CK2α and CK2α′ subunits differ in their sensitivity to 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.11.002

The goal of this study was to test the inhibitory activity of a series of tetrahalogenobenzimidazoles, including a number of novel derivatives, on individual catalytic subunits of human CK2. 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazoles and their newly obtained N¹- and 2-S-carboxyalkyl derivatives showed potent inhibitory activity against both these subunits. CK2α′ was up to 6 times more sensitive to the studied compounds than CK2α. The investigated iododerivatives showed, in most cases, stronger inhibitory properties than the respective brominated congeners, but the differences showed considerable dependence on the protein substrate used.

Full text of DOI:10.1016/j.ejmech.2011.11.002

European Journal of Medicinal Chemistry 47 (2012) 345e350
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
CK2a
and CK2a0 subunits differ in their sensitivity to 4,5,6,7-tetrabromo- and
4,5,6,7-tetraiodo-1H-benzimidazole derivatives
,
,
*
Monika Janeczko a, Andrzej Orzeszko b c, Zygmunt Kazimierczuk c, Ryszard Szyszka a, Andrea Baier a
a Department of Molecular Biology, Institute of Biotechnology, The John Paul II Catholic University of Lublin, Al. Krasnicka 102, 20-718 Lublin, Poland
b Military University of Technology, Nowoursynowska St.159C, 02-787 Warsaw, Poland
c Institute of Chemistry, Warsaw University of Life Sciences, Kaliskiego St.2, 00-908 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The goal of this study was to test the inhibitory activity of a series of tetrahalogenobenzimidazoles,
including a number of novel derivatives, on individual catalytic subunits of human CK2. 4,5,6,7-
tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazoles and their newly obtained N1- and 2-S-carbox-
yalkyl derivatives showed potent inhibitory activity against both these subunits. CK2a0 was up to 6 times
more sensitive to the studied compounds than CK2a. The investigated iododerivatives showed, in most
cases, stronger inhibitory properties than the respective brominated congeners, but the differences
showed considerable dependence on the protein substrate used.
Received 29 May 2011
Received in revised form
14 October 2011
Accepted 1 November 2011
Available online 13 November 2011
Keywords:
Protein kinase CK2 subunits
TBI derivatives
Ó 2011 Elsevier Masson SAS. All rights reserved.
TIBI derivatives
CK2 inhibitors
1. Introduction
CK2 is a highly ubiquitous and multifaceted serine/threonine
kinase described as a heterotetrameric holoenzyme generated by
Protein kinases, which are key regulators of many cellular
functions, constitute one of the largest human gene families. The
human genome encodes some 518 protein kinases that control
protein activity by catalyzing the addition of a negatively charged
phosphate group to proteins [1]. Protein kinases modulate a wide
variety of biological processes, especially those that transfer signals
from the cell membrane to intracellular targets and coordinate
the association of two
a and/or a
0 catalytic subunits with a dimer of
b
regulatory subunits [3e6]. Although human CK2a
and CK2a0 are
the products of expression of different genes, their catalytic
domains show about 90% identity, which may explain the similarity
in their enzymatic properties in vitro [5,7]. Notably, the C-termini of
CK2a
and CK2a0 are completely unrelated [7].
Protein kinase CK2 plays a global role in activities related to cell
growth, survival and death, and has a large number of potential
substrates localized in various cell compartments. In addition to its
involvement in cell growth and proliferation, CK2 is also a potent
suppressor of apoptosis, which characteristic underlies its impor-
tance for cancer cell phenotype [3e6].
To date, most of the publications on CK2 made no distinction
between the different isoenzymic forms of CK2 catalytic subunits.
However, knockout studies in mice and yeast have revealed some
degree of functional specialization [5]. Mice with knockout of cata-
complex biological functions.
A number of severe diseases,
including cancer, diabetes, and inflammation, are linked to
perturbations of kinaseemediated cell signaling pathways, thus
they represent some of the most promising drug targets. Protein
kinases share a catalytic domain conserved in the sequence and
structure, but are notably different in how their catalysis is regu-
lated. The ATP-binding pocket between the two lobes of the kinase
fold, together with less conserved surrounding pockets, has been
the focus of inhibitor design that had exploited differences in the
kinase structure and adaptability in order to achieve selectivity [2].
lytic
indicates that CK2
cannot compensate for CK2
a
and regulatory
b
subunits die in embryonic stage, which
are essential for viabilityandthat CK2a0
loss. The cellular functions of all CK2
a
and CK2
b
a
isoforms were also studied in human osteosarcoma cell lines
expressing active or inactive versions of each CK2 isoform. The
examination of these cell lines showed functional specialization of
both isoforms at the cellular level in mammals, and indicated that
Abbreviations: TBI, 4,5,6,7-tetrabromo-1H-benzimidazole; DMAT, 2-(dimethy-
lamino)-4,5,6,7-tetrabromo-1H-benzimidazole;
TIBI,
4,5,6,7-tetraiodo-1H-
benzimidazole.
* Corresponding author. Tel.: þ48 81 445 4603; fax: þ48 81 445 4611.
CK2a
0 isinvolvedinthecontrolofproliferationand/orcellsurvival[8].
E-mail address: baier@kul.pl (A. Baier).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.002

346
M. Janeczko et al. / European Journal of Medicinal Chemistry 47 (2012) 345e350
CK2 has been found to be deregulated in all cancers examined.
group were synthesized. For the syntheses, 4,5,6,7-tetrabromo- and
4,5,6,7-tetraiodo-benzimidazoles and their respective 2-
Those studies demonstrated increased CK2 expression and
enhanced nuclear localization in cancer cells compared to their
normal counterparts [9]. Overexpression of protein kinase CK2 is an
unfavorable prognostic marker in several cancers [10].
Consequently, CK2 has emerged as a relevant therapeutic target.
Several classes of ATP-competitive inhibitors have been identified,
showing variable effectiveness. In the beginning of the 1990s,
multihalogenated benzimidazoles were described as one of the
most powerful CK2 inhibitors that can cross the cell membranes
[11,12]. In addition, it was shown that some of these inhibitors can
distinguish between different CK2 isoforms both in vitro and in vivo
[13,14]. During last years, numerous small molecules were identi-
fied that inhibit CK2 activity by various mechanisms, e.g. allosteric
mercaptoderivatives (3aec) were alkylated with
u-bromoalkyl
esters in the presence of potassium carbonate in aprotic solvents to
yield the desired compounds 1aee and 4aeg. The obtained esters
were hydrolyzed in alkaline medium to yield the corresponding
carboxyalkyl compounds 2aee and 5aeg, respectively (Scheme 1).
The structures of the new compounds were confirmed by 1H NMR
and UV spectra and elemental analyses.
Recently we examined 4,5,6,7-tetrabromo-1H-benzotriazole
(TBB) and TBI as inhibitors of five CK2 isoforms from Saccharomyces
cerevisiae [21]. Interestingly, we have not only observed differences
between free catalytic subunits (CK2
a and CK2a
0) and holoenzymes
(
a2bb0 bb0 and aa0bb0), but also between both catalytic subunits.
, a’2
inhibitors, substrate-competitive inhibitors, CK2
itors, and CK2 /CK2 interaction inhibitors [15e20].
In this report we describe the effects of a series of halogenated
benzimidazoles on the activity of both
human CK2.
b
-targeting inhib-
Furthermore, we have shown the difference in Ki values using as
phosphoacceptor the well-known CK2 substrates, including mRNA
polyadenylation factor Fip1, survival factor Svf1, transcription
elongation factor Elf1 and acidic ribosomal protein P2B, or the
synthetic peptide RRRADDSDDDDD.
a
b
a and a
0 catalytic subunits of
In the present study we investigated 16 halogenated benz-
imidazole derivatives for their activity against human CK2
a and
2. Results and discussion
CK2a0 subunits. Both these subunits were over expressed and
purified to homogeneity by affinity chromatography as described in
the experimental section. The inhibitory effect was examined by
increasing concentrations of a given compound. All results ob-
tained using the synthetic peptide RRRADDSDDDDD or P2B as
a substrate are summarized in Tables 1e3. The introduction of S-
carboxymethyl (5a), S-carboxyethyl (5c) or S-carboxypropyl (5e)
For this study, besides from the most representative
benzimidazole-derived CK2 inhibitors like 4,5,6,7-tetrabromo-1H-
benzimidazole (TBI), 4,5,6,7-tetraiodo-1H-benzimidazole (TIBI), 2-
(dimethylamino)-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)
and their previously synthesized analogs, some new N1-carbox-
yalkyl and 2-S-carboxyalkyl derivatives with or without N1-methyl
R1
R1
R1
R1
R1
R1
R1
N
N
N
a
b
R2
R2
R2
N
H
N
R1
N
R1
R4
R3
R1
R1
R1
1a R1 = Br, R2 = H, R3 = CH2CO2Et [22]
1b R1 = Br, R2 = H, R3 = (CH2)3CO2Et
1c R1 = Br, R2 = N(CH3)2, R3 = CH2CO2Et [17]
1d R1 = Br, R2 = N(CH3)2, R3 = (CH2)3CO2Et
1e R1 = I, R2 = H, R3 = CH2CO2Et [22]
2a R1 = Br, R2 = H, R4 =CH2CO2H [22]
2b R1 = Br, R2 = H, R4 = (CH2)3CO2H
2c R1 = Br, R2 = N(CH3)2, R4 = CH2CO2H [17]
2d R1 = Br, R2 = N(CH3)2, R4 = (CH2)3CO2H
2e R1 = I, R2 = H, R4 = CH2CO2H [22]
R1 = Br, R2 = H (TBI)
R1 = Br, R2 = N(CH3)2 (DMAT)
R1 = I, R2 = H
(TIBI)
R1
R1
R1
H
N
R1
R1
R1
N
N
a
b
S
SR7
SR6
N
N
R1
N
R1
R1
R5
R5
R1
R5
R1
R1
3a R1 = Br, R5 = H
3b R1 = Br, R5 = CH3
3c R1 = I, R5 = H
5a R1 = Br, R5 = H, R7 = CH2CO2H [23]
5b R1 = Br, R5 = CH3, R7 = CH2CO2H [17]
5c R1 = Br, R5 = H, R7 = (CH2)2CO2H
5d R1 = Br, R5 = CH3, R7 = (CH2)2CO2H
5e R1 = Br, R5 = H, R7 = (CH2)3CO2H
5f R1 = Br, R5 = CH3, R7 = (CH2)3CO2H
5g R1 = I, R5 = H, R7 = CH2CO2H [22]
4a R1 = Br, R5= H, R6 = CH2CO2Et [23]
4b R1 = Br, R5 = CH3, R6 = CH2CO2Et [17]
4c R1 = Br, R5 = H, R6 = (CH2)2CO2Et
4d R1 = Br, R5 = CH3, R6 = (CH2)2CO2Et
4e R1 = Br, R5 = H, R6 = (CH2)3CO2Et
4f R1 = Br, R5 = CH3, R6 = (CH2)3CO2Et
4g R1 = I, R5 = H, R6 = CH2CO2Et [22]
Br
Br
N
CH3
N
Br
N
CH3
CH3
Br
1Me-DMAT [23]
Scheme 1. Synthesis of new 4,5,6,7-tetrabromo and 4,5,6,7-tetraiodo-1H-benzimidazole derivatives. a) K2CO3, butanone or acetone,
EtOH/H2O, NaOH, Hþ
u-bromoalkylcarboxylic acid ethyl ester, b)
.

M. Janeczko et al. / European Journal of Medicinal Chemistry 47 (2012) 345e350
347
Table 1
Derivatives of TBI and their Ki values (nM) for CK2
a
and CK2a0
.
Br
Br
Br
Br
N
N
SR2
R1
Compound
R1
R2
CK2
P2B
a
CK2a0
Peptide
P2B
Peptide
TBI
5a
5c
5e
5b
5d
5f
H
H
H
H
CH3
CH3
CH3
H
462 ꢀ 44
106 ꢀ 10
116 ꢀ 12
77 ꢀ 6
315 ꢀ 32
170 ꢀ 18
172 ꢀ 16
210 ꢀ 19
13 ꢀ 2
270 ꢀ 25
37 ꢀ 3
343 ꢀ 37
49 ꢀ 4
CH2CO2H
(CH2)2CO2H
(CH2)3CO2H
CH2CO2H
(CH2)2CO2H
(CH2)3CO2H
35 ꢀ 3
31 ꢀ 3
88 ꢀ 7
44 ꢀ 3
143 ꢀ 14
112 ꢀ 11
88 ꢀ 8
200 ꢀ 17
100 ꢀ 11
83 ꢀ 7
24 ꢀ 2
137 ꢀ 12
501 ꢀ 48
55 ꢀ 5
768 ꢀ 64
residue at position 2 of TBI resulted in an increase in the inhibitory
activity against CK2a0 for up to 8-fold and up to 11-fold, using the
synthetic peptide and P2B as a substrate, respectively, whereas the
respective increases in inhibitory activity of these derivatives
different free catalytic subunits, and we found that this is, in
general, the case (Table 2). This is particularly clear for the
compounds bearing carboxymethyl group at position
1
(compounds 2a and 2e). However, the compounds carrying S-car-
boxymethyl moiety at position 2 (e.g. 5a and 5g) did not follow this
rule (see Table 2).
against CK2a were markedly lower. Additional introduction of the
methyl group at position 1, which led to compounds 5b, 5d and 5f,
did not significantly changed the inhibitory activity (as compared to
those of 5a, 5c and 5e) using P2B as the protein substrate. However,
this substitution has brought mixed results when the synthetic
peptide was used for substrate (see Table 1). Compound 5c showed
significant selectivity towards CK2a0 subunit, as its inhibitory
Next, we tested DMAT, an analog of TBI with a dimethylamino
residue at position 2, and its derivatives as potential inhibitors of
free CK2 catalytic subunits (Table 3). DMAT was already shown to
exert higher inhibitory activity than TBI on CK2 holoenzyme [17].
The same was found for both free catalytic subunits in this study.
Introduction of a methyl group into position 1 of DMAT yielded
1Me-DMAT that was similarly or somewhat more effective against
potency against CK2a was 3e5 times lower (Fig. 1A). Noteworthy,
the inhibitory activity of 5b toward both CK2 subunits studied was
much higher than that of the parental compound TBI when using
the synthetic peptide as the substrate (Fig. 1B). Interestingly, 5f
showed much weaker inhibitory effect using the synthetic peptide
as the substrate than that using P2B as the substrate (Fig. 1C).
It has been previously reported that iodinated inhibitors are in
general more active than their brominated counterparts toward the
a2b2 holoenzyme [22]. We examined if this holds true also for
CK2a a, depending on
0, and was similarly or less potent towards CK2
the substrate used. To examine the influence of the length of the
residue at position 1, we have synthesized a 1Me-DMAT congener
with the carboxymethyl residue at this position (2c). This
compound showed a considerable inhibitory activity toward both
catalytic CK2 subunits irrespective of the substrate employed. In
case of CK2
a
0, its Ki value was 2 and 6 times lower than that of
Table 2
Derivatives of TBI and TIBI and their Ki values (nM) for CK2a .
and CK2a0
R1
R1
R1
N
R3
N
R2
R1
Compound
R1
R2
R3
CK2
P2B
a
CK2a0
Peptide
P2B
Peptide
TBI
TIBI
2a
5a
2e
Br
I
Br
Br
I
H
H
H
H
H
462 ꢀ 44
266 ꢀ 26
798 ꢀ 70
106 ꢀ 10
378 ꢀ 36
110 ꢀ 10
315 ꢀ 32
43 ꢀ 4
270 ꢀ 25
71 ꢀ 8
343 ꢀ 37
21 ꢀ 2
CH2CO2H
H
CH2CO2H
H
670 ꢀ 65
170 ꢀ 18
261 ꢀ 24
80 ꢀ 7
609 ꢀ 58
37 ꢀ 3
342 ꢀ 32
49 ꢀ 4
SCH2CO2H
H
SCH2CO2H
62 ꢀ 5
324 ꢀ 32
106 ꢀ 11
5g
I
78 ꢀ 6

348
M. Janeczko et al. / European Journal of Medicinal Chemistry 47 (2012) 345e350
Table 3
Derivatives of TBI and their Ki values (nM) for CK2
a
and CK2a0
.
Br
Br
Br
Br
N
N
R2
R1
Compound
R1
R2
H
N(CH3)2
N(CH3)2
H
CK2
P2B
a
CK2a0
Peptide
P2B
Peptide
TBI
DMAT
1Me-DMAT
2b
2c
2d
H
H
CH3
(CH2)3CO2H
CH2CO2H
(CH2)3CO2H
462 ꢀ 44
97 ꢀ 8
315 ꢀ 32
139 ꢀ 12
154 ꢀ 14
368 ꢀ 34
83 ꢀ 9
270 ꢀ 25
112 ꢀ 10
109 ꢀ 8
188 ꢀ 15
66 ꢀ 6
343 ꢀ 37
133 ꢀ 11
35 ꢀ 3
212 ꢀ 17
472 ꢀ 44
88 ꢀ 7
195 ꢀ 18
21 ꢀ 2
N(CH3)2
N(CH3)2
860 ꢀ 75
456 ꢀ 46
139 ꢀ 12
93 ꢀ 8
Fig. 1. Comparison of the results of phosphorylation assays. (A) Inhibition by 5c in relation to the choice of CK2 isoform and substrate; CK2a
e black bars, CK2a0 e white bars; (B)
Inhibition of peptide phosphorylation in relation to the choice of CK2 isoform and inhibitor; TBI e black bars, compound 5b e white bars; (C) Inhibition by 5f in relation to the
choice of CK2 isoform and substrate, P2B e black bars, synthetic peptide e white bars.
DMAT, using P2B or peptide as phosphate acceptor, respectively.
The effect on CK2 was similar but weaker (Table 3). Further
elongation of the carbon chain of the substituent at position 1 led to
a large reduction of the inhibitory effect, especially in case of CK2
(see compound 2d data). The opposite effects were observed for the
respective TBI derivative (2b), i.e. while it inhibited the phosphor-
subunit employed. The structural feature that underlies these
differences needs to be further investigated. Deduced from our
present and former studies [22], the most prominent characteris-
tics of effective human CK2 benzimidazole inhibitors include the
following: (1) iodination at the benzene ring of the benzimidazole
core, and (2) the presence of a polar substituent at position 2.
Crystallography studies may help to clarify the stronger inhibi-
a
a
ylation reaction mediated by CK2
a similarly to the parent
compound, its inhibitory activity against CK2a0 was much stronger.
It seems reasonable to assume that the differences described above
are related to the zwitterionic nature of the compounds 2c and 2d.
tion of CK2a
0. It is highly probable that the inhibitor/enzyme
complex differs between both catalytic subunits.
Our aim should be to analyze in more details the differences in
inhibition of individual catalytic subunits and the holoenzyme. It is
intriguing whether selectivity with respect to the subunits trans-
lates into differences in anti-cancer activity of human CK2
inhibitors.
3. Conclusions
In most cases, the human CK2a0 subunit compared to its CK2
a
counterpart is more sensitive to the inhibition by tetrahalogeno-
benzimidazole derivatives. Although comparisons to the results of
other scientific groups are difficult and not entirely reliable, it is
obvious that the inhibitory activity of these compounds against
4. Experimental
4.1. Chemistry
CK2a
, CK2a0 or holoenzyme can differ [17,22,23]. Small differences
between our values and from other research groups may also result
from different reaction conditions, like buffer composition, reaction
time and source of enzyme. As shown in this study, the effect of
modification of tetrahalogenobenzimidazoles on CK2 inhibitory
activity may yield different results depending on the substrate and
The following investigated compounds were prepared according
to previously described procedures: 4,5,6,7-tetraiodobenzimidazole
TIBI, (4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)-acetic acid (2a),
(4,5,6,7-tetraiodo-1H-benzimidazol-1-yl)-acetic acid (2e), (4,5,6,7-
tetraiodo-1H-benzimidazol-2-ylsulfanyl)-acetic acid (5g) [22]; 2-

M. Janeczko et al. / European Journal of Medicinal Chemistry 47 (2012) 345e350
349
dimethylamino-4,5,6,7-tetrabromobenzimidazol-1-yl)-acetic acid
(2c), (4,5,6,7-tetrabromo-1-methyl-1H-benzimidazol-2-ylsulfanyl)-
acetic acid (5b) [17]; 2-(dimethylamino)-4,5,6,7-tetrabromo-1H-
benzimidazole (DMAT), 2-(dimethylamino)-1-methyl-4,5,6,7-
tetrabromo-1H-benzimidazole (1Me-DMAT), (4,5,6,7-tetrabromo-
1H-benzimidazol-2-ylsulfanyl)-acetic acid 5a [23].
COOH). UV (MeOH): 240 (29 600), 274 (12 700), 313 (7700). Anal.
calcd for C13H13Br4N3O2 (562.88): C, 27.24; H, 2.33; N, 7.47. Found:
C, 27.16; H, 2.40; N, 7.39.
4.1.5. 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-2-ylsulfanyl)-
propionic acid ethyl ester (4c)
Melting points were determined on a Gallenkamp Melting Point
Apparatus, Mod. MFB 595 030G, in open capillary tubes. The 1H
NMR spectra were recorded on a Bruker AMX instrument (400 MHz
1H frequency) at 25 ꢁC. Chemical shifts are reported in ppm from
A mixture of 3a (470 mg, 1 mmol), potassium carbonate
(420 mg, 3 mmol) and 3-bromopropionic acid ethyl ester (360 mg,
2 mmol) in acetone (30 ml) was stirred at room temperature for 2 d.
Next, water (50 ml) was added and the mixture was left at 5 ꢁC. The
white precipitate that formed overnight was separated and crys-
tallized from ethanol to yield small needles (410 mg, 72%); mp.
183e185 ꢁC 1H NMR (DMSO-d6): 1.18 (t, 3H, J ¼ 7.1 Hz, CH3) 2.88 (t,
2H, J ¼ 6.8 Hz, CH2), 3.52 (t, 2H, J ¼ 6.8 Hz, CH2), 4.10 (q, 2H,
J ¼ 7.1 Hz, CH2), 13.3 (bs, 1H, HeN). Anal. calcd. for C12H10Br4N2O2S
(565.91): C, 25.47; H, 1.78; N, 4.95. Found: C, 25.41; H, 1.81; N, 4.86.
internal tetramethylsilane standard are given in d-units. The solvent
used for NMR spectra was deuteriodimethylsulfoxide. The UV
spectra were determined on Techcomp UV8500 spectrophotometer.
Elemental analyses were performed at the Faculty of Chemistry,
Warsaw Technical University using a Heraeus CHN Rapid Analyzer.
4.1.1. 4-(4,5,6,7-Tetrabromobenzimidazol-1-yl)-butyric acid ethyl
ester (1b)
4.1.6. 3-(1-Methyl-4,5,6,7-tetrabromobenzimidazol-2-ylsulfanyl)-
propionic acid ethyl ester (4d)
To the refluxed and stirred mixture of 4,5,6,7-
tetrabromobenzimidazole (1.3 g,
3
mmol) and potassium
Analogously as described for 4c, from 3b. Yield 84%, mp.
132e134 ꢁC 1H NMR (DMSO-d6):1.19 (t, 3H, J ¼ 7.1 Hz, CH3), 2.92 (t,
2H, J ¼ 6.8 Hz, CH2), 3.56 (t, 2H, J ¼ 6.7 Hz, CH2), 3.89 (s, 3H, CH3),
4.09 (q, 2H, J ¼ 7.1 Hz, CH2). Anal. calcd for C13H12Br4N2O2S
(579.93): C, 26.92; H, 2.09; N, 4.83. Found: C, 26.98; H, 2.18; N, 4.71.
carbonate 900 mg (6.5 mmol) in butanone (30 ml) 4-bromobutyric
acid ethyl ester (800 mg, 4.1 mmol) was added. The reflux was
continued for 24 h. The mixture was filtered and filter cake washed
with acetone. The filtrate was evaporated to dryness and the
residue crystallized from EtOH-H2O (2:1, v/v) to give 1.4 g (83%) of
colorless needles. m.p. 138e140 ꢁC 1H NMR (DMSO-d6): 1.12 (t, 3H,
CH3, J ¼ 6.3 Hz), 2.10 (m, 2H, CH2), 2.38 (t, 2H, CH2, J ¼ 7.3 Hz), 3.93
(q, 2H, CH2, J ¼ 7.1 Hz), 4.53 (t, 2H, CH2, J ¼ 7.1 Hz). 8.48 (s, 1H, CH).
Anal. calcd for C13H12Br4N2O2 (547.87): C, 28.50; H, 2.21; N, 5.11.
Found: C, 28.43; H, 2.25; N, 4.99.
4.1.7. 4-(4,5,6,7-Tetrabromo-1H-benzimidazol-2-ylsulfanyl)-
butyric acid ethyl ester (4e)
Analogously to described above for 4c; instead of 3-
bromopropionic acid ethyl ester 4-bromobutanoic acid ethyl ester
was used. m.p. 181e183 ꢁC, 82%. 1H NMR (DMSO-d6): 1.16 (t, 3H,
J ¼ 7.1 Hz, CH3), 2.00 (m, 2H, CH2), 2.46 (t, 2H, J ¼ 7.3 Hz, CH2), 3.38
(t, 2H, J ¼ 7.0 Hz, CH2), 4.05 (q, 2H, J ¼ 7.1 Hz, CH2), 13.3 (bs, 1H,
HeN). Anal. calcd. for C13H12Br4N2O2S (579.93): C, 26.92; H, 2.09; N,
4.83. Found: C, 26.98; H, 2.14; N, 4.74.
4.1.2. 4-(2-Dimethylamino-4,5,6,7-tetrabromobenzimidazol-1-yl)-
butyric acid ethyl ester (1d)
To the refluxed and stirred mixture of 2-dimethylamino-4,5,6,7-
tetrabromobenzimidazole (720 mg, 1.5 mmol) and potassium
carbonate 1.38 g (10 mmol) in butanone (30 ml) 4-bromobutyric
acid ethyl ester (1.56 g, 8 mmol) was added. The reflux was
continued for 20 h. The mixture was filtered and filter cake washed
with acetone. The filtrate was evaporated and the residue was
chromatographed on silica gel column (3 ꢂ 10 cm) with CHCl3
(200 ml) and CHCl3-MeOH (98:2). The product containing fractions
were evaporated to a viscous oil (740 mg, 84%). 1H NMR (DMSO-d6):
1.14 (t, 3H, CH3, J ¼ 7.1 Hz), 1.80 (m, 2H, CH2), 2.09 (t, 2H, CH2,
J ¼ 6.9 Hz), 2.98 (s, 6H, 2 X CH3), 3.98 (q, 2H, CH2, J ¼ 7.1 Hz), 4,33 (t,
2H, J ¼ 7.1 Hz). Anal. calcd for C15H17Br4N3O2 (590.94): C, 30.49; H,
2.90; N, 7.11. Found: C, 30.39; H, 2.95; N, 7.00.
4.1.8. 4-(1-Methyl-4,5,6,7-tetrabromobenzimidazol-2-ylsulfanyl)-
butyric acid ethyl ester (4f)
Analogously as described for 4c, from 3b. Yield 87%, m.p.
118e120 ꢁC (from ethanol). 1H NMR (DMSO-d6): 1.17 (t, 3H, CH3,
J ¼ 7.1 Hz), 2.05 (m, 2H, CH2,), 2.50 (t, 2H, CH2, overlap. DMSO), 3.42
(t, 2H, CH2, J ¼ 7.0), 3.93 (s, 3H, CH3), 4.06 (q, 2H, CH2, J ¼ 7.1 Hz).
Anal. calcd for C14H14Br4N2O2S (593.96): C, 28.31; H, 2.38; N, 4.72.
Found: C, 28.40; H, 2.33; N, 4.63.
4.1.9. 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-2-ylsulfanyl)-
propionic acid (5c)
A mixture of ester (4c) (570 mg, 1 mmol) and sodium hydroxide
(120 mg, 3 mmol) in ethanol-water solution (20 ml, 1:1) was stirred
at rt for 2 h. The mixture was brought to reflux and acidified with
acetic acid to pH w3. A white precipitate was filtered and crystal-
lized from ethanol-water to give amorphous powder (340 mg,
63%); m.p. 203e205 ꢁC 1H NMR (DMSO-d6): 2.81 (t, 2H, J ¼ 6.8 Hz,
CH2), 3.48 (t, 2H, J ¼ 6.8 Hz, CH2), 12.3e13.4 (bs, 2H, HeN and
COOH). UV (MeOH): 236 (36 700), 272 (12 500), 301 (11 800), 312
(14 700). Anal. calcd. for C10H6Br4N2O2S (537.85): C, 22.33; H, 1.12;
N, 5.21. Found: C, 22.40; H, 1.19; N, 5.13.
4.1.3. 4-(4,5,6,7-Tetrabromobenzimidazol-1-yl)-butyric acid (2b)
The mixture containing EtOH (20 ml), water (10 ml), NaOH
(200 mg, 5 mmol), and 1b (580 mg, 1.05 mmol) was stirred at room
temperature for 4 h. Next it was brought to the reflux and after
cooling adjusted to pH 4 with acetic acid and concentrated to 15 ml.
The white chromatographic pure precipitate was formed (495 mg,
91%). m.p. 194e196 ꢁC (from butanone-water; 3:1, v/v). 1H NMR
(DMSO-d6): 2.03 (m, 2H, CH2), 2.25 (t, 2H, CH2, J ¼ 7.3 Hz), 4.51 (t,
2H, CH2, J ¼ 7.1 Hz), 8.47 (s, 1H, CH), 12.2 (bs, 1H, COOH). UV
(MeOH): 230 (28 500), 269 (9700), 274 (9600), 303 (3500). Anal.
calcd for C11H8Br4N2O2 (519.81): C, 25.42; H, 1.55; N, 5.39. Found: C,
25.38; H, 1.60; N, 5.27.
4.1.10. 3-(1-Methyl-4,5,6,7-tetrabromobenzimidazol-2-ylsulfanyl)-
propionic acid (5d)
Analogously as described for 5c, from 4d. Yield: 82%, m.p.
>300 ꢁC (with decomp.) (from butanone-water; 3:1, v/v). 1H NMR
(DMSO-d6): 2.81 (t, 2H, CH2, J ¼ 6.8 Hz), 3.52 (t, 2H, CH2, J ¼ 6.7 Hz),
3.91 (s, 3H, CH3),12.3 (bs,1H, COOH). UV (MeOH): 233 (33 500), 277
(11 100), 302 (9200), 314 (10 700). Anal. calcd for C11H8Br4N2O2S
(551.88): C, 23.94; H, 1.46; N, 5.08. Found: C, 23,99; H, 1.36; N 4.97.
4.1.4. 4-(2-Dimethylamino-4,5,6,7-tetrabromobenzimidazol-1-yl)-
butyric acid (2d)
Analogously as described for 2b from 1d. Yield 96%, m.p.
220e222 ꢁC 1H NMR (DMSO-d6): 1.77 (m, 2H, CH2), 2.04 (t, 2H, CH2,
J ¼ 7.1 Hz), 2.98 (s, 6H, 2 X CH3), 4,33 (t, 2H, J ¼ 7.0 Hz). 12.1 (bs, 1H,

350
M. Janeczko et al. / European Journal of Medicinal Chemistry 47 (2012) 345e350
4.1.11. 4-(4,5,6,7-Tetrabromo-1H-benzimidazol-2-ylsulfanyl)-
butyric acid (5e)
phosphocellulose filters (Whatmann). Filters were washed with 5%
TCA three times and dried before counting in scintillation counter.
Inhibition studies were performed at fixed concentrations of
substrate and at variable concentrations of ATP in the absence or in
the presence of increasing concentrations of inhibitor. Kinetic
parameters were determined by non-linear-regression analysis
using GraphPad Prism 4.0 (GraphPad Software, Inc San Diego).
Analogously as described for 5c, from 4e. m.p.226e228 ꢁC (from
EtOH-H2O), (65%). 1H NMR (DMSO-d6): 1.99 (m, 2H, CH2), 2.39 (t,
2H, J ¼ 7.3 Hz, CH2), 3.36 (t, 2H, J ¼ 7.0 Hz, CH2), 12.1 (bs, 1H, COOH),
13.4 (bs, 1H, HeN). UV (MeOH): 238 (32 600), 272 (10 900), 301
(10 600), 313 (13 100). Anal. calcd. for C11H8Br4N2O2S (551.88): C,
23.94; H, 1.46; N, 5.08. Found: C, 23.98; H, 1.54; N, 5.01.
Acknowledgments
4.1.12. 4-(1-Methyl-4,5,6,7-tetrabromobenzimidazol-2-ylsulfanyl)-
butyric acid (5f)
This study (AO and ZK) was supported by the Ministry of Science
and Higher Education (Poland), grant N N209 371439.
Analogouslyasdescribed for 5c,from4f.Yield81%,m.p.153e155 ꢁC
(from butanone-water, 3:1, v/v).1H NMR (DMSO-d6): 2.00 (q, 2H, CH2,
J ¼ 7.2 Hz), 2.37 (t, 2H, CH2, J ¼ 7.4 Hz), 3.40 (t, 2H, CH2, J ¼ 7.2 Hz), 3.91
(s, 3H, CH3), 12.1 (bs, 1H, COOH). UV (MeOH): 235 (32 400), 278
(10 800), 302 (9000), 314 (10 600). Anal. calcd for C12H10Br4N2O2S
(565.91): C, 25.47; H, 1.78; N, 4.95. Found: C, 25.41; H, 1.83; N, 4.84.
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4.2.2. Substrates
Yeast ribosomal acidic protein P2B was overexpressed in E.coli
BL21(DE3) cells containing the plasmid pT7-7::6xHis-yP2B (kind
gift from J.P. Ballesta) and purified by affinity chromatography using
Ni-Affinity Gel (Sigma). The synthetic peptide RRRADDSDDDDD
was purchased from Sigma.
4.2.3. Phosphorylation assays
To estimate the inhibitory effect of new synthesized benzimid-
azole analogs two different assays were employed depending on
the substrate.
The reaction mixture containing 20 mM Tris/HCl pH 7.5, 15 mM
MgCl2, 2 pmol CK2
32P]ATP (Hartmann Analytics GmbH) and as substrate 10
or 50 M synthetic peptide RRRADDSDDDDD was incubated at
a
or CK2a0 (spec. act. 1
m
mol/min/mg), 20
mM
[
g-
mM P2B
m
37 ꢁC for 15 min. Reactions with P2B as substrate were stopped by
addition of SDS sample buffer. Samples were subjected to 12.5%
SDS/PAGE. After Coomassie Blue staining gels were dried and
exposed to Kodak X-ray film at ꢃ80 ꢁC overnight. The parts of the
gels corresponding to P2B were cut out and the phosphate incor-
poration was estimated by Cerenkov counting of 32P radioactivity in
scintillation counter. Phosphorylation of the peptide substrate was
terminated by addition of 10% TCA and aliquots were spotted onto