Hybrid-designed inhibitors of p38 MAP kinase utilizing N-arylpyridazinones

Article abstract of DOI:10.1021/jm025585h

Imidazo[1,2-α]pyridyl N-arylpyridazinones were hybridized from the classic pyridinylimidazoles and the more recent dual hydrogen bond acceptors, resulting in a new structural class of selective p38 MAP kinase inhibitors.

Full text of DOI:10.1021/jm025585h

J . Med. Chem. 2003, 46, 349-352
349
Historically, SmithKline Beecham demonstrated early
on that an orally active small molecule, SB203580 (1),
could reduce TNFR levels in vivo, validating the pyridi-
nylimidazole structural class.6 Significant improve-
ments to the kinase selectivity and whole blood potency
of 1 were subsequently accomplished at Merck with the
discovery of the (S)-sec-phenethylamine moiety repre-
sented in the second-generation pyridinylimidazole 2.7
Within a few years, Vertex demonstrated that even
greater kinase selectivity could be obtained with VX-
745 (3).8 Human clinical trials with 3 have been
reported,9 reviewed,10 and claimed to have achieved
proof of concept, although this press release lacks
scientifically reviewed data.11 In an effort to increase
the whole blood potency of 3, chemists at Merck
designed inhibitors such as 4 that retained the struc-
tural attributes of 3 in addition to the basic saturated
heterocycle present in 2.12
Design . The modeling view in Figure 2 is based on
the X-ray crystal structure of p38R in complex with the
soaked inhibitor 4.12 Hybrid 6 was energy-minimized
outside the protein and then docked into the enzyme
active site using the binding orientation of 4 as a
guide.13 The carbonyl oxygen of the urea moiety of 4 and
that of the pyridazinone ring of 6 are able to accept two
hydrogen bonds from the backbone amide NHs of
Met109 and Gly110. The 2-chloro-4-fluorophenyl ring
of 6 appears to superimpose quite well with the 2,4-
difluorophenyl ring of 4 near Thr106.14 However, the
tolyl moiety of 6 is situated in the binding pocket near
Val30 differently from the 2,6-dichlorophenyl ring of 4.
Within the MAP kinase family, the J NK Ser/Thr
protein kinases bear high homology to p38.15 Crystal
structure data reveal that inhibitors with a dual hy-
drogen bond motif at Met109 and Gly110 of p38R induce
a peptide flip at Gly110 (Figure 2). Since J NK1-3
kinases contain an aspartic acid at a position equivalent
to the position of Gly110 in p38R, the Ramachandran
space of this Asp residue is much more restricted. This
rotational restriction could result in a hindered peptide
flip, an inefficient dual hydrogen bond, and reduced
affinity for these inhibitors.16 VX-745 (3) was reported
to be more than 1000-fold selective for p38R over J NK
kinases.17 Since one of the closest homologues to p38R
kinase is JNK2â2, we counterscreened this JNK isozyme
for inhibitor selectivity.
This study describes a new class of p38 inhibitors
structurally hybridized from the prototypical pyridi-
nylimidazoles and the dual hydrogen bond acceptors
represented by 3 and 4. The pyridazinone moiety18 was
selected as a dual hydrogen bond acceptor because of
its diversity based on available arylhydrazines. The
imidazo[1,2-a]pyridine core19 was chosen for its basicity
to suppress serum albumin binding.20 On the basis of
this design, compounds such as 5 and 6 were anticipated
to be highly selective for p38 over J NK kinases and more
potent in whole blood relative to VX-745 (3).
Hybr id -Design ed In h ibitor s of p 38 MAP
Kin a se Utilizin g N-Ar ylp yr id a zin on es
Steven L. Colletti,* J essica L. Frie,
Elizabeth C. Dixon, Suresh B. Singh,
Bernard K. Choi, Giovanna Scapin,
Catherine E. Fitzgerald, Sanjeev Kumar,
Elizabeth A. Nichols, Stephen J . O’Keefe,
Edward A. O’Neill, Gene Porter, Koppara Samuel,
Dennis M. Schmatz, Cheryl D. Schwartz,
Wesley L. Shoop, Chris M. Thompson,
J ames E. Thompson, Ruixiu Wang, Andrea Woods,
Dennis M. Zaller, and J ames B. Doherty
Merck Research Laboratories, Merck & Co., Inc.,
Rahway, New J ersey 07065
Received September 16, 2002
Abstr a ct: Imidazo[1,2-a]pyridyl N-arylpyridazinones were
hybridized from the classic pyridinylimidazoles and the more
recent dual hydrogen bond acceptors, resulting in a new
structural class of selective p38 MAP kinase inhibitors.
In tr od u ction . TNFR is one of several cytokines that
plays a significant role in the development of an acute
or chronic inflammatory response. The success of the
soluble TNFR receptor fusion protein Enbrel (etaner-
cept)1 and the monoclonal TNFR antibody Remicade
(infliximab)2 in the treatment of rheumatoid arthritis
and Crohn’s disease has provided a proof of concept for
the treatment of these autoimmune diseases. These
biologics are generally well tolerated to date but have
drawbacks related to patient cost, efficiency of produc-
tion, and administration by injection. Therefore, an
orally active small-molecule drug that blocks or modu-
lates circulating TNFR remains an attractive therapy.
Of the different approaches toward this end, inhibition
of p38 MAP kinase results in the suppression of not only
TNFR but also IL-1â, another significant proinflamma-
tory cytokine.3
The biosynthesis of TNFR and IL-1â occurs predomi-
nately in activated monocytes and macrophages via an
intracellular signaling cascade that involves the dual
phosphorylation (via MKK3 and MKK6) of Thr180 and
Tyr182 within a TGY motif of p38 kinase. Phosphory-
lated p38 subsequently phosphorylates a variety of
substrates, including kinases and transcription factors.4
Two of these substrates that are activated by p38 are
MAPKAP kinases 2 and 3, which in turn phosphorylate
heat shock protein HSP 27, ultimately leading to gene
transcription and protein translation. This cascade of
events can be initiated by a wide variety of extracellular
stimuli or stresses such as endotoxin bacterial li-
popolysaccharide and cytokines, or osmotic shock, heat
shock, UV light, ionizing radiation, and oxidative stress.
Four isozymes of p38 have been cloned and character-
ized to include the ubiquitously expressed p38R and
p38â; p38γ is primarily expressed in skeletal muscle,
and p38δ is highly distributed within lung, kidney,
endocrine glandular, and small intestinal tissues.5
Ch em istr y. One highly convergent approach to the
synthesis of these molecules is shown in Scheme 1.
Intermediate 7 was prepared21 and then selectively
cyclized to the energetically preferred six-membered
* To whom correspondence should be addressed. Phone: 732-594-
6886. Fax: 732-594-9473. E-mail: steve_colletti@merck.com.
10.1021/jm025585h CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/20/2002

350 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Letters
Sch em e 2a
a
(a) n-Bu4NBr3, CH2Cl2, 0 °C f 23 °C, 3 h; (b) 2-aminopyridine,
EtOH, 60 °C, 14 h, ∼70% (two steps); (c) Me(MeO)NH/HCl,
i-PrMgCl, THF, -10 °C, 1 h, ∼74%; (d) MeMgBr, THF, 0 °C, 30
min, ∼97%; (e) LiHMDS, BrCH2CO2t-Bu, THF, -78 °C f 23 °C,
2 h; (f) TFA, CH2Cl2, 0 °C f 23 °C, 4 h, then HClg, MeOH, 0 °C,
1 h, ∼40% (two steps); (g) arylhydrazine hydrochloride, NaOAc,
HOAc, H2O, 130 °C, 20 h, ∼30%; (h) CuCl2, CH3CN, 85 °C, 72 h,
∼50%.
F igu r e 1. Selected p38 inhibitors.
form 10 in good overall yield from the starting benzoyl
acetates. The methyl ketones (10) were then homolo-
gated to the γ-ketoesters (11) in modest yield, followed
by cyclization with several different phenylhydrazines
to form the N-aryldihydropyridazinones. Oxidation
proved to be extremely slow and often incomplete, yet
absent of any byproducts to provide the desired py-
ridazinones (12).
In Vitr o SAR. The biological data for the p-fluoro
derivative 13 represent a baseline for SAR in the N-(2,6-
dichlorophenyl)pyridazinone series (A, Table 1). Unlike
pyridinyl imidazole 2, the m-trifluoromethyl group was
not tolerated in 14 at the level of the enzyme, indicating
a different binding orientation from 2. This result is
consistent with the activity of the dual hydrogen bond
acceptor inhibitors relative to the pyridinylimidazoles.
Likewise the o,m-dichloro analogues 15 and 16 dis-
played poor enzyme inhibition. As with 4,12 combined
substitution at the ortho and para positions was ideal
for enzyme affinity and is exemplified by the 2-chloro-
4-fluoroaryl analogues 17 and 18.
F igu r e 2. Modeling view of 6 (yellow) overlaid on the crystal
structure of p38R MAP kinase in complex with 4 (magenta).
Sch em e 1a
To reduce the lipophilicity of 17 and 18, the 2,4-
difluoroaryl analogues 5 and 19 were prepared.24 As
anticipated, the IC50 shift from enzyme inhibition to
THP-1 cellular activity was reduced with 5 relative to
17. However, the oxidation of 5 to 19 resulted in an
overall decrease in activity observed not only in whole
blood but also in the p38 inhibition assay.
a
(a) LiHMDS, succinic anhydride, THF, -78 °C f 23 °C, 2 h,
20%; (b) 2-chlorophenylhydrazine hydrochloride, NaOAc, HOAc,
110 °C, 2 h, 50%; (c) n-Bu4NBr3, 2:1 CH2Cl2/CCl4, 0 °C f 23 °C,
4 h, 99%.
Another approach to reduce the lipophilicity of 17 and
18 was to remove one chlorine atom and prepare the
N-(2-chlorophenyl)pyridazinone series (B, Table 1).
Although the IC50 shift between enzyme affinity and
THP-1 activity was attenuated for compounds 20 and
21 relative to 17 and 18, a corresponding reduction in
p38 affinity was also observed, resulting in THP-1
activity similar to that of 17 and 18. The m-trifluoro-
methylaryl analogues 22 and 23 displayed weak activity
similar to that of 14.
ring dihydropyridazinone. After bromination to form 8,
a Chichibabin based imidazopyridine synthesis was
envisioned using 2-aminopyridine. This reaction failed
repeatedly apparently because of the absence of any
microscopic tautomerization of 8 to the required R-bro-
moketone.
Subsequently a more linear synthesis was developed
that provided the targeted imidazopyridyl N-arylpy-
ridazinones (Scheme 2). Readily available benzoyl ace-
tates were brominated under ionic conditions, and the
R-monobromides were subjected to the Chichibabin
based imidazopyridine synthesis using 2-aminopyridine
to form haloaryl analogues of 9.22 The methyl esters
were converted to the Weinreb amides under basic
conditions,23 followed by methyl ketone generation to
On the basis of the patent literature25 and the SAR
of 4, the N-(2-chlorophenyl)pyridazinone (B) binding
subunit was replaced with an N-(2-tolyl)pyridazinone
moiety (C, Table 1). Although a reduction in lipophilicity
was anticipated, a potential reduction in p38 affinity
relative to the N-(2,6-dichlorophenyl)pyridazinone series

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 351
Ta ble 1. In Vitro Activity of N-Arylpyridazinone Analoguesa
IC50 (nM)
compd
R
bond order
p38Rb
p38âb
153
50% @ 5000
57% @ 5000
J NK2â2c
THP-1d
70
41% @ 4000
59% @ 4000
65% @ 4000
180
hWBe
(A) N-(2,6-Dichlorophenyl)pyridazinone Analogues
13
14
15
16
17
18
5
4-F
2
2
1
2
1
2
1
2
49
1000
2000
270
42
40% @ 10000
0% @ 10000
26% @ 5000
14% @ 5000
0% @ 10000
23% @ 10000
22% @ 10000
11% @ 5000
60% @ 4000
3-CF3
2,3-diCl
2,3-diCl
2-Cl-4-F
2-Cl-4-F
2,4-diF
2,4-diF
890
670
64
63
112
14
8
22
161
10
89% @ 4000
205
1000
19
(B) N-(2-Chlorophenyl)pyridazinone Analogues
20
21
22
23
2-Cl-4-F
2-Cl-4-F
3-CF3
1
2
1
2
208
76
171
344
2600
171
355
0% @ 10000
0% @ 10000
0% @ 10000
400
161
72% @ 4000
48% @ 4000
3-CF3
(C) N-(2-Tolyl)pyridazinone Analogues
24
25
26
6
27
28
2,3-diCl
2,3-diCl
2-Cl-4-F
2-Cl-4-F
2,4-diF
1
2
1
2
1
2
36% @ 100
33% @ 100
0% @ 1000
15% @ 10000
4% @ 5000
16% @ 5000
4% @ 10000
15% @ 5000
170
6
22
20
14
68
22
14
9
8
26
44
94
28
78
140
500
220
100
440
870
2,4-diF
a
b
Values are an average of two or greater individual assays. On average, repeat determinations differed by (20%. Filter plate binding
of compound versus [γ-33P]ATP in active recombinant murine FLAG-p38 fusion protein (GST-ATF2).7 c HTRF detection of GST-ATF2
d
phosphorylated by recombinant human flag-tagged J NK2â2 fusion protein. LPS-induced human TNFR inhibition detected by ELISA in
THP-1 cell supernatants. e LPS-induced human TNFR inhibition in human whole blood quantified by immunoassay.7
Ta ble 2. Biological Profiles for 3 and 6
(A) was a concern. In practice, the 2-tolyl analogues 24-
in vitro IC50 (nM)
compd p38R THP-1 hWB % inhibition plasma concn (nM)
ex vivo mMAPKAPa
26 and 6 were more active in functional assays and
enzyme inhibition relative to the corresponding 2,6-
dichlorophenyl analogues 15-18. Analogues 26 and 6
were also superior to the related 2-chlorophenyl deriva-
tives 20 and 21.
3
6
22
9
150
20
700
100
29
42
1734
810
a
BALB/c mice 3 (n ) 5) and 6 (n ) 5) were dosed ip at 3 mg/kg
in a vehicle of DMSO/EtOH/PEG400/saline (10:10:60:20). Blood
was collected for MAPKAP inhibition and plasma concentration
at 1 h after dosing.
For in the 2-tolyl series, attention was turned to the
5- to 10-fold IC50 shift observed between the THP-1 and
whole blood assays of 6 and 26. The 2,4-difluoroaryl
analogues were targeted again, this time toward the
preparation of 27 and 28. Once again, a reduction in
affinity and functional activity was observed between
the 2,4-difluoroaryl analogues 27 and 28, similar to that
between the 2,4-difluoroaryl analogues 5 and 19.26 Last,
the p38 inhibitors described in Table 1 show consistent
p38R selectivity relative to J NK2â2. The most active
hybrids, 5 and 6, demonstrated >1000-fold and >500-
fold kinase selectivity, respectively.
respectively, were measured from the same experiment
by an ex vivo p38 kinase inhibition assay.27
An additional comparison of 3 with 6 regarding in
vitro metabolism served to correlate these plasma
concentrations with microsomal stability. Both 3 and 6
were incubated with mouse liver microsomes, resulting
in a respective 46% and 9% compound presence after
10 min (Supporting Information). From both this mi-
crosomal data and the in vivo plasma concentrations,
it is apparent that 6 is more readily metabolized in the
mouse relative to 3, yet 6 appears to be equally, if not
slightly more, efficacious relative to 3 in the mMAPKAP
PD assay. This result is consistent with the greater in
vitro activity of 6 relative to 3 (Table 2).
Con clu sion . In summary, a new structural class of
p38 kinase inhibitors was designed based ultimately on
SB203580 and VX-745. Representative hybrids include
imidazopyridyl N-arylpyridazinones 5 and 6, both of
which demonstrated low nanomolar enzyme inhibition,
g1000-fold p38R/J NK2â2 kinase selectivity, and efficacy
P D P r ofile a n d in Vitr o Meta bolism of Com -
p ou n d 6. After the in vitro data shown in Table 1 were
obtained, analogue 6 was further evaluated against VX-
745 (3) in a murine MAPKAP pharmacodynamic (PD)
assay (Table 2). In this study, both 3 and 6 were each
dosed ip at 3 mg/kg in five mice, resulting in mMAPKAP
inhibition of 29% and 42%, respectively. The mMAPKAP
inhibition with 6 was statistically significant (p < 0.05)
relative to vehicle. Importantly, the compound exposure
in mice was different for 3 relative to 6. Plasma
concentrations of 1743 and 810 nM for 3 and 6,

352 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Letters
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in human whole blood. Analogue 6 was evaluated ex
vivo in mice, providing 42% inhibition of mMAPKAP
activity and further validating this class of p38 inhibi-
tors. Relative to the clinically used VX-745 (3), analogue
6 displayed similar efficacy in this pharmacodynamic
mouse model.
(16) p38 G110A and G110D mutants support this observation.
Fitzgerald, C. E.; O’Keefe, S. J .; Scapin, G.; et al. Manuscript in
preparation. Scapin, G. Selectivity in homologous protein fami-
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over J NK can also be achieved through exploiting the larger
hydrophobic pocket of p38R at T106 versus the smaller hydro-
phobic pocket of J NK, which has a methionine residue in this
position.14,15
(17) Salituro, F. G. Presented at the 27th National Medicinal
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a]pyridine is 6.8. Gilchrist, T. L. Heterocyclic Chemistry, 3rd ed.;
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(24) The observed IC50 shifts from enzyme inhibition to the THP-1
cellular assay to the suppression of TNFR in whole blood were
partly attributed to protein binding in these functional assays.
Serum protein binding measurements with related structural
classes supported this hypothesis.
(25) Salituro, F. G.; Bemis, G. W.; Cochran, J . E. (Vertex Pharma-
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N-aryl moiety. This increased planarity, based on a lowered
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energy barrier toward enzyme binding conformation that re-
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(13) See Supporting Information for modeling details. Since the
glycine-rich loop of p38 (containing Val30 and Tyr35) is known
to change conformation significantly, depending on the inhibitor
in the active site, the structure of p38 used here is only an
approximation of the active site for 6. Furthermore, it appears
that the protein would need to adjust at Val30 to accommodate
the tolyl moiety of 6.
(27) Blood plasma concentrations of 3 and 6 were calculated by
bioassay. The free compound p38 inhibition IC50 is multiplied
by 1 over the dilution factor of the plasma required to reach 50%
inhibition in the p38 assay (EC50). This method does not address
active metabolites.
J M025585H