Synthesis and studies of anticancer properties of lupane-type triterpenoid derivatives containing a cisplatin fragment

Article abstract of DOI:10.1016/j.ejmech.2014.01.031

Both betulinic acid 1 and cisplatin are promising antitumor agents, which induce apoptotic cell death of cancer cells. In the present investigation a new series of betulinic acid-cisplatin conjugates were synthesized and cytotoxicity and selectivity were assessed against five different tumor cell lines. The aim was to combine two structural units, both related with apoptosis induction. The derivatives exerted a dose-dependent antiproliferative action at micromolar concentrations and the effect of these structural variations on anticancer activity was studied and discussed. Several compounds revealed significant antitumor activity, as the most active substance 3-O-acetylbetulinic (2-(2-aminoethyl)aminoethyl)amide (IC₅₀ = 1.30-2.24 μM). Interestingly, Betulinic acid-cisplatin conjugates were less cytotoxic than the precursors.

Full text of DOI:10.1016/j.ejmech.2014.01.031

European Journal of Medicinal Chemistry 75 (2014) 460e466
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and studies of anticancer properties of lupane-type
triterpenoid derivatives containing a cisplatin fragment
a
a
b
c
Daniel Emmerich , Kranthi Vanchanagiri , Leopoldo C. Baratto , Harry Schmidt ,
Reinhard Paschke
a
a
,
*
Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale), Germany
Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Federal do Paraná, Centro Politécnico, 81531-970 Curitiba, PR, Brazil
Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, 06120 Halle (Saale), Germany
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 September 2013
Received in revised form
Both betulinic acid 1 and cisplatin are promising antitumor agents, which induce apoptotic cell death of
cancer cells. In the present investigation a new series of betulinic acidecisplatin conjugates were syn-
thesized and cytotoxicity and selectivity were assessed against five different tumor cell lines. The aim
was to combine two structural units, both related with apoptosis induction. The derivatives exerted a
dose-dependent antiproliferative action at micromolar concentrations and the effect of these structural
variations on anticancer activity was studied and discussed. Several compounds revealed significant
antitumor activity, as the most active substance 3-O-acetylbetulinic (2-(2-aminoethyl)aminoethyl)amide
14 January 2014
Accepted 20 January 2014
Available online 4 February 2014
Keywords:
(
IC50 ¼ 1.30e2.24
m
M). Interestingly, Betulinic acidecisplatin conjugates were less cytotoxic than the
Betulinic acid derivatives
Antitumor agent
Cisplatin conjugates
Cytotoxicity
precursors.
Ó 2014 Elsevier Masson SAS. All rights reserved.
SRB assay
1. Introduction
this approach. As a result of the combination of Wortmannin and
Cetuximab in a “double drug” concept, the antiproliferative activity
Betulinic acid (1) and its derivatives are pluripotent compounds
of both compounds could be improved [19]. Similarly the cytotoxic
and phototoxic properties of a RutheniumePorphyrine conjugate
are combined [20].
with numerous biological activities. Therefore they have been
investigated widely over the last few years [1e5], focusing in the
field of antitumor properties [6e10]. Since we have also success-
fully prepared highly active anticancer platinum complexes [11e
2. Results and discussion
1
3], we have developed a concept to combine both bioactive frag-
ments into one molecule. The aim was to find out if a combination
of two different apoptotic structures could lead to an increased
cytotoxicity. An insufficient process of apoptosis is not only an
important factor in the genesis of tumors, but also the main reason
for malignant tumors getting resistant against chemo- and radio-
therapies [14] (Fig. 1).
2
.1. Chemistry
The substances described in this work were prepared according
to known methods which were modified appropriately
Schemes 1e3). Compound 3 e an alkyl amide (polyamine) e was
(
prepared by reaction of 3-O-acetyl-betulinic acid (2) with dieth-
ylene triamine in dichloromethane (DCM) [21]. Platinum com-
Combinational therapy is common in the field of chemotherapy
[15e18]. The efficiency of this therapy depends strongly on the
plexes 3(PtClS), 5(PtCl
respective ligand molecules react with dichlorobis(dimethyl sulf-
oxide)platinum(II) in CH OH [22]. The complexes 3(PtCl ) and
(PtCl ) were prepared by a reaction of the appropriate DMSO
platinum complexes with an aqueous LiCl solution [23]; 3(PtCl
was also obtained by reaction with K [PtCl ] and KCl. 6(PtCl ) and
(PtClS) are platinum precursors used for the synthesis of 3(PtCl
and 3(PtClS) from compound 3, and 5(PtCl ) and 5(PtClS) from
compound 5. The diaminopropanol derivatives 4 and 5 (esters of
2
) and 6(PtClS) were formed by having the
nature of the single components: how they can be delivered, how
they are metabolized, and how and to which extent they can enter
the cell. Therefore it could be advantageous when the components
are covalently linked to each other. There are several examples for
3
2
6
2
2
)
2
4
2
6
2
)
*
Corresponding author. Tel.: þ49 3455521600.
2
E-mail address: reinhard.paschke@biozentrum.uni-halle.de (R. Paschke).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.031

D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
461
compound 2) and 6b have been prepared according to literature
24], as well as dichlorobis(dimethyl sulfoxide)platinum(II) [25].
[
2.2. Cytotoxicity
In the present study in vitro cytotoxic activity of betulinic acid 1
and its derivatives containing cisplatin similar ligands were studied
on five different cancer cell lines: 518A2 (melanoma), A2780
(
(
(
ovarian), A549 (lung carcinoma), MCF-7 (breast) and 8505C
anaplastic thyroid) as well as on one non-tumorous cell line
WWO70327) by SRB colorimetric assay method [26]. The
Fig. 1. Betulinic acid 1.
Scheme 1. Synthesis of 3-O-acetyl-betulinic acid derivatives. (a) Oxalyl chloride, diethylene triamine/DCM, 16 h rt, 6 h reflux; (b) dichlorobis(dimethyl sulfoxide)platinum(II)/
ꢀ
CH
3
OH, 2 h, rt; (c) LiCl/H
2
O, 2 h, 80 C; (d) oxalyl chloride, 6b/trimethylamine/DCM, 20 h rt, 30 min reflux; and (e) TFA/DCM, 1.5 h rt.

462
D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
platinum conjugates 5(PtCl
2
) and 5(PtClS) were less active than 5,
with IC50 of 13.13e35.66 M. It was observed that 5 was 1 to 3 times
m
more cytotoxic than its platinum derivatives.
The introduction of platinum ligands into Betulinic acid (1), i.e.
compound 3, led to no significant loss of activity in most cancer cell
lines. However, the presence of platinum groups did not have an
influence in such a way as to increase the cytotoxicity of the com-
pounds. In case of 5-platinum conjugates, loss of activity was
observed when compared to 1. Even though all compounds showed
broad spectrum activity, in most substances the antiproliferative
effect was pronounced against the A2780 cell line.
2 3
Scheme 2. Preparation of 6b: (a) Boc O/CH CN, 1 h rt.
2.3. Selectivity
compounds showed dose-dependent antitumoral activity against
the investigated cell lines. The IC50, defined as the concentration of
the compound at which 50 % cell inhibition is observed, was esti-
mated from the semi-logarithmic doseeresponse curves (Fig. 2.) by
GraphPad Prism5. The IC50 values were summarized in Table 1. The
compounds tested here were synthesized by derivatization at C-3
Selectivity of the compounds was assessed on human skin fi-
broblasts (WWO70327). The selectivity index was calculated by
IC50 value in fibroblast divided by IC50 value in cancer cell lines. The
results were summarized in Table 2. It was observed that com-
pound 1 was 3 to 5 times more selective towards cancer cells than
to fibroblasts, and similar selectivity was observed for 2. The most
active compound 3 and also compound 5 were the least selective
(
hydroxyl) and C-28 (carboxylic group) positions of Betulinic acid 1
and the structure activity relationship was developed in relation to
modifications at corresponding positions.
From the IC50 values it was observed that compounds 1e5
showed moderate to strong cytotoxicity against the investigated
substances towards all types of tumors. The selectivity of 3(PtCl
was between 2 and 3.5 times, and 5(PtCl ) was around 3 to 4 times
more selective towards cancer cells. Different selectivity patterns
were observed for 6(PtCl ). The doseeresponse curves for the test
2
)
2
2
cell lines. The lead compound 1 (IC50 ¼ 8e14
mM) shared similar
compounds towards tumor (A549) and non-tumor (WWO70327)
cells are given in Fig. 2.
range of IC50 values observed for its derivatives without platinum
ligands, showing higher cytotoxicity against A2780 cells. Com-
pound 2 was slightly more cytotoxic than 1; the presence of an
acetyl group at C3 seemed to influence the activity. Compound 3
3
. Conclusions
was the most active derivative (IC50 ¼ 1.3e2.24
mM) against all
In general when two cytotoxic groups covalently link in one
tested cancer types; the presence of both acetyl and (2-(2-
aminoethyl)aminoethyl) amide moieties at C3 and C28, respec-
tively, could be responsible for the high cytotoxicity. Even though
compounds 3 and 5 have symmetrical groups, it was found that 3
was 5 to 6 times more cytotoxic than 5. The reason could be the
linear alkyl amide (polyamine) group present at compound 3 that
strongly influences the ability of the substance to enter the cell
through the membrane as well as the capability to interact with cell
components. The C28 substitutions in compounds 4 and 5 retained
the cytotoxicity, but were not significant to produce more potent
derivatives.
molecule, the more pronounced cytotoxic activity can be expected.
But our results revealed that Betulinic acideCisplatin conjugates
more or less showed similar cytotoxicity to that of betulinic acid (1)
and in all the cases they were found to be less cytotoxic than
Cisplatin against all tumors. The property of double loading of two
cytotoxic groups in conjugates does not contribute to exhibit
improved cytotoxicity. On the other hand among the panel of de-
rivatives compound 3 was found to be highly cytotoxic with IC50 of
1.3e2.24 mM on all cancers and less selective towards tumors than
to normal cells. The order of cytotoxicity of all compounds on
investigated cancers from highest to lowest can be given below.
Compound 6(PtClS) was moderately active to inactive
(
IC50 ¼ 19.05 to >100
except for the A549 cell line. Probably the presence of chloride
groups in 6(PtCl ) contributes more favorably to the activity than
the sulfoxide groups in 6(PtClS). Compounds 3(PtCl ) and 3(PtClS)
were moderately active with IC50 values between 11.04 and
8.60 M; A549 cell line seemed to be more resistant to both
compounds than other cancer types. Even though compounds
(PtCl ) and 3(PtClS) were platinum derivatives of 3; they exhibi-
2
mM), while 6(PtCl ) was more cytotoxic,
3
> 2 >1 >5 > 4 > 3(PtCl
2 2
) > 3(PtClS) > 5(PtCl ) > 5(PtClS) >
6(PtCl ) > 6(PtClS)
2
2
2
In summary, the reason that the combination of the apoptotic
units doesn’t lead to an increase of cytotoxicity is not clear yet.
More extensive investigations for instance the exact mechanism of
anticancer action of the conjugates are necessary and this work is
currently in progress in our laboratory and the results will be re-
ported in due course. By this means, it should be possible to get a
clear idea about our findings.
2
m
3
2
ted cytotoxic properties similar to 1, except for the A549 cell line.
On the other hand, when compared to their precursor 3, those
compounds significantly lost activity. It was found that 3 was 5 to
14 times more cytotoxic than its platinum conjugates. In turn, the
4. Experimental
4.1. Chemistry
Chemicals and solvents were used as received; DCM was dried
over 4 Å molar sieves. The progress and result of every reaction
were monitored by TLC on neutral aluminum oxide 60 F254 plates,
made visible with 2% ceric ammonium sulfate solution. NMR
spectra were recorded on VNMRS 400 and INOVA 500 instruments;
the samples were dissolved in CDCl
3
, CD
3
OD or DMSO-d
Pt) as internal standard,
6
with TMS
Scheme 3. Synthesis of the diaminopropanol platinum complexes. (a) Dichlor-
ꢀ
1
13
195
obis(dimethyl sulfoxide) platinum(II)/CH
3
OH, 3 h rt; and (b) LiCl/water, 2 h, 80 C.
(for H and C) or Na
2
[PtCl
6
] (for

D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
463
Fig. 2. Survival of tumor (A549) and non-tumor (WW070327) cells determined by SRB test after 96 h of treatment with seven selected compounds.
respectively. Chemical shifts (
d
) are expressed in ppm; coupling
4.1.1. Preparation of the acid chloride of 3-O-acetylbetulinic acid [2]
constants in Hertz. ESI mass spectra were measured with a Thermo
Finnigan LCQ-Classic, in methanolic solution. Infrared spectra were
recorded on a Bruker Tensor 27 with ATR method. Betulinic acid 1
was gently supplied by BioSolutions Halle GmbH and 3-O-ace-
tylbetulinic acid 2 was prepared by an esterification of 1 with acetic
anhydride.
A solution of 2 (Scheme 2) ((1.0 g, 2.1 mmol) in dichloromethane
(DCM) reacted with an oxalyl chloride solution (2 M in DCM,
2.4 mL, 4.8 mmol), 30 min, rt, then the solvent was removed under
reduced pressure. The acid chloride of 2 e a crude white powder
obtained in quantitative yield e had been directly used for the
following reactions.
4
.1.2. Preparation of 3-O-acetylbetulinic (2-(2-aminoethyl)
Table 1
aminoethyl)amide [3]
IC50 ꢁ S.E for the 96 h of activity of investigated compounds on tumor cell lines
Diethylenetriamine (0.8 mL, 7.4 mmol) was dissolved in DCM
determined by SRB colorimetric assay.
(50 mL), and under vigorous stirring a solution of the acid chloride
Compound A549
A2780
8505C
518A2
MCF7
of 2 (1.0 g, 2.1 mmol) in DCM (10 mL) was added dropwise. The
mixture was stirred for 16 h and refluxed for 6 h, washed twice
with 10% aqueous K
1
2
3
3
3
4
5
5
5
6
6
13.3 ꢁ 0.82 8.75 ꢁ 0.96 12.63 ꢁ 1.67 14.8 ꢁ 0.6 14.03 ꢁ 1.21
10.52 ꢁ 0.01 5.95 ꢁ 0.42 10.40 ꢁ 0.40 10.95 ꢁ 0.12 10.72 ꢁ 0.40
2.05 ꢁ 0.34 1.52 ꢁ 0.29 2.24 ꢁ 0.22 1.30 ꢁ 0.03 1.59 ꢁ 0.20
17.32 ꢁ 0.00 12.66 ꢁ 4.66 11.57 ꢁ 0.00 12.12 ꢁ 1.63 12.41 ꢁ 0.58
28.69 ꢁ 0.12 12.63 ꢁ 3.73 11.04 ꢁ 3.77 12.45 ꢁ 2.13 14.23 ꢁ 1.83
16.91 ꢁ 0.19 8.32 ꢁ 0.86 15.50 ꢁ 0.37 12.72 ꢁ 0.25 15.06 ꢁ 0.30
12.58 ꢁ 0.24 6.60 ꢁ 0.49 12.12 ꢁ 0.25 11.5 ꢁ 0.16 11.79 ꢁ 0.27
17.85 ꢁ 1.87 13.13 ꢁ 0.42 22.64 ꢁ 0.44 29.83 ꢁ 1.80 15.98 ꢁ 0.23
14.57 ꢁ 0.94 19.05 ꢁ 0.43 20.13 ꢁ 1.89 35.66 ꢁ 0.16 19.31 ꢁ 0.59
27.12 ꢁ 0.00 6.55 ꢁ 1.10 9.69 ꢁ 0.00 11.89 ꢁ 1.66 10.47 ꢁ 0.00
2
CO
3
solution and brine. The organic phases
and evaporated under reduced pressure.
were dried over Na
2
SO
4
(PtCl
(PtClS)
2
)
Table 2
Selectivity index of the compounds towards tumor cells.
(PtCl
(PtClS)
(PtCl
(PtClS)
2
)
Compound
A549
A2780
8505c
518A2
MCF7
2
)
28.50 ꢁ 4.12 23.97 ꢁ 2.01 >100
23.87 ꢁ 2.54 19.05 ꢁ 1.76
1
2
3
3
5
5
6
3.27
2.76
1.36
2.30
1.27
3.05
1.06
4.98
4.89
1.76
3.15
2.33
4.15
4.39
3.45
2.78
1.19
3.44
1.27
2.40
2.96
2.94
2.65
2.06
3.29
1.34
3.0
3.10
2.71
1.68
3.21
1.3
Cisplatin
1.15 ꢁ 0.05 0.33 ꢁ 0.02 1.34 ꢁ 0.04 0.64 ꢁ 0.01 0.41 ꢁ 0.01
Values are derived from doseeresponse curves obtained by measuring the per-
centage of viable cells relative to untreated controls after 96 h exposure of the test
compounds to A549 (lung carcinoma), A2780 (ovarian cancer), 8505c (anaplastic
thyroid cancer), 518A2 (melanoma) and MCF-7 (breast cancer) cell lines using SRB
assay. Values are the average from at least three independent experiments.
(PtCl
2
)
(PtCl
(PtCl
2
2
)
)
3.41
2.74
1.22

464
D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
The crude product was dissolved in CH
again to give a white solid (1.17 g, 97% yield), H NMR (500 MHz,
CDCl ): 0.84 (3H, s, H-25), 0.85 (3H, s, H-24), 0.88 (3H, s, H-26), 0.96
3H, s, H-27), 1.00 (3H, s, H-23), 1.68 (3H, s, H-30), 1.82 (2H, m, H-2),
3
OH, filtered and evaporated
over Na
2 4
SO , and then concentrated under reduced pressure to give
1
a yellow powder (86 mg, 42% yield).
3
(
4.1.5.2. Method B. Compound 3(PtClS) (130 mg; 0.14 mmol) was
2
2
.04 (3H, s, eOAc), 2.11 (1H, m, H-13), 2.55 and 2.68 (1H each, m, H-
suspended in water (8 mL). LiCl (30 mg; 0.7 mmol) was added and
the mixture was stirred for 2 h at 80 C. The resulting precipitate
was filtered off, washed with water, and dried to yield a yellow
0
0
0
3
), 2.68 (2H, m, H-3 ), 2.74 (2H, m, H-4 ), 3.08 (1H, dt, H-19,
J
He
ꢀ
d
3 t
0
H
¼ 4.4 Hz, JHeH ¼ 10.8 Hz), 3.23 and 3.36 (1H each, m, H-1 ), 4.43
3
(
2
1H, dd, H-3, JHeH ¼ 11.3 Hz, 5.2 Hz), 4.57 and 4.69 (1H each, s, H-
powder (97 mg, 81% yield).
13
9), 5.47 (1H, s, -CONH). C NMR (125 MHz, CDCl
3
): 15.1 (C27), 16.8
1
6
H NMR (400 MHz, DMSO-d ): 0.76 (3H, s, H-25), 0.76 (3H, s, H-
0
0
(
(
(
(
(
(
C25), 16.8 (C26), 17.0 (C24), 19.3 (C6), 19.7 (C30), 21.2 (C2 ), 22.1
C12), 24.6 (C21), 26.9 (C15), 28.5 (C23), 30.6 (C16), 31.9 (C4), 34.1
C7), 35.5 (C10), 38.3 (C1), 38.8 (C22), 38.9 (C13), 39.3 (C1 ), 39.6
C4 ), 39.7 (C8), 41.8 (C14), 42.0 (C2 ), 43.5 (C3 ), 48.1 (C19), 51.4
C18), 51.9 (C9), 56.8 (C5), 57.0 (C17), 82.5 (C3), 110.0 (C29), 152.2
24), 0.78 (3H, s, H-26), 0.83 (3H, s, H-27), 0.90 (3H, s, H-23),1.61 (3H,
s, H-30), 1.97 (3H, s, eOAc), 2.13 (1H, m, H-2), 2.50e3.40 (9H, m, br,
0
0
0
0
0
3
H-19 þ H-1 þ H-2 þ H-3 þ H-4 ) 4.33 (1H, dd, H-3, JHeH ¼ 11.1 Hz,
0
0
0
13
4.8 Hz), 4.51 and 4.63 (1H each, s, H-29). C NMR (100 MHz, DMSO-
d
6
): 14.3 (C27), 15.9 (C24), 15.9 (C25), 16.4 (C26), 17.7 (C6), 19.0
0
0
C20), 172.9 (C1 ), 179.3 (C28). IR
gmax (neat): 2937, 2868, 1732,
00
(C30), 20.6 (C11), 20.9 (C2 ;), 23.3 (C2), 25.2 (C12), 27.7 (C23), 28.9
(C21), 30.3 (C15), 32.3 (C16), 33.8 (C4), 35.6 (C7), 36.6 (C10), 37.3
(C1), 37.6 (C22), 40.3 (C8), 41.9 (C14), 46.2 (C19), 49.6 (C18),
1
639,1452,1370,1316,1244,1195,1108, 1024, 978, 881, 543, 512. MS
þ
(
ESI): 585 (100, [M þ H] ). Analysis for C36
61 3 3
H N O (583.90): C,
74.05; H, 10.53; N, 7.20; found: C, 68.69; H, 9.59; N, 6.12.
4
9.8 (C9), 54.7 (C5), 54.9 (C17), 79.9 (C3), 109.3 (C29), 150.8 (C20),
0
0
170.1 (C1 ), 176.0 (C28). IR
gmax (neat): 2943, 1636, 1520. 1425,
4.1.3. Preparation of dichlorobis(dimethyl sulfoxide)platinum(II)
1374, 1318, 1255, 1197, 1028, 981, 883, 443. MS (ESI): 892.4
Dichlorobis(dimethyl sulfoxide)platinum(II) [25] was prepared
as described as follows: K [PtCl ] (0.5 g, 1.2 mmol) was dissolved in
2 4
þ
þ
(
C
100, [M þ H
2
O þ Na] ), 814.4 (21, [M ꢂ Cl] ). Analysis for
38 2 3 3
H61Cl N O Pt (873.92): C; 50.88, H; 7.23, N; 4.94, found: C;
water (4 mL), and DMSO (0.3 mL, 4.2 mmol) was added dropwise.
The solution was stirred for 5 min and then allowed to stand at
room temperature for 2 h. The forming solid was filtered off,
washed with water, ethanol, and ether and then dried to give pale
yellow crystals (448 mg, 88% yield).
51.64, H; 7.55, N; 4.54.
4
.1.6. Preparation of 1,3-bis(tert-butylcarboxyamino)2-propanol
6b)
Boc
16.6 mmol) were stirred in CH
(
2
O (2.9 g, 13.4 mmol) and 1,3-diamino-2-propanol (1.5 g,
CN (250 mL) for 1 h. The forming
0 00
.1.4. Preparation of kN ,N -(3-O-acetylbetulinic (2-(2-aminoethyl)
3
4
precipitate was filtered off and the solution concentrated under
aminoethyl)amide) chloro kS-dimethyl sulfoxide platinum(II)
chloride [3(PtClS)]
reduced pressure to yield a white solid (2.06 g, 53% yield).
1
3 3
H NMR (500 MHz, CDCl ): 1.34 (18H, s, eCH ), 3.10 (4H, m, e
Dichlorobis(dimethyl sulfoxide)platinum(II) (150 mg, 0.36 mmol)
was suspended in CH OH (10 mL) and a solution of 3 (207 mg,
.36 mmol) in CH OH (4 mL) was added dropwise. The mixture was
CH
2
e), 3.65 (1H, m, HOCH), 4.18 (1H, s, eOH), 5.41 (2H, s, eNHe).
C NMR (125 MHz, CDCl ): 28.2 (eCH ), 43.3 (eCH e), 70.2
HOCH), 79.4 (Cquart), 157.0 (C]O). IR max (neat): 3350, 2980, 2934,
3
1
3
3
3
2
0
3
(
g
stirred for2 hatrt, thenconcentrated underreducedpressure to3 mL
and stored in the refrigerator overnight. The precipitate was filtered
off and the solution was concentrated to 0.5 mL under reduced
pressure. The precipitate was filtered off, washed with water and
2917, 1664, 1526, 1443, 1392, 1365, 1158, 1116, 853, 574. MS (ESI):
þ
313.1 (100, [M þ Na] ).
1
dried to yield a yellow solid (299 mg, 91% yield). H NMR (400 MHz,
4.1.7. Preparation of 3-O-acetylbetulinic (1,3-bis(tert-butylcarboxy
3
CD OD): 0.84 (3H, s, H-25), 0.85 (3H, s, H-24), 0.89 (3H, s, H-26), 0.97
amino) 2-propyl)ester [4]
(
2
3H, s, H-27), 1.00 (3H, s, H-23), 1.68 (3H, s, H-30), 2.02 (3H, s, eOAc),
.15 (1H, m, H-22), 2.54 and 2.90 (1H each, m, H-2 ), 2.66 (6H, s,
Compound 6b (350 mg, 1.20 mmol) and NEt
3
(0.5 mL,
0
3.60 mmol) were dissolved in DCM (10 mL). A solution of 2a
(520 mg, 1.00 mmol) in DCM (10 mL) was added dropwise, the
mixture was stirred for 20 h, then refluxed for 30 min. The solution
was washed twice with 0.12 M hydrochloric acid (50 mL) and twice
with brine (50 mL), and then dried with Na SO . The solution was
2 4
concentrated under reduced pressure to give a pale yellowish solid
0
DMSO) 2.90 and 3.07 (2H, m, H-3 ), 3.07 (1H, m, H-19), 3.30 and 3.46
(
0
0
3
1H each, m, H-1 ), 3.46 (2H, m, H-4 ), 4.43 (1H, dd, H-3, J
He
13
H
(
¼ 10.8 Hz, 5.4 Hz), 4.58 and 4.70 (1H each, s, H-29). C NMR
OD): 15.2 (C27), 16.7 (C25), 16.8 (C26), 17.0 (C24), 19.3
100 MHz, CD
3
0
0
(
(
C6), 19.7 (C30), 21.2 (C2 ), 22.2 (C12), 24.7 (C21), 26.9 (C15), 28.5
C23), 30.7 (C16), 31.9 (C4), 34.0 (C7), 35.5 (C10), 38.3 (C1), 38.8 (C22),
(840 mg, 99% yield).
0
0
0
1
3
8.9 (C13), 39.3 (C1 ), 39.6 (C8), 39.9 (C4 ), 42.0 (C14), 42.1 (C2 ), 43.5
3
H NMR (500 MHz, CDCl ): 0.79 (3H, s, H-25), 0.80 (3H, s, H-24),
0
(C3 ), 48.1 (C19), 51.3 (C18), 51.9 (C9), 56.8 (C5), 57.0 (C17), 82.4 (C3),
0.81 (3H, s, H-26), 0.91 (3H, s, H-27), 0.93 (3H, s, H-23), 1.64 (3H, s,
H-30), 1.87 (2H, m, H-12), 2.00 (3H, s, eOAc), 2.17 (1H, m, H-2), 2.42
(1H, m, H13), 2.75 (1H, m, H-19), 3.13 and 3.20 (2H each, m, H-
00
195
1
CD
1
10.1 (C29), 152.2 (C20), 172.9 (C1 ), 180.1 (C28). Pt NMR (86 MHz,
3
OD): ꢂ3309 (SP-4-4), ꢂ3220 (SP-4-3). IR
gmax (neat): 2940, 2869,
635, 1449, 1371, 1253, 1196, 1131, 1024, 979, 948, 879, 733, 695, 441.
0
0
0
3
1 þ H-3 ), 3.71 (1H, m, H-2 ), 4.42 (1H, dd, H-3,
J
HeH ¼ 14.3 Hz,
þ
13
MS (ESI): 892.4 (100, [M ꢂ Cl] ). Analysis for C38
2 3 4
H67Cl N O PtS
4.8 Hz), 4.58 and 4.68 (1H each, s, H-29), 5.26 (2H, s, -NH). C NMR
(
3
928.03): C; 49.18, H; 7.28, N; 4.53, S; 3.45, found: C; 51.58, H; 7.83, N;
.29, S; 4.80.
(125 MHz, CDCl
(C6), 19.3 (C30), 20,8 (C11), 21.3 (C2 ), 25.3 (C12), 27.9 (C23), 28.4 (e
C(CH ), 29.6 (C21), 29.8 (C15), 32.2 (C16), 34.2 (C4), 36.2 (C7), 37.1
(C10), 37.7 (C13), 37.8 (C1), 38.4 (C22), 40.7 (C8), 42.4 (C14), 43.7
3
): 14.7 (C27), 15.9 (C25), 16.2 (C26), 16.5 (C24), 18.1
0
0
3 3
)
0
00
4
.1.5. Preparation of
k
N ,N -(3-O-acetylbetulinic (2-(2-aminoethyl)
)]
.1.5.1. Method A. Compound 3 (140 mg, 0.24 mmol) was dissolved
in CH OH (10 mL), and a solution of K [PtCl ] (100 mg, 0.24 mmol)
0
0
0
aminoethyl)amide) dichloro platinum(II) [3(PtCl
2
(C1 þ C3 ), 46.0 (C19), 49.7 (C18), 50.5 (C9), 55.5 (C5), 70.9 (C2 ),
79.8 (eOCMe ), 80.9 (C3), 110.3 (C29), 149.3 (C20), 157.1 (eNHCOOe
), 171.0 (C1 ), 177.3 (C28). IR max (neat): 2946, 2874, 1736, 1685,
4
3
0
0
3
2
4
g
in water (2 mL) was added. The mixture has been stirred under
light exclusion for 24 h, and then a 5% aqueous KCl solution (10 mL)
was added and stirred for another hour. The suspension was
extracted twice with DCM (30 mL), the organic phases were dried
1524, 1454, 1368, 1244, 1164, 1027, 978, 889, 856, 440. MS (ESI):
þ
þ
793.4 (100, [M þ Na] ), 693.4 (15, [M ꢂ OCOCMe
3
þ Na] ). Analysis
74 2 8
for C45H N O (771.093): C, 70.09; H, 9.67; N, 3.63; found: C, 65.87;
H, 9.58; N, 3.65.

D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
465
4
.1.8. Preparation of 3-O-acetylbetulinic (1,3-diamino 2-propyl)
ester [5]
Compound 6b (883 mg,1.05 mmol) was dissolved in DCM (5 mL).
CD
3
OD): 40.7 (eCH
OD): ꢂ3283. IR
2
e), 44.2 (DMSO), 66.3 (HOCH). 195Pt NMR
(86 MHz, CD
3
g
max (neat): 3142, 3071, 1315, 1239,
þ
1085, 1026, 853, 559, 438. MS (ESI): 399.1 (60, [M ꢂ Cl] ). Analysis
It was stirred and TFA (0.5 mL, 6.49 mmol) was added dropwise.
After about 1.5 h, the mixture was diluted with DCM (50 mL), then
5 2 2
for C H16ClN O PtS (366.738): C, 13.83; H, 3.71; N, 6,45; S, 7.38;
found: C, 14.38; H, 3.98; N, 6.16; S, 9.06.
washed with cold concentrated K
with brine (50 mL). The organic phase was dried over Na
concentrated under reduced pressure. The precipitate was dissolved
in CH OH, filtered, and the solution evaporated again to yield a white
2
CO
3
solution (50 mL) and twice
0
2
SO , then
4
4.1.11. Preparation of dichloro
platinum(II) [6(PtCl )]
k
N,N -(1,3-diamino-2-propanol)
2
3
Compound 6(PtClS) (200 mg, 0.46 mmol) was dissolved in wa-
solid (443 mg, 75% yield).
ter (5 mL) and LiCl (98 mg, 2.31 mmol) was added. The solution was
stirred for 2 h at 80 C, then concentrated and dried under reduced
1
ꢀ
H NMR (500 MHz, CDCl
3
): 0.84 (3H, s, H-25), 0.85 (3H, s, H-24),
0
.86 (3H, s, H-26), 0.94 (3H, s, H-27), 0.98 (3H, s, H-23), 1.70 (3H, s,
pressure. The remaining solid was suspended in water (1 mL) and
stored in the fridge for 20 h. The forming crystals were filtered off,
washed with water and ethanol, and dried to yield a pale yellow
H-30), 1.96 (2H, m, H-12), 2.05 (3H, s, eOAc), 2.25 (2H, m, H-2 þ H-
0
0
0
1
4
3), 3.01 (1H, m, H-19), 3.22 (4H, m, H-1 þ H-3 ), 3.75 (1H, m, H-2 ),
3
.48 (1H, dd, H-3, JHeH ¼ 10.5 Hz, 5.6 Hz), 4.61 and 4.74 (1H each, s,
powder (43 mg, 26% yield).
13
1
H-29), 5.31 (4H, s, eNH
2
). C NMR (125 MHz, CDCl
3
): 14.6 (C27),
H NMR (400 MHz, DMSO-d
6
): 2.58e2.76 (4H, m, eCH
2
e), 3.49
e), 65.4
13
16.0 (C25), 16.2 (C26), 16.5 (C24), 18.2 (C6), 19.3 (C30), 20,9 (C11),
(1H, m, HOCH). C NMR (100 MHz, DMSO-d
(HOCH).
6
): 47.5 (eCH
2
0
0
21.3 (C2 ), 23.7 (C2), 25.5 (C12), 28.0 (C23), 29.7 (C21), 30.7 (C15),
3
2.4 (C16), 34.3 (C4), 37.1 (C7), 37.2 (C10), 37.8 (C1), 38.3 (C22), 38.4
0
0
(
5
C13), 40.7 (C8), 42.5 (C14), 43.5 (C1 þ C3 ), 47.0 (C19), 49.3 (C18),
4.2. Biological studies
0
0.4 (C9), 55.4 (C5), 56.6 (C17), 70.9 (C2 ), 81.0 (C3), 109.6 (C29),
0
0
150.6 (C20), 171.1 (C1 ). IR
g
max (neat): 2942, 2871, 1453, 1367, 1244,
4.2.1. Cell lines and culture conditions
þ
1
162, 1025, 978, 881. MS (ESI): 671.3 (100, [M þ HOCOCMe
3
] ),
The cell lines 518A2 (melanoma), A2780 (ovarian), A549 (lung),
MCF-7 (breast) and 8505C (anaplastic thyroid tumor) were included
in this study. Cultures were maintained as monolayer in RPMI 1640
þ
571.5 (11, [M þ H] ). Analysis for C35
58 2 4
H N O (570.859): C, 73.64; H,
10.24; N, 4.91; found: C, 69.88; H, 9.31; N, 2.49.
(
PAA Laboratories, Pasching, Germany) supplemented with 10%
0
4
.1.9. Preparation of
propyl)ester) dichloro platinum(II) [5(PtCl
Dichlorobis(dimethylsulfoxide)platinum(II)(200 mg, 0.47 mmol)
was suspended in CH OH (10 mL), and a solution of 5 (300 mg,
.53 mmol) in CH OH (2 mL) was added dropwise. The mixture was
k
N,N -(3-O-acetylbetulinic (1,3-diamino 2-
heat-inactivated fetal bovine serum (SigmaeAldrich Chemie GmbH,
Germany) and penicillin/streptomycin (PAA Laboratories, Pasching,
Austria) at 37 C in a humidified atmosphere of 5% CO
2
)]
ꢀ
2
.
3
0
3
4.2.2. In vitro antitumoral studies
stirredfor2 h,theformingprecipitatewasfilteredoffandthesolution
was concentrated under reduced pressure onto 2 mL. The solution
was stored in the fridge for 40 h, then filtered again, and the solution
was concentrated onto 0.5 mL under reduced pressure. The forming
precipitate was filtered off, washed with water, and dried to give a
The cytotoxicity of all compounds was evaluated by
sulforhodamine-B (SRB) (SigmaeAldrich) microculture colori-
metric assay. Since the investigated compounds were insoluble in
water, they were initially dissolved in DMSO or DMF and further
diluted with culture medium for analysis. The final concentration of
DMSO or DMF never exceeded 0.5%, which was found to be non-
toxic to the cells. 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-
yellow powder (142 mg, 36% yield).
1
3
H NMR (400 MHz, CDCl ): 0.81 (3H, s, H-25), 0.83 (3H, s, H-24),
0
.84 (3H, s, H-26), 0.92 (3H, s, H-27), 0.96 (3H, s, H-23), 1.68 (3H, s,
H-30), 1.96 (2H, m, H-12), 2.03 (3H, s, eOAc), 2.17 (1H, m, H-2), 2.26
3
d
3 t
(
3
1H, m, H-13), 2.99 (1H, dt, H-19,
J
HeH ¼ 4.6 Hz,
J
HeH ¼ 10.7 Hz),
0
0
0
3
.53e3.10 (5H, m, H-1 þ H-2 þ H-3 ), 4.46 (1H, dd, H-3,
J
He
pounds (0e100 mM) for 96 h. The percentages of surviving cells
13
H
¼ 10.3 Hz, 6.0 Hz), 4.46 and 4.60 (1H each, s, H-29). C NMR
relative to untreated controls were determined 96 h after the
beginning of drug exposure. After 96 h treatment, the supernatant
medium from the 96-well plates was discarded and the cells were
fixed with 10% TCA. For a thorough fixation plates were allowed to
(
(
(
(
(
7
100 MHz, CDCl
3
): 14.6 (C27), 16.0 (C25), 16.1 (C26), 16.4 (C24), 18.1
00
C6), 19.3 (C30), 20,8 (C11), 21.3 (C2 ), 23.7 (C2), 25.4 (C12), 27.9
C23), 29.7 (C21), 30.6 (C15), 32.2 (C16), 34.2 (C4), 37.0 (C7), 37.1
C10), 37.8 (C1), 38.4 (C22), 38.4 (C13), 40.7 (C8), 42.4 (C14), 44.8
ꢀ
stand at 4 C for at least 2 h. After fixation the cells were washed in
0
0
C1 þ C3 ), 46.9 (C19), 49.3 (C18), 50.4 (C9), 55.4 (C5), 56.4 (C17),
a plate washer (Tecan Austria GmbH, Austria). The washing step
was done five times with water using alternate dispensing and
0
00
2.8 (C2 ), 81.0 (C3), 109.7 (C29), 150.4 (C20), 171.1 (C1 ), 182.2
max (neat): 2943, 2871, 1718, 1642, 1451, 1368, 1245, 1150,
(C28). IR
g
aspiration procedures. The plates were then dyed with 100
0.4% SRB for about 45 min. After dying the plates were washed with
% acetic acid to remove the dye and allowed to air-dry overnight.
mL of
1130, 1022, 978, 882, 752, 732, 438.
1
0
4.1.10. Preparation of chloro
k
N,N -(1,3-diamino 2-propanol)
Hundred microliters of 10 mM Tris base solution was added to each
well and the absorbance was measured at 570 nm using a plate
reader (TECAN Infinite F200 PRO, Tecan GmbH, Austria).
k
S-dimethyl sulfoxide platinum(II) chloride [6(PtClS)]
Dichlorobis(dimethyl sulfoxide)platinum(II)
.24 mmol) was suspended in CH
(100 mg,
OH (10 mL) and a solution of 1,3-
0
3
diamino-2-propanol (22 mg, 0.24 mmol) in CH
3
OH (2 mL) was
Acknowledgements
added dropwise. The mixture was stirred for 3 h, then concentrated
onto 2 mL under reduced pressure and stored in the fridge for 40 h.
The forming precipitate was filtered off and the solution was
allowed to dry under air. The forming crystals were washed care-
This work was supported by “Gruenderwerkstatt-Bio-
wissenschaften” and Univations from the University Halle.
fully with CH
3
OH and dried to yield colorless, hygroscopic crystals
Appendix A. Supplementary data
(
63 mg, 61 % yield).
1
H NMR (400 MHz, CD
3
OD): 2.66 (6H, s, dmso), 3.30 (1H, m,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.031.
13
HOCH), 3.45 and 3.48 (2H each, m, eCH
2
e). C NMR (100 MHz,

4
66
D. Emmerich et al. / European Journal of Medicinal Chemistry 75 (2014) 460e466
chemotherapeutic drugs, Martin-Luther-Universitaet Halle-Wittenberg, Ger-
References
many, 2005, p. 9, 2005-102005047308[102005047308]. DE. 30-9-2005.
14] Wilfried Roth, Apoptoseresistenz in malignen Tumoren. Neue Apoptose-
basierte Therapieansätze, Pathologe 30 (2010) 113e116.
15] I. Ben Sahra, K. Laurent, S. Giuliano, F. Larbret, G. Ponzio, P. Gounon, Y. Le
Marchand-Brustel, S. Giorgetti-Peraldi, M. Cormont, C. Bertolotto, M. Deckert,
P. Auberger, J.F. Tanti, F. Bost, Targeting cancer cell metabolism: the combi-
nation of metformin and 2-deoxyglucose induces p53-dependent apoptosis in
prostate cancer cells, Cancer Res. 70 (6) (2010) 2465e2475.
[
[
[
1] L. Bai, F. Meng, L. Zhao, M. Zhao, The anti-inflammatory activity of pentacyclic
triterpenes in vitro, Appl. Mech. Mater. 138e139 (Pt. 2) (2012) 1174e1178.
2] I.D. Bori, H.Y. Hung, K. Qian, C.H. Chen, S.L. Morris-Natschke, K.H. Lee, Anti-
AIDS agents 88. Anti-HIV conjugates of betulin and betulinic acid with AZT
prepared via clickchemistry, Tetrahedron Lett. 53 (15) (2012) 1987e1989.
3] W.F. de Moraes, P.M. Galdino, M. Nascimento, F.A. Vanderlinde, T.F. Bara
Maria, E.A. Costa, J.R. de Paula, Triterpenes involved in the anti-inflammatory
effect of ethanolic extract of Pterodon emarginatus Vogel stem bark, J. Nat.
Med. 66 (1) (2011).
4] P. Gonzalez, I. Mader, A. Tchoghandjian, S. Enzenmueller, S. Cristofanon,
F. Basit, K.M. Debatin, S. Fulda, Impairment of lysosomal integrity by B10, a
glycosylated derivative of betulinic acid, leads to lysosomal cell death and
converts autophagy into a detrimental process, Cell Death Differ. 19 (8) (2012)
[
[
[
[
16] G.q. Chen, Z.w. Yao, W.p. Zheng, L. Chen, H. Duan, Y. Shen, Combined anti-
tumor effect of ursolic acid and 5-fluorouracil on human esophageal carci-
noma cell Eca-109 in vitro, Chin. J. Cancer Res. 22 (1) (2010) 62e67.
17] W. Guo-Bao, C. Xiao-Qin, G. Qi-Rong, L. Jie, L. Gui-Nan, L. Yue, Arsenic Trioxide
overcomes cell adhesion-mediated drug resistance through down-regulating
the expression of beta(1)-integrin in K562 chronic myelogenous leukemia
cell line, Leuk. Lymphoma 51 (6) (2010) 1090e1097.
[
1337e1346.
[
18] J. Puente, A. Manzano, M. Martin, S. Lopez-Tarruella, E. az-Rubio, Breast can-
cer: complete response with the combination of sunitinib and trastuzumab in
a patient with grade III ductal carcinoma, Anti-Cancer Drugs 21 (Suppl. 1)
[
5] R. Graziose, P. Rojas-Silva, T. Rathinasabapathy, C. Dekock, M.H. Grace,
A. Poulev, M. Ann Lila, P. Smith, I. Raskin, Antiparasitic compounds from
Cornus florida L. with activities against Plasmodium falciparum and Leishmania
tarentolae, J. Ethnopharmacol. 142 (2) (2012) 456e461.
6] M. Bache, S. Bernhardt, A. Hein, S. Passin, M. Zschornak, R. Paschke,
D. Vordermark, Betulinic acid derivatives for in vitro glioblastoma therapy,
Exp. Strahlenther. Klin. Strahlenbiol. 22 (2013) 43e47.
7] H. Kommera, G.N. Kaluderovic, S. Dittrich, J. Kalbitz, B. Draeger, T. Mueller,
R. Paschke, Carbamate derivatives of betulinic acid and betulin with selective
cytotoxic activity, Bioorg. Med. Chem. Lett. 20 (11) (2010) 3409e3412.
8] H. Kommera, G.N. Kaluderovic, M. Bette, J. Kalbitz, P. Fuchs, S. Fulda, W. Mier,
(2010) S19eS22.
[
19] R.A. Smith, H. Yuan, R. Weissleder, L.C. Cantley, L. Josephson, A Wortmannin-
Cetuximab as a double drug, Bioconjugate Chem. 20 (11) (2009) 2185e2189.
20] T. Gianferrara, A. Bergamo, I. Bratsos, B. Milani, C. Spagnul, G. Sava, E. Alessio,
Ruthenium-porphyrin conjugates with cytotoxic and phototoxic antitumor
activity, J. Med. Chem. 53 (12) (2010) 4678e4690.
[
[
[
[
[
[
[
[
21] P. Lan, J. Wang, D.M. Zhang, C. Shu, H.H. Cao, P.H. Sun, X.M. Wu, W.C. Ye,
W.M. Chen, Synthesis and antiproliferative evaluation of 23-hydroxybetulinic
acid derivatives, Eur. J. Med. Chem. 46 (6) (2011) 2490e2502.
22] R. Paschke, J. Kalbitz, C. Paetz, Novel spacer linked bile acid-cisplatin com-
pounds as a model for specific drug delivery, synthesis and characterization,
Inorg. Chim. Acta 304 (2) (2000) 241e249.
2
ꢂ
R. Paschke, In vitro anticancer studies of Îꢁ and Î glucopyranose betulin
anomers, Chem. Biol. Interact 185 (2) (2010) 128e136.
9] H. Kommera, G.N. Kaluderovic, J. Kalbitz, R. Paschke, Lupane triterpenoids-
betulin and betulinic acid derivatives induce apoptosis in tumor cells,
Invest. New Drugs 29 (2) (2011) 266e272.
23] R.J. Holmes, M.J. McKeage, V. Murray, W.A. Denny, W.D. McFadyen, cis-
Dichloroplatinum(II)
complexes
tethered
to
9-aminoacridine-4-
[
10] M. Willmann, V. Wacheck, J. Buckley, K. Nagy, J. Thalhammer, R. Paschke,
T. Triche, B. Jansen, E. Selzer, Characterization of NVX-207, a novel betulinic
acid-derived anti-cancer compound, Eur. J. Clin. Invest. 39 (5) (2009) 384e394.
11] G.N. Kaluderovic, A. Dietrich, H. Kommera, J. Kuntsche, K. Maeder, T. Mueller,
R. Paschke, Liposomes as vehicles for water insoluble platinum-based po-
tential drug: 2-(4-(tetrahydro-2H-pyran-2-yloxy)-undecyl)-propane-1,3-
diamminedichloroplatinum(II), Eur. J. Med. Chem. 54 (2012) 567e572.
12] R. Paschke, A. Dietrich, T. Mueller, B. Kalinowski, G.N. Kaluderovic, Novel
platinum compound that overcomes cisplatin resistance and induces
apoptosis, Phys. Chem. (2010) 529e531. Proceedings of the Tenth Interna-
tional Conference on Fundamental and Applied Aspects of Physical Chemistry,
carboxamides: synthesis and action in resistant cell lines in vitro, J. Inorg.
Biochem 85 (2-3) (2001) 209e217.
[24] W. Wong. An optimized relaxivity and specificity hepatobiliary MRI contrast
agent. The University of Hong Kong, Peop. Rep. China. 2006-CN3127
[
[2007056961], 61 pp. WO. 21-11-2006.
[
25] J.H. Price, A.N. Williamson, R.F. Schramm, B.B. Wayland, Palladium(II) and
platinum(II) alkyl sulfoxide complexes. Examples of sulfur-bonded, mixed
sulfur- and oxygen-bonded, and totally oxygen-bonded complexes, Inorg.
Chem. 11 (6) (1972) 1280e1284.
[
[
[
26] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (13) (1990) 1107e
2010.
13] R. Paschke, W. Voigt, T. Mueller, J. Kalbitz, K. Maeder, A. Dietrich, Platinum (II)
compounds from 2-substituted propane-1,3-diamines for use as antitumor
1112.