Does one keto group matter? Structure-activity relationships of glycyrrhetinic acid derivatives modified at position C-11

Article abstract of DOI:10.1002/ardp.201000327

Several triterpenoic acids display a remarkable cytotoxicity on tumor cells. Glycyrrhetinic acid - the main content of the licorice root - possesses an apoptotic effect on tumor cells. Previous studies pointed out the presence of a keto group at position C-11 in glycyrrhetinic acid derivatives as the main reason for its apoptotic activity. Several pairs of derivatives were synthesized differing only at position C-11. These compounds were tested in a sulforhodamine B colorimetric assay for cytotoxicity screening on 12 tumor cell lines and mouse embryonic fibroblasts (NIH3T3). Our results show that there is no direct relation between the existence of the C-11 keto group and the apoptotic activity of the compounds. It remains disputable whether the C11 keto group in glycyrrhetinic acid derivatives is the main reason for their apoptotic effect. Copyright

Full text of DOI:10.1002/ardp.201000327

2
8
Arch. Pharm. Chem. Life Sci. 2012, 345, 28–32
Full Paper
Does One Keto Group Matter? Structure-Activity
Relationships of Glycyrrhetinic Acid Derivatives Modified
at Position C-11
Ren e´ Csuk, Stefan Schwarz, Ralph Kluge, and Dieter Str o¨ hl
Martin-Luther-Universit a¨ t Halle-Wittenberg, Bereich Organische Chemie, Halle (Saale), Germany
Several triterpenoic acids display a remarkable cytotoxicity on tumor cells. Glycyrrhetinic acid – the
main content of the licorice root – possesses an apoptotic effect on tumor cells. Previous studies pointed
out the presence of a keto group at position C-11 in glycyrrhetinic acid derivatives as the main reason
for its apoptotic activity. Several pairs of derivatives were synthesized differing only at position C-11.
These compounds were tested in a sulforhodamine B colorimetric assay for cytotoxicity screening on
12 tumor cell lines and mouse embryonic fibroblasts (NIH3T3). Our results show that there is no direct
relation between the existence of the C-11 keto group and the apoptotic activity of the compounds.
Keywords: Antitumor activity / Apoptosis / Glycyrrhetinic acid / Mouse embryonic fibroblasts
Received: November 1, 2010; Revised: November 23, 2010; Accepted: November 30, 2010
DOI 10.1002/ardp.201000327
Introduction
keto-enol tautomerism this keto group was proposed to be
involved in a mechanism producing an oxygen radical.
Among others, Fiore et al. [9] deduced that this should impel
mitochondrial permeability transition, finally leading to cell
death.
Known as antitumor agents for several years, triterpenoic
acids are still in the focus of scientific interest. Betulinic acid,
oleanoic acid, ursolic acid, boswellic acid or glycyrrhetinic
acid are well-known species of this class. They have a proven
cytotoxicity on tumor cells [1–4] by triggering apoptosis [4–8].
All of these triterpenes consist of five condensed cycloalkyl
rings which give them a high degree in molecular likeness.
Having a hydroxyl group on ring A and a carboxyl group on
ring E in common, glycyrrhetinic acid (1, Scheme 1), how-
ever, differs because of the presence of an a,b-unsaturated
keto group in position C-11 on ring C. Previous studies
pointed out the importance of both A and C ring
substitutions.
We examined three different pairs of derivatives to explore
the influence of a keto group in position C-11. In a first try, we
chose 1 and its deoxygenated analogue 2 (Scheme 1) for
comparison. To remove the keto group, 1 was subjected to
a Clemmensen reduction with zinc dust and conc. HCl in 1,4-
dioxane at room temperature to afford 2 [11]. A second pair of
compounds consisted of the respective methyl ester 3 and the
diene 4. Following the procedure of Su et al. [12], 1 was easily
transformed into the ester 3 by adding methyl iodide and
potassium carbonate to a solution of 1 in dry DMF. The
reaction of 3 with borane in THF followed by acidic workup
provided 4. Finally, a third couple consisted of the ethyl ester
Results and discussion
5
and the products of its reduction (borane/THF, basic work-
For glycyrrhetinic acid (1) several references suggested the
keto group in position C-11 is the major reason for the
cytotoxic and apoptotic effects [9, 10]. Based on a probable
up) 6 and 7, respectively.
To show a correlation between the biological activity of the
compounds and the presence or the absence of the keto
group a paired comparison was carried out. A sulforhod-
amine assay (SRB) [13] was conducted on 12 different tumor
cell lines as well as on a mouse embryonic fibroblast cell line
Correspondence: Prof. Dr. Ren e´ Csuk, Bereich Organische Chemie,
Martin-Luther-Universit a¨ t Halle-Wittenberg, Kurt-Mothes-Strasse 2,
D-06120 Halle (Saale), Germany.
E-mail: rene.csuk@chemie.uni-halle.de
Fax: þ49 (0) 345 5527030
(NIH3T3). Our first pair, 1 and 2, showed in a SRB assay almost
the same activity on tumor cells, i.e. the averaged IC50 (from
12 cell lines, Table 1) was 80.78 mM and 80.99 mM, respect-
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2012, 345, 28–32
Does One Keto Group Matter? Structure-Activity Relationships
29
ively. For the methyl ester 3 a slightly lower cytotoxicity
22.81 mM) on mouse embryonic fibroblast NIH3T3 cells
(
was established; the parent compound 1 displayed an activity
of 18.52 mM.
However, there are different effects to be established
between the non-tumorous cell line and the tumor cell lines
as suggested by the IC50 values. Compounds 3 and 4 possess a
comparable high cytotoxicity for the tumor cell lines.
Compared to ester 3, the deoxygenated compound 4 shows
a decreased toxicity [14] for the fibroblasts and most of the
tumor cell lines.
In accord with these results, a similar behaviour was found
for the third pair of compounds, 5 and 6. Their cytotoxic
effect on the tumor cells is similar to their effect on NIH3T3
cells. Unfortunately, we were not able to test compound 7 in
an SRB assay; compound 7 undergoes under screening con-
ditions an elimination reaction leading to the formation of 4.
As shown by NMR, significant deterioration of 7 advances
quickly in less than 1 day.
In acridine orange/ethidium bromide (AO/EB) tests (Fig. 1),
apoptotic cells are easily distinguished from necrotic cells. In
addition, Trypan blue staining followed by an automated cell
counting allows quantification of apoptotic behaviour.
As displayed in Fig. 1, in the AO/EB tests apoptotic cells are
stained in a green colour with bright spots whereas necrotic
cells appear in an orange-red colour. The results from these
experiments are summarized in Table 2.
Scheme 1. Reagents and conditions: a) zinc dust, conc. HCl,
Investigations by Fiore et al. [9] for 1 and by Salvi et al. [10]
for the antitumor active drug carbenoxolone suggest that the
presence of a keto group at C-11 was the main reason for the
cytotoxicity of these compounds on liver cells. Pairs of com-
pounds, 1 and 2 as well as 3 and 4, showed insignificant
differences in the extent of apoptosis. For compounds 5 and 6,
however, a significant difference is encountered (D% ¼ ca.
dioxane, 258C, 24 h; b) MeI, K
THF, THF, citric acid, 258C, 20 h; d) EtI, K
4 h; e) BH -THF, THF, Na CO , 258C, 4 d.
2
CO
3
, DMF, 258C, 24 h; c) BH
3
-
2
CO , DMF, 258C,
3
2
3
2
3
Table 1. Results of SRB assay: The values for melanoma (518A2),
cervical cancer (A431), lung carcinoma (A549), ovarian cancer
(
A2780), colon cancer (DLD-1, HCT-8, HCT-116, HT-29), anaplastic
3
5%). Interestingly enough, treatment of the cells with 6
thyroid cancer (8505C, SW-1736), mamma carcinoma (MCF-7) and
liposarcoma cells were obtained by an SRB assay after 96 h of
treatment and are the average from at least three independent
experiments; standard error 5%.
(with a missing carbonyl group) results in the formation of
more apoptotic cells compared to compound 5.
In addition, our results show that 1 has a much stronger
activity on fibroblasts than on tumor cells. This effect is lost
after removal of the 11-keto group. Upon removal of this
group, the cytotoxicity on tumor cell lines and on fibroblasts
becomes comparably high. Except for glycyrrhetinic acid (1),
all the compounds holding an 11-keto group show similar
IC50 values both for the tumor cell lines and for the fibroblast
cells. One would expect a significant loss of cytotoxicity after
removal of the keto group; however, the activity remains
unaltered. The differences in the activity of the compounds
Compound
1
2
3
4
5
6
5
8
18A2
505C
83.92
86.50
74.57
79.58
82.76
81.21
71.49
78.52
62.78
86.13
79.13
90.50
87.70
88.76
90.30
73.88
90.19
72.47
68.70
27.54
26.07
25.54
25.28
23.50
26.12
22.10
24.36
27.54
20.47
22.14
34.87
22.81
34.54
33.88
23.58
33.55
31.59
31.73
31.82
31.34
23.89
34.81
34.37
32.35
42.22
25.23
24.58
26.96
23.45
22.74
28.14
21.58
43.42
22.14
27.66
18.61
13.37
23.66
51.52
52.80
57.01
46.55
48.97
52.80
47.78
44.32
44.32
52.80
48.97
45.48
43.16
A2780
A431
A549
DLD-1
HCT-116 78.83
HCT-8
HT-29
LIPO
MCF-7
SW1736
NIH 3T3
78.85
80.09
81.44
84.70
76.93
18.52
5
and 6 might be based on the presence of an extra hydroxyl
group. Introducing such a polar group creates a different
polarity pattern in the molecule. Decreasing the number of
free polar groups in the molecule, for example, by esterifi-
cation, increases the cytotoxicity of the compounds.
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R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 28–32
Figure 1. Results from the AO/EB test: Treatment of A549 cells with (from left to right): 1 (90 mM) and 2 (90 mM), 3 (35 mM) and 4 (35 mM),
(60 mM) and 6 (60 mM).
5
Table 2. Apoptotic effect of GA derivatives in A549 cells (in %, standard error 5%, 6 independent experiments); cells were treated with 1
90 mM), 2 (90 mM), 3 (35 mM), 4 (35 mM), 5 (60 mM) or 6 (60 mM).
(
Compound
1
2
3
4
5
6
Apoptosis [%]
73.73 ꢀ 1.40
76.43 ꢀ 1.78
70.64 ꢀ 0.74
66.93 ꢀ 5.80
44.92 ꢀ 4.13
80.62 ꢀ 2.67
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
Therefore, the presence of an extra hydroxyl group at pos-
ition C-11 would be responsible for the drop of activity.
In summary, it is disputable whether the C-11 keto group is
the main reason for the apoptotic effect of glycyrrhetinic acid
and derivatives.
1
0% 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 dyeing the plates were washed with 1% acetic acid to
remove the excess of the dye and allowed to air dry overnight.
Experimental
General
1
00 mL of 10 mM Tris base solution was added to each well
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
and absorbance was measured at 570 nm (using a 96-well plate
reader, Tecan Spectra, Crailsheim, Germany). The IC50 was
estimated from the semi-logarithmic dose-response curves.
Apoptosis test – dye exclusion test
(
film or KBr pellet) on a Perkin-Elmer FT-IR spectrometer
Apoptotic cell death was analysed by Trypan blue dye (Sigma
Aldrich, Germany) on A431 and A2780 cell lines. The cell culture
flasks with 70–80% confluence were treated with IC90 doses of the
compounds for 24h. The supernatant medium with floating cells
was collected after treatment and centrifuged to collect dead and
apoptotic cells. This pellet was re-suspended in serum free media.
Equal amounts of cell suspension and Trypan blue were mixed
and analysed under a microscope. Viable cells exclude the dye
and appear colourless whereas cells whose cell membrane is
destroyed are stained in blue colour. If there are more colourless
cells than stained cells, then death of the cells can be character-
ised as apoptotic.
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.
Cell lines and culture conditions
The cell lines 518A2, 8505C, A253, A2780, A431, A549, DLD-1,
FaDu, HCT-116, HCT-8, HT-29, LIPO, MCF-7, SW1736, and SW480
were included in this study. Cultures were maintained as mono-
layer in RPMI 1640 (PAA Laboratories, Pasching, Germany)
supplemented with 10% heat inactivated fetal bovine serum
(
(
Biochrom AG, Berlin, Germany) and penicillin/streptomycin
PAA Laboratories) at 378C in a humidified atmosphere of 5%
Apoptosis test – acridine orange/ethidium bromide
(AO/EB)
2
CO /95% air.
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 centri-
fuged; 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
coloured nucleus indicates necrotic cells.
Cytotoxicity assay
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
9
6-well plates on day 0 at the appropriate cell densities to prevent
confluence of the cells during the period of experiment. After
4 h, the cells were treated with serial dilutions of the com-
2
pounds (0–300 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
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Arch. Pharm. Chem. Life Sci. 2012, 345, 28–32
Does One Keto Group Matter? Structure-Activity Relationships
31
Apoptosis test – Trypan blue cell counting
Methyl 3b-hydroxy-oleana-9(11),12-dien-30-oate (4)
Approx. 500,000 cells (A549) were seeded in cell culture flasks
and were allowed to grow for 1 day. After removal 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; the cell pellet was sus-
pended in PBS and centrifuged again. Equal amounts of Trypan
blue solution (0.4% in phosphate-buffer saline, pH 7.2) and sus-
pension of the pellet in PBS were mixed and put on chamber
To a solution of 3 (240 mg, 0.49 mmol) in dry THF (10 mL),
borane (1 M in THF, 7 mL, 7 mmol) was slowly added. After
20 h of stirring at 258C, ethyl acetate (25 mL) was added and
the organic layer was washed with a solution of citric acid (10%),
a saturated solution of sodium hydrogen carbonate and water.
After drying (Na SO ), filtration and evaporation, the residue was
2 4
subjected to chromatography (silical gel, hexane/ethyl acetate
7:3) to yield 4 (162 mg, 71%) as colourless crystals. Mp 207–2108C
TM
TM
slides (Invitrogen ). Automatic cell counter (Invitrogen
(lit. 243–2458C [20]);
R
f
¼ 0.60 (hexane/ethyl acetate 7:3);
1
countess automated cell counter) was used for counting the
cells, differentiating between cells with an intact cell membrane
and cells without.
[a]
D
¼ 296.708 (c 0.50, CHCl
3
).
Ethyl 3b-hydroxy-11-oxo-olean-12-en-30-oate (5)
Following the procedure given for 3, 5 was obtained from 1 and
iodoethane (4.0 mL, 49.5 mmol) as a white crystalline solid
3
b-Hydroxy-olean-12-en-30-acid (2)
To a mixture of 1 (490 mg, 1.04 mmol) and zinc dust (160 mg,
.45 mmol) in 1,4-dioxane (15 mL), conc. HCl was slowly added
(1.75 g, 82%). Mp 220–2248C (lit. 93–948C [12]); R
ane/ethyl acetate 7:3); [a]
¼ 144.358 (c 0.48, CHCl
(methanol):
f
¼ 0.40 (hex-
2
D
3
); UV-vis
until the reaction of the zinc was complete. After 1 day of stirring
at 258C, the solvent was evaporated and the residue subjected
to chromatography (silical gel, hexane/ethyl acetate 7:3).
Recrystallization from acetic acid gave 2 (180 mg, 38%) as colour-
less needles; mp 308–3138C (lit. 323–3258C [15], 322–3258C
l
max (log e) ¼ 267 nm (3.90); MS (ESI): m/z
þ þ
(%) ¼ 499.5 ([MþH] , 90), 521.4 ([MþNa] , 13), 552.9
þ
([MþNaþMeOH] , 100); IR (KBr): n ¼ 3543 br, 2939 s, 2869 m,
1726 s, 1651 s, 1616 m, 1455 m, 1389 m, 1364 w, 1330 m,
1312 w, 1278 w, 1257 m, 1210 m, 1172 s, 1135 w, 1086 m,
1042 m, 1028 m, 994 m, 920 w, 880 w, 767 w, 704 w, 671 w,
[
(
16]);
R
c 0.34, methanol).
f
¼ 0.77 (ethyl acetate/hexane 1:1); [a] ¼ 116.978
D
ꢂ1
1
604 w cm ; H NMR (400 MHz, CDCl
.16 (dq, 1 H, methylene-HH’, J ¼ 10.8 Hz, 7.1), 4.10 (dq, 1 H,
methylene-HH’, J ¼ 10.8, 7.1 Hz), 3.19 (dd, 1 H, H-3, J ¼ 11.1,
3
): d ¼ 5.61 (s, 1 H, H-12),
4
Methyl 3b-hydroxy-11-oxo-olean-12-en-30-oate (3)
5
.1 Hz), 2.76 (ddd, 1 H, H-1, J ¼ 13.5, 3.6, 3.6 Hz), 2.31 (s, 1 H,
A solution of 1 (31.00 g, 65.9 mmol) in dry DMF (150 mL) con-
taining potassium carbonate (15.34 g, 111.0 mmol) was stirred
at room temperature for 30 min. Iodomethane (4.94 mL,
H-9), 2.07 (ddd, 1 H, H-18, J ¼ 12.5, 4.1, 1.1 Hz), 2.00 (ddd, 1 H,
H-15, J ¼ 13.7, 13.7, 4.5 Hz), 1.96 (m, 1 H, H-21), 1.88 (ddd, 1 H,
H-19, J ¼ 13.5, 4.2, 2.8 Hz), 1.80 (ddd, 1 H, H-16, J ¼ 13.7, 13.7,
7
2
9.0 mmol) was added and the mixture was further stirred for
h. The solvents were evaporated and the crude residue was
4
.3 Hz), 1.64 (m, 1 H, H-2), 1.61 (m, 1 H, H-7), 1.58 (m, 1 H, H-2’), 1.57
(
3
1
m, 1 H, H-19’), 1.56 (m, 1 H, H-6), 1.42 (ddd, 1 H, H-6’, J ¼ 12.4, 3.1,
dissolved in a mixture of dichloromethane (300 mL) and hydro-
chloric acid (50 mL, 1.0 M). The aqueous layer was extracted with
dichloromethane (3 ꢁ 50 mL), the combined organic layers were
.1 Hz), 1.38 (m, 1 H, H-7’), 1.35 (m, 1 H, H-22), 1.34 (s, 3 H, H-27),
.28 (m, 1 H, H-22’), 1.26 (m, 1 H, H-21’), 1.23 (t, 3 H, ethyl,
J ¼ 7.1 Hz), 1.15 (m, 1 H, H-16’, J ¼ 13.7, 4.5, 2.7 Hz), 1.11 (s,
2 4
washed with brine (50 mL), dried (Na SO ), filtered and evapor-
3
H, H-28), 1.11 (s, 3 H, H-25), 1.10 (s, 3 H, H-26), 0.99 (m, 1 H,
ated. Recrystallization from methanol yielded 3 (29.09 g, 91.1%)
as a colourless crystalline solid; mp 254–2588C (lit. 254–2588C
H-15’), 0.97 (s, 3 H, H-23), 0.95 (ddd, 1 H, H-1’, J ¼ 10.4, 4.4, 4.4 Hz),
0
1
1
.78 (s, 3 H, H-24), 0.78 (s, 3 H, H-29), 0.67 (dd, 1 H, H-5, J ¼ 11.5,
13
3
[
12], 254–2568C [17], 248–2508C [18, 19]); R
f
¼ 0.48 (hexane/ethyl
.6 Hz); C NMR (125 MHz, CDCl
): d ¼ 200.2 (C-11), 176.3 (C-30),
acetate 7:3); [a]
l
5
D
¼ 141.188 (c 0.48, CHCl
3
). UV-vis (methanol):
max (log e) ¼ 270 nm (4.15); MS (ESI): m/z (%) ¼ 485.5 ([MþH] ,
69.2 (C-13), 128.5 (C-12), 78.7 (C-3), 61.8 (C-9), 60.3 (ethylene), 54.9
þ
(
(
(
C-5), 48.4 (C-18), 45.4 (C-14), 43.8 (C-20), 43.2 (C-8), 41.1 (C-19), 39.1
C-1), 39.1 (C-4), 37.7 (C-22), 37.1 (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), 27.3 (C-2), 26.5 (C-16),
þ
þ
5), 507.5 ([MþNa] , 12), 539.1 ([MþNaþMeOH] , 100); IR (KBr):
n ¼ 3614 br, 2970 s, 2955 s, 2875 m, 1726 s, 1659 s, 1466 m,
1
9
3
1
450 m, 1364 w, 1216 m, 1189 m, 1136 w, 1085 w, 1040 w,
): d ¼ 5.65 (s, 1 H, H-12),
2
6.4 (C-15), 23.4 (C-27), 18.7 (C-26), 17.5 (C-6), 16.3 (C-25), 15.5
ꢂ1
1
92 w cm ; H NMR (400 MHz, CDCl
3
50 4
(C-24), 14.3 (methyl); anal. calcd. for C32H O (498.74): C, 77.06;
.67 (s, 3 H, methyl), 3.21 (dd, 1 H, H-3, J ¼ 10.8, 5.7 Hz), 2.78 (ddd,
H, H-1, J ¼ 13.3, 3.3, 3.3 Hz), 2.32 (s, 1 H, H-9), 2.06 (dd, 1 H, H-18,
H, 10.10; found: C, 76.82; H, 10.21.
J ¼ 13.3, 3.8 Hz), 2.00 (m, 1 H, H-15), 1.95 (m, 1 H, H-21), 1.90 (dd, 1
Ethyl 3b,11b-dihydroxy-olean-12-en-30-oate (6) and ethyl
3b,11a-dihydroxy-olean-12-en-30-oate (7)
H, H-19, J ¼ 13.8, 3.9 Hz), 1.83 (ddd, 1 H, H-16, J ¼ 14.3, 14.3,
5
.2 Hz), 1.65 (m, 1 H, H-2), 1.62 (m, 1 H, H-7), 1.58 (m, 1 H, H-2’), 1.57
(
H-7’), 1.36 (m, 1 H, H-22), 1.34 (s, 3 H, H-27), 1.30 (m, 1 H, H-22’),
m, 1 H, H-19’), 1.57 (m, 1 H, H-6), 1.43 (m, 1 H, H-6’), 1.38 (m, 1 H,
Compound 2 (2.00 g, 4.02 mmol) was dissolved in anhydrous
THF (80 mL). After addition of borane (1 M in THF, 12 mL,
12 mmol), the mixture was stirred for 4 days at 258C. The mix-
ture was poured into a saturated solution of sodium bicarbonate
(50 mL) and extracted with chloroform. The combined organic
layers were concentrated in vacuo; the residue was purified by
chromatography (silica gel, hexane/ethyl acetate 7:3) to yield 6
and 7.
1
3
0
.28 (m, 1 H, H-21’), 1.17 (m, 1 H, H-16’), 1.13 (s, 3 H, H-28), 1.12 (s,
H, H-25), 1.11 (s, 3 H, H-26), 1.00 (m, 1 H, H-15’), 0.99 (s, 3 H, H-23),
.96 (m, 1 H, H-1’), 0.79 (s, 3 H, H-24), 0.79 (s, 3 H, H-29), 0.68 (m, 1 H,
13
H-5); C NMR (125 MHz, CDCl
69.0 (C-13), 128.6 (C-12), 78.8 (C-3), 61.9 (C-9), 55.1 (C-5), 51.8
methyl), 48.5 (C-18), 45.5 (C-14), 44.1 (C-20), 43.3 (C-8), 41.2 (C-19),
3
): d ¼ 200.0 (C-11), 176.8 (C-30),
1
(
3
3
9.2 (C-1), 39.2 (C-4), 37.8 (C-22), 37.2 (C-10), 32.9 (C-7), 31.9 (C-17),
1.2 (C-21), 28.6 (C-29), 28.4 (C-23), 28.2 (C-28), 27.4 (C-2), 26.6
Data for 6: colourless solid (1.18 g, 59%); mp 157–1608C;
R
f
¼ 0.38 (hexane/ethyl acetate 7:3); [a]
D
¼ 112.388 (c 0.55,
(
C-16), 26.6 (C-15), 23.5 (C-27), 18.8 (C-26), 17.6 (C-6), 16.4 (C-25),
5.6 (C-24).
CHCl
m/z (%) ¼ 523.4 ([MþNa] , 27), 554.9 ([MþNaþMeOH] , 100); IR
3
); UV-vis (methanol): lmax (log e) ¼ 298 nm (3.44); MS (ESI):
þ þ
1
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

3
2
R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 28–32
(
KBr): n ¼ 3440 br, 2932 s, 1728 s, 1636 w, 1465 m, 1383 m,
Schwarz. The cell lines were kindly provided by Dr. Thomas M u¨ ller
(Dept. of Haematology/Oncology, Univ. Halle).
ꢂ1
1
313 m, 1218 m, 1173 s, 1086 m, 1029 m, 996 w, 755 w cm
;
1
H NMR (400 MHz, CDCl
dd, 1 H, H-11, J ¼ 4.4, 4.4 Hz), 4.16 (dq, 1 H, methylene-HH’,
J ¼ 10.8, 7.1 Hz), 4.13 (dq, H, methylene-HH’, J ¼ 10.8,
.1 Hz), 3.23 (dd, 1 H, H-3, J ¼ 11.1, 5.0 Hz), 2.05 (ddd, 1 H, H-1,
3
): d ¼ 5.37 (d, 1 H, H-12, J ¼ 4.0 Hz), 4.33
(
The authors have declared no conflict of interest.
1
7
J ¼ 13.0, 3.4, 3.4 Hz), 2.00 (dd, 1 H, H-18, J ¼ 13.6, 4.0 Hz), 1.95 (m,
References
1
2
H, H-21), 1.93 (m, 1 H, H-15), 1.87 (ddd, 1 H, H-19, J ¼ 13.6, 4.3,
.7 Hz), 1.80 (ddd, 1 H, H-16, J ¼ 13.6, 13.6, 4.6 Hz), 1.72 (m, 1 H,
[
[
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H-2), 1.68 (m, 1 H, H-2’), 1.61 (dd, 1 H, H-19’, J ¼ 13.6, 13.6 Hz), 1.59
1
(
3
(
m, 1 H, H-7), 1.55 (m, 1 H, H-6), 1.52 (ddd, 1 H, H-6’, J ¼ 12.4, 3.1,
.1 Hz), 1.40 (s, 1 H, H-9), 1.40 (s, 3 H, H-25), 1.34 (m, 1 H, H-22), 1.32
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ethyl, J ¼ 7.1 Hz), 1.22 (s, 3 H, H-26), 1.19 (m, 1 H, H-1’), 1.12 (s, 3 H,
[
H-28), 1.09 (s, 3 H, H-27), 1.05 (ddd, 1 H, H-16’, J ¼ 13.6, 4.2, 2.4 Hz),
4
0
.99 (s, 3 H, H-23), 0.91 (ddd, 1 H, H-15’, J ¼ 13.6, 4.3, 2.0 Hz), 0.82
(
s, 3 H, H-24), 0.80 (s, 3 H, H-29), 0.70 (dd, 1 H, H-5, J ¼ 10.7, 2.7 Hz);
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1
3
C NMR (125 MHz, CDCl
C-12), 79.0 (C-3), 66.7 (C-11), 60.1 (ethylene), 55.7 (C-5), 52.5 (C-9),
3
): d ¼ 176.9 (C-30), 147.8 (C-13), 126.2
(
[
[
[
5] M. L. Schmidt, K. L. Kumanoff, L. Ling-Indeck, J. M. Pezzuto,
Eur. J. Cancer 1997, 33, 2007–2010.
4
3
2
7.9 (C-18), 44.0 (C-20), 42.7 (C-8), 42.3 (C-19), 39.5 (C-14), 38.7 (C-1),
8.7 (C-22), 38.3 (C-10), 38.2 (C-4), 33.2 (C-7), 31.7 (C-17), 31.2 (C-21),
8.4 (C-23), 28.4 (C-29), 28.3 (C-28), 27.0 (C-2), 26.8 (C-15), 26.5
6] P. Zhang, H. Li, D. Chen, J. Ni, Y. Kang, S. Wang, Acta Biochim.
Biophys. Sin. 2007, 39, 803–809.
(
C-16), 25.2 (C-27), 19.3 (C-26), 18.7 (C-6), 18.1 (C-25), 15.7 (C-24),
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Yang, Y.-S. Pu, C.-N. Lin, Bioorg. Med. Chem. 2009, 17, 7265–
1
1
4.3 (methyl); anal. calcd. for C33
0.57; found: C, 76.73; H, 11.07.
45 4
H O (514.78): C, 76.99; H,
7
274.
Data for 7: White crystalline solid (167 mg, 8%); mp 161–
¼ 0.21 (hexane/ethyl acetate 7:3); [a] ¼ 82.748 (c 0.38,
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1
658C; R
f
CHCl
3
); UV-vis (methanol): lmax (log e) ¼ 224 nm (3.85); MS (ESI):
þ
m/z (%) ¼ 523.5 ([MþNa] , 100); IR (KBr): n ¼ 3405 br, 2945 s,
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195–201.
1
1
(
727 m, 1715 m, 1618 w, 1466 m, 1390 w, 1314 w, 1218 m,
ꢂ1
1
152 m, 1109 w, 1085 w, 1040 w, 858 w, 620 w cm
): d ¼ 5.11 (d, 1 H, H-12, J ¼ 3.3 Hz), 4.12 (dq,
H, methylene-HH’, J ¼ 10.8, 7.1 Hz), 4.05 (dq, 1 H, methylene-
; H NMR
400 MHz, DMSO-d
6
[
[
[
10] M. Salvi, C. Fiore, V. Battaglia, M. Palermo, D. Armanini, A.
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1
HH’, J ¼ 10.8, 7.1 Hz), 3.96 (dd, 1 H, H-11, J ¼ 7.5, 3.3 Hz), 2.97 (m,
11] Jpn. Kokai Tokkyo Koho, Jpn. Pat. No. 59070638, Apr. 21,
1
1
H, H-3), 2.05 (ddd, 1 H, H-1, J ¼ 14.1, 3.1, 3.1 Hz), 1.97 (m, 1 H,
984; Chem. Abstr. 1984, 101, 171562t.
H-15), 1.77 (m, 1 H, H-21), 1.77 (ddd, 1 H, H-19, J ¼ 13.6, 4.3,
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2
4
1
1
1
3
0
3
0
1
5
4
3
.7 Hz), 1.76 (m, 1 H, H-18), 1.57 (ddd, 1 H, H-16, J ¼ 13.6, 13.6,
.6 Hz), 1.57 (dd, 1 H, H-19’, J ¼ 13.6, 13.6 Hz), 1.49 (m, 1 H, H-6),
.48 (d, 1 H, H-9, J ¼ 7.9 Hz), 1.44 (m, 1 H, H-2), 1.40 (m, 1 H, H-2’),
.39 (m, 1 H, H-7), 1.33 (m, 1 H, H-21’), 1.30 (m, 1 H, H-6’), 1.28 (m,
H, H-22), 1.20 (m, 1 H, H-7’), 1.18 (t, 3 H, ethyl, J ¼ 7.1 Hz), 1.17 (s,
H, H-27), 1.16 (m, 1 H, H-22’), 1.10 (m, 1 H, H-1’), 1.07 (s, 3 H, H-28),
.95 (s, 3 H, H-25), 0.93 (m, 1 H, H-26), 0.92 (s, 3 H, H-16’), 0.89 (s,
H, H-23), 0.83 (m, 1 H, H-15’), 0.72 (s, 3 H, H-29), 0.68 (s, 3 H, H-24),
[13] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D.
Vistica, J. T. Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Nat.
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Loppinet, S. Visvikis, Chem. Pharm. Bull. 2004, 52, 1436–1439.
1
3
.67 (dd, 1 H, H-5); C NMR (125 MHz, DMSO-d
6
): d ¼ 175.9 (C-30),
[
15] J.-P. Yong, J.-W. Wang, H. Aisa, L.-J. Liu, Chem. Nat. Comp. 2008,
4, 194–196.
44.6 (C-13), 127.5 (C-12), 76.7 (C-3), 65.4 (C-11), 59.6 (ethylene),
5.0 (C-5), 54.7 (C-9), 47.0 (C-18), 43.4 (C-20), 42.6 (C-8), 41.7 (C-19),
1.0 (C-14), 40.3 (C-1), 38.7 (C-10), 37.8 (C-22), 37.6 (C-4), 32.9 (C-7),
1.4 (C-17), 30.5 (C-21), 28.3 (C-23), 28.1 (C-29), 27.9 (C-28), 27.2
4
[16] L. Mikhailova, L. Baltina, R. Kondratenko, O. Kunert, L.
Spirikhin, F. Galin, G. Tolstikov, Chem. Nat. Prod. 2006, 42,
5
53–557.
(
1
(
C-2), 26.3 (C-15), 25.8 (C-16), 25.2 (C-27), 18.1 (C-6), 17.8 (C-26),
6.5 (C-25), 16.0 (C-24), 14.2 (methyl); anal. calcd. for C33
514.78): C, 76.99; H, 10.57; found: C, 76.78; H, 10.69.
[
[
[
17] G. S. R. Subba Rao, P. Kondaiah, S. K. Singh, P. Ravanan, M. B.
Sporn, Tetrahedron, 2008, 64, 11541–11548.
45 4
H O
18] L. Mikhailova, M. Khudobko, L. Baltina, O. Kukovinets, V.
Mavrodiev, F. Galin, Chem. Nat. Comp. 2007, 43, 571–575.
19] A. Presser, A. H u¨ fner, Monatsh. Chemie 2004, 135, 1015–1022.
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
[20] L. Mikhailova, M. Khudobko, L. Baltina, L. Spirikhin, R.
Kondratenko, L. Baltina, Chem. Nat. Comp. 2009, 45, 393–397.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com