Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent inhibition of TNF-α production in human whole blood

Article abstract of DOI:10.1016/j.bmcl.2009.05.009

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-α (TNF-α) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-a

Full text of DOI:10.1016/j.bmcl.2009.05.009

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3485–3488
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent
inhibition of TNF-a production in human whole blood
a
a
a
a
a
Junjun Wu a, , Neal Green , Rajeev Hotchandani , Yonghan Hu , Jeffrey Condon , Adrian Huang ,
Neelu Kaila a, Huan-Qiu Li a, Satenig Guler a, Wei Li a, Steve Y. Tam a, Qin Wang b,c, Jeffrey Pelker d,
Suzana Marusic d, Sang Hsu d, J. Perry Hall d, Jean-Baptiste Telliez d, Junqing Cui d, Lih-Ling Lin d
*
a Chemical and Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
b Drug Safety and Metabolism, Wyeth Research, Building G, One Burtt Road, Andover, MA 01810, USA
c Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
d Inflammation Signaling, Wyeth Research, 200 Cambridge Park 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:
Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor
Received 27 March 2009
Revised 4 May 2009
Accepted 4 May 2009
Available online 7 May 2009
Necrosis Factor-
oro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent
inhibitors of this enzyme. In order to improve the inhibition of TNF- production in LPS-stimulated
human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied.
We found that a cyclic amine group appended to the triazole ring could considerably enhance potency,
aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to
the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-
triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model,
a (TNF-a) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-flu-
a
Keywords:
Tpl2 kinase
TNF-a
Triazole
Quinoline-3-carbonitrile
compound 34 showed good efficacy in inhibiting TNF-
a
production.
Ó 2009 Elsevier Ltd. All rights reserved.
Studies on the complex biology of Tumor Necrosis Factor-
a
thus could potentially have the double benefits of blocking both
(TNF- ) have revealed that this cytokine is involved in the inflam-
a
TNF-a
production and signaling.
mation process in several disease states such as rheumatoid arthri-
tis (RA), psoriasis, inflammatory bowel disease, and multiple
Cl
F
Cl
sclerosis.1 TNF-
a
also stimulates the production of other pro-
F
inflammatory cytokines such as IL-1 and IL-6, and induces the
HN
H
HN
N
HN
activity of degradative enzymes such as the matrix metalloproteas-
H
N
es (MMPs).2 Inhibition of TNF-
a with a number of biological agents
6
N
CN
6
CN
N
N
3
such as etanercept (Enbrel),3 infliximab (Remicade),4 and ada-
limumab (Humira)5 has been a major therapeutic advance in treat-
ing RA. These protein therapeutics have changed the treatment
paradigm and improved the quality of life of RA patients. Small
molecule drug can usually be less expensive and more conve-
niently administered (orally available) than proteins. Our desire
for such therapeutics prompted our investigation of small mole-
3
O
N
N
7
IC50 (Tpl-2) 1200 nM
IC50 (Tpl-2)
19 nM
IC50 (hWB) 6800 nM
IC50 (hWB) 3300 nM
1
2
Cl
Cl
F
F
R
cules to inhibit TNF-
Tpl2 (cot/MAP3K8), a serine/threonine kinase, plays a pivotal
role in TNF- production through the MEK-Erk pathway. Studies
with Tpl2 knockout mice demonstrated that Tpl2 is essential for
lipopolysaccharide (LPS) induced TNF-
production.6 Furthermore,
Tpl2 kinase is also required for TNF-
signaling.7 Inhibition of Tpl2
a production.
HN
H
HN
H
N
N
N
N
6
CN
N
6
CN
N
N
a
3
3
N
N
8
8
X = Br or Cl
30 nM
a
X
X
3a: X= Br IC50 (Tpl-2)
a
IC50 (hWB) 6300 nM
3
4
* Corresponding author. Tel.: +1 617 665 5673; fax: +1 617 665 5682.
E-mail address: jjwu@wyeth.com (J. Wu).
Figure 1. Tpl2 kinase inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.009

3486
J. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3485–3488
Our preparation of 8-halo-4-anilino-6-[(1H-[1,2,3]triazol-4-yl-
H
EtO
EtO
N
a, b
R
methyl)-amino]-quinoline-3-carbonitriles employed the synthetic
route outlined in Scheme 1. Aldehyde 5 was synthesized through
a [3+2] dipolar cyclo-addition of 3,3-diethoxyprop-1-yne and an
organic azide,11 which was either purchased commercially or pre-
pared from the azide displacement of an alkyl halide. Under reduc-
tive alkylation conditions, aldehyde 5 reacted with 6-amino-8-
halo-4-(3-chloro-4-fluorophenylamino)-quinoline-3-carbonitrile
6, which was prepared through a previously reported multi-step
synthesis,10 to give the target compounds 7–48.
N
R
N+
N-
O
N N
5
F
HN
N
Cl
H
H2N
CN
c
R
+
N
O
N N
X
All compounds prepared for this study were tested for inhibi-
tion of Tpl2 kinase using an ELISA assay that measures the amount
of direct phosphorylated substrate MEK kinase.8,9 Functional inhi-
6
F
R
bition of TNF-a was measured using LPS-stimulated human mono-
N
HN
Cl
cytes and LPS-stimulated human whole blood. As illustrated in
Table 1, compounds with an unsubstituted triazole at the 6-posi-
tion (7 and 8) are potent in Tpl2 inhibition (IC50 of 40 nM and
H
N
6
N
CN
3
N
X = Cl or Br
N
8
18 nM, respectively). They inhibit TNF-
lated human whole blood with IC50s of 4.2
a
production in LPS-stimu-
M, and 7.0 M, respec-
X
l
l
7-48
tively. Compared to 3, these compounds show no significant
improvement in hWB potency. In addition, 7 and 8 suffer from very
low aqueous solubility and permeability.
Scheme 1. Reagents and conditions: (a) sodium L-ascorbate, copper(II) sulfate; (b)
hydrochloric acid; (c) sodium triacetoxyborohydride, 1,2-dichloroethane, rt, 4 h,
20–80%.
In order to improve the potency and physical properties of 7 and
8, a variety of groups were introduced to the triazole ring (Table 1).
The in vitro data suggest that incorporation of hydrophobic groups
on the triazole moiety is not beneficial to the binding activity. For
example, in both the C-8 chloride and bromide series, compounds
with a hydrophobic group (9–11 and 15–16) demonstrated more
than three-fold loss of potency in Tpl-2 binding affinity; in human
blood assay, the IC50s of these compounds were greater than
Previous studies in our laboratories have shown that 1,7-naph-
thyridine-3-carbonitriles (1, Fig. 1)8 and 4,6-diaminoquinoline-3-
carbonitriles (2)9 were potent inhibitors of Tpl2 kinase. We subse-
quently reported improved selectivity and in vivo efficacy with 8-
substituted-4-anilino-6-aminoquinoline-3-carbonitriles such as 3
in models of inflammation.10 8-Bromo-4-anilino-6-aminoquino-
line-3-carbonitriles (3a) were over 1000-fold selective against a
panel of serine–threonine and tyrosine kinases; however, these
compounds show similar potency as compounds 1 and 2 in the
20 lM in hWB. However, when an acidic group such as a carboxylic
acid was employed, the resulting analogs were highly potent in
Tpl2 inhibition as indicated by 12 and 17, which showed Tpl2
inhibition IC50s of 9.6 nM and 7.0 nM, respectively, four-fold more
potent than 7 and 8. In addition, both compounds also have much
improved aqueous solubility and 17 exhibited high permeability
in PAMPA assay (1.8 Â 10À6 cm/s). Unfortunately these improved
potencies and physical properties did not translate into increased
inhibition of TNF-a production from LPS-stimulated human whole
blood (hWB). As part of our continuing efforts toward the optimi-
zation of Tpl2 inhibitors, we herein report on a series of 8-halo-4-
anilino-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-car-
bonitriles (4) with improved potency in human blood.
potency in hWB as neither 12 nor 17 had IC50s of below 20 lM.
Table 1
Inhibition of Tpl2 kinase by compounds with N1-substitutions at the triazole tail
F
R
N
HN
N
Cl
H
N
N
N
CN
X
Inhibition of LPS-induced TNF-
a
production
IC50 (hWB) (
Compound
X
R
Tpl2 IC50 (nM)
IC50 (monocytes) (
lM)
lM)
Solubility (lg/mL, pH 7.4)
PAMPA (Â10À6 cm/s)
7
8
9
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
H
H
40
18
0.39
1.8
0.69
3.9
5.0
5.0
0.27
0.05
1.8
NA
5.0
7.0
0
0
1.0
1.0
0
0
0.1
0
4.2
>20
>20
>20
>20
4.4
5.5
>20
NA
t-Butyl
Phenyl
Benzyl
CH2COOH
2-Pyrrolidinylethyl
3-Pyridinylmethyl
t-Butyl
170
120
140
9.6
40
10
61
40,000
7.0
23
10
11
12
13
14
15
16
17
18
19
73
28.8
34.7
0.04
0.57
0.18
Phenyl
0
64
35.7
28.2
1.2
1.8
0.72
0.13
CH2COOH
2-Pyrrolidinylethyl
3-Pyridinylmethyl
>20
5.9
3.9
0.32
0.22
20

J. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3485–3488
3487
Table 2
solubility and permeability, 14, with a 3-pyridinylmethyl group at
the triazole, is moderately soluble in aqueous buffer (34.7 g/mL at
Inhibition of Tpl2 kinase by compounds with N1-substitutions at the triazole tail
l
pH 7.4) and showed moderate cell permeability (0.18 Â 10À6 cm/s
in PAMPA). In addition, 14 is four-fold more potent than 7 in the
enzymatic assay of Tpl2 inhibition. Further optimization of 14
led to 20 (Table 2), which showed a 10-fold increase in Tpl2 inhi-
bition and two-fold improvement in human whole blood activity.
However, in a pharmacokinetic study, 20 showed high clearance
(88 mL/min/kg) in rats. One of the metabolic soft spots was the
benzylic methylene between the pyridyl and triazole rings. To
avoid the methylene linker, the pyridine ring was directly con-
nected to the triazole ring and 23 was prepared. Without the meth-
ylene between the pyridine ring and triazole, 23 showed lower
clearance (28 mL/min/kg) in rats than 20, however, this compound
showed diminished activity in human monocytes and hWB, de-
spite the improved potency (IC50 = 3.0 nM) in the enzymatic assay.
We then turned our attention to amine groups as substitutions
on the triazole. A series of compounds with a cyclic or non-cyclic
amine moiety at the triazole were synthesized and studied. Some
representative examples (compounds 24–37) are listed in Table
2. We found that compounds with an acyclic amine substitute on
the triazole had reduced activity in hWB (for example, IC50s for
F
R
N
HN
N
Cl
H
N
N
CN
N
Cl
Compounds
R
IC50 (nM) Tpl2
4
IC50
2.8
(lM) (hWB)
20
N
N
21
22
23
24
25
26
27
2.0
7.6
3
9
N
>20
>20
19
N
17
20
13
22
H2N
24, 25, and 26 are greater than 10 lM). On the other hand, cyclic
amine analogs (27–37) were generally more potent in both assays.
Some of them showed significantly improved potency in hWB (34–
37). Specifically, 34, with a 1-ethyl-4-piperidinyl substitution at
the triazole, was 25-fold more potent than 7 in Tpl-2 binding in
the enzymatic assay (IC50 1.6 nM) and 23-fold more potent in
12.8
10.0
5.4
H2N
N
hWB (IC50 0.3 lM). In addition, compared to 7, this series of com-
pounds (31–37) exhibited moderate to significant improvements
in aqueous solubility and cell membrane permeability (Table 3).
For example, 35, with a 4-piperidinyl group at the triazole, is quite
N
28
29
30
28
8.0
22
5.7
7.1
3.1
N
N
soluble in aqueous buffer (50 lg/mL at pH 7.4) and showed mod-
erate cell permeability (0.48 Â 10À6 cm/s in PAMPA).
Several active compounds from Table 2 were tested in a panel of
O
N
kinases, including some known to be involved in TNF-a production
(e.g., p38 and MK2) (Table 4). All the compounds reported here are
highly selective against these kinases. It is noteworthy to mention
that even though 7 weakly inhibits EGFR with an IC50 of 1.0 lM,
compounds with N-1 substitutions at the triazole ring showed
much improved selectivity (10–30-fold improvement) between
Tpl2 and this receptor tyrosine kinase. The selectivity of Tpl2
against EGFR for compound 34 is 6875-fold.
In a pharmacokinetics study, compound 34 was orally adminis-
tered in rats with 100 mg/kg dosing and showed a Cmax of 517 ng/
N
31
32
33
34
35
36
37
18
5.5
3.9
4.1
0.3
0.9
1.1
0.4
HN
N
5.3
2.2
1.6
8.2
9.1
10
mL (0.89
compound was then tested in the LPS-induced TNF-
model in female Sprague-Dawley rats. With a 25 mg/kg po dose,
l
M) and AUC of 4841 ng h/mL, above its IC50 in hWB. The
a
production
N
34 inhibited LPS-induced TNF-a production by 83% compared to
the vehicle group.
N
Table 3
Physical properties of selected 8-bromo-4-(3-chloro-4-fluoro-phenylamino)-6-{[1-
substituted-1-[1,2,3]triazol-4-ylmethyl]-amino}-quinoline-3-carbonitriles
N
Compound Solubilitya
4.5)
(l
g/mL, pH Solubilitya
7.4)
(lg/mL, pH PAMPA
(Â10À6 cm/s)
N
7
3.0
0
0
32
33
34
35
37
>100
N/A
N/A
>100
22
50
1.0
4.0
50
1.0
0.03
0.39
0.36
0.48
0.43
On the other hand, compounds with groups containing a basic
nitrogen atom showed comparable or slightly increased potency
over 7 and 8 in both the enzymatic assay and secondary assays.
Specifically, when a pyridine moiety was applied, compounds
tended to show better aqueous solubility and cell permeability
profiles (14 and 19). For example, while compound 7 has very poor
a
The compound to be tested was dissolved in DMSO (20 mg/ml) and a small
portion of the solution was added to an aqueous buffer at either pH of 4.5 or 7.4. The
mixture was incubated for 18 h at ambient temperature prior to quantitation using
a UV plate reader.12

3488
J. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3485–3488
Table 4
References and notes
In vitro selectivity profile of selected 8-bromo-4-(3-chloro-4-fluoro-phenylamino)-6-
{[1-substituted-1-[1,2,3]triazol-4-ylmethyl]-amino}-quinoline-3-carbonitriles
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M)
P38
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Src
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CAMKII
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MK2
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PKA
M)
PKC
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(l
(
l
(
l
(
l
(
l
(
l
(
l
8
1.0
4
16
>200
4.7
16
41
53
72
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110
>100
39
2.6
75
49
63
>200
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compounds with moderate improvements in physicochemical
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ally efficacious in reducing TNF-a production in a LPS-induced rat
model. This series of inhibitors demonstrates potential as orally
available compounds for the treatment of RA and other inflamma-
tory diseases.
Acknowledgments
The authors thank Dr. Katherine Lee for reviewing this Letter.
We also thank Drs. Nelson Huang, Walt Massefski, and Ms. Ning
Pan for technical support.
12. Kerns, E. H.; Di, L. Drug Discovery Today 2003, 8, 316.