Halogenated naphthochalcones and structurally related naphthopyrazolines with antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2016.09.076

Three 3-(3-halo-4,5-dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-ones 1 and three structurally related 2-pyrazolines 2 were prepared and assessed in vitro for anticancer activity. The chalcones 1 were antiproliferative with low double-digit micromolar IC

Full text of DOI:10.1016/j.bmcl.2016.09.076

Accepted Manuscript
Halogenated naphthochalcones and structurally related naphthopyrazolines
with antitumor activity
Florian Schmitt, Heidrun Draut, Bernhard Biersack, Rainer Schobert
PII:
S0960-894X(16)31025-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.076
BMCL 24300
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
28 July 2016
28 September 2016
29 September 2016
Please cite this article as: Schmitt, F., Draut, H., Biersack, B., Schobert, R., Halogenated naphthochalcones and
structurally related naphthopyrazolines with antitumor activity, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.076
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Halogenated naphthochalcones and structurally related naphthopyrazolines with
antitumor activity
Florian Schmitta, Heidrun Drauta, Bernhard Biersacka, and Rainer Schoberta, *
aOrganic Chemistry Laboratory, University Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany
ARTICLE INFO
ABSTRACT
Article history:
Three 3-(3-halo-4,5-dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-ones 1 and three structurally
related 2-pyrazolines 2 were prepared and assessed in vitro for anticancer activity. The
chalcones 1 were antiproliferative with low double-digit micromolar IC50 values against six
tumor cell lines whereas the pyrazolines 2 showed low single-digit micromolar IC50 values
against this panel. The pyrazolines inhibited ATP-binding cassette efflux transporters of types P-
gp and BCRP while the chalcones inhibited selectively BCRP. All test compounds induced an
accumulation of HT-29 colon carcinoma cells in the G2/M phase of the cell cycle and they
interfered with the microtubule and F-actin dynamics, but only the chalcones induced apoptosis
in 518A2 melanoma cells after 24 h.
Keywords:
Chalcones
Pyrazolines
BCRP
2016 Elsevier Ltd. All rights reserved
.
P-gp
TUNEL
A critical hurdle to a successful chemotherapy is multidrug
resistance (MDR) of cancer cells which may be inherent or
acquired during previous lines of therapy. The energy-dependent
efflux of xenobiotics, mediated by adenosine triphosphate-
binding cassette (ABC) transporters, plays a major role in the
process of cellular resistance to chemotherapeutics.1 An
overexpression of these transporters in tumor cells decreases the
efficacy of drugs by reducing their intracellular concentration.
The new chalcones 1a and 1c were obtained as colorless solids
from a Claisen-Schmidt condensation of 2-acetylnaphthalene and
3,4-dimethoxy-5-chloro-benzaldehyde or 3,4-dimethoxy-5-iodo-
benzaldehyde, respectively (Scheme 1).6 The synthesis of the
chalcones 1b and 1d was reported previously.7,8,9 The new
acetylpyrazolines 2a-c were prepared by reaction of 1a-c with
hydrazine hydrate in acetic acid and obtained as colorless solids
(Scheme 1).6
The multidrug resistance-associated protein
1 (MRP1), P-
The effect of the chalcones 1a-d and of the 2-pyrazolines 2a-c on
the growth of cancer cells of five different entities was assessed
using the MTT assay.10 All compounds showed dose-dependent
cell growth inhibition against the entire panel of cell lines (Table
1). The chalcones featured low double-digit micromolar IC50
concentrations with the 3-halophenyl chalcones 1a-c being on
average slightly more active than the trimethoxyphenyl derivative
1d. It is known that 3,4-dimethoxy substituted chalcones inhibit
ABC efflux transporters of the BCRP type.2 Hence, it is not
surprising that the multidrug-resistant cancer cell line MCF-
7/Topo, which overexpresses these particular efflux pumps,11 was
approximately 10-times more sensitive to the chalcones than the
other cell lines, with IC50 values in the low single-digit
glycoprotein (P-gp), and the breast cancer resistance protein
(BCRP) are the most important ABC-transporters involved in
MDR.2 Effective and selective ABC-transporter inhibitors can
help to restore the impact of anticancer drugs in MDR tumors. A
good deal of 1,3-diphenyl-prop-2-en-1-ones (a.k.a. chalcones),
precursors in the biosynthesis of flavonoids3, are known to inhibit
ABC-transporters.1,2,4 We recently reported a combretastatin A4
derived chalcone and its platinum complex that inhibited BCRP
and P-gp.4 Structure–activity relationship (SAR) studies by
others showed that chalcones bearing basic functional groups are
likely to be P-gp inhibitors,5 whereas non-basic chalcones
displayed no P-gp inhibition but selectively inhibited BCRP.2 A
strong BCRP inhibition was noted for naphthylchalcones with a
chloro substituted 3-phenyl ring. Moreover, chalcones may also
exert other cancer-relevant effects, including apoptosis induction,
antiproliferative effects, and an interference with the cell cycle
progression.3 Herein, we report on a series of halogenated
naphthylchalcones 1 and structurally related 2-pyrazolines 2, on
their potential as selective ABC-transporter inhibitors, and on
further anticancer properties.
micromolar
range.
The
3-halophenyl
substituted
napthylpyrazolines 2 were more active compared with their
chalcone congeners. They showed low single-digit micromolar
IC50 values against all tested cancer cell lines including the
multidrug-resistant HT-29 colon, MCF-7/Topo breast, and KB-
V1/Vbl cervix carcinoma cells.
*corresponding author. e-mail: Rainer.Schobert@uni-bayreuth.de

Scheme 1. Synthesis of chalcones 1a-d and of acetylpyrazolines 2a-c. Reagents and conditions: (i) NaOH, MeOH/H2O, r.t., 16 h, 80-
85%; (ii) N2H4 × H2O, AcOH, reflux, 3 h, 32-71%.
Table 1. Inhibitory concentrations IC50 (µM, 72 h) of compounds 1a-d and 2a-c when applied to human cancer cell lines.a
Cell line /
compound
HT-29
HCT-116
518A2
MCF-7/Topo
Panc-1
KB-V1/Vbl
1a
1b
1c
1d7
2a
2b
2c
23.9 2.5
22.8 4.8
13.3 1.3
28.1 7.4
3.5 0.1
2.5 0.1
2.3 0.6
12.9 0.7
11.2 1.3
14.4 1.3
13.7 2.7
3.8 0.2
2.7 0.2
1.6 0.1
16.4 1.6
20.2 2.5
18.1 1.3
22.9 5.6
4.6 0.6
3.8 0.9
3.8 0.5
1.5 0.4
2.3 0.3
1.3 0.4
5.6 1.0
2.1 0.1
2.6 0.4
2.5 0.4
10.3 0.2
9.3 0.2
10.4 0.1
13.3 0.4
3.1 0.1
1.5 0.1
1.1 0.1
13.1 1.7
10.5 0.7
16.5 1.0
13.9 2.4
4.7 0.2
2.2 0.1
1.9 0.1
a
Human cancer cell lines: HT-29 and HCT-116 colon carcinomas, 518A2 melanoma, MCF-7/Topo breast cancer adenocarcinoma, Panc-1 pancreatic ductular
adenocarcinoma and KB-V1/Vbl cervix carcinoma. Values are the means SD determined in four independent experiments and derived from dose-response
curves (percentage of viable cells relative to untreated controls) after 72 h incubation using the MTT assay.
The chalcones 1a-d and the pyrazolines 2a-c were then screened
for inhibition of the ABC-transporters P-gp/ABCB1 and
BCRP/ABCG2 by means of the calcein-AM accumulation assay
in KB-V1/Vbl cells and the mitoxantrone accumulation assay in
MCF-7/Topo cells, respectively (Table 2).12,13 MCF-7/Topo
breast cancer cells, which overexpress BCRP, were treated with
the test compounds (1a-d, 2a-c; 0-100 µM) or the known BCRP
inhibitor fumitremorgin C (0.1-100 µM) for 30 min in the
presence of the fluorescent BCRP substrate mitoxantrone. The
mitoxantrone fluorescence intensity was taken as a measure of
the degree of BCRP inhibition and used to obtain dose-response
curves which allow the determination of IC50 values. The
halophenyl chalcones 1a-c and their pyrazoline analogues 2a-c
proved to be equally effective inhibitors of BCRP on average
with IC50 values in the range of 5–9 µM, while the trimethoxy-
substitued chalcone 1d was two to three times more active. These
findings are in line with the results of the MTT assay and the
lower IC50 values of the chalcones against MCF-7/Topo breast
cancer cells compared with the other tested cell lines. P-gp
overexpressing KB-V1/Vbl cervix carcinoma cells were also
treated with the test compounds (1a-d, 2a-c; 0-500 µM) or the P-
gp inhibitor verapamil (0.1-500 µM) for 15 min in the presence
of the non-fluorescent P-gp substrate calcein acetoxymethyl ester
(calcein-AM). Inhibition of P-gp impedes the efflux of calcein-
AM and allows esterases to hydrolyze calcein-AM to give the
intensely fluorescent calcein which is not a P-gp substrate
anymore and thus accumulates in the cytosol.14 The calcein
fluorescence intensity as a measure of P-gp inhibition was used
to calculate the IC50 values of the test compounds. In line with
the weaker antiproliferative activities of the halophenyl chalcones
1a-c against KB-V1/Vbl cancer cells in the MTT assays,
compared with those of their 2-pyrazoline analogues, no P-gp
inhibition was observed for the former, whereas the inhibitory
activities of the latter even exceeded that of the established P-gp
inhibitor verapamil, clinically used to re-sensitize resistant
tumors.15 Interestingly, the selectivity for BCRP was lost when
the substituent in the meta position of the phenyl ring was
changed from halide (1a-c) to methoxy (1d). In terms of IC50

values, 1d and the 2-pyrazolines 2a-c were on
five times better inhibitors of P-gp when compare
a
a
verage four to
d to verapamil.
d
Table 2. Concentrations IC50 [µM]a for the inhi
and P-gp transporters by the test compounds 1a-d and 2a-c, the
bbition of BCRP
specific BCRP inhibitor fumitremorgin C, and t
inhibitor verapamil.
h
h
e specific P-gp
BCRP
P-gp
-
0.96 0.24b
fumitremorgin C
-
65.6 10.1
>500
verapamil
8.7 1.3
4.9 0.3
7.8 1.6
2.4 0.5
7.4 0.3
8.9 1.9
7.1 1.8
1a
1b
1c
1d
2a
2b
2c
Figure 2. Flow cytometric analyses of apoptosis in 518A2
melanoma cells visualized by TUN L technique. Cells were
treated with vehicle (DMSO), 25 µM oof 1a or 1d, or 7.5 µM of
2a for 24 h and stained by transfer se-mediated fluorescein-
dUTP nick end labeling of 3’-OH ends oof fragmented DNA.
>500
EE
>500
a
1
1
3.0 1.6
2.1 0.1
2.6 1.6
6.3 1.4
11
11
11
Since the G2/M arrest might be induc
eed by the test compounds’
interference with the tubulin dynamic
c
ss causing a retarded cell
a
determined in MCF-7/Topo breast cancer cells after 30 min or in KB-
V1-V1/Vbl cervix carcinoma cells after 15 min ex osure to the
compounds. The results are the mean SD of thr e independent
division and mitotic progression, we innvestigated the effects of
the test compounds on the cytoskelletal organization of the
p
e
RR
b
microtubules and of the microfilam
ee
nts (F-actin) in 518A2
experiments and derived from dose-response curves.
independent experiments.
esults of two
melanoma cells. The distribution of mi
i
ccrotubules in 518A2 cells
treated with 1a, 1d or 2a for 24 h did not differ much from
untreated controls when visualized by immunofluorescence (cf.
Supplementary Data). In contrast, a mooderate inhibitory effect of
the test compounds 1a, 1c, 2a and 2c wwas observed in a cell-free
polymerization assay with purified pig brain tubulin (cf.
Supplementary Data). This was further confirmed by quantifying
the fractions of intact microtubules in treated 518A2 melanoma
cells (Fig. 3). Any interference of the test compounds with the
tubulin dynamics increases the proporrtion of detergent-soluble
tubulin heterodimers which are partt of the supernatant, in
contrast to the intact tubulin polymerss which can be separated
from the former fraction by centrifuggation. In line with these
results, the quantification of the intactt microtubule fraction by
Western blot analyses confirmed a significant reduction of this
fraction with increasing concentrations oof 1c and 2c.
The interference of the test compounds 1a-d an
cell cycle progression of HT-29 colon carcin
determined by flow cytometry using propidium iodide staining.
Treatment of the cells with 15 µM of th chalcones or
d
d
2a-c with the
ooma cells was
ee
pyrazolines led to a significant accumulation of cells in G2/M
phase of the cell cycle whereas the population of cells in G1-
phase was drastically reduced (Fig. 1). This efffect was more
pronounced for the pyrazolines.
The extent of apoptosis in 518A2 melanoma c
ells treated with
1a, 1d, or 2a for 24 h, was analyzed by flow cytoometry using the
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end
labeling) technique (Fig. 2) to visualize DN
typical of apoptosis. Interestingly, apoptosis was oonly induced by
A
fragmentation
the chalcones 1a and 1d, whereas cells tr
pyrazoline 2a did not differ from untreated contr
e
ated with the
ol cells. These
findings were confirmed by DNA ladder assays using agarose gel
electrophoresis to visualize the DNA ladder patttern typical of
apoptosis (cf. Supplementary Data).
Figure 3. Decrease of intact tubullin polymers in 518A2
melanoma cells upon treatment with 11c or 2c. A) Fractions of
detergent-insoluble 518A2 cell lysatees, treated for 4 h with
various concentrations of 1c or 2c, were subjected to SDS-PAGE
and the tubulin content was visualizedd by immunoblotting for
alpha-tubulin (55 kDa). B) Densitomeetric analyses of Western
blots from at least two independent eexperiments (mean SD)
Figure 1. Percentage of HT-29 colon carcinomaa cells in G1, S
and G2/M cell cycle phases and the proportion of dead cells (sub-
G1) as determined by flow cytometry. Cells were treated with 15
µM of 1a-d, or 2a-c, or DMSO (control cells) f
are the means SD of three independent experim
o
o
r 24 h. Values
were used to quantify the concentraation-dependent levels of
tubulin polymers in detergent-insoluble lysate fractions.
e

In addition, we investigated potential morphological changes of
the F-actin cytoskeleton of 518A2 melanoma cells upon
treatment with compounds 1a, 1d or 2a for 24 h. We observed
the formation of stress fibers traversing the whole cell body,
which is a typical stress response of cells to exposure to tubulin-
binding agents (Fig. 4). Untreated control cells showed the
normal cortical actin fiber network with tenuous filaments across
the cells and a greater number of intercellular connections.
Transferase, Topo
desoxyribonucleotide Transferase-mediated dUTP Nick End
Labelling, Vbl - Vinblastin.
–
Topotecan, TUNEL
–
Terminal
Supplementary data. Details of the syntheses and physical data
of compounds 1a, 1c, 2a-c, on cell culture conditions, biological
assays (MTT, mitoxantrone, calcein-AM, TUNEL, fluorescence
labeling of F-actin, cell cycle analysis, DNA fragmentation,
fluorescence labeling of microtubules, in vitro tubulin
polymerization, quantification of polymeric tubulin fractions) can
be found, in the online version, at doi:xxxx.
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Graphical Abstract
Halogenated naphthochalcones and structurally
related naphthopyrazolines with antitumor
activity
Leave this area blank for abstract info.
Florian Schmitt, Heidrun Draut, Bernhard Biersack, and Rainer Schobert

Highlights
Halogenated naphthochalcones and structurally related naphthopyrazolines
with antitumor activity
Highlights
•
•
•
•
3-Halophenyl-1-naphthylchalcones 1 are active against cancer cells
Chalcones 1 selectively inhibit BCRP transporters
Analogous pyrazolines 2 are more cytotoxic and inhibit BCRP and P-gp
Compounds 1 and 2 interfere with the dynamics of microtubules and F-actin