 1H, C22HR), 2.46 (d, J ) 18.7 Hz, 1H, C22Hâ), 2.48
(ddd, J ) 13.4 Hz, J ′ ) 4.1 Hz, J ′′ ) 2.6 Hz, 1H, C16Hâ), 2.67
(dd, J ) 12.7 Hz, J ′ ) 3.7 Hz, 1H, C13Hâ), 3.19 (sept., J ) 7.0
Hz, 1H, C20H), 4.49 (dd, J ) 11.0 Hz, J ′ ) 5.4 Hz, 1H, C3HR),
5.66 (d, J ) 5.5 Hz, 1H, OCH2O), 5.84 (d, J ) 5.5 Hz, 1H,
OCH2O). EIMS m/z 626 (M+, 6), 611 (1), 596 (28), 583 (4), 566
21,22-Dioxolu p -18-en e-3â,28-d iyl Dia ceta te (4a ). A solu-
tion of ketone diacetate 3a (2 g, 3.7 mmol) and SeO2 (1.6 g,-
14.4 mmol) in dioxane (40 mL), AcOH (20 mL), and Ac2O (2

5410 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
mL) was heated under reflux until reaction was complete by
TLC (PhMe/Et2O 10:1). After cooling, precipitated selenium
was removed by filtration and the filtrate was poured slowly
into an excess of vigorously stirred water. The red-orange
precipitate was filtered under reduced pressure, washed
carefully with H2O, and air-dried. The crude product was
dissolved in CHCl3 and the solution was filtered through a
column of alumina. The column was eluted with CHCl3 and
the filtrate was evaporated under reduced pressure. The
residue was crystallized from MeOAc to afford 4a as pale-
orange crystals (1.45 g, 82%, mp 267-270 °C): [R]D -127° (c
vigorously at room temperature for 4 h, at which time reaction
was complete by TLC (PhMe/Et2O 6:1). EtOH was added, the
mixture was filtered, and the organic layer was chromato-
graphed on a short SiO2 column (EtOAc). The eluate was
evaporated under reduced pressure and the residue was
crystallized from Me2CO to afford 5a as gold petals (339 mg,
1
64%, mp 226-228 °C): [R]D +71° (c 0.39); H NMR δ 0.84 (s,
3H, CH3), 0.85 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.92 (s, 3H,
CH3), 1.13 (s, 3H, CH3), 1.13 (d, J ) 6.9 Hz, 3H, CH3), 1.16 (d,
J ) 6.9 Hz, 3H, CH3), 1.84 (ddd, J ) 13.9 Hz, J ′ ) 4.4 Hz, J ′′
) 2.2 Hz, 1H, C16Hâ), 2.05 (s, 6H, 2 OCOCH3), 2.31 (td, J )
13.9 Hz, J ′ ) 13.9 Hz, J ′′ ) 4.6 Hz, 1H, C16HR), 2.68 (dd, J )
11.6 Hz; J ′ ) 3.3 Hz, 1H, C13Hâ), 2.45 (d, J ) 16.6 Hz, 1H,
C22HR), 3.25 (d, J ) 16.6 Hz, 1H, C22Hâ), 3.38 (sept., J ) 6.9
Hz, 1H, C20H), 4.20 (d, J ) 11.3 Hz, 1H, C28HR), 4.48 (d, J )
11.3 Hz, 1H, C28Hâ), 4.48 (dd, J ≈ 10 Hz, J ′ ≈ 6 Hz, 1H, C3HR);
EIMS m/z 572 (M+, 0.2), 557 (0.4), 501 (15), 459 (16), 441 (53),
399 (8), 381 (32), 371 (15), 353 (1), 339 (1), 215 (6), 201 (17),
189 (50), 71 (100). Anal. (C34H52O7) C, H.
1
0.32); H NMR δ 0.85 (s, 3H, CH3), 0.86 s, 3H, CH3), 0.94 (s,
3H, CH3), 0.97 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.24 (d, J ) 7.0
Hz, 3H, CH3), 1.26 (d, J ) 7.0 Hz, 3H, CH3), 1.91 (s, 3H,
OCOCH3), 2.06 (s, 3H, OCOCH3), 3.12 (dd, J ) 12.5 Hz, J ′ )
3.5 Hz, 1H, C13Hâ), 3.36 (sept., J ) 7.0 Hz, 1H, C20H), 4.02 (d,
J ) 11.0 Hz, 1H, C28HR), 4.49 (dd, J ) 11.0 Hz, J ′ ) 5.5 Hz,
1H, C3HR), 4.84 (d, J ) 11.0 Hz, C28HR). Ref.16 mp 269-271
°C, [R]D -136° (c 0.6).
Meth yl 3â-Acetoxy-21,22-d ioxolu p -18-en -28-oa te (4b).
Ketomethyl ester 3d (500 mg, 0.98 mmol) was oxidized with
SeO2 (400 mg, 3.6 mmol) in a mixture of dioxane (10 mL),
AcOH (5 mL), and Ac2O (0.5 mL) in the same manner as in
the preparation of diketone diacetate 4a . Crystallization from
dimethyl carbonate afforded 4b as pale-orange needles (387
Meth yl 3â-Acetoxy-18,19,21-tr ioxo-18,19-secolu p a n -28-
oa te (5b). Ketomethyl ester 3d (400 mg; 0.76 mmol) was
oxidized with Ru(VIII)O4 (from Ru(IV)O2‚H2O; 13 mg, 0.1
mmol) and NaIO4 (1.1 g) in EtOAc (18 mL), H2O (15 mL), and
CF3COOH (0.4 mL) in the same manner as in the preparation
of triketone diacetate 5a . Crystallization from MeOH afforded
5b as yellow petals (259 mg, 61%, mp 173-175 °C): [R]D +43°
1
mg, 73%, mp 228-234 °C dec): [R]D -100° (c 0.43); H NMR
1
δ 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.97
(s, 3H, CH3), 1.06 (s, 3H, CH3), 1.26 (d, J ) 7.0 Hz, 3H, CH3),
1.29 (d, J ) 7.0 Hz, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.54 (ddd,
J ) 13.5 Hz, J ′ ) 4.4 Hz, J ′′ ) 2.2 Hz, 1H, C16Hâ), 2.78 (dd, J
) 12.7 Hz, J ′ ) 3.3 Hz, 1H, C13Hâ), 3.36 (sept., J ) 7.0 Hz,
1H, C20H), 3.72 (s, 3H, OCH3), 4.49 (dd, J ) 11.2 Hz, J ′ ) 5.3
Hz, 1H, C3HR); EIMS m/z 540 (M+, 1), 525 (1), 512 (3), 501 (1),
484 (100), 469 (37), 279 (3), 217 (4), 203 (7), 191 (17). Anal.
(C33H48O6) C, H.
(c 0.45); H NMR δ 0.82 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.85
(s, 3H, CH3), 0.89 (s, 3H, CH3), 1.07 (s, 3H, CH3), 1.12 (d, J )
7.0 Hz, 3H, CH3), 1.14 (d, J ) 7.0 Hz, 3H, CH3), 2.04 (s, 3H,
OCOCH3), 2.87 (d, J ) 16.5 Hz, 1H, C22HR), 2.99 (dd, J ) 11.9
Hz, J ′ ) 3.5 Hz, 1H, C13Hâ), 3.15 (d, J ) 16.5 Hz, 1H, C22Hâ),
3.36 (sept., J ) 7.0 Hz, 1H, C20H), 3.78 (s, 3H, OCH3), 4.47
(dd, J ) 11.0 Hz, J ′ ) 5.4 Hz, 1H, C3HR); EIMS m/z 558 (M+,
4), 527 (1), 487 (17), 444 (4), 427 (98), 395 (6), 367 (6), 277 (5),
249 (8), 231 (8), 215 (11), 201 (32), 189 (100). Anal. (C33H50O7)
C, H.
(P iva loyloxy)m eth yl 3â-Acetoxy-18,19,21-tr ioxo-18,19-
secolu p a n -28-oa te (5c). Keto-Pom ester 3e (400 mg, 0.64
mmol) was oxidized with Ru(VIII)O4 (from Ru(IV)O2‚H2O; 13
mg, 0.1 mmol) and NaIO4 (2.1 g) in EtOAc (16 mL), H2O (14
mL), and CF3COOH (0.4 mL) in the same manner as in the
preparation of triketone diacetate 5a . Crystallization from
MeOH afforded 5c as yellow petals (273 mg, 65%, mp 152-
154 °C): [R]D +40° (c 0.38); 1H NMR δ 0.81 (s, 3H, CH3), 0.84
(s, 3H, CH3), 0.84 (s, 3H, CH3), 0.89 (s, 3H, CH3), 1.08 (s, 3H,
CH3), 1.11 (d, J ) 7.0 Hz, 3H, C30H3), 1.14 (d, J ) 7.0 Hz, 3H,
C30H3), 1.22 (s, 9H, C(CH3)3), 2.04 (s, 3H, OCOCH3), 2.36 (br.
m, 1H, C16HR), 2.88 (d, J ) 16.8 Hz, 1H, C22HR), 3.06 (dd, J )
11.9 Hz, J ′ ) 3.5 Hz, 1H, C13Hâ), 3.18 (d, J ) 16.9 Hz, 1H,
C22Hâ), 3.35 (sept., J ) 7.0 Hz, 1H, C20H), 4.47 (dd, J ) 11.0
Hz, J ′ ) 5.5 Hz, 1H, C3HR); 5.75 (d, J ) 5.5 Hz, 1H, OCH2O),
5.85 (d, J ) 5.5 Hz, 1H, OCH2O); EIMS m/z 658 (M+, 1), 612
(2), 598 (7), 584 (2), 544 (3), 498 (16), 484 (6), 470 (32), 438
(7), 413 (23), 349 (13), 249 (8), 235 (20), 217 (8), 203 (20), 189
(100). Anal. (C38H58O9) C, H.
P iva loyloxym eth yl 3â-Acetoxy-21,22-d ioxolu p -18-en -
28-oa te (4c). The keto-Pom ester 3e (200 mg, 0.32 mmol) was
oxidized with SeO2 (160 mg, 1.4 mmol) in dioxane (4 mL),
AcOH (2 mL), and Ac2O (0.2 mL) in the same manner as in
the preparation of diketone diacetate 4a . Lyophilization from
benzene afforded 4c as a pale-orange powder (145 mg, 71%,
1
mp 104-108 °C): [R]D -135° (c 0.43); H NMR δ 0.85 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.97 (s, 3H, CH3),
1.09 (s, 3H, CH3), 1.19 (s, 9H, C(CH3)3), 1.26 (d, J ) 7.0 Hz,
3H, CH3), 1.26 (d, J ) 7.0 Hz, 3H, CH3), 2.05 (s, 3H, OCOCH3),
2.54 (ddd, J ) 13.5 Hz, J ′ ) 4.5 Hz, J ′′ ) 2.2 Hz, 1H, C16Hâ),
2.80 (dd, J ) 12.4 Hz, J ′ ) 3.5 Hz, 1H, C13Hâ), 3.34 (sept., J )
7.0 Hz, 1H, C20H), 5.55 (d, J ) 5.4 Hz, 1H, OCH2O), 5.91 (d,
J ) 5.4 Hz, 1H, OCH2O); EIMS m/z 640 (M+, 2), 610 (2), 582
(2), 565 (1), 550 (1), 542 (1), 506 (1), 498 (45), 481 (6), 470 (100),
455 (12), 439 (9), 217 (6), 203 (9), 189 (12). Anal. (C38H56O8) C,
H.
Acetoxym eth yl 3â-Acetoxy-21,22-d ioxolu p -18-en e-28-
oa te (4d ). Keto-Acm ester 3f (250 mg, 0.43 mmol) was oxidized
with SeO2 (200 mg, 1.8 mmol) in dioxane (5 mL), AcOH (2.5
mL), and Ac2O (0.25 mL) in the same manner as in the
preparation of diketone diacetate 4a . Crystallization from
butanone afforded 4d as pale-orange needles (176 mg, 69%,
Acetoxym eth yl 3â-Acetoxy-18,19,21-tr ioxo-18,19-seco-
lu p a n -28-oa te (5d ). Keto-Acm ester 3f (400 mg, 0.68 mmol)
was oxidized with Ru(VIII)O4 (from Ru(IV)O2‚H2O; 13 mg, 0.1
mmol) and NaIO4 (2.1 g) in EtOAc (16 mL), H2O (14 mL), and
CF3COOH (0.4 mL) in the same manner as in the preparation
of the triketone diacetate 5a . Crystallization from MeOH
afforded 5d as yellow petals (273 mg, 65%, mp 215-235 °C
1
mp 227-228 °C): [R]D -177° (c 0.43); H NMR δ 0.85 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.97 (s, 3H, CH3),
1.07 (s, 3H, CH3), 1.26 (d, J ) 7.0 Hz, 3H, CH3), 1.27 (d, J )
7.0 Hz, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.09 (s, 3H, OCOCH3),
2.55 (ddd, J ) 13.7 Hz, J ′ ) 4.5 Hz, J ′′ ) 2.4 Hz, 1H, C16Hâ),
2.79 (dd, J ) 12.6 Hz, J ′ ) 3.4 Hz, 1H, C13Hâ), 3.35 (sept., J )
7.0 Hz, 1H, C20H), 4.49 (dd, J ) 11.1 Hz, J ′ ) 5.3 Hz, 1H,
C3HR), 5.62 (d, J ) 5.5 Hz, 1H, OCH2O), 5.82 (d, J ) 5.5 Hz,
1H, OCH2O); EIMS m/z 598 (M+, 8), 568 (2), 540 (10), 498 (40),
470 (100), 455 (18), 439 (6), 285 (5), 259 (5), 243 (10), 217 (20),
203 (29), 190 (46). Anal. (C35H50O8) C, H.
1
dec): [R]D +32° (c 0.44); H NMR δ 0.81 (s, 3H, CH3), 0.84 (s,
3H, CH3), 0.84 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.08 (s, 3H,
CH3), 1.11 (d, J ) 7.0 Hz, 3H, CH3), 1.14 (d, J ) 7.0 Hz, 3H,
CH3), 2.04 (s, 3H, CH3), 2.12 (s, 3H, 2 OCOCH3), 2.37 (br. m,
1H, C16HR), 2.85 (d, J ) 16.8 Hz, 1H, C22HR), 3.06 (dd, J )
11.9 Hz, J ′ ) 3.4 Hz, 1H, C13Hâ), 3.21 (d, J ) 16.8 Hz, 1H,
C22Hâ), 3.35 (sept., J ) 7.0 Hz, 1H, C20H), 4.47 (dd, J ) 11.0
Hz, J ′ ) 5.5 Hz, 1H, C3HR), 5.76 (d, J ) 5.5 Hz, 1H, OCH2O),
5.83 (d, J ) 5.5 Hz, 1H, OCH2O); EIMS m/z 616 (M+, 1), 574
(1), 557 (1), 515 (4), 485 (5), 473 (9), 455 (8), 413 (25), 395 (18),
367 (15), 275 (3), 249 (11), 215 (8), 189 (100). Anal. (C35H52O9)
C, H.
18,19,21-Tr ioxo-18,19-secolu p a n -3â,28-d iyl Dia ceta te
(5a ). A solution of ketone diacetate 3a (500 mg, 0.93 mmol)
in EtOAc (21 mL) was added to a mixture of Ru(IV)O2‚H2O
(15 mg, 0.11 mmol), NaIO4 (1.3 g, 6.1 mmol), H2O (18 mL),
and CF3COOH (0.5 mL). The biphasic mixture was stirred

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5411
18,19,21,22-Tet r a oxo-18,19-secolu p a n -3â,28-d iyl Di-
a ceta te (6). A solution of diketone diacetate 4a (500 mg, 0.93
mmol) in EtOAc (20 mL) and MeCN (5 mL) was added to a
mixture of Ru(IV)O2‚H2O (20 mg, 0.15 mmol), NaIO4 (3.5 g,
16.4 mmol), H2O (18 mL), and CF3COOH (0.5 mL). The
biphasic mixture was stirred vigorously at room temperature
for 20 h, when TLC (PhMe/EtOAc 20:1) indicated complete
reaction. EtOH was added, the mixture was filtered, the
phases were separated, and the organic layer was dried and
evaporated under a reduced pressure. The residue (643 mg),
which according to 1H NMR analysis contained tetraketone 6
and â-keto acid 8a in a ratio of approximately 1:1, was
chromatographed on SiO2 (50 g) in PhMe/EtOAc (20:1).
Crystallization from Me2CO/isopentane afforded pure 6 as
yellow-orange crystals (190 mg, 36%, mp 202-205 °C): [R]D
diazomethane and then acetylated with Ac2O (2 mL) in
pyridine (2 mL). The usual workup gave a white foamy residue.
Purification by preparative TLC (PhMe/Et2O 10:1) and crystal-
lization from CHCl3/MeOH afforded 7c (76 mg, 58%, mp 192-
194 °C): [R]D +11° (c 0.50); 1H NMR δ 0.79 (s, 3H, CH3), 0.84
(s, 3H, CH3), 0.85 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.12 (s, 3H,
CH3), 2.04 (s, 3H, OCOCH3), 2.13 (dd, J ) 16.0 Hz, J ′ ) 5.5
Hz, 1H, C22HR), 2.56 (dd, J ) 12.4 Hz, J ′ ) 3.7 Hz, 1H, C13Hâ),
2.73 (dd, J ) 16.0 Hz, J ′ ) 7.3 Hz, 1H, C22Hâ), 2.78 (br. m, ΣJ
≈ 28 Hz, 1H, C17H), 3.67 (s, 3H, OCH3), 4.48 (dd, J ) 11.2 Hz,
J ′ ) 5.3 Hz, 1H, C3HR). Ref.19 mp 193-195 °C, [R]D +12°.
28-Meth yl-21-h yd r ogen 3â-Acetoxy-18-oxo-19,20,29,30-
tetr a n or lu p a n e-21,28-d ioa te (7d ). Triketomethyl ester 5b
(200 mg, 0.36 mmol) was oxidized with AcOOH (3 mL of a 32%
solution in glacial acetic acid) in CHCl3 (30 mL) in the same
manner as in the preparation of the keto acid 7a . Crystalliza-
tion from Et2O afforded 7d as small white crystals (129 mg,
1
+37° (c 0.27); H NMR δ 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3),
0.89 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.21 (d,
J ) 6.9 Hz, 3H, CH3), 1.22 (d, J ) 6.9 Hz, 3H, CH3), 2.02 (s,
3H, OCOCH3), 2.05 (s, 3H, OCOCH3), 2.43 (br. m, 1H, C16Hâ),
2.84 (dd, J ) 11.6 Hz, J ′ ) 3.5 Hz, 1H, C13Hâ), 3.41 (sept. J )
7.0 Hz, 1H, C20H), 4.48 (m, ΣJ ≈ 15 Hz, 1H, C3HR), 4.48 (d, J
) 11.1 Hz, 1H, C28HR), 4.77 (d, J ) 11.1 Hz, 1H, C28Hâ); EIMS
m/z 586 (M+, 3), 544 (2), 526 (4), 514 (33), 513 (9), 498 (2), 487
(78), 474 (12), 459 (17), 444 (11), 427 (7), 417 (6), 400 (27), 383
(11), 367 (8), 357 (31), 339 (28), 322 (12), 297 (10), 276 (12),
249 (20), 229 (13), 216 (17), 203 (50), 189 (100). Anal.
(C34H50O8) C, H.
1
71%, mp 177-180 °C): [R]D,+64° (c 0.44); H NMR δ 0.82 (s,
3H, CH3), 0.84 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.89 (s, 3H,
CH3), 1.08 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.46 (ddd, J )
14.0 Hz, J ′ ) 4.1 Hz, J ′′ ) 2.7 Hz, 1H, C16Hâ), 2.62 (d, J )
16.3 Hz, 1H, C22HR), 2.85 (dd, J ) 12.0 Hz, J ′ ) 3.5 Hz, 1H,
C13Hâ), 2.85 (d, J ) 16.3 Hz, 1H, C22Hâ), 3.73 (s, 3H, OCH3);
4.47 (dd, J ) 11.0 Hz, J ′ ) 5.3 Hz, 1H, C3HR); EIMS m/z 504
(M+, 2), 461 (1), 444 (20), 429 (8), 401 (10), 385 (1), 335 (3),
309 (4), 264 (7), 254 (6), 241 (59), 228 (8), 223 (30), 216 (8),
204 (15), 197 (10), 189 (100). Anal. (C29H44O7) C, H.
3â,28-Dia cet oxy-18-oxo-19,20,29,30-t et r a n or lu p a n -21-
oic Acid (7a ). A solution of triketone 5a (300 mg, 0.52 mmol)
and AcOOH (5 mL of a 32% solution in glacial acetic acid) in
CHCl3 (40 mL) was stirred at room temperature for 24 h, when
TLC (PhMe/Et2O 6:1) indicated complete reaction. The color-
less reaction mixture was diluted with cold H2O and was
extracted with CHCl3. The combined organic layers were
washed successively with 5% aqueous KI solution (400 mL),
saturated aqueous sodium sulfite solution (200 mL), and brine
(2 × 200 mL) and then dried and evaporated. According to 1H
NMR analysis the resulting pale-yellow oil (268 mg) was a
mixture of the keto acid 7a and spirolactone 1419 in a ratio of
approximately 4:1. Preparative TLC (CHCl3/EtOAc/AcOH 100:
10:1), followed by crystallization from Et2O/hexane, afforded
7a as a white crystalline powder (165 mg, 61%, mp 152-155
Dim eth yl 3â-Acetoxy-18-oxo-19,20,29,30-tetr an or lu pan e-
21,28-d ioa te (7e). This compound was obtained by reaction
of acid 7d with diazomethane (mp 127-129 °C, MeOH): [R]D
1
+74° (c 0.40); H NMR δ 0.83 (s, 3H, CH3), 0.84 (s, 3H, CH3),
0.84 (s, 3H, CH3), 0.89 (s, 3H, CH3), 1.07 (s, 3H, CH3), 2.04 (s,
3H, OCOCH3), 2.43 (ddd, J ) 13.9 Hz, J ′ ) 4.2 Hz, J ′′ ) 2.7
Hz, 1H, C16Hâ), 2.60 (d, J ) 16.0 Hz, 1H, C22HR), 2.81 (d, J )
16.0 Hz, 1H, C22Hâ), 2.83 (dd, J ) 12.0 Hz, J ′ ) 3.6 Hz, 1H,
C13Hâ), 3.66 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 4.47 (dd, J )
11.0 Hz, J ′ ) 5.5 Hz, 1H, C3HR); EIMS m/z 518 (M+, 26), 458
(100), 443 (52), 427 (39), 415 (66), 353 (27), 255 (61), 223 (27),
189 (35). Anal. (C30H46O7) C, H.
21-Meth yl-28-(p iva loyloxy)m eth yl 3â-Acetoxy-18-oxo-
19,20,29,30-tetr a n or lu p a n e-21,28-d ioa te (7f). The triketo-
Pom ester 5c (200 mg, 0.30 mmol) was oxidized with AcOOH
(10 mL of a 32% solution in glacial acetic acid) in CHCl3 (30
mL) in the same manner as in the preparation of keto acid
7a . The resulting colorless solid (157 mg) contained a polar
compound as the major component and a less polar impurity
according to TLC (PhMe/Et2O 6:1). Crude methyl ester 7f was
obtained by treatment with diazomethane. Preparative HPLC
(15% EtOAc in hexane) and crystallization from MeOH af-
forded pure 7f (83 mg, 44%, mp 153-156 °C): [R]D +63° (c
1
°C and 185-189 °C dec): [R]D +26° (c 0.30); H NMR δ 0.84
(s, 3H, CH3), 0.85 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.90 (s, 3Hm
CH3), 1.14 (s, 3H, CH3), 2.03 (s, 3H, OCOCH3), 2.05 (s, 3H,
OCOCH3), 2.53 (d, J ) 15.6 Hz, 1H, C22HR), 2.60 (d, J ) 15.6
Hz, 1H, C22Hâ), 2.81 (dd, J ) 11.9 Hz, J ′ ) 3.5 Hz, 1H, C13Hâ),
4.20 (d, J ) 11.4 Hz, 1H, C28HR), 4.48 (dd, J ) 11.0 Hz, J ′ )
5.3 Hz, 1H, C3HR), 4.60 (d, J ) 11.4 Hz, 1H, C28Hâ); EIMS m/z
518 (M+, 3), 503 (1), 486 (2), 458 (100), 443 (38), 430 (6), 415
(60), 398 (27), 383 (16), 355 (17), 323 (7), 255 (32), 241 (16),
204 (15), 189 (15). Anal. (C30H46O7) C, H.
Meth yl 3â,28-Dia cetoxy-18-oxo-19,20,29,30-tetr a n or lu -
p a n -21-oa te (7b). This compound was obtained by esterifi-
cation of keto acid 7a with diazomethane (mp 209-211 °C,
MeOH): [R]D +52° (c 0.46); 1H NMR δ 0.84 (s, 3H, CH3), 0.85
(s, 3H, CH3), 0.90 (s, 3H, CH3), 0.91 (s, 3H, CH3), 1.13 (s, 3H,
CH3), 2.03 (s, 3H, OCOCH3), 2.05 (s, 3H, OCOCH3), 2.43 (d, J
) 15.9 Hz, 1H, C22HR), 2.66 (d, J ) 15.9 Hz, 1H, C22Hâ), 2.75
(dd, J ≈ 12 Hz, J ′ ≈ 4 Hz, 1H, C13Hâ), 3.66 (s, 3H, OCH3), 4.19
(d, J ≈ 11 Hz, 1H, C28HR), 4.48 (dd, J ≈ 10 Hz, J ′ ≈ 6 Hz, 1H,
C3HR), 4.56 (d, J ≈ 11 Hz, 1H, C28Hâ); EIMS m/z 532 (M+, 3),
517 (1), 501 (1), 472 (40), 457 (11), 441 (4), 429 (17), 412 (3),
397 (4), 381 (3), 369 (2), 339 (3), 269 (91), 255 (23), 204 (14),
195 (30), 189 (100). Anal. (C31H48O7) C, H.
Meth yl 3â-Acetoxy-18-oxo-19,20,28,29,30-p en ta n or lu -
p a n -21-oa te (7c). A suspension of methyl keto ester 7b (140
m, 0.27 mmol) in a solution of KOH (140 mg, 2.43 mmol) in
EtOH (7.5 mL) was refluxed for 3 h, at which time all starting
material had dissolved. After cooling, the reaction mixture was
poured into H2O (50 mL). The resulting solution was acidified
with 10% aqueous HCl (2 mL) and was extracted twice with
Et2O. The combined organic extracts were washed with H2O,
dried, and evaporated. The residue (102 mg) was treated with
1
0.30); H MNR δ 0.82 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.84 (s,
3H, CH3), 0.89 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.21 (s, 9H,
(C(CH3)3), 2.04 (s, 3H, OCOCH3), 2.42 (ddd, J ) 13.9 Hz, J ′ )
4.2 Hz, J ′′ ) 2.7 Hz, 1H, C16Hâ), 2.61 (d, J ) 16.2 Hz, 1H,
C22HR), 2.82 (d, J ) 16.2 Hz, 1H, C22Hâ), 2.90 (dd, J ) 12.1
Hz, J ′ ) 3.7 Hz, 1H, C13Hâ), 3.66 (s, 3H, OCH3), 4.47 (dd, J )
11.0 Hz, J ′ ) 5.5 Hz, 1H, C3HR), 5.69 (d, J ) 5.5 Hz, 1H,
OCH2O), 5.87 (d, J ) 5.5 Hz, 1H, OCH2O); EIMS m/z 618 (M+,
4), 558 (69), 543 (16), 515 (21), 486 (26), 427 (65), 383 (29),
355 (100), 337 (21), 269 (29), 223 (27), 189 (29). Anal.
(C35H54O9) C, H.
28-Ace t oxym e t h yl-21-m e t h yl
3â-Ace t oxy-18-oxo-
19,20,29,30-tetr a n or lu p a n e-21,28-d ioa te (7 g). Triketo-Acm
ester 5d (200 mg, 0.32 mmol) was oxidized with AcOOH (10
mL of a 32% solution in glacial acetic acid) in CHCl3 (30 mL)
in the same manner as in the preparation of the keto acid 7a .
The resulting colorless solid (143 mg) contained a polar
compound as the major component and a less polar impurity
according to TLC (PhMe/Et2O 6:1). Crude methyl ester 7g was
obtained by treatment with diazomethane. Preparative HPLC
(25% EtOAc in hexane) and crystallization from MeOH af-
forded pure 7g (73 mg, 39%, mp 127-129 °C): [R]D +66° (c
1
0.27); H NMR δ 0.82 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.84 (s,
3H, CH3), 0.90 (s, 3H, CH3), 1.08 (s, 3H, CH3), 2.04 (s, 3H,

5412 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
OCOCH3), 2.11 (s, 3H, OCOCH3), 2.42 (ddd, J ) 13.9 Hz, J ′ )
4.2 Hz, J ′′ ) 2.7 Hz, 1H, C16Hâ), 2.61 (d, J ) 16.2 Hz, 1H,
C22HR), 2.83 (d, J ) 16.2 Hz, 1H, C22Hâ), 2.91 (dd, J ) 12.1
Hz, J ′ ) 3.5 Hz, 1H, C13Hâ), 3.66 (s, 3H, OCH3), 4.47 (dd, J )
11.0 Hz, J ′ ) 5.5 Hz, 1H, C3HR), 5.76 (d, J ) 5.6 Hz, 1H,
OCH2O), 5.83 (d, J ) 5.6 Hz, 1H, OCH2O); EIMS m/z 576 (M+,
7), 552 (3), 528 (3), 516 (100), 501 (22), 473 (42), 427 (47), 383
(22), 353 (25), 313 (13), 223 (20), 189 (28). Anal. (C32H48O9) C,
H.
0.30 mmol) was added to the solution of â-keto acid 8a (100
mg, 0.20 mmol) and bromomethyl acetate (34 mg, 0.22 mmol)
in dry DMF (5 mL) and this heterogeneous mixture was stirred
at room temperature for 2 d, when TLC (PhMe/Et2O 6:1)
indicated complete reaction. The reaction mixture was filtered,
poured into cold H2O (50 mL), and extracted with CHCl3. The
combined extracts were washed twice with brine, dried, and
evaporated. The resulting gray oil (131 mg) was purified by
preparative TLC (PhMe/Et2O 5:1) and the product was crystal-
lized from butanone to afford pure 8d as white needles (45
mg, 39%, mp 234-236 °C): [R]D +69° (c 0.46); 1H NMR δ 0.85
(s, 3H, CH3), 0.86 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.93 (s, 3H,
CH3), 1.12 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.05 (s, 3H,
OCOCH3), 2.13 (s, 3H, OCOCH3), 2.44 (m, 1H, C16HR), 2.60
(dd, J ) 11.8 Hz, J ′ ) 3.6 Hz, 1H, C13Hâ), 4.43 (d, J ) 11.2
Hz, 1H, C28HR), 4.48 (dd, J ) 11.1 Hz, J ′ ) 5.2 Hz, 1H, C3HR),
4.56 (d, J ) 11.2 Hz, C28Hâ), 5.79 (d, J ) 5.5 Hz, 1H, OCH2O),
5.80 (d, J ) 5.5 Hz, 1H, OCH2O); EIMS m/z 576 (M+, 2), 534
(2), 516 (100), 501 (25), 473 (32), 444 (16), 383 (14), 338 (13),
313 (30), 223 (27), 209 (13), 189 (52). Anal. (C32H48O9) C, H.
3â,28-Dia cetoxy-18-oxo-19,20,21,29,30-p en ta n or lu p a n -
22-oic Acid (8a ). F r om Tetr a k eton e 6. A solution of 6 (102
mg, 0.17 mmol) and AcOOH (6 mL of a 32% solution in glacial
acetic acid) in CHCl3 (15 mL) was stirred at room temperature
for 17 h when TLC (PhMe/Et2O 6:1) indicated complete
reaction. The colorless reaction mixture was diluted with cold
H2O and was extracted with chloroform (3 × 20 mL). The
combined extracts were washed successively with 5% aqueous
KI, saturated aqueous Na2SO3, and brine, dried, and evapo-
rated. The resulting colorless solid (96 mg) contained a polar
compound as the major component according to TLC (PhMe/
Et2O 6:1). Preparative TLC (CHCl3/EtOAc/AcOH 100:10:1) and
crystallization from CH2Cl2/Et2O afforded pure 8a as small
white needles (36 mg, 41%, mp 137-140 °C dec): [R]D +40° (c
0.37); 1H NMR δ 0.85 (s, 3 H, CH3), 0.86 (s, 3 H, CH3), 0.91 (s,
6 H, CH3), 1.14 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.06 (s,
3H, OCOCH3), 2.79 (dd, J ) 11.7 Hz, J ′ ) 3.6 Hz, 1H, C13Hâ),
4.46 (d, J ≈ 1 Hz, 1H, C28HR), 4.48 (dd, J ≈ 11 Hz, J ′ ≈ 6 Hz,
1H, C3HR), 4.64 (d, J ≈ 11 Hz, 1H, C28Hâ); EIMS m/z 444
(M+ - 60, 1), 416 (3), 414 (3), 400 (22), 382 (2), 354 (6), 340
(15), 325 (10), 297 (9), 291 (7), 281 (7), 278 (9), 276 (31), 271
(7), 264 (27), 257 (7), 231 (7), 216 (33), 204 (40), 189 (100).
Anal. (C29H44O7) C, H.
An h yd r id e of 3â,28-Dia cetoxy-21,22-secolu p -18-en e-
21,22-d ioic Acid (9a ). A solution of diketone diacetate 4a (1
g, 1.81 mmol) and AcOOH (20 mL of a 32% solution in glacial
acetic acid) in CHCl3 (120 mL) was stirred at room tempera-
ture for 7 d when TLC (PhMe/Et2O 6:1) indicated complete
reaction. The colorless reaction mixture was diluted with cold
H2O and extracted with CHCl3. The combined extracts were
washed successively with 5% aqueous KI, saturated aqueous
Na2SO3, and brine and then dried and evaporated. The
resulting pale-yellow oil (1.2 g) was crystallized from CHCl3/
MeOH to afford 9a as a white crystalline powder (875 mg, 85%,
1
mp 306-309 °C): [R]D +88° (c 0.45); H NMR δ 0.86 (s, 6H,
CH3), 0.90 (s, 3H, CH3), 0.91 (s, 3H, CH3), 1.11 (s, 3H, CH3),
1.15 (d, J ) 6.7 Hz, 3H, CH3), 1.31 (d, J ) 7.0 Hz, 3H, CH3),
2.02 (s, 3H, OCOCH3), 2.05 (s, 3H, OCOCH3), 2.53 (dt, J )
14.4 Hz, J ′ ) J ′′ ) 3.5 Hz, 1H, C16Hâ), 2.72 (dd, J ) 12.7 Hz,
J ′ ) 2.9 Hz, 1H, C13Hâ), 3.26 (sept., J ) 6.9 Hz, 1H, C20H),
3.89 (d, J ) 11.0 Hz, 1H, C28HR), 4.47 (dd, J ) 11.3 Hz, J ′ )
5.0 Hz, 1H, C3HR), 4.54 (d, J ) 11.0 Hz, 1H, C28Hâ). Ref.18 mp
308-310 °C, [R]D +87° (c 0.60).
F r om An h yd r id e 9a . A suspension of 9a (500 mg, 0.88
mmol) in EtOAc (20 mL) and MeCN (5 mL) was added to a
mixture of Ru(IV)O2‚H2O (40 mg, 0.30 mmol), NaIO4 (7.5 g,
35.1 mmol), H2O (18 mL) and CF3COOH (0.5 mL). The
biphasic mixture was stirred vigorously at room temperature
for 6 d, after which time TLC (PhMe/EtOAc 20:1) indicated
complete reation. EtOH was added, the mixture was filtered,
and the organic layer was dried and evaporated under reduced
pressure. The white residue (643 mg) was crystallized from
CH2Cl2/Et2O to afford 8a as small white needles (237 mg, 54%,
mp 138-140 °C): [R]D +40° (c 0.37). This material was
identical to the sample obtained from the procedure described
above.
An h yd r id e of 3â-Acet oxy-17â-m et h oxyca r b on yl-28-
n or -21,22-secolu p -18-en e-21,22-d ioic Acid (9b ). Diketo-
methyl ester 4b (500 mg, 0.93 mmol) was oxidized by treat-
ment with AcOOH (10 mL of a 32% solution in glacial acetic
acid) in CHCl3 (55 mL) in the same manner as in the
preparation of the anhydride 9a . Crystallization from CHCl3/
MeOH afforded 9b as small white crystals (355 mg, 69%, mp
Meth yl 3â,28-Dia cetoxy-18-oxo-19,20,21,29,30-p en ta n -
or lu p a n -22-oa te (8b). A solution of diene diacetate 10 (500
mg, 0.95 mmol) in EtOAc (15 mL) and MeCN (5 mL) was added
to a mixture of Ru(IV)O2‚H2O (20 mg, 0.15 mmol), NaIO4 (3.5
g, 16.4 mmol), H2O (15 mL), and CF3COOH (0.5 mL). The
biphasic mixture was stirred vigorously at room temperature
for 60 h, after which time TLC (PhMe/Et2O 10:1) indicated
complete reaction. EtOH ethanol was added, the mixture was
filtered, and the organic layer was separated and filtered
through a short column of silica gel. The column was eluted
with EtOAc and the eluate was evaporated under reduced
pressure. The resulting red-brown solid (437 mg) contained,
according to TLC (PhMe/Et2O 6:1), a polar compound as the
major component and a less polar impurity. The crude â-ke-
tomethyl ester 8b was obtained by treatment with diazo-
methane. Preparative TLC (PhMe/Et2O 10:1) and crystalliza-
tion from CHCl3/MeOH afforded pure 8b (232 mg, 47%, mp
1
231-237 °C dec): [R]D +116° (c 0.49); H NMR δ 0.85 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.92 (s, 3H, CH3),
1.12 (s, 3H, CH3), 1.16 (d, J ) 6.7 Hz, 3H, CH3), 1.34 (d, J )
7.0 Hz, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.38 (ddd, J ) 14.5
Hz, J ′ ) 12.1 Hz, J ′′ ) 3.7 Hz, 1H, C16HR), 2.49 (ddd, J ) 14.5
Hz, J ′ ) 5.3 Hz, J ′′ ) 4.0 Hz, 1H, C16Hâ), 2.85 (dd, J ) 12.6
Hz, J ′ ) 3.0, 1H, C13Hâ), 3.32 (sept., J ) 6.9 Hz, 1H, C20H),
3.76 (s, 3H, OCH3), 4.47 (dd, J ) 11.1 Hz, J ′ ) 5.2 Hz, 1H,
C3HR); EIMS m/z 556 (M+,7), 541 (4), 510 (1), 496 (7), 484 (5),
481 (3), 469 (7), 453 (9), 437 (5), 409 (2), 315 (1), 293 (10), 279
(8), 249 (8), 236 (10), 221 (9), 203 (23), 189 (100). Anal.
(C33H48O7) C, H.
3â-H y d r o x y -19,20,21,22,28,29,30-h e p t a n o r lu p a n -
18-on e (11a ). â-Keto acid 8a (150 mg, 0.30 mmol) was added
to a solution of KOH (160 mg, 2.86 mmol) in dioxane (5.3 mL)
and H2O (3.7 mL) and the mixture was refluxed for 3 h under
Ar. All starting material had dissolved during this time. After
cooling, the reaction mixture was poured into H2O (50 mL);
the resulting solution was acidified with 10% aqueous HCl (2
mL) and was extracted twice with Et2O. The combined extracts
were washed with H2O, dried, and evaporated. The resulting
white crystalline solid (96 mg) contained a polar compound
by TLC (PhMe/Et2O 1:1). This was purified by preparative TLC
(PhMe/Et2O 1:1) and crystallization from CHCl3/MeOH to
afford pure 11a as a white crystalline powder (67 mg, 61%,
1
255-258 °C dec): [R]D +68° (c 0.26); H NMR δ 0.85 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.94 (s, 3H, CH3),
1.13 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.06 (s, 3H, OCOCH3),
2.45 (m, ΣJ ) 34 Hz, 1H, C16HR), 2.61 (dd, J ) 11.7 Hz, J ′ )
3.6 Hz, 1H, C13Hâ), 3.77 (s, 3H, OCH3), 4.44 (d, J ) 11.2 Hz,
1H, C28HR), 4.48 (dd, J ≈ 11 Hz, J ′ ≈ 6 Hz, 1H, C3HR), 4.56 (d,
J ) 11.2 Hz, 1H, C28Hâ); EIMS m/z 487 (M+ - 31, 3), 458 (45),
443 (15), 440 (3), 430 (9), 415 (21), 398 (6), 389 (5), 376 (6),
370 (7), 349 (4), 323 (4), 255 (95), 241 (23), 223 (23), 204 (18),
199 (18), 189 (100). Anal. (C30H46O7) C, H.
1
Acetoxym eth yl 3â,28-Dia cetoxy-18-oxo-19,20,21,29,30-
p en ta n or lu p a n -22-oa te (8d ). Well-powdered Ag2CO3 (83 mg,
mp 191-193 °C sublim): [R]D +10° (c 0.24); H NMR δ 0.77
(s, 3H, CH3), 0.83 (s, 3H, CH3), 0.87 (d, J ) 0.9 Hz, 3H, CH3),

Triterpenoids with Anticancer Activity
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5413
0.97 (s, 3H, CH3), 1.11 (s, 3H, CH3), 2.16-2.34 (m, 2H, C17H2),
2.49 (dd, J ≈ 12 Hz, J ′ ≈ 4 Hz, 1H, C13Hâ), 3.20 (dd, J ) 10.8
Hz, J ′ ) 5.4 Hz, 1H, C3HR); EIMS m/z 346 (M+, 17), 328 (15),
313 (5), 310 (2), 295 (1), 285 (2), 234 (14), 222 (8), 216 (7), 207
(78), 204 (17), 189 (43), 135 (37), 121 (33), 111 (100). Anal.
(C23H38O2) C, H, N.
(15 mL), and CF3COOH (0.5 mL). The reaction mixture was
stirred vigorously at room temperature for 3 d, after which
time TLC (CHCl3/EtOAc/AcOH 100:10:1) indicated complete
reaction. EtOH ethanol was added, the mixture was filtered,
and the organic layer was separated and filtered through a
short column of silica gel. The column eluted with EtOAc and
the eluate was evaporated under reduced pressure. The
resulting brown solid (379 mg) contained, according to TLC
(CHCl3/EtOAc/AcOH 100:10:1), a polar compound as the major
component and a less polar impurity. Preparative TLC (CHCl3/
EtOAc/AcOH 100:10:1) and crystallization from Et2O/hexane
afforded 15a (122 mg, 27%, mp 247-248 °C dec): [R]D -4° (c
18-Oxo-19,20,21,22,28,29,30-h ep ta n or lu p a n -3â-yl Ace-
ta te (11b). This was obtained by acetylation of 11a with Ac2O
in pyridine and the usual workup procedure (mp 215-218 °C,
CHCl3/MeOH): [R]D +11° (c 0.48); 1H NMR δ 0.83 (s, 3H, CH3),
0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.11 (s,
3H, CH3), 2.04 (s, 3H, OCOCH3), 2.16-2.35 (m, 2H, C17H2),
2.48 (dd, J ≈ 12 Hz, J ′ ≈ 3 Hz, 1H, C13Hâ), 4.48 (dd, J ≈ 11
Hz, J ′ ≈ 6 Hz, 1H, C3HR); EIMS m/z 388 (M+, 22), 373 (2), 370
(12), 328 (49), 313 (17), 310 (5), 295 (5), 285 (24), 276 (9), 264
(24), 259 (12), 246 (6), 216 (20), 204 (27), 189 (100), 111 (68).
Anal. (C25H40O3) C, H, N. Ref.15 mp 200-203 °C, [R]D +22°.
18-Oxo-19,20,21,22,29,30-h exa n or lu p -17(28)-en -3â-yl
Aceta te (12). A solution of â-keto acid 8a (1 g 1.98 mmol) in
diglyme (10 mL) was refluxed for 10 min, after which time
TLC (PhMe/Et2O 6:1) indicated complete reaction. After cool-
ing, the reaction mixture was poured into excess H2O and this
was extracted with CHCl3. The combined extracts were washed
with H2O, dried, and evaporated. According to TLC (PhMe/
Et2O 6:1) the resulting pale-yellow oil (1.5 g) was a mixture of
three major components (heptanor ketone 11b, methylene
ketone 12, and an unidentified compound) in the ratio of
approximately 1:1:1. This mixture was separated by prepara-
tive HPLC (10% EtOAc in hexane).
P u r e 12 (72 mg, 9%, mp 268-269 °C, lyophilisate from
benzene): [R]D +32° (c 0.37); 1H NMR δ 0.85 (s, 3H, CH3), 0.86
(s, 3H, CH3), 0.90 (s, 3H, CH3), 0.91 (s, 3H, CH3), 1.06 (s, 3H,
CH3), 2.05 (s, 3H, OCOCH3), 2.46 (dd, J ) 12 Hz, J ′ ) 4 Hz,
1H, C13Hâ), 2.49-2.65 (br. m, 2H, C16H2), 4.49 (m, ΣJ ) 16.5
Hz, 1H, C3HR), 5.06 (dt, J ) 1.2 Hz, J ′ ) J ′′ ) 2.1 Hz, 1H,
C28HR), 5.72 (dt, J ) 0.8 Hz, J ′ ) J ′′ ) 2.0 Hz, 1H, C28Hâ);
EIMS m/z 400 (M+, 48), 337 (100), 320 (38), 297 (24), 283 (25),
276 (97), 264 (63), 249 (24), 189 (19). Anal. (C26H40O3) C, H.
Hep ta n or k eton e a ceta te 11b (131 mg, 17%) was identical
with an authentic sample.
17r-Eth oxym eth yl-18-oxo-19,20,21,22,28,29,30-h ep ta n -
or lu p a n -3â-yl Aceta te (13b). A suspension of â-keto acid 8a
(120 mg, 0.24 mmol) in a solution of KOH (120 mg, 2.14 mmol)
in EtOH (6.3 mL) was refluxed for 3 h. All starting material
had dissolved during this time. After cooling, the reaction
mixture was poured into H2O (50 mL) and the resulting
solution was acidified with 10% aqueous HCl (2 mL). This was
extracted twice with Et2O. The combined extracts were washed
with H2O, dried, and evaporated. According to 1H NMR
analysis, the resulting pale-yellow oil (78 mg) was a mixture
of heptanor ketone 11a and ethoxyheptanor ketone 13a in a
ratio of approximately 1:1. The residue was acetylated with
Ac2O (1.5 mL) in pyridine (1.5 mL). Usual workup gave a white
foamy residue, which was fractionated by preparative HPLC
(10% EtOAc in hexane).
1
0.8, dioxane); H NMR (in the mixture of CDCl3 and CD3OD)
δ 0.84 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.87 (s, 3H, CH3), 1.00
(s, 3H, CH3), 1.09 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3), 2.29
(br. m, 2H, C16H2), 2.64 (dd, J ) 11.9 Hz, J ′ ) 4.7 Hz, 1H,
C13Hâ), 4.48 (dd, J ) 11.2 Hz, J ′ ) 5.1 Hz, 1H, C3HR). Ref.25
mp 246-248 °C, hexane/Me2CO); [R]D -4.7° (c 0.42, CHCl3).
F r om Acid 3c. A solution of 3c (200 mg, 0.39 mmol) in
EtOAc (5 mL), MeOAc (15 mL), and MeCN (5 mL) was added
to a mixture of Ru(IV)O2‚H2O (15 mg, 0.11 mmol), NaIO4 (1.8
g, 8.4 mmol), H2O (15 mL), and CF3COOH (0.5 mL). The
biphasic reaction mixture was stirred vigorously at room
temperature for 60 h. Then the reaction mixture was worked
up as in the previous example. Diacid 15a (58 mg 33%, mp
245-247 °C dec): [R]D -6° (c 0.42, dioxane), was identical to
an authentic sample.
F r om Meth ylen e Keton e 12. A solution of 12 (50 mg; 0.12
mmol) in EtOAc (2 mL) and MeCN (0.5 mL) was added to a
mixture of Ru(IV)O2‚H2O (1.5 mg, 0.01 mmol), NaIO4 (150 mg,
0.7 mmol), H2O (2 mL), and CF3COOH (50 µL) was stirred
vigorously at room temperature for 20 h. The reaction mixture
was worked up as in the previous example. The diacid 15a
(22 mg, 39%, mp 244-246 °C dec): [R]D -3° (c 0.3, dioxane),
was identical to an authentic sample.
Dim et h yl 3â-Acet oxy-19,20,21,22,28,29,30-h ep t a n or -
17,18-secolu p a n -17,18-d ioa te (15b). This compound was
obtained by reaction of diacid 15a with diazomethane (mp
1
143-145 °C, MeOH): [R]D +10° (c 0.33); H NMR δ 0.84 (s,
3H, CH3), 0.85 (s, 3H, CH3), 0.87 (d, J ) 0.8 Hz, 3H, CH3),
0.99 (s, 3H, CH3), 1.04 (s, 3H, CH3), 2.05 (s, 3H, OCOCH3),
2.62 (dd, J ) 12.7 Hz, J ′ ) 4.0 Hz, 1H, C13Hâ), 3.63 (s, 3H,
OCH3), 3.65 (s, 3H, OCH3), 4.48 (dd, J ) 11.3 Hz, J ′ ) 5.2 Hz,
1H, C3HR). Ref.16 mp 140-144 °C, EtOH): [R]D +12.2° (c 1,
CHCl3).
Bis[(pivaloyloxy)m eth yl]3â-Acetoxy-19,20,21,22,28,29,30-
h ep t a n or -17,18-secolu p a n e-17,18-d ioa t e (15c). DBU (17
µL, 0.11 mmol) and Pom-Cl (16 µL, 0.11 mmol) were added to
a solution of diacid 15a (25 mg, 57 µmol) in CH2Cl2 (300 µL)
and MeCN (120 µL). The mixture was stirred at room tem-
perature for 3 h and was then diluted with ice-cold H2O and
extracted with CHCl3. The combined extracts were washed
with cold brine. After drying and evaporating, a viscous pale-
brown oil (37 mg) was obtained. This was chromatographed
on SiO2 (PhMe/Et2O 10:1). Pure 15c (28 mg, 72%) was obtained
1
Hep ta n or k eton e a ceta te 11b (14 mg, 15%, mp 216-219
°C, CHCl3/MeOH): [R]D +11° (c 0.48), was identical to an
authentic sample.
as a colorless oil: [R]D +19° (c 0.46); H NMR δ 0.84 (s, 3H,
CH3), 0.85 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.99 (s, 3H, CH3),
1.06 (s, 3H, CH3), 1.21 (s, 18H, C(CH3)3), 2.05 (s, 3H, OCOCH3),
2.12-2.30 (br m, 2H, C16H2), 2.65 (dd, J ) 12.0 Hz, J ′ ) 4.6
Hz, 1H, C13Hâ), 4.48 (dd, J ) 11.3 Hz, J ′ ) 5.0 Hz, 1H, C3HR),
5.66 (d, J ) 5.5 Hz, 1H, OCH2O), 5.71 (d, J ) 5.5 Hz, 1H,
OCH2O), 5.74 (d, J ) 5.5 Hz, 1H, OCH2O), 5.76 (d, J ) 5.5
Hz, 1H, OCH2O); EIMS m/z 678 (M+, 0), 647 (6), 604 (2), 587
(63), 503 (13), 489 (16), 444 (18), 419 (11), 375 (19), 359 (13),
331 (7), 315 (21), 276 (21), 249 (9), 229 (16), 203 (8), 189 (100).
Anal.(C37H60O10) C, H.
Eth oxyh ep ta n or k eton e a ceta te 13b (18 mg, 17%. mp
139-144 °C, CHCl3/MeOH): [R]D +23° (c 0.32). 1H NMR δ 0.80
(s, 3H, CH3), 0.85 (s, 6H, CH3), 0.90 (s, 3H, CH3), 1.10 (s, 3H,
CH3), 1.18 (t, J ) 7.0 Hz, 3H, OCH2CH3), 2.05 (s, 3H,
OCOCH3), 2.43-2.55 (m, 2H, C13H & C17H), 3.28 (dd, J ) 9.6
Hz, J ′ ) 7.5 Hz, 1H, C28HR), 3.40-3.58 (m, 2H, OCH2CH3),
3.77 (dd, J ) 9.6 Hz, J ′ ) 5.1 Hz, 1H, C28Hâ), 4.48 (m, ΣJ )
16.5 Hz, 1H, C3HR); EIMS m/z 446 (M+, 49), 431 (1), 428 (1),
417 (3), 402 (7), 387 (4), 386 (4), 371 (7), 343 (7), 276 (6), 264
(5), 216 (7), 204 (8), 189 (47), 183 (77), 169 (68), 114 (100).
Anal. (C28H46O4) C, H.
3â-Acetoxy-19,20,21,22,28,29,30-h ep ta n or -17,18-secolu -
p a n -17,18-d ioic Acid (15a ). F r om Hyd r oxy Keton e 3b. A
solution of 3b (500 mg, 1.00 mmol) in EtOAc (10 mL), MeOAc
(10 mL), and MeCN (5 mL) was added to a mixture of Ru(IV)-
O2‚H2O (20 mg, 0.15 mmol), NaIO4 (3.5 g, 16.4 mmol), H2O
Cr ysta llogr a p h y. Single crystals of â-keto acid 8a were
grown from a CHCl3/EtOAc/petroleum ether/MeOH solvent
system. Crystal structure data: unit cell parameters, a )
7.4459(9) Å, R ) 90°, b ) 11.0454(9) Å, â ) 94.002(11)°, c )
36.178(4) Å, γ ) 90°; C30.50H50O8.50 (C29H44O7‚1.5MeOH) )
552.70, two unit formulas per unit cell; calculated density
1.237 Mg m3; space group P21; λ ) 1.54178 Å, 10 322
independent reflections collected; the structure was solved by

5414 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Sˇ arek et al.
direct methods and refined by full-matrix least-squares against
F2 (SHELXS-97); the program used for refinement was
SHELXL-97; refinement with statistical weights [w ) 1/σ2(F2)]
gave a final Flack parameter of -0.05(9); the final R value
was 3.63%. Full crystallographic data are included in the
Supporting Information.
phological markers of apoptosis: cytoplasmic membrane blab-
bing, cellular fragmentation, and formation of apoptotic bodies.
Sta tistics. The cytotoxic activities of BA (2b) and the most
effective compounds, 16, 4b, and 6, listed in Table 1 were
mutually compared in order to investigate for a common
mechanism of action. The nonparametric Spearman correlation
analysis was performed using software Prisma, version 4.0.
The correlation diagrams, correlation coefficients, and statisti-
cal significances are shown in Figure 3.
Cell Lin es. All cells were purchased from the American
Tissue Culture Collection (ATTC), unless otherwise indicated.
Cladribine-, gemcitabine-, cytosinarabinoside-, and fludara-
bine-resistant sublines of K562 cells (K562-CdA, K562-GEM,
K562-ARA-C, K562-FLUD) were kindly provided by Dr. J .
Dummont (University of Lyon, Lyon, France). The drug-
resistant sublines of CEM cells (CEM-DNR bulk, CEM-DNR
1/C2, CEM-VCR bulk, CEM-VCR 1/F3, CEM-VCR 3/D5) were
selected in our laboratory by the cultivation of maternal cell
lines in increasing concentrations of daunorubicine or vinc-
ristine, respectively.27 The human T-lymphoblastic leukemia
cell line, CEM, was used for routine screening of compounds.
To prove a common mechanism of action, selected compounds
that showed activity in the screening assay were tested in a
panel of cell lines (Table 1). These lines were from different
species and of various histogenetic origin, and they possess
various alterations in their cell cycle-regulatory proteins and
hormone receptor status (Table 1). The cells were maintained
in Nunc/Corning 80 cm2 plastic tissue culture flasks and
cultured in cell culture medium (DMEM/RPMI 1640 with 5
g/L glucose, 2 mM glutamine, 100 U/mL penicillin, 100 µg/
mL streptomycin, 10% fetal calf serum, and NaHCO3).
Cytotoxic MTT Assa y.28 Cell suspensions were prepared
and diluted according to the particular cell type and the
expected target cell density (2500-30 000 cells/well based on
cell growth characteristics). Cells were added by pipet (80 µL)
into 96-well microtiter plates. Inoculates were allowed a
preincubation period of 24 h at 37 °C and 5% CO2 for
stabilization. Four-fold dilutions, in 20-µL aliquots, of the
intended test concentration were added at time zero to the
microtiter plate wells. All test compound concentrations were
examined in duplicate. Incubation of the cells with the test
compounds lasted for 72 h at 37 °C, in a 5% CO2 atmosphere
at 100% humidity. At the end of the incubation period, the
cells were assayed using MTT. Aliquots (10 µL) of the MTT
stock solution were pipetted into each well and incubated for
Ack n ow led gm en t. This study was supported in
part by the Ministry of Education of the Czech Republic
(MSM 151100001 and MSM 113100001), the Grant
Agency of the Czech Republic (301/03/1570), and the
Cancer Research Foundation in Olomouc. We are grate-
ful to Veronika Drabkova, Lenka Surzynova, and J iri
Lipert for excellent technical assistance. The editorial
help of Sergej Kuzmin and the electron microscopy
services provided by Dr. Radek Novotny are greatly
appreciated. We thank also Iva Tislerova for measure-
ment of NMR spectra, Martin Sticha for the mass
spectral measurements, and Stanislav Hilgard for mea-
surement of IR spectra. Special thanks to Bohunka
Sperlichova for measurement of optical rotations.
Su p p or t in g In for m a t ion Ava ila b le: Full crystallo-
graphic information of â-keto acid 8a and 13C NMR data and
expanded cytotoxic activity of all unpublished compounds. This
material is available free of charge via Internet at http://
pubs.acs.org.
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a
further 1-4 h. After this incubation period formazan
produced was dissolved by the addition of 100 µL/well of 10%
aqueous SDS (pH ) 5.5), followed by a further incubation at
37 °C overnight. The optical density (OD) was measured at
540 nm with a Labsystem iEMS Reader MF. Tumor cell
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Ap op tosis a n d Cell Cycle An a lysis by F ACS.29 CEM
cells (1 × 106/mL) were cultured in 6-well plates, with or
without 10 µM concentration of BA (2b), betulinines, 16, 4b,
8a , and paclitaxel (1µM) as a reference drug, at 37 °C and 5%
CO2 for 3-24 h. Following the incubation, cells were pelleted,
washed in Hank’s buffered salt solution (HBSS), and fixed in
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Mor p h ologica l An a lysis of Ap op tosis by Sca n n in g
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(2b), betulinines, 16, 4b, and 8a , at 37 °C and 5% CO2 for 3-24
h. Following incubation, cells were pelleted, washed in Hank’s
buffered salt solution, and fixed in 2% glutaraldehyde/HBSS
at 4 °C overnight. Cellular suspensions were cytocentrifuged
on microscopic slides, dried, and covered with gold under
vacuum. The surfaces of (un)treated cells were examined by
scanning electron microscope (Tesla BS340) for typical mor-

Triterpenoids with Anticancer Activity
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