Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-α production

Article abstract of DOI:10.1016/j.bmcl.2009.01.088

COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-α and other pro-inflammatory cytokines such as IL-1β via the ERK/MAP kinase pathway. As TNF-α and IL-1β are clinically validated targets for therapeutic intervention in

Full text of DOI:10.1016/j.bmcl.2009.01.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1722–1725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase
and TNF-a production
*
Kevin Cusack , Hamish Allen, Agnieszka Bischoff, Anca Clabbers, Richard Dixon, Shannon Fix-Stenzel,
Michael Friedman, Yvette Gaumont, Dawn George, Thomas Gordon, Pintipa Grongsaard, Bernd Janssen,
Yong Jia, Maria Moskey, Christopher Quinn, Andres Salmeron, Christine Thomas, Grier Wallace,
Neil Wishart, Zhengtian Yu
Abbott Laboratories, Medicinal Chemistry, 100 Research Drive, Worcester, MA 01605, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-a and other pro-
inflammatory cytokines such as IL-1b via the ERK/MAP kinase pathway. As TNF-a and IL-1b are clinically
validated targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT provides a
potential avenue for amelioration of disease. Herein we describe identification of a cellular active selec-
tive small molecule inhibitor of COT kinase.
Received 2 December 2008
Revised 23 January 2009
Accepted 27 January 2009
Available online 30 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Thienopyridine
COT
Tpl2
Kinase
TNF
RA
Rheumatoid arthritis
Elevated levels of TNF-
a
and IL-1b have been implicated as
via injection or infusion and identification of an orally available
small molecule therapy would provide an additional benefit to
patients. Identification of an inhibitor of COT kinase would alter
mediators of a number of autoimmune diseases, in particular, the
pain and joint destruction characteristic of rheumatoid arthritis
(RA).1 COT kinase (Tpl2 in mice) is a serine/threonine MAP3 kinase
production of pro-inflammatory cytokines such as TNF-
a and IL-
that regulates production of TNF-
a
and other pro-inflammatory
1b and as such have the potential to be a novel and effective small
molecule therapy for the treatment of RA. To date, 1,7-napthyri-
dine-3-carbonitriles, quinoline-3-carbonitriles, and thienopyri-
dines have been reported as COT kinase inhibitors.7,8 Herein we
describe additional small molecule inhibitors of COT kinase from
within the thieno[2,3-c]pyridine series and the first confirmed cell
active, selective small molecule inhibitor of COT kinase which may
serve as an important tool for further investigation of this target.
COT has a low homology to other kinases which provides a
unique opportunity to achieve selectivity across the kinome. How-
ever low homology coupled with an inability to obtain crystal
structures can also present a challenge in proceeding with a struc-
ture-based or in silico based drug design strategy as surrogate
structures may not be predictive enough to translate to the target
protein. Efforts to obtain a crystal structure of this protein have
been unsuccessful to date. Contributing to this difficulty may be
the stability of free COT. COT is now known to be part of a ternary
complex with p105 and ABIN2 in the resting state9 while free COT
is believed to be the active form and is rapidly degraded in vitro.
cytokines such as IL-1b via the ERK-MAP kinase pathway.2 In par-
ticular, COT/Tpl2 is essential for the activation of the MAPK path-
way in macrophages stimulated by TLR agonists such as
lipopolysaccharide (LPS)3 and has also been suggested to be re-
quired for TNF-R signaling.4 COT KO mice are viable and display
a significantly delayed onset of Crohn’s disease when crossed with
D
ARE mutant mice.5 In addition, downstream cytokines such as
TNF-
a and IL-1b are clinically validated targets for therapeutic
intervention in RA.6 Therefore blocking COT could provide a poten-
tial avenue for amelioration of disease.
Therapies that have successfully targeted TNF-a or IL-1b in man
include biologics directed towards these cytokines such as
adalimumab (Humira), etanercept (Enbrel) and infliximab
(Remicade) which have demonstrated significant efficacy in the
treatment of RA.6 However these agents require administration
* Corresponding author. Tel.: +1 508 688 8006; fax: +1 508 688 8100.
E-mail address: kevin.cusack@abbott.com (K. Cusack).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.088

K. Cusack et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1722–1725
1723
for palladium mediated Buchwald and Suzuki type coupling reac-
tions (Scheme 1) allowed utilization of intermediate 2 for installa-
tion of both N-linked or direct attached biphenyl northern
domains, respectively.
Northern
Domain
I
O
4
Of the ligands evaluated, Xanthphos proved to be superior for
Buchwald coupling to bromoester 2. Use of Cs2CO3 as base allowed
the reaction to proceed without significant hydrolysis of the
methyl ester. Suzuki coupling was facilitated by Pd on solid sup-
port allowing simple removal of Pd via filtration following reaction
in the microwave. The methyl ester was either hydrolyzed to the
corresponding carboxylate or converted to the corresponding
amide with ammonia (Scheme 2).
O
2
N
S
OH
1
Figure 1. High throughput screening (HTS) hit.
The C-2 amides could be further dehydrated then reacted with
sodium azide to form the corresponding C-2 tetrazoles (Scheme 3).
Isolation of the tetrazole is based on a procedure from Wittenber-
ger et al., whereby the reaction is quenched with a large excess of
acetonitrile.11 CH3CN sequesters excess azide as methyl tetrazole
(soluble in the aqueous layer during workup) providing a safer,
more efficient workup procedure.
Compounds were evaluated for inhibition of COT kinase activity
and selectivity versus a limited kinase panel using an HTRF-based
assay.12 SAR indicated acidic C-2 groups were well tolerated in
agreement with O-linked analogs previously described (included
NH
O
O
i
Br
N
S
O
O
3
N
S
2
ii
O
O
N
S
4
NH
NH
Scheme 1. Installation of direct attached and N-linked northern domains. Reagents
and conditions: (i) 2.4 equiv Cs2CO3, p-PhPhNH2, Pd2dba3/Xanthphos (1:2, 6 mol%),
dioxane, 100 °C, 18 h; (ii) 2 equiv Cs2CO3, m-PhPhB(OH)2, 4 mol% Pd FibreCatÒ
(Strem), EtOH/H2O, 110 °C, 15 min, microwave.
O
N
i, ii
N
N
N
N
S
N
H
S
NH2
3c
3b
The program has therefore had to rely on molecular modeling and
classical SAR in order to rationalize interactions with the protein.
Thieno[2,3-c]pyridine, 1 (Fig. 1), was identified as our lead
structure following screening of the Abbott compound collection
i, ii
(IC50 = 1 l
M).8
O
N
N
N
Previous exploration of SAR around 1 revealed a preference for a
C-4 biphenyl ether moiety and a slight preference for an acidic C-2
group.8 Therefore the biphenyl system was held constant while
exploration around the linker ether oxygen and C-2 carboxylate
was initiated. Synthesis was initiated from previously described
4-bromo-thienopyridine 2.10 Identification of optimal conditions
N
N
S
N
H
S
NH2
4c
4b
Scheme 3. Conversion of the amide head group to the corresponding tetrazole.
Reagents and conditions: (i) TFAA, pyridine, 25 °C. 6 h; (ii) 1.3 equiv NaN3, 1.3 equiv
NH4Cl, DMF, 80 °C, 18 h.
NH
NH
NH
ii
i
O
O
O
O
N
N
N
S
S
OH
S
NH2
3a
3b
3
ii
i
O
O
O
N
N
N
S
S
S
OH
O
NH2
4
4b
4a
Scheme 2. Conversion of the ester head group. Reagents and conditions: (i) NaOH (1 N), 70 °C, 12 h; (ii) NH3, MeOH, 110 °C.

1724
K. Cusack et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1722–1725
Table 1
Br
Br
Cl
SAR of C-2 and linker for biaryl analogs 3–7
O
i, ii
O
O
iii
N
N
S
O
S
2
L
8
R
N
S
a
Compound
Linker
R
COT HTRF IC50 (lM)
3a
3b
3c
4a
4b
4c
5
N
N
N
Bond
Bond
Bond
O
O
O
CO2H
C(O)NH2
Tetrazole
CO2H
C(O)NH2
Tetrazole
CO2H
C(O)NH2
Tetrazole
0.14
0.28
0.21
0.05
2.9
0.08
0.31
>50
O
O
iv, v
O
R
N
N
S
S
Cl
NH2
9
10a (R = OH)
10b (R = NH2)
6
7
0.13
Scheme 4. Introduction of a C-7 amino group. Reagents and conditions: (i) mCPBA,
CH2Cl2, (ii) POCl3, (iii) 3-biphenylboronic acid, PdCl2 dppf, Cs2CO3, 10:1 DME:H2O,
(iv) Benzophenone imine, Pd2dba3, Xanthphos, Cs2CO3, dioxane, 100 °C, 5 h, then
2 N HCl, rt, (v) NaOH (1 N) 70 °C, 12 h (R = OH), or 7 M NH3, 70 °C (R = NH2).
a
Details are provided in Supplemental material.
in Table 1 as comparators, entries 5 and 7) while the C-2 amide
group appeared to be less consistently tolerated (Table 1, entries
3b, 4b and 6). Direct attachment of the northern domain biphenyl
provided slightly more potent C-2 acidic analogs (Table 1, entries
4a and 4b) and led to further exploration of this subseries. Thieno-
pyridines in general showed good selectivity over MEK and ERK,
MAP kinases downstream of COT.8
In order to probe additional interactions with the protein, an
amino group ortho to the pyridine nitrogen of the thienopyri-
dine core was targeted. The direct attached series was chosen
in order to avoid a 1,4-heteroatom relationship across the aro-
matic ring.
A model of compound 10a bound to COT kinase is shown in
Figure 2. The biphenyl moiety projects into the solvent exposed
region adjacent to the hinge, making VdW contact with the Gly-rich
loop in accord with previously reported interactions between the
ligand and the ATP-binding.8 A C5-amino analog (inactive, data not
shown) does not fit into the active site well because of a) a mis-
aligned H-bond with either of Gly 210 or Lys 167, and/or b) an
increased biaryl torsion of the pendent phenyl ring due to the ortho
amino group which results in a poor steric fit into the active site.
Starting from intermediate 2, the aza-nitrogen was oxidized to
the corresponding N-oxide with mCPBA (Scheme 4). Reaction with
phosphorous oxychloride resulted in regioselective C-7 ortho chlo-
rination to provide intermediate 8. Suzuki coupling to install the
biphenyl moiety followed by Pd-mediated coupling with benzo-
phenone imine and acidic hydrolysis provided the aminopyridine
ester. The ester was either hydrolyzed to the corresponding acid
10a or converted to the corresponding amide 10b via aminolysis.
Analog 10a makes a productive hydrogen bond between the
amino group and the hinge (Fig. 2) resulting in a 2–3Â gain in
potency versus 4a (Table 2). In addition a corresponding 18-fold
gain in potency is observed for amino analog 10b versus 4b.
The most potent analogs were further profiled in a cellular
assay measuring the inhibition of TNF production in peripheral
human blood monocytic cells (PBMCs) stimulated with LPS (Table
2).12 Analogs bearing a C-2 acidic group, while potent against the
enzyme, did not translate to equipotent cell active analogs. How-
ever, analog 10b (Table 2) with a neutral C-2 amide demonstrated
a good correlation between enzyme and cellular potency.
In order to understand the cellular selectivity profile analog 10b
was further assessed in macrophages. Two different forms of COT
Table 2
SAR of C-7 amino biaryl direct attached analogs
R2
N
S
R1
R1
R2
COT IC50
(lM)
PBMC TNF inhibitiona
(lM)
a
Compound
4a
10a
4b
H
NH2
H
CO2H
CO2H
C(O)NH2
C(O)NH2
0.05
0.02
2.9
3.7
2.3
NDb
0.37
10b
NH2
0.16
Figure 2. Model of compound 10a bound to homology model of COT/Tpl-2 kinase.
Hydrogen bonds between the ligand and Gly 210 N–H (3.2 Å), Glu 208 C@O (3.0 Å),
and Lys 167 NH3+ (3.0 Å) are shown as black dotted lines.
a
Details are provided in Supplemental material.
Low solubility and non-reproducible results.
b

K. Cusack et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1722–1725
1725
Finally, COT 10b was assessed for selectivity and its ability
to inhibit TNF- production in human whole blood (Table 3).
+LPS
min
WT
KO
a
0
5 15 30 60
S
0
5 15 30 60 S
Though selective and potent in isolated cells, 10b was not po-
tent in human whole blood due in part to high plasma protein
binding.
Cot
COT(1-467)
COT(30-467)
pMEK
pERK
In summary, a novel series of 7-amino substituted thieno
[2,3-c]pyridines have been identified as COT enzyme inhibitors.
Analog 10b is demonstrated to selectively inhibit the COT pathway
following LPS stimulation in macrophages and provides an impor-
tant tool molecule for further studies.
pp38
ERK
Acknowledgement
Figure 3. Bone marrow derived macrophages (BMDM) from wild type and COT
deficient mice. Cells are plated and serum starved (0.5% FBS) for 16 h prior to
stimulation. Cells are stimulated with LPS (100 ng/ml) (L) from 5 to 60 min or 20%
FBS (S) for 15 min. Western blots are carried out with indicated antibodies. Long
form of COT (1–467) is phosphorylated and degraded with kinetics similar to
phosphorylation/activation of ERK and MEK in the wild type animal BMDMs while
the alternatively translated form of COT (30–467) is not degraded upon LPS
stimulation. COT KO cells show complete failure to induce MEK/ERK phosphory-
lation upon LPS stimulation. p38 kinase is phosphorylated in both wild-type and
COT deficient cells with little quantitative defect.
The authors thank Kent Stewart for helpful discussions about
the COT model.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.088.
References and notes
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Figure 4. BMDM cells were stimulated with LPS or PMA in the presence of
increasing concentration of compound 10b (lM). PMA is able to stimulate MEK
activation and TNF production in a PKC dependent manner but is not able to induce
phosphorylation/degradation of COT (1–467). LPS induces phosphorylation/degra-
dation of COT (1–467) as well as COT dependent MEK phosphorylation and both
processes are inhibited by compound 10b in a dose dependent manner.
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Table 3
Profile of compound 10b
Compound COT
MEK
IC50
ERK
MK2
IC50
p38
IC50
PBMC
TNF IC50
Hu WB
TNF IC50
a
a
a
a
a
a
a
IC50
M)
IC50
M)
(
l
(
lM)
(l
(lM)
(lM)
(lM)
(lM)
10b
0.17
13
4.3
>50
>50
0.37
34
a
Details are provided in the Supplemental material.
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Belich, M. P.; Walker, P. A.; Howell, S.; Smerdon, S. J.; Gamblin, S. J.; Ley, S. C.
Mol. Cell. Biol. 2003, 23, 4739; (c) Waterfield, M. R.; Zhang, M.; Norman, L. P.;
Sun, S.-C. Mol. Cell 2003, 11, 685.
are present due to alternative translation initiation (M-1 or M-30).
The long form (COT (1–467)) is activated and degraded following
LPS stimulation (Fig. 3). This effect is not observed when 20% FBS
is added as stimulus following starvation. The kinetics of activation
and degradation of COT (1–467) are consistent with the activation
of MEK and ERK in COT WT cells. MEK and ERK activation is totally
absent in COT KO cells upon LPS stimulation with little effect on
p38 activation. Alternatively, stimulation with PMA activates
10. Dunn, A. D.; Norrie, R. J. Prakt. Chem./Chem-Ztg 1992, 334, 483.
11. Wittenberger, S. J.; Narayanan, B. A.; Haight, A. R.; Scarpetti, D., WO9407872
Process for the Preparation of Tetrazoles.
TNF-
a production via a COT independent pathway and as such
12. All assays are described in the supplemental materials.
serves as a cellular readout of selectivity (Fig. 4). Compound 10b
shows COT pathway specificity via lack of inhibition of PMA
induced MEK phosphorylation.13
13. Hall, J. P.; Kurdi, Y.; Hsu, S.; Cuuzzo, J.; Liu, J.; Telliez, J.-B.; Seidl, K. J.; Winkler,
A.; Hu, Y.; Green, N.; Askew, G. R.; Tam, S.; Clark, J. D.; Lin, L.-L. J. Biol. Chem.
2007, 282, 33295.