Design, synthesis and biological evaluation of 2-aminopyrimidinones and their 6-aza-analogs as a new class of CK2 inhibitors

Article abstract of DOI:10.3109/14756366.2013.837898

In order to find the new potent CK2 inhibitors the 60 derivatives of 2-aminopyrimidinone and their 6-aza-substituted analogs were synthesized and tested in vitro. Among them, the most efficient inhibitor 2-hydroxy-5-[4-(4-methoxyphehyl)-6-oxo-1,6-dihydropyrimidin-2-ylamino] benzoic acid was identified (IC50=1.1μM). The structure - activity relationship study of newly synthesized derivatives was carried out and their binding mode with adenosine triphosphate-acceptor site of CK2 was proposed.

Full text of DOI:10.3109/14756366.2013.837898

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.837898
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ORIGINAL ARTICLE
Design, synthesis and biological evaluation of 2-aminopyrimidinones
and their 6-aza-analogs as a new class of CK2 inhibitors
Maksym O. Chekanov1,2,3, Olga V. Ostrynska1,2, Sergii S. Tarnavskyi1,2, Anatoliy R. Synyugin1,2, Nadiia V.
Briukhovetska1, Volodymyr G. Bdzhola1,2, Alexander E. Pashenko3, Andrey A. Fokin3, and Sergiy M. Yarmoluk1,2
2
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine, Otava Ltd., 65 Ellerslie Ave., Suite 560, Toronto, ON, Canada, and
3Department of Organic Chemistry, National Technical University of Ukraine ‘‘Kiev Polytechnic Institute’’, Kiev, Ukraine
Abstract
Keywords
In order to find the new potent CK2 inhibitors the 60 derivatives of 2-aminopyrimidinone and
their 6-aza-substituted analogs were synthesized and tested in vitro. Among them, the most
efficient inhibitor 2-hydroxy-5-[4-(4-methoxyphehyl)-6-oxo-1,6-dihydropyrimidin-2-ylamino]
benzoic acid was identified (IC50 ¼ 1.1 mM). The structure–activity relationship study of newly
synthesized derivatives was carried out and their binding mode with adenosine triphosphate-
acceptor site of CK2 was proposed.
Aza-pyrimidinones, CK2 inhibitors,
combinatorial synthesis, chemical
optimization, molecular docking,
pyrimidinones, receptor-based virtual
screening
History
Received 20 May 2013
Revised 21 June 2013
Accepted 20 August 2013
Published online 1 October 2013
Introduction
Experimental
Protein kinase CK2 (CK2) is a constitutively active pleiotropic General
kinase. It controls the key cellular activities and is involved in
Starting materials and solvents were purchased from commercial
suppliers and were used without further purification. 1HNMR
spectrawere recorded on a Varian VXR 400 instrument at 400 MHz
(Agilent Technologies, Santa Clara, CA). 13C NMR spectra were
recorded on a Varian VXR 400 instrument at 100 MHz. Chemical
shifts are described as parts per million (ꢀ) downfield from an
internal standard of tetramethylsilane, and spin multiplicities are
given as s (singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet) or m (multiplet). High-performance liquid chromatogra-
phy-mass spectra (HPLC-MS) analysis was performed using the
Agilent 1100 LC/MSD SL (Agilent Technologies) separations
module and Mass Quad G1956B mass detector with electrospray
ionization (Agilent Technologies) (þve or ꢀve ion mode as
indicated) and with HPLC performed using Zorbax SB-C18
(Agilent Technologies), Rapid Resolution HT Cartridge
4.6 ꢁ 30 mm 1.8-m (Agilent Technologies P/N:823975-902) i.d.
column, at a temperature of 40ꢂC with gradient elution of 0–100%
CH3CN (with 1 mL/L HCOOH): H2O (with 1 mL/L HCOOH) at a
flow rate of 3 mL/min and a run time of 2.8 min. Compounds were
detected at 215 nm using a Diode Array G1315B detector. All
tested compounds had ꢃ90% purity as determined by this method.
All purified synthetic intermediates had ꢃ95% purity as deter-
mined by this method.
majority of signal transduction pathways. CK2 phosphorylates
many substrates and participates in the complex series of cellular
functions such as progression of the cell cycle, apoptosis, cell
differentiation and transcription processes1. CK2 overexpression
and hyper-activation are observed in a large number of tumors2,
including breast cancer3, lung4, prostate5, colon6, kidney7 and
hematological malignancies8. Recently, it was reported that the
decrease in CK2 regulation plays a critical role in inflammatory9
and infectious diseases10. Altogether, these facts raised the
importance of finding pharmacologically useful inhibitors of
this kinase. As a result, a large number of publications are devoted
recently for the development of new CK2 inhibitors11.
Accordingly to our previous report, the derivatives of fused
pyrimidinone (Figure 1) are potent inhibitors of CK212.
The aim of this study was the de novo design of protein
kinase CK2 inhibitors based on 2-aminopyrimidinone and its
6-aza-analogues employing combinatorial chemistry techniques
and molecular modeling methods. Considering the fact that most
inhibitors of protein kinase CK2 published up to date12–16 have
in their structure carboxyl and/or phenol groups, we focused our
study only on derivatives containing these substituents (Figure 2).
Address for correspondence: Sergiy M. Yarmoluk, Department of
Medicinal Chemistry, Institute of Molecular Biology and Genetics,
NAS of Ukraine, 150 Zabolotnogo Str., 03680 Kyiv, Ukraine. Tel: +38
Virtual screening
For virtual screening of a combinatorial library we used DOCK
044 526 1129. Fax: +38 044 526 0759. E-mail: yarmoluksm@gmail.com 4.0 package (Irwin ‘‘Tack’’ Kuntz’s Group, San Francisco,

2
M. O. Chekanov et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
incorporated radioactivity in the presence of inhibitor to the
radioactivity incorporated in control reactions, i.e. in the absence
of inhibitor. Serial dilutions of inhibitor stock solution were used
to determine its IC50 concentration. The IC50 values represent
means of triplicate experiments with standard error of the mean
never exceeding 15%.
Chemistry
Accordingly to the synthetic procedures27, the series of building-
blocks 2a–2u were synthesized. The structures of synthesized
compounds are listed in Table 1. Their spectral properties are
available online in Supplemental Information section.
Figure 1. Potent CK2 inhibitor representing fused pyrimidinone family.
General procedure for the combinatorial synthesis
of compounds 3–62
The solution of corresponding building-blocks 2a–2u (2.0 mmol)
and corresponding aminophenol/amino acid (4.0 mmol) in
pyridine was being heated for 8 h at 100ꢂC. After that the
reaction mixture was quenched by isopropyl alcohol (4 mL) and
cooled down to room temperature. The precipitate was filtered,
washed with isopropyl alcohol (2.0 mL) and air dried.
The structures of synthesized compounds are summarized in
Tables 2–4. The spectra of the four selected compounds
are presented below. The spectra of other compounds of the
combinatorial library are available online in Supplemental
Information section.
Figure 2. 2-Aminopyrimidinone scaffold.
4-Methyl-3-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino]-
benzoic acid (4)
CA)17–20. Calculations of ligand geometry were performed using
universal molecular mechanic force field (YFF) described previ-
ously21,22. Partial atomic charges of the ligands were calculated
employing the Kirchhoff method23. Minimization of CK2 crystal
structure (protein data bank (PDB) ID: 2ZJW) was performed
with GROMACS (Biophysical Chemistry Department of
University of Groningen, Netherlands)24 package (steepest des-
cent algorithm, GROMACS force field). Partial atomic charges of
the receptor were calculated using assisted model building with
energy refinement (http://en.wikipedia.org/wiki/AMBER) force
field. Flexible docking and scoring were performed as reported
elsewhere25.
Yield 58% (light gray solid). M.p. 227ꢂC. LC-MS m/z 259
[M þ Hþ], Rt ¼ 0.96 min. 1H NMR (DMSO-d6) ꢀ 2.06 (s, 3H,
CH3), 2.30 (s, 3H, CH3), 5.51 (s, 1H, CH2), 7.22 (d, 1H), 7.60 (d,
1H), 8,32 (s, 1H).
3-[(4-Oxo-3,4-dihydroquinazolin-2-yl)amino]benzoic acid (7)
Yield 64% (light gray solid). M.p. 4300ꢂ C dec. LC-MS m/z 285
[M þ Hþ], Rt ¼ 0.85 min. 1H NMR (DMSO-d6) ꢀ 7.19 (t, 1H),
7.38–7.42 (m, 2H), 7.59–7.62 (m, 2H), 7.96 (dd, 1H), 8.08 (dd,
1H), 8.21 (s, 1H), 8.71(br. s., 1H, NH), 10.69 (br. s., 1H, NH),
12.54 (br. s., 1H, OH).
5-[(4-Butyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino]-2-
hydroxybenzoic acid (21)
Biological testing
Selected compounds were tested using in vitro kinase assay. Each
test was performed twice in a total reaction volume of 30 mL,
containing 6 mg of peptide substrate RRRDDDSDDD (New
England Biolabs Ltd., Herts, UK); 10 units of recombinant
human CK2 holoenzyme (New England Biolabs); 50 mM
adenosine triphosphate (ATP) and g-labeled 32P ATP, diluted to
specific activity 100 mCi/mM; CK2 buffer (20 mM Tris-HCl, pH
7.5; 50 mM KCl; 10 mM MgCl2) and inhibitor in varying
concentrations. Incubation time was 20 min at 30ꢂC. The reaction
was stopped by adding an equal volume of 10% o-phosphoric
acid and the reaction mixture was loaded onto 20-mm discs of
phosphocellulose paper (Whatman plc, Kent, UK). Disks were
washed three times with 1% o-phosphoric acid solution, air dried
at room temperature and the level of radioactive signal was
measured according to the Cherenkov method in a beta-counter
(LKB Instruments; http://www.lkb.com.au/). As negative control
Yield 51% (white solid). M.p. 4260ꢂC dec. LC-MS m/z 303
[M þ Hþ], Rt ¼ 0.76 min. 1H NMR (DMSO-d6) ꢀ 0.93 (t, 3H,
CH3), 1.30–1.40 (m, 2H, CH2), 1.59–1.66 (m, 2H, CH2), 2.34 (t,
2H, CH2), 5.51 (s, 1H), 6.82 (d, 1H), 7.65 (dd, 1H), 8.06 (d,1H),
11.01 (br. s., 1H, NH).
2-Hydroxy-5-{[4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-
2-yl]amino}benzoic acid (25)
Yield 56% (light gray solid). M.p. 4300ꢂC dec. LC-MS m/z 353
[M þ Hþ], Rt ¼ 0.76 min. 1H NMR (DMSO-d6) ꢀ 3.81 (s, 3H,
OCH3), 6.33 (s, 1H), 6.95–7.00 (m, 3H), 7.58 (dd, 1H), 8.04
(d, 2H), 8.60 (s, 1H), 8.82 (br. s., 1H, NH), 10.97 (br. s., 1H, NH).
Results and discussion
an equal volume of dimethyl sulfoxide (DMSO) was added to To identify the potent inhibitors of CK2 among the 2-
the reaction mixture. Quercetin, a known CK2 inhibitor26, was aminopyrimidinones and their 6-aza-analogues, the receptor-
used as inhibition positive control. The final concentration of based virtual screening of the virtual library (1207 compounds)
quercetin was 0.55 mM (which inhibited CK2 activity by 50%). was carried out. The molecular docking of ligands was performed
Percent inhibition was calculated as ratio of substrate- by DOCK 4.0 in ATP-binding site19,20,28 of CK2. Using the

DOI: 10.3109/14756366.2013.837898
Design, synshesis and biological evaluation of 2-aminopyrimidinones
3
Table 1. Chemical structures of building-blocks 2a–2u.
No
2a
No
2h
No
2o
Structure
Structure
Structure
O
O
O
HN
HN
HN
N
S
N
S
N
S
O
O
N
O
HN
N
HN
HN
2b
2c
2i
2j
2p
2q
N
S
N
S
N
S
O
O
O
HN
HN
HN
S
N
N
S
S
N
O
S
N
O
N
O
HN
HN
HN
2d
2e
2k
2l
2r
S
N
N
O
S
N
O
O
O
HN
HN
HN
2s
N
S
N
O
S
N
N
S
N
O
O
HN
HN
NH
N
2f
2m
2n
2t
S
N
O
S
N
S
S
O
O
HN
N
NH
N
HN
2g
2u
S
N
Cl
S
N
above-mentioned software, the 13 most promising compounds between the carboxyl oxygen of inhibitor and the side chain
which had the best estimated binding energies were selected for nitrogen of Lys68. It is worth to mention that this kind of
further synthesis and biochemical evaluation.
interaction is observed in a number of small-molecule CK2
The selected compounds were synthesized accordingly to the inhibitors29–33. Also, compound 7 forms additional hydrogen
combinatorial chemistry procedures (Scheme 1). To synthesize bond with Val116 in the hinge region of ATP-acceptor pocket.
the derivatives of 2-aminopyrimidinone and their 6-aza-analogs, As it was previously published the bonding with that region is
the series of building-blocks 2a–2u (Table 1) were synthesized prerequisite to inhibitory activity34. The H-bond is formed
accordingly to the previously reported procedures27.
between the oxygen of pyrimidinone heterocycle and the
The newly synthesized compounds 3–15 were tested in vitro. backbone NH of Val116. It was found that the same
Their chemical structures and results of the biochemical tests are interaction exists between inhibitor 4 and CK2.
shown in Table 2.
Thus, the analysis of obtained complexes showed that
The biochemical testing revealed that compounds 4 and 7 were compounds 4 and 7 are complementary to the CK2 ATP-binding
the most potent inhibitors of CK2 among 3–15 (IC50 is 28 and site. One of the key substituents that define the compounds
24 mM, respectively).
activity is a carboxyl group at the third position of the ring A. For
The analysis of a complex ‘‘Inhibitor 7-CK2’’ (Figure 3) instance, compound 4 with a carboxyl group in this position has
obtained by the molecular docking method shows that IC50 value 28 mM, while compound 3 with this substituent in the
stabilization of the ligand in ATP-binding site is achieved position 4 of the ring A is not active.
by the hydrophobic contacts with Leu45, Val53, Val66,
In order to increase the biological activity of the ligands, we
Val116, Ile174 and stacking interaction with Phe113. carried out chemical optimization of 2-aminopirymidinons and
Moreover, the hydrogen bond is present between ligand and their 6-aza-analogues according to the models obtained by
Lys68 located deeply in the ATP-binding pocket. It is formed molecular modeling methods. To perform the optimization of

4
M. O. Chekanov et al.
Table 2. Chemical structures and in vitro activities of compounds 3–15.
J Enzyme Inhib Med Chem, Early Online: 1–8
O
R
4
4
5
R
R
1
6
N
X
Y
B
Z
R
HO
A
3
1
N
2
2
3
No
R1
R2
R3
X
Y
Z
R4
COOH position
IC50 (mM)
3
4
5
6
7
H
H
H
Me
Me
n-Pr
–
–
–
–
–
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
H
6-Me
H
H
H
4
3
4
4
3
430
28
430
430
24
–(CH2)4–
X
Y
8
9
Me
Me
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
H
H
H
Me
Me
Me
C
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
H
H
H
H
6-Me
H
4
3
4
3
3
4
3
3
430
430
430
430
430
430
430
430
10
11
12
13
14
15
H
5-COOH
Table 3. Chemical structures and in vitro activities of compounds 16–34.
O
5
HO
O
R
R
4
3
1
6
N
X
Y
B
Z
R
A
2
HO
1
N
2
3
No
R1
R2
H
Me
Et
n-Pr
i-Pr
n-Bu
Me
R3
X
Y
Z
IC50 (mM)
16
17
18
19
20
21
22
23
24
H
H
H
H
H
–
–
–
–
–
–
–
–
–
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
430
20
430
430
430
9,5
17
12
430
H
Me
–(CH2)4–
X
Ph
N
Y
25
26
27
28
29
H
Bn
4-Me-Bn
4-Cl-Bn
4-MeO-Ph
Me
Me
–
–
–
–
–
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
1,1
430
430
430
19
Me
X
Y
30
31
32
33
34
H
Me
t-Bu
Ph
–
–
–
–
–
–
–
–
–
Me
C
C
C
C
N
N
N
N
N
N
N
N
N
N
C
430
430
430
430
430
–

DOI: 10.3109/14756366.2013.837898
Design, synshesis and biological evaluation of 2-aminopyrimidinones
5
Table 4. Chemical structures and in vitro activities of compounds 35–62.
O
R
4
4
5
R
R
1
6
N
X
Y
B
Z
R
HO
A
3
1
N
2
2
3
No
R1
R2
R3
X
Y
Z
R4
H
2-Me
H
OH position
IC50 (mM)
35
36
37
38
39
40
41
42
43
44
H
H
H
H
H
H
H
Me
Me
Me
n-Pr
n-Pr
n-Pr
i-Pr
–
–
–
–
–
–
–
–
–
–
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
4
4
3
4
4
3
4
4
4
4
23
430
430
20
430
430
430
430
15
H
2-Me
4-Me
2-Me
2-Me
2-Me
H
–(CH2)4–
H
Ph
27
X
Y
45
–
C
C
N
H
3
430
X
Y
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
H
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
H
H
H
H
H
H
Me
Me
Me
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
H
2-Me
H
2-Me
H
4-Me
H
H
H
H
H
4-Me
5-Me
5-Cl
H
2-Me
H
4
4
4
4
3
3
4
3
4
3
2
2
2
2
4
4
3
430
430
430
430
430
430
430
430
430
430
430
430
430
430
430
430
430
Me
Me
Me
Me
Ph
Ph
–
–
–
–
–
–
–
–
–
Scheme 1. Combinatorial synthesis of 2-aminopyrimidinones and their 6-aza-analogs.
above-mentioned derivatives the following approaches were (3) to synthesize ligands substituted with the nitrogen in a ring B
suggested:
at positions X, Y and Z (at the same time), X and Y (at the
same time) (see Figure 2). This was suggested in order to
evaluate the influence of number and position of nitrogen
atoms on inhibitors activity and
(1) to synthesize compounds with carboxyl residue at position
3 and hydroxyl residue at position 4 of the ring A (Figure
2). It was expected that two acceptor groups (carboxyl
and hydroxyl) in ortho-position will be favorable to (4) to synthesize the derivatives containing hydrophobic ali-
formation stronger H-bonds with Lys68 of CK2 ATP-
binding site;
phatic and aromatic substituents: R1 (methyl, butylene,
t-butyl, benzyl, phenyl, phenylene, etc.), R2 (methyl, ethyl,
n-propyl, iso-propyl, n-butyl, butylene, phenyl, phenylene,
etc.) and R3 (methyl) in the ring B (Figure 2). It was
important to investigate the interaction between substituents
and hydrophobic region II of CK2 ATP-acceptor pocket.
It should be noted that these substituents may promote the
(2) to synthesize derivatives with the acceptor groups at ortho-,
meta- or para-position of the ring A (Figure 2). The aim of
these modifications was to study influence of the acceptors
and their position on hydrogen bonding between inhibitors
and Lys68 of the ATP-binding site;

6
M. O. Chekanov et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
p-chlorobenzyl, respectively, were inactive. At the same time,
compounds 17, 22, 23 and 29 with smaller R1 substituents
(hydrogen, methyl, butylene and phenylene) have IC50 values 20,
17, 12 and 19 mM, respectively. Thus, the small groups such as
hydrogen or methyl are required at position X of the ring B.
Among aminophenol derivatives of all four active compounds
(35, 38, 43 and 44) have hydroxyl group at position 4 of the ring A
(Table 4). Noteworthy, insertion of OH group at position 3 of the
ring A clearly led to the complete loss of activity which is evident
in compound pairs 7 and 45, 35 and 37, 44 and 45 (IC50 are 24 mM
and 430, 23 mM and 430, 27 mM and 430 mM, respectively).
Exactly as in the case of aminosalicylic acid derivatives, the
inhibitory activity of aminophenol derivatives was influenced by
the structure of substituents R1 and R2 of the ring B and the same
trends were observed. For example, all active aminophenol
derivatives (35, 38, 43 and 44) have small substituent R1 at
position X of the ring B. Furthermore, the same as in the case
of aminosalicylic acid derivatives, increase in the size of R2 at
position Y of the ring B improved the activities (row 35, 38
and 43). The compound 43 with the phenyl group was more
active (IC50 ¼ 15 mM) than the compound 38 with the n-propyl
group (IC50 ¼ 20 mM) and compound 35 with the methyl group
(IC50 ¼ 23 mM).
Figure 3. Complex ‘‘compound 7-CK2’’ obtained by the molecular
docking method. Intermolecular H-bonds are shown as dotted lines.
The inhibitory properties of ligands were also affected by a
substituent R4 at position 2 of the ring A. Compounds 35 and 38
substituted by hydrogen at this position showed the corresponding
activities of 23 and 20 mM, whereas compounds 36 and 39
substituted by methyl groups were inactive.
In overall, the presence of carboxyl group in position 3 of the
ring A and a hydroxyl group at position 4 had a mixed influence
on inhibitory activities. In the one case, the presence of carboxyl
groups led to the disappearance of activity (compounds 19 Ta 38,
IC50 was 430 and 20 mM, respectively), in the other case
the inhibitory properties of the ligands were almost the same
Figure 4. Structure of the most active CK2 inhibitor obtained as a result
of chemical optimization.
formation of new hydrophobic contacts between the inhibitor (compounds 17 Ta 35, IC50 20 and 23 mM, respectively), in
and amino acid residues in the CK2-binding site (leading another instance it increased the activity (compounds 29 and 44,
to increased inhibitory activity) and, in the same time, IC50 was 19 and 27 mM, respectively).
create steric hindrances destabilizing the ligand in CK2 ATP-
acceptor pocket.
It is important to add that all aza-analogs of 2-aminopyr-
imidinone containing nitrogen at position X and/or Y were not
According to the chemical optimization plan, using building- active (ligands 10–15, 30–34 and 54–62). Accordingly to the
blocks 2a–2u (Table 1), the 47 new compounds 16–62 were calculations performed using Instant JChem35, it was found that
synthesized (Scheme 1) and assayed in vitro (Tables 3 and 4).
electronegative charge of oxygen atom in pyrimidinone hetero-
The results of in vitro testing show that 10 optimized cycle is decreased when additional atom of nitrogen is inserted
compounds inhibit the protein kinase CK2. 5-Aminosalicylic into the heterocycle (Figure 5). Thus, the following values were
acid derivatives (17, 21, 22, 23, 25 and 29) with the carboxyl obtained for 2-aminopyrimidin-4-ones, 2-amino-6-azapyrimidin-
group at position 3 and hydroxyl group at position 4 of the 4-ones and 4-amino-6-azapyrimidin-2-ones ꢀ0.51, ꢀ0.45 and
ring A have the activities in the range of 1.1–20 mM (Table 3). ꢀ0.47, respectively. The oxygen of pyrimidinone cycle is forming
Among aminophenol derivatives, the most active ligands were 35, important hydrogen bonds with amino acid residues of the kinase
38, 43 and 44. These inhibitors show activity in the range of 15– (Figures 3 and 6). Therefore, the decrease in negative charge
27 mM and have a hydroxyl group at position 4 of the ring A on pyrimidinone’s oxygen a likely is reducing the stability of
(Table 4).
ligand-receptor complex. As a result, all the above-mentioned
Activities of the six aminosalicylic acid derivatives depend aza-analogs of 2-aminopyrimidine are binding with ATP-acceptor
on the size of substituents R1 and R2 in the ring B. Ligands with pocket weaker than 2-aminopyrimidines themselves and are not
short substituents R2 such as ethyl, n-propyl and i-propyl were active.
found inactive (compounds 18, 19 and 20) or showed low activity
To explain the obtained structure–activity relationships, the
(compound 17, IC50 ¼ 20 mM). At the same time, ligands complexes of the ligands and CK2 were generated by molecular
with bigger substituents R2 made positive contributions to docking methods. It was shown that the ligands are complemen-
activity. For example, n-butyl-substituted compound 21 has the tary to the ATP-binding site of CK2 and located similarly to
activity IC50 ¼ 9.5 mM. Moreover, compound 25 (Figure 4) with compounds 3–15 (see above). The compound 25 (Figure 6) which
4-methoxyphenyl as R2 was the most potent among all tested has the highest inhibitory activity among the other optimized
ligands (IC50 ¼ 1.1 mM). Thus, the large hydrophobic groups such ligands forms a number of additional intermolecular bonds with
as n-butyl, butylene, phenyl, phenylene and 4-methoxyphenyl the CK2 catalytic subunit. The first additional bond is formed
are required at position Y of the ring B.
between hydroxyl group of the ligand and Lys68 while the bond
On the contrary, increasing the size of the substituent R1 led between the ligand’s carboxyl group and this amino acid is
to lower activities. For instance, compounds 26, 27 and 28 remained. The second bond is formed with Glu114 in the hinge
with relatively large groups such as benzyl, p-methylbenzyl and region of CK2. In addition, the 4-methoxyphenyl substituent of

DOI: 10.3109/14756366.2013.837898
Design, synshesis and biological evaluation of 2-aminopyrimidinones
7
Figure 5. Calculated electronegative charges
at the oxygen atoms for (a) 2-aminopyrimi-
din-4-one, (b) 2-amino-6-azapyrimidin-4-one
and (c) 4-amino-6-azapyrimidin-2-one.
aminopyrimidinone derivatives and their 6-aza-analogs were
developed by methods of combinatorial chemistry and molecular
docking. Among in vitro tested compounds, the most active ligand
2-hydroxy-5-[4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-
2-ylamino] benzoic acid 25 was identified (IC50 ¼ 1.1 mM).
The relationship ‘‘chemical structure-biological activity’’ for
2-aminopyrimidinone derivatives and their 6-aza-analogs was
investigated and the binding mode of the synthesized ligands
with CK2 acceptor site was proposed. It was established that
only 2-aminopyrimidinone derivatives inhibited protein kinase
CK2 with the activities in the range of 1.1–30 mM while their
6-aza-analogs were not active. Probably, it is associated with the
lower ability of carbonyl group of 6-aza-2-aminopyrimidinone
ring to form hydrogen bonds with amino acid residues of CK2
ATP-binding site.
Declaration of interest
Figure 6. Complex of ligand 25 with CK2. Complex is obtained using
a molecular docking method. Intermolecular H-bonds are shown as
dotted lines.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
References
25 is responsible for rigid ligand fixation in a hydrodrophobic
pocket-2 of kinase ATP-binding site. Presumably, the summation
of the above-mentioned additional interactions is responsible
for higher inhibitory activity of 25 comparing to the other
synthesized ligands.
As it was noticed before, the absence of activity in compounds
26, 27 and 28 is associated with presence of the large substituents
R1 at position X of the ring A. The analysis of the obtained
complexes showed that insertion of hydrophobic groups to this
position leads to increased steric hindrances in the middle of the
ATP-binding site which results in breaking of hydrogen bond with
Val116. The substituent R4 of compounds 35–62 (Table 4) is
causing conformationally disfavored orientation of the ligands
in the middle of the ATP-binding site. As a result, the hydrogen
bond with Lys68 is broken. This causes the complete loss of
inhibitory activity of compounds.
Thus, there are two key structural feature required for activity
of 2-aminopyrimidinone derivatives. The presence of acceptor
group in the ring A promotes stronger interaction with Lys68
which improves inhibitory properties of 2-aminopyrimidinone
derivatives. And additional stabilization of the complex is
provided by the large substituents oriented into hydrophobic
pocket-II of the ATP-acceptor site of CK2 (Figure 6).
Therefore, the 2-aminopyrimidinone derivatives are hopeful
for further study and optimization which should be achieved by
the formation of additional interactions with Glu114 and Asp175
as well as by enhancement of the complex stability via addition of
hydrophobic groups into the pyrimidinone ring.
1. Volynets GP, Golub AG, Bdzhola VG, et al. The role of
protein kinase CK2 in the process of oncogenesis, regulation of
apoptosis and cellular stress response. Ukr Bioorg Acta 2007;2:
25–32.
2. Ruzzene M, Pinna LA. Addiction to protein kinase CK2: a common
denominator of diverse cancer cells? Biochim Biophys Acta 2010;3:
499–504.
3. Landesman-Bollag E, Romieu-Mourez R, Song DH, et al. Protein
kinase CK2 in mammary gland tumorigenesis. Oncogene 2001;20:
3247–57.
4. Lin YC, Hung MS, Lin CK, et al. CK2 inhibitors enhance the radio
sensitivity of human non-small cell lung cancer cells through
inhibition of stat3 activation. Cancer Biother Radiopharm 2011;26:
381–8.
5. Laramas M, Pasquier D, Filhol O, et al. Nuclear localization of
protein kinase CK2 catalytic subunit (CK2-alpha) is associated with
poor prognostic factors in human prostate cancer. Eur J Cancer
2007;43:928–34.
6. Zou J, Luo H, Zeng Q, et al. Protein kinase CK2a is overexpressed
in colorectal cancer and modulates cell proliferation and invasion via
regulating EMT-related genes. J Transl Med 2011;9:97.
7. Stalter G, Siemer S, Becht E, et al. Asymmetric expression of
protein kinase CK2 subunits in human kidney tumors. Biochem
Biophys Res Commun 1994;202:141–7.
8. Piazza F, Manni S, Ruzzene M, et al. Protein kinase CK2 in
hematologic malignancies: reliance on a pivotal cell survival
regulator by oncogenic signaling pathways. Leukemia 2012;26:
1174–9.
9. Singh NN, Ramji DP. Protein kinase CK2, an important regula-
tor of the inflammatory response? J Mol Med (Berl) 2008;86:
887–97.
10. Ivanov KI, Puustinen P, Gabrenaite R, et al. Phosphorylation of the
potyvirus capsid protein by protein kinase CK2 and its relevance for
virus infection. Plant Cell 2003;15:2124–39.
Conclusions
11. Cozza G, Pinna LA, Moro S. Protein kinase CK2 inhibitors: a patent
review. Expert Opin Ther Pat 2012;22:1081–97.
12. Kotey IM, Balanda AO, Bdzhola VG, et al. Synthesis and biological
evaluation of thienopyrimidinones as CK2 protein kinase inhibitors.
Ukr Bioorg Acta 2008;2:3–9.
It was established that 2-aminopyrimidinone derivatives are
promising class of compounds for the development of new
potent inhibitors of protein kinase CK2. In order to find new and
effective inhibitors of human protein kinase CK2, the new 2-

8
M. O. Chekanov et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
13. Pagano MA, Poletto G, Di Maira G, et al. Tetrabromocinnamic acid 25. Prykhod’ko AO, Yakovenko OY, Golub AG, et al. Evaluation of 4H-
(TBCA) and related compounds represent a new class of specific
protein kinase CK2 inhibitors. ChemBioChem 2007;8:129–39.
14. Sarno S, De Moliner E, Ruzzene M, et al. Biochemical and three-
dimensional-structural study of the specific inhibition of protein
kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]
acetic acid (IQA). Biochem J 2003;374:639–46.
15. Golub AG, Yakovenko OY, Prykhod’ko AO, et al. Evaluation
of 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as inhibitors
of human protein kinase CK2. Biochim Biophys Acta 2008;1784:
143–9.
16. Golub AG, Yakovenko OY, Bdzhola VG, et al. Evaluation of
3-carboxy-4(1H)-quinolones as inhibitors of human protein kinase
CK2. J Med Chem 2006;49:6443–50.
17. Shoichet BK, Stroud RM, Santi DV, et al. Structure-based
discovery of inhibitors of thymidylate synthase. Science 1993;259:
1445–50.
4-chromenone derivatives as inhibitors of protein kinase CK2.
Biopolym Cell 2005;21:1–6.
26. Meggio F, Pagano M, Moro S, et al. Inhibition of protein kinase
CK2 by condensed polyphenolic derivatives. An in vitro and in vivo
study. Biochemistry 2004;43:12931–6.
27. Chekanov MO, Tarnavskiy SS, Synyugin AR, et al. Solution
phase combinatorial synthesis of a diverse library of 2-aminopyr-
imidinones and their 6-aza-analogs. Ukr Bioorg Acta 2011;10:
29–40.
28. Ewing TJ, Kuntz ID. Critical evaluation of search algorithms for
automated molecular docking and database screening. J Comput
Chem 1996;18:1175–89.
29. Suzuki Ya, Oishi Sh, Takei Y, et al. Design and synthesis of a novel
class of CK2 inhibitors: application of copper- and gold-catalysed
cascade reactions for fused nitrogen heterocycles. Org Biomol Chem
2012;10:4907–15.
18. Bodian DL, Yamasaki RB, Buswell RL, et al. Inhibition of the
fusion-inducing conformational change of influenza hemagglutinin
by benzoquinones and hydroquinones. Biochemistry 1993;32:
2967–78.
19. Ring CS, Sun E, McKerrow JH, et al. Structure-based inhibitor
design by using protein models for the development of antiparasitic
agents. Proc Natl Acad Sci USA 1993;90:3583–7.
20. Ewing TJ, Makino S, Skillman AG, et al. DOCK 4.0: search
strategies for automated molecular docking of flexible molecule
databases. J Comput Aided Mol Des 2001;15:411–28.
21. Yakovenko OY, Oliferenko AA, Golub AG, et al. The new method
of distribution integrals evaluations for high throughput virtual
screening. Ukr Bioorg Acta 2007;1:52–62.
22. Yakovenko OY, Li YY, Oliferenko AA, et al. Ab initio parameter-
ization of YFF1, a universal force field for drug-design applications.
J Mol Model 2012;18:663–73.
30. Fuchi N, Iura Y, Kaneko H, et al. Discovery and structure–activity
relationship of 2,6-disubstituted pyrazines, potent and selective
inhibitors of protein kinase CK2. Bioorg Med Chem Lett 2012;22:
4358–61.
31. Golub AG, Bdzhola VG, Briukhovetska NV, et al. Synthesis and
biological evaluation of substituted (thieno[2,3-d]pyrimidin-4-
ylthio)carboxylic acids as inhibitors of human protein kinase CK2.
Eur J Med Chem 2011;46:870–6.
32. Golub AG, Bdzhola VG, Kyshenia YV, et al. Structure-based
discovery of novel flavonol inhibitors of human protein kinase CK2.
Mol Cell Biochem 2011;356:107–15.
33. Ferguson AD, Sheth PR, Basso AD, et al. Structural basis of
CX-4945 binding to human protein kinase CK2. FEBS Lett 2011;
585:104–10.
34. Backes AC, Zech B, Felber B, et al. Small-molecule inhibitors
binding to protein kinases. Part I: exceptions from the traditional
pharmacophore approach of type I inhibition. Expert Opin Drug
Discov 2008;3:1409–25.
35. Instant JChem, release version 5.5.1. ChemAxon. Available from:
http://www.chemaxon.com
23. Yakovenko OY, Oliferenko AA, Bdzhola VG, et al. Kirchhoff atomic
charges fitted to multipole moments: implementation for a virtual
screening system. J Comput Chem 2008;29:1332–43.
24. Lindahl E, Hess B, van der Spoel D. GROMACS 3.0: a package
for molecular simulation and trajectory analysis. J Mol Mod 2001;7:
306–17.
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