Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type i receptor kinase

Article abstract of DOI:10.1016/j.bmcl.2012.01.066

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzy

Full text of DOI:10.1016/j.bmcl.2012.01.066

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2024–2029
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors
of transforming growth factor-b type I receptor kinase
Hideaki Amada a, , Yoshinori Sekiguchi a, Naoya Ono a, Yuko Matsunaga a, Takeshi Koami a,
⇑
Hajime Asanuma a, Fumiyasu Shiozawa a, Mayumi Endo a, Akiko Ikeda b, Mari Aoki b,
Natsuko Fujimoto b, Reiko Wada b, Masakazu Sato a
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama, Saitama 331-9530, Japan
b Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-b (TGF-b) type I
receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were
evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-b-induced Smad2/3
phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,
3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited
good enzyme inhibitory activity (IC50 = 8.2 nM) as well as inhibitory activity against TGF-b-induced
Smad2/3 phosphorylation at a cellular level (IC50 = 32 nM).
Received 6 December 2011
Revised 5 January 2012
Accepted 10 January 2012
Available online 28 January 2012
Keywords:
Transforming growth factor-b
ALK5 inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
4-Thiazolylimidazoles
Transforming growth factor-b (TGF-b) is a cytokine that plays
important roles in the regulation of a variety of physiological
processes. It forms part of a family of cytokines that are involved
in cell growth and differentiation, matrix expression, and embry-
onic development. TGF-b belongs to the TGF-b superfamily, which
includes TGF-b1, TGF-b2, TGF-b3, activins, inhibins, and bone mor-
phogenetic proteins. TGF-b signals through two types of transmem-
brane serine/threonine kinase receptors, namely the TGF-b type I
receptor and the type II receptor (TGF-bRI and TGF-bRII, respec-
tively). TGF-bRI is also known as activin receptor-like kinase 5
(ALK5). TGF-b binding to TGF-bRII recruits and is followed by its
association with ALK5. Activated ALK5 in turn phosphorylates and
activates transcription factors Smad2/3, allowing them to bind to
the commonly mediated Smad4. These Smad complexes translocate
into the nucleus to affect gene transcription.1–3 The deregulation of
TGF-b signaling has been implicated in various human diseases,
such as, fibrosis,4 atherosclerosis,5 and cancer.6 Therefore, the inhi-
bition of ALK5 seems to be a good strategy for the treatment of these
diseases.
we took into consideration of the hydrogen bond ability and the
van der Waals’ radius and designed five- and six-membered hetero-
cycles as an alternative for 2-pyridyl group. Using a docking model,
which we built based on the X-ray structure of ALK5 co-crystallized
with its inhibitors,13,16 we predicted that the thiazolyl group might
be able to bind in a manner similar to that of the 2-pyridyl group
(Fig. 2). Therefore, we synthesized a series of 4-thiazolylimidazoles
to verify this hypothesis.
The compounds appearing in Table 1 were synthesized accord-
ing to Scheme 1. Commercially available 5-ethynyl-1,3-benzodiox-
ole 1 was used as the starting material and was reacted with either
bromothiazoles (2a, 2c–e) or iodothiazole 2b under reflux to give
acetylenes 3a–e. The oxidation of acetylenes 3a–e with DMSO
and PdCl2 afforded
a-diketones 4a–e. The resulting a-diketones
4a–e were reacted with 4-formylbenzonitrile and ammonium ace-
tate in acetic acid under reflux to give imidazole analogues 5a–e.
The cyano group in 5a–e was hydrolyzed with KOH in t-BuOH un-
der reflux to afford benzamide analogues 6a–e.
The synthesis of 2-substituted-4-(4-methylthiazol-2-yl)imidaz-
ole compounds is summarized in Schemes 2 and 3. Cyclization of
Many research groups have reported small-molecule inhibitors
of ALK5 (Fig. 1).3,7–15 According to the published literature on this
topic, a typical hydrogen bond acceptor, usually the 2-pyridyl group,
forms a water-mediated hydrogen bond with the side chains of
Tyr-249 and Glu-245 as well as the backbone of Asp-351. Initially,
a-diketone 4b with aldehydes 7a–c and ammonium acetate under
reflux followed by deprotection of the phthalimido group with
hydrazine monohydrate gave amine analogues 8a–c. The resulting
amines 8a–c were coupled to carboxylic acids (R2-CO2H) in the
presence of EDCÁHCl or DCC and HOBtÁH2O to give reverse amide
analogues 9a–d. The reaction of
a-diketone 4b with aldehydes
10a–d and ammonium acetate under reflux followed by hydrolysis
with NaOH gave carboxylic acids 11a–d. The carboxylic acids
⇑
Corresponding author. Tel.: +81 48 669 3029; fax: +81 48 652 7254.
E-mail address: hideaki.amada@po.rd.taisho.co.jp (H. Amada).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.066

H. Amada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2024–2029
2025
CH3
N
CH3
N
CH3
N
CH3
N
N
F
O
N
O
N
N
N
N
N
H
NH2
O
NH
NH
N
N
N
H
N
H
N
O
O
NH
O
N
N
SB-431542
SB-505124
GSK
GSK
Abbott
N
CH3
N
CH3
N
CH3
N
CN
N
N
N
HN
HN
S
N
N
N
N
N
N
H
N
N
N
LY550410
LY580276
IN-1166
A-83-01
F
N
Figure 1. Representative ALK5 inhibitors.
11a–d were reacted with amines (R4-NH2) to afford amide ana-
logues 12a–f. Alternatively, the carboxylic acid 11d was reacted
with SOCl2 under reflux to give the corresponding acid chloride.
The reaction of the resulting acid chloride with aqueous ammonia
gave pentanamide analogue 12g (Scheme 2).
Commercially available 2-(1,3-benzodioxol-5-yl)acetic acid 13
was converted to Weinreb amide. The Weinreb amide was reacted
with an anion of 4-methylthiazole generated by treatment with
n-BuLi to obtain ketone 14. Treatment of 14 with copper(II)
bromide followed by reaction with N-acetylguanidine under reflux
gave imidazole analogue 15. Deprotection of the acetyl group
under acidic conditions gave 2-aminoimidazole analogue 16. Acyl-
ation of the amino group in 16 with the corresponding acid chlo-
ride (R5-COCl) gave 17a and 17b (Scheme 3).
The imidazole analogues 18–20 were prepared from 2-ethynyl-
4-methylthiazole 21, 1-ethynyl-4-fluorobenzene 22, 6-ethynyl-
1,3-benzothiazole 23, and the corresponding reagents using the
same reaction conditions shown in Schemes 1 and 2. However,
the reaction of acetylene 24c with DMSO in the presence of PdCl2
Figure 2. X-ray crystal structure of LY580276 (brown) (PDB code: 1RW813) and the
predicted binding mode of 6b (orange) bound to the ATP site of ALK5.
gave
a-diketone 25c in a very low yield (6%). The oxidation of 24c
was accomplished by reaction with KMnO4 in a mixed solvent of
Table 1
acetone and buffer (NaHCO3, MgSO4 in H2O) to afford
a-diketone
Inhibitory profile of 4-thiazolylimidazoles 6a–e
25c in 73% yield (Scheme 4).17
All the compounds were evaluated for their ALK5 inhibitory
activity in an enzyme assay18 and their TGF-b-induced Smad2/3
phosphorylation inhibitory activity in a cell-based assay.19 The
R1
O
N
N
H
NH2
O
unsubstituted thiazole ring analogue 6a showed
a tolerable
potency. The introduction of a methyl group at the 4-position of
the thiazole ring markedly increased ALK5 inhibition in the enzyme
inhibitory activity and cellular activity assays (6b). The introduction
of either an ethyl group or a bromo group at this position resulted in
a moderate potency (6c and 6d). Replacement of the 4-methylthia-
zol-2-yl group with a 2-methylthiazol-4-yl group led to a decrease
in the enzyme inhibitory activity and the cellular activity. Therefore,
we identified 6b as the initial lead compound (Table 1).
Subsequently, we focused on developing SAR with respect to
the benzamide moiety of the initial lead 6b. Replacement of the
benzamide moiety was expected to improve the physicochemical
properties. The SAR observed with a substituent at the 2-position
of the imidazole ring are summarized in Table 2. The introduction
of aminoalkyl groups significant decreased the inhibition activity,
even though the potency of the 2-aminoimidazole analogue 16
was maintained (8a–c vs 16). These results suggest that the aro-
matic amine 16 with a weak basicity is stronger for ALK5 kinase
inhibition than the aliphatic amines 8a–c with basicity. Conversion
from the amino group in 16 to either a benzamido group or an
O
Compound
R1
IC50 (nM)
Smad2/3a
ALK5a
1100
N
S
6a
6b
6c
6d
6e
2700
170
760
390
680
H3C
N
S
54
340
270
370
N
S
H3C
Br
N
S
H3C
N
S
a
Values are the mean of two or more separate experiments.

2026
H. Amada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2024–2029
R1
O
O
R1
O
O
a or b or c
O
O
O
O
d
e
X
R1
2a-e
+
1
3a-e
4a-e
R1
R1
O
N
N
f
CN
N
H
NH2
N
H
O
O
5a-e
6a-e
O
O
Scheme 1. Reagents and conditions: (a) preparation of 3a: PdCl2(PPh3)2, CuI, NEt3, CHCl3, reflux; (b) preparation of 3b, 3c, and 3e: Pd(PPh3)4, NEt3, CH3CN, reflux; (c)
preparation of 3d: Pd(PPh3)4, CuI, NH(i-Pr)2, THF, rt; (d) PdCl2, DMSO, 125 °C; (e) 4-formylbenzonitrile, NH4OAc, AcOH, reflux; (f) KOH, t-BuOH, reflux.
H3C
H3C
O
N
N
R2
O
OHC
( ) N
NH2
n
N
HN
( )
N
n
S
c
S
( )
n
O
N
H
N
H
O
O
9a (n=1, R2=Ph)
9b (n=1, R2=n-Pr)
9c (n=2, R2=n-Pr)
9d (n=3, R2=n-Pr)
7a (n=1)
8a (n=1)
8b (n=2)
7b (n=2)
O
O
7c
(n=3)
8c
(n=3)
a, b
4b
H3C
H3C
d, e
O
N
N
O
O
R4
NH
OH
N
N
S
f or g
S
( )
( )
OHC
OR3
n
n
( )
N
H
N
H
n
O
O
4
3
11a
12a
12b
10a
(n=0)
(n=0, R =Ph)
(n=0, R =Et)
O
O
4
10b (n=2, R3=Me)
10c (n=3, R3=Me)
10d (n=4, R3=Me)
(n=0, R =n-Pr)
11b (n=2)
11c (n=3)
12c (n=2, R4=n-Pr)
12d (n=3, R4=n-Pr)
12e (n=4, R4=Ph)
12f (n=4, R4=n-Pr)
11d
(n=4)
(n=4, R4=H)
12g
Scheme 2. Reagents and conditions: (a) 7a–c, NH4OAc, THF-MeOH, reflux; (b) N2H4ÁH2O, EtOH, reflux; (c) R2-CO2H, EDCÁHCl or DCC, HOBtÁH2O, DMF, rt; (d) 10a–d, NH4OAc,
THF–MeOH, rt or reflux; (e) NaOH, MeOH–H2O, reflux; (f) preparation of 12a–f: R4-NH2, EDCÁHCl or DCC, HOBtÁH2O, DMF, rt; (g) preparation of 12g: (i) SOCl2, CHCl3, reflux;
(ii) NH4OH, CHCl3, rt.
H3C
H3C
H3C
N
S
H3C
N
S
a, b
HO
O
I
N
N
O
N
O
O
S
N
S
a, b, c
d, e
c
NH
Ac
2b
21
N
H
O
O
H3C
N
S
d
R7
13
O
14
15
R7
O
H3C
H3C
7
7
22
24a
24b
: R = 4-fluorophenyl
: R = 4-hydroxyphenyl
N
7 = 1,3-benzothiazol-6-yl
: R7 = 4-fluorophenyl
O
23:
R
R5
24c: R7 =1,3-benzothiazol-6-yl
N
N
S
S
f
g
NH
NH2
H3C
N
H
17a
17b
N
H
O
O
N
O
accordance with
scheme 2
5
16
(R =Ph)
(R =n-Pr)
e or f
O
O
S
5
18-20
R7
O
: R7 = 4-hydroxyphenyl
: R7 = 4-fluorophenyl
Scheme 3. Reagents and conditions: (a) SOCl2, toluene, 60 °C; (b) N,O-dim-
ethylhydroxylamine hydrochloride, NaOH, H2O–toluene, 0 °C-rt; (c) 4-methylthia-
zole, n-BuLi, THF, À78 °C; (d) CuBr2, AcOEt, reflux; (e) N-acetylguanidine, CH3CN,
reflux; (f) H2SO4, MeOH–H2O, reflux; (g) R5-COCl, pyridine, rt or reflux.
25a
25b
25c: R7 = 1,3-benzothiazol-6-yl
Scheme 4. Reagents and conditions: (a) trimethylsilylacetylene, PdCl2(PPh3)2, CuI,
NEt3, reflux; (b) K2CO3, MeOH, rt; (c) 4-iodophenol, PdCl2(PPh3)2, CuI, NEt3, reflux;
(d) 2b, Pd(PPh3)4, NEt3, CH3CN, reflux; (e) PdCl2, DMSO, 125 °C; (f) KMnO4, acetone-
buffer (NaHCO3, MgSO4, H2O), rt.
alkanamido group resulted in a large loss in enzyme and cellular
potency (15, 17a, and 17b). Interestingly, replacement of the amino
group in 8a with either a benzamido or a butanamido group that
had a non-basicity resulted in good potency (9a and 9b). However
butanamide analogues 9c and 9d with a longer linker chain
compared with 9b showed a decreased potency. On the other hand,
the aromatic carboxylic acid analogue 11a showed a moderate
enzyme inhibitory activity. As the linker chain grew in length,
the aliphatic carboxylic acid analogues 11b–d displayed an

H. Amada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2024–2029
2027
Table 2
Inhibitory profile of 4-(4-methylthiazol-2-yl)imidazoles 8a–c, 9a–d, 11a–d, 12a–g, 15, 16, 17a, and 17b
H3C
N
N
S
R6
N
H
O
O
Compound
R6
IC50 (nM)
ALK5a
Smad2/3a
8a
8b
8c
–CH2NH2
–(CH2)2NH2
–(CH2)3NH2
O
790
2200
810
3600
1900
1000
9a
150
680
N
H
O
9b
9c
9d
63
350
N
H
CH3
CH3
H
N
420
600
1600
1400
O
O
N
H
CH3
11a
11b
11c
11d
–CO2H
190
530
130
30
NEb
NEb
NEb
NEb
–(CH2)2CO2H
–(CH2)3CO2H
–(CH2)4CO2H
O
12a
12b
12c
12d
>1000c
>1000c
380
1700
3900
1000
1700
N
H
O
CH3
N
H
O
CH3
N
H
H
N
CH3
510
O
O
O
O
12e
170
830
N
H
CH3
12f
130
110
750
730
N
H
12g
NH
2
H
N
CH3
15
16
1100
90
1900
280
O
–NH2
H
N
17a
17b
>4000
>4000
NEb
O
H
N
CH3
NH2
2100
O
O
6b
54
170
a
b
c
Values are the mean of two or more separate experiments.
Not evaluated.
The right IC50 values could not be calculated because of their low solubility.

2028
H. Amada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2024–2029
Table 3
showed a potent enzyme inhibitory activity (18). This result might
be due to its low membrane permeability. The introduction of a
4-fluorophenyl group displayed poor activities in both the enzyme
and the cell-based assay (19). The introduction of a 1,3-ben-
zothiazol-6-yl group resulted in an approximately eightfold
increase in the enzyme inhibition activity and an elevenfold
increase in the cellular activity, compared with the 1,3-ben-
Inhibitory profile of 4-(4-methylthiazol-2-yl)imidazoles 18–20
H3C
CH3
N
HN
N
S
O
R7
N
H
zodioxol-5-yl group (20 vs 9b). Compound 20 (18.1 lg/mL) had a
solubility in water that was about 200 times higher than that of
the initial lead 6b.
Compound
R7
IC50 (nM)
ALK5a
130
Smad2/3a
To examine the binding mode of 20 in the ATP binding site of
ALK5, 20 was docked into the molecular model.13,16 As shown in
Figure 3, the benzothiazole ring binds to the hinge region and
accepts a hydrogen bond from the backbone NH of His-283. The
4-methylthiazol-2-yl nitrogen atom forms water-mediated
networks of hydrogen bonds with the carboxy oxygen of Glu-245,
the hydroxy hydrogen of Tyr-249, and the backbone NH of Asp-
351. The amide moiety at the 2-position of the imidazole ring is
assumed to form a ring through an intramolecular hydrogen bond
in the sugar region. Alternatively, many polar residues exist near
the entrance of the pocket, and the amide moiety is considered to
be committed to an additional hydrogen bond to polar residues or
a water-mediated hydrogen bond to them without ring formation.20
Compound 20, the most potent analogue, was evaluated for
selectivity using a diverse kinase panel.21 Compound 20 was highly
selective against most of the kinases; however, moderate inhibi-
tion was observed for KDR (94%), HGK (75%), CK1d (63%), LYN
18
19
HO
F
1200
1800
6400
N
20
9b
8.2
32
S
O
63
350
O
a
Values are the mean of two or more separate experiments.
increase in potency. The amide group, which was directly linked to
the imidazole ring, displayed a significant loss in enzyme and
cellular potency (12a and 12b). Conversion from the most potent
aliphatic carboxylic acid analogue 11d to pentanamide analogue
12g was tolerable with regard to the enzyme inhibition activity.
The introduction of either a phenyl group or a n-propyl group to
a terminally carbamoyl group in 12g resulted in a slight decrease
in potency (12g vs 12e and 12f). The N-propylamide analogues
12c and 12d showed weak potency compared with the corre-
sponding 12f. We thought that the position of the amide linkage
and an appropriate number of atoms in the linker were very impor-
tant for potency (9b vs 9c, 9d, 12b–d, 12f, 15, and 17b). Compound
(60%), LCK (58%) and p38a (57%) at 10 lM.
In conclusion, we have described that the synthesis, enzyme
inhibitory activity, inhibitory activity against TGF-b-induced
Smad2/3 phosphorylation at the cellular level, SAR, and proposed
ALK5 binding mode of a novel series of 4-thiazolylimidazoles. We
found that the thiazolyl group could be replaced with 2-pyridyl
group. The improvement of the physicochemical properties was
achieved by replacement of the benzamide moiety with an
alkylamide moiety. Compound 20 showed the most ALK5
inhibitory activity and also was highly selective for other kinases.
9b maintained the inhibitory activities; furthermore, 9b (15.7
mL) was about 170 times more soluble in water than 6b
(0.09 g/mL).
lg/
Supplementary data
l
Next, we investigated the effect of the substituent at the 5-posi-
tion of the imidazole ring. The ALK5 inhibitory activities of these
compounds are summarized in Table 3. Replacement of the 1,
3-benzodioxol-5-yl group with a 4-hydroxyphenyl group led to
the loss of inhibition activity in a cell-based assay, even though it
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.066.
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Figure 3. Predicted binding mode of 20 bound to the ATP site of ALK5. Hydrogen
bonds are shown as dotted lines. The protein surface is colored according to the
residue type (magenta, polar; green, hydrophobic; red, exposed).

H. Amada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2024–2029
2029
16. The binding model was examined and visualized using MOE™ (Molecular
Operating Environment) Version 2009.10., Chemical Computing Group:
Montreal, Canada.
19. A description of the assay conditions for inhibitory activity against TGF-b-
induced Smad2/3 phosphorylation in whole cells can be found in
Supplementary data.
17. Srinivasan, N. S.; Lee, D. G. J. Org. Chem. 1979, 44, 1574.
18. A description of the assay conditions for ALK5 inhibitory activity at the enzyme
level can be found in Supplementary data.
20. The calculated conformation of 20 can be found in Supplementary data.
21. A description of the assay conditions for the kinase selectivity profile can be
found in Supplementary data.