Pt(II) complexes of a combretastatin A-4 analogous chalcone: Effects of conjugation on cytotoxicity, tumor specificity, and long-term tumor growth suppression

Article abstract of DOI:10.1021/jm801001d

Three dichlorido(6-aminomethylnicotinate)platinum complexes 6 comprising a combretastatin A-4 analogous chalcone were tested on a panel of 21 tumor cell lines from 9 entities. Parent chalcone 1a and the directly linked conjugate 6a exhibited excellent ant

Full text of DOI:10.1021/jm801001d

J. Med. Chem. 2009, 52, 241–246
241
Articles
Pt(II) Complexes of a Combretastatin A-4 Analogous Chalcone: Effects of Conjugation on
Cytotoxicity, Tumor Specificity, and Long-Term Tumor Growth Suppression
Rainer Schobert,*,† Bernhard Biersack,† Andrea Dietrich,§ Sebastian Knauer,† Miroslava Zoldakova,† Angelika Fruehauf,§ and
Thomas Mueller§
Organic Chemistry Laboratory, UniVersity of Bayreuth, UniVersita¨tsstrasse 30, 95440 Bayreuth, Germany, and Department of Internal
Medicine IV, Oncology/Hematology, Research Laboratory, Martin Luther UniVersity Halle-Wittenberg, 06099 Halle, Germany
ReceiVed August 7, 2008
Three dichlorido(6-aminomethylnicotinate)platinum complexes 6 comprising a combretastatin A-4 analogous
chalcone were tested on a panel of 21 tumor cell lines from 9 entities. Parent chalcone 1a and the directly
linked conjugate 6a exhibited excellent antiproliferative activities, similar in magnitude [average log(IC50)
values of -7.3 (1a) and -7.0 (6a)] and cell line specificity but slightly different in the mechanism of
apoptosis induction. While 1a and 6a caused an equally fast rise in caspase-9 in the tested cancer cell lines,
the downstream effector caspase-3 built up faster in cells treated with 1a compared to 6a, yet reached an
equal end level. They also had different long-term effects on the regrowth of cancer cells treated with a
single dose. In contrast, conjugates 6b,c featuring longer spacers between the Pt complex and the chalcone
moieties were less antiproliferative than 6a.
Introduction
The anticancer agent combretastatin A-4 (Figure 1), a
naturally occurring cis-stilbene from the bark of the South
African Cape Bushwillow (Combretum caffrum), binds to the
colchicine binding site of ꢀ-tubulin and interferes with the
polymerization of tubulin. Its water soluble phosphate ester has
already entered clinical phase III trials. Combretastatin A-4 is
strongly angiotoxic and also active in MDRa positive tumors.1
Figure 1. Combretastatin A-4 and chalcone analogues 1a,b: potent
Since the thermodynamically favored trans isomer of combre-
tastatin A-4 is much less active, a search for chemically more
stable active analogues began that eventually led to the methoxy
functionalized chalcones 1a and 1b. Both showed high activity
in K562 leukemia cells.2 The biological potential of chalcones
is amazing because of their tubulin binding and alkylating
properties and their possible interactions with various proteins
related to cell apoptosis and proliferation. However, systematic
studies of chalcones have only started to emerge.3-6 Some
chalcones were found to increase the level of the tumor
suppressor protein p53 in various cancer cell lines by disrupting
itscomplexeswiththeoncoproteinMDM2.3Simplestructure-activity
studies were undertaken in order to optimize the antiproliferative
efficacy of libraries of synthetic chalcones in HT-29 colon cancer
cells where the best performers reached IC50 values below 10
µM.4 Heteroaryl substituted chalcones were developed as
inhibitors of vascular cell adhesion molecule-1 (VCAM-1)
expression for the treatment of chronic inflammatory diseases.5
inhibitors of tubulin polymerization.
But hitherto, apart from N-mustards and DNA minor groove
binding pyrrolo[2,1-c][1,4]benzodiazepines,7 no conjugates have
been disclosed of combretastatin A-4 or related chalcones with
clinically established anticancer drugs that operate by a different
mode of action such as DNA-targeting platinum(II) complexes.
We now prepared such conjugates of chalcone 1a by our
protocol based upon the esterification of 6-aminomethylnico-
tinate ligands.8-11 The product chalcone-Pt(II) complex con-
jugates 6 were tested on a representative panel of 21 tumor cell
lines and nonmalignant fibroblasts. Their efficacy, specificity,
regrowth retardation, and mechanism of induction of apoptosis
were compared with those of free parent chalcone 1a in order
to pinpoint beneficial synergisms of conjugating DNA-seeking
platinum and tubulin-targeting chalcone moieties.
Results and Discussion
Chemistry. Chalcone 1a was prepared from isovanillin and
3,4,5-trimethoxyacetophenone in one step according to a modi-
fied literature procedure.2 The hydroxyalkyl functionalized
chalcones 3 were synthesized via Williamson etherification of
the chalcone 1a with tetrahydropyranyloxyalkyl bromide in the
presence of K2CO3 and tetrabutylammonium iodide (TBAI)
followed by deprotection of the intermediate THP ethers 2 with
PPTS in ethanol (Scheme 1). Boc-protected 6-aminomethylni-
cotinic acid8 was connected to the chalcones 1a and 3 by
* To whom correspondence should be addressed. Phone: +49(0)921
552679. Fax: +49(0)921 552671. E-mail: Rainer.Schobert@uni-bayreuth.de.
†
University of Bayreuth.
§
Martin Luther University Halle-Wittenberg.
a Abbreviations: DIABLO, direct IAP-binding protein with low isoelectric
point; DMAP, 4-dimethylaminopyridine; IAP, inhibitor of apoptosis protein;
MDR, multidrug resistance; PARP, poly(ADP-ribose) polymerase; PPTS,
pyridinium p-toluenesulfonate; Smac, second mitochondria-derived activator
of caspase; SRB, sulforhodamine-B; TBAI, tetrabutylammonium iodide;
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end
labeling.
10.1021/jm801001d CCC: $40.75
2009 American Chemical Society
Published on Web 12/22/2008

242 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Schobert et al.
Table 1. Inhibitory Concentrations IC50 (nM)a for Compounds 1a and
6a-c
Scheme 1. Synthesis of Chalcone-Platinum(II) Complex
Conjugates 6a
cell panel
germ cell
cell line
1a
6a
6b
6c
H12.1
33.6 ( 13
31.4 ( 5
141.8 ( 41 283.2 ( 90
56.0 ( 10
61.6 ( 12
49.0 ( 3
4200
2102EP
1411HP
HCT-8
11000 4400
14000 5300
11000 3300
12000 4800
21000 5300
13000 5000
11000 8400
14000
colon
167.0 ( 20
61.7 ( 9
HCT-116 25.1 ( 4
HT-29
DLD-1
SW480
2600 ( 500 >10000
32.3 ( 2.8
102 ( 35
67.5 ( 14
62.0 ( 10
93 ( 106
132.4 ( 32
non-small-cell A549
lung
A427
H322
H358
51.2 ( 14
39.0 ( 2
17.1 ( 2
41.0 ( 11
22.8 ( 3
15.0 ( 3
22.3 ( 12
92 ( 68
12000 4400
11000
7100
4300 6000
13500
6300 3000
8800 4000
58.0 ( 7
26.2 ( 6
57.6 ( 7
49.7 ( 8
24.7 ( 2
144.1 ( 60
head-and-neck FADU
A253
cervix
mamma
A431
MCF-7
BT474
A2780
518A2
8505C
193.2 ( 38 8500 ( 2300 18000 7000
ovarian
melanoma
thyroidal
15.6 ( 3
33.6 ( 3
49.7 ( 3
28.7 ( 5
10500 4200
14000 5000
17000 5500
16000 5700
57.2 ( 11
88.1 ( 19
53.4 ( 10
SW1736 27.5 ( 4
a Values are derived from dose-response curves obtained by measuring
the percentage of surviving cells relative to untreated controls after 96 h of
exposure of cells to test compounds using the SRB assay. Values for 1a
and 6a represent mean values from three independent experiments. Values
for 6b and 6c are the average of two experiments.
a Reagents and conditions: (i) K2CO3, THP-alkyl bromide, TBAI, DMF,
24 h, room temp, 95-99%; (ii) PPTS, ethanol, 1 h, reflux, 81-84%; (iii)
Et3N, C6H2Cl3COCl, DMAP, alcohol 1a or 3, DMF/toluene (1:10), 16 h,
room temp; (iv) 4 M HCl/dioxane, 1 h, room temp; (v) K2PtCl4, aqueous
THF, pH 5-6, 24 h, room temp.
Yamaguchi esterification to give the respective esters 4.8,9
Deprotection of the amino group in HCl/dioxane and treatment
of the resulting ammonium chlorides 5 with K2PtCl4 in aqueous
THF under slightly acidic conditions (pH 5-6) gave the Pt(II)
complexes 6.8-11
Biological Evaluation. Chalcone 1a and the directly linked
conjugate 6a showed a similar spectrum of antiproliferative
activity (Table 1) and specificity (Figure 2) in the tested cell
lines. Chalcone 1a was generally more active than the platinum
conjugate 6a. This corroborated the assumption that compounds
6 might act as intact conjugates within the cells. However, while
they proved stable in PBS at 37 °C for more than 96 h, we
cannot exclude their intracellular enzymatic hydrolysis. The
observed activities of 1a and 6a were extraordinary by any
standard even in cell lines insensitive or refractory to cisplatin
(H322, H358, SW1736, 8505C, DLD-1) or to 5-fluorouracil and/
or oxaliplatin (DLD-1, SW480) (data not shown). The log(IC50/
96 h) values averaged over all 21 cell lines were -7.3 (1a) and
-7.0 (6a); i.e., IC50 values lay mostly in the double-digit
nanomolar range. The similarities in their activity profiles speak
for the two compounds probably acting on the same targets in
most cell lines. This would also be in line with data recently
published by Tron et al., who investigated combretastatin A-4
conjugates with various N-mustards.12 They found a generally
high activity with combretastatin A-4 being more potent than
the corresponding chlorambucil ester except for one particularly
sensitive neuroblastoma cell line (SH-SY5Y) in which the
conjugate showed enhanced potency. The cell line most
susceptible to compounds 1a and 6a (IC50/96 h ≈ 20 nM) was
the cervical carcinoma cell line A431, whereas HT-29 colon
carcinoma cells and breast tumor BT474 cells were least
sensitive.
Figure 2. Cell line specificities of chalcone 1a (left) and Pt conjugate
6a (right) as deviation of the log(IC50/96 h) of individual cell lines from
the mean over all cell line log(IC50/96 h) values. Negative values
indicate higher and positive values lower than average activities. Mean
log(IC50/96 h) values are -7.3 (1a) and -7.0 (6a).
Increasing the distance between platinum complex and
chalcone moieties by introducing a spacer had a dramatic effect
on the cytotoxicity (Table 1) and to some extent also on the
cell line specificity profile, possibly by curtailing some syner-
getic modes of action or by addressing other cellular targets or
by leading to differerent hydrolysis products with divergent
activities. The alkyl spacered conjugates 6b and 6c had up to
1000 times greater IC50 values than complex 6a. The hexyl-
tethered conjugate 6c proved generally more active than the

Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 243
Figure 4. Effects of 3 µM each of compounds 1a, 6a, vincristine, and
paclitaxel on the polymerization of tubulin as ascertained with a
fluorescence-based assay kit from Cytoskeleton. Data are representative
of three independent experiments. RFU ) relative fluorescence units.
Figure 3. Regrowth of cell cultures of A427 non-small-cell lung cancer
cell line (top) and A431 cervical cancer cell line (bottom) over a 10-
day period following treatment with 1a or 6a at the indicated
concentrations for 96 h. Data are representatitive of three independent
experiments.
concentrations either do not bind to plasmid DNA or upon
binding do not change the DNA morphology. Then the metal
content of salmon sperm DNA treated with high doses (100
µM) of platinum complexes 6a and 6c was quantified by
inductive coupled plasma optical emission spectroscopy (ICP-
OES). This method furnished Pt content in the DNA samples
of 0.05% for 6a and 0.037% for 6c, which is about 4 times less
than the 0.187% we found for the good DNA-binder cisplatin.
Compounds 1a and also 6a to a lesser extent inhibited the
polymerization of tubulin when applied in vitro at physiologi-
cally meaningful concentrations of 3 µM using the tubulin
polymerization assay kit by Cytoskeleton. Figure 4 shows the
time dependency of this process in samples containing 1a, 6a,
or the reference compounds paclitaxel, a known accelerator, or
vincristine, a known inhibitor of microtubule formation. The
differences in the tubulin polymerization rates of 1a and 6a
roughly correlate with their average antiproliferative effects in
tumor cells.
Compounds 1a and 6a are inducers of apoptosis in cancer
cells, detectable in its late stages by means of the TUNEL assay
that labels the 3′-OH ends of typical DNA fragments with
fluorescein-tagged nucleotides.13 It was conducted employing
the in situ cell death detection kit (Roche) with HL-60 leukemia
and 518A2 melanoma cells that had been exposed to 10 µM
concentrations of compounds 1a and 6a for 15 h. Fluorescence
microscopy revealed apoptotic cells as green fluorescent spots
that were counted and compared with the overall cell numbers.
At the end of the exposure period about 50-60% of the 518A2
cells and about 40-50% of the HL-60 cells were apoptotic.
To determine whether apoptosis was initiated by compounds
1a and 6a via the same mechanism, we carried out immuno-
blotting assays for caspase-9, a central enzyme in the intrinsic
mitochondrial apoptosis pathway, as well as for the downstream
effector caspase-3 and for the PARP protein which plays a
pivotal role in the nucleotide excision repair (NER) of DNA
lesions and which is cleaved during apoptosis. Figure 5 shows
Western blots for caspase-9 in HL-60 leukemia cells pre-exposed
to the compounds and then lysed after 0-30 h periods. The
cell protein was separated by electrophoresis, blotted onto
PVDF, treated with antibodies against caspase-9, and visualized.
Both compounds gave a congruent time-dependent profile of
procaspase-9 and cleaved/activated caspase-9 concentrations
with a maximum of the latter after ∼7 h past exposure,
indicating an activated mitochondrial apoptosis.
propyl-linked 6b (except for FaDu human head-and-neck
squamous cell carcinoma cells).
Beside low IC50 values, other qualities of potential new
anticancer drug candidates matter as well, such as their ability
to suppress a regrowth of cancer cells following a single-dose
impact. Tron et al. reported that combretastatin A-4 and its
chlorambucil ester were unable to kill all cells of SH-SY5Y
neuroblastoma cultures even at high concentrations despite their
relatively low IC50 values. A significant cell viability of ∼10%
persisted.12 We observed a similar behavior of the chalcones
1a and 6a for certain cell lines. While the dose-response curves
of sensitive cell lines such as A431, A2780, HCT-116, and
DLD-1 crossed the IC90 threshold (10-100 nM) early on, those
of the less sensitive 1411HP, BT474, SW480, and HT-29 cells
leveled off considerably toward higher concentrations. For 5
tumor cell lines out of the panel of 21 we also observed distinct
differences in the long term regrowth retardation by chalcone
1a and its conjugate 6a. These tumor cell lines were treated
once with drug concentrations around the IC90 values for 96 h
and then were rinsed drug-free and resuspended with fresh
media. Their regrowth was monitored over the following 10
days. A one-off treatment with 0.1 µM conjugate 6a prevented
regrowth of A427 non-small-cell lung cancer cells and cisplatin-
resistant 1411HP germ cell tumor cells more effectively than a
comparable exposure to the free chalcone 1a, while the latter
performed better at low concentrations in eradicating A253 head-
and-neck carcinoma cells, A431 cervical carcinoma cells, and
A2780 ovarian carcinoma cells. The growth kinetics of treated
cells of A427 and A431 are depicted in Figure 3. There are
several plausible explanations for these divergent long-term
effects such as a shift in targets, or differences in cellular storage
or induction of resistance. Alternatively, cleavage of 6a in cells
of A427 to give 1a and auxiliary platinum complexes could be
responsible for its superior effect, while in cells of A431 either
such cleavage occurs to a lesser extent or the mixture of cleavage
products is less efficacious for unknown reasons.
Next we addressed the effects of compounds 1a and 6 on
the potential targets DNA and tubulin. When the compounds
were incubated at concentrations of 5-60 µM with pBR322
plasmid DNA, no effect on the electrophoretic mobilities of the
various morphologically different DNA forms present was
visible. This means the tested compounds at relatively low
Next, A2780 ovarian cancer cells, 518A2 melanoma cells,
and DLD-1 colon carcinoma cells as well as nonmalignant

244 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Schobert et al.
Figure 5. Time-dependent processing of procaspase-9 (∼46 kDa) to
activated caspase-9 (∼35 kDa) in HL-60 leukemia cells treated with
10 µM compounds 1a (left) and 6a (right) for 0, 3, 7, 24, and 30 h.
Shown is ECL-visualization of lysates immunoblotted with prim./sec.
antibodies to caspase-9 from Calbiochem. Data are representative of
three independent experiments.
Figure 7. Initial rates of cleavage of substrate Ac-DEVD-(p-nitroa-
nilide) by caspase-3 isolated from HL-60 leukemia cells treated with 1
µM 1a or 6a or a control (C) for the indicated times, calculated from
the initial slope of the absorbance vs time curves. Data represent the
mean values of three experiments.
binding to and inhibiting caspase-3 but not the initiator caspase-
9.17 Possibly, chalcone 1a caused a faster release than conjugate
6a of IAP-inactivating proteins, such as Smac/DIABLO. This is
remarkable, as platinum drugs were themselves reported to decrease
IAP levels and to induce apoptosis in sensitive cancer cells.18
Figure 6. Western blots for analysis of cleavage/activation of caspase-
3, cleavage of PARP and actin as loading control in A2780 ovarian
cancer cells, 518A2 melanoma cells, DLD-1 colon cancer cells, and
nonmalignant human WW070327 fibroblasts after a 24 h treatment with
1 µM 1a or 1 µM 6a; c means untreated control cells. Data are
representative of three independent experiments.
Conclusions
The chalcone-platinum complex conjugate 6a exhibited
excellent tumor growth inhibition across a panel of 21 cell lines,
similar in magnitude and cell line specificity to the activity of
the free chalcone 1a. However, cell line specific differences were
found in the long term effect of 1a and 6a on the regrowth of
cancer cells treated with a single dose. At low 0.1 µM
concentration, 6a prevented regrowth of A427 non-small-cell
lung cancer cells and 1411HP germ cell tumor cells more
effectively than 1a while the latter performed better in eradicat-
ing A253 head-and-neck carcinoma cells, A431 cervical car-
cinoma cells, and A2780 ovarian cancer cells at very low 0.03
µM concentrations. Whether a shift to other cellular targets or
the combined effect of conceivable hydrolysis products of 6a
plays a role here remains unclear. In preliminary in vitro
experiments, the Pt complexes 6 interacted with various forms
of DNA, though less tightly than cisplatin, achieving significant
metalation only at superclinical concentrations. Tubulin seemed
to be the primary target of both compounds 1a and 6a, which
at different rates inhibited its polymerization in vitro at
physiologically meaningful concentrations. These rate differ-
ences would match the different cytotoxicity profiles of 1a and
6a and yet cannot explain the subtle differences in cell line
specific regrowth retardations and in caspase kinetics. The mode
of cell death initiated by chalcones 1a and 6a in cells of various
cancer cell lines was predominantly apoptotic as indicated by
the activation of caspases-9 and -3, by cleavage of PARP, and
by TUNEL. We found a slower progression from caspase-9 to
caspase-3 in cancer cells treated with 6a compared to those
treated with 1a. This could be due to an intracellular cleavage
of 6a to give the more active 1a and platinum complexes or to
a slower release of proapaptotic proteins such as Smac/DIABLO
from the mitochondria exposed to 6a when compared to 1a. A
series of immunoblotting kinetics for potentially involved pro-
and antiapoptotic proteins affecting the caspase-9 to caspase-3
progression are currently underway. HT-29 colon cancer cells
and BT474 breast cancer cells were conspicuously insensitive
to compounds 1a and 6, which might have to do with the
human fibroblasts WW070327 were treated for 24 h with 1 µM
concentrations of 1a and 6a, which amounts to 30-60 times
the IC50 in the case of the tested cancer cell lines. Then the cell
samples were analyzed by Western blotting for cleavage/
activation of caspase-3 and for cleavage of PARP. The cyto-
skeleton component actin was used as a loading control. Figure
6 shows that there was virtually no difference in apoptosis
induction of the three cancer cell lines treated with either 1a or
6a. Both compounds caused a significant increase of the
activated form of caspase-3 and of the cleaved form of PARP
relative to an untreated control (C). This is typical of an
apoptotic cell death. In contrast, the nonmalignant fibroblasts
showed no signs of apoptosis when treated with the same 1
µM doses of 1a or 6a. This is a pharmacologically promising
aspect.
We also monitored the progress of the caspase-3 rise in the
hours immediately following the exposure of HL-60 leukemia
cells to compounds 1a and 6a by means of the colorimetric
CASPASE-3 cellular activity assay kit PLUS from Biomol. The
cells were lysed, and caspase-3 activity was measured with a
specific dye-labeled substrate, i.e., Ac-DEVD-(p-nitroanilide).
The initial rate of the cleavage of this substrate was ascertained
by measuring the extinction of the samples, which was taken
as equivalent to the respective concentration of caspase-3.
Interestingly, levels of caspase-3 in cells treated with the two
compounds were comparable only from 15 h exposure time on
(Figure 7). Until then, the caspase-3 concentration grew more
slowly in cells exposed to the conjugate 6a when compared to
1a. Some biosynthetically close relatives of 1a such as curcumin,
resveratrol, and quercetin are known to sensitize cancer cells
to proapoptotic stimuli by inducing proteins that negatively
regulate apoptosis-inhibiting proteins (IAP).14-16Some members
of the IAP family, such as survivin, block apoptosis by specifically

Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 245
overexpression of the cellular apoptosis susceptibility (CAS)
gene in these cell lines.19 CAS functions in the mitotic spindle
checkpoint and its overexpression was shown to correlate with
tumor progression rather than apoptosis.20 Nonmalignant fibro-
blasts were far less prone to apoptosis induced by even high
concentrations of 1a or 6a as ascertained by the absence of the
typical apoptotic features. This tolerance of fibroblasts when
compared to cancer cells is a pharmacologically interesting
aspect.
Biological Studies. 1. Cytotoxicity Assays. Cell Lines and
Culture Conditions. Twenty-one cell lines from nine entities
(summarized in Table 1) were used for the evaluation of the
cytotoxicity of the chalcones. Most of them were obtained from
the American Type Culture Collection (ATCC), Rockville, MD,
and from the German National Resource Center for Biological
Material (DSMZ), Braunschweig, Germany. Cell lines were main-
tained as monolayer cultures in RPMI 1640 supplemented with 10%
heat inactivated fetal bovine serum (Biochrom KG Seromed,
Germany) and streptomycin/penicillin (GIBCO, Germany). Cultures
were grown at 37 °C in a humidified atmosphere of 5% CO2/95%
air.
Experimental Section
SRB Assay and Regrowth Analyses. Dose-response curves
of the cell lines exposed to drug concentrations of 0.001-10 µM
were established using the sulforhodamine-B (SRB) microculture
colorimetric assay21 and performed as previously described.22
Briefly, cells were seeded into 96-well plates on day 0 at cell
densities previously determined to ensure exponential cell growth
during the period of the experiment. On day 1, cells were treated
with the respective concentration range of the drugs, and the
percentage of surviving cells relative to untreated controls was
determined on day 5. For analysis of tumor cell regrowth after drug
treatment, the SRB assay as decribed above was extended. After
96 h of exposure to the serial dilutions of drugs the cells were either
fixed (day 0 after treatment) or washed drug-free and grown in
fresh media for additional spells of time (2, 4, 6, 10 days).
Appropriate concentrations were chosen to unveil differences in
regrowth kinetics.
2. Immunoblotting. With Caspase-9 Antibodies. HL-60 cells
(0.5 × 106 cells/mL) were incubated (37 °C, 5% CO2, 95%
humidity) with 10 µM test compound (in 1% DMF/99% PBS) for
up to 30 h. At specified time intervals 2 mL aliquots were
withdrawn. The cells were centrifuged and suspended in 100 µL
of lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100,
2% protease inhibitor cocktail Set III (Calbiochem), pH 7.4],
vortexed, and incubated on ice for 15 min.23 The proteins were
separated on a 12% SDS-PAGE using 60 µg of protein per
pocket24 and blotted on a PVDF membrane. The membrane was
then blocked by treatment with 10% milk powder in AP-T (0.1 M
Tris-HCl, 0.1 M NaCl, 0.25 mM MgCl2, 1% Tween-20, pH 7.4)
for 1 h, washed three times for 10 min with AP-T, and finally
incubated with the primary antibody (Calbiochem, 1:10.000 in AP-
T). After another three washing steps the secondary antibody
conjugated with horseradish-peroxidase (Calbiochem, 1:10.000 in
AP-T) was applied. After another three washing steps visualization
was conducted using Roti-Lumin (Roth) and Amersham Hyperfilm
ECL (GE Healthcare). The film was exposed for 10 min.
With Antibodies for Caspase-3, PARP, and Actin. Cells were
harvested, rinsed twice with PBS, and lysed in RIPA buffer [50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1%
SDS] supplemented with a protease inhibitor cocktail (Sigma).
Insoluble components were removed by centrifugation, and protein
concentrations were measured (BIO-RAD protein assay, Bio-Rad,
Germany). After boiling for 5 min in SDS-loading buffer (62.5
mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 100 mM DTT) 40
µg of protein per lane was separated by SDS-PAGE and elec-
troblotted onto nitrocellulose membrane (Bio-Rad). Equal protein
loadings were ensured by Ponceau S staining (Sigma). Membranes
were blocked with 5% milk powder in PBST for 1 h and probed
for 2 h with the following antibodies diluted in PBST/5% milk:
actin (C-2) mouse monoclonal (0.5 µg/mL) (Santa Cruz Biotech-
nology), caspase-3 (Clon 33) mouse monoclonal (0.5 µg/mL) (MBL
Biozol), PARP mouse monoclonal (4C10-5) (BD Pharmingen).
Immunocomplexes were visualized by enhanced chemiluminescence
(Amersham Pharmacia Biotech, U.K.) using horseradish peroxidase-
conjugated antimouse or antirabbit IgG (Santa Cruz Biotechnology).
3. Colorimetric Caspase-3 Assays. These assays were per-
formed in accordance with the manufacturer’s recommendations
using the CASPASE-3 cellular activity assay kit PLUS (Biomol,
Plymouth Meeting, PA). This employs N-acetyl-Asp-Glu-Val-Asp-
p-nitroaniline (Ac-DEVD-pNA) as a substrate for caspase-3 enzyme
Chemistry. (2E)-3-[4′-Methoxy-3′-(tetrahydropyranyloxypropyl)]phenyl-
1-(3′,4′,5′-trimethoxyphenyl)prop-2-en-1-one (2a). Typical
Procedure. Anhydrous K2CO3 (922 mg, 6.76 mmol) was added to
a stirring solution of chalcone 1a2 (222 mg, 0.65 mmol) in dry
DMF (10 mL) whereupon the color of the solution turned red. After
10 min 3-tetrahydropyranyloxypropyl 1-bromide (1.44 g, 6.46
mmol) and TBAI (50 mg) were added and the reaction mixture
was stirred under argon at room temperature for 24 h. It was diluted
with ethyl acetate and brine, the water phase was extracted twice
with ethyl acetate, and the combined organic phases were washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuum.
The oily residue was purified by column chromatography on silica
gel 60 to leave 2a as a yellow oil (300 mg, 0.62 mmol, 95%): Rf
) 0.43 (hexane/ethyl acetate 1:1).
(2E)-3-[4′-Methoxy-3′-(3′′-hydroxypropyl)]phenyl-
1-(3′,4′,5′-trimethoxyphenyl)prop-2-en-1-one (3a). Typical
Procedure. THP ether 2a (300 mg, 0.62 mmol) was dissolved in
ethanol (50 mL), PPTS (100 mg) was added, and the resulting
mixture was stirred under reflux for 1 h. The solvent was
evaporated, the oily residue thus obtained was taken up in ethyl
acetate, washed once with water, dried, filtered, concentrated in
vacuum, and purified by column chromatography (hexane/ethyl
acetate 1:2), leaving the product as a yellow oil (210 mg, 0.52 mmol,
84%) which solidified upon standing: mp 116 °C; Rf ) 0.26.
(E)-3-(4′-Methoxyphenyl)-1-(3′,4′,5′-trimethoxyphenyl)-
prop-2-en-1-one-3′-yl
6-(tert-butoxy-carbonylaminome-
thyl)nicotinate (4a). Typical Procedure. 6-(tert-Butoxycarbo-
nylaminomethyl)nicotinic acid8 (140 mg, 0.56 mmol) was dissolved
in dry DMF (2 mL) and treated with Et3N (90 µL, 0.65 mmol) and
2,4,6-trichlorobenzoyl chloride (100 µL, 0.65 mmol). The resulting
suspension was stirred under argon at room temperature for 20 min.
A solution of chalcone 1a (190 mg, 0.55 mmol) and DMAP (138
mg, 1.12 mmol) in dry DMF/toluene (1:9, 10 mL) was added, and
the resulting mixture was stirred under argon at room temperature
for 16 h. After dilution with ethyl acetate and washing with water
the organic phase was dried over Na2SO4 and concentrated in
vacuum. The residue was purified by column chromatography on
silica gel 60 (hexane/ethyl acetate 1:1) to leave 4a as a yellow oil
(220 mg, 0.38 mmol, 68%): Rf ) 0.25.
(E)-2-Methoxy-5-[3′-oxo-3′-(3′′,4′′,5′′-trimethoxyphenyl)-
prop-1′-enyl]phenyl 6-aminomethylnicotinate bis(hydrochlo-
ride) (5a). Typical Procedure. Compound 4a (210 mg, 0.36
mmol) was treated with 4 M HCl/dioxane (10 mL) at room
temperature for 1 h. The formed precipitate was collected, washed
with diethyl ether, dried, and used as such for the next step. Yield,
166 mg (0.30 mmol, 83%); light-brown solid of mp 165 °C.
cis-{(E)-2-Methoxy-5-[3′-oxo-3′-(3′′,4′′,5′′-trimethoxyphenyl)
prop-1′-enyl]phenyl 6-aminomethylnicotinate}dichloridoplatinum(II)
(6a). Typical Procedure. Ligand 5a (160 mg, 0.29 mmol) was
dissolved in THF (5 mL) and treated with an aqueous solution of
K2PtCl4 (121 mg, 0.29 mmol). The resulting colorless precipitate
was redissolved by addition of THF. The pH value was adjusted
to 5-6 with aqueous NaOH, and the reaction mixture was stirred
at room temperature for 24 h. The formed precipitate was collected,
washed in turn with water and diethyl ether, and finally dried in
vacuum. Yield, 166 mg (0.22 mmol, 77%) of 6a; yellow solid of
mp >250 °C (dec).

246 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Schobert et al.
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with or without inhibitor Ac-DEVD-CHO added. Briefly, HL-60
cells were treated with test compound 1a or 6a for the indicated
periods of time, harvested, and then lysed with the buffer provided
in the kit (supplemented with 0.1% Triton X-100). The cleavage
of the substrate DEVD bonded to the chromophore p-nitroanilide
(pNA) was measured by reading the absorbance (at 405 nm
wavelength) of the samples (maintained at 37 °C) at 15 min
increments for 90 min. The rate of cleavage of pNA [(pmol/min)/
µg of protein] was calculated from the initial slope of the absorbance
vs time curves.
4. Tubulin Polymerization Assay. Analysis of tubulin polym-
erization was performed with a tubulin polymerization assay kit
(Cytoskeleton) according to the manufacturer’s instructions. The
assay is fluorescence-based, and tubulin polymerization was
monitored by measuring RFU (relative fluorescence units) on a
SpectraFluorPlus (Tecan, Switzerland) using the following filters:
excitation 360 nm, emission 465 nm.
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Acknowledgment. This work was supported by a grant from
the Deutsche Forschungsgemeinschaft (Grant Scho 402/8-2).
We are indebted to Franziska Reipsch for technical assistance
and to Dr. Florenz Sasse from the Helmholtz Center for Infection
Research, Braunschweig (Germany), for additional tubulin
binding experiments.
Supporting Information Available: Purity data for new com-
pounds, spectroscopic data of compounds 2a, 3a, 4a, 5a, 6a,
syntheses and characterization of derivatives 2b, 3b, 4b, 4c, 5b,
5c, 6b, 6c, an experimental description of the TUNEL assay, and
various experiments on the interactions of 1a and 6 with DNA.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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