Synthesis and biological activity of some antitumor active derivatives from glycyrrhetinic acid

Article abstract of DOI:10.1016/j.ejmech.2010.09.028

Aminoalkyl substituted derivatives were synthesized starting from glycyrrhetinic acid methyl ester and screened for antitumor activity in a panel of 15 human cancer cell lines by an SRB assay. The most compound 7 possesses an aminohexyl side chain, induces apoptosis and shows IC₅₀ values of 0.6-3.0 μM.

Full text of DOI:10.1016/j.ejmech.2010.09.028

European Journal of Medicinal Chemistry 45 (2010) 5718e5723
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of some antitumor active derivatives from
glycyrrhetinic acid
*
René Csuk , Stefan Schwarz, 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:
Received 6 July 2010
Received in revised form
1 September 2010
Accepted 1 September 2010
Available online 17 September 2010
Aminoalkyl substituted derivatives were synthesized starting from glycyrrhetinic acid methyl ester and
screened for antitumor activity in a panel of 15 human cancer cell lines by an SRB assay. The most
compound 7 possesses an aminohexyl side chain, induces apoptosis and shows IC50 values of 0.6e3.0
mM.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Glycyrrhetinic acid
Antitumor activity
Apoptosis
1. Introduction
Reaction of 3 with a variety of
furnished compounds 4e11.
a,u-alkanediamines (Scheme 2)
Glycyrrhetinic acid (GA) got into the focus of scientific interest at
least for its antitumor activity; on-going investigations for the last
two decades confirmed an apoptotic effect of GA on tumor cells
[1e4]. Its vast abundance in natural sources is another important
argument in the search of GA derived antitumor agents. Thus, the
extract of licorice contains up to 15% of the glycoside glycyrrhizin
[5,6] whereas other antitumor active triterpenoids, e.g. betulinic
acid (2.4%) [7] or oleanoic acid (ca. 3.1%) [7,8] are more difficult to
obtain from their natural sources. One of our goals was increasing
the activity of GA by “simple” transformationsebut retaining the
advantages residing in the parent compound GA.
3. Results
The compounds 1e11 were tested for antitumor activity in
a panel of 15 human cancer cell lines using a sulforhodamine B
assay (SRB). The results from these tests are summarized in Table 1.
Screening of compounds 2 and 3 in the SRB assay revealed that
a free carboxylic group in position C(30) results in a lower inhibi-
tion of cell growth compared to the corresponding esters. Esterifi-
cation results in an approx. 3-fold higher cytotoxicity. Additional
chloroacetylation afforded an analogue 3 showing a ca. 10-fold
higher cytotoxicity than the parent compound GA.
In the SRB assay all of the amino compounds 4e11 showed
significantly improved IC50 values compared to GA. Compound 7
carrying a diaminohexyl chain was the most active derivative
2. Chemistry
Preliminary results indicated that changing of the polarity
pattern of GA might be of some advantage in obtaining better
cytotoxicity; hence, preparing an ester of GA (for gain in lipophility)
and introducing an extra amino group (for gain in polarity) seemed
appropriate. Starting from GA, the methyl ester 1 was prepared [9]
in 91% yield by reacting GA with methyl iodide and potassium
carbonate (Scheme 1). GA as well as its ester 1 were converted into
the corresponding chloroacetyl derivatives 2 and 3, respectively.
showing IC50 values between 0.59 and 3.0 mM. This correspondsein
averageeto an approx. 60 times higher cytotoxicity than GA. In
addition, the length of the alkyl chain seems to be of importance
(Fig. 1). As depicted in Fig. 1, compound 7 inhibits the growth of the
tumor cells best. Our findings parallel previous results [10e13]
showing that a methyl esterification of the C-30 carboxylic group
significantly improves the cytotoxic activities of GA. Nitrogen-con-
taining substituents directly attached to the A-ring or close by seem
to improve [10,14] the antiproliferative effect of GA derivatives.
Supposing an extrinsic mechanism of apoptotic action [15] one
might reason that these compounds interact with a membrane
* Corresponding author. Tel.: þ49 345 55 25660; fax: þ 49 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.028

R. Csuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5718e5723
5719
5.2. Cell lines and culture conditions
The cell lines 518A2, 8505C, A253, A2780, A431, A549, DLD-1,
FaDu, HCT-116, HCT-8, HT-29, LIPO, MCF-7, SW1736, SW480 were
included in this study. All these cell lines were kindly provided by
Dr. Thomas Müller, Department of Hematology/Oncology, Martin-
Luther-University Halle-Wittenberg, Halle (Saale), Germany.
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.
Scheme 1. Synthesis of the chloroacetyl derivatives: reagents and conditions: (a)
K2CO3, CH3I, DMF, 24 h, 25 ꢀC, 91%; (b) CleCH2COCl, Et3N, THF (or CH2Cl2), 25 ꢀC, 12 h,
45% (for 2) and 86.9% (for 3).
located receptor that belongs to the TNF family. The data presented
in Fig. 1 seem to support this assumption since an optimized length
of the aminoalkyl side chain should fit into the binding pocket of
the receptor best. But these assumptions, however, cannot explain
the lowered cytotoxicity associated with compounds 1e3; espe-
cially 3 show a high cytotoxicity but differs in its structure signifi-
cantly from 4e11. Hence, explaining our findings by evoking an
intrinsic mechanism of action [1,16] seems more likely.
As shown by an extra dye exclusion test (trypan blue) and DNA
laddering experiments (by gel electrophoresis) compound 7 trig-
gers apoptosis. Quite recently, Yamaguchi et al. [17] were able to
show that GA exhibited a tumor cell-selective toxicity through
H-Ras down-regulation but also induces actin disruption [18]. In
addition, GA suppresses [19] colon carcinogenesis. Further studies
concerning the apoptotic mechanism are presently under investi-
gation in our laboratory.
5.3. Cytotoxicity assay
The cytotoxic activities of our compounds were evaluated using
the sulforhodamine B (SRB) [20] (SigmaeAldrich) 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
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
4. Conclusions
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 dose-response curves.
In this study we have synthesized aminoalkyl modified GA
methyl esters; screening for antitumor activity in a panel of 14
human cancer cell lines by an SRB assay showed for the most active
compound 7 an IC50 values between 0.6 and 3.0 mM. Cell death is
triggered by apoptosis as shown by a dye-exclusion test and DNA-
laddering experiments.
5.4. Apoptosis tests [21]
5. Experimental
5.4.1. Dye exclusion test
5.1. General
Apoptotic cell death was analyzed by trypan blue dye (Sigma
Aldrich, Germany) on A431 and A2780 cell lines. The cell culture
flasks with 70e80% confluence were treated with IC 90 doses of the
compounds for 24 h. After treatment, the supernatant medium
with floating cells was collected and centrifuged to collect the dead
and apoptotic cells. The cell pellet was suspended in serum free
media. Equal amounts of cell suspension and trypan blue were
mixed and analyzed by microscope. The viable cells exclude the dye
and are colourless and cells whose cell membrane was destroyed
are turning into blue. When there were more colourless cells than
colored, death is characterised as apoptotic.
Melting points are uncorrected (Leica hot stage microscope),-
NMR spectra were recorded using the Varian spectrometers Gemini
200, Gemini 2000 or Unity 500 (d given in ppm, J in Hz, internal
Me4Si), IR spectra (film or KBr pellet) on a PerkineElmer FT-IR
spectrometer Spectrum 1000, MS spectra were taken on an Intectra
GmbH AMD 402 (electron impact, 70 eV) or on a Finnigan MAT TSQ
7000 (electrospray, voltage 4.5 kV, sheath gas nitrogen) instrument.
TLC was performed on silica gel (Merck 5554, detection by UV
absorption). The solvents were dried according to usual procedures.
Scheme 2. Synthesis of the aminoalkyl derivatives: reagents and conditions: (a) H2N-(CH2)n-NH2, DMF, K2CO3, 12 h, 25 ꢀC, 21e73%.

5720
R. Csuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5718e5723
Table 1
Cytotoxicity (IC50 values in mmol) for 1e11 in a panel of various cancer cell lines.
GAa
1
2
3
4
5
6
7
8
9
10
11
518A2
8505C
A253
A2780
A549
DLD-1
FADU
HCT-116
HCT-8
HT-29
LIPO
83.92
86.50
80.78
74.57
82.76
81.21
84.55
78.83
78.85
80.09
81.44
84.70
76.93
86.80
27.54
26.07
19.42
25.54
23.50
26.12
23.41
22.10
24.36
27.54
20.47
22.14
34.87
16.08
25.43
26.08
25.54
23.77
24.80
17.36
23.56
14.41
13.39
16.91
25.39
25.11
16.42
25.91
5.24
3.79
3.37
3.64
4.39
5.15
4.39
5.57
4.30
2.37
2.90
3.89
3.55
6.05
3.68
2.55
2.12
2.56
2.43
3.31
2.66
3.51
2.41
1.51
1.69
2.57
2.45
3.30
2.54
2.02
1.78
2.27
2.00
2.52
2.40
3.30
2.19
1.38
1.28
1.93
1.79
2.69
1.91
1.09
1.68
1.12
1.36
1.59
0.91
1.78
1.17
0.62
0.59
1.59
1.17
1.61
2.25
1.27
2.13
1.74
1.14
2.21
1.25
2.20
1.70
0.89
0.86
1.44
0.98
2.24
2.24
3.49
3.35
3.01
2.80
4.08
3.96
4.26
3.53
2.92
2.76
4.36
3.89
4.09
3.93
3.12
6.18
4.65
3.30
2.23
4.50
5.54
3.44
2.42
2.06
5.48
3.33
3.30
5.74
4.33
7.6
15.86
6.19
6.01
8.39
6.13
12.44
5.13
3.97
5.34
14.55
6.69
3.14
8.92
5.48
3.63
5.16
5.53
5.65
3.86
4.07
2.73
6.93
2.68
3.73
4.73
MCF-7
SW1736
SW480
a
From SRB assays after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ꢄ 5%.
5.4.2. DNA Fragmentation assay
H-1, J ¼ 13.3, 3.3, 3.3 Hz), 2.32 (s,1H, H-9), 2.06 (dd,1H, H-18, J ¼ 13.3,
3.8 Hz), 2.00 (m, 1H, H-15), 1.95 (m, 1H, H-21), 1.90 (dd, 1H, H-19,
J ¼ 13.8, 3.9 Hz),1.83 (ddd,1H, H-16, J ¼ 14.3,14.3, 5.2 Hz),1.65 (m,1H,
H-2),1.62 (m,1H, H-7),1.58 (m,1H, H-20),1.57 (m,1H, H-190),1.57 (m,
1H, H-6),1.43 (m,1H, H-60),1.38 (m,1H, H-70),1.36 (m,1H, H-22),1.34
(s, 3H, H-27), 1.30 (m, 1H, H-220), 1.28 (m, 1H, H-210), 1.17 (m, 1H, H-
160),1.13 (s, 3H, H-28),1.12 (s, 3H, H-25),1.11 (s, 3H, H-26),1.00 (m,1H,
H-150), 0.99 (s, 3H, H-23), 0.96 (m,1H, H-10), 0.79(s, 3H, H-24), 0.79 (s,
3H, H-29), 0.68 (m, 1H, H-5) ppm; 13C NMR (125 MHz, CDCl3):
Determination of apoptotic cell death was performed by DNA
gel electrophoresis. Briefly, A431 and A2780 were treated with
respective IC 90 doses of the compounds for 24 h. Floating cells
(induced by drug exposure) were collected, washed with PBS and
lysed with lysis buffer (100 mM Tris-HCL Ph 8.0; 20 mM EDTA; 0.8%
SDS) (all from SigmaeAldrich). Then they are treated with RNAse A
at 37 ꢀC for 2 h and proteinase K at 50 ꢀC (both from Roche Diag-
nostics chemical company, Mannheim, Germany). DNA laddering
was observed by running the samples on 2% agarose gel followed
by ethidium bromide (SigmaeAldrich) staining.
d
¼ 200.0 (C-11), 176.8 (C-30), 169.0 (C-13), 128.6 (C-12), 78.8 (C-3),
61.9 (C-9), 55.1 (C-5), 51.8 (COOCH3), 48.5 (C-18), 45.5 (C-14), 44.1 (C-
20), 43.3 (C-8), 41.2 (C-19), 39.2 (C-1), 39.2 (C-4), 37.8 (C-22), 37.2 (C-
10), 32.9 (C-7), 31.9 (C-17), 31.2 (C-21), 28.6 (C-29), 28.4 (C-23), 28.2
(C-28), 27.4 (C-2), 26.6 (C-16), 26.6 (C-15), 23.5 (C-27), 18.8 (C-26),
17.6 (C-6), 16.4 (C-25), 15.6 (C-24) ppm; MS (ESI): m/z (%) ¼ 485.5
([Mþ H]þ, 55), 507.5 ([M þ Na]þ,12), 539.1 ([M þNaþ MeOH]þ,100).
5.5. Methyl 3b-hydroxy-11-oxo-olean-12-en-30-oate (1)
To a solution of GA (31.00 g, 65.9 mmol) in dry DMF (150 ml),
potassium carbonate (15.34 g, 111.0 mmol) was added. After 30 min
of stirring 25 ꢀC, iodomethane (4.94 ml, 79.0 mmol) was added and
stirring continued for 2 h. The solvents were evaporated and the
crude residue was dissolved in a mixture of dichloromethane
(300 ml) and hydrochloric acid (50 ml,1.0 M). The aqueous layer was
extracted with dichloromethane (3 ꢁ 50 ml), the combined organic
layers were washed with brine (50 ml), dried (Na2SO4), filtered and
the solvent was evaporated. Re-crystallization from methanol yiel-
ded 1 (29.09 g, 91.1%) as a colourless crystalline solid. Mp
254e258 ꢀC (lit. 254e258 ꢀC [9]); RF ¼ 0.48 (hexane/ethyl acetate
5.6. 3b-(Chloroacetyl)-11-oxo-olean-12-en-30-oic acid (2)
GA (0.76 g, 1.62 mmol) was dissolved in mixture of dry THF
(15 ml) and dry triethylamine (1 ml). Chloro-acetylchloride (142 l,
m
1.78 mmol) was added dropwise and the solution was stirred at
25 ꢀC for 12 h. The solvent was removed and the residue dissolved
in dichloromethane (15 ml). After washing with hydrochloric acid
(10 ml, 1.0 M), the aqueous layer was extracted with dichloro-
methane (3 ꢁ 10 ml), followed by washing the combined organic
layers with brine (10 ml). They were dried (Na2SO4), filtered and
evaporated. The crude residue was subjected to chromatography
(silica gel, hexane/ethyl acetate 7:3) to afford 2 (0.40 g, 45.3%) as
colourless crystals. Mp 259e262 ꢀC; RF ¼ 0.20 (hexane/ethyl acetate
7:3); ½a 2D0
ꢂ
¼ 141:18ꢀ (c ¼ 0.48, CHCl3); UVevis (methanol): lmax
(log
3
) ¼ 270 nm (4.15); IR (KBr): ¼ 3614br, 2970s, 2955s, 2875m,
n
1726s, 1659s, 1466m, 1450m, 1364w, 1216m, 1189m, 1136w, 1085w,
1040w, 992wcmꢃ1; 1H NMR (400 MHz, CDCl3):
¼ 5.65 (s,1H, H-12),
3.67 (s, 3H, COOCH3), 3.21 (dd,1H, H-3, J ¼ 10.8, 5.7 Hz), 2.78 (ddd,1H,
d
7:3); ½a 2D0
ꢂ
¼ 117:21ꢀ (c ¼ 0.5, CHCl3); UVevis (methanol): lmax
(log
3
) ¼ 267 nm (4.01); IR (KBr):
n
¼ 3385br, 2960s, 1738s, 1652s,
1466w, 1387m, 1326m, 1214m, 985w, 789w cmꢃ1
(500 MHz, CDCl3):
;
1H NMR
¼ 5.69 (s, 1H, H-12), 4.59 (dd, 1H, H-3, J ¼ 11.8,
d
4.9Hz), 4.06 (d, 1H, CHH0Cl, J ¼ 16.8 Hz), 4.02 (d, 1H, CHH0Cl,
J ¼ 16.8 Hz), 2.82 (ddd,1H, H-1, J ¼ 13.7, 3.6, 3.6 Hz), 2.35 (s,1H, H-9),
2.17 (dd,1H, H-18, J ¼ 13.4, 3.8 Hz), 2.02 (ddd,1H, H-15, J ¼ 13.4,13.4,
4.5 Hz), 1.98 (m, 1H, H-21), 1.92 (dd, 1H, H-19, J ¼ 13.8, 3.9 Hz), 1.82
(ddd, 1H, H-16, J ¼ 13.5, 13.5, 4.5 Hz), 1.72 (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 Hz), 1.57 (m, 1H,
H-6), 1.44 (m, 1H, H-60), 1.41 (m, 1H, H-70), 1.39 (m, 1H, H-22), 1.36 (s,
3H, H-27), 1.31 (m, 1H, H-220), 1.28 (m, 1H, H-210), 1.21 (s, 3H, H-28),
1.19 (m, 1H, H-160), 1.15 (s, 3H, H-25), 1.12 (s, 3H, H-26), 1.05 (m, 1H,
H-150), 0.97 (m, 1H, H-10), 0.89 (s, 6H, H-23, H-24), 0.82 (s, 3H, H-
29), 0.80 (m, 1H, H-5) ppm; 13C NMR (125 MHz, CDCl3):
(C-11), 181.2 (C-30), 169.3 (C-13), 167.1 (CH2ClCOO), 128.5 (C-12),
83.1 (C-3), 61.7 (C-9), 55.0 (C-5), 48.3 (C-18), 45.5 (C-14), 43.8
d
¼ 200.0
Fig. 1. IC50 values (
mM) as a function of the alkyl chain length; lowest, highest and an
averaged value (mean of 14 cell lines) are given.

R. Csuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5718e5723
5721
(C-20), 43.3 (C-8), 41.2 (CH2ClCOO), 40.9 (C-19), 38.7 (C-1), 38.3 (C-
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 93:49ꢀ (c
¼
0.43, CHCl3);
) ¼ 267 nm (4.03); IR (KBr):
4), 37.7 (C-22), 37.0 (C-10), 32.7 (C-7), 31.9 (C-17), 31.0 (C-21), 28.6
(C-29), 28.4 (C-23), 28.1 (C-28), 26.5 (C-16), 26.5 (C-15), 23.5 (C-2),
23.4 (C-27), 18.8 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS
(ESI): m/z (%) ¼ 547.6 ([M þ H]þ, 31), 569.4 ([M þ Na]þ, 100), 601.1
([M þ Na þ MeOH]þ, 32); anal. calcd for C32H47ClO5 (547.17): C,
70.24; H, 8.66; Cl, 6.48; found: C, 70.11; H, 8.78; Cl, 6.31.
UVevis (methanol): lmax (log
3
n
¼ 3432br, 2934s, 1731s, 1660s, 1464m, 1387m, 1322m, 1217m,
1154m, 1086w, 1022w, 985m, 880w, 769w cmꢃ1; 1H NMR (500 MHz,
CDCl3):
¼ 5.64 (s, 1H, H-12), 4.56 (m, 1H, H-3), 3.66 (s, 3H,
d
COOCH3), 3.38 (s, 2H, CH2COO), 2.90 (t, 2H, chain-3, J ¼ 6.3 Hz), 2.79
(ddd, 1H, H-1, J ¼ 13.5, 3.2, 3.2 Hz), 2.71 (t, 2H, chain-1, J ¼ 6.4 Hz),
2.34 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.01 (m, 1H, H-15), 1.97 (m, 1H,
H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.6, 3.6, 2.1 Hz), 1.80 (m, 1H, H-16),
1.71, (m, 1H, H-2), 1.70 (m, 2H, chain-2), 1.64 (m, 1H, H-7), 1.61 (m,
1H, H-20), 1.59 (dd, 1H, H-190, J ¼ 12.4, 12.4 Hz), 1.54 (m, 1H, H-6),
1.46 (m, 1H, H-60), 1.41 (m, 1H, H-70), 1.38 (m, 1H, H-22), 1.34 (s, 3H,
H-27), 1.30 (m, 1H, H-220), 1.28 (m, 1H, H-210), 1.19 (m, 1H, H-160),
1.14 (s, 3H, H-25), 1.13 (s, 3H, H-23), 1.10 (s, 3H, H-26), 1.04 (ddd, 1H,
H-10, J ¼ 13.1, 13.1, 3.7 Hz), 0.99 (m, 1H, H-150), 0.85 (s, 6H, H-24, H-
28), 0.79 (m, 1H, H-5), 0.78 (s, 3H, H-29) ppm; 13C NMR (125 MHz,
5.7. Methyl 3b-(chloroacetyl)-11-oxo-olean-12-en-30-oate (3)
Compound 1 (2.00 g, 4.1 mmol) was dissolved in dry dichloro-
methane (20 ml) and mixed with dry triethylamine (1 ml). Chloro-
acetylchloride (360ml, 4.5 mmol) was added dropwise and stirring at
25 ꢀC continued for 12 h. After completion of the reaction (as
monitored by TLC), the mixture was washed with hydrochloric acid
(10 ml, 1.0 M). The aqueous layer was extracted with dichloro-
methane (3 ꢁ10 ml), the combined organic layers werewashed with
brine (15 ml), dried (Na2SO4), filtered and the solvents were evap-
orated. The residue was subjected to chromatography (silica gel,
hexane/ethyl acetate 9:1) to afford 3 (2.30 g, 86.9%) as colourless
crystals. Mp 243e246 ꢀC; RF ¼ 0.77 (hexane/ethyl acetate 7:3);
a 20
CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30), 172.4 (CH2COO), 169.2 (C-13),
128.5 (C-12), 81.4 (C-3), 61.7 (C-9), 55.0 (C-5), 51.7 (COOCH3), 50.9
(CH2COO), 48.4 (C-18), 47.8 (chain-1), 45.4 (C-14), 44.0 (C-20), 43.2
(C-8), 41.1 (C-19), 40.6 (chain-3), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22),
36.9 (C-10), 32.7 (C-7), 31.8 (C-17), 31.3 (chain-2), 31.1 (C-21), 28.5
(C-29), 28.3 (C-23), 28.1 (C-28), 26.4 (C-16), 26.4 (C-15), 23.6 (C-2),
23.3 (C-27), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS
½ ꢂD ¼ 115:53ꢀ (c
¼
0.31, CHCl3); UVevis (methanol): lmax
(log
3
) ¼ 267 nm (4.08); IR (KBr):
n
¼ 3432br, 2951s, 1732s, 1645m,
1540m, 1463s, 1371s, 1331m, 1250s, 1210s, 1179s, 1062m, 1025m,
978m, 945w, 888m, 831m, 778w, 749w cmꢃ1 1H NMR (500 MHz,
CDCl3):
¼ 5.65 (s,1H, H-12), 4.58 (dd,1H, H-3, J ¼ 11.6, 4.8 Hz), 4.05
(ESI): m/z (%) ¼ 599.3 ([M þ H]þ, 100), 625.3 ([2M þ Na þ MeOH]2þ
,
;
95); anal. calcd for C36H58N2O5 (598.86): C, 72.20; H, 9.76; N, 4.68;
found: C, 72.06; H, 9.84; N, 4.51.
d
(d,1H, CHH0Cl, J ¼ 17.1 Hz), 4.01 (d,1H, CHH0Cl, J ¼ 17.1 Hz), 3.67 (s, 3H,
COOCH3), 2.81 (ddd, 1H, H-1, J ¼ 13.7, 3.6, 3.6 Hz), 2.34 (s, 1H, H-9),
2.06 (dd,1H, H-18, J ¼ 13.5, 3.5 Hz), 2.01 (m,1H, H-15),1.97 (m,1H, H-
21), 1.90 (ddd, 1H, H-19, J ¼ 13.5, 4.1, 2.7 Hz), 1.80 (ddd, 1H, H-16,
J ¼ 13.7, 13.7, 4.4 Hz), 1.73 (ddd, 1H, H-2, J ¼ 11.8, 3.3, 1.5 Hz), 1.65 (m,
1H, H-7), 1.63 (m, 1H, H-20), 1.59 (dd, 1H, H-190, J ¼ 13.5 Hz), 1.57 (m,
1H, H-6),1.45 (m,1H, H-60),1.40 (m,1H, H-70),1.37 (m,1H, H-22),1.34
(s, 3H, H-27), 1.29 (m, 1H, H-220), 1.28 (m, 1H, H-210), 1.16 (m, 1H, H-
160), 1.14 (s, 3H, H-28), 1.13 (s, 3H, H-25), 1.10 (s, 3H, H-26), 1.04 (ddd,
1H, H-10, J ¼ 13.4, 13.4, 3.5 Hz), 0.99 (m, 1H, H-150), 0.88 (s, 6H, H-23,
H-24), 0.79 (m,1H, H-5), 0.78 (s, 3H, H-29) ppm; 13C NMR (125 MHz,
5.8.2. Methyl 3b-3-{[N-(4-aminobutyl)glycyl]oxy}-11-oxo-olean-
12-en-30-oate (5)
Following the general procedure, 5 was obtained from 1,4-
butanediamine; yield: 120 mg, 38.5%; mp 219e222 ꢀC; RF ¼ 0.71
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 82:38ꢀ (c
¼
0.32, CHCl3);
UVevis (methanol): lmax (log
3
) ¼ 267 nm (4.01); IR (KBr):
n
¼ 3423br, 2949s, 1730s, 1660s, 1465m, 1388m, 1217m, 1153m,
1086w, 986w, 769w cmꢃ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.65 (s,
1H, H-12), 4.56 (m, 1H, H-3), 3.67 (s, 3H, COOCH3), 3.38 (s, 2H,
CH2COO), 2.88 (m, 2H, chain-4), 2.80 (ddd, 1H, H-1, J ¼ 13.5, 3.2,
3.2 Hz), 2.66 (t, 2H, chain-1, J ¼ 6.2 Hz), 2.34 (s,1H, H-9), 2.06 (m,1H,
H-18), 2.01 (m, 1H, H-15), 1.97 (m, 1H, H-21), 1.90 (m, 1H, H-19), 1.81
(m,1H, H-16),1.72 (m, 2H, chain-3),1.69 (m,1H, H-2),1.64 (m,1H, H-
7), 1.63 (m, 2H, chain-2), 1.62 (m, 1H, H-20), 1.59 (dd, 1H, H-190,
J ¼ 13.6,13.6 Hz),1.54 (m, 1H, H-6), 1.46 (m, 1H, H-60), 1.41 (m, 1H, H-
70), 1.38 (m, 1H, H-22), 1.35 (s, 3H, H-27), 1.31 (m, 1H, H-220), 1.28 (m,
1H, H-210), 1.19 (m, 1H, H-160), 1.14 (s, 3H, H-25), 1.13 (s, 3H, H-23),
1.11 (s, 3H, H-26), 1.02 (ddd, 1H, H-10, J ¼ 13.1, 13.1, 3.7 Hz), 0.99 (m,
1H, H-150), 0.86 (s, 6H, H-24, H-28), 0.80 (m, 1H, H-5), 0.79 (s, 3H, H-
CDCl3):
d
¼ 200.1 (C-11),176.9 (C-30),169.5 (C-13),167.1 (CH2ClCOO),
128.4 (C-12), 83.0 (C-3), 61.6 (C-9), 55.0 (C-5), 51.8 (COOCH3), 48.4
(C-18), 45.4 (C-14), 44.0 (C-20), 43.2 (C-8), 41.2 (CH2ClCOO), 41.1
(C-19), 38.7 (C-1), 38.2 (C-4), 37.7 (C-22), 36.9 (C-10), 32.6 (C-7), 31.8
(C-17), 31.1 (C-21), 28.5 (C-29), 28.3 (C-23), 28.0 (C-28), 26.4 (C-16),
26.4 (C-15), 23.4 (C-2), 23.3 (C-27),18.6 (C-26),17.3 (C-6),16.6 (C-24),
16.4 (C-25) ppm; MS (ESI): m/z (%) ¼ 561.4 ([M þ H]þ, 80), 583.3
([M þ Na]þ, 70), 614.8 ([M þ Na þ MeOH]þ, 100), 1121.1 ([cluster
2M þ H]þ, 36), 1143.1 (cluster [2M þ Na]þ, 72); anal. calcd for
C33H49ClO5 (561.19): C, 70.63; H, 8.80; found: C, 70.51; H, 8.96.
29) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30),
172.4 (CH2COO), 169.2 (C-13), 128.5 (C-12), 81.2 (C-3), 61.7 (C-9),
55.0 (C-5), 51.8 (COOCH3), 51.1 (CH2COO), 48.9 (chain-1), 48.4 (C-
18), 45.4 (C-14), 44.0 (C-20), 43.2 (C-8), 41.1 (C-19), 40.7 (chain-4),
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-29), 28.3 (C-23), 28.1 (C-28), 28.2 (chain-3), 27.8
(chain-2), 26.4 (C-16), 26.4 (C-15), 23.6 (C-2), 23.3 (C-27), 18.7 (C-
26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS (ESI): m/z
(%) ¼ 613.5 ([M þ H]þ, 100); anal. calcd for C37H60N2O5 (612.88): C,
72.51; H, 9.87; N, 4.57; found: C, 72.37; H, 9.98; N, 4.34.
5.8. General procedure for the preparation of the amines
To a solution of 3 (1 eq.) in dry DMF (15 ml per eq.), potassium
carbonate (2 eq.) and the respective 1,u-alkandiamine (2 eq.) was
added. After 12 h of stirring at 25 ꢀC, the solvent was removed
under reduced pressure and the crude residue was dissolved in
a mixture of dichloromethane and water. The aqueous layer was
extracted with dichloromethane, the combined organic layers were
washed with brine, dried (Na2SO4), filtered and evaporated. The
remaining residue was subjected to chromatography (silica gel,
loaded with methanol, unloaded with methanol/diethylamine 9:1)
to afford the final products (4e11).
5.8.3. Methyl 3b-3-{[N-(5-aminopentyl)glycyl]oxy}-11-oxo-olean-
12-en-30-oate (6)
Following the general procedure, 6 was obtained from 1,5-
pentanediamine; yield: 110 mg, 21.2%; mp 214e216 ꢀC; RF ¼ 0.71
5.8.1. Methyl 3b-3-{[N-(3-aminopropyl)glycyl]oxy}-11-oxo-olean-
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 94:52ꢀ (c
¼
0.28, CHCl3);
) ¼ 267 nm (3.98); IR (KBr):
12-en-30-oate (4)
UVevis (methanol): lmax (log
3
Following the general procedure, 4 was obtained from 1,3-pro-
n
¼ 3432br, 2934s, 1731s, 1660s, 1464m, 1387m, 1322w, 1217s,
panediamine; yield: 140 mg, 27.0%; mp 191e195 ꢀC; RF ¼ 0.71
1154m, 1086w, 1022w, 985w, 880w, 769w cmꢃ1; 1H NMR (500 MHz,

5722
R. Csuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5718e5723
CDCl3):
d
¼ 5.64 (s,1H, H-12), 4.56 (dd,1H, H-3, J ¼ 11.5, 4.5 Hz), 3.66
J ¼ 13.6, 3.2, 3.2), 2.65 (t, 2H, chain-7, J ¼ 7.1), 2.57 (t, 2H, chain-1,
J ¼ 7.2), 2.34 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.00 (m, 1H, H-15), 1.97
(m, 1H, H-21), 1.90 (m, 1H, H-19), 1.80 (m, 1H, H-16), 1.70 (m, 1H, H-
2), 1.63 (m, 1H, H-7), 1.62 (m, 1H, H-20), 1.59 (dd, 1H, H-190, J ¼ 13.1,
13.1), 1.55 (m, 1H, H-6), 1.47 (m, 2H, chain-3), 1.44 (m, 2H, chain-6),
1.43 (m, 1H, H-60), 1.40 (m, 1H, H-70), 1.38 (m, 1H, H-22), 1.34 (s, 3H,
H-27), 1.31e1.28 (m, 8H, H-210, H-220, chain-2, chain-4, chain-5),
1.20 (m, 1H, H-160), 1.14 (s, 3H, H-25), 1.13 (s, 3H, H-23), 1.10 (s, 3H,
H-26), 1.03 (m, 1H, H-10), 0.98 (m, 1H, H-150), 0.86 (s, 6H, H-24, H-
28), 0.79 (m, 1H, H-5), 0.78 (s, 3H, H-29); 13C NMR (125 MHz,
(s, 3H, COOCH3), 3.36 (s, 2H, CH2COO), 2.79 (ddd, 1H, H-1, J ¼ 13.7,
3.6, 3.6 Hz), 2.72 (t, 2H, chain-5, J ¼ 5.4 Hz), 2.59 (m, 2H, chain-1),
2.34 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.00 (m, 1H, H-15), 1.97 (m, 1H,
H-21), 1.90 (m, 1H, H-19), 1.80 (m, 1H, H-16), 1.68 (m, 1H, H-2), 1.62
(m, 1H, H-7), 1.59 (dd, 1H, H-190, J ¼ 13.7, 13.7 Hz), 1.58 (m, 1H, H-20),
1.53 (m, 1H, H-6), 1.49 (m, 4H, chain-2, chain-4), 1.41 (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), 1.28 (m, 1H, H-210), 1.18 (m, 1H, H-160), 1.14 (s, 3H, H-25), 1.12
(s, 3H, H-23),1.10 (s, 3H, H-26),1.02 (m,1H, H-10),1.01 (m, 2H, chain-
3), 0.98 (m, 1H, H-150), 0.85 (s, 6H, H-24, H-28), 0.79 (m, 1H, H-5),
CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30), 172.4 (CH2COO), 169.2 (C-13),
0.78 (s, 3H, H-29) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.0 (C-
128.5 (C-12), 81.1 (C-3), 61.7 (C-9), 55.0 (C-5), 51.7 (COOCH3), 51.2
(CH2COO), 49.6 (chain-1), 48.4 (C-18), 45.4 (C-14), 44.0 (C-20), 43.2
(C-8), 42.2 (chain-7), 41.1 (C-19), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22),
36.9 (C-10), 33.8 (chain-6), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 30.0
(chain-3), 29.3 (chain-4), 28.5 (C-29), 28.3 (C-23), 28.1 (C-28), 27.2
(chain-2), 26.8 (chain-5), 26.5 (C-16), 26.4 (C-15), 23.6 (C-2), 23.3
(C-27), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25); MS (ESI): m/z
(%) ¼ 328.6 ([Mþ2H]2þ, 9), 655.5 ([M þ H]þ, 100); anal. calcd for
C40H66N2O5 (654.96): C, 73.35; H, 10.16; N, 4.28; found: C, 73.22; H,
10.29; N, 4.11.
11), 176.9 (C-30),172.4 (CH2COO),169.2 (C-13), 128.5 (C-12), 81.2 (C-
3), 61.7 (C-9), 55.0 (C-5), 51.7 (COOCH3), 51.1 (CH2COO), 49.4 (chain-
1), 48.4 (C-18), 45.4 (C-14), 44.0 (C-20), 43.2 (C-8), 41.6 (chain-5),
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), 29.7 (chain-2), 29.7 (chain-4), 28.5 (C-29),
28.3 (C-23), 28.1 (C-28), 26.4 (C-16), 26.4 (C-15), 24.4 (chain-3), 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
627.4 ([M
þ
H]þ, 100), 653.3
([2M þ Na þ MeOH]2þ, 24); anal. calcd for C38H62N2O5 (626.91): C,
72.80; H, 9.97; N, 4.47; found: C, 72.63; H, 10.11; N, 4.26.
5.8.6. Methyl 3b-3-{[N-(8-aminooctyl)glycyl]oxy}-11-oxo-olean-
5.8.4. Methyl 3
b
-3-{[N-(6-aminohexyl)glycyl]oxy}-11-oxo-olean-
12-en-30-oate (9)
12-en-30-oate (7)
Following the general procedure, 9 was obtained from 1,8-
Following the general procedure, 7 was obtained from 1,6-
octanediamine; yield: 300 mg, 45.2%; mp 249e252 ꢀC; RF ¼ 0.71
hexanediamine; yield: 260 mg, 47.9%; mp 141e144 ꢀC; RF ¼ 0.71
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 97:05ꢀ (c
¼
0.63, CHCl3);
) ¼ 266 nm (4.00); IR (KBr):
(MeOH/diethylamine 9:1); ½a D20
ꢂ
¼ 108:05ꢀ (c ¼ 0.54, CHCl3);
) ¼ 266 nm (4.00); IR (KBr):
UVevis (methanol): lmax (log
3
UVevis (methanol): lmax (log
3
n
¼ 3384br, 2928s, 2856s, 1732s, 1655s, 1465m, 1387m, 1216s,
1154m, 1086w, 1049w, 985m, 880w, 770 (w) cmꢃ1 1H NMR
(500 MHz, CDCl3):
¼ 5.64 (s, 1H, H-12), 4.57 (dd, 1H, H-3, J ¼ 11.7,
n
¼ 3422br, 2931s, 2857s, 1732s, 1659s, 1464m, 1388m, 1322m,
;
1280m, 1216s, 1154s, 1086m, 1021w, 985m, 917w, 879w, 769w, 670w
cmꢃ1 1H NMR (500 MHz, CDCl3):
¼ 5.64 (s, 1H, H-12), 4.57 (dd,
d
;
d
4.8), 3.67 (s, 3H, COOCH3), 3.37 (s, 2H, CH2COO), 2.79 (ddd, 1H, H-1,
J ¼ 13.7, 3.4, 3.4), 2.65 (t, 2H, chain-8, J ¼ 7.0), 2.57 (t, 2H, chain-1,
J ¼ 7.2), 2.34 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.01 (m, 1H, H-15), 1.97
(m, 1H, H-21), 1.90 (m, 1H, H-19), 1.82 (m, 1H, H-16), 1.70 (m, 1H, H-
2), 1.64 (m, 1H, H-7), 1.62 (m, 1H, H-20), 1.59 (dd, 1H, H-190, J ¼ 13.2,
13.2), 1.54 (m, 1H, H-6), 1.47 (m, 2H, chain-7), 1.44 (m, 1H, H-60), 1.41
(m, 1H, H-70), 1.38 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.30 (m, 1H, H-
210), 1.29e1.26 (m, 11H, H-220, chain-2, chain-3, chain-4, chain-5,
chain-6), 1.18 (m, 1H, H-160), 1.14 (s, 3H, H-25), 1.13 (s, 3H, H-23), 1.10
(s, 3H, H-26), 1.01 (m, 1H, H-10), 0.98 (m, 1H, H-150), 0.86 (s, 6H, H-
24, H-28), 0.80 (m, 1H, H-5), 0.78 (s, 3H, H-29); 13C NMR (125 MHz,
1H, H-3, J ¼ 11.6, 4.7 Hz), 3.67 (s, 3H, COOCH3), 3.37 (s, 2H, CH2COO),
2.79 (ddd, 1H, H-1, J ¼ 13.7, 3.5, 3.5 Hz), 2.66 (t, 2H, chain-6,
J ¼ 7.0 Hz), 2.58 (t, 2H, chain-1, J ¼ 7.2 Hz), 2.34 (s, 1H, H-9), 2.06 (m,
1H, H-18), 2.01 (m, 1H, H-15), 1.97 (m, 1H, H-21), 1.87 (m, 1H, H-19),
1.82 (m, 1H, H-16), 1.70 (m, 1H, H-2), 1.63 (m, 1H, H-7), 1.62 (m, 1H,
H-20), 1.59 (dd, 1H, H-190, J ¼ 13.5, 13.5 Hz), 1.55 (m, 1H, H-6), 1.48
(m, 2H, chain-3), 1.45 (m, 2H, chain-5), 1.42 (m, 1H, H-60), 1.40 (m,
1H, H-70), 1.38 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.33e1.27 (m, 6H, H-
210, H-220, chain-2, chain-4), 1.19 (m, 1H, H-160), 1.14 (s, 3H, H-25),
1.13 (s, 3H, H-23), 1.11 (s, 3H, H-26), 1.03 (m, 1H, H-10), 0.98 (m, 1H,
H-150), 0.86 (s, 6H, H-24, H-28), 0.79 (m, 1H, H-5), 0.78 (s, 3H, H-29)
CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-30), 172.4 (CH2COO), 169.2 (C-13),
ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 200.2 (C-11), 177.1 (C-30),
128.5 (C-12), 81.1 (C-3), 61.7 (C-9), 55.0 (C-5), 51.7 (COOCH3), 51.2
(CH2COO), 49.6 (chain-1), 48.4 (C-18), 45.4 (C-14), 44.0 (C-20), 43.2
(C-8), 41.1 (C-19), 39.1 (chain-8), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22),
36.9 (C-10), 33.7 (chain-7), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 30.0
(chain-3), 29.5 (chain-5), 29.4 (chain-4), 28.5 (C-29), 28.3 (C-23),
28.1 (C-28), 27.2 (chain-2), 26.8 (chain-6), 26.5 (C-16), 26.4 (C-15),
23.6 (C-2), 23.3 (C-27), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-
25); MS (ESI): m/z (%) ¼ 335.6 ([Mþ2H]2þ, 100), 669.4 ([M þ H]þ,
84); anal. calcd for C41H68N2O5 (668.99): C, 73.61; H, 10.25; N, 4.19;
found: C, 73.42; H, 10.39; N, 4.09.
172.3 (CH2COO), 169.1 (C-13), 128.5 (C-12), 81.2 (C-3), 61.8 (C-9),
55.2 (C-5), 51.9 (COOCH3), 51.4 (CH2COO), 49.7 (chain-1), 48.6 (C-
18), 45.5 (C-14), 44.2 (C-20), 43.3 (C-8), 42.3 (chain-6), 41.3 (C-19),
38.9 (C-1), 38.3 (C-4), 37.9 (C-22), 37.1 (C-10), 33.9 (chain-5), 32.9
(C-7), 32.0 (C-17), 31.3 (C-21), 30.2 (chain-2), 28.7 (C-29), 28.5 (C-
23), 28.3 (C-28), 27.3 (chain-3), 27.0 (chain-4), 26.7 (C-16), 26.7 (C-
15), 23.8 (C-2), 23.5 (C-27), 18.9 (C-26), 17.6 (C-6), 16.9 (C-24), 16.6
(C-25) ppm; MS (ESI): m/z (%) ¼ 641.5 ([M þ H]þ, 100), 667.4
([2M þ Na þ MeOH]2þ, 54); anal. calcd for C39H64N2O5 (640.94): C,
73.08; H, 10.06; N, 4.37; found: C, 72.95; H, 10.18; N, 4.26.
5.8.7. Methyl 3b-3-{[N-(9-aminononyl)glycyl]oxy}-11-oxo-olean-
5.8.5. Methyl 3
b
-3-{[N-(7-aminoheptyl)glycyl]oxy}-11-oxo-olean-
12-en-30-oate (10)
12-en-30-oate (9)
Following the general procedure, 10 was obtained from 1,9-
Following the general procedure, 9 was obtained from 1,7-
nonanediamine; yield: 410 mg, 72.6%; mp 93e97 ꢀC; RF ¼ 0.71
heptanediamine; yield: 160 mg, 30.0%; mp 130e132 ꢀC; RF ¼ 0.71
(MeOH/diethylamine 9:1); ½a D20
ꢂ
¼ 53:17ꢀ (c ¼ 0.48, MeO); UVevis
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 97:66ꢀ (c
¼
0.34, CHCl3);
) ¼ 266 nm (4.00); IR (KBr):
(methanol): lmax (log
3
) ¼ 266 nm (3.77); IR (KBr):
n
¼ 3326br,
UVevis (methanol): lmax (log
3
2925s, 2853s, 2153w, 1732 m, 1652 m, 1571s, 1489s, 1386s, 1339 m,
1312s, 1223 m, 1156 m, 986w, 878w, 817w, 770w, 746w, 720w, 686
n
¼ 3421br, 2929s, 2856s, 1732s, 1655s, 1464m, 1386m, 1322m,
1216s, 1154m, 1086w, 1022w, 985m, 879w, 770 (w) cmꢃ1; 1H NMR
(500 MHz, CDCl3):
¼ 5.64 (s, 1H, H-12), 4.57 (dd, 1H, H-3, J ¼ 11.7,
4.6), 3.66 (s, 3H, COOCH3), 3.36 (s, 2H, CH2COO), 2.79 (ddd, 1H, H-1,
(w) cmꢃ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.64 (s, 1H, H-12), 4.56
d
(dd, 1H, H-3, J ¼ 11.8, 4.7), 3.66 (s, 3H, COOCH3), 3.36 (s, 2H,
CH2COO), 2.79 (ddd, 1H, H-1, J ¼ 13.9, 3.4, 3.4), 2.65 (t, 2H, chain-9,

R. Csuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5718e5723
5723
J ¼ 7.0), 2.56 (t, 2H, chain-1, J ¼ 7.2), 2.34 (s, 1H, H-9), 2.06 (m, 1H, H-
18), 2.00 (m, 1H, H-15), 1.97 (m, 1H, H-21), 1.92 (m, 1H, H-19), 1.80
(m, 1H, H-16), 1.70 (m, 1H, H-2), 1.64 (m, 1H, H-7), 1.62 (m, 1H, H-20),
1.58 (dd, 1H, H-190, J ¼ 13.5, 13.5), 1.53 (m, 1H, H-6), 1.48 (m, 2H,
chain-8), 1.43 (m, 1H, H-60), 1.40 (m, 1H, H-70), 1.38 (m, 1H, H-22),
1.34 (s, 3H, H-27), 1.30 (m, 1H, H-210), 1.28e1.25 (m, 13H, H-220,
chain-2, chain-3, chain-4, chain-5, chain-6, chain-7), 1.19 (m, 1H, H-
160), 1.14 (s, 3H, H-25), 1.12 (s, 3H, H-23), 1.10 (s, 3H, H-26), 1.02 (m,
1H, H-10), 0.98 (m, 1H, H-150), 0.85 (s, 6H, H-24, H-28), 0.79 (m, 1H,
screening of the compounds, and to Dr. Th. Müller, Dept. Haema-
tology/Oncology (Univ. Halle) for providing the cell lines.
References
[1] C.S. Lee, Y.J. Kim, M.S. Lee, E.S. Han, S.J. Lee, 18 beta-glycyrrhetinic acid induces
apoptotic cell death in SiHa cells and exhibits a synergistic effect against
antibiotic anti-cancer drug toxicity, Life Sci. 83 (2008) 481e489.
[2] I. Jutooru, G. Chadalapaka, S. Chintharlapalli, S. Papineni, S. Safe, Induction of
apoptosis and nonsteroidal anti-inflammatory drug-activated gene
1 in
pancreatic cancer cells by a glycyrrhetinic acid derivative, Mol. Carcinogen 48
(2009) 692e702.
H-5), 0.78 (s, 3H, H-29); 13C NMR (125 MHz, CDCl3):
d
¼ 200.0 (C-
11), 176.9 (C-30), 172.4 (CH2COO), 169.2 (C-13), 128.5 (C-12), 81.1 (C-
3), 61.7 (C-9), 55.0 (C-5), 51.8 (COOCH3), 51.2 (CH2COO), 49.6 (chain-
1), 48.4 (C-18), 45.4 (C-14), 44.0 (C-20), 43.2 (C-8), 42.2 (chain-9),
41.1 (C-19), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22), 36.9 (C-10), 33.8
(chain-8), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 30.1 (chain-3), 29.6
(chain-6), 29.5 (chain-5), 29.4 (chain-4), 28.5 (C-29), 28.3 (C-23),
28.1 (C-28), 27.2 (chain-2), 26.9 (chain-7), 26.5 (C-16), 26.4 (C-15),
23.6 (C-2), 23.3 (C-27), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-
25); MS (ESI): m/z (%) ¼ 342.6 ([Mþ2H]2þ, 100), 683.4 ([M þ H]þ,
40); anal. calcd for C42H70N2O5 (638.02): C, 73.86; H, 10.33; N, 4.10;
found: C, 73.65; H, 10.54; N, 4.03.
[3] H. Hibasami, H. Iwase, K. Yoshioka, H. Takahashi, Glycyrrhetic acid (a meta-
bolic substance and aglycon of glycyrrhizin) induces apoptosis in human
hepatoma, promyelotic leukemia and stomach cancer cells, Int. J. Mol. Med. 17
(2006) 215e219.
[4] Y. Satomi, H. Nishino, S. Shibata, Glycyrrhetinic acid and related compounds
induce G1 arrest and apoptosis in human hepatocellular carcinoma HepG2,
Anticancer Res. 25 (2005) 4043e4047.
[5] D.R. Lauren, D.J. Jensen, J.A. Douglas, J.M. Follett, Efficient method for deter-
mining the glycyrrhizin content of fresh and dried roots, and root extracts, of
Glycyrrhiza species, Phytochem. Anal. 12 (2001) 332e335.
[6] L.A. Baltina, Chemical modification of glycyrrhizic acid as a route to new
bioactive compounds for medicine, Curr. Med. Chem. 10 (2003) 155e171.
[7] S. Jäger, H. Trojan, T. Kopp, M.N. Laszczyk, A. Scheffler, Pentacyclic triterpene
Distribution in various plants - rich sources for a new group of multi-potent
plant extracts, Molecules 14 (2009) 2016e2031.
[8] Y. Allouche, A. Jimenez, M. Uceda, M.P. Aguilera, J.J. Gaforio, G. Beltran, Tri-
terpenic content and chemometric analysis of virgin olive oils from forty olive
cultivars, J. Agr. Food Chem. 57 (2009) 3604e3610.
5.8.8. Methyl 3b-3-{[N-(10-aminodecl)glycyl]oxy}-11-oxo-olean-
12-en-30-oate (11)
[9] X.D. Su, H. Lawrence, D. Ganeshapillai, A. Cruttenden, A. Purohit, M.J. Reed,
N. Vicker, B.V.L. Potter, Novel 18 beta-glycyrrhetinic acid analogues as potent
and selective inhibitors of 11 beta-hydroxysteroid dehydrogenases, Bioorgan.
Med. Chem. 12 (2004) 4439e4457.
[10] Y. Gao, X. Guo, X. Li, D. Liu, D. Song, Y. Xu, M. Sun, Y. Jing, L. Zhao, The
synthesis of glycyrrhetinic acid derivatives containing a nitrogen heterocycle
and their antiproliferative effects in human leukemia cells, Molecules 15
(2010) 4439e4449.
[11] D. Song, Y. Gao, R. Wang, D. Liu, L.X. Zhao, Y.K. Jing, Downregulation of c-FLIP,
XIAP and Mcl-1 protein as well as depletion of reduced glutathione contribute
to the apoptosis induction of glycyrrhetinic acid derivatives in leukema cells,
Cancer Biol. Ther. 9 (2010) 96e108.
[12] G.S.R. Subba Rao, P. Kondaiah, S.K. Singh, P. Ravanan, M.B. Sporn, Chemical
modifications of natural triterpenes - glycyrrhetinic and boswellic acids:
evaluation of their biological activity, Tetrahedron 64 (2008) 11541e11548.
[13] G. Chadalapaka, I. Jutooru, A. McAlees, T. Stefanac, S. Safe, Structure-depen-
dent inhibition of bladder and pancreatic cancer cell growth by 2-substituted
glycyrrhetinic and ursolic acid derivatives, Bioorg. Med. Chem. Lett. 18 (2008)
2633e2639.
[14] D. Liu, D.D. Song, G. Guo, R. Wang, J.L. Lv, Y.K. Jing, L.X. Zhao, The synthesis of
18 beta-glycyrrhetinic acid derivatives which have increased antiproliferative
and apoptotic effects in leukemia cells, Bioorgan. Med. Chem. 15 (2007)
5432e5439.
[15] W.S. Suh, Y.S. Kim, A.D. Schimmer, S. Kitada, M. Minden, M. Andreeff, N. Suh,
M. Sporn, J.C. Reed, Synthetic triterpenoids activate a pathway for apoptosis in
AML cells involving downregulation of FLIP and sensitization to TRAIL,
Leukemia 17 (2003) 2122e2129.
[16] S. Inoue, R.T. Snowden, M.J.S. Dyer, G.M. Cohen, CDDO induces apoptosis via
the intrinsic pathway in lymphoid cells, Leukemia 18 (2004) 948e952.
[17] T. Ju, H. Yamaguchi, T. Noshita, Y. Kidachi, H. Umetsu, K. Ryoyama, Selective
cytotoxicity of glycyrrhetinic acid against tumorigenic r/m HM-SFME-1 cells:
potential involvement of H-Ras downregulation, Tox. Lett. 192 (2010) 425e430.
[18] H. Yamaguchi, T. Noshita, T. Yu, Y. Kidachi, K. Kamiie, H. Umetsu, K. Ryoyama,
Novel effects of glycyrrhetinic acid on the central nervous system tumorigenic
progenitor cells: induction of actin disruption and tumor cell-sensitive
toxicity, Eur. J. Med. Chem. 45 (2010) 2943e2948.
Following the general procedure, 11 was obtained from 1,10-
decanediamine; yield: 290 mg, 41.6%; mp 82e85 ꢀC; RF ¼ 0.71
(MeOH/diethylamine 9:1);
½
a 2D0
ꢂ
¼ 65:77ꢀ (c
¼
0.45, MeOH);
) ¼ 266 nm (3.83); IR (KBr):
UVevis (methanol): lmax (log
3
n
¼ 3336br, 2923s, 2852s, 1732s, 1652s, 1577m, 1466s, 1387m,
1317m, 1217m, 1154m, 1086w, 987w, 878w, 818w, 721 (w) cmꢃ1; 1H
NMR (500 MHz, CDCl3):
¼ 5.64 (s, 1H, H-12), 4.57 (dd, 1H, H-3,
d
J ¼ 11.6, 4.8), 3.67 (s, 3H, COOCH3), 3.37 (s, 2H, CH2COO), 2.79 (ddd,
1H, H-1, J ¼ 13.3, 3.7, 3.7), 2.65 (t, 2H, chain-10, J ¼ 7.0), 2.57 (t, 2H,
chain-1, J ¼ 7.2), 2.34 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.00 (m, 1H, H-
15),1.97 (m,1H, H-21),1.90 (m,1H, H-19),1.82 (m,1H, H-16),1.70 (m,
1H, H-2), 1.63 (m, 1H, H-7), 1.62 (m, 1H, H-20), 1.59 (dd, 1H, H-190,
J ¼ 13.7,13.7), 1.55 (m,1H, H-6), 1.47 (m, 2H, chain-9), 1.43 (m,1H, H-
60), 1.40 (m, 1H, H-70), 1.38 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.30 (m,
1H, H-210), 1.29e1.24 (m, 15H, H-220, chain-2, chain-3, chain-4,
chain-5, chain-6, chain-7, chain-8), 1.20 (m, 1H, H-160), 1.14 (s, 3H,
H-25), 1.13 (s, 3H, H-23), 1.10 (s, 3H, H-26), 1.02 (m, 1H, H-10), 0.98
(m, 1H, H-150), 0.86 (s, 6H, H-24, H-28), 0.80 (m, 1H, H-5), 0.78 (s,
3H, H-29); 13C NMR (125 MHz, CDCl3):
d
¼ 200.0 (C-11), 176.9 (C-
30), 172.5 (CH2COO), 169.2 (C-13), 128.5 (C-12), 81.0 (C-3), 61.7 (C-
9), 55.0 (C-5), 51.8 (COOCH3), 51.3 (CH2COO), 49.7 (chain-1), 48.4
(C-18), 45.4 (C-14), 44.0 (C-20), 43.2 (C-8), 42.3 (chain-10), 41.1 (C-
19), 38.7 (C-1), 38.1 (C-4), 37.7 (C-22), 36.9 (C-10), 33.8 (chain-9),
32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 30.1 (chain-3), 29.5 (chain-7),
29.5 (chain-5), 29.5 (chain-6), 29.5 (chain-4), 28.5 (C-29), 28.3 (C-
23), 28.1 (C-28), 27.2 (cahin-2), 26.9 (chain-8), 26.5 (C-16), 26.4 (C-
15), 23.6 (C-2), 23.3 (C-27), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4
(C-25); MS (ESI): m/z (%) ¼ 349.5 ([Mþ2H]2þ, 100), 697.4 ([M þ H]þ,
22); anal. calcd for C43H72ClO5 (697.04): C, 74.09; H, 10.41; N, 4.02;
found: C, 73.86; H, 10.61; N, 3.95.
[19] M.-Z. Zhang, J. Xu, B. Yao, H. Yin, Q. Cai, M.J. Shrubsole, X. Chen, V. Kon,
W. Zheng, A. Pozzi, R.C. Harris, Inhibition of 11
b-hydroxysteroid dehydroge-
nase type II selectively blocks the tumor COX-2 pathway and suppresses colon
carcinogenesis in mice and humans, J. Clin. Invest. 119 (2009) 876e885.
[20] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
Acknowledgments
[21] J. Gong, F. Draganos, Z. Darzynkiewicz,
A selective procedure for DNA
Many thanks are due to Dr. R. Paschke and Dr. H. Kommera,
Biozentrum (Univ. Halle) for assistance with the biological
extraction from apoptotic cells applicable for gel electrophoresis and flow
cytometry, Anal. Biochem. 218 (1994) 314e319.