Tormentic acid derivatives: Synthesis and apoptotic activity

Article abstract of DOI:10.1016/j.ejmech.2012.08.032

Several derivatives of tormentic acid have been prepared and tested for their antitumor activity. The dichloroacetate 14 is an excellent antitumor active agent acting by an apoptose inducing pathway as demonstrated by OA/PI staining, DNA laddering experiments as well as by an annexin V binding assay.

Full text of DOI:10.1016/j.ejmech.2012.08.032

European Journal of Medicinal Chemistry 56 (2012) 237e245
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tormentic acid derivatives: Synthesis and apoptotic activityq
*
René Csuk , Bianka Siewert, Christian Dressel, Renate Schäfer
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:
Several derivatives of tormentic acid have been prepared and tested for their antitumor activity. The
dichloroacetate 14 is an excellent antitumor active agent acting by an apoptose inducing pathway as
demonstrated by OA/PI staining, DNA laddering experiments as well as by an annexin V binding assay.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 10 July 2012
Received in revised form
22 August 2012
Accepted 22 August 2012
Available online 31 August 2012
Keywords:
Tormentic acid
Antitumor activity
Apoptosis
Acridine orange/propidium iodide assay
Annexin V
DNA laddering
1. Introduction
As early as 1915 tormentol (tormentoside) [1] was isolated as
the
b-D-glucopyranosyl ester of tormentic acid the structure of
Cancer is still one of the leading causes of death. The index of
cancer cure is often low and its treatment is still a challenge. Cancer
cells hold the ability to evade death, and expressing multidrug
resistance is an important draw-back in the chemotherapy of
cancer.
Natural products have been used to treat diseases for thousands
of years. They still play an important role in development of new
drugs. Among them, triterpenes represent a class of most signifi-
cant compounds. They have been shown to possess a broad variety
of medicinal properties. In continuation of our previous studies on
betuline, betulinic, glycyrrhetinic and boswellic acid derivatives as
antitumor active compounds, we became interested in tormentic
acid as a lead compound in the synthesis of antitumor active
derivatives.
Common tormentil (bloodroot, Potentilla erecta), also known as
shepherd’s knot, is a low, clump-forming plant growing wild all
over northern Europe and all over Asia. Extracts prepared from the
dried root have been used to treat bleedings and diarrhea (because
of its high content in tannins acting as adstringents) or to dye
leather red (because of the presence of phlobaphenes).
which was established in 1966 [2e4]. Tormentic acid, i.e. (2 R, 3 R,
19 R) 2,3,19-trihydroxy-urs-12-en-28-carboxylic acid (1), can be
extracted [5e17] from various plants, among them Myrianthus
serratus, Perilla frutescens, Cotoneaster simsonsii, Rubus sieboldii but
also from species of Potentilla, e.g. Potentilla anserina, Tormentilla
tormentilla or P. erecta.
There are ample examples for the antitumor activity of
pentacyclic triterpenes; less is known, however, about the bio-
logical activity of 1 and even fewer data have been reported for
derivatives of 1. Thus, 1 is able to inhibit in vitro platelet
aggregation [18], and the influence of 1 on forming atheroscle-
rotic plaques [19] in mice has been investigated. In addition, TA
reduced vascular smooth muscle cell proliferation [20] and
possesses [21,22] some anti-inflammatory activity. Compound 1
reduced also the viability of human gastric cells [13] by an
inhibition [13,23,24] of a- and b-DNA polymerases. Only a weak
cytotoxic activity has been established [25,26] for different
tumor cell lines; some anticancer activity has been found for 1
for lymphocytic leukemua cells [27]. Interesting to note that 1
shows little toxicity [13] to normal cells, and 1 has been sug-
gested [20] to be developed for the treatment of post-
angioplasty re-stenosis. Recently, 1 methyl ester (2) has been
shown [28] to act as a selective, low micromolar inhibitor of
q
Dedicated to Prof. Dr. Rainer Beckert, Friedrich-Schiller Universität Jena, on the
occasion of his 60th birthday. Ad multos annos!
11
b-hydroxysteroid dehydrogenase and to display anti-
* Corresponding author. Tel.: þ49 0 345 55 25660; fax: þ49 0 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
inflammatory effects [29,30].
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.032

238
R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
2. Results
Acetylation of 1 (Scheme 2) with acetic anhydride in dry pyri-
dine for 24 h yielded 66% of the diacetate 9. If the reaction was
stopped after 3 h, the 2-O-acetyl derivative 10 and the 3-O-acetyl
derivative 11 were isolated in 56% and 21%, respectively. The
monoacetates 10 and 11 were previously isolated [25] from
Cecropia lyratiloba, and shown to be effective inhibiting the viability
of a chronic myeloid leukemia blast crisis cell line by inducing
apoptosis [2]. Acetylation of 3 under similar conditions provided
acetate 12 whereas from compound 4 mono-acetylated 13 was
formed. Acylation of 2 with chloroacetyl chloride yielded the 2,3-
bis(chloroacetyloxy)-compound 14, the 2-O-chloro acetate 15 and
the 3-O-chloro acetate 16.
2.1. Chemistry
Quite recently, we became aware that small structural modifi-
cations, e.g. esterification or acylation of a triterpenoid skeleton
[31e38], might result in obtaining compounds of improved cyto-
toxicity. Thus, 1 was used as an easy accessible starting material for
the synthesis of “simple” derivatives.
Treatment of 1 (Scheme 1) with MeI/K2CO3 gave the methyl ester
2 [4,28,39,40] in almost quantitative yield. TEMPO oxidation [41,42]
of 2 yielded the 2-oxo compound 3 [1] in 83% yield; compound 3 is
characterized by the presence of a carbonyl signal in its 13C NMR
spectra at
d
¼ 211.0 ppm. This oxidation advances in a regioselective
2.2. Biology
way; no oxidation at position C-3 could be noted. The reason for this
regioselectivity might be the steric hindrance at position C-3
because of the presence of the two geminal methyl groups at C-4.
Oxidation of 2 using bis(tri-n-butyl-tin)oxide [43,44] in the
presence of bromine at 0 ꢀC, however, yielded the 3-oxo compound
4 whose carbonyl group can be found in the 13C NMR spectrum at
Thus, contrary to previous findings with betulinic acid, neither
an esterification nor an acetylation resulted in products of signifi-
cantly increased cytotoxicity (Table 1). Mono- or diacetylated
products 9e11 showed only moderate cytotoxicity (IC50 > 30 mmol)
for all human tumor cell lines tested. A similar behavior can be
found for the mono-acetylated keto compounds 12 [1] and 13 and
for the keto compounds 3, 4 and 5. An improvement was observed
for the mono-chloroacetylated compounds 15 and 16; a signifi-
cantly improved cytotoxicity, however, was observed for bis-
chloroacetylated 14.
Cell death can occur [46,47] either necrotic or programmed by
a variety of different forms being known for the latter. Apoptosis is
characterized [46] inter alia by cell shrinking, membrane blebbing,
an enhanced activity of caspases, a translocation of phosphati-
dylserine and DNA fragmentation. Previous work of Rocha et al.
[2,25] and Fogo et al. [20] gave evidence for 1 and several alkynic
derivatives thereof for acting by inducing apoptosis. To evaluate the
ability of our compounds, tormentic acid methyl ester 2 and the
d
¼ 216.6 ppm. Using a prolonged reaction time and an excess of
oxidizing agent gave the 3-oxo-1,12-diene 5 in 57% yield. Reduction
of 3 with sodium borohydride proceeded in a stereoselective way
and provided the 2-epi compound 6; compound 6 represents a 2,3-
bis epimer to euscaphic ester 8; the latter is easily obtained from
naturally occurring euscapic acid (7) by esterification with diazo-
methane. As an alternative, reduction of 5 under the same condi-
tions gave a 70% yield of 6. Compound 8 was oxidized in
a regioselective manner to afford 4.
For betulin and betulinic acid, several acylated derivatives
showed a higher antitumor activity than their parent compounds
[38,45]. Therefore, it seemed of interest to prepare several acylated
analogs of 2 and to compare their biological activity with parent 2.
Scheme 1. a) TEMPO, NaOCl, CH2Cl2, 8 h, 63%; b) [(nBu)3Sn]2O, Br2, 0 ꢀC, 1 min, 62%; c) [(nBu)3Sn]2O, Br2, 0 ꢀC, 15 min, 57%; d) NaBH4, MeOH, reflux, 1 h, 82%; e) [(nBu)3Sn]2O, Br2,
ꢀC, 1 min, 67%; f) NaBH4, MeOH, reflux, 1 h, 70%; g) CH2N2, MeOH, 95%.
0

R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
239
Scheme 2. a) Ac2O, pyridine, CH2Cl2, 24 ꢀC, 3e24 h, 9: 66%, 10: 56%, 11: 21%, 12: 66%, 13: 68%; b) ClCH2COCl, pyridine, CH2Cl2, 24 ꢀC, 4 h, 14: 26%, 15: 40%, 16: 11%.
most active compound of this series, bis-chloroacetyl 14 were
tested in more detail.
a characteristic feature of necrosis [leading to deep red light
emission from the propidium iodide (PI)], an intact membrane
indicates a programmed cell death since PI e as a double charged
molecule e cannot enter the cell as long as the cell membrane is
intact [59]. All compounds used in this AO/PI assay induced
As indicated above, programmed cell death is characterized [47]
by different morphological changes. Thus, living cells (518A2) were
stained with acridine orange (AO) and investigated by fluorescence
microscopy. AO is an uncharged cationic dye; it binds to nucleic
acids. AO and nucleic acids form either monomeric complexes with
double stranded nucleic acids (exhibiting a green fluorescence) or
dimers with single stranded nucleic acids (emitting orange light).
Maslinic acid (MA) is well known for its ability to induce apoptosis
in cancer cells and was used as a control [22,27,48e57]. A typical
condensation of the chromatin as well as blebbing of the nuclear
membrane and a shrinking of the cells was observed. Compound 2,
however, shows only a weak ability to induce cell death for 518A2
a
controlled cell death hence paralleling previous findings
[2,25,50,60] for other triterpenoic acids.
To gain a deeper insight, additional experiments were called for.
Phosphatidylserine e a label for cell death e switches from the
inner to the outer cell membrane during the cascade of apoptosis
[47]. Annexin V, a cellular protein of the annexin group, selectively
binds to phosphatidylserine. By a combination of the protein with
fluorescine isothiocyanate (FITC) a fluorescence active dye is
formed. In this assay 8505C cancer cells emitted green light hence
having bound annexin V-FITC and thus indicating that these cells
died by apoptosis [61]. The same was true for experiments
employing 518A2 cancer cells. Fig. 2 depicts the results from the
annexin V-FITC/PI stained cells by FACS-analysis.
Another typical hallmark of apoptosis is an exactly determined
cutting of the DNA by endonucleases into multiple 180 bp frag-
ments (and multiples thereof) [62,63]. All tested compounds gave
the characteristic DNA ladders. To evaluate the cancer-to-control
selectivity of some of our compounds, additional experiments
cells at a concentration of 30 mM. The condensation of chromatin as
well as the blebbing of the nuclear membrane indicates a pro-
grammed cell death process. The same phenomenon was observed
for 8505C human thyroid carcinoma cell cells. Some red dots were
seen during microscopy; these were assigned to proteasomes or are
due to lysosomal activity [58].
Additional investigations using an AO/PI exclusion dye assay
(Fig. 1) showed that the majority of the dead cells still possess an
intact cell membrane. While membrane disruption is
Table 1
Cytotoxicity (IC50 in mmol; SRB assay) for tormentic acid (1) and compounds 2e16 in a panel of various cancer cell lines [518A2 (melanoma), 8505C (anaplastic thyroid), A253
(head), A2780 (ovarian), A549 (lung), DLD1 (colon), MCF7 (mamma)], non malignant mouse fibroblast (NiH 3T3), and human fibroblast primary culture cells (WW030272).
Values were obtained from SRB assays after 96 h of treatment; the values are averaged from at least 5 independent experiments (n.d. not determined).
518A2
8505C
A253
A2780
A549
DLD-1
MCF7
NiH 3T3
WW030272
1
2
3
4
5
6
8
9
10
11
12
13
14
15
16
>30
23.4 ꢁ 0.8
42.0 ꢁ 5.2
>30
12.9 ꢁ 0.2
25.0 ꢁ 0.9
>30
>30
>30
31.0 ꢁ 0.1
n.d.
n.d.
31.0 ꢁ 0.2
37.4 ꢁ 1.0
>30
32.3 ꢁ 2.5
31.3 ꢁ 3.4
28.7 ꢁ 0.5
9.4 ꢁ 0.5
26.0 ꢁ 5.4
22.6 ꢁ 1.0
17.8 ꢁ 4.5
20.4 ꢁ 3.7
25.3 ꢁ 2.6
26.2 ꢁ 0.7
8.2 ꢁ 0.9
14.8 ꢁ 0.4
1.5 ꢁ 0.8
2.6 ꢁ 0.3
6.9 ꢁ 0.7
>30
47.7 ꢁ 1.1
47.5 ꢁ 1.1
n.d.
n.d.
n.d.
31.3 ꢁ 3.9
17.0 ꢁ 2.0
23.9 ꢁ 4.3
17.8 ꢁ 1.9
4.9 ꢁ 0.4
>30
15.6 ꢁ 5.0
19.2 ꢁ 0.6
7.7 ꢁ 2.0
>30
>30
>30
>30
n.d.
8.9 ꢁ 0.5
17.2 ꢁ 2.7
>30
6.8 ꢁ 1.8
>30
15.3 ꢁ 2.0
16.4 ꢁ 12.6
27.3 ꢁ 3.5
>30
11.8 ꢁ 0.2
18.0 ꢁ 0.1
>30
>30
18.9 ꢁ 3.5
12.8 ꢁ 1.5
17.6 ꢁ 4.9
24.3 ꢁ 9.0
28.1 ꢁ 0.9
4.4 ꢁ 0.4
9.0 ꢁ 1.4
0.8 ꢁ 0.4
2.6 ꢁ 0.3
4.1 ꢁ 0.4
>30
>30
n.d.
27.7 ꢁ 0.2
29.3 ꢁ 2.5
30.6 ꢁ 0.4
35.8 ꢁ 1.8
23.4 ꢁ 1.7
>30
>30
>30
>30
32.3 ꢁ 0.1
n.d.
>30
>30
>30
>30
>30
>30
>30
>30
>30
n.d.
n.d.
n.d.
n.d.
>30
>30
5.6 ꢁ 1.2
28.9 ꢁ 0.7
1.1 ꢁ 0.2
4.5 ꢁ 0.4
5.5 ꢁ 0.6
7.0 ꢁ 0.2
6.7 ꢁ 1.1
18.5 ꢁ 0.4
1.6 ꢁ 0.9
4.6 ꢁ 0.5
4.1 ꢁ 0.4
7.8 ꢁ 0.4
31.3 ꢁ 0.4
1.2 ꢁ 0.5
9.7 ꢁ 0.6
10.0 ꢁ 1.0
13.5 ꢁ 0.4
28.2 ꢁ 0.5
2.2 ꢁ 0.2
3.7 ꢁ 0.4
6.0 ꢁ 0.6
6.7 ꢁ 1.1
25.1 ꢁ 4.6
1.1 ꢁ 0.1
2.1 ꢁ 0.2
4.2 ꢁ 0.4
>30
1.6 ꢁ 0.7
4.6 ꢁ 0.5
7.5 ꢁ 0.8
3.4 ꢁ 1.1
n.d.
n.d.

240
R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
Fig. 1. AO/PI assay of dead 8505C cells. The cells were treated with MA (A), tormentic acid (B), 2 (C) and 14 (D); green cells indicate a controlled cell death, deep red cells a necrotic
way of exitus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
using human fibroblast primary culture cells WW030272 were
(PAA Laboratories) at 37 ꢀC in a humidified atmosphere of 5% CO2/
used. The results of these experiments are included in the table.
95% air.
3. Conclusion
4.1.2. Cytotoxicity assay
The cytotoxicity of the compounds was evaluated using the
In summary, tormentic acid (1) as well as its methyl ester 2 are
adequate and its dichloroacetate 14 is an excellent antitumor active
agent acting by an apoptose inducing pathway as demonstrated by
OA/PI staining, DNA laddering experiments as well as by an annexin
V binding assay.
sulforhodamine-B
(SRB)
(SigmaeAldrich)
microculture
colorimetric assay. In short, exponentially growing cells were
seeded into 96-well plates on day 0 at the appropriate cell densi-
ties to prevent confluence of the cells during the period of exper-
iment. 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 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 4 ꢀC. After
fixation, the cells were washed in a strip washer. The washing was
done five times with water using alternate dispensing and aspira-
4. Experimental
4.1. Biological material
4.1.1. Cell lines and culture conditions
The cell lines 518A2, 8505C, A253, A2780, A549, DLD-1, MCF-7,
NiH 3T3 and WW030272 were included in this study. Cultures were
maintained as monolayer in RPMI 1640 (PAA Laboratories, Pasch-
ing, Germany) supplemented with 10% heat inactivated fetal bovine
serum (Biochrom AG, Berlin, Germany) and penicillin/streptomycin
tion procedures. Afterward the plates were dyed with 100
ml of 0.4%
SRB (sulforhodamine B) for about 30 min. The plates were washed
Fig. 2. FACS analysis of 8505C cells after 6 h incubation with 1 (left) or compound 14 (right).

R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
241
with 1% acetic acid to remove the excess of the dye and allowed to
air dry overnight. Tris base solution (100 l of 10 mM) was added to
each well and absorbance was measured at 570 nm (using a 96 well
plate reader, Tecan Spectra, Crailsheim, Germany). The IC50 was
estimated from the doseeresponse curves.
a FINNIGAN MAT TSQ 7000 (electrospray, voltage 4.5 kV, sheath gas
nitrogen) instrument. Elemental analyses were measured on
a Foss-Heraeus Vario EL unit. IR spectra were recorded on a Perkine
Elmer FT-IR spectrometer Spectrum 1000, optical rotations on
a PerkineElmer 341 polarimeter (1 cm micro cell, 25 ꢀC) and UVe
vis spectra on a PerkineElmer unit, Lambda 14. TLC was performed
on silica gel (Merck 5554, detection by UV absorption). Solvents
were dried according to usual procedures. The purity of the
compounds was checked by HPLC/DAD and found to be >98% for
each compound.
m
4.1.3. Morphological investigation of living cells
In an eight-well chamber slide (SigmaeAldrich) 10.000 cells of
the human thyroid cancer cell line 8505C or 10.000 cells of the
melanoma cell line 518A2 were seeded. After 24 h of incubation, the
medium was removed, and the cells were treated with maslinic
acid (MA), tormentic acid (1) or tormentic acid methyl ester (2)
4.3. (3 R, 19 R) methyl 3,19-dihydroxy-2-oxo-urs-12-en-28-
(4 ml, 30
m
M). On the next day the supernatant medium was
carboxylate (3)
removed, and the cells were washed with PBS (w(o, 1 ml)) and
stained with acridine orange (5.10ꢂ6 mol). Visual inspection was
performed using a fluorescence microscope (Zeiss Axioskop).
To a solution of 2 (376 mg, 0.75 mmol) and TEMPO (2 mg,
0.01 mmol) in dichloromethane (20 ml), a solution of KBr (9 mg,
0.075 mmol) and (n-Bu)4NBr (120 mg, 0.37 mmol), in an aq. solu-
tion of NaHCO3 (5%, 3 ml) was added. Under vigorous stirring an aq.
solution of NaOCl (1 M, 0.8 ml) was slowly added with 2 h (no
further discoloration of the reaction mixture), and stirring was
continued for another 6 h. The reaction was quenched by the
addition of water (50 ml), and extracted with dichloromethane
(4 ꢃ 40 ml). The combined organic phases were washed with brine
(2 ꢃ 30 ml), dried (Na2SO4), and the solvent was evaporated. The
residue was subjected to chromatography (silica gel, toluene/ethyl
acetate/formic acid/n-heptane 80:20:3:10) to yield 3 (237 mg, 63%)
4.1.4. Apoptosis assay of dead cells by AO/PI dye exclusion test and
annexin V-FITC
The death of the cells was analyzed employing an OA/PI assay as
well as with annexin V-FITC dye using fluorescence microscopy and
human cancer cell lines 518A2 and 8505C, respectively. Approx.
1*106 cells were seeded in cell culture flasks (25 cm2), and the cells
were allowed to grow up to 80%. After removing of the used
medium, the substance loaded fresh medium was reloaded (or
a blank new medium as a control). After 24e48 h, the supernatant
medium was collected and centrifuged (300 g, 4 ꢀC), the pellet was
gently suspended in phosphate-buffered saline (PBS, 1 ml) and
centrifuged again. The PBS was removed, and the pellet again
as a colorless solid; mp 104e106 ꢀC; ½a D20
ꢄ
¼ þ37.3ꢀ (c ¼ 0.47, CHCl3);
RF
¼
0.47 (toluene/ethyl acetate/formic acid/n-heptane
80:20:3:10); IR (KBr):
n
¼ 3488 br, 2949 s, 1717 s, 1458 m, 1394 m,
gently suspended in PBS (100
performed using a fluorescence microscope after having mixed the
cell suspension (10 l) with a solution of AO/PI (10 l). A green
ml). The analysis of the cells was
1234 m, 1208 m, 1153 m, 1117 m, 1057 m, 1034 m, 970 w, 772 w,
733 w cmꢂ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.28 (dd, J11 , 12 ¼ 3.1,
0
00
,
m
m
J11
¼ 3.1 Hz, 1 H, H-12), 3.83 (s, 1 H, H-3ax), 3.53 (s, 3 H, H-31),
12
fluorescence indicates apoptosis whereas a red colored cell indi-
cates necrosis.
2.54 (m, 1 H, H-16ax), 2.39 (d, J1eq, 1ax ¼ 12.5 Hz, H-1eq), 2.03 (d, J1ax,
¼ 12.5 Hz, 1 H, H-1ax), 1.89 (m, 3 H, H-9, H-110 and H-1100), 1.67
1eq
For the investigations using annexin V-FITC, the cells were
washed with annexin V binding buffer after the treatment with
PBS, then centrifuged and dyed for 15 min using an annexin V
staining buffer. Analyses were performed using a fluorescence
microscope; green colored cells indicate cells with phosphati-
dylserine on the outer cell membrane, a phenomenon that is typical
for apoptosis.
(m, 1 H, H-2200), 1.66e1.51 (m, 6 H, H-600, H-700, H-15ax, H-16eq, H-2100
and H-220), 1.39 (m, 2 H, H-60, H-5), 1.37e1.30 (m, 2 H, H-70, H-20),
1.24 (s, 3 H, H-27), 1.19 (m, 1 H, H-210), 1.14 (s, 3 H, H-29), 1.13 (s, 3 H,
H-23), 0.97 (m, 1 H, H-15eq), 0.87 (d, J20, 30 ¼ 6.5 Hz, 3 H, H-30), 0.81,
0.63 and 0.62 (each s, 9 H, H-24, H-25, H-26) ppm; 13C NMR
(125 MHz, CDCl3):
d
¼ 211.0 (C2), 178.2 (C28), 138.4 (C13), 128.2
(C12), 82.6 (C3), 73.1 (C19), 54.4 (C5), 53.2 (C18), 53.1 (C1), 51.6
(C31), 47.8 (C17), 47.2 (C9), 45.7 (C4), 43.7 (C10), 41.3 (C14), 41.1
(C20), 40.3 (C8), 37.3 (C22), 32.4 (C7), 29.4 (C23), 28.2 (C15), 27.4
(C29), 25.9 (C21), 25.4 (C16), 24.3 (C27), 23.6 (C11), 21.0 (C6), 18.6,
16.5, 16.1 (C24, C25, C26), 16.2 (C30) ppm; MS (ESI, MeOH): m/z
(%) ¼ 501.4 ([M þ H]þ,10), 518.4 ([M þ NH4]þ,10), 523.3 ([M þ Na]þ,
100), 539.3 ([M þ K]þ, 19); analysis for C31H48O5 (500.71): C, 74.36;
H, 9.66; found: C, 74.21; H, 9.82.
4.1.5. DNA laddering experiments
Approximately 1*106 cells (518A2 or 8505C) were seeded in cell
culture flasks (25 cm2), and the cells were allowed to grow up to
80%. After removing of the used medium, the substance loaded
medium was reloaded (or a blank fresh medium as a control). After
24e48 h, the supernatant medium was collected and centrifuged
(300 g, 4 ꢀC). The pellet was gently suspended in phosphate-
buffered saline (PBS 1 ml) and centrifuged again. The PBS was
4.4. (2 R, 19 R) methyl 2,19-dihydroxy-3-oxo-urs-12-en-28-
carboxylate (4)
removed and lyses buffer (30
m
l, 0 ꢀC, 10 min) was added. The cells
l,
ꢀC, 10 min) and for 12 h at 50 ꢀC after having been treated with
were incubated for 2 h (37 ꢀC) after treatment with RNAse (10
m
0
From 2: To a solution of 2 (500 mg, 0.99 mmol) in dry chloro-
form (20 ml) at 0 ꢀC [(n-Bu)3Sn]2O (0.5 ml, 0.99 mmol) and bromine
proteine kinase K (10 ml). The extract was mixed with DNA-ladder
dye (10
ml) and analyzed by gel electrophoresis (agarose,
(51 ml, 0.99 mmol) were added. After stirring for 1 min, NEt3
150 mV, 2 h).
(0.10 ml) was added, the solvents were removed under diminished
pressure, and the residue was subjected to chromatography (silica
gel, toluene/ethyl acetate/formic acid/n-heptane, 80:20:3:10) to
afford 4 (310 mg, 62%) as a colorless solid.
4.2. General e chemistry
Reagents were bought from commercial suppliers without any
further purification. Melting points were measured with a LEICA
hot stage microscope and were not corrected. NMR spectra were
recorded on VARIAN Gemini 200, Gemini 2000 or Unity 500
spectrometers at 27 ꢀC with trimethylsilane as an internal standard,
From 8: Analogous synthesis starting from 8 (673 mg,
0.99 mmol) gave 4 (452 mg, 67%) as a colorless solid; mp 114e
117 ꢀC; ½a 2D0
ꢄ
¼ þ31.3ꢀ (c ¼ 0.42, CHCl3); RF ¼ 0.46 (toluene/ethyl
acetate/formic acid/n-heptane, 80:20:3:10); IR (KBr):
n
¼ 3483 br,
2935 s, 1720 s, 1458 m, 1390 m, 1263 m, 1232 m, 1207 m, 1192 m,
d
are given in ppm and J in Hz. Mass spectra were taken on
1153 s, 1094 m, 1056 m, 961 w, 866 w, 771 w cmꢂ1 1H NMR
;

242
R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
0
00
(500 MHz, CDCl3):
d
¼ 5.32 (dd, 1 H, J12, 11 ¼ 3.5, J12, 11 ¼ 3.5 Hz, H-
106e107 ꢀC; ½a 2D0
ꢄ
¼ þ50.7ꢀ (c ¼ 0.6, CHCl3) (lit.: [3] 16.3ꢀ);
12), 4.51 (dd,1 H, J2ax, 1ax ¼ 12.5, J2ax, 1eq ¼ 6.5 Hz,1 H, H-2ax), 3.58 (s,
RF
¼
0.26 (toluene/ethyl acetate/formic acid/n-heptane,
3 H, H-31), 2.57 (s, 1 H, H-18), 2.51 (m, 1 H, H-16ax), 2.40 (dd, J1eq
,
80:20:3:10); IR (KBr):
n
¼ 3510 br, 2929 s, 1721 s, 1648 w, 1458 m,
¼ 12.5, J1eq, 2ax ¼ 6.5 Hz, 1 H, H-1eq), 2.04e2.01 (m, 2 H, H-110, H-
1380 m, 1368 m, 1322 m, 1262 m, 1229 m, 1208 m, 1152 s, 1114 m,
1095 m, 1050 m, 1030 m, 1000 m, 973 w, 932 w, 900 w, 867 w,
1ax
1100), 1.72e1.28 (m, 12 H, H-60, H-600, H-70, H-700, H-9, H-15ax, H-16eq
,
H-20, H-210, H-2100, H-220, H-2200), 1.25 (s, 3 H, H-25), 1.22 (s, 3 H, H-
27),1.20 (s, 3 H, H-29),1.16 (s, 3 H, H-24),1.16e1.13 (m, 2 H, H-1ax, H-
5), 1.11 (s, 3 H, H-23), 1.01 (m, 1 H, H-15eq), 0.93 (d, J30, 20 ¼ 7.0, 3 H,
806 w, 787 w, 772 w, 706 w, 684 w, 654 w cmꢂ1 1H NMR
;
0
00
(500 MHz, CDCl3):
d
¼ 5.35 (dd, 1 H, J12, 11 ¼ 3.4, J12, 11 ¼ 3.4 Hz, H-
12), 4.07 (ddd, 1 H, J2eq, 3ax ¼ 4.0, J2eq, 1ax ¼ 3.6, J2eq, 1eq ¼ 2.8 Hz, H-
2eq), 3.58 (s, 3 H, H-31), 3.20 (d, 1 H, J3ax, 2eq ¼ 4.0 Hz, H-3ax), 2.58
(s, 1 H, H-18), 2.48 (m, 1 H, H-16ax), 2.08 (dd, 1 H, J1eq, 1ax ¼ 14.5,
J1eq, 2eq ¼ 2.8 Hz, H-1eq), 2.02e1.99 (m, 2 H, H-110, H-1100), 1.72e
1.47 (m, 9 H, H-60, H-600, H-700, H-9, H-15ax, H-16eq, H-2100, H-220,
H-2200), 1.39 (m, 1 H, H-20), 1.29 (m, 1 H, H-210), 1.26 (m, 1 H, H-70),
1.23 (s, 3 H, H-27), 1.21 (s, 3 H, H-25), 1.19 (s, 3 H, H-29), 1.14 (dd,
1 H, J1ax, 1eq ¼ 14.5, J1ax, 2eq ¼ 3.6 Hz, H-1ax), 1.01 (m, 1 H, H-15eq),
0.99 and 0.98 (each s, 6 H, H-23, H-24), 0.92 (d, 3 H, J30,
H-30), 0.73 (s, 3 H, H-26) ppm; 13C NMR (CDCl3,125 MHz):
d
¼ 216.6
(C3), 178.3 (C28), 138.3 (C13), 128.5 (C12), 73.1 (C19), 69.1 (C2), 57.6
(C5), 53.1 (C18), 51.6 (C31), 49.5 (C1), 47.8, 47.7 (C4, C17), 46.9 (C9),
41.2 (C14), 41.1 (C20), 40.0 (C8), 37.6 (C10), 37.3 (C22), 32.4 (C7),
28.2 (C15), 27.4 (C29), 25.9 (C21), 25.4 (C16), 24.7 (C23), 24.4 (C27),
23.8 (C11), 21.6 (C24), 19.2 (C6), 16.8 (C26), 16.1 (C30), 15.9 (C25)
ppm; MS (ESI, MeOH): m/z (%) ¼ 501.4 ([M þ H]þ, 116), 518.4
([M þ NH4]þ, 10), 523.3 ([M þ Na]þ, 100), 539.3 ([M þ K]þ, 17);
analysis for C31H48O5 (500.71): C, 74.36; H, 9.66; found: C, 74.25;
H, 9.81.
¼ 6.6 Hz, H-30), 0.81 (m, 1 H, H-5), 0.68 (s, 3 H, H-26) ppm; 13
C
20
NMR (125 MHz, CDCl3, 125 MHz):
d
¼ 178.3 (C28), 138.1 (C13),
129.3 (C12), 78.5 (C3), 73.2 (C19), 71.1 (C2), 55.1 (C5), 53.2 (C18),
51.6 (C31), 47.9 (C17), 47.6 (C9), 44.0 (C1), 41.3 (C14), 41.1 (C20),
40.0 (C8), 38.1 (C4), 37.4 (C22), 36.7 (C10), 32.7 (C7), 29.7 (C23),
28.1 (C15), 27.4 (C29), 26.0 (C21), 25.5 (C16), 24.6 (C27), 23.7 (C11),
18.2 (C6), 17.3 (C24), 16.6 (C26), 16.2 (C25), 16.1 (C30) ppm; MS
4.5. (19 R) methyl 2,19-dihydroxyursa-3-oxo-1,12-dien-28-
carboxylate (5)
Following the procedure as described above (15 min reaction
time), from 2 (214 mg, 0.43 mmol), [(n-Bu)3Sn]2O (0.44 ml,
(ESI, MeOH): m/z (%)
¼
525.5 ([M
þ
Na]þ, 100), 556.9
0.86 mmol) and bromine (44
m
l, 86 mmol), compound 5 (121 mg,
([M þ MeOH] þ, 21); analysis for C31H50O5 (502.73): C, 74.06; H,
57%) was obtained as
a
white solid; mp 115e118 ꢀC;
10.02; found: C, 73.96; H, 10.14.
½
a 2D0
ꢄ
¼ þ62.8ꢀ (c ¼ 0.47, CHCl3); RF ¼ 0.65 (toluene/ethyl
acetate/formic acid/n-heptane 80:20:3:10); IR (KBr):
n
¼ 3436 br,
4.7. Euscaphic acid methyl ester (8)
2933 s, 2876 s, 1725 s, 1669 s, 1648 m, 1458 m, 1404 m, 1383 s,
1238 s, 1208 s, 1152 s, 1091 m, 1054 m, 1034 m, 970 w, 930 w,
From the esterification of euscaphic acid (7) with diazomethane;
mp: 120e122 ꢀC (lit.: [40] 130e132 ꢀC; [64] 122e124 ꢀC; [65]
865 w, 786 w, 772 w, 753 w, 538 w cmꢂ1 1H NMR (500 MHz,
;
CDCl3):
110
d
¼ 6.34 (s, 1 H, H-1), 5.93 (s, 1 H, OH), 5.40 (dd, 1 H, J12,
140 ꢀC); ½a 2D0
ꢄ
¼ þ31.6ꢀ (c ¼ 0.46, CHCl3) (lit: þ31ꢀ [66]); RF ¼ 0.19
00
¼ 3.5, J12, 11 ¼ 3.5 Hz, H-12), 3.61 (s, 3 H, H-31), 2.62 (s, 1 H, H-
(toluene/ethyl acetate/formic acid/n-heptane, 80:20:3:10); MS (ESI,
MeOH): m/z (%) ¼ 503.4 ([M þ H]þ,10), 520.3 ([M þ NH4]þ, 6), 525.5
([M þ Na]þ, 100), 556.9 ([M þ MeOH]þ, 46).
00
00
18), 2.52 (m, 1 H, H-16ax), 2.21 (dd, 1 H, J11 ,
¼ 6.6, J11 ,
9
¼ 3.5 Hz, H-1100), 2.13 (dd, 1 H, J11 , 9 ¼ 11.2, J11 , 12 ¼ 3.5 Hz, H-
0
0
12
110), 1.96 (dd, 1 H, J9, 11 ¼ 11.2, J9, 11 ¼ 6.6 Hz, H-9), 1.76e1.52 (m,
9 H, H-5, H-60, H-600, H-700, H-15ax, H-16eq, H-2100, H-220, H-2200),
1.43e1.37 (m, 2 H, H-70, H-20), 1.26 (s, 3 H, H-27), 1.24 (m, 1 H, H-
210), 1.22, 1.21, 1.12 (each s, 12 H, H-23, H-24, H-25, H-29), 1.04 (m,
1 H, H-15eq), 0.94 (d, 3 H, J30, 20 ¼ 7.0 Hz, H-30), 0.77 (s, 3 H, H-26)
0
00
4.8. (2 R, 3 R, 19 R) 2,3-Bis(acetyloxy)-19-hydroxyurs-12-en-28-
carboxylic acid (9)
Acetylation of 1 (150 mg, 0.31 mmol) in dry pyridine (6 ml) with
acetic anhydride for 12 h at 24 ꢀC yielded after usual work-up and
re-crystallization from toluene 9 as a colorless solid; mp 178e
ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 201.1 (C3), 178.3 (C28), 143.6
(C2), 138.6 (C13), 128.4 (C12), 128.1 (C1), 73.1 (C19), 53.8 (C5),
53.3 (C18), 51.6 (C31), 47.9 (C17), 43.9 (C4), 42.6 (C9), 41.6 (C10),
42.0 (C20), 40.6 (C14), 38.4 (C8), 37.3 (C22), 32.6 (C7), 28.2 (C15),
27.4 (C29), 27.1 (C23), 26.0 (C21), 25.4 (C16), 24.5 (C27), 23.6
(C11), 21.8, 19.4 (C24, C25), 18.7 (C6), 17.1 (C26) 16.0 (C30) ppm;
MS (ESI, MeOH): m/z (%) ¼ 499.7 ([M þ H]þ, 13), 521.5 ([M þ Na]þ,
100), 537.4 ([M þ K]þ, 75), 553.2 ([M þ MeOH]þ, 98), 569.3
([M þ K þ MeOH]þ, 34); analysis for C31H46O5 (498.69): C, 74.66;
H, 9.29; found: C, 74.53; H, 9.38.
180 ꢀC (lit.: [55] 186e189 ꢀC); ½a 2D0
ꢄ
¼ þ5.8ꢀ (c ¼ 0.51, CHCl3)
(lit.: þ12ꢀ [25]; þ6ꢀ [67]); RF ¼ 0.45 (toluene/ethyl acetate/formic
acid/n-heptane, 80:20:3:10); IR (KBr):
n
¼ 3433 br, 2937 s, 1743 s,
1456 m, 1369 s, 1252 s, 1154 m, 1109 w, 1033 m, 965 m, 932 w,
866 w, 759 w, 642 w, 598 w cmꢂ1 1H NMR (500 MHz, CDCl3):
¼ 5.31 (dd, 1 H, J12, 11 ¼ 3.2, J12, 11 ¼ 3.2 Hz, H-12), 5.08 (ddd, 1 H,
;
0
00
d
J2ax, 1ax ¼ 11.1, J2ax, 3ax ¼ 10.3, J2ax, 1eq ¼ 4.7 Hz, H-2ax), 4.73 (d, 1 H,
J3ax, 2ax ¼ 10.3 Hz, H-3ax), 2.52 (m, 2 H, H-18, H-16ax), 2.03 (s, 3 H, H-
32 or H-34), 2.02 (m, 1 H, H-1eq), 1.96 (m, 5 H, H-32 or H-34 and H-
110, H-1100), 1.79e1.47 (m, 8 H, H-600, H-700, H-9, H-15ax, H-16eq, H-
2100, H-220, H-2200), 1.42e1.35 (m, 2 H, H-60, H-20), 1.32e1.27 (m, 2 H,
H-70, H-210), 1.23 (s, 3 H, H-27), 1.18 (s, 3 H, H-29), 1.09 (m, 1 H, H-
1ax),1.04 (s, 3 H, H-25), 0.99 (m, 1 H, H-15eq), 0.96 (m,1 H, H-5), 0.93
(d, 3 H, J30, 20 ¼ 6.6 Hz, H-30), 0.88 (s, 6 H, H-23, H-24), 0.70 (s, 3 H,
4.6. (2 S, 3 R, 19 R) methyl 2,3,19-trihydroxyurs-12-en-28-
carboxylate (6)
From 3: To a solution of NaBH4 (53 mg, 1.41 mmol) in MeOH
(2 ml), a solution of 3 (235 mg, 0.47 mmol) was added drop-wise
and heated under reflux for 1 h. After quenching with an aqueous
solution of NH4Cl (satd., 5 ml), dilution with water (20 ml), the
mixture was extracted with ethyl acetate (3 ꢃ 20 ml), the combined
extracts were washed (2 ꢃ 20 ml) and dried (Na2SO4). The solvent
was removed and the residue subjected to chromatography (silica
gel, n-pentane/ethyl acetate, 2:1) to afford 6 (193 mg, 82%) as
a colorless solid.
H-26) ppm; 13C NMR (125 MHZ, CDCl3):
d
¼ 184.2 (C28),170.8,170.6
(C31, C33), 138.0 (C13), 128.8 (C12), 80.6 (C3), 73.0 (C19), 70.0 (C2),
54.7 (C5), 52.8 (C18), 47.7 (C17), 47.1 (C9), 43.9 (C1), 41.1 (C14), 41.0
(C20), 39.9 (C8), 39.3 (C4), 38.1 (C10), 37.4 (C22), 32.4 (C7), 28.4
(C23), 28.1 (C15), 27.3 (C29), 25.9 (C21), 25.2 (C16), 24.4 (C27), 23.7
(C11), 22.0, 20.9 (C32, C34), 18.2 (C6), 17.6 (C24), 16.9 (C26), 16.3
(C25), 16.1 (C30) ppm; MS (ESI, MeOH): m/z (%) ¼ 571.4 ([M ꢂ H]ꢂ,
100), 616.9 ([M þ HCO2]ꢂ, 27); analysis for C34H52O7 (572.77): C,
71.30; H, 9.15; found: C, 71.18; H, 9.23.
From 5: In analogous manner from 5 (80 mg, 0.16 mmol)
compound 6 (56 mg, 70%) was obtained as a colorless solid; mp

R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
243
4.9. (2 R, 3 R, 19 R) 2-acetyloxy-3,19-dihydroxyurs-12-en-28-
carboxylic acid (10) and (2 R, 3 R, 19 R) 3-acetyloxy-2,19-
dihydroxyurs-12-en-28-carboxylic acid (11)
2935 s, 1721 s, 1458 m, 1396 m, 1371 m, 1292 m 1236 s, 1151 m, 1096
w, 1053 m, 1034 m, 1009 m, 969 w, 930 w, 866 w, 772 w, 691 w,
499 w cmꢂ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.28 (dd, 1 H, J12,
00
110
¼ 2.5, J12, 11 ¼ 2.5 Hz, H-12), 4.88 (s, 1 H, H-3ax), 3.53 (s, 3 H, H-
Acetylation of 1 (300 mg, 0.61 mmol) in dichloromethane
(30 ml) containing dry pyridine (2 ml) with acetic anhydride (1 ml)
for 3 h at 24 ꢀC followed by usual aqueous work-up and chroma-
tography (silica gel, n-pentane/ethyl acetate/ethanol, 17:10:1)
afforded 10 (182 mg, 56%) and 11 (67 mg, 21%).
31), 2.54 (s, 1 H, H-18), 2.46 (m, 1 H, H-16ax), 2.34 (d, 1 H, J1eq,
¼ 12.2 Hz, H-1eq), 2.12 (d, 1 H, J1ax, 1eq ¼ 12.2 Hz, H-1ax), 2.11 (s,
1ax
3 H, H-33), 1.88e1.85 (m, 3 H, H-9, H-110, H-1100), 1.68e1.51 (m, 7 H,
H-60, H-600, H-15ax, H-16eq, H-2100, H-220, H-2200), 1.46 (m, 1 H, H-5),
1.37e1.30 (m, 3 H, H-70, H-700, H-20), 1.24 (s, 3 H, H-27), 1.21 (m, 1 H,
H-210), 1.15 (s, 3 H, H-29), 1.04 (s, 3 H, H-23), 0.99 (m, 1 H, H-15eq),
0.88 (d, 3 H, J30, 20 ¼ 6.7 Hz, H-30), 0.84, 0.79, 0.62 (each s, 9 H, H-24,
Data for 10: colorless solid; mp 171e174 ꢀC; ½a D20
ꢄ
¼ þ4.7ꢀ
(c ¼ 0.51, CHCl3); RF ¼ 0.23 (toluene/ethyl acetate/formic acid/n-
heptane, 80:20:3:10); IR (KBr):
n
¼ 3510 br, 2937 s, 1724 s, 1457 m,
H-25, H-26) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 204.2 (C2), 178.2
1369 m, 1255 s, 1155 m, 1095 m, 1031 m, 961 m, 933 w, 865 w,
(C28), 170.5 (C32), 138.3 (C13), 128.2 (C12), 84.1 (C3), 73.1 (C19),
55.2 (C5), 53.9 (C1), 53.2 (C18), 51.6 (C31), 47.9 (C17), 47.1 (C9), 43.6,
43.1 (C4, C10), 41.3 (C14), 41.1 (C20), 40.3 (C8), 37.3 (C22), 32.4 (C7),
29.0 (C23), 28.2 (C15), 27.4 (C29), 26.0 (C21), 25.4 (C16), 24.3 (C27),
23.6 (C11), 20.6 (C33) 18.6 (C6), 17.5, 16.1, 15.9 (C24, C25, C26), 16.2
(C30); MS (ESI, MeOH): m/z (%) ¼ 543.4 ([M þ H]þ, 39), 560.6
([M þ NH4]þ, 38), 565.5 ([M þ Na]þ,100), 581.4 ([M þ K]þ,18), 597.0
([M þ Na þ MeOH]þ, 25); analysis for C33H50O6 (542.75): C, 73.03;
H, 9.29; found: C, 72.87; H, 9.41.
766 w, 660 w cmꢂ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.30 (dd,1 H, J12,
00
110
¼ 3.2, J12, 11 ¼ 3.2 Hz, H-12), 4.92 (ddd, 1 H, J2ax, 1ax ¼ 10.9, J2ax,
¼ 10.0, J2ax, 1eq ¼ 4.4 Hz, H-2ax), 3.18 (d, 1 H, J3ax, 2ax ¼ 10.0 Hz, H-
3ax
3ax), 2.51 (s, 1 H, H-18), 2.45 (m, 1 H, H-16ax), 2.04 (s, 3 H, H-33), 1.99
(m, 1 H, H-1eq), 1.99e1.94 (m, 2 H, H-110, H-1100), 1.78e1.45 (m, 8 H,
H-600, H-700, H-9, H-15ax, H-16eq, H-2100, H-220, H-2200), 1.40e1.26 (m,
3 H, H-70, H-20, H-210), 1.22 (s, 3 H, H-27), 1.17 (s, 3 H, H-29), 1.03,
1.01 (each s, 6 H, H-23, H-25), 1.00 (m, 1 H, H-15eq), 0.96 (m, 1 H, H-
1ax), 0.92 (d, 3 H, J30, 20 ¼ 6.6 Hz, H-30), 0.86 (m, 1 H, H-5), 0.83 (s,
3 H, H-24), 0.69 (s, 3 H, H-26) ppm; 13C NMR (125 MHz, CDCl3):
4.11. (2 R, 19 R) methyl 2-acetyloxy-19-hydroxy-3-oxo-urs-12-en-
28-carboxylate (13)
d
¼ 184.1 (C28), 171.6 (C31), 138.0 (C13), 129.0 (C12), 80.8 (C3), 73.3
(C2), 73.1 (C19), 55.0 (C5), 52.8 (C18), 47.7 (C17), 47.1 (C9), 43.7 (C1),
41.1 (C14), 41.0 (C20), 40.0 (C8), 39.7 (C4), 38.3 (C10), 37.4 (C22),
32.5 (C7), 28.5 (C23), 28.1 (C15), 27.3 (C29), 25.9 (C21), 25.3 (C16),
24.5 (C27), 23.7 (C11), 21.3 (C32), 18.3 (C6), 17.0, 16.6, 16.3 (C24, C25,
C26), 16.1 (C30) ppm; MS (ESI, MeOH): m/z (%) ¼ 529.7 ([M ꢂ H]ꢂ,
100), 575.3 ([M þ HCO2]ꢂ, 13); analysis for C32H50O6 (530.74): C,
72.42; H, 9.50; found: C, 72.36; H, 9.58.
Following the procedure given for 12, from
4 (100 mg,
0.20 mmol), pyridine (4 ml) and acetic anhydride (8 ml) 13 (74 mg,
68%) was obtained as
a
colorless solid; mp 101e104 ꢀC;
½
a 2D0
ꢄ
¼ þ39.3ꢀ (c ¼ 0.46, CHCl3); RF ¼ 0.61 (toluene/ethyl acetate/
formic acid/n-heptane, 80:20:3:10); IR KBr:
n
¼ 3545 br, 2936 s,
2878 m, 1724 s, 1458 m, 1371 s, 1235 s, 1152 s, 1093 m, 1032 m,
1011 m, 960 m, 931 w, 906 w, 866 w, 804 w, 772 w, 704 w, 655 w,
Data for 11: mp 190e192 ꢀC; ½a D20
ꢄ
¼ þ0.99ꢀ (c ¼ 0.41, MeOH);
RF
¼
0.20 (toluene/ethyl acetate/formic acid/n-heptane,
601 w, 485 w cmꢂ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.59 (dd, 1 H,
80:20:3:10); (KBr):
1461 m, 1369 s, 1253 s, 1158 m, 1104 m, 1049 m, 1031 m, 1005 m,
959 m, 934 w, 907 w, 868 w, 769 w, 650 w, 562 w cmꢂ1 1H NMR
¼ 5.29 (dd, 1 H, J12, 11 ¼ 3.3, J12, 11 ¼ 3.3 Hz, H-
12), 4.51 (d, 1 H, J3ax,
n
¼ 3576 m, 3432 br, 2930 s, 1737 s, 1689 s,
0
J2ax, 1ax ¼ 13.3 Hz, J2ax, 1eq ¼ 6.5 Hz, H-2ax), 5.33 (dd, 1 H, J12, 11 ¼ 3.3,
00
J12, 11 ¼3.3 Hz, H-12), 3.59 (s, 3 H, H-31), 2.58 (s,1 H, H-18), 2.49 (m,
;
1 H, H-16ax), 2.19 (dd, 1 H, J1eq, 1ax ¼ 12.5 Hz, J1eq, 2ax ¼ 6.5 Hz, H-
1eq), 2.12 (s, 3 H, H-33), 2.02 (m, 2 H, H-110, H-1100), 1.73e1.50 (m,
8 H, H-600, H-700, H-9, H-15ax, H-16eq, H-2100, H-220, H-2200), 1.41e1.32
(m, 5 H, H-1ax, H-60, H-70, H-20, H-210), 1.27 (s, 3 H, H-25), 1.22 (s,
3 H, H-27), 1.18 (s, 3 H, H-29), 1.15 (m, 1 H, H-5), 1.13 (s, 3 H, H-24),
1.10 (s, 3 H, H-23), 1.02 (m, 1 H, H-15eq), 0.92 (d, 3 H, J30, 20 ¼ 6.7 Hz,
H-30), 0.73 (s, 3 H, H-26) ppm; 13C NMR (125 MHz, CDCl3):
0
00
(500 MHz, CD3OD):
d
¼ 9.9 Hz, H-3ax), 3.76 (ddd, 1 H, J2ax,
2ax
¼ 10.9, J2ax, 3ax ¼ 9.9, J2ax, 1eq ¼ 4.4 Hz, H-2ax), 2.58 (ddd,1 H, J16ax,
1ax
¼ 14.3, J16ax, 15ax ¼ 12.8, J16ax, 15eq ¼ 4.3 Hz, H-16ax), 2.50 (s, 1 H,
16eq
H-18), 2.09 (s, 3 H, H-32), 2.04e1.97 (m, 3 H, H-1eq, H-110, H-1100),
1.83e1.41 (m, 9 H, H-600, H-700, H-9, H-15ax, H-16eq, H-20, H-2100, H-
220, H-2200), 1.35 (s, 3 H, H-27), 1.33e1.30 (m, 3 H, H-60, H-70, H-210),
1.19 (s, 3 H, H-29), 1.03 (s, 3 H, H-23), 1.01e0.96 (m, 3 H, H-1ax, H-5,
H-15eq), 0.93 (d, 3 H, J30, 20 ¼ 6.7 Hz, H-30), 0.88, 0.87 (each s, 6 H,
H-24, H-25), 0.80 (s, 3 H, H-26) ppm; 13C NMR (125 MHz, CD3OD):
d
¼ 209.3 (C3),178.3 (C28),170.2 (C32),138.5 (C13),128.3 (C12), 73.1
(C19), 71.7 (C2) 57.0 (C5), 53.2 (C18), 51.6 (C31), 48.7 (C4), 47.8 (C17),
46.9 (C9), 45.6 (C1), 41.3 (C20), 41.1 (C14), 40.0 (C8), 37.8 (C10), 37.3
(C22), 32.4 (C7), 28.2 (C15), 27.4 (C29), 25.9 (C16), 25.4 (C21), 24.8
(C23), 24.4 (C27), 23.8 (C11), 21.3 (C24), 20.7 (C33), 19.2 (C6), 16.8
(C26), 16.0 (C30), 15.8 (C25) ppm; MS (ESI, MeOH): m/z (%) ¼ 543.3
([M þ H]þ 34), 560.4 ([M þ NH4]þ, 42), 565.5 ([M þ Na]þ, 100),
581.4 ([M þ K]þ, 43), 597.1 ([M þ Na þ MeOH]þ, 27); analysis for
C33H50O6 (542.75): C, 73.02; H, 9.29; found: C, 72.88; H, 9.31.
d
¼ 180.8 (C28), 171.9 (C31), 138.7 (C13), 127.7 (C12), 84.4 (C3), 72.1
(C19), 66.1 (C2), 55.0 (C5), 53.6 (C18), 48.1 (C17), 47.1 (C9), 46.9 (C1),
41.7 (C20), 41.2 (C14), 39.7 (C8), 38.9 (C4), 37.7 (C10), 37.6 (C22),
32.6 (C7), 28.1 (C15), 27.7 (C24), 25.9 (C21), 25.6 (C29), 25.2 (C16),
23.4 (C27), 23.3 (C11), 19.7 (C32), 18.1 (C6), 16.7 (C25), 16.0 (C26),
15.6 (C23), 15.2 (C30) ppm; MS (ESI, MeOH): m/z (%) ¼ 529.7
([M ꢂ H]ꢂ, 100), 575.3 ([M þ HCO2]ꢂ, 15); analysis for C32H50O6
(530.74): C, 72.41; H, 9.50; found: C, 72.31; H, 9.66.
4.12. (2 R, 3 R, 19 R) methyl 2,3-bis(chloroacetyloxy)-19-hydroxy
urs-12-en-28-carboxylate (14), (2 R, 3 R, 19 R) methyl 2-chloro
acetyloxy-3,19-dihydroxyurs-12-en-28-carboxylate (15), and (2 R,
3 R, 19 R) methyl 3-chloroacetyloxy-2,19-dihydroxyurs-12-en-28-
carboxylate (16)
4.10. (3 R, 19 R) methyl 3-acetyloxy-19-hydroxy-2-oxo-urs-12-en-
28-carboxylate (12)
To a solution of 3 (100 mg, 0.20 mmol) in dry pyridine (4 ml)
acetic anhydride (8 ml) was slowly added and stirring at 24 ꢀC was
continued for 24 h. The reaction mixture was poured into ice-cold
water, and the precipitate was filtered off. Re-crystallization from
methanol yielded 12 (72 mg, 66%) as a colorless solid; mp 218e
Compound 2 (1.03 g, 2.05 mmol) was acylated at 24 ꢀC for 4 h
with chloroacetyl chloride (282 mg, 2.5 mmol) and pyridine
(0.2 ml) in dry dichloromethane (30 ml). After usual aqueous work-
up and chromatography (silica gel, toluene/ethyl acetate/formic
acid/n-heptane, 80/20/3/10), compounds 14 (341 mg, 26%), 15
(463 mg, 40%) and 16 (124 mg, 11%) were obtained.
220 ꢀC; ½a 2D0
ꢄ
¼ þ72.2ꢀ (c ¼ 0.67, CHCl3); RF ¼ 0.56 (toluene/ethyl
acetate/formic acid/n-heptane, 80:20:3:10); IR (KBr):
n
¼ 3511 br,

244
R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
Data for 14: colorless solid; mp 92e93 ꢀC; ½a D20
ꢄ
¼ ꢂ8.11ꢀ
CDCl3):
d
¼ 178.3 (C28), 168.3 (C32), 138.2 (C13), 128.7 (C12), 87.1
(c ¼ 0.39, CHCl3); RF ¼ 0.77 (toluene/ethyl acetate/formic acid/n-
(C3), 73.2 (C19), 67.3 (C2), 55.0 (C5), 53.2 (C18), 51.6 (C31), 47.8
(C17), 47.6 (C1), 47.1 (C9), 41.2 (C14), 41.1 (C20, C33), 39.9 (C8), 39.4
(C4), 38.1 (C10), 37.3 (C22), 32.5 (C7), 28.5 (C23), 28.1 (C15), 27.3
(C29), 25.9 (C21), 25.4 (C16), 24.4 (C27), 23.7 (C11), 18.3 (C6), 17.5,
16.6, 16.4 (C24, C25, C26), 16.1 (C30) ppm; MS (ESI, MeOH): m/z
(%) ¼ 601.4 ([M þ Na]þ, 100); analysis for C33H51ClO6 (579.21): C,
68.43; H, 8.88; found: C, 68.27; H, 8.99.
heptane, 80/20/3/10); IR (KBr):
n
¼ 3568 br, 2948 s, 2878 m, 1736 s,
1457 m, 1412 m, 1397 m, 1370 m, 1310 s, 1262 s, 1168 s, 1071 w,
1023 m, 1001 m, 968 m, 928 m, 865 w, 791 m, 772 w, 696 w, 656 w,
587 w cmꢂ1
;
1H NMR (500 MHz, CDCl3):
00
d
¼ 5.32 (dd, 1 H, J12,
110
¼ 3.5, J12, 11 ¼ 3.5 Hz, H-12), 5.17 (ddd, 1 H, J2ax, 1ax ¼ 11.4, J2ax,
¼ 10.3, J2ax,
¼ 4.7 Hz, 1 H, H-2ax), 4.84 (d, 1 H, J3ax,
3ax
2ax
1eq
¼ 10.3 Hz, H-3ax), 4.02, 3.93 (each s, 4 H, H-33, H-35), 3.58 (s,
3 H, H-31), 2.57 (s, 1 H, H-18), 2.49 (m, 1 H, H-16ax), 2.07 (dd, 1 H,
J1eq, 1ax ¼ 12.3, J1eq, 2ax ¼ 4.7, H-1eq), 1.98e1.95 (m, H, H-110, H-1100),
1.73e1.51 (m, 8 H, H-600, H-700, H-9, H-15ax, H-16eq, H-2100, H-220, H-
2200), 1.45e1.26 (m, 4 H, H-60, H-70, H-20, H-210), 1.23 (s, 3 H, H-27),
1.18 (s, 3 H, H-29), 1.13 (m, 1 H, H-1ax), 1.05 (s, 3 H, H-25), 1.02e0.98
(m, 2 H, H-5, H-15eq), 0.92 (d, 3 H, J30, 20 ¼ 6.4, H-30), 0.92 (s, 6 H, H-
23, H-24), 0.67 (s, 3 H, H-26) ppm; 13C NMR (125 MHz, CDCl3):
Acknowledgments
We like to thank Dr. G. Kaluderovic for many helpful discussions
and Dr. St. Schwarz for performing some preliminary biological
testing. Many thanks are due to Dr. R. Kluge for the measurement of
the MS spectra, to Dr. D. Ströhl for recording the NMR spectra, and
to Dr. Th. Müller, Dept. Haematology/Oncology (Univ. Halle), for
providing the cell lines. Support by “Gründerwerkstatt e Bio-
wissenschaften” is gratefully acknowledged.
d
¼ 178.2 (C28), 167.2, 166.9 (C32, C34), 138.3 (C13), 128.4 (C12),
82.3 (C3), 73.1 (C19), 72.2 (C2), 54.7 (C5), 53.1 (C18), 51.6 (C31), 47.8
(C17), 47.1 (C9), 43.6 (C1), 41.2 (C14), 41.1 (C20), 40.8 (C33, C35),
39.9 (C8), 39.6 (C4), 38.2 (C10), 37.3 (C22), 32.5 (C7), 28.3 (C23), 28.1
(C15), 27.4 (C29), 26.0 (C21), 25.4 (C16), 24.4 (C27), 23.7 (C11), 18.2
(C6), 17.5 (C24), 16.6 (C26), 16.3 (C25), 16.1 (C30) ppm; MS (ESI,
MeOH): m/z (%) ¼ 672.3 ([M þ NH4]þ, 14), 677.4 ([M þ Na]þ, 100);
analysis for C35H52Cl2O7 (655.69): C, 64.11; H, 7.99; found: C, 64.00;
H, 8.09.
References
[1] M.S. Kemp, P.J. Holloway, R.S. Burden, 3b,19a-Dihydroxy-2-oxours-12-en-28-
oic acid: a pentacyclic triterpene induced in the wood of Malus pumila Mill.
infected with Chondrostereum purpureum (Pers. ex Fr.) Pouzar., and a constit-
uent of the cuticular wax of apple fruits, J. Chem. Res. Synop. (1985) 154e155.
Data for 15: colorless solid; mp 93e95 ꢀC; ½a D20
ꢄ
¼ ꢂ3.42ꢀ
[2] G.d.G. Rocha, M. Simoes, R.R. Oliveira, M.A.C. Kaplan, C.R. Gattass, 3b-acetyl
tormentic acid induces apoptosis of resistant leukemia cells independently of
P-gp/ABCB1 activity or expression, Invest. New Drugs 30 (2012) 105e113.
[3] G. Delgado, J. Hernandez, R. Pereda-Miranda, Triterpenoid acids from Cunila
lythrifolia, Phytochemistry 28 (1989) 1483e1485.
(c ¼ 0.50, CHCl3); RF ¼ 0.46 (toluene/ethyl acetate/formic acid/n-
heptane, 80/20/3/10); IR (KBr):
1457 m, 1380 m, 1310 s, 1263 m, 1231 m, 1192 s, 1168 s, 1074 m,
1046 m, 1033 m, 1015 m, 964 m, 865 w, 791 w, 772 w, 661 w cmꢂ1
1H NMR (500 MHz, CDCl3)
n
¼ 3528 s, 2947 s, 2877 s, 1727 s,
[4] P. Potier, B. Das, A.M. Bui, M.M. Janot, A. Pourrat, H. Pourrat, Structure of
;
tormentic acid,
a pentacyclic triterpene acid isolated from the roots of
110
d
¼ 5.34 (dd, 1 H, J12,
¼ 3.5, J12,
Potentilla tormentilla, Bull. Soc. Chim. Fr. (1966) 3458e3465.
1100
¼ 3.5 Hz, H-12), 5.03 (ddd, 1 H, J2ax, 1ax ¼ 11.5, J2ax, 3ax ¼ 10.0, J2ax,
¼ 4.5 Hz, H-2ax), 4.06 (s, 2 H, H-33), 3.60 (s, 3 H, H-31), 3.25 (d,
[5] D. Lontsi, N.F. Ngounou, A.L. Tapondjou, B.L. Sondengam, B. Bodo, M.-T. Martin,
An E-ring g-lactone pentacyclic triterpene from Myrianthus serratus, Phyto-
chemistry 49 (1998) 2473e2476.
1eq
1 H, J3ax, 2ax ¼ 10.0 Hz,1 H, H-3ax), 2.59 (s,1 H, H-18), 2.50 (m,1 H, H-
16ax), 2.03 (dd, 1 H, J1eq, 1ax ¼ 12.2, J1eq, 2ax ¼ 4.5 Hz, H-1eq), 1.99e
1.96 (m, 2 H, H-110, H-1100), 1.74e1.48 (m, 8 H, H-600, H-700, H-9, H-
15ax, H-16eq, H-2100, H-220, H-2200), 1.45e1.39 (m, 2 H, H-60, H-20),
1.33 (m,1 H, H-70),1.31 (m,1 H, H-210),1.25 (s, 3 H, H-27),1.19 (s, 3 H,
H-29), 1.06, 1.03 (each s, 6 H, H-23, H-25), 1.02 (m, 1 H, H-1ax), 0.99
(m, 1 H, H-15eq), 0.93 (d, 3 H, J30, 20 ¼ 6.7 Hz, H-30), 0.89 (m, 1 H, H-
5), 0.87 (s, 3 H, H-24), 0.68 (s, 3 H, H-26) ppm; 13C NMR (125 MHz,
[6] A.L. Tapondjou, N.F. Ngounou, D. Lontsi, B. Sondengam, M.-T. Martin, B. Bodo,
Pentacyclic triterpenes from Myrianthus liberecus, Phytochemistry 40 (1995)
1761e1764.
[7] T. Akihisa, S. Kamo, T. Uchiyama, H. Akazawa, N. Banno, Y. Taguchi,
K. Yasukawa, Cytotoxic activity of Perilla frutescens var. japonica leaf extract is
due to high concentrations of oleanolic and ursolic acids, J. Nat. Med. 60
(2006) 331e333.
[8] N. Banno, T. Akihisa, H. Tokuda, K. Yasukawa, H. Higashihara, M. Ukiya,
K. Watanabe, Y. Kimura, J.-i. Hasegawa, H. Nishino, Triterpene acids from the
leaves of Perilla frutescens and their anti-inflammatory and antitumor-
promoting effects, Biosci. Biotechnol. Biochem. 68 (2004) 85e90.
[9] J.H. Chen, Z.H. Xia, R.X. Tan, High-performance liquid chromatographic anal-
ysis of bioactive triterpenes in Perilla frutescens, J. Pharm. Biomed. Anal. 32
(2003) 1175e1179.
[10] F. Matsuyama, T. Yoshida, T. Hatano, S. Taniguchi, Cell Culture for Producing
Triterpenes. WO 2005063227A1 20050714 (2005).
[11] E. Palme, A.R. Bilia, I. Morelli, Flavonols and isoflavones from Cotoneaster
simonsii, Phytochemistry 42 (1996) 903e905.
[12] H. Tabata, Y. Kato, Y. Naohara, Method for Producing Extract Containing Tri-
terpenes or Flavonoids, and Composition Containing the Same. JP 2011026265
A20110210 (2011).
[13] C. Murakami, K. Ishijima, M. Hirota, K. Sakaguchi, H. Yoshida, Y. Mizushina,
Novel anti-inflammatory compounds from Rubus sieboldii, triterpenoids, are
inhibitors of mammalian DNA polymerases, Biochim. Biophys. Acta Protein
Struct. Mol. Enzymol. 1596 (2002) 193e200.
[14] L. Chu, L. Wang, Z. Zhang, H. Gao, J. Huang, B. Sun, L. Wu, Studies on the
chemical constituents of Potentilla anserine L, Mod. Chin. Med. 10 (2008)
10e12.
[15] P. Liu, H. Duan, Q. Pan, Y. Zhang, Z. Yao, Triterpenes from herb of Potentilla
chinensis, China J. Chin. Mater. Med. 31 (2006) 1875e1879.
[16] H. Luo, X. Zhao, Method for Extracting Total Triterpenoids from Potentilla
Plant. CN101172131 A20080507 (2008).
[17] X. Zhao, H. Luo, Medical Application of Total Triterpene Extract of Potentilla
Plant to Prepare the Medicine for Treating and Preventing Type II Diabetes
Mellitus And/or Hyperlipemia. CN101172109 A20080507 (2008).
[18] J.L. Jin, Y.Y. Lee, J.E. Heo, S. Lee, J.M. Kim, H.S. Yun-Choi, Anti-platelet penta-
cyclic triterpenoids from leaves of Campsis grandiflora, Arch. Pharm. Res. 27
(2004) 376e380.
[19] Q. Zhang, Z. Chang, Q. Wang, Ursane triterpenoids inhibit atherosclerosis and
xanthoma in LDL receptor knockout mice, Cardiovasc. Drugs Ther. 20 (2006)
349e357.
CDCl3):
d
¼ 178.3 (C28), 167.5 (C32), 138.2 (C13), 128.7 (C12), 80.5
(C3), 75.6 (C2), 73.1 (C19), 55.0 (C5), 53.2 (C18), 51.6 (C31), 47.9
(C17), 47.1 (C9), 43.4 (C1), 41.2 (C14), 41.1 (C20, C33), 39.9 (C4, C8),
38.3 (C10), 37.3 (C22), 32.6 (C7), 28.5 (C23), 28.1 (C15), 27.4 (C29),
26.0 (C21), 25.4 (C16), 24.5 (C27), 23.7 (C11), 18.4 (C6), 16.6, 16.2
(C24, C25, C26), 16.1 (C30) ppm; MS (ESI, MeOH): m/z (%) ¼ 601.4
([M þ Na]þ,100); analysis for C33H51ClO6 (579.21): C, 68.43; H, 8.88;
found: C, 68.32; H, 9.03.
Data for 16: colorless solid; mp 108e113 ꢀC; ½a D20
ꢄ
¼ þ13.82ꢀ
(c ¼ 0.47, CHCl3); RF ¼ 0.33 (toluene/ethyl acetate/formic acid/n-
heptane, 80/20/3/10); IR (KBr):
n
¼ 3528 br, 2946 s, 2878 s, 1725 s,
1669 m, 1456 m, 1380 m, 1311 s, 1263 s, 1230 s, 1192 s, 1152 s,
1095 m,1043 m, 1016 m, 988 m, 969 m, 929 m, 866 w, 788 w, 772 w,
698 w, 659 w, 465 w cmꢂ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.34 (dd,
110
1100
1 H, J12,
¼ 3.5, J12,
¼ 3.5 Hz, H-12), 4.58 (d, 1 H, J3ax,
¼ 10.0 Hz, H-3ax), 4.14 (s, 2 H, H-33), 3.82 (ddd, 1 H, J2ax,
¼ 11.5, J2ax, 3ax ¼ 10.0, J2ax, 1eq ¼ 4.4 Hz, H-2ax), 3.59 (s, 3 H, H-31),
2ax
1ax
2.59 (s, 1 H, H-18), 2.51 (m, 1 H, H-16ax), 2.07 (dd, 1 H, J1eq, 1ax ¼ 12.6,
J1eq, 2ax ¼ 4.4,1 H, H-1eq), 2.02e1.98 (m, 2 H, H-110, H-1100),1.74e1.48
(m, 8 H, H-600, H-700, H-9, H-15ax, H-16eq, H-2100, H-220, H-2200), 1.43e
1.38 (m, 2 H, H-60, H-20), 1.33 (m, 1 H, H-70), 1.26 (s, 3 H, H-27), 1.24
(m, 1 H, H-210), 1.21 (s, 3 H, H-29), 1.06e1.01 (m, 3 H, H-1ax, H-5, H-
15ax), 0.99, 0.91, 0.89 (each s, 9 H, H 23, H-24, H-25), 0.93 (d, 3 H, J30,
¼ 6.7 Hz, H-30), 0.68 (s, 3 H, H-26) ppm; 13C NMR (125 MHz,
20

R. Csuk et al. / European Journal of Medicinal Chemistry 56 (2012) 237e245
245
[20] A.S. Fogo, E. Antonioli, J.B. Calixto, A.H. Campos, Tormentic acid reduces
vascular smooth muscle cell proliferation and survival, Eur. J. Pharmacol. 615
(2009) 50e54.
[21] N. Zeng, Y. Shen, L.-Z. Li, W.-H. Jiao, P.-Y. Gao, S.-J. Song, W.-S. Chen, H.-W. Lin,
Anti-inflammatory triterpenes from the leaves of Rosa laevigata, J. Nat. Prod.
74 (2011) 732e738.
[22] H. Gao, L. Wu, M. Kuroyanagi, K. Harada, N. Kawahara, T. Nakane, K. Umehara,
A. Hirasawa, Y. Nakamura, Antitumor-promoting constituents from Chaeno-
meles sinensis Koehne and their activities in JB6 mouse epidermal cells, Chem.
Pharm. Bull. 51 (2003) 1318e1321.
[43] Y.Tsuda,M. Hanajima, K.Yoshimoto,Utilizationofsugarsinorganic-synthesis.13.
Regioselective oxidation of a
-arabinopyranoside via cyclic tin intermediate e
b-L
facile synthesis of 4-amino-4-deoxy-l-arabinose, an amino-sugar found in lipo-
polysaccharides of some Salmonella R-mutant strains, Chem. Pharm. Bull. 31
(1983) 3778e3780.
[44] Y. Ueno, M. Okawara, Oxidation using distannoxane .1. Selective oxidation of
alcohols, Tetrahedron Lett. (1976) 4597e4600.
[45] H. Kommera, G.N. Kaluderovic, J. Kalbitz, R. Paschke, Synthesis and anticancer
activity of novel betulinic acid and betulin derivatives, Arch. Pharm. 343
(2010) 449e457.
[23] Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki,
M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai,
K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA poly-
[46] Z. Darzynkiewicz, G. Juan, X. Li, W. Gorczyca, T. Murakami, F. Traganos,
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death
(necrosis), Cytometry 27 (1997) 1e20.
merase
l
-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935e1944.
[47] D. Wlodkowic, J. Skommer, Z. Darzynkiewicz, Cytometry in cell necrobiology
revisited. Recent advances and new vistas, Cytometry A 77A (2010) 591e606.
[48] M.E. Juan, U. Wenzel, V. Ruiz-Gutierrez, H. Daniel, J.M. Planas, Olive fruit
extracts inhibit proliferation and induce apoptosis in HT-29 human colon
cancer cells, J. Nutr. 136 (2006) 2553e2557.
[24] Y. Mizushina, F. Sugawara, K. Sakaguchi, H. Yoshida, DNA polymerase
l
inhibitors, anticancer agents, and antiinflammatory agents containing
phenols, and method for screening of anticancer agents or antiinflammatory
agents by testing of inhibitory activity against DNA polymerase
2005029571 A 20050203 (2005).
l. JP
[49] F.J. Reyes, J.J. Centelles, J.A. Lupianez, M. Cascante, (2
a,3b)-2,3-
[25] G.d.G. Rocha, M. Simoes, K.A. Lucio, R.R. Oliveira, K.M.A. Coelho, C.R. Gattass,
Natural triterpenoids from Cecropia lyratiloba are cytotoxic to both sensitive
and multidrug resistant leukemia cell lines, Bioorg. Med. Chem. 15 (2007)
7355e7360.
[26] J.-J. Cheng, L.-J. Zhang, H.-L. Cheng, C.-T. Chiou, I.J. Lee, Y.-H. Kuo, Cytotoxic
hexacyclic triterpene acids from Euscaphis japonica, J. Nat. Prod. 73 (2010)
1655e1658.
dihydroxyolean-12-en-28-oic acid, new natural triterpene from Olea
europea, induces caspase dependent apoptosis selectively in colon adeno-
carcinoma cells, FEBS Lett. 580 (2006) 6302e6310.
a
[50] F.J. Reyes-Zurita, G. Pachon-Pena, D. Lizarraga, E.E. Rufino-Palomares,
M. Cascante, J.A. Lupianez, The natural triterpene maslinic acid induces
apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism,
BMC Cancer 11 (2011) 154.
[27] A. Numata, P. Yang, C. Takahashi, R. Fujiki, M. Nabae, E. Fujita, Cytotoxic tri-
terpenes from a Chinese medicine, Goreishi, Chem. Pharm. Bull. 37 (1989)
648e651.
[51] F.J. Reyes-Zurita, E.E. Rufino-Palomares, J.A. Lupianez, M. Cascante, Maslinic
acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29
human colon-cancer cells via the mitochondrial apoptotic pathway, Cancer
Lett. 273 (2009) 44e54.
[28] J.M. Rollinger, D.V. Kratschmar, D. Schuster, P.H. Pfisterer, C. Gumy,
E.M. Aubry, S. Brandstoetter, H. Stuppner, G. Wolber, A. Odermatt, 11
b
-
[52] C. Li, Z. Yang, C. Zhai, W. Qiu, D. Li, Z. Yi, L. Wang, J. Tang, M. Qian, J. Luo, M. Liu,
Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor
alpha by inhibiting NF-kappaB signaling pathway, Mol. Cancer 9 (2010) 73.
[53] C.R. Pungitore, J.M. Padron, L.G. Leon, C. Garcia, G.M. Ciuffo, V.S. Martin,
C.E. Tonn, Inhibition of DNA topoisomerase I and growth inhibition of human
cancer cell lines by an oleanane from Junellia aspera (Verbenaceae), Cell. Mol.
Biol. 53 (2007) 13e17.
Hydroxysteroid dehydrogenase inhibiting constituents from Eriobotrya
1
japonica revealed by bioactivity-guided isolation and computational
approaches, Bioorg. Med. Chem. 18 (2010) 1507e1515.
[29] H.-J. An, I.-T. Kim, H.-J. Park, H.-M. Kim, J.-H. Choi, K.-T. Lee, Tormentic acid, a tri-
terpenoid saponin, isolatedfromRosarugosa, inhibited LPS-inducediNOS, COX-2,
and TNF-a expression through inactivation of the nuclear factor-kb pathway in
RAW 264.7 macrophages, Int. Immunopharmacol. 11 (2011) 504e510.
[30] C.-T. Chang, S.-S. Huang, S.-S. Lin, S. Amagaya, H.-y. Ho, W.-C. Hou, P.-H. Shie,
J.-B. Wu, G.-J. Huang, Anti-inflammatory activities of tormentic acid from
suspension cells of Eriobotrya japonica ex vivo and in vivo, Food Chem. 127
(2011) 1131e1137.
[31] R. Csuk, A. Barthel, R. Kluge, D. Ströhl, Synthesis, cytotoxicity and liposome
preparation of 28-acetylenic betulin derivatives, Bioorg. Med. Chem. 18
(2010) 7252e7259.
[54] Y.W. Hsum, W.T. Yew, P.L. Hong, K.K. Soo, L.S. Hoon, Y.C. Chieng, L.Y. Mooi,
Cancer chemopreventive activity of maslinic acid: suppression of COX-2
expression and inhibition of NF-kappaB and AP-1 activation in Raji cells,
Planta Med. 77 (2011) 152e157.
[55] T. Yamagishi, D.C. Zhang, J.J. Chang, D.R. McPhail, A.T. McPhail, K.H. Lee,
Antitumor agents. Part 94. The cytotoxic principles of Hyptis capitata and the
structures of the new triterpenes hyptatic acid A and B, Phytochemistry 27
(1988) 3213e3216.
[32] R. Csuk, A. Barthel, R. Kluge, D. Ströhl, H. Kommera, R. Paschke, Synthesis and
biological evaluation of antitumour-active betulin derivatives, Bioorg. Med.
Chem. 18 (2010) 1344e1355.
[33] R. Csuk, A. Barthel, S. Schwarz, H. Kommera, R. Paschke, Synthesis and bio-
logical evaluation of antitumor-active gamma-butyrolactone substituted
betulin derivatives, Bioorg. Med. Chem. 18 (2010) 2549e2558.
[34] R. Csuk, A. Barthel, R. Sczepek, B. Siewert, S. Schwarz, Synthesis, encapsulation
and antitumor activity of new betulin derivatives, Arch. Pharm. 344 (2011)
37e49.
[35] R. Csuk, S. Schwarz, R. Kluge, D. Ströhl, Improvement of the cytotoxicity and
tumor selectivity of glycyrrhetinic acid by derivatization with bifunctional
aminoacids, Arch. Pharm. 344 (2011) 505e513.
[36] R. Csuk, S. Schwarz, B. Siewert, R. Kluge, D. Ströhl, Synthesis and antitumor
activity of ring A-modified glycyrrhetinic acid derivatives, Z. Naturforsch. B 66
(2011) 521e532.
[37] S. Schwarz, R. Csuk, Synthesis and antitumour activity of glycyrrhetinic acid
derivatives, Bioorg. Med. Chem. 18 (2010) 7458e7474.
[38] H. Kommera, G.N. Kaluderovic, J. Kalbitz, B. Dräger, R. Paschke, Small struc-
tural changes of pentacyclic lupane type triterpenoid derivatives lead to
significant differences in their anticancer properties, Eur. J. Med. Chem. 45
(2010) 3346e3353.
[39] D. Lontsi, B.L. Sondengam, M.T. Martin, B. Bodo, Cecropioic acid, a pentacyclic
triterpene from Musanga cecropioides, Phytochemistry 48 (1998) 171e174.
[40] C.M. Ojinnaka, J.I. Okogun, D.A. Okorie, Triterpene acids from Myrianthus
arboreus, Phytochemistry 19 (1980) 2482e2483.
[41] Z.-j. Ma, Z.-j. Zhao, Studies on chemical constituents from stem barks of
Fraxinus paxiana, China J. Chin. Mater. Med. 33 (2008) 1990e1993.
[42] Z. Hong, W. Chen, J. Zhao, Z. Wu, J. Zhou, T. Li, J. Hu, Hepatoprotective effects of
Rubus aleaefolius Poir. and identification of its active constituents,
J. Ethnopharmacol. 129 (2010) 267e272.
[56] M.E. Juan, J.M. Planas, V. Ruiz-Gutierrez, H. Daniel, U. Wenzel, Anti-
proliferative and apoptosis-inducing effects of maslinic and oleanolic acids,
two pentacyclic triterpenes from olives, on HT-29 colon cancer cells, Br. J.
Nutr. 100 (2008) 36e43.
[57] R. Martin, J. Carvalho-Tavares, E. Ibeas, M. Hernandez, V. Ruiz-Gutierrez,
M.L. Nieto, Acidic triterpenes compromise growth and survival of astrocytoma
cell lines by regulating reactive oxygen species accumulation, Cancer Res. 67
(2007) 3741e3751.
[58] V. Alexander, Chapter nine e acridine orange as a probe for cell and molecular
biology, in: W.T. Mason (Ed.), Fluorescent and Luminescent Probes for Bio-
logical Activity, second ed., Academic Press, London, 1999, pp. 117e135.
[59] H.M. Shapiro, Practical Flow Cytometry, third ed., Wiley-Liss, 1995.
[60] J.A.R. Salvador, Pentacyclic Triterpenes as Promising Agents in Cancer, Nova
Science Pub Inc, 2010.
[61] E. Bossy-Wetzel, D.R. Green, Detection of apoptosis by annexin V labeling,
Methods Enzymol. 322 (2000) 15e18.
[62] A.J. McGahon, S.J. Martin, R.P. Bissonnette, A. Mahboubi, Y. Shi, R.J. Mogil,
W.K. Nishioka, D.R. Green, The end of the (cell) line: methods for the study of
apoptosis in vitro, Methods Cell. Biol. 46 (1995) 153e185.
[63] A.H. Wyllie, Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation, Nature 284 (1980) 555e556.
[64] J.M. Fang, K.C. Wang, Y.S. Cheng, The chemical constituents from the aerial
part of Rosa laevigata, J. Chin. Chem. Soc. (Taipei) 38 (1991) 297e299.
[65] G. Ruecker, R. Mayer, J.S. Shin-Kim, Triterpene saponins from the Chinese drug
“Daxueteng” (Caulis sargentodoxae), Planta Med. 57 (1991) 468e470.
[66] S.-S. Lee, S.-N. Shy, K.C.S. Liu, Triterpenes from Paliurus hemsleyanus, Phyto-
chemistry 46 (1997) 549e554.
[67] C. Terreaux, M.P. Maillard, M.P. Gupta, K. Hostettmann, Triterpenes and tri-
terpene glycosides from Paradrymonia macrophylla, Phytochemistry 42 (1996)
495e499.