2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein kinase 1 gamma (CK1γ) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.046

Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2012.07.046

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5392–5395
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein
kinase 1 gamma (CK1c) inhibitors
Zihao Hua a, , Xin Huang b, , Howard Bregman a, Nagasree Chakka a, Erin F. DiMauro a,
⇑
⇑
Elizabeth M. Doherty c, Jon Goldstein d, Hakan Gunaydin b, Hongbing Huang a, Stephanie Mercede c,
John Newcomb d, Vinod F. Patel a, Susan M. Turci d, Jie Yan a, Cindy Wilson d, Matthew W. Martin a
a Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
b Molecular Structure, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
c Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Oncology Research, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benz-
imidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1
isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h
which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity,
and acceptable pharmacokinetic properties.
Received 1 June 2012
Revised 10 July 2012
Accepted 12 July 2012
Available online 20 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Wnt pathway
Casein kinase 1
b-Catenin
GSK3b
The casein kinase 1 (CK1) family of highly conserved serine/thre-
onine protein kinases has six human isoforms ( , d, 1, 2 and 3)
that regulate several cellular growth and survival processes includ-
ing Wnt signaling, cell cycle control, DNA repair, and apoptosis.1
Several studies suggest that CK1 plays an important role in onco-
genesis resulting from the deregulation of these cellular processes.
Potent and specific small molecule modulators of CK1 family
members are crucial tools to tease out their redundant versus dis-
tinct roles in vivo and to improve our current therapeutic strate-
gies that target CK1 in human cancer. Pyrvinium, an FDA-
approved anti-helminthic drug, was recently identified as a very
a
e,
c
c
c
potent and specific activator of CK1a that inhibits the Wnt/b-cate-
CK1
pathway. It binds to axin, and phosphorylates b-catenin, ultimately
leading to the degradation of b-catenin.2 CK1d and CK1
, two clo-
a
is an essential component of the Wnt/b-catenin signaling
nin pathway and decreases cell viability.7 On the other hand, PF-
670462, which inhi
e
bits CK1d/
modest effect on cancer cell survival despite being a potent inhib-
itor of Wnt signaling.8 Potent and selective inhibitors of CK1
e with great potency and selectivity, displayed only
sely related CK1 isoforms, have been shown to be necessary for
proper activation of Wnt/b-catenin signaling3 and have been impli-
cated in the progression of colon, pancreatic, and breast cancer.4
c
,
however, have not been reported to date. Herein, we present the
discovery and optimization of benzimidazole compounds as potent
CK1
way, coupling Wnt receptor activation to cytoplasmic signal trans-
duction.5 CK1
phosphorylates the cytoplasmic domain of the Wnt
c is also a key regulator of the Wnt/b-catenin signaling path-
and selective CK1
The benzimidazole compound 1a (Fig. 1) was identified as a po-
tent inhibitor of CK1 from a high-throughput screen of the Amgen
compound library. Compound 1a showed good CK1 potency
(IC50 = 0.14 M) and no inhibitory activity against an Ambit panel
of 399 kinases (>50 POC at 1
M)9, including glycogen synthase ki-
c inhibitors.
c
co-receptor LRP5 and LRP6 upon Wnt ligand binding which leads to
axin recruitment to the membrane and ultimately to the inhibition
of b-catenin degradation and Wnt pathway activation. In addition,
c
c
l
CK1c2 has recently been discovered to inhibit TGF-b function
l
through phosphorylation and subsequent degradation of activated
nase 3b (GSK3b) which downregulates the Wnt signaling path-
SMAD3.6
way.10 More importantly, compound 1a demonstrated excellent
selectivity over other CK1 isoforms such as CK1a and CK1d. How-
ever, compound 1a showed only modest potency in the LRP6 phos-
phorylation cell assay11 and poor pharmaceutical properties (high
metabolic clearance and low solubility).
⇑
Corresponding authors. Tel.: +1 617 444 5233; fax: +1 617 577 9822 (Z.H.); tel.: +1
617 444 5045; fax: +1 617 577 9511 (X. H).
E-mail addresses: zihao.hua@amgen.com (Z. Hua), hxin@amgen.com (X. Huang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.046

Z. Hua et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5392–5395
5393
N
In order to understand the origin of its potency and selectivity, a
co-crystal structure of 1a with CK1 3 was obtained. As shown in
Figure 1,12 the cyano benzimidazole moiety of 1a binds deeply into
the ATP binding pocket of CK1 3 with the cyano group involved in
a hydrogen bond interaction with the side chain of Lys72 through
one water molecule and with the side chains of Glu86 and Tyr90
through another water molecule. The 2-amino-benzimidazole por-
tion is hydrogen bonded to the main chain nitrogen and carbonyl
HN
N
c
N
H
N
c
CK1
CK1
CK1
γ
α
δ
IC50
IC50
IC50
IC50
PO4-LRP6 IC50
:
0.14 μm
12.1 μm
5.8 μm
30.8 μm
6.6 μm
O
:
:
1a
F3C
GSK3
β
:
:
oxygen of Leu119 from the linker of CK1c3. The phenyl group is in-
volved in a ‘face-to-face’ hydrophobic interaction with Pro333
while the pyridyl group is involved in an ‘edge-to-face’ hydropho-
bic interaction with Pro331. Both prolines are part of the tail in
CK1
close to the ATP binding site and are exclusively/uniquely con-
served in the CK1 isoforms (CK1 1, CK1 2, and CK1 3). They can-
c3 immediately C-terminal to the kinase domain that comes
c
c
c
c
not be found in other CK1 isoforms and kinases. Thus, it could be
argued that the observed selectivity of compound 1a against other
CK1 isoforms and kinases could stem from its hydrophobic interac-
tion with these two prolines.
The binding mode of 1a with CK1c3 led us to the initiate struc-
ture–activity relationship (SAR) studies focusing initially on the
variation of the aniline moiety in order to improve both enzyme
and cell potency while maintaining good CK1 isoform selectivity
and GSK3b selectivity (Table 1).13 With improved pharmaceutical
properties also as a goal, the strategy was to target smaller and less
lipophilic molecules. Ligand efficiency (LE)14 and lipophilic effi-
ciency (LipE)15 were tracked to monitor the binding effectiveness
Figure 1. Selective CK1c inhibitor 1a and co-crystal structure with CK1c3.
Table 1
Left-hand-side SAR of 2-phenylamino-6-cyano-1H-benzimidazoles
N
HN
N
H
N
R
d
Compd
R
c Log Pa
CK1cb IC50
M)
LEc
LipE
CK1ab IC50
M)
CK1db IC50
M)
GSK3be IC50
M)
PO4-LRP6f IC50
M)
Synthetic
routeg
(
l
(
l
(
l
(
l
(l
F3C
O
N
1a
1b
5.32
5.78
0.14
0.27
0.32
0.36
1.54
0.79
12.1
8.82
5.77
3.17
30.8
ND
6.56
Und.
A
A
O
1c
1d
1e
5.57
4.33
3.70
0.099
0.026
0.046
0.40
0.41
0.42
1.43
3.25
3.64
5.49
4.67
3.54
2.18
1.63
1.08
30.8
24.2
Und.
4.89
1.17
1.45
A
A
A
N
F
N
N
1f
5.51
4.19
3.36
0.029
0.060
0.018
0.47
0.52
0.48
2.02
3.04
4.39
7.58
19.7
9.18
2.62
3.61
2.32
44.5
30.1
60.0
1.51
4.54
0.70
A
A
A
Me
HO
1g
1h
1i
1j
5.69
6.01
0.079
0.86
0.42
0.36
1.41
0.06
Und.
Und.
2.18
4.37
Und.
Und.
10.1
ND
B
B
F
Me
ND = not determined.
Und. = undefined.
a
Calculated logarithm of octanol/water distribution coefficient.
Inhibition of kinase activity (Lance).
b
c
d
e
f
LE (ligand efficiency) = ꢀ1.36 log Ki/N (N = number of non H atoms).
LipE = pIC50 — c Log P.
Inhibition of kinase activity (Alpha-Screen).
Cell assay measuring phosphorylation of LRP6 in HEK293 cell.
See Scheme 1 for detailed synthetic routes.
g

5394
Z. Hua et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5392–5395
Table 2
Modifications of the cyanobenzimidazole core.
R
HN
Compd
R
CK1ca
CK1da
M) IC50 (
PO4-LRPb
M) IC50 ( M)
Syn.
IC50 (
l
l
l
routec
N
1f
2
0.029
2.62
1.51
Und.
2.34
A
B
A
N
H
N
N
5.80
0.50
Und.
2.88
N
H
N
Me
3
N
H
N
NO2
N
Figure 2. Overlay of the co-crystal structure of 1a (green) and 1f (yellow) with
CK1c3.
4
8.52
Und.
Und.
A
N
H
HN
N
O
5
6
7
0.42
0.53
0.14
Und.
Und.
3.97
Und.
Und.
13.0
A
A
A
N
H
N
of molecules.16 Replacing the trifluoromethylpyridine in 1a
(LE = 0.32, LipE = 1.54) with a phenyl ring (1b) resulted in a slight
loss in both enzyme and cell activity. Further replacement of the
diaryl ether with a diaryl moiety (1c–1e) led to improved enzyme
and cell potency relative to 1a, while maintaining good selectivity
N
CN
Me
N
H
N
N
H
CN
over CK1a, CK1d and GSK3b. Among these inhibitors, 1d with the
N
lowest c Log P (3.70) demonstrated improved ligand efficiency
(LE = 0.42) and lipophilic efficiency (LipE = 3.64) compared to 1a.
Finally, the diaryl-group was replaced by a para-substituted phenyl
ring. Although compounds with a tert-butyl group (1f) or a methyl
group (1g) in para-position showed similar Ck1c potency as 1c–1e
and improved ligand efficiency, the lower LipE (2.02 for 1f, 3.04 for
8
0.18
2.37
1.66
3.17
14.9
3.83
9.10
B
O
N
CN
CN
CN
9
0.039
0.033
B
S
N
10
Cd
N
1g) indicated that the gain in potency was not attractive relative to
lipophilicity cost. Co-crystal structure of 1f with CK1c3 revealed
that the tert-butyl phenyl moiety was engaged in hydrophobic
interaction with Pro331 and Pro333 (Fig. 2).
Modulation of the physicochemical properties of 1f by replacing
one of the methyl groups with a hydroxyl group led to compound
1h (c Log P = 3.36) demonstrating similar enzyme potency as 1f
Und. = undefined.
a
Inhibition of kinase activity (Lance).
b
c
Cell assay measuring phosphorylation of LRP6 in HEK293 cell.
See Scheme 1 for detailed synthetic routes.
See Scheme 2 for detailed synthesis.
d
Route A:
and good selectivity over CK1a, CK1d and GSK3b. More impor-
tantly, compound 1h exhibited significantly improved lipophilic
efficiency (LipE = 4.39) relative to 1f and good cellular potency
S
C
N
NH2
N
HN
N
H
a
(IC50 = 0.70
l
M). Substituents at the ortho position of the aniline,
CN
b
which would affect the ‘face-to-face’ hydrophobic interaction with
Pro333 by distorting the orientation of the aniline, were expected
to be detrimental to CK1 isoform selectivity and GSK3b selectivity.
Introduction of a fluorine at this position (1i) resulted in a three-
1
R
R
R
11
12
e
fold decrease of CK1
similar potency on CK1
at this position (1j) further decreased CK1
(IC50 = 0.86 M).
An additional SAR study was conducted by synthesizing com-
pounds derived from 1f with focus on the modification of the
cyanobenzimidazole core (Table 2). Replacing the cyano group
with a methyl group (2) eroded the activity and highlighted the
importance of the water mediated hydrogen bond interaction be-
tween the cyano group and the side chains of Lys72, Glu86 and
Tyr90. Further attempts to replace the cyano group and/or mimic
its role, such as nitro 3, imidazole 4, isoxazole 5, or isobutyronitrile
c
potency relative to 1f while maintaining
, CK1d and GSK3b. A larger methyl group
potency
Route B:
a
H
N
c
N
H2N
d
c
Cl
l
O
N
H
H2N
CN
N
H
CN
CN
13
14
15
Scheme 1. Synthesis of benzimidazoles (1–7). Reagents and conditions: (a)
thiophosgene, K2CO3, CHCl3, room temperature; (b) 3,4-diamino benzonitrile,
EDC, THF, 70 °C, 35–50% for two steps; (c) 1,10-carbonyldiimidazole, CH2Cl2, room
temperature; (d) POCl3, DMF, 110 °C; (e) RNH2, MeSO3H, CH3CN, microwave, 140 °C,
39–62%.
6, all resulted in significant loss of CK1c activity. Introduction of a
methyl group next to the cyano group resulted in a five-fold less
active compound (7) than 1f. This is most likely due to a distortion
of the conformation of the benzimidazole avoiding a steric clash of
benzoxazole analog 8 was six-fold less active than 1f but there
was only a slight loss in CK1c activity for the benzothiazole
the methyl group with the gatekeeper Leu116 of CK1c3. The
compound 9. Imidazo[1,2–a]pyridine compound 10, a regioisomer

Z. Hua et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5392–5395
5395
In summary, a class of 2-phenylamino-6-cyano-1H-benzimi-
dazoles was discovered as potent CK1 inhibitors with excellent
Route C:
H2N
c
HN
HO2C
N
N
a
broad kinase and CK1 isoform selectivity starting from a high-
throughput screen. SAR efforts delivered compound 1h, which dis-
played good cellular potency and modest pharmacokinetic proper-
ties in rodents. The structural and SAR information obtained from
this study should provide useful insights into the future design of
b
Cl
N
N
N
Br
Br
Br
16
17
18
N
other isoform selective CK1c inhibitors with requisite pharmacoki-
netic properties for pharmacodynamic studies.
HN
CN
N
c
d
Cl
N
Acknowledgments
CN
19
10
The authors would like to acknowledge Liyue Huang and Daniel
Waldon for PKDM support. We thank Margaret Chu-Moyer for her
support and helpful discussions.
Scheme 2. Synthesis of Imidazo[1,2-a]pyridine compound 10. Reagents and
conditions: (a) chloroacetic acid, TEA, CH3CN, 85 °C, 53%; (b) POCl3, CH3CN, 85 °C,
60%; (c) Pd(PPh3)4, Zn(CN)2, 90 °C, DMF, 77%; (d) Pd2(dba)3, Me4t-BuXPhos, Cs2CO3,
150 °C, t-BuOH, 10%.
References and notes
1. Knippschild, U.; Gocht, A.; Wolff, S.; Huber, N.; Lohler, J.; Stoter, M. Cell.
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of benzimidazole 1f, exhibited similar CK1c activity but signifi-
2. Liu, C.; Li, Y.; Semenov, M.; Han, C.; Baeg, G. H.; Tan, Y.; Zhang, Z.; Lin, X.; He, X.
Cell 2002, 108, 837.
cantly decreased cell potency.
The isoform selective CK1c inhibitor 1h emerged from the SAR
studies with promising cellular potency and lipophilic efficiency
3. (a) Peters, J. M.; McKay, R. M.; McKay, J. P.; Graff, J. M. Nature 1999, 401, 345; (b)
Sakanaka, C.; Leong, P.; Xu, L.; Harrison, S. D.; Williams, L. T. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 12548; (c) Swiatek, W.; Tsai, I. C.; Klimowski, L.; Pepler, A.;
Barnette, J.; Yost, H. J.; Virshup, D. M. J. Biol. Chem. 2004, 279, 13011.
4. (a) Brockschmidt, C.; Hirner, H.; Huber, N.; Eismann, T.; Hillenbrand, A.;
Giamas, G.; Radunsky, B.; Ammerpohl, O.; Bohm, B.; Henne-Bruns, D.; Kalthoff,
H.; Leithauser, F.; Trauzold, A.; Knippschild, U. Gut. 2008, 57, 799; (b) Kim, S. Y.;
Dunn, I. F.; Firestein, R.; Gupta, P.; Wardwell, L.; Repich, K.; Schinzel, A. C.;
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Hahn, W. C. PLoS One 2010, 5, e8979.
and was therefore selected for further characterization. Compound
1h showed no inhibitory activity against an internal panel of 48 ki-
9
nases (>50 POC at 1
l
M) and was stable in both rat and human
liver microsomes (Clint <14 lL/min/mg). When dosed intrave-
nously in rat (0.5 mg/kg) compound 1h demonstrated moderate
clearance (1.3 L/h/kg) and half-life (1.5 h).17 However, high clear-
ance (14.4 L/h/kg) and short half-life (0.6 h) were observed when
dosed intravenously in mouse (1.5 mg/kg).
5. Davidson, G.; Wu, W.; Shen, J.; Bilic, J.; Fenger, U.; Stannek, P.; Glinka, A.;
Niehrs, C. Nature 2005, 438, 867.
6. Guo, X.; Waddell, D. S.; Wang, W.; Wang, Z.; Liberati, N. T.; Yong, S.; Liu, X.;
Wang, X. F. Oncogene 2008, 27, 7235.
The benzimidazole derivatives 1–7 were synthesized using two dif-
ferentapproachesshown in Scheme 1. Route Astartswith the synthesis
of arylisothiocyanates 12 prepared from the corresponding anilines 11
by reaction with thiophosgene in the presence of potassium carbonate.
In a one-pot transformation, the crude arylisothiocyanates 12 were
subsequently treated with 3,4-diaminobenzonitrile (13) in the pres-
ence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) to
deliver the desired benzimidazole derivatives 1–7 in yields ranging
from35%to 50%fortwo steps.18 AnalternativesyntheticrouteB started
with the conversion of 3,4-diaminobenzonitrile (13) to benzimidazo-
lone 14 by using 1,10-carbonyldiimidazole as carbonylating reagent.
Chlorination of the benzimidazolone 14 with phosphorous oxychloride
provided the necessary 2-choloro-1H-benzimidzaole intermediate
15.19 Finally, microwave assisted coupling of 2-choloro-1H-ben-
zimidzaole 15 with desired anilines in the presence of methanesulfonic
acid gave benzimidazole derivatives 1 in 39–62% yield. Amination of 2-
chloro-6-cyanobenzoxazole and 2-chloro-6-cyanobenzothiazole with
4-(tert-butyl)aniline provided benzoxazole 8 and benzothiazole 9,
respectively (similar to route B for the synthesis of benzimidazoles).
The synthesis of imidazo[1,2-a]pyridine compound 10 is illus-
trated in Scheme 2. Reaction of 2-amino-5-bromo-pyridine 16 with
chloroacetic acid in the presence of triethylamine provided imino-
pyridine acetic acid 17 in good yield. Conversion of 17 to desired
2-chloro-6-bromo-imidazolpyridine (18) was accomplished by
treatment with phosphorous oxychloride in refluxing acetoni-
trile.20 Selective Pd-catalyzed cynation of intermediate 18 afforded
2-chloro-6-cyano-imidazolpyridine 19. Amination of 19 using
palladium-catalyzed coupling with 4-(tert-butyl)aniline provided
desired compound 10 in 10% unoptimized yield.
7. Thorne, C. A.; Hanson, A. J.; Schneider, J.; Tahinci, E.; Orton, D.; Cselenyi, C. S.;
Jernigan, K. K.; Meyers, K. C.; Hang, B. I.; Waterson, A. G.; Kim, K.; Melancon, B.;
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E. Nat. Chem. Biol. 2010, 6, 829.
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Virshup, D. M. Oncogene 2011, 30, 2558.
9. Kinase binding assay, POC =% of control.
10. Takahashi-Yanaga, F.; Sasaguri, T. J. Pharm. Sci. 2009, 109, 179.
11. The cellular assay is an MSD assay that uses a PO4-LRP5/6 specific antibody
(pThr1479) and
a total LRP6 specific antibody in LRP6 and CK1c2
overexpressing HEK-293 cells.
12. Atomic coordinates for the co-crystal structures of 1a and 1f have been
deposited in the RCSB with PDB codes 4G16 and 4G17, respectively.
13. All the biological data presented in this paper is an average of replicates.
14. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
15. Lesson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
16. (a) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.;
Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Bioorg. Med. Chem. Lett. 2009, 19,
4406; (b) Cui, J. J.; Tran-Dube, M.; Shen, H.; Nambu, M.; Kung, P.-P.; Pairish, M.;
Jia, L.; Meng, J.; Funk, L.; Botrous, I.; McTigue, M.; Grodsky, N.; Ryan, K.;
Padrique, E.; Alton, G.; Timofeevski, S.; Yamazaki, S.; Li, Q.; Zou, H.;
Christensen, J.; Mroczkowski, B.; Bender, S.; Kania, R. S.; Edwards, M. P. J.
Med. Chem. 2011, 54, 6342.
17. Compound 1f having similar potency but lower LipE to compound 1h
demonstrated high clearance (6.48 L/h/kg) when dosed intravenously in rat
(0.58 mg/kg).
18. Carpenter, R. D.; Andrei, M.; Lau, E. Y.; Lightstone, F. C.; Liu, R.; Lam, K. S.; Kurth,
M. J. J. Med. Chem. 2007, 50, 5863.
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Gore, V. K.; Klionsky, L.; Ma, V. V.; Qian, Y -X.; Tamir, R.; Wang, X.; Xi, N.; Xu, S.;
Zhu, D.; Gavva, N. R.; Treanor, J. J. S.; Norman, M. H. J. Med. Chem. 2006, 49,
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