Novel 3-aminopyrazole inhibitors of MK-2 discovered by scaffold hopping strategy

Article abstract of DOI:10.1016/j.bmcl.2009.10.138

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFα release in cells. In addition, selected derivative 1

Full text of DOI:10.1016/j.bmcl.2009.10.138

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1293–1297
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 3-aminopyrazole inhibitors of MK-2 discovered by scaffold
hopping strategy
*
Juraj Velcicky , Roland Feifel, Stuart Hawtin, Richard Heng, Christine Huppertz, Guido Koch,
Markus Kroemer, Henrik Moebitz, Laszlo Revesz, Clemens Scheufler, Achim Schlapbach
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The
Received 22 September 2009
Revised 29 October 2009
Accepted 30 October 2009
Available online 3 November 2009
new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNF
a
release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNF release in vivo.
a
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MK2
TNFa-inhibition
3-Aminopyrazole
Scaffold hopping
Induced-fit
X-ray
Successful use of anti-TNF
a
biologics etanercept (EnbrelÒ), inf-
pyrrole ring of 1 with a six-membered phenyl ring. In order to fit
the new structure (distance and geometry) into the MK2-pharma-
cophore, the six-membered pyridine ring in 1 had to be substituted
with a five-membered heterocycle. General structure of such a new
scaffold is represented by a formula 2.
liximab (RemicadeÒ) and adalimumab (HumiraÒ) in the clinic
demonstrates a key-role of this cytokine in triggering and sustain-
ing the inflammation in autoimmune diseases such as rheumatoid
arthritis, psoriasis, Crohn’s disease and others.1 TNF
a is mainly
produced by macrophages and its biosynthesis is initiated upon
extracellular activation (e.g., LPS) of signaling pathways leading
to activation of mitogen-activated protein kinases (MAPKs) such
as p38 MAPK (p38).2 Some time ago, it was shown that MAPKAP
kinase-2 (MK2),3 a direct downstream substrate of p38, plays a
By screening for a suitable five-membered heterocycle, we have
found that pyrazole represented by compound 3b fitted best to
such an exercise (Table 1). Although a simple pyrazole only weakly
inhibits MK2 kinase activity, an additional amino group in the 3-
position of pyrazole-ring (3b) gained potency at MK2 by one log
unit (3b vs 3a). An amino group in 5-position of pyrazole (3c) led
to decreased inhibition of MK2, indicating that the 3-aminopyra-
zole ring is well positioned for binding to this kinase.
crucial role in the signaling and synthesis of TNFa. Evidence using
MK2 k.o. mice showed decreased TNF
a
levels in vivo and ex vivo
upon LPS stimulation.3a Furthermore, these mice were also resis-
tant to collagen-induced arthritis.4 As MK2 is downstream of
p38, less side effects are predicted for MK2 inhibitors compared
to p38, whilst retaining the beneficial properties, that is, preven-
Pyrazole 3a was prepared in four steps starting with an acidic
formation of the pyrazole-ring by a condensation of p-methoxy-
phenyl hydrazine with 1,1,3,3-tetramethoxy-propane (Scheme 1).
tion and treatment of diseases mediated by TNFa. This anticipation
led various groups to the search for potent MK2 inhibitors.5
Recently, we reported our initial chemical efforts in targeting this
kinase5a and in this study we wish to disclose our strategy towards
further modification leading to aminopyrazole series.
In order to substantially modify the pyrrolo-pyrimidinone
structure of 1 we employed a scaffold hopping strategy6 (Fig. 1).
Using this approach, we aimed to replace the five-membered
O
O
NH2
NH
X
N
N
N
H
N
X
X
R
Ar
1
2
* Corresponding author.
E-mail address: juraj.velcicky@novartis.com (J. Velcicky).
Figure 1. Scaffold hopping strategy in derivatization of scaffold 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.138

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J. Velcicky et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1293–1297
Table 1
with 3-methoxyacrylonitrile followed by NBS bromination) and
Modification of five-membered heterocycle
boronic esters 11a–d (obtained by palladium catalyzed borona-
tion of the corresponding bromides or triflates known in the
literature).9 Sulfonamide 7 was obtained by Suzuki coupling of
O
Het
bromopyrazole
9 with commercially available 4-sulfamoyl-
NH2
phenylboronic ester.
Interestingly, while nitrile 6a and sulfonamide 7 are weaker
MK2-inhibitors than amide 3b (Table 2), freezing the rotation of
the amide group by its transformation to a lactame such as 8b signif-
icantly improved the potency at MK2 (IC50 of 3b/8b ꢀ25). Quite sur-
prisingly, the lactame ring size was found to be of great importance
3
O
Compound
Het
MK2a
(lM)
1a
R in 1: p-MeO-Ph
0.54 0.09
N
N
3a
3b
22.6 7.4
for this scaffold. While
tency (8c being in a range of the amide 3b),
x
-lactames 8c and 8d show a drop in the po-
-lactame 8a is even less
c
NH2
tolerated than the primary amide 3b (Table 2). Unfortunately, none
of the inhibitors described above showed any effect in our cellular
2.0 0.4
N
N
assays (inhibition of TNFa11 release in human PBMC cells or phos-
phorylation of hsp2712 (p-hsp27) in the monocytic cell line
THP-1). This did not appear to be due to the lack of cell penetration
as compounds showed reasonable permeability (e.g., 8b: PAMPA
N
N
3c
22.5 1.5
NH2
(log Pe pH 6.8: À4.4); Caco-2 (Papp(A À B): 48.3 Â 10À6 cm sÀ1
P(B À A)/P(AÀB): 0.31)).
;
a
Inhibition of kinase activity (IC50 values) were measured using a FRET based
assay10 and are reported as a mean of P2 measurements.
In the search for compounds with cellular potency, we have
found that m-phenol 14a shows potency in both cellular assays
(TNFa and p-hsp27). Therefore, we explored this further by search-
ing for its biomimetics13 with less potential to fast in vivo clear-
ance (Table 3).
Following bromination, Suzuki reaction with p-cyanophenyl-boro-
nic acid and hydrolysis of the nitrile led to an amide 3a. Unfortu-
nately, the synthetic precursor 6a for preparation of the more
potent 3-aminopyrazole 3b was obtained only as a minor product
(6a/6b = 1:15) under acidic cyclization7 of the hydrazine with enol
5a. However, cyclization of the hydrazine with methyl ether 5b un-
der basic conditions,8 completely reverts the regioselectivity of this
reaction and 3-aminopyrazole intermediate 6a could be obtained
in excellent yield (93%) as a single isomer.
Since the corresponding anisole derivative 14b is inactive in the
p-hsp27 assay, the hydrogen donor of the phenol seems to be impor-
tant for cellular activity. This observation was further supported by
sulfonamide 14c, benzimidazole 14d or indole 14e, latter being the
best compound obtained in terms of MK2 activity as well as cellular
potency (Table 3). N-Methylation of indole (14e) resulted in a cellu-
laryinactivecompound14f. Interestingly, the2-indolonecompound
We next aimed to improve the MK2 binding affinity of the 3-ami-
nopyrazole scaffold. Since initial derivatization of N-aryl substituent
proved to be unsuccessful, we focused on the benzamide portion.
For this purpose, diverse lactames were prepared (Scheme 2)
by Suzuki coupling of 3-amino-4-bromopyrazole 9 (reached in
two steps by basic condensation of p-MeO-phenyl hydrazine
14g (having a hydrogen donor) was inactive for p-hsp27 (>30
well, indicating that the carbonyl group may negatively interfere
with the enzyme (MK2: 0.22 M).
An insight into the binding of such compounds was obtained by
X-ray crystallography of 14e with MK2 (47–364)14 ( Fig. 2). As
expected, the aminopyrazole binds to the hinge region of MK2,
lM) as
l
O
Br
NH2
NH2.HCl
HN
N
O
N
N
a-b)
c-d)
O
+
N
O
O
O
O
O
4
3a
O
N
N
N
NH2
X
H
N
X
.HCl
NH2
O
N
e)
N
d)
N
N
Y
(f) or (g)
Y
N
O
R
N
O
O
5a: R = H
6a: X = NH2, Y = H
6b: X = H, Y = NH2
3b: X = NH2, Y = H
3c: X = H, Y = NH2
5b: R = Me
Scheme 1. Reagents and conditions: (a) EtOH, reflux, 1 h (95%); (b) NBS, THF, 23 °C, 3.5 h (95%); (c) p-CN-Ph-B(OH)2, PdCl2(PPh3)2 (cat.), Na2CO3, n-PrOH/H2O; 160 °C, 15 min,
microwave (50%); (d) H2O2, NaOH, H2O/MeOH/DMF, 23 °C, 16h (3a: 70%; 3b: 56%; 3c: 90%); (e) HCOOEt, for 5a: NaH, DME, 60 °C, 3h (96%); for 5b: NaOEt, EtOH, 50 °C, 3h, then
MeI, DMF, 40 °C, 2h (86%); (f) 5a, EtOH, AcOH, reflux, 3h (6a: 3%, 6b: 46%); (g) 5b, NaOEt/EtOH, reflux, 4h (6a: 93%).

J. Velcicky et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1293–1297
1295
O
O
O
NH2
S
O
A
NH
( )n
NH2
c)
N
NH
( )n
O
B
O
A
N
N
X
d)
e)
10a: n = 0, A = CH2, X = Br
10b: n = 1, A = CH2, X = Br
10c: n = 2, A = CH2, X = OTf
10d: n = 2, A = O, X = OTf
11a-d
7
O
O
NH2
NH
( )n
N
A
N
NH2
Br
H
N
.HCl
NH2
N
a-b)
O
O
N
+
O
9
8a-d
O
O
NH2
H2N
N
H2N
Br
N
NH2.HCl
Br
NH
HN
N
N
a)
e, b)
for indole: f)
11b, e)
N
N
N
+
O
Br
Ar
Ar
12
13a-g
14a-g
Scheme 2. Reagents and conditions: (a) NaOEt, EtOH, reflux, 20 h (9: 73%; 12: 91%); (b) NBS, THF, 23 °C, 16 h (9: 87%; 13a–g: 51–96% over two steps); (c) pinacolatodiboron,
Pd(dppf)Cl2/dppf (cat.), KOAc, dioxane, 80 °C, 16 h (87–97%); (d) 4-boronobenzenesulfonamide pinacol ester, PdCl2(PPh3)2 (cat.), Na2CO3, n-PrOH/H2O, 150 °C, 15 min,
microwave (29%); (e) PdCl2(PPh3)2 (cat.), Na2CO3, n-PrOH/H2O; 150 °C, 15 min, microwave or for 11b:80 °C, 16 h (8a–d: 17–63%; 14a–g: 9–36%); (f) NaH, Boc2O, DMF, rt, 16h
(99%).
Table 2
Table 3
Modification of the amide portion
Search for m-phenol biomimetics
NH2
Ar
N
NH2
O
N
N
N
O
Ar
Compound
Ar
MK2a
(lM)
NH
O
Compound Ar
MK2a
(lM)
TNFab
(l
M) p-hsp27c
3.5 1.0
(lM)
3b
1.98 0.37
31.2 1.2
15.4 0.6
NH2
N
14a
0.18 0.02
0.18 0.01
2.8 0.7
HO
6a
7
14b
4.5 0.4
>10
>30
O
O
O
S
NH2
O
O
S
14c
14d
0.18 0.07
P11.3
N
H
O
8a
8b
6.0 1.0
N
NH
0.082 0.042 5.3 0.9
0.061 0.006 2.5 0.8
4.1 0.3
N
H
O
0.084 0.029
NH
14e
14f
14g
2.0 0.8
>30
N
H
O
NH
NH
8c
1.1 0.4
0.29 0.07
0.22 0.12
>10
N
O
O
P3.8
>30
N
H
8d
0.35 0.22
a
IC50 values are measured using FRET kinase assay10 and are reported as a mean
O
of P2 measurements.
IC50 values are measured using FRET kinase assay10 and are reported as a mean
a
b
Inhibition of LPS stimulated release of TNFa
from human PBMCs.11
of P2 measurements.
c
Inhibition of anisomycin stimulated phosphorylation of hsp27 in THP-1 cells.12

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J. Velcicky et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1293–1297
MK2 kinase inhibitor 14e. This compound showed potent inhibi-
tion of MK2 activity and reasonable cellular activity (inhibition of
TNF and phoshorylation of hsp27). In addition, the compound
was effective in vivo for inhibition of LPS-induced TNF release
a
a
in mice. Interestingly, a novel binding pocket behind the hinge re-
gion which was induced by the ligand was discovered which seems
to improve binding to MK2 and also kinase selectivity.
Acknowledgments
We are very grateful to our colleagues from the NIBR kinase
platform for providing us with in vitro kinase and selectivity data
as well as the NIBR Prep Labs for the syntheses of valuable
intermediates.
References and notes
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Figure 2. Crystal structure of 14e bound to MK2.14
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interacting with backbone of E139 and L141, and the lactame por-
tion makes a dual hydrogen bond interaction with residues of con-
served K93 and D207. Surprisingly, an unusual binding of the
indole-ring to a new, ligand induced hydrophobic pocket behind
MK2-hinge region was observed. The indole-NH group forms an
additional hydrogen bond interaction with the backbone carbonyl
of F90, highlighting the importance of an hydrogen donor at this
position. The induced-fit of MK2 with compound 14e most proba-
bly favors the observed high selectivity (IC50 >10
itor against 29 different kinases in our in house kinase panel
including CDK2, JNK1, JNK2 and p38 as well as its low toxicity
in THP-1 cell proliferation assay (IC50 >20 M).
In addition, compound 14e also inhibited LPS-induced TNF
lease in mice.15 Although good inhibition (68%) was observed at
100 mg/kg po dosage (blood levels of 17.4 M), no inhibition was
observed at a lower 30 mg/kg po dose. This was not surprising as
the blood levels at the lower dose (1.7 M) did not reach levels ex-
lM) of this inhib-
a
l
a
re-
l
l
pected to exert an effect as demonstrated using human PBMCs.
One possible explanation for the lack of linearity in the dose/blood
levels could be due to the low solubility of this compound (<2 mg/L
at pH 1, 4 and 6.8). In an additional study, a concentration depen-
dant effect was observed when TNFa levels were measured at dif-
ferent time points after compound dosing. Thus, dosing the
6. (a) Schneider, G.; Neidhart, W.; Giller, T.; Schmid, G. Angew. Chem., Int. Ed. 1999,
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H. Drug Discovery Today 2007, 12, 149.
7. (a) Silvestri, R.; Cascio, M. G.; La Regina, G.; Piscitelli, F.; Lavecchia, A.; Brizzi, A.;
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Res., Synop. 1997, 11, 420.
compound 1 h before LPS challenge yields in 66% inhibition at
14.1
6.8
l
M
blood exposure; 4 h before LPS challenge (23% at
lM) and 6 h (67% at 25.8 M). This study also indicates a sec-
l
8. Iida, T.; Satoh, H.; Maeda, K.; Yamamoto, Y.; Asakawa, K.; Sawada, N.; Wada, T.;
Kadowaki, C.; Itoh, T.; Mase, T.; Weissman, S. A.; Tschaen, D.; Krska, S.; Volante,
R. P. J. Org. Chem. 2005, 70, 9222.
ond absorption peak at 4–6 h, which may be caused by the low sol-
ubility of this compound. Therefore, we are currently further
searching for more potent and more soluble analogs of 14e.
In a summary, we have discovered and profiled novel 3-amino-
pyrazole MK2-inhibitors. This new class was revealed by scaffold
hopping strategy and could be developed to obtain a selective
9.
a
c
-lactame-bromide: Clayton, J.; Ma, F.; Van Wagenen, B.; Ukkiramapandian,
R.; Egle, I.; Empfield, J.; Isaac, M.; Slassi, A.; Steelman, G.; Urbanek, R.; Walsh, S.
WO 2006/020879.; d-lactame-bromide: Coppola, G. M.; Damon, R. E.;
Kukkola, P. J.; Stanton, J. L. WO 2004/065351.; -lactame-triflates were
obtained by triflation (11c: Tf2O, Et3N, DMAP (cat.), CH2Cl2, À78 °C to 23 °C,
b
x

J. Velcicky et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1293–1297
1297
18 h, 67%; 11d: Tf2O, pyridine, 23 °C, 18 h, 35%) of the literature known
phenols: (c) Shtacher, G.; Erez, M.; Cohen, S. J. Med. Chem. 1973, 16, 516.
10. MK2 was pre-activated in kinase buffer (25 mM Tris–HCl, pH 7.5, 25 mM
incubated for 10 min at 37 °C. Plates were briefly centrifuged (720 g for 5 min at
4 °C) prior to careful aspiration of media on ice. Cells were permeabilised with
the addition of 1 ml ice-cold 90% (v/v) methanol, centrifuged (720g for 5 min at
room temperature) following the addition of wash buffer (0.5 ml) (PBS
containing 1% v/v FCS). Cells were washed twice (1.5 ml) with careful
aspiration of media and repeated centrifugation steps. The primary antibody
b-glycerophosphate, 0.1 mM Na3VO4, 25 mM MgCl2, 20
M ATP, 150 g/ml human MK2, 30 g/ml active human p38
22 °C. For MK2 inhibition, reactions contained compound (10
lM DTT) containing
5
l
l
l
a for 30 min at
l; 0.5% DMSO
l
final) or vehicle control, 250 nM hsp27 peptide biotinyl-AYSRALSRQLSSGVSEIR-
COOH as substrate (10 l) and activated MK2 mix (10 l) containing ATP (5
final). Following incubation at 22 °C for 45 min, reactions were terminated with
125 M EDTA (10 l). Samples (10 l) were transferred to black 384-well plates
for detection of p-hsp27 by time-resolved fluorescence resonance energy
transfer using an antibody mix (10 l) containing a rabbit anti-phospho-hsp27
anti-phospho (Ser78) hsp27 diluted to 1:125 in wash buffer (25
ll/well) was
l
l
lM
incubated on cells for 60 min at room temperature. Cells were washed, prior to
incubation with secondary goat anti-rabbit IgG ALEXA (fluor) 647-conjugated
l
l
l
antibody diluted 1:5000 in wash buffer (50
temperature in the dark. Cells were washed as described above, prior to careful
resuspension in wash buffer (50 l) for FACS (fluorescence activated cell sorting)
ll/well) for 60 min at room
l
l
(Ser82) antibody (2.5 M, Upstate), and anti-rabbit europium-labeled secondary
antibody LANCE Eu-W1024 (3.6 nM; Perkin Elmer) as fluorescence donor along
with streptavidin SureLight-APC (6.25 nM; Perkin Elmer) as acceptor. Following
incubation at 22 °C for 90 min, the FRET ratio 665/620 nm was determined.
11. Human peripheral blood mononuclear cells (hPBMCs) were prepared from
peripheral blood of healthy volunteers using Ficoll-Plaque Plus (Amersham)
density separation. Cells were seeded at a 1 Â 105 cells/well in 96-well plates
in RPMI 1640 medium (Invitrogen) containing 10% (v/v) fetal calf serum. After
pre-incubation with serial dilutions of test compound (0.25% v/v DMSO final)
analysis. Cells were transferred to ‘V’ bottom 96-well plates and analysed using a
FACSCaliburTM cytometer (Becton Dickinson) equipped with red-diode laser
(excitation 635 nm). Gating of cells according to forward and side scatter, mean
fluorescent intensity (MFI) was calculated at 653–669 nm (emission).
13. (a) Wermuth, C. G.. In The Practice of Medicinal Chemistry; Academic Press:
London, 2003; Vol. 2; see also: (b) Lima, L. M.; Barreiro, E. J. Curr. Med. Chem.
2005, 12, 23; (c) Chen, X.; Wang, W. Ann. Rep. Med. Chem. 2003, 38, 333.
14. X-ray crystal structure was obtained with 2.55 Å resolution. The X-ray
coordinates are deposited with RCSB Protein Data Bank, deposition code is
3KGA; The protein used in this study is a segment of MK2 containing residues
for 30 min at 37 °C, cells were stimulated with the addition of IFN
and lipopolysaccharide (LPS) (5 g/ml) per well and incubated for 3 h at 37 °C.
Following a brief centrifugation, supernatant (10 l) sample from each well
was quantified against TNF calibration curve using HTRF TNF kit (CisBio).
12. THP-1 cells were stimulated with anisomycin (25 l) diluted in low-serum RPMI
media (150 ng/ml final) for 15 min at 37 °C. Following stimulation, cells were
fixed with 10% (w/v) paraformaldehyde (34 l; 1.8% v/v final), briefly mixed and
c (10 ng/ml)
l
47–364D(216–237)G.
l
15. The MK2 compound was administered po to OF1 mice (female, 8 weeks old),
followed by LPS injection (20 mg/kg) 1 h later. 1 h post LPS injection the
experiment was terminated and blood withdrawn. Compound blood levels
a
a
l
were determined by LC–MS/MS and plasma levels of mouse TNF
a determined
l
by ELISA.