Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.02.017

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC₅₀ in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)- dione) inhibited human CK2 with an IC₅₀ of 0.11 μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a K _i of 0.06 μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.

Full text of DOI:10.1016/j.bmc.2012.02.017

Bioorganic & Medicinal Chemistry 20 (2012) 2282–2289
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase
CK2 inhibitors
Claas Hundsdörfer a, Hans-Jörg Hemmerling b, Claudia Götz c, Frank Totzke d, Patrick Bednarski e,
Marc Le Borgne 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 Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Gebäude 44, D-66424 Homburg/Saar, 66421 Homburg, Germany
d ProQinase GmbH, Breisacher Str. 117, 79106 Freiburg, Germany
e Institut für Pharmazie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany
f Institut des Sciences Pharmaceutiques et Biologiques, Université Claude Bernard Lyon 1, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of
potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a conve-
nient and straightforward synthesis protocol. The compounds were tested for inhibition of human
protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC50 in
the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroinde-
Received 25 November 2011
Revised 31 January 2012
Accepted 4 February 2012
Available online 13 February 2012
no[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC50 of 0.11
cantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive
inhibition by compound 4b was shown and a Ki of 0.06 M was determined. Our findings indicate that
indeno[1,2-b]indoles are a promising starting point for further development and optimization of human
protein kinase CK2 inhibitors.
lM and did not signifi-
Keywords:
Indeno[1,2-b]indoles
Inhibitors
Human protein kinase
CK2
l
Ó 2012 Elsevier Ltd. All rights reserved.
Cancer
1. Introduction
colon, breast and lung,7 and its impact in cell survival and neopla-
sia is supported by a growing amount of evidence.7,8
Protein kinases are of high importance in metabolic and regula-
tory processes. Usually, peptides or proteins are phosphorylated by
kinases at the hydroxyl groups of serine, threonine, or tyrosine res-
idues. Protein kinases play major roles in signal transduction and
control fundamental cellular functions such as cell–cell-communi-
cation, differentiation, migration, the cell cycle and apoptosis.1
Human CK2 is a second-messenger- and phosphorylation inde-
pendent constitutively active S/T protein kinase, with a growing
substrate list of more than 400 potential physiological targets
reported in the literature up to date.2 CK2 is a heterotetrameric
In the last three decades numerous inhibitors addressed to the
target CK2 have been identified: One of the first reported inhibitors
dichlororibobenzimidazole (DRB) showed a rather weak IC50 of
15 l
M.9 Tetrabromobenzotriazole (TBB) revealed an IC50 of
1.6
l
M10 and the anthraquinone emodin was reported to have an
M.11 Further inhibitors with IC50 in the upper sub-
IC50 of 0.89
micromolar range include the polybrominated benzimidazoles
(IC50 from 0.93 to 0.49
M) described by Andrzejewska et al.12
l
l
and their polyiodinated derivatives, which are around tenfold more
potent.13 The potent and selective indoloquinazoline acetic acid
protein composed of two catalytic
a
(or a0) subunits and two reg-
derivative IQA revealed an IC50 of 0.08
polyphenol ellagic acid an IC50 of 0.04 l
M was found (Fig. 1).15
l
M,14 and for the natural
ulatory b subunits.3 It shows ‘dual cosubstrate specificity’, that is,
the ability to utilize adenosine triphosphate (ATP) or guanosine tri-
phosphate (GTP) as the cofactor.4,5 Besides its role in a variety of
non-cancer related diseases (such as neurodegenerative disorders,
inflammatory processes, angiogenesis-related diseases and viral
infections),6 there is a strong focus on its involvement in cancer.
Increased activity of the human protein kinase CK2 has been
detected in many different tumors, including those of the prostate,
Thus far only one very potent and highly selective inhibitor, the
benzonaphthyridine derivative CX-4945 (Fig. 1), with an IC50 of
0.001
of cancer.16
Besides the concept of alternative inhibition modes17 (e.g.,
lM, has reached advanced clinical trials for the treatment
CK2b-targeted inhibitors18 or inhibitors disturbing CK2
a/CK2b-
subunit interaction19), most of the published inhibitors share
common structural features: they consist of small and planar het-
erocyclic scaffolds, able to fit into the nucleotide-binding pocket of
⇑
Corresponding author. Tel.: +49 251 8332200; fax: +49 251 8332211.
E-mail address: joachim.jose@uni-muenster.de (J. Jose).
CK2a. They serve as ATP/GTP-competitive inhibitors of CK2 and
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.017

C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
2283
O
OH
O
OH
Cl
OH
OH
O
O
N
(a)
(b)
HO
HO
NH
N
N
O
O
HN
O
CX-4945
IQA
Ellagic acid
H3C
H
O
N
O
O
O
N
N
H
S36888
Figure 1. (a) Known CK2 inhibitors IQA, ellagic acid and CX-4945. (b) S36888, a topoisomerase II inhibitor of the indeno[1,2-b]indole type.
displace the cofactor.16,17,20,21 These structural features are also
found in indeno[1,2-b]indoles such as S36888 (Fig. 1). These com-
pounds have previously been shown to act as DNA-intercalators
and inhibitors of topoisomerase II with considerable cytotoxic
activity towards cancer cells.22
O
O
O
H
R4
R3
OH
OH
H
N
R2
Based on the assumption, that the small and planar heterocyclic
indeno[1,2-b]indoles could fit into the ATP/GTP-binding pocket of
the CK2, we aimed to develop a new class of CK2 inhibitors. Within
19 indeno[1,2-b]indoles synthesized, four potent CK2 inhibitors
were found. One of them, 5-isopropyl-7,8-dihydroindeno[1,2-b]-
indole-9,10(5H,6H)-dione 4b, inhibited the human CK2 in the
sub-micromolar range. As it displayed only low inhibition towards
a panel of 22 other human kinases, our results suggest 4b to be a
rather selective inhibitor of CK2. The mode of inhibition was
shown to be ATP-competitive and we present evidence of cell
permeability.
R1
1
2
i
O
O
O
O
ii
OH
H
H
R4
R3
R4
R3
HO
N
N
R2
R2
R1
R1
2. Results and discussion
2.1. Synthesis
3
4
Scheme 1. Preparation of indeno[1,2-b]indoles 4. For R1, R2, R3 and R4 refer to Table
1. Reagents and conditions: (i) CHCl3 or MeOH, rt, 8–24 h; (ii) DMF, acetic acid,
TMTA, rt, 3-12 h.
Compounds of type 4 were synthesized according to a recently
published deoxygenation procedure of vic-dihydroxyindeno[1,2-
b]indoles 3 using N,N,N0,N0-tetramethylsulfuric acid diamide (tetra-
methyl-thionylamide, TMTA) as a novel reagent. This resulted in
moderate to very good yields of 60–95% (Scheme 1).23,24 Com-
pounds 3 were prepared by condensation of indanetrione hydrate
1 and 3-amino-cyclohex-2-enones 2 in yields ranging from 40% to
90%. Precursor 1 is commercially available and 2 is readily avail-
able by refluxing a solution of cyclohexane-1,3-diones and primary
amines in benzene with azeotropic removal of water.23,24
the amines can occur at both carbonyls with the same probability.
Thus, a racemic mixture of both enantiomers of 4n and 4o is
formed and was used for primary testing. Due to low activity of
the racemate in CK2 testing, the isomers were not separated.
For derivative 4n, 1,2-diaxial coupling constants between H-7
and H-6/H-8 of 10.7 Hz (3JH6–H7 and 3JH7–H8) were observed, giving
evidence of the equatorial position of the phenyl ring at C-7 for
both enantiomers.
For the chiral derivatives 4l and 4m, a coupling constant of
10.5 Hz (3JH7–H8) between H-7 and H-8 was observed, showing a
1,2-diaxial coupling of the protons at C-7 and C-8. This results in
the conformers with both the methyl and the carbomethoxy
groups at C-7 and C-8 in equatorial position on the half-chair,
which is in accordance with the crystal structure for the enami-
none precursor described by Greenhill et al.25 with the (S)-config-
uration at C-7 and the (R)-configuration at C-8.
Also for 4o, H-7 shows large couplings with the vicinal protons
H-6/H-8 of 10.1 and 11.1 Hz (3JH6–H7 and 3JH7–H8) proving the equa-
torial position of the methyl group at C-7 for both enantiomers.
In order to convert the 7,8-dihydroindeno[1,2-b]indole-9,10
(5H,6H)-diones 4 into the aromatic 9-hydroxyindeno[1,2-b]indole-
10(5H)-ones 5 some representative dehydrogenation reactions
were explored. The reaction with mercury(II)acetate in boiling
acetonitrile according to Iida et al.26 did not give the desired prod-
ucts. Efforts to aromatize 4 by dehydrogenation in the presence of
10% Pd/C in boiling xylene27 resulted in the formation of 5,
The enaminone precursors of 4n and 4o were prepared from
symmetric 1,3-cyclohexanediones. Condensation of the dione with

2284
C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
concentration of 10
4d) revealed IC50 in the sub-micromolar range (0.11 and 0.82
l
M (4b, 4d, 4f and 4g). Two of them (4b and
M).
O
O
OH
O
l
iii
R4
R2
H
The CK2 inhibition by compounds 4 was in most cases (4b, 4c, 4j
vs 5b, 5c, 5j) stronger than their dehydrogenated counterparts 5.
An exception was the phenolic indeno[1,2-b]indole 5n, which
showed higher inhibition than its analog 4n. Both derivatives bear
the sterically demanding phenyl ring at R2, which is shifted in-plane
in 5n. Additionally, the lower activity of 4n could be, at least in part,
due to the use of the racemic mixture of both isomers, assuming that
onlyoneisomerwasactivetowardsCK2. Ingeneralforcompounds4,
substitutions at R2 or R3, with one or two methyl groups (4k, 4o) or a
phenyl ring (4n), lowered the inhibitory activity of the compounds.
An ester groupat R4 (4l, 4m) had barely any effect onpotency. Phenyl
or phenylalkyl residues at the indole nitrogen led to a stronger inhi-
bition (4d, 4f and 4g), but for longer alkyl-linkers (4e) inhibitory
activity was lost again. N-substitution with 2-pyridyl-alkyl and
methoxy-substituted phenyl-alkyl residues led to a somewhat les-
ser activity compared to the phenyl-alkyl derivatives with the same
chain-length. Best effects for the cyclohexeneone derivatives 4 were
achieved with an isopropyl residue at the indole nitrogen (4b),
leading to a sevenfold stronger inhibition than the most potent
phenylalkyl derivative. Within the phenolic derivatives 5, N-substi-
tution with the isopropyl group also led to the strongest inhibition
(48% for 5b).
R4
H
N
N
R2
R1
R1
5
4
Scheme 2. Preparation of indeno[1,2-b]indoles 5. For R1, R2 and R4 refer to Table 1.
Reagents and conditions: (iii) 1,4-dioxane, DDQ, 60–70 °C, 24–72 h.
contaminated by a number of additional compounds such as
dimerization products, which made the separation difficult. Final-
ly, a slightly modified version of Rebeks approach to mitomycins
by means of DDQ gave the best results.28,29 The reaction in 1,4-
dioxane at 60-70 °C gave 5 in moderate to good yields of 54–74%
(Scheme 2). However, the products were strongly dependent on
the reaction conditions. In refluxing 1,4-dioxane as well as in
refluxing acetonitrile a lot of additional byproducts were formed.
A total of 19 indeno[1,2-b]indole-derivatives were prepared in
yields that ranged from moderate to very good (Table 1).
2.2. Inhibition of human CK2 holoenzyme
The indeno[1,2-b]indole derivatives were tested for their inhib-
itory activity towards human CK2 holoenzyme (Table 1). The syn-
thetic peptide RRRDDDSDDD was used as the substrate, which is
reported to be most efficiently phosphorylated by CK2.30 For initial
testing, inhibition was determined relative to the controls at inhib-
2.3. Mode of inhibition
To determine the mode of inhibition, we measured CK2 activity
using the non-radiometric capillary electrophoresis (CE) method of
Gratz et al.31 For the strongest inhibitor 4b, IC50 were determined
at six different ATP concentrations ranging from approximately
1/10 to 10 times the Km for ATP. At an ATP concentration at the
Km which was also used for the radiometric measurements, the
itor concentrations of 10 lM in DMSO as the solvent. The radioac-
tivity of the pure solvent without inhibitor was used as negative
control and set to 0% inhibition; Reactions without CK2 were used
as positive control and set to 100% inhibition. Compounds with
more than 50% inhibition at a concentration of 10
jected to an IC50 determination. For this purpose, inhibition was
measured at final concentrations ranging from 0.01 to 30 M in
appropriate intervals. IC50 were calculated from the resulting
dose–response curves. Each value was determined at least in trip-
licate in independent experiments. For the known inhibitor emo-
lM were sub-
IC50 for 4b was 0.36
radiometrically determined IC50 of 0.11
l
M, which is in good agreement with the
M. A linear increase of
l
l
the IC50 with increasing ATP could be observed (Fig. 2), showing
the assumed ATP competitivity.32
din we measured an IC50 of 0.46
good agreement with the reported value of 0.89
l
M in this assay, which is in
2.4. Determination of Ki
l
M.11
From the 19 synthesized indeno[1,2-b]indoles, we found four po-
With the demonstrated mode of inhibition, the inhibition con-
tent compounds with an inhibition of more than 50% at a
stant (Ki) can be calculated from the IC50 using the Cheng–Prusoff
Table 1
Synthesized indeno[1,2-b]indole derivatives and inhibition of human CK2 holoenzyme
Compound
R1
R2
R3
R4
Inhibition (%)a
IC50 (l
M)b
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4nc
4oc
5b
5c
5j
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
—
—
—
—
H
H
H
H
H
H
H
H
19
93.4
48
67
27
66
60
30
41
50
10
28
15
3
n.d.
0.11
n.d.
0.82
n.d.
4.66
1.44
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
iso-C3H7
CH2C6H5
(CH2)2C6H5
(CH2)3C6H5
CH(CH3)C6H5
C6H5
CH2-2-C5H4N
CH2C6H4-4-OCH3
(CH2)2C6H3-3,4-(OCH3)2
CH2C6H5
H
H
H
H
H
CH3
CH3
CH3
C6H5
CH3
H
H
H
C6H5
H
CO2CH3
CO2CH3
CH2C6H5
CH2C6H5
CH2C6H5
iso-C3H7
CH2C6H5
(CH2)2C6H3-3,4-(OCH3)2
CH2C6H5
H
H
H
H
H
H
11
48
25
34
31
5n
a
b
c
Given is the average percent inhibition at 10
n.d.: not determined.
A racemic mixture of both enantiomers was used for testing.
lM.

C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
2285
Figure 4. Plot of Kampp against the concentration of 4b, for the determination of Ki.
Figure 2. Graphical determination of the inhibition modality. IC50 were plotted
against the corresponding ATP concentrations. The abscissa was plotted on
logarithmic scale for clarity.
a
Poor absorption and low permeability is predicted for drugs with
ClogP P 5 and TPSA P 140 Å2.36–38
To support this prediction, cytotoxicity of 4b towards a human
cancer cell line was evaluated. Therefore, growth inhibition by
compound 4b was measured using the human esophageal cancer
cell line KYSE-70. At a concentration of 20 lM, a growth inhibition
of 25% ( 3%, n = 3) was observed. Although the effect was weak,
this demonstrates membrane permeability of the compound. The
low effects of 4b towards the cancer cell line could be due to
biological conversion of the compounds into inactive derivatives.
Alternatively, multidrug resistance efflux transporters as P-glyco-
protein (P-gp), which are known to be responsible for the drug
resistance of tumors,39 could have lead to a reduction of intracellu-
lar concentrations of the active compound.
2.6. Selectivity profiling
Compound 4b, which showed the highest inhibition of CK2
(IC50 = 0.11 lM), was screened for inhibitory activity against a pa-
Figure 3. Determination of Kampp for different concentrations of the inhibitor 4b.
Double reciprocal plot of reaction velocity against the cosubstrate concentration.
nel of 22 different human kinases (Table 2). IC50 were measured by
testing 10 concentrations of each compound in singlicate and cal-
culated from the resulting dose–response curves. The known
inhibitor ellagic acid, with a reported IC50 towards the CK2 of
equation for competitive inhibition.33 Based on the measurements
from the radiometric method, the Ki for the strongest inhibitor 4b
was 0.06 lM.
0.04 l
M,15 was used as a reference.
Compound 4b showed selectivity towards CK2 in the panel
tested. None of the 22 other kinases were significantly inhibited
by 4b (IC50 >35 lM). The inhibition of CK2 by this compound is
For comparison, Ki for 4b was also determined directly by the
method described by Copeland.34 Therefore, reaction velocities
therefore 300-fold higher than any other kinase tested. Preliminary
studies against a panel of 11 kinases suggest that ellagic acid is a
strong and selective inhibitor of CK2.15 Our results suggest that
4b is a much more selective inhibitor than ellagic acid, which also
(v) at different inhibitor and ATP concentrations were measured
with the CE-based CK2 activity assay31 and were plotted in a
Lineweaver–Burk diagram against the varied ATP concentrations
(Fig. 3). 1/vmax is given by the intersections with the ordinate,
ꢀ1/Kampp are given by the intersections with the abscissa. As already
shown in Section 2.3, competitive inhibition is demonstrated by
inhibited eight other kinases with IC50 values below 1
EGF-R, IGF1R, SRC, VEGF-R2, INS-R, MET and TIE2).
lM (ARK5,
the increasing Kampp with increasing inhibitor concentrations and
34
3. Conclusions and outlook
the unaffected vmax
.
For the calculation of Ki, Kapp were plotted against the corre-
m
We show that substituted indeno[1,2-b]indole derivatives are a
novel class of potent inhibitors of the human protein kinase CK2. A
set of 19 indeno[1,2-b]indoles was prepared in moderate to very
good yields and tested for their inhibitory activity and selectivity.
Four compounds with good inhibitory activity were found. The
sponding inhibitor concentrations (Fig. 4). The negative Ki is given
by the intersection of the linear regression with the abscissa. For
the CE-based measurements, a Ki of 0.15
mined, which is in good agreement with the calculated Ki of
0.06 M from the radiometric measurements.
lM for 4b was deter-
l
most active derivative, 4b, with an IC50 of 0.11 lM showed selec-
tivity towards the human CK2, as judged by low inhibition of 22
different human kinases. Competitive inhibition of compound 4b
was demonstrated. Inhibition constants were determined by the
standard radiometric activity assay and a novel capillary electro-
phoresis based method, giving comparable Ki of 0.06 and
2.5. Cell permeability
For an evaluation of the cell permeability of 4b, fragment-based
partition coefficients (ClogP) and topological polar surface areas
(TPSA) were calculated. The data suggest, that 4b should be able
to penetrate cell membranes, with a ClogP of 3.317 and a TPSA
of 39.076 Å2 (calculated by the Molinspiration web services35).
0.15
lM, respectively. Membrane permeability of the strongest
inhibitor 4b could be demonstrated by theoretical predictions

2286
C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
Table 2
Inhibitiona of 22 different protein kinases by indeno[1,2-b]indole 4b and the known inhibitor ellagic acid (EA)
Kinase
AKT1
ARK5
Aurora-A
Aurora-B
B-RAF
CDK2/CycA
CDK4/CycD1
EGF-R
EPHB4
ERBB2
FAK
4b
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
EA
3.34
0.51
2.48
2.50
1.76
3.39
1.09
0.69
1.83
3.54
3.11
Kinase
IGF1R
SRC
VEGF-R2
VEGF-R3
INS-R
MET
PDGFR -b
PLK1
SAK
TIE2
COT
4b
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
EA
0.25
0.80
0.79
1.54
0.34
0.58
1.30
3.34
2.97
0.26
2.37
a
Given are IC50 in lM.
and the cytotoxic potential of this compound towards an esopha-
geal cancer cell line. Taken together, the results show that indeno
[1,2-b]indoles, such as 4b, are a promising starting point for the
further development of potent and selective CK2 inhibitors, leading
to potential drugs for the treatment of malignancies and non-can-
cer related diseases. Synthesis and evaluation of further deriva-
tives, in particular N-isopropyl substituted derivatives, is in
progress.
to a small volume and worked up as described under General Pro-
cedure A.
4.2.1. 5-Isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-
dione (4b)
General Procedure B. Reaction time: 3 h. Yield: 89%. Mp: 201 °C
(EtOAc). IR (KBr):
m
(cmꢀ1) = 2944, 1707, 1665, 1609, 1520, 1485,
1426, 1319, 1297. 1H NMR (200 MHz, TMS, CDCl3):
d
(ppm) = 1.65 (d, J = 7 Hz, 6H, 2CH3), 2.1–2.2 (m, 2H, H-7), 2.4–2.5
(m(t), 2H, CH2), 2.8–2.9 (m(t), 2H, CH2), 4.5– 4.7 (m, 1H, CH),
7.0–7.5 (m, 4H, Ar-H). EI-MS (70 eV): m/z (%) = 279 (49) [M+],
278 (24), 264 (10), 237 (11), 223 (10), 209 (25), 208 (22), 181
(43), 180 (18), 153 (22), 152 (19), 130 (15), 126 (13), 125 (10),
102 (11), 78 (14), 77 (16), 75 (13), 69 (11), 51 (13), 43 (98), 42
(16), 41 (100). Anal. Calcd for C18H17NO2 (279.34): C, 77.40; H,
6.13; N, 5.02. Found: C, 76.93; H, 6.00; N, 4.90.
4. Experimental section
4.1. General
Melting points: Büchi melting point apparatus by Dr. Tottoli;
not corrected. IR spectra: Perkin Elmer FT-IR 1600, using KBr discs.
NMR spectra: Bruker AC 200F (1H: 200 MHz/13C: 50 MHz) with
TMS as the internal standard and Varian AS 400 Mercuryplus
(
13C: 100 MHz) with the solvent peak as the internal standard in
4.2.2. 5-Benzyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-
dione (4c)
the designated solvents, using d (ppm) scale; signals, labeled by ꢁ
exchanged by addition of D2O. 13C NMR spectra of 5j could not
be obtained due to insufficient solubility in all standard deuterated
solvents. EI mass-spectra: Finnigan 4200 quadrupole mass-
General Procedure B. Reaction time: 3 h. Yield: 95%. Mp: 185 °C
(EtOAc). IR (KBr):
m
(cmꢀ1) = 1703, 1666, 1607, 1526, 1499, 1453,
1443, 1431, 1293. 1H NMR (200 MHz, TMS, CDCl3):
d
(ppm) = 2.0–2.2 (m, 2H, H-7), 2.41 (m(t), 2H, CH2) 2.62 (m(t), 2H,
CH2), 5.17 (s, 2H, NCH2), 6.68–6.72 (m, 1H), 7.00–7.2 (m, 4H),
spectrometer, equipped with
a MASPEC data system; 70 eV
ionizing potential. Microanalyses: Perkin Elmer Elemental
Analyzer 2400/II. MPLC or flash chromatography was performed
on 230–400 mesh silica (Merck). Solvents were purified by stan-
dard methods and dried over molecular sieves or sodium. Yields
refer to the amount of the products after one recrystallization
and are not optimized.
7.29–7.41(m, 4H). 13C NMR (50 MHz, TMS, CDCl3):
d
(ppm) = 21.70, 22.85, 37.71, 49.14, 117.24, 117.88, 119.81,
123.43, 125.91, 125.98, 128.21, 128.25, 129.28, 132.44, 134.71,
134.89, 138.66, 150.66, 153.01, 184.18, 192.25. EI-MS (70 eV):
m/z (%) = 327 (38) [M+], 326 (12), 299 (4), 298 (3), 272 (3), 271
(5), 270 (6), 250 (5), 208 (5), 180 (4), 152 (4), 92 (8), 91 (100). Anal.
Calcd for C22H17NO2 (327.38): C, 80.71; H, 5.23; N, 4.28. Found: C,
80.81; H, 5.20; N, 4.24.
4.2. Synthesis of 7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-
diones (4)23,24,40
4.2.3. 5-Phenethyl-7,8-dihydroindeno[1,2-b]indole-
9,10(5H,6H)-dione (4d)
The synthesis and characterization of compounds 4 of Table 1
has been previously described by Hemmerling and Reiss.23 We de-
scribe here again the methods to obtain compounds 4 that were
shown to be pharmacologically most active (4b, 4d). We also de-
scribe the compounds that served as reactants (4c, 4j, 4n) in the
reactions to prepare compounds 5.
General Procedure A: Compound 3 (5.0 mmol) was dissolved in
15 mL DMF and 3 mL acetic acid was added. After addition of TMTA
(20 mmol) the solution was stirred for the time indicated. After
this time a precipitate has deposited, which was separated by suc-
tion. The filtrate was evaporated in vacuo, the oily residue diluted
with H2O, and the solution was basified with NaHCO3. Extraction
with CHCl3 gave a second crop, which was purified by MPLC on
SiO2/EtOAc-hexane or CHCl3-EtOAc.
General Procedure B: Compound 3 (5.0 mmol) was dissolved in
15 mL DMF and 3 mL acetic acid was added. After addition of TMTA
(20 mmol) the solution was stirred for the time indicated. After
this time, the solution was poured into 500 mL H2O. The suspen-
sion was stirred for 3 h and the solids separated by filtration. The
solid was air dried and crystallized. The filtrate was evaporated
General Procedure A. Reaction time: 5 h. Yield: 62%. Mp: 189–
190 °C (EtOAc). IR (KBr):
1499, 1476, 1296. 1H NMR (200 MHz, TMS, DMSO-d6):
m
(cmꢀ1) = 1697, 1659, 1606, 1524,
d
(ppm) = 1.7–1.9 (m, 2H, CH2), 2.2–2.4 (m, 4H, 2CH2), 2.9–3.1 (m,
2H, CH2), 4.3–4.4 (m, 2H, NCH2), 7.0–7.4 (m, 9H, Ar-H). EI-MS
(70 eV): m/z (%) = 341 (100) [M+], 285 (12), 250 (8), 249 (10), 237
(38), 209 (37), 194 (19), 121 (25), 105 (19), 104 (15), 91 (11). Anal.
Calcd for C23H19NO2 (341.40): C, 80.92; H, 5.61, N, 4.10. Found: C,
80.92; H, 5.39; N, 4.04.
4.2.4. 5-(3,4-Dimethoxyphenethyl)-7,8-dihydroindeno[1,2-
b]indole-9,10(5H,6H)-dione (4j)
General Procedure B. Reaction time: 8 h. Yield: 90%. Mp: 189–
190 °C (DMF/H2O). IR (KBr):
m
(cmꢀ1) = 1699, 1663, 1605, 1516,
1502, 1450, 1430, 1292, 1264, 1245. 1H NMR (200 MHz, TMS,
CDCl3): d (ppm) = 1.8–1.95 (m, 2H, CH2), 2.07 (t, J = 6.0 Hz, 2H,
CH2), 2.35 (t, J = 6.0 Hz, 2H, CH2), 3.02 (t, J = 6.0, 2H, CH2), 3.74,
3.82 (2 s, 6H, 2OCH3), 4.16 (t, J = 6.0, 2H, NCH2), 6.42 (d, J = 1.7,

C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
2287
1H, H20), 6.53 (dd, J1 = 1.7 Hz, J2 = 8.0 Hz, 1 H, H6’), 6.75 (d,
J = 8.0 Hz, 1H, H5), 6.85–7.5 (m, 4H, Ar-H). EI-MS (70 eV): m/z
(%) = 401 (22) [M+], 400 (7), 399 (10), 165 (25), 164 (29), 163 (5),
151 (100), 150 (12), 149 (17), 107 (14), 105 (11), 102 (11). Anal.
Calcd for C25H23NO4 (401.45): C, 74.79; H, 5.77; N, 3.49. Found:
C, 74.63; H, 5.84; N, 3.37.
1430, 1262, 1237, 1158, 1028. 1H NMR (200 MHz, TMS, CD2Cl2):
d(ppm) = 3.09 (t, J = 6.6 Hz, 2H, CH2), 3.62, 3.69 (2s, 6H, 2OCH3),
4.40 (t, J = 6.7 Hz, 2H, NCH2), 6.4 (m, 10H, Ar-H). EI-MS (70 eV):
m/z (%) = 399 (62) [M+], 248 (26), 165 (18), 151 (100). Anal. Calcd
for C25H21NO4 (399.44): C, 75.17; H, 5.30; N, 3.51. Found: C,
75.44; H, 5.24; N, 3.53.
4.2.5. 5-Benzyl-7-phenyl-7,8-dihydroindeno[1,2-b]indole-
9,10(5H,6H)-dione (4n)
4.3.4. 5-Benzyl-9-hydroxy-7-phenylindeno[1,2-b]indole-10(5H)-
one (5n)
General Procedure A. Reaction time: 12 h. Yield: 80%. Mp:
252 °C (Toluene). IR (KBr):
Preparation according to 5b with 4n. Reaction temperature:
60 °C. Reaction time: 48 h. Purification by MPLC on silica: (Eluent:
EtOAc/hexane = 40:60 (v/v)). Yield: 74%. Mp: 254–255 °C (CH3CN)
m
(cmꢀ1) = 1705, 1669, 1605, 1499. 1H
NMR (200 MHz, TMS, CDCl3): d (ppm) = 2.64–3.01 (m, 4H, H-6,
H-8), 3.42–3.57 (m, 1H, H-7), 5.22 (s, 2H, NCH2), 6.73–6.82 (m,
1H, H-4), 7.06–7.52 (m, 13H, Ar-H). 13C NMR (50 MHz, TMS,
CDCl3): d (ppm) = 29.8, 41.4, 44.7, 49.2, 117.2, 117.8, 120.0,
123.9, 125.3, 125.9, 126.8, 127.2, 128.2, 128.4, 128.5, 128.8,
129.0, 129.4, 132.4, 134.6, 134.8, 138.7, 142.5, 149.2, 153.4,
184.1, 191.0. EI-MS (70 eV): m/z (%) = 403 (4) [M+], 312 (3),
299(12), 271 (13), 208 (13), 180 (4), 152 (5), 104 (4), 91 (100). Anal.
Calcd for C28H21NO2 (403.49): C, 83.35; H, 5.25; N, 3.47. Found: C,
83.54; H, 5.27; N, 3.33.
IR (KBr):
m
(cmꢀ1) = 3426, 1667, 1636, 1601, 1576, 1519, 1505,
1496, 1482, 1444, 1428, 1421, 1352, 1273. 1H NMR (200 MHz,
TMS, CDCl3): d (ppm) = 5.47 (s, 2H, NCH2), 6.47ꢁ (s, 1 H, OH),
6.94–7.55 (m, 16H, Ar-H). 13C NMR (50 MHz, TMS, DMSO-d6): d
(ppm) = 47.8, 101.4, 107.2, 112.1, 113.8, 119.0, 122.4, 126.4,
126.5, 127.1, 127.5, 128.8, 128.8, 129.5, 132.4, 134.1, 136.8,
137.0, 140.1, 140.5, 144.9, 151.3, 157.7, 182.8. EI-MS (70 eV): m/z
(%) = 401 (10) [M+], 310 (19), 282 (10), 253 (5), 226 (4), 151 (2),
102 (5), 92 (7), 91 (100), 77 (7), 65 (20). Anal. Calcd, for
C28H19NO2 (401.46): C, 83.77; H, 4.77; N, 3.49. Found: C, 83.87;
4.3. Synthesis of 9-hydroxyindeno[1,2-b]indole-10(5H)-ones (5)
H, 4.85; N, 3.49.
4.3.1. 9-Hydroxy-5-isopropylindeno[1,2-b]indole-10(5H)-one
(5b)
4.4. Preparation of recombinant human CK2 enzyme and
testing of inhibitors
Compound 4b (1.0 g, 3.6 mmol) was dissolved in 1,4-dioxane
(150 mL) and a solution of DDQ (0.91 g, 4.0 mmol) was added.
The mixture was heated to 70 °C and stirred for 24 h with TLC
monitoring. Half of the solvent was then evaporated in vacuo
and after cooling to rt the solid that separated was removed by suc-
tion. The filtrate was evaporated to dryness and the solid residue
subjected to MPLC at silica (eluent: EtOAc) gave 0.8 g of 5b. Yield:
As described earlier,41
a-subunit (CSNK2A1) and b-subunit
(CSNK2B) of protein kinase CK2 were expressed separately in
E. coli (BL21(DE3) pT7-7). The starter cultures were grown over-
night at 37 °C until the stationary phase was reached. With the
separate starter cultures for each subunit, fresh medium was inoc-
ulated and cultivated until an OD500 of 0.6. For induction of protein
expression IPTG was added to a final concentration of 1 mM and
was run at 30 °C for 5-6 h for CSNK2A1 and at 37 °C for 3 h for
CSNK2B. Bacterial cells were harvested by centrifugation
(6000ꢂg at 4 °C for 10 min) and disrupted by sonification (3 times
30 s on ice). Cell debris was removed by centrifugation and the
bacterial extracts for both subunits were combined and subjected
to a three-column purification. Activity of the fractions was deter-
mined by measurements of the conversion of a peptide substrate
(RRRDDDSDDD). Fractions exhibiting CK2 enzymatic activity were
combined and analyzed by SDS-PAGE and Western Blot. They were
stored in aliquots at ꢀ80 °C until used for testing. For testing of
65%. Mp: 182 °C (MeOH). IR (KBr):
m
(cmꢀ1) = 2438, 1662, 1604,
1492, 1449, 1429, 1420, 1230. 1H NMR (200 MHz, TMS, CDCl3): d
(ppm) = 1.72 (d, J = 7 Hz, 6H, 2 CH3), 4.79–4.93 (m, 1H, CH), 6.66–
6.70 (m, 1H, Ar-H), 6.75ꢁ (s, 1H, OH), 6.9–7.5 (m, 6H, Ar-H). 13C
NMR (100 MHz, DMSO-d6): d (ppm) = 182.80, 156.12, 151.22,
142.70, 140.28, 134.51, 132.47, 129.38, 124.26, 122.43, 120.06,
113.21, 109.33, 107.76, 104.73, 49.21, 21.22. EI-MS (70 eV): m/z
(%) = 277 (99) [M+], 262 (11), 235 (100), 220 (12), 207 (12), 206
(22), 179 (30), 178 (16), 177 (11), 151 (16), 150 (10), 76 (22). Anal.
Calcd for C18H15NO2 (277.32): C, 77.96; H, 5.45; N, 5.05. Found: C,
78.18; H, 5.66; N, 5.00.
inhibitors, 0.5
incubated at room temperature for 10 min. The inhibitors (in
DMSO) were then added to a final concentration of 10 M in
20 L of kinase buffer (50 mM Tris/HCl, pH 7.5, 100 mM NaCl,
10 mM MgCl2, 1 mM DTT). DMSO alone was used as negative con-
trol. The reaction was started by the addition of 30 L assay buffer
(25 mM Tris/HCl, pH 8.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT,
100 M ATP, 0.19 mM peptide substrate, 0.6 Ci [
32P]ATP). Final
concentrations in the reaction mixture were 60 M for the cosub-
strate ATP and 114 M for the substrate peptide. The reaction was
lL (0.25 U/mL) of CK2 holoenzyme was pre-
4.3.2. 5-Benzyl-9-hydroxyindeno[1,2-b]indole-10(5H)-one (5c)
Preparation according to 5b with 4c. Reaction temperature:
65 °C. Reaction time: 72 h. Yield: 54%. Mp: 198–199 °C (EtOH). IR
l
l
(KBr):
m
(cmꢀ1) = 3426, 1666, 1604, 1523, 1504, 1488, 1448,
1440, 1430, 1422, 1238. 1H-NMR (200 MHz, TMS, CDCl3): d
(ppm) = 5.49 (s, 2 H, NCH2), 6.54ꢁ (s, 1H, OH), 6.7–7.6 (m, 12H,
Ar-H). 13C NMR (50 MHz, TMS, CDCl3): d (ppm) = 49.23, 103.43,
108.16, 113.12, 115.38, 118.83, 123.54, 125.66, 126.34, 128.27,
129.20, 129.56, 132.44, 135.33, 135.69, 140.35, 144.02, 149.93,
156.80, 185.97. EI-MS (70 eV): m/z (%) = 325 (20) [M+], 248 (7),
234 (5), 206 (4), 177 (3), 151 (5), 92 (8), 91 (100). Anal. Calcd for
l
l
l
c-
l
l
carried out at 37 °C for 15 min then the mixture was spotted onto a
P81 ion exchange paper (Whatman). After washing thrice with
excess phosphate (85 mM H3PO4) and subsequently with ethanol,
the filter was dried and the adhering radioactivity measured by a
scintillation counter (Packard). Initial inhibition at a fixed concen-
C22H15NO2 (325.37): C, 81.21; H, 4.65; N, 4.30. Found: C, 80.98;
H, 4.91; N, 4.37.
4.3.3. [2-(3,4-Dimethoxyphenyl)-ethyl]-9-hydroxyindeno[1,2-
b]indole-10(5H)-one (5j)
tration of 10 lM was determined relative to the controls. Pure sol-
vent without inhibitor was used as negative control (0 %
inhibition), assays without CK2 were used as positive control
(100 % inhibition). For IC50 determination inhibition was measured
Preparation according to 5b with 4j. Reaction temperature:
65 °C. Reaction time: 48 h. Yield: 58%. Mp: 137–138 °C (Toluene/
Cyclohexane). IR (KBr):
m
(cmꢀ1) = 3422, 1669, 1604, 1516, 1448,
at final concentrations ranging from 0.01 to 30 lM in appropriate

2288
C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
intervals. IC50 were calculated from the resulting dose-response
curves at least in triplicate in independent experiments.
was measured in a 50
from Perkin–Elmer/NEN (Boston, MA). The reaction contained
20 L assay buffer, 5 L aqueous ATP solution, 5 L inhibitor (in
10% DMSO), 10 L of the substrate, and 10 L purified recombinant
human protein kinase. The assay for all enzymes contained 60 mM
HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 M sodium
and
lL reaction volume in 96-well FlashPlates
l
l
l
4.5. Mode of inhibition
l
l
For the determination of the mode of inhibition, the recently
established capillary electrophoretic CK2 activity assay was used.31
Enzymatic reactions were performed as described in Section 4.2.2,
l
orthovanadate, 1.2 mM dithiothreitol, 50 lg/mL PEG20000,
1
lM [
c
-
33P]ATP (approx. 5 ꢂ 105 cpm/well). Used substrates:
but without the use of the [
c-
32P] labeled ATP in the assay buffer.
GSK3(14-27) for AKT1; tetra(LRRWSLG) for Aurora 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 INS-R,
MET and PDGF-Rb; Casein for PLK1; p38-alpha-KRKR for SAK;
Autophosphorylation was measured for ARK5 and COT. All protein
kinases were expressed in Sf9 insect cells as His-tagged or GST-fu-
sion proteins and purified by affinity chromatography. Reactions
were incubated at 30 °C for 80 minutes. The reaction was quenched
Total reaction volume was scaled up to 200
CK2-supplemented kinase buffer and 120
tions were stopped by the addition of 4
l
l
L, composed of 80
L assay buffer. Reac-
lL EDTA (0.5 M) and the
lL
reaction mixture was analyzed by a PA800 capillary electrophore-
sis from Beckman Coulter (Krefeld, Germany). For electrophoretic
separation, 2 M acetic acid was used as the electrolyte. The sepa-
rated substrate and product peptide were detected and quantified
using a DAD-detector at 214 nm. Measurements were performed
using six ATP concentrations ranging from approximately a tenth
with 50
lL of 2% (v/v) H3PO4, plates were aspirated and washed
to ten times the Km (75
were determined using nine inhibitor concentrations ranging from
l
M) and for each ATP concentration IC50
twice with 200 lL of 0.9% (w/v) NaCl or 200 lL H2O. Incorporation
of 33Pi was measured with a microplate scintillation counter
(Microbeta Trilux, Wallac). Assays were performed with a Beck-
manCoulter/Sagian robotic system.
0.001 lM to 100 lM. For the analysis of inhibition modality IC50
were plotted versus the ATP concentration.32
4.6. Determination of Ki
Acknowledgments
The Ki of 4b for the radiometric measurements was calculated
from IC50 with the Cheng–Prusoff equation for competitive inhibi-
tion (IC50 = Ki(1+([ATP]/Km)).33
We thank P. Proksch, Heinrich-Heine-University Düsseldorf,
and A. Bollacke, Westfälische-Wilhelms-University Münster, for
their helpful support.
For determination of Ki the method described by Copeland was
used.34 Reaction velocities were determined at different inhibitor
and cosubstrate concentrations with the CE-Method described in
Section 4.2.3. Reaction velocities were plotted in a Lineweaver–
Burk plot against the varied ATP concentrations where ꢀ1/Kmapp
are given by the intersections with the abscissa. Kampp were then
plotted against the corresponding inhibitor concentrations and
ꢀKi is given by the intersection of the linear regression with the
abscissa.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.017. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
4.7. Cell permeability
1. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science
1912, 2002, 298.
2. Salvi, M.; Sarno, S.; Cesaro, L.; Nakamura, H.; Pinna, L. A. Biochim. Biophys. Acta
(BBA)–Mol. Cell Res. 2009, 1793, 847.
3. Niefind, K.; Guerra, B.; Ermakowa, I.; Issinger, O.-G. EMBO J. 2001, 20, 5320.
4. Niefind, K.; Putter, M.; Guerra, B.; Issinger, O.-G.; Schomburg, D. Nat. Struct. Mol.
Biol. 1999, 6, 1100.
Fragment-based partition coefficients (ClogP) and topological
polar surface areas (TPSA) were calculated by the molinspiration
web services.35
The ability of the compounds to permeate cells was evaluated
indirectly with the Crystal Violet staining method as previously de-
scribed.42,43 With this method, growth inhibition of adherent can-
cer cell lines is determined in 96-well microtiter plates. The human
esophageal cancer cell line used (KYSE-70) was obtained from the
DSMZ (German Collection of Microorganisms and Cell Cultures)
and was grown in streptomycin and penicillin G supplemented
RPMI 1640 medium containing 10% FCS (Sigma). Cells were incu-
bated for 96 h with the compounds dissolved in DMF. Final concen-
tration of the solvent was 0.1%. After incubation, cells were fixed
20 min with 1% glutaraldehyde and then stained for half an hour
with 0.02% Crystal Violet in water. Following washing for half an
hour in water, the residual cell-bound dye was dissolved with eth-
anol/water (7:3 (v/v)) and the absorption (570 nm) was measured
with an Anthos 2010 plate reader (Anthos, Salzburg, Austria).
5. Faust, M.; Montenarh, M. Cell Tissue Res. 2000, 301, 329.
6. Guerra, B.; Issinger, O.-G. Curr. Med. Chem. 1870, 2008, 15.
7. Ruzzene, M.; Pinna, L. A. Biochim. Biophys. Acta, ProteinsProteomics 2010, 1804, 499.
8. Ahmad, K. A.; Wang, G.; Unger, G.; Slaton, J.; Ahmed, K. Adv. Enzyme Regul.
2008, 48, 179.
9. Zandomeni, R. J. Biol. Chem. 1986, 261, 3414.
10. Sarno, S.; Reddy, H.; Meggio, F.; Ruzzene, M.; Davies, S. P.; Donella-Deana, A.;
Shugar, D.; Pinna, L. A. FEBS Lett. 2001, 496, 44.
11. Sarno, S.; de Moliner, E.; Ruzzene, M.; Pagano, M. A.; Battistutta, R.; Bain, J.;
Fabbro, D.; Schoepfer, J.; Elliott, M.; Furet, P.; Meggio, F.; Zanotti, G.; Pinna, L. A.
Biochem. J. 2003, 374, 639.
12. Andrzejewska, M.; Pagano, M. A.; Meggio, F.; Brunati, A. M.; Kazimierczuk, Z.
Bioorg. Med. Chem. 2003, 11, 3997.
13. Gianoncelli, A.; Cozza, G.; Orzeszko, A.; Meggio, F.; Kazimierczuk, Z.; Pinna, L. A.
Bioorg. Med. Chem. 2009, 17, 7281.
14. Vangrevelinghe, E.; Zimmermann, K.; Schoepfer, J.; Portmann, R.; Fabbro, D.;
Furet, P. J. Med. Chem. 2003, 46, 2656.
15. Cozza, G.; Bonvini, P.; Zorzi, E.; Poletto, G.; Pagano, M. A.; Sarno, S.; Donella-
Deana, A.; Zagotto, G.; Rosolen, A.; Pinna, L. A.; Meggio, F.; Moro, S. J. Med.
Chem. 2006, 49, 2363.
16. Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.;
Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J.
P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Anderes, K.; Bliesath, J.; Drygin, D.;
Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Trent, K.; Rice, W. G.; Ryckman, D. M.
J. Med. Chem. 2011, 54, 635.
4.8. Kinase selectivity
The selectivity of 4b towards CK2 was investigated using a com-
mercial radiometric protein kinase assay, which tested this inhibi-
tor against a panel of 22 purified recombinant human kinases
17. Prudent, R.; Cochet, C. Chem. Biol. 2009, 16, 112.
18. Martel, V.; Filhol, O.; Colas, P.; Cochet, C. Oncogene 2006, 25, 7343.
19. Laudet, B.; Moucadel, V.; Prudent, R.; Filhol, O.; Wong, Y.-S.; Royer, D.; Cochet,
C. Mol. Cell. Biochem. 2008, 316, 63.
(
33PanQinaseÒ Activity Assay, ProQinase, Freiburg, Germany). The
assay was conducted as previously described.44 Briefly, inhibition

C. Hundsdörfer et al. / Bioorg. Med. Chem. 20 (2012) 2282–2289
2289
20. Sarno, S.; Pinna, L. A. Mol. Biosyst. 2008, 4, 889.
21. Sarno, S.; Salvi, M.; Battistutta, R.; Zanotti, G.; Pinna, L. A. Biochim. Biophys. Acta,
Proteins Proteomics 2005, 1754, 263.
22. Bal, C.; Baldeyrou, B.; Moz, F.; Lansiaux, A.; Colson, P.; Kraus-Berthier, L.;
Léonce, S.; Pierré, A.; Boussard, M.-F.; Rousseau, A.; Wierzbicki, M.; Bailly, C.
Biochem. Pharmacol. 1911, 2004, 68.
23. Hemmerling, H. J.; Reiss, G. Synthesis 2009, 985.
24. Hemmerling, H.-J.; Merschenz-Quack, A.; Wunderlich, H. Z. Naturforsch., B:
Chem. Sci. 2004, 59, 1143.
25. Greenhill, J. V.; Chaaban, I.; Steel, P. J. J. Heterocycl. Chem. 1992, 29, 1375.
26. Iida, H.; Yuasa, Y.; Kibayashi, C. Synthesis 1982, 471.
27. Bouaziz, Z.; Nebois, P.; Poumaroux, A.; Fillion, H. Heterocycles 2000, 52, 977.
28. Rebek, J.; Shaber, S. H.; Shue, Y. K.; Gehret, J. C.; Zimmerman, S. J. Org. Chem.
1984, 49, 5164.
33. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
34. Copeland, R. A. Enzymes A Practical Introduction to Structure, Mechanism and
Data Analysis; John Wiley & Sons: Hoboken, 2000.
35. http://www.molinspiration.com/cgi-bin/properties. Accession date: January
12, 2012.
36. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Del. Rev.
2001, 46, 3.
37. Clark, D. E. J. Pharm. Sci. 1999, 88, 807.
38. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
39. Gottesman, M. M. Annu. Rev. Med. 2002, 53, 615.
40. De Jonge, C.; Hagemann, H.; Hoentjen, G.; Mijs, W. Org. Synth. 1988, Coll. Vol. 6,
412.
41. Olgen, S.; Gotz, C.; Jose, J. Biol. Pharm. Bull. 2007, 30, 715.
42. Bracht, K.; Boubakari, G.; Grunert, R.; Bednarski, P. J. Anti-Cancer Drugs 2006,
17, 41.
29. Edstrom, E. D. Synlett 1995, 49.
30. Kuenzel, E. A.; Mulligan, J. A.; Sommercorn, J.; Krebs, E. G. J. Biol. Chem. 1987,
262, 9136.
43. Saczewski, F.; Reszka, P.; Gdaniec, M.; Grunert, R.; Bednarski, P. J. J. Med. Chem.
2004, 47, 3438.
31. Gratz, A.; Götz, C.; Jose, J. Electrophoresis 2010, 31, 634.
32. Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug Discovery; John Wiley &
Sons: Hoboken, 2005.
44. Aly, A. H.; Edrada-Ebel, R.; Indriani, I. D.; Wray, V.; Müller, W. E. G.; Totzke, F.;
Zirrgiebel, U.; Schächtele, C.; Kubbutat, M. H. G.; Lin, W. H.; Proksch, P.; Ebel, R.
J. Nat. Prod. 2008, 71, 972.