Discovery of indazoles as inhibitors of Tpl2 kinase

Article abstract of DOI:10.1016/j.bmcl.2011.06.065

Synthesis, modeling and structure-activity relationship of indazoles as inhibitors of Tpl2 kinase are described. From a high throughput screening effort, we identified an indazole hit compound 5 that has a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the indazole, we discovered compound 31 with good potency in LANCE assay and cell-based p-Erk assay.

Full text of DOI:10.1016/j.bmcl.2011.06.065

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4758–4761
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of indazoles as inhibitors of Tpl2 kinase
Yonghan Hu a, , Derek Cole b, Rajiah Aldrin Denny a, David R. Anderson a, Manus Ipek a, Yike Ni a,
⇑
Xiaolun Wang a, Suvit Thaisrivongs a, Timothy Chamberlain c, J. Perry Hall c, Julie Liu c,
Michael Luong c, Lih-Ling Lin c, Jean-Baptiste Telliez c, Ariamala Gopalsamy a
a Worldwide Medicinal Chemistry, Pfizer Global Research and Development, 200 CambridgePark Drive, Cambridge, MA 02140, USA
b Department of Chemistry, Takeda San Diego, 10410 Science Center Drive, San Diego, CA 92121, USA
c Inflammation & Immunology, Pfizer Global Research and Development, 200 CambridgePark Drive, Cambridge, MA 02140, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, modeling and structure–activity relationship of indazoles as inhibitors of Tpl2 kinase are
described. From a high throughput screening effort, we identified an indazole hit compound 5 that has
a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the inda-
zole, we discovered compound 31 with good potency in LANCE assay and cell-based p-Erk assay.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 16 May 2011
Revised 14 June 2011
Accepted 15 June 2011
Available online 22 June 2011
Keywords:
Tpl2 kinase
Indazole
Rheumatoid arthritis
Rheumatoid arthritis (RA) is an autoimmune disorder that
causes chronic inflammation of the joints. It affects 20-million peo-
ple worldwide, particularly women.1 Inflammatory cytokines such
compound 5 (Fig. 2) with Tpl2 IC50 of 3.2 lM. In this manuscript,
we report the synthesis and structure–activity relationship of a
new series of indazole-based Tpl2 inhibitors.
as tumor necrosis factor
leukin-6 (IL-6) are believed to be major contributors to this debil-
itating systemic disease. The clinical success of anti-TNF agents,
notably ENBRELÒ (sTNFR ), RemicadeÒ and HumiraÒ, has validated
TNF as an important therapeutic target for RA.
Major pharmaceutical companies have focused on the inhibi-
a
(TNF
a
), interleukin-1 (IL-1) and inter-
To our knowledge no crystal structure is available for Tpl2. This
is partially due to the low yield for the expression of Tpl2 and the
difficulty in isolating stable and active protein. Tpl2 has low se-
quence similarity to other kinases and therefore this presents
a
a
a
tion of TNF
a
production through either inhibiting TNF
a
converting
Cl
R
N
HN
N
enzyme2 or the disruption of signal transduction pathways that in-
clude several members of the mitogen activated protein kinase
(MAP kinase) family, particularly p38 kinase.3
H
N
F
N
CN
F
N
HN
N
N
H
N
N
CN
Tpl2 kinase is a serine/threonine kinase in the MAP3K family. It
activates the translation of the TNF
production through the phosphorylation of MEK/ERK. Tpl2 is
N
Cl
a messenger RNA and TNFa
R =
Cl
important for both TNF
a production and signaling. Therefore,
Tpl2 has become a highly desirable target for treating RA.4
We5 and others6 have recently disclosed a few series of Tpl2
inhibitors of different scaffolds (Fig. 1), including 1,7-naphthyri-
dine-3-carbonitrile (1), 8-substituted-4-anilino-6-amiquionline-3-
carbonitrile (2) and thienopyridines (3 and 4).
2
1
O
H2N
O
With our continuous interest in the Tpl2 inhibition for the treat-
ment of rheumatoid arthritis, we conducted a high throughput
screening of our internal compound collection and identified
O
N
N
N
S
NH2
NH
NH2
N
N
S
4
3
⇑
Corresponding author. Tel.: +1 617 665 5621; fax: +1 617 665 5682.
E-mail address: Fred.Hu@Pfizer.com (Y. Hu).
Figure 1. Published Tpl2 inhibitors.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.065

Y. Hu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4758–4761
4759
N
O
O
O
O
1.5 eq.
B
B
N
I
H
N
Ar
1.
2.
PdCl2 (dppf) 0.05 eq.,
5
KOAc 3 eq., DMA, 80 oC, 2h
N
3
O
N
H
O
N
N
N
Ar
X
N
NH
NH
1
THP
Pd(PPh3)4 0.05 eq., K3PO4 1.2 eq.
DMA-H2O, 90 oC, 5h
5
6
7
50% two steps
Figure 2. Indazole inhibitor of Tpl2 kinase.
R1
NH2
NH2
R2
significant challenges to perform structure-based drug design.
Though there is no mutagenesis data available, there are SAR data
points available internally and in the public domain.5,6 We decided
to use these available SAR data to build and validate homology
model to drive our structure-based design efforts.
Tpl2 kinase domain was blasted against PDB to find MST,
Aurora, and PAK family of Serine-Threonine kinases as close ano-
logs (with >30% sequence identity and >45% homology). Near the
ATP binding site we determined that MST4 and MST3 have 15/27
residues identical. Homology model was built using MST4 (PDB:
3GGF/2.35A) and MST3 (PDB: 3A7H/1.96A) as templates using
Modeller.7 The best models were refined further and the side
chains optimized to make sure the main chain conformations fall
under the acceptable regions in the Ramachandran map and side
chain conformations correspond to those in the rotamer libary.8
The refined models were tested to make sure active kinase form
signatures are preserved, such as salt bridge interaction between
R3
8
R4
1 eq.
R1
R2
Ar
NaHSO3 2 eq,
DMA, 120 oC, 21h
80-90%
N
R3
N
R4
H
N
NH
9
Scheme 1. Synthesis of indazole-based Tpl2 inhibitors.
Docking of compound 5 in the homology model (Fig. 3) indi-
cated that indazole ring picks up donor-acceptor interactions with
Gly107 and Glu105 hinge. 30-Pyridyl nitrogen at C-5 of the indazole
has the right orientation to have a hydrogen-bond interaction with
Lys64. The methyl in the pyridyl 60-position points towards the
hydrophobic pocket created by residues Ile41, Pro42, and Glu114.
The aminoethyl basic nitrogen interacts with Arg43 backbone in
the G-loop. The benzimidazole ring points to the solvent front with
the flat rings having van der Waals interaction to the hydrophobic
pocket. In the SAR modifications below, we keep the key interac-
tions from the indazole nitrogen donor-acceptor system, C5-pyri-
dine and basic secondary amines.
Glu in
a-helix and Lys in b3, DFG orientation and distance from
the HBA in hinge.9 Glide docking models10 were generated using
the refined homology structures. The validity of the models was
further tested against the SAR data of Tpl2 inhibitors of cyanoquin-
oline scaffold and the inability of the Staurosporine binding to
Tpl2.5
The synthesis of Indazole-based Tpl2 inhibitors is shown in
Scheme 1. The synthesis of intermediate 6 has been previously re-
ported.11 Palladium catalyzed boronic ester formation followed by
Suzuki coupling with aryl halide gave compound 7. Condensation
of aldehyde 7 with dianiline 8 gave final compound 9.
Table 1
SAR on substitution at the benzimidazole ring
N
N
R1
H
R2
N
R3
N
H
N
NH
Compound
R1
5
10
11
12
13
H
H
H
H
H
R2
R3
Me
Me
Me
H
Br
N
H
H
H
H
Tpl2
lM
3.200
0.240
0.418
0.109
1.126
14
15
16
17
18
R1
H
H
H
H
H
R2
R3
Tpl2
Cl
Me
1.734
tBu
H
1.995
CH2OiPr
H
1.460
CN
H
2.669
CN
F
4.943
l
M
M
19
20
21
22
23
R1
R2
R3
Tpl2
F
F
H
H
F
F
Cl
Cl
H
Cl
H
(CH3)2N
H
Cf3H
Figure 3. Modeling of compound 5 with homology model of Tpl2 kinase. Hydrogen
bonds are represented with yellow and red line in 2D and 3D depictions.
l
20.690
21.395
10.280
19.300
2.390

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Y. Hu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4758–4761
Table 2
SAR on imidazole ring substitution
N
N
H
R1
N
R2
N
H
N
NH
Compound
24
25
26
27
28
29
30
F
N
R1
R2
H
Ph
Imadazole repleced by tetrazole
>100
H
25
Me
18.7
Me
3.0
H
4.5
Tpl2
lM
38
28.3
Table 3
Indazole C-5 modifications
Waals interaction at the R1 position of imidazole, compounds 29
and 30 both regain activity to low single digit M range (cf 24).
l
R
We next explored SAR on the C5 position of the indazole (Table
3). Compared to compound 12, a hydroxyethyl group (31) im-
proves activity by two-fold. An ortho-Cl reduced Tpl2 activity (33
vs 12). Compounds with an H-bond without a methylene spacer
(34), an amide functionality (35), or compounds without an NH
N
N
H
N
NH
Compound 12
310
32
functionality (36–38) all render IC50 >100 lM. These results are
N
N
N
in line with modeling results from the homology model previously
discussed. A bulkier group (32) resulted in significant loss of
potency.
N
H
N
H
N
H
OH
R
Compounds 11, 12 and 31 were evaluated for inhibition of
phospho-Erk in human monocytes. These compounds show good
Tpl2
lM
0.109
0.047
16
33
34
35
cell-based activities with an IC50 of 0.512, 0.272, 0.079
respectively.
lM,
H
O
N
N
N
N
H
N
NH2
R
Cl
Compound 12 was evaluated for kinase selectivity and it dem-
onstrated good selectivity (<5% inhibition at 1 M) against IKKb,
JNK1 , p38, EGFR, CK1 , while showing less selectivity against
l
a
a
Tpl2
l
M
M
9.26
>100
>100
other kinases, including CDK2, JAK3, MST4, GSK3b.
36
37
38
In summary, we have discovered a series of indazole-based Tpl2
inhibitors with good activities in LANCE and cell-based p-Erk as-
says. We are currently investigating an avenue to improve kinase
selectivity of this indazole scaffold.
N
N
N
OH
N
R
O
Tpl2
l
>100
>100
>100
Acknowledgment
Compounds synthesized as shown in Scheme 1 were evaluated
in LANCE™ assay12 against Tpl2 kinase and the data are shown in
Tables 1–3.
The authors thank Cristina Grosanu for her help in compound
solubilization.
Compared to compound 5 (Table 1), di- (10) and mono-methyl
(11) substitutions have improved activities by 10-fold. Compound
without substitutions on the benzene ring of the benzimidazole
functionality (12), has a further improvement of Tpl2 activity
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