Discovery and structure-activity relationship of 2,6-disubstituted pyrazines, potent and selective inhibitors of protein kinase CK2

Article abstract of DOI:10.1016/j.bmcl.2012.05.006

We report the discovery and structure-activity relationship of 2,6-disubstituted pyrazines, which are potent and selective CK2 inhibitors. Lead compound 1 was identified, and derivatives were prepared to develop potent inhibitory activity. As a result, we obtained compound 7, which was the smallest unit that retained potency. Then, introducing an aminoalkyl group at the 6-position of the indazole ring resulted in improved efficacy in both enzymatic and cell-based CK2 inhibition assays. Moreover, compound 13 showed selectivity against other kinases and in vivo efficacy in a rat nephritis model. These results show that 2,6-disubstituted pyrazines have potential as therapeutic agents for nephritis.

Full text of DOI:10.1016/j.bmcl.2012.05.006

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4358–4361
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and structure–activity relationship of 2,6-disubstituted pyrazines,
potent and selective inhibitors of protein kinase CK2
⇑
Nobuhiro Fuchi , Yosuke Iura, Hiroaki Kaneko, Aiko Nitta, Kazuharu Suyama, Hiroshi Ueda,
Shinichi Yamaguchi, Kazumi Nishimura, Shigeo Fujii, Yumiko Sekiya, Masateru Yamada, Toshiya Takahashi
Toray Industries, Inc., 10-1-6 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery and structure–activity relationship of 2,6-disubstituted pyrazines, which are
potent and selective CK2 inhibitors. Lead compound 1 was identified, and derivatives were prepared to
develop potent inhibitory activity. As a result, we obtained compound 7, which was the smallest unit that
retained potency. Then, introducing an aminoalkyl group at the 6-position of the indazole ring resulted in
improved efficacy in both enzymatic and cell-based CK2 inhibition assays. Moreover, compound 13
showed selectivity against other kinases and in vivo efficacy in a rat nephritis model. These results show
that 2,6-disubstituted pyrazines have potential as therapeutic agents for nephritis.
Received 27 February 2012
Revised 17 April 2012
Accepted 2 May 2012
Available online 9 May 2012
Keywords:
CK2
Kinase inhibitor
Docking study
Cu-catalyzed N-arylation
Nephritis
Ó 2012 Elsevier Ltd. All rights reserved.
Protein kinase CK2 (formerly known as casein kinase II) is an
extremely well-conserved pleiotropic protein kinase with a grow-
ing list of >300 substrates, the majority of which are proteins
implicated in signal transduction gene expression, and transcrip-
tion-related functions.1–6 Protein kinase CK2 is a ubiquitous het-
Representative inhibitors of CK2 are shown in Figure 1. Flavo-
M) but lack
noids such as emodin have good potency (IC50 = 2
l
specificity.15 Polyhalogenated heteroaryls such as tetrabromobenz-
imidazole (TBB),16 which shows higher potency and selectivity in
comparison with emodin, are useful for evaluating the role of
CK2. However, polyhalogenated aromatics are not suitable for
use as pharmaceutical agents because of their toxicity. Vangrev-
erotetrameric serine/threonine protein kinase made up of two
a
or a0 catalytic subunits and two b-regulatory subunits. CK2 is acti-
vated during cell division, cellular differentiation, and embryogen-
esis, and plays an important role in transducing signals between
extracellular growth factors and nuclear responses.1–6 Overexpres-
sion and activation of CK2 have been observed in a wide variety of
cancers,7–10 and the regulation of the protein has been reported to
be deeply involved in inflammatory disorders,11 pain,12 and viral
infections,13 indicating that the potential pharmacological use of
CK2 inhibitors extends beyond oncology.
Br
HN
Cl
OH
O
OH
Br
Br
N
N
N
N
H
HO
From cDNA microarray analysis results, Yamada et al. reported
that CK2 plays a critical role in the progression of immunogenic re-
nal injury; administration of either antisense oligodeoxynucleotide
Br
O
CO2H
TBB
Emodin
CX-4945
O
against CK2a or low molecular weight CK2 inhibitor (emodin,
N
Fig. 1) effectively prevented the progression of renal pathology in
rat glomerulonephritis models.14 Against this background, we be-
gan research to develop a novel CK2 inhibitor as a therapeutic
agent for nephritis.
NH
COOH
N
N
N
N
COOH
HOOC
IQA
1
⇑
Corresponding author. Tel.: +81 467 32 9562; fax: +81 467 32 9632.
E-mail address: Nobuhiro_Fuchi@nts.toray.co.jp (N. Fuchi).
Figure 1. Representative CK2 inhibitors and lead compound 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.006

N. Fuchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4358–4361
4359
Table 1
N
N
N
N
b
b
Inhibitory profile of 2,6-disubstituted pyrazines 1–7
X
X
EtO2C
A
RO2C
A
X
B
N
X = Cl
X = I
a
2
A = indole-3-yl, B = indole
X
N
Y
N
N
3 A = indole-3-yl, B = pyrrole
4 A = pyrrole-3-yl, B = indole
5
A = pyrrole-3-yl, B = imidazole
RO2C
A
B
6 A = pyrrole-3-yl, B = indoline
7 A = pyrrole-3-yl, B = indazole
Compound
X
Y
CK2a IC50, nM
R = Et
R = H
c
Scheme 1. Reagents and conditions: (a) NaI, 15-crown-5, p-TsOH, sulfolane, 150 °C,
35%; (b) CuI (5 mol %), trans-1,2-diaminocyclohexane (20 mol %), K3PO4, dioxane,
110 °C, 24 h; (c) NaOH, H2O, dioxane, rt.
N
N
1
300
500
COOH
N
HOOC
N
elinghe et al. identified an inhibitor with novel structure
(5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl) acetic acid (IQA)
through a high-throughput docking study.17 IQA demonstrated
strong inhibitory activity (IC50 = 80 nM), but decomposed under
aqueous conditions, precluding its use as a drug. CX-4945 is the
only CK2 inhibitor in clinical trials for the treatment of cancer.18
From an N-aryl indole-3-acetic acid library, we have found com-
pound 1 as an initial hit that had strong potency (IC50 = 300 nM) for
CK2 inhibition.19 Suzuki et al. reported that several 2,6-disubsti-
tuted pyrazine derivatives bearing pyrrole-3-acetic acid and mon-
osubstituted aniline possess inhibitory activities.20 Herein we
report the discovery and structure–activity relationship (SAR) of
2,6-disubstituted pyrazine derivatives and the evaluation of novel
therapeutic targets for nephritis.
Compounds 2–7 were efficiently synthesized via sequential Cu-
catalyzed N-arylation of 2,6-diiodopyrazines (Scheme 1). To in-
crease the reactivity, 2,6-dichloropyrazine was converted to 2,6-
diiodopyrazine.21 Through the protocol developed by Buchwald
and co-workers,22 the first N-arylation of ethyl indole-3-acetate
or ethyl pyrrole-3-acetate was accomplished by using trans-1,2-
cyclohexanediamine and catalysts derived from CuI. The desired
mono-N-arylated pyrazine20 was easily separated from the mix-
ture of dimer and substrate by silica gel flash chromatography.
The second N-arylation, using commercially available heterocycles
(indole, pyrrole, imidazole, indoline, and indazole) under the same
reaction conditions, yielded 2,6-disubstituted pyrazines. Finally,
compounds 2–7 were obtained by hydrolysis of ethyl ester under
aqueous basic conditions.
2
3
COOH
N
N
3000
COOH
N
N
N
4
5
6
7
100
100
50
COOH
N
N
COOH
N
N
COOH
N
N
N
30
COOH
Ethyl pyrrole-3-acetate in Scheme 1 was prepared from pyrrole
in four steps (Scheme 2). After TIPS protection of the pyrrole nitro-
gen, an ethyl oxalate chain was regioselectively introduced at the
3-position of the pyrrole. The desired product was obtained in good
yield via transfer hydrogenolysis of pyrroleglyoxalate23 in the pres-
ence of NaH2PO2, followed by removal of the TIPS group.
The prepared compounds (1–7) were evaluated in an enzymatic
CK2 inhibition assay.24 The data summarized in Table 1 showed
that when one acetic acid group was removed from 1, CK2 inhibi-
tory activity was maintained (compound 2). Removal of the ben-
zene ring from the indole at position Y (compound 3) resulted in
a
Values are the mean of two or more separate experiments.
low potency. On the other hand, upon introducing pyrrole-3-acetic
acid at position X, 4 showed good potency. This result means that
compound 4 is the smallest unit to retain CK2 inhibitory activity.
The SAR of heteroaryl groups at position Y is summarized for com-
pounds 4–7. Benzimidazole 5 and non-aromatic indoline 6 retain
inhibitory activity. With the introduction of indazole 7, inhibitory
activity improved to three-fold that of indole 4. By altering the
two indole-3-acetic acid groups in lead compound 1, the more po-
tent and simple structure 7 was obtained.
To evaluate inhibitory activity, we introduced substituents on
the indazole ring. The compounds appearing in Table 2 were syn-
thesized according to Scheme 3. After the first N-arylation of ethyl
pyrrole-3-acetate, an iodopyrazine intermediate was coupled with
the indazoles containing substituents at the 3- and 6-positions.
Moreover, reductive amination with the intermediate containing
a 6-amino group and corresponding ketone afforded aminoalkyl
derivatives. Finally, compounds 8–14 were obtained via ethyl ester
hydrolysis.
TIPS
H
H
N
N
N
a,b
c,d
O
O
OEt
OEt
O
Scheme 2. Reagents and conditions: (a) NaH, TIPS-Cl, DMF, quant.; (b) ethyl oxalyl
chloride, pyridine, CH2Cl2; (c) TBAF, THF, 50% (2 steps); (d) Pd/C (20 wt %), NaH2PO2,
1,4-dioxane, reflux, 85%.

4360
N. Fuchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4358–4361
Table 2
CK2 inhibitory profile of 2,6-disubstituted pyrazines 7–14
R2
N
N
N
N
N
R1
COOH
Compound
R1
R2
CK2a (enzyme) IC50, nM
CK2a (cell) IC50
, lM
7
8
9
10
11
12
H
H
H
H
NH2
Cl
30
12
8.0
25
16
22
N.D.b
30
Cl
NH2
H
H
H
15
N.D.b
4.0
CN
11
H
N
13
14
H
H
9.3
8.0
0.9
1.0
H
N
a
Values are the mean of two or more separate experiments.
Not determined.
b
R2
groups maintained or slightly increased enzymatic CK2 inhibitory
activity. In particular, compounds 13 and 14 (alkylamino group
at R2) showed high inhibitory activity in both enzymatic and
cell-based CK2 inhibition assays.
The results in Table 2 can be explained by a docking simulation
of compound 7 complexed with CK2 protein (Fig. 2). Compound 7
was manually placed in the binding pocket (PDB structure 1JWH of
N
N
N
a
N
N
N
N
N
I
R1
N
R1
R2
N
H
CO2R
CO2R
R = Et
R = H
human CK2a used as a template) and subjected to in situ minimi-
c
8
9
R1 = Cl, R2 = H
zation. As a result, two hydrogen bonds between carboxylic acid
and Lys68 and Asp175 and between the N-4 pyrazine atom and
Val116 were observed in the binding pocket. The pyrrole and pyr-
azine are close to the kinase protein, so the introduction of substit-
uents is expected to decrease inhibitory activity. On the other
hand, there is sufficient space around the indazole to introduce a
substituent. This hypothesis is consistent with the results for com-
pounds 7–14. Almost all known CK2 inhibitors have fused-ring pla-
ner structures; the investigated compounds contain three rings
that are directly connected. Two heteroaryl rings connected at
the 2- and 6-positions of the pyrazine ring enable the compounds
to adopt a planer conformation in the ATP binding site of CK2. Re-
cently, the binding of compounds 13 and 14 to the hydrophobic
R1 = NH2, R2 = H
10 R1 = H, R2 = NH2
R1 = H, R2 = cyclopentylamino
R1 = H, R2 = isopropylamino
13
14
b
1 = H, R2 = Cl
1 = H, R2 = CN
10
11
12
R
R
Scheme 3. Reagents and conditions: (a) CuI (5 mol %), trans-1,2-diaminocyclohex-
ane (20 mol %), K3PO4, dioxane, 110 °C, 24 h; (b) acetone or cyclopentanone,
NaBH3CN, AcOH, DMF, 65 °C; (c) NaOH, H2O, dioxane, rt.
We investigated the effects of substituents at the 6- and 3-posi-
tions of the indazole ring. Compounds 7–14 were evaluated in
enzymatic and cell-based CK2 inhibition assays.24,25 The data sum-
marized in Table 2 show that both R1 (chloro and amino) and R2
(amino, chloro, cyano, cyclopentylamino and isopropylamino)
pocket adjacent to the ATP binding site of human CK2
a was veri-
fied by X-ray crystallography (PDB structure 3at3 and 3at4).26
Figure 2. Docking study of compound 7 and CK2 protein (PDB: 1JWH): top view (left), side view (middle) and diagram showing the interaction of compound 7 and CK2
protein (right).

N. Fuchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4358–4361
4361
Table 3
amount of proteinuria derived from the anti-GBM glomerulone-
phritis-induced renal dysfunction. These results show that CK2
warrants further study as a therapeutic target for treatment of
nephritis.
Protein kinase selectivity profile of compound 13
HN
N
N
References and notes
N
N
N
1. Meggio, F.; Pinna, L. A. FASEB J. 2003, 17, 349.
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Cardiff, R. D.; Seldin, D. C. Oncogene 2001, 20, 3247.
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Rao, P.; Singh, B. Clin. Cancer Res. 2004, 10, 5792.
Compound CK2 IC50, nM
Ratio for CK2 IC50 value
CAMK2 S6 PKA ERK
>740 162 626 >740 >740 >740
PKC
p38b2
13
9.3
10. Laramas, M.; Pasquier, D.; Filhol, O.; Ringeisen, F.; Descotes, J. L.; Cochet, C. Eur.
J. Cancer 2007, 43, 928.
11. Singh, N. N.; Ramji, D. P. J. Mol. Med. 2008, 17, 887.
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Commun. 2000, 275, 434.
14. Yamada, M.; Katsuma, S.; Adachi, T.; Hirasawa, A.; Shiojima, S.; Kadowaki, T.;
Okuno, Y.; Koshimizu, T.; Fujii, S.; Sekiya, Y.; Miyamoto, Y.; Tamura, M.;
Yumura, W.; Nihei, H.; Kobayashi, M.; Tsujimoto, G. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 7736.
p<0.05
100
50
0
15. Yim, H. L.; Lee, Y. H.; Lee, C. H.; Lee, S. K. Planta Med. 1995, 65, 9.
16. Pagano, M. A.; Andrzejewska, M.; Ruzzene, M.; Sarno, S.; Cesaro, L.; Bain, J.;
Elliott, M.; Meggio, F.; Kazimierczuk, Z.; Pinna, L. A. J. Med. Chem. 2004, 47,
6239.
17. Vangrevelinghe, E.; Zimmermann, K.; Schoepfer, J.; Portmann, R.; Fabbro, D.;
Furet, P. J. Med. Chem. 2003, 46, 2656.
18. Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.;
Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J.
P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Anderes, K.; Bliesath, J.; Drygin, D.;
Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Trent, K.; Rice, W. G.; Ryckman, D. M.
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13
19. Takahashi, T.; Fuchi, N.; Yamada, M.; Nitta, A. Jpn. Kokai Tokkyo Koho,
JP2006045119.
20. Suzuki, Y.; Cluzeau, J.; Hara, T.; Hirasawa, A.; Tsujimoto, G.; Oishi, S.; Ohno, H.;
Fujii, N. Arch. Pharm. Chem. Life Sci. 2008, 341, 554.
Figure 3. In vivo effect of compound 13 in anti-GBM nephritis rat.
21. Turck, A.; Ple, N.; Dognon, D.; Harmoy, C.; Queguiner, G. J. Heterocycl. Chem.
1994, 31, 1449.
22. Antilla, J. C.; Klapars, A.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 11684.
23. Demopoulos, V. L. Synth. Commun. 1989, 19, 2585.
24. Enzymatic protein kinase assays were performed using CK2, protein kinase C
(PKC), calcium/calmodulin-dependent protein kinase II (CAMK2), p70
ribosomal protein S6 kinase (S6), cyclic AMP-dependent protein kinase
Consistent with the results of docking simulation, three rings were
taking a planar structure.
Compound 13 was screened against other protein kinases to as-
sess its selectivity profile (Table 3).24 As anticipated, compound 13
displayed excellent selectivity with more than 100-fold selectivity
toward six protein kinases.
We further examined the in vivo effect of compound 13 on
nephritis (Fig. 3).27 Administration of compound 13 (15 mg/kg,
intraperitoneally, once daily) significantly improved the amount
of proteinuria in anti-GBM glomerulonephritis rats. This result is
consistent with the results for emodin,14 indicating that CK2 is a
promising therapeutic target for treatment of nephritis.
In conclusion, we have described the synthesis and SAR of 2,6-
disubstituted pyrazines, and we found them to be potent CK2
inhibitors. From the lead compound (1), we minimized the struc-
ture to generate indazole 7. Introducing a cyclopentylamino group
at the 6-position of the indazole ring gave 13, which exhibited high
CK2 inhibitory activity in both enzymatic and cell-based assays.
Compound 13 showed excellent selectivity and improved the
(PKA), extracellular signal-regulated kinase
2 (ERK), p38 beta mitogen-
activated protein kinase (p38b2).
25. Cell-based CK2 inhibition assays were performed using mammalian HEK293
cell culture system expressing a serum-responsive element-luciferase reporter
gene responsive CK2.
26. 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.
27. Anti-GBM (glomerular basement membrane) glomerulonephritis was induced
in rats (Wistar–Kyoto, male, body weight 200–250 g) by a single intravenous
injection of rabbit anti-GBM serum (0.3 mL/kg) at day 0. Compound 13 was
administered intraperitoneally at 15 mg/kg of body weight once daily after an
injection of anti-GBM serum. At day 7, the 24 h urine samples were obtained,
with each rat being kept in an individual metabolic cage with free access to
water and food. The amount of urinary protein was determined by the
Pyrogallol red method and expressed as mg/day of urine. The animal
experiments were conducted according to the Guidelines for Animal
Experiments, Research and Development Division, Toray Industries, Inc.