Aminocyanopyridine inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

Article abstract of DOI:10.1016/j.bmcl.2005.01.067

A class of inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2) was discovered. These compounds have demonstrated activity against the enzyme with IC₅₀ values as low as 130 nM and suppress the expression of TNFα in U

Full text of DOI:10.1016/j.bmcl.2005.01.067

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1587–1590
Aminocyanopyridine inhibitors of mitogen activated protein
kinase-activated protein kinase 2 (MK-2)
a,
a,
David R. Anderson,a,* Shridhar Hegde, Emily Reinhard, Leslie Gomez,a,à
William F. Vernier,a,§ Len Lee,a Shuang Liu,b Aruna Sambandam,b
Patricia A. Snidera,– and Liaqat Masihb
*
aPfizer Global Research and Development, 700 Chesterfield Parkway W, Chesterfield, MO 63017, USA
bAlbany Molecular Research, Inc., 21 Corporate Circle, Albany, NY 12212, USA
Received 7 December 2004; revised 26 January 2005; accepted 27 January 2005
Abstract—A class of inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2) was discovered. These com-
pounds have demonstrated activity against the enzyme with IC50 values as low as 130 nM and suppress the expression of TNFa in
U937 cells. These represent the first small molecule inhibitors of MK-2 to be reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
TNFa has been implicated in several inflammatory dis-
ease states in humans.1 Biologic anti-TNFa therapy has
been shown to be effective in the treatment of diseases
such as rheumatoid and psoriatic arthritis.2 Numerous
potential biological targets have been identified for inhi-
bition to attenuate the biosynthesis of TNFa and hence
be efficacious in the treatment of inflammatory disease.
Among the best documented of these targets is p38
MAPK.3,4 It was reported that the p38 kinase inhibitor
VX-745 has demonstrated efficacy in rheumatoid arthri-
tis patients in a clinical study.5 Mitogen-activated pro-
tein kinase-activated protein kinase 2 (MK-2) is a
direct substrate of p38 kinase and is linked to TNFa
production.6,7 An MK-2 knockout mouse has been re-
ported to be resistant to disease in arthritis models.6
Administration of lipopolysaccharide (LPS) to MK-2
knockout mice results in the rise of serum TNFa, but
only to a maximum of 10–20% of the wild type control
animals. Moreover, the MK-2 knockout mice were re-
ported to be fertile and healthy. These reports suggest
that MK-2 is an attractive target for the safe and effec-
tive treatment of TNFa mediated disease.
Through high throughput screening and early explor-
atory synthesis, we identified a series of benzopyrano-
pyridines represented by structure
1 as modest
inhibitors of MK-2 (Fig. 1). For example, compound
1a exhibited an IC50 value of 1.94 lM8 and was deter-
mined to bind in a competitive manner with ATP. Like-
wise, closely related analogs 1b–e (Table 1) also showed
MK-2 inhibition with low micromolar IC50 values.
Compounds 1a–e were readily accessible via stepwise
condensation of salicylaldehydes with 3 equiv of malon-
onitrile as shown in Scheme 1. Although these were
interesting lead molecules, the malononitrile group in
the 5-position was deemed undesirable as a potential
Keywords: MK-2; Kinase inhibitor; TNFa.
*
Corresponding authors. Tel.: +1 636 247 7651; fax: +1 636 247 5400;
e-mail addresses: david.r.anderson@pfizer.com; shridhar.g.hegde@
pfizer.com
NC
CN
NH2
Present address: Synaptic Pharmaceutical, 215 College Rd., Paramus,
NJ 07652, USA.
R2 NH2
CN
6
à Present address: Johnson and Johnson PRD, 3210 Merryfield Row,
San Diego, CA 92129, USA.
CN
7
R1
8
5
MeO
O
N
NH2
§ Present address: Pfizer Global Research and Development, 10770
Science Center Drive, San Diego, CA 92121, USA.
– Present address: Albany School of Pharmacy, 106 New Scotland
Ave., Albany, NY 12208, USA.
O
N
NH2
9
1a
1
Figure 1. Benzopyranopyridine MK-2 inhibitors.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.067

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D. R. Anderson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1587–1590
Table 1. MK-2 inhibition for compounds 1a–g
Table 2. IC50 values for aminocyanopyridine inhibitors against MK-2
and cellular TNFa expression
R2 NH2
6
NH2
CN
7
5
6
CN
7
R1
8
R1
8
O
N
NH2
9
O
N
NH2
9
1 a-g
2-a-m
Compd
R1
R2
MK-2 Inhibition IC50 (lM)a
Compd R1
MK-2
Inhibition
IC50 (lM)a
U937 TNFa
IC50 (lM)b
1a
1b
1c
1d
1e
1f
8-OMe
8-OH
7-OMe
7-Br
CH(CN)2
CH(CN)2
CH(CN)2
CH(CN)2
CH(CN)2
Ph
1.94
12.8
6.18
2a
2b
2c
2d
2e
2f
7,8-Di-OH
8-OH
7-OH
0.13
0.18
0.47
0.33
0.46
0.49
0.53
0.56
0.62
0.69
0.77
0.85
0.92
1.08
4.22
3.8
7-OH
8-OH
8-OMe
25.4
64.8
>200
>200
7,8-Di-OMe
9-OH
7,8-O-CH2CH2O-
1g
Ph
7.12
a IC50 values determined by method in Ref. 8.
55.7
2g
2h
2i
7-NH2
H
>100
>100
11.9
7-OH,8-Et-O-(CH2)2O
6,8-Di-OH
7-OH,8-EtO
CHO
2j
>100
9.4
R
2k
2l
OH
8,9-Di-OH
6.92
2.91
a
2m
8-O-(CH2)2-pyrrolodine 1.68
c
a IC50 values determined by method in Ref. 8.
b IC50 values determined by method in Ref. 9.
NC
CN
CN
Ph
CN
R
R
O
NH2
O
NH2
a
R
R
Et
N
Et
b
d
OH
OH
NC
CN
H3C
NC
R
Et
Ph NH2
b
N+
NH2
N
I-
H2N
+
CN
CN
CN
CN
Et
OH
R
R
O
N
NH2
O
NH2
c
1 f-g
NH2
1 a-e
R
CN
NH2
Scheme 1. Reagents and conditions: (a) CH2(CN)2 (2 equiv), cat.
piperidine/EtOH, rt; (b) CH2(CN)2, cat. piperidine/EtOH, reflux; (c)
Ph–C@C(CN)2, cat. piperidine, EtOH, reflux; (d) CH2(CN)2, pyridine,
reflux.
O
N
Scheme 2. Reagents and conditions: (a) Et2NH, CH2O; (b) CH3I/
acetone; (c) EtOH, cat. piperidine, heat.
toxicophore as well as a chemically reactive functional
group. Hence our initial efforts of optimizing structure
1 were focused on either replacing the malononitrile
group with alkyl or aryl groups or excising it completely.
Analogs 1f and 1g bearing a phenyl group in the 5-posi-
tion were prepared via sequential condensation of sali-
cylaldehydes with benzylidenemalononitrile and
malononitrile as shown in Scheme 1. Attempts to pre-
pare 5-alkyl substituted analogs using this methodology
were unsuccessful. Both 1f and 1g were completely de-
void of MK-2 inhibition (Table 1).
tiary amine to a quaternary salt was followed by
reaction with malononitrile dimer to produce the desired
target compounds.
This method was limited to the para substituted phenols
as starting materials, however, and alternative methods
were sought to prepare a wider range of analogs. A
modified synthetic scheme outlined in Scheme 3 allows
for the incorporations of a wider variety of substitution
patterns. Thus, malononitrile dimer was condensed with
substituted salicyaldehydes in acetic acid and ethanol
(1:1). The solvents were evaporated and the residue
taken up in trifluoroacetic acid and reduced with tri-
ethylsilane. The products usually precipitated out of the
reaction mixture as pure TFA salts. In cases where the
products were not pure, the final products were purified
by reverse phase chromatography. This method allowed
for the rapid synthesis of analogs using parallel
synthesis.
Significant improvement in MK-2 inhibition was
achieved with 5-unsubstituted analogs. For example,
compound 2d exhibited an MK-2 IC50 value of
0.33 lM (Table 2). These compounds can be prepared
by the method described in Scheme 2. The Mannich
reaction of a para substituted phenol with diethylamine
and formaldehyde is used to introduce a diethylaminom-
ethyl group in the ortho position. Conversion of the ter-

D. R. Anderson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1587–1590
1589
Table 3. IC50 values for aminocyanopyridine inhibitors against MK-2
NH2
O
NC
a, b
CN
R1
CN
CN
+
H2N
H
NH
R
R
HO
HO
CN
R2
O
N
NH2
OH
Scheme 3. Reagents and conditions: (a) EtOH/AcOH (1:1); (b) TFA/
Et3SiH.
O
N
N
H
3a-d
Compd
R1
R2
MK-2 Inhibition IC50 (lM)a
Using these routes, potent inhibitors of MK-2 were pre-
pared. Table 2 provides some illustrative examples. The
most potent compounds discovered in this series have a
hydroxyl group at either the 8- (compound 2b) or at
both the 7- and 8-positions (compound 2a) of the phenyl
ring. The corresponding 7-hydroxy analog (compound
2c) was less potent, as was the 9-hydroxy analog (com-
pound 2e). It appears that hydroxyl groups in the 7-
and 8-positions are involved in hydrogen bonding to
the target. Compound 2e apparently cannot engage in
any such interaction because it has the same potency
as the unsubstituted analog 2h. Methoxy substitution
is also tolerated as demonstrated by example 2d, but
an amine (compound 2g) is essentially equipotent with
the unsubstituted phenyl. Similarly, tying the oxygens
of compound 2a into a ring resulted in a compound
(2f) no more potent than the unsubstituted phenyl
(2h). Further elaboration of the phenols resulted in
losses in potency of MK-2 inhibition. For example,
appending a 1-pyrrolidinylethyl group to the 8-hydroxy
compound (compound 2m) resulted in a large drop in
potency (ꢀ10·). The most potent analogs found were
also active in a cellular assay (Table 2). U937 cells were
treated with compound 2a for 1 h and then challenged
with LPS.9 Several compounds in this series were found
to be active in this assay. These results indicate that
small molecule MK-2 inhibitors are effective in reducing
cellular TNFa expression.
3a
3b
3c
3d
CH2-CH2-OH
H
H
H
Et
0.237
0.403
0.419
>200
Pr
Et
Et
a Values determined by method in Ref. 8.
critical hydrogen bonding to the target or there is no
room to accommodate an alkyl group at this position.
The heteroatom of the central ring was also replaced.
The nitrogen and sulfur analogs were prepared starting
from the corresponding ortho-amino or mercapto ben-
zaldehydes in accordance with Scheme 3. The nitrogen
analog required further substitution to avoid aromatiza-
tion. It was found that neither of these substitutions was
well tolerated (Table 4). The sulfur analog was inactive
against the target and the N-methyl analog (compound
4c) was only weakly active compared to compound 2h.
The core of these compounds can be truncated as well.
Commercially available 5 was also found to be an inhib-
itor of MK-2 (IC50 = 2 lM). This suggests that the
amino cyano pyridine substructure is an important part
of the MK-2 binding pharmacophore. While the benzo-
pyran portion of the active structures described herein
also appears to contribute significantly to binding, the
above result further suggests that it may be optimized
in a variety of ways.
Substitution at the 4-amino position with small alkyl
groups can be accomplished by treating 2d with sodium
hydroxide in DMSO with an alkyl halide (Scheme 4).
The methoxy groups were demethylated with BBr3 to
produce the final compounds. The activities of these
compounds are reported in Table 3.
NH2
CN
F3C
O
N
NH2
5
In general these compounds are only slightly less active
than the unsubstituted analogs, suggesting that both
hydrogens of the 4-amino group are not involved in
hydrogen bonding and that there may be some space
for further elaboration. Substitution at both the 4- and
2-amino groups (compound 3d) resulted in a compound
with no measurable activity. This suggests that either
both hydrogens of the 2-amino group are involved in
Compounds that were active in the cellular assay were
also tested in vivo. Compound 2a was found to be active
in a rat LPS assay (20 mpk, IP, 68% inhibition) and 2m
Table 4. IC50 values for aminocyanopyridine inhibitors against MK-2
NH2
CN
R
NH2
N
HN
N
X
N
NH2
O
O
CN
HO
HO
CN
a, b
4a-c
Compd
X
MK-2 Inhibition IC50 (lM)a
O
NH2
O
NH2
4a
4b
4c
S
>200
>200
70
2d
3a-d
SO2
N-Me
Scheme 4. Reagents and conditions: (a) NaOH, DMSO, R-Br; (b)
BBr3/DMSO.
a Values determined by method in Ref. 8.

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D. R. Anderson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1587–1590
scint-40 (Packard) was added and the amount of phos-
phorylated-peptide was determined. The assay is
performed at a final concentration of 2% DMSO. The
error for this assay is taken to be 0.2 log units.
was found to be orally active (20 mpk, PO, 60%
inhibition).10
In conclusion, a class of small molecule MK-2 inhibitors
has been discovered and the basic SAR of the series has
been described. These inhibitors are effective in attenuat-
ing TNFa production in cellular assays and are active in
vivo as well.
9. The human monocyte-like cell line, U937 (ATCC #CRL-
1593.2), is cultured in RPMI1640 media with 10% heat-
inactivated fetal calf serum (GIBCO), glutamine and pen/
strep at 37 ꢁC and 5% CO2. Differentiation of U937 to
monocytic/macrophage-like cells is induced by the addi-
tion of phorbol12-myristate 13-acetate (Sigma) at final
concentration of 20 ng/mL to a culture of U937 cells at
ꢀ0.5 million cells/mL and incubated for 24 h. The cells are
centrifuged, washed with PBS and resuspended in fresh
media without PMA and incubated for 24 h. Cells
adherent to the culture flask are harvested by scraping,
centrifugation, and resuspended in fresh media to 2 mil-
lion cells/mL, and 0.2 mL is aliquoted to each of 96 wells
in flat-bottom plate. Cells are then incubated for an
additional 24 h to allow for recovery. The media is
removed from the cells, and 0.1 mL of fresh media is
added per well. 0.05 mL of serially diluted compound or
control vehicle (Media with DMSO) is added to the cells.
The final DMSO concentration does not exceed 1%. After
1 h incubation, 0.05 mL of 400 ng/mL LPS (E. coli
serotype 0111:B4, Sigma) in media is added for final
concentration of 100 ng/mL. Cells are incubated at 37 ꢁC
for 4 h. After 4 h incubation, supernatants are harvest and
assayed by ELISA for the presence of TNFa. The error for
this assay is taken to be 0.5 log units.
10. Adult male 225–250 g Lewis rats (Harlan Sprague–
Dawley) were used. Rats were fasted 18 h prior to oral
dosing, and allowed free access to water throughout the
experiment. Each treatment group consisted of five
animals. Compounds were prepared as a suspension in a
vehicle consisting of 0.5% methylcellulose, 0.025% Tween-
20 in PBS. Compounds or vehicle were orally administered
in a volume of 1 mL using an 18-gauge gavage needle. LPS
(E. coli serotype 0111:B4, Lot #39H4103, Cat. # L-2630,
Sigma) was administered 1–4 h later by injection into the
penile vein at a dose of 1 mg/kg in 0.5 mL sterile saline.
Blood was collected in serum separator tubes via cardiac
puncture 1.5 h after LPS injection, a time point corre-
sponding to maximal TNFa production. After clotting,
serum was withdrawn and stored at À20 ꢁC until assay by
ELISA.
References and notes
1. Camussi, G.; Lupia, E. Drugs 1998, 55, 613.
2. (a) Richard-Miceli, C.; Dougados, M. BioDrugs 2001, 15,
251; (b) Braun, J.; Sieper, J. BioDrugs 2003, 17, 187.
3. Chen, Z.; Gibson, T. B.; Robinson, F.; Silvestro, L.;
Pearson, G.; Xu, B.; Wright, A.; Vanderbilt, C.; Cobb, M.
Chem. Rev. 2001, 101, 2449.
4. Pargellis, C.; Regan, J. Curr. Opin. Invest. Drugs 2003, 4,
566.
5. Haddad, J. Curr. Opin. Invest. Drugs 2001, 2, 1070.
6. Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert, R.;
Birchmeier, C.; Volk, H. Nat. Cell Biol. 1999, 1, 94.
7. Neininger, A.; Kontoyiannis, D.; Kotlyarov, A.; Winzen,
R.; Eckert, R.; Volk, H. J. Biol. Chem. 2002, 277, 3065.
8. MK-2 IC50 value determination: Recombinant MAP-
KAPK2 was phosphorylated at a concentration of 42–
78 mM by incubation with 0.23 mM of active p38a in
50 mM HEPES, 0.1 mM EDTA, 10 mM magnesium
acetate, and 0.25 mM ATP, pH 7.5 for 1 h at 30 ꢁC. The
phosphorylation of HSP-peptide (KKKALSRQLSVAA)
by MAPKAPK2 was measured using an anion exchange
resin capture assay method. The reaction was carried out
in 50 mM b-glycerolphosphate, 0.04% BSA, 10 mM mag-
nesium acetate, 2% DMSO, and 0.8 mM dithiotheritol,
pH 7.5 in the presence of the HSP-peptide with 0.2 lCi
[g33P]ATP and 0.03 mM ATP. The reaction was initiated
by the addition of 15 nM MAPKAPK2 and was allowed
to incubate at 30 ꢁC for 30 min. The reaction was
terminated and [g33P]ATP was removed from solution
by the addition of 150 mL of AG 1X8 ion exchange resin
in 900 mM sodium formate pH 3.0. A 50 mL aliquot of
head volume was removed from the quenched reaction
mixture and added to a 96-well plate, 150 mL of Micro-