Synthesis and SAR of 2-Phenoxypyridines as novel c-Jun N-terminal kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.090

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK3) inhibitors is described. The development and optimization of the 2-phenoxypyridine series was carried out from an earlier pyrimidine series of JNK1 inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.

Full text of DOI:10.1016/j.bmcl.2011.09.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7072–7075
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of 2-Phenoxypyridines as novel c-Jun N-terminal
kinase inhibitors
Xinyi Song, Weimin Chen, Li Lin, Claudia H. Ruiz, Michael D. Cameron, Derek R. Duckett,
⇑
Theodore M. Kamenecka
Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #A2A, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of a novel series of c-jun N-terminal kinase (JNK3) inhibitors is described. The
development and optimization of the 2-phenoxypyridine series was carried out from an earlier pyrimi-
dine series of JNK1 inhibitors. Through the optimization of the scaffold 2, several potent compounds with
good in vivo profiles were discovered.
Received 26 August 2011
Revised 20 September 2011
Accepted 21 September 2011
Available online 29 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
c-Jun
JNK
Kinase inhibitor
Phenoxypyridine
The c-Jun N-terminal kinases (JNKs) are members of mitogen-
activated protein kinase (MAPK) family which are activated in re-
sponse to dual phosphorylation on threonine and tyrosine when
exposed to extracellular stimuli such as stress and cytokines.1–6
Activation of the JNK pathway leads to the phosphorylations of a
number of transcription factors involved in cell proliferation and
apoptosis such as c-jun,7,8 ATF-2,9,10 nuclear factor of activated T
cells (NFAT),11 and tumor suppressor p53.1 Ten different JNK iso-
forms derived from three distinct genes (jnk1, jnk2, and jnk3) have
been identified.12 JNK1 and JNK2 are ubiquitously expressed in
most tissues, while JNK3 is primarily expressed in the brain and,
to a lesser extent, in the heart and testis.13,14 This variably localized
expression, together with the activation by different biochemical
pathways, indicates that different JNK isoforms have distinct
biological functions.
In recent studies, JNK-1, often in concert with JNK-2, has been
suggested to play a central role in the development of obesity
-induced insulin resistance which implies therapeutic inhibition
of JNK1 may provide a potential solution in type-2 diabetes melli-
tus.15–17 JNK2 has been described in the pathology of autoimmune
disorders such as rheumatoid arthritis and asthma, and it also has
been implicated to play a role in cancer, as well as in a broad range
of diseases with an inflammatory component.18–22 JNK3 has been
shown to play important roles in the brain to mediate neurodegen-
eration, such as beta amyloid processing, Tau phosphorylation and
neuronal apoptosis in Alzheimer’s disease, as well as the mediation
of neurotoxicity in a rodent model of Parkinson’s disease.23–25 JNK3
is almost exclusively found in the brain. Therefore, developing JNK
isoform-specific inhibitors as therapeutics has gained considerable
interest over the past few years.26–46 As part of our medicinal
chemistry research program, we initiated a JNK3 project with a
key objective of identifying brain penetrant compounds with good
JNK3 potency. We previously reported on the synthesis and SAR of
4-pyrazole substituted pyridines.44 Compounds in this class had
moderate in vitro potency, but good in vivo profiles (rodent pk
and brain penetration). In the continuous development and opti-
mization of JNK3 inhibitors, we found 2-phenoxy substituted pyr-
idines to be potent JNK3 inhibitors with excellent in vivo profiles.
In a recent publication on the development of 4-anilinopyrimi-
dines as JNK1 inhibitors by a group at Abbott, the authors reported
a series of 2-alkoxypyrimidines during the course of their SAR
studies (Fig. 1, 1, 2).47 Compound 2 was the most potent of the
eight alkoxy analogs described. The authors noted that further
modification of the phenol ring in 2 was detrimental to JNK1
H2NOC
HN
H2NOC
HN
N
N
N
O
N
OPh
1 JNK1 IC50 = 1.9 µM
2 JNK1 IC50 = 0.3 µM
⇑
Corresponding author.
E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
Figure 1. 2-Alkoxypyrimidine JNK 1 inhibitor leads.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.090

X. Song et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7072–7075
7073
Our strategy was based on scaffold 5 and more precisely on the
maintenance of the core 4-anilinopyridine wherein we could vary
the R1, R2 and R3 groups by modification or introduction of substit-
uents. The analogues 9–12 were synthesized in straightforward
fashion starting from commercially available tri-substituted pyri-
dines (6). Buchwald amination of 2,4-dichloropyridines occurred
regiospecifically at the 4-position affording 4-anilinopyridines
7.51 A subsequent Buchwald arylation at C2 with substituted phe-
nols gave 2-phenoxy pyridines 8. Finally, conversion of the 2-cyano
group to the corresponding amide yielded final products (9–12).
While 2-aminobenzamides could be used in the first Buchwald
amination step, the primary amide group interfered with the phen-
oxy coupling in step two, forcing us to resort to carrying a masked
amide (as a cyano) through the synthesis. Interestingly, this was
not an issue if we coupled an aniline at C2 instead of a phenol.
Given the report that 5-substitution on the pyridine ring im-
proved in vitro potency, we did a quick scan of the 5-position
(Table 1).47 Compounds were synthesized as described in Scheme
1, using the appropriately substituted pyridines as starting materi-
als along with 4-methoxyphenol. We were surprised at the boost
in potency observed, as we were now approaching single digit
nM inhibitors of JNK3 with even better activity against JNK1. We
continued to SAR the 2-phenoxypyridines this time varying the
substituent at the C2-position (Table 2). Chemistry was carried
out on the 5-CF3-analog (9c) instead of the 5-chloro derivative
(9b) despite the slight drop in potency. Synthesis of all intermedi-
ates and final products wherein R1 = CF3 were higher yielding and
synthetically cleaner versus compound synthesis wherein R1 = Cl.
SAR of the C2 position (R3) is shown in Table 2.
H2NOC
HN
H2NOC
HN
R2
H2NOC
NH
HN
R1
N
R3
N
O
N
O
N
O
3
JNK1 IC50 = 0.33 µM
JNK3 IC50 = 0.52 µM
4 JNK1 IC50 = 0.31 µM
JNK3 IC50 = 0.07 µM
5
Figure 2. 2-Phenoxypyridine JNK inhibitors.
Table 1
5-Substituted 2-phenoxypyridines as JNK inhibitors
H2NOC
HN
R1
OMe
N
O
Compound
R1
JNK1 IC50a
(l
M)
JNK3 IC50
(
l
M)
a
9a
9b
9c
F
Cl
CF3
0.026
0.008
0.027
0.048
0.015
0.037
All standard deviations < 20%.
a
The values are averages of two or more experiments.
Interestingly, there was a specific preference for substitution at
the 3- and 4-positions of the phenoxy group (9c,10a). Substitution
at the 2-position resulted in a large decrease in potency (10b, 10k).
The size and polarity of the substitution at the 4-position also had
an effect on potency. While a 4-methoxy group seemed ideal with
regards to in vitro potency, larger groups led to a loss in activity
potency and any additional substitution led to loss of JNK1 activ-
ity.48 During the course of our investigations on 2,4-bisanilinopyri-
dines as JNK3 inhibitors,49 we were curious to see the effect of
2-phenoxy substitution on JNK3 activity. The first synthesized
compound (3) had similar JNK1 activity as reported by the
scientists at Abbott, and was roughly equipotent on JNK3 (Fig. 2).
Interestingly, further substitution on the phenoxy ring did little
to JNK1 activity, but greatly improved potency on JNK3 (i.e., 4).
Encouraged by the improvement in JNK3 potency, we initiated
the structure–activity relationship (SAR) study described herein.50
Table 2
2-Phenoxypyridine SAR
H2NOC
HN
F3C
R1
R3
NC
N
O
Cl
HN
R
R
a
a
a
Compound
R3
JNK1 IC50
(l
M)
JNK3 IC50 (lM)
9c
4-MeOPh
3-MeOPh
2-MeOPh
4-CF3OPh
4-EtOPh
4-n-PrOPh
4-t-BuOPh
4-FPh
4-BrPh
3-ClPh
3,4-ClPh
2-Cl,4-MeOPh
1-Naphthyl
2-Naphthyl
3,4-MethylenedioxyPh
4-Triazole-Ph
0.027
0.038
3.2
0.035
0.035
0.10
0.022
0.10
0.48
NT
0.68
0.74
0.65
NT
0.044
NT
0.037
0.070
3.5
0.035
0.11
0.14
0.027
0.20
0.38
0.20
0.10
0.10
0.99
0.32
0.048
0.080
0.034
0.072
N
Cl
N
Cl
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
6
7
R = CF3, Cl, F, H
R1
b
R1
H2NOC
NC
HN
HN
R
c
R
R2
R2
N
O
N
O
9-12
8
4-Piperazine-Ph
3-Pyridyl-4-Ph
NT
0.053
Scheme 1. Reagents and conditions: (a) R1-susbtituted 2-cyanoaniline, Pd(OAc)2,
Xantphos, Cs2CO3, dioxane, microwave 130 °C, 1 h; (b) R2-substituted phenol,
Pd(OAc)2, Xantphos, dioxane 120 °C, 12 h; (c) KOH, DMSO, H2O2, EtOH.
All standard deviations < 20%.
a
The values are averages of two or more experiments.

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X. Song et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7072–7075
Table 3
Table 4
2-Aminobenzamide ring SAR
In vivo profile of selected JNK3 2-phenoxy analogs
b
[Plasma]c [Brain]
M) M)
Bpd
R2
Compound t1/2
(h)
Clp (mL/
min/kg)
AUC
M h)
F
(%)
H2NOC
(
l
(l
(l
4
9b
9c
10o
12d
1.5
1
1
1
0.5
38
13
3
6
22
0.3
6.3
20.1
9.1
1.0
14
90
79
73
25
NTe
2.4
10
15.7
NT
NT
1.8
5.2
2.5
NT
NT
74
51
16
NT
HN
N
F3C
OMe
O
a
b
c
The values are averages of two or more experiments.
Rat pharmacokinetics, 1 mg/kg iv, 2 mg/kg po.
Compound dosed 10 mg/kg IP in mice, plasma and brain levels at t = 2 h.
Bp = brain penetration.
NT = not tested.
Compound
R2
JNK3 (IC50 lM)
d
e
9c
H
2-Cl
0.037
2.2
11a
11b
11c
11d
11e
11f
11g
11h
11i
2-OMe
3-F
3-Cl
4-F
4-Cl
4-OMe
5-F
2.2
0.045
0.030
0.049
0.078
0.017
0.060
0.16
bond with the amide at C5 positioning the amide for binding in
the ATP site of the enzyme.47 We probed this by N-methylation
(12a), which clearly resulted in complete loss in potency. We also
made the corresponding secondary amide in the 4-aminobenzam-
ide group (12b). This resulted in a 116-fold drop in JNK1 potency,
and only an 11-fold drop in JNK3 activity. Despite the high se-
quence homology between the enzymes, the binding pockets are
lined with different amino acids, which make it possible to synthe-
size selective ligands for each. JNK1 obviously feels the effect of the
secondary amide more so than JNK3, and may provide a path for-
ward in this particular series to further design JNK3-selective li-
gands. In 12c, we have replaced the primary amide with a
triazole. There is only a slight drop (10–15-fold) in in vitro activity,
so this modification is somewhat more tolerated. Further investi-
gation into additional amide bioisosteres is being investigated to
see if this provides any benefit with regards to in vitro or in vivo
activity. Lastly, we reverted to the Abbott lead (2) with our potency
improving modifications to see the effect of pyridine versus pyrim-
idine as the core (12d). Interestingly, the additional ring nitrogen
atom provided a twofold boost in both JNK1 and JNK3 potency,
and represents the most potent 2-phenoxy analog synthesized.
Several compounds from this series were profiled in vivo (Table
4).52 Our initial lead 4, lacking substitution at C5 of the pyridine
ring had a poor rat pk profile with moderate clearance and low oral
absorption. We found that installation of a group at C5 (9b, c, 10o)
resulted in a vast improvement in in vivo exposure, with much
lower clearance rates and excellent oral bioavailability. Interest-
ingly, pyrimidine (12d), the most potent analog, and only a slight
deviation from 9b, had a poorer in vivo profile. Phenoxypyridine
9c exhibited the best overall profile with excellent rat pk, good
brain penetration and exposure, and good in vitro potency.
5-Cl
The values are averages of two or more experiments. All standard deviations < 20%.
H2NOC
N
MeHNOC
HN
Cl
Cl
OMe
OMe
N
O
N
O
12a JNK3 IC50 = >20 µM
12b JNK1 IC50 =0.93 µM
JNK3 IC50 = 0.17 µM
HN
N
H2NOC
N
HN
HN
Cl
OMe
Cl
OMe
N
N
O
N
O
12c JNK1 IC50 = 0.12 µM
JNK3 IC50 = 0.14 µM
12d JNK1 IC50 = 0.004 µM
JNK3 IC50 = 0.007 µM
Figure 3. Analogs of 9b as JNK inhibitors.
(10d–e). Most surprising was the fact that the 4-O-t-Buoxy substi-
tuted analog 10f was equipotent as 9c. Also, 4-halogenated deriv-
atives (10g–j) as well as naphthyl analogs (10l–m) were not as
potent as those bearing hydrogen bond acceptors (10o–q).
With 9c showing a good balance between potency and ease of
synthesis, this core was maintained while we investigated substi-
tution on the 2-aminobenzamide phenyl ring (Table 3). We had
hoped that substitution might not only affect in vitro potency,
but also improve metabolic stability and hence, improve in vivo
pharmacokinetics. Much like SAR of the phenoxy ring, it appeared
that substitution was tolerated at positions 3–5 (11c–i), with only
2-substitution leading to loss of activity (11a–b). It’s not entirely
clear the reason for this, but may derive from the ortho-substitu-
tents effect on the aminobenzamide and pyridine rings
maintaining planarity (A1,2 strain).
In summary, a series 2-phenoxypyridines has been developed.
Optimization of this scaffold by modification of three substituents
(R1, R2 and R3) afforded a series of potent JNK3 inhibitors. Several
compounds from this class have excellent pharmacokinetics and
brain penetration. Evaluation of compounds of this type in vivo
in CNS models of human disease (Parkinson’s and Alzheimer’s
disease, stroke) is ongoing and will be reported in due course.
Acknowledgment
We thank Mr. Tomas Vojkovsky for the synthesis of compounds
3 and 4.
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structure shows that this NH forms an intramolecular hydrogen
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diaminopyrimidine in JNK1 showing the diaminopyrimidine binding to the
hinge group of the enzyme. There are no X-ray crystal structures of the
corresponding 2-alkoxypyrimidines, but it is still possible that they bind in a
similar fashion despite lacking the bidentate interaction.
49. Data to be reported elsewhere. Manuscript in progress.
50. Biochemical Assays Measuring JNK Activity: Biochemical IC50’s for JNK3 and
JNK1 were determined using HTRF. Briefly, final assay concentrations of JNK3,
biotinylated-ATF2 and ATP were 0.015 nM, 200 nM, and 500 nM, respectively.
Final assay concentrations of JNK1, biotinylated-ATF2, and ATP were 0.031 nM,
200 nM, and 500 nM, respectively. In both assays, the phosphor-Thr53-ATF-2
product was detected by a Europiumcryptate-labeled anti-phospho-Thr53-
ATF-2 antibody (PerkinElmer, TRF0212-M). Streptavidin-allophycocyanin-XL
665 (Cisbio, 610SAXLB) was used as the acceptor. A 10-point dose-response
curve for each compound was generated in duplicate and data were fit to a four
parameter logistic.
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containing tubes and plasma was generated using standard centrifugation
techniques. Plasma proteins were precipitated with acetonitrile and drug
concentrations were determined by LC–MS/MS. Data was fit by WinNonLin
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using
a noncompartmental model and basic pharmacokinetic parameters
including peak plasma concentration (Cmax), oral bioavailability, exposure
(AUC), half-life (t1/2), clearance (CL), and volume of distribution (Vd) were
calculated. CNS exposure was evaluated in C57Bl6 mice (n = 3). Compounds
were dosed at 10 mg/kg intraperitoneally and after 2 h blood and brain were
collected. Plasma was generated and the samples were frozen at À80 °C. The
plasma and brain were mixed with acetonitrile (1:5 v:v or 1:5 w:v,
respectively). The brain sample was sonicated with a probe tip sonicator to
break up the tissue, and samples were analyzed for drug levels by LC–MS/MS.
Plasma drug levels were determined against standards made in plasma and
brain levels against standards made in blank brain matrix. All procedures were
approved by the Scripps Florida IACUC.