Synthesis of novel halogenated heterocycles based on o‐phenylenediamine and their interactions with the catalytic subunit of protein kinase ck2

Article abstract of DOI:10.3390/molecules26113163

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER‐stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low‐mass ATP‐competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5‐dihalo‐benzene‐1,2‐diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP‐binding site of CK2. HPLC‐derived ligand hydrophobicity data are compared with the binding affinity assessed by low‐volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.

Full text of DOI:10.3390/molecules26113163

molecules
Article
Synthesis of Novel Halogenated Heterocycles Based on
o-Phenylenediamine and Their Interactions with the Catalytic
Subunit of Protein Kinase CK2
Maria Winiewska-Szajewska , Agnieszka Monika Maciejewska , El z˙ bieta Speina , Jarosław Pozna n´ ski *
and Daniel Paprocki *
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi n´ skiego 5a, 02-106 Warsaw, Poland;
mwin@ibb.waw.pl (M.W.-S.); agniesza@ibb.waw.pl (A.M.M.); elasp@ibb.waw.pl (E.S.)
*
Correspondence: jarek@ibb.waw.pl (J.P.); dpaprocki@ibb.waw.pl (D.P.)
Abstract: Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating
hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability,
apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is
found perturbed in many pathological states, including cancers, it becomes an attractive target for
the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed,
the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic
ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand
series were selected to enable the separation of the scaffold effect from the hydrophobic interactions
attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied
to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand
hydrophobicity data are compared with the binding affinity assessed by low-volume differential
scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have
not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
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ꢁꢂꢃꢄꢅꢆ
Citation: Winiewska-Szajewska, M.;
Maciejewska, A.M.; Speina, E.;
Pozna n´ ski, J.; Paprocki, D. Synthesis
of Novel Halogenated Heterocycles
Based on o-Phenylenediamine and
Their Interactions with the Catalytic
Subunit of Protein Kinase CK2.
activity against CK2α and toxicity against four reference cell lines have been determined for eight
compounds identified as the most promising in nanoDSF assay.
Molecules 2021, 26, 3163. https://
doi.org/10.3390/molecules26113163
Keywords: kinase CK2; differential scanning fluorimetry; molecular modeling; halogenated
heterocycles; hydrophobic contribution; ligand binding; inhibitory activity; cell toxicity
Academic Editor: Pascal Marchand
Received: 27 February 2021
Accepted: 22 May 2021
Published: 25 May 2021
1
. Introduction
Protein kinase CK2 is considered as one of the essential human proteins, which is
involved in numerous cellular processes. This is a highly pleiotropic kinase, which can
phosphorylate hundreds of protein substrates including transcriptional factors, proteins
affecting the structure of DNA/ RNA, and/or involved in RNA synthesis and translation,
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iations.
signaling proteins and also pathogenic virus replication machinery proteins [1]. The nu-
merous natural substrates of kinase CK2 make it indispensable for many cellular functions
including cell viability, apoptosis, cell proliferation and survival, angiogenesis, DNA dam-
age and repair, the ER-stress response, the regulation of carbohydrate metabolism, and in
the nervous system [2]. Kinase CK2 is the holoenzyme that acts in vivo as a heterotetramer,
consisting of two catalytic (
as a monomeric kinase alone, the regulatory subunit only improving the activity [3].
Since kinase CK2 was found to be overexpressed in many forms of human cancers [
its role in cancer pathogenesis is thoroughly investigated. CK2 could promote cell survival
by phosphorylation of sites adjacent to caspase cleavage sites, which blocks its activity [ ].
Kinase CK2 is also involved in DNA damage response and DNA repair pathways, which
may additionally contribute to the regulation of cancer cell survival [ ]. Despite the fact
that the exact function of hCK2 in oncogenesis remains unknown and also its contribution
α
- and/or ’) and two regulatory ( ) subunits, but also may act
α
β
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4],
4–7
8
4
.0/).
Molecules 2021, 26, 3163. https://doi.org/10.3390/molecules26113163
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3163
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may be different among various carcinomas [
in targeting this kinase therapeutically [12].
9–
11], widespread interest has been expressed
Dozens of kinase CK2 inhibitors have been already described. Among them, ATP-
competitive inhibitors, halogenated benzotriazole, and benzimidazole derivatives play
an important role. Following 5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (DRB) [13],
4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz)
were identified as strong and selective kinase CK2 inhibitors [14]. Further studies on these
compounds resulted in the synthesis of numerous potent kinase CK2 inhibitors, includ-
ing 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole (DMAT) [15]. Besides, studies
0
also showed that derivatives of 4,5,6,7-tetraiodo-1H-benzimidazole (TIBz) inhibit
α and α
subunits of CK2 much more than TBBz does [16]. Sulfurated derivatives of TBBz are also ef-
ficient kinase CK2 inhibitors, e.g., 4,5,6,7-tetrabromo-1H-benzoimidazole-2(3H)-thione (K22)
and 4,5,6,7-tetrabromo-2-(methylthio)-1H- benzoimidazole (K37) inhibit kinase stronger
than TBBt [17,18]. 5,6,7,8-tetrabromo-1-methyl-2,3-dihydro-1H-benzoimidazo[1,2-a] imida-
zole (K44), containing an additional imidazolidine ring in its structure, was also reported to
strongly inhibit CK2 [17,18]. Structures of all these CK2 inhibitors are shown in Figure 1.
Figure 1. Structures of potent inhibitors of kinase CK2. Bolded fragments represent the common o-phenylenediamine substructure.
The majority of halogenated ligands target the ATP-binding site of a protein kinase;
however, their poses differ significantly [19–23]. Recent studies have indicated that the
binding mode of halogenated benzotriazoles and benzimidazoles is generally driven by a
competition between a hydrogen bond or salt bridge and halogen bonding [20], while ligand
hydrophobicity contributes significantly to the binding affinity [23,24]. Systematic structural
studies on complexes of halogenated ligands pointed the preferred geometry [25
–27] and

Molecules 2021, 26, 3163
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topology [21,28] of a halogen bond in protein-ligand systems, however, the thermodynamic
contribution of a halogen bond to free energy of ligand binding remains debatable, being
estimated in the range of 0.2 [29] to 5 kcal/mol [30].
Our long-term systematic studies on protein kinase CK2 ligands have demonstrated
that halogenated benzotriazole derivatives showed moderate inhibitory activity against
CK2, which is closely related to the physicochemical properties of the ligand [31–36]. We
have also analyzed the effect of substitution pattern, demonstrating that benzotriazole
derivatives carrying halogen atoms at positions 5 and 6 interact with CK2 much stronger
than those substituted at positions proximal to the triazole ring (positions 4 and 7) [34,35].
What is more, the presence of the iodine atom in the structure significantly enhances
the affinity [37], which is in line with results obtained by Janeczko et al. for massively
iodinated benzimidazole derivatives [16]. Both benzotriazoles and benzimidazoles possess
o-phenylenediamine fragment in their structure (see Figure 1). Looking for the novel
groups of compounds as putative kinase CK2 inhibitors, we decided to synthesize groups
of similar compounds, based on o-phenylenediamine configuration.
Here, we synthesized and described six series of heterocyclic halogenated ligands
derived from 4,5-dihalogeno-benzene-1,2-diamines. These ligands were selected to enable
the separation of the scaffold effect from the hydrophobic interactions attributed directly
to the halogen atoms. We measured the hydrophobicity using reverse-phase HPLC. The
binding affinities to the catalytic subunit of human protein kinase CK2 (hCK2α) were
initially assessed using low-volume differential scanning fluorimetry (nanoDSF), and
the dominating modes of binding were analyzed using a molecular modeling approach.
Cytotoxicity against four reference cell lines was tested for the most promising compounds
along with their inhibitory activity against CK2.
2. Results
2.1. Chemistry
We synthesized six series of halogenated heterocyclic compounds starting from 4,5-
dihalogenobenzene-1,2-diamines (1a ). While the fluorinated, chlorinated, and bromi-
–
e
nated amines are commercially available, 4,5-diiodobenzene-1,2-diamine was synthesized,
according to the protocol described previously, using a two-step procedure starting from 1,2-
dinitrobenzene [37]. 2,1,3-Benzothiadiazole derivatives (2a
corresponding diamines during reaction with thionyl chloride and triethylamine performed
in dichloromethane [38]. 2-Trifluoromethyl-1H-benzimidazole derivatives (3a ) were syn-
thesized by heating corresponding amine 1a in trifluoroacetic acid in the presence of
the catalytic amount of conc. HCl [39]. 2-hydroxymethyl-1H-benzimidazole derivatives
4a ) were obtained by heating diamine with 2-hydroxyacetic acid in hydrogen chloride
–e) were synthesized from the
–
e
–
e
(
,c–e
solution [40]. 1H-benzo[d]imidazol-2(3H)-ones (5a
N,N -carbonyldiimidazole (CDI) mixed in DMF [41]. Quinoxaline derivatives (6a
,
c
–
e
) were synthesized from 1a
–
e
and
0
,
c–
e
) were
obtained by heating substrate 1a–e with glyoxal solution in ethanol. Quinoxaline-2,3-diol
derivatives (7a ) were obtained by heating diamine 1a with oxalic acid in DMF. The
synthesis strategy of compounds 2–7 is summarized in Scheme 1.
–
e
–e
2.2. Hydrophobicity Data
RP-HPLC method, a fast and efficient alternative for standard logD determination
in octan-1-ol/water system [42], was used to assess the hydrophobicity of all synthesized
compounds. This method has been successfully used in our laboratory for halogenated
benzotriazole derivatives, including the evaluation of the effect of hydrophobicity on their
binding to the catalytic subunit of protein kinase CK2 [24,43].
As expected, the substitution of two hydrogen atoms with the larger halogen atoms:
fluorine, chlorine, bromine, or iodine, respectively, always results in an increase of apparent
hydrophobicity (Figure 2A). This trend is generally independent on the scaffold of the
molecule (Figure 2B).

Molecules 2021, 26, 3163
4 of 16
◦
Scheme 1. Synthesis of compounds
reflux; ( ) HOCH COOH, HCl, H O, reflux; (
f) oxalic acid, DMF, 120 C.
2
–
7
. Conditions: (
a
) SOCl , Et N, DCM, 40 C; (
b
) TFA, HCl
2
3
c
d
) CDI, DMF, RT; (e) 40% glyoxal solution, EtOH, reflux;
2
2
◦
(
Figure 2. Effect of substitution at the two distant positions of the benzene ring with a halogen atom
on HPLC-derived hydrophobicity index (log( )) ( ). The same values are represented relative to the
unsubstituted compound of the same scaffold (B).
τ
A
Indeed, regardless of the scaffold, the increase of hydrophobicity upon halogen sub-
stitution in distant positions of the benzene ring of 2-trifluoromethyl- 1H- benzimidazole
(
3a), 2-hydroxymethyl-1H-benzimidazole (4a), and 1H-benzo[d]imidazol-2(3H)-one (5a) is
identical. A similar trend is also observed in the case of quinoxaline-2,3-diol (7a) and benzo-
,2-diamine (1a), however, for the latter, the effect of halogenation is shifted downwards by
.1 log( ) units. The largest changes in hydrophobicity experience quinoxaline (6a) deriva-
1
0
τ
tives, which should be directly attributed to the lack of the effect of electronegative halogen
atom on the proximal proton-donating groups. Such effect, partially compensating halogen
hydrophobicity, was evidenced in protein-ligand structures, in which hydrogen bonds
proximal to a halogen atom were statistically shortened (so must be regarded as stronger)
when a nitrogen atom close to the halogen atom (less than six chemical bonds) was a
hydrogen bond donor [44]. No such configuration is present in 6a, so the effect of halogen
substitution is larger because it cannot be compensated in 6c–e by the halogen-induced

Molecules 2021, 26, 3163
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strengthening of solute-solvent interaction. Finally, the effect of halogen substitution in the
most hydrophobic 2,1,3-benzothiadiazole (2a) remains irregular, which putatively results
from an uncontrolled aggregation of these solutes.
2.3. Binding Affinity—Thermal Shift Assay
Binding affinities of the newly synthesized compounds towards hCK2α were assessed
with the low-volume differential scanning fluorimetry (nanoDSF). The shift of the tempera-
ture of thermal denaturation in the presence of 10-fold excess of a particular ligand relative
to the apo form of hCK2α (∆T ) can be used as a semi-qualitative measure of the binding
m
affinity [35] or inhibitory activity [45].
Groups of ligands with different scaffolds vary visibly in their affinity to the target
protein (see Table 1 and Figure 3). Interestingly, two of the most hydrophobic groups
of compounds, 2,1,3-benzothiadiazoles (2a
interact with hCK2 —for all these compounds,
,2-diamine derivatives (1a
–
e
) and quinoxalines (6a
–
e
), virtually do not
◦
α
∆
T
m
value never exceeds 1 C. Benzene-
◦
1
–
e
) also interact weakly with hCK2
α
(
∆
T
m
value 1.7 C for
1e), but this effect should be attributed directly to the size of the ligand—the absence
of the second aromatic ring in the scaffold that appears to be necessary for the efficient
ligand-binding to the kinase [20]. There is no general correlation between hydrophobicity
and binding affinity, however, when one analyzes individual series representing the same
m
scaffold. A comparison of log(τ) and ∆T for the protein–ligand complexes indicates that
any increase in ligand hydrophobicity is clearly reflected by an increase in binding affinity,
as long as the ligand binds to the target (see Figure 3). Moreover, it seems that the effect
of Cl, Br, and I substitution on the stabilization of the protein–ligand complexes not only
does not depend on the structure of the heterocyclic ring but is directly proportional to the
hydrophobicity of the halogen. The latter is evidenced by almost parallel lines representing
the contribution of halogen atoms in individual ligands. It could be thus anticipated that for
the studied series of ligands, the thermodynamic contribution of non-specific hydrophobic
interactions of halogen atoms to the free energy of ligand binding remains separated from
that of the direct electrostatic interactions involving a more polar heterocyclic ring. This
hypothesis was tested with in silico modeling of protein–ligand structures.
m
Figure 3. Correlation between HPLC-derived hydrophobicity index (log(τ)) and the ∆T determined
using nanoDSF.

Molecules 2021, 26, 3163
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Table 1. NanoDSF-derived
m
∆T , HPLC-derived hydrophobicity index log(τ) values and retention times Tret of tested
compounds, and Gibbs free energy of binding adopted from the docking scores.
◦
Entry
Structure
R
Compound
∆Tm [ C]
Tret [min]
log(τ)
∆Gbind [kJ/mol]
1
2
3
4
5
H
F
1a
1b
1c
−0.1 ± 0.1
−0.1 ± 0.1
0.1 ± 0.1
0.2 ± 0.1
1.7 ± 0.1
0.0 ± 0.1
−0.3 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
0.4 ± 0.1
0.8 ± 0.1
1.2 ± 0.1
2.4 ± 0.1
4.3 ± 0.1
0.4 ± 0.1
0.7 ± 0.1
1.9 ± 0.1
3.7 ± 0.1
0.8 ± 0.1
0.8 ± 0.1
1.8 ± 0.1
2.4 ± 0.1
−0.1 ± 0.1
0.1 ± 0.1
0.4 ± 0.1
0.4 ± 0.1
0.1 ± 0.1
0.2 ± 0.1
1.2 ± 0.1
2.7 ± 0.1
4.1 ± 0.1
6.12
6.28
7.52
8.10
9.03
−0.651
−0.591
−0.298
−0.208
−0.093
0.009
0.169
0.179
0.461
0.468
25.9
31.4
28.5
29.2
28.9
Cl
Br
I
1d
1e
6
7
8
9
H
F
Cl
Br
I
2a
2b
2c
2d
2e
10.10
12.38
12.55
19.45
19.70
28.3
33.5
30.8
29.9
29.9
1
0
11
12
13
14
15
H
F
Cl
Br
I
3a
3b
3c
3d
3e
7.68
9.02
13.57
15.20
17.83
−0.270
−0.095
0.234
0.310
0.409
36.6
40.5
37.9
35.6
40.9
16
17
18
19
H
Cl
Br
I
4a
4c
4d
4e
6.32
9.17
10.12
11.12
−0.579
−0.079
0.010
31.4
33.8
32.7
32.2
0.088
20
21
22
23
H
Cl
Br
I
5a
5c
5d
5e
6.28
9.12
10.03
11.03
−0.591
−0.084
0.003
32.4
33.0
33.3
32.7
0.082
24
25
26
27
H
Cl
Br
I
6a
6c
6d
6e
7.53
20.62
24.37
25.73
−0.295
0.495
0.588
0.618
29.7
31.1
31.2
31.1
28
29
30
31
32
H
F
Cl
Br
I
7a
7b
7c
7d
7e
6.00
6.42
7.97
8.35
8.82
−0.699
−0.548
−0.227
−0.174
−0.117
34.5
40.6
38.1
38.5
38.5
2.4. Inhibitory Activity
Since in the thermal shift assay, most of the studied compounds seem to have a
negligible affinity towards hCK2
α
, only eight compounds were further subjected to the
◦
direct measurement of IC —solely compounds with the thermal shift above 2 C may be
50
considered as at least moderate inhibitors of hCK2α.
Comparison of thermal shift assay data ( Tm) with the directly measured IC50 con-
∆
firms that nanoDSF is an attractive method for inhibitors screening, however, for ligands
with different scaffolds, hence different modes of binding, this might not be directly re-
flected in the biochemical assay.
Finally, it should be mentioned that the inhibitory activity of two compounds found
the most promising according to nanoDSF (i.e., 5,6-diiodo-2-trifluoromethyl-1H- benz-
imidazole, 3e, and 6,7-diiodooquinoxaline-1,3-diol,7e) also exhibit the highest inhibitory
activity with micromolar IC50 values (see Table 2).
2
.5. Cell Toxicity
The same four bromo and four iodo- derivatives that, according to nanoDSF assay,
interact with the catalytic subunit of CK2 (scaffolds 3, 4, 5, 7) were tested against the reference
cancer cell lines: epidermoid carcinoma A-431, colorectal carcinoma HCT116, HCT116p53
and normal fibroblasts BJ. All of them displayed moderate activity to reduce cell viability
<100 M). Interestingly, compound 3e was identified as even more active than 3d, the
promising activity of which has been already reported [46]. In this study compound, 3d
displayed IC80 values between 2 and 29.1 M tested for five cancer cell lines (IM-9, MOLT3,
U-937, MCF-7, and PC-3), and 8.7 M on phytohemagglutinin-stimulated normal human
lymphocytes. It was especially active against two cell lines: MCF-7 (breast carcinoma) and
PC-3 (prostate carcinoma), with IC80 values below 3 M. Our data show the selectivity index
α
−/−
,
(
µ
µ
µ
µ

Molecules 2021, 26, 3163
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of 3e
~10 (relative to BJ fibroblasts), which makes this compound worth further investigations.
We were unable to determine the cytotoxicity for 5e, for which the observed nonmonotonic
dose-response effect was most probably caused by uncontrolled aggregation. All cell toxicity
data are summarized in Table 2, and the analyses of viability are shown in Supplementary
Figures S30–S37. The toxicity is generally uncorrelated with the inhibitory activity; however,
this effect can be directly assigned to membrane permeability. This hypothesis is strongly
supported by a clear correlation between compound hydrophobicity (log (τ)) and IC50 values
estimated from viability data (Figure 4).
Table 2. Activity of bromo (d) and iodo (e) derivatives against hCK2α and four human cell lines.
IC50 [µM]
Compound
−
/−
hCK2α Assay
15 ± 3
7 ± 4
A-431
13 ± 2
7 ± 1
45 ± 5
39 ± 10
180 ± 40
-
BJ
HTC116
10 ± 2
6 ± 1
HCT116p53
12 ± 2
6 ± 1
3d
55 ± 6
55 ± 5
145 ± 15
15 ± 2
40 ± 5
-
3e
4d
>1000
21 ± 1
20 ± 2
18 ± 3
-
28 ± 3
23 ± 2
22 ± 3
-
4e
25 ± 5
>1000
5d
5e
13 ± 2
13 ± 4
2 ± 1
7d
385 ± 82
94 ± 14
16 ± 2
128 ± 13
195 ± 35
41 ± 4
850 ± 300
70 ± 10
7e
Figure 4. Correlation between cell viability, IC , and hydrophobicity of tested compounds, log(τ).
5
0

Molecules 2021, 26, 3163
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Interestingly, contrary to the enzymatic assay, the activity of 7e remains marginal
IC50 ~100 µM), but such a low value should be assigned to a moderate hydrophobicity
(
of this compound. In this context, one can expect that esterification of the two hydroxyl
groups of 7e would improve its apparent activity in viability tests.
2.6. Molecular Modeling
Molecular modeling was initially performed to assess whether a given scaffold could
be harbored at the ATP binding site. Free energy of ligand binding (∆Gbind) was estimated
with the aid of the VINA-AutoDock algorithm implemented in Yasara Structure [47]. The
obtained ensembles of structures agreed qualitatively with our knowledge concerning
the binding of halogenated ligands at the ATP-binding site of CK2α [21,48,49]. Thus,
all chlorinated, brominated, and iodinated ligands preferably adopt the orientation, in
which both halogen atoms are solvent-protected, being close to the hinge region, while
the heterocyclic ring points towards the polar region of the ATP-binding site formed
by sidechains of Lys68, Glu81, and Asp175. The competition between hydrophobic and
electrostatic interactions is visible for the majority of tested ligands, again clearly identifying
the balance of hydrophobic and electrostatic interactions as the main driving force.
Interaction between a fluorinated ligand (1b, 2b, 3b, 7b) and the protein is found
generally overestimated (Figure 5, red points). Contrarily, the binding affinity of the
majority of iodinated and some brominated ligands is underestimated (Figure 5, blue and
green points). However, the latter effect may reflect the absence of the parameterization of
halogen bonding interactions in the classical force-field used in the scoring function [50].
On the other hand, these discrepancies can be treated as indicators for the existence of
halogen bonding in these complexes, which mainly applies to 3e
despite the discrepancy in the estimated binding affinity, most of the low-energy poses of
,6-diiodo-, 5,6-dibromo, and 5,6-dichloro- derivatives preserve halogen atoms pointing
, 4e, and 7e. Interestingly,
5
toward the hinge region, thus, in the orientation, enabling halogen-bonding, with the
heterocyclic ring close to Lys68 (see Supplementary Table S1 and Figures S40 and S41).
Figure 5. Correlation between the calculated Gibbs free energy of binding of synthesized compounds
to CK2 obtained from molecular modeling (∆Gbind) and the ∆T , estimated with the aid of nanoDSF.
m

Molecules 2021, 26, 3163
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3. Discussion
We have synthesized six groups of halogenated heterocycles, based on benzene-1,2,-
diamine derivatives. To analyze their applicability as the precursors of potential hCK2
inhibitors, we used molecular modeling together with the nanoDSF method, which enabled
qualitative comparison of their binding affinity. Many studies have been carried out on CK2
Type I inhibitors and some preferences of ligands in CK2 ATP-binding sites are already well
known [20
driving forces of binding ligand at the ATP- binding site of CK2
electrostatic contribution together with hydrophobic effect [20
ligands commonly bind deeper in the CK2
Additionally, the substitution with hydrophobic halogen atoms significantly increases the
affinity towards the CK2 by a favorable change in the physico-chemical properties of the
,33,35,48,51]. Besides the obvious steric hindrance of the scaffold, the two main
α
are clearly identified: the
35]. Negatively charged
,
33
,
α
pocket, preferably interacting with Lys68 [20].
α
ligand. The latter also includes the possible formation of halogen bonds with the hinge
region [48].
Using these different groups of compounds, we analyzed the interplay between the
scaffold and the effect of substitution with halogen atoms. For smaller scaffolds, like
benzotriazole/benzimidazole, unsubstituted on triazole/imidazole ring, a salt bridge or
hydrogen bond formation with Lys68 and halogen bonding with the hinge region are largely
mutually exclusive because these two regions are in the hCK2
interactions to occur simultaneously [20 48]. This is why we tested larger scaffolds with
either a larger heterocyclic ring (6a 7a ) or substitution in the imidazole, both of them to
α, too far apart for these
,
–
e
,
–e
preserve possible interactions with a hinge. We analyzed solely dihalogenated compounds
of the same pattern of halogen atoms at the benzene ring to assure that predominating
interactions with the hinge region (occurring with more halogen atoms) and configuration
effect (depending on the halogenation pattern) will not predominate or interfere with
the interdependence we were analyzing [24,34,43]. Interestingly, for the tested halogen
configuration, we can notice that only ligands with uneven charge distribution on the
non-benzene ring (groups 3, 4, 5, 7) exhibit measurable affinity towards CK2, while ligands
even much hydrophobic but without proton-donating groups (series 2 and 6) do not
◦
stabilize the complex (i.e.,
∆
T
m
< 1 C). This observation indicates that two halogen atoms
are insufficient to stabilize the ligand in the complex with a position close to the linker
region, and the formation of hydrogen bonds with the lysine seems to be necessary. Such
dependence of ligand position on the number of halogen atoms was observed for the
halogenated benzotriazoles, where 5,6 dibromobenzotriazole interacts with Lys68 (pdb
6
TLP), while tetrabromobenzotriazole interacts either with the hinge region or with Lys68
(
pdb 6TLL). What is more, an inspection of in silico determined structures for complexes
◦
with ligands that bind to the protein (
∆
T
m
< 1 C) points out to this unique interplay, as
ligands with no halogen atom or with fluorine (which is not capable for the formation of
halogen bond [52]) display in almost all cases different orientation relative to the hinge
region (see Figures S40 and S41), possibly forming hydrogen bonds. The substitution with
other halogen atoms switches the preferable orientation of the ligand, which has an effect
that is somehow explicable by hydrophobic interactions. However, since the force-field
used in the docking procedure does not include the explicit potential for a halogen bond,
these interactions are underestimated.
These different orientations of chlorinated, brominated, and iodinated compounds
might explain why the relation of measured log(τ) and ∆T values for series represent-
m
ing the same compound scaffold is linear solely for these three substituents, but not for
hydrogen and fluorine (see Figure 3).
Six out of eight compounds with the highest shift in nanoDSF assay displayed at least
moderate inhibitory activity in the enzymatic assay; among them, 3e and 7e were the most
active in both experiments. Some of tested scaffolds have already been reported in the liter-
ature. Thus, 1H-benzo[d]imidazol-2(3H)-one derivatives (5a
–
d
) moderately interact with
◦
CK2 (
∆
T
m
values are up to 2.4 C for iodinated derivative with and IC50 = 13
±
2 µM, while
4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2(3H)-one inhibits CK2 stronger (IC = 0.39 µM
50

Molecules 2021, 26, 3163
10 of 16
and K = 0.20
µM) [15]. 2-Trifluoromethyl-1H-benzimidazole derivatives have been also
i
reported: compound 3d with IC50 value against CK2 of 28
surement by us, 15 M) and 4,5,6-tribrominated and tetrabrominated analogs with the
inhibitory activity of 1.2 M and 0.6 M, respectively [53]. Replacement of bromine atoms
µM (being in line with mea-
±
3 µ
µ
µ
with iodine additionally improves the activity up to 0.12 µM [54].
It is worth noting that for these two 5,6-halogenated scaffolds, the observed tendency
is opposite to that observed for their tetrahalogenated analogs. Thus, 2-trifluoromethyl-
1H-benzimidazole interacts stronger than the 1H-benzo[d]imidazol-2(3H)-one counterpart
(
7 vs. 13 M). This again points out the subtle interplay between the scaffold and the
µ
substitution with halogen atoms, indicating that the interaction of 2-trifluoromethyl-1H-
benzimidazole with Lys68 is slightly stronger than for its isostructural analog-carrying
carbonyl group.
In this context, quinoxaline-2,3-diol derivatives (7d,e) might lead to novel potent
CK2 inhibitors, as both inhibit hCK2
derivatives (3d,e), stabilizing the complex for approx. 4 C (IC50 = 13 and 2 vs. 15 and
α
comparable to 2-trifluoromethyl-1H-benzimidazole
◦
7
µM, respectively), but have not been reported as kinase CK2 inhibitors yet.
Finally, the viability tests performed for reference cell lines showed moderate activity
for some compounds. Interestingly, IC50 values determined in this test are correlated with
a compound’s hydrophobicity rather than the inhibitory activity in the CK2 assay. This
is
α
indicates a dominant role of membrane permeability, but it may also indicate that CK2
not a major target for studied test compounds.
α
4. Materials and Methods
4.1. Chemistry
All starting materials and solvents for reactions were purchased from Sigma Aldrich
now Merck KGaA, Darmstadt, Germany), Fluorochem (Hadfield, UK), ABCR (Karlsruhe,
(
´
1
Germany), or Chempur (Piekary Sla˛skie, Poland). H-NMR spectra were recorded with
Varian 500 MHz spectrometer. TMS or the residual solvent signal were used as the internal
standard. High-resolution mass spectrometry spectra were recorded on a TQ OrbitrapVelos
instrument (Thermo Scientific, Waltham, MA, USA). The reaction progress was monitored
by the thin-layer chromatography analysis using silica gel plates (Kieselgel 60F254. Merck,
Darmstadt, Germany). Column chromatography was performed on Silica Gel 60 (0.040–
0.063 mm. Merck, Darmstadt, Germany).
Detailed experimental data, as well as spectral data for all synthesized compounds,
are available in the Supplementary Materials.
Synthesis of 2,1,3-benzothiadiazole derivatives (2a–e)
Benzene-1,2-diamine derivative (5 mmol) was dissolved in dry DCM (15 mL). Triethylamine
(
3 mL) was added, then thionyl chloride (15 mmol, 1.1 mL) dissolved in 3 mL of dry DCM
◦
was added dropwise at 0 C. The reaction mixture was allowed to reach room temperature,
then it was heated at 40 C overnight. Afterward, the reaction mixture was filtered. The
◦
filtrate was evaporated, the crude product was purified by column chromatography on
silica gel using DCM as eluent.
1
2
,1,3-benzothiadiazole (2a) Yield = 66%. H-NMR (400 MHz, d-DMSO)
δ
7.62–7.72 (2H,
13
m, ArH), 7.99–8.80 (2H, m, ArH); C-NMR (100 MHz, d-DMSO)
δ 121.24, 129.83, 154.19;
+
HRMS calcd. for C H N S [M + H] : 137.01680, found: 137.01673.
6
5
2
5
8
,6-difluoro-2,1,3-benzothiadiazole (2b) Yield = 66%. 1H-NMR (500 MHz, d-DMSO)
δ
.18–8.27 (2H, m, ArH); 13C-NMR (125 MHz, d-DMSO)
δ
106.37, 106.54, 150.46, 1651.77,
+
151.94, 153.81, 153.97; HRMS calcd. for C H F N S [M + H] : 172.99795, found: 172.99803.
6 3 2 2
1
5
(
,6-dichloro-2,1,3-benzothiadiazole (2c) Yield = 75%. H-NMR (500 MHz, d-DMSO)
121.75, 133.54, 152.61; HRMS calcd. for
C H Cl N S [M + H] : 204.93885, found: 204.93872.
δ 8.53
2H, s, ArH); 13C-NMR (125 MHz, d-DMSO)
δ
+
6
3
2
2

Molecules 2021, 26, 3163
11 of 16
1
5
(
,6-dibromo-2,1,3-benzothiadiazole (2d) Yield = 78%. H-NMR (500 MHz, d-DMSO)
δ
δ
8.67
8.79
2H, s, ArH); 13C-NMR (125 MHz, d-DMSO)
δ
124.95, 126.26, 153.32; HRMS calcd. for
+
C H Br N S [M + H] : 292.83782, found: 292.83741.
6
3
2
2
1
5
(
,6-diiodo-2,1,3-benzothiadiazole (2e) Yield = 47%. H-NMR (500 MHz, d-DMSO)
2H, s, ArH); 13C-NMR (125 MHz, d-DMSO)
δ
111.39, 130.38, 154.22; HRMS calcd. for
+
C H I N S [M + H] : 388.81008, found: 388.30973.
6
3 2
2
Synthesis of 2-trifluoromethylo-1H-benzimidazole derivatives (3a–e)
Benzene-1,2-diamine derivative (2 mmol) was dissolved in trifluoroacetic acid (2 mL) and
a catalytic amount of concentrated HCl was added. The reaction mixture was heated at
reflux overnight. The reaction was quenched by the addition of 50 mL of concentrated
NaHCO solution; afterwards, it was extracted by ethyl acetate (3
×
30 mL). Combined
3
organic layers were dried by MgSO , then the solvent was evaporated. The crude product
4
was purified by column chromatography on silica gel using 85:15 (hexane:AcOEt) as eluent
(
compounds 3d and 3e) or by recrystallization from hexane (compounds 3a, 3b, and 3c).
1
2
7
1
1
-trifluoromethylo-1H-benzimidazole (3a) Yield = 73%. H-NMR (500 MHz, d-DMSO)
δ
1
3
.34–7.42(2H, m, ArH), 7.68-7.77(2H, m, ArH); C-NMR (125 MHz, d-DMSO)
δ
115.88,
+
18.02, 120.17, 122.32, 124.09, 139.90, 140.22; HRMS calcd. for C H F N [M + H] :
8
6
3
2
87.04776, found: 187.04773.
1
5
,6-difluoro-2-trifluoromethylo-1H-benzimidazole (3b) Yield = 67%. H-NMR (500 MHz,
+
d-DMSO)
δ
7.71–7.85 (2H, m, ArH); HRMS calcd. for C H F N [M + H] : 223.02892,
8
4
5
2
found: 223.02882.
1
5
,6-dichlorlo-2-trifluoromethylo-1H-benzimidazole (3c) Yield = 85%. H-NMR (500 MHz,
+
d-DMSO)
δ
8.00 (2H, s, ArH); HRMS calcd. for C H F Cl N [M + H] : 254.96981,
8
4
3
2
2
found: 254.96976.
1
5
,6-dibromo-2-trifluoromethylo-1H-benzimidazole (3d) Yield = 40%. H-NMR (500 MHz,
13
d-DMSO)
δ
8.16 (2H, s, ArH); C-NMR (125 MHz, d-DMSO)
δ
115.88; HRMS calcd. for
+
C H F Br N [M + H] : 342.86878, found: 342.86896.
8
4
3
2
2
1
5
,6-diiodo-2-trifluoromethylo-1H-benzimidazole (3e) Yield = 33%. H-NMR (500 MHz,
+
d-DMSO)
found: 438.84123.
Synthesis of 2-hydroxymethylo-1H-benzimidazole derivatives (4a,c–e)
δ
8.32 (2H, m, ArH); HRMS calcd. for C H F I N [M + H] : 438.84105,
8
4
3 2
2
Benzene-1,2-diamine derivative (5 mmol) and hydroxyacetic acid (15 mmol, 1.14 g) were
dissolved in water (1.5 mL) and concentrated HCl (0.5 mL) was added. The reaction
mixture was heated at reflux overnight. The reaction was cooled to RT and then 20% NaOH
solution was added until pH = 13. The formed participate was filtered and washed several
times with water. Compound 4e was additionally purified by recrystallization from MeOH.
1
2
-hydroxymethylo-1H-benzimidazole (4a) Yield = 85%. H-NMR (500 MHz, d-DMSO) δ
4
.70 (2H, s, CH ), 5.69 (1H, s br, OH), 7.11-7.15 (2H, m, ArH), 7.49 (2H, s br, ArH), 12.30
2
1
3
(1H, s br, NH); C-NMR (125 MHz, d-DMSO)
δ
57.74, 121.25, 155.02; HRMS calcd. for
+
C H N O [M + H] : 149.07094, found: 149.07089.
8
9
2
1
5
,6-dichloro-2-hydroxymethylo-1H-benzimidazole (4c) Yield = 84%. H-NMR (500 MHz,
d-DMSO)
δ 4.70 (2H, s, CH ), 5.81 (1H, s br, OH), 7.73 (2H, s br, ArH), 12.04 (1H, s br,
2
NH); 13C-NMR (125 MHz, d-DMSO)
δ
57.57, 123.71, 158.10; HRMS calcd. for C H Cl N O
8
7
2
2
+
[
M + H] : 216.99299, found: 216.99308.
1
5
,6-dibromo-2-hydroxymethylo-1H-benzimidazole (4d) Yield = 81%. H-NMR (500 MHz,
13
d-DMSO)
δ
4.70 (2H, s, CH ), 5.83 (1H, s br, OH), 7.88 (2H, s br, ArH); C-NMR (125 MHz, d-
2
+
DMSO)
found: 304.89222.
,6-diiodo-2-hydroxymethylo-1H-benzimidazole (4e) Yield = 66%. H-NMR (500 MHz,
d-DMSO) 4.68 (2H, s, CH ), 5.80 (1H, s br, OH), 8.07 (2H, s br, ArH), 12.52 (1H, s br,
δ
57.50, 115.47, 119.18, 157.91; HRMS calcd. for C H Br N O [M + H] : 304.89196,
8
7
2
2
1
5
δ
2

Molecules 2021, 26, 3163
12 of 16
NH); 13C-NMR (125 MHz, d-DMSO)
δ
57.48, 98.13, 157.17; HRMS calcd. for C H I N O
8
7 2
2
+
[
M + H] : 400.86423, found: 400.86446.
Synthesis of 1H-benzo[d]imidazol-2(3H)-one derivatives (5a,c–e)
0
Benzene-1,2-diamine derivative (3 mmol) and N,N -carbonyldiimidazole (CDI) (3 mmol,
4
86 mg) were mixed in DMF (4 mL) overnight at RT. The reaction was quenched by the
addition of water (20 mL). The formed participate was filtered and washed several times
with water. Compound 5d was additionally purified by recrystallization from ethyl acetate,
then MeOH.
1
1H-benzo[d]imidazol-2(3H)-one (5a) Yield = 45%. H-NMR (500 MHz, d-DMSO)
δ
6.91
13
(4H, s br, ArH), 10.56 (2H, s br, NH); C-NMR (125 MHz, d-DMSO)
δ
108.46, 120.40, 129.65,
+
155.25; HRMS calcd. for C H N O [M + H] : 135.05529, found: 135.05526.
7
7
2
5
,6-dichloro-1H-benzo[d]imidazol-2(3H)-one (5c) Yield = 67%. 1H-NMR (500 MHz, d-
13
DMSO)
δ
δ
7.08 (2H, s br, ArH), 10.91 (2H, s br, NH); C-NMR (100 MHz, d-DMSO)
+
109.73, 122.35, 129.83, 155.18; HRMS calcd. for C H Cl N O [M + H] : 202.97734,
7
5
2
2
found: 202.97743.
1
5
,6-dibromo-1H-benzo[d]imidazol-2(3H)-one (5d) Yield = 67%. H-NMR (500 MHz, d-
1
3
DMSO)
δ
7.20 (2H, s br, ArH), 10.90 (2H, s br, NH); C-NMR (100 MHz, d-DMSO)
+
δ
112.66, 113.88, 130.63, 154.93; HRMS calcd. for C H Br N O [M + H] : 290.87631,
7
5
2
2
found: 290.87626.
1
5
δ
,6-diiodo-1H-benzo[d]imidazol-2(3H)-one (5e) Yield = 67%. H-NMR (500 MHz, d-DMSO)
7.41 (2H, s br, ArH), 10.80 (2H, s br, NH); 13C-NMR (100 MHz, d-DMSO)
δ
96.40, 118.00,
+
131.46, 154.58; HRMS calcd. for C H I N O [M + H] : 386.84858, found: 386.84861.
7
5 2
2
Synthesis of quinoxaline derivatives (6a,c–e)
Benzene-1,2-diamine derivative (3 mmol) and 40% glyoxal solution in water (9 mmol,
.3 mL) were heated at reflux in EtOH (6 mL) overnight. The reaction was quenched by the
addition of water (50 mL). The formed participate was filtered and washed several times
with water (compound 6a was extracted with AcOEt (3 30 mL). Compounds 6c and 6d
1
×
were purified by recrystallization from MeOH. Compounds 6a and 6e were purified by
column chromatography on silica gel, using 8:2 (hexane:AcOEt) as eluent.
1
quinoxaline (6a) Yield = 70%. H-NMR (500 MHz, d-DMSO)
δ 7.84–7.92 (2H, m, ArH),
13
8
.08–8.16 (2H, m, ArH), 8.96 (2H, s br, ArH); C-NMR (125 MHz, d-DMSO)
42.23, 145.72; HRMS calcd. for C H N [M + H] : 131.06037, found: 131.06042.
δ
129.14, 130.21,
+
1
8
7
2
1
6
9
,7-dichloroquinoxaline (6c) Yield = 99%. H-NMR (500 MHz, d-DMSO)
δ
8.46 (2H, s, ArH),
.02 (2H, s, ArH); 13C-NMR (125 MHz, d-DMSO)
calcd. for C H Cl N [M + H] : 198.98234, found: 198.98252.
δ 130.13, 133.01, 141.15, 147.13; HRMS
+
8
5
2
2
1
6
,7-dibromoquinoxaline (6d) Yield = 89%. H-NMR (500 MHz, d-DMSO)
δ
8.56 (2H, s,
13
ArH), 9.01 (2H, s, ArH); C-NMR (125 MHz, d-DMSO)
HRMS calcd. for C H Br N [M + H] : 286.88140, found: 286.88165.
δ 125.77, 133.31, 141.56, 147.14;
+
8
5
2
2
1
6
8
,7-diiodoquinoxaline (6e) Yield = 25%. H-NMR (500 MHz, d-DMSO)
δ
8.68 (2H, s, ArH),
.96 (2H, s, ArH); 13C-NMR (125 MHz, d-DMSO)
calcd. for C H I N [M + H] : 382.85366, found: 382.85426.
δ 111.06, 138.74, 141.92, 1467.83; HRMS
+
8
5 2
2
Synthesis of quinoxaline-2,3-diol derivatives (7a–e)
Benzene-1,2-diamine derivative (3 mmol) and oxalic acid (9 mmol, 810 mg) were heated
◦
at 120 C in DMF (1 mL) overnight. The reaction was quenched by the addition of wa-
ter (20 mL). The formed participate was filtered and washed several times with water
(
compound 6a was extracted with AcOEt (3
recrystallization from EtOH.
quinoxaline-1,3-diol (7a) Yield = 57%. H-NMR (500 MHz, d-DMSO)
×
10 mL)). Crude products were purified by
1
δ
7.02–7.10 (2H, m,
13
ArH), 7.10–7.16 (2H, m, ArH), 11.90 (2H, s, 2xOH); C-NMR (125 MHz, d-DMSO)
1
δ 115.12,
+
23.00, 125.60, 155.17; HRMS calcd. for C H N O [M + H] : 163.05020, found: 163.05011.
8
7
2
2

Molecules 2021, 26, 3163
13 of 16
1
6
7
,7-difluoroquinoxaline-1,3-diol (7b) Yield = 68%. H-NMR (500 MHz, d-DMSO)
δ
7.00–
13
.11 (2H, m, ArH), 11.92 (2H, s2xOH); C-NMR (125 MHz, d-DMSO)
δ
103.63, 103.74,
1
03.81, 122.26, 122.32, 143.91, 144.04, 145.83, 145.96, 154.70; HRMS calcd. for C H F N O
8 5 2 2 2
+
[
M + H] : 199.03136, found: 199.03138.6,7-dichloroquinoxaline-1,3-diol
H-NMR (500 MHz, d-DMSO)
(
7c) Yield = 86%.
1
13
δ
7.21 (2H, s, ArH), 12.00 (2H, s, 2xOH); C-NMR (100 MHz,
115.99, 124.29, 126.03, 154.71; HRMS calcd. for C H Cl N O [M + H] :
+
d-DMSO)
δ
8
5
2
2
2
2
30.97226, found: 230.97218.
,7-dibromooquinoxaline-1,3-diol (7d) Yield = 91%. H-NMR (500 MHz, d-DMSO)
116.15, 118.96, 126.64,
54.73; HRMS calcd. for C H Br N O [M + H] : 318.87123, found: 318.87105.
1
6
(
δ
7.38
2H, s, ArH), 11.99 (2H, s, 2xOH); 13C-NMR (100 MHz, d-DMSO)
δ
+
1
8
5
2
2
2
1
6
(
1
,7-diiodooquinoxaline-1,3-diol (7e) Yield = 63%. H-NMR (400 MHz, d-DMSO)
δ
7.51
13
2H, s, ArH), 11.77 (2H, s br, 2xOH); C-NMR (100 MHz, d-DMSO)
55.49; HRMS calcd. for C H I N O [M + H] : 414.84349, found: 414.84366.
δ
99.11, 124.80, 128.06,
+
8
5 2
2
2
4.2. NanoDSF
hCK2 was expressed and purified according to the method described previously [35].
α
Thermal stability of 1:10 protein–ligand complexes was monitored on Prometheus (Nanotem-
per Technologies, München, Germany) using PR-C002 Prometheus NT.48 Standard Capillaries
◦
◦
using temperature ramp of 20 to 80 C with 1 C/min heating rate. Temperature-induced
variation in ratio fluorescence intensity at 330 and 350nm was globally analyzed using the
procedure described before [37]. All experiments were performed in 25 mM Tris–HCl (pH 8,
0.5 M NaCl) with 2% (v/v) DMSO. Protein was kept in constant 2.5 µM concentration.
4.3. Inhibitory Activity against Human CK2α
The inhibitory effect of 8 selected compounds was monitored based on the luminescence
measured with the SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San
Jose, CA, USA), using the previously described method with the aid of ADP-Glo kinase assay
(Promega, Walldorf, Germany) [43]. Inhibitor concentration varied in the range of 1 nM–1 mM
(see Supplementary Figure S38). Each experiment was repeated in triplicate and IC50 values
were further estimated globally using the sigmoidal dose-response equation implemented in
Origin 9.4 package (www.originlab.com, upgraded 20.07.27).
4.4. Cell Viability Assay
The following human cell lines were used in this study: (i) epidermoid carcinoma
®
®
A-431 (ATCC CRL-1555), (ii) colorectal carcinoma HCT116 (ATCC CCL-247), (iii) col-
−
/−
®
orectal carcinoma HCT116p53
, and (iv) normal fibroblasts BJ (ATCC CRL-2522). The
cell line was a kind gift from professor Bert Vogelstein (John Hopkin’s
University, Baltimore, MD, USA). The remaining cell lines were purchased from ATCC
−
/−
HCT116p53
(
(
Manassas, VA, USA) or Sigma Aldrich by Merck (Darmstadt, Germany).
Cells were cultured in Dulbecco’s modified Eagle’s or Roswell Park Memorial Institute
RPMI)-1640 medium supplemented with 10% or 15% fetal bovine serum (Gibco by Thermo
◦
Fisher Scientific, Waltham, MA, USA) under standard culture conditions (37 C, 5% CO )
2
in a humidified incubator with atmospheric oxygen.
The viability of cells was assayed by measuring the conversion of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the formazan (the rate of this
reaction is proportional to the number of surviving cells). Cells were seeded in a 96-well
plate at a density of 1500–2500 cells per well, 24 h before treatment. Treatment with tested
compounds and cisplatin (0.25–160
µM) was performed for 72 h. MTT stock solution
(
3
Sigma-Aldrich) was added at the final concentration 0.5 mg/mL. After 2 h of incubation at
◦
7 C, water-insoluble formazan was dissolved in a lysis buffer containing 20% SDS, 50%
DMF, 2.5% hydrochloric acid, and 2.5% acetic acid. Optical densities were measured at
70 nm using a scanning multi-well spectrophotometer (PARADIGM Detection Platform;
Beckman Coulter, Brea, CA, USA).
5

Molecules 2021, 26, 3163
14 of 16
4.5. Chromatographic Hydrophobicity Index
Chromatographic hydrophobicity index (log(
τ
)) was determined with the reverse
phase HPLC (RP-HPLC). Column: Nova-pak® C18 4
µm, 4.6
×
250 mm. Eluent: 2:1
methanol: 20 mM aqueous ammonium formate (pH = 8) (v/v). Flow: 0.7 mL/min. Detector
wavelength: 280 nm. The individual retention times (Tret) were converted to a hydrophobic-
ity scale according to the formula
τ
= (Tret − T )/T . where the retention time of unretained
0
0
solvent (T ) was estimated to 300 s [24].
0
4.6. Molecular Modeling
Docking of all tested ligands at the binding site of the catalytic subunit of human
protein kinase CK2 was performed with VINA algorithm implemented in Yasara Structure
package (www.yasara.com, version 20.12.24) using amber14 force-field with 8 Å cutoff
for long-range electrostatic interactions. We used 8 just-resolved structures of complexes
of hCK2
TLV, 6TLU, 6TLS, 6TLR, 6TLP, 6TLO, and 6TLL) as the reference protein structures.
This, including alternative protein conformations, stated 18 structures to be tested as the
α with variously brominated benzotriazoles (Protein Data Bank records 6TLW,
6
‘
receptor’. Such an extended set of template protein structures was chosen to allow the
sampling of a larger conformational space that allows multiple ligand orientations at the
binding site (see Supplementary Figure S39). In silico screening procedure was restricted to
the cuboid region that contained all residues from the ATP binding site, further additionally
extended by 2 Å in each direction. During simulations, the sidechain atoms of protein
residues proximal to the location of the original ligand (4 Å threshold) were flexible, while
coordinates of all other protein atoms were kept fixed. Finally, each of 35 ligands was
subjected to 25 independent cycles of the docking procedure using 18 structural templates.
The resulting structures were then clustered, and for each ligand, the highest scored one
was used as the representative. This score was used as a rough estimate of the free energy
of ligand binding.
Supplementary Materials: The following are available online. Figures S1–S29: NMR spectra of
the synthesized compounds, Figures S30–S37: cell viability data, Figure S38, kinase inhibition data,
Figures S39–S41 and Table S1, molecular modeling data.
Author Contributions: Conceptualization: D.P. and J.P.; methodology: D.P., M.W.-S., and J.P.; soft-
ware: J.P.; resources: J.P. and A.M.M.; compound synthesis: D.P.; DSF: D.P. and M.W.-S.; HPLC:
A.M.M.; molecular modeling: J.P.; inhibitory activity: M.W.-S.; toxicity to cell cultures: E.S.; writing—
original draft preparation: D.P. and M.W.-S.; writing—review and editing: J.P., D.P., M.W.-S., A.M.M.,
and E.S.; supervision: J.P.; project administration: J.P.; funding acquisition: J.P. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by NCN grant 2017/25/B/ST4/01613. The equipment used was
sponsored in part by the Centre for Preclinical Research and Technology (CePT).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: 2a–e, 3a–e, 4a,c–e, 5a,c–e, 6a,c–e, 7a–e are available from the authors.
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