Synthesis and biological evaluation of novel 2 (4'-hydroxynaphthyl) chromen-4-one as a CK2 inhibitor

Article abstract of DOI:10.1681/ph.2018.7971

Protein kinase CK2 is a potential drug target for many diseases including cancer, inflammatory disorders, Alzheimer's disease, Parkinson's disease and viral infections. Significant efforts have been made for the discovery of potent inhibitors of this enzyme. Herein, we report on the synthesis, characterization, and biological evaluation of novel flavonoid compounds as CK2 inhibitors. The tested compounds were 2 (4'-hydroxynaphthyl) chromen-4-one which is a naphthyl backbone flavonoid with an IC₅₀ value of 0.45±0.059 μM and 2(4-hydroxyphenyl)-4Hchromen-4-one a phenyl based derivative with an IC₅₀ value of 0.33±0.048 μM. Cell viability was tested using MCF-7 cells. Both compounds were able to reduce the cell viability around 50 % in concentration of 100 μM after 48 h. Molecular modeling studies were performed to understand the binding mode of both compounds.

Full text of DOI:10.1681/ph.2018.7971

ORIGINAL ARTICLES
Institut für Pharmazeutische und Medizinische Chemie1, PharmaCampus, Westfälische Wilhelms-Universität Münster,
Germany, Department of Pharmaceutical Chemistry and Drug Quality Control2, Faculty of Pharmacy, Damascus University,
Syria
Synthesis and biological evaluation of novel 2 (4`-hydroxynaphthyl)
chromen-4-one as a CK2 inhibitor
2
2
1
1
S. HAIDAR1, 2,*, M. JABBOUR , M. A. AL-KHAYAT , D. AICHELE , J. JOSE
Received November 14, 2017, accepted December 18, 2017
*Corresponding author: Prof. Dr. Samer Haidar, Institut für Pharmazeutische und Medizinische Chemie, Pharma-
Campus, Westfälische Wilhelms-Universität Münster, Corrensstr. 48, 48149 Münster, Germany
shaid_01@uni-muenster.de
Pharmazie 73: 191–195 (2018)
doi: 10.1681/ph.2018.7971
Protein kinase CK2 is a potential drug target for many diseases including cancer, inflammatory disorders, Alzhei-
mer’s disease, Parkinson’s disease and viral infections. Significant efforts have been made for the discovery of
potent inhibitors of this enzyme. Herein, we report on the synthesis, characterization, and biological evaluation
of novel flavonoid compounds as CK2 inhibitors. The tested compounds were 2 (4`-hydroxynaphthyl) chromen-
4-one which is a naphthyl backbone flavonoid with an IC50 value of 0.45 0.059 μM and 2(4-hydroxyphenyl)-4H-
chromen-4-one a phenyl based derivative with an IC50 value of 0.33 0.048 μM. Cell viability was tested using
MCF-7 cells. Both compounds were able to reduce the cell viability around 50 % in concentration of 100 μM after
48 h. Molecular modeling studies were performed to understand the binding mode of both compounds.
1. Introduction
Casein kinase 2, CK2, a heterotetrameric holoenzyme, is a ubiq-
uitously expressed serine/threonine protein kinase in mammalian
cells. CK2 enhances cancer phenotype by blocking apoptosis or
stimulating cell growth. Abnormal high levels of this enzyme
were detected in several types of cancer including breast, lung,
and colorectal, thus, inhibition of this kinase can induce the phys-
iological process of apoptosis, leading to tumor cell death. CK2
is now considered as an important drug target for cancer therapy,
and quite a number of CK2 inhibitors from different chemical
classes has been developed, such as anthraquinone/xanthenone,
benzofuran derivatives, indenoindoles, coumarins and flavonoids
(Cozza et al. 2012; Alchab et al. 2015; Haidar et al. 2015; Cozza
and Pinna 2016; Haidar et al. 2017). More than 4000 compounds
belonging to the group of natural botanic flavonoids are known
so far (Ren et al. 2003). These compounds have many biological
effects, such as, antioxidative, anti-mutagenic, anti-inflammatory,
and antiviral properties (Patel et al. 2007). Many studies have
demonstrated the anticancer effect of the flavonoids both in vitro
and in vivo. Some flavonoids or their derivatives were proven to
be active CK2 inhibitors. Fig. 1 presents the chemical structures
of some known CK2 inhibitors with flavonoid structure. Different
chemical methods were applied to synthesize flavonoids, among
them the Baker-Venkatraman rearrangement, oxidative cyclization
of 2-hydroxy chalcones (Cabrera et al. 2007; Thejan 2011), or
hydrolysis of flavylium salts (Dorofeenko et al. 1977; Yakovenko
Fig. 2: Chemical structure of compound 2
et al. 1981; Golub et al. 2013). Flavylium salts can be prepared by
aldol condensation of 2`-hydroxyacetophenone and benzaldehyde
derivatives in the presence of tri-ethyl orthoformate and perchloric
acid (Dorofeenko et al. 1977; Yakovenko et al. 1981; Golub et al.
2013). In this work we were able to prepare 4`-hydroxyflavonoid
derivatives with naphthalene backbones instead of benzene, using
4-hydroxynaphthalin-1-carbaldehyde as starting material. This
was based on the assumption that the extra aromatic ring might
enhance the affinity to the hydrophobic pocket in the active site of
CK2 according to the fact that the aromatic and/or apolar portions
of CK2 inhibitors are responsible for the major interactions with
the active site of the enzyme. The phenyl based analog was resyn-
thesized for comparison since it was never tested as CK2 inhibitor
before. Here, we describe the synthesis and characterization as well
Myricetin
0.92 0.13
Quercetin
0.55 0.08
Apigenin
0.80 0.12
Fig. 1: Structures of some CK2 flavonoid inhibitors with their IC50 values
Pharmazie 73 (2018)
191

ORIGINAL ARTICLES
Scheme:
Synthesis pathway to compound 4: 2 (4` hydroxynaphthyl) chromen-4-one.
as the biological evaluation of novel flavonoid derivatives as CK2
inhibitors, together with molecular modeling study to illustrate
their binding mode to the active site; also, our recently developed
structure based pharmacophore was used to evaluate the results.
2. Investigations, results and discussion
2.1. Synthesis
A two-steps synthesis procedure was applied to synthesize
compound 4, analogues to the known compound 2 which was
resynthesized to evaluate it activity against CK2. The first step
was an aldol condensation in acidic media between 2`-hydroxy-
acetophenone and aromatic aldehydes, in the presence of triethyl
orthoformate to prepare flavylium salts. The salt undergoes
hydrolysis in the second step to produce flavonoid derivatives. The
aromatic aldehydes used in this study are 4`-hydroxybenzaldehyde
and 4` hydroxy 1-naphthaldehyde in order to prepare compounds
2 and 4, respectively. Figure 2 presents the chemical structures of
compound 2 while the scheme presents the synthesis pathway to
compounds 4.
Fig. 3: Chemical structure of the known CK2 inhibitor emodin
2. 3. Effect of CK2 inhibitors 2 and 4 on cell prolifera-
tion
Cell viability was tested using the MTT, (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenylte- tetrazolium bromide) assay. This assay
is a colorimetric assay, which measures the conversion of MTT into
violet formazan which is produced by succinate dehydrogenase of
the intact mitochondria in viable cells. As it is clear from Fig. 4,
compounds 2 and 4 were able to reduce the cell viability around
50 % after 48 h of incubation and with concentration of 100 μM.
No effects were observed with 20 μM of inhibitors concentration
(data not shown). These results indicate that the tested compounds
showed a moderate effect in cell viability. Actually this finding
does not contradict the in vitro results, as other factors are involved
in such assays as cell permeability and type of cell lines.
2.2. Inhibition of human protein kinase CK2
The new synthesized compound (4`-hydroxynaphthyl) chromen-
4-one (4) was tested for its inhibitory activity towards the human
CK2 holoenzyme and compared with the activity of compound 2
following the procedure described earlier (Olgen et al. 2007). It is
important to mention that compound 2 is a commercially available
compound which was to the best of our knowledge never tested as
CK2 inhibitor. The synthetic peptide RRRDDDSDDD was used as
substrate, which is reported to be most efficiently phosphorylated
by CK2. The purity of the CK2 holoenzyme was superior to 99 %.
For initial testing, inhibition was determined at inhibitor concen-
trations of 10 μM in DMSO as solvent. The reaction with pure
solvent without inhibitor was used as positive control and set to 0%
inhibition. Reactions without CK2 were used as negative control
and were taken as 100% inhibition, both compounds showed more
than 80% inhibition in this initial test. IC values were determined
by measuring CK2 inhibition at eight50different concentrations
ranging from 0.001 to 100 μM in appropriate intervals and calcu-
lated from the resulting dose-response curve (Gratz et al. 2010).
The IC50 value of compound 4 turned out to be 0.45 0.059 μM,
while the IC50 value of compound 2 was 0.33 0.048 μM. However,
the difference between the IC values of both compounds was
statistically not significant (P >500 .05). Extending the structure of 2
with an extra aromatic ring did not affect the activity considerably.
As a control the well-known inhibitor of CK2, emodin (Fig. 3) was
subjected to the same test and showed an IC value of 0.58 0.05
μM. The three compounds presented in Fig. 510 namely: myricetin,
quercetin, and apigenin were not tested by us, for that reason we
cannot compare their activity with the activity of our compounds
correctly, even though compounds 2 and 4 seems to be in the same
range of activity.
Fig. 4: Cell viability was evaluated by MTT assay using MCF-7 cells after 24 and 48
h treatment of the compounds with 100μM concentration. Values were shown
as mean SD, n= 3
2.4. Molecular docking of compounds 2 and 4
In order to further understand the binding mode of the inhibi-
tors, compounds 2 and 4 were docked in the crystal structure of
CK2 co-crystalized with apigenin (PDB ID: 4DGM), using the
parameters described in the experimental section. Figure 5 shows
192
Pharmazie 73 (2018)

ORIGINAL ARTICLES
cules. For comparison apigenin can form two bonds with aspartate
175 beside two indirect bonds via water molecules. Recently we
described a structure-based pharmacophore model for a set of
flavonoids as CK2 inhibitors (Jabbour et al. 2016), compounds 2
and 4 fit well with all features of the newly developed pharmaco-
phore as described and shown in Fig. 7 which indicates that both
compounds has similar features to the active flavonoids.
(A)
(B)
(A)
Fig. 7: Alignment of the
formerly developed
structure-based
(C)
pharmacophore
model with com-
pounds 2 (A) and 4
(B). Acc: H-bond
acceptor, Aro: Aro-
matic center, Hyd:
Hydrophobic cen-
troid, Don&Acc:
Fig. 5: Snapshots representing the docked complexes of (A) apigenin (B) compound
2 and (C) compound 4 with the ATP binding site of CK2
H-bond donor and
H-bond acceptor.
(B)
the best obtained conformation and orientation of compounds 2
and 4 (in 3D form) in the ATP active site of CK2, with S score
equals to -9.696 and -13.3367 respectively, while apigenin has S
score of –11.423 after extraction and redocking. Figure 6 shows
the 2D-interactions between each compound and the amino acid
residues in the ATP binding site of CK2. Compound 2 forms an
interaction network with the active site of the enzyme, a direct
hydrogen bond with two aspartate 75, while compound 4 had two
arene – arene interactions with histidine 160 and methionine 163
beside two indirect bonds with 4`-hydroxyl group via water mole-
In this work we were able to synthesize a novel flavone deriva-
tive compound 4 with a good in vitro activity against CK2 in the
sub-micro molar range in order to expand the boundaries of appli-
cation of flavonoid synthesis beyond benzaldehyde derivatives,
and also to explore the ability of naphthyl moiety to create a more
stable and active inhibitors of this kinase. As a matter of fact the
activity of compound 4 was in the same rang of the activity of
compound 2 and even better than other known CK2 inhibitors such
(a)
(b)
(c)
Fig. 6: Two - dimensional representation of the interactions between the ATP binding site of CK2 and apigenin (a), compounds 2 (b), and compound 4 (c)
Pharmazie 73 (2018)
193

ORIGINAL ARTICLES
Western Blot. The capillary electrophoresis based assay was used for testing the
as emodin, also the binding mode was illustrated by a molecular
modeling approach.
inhibitors of the human CK2 as described earlier, 2 μL of the dissolved inhibitors
(stock solution concertation: 10 μM in DMSO) were mixed with 78 μL of CK2
supplemented kinase buffer which was composed of 1 μg CK2 holoenzyme, 50 mM
Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT. The reaction was
initiated by the addition of 120 μL assay buffer, which contains 25 mM Tris/HCl
(pH 8.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μM ATP and 190 μM of the
substrate peptide RRRDDDSDDD. The reaction was carried out for 15 min at 37 °C
and stopped by the addition of 4 μL EDTA (0.5 M). Subsequently the reaction mixture
was analyzed by a PA800 capillary electrophoresis from Beckman Coulter (Krefeld,
Germany). Acetic acid (2 M, adjusted with conc. HCl to a pH of 2.0) was used as the
electrolyte for electrophoretic separation. The separated substrate and product peptide
were detected at 214 nm using a DAD-detector. IC50 values were calculated from the
resulting dose–response curves.
3. Experimental
3.1. Synthesis
Starting materials, solvents, and reagents were purchased from commercial sources
and were used without further purification. The reactions were monitored by TLC on
silica-gel plates (Merck 60F254). Melting point was determined using electro thermal
apparatus 9100, and used without calibration. IR spectra were recorded on KBr on
Jasco FT/IR-4200 apparatus. UV spectra were recorded using Shimadzu UV-BC
3101. 1H NMR and 13C NMR spectra were recorded on a Bruker Ultra Shield 400
instrument (400 MHz for 1H, 100 MHz for 13C). Elemental analyses were performed
on EURO-EA instrument. CK2 activity was analyzed by a PA800 capillary electro-
phoresis from Beckman Coulter (Krefeld, Germany).
3.2.2. Cell viability assay
The effect of CK2 inhibitors on the viability of MCF-7 cells was evaluated using
MTT assay (Mosmann 1983). MCF-7 breast cancer cells (kindly provided by the
Department of Clinical Radiology of the University Hospital Muenster, Germany),
were cultured in RPMI 1640 medium containing GlutaMax (Life Technologies) and
10% fetal calf serum. MTT assay was performed in 96-well plates. Cells were seeded
at a density of 1 x 105 cells per well. Cells were incubated for 24 or 48 h at 37 °C in
a humidified atmosphere (5% CO2). After overnight incubation, seeding medium was
removed and replaced with fresh medium containing the inhibitor at 20 or 100 μM.
DMSO, at a final concentration of 1%, served as a control. Afterwards MTT reagent
(Sigma Aldrich, Germany) was added at a final concentration of 0.5 mg/mL. After
incubation for 2 h at 37 °C medium was discarded and 200 μL DMSO were added for
solubilization the formazan. After mixing, the absorption was determined at 570 nm
with a reference wavelength of 630 nm using a microplate reader. CK2 inhibitors were
assayed in triplicates, and the experiments were repeated three times.
3.1.1. General procedure for the preparation of the flavylium salts
In 25 ml round bottom flask, a mixture of 1 mM of 2`-Hydroxyacetophenone, 2 mM
mole of the aromatic aldehyde, and 1.4 ml triethyl orthoformate was prepared. Then,
0.1 ml of perchloric acid 70% was added drop wise, and the mixture was stirred at
room temperature for 3 h. The precipitate was removed by filtration, washed with dry
ether then recrystallized using glacial acetic acid to gain the corresponding flavylium
salt.
3.1.2. General procedure for the hydrolysis of the flavylium salts
The pure flavylium salt was refluxed with 25 ml of distilled water for 5-10 min, the
precipitated flavonoid was removed by filtration, recrystallized using ethanol.
3.1.3. Characterization of compound
3.3. Statistical analysis
4-Ethoxy-2-(4-hydroxyphenyl) chromenium perchlorate (1):
Prism 6 (GraphPad Software) was used to evaluate the IC50 values and their statistical
significance with Student’s t test. Values were considered statistically significant at
p below 0.05.
Orange powder. Yield 63.8 %, mp 254-256 ºC, IR (ν cm-1): 3438, 3079, 1103, 1598,
1
1531, 1618. H NMR (CD3OD, 400 MHz, 298K) δ/ppm: 10.350 (s, 1H, OH), 8.51-
8.44 (m, 2H, H2’, H6’), 8.36-8.33 (m, 1H, Ar-H), 8.22-8.10 (m, 2H, Ar-H), 8.03 (s, 1H,
H3), 7.83 (ddd, J = 8.19, 6.24, 1.96 Hz, 1H, Ar-H), 7.20-6.99 (m, 2H, H3’, H5’), 3.59
(q, J = 7.06, 7.06, 7.05 Hz, 2H, CH2), 1.16 (t, J = 7.04, 7.04 Hz, 3H, CH3). 13C NMR
(CD3OD, 100 MHz, 298K) δ/ppm: 134.239, 132.726, 128.695 (C2’, C6’), 125.459,
125, 124.354, 122.570, 120.469, 118.311, 117.13, 116.019 (C3’, C5’), 98.038 (C4),
70.501 (C3), 66.310 (CH2), 13.416 (CH3).
3.4. Computational study
Computational work was performed on Intel (R) Core (TM) processor 3.20 GHz,
using Molecular Operating Environment software package (MOE, Chemical
Computing Group, Montreal, Canada) (Molecular Operating Environment (MOE)).
2-(4-Hydroxyphenyl)chromen-4-one (2):
Pale yellow solid. Yield: 75.84 %, mp 274-276 ºC (Lit.: 270-273 ºC) (Kshatriya et al.
2014), UVλmax (methanol, nm): 219.5, 253, 326. IR (ν, cm-1): 3434.6, 3200, 1633.41,
1
3.4.1. Protein preparation
1600, 1565.92, 1456.9. H NMR (DMSO-d6, 400 MHz, 298K) δ/ppm: δ 10.340 (S,
1H, OH), δ 8.031 (dd, J = 7.94, 1.48 Hz, 1H, H5), δ 8.00-7.94 (m, 2H, H2’, H6’), δ 7.814
(ddd, J = 8.58, 6.99, 1.69 Hz, 1H, H7), δ7.753 (dd, J = 8.52, 0.92 Hz, 1H, H8), δ 7.482
(ddd, J = 8.02, 6.99, 1.20 Hz ,1H, H6), δ 7.00-6.90 (m, 2H, H5’, H3’), δ 6.88 (S, 1H, H3).
13C NMR (DMSO-d6, 100 MHz, 298K) δ/ppm: 177.283 (C4), 163.540 (C2), 161.267
(C4’), 155.806 (C10), 134.588 (C7), 129.127 (C2’, C6’), 125.793 (C6), 125.199 (C5),
123.787 (C9), 122.047 (C1’), 118.837 (C8), 116.419 (C3’, C5’), 105.280 (C3). Elemental
analysis: calcd: C, 75.623%, H, 4.22%, found: C 75.99%, H 3.85%.
Three dimensional 3D structure of the CK2 complex with apigenin was obtained from
the Protein Data Bank (PDB) using PDB ID: (4DGM) having a resolution of 1.65 Å.
The structure was optimized by using QuickPrep function implemented in the soft-
ware. Then water molecules were removed from the structure and 3D protonation was
done to change the state into ionization level. In the second step, energy minimization
was performed using defaults parameters, where the force field was Amber 99.
4-Ethoxy-2-(4-hydroxynaphthalen-1-yl) chromenium perchlorate (3):
º
Dark brown powder. Yield: 91.7 %, mp 233-235 C, IR (ν, cm-1): 3427, 1114, 1619,
3.4.2. Docking study
1535, 1597. 1H NMR (CD3CN, 400 MHz, 298K) δ/ppm: 9.17 (s, 1H, OH), 8.55 (m,
1H, Ar-H), 8.45-8.34 (m, 2H,Ar-H), 8.27 (d, J= 8.33, 1H, H2’), 8.19 (ddd, J= 8.71,
7.13, 1.58 Hz, 1H, Ar-H), 8.09 (m, 1H, Ar-H),7.87 (ddd, J = 8.17, 7.19, 1.07 Hz, 1H,
Ar-H), 7.81 (ddd, J= 8.54, 6.88, 1.42 Hz, 1H, Ar-H), 7.77 (s, 1H, H3), 7.71-7.66 (m,
1H, Ar-H), 7.17 (d, J= 8.33 Hz, 1H, H3’), 4.83 (q, J= 7.03, 7.03, 7.03 Hz, 2H, CH2),
1.67 (t, J= 7.02, 7.02 Hz, 3H, CH3). 13C NMR (CD3CN, 100 MHz, 298K) δ/ppm:
206.9, 177.337, 174.422, 160.843, 156.662, 138.036, 136.176, 131.837, 130.274,
129.056, 126.768, 124.95,124.479, 124.305, 123.432, 119.161, 118.783, 108.725,
102.519, 70.612(CH2), 13.328(CH3). Elemental analysis: calcd: C, 60.5%, H, 4.08%,
found: C, 60.1%, H, 3.65%.
The docking of the selected compounds from the database (compounds) into the
active site of CK2 enzyme (4DGM) was achieved using MOE-Dock implemented
on MOE. The docking parameters were set as Rescoring 1: London dG, Placement:
triangle matcher, Retain 30, Refinement Force field, and Rescoring 2: GBVI/WSA
dG. Docking part of MOE can give correct conformation of the ligand to obtain
minimum energy structure. The top conformation for each compound was selected
based on the S score and visual inspection was carried out by Lgplot implemented
in MOE. Compounds showing significant interaction with the residues of binding
pocket of CK2 were picked as promising hits. Prior to dock, the initial ligand from the
complex structure was extracted. For the scoring function, lower scores indicate more
favorable poses. The unit for the scoring function is Kal/mol, and the S score refers to
the final score, which is the score of the last stage that was not set to None. The Lig X
function in MOE was used for conducting interactive ligand modification and energy
minimization in the active site of the receptor.
2 (4`-Hydroxynaphthyl) chromen-4-one (4):
º
Golden brown crystals. Yield: 81.25 %, mp 240-241 C, UV λ
(methanol, nm):
max
214.5, 234, 308, 348. IR (ν, cm1): 3434.6, 3123, 1617.02, 1508.06, 1476.02, 1559.17.
1H NMR (DMSO-d6, 400 MHz, 298K) δ/ppm: 10.98 (s, 1H, OH), 8.28 (dd, J = 8.29,
0.99 Hz ,1H, H5), 8.11 (m, 2H, H5’, H8’), 7.84 (ddd, J = 8.70, 7.15, 1.73 Hz, 1H, H7),
7.75 (d, J = 7.95 Hz ,1H, H2’), 7.69 (dd, 1H, H8), 7.62 (ddd, J = 8.45, 6.81, 1.54 Hz,
1H, H6), 7.55 (dddd, J = 8.98, 8.05, 7.06, 1.18 Hz, 2H, H6’, H7’), 7.01 (d, J = 7.97 Hz
,1H, H3’), 6.59 (s, 1H, H3). 13C NMR (DMSO-d6, 100 MHz, 298K) δ/ppm: 177.403
(C4), 166.187 (C2), 156.855 (C4’), 156.592 (C10), 134.715 (C7), 131.8 (C9’), 130.448
(C2’), 128.371 (C6), 125.996 (C7’), 125.777 (C6’), 125.369 (C8’), 125.0 (C10’), 124.990
(C5’), 123.846 (C9), 123.176 (C5), 120.961 (C1’), 119 (C8), 111.830 (C3), 107.969 (C3’).
Elemental analysis: calcd: C, 79.1%, H, 4.1%, found: C, 78.679%, H, 3.714%.
Acknowledgements: Many thanks to M. Ktaifani, for his help and support.
Conflicts of interest: None declared.
References
Alchab F, Alchab F, Ettouati L, Bouaziz Z, Bollacke A, Delcros JG, Gertzen CG,
Gohlke H, Pinaud N, Marchivie M, Guillon J, Fenet B, Jose J, Borgne ML (2015)
Synthesis, biological evaluation and molecular modeling of substituted inde-
no[1,2-b]indoles as inhibitors of human protein kinase CK2. Pharmaceuticals 8:
279-302.
3.2. Biological evaluation
3.2.1. Inhibition of human CK2 holoenzyme
Cabrera M, Cabrera M, Simoens M, Falchi G, Lavaggi ML, Piro OE, Castellano EE,
Vidal A, Azqueta A, Monge A, de Ceráin AL, Sagrera G, Seoane G, Cerecetto H,
González M (2007) Synthetic chalcones, flavanones, and flavones as antitumoral
agents: biological evaluation and structure-activity relationships. Bioorg Med
Chem 15: 3356-3367.
The preparation of the human recombinant CK2 holoenzyme was performed
according to a protocol previously described (Gratz et al. 2010). For the expres-
sion of the α-subunit (CSNK2A1) and β-subunit (CSNK2B) of the human protein
kinase CK2, the pT7-7 expression system in Escherichia coli BL21 (DE3) was used.
Fractions exhibiting CK2 activity were combined and analyzed by SDS-PAGE and
194
Pharmazie 73 (2018)

ORIGINAL ARTICLES
Cozza G, Pinna LA (2016) Casein kinases as potential therapeutic targets. Expert
Opin Ther Targets 20: 319-340.
Cozza G, Pinna LA, Moro S (2012) Protein kinase CK2 inhibitors: a patent review.
Expert Opin Ther Pat 22: 1081-1097.
Kshatriya J, Machhi JK, Nazeruddin GM (2014) Regioselectivity and chemoselec-
tivity in the synthesis of flavones. Online Int Interdiscipl Res J 4: 196-205.
Molecular Operating Environment (MOE), C. C. G. I., 1010 Sherbooke St. West,
Suite #910, Montreal, QC, Canada, H3A 2R7, 2017.
Dorofeenko GN, Tkachenko VV, Yakovenko VI, Ezheritskii VVM, Oganesyan ET
(1977) Synthesis of 4-ethoxyflavylium salts and flavones. Chem Heterocycl Comp
13: 149-152.
Golub AG, Golub AG, Bdzhola VG, Ostrynska OV, Kyshenia IV, Sapelkin VM, Pryk-
hod’ko AO, Kukharenko OP, Yarmoluk SM (2013) Discovery and characterization
of synthetic 4’-hydroxyflavones-New CK2 inhibitors from flavone family. Bioorg
Med Chem 21: 6681-6689.
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: applica-
tion to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
Olgen S, Götz C, Jose J (2007) Synthesis and biological evaluation of 3-(substitut-
ed-benzylidene)-1,3-dihydro-indolin derivatives as human protein kinase CK2 and
p60(c-Src) tyrosine kinase inhibitors. Biol Pharm Bull 30: 715-718.
Patel D, Shukla S, Gupta S (2007) Apigenin and cancer chemoprevention: progress,
potential and promise (review). Int J Oncol 30: 233-245.
Gratz A, Götz C, Jose J (2010). A CE-based assay for human protein kinase CK2
activity measurement and inhibitor screening. Electrophoresis 31: 634-640.
Haidar S, Haidar S, Bouaziz Z, Marminon C, Laitinen T, Poso A, Le Borgne M, Jose J
(2017) Development of pharmacophore model for indeno[1,2-b]indoles as human
protein kinase CK2 inhibitors and database mining. Pharmaceuticals 10: pii E8.
Haidar S, Meyers A, Bollacke A, Jose J (2015) Synthesis and biological evaluation of
2,6-di(furan-3-yl)anthracene-9, 10-dione as an inhibitor of human protein kinase
CK2. Pharmazie 70: 772-776.
Ren W, Qiao Z, Wang H, Zhu L, Zhang L (2003). Flavonoids: promising anticancer
agents. Med Res Rev 23: 519-534.
Thejan D, Tulasi PC, Madhusudhana RI, Gupta A, Reddy K (2011) A facile synthesis
of flavone derivatives used as potent anti inflammatory agents. Int J Pharm Pharm
Sci 3: 51-54.
Yakovenko VI, Oganesyan NT, Dorofeenko GN (1981) One-step synthesis of
4-ethoxy-6-bromo-7-hydroxyflavylium salts and the corresponding flavones.
Chem Heterocycl Comp 17: 115-118.
Jabbour M et al. (2016) Pharmacophore model mapping for some CK2 inhibitors
from flavonoid family. Arab J Pharm Sci 5(4): 47-65.
Pharmazie 73 (2018)
195