Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase CK2 with antiproliferative activity towards a broad panel of cancer cell lines

Article abstract of DOI:10.1016/j.bbrc.2012.06.068

We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloquinones 6b and 6c, and demonstrated inhibition of th

Full text of DOI:10.1016/j.bbrc.2012.06.068

Biochemical and Biophysical Research Communications 424 (2012) 71–75
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase
CK2 with antiproliferative activity towards a broad panel of cancer cell lines
Claas Hundsdörfer a, Hans-Jörg Hemmerling b, Janina Hamberger c, Marc Le Borgne d, Patrick Bednarski c,
Claudia Götz e, Frank Totzke f, Joachim Jose a,
⇑
a Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Hittorfstraße 58-62, 48149 Münster, Germany
b Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
c Institut für Pharmazie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany
d Institut des Sciences Pharmaceutiques et Biologiques, Université Claude Bernard Lyon 1, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France
e Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Gebäude 44, 66424 Homburg/Saar, Germany
f ProQinase GmbH, Breisacher Str. 117, D-79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2012
Available online 21 June 2012
We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein
kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloqui-
nones 6b and 6c, and demonstrated inhibition of the human CK2 by the compounds. Furthermore, we
showed substantial antiproliferative activity of both compounds towards a broad panel of human cancer
cell lines in the low micromolar range. Whereas the earlier indeno[1,2-b]indoles have been shown to be
selective for CK2, the indeno[1,2-b]indoloquinones 6b and 6c also inhibited the AMPK activated protein
kinase ARK5, potentially contributing to the anti-cancer effects of the compounds. In addition, with com-
pound 6b we found a very potent inhibitor of the leukemia-associated receptor tyrosine kinase FLT3, with
Keywords:
Inhibitors
Protein kinase
CK2
Cancer
ARK5
an IC50 of 0.18 lM.
Ó 2012 Elsevier Inc. All rights reserved.
FLT3
1. Introduction
neurodegenerative diseases and in particular in cancer [7,8]. Its
implication in cancer has been underlined by a number of facts
and in a variety of cancers an elevated activity of CK2 has been re-
ported [9,10]. Furthermore, an induction of apoptosis in cancer
cells by the downregulation of CK2 activity (e.g. by CK2 inhibitors,
Protein kinases act as key regulators of numerous cellular pro-
cesses such as transcription, metabolism, differentiation, cell–cell
communication, migration, cell cycle control and apoptosis. They
mediate most of the signal transduction in eukaryotes by phos-
phorylation of client proteins at their serine, threonine or tyrosine
residues [1]. Except for a small number of dual specificity kinases
that are capable to phosphorylate all three residues, protein ki-
nases are predominantly specific either for serine and threonine
or for tyrosine residues only [2]. The human protein kinase CK2,
earlier reported to be solely a Ser/Thr kinase, is one of these dual
specificity kinases that is also able to phosphorylate tyrosine resi-
dues [3] with an enormous list of over 400 potential substrates [4].
Moreover, it also shows dual cosubstrate specificity, as it is able to
use ATP or GTP as the phosphate group donor [5,6]. CK2 has been
reported to play a part in various diseases as viral infections, par-
asitosis, inflammatory processes, angiogenesis related diseases,
CK2a siRNA) has been shown [11], emphasizing the importance of
the development of CK2 inhibitors for the treatment of cancers.
We recently found that indeno[1,2-b]indoles (Fig. 1A) are po-
tent inhibitors of the human protein kinase CK2, with an IC50 of
0.11 lM for the strongest inhibitor [12]. The earlier prepared inde-
no[1,2-b]indoles have also shown to be moderate inhibitors of the
growth of esophageal cancer cells [12]. Additionally, as recently re-
ported by Kashyap et al., structurally closely related indenoindol-
ones (Fig. 1B) show higher anti-cancer activity than the
chemotherapeutic drugs etoposide and 5-fluorouracil (5-FU) in
kidney cancer cells [13].
To expand the structural diversity of our earlier reported inde-
no[1,2-b]indoles as inhibitors of the human CK2, we prepared two
novel derivatives of the compounds, the quinoid indeno[1,2-
b]indoloquinones (Fig. 2), by a new and convenient synthesis
protocol.
⇑
Corresponding author. Fax: +49 251 8332211.
E-mail addresses: claas.hundsdoerfer@wwu.de (C. Hundsdörfer), h-j.hemmer
ling@web.de (H.-J. Hemmerling), janina-hamberger@gmx.de (J. Hamberger), mar
c.le-borgne@adm.univ-lyon1.fr (M. Le Borgne), bednarsk@uni-greifswald.de
(P. Bednarski), claudia.goetz@uniklinikum-saarland.de (C. Götz), f.totzke@proqina
se.com (F. Totzke), joachim.jose@uni-muenster.de (J. Jose).
We evaluated their potential as inhibitors of the human CK2
and their ability to inhibit the growth of different tumor cell lines.
Furthermore, we investigated their selectivity towards a panel of
23 other kinases.
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.06.068

72
C. Hundsdörfer et al. / Biochemical and Biophysical Research Communications 424 (2012) 71–75
(%) = 339 (10) [M+], 151 (3), 127 (4), 114 (2), 91 (100), 65 (23);
HRMS (EI): m/z [M]+ calcd.: 339.0895, found: 339.0896.
2.2. Testing of inhibitors of the human CK2 [12]
The preparation of the human recombinant CK2 holoenzyme
was performed according to a protocol described earlier [15]. For
testing, 0.5
room temperature for 10 min. Stock solutions of the Inhibitors (in
DMSO) were diluted 1:1000 in 20 l of kinase buffer (50 mM Tris/
HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT) to the final
concentrations. The reaction was started by the addition of 30 l as-
say buffer (25 mM Tris/HCl, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT,
100 M ATP, 0.19 mM peptide substrate (RRRDDDSDDD), 0.6 Ci
32P]ATP). The reaction was performed at 37 °C for 15 min and
ll (0.25 U/ml) of CK2 holoenzyme was incubated at
Fig. 1. (A) Compound 4b as the most potent CK2 inhibitor of the indeno[1,2-
b]indole type. (B) A structurally closely related indenoindolone with higher anti-
cancer potency than 5-FU and etoposide.
l
l
l
l
[c-
subsequently spotted onto a P81 ion exchange paper (Whatman).
After washing with excess phosphate (three times with 85 mM
H3PO4) and subsequently with ethanol, the filter was dried and
radioactivity measured by a scintillation counter (Packard). For
IC50 determination, inhibition was measured at ten final concentra-
tions from 0.01 to 30 lM in triplicate in independent experiments.
Fig. 2. The two novel indeno[1,2-b]indoloquinones 6b and 6c.
IC50 were calculated from the resulting dose-response curves.
2. Materials and methods
2.3. Cell culture
2.1. Synthesis
As previously described [16,17], measurement of growth inhibi-
tion of adherent cell lines was performed by the use of Crystal Vio-
let staining in 96-well microtiter plates. Eight human cancer cell
lines (5637, SISO, KYSE-70 used in primary testing, additionally
MCF-7, DAN-G, LCLC, A427, RT-4 used in secondary testing) were
obtained from the DSMZ (German Collection of Microorganisms
and Cell Cultures). These were grown in RPMI 1640 medium con-
taining 10% FCS (Sigma), supplemented with penicillin G and strep-
tomycin. Cells were incubated for 96 h with the compounds in
DMF with a final solvent concentration of 0.1% in each well. Prein-
cubated cells were fixed (20 min, 1% glutaraldehyde) and subse-
quently stained for 30 min with 0.02% Crystal Violet in water.
After washing for 30 min in water, the remaining cell-bound dye
was redissolved with ethanol/water (7:3) and the absorption at
570 nm was measured with an Anthos 2010 plate reader (Anthos,
Salzburg, Austria).
2.1.1. General
Melting points: Büchi melting point apparatus, uncorrected.
NMR spectra: Bruker AC 200F (1H: 200 MHz/13C: 50 MHz) with
TMS as the internal standard using d (ppm) scale. IR spectra: Perkin
Elmer FT-IR 1600. Mass-spectra: Finnigan 4200 quadrupole mass-
spectrometer, equipped with a MASPEC data system. HRMS: Bru-
ker micrOTOF-Q II (APCI) and Finnigan MAT 8200 (EI). MPLC was
performed on 230–400 mesh silica (Merck) with EtOAc as the elu-
ent. Solvents were purified by standard methods and dried over
molecular sieves. Yields refer to the amount of the products after
one recrystallization and are not optimized. Synthesis and charac-
terization of the precursors 4 and 5 has been published earlier
[12,14].
2.1.2. 5-Isopropyl-5H-indeno[1,2-b]indole-6,9,10-trione 6b
2.4. Selectivity studies
Compound 5b (1.5 g, 5.4 mmol) was dissolved in dry DMF
(100 ml) and Salcomine (80 mg, Merck) was added. The mixture
was treated with dry O2 at 70 °C. After 8 h the mixture was filtered
and the filtrate evaporated to dryness. The residue was dissolved in
CHCl3 and purified by MPLC to give 6 as a red powder (2.4 mmol,
45%). Mp: 254 °C (CH3CN); 1H NMR (200 MHz, DMSO-d6):
d = 1.70 (d, J = 7 Hz, 6H, 2CH3), 5.6–6.1 (broad, 1 H, CH), 6.62,
6.63 (AB, 2H, H-7, H-8), 7.25–7.66 ppm (m, 4H, Ar-H); 13C NMR
(50 MHz, CDCl3): d = 21.8, 51.1, 122.4, 125.5, 130.8, 134.1, 134.5,
135.0, 136.4, 138.7, 140.9, 155.6, 178.8, 182.5, 184.2 ppm; IR
A radiometric protein kinase assay (33PanQinaseÒ Activity As-
say, ProQinase, Freiburg, Germany) was used for measuring the ki-
nase activity as described by Aly et al. [18]. Therefore, inhibition
was measured in a total reaction volume of 50
Plates. The reaction was composed of 20 l assay buffer, 5
ous ATP solution, 5 l inhibitor (dissolved in 10% DMSO), 10
purified recombinant human protein kinase and 10 l of the sub-
strate. The reaction for all enzymes contained 60 mM HEPES-
NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 M Na-orthovanadate,
1.2 mM DTT, 50 g/ml PEG20000, and 1 M [
33P]ATP (approx.
l
l in 96-well Flash-
l
ll aque-
l
ll
l
l
(KBr):
m
= 1715, 1662, 1649, 1599, 1518, 1441, 1289 cmÀ1; EI-MS
l
l
c-
(EI, 70 eV): m/z (%) = 291 (57)[M+], 276 (10), 249 (100), 221 (21),
195 (10); HRMS (APCI): m/z [M + 1]+ calcd: 292.0968, found:
292.0970.
5 Â 105 cpm/well). Substrates used for the enzymatic reactions
were as follows: GSK3(14-27) for AKT1; tetra(LRRWSLG) for Aur-
ora A and B; MEK1 KM for B-RAF; Histone H1 for CDK2/CycA;
RB-CTF for CDK4/CycD1; Poly(E,Y)4:1 for EGF-R, EPHB4, ERBB2,
FAK, IGF1-R, SRC, VEGF-R2, VEGF-R3 and TIE2; Poly(A,E,K,Y)6:2:5:1
for FLT3, INS-R, MET and PDGFR-b; Casein for PLK1; p38-alpha-
KRKR for SAK; Autophosphorylation was used for ARK5 and COT.
Protein kinases were expressed as His-tagged or GST-tagged fusion
proteins in Sf9 insect cells and purified by affinity chromatography.
Reactions were incubated for 80 min at 30 °C. The reactions were
2.1.3. 5-Benzyl-5H-indeno[1,2-b]indole-6,9,10-trione 6c
Preparation according to 6b with 5c. Yield: 62%; mp: 224–
226 °C (EtOH); 1H NMR (200 MHz, CD2Cl2): d = 5.84 (s, 2H, CH2),
6.54, 6.59, 6.60, 6.65 (AB, 2H, H-7, H-8), 7.1–7.6 ppm (m, 9H, Ar-
H); 13C NMR (50 MHz, CDCl3): d = 50.7, 119.6, 120.8, 122.7, 124.7,
126.6, 128.4, 129.2, 130.3, 133.4, 133.5, 133.7, 134.8, 136.3,
137.2, 139.8, 155.8, 178.1, 181.6, 183.4 ppm; IR (KBr):
m
= 1719,
quenched with 50
rated and washed two times with 200
l
l of 2% (v/v) phosphoric acid, plates were aspi-
1665, 1646, 1526, 1461, 1386, 1252 cmÀ1; EI-MS (70 eV): m/z
ll of 0.9% (w/v) NaCl or

C. Hundsdörfer et al. / Biochemical and Biophysical Research Communications 424 (2012) 71–75
73
200 l
l H2O. Incorporation of 33Pi was measured with a microplate
Table 1
Inhibition of the human CK2 by indeno[1,2-b]indoloquinones.
scintillation counter (Microbeta Trilux, Wallac).
Compound
Ra
IC50 (l
M)b
6b
6c
CH(CH3)2
CH2C6H5
5.05
1.49
3. Results and discussion
a
According to Fig. 3.
Determinations were performed in triplicate in independent
As described earlier, we prepared the precursors 4 and 5 by a con-
venient straightforward synthesis protocol [12,14]. Condensation of
indanetrione hydrate 1 with the 3-(isopropylamino)cyclohex-2-
enones 2 gave the vic-dihydroxyindeno[1,2-b]indoles 3, which were
then converted to the 5-isopropy-5,6,7,8-tetrahydro-indeno[1,2-
b]indole-9,10-diones 4 by the means of tetramethyl-thionylamide
(TMTA). The 9-Hydroxy-5H-indeno[1,2-b]indole-10-ones 5 were
then prepared by the oxidation of 4 with 2,3-dichloro-5,6-dicy-
ano-1,4-benzoquinone (DDQ) in 1,4-dioxane [12], with subsequent
oxidation with molecular oxygen in the presence of Salcomine [19]
to give the 5H-indeno[1,2-b]indole-6,9,10-triones 6 (Fig. 3).
b
experiments.
tumor cell lines. This included two bladder cancer cell lines
(5637 and RT-4), a cervical cancer (SISO), an esophagus cancer cell
line (KYSE-70), a breast cancer cell line (MCF-7), a pancreas line
(DAN-G) and two lung cancer lines (LCLC and A427). Measure-
ments were performed at five different inhibitor concentrations.
GI50 were obtained by a linear least-squares regression of the
growth values vs. the logarithm of the inhibitor concentration (Ta-
ble 2).
Two novel indeno[1,2-b]indoloquinones 6 were synthesized
and tested for their inhibition of the human CK2 (Table 1). For
IC50 determination, inhibition was measured at final concentra-
6b showed very good activity towards six out of eight cell lines
with GI50 of 1.3 to 1.7
A427), whereas DAN-G and RT-4 were unaffected by this com-
pound (GI50 > 10 M). The same growth inhibition pattern was ob-
tained with compound 6c, but with somewhat higher GI50 ranging
from 2.8–4.1 M with tumor cell lines 5637, SISO, KYSE-70, MCF-7,
LCLC and A427, respectively. No activity was seen towards tumor
cell lines DAN-G and RT-4 (GI50 > 20 M).
Due to the unaffected growth of some cell lines (GI50 > 10
lM (5637, SISO, KYSE-70, MCF-7, LCLC,
tions ranging from 0.01 to 30 lM in appropriate intervals and
l
IC50 were calculated from the resulting dose-response curves. Pure
DMSO was used as negative control (0% inhibition); reactions de-
void of CK2 were used as positive control (100% inhibition). For
l
the known inhibitor Emodin, with a reported IC50 of 0.89
lM
l
[20], we determined an IC50 of 0.46 M. Both new compounds
l
lM) a
showed inhibitory activity towards the recombinant CK2 holoen-
general cytotoxic effect of the compounds (e.g. by ROS formation or
Michael type reactions due to the quinoid substructure [21]) ap-
pears to be unlikely and mechanisms specific to tumor growth like
kinase inhibition seem to account for the observed inhibitory activ-
ity. However, we did not see a clear correlation between the degree
of inhibition of the CK2 by the compounds and the antiproliferative
zyme. The N-isopropyl substituted indeno[1,2-b]indoloquinone
6b revealed an IC50 of 5.05
was three times more potent with an IC50 of 1.49
l
M and the N-benzyl derivative 6c
M.
l
For further evaluation, synthesized compounds were investi-
gated in cell proliferation experiments. For primary testing, rela-
tive growth inhibition of three adherent tumor cell lines (5637,
effects. The weaker inhibitor of CK2 (6b, with an IC50 of 5.05
was the more potent inhibitor of growth in the cancer cell lines
(GI50 of 1.3–1.7 M). In contrast, the stronger inhibitor of CK2
(6c, with an IC50 of 1.49 M) was a weaker inhibitor of cell growth
in the cancer lines tested (GI50 of 2.8–4.1 M).
lM)
SISO and KYSE-70) at a concentration of 20 lM was evaluated.
We previously reported that the indeno[1,2-b]indole 4b is a strong
CK2 inhibitor but only a moderate inhibitor of the growth of the
esophageal KYSE-70 line (25% inhibition at 20 lM) [12]. In con-
l
l
l
trast, we found in this study that the indeno[1,2-b]indoloquinones
To exclude a difference in the cell permeability of the com-
pounds as the cause for the lower antiproliferative effects of 6c,
we predicted permeability properties by three different ap-
proaches. Fragment-based partition coefficients (ClogP) and topo-
showed nearly complete inhibition of growth (88–100%) of the
three tumor lines at a concentration of 20 lM.
We then determined the GI50 (concentration required for 50%
inhibition of growth) of these compounds towards eight adherent
Fig. 3. Preparation of the indeno[1,2-b]indoloquinones 6b and 6c. For R refer to Table 1. Reagents and conditions: (i) MeOH, r.t., 3–8 h; (ii) DMF, acetic acid, TMTA, r.t., 3 h; (iii)
DDQ, 1,4-dioxane, 65–70 °C, 24–72 h; (iv) O2, Salcomine, DMF, 70 °C, 8 h.

74
C. Hundsdörfer et al. / Biochemical and Biophysical Research Communications 424 (2012) 71–75
Table 2
potent inhibitor of growth in the cancer cell lines (GI50 of
1.3–1.7 M). Correspondingly, the weaker inhibitor of ARK5 (6c,
with an IC50 of 3.7 M) inhibited the growth of the cancer lines
to the same extent (GI50 of 2.8–4.1 M). Thus, ARK5 seems to be
Determination of GI50 in cell culture.
l
l
6b
6c
l
Cell line
GI50 in l
Ma
a potential candidate contributing to the effects observed in the tu-
mor lines. ARK5 is a family member of the AMP activated Ser/Thr
protein kinases (AMPKs) and is activated by the oncogene Akt
[28]. Little is reported about the protein kinase ARK5, but it has
been shown by Suzuki and coworkers to be involved in tumor
malignancy through the repression of caspase 8 activity, resulting
in the prevention of cell death [29]. Compared to the other tested
bladder cell line 5637, decreased expression of apoptosis-related
genes in RT-4 has been shown [30]. This would explain the insen-
sitivity of the bladder cancer cell line RT-4 towards the inhibitors.
Besides its role in the prevention of cell death, it was suggested
that ARK5 plays a role in metastasis [31], and ARK5 mediated inva-
sion of cancers has been shown [32], making this kinase an attrac-
tive target for the development of novel therapeutics for the
treatment of cancers.
5637
SISO
KYSE-70
MCF-7
DAN-G
LCLC
1.7 0.4
1.5 0.2
1.5 0.4
1.3 0.1
>10
1.5 0.2
1.4 0.2
>10
2.8 0.6
2.8 0.6
2.8 0.3
3.3 0.4
>20
4.1 1.0
3.7 0.7
>20
A427
RT-4
a
Given is the concentration for growth inhibition of
50% of the corresponding cell line SD. GI50 are the
average of three to seven independent experiments.
Table 3
ClogP, TPSA and Caco-2 cell permeability coefficients of the tested compounds.
ClogPa
TPSAa in Å2
PCaco-2 in nm/s
b
The insensitivity of the pancreatic adenocarcinoma line DAN-G
towards the compounds could be due to a high expression of the
multidrug resistance associated protein 5 (MRP5). A significant
correlation between the expression of MRP5 in eight pancreatic
adenocarcinoma cell lines (Colo-357, T3M4, Aspc-1, DAN-G,
Panc-1, Patu-89O2, Patu-8988T and Patu-8988S) and the sensitiv-
ity of these cell lines towards the chemotherapeutic agent 5-fluo-
rouracil (5-FU) has been reported. DAN-G showed the lowest
sensitivity towards 5-FU while expressing high levels of MDR5
[33]. This would explain the insensitivity of DAN-G cells towards
the inhibitors.
6b
6c
2.9
3.7
56.1
56.1
13
22
a
Calculated by the molinspiration web services [22].
Calculated by the PreADMET webserver [23].
b
logical polar surface areas (TPSA) were calculated using the Molin-
spiration web services [22] and permeability coefficients towards
Caco-2 cells (PCaco-2) were determined with the PreADMET web-
server (Table 3) [23].
The data suggest that there are only minor differences in the
membrane permeability of the compounds. Low membrane per-
meability is predicted for any compounds with ClogP P 5 [24],
TPSA P 140 Å2 [25,26] and PCaco-2 < 4 nm/s [27], which means that
both derivatives should be able to permeate cell membranes. Com-
pared to 6b with a PCaco-2 of 13 nm/s, an even higher PCaco-2 of
22 nm/s is predicted for the less active derivative 6c, making it un-
likely that membrane permeability properties are the cause for the
differing effects of the two compounds in cell culture.
To evaluate potential inhibition of other kinases beyond CK2,
we tested the compounds in a selectivity study towards a panel
of 23 different kinases (Table 4).
Seven kinases (ARK5, FLT3, SRC, VEGF-R2, VEGFR-3, INS-R and
SAK) were inhibited by the indeno[1,2-b]indoloquinone 6b with
With an IC50 of 0.18 lM, 6b is also a potent inhibitor of the hu-
man receptor tyrosine kinase FLT3. The inhibition of FLT3 does not
explain the antiproliferative effects of the compounds due to two
reasons: firstly, derivative 6c strongly inhibited the growth of the
cancer cell lines, but did not inhibit the kinase FLT3 (IC50 > 20 lM)
and secondly, expression of FLT3 in human cancers has been
shown to be restricted to cancer cells of the hematopoietic system
[34]. The human FLT3 (FMS-like tyrosine kinase 3) belongs to the
family of subclass III receptor tyrosine kinases, along with the
platelet-derived growth factor receptor (PDGFR), the steel factor
receptor (cKIT) and the macrophage colony-stimulating factor
receptor (M-CSFR also named FMS) [35]. The receptor tyrosine
kinase FLT3 plays a crucial role in hematopoietic malignancies.
Elevated levels of FLT3 have been found in 100% of patients with
B-lineage acute lymphoid leukemia (B-lineage ALL), in 88%
with acute myeloid leukemia (AML) and in 25% with T-cell acute
lymphoid leukemia (T-ALL) [36]. Gain of function mutations of
FLT3 are most frequently in AML patients [37], which are found
in 25% [35] to 35% [38] of all cases. Although FLT3 was recognized
as a potential target for the treatment of AML a decade ago [39],
few potent and selective inhibitors are available, with exceptions
being MLN-518 and AC220 [40]. Thus, compound 6b seems to be
an interesting starting point for the development of further FLT3
inhibitors for the treatment of acute leukemias.
IC50 below 10
IC50 of 0.57
0.18 M was found. Compound 6c inhibited 5 kinases (ARK5,
EGF-R, IGF1-R, SRC and VEGF-R2) with IC50 below 10 M. The
strongest inhibition was observed with ARK5 and SRC with IC50
of 3.7 and 2.4 M, respectively.
l
M. Thereof, ARK5 was inhibited with a considerable
l
M and for FLT3 an even three times lower IC50 of
l
l
l
Besides inhibition of CK2, inhibition of ARK5 by the indeno[1,2-
b]indoloquinones could contribute to the antiproliferative effects
of the compounds in the cell culture experiments. The stronger
inhibitor of ARK5 (6b, with an IC50 of 0.57 lM) was the more
Table 4
Inhibition of 23 different protein kinases by indeno[1,2-b]indoloquinones 6b and 6c.
Kinase
IC50
AKT1
ARK5
Aurora-A
Aurora-B
B-RAF
CDK2/CycA
CDK4/CycD1
EGF-R
EPHB4
ERBB2
FAK
FLT3
(
l
M)
6b
6c
n.i.a
n.i.
0.57
3.7
20
n.i.
19
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
7.4
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
0.18
n.a.
Kinase
IC50
IGF1-R
SRC
VEGF-R2
VEGF-R3
INS-R
MET
PDGFR-b
PLK1
SAK
TIE2
COT
(
l
M)
6b
6c
10
7.2
5.6
2.4
6.4
9.6
3.3
n.i.
5.5
19
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
5.9
12
11
14
n.i.
13
a
n.i.: no inhibition, IC50 > 20 lM.

C. Hundsdörfer et al. / Biochemical and Biophysical Research Communications 424 (2012) 71–75
75
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In conclusion, we prepared two novel indeno[1,2-b]indoloqui-
nones with considerable inhibitory activity towards the human
protein kinase CK2. The compounds showed significant antiprolif-
erative activity towards a broad panel of human tumor cell lines.
Besides the inhibition of CK2, both compounds inhibited the hu-
man protein kinase ARK5, which may be an additional cause for
the antiproliferative effects of the compounds. In addition, com-
pound 6b turned out to be a strong inhibitor of the human receptor
tyrosine kinase FLT3. The results indicate that indeno[1,2-b]indolo-
quinones are a valuable starting point for the development of
CK2-inhibitors with antiproliferative activity towards a variety
of cancer cells. Additionally, the indeno[1,2-b]indoloquinone 6b,
could provide a new lead structure for future FLT3 inhibitors for
the treatment of acute leukemias. Further derivatives of the inde-
no[1,2-b]indoloquinones will be synthesized and tested for inhibi-
tion of the human protein kinase CK2 and their antiproliferative
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Acknowledgments
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