Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a] pyrimidine-6-carboxylate analogues as a new scaffold for protein kinase casein kinase 2 inhibitor

Article abstract of DOI:10.1016/j.bmc.2014.07.037

Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/threonine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000 chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased) and performed in vitro kinase assay-based screening using hits found from the first screening. We identified compound 24 (C24)[(Z)-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-oxo-3, 5-dihydro-2H-thiazolo[3,2-a] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half maximal inhibitory concentration (IC₅₀) = 0.56 μM] inhibited PKCK2 2.2-fold more efficiently than did TBB (IC₅₀ = 1.24 μM), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31 human protein kinases. The K _i values of compound 24 and TBB for PKCK2 were 0.78 μM and 2.70 μM, respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular docking of compound 24 bound to PKCK2α versus previously reported complexes of PKCK2 with other inhibitors, our findings suggest a new scaffold for specific PKCK2α inhibitors. Thus, compound 24 appears to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization.

Full text of DOI:10.1016/j.bmc.2014.07.037

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,
2-a]pyrimidine-6-carboxylate analogues as a new scaffold for protein
kinase casein kinase 2 inhibitor
Cheng-Hao Jin a, , Kyu-Yeon Jun b, , Eunjung Lee a, Seongrak Kim a,c, Youngjoo Kwon b, Kunhong Kim a,c,
,
⇑
Younghwa Na d,
⇑
a Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 120-750, Republic of Korea
b College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
c Integrated Genomic Research Center for Metabolic Regulation, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea
d College of Pharmacy, CHA University, Pocheon 487-010, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/thre-
onine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To
develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000
chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased)
and performed in vitro kinase assay-based screening using hits found from the first screening. We
identified compound 24 (C24)[(Z)-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-
oxo-3,5-dihydro-2H-thiazolo[3,2-a] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more
potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half max-
Received 18 May 2014
Revised 20 July 2014
Accepted 22 July 2014
Available online xxxx
Keyword:
Protein kinase casein kinase 2 (PKCK2)
Selective PKCK2 inhibitor
ATP binding site
imal inhibitory concentration (IC50) = 0.56
(IC50 = 1.24 M), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31
human protein kinases. The Ki values of compound 24 and TBB for PKCK2 were 0.78 M and 2.70 M,
lM] inhibited PKCK2 2.2-fold more efficiently than did TBB
l
l
l
respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed
anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more
efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis
inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular
docking of compound 24 bound to PKCK2
inhibitors, our findings suggest a new scaffold for specific PKCK2
a
versus previously reported complexes of PKCK2 with other
inhibitors. Thus, compound 24 appears
a
to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization.
Ó 2014 Published by Elsevier Ltd.
1. Introduction
(to give a heterotetrameric holoenzyme), which appear to play
roles in targeting and substrate recruiting rather than controlling
Protein kinase casein kinase 2 (PKCK2) is a highly pleiotropic
serine/threonine-specific kinase ubiquitously expressed in all
types of eukaryotic cells.1 There are more than 300 protein
substrates already identified so far, and their number is increasing
rapidly.2 Unlike the majority of the protein kinases family, which
are turned on only in response to specific stimuli, the two catalytic
catalytic activity.3,4 PKCK2 plays a central role in controlling nearly
all cellular functions, with special reference to signal transduction,
gene expression, DNA repair, RNA and protein synthesis,2,4 and
participates in a wide variety of cellular processes including cell
cycle control, cell differentiation, proliferation, survival, protection
of cells from apoptosis, and tumorigenesis.4–8 Abnormally elevated
PKCK2 activity has been documented in a number of cancers
including that of kidney,9 lung,10 head and neck,11 prostate,12 and
mammary gland,13 and in a number of experimental models.14
Coincidental arguments support the notion that PKCK2 promotes
cell survival through the regulation of oncogenes and plays a global
anti-apoptotic role.14 Those actions of PKCK2 in tumorigenesis are
through the regulation of key oncogenes and tumor suppressor
(
a
and/or a0) subunits of PKCK2 are in fact constitutively active
either alone or in combination with the two regulatory (b) subunits
⇑
Corresponding authors. Tel.: +82 2 2228 1680; fax: +82 2 312 5041 (K.K.); tel.:
+82 31 8017 9416; fax: +82 31 8017 9420 (Y.N.).
E-mail addresses: kimkh34@yuhs.ac (K. Kim), yna7315@cha.ac.kr (Y. Na).
These authors equally contributed to this work.
http://dx.doi.org/10.1016/j.bmc.2014.07.037
0968-0896/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Jin, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.037

2
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
proteins within various prosurvival pathways, including the Wnt15
and nuclear factor-
B networks.16 The feasibility of targeting
currently available PKCK2 inhibitors lack the potency and the phar-
macological properties necessary to be suitable and successful in
clinical applications. Accordingly, the purpose of this study was
to develop efficient, selective, and cell-permeable inhibitors of
PKCK2.
j
PKCK2 for cancer therapy6,17–20 has been shown by experiments
where successful disruption of PKCK2 activity in vitro with anti-
sense PKCK2 resulted in the induction of cell apoptosis. This con-
cept has been validated in vivo in prostate cancer xenograft.20
Furthermore, the reports that many viral proteins are PKCK2 sub-
strates indicate that this kinase may play a role in viral infections
as well.2 For these reasons, there is interest in PKCK2 as a potential
target for anti-neoplastic and anti-infectious compounds, with the
obvious caveat that its activity cannot be fully, permanently, and
ubiquitously inhibited without lethal consequences.
2. Results and discussion
2.1. Screening compounds library for PKCK2 inhibitors
Previously, we reported that PKCK2 inhibition could sensitize
tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-
Cl
I
I
Br
N
N
O
O
HN
Cl
O
I
I
Br
Br
CH3
OH
N
N
Cl
O
N
N
H
HO
O
Br
OH
HO
DRB
OH
CX-4945
TID
TBB
Cl
N
O
O
O
CO2H
Br
H
N
Br
Br
EtO
H3C
N
OH
Br
O
Br
Br
N
S
N(CH3)2
Br
N
N
H
Br
Br
H3CO
OH
IQA
TBCA
DMAT
773 (Compound 22)
Currently, a number of PKCK2 inhibitors are known, including CX-
resistant cancer cells to TRAIL.8 Therefore, we first conducted
cell-based phenotypic screening using 4000 chemicals (2000: ran-
domly selected; 2000: kinase-biased) purchased from ChemDiv to
identify potential inhibitors of PKCK2. Cell viability was evaluated
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay using TRAIL-resistant HCE4 cells. The cells were
4945,21 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones (TID),22
tetrabromobenzotriazole (TBB),23 5,6-dichloro-1-b-
D-ribofuranosyl
benzimidazole (DRB),24 2-dimethylamino-4,5,6,7-tetrabromo-1H-
benzimidazole (DMAT),9 emodin,25 apigenin,26 [5-oxo-5,6-dihydr-
oindolo-(1,2-a)quinazolin-7-yl]acetic
acid
(IQA),27
and
tetrabromocinnamic acid (TBCA).28 However, except for CX-4945,
incubated with each chemical (10 lM) for 24 h and subsequently
O
R1
N
O
R1
O
R1-CHO
+
(A)
ClCH2CO2H
Thiourea
EtO
H3C
N
EtO
H3C
NH
+
R2-CHO
c-HCl
EtOH
S
NaOAc
R2
H3C
N
H
S
Acetic anhydride
AcOH
O
O
5-9, 11-18
EtO
1 R1 = 4-Methoxyphenyl
2 R1 = 4-Chlorophenyl
3 R1 = 3-Furanyl
Pyrrolidine
O
4 R1 = 3-Pyridyl
10 R1 = 4-Hydroxy-3-methoxyphenyl
O
R1
N
EtO
H3C
N
S
R3
19 - 23
Cl
Cl
(B)
O
O
O
OH
ClCH2CO2H
EtO
H3C
N
HO
R
EtO
H3C
NH
+
S
NaOAc
AcOH
N
R
N
H
S
CHO
R = H, OH
HO
OH
24 R = H
25 R = OH
2
Scheme 1. Synthetic methods for benzylidenethiazolopyrimidones 5–9 and 11–25.
Please cite this article in press as: Jin, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.037

C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
treated or untreated with TRAIL (200 ng/mL) for 2 h. A ‘hit’ was
defined as a chemical that showed >90% of cell viability in the pres-
ence of the chemical alone and <50% of cell viability in the presence
of both TRAIL and each chemical (Fig. S1). We performed in vitro
kinase assays against human recombinant PKCK2a (rhCK2a) using
the positive ‘hit’ chemicals. As shown in Figure S2, among the hits,
sponding aldehyde, using c-HCl as catalyst in ethanol solvent.30,31
Compounds 2, however, showed a better reaction yield with chlo-
roacetic acid as catalyst without solvent instead of the c-HCl.32 All
the intermediate compounds were confirmed by the spectroscopic
methods and compared with reported information.29–32 Prepared
compounds 1–4 were condensed with 2,4-dimethoxy-5-bromo-
benzaldehyde (or 5-(3,4-dichlorophenyl)furfural) and chloroacetic
acid in acetic anhydride and AcOH to afford the desired bicyclic
compounds 5–9. In the 1H NMR spectra, compounds 5–9 showed
a singlet peak around d 6.14–6.24 corresponding to the methine
proton of the thiazolopyrimidone skeleton, and the alkenyl proton
of benzylidene appeared at around d 7.60 as singlet. Synthetic
methods for compounds 1–9 were described in Scheme 1. But
the aldehyde or arylthiazolopyrimidine compounds possessing a
hydroxyl group, generated O-acetylated benzylidenethiazolopyr-
imidines compounds 11–18. 1H NMR spectra showed a methyl
peak of an acetyl group at around d 2.30 as a singlet, and in the
one lead chemical (773) exhibited a remarkable inhibitory activity
against PKCK2
a.
2.2. Chemistry
To optimize the inhibitory activity of 773 against PKCK2, we
synthesized 19 derivatives that have structural similarity. Synthe-
sis for the benzylidenethiazolopyrimidine derivatives was accom-
plished by the reported method.29 First, according to the Biginelli
method, the key starting arylthiazolopyrimidine compounds 1–4
were prepared from ethylacetoacetate, thiourea, and the corre-
Table 1
Structures of synthesized compounds
O
EtO
R1
N
O
N
S
R2
H3C
Compd
R1
R2
Compd
R1
R2
MeO
Cl
OCH3
Cl
Cl
Cl
Cl
OMe
OMe
OMe
OAc
5
16
Br
Br
Br
Br
OMe
OAc
MeO
MeO
MeO
6
7
17
18
OAc
OAc
O
N
OAc
OAc
OH
OMe
O
OMe
Cl
8
9
19
20
21
Cl
OH
OMe
Cl
O
OMe
O
Br
OAc
Cl
Cl
Cl
Cl
Cl
OH
OMe
11
OMe
Br
OAc
OMe
OH
12
13
14
15
22
23
24
25
OMe
OMe
Cl
Br
OMe
Cl
OAc
OH
OMe
OH
OMe
Br
OAc
OMe
OH
OH
Br
Cl
OAc
OMe
OH
OH
Please cite this article in press as: Jin, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.037

4
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. PKCK2 inhibitory efficacy of derivatives of lead compound 773. In vitro kinase assay was performed in the presence of 773 derivatives (10 lM) as described in
Section 4. After incubation, samples were subjected to SDS–PAGE followed by staining with Coomassie brilliant blue and autoradiography. 32P-GST-CS represents
phosphorylated GST-CS and GST-CS represents Coomassie blue stained input GST-CS.
13C NMR spectra, carbon signals of an acetyl group appeared at
Table 2
Kinase selectivity profiles of TBB, compounds 24, and 25
around 20 ppm and 168 ppm. All other analytical data confirmed
the introduction of acetyl group to the phenolic OH group. Depro-
tection of the acetyl group was achieved by treatment with pyrrol-
idine instead of with strong bases (i.e., KOH or NaOH). The acetyl
groups were successfully removed in good reaction yields, 80–
100%, under this condition. Synthetic methods for the reaction
were shown in Scheme 1A. Compounds 17 and 18 have provided
a complicated product mixture under the pyrrolidine deprotection
condition. To overcome this problem, an alternative method was
employed for compounds 24 and 25. To prepare 24 and 25, the
reaction was conducted with the same methods for compounds
11–18 without acetic anhydride. In the 1H and 13C NMR spectra,
the signals of acetyl groups did not appear and other peaks corre-
sponding to the desired structures were observed (Scheme 1B).
Structures of the synthesized compounds are described in Table 1.
Number
Kinase
TBB
(10
C24
(10
C25
(10
l
M)
l
M)
l
M)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
AMPK
AMPK
a
a
1
2
75
83
49
149 12
51
6
2
3
0
82
65
69
117
50
0
82
14
98
87
52 10
101
91
57
20
85
98
117
127
78
130
90 11
91
36
89 12
8
6
7
0
1
2
8
2
8
2
64
48
65
127
77
54
54
118
75
87
51
92
92
61
5
87
86
122
124
69
92
50 11
91
28
6
1
3
0
2
1
5
9
1
4
9
0
6
2
1
4
7
4
5
1
6
CDK2/cyclinA
CHK1
CK1
CK2
CSK
DYRK2
Fgr
GCK
7
1
4
4
2
5
2
1
5
6
1
4
2
2
5
8
116
2
102
92
42
51
50
78
113
104
78
95
66
93
GSK3b
JNK1
JNK2
Lck
a
a
1
2
7
3
2
5
3
7
2
1
3
1
Lyn
MAPK2
MAPKAP-K2
MAPKAP-K3
MSK1
PDK1
PKA
2.3. Inhibitory effect of new PKCK2 inhibitors
To evaluate the inhibitory effect of new PKCK2 inhibitors,
in vitro kinase assays were performed. As shown in Figure 1,
in vitro kinase assays were conducted in the presence or absence
of rhCK2a. TBB and DMAT (reference PKCK2 inhibitors) are com-
mercially available, selective, cell-permeable, and fairly potent
PKCK2 inhibitors. The phosphorylation of GST-CS (GST-tagged
recombinant CK2 Substrate) was partially inhibited by 10 lM of
TBB, DMAT, or 773 (compound 22) treatments. Among exploited
PKCK2 inhibitors, compounds 24 and 25 strongly inhibited phos-
phorylation of GST-CS, and their PKCK2 inhibitory activity was
greater than that of 22, TBB, or DMAT.
109 14
PKB
PKC
a
a
61
106
60
73
99
112
29
106
65
1
5
2
0
0
6
3
4
4
5
7
3
3
3
5
6
PRAK
ROCK-II
SAPK2a
SAPK2b
SAPK3
SAPK4
SGK
108
59
101 11
63
76
61
28
72
114
69
79
85
5
3
0
0
5
6
1
3
1
3
Syk
87
77
⁄ Residual kinase activities were determined in the presence of the indicated con-
centrations of inhibitors and are expressed as a percentage of the control without
inhibitor.
2.4. Selectivity of new PKCK2 inhibitors
Protein kinases are central components of signal transduction
cascades often dysregulated in cancer, and they represent some
of the most promising drug targets. However, target selectivity is
a major concern because most characterized kinase inhibitors
target the highly conserved ATP-binding pocket. To determine
the selectivity of the new inhibitors against PKCK2, TBB,
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C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
inhibitory activity (residual activity was approximately 54 1%),
and it also drastically reduced the activities of Lyn (residual
activity was approximately 5 1%), PRAK (residual activity was
approximately 28 3%), and Syk (residual activity was approxi-
mately 28 3%; Table 2).
TBB
C24
C25
100
80
60
40
20
0
As shown in Figure 2, compound 24 exhibited remarkable
inhibition against PKCK2. While the IC50 value of compound 24
against PKCK2 calculated under comparable conditions (15
ATP concentration) was 0.56 M, the IC50 values of TBB and com-
pound 25 were 1.24 M and 8.79 M, respectively. The Ki values
lM of
l
l
l
of these compounds against PKCK2 were also calculated (Fig. 3).
TBB displayed an ATP-competitive pattern with moderate Ki value
(approximately 2.70
lM) against PKCK2 and Ki value of compound
24 was 0.78 M. These results indicate that compound 24 may be a
l
promising candidate for developing highly specific, selective, and
potent inhibitor targeting PKCK2.
-9
-8
-7
-6
-5
-4
Log10 conc (M)
2.4.1. Cellular inhibitory activity of new PKCK2 inhibitors
The usefulness of a protein kinase inhibitor is also highly
dependent on its cell permeability; the greater the permeability
the better the likelihood of dissecting cellular functions affected
by the kinase of interest. Firstly, to prove the in vitro efficacy of
TBB and compound 24 for direct inhibition of PKCK2, in vitro
kinase assays were performed. Compound 24 drastically inhibited
PKCK2 in a dose-dependent manner, which decreased phosphory-
lation of GST-CS more effectively than did TBB (Fig. 4A).
Figure 2. IC50 of TBB, compounds 24, and 25. PKCK2 activities were determined, as
described in Section 4, in the presence of the indicated concentration of inhibitors.
The data represent the means of experiments run in triplicate. Standard deviation
(SD) did not exceed 10%.
compounds 24, and 25 were tested at 10 lM concentrations for
their ability to inhibit a panel of 31 protein kinases. As shown in
Table 2, TBB was sufficient to inhibit PKCK2 activity more than
90% (residual activity was approximately 6 1%). However, there
were a handful of kinases that were sensitive to the inhibitor,
including DYRK2, which was inhibited to nearly the same extent
as PKCK2 (residual activity was approximately 2 4%), whereas
SAPK3 was inhibited to a lesser extent (residual activity was
approximately 29 3%). On the other hand, compound 24 exhib-
ited higher selectivity for PKCK2 than did TBB, and PKCK2 activity
was completely suppressed (residual activity was approximately
To examine whether compound 24 effectively inhibits PKCK2
in vivo, TE2-CK2a (PKCK2a catalytic subunit over-expressing TE2
cells)33 was treated with compound 24 or TBB. We found that com-
pound 24 more efficiently inhibited intracellular PKCK2 activity
than did TBB in a dose-dependent manner (Fig. 4B). Thus, the rea-
sonable potency and the efficient inhibition of endogenous PKCK2
activity in a cancer cell line make compound 24 a promising candi-
date for targeting PKCK2. In addition, this observation, in conjunc-
tion with the remarkable selectivity of compound 24, paved the
road toward the widespread usage of this compound for studies
aimed at unraveling the functional implications of PKCK2 activity.
0
2%). All the other kinases were either unaffected or partially
inhibited by compound 24, except DYRK2 (residual activity was
approximately 14 2%) and Lyn (residual activity was approxi-
mately 20 5%). With the observation that TBB also inhibits DYRK2
as efficiently as it does PKCK2, DYRK2 appears to be susceptible to
PKCK2 inhibitors. We found that DYRK2 was much less susceptible
to compound 24 than was PKCK2, which suggests that compound
24 can discriminate between PKCK2 and DYRK2. Collectively taken,
the data indicate that compound 24 selectivity over 31 kinases is
almost the same or better than TBB. Unfortunately, compound 25
was less selective than TBB or compound 24, having lower PKCK2
2.4.2. Pro-apoptotic efficacy of new PKCK2 inhibitors in human
stomach and liver cancer cell lines
Cytotoxicity of compound 24 was examined by the MTT assay,
using cancer cells including eight human stomach cancer cells
(AGS, MKN-1, MKN-28, MKN-45, MKN-74, NCI-N87, SNU484, and
SNU668) and six liver cancer cells (HepG2, Hep3B, SK-HEP-1,
SNU739, SNU761, and SNU878). The inhibition of PKCK2 by TBB
0.0035
0.004
0.00025
0.00025
A
B
0.00020
0.00020
0.0030
0.00015
0.00015
0.003
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
0.00010
0.00005
0.00000
0.00010
0.00005
Ki=0.78 µM
Ki = 2.7 µM
0.00000
-0.2
-3
-2
-1
0
1
2
-0.8
-0.6
-0.4
0.0
0.2
0.4
0.6
0.002
0.001
0.000
[TBB] µM
[C24] (µM)
[TBB] = 1.0 µM
[TBB] = 0.2 µM
[C24] = 0.5 µM
[C24] = 0.1 µM
[C24] = 0.0 µM
[TBB] = 0.0 µM
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
-1
1/[ATP] (mM
-1
)
1/[ATP] (mM )
Figure 3. Kinetic analysis of PKCK2 inhibition by compound 24. Competitive inhibition of PKCK2 by TBB (A) and 24 (B). Kinetic analysis was performed, as described in
Section 4, in the presence of the indicated concentration of inhibitors. Ki was calculated by linear regression analysis of Km/Vmax against inhibitor concentration plots. The data
represent the means of experiments run in triplicate. Standard deviation (SD) did not exceed 10%.
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6
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 4. In vitro and in vivo inhibition of PKCK2 by C24. PKCK2 inhibitory activity of TBB and compound 24 in vitro (A) and in vivo (B). For in vivo experiments, TE2-CK2
cells were treated with the indicated concentration of TBB or compound 24 for 24 h followed by in vitro kinase assay.
a
or compound 24 showed cytotoxicity toward all the cancer cell
lines examined, and compound 24 was more cytotoxic than TBB
(Fig. 5A and B). The increase in cytotoxicity was attributed to
increase in cellular apoptosis (Fig. 5C). The same results were
evaluate whether compound 24 could sensitize TRAIL-resistant
cancer cell lines, the cells were treated or untreated with TRAIL
in the presence or absence of compound 24 pre-treatment. As
shown in Figure 6, the cells were TRAIL resistant but underwent
apoptosis in the presence of compound 24, and cell death increased
synergistically by the combination of compound 24 and TRAIL.
These results suggest that compound 24 can be used alone or in
combination with TRAIL for inducing cancer cell apoptosis.
obtained from TE2-GFP and TE2-CK2a cells (Supplemental Fig. 3).
2.4.3. Compound 24 sensitize TRAIL resistant cancer cells to
TRAIL
Previously, we reported a novel mechanism by which PKCK2
inhibition sensitizes TRAIL-resistant cancer cells to TRAIL.8 We
evaluated TRAIL sensitivities and endogenous PKCK2 activities of
eight human cancer cell lines. We found that six cancer cell lines
(AGS, MKN-1, MKN-28, Hep3B, SK-HEP-1, and SNU739) showed
higher PKCK2 activity as compared with two cancer cell lines
(MKN45 and SNU886), and the cell lines with greater PKCK2
activity showed TRAIL resistance (Supplemental Fig. 4). To further
2.5. Compound 24 potentially binds to the ATP binding site in
PKCK2
a
Docking studies were carried out for compound 24 with
PKCK2 to examine the mode of action. An analysis of the docked
compound 24 to the ATP binding site of PKCK2 showed that 24
fits into the ATP binding site. Compound 24 has hydrophobic
a
a
Figure 5. Anti-proliferative and pro-apoptotic efficacy of compound 24 on stomach- and liver-cancer cells. Anti-proliferative efficacy of compound 24 on stomach cancer cells
(A) and on liver cancer cells (B). (C) Pro-apoptotic efficacy of TBB or compound 24 on AGS and HepG2 cancer cell lines. Cells were treated with TBB (10 M) or compound 24
(10 M) for 24 h. Cell viability and apoptosis were examined using MTT assay and Annexin-V-FLUOS staining, respectively. The data are expressed as mean SD. Experiments
run in quadruplicate.
l
l
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C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 6. C24 sensitizes TRAIL-resistant cancer cells to TRAIL. TRAIL cytotoxicity toward stomach (A) or liver (B) cancer cells. Cell viability (left panel) was evaluated by the
MTT assay and apoptosis (right panel) was examined by Annexin-V-FLUOS staining using cancer cells incubated with or without compound 24 (10 lM) for 24 h and
subsequently treated with TRAIL (200 ng/mL) for 2 h. The data are expressed as mean SD.
interactions with Ser51, Val53, Val66, Ile95, Phe113, Val116,
Asn118, Asp120, Ile174, and Asp175. The oxygen of the carboxylate
group has hydrogen bonding interaction with the conserved water
molecule. This water molecule forms a bridge between the inhibi-
tors and residue Asp175. The hydroxyl group on the benzylidene
ring on position 4 of compound 24 also forms a hydrogen bond
with residue Asn118, which constitutes the protein kinase hinge
region (Fig. 7). There are 22 structures of ATP-competitive PKCK2
inhibitors in complexes with PKCK2, reported in PDB,34 including
ellagic acid, benzimidazole derivatives, carboxylic acid derivatives,
anthraquinone, xanthenone, fluorenone, and pyrazolotriazines.
These compounds are mostly planar and fill the ATP binding pocket
with hydrophobic interactions with residues Val53, Val66, Lys68,
Ile95, Phe113, Val116, Met163, and Ile174. Some of the inhibitors
have additional hydrogen bonds with the residues (from Glu114
to Val117) comprising the hinge region of PKCK2a. These interac-
tions may improve the activity of the inhibitors but they are not
essential because some only exhibit hydrophobic interactions
between ligand and PKCK2a. Based on the comparison of com-
pound 24 bound to PKCK2
a
with previously reported PKCK2
Figure 7. Molecular docking of compound 24 with PKCK2. (A) The graphical representation of compound 24 docked to the active site of CK2a. The protein is represented as a
ribbon diagram and the compound is shown in stick representation, colored by atom type (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow; hydrogen, white; halides,
green). (B) The close up view of the docked complex. The residues of the active site of CK2 that have van der Waals contacts with compound 24 are shown. The stick
representation of the complex is shown. The carbon atoms of the protein and complex are colored in gray and cyan, respectively. The rest of the atoms are colored as follows
(oxygen, red; nitrogen, blue; hydrogen, cyan; and halides, green). Dotted green lines indicate hydrogen bonding interactions.
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8
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
inhibitor complexes, our compounds suggest a novel scaffold for
specific PKCK2 inhibitors. The compound 24-bound complex
4.2. General synthetic methods for
benzylidenethiazolopyrimidone compounds 5–9
a
showed some similarities to known PKCK2 inhibitor complexes
in terms of the residues: in the ATP binding pocket involved in
hydrophobic interactions and in hydrogen bonding interactions
involving the hinge region and the conserved water molecule.
However, complexes with compound 24 are unique because the
orientation of the chlorophenyl ring, which is almost perpendicular
to the thiazolopyrimidine ring, leads to a different binding plane
with respect to the adenine ring of ATP, whereas the planar ring
parts of other known PKCK2 inhibitors are superimposed to the
purine binding plane.35
A reaction mixture of thiopyrimidone (1 equiv), 2,4-dimethoxy-
5-bromobenzaldehyde (1 equiv), chloroacetic acid (1.025 equiv)
and NaOAc (1 equiv) in acetic anhydride and acetic acid was
refluxed for 6–16 h. The reaction mixture was cooled to room tem-
perature and poured into ice water and, subsequently extracted
with CH2Cl2. Combined organic solvent was washed with NaHCO3
and dried over Na2SO4. Solvent was removed under reduced pres-
sure, and residue was purified by silica gel column chromatogra-
phy or treatment with proper solvents.
4.2.1. (Z)-Ethyl 2-(5-bromo-2,4-dimethoxybenzylidene)-5-(4-
methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylate (5)
3. Conclusion
In summary, screening of the compound library in an in vitro
kinase assay permitted the discovery of two leads for PKCK2 inhib-
itors, 773 and 1062. Structural modification of the initial 773 fur-
ther allowed us to identify two candidates for PKCK2 inhibitors
(compounds 24 and 25). Of particular interest is compound 24,
which is able to inhibit PKCK2 activity with remarkable selectivity
and fairly high efficiency. Compound 24 is cell permeable, down-
regulates endogenous PKCK2 activity, and displays remarkable
pro-apoptotic efficacy when tested on a variety of cancer cell lines.
In addition, compound 24 can sensitize TRAIL-resistant cancer cells
to TRAIL. Molecular docking study of the active compounds in
PKCK2 revealed possible binding modes, suggesting
scaffold for specific PKCK2 inhibitors. These docking studies
may provide valuable information for the rational design of new
derivatives with improved inhibitory properties. Based on our
observations, compound 24 may be a candidate for the develop-
ment and exploitation of anti-cancer drug targeting PKCK2.
A reaction mixture of ethyl 4-(4-methoxyphenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (0.62 g, 2 mmol),
2,4-dimethoxy-5-bromobenzaldehyde (1) (0.49 g, 2 mmol), chloro-
acetic acid (0.19 g, 2.05 mmol) and NaOAc (0.16 g, 2 mmol) in
acetic anhydride (3 mL) and acetic acid (4 mL) was refluxed for
10 h. The residue was purified by silica gel column chromatogra-
phy (eluent ethyl acetate/CH2Cl2 = 2:3) to give compound 5 as a
brown solid (0.90 g, 78.3%). mp: 216–218 °C; Rf 0.33 (ethyl
acetate/n-hexane = 1:1); HPLC: RT 14.76 min (purity; 96%); 1H
NMR (CDCl3, 400 MHz) d 1.19 (t, J = 7.2 Hz, 3H), 2.51 (s, 3H), 3.76
(s, 3H), 3.88 (s, 3H), 3.95 (s, 3H), 4.10 (dq, J = 2.8, 7.2 Hz, 2H),
6.14 (s, 1H), 6.44 (s, 1H), 6.81 (d, J = 8.2 Hz, 2H), 7.33 (d,
J = 8.8 Hz, 1H), 7.56 (s, 1H), 7.93 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 14.3, 23.0, 55.0, 55.4, 56.0, 56.6, 60.6, 96.1, 103.0,
109.1, 114.1, 116.6, 118.9, 127.8, 129.6, 132.8, 133.7, 152.6,
156.7, 159.0, 159.5, 159.8, 165.7, 165.9 ppm; HRMS (ESI) [M+H]+
C26H26BrN2O6S calcd 573.0689, found 573.0687.
a novel
a
4. Experimental methods
4.1. Chemistry general
4.2.2. (Z)-Ethyl 2-(5-bromo-2,4-dimethoxybenzylidene)-5-(4-
chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylate (6)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
Most of the chemicals and reagents used were obtained from
Aldrich Chemical Co. and others were from companies like Jun-
sei, Acros Organics, and Tokyo Chemicals. Melting points were
measured without correction in open capillaries with Barnstead
Electrothermal melting point apparatus, Manual MEL-TEMP
(Model No: 1202D). Chromatographic separations were moni-
tored by thin-layer chromatography using a commercially avail-
able pre-coated Merck Kieselgel 60 F254 plate (0.25 mm) and
detected by visualizing under UV at 254 and 365 nm. Silica gel
column chromatography was carried out with Merck Kiesel gel
60 (0.040–0.063 mm). All solvents used for chromatography
were directly used without distillation. The purity was assessed
by HPLC (Shimadzu LC-6AD) analysis under the following condi-
tions; column, Synergi 4u Fusion-RP 80A (Phenomenex, 4.6 mm
i.d. ꢀ 250 mm, 4 micron); mobile phase, isocratic elution of 80%
acetonitrile for compounds 5–9, 20, and 21, 60% acetonitrile
for compounds 19 and 23 and 40% acetonitrile for compounds
22, 24, and 25; flow rate, 1.0 mL/min; detection, UV detector
(Shimadzu SPD-M20A diode array detector, 400 nm). The purity
of compound is described as percent (%). NMR spectra were
recorded on Varian AS 400 (1H NMR at 400 MHz and 13C NMR
at 100 MHz) with tetramethyl silane as an internal standard.
Chemical shift (d) values are expressed in ppm and coupling
constant (J) values in hertz (Hz). Mass spectral investigations
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.59 g,
2 mmol), 2,4-dimethoxy-5-bromobenzaldehyde (0.49 g, 2 mmol),
chloroacetic acid (0.19 g, 2.05 mmol) and NaOAc (0.16 g, 2 mmol)
in acetic anhydride (3 mL) and acetic acid (4 mL) was refluxed
for 16 h. The residue was triturated with ethyl acetate/CH2Cl2
and solid was collected and washed with same solvent. After vac-
uum drying, compound 6 was obtained as a brown solid (0.60 g,
52.0%). Mp: 222–224 °C; Rf 0.56 (ethyl acetate/n-hexane = 1:1);
HPLC: RT 20.66 min (purity; 90%); 1H NMR (CDCl3, 400 MHz) d
1.19 (t, J = 7.2 Hz, 3H), 2.51 (s, 3H), 3.89 (s, 3H), 3.95 (s, 3H), 4.10
(dq, J = 2.0, 7.2 Hz, 2H), 6.15 (s, 1H), 6.44 (s, 1H), 7.27 (d,
J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.56 (s, 1H), 7.93 (s, 1H);
13C NMR (CDCl3, 100 MHz) d 14.3, 23.1, 54.9, 56.0, 56.6, 60.8,
96.1, 103.0, 108.5, 116.4, 118.4, 128.3, 129.0, 129.7, 133.8, 134.7,
138.9, 153.2, 156.8, 159.2, 159.6, 165.5, 165.6 ppm; HRMS (ESI)
[M+H]+ C25H23BrClN2O5S calcd 577.0194, found 577.0196.
4.2.3. (Z)-Ethyl 2-(5-bromo-2,4-dimethoxybenzylidene)-5-
(furan-3-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylate (7)
A reaction mixture of ethyl 4-(furan-3-yl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (3) (0.27 g, 1 mmol),
2,4-dimethoxy-5-bromobenzaldehyde (0.25 g, 1 mmol), chloroace-
tic acid (0.10 g, 1.03 mmol) and NaOAc (0.08 g, 1 mmol) in acetic
anhydride (1.5 mL) and acetic acid (2.0 mL) was refluxed for 6 h.
The residue was triturated with ethyl acetate/n-hexane and the
solid was collected and washed with the same solvent. After vac-
uum drying, compound 7 was obtained as a brown solid (0.38 g,
were
run
on
electron
ionization
method
using
GC:7890AMS:5975C MSD (Agilent) or liquid chromatography–
electrospray ionization-time of flight mass spectrometry (Agi-
lent) in a positive mode.
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C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
71.2%). mp: 194–196 °C; Rf 0.43 (ethyl acetate/n-hexane = 1:1);
HPLC: RT 13.19 min (purity; 97%); 1H NMR (CDCl3, 400 MHz) d
1.25 (t, J = 7.2 Hz, 3H), 2.49 (s, 3H), 3.92 (s, 3H), 3.96 (s, 3H), 4.18
(dq, J = 4.0, 7.2 Hz, 2H), 6.24 (s, 1H), 6.36 (s, 1H), 6.46 (s, 1H),
7.30 (s, 1H), 7.44 (s, 1H), 7.59 (s, 1H), 8.00 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 14.4, 23.0, 47.0, 56.0, 56.7, 60.7, 96.1, 103.1, 107.8,
109.6, 116.5, 118.9, 133.8, 141.3, 143.6, 153.5, 156.8, 159.1,
159.6, 165.7, 165.8 ppm. Two other peaks were not observed;
HRMS (ESI) [M+H]+ C23H22BrN2O6S calcd 533.0376, found
533.0377.
was purified by silica gel column chromatography or treatment
with proper solvents.
4.3.1. (Z)-Ethyl 2-(4-Acetoxy-3-methoxybenzylidene)-5-(furan-
2-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimi
dine-6-carboxylate (11)
A reaction mixture of ethyl 4-(furan-2-yl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (3)(0.27 g, 1.0 mmol),
4-hydroxy-3-methoxybenzaldehyde (0.15 g, 1.0 mmol), chloroace-
tic acid (0.10 g, 1.03 mmol), and NaOAc (0.08 g, 1.0 mmol) in acetic
anhydride (3 mL) and acetic acid (4 mL) was refluxed for 12 h. The
residue was purified by silica gel column chromatography (ethyl
acetate/n-hexane = 1:2 ? 1:1) to afford compound 11 as an orange
solid (0.33 g, 75.0%). mp: 166–168 °C; Rf 0.59 (ethyl acetate/n-hex-
ane = 1:1); 1H NMR (CDCl3, 400 MHz) d 1.25 (t, J = 7.2 Hz, 3H), 2.33
(s, 3H), 2.51 (s, 3H), 3.88 (s, 3H), 4.11–4.23 (m, 2H), 6.29 (br s, 1H),
6.35 (d, J = 3.6 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 7.08 (s, 1H), 7.10 (d,
J = 8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.31 (dd, J = 0.8, 1.6 Hz, 1H),
7.77 (s, 1H); 13C NMR (CDCl3, 100 MHz) d 14.4, 20.9, 23.0, 48.7,
56.1, 60.8, 106.4, 109.2, 110.7, 113.4, 120.6, 123.5, 123.9, 132.3,
133.1, 141.7, 143.1, 151.5, 151.9, 154.0, 156.0, 165.2, 165.5,
168.8 ppm; GC–MS (EI): m/e 482.2 [M]+.
4.2.4. (Z)-Ethyl 2-(5-bromo-2,4-dimethoxybenzylidene)-5-(pyri
din-3-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrim
idine-6-carboxylate (8)
A reaction mixture of ethyl 4-(pyridind-3-yl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(4)
(0.50 g,
1.8 mmol), 2,4-dimethoxy-5-bromobenzaldehyde (1) (0.44 g,
1.8 mmol), chloroacetic acid (0.17 g, 1.85 mmol) and NaOAc
(0.15 g, 1.8 mmol) in acetic anhydride (3 mL) and acetic acid
(4 mL) was refluxed for 9 h. The residue was purified by silica gel
column
chromatography
(eluent:
ethyl
acetate/n-hex-
ane = 1:5 ? 1:3) to give compound 8 as an orange solid (0.05 g,
5.1%). mp: 188–190 °C; HPLC: RT 8.83 min (purity; 97%); 1H NMR
(CDCl3, 400 MHz) d 1.19 (t, J = 7.2 Hz, 3H), 2.51 (s, 3H), 3.90 (s,
3H), 3.95 (s, 3H), 4.11 (dq, J = 1.2, 7.2 Hz, 2H), 6.20 (s, 1H), 6.45
(s, 1H), 7.25 (dd, J = 8.0, 5.6 Hz, 1H), 7.57 (s, 1H), 7.71 (ddd,
J = 2.0, 2.0, 8.0 Hz, 1H), 7.94 (s, 1H), 8.52 (dd, J = 1.6, 5.6 Hz, 1H),
8.69 (d, J = 2.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 14.3., 23.2,
53.5, 56.0, 56.7, 60.9, 96.1, 103.1, 107.8, 116.3, 118.3, 123.8,
128.6, 133.9, 135.8, 136.1, 149.8, 150.0, 153.9, 157.0, 159.3,
159.6, 165.4, 165.5 ppm; HRMS (ESI) [M+H]+ C24H23BrN3O5S calcd
544.0536, found 544.0538.
4.3.2. (Z)-Ethyl 2-(4-Methoxybenzylidene)-5-(4-acetoxy-3-meth
oxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyri
midine-6-carboxylate (12)
A reaction mixture of ethyl 4-(4-hydroxy-3-methoxyphenyl)-6-
methyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (10)
(0.32 g, 1.0 mmol), 4-methoxybenzaldehyde (0.14 g, 1.0 mmol),
chloroacetic acid (0.10 g, 1.03 mmol), and NaOAc (0.08 g,
1.0 mmol) in acetic anhydride (2 mL) and acetic acid (3 mL) was
refluxed for 11 h. The residue was purified by silica gel column
chromatography (ethyl acetate/n-hexane = 1:3) to afford com-
pound 12 as an orange solid (0.37 g, 70.8%). mp: 194–196 °C;
Rf 0.51 (ethyl acetate/n-hexane = 1:1); 1H NMR (CDCl3, 400 MHz)
d 1.20 (t, J = 7.2 Hz, 3H), 2.29 (s, 3H), 2.52 (s, 3H), 3.81 (s, 3H),
3.86 (s, 3H), 4.13 (dq, J = 4.0, 7.2 Hz, 1H), 6.20 (s, 1H), 6.95
(d, J = 1.2 HZ,2H), 6.98 (d, J = 8.8 Hz, 2H), 7.06 (s,1H), 7.44
(d, J = 8.8 Hz, 2H), 7.73 (S,1H); 13C NMR (CDCl3, 100 MHz) 14.3,
20.9, 22.9, 55.1, 55.7, 56.1, 60.8, 108.7, 112.8, 115.0, 117.4, 120.3,
123.1, 126.0, 132.3, 139.0, 140.0, 151.2, 153.1, 156.9, 161.7,
165.6, 165.7, 169.0 ppm. One peak is missed; GC–MS (EI): m/e
522.2 [M]+.
4.2.5. (Z)-Ethyl 5-(4-chlorophenyl)-2-((5-(3,4-dichlorophenyl)fu
ran-2-yl)methylene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (9)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.5 g,
1.61 mmol), 5-(3,4-dichlorophenyl) furfural (0.39 g, 1.61 mmol),
chloroacetic acid (0.17 g, 1.83 mmol), and NaOAc (0.14 g,
1.75 mmol) in acetic anhydride (8 mL) and acetic acid (12 mL)
was refluxed for 16 h. Ethanol was added to the residue and the
mixture was heated to around 60 °C. The solid was filtered and
washed with EtOH to give compound 9 as a reddish brown solid
(0.80 g, 86.7%). mp: 238–240 °C; Rf 0.54 (ethyl acetate/n-hex-
ane = 1:3); HPLC: RT 4.03 min (purity; 100%); 1H NMR (CDCl3,
400 MHz) d 1.21 (t, J = 7.2 Hz, 3H), 2.54 (s, 3H), 4.12 (q, J = 7.2 Hz,
2H), 6.16 (s, 1H), 6.85 (s, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.35 (d,
J = 8.0 Hz, 2H), 7.48 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.58 (d,
J = 8.4 Hz, 1H), 7.77 (s, 1H); 13C NMR (CDCl3, 100 MHz) 14.3, 20.3,
55.1, 60.9, 108.7, 110.1, 118.3, 118.7, 120.3, 123.8, 126.2, 129.0,
129.1, 129.6, 131.4, 133.2, 133.7, 134.8, 138.7, 149.9, 152.8,
155.6, 157.1, 165.0, 165.4 ppm; HRMS (ESI) [M+H]+ C27H20Cl3N2O4S
calcd 573.0204, found 573.0204.
4.3.3. (Z)-Ethyl 2-(4-acetoxy-3-bromo-5-methoxybenzylidene)-
5-(4-methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (13)
A reaction mixture of ethyl 4-(4-methoxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (1) (0.31 g,
1.0 mmol), 3-bromo-4-hydroxy-5-methoxybenzaldehyde (0.23 g,
1.0 mmol), chloroacetic acid (0.10 g, 1.03 mmol), and NaOAc
(0.08 g, 1.0 mmol) in acetic anhydride (3 mL) and acetic acid
(4 mL) was refluxed for 7 h. The residue was purified by silica gel
column chromatography (ethyl acetate/n-hexane = 1:3 ? 1:2) to
afford compound 13 as an orange solid (0.19 g, 31.6%); mp: 86–
88 °C; Rf 0.19 (ethyl acetate/n-hexane = 1:3); 1H NMR (CDCl3,
400 MHz) d 1.19 (t, J = 7.2 Hz, 3H), 2.37 (s, 3H), 2.52 (s, 3H), 3.78
(s, 3H), 3.86 (s, 3H), 4.11 (dq, J = 2.8, 7.2 Hz, 2H), 6.16 (s, 1H),
6.83 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 2.0 Hz,
1H), 7.33 (d, J = 8.8 Hz, 2H), 7.60 (s,1H); 13C NMR (CDCl3,
100 MHz)14.3, 20.6, 22.9, 55.3, 56.5, 60.8, 109.8, 111.9, 114.2,
118.4, 122.3, 126.6, 129.6, 131.2, 132.3, 133.0, 139.5, 152.0,
153.0, 155.4, 160.0, 165.0, 165.7, 167.8 ppm.; GC–MS (EI): m/e
600.2 [M]+.
4.3. General synthetic method for O-acetylated benzylidenethia
zolopyrimidone compounds 11–18
A reaction mixture of thiopyridmidone (1 equiv), corresponding
aldehyde (1 equiv), chloroacetic acid (1.025 equiv), and NaOAc
(1 equiv) in acetic anhydride and acetic acid was refluxed for 6–
16 h. The reaction mixture was cooled to room temperature and
poured into iced water, which was extracted with CH2Cl2. Com-
bined organic solvent was washed with NaHCO3 and dried over
Na2SO4. Solvent was removed under reduced pressure and residue
Please cite this article in press as: Jin, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.037

10
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.3.4. (Z)-Ethyl 2-(4-acetoxy-3-bromo-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (14)
1.61 mmol), 3,4-dihydroxybenzaldehyde (0.22 g, 1.61 mmol), chlo-
roacetic acid (0.17 g, 1.83 mmol) and NaOAc (0.14 g, 1.75 mmol) in
acetic anhydride (10 mL) and acetic acid (15 mL) was refluxed for
16 h. Ethanol was added to the residue and the mixture was heated
to around 50 °C. The solid was filtered and washed with EtOH to
give compound 17 as a gray-orange solid (0.47 g, 52.6%). mp:
98–100 °C; Rf 0.20 (ethyl acetate/n-hexane = 1:3); 1H NMR (CDCl3,
400 MHz) d 1.20 (t, J = 6.8 Hz, 3H), 2.31 (s, 6H), 2.51 (s, 3H), 4.11 (q,
J = 6.8 Hz, 2H), 6.16 (s, 1H), 7.27–7.33 (m, 7H), 7.66 (s, 1H); 13C
NMR (CDCl3, 100 MHz) 14.3, 20.9, 23.0, 23.1, 55.2, 60.9, 109.0,
121.5, 124.6, 125.1, 128.3, 129.1, 129.7, 131.8, 131.9, 132.0,
134.9, 138.5, 142.8, 143.8, 152.8, 165.1, 165.5, 168.0, 168.1 ppm;
HRMS (ESI) [M+H]+ C27H24ClN2O7S calcd 555.0987, found
555.0990.
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.17 g,
0.56 mmol), 3-bromo-4-hydroxy-5-methoxybenzaldehyde (0.12 g,
0.56 mmol), chloroacetic acid (0.05 g, 0.57 mmol), and NaOAc
(0.05 g, 0.56 mmol) in acetic anhydride (2 mL) and acetic acid
(3 mL) was refluxed for 16 h. The residue was purified by silica
gel column chromatography (ethyl acetate/n-hexane = 1:4) to
afford compound 14 as an orange solid (0.20 g, 59.4%). mp: 198–
200 °C; Rf 0.28 (ethyl acetate/n-hexane = 1:3); 1H NMR (CDCl3,
400 MHz) d 1.20 (t, J = 7.2 Hz, 3H), 2.37 (s, 3H), 2.52 (s, 3H), 3.86
(s, 3H), 4.12 (dq, J = 1.6, 7.2 Hz, 2H), 6.17 (s, 1H), 6.96 (d,
J = 1.6 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 1.6 Hz, 1H),
7.35 (d, J = 8.4 Hz, 2H), 7.61 (s, 1H); 13C NMR (CDCl3,
100 MHz)14.3, 20.7, 23.0, 55.2, 56.5, 60.9, 109.1, 111.9, 118.5,
121.9, 126.6, 129.1, 129.7, 131.6, 132.9, 134.9, 138.4, 139.6,
152.7, 153.1, 155.5, 164.9, 165.4, 167.8 ppm.; GC–MS (EI): m/e
562.1[M-CH2CO]+.
4.3.8. (Z)-Ethyl 5-(4-chlorophenyl)-7-methyl-3-oxo-2-(3,4,5-
triacetoxybenzylidene)-3,5-dihydro-2H-thiazolo[3,2-a]pyri-
midine-6-carboxylate (18)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.1 g,
0.34 mmol), 3,4,5-trihydroxybenzaldehyde (49.6 mg, 0.34 mmol),
chloroacetic acid (33.0 mg, 0.35 mmol), and NaOAc (27.9 mg,
0.34 mmol) in acetic anhydride (2 mL) and acetic acid (3 mL) was
refluxed for 16 h. Ethanol was added to the residue and the mix-
ture was heated to around 50 °C. The solid was filtered and washed
with EtOH to give a gray-orange solid (96.0 mg, 48.7%). mp: 168–
170 °C; Rf 0.81 (ethyl acetate/n-hexane = 1:1); 1H NMR (CDCl3,
400 MHz) d 1.20 (t, J = 7.2 Hz, 3H), 2.31 (s, 9H), 2.51 (s, 3H), 4.11
(q, J = 7.2 Hz, 2H), 6.16 (s, 1H), 7.24 (s, 2H), 7.29 (d, J = 8.4 Hz,
2H), 7.34 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H); 13C NMR (CDCl3,
100 MHz) 14.3, 20.4, 20.9, 23.0, 55.2, 60.9, 109.1, 122.1, 122.6,
129.1, 129.7, 131.0, 131.1, 131.6, 134.9, 136.2, 138.4, 144.2,
152.8, 155.5, 164.9, 165.4, 166.8, 167.7 ppm; HRMS (ESI) [M+H]+
C29H26ClN2O9S calcd 613.1042, found 613.1044.
4.3.5. (Z)-Ethyl 2-(4-Acetoxy-2-bromo-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (15)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.62 g,
2.12 mmol), 2-bromo-4-hydroxy-5-methoxybenzaldehyde (0.49 g,
2.12 mmol), chloroacetic acid (0.21 g, 2.18 mmol), and NaOAc
(0.17 g, 2.12 mmol) in acetic anhydride (6 mL) and acetic acid
(9 mL) was refluxed for 16 h. The residue was triturated with ethyl
acetate/n-hexane and solid was collected and washed with same
solvent. After vacuum drying, compound 15 was obtained as an
orange solid (0.69 g, 53.3%). mp: 196–198 °C; 1H NMR (CDCl3,
400 MHz) d 1.20 (t, J = 7.2 Hz, 3H), 2.32 (s, 3H), 2.52 (s, 3H), 3.85
(s, 3H), 4.11 (dq, J = 2.4, 7.2 Hz, 2H), 6.17 (s, 1H), 7.10 (s, 1H),
7.29 (d, J = 8 .0 Hz, 2H), 7.35 (s, 1H), 7.36 (d, J = 8.0 Hz, 2H),
7.97(s, 1H); 13C NMR (CDCl3, 100 MHz)14.3, 20.7, 23.0, 52.2, 56.4,
60.9, 109.2, 112.0, 116.9, 123.1, 128.2, 129.1, 129.8, 131.7, 132.0,
135.0, 138.3, 141.6, 151.2, 152.6, 155.5, 164.6, 165.4, 168.4 ppm;
GC–MS (EI): m/e 604.1 [M]+.
4.4. General synthetic method for benzylidenethiazolopyrimidone
compounds 19–23
Corresponding O-acetylbenzylidenethiazolopyrimidone was
dissolved in pyrrolidine and the reaction mixture was kept stirring
for 10–20 min, at room temperature. After dilution of mixture with
ethyl acetate, the organic layer was sequentially washed with 1 M
H2SO4, satd NH4Cl, and brine and dried over Na2SO4. Solvent was
removed under reduced pressure and the residue was triturated
with ethyl acetate/n-hexane.
4.3.6. (Z)-Ethyl 2-(4-Acetoxy-3-chloro-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (16)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.10 g,
0.34 mmol), 3-chloro-4-hydroxy-5-methoxybenzaldehyde (0.06 g,
0.34 mmol), chloroacetic acid (0.03 g, 0.35 mmol), and NaOAc
(0.03 g, 0.34 mmol) in acetic anhydride (2 mL) and acetic acid
(3 mL) was refluxed for 16 h. The residue was purified by silica
gel column chromatography (ethyl acetate/n-hexane = 1:3) to
afford compound 16 as an orange solid (0.09 g, 44.6%). mp: 184–
186 °C; Rf 0.32 (ethyl acetate/n-hexane = 1:3); 1H NMR (CDCl3,
400 MHz) d 1.21 (t, J = 7.2 Hz, 3H), 2.38 (s, 3H), 2.53 (s, 3H), 3.87
(s, 3H), 4.12 (d, J = 7.2 Hz, 2H), 6.18 (s, 1H), 6.94 (d, J = 1.6 Hz,
1H),7.15 (d, J = 1.6 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.35 (d,
J = 8.4 Hz, 2H),7.62 (s, 1H); 13C NMR (CDCl3, 100 MHz) 14.3, 20.5,
24.7, 55.2, 56.6, 60.9, 109.2, 111.4, 118.3, 122.0, 123.7, 129.1,
129.4, 129.7, 131.8, 132.3, 134.9, 138.4, 152.7, 153.2, 155.5,
164.9, 165.4, 167.8 ppm; GC–MS (EI): m/e 518.2 [M-CH2CO]+.
4.4.1. (Z)-Ethyl 2-(4-hydroxy-3-methoxybenzylidene)-5-(furan-
2-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimid
ine-6-carboxylate (19)
Ethyl
2-(4-acetoxy-3-methoxybenzylidene)-5-(furan-2-yl)-
7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-car-
boxylate (11) (53.3 mg, 0.10 mmol) was dissolved in pyrrolidine
(0.5 mL) and the reaction mixture was kept stirring for 10 min.
After evaporation of solvent, compound 19 was obtained as an
orange solid (50.5 mg, quantitative). Mp: 224–226 °C; Rf 0.53
(ethyl acetate/n-hexane = 1:1); HPLC: RT 12.37 min (purity;
100%); 1H NMR (CDCl3, 400 MHz) d 1.25 (t, J = 7.2 Hz, 3H), 2.51(s,
3H), 3.93 (s, 3H), 4.10–4.25 (m, 2H), 6.28 (dd, J = 1.6, 3.6 Hz, 1H),
6.34 (d, J = 3.6 Hz, 1H), 6.35 (s, 1H), 6.99 (d, J = 1.6 Hz, 1H), 7.00
(d, J = 8.4 Hz, 1H), 7.07 (dd, J = 1.6, 8.4 Hz, 1H), 7.30 (dd, J = 0.8,
0.8 Hz, 1H), 7.75 (s, 1H); 13C NMR (CDCl3, 100 MHz) d 14.4, 23.0,
48.6, 56.1, 60.8, 106.0, 109.0, 110.7, 111.9, 115.6, 117.2, 125.7,
125.9, 134.3, 142.9, 147.2, 148.5, 151.8, 154.2, 156.6, 165.5,
165.6 ppm; HRMS (ESI) [M+H]+ C22H21N2O6S calcd 441.1115, found
441.1115.
4.3.7. (Z)-Ethyl 5-(4-chlorophenyl)-2-(3,4-diacetoxybenzylidene)
-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-
carboxylate (17)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(0.5 g,
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C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
4.4.2. (Z)-Ethyl 2-(4-methoxybenzylidene)-5-(4-hydroxy-3-
methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylate (20)
afford compound 23 as an orange solid (16.5 mg, 90.5%). Mp:
212–214 °C; Rf 0.61 (ethyl acetate/n-hexane = 1:1); HPLC: RT
9.83 min (purity; 99%); 1H NMR (CDCl3, 400 MHz) d 1.18 (t,
J = 7.2 Hz, 3H), 2.50 (s, 3H), 3.93 (s, 3H), 4.11 (q, J = 7.2 Hz, 2H),
6.14 (s, 1H), 6.86 (s, 1H), 7.10 (s, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.33
(d, J = 8.0 Hz, 2H), 7.57 (s, 1H); 13C NMR (CDCl3, 100 MHz) 14.3,
23.1, 55.1, 56.7, 60.9, 108.9, 110.2, 118.9, 120.8, 125.3, 125.9,
129.1, 129.7, 132.8, 134.8, 138.6, 144.5, 147.9, 152.9, 156.0,
165.2, 165.5 ppm; HRMS (ESI) [M+H]+ C24H21Cl2N2O5S calcd
519.0543, found 519.0550.
Ethyl
2-(4-Methoxybenzylidene)-5-(4-acetoxy-3-methoxy-
phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxylate (12) (21 mg, 0.04 mmol) was dissolved in pyrrolidine
(0.5 mL) and the reaction mixture was kept stirring for 10 min.
After evaporation of solvent, compound 20 was obtained as an
orange solid (18.0 mg, 93.3%). Mp: 188–190 °C; Rf 0.12 (ethyl ace-
tate/n-hexane = 1:3); HPLC: RT 6.38 min (purity; 100%); 1H NMR
(CDCl3, 400 MHz) d 1.23 (t, J = 7.2 Hz, 3H), 2.53 (s, 3H), 3.87
(s, 3H), 3.88 (s, 3H), 4.13 (dq, J = 2.0, 7.2 Hz, 2H), 5.62 (s, 1H),
6.15 (s, 3H), 6.83 (d, J = 8.4 Hz, 1H), 6.90 (dd, J = 2.0, 8.4 Hz, 1H),
6.95 (d, J = 2.0 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz,
2H), 7.72 (s, 1H); 13C NMR (CDCl3, 100 MHz) d 14.4, 23.0, 55.4,
55.7, 56.2, 60.7, 109.2, 111.1, 114.6, 115.0, 117.5, 121.2, 126.1,
132.3, 132.4, 133.6, 146.1, 146.5, 152.5, 156.6, 161.7, 165.8,
165.9 ppm; HRMS (ESI) [M+H]+ C25H25N2O6S calcd 481.1428, found
481.1438.
4.4.6. (Z)-Ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)
-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-
carboxylate (24)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-thi-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate
(2)
(10.0 g,
32.2 mmol), 3,4-dihydroxybenzaldehyde (4.44 g, 32.1 mmol), chlo-
roacetic acid (3.04 g, 32.2 mmol), and NaOAc (7.92 g, 96.6 mmol) in
acetic acid (100 mL) was refluxed (140 °C) for 5 h. The reaction
mixture was cooled to room temperature and poured into ice
water. Solid was collected and washed with water and n-hexane
and dissolved in EtOAc. Organic solvent was dried over Na2SO4
and solvent was removed under reduced pressure. The residue
was treated with CH2Cl2 and small amount of n-hexane to give
compound 24 as a weak green solid (9.2 g, 60.7%). Mp: 272–
274 °C; HPLC: RT 8.66 min (purity; 100%); 1H NMR (DMSO-d6,
400 MHz) d 1.12 (t, J = 7.2 Hz, 3H), 2.34 (s, 3H), 4.08 (q, J = 7.2 Hz,
2H), 6.01 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H),
7.00 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz,
2H), 7.60 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) 13.9, 22.6, 54.3,
60.2, 107.8, 114.7, 116.4, 116.5, 124.1, 124.2, 128.7, 129.5, 133.1,
134.3, 139.5, 146.0, 149.2, 152.0, 156.2, 164.5, 164.8 ppm: HRMS
(ESI) [M+H]+ C23H20ClN2O5S calcd 471.0776, found 471.0779.
4.4.3. (Z)-Ethyl 2-(4-hydroxy-3-bromo-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,
2-a]pyrimidine-6-carboxylate (21)
Ethyl 2-(4-Acetoxy-3-bromo-5-methoxybenzylidene)-5-(4-chlo
rophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimi-
dine-6-carboxylate (14)(36 mg, 0.06 mmol) was dissolved in pyrrol-
idine (0.5 mL) and the reaction mixture was kept stirring for 10 min.
The residue was treated with EtOAc/n-hexane to afford compound
21 as an orange solid (28.0 mg, 83.6%). Mp: 232–233 °C; Rf 0.57
(ethyl acetate/n-hexane = 1:1); HPLC: RT 10.48 min (purity;
99%);1H NMR (CDCl3, 400 MHz) d 1.21 (t, J = 7.2 Hz, 3H), 2.52 (s,
3H), 3.95 (s, 3H), 4.12 (dq, J = 1.2, 7.2 Hz, 2H), 6.17 (s, 1H), 6.91 (d,
J = 1.6 Hz, 1H), 7.27 (d, J = 1.6 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.35
(d, J = 8.4 Hz, 2H), 7.60 (s, 1H); 13C NMR (CDCl3, 100 MHz) d 14.3,
23.0, 55.1, 56.6, 60.9, 108.9, 109.3, 110.7, 118.9, 126.6, 128.2,
129.1, 129.7, 132.6, 134.8, 138.6, 145.6, 147.6, 152.9, 156.1, 165.2,
165.5 ppm; HRMS (ESI) [M+H]+ C24H21BrClN2O5S calcd 563.0038,
found 563.0047.
4.4.7. (Z)-Ethyl 5-(4-chlorophenyl)-7-methyl-3-oxo-2-(3,4,5-trih
ydroxybenzylidene)-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-
6-carboxylate (25)
A reaction mixture of ethyl 4-(4-chlorophenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (2) (0.2 g,
0.64 mmol), 3,4,5-trihydroxybenzaldehyde (99.0 mg, 0.64 mmol),
chloroacetic acid (91.2 mg, 0.97 mmol), and NaOAc (105.5 g,
1.29 mmol) in acetic acid (5 mL) was refluxed (140 °C) for 6 h.
The reaction mixture was cooled to room temperature and poured
into ice water. Solid was collected and washed with water and
n-hexane and dissolved in EtOAc. Organic solvent was dried over
Na2SO4 and solvent was removed under reduced pressure. The res-
idue was treated with CH2Cl2 and small amount of n-hexane to
give compound 25 as a weak green solid (136.0 mg, 30.3%). mp:
256–258 °C; HPLC: RT 4.30 min (purity; 100%); 1H NMR (DMSO-
d6, 400 MHz) d 1.12 (t, J = 6.8 Hz, 3H), 2.38 (s, 3H), 4.04 (brs, 2H),
6.02 (s, 1H), 6.58 (s, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.41 (d,
J = 8.4 Hz, 2H), 7.51 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) 14.5,
23.2, 54.9, 60.8, 108.4, 110.4, 115.3, 123.6, 129.3, 130.1, 133.7,
135.2, 138.0, 140.1, 147.1, 152.6, 156.9, 165.1, 165.4 ppm; HRMS
(ESI) [M+H]+ C23H20ClN2O6S calcd 487.0720, found 487.0733.
4.4.4. (Z)-Ethyl 2-(4-hydroxy-2-bromo-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,
2-a]pyrimidine-6-carboxylate (22)
Ethyl 2-(4-acetoxy-2-bromo-5-methoxybenzylidene)-5-(4-
chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyri-
midine-6-carboxylate (15) (0.63 g, 1.04 mmol) was dissolved in
pyrrolidine (5 mL) and the reaction mixture was kept stirring for
20 min.The residue was treated with CH2Cl2/n-hexane to afford
compound 22 as an orange solid (0.54 g, 92.0%). Mp: 234–235 °C;
Rf 0.53 (ethyl acetate/n-hexane = 1:1); HPLC: RT 4.99 min (purity;
99%); 1H NMR (CDCl3, 400 MHz) d 1.11 (t, J = 7.2 Hz, 3H), 2.38 (s,
3H), 3.85 (s, 3H), 4.03 (dq, J = 3.2, 7.2 Hz, 2H), 6.01 (s, 1H), 7.01
(s, 1H), 7.13 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz,
2H), 7.77 (s, 1H); 13C NMR (CDCl3, 100 MHz) 13.9, 22.5, 54.5,
55.8, 60.3, 108.2, 111.3, 117.9, 119.0, 119.9, 122.5, 128.8, 129.6,
131.2, 133.2, 139.1, 147.8, 150.6, 151.6, 155.5, 164.1, 164.7 ppm;
HRMS (ESI) [M+H]+ C24H21BrClN2O5S calcd 563.0038, found
563.0040.
4.5. In vitro kinase assay
4.4.5. (Z)-Ethyl 2-(4-hydroxy-3-chloro-5-methoxybenzylidene)-
5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,
2-a]pyrimidine-6-carboxylate (D)
Ethyl 2-(4-Acetoxy-3-chloro-5-methoxybenzylidene)-5-(4-
chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyri-
midine-6-carboxylate (16) (19.7 mg, 0.04 mmol) was dissolved in
pyrrolidine (0.5 mL) and the reaction mixture was kept stirring
for 10 min. The residue was treated with to CH2Cl2/n-hexane
Inhibitory effects of PKCK2 inhibitors were measured using
in vitro kinase assay as previously described.35 Briefly, 3
lg of bac-
terially expressed GST-CS (CK2 Substrate) protein was incubated
with glutathione Sepharose 4B beads for 60 min and washed twice
with 1ꢀ kinase buffer (4 mM MOPS, pH 7.2, 5 mM b-glycerolphos-
phate, 1 mM EGTA, 200 lM sodium orthovanadate, and 200 lM
DTT). The beads were incubated with 100
lg cell lysate in a final
volume of 50 L kinase reaction buffer [10
l
l
L of 5ꢀ kinase buffer,
Please cite this article in press as: Jin, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.037

12
C.-H. Jin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
10
l
L magnesium/ATP cocktail solution (90
L (100 Ci)
-[32P]-ATP] for 20 min at
30 °C. Reactions were stopped by washing twice with 1ꢀ kinase
buffer. Samples were resuspended with 30
l
L of 75 mM MgCl2/
4.10. Apoptosis detection
500 mM ATP plus 10
l
l
c
The apoptotic effect of PKCK2 inhibitors on stomach and liver
cancer cell lines were examined by Annexin-V-FLUOS staining kits
(Roche Diagnostics, Mannheim, Germany), as previously
described.36 In brief, the cells were treated with TBB or compound
24 as described above, washed with phosphate-buffered saline,
l
L of 2ꢀ sodium dode-
cyl sulfate (SDS) sample loading buffer, subjected to 12% SDS–poly-
acrylamide gel electrophoresis (PAGE) followed by staining with
Coomassie brilliant blue, and dried on Whatman paper. Incorpora-
tion of 32P was detected by autoradiography.
and stained with 100 lL Annexin-V-FLUOS/propidium iodide mix-
ture for 15 min. Finally, apoptotic cells were analyzed by fluores-
4.6. Kinase selectivity
cence microscopy (Nikon TE2000U).
To examine kinase selectivity, we used KinaseProfiler service
(Millipore). 773, TBB, compounds 24, and 25 were screened against
31 human kinases. The kinases were tested for inhibition with
4.11. Molecular docking studies
The coordinates for the catalytic subunit of human CK2 (CK2a)
10
lM concentrations of each inhibitor. Radioactive phosphory-
were retrieved from the Protein Data Bank (PDB code 1JWH).37 All
the water molecules, ligands, and cofactors were removed except
for the conserved water molecule as following the previous work38
and the hydrogen atoms were added. The structures of compound
24 were constructed by Sybyl 8.0 and minimized energetically
using a Tripos force field with Gasteiger-Huckel charges. The
receptor and ligand file were prepared according to the original
publication protocols.38,39 Docking was carried out with Autodock
4.0/ADT using the Lamarckian genetic algorithm search
parameters.40
lated product was quantitated with and without inhibitor. Kinase
activity is expressed as a percentage of the mean kinase activity
in the positive control samples. Positive-control wells contain all
components of the reaction except inhibitors. DMSO is included
in these wells to control for solvent effects. IC50 of TBB, compounds
24, and 25 for inhibition of PKCK2 were determined using the IC50
profiler assay service (Millipore).
4.7. Kinetic determination
For kinetic analysis, increasing TBB and compound 24 concen-
trations were applied. Initial velocities were determined at each
of the substrate concentration tested. Kinetic parameters (Km and
Vmax) were calculated either in the presence or absence of increas-
ing concentrations of inhibitor using the sigma plot program
(enzyme kinetics module). Inhibition constants were subsequently
calculated by linear regression analyses of Km/Vmax against inhibi-
tor concentration plots. Ki values were determined based on these
averages. The rationale underlying this approach was that kinase
inhibition is competitive with respect to ATP.
Acknowledgments
This work was supported by (FG09-31-05) of the 21C Frontier
Functional Human Genome Project from the Ministry of Education,
Science and Technology in Korea and was supported by a Faculty
Research Grant of Yonsei University College of Medicine (6-2008-
0227). K.Y. Jun was supported by RP-Grant 2013 of Ewha Womans
University.
Supplementary data
4.8. Cell culture and transfection
Supplementary data (compounds library screening data for
PKCK2) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2014.07.037.
The human esophageal cancer cell lines HCE4, TE2-GFP, and
TE2-CK2a
were grown as previously described.33 Human stomach
cancer cell lines AGS, MKN-1, MKN-28, MKN-45, MKN-74, NCI-
N87, SNU484, and SNU668 and human liver cancer cell lines
HepG2, Hep3B, SK-HEP1, SNU739, SNU761, and SNU878 were
obtained from the Korea Cell Line Bank (KCLB; Seoul, Korea).
MKN-1, MKN-28, MKN-45, MKN-74, NCI-N87, SNU484, SNU668,
SK-HEP1, SNU739, SNU761, and SNU878 cells were maintained
in RPMI1640 medium (Gibco, Gaithersburg, MD). AGS and HepG2
cells were maintained in Modified Eagle’s Medium (Gibco). Hep3B
and SK-HEP-1 cells were maintained in Dulbecco’s Modified Eagle’s
Medium (Gibco). Each medium was supplemented with 10% (v/v)
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