Inhibitors of unactivated p38 MAP kinase

Article abstract of DOI:10.1016/j.bmcl.2006.08.101

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based

Full text of DOI:10.1016/j.bmcl.2006.08.101

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6102–6106
Inhibitors of unactivated p38 MAP kinase
James Bullington,a,* Dennis Argentieri,a Kristin Averill,a Demetrius Carter,a
Druie Cavender,a Bohumila Fahmy,a Xiaodong Fan,a Daniel Hall,a
Geoffrey Heintzelman,a Paul Jackson,a Wai-Ping Leung,b Xun Li,a Ping Ling,b
Gilbert Olini,a Thomas Razler,a Michael Reuman,a Kenneth Rupert,a Ronald Russell,a
John Siekierka,a Scott Wadsworth,a Russell Wolff,a Bangping Xianga and Yue-Mei Zhanga
aJohnson and Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202, Raritan, NJ 08869, USA
bJohnson and Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, LoJolla, CA 92121, USA
Received 26 June 2006; revised 23 August 2006; accepted 28 August 2006
Available online 12 September 2006
Abstract—Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The
first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-
based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyrid-
inylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly
inhibiting activated p38 kinase activity in vitro.
Ó 2006 Elsevier Ltd. All rights reserved.
With the success of anti-TNF-a biologics like Remicade,
Enbrel, and Humira the desire for small molecule inhib-
itors of tumor necrosis factor-a (TNF) production has
increased.1 These drugs have demonstrated that block-
ing TNF production has a beneficial effect on rheuma-
toid arthritis (RA), ankylosing, spondylitis and
systemic lupus erythematosus.
Previous work in our laboratories on pyridylpyrroles as
p38 kinase inhibitors was impeded by cytochrome p450
inhibition.4 (Fig. 1) The most promising structures of
these early pyrroles potently inhibited both activated
p38 kinase activity and LPS-induced TNF production
in human peripheral blood mononuclear cells (PBMCs)
in the single digit nanomolar range. They also inhibited
TNF production orally in mice with ED50s less than
10 mg/kg. However, this series potently inhibited multi-
ple cytochrome p450 enzymes in the 100–300 nM range.
Our goal was to reduce CYP450 inhibition while main-
taining these biological profiles against p38.
Since secretion of TNF is activated through the p38
MAP kinase pathway, many series of p38 MAP kinase
inhibitors have been explored.2 To date, no p38 inhibi-
tor has advanced to the marketplace although several
inhibitors have been clinically tested in humans and
have advanced as far as Phase II.3 Recent work has
investigated new structural types and different binding
modes.2e We now report potent pyridinylimidazole-
based inhibitors of the p38 pathway which, unlike
previous structures, have little effect on activated p38
catalytic activity in vitro. Instead, they potently bind
to unphosphorylated p38 enzyme, consequently inhibit-
ing its activation.
It has been previously suggested that CYP inhibition
can be reduced by introducing polarity on the side chain
ortho to the pyridine ring.5 We proposed applying this
method to our pyrroles by replacing the propylphenyl
F
F
N
N
N
R
N
R
N
HN
Keywords: p38; Kinase; TNF; Pyrrole; Toxicity.
*
Early Series
Proposed series
Corresponding author. Tel.: +1 609 586 8123; e-mail: jbullington@
ruxtonrx.com
Figure 1. Changing the propylphenyl to piperazine.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.101

J. Bullington et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6102–6106
6103
group of our earlier series with a piperazine (Fig. 1) to
address CYP450 inhibition and increase solubility.
was also orally potent in our in vivo mouse model
(ED50 = 7.5 mg/kg). We were again surprised to discov-
er that compound 4 did not inhibit activated p38 kinase
activity in vitro at concentrations up to 10 lM. Substitu-
tions ortho to the phenyl ring typically reduce the poten-
cy of inhibitors of activated p38.2d Since our compounds
were not inhibiting the activated p38 enzyme, the ester
substitution was tolerated. The selectivity profile of
compound 4 was excellent with IC50s over 10 lM
against 21 in-house targets and greater than 1 lM in a
Cerep panel with the exception of 5-HT3 receptor
(IC50 = 275 nM). Unfortunately, when we attempted a
5-day hepatomegaly study, this compound was found
to be acutely toxic in mice (n = 5) at 200 mg/kg.
In order to selectively substitute the pyrrole ring we used
an ester to block one side of the pyrrole, which later
could be easily removed to give our target structures.
The synthesis of a key intermediate pyrrole (4) was ini-
tiated by a base condensation of 4-pyridylacetonitrile
with a substituted benzaldehyde using known methods
to form the a,b-unsaturated nitrile (Scheme 1). The pyr-
role ring (1) was formed by treating the unsaturated ni-
trile with methyl isocyanoacetate in base.6 Alkylation of
the pyrrole nitrogen was achieved in high yield by
deprotonation with sodium hydride in DMSO at 20 °C
and rapid addition of methyl iodide in one portion.
Slower addition of MeI resulted in alkylation of the pyr-
idine nitrogen to give the quaternary salt as a by-prod-
uct. Next, bromination with NBS proceeded easily in
moderate yield to give compound 2. The bromide was
converted to structure 3 via a palladium catalyzed ami-
nation utilizing Pd2(dba)3, BINAP, and Cs2CO3 in tolu-
ene. Many different palladium conditions were used in
an attempt to increase the yield, but all results were infe-
rior to the conditions stated above. Removal of the Boc
group with TFA produced the unprotected piperazine
(4), which although was an intermediate was screened
for TNF inhibition.
This prompted us to further explore the SAR in order to
eliminate toxicity while maintaining the biological pro-
file of this new series. We rapidly made some simple
modifications to identify which regions of the molecule
were responsible for the observed toxicity. Changing
the substituents on the phenyl ring did not affect toxicity
as shown by compounds 5–7 (Table 1). All compounds
were still potent in the cellular and in vivo assays, but
were also toxic.
Further modification of compound 4 was accomplished
by methylation of the piperazine nitrogen via reductive
amination using sodium triacetoxyborohydride and
formaldehyde to give compound 8 (Table 2). This mol-
ecule was active in PBMCs and in the mouse model,
inhibiting TNF production by 89 percent at 10 mg/kg.
Although compound 8 was not toxic at 200 mg/kg, some
lethargy was observed in the first few hours which
Surprisingly, we found that this intermediate (4) potent-
ly inhibited TNF production in the PBMC assay
(IC50 = 33 nM). In addition, this compound had no ef-
fect on cytochrome p450 enzymes below 10 lM with
the exception of the BFC substrate of 3A4 (1.3 lM). It
F
F
F
N
N
N
(i)
(ii), (iii)
CO2Me
(v)
CO2Me
F
N
H
1
Br
NC
N
2
F
N
N
(iv)
CO2Me
N
N
CO2Me
N
N
N
Boc
HN
3
4
Scheme 1. Synthesis of compound 4. Reagents and conditions: (i) CNCH2CO2Me, t-BuOK, THF, 0 °C, 89%; (ii) NaH, MeI, DMSO, 25 °C, 92%;
(iii) NBS, CH2Cl2, 46%; (iv) Pd2 (dba)3, Boc-piperazine, BINAP, Cs2CO3, toluene, 100 °C, 40% (63% based on recovered SM); (v) TFA, CH2Cl2.
Table 1. Changing the aromatic substitution does not affect toxicity
R
N
CO2Et
N
N
HN
Compound
R
p38 enzyme IC50 (nM)
TNF % inhibition (cell) IC50 (nM)
TNF % inhibition (mouse) 10 mg/kg
5
6
7
3-CF3
4-Me
3,5-F
>10,000
3939
>10,000
21
60
93
83
79
79

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J. Bullington et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6102–6106
Table 2. N-Alkyl derivatives of the piperazine reduce toxicity
Table 4. Acyclic analogs of the piperazine
F
F
N
N
CO2Me
CO2Me
N
R1
N
N
N
R
N
R
Compound
R
TNF % inhibition (cell) IC50 (nM)
Compound
R
R1
H
TNF % inhibition
(cell) IC50 (nM)
8
9
Me
Et
95
135
16
98
92
50
38
10
11
n-Pr
i-Pr
901
1981
17
18
19
H
diminished with time. Since methylation reduced toxici-
ty, reductive aminations were carried out to prepare the
ethyl, n-propyl, and i-propyl piperazine derivatives
(compounds 9–11). We observed a decrease of TNF
inhibition in the cell assay as the alkyl group increased
in size. Similar to compound 8, compound 9 demon-
strated lethargy in the mouse at 200 mg/kg.
H
Me
The reduced toxicity of compounds 8 and 9 prompted us
to further investigate other functional groups at the 5
position of the pyrrole. We initially kept the methyl ester
constant while replacing the piperazine with other het-
erocyclic rings (Table 3). Although pyrrolidine 12 and
morpholine 13 were potent in the cell assay they did
not show potency in vivo. Compound 14 was potent
in the mouse model, inhibiting TNF production by
96% at 10 mg/kg, but again we observed lethargy in mice
when dosed at 200 mg/kg. Compound 15 which intro-
duced an amino linker was not potent in the cell assay
and did not warrant further testing.
3 mg/kg in the in vivo mouse model. Unfortunately, this
compound was found to be rapidly metabolized in
human liver microsomes. In order to account for metab-
olism issues we injected LPS 2 h after dosing mice in-
stead of our original 0.5 h. This helped us to filter out
potential metabolically unstable analogs through in vivo
potency in mice. For example, compound 19 was ineffec-
tive with this new time course.
Examining the importance of the ester with respect to
toxicity, we saponified compound 3 to the acid, which
spontaneously decarboxylated at room temperature.
This structure was deprotected with TFA to produce
compound 20 (Scheme 2), which lacked the ester of
our lead structure 4 (Scheme 1). Compound 20 had an
IC50 of 15 nM in the cell assay and inhibited TNF pro-
duction by 75 percent in the in vivo mouse model at
10 mg/kg. Mice treated at 200 mg/kg survived, showing
that the ester also plays a role in the toxicity of com-
pound 4.
Additionally, we looked at acyclic analogs of the piper-
azine ring. These compounds were good inhibitors of
TNF production in human PBMCs (Table 4). However
compounds 16–18 did not potently inhibit TNF in vivo.
Compound 19, which is the methylated analog of com-
pound 18, inhibited TNF 78% and had an ED50 of
Furthermore, we replaced the methyl ester with ketones
and nitriles (21–24) which maintained good inhibition in
the cellular assay. Unlike ketones 21–23 the nitrile
showed good in vivo inhibition in mice (Table 5). Com-
pound 21 showed lethargy in the mouse toxicity screen,
but compound 22 did not show any signs of toxicity at
200 mg/kg. The bulkier aryl ketone and amides (25–27)
possessed significantly reduced potency of TNF inhibi-
tion in the cell assays.
Table 3. Replacing the piperazine ring with other heterocycles
F
N
CO2Me
R
N
Compound
R
TNF % inhibition TNF % inhibition
(cell) IC50 (nM)
(mouse) 10 mg/kg
12
13
14
15
17
ns
F
47
72
ns
96
nt
N
(i), (ii)
Compound 3
N
N
HN
315
20
Scheme 2. Reagents: (i) KOH, MeOH, NH4Cl; (ii) TFA, CH2Cl2.
ns, not significant; nt, not tested.

J. Bullington et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6102–6106
6105
Table 5. Ester replacements
F
F
N
N
F
(i)
(ii)
N
2
CN
CN
N
Br
N
N
AcN
N
R
N
HN
31
Compound
R
TNF % inhibition TNF % inhibition
(mouse) 10 mg/kg
Scheme 3. Synthesis of compound 31. Reagents and conditions: (i)
compound 2, NH3, (CH3)3Al, 125 °C, 68%; (ii) Pd(OAc)2, acetylpip-
erazine, BINAP, Cs2CO3, toluene, 100 °C, 93%.
(cell) IC50 (nM)
21
22
23
24
25
26
27
COCOMe
COMe
COEt
28
40
52
39
67
18
32
a
The synthesis of compound 31 is shown in Scheme 3.
Starting with intermediate 2 (Scheme 1), a one-step con-
version of the ester to a nitrile was accomplished by
heating to 125 °C with trimethylaluminum in ammonia.
This nitrile was used in a palladium amination reaction
using acetylpiperazine in conditions similar to reaction
(iv) in Scheme 1 to give structure 31.
CN
COPh
CONHMe
660
779
nt
nt
nt
CONH-iPr 10,000
a ED50, 5 mg/kg; nt, not tested.
Since the ketones and nitriles looked promising, we
examined modifications to the piperazine along with
these ester replacements. We hoped to achieve an addi-
tive effect from lowering toxicity on two regions of the
molecule (Table 6). Compound 28 shows good inhibi-
tion in the cell assay and also in the in vivo model when
tested in the original 0.5 h assay, but when tested in the
2 h assay the potency was reduced indicating a possible
short half-life. This compound was non-toxic at 200 mg/
kg, but with the potential metabolism issue the com-
pound may not be present in high enough concentra-
tions to show toxicity in this model. Unlike the ester
derivative (8) methylation of the piperazine when a
methyl ketone is present (29) decreased potency dramat-
ically when compared to its unsubstituted analog (com-
pound 22). Methylation of the nitrile derivative (30)
follows similar SAR to the ester series.
This molecule did not inhibit cytochrome p450 enzymes
below 15 lM for CYP1A2, 2D6, and 3A4 (BQ, RBE,
DBF, and BFC substrates). It weakly inhibited CYP2C9
and CYP2C19 with an IC50 of 4, 8 lM, respectively. The
hydrochloride salt of this molecule easily dissolved in
water at 100 mg/mL and had good bioavailability of
70–100% in mice, rats, dogs, and monkeys. When tested
in vivo, compound 31 showed a 50–60% reduction of the
clinical score in the collagen-induced arthritis (CIA)
mouse at 30 mg/kg (po). It also reduced swelling 91%
in the adjuvant-induced arthritis rat at 30 mg/kg (po).
Compound 31 also had an ED50 of 3 mg/kg in the car-
rageenan hind paw edema model. These data supported
the advancement of compound 31 to our preclinical
development pipeline.
References and notes
We were pleased to find that compound 31 was potent in
the TNF mouse model with an ED50 of 0.5 mg/kg and
also non-toxic at 200 mg/kg. It weakly inhibited activat-
ed p38 catalytic activity (IC50 = 2 lM) and bound with
high affinity to unphosphorylated p38a (Kd = 100 nM).
Furthermore, compound 31 inhibited p38 phosphoryla-
tion by the upstream kinases.
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Table 6. Combining the R and R1 effects of reducing toxicity
F
N
R1
N
N
N
R
Compound
R
R1
TNF %
inhibition
(cell) IC50 (nM)
TNF %
inhibition
(mouse)
10 mg/kg
0.5 h 2.0 h
28
29
30
31
COMe COMe
43
COMe 529
61
28
40
47
81
Me
Me
CN
80
123
COMe CN

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