Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2

Article abstract of DOI:10.1016/j.bmc.2009.08.047

A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-t

Full text of DOI:10.1016/j.bmc.2009.08.047

Bioorganic & Medicinal Chemistry 17 (2009) 7281–7289
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2
Alessandra Gianoncelli a, Giorgio Cozza a, Andrzej Orzeszko b, Flavio Meggio a, Zygmunt Kazimierczuk c,
,
*
Lorenzo A. Pinna a,
*
a Department of Biological Chemistry and CNR Institute of Neurosciences, University of Padova, viale G. Colombo 3, 35-121 Padova, Italy
b Military University of Technology, Kaliskiego St. 2, 00-908 Warsaw, Poland
c Laboratory of Experimental Pharmacology, Polish Academy of Sciences Medical Research Center, Pawinskiego St. 5, 02-106 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2009
Revised 20 July 2009
Accepted 21 August 2009
Available online 27 August 2009
A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole
with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-car-
boxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of
comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the
new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiod-
obenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular
modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the
binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodo-
benzimidazole (TIBI) is one of the most efficient CK2 inhibitors (Ki = 23 nM) described to date.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Tetraiodobenzimidazoles
Tetrabromobenzimidazoles
CK2 inhibitors
Molecular modeling
1. Introduction
is ubiquitously distributed, constitutively active and participates
in a wide variety of cellular processes including gene expression,
The crucial role of protein kinases in nearly all cellular processes
and the growing list of kinases whose dysregulation underlies
pathological conditions (www.cellsignal.com/reference/kinase_dis
ease.html) highlight the importance of kinases as drug targets.1,2
Protein kinases have been actually implicated not only in oncology
but also in a number of non-oncology indications including central
nervous system disorders, autoimmune disease, post-transplant
immunosuppression, osteoporosis, and several metabolic disorders
like diabetes. Of special interest, among the large family of protein
kinases, appears the regulation of kinases implicated in fundamen-
tal processes of cell life like proliferation, survival and apoptosis
owing to the crucial commitment with the development and pro-
gression of cancer. The search of small molecules capable of down-
regulating kinase activity was primarily addressed toward the
nucleotide pocket of the kinase3 exploiting structural differences
in this region for molecular recognition.4 However, due to the high
conservation of the ATP-binding site, development of absolutely
specific ATP-competitive inhibitors is hard to attain and the possi-
bility to develop allosteric inhibitors is being explored.4
cell differentiation and proliferation and its activity has been asso-
ciated with a number of pathologies including inflammation, infec-
tion, and tumorigenesis.6–10 CK2 overexpression has been
invariably observed in transformed tissues and it appears to repre-
sent an unfavorable prognostic marker in some types of cancer
(e.g., prostate and lung carcinomas and in acute myeloid leuke-
mia).11,12 The nucleotide pocket of CK2, which can utilize both
ATP and GTP as phosphate donors, displays unique features for
being smaller than it is in most other kinases due to the presence
of some unique bulky residues hampering the entrance of large
molecules like staurosporine, a very powerful, highly promiscuous
inhibitor of most protein kinases but weakly effective on CK2.13,14
By contrast, smaller molecules which are not retained in the hydro-
phobic pocket of most kinases are firmly entrapped in that of CK2,
resulting in efficient inhibition. The molecular structure of CK2 in
mammals invariably consists of a heterotetrameric holoenzyme
composed of two catalytic (
a
and/or a0) and two regulatory (b)
subunits although emerging evidence would indicate specific roles
also for the isolated subunits under certain circumstances.15
Searching for CK2 inhibitors, just in the late 1980s polyhalogenated
benzimidazoles were found to represent a valuable scaffold to
effectively compete with ATP binding16 and 4,5,6,7-tetra-
bromobenzotriazole (TBB) was later demonstrated to be one of
the most powerful and selective cell permeable inhibitors of
CK2.17,18 The presence of four bromine atoms on the benzene ring
of TBB is critical to fill the CK2 hydrophobic pocket adjacent to the
ATP-binding site and to assure optimal apolar interactions with
Protein kinase CK2, formerly known as casein kinase-2 or II, is a
multifunctional serine/threonine protein kinase that specifically
phosphorylates residues located in acidic sequences of more than
300 substrates most of which are phosphorylated also in vivo.5 It
* Corresponding authors. Tel./fax: +48 22 822 1848.
E-mail addresses: ZKazimierczuk@gmail.com (Z. Kazimierczuk), lorenzo.pinna@
unipd.it (L.A. Pinna).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.047

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A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
some bulky side chains, in particular those of Val66 and Ile174.13,14
A direct correlation was also recently established for TBB deriva-
tives between the log Ki and the variation in the accessible surface
area upon binding on CK2.13 From this point of view, it was known
since longtime that the reduction of the number of bromine atoms
or their replacement by less bulky halogens like chlorine and fluo-
rine severely impairs the inhibitory efficiency.16,19 However, the
replacement of all four bromine atoms of the benzoimidazole or
benzotriazole moiety by bulkier iodines was hard to attain and
iodo-bromo or iodo-chloro benzimidazoles were unknown. Re-
cently, structurally related tetraiodinated-isoindole derivatives
were synthesized and found to inhibit protein kinase CK2 in an
ATP-competitive manner with IC50 values ranging between 0.15
Of the obtained tetraiodinatedbenzimidazoles, 2-bromo-4,5,
6,7-tetraiodobenzimidazole (13a) was found particularly useful
for further modifications. This compound was converted to 2-mer-
captoderivative 15 in the reaction with thiourea, whereas substitu-
tion of its bromine atom with ethanoloamine provided 2-
ethanoloamino-4,5,6,7-tetraiodobenzimidazole (14), and heating
of the compound with sodium acetate in acetic acid yielded the
2-oxoderivative (17) The sulfur atom in 15 can be easily alkylated
by halogenated compounds; for example, we used bromoacetic
acid to obtain the respective 2-carboxymethylthioderivative 16
(Scheme 2).
The ethyl esters of 1-carboxymethyl-substituted tetrahalogen-
ated benzimidazoles (18 and 19) were obtained from 4,5,6,7-tet-
rabromo- (TBI) and 4,5,6,7-tetraiodobenzimidazole 1a (TIBI) in
the reaction with ethyl bromoacetate, using potassium carbonate
as base catalyst. The esters were then subject to further modifica-
tions, such as alkaline hydrolysis—to provide the respective car-
boxylic acids 20 and 21. Acetamido derivatives of 4,5,6,7-
tetrahalogeneobenzimidazoles 22 and 23 were obtained by two
synthetic procedures: (a) by alkylation of the respective benzimid-
azole with iodoacetamide, or (b) by amidation with methanolic
ammonia. A nucleophilic substitution of above mentioned esters
by methylamine or hydrazine yielded the respective N-methyla-
mides 24 and 25 or hydrazides, 26 and 27 (Scheme 3).
and 1.5 l
M.20 The highest efficiency documented in the literature
for a CK2 inhibitor belongs to a class of pyrazolo-triazine deriva-
tives reported to affect CK2 with Ki values in the nanomolar and
subnanomolar range.21,22 Regrettably, however, the experimental
conditions for these assays were not detailed; this hampers reliable
comparison with other inhibitors. In this paper the synthesis of a
number of tetraiodinatedbenzimidazoles is described for the first
time together with a structure–activity analysis in comparison
with the corresponding tetrabrominated derivatives. In most cases
the replacement of bromines by iodines increases the potency of
the inhibitor reaching with some derivatives IC50 values in the
low nanomolar range.
2.2. Biological activity
2. Results and discussion
2.1. Chemical syntheses
Table 1 shows the inhibitory potency of all new derivatives to-
ward protein kinase CK2. Whenever applicable, Ki values of new
iodinated inhibitors are reported in comparison with the analogous
brominated compounds either newly synthesized and tested here
or drawn from the literature. As a main outcome of these determi-
nations we observed firstly that the iodinated inhibitors behave in
general more efficiently than the corresponding brominated ones
with Ki values for CK2 which, in some cases, reach the low nano-
molar range. In particular, the inhibition constants of tetraiodinat-
ed compounds 1a, 7a, 9a, and 14 (23, 24, 19, and 27 nM,
respectively) are among the lowest values ever reported in litera-
ture for CK2 inhibitors. Also these compounds, as it was previously
found with similar polyhalogenated derivatives,28 behave as com-
petitive inhibitors with respect to the phosphodonor nucleotide,
In this study, we made an effort to use an iodine-periodic acid
system to obtain three series of iodinated heterocyclic compounds,
namely iodinated benzimidazoles, benzotriazoles, and indazoles.
We succeeded only in the case of benzimidazoles, while the other
heterocycles tested, that is, benzotriazoles and indazoles, decom-
posed under the reaction conditions. The series of variously substi-
tuted benzimidazoles 1–13 that are commercially available or
were obtained according to the previously described methods
(e.g., see23–28) were used for the synthesis of the respective tetraio-
dinated derivatives 1a–13a using, with minor modifications, the
procedure described for aromatic compounds29 (Scheme 1).
X5
X5'
X4
X4'
N
N
a
X1
X1
N
N
X3
X3'
H
H
X2
X2'
1-13
1a - 13a
X1
H
H
H
H
X2
H
H
H
H
H
H
H
H
H
H
H
H
H
X3
H
H
X4
H
Cl
Br
Cl
H
H
H
Cl
H
H
X5
H
H
H
H
Cl
Br
H
H
H
H
H
H
H
X1
H
H
H
H
X2’ X3’ X4’ X5’
1
2
3
4
5
6
7
8
9
10
11
12
13
1a
2a
3a
4a
5a
6a
7a
8a
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Cl
Cl
Br
I
Cl
I
I
I
I
I
I
Cl
Br
Cl
I
I
I
Cl
I
I
I
I
I
H
Cl
Cl
Br
H
Cl
H
H
H
H
H
I
H
H
H
H
Cl
Br
I
I
I
I
I
I
I
CH3
CH3
C2H5
CF3
C2F5
Cl
CH3
CH3
9a C2H5
10a CF3
11a C2F5
H
H
H
I
I
I
12a
13a
Cl
Br
Br
Scheme 1. Reagents and conditions: (a) I2, H5IO6, H2SO4, 3 h, 5–60 °C.

A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
7283
I
I
I
I
I
I
N
N
a
Br
NHCH2CH2OH
N
H
N
H
I
I
13a
14
b
d
I
I
I
H
H
N
I
I
I
I
I
I
N
N
c
O
S
SCH2COOH
N
N
H
N
H
H
I
I
I
17
15
16
Scheme 2. Reagents and conditions: (a) 2-aminoethanol, EtOH; (b) thiourea, MeOEtOH; (c) BrCH2COOH, K2CO3, 2-butanone, reflux, 3 h; (d) acetic acid, sodium acetate, reflux,
20 h.
X
X
X
X
X
X
X
X
X
N
N
N
N
N
a
b
N
H
CH2COOEt
CH2COOH
X
X
X
18 X = Br
19 X = I
TBBz X = Br
1a X = I
20 X = Br
21 X = I
e
c
d
X
X
X
X
X
X
X
X
N
N
N
N
N
N
X
CH2CONHNH2
CH2CONH2
CH2CONHCH3
X
X
X
22 X = Br
23 X = I
24 X = Br
25 X = I
26 X = Br
27 X = I
Scheme 3. Reagents and conditions: (a) BrCH2COOEt, K2CO3, 2-butanone, reflux, 3 h; (b) NaOH, EtOH–H2O, 50 °C, 3 h; (c) ICH2CONH2, K2CO3, 2-butanone, reflux, 3 h; (d)
H2NCH3, EtOH, rt, 2d; (e) H2NNH2ꢀH2O, EtOH or MeOEtOH.
displaying a classical double-reciprocal plot allowing to calculate,
in the case of 4,5,6,7-tetraiodobenzimidazole (1a or TIBI), a Ki value
of 23 nM (Fig. 1A). Conversely, inhibition is non-competitive with
respect to the peptide phosphoacceptor substrate (Fig. 1B) further
supporting a mechanism of action implying the occupancy of the
nucleotide binding pocket in competition with ATP. Moreover,
the performance of compound 1a allows to definitely rank the effi-
ciency of halogens as substituents of the benzene ring, iodine being
tetraiodinated versus tetrabrominated compounds highlights that
the presence of an additional substitution on the imidazole ring
may affect not only the absolute potency of the inhibitor but also
its relative efficacy with respect to the brominated homologue. In
fact, all the four compounds bearing a substitution at position 1
of the imidazole ring (compounds 21, 23, 25 and 27 of Table 1) dis-
play a Ki value about one order of magnitude higher than that of
derivative 1a having no substitution at that position. Nevertheless,
they are still 10-fold more potent than the correspondent bromi-
nated derivatives as it was observed in the case of the un-substi-
tuted compound 1a. On the contrary, the added value of the
presence of four iodine atoms on the benzene moiety appears to
be variably affected by substitutions at position 2 of the imidazole
ring. These latter compounds, with very few exceptions, display
inhibition constants significantly higher and close to those of the
corresponding brominated derivatives.
the best, followed by bromine (0.023 vs 0.3 lM by comparing,
compounds 1a and K17 (also termed TBI) of Table 1) which in turn
proved to be about two orders of magnitude more effective than
chlorine, 4,5,6,7-tetrachlorobenzimidazole (TCI) exhibiting a Ki va-
lue of 21 l
M under comparable conditions.30 This came not as a
surprise considering the data already available for di-substituted
benzimidazole derivatives in which fluorine was found to be the
least effective.16,19 However, a deeper analysis of the Ki values of

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A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
Table 1
Inhibition of protein kinase CK2 by iodinated benzimidazole derivatives (A) in comparison to respective brominated compounds (B)
A
Ki (lM)
B
Ki (
lM)
Ratio B/A
Imidazole substitution
Iodinated compounds
Brominated compounds
1a (K88, TIBI)
2a (K105)
3a (K104)
4a (K98)
5a (K106)
6a (K95)
7a (K92)
8a (K99)
9a (K93)
10a (K89)
11a (K94)
12a (K103)
13a (K96)
14 (K100)
15 (K97)
16 (K101)
17 (K112)
21 (K107)
23 (K108)
25 (K109)
27 (K110)
0.023
0.16
0.10
0.46
0.59
0.075
0.024
0.33
0.019
0.12
0.07
0.12
0.09
0.027
0.05
0.054
0.05
0.14
0.13
0.18
0.16
K17a,b (TBI)
—
—
—
—
—
—
—
—
0.30
—
—
—
—
—
—
—
—
0.37
0.20
0.25
0.37
0.14
0.20
0.12
0.18
1.34
1.45
1.91
1.51
13.04
—
—
—
—
—
—
—
—
3.08
2.87
2.08
4.11
5.18
4.00
2.22
3.60
9.57
11.15
10.60
9.40
None
None
None
None
None
None
2-CH3
2-CH3
2-C2H5
2-CF3
2-C2F5
2-Cl
2-Br
2-NH(CH2)2OH
2-SH
2-SCH2COOH
2@O
1-CH2COOH
1-CH2CONH2
1-CH2CONHCH3
1-CH2CONHNH2
K10a
K11a
K21a
K20a
K30b
K22a
K33b
K32b
20 (K68)
22 (K83)
24 (K84)
26 (K85)
Bold labels refer to compounds whose synthesis is described in this paper. The corresponding nomenclature (K) adopted in previous publications is also reported as a
reference.
a
Drawn from Andrzejewska et al.27
Drawn from Pagano et al.28
b
2.3. Molecular modeling studies
smaller ones (V66I174AA) undergoes a 70- and 16-fold reduced
inhibition by compounds 1a and TBI, respectively (data not
shown). This corroborates the view that, a part from halogen
bonds, occupancy and hydrophobic interactions play a crucial role
in optimizing the performance of tetraiododerivatives.
Searching for a plausible structural explanation of the superior-
ity of tetraiodinated derivatives as CK2 inhibitors, a molecular
modeling approach was undertaken. Starting from the crystal
structure of K17 in complex with ZmCK2
a subunit (PDB code
2OXY)13 we determined the binding mode of compound 1a by a
molecular docking strategy, in comparison with that of the tetrab-
romo (TBI, K17) and tetrachloro (TCI) homologues, using human
CK2 as target protein (PDB code: 1JWH)31 (Fig. 2). All three inhib-
itors lay inside the CK2 binding cleft exactly at the same position as
3. Experimental
3.1. Chemical synthesis
3.1.1. General procedure
K17 in ZmCK2a, revealing identical binding modes. However, com-
All chemicals and solvents were purchased from Sigma–Aldrich.
Melting points (uncorr.) were measured in open capillary tubes on
a Gallenkamp-5 melting point apparatus. Ultraviolet absorption
spectra were recorded on Kontron Uvikon 940 spectrophotometer.
1H NMR spectra (in ppm) were measured with Varian Gemini
200 MHz (or Varian UNITYplus 500 MHz) spectrometer at 298 K
in DMSO-d6 using tetramethylsilane as internal standard. Mass-
spectra (70 eV) were obtained with AMD-604 (Intectra) spectrom-
eter. Flash chromatography was performed with Merck silica gel
60 (200–400 mesh). Elemental analyses of the new compounds
were within 0.4% of the respective theoretical values.
pound 1a is able to fill better the CK2 binding cleft due to the in-
creased dimensions of iodine substituents with respect to
bromine and chlorine. To better validate this hypothesis we calcu-
lated the percentage of human CK2 ATP-binding cleft occupancy
(occupancy factor, OF) by the three analogs. The CK2 ATP-binding
pocket volume was calculated as described in Section 3 by using
the apo crystal structure 1JWH plus one conserved water mole-
cule.13 The OF values were calculated relative to the binding cleft
and the inhibitor’s volume. As also shown in Fig. 2, TCI shows a
low binding cleft OF value (69%), and tetrabromine substitution
is responsible of an increased occupancy up to 75% while the most
potent inhibitor 1a (TIBI), bearing the iodine substitution, fills by
86% the CK2 ATP-binding pocket. Almost identical conclusions
can be drawn by comparing the increased hydrophobicity with
the increased inhibitory potency of the tetrahalogenated deriva-
tives. Moreover, iodine atoms of derivative 1a are able to perform
stronger halogen bonds with the binding cleft, in particular with
the backbone carbonyls of Glu114 and Val116 of the hinge re-
gion.13 In fact, even if all four halogen atoms are capable of acting
as halogen bond donors, iodine usually forms the strongest interac-
tions, followed by bromine and chlorine atoms, respectively.32 The
same arguments apply also to the other tetraiodobenzimidazole
derivatives considered in this paper, all displaying in general an
inhibitory efficiency higher than that of their brominated counter-
parts. Note, however, that a double mutant of CK2 in which two
bulky residues of the hydrophobic pocket have been replaced by
3.1.2. General procedure for iodination of benzimidazole
derivatives
The amounts of reagents used for the preparation of tetraiodo-
benzimidazoles were according below described procedure. For
diiodo- or triiodo-derivatives amounts of iodine and periodic acid
were, respectively, reduced.
Periodic acid (2.28 g, 10 mmol) was dissolved in concd H2SO4
(50 mL). Finely powdered iodine (10.1 g, 40 mmol) was added to
the solution. After 20 min of stirring the homogenous mixture
was placed in ice bath. Next, the respective benzimidazole
(10 mmol) was slowly added (exothermic reaction). The reaction
mixture was stirred at room temperature for 1 h and then has been
heated to the final (60 °C) temperature. After next 2 h the mixture
was poured onto crushed ice. The resulting dark solid was filtered
off, added to the solution of sodium dithionite (25 mL, 10%) and

A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
7285
Figure 1. Double-reciprocal plot analysis of CK2 inhibition by compound 1a (TIBI). CK2 activity was determined as described in Section 3 either in the absence or in the
presence of the indicated fixed concentrations of inhibitor. In A the concentration of the peptide substrate was constant (100 M) at increasing concentrations of ATP while in
B the ATP concentration was constant (80 M) at increasing concentrations of peptide substrate. The data represent the mean of triplicate experiments with SEM never
l
l
exceeding 15%. The Ki of the inhibitor competitive with respect to ATP was then calculated by linear regression analysis of Km/Vmax versus inhibitor concentration plot shown
in the inset of panel A.
stirred for 10 min. The crude product was dissolved in EtOH/2 M
NaOH (1:1) and filtered. The pale yellow or green filtrate was
brought to pH 4–5 with acetic acid to yield respective iodinated
benzimidazole as yellow or yellowish chromatographically pure
precipitate. For analysis and biological investigation small amounts
were crystallized from EtOH, MeOEtOH, dioxane, DMF/dioxane or
purified by alkalization and precipitation with acetic acid from or-
ganic solvent–water mixtures.
3.1.4. 5-Chloro-4,6,7-triiodo-1H-benzimidazole (2a)
As described above starting from 5-chloro-benzimidazole. Mp
256–259 °C (with decomp.), 65%. A sample for analysis was crys-
tallized from EtOH. UV (MeOH): 230 (23,300), 281 (14,300), 305
(sh, 5800). 1H NMR (Me2SO-d6) d (ppm): 8.32 (s, H–C), 13.05
(br s, H–N). MS m/z: 532 (33, M++2), 530 (100, M+), 440 (19,
M+ꢁ70), 438 (22, M+ꢁ22), 403 (M+ꢁ127). Anal. Calcd for
C7H2ClI3N2 (530.27): C, 15.86; H, 0.38; N, 5.28. Found: C, 15.88;
H, 0.40; N, 5.32.
3.1.3. 4,5,6,7-Tetraiodo-1H-benzimidazole (1a)
From benzimidazole as described above: (mp 296–299 °C (de-
comp.) I2 evol. >250 °C, 77%. A sample for analysis was crystallized
from DMF/dioxane. UV (MeOH): 233 (28,600), 275 (9900), 295
(10,300). 1H NMR (Me2SO-d6) d (ppm): 8.21 (s, 1H, H–C), 12.85
(br s, 1H, H–N). MS m/z: 623 (9, M++1), 622 (100, M+), 495 (16,
M+ꢁ127), 368 (22, M+ꢁ254). Anal. Calcd for C7H2I4N2 (621.72): C,
13.52; H, 0.32; N, 4.51. Found: C, 13.67; H, 0.44; N, 4.40.
3.1.5. 5-Bromo-4,6,7-triiodo-1H-benzimidazole (3a)
As described above starting from 5-bromo-benzimidazole. Mp
253–255 °C (with decomp.), 65%. A sample for analysis was crystal-
lized from methoxyethanol. UV (MeOH): 236 (19,500), 282
(12,900), 305 (sh, 5600). 1H NMR (Me2SO-d6) d (ppm): 8.28 (s,
1H, H–C), 13.0 (br s, 1H, H–N). MS m/z: 576 (M++1), 574 (M+ꢁ1),
482 (32, M+ꢁ93), 449 (15, M+ꢁ126), 447 (M+ꢁ128). Anal. Calcd

7286
A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
Figure 2. Molecular docking of the tetraiodinated (A), tetrabrominated (B), and tetrachlorinated (C) benzimidazole derivatives bound to the active site of protein kinase CK2.
Computational analysis was performed as described in Section 3. Connolly’s distribution surfaces of the three inhibitors inside the nucleotide pocket are represented in light
green. The occupancy factor (OF) values, discussed in the text, are also indicated.
for C7H2BrI3N2 (574.72): C, 14.63; H, 0.35; N, 4.87. Found: C, 14.50;
H, 0.42; N, 4.76.
UV (MeOH): 234 (29,300), 277 (10,300), 292 (10,500). 1H NMR
(Me2SO-d6) d (ppm): 2.47 (s, 3H, H3C), 12.5 (br s, 1H, H–N). MS
m/z: 637 (10, M++1), 636 (100, M+), 509 (17 (M+ꢁ127), 382
(M+ꢁ254). Anal. Calcd for C8H4I4N2 (635.75): C, 15.11; H, 0.63; N,
4.41. Found: C, 15.09; H, 0.73; N, 4.27.
3.1.6. 5,6-Dichloro-4,7-diiodo-1H-benzimidazole (4a)
As described above from 5,6-dichloro-benzimidazole. Mp 271–
273 °C (with decomp.), 70%. A sample for analysis was crystallized
from EtOH. UV (MeOH): 229 (23,000), 278 (19,000), 302 (9500). 1H
NMR (Me2SO-d6) d (ppm): 8.40 (s, 1H, H–C), 13.2 (br s, 1H, H–N).
MS m/z: 442 (10, M++4), 440 (63, M++2), 438 (100, M+), 311
(M+ꢁ127). Anal. Calcd for C7H2Cl2I2N2 (438.82): C, 19.16; H, 0.46;
N, 6.38. Found: C, 19.19; H, 0.55; N, 6.23.
3.1.10. 5,6-Dichloro-4,7-diiodo-2-methyl-1H-benzimidazole
(8a)
As described above from 5,6-dichloro-2-methylbenzimidazole.
Mp 288–290 °C (with decomp.), 52%. A sample for analysis was
crystallized from EtOH. UV (MeOH): 234 (23,500), 275 (18,300),
303 (8500). 1H NMR (Me2SO-d6) d (ppm): 2.52 (s, 3H, H3C), 12.8
(br s, 1H, H–N). MS m/z: 456 (11, M++4), 454 (56, M++2), 452
(100, M+), 311 (M+ꢁ127). Anal. Calcd for C8H4Cl2I2N2 (452.85): C,
21.22; H, 0.89; N, 6.19. Found: C, 21.15; H, 0.95; N, 6.08.
3.1.7. 4,6-Dichloro-5,7-diiodo-1H-benzimidazole (5a)
As described above from 4,6-dichloro-benzimidazole. Mp 254–
256 °C, 42%. A sample for analysis was crystallized from EtOH. UV
(MeOH): 236 (22,500), 273 (8700), 302 (4400). 1H NMR (Me2SO-d6)
d (ppm): 8.39 (s, 1H, H–C), 13.08 and 13.48 (br s, 1H, H–N). MS m/z:
440 (28, M++1), 438 (100, M+ꢁ1), 313 (12, M+ꢁ126), 311 (13,
M+ꢁ128). Anal. Calcd for C7H2Cl2I2N2 (438.82): C, 19.16; H, 0.46;
N, 6.38: Found: C, 19.14; H, 0.44; N, 6.34.
3.1.11. 2-Ethyl-4,5,6,7-tetraiodo-1H-benzimidazole (9a)
As described above from 2-ethyl-benzimidazole. Mp 261–
262 °C (from EtOH), 68%. UV (MeOH): 237 (28,500), 277 (9700),
293 (11,100). 1H NMR (Me2SO-d6) d (ppm): 1.28 (t, 3H, H3C,
J = 7.6 Hz), 2.82 (q, 2H, H2C, J = 7.6 Hz), 12.6 (br s, 1H, H–N). MS
m/z: 637 (10, M++1), 636 (100, M+), 509 (17 (M+ꢁ127), 382
(M+ꢁ254). Anal. Calcd for C9H6I4N2 (649.76): C, 16.64; H, 0.93; N,
4.31. Found: C, 16.59; H, 0.99; N, 4.20.
3.1.8. 4,6-Dibromo-5,7-diiodo-1H-benzimidazole (6a)
As described above from 4,6-dibromo-benzimidazole. Mp 309–
311 °C (with decomp.), 70%. A sample for analysis was crystallized
from dioxane. UV (MeOH): 236 (21,300), 277 (11,700), 303 (sh,
4200). 1H NMR (Me2SO-d6) d (ppm): 8.34 (s, 1H, H–C), 13.09 and
13.23 (br s, 1H, H–N). MS m/z: 529 (47 (M++2), 527 (100, M+), 525
(48, M+ꢁ2), 400 (M+ꢁ127). Anal. Calcd for C7H2Br2I2N2 (527.72): C,
15.93; H, 0.38; N, 5.31. Found: C, 15.90; H, 0.47; N, 5.22.
3.1.12. 4,5,6,7-Tetraiodo-2-trifluoromethyl-1H-benzimidazole
(10a)
As described above from 2-trifluoromethylbenzimidazole Mp
240–242 °C (with decomp.) (from MeOEtOH), 38%. UV (MeOH):
237 (35,200), 283 (10,000), 303 (9900). 1H NMR (Me2SO-d6) d
(ppm): 14.4 (br s, H–N). MS m/z: 690 (5, M++1), 689 (100, M+), 563
(16, M+ꢁ126), 436 (20, M+ꢁ253). Anal. Calcd for C8HF3I4N2
(689.72): C, 13.93; H, 0.15; N, 4.31. Found: C, 13.78; H, 0.24; N, 4.20.
3.1.9. 2-Methyl-4,5,6,7,-tetraiodo-1H-benzimidazole (7a)
As described above from 2-methyl-benzimidazole: Mp 289–
291 °C, 69%. A sample for analysis was crystallized from dioxane.

A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
7287
3.1.13. 2-Pentafluoroethyl-4,5,6,7-tetraiodo-1H-benzimidazole
(11a)
2,87 mmol) in butanone (45 mL) was refluxed for 6 h. The insolubil-
ities were filtered off and washed with 50 mL of hot butanone. The
filtrate was evaporated to dryness. The residue was crystallized from
dioxane; mp 274–276° (decomp. >240); yield 70% (4.98 g). UV
(MeOH: 254 (13,100), 309 (12,300). 1H NMR (Me2SO-d6) d (ppm):
4.16 (s, 2H, H2C), 12.9 (br s, 1H, HO). TOF MS ES+: 712.6257
(M++H+, 712.6251). Anal. Calcd for C9H4I4N2O2S (711.83): C, 15.19;
H, 0.57; N, 4.50. Found: C, 15.20; H, 0.51; N, 4.43.
As described above from 2-pentafluoroethylbenzimidazole. Mp
247–249 °C (with decomp.), 48% (from EtOH). UV (MeOH): 237
(36,100), 284 (11,000), 304 (11,200). 1H NMR (Me2SO-d6) d
(ppm): 14.3 (br s, 1H, H–N). MS m/z: 741 (10, M++1), 740 (100,
M+), 613 (16, M+ꢁ127), 486 (20, M+ꢁ254). Anal. Calcd for
C9HF5I4N2 (739.73): C, 14.61; H, 0.14; N, 3.79. Found: C, 14.51; H,
0.22; N, 3.64.
3.1.19. 4,5,6,7-Tetraiodo-1H,3H-dihydro-2H-benzimidazole-2-
one (17)
3.1.14. 2-Chloro-4,5,6,7-tetraiodo-1H-benzimidazole (12a)
As described above from 2-chlorobenzimidazole. Mp 303–
306 °C (with decomp., >250 °C I2 evol.), 37%. A sample for analysis
was crystallized from DMF/dioxane. UV (MeOH): 235 (35,200), 275
(12,200) 295 (12,000), 315 (sh). 1H NMR (Me2SO-d6) d (ppm): 13.7
(br s, 1H, H–N). MS m/z: 658 (42, M++2), 656 (100, M+), 529 (24,
M+ꢁ127), 402 (35, M+ꢁ254). Anal. Calcd for C7HClI4N2 (656.17):
C, 12.81; H, 0.15; N, 4.00. Found: C, 12.75; H, 0.20; N, 3.92.
The stirred mixture of 13a (700 mg, 1 mmol), anhyd sodium
acetate (410 mg, 5 mmol) in anhyd acetic acid was refluxed for
20 h. The mixture was cooled, the formed precipitate was filtered
and washed few times with EtOH–water (1:1). The product
(608 mg, 95%) was chromatographically pure. For analytical and
biological investigation small amount was crystallized from
EtOH/dioxane; mp >310 °C (decomp.) UV (MeOH): 236 (36,900),
306 (6400). 1H NMR (Me2SO-d6) d (ppm): 11.04 (s, H–N-imid.).
MS m/z: 639 (9, M++1), 638 (100, M+), 512 (25 (M+ꢁ128), 384
(27, M+ꢁ256). Anal Calcd for C7H2N2I4O (637.72): C, 13.18; H,
0.32; N, 4.39. Found: C, 13.37; H, 0.38; N, 4.26.
3.1.15. 2-Bromo-4,5,6,7-tetraiodo-1H-benzimidazole (13a)
As described above from 2-bromobenzimidazole: Mp 296–
299 °C, (I2 evol. >230 °C), 88%. A sample for analysis was crystal-
lized from DMF/dioxane. UV (MeOH): 239 (28,100), 273 (12,100),
300 (12,700). 1H NMR (Me2SO-d6) d (ppm): 13.6 (br s, 1H, H–N).
MS m/z: 701 (96, M+), 699 (M+ꢁ2), 574 (19, M+ꢁ127), 572 (17,
M+ꢁ129). Anal. Calcd for C7HBrI4N2 (700.62): C, 12.00; H, 0.14;
N, 4.00. Found: C, 12.00; H, 0.19; N, 3.84.
3.1.20. (4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)-acetic acid
ethyl ester (18)
Similarly to 16 butanone was used as a solvent at reflux condi-
tion for 5 h. Instead of bromoacetic acid ethyl ester of this acid was
used (mp 199–200 °C, 80%). UV (MeOH): 228 (35,900), 264
(11,900), 272 (11,800), 302 (4100). 1H NMR (Me2SO-d6) d (ppm):
1.23 (t, 3H, H3C, J = 7.1 Hz), 4.19 (q, 2H, H2C, J = 7.1 Hz), 5.29 (s,
2H, H2C–N), 8.32 (s, 1H, H–C). MS m/z: 524 (16, M++4), 522 (65,
M++2), 520 (100, M+), 518 (67, M+ꢁ2), 516 (17, M+ꢁ4). Anal. Calcd
for C11H8Br4N2O2 (519.81): C, 25.42; H, 1.55; N, 5.39. Found: C,
25.44; H, 1.56; N, 5.35.
3.1.16. 2-[(2-Hydroxyethyl)amino)]-4,5,6,7-tetraiodo-1H-
benzimidazole (14)
To a mixture of 13a (600 mg, 0.85 mmol) in MeOEtOH (15 mL),
ethanoloamine (2 g, 2 mL, 33 mmol) was added. The reaction mix-
ture was stirred and heated at 110–115 °C (bath temp) for 10 h.
The reaction mixture was diluted with water (20 mL) and brought
to pH 4–5. The precipitate was filtered and deposited on silica gel
column (2 ꢂ 12 cm). Chromatography was performed with CHCl3
(200 mL) and CHCl3–MeOH (95:5, v/v). The product containing
fractions were evaporated to dryness and the residue crystallized
from dioxane–water to give pale yellow powder (320 mg, 55%);
mp 249–251 °C. UV (MeOH): 244 (35,200), 274 (13,300), 315
3.1.21. (4,5,6,7-Tetraiodo-1H-benzimidazol-1-yl)-acetic acid
ethyl ester (19)
Similarly to 18 butanone and bromoacetic acid ethyl ester were
used at reflux condition for 5 h: (mp 224 °C, 77%). UV (MeOH): 236
(32,500), 279 (11,400), 299 (10,800). 1H NMR (Me2SO-d6) d (ppm):
1.23 (t, 3H, H3C, J = 7.1 Hz), 4.19 (q, 2H, H2C, J = 7.1 Hz), 5.29 (s, 2H,
H2C–N), 8.32 (s, 1H, H–C). MS m/z: 709 (13, M++1), 708 (100, M+),
635 (14, M+ꢁ73), 553 (20, M+ꢁ155). Anal. Calcd for C11H8I4N2O2
(707.82): C, 18, 67; H, 1.14; N, 4.52. Found: C, 18.65; H, 1.18; N, 4.39.
(12,200). 1H NMR (Me2SO-d6)
d (ppm): 3.40 (q, 2H, H2C,
J = 5.6 Hz), 3.57 (t, 2H, H2C, J = 5.5 Hz), 5.01 (br s, 1H, HO), 6.45 (t,
1H, H–N–CH2, J = 6.6 Hz), 10.9 (br s, 1H, H–N-imid.). MS m/z: 681
(5, M++1), 680 (100, M+), 662 (19, M+ꢁ18), 637 (100, M+ꢁ43),
510 (60, M+ꢁ170). Anal Calcd for C9H7N3I4O (680.79): C, 15.88;
H, 1.04; N, 6.17. Found: 15.91; H, 1.15; N, 6.06.
3.1.22. (4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)-acetic acid
(20)
3.1.17. 4,5,6,7-Tetraiodo-1H,3H-dihydro-2H-benzimidazole-2-
thione (15)
The suspension of 18 (520 mg, 1 mmol) in solution of KOH
(280 mg, 5 mmol) in ethanol/water (3:1, v/v, 16 mL) was stirred
at room temperature for 3 h. After two hours the mixture became
clear. The solution was brought to pH 2–3 with concd HCl. The
white chromatographically pure precipitate was filtered and
washed with water (390 mg, 79%). A sample for analysis and bio-
logical purposes was crystallized from dioxane–water gave white
crystals with no melting point until 300 °C (melt. and decomp.
>315 °C). UV (MeOH): 228 (28,500), 265 (9800), 272 (9900), 302
(3500). 1H NMR (DMSO-d6): 5.36 (s, CH2), 8.32 (s, H–C), 13.4 (br
s, OH). MS m/z: 496 (16, M++4), 494 (64, M++2), 492 (100, M+),
490 (66, M+ꢁ2), 488 (M+ꢁ4), 447 (70, M+ꢁ45). Anal. Calcd for
C9H4Br4N2O2 (491.76): C, 21.98; H, 0.82; N, 5.70. Found: C, 22.15;
H, 0.95; N, 5.51.
To the stirred solution of 13a (700 mg, 1 mmol) in MeOEtOH
(25 mL) thiourea (156 mg, 2 mmol) was added. The reaction mix-
ture was stirred and heated at 105–110 °C (bath temp) for 14 h.
A precipitate formed was filtered, dissolved in EtOH/ 0.2 M aq.
NaOH (1:1, v/v) and precipitated by addition of acetic acid to pH
4–5 to give as pale yellow chromatographically pure precipitate
(520 mg, 80%); mp (decomp. >290 °C with I2 evol.) (for analysis
crystallized from MeOEtOH–water). UV (MeOH): 222 (18,600),
264 (35,450), 336 (20,500). 1H NMR (Me2SO-d6) d (ppm): 12.8 (br
s, 1H, H–N). MS m/z: 655 (5, M++2), 654 (9, M++1), 653 (100, M+),
527 (M+ꢁ126), 400 (M+ꢁ253). Anal Calcd for C7H2N2I4S (653.78):
C, 12.86; H, 0.31; N, 4.28. Found: C, 12.95; H, 0.42; N, 4.13.
3.1.18. (4,5,6,7-Tetraiodo-1H-benzimidazol-2-ylsulfanyl)-acetic
acid (16)
3.1.23. (4,5,6,7-Tetraiodo-1H-benzimidazol-1-yl)-acetic acid
(21)
The mixture of 2-mercapto-4,5,6,7-tetraiodo-1H-benzimidazole
(751 mg, 1.15 mmol), K2CO3 (1 g), bromoacetic acid (400 mg,
Similarly to 20 starting from 19, however stirred at 50 °C: (mp
250–252 °C (decomp. with gas evol.), 70%). UV (MeOH): 238

7288
A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
(32,400), 279 (9100), 298 (8400). 1H NMR (Me2SO-d6) d (ppm):
5.21 (s, H2C), 8.37 (s, H–C), 13.3 (br s, OH). TOF MS ES+: 680.6526
(M++H+, 680.6530) Anal. Calcd for C9H4I4N2O2 (679.76): C, 15.90;
H, 0.59; N, 4.12. Found: C, 15.91; H, 0.58; N, 4.09.
(220 mg, 72%). A sample for analysis was crystallized from dioxane.
Mp >305 °C (decomp.). UV (MeOH): 232 (30,600), 267 (11,800),
273 (11,600), 302 (3800). 1H NMR (Me2SO-d6) d (ppm): 4.29
(br s, H2N), 5.50 (s, H2C), 8.44 (s, H–N), 9.37 (s, H–C). MS m/z:
510 (12, M++4), 508 (46, M++2), 506 (71, M+), 504 (47, M+ꢁ2),
502 (12, M+ꢁ4), 425 (100, M+ꢁ126). Anal. Calcd for C9H6Br4N4O
(505.79): C, 21.37; H, 1.20; N, 11.08. Found: C, 21.44; H, 1.30; N,
11.26.
3.1.24. 2-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)-
acetamide (22)
Method A: The suspension of 19 (260 mg, 0.5 mmol) in metha-
nolic ammonia (10 mL, saturated at 0 °C, diluted with 10 mL
MeOH) was stirred at room temperature for one week. The precip-
itate was filtered off and crystallized from DMF/dioxane gave
white powder (195 mg, 80%). Mp >325 °C.
Method B: To the stirred and refluxed mixture of TBI (260 mg,
0.5 mmol), anhyd K2CO3 (600 mg) in dioxane iodoacetamide
(130 mg, 0.7 mmol) was added. The stirring and reflux was contin-
ued for 2 h. The mixture was chilled and water (20 mL) was added.
The precipitate was filtered off and crystallized from DMF/dioxane
gave white powder (180 mg, 73%). Mp >325 °C. UV (MeOH): 230
(30,100), 267 (10,800), 273 (10,400), 302 (3500). 1H NMR
(Me2SO-d6) d (ppm): (s, H2C), 7.37 and 7.72 (2s, H2N), 8.43 (s,
HC). MS m/z: 495 (6), 493 (22, M++4), 491 (36, M++2), 489 (26,
M+ꢁ2), 487 (7, M+ꢁ4), 447 (65, M+ꢁ44), 410 (100, M+ꢁ81). Anal.
Calcd for C9H5Br4N3O (490.78): C, 22.03; H, 1.03; N, 8.58. Found:
C, 21.88; H, 1.12; N, 8.43.
3.1.29. (4,5,6,7-Tetraiodo-1H-benzimidazol-1-yl)-acetic acid
hydrazide (27)
Similarly to 26, however instead ethanol methoxyethanol at
100 °C (bath temp) was used. Mp 264–266 (decomp. with gas
evol.), 75%. A sample for analysis was crystallized from dioxane/
ethanol. UV (MeOH): 236 (40,100), 282 (10,500), 299 (10,300).
1H NMR (Me2SO-d6) d (ppm):4.23 (br s, 2H, H2N), 5.05 (s, 2H,
H2C), 8.27 (s, 1H, H–N), 9.31 (s, 1H, H–C). MS m/z: 694 (7, M+),
621 (100, M+ꢁ73), 496 (87, M+ꢁ198). Anal. Calcd for C9H6I4N4O
(693.79): C, 15.58; H, 0.87; N, 8.08. Found: C, 15.55; H, 0.89,
8.05.
4. Biological evaluation
4.1. Materials
4.1.1. Phosphorylation
3.1.25. 2-(4,5,6,7-Tetraiodo-1H-benzimidazol-1-yl)-acetamide
(23)
CK2 activity was tested in a final volume of 25
50 mM Tris–HCl pH 7.5, 100 mM NaCl, 12 mM MgCl2, 100
thetic peptide substrate RRRADDSDDDDD and 0.02 mM [
ATP] (500–1000 cpm/pmol), unless otherwise indicated, and incu-
bated for 10 min at 37 °C. Native CK2 purified from rat liver (0.5–
1 pmol) was usually the phosphorylating enzyme. Assays were
l
l containing
M syn-
33P-
l
Similarly to 22 (method B) starting from 19: (mp 291–301 °C
(decomp. with I2 evol.), 65%). UV (MeOH): 236 (29,100), 278
(8100) 300 (8400). 1H NMR (Me2SO-d6) d (ppm): 5.05 (s, H2C),
7.34 and 7.68 (2s, H2N), 8.29 (s, H–C). MS m/z: 679 (9, M+), 622
(48, M+ꢁ57), 553 (49, M+ꢁ126), 496 (100, M+ꢁ183). Anal. Calcd
for C9H5I4N3O (678.78): C, 15.93; H, 0.74; N, 6.19. Found: C,
15.99; H, 0.77; N, 6.13.
c
-
stopped by addition of 5 ll of 0.5 M orthophosphoric acid before
spotting aliquots onto phosphocellulose filters. Filters were
washed in 75 mM phosphoric acid (5–10 mL/each) four times then
once in methanol and dried before counting. Inhibition constants
were determined from Lineweaver–Burk double-reciprocal plots
of data obtained in experiments run at fixed concentration of pep-
tide substrate and at increasing concentrations of ATP either in the
absence or in the presence of variable concentrations of inhibitor.
Alternatively inhibition constants were also deduced from the
IC50/Ki Cheng–Prusoff relationship.33
3.1.26. N-Methyl-2-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)-
acetamide (24)
The suspension of 18 (260 mg, 0.5 mmol) in ethanolic methyl-
amine (25 mL, 20%) was stirred at room temperature for 2 d. The
precipitate was filtered off and crystallized from dioxane gave pure
white crystalline powder (230 mg, 91%); mp 322–325 °C (with
dec.). UV (MeOH): 232 (27,900), 267 (10,800), 273 (10,600), 302
(3600). 1H NMR (Me2SO-d6) d (ppm): 2.63 (d, 3H, H3C, J = 4.6 Hz),
5.16 (s, 2H, H2C), 8.12 (q, 1H, H–N, J = 4.7 Hz), 8.43 (s, 1H, H–C).
MS m/z: 507 (9, M++2), 505 (13, M+), 503 (10, M+ꢁ2), 426 (100,
M+ꢁ79). Anal. Calcd for C10H7Br4N3O (504.80): C, 23.79; H, 1.40;
N, 8.32. Found: C, 23.64; H, 1.52; N, 8.29.
4.1.2. Molecular modeling
The human CK2a subunit was retrieved from the PDB (code
1JWH)31 and processed in order to remove the ligands and all
water molecules except to the one conserved.13 Hydrogen atoms
were added to the protein structure using standard geometries
with the MOE program.34 To minimize contacts between hydrogens,
the structure were subjected to Amber99 force-field minimization
until the rms (root mean square) of conjugate gradient was
<0.1 kcal molꢁ1 Åꢁ1 (1 Å = 0.1 nm) keeping the heavy atoms fixed
at their crystallographic positions. To strictly validate the model
generated and to calibrate the high-throughput docking protocol,
a small database of known CK2 inhibitors was built and a set of
3.1.27. N-Methyl-2-(4,5,6,7-tetraiodo-1H-benzimidazol-1-yl)-
acetamide (25)
Similarly to described for 23 however stirred for 3 d: (mp 302–
304 °C (decomp. with I2 evol.) 83%). UV (MeOH): 237 (29,400), 278
(8200), 299 (7500). 1H NMR (Me2SO-d6) d (ppm): 3.29 (d, 3H, H3C,
J = 4.6 Hz), 5.05 (s, 2H, H2C), 8.06 (q, 1H, H–N, J = 4.7 Hz), 8.28 (s,
1H, H–C). MS m/z: 693 (31, M+), 566 (100, M+ꢁ127), 439 (41,
M+ꢁ254). Anal. Calcd for C10H7I4N3O (692.80): C, 17, 34; H, 1.02;
N, 6.07. Found: C, 17.30; H, 0.99; N, 6.01.
docking runs was performed. After the calibration phase, semiflex-
34
ible ligand-docking steps with three different programs, MOEDOCK
,
35
36
GLIDE
,
and GOLD
,
were performed essentially as described previ-
a ATP-binding pocket volume was calculated
ously.37 Human CK2
starting from the apo crystal structure 1JWH plus one conserved
water molecule; firstly a set of MOE dummy atoms defining the ac-
tive site was created, secondly the volume was calculated using a
water molecule probe.34 On the other hand 1a, K17 and TCI struc-
tures and volumes were calculated with Spartan 08, using a Semi-
empirical Quantum Mechanical Methods AM1.38
3.1.28. (4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)-acetic acid
hydrazide (26)
The mixture of 18 (320 mg, 0.6 mmol) and hydrazine hydrate
(500 mg, 10 mmol) in ethanol (15 mL) was stirred and refluxed
for 2 h. The mixture was chilled and left for the crystallization.
The white chromatographically pure precipitate was obtained

A. Gianoncelli et al. / Bioorg. Med. Chem. 17 (2009) 7281–7289
16. Meggio, F.; Shugar, D.; Pinna, L. A. Eur. J. Biochem. 1990, 187, 89–94.
7289
Acknowledgments
17. Sarno, S.; Reddy, H.; Meggio, F.; Ruzzene, M.; Davies, S. P.; Donella-Deana, A.;
Shugar, D.; Pinna, L. A. FEBS Lett. 2001, 496, 44.
This work was supported by grants to L.A.P. from AIRC, Euro-
pean Commission (PRO-KINASERESEARCH 503467), and by the
Italian Cystic Fibrosis Research Foundation (Grant FFC#4/2007)
with the contribution of ‘Banca Popolare di Verona e Novara’ and
‘Fondazione Giorgio Zanotto’. The Molecular Modelling Section
(MMS) coordinated by Professor S. Moro (Padova, Italy) is grate-
fully acknowledged. The authors (A.O. and Z.K.) are grateful to
Foundation for the Development Diagnostics and Therapy for par-
tial financial support.
18. Zien, P.; Duncan, J. S.; Skierski, J.; Bretner, M.; Litchfield, D. W.; Shugar, D.
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