Biological properties and structural study of new aminoalkyl derivatives of benzimidazole and benzotriazole, dual inhibitors of CK2 and PIM1 kinases

Article abstract of DOI:10.1016/j.bioorg.2018.06.022

The new aminoalkyl-substituted derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) were synthesized, and their influence on the activity of recombinant human CK2 α, CK2 holoenzyme and PIM1 kinases was evaluated. All derivatives inhibited the activity of studied kinases and the most efficient were aminopropyl-derivatives 8b and 14b. These compounds also exerted inhibition of cancer cell lines – CCRF-CEM (acute lymphoblastoid leukemia), MCF-7 (human breast cancer), and PC-3 (prostate cancer) proliferation and their EC₅₀ is comparable with the value for clinically studied CK2 inhibitor CX-4945. Preliminary structure activity relationship analysis indicated that the spacer length affected antitumor potency, and two to three methylene units were more favorable. The complex of CK2 α^1-335/8b was crystallized, both under high-salt conditions and under low-salt conditions giving crystals which diffracted X-rays to about 2.4 ? resolution, what enabled the determination of the corresponding 3D-structures.

Full text of DOI:10.1016/j.bioorg.2018.06.022

Accepted Manuscript
Biological properties and structural study of new aminoalkyl derivatives of ben-
zimidazole and benzotriazole, dual inhibitors of CK2 and PIM1 kinases
K. Chojnacki, P. Wińska, M. Wielechowska, E. Łukowska-Chojnacka, C.
Tölzer, K. Niefind, M. Bretner
PII:
S0045-2068(18)30274-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.022
YBIOO 2402
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
20 March 2018
15 June 2018
18 June 2018
Please cite this article as: K. Chojnacki, P. Wińska, M. Wielechowska, E. Łukowska-Chojnacka, C. Tölzer, K.
Niefind, M. Bretner, Biological properties and structural study of new aminoalkyl derivatives of benzimidazole and
benzotriazole, dual inhibitors of CK2 and PIM1 kinases, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.06.022
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Biological properties and structural study of new aminoalkyl derivatives of benzimidazole and
benzotriazole, dual inhibitors of CK2 and PIM1 kinases.
K. Chojnackia, P. Wińskaa, M. Wielechowskaa, E. Łukowska-Chojnackaa C. Tölzerb, K.
Niefindb, M. Bretnera
a Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw,
Poland
b Department für Chemie, Institut für Biochemie, Universtät zu Köln, Zülpicher Straße 47, D-
50674 Köln, Germany
Abstract
The new aminoalkyl- substituted derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1H-
benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) were synthesized,
and their influence on the activity of recombinant human CK2, CK2 holoenzyme and PIM1
kinases was evaluated. All derivatives inhibited the activity of studied kinases and the most
efficient were aminopropyl- derivatives 8b and 14b. These compounds also exerted inhibition
of cancer cell lines - CCRF-CEM (acute lymphoblastoid leukemia), MCF-7 (human breast
cancer), and PC-3 (prostate cancer) proliferation and their EC50 is comparable with the value
for clinically studied CK2 inhibitor CX-4945. Preliminary structure activity relationship
analysis indicated that the spacer length affected antitumor potency, and two to three
methylene units were more favorable. The complex of CK2α1-335/8b was crystallized, both
under high-salt conditions and under low-salt conditions giving crystals which diffracted X-
rays to about 2.4 Å resolution, what enabled the determination of the corresponding 3D-
structures.
Keywords: casein kinase 2 CK2; protein kinase PIM1; ATP-competitive inhibitors;
antiproliferative activity, structural study
1. Introduction
Protein kinases catalyze the transfer of phosphate groups from adenosine triphosphate
(ATP) to serine, threonine, or tyrosine residues of target proteins. This phosphorylation is an
important stage in regulation of cell growth, cellular signal transduction, cell differentiation,
and influences apoptotic mechanisms. Deregulation of protein kinases activity or expression
1

is implicated in a number of diseases, including cancer, diabetes, and inflammation. Thus,
targeted inhibition of the deregulated protein kinases has become an attractive therapeutic
strategy in cancer therapy. Casein kinase 2 (CK2) and PIM kinase (Proviral Integration site of
Moloney Virus) belong to the serine/threonine kinases family and their overexpression is
frequently associated with acute myeloid leukemia, and a variety of cancers including
prostate, breast or/and lung cancers [1,2]. The high activity of these kinases in cancer cells is
associated with inhibition of apoptosis, suggesting a protective role of CK2 and PIM1 in
programmed cell death. Recent studies have shown that the contribution of CK2 and PIM1
kinases in regulation of transcription, differentiation, or signalling of DNA damage/repair
systems is achieved by regulating survival pathways. For example, both kinases participate in
an activation of the transcriptional factor NF-κB, observed in transformed cells [3, 4].
Elevated level of CK2 induces abnormal activation of NF-κB, which in turn contributes to the
development of breast cancer [5]. Moreover, the tumor transformation of lymphocytes with
PIM involvement is dependent on activation of NF-κB [4]. Downregulation of these kinases
by chemical methods promotes apoptosis in cells [6], indicating CK2 and PIM1 kinases as
molecular targets in the development of new therapeutic agents.
Many PIM inhibitors have been reported to date [7, 8, 9,] however, none of them has
been marketed so far. SGI-1776 is a representative first-generation PIM inhibitor, which had
been under clinical trials for leukemia and prostate cancer [10]. While most of the first-
generation PIM inhibitors are PIM1-selective, there is currently great interest in the potential
of pan-PIM inhibitors to treat cancer because the three PIM kinases are reported to function
redundantly [11]. Representative pan-PIM inhibitors are AZD1208 from AstraZeneca, [12]
and PIM447 from Novartis [11]. Clinical trials of PIM447 are underway [11].
Among several classes of CK2 inhibitors effectively inhibiting the growth of tumor
cells in vitro at low micromolar ranges are derivatives of benzotriazole and benzimidazole e.g.
4,5,6,7-tetrabromo-1H-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi),
4,5,6,7-tetrabromo-1H-benzimidazole-2-N,N-dimethylamine (DMAT), 4,5,6,7-tetraiodo-1H-
benzimidazole (TIBI), (Ki 23 nM), and one of the most efficient CK2 and PIM dual inhibitors
TDB (Ki 32 nM, and 86 nM respectively) [13, 14].
2

Br
Br
Br
Br
Br
I
I
H
N
H
N
H
N
H
N
Br
Br
Br
Br
Br
Br
I
I
N
N
N
N
N
N
Br
Br
TBBt
TBBi
DMAT
TIBi
Br
Br
N
N
HN
Cl
O
N
Br
N
O
OH
TDB
CX-4945
OH
OH
Figure 1. The known CK2 inhibitors, derivatives of 1H-benzotriazole, 1H-
benzimidazole and CX-4945
Studies conducted by us [15], as well as others [14] show that CK2 inhibitors
frequently exhibit similar inhibitory activity against the PIM1 kinase due to structural
similarities within the structures of both kinases [16]. CX-4945 (Silmitasertib), the first ATP-
competitive CK2 kinase inhibitor that is currently in Phase I/II clinical trials, also inhibits in
vitro not only CK2 kinase activity, but also PIM1 and PIM2 kinase with IC50 values of 0.048
and 0.186 μM, respectively [7,17]. Recent studies have shown that the use of dual inhibitors
(CK2 and PIM inhibition) results in reduced proliferation and induction of apoptosis in human
T lymphoblastoid cells (CML) and their multidrug resistant variant (R-CEM), human cervical
cancer cells (HeLa) [14] as well as some other cell lines [18].
Continuing our search for CK2 inhibitors we synthesized the new azidoalkyl-, and
aminoalkyl- substituted derivatives of known inhibitors TBBi and TBBt. The influence of
new derivatives on activity of CK2and CK2 holoenzyme was evaluated. We also examined
their influence on the kinase PIM1 activity, and on few other kinases to explore the specificity
of new compounds. Simultaneous inhibition of the activity of several kinases is a
disadvantage when examining the role of different kinases in the cell metabolism or
neoplastic transformation, but it can be beneficial in the search for potential anti-cancer drugs.
3

We previously found that the introduction of a methyl group on the C2 atom of TBBi
led to the 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole (2MeTBBi) with a promising
inhibitory activity of CK2 and PIM1 and with increased cytotoxicity against CCRF-CEM and
MCF-7 cells [19]. Here we report the experimental details of the synthesis of a new series
aminoalkyl derivatives of TBBt, TBBi and 2MeTBBi. The inhibitory activity of the
synthesized compounds was tested using a standard isoptopic kinase assay and the selectivity
of selected compounds against a panel of 8 kinases was determined and compared with
selectivity of known inhibitors. The Antiproliferative activity of new compounds was tested
against three different cancer cell lines. A crystallographic study of CK2-inhibitor
complexes was performed in order to investigate the enzyme/inhibitor interactions.
2. Results and discussion
2.1. Chemistry
The synthesis of the halogenoalkyl derivatives 2a-d, 3a-d and 4b-d was carried out using
as starting materials the 4,5,6,7-tetrabromo-1H-benzimidazole 1a, 4,5,6,7-tetrabromo-1H-
benzotriazole 1b (Scheme 1), or 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole 11, (Scheme
2). Alkylation of 1a, 1b or 11 with the 1,2-dibromoethane, 1,4-dibromobuthane or 1,5-
dibromopenthane in acetonitrile in the presence of DBU as a base gave 2a,c-d, 3a,c-d, 4c-d
and 12a,c-d, or with the 1-bromo-3-chloropropane gave derivatives 2b, 3b, 4b and 12b. The
azidoalkyl- derivatives with different length of the alkyl chain 5a-d, 6a-d, 7a-d and 13a-d,
were obtained according to known procedures [15] by treatment appropriate halogenoalkyl-
derivative with sodium azide in DMF.
4

Scheme 1. Synthetic route to the TBBi and TBBt derivatives: i) dibromoalkyl or
chlorobromoalkyl, DBU, CH3CN, rt, 72 h; ii) NaN3, DMF, 40°C, 24 h; iii) Ph3P,
THF/H2O, rt, 48 h.
Scheme 2. Synthetic route to the 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole
derivatives: i) Br2, H2O. reflux, 72 h; ii) dibromoalkyl, DBU, CH3CN, rt, 2-9 days;
iii) NaN3, DMF, 60°C, 24 h; iv) Ph3P, THF/H2O, rt, 48 h.
The reduction of alkylazido derivatives was performed with the use of Ph3P in THF/H2O.
After completion of the reaction products were purified by column chromatography to obtain
TBBi aminoalkyl- derivatives 8a-d in good yields (70-86%), TBBt aminoalkyl- derivatives
9c-d and 10b-d in yields ranging from 44% to 93% and 2MeTBBi aminoalkyl- derivatives
14a-d in yields ranging from 64% to 96%. The synthesis of compounds 9a-b and 10a was
unsuccessful with the use of this method. The structures of the new compounds were
1
confirmed by analytical data, including high resolution mass spectra (MS or FTMS), H and
13C NMR.
2.2. Evaluation of biological activity
2.2.1. Kinases inhibition study
The influence of azidoalkyl- derivatives 5a-d, 6a-d, 7a-d, and aminoalkyl- derivatives
8a-d, 9c-d, 10b-d, 14a-d on the human CK2and PIM1 activity was evaluated using
radiometric assay, and the results are shown in Table 1. The synthetic peptide
RRRADDSDDDDD was used as the substrate of CK2 and peptide ARKRRRHPSGPPTA as
the substrate of PIM1. Screening studies of inhibitory activity of obtained derivatives were
performed in the presence of 5 M and 10 M compounds and subsequently, the percent of
residual activity of CK2 and PIM1 was calculated. Ki values were determined for the most
active compounds for both human CK2α and CK2 holoenzyme, as well as for PIM1.
5

Among derivatives with azidoalkyl- substituents the CK2 activity was inhibited most
effectively by the TBBi derivative 5a with the azido group linked by a two-carbon alkyl chain
to benzimidazole scaffold, but it was less effective than parent TBBi (Ki 2.3 M versus 0.58
M). Furthermore, the activity of the PIM1 kinase was inhibited with the same efficiency by
compound 5a and TBBi. On the other hand azidoalkyl- TBBt derivatives 6a-d and 7a-d
showed a very weak inhibition of both CK2 and PIM kinase activity.
The reduction of azido groups to amine led to the new derivatives with aminoalkyl-
substituents, which appeared to be more effective inhibitors of both kinases. Among the
synthesized new aminoalkyl-TBBi derivatives, we found three potent inhibitors of CK2 which
inhibited more than 50% activity of enzyme at a concentration of 5 M (8b,c,d), with Ki for
CK2 in the range 0.75-1.35 M and Ki for holoenzyme CK2 in the range of 0.17-0.23 M,
comparable to Ki 0.15 M for TBBi. These TBBi derivatives appeared to be also potent
inhibitors of kinase PIM1, with an inhibition more than 90% activity at a concentration of 5
M and Ki for PIM1 in the range 0.19-0.27 M also comparable to Ki for TBBi 0.27 M.
Moreover, replacement of TBBi by TBBt led to less potent inhibitors. N1-TBBt
derivatives 9c-d at 5M concentration inhibited 40-50% activity of both enzymes, while N2-
TBBt derivatives 10b-d were found to inhibit more effectively PIM1 activity than
CK2activity.
Table 1. The residual activity (%) of CK2,holoenzyme CK2 and PIM1 in the presence
of alkylazido-TBBi (5a-d) alkylazido-TBBt (6a-d, 7a-d), aminoalkyl-TBBi (8a-d),
aminoalkyl-TBBt derivatives (9c,d; 10b-d) and akylamino-2-MeTBBi (14a-d) derivatives.
Residual activity of
Residual activity of PIM1 [%]
CK2
holoenzyme
Ki* M]
CK2 or Ki*[M]
or Ki* [M]
10M Ki* M]
5M
12.5
22
10M
10.5
9.3
Ki* 
Compd.
5a
5M
48.6
54.3
62.2
62
36.6
44.4
34
2.30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.15
5b
-
-
-
-
-
-
-
31.7
45
15.4
33.2
73.5
81
5c
50
5d
87
73
78.5
97
6a
95
90
6b
99
86
89
87
6c
100
96
98
99.7
85
94
6d
95
83.5
7a
6

81
88
100
75
34
24
36
58
60
67
85
64
34
23
17
79
87
-
-
94
89
93
87
-
7b
7c
-
-
-
100
50
-
-
97.8
10.2
9.6
9.5
8.1
54
90
-
7d
-
1.20
0.77
1.35
-
0.77
0.17
0.19
0.23
0.71
-
5.8
4.5
4.7
4.6
34
0.48
0.19
0.27
0.22
-
8a
20
8b
12
8c
23
8d
43
9c
46
-
47
25
-
9d
49
-
0.55
1.2
-
24.7
34
14
0.80
1.10
-
10b
10c
10d
14a
14b
14c
62
-
21
50
-
28
14
19.5
13
1.81
0.82
0.78
0.44
0.18
0.22
2.7
2.1
3.2
1.5
1.5
1.3
0.09
0.08
0.10
8.6
30
19
1.01
0.44
1.2
0.6
0.05
14d
-
-
1.07
0.58
0.17
-
6[19]
14
-
7.6
9
-
11
20
4.6
12
2.2
0.15
0.04
0.27
0.50
TBBi
TBBt
19.6
*Ki values were calculated using Cheng and Prusoff equation: Ki = IC50/(1+ [S]/Km) [20]
The introduction of additional methyl group to C2 carbon in TBBi had little effect on
inhibitory properties towards CK2 kinase (Ki in the range 0.78-1.81 M) and holoCK2 (Ki
0.18-0.44 M for 14a-d vs 0.17-0.77 M for 8a-d) but significantly affected PIM1 inhibition
(Ki 0.05-0.10 M for 14a-d vs 0.19-0.48 M for 8a-d). Among the aminoalkyl- derivatives
14a-d the most potent inhibitors of both CK2 and PIM1 kinases were compounds 14b and
14c, which displayed similar Ki as TBBi for CK2 and holoCK2, and lower Ki than
compound 11, and much lower Ki for PIM1 (0.08 – 0.10 M vs 0.27 M for TBBi).
2.2.2. Kinase profile
The effect of some new derivatives on the activity of other kinases, especially those,
which activity is usually inhibited by the TBBi, TBBt and their derivatives, was also
examined. Specificity studies were performed in ProQinase GmbH, Freiburg, Germany (8b,
7

14b), and data for compounds TBBi, TBBt and CX-4945 are taken from a previous
publications [21, 22].
Table 2. In vitro residual enzymatic activity (%) of 9 kinases in the presence of 8b and 14b
derivatives (selectivity profile)
Kinase
group
Other
CK1
CAMK
CMGC
CMGC
CMGC CMGC CAMK
CLK2 HIPK2 IKKa
AGC
CAMK
PKD2
PIM1
Kinase
Compound
TBBi[21]
CK2
CK1-1
CDK2
DYRK1A
PKA
Residual activity (%) in the presence of 5 M compounds (or 10 M, comp. TBBi and TBBt)[21] and
(0.5 M CX-4945[22]
)
14
6
48
84
23
20
22
21
48
40
3
-
3
75
113
70
5[PKD1]
20
37[IKK
-
2MeTBBi[19] Ki 1.11
17
31
-
15
43
34
30
5
14
8b
14b
TBBt[21]
CX-4945[22]
34
23
4.6
0
42
9.6
2.1
19.6
6
28
39
63
47
10
29
84
35
37[PKD1]
-
61
4
-
58[IKK
102
45
95[CK1
5[DYRK2]
0[CLK3]
15
78
As shown at Table 2, compounds 8b and 14b exhibited similar inhibitory activity to
the studied kinases as other TBBi or TBBt derivatives reported previously [21], 8b inhibits
more efficiently PIM1 (Table 1) CDK2, CLK2 and less efficiently CK1and PKA kinases than
CK2, 14b inhibits more successfully PIM1 (Table 1), CLK2 and less CK1, DYRK1A and
PKA kinases than CK2, but the differences in the rate of inhibition are not big; and CX-
4945 at 0.5 M inhibits the activity of DYRK2, CLK3 and PIM1 (6%, [22]).
2.2.3. Antiproliferative activity
To determine how inhibition of CK2 and PIM1 kinases affects the viability of
different cancer cell lines: CCRF-CEM (leukemia), MCF-7 (breast) and PC-3 (prostate), we
selected the most active inhibitors 8a-d, 10b and 14a-d for in vitro studies and compared their
influence with the known potent and selective inhibitor of CK2, CX-4945. The cells were
treated with different concentration of compounds for 48 h.
The EC50 values were determined to express the concentration of compound which
lowered the viability of the cells to 50% in comparison to cells cultures not treated with
inhibitors and the results are collected in Table 3.
8

Table 3. The influence of new aminoalkyl- derivatives 8a-d and 14a-d on the viability of
CCRF-CEM, MCF-7 and PC-3 cell lines
compound
CCRF-CEM
EC50[M]
4.3 ±0.8
6.3 ±0.1
8.0 ±0.9
6.7 ±0.1
9.0 ±0.3
0.7 ±0.02
2.1 ±0.3
7.5 ±1.7
4.8 ± 0.5
14.2 ±2.8
MCF-7
PC-3
clogPa
soluba
mg/mL
EC50[M]
EC50[M]
8.2±0.4
12.1±1.1
16.1±2.1
10.2±0.9
16.7±2.3
1.4±0.4
4.4±0.4
14.2± 0.8
8.9±0.8
17.5±0.1
6.0 ±0.1
3.76
3.82
4.34
4.78
3.82
3.8
0.47
1.69
1.41
0.87
7.33
1.35
4.89
4.13
2.56
<0.01
8a
8b
8.8 ±0.3
12.7 ±0.4
8.2 ±0.4
9.6 ±0.4
0.9 ±0.03
3.4 ± 0.2
10 ±0.4
8c
8d
10b
14a
14b
14c
14d
TBBi
3.94
4.46
4.91
4.33
6.8 ±0.1
15.7 ±0.5
>50
<10
>50
> 20
>50
n.d.
4.38
4.46
3.29
<0.01
<0.01
0.37
TBBt
2MeTBBi
CX-4945
3.1 ± 0.50
8.4 ± 0.35
10.4± 0.1
The reported values represent the means ± SD from three parallel experiments.
aCalculator plugins were used for clogP and solubility at pH 7.4 ChemAxon
http://chemicalize.com
All new derivatives reduced the viability of studied cancer cell lines at lower
concentrations that their parent compounds TBBi, TBBt and 2MeTBBi. The most sensitive
was the CCRF-CM cell line with EC50 values for compounds 8a-d in the range of 4-8 μM,
and for derivatives 14a-d in the range of 0.7-7.5 μM, while for CX-4945 EC50 was 3.1 M.
(Table 3). TBBt derivative (compound 10b) presented rather a moderate cytotoxic activity
against studied cell lines (EC50 values in the range 9-16.7 μM) but still much higher than
TBBt (EC50 > 50 M) or TBBi (EC50 14.2-17.5 M). More efficient in inhibiting of cell
viability were derivatives of 2MeTBBi (14a-d) than TBBi derivatives (8a-d) also in relation
to the viability of other tested cell lines.
The determined EC50 value for the most potent compounds were as follow: for 14a
0.7±0.02 M; 0.9±0.03 M; 1.4±0.4 M and for 14b 2.1±0.3 M; 3.4±0.2 M; 4.4±0.4 M,
while for CX-4945 were 3.1±0.50 M; 8.4±0.35 M; 10.4±0.1 M for CCRF-CEM, MCF-7
9

and PC-3 cells, respectively. The higher activity of new derivatives having a methyl group on
carbon 2 in the imidazole ring (compounds 14a-d) may be associated with higher solubility of
these derivatives in aqueous solutions (see Table 3).
We also examined the influence of the spacer length of aminoalkyl derivatives on
cytotoxic activities. The data collected in Table 3 showed that compounds 8c,d and 14c,d
with a spacer of four to five methylene units exhibited lower cytotoxic activities, than
compounds 8a,b and 14a,b with the spacer of two or three methylene units.
2.3. Structural study
In order to clarify the binding mode of 8b (Figure 2) to the active site of CK2α we co-
crystallized CK2α1-335, a C-terminally truncated version of human CK2α [23], with the
inhibitor. Previous comparative structure analyses with different CK2 inhibitors [24, 25] had
shown that the local enzyme conformation next to the ligand or the orientation and
conformation of the ligand or both can vary to a certain degree on the salt content of the
crystallization medium and that in particular halogen bonds with aromatic rings can be
strongly salt-dependent [24, 25]. Therefore, we crystallized the CK2α1-335/8b complex, both
under high-salt conditions (with NaCl as precipitant; structure no. 1 in Table 4) and under
low-salt conditions (with PEG3350 as a precipitating agent; structure no. 2 in Table 4). Both
types of crystals diffracted X-rays to about 2.4Å resolution and enabled the determination of
the corresponding 3D-structures (Figure 2A).
Table 4. Crystallization, X-ray diffraction data and refinement statistics
Structure No.
PDB code
1
2
5OWH
Crystallization
5OWL
Method
sitting drop variant of vapour diffusion
20 °C
Temperature
4.4M NaCl, 0.1M citric acid, 25% (w/v) PEG3350, 0.2M ammo-
pH 5.5 nium sulfate, 0.1M BIS-TRIS, pH 6.5
Reservoir composition
1µL reservoir + 1µL enzyme/inhibitor mixture (48µL 7.5 mg/mL
enzyme, 0.5M NaCl, 25mM Tris/HCl, pH 8.5, mixed and pre-
equilibrated with 2 µL 20 mM inhibitor in DMSO)
Sitting drop composition before
equilibration
X-ray Diffraction Data Collection
Wavelength [Å]
1.0000
0.9660
Temperature [K]
100
Synchrotron (beamline)
ESRF (ID23-1)
ESRF (ID23-1)
10

Space group
Unit cell
P43212
P43212
a, b, c [Å]
71.66, 71.66, 133.11
126.31, 126.31, 125.21
α, β, γ [°]
90.0, 90.0, 90.0
1
90.0, 90.0, 90.0
2
Protomers per asymmetric unit
Resolution [Å] (highest res. shell)
Rsym [%]
1
1
63.100 – 2.376 (2.461 – 2.376) 72.710 – 2.391 (2.477 – 2.391)
1
1
9.9 (143.0)
7.2 (106.7)
1
1
CC1/2
0.998 (0.536)
0.999 (0.665)
1
1
Signal-to-noise ratio (I/σI)
No. of unique reflections
Completeness [%]
11.92 (1.07)
14.56 (1.51)
1
1
1
14462 (1391)
98.72 (96.60)
40314 (3831)
99.16 (95.67)
1
1
1
Multiplicity
7.2 (7.1)
5.0 (4.9)
2
Wilson B-factor [Å ]
55.07
54.14
Structure Refinement
13436/1026
No. of reflections for Rwork/Rfree
Rwork/Rfree [%]
39250/1037
19.94/23.50
5795
21.45/25.96
2886
Number of non-H-atoms
Protein 2817
5608
Ligand/ion 18
Water 51
65
122
2
Aver. B-factor [Å ]
67.25
62.70
Protein 67.32
Ligand/ion 105.78
water 49.87
62.26
114.58
55.10
RMS deviations
Bond lengths [Å] 0.003
Bond angles [°] 0.53
0.003
0.54
Ramachandran plot
favoured (%) 96.08
allowed (%) 3.92
outliers (%) 0.00
96.18
3.19
0.00
1
The data in brackets refer to the highest resolution shell.
The low-salt structure contains two independent protein chains per asymmetric unit,
the high-salt structure only one. In each of the three protomers an 8b inhibitor molecule is
bound to the ATP site at the interface of the two major domains of the enzyme (Figure 2A).
This is consistent to the fact that the ATP site of CK2α – compared to many other eukaryotic
protein kinases – is particularly narrow and hydrophobic [26] and thus prone to accommodate
flat and non-polar molecular scaffolds [27].
11

In the high-salt structure this hydrophobicity driven affinity is supported by five polar
interactions of peripheral substituents of 8b (Figure 2 B) with the protein: three bromine
atoms form halogen bonds, namely Br(C7) with the side chain oxygen of Asn118, Br(C6)
with the peptide carbonyl oxygen of Val116 and Br(C4) with the aromatic side chain of
Phe113; in addition the positively charged amino propyl group forms an ionic contact to the
side chain of Asp120 and is hydrogen-bonded to the carbonyl-oxo group of His160 (Figure
1B). Two of these interactions – those with the side chains of Asn118 and Asp120 – are only
possible if these side chains are closely enough to the centre of the ATP site, meaning they
require a particular local conformation of the enzyme’s interdomain hinge/helix αD region
referred to as “closed” [28] or “Phe121-in” conformation [29] in the literature. Crystallization
conditions dominated by precipitating kosmotropic salts support this closed conformation
irrespective of the ATP-site ligand [30] because this optimizes the hydrophic packing within
the protein [31]. Insofar, the high sodium chloride concentration in the crystallization medium
used to obtain structure 1 indirectly – namely by stabilizing the closed conformation of the
hinge/helix αD region – favours the binding of 8b with an orientation in which the amino
propyl moiety points outwards (Figure 2A/B).
Figure 2: Binding of inhibitor 8b to CK2α
A) Overview of the high-salt (green) and the low-salt (red) complex structures after 3D-fit.
The bound inhibitor is drawn with yellow-green C-atoms in its high-salt orientation and with
magenta-coloured C-atoms in its low-salt orientation. B/C) ATP-site with bound inhibitor 8b
in the high-salt structure with a section of the hinge region in the low-structure drawn for
comparison with magenta-coloured C-atoms (B) and in the low-salt structure (C) covered
with the respective polder OMIT map [32] at a contouring level of 3 σ. Halogen bonds are
indicated either with black dotted lines (carbonyl oxygen atoms as Lewis bases) or with
12

orange-coloured dotted lines (phenyl ring π-system as Lewis base) and hydrogen bonds with
blue dotted lines. In the case of the π-halogen bonds three typical parameters are given: the
distance d (between the bromine atom and the aromatic ring centroid) and the angle θ
(between the bromine-carbon bond and the aromatic ring centroid) as defined by Lu et al. [33]
and the d2-distance between the two aromatic ring centres defined by Matter et al. [34].
Against this background it is not surprising that the binding mode of 8b under low-salt
crystallization conditions (Figure 2 C) in which the hinge region adopts an open conformation
(Figure 2 C and also visible for comparison as hinge region section with magenta-coloured C-
atoms in Figure 2 B) is different from that of the high-salt state (Figure 2 B). The side chain
contacts to Asn118 and Asp120 are no longer possible; consequently, the inhibitor is turned
by about 180 degrees around a pseudo-twofold axis running lengthwise through the C2 atom
and the two-ring system. Due to this rotation the amino propyl substituent no longer points
outwards to the hinge, but inwards to the Ph113 side chain. Three halogen bonds are still
present, but now two backbone carbonyl groups are involved (Glu114 and Val116; Figure 2
C). Further, the parameters d, d2 and θ fit better to the typical values of π-Br interactions than
in the high-salt case where in particular the d distance is with 4.4Å (Figure 2 B) rather large.
Noteworthy, under low-salt conditions the Br(C4) atom of the inhibitor is covered
neither by the polder OMIT map (Figure 2 C) nor by the normal electron density (not shown).
Rather there is a significant piece of density next to this atom which is not suitable for filling
with a water molecule or another small molecule. Most probably, this is a radiation damage
artefact occurring during X-ray diffraction data collection and leading to a break of the
covalent bond between the carbon atom C4 and the bromine atom Br(C4) which is not
protected in this structure by a halogen bond. A similar phenomenon was observed in a
complex structure between the paralogous subunit CK2α’ and a tetrabromo benzotriazole
derivative [35] (PDB code 3OFM) in which the enzyme/inhibitor arrangement is quite similar
to the low-salt state found here (Figure 2 C).
3. Conclusions
In summary, a series of new aminoalkyl- TBBi and TBBt derivatives were synthesized by
reduction of the corresponding azidoalkyl- derivatives with quite reasonable yields. The novel
aminoalkyl- TBBi derivatives 8b-c inhibit the activity of CK2 holoenzyme and the PIM1 as
effectively as TBBi, while the derivatives of 2MeTBBi (11), compounds 14a-b exerted
superior inhibition of PIM1 kinase. The aminoalkyl-TBBt derivatives 9c-d and 10b-d
13

appeared to be weaker inhibitors of CK2 but a little stronger inhibitors of PIM1 than parent
TBBt. Evaluation of anti-proliferative activities against CCRF-CEM, MCF-7 and PC-3
human cancer cell lines provided however very interesting results. New alkylamine-
derivatives of TBBi and TBBt reduce the viability of cancer cells much more strongly than
their parent compounds. Particularly interesting properties have shown derivatives of
2MeTBBi (11), compounds 14a-b, which inhibit the viability of tumor cells even more
effectively than the known and approved for clinical trials compound CX-4945. These studies
provide evidence for the effectiveness of developing therapies based on the mechanism of a
combination of CK2 and PIM kinase inhibitors in the treatment of cancer.
4. Experimental
4.1. Chemistry
4.1.1 General
Commercially obtained reagents were used as received without additional purification.
Melting points were determined with an MPA100 Optimelt SRS apparatus and are
uncorrected. NMR spectra were recorded with tetramethylsilane as an internal standard using
a Varian NMR System 500 MHz or Varian Mercury 400 MHz or Bruker 500 MHz
spectrometers. Chemical shifts are reported in ppm from internal tetramethylsilane standard,
splitting patterns have been designated as follows: s = singlet; bs = broad singlet; d = doublet;
t = triplet; q = quartet; dd = double doublet; m = multiplet, quin = quintet. Thin layer
chromatography (TLC) was carried out on aluminium plates with silica gel Kieselgel 60 F254
(Merck) (0.2 mm thickness film) and detection of compounds was performed after UV light at
254nm. Silica gel with grain size 40-63μm was used for column chromatography. Mass
spectrometry were recorded on Micro-mass ESI Q-TOF Premier Waters Spectrometer, and
FTMS were recorded on Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, ESI
(electrospray) with spray voltage 4.00 kV. The most intensive signal are reported.
The following compounds were prepared according to previously described by us
procedures: 4,5,6,7-tetrabromo-1-(2-bromoethyl)-1H-benzimidazole (2a) [15], 4,5,6,7-
tetrabromo-1-(3-chloropropyl)-1H-benzimidazole (2b) [36], 4,5,6,7-tetrabromo-1-(2-
bromoethyl)-1H-benzotriazole
(3a)
[36],
4,5,6,7-tetrabromo-1-(2-bromoethyl)-2H-
benzotriazole (4a) [36], 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzotriazole (3b) and
4,5,6,7-tetrabromo-1-(3-chloropropyl)-2H-benzotriazole (4b) [36], 1-(2-azidoethyl)-4,5,6,7-
tetrabromo-1H-benzimidazole
(5a)
[36],
1-(3-azidopropyl)-4,5,6,7-tetrabromo-1H-
14

benzimidazole (5b) [36], 1-(2-azidoethyl)-4,5,6,7-tetrabromo-1H-benzotriazole (6a) [36], 1-
(3-azidopropyl)-4,5,6,7-tetrabromo-1H-benzotriazole (6b) [36], 2-(2-azidoethyl)-4,5,6,7-
tetrabromo-2H-benzotriazole
(7a)
[36],
2-(3-azidopropyl)-4,5,6,7-tetrabromo-2H-
benzotriazole (7b) [36], and the properties are in agreement with published previously.
4.1.2. Synthesis of 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole (11).
To the suspension of 2-methyl-1H-benzimidazole (1.32 g, 10.0 mmol) in water (80 mL),
bromine (10 mL 195 mmol, 19.5 eq.) was added and refluxed over 72 h. After cooling, the
precipitate was filtered and washed with water. Crude product was purified by crystallization
+
from methanol. Yield: 4.32 g (96%); Mp. 330°C; MS [M+H]+ m/z calcd for C8H5Br4N2
448.7812; found 448.6709. 1H NMR (500 MHz, DMSO-d6) δ ppm: 2.55 (s, 3H, CH3).
4.1.3. General method for the synthesis of aminoalkyl derivatives (8a-d; 9c-d; 10b-d; 14a-d):
A three-step reaction sequence was utilized to prepare title products.
A solution of 1a, 1b or 11 (2.3 mmol) in acetonitrile (20 mL) was stirred with DBU (5.7
mmol) and 1-2-dibromoethane, 1-bromo-3-chloropropane, 1,4-dibromobuthane or 1,5-
dibromopenthane (16 mmol) at room temperature. After completion of the reaction the
reaction mixture was concentrated under reduced pressure. The residue was diluted with water
(25 mL) and extracted with chloroform (3 x 25 mL). The combined organic layers were
washed with water (50 mL), dried over anhydrous MgSO4, filtered, and the solvent was
removed under reduced pressure. The residue was purified as specified in each case.
A solution of appropriate halogenoalkyl derivative 2a-d, 3a-d, 4a-d, 12a-d (1.0 mmol)
and NaN3 (1.3 mmol) in DMF (10 mL) was stirred at 40°C for 24 h. After completion of the
reaction (TLC) the reaction mixture was diluted with ethyl acetate (30 mL), washed with
water (3 x 30 mL), dried over anhydrous MgSO4, filtered, and the solvent was removed under
reduced pressure. Products were purified by crystallization or column chromatography.
A solution of appropriate azide 5a-d, 6a-d, 7a-d, 13a-d (0.56 mmol) and Ph3P (0.62
mmol) in THF (6 mL) and H2O (0.6 mL) was stirred for 48 h at room temperature. After
completion of the reaction (TLC) the reaction mixture was evaporated under reduced
pressure. The residue was suspended in sat. NaHCO3aq. (25 mL) and extracted with
chloroform (3 x 25 mL). The organic layer was dried over anhydrous MgSO4, filtered, and the
solvent was removed under reduced pressure. Products were purified by column
chromatography.
15

4.1.3.1. 2-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)ethanamine (8a).
From 5a; the product was purified by column chromatography (DCM/MeOH/TEA,
15/1/0.1) to afford 8a as white solid (72%), Mp. T>253°C decomp.; FTMS [M+H]+ m/z calcd.
+
for C9H8Br4N3 477.7405, found 477.7403; 1H NMR (500 MHz, DMSO-d6) δ ppm: 3.14 (t,
13
2H, J = 5.9 Hz), 4.62 (t, 2H, J = 5.9 Hz), 8.46 (s, 1H); C NMR (125 MHz, DMSO-d6) δ
ppm: 41.65, 46.91, 106.95, 116.96, 120.79, 122.68, 131.96, 144.20, 149.59.
4.1.3.2. 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-1-amine (8b).
From 5b; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5) to afford 8b as white solid (70%), Mp. 132-134°C; FTMS [M+H]+ m/z calcd. for
+
C10H10Br4N3 491.7562, found 491.7559; 1H NMR (500 MHz, DMSO-d6) δ ppm: 1.58 (quin,
2H, J = 6.9 Hz), 2.50 – 2.53 (m, 2H), 4.53 (t, 2H, J = 6.9), 8.48 (s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ ppm: 35.32, 38.58, 44.82, 107.07, 116.97, 120.81, 122.77, 131.78, 144.15,
149.41.
4.1.3.3. 4-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)butan-1-amine (8c).
From 1a, after work-up and purification by column chromatography (DCM) 2c was
obtained as a white solid; Yield 77%; Mp. 148-149°C; MS [M+H]+ m/z calcd. for
+
C11H10Br5N2 568.6715, found 568.6274.
5c from 2c was purified by crystallization from methanol. White crystals; Yield 87%;
+
Mp. 101-102°C; MS [M+H]+ m/z calcd. for C11H10Br4N5 531.7624, found 531.7014.
8c from 5c; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5 – 8/2/0.5) to afford 8c as white solid. Yield 86%; Mp. 120-122°C; FTMS [M+H]+ m/z
+
1
calcd. for C11H12Br4N3 505.7718, found 505.7714; H NMR (500 MHz, DMSO-d6) δ ppm:
1.29-1.35 (m, 2H), 1.76-1.82 (m, 2H), 2.53 (t, 2H, J = 6.9), 4.47 (t, 2H, J = 7.3), 8.49 (s, 1H);
13C NMR (125 MHz, DMSO-d6) δ ppm: 29.53, 30.01, 41.41, 46.86, 107.01, 116.99, 120.80,
122.80, 131.71, 144.12, 149.31.
4.1.3.4. 5-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)pentan-1-amine (8d).
From 1a, after work-up and purification by column chromatography (DCM) 2d was
obtained as a white solid; yield 79%; Mp. 111-114°C; MS [M+H]+ m/z calcd. for
+
C12H12Br5N2 582.6871, found 582.6476.
16

5d from 2d was purified by column chromatography (DCM/MeOH, 0-2% MeOH) to
+
afford white solid, yield 68%; Mp. 148-149°C; MS [M+H]+ m/z calcd. for C12H12Br4N5
545.7780, found 545.7087.
8d from 5d; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5 – 8/2/0.5) to afford 8d as a white solid. Yield 87%, Mp. 144-147°C; FTMS [M+H]+
+
1
m/z calcd. for C12H14Br4N3 519.7875, found 519.7876; H NMR (500 MHz, DMSO-d6) δ
ppm: 1.24-1.31 (m, 2H), 1.32-1.39 (m, 2H), 1.73-1.81 (m, 2H), 2.49 – 2.52 (m, 2H), 4.46 (t,
2H, J = 7.3), 8.49 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ ppm: 23.52, 31.90, 32.81, 41.71,
46.92, 107.02, 116.99, 120.81, 131.73, 144.13, 149.32.
4.1.3.5. 4-(4,5,6,7-Tetrabromo-1H-benzotriazol-1-yl)butan-1-amine (9c).
From 1b; after work-up and purification by column chromatography (hexane:DCM, 40-
70% DCM) 3c was obtained as a white solid, yield 14%; Mp. 89-91°C; MS [M+H]+ m/z
+
calcd. for C10H9Br5N3 569.6667, found 569.6144.
6c from 3c; the product was purified by crystallization from methanol/chloroform 10/1,
affording 6c as white crystals, yield 65%; Mp. 92-93°C; MS [M+H]+ m/z calcd. for
+
C10H9Br4N6 532.7576, found 532.7770.
9c from 6c; the product was purified by column chromatography (DCM/MeOH/TEA,
100/7/0.6) to afford 9c as white solid, yield 52%; Mp. >211°C decomp.; MS [M+H]+ m/z
+
1
calcd. for C10H11Br4N4 506.7671, found 506.7355; H NMR (500 MHz, DMSO-d6) δ ppm:
1.49-1.64 (m, 2H), 1.90-2.06 (m, 2H), 2.70-2.87 (m, 2H), 4.93 (t, 2H, J = 6.8 Hz); 13C NMR
(125 MHz, DMSO-d6) δ ppm: 24.35, 27.97, 38.58, 49.87, 107.34, 116.19, 124.02, 132.13,
145.39.
4.1.3.6. 5-(4,5,6,7-Tetrabromo-1H-benzotriazol-1-yl)pentan-1-amine (9d).
From 1b; after work-up and purification by column chromatography (hexane:DCM, 40-
70% DCM) 3d was obtained as white solid, yield 20%; Mp. 104-106°C; MS [M+H]+ m/z
+
calcd. for C11H11Br5N3 585.6803, found 585.6236.
6d from 3d; the product was purified by column chromatography (hexane/DCM, DCM
70-100%), affording 6d as a white solid, yield 53%; Mp. 81-83°C; MS [M+H]+ m/z calcd. for
+
C11H11Br4N6 546.7733, found 546.7888.
9d from 6d; the product was purified by column chromatography (DCM/MeOH/TEA,
100/7/0.6) to afford 9d as a white solid, yield 44%; Mp. >266°C decomp.; FTMS [M+H]+ m/z
+
1
calcd. for C11H13Br4N4 520.7827, found 520.7829; H NMR (500 MHz, DMSO-d6) δ ppm:
17

1.31-1.42 (m, 2H), 1.55-1.67 (m, 2H), 1.85-2.00 (m, 2H), 2.58 - 2.66 (m, 2H), 4.89 (t, 2H, J =
13
7.1 Hz); C NMR (125 MHz, DMSO-d6) δ ppm: 23.01, 26.78, 30.53, 38.83, 50.10, 107.36,
116.18, 123.95, 132.10, 145.37.
4.1.3.7. 3-(4,5,6,7-Tetrabromo-2H-benzotriazol-2-yl)propan-1-amine (10b).
From 7b; the product was purified by column chromatography (DCM/MeOH/TEA,
80/2.5/0.4) to afford 10b as a white solid, yield 68%; Mp. >242°C decomp.; FTMS [M+H]+
+
m/z calcd. for C9H9Br4N4 492.7514, found 492.7517; 1H NMR (500 MHz, DMSO-d6) δ ppm:
13
2.18 (quin, 2H, J = 6.9 Hz), 2.72 (t, 2H, J = 6.9 Hz), 4.88 (t, 2H, J = 6.9 Hz); C NMR (125
MHz, DMSO-d6) δ ppm: 31.07, 37.99, 55.13, 114.01, 125.97, 142.97.
4.1.3.8. 4-(4,5,6,7-Tetrabromo-2H-benzotriazol-2-yl)butan-1-amine (10c).
From 1b; after work-up and purification by column chromatography (hexane:DCM, 40-
70% DCM) 4c was obtained as a white solid, yield 43%; Mp. 155-156°C.
7c from 4c; the product was purified by crystallization from methanol/chloroform 10/1,
affording 7c as a white crystals, yield 88%; Mp. 106-108°C.
10c from 7c; the product was purified by column chromatography (DCM/MeOH/TEA,
80/2.5/0.4) to afford 10c as a white solid; yield 84%; Mp. >264°C decomp.; FTMS [M+H]+
+
1
m/z calcd. for C10H11Br4N4 506.7671, found 506.7670; H NMR (500 MHz, DMSO-d6) δ
ppm: 1.35 – 1.50 (m, 2H), 2.06 (quin, 2H, J = 7.3 Hz), 2.64 (t, 2H, J = 6.9 Hz), 4.80 (t, 2H, J
= 7.1 Hz); 13C NMR (125 MHz, DMSO-d6) δ ppm: 27.12, 28.28, 57.26, 114.01, 125.96,
142.94.
4.1.3.9. 5-(4,5,6,7-Tetrabromo-2H-benzotriazol-2-yl)pentan-1-amine (10d).
From 1b; after work-up and purification by column chromatography (hexane:DCM, 40-
70% DCM) 4d was obtained as a white solid, yield 63%; Mp. 163-164°C.
7d from 4d; the product was purified by crystallization from methanol/chloroform 10/1,
affording 7d as a white crystals, yield 90%; Mp. 88-90°C.
10d from 7d; the product was purified by column chromatography (DCM/MeOH/TEA,
95/5/0.5) to afford 10d as a white solid, yield 93%; Mp. 232-235°C; FTMS [M+H]+ m/z
+
1
calcd. for C11H13Br4N4 520.7827, found 520.7828; H NMR (500 MHz, DMSO-d6) δ ppm:
1.24 – 1.41 (m, 2H), 1.47 – 1.65 (m, 2H), 1.90 – 2.14 (m, 2H), 2.73 (t, 2H, J = 7.3 Hz); 4.79
13
(t, 2H, J = 6.8); C NMR (125 MHz, DMSO-d6) δ ppm: 23.15, 26.84, 29.05, 38.87, 57.23,
114.04, 125.95, 142.97.
18

4.1.3.10. 2-(4,5,6,7-Tetrabromo-2-methyl-1H-benzimidazol-1-yl)ethan-1-amine (14a).
From 11; after work-up and purification by column chromatography (DCM) 12a was
obtained as a white solid; yield 29%; Mp. 174-176°C; MS [M+H]+ m/z calcd. for
+
C10H8Br5N2 554.6558, found 554.6149.
13a from 12a; the product was purified by crystallization from methanol affording 13a as
+
a white crystals; yield 96%; Mp. 173-177°C; MS [M+H]+ m/z calcd. for C10H8Br4N5
517.7467, found 517.7076;
14a from 13a; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5) to afford 14a as a white solid, yield 70%; Mp. 149-152°C; FTMS [M+H]+ m/z
+
calcd. for C10H10Br4N3 491.7562, found 491.7564; 1H NMR (500 MHz, DMSO-d6) δ ppm:
2.60 (s, 3H), 2.89 (t, 2H, J = 6.6), 4.35 (t, 2H, J = 6.6 Hz);13C NMR (125 MHz, DMSO-d6) δ
ppm: 15.09, 43.24, 48.11, 106.31, 115.65, 120.18, 121.77, 132.62, 157.76.
4.1.3.11. 3-(4,5,6,7-Tetrabromo-2-methyl-1H-benzimidazol-1-yl)propan-1-amine (14b).
From 11; after work-up and purification by column chromatography (DCM) product 12b
was obtained as
a
mixture of 4,5,6,7-tetrabromo-1-(3-chloropropyl)-2-methyl-1H-
benzimidazole and 4,5,6,7-tetrabromo-1-(3-bromopropyl)-2-methyl-1H-benzimidazole; MS
+
+
[M+H]+ m/z calcd. for C11H10Br4ClN2 524.7220, found 524.6685 and C11H10Br5N2
568.6715, found 568.6478 and was used without further purification.
13b from 12b; the product was purified by crystallization from methanol affording 13b as
+
a white solid, yield 37% from 11; Mp. 168-170°C; MS [M+H]+ m/z calcd. for C11H10Br4N5
531.7624, found 531.7864;
14b from 13b; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5) to afford 14b as a white solid, yield 96%; Mp. >131°C deccomp.; FTMS [M+H]+ m/z
+
1
calcd. for C11H12Br4N3 505.7718, found 505.7717; H NMR (500 MHz, DMSO-d6) δ ppm:
1.83-1.92 (m, 2H); 2.60 (s, 3H), 2.70 (t, 2H, J = 6.7), 4.47 (t, 2H, J = 6.7 Hz); 13C NMR (125
MHz, DMSO-d6) δ ppm: 14.64, 33.43, 38.16, 52.48, 109.99, 115.73, 120.36, 121.97, 132.55,
143.16, 157.12.
4.1.3.12. 4-(4,5,6,7-Tetrabromo-2-methyl-1H-benzimidazol-1-yl)butan-1-amine (14c).
From 11; after work-up and purification by column chromatography (DCM/MeOH 99/1)
12c was obtained as a white solid; yield 59%; Mp. 148-152°C; MS [M+H]+ m/z calcd. for
+
C12H12Br5N2 582.6871, found 582.6865;
19

13c from 12c; the product was purified by crystallization from methanol affording 13c as
+
a white crystals, yield 99%; Mp. 90-95°C; MS [M+H]+ m/z calcd. for C12H12Br4N5
545.7780, found 545.7614;
14c from 13c; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5) to afford 14c as a white solid, yield 64%; Mp. >112°C deccomp.; FTMS [M+H]+ m/z
+
1
calcd. for C12H14Br4N3 519.7875, found 519.7879; H NMR (500 MHz, DMSO-d6) δ ppm:
1.40-1.54 (m, 2H), 1.66-1.83 (m, 2H), 2.53-2.71 (m, 5H), 4.25-4.48 (m, 2H); 13C NMR (125
MHz, DMSO-d6) δ ppm: 14.62, 28.81, 29.00, 41.00, 52.47, 106.36, 115.74, 120.35, 122.01,
132.49, 143.11, 157.00.
4.1.3.13. 5-(4,5,6,7-Tetrabromo-2-methyl-1H-benzimidazol-1-yl)pentan-1-amine (14d).
From 11; after work-up and purification by column chromatography (DCM) 12d was
obtained as a white solid; yield 58%; Mp. 149-155°C; MS [M+H]+ m/z calcd. for
+
C13H14Br5N2 596.7028, found 596.6276.
13d from 12d; the product was purified by column chromatography (DCM) affording
+
13d as a white solid; yield 99%; Mp. 117-120°C; MS [M+H]+ m/z calcd. for C13H14Br4N5
559.7937, found 559.7260;
14d from 13d; the product was purified by column chromatography (DCM/MeOH/TEA,
9/1/0.5) to afford 14d as a white solid; yield 86%, Mp. > 92°C deccomp.; FTMS [M+H]+ m/z
+
1
calcd. for C13H16Br4N3 533.8031, found 533.8032; H NMR (500 MHz, DMSO-d6) δ ppm:
1.33-1.47 (m, 4H), 1.65-1.75 (m, 2H), 2.54 (t, 2H, J = 6.4 Hz), 2.58 (s, 3H), 4.29-4.41 (m,
2H); 13C NMR (125 MHz, DMSO-d6) δ ppm: 14.61, 23.58, 31.25, 32.66, 41.62, 52.47,
106.30, 115.73, 120.32, 121.97, 132.45, 143.10, 156.94.
4.2. Crystallization
4.2.1. CK2 Protein for crystallization
The C-terminal truncated version of human CK2α (CK2α1-335) was expressed and
purified as described previously [23] with the exception that the P11 phosphocellulose
chromatography step was replaced with a batch purification step using the same resin
material. Purified CK2α1-335 was rebuffered in storage solution (25 mM Tris/HCl, 500 mM
NaCl, pH 8,5). The final CK2α1-335 protein concentration was 7 mg/mL as determined via UV-
absorption at 280 nm.
20

The CK2α1-335 solution (90 µL) were mixed with 10 µL of a 10 mM 8b solution in
DMSO. The mixture incubated for 30 min at room temperature; afterwards precipitated
material was removed by centrifugation. CK2α1-335/8b co-crystals were obtained in high-salt
and in low-salt conditions using the sitting drop variant of vapor diffusion at 20°C. The
optimized drop composition was 1 µL of the preincubated CK2α1-335/8b mixture and 1 µL
reservoir solution. The optimized reservoir solutions were 4.4 M NaCl, 0.1 M sodium acetate,
pH 5.5, (high-salt case) and 25% (w/v) PEG3350, 0.2 M ammonium sulfate, 0.1 M BIS-TRIS
buffer, pH 5.5 (low-salt case).
4.2.2. X-Ray Diffractometry
Suitable CK2α1-335/8b co-crystals were flash-frozen in liquid nitrogen. Cryo conditions
for the low-salt CK2α1-335/8b crystals were obtained by adding (R)-butane-1,3-diol into the
drop. The high-salt condition was already a cryo condition. Preliminary X-ray diffraction data
were collected at the beamline X06DA of the Swiss Light Source (SLS), Paul Scherrer
Institut, in Villigen, Switzerland. The final diffraction data sets were obtained at the beamline
ID23-1 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, using
wavelengths of 1 Å (high-salt structure) and 0.9660 Å (low-salt structure) and a temperature
of 100 K. The diffraction data sets were integrated with XDS [37] and scaled with AIMLESS
[38] from the CCP4 sofware suite [39].
4.2.3. Structure Solution, Refinement, Validation, Deposition and Illustration
The complex structures were solved by molecular replacement with PHASER [40]
using the protein chain of the CK2α1-335 structure 5CQU [41] as a search model. The
structures were refined and validated using PHENIX [42] which also served to generate the
topology of the 8b molecule. COOT [43] was used for manual corrections of the structures.
The final structures are available at the Protein Data Bank under the accession codes 5OWH
(high-salt structure) and 5OWL (low-salt structure). The structure illustrations in Figure 2
were prepared with PYMOL [44].
4.3 Biological assays
4.3.1. Cloning , expression and purification of human CK2 and CK2 kinase
The coding regions of human CK2α and CK2β were amplified by PCR using the
following primers: 5’- CCG GAA TTC TCG GGA CCC GTG CCA AGC AGG (CK2αF),
21

5’- TAA AGC GGC CGC TGC TGA GCG CCA GCG G (CK2αR), 5’- GGA ATT CCA
TAT GAG CAG CTC AGA GGA GGT GTC C-3’ (CK2βF) and 5’- CCC AAG CTT CAA
ACC CGT TTA GAG GCC C-3’ (CK2βR) and IMAGE clones (GenBank entry BC053532.1
and BC112017.1) as a template. The CK2α PCR-product was digested with EcoRI and NotI
and ligated into pGEX4T1 vector (GE Healthcare) carrying a N-terminal GST tag. The CK2β
PCR-product was digested with NdeI and HindIII and ligated into pET28a vector (Novagen)
carrying a N-terminal His tag. The cloned CK2α and CK2β genes were verified by
sequencing.
GST-CK2α and His-CK2β were overproduced separately in the bacterial strain
BL21(DE3)pLysS (Invitrogen) growing in Super Broth medium [13] after induction with
1 mM IPTG for 20 h at 20°C. Pellets were resuspended in extraction buffer (20 mM
NaH2PO4, pH 7.9) 500 mM NaCl, 50 mM imidazole, protease inhibitors (Complete EDTA-
free, Roche), mixed (2:1 pellet α:β mass ratio) and sonicated. The cleared lysate was loaded
onto HisTrap HP 5 mL column mounted on a AKTA Purifier 10 FPLC system (GE
Healthcare). CK2 holoenzyme was eluted with imidazole gradient in extraction buffer (50-
500 mM). Fractions containing His-tagged CK2 were dialyzed against 20 mM NaH2PO4 (pH
7.9), 200 mM NaCl and loaded onto GSTTrap HP 5 mL column (GE Healthcare). GST-
tagged CK2 was eluted with 10 mM glutathione in dialysis buffer. Finally, fractions
containing CK2 holoenzyme were dialyzed overnight against 25 mM Tris-HCl pH 8.5, 200
mM NaCl, 1 mM DTT, 0-25% glycerol and stored at -20°C. The protein concentration in final
solution was 1.61 mg/mL (determined by Bradford method with bovine serum albumin as a
standard) [45].
4.3.2. Cloning, expression and purification of human kinase PIM1
The coding region of human PIM1 was amplified by PCR using the following primers:
5’- GGA ATT CCA TAT GCT CTT GTC CAA AAT CAA CTC GCT TG (PIM1F) and
5’- CAA GCT TGT TTG CTG GGC CCC GGC GA (PIM1R) and IMAGE clone (GenBank
entry BC0020224.1) as a template. The PIM1 PCR-product was digested with NdeI and
HindIII and ligated into pET28a vector (Novagen) carrying a N-terminal His tag. The cloned
PIM1 gene was verified by sequencing.
Expression of N-terminal histidine-tagged PIM1 was done in the bacterial strain
BL21(DE3)pLysS (Invitrogen) growing in Super Broth medium after induction with 1 mM
IPTG for 20 h at 20oC. The cell pellet from 800 mL of bacterial culture was resuspended in
extraction buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole, protease
22

inhibitors (Complete EDTA-free, Roche)) and sonicated. The cleared lysate was loaded onto
HisTrap HP 5 mL column mounted on a AKTA Purifier 10 FPLC system (GE Healthcare).
PIM1 was eluted with imidazole gradient in extraction buffer (20-500 mM). Fractions
containing His-tagged PIM1 were dialysed against 50 mM Tris-HCl pH 7.5, 150 mM NaCl,
2 mM DTT, 0.5 mM PMSF, 0-25% glycerol and stored at -20°C. The protein concentration in
final solution was 3.0 mg/mL (determined by Bradford method with bovine serum albumin as
a standard) [45].
4.3.3. Phosphorylation assays
The new TBBi and TBBt derivatives were tested for their inhibitory activity toward
human CK2, human CK2 holoenzyme and PIM1 (Table 3) using P81 filter isotopic assay
[46]. CK2α activity was tested in final volume of 50 µL containing CK2α (21.2 nM), Tris-
HCl (pH 7.5; 23 mM), MgCl2 (20 mM), DTT (0.4 mM), synthetic peptide substrate
(RRRADDSDDDDD 40 µM; Biaffin GmbH), [-32P]-ATP (10 µM, 200-300 cpm/pmol) and
appropriate concentrations of inhibitor in 2 L DMSO or 2 L DMSO as control. After 15
min incubation at 30°C, 5 L of the assay mixture was spotted onto P81 paper, which was
subsequently washed with 0.6% o-phosphoric acid three times and allowed to dry.
CK2 holoenzyme activity was tested in final volume of 50 µL containing CK2
holoenzyme (28.6 nM), Tris-HCl (pH 7.5, 23 mM), NaCl (100 mM), MgCl2 (20 mM), DTT
(0.4 mM), peptide substrate RRRADDSDDDDD (40 µM) and-32P]-ATP (20 µM, 200-300
cpm/pmol) and appropriate concentrations of inhibitor in 2 L DMSO or 2 L DMSO as
control. The reaction mixture was incubated for 15 min at 30°C, and treated as above.
PIM1 activity was tested in final volume of 50 µL containing PIM1 (25.30 nM), Tris-HCl
(pH 7.5, 23 mM), MgCl2 (20 mM), DTT (0.4 mM), [32P]-ATP (20 µM, 200-300
cpm/pmol), and 30 µM synthetic peptide substrate ARKRRRHPSGPPTA. The reaction was
initiated with enzyme, incubated for 20 min at 30°C, and next 5 µL of the reaction mixture
was spotted onto P81 paper. The filter papers were washed 3 x with 0.6% o-phosphoric acid
and once with 95% ethanol before counting in a scintillation counter (Canberra-Packard).
The IC50 values for studied compounds were determined at 4% DMSO with 8
concentrations of each tested inhibitor at the range of 0.064-1000 µM and calculated by fitting
the data to sigmoidal dose-response (variable slope) Y = Bottom + (Top-Bottom) /
(1+10^((LogIC50-X)*HillSlope) equation in GraphPad Prism.
23

4.3.4 Selectivity profile
Specificity studies for compounds 8b and 14b at 5 M concentration were performed in
ProQinase GmbH, Freiburg, Germany.
4.3.5 Cell culture and treatment
CCRF-CEM (human Caucasian acute lymphoblastic leukemia, ECACC 85112105) were
obtained from ECACC, MCF-7 (human breast cancer cell line, authentication certificate,
ATCC STRA4788) was a gift from Nencki Institute of Experimental Biology in Warsaw,
PAS. PC-3 (human prostate cancer) was a gift from National Medicines Institute, Warsaw,
Poland. MCF-7 were cultured in DMEM with high glucose medium (Lonza) with 10 µg/mL
of human recombinant insulin. CCRF-CEM suspension cells and PC-3 cells were cultured in
RPMI 1640 medium (Lonza). All cell lines were supplemented with 10% fetal bovine serum
(EuroClone), 2 mM L-glutamine and antibiotics (100 U/mL penicillin, 100 µg/mL
streptomycin) and grown in 75 cm2 cell culture flasks (Sarstedt), in a humidified atmosphere
of CO2/air (5/95%) at 37°C.
4.3.6 Cell viability assays (MTT-based) and EC50 determination
Before the treatment, adherent cells (MCF-7, and PC-3) were trypsinized in 0.25%
trypsin-EDTA solution (Sigma-Aldrich) and seeded into 96-well microplates at 0.6 x 104
cells/well. CCRF-CEM were seeded at 2 x 104 cells/well. Leukemia cells after seeding and
adherent cells 24 h after plating (at 70% of confluency) were treated with tested compounds at
the appropriate concentrations or DMSO at 0.5% final concentration. EC50 values were
determined at eight concentrations in the range of 0.39-50 µM for each inhibitor for PC-3, and
0.78-100 µM for MCF-7 and 1.2-20 µM or 0.1-2.0 µM (14a) for CCRF-CEM. The
experimental data were fitted to sigmoidal dose-response (variable slope) Y = Bottom + (Top-
Bottom) / (1+10^((LogIC50-X)*HillSlope) equation in GraphPad Prism.
After 48 h incubation with compounds or DMSO, suspension cells were centrifuged and
supernatants of CCRF-CEM, MCF-7 and PC-3 cells were discarded; subsequently MTT stock
solution (Sigma-Aldrich) was added to each well to a final concentration of 0.5 mg/mL. After
2 h of incubation at 37°C, water-insoluble dark blue formazan crystals were dissolved in
DMSO (200 µL) (37°C/10 min incubation). Optical densities were measured at 570 nm using
BioTek microplate reader. All measurements were carried out in triplicate and the results are
expressed in percentage of cell viability relative to control (cells without inhibitor in 0.5%
DMSO).
24

1
Supplementary Materials: HRMS spectra, H-NMR, and 13C-NMR spectra are available
online at
Declaration of interest
The authors report no conflicts of interest.
Acknowledgments: This work was supported by an NSC Poland grant
2014/13/B/NZ7/02273, by Warsaw University of Technology and by the Deutsche
Forschungsgemeinschaft (grant NI 643/4-2).
We thank the staff of the beamline X06DA at the SLS (Villigen, Switzerland) and of the
beamline ID23-1 at the ESRF (Grenoble, France) for support with X-ray data collection. We
are grateful to Professor Ulrich Baumann (University of Cologne) for access to protein
crystallography infrastructure.
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