Design and biological evaluation of imidazo[1,2-a]pyridines as novel and potent ASK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.084

Imidazo[1,2-a]pyridine derivatives were designed, synthesized, and evaluated as inhibitors of the apoptosis signal-regulating kinase 1 (ASK1). These were based on a benzothiazole derivative that was discovered from high-throughput screening of our compound library. As a result, we identified potent, selective, and orally bioavailable ASK1 inhibitors for wide range of therapeutic targets.

Full text of DOI:10.1016/j.bmcl.2012.10.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7326–7329
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and biological evaluation of imidazo[1,2-a]pyridines as novel
and potent ASK1 inhibitors
⇑
Yoshito Terao , Hideo Suzuki, Masato Yoshikawa, Hiroaki Yashiro, Shiro Takekawa, Yasushi Fujitani,
Kengo Okada, Yoshihisa Inoue, Yoshio Yamamoto, Hideyuki Nakagawa, Shuhei Yao, Tomohiro Kawamoto,
Osamu Uchikawa
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Imidazo[1,2-a]pyridine derivatives were designed, synthesized, and evaluated as inhibitors of the apop-
tosis signal-regulating kinase 1 (ASK1). These were based on a benzothiazole derivative that was discov-
ered from high-throughput screening of our compound library. As a result, we identified potent, selective,
and orally bioavailable ASK1 inhibitors for wide range of therapeutic targets.
Received 20 August 2012
Revised 16 October 2012
Accepted 17 October 2012
Available online 23 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
ASK1
Imidazo[1,2-a]pyridine
LLE
Apoptosis signal-regulating kinase 1 (ASK1) was originally iden-
tified as a member of the mitogen-activated protein kinase kinase
kinase (MAP3K) family that activates both p38 MAP kinase and
c-Jun N-terminal kinase (JNK) pathways.1 ASK1 is stimulated by
various cell stressors including cytotoxic cytokines, reactive
oxygen species (ROS), and endoplasmic reticulum stress. Recent
studies revealed that ASK1 contributes not only to the regulation
of cell death, but also to cytokine responses, cell differentiation,
and immune regulation. Therefore, ASK1 inhibitors are thought
to have potential for the protection of cells from various stresses
in wide-ranging pathological situations such as autoimmune dis-
ease, diabetes, cardiovascular disease, neurodegenerative disor-
ders, and inflammatory disorders.2,3 For example, ASK1 is
To obtain useful compounds for exploring ASK1 inhibition, we
focused our early efforts on the discovery of potent, selective,
and orally bioavailable ASK1 inhibitors. We initially identified ben-
zothiazole 1 (IC50: 260 nM), with inhibitory activity against ASK1,
by high-throughput screening of our in-house compound library
(Fig. 1). On the basis of our speculation that compound 1 is as an
ATP-competitive inhibitor, the essential interactions were assumed
to be hydrogen bonds between the aminothiazole moiety and
Val757 in the hinge region of ASK1.11 We also speculated that
the tert-butylphenyl moiety of compound 1 was directed to the
solvent-exposed region, while the benzothiazole was located at
the inner binding pocket (Fig. 2). On the other hand, compound 1
showed high lipophilicity (logD, measured at pH 7.4) and low
ligand-lipophilicity efficiency (LLE, pIC50–logD), which often raises
the issue of undesirable physical properties and ADME (absorption,
distribution, metabolism, and elimination) profiles and promotes
interactions with other protein kinases and adverse biological
activities.12 To avoid these issues, our approach was aimed at the
enhancement of the LLE and the reduction of the lipophilicity
through the transformation of the benzothiazole core and the
introduction of hydrophilic moieties on compound 1. First, the
2-aminoimidazo[1,2-a]pyridine derivative was designed to reduce
the lipophilicity of compound 1. This scaffold was reported as a
new class of ATP-competitive kinase inhibitors.13,14 As a result,
compound 3a showed a reduction in lipophilicity (logD: 3.78).
Interestingly, compound 3a (IC50: 22 nM) was 10 times more
potent than compound 1, which has the benzothiazole with the
same substituents as 3a. This improvement is presumably due to
activated by the tumor necrosis factor a, which impairs insulin ac-
tion.4 A high glucose level also induces activation of the ASK1 sig-
nal that mediates cellular senescence, which is assumed to lead to
vascular aging in diabetic patients.5 Thus, ASK1 is related to the
pathological situation occurring in relation to diabetes. In addition,
amyloid b induces neuronal cell death, which plays a central role in
Alzheimer’s disease through ROS-mediated ASK1 activation.6 Fur-
thermore, ASK1 knockout mice inhibited Angiotensin II-induced
cardiac hypertrophy.7 Therefore, inhibition of ASK1 could have
great therapeutic potential in terms of the treatment of these dis-
eases. However, there have so far been few reports on selective
ASK1 inhibitors.8–10
⇑
Corresponding author. Tel.: +81 466 32 1058.
E-mail address: yoshito.terao@takeda.com (Y. Terao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.084

Y. Terao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7326–7329
7327
N
S
presence of a catalytic amount of CuI gave the N–H arylated com-
pound 9. Imidazole was also used as a coupling substrate to give
compound 10. The arylation of isobutyraldehyde with aryl bromide
HN
O
Me
Me
Me
Me
11 in the presence of Pd(OAc)2-P(t-Bu)3 using Cs2CO3 gave the
a-
log D 5.08
1, IC50 260 nM
arylated aldehyde 12, which led to the propanol derivative 16
(Scheme 2).17 Treatment of ester 17, which was synthesized in a
same manner as the procedure in compound 10, with MeMgBr
gave tert-alcohol 18.
LLE 1.51
Figure 1. Benzothiazole derivative 1 (hit compound).
Among the halogen-substituted compounds (3b–d), the Cl
derivative 3c (IC50: 16 nM) showed potent inhibitory activity
(Table 1). Introduction of a hydroxymethyl moiety (5) for the
reduction of log D led to a decrease in inhibitory activity, while
the LLE value was comparable to that of compound 3a, suggesting
that the introduction of a polar moiety was tolerable in terms of
LLE. To explore the potential of aromatic substituents, we synthe-
sized and evaluated several compounds. The phenyl compound
7 (IC50: 82 nM) had a reduced LLE value (2.26). On the other hand,
the 3-pyridyl derivative 8 (IC50: 21 nM) with polar substituents
showed not only potent inhibitory activity, but also an efficient
LLE value (4.01). The 1-imidazolyl derivative 10 (LLE: 4.69) showed
a higher LLE value than the 1-pyrrolyl derivative 9 (LLE: 3.28).
These results suggested that the nitrogen atom at the 3-position
of the pyridyl and imidazolyl moieties might be involved in an
interaction with ASK1. This hypothesis was confirmed when the
co-crystal structure of compound 10 with ASK1 revealed direct
interaction between the imidazole nitrogen and the side chain of
Lys709 (Fig. 3).18 In addition, several other interactions were ob-
served, including the water-mediated interaction between the
amide carbonyl of the compound and Ser761, as shown in Figure 3
(dotted lines). As we expected, two hydrogen bonds interactions
between the imidazo[1,2-a]pyridine and Val757 were detected in
the hinge region. The protein–ligand complex also suggested that
Hinge region
N-terminal
domain
Val757
H
N
C-terminal
domain
O
N
H
inner
binding
pocket
O
N
Me
HN
6
Me
solvent-
exposed
region
S
Me
Me
O
Figure 2. Binding mode of compound 1 in ASK1.
the increase of the pKa (calc) value on the nitrogen at the 1-posi-
tion compared to the benzothiazole (1: 5.43 versus 3a: 10.07),
enhancing the electron-donating ability. The LLE value also in-
creased dramatically from 1.51 to 3.88, suggesting that the core
structure has the potential for further optimization as a proficient
ASK1 inhibitor.
2-Aminoimidazo[1,2-a]pyridines15 with several kinds of sub-
stituents at the 6-position (3, 5–10) and on the tert-butyl moiety
(16 and 18) were prepared for the investigation of the structure–
activity relationship (SAR) (Scheme 1). Aldehyde 4, leading to com-
pounds 5 and 6, was prepared from aryl iodide 3e and i-PrMgCl by
halogen–metal exchange followed by the addition of DMF as an
electrophile.16 The reaction of aryl iodide 3e and pyrrole in the
N
N
a
Me
Me
Me
HN
O
H2N
N
N
R1
R1
2a (R1 = Me)
2b (R1 = F)
2c (R1 = Cl)
2d (R1 = Br)
2e (R1 = I)
3a (R1 = Me)
3b (R1 = F)
3c (R1 = Cl)
3d (R1 = Br)
3e (R1 = I)
N
N
N
b
c
Me
Me
Me
HN
Me
Me
HN
3e
N
H
N
N
OH
O
Me
O
O
4
5
Me
NH
d
Me
HN
Me
Me
O
6
N
e
Me
Me
Me
HN
O
3d
3e
N
R1
7 (R1 = Ph), 67%
8 (R1 = 3-pyridyl), 38%
N
f
Me
Me
Me
HN
N
N
X
O
9 (X = CH), 20%
g
10 (X = N)
, 48%
10•HCl
Scheme 1. Synthesis of 6-substituted imidazo[1,2-a]pyridine derivatives. Reagents and conditions: (a) 4-t-butylbenzoyl chloride, NEt3, THF, room temp, 10–69%;
(b) i-PrMgCl, DMF, THF; (c) LiBH4, THF, room temp, 18% (2 steps); (d) MeNH2, NaBH(OAc)3, AcOH, CH2Cl2–DMF, room temp, 25% (2 steps); (e) ArB(OH)2, Na2CO3, Pd(PPh3)4,
DME-H2O, 100 °C; (f) imidazole, K2CO3, CuI, DMF, reflux, 43%; (g) HCl in ethyl acetate, room temp, 60%.

7328
Y. Terao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7326–7329
H
OEt
O
O
Me
Me
OEt
O
HO
Me
Me
OEt
O
a
b
c
Br
11
12
13
TBDMS
TBDMS
N
O
Me
Me
OEt
O
O
Me
Me
OH
HO
Me
Me
HN
O
d
e, f, g
N
N
N
O
14
15
16
N
N
HN
h
HN
O
HO
Me
Me
N
N
N
N
N
N
EtO
O
O
17
18
Scheme 2. Synthesis of compounds 16, 18. Reagents and conditions: (a) isobutyraldehyde, Cs2CO3, Pd(OAc)2–P(t-Bu)3, dioxane, reflux; (b) NaBH4, THF, room temp, 44% (2
steps); (c) TBDMSOTf, 2,6-lutidine, CH2Cl2, room temp, quant; (d) KOH (aq), MeOH-THF, room temp, 82%; (e) (COCl)2, DMF, CH2Cl2 and then 3e, NEt3, THF, room temp, 35%; (f)
imidazole, K2CO3, CuI, DMF, reflux, 54%; (g) TBAF, THF, room temp, 74%; (h) MeMgBr, THF, reflux, 41%.
Table 1
Table 2
In vitro activities of imidazo[1,2-a]pyridine derivatives
Selectivity profile of compound 10ÁHCl for representative kinases
Kinase
IC
Kinase
IC
(lM)
50
(l
M)
N
50
R2
Me
Me
HN
O
ASK2
MEKK1
TAK1
IKKb
0.51
>10
>10
>10
>10
JNK1
>10
>10
>10
>10
>10
N
R1
p38a
gsk3b
PKCh
B-raf
Compound
R1
R2
IC50 (nM)a
logDb
LLEc
ERK1
3a
3b
3c
3d
5
6
7
8
9
Me
F
Cl
Br
CH2OH
CH2NHMe
1-Phenyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
22
34
16
3.78
3.55
4.04
4.17
2.83
2.40
4.83
3.60
4.24
3.16
1.62
1.27
3.88
3.92
3.76
5.51
4.09
3.66
2.26
4.01
3.28
4.69
5.87
5.88
were designed to occupy the hydrophilic space. Consequently,
these compounds showed very high LLE values and tolerable IC50
values, demonstrating the potential of additional interactions.
Further in vitro profiles were evaluated using the 1-imidazoly
derivatives. For preliminary kinase selectivity among representa-
tive kinases, compound 10ÁHCl was found to be a selective inhibi-
tor, except for ASK2 (Table 2).19 As a cell assay of ASK1 inhibitors,
the downstream phosphorylation of JNK and p38 was examined
using compound 10. Compound 10 was found to inhibit streptozo-
21
129
870
82
21
30
14
32
70
3-Pyridyl
1-Pyrrolyl
1-imidazolyl
1-imidazolyl
1-imidazolyl
10
16
18
HOCH2
HO
a
b
c
All values are averages of n = 2.
The logD values were determined at pH 7.4.
The LLE values were determined by pIC50–logD.
tocin (STZ)-induced JNK in INS-1 pancreatic b cells from 0.3 lM
(Fig. 4).20 Phosphorylation of p38 was also inhibited in a dose-
dependent manner. In addition, pharmacokinetic profiles in rats
were tested for the representative compounds. Good oral bioavail-
ability was detected for compounds 10ÁHCl and 16 compared to
that of the benzothiazole derivative 1 (Table 3). These findings
the nitrogen atoms of the Lys769 and Arg705 side chains were
present near the t-butyl group in the outer pocket. Compounds
16 and 18, with a hydroxyl group at the terminal lipophilic moiety,
N
Me
Me
Me
HN
O
N
N
N
10
Figure 3. Co-crystal structure of ASK1 and compound 10.

Y. Terao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7326–7329
7329
References and notes
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(review article).
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Signaling Gateway, (review article) 2010, doi:http://dx.doi.org/10.1038/
mp.a002816.01.
4. Imoto, K.; Kukidome, D.; Nishikawa, T.; Matsuhisa, T.; Sonoda, K.; Fujisawa, K.;
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Araki, E. Diabetes 2006, 55, 1197.
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Kawamoto, H.; Ichijo, H.; Ogihara, T. Diabetes 2006, 55, 1660.
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Masutani, H.; Yodoi, J.; Urano, Y.; Nagano, T.; Ichijo, H. Cell Death Differ. 2005,
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Ichijo, H.; Iwao, H. Circ. Res. 2003, 93, 874.
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D.; Jorand-Lebrun, C.; Muzerelle, M.; Vitte, P. A.; Rückle, T.; Kimura, A.;
Kohyama, K.; Matsumoto, Y.; Ichijo, H.; Harada, T. EMBO J. 2010, 2, 504.
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Nagano, T. Bioorg. Med. Chem. 2011, 19, 486.
Figure 4. Inhibiton of STZ-induced MAPK phosphorylation in INS-1 cells.
10. Volynets, G. P.; Chekanov, M. O.; Synyugin, A. R.; Golub, A. G.; Kukharenko, O.
P.; Bdzhola, V. G.; Yarmoluk, S. M. J. Med. Chem. 2011, 54, 2680.
11. Recently, two different groups, Nagano9 et al. and Yarmoluk10 et al., have
reported the importance of the hydrogen bonding between Val757 and ASK1
inhibitors according to in silico modeling using 9H-purin-2-amine and 3,4-
dihydronaphthalen-1(2H)-one scaffolds, respectively.
12. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
13. Hamdouchi, C.; Zhong, B.; Mendoza, J.; Collins, E.; Jaramillo, C.; De Diego, J. E.;
Robertson, D.; Spencer, C. D.; Anderson, B. D.; Watkins, S. A.; Zhang, F.; Brooks,
H. B. Bioorg. Med. Chem. Lett. 2005, 15, 1943.
Table 3
Pharmacokinetic parameters of compoundsa
compound
Cmax (ng/mL)
Tmax (h)
AUCpo,0À8h (ng.h/mL)
F (%)
1
—
—
<10
275.4
407.4
—
41.1
41.0
10ÁHCl
285.1
101.3
1.67
1.00
16
14. Stec, M. M.; Andrews, K. L.; Booker, S. K.; Caenepeel, S.; Freeman, D. J.; Jiang, J.;
Liao, H.; McCarter, J.; Mullady, E. L.; SanMiguel, T.; Subramanian, R.; Tamayo,
N.; Wang, L.; Yang, K.; Zalameda, L. P.; Zhang, N.; Hughes, P. E.; Norman, M. H. J.
Med. Chem. 2011, 54, 5174.
Average of three rats. F means bioavailability.
a
Rat cassette doing at 0.1 mg/kg, iv and 1 mg/kg, po.
15. Hamdouchi, C.; de Blas, J.; del Prado, M.; Gruber, J.; Heinz, B. A.; Vance, L. J. Med.
Chem. 1999, 42, 50.
16. Jaramillo, C.; Carretero, J. C.; de Diego, J. E.; del Prado, M.; Hamdouchi, C.;
Roldan, J. L.; Sanchez-Martinez, C. Tetrahedron Lett. 2002, 43, 9051.
17. Terao, Y.; Fukuoka, Y.; Satoh, T.; Miura, M.; Nomura, M. Tetrahedron Lett. 2002,
43, 101.
18. The crystal structure of compound 10 bound to ASK1 has been deposited in the
Protein Data Bank with deposition code 3VW6.
19. Compound 10 HCl was also tested for its inhibitory activities toward a panel of
indicated that the lowered lipophilicity might contribute to the
improvement of the PK profiles.
In summary, we have identified potent ASK1 inhibitors with
high LLE values. This discovery could serve to provide promising
and useful compounds for elucidating potential ASK1 inhibitors
for medicinal applications; this will be addressed in future studies.
195 kinases (one dose assay at 1
kinases were less than 50%.
lM). The inhibitory activities towards 187
20. See protocol given in supplementary data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.084.