Design and synthesis of 6-anilinoindazoles as selective inhibitors of c-Jun N-terminal kinase-3

Article abstract of DOI:10.1016/j.bmcl.2005.06.083

The structure-based design and synthesis of a new series of c-Jun N-terminal kinase-3 inhibitors with selectivity against JNK1 and p38α is reported. The novel series of substituted 6-anilinoindazoles were designed based on a combination of hits from high

Full text of DOI:10.1016/j.bmcl.2005.06.083

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5095–5099
Design and synthesis of 6-anilinoindazoles as selective inhibitors
of c-Jun N-terminal kinase-3
Britt-Marie Swahn,a,* Fernando Huerta,a Elisabet Kallin,a Jonas Malmstro¨m,a
a
Tatjana Weigelt, Jenny Viklund, Patrick Womack, Yafeng Xue and Liselotte Ohberg
a
a
b
a
¨
aDepartment of Medicinal Chemistry, AstraZeneca R&D So¨derta¨lje, S-15185 So¨derta¨lje, Sweden
bStructural Chemistry Laboratory, AstraZeneca R&D Mo¨lndal, S-43183 Mo¨lndal, Sweden
Received 12 April 2005; revised 23 June 2005; accepted 27 June 2005
Available online 1 September 2005
Abstract—The structure-based design and synthesis of a new series of c-Jun N-terminal kinase-3 inhibitors with selectivity against
JNK1 and p38a is reported. The novel series of substituted 6-anilinoindazoles were designed based on a combination of hits from high
throughput screening and X-ray crystal structure information of the compounds crystallized into the JNK3 ATP binding active site.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The c-Jun N-terminal kinases (JNKs), also called stress
activated protein kinases, belong to the mitogen-activat-
ed protein kinase (MAPK) family, which regulate signal
transduction in response to environmental stress. Three
distinct genes encoding JNKs have been identified (jnk1,
jnk2, and jnk3), and at least 10 different splicing iso-
forms exist in mammalian cells.1 JNK1 and JNK2 are
widely expressed in a variety of tissues, whereas JNK3
is selectively expressed in the brain and to a lesser extent
in the heart and testis.1,2
is >98% homologous. It has, therefore, been a challenge
to find JNK3 isoform selective ATP competitive inhibi-
tors, and so far there is no successful report of such com-
pounds even though some JNK3 inhibitors have been
reported.8,9
As part of a drug discovery program, a high throughput
screening to find JNK3 inhibitors was conducted. Sever-
al hits were found and some of these were co-crystallized
with JNK3 and the X-rays of the complexes were
collected.
The JNK3 knockout mice develop normally and reveal
no apparent abnormality but have been shown to be
resistant to kainic acid-induced excitotoxicity and asso-
ciated apoptotic cell death.3 Mice lacking the jnk3 gene
have also been shown to be protected from brain injury
after cerebral ischemia–hypoxia, as well as MPTP
(1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine)-induced
dopaminergic cell death.4,5 In contrast, the loss of jnk1/
jnk2 genes lead to neuronal tube defects and embryonic
lethality.6,7 For treating neurodegenerative diseases such
as stroke, AlzheimerÕs, and ParkinsonÕs disease it could,
therefore, be beneficial to find JNK3 isoform selective
compounds. The three JNK isoforms share more than
90% amino acid sequence identity and the ATP pocket
The crystal structures of JNK1 and JNK3 have previ-
ously been reported,9–11 but interestingly we found that
the known p38 inhibitor, SB203580,12,13 1 (Fig. 1)
bound into JNK3 in an induced-fit manner.14
The methylthioethyl chain of Met 146 had moved to
accommodate the p-fluorophenyl group of this ligand
compared to its position when the ATP analogue ADP
2 was bound (n.b. AMP–PMP and adenylyl imidodi-
phosphate were used in the crystallization experiment,
but only the ADP portion was well-defined in the elec-
tron density map). Thus, the p-fluorophenyl group
entered into what is called the selectivity pocket of ki-
nases. Importantly, we could notice a slight difference
in activity for compound 1 in JNK3 compared to
JNK1 with IC50 = 0.1 and 1.6 lM, respectively.15
Keywords: Structure based design; 6-Anilinoindazoles; c-Jun N-termi-
nal kinase-3; JNK3 inhibitors.
*
We concluded that this structural and activity informa-
tion could be used to increase the selectivity for
Corresponding author. Tel.: +46 8 553 26474; fax: +46 8 553 28
877; e-mail: britt-marie.swahn@astrazeneca.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.083

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B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5095–5099
should be able to bind in the ATP pocket of JNK3 in
an induced-fit manner.
Glu147
The synthesis of compound 4 was conducted as shown
in Scheme 1.19 The starting material was commercially
available 6-nitroindazole 5, which was first iodinated
and then N-protected with a Boc group under standard
conditions.20 Compound 6 was then subjected to a Suzu-
ki coupling with a phenyl boronic acid to yield 7.20a,21
Thereafter, the nitro group was catalytically reduced
and the amine 8 was reacted under Buchwald condi-
tions22 with an arylbromide to yield 9. After deprotec-
tion of the Boc group, the desired compound 4 was
assessed for inhibition of JNK3 and JNK1 as well as
co-crystallized with JNK3 to produce the X-ray struc-
ture of the complex.
Lys93
Met146
Asp150
Asn152
Figure 1. Overlay of X-ray crystal structures of p38 inhibitor 1 (blue)
and ADP 2 in the JNK3 ATP binding site.16,17
In the X-ray of compound 4a in the ATP pocket of JNK3
(Fig. 3), it was apparent that our compound bound in an
induced-fit manner. The aniline part of the molecule fits
inhibition of JNK3 compared to the inhibition of JNK1.
From the high throughput screening campaign, we also
found that 3-(4-pyridyl)indazole 3 (Fig. 2) was an inhib-
itor of JNK3 as well as JNK1.18 Thus, we anticipated
that the 3-arylindazole nucleus would be an interesting
scaffold for introducing a substituent that could bind
into the selectivity pocket. We predicted by structural
comparison that the 6-anilino group in compound 4
Glu147
Lys93
Met146
F
NH
H
N
H
Asp150
N
N
NH
N
N
N
Asn152
O
S
N
1
4
3
Figure 3. X-ray crystal structure of 4a (green) in the JNK3 active site
overlaid with ADP.
Figure 2. Design of the 6-anilino series of compounds.
O
O
O
O
H
NO2
N
NO2
N
NO2
N
c
a
N
N
N
b
I
5
6
7
R1
R2
R2
O
O
O
O
NH2
H
NH
N
N
NH
N
d
e
f
N
N
N
R1
R1
4
8
9
R1
Scheme 1. Reagents: (a) I2, KOH, DMF, 78%; (b) (Boc)2O, DMAP, Et3N, CH3CN/CH3OH, 82%; (c) Ph-B(OH)2, PdCl2(dppf), 89%; (d) H2, PtO2,
100%; (e) Ph-Br, Pd(OAc)2, S-BINAP, 32%; and (f) HCl, Et2O/MeOH, 55%. Yields are indicated for compound 4a in which R1 = R2 = H.

B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5095–5099
Table 1. IC50 values for compounds 4a–j against JNK3, JNK1, and p38a24
5097
Compound
R1
R2
JNK3 IC50 (nM)
JNK1 IC50 (nM)
p38a IC50 (nM)
4a
4b
4c
4d
4e
4f
H
H
48
30
>10,000
5350
1600
246
45
30
18
46
13
H
2-Cl
2-OMe
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
H
4-COOH
202
32
4-CONH(CH2)2N(CH3)2
3-CONH2
3-COOH
1.9
3.3
5.3
3.4
21
8.7
3.2
24
3.7
16
4.4
81
61
4g
4h
4i
3-CONH(CH2)3-4-morfolinyl
3-CONH(CH2)2N(CH3)3
3-CONH-4-piperidinyl
228
698
71
4j
1.4
into the selectivity pocket of JNK3 because the side chain
of the gatekeeper amino acid Met146 has moved to
accommodate the ligand. The indazole nitrogens of com-
pound 4a displayed hydrogen bonds to backbone amino
acids Met149 and Glu147, and the anilino NH hydrogen
bond to Lys93 via a water molecule.
A
Glu147
Met146
Lys93
Compound 4a also possessed an excellent selectivity
against JNK1 with an IC50 value of 0.048 lM for JNK3
and >10 lM for JNK1. These results encouraged us to
synthesize more analogues according to Scheme 1 with
different R1 and R2 substituents. The corresponding
IC50 values for JNK3 and JNK1 are shown in Table 1.
Asp150
Asn152
Gln155
The overall kinase selectivity for compounds 4 was good
as evaluated against a range of kinases and exhibited no
significant activity at 10 lM.25 Even though we could
achieve a large selectivity against JNK1 and many other
kinases, a disturbing inhibition of the MAP kinase p38a
still remained for compounds 4. By comparing the crys-
tal structures of compounds 4g and 4h in JNK3, we no-
ticed that the carboxylic acid and the amide derivatives
bound in different directions (Fig. 4). The 3-carboxylic
acid has a hydrogen bond to Asn152, whereas the 3-am-
ide substituents with a sufficiently long chain bearing a
basic amine reached out to interact with Asp150. We
anticipated that it could be possible to achieve selectivity
against p38 by combining these substituents in the same
molecule. In p38, the corresponding amino acid to
Asn152 is Asp112, that is an acidic amino acid, which
would probably reduce the affinity for a negatively
charged substituent on the ligand. To test this hypothe-
sis, we decided to synthesize compound 10 which was
executed according to Scheme 2.
B
Glu147
Lys93
Met146
Asp150
Asn152
Figure 4. (A) Overlay of X-ray crystal structures of 4g (magenta) and
ADP 2 in the JNK3 ATP binding site. (B) Overlay of X-ray crystal
structures of 4h (purple) and ADP 2 in the JNK3 ATP binding site.
The SEM protected derivative 1126 was found to be a
better starting material than the Boc-protected com-
pound 6 because the Boc group was unstable during
the subsequent reactions. Compound 11 was treated
with 3-amino-5-(methoxycarbonyl)-phenyl boronic acid
under Suzuki reaction conditions to yield 12.20a,21 The
amino group was acylated with 4-dimethylamino butyric
acid in the presence of a coupling reagent to afford 13.
Thereafter, the nitro group was reduced and reacted
with 1-bromo-2-chlorobenzene under Buchwald reac-
tion conditions to yield 14.22 Deprotection of the SEM
group with TBAF and then hydrolysis of the methyl
ester with LiOH gave the desired compound 10.
Compound 10 was then examined for inhibitory effect on
JNK3, JNK1, and p38. The compound exhibited an
excellent selectivity for JNK3 compared to JNK1 and
p38 [JNK3 IC50 = 3 nM, JNK1 IC50 = 101 nM (30·),
and p38a IC50 = 903 nM (300·)]. Compound 10 was
co-crystallized with JNK3 and the X-ray of the complex
confirmed the predicted binding mode (Fig. 5A). The
carboxylic acid group displayed a hydrogen bond to
Asn152 and the basic amine interacted with Asp150.
In conclusion, the structure-based design of a new ser-
ies of JNK3 selective 6-anilinoindazoles was described.

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B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5095–5099
SEM
SEM
N
NO
2
SEM
NO
N
2
NO
N
b
2
N
N
a
N
N
O
I
N
H
N
2
H
O
O
11
12
O
13
O
Cl
SEM
Cl
NH
e
f
c
N
H
N
NH
N
d
N
N
O
N
O
N
H
N
O
H
O
O
14
10
HO
Scheme 2. Reagents: (a) 3-amino-5-(methoxycarbonyl)phenyl boronic acid, PdCl2(dppf), 54%; (b) 4-dimethylamino butyric acid, HATU, 89% (c) H2,
PtO2, 99%; (d) 1-bromo-2-chlorobenzene, Pd(OAc)2, S-BINAP, 41%; (e) TBAF, 67%; and (f) LiOH in THF, 72%.
A
acid sequence in the selectivity pocket of JNK3 differs
somewhat from JNK1 and is probably the explanation
for the difference in inhibitory activity of these ligands
in JNK3 and JNK1.
Glu147
Lys93
Met146
Acknowledgments
Asp150
The authors thank Dr. Johanna Lindquist, Department
of Molecular Pharmacology AstraZeneca R&D So¨der-
ta¨lje, for determining the IC50 values for JNK1, JNK3,
and p38a, and Dr. Stefan Geschwindner, Structural
Chemistry Laboratory, AstraZeneca R&D Mo¨lndal,
for his support in protein production and initial crystal-
lization studies.
Asn152
B
Met146
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