Synthesis and antitumor activity of ring A modified glycyrrhetinic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.08.038

Triterpenoic acids show many pharmacological effects, among them an antiinflammatory or an antitumor activity. One of these, glycyrrhetinic acid (1) is of interest because of its antitumor profile. Glycyrrhetinic acid is not only cytotoxic but also triggers apoptosis in various human tumor cell lines. To improve the cytotoxicity of parent 1 we set out to synthesize new derivatives of it - differing in structure and lipophilicity. These compounds were tested in a sulforhodamine B assay for cytotoxicity, and screened for their ability to induce apoptosis using an acridine orange/ethidium bromide assay and trypan blue staining. The most active compound, 34, a benzyl glycyrrhetinate holding an extra 3-N-(3-aminopropyl)glycyl substituent showed IC₅₀ between 1.96 and 5.14 μm for five human cancer cell lines and triggers apoptosis in 80% of the cells.

Full text of DOI:10.1016/j.ejmech.2011.08.038

European Journal of Medicinal Chemistry 46 (2011) 5356e5369
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of ring A modified glycyrrhetinic acid derivatives
*
René Csuk , Stefan Schwarz, Bianka Siewert, Ralph Kluge, Dieter Ströhl
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Triterpenoic acids show many pharmacological effects, among them an antiinflammatory or an anti-
tumor activity. One of these, glycyrrhetinic acid (1) is of interest because of its antitumor profile. Gly-
cyrrhetinic acid is not only cytotoxic but also triggers apoptosis in various human tumor cell lines. To
improve the cytotoxicity of parent 1 we set out to synthesize new derivatives of it e differing in structure
and lipophilicity. These compounds were tested in a sulforhodamine B assay for cytotoxicity, and
screened for their ability to induce apoptosis using an acridine orange/ethidium bromide assay and
trypan blue staining. The most active compound, 34, a benzyl glycyrrhetinate holding an extra 3-N-(3-
Received 15 July 2011
Received in revised form
25 August 2011
Accepted 26 August 2011
Available online 31 August 2011
Keywords:
aminopropyl)glycyl substituent showed IC50 between 1.96 and 5.14
lines and triggers apoptosis in 80% of the cells.
mm for five human cancer cell
Glycyrrhetinic acid
Antitumor activity
Apoptosis
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
a-epimer was prepared in a two-step synthesis [9] as depicted in
Scheme 1. Thus, oxidation of 1 using CrO3/acetone provided ketone
2 [3] whose stereoselective reduction [9] with L-selectride [10]
at ꢀ75 ꢁC gave the 3-epi derivative 3.
While betulinic acid and oleanoic acid and their derivatives have
already been studied extensively, there are still some gaps in the
knowledge about glycyrrhetinic acid (1, Fig. 1). Compound 1 shows
a modest cytotoxicity but is able to trigger apoptosis in tumor cells
[1e4]. Also, 1 can be accessed easily from the roots of licorice in
high yields of up to 24% [5,6]. Nevertheless, the cytotoxicity of 1
needs to be improved: in a previous study we were able to deter-
mine the IC50 value of 1 for 15 different human tumor cells lines,
To alter the lipophilicity of the molecule and to improve
bioavailability, the carboxylic acid at position C-30 was transformed
into different esters either by alkylation with alkyl iodides [7,11] in
the presence of finely grounded K2CO3 in DMF (/4 [12]) or via
a DCC mediated coupling in DCM (/6). The hydrogen succinate 5
was obtained from 1 using succinic anhydride in pyridine [13], and
the ether 7 was obtained from 4 with sodium hydride/methyl
iodide in THF [11]. The tosylate 8 [14] of methyl glycyrrhetinate (4)
was accessed by tosylation of 4.
and for all tumor cell lines IC50 > 80
mM [7] were measured; this is
poor compared to betulinic acid (IC50 ¼ 7e14
mM [8]).
To improve the cytotoxicity of 1 it seems necessary to change
the molecule at various positions. Our focus in this study was on
ring A, especially positions O-3 and C-2 seemed of interest; this
should lead to compounds displaying an altered pattern of lip-
ophilicity compared to parent 1. IC50 values were determined in
sulforhodamine B assays as a measure of the cytotoxicity using
different human tumor cell lines. Additional trypan blue staining
experiments as well as acridine orange/ethidium bromide assays
on human alveolar basal epithelial cells A549 were used to deter-
mine their ability of inducing apoptosis in this cancer cell line.
To improve cytotoxicity, we considered the synthesis of deriv-
atives possessing an extra nitrogen-containing moiety. Thus,
compound 4 was oxidized at O-3 by a Jones oxidation [9] to yield 11
(Scheme 3). A derivative possessing an anellated pyrazine ring at
positions C-2 and C-3 (of ring A) was obtained from the reaction of
ketone 11 with ethylenediamine and sulfur in morpholine at 130 ꢁC
[15]. The corresponding 3-amino epimers resulted from a reductive
amination of 11 with ammonium acetate in ethanol [16] followed
by reducing the imine using sodium cyanoborohydride [17] in
a one-pot reaction. Both epimers (13 [18] and 14 [19]) were
obtained from this reduction with the
product under these conditions.
b-epimer 14 being the major
2. Chemistry
In addition, compound 14 served as a starting material for
several derivatives: Steglich esterification of 14 with Boc- -Ala
To determine whether a configurational inversion at position
C-3 has an influence on the cytotoxicity of 1, the corresponding
L
furnished 15 whose deprotection with trifluoroacetic acid in DCM
yielded amine 16. The synthesis of the oximes 17e19 started from
11. Thus, reaction of 11 with hydroxylamine hydrochloride in
* Corresponding author. Tel.: þ49 (0) 345 55 25660; fax: þ49 (0) 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.038

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5357
improvement of the antitumor activity is connected with these
structural changes.
Transforming the carboxylic acid into an ester (Table 2, Scheme
2), however, did not improve cytotoxicity. Except for compounds 6
(IC50 ¼ 28.59
m
M on A253 cells) and 7 (IC50 ¼ 28.99
mM in 518A2
M. For the methyl ether 7 an
cells), all IC50 values were above 30
m
IC50 could not be determined because of its insolubility in solvents
usually used in biological tests (DMSO, DMF or MeOH).
Whereas the pyrazine derivative 12 gave IC50 > 30 mM (Table 3,
Scheme 3), the amines 13 and 14 showed an excellent cytotoxicity
for the tumor cell lines being about 10 times higher than the
activity of parent compound 4.
Fig. 1. Structure of glycyrrhetinic acid (1).
pyridine followed by an acid work-up furnished 17; the corre-
sponding alkylated products 18e20 were obtained from the
alkylation of 17 with an alkyl halide in THF. Compound 20 was
obtained from 17; its deprotection did not result in the formation of
a free amine but a seconitrile, hence paralleling previous findings of
Askam and Bradley for an analogous tosylate [20].
Another option to modify the lipophilicity and/or polarity
pattern was accomplished by the incorporation of a thiol moiety.
While a direct sulfurization at position C-11 with Lawesson’s
reagent [21] failed under a variety of different conditions, dimeric
compound 22 was obtained from 4 by a DCC mediated coupling
reaction of a bis-Boc protected cysteine followed by the removal of
the Boc group with trifluoroacetic acid in DCM. A selective depro-
tection of the amino group in 23 was achieved in 86% resulting in
compound 24 possessing a free amino group as well as a protected
side chain.
Ring opening reactions concerning ring A (Scheme 5) started
from compound 25 [22] (obtained from 9), and ring A was
expanded by an insertion of oxygen using a BaeyereVilliger
oxidation [23] to yield 26. Ring opening under basic conditions
(KOH/EtOH) yielded the hydroxyl-substituted acid 27 whereas an
elimination reaction occurred when 26 was treated with hydro-
chloric acid in EtOH, and 28 was obtained. Prolonged heating under
reflux led to the formation of ethyl ester 29.
Fluorine’s special properties (small size, high electronegativity)
contribute to its importance in medicinal chemistry. The effects of
fluorine substitution on the biological behavior of biologically
active molecules have been used effectively in the development of
new drugs. To introduce a fluorine substituent on ring A, epoxide 31
[24,25] was reacted with Olah’s reagent [26] and 32 was obtained.
Nucleophilic ring opening of the epoxide occurred at the less
hindered carbon C-2 and followed FürstePlattner’s [27] rule.
Compound 32 is characterized in its 1H NMR spectrum by a chem-
For the oxime 17 IC50 values of 12e19
mM were determined; for
the human ovarian tumor cell line A2780 a low IC50 ¼ 6.18
m
M was
determined. Incorporation of alkyl chains in oxime 17, however, led
to a decrease of cytotoxicity.
The cytotoxicity of compounds 21e24 (Table 4, Scheme 4) was
determined, and their activity was similar to 4, except for
compounds 22 and 24 being about twice as active as their parent
compound 4.
As far as products with an opened ring A are concerned, except
for derivative 29, none of these compounds showed increased
cytotoxicity (Table 5, Scheme 5). The IC50 values of all compounds
were higher than that of parent compound 9. Compounds 26 and
29, however, exhibited lower IC50 values for A2780 cells
(IC50 ¼ 15.00
m
M for 26 and IC50 ¼ 5.89
mM for 29) as well as for
518A2 cells (IC50 ¼ 9.28
m
M for 29).
Fluorine substitution, however, did not lead to compounds of
higher cytotoxicity (Table 6, Scheme 6) that parent compound 9.
Better results were obtained when benzylester 33 was trans-
formed into the aminopropyl derivative 34. The IC50 values of
compound 34 (Table 7, Scheme 7) were about 3e5 times better
than those of its parent compound 33.
Glycyrrhetinic acid is known for its ability to trigger apoptosis.
To determine the extent of apoptosis, trypan blue staining and
counting experiments were performed the results of which are
summarized in Table 8. For parent glycyrrhetinic acid an extent of
ca. 74% was determined. Most of the compounds gave a positive
result in these experiments except for compounds 17 and 22.
Apoptotic effect [in %] of derivatives of 1 on A549 cells (þ/ꢀ
standard error, 6 experiments each); cells were treated with 1
(90
(20
m
m
M), 13 (4
M), 32 (60
Additional AO/EB tests support these results. In this test, green
m
M), 14 (4
m
M), 16 (8
mM), 17 (20 mM), 22 (20 mM), 24
mM), and 34 (8
mM), respectively.
fluorescent cells were found, hence indicating an apoptotic
behavior of the compounds. On principle, an AO/EB assay does not
allow quantification of the extent of apoptosis but confirms the
results from the trypan blue staining experiments (Fig. 2).
ical shift of H-2 at
d
¼ 4.64 ppm showing JH-1, H-2 ¼ 5.8 Hz, JH-2, H-
¼ 6.4 Hz and JH-2, F ¼ 49.7 Hz.
3
Reaction of 1 with benzyl chloride in the presence of finely
grounded potassium carbonate gave benzylester 33 [7,11] that was
transformed into the aminopropyl derivative 34.
4. Conclusions
3. Results
Herein we synthesized 25 derivatives of 1 differing in lip-
ophilicity and structure at ring A, and screened them for their
cytotoxicity. Neither configurational inversion at position C-2 nor
structural modifications at position C-3 led to higher cytotoxicity.
The introduction of at least one nitrogen-containing substituent,
however, was quite promising. Thus, derivatives possessing
a primary amino group (13e15) showed the highest activities
whereas substitution (12, 17e20) decreased cytotoxicity. A similar
behavior was established for compounds 21e24. In addition,
introduction of sulfur decreased cytotoxicity.
Screening of the compounds 1e3 (Scheme 1) in an SRB assay
using 11 different human cancer cell lines (Table 1) revealed that no
Structural modification of ring A, i.e., expanding (/26) or ring
opening (/27e29) by and large decreased cytotoxicity, except for
29 on A2780 cells. It seems that the presence of an intact ring A is
essential for cytotoxicity.
Scheme 1. Inversion of configuration at C-3: reagents and conditions: a) CrO3, acetone,
25 ꢁC, 1 h, 78%; b) L-selectride, THF, ꢀ75 ꢁC, 2 h, 61%.

5358
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
Table 1
Cytotoxicity (IC50 values in mmol) for 1e3 in a panel of various cancer cell lines.
518A2
A253
80.78
>30
>30
A431
79.58
>30
>30
A549
82.76
>30
>30
DLD-1
HCT-116
HCT-8
HT-29
Lipo
81.44
>30
>30
MCF-7
SW 1736
1*
2
3
83.92
>30
>30
81.21
>30
>30
78.33
>30
>30
78.85
>30
>30
80.09
>30
>30
84.70
>30
>30
76.93
>30
>30
From SRB assays after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ꢃ 5e7%; * data from a previous study [7].
Attachment of a diaminoalkyl moiety at position 3 of a lipophilic
ester of glycyrrhetinic acid, has an increasing effect on the cyto-
toxicity and was most rewarding: compound 34 exhibits IC50 values
from the 96-well plates was discarded and the cells were fixed with
10% TCA. For a thorough fixation, the plates were allowed to stand
at 4 ꢁC. After fixation, the cells were washed in a strip washer for
four times with water using alternate dispensing and aspiration
between 1.96 and 5.14
mM. Hence, compound 34 was the most
active compound of this study, thus indicating a feasible route for
the development of glycyrrhetinic acid derivatives showing
promising cytotoxicity.
procedures. The plates were dyed with 100 ml of 0.4% SRB (sulfo-
rhodamine B) for about 20 min. After dying, the plates were washed
with 1% acetic acid to remove the dye and allowed to air dry
overnight. Then 100 ml of 10 mM Tris base solution were added to
each well and absorbance was measured at 570 nm using a 96-well
plate reader (Tecan Spectra, Crailsheim, Germany). The IC 50 was
calculated from the semi-logarithmic doseeresponse curves.
5. Experimental
5.1. General
Melting points are uncorrected (Leica hot stage microscope),
5.4. Apoptosis tests
NMR spectra were recorded using the Varian spectrometers Gemini
200, Gemini 2000 or Unity 500 (d given in ppm, J in Hz, internal
5.4.1. Acridine orange/ethidium bromide (AO/EB) [29]
Me4Si), IR spectra (film or KBr pellet) on a PerkineElmer FT-IR
spectrometer Spectrum 1000, MS spectra were taken on an Intec-
tra 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, detection
by UV absorption). The solvents were dried according to usual
procedures.
Apoptotic cell death was analyzed by acridine orange/ethidium
bromide dye using fluorescence microscopy on A549 cells. There-
fore, approx. 500,000 cells were seeded in cell culture flasks and
were allowed to grow for 24 h. The medium was removed and the
substance loaded medium was added. After 24e48 h, the super-
natant medium was collected and centrifuged; the pellet was sus-
pended in phosphateebuffer saline (PBS) and centrifuged again.
The liquid was removed, and the pellet was suspended in PBS. After
mixing the suspension with a solution of AO/EB, analysis was per-
formed under a fluorescence microscope. While a green fluores-
cence shows apoptosis, a red colored nucleus indicates necrotic
cells.
5.2. Cell lines and culture conditions
Cultures were maintained as monolayer in RPMI 1640 (PAA
Laboratories, Pasching, Germany) supplemented with 10% heat
inactivated fetal bovine serum (Biochrom AG, Berlin, Germany) and
penicillin/streptomycin (PAA Laboratories) at 37 ꢁC in a humidified
atmosphere of 5% CO2/95% air.
5.4.2. Trypan blue cell counting
Approx. 500,000 cells (A549) were seeded in cell culture flasks
and were allowed to grow for 1 day. After removing of the medium,
the substance loaded medium was introduced and the flasks were
incubated for about 24e48 h. The supernatant medium was
collected and centrifuged; cell pellet was suspended in PBS and
centrifuged again. Equal amounts of Trypan blue solution (0.4% in
phosphate-buffer saline, pH 7.2) and suspension of the pellet in PBS
were mixed and put on chamber slides (invitrogenÔ). Automatic
cell counter (invitrogenÔ countessÒ automated cell counter) was
used for counting the cells, differing between cells with an intact
cell membrane and cells without.
5.3. Cytotoxicity assay [28]
The cytotoxic activities of our compounds were evaluated using
the sulforhodamine B (SRB) [21] (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 confluence of the cells during the period of experiment.
After 24 h, the cells were treated with serial dilutions of the
compounds (0e30 mM) for 96 h. The final concentration of DMSO or
DMF as a solvent never exceeded 0.5%, which was shown to be non-
toxic to the cells. The percentages of surviving cells compared to
untreated controls were determined 96 h after the beginning of
drug exposure. After 96 h of treatment, the supernatant medium
5.5. (3a)-3-Hydroxy-11-oxoolean-12-en-30-oic acid (3) [9]
To a solution of 2 (450 mg, 0.96 mmol) in dry THF (30 ml)
at ꢀ75 ꢁC, L-Selectride (1 M in THF, 10 ml, 10 mmol) was added, and
the mixture was stirred at ꢀ75 ꢁC for 2 h. After warming to room
temperature, hydrochloric acid (1 M) was added until the pH ¼ 2.
The aqueous layer was extracted with chloroform (3 ꢂ 15 ml), the
combined extracts were washed with water (20 ml), dried (Na2SO4)
Table 2
Cytotoxicity (IC50 values in mmol) for 4-10 in a panel of various cancer cell lines.
518A2
8505C
A253
19.42
>30
28.59
>30
25.04
>30
A549
23.50
>30
>30
>30
DLD-1
Lipo
20.47
>30
>30
>30
4*
5
6
8
9*
10
27.54
>30
>30
>30
25.23
28.99
26.07
>30
>30
>30
24.58
>30
26.12
>30
>30
>30
28.14
>30
and evaporated. Re-crystallization from methanol afforded
3
(170 mg, 38%) as colorless crystals; mp 308e310 ꢁC (lit. >325 ꢁC
[24]); Rf ¼ 0.32 (hexane/ethyl acetate 7:3); [
a
]
¼ þ114.66ꢁ (c 0.31,
D
CHCl3); UVevis (methanol): lmax (log
3
) ¼ 250 nm (3.95); IR (KBr):
22.74
>30
27.66
>30
n
¼ 3424br, 2960s, 1717m, 1645s, 1458w, 1386m, 1328w, 1253w,
1208w, 1159m, 1088w, 1062w, 1028w, 1003w cmꢀ1 1H NMR
(500 MHz, DMSO-d6):
¼ 12.11 (s, 1H, COOH), 5.38 (s, 1H, H-12),
;
From SRB assays after 96 h of treatment; the values are averaged from at least 3
independent experiments; variation ꢃ 5e7%; * data from a previous study [7].
d

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5359
Scheme 2. Derivatization of 1; reagents and conditions: a) K2CO3/MeI, DMF, 25 ꢁC, 2 h [7,11]; b) succinic anhydride, pyridine, 85 ꢁC, 48 h, 64%; c) Boc-L-Ser(OTBTMS)eOH, DCC,
DMAP, DCM, 25 ꢁC, 20 h, 22%; d) NaH (60% in mineral oil), MeI, THF, reflux, 30 min, 34%; e) p-TsCl, pyridine, 0 ꢁC, 4 h, 50%; K2CO3/EtI, DMF, 25 ꢁC, 2 h [7,11]; g) succinic anhydride,
pyridine, 85 ꢁC, 48 h, 86%.
4.17 (d,1H, OH, J ¼ 4.3 Hz), 3.15 (d,1H, H-3, J ¼ 3.5 Hz), 2.37 (s,1H, H-
9), 2.27 (ddd, 1H, H-1, J ¼ 13.1, 3.5, 3.5 Hz), 2.08 (m, 1H, H-15), 2.05
(m, 1H, H-18), 1.85 (m, 1H, H-2), 1.78 (m, 1H, H-21), 1.71 (m, 1H, H-
16), 1.64 (m, 1H, H-19), 1.62 (m, 1H, H-7), 1.37 (m, 1H, H-6), 1.35 (s,
3H, H-27), 1.34 (m, 1H, H-210), 1.32 (m, 1H, H-22), 1.31 (m, 1H, H-20),
1.30 (m, 1H, H-70), 1.29 (m, 1H, H-10), 1.27 (m, 1H, H-60), 1.25 (m, 1H,
H-220), 1.18 (m, 1H, H-5), 1.13 (m, 1H, H-160), 1.08 (s, 3H, H-29), 1.02
(s, 6H, H-26 and H-25), 0.94 (m, 1H, H-150), 0.82 (s, 3H, H-28), 0.75
(s, 3H, H-24), 0.74 (s, 3H, H-23) ppm; 13C NMR (125 MHz, DMSO-
n
¼ 3433br, 2952s, 2875m, 1731s, 1654s, 1465m, 1388m, 1363m,
1329w, 1280m, 1217s, 1167s, 1087w, 1049w, 1021w, 989m cmꢀ1; 1H
NMR (500 MHz, CDCl3):
¼ 5.67 (s, 1H, H-12), 4.55 (dd, 1H, H-3,
J ¼ 11.6, 4.8 Hz), 3.69 (s, 3H, CH3), 2.80 (ddd, 1H, H-1, J ¼ 13.7, 3.3,
3.3 Hz), 2.69 (m, 2H, chain- -CH2), 2.64 (m, 2H, chain- -CH2), 2.36
d
g
b
(s, 1H, H-9), 2.08 (dd, 1H, H-18, J ¼ 13.9, 3.5 Hz), 2.03 (m, 1H, H-15),
1.99 (m, 1H, H-21), 1.93 (ddd, 1H, H-19, J ¼ 13.5, 3.8, 2.6 Hz), 1.82
(ddd, 1H, H-16, J ¼ 13.3, 13.3, 4.2 Hz), 1.71 (m, 1H, H-2), 1.66 (m, 1H,
H-7),1.62 (m,1H, H-20),1.61 (dd,1H, H-190, J ¼ 13.6,13.6 Hz),1.58 (m,
1H, H-6), 1.48 (m, 1H, H-60), 1.42 (m, 1H, H-70), 1.40 (m, 1H, H-22),
1.36 (s, 3H, H-27),1.31 (m, 1H, H-220),1.31 (m,1H, H-210), 1.18 (m,1H,
H-160), 1.16 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.13 (s, 3H, H-26), 1.08
(m, 1H, H-10), 1.02 (m, 1H, H-150), 0.88 (s, 3H, H-24), 0.87 (s, 3H, H-
23), 0.81 (s, 3H, H-28), 0.79 (m, 1H, H-5) ppm; 13C NMR (125 MHz,
d6):
d
¼ 199.1 (C11), 177.6 (C30), 169.5 (C13), 127.3 (C12), 73.5 (C3),
61.1 (C9), 48.0 (C18), 47.5 (C5), 45.0 (C8), 43.0 (C20), 42.9 (C14), 40.6
(C19), 37.5 (C22), 37.1 (C10), 36.7 (C4), 33.0 (C1), 32.1 (C7), 31.5
(C17), 30.3 (C21), 28.7 (C28), 28.3 (C23), 27.8 (C29), 25.9 (C16), 25.8
(C15), 25.1 (C2), 23.1 (C27), 22.2 (C24), 18.3 (C26), 16.9 (C6), 16.1
(C25) ppm; MS (ESI): m/z (%) ¼ 471.5 ([M þ H]þ, 70), 493.5
([M þ Na]þ, 25), 525.1 ([M þ MeOH þ Na]þ, 100).
CDCl3):
(chain-
d
¼ 200.1 (C-11), 177.1 (chain-
d-COOH), 176.9 (C-30), 171.8
a
-COO), 169.3 (C-13),128.5 (C-12), 81.2 (C-3), 61.7 (C-9), 55.0
(C-5), 51.7 (OCH3), 48.4 (C-18), 45.4 (C-8), 44.0 (C-20), 43.2 (C-14),
41.1 (C-19), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22), 36.9 (C-10), 32.7 (C-7),
31.8 (C-17), 31.1 (C-21), 29.3 (chain-g-CH2), 28.9 (chain-b-CH2), 28.5
(C-28), 28.3 (C-29), 28.0 (C-23), 26.4 (C-16), 26.4 (C-15), 23.5 (C-2),
23.3 (C-27), 18.7 (C-26), 17.3 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS
(ESI): m/z (%) ¼ 585.4 ([M þ H]þ, 12), 607.5 ([M þ Na]þ, 54), 899.4
([3Mþ2Na]2þ, 88), 1169.2 ([2M þ H]þ, 18), 1191.6 ([2M þ Na]þ, 100),
583.2 ([M ꢀ H]ꢀ, 32), 629.0 ([M þ HCO2]ꢀ, 96), 1167.0 ([2M ꢀ H]ꢀ,
100).
5.6. Methyl (3
Compound 4 was prepared according to [12].
5.7. 4-{[(3 )-30-Methoxy-11,30-dioxoolean-12-en-3-yl]oxy}4-
b) 3-hydroxy-11-oxo-olean-12-en-30-oate (4)
b
oxobutanoic acid (5) [13]
To a solution of 4 (960 mg, 1.98 mmol) in dry pyridine (20 ml),
succinic anhydride (430 mg, 4.3 mmol) was added, and the mixture
was stirred at 85 ꢁC for 2 days. The solvent was removed, DCM
(30 ml) added, and the reaction was washed with hydrochloric acid
(1 M, 20 ml), extracted with DCM (3 ꢂ 20 ml), and the combined
organic layers were dried (Na2SO4), filtered and evaporated. The
residue was subjected to chromatography (silica gel, hexane/ethyl
acetate 1:1) to afford 5 (740 mg, 64%) as a colorless powder; mp
260e263 ꢁC (lit. 262e264 ꢁC [13]); Rf ¼ 0.14 (hexane/ethyl acetate
5.8. Methyl (3
butyl(dimethyl)silyl]-
b
)-3-({N-(tert-butoxycarbonyl9)-O-[tert-
L
-seryl}oxy)-11-oxoolean-12-en-30-oate (6)
Compound 4 (410 mg, 0.85 mmol), Boc-L-Ser(OTBDMS)eOH
(320 mg, 1.00 mmol) and DMAP (30 mg, 0.25 mmol) were dissolved
in dry DCM (25 ml). DCC (210 mg, 1.02 mmol) was added and the
solution was stirred at 25 ꢁC for 20 h. The precipitate was filtered off
and the filtrate evaporated. Purification via chromatography (silica
gel, CHCl3/ether 9:1) yielded 6 (150 mg, 22%) as a colorless powder;
1:1); [
a]
¼ þ154.55ꢁ (c 0.21, CHCl3) (lit. þ156 ꢃ 2ꢁ (c 0.05, CHCl3)
D
[13]); UVevis (methanol): lmax (log
3 ) ¼ 249 nm (4.09); IR (KBr):
Table 3
Cytotoxicity (IC50 values in
mmol) for 4, 11e20 in a panel of various cancer cell lines.
518A2
8505C
A253
A2780
A549
DLD-1
Lipo
MCF-7
SW1736
4*
27.54
>30
>30
2.74
2.52
5.55
18.93
>30
26.07
>30
>30
2.33
2.45
4.46
17.18
>30
19.42
>30
>30
2.44
2.36
4.46
13.56
>30
25.54
>30
>30
3.42
3.32
5.39
6.18
>30
23.50
>30
>30
3.33
2.51
5.38
17.40
>30
26.12
>30
>30
3.21
2.56
6.44
10.46
>30
20.47
>30
>30
3.30
2.74
5.82
18.34
>30
22.14
>30
>30
2.55
2.47
5.08
15.95
>30
26.35
15.17
>30
29.09
>30
2.84
2.62
5.25
13.00
>30
17.77
14.61
11
12
13
14
16
17
18
19
20
>30
16.49
>30
15.47
>30
14.12
19.73
12.03
>30
15.83
>30
19.10
>30
18.99
From SRB assays after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ꢃ 5e7%; * data from a previous study [7].

5360
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
Scheme 3. Derivatization of 4 or 11; reagents and conditions: a) CrO3, acetone, 25 ꢁC, 20 min [9]; b) S, ethylendiamine, morpholine, 130 ꢁC, 4 h, 39%; c) H3CCOONH4, MeOH, 25 ꢁC,
10 min followed by NaBH3CN, MeOH, 25 ꢁC, 24 h yielding 13 (20%) and 14 (52%); d) Boc-L-Ala, DCC, DMAP, DCM, 25 ꢁC, 16 h, 57%; F3CO2H, DCM, 25 ꢁC, 1 h, 60%; f) H2NOH.HCl,
pyridine, 60 ꢁC, 3 h, 80%; i-PropI, KOH, THF, reflux, 24 h, 37%; Oct-Br, KOH, THF, reflux, 24 h, 29%; Boc-L-Ala, DCC, DMAP, DCM, 25 ꢁC, 16 h, 48%.
mp 120e123 ꢁC (decomp.); Rf ¼ 0.68 (hexane/ethyl acetate 7:3);
28.5 (C-28), 28.4 (Boc-CH3), 28.4 (C-29), 28.0 (C-23), 26.5 (C-16),
26.4 (C-15), 25.8 (TBDMS-CH3), 23.6 (C-2), 23.3 (C-27), 18.7 (C-26),
18.2 (TBDMS-quart.eC), 17.3 (C-6), 16.7 (C-24), 16.3 (C-25), ꢀ5.5
[
a]
¼
þ74.12ꢁ (c 0.57, CHCl3); UVevis (methanol): lmax
D
(log
3
) ¼ 249 nm (4.05); IR (KBr):
n
¼ 3448br, 2952s, 2858s, 1732s,
1662s, 1498s, 1465m, 1389m, 1367m, 1257m, 1216s, 1167s, 1115s,
(SieCH3), ꢀ5.6 (SieCH3) ppm; 29Si NMR (100 MHz, CDCl3):
¼ 21.4
d
1062m, 986m, 837w, 779m, 723m cmꢀ1; 1H NMR (500 MHz, CDCl3):
(TBDMS-Si) ppm; MS (ESI): m/z (%) ¼ 786.2 ([M þ H]þ, 11), 808.4
([M þ Na]þ, 100), 824.3 ([M þ K]þ, 6), 1201.1 ([3Mþ2Na]2þ, 8),
1593.1 ([2M þ Na]þ, 16); analysis for C45H75NO8Si (786.2): C, 68.75;
H, 9.62, N, 1.78; found; C, 68.65; H, 9.81; N, 1.63.
d
¼ 5.66 (s, 1H, H-12), 5.34 (d, 1H, NH, J ¼ 8.1 Hz), 4.59 (dd, 1H, H-3,
J ¼ 11.7, 4.8 Hz), 4.30 (m, 1H, Ser-CH), 4.07 (dd, 1H, Ser-CHH0,
J ¼ 10.0, 2.1 Hz), 3.87 (dd, 1H, Ser-CHH0, J ¼ 10.0, 2.1 Hz), 3.69 (s, 3H,
OCH3), 2.81 (ddd,1H, H-1, J ¼ 13.6, 3.5, 3.5 Hz), 2.36 (s,1H, H-9), 2.08
(dd,1H, H-18, J ¼ 13.3, 3.7 Hz), 2.03 (m,1H, H-15),1.99 (m,1H, H-21),
1.93 (ddd,1H, H-19, J ¼ 13.5, 4.2, 2.7 Hz),1.82 (ddd,1H, H-16, J ¼ 13.7,
13.7, 4.4 Hz), 1.73 (m, 1H, H-2), 1.66 (m, 1H, H-7), 1.63 (m, 1H, H-20),
1.61 (dd, 1H, H-190, J ¼ 13.6, 13.6 Hz), 1.57 (m, 1H, H-6), 1.48 (m, 1H,
H-60), 1.45 (s, 9H, Boc-CH3), 1.43 (m, 1H, H-70), 1.40 (m, 1H, H-22),
1.36 (s, 3H, H-27),1.31 (m,1H, H-220), 1.31 (m,1H, H-210), 1.18 (m,1H,
H-160), 1.16 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.13 (s, 3H, H-26), 1.06
(m, 1H, H-10), 1.01 (m, 1H, H-150), 0.90 (s, 3H, H-24), 0.89 (s, 3H, H-
23), 0.86 (s, 9H, TBDMS-CH3), 0.80 (s, 3H, H-28), 0.80 (m, 1H, H-5),
0.04 (s, 3H, SieCH3), 0.02 (s, 3H, SieCH3) ppm; 13C NMR (125 MHz,
5.9. Methyl (3b)-3-(methoxy)-11-oxo-olean-12-en-30-oate (7)
To a solution of 4 (200 mg, 0.41 mmol) in dry THF (10 ml),
sodium hydride (60% in mineral oil, 30 mg, 0.75 mmol) was added,
and the mixture was refluxed for 30 min. Methyl iodide was added
and refluxing was continued for another 30 min. After cooling to
25 ꢁC, water (10 ml) was added dropwise, and the mixture was
extracted with DCM (3 ꢂ 10 ml). The organic layers were washed
with brine (20 ml), dried (Na2SO4), filtered and evaporated. The
residue was subjected to chromatography (silica gel, hexane/ethyl
acetate 9:1) to afford 7 (70 mg, 34%) as a colorless powder; mp
>300 ꢁC (lit. >300 ꢁC [3]); Rf ¼ 0.75 (hexane/ethyl acetate 7:3);
CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30), 170.4 (Ser-COO), 169.2 (C-13),
155.2 (Boc-COO), 128.5 (C-12), 81.8 (C-3), 79.6 (Boc-quart.eC), 63.8
(Ser-CH2), 61.7 (C-9), 55.9 (Ser-CH), 55.0 (C-5), 51.7 (OCH3), 48.4 (C-
18), 45.4 (C-8), 44.0 (C-20), 43.2 (C-14), 41.1 (C-19), 38.7 (C-1), 38.2
(C-4), 37.7 (C-22), 36.9 (C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21),
[
a
]
¼
þ160.78ꢁ (c 0.63, CHCl3); UVevis (methanol): lmax
D
(log
3
) ¼ 250 nm (4.02); IR (KBr): ¼ 3442br, 2930s, 2872s, 2856m,
n
2816m, 1732s, 1654s, 1616w, 1463m, 1388m, 1358m, 1324m, 1279w,
1264w, 1246w, 1220s, 1184m, 1156s, 1101s, 1087m, 1048w,
1027w cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.65 (s, 1H, H-12),
Table 4
3.67 (s, 3H, OCH3), 3.34 (s, 3H, CH3O), 2.81 (ddd, 1H, H-1, J ¼ 13.2,
3.7, 3.7 Hz), 2.65 (dd, 1H, H-3, J ¼ 11.6, 4.4 Hz), 2.31 (s, 1H, H-9),
2.06 (dd, 1H, H-18, J ¼ 14.8, 3.9 Hz), 2.01 (m, 1H, H-15), 1.98 (m, 1H,
H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.7, 3.9, 2.5 Hz), 1.81 (m, 1H, H-16),
1.78 (m, 1H, H-2), 1.62 (m, 1H, H-7), 1.59 (dd, 1H, H-190, J ¼ 13.5,
13.5 Hz), 1.55 (m, 1H, H-6), 1.49 (m, 1H, H-20), 1.42 (m, 1H, H-60),
1.38 (m, 1H, H-70), 1.37 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.30 (m, 1H,
H-220), 1.30 (m, 1H, H-210), 1.16 (m, 1H, H-160), 1.13 (s, 3H, H-25),
1.13 (s, 3H, H-29), 1.11 (s, 3H, H-26), 1.00 (m, 1H, H-150), 0.97 (s, 3H,
Cytotoxicity (IC50 values in mmol) for 4, 21e24 in a panel of various cancer cell lines.
518A2
8505C
A253
19.42
>30
19.07
>30
15.84
A549
23.50
>30
18.75
>30
17.90
DLD-1
Lipo
20.47
>30
22.60
>30
16.72
SW1736
4*
27.54
>30
24.54
>30
26.07
>30
15.10
>30
26.12
>30
>30
>30
>30
>30
>30
16.82
>30
15.69
21
22
23
24
16.78
15.45
From SRB assays after 96 h of treatment; the values are averaged from at least 3
independent experiments; variation ꢃ 5e7%; * data from a previous study [7].

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5361
Scheme 4. Derivatization of 4 leading to sulfur-containing derivatives 22e24; reagents and conditions: a) Boc-L-Cys(SBoc)eOH, DCC, DMAP, DCM, 25 ꢁC, 16 h, 93%; b) F3CCO2H,
DCM, 25 ꢁC, 24 h, 43 and 86%; c) Boc-L-Cys(SBn)eOH, DCC, DMAP, DCM, 25 ꢁC, 16 h, 57%; F3CCO2H, DCM, 25 ꢁC, 24 h, 86%.
H-23), 0.88 (m, 1H, H-10), 0.79 (s, 3H, H-28), 0.77 (s, 3H, H-24), 0.67
(m, 1H, H-5) ppm; 13C NMR (125 MHz, CDCl3):
1.58 (m, 1H, H-20), 1.56 (dd, 1H, H-190, J ¼ 13.4, 13.4 Hz), 1.54 (m, 1H,
H-6), 1.41 (m, 1H, H-60), 1.39 (m, 1H, H-70), 1.36 (m, 1H, H-22), 1.31 (s,
3H, H-27), 1.26 (m, 2H, H220 and H210), 1.14 (m, 1H, H-160), 1.11 (s,
3H, H-29), 1.06 (s, 3H, H-25), 1.05 (s, 3H, H-26), 0.97 (m, 1H, H-150),
0.90 (ddd, 1H, H-10, J ¼ 13.7, 13.7, 2.9 Hz), 0.84 (s, 3H, H-23), 0.8 (s,
3H, H-24), 0.76 (s, 3H, H-28), 0.70 (d, 1H, H-5, J ¼ 11.5, 1.5) ppm; 13C
d
¼ 200.3 (C-11),
176.9 (C-30), 169.1 (C-13), 128.6 (C-12), 88.3 (C-3), 61.8 (C-9), 57.4
(CH3O), 55.5 (C-5), 51.7 (OCH3), 48.4 (C-18), 45.4 (C-8), 44.0 (C-20),
43.2 (C-14), 41.1 (C-19), 39.0 (C-1), 39.0 (C-4), 37.7 (C-22), 37.1 (C-
10), 32.8 (C-7), 31.8 (C-17), 31.1 (C-21), 28.5 (C-28), 28.3 (C-29),
28.1 (C-23), 26.5 (C-16), 26.4 (C-15), 23.3 (C-27), 22.0 (C-2), 18.7
(C-26), 17.4 (C-6), 16.3 (C-24), 16.3 (C-25) ppm; MS (ESI): m/z
NMR (125 MHz, CDCl3):
d
¼ 199.7 (C11), 176.8 (C30), 169.4 (C13),
144.2 (aromatic-C4), 135.0 (aromatic-C1), 129.6 (aromatic-C2),
128.3 (C12), 127.5 (aromatic-C3), 90.6 (C3), 61.5 (C9), 55.2 (C5), 51.7
(OCH3), 48.3 (C18), 45.3 (C8), 44.0 (C20), 43.1 (C14), 41.1 (C19), 38.8
(C1), 37.7 (C22), 36.7 (C10), 32.6 (C7), 31.8 (C17), 31.1 (C21), 28.5
(C28), 28.3 (C29), 27.9 (C28), 26.4 (C16), 26.3 (C15), 24.5 (C2), 23.3
(C27), 21.6 (aromatic-CH3), 18.6 (C26), 17.5 (C6), 16.3 (C24), 16.3
(C25) ppm; MS (ESI): m/z (%) ¼ 639.4 ([M þ H]þ, 20), 661.1
([M þ Na]þ,100), 677.1 ([M þ K]þ, 8), 980.8 ([3Mþ2Na]2þ, 8),1299.0
([2M þ Na]þ, 8).
(%)
¼
499.5 ([M
þ
H]þ, 56), 521.5 ([M
þ
Na]þ, 6), 539.1
([M þ Na þ H2O]þ, 11), 552.9 ([M þ Na þ MeOH]þ, 100), 997.2
([2M þ H]þ, 20), 1019.3 ([2M þ Na]þ, 42).
5.10. Methyl (3b)-3-{[(4-methylphenyl)sulfonyl]oxy}-11-oxolean-
12-en-30-oate (8) [14]
Compound 4 (300 mg, 0.62 mmol) was dissolved in dry pyridine
(10 ml) and cooled to 0 ꢁC. 4-Toluenesulfonyl chloride (160 mg,
0.81 mmol) was added, and the solution was stirred for 4 h. After
usual aqueous work-up, extraction with ethyl acetate (3 ꢂ 10 ml)
5.11. Ethyl (3
Compound 9 was prepared according to [7].
5.12. 4-{(3 )-30-Ethoxy-11,30-dioxo-olean-12-en-3-yl]oxy}-4-
b)-3-hydroxy-11-oxoolean-12-en-30-oate (9)
and chromatography (silica gel, hexane/ethyl acetate 7:3)
8
(200 mg, 50%) was obtained as a colorless powder; mp 205e207 ꢁC;
Rf ¼ 0.57 (hexane/ethyl acetate 7:3); [
UVevis (methanol): lmax (log
(4.02); IR (KBr):
a
]
¼ þ106.7ꢁ (c 0.53, CHCl3);
b
D
3
) ¼ 196 (4.58), 228 (4.16), 249 nm
oxobutanoic acid (10)
n
¼ 3439br, 2950s, 2869s, 1920w, 1731s, 1659s,
1621m, 1598w, 1466s, 1386m, 1338s, 1293m, 1279m, 1261m, 1246m,
1216s, 1189s, 1169s, 1098m, 1088m, 1048w, 1030w, 1018w, 983m,
943s, 930s, 913s, 881s, 840m, 808m, 794m, 768w, 714w, 703w, 690s,
Following the procedure given for 5, from 9 (310 mg, 0.62 mmol),
succinic anhydride (60 mg, 0.62 mmol) and dry pyridine (10 ml), 10
(320 mg, 86%) was obtained as colorless solid; mp 221e224 ꢁC;
629w, 610w, 582w, 559m, 547m, 538m, 475w cmꢀ1
;
1H NMR
Rf ¼ 0.08 (hexane/ethyl acetate 1:1); [
a
]
¼ þ119.54ꢁ (c 0.31, CHCl3);
D
(500 MHz, CDCl3):
d
¼ 7.76 (d, 2H, aromatic-H2, J ¼ 8.1 Hz), 7.28 (d,
UVevis (methanol): lmax (log
3
) ¼ 249 nm (4.08); IR (KBr):
2H, aromatic-H3, J ¼ 8.0 Hz), 5.60 (s, 1H, H-12), 4.23 (dd, 1H, H-3,
J ¼ 12.1, 4.6 Hz), 3.64 (s, 3H, CH3), 2.73 (ddd, 1H, H-1, J ¼ 13.6, 3.8,
3.8 Hz), 2.40 (s, 3H, aromatic-CH3), 2.26 (s, 1H, H-9), 2.04 (dd, 1H, H-
18, J ¼ 13.2, 3.3 Hz), 2.00 (m, 1H, H-15), 1.95 (m, 1H, H-21), 1.87 (m,
1H, H-19), 1.78 (m, 1H, H-2), 1.77 (m, 1H, H-16), 1.61 (m, 1H, H-7),
n
¼ 3430br, 2953s, 1728s, 1657s, 1466s, 1387s, 1315m, 1291m, 1215s,
1176s,1086m,1022m, 987m, 958m cmꢀ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.64 (s, 1H, H-12), 4.55 (dd, 1H, H-3, J ¼ 11.6, 4.8 Hz), 4.19 (dt, 1H,
Et-CHH0, J ¼ 10.8, 7.1 Hz), 4.12 (dt, 1H, Et-CHH0, J ¼ 10.8, 7.1 Hz), 2.80
(ddd,1H, H-1, J ¼ 13.7, 3.3, 3.3 Hz), 2.69 (m, 2H, chain-
g-CH2), 2.64 (m,
Table 5
Cytotoxicity (IC50 values in
mmol) for 9, 25e29 in a panel of various cancer cell lines.
518A2
8505C
A253
25.04
>30
28.73
>30
>30
>30
A2780
A549
22.74
>30
>30
>30
>30
>30
DLD-1
Lipo
27.66
>30
>30
>30
>30
>30
MCF-7
SW1736
9*
25.23
>30
>30
>30
>30
24.58
>30
>30
>30
>30
26.96
>30
15.00
>30
>30
5.89
28.14
>30
>30
>30
>30
18.61
>30
20.58
>30
>30
25.42
13.37
>30
12.71
>30
>30
26.74
25
26
27
28
29
9.28
>30
>30
From SRB assays after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ꢃ 5e7%; * data from a previous study [7].

5362
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
Scheme 5. Ring A modifications; reactions and conditions: a) CrO3, acetone, 25 ꢁC,
20 min [9]; b) m-CPBA, CHCl3, 55 ꢁC, 24 h, 81%; c) NaOH, EtOH, 25 ꢁC, 24 h, 20%; d) HCl,
EtOH, 25 ꢁC, 5 min, 96%; e) HCl, EtOH, reflux, 30 min, 95%.
Scheme 6. Fluorination at position C-2; reactions and conditions: a) PPh3, 3,3-
dimethyl glutarimide, DEAD,THF, 25 ꢁC, 24 h, 82%; b) m-CPBA, CH2Cl2, 25 ꢁC, 20 h,
77%; c) Olah’s reagent, 25 ꢁC, 5 h, 19%.
2H, chain-
b
-CH2), 2.36 (s, 1H, H-9), 2.10 (dd, 1H, H-18, J ¼ 13.5,
3.9 Hz), 2.03 (ddd,1H, H-15, J ¼ 13.5,13.5, 4.2 Hz),1.99 (m,1H, H-21),
1.93 (ddd,1H, H-19, J ¼ 13.6, 4.0, 2.8 Hz),1.82 (ddd,1H, H-16, J ¼ 13.6,
13.6, 4.3 Hz), 1.71 (m, 1H, H-2), 1.66 (m, 1H, H-7), 1.63 (m, 1H, H-20),
1.61 (dd,1H, H-190, J ¼ 13.5,13.5 Hz),1.58 (m,1H, H-6),1.48 (m,1H, H-
60), 1.43 (m, 1H, H-70), 1.39 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.33 (m,
1H, H-220),1.33 (m,1H, H-210),1.26 (t, 3H, Et-CH3, J ¼ 7.1 Hz),1.18 (m,
1H, H-160),1.16 (s, 3H, H-25),1.14 (s, 3H, H-29),1.12 (s, 3H, H-26),1.04
(ddd,1H, H-10, J ¼ 13.8,13.8, 3.6 Hz),1.02 (m,1H, H-150), 0.88 (s, 3H, H-
24), 0.87 (s, 3H, H-23), 0.80 (s, 3H, H-28), 0.79 (m, 1H, H-5) ppm; 13C
combined organic extracts were dried (Na2SO4), filtered and
evaporated. Chromatographic purification (silica gel, chloroform/
ether 7:3) gave 12 (200 mg, 39%) as an off-white powder; mp
315 ꢁC; Rf ¼ 0.16 (chloroform/ether 7:3); [
a]
¼ þ191.8ꢁ (c 0.36,
D
CHCl3); UVevis (methanol): lmax (log
3
) ¼ 226 (3.97), 253 (4.14)
nm; IR (KBr):
1361w, 1323w, 1280m, 1209m, 1186s, 1153w, 1120m, 1108m, 1085w,
1063w, 1028w cmꢀ1 1H NMR (500 MHz, CDCl3):
¼ 8.38 (d, 1H,
n
¼ 3432br, 2941s,1731s,1654s,1451m,1400m,1387w,
;
d
pyrazine-H, J ¼ 2.2 Hz), 8.30 (d, 1H, pyrazine-H, J ¼ 2.2 Hz), 5.75 (s,
1H, H-12), 4.15 (d, 1H, H-1, J ¼ 17.0 Hz), 3.67 (s, 3H, CH3), 2.54 (s, 1H,
H-9), 2.54 (d, 1H, H-10, J ¼ 17.0 Hz), 2.10 (dd, 1H, H-18, J ¼ 13.3,
3.1 Hz), 2.03 (ddd,1H, H-15, J ¼ 13.6,13.6, 4.3 Hz),1.96 (m,1H, H-21),
1.93 (m, 1H, H-19), 1.85 (ddd, 1H, H-16, J ¼ 13.7, 13.7, 4.1 Hz), 1.75 (m,
1H, H-7), 1.67 (m, 1H, H-6), 1.59 (dd,1H, H-190, J ¼ 13.5, 13.5 Hz), 1.52
(ddd, 1H, H-70, J ¼ 12.6, 2.8, 2.8 Hz), 1.43 (dd, 1H, H-5, J ¼ 11.3,
2.1 Hz), 1.41 (m, 1H, H-22), 1.38 (m, 2H, H-220 and H-210), 1.38 (s, 3H,
H-24), 1.31 (s, 3H, H-27), 1.29 (s, 3H, H-23), 1.22 (m, 1H, H-160), 1.18
(s, 3H, H-26), 1.12 (s, 3H, H-29),1.10 (s, 3H, H-25),1.01 (m,1H, H-150),
NMR (125 MHz, CDCl3):
d
¼ 200.1 (C-11),177.1 (chain-
d-COOH),176.4
(C-30), 171.8 (chain- -COO), 169.5 (C-13), 128.4 (C-12), 81.2 (C-3),
a
61.7 (C-9), 60.3 (Et-CH2), 55.0 (C-5), 48.4 (C-18), 45.4 (C-8), 43.8 (C-
20), 43.2 (C-14), 41.1 (C-19), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22), 36.9
(C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 29.3 (chain-
g-CH2), 28.9
(chain- -CH2), 28.6 (C-28), 28.3 (C-29), 28.0 (C-23), 26.5 (C-16), 26.4
b
(C-15), 23.5 (C-2), 23.3 (C-27),18.7 (C-26),17.4 (C-6),16.7 (C-24),16.4
(C-25), 14.3 (Et-CH3) ppm; MS (ESI): m/z (%) ¼ 599.5 ([M þ H]þ, 20),
621.4 ([M þ Na]þ, 68), 921.0 ([3Mþ2Na]2þ, 58), 1197.3 ([2M þ H]þ,
22),1219.4 ([2M þ Na]þ,100);analysis for C36H54O7 (598.8): C, 72.21;
H, 9.09; found: C, 72.03; H, 9.24.
0.80 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 198.9 (C11),
176.8 (C30), 169.4 (C13), 158.8 (C3), 150.7 (C2), 142.1 (pyrazine),
141.7 (pyrazine), 128.6 (C12), 59.6 (C9), 53.0 (C5), 51.7 (OCH3), 48.7
(C1), 48.4 (C18), 45.0 (C8), 44.0 (C20), 43.3 (C14), 41.2 (C19), 39.2
(C10), 37.7 (C22), 36.5 (C4), 31.8 (C23), 31.7 (C17), 31.7 (C7), 31.1
(C21), 28.5 (C28), 28.3 (C29), 26.5 (C16), 26.4 (C15), 24.2 (C27), 23.3
(C24), 19.3 (C6), 18.2 (C26), 15.8 (C25) ppm; MS (ESI): m/z
(%) ¼ 519.5 ([M þ H]þ, 89), 572.9 ([M þ MeOH þ Na]þ, 100), 1037.2
([2M þ H]þ, 39); analysis for C33H46N2O3 (518.7): C, 76.41; H, 8.94;
N, 5.40; found: C, 76.27; H, 9.11; N, 5. 32.
5.13. Methyl 3,11-dioxoolean-12-en-30-oate (11)
Compound 11 was prepared according to [9].
5.14. Methyl (2S,4aS,6aS,6bR,14aS,16bR)-2,4a,6a,6b,9,9,14a-hepta-
methyl-15-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,14,14a,14b,15,16b-octa-
decahydrochryseno[1,2-g]quinoxaline-2-carboxylate (12)
Compound 11 (0.5 g, 1.00 mmol) was dissolved in morpholine
(20 ml), sulfur (powder, 150 mg, 2.50 mmol) and ethylenediamine
(15 ml, 2.20 mmol) was added. The mixture was stirred at 130 ꢁC for
4 h. After cooling, the mixture was poured into ice water (50 ml),
the aqueous layer was extracted with CHCl3 (3 ꢂ 50 ml). The
5.15. Methyl (3
a)-3-amino-11-oxo-olean-12-en-30-oate (13) and
methyl (3 )-3-amino-11-oxo-olean-12-en-30-oate (14)
b
To a solution of 11 (1.00 g, 2.06 mmol) in dry methanol (60 ml),
ammonium acetate (1.60 g, 20.6 mmol) was added, and the mixture
Table 6
Cytotoxicity (IC50 values in
m
mol) for 9, 30e32 in a panel of various cancer cell lines.
A253 A431 A549
25.04 23.45 22.74
518A2
DLD-1
HCT-116
HT-29
Lipo
27.66
>30
>30
>30
MCF-7
9*
25.23
>30
>30
>30
28.14
>30
>30
21.58
>30
>30
22.14
>30
>30
18.61
>30
>30
30
31
32
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
24.72
>30
From SRB assays after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ꢃ 5e7%; * data from a previous study [7].

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5363
Table 7
NMR (125 MHz, CDCl3):
d
¼ 199.6 (C11), 176.7 (C30), 169.0 (C13),
Cytotoxicity (IC50 values in mmol) for 33 and 34 in a panel of various cancer cell lines.
128.3 (C12), 61.3 (C9), 59.6 (C3), 55.1 (C5), 51.5 (OCH3), 48.2 (C18),
45.1 (C8), 43.8 (C20), 43.0 (C14), 41.0 (C19), 38.9 (C4), 37.6 (C1), 37.0
(C10), 36.7 (C22), 32.4 (C7), 31.6 (C17), 31.0 (C21), 28.3 (C29), 28.1
(C28), 28.1 (C23), 26.4 (C16), 26.4 (C15), 24.2 (C2), 23.1 (C27), 18.5
(C24), 17.3 (C6), 16.0 (C26), 15.8 (C25) ppm; MS (ESI): m/z
(%) ¼ 484.3 ([M þ H]þ, 100).
518A2
8505C
A253
A549
DLD-1
Lipo
33*
34
18.19
5.14
8.10
2.07
10.67
1.96
6.15
4.74
22.69
4.96
11.54
2.99
From SRB assays after 96 h of treatment; the values are averaged from at least 3
independent experiments; variation ꢃ 5e7%; * data from a previous study [7].
5.16. Methyl (3
oxoolean-12-en-30-oate (15)
b) 3-{[N-(tert-butoxycarbonyl)-L-alanyl]amino}-11-
was stirred at 25 ꢁC for 10 min. Sodium cyanoborohydride (130 mg,
2.06 mmol) was added, and stirring was continued for 24 h. The
mixture was concentrated to 20 ml and acidified with conc.
hydrochloric acid. The precipitate was filtered off and washed with
water. Purification by chromatography (silica gel, chloroform/
methanol 9:1) gave 13 (200 mg, 20%) and 14 (520 mg, 52%) each as
a colorless powder.
Following the procedure given for 20, compound 15 was
obtained from 14 (160 mg, 0.32 mmol), DCC (80 mg, 0.39 mmol),
DMAP (10 mg, 0.08 mmol) and Boc-L-alanine (80 mg, 0.42 mmol).
Purification by chromatography (silica gel, hexane/ethyl acetate
3:7) and recrystallization from ethyl acetate/hexane gave 15
(120 mg, 57%) as colorless crystals; mp 198e203 ꢁC; Rf ¼ 0.1
Data for 13: mp 212e215 ꢁC; Rf ¼ 0.71 (chloroform/methanol
8:2); [
(log
a
]
¼ þ114.3ꢁ (c 0.35, CDCl3); UVevis (methanol): lmax
D
(hexane/ethyl acetate 7:3); [
(methanol): lmax (log
a
]
¼ þ49.6ꢁ (c 0.50, CHCl3); UVevis
D
3
) ¼ 249 (4.02) nm; IR (KBr): ¼ 3422br, 2950s, 2361w, 1732s,
n
3
) ¼ 249 (3.92) nm; IR (KBr):
n
¼ 3328br,
1660s, 1618m, 1519m, 1463m, 1385m, 1315w, 1280w, 1259w, 1216m,
1191m, 1155m, 1133w, 1084w, 1063w, 1031w cmꢀ1 1H NMR
(500 MHz, CDCl3):
¼ 5.66 (s, 1H, H-12), 3.69 (s, 3H, CH3), 3.03 (m,
2930s, 2852s, 1731s, 1662s, 1626s, 1575m, 1534m, 1456m, 1388m,
1367m, 1313m, 1245m, 1218m, 1166m cmꢀ1 1H NMR (500 MHz,
CDCl3):
¼ 6.02 (d,1H, NH), 5.59 (s,1H, H-12), 4.88 (m,1H, NH), 3.62
;
;
d
d
1H, H-3), 2.73 (s, 1H, H-9), 2.60 (ddd, 1H, H-1, J ¼ 15.3, 3.4, 3.4 Hz),
2.07 (m, 1H, H-18), 2.04 (m, 1H, H-6), 2.02 (m, 1H, H-15), 1.99 (m, 1H,
H-21), 1.92 (m, 1H, H-19), 1.83 (m, 1H, H-60), 1.80 (m, 1H, H-16), 1.78
(m, 1H, H-7), 1.63 (dd, 1H, H-190, J ¼ 13.8, 13.8 Hz), 1.49 (m, 1H, H-5),
1.44 (m, 1H, H-2), 1.43 (s, 3H, H-27), 1.38 (m, 1H, H-20), 1.37 (m, 1H,
H-10), 1.36 (m, 1H, H-22), 1.35 (m, 1H, H-70), 1.31 (m, 2H, H-220 and
H-210), 1.17 (m, 1H, H-160), 1.14 (s, 3H, H-29), 1.13 (s, 3H, H-25), 1.09
(s, 3H, H-26),1.07 (s, 3H, H-29),1.01 (m,1H, H-15), 0.95 (s, 3H, H-24),
(s, 3H, CH3), 3.59 (dd, 1H, H-3, J ¼ 11.0, 4.4 Hz), 3.47 (m, 1H, Ala-CH),
2.70 (ddd, 1H, H-1, J ¼ 13.2, 2.8, 2.8 Hz), 2.32 (s, 1H, H-9), 2.01 (dd,
1H, H-18, J ¼ 13.7, 3.5 Hz), 1.95 (ddd, 1H, H-15, J ¼ 13.5, 13.5, 4.1 Hz),
1.93 (m, 1H, H-21), 1.84 (m, 1H, H-19), 1.75 (ddd, 1H, H-16, J ¼ 13.7,
13.7, 4.3 Hz), 1.62 (m, 1H, H-7), 1.55 (dd, 1H, H-190, J ¼ 13.6, 13.6 Hz),
1.58 (m,1H, H-6),1.56 (m,1H, H-2),1.46 (m,1H, H-20),1.43 (m,1H, H-
60), 1.38 (s, 9H, Boc-CH3), 1.37 (m, 1H, H-70), 1.34 (m, 1H, H-22), 1.30
(s, 3H, H-27), 1.28 (s, 3H, Ala-CH3), 1.25 (m, 2H, H-220 and H-210),
1.10 (m, 1H, H-160), 1.08 (s, 3H, H-29), 1.07 (s, 3H, H-25), 1.05 (s, 3H,
H-26), 1.00 (m, 1H, H-10), 0.95 (m, 1H, H-150), 0.83 (s, 3H, H-23), 0.81
(m, 1H, H-5), 0.73 (s, 3H, H-28), 0.72 (s, 3H, H-24) ppm; 13C NMR
0.80 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.0 (C11),
177.0 (C30), 169.1 (C13), 128.3 (C12), 60.7 (C9), 58.0 (C3), 51.8
(OCH3), 48.4 (C18), 47.4 (C5), 45.5 (C8), 44.0 (C20), 43.5 (C14), 41.1
(C19), 37.7 (C22), 36.9 (C4), 35.7 (C10), 32.7 (C1), 32.4 (C7), 31.8
(C17), 31.2 (C21), 28.8 (C23), 28.5 (C28), 28.3 (C29), 26.5 (C16), 26.4
(C15), 24.3 (C27), 22.7 (C24), 22.1 (C6), 18.6 (C26), 17.4 (C2), 16.5
(C25) ppm; MS (ESI): m/z (%) ¼ 484.3 ([M þ H]þ, 100).
(125 MHz, CDCl3):
d
¼ 200.2 (C11), 177.0 (C30), 172.0 (Ala-COO),
169.2 (C13), 156.7 (Boc-COO), 128.5 (C12), 80.1 (Boc-quart.eC), 60.7
(C9), 58.0 (C3), 51.8 (OCH3), 48.4 (C18), 47.4 (C5), 45.4 (C8), 44.0
(C20), 43.2 (C14), 41.1 (C19), 39.6 (C1), 38.2 (C4), 37.7 (C22), 36.9
(C10), 32.7 (C7), 31.8 (C17), 31.1 (C21), 28.5 (C28), 28.4 (C29), 28.3
(C23), 28.3 (Boc-CH3), 26.4 (C16), 26.4 (C15), 25.6 (C2), 23.3 (C27),
18.6 (C26), 17.9 (Ala-CH3), 17.7 (C6), 16.5 (C24), 16.2 (C25) ppm; MS
(ESI): m/z (%) ¼ 655.3 ([M þ H]þ, 32), 677.4 ([M þ Na]þ, 100),
1004.82 ([3Mþ2Na]2þ, 12), 1332.2 ([2M þ Na]þ, 25); analysis for
C39H62N2O6 (654.9): C, 71.52; H, 9.54; N, 4.28; found: C, 71.41; H,
9.73; N, 4.13.
Data for 14: mp 206 ꢁC; Rf ¼ 0.64 (chloroform/methanol 8:2);
[
a]
¼
þ121.8ꢁ (c 0.57, CDCl3); UVevis (methanol): lmax
D
(log
3 ) ¼ 249 (4.02) nm; IR (KBr):
n
¼ 3431br, 2950s, 1731s, 1659s,
1551s, 1465s, 1387s, 1328m, 1278m, 1249m, 1217s, 1190m, 1154s,
1088m, 1014m, 993m cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 5.65 (s,
d
1H, H-12), 3.68 (s, 3H, CH3), 2.84 (ddd, 1H, H-1, J ¼ 13.0, 2.9, 2.9 Hz),
2.74 (m, 1H, H-3), 2.33 (s, 1H, H-9), 2.07 (dd, 1H, H-18, J ¼ 13.4,
4.3 Hz), 2.01 (ddd, 1H, H-15, J ¼ 9.8, 9.8, 3.8 Hz), 1.99 (m, 1H, H-21),
1.92 (ddd, 1H, H-19, J ¼ 13.5, 4.3, 2.8 Hz), 1.90 (m, 1H, H-2), 1.81 (m,
1H, H-20),1.78 (m,1H, H-16),1.64 (m,1H, H-7),1.62 (m,1H, H-6),1.60
(dd,1H, H-190, J ¼ 13.5,13.5 Hz),1.44 (m,1H, H-60), 1.41 (m,1H, H-70),
1.38 (m, 1H, H-22), 1.35 (s, 3H, H-27), 1.28 (m, 2H, H-220 and H-210),
1.17 (m, 1H, H-160), 1.14 (s, 6H, H-25 and H-29), 1.12 (s, 3H, H-24),
1.10 (s, 3H, H-23), 0.99 (m, 1H, H-150), 0.96 (m, 1H, H-10), 0.94 (s, 3H,
H-26), 0.80 (s, 3H, H-28), 0.74 (dd, 1H, H-5, J ¼ 11.9, 1.3 Hz) ppm; 13C
5.17. Methyl (3b) 3-(L-alanylamino)-11-oxo-olean-12-en-30-oate
(16)
Compound 15 (100 mg, 0.15 mmol) was dissolved in DCM
and trifluoroacetic acid (2 ml; 25.96 mmol) was added. After
1 h of continuous stirring, a saturated solution of sodium
Scheme 7. Synthesis of compound 34; reactions and conditions: a) benzyl chloride, K2CO3, DMF, 25 ꢁC, 2 h [7,11]; b) chloroacetyl chloride, DCM, 25 ꢁC, 24 h, then diaminopropane,
K2CO3, DMF, 25 ꢁC, 2 h, 82%.

5364
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
Table 8
Apoptotic effect [in %] of derivatives of 1 on A549 cells.
Compound 13 14
Apoptosis [%] 73.73 ꢃ 1.40 64.88 ꢃ 3.60 74.21 ꢃ 1.85 56.77 ꢃ 4.56 32.43 ꢃ 3.40 68.54 ꢃ 2.40 40.91 ꢃ 3.43 60.64 ꢃ 3.16 80.32 ꢃ 1.68 80.57 ꢃ 3.23
1
16
17
20
22
24
32
34
hydrogencarbonate (10 ml) was added, and the organic layer was
washed with water (20 ml) afterward. The solvent was removed,
and the residue was subjected to chromatography (silica gel,
chloroform/methanol 8:2) to yield 16 (50 mg, 60%) as a colorless
powder; mp 183e185 ꢁC; Rf ¼ 0.50 (chloroform/methanol 8:2);
acid (1 M, 10 ml). Usual aq. work-up followed by chromatography
(silica gel, hexane/ethyl acetate 7:3) gave 17 (835 mg, 80%) as
a colorless powder; mp 283 ꢁC (lit. 289e290 ꢁC [20]); Rf ¼ 0.66
(hexane/ethyl acetate 8:2); [
a]
¼ þ101.5ꢁ (c 0.68, CHCl3) (lit.
D
[
a
]
¼ D106.7ꢁ (c 2.0, CHCl3) [20]); UVevis (methanol): lmax
D
UVevis (methanol): lmax (log
3
) ¼ 248 (3.57) nm; IR (KBr):
(log
3
) ¼ 249 nm (4.11); IR (KBr):
n
¼ 3278br, 2932s, 2870m, 1729s,
n
¼ 3330br, 2930s, 2852m, 1730s, 1659s, 1539s, 1451m, 1385m,
1314w, 1246m, 1218m, 1154w, 1088w, 1030w cmꢀ1 1H NMR
(500 MHz, methanol-d4):
¼ 5.48 (s, 1H, H-12), 3.60 (s, 3H, CH3),
1655s, 1618w, 1464w, 1387s, 1366m, 1320s, 1281m, 1248w, 1218w,
1190w, 1153w, 1087w, 1030w cmꢀ1 1H NMR (500 MHz, CDCl3):
¼ 5.61 (s, 1H, H-12), 3.62 (s, 3H, CH3), 2.99 (ddd, 1H, H-2, J ¼ 15.5,
;
;
d
d
3.50 (dd, 1H, H-3, J ¼ 12.6, 4.0 Hz), 3.39 (m, 1H, Ala-CH), 2.67
(ddd, 1H, H-1, J ¼ 13.0, 3.2, 3.2 Hz), 2.42 (s, 1H, H-9), 2.07 (dd, 1H,
H-18, J ¼ 13.9, 4.3 Hz), 2.03 (ddd, 1H, H-15, J ¼ 13.8, 4.1, 4.1 Hz),
1.87 (ddd, 1H, H-21, J ¼ 13.6, 5.2, 3.0 Hz), 1.79 (m, 1H, H-16), 1.77
(m, 1H, H-19), 1.70 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.65 (dd, 1H, H-
190, J ¼ 13.5, 13.5 Hz), 1.62 (ddd, 1H, H-20, J ¼ 13.9, 9.1, 3.2 Hz), 1.60
(m, 1H, H-6), 1.44 (m, 1H, H-60), 1.43 (m, 1H, H-70), 1.38 (m, 1H, H-
22), 1.34 (s, 3H, H-27), 1.31 (m, 2H, H-220 and H-210), 1.18 (d, 3H,
Ala-CH3, J ¼ 7.0 Hz), 1.15 (m, 1H, H-160), 1.06 (s, 3H, H-25), 1.05 (s,
3H, H-26), 1.05 (s, 3H, H-29), 1.00 (m, 1H, H-10), 0.95 (m, 1H, H-
150), 0.83 (dd, 1H, H-5, J ¼ 12.1, 1.2 Hz), 0.77 (s, 3H, 23), 0.75 (s,
3H, H-24), 0.73 (s, 3H, H-28) ppm; 13C NMR (125 Hz, methanol-
4.2, 4.2 Hz), 2.81 (ddd,1H, H-1, J ¼ 13.2, 5.3, 3.7 Hz), 2.31 (s,1H, H-9),
2.22 (ddd,1H, H-20, J ¼ 15.6,12.9, 5.7 Hz), 2.02 (dd,1H, H-18, J ¼ 13.2,
2.8 Hz), 1.95 (m, 1H, H-15), 1.92 (m, 1H, H-21), 1.84 (ddd, 1H, H-19,
J ¼ 13.5, 3.7, 2.8 Hz), 1.77 (ddd, 1H, H-16, J ¼ 13.4, 13.3, 4.2 Hz), 1.60
(ddd, 1H, H-7, J ¼ 12.6, 12.6, 3.5 Hz), 1.54 (dd, 1H, H-190, J ¼ 13.7,
13.7 Hz), 1.54 (m, 1H, H-6), 1.45 (ddd, 1H, H-60, J ¼ 12.5, 12.5, 2.5 Hz),
1.37 (ddd, 1H, H-70, J ¼ 12.5, 2.9, 2.9 Hz), 1.32 (ddd, 1H, H-22, J ¼ 10.0,
10.0, 3.0 Hz), 1.28 (s, 3H, H-29), 1.25 (m, 2H, H-210 and H-220), 1.19 (s,
3H, H-25), 1.12 (s, 3H, H-23), 1.12 (m, 1H, H-160), 1.09 (s, 3H, H-26),
1.08 (s, 3H, H-28), 1.03 (s, 3H, H-24), 0.98 (m, 1H, H-10), 0.94 (m, 1H,
H-150), 0.91 (m, 1H, H-5), 0.74 (s, 3H, H-27) ppm; 13C NMR
(125 MHz, CDCl3):
d
¼ 199.6 (C11), 176.9 (C30), 169.4 (C13), 167.2
d4):
d
¼ 202.2 (C11), 178.4 (C30), 177.1 (Ala-COO), 172.4 (C13),
(C3), 128.5 (C12), 61.3 (C9), 55.6 (C5), 51.7 (OCH3), 48.4 (C18), 45.3
(C8), 44.0 (C20), 43.2 (C14), 41.1 (C19), 40.5 (C4), 39.0 (C1), 37.7
(C22), 37.0 (C10), 32.4 (C7), 31.8 (C17), 31.1 (C21), 28.5 (C28), 28.3
(C29), 27.1 (C23), 26.5 (C16), 26.4 (C15), 23.3 (C27), 23.2 (C24), 18.6
(C26), 18.2 (C6), 17.4 (C2), 15.7 (C25) ppm; MS (ESI): m/z (%) ¼ 498.5
([M þ H]þ, 58), 520.3 ([M þ Na]þ, 6), 552.0 ([M þ MeOH þ Na]þ,
100), 995.0 ([2M þ H]þ, 42), 1017.3 ([2M þ H]þ, 43).
128.8 (C12), 63.0 (C9), 57.9 (C3), 56.7 (C5), 52.3 (Ala-CH), 51.4
(OCH3), 49.9 (C18), 46.7 (C8), 45.3 (C20), 44.7 (C14), 42.4 (C19),
40.9 (C1), 39.6 (C4), 39.0 (C22, CH2), 38.4 (C10), 33.7 (C7), 33.0
(C17), 32.0 (C21), 29.2 (C28), 29.2 (C23), 28.5 (C29), 27.6 (C16),
27.4 (C15), 26.0 (C2), 23.9 (C27), 21.8 (Ala-CH3), 19.3 (C26), 18.9
(C6), 17.2 (C24), 16.9 (C25) ppm; MS (ESI): m/z (%) ¼ 555.4
([M þ H]þ, 100); analysis for C34H54N2O4 (554.8): C, 73.61; H,
9.81; N, 5.05; found: C, 73.49; H, 9.99; N, 4.88.
5.19. Methyl 3-(i-propoxyimino)-11-oxo-olean-12-en-30-oate (18)
5.18. Methyl 3-(hydroxyimino)-11-oxo-olean-12-en-30-oate (17)
To a solution of 17 (500 mg, 0.60 mmol) in dry THF (20 ml),
potassium hydroxide (0.16 g, 3.00 mmol) and i-propyl iodide
(340 mg, 2.00 mmol) was added. After refluxing for 24 h, the
mixture was cooled and poured into water (40 ml). The aqueous
layer was extracted with chloroform (3 ꢂ 50 ml), the extracts were
To a solution of 11 (1.00 g, 2.10 mmol) in dry pyridine (10 ml),
hydroxylamine hydrochloride (348 mg, 4.00 mmol) was added. The
mixture was stirred at 60 ꢁC for 3 h and acidified with hydrochloric
Fig. 2. Results from the AO/EB tests: A549 cells treated with (left to right, upper row then lower row): 13 (4
(20 M), 32 (60 M), 34 (8 M).
mM), 14 (4 mM), 16 (8 mM), 17 (20 mM), 20 (20 mM), 22 (20 mM), 24
m
m
m

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5365
dried (Na2SO4), filtered and evaporated. Purification by chroma-
5.21. Methyl 3-[6-{(2L)-2-[tert-butoxycarbonylamino]propanoyl}-
tography (silica gel, chloroform/ether 8:2) afforded 18 (120 mg,
oxyimino]-11-oxo-olean-12-en-30-oate (20)
37%) as a colorless powder; mp 145 ꢁC; Rf ¼ 0.77 (chloroform/
ether 9:1); [
(log
a
]
¼ 105.42ꢁ (c 0.32, CHCl3); UVevis (methanol): lmax
To a solution of 17 (250 mg, 0.50 mmol) and DMAP (10 mg,
0.08 mmol) in dry DCM (20 ml), Boc-L-alanine (120 mg, 0.63 mmol)
D
3
) ¼ 257 nm (4.26), IR (KBr):
n
¼ 3446s, 2972s, 2870s, 1732s,
1662s, 1622w, 1458m, 1386m, 1366w, 1325w, 1281w, 1262w, 1218m,
1189m, 1153m, 1088w, 1028w, 963m cmꢀ1 1H NMR (500 MHz,
CDCl3):
¼ 5.66 (s, 1H, H-12), 4.26 (qq, 1H, chain-CH, J ¼ 6.2,
and DCC (120 mg, 0.60 mmol) were added. The mixture was stirred
at 25 ꢁC for 16 h. The solvent was removed under reduced pressure,
the residue purified by chromatography (silica gel, hexane/ethyl
acetate 7:3) to afford 20 (160 mg, 48%) as a colorless powder; mp
;
d
6.2 Hz), 3.67 (s, 3H, CH3), 2.89 (ddd, 1H, H-2, J ¼ 15.7, 5.0, 4.0 Hz),
2.76 (ddd, 1H, H-1, J ¼ 13.3, 5.7, 4.0 Hz), 2.36 (s, 1H, H-9), 2.24 (ddd,
1H, H-20, J ¼ 15.6, 12.2, 5.9 Hz), 2.06 (dd, 1H, H-18, J ¼ 13.5, 3.3 Hz),
1.99 (m, 1H, H-15), 1.97 (m, 1H, H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.5,
3.6, 2.7 Hz), 1.82 (ddd, 1H, H-16, J ¼ 13.6, 13.6, 4.4 Hz), 1.69 (m, 1H,
H-7), 1.59 (dd, 1H, H-190, J ¼ 13.5, 13.5 Hz), 1.56 (m, 1H, H-6), 1.52
(m, 1H, H-60), 1.44 (m, 1H, H-70), 1.39 (m, 1H, H-22), 1.33 (s, 3H, H-
27), 1.30 (m, 2H, H-220 and H-210), 1.21 (s, 3H, H-25), 1.20 (m, 1H,
H-160), 1.19 (m, 6H, chain-CH3), 1.16 (s, 3H, H-23), 1.13 (s, 6H, H-26
and H-29), 1.04 (s, 3H, H-24), 1.03 (m, 1H, H-10), 1.01 (m, 1H, H-5),
1.00 (m, 1H, H-150), 0.78 (s, 3H, H-28) ppm; 13C NMR (125 MHz,
116e118 ꢁC; Rf ¼ 0.65 (hexane/ethylacetate 7:3); [
a
]
¼ þ95.5ꢁ (c
D
0.40, CHCl3); UVevis (methanol): lmax (log
3
) ¼ 249 (4.11) nm; IR
(KBr):
1619w, 1509m, 1456s, 1389m, 1367m, 1345m, 1249m, 1218m, 1161m,
1109s, 1064w, 1026w cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 5.67 (s,
n
¼ 3385br, 2979s, 2872m, 2361w, 1763m, 1727s, 1660s,
d
1H, H-12), 5.09 (d, 1H, Ala-NH, J ¼ 7.0 Hz), 4.47 (m, 1H, Ala-CH), 3.67
(s, 3H, CH3), 2.88 (m, 1H, H-2), 2.85 (m, 1H, H-1), 2.41 (m, 1H, H-20),
2.37 (s, 1H, H-9), 2.08 (dd, 1H, H-18, J ¼ 13.2, 3.3 Hz), 2.03 (m, 1H, H-
15), 1.98 (m, 1H, H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.9, 4.0, 2.7 Hz), 1.82
(ddd, 1H, H-16, J ¼ 13.5, 13.5, 4.5 Hz), 1.69 (m, 1H, H-7), 1.64 (m, 1H,
H-6),1.58 (dd,1H, H-190, J ¼ 13.8,13.8 Hz),1.51 (m,1H, H-60),1.42 (m,
1H, H-70), 1.41 (s, 12H, Boc-CH3 and Ala-CH3), 1.39 (m, 1H, H-22),
1.33 (s, 3H, H-27), 1.29 (m, 2H, H-220 and H-210), 1.26 (s, 3H, H-23),
1.25 (s, 3H, H-25), 1.18 (m, 1H, H-160), 1.14 (s, 3H, H-24), 1.14 (m, 1H,
H-10), 1.13 (m, 1H, H-5), 1.12 (s, 6H, H-26 and H-29), 1.01 (m, 1H, H-
CDCl3):
d
¼ 199.6 (C11), 176.7 (C30), 169.0 (C13), 164.5 (C3), 128.6
(C12), 74.3 (chain-CH), 61.5 (C9), 55.6 (C5), 51.8 (OCH3), 48.6 (C18),
45.5 (C8), 44.2 (C20), 43.4 (C14), 41.3 (C19), 40.3 (C4), 39.1 (C1),
37.9 (C22), 37.1 (C10), 32.6 (C7), 32.0 (C17), 31.3 (C21), 28.7 (C28),
28.5 (C29), 27.8 (C23), 26.7 (C16), 26.7 (C15), 23.7 (C24), 23.5
(C27), 21.8 (chain-CH3), 18.8 (C26), 18.5 (C6), 18.2 (C2), 15.8 (C25)
150), 0.79 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 199.2
ppm; MS (ESI): m/z (%)
¼
540.5 ([M
þ
H]þ, 84), 593.5
(C11), 176.7 (C30), 175.8 (Ala-COO), 171.4 (C3), 169.5 (C13), 154.9
(Boc-COO), 128.4 (C12), 79.9 (Boc-quart.eC), 61.4 (C9), 55.6 (C5),
51.7 (OCH3), 48.6 (C18), 47.8 (Ala-CH), 45.4 (C8) 44.2 (C20), 43.5
(C14), 41.7 (C4), 41.3 (C19), 39.4 (C1), 37.9 (C22), 37.1 (C10), 32.5
(C7), 32.0 (C17), 31.3 (C21), 28.7 (C28), 28.3 (Boc-CH3), 28.4 (C29),
27.4 (C23), 26.7 (C16), 26.6 (C15), 23.5 (C27), 23.2 (C24), 19.9 (C2),
19.0 (Ala-CH3), 18.8 (C26), 18.4 (C6), 16.1 (C25) ppm; MS (ESI): m/z
(%) ¼ 669.1 ([M þ H]þ, 5), 686.2 ([M þ NH4]þ, 5), 691.3 ([M þ Na]þ,
100), 1359.1 ([2M þ Na]þ, 24); analysis for C39H60N2O7 (668.9): C,
70.03; H, 9.04; N, 4.19; found: C, 69.85; H, 9.25; N, 3.98.
([M þ MeOH þ Na]þ, 100), 1101.3 ([2M þ Na]þ, 40); analysis for
C34H53NO4 (539.8): C, 75.65; H, 9.90; N, 2.59; found: C, 75.47; H,
10.02; N, 2.37.
5.20. Methyl 3-[(octyloxy)imino]-11-oxo-olean-12-en-30-oate (19)
To a solution of 17 (300 mg, 0.60 mmol) in dry THF (20 ml),
potassium hydroxide (0.1 g,1.8 mmol) and n-bromooctane (230 mg,
1.20 mmol) was added. After refluxing for 24 h, the mixture was
cooled and filtered. The solvent was removed and the residue was
subjected to chromatography (silica gel, chloroform/ether 9:1) to
yield 19 (106 mg, 29%) as a colorless powder; mp 92e96 ꢁC;
5.22. Methyl (3b)-3-({[(2L)-(tert-butoxycarbonyl)amino][(tert-but-
oxycarbonyl)thio] acetyl}oxy)-11-oxoolean-12-en-30-oate (21)
Rf ¼ 0.81 (hexane/ethyl acetate 95:5); [
UVevis (methanol): lmax (log
(KBr):
1363m, 1321w, 1280w, 1262w, 1217w, 1189w, 1155m, 1088m, 1050m,
1028w cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 5.64 (s, 1H, CH (12)),
a
]
¼ þ81.6ꢁ (c 0.44, CHCl3);
D
3
) ¼ 200 (4.04), 249 (4.03) nm; IR
Following the procedure for 20, 21 (740 mg, 93%) was obtained
from 4 (490 mg, 1.01 mmol), Boc-L-Cys(SBoc)eOH (380 mg,
n
¼ 3440br, 2927m, 2858s, 1731s, 1655s, 1464s, 1386m,
1.18 mmol), DCC (380 mg, 1.84 mmol) and DMAP (30 mg,
0.25 mmol) after purification by chromatography (silica gel, chlo-
roform/ether 9:1) as a colorless powder; mp 130e133 ꢁC; Rf ¼ 0.70
d
3.95 (t, 2H, chain-1, J ¼ 6.7 Hz), 3.65 (s, 3H, CH3), 2.91 (ddd, 1H, H-2,
J ¼ 15.7, 5.2, 3.8 Hz), 2.76 (ddd, 1H, H-1, J ¼ 13.3, 5.8, 3.8 Hz), 2.34 (s,
1H, H-9), 2.21 (ddd, 1H, H-20, J ¼ 15.7, 12.3, 5.8 Hz), 2.08 (dd, 1H, H-
18, J ¼ 13.5, 3.5 Hz), 1.98 (m, 1H, H-15), 1.94 (m, 1H, H-21), 1.87 (ddd,
1H, H-19, J ¼ 13.6, 4.0, 2.6 Hz), 1.82 (m, 2H, H-16 and H-7), 1.58 (dd,
1H, H-190, J ¼ 13.5, 13.5 Hz), 1.63 (m, 2H, chain-4), 1.57 (m, 2H,
chain-2), 1.41 (m, 1H, H-70), 1.36 (m, 1H, H-22), 1.31 (s, 3H, H-24),
1.30e1.28 (m, 9H, H-220, H-210, chain-6, chain-5, chain-3, H-6), 1.19
(s, 3H, H-25), 1.13 (s, 3H, H-23), 1.11 (s, 3H, H-29), 1.10 (s, 3H, H-26),
1.02 (s, 3H, H-27), 1.02e0.97 (m, 3H, H-10, H-160, H-150), 0.84 (m, 3H,
chain-8), 0.77 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
(hexane/ethyl acetate 7:3); [
a
]
¼ þ78.66ꢁ (c 0.41, CHCl3); UVevis
D
(methanol): lmax (log
3
) ¼ 250 nm (4.00); IR (KBr):
n
¼ 3439br,
2978s, 2875m, 1727s, 1661s, 1500m, 1457m, 1392m, 1369s, 1249m,
1216s, 1168s, 1128s, 1087m, 1050m, 1021m, 986m cmꢀ1 1H NMR
(500 MHz, CDCl3):
¼ 5.67 (s, 1H, H-12), 5.30 (d, 1H, NH, J ¼ 7.6 Hz),
;
d
4.58 (dd, 1H, H-3, J ¼ 11.8, 4.8 Hz), 4.54 (m, 1H, Cys-CH), 3.69 (s, 3H,
OCH3), 3.40 (dd, 1H, Cys-CHH0, J ¼ 14.0, 4.0 Hz), 3.19 (dd, 1H, Cys-
CHH0, J ¼ 14.0, 6.6 Hz), 2.82 (ddd, 1H, H-1, J ¼ 13.7, 3.2, 3.2 Hz), 2.35
(s, 1H, H-9), 2.08 (dd, 1H, H-18, J ¼ 13.0, 3.4 Hz), 2.03 (ddd, 1H, H-15,
J ¼ 13.4,13.4, 4.3 Hz),1.99 (m,1H, H-21),1.92 (ddd,1H, H-19, J ¼ 13.6,
3.5, 2.9 Hz), 1.82 (ddd, 1H, H-16, J ¼ 13.7, 13.7, 4.5 Hz), 1.74 (m, 1H, H-
2), 1.66 (m, 1H, H-7), 1.62 (m, 1H, H-20), 1.61 (dd, 1H, H-190, J ¼ 13.5,
13.5 Hz), 1.59 (m, 1H, H-6), 1.46 (s, 9H, S-Boc-CH3), 1.45 (m, 1H, H-6),
1.44 (s, 9H, O-Boc-CH3), 1.43 (m, 1H, H-70), 1.39 (m,1H, H-22),1.36 (s,
3H, H-27),1.31 (m,1H, H-220), 1.31 (m,1H, H-210),1.18 (m,1H, H-160),
1.16 (s, 3H, H-25),1.15 (s, 3H, H-29),1.13 (s, 3H, H-26),1.04 (m,1H, H-
10), 1.01 (m, 1H, H-150), 0.89 (s, 3H, H-23), 0.89 (s, 3H, H-24), 0.80 (s,
3H, H-28), 0.79 (m, 1H, H-5) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 199.7 (C11), 176.8 (C30), 169.1 (C13), 165.1 (C3), 128.5 (C12), 73.3
(chain-1), 61.3 (C9), 55.5 (C5), 51.7 (OCH3), 48.3 (C18), 45.2 (C8),
44.0 (C20), 43.2 (C14), 41.1 (C19), 40.1 (C4), 39.0 (C1), 37.7 (C22),
36.9 (C10), 32.8 (C7), 32.4 (C17), 31.8 (C21), 31.1 (chain-6), 29.4
(chain-2), 29.2 (chain-5), 29.1 (chain-4), 28.5 (C28), 28.2 (C29), 28.1
(chain-3), 27.4 (C23), 26.4 (C15), 26.3 (C16), 23.4 (C27), 23.3 (C24),
22.6 (C2),18.6 (C26),18.2 (chain-7),17.8 (C6),15.6 (C25),14.1 (chain-
8) ppm; MS (ESI): m/z (%) ¼ 610.5 ([M þ H]þ, 88), 632.5 ([M þ Na]þ,
100), 1219.2 ([2M þ H]þ, 20), 1241.4 ([2M þ Na]þ, 44); analysis for
C39H63NO4 (609.9): C, 76.80; H, 10.41; N, 2.30; found: C, 76.54; H,
10.69; N, 2.11.
d
¼ 199.9 (C-11), 176.9 (C-30), 170.2 (Cys-COO), 169.2 (C-13), 168.4
(SeBoceCOO), 155.0 (OeBoceCOO), 128.5 (C-12), 85.4 (S-Boc-
quart.eC), 82.5 (C-3), 79.8 (Boc-quart.eC), 61.7 (C-9), 55.0 (C-5),

5366
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
53.9 (Cys-CH), 51.8 (OCH3), 48.4 (C-18), 45.4 (C-8), 44.0 (C-20), 43.2
(C-14), 41.1 (C-19), 38.7 (C-1), 38.2 (C-4), 37.7 (C-22), 36.9 (C-10),
33.0 (Cys-CH2), 32.7 (C-7), 31.8 (C-17), 31.2 (C-21), 28.5 (C-28), 28.3
(3ꢂ O-Boc-CH3), 28.3 (C-29), 28.1 (3ꢂ SeBoceCH3), 28.1 (C-23),
26.5 (C-16), 26.4 (C-15), 23.6 (C-2), 23.4 (C-27), 18.7 (C-26), 17.4 (C-
6), 16.8 (C-24), 16.4 (C-25) ppm; MS (ESI): m/z (%) ¼ 788.2
([M þ H]þ, 10), 810.3 ([M þ Na]þ, 100), 1204.5 ([3Mþ2Na]2þ, 6),
1597.2 ([2M þ H]þ, 12); analysis for C44H69NO9S (788.1): C, 67.06; H,
8.82; N, 1.78; S, 4.07; found: C, 66.88; H, 9.01; N, 1.64; S, 3.86.
(m, 1H, H-7), 1.66 (m, 1H, H-6), 1.64 (m, 1H, H-20), 1.61 (dd, 1H, H-190,
J ¼ 13.6,13.6 Hz),1.58 (m,1H, H-60),1.46 (s, 9H, Boc-CH3),1.43 (m,1H,
H-70), 1.38 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31 (m, 1H, H-220), 1.31
(m,1H, H-210),1.18 (m,1H, H-160),1.16 (s, 3H, H-25),1.15 (s, 3H, H-29),
1.13 (s, 3H, H-26),1.08 (m,1H, H-150),1.02 (m,1H, H-10), 0.87 (s, 3H, H-
23), 0.85 (s, 3H, H-24), 0.81 (s, 3H, H-28), 0.79 (m, 1H, H-5) ppm; 13C
NMR (125 MHz, CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30), 170.8 (Cys-
COO), 169.3 (C-13), 157.6 (Boc-COO), 137.7 (Car), 130.1 (Car), 128.9
(Car), 128.6 (Car), 128.5 (C-12), 128.4 (Car), 127.1 (Car), 82.4 (C-3), 79.7
(Boc-quart.eC), 61.6 (C-9), 55.0 (C-5), 53.5 (Cys-CH), 51.7 (OCH3),
48.4 (C-18), 45.4 (C-8), 44.0 (C-20), 43.2 (C-14), 41.1 (C-19), 38.7 (C-
1), 38.1 (C-4), 37.7 (C-22), 36.9 (C-10), 36.8 (Bn-CH2), 33.7 (Cys-CH2),
32.6 (C-7), 31.8 (C-17), 31.1 (C-21), 28.5 (C-28), 28.3 (3 x Boc-CH3),
28.3 (C-29), 28.1 (C-23), 26.4 (C-16), 26.4 (C-15), 23.5 (C-2), 23.3 (C-
27),18.7 (C-26),17.3 (C-6),16.7 (C-24),16.3 (C-25) ppm; MS (ESI): m/z
(%) ¼ 778.1 ([M þ H]þ,10), 795.3 ([M þ NH4]þ,11), 800.4 ([M þ Na]þ,
100), 816.3 ([M þ K]þ, 20), 832.7 ([M þ Na þ MeOH]þ, 13), 1189.7
([3Mþ2Na]2þ, 12), 1578.3 ([2M þ H þ Na]þ, 20); analysis for
C46H67NO7S (778.1): C, 71.01; H, 8.68; N,1.80; S, 4.12; found: C, 70.88;
H, 8.81; N, 1.55; S, 4.00.
5.23. Dimethyl (3b b
,30 )-3,30-(dithiobis{[(2S)-2-amino-1-oxoetha-
ne-2,1-diyl]oxy}) bis(11-oxoolean-12-en-30-oate) (22)
To a solution of 21 (640 mg, 0.81 mmol) in dry DCM (15 ml),
trifluoroacetic acid (4 ml, 51.92 mmol) was added, and the mixture
was stirred at room temperature for 1 day. A saturated solution of
NaHCO3 (10 ml) was slowly added, and the mixture was extracted
with DCM (3 ꢂ 10 ml). The organic layers were washed with water
(20 ml) and brine (20 ml), dried (Na2SO4) and filtered. The solvent
was removed and 22 (410 mg, 43%) was obtained as a slightly
yellowish powder; mp 220e224 ꢁC; Rf ¼ 0.95 (chloroform/meth-
anol 9:1); [
(log
a
]
¼ þ103.37ꢁ (c 0.49, CHCl3); UVevis (methanol): lmax
5.25. Methyl (3b)-3-{[(2S)-2-amino-2-(benzylthio)acetyl]oxy}-11-
oxoolean-12-en-30-oate (24)
D
3
) ¼ 249 nm (4.34); IR (KBr):
n
¼ 3432br, 2950s, 1732s, 1660s,
1465m, 1388m, 1324w, 1216s, 1087w cmꢀ1
;
1H NMR (500 MHz,
¼ 5.67 (s,1H, H-12), 4.59 (dd,1H, H-3, J ¼ 11.7, 4.6 Hz), 3.87
CDCl3):
d
Following the procedure given for 22, from 23 (190 mg,
0.24 mmol) and trifluoroacetic acid (1 ml, 12.98 mmol) 24 (140 mg,
86%) was obtained as a colorless powder; mp 128e131 ꢁC; Rf ¼ 0.66
(m, 1H, Cys-CH), 3.68 (s, 3H, OCH3), 3.22 (m, 1H, Cys-CHH0), 2.97 (m,
1H, Cys-CHH0), 2.82 (ddd, 1H, H-1, J ¼ 13.7, 3.5, 3.5 Hz), 2.36 (s, 1H,
H-9), 2.22 (m, 2H, NH2), 2.08 (dd, 1H, H-18, J ¼ 13.3, 3.5 Hz), 2.02
(ddd, 1H, H-15, J ¼ 13.6, 13.6, 4.2 Hz), 1.99 (m, 1H, H-21), 1.92 (ddd,
1H, H-19, J ¼ 13.7, 3.6, 2.5 Hz), 1.82 (ddd, 1H, H-16, J ¼ 13.5, 13.5,
4.4 Hz), 1.73 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.65 (m, 1H, H-20), 1.61
(dd, 1H, H-190, J ¼ 13.6, 13.6 Hz), 1.57 (m, 1H, H-6), 1.45 (m, 1H, H-60),
1.40 (m, 1H, H-70), 1.38 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31 (m, 1H,
H-220), 1.31 (m, 1H, H-210), 1.18 (m, 1H, H-160), 1.16 (s, 3H, H-25), 1.15
(s, 3H, H-29), 1.13 (s, 3H, H-26), 1.05 (m, 1H, H-10), 1.01 (m, 1H, H-
150), 0.90 (s, 3H, H-23), 0.90 (s, 3H, H-24), 0.81 (m, 1H, H-5), 0.80 (s,
(chloroform/methanol 9:1); [
(methanol): lmax (log
2929s, 1732s, 1660s, 1570w, 1454m, 1387m, 1324w, 1217s, 1155m,
1087w, 1028w cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 7.36e7.20 (m,
a
]
¼ þ62.38ꢁ (c 0.43, CHCl3); UVevis
D
3
) ¼ 249 nm (4.31); IR (KBr):
n
¼ 3406br,
d
5H, HeAr), 5.66 (s,1H, H-12), 4.58 (dd, 1H, H-3, J ¼ 11.8, 4.7 Hz), 3.93
(m, 1H, Cys-CH), 3.82 (s, 2H, Bn-CH2), 3.69 (s, 3H, OCH3), 3.02 (dd,
1H, Cys-CHH0, J ¼ 13.9, 4.4 Hz), 2.91 (dd, 1H, Cys-CHH0, J ¼ 13.9,
7.1 Hz), 2.81 (m, 1H, H-1), 2.34 (s, 1H, H-9), 2.09 (dd, 1H, H-18,
J ¼ 13.5, 3.7 Hz), 2.03 (m, 1H, H-15), 1.99 (m, 1H, H-21), 1.93 (m, 1H,
H-19), 1.82 (ddd, 1H, H-16, J ¼ 13.5, 13.5, 3.9 Hz), 1.71 (m, 1H, H-2),
1.68 (m, 1H, H-7), 1.66 (m, 1H, H-6), 1.63 (m, 1H, H-20), 1.61 (dd, 1H,
H-190, J ¼ 13.5, 13.5 Hz), 1.56 (m, 1H, H-60), 1.42 (m, 1H, H-70), 1.38
(m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31 (m, 1H, H-220), 1.31 (m, 1H, H-
210), 1.18 (m, 1H, H-160), 1.15 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.12 (s,
3H, H-26), 1.09 (m, 1H, H-150), 1.02 (m, 1H, H-10), 0.85 (s, 3H, H-23),
0.83 (s, 3H, H-24), 0.81 (s, 3H, H-28), 0.77 (m, 1H, H-5) ppm; 13C
3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 199.9 (C-11), 176.9
(C-30), 172.9 (Cys-COO), 169.3 (C-13), 128.5 (C-12), 82.2 (C-3), 61.7
(C-9), 55.0 (C-5), 54.0 (Cys-CH), 51.8 (OCH3), 48.4 (C-18), 45.4 (C-8),
44.0 (C-20), 44.0 (Cys-CH2), 43.2 (C-14), 41.1 (C-19), 38.7 (C-1), 38.1
(C-4), 37.7 (C-22), 36.9 (C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21),
28.5 (C-28), 28.3 (C-29), 28.3 (C-23), 26.5 (C-16), 26.4 (C-15), 23.6
(C-2), 23.4 (C-27), 18.7 (C-26), 17.4 (C-6), 16.9 (C-24), 16.4 (C-25)
ppm; MS (ESI): m/z (%)
¼
587.4 ([Mþ2H]2þ
,
100), 1173.3
NMR (125 MHz, CDCl3):
d
¼ 199.8 (C-11), 176.9 (C-30), 169.2 (Cys-
([2Mþ2H]2þ, 38); analysis for C68H104N2O10S2 (1173.1): C, 69.59; H,
COO), 169.2 (C-13), 137.6 (Car), 129.0 (Car), 129.0 (Car), 128.6 (Car),
128.5 (C-12),128.5 (Car), 127.2 (Car), 83.1 (C-3), 61.6 (C-9), 55.0 (C-5),
51.7 (OCH3), 49.2 (Cys-CH), 48.4 (C-18), 45.4 (C-8), 44.0 (C-20), 43.2
(C-14), 41.1 (C-19), 38.6 (C-1), 38.1 (C-4), 37.7 (C-22), 36.9 (C-10),
36.7 (Bn-CH2), 33.9 (Cys-CH2), 32.6 (C-7), 31.8 (C-17), 31.1 (C-21),
28.5 (C-28), 28.3 (C-29), 28.2 (C-23), 26.4 (C-16), 26.4 (C-15), 23.5
(C-2), 23.4 (C-27), 18.7 (C-26), 17.3 (C-6), 16.8 (C-24), 16.3 (C-25)
8.93; N, 2.39; S, 5.46; found: C, 69.37; H, 9.04; N, 2.21; S, 5.41.
5.24. Methyl (3b)-3-({(benzylthio)[(tert-butoxycarbonyl)-(2L)-
amino]acetyl}oxy)-11-oxoolean-12-en-30-oate (23)
Following the procedure given for 20, from 4 (730 mg,
1.51 mmol), Boc-
L-Cys(SBn)eOH (720 mg, 2.32 mmol), DCC (390 mg,
ppm; MS (ESI): m/z (%) ¼ 678.3 ([M þ H]þ, 100),1017.2 ([3Mþ2H]2þ
,
1.89 mmol) and DMAP (30 mg, 0.25 mmol), followed by chroma-
tography (silica gel, hexane/ethyl acetate 95:5) 23 (670 mg, 57%) was
obtained as a colorless powder; mp 112e115 ꢁC; Rf ¼ 0.64 (hexane/
6), 1355.6 ([2M þ H]þ, 2); analysis for C41H59NO5S (678.0): C, 72.63;
H, 8.77; N, 2.07; S, 4.73; found: C, 72.46; H, 8.88; N, 1.97; S, 4.62.
ethyl acetate 7:3); [
anol): lmax (log
a
]
¼ þ57.94ꢁ (c 0.43, CHCl3); UVevis (meth-
5.26. Ethyl 3,11-dioxo-olean-12-en-30-oate (25)
Compound 25 was prepared according to [9,22].
D
3
) ¼ 248 nm (4.01); IR (KBr):
n
¼ 3329br, 2930s,
2852m, 1719s, 1654s, 1627s, 1576m, 1508m, 1455m, 1390m, 1367m,
1341m,1246m,1216s,1167s,1088w,1063wcmꢀ1; 1H NMR (500 MHz,
CDCl3):
d
¼ 7.32e7.20 (m, 5H, HeAr), 5.67 (s, 1H, H-12), 5.29 (d, 1H,
5.27. Ethyl (7aR,7bS,9aS,12S,13aR,15bS)-5,5,7a,7b,9a,12,15b-hepta-
methyl-3,15-dioxo9a,10,11,12,13,13a,15,15a,15b-icosahydrochryseno
[2,1-c]oxepine-12-carboxylate (26)
NH, J¼ 7.9 Hz), 4.57(dd,1H, H-3, J ¼11.7, 4.8Hz), 4.51 (m,1H, Cys-CH),
3.75 (s, 2H, Bn-CH2), 3.69 (s, 3H, OCH3), 2.88 (m, 1H, Cys-CHH0), 2.84
(m,1H, Cys-CHH0), 2.82 (m,1H, H-1), 2.35 (s,1H, H-9), 2.09 (dd,1H, H-
18, J ¼ 13.5, 3.5 Hz), 2.03 (m,1H, H-15),1.99 (m,1H, H-21),1.93 (m,1H,
H-19),1.83 (ddd,1H, H-16, J ¼ 13.7,13.7, 3.7 Hz),1.74 (m,1H, H-2),1.72
To a solution of 25 (2.56 g, 5.20 mmol) in chloroform (25 ml), 4-
chloroperbenzoic acid (2.63 g, 15.40 mmol) was added. The mixture

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5367
was stirred at 55 ꢁC for 24 h, followed by the addition of saturated
solution of NaHCO3 (30 ml). The aqueous layer was extracted with
DCM (3 ꢂ 30 ml), the combined extracts were dried (Na2SO4),
filtered and evaporated. The residue was purified by chromatog-
raphy (silica gel, chloroform/ether 9:1) to yield 26 (2.15 g, 81%) as
a colorless powder; mp 181e185 ꢁC (lit. 166e171 ꢁC [30]); Rf ¼ 0.42
([M þ H]þ, 100), 553.5 ([M þ Na]þ, 73); analysis for C32H50O6
(530.7): C, 72.42; H, 9.50; found: C, 72.35; H, 9.71.
5.29. 3-[(1S,4aR,4bS,6aS,9S,10aR)-9-(Ethoxycarbonyl)-2-isopropen
yl-1,4a,4b,6a,9-pentamethyl-12-oxo-1,2,3,4,4a,4b,5,6,6a,7,8,9,10,
10a,12,12a-hexadecahydrochrysen-1-yl]propanoic acid (28)
(hexane/ethyl acetate 7:3); [
a
]
¼ þ159.73ꢁ (c 0.23, CHCl3) (lit.
D
[
a
]
¼ þ189ꢁ (c 0.1, CHCl3) [30]); UVevis (methanol): lmax
The pH of a solution of 27 (357 mg, 0.67 mmol) in ethanol
(20 ml) was adjusted to 2 by adding diluted hydrochloric. The
mixture was stirred at room temperature for 5 min, followed by
extraction with DCM (3 ꢂ 15 ml); the organic layer was dried
(Na2SO4), filtered and evaporated to afford 28 (331 mg, 96%) as
a colorless powder; mp 256e260 ꢁC (decomp.); Rf ¼ 0.38 (hexane/
D
(log
3
) ¼ 250 nm (4.07); IR (KBr): ¼ 3432br, 2964s, 2934s, 2866m,
n
1728s, 1651s, 1618w, 1460m, 1386m, 1329m, 1314m, 1281m, 1251m,
1219m, 1172s, 1152s, 1115s, 1087m, 1017m, 980m cmꢀ1 1H NMR
;
(500 MHz, CDCl3):
d
¼ 5.66 (s, 1H, H-12), 4.17 (dq, 1H, Et-CHH0,
J ¼ 10.8, 7.1 Hz), 4.10 (dq, 1H, Et-CHH0, J ¼ 10.8, 7.2 Hz), 2.69 (m, 1H,
H-1), 2.59 (m, 1H, H-2), 2.53 (s, 1H, H-9), 2.11 (dd, 1H, H-18, J ¼ 13.3,
3.3 Hz), 2.01 (ddd, 1H, H-15, J ¼ 13.7, 13.7, 4.6 Hz), 1.97 (m, 1H, H-21),
1.90 (ddd,1H, H-19, J ¼ 13.7, 4.2, 2.5 Hz),1.81 (ddd,1H, H-16, J ¼ 13.7,
13.7, 4.6 Hz), 1.69 (m, 1H, H-7), 1.63 (m, 1H, H-6), 1.58 (dd, 1H, H-190,
J ¼ 13.7, 13.7 Hz), 1.57 (m, 1H, H-5), 1.53 (m, 1H, H-60), 1.51 (m, 1H, H-
10), 1.46 (s, 3H, H-23), 1.44 (m, 1H, H-20), 1.43 (s, 3H, H-24), 1.41 (m,
1H, H-70), 1.38 (m, 1H, H-22), 1.36 (s, 6H, H-25 and H-27), 1.32 (m,
1H, H-220),1.29 (m,1H, H-210),1.24 (t, 3H, Et-CH3, J ¼ 7.1 Hz),1.18 (m,
1H, H-160), 1.14 (s, 3H, H-26), 1.12 (s, 3H, H-29), 1.01 (m, 1H, H-150),
ethyl acetate 7:3); [
anol): lmax (log
a
]
¼ þ99.91ꢁ (c 0.41, CHCl3); UVevis (meth-
D
3
) ¼ 249 nm (3.99); IR (KBr):
n
¼ 3432br, 2976s,
1727s, 1657s, 1561s, 1455m, 1386s, 1314m, 1218m, 1175w, 1087w,
1032w cmꢀ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.60 (s, 1H, H-12), 4.84
(s, 1H, H-23), 4.68 (s, 1H, H-230), 4.18 (dq, 1H, Et-CHH0, J ¼ 10.8,
7.1 Hz), 4.11 (dq, 1H, Et-CHH0, J ¼ 10.8, 7.1 Hz), 2.72 (s, 1H, H-9), 2.44
(m, 1H, H-1), 2.15 (m, 1H, H-2), 2.10 (m, 1H, H-18), 2.01 (ddd, 1H, H-
15, J ¼ 13.7, 13.7, 3.7 Hz), 1.97 (m, 1H, H-2), 1.94 (m, 1H, H-21), 1.92
(m, 1H, H-5), 1.88 (m, 1H, H-19), 1.79 (m, 1H, H-6), 1.76 (m, 1H, H-16),
1.73 (s, 3H, H-24), 1.67 (m, 1H, H-10), 1.62 (m, 1H, H-7), 1.58 (dd, 1H,
H-190, J ¼ 13.7, 13.7 Hz), 1.39 (m, 1H, H-60), 1.37 (m, 1H, H-22), 1.34
(m, 1H, 70), 1.29 (m, 2H, H-220 and H-210), 1.37 (s, 3H, H-27), 1.25 (t,
3H, Et-CH3, J ¼ 7.1 Hz), 1.18 (m, 1H, H-160), 1.13 (s, 3H, H-26), 1.12 (s,
3H, H-25), 1.08 (s, 3H, H-29), 0.98 (m, 1H, H-150), 0.78 (s, 3H, H-28)
0.79 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 198.8 (C11),
176.3 (C30), 175.5 (C3), 169.4 (C13), 128.6 (C12), 85.6 (C4), 61.3 (C9),
60.3 (Et-CH2), 54.5 (C5), 48.3 (C18), 45.3 (C8), 43.8 (C20), 43.4 (C14),
41.1 (C19), 39.6 (C10), 38.7 (C1), 37.7 (C22), 32.3 (C2), 32.2 (C23),
32.0 (C7), 31.8 (C17), 31.1 (C22), 28.6 (C28), 28.3 (C29), 26.4 (C16),
26.4 (C15), 25.9 (C24), 23.1 (C27), 22.1 (C6), 18.2 (C26), 17.5 (C25),
14.3 (Et-CH3) ppm; MS (ESI): m/z (%) ¼ 513.4 ([M þ H]þ, 100), 535.7
([M þ Na]þ, 7), 567.0 ([M þ MeOH þ Na]þ, 46).
ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.8 (C11), 181.5 (C3), 176.5
(C30), 169.8 (C13), 147.0 (C4), 128.6 (C12), 113.9 (C23), 60.4 (Et-CH2),
52.8 (C9), 50.6 (C5), 48.2 (C18), 45.1 (C8), 43.9 (C20), 43.7 (C14), 41.0
(C19), 39.0 (C10), 37.8 (C22), 36.2 (C1), 31.8 (C17), 31.8 (C2), 31.4
(C7), 31.2 (C21), 28.6 (C28), 28.3 (C29), 26.6 (C16), 26.5 (C15), 24.0
(C6), 23.8 (C24), 23.3 (C27), 19.9 (C25), 18.8 (C26), 14.4 (Et-CH3)
ppm; MS (ESI): m/z (%) ¼ 511.5 ([M ꢀ H]ꢀ, 100), 557.2 ([M þ HCO2]ꢀ,
40); analysis for C32H48O5 (512.7): C, 74.96; H, 9.44; found: C, 74.77;
H, 9.51.
5.28. 3-[(1S,4aR,4bS,6aS,9S,10aR)-9-(Ethoxycarbonyl)-2-(1-hydro-
xy-1-methylethyl)-1,4a,4b,6a,9-pentamethyl-12-oxo-1,2,3,4,4a,4b,5,
6,6a,7,8,9,10,10a,12,12a-hexadecahydrochrysen-1-yl]propanoic acid
(27)
Compound 26 (2.15 g, 4.19 mmol) was dissolved in ethanol
(20 ml), potassium hydroxide (1.14 g, 20.32 mmol in 5 ml water)
was added. The mixture was stirred at room temperature for 24 h,
the pH was adjusted to 7 by adding diluted hydrochloric acid, and
the solvents were removed under diminished pressure. Water
(20 ml) and ethyl acetate (20 ml) were added, the aqueous layer
was extracted with ethyl acetate (3 ꢂ 20 ml), the combined organic
layers were dried (Na2SO4), filtered and evaporated to yield 27
(453 mg, 20%) as colorless crystals; mp 115e119 ꢁC; Rf ¼ 0.16
5.30. Ethyl (3S,4aR,7S,10aR,10bS,12aS)-7-(3-ethoxy-3-oxopropyl)-
8-isopropenyl-3,7,10a,10b,12a-pentamethyl-6-oxo-1,2,3,4,4a,6,6a,7,
8,9,10,10a,10b,11,12,12a-hexadecahydrochrysene-3-carboxylate (29)
Compound 27 (543 mg, 1.02 mmol) was dissolved in ethanol
(50 ml), hydrochloric acid (conc., 2 ml) was added. After refluxing
for 30 min, the solvent was removed under reduced pressure, and
the residue was subjected to chromatography (silica gel, hexane/
ethyl acetate 8:2) to afford 29 (526 mg, 95%) as a colorless powder;
(hexane/ethyl acetate 7:3); [
(methanol): lmax (log
a
]
¼ þ99.01ꢁ (c 0.79, CHCl3); UVevis
D
3
) ¼ 249 nm (4.01); IR (KBr):
n
¼ 3432br,
mp 101e104 ꢁC; Rf ¼ 0.79 (hexane/ethyl acetate 7:3); [
a]
¼
D
2976s, 1727s, 1660s, 1560w, 1457m, 1385s, 1313m, 1247w, 1216s,
1175s, 1086m, 1030w cmꢀ1; 1H NMR (500 MHz, DMSO-d6):
þ125.70ꢁ (c 0.44, CHCl3); UVevis (methanol): lmax (log
) ¼ 250 nm
3
d
¼ 5.36
(4.14); IR (KBr):
n
¼ 3424br, 2977s, 2942s, 1724s, 1645s, 1611m,
(s, 1H, H-12), 4.11 (dq, 1H, Et-CHH0, J ¼ 10.8, 7.1 Hz), 4.03 (m, 1H, Et-
CHH0), 2.69 (s, 1H, H-9), 2.32 (m, 2H, H-2), 2.22 (m, 1H, H-1), 2.04
(ddd, 1H, H-15, J ¼ 13.7, 13.7, 4.2 Hz), 1.95 (dd, 1H, H-18, J ¼ 12.0,
5.4 Hz), 1.87 (m, 1H, H-10), 1.79 (m, 1H, H-21), 1.72 (m, 1H, H-19), 1.70
(m, 1H, H-190), 1.67 (m, 1H, H- 16), 1.55 (m, 1H, H-7), 1.37 (m, 1H, H-
210),1.35 (m, 2H, H-6 and H-60),1.33 (s, 3H, H-27),1.31 (m,1H, H-22),
1.72 (m, 1H, H-70), 1.26 (s, 3H, H-25), 1.25 (m, 1H, H-5), 1.20 (m, 1H,
H-220), 1.16 (t, 3H, Et-CH3, J ¼ 7.1 Hz), 1.13 (m, 1H, H-160), 1.12 (s, 3H,
H-23), 1.08 (s, 3H, H-29), 1.06 (s, 3H, H-24), 1.01 (s, 3H, H-26), 0.92
(m, 1H, H-150), 0.71 (s, 3H, H-28) ppm; 13C NMR (125 MHz, DMSO-
1460m, 1386m, 1364w, 1330m, 1310w, 1278m, 1260m, 1213m, 1171s,
1115w, 1089w, 1064w, 1024w cmꢀ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 5.64 (s, 1H, H-12), 4.87 (dd, 1H, H-23, J ¼ 1.3, 1.3 Hz), 4.67 (d, 1H,
H-230, J ¼ 1.2 Hz), 4.17 (dq, 1H, C30-CHH0, J ¼ 10.8, 7.1 Hz), 4.10 (dq,
1H, C30-CHH0, J ¼ 10.8, 7.1 Hz), 4.05 (q, 2H, C3eCH2, J ¼ 7.1 Hz), 2.59
(ddd, 1H, H-1, J ¼ 14.1, 12.0, 6.2 Hz), 2.58 (s, 1H, H-9), 2.27 (ddd, 1H,
H-2, J ¼ 14.1, 12.0, 4.2 Hz), 2.09 (m, 1H, H-18), 2.03 (m,1H, H-20), 1.99
(m, 1H, H-15), 1.97 (m, 1H, H-21), 1.94 (dd, 1H, H-5, J ¼ 12.0, 2.5 Hz),
1.91 (m, 1H, H-19), 1.83 (m, 1H, H-6), 1.80 (m, 1H, H-16), 1.74 (s, 3H,
H-24), 1.72 (m, 1H, H-10), 1.68 (m, 1H, H-7), 1.59 (dd, 1H, H-190,
J ¼ 13.3, 13.3 Hz), 1.42 (m, 1H, H-60), 1.37 (m, 1H, H-22), 1.35 (m, 1H,
H-70), 1.32 (m, 1H, H-220), 1.28 (m, 1H, H-210), 1.37 (s, 3H, H-27), 1.25
(t, 3H, C30eCH3, J ¼ 7.1 Hz), 1.21 (m, 1H, H-160), 1.20 (t, 3H, C3eCH3,
J ¼ 7.1 Hz), 1.15 (s, 3H, H-26), 1.15 (s, 3H, H-25), 1.12 (s, 3H, H-29),
1.00 (m, 1H, H-150), 0.79 (s, 3H, H-28) ppm; 13C NMR (125 MHz,
d6):
d
¼ 198.8 (C11), 176.3 (C30), 175.6 (C3), 168.1 (C13), 127.8 (C12),
73.6 (C4), 59.8 (Et-CH2), 52.5 (C9), 49.9 (C5), 48.0 (C18), 44.8 (C8),
43.4 (C20), 43.3 (C14), 40.8 (C10), 40.4 (C19), 37.3 (C22), 34.8 (C2),
33.0 (C23), 31.6 (C17), 31.6 (C7), 31.3 (C1), 30.3 (C21), 28.3 (C28),
27.8 (C29), 27.6 (C24), 26.0 (C16), 25.7 (C15), 22.5 (C27), 21.2 (C6),
19.3 (C25), 18.1 (C26), 14.1 (Et-CH3) ppm; MS (ESI): m/z (%) ¼ 531.3
CDCl3):
d
¼ 199.5 (C11), 176.3 (C30), 173.8 (C3), 169.4 (C13), 146.6

5368
R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
(C4), 128.4 (C12), 114.1 (C23), 60.3 (C30eCH2), 60.2 (C3eCH2), 52.8
(C9), 50.8 (C5), 48.3 (C18), 45.1 (C8), 43.8 (C20), 43.7 (C14), 41.2
(C19), 38.8 (C10), 37.7 (C22), 34.4 (C1), 31.8 (C17), 31.4 (C7), 31.1
(C21), 29.4 (C2), 28.6 (C28), 28.3 (C29), 26.5 (C16), 26.4 (C15), 23.8
(C6), 23.5 (C24), 23.3 (C27), 19.5 (C25), 18.6 (C26), 14.3 (C30eCH3),
14.2 (C3eCH3) ppm; MS (ESI): m/z (%) ¼ 541.5 ([M þ H]þ,100), 558.3
([M þ NH4]þ, 13), 563.6 ([M þ Na]þ, 16); analysis for C34H52O5
(540.8): C, 75.51; H, 9.69; found: C, 75.41; H, 9.82.
(d, 1H, H-3, J ¼ 3.7 Hz), 2.29 (s, 1H, H-9), 2.09 (dd, 1H, H-18,
J ¼ 13.3, 4.2 Hz), 1.99 (ddd, 1H, H-15, J ¼ 13.3, 13.3, 4.6 Hz), 1.96
(m, 1H, H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.7, 4.2, 2.9 Hz), 1.78 (ddd,
1H, H-16, J ¼ 13.7, 13.7, 5.0 Hz), 1.61 (m, 1H, H-7), 1.57 (dd, 1H, H-
190, J ¼ 13.7, 13.7 Hz), 1.48 (m, 1H, H-6), 1.39 (m, 1H, H-21), 1.37
(m, 1H, H-60), 1.35 (m, 1H, H-22), 1.33 (m, 1H, H-10), 1.30 (s, 3H, H-
27), 1.29 (m, 1H, H-220), 1.27 (m, 1H, H-210), 1.24 (t, 1H, J ¼ 7.1 Hz,
Me), 1.17 (m, 1H, H-160), 1.13 (s, 3H, H-26), 1.11 (s, 3H, H-28), 1.09
(s, 3H, H-23), 1.07 (s, 3H, H-25), 1.02 (s, 3H, H-24), 0.93 (m, 1H, H-
150), 0.92 (dd, 1H, H-5, J ¼ 11.6, 2.9 Hz), 0.78 (s, 3H, H-29) ppm;
5.31. Ethyl 11-oxo-olean-2,12-dien-30-oate (30)
13C NMR (125 MHz, CDCl3):
d
¼ 199.7 (C11), 176.3 (C30), 169.7
A mixture of 9 (1.31 g, 2.6 mmol), triphenyl phosphane (2.78 g,
10.6 mmol) and 3,3-dimethyl glutarimide (1.49 g, 10.6 mmol) in dry
THF (25 ml) was cooled to 0 ꢁC. Under continuous stirring, DEAD
(1.65 ml, 10.4 mmol) was added dropwise, and stirring was
continued at 25 ꢁC for 24 h. After concentration to dryness, the
residue was subjected to chromatography (silica gel, chloroform/
ether 9:1) to yield 30 (1.02 g, 82%) as colorless crystals; mp
(C13), 128.5 (C12), 61.3 (C3), 60.4 (C9), 60.3 (Et-CH2), 52.6 (C2),
48.4 (C18), 46.6 (C5), 45.1 (C8), 43.8 (C20), 43.3 (C14), 41.1 (C19),
40.6 (C1), 37.7 (C22), 35.9 (C4), 32.6 (C10), 31.9 (C7), 31.8 (C17),
31.1 (C21), 28.6 (C28), 28.3 (C29), 28.2 (C23), 26.4 (C16), 26.4
(C15), 23.2 (C27), 22.0 (C24), 18.3 (C26), 17.9 (C6), 17.9 (C25), 14.3
(Me); MS (ESI): m/z (%) ¼ 497.6 ([M þ H]þ, 92), 519.4 ([M þ Na]þ,
10), 551.0 ([M þ MeOH þ Na]þ, 62), 767.4 (3Mþ2Na]2þ, 6), 993.3
([2M þ H]þ, 94), 1015.4 ([2M þ Na]þ, 100), 1031.3 ([2M þ K]þ, 12);
analysis for C32H48O4 (496.72): C, 77.38; H, 9.74; found: C, 77.26;
H, 9.92.
138e142 ꢁC; Rf ¼ 0.87 (hexane/ethyl acetate 7:3); [
a
]
¼ 216.97ꢁ (c
D
0.33, CHCl3); UVevis (methanol): lmax (log
3
) ¼ 249 nm (4.02); IR
(KBr):
1360w, 1348w, 1328w, 1310w, 1277w, 1256m, 1210m, 1169s, 1134m,
1088w, 1062w, 1031m cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 5.67 (s,
n
¼ 3422br, 2960s, 2872s,1723s,1648s,1612w,1458m,1386m,
d
5.33. Ethyl (2b,3a) 2-fluoro-3-hydroxy-11-oxo-olean-12-en-30-
1H, 12-H), 5.43 (ddd, 1H, H-2, J ¼ 10.1, 6.1, 1.7 Hz), 5.37 (dd, 1H, H-3,
J ¼ 10.1, 2.3 Hz), 4.19 (dq, 1H, Et-CHH0, J ¼ 10.8, 7.2 Hz), 4.12 (dq, 1H,
Et-CHH0, J ¼ 10.8, 7.2 Hz), 3.04 (dd, 1H, H-1, J ¼ 17.5, 6.0 Hz), 2.41 (s,
1H, H-9), 2.11 (dd, 1H, H-18, J ¼ 12.8, 4.3 Hz), 2.03 (ddd, 1H, H-15,
J ¼ 13.4,13.4, 4.7 Hz),1.99 (m,1H, H-21),1.92 (ddd,1H, H-19, J ¼ 13.9,
4.1, 2.9 Hz), 1.83 (ddd, 1H, H-16, J ¼ 13.6, 13.6, 4.3 Hz), 1.70 (m, 1H, H-
7), 1.65 (m, 1H, H-10), 1.61 (dd, 1H, H-190, J ¼ 13.5, 13.5 Hz), 1.56 (m,
1H, H-6), 1.48 (ddd, 1H, H-60, J ¼ 12.5, 12.5, 3.2 Hz), 1.43 (m, 1H, H-
70),1.39 (m,1H, H-22),1.33 (m,1H, H-210),1.30 (m,1H, H-220),1.36 (s,
3H, H-27), 1.26 (t, 3H, Me, J ¼ 7.2 Hz), 1.21 (ddd, 1H, H-160, J ¼ 13.9,
4.4, 2.4 Hz), 1.16 (s, 3H, H-25), 1.16 (s, 3H, H-26), 1.14 (s, 3H, H-29),
1.12 (m, 1H, H-5), 1.02 (m, 1H, H-150), 0.96 (s, 3H, H-23), 0.91 (s, 3H,
oate (32)
Compound 31 (619 mg, 1.25 mmol) was dissolved in dry DCM
(10 ml) and Olah’s-reagent [26] (1 ml, 4.35 mmol) was slowly
added. The mixture was stirred at room temperature for 5 h, and
crushed ice was added. The aqueous layer was extracted with DCM
(3 ꢂ 20 ml), the extracts were dried (Na2SO4), filtered and evapo-
rated. The residue was subjected to chromatography (silica gel,
chloroform/ether 9:1) to afford 32 (120 mg, 19%) as a colorless
powder; mp 152e155 ꢁC; Rf ¼ 0.48 (hexane/ethyl acetate ¼ 7:3);
[
a
]
¼
þ113.77ꢁ (c 0.64, CHCl3); UVevis (methanol): lmax
D
(log
3
) ¼ 249 nm (3.98); IR (KBr):
n
¼ 3448br, 2959s, 2873s, 1727s,
H-24), 0.82 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.1
1659s, 1456m, 1389s, 1365m, 1314m, 1280m, 1246m, 1218s, 1174s,
1121m, 1089m, 1048m, 1021s cmꢀ1 1H NMR (500 MHz, CDCl3):
¼ 5.67 (s, 1H, H-12), 4.64 (ddd, 1H, H-2, J ¼ 49.7, 6.4, 5.6 Hz), 4.18
(C11), 176.4 (C30), 169.4 (C13), 137.0 (C3), 128.6 (C12), 121.9 (C2),
60.5 (C9), 60.3 (Et-CH2), 51.8 (C5), 48.4 (C18), 45.3 (C14), 43.8 (C20),
43.3 (C8), 41.5 (C1), 41.2 (C19), 37.7 (C22), 36.2 (C4), 34.3 (C10), 31.9
(C7), 31.9 (C23), 31.8 (C17), 31.1 (C21), 28.6 (C28), 28.3 (C29), 26.5
(C16), 26.5 (C15), 23.3 (C27), 23.0 (C24), 18.7 (C6), 18.3 (C26), 16.1
(C25), 14.3 (Me) ppm; MS (ESI): m/z (%) ¼ 481.5 ([M þ H]þ, 100),
503.3 ([M þ Na]þ, 7), 534.9 ([M þ MeOH þ Na]þ, 50), 961.3
([2M þ H]þ, 66), 983.4 ([2M þ Na]þ, 54), 999.2 ([2M þ K]þ, 4);
analysis for C32H48O3 (480.72): C, 79.95; H, 10.06; found: C, 79.68;
H, 10.18.
;
d
(dq, 1H, Et-CHH0, J ¼ 10.8, 7.1 Hz), 4.12 (dq, 1H, Et-CHH0, J ¼ 10.8,
7.1 Hz), 3.73 (dd, 1H, H-3, J ¼ 8.6, 6.4 Hz), 2.91 (ddd, 1H, H-1, J ¼ 15.4,
15.4, 5.5 Hz), 2.43 (s, 1H, H-9), 2.11 (ddd, 1H, H-18, J ¼ 13.5, 4.2,
1.2 Hz), 2.02 (ddd,1H, H-15, J ¼ 13.6,13.6, 4.6 Hz),1.98 (m,1H, H-21),
1.91 (ddd, 1H, H-19, J ¼ 13.6, 4.3, 2.8 Hz), 1.82 (ddd, 1H, H-16,
J ¼ 13.6, 13.6, 4.3 Hz), 1.68 (ddd, 1H, H-10, J ¼ 15.4, 15.4, 5.7 Hz), 1.64
(m, 1H, H-7), 1.59 (dd, 1H, H-190, J ¼ 13.5, 13.5 Hz), 1.53 (m, 1H, H-6),
1.50 (m, 1H, H-60), 1.40 (m, 1H, H-70), 1.38 (m, 1H, H-22), 1.35 (s, 3H,
H-27), 1.33 (m, 2H, H-220 and H-210), 1.26 (t, 3H, Et-CH3, J ¼ 7.1 Hz),
1.20 (m, 1H, H-160),1.15 (m, 1H, H-5),1.13 (s, 3H, H-29),1.13 (s, 3H, H-
25), 1.11 (s, 3H, H-26), 1.04 (s, 3H, H-24), 1.03 (m, 1H, H-150), 0.98 (s,
3H, H-23), 0.80 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
5.32. Ethyl (2a,3a)-2,3-epoxy-11-oxo-olean-12-en-31-oate (31)
Compound 30 (1.01 g, 2.1 mmol) was dissolved in dry
dichloromethane (20 ml), m-CPBA (1.14 g, 4.68 mmol) was added,
and the mixture was stirred at room temperature for 20 h. An aq.
solution of potassium hydrogensulfate (satd., 10 ml) was added,
the aqueous layer extracted with dichloromethane (3 ꢂ 15 ml),
and the combined organic layers were dried (Na2SO4), filtrated
and evaporated to dryness. Chromatographic purification (silica
gel, chloroform/ether 9:1) afforded 31 (806 mg, 77%) as a color-
less powder; mp 191e193 ꢁC; Rf ¼ 0.71 (hexane/ethyl acetate
d
¼ 199.5 (C11), 176.4 (C30), 169.9 (C13), 128.3 (C12), 92.8 (d, C2,
J ¼ 172 Hz), 75.2 (d, C3, J ¼ 21 Hz), 62.5 (C9), 60.3 (Et-CH2), 49.1 (C5),
48.3 (C18), 45.3 (C8), 43.8 (C20), 42.5 (d, C1, J ¼ 18 Hz), 42.2 (C14),
41.2 (C19), 37.7 (C22), 36.9 (C4), 36.8 (C10), 32.1 (C7), 31.8 (C17), 31.1
(C21), 28.6 (C28), 28.3 (C29), 26.6 (C23), 26.4 (C16), 26.4 (C15), 23.5
(C27), 22.1 (C24), 19.5 (C26), 18.4 (C25), 18.0 (C6), 14.3 (Et-CH3)
ppm; 19F NMR (190 MHz, CDCl3):
d
¼ ꢀ180.9 (m, 1F, F-2) ppm; MS
(ESI): m/z (%) ¼ 517.5 ([M þ H]þ, 100), 539.5 ([M þ Na]þ, 52), 571.1
([M þ MeOH þ Na]þ, 22); analysis for C32H49FO4 (516.7): C, 74.38;
H, 9.56; found: C, 74.21; H, 9.74.
7:3); [
(log
a
]
¼ 143.65ꢁ (c 0.48, CHCl3); UVevis (methanol): lmax
D
3
) ¼ 251 nm (4.09); IR (KBr):
n
¼ 3416br, 2978s, 2955s, 1736s,
1718s, 1645s, 1614w, 1458m, 1385m, 1314m, 1301m, 1285m,
1260m, 1222s, 1163s, 1113m, 1091m, 1039m, 1014w; 1H NMR
(500 MHz, CDCl3):
d
¼ 5.63 (s, 1H, H-12), 4.16 (dq, 1H, Et-CHH0,
5.34. Benzyl (3
a) 3-hydroxy-11-oxo-olean-12-en-30-oate (33)
J ¼ 10.8, 7.1 Hz), 4.10 (dq, 1H, Et-CHH0, J ¼ 10.8, 7.1 Hz), 3.19 (dd,
1H, H-2, J ¼ 6.6, 3.7 Hz), 3.13 (dd, 1H, H-1, J ¼ 14.9, 6.6 Hz), 2.79
Compound 33 was prepared according to [7,11].

R. Csuk et al. / European Journal of Medicinal Chemistry 46 (2011) 5356e5369
5369
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To a solution of 33 (290 mg, 0.51 mmol) in dry DCM (15 ml),
chloroacetyl chloride (50 ml, 0.61 mmol) was added. After stirring at
25 ꢁC for 24 h and usual aqueous work-up, the crude product was
dissolved in dry DMF (10 ml), finely grounded K2CO3 (700 mg,
5.07 mmol) and 1,3-diaminopropane (0.5 ml, 5.95 mmol) were
added, and the mixture was stirred at 25 ꢁC for 2 h. The solvent was
removed under reduced pressure, and water (30 ml) was added.
After the extraction with DCM (3 ꢂ 20 ml), the combined organic
layers were washed with water (20 ml) and brine (20 ml), dried
(Na2SO4), filtered, and the solvent was evaporated. Purification by
chromatography (silica gel, load with methanol, unload with
methanol/diethylamine 9:1) gave 34 (270 mg, 82%) as a slight
yellowish powder; mp 154e157 ꢁC; Rf ¼ 0.02 (chloroform/meth-
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anol 9:1); [
(log
a
]
¼ þ100.62ꢁ (c 0.49, CHCl3); UVevis (methanol): lmax
D
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(1982) 993e1001.
3
) ¼ 249 nm (3.99); IR (KBr):
n
¼ 3433br, 2945s, 1728s, 1660s,
1464m, 1387m, 1306w, 1279m, 1248m, 1213s, 1174m, 1145s, 1083m,
1023w, 985m cmꢀ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 7.38e7.29 (m,
5H, HeAr), 5.52 (s, 1H, H-12), 5.18 (d, 1H, Bn-CHH0, J ¼ 12.3 Hz), 5.07
(d, 1H, Bn-CHH0, J ¼ 12.3 Hz), 4.55 (m, 1H, H-3), 3.37 (s, 2H,
CH2COO), 2.78 (m, 1H, H-1), 2.77 (t, 2H, chain-3, J ¼ 6.8 Hz), 2.67 (t,
2H, chain-1, J ¼ 6.8 Hz), 2.32 (s, 1H, H-9), 2.02 (m, 1H, H-18), 1.99 (m,
1H, H-15), 1.97 (m, 1H, H-21), 1.92 (m, 1H, H-19), 1.78 (m, 1H, H-16),
1.73 (m, 1H, H-2), 1.67 (m, 1H, H-7), 1.63 (m, 2H, chain-2), 1.62 (m,
1H, H-20), 1.61 (dd, 1H, H-190, J ¼ 13.9, 13.9 Hz), 1.55 (m, 1H, H-6),
1.46 (m, 1H, H-60), 1.40 (m, 1H, H-70), 1.36 (m, 1H, H-22), 1.33 (s, 3H,
H-27), 1.29 (m, 1H, H-220), 1.29 (m, 1H, H-210), 1.16 (m, 1H, H-160),
1.14 (s, 6H, H-25 & H-29), 1.09 (s, 3H, H-26), 1.03 (m, 1H, H-10), 0.97
(m, 1H, H-150), 0.87 (s, 3H, H-24), 0.86 (s, 3H, H-23), 0.79 (m, 1H, H-
5), 0.71 (s, 3H, H-28) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 199.9 (C-
11), 176.2 (C-30), 172.4 (CH2COO), 169.0 (C-13), 136.1 (Car), 128.5
(Car), 128.5 (Car),128.4 (C-12),128.3 (Car),128.2 (Car),127.8 (Car), 81.2
(C-3), 66.2 (Bn-CH2), 61.6 (C-9), 55.0 (C-5), 51.2 (CH2COO), 48.2 (C-
18), 47.4 (chain-1), 45.3 (C-8), 44.0 (C-20), 43.2 (C-14), 41.0 (C-19),
40.3 (chain-3), 38.7 (C-1), 38.1 (C-4), 37.6 (C-22), 36.9 (C-10), 33.4
(chain-2), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.4 (C-28), 28.2 (C-
29), 28.1 (C-23), 26.4 (C-16), 26.4 (C-15), 23.6 (C-2), 23.3 (C-27),18.6
(C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS (ESI): m/z
(%) ¼ 675.5 ([M þ H]þ, 4), 701.5 ([M þ Na]þ, 100); analysis for
C42H62N2O5 (675.0): C, 74.74; H, 9.26; N, 4.15; found: C, 74.57; H,
9.41; N, 4.00.
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(E)-ethyl 3-keto-18-
(2007) 1013e1016.
b-glycyrrhetinate-3-oxime, Chin. J. Struct. Chem. 26
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A. Gonzalez-Coloma, Biovalorization of friedelane triterpenes derived from
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3566e3571.
Acknowledgments
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potent inhibitors of Naþ Kþ-ATPase
, e synthesis and structureeactivity-
relationships, Eur. J. Med. Chem. 27 (1992) 345e351.
We like to thank Dr. Harish Kommera and PD Dr. Reinhard
Paschke from Biosolutions Halle GmbH for support. We are grateful
to the Stiftung der Deutschen Wirtschaft e.V. (SDW) for a personal
scholarship to Stefan Schwarz. The cell lines were kindly provided
by Dr. T. Müller (Dept. of Haematology/Oncology, Univ. Halle).
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and 2,3-thiirane intermediates, Tetrahedron 66 (2010) 4390e4402.
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methods and reactions. 63. Pyridinium poly-(hydrogen fluoride) (30-percent
pyridine 70-percent hydrogen-fluoride) e convenient reagent for organic
fluorination reactions, J. Org. Chem. 44 (1979) 3872e3881.
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