Structure–activity relationship study of 4-(thiazol-5-yl)benzoic acid derivatives as potent protein kinase CK2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.01.043

Two classes of modified analogs of 4-(thiazol-5-yl)benzoic acid-type CK2 inhibitors were designed. The azabenzene analogs, pyridine- and pyridazine-carboxylic acid derivatives, showed potent protein kinase CK2 inhibitory activities [IC₅₀ (CK2α) = 0.014–0.017 μM; IC₅₀ (CK2α′) = 0.0046–0.010 μM]. Introduction of a 2-halo- or 2-methoxy-benzyloxy group at the 3-position of the benzoic acid moiety maintained the potent CK2 inhibitory activities [IC₅₀ (CK2α) = 0.014–0.016 μM; IC₅₀ (CK2α′) = 0.0088–0.014 μM] and led to antiproliferative activities [CC₅₀ (A549) = 1.5–3.3 μM] three to six times higher than those of the parent compound.

Full text of DOI:10.1016/j.bmc.2016.01.043

Accepted Manuscript
Structure–activity relationship study of 4-(thiazol-5-yl)benzoic acid derivatives
as potent protein kinase CK2 inhibitors
Hiroaki Ohno, Daiki Minamiguchi, Shinya Nakamura, Keito Shu, Shiho
Okazaki, Maho Honda, Ryosuke Misu, Hirotomo Moriwaki, Shinsuke
Nakanishi, Shinya Oishi, Takayoshi Kinoshita, Isao Nakanishi, Nobutaka Fujii
PII:
S0968-0896(16)30043-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.043
BMC 12786
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 December 2015
20 January 2016
21 January 2016
Please cite this article as: Ohno, H., Minamiguchi, D., Nakamura, S., Shu, K., Okazaki, S., Honda, M., Misu, R.,
Moriwaki, H., Nakanishi, S., Oishi, S., Kinoshita, T., Nakanishi, I., Fujii, N., Structure–activity relationship study
of 4-(thiazol-5-yl)benzoic acid derivatives as potent protein kinase CK2 inhibitors, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.043
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Bioorganic & Medicinal Chemistry
Structure–activity relationship study of 4-(thiazol-5-yl)benzoic acid derivatives as
potent protein kinase CK2 inhibitors
a,
a
b
a
a
a
Hiroaki Ohno , Daiki Minamiguchi , Shinya Nakamura , Keito Shu , Shiho Okazaki , Maho Honda ,
a
b
b
a
c
Ryosuke Misu , Hirotomo Moriwaki , Shinsuke Nakanishi , Shinya Oishi , Takayoshi Kinoshita , Isao
b
a,
Nakanishi , Nobutaka Fujii *
a
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Faculty of Pharmacy, Department of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
b
c
Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Two classes of modified analogs of 4-(thiazol-5-yl)benzoic acid-type CK2 inhibitors were designed.
The azabenzene analogs, pyridine- and pyridazine-carboxylic acid derivatives, showed potent protein
kinase CK2 inhibitory activities [IC50 (CK2α) = 0.014–0.017 μM; IC50 (CK2α') = 0.0046–0.010 μM].
Introduction of a 2-halo- or 2-methoxy-benzyloxy group at the 3-position of the benzoic acid moiety
maintained the potent CK2 inhibitory activities [IC50 (CK2α) = 0.014–0.016 μM; IC50 (CK2α') =
Available online
0
.0088–0.014 μM] and led to antiproliferative activities [CC50 (A549) = 1.5–3.3 μM] three to six
Keywords:
Protein kinase CK2
Kinase inhibitor
times higher than those of the parent compound.
2
009 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Rational design
2
4
1
. Introduction
on the crystal structure of the CK2α–AMP-PNP complex.
Structural optimization of the azole and amide moieties led to
identification of amido-substituted thiazolyl- and pyrazolyl-
benzoic acid derivatives 1 and 2. We then investigated the
Protein kinase CK2 (previously known as casein kinase 2) is a
ubiquitous, highly pleiotropic serine/threonine-specific protein
25
1
,2
kinase. It is often present as a heterotetramer composed of two
introduction of ring fusion for conformational restriction, to
catalytic α subunits (α or α') and two regulatory β subunits in
obtain the planar horseshoe-shaped conformation required for
3
,4
various combinations. The CK2α' subtype is exclusively found
in brain and testis, whereas the CK2α subtype is ubiquitously
26,27
binding of CC-4791 to CK2α
and to reduce entropic loss
28
during binding. Benzindazole derivative 3 and indolopyrazole
, derived from the pyrazole-based inhibitor 2, were identified
5
expressed. CK2 is a key regulator of various cellular events,
29
4
including signal transduction, transcriptional control, apoptosis,
as potent CK2 inhibitors. Further structural optimization was
achieved by modification of the benzoic acid moiety. In this
study, the effects of replacement of benzoic acid with a pyridine-
or pyridazine-carboxylic acid and introduction of hydrophobic
substituents were investigated to obtain further structure–activity
relationship information.
6
–8
and cell cycle. More importantly, overexpression and elevated
9
–13
activity of CK2 are closely related to many human diseases,
9
10
11
including cancers of the breast, 13lung,
and pancreas,
1
2
leukemia, and glomerulonephritis. CK2 is therefore an
1
4–16
important pharmacological target.
Inhibition of CK2 decreases cellular proliferation and induces
apoptosis in cancer cells. Several types of CK2 inhibitors,
including natural products, have been reported, such as
1
7
18
emodine, 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), (5-oxo-
5
1
9
,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid (IQA), CX-
4
945 (orally bioactive inhibitor, currently undergoing clinical
2
0,21
22,23
trials for cancer treatment),
and CC-4791
(Fig. 1).
Recently, our group identified CK2 inhibitors with a phenylazole
scaffold through virtual screening of a compound database, based

Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: hohno@pharm.kyoto-u.ac.jp (H. Ohno); nfujii@pharm.kyoto-u.ac.jp (N. Fujii).

CO H
Many CK2 inhibitors have common substructures, including
sp -hybridized heteroatom(s) (C=N or C=O) and acidic
2
OH
O
O
OH
Br
2
Br
Br
N
functional group(s) (CO H, ArOH, or triazole) (Figs. 1 and 2).
N
N
NH
2
N
H
Our previous studies showed that these motifs in CC-4791 and
the compound 1 are important for the formation of hydrogen
bonds with the backbone amide group (Val116) or electrostatic
HO
Me
O
Br
TBB
Emodin
IQA
2
5–27
N
N
N
interaction with Lys68 of CK2 (Fig. 4).
Our inhibitors 1–4
lack the hydrophobic group present in CX-4945 (chlorophenyl
group) and CC-4791 (indazole ring bearing a cyclopentylamino
group). Binding mode analyses of CX-4945 and CC-4791
suggested that these groups occupy the hydrophobic pocket of
CK2; this helps to improve the binding efficacy and decrease the
desolvation energy. Based on these findings, we examined the
introduction of ring fusion or a hydrophobic group into the
benzoic acid moiety. Naphthoic acid derivative 6 and 3-
substituted benzoic acid derivatives 7a–d (R = OPh, OBn,
CH OPh, or OCH CH Ph) were designed (Fig. 3).
N
N
N
HO2C
N
NH
HO C
2
NH
CC-4791
Cl
CX-4945
Figure 1. Representative CK2 inhibitors.
2
2
2
N
N
HN
NH
NH
S
Ar
Ar
O
O
HO2C
1
HO2C
2
Ar = C6H4(4-OMe)
Ar = C6H4(4-OMe)
HN
N
H
N
N
Ph
S
N
HO2C
HO C
2
H
3
4
Figure 2. CK2 inhibitors developed by our group.
2
2
. Results and discussion
.1. Design
Figure 4. Superposition of CX-4945 (blue), CC-4791 (yellow), and
inhibitor 1 (magenta) at ATP binding site. PDBIDs for CK2 complexes with
CX-4945, CC-4791, and inhibitor 1 are 3PE1, 3AT3, and 5B0X, respectively.
We designed the aza analogs 5a–c (Fig. 3), based on binding
affinity predictions, of CK2α–inhibitor complexes by the
thermodynamic integration (TI) method using Amber. The
calculations suggested that replacement of the carbon atom at the
3
0
2
- or 3-position of the benzoic acid moiety of 1 with a nitrogen
2
.2. Synthesis
atom (X or Y = N: 5a and 5b) would increase the affinities five-
to ten-fold. Replacement of both carbon atoms with nitrogen
atoms (X = Y = N: 5c) might further improve the binding affinity.
We also expected that the aqueous solubilities of the aza analogs
would be better than that of the parent benzoic acid 1 because of
additional hydrophilic interactions of the pyridine-type
nitrogen(s). Our findings that nicotinic acid is a potential CK2
The aza analogs 5a–c were prepared from 5-bromothiazol-2-
3
2
amine hydrochloride 8 (Scheme 1). A reported procedure was
used to protect the amino group of 8 with Boc and 2-
(trimethylsilyl)ethoxymethyl (SEM) groups. Conversion of 9 to
the corresponding pinacol (pin) borane by treatment with n-BuLi
and i-PrOBpin, followed by Suzuki–Miyaura cross coupling with
aryl halides 10a–c bearing a methoxycarbonyl group at the 4-
position, afforded the coupling products 11a–c. Removal of the
Boc and SEM groups, acylation, and hydrolysis gave the desired
aza analogs 5a–c.
3
1
binder support this design concept.
N
NH
O
introduction of
nitrogen atom(s)
S
Ar
Y
1
(a) n-BuLi
Boc (b) i-PrOBpin
HO C
X
N
N
2
ref 32
5a: X = N, Y = CH
Br
NH2·HCl
Br
N
SEM
introduction of
hydrophobic moiety
5b: X = CH, Y = N
5c: X = N, Y = N
S
S
(
c) 10, Pd(PPh3)4
8
9
K2CO3
N
Boc
N
SEM
N
N
N
(a) 4N HCl
NH
O
NH
NH
S
S
S
S
Ar
Ar
Ar
(
b) ArCOCl
Y
Y
O
O
MeO C
X
Et N
RO C
X
HO2C
HO2C
R
2
3
2
7
7
a: R = OPh
b: R = OBn
11a–c
12a–c: R = Me
5a–c: R = H
LiOH·H2O
THF/H2O
6
7c: R = CH OPh
2
7d: R = OCH CH Ph
Ar = C6H4(4-OMe); SEM = TMSCH2CH2OCH2
Z
2
2
1
0a: X = N, Y = CH, Z = Br
Figure 3. Design of CK2 inhibitors 5, 6, and 7.
1
0b: X = CH, Y = N, Z = Br
Y
MeO2C
X
10c: X = N, Y = N, Z = I

Scheme 1. Synthesis of aza analogs 5a–c.
CC50 values were determined by the MTS assay against the A549
lung cancer cells after 72 h exposure to the compound.
The synthesis of 3-substituted benzoic acid derivatives 7a–d is
shown in Scheme 2. Commercially available thiazol-2-amine 13
was converted to stannane 14 through acylation of the amino
group and regioselective lithiation–stannylation at the 5-position.
Migita–Kosugi–Stille cross coupling of 14 with halides 15a–d
followed by hydrolysis of the ester gave the substituted benzoic
acid derivatives 7a–d. Other substituted derivatives, i.e., 6 and
Table 2
CK2 inhibitory and cell antiproliferative activities of benzoic
acid derivatives 6 and 7a–d
N
N
NH
O
NH
O
S
S
Ar
Ar
1
7a–h, were prepared in a similar manner (see supplementary
data).
HO C
HO C
R
2
2
6
1, 7a–d
(
a) ArCOCl
Pd(PPh3)
4
Ar = C6H4(4-OMe)
N
N
S
Et N
LiCl
3
(
n-Bu) Sn
NH
NH2
3
S
a
(b) n-BuLi
c) (n-Bu)3SnCl
X
R
IC50 (μM)
CC50
b
Ar
(
Compound
R
13
14
O
(
μM)
CK2α
0.019
0.020
1.3
CK2α'
0.011
0.011
0.28
MeO2C
Ar = C6H4(4-OMe)
1
5a–d (X = Br or I)
CX-4945
–
H
–
9.9
8.5
N
N
1
6
NH
S
NH
O
LiOH·H2O
THF/H2O
S
Ar
Ar
>30
4.7
O
MeO C
R
HO C
R
2
2
7
7
a
OPh
OBn
0.015
0.019
0.010
0.012
0.012
0.011
1
6a–d
7a–d
a: R = OPh
b: R = OBn
b
1.5
21.9
3.6
c: R = CH OPh
2
7
c
CH OPh
0.028
0.015
2
d: R = OCH CH Ph
2
2
7
d
OCH CH Ph
2
2
Scheme 2. Synthesis of benzoic acid derivatives 7a–d.
a
IC50 values were derived from the dose–response curves
b
generated from duplicate data points of the CK2 kinase assay.
CC50 values were determined by the MTS assay against the A549
lung cancer cells after 72 h exposure to the compound.
2
.3. CK2 inhibitory and cell antiproliferative activities
The in vitro inhibitory activities of the aza analogs 5a–c
toward two subtypes of the catalytic subunit of CK2 are shown in
Table 1. As predicted by the TI calculations, 5a–c showed highly
potent inhibitory activities toward CK2α (IC50 = 0.014–0.017
μM) and CK2α' (0.0046–0.010 μM). These are comparable to the
activities of CX-4945 and the parent compound 1, but significant
improvements were not observed. However, 5a–c did not inhibit
proliferation of lung cancer cells A549 at 30 μM. This is partly
because the basic pyridine or pyridazine moiety decreases the
We next focused on benzoic acid derivatives bearing an
additional hydrophobic moiety (Table 2). Replacement of the
benzene ring of 1 by a naphthalene ring to give 6 significantly
decreased the inhibitory activity toward both CK2α (IC50 = 1.3
μM) and CK2α' (0.28 μM). In contrast, introduction of an alkoxy
group retained the potent inhibitory activities toward CK2: the
phenoxy (7a), benzyloxy (7b), phenoxymethyl (7c), and 2-
phenylethoxy (7d) derivatives were highly potent toward CK2α
3
3
membrane permeabilities of 5a–c.
(
IC50 = 0.015–0.028 μM) and CK2α' (0.010–0.012 μM).
Promising cell antiproliferative activities against A549 were
observed with 7a (CC50 = 4.7 μM), 7b (1.5 μM), and 7d (3.6 μM).
Encouraged by these results, we further optimized the structure
of the 3-substituted benzoic acid derivatives. The 3-benzyloxy
derivative 7b was selected as the lead compound for further
optimization, because of its higher cell antiproliferative activity
and ease of derivatization because of the wide availability of
substituted benzyl halides.
Table 1
CK2 inhibitory and cell antiproliferative activities of pyridine-
and pyridazine-carboxylic acid derivatives 5a–c
N
NH
S
OMe
Y
O
HO2C
X
1, 5a–c
A series of 3-benzyloxy-substituted analogs 17a–h were
prepared by Migita–Kosugi–Stille cross coupling using a variety
of substituted 4-iodobenzoate derivatives of type 15 (see
supplementary data). The biological activities of 17a–h are
summarized in Table 3. Among the chlorinated benzyl ethers
a
IC50 (μM)
CC50
b
Compound
X
Y
(
μM)
CK2α
CK2α'
0.011
0.011
0.0046
0.010
0.0096
CX-4945
–
CH
N
–
CH
CH
N
0.019
0.020
0.017
0.014
0.014
9.9
8.5
1
7a–c, the inhibitory activities of the 2-chloro derivative 17a
1
toward CK2α (IC50 = 0.016 μM) and CK2α' (0.0098 μM) were
higher than those of 7b. The inhibitory activities of 4-chloro
derivative 17c toward CK2α (IC50 = 0.026 μM) and CK2α' (0.019
μM) were lower than those of 17a. For a reason that is unclear, 3-
chloro derivative 17b showed significantly lower CK2 inhibitory
activities [IC50 (CK2α) = 0.33 μM; IC50 (CK2α') = 0.16 μM] and
cell antiproliferative activity (CC50 = 18.7 μM). Highly potent
inhibitory activities against CK2 were observed with 2-
5
5
a
>30
>30
>30
b
CH
N
5
c
N
a
IC50 values were derived from the dose–response curves
generated from duplicate data points of the CK2 kinase assay.
b

halophenyl derivatives 17d (fluorine) and 17e (bromine). Other
-substituted derivatives, including 2-methoxyphenyl (17f), 2-
than that of the parent compound 1, are promising potent CK2
inhibitors, although the solubility problem needs to be solved.
2
cyanophenyl (17g), and 2-pyridyl (17h) derivatives showed
potent CK2 inhibitory activities [IC50 (CK2α) = 0.014–0.016 μM;
IC50 (CK2α') = 0.0095–0.013 μM], although no antiproliferative
activities were detected with 17g and 17h at 30 μM. All the 2-
halo derivatives and the 2-methoxy-substituted one showed good
antiproliferative activities against A549 (CC50 = 1.5–3.3 μM).
Docking simulation of the potent inhibitors 17a, 17e, and 17f
suggested that these derivatives bind to CK2 at ATP binding site
in a similar manner to CX-4945, CC-4791, and the inhibitor 1
Table 4
Aqueous solubilities and biological activities of 5a–c and 17f
N
NH
N
S
Ar
NH
S
O
Ar
HO2C
O
Y
O
HO2C
X
(
Figure S1, supplementary data). As we expected, the substituted
1
, 5a–c
phenyl group of 17 newly introduced in this study occupies the
hydrophobic pocket of CK2.
Ar = C H (4-OMe)
MeO
17f
6
4
Solubility
IC50
μM)
CC50
c
(μM)
Compound
X
Y
in buffer
b
(
Table 3
a
(μg/mL)
CK2 inhibitory and cell antiproliferative activities of
benzyloxy-substituted derivatives 17a–h
CX-4945
–
CH
N
–
CH
CH
N
57
2.7
0.019
0.020
0.017
0.014
0.014
0.014
9.9
8.5
N
1
NH
S
5a
14
>30
>30
>30
OMe
O
HO2C
O
5
b
CH
N
89
Ar
7b, 17a–h
5
c
N
1025
1.4
a
1
7f
–
–
2.0
IC50 (μM)
CC50
Compound
Ar
b
a
b
Solubility in 67 mM phosphate buffer (pH 7.4). IC50 values
(μM)
CK2α
0.019
0.020
0.019
0.016
0.33
CK2α'
0.011
0.011
0.012
0.0098
0.16
were derived from the dose–response curves generated from
duplicate data points of the CK2α kinase assay. CC50 values
were determined by the MTS assay against the A549 cells after
2 h exposure to the compound.
c
CX-4945
–
–
9.9
8.5
1.5
1.6
1
7
7
b
C H
6
5
1
7a
7b
C H (2-Cl)
6 4
3
. Conclusions
1
C H (3-Cl)
18.7
2.5
3.3
1.5
2.0
6
4
Modification of the benzoic acid moiety of 4-(thiazol-5-
1
7c
7d
7e
7f
C H (4-Cl)
0.026
0.014
0.017
0.014
0.016
0.015
0.019
0.0088
0.014
0.013
0.011
0.0095
6
4
yl)benzoic acid-type CK2 inhibitor was investigated. As the TI
calculations predicted, replacement of the benzoic acid with
pyridine- or pyridazine-carboxylic acid maintained the highly
potent inhibitory activities toward CK2. However, no
antiproliferative activities at 30 μM were observed with these aza
analogs. In contrast, the introduction of 2-halo- and 2-methoxy-
substituted benzyloxy groups into the benzoic acid moiety was
highly effective in improving the antiproliferative activity,
leading to the identification of promising CK2 inhibitor
candidates.
1
C H (2-F)
6 4
1
C H (2-Br)
6 4
1
C H (2-OMe)
6 4
1
7g
7h
C H (2-CN)
>30
>30
6
4
1
2-pyridyl
a
IC50 values were derived from the dose–response curves
b
generated from duplicate data points of the CK2 kinase assay.
CC50 values were determined by the MTS assay against the A549
cells after 72 h exposure to the compound.
4
. Experimental
General Methods. H NMR spectra were recorded using a
1
2
.4. Aqueous solubility
JEOL AL-500 spectrometer at 500 MHz frequency. Chemical
shifts are reported in δ (ppm) relative to Me Si (in CDCl ) as
internal standard. C NMR spectra were recorded using a JEOL
4
3
The aqueous solubilities of representative inhibitors were
13
evaluated (Table 4). The 2-aza (5a) and 3-aza analogs (5b) are
five to 33 times more soluble (14.3–89.9 μg/mL) in phosphate
buffer at pH 7.4 than 1 (2.7 μg/mL). The introduction of two
nitrogen atoms (5c) significantly improves the solubility (1.025
mg/mL). In contrast, the introduction of 2-methoxybenzyl ether
AL-500 and referenced to the residual CHCl signal. IR spectra
3
were obtained on a JASCO FT/IR-4100 spectrometer. Exact
mass (HRMS) spectra were recorded on a JMS-HX/HX 110A
mass spectrometer or Shimadzu LC-ESI-IT-TOF-MS equipment
(ESI). Melting points were measured by a hot stage melting
(17f) does not have a positive effect on the aqueous solubility
points apparatus (uncorrected). For column chromatography,
Wakogel C-300E (Wako), Chromatorex NH-DM1020 (Fuji
Silysia), or Aluminum oxide 90 (Merck-Millipore) was
employed. Several benzoic acid derivatives (7a, 17b and 17g)
were further purified by HPLC [Cosmosil 5C18-ARII column
(1.4 μg/mL). As described above, the aza analogs 5a–c showed
no antiproliferative activities at 30 μM. The 2-halo- (17a, 17d,
and 17e) and 2-methoxy- (17f) benzyl ether derivatives, which
have antiproliferative activities three to six times more potent
(20 × 250 mm, Nacalai Tesque, Inc.); water/acetonitrile

containing 0.1% NH ; linear gradient; flow rate of 8 mL/min; UV
3.87 (s, 3H, OCH ), 7.08 (d, J = 8.6 Hz, 2H, Ar), 8.08 (d, J = 7.4
3
3
detector at 254 nm] to afford their ammonia salts as lyophilized
powders.
Hz, 1H, Ar), 8.13 (d, J = 8.6 Hz, 2H, Ar), 8.19–8.21 (m, 2H, Ar),
1
3
9.01 (s, 1H, Ar); C NMR (125 MHz, DMSO-d , 60 C) δ: 55.3,
6
1
1
13.7 (2C), 123.7, 124.8, 126.5, 130.1 (2C), 130.8, 133.4, 136.6,
General Procedure for Suzuki-Miyaura Cross Coupling:
+
45.6, 146.2, 159.6, 162.7, 164.5, 165.3; HRMS (FAB ) calcd
Synthesis
(
(
of
trimethylsilyl)ethoxy]methyl]amino)thiazol-5-yl]picolinate
11a). n-BuLi (1.55 M solution in hexane; 0.484 mL, 0.75 mmol)
Methyl
5-{2-((tert-Butoxycarbonyl){[2-
+
for C H N O S [M + H] : 356.0700; found: 356.0705.
1
7
14
3
4
CK2 Kinase Assay. CK2 inhibitory activities were evaluated
3
1
®
was added slowly to a mixture of bromide 9 (204 mg, 0.5
mmol) in THF (5 mL) at –78 C under argon. i-PrOBpin (152
by the off-chip mobility shift assay by the QuickScout service
from Carna Bioscience (Kobe, Japan). Human GST-fusion
CK2α(1-391) or CK2α’(1-350) was co-expressed with His-
tagged human CK2β(1-215) using baculovirus expression
system. GST-CK2α and GST-CK2α’ were purified by using
glutathione sepharose chromatography. Each chemical in DMSO
at different concentrations was diluted fourfold with reaction
buffer [20 mM HEPES (pH 7.5), 0.01% Triton X-100, 2 mM
DTT]. For CK2 reactions, a combination of the compound, 1 μM

L, 0.75 mmol) was added to the mixture, and the resulting
mixture was then allowed to warm to 0 C. 2 N NH Cl was added
4
to the mixture. The mixture was partitioned between EtOAc and
brine, and the layers were separated. The organic layer was dried
over MgSO and concentrated in vacuo to give a residual oil. A
4
3
4
mixture of this crude borate, bromopyridine 10a (129 mg, 0.6
mmol), Pd(PPh ) (5.8 mg, 0.005 mmol), and K CO (207 mg,
3
4
2
3
1
.5 mmol) in DMF (5 mL) under argon was stirred at 80 C
CK2tide, 5 mM MgCl , 5 μM ATP in reaction buffer (20 μL)
2
overnight. After being cooled to room temperature, the solvent
was removed under reduced pressure and the residue was diluted
with CH Cl and H O. The organic phase was washed with brine
were incubated with each CK2 in PP 384-well plates at room
temperature for 1 h (n = 2). The reaction was terminated by
addition of 60 μL of termination buffer (Carna Biosciences).
Substrate and product were separated by electrophoretic means
using the LabChip3000 system. The kinase reaction was
evaluated by the product ratio, which was calculated from the
peak heights of the substrate (S) and product (P): [P/(P+S)].
Inhibition data were calculated by comparing with no-enzyme
controls for 100% inhibition and no-inhibitor reactions for 0%
inhibition. IC50 values were calculated using GraphPad Prism
software.
2
2
2
and dried over MgSO . The filtrate was concentrated under
4
reduced pressure, and the resulting residue was purified by flash
chromatography over silica gel with EtOAc/hexane to give 11a
1
(
88.6 mg, 38%) as a white solid: mp 128–130 C; H-NMR (500
MHz, CDCl , 40 C) δ: –0.02 (s, 9H, Si(CH ) ), 0.95 (t, J = 8.3
3
3 3
Hz, 2H, CH ), 1.60 (s, 9H, C(CH ) ), 3.69 (t, J = 8.3 Hz, 2H,
2
3 3
OCH ), 4.00 (s, 3H, OCH ), 5.57 (s, 2H, NCH O), 7.78 (s, 1H,
2
3
2
Ar), 7.92 (dd, J = 8.0, 1.7 Hz, 1H, Ar), 8.11 (d, J = 8.0 Hz, 1H,
1
3
Ar), 8.88 (d, J = 1.7 Hz, 1H, Ar); C NMR (125 MHz, CDCl3,
Growth Inhibition Assay. A549 cell was cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Sigma),
supplemented with 10% (v/v) fetal bovine serum at 37 °C in a
4
1
0 C) δ: –1.4 (3C), 18.2, 28.1 (3C), 52.9, 67.1, 75.7, 84.4, 125.4,
28.7, 131.6, 133.2, 136.1, 146.0, 146.5, 152.8, 161.8, 165.3;
+
+
HRMS (FAB ) calcd for C H N O SSi [M + H] : 466.1826;
2
1
32
3
5
5
% CO -incubator. Growth inhibition assays using these cells
2
found: 466.1832.
were performed in 96-well plates (BD Falcon). A549 cell was
seeded at 500 cells/well in 60 μL of culture media, and were
cultured for 6 h. Then, 60 μL of the chemical diluents were added,
and the cells under chemical treatment were incubated for further
72 h. The final volume of DMSO in the medium was equal to
0.3% (v/v). The wells in the plates were washed twice with the
cultured medium without phenol-red. After 1 h incubation with
100 μL of the medium, the cell culture in each well was
supplemented with 20 μL of the MTS reagent (Promega),
followed by incubation for additional 40 min. Absorbance at 490
nm of each well was measured using a EnVision Xcite Multilabel
Reader (Perkin Elmer). CC50 values were calculated using
GraphPad Prism software (n = 2).
General Procedure for Deprotection and N-Acylation:
Synthesis of Methyl 5-[2-(4-Methoxybenzamido)thiazol-5-
yl]picolinate (12a). HCl (4 N in dioxane; 8 mL) was added to a
solution of 11a (233 mg, 0.5 mmol) in dioxane (2 mL), and the
mixture was stirred at room temperature for 16 h. After
evaporation of the solvent, a mixture of this crude product, 4-
methoxybenzoyl chloride (101 L, 0.75 mmol) and Et N (208
3

L, 3.0 mmol) in THF (10 mL) was stirred at 0 C overnight.
The solvent was removed under reduced pressure. The residue
was dissolved in EtOAc and H O. The organic phase was washed
2
with brine, and dried over MgSO . The filtrate was concentrated
4
under reduced pressure, and the resulting residue was purified by
flash chromatography over NH-silica gel with CHCl –MeOH to
3
Thermodynamic Solubility in Aqueous Solution. An equal
volume of a mixture of 1/15 M phosphate buffer (pH 7.4, 0.5
mL) and EtOH (0.5 mL), or 1/15 M phosphate buffer (pH 7.4,
give 12a (160 mg, 86%) as a pale yellow solid: mp 263–265 C;
1
H-NMR (500 MHz, CDCl , 40 C) δ: 3.90 (s, 3H, OCH ), 4.03
3
3
(
s, 3H, OCH ), 7.03 (d, J = 8.6 Hz, 2H, Ar), 7.67 (s, 1H, Ar),
3
1
.0 mL) was added to a compound in a vial. The suspension was
7
.92–7.99 (m, 3H, Ar), 8.15 (d, J = 8.0 Hz, 1H, Ar), 8.92 (s, 1H,
13
then shaken for 24 h at 37 C, and undissolved material was
separated by filtration. m-Cresol was added as an internal
standard (final concentration: 0.05 mg/mL) and the mixture was
Ar), 10.77 (br s, 1H, NH); C NMR (125 MHz, CDCl , 40 C) δ:
3
5
1
2.9, 55.6, 114.4 (2C), 124.0, 125.5, 128.2, 129.8 (2C), 131.4,
33.5, 135.6, 146.5, 146.6, 160.1, 163.8, 164.5, 165.3; HRMS
+
+
diluted in 0.1% NH and injected onto the HPLC column. The
3
(
FAB ) calcd for C H N O S [M + H] : 370.0856; found:
18 16 3 4
peak area ratio of the sample to the standard was recorded by UV
detection at 254 nm. The concentration of the sample solution
was calculated using a previously determined calibration curve,
corrected for the dilution factor of the sample.
3
70.0861.
General Procedure for Hydrolysis: Synthesis of 5-[2-(4-
Methoxybenzamido)thiazol-5-yl]picolinic Acid (5a). 1 N LiOH
750 L, 0.75 mmol) was added to a stirred mixture of 12a (92.3
(
mg, 0.25 mmol) in THF (5 mL) and water (5 mL), and the
mixture was stirred for 24 h at room temperature. The mixture
was acidified by 1 N HCl until pH < 2, then cooled to 0 C. The
yellow precipitate was collected by filtration, washed with water,
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific
Research (B) (N.F.) and for the Encouragement of Young
Scientists (A) (H.O.) from JSPS, Japan, and the Platform for
and dried under vacuum to give 5a (71.5 mg, 81%) as a yellow
1
solid: mp 274–276 C; H-NMR (500 MHz, DMSO-d , 60 C) δ:
6

2
5. Hou, Z.; Nakanishi, I.; Kinoshita, T.; Yasue, M.; Suzuki, Y.;
Kanemitsu, M.; Kure, T.; Ohno, H.; Murata, K.; Kitaura, K.;
Hirasawa, A.; Tsujimoto, G.; Oishi, S.; Fujii, N. J. Med. Chem.
Drug Design, Discovery, and Development from MEXT, Japan.
Preliminary experiments and diffraction data collection were
carried out at the beam line BL17A of the Photon Factory
2
012, 55, 2899–2903.
(2008G001) or at the Osaka University beam line BL44XU of
2
2
6. Nakaniwa, T.; Kinoshita, T.; Sekiguchi, Y.; Tada, T.; Nakanishi,
I.; Kitaura, K.; Suzuki, Y.; Ohno, H.; Hirasawa, A.; Tsujimoto, G.
Acta Cryst. 2009, F65, 75–79.
7. Kinoshita, T.; Sekiguchi, Y.; Fukada, H.; Nakaniwa, T.; Tada, T.;
Nakamura, S.; Kitaura, K.; Ohno, H.; Suzuki, Y.; Hirasawa, A.;
Nakanishi, I.; Tsujimoto, G. Mol. Cell. Biochem. 2011, 356, 97–
SPring-8 (2013A6818 and 2013B6818).
Supplementary data
Supplementary data associated with this article (provided by
the authors including experimental procedures and
characterization data for all new compounds) can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc...
1
05.
28. Suzuki, Y.; Oishi, S.; Takei, Y.; Yasue, M.; Misu, R.; Naoe, S.;
Hou, Z.; Kure, T.; Nakanishi, I.; Ohno, H.; Hirasawa, A.;
Tsujimoto, G.; Fujii, N. Org. Biomol. Chem. 2012, 10, 4907–
4
915.
2
3
9. Hou, Z.; Oishi, S.; Suzuki, Y.; Kure, T.; Nakanishi, I.; Hirasawa,
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Graphical Abstract
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Structure–activity relationship study of 4-
thiazol-5-yl)benzoic acid derivatives as
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(
potent protein kinase CK2 inhibitors
Hiroaki Ohno, Daiki Minamiguchi, Shinya Nakamura, Keito Shu, Shiho Okazaki, Maho Honda, Ryosuke Misu,
Hirotomo Moriwaki, Shinsuke Nakanishi, Shinya Oishi, Takayoshi Kinoshita, Isao Nakanishi, Nobutaka Fujii
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Faculty of
Pharmacy, Department of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan;
Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
N
NH
N
S
OMe
NH
O
O
S
HO 2C
O
OMe
HO2C
modification
CK2 inhibitor
MeO
IC50 (CK2) = 0.020 M
CC50 (A549) = 8.5 M
IC50 (CK2) = 0.014 M
CC50 (A549) = 2.0 M