Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.08.037

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Full text of DOI:10.1016/j.bmcl.2008.08.037

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4952–4955
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of thieno[2,3-c]pyridines as potent COT inhibitors
*
Dawn George , Michael Friedman, Hamish Allen, Maria Argiriadi, Claude Barberis, Agnieszka Bischoff,
Anca Clabbers, Kevin Cusack, Richard Dixon, Shannon Fix-Stenzel, Thomas Gordon, Bernd Janssen, Yong Jia,
Maria Moskey, Christopher Quinn, Jose-Andres Salmeron, Neil Wishart, Kevin Woller, Zhengtian Yu
Abbott Bioresearch Center, 381 Plantation Street, 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:
Received 2 July 2008
Revised 11 August 2008
Accepted 11 August 2008
Available online 14 August 2008
Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubsti-
tuted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2-
and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Thienopyridine
COT
Tpl2
MAP3K8
COT/Tpl2/MAP3K8 is a mitogen-activated serine/threonine ki-
nase kinase kinase (MAP3K) that is essential for lipopolysaccharide
(LPS)-induced activation of the ERK MAPK cascade in macro-
phages.1 COT kinase activation is believed to play an integral role
in the production of pro-inflammatory cytokines such as TNF and
IL-1b.2 Elevated levels of these cytokines have been clinically
implicated as mediators of a number of autoimmune diseases,3 in
particular, the pain and joint destruction characteristic of rheuma-
toid arthritis. By inference, pharmaceutical agents that inhibit COT
kinase have the potential to be a novel and effective therapy for the
treatment of a range of immunological diseases, such as rheuma-
toid arthritis. To date, only 1,7-naphthyridine-3-carbonitriles4
and quinoline-3-carbonitriles5 have been reported as COT kinase
inhibitors. Herein, we report a new class of potent COT inhibitors
based on a thieno[2,3-c]pyridine core.
Our initial hit came from a high throughput screen of the Abbott
compound collection using a 33P gel-permeation assay in the pres-
ence of 30
lected based upon its moderate potency for the COT HTRF kinase
assay (IC50 = 1
M),6 good selectivity across a panel of 15 kinases
lM ATP. The resulting thieno[2,3-c]pyridine, 1, was se-
l
(representing all branches of the human kinome), and its concur-
rence with internal hit criteria. Interestingly, 1 had been identified
internally as a member of a series that inhibited TNF stimulated
ICAM-1 and E-selectin expression; however, the specific molecular
target was unknown.7
While COT has a low homology to other kinases, providing a un-
ique opportunity to achieve selectivity across the kinome, it also
presented a challenge in terms of applying structure-based drug
design techniques. To date, no crystal structure of this protein is
known. The optimization of 1 focused primarily upon SAR explora-
tion of the 2- and 4-substituents of the thieno[2,3-c]pyridine core
based on modeling hypotheses. In this letter, we describe the opti-
mization of these thienopyridines that resulted in the identifica-
tion of cell-active inhibitors of COT.
Preparation of the versatile intermediates 3–6 was carried out
from commercially available 3,5-dibromo-4-pyridine carboxalde-
hyde (Scheme 1). Cyclization of aldehyde 2 with methyl thioglyco-
late or 2-mercaptoacetamide provided either the ester 37 or
primary amide 4, respectively. The tetrazole moiety 6 was intro-
duced by dehydration of the amide to the nitrile 5 and by subse-
quent reaction with sodium azide.
Functionalization of the thienopyridines at the 4-position
provided access to a wide variety of substituted aryl ether
analogs. This transformation was accomplished via an Ulmann
* Corresponding author. Tel.: +1 508 688 8001; fax: +1 508 688 8100.
E-mail address: dawn.george@abbott.com (D. George).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.037

D. George et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4952–4955
4953
Scheme 1. Reagents and conditions: (a) HSCH2CO2CH3, Cs2CO3, THF, 60 °C, 2 h; (b)
HSCH2CONH2, Cs2CO3, THF, 60 °C, 2 h; (c) TFAA, pyridine, rt, 6 h; (d) 1.3 equiv NaN3,
1.3 equiv NH4Cl, DMF, 80 °C, 4–18 h.
Scheme 4. Reagents and conditions: (a) NaOH, water, dioxane, 100 °C, 1 h; (b)
(PhO)2OPN3, Et3N, tBuOH, reflux, 16 h; (c) Ac2O, EtPr2N, THF, rt, 16 h; (d) NH3,
MeOH, sealed tube, 100 °C, 1 h; (e) TFAA, pyridine, rt, 6 h; (f) 1.3 equiv NaN3, 1.3
equiv NH4Cl, DMF, 80 °C, 4–18 h.
coupling8 of bromide 6 with substituted phenols, as shown in
Scheme 2.
An alternate route was utilized to access halogen-substituted
aryl ether moieties off C4 of the thienopyridine core. Treatment
of 3,5-dichloro-4-pyridine carboxaldehyde with a substituted phe-
nol and subsequent reaction with methyl thioglycolate provided
direct access to phenoxy compound 9 (Scheme 3). Suzuki coupling
of the aryl halide with boronic acids generated biphenyl-substi-
tuted analogs 10.
Final product elaboration to the acid 1 was accomplished by es-
ter hydrolysis. Curtius rearrangement of the acid provided the C2
amine 11 which could be further elaborated by acylation as de-
picted in Scheme 4. The corresponding nitrile 14 was prepared
by treatment of ester 9 with ammonia to generate amide 13 fol-
lowed by dehydration. Reaction with sodium azide generated tet-
razole 15.
Alternatively, oxazolidinone 17 was synthesized via reaction of
hydroxylamine hydrochloride with nitrile 14 followed by cycliza-
tion in the presence of CDI (Scheme 5). Reaction of acid chloride
Scheme 5. Reagents and conditions: (a) NH2OH-HCl, i-Pr2EtN, DMSO, 70 °C, 1.5 h;
(b) CDI, DMF, 100 °C, 1.5 h.
18 with trimethylsilyl hydroxylamine and subsequent liberation
of the silyl group during reverse-phase (RP) HPLC purification gen-
erated the hydroxamic acid 20 (Scheme 6).
Our initial binding mode hypothesis for 1 (Fig. 1) had the 6-N of
the thieno[2,3-c]pyridine core bound to the hinge of COT kinase
(Gly 210), thus presenting the carboxylic acid toward the water-
accessible ribose pocket and the 4-substituent in a hydrophobic
domain abutting the gatekeeper residue (Met 207). In order to test
Scheme 2. Reagents and conditions: (a) RPhOH, Cu(I)Cl, Cs2CO3, 2,2,6,6-tetra-
methyl-3,5-heptanedione, NMP, 220 °C, microwave, 10 min.
Scheme 3. Reagents and conditions: (a) 4-I-PhOH, KOtBu, THF, rt, 16 h; (b) HSCH2CO2CH3, Cs2CO3, THF, 60 °C, 2 h; (c) RPhB(OH)2, Pd(PPh3)4, Cs2CO3, DME/EtOH/H2O, 135 °C,
microwave, 6 min.

4954
D. George et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4952–4955
Table 1
Inhibition of COT kinase by representative C-2 analogs
Compound
R
COT HTRF IC50, l
Ma
Scheme 6. Reagents and conditions: (a) (COCl)2, CH2Cl2, cat DMF, rt, 1 h; (b)
Me3SiONH2, CH2Cl2, 60 h; (c) RP-HPLC.
1
11
12
–CO2H
–NH2
–NHCOCH3
0.94
>50
>50
17
15
1.12
0.41
0.54
20
–CONHOH
a
See Ref. 6 for assay conditions.
Table 2
Inhibition of COT kinase by selected C-4 analogs
Compound
R
COT HTRF IC50, l
Ma
21
22
23
24
25
26
27
28
H
2.2
15
1.7
0.15
7.5
1.6
Figure 1. Model of 1 bound to homology model of COT/Tpl-2 kinase. A hydrogen
bond between the ligand and the backbone NH of Gly 210 (3.1 Å) is shown as a
black dotted line.
2-Ph
3-Ph
4-Ph
4-Me
4-Br
4-Cl
4-tBu
0.71
0.61
this proposal, we initially evaluated the properties of the presum-
ably solvent exposed 2-substituent and its impact on enzyme po-
tency (Table 1). Compounds were evaluated for inhibition of COT
kinase activity using an HTRF-based assay.6 The neutral 2-amino
analogs 11 and 12 were inactive against COT; however, replace-
ment of the acid group with small acidic heterocycles9 was toler-
ated. For example, the oxazolidinone (estimated pKa ꢀ6.1) 17
and tetrazole (estimated pKa ꢀ5.3) 15 maintained potency similar
to that of the carboxylic acid 1. The hydroxamic acid moiety in 20
(estimated pKa ꢀ6.9) also served as an equitable replacement of
the carboxylic acid.
Optimization of the 4-position of the thieno[2,3-c]pyridine fo-
cused on a range of hydrophobic residues. To the best of our
knowledge, no kinase inhibitors have successfully circumvented
a bulky methionine gatekeeper to access the rear ‘hydrophobic’
selectivity pocket, hence we focused primarily on optimizing the
van der Waal interactions between the 4-substituent and the
Gly-rich loop. For comparative purposes, the tetrazole moiety
was held constant at the 2-position, and initially a range of substi-
tuted phenoxy analogs were prepared (Table 2). The unsubstituted
phenoxy derivative 21 was less potent than the lead compound 1.
Preparation of the 2-, 3-, and 4-phenyl-substituted analogs (22–24)
established the preference for substitution at the 4-position. Small
lipophilic acyclic substituents were also examined in this region.
Compared to the 4-Ph derivative 24, the 4-Me and 4-Br analogs
25 and 26 had significantly reduced potency. Interestingly, both
a
See Ref. 6 for assay conditions.
the 4-Cl and 4-tBu analogs (27, 28) were moderately active but still
less potent than 24.
Since the effort to replace the distal phenyl group failed to
maintain or improve potency, the biphenyl moiety was retained
within the core pharmacophore and further optimization was fo-
cused on the substitution pattern of the distal ring (Table 3). In
general, substitution at the 4-position (31, 34–41) afforded analogs
with a trend toward greater potency compared to those substi-
tuted at either the 2- or 3-positions (comparative groupings are
29–31 as well as 32–34). Both electron-donating and electron-
withdrawing groups were tolerated. Of the substituents evaluated
at the 4-position of the distal phenyl ring, the analogs that pro-
vided a marked improvement in potency were those capable of
forming H-bonds with the protein, such as 39–41. Since the pri-
mary amide showed improved potency over the N,N-dimethyl
derivative, we speculate that both an H-bond donor and an H-bond
acceptor are required for optimal potency.
A proposed model for an alternative binding mode of compound
1 bound to COT kinase is shown in Figure 2. The inhibitor was
docked manually to optimize hydrogen bonds between the ligand
and both the hinge and salt-bridge Lys residue within the ATP-

D. George et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4952–4955
4955
Table 3
the thienopyridines showed good selectivity over these kinases,
suggesting that cell potency is due to the inhibition of COT (Table
3).
Inhibition of COT kinase by selected C-4 analogs
Selected compounds were profiled in a cellular activity assay
measuring the inhibition of pERK production in murine peritoneal
exudates cells (PECs) stimulated with lipopolysaccharide (LPS), Ta-
ble 3.10 Parent compound 24 demonstrated low micromolar po-
tency in agreement with enzyme data. This was also the case for
the 4-Cl and 4-F congeners 34 and 36. However, the 4-CF3-substi-
tuted analog 38 showed approximately a 10-fold increase in cellu-
lar potency, possibly indicating better cell penetration or reduced
plasma protein binding. Further improvement with the 4-carbox-
amide (41) was observed, again in line with enzyme data.
In summary, we have described a novel series of substituted
thieno[2,3-c]pyridines as potent COT enzyme inhibitors. Further
profiling and optimization of this chemotype will be the discussion
of a subsequent publication.
Compound
R
COT
MEK
IC50
ERK
COT cell
IC50
IC50
,
l
Ma
,
l
Mb
IC50
,
l
Mb
, l
Mb
24
29
30
31
32
33
34
35
36
37
38
39
40
41
H
0.15
0.32
0.24
0.13
0.15
0.12
0.09
0.31
0.17
0.23
0.28
0.05
0.04
0.01
9.7
21.8
23
12
4.3
2-OMe
3-OMe
4-OMe
2-Cl
3-Cl
4-Cl
49
1.5
1.9
2.4
21.2
7.6
1.5
1.8
48
>50
>50
>50
47
11.3
1.1
2.1
6.5
4-CN
4-F
Acknowledgment
4-NH2
4-CF3
4-CONMe2
4-CH2OH
4-CONH2
1.7
>50
0.56
0.13
The authors thank Kent Stewart for helpful discussions about
the COT model.
14
18
a
See Ref. 6 for assay conditions.
See Ref. 10 for assay conditions.
Supplementary data
b
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.037.
References and notes
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Figure 2. Model of 1 bound to homology model of COT/Tpl-2 kinase.
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binding site. The aryl-oxy unit at the 4-position of the thienopyr-
idyl core ring projects along the protein surface and has some
exposure to solvent, in accord with the SAR in which a wide variety
of substituents gave rise to active analogs. We speculate that the
high activity of compounds 39–41 was due to polar interactions
in this extended hinge region, but the exact binding mode and
interactions will need confirmation by crystallographic analysis
of an inhibitor bound to the enzyme.
Several compounds were evaluated for selectivity over MEK and
10. Details of the MEK1 cascade HTRF assay, the ERK HTRF assays, and the COT
cellular assays are provided in the supporting information.
ERK, two kinases downstream of COT in its pathway.10 In general,