Improvement of the cytotoxicity and tumor selectivity of glycyrrhetinic acid by derivatization with bifunctional aminoacids

Article abstract of DOI:10.1002/ardp.201100030

Glycyrrhetinic acid (GA) is a major ingredient of the dried extract of licorice roots; its antitumor activity is low compared to other members of the triterpenoic family. For example, oleanolic acid, betulin or betulinic acid are more cytotoxic with a pro

Full text of DOI:10.1002/ardp.201100030

Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
505
Full Paper
Improvement of the Cytotoxicity and Tumor Selectivity of
Glycyrrhetinic Acid by Derivatization with Bifunctional
Aminoacids
´
Rene Csuk, Stefan Schwarz, Ralph Kluge, and Dieter Strohl
¨
¨
Martin-Luther-Universitat Halle-Wittenberg, Bereich Organische Chemie, Halle (Saale), Germany
Glycyrrhetinic acid (GA) is a major ingredient of the dried extract of licorice roots; its antitumor
activity is low compared to other members of the triterpenoic family. For example, oleanolic
acid, betulin or betulinic acid are more cytotoxic with a pronounced activity for tumor cells. GA,
however, is easily to earn, cheap and shows apoptotic effects on tumor cells – like the other
triterpenoic acids. These facts bring GA and derivatives in the focus of our scientific interest. Here
we tried to improve the poor cytotoxicity of GA by simple derivatization. Thus, we selected various
glutamyl and aspartyl substituents for the synthesis of C(3) esters of GA methyl ester. A short (3-5 steps)
synthesis was elaborated that allowed to access more effective compounds. One compound, methyl
3b 3-(O-benzyl-L-glutamyl)-11-oxo-olean-12-en-30-oate (5), having a glutamyl substituent with a
benzyl protected side chain showed up to 67-fold higher cytoxicity and an up to 140-fold better
selectivity towards tumor cells than parent GA. All compounds were evaluated by a sulforhodamine B
assay as well as by a trypan blue test and extra acridine orange/ethidium bromide tests for
apoptosis.
Keywords: Antitumor activity / Apoptosis / Glycyrrhetinic acid / Mouse embryonic fibroblasts
Received: January 27, 2011; Revised: March 21, 2011; Accepted: April 6, 2011
DOI 10.1002/ardp.201100030
Introduction
the cytotoxicity and to improve the selectivity. From prelimi-
nary screenings, aspartyl and glutamyl substituted deriva-
tives of GA methyl ester (1) appeared most promising. Thus,
these substances were evaluated in a sulforhodamine B assay
(SRB) as well as by acridine orange/ethidium bromide (AO/EB)
and trypan blue tests.
Searching for an effective antitumor drug does not necessarily
mean to look for the most cytotoxic molecules. Although, a
high toxicity is compulsory, other features – among them the
ability to induce apoptosis and a high selectivity between
tumor cells and normal cells – are important.
Glycyrrhetinic acid (GA), a triterpenoic acid has a proven Results and discussion
[1–5] cytotoxic effect on tumor cells. It is also known [1–3, 6]
to induce apoptosis. Because of the high content of GA (up to
24%) found in the roots of licorice (Glycyrrhiza glabra) [7, 8], GA
is still in the focus of scientific interest. However, the cyto-
toxicity of GA is significantly lower [4] than those of other
triterpenoic acids (for example betulinic acid, oleanolic acid,
ursolic acid). In addition, GA displayed only a poor selectivity
[6] towards tumor cells. Hence, our main aim was to increase
Four pairs of different aspartic and glutamic acid substituted
GA derivatives (see Scheme 1) were investigated in a SRB assay
to determine their cytotoxicity on various tumor cell lines.
The protected compounds 8 and 9 did not show any signifi-
cant activity on the cells in the applied range of concen-
trations. Derivatives 6 and 7, however, still protected by a
N-Boc group, showed lower IC50 values on eight tumor cell
lines (cf. Table 1) than the methyl ester [6] of GA. The tumor-to-
control selectivity (as estimated dividing the IC50 for NiH3T3
(mouse embryonic fibroblasts) by an IC50 value (averaged for
all tumor cell lines) increased from 1.09 (for 1) to 1.35 (for 5)
and 1.36 (for 6).
´
Correspondence: Prof. Rene Csuk, Bereich Organische Chemie, Martin-
Luther-Universitat Halle-Wittenberg, Kurt-Mothes-Strasse 2, D-06120
¨
Halle (Saale), Germany.
E-mail: rene.csuk@chemie.uni-halle.de
Fax: þ49 (0) 345 5527030
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R. Csuk et al.
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Scheme 1. Reagents and conditions: a) DCC, DMAP, Boc-Asp(OBzl)OH or Boc-Glu(OBzl)OH, DCM, 12 h, 258C; b) TFA, DCM, 12 h,
-
258C; NH4þHCO2 , Pd/C (10%), THF/MeOH, 12 h, 258C; d) HCl (gas), DCM, 12 h, 258C; e) CH3I, K2CO3, DMF, 2 h, 258C.
Compounds 4 and 5 as well as compounds 12 and 13 still
carry a free amino group but a protected side chain. The two
methyl esters 12 and 13 showed an insignificant activity,
except for compound 12 on the human breast adenocarci-
noma cell line MCF-7. Selectivity switches: again, except for
the MCF-7 cells, the IC50 value for NiH3T3 cells is lower than
for the human tumor cell lines, therefore resulting in a
selectivity index of 0.55 for 12 and 0.72 for 13.
IC50 of 15-19 mM. For MCF-7 cells, compound 4 showed the
highest activity (IC50 ¼ 7.35 mM), hence doubling the selec-
tivity towards the tumor cell line. Compound 5, being the
most active derivative of this series gave IC50 values between
1.27-2.33 mM. Compared to other compounds, 5 displayed an
extraordinary selectivity F ¼ 23 in average. For A253 human
head and neck carcinoma cells a 30 times lower IC50 value for
the tumor cells than for the NiH3T3 fibroblasts is observed.
To rule out an alleged apoptotic behavior, an AO/EB test
was performed. For all of the compounds, except for 8 and 9
(that did not show a significant activity in the SRB assay) a
sufficient number of cells undergoing apoptosis were
The use of a benzyl group to protect the side chain led to
compounds showing the best results in the SRB assay. The
aspartic acid derivative 4 showed the same selectivity (1.35) as
the Boc-protected compound 6 (with a free side chain) but an
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Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
Glycyrrhetinic Acid Derivatives
507
Table 1. Results of the SRB-assay on various tumor cell lines and mouse embryonic fibroblasts (NiH3T3): Results (IC50) are given in mM; F
indicates the quotient of the averaged IC50 value (tumor cells) divided by the IC50 (mouse fibroblasts).
substance/cell line
4
5
6
7
8
9
12
13
518A2
8505C
A253
A2780
A549
Lipo
MCF-7
SW1736
average
NiH3T3
F
10.90 ꢀ 0.55
12.97 ꢀ 0.65
7.99 ꢀ 0.40
8.84 ꢀ 0.44
10.94 ꢀ 0.55
11.35 ꢀ 0.57
7.35 ꢀ 0.36
16.68 ꢀ 0.83
10.88 ꢀ 0.54
14.74 ꢀ 0.74
1.35
1.75 ꢀ 0.09
1.76 ꢀ 0.09
1.28 ꢀ 0.06
1.65 ꢀ 0.08
1.77 ꢀ 0.09
1.74 ꢀ 0.09
1.27 ꢀ 0.06
2.33 ꢀ 0.12
1.69 ꢀ 0.08
39.09 ꢀ 1.95
23.13
17.19 ꢀ 0.86
15.82 ꢀ 0.79
15.07 ꢀ 0.75
17.29 ꢀ 0.86
19.82 ꢀ 0.99
16.67 ꢀ 0.83
17.47 ꢀ 0.87
17.13 ꢀ 0.86
17.06 ꢀ 0.85
23.09 ꢀ 1.15
1.35
17.94 ꢀ 0.90
17.00 ꢀ 0.85
13.80 ꢀ 0.69
18.24 ꢀ 0.91
21.20 ꢀ 1.06
18.78 ꢀ 0.94
16.96 ꢀ 0.85
19.24 ꢀ 0.96
17.90 ꢀ 0.90
24.42 ꢀ 1.22
1.36
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
39.24 ꢀ 1.96
45.36 ꢀ 2.27
30.47 ꢀ 1.52
22.44 ꢀ 1.12
31.59 ꢀ 1.58
40.62 ꢀ 2.03
16.89 ꢀ 0.84
20.85 ꢀ 1.04
30.93 ꢀ 1.55
16.89 ꢀ 0.84
0.55
47.72 ꢀ 2.39
61.57 ꢀ 3.08
53.07 ꢀ 2.65
29.19 ꢀ 1.46
60.96 ꢀ 3.05
54.77 ꢀ 2.74
29.26 ꢀ 1.46
38.50 ꢀ 1.93
46.88 ꢀ 2.34
33.63 ꢀ 1.68
0.72
detected as depicted in Fig. 1. In this assay apoptotic cells
appear in a green color with bright fluorescent parts whereas
necrotic cells exhibit an orange color.
To determine the extent of apoptosis, A549 lung carcinoma
cells were incubated with our compounds at a concentration
>IC50, a trypan blue test was performed and quantification of
apoptosis was done by using an automated cell counter
(Table 2). In this test, GA has a default value of approx.
74%; all of our compounds showed levels of apoptosis
between 65-86%.
In this study a number of derivatives of GA methyl ester (1)
were synthesized. For compound 5 a IC50 ¼ 1.27 mM for
MCF-7 cells was established; the toxicity of 5 for A549
cells (IC50 ¼ 1.77 mM) is higher than that of betulinic acid
(IC50 ¼ 10.3 mM [9] or 11.9 mM [10], oleanolic acid (IC50
¼
27 mM [9], betulin [IC50 ¼ 3.8 mM [9]) or parent glycyrrhetinic
acid (IC50 ¼ 63.2 mM [2] or IC50 ¼ 82.8 mM [6]). In addition,
previously for derivatives of GA similar to CDDO [11] a
significantly lower activity on various tumor cells has been
reported.
A comparison of the aspartyl and glutamyl substituted
derivatives revealed an interesting behavior. For the Boc-
protected compounds having an unprotected side chain
(compounds 6 and 7) no significant difference in the IC50
value is observed. Both compounds show IC50 values of circa
17-18 mM with a slightly higher value for NiH3T3 mouse
fibroblasts (23.09 mM and 24.42 mM, respectively). The depro-
tected derivatives 8 and 9, however, did not show any activity
Figure 1. Results from the AO/EB test: treatment of A549 cells
(from left to right): 4 (15 mM), 5 (3 mM), 6 (25 mM), 7 (25 mM), 12
(35 mM) and 13 (80 mM).
Table 2. Apoptotic effect of GA derivatives in A549 cells in % (ꢀ standard error, 6 experiments), cells were treated with GA (90 mM), 4
(15 mM), 5 (3 mM), 6 (30 mM), 7 (30 mM), 12 (40 mM) and 13 (80 mM).
Compound
GA
4
5
6
7
12
13
Apoptosis [%] 73.73 ꢀ 1.40
80.53 ꢀ 2.93
86.10 ꢀ 2.98
65.50 ꢀ 3.08
82.63 ꢀ 3.36
76.76 ꢀ 3.20
79.44 ꢀ 2.17
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Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
at concentrations <100 mM at all. They are neither cytotoxic
for tumor cells nor for the mouse embryonic fibroblasts.
Pairs of side chain protected derivatives show anomalous
activity with respect to the protecting group. The methyl
esters 12 and 13 show higher IC50 values than compounds
6 and 7 – except for 12 on MCF-7 cells. Furthermore, the
aspartyl substituent (in 12) seems to be more effective than
a glutamyl moiety. For 12 a lower IC50 value is found for each
of the cell lines including the fibroblasts. The selectivity of
both compounds, however, is worse than for all other
compounds.
Cell lines and culture conditions
The cell lines 518A2, 8505C, A253, A2780, A549, LIPO, MCF-7,
NiH3T3, and SW1736 were included in this study. 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 penicil-
lin/streptomycin (PAA Laboratories) at 378C in a humidified
atmosphere of 5% CO2/95% air.
Cytotoxicity Assay [13]
The cytotoxicity of the compounds was evaluated using the
sulforhodamine B (SRB) (Sigma Aldrich) microculture colorimet-
ric 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 com-
pounds (0–100 mM) for 96 h. The final concentration of DMSO or
DMF solvent never exceeded 0.5% which was non-toxic to the
cells. The percentages of surviving cells relative to untreated
controls were determined 96 h after the beginning of drug
exposure. After a 96 h treatment, the supernatant medium from
the 96 well plates was thrown away and the cells were fixed with
10% TCA. For a thorough fixation, the plates were allowed to rest
at 48C. After fixation, the cells were washed in a strip washer. The
washing was done four times with water using alternate dis-
pensing and aspiration procedures. The plates were then dyed
with 100 mL of 0.4% SRB (sulforhodamine B) for about 20 min.
After dying, the plates were washed with 1% acetic acid to
remove the excess of the dye and allowed to air dry overnight.
100 mL of 10 mM Tris base solution were added to each well and
absorbance was measured at n ¼570 nm (using a 96 well plate
reader, Tecan Spectra, Crailsheim, Germany). The IC50 was esti-
mated by linear regression between the value before and after
the 50% line is crossed in a dose-response curve.
The benzyl esters 4 and 5 are the most effective cytotoxic
compounds of this study. As indicated in a previous investi-
gation [6], combining a free amino group and an unprotected
carboxylic group creates inactive compounds – as exempli-
fied for 8 and 9. The derivatives 6 and 7 are more active than
12 and 13; obviously an unprotected amino group is not that
important for retaining cytotoxicity as previously assumed.
Changing a methyl ester to a benzyl ester (as exemplified in 3
and 4) increases activity. Hence, a free amino group seems
necessary for gaining cytotoxicity; a benzyl group seems to
add to stability under biological conditions and to higher
lipophilicity.
Glutamyl substituted 5 is more active than aspartyl sub-
stituted 4. The reason for this behavior remains unclear up to
now and has to be subject of further investigations. The
selectivities of these compounds are better than for parent
GA but lower [12] as for betulinic acid. Derivative 5, however,
combines a higher cytotoxicity and an outstanding selectiv-
ity of approx. 30 (for MCF-7 cells). Derivatization does not
affect apoptosis as deduced from the AO/EB test.
Apoptosis Test – Acridine Orange/Ethidium Bromide
(AO/EB)
In conclusion, we were able to increase the cytotoxicity of
GA by introducing glutamyl and aspartyl substituents.
Compound 5 showed an IC50 ¼ 1.27 mM for MCF-7 cells
(67-fold better than GA) and a tumor-to-control selectivity
of approx. 30.8 (for MCF-7 vs. NiH3T3; 140-fold better than
GA). Hence, for MCF-7 cells, compound 5 is more effective but
also more selective than gold standard betulinic acid.
Apoptotic cell death was analysed by acridine orange/ethidium
bromide dye using fluorescence microscopy on A549 cells.
Therefore 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 24–48 h, the
supernatant medium was collected and centrifuged; the pellet
was suspended in phosphate-buffer 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 performed under a fluorescence microscope. While
a green fluorescence shows apoptosis, a red colored nucleus
indicates necrotic cells.
Experimental
General
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), optical rotations were obtained using a
Perkin-Elmer 341 polarimeter (1 cm micro cell, 258C), IR spectra
(film or KBr pellet) on a Perkin-Elmer 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.
Apoptosis test – 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 24–48 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
(InvitrogenTM). Automatic cell counter (InvitrogenTM countess1
automated cell counter) was used for counting the cells,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
Glycyrrhetinic Acid Derivatives
509
differing between cells with an intact cell membrane and cells
without.
added. After 30 min of stirring at room temperature, iodo-
methane (1.1 eq.) was added and the mixture was stirred for an
additional 2 h. The solvents were evaporated and the crude res-
idue was dissolved in a mixture of DCM (30 mL) and aq. hydro-
chloric acid (1.0 M). The aqueous layer was extracted with DCM
(3 ꢁ 25 mL), the combined organic layers were washed with
brine, dried (Na2SO4), filtered and the solvent was evaporated.
Purification by flash chromatography (SiO2, hexane/ethyl acetate
8:2) yielded the ester.
Apoptosis test — 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 24–48 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
(InvitrogenTM). Automatic cell counter (InvitrogenTM countess1
automated cell counter) was used for counting the cells,
differing between cells with an intact cell membrane and cells
without.
Methyl 3b 3-(Boc-O-benzyl-L-asparagyl)-11-oxo-olean-12-
en-30-oate (2)
Obtained from 1 (530 mg, 1.1 mmol) and Boc-L-Asp(OBzl)OH
(410 mg, 1.27 mmol) by method A as a colorless powder; yield:
720 mg, 83%; mp 74–768C; RF ¼ 0.71 (hexane/ethyl acetate 7:3);
[a]D ¼ 77.688 (c ¼ 0.27, CHCl3); UV-VIS (methanol): lmax
(log e) ¼ 206 nm (4.08), 249 nm (3.95); IR (KBr): n ¼ 3439 (br),
2976 (m), 2361 (w), 1731 (s), 1659 (m), 1499 (m), 1456 (m), 1389
(m), 1367 (m), 1217 (s), 1166 (s), 1048 (w), 1025 (w), 986 (w), 863
General procedure for the esterifications in position
C(3) (method A)
1
(w), 752 (w), 698 (w) cmꢂ1; H-NMR (500 MHz, CDCl3): d ¼ 7.38–
The starting material (1 eq.) was dissolved in dry DCM (15 mL),
DMAP (20 mg, 0.16 mmol) and the protected amino acid (1.2 eq.)
were added. After addition of DCC (1.2 eq.) the mixture was stirred
at room temperature for 12 h, filtered. The filtrate was washed
with water and brine (15 mL each), dried over sodium sulfate,
filtered and the solvent was evaporated. Purification was per-
formed by flash chromatography (SiO2, hexane/ethyl acetate 8:2).
7.31 (m, 5H, H-Ar), 5.67 (s, 1H, H-12), 5.51 (d, 1H, NH, J ¼ 8.9 Hz),
5.11 (s, 2H, Bzl-CH2), 4.56 (m, 2H, Asp-CH), 4.55 (dd, 1H, H-3,
J ¼ 11.5, 4.9 Hz), 3.70 (s, 3H, OMe), 3.05 (ddd, 1H, Asp-CHH’,
J ¼ 15.9, 15.9, 3.9 Hz), 2.87 (ddd, 1H, Asp-CHH’, J ¼ 17.1, 4.7,
1.9 Hz), 2.81 (ddd, 1H, H-1, J ¼ 13.7, 3.5, 3.5 Hz), 2.35 (s, 1H,
H-9), 2.09 (dd, 1H, H-18, J ¼ 14.0, 3.6 Hz), 2.03 (m, 1H, H-15),
2.00 (m, 1H, H-21), 1.93 (ddd, 1H, H-19, J ¼ 13.7, 3.6, 2.4 Hz),
1.83 (ddd, 1H, H-16, J ¼ 13.7, 13.7, 4.3 Hz), 1.70 (m, 1H, H-2), 1.66
(m, 1H, H-7), 1.63 (m, 1H, H-2’), 1.59 (dd, 1H, H-19’, J ¼ 13.5,
13.5 Hz), 1.57 (m, 1H, H-6), 1.44 (s, 9H, Boc-CH3), 1.42 (m, 1H,
H-6’), 1.39 (m, 1H, H-7’), 1.38 (m, 1H, H-22), 1.37 (s, 3H, H-27), 1.32
(m, 1H, H-22’), 1.32 (m, 1H, H-21’), 1.18 (m, 1H, H-16’), 1.15 (s, 3H,
H-25), 1.15 (s, 3H, H-29), 1.13 (s, 3H, H-26), 1.03 (m, 1H, H-1’), 1.02
(m, 1H, H-15’), 0.84 (s, 3H, H-24), 0.81 (s, 3H, H-23), 0.81 (s, 3H,
H-28), 0.78 (m, 1H, H-5) ppm; 13C-NMR (125 MHz, CDCl3):
d ¼ 200.0 (C-11), 176.9 (C-30), 170.9 (Asp-COOBzl), 170.7 (Asp-
COO), 169.2 (C-13), 155.4 (Boc-COO), 135.7 (Car), 128.6 (Car),
128.6 (Car), 128.5 (Car), 128.4 (C-12), 128.3 (Car), 128.3 (Car), 82.3
(C-3), 80.0 (Boc-quart.-C), 66.8 (Bzl-CH2), 61.7 (C-9), 55.0 (C-5), 51.8
(OMe), 50.2 (Asp-CHNH2), 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
(Asp-CH2), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.5 (C-28), 28.3 (C-29),
28.3 (Boc-CH3), 27.9 (C-23), 26.5 (C-16), 26.4 (C-15), 23.4 (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,
MeOH): m/z (%) ¼ 790.1 ([M þ H]þ, 10), 812.3 ([M þ Na]þ, 100);
anal. calcd. for C47H67O9 (790.03): C, 71.45; H, 8.55; N, 1.77; found:
C, 71.32; H, 8.67; N, 1.59.
General procedure for deprotection (method B)
To a solution of the Boc-protected compound in dry DCM
(10 mL/mmol), trifluoroacetic acid (1 mL/10 mL DCM) was added.
The mixture was allowed to stir at room temperature for 12 h.
After completion of the reaction (as monitored by tlc), the
solution was washed with a satd. aq. solution of sodium hydro-
gen carbonate (20 mL). The aqueous layer was extracted with
DCM (3 ꢁ 25 mL), the combined organic extracts were washed
with brine (20 mL), dried over sodium sulfate, filtrated and
evaporated to yield the amine.
General procedure for deprotection (method C) [14]
The solution of the benzyl ester (1 eq.) in a mixture of MeOH and
THF (1:2, 50 mL/mmol) was purged with argon for 3 min, followed
by the addition of ammonium formate (5 eq.). Palladium on
activated coal (10%; 100 mg/mmol) was added and the mixture
was stirred at room temperature for 12–14 h. The solvents were
removed under reduced pressure and the residue was dissolved in
DCM (25 mL). Usual aqueous work-up followed by chromatog-
raphy (SiO2, hexane/ethyl acetate 1:1) yielded the product.
General procedure for the deprotection (method D)
The Boc-protected compound was dissolved in dry DCM
(10 mL/mmol). After saturation with dry hydrogen chloride gas
for 15 min stirring at room temperature was continued for 12 h.
After completion of the reaction (as monitored by tlc), the solvent
was removed under reduced pressure. The residue was washed
with ethyl acetate until no parent substance could be detected;
analytical samples were obtained by re-crystallization.
Methyl 3b 3-(Boc-O-benzyl-L-glutamyl)-11-oxo-olean-12-
en-30-oate (3)
Obtained from 1 (560 mg, 1.16 mmol) and Boc-L-Glu(OBzl)OH
(440 mg, 1.28 mmol) by method A as a colorless powder; yield:
680 mg, 73%; mp 139–1438C; RF ¼ 0.41 (hexane/ethyl acetate
7:3); [a]D ¼ 75.928 (c ¼ 0.28, CHCl3); UV-VIS (methanol): lmax
(log e) ¼ 205 nm (4.30), 249 nm (4.18); IR (KBr): n ¼ 3386 (br),
2950 (s), 1732 (s), 1659 (m), 1500 (m), 1455 (m), 1390 (m), 1367 (m),
1257 (m), 1216 (m), 1165 (s), 1086 (w), 1049 (w), 1027 (w), 986 (w),
1
751 (w), 698 (w) cmꢂ1; H-NMR (500 MHz, CDCl3): d ¼ 7.39–7.29
General procedure for the esterification (method E)
To a solution of the starting material (1 eq.) in dry DMF
(15 mL/mmol), finely grounded potassium carbonate (2 eq.) was
(m, 5H, H-Ar), 5.67 (s, 1H, H-12), 5.12 (s, 2H, Bzl-CH2), 5.10 (m, 1H,
Glu-NH), 4.56 (dd, 1 H, H-3, J ¼ 11.6, 4.7 Hz), 4.33 (m, 1 H, Glu-
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R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
CHN), 3.69 (s, 3H, OMe), 2.82 (ddd, 1H, H-1, J ¼ 13.8, 3.4, 3.4 Hz),
2.51 (m, 1H, Glu-CHH’COOBzl), 2.44 (m, 1H, Glu-CHH’COOBzl),
2.35 (s, 1H, H-9), 2.24 (m, 1H, Glu-CHH’CHN), 2.08 (dd, 1H, H-18,
J ¼ 13.7, 3.5 Hz), 2.03 (m, 1H, H-15), 1.99 (m, 1H, H-21), 1.96 (m,
1H, Glu-CHH’CHN), 1.92 (m, 1H, H-19), 1.83 (ddd, 1H, H-16,
J ¼ 13.9, 13.9, 4.4 Hz), 1.73 (m, 1H, H-2), 1.66 (m, 1H, H-7), 1.63
(m, 1H, H-2’), 1.61 (dd, 1H, H-19’, J ¼ 13.3, 13.3 Hz), 1.58 (m, 1H,
H-6), 1.48 (m, 1H, H-6’), 1.43 (s, 9H, Boc-CH3), 1.41 (m, 1H, H-7’),
1.39 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31 (m, 2H, H-22’ and H-21’),
1.18 (m, 1H, H-16’), 1.16 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.13 (s, 3H,
H-26), 1.08 (m, 1H, H-1’), 1.02 (m, 1H, H-15’), 0.88 (s, 3H, H-24), 0.86
(s, 3H, H-23), 0.81 (s, 3H, H-28), 0.79 (m, 1H, H-5) ppm; 13C-NMR
(125 MHz, CDCl3): d ¼ 199.9 (C-11), 176.9 (C-30), 172.6 (Glu-
COOBzl), 171.9 (Glu-COO), 169.2 (C-13), 155.4 (Boc-COO), 135.8
(Car.), 128.5 (Car.), 128.5 (Car.), 128.5 (C-12), 128.2 (Car.), 128.2 (Car.),
128.2 (Car.), 82.2 (C-3), 79.9 (Boc-quart.-C), 61.7 (C-9), 55.0 (C-5), 53.3
(Glu-CHNH), 51.8 (OMe), 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), 30.4 (Glu-CH2COOBzl), 28.5 (C-28),
28.3 (C-29), 28.3 (Boc-CH3), 28.1 (C-23), 27.9 (Glu-CH2CHNH),
26.5 (C-16), 26.4 (C-15), 23.5 (C-2), 23.3 (C-27), 18.7 (C-26), 17.3
(C-6), 16.8 (C-24), 16.4 (C-25) ppm; MS (ESI, MeOH): m/z (%) ¼ 804.1
([M þ H]þ, 8), 826.3 ([M þ Na]þ, 100); anal. calcd. for C48H69NO9
(804.06): C, 71.70; H, 8.65; N, 1.74; found: C, 71.56; H, 8.81; N,
1.62.
Methyl 3b 3-(O-benzyl-L-glutamyl)-11-oxo-olean-12-en-
30-oate (5)
Obtained from 3 (150 mg, 0.19 mmol) and TFA (1 mL, 13 mmol)
by method B as a colorless powder; yield: 90 mg, 64%; mp 282–
2888C (dec.); RF ¼ 0.74 (dichloromethane/methanol 9:1);
[a]D ¼ 100.518 (c ¼ 0.66, CHCl3); UV-VIS (methanol): lmax (log
e) ¼ 249 nm (3.99); IR (KBr): n ¼ 3398 (br), 2935 (s), 1729 (s),
1660 (s), 1464 (m), 1387 (m), 1322 (m), 1280 (m), 1217 (s), 1152
(s), 1084 (m), 1024 (m), 986 (m), 916 (w), 870 (w), 822 (w), 768 (w),
737 (w), 699 (m), 590 (w), 543 (w), 481 (w) cmꢂ1 1H-NMR
;
(500 MHz, CDCl3): d ¼ 7.39–7.29 (m, 5H, H-Ar), 5.67 (s, 1H,
H-12), 5.12 (s, 2H, Bzl-CH2), 4.57 (dd, 1H, H-3, J ¼ 11.8, 4.7 Hz),
4.22 (m, 1H, Glu-CHN), 3.67 (s, 3H, OMe), 2.81 (ddd, 1H, H-1,
J ¼ 13.6, 3.7, 3.7 Hz), 2.57 (m, 1H, Glu-CHH’COOBzl), 2.47
(m, 1H, Glu-CHH’COOBzl), 2.33 (s, 1H, H-9), 2.21 (m, 1H,
Glu-CHH’CHN), 2.07 (m, 1H, H-18), 2.01 (m, 1H, H-15), 1.97
(m, 1H, H-21), 1.97 (m, 1H, Glu-CHH’CHN), 1.90 (ddd, 1H, H-19,
J ¼ 13.7, 4.2, 2.2 Hz), 1.81 (ddd, 1H, H-16, J ¼ 14.0, 14.0, 4.2 Hz),
1.72 (m, 1H, H-2), 1.66 (m, 1H, H-7), 1.61 (m, 1H, H-2’), 1.59 (dd, 1H,
H-19’, J ¼ 13.4, 13.4 Hz), 1.57 (m, 1H, H-6), 1.45 (m, 1H, H-6’), 1.40
(m, 1H, H-7’), 1.38 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.29 (m, 2H,
H-22’ and H-21’), 1.16 (m, 1H, H-16’), 1.15 (s, 3H, H-25), 1.13 (s, 3H,
H-29), 1.11 (s, 3H, H-26), 1.06 (m, 1H, H-1’), 1.00 (m, 1H, H-15’), 0.86
(s, 3H, H-24), 0.86 (s, 3H, H-23), 0.78 (s, 3H, H-28), 0.77 (m, 1H, H-5)
ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 199.9 (C-11), 177.4 (Glu-
COOBzl), 176.9 (C-30), 171.6 (Glu-COO), 169.2 (C-13), 135.8 (Car.),
128.6 (Car.), 128.6 (Car.), 128.5 (C-12), 128.2 (Car.), 128.2 (Car.), 128.2
(Car.), 82.2 (C-3), 61.7 (C-9), 55.0 (C-5), 53.6 (Glu-CHNH), 51.7 (OMe),
48.4 (C-18), 45.3 (C-8), 44.0 (C-20), 43.2 (C-14), 41.1 (C-19), 38.6 (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),
30.5 (Glu-CH2COOBzl), 29.3 (Glu-CH2CHNH), 28.5 (C-28), 28.3
(C-29), 28.1 (C-23), 26.5 (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), 16.4 (C-25) ppm; MS (ESI, MeOH):
m/z (%) ¼ 704.3 ([M þ H]þ, 100); anal. calcd. for C43H61NO7
(703.95): C, 73.37; H, 8.73; N, 1.99; found: C, 73.22; H, 8.99; N,
1.85.
Methyl 3b 3-(O-benzyl-L-asparagyl)-11-oxo-olean-12-en-
30-oate (4)
Obtained from 2 (150 mg, 0.19 mmol) and TFA (1 mL, 13.0 mmol)
by method B as a colorless powder; yield: 120 mg, 73%; mp 172–
1798C; RF ¼ 0.78 (dichloromethane/methanol 9:1); [a]D ¼ 86.228
(c ¼ 0.35, CHCl3); UV-VIS (methanol): lmax (log e) ¼ 207 nm
(4.18), 249 nm (4.08); IR (KBr): n ¼ 3414 (br), 2960 (s), 1728 (s),
1652 (s), 1616 (m), 1456 (m), 1390 (s), 1354 (m), 1324 (m), 1264 (s),
1216 (s), 1172 (s), 1086 (m), 1028 (m), 985 (m), 971 (m), 910 (w), 869
(w), 826 (w), 752 (m), 699 (m), 589 (w), 546 (w), 508 (w) cmꢂ1
;
1H-NMR (500 MHz, CDCl3): d ¼ 7.36–7.29 (m, 5H, H-Ar), 5.64 (s, 1H,
H-12), 5.12 (s, 2H, Bzl-CH2), 4.55 (dd, 1H, H-3, J ¼ 11.7, 4.9 Hz),
3.82 (dd, 2H, Asp-CH, J ¼ 5.5, 5.5 Hz), 3.67 (s, 3H, OMe), 2.88 (dd,
1H, Asp-CHH’, J ¼ 16.5, 4.6 Hz), 2.80 (m, 1H, H-1), 2.76 (m, 1H,
Asp-CHH’), 2.33 (s, 1H, H-9), 2.06 (m, 1H, H-18), 2.00 (m, 1H, H-15),
1.97 (m, 1H, H-21), 1.90 (ddd, 1H, H-19, J ¼ 13.4, 3.6, 2.8 Hz), 1.80
(ddd, 1H, H-16, J ¼ 13.7, 13.7, 4.3 Hz), 1.67 (m, 1H, H-2), 1.65 (m,
1H, H-7), 1.62 (m, 1H, H-2’), 1.59 (dd, 1H, H-19’, J ¼ 13.5, 13.5 Hz),
1.55 (m, 1H, H-6), 1.42 (m, 1H, H-6’), 1.39 (m, 1H, H-7’), 1.36 (m, 1H,
H-22), 1.34 (s, 3H, H-27), 1.29 (m, 1H, H-22’), 1.29 (m, 1H, H-21’),
1.16 (m, 1H, H-16’), 1.13 (s, 3H, H-25), 1.13 (s, 3H, H-29), 1.10 (s, 3H,
H-26), 1.02 (ddd, 1H, H-1’, J ¼ 14.1, 14.1, 3.7 Hz), 1.00 (m, 1H,
H-15’), 0.83 (s, 3H, H-24), 0.81 (s, 3H, H-23), 0.78 (s, 3H, H-28), 0.77
(m, 1H, H-5) ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 199.9 (C-11),
176.9 (C-30), 171.0 (Asp-COO), 171.0 (Asp-COOBzl), 169.2 (C-13),
135.5 (Car), 128.6 (Car), 128.6 (Car), 128.5 (C-12), 128.3 (Car), 128.3
(Car), 128.2 (Car), 81.9 (C-3), 66.7 (Bzl-CH2), 61.6 (C-9), 55.0 (C-5), 51.7
(OMe), 51.2 (Asp-CHNH2), 48.4 (C-18), 45.4 (C-8), 44.0 (C-20), 43.2
(C-14), 41.1 (C-19), 38.9 (Asp-CH2), 38.7 (C-1), 38.1 (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-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),
18.6 (C-26), 17.3 (C-6), 16.8 (C-24), 16.3 (C-25) ppm; MS (ESI, MeOH):
m/z (%) ¼ 690.2 ([M þ H]þ, 100); anal. calcd. for C42H59NO7
(689.92): C, 73.12; H, 8.62; N, 2.03; found: C, 73.02; H, 8.72; N,
1.87.
Methyl 3b 3-(Boc-L-asparagyl)-11-oxo-olean-12-en-30-
oate (6)
Obtained from 2 (390 mg, 0.49 mmol) by method C as a colorless
powder; yield: 310 mg, 90%; mp 213–2158C; RF ¼ 0.22 (hexane/
ethyl acetate 1:1); [a]D ¼ 68.908 (c ¼ 0.90, CHCl3); UV-VIS (meth-
anol): lmax (log e) ¼ 249 nm (4.10); IR (KBr): n ¼ 3366 (br), 2974
(s), 2874 (m), 1732 (s), 1654 (s), 1521 (s), 1456 (m), 1392 (s), 1369 (s),
1288 (m), 1216 (s), 1166 (s), 1087 (m), 1043 (m), 1025 (m), 986 (m),
971 (m), 919 (w), 903 (w), 882 (w), 867 (w), 824 (w), 770 (w), 670 (w),
541 (w) cmꢂ1
;
1H-NMR (500 MHz, CDCl3): d ¼ 5.65 (s, 1H, H-12),
5.47 (m, 1H, NH), 4.55 (m, 2H, H-3 and Asp-CH), 3.67 (s, 3H, OMe),
3.05 (m, 1H, Asp-CHH’), 2.87 (m, 1H, Asp-CHH’), 2.81 (m, 1H, H-1),
2.33 (s, 1H, H-9), 2.06 (m, 1H, H-18), 1.99 (m, 1H, H-15), 1.97 (m, 1H,
H-21), 1.93 (m, 1H, H-19), 1.82 (m, 1H, H-16), 1.72 (m, 1H, H-2), 1.68
(m, 1H, H-2’), 1.63 (m, 1H, H-7), 1.59 (dd, 1H, H-19’, J ¼ 13.7,
13.7 Hz), 1.55 (m, 1H, H-6), 1.43 (s, 9H, Boc-CH3), 1.42 (m, 1H,
H-6’), 1.40 (m, 1H, H-7’), 1.37 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.30
(m, 1H, H-22’), 1.28 (m, 1H, H-21’), 1.18 (m, 1H, H-16’), 1.14 (s, 3H,
H-25), 1.13 (s, 3H, H-29), 1.10 (s, 3H, H-26), 1.03 (m, 1H, H-1’), 0.98
(m, 1H, H-15’), 0.85 (s, 3H, H-24), 0.84 (s, 3H, H-23), 0.78 (s, 3H, H-
28), 0.77 (m, 1H, H-5) ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 200.1
(C-11), 176.9 (C-30), 176.9 (Asp-COOH), 170.8 (Asp-COO), 169.4 (C-
13), 155.5 (Boc-COO), 128.5 (C-12), 82.7 (C-3), 80.2 (Boc-quart.-C),
61.7 (C-9), 55.0 (C-5), 51.8 (OMe), 50.2 (Asp-CHNH2), 48.4 (C-18),
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Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
Glycyrrhetinic Acid Derivatives
511
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), 36.5 (Asp-CH2), 32.7 (C-7), 31.8 (C-17), 31.1
(C-21), 28.5 (C-28), 28.3 (C-29), 28.3 (Boc-CH3), 27.8 (C-23), 26.5
(C-16), 26.4 (C-15), 23.4 (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, MeOH): m/z (%) ¼ 722.4
([M þ Na]þ, 100), 1072.3 ([3 M þ 2 Na]2þ, 11); anal. calcd.
for C40H61NO9 (699.91): C, 68.64; H, 8.78; N, 2.00; found: C,
68.49; H, 8.87; N, 1.77.
H-7’), 1.47 (m, 1H, H-22), 1.44 (s, 3H, H-27), 1.41 (m, 2H, H-22’ and
H-21’), 1.29 (m, 1H, H-16’), 1.18 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.15
(s, 3H, H-26), 1.13 (m, 1H, H-1’), 1.05 (m, 1H, H-15’), 0.94 (s, 3H,
H-23), 0.93 (s, 3H, H-24), 0.91 (m, 1H, H-5), 0.83 (s, 3H, H-28) ppm;
13C-NMR (125 MHz, MeOH-d4): d ¼ 202.0 (C-11), 178.5 (C-30), 172.6
(Asp-COOMe), 172.5 (Asp-COO), 169.0 (C-13), 128.8 (C-12), 85.3
(C-3), 62.9 (C-9), 56.1 (C-5), 52.4 (OMe), 50.9 (Asp-CHNH), 49.9
(C-18), 46.8 (C-8), 45.4 (C-20), 44.7 (C-14), 42.5 (C-19), 39.7 (C-1),
39.3 (C-22), 39.1 (C-4), 38.3 (C-10), 34.8 (Asp-CH2), 33.7 (C-7), 33.0
(C-17), 32.1 (C-21), 29.2 (C-28), 28.6 (C-23), 28.6 (C-29), 27.7 (C-16),
27.5 (C-15), 24.5 (C-2), 23.9 (C-27), 19.4 (C-26), 18.5 (C-6), 17.3 (C-24),
17.0 (C-25) ppm; MS (ESI, MeOH): m/z (%) ¼ 600.4 ([M þ H]þ, 100);
anal. calcd. for C35H54NO7Cl (636.26): C, 66.07; H, 8.55; N, 2.20; Cl,
5.57; found: C, 65.86; H, 8.73; N, 2.11; Cl, 5.64.
Methyl 3b 3-(Boc-L-glutamyl)-11-oxo-olean-12-en-30-oate
(7)
Obtained from 3 (210 mg, 0.26 mmol) by method C as a colorless
powder; yield: 190 mg, 96%; mp 212–2148C; RF ¼ 0.13 (hexane/
ethyl acetate 1:1); [a]D ¼ 123.608 (c ¼ 0.54, MeOH); UV-VIS (meth-
anol): lmax (log e) ¼ 250 nm (4.13); IR (KBr): n ¼ 3259 (br), 2956
(s), 1724 (s), 1652 (s), 1582 (w), 1466 (m), 1389 (m), 1365 (m), 1322
(w), 1233 (s), 1164 (m), 1105 (m), 1022 (w), 982 (m), 919 (w), 850
Methyl 3b 3-(L-glutamyl)-11-oxo-olean-12-en-30-oate
hydrochloride (9)
(w), 750 (w), 682 (w), 589 (w), 541 (w) cmꢂ1
;
1H-NMR (500 MHz,
Obtained from 7 (560 mg, 0.78 mmol) by method D as a colorless
powder; yield: 470 mg, 93%; mp 236–2398C (dec.); RF ¼ 0.68
(methanol); [a]D ¼ 125.688 (c ¼ 0.56, methanol); UV-VIS (metha-
nol): lmax (log e) ¼ 250 nm (4.11); IR (KBr): n ¼ 3438 (br), 2950 (s),
1732 (s), 1660 (s), 1619 (m), 1509 (m), 1465 (s), 1387 (s), 1355 (m),
1323 (m), 1279 (s), 1218 (s), 1153 (s), 1086 (m), 1048 (w), 987 (m),
948 (m), 914 (w), 868 (w), 823 (w), 769 (w), 748 (w), 714 (w), 701 (w),
672 (w), 619 (w), 590 (w), 546 (w), 505 (w), 456 (w) cmꢂ1; 1H-NMR
(500 MHz, MeOH-d4): d ¼ 5.59 (s, 1H, H-12), 4.69 (dd, 1H, H-3,
J ¼ 11.7, 4.7 Hz), 4.15 (dd, 1H, Glu-CHN, J ¼ 6.4, 6.4 Hz), 3.70
(s, 3H, OMe), 2.81 (ddd, 1H, H-1, J ¼ 13.7, 3.4, 3.4 Hz), 2.60 (m, 1H,
Glu-CHH’COOMe), 2.54 (m, 1H, Glu-CHH’COOMe), 2.51 (s, 1H, H-9),
2.28 (m, 1H, Glu-CHH’CHN), 2.17 (m, 1H, Glu-CHH’CHN), 2.16 (m,
1H, H-15), 2.13 (m, 1H, H-18), 1.97 (m, 1H, H-21), 1.89 (m, 1H, H-19),
1.87 (m, 1H, H-16), 1.79 (m, 1H, H-2), 1.76 (m, 1H, H-7), 1.74 (dd,
1H, H-19’, J ¼ 13.5, 13.5 Hz), 1.72 (m, 1H, H-2’), 1.66 (m, 1H, H-6),
1.55 (m, 1H, H-6’), 1.48 (m, 1H, H-7’), 1.46 (m, 1H, H-22), 1.44 (s, 3H,
H-27), 1.41 (m, 2H, H-22’ and H-21’), 1.29 (m, 1H, H-16’), 1.19 (s, 3H,
H-25), 1.16 (s, 3H, H-29), 1.15 (s, 3H, H-26), 1.13 (m, 1H, H-1’), 1.05
(m, 1H, H-15’), 0.97 (s, 3H, H-23), 0.96 (s, 3H, H-24), 0.93 (m, 1H,
H-5), 0.83 (s, 3H, H-28) ppm; 13C-NMR (125 MHz, MeOH-d4):
d ¼ 202.2 (C-11), 178.6 (C-30), 175.2 (Glu-COOMe), 172.8 (Glu-
COO), 170.2 (C-13), 128.9 (C-12), 85.2 (C-3), 62.9 (C-9), 56.0 (C-5),
53.7 (Glu-CHNH), 52.3 (OMe), 49.9 (C-18), 46.7 (C-8), 45.3 (C-20),
44.7 (C-14), 42.4 (C-19), 39.6 (C-1), 39.2 (C-22), 39.0 (C-4), 38.2 (C-10),
33.6 (C-7), 33.0 (C-17), 32.0 (C-21), 30.4 (Glu-CH2COOMe), 29.1
(C-28), 28.7 (C-23), 28.5 (C-29), 27.6 (C-16), 27.3 (C-15), 26.8
(Glu-CH2CHNH), 24.4 (C-2), 23.8 (C-27), 19.3 (C-26), 18.4 (C-6),
17.3 (C-24), 16.9 (C-25) ppm; MS (ESI, MeOH): m/z (%) ¼ 614.3
CDCl3): d ¼ 5.64 (s, 1H, H-12), 5.14 (d, 1H, Glu-NH, J ¼ 7.5 Hz),
4.53 (dd, 1H, H-3, J ¼ 11.8, 4.7 Hz), 4.31 (m, 1H, Glu-CHN), 3.67
(s, 3H, OMe), 2.79 (ddd, 1H, H-1, J ¼ 13.5, 3.4, 3.4 Hz), 2.46 (m, 1H,
Glu-CHH’COOMe), 2.42 (m, 1H, Glu-CHH’COOMe), 2.32 (s, 1H, H-9),
2.20 (m, 1H, Glu-CHH’CHN), 2.06 (dd, 1H, H-18, J ¼ 13.5, 3.3 Hz),
1.99 (ddd, 1H, H-15, J ¼ 13.7, 13.7, 4.3 Hz), 1.96 (m, 1H, H-21), 1.94
(m, 1H, Glu-CHH’CHN), 1.89 (m, 1H, H-19), 1.79 (ddd, 1H, H-16,
J ¼ 13.8, 13.8, 4.4 Hz), 1.72 (m, 1H, H-2), 1.66 (m, 1H, H-7), 1.61 (m,
1H, H-2’), 1.61 (dd, 1H, H-19’, J ¼ 13.6, 13.6 Hz), 1.56 (m, 1H, H-6),
1.45 (m, 1H, H-6’), 1.41 (s, 9H, Boc-CH3), 1.38 (m, 1H, H-7’), 1.37
(m, 1H, H-22), 1.33 (s, 3H, H-27), 1.28 (m, 2H, H-22’ and H-21’), 1.15
(m, 1H, H-16’), 1.13 (s, 3H, H-25), 1.12 (s, 3H, H-29), 1.09 (s, 3H,
H-26), 1.04 (m, 1H, H-1’), 0.98 (m, 1H, H-15’), 0.86 (s, 3H, H-24), 0.84
(s, 3H, H-23), 0.77 (s, 3H, H-28), 0.76 (m, 1H, H-5) ppm; 13C-NMR
(125 MHz, CDCl3): d ¼ 200.0 (C-11), 177.2 (Glu-COOH), 176.9
(C-30), 171.8 (Glu-COO), 169.3 (C-13), 155.6 (Boc-COO), 128.4
(C-12), 82.4 (C-3), 80.1 (Boc-quart.-C), 61.6 (C-9), 55.0 (C-5), 53.1
(Glu-CHNH), 51.8 (OMe), 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), 32.6
(C-7), 31.8 (C-17), 31.1 (C-21), 30.1 (Glu-CH2COO), 28.5 (C-28), 28.3
(C-29), 28.3 (Boc-CH3), 28.1 (C-23), 28.0 (Glu-CH2CHNH), 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, MeOH): m/z (%) ¼ 628.3 ([M þ H]þ, 100);
anal. calcd. for C41H63NO9 (713.94): C, 68.97; H, 8.89; N, 1.96:
found: C, 68.81; H, 9.02; N, 1.81.
Methyl 3b 3-(L-asparagyl)-11-oxo-olean-12-en-30-oate
hydrochloride (8)
([M þ H]þ, 100), 921.1 ([3 M þ 2 H]2þ
,
4); anal. calcd.
Obtained from 6 (340 mg, 0.49 mmol) by method D as a colorless
powder; yield: 280 mg, 91%; mp 272–2768C (dec.); RF ¼ 0.66
(methanol); [a]D ¼ 122.888 (c ¼ 0.44, MeOH); UV-VIS (methanol):
lmax (log e) ¼ 250 nm (4.07); IR (KBr): n ¼ 3439 (br), 2952 (s), 2362
(w), 1732 (s), 1660 (s), 1520 (m), 1466 (m), 1387 (m), 1322 (m), 1255
(s), 1217 (s), 1153 (m), 1085 (m), 1049 (w), 987 (w), 946 (w), 901 (w),
for C36H56NO7Cl (650.29): C, 66.49; H, 8.68; N, 2.15; Cl, 5.45;
found: C, 66.28; H, 8.85; N, 2.01; Cl, 5.67.
Methyl 3b 3-(Boc-O-methyl-L-asparagyl)-11-oxo-olean-
12-en-30-oate (10)
878 (w), 823 (w), 800 (w), 688 (w), 670 (w), 638 (w), 545 (w) cmꢂ1
;
Obtained from 6 (250 mg, 0.36 mmol) by method E as a colorless
powder; yield: 240 mg, 93%; mp 213–2178C (dec.); RF ¼ 0.52 (hex-
ane/ethyl acetate 7:3); [a]D ¼ 99.898 (c ¼ 0.54, CHCl3); UV-VIS
(methanol): lmax (log e) ¼ 250 nm (4.07); IR (KBr): n ¼ 3423
(br), 2954 (s), 2875 (m), 1747 (s), 1709 (s), 1652 (s), 1615 (w),
1506 (m), 1455 (m), 1433 (m), 1390 (m), 1364 (m), 1338 (m),
1287 (m), 1217 (s), 1162 (s), 1086 (m), 1052 (m), 1003 (m), 986
(m), 957 (w), 916 (w), 879 (w), 866 (m), 826 (w), 771 (w), 670 (w), 590
1H-NMR (500 MHz, MeOH-d4): d ¼ 5.58 (s, 1H, H-12), 4.67 (dd, 1H,
H-3, J ¼ 11.7, 4.7 Hz), 4.32 (dd, 1H, Asp-CHN, J ¼ 4.9, 4.9 Hz), 3.70
(s, 3H, OMe), 3.07 (dd, 1H, Asp-CHH’, J ¼ 18.2, 5.5 Hz), 2.97 (dd,
1H, Asp-CHH’, J ¼ 18.2, 4.5 Hz), 2.80 (ddd, 1H, H-1, J ¼ 13.7, 3.7,
3.7 Hz), 2.50 (s, 1H, H-9), 2.15 (m, 1H, H-15), 2.13 (m, 1H, H-18),
1.97 (m, 1H, H-21), 1.89 (m, 1H, H-19), 1.87 (m, 1H, H-16), 1.80 (m,
1H, H-2), 1.77 (m, 1H, H-7), 1.74 (dd, 1H, H-19’, J ¼ 13.4, 13.4 Hz),
1.72 (m, 1H, H-2’), 1.65 (m, 1H, H-6), 1.54 (m, 1H, H-6’), 1.50 (m, 1H,
(w), 540 (w) cmꢂ1
;
1H-NMR (400 MHz, CDCl3): d ¼ 5.66 (s, 1H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

512
R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
H-12), 5.50 (d, 1H, NH, J ¼ 8.6 Hz), 4.56 (m, 2H, H-3 and Asp-CH),
3.69 (s, 3H, OMe), 3.68 (s, 3H, Asp-OMe), 3.03 (dd, 1H, Asp-CHH’,
J ¼ 17.1, 3.9 Hz), 2.83 (m, 1H, Asp-CHH’), 2.80 (m, 1H, H-1), 2.35
(s, 1H, H-9), 2.08 (dd, 1H, H-18, J ¼ 13.4, 3.3 Hz), 2.03 (m, 1H, H-
15), 1.99 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J ¼ 13.8, 4.0, 2.6 Hz),
1.82 (ddd, 1H, H-16, J ¼ 13.6, 13.6, 4.1 Hz), 1.73 (m, 1H, H-2), 1.67
(m, 1H, H-2’), 1.63 (m, 1H, H-7), 1.60 (dd, 1H, H-19’, J ¼ 13.5,
13.5 Hz), 1.58 (m, 1H, H-6), 1.45 (s, 9H, Boc-CH3), 1.43 (m, 1H,
H-6’), 1.41 (m, 1H, H-7’), 1.38 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31
(m, 1H, H-22’), 1.31 (m, 1H, H-21’), 1.18 (m, 1H, H-16’), 1.16 (s, 3H,
H-25), 1.15 (s, 3H, H-29), 1.12 (s, 3H, H-26), 1.06 (m, 1H, H-1’), 1.02
(m, 1H, H-15’), 0.87 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.80 (m, 1H,
H-5), 0.79 (s, 3H, H-28) ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 199.9
(C-11), 176.9 (C-30), 171.5 (Asp-COOMe), 170.7 (Asp-COO), 169.2
(C-13), 155.4 (Boc-COO), 128.5 (C-12), 82.4 (C-3), 79.9 (Boc-quart.-C),
61.6 (C-9), 55.0 (C-5), 51.9 (Asp-OMe), 51.7 (OMe), 50.2 (Asp-CHNH2),
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), 36.6 (Asp-CH2), 32.7 (C-7), 31.8
(C-17), 31.1 (C-21), 28.5 (C-28), 28.3 (C-29), 27.8 (C-23), 26.5 (C-16),
26.4 (C-15), 23.4 (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, MeOH): m/z (%) ¼ 714.2 ([M þ H]þ, 5),
736.4 ([M þ Na]þ, 100), 1093.3 ([3 M þ 2 Na]2þ, 12), 1450.0
([2 M þ Na]þ, 22); anal. calcd. for C41H63NO9 (713.94): C, 68.97;
H, 8.89; N, 1.96; found: C, 68.76; H, 9.04; N, 1.79.
Methyl 3b 3-(O-methyl-L-asparagyl)-11-oxo-olean-12-en-
30-oate (12)
Obtained from 10 (550 mg, 0.79 mmol) by method B as a color-
less powder; yield: 360 mg, 83%; mp 226–2298C (dec.); RF ¼ 0.73
(dichloromethane/methanol 9:1); [a]D ¼ 111.728 (c ¼ 0.47,
CHCl3); UV-VIS (methanol): lmax (log e) ¼ 249 nm (3.62); IR
(KBr): n ¼ 3365 (br), 2962 (m), 2874 (m), 1739 (s), 1727 (s), 1652
(s), 1614 (w), 1438 (m), 1389 (m), 1318 (w), 1264 (m), 1218 (m),
1186 (m), 1161 (m), 1086 (w), 1026 (m), 988 (w), 959 (w), 904 (w),
879 (w), 851 (w), 826 (w), 771 (w), 670 (w), 590 (w), 543 (w) cmꢂ1
;
1H-NMR (400 MHz, CDCl3): d ¼ 5.67 (s, 1H, H-12), 4.58 (dd, 1H,
H-3, J ¼ 11.6, 4.9 Hz), 3.80 (dd, 2H, Asp-CH, J ¼ 7.1, 4.7 Hz), 3.70
(s, 3H, OMe), 3.69 (s, 3H, Asp-OMe), 2.83 (m, 1H, Asp-CHH’), 2.81
(m, 1H, H-1), 2.72 (dd, 1H, Asp-CHH’, J ¼ 16.5, 7.2 Hz), 2.36 (s, 1H,
H-9), 2.08 (dd, 1H, H-18, J ¼ 12.8, 3.6), 2.03 (m, 1H, H-15), 1.99
(m, 1H, H-21), 1.92 (ddd, 1H, H-19, J ¼ 13.5, 3.9, 2.7 Hz), 1.82 (ddd,
1H, H-16, J ¼ 13.6, 13.6, 4.2 Hz), 1.73 (m, 1H, H-2), 1.69 (m, 1H, H-
7), 1.63 (m, 1H, H-2’), 1.61 (dd, 1H, H-19’, J ¼ 13.5, 13.5 Hz), 1.57
(m, 1H, H-6), 1.48 (m, 1H, H-6’), 1.43 (m, 1H, H-7’), 1.40 (m, 1H,
H-22), 1.36 (s, 3H, H-27), 1.31 (m, 1H, H-22’), 1.31 (m, 1H, H-21’),
1.18 (m, 1H, H-16’), 1.16 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.13 (s, 3H,
H-26), 1.06 (m, 1H, H-1’), 1.01 (m, 1H, H-15’), 0.87 (s, 3H, H-24), 0.87
(s, 3H, H-23), 0.81 (m, 1H, H-5), 0.80 (s, 3H, H-28) ppm; 13C-NMR
(125 MHz, CDCl3): d ¼ 199.9 (C-11), 176.9 (C-30), 173.9 (Asp-COO),
171.6 (Asp-COOMe), 169.2 (C-13), 128.5 (C-12), 81.8 (C-3), 61.6 (C-9),
55.0 (C-5), 51.8 (Asp-OMe), 51.7 (OMe), 51.5 (Asp-CH), 48.4 (C-18),
45.4 (C-8), 44.0 (C-20), 43.2 (C-14), 41.1 (C-19), 38.7 (C-1), 38.6 (Asp-
CH2), 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), 28.0 (C-23), 26.5 (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, MeOH): m/z (%) ¼ 614.2 ([M þ H]þ, 100), 636.3
([M þ Na]þ, 4); anal. calcd. for C36H55NO7 (613.82): C, 70.44;
H, 9.03; N, 2.28; found: C, 70.25; H, 9.19; N, 2.14.
Methyl 3b 3-(Boc-O-methyl-L-glutamyl)-11-oxo-olean-12-
en-30-oate (11)
Obtained from 7 (500 mg, 0.70 mmol) by method E as a colorless
powder; yield: 390 mg, 77%; mp 202–2058C; RF ¼ 0.38 (hexane/
ethyl acetate 7:3); [a]D ¼ 95.048 (c ¼ 0.66, CHCl3); UV-VIS (meth-
anol): lmax (log e) ¼ 250 nm (4.10); IR (KBr): n ¼ 3381 (br), 2951
(s), 2874 (m), 1748 (s), 1726 (s), 1711 (s), 1652 (s), 1509 (m), 1453
(m), 1390 (m), 1366 (m), 1354 (m), 1326 (m), 1264 (m), 1216 (s),
1163 (s), 1087 (w), 1076 (w), 1050 (w), 1026 (m), 984 (m), 918 (w),
882 (w), 825 (w), 772 (w), 538 (w) cmꢂ1; 1H-NMR (400 MHz, CDCl3):
d ¼ 5.67 (s, 1H, H-12), 5.09 (d, 1H, Glu-NH, J ¼ 7.9 Hz), 4.56 (dd,
1H, H-3, J ¼ 11.6, 4.8 Hz), 4.32 (m, 1H, Glu-CHN), 3.69 (s, 3H, Glu-
OMe), 3.68 (s, 3H, OMe), 2.82 (ddd, 1H, H-1, J ¼ 13.7, 3.4, 3.4 Hz),
2.45 (m, 1H, Glu-CHH’COOMe), 2.39 (m, 1H, Glu-CHH’COOMe),
2.36 (s, 1H, H-9), 2.23 (m, 1H, Glu-CHH’CHN), 2.08 (dd, 1H, H-18,
J ¼ 13.7, 3.5 Hz), 2.03 (m, 1 H, H-15), 1.99 (m, 1H, H-21), 1.96
(m, 1H, Glu-CHH’CHN), 1.92 (ddd, 1H, H-19, J ¼ 13.3, 3.3, 2.3 Hz),
1.83 (ddd, 1H, H-16, J ¼ 13.7, 13.7, 4.2 Hz), 1.74 (m, 1H, H-2), 1.66
(m, 1H, H-7), 1.63 (m, 1H, H-2’), 1.61 (dd, 1H, H-19’, J ¼ 13.5,
13.5 Hz), 1.57 (m, 1H, H-6), 1.46 (m, 1H, H-6’), 1.44 (s, 9H, Boc-
CH3), 1.42 (m, 1H, H-7’), 1.39 (m, 1H, H-22), 1.36 (s, 3H, H-27), 1.31
(m, 2H, H-22’ and H-21’), 1.18 (m, 1H, H-16’), 1.17 (s, 3H, H-25), 1.15
(s, 3H, H-29), 1.13 (s, 3H, H-26), 1.07 (m, 1H, H-1’), 1.02 (m, 1H,
H-15’), 0.90 (s, 3H, H-24), 0.88 (s, 3H, H-23), 0.81 (s, 3H, H-28), 0.80
(m, 1H, H-5) ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 199.9 (C-11),
173.2 (Glu-COOMe), 176.9 (C-30), 171.6 (Glu-COO), 169.2 (C-13),
155.3 (Boc-COO), 128.5 (C-12), 82.2 (C-3), 79.9 (Boc-quart.-C), 61.7 (C-
9), 55.0 (C-5), 53.3 (Glu-CHNH), 51.8 (OMe), 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), 30.2 (Glu-
CH2COOMe), 28.5 (C-28), 28.3 (C-29), 28.3 (Boc-CH3), 28.1 (C-23),
28.0 (Glu-CH2CHNH), 26.5 (C-16), 26.4 (C-15), 23.6 (C-2), 23.3 (C-27),
18.7 (C-26), 17.3 (C-6), 16.8 (C-24), 16.4 (C-25) ppm; MS (ESI, MeOH):
m/z (%) ¼ 750.4 ([M þ Na]þ, 100), 1114.3 ([3 M þ 2 Na]2þ, 22),
1478.1 ([2 M þ Na]þ, 24); anal. calcd. for C42H65NO9 (727.97):
C, 69.30; H, 9.00; N, 1.92; found: C, 69.11; H, 9.24; N, 1.68.
Methyl 3b 3-(O-methyl-L-glutamyl)-11-oxo-olean-12-en-
30-oate (13)
Obtained from 11 (320 mg, 0.44 mmol) by method B as a color-
less powder; yield: 260 mg, 94%; mp 288–2908C (dec.); RF ¼ 0.73
(dichloromethane/methanol 9:1); [a]D ¼ 123.608 (c ¼ 0.54, meth-
anol); UV-VIS (methanol): lmax (log e) ¼ 250 nm (4.13); IR (KBr):
n ¼ 3259 (br), 2956 (s), 1724 (s), 1652 (s), 1582 (w), 1466 (m), 1389
(m), 1365 (m), 1322 (w), 1233 (s), 1164 (m), 1105 (m), 1022 (w), 982
(m), 919 (w), 850 (w), 750 (w), 682 (w), 589 (w), 541 (w) cmꢂ1
;
1H-NMR (400 MHz, CDCl3): d ¼ 5.66 (s, 1H, H-12), 4.56 (dd, 1H,
H-3, J ¼ 11.8, 4.7 Hz), 3.67 (s, 3H, Glu-OMe), 3.66 (s, 3H, OMe), 3.43
(dd, 1H, Glu-CHN, J ¼ 8.4, 4.7 Hz), 2.80 (ddd, 1H, H-1, J ¼ 13.7,
3.3, 3.3 Hz), 2.47 (dd, 2H, Glu-CH2COOMe, J ¼ 7.6, 7.2 Hz), 2.34
(s, 1H, H-9), 2.11 (m, 1H, Glu-CHH’CHN), 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 13.7, 3.3,
2.5 Hz), 1.83 (m, 1H, Glu-CHH’CHN), 1.79 (m, 1H, H-16), 1.70
(m, 1H, H-2), 1.65 (m, 1H, H-7), 1.61 (m, 1H, H-2’), 1.59 (dd, 1H,
H-19’, J ¼ 13.3, 13.3 Hz), 1.55 (m, 1H, H-6), 1.45 (m, 1H, H-6’), 1.40
(m, 1H, H-7’), 1.35 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.29 (m, 2H,
H-22’ and H-21’), 1.16 (m, 1H, H-16’), 1.14 (s, 3H, H-25), 1.13 (s, 3H,
H-29), 1.11 (s, 3H, H-26), 1.04 (m, 1H, H-1’), 0.99 (m, 1H, H-15’), 0.88
(s, 3H, H-24), 0.86 (s, 3H, H-23), 0.80 (m, 1H, H-5), 0.78 (s, 3H, H-28)
ppm; 13C-NMR (125 MHz, CDCl3): d ¼ 200.0 (C-11), 176.9 (C-30),
173.6 (Glu-COOMe), 171.6 (Glu-COO), 169.2 (C-13), 128.5 (C-12),
81.5 (C-3), 61.7 (C-9), 55.0 (C-5), 54.1 (Glu-CHNH), 51.7 (OMe),
51.6 (Glu-OMe), 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
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 505–513
Glycyrrhetinic Acid Derivatives
513
(C-7), 31.8 (C-17), 31.1 (C-21), 30.5 (Glu-CH2COOMe), 29.6 (Glu-
CH2CHNH), 28.5 (C-28), 28.3 (C-29), 28.1 (C-23), 26.5 (C-16), 26.4
(C-15), 23.6 (C-2), 23.3 (C-27), 18.7 (C-26), 17.3 (C-6), 16.8 (C-24), 16.4
(C-25) ppm; MS (ESI, MeOH): m/z (%) ¼ 628.3 ([M þ H]þ, 100); anal.
calcd. for C37H57NO7 (627.85): C, 70.78; H, 9.15; N, 2.23; found: C,
70.59; H, 9.25; N, 2.07
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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. Thomas Mu¨ller
(Dept. of Haematology/Oncology, Univ. Halle).
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Bioorg. Med. Chem. 2010, 18, 2549–2558.
The authors have declared no conflict of interest.
[11] G. Chadalapaka, I. Jutooru, A. McAlees, T. Stefanac, S. Safe,
Bioorg. Med. Chem. Lett. 2008, 18, 2633–2639.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com