Discovery and characterization of synthetic 4′-hydroxyflavones - New CK2 inhibitors from flavone family

Article abstract of DOI:10.1016/j.bmc.2013.08.013

Human protein kinase CK2 is one of the most intriguing enzymes, which functional role still remains unclear despite of decades of studying. At present there is abundant evidence pointing to the fact that inhibitors of CK2 could be used as pharmaceutical a

Full text of DOI:10.1016/j.bmc.2013.08.013

Bioorganic & Medicinal Chemistry 21 (2013) 6681–6689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and characterization of synthetic 40-hydroxyflavones—New
CK2 inhibitors from flavone family
Andriy G. Golub b, Volodymyr G. Bdzhola a, Olga V. Ostrynska a, Iaroslav V. Kyshenia a,
Vladislav M. Sapelkin a, Andriy O. Prykhod’ko a, Olexander P. Kukharenko a, Sergiy M. Yarmoluk a,
⇑
a Department of Medicinal Chemistry, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotnogo str., 03680 Kyiv, Ukraine
b Otava Ltd, 65 Ellerslie Ave., Suite 560, Toronto, Ontario M2N 1Y1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Human protein kinase CK2 is one of the most intriguing enzymes, which functional role still remains
unclear despite of decades of studying. At present there is abundant evidence pointing to the fact that
inhibitors of CK2 could be used as pharmaceutical agents to treat cancer, viral infections and inflamma-
tory diseases. Here we report novel synthetic flavone inhibitors, 40-hydroxyflavones, possessing high
activity towards CK2. These compounds were identified with receptor-based virtual screening and then
chemically optimized on the base of rationale derived from biochemical screening and molecular mod-
eling. It has been demonstrated that synthetic flavone derivatives are much more potent CK2 inhibitors
than the natural ones, and we believe that their further examination will be helpful for studying biolog-
ical role of CK2 as well as for development of new kinase-oriented drugs.
Received 29 May 2013
Revised 1 August 2013
Accepted 5 August 2013
Available online 13 August 2013
Keywords:
Protein kinase CK2
40-Hydroxyflavone
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
Drug design
Docking
Molecular modeling
1. Introduction
process of apoptosis leading to tumor cell death.10,11 In some cases,
specific inductors of apoptosis could be used as supplementary
Signal transduction via protein phosphorylation/dephosphoryl-
ation is widely known mechanism which regulates most physio-
logical processes of living cell. This mechanism is served by
protein kinases—a numerous group of enzymes that catalyze phos-
phate transfer to physiological substrate(s). It has been proven that
kinase malfunction leads to various disorders, especially to
tumorogenesis.1 This, in particular, is well-defined for protein
kinase CK2 (‘casein kinase 2’)—extremely pleiotropic and constitu-
tively active Ser/Thr kinase that is involved in almost all cellular
events.2 There is a growing body of evidence that CK2 displays ele-
vated activity and expression in various types of tumors,3–5 pro-
motes development of viral infections by phosphorylation of viral
proteins,6 and involved in transduction of inflammatory signals.7
For a long time CK2 has been excluded from group of druggable ki-
nases due to its ‘undesirable’ features like constitutive activity and
ability to phosphorylate vast number (more than 300) of physio-
logical substrates. However, nowadays it is considered as a prom-
ising drug target.8 In case of tumorogenesis, CK2 enhances cancer
phenotype by blocking of apoptosis and simultaneous stimulation
of cell growth.9 It is currently accepted that therapeutic effect from
CK2 inhibition is primarily based on induction of the physiological
treatment.12–14 CK2 is essential for cell life span,15,16 therefore
‘switching off’ its activity will cause death of both tumor and nor-
mal cells. Fortunately, it was shown that lowering CK2 activity by
30–40% with small molecule inhibitors or anti-sense RNA was en-
ough for apoptosis induction in tumors.10,17
There is a number of CK2 inhibitors discovered and character-
ized over the past decades.18–20 In 2009, the first orally adminis-
tered highly selective and potent CK2 inhibitor, CX-4945, was
introduced in phase I clinical trials due to its broad spectrum anti-
proliferative activity in multiple cancer cell lines including inflam-
matory breast cancer.
Chemical class of flavones discussed in this paper has been al-
ready known for its ability to inhibit various protein kinases. These
inhibitors are natural, low-toxic compounds with broad biological
activity.21 It has been shown that flavones have antioxidant,22,23
antiinflammatory,24 antiviral,25 antimutagenic26,27 and cyto-
toxic28–31 activity. Among this class there are compounds with
inhibitory activity towards CK2.32 For example, 40,5,7-threehydr-
oxyflavone (apigenin), which is natural component of herbal cham-
omile as well as of many fruits and vegetables, displays high
inhibitory activity towards CK2 (IC50 = 0.8 lM). Another flavone,
3,30,40,5,7-pentahydroxyflavone (quercetin), differs from apigenin
by the presence of two hydroxyl groups in positions 3 and 30.
⇑
Corresponding author. Tel./fax: +380 44 522 2458.
E-mail address: sergiy@yarmoluk.org.ua (S.M. Yarmoluk).
Quercetin is known inhibitor of PI3-kinase (IC50 = 3.8 lM),
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.013

6682
A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
phospholipase A2 (IC50 = 2
l
M) and CK2 (IC50 = 0.55
l
M). Thus, the
40-Hydroxyl group, a common substituent for all the investi-
gated active compounds, is located in the back pocket nearby
two polar residues Lys68 and Asp175. Besides the formation of
hydrogen bond with side chain of Lys68, an additional bond is pre-
sumed to be formed between 40-hydroxyl group and the main
chain of Asp175 according to favorable location of corresponding
presence of two hydroxyl groups increases inhibitory activity of
quercetin in comparison to apigenin. However, addition of an extra
hydroxyl group in position 50 (myrecetin) decreases inhibitory
activity against CK2 (IC50 value for myrecetin is 0.92 lM). Flavones
chrysin and morin have 10 times lower IC50 values if compared to
apigenin and quercetin. This could be explained by the absence of a
hydroxyl group in 2-Ph ring (chrysin) and presence of 20-OH group
in phenyl ring (morin).
The most active CK2 inhibitor among flavones is fisetin with
IC50 = 0.35 lM. It should be noted that in experiments with mu-
tated CK2 (Val66Ala) IC50 values of fisetin and quercetin remained
the same, whereas IC50 values of chrysin, morin and apigenin in-
creased by several fold. Based on these data, authors31 suppose
that different flavones are oriented differently in the CK2 active
site due to their polyphenolic structure.
All the studied flavone inhibitors of CK2 are natural compounds,
but this article is focused on synthetic flavone derivatives. Initially,
they were selected from in-house compound collection (Otava Ltd)
via virtual screening, and then a number of additional derivatives
were synthesized during the structure optimization stage. For re-
ported inhibitors, the binding mode in CK2 active site has been
developed and the selectivity issues have been analyzed.
donor and acceptor (the distance between corresponding nitrogen
0
and oxygen atoms in FNH28–CK2 complex is 3.38 ÅA).
Proposed binding mode is in good agreement with the biologi-
cal activity data and it explains the influence of chemical substitu-
ents on the inhibitory effect. However, taking into consideration
that the formation of hydrogen bond with Lys68 is sterically possi-
ble both for 40-hydroxyl group and 30-hydroxyl group, it was still
ambiguous which one is more preferable to be taken as a part of
the core for further optimization. Also, the role of substituent next
to the hydroxyl group was unclear. To elucidate these issues, com-
pounds FNH50–FNH55 have been synthesized and tested in vitro
towards CK2 (Table 2).
As a result, it was determined that FNH53 containing 40-hydro-
xyl group is slightly more active than FNH52 containing 30-hydro-
xyl group (IC50 = 1600 nM and 2200 nM, respectively). At the same
time, the activity of FNH55 having both 30- and 40-hydroxyl groups
is much higher (IC50 = 450 nM). This could be explained by simul-
taneous formation of intermolecular hydrogen bonds both with
Lys68 and/or Asp175. Moreover, there is an evidence that two
adjacent hydroxyl groups increase acidity of each other. Therefore,
in this case the negatively charged cluster of two hydroxyl groups
causes an additional electrostatic attraction of positively charged
NH3 group of Lys68.
2. Results and discussion
Combinatorial library of 150,000 organic compounds (Otava
Ltd) has been used for receptor-based virtual screening towards
human CK2. Followed in vitro tests revealed that 19 flavones out
of 49 selected by virtual screening display ability to inhibit activity
The presence of methoxy group (in position 30) in immediate
vicinity to 40-hydroxyl group increases flavone activity by more
than an order of magnitude (IC50 for compounds FNH28 and
FNH53 is 100 nM and 1600 nM, respectively). However, presence
of more bulky substituents in position 30 decreases the activity dra-
matically (FNH12, IC50 = 800 nM; FNH13, IC50 = 7500 nM) which,
most likely, is the matter of steric hindrance. Interesting, the pres-
ence of 40-methoxygroup ‘deactivates’ flavone FNH50 completely
of CK2 (IC50 <30 lM, Table 1). The most active compound among
those listed in the table is 6,8-dimethyl-40-hydroxy-30-methoxyf-
lavone FNH28.
It should be emphasized that the identified flavone FNH28 ap-
peared to be 5 times more active than quercetin and 3 times
more active than the most active flavone inhibitor known so
far—fisetin. Moreover, the lack of polyphenolic structure (which
is typical for natural flavones) suggests that these inhibitors could
be selective towards CK2. Indeed, in vitro tests on two Ser/Thr ki-
nases Rho-associated protein kinase 1 (ROCK1) and Apoptosis sig-
(IC50 >30 lM).
Activity of the flavones is also considerably affected by substit-
uents of the ring A. According to the binding mode the presence of
methyl groups is probably the cause of additional hydrophobic
interactions with CR2 residues Leu45, Val53 and Asn118 and, in
such a way, increase the inhibitory activity (FNH28, IC50 = 100
nM; FNH12, IC50 = 800 nM). Thus, we decided to use 40-hydroxyf-
lavone core as a template for further structural optimization.
Two strategies have been applied for the optimization. The first
one is the introduction of hydrophobic substituents in the ring A
that, according to the model, is aimed at increasing binding affinity
in the region of ATP binding site formed by residues Leu45, Val53
and Met163. The second one—variation of substituents in the ring
B, where the introduction of electron withdrawing group(s) should
increase acidic properties of 40-hydroxyl group, and, consequently,
should increase the energy of hydrogen bond with the Lys68.
Data on the newly synthesized compounds FNH56–FNH83 rep-
resented in Table 2 display a number of substantially more active
flavone derivatives identified. There is an incredibly high hit
rate—26 out of 28 new flavones have submicromolar activity
towards CK2.
nal-regulating kinase
1
(ASK1) showed that flavone FNH28
M. Thus,
revealed negligible inhibition of their activity at 10
these results motivated us to perform further investigation of this
class of compounds.
l
According to the data represented in Table 1, compounds con-
taining hydroxyl group in 40-position of the phenyl (FNH12,
FNH13, FNH16 and FNH28) demonstrate the highest inhibitory
activity. In order to inspect their binding mode and to figure out
the structure–activity relationships, we analyzed CK2-ligand com-
plexes generated by molecular docking. It turned out that all active
compounds have quite similar binding mode. We distinguished 7
amino acid residues (Leu45, Val53, Val66, Ile 95, Phe113, Met163
and Ile174) that contribute the most to the hydrophobic interac-
tions with the inhibitors. Interaction of the phenyl ring B of ligand’s
heterocyclic core with Phe113, Ile95 and Ile174 is of special impor-
tance, at that the contact with Phe113 is a stacking-like interaction
which apparently plays significant role in inhibitor fixation in the
ATP-binding site (Fig. 1).
As for other key contacts, there are 2 intermolecular hydrogen
bonds involving hinge region (Val116) and back pocket (Lys68)
of the CK20s active site. Obtained binding mode correlates well
with recent X-ray data33 obtained for natural 40-hydroxyflavone
Kinetic studies of the two most active derivatives, FNH68 and
FNH79, showed competitive mode of inhibitory action (Fig. 2).
The corresponding Ki values are 3.5 nM and 1.8 nM.
Analysis of structure–activity relationships of these compounds
resulted in the following findings. Increasing of the hydrophobic
potential of substitiuent in position 6 of the flavones leads to
increasing of the inhibitory activity. This is observed in the range
of Me < Cl 6 MeO < NAc < Et 6 Br, IC50 values for compounds
derivatives and Z. maize CK2a. Therefore, flavones show good
chemical complementarity to ATP-binding site of CK2 and their
structural optimization could generate series of more active
compounds.

A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
6683
Table 1
Structure and activity of flavone derivatives
R5
R8
O
O
R6
R7
R3
R6'
R3'
R5'
R4'
R2'
R20
R30
R40
R50
R60
ID
R3
R5
R6
R7
R8
Residual activity of CK2 (%) IC50 (lM)
FNH1
FNH2
FNH3
FNH4
FNH5
FNH6
FNH7
FNH8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
Br
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
OMe
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OAc
H
H
H
OH
H
H
OH
OH
OH
H
H
H
OH
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
OMe
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
68
73
49
89
65
92
58
73
86
66
77
5
21
80
81
18
38
60
56
39
45
50
47
57
64
65
>30
>30
30
Me
OH
OAc
Me
Me
H
H
H
H
H
>30
>30
>30
>30
>30
>30
>30
>30
0.8
7.5
>30
>30
8
OMe OMe
H
H
H
NMe2
NMe2
OMe
FNH9
FNH10
FNH11
FNH12
FNH13
FNH14
FNH15
FNH16
FNH17
FNH18
FNH19
FNH20
OMe OMe
H
OMe OH
OEt
H
OMe OMe
OEt
H
H
H
H
H
H
OMe
H
OMe OMe
OMe OMe
H
OMe OH
OMe OMe
F
H
H
OH
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
OH
OH
H
OH
Cl
F
Me
i-Pr
H
OMe
H
23
>30
>30
26
28
28
FNH21 OH
FNH22
FNH23
FNH24
FNH25
FNH26
FNH27
FNH28
FNH29
FNH30
FNH31
FNH32
FNH33
H
H
H
H
H
H
H
H
H
H
H
H
25
OEt
>30
>30
>30
21
0.1
>30
19
26
>30
>30
Me
Me 35
Me
Me 87
H
H
H
H
2
Br
H
29
45
66
95
OMe OMe
OMe OMe
H
H
H
H
H
OCH2CO
Morfoline
OCH2CO2
H
OCH2CO2
H
Me
FNH34
FNH35
H
H
Me
OMe
H
H
H
H
H
H
Me
Me
H
H
H
H
83
77
>30
>30
FNH36
FNH37
H
H
H
H
Me
Me
H
H
H
H
H
H
H
OH
H
H
H
H
H
35
97
19
>30
OCH2CO
Morfoline
OCH2CO
Morfoline
OCH2CO2
H
FNH38 Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
97
>30
28
FNH39
H
Me
Me
Me 50
FNH40
FNH41
FNH42
H
H
H
H
H
H
OMe
OMe
OMe
H
H
H
H
H
H
OSO2Me
Me
H
OH
H
Me
H
Me 46
H
H
28
21
>30
OCO(1,4-Benzodioxane-2-yl) Me
H
37
73
CH2NMe
Benzyl
FNH43
FNH44
FNH45
H
H
H
H
OMe
H
OMe
OMe
H
H
H
H
H
H
CH2(4-Me-1-Pyperidyl)
CH2(4-Et-1-Pyperazinyl)
CH2NMe
OH
OH
OH
H
H
H
H
H
H
68
77
73
>30
>30
>30
OMe OMe
OMe OMe
Benzyl
FNH46
FNH47
FNH48
FNH49
H
H
H
H
OMe OMe
OMe
H
H
H
H
H
H
H
H
H
H
CH2(1-Pyperidyl)
CH2Morfoline
CH2N(2-furfuryl)CH2O
CH2N(3-
OH
OH
H
H
H
H
H
H
H
H
74
79
17
18
>30
>30
12
H
H
H
OMe
OMe
OMe
H
15
trifluoromethylphenyl)CH2O
FNH57, FNH59, FNH58, FNH61, FNH60 and FNH62 are 320 nM,
240 nM, 200 nM, 150 nM, 120 nM and 120 nM, respectively.
Also, introduction of additional hydrophobic substituent in
position 8 positively affects the activity (IC50 value of compounds
FNH57 and FNH28 is 320 nM and 100 nM, respectively). This effect
can also be seen when compare compounds FNH59 (IC50 = 240
nM), FNH76 (IC50 = 40 nM) and FNH68 (IC50 = 10 nM) where the
presence of chlorine atom is more favorable than methyl group.

6684
A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
O
If compare the influence of substituents in positions 6 and 7, it
Val53
Val66
can be seen that 7-methoxy substituted flavone FNH83 displays
noticeable increase in activity (FNH83, IC50 = 175 nM; FNH58,
IC50 = 200 nM). The similar situation was detected for 6- and
7-methyl derivatives (FNH63, IC50 = 90 nM; FNH57, IC50 = 320
nM). Accordingly, activity of 5,7-dimethyl substituted flavone
FNH71 (IC50 = 30 nM) is higher than activity of 6,8-dimethyl
substituted flavone FNH28 (IC50 = 100 nM).
O
Leu45
A
N
Phe113
N
O
C
Lys68
N
B
O
O
Val116
The activity of 6,7 disubstituted flavones is unexpectedly low if
compare to 5,7 and 6,8 disubstituted ones. Similarly, 5,6,7-three-
N
Ile95
Met163
Asp175
O
N
substituted compound FNH81 is also showed low activity (IC50
=
O
Ile174
2000 nM).
As to position 30 of the ring B, it was revealed that the activity
increases in the range MeO < Br < NO2 (IC50 for compounds
FNH28, FNH74 and FNH70 is 100, 50 and 12 nM, respectively).
The indicated substituents of the ring B considerably affect acidity
of hydroxyl group of the phenol taking into account calculated pKa
values for compounds FNH28, FNH74 and FNH70 (9.98, 8.44 and
6.77, respectively). This correlates well with the observation that
the activity of the flavones increases along with increasing of acid-
ity of 40-hydroxyl group. Finally, introduction of two substituents
in 30 and 50 positions of 40-hydroxyflavone slightly decreases the
inhibitory activity.
O
O
Figure 1. Schematic representation of binding mode of the active flavone deriv-
atives in CK2 active site. Data obtained with molecular docking. Intermolecular
hydrogen bonds (dashed lines), supposed hydrogen bond with main chain of
Asp175 (wavy line) and stacking interaction with Phe113 (arrows) are indicated.
The strict dependence of the activity on hydrophobicity of substit-
uents in positions 6 and 8 is observed for compounds FNH28,
FNH68 an FNH79 (IC50 is 100 nM, 10 nM and 4 nM, respectively).
The bromine atoms presented here cause an additional hydropho-
bic potential which increase the binding affinity.
Table 2
Structure of newly synthesized flavone derivatives and data on their inhibitory activity towards CK2
R5
R8
O
R6
R7
R3
R6'
R3'
R5'
R4'
O
R2'
R20
R30
R40
R50
R60
ID
R3
R5
R6
R7
R8
IC50 (lM)
FNH50
FNH51
FNH52
FNH53
FNH54
FNH55
FNH56
FNH57
FNH58
FNH59
FNH60
FNH61
FNH62
FNH63
FNH64
FNH65
FNH66
FNH67
FNH68
FNH69
FNH70
FNH71
FNH72
FNH73
FNH74
FNH75
FNH76
FNH77
FNH78
FNH79
FNH80
FNH81
FNH82
FNH83
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OMe
OH
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
H
H
H
Cl
H
H
NO2
Cl
H
Br
H
H
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
H
Me
Me
Me
Me
Me
Me
OH
Me
OMe
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
Me
Me
Me
Me
H
Me
H
H
H
H
H
H
H
H
Me
Me
H
H
Cl
Cl
Me
H
>30
>30
2.2
1.6
8
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
0.45
0.25
0.32
0.20
0.24
0.12
0.15
0.12
0.09
0.09
0.65
0.79
3
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NO2
OMe
OMe
Cl
Et
NAc
Br
H
Me
Me
Cl
Me
Cl
Cl
Me
H
Me
Me
Me
Me
Cl
H
Br
Br
H
Cl
H
OMe
H
Me
Me
H
H
H
Me
H
H
H
H
0.01
0.02
0.012
0.03
0.066
0.078
0.05
0.18
0.04
0.028
0.018
0.004
0.087
2
Me
Me
Me
Me
Me
Br
Br
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
C4H4
H
Br
Br
H
C4H4
Me
Me
H
H
Me
H
0.3
0.175

A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
6685
7000
6000
5000
10000
8000
6000
4000
3000
2000
1000
0
4000
Ki = 3,5 nM
Ki = 1,8 nM
[I] = 20 nM
1000
2000
[I] = 40 nM
700
600
500
400
300
200
100
0
0
-5
0
5
10
15
20
25
-15 - 10 -5
-1000
0
5
10 15 20 25 30 35 40 4 5
[I], nM
[I], nM
-2000
800
600
400
200
0
[I] = 10 nM
[I] = 5 nM
[I] = 2,5 nM
[I] = 0 nM
[I] = 20 nM
[I] = 5 nM
[I] = 0 nM
-0,04 -0,02 0,00 0,02 0,04 0,06 0,08 0,10
-0,04 -0,02 0,00 0,02 0,04 0,06 0,08 0,10
-100
1/[ATP], μΜ−1
1/[ATP], μΜ−1
-200
-200
a
b
Figure 2. Lineweaver–Burk plots of CK2 inhibition by FNH68 (a) and FNH79 (b). Ki values are 3.5 nM and 1.8 nM, respectively. Concentration of inhibitors varied from 0 to
40 nM. Enzyme activities were tested as described in Section 4.
Complexes of FNH68 and FNH79 with CK2 obtained by molec-
ular docking revealed strong binding of newly synthesized flavones
caused by interactions with hydrophobic residues of the ATP-
acceptor site and formation of intermolecular hydrogen bonding
network (Fig. 3). One hydrogen bond forms between hydroxyl
and/or metoxy group of the inhibitor and positively charged resi-
due Lys68. Another one forms between inhibitor’s carboxyl group
and main chain nitrogen atom of hinge region residue Val116. It
should be mentioned that observable intermolecular contacts are
the same as described for above mentioned compounds FNH1–
FNH49 including stacking interaction with Phe113. As earlier, tak-
ing into account dynamics of the active site residues, we presume
formation of hydrogen bond(s) between carboxyl or methoxy
group of the inhibitor and Asp175.
also a number of crystal structures proving it.38,39 One can suppose
the presence of intermolecular halogen bonds there,40 however,
accordingly to modeling data they have not been identified.41
The higher activity of bromine-substituted derivative could be
explained by increased hydrophobicity of FNH79 (cLogP = 4.58)
if compare to FNH68 (cLogP = 4.42), so the binding enhancement
arises from higher hydrophobic contribution in the contact surface
area between the inhibitor and CK2. Also, it can be presumed that
FNH79 adjusts the steric volume available for inhibitor binding
more effectively than chlorine-substituted flavone FNH68.
Initial selectivity tests of FNH76, FNH78, FNH79 and FNH68
using four serine/threonine (ASK1, c-Jun N-terminal kinase 3
(JNK3), serine/threonine-protein kinase 6 (Aurora A) and ROCK1)
and three tyrosine protein kinases (Fibroblast growth factor recep-
tor 1 (FGFR1), Met receptor tyrosine kinase (Met) and TEK tyrosine
kinase, endothelial (Tie2)) as targets have revealed negligible
Position and role of the halogen atoms is a point at issue.34 We
have described similar binding mode previously,35–37 and there is
Val53
Leu45
Val53
Leu45
Lys68
Lys68
His115
Val66
His115
Val66
Phe113
Phe113
Val116
Val116
Asp175
Asp175
Ile95
Ile95
Asn118
Asn118
Met163
Met163
Ile174
Ile174
a
b
Figure 3. Binding mode of flavones FNH68 (a) and FNH79 (b) obtained with molecular docking. Intermolecular hydrogen bonds are indicated with dotted lines.

6686
A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
Table 3
Specificity spectrum of FNH76, FNH78, FNH79 and FNH68*
ID
Residual activity Residual activity Residual activity
Residual
activity of Met
(%)
Residual activity of Residual
Residual
activity of Tie2
(%)
Residual activity
of ROCK1 (%)
of ASK1 (%)
of Jnk3 (%)
of FGFR1 (%)
Aurora A (%)
activity of CK2
(%)
FNH76 116
FNH78 124
FNH79 126
FNH68 126
102
103
103
99
81
54
74
74
108
54
87
57
19
55
54
2.3
1.3
0.8
1.1
112
36
85
113
121
89
126
93
120
*
Kinase residual activity, determined in the presence of 10
experiment was 100 M.
lM of inhibitor is expressed as a percentage of the control without inhibitor. Final concentration of ATP in the
l
inhibitory activity of FNH76, FNH79 and FNH68 at concentration
10 M (see Table 3) and considerable cross reactivity for FNH78,
which inhibits Aurora A and Tie 2 kinases by 81% and 64%, respec-
tively. Although the number of kinases used in the assay is limited,
these tests, however, allowed us to suggest at least some level of
selectivity for these flavone inhibitors towards CK2.
chosen. DOCK employs an incremental reconstruction algorithm
where rigid anchor fragments are identified first. At the next step,
the selected fragment is placed into the active site of the receptor
using a sphere-matching procedure. The complete ligand is con-
structed by adding the remaining components step by step. At each
step of reconstruction a specified number of optimal partial solu-
tions are selected for the next extension step. Solutions are scored
using energy score (sum of van der Waals and electrostatic
components). The docking scores have been obtained in the range
from –14 to –51 kcal/mol. 3500 compounds with scores less than –
42 kcal/mol have been chosen and have been inspected for steric
clashes and unfavourable contacts. Finally, 800 compounds from
different chemical classes were considered as promising (including
49 40-Hydroxyflavone derivatives) and tested in vitro.
l
3. Conclusions
Here we report design, synthesis and characterization of novel
CK2 inhibitors—40-hydroxyflavones, which possess much more
higher activity towards CK2 than their natural derivatives. IC50
value of the 40-hydroxyflavone FNH79 is nearly 100 times higher
than IC50 of fisetin, the most active natural flavone known to inhi-
bit CK2. Taking into account that developed 40-hydroxyflavones are
not polyphenolic in contrast to their natural derivatives, we sug-
gest that they bind active sites of different kinases in similar way
which is important for structure optimization and specificity
reasons.
4.2. Biological testing
Selected compounds were tested using in vitro kinase assay.
Each test was performed twice in a total reaction volume of 30
l
L, containing 6
land Biolabs); 10 units of recombinant human CK2 holoenzyme
(New England Biolabs); 50 M ATP and
-labeled 32P ATP, diluted
to specific activity 100 Ci/ M; CK2 buffer (20 mM Tris–HCl, pH
lg of peptide substrate RRRDDDSDDD (New Eng-
4. Experimental section
4.1. Virtual screening
l
c
l
l
7.5; 50 mM KCl; 10 mM MgCl2) and inhibitor in varying concentra-
tions. Incubation time was 20 min at 30 °C. 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 phosphocel-
lulose paper (Whatman). Disks were washed three times with 1%
o-phosphoric acid solution, air-dried at room temperature, and
counted by the Cherenkov method in a beta-counter (LKB). As neg-
ative control an equal volume of DMSO (dimethyl sulfoxide) was
added to the reaction mixture. Quercetin, a known CK2 inhibitor,46
was used as inhibition positive control. The final concentration of
For high-throughput virtual screening we used compounds
from commercially available library (Otava Ltd) that comprises
ꢀ250,000 compounds from different chemical classes. Initially,
all compounds were filtered by Lipinski’s rule of 5; compounds
with reactive groups and biologically unstable compounds were
removed. These calculations resulted in ꢀ150,000 drug-like com-
pounds that were further taken for virtual screening.
The crystal structure of human protein kinase CK2 was obtained
from the Brookhaven Protein Data Bank (PDB ID: 1JWH). The
receptor molecule has been minimized in water with GROMACS
molecular dynamics simulation package (GROMACS force field,
steepest descent algorithm, 1000 steps, em_tolerance = 100, em_-
step = 0.001). Active site spheres were calculated with DOCK sph-
gen software. 33 spheres from the biggest cluster of 40 spheres
were selected to fill receptor active site. Seven spheres were de-
leted manually since their positions were outside of the active site
cavity. Connolly MS (http://www.net-sci.org/Science/Compchem/
featurel4.html) and Grid programs from DOCK package were used
to generate receptor Connolly surface and energy grids. Surface
and grid calculations were performed with parameter settings as
in,42 except for grid spacing that was set to 0.3.
quercetin was 0.55 lM (which inhibited CK2 activity by 50%). Per-
cent inhibition was calculated as ratio of substrate-incorporated
radioactivity in the presence of inhibitor to the radioactivity incor-
porated in control reactions, that is, in the absence of inhibitor. Se-
rial dilutions of inhibitor stock solution were used to determine its
IC50 concentration. The IC50 values represent means of triplicate
experiments with SEM never exceeding 15%.
Selectivity tests were performed on protein kinases ASK1, JNK3,
Met, Aurora A, Rock 1, FGFR1 and Tie2 according to the supplier’s
recomendations (Millipore).
4.3. Chemistry
Calculations of ligand geometry were performed using YFF force
field developed by the authors.43,44 Partial atomic charges of the
ligands were calculated with Kirchhoff method.45
For receptor–ligand flexible docking the DOCK program has
been used. DOCK input parameters were set as in42 with some
exceptions to increase the calculations accuracy: the minimum of
heavy atoms in the anchor was set to 6, the maximum number
of orientations was set to 1000, and the ‘all atoms’ model has been
Compounds FNH50–FNH83 were synthesized by two-step pro-
cedure47,48 (Scheme 1). Reaction of 20-hydroxy acetophenones and
appropriate benzaldehyde derivatives in triethylortoformate pro-
duces flavilium salts, which are readily hydrolysed to flavones. Suf-
ficient yield of compounds was obtained. Purity of all the
synthesized compounds was at least 95% which has been con-
firmed by HPLC and elemental analysis.

A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
6687
4.3.1. 2-(3-Hydroxy-4-methoxyphenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH50)
7.599 (d, 1H, J1 = 9.3 Hz), 9.569 (s, 1H) Anal. (C17H14O5)C,H. Anal.
Calcd for C17H14O5: C, 68.45, H, 4.73. Found: C, 68.37, H, 4.71.
Mp 203–205 °C. 1H NMR (DMSO-d6) d 2.416 (s, 3H), 2.659 (s,
3H), 3.888 (s, 3H), 6.644 (s, 1H), 7.007 (d, 1H, J = 6,3 Hz), 7.399
(m, 2H), 7.449 (dd, 1H, J1 = 8.3 Hz, J2 = 2.2 Hz), 7.628 (s, 1H),
9.211 (s, 1H) Anal. (C18H16O4)C,H. Anal. Calcd for C18H16O4: C,
72.96, H, 5.44. Found: C, 73.10, H, 5.36.
4.3.10. 6-Chloro-2-(4-hydroxy-3-methoxy-phenyl)-4H-
chromen-4-one (FNH59)
Mp 247–249 °C. 1H NMR (DMSO-d6) d 2.420 (s, 3H), 2.571 (s,
3H), 3.968 (s, 3H), 6.900 (s, 1H), 7.388 (s, 1H), 7.487 (s, 1H),
7.570 (s, 1H), 7.634 (s, 1H), 9.974 (s, 1H) Anal. (C16H11ClO4)C,H,Cl.
Anal. Calcd for C16H11ClO4: C, 63.48, H, 3.66, Cl, 11.71. Found: C,
63.31, H, 3.60, Cl, 11.82.
4.3.2. 2-(2-Hydroxy-3-methoxyphenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH51)
Mp 211–213 °C. 1H NMR (DMSO-d6) d 2,414 (s, 3H), 2.567 (s,
3H), 3.890 (s, 3H), 6.633 (s, 1H), 6.999 (d, 1H, J = 8,5 Hz), 7.371 (s,
1H), 7.406 (d, 1H, J2 = 2.2 Hz), 7,440(dd, 1H, J1 = 8.3 Hz,
J2 = 2.2 Hz), 7.629 (s, 1H), 9.183 (br s, 1H) Anal. (C18H16O4)C,H.
Anal. Calcd for C18H16O4: C, 72.96, H, 5.44. Found: C, 73.08, H, 5.37.
4.3.11. 6-Ethyl-2-(4-hydroxy-3-methoxy-phenyl)-4H-chromen-
4-one (FNH60)
Mp 79–81 °C. 1H NMR (DMSO-d6) d 1.299 (t, 3H, J1 = 7.3 Hz),
2.767 (q, 2H, J1 = 7.3 Hz), 3.926 (s, 3H), 6.797 (s, 1H), 6.896 (m,
1H), 7.490 (m, 2H), 7.556 (m, 2H), 7.824 (s, 1H), 9.561 (s, 1H) Anal.
(C18H16O4)C,H. Anal. Calcd for C18H16O4: C, 72.96, H, 5.44. Found: C,
73.16, H, 5.36.
4.3.3. 2-(3-Hydroxyphenyl)-6,8-dimethyl-4H-chromen-4-one
(FNH52)
Mp 255–258 °C. 1H NMR (DMSO-d6) d 2.423 (s, 3H), 2.575 (s,
3H), 6.714 (s, 1H), 6.936 (m, 1H), 7.303 (m, 1H), 7.388 (m, 3H),
7.651 (s, 1H), 9.6 02 (br s, 1H) Anal. (C17H14O3)C,H. Anal. Calcd
for C17H14O3: C, 76.68, H, 5.30. Found: C, 76.82, H, 5.21.
4.3.12. N-[2-(4-Hydroxy-3-methoxyphenyl)-4-oxo-4H-
chromen-6-yl]acetamide (FNH61)
Mp 263–265 °C. 1H NMR (DMSO-d6) d 2.081 (s, 3H), 3.921(s,
3H), 6.795 (s, 1H), 6.895 (d, 1H, J1 = 9.3 Hz), 7.498 (m, 2H), 7.574
(d, 1H, J1 = 9.3 Hz), 8.021 (d, 1H, J1 = 8.3 Hz), 8.161 (d, 1H,
J2 = 2.0 Hz), 9.588 (s, 1H), 10.099 (s, 1H) Anal. (C18H15NO5)C,H.
Anal. Calcd for C18H15NO5: C, 66.46, H, 4.65, N, 4.31. Found: C,
66.32, H, 4.69, N, 4.4.
4.3.4. 2-(4-Hydroxyphenyl)-6,8-dimethyl-4H-chromen-4-one
(FNH53)
Mp 260–263 °C (decomp.). 1H NMR (DMSO-d6) d 2.415 (s, 3H),
2.558 (s, 3H), 6.664 (s, 1H), 6.894 (d, 1H, J1 = 8.5 Hz), 7.366 (s,
1H), 7.630 (s, 1H), 7.826 d,1H, J1 = 8.5 Hz), 9.981 (br s, 1H) Anal.
(C17H14O3)C,H. Anal. Calcd for C17H14O3: C, 76.68, H, 5.30. Found:
C, 76.55, H, 5.36.
4.3.13. 6-Bromo-2-(4-hydroxy-3-methoxy-phenyl)-4H-
chromen-4-one (FNH62)
Mp 233–235 °C. 1H NMR (DMSO-d6) d 3.922 (s, 3H), 6.897 (s,
1H), 6.898 (d, 1H, J1 = 8.8 Hz), 7.517 (m, 2H), 7.668 (d, 1H,
J1 = 8.8 Hz), 7.853 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.104 (d, 1H,
J2 = 2.4 Hz), 9.650 (s, 1H) Anal. (C16H11BrO4)C,H,Br. Anal. Calcd for
4.3.5. 2-(4-Hydroxyphenyl)-6-methyl-4H-chromen-4-one
(FNH54)
Mp 243–246 °C. 1H NMR (DMSO-d6) d 2.460 (s, 3H), 6.687 (s,
1H), 6.890 (d, 2H, J1 = 8.8 Hz), 7.523 (m, 2H), 7.802 (s, 1H), 7.843
(d, 2H, J1 = 8.8 Hz), 10.035 (s, 1H) Anal. (C16H12O3)C,H. Anal. Calcd
for C16H12O3: C, 76.18, H, 4.79. Found: C, 76.30, H, 4.72.
C16H11BrO4: C, 55.36, H, 3.19, Br, 23.02. Found: C, 55.41, H, 3.26,
Br, 22.90.
4.3.14. 2-(4-Hydroxy-3-methoxy-phenyl)-7-methyl-4H-
chromen-4-one (FNH63)
4.3.6. 2-(3,4-Dihydroxyphenyl)-6,8-dimethyl-4H-chromen-4-
one (FNH55)
Mp 231–234 °C. 1H NMR (DMSO-d6) d 3.922 (s, 3H), 6.898 (d,
1H, J1 = 8.8 Hz), 6.901 (s, 1H), 7.517 (m, 2H), 7.670 (d, 1H,
J1 = 8.8 Hz), 7.854 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.103 (d, 1H,
J2 = 2.4 Hz), 9.664 (s, 1H) Anal. (C17H14O4)C,H. Anal. Calcd for
Mp 258–270 °C. 1H NMR (DMSO-d6) d 2.414 (s, 3H), 2.567 (s,
3H), 6.564 (s, 1H), 6.857 (dd, 1H, J1 = 8.3 Hz, J2 = 1 Hz), 7.319 (dd,
1H, J1 = 8.3 Hz, J2 = 2.2 Hz), 7.371 (m, 2H), 7.623 (s, 1H), 9.138 (s,
1H), 9.494 (s, 1H) Anal. (C17H14O4)C,H. Anal. Calcd for C17H14O4:
C, 72.33, H, 5.00. Found: C, 72.48, H, 4.87.
C17H14O4: C, 72.33, H, 5.00. Found: C, 72.19, H, 4.93.
4.3.15. 2-(3-Chloro-4-hydroxy-5-methoxyphenyl)-6,8-dimethyl-
4H-chromen-4-one (FNH64)
4.3.7. 6-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-
chromen-4-one (FNH56)
Mp 256–259 °C. 1H NMR (DMSO-d6) d 2.420 (s, 3H), 2.571 (s,
3H), 3.968 (s, 3H), 6.900 (s, 1H), 7.388 (s, 1H), 7.489 (d, 1H,
J2 = 1,7 Hz), 6.837 (s, 1H), 7.636 (s, 1H), 9.974 (s, 1H) Anal. (C18H15-
ClO4)C,H,Cl. Anal. Calcd for C18H15ClO4: C, 65.36, H, 4.57, Cl, 10.72.
Found: C, 65.48, H, 4.61, Cl, 10.62.
Mp 142–145 °C. (decomp.) 1H NMR (DMSO-d6) d 6.728 (s, 1H),
6.886 (m, 1H), 7.149(dd, 1H, J1 = 9.3 Hz, J2 = 3.2 Hz), 7.291 (d, 1H,
J = 3.2 Hz), 7.473 (m, 3H), 9.527 (s, 1H), 9.674 (s, 1H) Anal. (C16H12-
O5)C,H. Anal. Calcd for C16H12O5: C, 67.60, H, 4.25. Found: C, 67.72,
H, 4.18.
4.3.16. 2-(3-Bromo-4-hydroxy-5-methoxyphenyl)-6,8-dimethyl-
4H-chromen-4-one (FNH65)
4.3.8. 2-(4-Hydroxy-3-methoxyphenyl)-6-methyl-4H-chromen-
4-one (FNH57)
Mp 269–271 °C. 1H NMR (DMSO-d6) d 2.422 (s, 3H), 2.573 (s,
3H), 3.969 (s, 3H), 6.899 (s, 1H), 7.393 (m, 1H), 7.527 (m. 1H),
7.639 (m, 1H), 7.715 (m, 1H), 10.037 (s, 1H) Anal. (C18H15BrO4)-
C,H,Br. Anal. Calcd for C18H15BrO4: C, 57.62, H, 4.03, Br, 21.30.
Found: C, 57.75, H, 4.10, Br, 21.18.
Mp 192–194 °C. 1H NMR (DMSO-d6) d 2.467 (s, 3H), 3.921 (s,
3H), 6.800 (s, 1H), 6.893 (m, 1H), 7.495 (m, 1H), 7.545 (m, 2H),
7.806 (s, 1H), 9.589 (s, 1H) Anal. (C17H14O4)C,H. Anal. Calcd for
C17H14O4: C, 72.33, H, 5.00. Found: C, 72.50, H, 4.95.
4.3.9. 2-(4-Hydroxy-3-methoxyphenyl)-6-methoxy-4H-
chromen-4-one (FNH58)
4.3.17. 6-Chloro-2-(4-hydroxy-3-methoxyphenyl)-7-methyl-4H-
chromen-4-one (FNH66)
Mp 208–210 °C. 1H NMR (DMSO-d6) d 3.894 (s, 3H), 3.924 (s,
3H), 6.802 (s, 1H), 6.893 (d, 2H, J1 = 8.8 Hz), 7.297 (dd, 1H,
J1 = 9.3 Hz, J2 = 2.9 Hz), 7.409 (d, 1H, J2 = 3.2 Hz), 7.489 (m, 2H),
Mp 273–275 °C. 1H NMR (DMSO-d6) d 2.513 (s, 3H), 3.920
(s, 3H), 6.837 (s, 1H), 6.,893 (d, 1H, J1 = 9.0 Hz), 7.486 (m, 2H),
7.676 (s, 1H), 7.928 (s, 1H), 9.632 (s, 1H) Anal. (C17H13ClO4)C,H,Cl.

6688
A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
O
O
O
O
b
a
R1
R1
R1
R2
R2
O+
O
OH
O
Cl
O
OH
OH
R3
FNH50 - FNH83
R3
O
Scheme 1. Synthesis of 40-hydroxyflavones. Reagents and conditions: (a) CH(OEt)3, 4-OH Benzaldehyde derivative, HClO4; (b) i-PrOH, H2O, reflux.
Anal. Calcd for C17H13ClO4: C, 64.47, H, 4.14, Cl, 11.19. Found: C,
64.59, H, 4.19, Cl, 11.06.
4.3.24. 2-(3,5-Dichloro-4-hydroxyphenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH73)
1H NMR (DMSO-d6) d 2.414 (s, 3H), 2.557 (s, 3H), 6.865 (s, 1H),
7.406 (s, 1H), 7.630 (s, 1H), 7.934 (s, 1H), 10.773 (br s, 1H) Anal.
(C17H12Cl2O3)C,H,Cl. Anal. Calcd for C17H12Cl2O3: C, 60.92, H, 3.61,
Cl, 21.15. Found: C, 61.03, H, 3.65, Cl, 21.01
4.3.18. 2-(4-Hydroxy-3-methoxyphenyl)-6,7-dimethyl-4H-
chromen-4-one (FNH67)
Mp 223–226 °C. 1H NMR (DMSO-d6) d 2.366 (s, 3H), 2.405 (s,
3H), 3.921 (s, 3H), 6.734 (s, 1H), 6.886 (d, 1H, J1 = 8.3 Hz), 7.420
(s, 1H), 7.459 (m, 2H), 7.740 (s, 1H), 9.533 (s, 1H) Anal. (C18H16O4)-
C,H. Anal. Calcd for C18H16O4: C, 72.96, H, 5.44. Found: C, 72.85, H,
5.48.
4.3.25. 2-(3-Bromo-4-hydroxy-phenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH74)
Mp 284–289 °C. 1H NMR (DMSO-d6) d 2.417 (s, 3H), 2.561 (s,
3H), 6.729 (s, 1H), 7.078 (d, 1H, J1 = 8.8 Hz), 7.382 (s, 1H), 7.631
(s, 1H), 7.814 (dd,1H, J1 = 8.3 Hz, J2 = 2.0 Hz), 8.067 (d, 1H,
J2 = 2,0 Hz) Anal. (C17H13BrO3)C,H,Br. Anal. Calcd for C17H13BrO3:
C, 59.15, H, 3.80, Br, 23.15. Found: C, 59.25, H, 3.77, Br, 23.07.
4.3.19. 6,8-Dichloro-2-(4-hydroxy-3-methoxyphenyl)-4H-
chromen-4-one (FNH68)
Mp 213–215 °C. 1H NMR (DMSO-d6) d 3.914 (s, 3H), 6.919
(d, 1H, J1 = 8.3 Hz), 7.003 (s, 1H), 7.566 (m, 2H), 7.907 (m,
2H), 9.741 (s, 1H) Anal. (C16H10Cl2O4)C,H,Cl. Anal. Calcd for C16-
H10Cl2O4: C, 57.00, H, 2.99, Cl, 21.03. Found: C, 56.82, H, 2.90,
Cl, 21.14.
4.3.26. 2-(3,5-Dibromo-4-hydroxyphenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH75)
Mp >300 °C (decomp.). 1H NMR (DMSO-d6) d 2.415 (s, 3H),
2.556 (s, 3H), 6.826 (s, 1H), 7.393 (s, 1H), 7.633 (s, 1H), 8.098 (s,
1H), 10.535 (br s, 1H) Anal. (C17H12Br2O3)C,H,Br. Anal. Calcd for C17-
H12Br2O3: C, 48.15, H, 2.85, Br, 37.68. Found: C, 48.27, H, 2.93, Br,
37.51.
4.3.20. 6,8-Dichloro-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-
4H-chromen-4-one (FNH69)
Mp 273–275 °C. 1H NMR (DMSO-d6) d 3.966(s, 3H), 7.147 (s,
1H), 7.561 (s, 1H), 7.672(d, 1H, J2 = 1,7 Hz), 7.929 (dd, 2H,
J1 = 11.3 Hz, J2 = 2.4 Hz), 10.176 (s, 1H) Anal. (C16H9Cl3O4)C,H,Cl.
Anal. Calcd for C16H9Cl3O4: C, 51.72, H, 2.44, Cl, 28.62. Found: C,
51.85, H, 2.49, Cl, 28.50.
4.3.27. 6-Chloro-2-(4-hydroxy-3-methoxy-phenyl)-8-methyl-
4H-chromen-4-one (FNH76)
Mp 231–233 °C. 1H NMR (DMSO-d6) d 2.586 (s, 3H), 3.907 (s,
3H), 6.903 (m, 2H), 7.497 (m, 2H), 7.599 (s, 1H), 7.766 (s, 1H),
9.697 (s, 1H) Anal. (C17H13ClO4)C,H,Cl. Anal. Calcd for C17H13ClO4:
C, 64.47, H, 4.14, Cl, 11.19. Found: C, 64.36, H, 4.08, Cl, 11.31.
4.3.21. 2-(4-Hydroxy-3-nitrophenyl)-6,8-dimethyl-4H-
chromen-4-one (FNH70)
Mp 274–279 °C. (decomp.) 1H NMR (DMSO-d6) d 2.419 (s, 3H),
2.567 (s, 3H), 6.876 (s, 1H), 7.275 (d, 1H, J1 = 9 Hz), 7.401 (s, 1H),
7.639 (s, 1H), 8.141 (dd, 2H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.482 (d,1 H,
J2 = 2,4 Hz), 11.546 (s, 1H) Anal. (C17H13NO5)C,H,N. Anal. Calcd for
4.3.28. 2-(4-Hydroxy-3-methoxy-phenyl)-benzo[h]-4H-
chromen-4-one (FNH77)
Mp 231–233 °C. 1H NMR (DMSO-d6) d 3.968 (s, 3H), 6.982 (d,
1H, J1 = 8,3 Hz), 7.028 (s, 1H), 7.601 (s, 1H), 7.675 (d, 1H,
J1 = 8,1 Hz), 7.761 (m, 2H), 7.850 (d, 1H, J1 = 8.8 Hz), 7.996 (d, 1H,
J1 = 8.8 Hz), 8.037 (m, 1H), 8.625 (m, 1H), 9.671 (s, 1H) Anal. (C20-
H14O4)C,H. Anal. Calcd for C20H14O4: C, 75.46, H, 4.43. Found: C,
75.58, H, 4.39.
C17H13NO5: C, 65.59, H, 4.21, N, 4.50. Found: C, 65.50, H, 4.17, N,
4.53.
4.3.22. 2-(4-Hydroxy-3-methoxyphenyl)-5,7-dimethyl-4H-
chromen-4-one (FNH71)
4.3.29. 6,8-Dibromo-2-(3-chloro-4-hydroxy-5-methoxy-
phenyl)-4H-chromen-4-one (FNH78)
Mp 217–219 °C. 1H NMR (DMSO-d6) d 2.416 (s, 3H), 2.560 (s,
3H), 3.913 (s, 3H), 6.783 (s, 1H), 6.901 (m, 1H), 7.368 (s, 1H),
7.479 (m, 2H), 7.629 (s, 1H), 9.567 (s, 1H) Anal. (C18H16O4)C,H.
Anal. Calcd for C18H16O4: C, 72.96, H, 5.44. Found: C, 73.05, H,
5.50.
Mp 276–278 °C. 1H NMR (DMSO-d6) d 3.963 (s, 3H), 7.139 (s,
1H), 7.582 (s, 1H), 7.703 (s, 1H), 8.078 (d, 1H), 8.181 (d, 1H),
10.196 (s, 1H) Anal. (C16H9Br2ClO4)C,H. Anal. Calcd for C16H9Br2-
ClO4: C, 41.73, H, 1.97. Found: C, 41.65, H, 1.91.
4.3.23. 2-(4-Hydroxy-3-methoxy-5-nitrophenyl)-6,8-dimethyl-
4H-chromen-4-one (FNH72)
4.3.30. 6,8-Dibromo-2-(4-hydroxy-3-methoxyphenyl)-4H-
chromen-4-one (FNH79)
Mp >300 °C. 1H NMR (DMSO-d6) d 2.391 (s, 3H), 2.552 (s, 3H),
4.018 (s, 3H), 7.165 (s, 1H), 7.516 (d, 1H), 7.655 (d, 1H), 7.842 (d,
1H, J2 = 1.7 Hz), 8.150 (d, 1H, J2 = 1.7 Hz), 11.289 (br s, 1H) Anal.
(C18H15NO6)C,H,N. Anal. Calcd for C18H15NO6: C, 63.34, H, 4.43, N,
4.10. Found: C, 63.22, H, 4.39, N, 4.21.
Mp 238–240 °C. 1H NMR (DMSO-d6) d 3.909 (s, 3H), 6.918 (d,
1H, J1 = 8.8 Hz), 7.006 (s, 1H), 7.581 (m, 2H), 8.070 (d, 1H,
J2 = 2.0 Hz), 8.161 (d, 1H, J2 = 2.0 Hz), 9.796 (s, 1H) Anal. (C16H10Br2-
O4)C,H,Br. Anal. Calcd for C16H10Br2O4: C, 45.11, H, 2.37, Br, 37.51.
Found: C, 45.03, H, 2.35, Br, 37.61.

A. G. Golub et al. / Bioorg. Med. Chem. 21 (2013) 6681–6689
6689
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4.3.31. 2-(3-Chloro-4-hydroxy-5-methoxy-phenyl)-benzo[h]-
4H-chromen-4-one (FNH80)
Mp >300 °C. 1H NMR (DMSO-d6) d 4.022 (s, 3H), 7.134 (s, 1H),
7.778 (m, 3H), 7.863 (d, 1H, J1 = 8.8 Hz), 7.994 (d, 1H, J1 = 8.8 Hz),
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4.3.33. 2-(4-Hydroxy-3-methoxy-phenyl)-7,8-dimethyl-4H-
chromen-4-one (FNH82)
Mp 227–229 °C. 1H NMR (DMSO-d6) d 2.406 (s, 3H), 2.462 (s,
3H), 3.896 (s, 3H), 6.921 (s, 1H), 6.964 (d, 1H, J1 = 8.1 Hz), 7.273
(d, 1H, J1 = 8.1 Hz), 7.578 (m, 2H), 7.751 d, 1H, J1 = 8.1 Hz), 9.908
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5.44. Found: C, 72.85, H, 5.50.
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4.3.34. 2-(4-Hydroxy-3-methoxy-phenyl)-7-methoxy-4H-
chromen-4-one (FNH83)
Mp 217–219 °C. 1H NMR (DMSO-d6) d 3.906 (s, 3H), 3.922 (s,
3H), 6.893 (s, 1H), 6.940 (d, 1H, J1 = 8.8 Hz), 7.052 (dd, 1H,
J1 = 9.0 Hz, J2 = 2.2 Hz), 7.315 (d, 1H, J2 = 2,0 Hz), 7.589 (m, 2H),
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Calcd for C17H14O5: C, 68.45, H, 4.73. Found: C, 68.55, H, 4.79.
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Acknowledgements
This research was supported by grants from the National Acad-
emy of Sciences of Ukraine (0107U003345 and 0107U004939).
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