Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part 1

Article abstract of DOI:10.1002/cmdc.201900129

We identified a lead series of p38 mitogen-activated protein kinase inhibitors using a structure-based design strategy from high-throughput screening of hit compound 1. X-ray crystallography of 1 with the kinase showed an infrequent flip of the peptide bond between Met109 and Gly110, which was considered to lead to high kinase selectivity. Our structure-based design strategy was to conduct scaffold transformation of 1 with maintenance of hydrogen bond interactions with the flipped hinge backbone of the enzyme. In accordance with this strategy, we focused on scaffold transformation to identify imidazo[4,5-b]pyridin-2-one derivatives as potent inhibitors of the p38 MAP kinase. Of the compounds evaluated, 21 was found to be a potent inhibitor of the p38 MAP kinase, lipopolysaccharide-induced tumor necrosis factor-α (TNF-α) production in human monocytic leukemia cells, and TNF-α-induced production of interleukin-8 in human whole blood cells. Herein we describe the discovery of potent and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors that suppressed cytokine production in a human whole blood cell-based assay.

Full text of DOI:10.1002/cmdc.201900129

Accepted Article
Title: Structure-based Design, Synthesis, and Biological Evaluation of
Imidazo[4,5-b]pyridin-2-one-based p38 MAP Kinase Inhibitors:
Part 1
Authors: Akira Kaieda, Masashi Takahashi, Hiromi Fukuda, Rei
Okamoto, Shinji Morimoto, Masayuki Gotoh, Takahiro
Miyazaki, Yuri Hori, Satoko Unno, Tomohiro Kawamoto,
Toshimasa Tanaka, Sachiko Itono, Terufumi Takagi, Hiroshi
Sugimoto, Kengo Okada, Gyorgy Snell, Ryan Bertsch,
Jasmine Nguyen, Bi-Ching Sang, and Seiji Miwatashi
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900129
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Structure-based Design, Synthesis, and
Biological Evaluation of Imidazo[4,5-b]pyridin-
2-one-based p38 MAP Kinase Inhibitors: Part 1
Akira Kaieda,*[a] Masashi Takahashi,[a] Hiromi Fukuda,[a] Rei Okamoto,[a] Shinji Morimoto,[a] Masayuki
Gotoh,[a] Takahiro Miyazaki,[a] Yuri Hori,[a] Satoko Unno,[a] Tomohiro Kawamoto,[a] Toshimasa Tanaka,[a]
Sachiko Itono,[a] Terufumi Takagi,[a] Hiroshi Sugimoto,[a] Kengo Okada,[a] Gyorgy Snell,[b] Ryan
Bertsch,[b] Jasmine Nguyen,[b] Bi-Ching Sang,[b] and Seiji Miwatashi[a]
[a]
[b]
A. Kaieda, M. Takahashi, H. Fukuda, R. Okamoto, S. Morimoto, M. Gotoh, T. Miyazaki, Y. Hori, S. Unno, T. Kawamoto, T. Tanaka, S. Itono, T. Takagi, H.
Sugimoto, K. Okada, S. Miwatashi
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited., 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
E-mail: akira.kaieda@takeda.com
G. Snell, R. Bertsch, J. Nguyen, B. Sang
Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
Abstract: We identified a lead series of p38 mitogen-activated
protein kinase inhibitors using a structure-based design strategy
from high-throughput screening of hit compound 1. X-ray
crystallography of 1 with the kinase showed an infrequent flip of the
peptide bond between Met109 and Gly110, which was considered
to lead to high kinase selectivity. Our structure-based design
analysis of differential expression, localization, and activation of
these MAP kinases in synovial tissue and cells extracted from
patients with rheumatoid arthritis (RA), indicated the
overactivation of the p38α isoform within the inflammatory
tissue.[11-13] Therefore, the development of p38 MAP kinase
inhibitors has been considered a promising solution for RA
treatment.[14-18]
strategy was to conduct scaffold transformation of
1 with
maintenance of hydrogen bond interactions with the flipped hinge
backbone of the enzyme. In accordance with this strategy, we
focused on scaffold transformation to identify imidazo[4,5-b]pyridin-
2-one derivatives as potent inhibitors of the p38 MAP kinase. Of the
compounds evaluated, 21 was a potent inhibitor of the p38 MAP
kinase, lipopolysaccharide-induced tumor necrosis factor-α(TNF-α)
production in human monocytic leukemia cells, and TNF-α-induced
production of interleukin-8 in human whole blood cells. Herein, we
have described the discovery of potent and orally bioavailable
imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors that
suppressed cytokine production in a human whole blood cell-based
assay.
Numerous preclinical studies have reported that the
inhibition of p38 MAP kinase effectively suppressed TNF-α
production both in vitro and in vivo, and multiple chemical
classes of p38 MAP kinase inhibitors have been discovered
(Figure 1, 2, and 3). Unfortunately, BIRB-796[19,20], Scio-
469[19,21], BMS-582949[19,22], VX-702[19,23,24], Ro-4402257[19,25]
and TAK-715[16,26] have been discontinued, mainly owing to lack
of efficacy. However, other inhibitors, including VX-745[19,27]
,
,
BCT-197, LY-2228820, GW856553[19,28,29], PH-797804, AZD-
7624, CHF-6297, FX-005, and ARRY-797[19]
examined in Phase II clinical trials.[30]
, have been
Introduction
The p38α mitogen-activated protein (MAP) kinase, a member of
the family of serine/threonine protein kinases, is widely
expressed in endothelial, immune, and inflammatory cells and
plays a critical role in the regulation of the biosynthesis of
proinflammatory cytokines, including tumor necrosis factor-
TNF-α), interleukin-1β, and interleukin-6.[1,2] Selective
biological agents against each of these cytokines have proven
efficacious for the treatment of inflammatory diseases, including
rheumatoid arthritis (RA), psoriasis, and inflammatory bowel
disease.[3,4] TNF-α monoclonal antibodies (infliximab[5-7]
adalimumub[8,9]
and the TNF-α receptor fusion protein,
etanercept[10], are currently used as effective anti-RA agents;
these have shown good efficacy in patients with active RA as
alternative treatments to disease modifying antirheumatic drugs
(DMARDs).
,
)
Figure 1. Representative discontinued p38 MAP kinase inhibitors.
The p38α also plays a central regulatory in the production
of proinflammatory cytokines from microglia in the central
nervous system (CNS). Human genetic studies and other
biologic data suggest that the major drivers of Alzheimer’s
disease (AD) include dysregulated microglia[31,32] and
The MAP kinases include extracellular signal-regulated
kinase, c-JUN N-terminal kinase, and p38 MAP kinase. The
1
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neuroinflammation[33,34]. In cognitively-impaired aged rats, VX-
745 resulted in improved performance in the Morris water maze
(MWM) test for cognitive performance.[35] The clinical
development of VX-745 for the treatment of AD was recently
started. Therefore, the inhibition of p38 MAP kinase is an
attractive therapeutic target, for not only peripheral
inflammation diseases, but also CNS diseases such as AD.
imidazo[1,2-b]pyridazine series (Figure 4). The hit compound 1
was successfully co-crystallized with the ATP binding domain of
p38 MAP kinase (Figure 5). On the basis of the three-
dimensional structure of the protein-ligand complex, we applied
a structure-based approach for designing novel type of p38
MAP kinase inhibitors. For the inhibition of p38 MAP kinase, it
is known that there is often a hydrogen bonding interaction
observed between the inhibitor and the main chain amino acid
in the hinge region of the ATP binding site; specifically,
between the carbonyl oxygen and N-H of Met109[36]
.
Furthermore, in the co-crystal structure of the complex of
compound 1 with p38 MAP kinase, unusual hydrogen bonding
interaction between 1 and the hinge region was observed.
Herein, we have reported a series of imidazo[4,5-b]pyridin-2-
ones with greatly improved in vitro potency and bioavailability
based on the design of compound 1. From the X-ray
crystallographic analysis of the complex of 1 bound to p38 MAP
kinase, we designed and synthesized a series of scaffolds with
hydrogen bonding acceptors able to interact with Met109 and
Gly110 in a bidentate manner (Figure 6). Through scaffold
hopping and SAR study, compound 21 was identified as a lead
which exhibited excellent enzymatic inhibitory activity (IC50
=
9.6 nM) and potent cellular activity, with good oral bioavailability
in rats. In this article, the design, synthesis, and biological
activity of imidazo[4,5-b]pyridin-2-one derivatives are described
as a lead series for another part of our overall strategy to
advance multiple p38 MAP kinase inhibitors with various
scaffolds.
Results and Discussion
Figure 2. p38 MAP kinase inhibitors in Phase II clinical trials.
High-throughput screening hit
1 showed moderate p38
inhibitory activity (IC50 = 140 nM) and was successfully co-
crystallized with the ATP binding domain of the p38 MAP
kinase (Figure 5). At a resolution of 1.80 Å, the co-crystal
structure of 1 bound to p38 MAP kinase suggested that the
carbonyl group of 1 interacted with the enzyme through two
hydrogen bonds with the backbone amide of Met109 and
Gly110 and that the hinge backbone conformation was different
from that typically seen in protein kinases. Normally, the
carbonyl of His107 and the NH and carbonyl of Met109 in the
backbone are directed toward the ATP binding site and
available for hydrogen bonding interactions with ligands.
However, in this crystal structure, a flip of the peptide bond
between Met109 and Gly110 led to a switching of the
hydrogen-bond acceptor and donor distribution around the
peptide plane. The peptide flip is considered energetically
favorable because of the small size of the Gly110 side chain;
bulkier residues would make the peptide flip much more
energetically unfavorable.[36,37]
Only approximately 9.2% of all human protein kinases
have glycine at the equivalent sequence position X + 4, where
X is the gate-keeper residue.[37,38] It is supposed that the
induction of the peptide-flipped hinge conformation is important
for high kinase selectivity.[37] The hypothesis allowed the design
of scaffold hopping with a cyclized carbonylpiperidine moiety,
as shown in Figure 6. The benzimidazolone-based p38 MAP
kinase inhibitors, initially reported by a research group at
Boehringer Ingelheim, made our design attractive[39], while the
carbonyl group only interacted with not Gly110 but Met109 of
Figure 3. Structure of 1,3-thiazole and imidazo[1,2-b]pyridazine derivatives
as p38 MAP kinase inhibitors.
Figure 4. Structure of HTS hit 1 and lead compound 21 as p38 MAP kinase
inhibitors.
We have previously reported the design and synthesis of
p38 MAP kinase inhibitors based on 1,3-thiazole[16] and
imidazo[1,2-b]pyridazine[26] (Figure 3). In our continued efforts
to discover alternative classes of p38 MAP kinase inhibitors, we
conducted high-throughput screening for p38 MAP kinase
inhibition and identified a hit compound 1 (IC50 = 140 nM) with a
completely different structure from the 1,3-thiazole or
2
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the normal, not flipped, hinge backbone, and the NH group
interacted with His107 of the normal hinge. This design of
compound, with an exocyclic hydrogen binding acceptor, is
intended to allow additional interaction with the carbonyl of
His107; that is, hydrogen binding donor X can interact with
His107. We therefore explored several scaffolds, i.e., oxindole,
azaoxindole, benzimidazolone, or imidazopyridin-2-one with an
exocyclic hydrogen binding acceptor able to interact with
Met109 and Gly110 in a bidentate manner and to potentially
lead to high kinase selectivity and discovered that imidazo[4,5-
b]pyridin-2-one 2 resulted in moderate inhibition of the p38
MAP kinase (IC50 = 390 nM).
in inhibitory activity. The para-fluoro group was therefore
considered to be a good fit into the shallow pocket. In addition,
the difluoro analogs 7 and 8 were synthesized and evaluated.
Although the 2,4-difluoro compound 7 had lower inhibitory
activity, the 2,6-difluoro compound 8 displayed more potent
inhibitory activity against p38 than 5. Furthermore, to confirm
the effect of the ortho-substituent on the R1 phenyl group, the
2,6-dichloro compound 9, in which the fluoro groups were
substituted for the chloro groups, was evaluated and was found
to exhibit more potent inhibitory activity with an IC50 value of 5.6
nM. These results suggested that the steric effect of the ortho-
position on the phenyl group as a R1 substituent was important
for potent inhibitory activity, rather than the electron
withdrawing effect. It is of note that the phenyl group for R1 and
the imidazo[4,5-b]pyridin-2-one scaffold should be in mutually
orthogonal planes. This consideration encouraged us to further
explore substituent effects at the R1 position. The benzyl
compound 10 showed moderate inhibitory activity; however the
phenethyl compound 11 reduced inhibitory activity compared
with 4. To control the direction of the phenyl group of the benzyl
moiety of 10, one or two methyl groups were introduced to the
benzyl position (12, 13). The mono-methyl substituted 12
showed tolerable inhibitory activity and the di-methyl substituted
13 showed decreased potency. A fluoro group, as an electron-
withdrawing substituent, was introduced to the ortho-, meta-, or
para-position (14-16) of the phenyl group of the benzyl moiety.
Compared with 10, the meta- and para-fluoro analogs 15 and
16 showed moderate potency and the ortho-fluoro 14 exhibited
more potent inhibitory activity, with an IC50 value of 15 nM for
the inhibition of the p38 MAP kinase. The introduction of
another electron-withdrawing chloro group substituent (17) at
an ortho-position on the phenyl group of the benzyl moiety
reduced inhibitory activity to an IC50 value of 100 nM. This
result suggested that the relatively small size of the fluorine
atom was important for the inhibitory activity against the p38
MAP kinase. The 2,6-difluoro compound 18 slightly increased
inhibitory activity, with an IC50 value of 12 nM. As the mono-
substituted compound 12 was tolerable and the di-substituted
compound 13 showed reduced activity, we explored some
branched alkyl groups for R1. The ipropyl compound 19
exhibited more potent inhibitory activity than 12, which
suggested that the phenyl group at the R1 position of 12 would
be replaceable with the methyl group, which led to enhanced
Figure 5. Binding interactions between 1 and p38 MAP kinase determined by
X-ray crystallographic analysis (left: PDB code 6M95). Typical hinge
conformation and binding mode of
conformation (right).
1 with the peptide flipped hinge
i
ligand efficiency. The butyl compound 20 showed tolerable
Figure 6. Scaffold hopping for discovery of new chemotypes.
inhibitory activity, and the sbutyl 21 and 3-pentyl 22 compounds
had enhanced inhibitory activity against the p38 MAP kinase,
with IC50 values of 9.6 and 6.7 nM, respectively. Given the
properties of 20 and 21, we determined that the methyl group at
an adjacent position to the nitrogen atom would be effective for
strong inhibitory activity against the p38 MAP kinase. This
result also suggested that the branched alkyl group for R1 and
the imidazo[4,5-b]pyridin-2-one scaffold should be in mutually
orthogonal planes. This was supported by the conformation of
the benzyl group of 10, as indicated in the X-ray crystal
structure of the complex of 10 bound with the p38 MAP kinase
(Figure 7).
As described in the introduction part, the development of
the preceding multiple p38 MAP kinase inhibitors have often
been discontinued due to lack of efficacy. Since not only strong
enzyme inhibitory activity but also physicochemical properties
are considered to be an important factor for a druglike lead
The inhibitory potencies (IC50) of the synthesized
compounds were estimated by using a p38 MAP kinase assay
(Table 1). As shown in Figure 5, the benzyl group is directed
toward the shallow inner hydrophobic pocket defined by the
residues Thr106 and Leu104. The previous research proved
that the R2 group was important for potent inhibitory activity and
that smaller substituents, such as a methyl or fluoro group,
were preferable.[16,26,38] Therefore, the introduction of small
substituents for the R2 group was focused on. Although the 3-
methyl 3 showed much weaker inhibition of the p38 MAP
kinase than 2, the 2,4-difluoro derivative 4 exhibited more
potent activity than 2. Compared with 4, the ortho-fluoro 5
showed more potent inhibition of the p38 MAP kinase, with an
IC50 value of 27 nM. The replacement of the 2,4-difluorophenyl
group in 5 with a 2,6-difluorophenyl group (6) led to a reduction
3
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series, we analyzed structure-solubility relationship among
these compounds (Table 1). The compounds 4, 5, 7 and 8 in
which R1 is an aryl group indicated a moderate solubility. The
benzyl series 10 and 14-18 had relatively low solubility. On the
other hand, it was found that the mono-methyl substituent on
compound 12 greatly improved solubility. For the alkyl series,
the isobutyl 20 was poorly-soluble, while isopropyl 19,
secondary butyl 21 and 3-pentyl 22 showed high solubility.
From these facts, it was found that the water solubility tended to
be higher when the adjacent position of the nitrogen on the
imidazole ring was branched. It is worthy noted that the
structural modification which leads to increase solubility is
consistent with the direction in which the enzyme inhibitory
activity enhances. Considering these observations, selected
compounds were further evaluated on cellular level activity.
Table 1. Inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives against the p38 MAP kinase.
ID
2
R1
R2
R3
IC50[a] (nM)
390 (330-470)
> 10000
Solubility[b] (µg/mL)
Ph
2-Me
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NCH3
NHCH2
0.46
0.34
4.7
3
Ph
3-Me
4
Ph
2,4-diF
2,4-diF
2,6-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
2,4-diF
63 (55-72)
5
2-F-Ph
27(24-30)
3.3
6
2-F-Ph
87 (76-99)
0.69
4.5
7
2,4-diF-Ph
2,6-diF-Ph
2,4-diCl-Ph
PhCH2
100 (90-110)
9.9 (8.9-11)
5.6 (4.9-6.4)
29 (26-32)
8
1.7
9
< 0.12
1.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
PhCH2CH2
PhCH(CH3)
PhC(CH3)2
2-F-PhCH2
3-F-PhCH2
4-F-PhCH2
2-Cl-PhCH2
2,6-diF-PhCH2
iPropyl
110 (90-120)
31 (26-36)
0.26
10
150 (130-160)
15 (13-16)
3.0
0.49
1.8
37 (33-42)
52 (44-60)
0.81
0.14
0.11
5.7
100 (91-120)
12 (11-13)
18 (16-19)
iButyl
26 (23-30)
0.69
11
sButyl
9.6 (8.5-11)
6.7 (6.0-7.5)
110 (92-120)
9500 (8300-11000)
3-Pentyl
sButyl
42
1.6
sButyl
49
[a] The IC50 values shown are the mean values of quadruple measurements; the numbers in parentheses represent the 95% confidence intervals. [b] Solubility in
pH = 6.8.
resolution, revealed that compound 10 binds to the p38 MAP
As indicated in Figure 7, the X-ray structure of the
kinase as expected. As observed in the crystal structure of hit
complex of 10 with the p38 MAP kinase, determined at 2.5 Å
compound 1 with p38, the exocyclic carbonyl oxygen of 10
4
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undergoes bidentate coordination with Met109 and Gly110 in
the hinge region. Furthermore, the additional hydrogen bonding
interaction between the NH hydrogen of imidazo[4,5-b]pyridin-
2-one ring and the carbonyl oxygen of His107 was observed, as
expected. The aniline hydrogen binds to Lys53 and Asp168 of
the enzyme through water-mediated hydrogen bonding. In
addition to these electrostatic interactions, the hydrophobic
interaction of the R2 groups contributes to the increased
inhibition of p38, as shown by the SAR study (Table 1). The
2,4-difluorophenyl group fills the inner shallow hydrophobic
pocket of the p38 MAP kinase consisting of Thr106 and Leu104.
The SAR study of the R1 groups suggests that increased
lipophilicity of this part (8, 9, 21, and 22) changes the
orientation of R1 substituents into the direction of hydrophobic
pocket consisted of Ala157 and efficiently interact with protein
surface compared with the benzyl group of 10 which does not
occupy this pocket as shown in Figure 7. In a kinase panel
study to probe potential off-target liabilities, while 1 showed
negligible inhibition at 10 M against 13 kinases, 8 and 22
exhibited excellent selectivity over 30 kinases (The evaluated
kinase list and data for 1, 8, and 22 are given in the Supporting
Information).
representative compounds which were selected as the poorly or
highly water soluble compounds with the potent enzymatic
activities are summarized in Table 2. These compounds, 8, 9,
21, and 22, which showed similar inhibition of the p38 MAP
kinase, inhibited the lipopolysaccharide (LPS)-induced
production of TNF-α in THP-1 cells with IC50 values between
12–46 nM. The poorly-soluble aryl series 8 and 9 showed a
slight decrease, in parallel, between p38 enzymatic inhibition
and THP-1 cell-based inhibition; this was also seen in the
highly-soluble alkyl series 21 and 22. In addition, the
compounds 8, 9, 21, and 22 suppressed the TNF-α-induced
production of interleukin-8 (IL-8) in hWB cells, but the potency
of the highly-soluble alkyl series 21 and 22 was much greater
than the poorly-soluble aryl series 8 and 9. Although the
explanation of this discrepancy between the aryl and alkyl
series in the WB assay was unclear, it was considered that the
physicochemical properties of 21 and 22 resulted in relatively
potent activity in the WB cell-based assay due to contributions
from their higher solubility as we anticipated. The
pharmacokinetic properties of the lead compounds 21 and 22 in
rats are shown in Table 3; higher oral exposure (AUC = 223.2
ng·h/mL) and bioavailability (F% = 49.2) was found for
compound 21, although lower oral exposure and bioavailability
(AUC = 37.5 ng·h/mL; F% = 14.4) was found for compound 22.
The absorption and exposure of compound 21 were favorable
in rats at a dose of 1 mg/kg and were sufficient for further study
as a representative lead compound.
Table 2. The inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives on
TNF-α production in human THP-1 cells, IL-8 production in human whole
blood cells, and solubility.
[a]
[a]
Solubility[b]
(µg/mL)
Figure 7. X-ray crystal structure of the complex of 10 and the p38 MAP
kinase (left: PDB code 6M9L). Surface representation of the p38 MAP kinase
illustrating the binding cavity. The inhibitor 10 is buried into this cavity. The
pocket close to Ala157 is unoccupied by 10 (right).
ID
p38 IC50
(nM)
THP-1 IC50
(nM)
hWB IC50
(nM)
8
9.9
5.6
9.6
6.7
36 (21-60)
21 (12-35)
46 (26-81)
12 (6.5-23)
93 (85-100)
200 (160-250)
15 (11-20)
1.7
9
< 0.12
11
The human monocytic leukemia cells (THP-1 cells) were
used for the assessment of the effects of p38 MAP kinase
inhibitors on cytokine production. In addition, the human whole
blood (hWB) cell-based assay was conducted to evaluate the
potential of the p38 inhibitors as a lead chemotype. It is
supposed that suppression of cytokine production in the hWB
assay reflects in vivo efficacy owing to the relative complexity of
the cell-based assay. The inhibitory potencies (IC50) of the
21
22
20 (9.1-31)
42
[a] The IC50 values shown are the mean values of quadruple measurements;
the numbers in parentheses represent the 95% confidence intervals. [b]
Solubility in pH = 6.8.
Table 3. Pharmacokinetic parameters of 21 and 22 in rats[a]
.
ID
Cmax[b]
(ng/mL)
Tmax[b]
(h)
AUC[b]
(n·gh/mL)
MRT[b]
(h)
Vd
(mL/kg)
CL
(mL/h/kg)
F
(%)
21
22
55.8
14.1
2.7
223.2
37.5
2.9
1.8
1894
2905
2240
3855
49.2
14.4
0.80
[a] Mean values of measurements conducted in three animals; i.v. 0.1 mg/kg, p.o. 1.0 mg/kg in 0.5% methyl cellulose suspension. [b] p.o. data.
5
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Chemistry
The synthesis of imidazo[4,5-b]pyridin-2-one analogs 2–
24 followed a general route, as outlined in Schemes 1–3
(experimental procedures for compounds 2–24 and
intermediates are given in the supporting information). During
the synthesis of the designed p38 MAP kinase inhibitors, we
developed a highly regio-selective method of the introduction of
amines into the tri-substituted pyridines 25 and 26. In Scheme 1,
chloropyridine 25 was treated with cesium fluoride to afford
fluoropyridine 26. Aromatic nucleophilic substitution reaction
with the appropriate amines R1NH2 produced the corresponding
compounds 27a–o. For the introduction of the second amine,
the Buchwald reaction using the appropriate amines R2NH2 led
to the corresponding key intermediates 28a–s. The subsequent
alkaline hydrolysis of 28a–s produced the carboxylic acids 29a–
Scheme 2. Synthesis of compound 7. Reagents and conditions: (a) 2,4-
Difluoroaniline, Xphos, Pd2(dba)3, Cs2CO3, 60 °C; (b) 1 N NaOH, THF-
MeOH-H2O, rt; (c) DPPA, Et3N, toluene, 100 °C.
s
as a cyclization precursor. The final cyclization was
successfully accomplished with treatment of 29a–s with DPPA.
The reaction mechanism of the cyclization step was considered
to include multistep reactions, which consisted of the formation
of acyl azide, the production of isocyanate via Curtius
rearrangement, and intramolecular cyclization, in which the
isocyanate was trapped by an adjacent amino group. As
indicated in Scheme 2, for the introduction of the same
substituent for the R1 and R2 groups, Buchwald reaction
Scheme 3. Synthesis of compound 6, 8, and 9. Reagents and conditions: (a)
R2NH2, Xantphos, Pd2(dba)3, Cs2CO3, toluene, 100 °C; (b) R1NH2, BINAP,
Pd(OAc)2, Cs2CO3, toluene, MW, 150 °C; (c) 1 N NaOH, THF-MeOH-H2O, rt;
(d) DPPA, Et3N, toluene, 100 °C.
conditions using excess 2,4-difluoroaniline (more than
2
equivalents) with 26 afforded 28t, which led to 7 in a similar
manner to that described in Scheme 1. Next, we tried to change
the order of the introduction of R1 and R2 groups. In Scheme 3,
we succeeded in a highly regio-selective Buchwald reaction
using Xantphos and Pd2(dba)3 as the catalysts to accomplish
the synthesis of 30a–b without any impact of the chloride group
at the 2-position. The subsequent Buchwald reaction using
BINAP and Pd(OAc)2 as a catalyst produced the tri-substituted
pyridines 28u–w modified by the desired appropriate
substituents. The tri-substituted pyridines 28u–w led to the
corresponding compounds 6, 8, and 9 in a similar manner to
that described in Scheme 1.
Conclusions
Based on the X-ray crystallographic analysis of the complex of
1 with the p38 MAP kinase, we focused on the scaffold
transformation of the carbonylpiperidine moiety of 1 while
maintaining the binding mode in which 1 was coordinated in a
bidentate manner. To investigate a scaffold with an exocyclic
hydrogen binding acceptor, imidazo[4,5-b]pyridin-2-one
derivatives were designed, synthesized, and identified as
potent inhibitors of the p38 MAP kinase. Through the extensive
SAR analysis and structure-solubility relationship analysis,
compound 21 was discovered to exhibit excellent enzymatic
inhibitory activity (IC50 = 9.6 nM), suppress IL-8 production in
human WB cells (IC50 = 15 nM), and have good oral
bioavailability in rats. Collectively, we identified that 21 was
appropriate for further study as a lead compound of
imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.
Experimental Section
Scheme 1. Synthesis of compounds 2-5 and 10-24. Reagents and
conditions: (a) CsF, DMSO, 50 °C; (b) R1NH2, DMSO, 100 °C; (c) R2NH2,
XPhos, Pd2(dba)3, Cs2CO3, toluene, 100 °C; (d) 1 N NaOH, THF-MeOH-H2O,
rt; (e) DPPA, Et3N, toluene, 100 °C.
Chemistry: Melting points were determined with a Yanagimoto melting
point apparatus or a Büchi melting point apparatus B-545 and are
uncorrected. 1H NMR spectra were obtained at 200 or 300 MHz on a
Varian Gemini-200, a Varian Ultra-300, or a Bruker DPX-300
spectrometer. Chemical shifts are given in δ values (ppm) using
6
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10.1002/cmdc.201900129
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tetramethylsilane as the internal standard. Peak multiplicities are
expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublet; br, broad; brs, broad singlet; m, multiplet. Elemental
analyses were carried out by Takeda Analytical Laboratories Ltd.
Reactions were followed by TLC on Silica gel 60 F 254 precoated TLC
plates (E. Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.).
Chromatographic separations were carried out on silica gel 60 (0.063–
0.200 or 0.040–0.063 mm, E. Merck) or basic silica gel (Chromatorex®
NH, 100–200 mesh, Fuji Silysia Chemical Ltd.) using the indicated
eluents. Yields are unoptimized. Column chromatography was
performed using Merck silica gel 60 (70–230 mesh). Thin-layer
chromatography (TLC) was performed on Merck silica gel plates
60F254. LC-MS analysis was performed on a Shiseido CAPCELL PACK
C-18 UG120 S-3 column (1.5 mmϕ × 35 mm) in a Waters Alliance 2795
or an Agilent 1100 LC system equipped with a Waters 2487 absorbance
detector and a Micromass ZQ2000 mass spectrometer. Analytes were
eluted using a linear gradient of water (0.05% TFA)/acetonitrile (0.04%
TFA) from 90:10 to 0:100 over 4 min at a flow rate of 0.5 mL/min. UV
detection was at 220 nm. Preparative HPLC was performed on a
Shiseido CAPCELL PACK C-18 UG120 S-5 column (20 mmϕ × 50 mm),
eluting at 25 mL/min with a gradient of water (0.1% TFA)/acetonitrile
(0.1% TFA). UV detection was at 220 nm. The purity of all compounds
used in biological studies was determined to be ≥ 95% by elemental
analysis or a HPLC with Corona CAD (Charged Aerosol Detector), Nano
quantity analyte detector (NQAD), or photo diode array detector.
Elemental analyses were performed by Takeda Analytical Research
Laboratories, Ltd. Experimentally determined hydrogen, carbon, and
nitrogen composition by elemental analysis was within ±0.4% of the
expected value, implying a purity of ≥ 95%. For a HPLC analysis, the
column was a Capcell Pak C18AQ (50 mm × 3.0 mm ID, Shiseido,
Japan) or L-column 2 ODS (30 mm × 2.0 mm ID, CERI, Japan) with a
temperature of 50 °C and a flow rate of 0.5 mL/min. Mobile phases A
and B under a neutral condition were a mixture of 50 mM ammonium
acetate, H2O, and CH3CN (1:8:1, v/v/v) and a mixture of 50 mM
ammonium acetate and CH3CN (1:9, v/v), respectively. The ratio of
mobile phase B was increased linearly from 5% to 95% over 3 min, 95%
over the next 1 min. Mobile phases A and B under an acidic condition
were a mixture of 0.2% formic acid in 10 mM ammonium formate and
0.2% formic acid in CH3CN, respectively. The ratio of mobile phase B
was increased linearly from 14% to 86% over 3 min, 86% over the next
1 min. All experiments using animals were reviewed and approved by
the Internal Animal Care and Use Committee of Takeda Pharmaceutical
Research Division. The experimental procedures for representative
compounds 2, 7 and 6 were as follows (experimental procedures for
compounds 2–24 and intermediates are given in the supporting information).
Methyl 5-bromo-2-phenylaminopyridine-3-carboxylate (27a). A
solution of 26 (577 mg, 2.47 mmol) and aniline (276 mg, 2.96 mmol) in
DMSO (4 mL) was stirred at 100 °C for 15 h and cooled to room
temperature. To the mixture was added H2O. The resulting solid was
collected on a filter, washed with H2O, and dried under reduced
pressure to give 27a as a pale yellow powder (670 mg, 80%). 1H NMR
(300 MHz, DMSO-d6) δ: 3.91 (3H, s), 7.07 (1H, t, J = 7.3 Hz), 7.29-7.39
(2H, m), 7.62-7.70 (2H, m), 8.33 (1H, d, J = 2.4 Hz), 8.51 (1H, d, J = 2.6
Hz), 10.01 (1H, s). MS (ESI+): 308 (M+H).
Methyl 2-anilino-5-(2-methylanilino)pyridine-3-carboxylate (28a). A
solution of 27a (307 mg, 1.0 mmol), 2-methylaniline (0.13 mL, 1.2 mmol),
Pd2(dba)3 (9.2 mg, 0.01 mmol), Xphos (9.5 mg, 0.02 mmol), and cesium
carbonate (652 mg, 2.0 mmol) in toluene (5 mL) was stirred at 100 °C
for overnight under argon atmosphere. To the resulting mixture was
added H2O. The mixture was extracted with ethyl acetate, washed with
H2O and brine, dried over magnesium sulfate, and concentrated in
vacuo. The residue was purified with a silica gel column
chromatography (hexane: ethyl acetate= 10: 0- 2: 8) to give 28a as a
yellow powder (293 mg, 92%). 1H NMR (300 MHz, DMSO-d6) δ: 2.22
(3H, s), 3.83 (3H, s), 6.81 (1H, t, J = 7.3 Hz), 6.89-7.02 (2H, m), 7.16
(1H, d, J = 7.2 Hz), 7.23-7.36 (3H, m), 7.69 (2H, d, J = 7.5 Hz), 7.94 (1H,
d, J = 3.0 Hz), 8.25 (1H, d, J = 3.0 Hz), 9.86 (1H, s).
2-Anilino-5-(2-methylanilino)pyridine-3-carboxylic acid (29a). A
solution of 28a (290 mg, 0.87 mmol) and 1N NaOH aqueous solution
(3.57 mL, 3.57 mmol) in tetrahydrofuran (5 mL), methanol (5 mL) and
H2O (1 mL) was stirred at room temperature for 3 h. To the resulting
mixture was added 1N HCl aqueous solution (3.57 mL, 3.57 mmol) at
room temperature. After being concetrated in vacuo, the residue was
diluted with ethyl acetate, washed with H2O and brine, dried over
magnesium sulfate, and concentrated in vacuo. The residue was
purified with a silica gel column chromatography (hexane: ethyl acetate=
7: 3- 1: 9) to give 29a as a yellow powder (120 mg, 43%). 1H NMR (300
MHz, DMSO-d6) δ: 2.23 (3H, s), 6.79 (1H, td, J = 7.3, 0.9 Hz), 6.90-6.99
(2H, m), 7.02-7.10 (1H, m), 7.15 (1H, d, J = 7.2 Hz), 7.22 (1H, s), 7.23-
7.35 (2H, m), 7.69 (2H, d, J = 7.6 Hz), 7.94 (1H, d, J = 2.7 Hz), 8.21 (1H,
d, J = 2.7 Hz), 10.23 (1H, brs), 13.60 (1H, brs).
6-(2-Methylanilino)-3-phenyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-
2-one (2). A solution of 29a (120 mg, 0.38 mmol), DPPA (0.084 mL,
0.39 mmol), and Et3N (0.059 mL, 0.42 mmol) in toluene (10 mL) was
stirred at 100 °C for 2 h. To the resulting mixture was added H2O. The
mixture was extracted with ethyl acetate, washed with H2O and brine,
dried over magnesium sulfate, and concentrated in vacuo. The residue
was purified with a silica gel column chromatography (hexane: ethyl
acetate= 7: 3- 4: 6) to give a brown powder. The powder was
Methyl 5-bromo-2-fluoropyridine-3-carboxylate (26). To a solution of
methyl 5-bromo-2-chloropyridine-3-carboxylate (25) (100 g, 39.9 mmol)
in DMSO (1.33 L) was added cesium fluoride (84.9 g, 559 mmol) at
45 °C. The mixture was stirred at 50 °C for 48 h and cooled to room
temperature. To the mixture was added ethyl acetate. The mixture was
washed with H2O. The aqueous layer was extracted with ethyl acetate.
The combined organic layers was washed with brine, dried over sodium
sulfate, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane: ethyl acetate= 100: 0- 50: 1) to
give 26 as a white powder (81.3 g, 87%). 1H NMR (300 MHz, DMSO-d6)
δ: 3.89 (3H, s), 8.56 (1H, dd, J = 8.2, 2.5 Hz), 8.66 (1H, dd, J = 2.6, 1.3
Hz).
recrystallized from ethanol to give 2 as a pale white powder (75 mg,
62%). mp: 283–284 °C (ethanol). 1H NMR (300 MHz, DMSO-d6) δ: 2.22
(3H, s), 6.82-6.89 (1H, m), 6.96-7.13 (3H, m), 7.18 (1H, d, J = 7.5 Hz),
7.30-7.42 (2H, m), 7.52 (2H, t, J = 7.7 Hz), 7.63-7.76 (3H, m), 11.15 (1H,
s). MS (ESI+): 317 (M+H). Anal. Calcd for C19H16N4O·0.1H2O: C, 71.73;
H, 5.13; N, 17.61. Found: C, 71.73; H, 5.01; N, 17.48.
2,5-Bis(2,4-difluoroanilino)pyridine-3-carboxylic acid (29t). A
solution of 26 (300 mg, 1.3 mmol), 2,4-difluoroaniline (199 mg, 1.5
7
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10.1002/cmdc.201900129
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mmol), Pd2(dba)3 (11.7 mg, 0.01 mmol), Xphos (12.2 mg, 0.03 mmol),
and cesium carbonate (835 mg, 2.6 mmol) in toluene (30 mL) was
stirred at 100 °C for 15 h under argon atmosphere. To the mixture were
added 2,4-difluoroaniline (199 mg, 1.5 mmol), Pd2(dba)3 (11.7 mg, 0.01
mmol), Xphos (12.2 mg, 0.03 mmol), and cesium carbonate (835 mg,
2.6 mmol). The mixture was stirred at 100 °C for 15 h under argon
atmosphere. The mixture was filtered through celite pad. To the filtrate
was added H2O. The mixture was extracted with ethyl acetate, washed
with H2O and brine, dried over sodium sulfate, and concentrated in
vacuo. The residue was purified with a silica gel column
DMSO-d6) δ: 6.87-7.01 (1H, m), 7.06-7.29 (5H, m), 7.65-7.72 (1H, m),
7.99 (1H, s), 8.07 (1H, d, J = 3.0 Hz), 8.59 (1H, td, J = 8.3, 1.5 Hz),
10.35 (1H, brs), 13.69 (1H, brs). MS (ESI+): 360 (M+H).
6-(2,6-Difluoroanilino)-3-(2-fluorophenyl)-1,3-dihydro-2H-
imidazo[4,5-b]pyridin-2-one (6). This compound was prepared from
29u as described in the synthesis of 2 as a white powder (44%). mp:
296–297 °C. 1H NMR (300 MHz, DMSO-d6) δ: 6.78 (1H, s), 7.08-7.25
(3H, m), 7.27-7.67 (5H, m), 7.95 (1H, s), 11.16 (1H, s). MS (ESI+): 357
(M+H). Anal. Calcd for C18H11N4F3O: C, 60.68; H, 3.11; N, 15.72.
Found: C, 60.39; H, 3.21; N, 15.53.
chromatography (hexane: ethyl acetate= 98: 2- 70: 30) to give methyl
2,5-bis(2,4-difluoroanilino)pyridine-3-carboxylate (28t) as a brown
powder (127 mg, 25%), and used in the next reaction. This compound
was prepared from 28t as described in the synthesis of 29a as a brown
powder (122 mg, quant). 1H NMR (300 MHz, DMSO-d6) δ: 6.85-7.18
(3H, m), 7.19-7.43 (2H, m), 7.79 (1H, s), 7.92 (1H, d, J = 3.0 Hz), 8.16
(1H, d, J = 2.6 Hz), 8.44-8.62 (1H, m).
Biology: p38 MAP kinase enzyme assay: The FLAG-tagged human
p38α protein was expressed with a baculovirus system and activated by
constitutive active human MKK3. Then, the recombinant p38α protein
was purified using the anti-FLAG antibody affinity agarose gel (Sigma).
Kinase reactions were evaluated with LanthaScreen assay system (Life
Technologies, USA); 2.5 μL of test compounds, diluted with DMSO (final
concentration 1% DMSO), were added to the reaction mixture (25 mM
HEPES [pH7.5], 10 mM Mg acetate, 1 mM DTT, 0.01% Tween-20, and
0.01% BSA) containing 125 pg human p38α protein and 8 nM GFP-
ATF2 (19-96) (Life Technologies) in 384-well plates (Nunc, USA). After
a 5 min incubation at room temperature, the reaction was started by
adding 5 μL of 580 μM ATP. After a 20 min incubation, the reaction was
terminated by adding 5 μL of 80 mM EDTA. Then, 5 μL of the Tb-anti-
phospho ATF2 (pThr71) antibody (Life Technologies) was added and
incubated for 60 min at room temperature. The time-resolved
6-(2,4-Difluoroanilino)-3-(2,4-difluorophenyl)-1,3-dihydro-2H-
imidazo[4,5-b]pyridin-2-one (7). This compound was prepared from
29t as described in the synthesis of 2 as a white powder (7%). mp: 245–
247 °C. 1H NMR (300 MHz, DMSO-d6) δ: 6.87-7.07 (2H, m), 7.12-7.35
(3H, m), 7.46-7.60 (1H, m), 7.60-7.73 (2H, m), 7.85 (1H, s), 11.26 (1H,
s). MS (ESI+): 375 (M+H). HPLC purity: 100%.
Methyl 2-chloro-5-(2,6-difluoroanilino)pyridine-3-carboxylate (30a).
A solution of 25 (1500 mg, 6.0 mmol), 2,6-difluoroaniline (0.831 mL, 7.2
mmol), Pd2(dba)3 (137 mg, 0.15 mmol), Xantphos (174 mg, 0.3 mmol),
and cesium carbonate (6800 mg, 21 mmol) in toluene (20 mL) was
stirred at 100 °C under nitrogen atmosphere for overnight. To the
resulting mixture was added H2O. The mixture was extracted with ethyl
acetate, washed with H2O and brine, dried over magnesium sulfate, and
concentrated in vacuo. The residue was purified with a silica gel column
chromatography (hexane: ethyl acetate= 4: 1) to give 30a as a yellow
powder (896 mg, 50%). 1H NMR (300 MHz, DMSO-d6) δ: 3.84 (3H, s),
7.16-7.35 (3H, m), 7.39-7.46 (1H, m), 8.03 (1H, d, J = 3.0 Hz), 8.62 (1H,
s). MS (ESI+): 299 (M+H).
fluorescence resonance energy transfer (TR-FRET) signal was
measured using EnVision Multilabel Plate Reader (PerkinElmer).
TNF-α production assay in THP-1 cells: THP-1 cells were suspended
in RPMI 1640 medium (Life Technologies) containing 1% fetal bovine
serum (Morgate, Australia). Then, 40 μL of the cell suspension
(0.625X106 cells/mL) was added to 384-well plates (Corning, USA) and
mixed with 5 μL of test compounds diluted with 10% DMSO. After 60
min incubation at 37 °C and 5% CO2, the cells were stimulated with 5 µL
of 100 μg/mL LPS (Wako). After incubating for 4 h at 37°C and 5% CO2,
the concentration of TNF-α in the medium was measured with the TNF-
α HTRF kit (CisBio, USA). The TR-FRET signal was measured with
EnVision Multilabel Plate Reader (PerkinElmer).
Methyl 5-(2,6-difluoroanilino)-2-(2-fluoroanilino)pyridine-3-
carboxylate (28u). A solution of 30a (448 mg, 1.5 mmol), 2-fluoroaniline
(0.174 mL, 1.8 mmol), palladium acetate (16.8 mg, 0.075 mmol), BINAP
(56.0 mg, 0.090 mmol), and cesium carbonate (1220 mg, 3.8 mmol) in
toluene (10 mL) was stirred at 150 °C under microwave radiation for 2 h.
After being cooled to room temperature, the mixture was diluted with
ethyl acetate and filtered through celite pad. The filtrate was washed
with H2O and brine, dried over sodium sulfate, and concentrated in
vacuo. The residue was purified with a silica gel column
TNF-α production assay in human whole blood cells: Human whole
blood was diluted with RPMI 1640 medium (Nikken-bio) to be 2.5 times.
Then, 160 μL of the diluted blood was added to 96-well plates and
mixed with 20 μL of test compounds diluted with 10% DMSO. After 60
min incubation at 37 °C and 5% CO2, the blood was stimulated with
20µL of 300 ng/mL human TNF-α. After incubating for 18-24 h at 37°C
and 5% CO2, the concentration of IL-8 in the medium was measured
with the IL-8 ELISA kit (R&D systems, USA). The absorbance was
measured with Wallac 1420 Plate Reader (PerkinElmer).
chromatography (hexane: ethyl acetate= 4: 1) to give 28u as a yellow
powder (466 mg, 83%). 1H NMR (300 MHz, DMSO-d6) δ: 3.88 (3H, s),
6.90-7.05 (1H, m), 7.08-7.32 (5H, m), 7.70 (1H, d, J = 2.7 Hz), 8.03 (1H,
s), 8.10 (1H, d, J = 2.7 Hz), 8.56 (1H, dt, J = 8.3, 1.5 Hz), 10.07 (1H, d, J
= 3.4 Hz). MS (ESI+): 374 (M+H).
Solubility: Small volumes of the compound solution dissolved in DMSO
were added to the aqueous buffer solution (pH 6.8). After incubation,
precipitates were separated by filtration. The solubility was determined
by HPLC analysis of each filtrate.
5-(2,6-Difluoroanilino)-2-(2-fluoroanilino)pyridine-3-carboxylic acid
(29u). This compound was prepared from 28u as described in the
synthesis of 29a as a yellow powder (quant). 1H NMR (300 MHz,
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900129
ChemMedChem
FULL PAPER
[10] Moreland, L. W.; Baumgartner, S. W.; Schiff, M. H.; Tindall, E. A.;
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Pharmacokinetics: Rat cassette BA: Test compounds were
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[11] Hale, K. K.; Trollinger, D.; Rihanek, M.; Manthey, C. L. Differential
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Acknowledgements ((optional))
The authors thank Drs. Shigenori Ohkawa, Yuji Ishihara, Fumio
Itoh, Osamu Uchikawa and Yoshinori Ikeura for their valuable
discussions of this work, and Drs. Takashi Ichikawa and
Shinichiro Matsunaga for their critical review of the manuscript.
We also thank the members of the Takeda Analytical Research
Laboratories, Ltd. for elemental analyses. We would like to
thank the staff at the Berkeley Center for Structural Biology,
Lawrence Berkeley National Laboratory, which operates the
Advanced Light Source beamline 5.0.3, where the x-ray
diffraction data was collected. The Berkeley Center for
Structural Biology is supported in part by the National Institutes
of Health, National Institute of General Medical Sciences, and
the Howard Hughes Medical Institute. The Advanced Light
Source is a Department of Energy Office of Science User
Facility under Contract No. DE-AC02-05CH11231.
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and cells in rheumatoid arthritis. Arthritis Rheum. 2000, 43, 2501–
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[16] Miwatashi, S.; Arikawa, Y.; Kotani, E.; Miyamoto, M.; Naruo, K.;
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MAP Kinase as an Anti-TNF- drug: Discovery of N-[4-[2-ethyl-4-(3-
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Keywords: p38 mitogen-activated protein kinase inhibitors •
rheumatoid arthritis • structure-based design • imidazo[4,5-
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Entry for the Table of Contents
Scaffold hopping: High-throughput screening identified the carbonylpiperidine derivative as a hit compound of p38 MAP kinase
inhibitors. Based on the X-ray crystallographic analysis of the complex of the hit with the enzyme, structure-based design has led to
potent and orally available imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.
11
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