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

Article abstract of DOI:10.1002/cmdc.201900373

We identified novel potent inhibitors of p38 mitogen-activated protein (MAP) kinase using a structure-based design strategy, beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroanilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To enhance the inhibitory activity of 1 against production of tumor necrosis factor-α (TNF-α) in human whole blood (hWB) cell assays, we designed and synthesized hybrid compounds in which the imidazo[4,5-b]pyridin-2-one core was successfully linked with the p-methylbenzamide fragment. Among the compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)benzamide (25) exhibited potent p38 inhibition, superior suppression of TNF-α production in hWB cells, and also significant in vivo efficacy in a rat model of collagen-induced arthritis (CIA). In this paper, we report the discovery of potent, selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.

Full text of DOI:10.1002/cmdc.201900373

Full Papers
DOI: 10.1002/cmdc.201900373
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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 2
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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]
Weston Lane,[b] Bi-Ching Sang,[b] Kumar Saikatendu,[b] Shinichiro Matsunaga,[a] and
Seiji Miwatashi[a]
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We identified novel potent inhibitors of p38 mitogen-activated
protein (MAP) kinase using a structure-based design strategy,
beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroa-
nilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To en-
hance the inhibitory activity of 1 against production of tumor
necrosis factor-α (TNF-α) in human whole blood (hWB) cell
assays, we designed and synthesized hybrid compounds in
which the imidazo[4,5-b]pyridin-2-one core was successfully
linked with the p-methylbenzamide fragment. Among the
compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imi-
dazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)
benzamide (25) exhibited potent p38 inhibition, superior
suppression of TNF-α production in hWB cells, and also
significant in vivo efficacy in a rat model of collagen-induced
arthritis (CIA). In this paper, we report the discovery of potent,
selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-
based p38 MAP kinase inhibitors.
Introduction
overactive in the inflammatory tissues.[11–13] Therefore, the
development of p38 MAP kinase inhibitors has been considered
to be a promising solution for the treatment of RA.[14–18]
The p38α mitogen-activated protein (MAP) kinase is widely
expressed in endothelial, immune, and inflammatory cells, and
plays a critical role in regulating the biosynthesis of proin-
flammatory cytokines.[1,2] Selective biological agents targeting
these proinflammatory cytokines have been proven to be
efficacious in the treatment of inflammatory diseases, including
rheumatoid arthritis (RA), psoriasis, and inflammatory bowel
disease.[3,4] Anti-tumor necrosis factor-α (TNF-α) monoclonal
antibodies, including infliximab[5–7] and adalimumab,[8,9] and the
TNF-α receptor fusion protein, etanercept,[10] are currently being
used as effective anti-RA agents, and have shown good efficacy
in patients with active RA.
Numerous preclinical studies have reported that the inhib-
ition of p38 MAP kinase effectively suppresses the production
of TNF-α both in vitro and in vivo.[19] Multiple chemical classes of
p38 MAP kinase inhibitors have been discovered, including VX-
745,[20] BCT-197, LY-2228820, GW856553,[21,22] PH-797804, AZD-
7624, CHF-6297, FX-005, and ARRY-797, which have been
examined in phase II clinical trials, and are represented in
Figure 1.[23] Another approach for suppression of the production
of TNF-α has reported, CBS-3595 in phase I has been discovered
as a dual inhibitor of p38 MAP kinase and phosphodiesterase
4.[24]
We have previously reported the design and synthesis of
p38 MAP kinase inhibitors derived from 1,3-thiazole[16] and
imidazo[1,2-b]pyridazine[25] (Figure 2). In our continued efforts
to develop alternate classes of p38 MAP kinase inhibitors, we
designed and reported the imidazo[4,5-b]pyridin-2-one series[26]
of lead compounds with structures entirely different from those
of the 1,3-thiazole series or the imidazo[1,2-b]pyridazine series.
In addition to structural differentiation, as we reported in
previous paper,[26] the lead compound 1 can bind to Met109
and Gly110 in the peptide-flipped hinge region. It is supposed
that the induction of the peptide-flipped hinge conformation
contributes to high kinase selectivity.[27] Lead compound 1
showed potent p38 MAP kinase inhibition (IC50 =9.6 nM) and
suppressed the TNF-α-induced production of interleukin-8 (IL-8)
in human whole blood (hWB) cell assay (IC50 =15 nM), as shown
in Figure 3. However, its ability to suppress the lipopolysacchar-
The MAP kinase family includes the extracellular signal-
regulated kinases, c-JUN N-terminal kinases, and p38 MAP
kinases. Analyses of MAP kinases from the synovial tissues and
cells of patients with RA indicated that the p38α isoform is
[a] A. Kaieda, Dr. M. Takahashi, H. Fukuda, Dr. R. Okamoto, Dr. S. Morimoto,
M. Gotoh, T. Miyazaki, Y. Hori, S. Unno, Dr. T. Kawamoto, Dr. T. Tanaka,
Dr. S. Itono, T. Takagi, Dr. H. Sugimoto, Dr. K. Okada, Dr. S. Matsunaga,
Dr. 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
[b] Dr. W. Lane, Dr. B.-C. Sang, Dr. K. Saikatendu
Takeda California
10410 Science Center Drive
San Diego, CA 92121 (USA)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.201900373
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Figure 3. Inhibitory activities of 1 on enzymatic and cellular activities.
order to validate this hypothesis, we proceeded to modify
compound 1 by employing a structure-based design strategy.
In order to confirm the increase in cellular activity, human
monocytic leukemia cells (THP-1 cells) were used instead of
hWB cells to evaluate the suppression in the LPS-induced
production of TNF-α.
Imidazo[4,5-b]pyridin-2-one (2) was successfully co-crystal-
lized with the ATP binding domain of p38 MAP kinase (Figure 4
upper part).[26] Using information from the three-dimensional
structure of the protein-ligand complex, we employed an
expanding approach by targeting some of the interactions of
the ligand with the back pocket of the enzyme for generating a
novel p38 MAP kinase inhibitor scaffold. A research group at
Eberhard-Karls-University Tubingen previously reported this
approach to enhance enzymatic inhibitory activity of dibenzepi-
none-based p38 MAP kinase inhibitors.[28] As highlighted in
Figure 1, the structures of BMS-582949,[29] BCT-197, GW856553,
PH-797804, and AZD-7624 share common fragments, including
Figure 1. Representative p38 MAP kinase inhibitors.
Figure 2. Structure of 1,3-thiazole and imidazo[1,2-b]pyridazine derivatives
as p38 MAP kinase inhibitors.
Figure 4. Structure of 2 (upper left) and X-ray crystal structure of the
complex of 2 and the p38 MAP kinase (upper right: PDB code 6M9L). The
purple arrow shows the direction of formation of direct hydrogen bonding
with the enzyme back pocket residues. Structure (lower left) and X-ray
crystal structure of the complex (lower right) of a representative of GSK’s p-
methylbenzamide derivatives.
ide (LPS)-induced production of TNF-α was found to be
drastically attenuated in the hWB cell assay (IC50 =2600 nM). We
therefore hypothesized that a more potent enzymatic inhibition
would be necessary for further enhancing cellular activity. In
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the p-methylbenzamide moiety, which frequently binds to the
back pocket of p38 MAP kinase. Using X-ray crystallography, a
group of researchers at GSK demonstrated that the amide
group of the methylbenzamide moiety of biphenyl amide
derivatives has hydrogen bonding interactions with Glu71 and
Asp168 in the back pocket of the enzyme (Figure 4 lower
part).[30,31] Based on the information on the binding properties
of compound 2 combined with the data on the biphenyl amide
derivatives developed by GSK, we designed and synthesized
hybrid compounds from compound 1 that could interact
directly with the back pocket of p38 MAP kinase (Figure 5).
Herein, we report imidazo[4,5-b]pyridin-2-one core was success-
fully linked with the p-methylbenzamide fragment. We sub-
sequently discovered compound 25, through structure activity
relationship (SAR) studies and optimization, which exhibited
exceptional enzymatic inhibition (IC50 =0.42 nM), potent cellular
activity, and significant dose-dependent in vivo efficacy in rats.
Lead optimization, including the design, synthesis, and evalua-
tion of the biological activity of imidazo[4,5-b]pyridin-2-one
derivatives are also discussed herein for advancing the develop-
ment of p38 MAP kinase inhibitors with variable scaffolds.
activity (9.6 nM), as shown in Figure 3. However, since the
suppression of the LPS-induced production of TNF-α in the
hWB cell assay by compound 1 was relatively poor (IC50 =
2600 nM), we hypothesized that a further increase in the
enzymatic activity would enhance cellular activity. Co-crystal-
lization study of the analogous compound 2 with the ATP
binding site of p38 MAP kinase (Figure 4 upper right)[26]
revealed that the NH group of aniline interacts with the protein
back pocket site, via water-mediated hydrogen bonds with
Lys53 and Asp168. In contrast, X-ray crystallography of p-
methylbenzamide derivatives complexed with p38 MAP kinase
revealed that the amide moiety directly interacts with Glu71
and Asp168 in the enzyme back pocket via hydrogen bonds
(Figure 4 lower right).[30] Based on the information from the co-
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crystallization studies of compound
2 and the meth-
ylbenzamide derivatives, we designed and synthesized the
hybrid compounds from compound 1, that would directly
interact with the back pocket of p38 MAP kinase (Figure 5).
The inhibitory properties, measured by the IC50 values, of
the synthesized compounds were evaluated by using the p38
MAP kinase assay. Human monocytic leukemia cells (THP-1
cells) were used to assess the effects of the p38 MAP kinase
inhibitors on cytokine production. As expected, the p38 MAP
kinase inhibitory activity of the biaryl derivative, compound 3,
dramatically increased by more than 10-fold in comparison with
that of the lead compound 1, with the IC50 value being 0.73 nM
(Table 1). Furthermore, the suppression in TNF-α production
Results and Discussion
We recently reported the imidazo[4,5-b]pyridin-2-one derivative,
compound 1, which exhibited potent p38 MAP kinase inhibition
Figure 5. Expanding approach for direct hydrogen bond formation with the back pocket residues of the p38, and the design concept using p-
methylbenzamide moiety.
Table 1. Inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives 3–6 against the p38 MAP kinase and production of TNF-α in human THP-1 cells.
ID
Position
X
p38 IC50[a] (nM)
THP-1 IC50[a] (nM)
3
4
5
6
6
6
7
7
bond
NH
bond
NH
0.73 (0.68–0.78)
41 (36–46)
82[b]
7.7 (4.6–13)
370 (160–840)
ND[c]
3.5 (3.2–3.8)
65 (34–130)
[a] The IC50 values shown are the mean values of quadruple measurements; the numbers in parentheses represent the 95% confidence intervals. [b] The IC50
value shown is the value of single measurements [c] No data.
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(IC50 =7.7 nM) by the biaryl compound 3 was higher in THP-1
cells than that induced by compound 1. In order to obtain the
SAR of compound 3, we synthesized and evaluated compounds
4–6 with modified linkers and observed that the inhibitory
activity of the biaryl compound 3 against p38 MAP kinase and
the production of TNF-α in THP-1 cells were the highest among
these compounds. Based on these results, we proceeded to
optimize the benzamide moiety of compound 3.
(9), fluoro (10), ethyl (11), and cyano (12) group. Compound 9,
bearing the chloro substituent, retained the inhibitory activity
against p38, whereas compounds 10, 11, and 12, bearing the
fluoro, ethyl, and cyano substituents exhibited moderate but
weaker inhibitory potencies than compound 3, which bore the
methyl substituent. These results further confirmed that the
presence of a methyl substituent at the ortho position positively
contributed to a strong inhibitory activity. It could be further
inferred that the strength of the inhibitory activity against p38
MAP kinase was affected by the size of the ortho substituent
and not by its electrostatic effect, since the bulkiness of the
methyl or chloro group adequately corresponded to the space
between the enzyme and the ligand. The replacement of the
phenyl group in compound 3 by a thiophen group (13), which
is a bioisostere of phenyl group, reduced the inhibitory activity.
The reason behind the decrease in the inhibitory activity of
compound 13 could be attributed to the fact that the SÀ O
interaction between the sulfur of thiophen and the oxygen of
the carbonyl group altered the direction of the amide bond in
an unfavorable manner.
1
2
3
4
5
6
7
8
9
As indicated in Table 2, the m-methyl compound
7
exhibited an approximate 600-fold decrease in inhibitory
activity, in comparison to that of compound 3. The o-methyl
group of compound 3 was found to be important for p38 MAP
kinase inhibition. The o-methyl group induced a torsion
between the imidazo[4,5-b]pyridin-2-one ring and the phenyl
ring, and this torsion was considered to be sufficient to induce
the benzamide moiety to interact with the back pocket of the
enzyme. Furthermore, since the p-amide compound 8 also
showed no inhibitory activity up to a concentration of 10 μM, it
was concluded that the orientation of substituents on the
phenyl group of compound 3 was essential for the inhibitory
activity. In order to confirm the effect of the o-methyl group on
the phenyl group, the methyl group was substituted by a chloro
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The effects of the substituent on the inhibitory activities of
the compounds are depicted in Table 3. Our previous study
Table 2. The inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives 7–13 against the p38 MAP kinase.
ID
R1
R2
R3
R4
IC50[a] (nM)
3
7
8
9
Me
H
Me
Cl
H
Me
H
H
H
CONHcPr
CONHcPr
H
0.73
440 (390–500)
>10000
0.79 (0.72–0.87)
16 (15–18)
11 (9.6–12)
17 (15–19)
1300 (1200–1500)
CONHcPr
H
H
CONHcPr
CONHcPr
CONHcPr
CONHcPr
NA[b]
10
11
12
13
F
Et
H
H
H
H
CN
H
H
NA[b]
NA[b]
NA[b]
[a] The IC50 values shown are the mean values of quadruple measurements; the numbers in parentheses represent the 95% confidence intervals. [b] Not
applicable.
Table 3. The inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives 14–21 against the p38 MAP kinase.
ID
R1
IC50[a] (nM)
ID
R1
IC50[a] (nM)
14
15
16
17
3-Pen
tBu
0.79 (0.74–0.86)
0.77 (0.72–0.83)
1.3 (1.2–1.4)
18
19
20
21
C(Me)2CH2OH
4-pyranyl
1.1 (1.0–1.3)
2.1 (1.9–2.2)
1.4 (1.3–1.6)
5.1 (4.4–5.9)
iBu
1-Me-cPr
c
CH2 Pr
1.8 (1.6–2.0)
1-Me-oxetanyl
[a] The IC50 values shown are the mean values of quadruple measurements; the numbers in parentheses represent the 95% confidence intervals.
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demonstrated that the R1 substituent on the nitrogen atom of
imidazo[4,5-b]pyridin-2-one is important since it influences not
only the inhibitory activity of the compound, but also its water
solubility.[26] We further identified that branched alkyls such as
secondary butyl or 3-pentyl groups increases the inhibitory
activity of the compound and its water solubility, making them
more preferable substituents. We therefore focused on using
branched R1 substituents in this study. The inhibitory activities
of compounds 14 and 15, bearing 3-pentyl and tertiary butyl
substituents, respectively, were comparable to that of com-
pound 3, bearing a secondary butyl substituent, whereas the
inhibitory activities of compounds 16 and 17, carrying isobutyl
and cyclopropylmethyl substituents, respectively, were slightly
reduced in comparison to that of compound 3. These results
were consistent with those of our previous study, in which the
presence of a alkyl group branched at the alpha-carbon
adjacent to the nitrogen atom on imidazo[4,5-b]pyridin-2-one
conferred a strong p38 MAP kinase inhibitory activity.[26] It
should also be noted that an achiral substituent such as the 3-
pentyl group of compound 14 or the tertiary butyl group of
compound 15 facilitates the optimization process compared to
the racemate secondary butyl substituent of compound 3. The
addition of a hydroxyl group (18) to the tertiary butyl group
slightly decreased its inhibitory activity. Substitutions with 4-
pyranyl, 1-methylcyclopropyl, and 1-methyloxetanyl groups in
compounds 19, 20, and 21, respectively, also decreased their
inhibitory activity.
We then proceeded to optimize the amide side chain of the
benzamide moiety of compound 15. Substitutions with alkyl
groups, namely, methyl, cyclobutyl, or isopropyl groups in
compounds 22, 23, and 24, respectively, decreased their
inhibitory potential. The observation that compound 15, with
the cyclopropyl substituent, was more potent than compound
24, bearing the isopropyl substituent, led us to the idea that
sp2 character of cyclopropane ring could play an important role
in enhancing the p38 MAP kinase inhibitory activity. It was
previously reported by a research group at BMS that the p38
MAP kinase inhibitory activity is retained upon substitution of
the amide side chain by an azole group.[32] The effect of
replacing the cyclopropyl group with an azole group on the
inhibitory activity was explored. It was observed that substitu-
tion with 4-isoxazolyl 26 decreased the inhibitory activity;
however, substitution with 3-isoxazolyl 25, pyrazolyls 27–28
and 5-thiazol 29 retained their potent p38 MAP kinase
inhibitory activity. Compound 25, with the 3-isoxazolyl sub-
stituent, exhibited the most potent inhibition, with an IC50 value
of 0.42 nM, and was successfully co-crystallized with the ATP
binding domain of p38 MAP kinase at a resolution of 2.5 Å
(Figure 6). The X-ray structure of the complex of compound 25
with the enzyme revealed that compound 25 binds to the
enzyme as expected, and the flipped amide bond between
Met109 and Gly110 was also observed.[26] The carbonyl oxygen
and the 3-NH hydrogen of the imidazo[4,5-b]pyridin-2-one ring
interact with the hinge region via hydrogen bonds with Gly110,
Met109, and His107 (Figure 6, Left). The carbonyl oxygen and
the NÀ H group of the benzamide moiety form direct hydrogen
bonding interactions with Asp168 and Glu71, respectively. In
addition to these electrostatic interactions, the terminal
isoxazole ring stacks perpendicularly with Phe169 of DFG which
adopts DFG-in conformation. These CH-π interactions of the
azole groups, such as the R1 group of compound 25 and
compounds 27–29 contribute to the increase in their inhibitory
activity, as shown by the SAR study (Table 4). Additionally, the
methylbenzene group filled the inner shallow hydrophobic
pocket of the p38 MAP kinase, comprising Thr106 and Leu104.
The imidazo[4,5-b]pyridin-2-one ring was found to be sand-
wiched between Ala51/Val38 of the N-lobe and Leu167 of the
C-lobe (Figure 6, Right). Compound 25 exhibited excellent
selectivity among the 28 kinases in a kinase panel study aimed
to probe the potential off-target liabilities (Table S1). It is also
worth to note that compound 25 exhibited excellent selectivity
against the 312 kinases in the TR-FRET based competitive
binding assay (Kinome MAP (Figure S1) are provided in the
supporting information).[33] Compound 25 showed the inhib-
itory activities against BRAF, LYN, RET, p38δ (6<pIC50 <7),
RIPK2 (7<pIC50 <8), p38α, p38β, p38γ (pIC50 >8).
1
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The potential of the p38 MAP kinase inhibitors were
evaluated by hWB cell-based assays.[27] Owing to the relative
Figure 6. X-ray crystal structure of the complex of 25 and the p38 MAP kinase (PDB code 6OHD). Left: top view, Right: side view.
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Table 4. The inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives 22–29 against the p38 MAP kinase.
1
2
3
4
5
6
7
8
9
ID
R1
IC50[a] (nM)
ID
R1
IC50[a] (nM)
22
23
24
25
Me
2.7 (2.2–3.3)
1.2 (1.1–1.4)
2.6 (2.2–3.0)
0.42 (0.36–0.48)
26
27
28
29
4-isoxazolyl
2.2 (1.9–2.5)
cBu
1-Me-3-pyrazolyl
1-Me-5-pyrazolyl
5-thiazolyl
0.86 (0.78–0.94)
0.78 (0.71–0.86)
0.81 (0.72–0.91)
iPr
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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3-isoxazolyl
[a] The IC50 values shown are the mean values of quadruple measurements; the numbers in parentheses represent the 95% confidence intervals.
complexity of the cell-based assay, the suppression of cytokine
production in the hWB assay is considered to reflect the in vivo
efficacy of the compound. The inhibitory potencies, measured
in terms of the IC50 values, of the representative compounds are
summarized in Table 5. These compounds, namely, compounds
3, 9, 14, 15, 25, 27, 28, and 29, which exhibited highly potent
p38 MAP kinase inhibition, inhibited the LPS-induced produc-
tion of TNF-α in THP-1 cells with IC50 values ranging between
0.49 and 18 nM. Interestingly, compounds 3, 9, and 14
exhibited more than 10-fold decrease between p38 inhibition
and THP-1 cell-based inhibition, whereas the tert-butyl com-
pounds, namely, compounds 15, 25, and 27–29 exhibited only
a relatively slight decrease. Additionally, these compounds
suppressed the TNF-α-induced production of IL-8 in hWB cells.
Unlike the results of the THP-1 cell assay, compounds 3, 14, 15,
and 25 exhibited a strong suppression in IL-8 production.
Compounds 15, 25, and 28 exhibited a potent inhibition of the
LPS-induced production of TNF-α. The explanation on discrep-
ancy in the inhibitory activity of the compounds between the
two cell-based assays was still ambiguous, and it was also not
possible to explain the differences in the SAR of the compounds
between the two cell-based assays. However, compounds 15
and 25 exhibited highly potent cytokine suppression in both
the cell-based assays. The pharmacokinetic properties of
compounds 15 and 25 in rats are shown in Table 6. It was
observed that compound 25 exhibited a higher oral exposure
(AUC=370.4 ngh/mL) and bioavailability (F%=41.6); however,
compound 15 exhibited a lower oral exposure and bioavail-
ability (AUC=194.6 ngh/mL; F%=20.7). At a dose of 1 mg/kg,
the absorption and exposure of compound 25 were favorable
in rats and were sufficient for further in vivo pharmacological
testing.
Compound 25 was evaluated in a pseudoestablished rat
model of collagen-induced arthritis (CIA) (Figure 7). The rats
were orally administered compound 25, with b.i.d. dosing, at
doses of 0.1, 0.3, and 1 mg/kg for 2 days. The swelling of the
paws was measured on day 14. Compound 25 exhibited dose-
dependent efficacy in markedly reducing paw swelling at doses
of 0.1–1.0 mg/kg. At doses of 0.1, 0.3, and 1.0 mg/kg, the
Table 5. The inhibitory activities of imidazo[4,5-b]pyridin-2-one derivatives 3, 9, 14, 15, 25, and 27–29 on TNF-α production in human THP-1 cells, and IL-8
production and TNF-α production in human whole blood cells.
ID
p38 MAPK
IC50[a] (nM)
THP-1
hWB IL-8
hWB TNF-α
Solubility
pH=6.8 (μg/mL)
IC50[a] (nM)
IC50[a] (nM)
IC50[a] (nM)
3
9
0.73
0.79
0.79
0.77
0.42
0.86
0.78
0.81
7.7
2.4 (2.0–2.7)
8.1 (6.1–10)
2.6 (1.3–4.0)
2.0 (0.96–3.1)
2.5 (1.7–3.4)
7.4 (4.8–10)
7.1 (4.5–9.8)
93 (23–160)
170 (130–210)
450 (200–700)
150 (71–220)
61 (40–82)
30 (28–33)
150 (78–210)
6.5
3.1
2.0
3.6
5.5
3.2
49
18 (10–30)
9.4 (5.5–16)
1.4 (0.95–2.0)
0.49 (0.23–1.0)
1.5 (0.95–1.3)
1.4 (0.86–2.2)
3.1 (1.7–5.4)
14
15
25
27
28
29
70 (42–97)
510 (490–530)
0.76
[a] The IC50 values shown are the mean values of more than double measurements; the numbers in parentheses represent the 95% confidence intervals.
Table 6. Pharmacokinetic parameters of 15 and 25 in rats[a].
[b]
[b]
ID
Cmax
Tmax
(h)
AUC[b]
(n·gh/mL)
MRT[b]
(h)
Vd
(mL/kg)
CL
(mL/h/kg)
F
(%)
(ng/mL)
15
25
55.2
98.5
1.0
1.3
194.6
370.4
2.5
2.5
2145
1947
1067
1169
20.7
41.6
[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.
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Figure 7. Anti-inflammatory effects of 25 in the model of collagen-induced arthritis in the rat (bid, po, 2 days treatment). Left: Time course; Right: pow volume
at day 14. Data represent the mean � S.E. for 5 or 8 animals/group. #: p<0.025 vs. control by Williams test.
reduction in paw swelling was found to be statistically
significant.
3–29 and intermediates are provided in the supporting
information). In Scheme 1, chloropyridine 30 was treated with
cesium fluoride to afford fluoropyridine 31.[26] An aromatic
nucleophilic substitution reaction with the appropriate amines
R1NH2 produced the corresponding compound 32. The alkaline
hydrolysis of 32 produced the carboxylic acid 33 as a cyclization
precursor. Subsequent cyclization was successfully accom-
plished by treating compound 33 with DPPA. Amidation of the
carboxylic acid 35 and subsequent Miyaura-borylation of 36 led
to the generation of compound 37. The Suzuki-Miyaura cross
coupling reaction of compounds 34 and 37 finally led to the
generation of compound 3.
Chemistry
The synthesis route of compound 3 is indicated in Scheme 1 as
a representative route for the synthesis of imidazo[4,5-b]
pyridin-2-one analogs (experimental procedures for compounds
Conclusions
In our structure-based design strategy beginning from lead
compound 1, we focused on the design and synthesis of hybrid
compounds in which the imidazo[4,5-b]pyridin-2-one core was
successfully linked with the p-methylbenzamide fragment, to
enhance the suppression of the LPS-induced production of
TNF-α in the hWB cell assay. The SAR analyses revealed that
compound 25 had excellent p38 MAP kinase inhibitory activity
(IC50 =0.42 nM), suppressed the production of TNF-α in hWB
cells (IC50 =30 nM), and had good oral bioavailability in rats. It
was further observed that compound 25 exhibited dose-
dependent efficacy in a rat model of CIA at doses between 0.1–
1.0 mg/kg.
Experimental Section
Biology: p38 MAP kinase enzyme assay.[25,26] According to
previously reported method, the FLAG-tagged human p38-α
protein was expressed, activated, and purified. Kinase reactions
were evaluated with LanthaScreen assay system; 2.5 μL of test
compounds, diluted with DMSO (final concentration 1% DMSO),
were added to the reaction mixture (25 mM HEPES [pH 7.5], 10 mM
Scheme 1. Synthesis of compounds 3. Reagents and conditions: (a) CsF,
°
°
DMSO, 50 C; (b) 1-methylpropylamine, DMSO, 100 C; (c) 8 N NaOH, THF-
°
MeOH, rt; (d) DPPA, Et3N, toluene, 100 C; (e) cyclopropylamine, WSC, HOBt,
Et3N, DMF rt; (f) (Bpin)2, PdCl2(dppf)·CH2Cl2, NaOAc, DME, reflux; (g)
°
PdCl2(dppf)·CH2Cl2, Na2CO3, DME-H2O, 100 C.
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Mg acetate, 1 mM DTT, 0.01% Tween-20, and 0.01% BSA)
containing 125 pg human p38-α protein and 8 nM GFP-ATF2. After
a 5 min incubation at room temperature, 5 μL of 580 μM ATP was
added to the reaction mixture. After a 20 min incubation, 5 μL of
80 mM EDTA was added to the reaction mixture. Then, 5 μL of the
Tb-anti-phospho ATF2 (pThr71) antibody (Life Technologies) was
added and subsequently incubated for 60 min at room temper-
ature. The time-resolved fluorescence resonance energy transfer
(TR-FRET) signal was measured using EnVision Multilabel Plate
Reader (PerkinElmer).
using animals were reviewed and approved by the Internal Animal
Care and Use Committee of Takeda Pharmaceutical Research
Division.
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. A. K., M. T., H. F., R. O., S. M., S. M. and S. M.
contributed design and synthesis of compounds; M. G., T. M., Y.
H., S. U., T. K., and H. S. conducted in vitro and in vivo study; T.
T., S. I., T. T., K. O., W. L, B. S. and K. S. contributed x-ray
crystallography.
TNF-α production assay in THP-1cells.[25,26] THP-1 cells were
suspended in RPMI 1640 medium (Life Technologies) containing
1% fetal bovine serum (Morgate, Australia) according to previously
reported method. Then, 40 μL of the cell suspension
(0.625X106 cells/mL) was incubated with 5 μL of test compounds
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°
diluted with 10% DMSO. After 60 min incubation at 37 C under 5%
CO2, the cells were activated with 5 μL of 100 μg/mL LPS (Wako).
°
After incubating for 4 h at 37 C under 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 detected with EnVision
Multilabel Plate Reader.
Abbreviations
MAP mitogen-activated protein
TNF-α tumor necrosis factor-α
IL-1β interleukin-1β
IL-8or TNF-α production assay in human whole blood cells.[26]
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
IL-8
RA
LPS
interleukin-8
rheumatoid arthritis
lipopolysaccharide
°
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 or
°
20 μL of 10 ng/mL LPS. After incubating for 18–24 h at 37 C and
hWB human whole blood
CIA collagen-induced arthritis
SAR structure-activity relationships
5% CO2, the concentration of IL-8 or TNF-α in the medium was
measured with the IL-8 or TNF-α ELISA kit (R&D systems, USA). The
absorbance was measured with Wallac 1420 Plate Reader (Perki-
nElmer).
Acknowledgements
Solubility:[25,26] According to the standard screening method in
Takeda Pharmaceutical Company Limited, small volumes of the
compound solution dissolved in DMSO were added to the aqueous
buffer solution (pH 6.8). After incubation, precipitates were sepa-
rated by filtration. The solubility was determined by HPLC analysis
of each filtrate.
The authors express their gratitude to Drs. Shigenori Ohkawa, Yuji
Ishihara, Fumio Itoh, Osamu Uchikawa, and Yoshinori Ikeura for
their valuable discussions, and to Dr. Takashi Ichikawa for
critically reviewing the manuscript. The authors remain grateful to
the members of the Takeda Analytical Research Laboratories, Ltd.,
for performing the elemental analyses. We thank the staff at
Berkeley Center for Structural Biology, Lawrence Berkeley National
Laboratory, wherefrom the X-ray diffraction data were collected
using the Advanced Light Source beamline 5.0.3. 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.
Pharmacokinetics: Rat cassette BA:[26] According to the standard
method in Takeda Pharmaceutical Company Limited, the com-
pounds were administered intravenously (0.1 mg/kg) or orally
(1 mg/kg) by cassette dosing to non-fasted rats. After administra-
tion, blood samples were collected and centrifuged to obtain the
plasma fraction. The obtained plasma samples were deproteinized
by mixing with acetonitrile followed by centrifugation. The
compound concentrations in the supernatant were measured by
LC-MS/MS. All the experiments using animals were reviewed and
approved by the Internal Animal Care and Use Committee of
Takeda Pharmaceutical Research Division.
Rat collagen model of induced arthritis.[25] Rat CIA was induced
according to previously reported method. On day 6 and 11, the
volumes of both hind paws were measured using a Plethysmom-
eter (UGO BASIL, Varese, Italy). The CIA rats were divided based on
body weight and increase in paw volume (n=8 per group) on day
11: one group received 0.5% methyl cellulose (MC), while the other
five groups received compounds suspended in 0.5% MC at a
dosage of 0.1–1 mg/kg. The increase in paw volume was expressed
as the value of paw volume measured on day 11, minus the value
of the paw volume measured on day 6, in each CIA rat. The
compounds were administered to rats twice a day from day 12 to
day 13. The same paw was subsequently measured on day 14.
Differences between the means of the control and drug-treated
groups were analyzed using the Williams test. All the experiments
Conflict of Interest
The authors declare no conflict of interest.
Keywords: p38 mitogen-activated protein kinase inhibitors ·
rheumatoid arthritis · structure-based design · imidazo[4,5-b]
pyridin-2-one derivatives
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Version of record online: ■■■, ■■■■
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A. Kaieda*, Dr. M. Takahashi, H.
Fukuda, Dr. R. Okamoto, Dr. S.
Morimoto, M. Gotoh, T. Miyazaki, Y.
Hori, S. Unno, Dr. T. Kawamoto,
Dr. T. Tanaka, Dr. S. Itono, T. Takagi,
Dr. H. Sugimoto, Dr. K. Okada, Dr. W.
Lane, Dr. B.-C. Sang, Dr. K. Saikaten-
du, Dr. S. Matsunaga, Dr. S.
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Miwatashi
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Structure-Based Design,
Synthesis, and Biological Evalua-
tion of Imidazo[4,5-b]Pyridin-2-
one-Based p38 MAP Kinase Inhibi-
tors: Part 2
Scaffold hopping: Structure-based
design with X-ray crystallographic
analysis of the complex between lead
compound 1 and p38 MAP kinase,
and expanding an approach for direct
hydrogen bond formation with the
back pocket residues of the kinase,
led to potent p38 MAP kinase
inhibitor 25, which was found to
exhibit strong suppression of TNF-α
production in human whole blood
and to have excellent in vivo efficacy
in a rat CIA model.