4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2)

Article abstract of DOI:10.1016/j.bmcl.2010.06.107

A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-μM IC₅₀ values, and suppresses LPS-induced TN

Full text of DOI:10.1016/j.bmcl.2010.06.107

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4738–4740
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BMCL Digest
4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein
kinase-activated protein kinase 2 (MK2)
Henric Olsson a, Peter Sjö b, Oguz Ersoy b, Anna Kristoffersson b, Joakim Larsson b, Bo Nordén b,
*
a Department of Biological Sciences, AstraZeneca R&D Lund, S-22187 Lund, Sweden
b Department of Medicinal Chemistry, AstraZeneca R&D Lund, S-22187 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via
high-throughput screening. This compound class demonstrates activity against the enzyme with sub-
Received 23 March 2010
Revised 17 June 2010
Accepted 19 June 2010
Available online 25 June 2010
l
M IC50 values, and suppresses LPS-induced TNF
ments indicated mixed binding approaching non-ATP competitive inhibition.
Ó 2010 Elsevier Ltd. All rights reserved.
a levels in THP-1 cells. MK2 inhibition kinetic measure-
Keywords:
MK2
Kinase inhibitor
TNFa
Non-ATP competitive
Mitogen activated protein kinase-activated protein kinase 2
(MK2) is implicated in the regulation of synthesis of key pro-inflam-
matory cytokines such as TNFaand IL-6. By inhibiting MK2 therapeu-
tors of MK2 exemplified by the compound 1e, with an IC50 value of
12
M (Fig. 1).6
To further explore the potential residing in this class of MK2
l
tic benefits are expected in a wide range of inflammatory diseases.
MK2 is a distal kinase in the p38 MAPK signaling module where it is
inhibitors we initiated a synthetic program to explore the struc-
ture–activity relationships. We measured inhibition constants to
get kinetics related information.
phosphorylated and activated preferentially by the p38
a and b iso-
forms. The role of the p38 pathway in inflammation is well estab-
The preparation of the 6-aryl-4-anilinoquinolines was readily
accomplished starting from 6-bromo-4-chloroquinoline7 according
to Scheme 1.
lished as defined by the anti-inflammatory effects of the p38ab
inhibitor SB203580 and related compounds.1 MK2, being a more dis-
talcomponent ofthep38cascade,appearsasahighlyattractivetarget
since it has been shown that MK2 is essential for LPS-induced biosyn-
thesis of several pro-inflammatory cytokines in macrophages.2
Furthermore, it was recently shown that MK2 regulates biosyn-
The coupling reaction was performed using standard Suzuki con-
ditions, adding the boronic acid in slight excess, with Pd(dppf)2Cl2
being the preferred catalyst. No coupling was observed in the quin-
oline 4-position, and yields were typically in the 70% range. The
nucleophilic aromatic substitution in the 4-position proceeded
smoothly using equimolar amounts of aniline in a sealed tube at
200 °C using N-methyl pyrrolidone (NMP) as solvent. Among the
first set of compounds synthesized little improvement of potency
was observed, Table 1. Most compounds showed an inhibition equal
to or inferior to that observed for the initial hit compound 1e. Inter-
thesis of TNF
tional levels. Whereas MK2 regulates biosynthesis of IL-6 at the
level of mRNA stabilization, TNF production is mainly controlled
through translational control.3 Using MK2 deficient mice, LPS-in-
a and IL-6 independently at different post-transcrip-
a
duced production of TNF
a and NO were inhibited, and the phos-
phorylation of Hsp25/27 was severely affected.4 These results
suggest that MK2 may be a relevant target of the SAPK2 (stress-
activated protein kinase 2) pathway in inflammation.
Recently, several reports of MK2 inhibitors have been
published.5
COOH
R2
R1
Cl
N
N
HN
N
From screening the AstraZeneca core compound collection we
identified a novel series of 6-aryl-4-anilinoquinoline based inhibi-
1
1e
* Corresponding author. Tel.: +46 46 337483; fax: +46 46 337119.
E-mail address: bo.norden@astrazeneca.com (B. Nordén).
Figure 1. 6-Aryl-4-anilinoquinoline MK2 inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.107

H. Olsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4738–4740
4739
R1
Table 2
Cl
N
Cl
MK2 IC50’s for compounds 2a–r
Br
a
N
N
N
R2
HN
N
R1
R1
HN
N
b
1a - 1q
Compd
R1
MK2 IC50 (lM)
Scheme 1. Reagents and conditions: (a) ArB(OH)2, Pd(dppf)2Cl2, Na2CO3, EtOH/
toluene, 120 °C. (b) ArNH2, NMP, 200 °C, sealed tube. Overall yields are ꢀ70%.
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
3-Thiophene
H
Br
2-Thiophene
p-Br-phenyl
p-Cl-phenyl
p-F-phenyl
p-COOH-phenyl
m-NO2-phenyl
m-NH2-phenyl
m-CF3-phenyl
m-Cl-phenyl
m-CH3O-phenyl
p-CHO-phenyl
p-CF3-phenyl
p-CH3S-phenyl
p-CH3-phenyl
m-CH3-phenyl
26
>200
>200
>200
0.4
0.6
0.7
>200
9.0
14
Table 1
MK2 inhibition6 for compounds 1a–q
R2
R1
HN
1.5
2.6
8.0
1.8
1.4
0.7
0.8
1.4
N
Compd
R1
R2
MK2 IC50 (lM)
1a
1b
1c
1d
1e
1f
H
H
H
H
Cl
Cl
p-H
p-(CH2)2OH
p-Et
p-Morpholine
p-COOH
p-OH
>200
22
41
120
12
23
N
N
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
Cl
Cl
Cl
Cl
CH3
CH3
CH3
CH3
H
p-Morpholine
p-(CH2)2OH
p-CN
p-CO-piperazine-4-Me
2-OH, 5-Cl
p-OH
24
29
27
15
33
25
27
35
N
N
Cl
N
HN
a
Br
Br
p-CN
N
2-CH3, 3-CH3
2-OH, 5-Cl
2-F, 4-Cl
43
121
5.2
Cl
H
p-(N-methyl piperazine)
HN
N
b
R1
estinglytheadditionof p-(N-methylpiperazine)substitutedanilines
provided a twofold improvement of the potency (compound 1q).
Substituent containing o- and m-substituted anilines as well as
bicyclical anilines, for example, indole, indane, benzodioxole, and
benzofuranone showed significantly reduced capacity to inhibit
MK2 activity.
2a - 2r
Scheme 2. Reversed reaction order compared to Scheme 1. Reagents and condi-
tions: (a) ArNH2, NMP, 200 °C, sealed tube. (b) ArB(OH)2, Pd(dppf)2Cl2, Na2CO3,
EtOH/toluene, 120 °C. Overall yields are in the 50–65% range.
The improved potency observed for the compound 1q prompted
us to further investigate the N-methyl piperazine based sub-series.
Indeed, expansion led to the identification of significantly improved
MK2 inhibitors, Table 2. In this series para-substituted phenyl
groups in the quinoline 6-position appear to provide the most potent
80
20
inhibitors, with IC50’s down to 0.4 lM (compound 2e).
The N-methyl piperazine derivatives were prepared according
to Scheme 2.
To maximize the overall yields an excess of boronic acid was
used in the Suzuki couplings (Schemes 1a and 2b, respectively).
Aryl substitution on the quinoline 2-, 5-, 7- and 8-positions ren-
dered less active compounds than the 6-substituted quinolines (2).
The most potent compounds in this series, compounds 2e–2g were
0.1
1
10
Conc./µM
Figure 2. Inhibition of LPS-induced TNFa release from THP-1 cells by compound 2g.
also found to be active to suppress LPS-induced TNF
a release from
THP-1 cells,8, for example, 2g was found to have an EC50 of 4.3
lM
to bad permeability since compounds 2e–g, 2k, 2p–r, and 1q were
measured to have a Papp of 0.3 or less using a human Caco-2 perme-
ability assay.
(Fig. 2) which is almost identical to the value obtained for the other
compounds. The lower activity in the cell based assay might be due

4740
H. Olsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4738–4740
cated a case of mixed inhibition with a dominating element of
uncompetitive inhibition (Fig. 3).
[I]/µM
0
13000
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Global analysis of the statistical parameters for goodness of fit
confirmed that the models for mixed and uncompetitive inhibition
best described the complete data set with better R2 values than for
competitive or non-competitive inhibition (Table 3). Using the
model for mixed inhibition Kic ꢁ Kiu, for example, the affinity for
compound 2f is almost 20-fold higher at the uncompetitive site
than in the competitive (ATP-binding) site (Table 3).9
Compound 2f inhibited MK2 more efficiently with increasing
ATP concentrations (Fig. 4), further supporting the conclusion that
it is mainly uncompetitive in nature.
0.25
0.5
1
2
In summary, we have reported on a set of small molecule MK2
enzyme inhibitors, also which may attenuate TNF
a production in
0
10
20
30
40
50
60
70
80
THP-1 cells. The measurement of inhibition kinetics of these inhib-
itors strongly indicates an uncompetitive binding mechanism.
However, this class of amino-aryl-quinoline compounds were con-
sidered to be of less importance as starting points for lead optimi-
zation. Although many of the compounds are stable in the rat HW
[ATP]/µM
Figure 3. MK2 activity versus ATP concentration in the presence of 0, 0.25, 0.5, 1,
and 2 M of compound 2f fitted with the equation for mixed inhibition using the
l
EnzFitter software.
microsome metabolism assay (<10 ll/min/mg), less stability is
usually found in human microsomes. Also this structural class
shows a general cytotoxicity in human THP-1 cells using WST-1
dye and recording cell proliferation and viability, respectively
Table 3
Inhibition constants and goodness of fit obtained from the different models of
inhibitor modality
(usually below 4
lM).
Mixed
Uncompetitive
13.3
Non-competitive
Competitive
Km
Kic
Kiu
R2
(
l
M)
M)
M)
12.6
5.6
0.32
0.99
8.80
0.48
0.48
0.97
7.21
0.096
(
l
Acknowledgment
(l
0.31
0.99
0.83
We would like to thank Britta Lundquist and Susanna Olsson for
skillful experimental support.
Km, dissociation constant for the MK2-ATP complex; Kic, competitive inhibition
constant; Kiu, uncompetitive inhibition constant.9
References and notes
1.8
1.6
1.4
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3. Neininger, A.; Kontoyiannis, D.; Kotlyarov, A.; Winzen, R.; Eckert, R.; Volk, H. D.;
Holtmann, H.; Kollias, G.; Gaestel, M. J. Biol. Chem. 2002, 277, 3065.
4. Kotlyarov, A.; Yannoni, Y.; Fritz, S.; Laass, K.; Telliez, J. B.; Pitman, D.; Lin, L. L.;
Gaestel, M. Mol. Cell Biol. 2002, 22, 4827.
5. Anderson, D. R.; Meyers, M. J.; Kurumbail, R. G.; Caspers, N.; Poda, G. I.; Long, S.
A.; Pierce, B. S.; Mahoney, M. W.; Mourey, R. J. Bioorg. Med. Chem. Lett. 2009.
doi:10.1016/j.bmcl.2009.02.015, and references cited therein.
1.2
1
0.8
0.6
0.4
0.2
0
6. MK2 activity (0.4 U/ml of activated MK2) was measured in 96-well micro titer
plates in 8 mM MOPS, pH 7.0, 10 mM MgAc, 0.2 mM EDTA, 0.002% Brij 35, 1%
0
20
40
[ATP]/
60
M
80
100
glycerol, 0.04% BSA, 0.1% b-mercaptoethanol in the presence of 10
(400 dpm/pmol) and 3 M glycogen synthase peptide substrate (KKLNRTLSVA)
in a total volume of 25 l. After 30 min at room temperature reactions were
quenched by the addition of 25 l of 10% HAc, transferred to pre-wet Millipore
P81 filter plates and incubated for 30 min at room temperature. Filters were
l
M [33P]-ATP
µ
l
l
Figure 4. Inhibition of MK2 by 2f at increasing ATP concentrations.
l
washed with 5% Hac and counted (TriLux Microbeta) after adding 25
scintillation cocktail (OptiPhase SuperMix, Perkin Elmer).
7. Lin, A. J.; Loo, T. L. J. Med. Chem. 1978, 21, 568.
ll/filter of
However, the indication is that this compound class may retain
the activity also in a cell based context, in a manner consistent
with MK2 enzyme inhibition.
To understand inhibition modality and to determine an inhibi-
tion constant, the plots of reaction velocity versus ATP concentra-
tion at various fixed inhibitor concentrations (2f) were fitted
globally to the equations for competitive, non-competitive, and
uncompetitive inhibition, respectively. This analysis strongly indi-
8. TNFa levels in THP-1 cell:
The assay was performed in a round bottom well format, where 100,000 THP-1
cells were incubated with or without compound for 45 min followed by a 5 h
stimulation with 10 ng/ml of LPS at 37 °C. Cell-free supernatants were then
collected and stored at À80 °C until use. TNF
a concentrations in the
supernatants were analyzed using TNF ELISA kit from R&D Systems
a
a
according to the instructions of the manufacturer.
9. Copeland, R. A. In Enzymes, 2nd ed.; Wiley InterScience, 2000; p 282.