Pyrrolo-pyrimidones: A novel class of MK2 inhibitors with potent cellular activity

Article abstract of DOI:10.1016/j.bmcl.2008.10.039

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimul

Full text of DOI:10.1016/j.bmcl.2008.10.039

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6142–6146
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolo-pyrimidones: A novel class of MK2 inhibitors with potent
cellular activity
*
Achim Schlapbach , Roland Feifel, Stuart Hawtin, Richard Heng, Guido Koch, Henrik Moebitz, Laszlo Revesz,
Clemens Scheufler, Juraj Velcicky, Rudolf Waelchli, Christine Huppertz
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:
Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2
inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show
potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to
inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel
of 28 kinases.
Received 11 September 2008
Revised 1 October 2008
Accepted 2 October 2008
Available online 11 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
MK2
MAPKAPK-2
TNFa inhibition
Inflammation
Rheumatoid arthritis
The first anti-TNF
a
(tumor necrosis factor alpha) biologicals
In a first effort to optimize this series we evaluated position R1
utilizing aryl, heteroaryl and styrene substituents. For substituent
R2 small alkyl, benzyl and amino groups were explored (Table 1).
Whereas aryl substituents in position R1 yielded compounds which
inhibit MK29 in the micromolar range, potency improved signifi-
cantly when the more flexible phenylethenyl (styryl) substituents
were introduced. In particular, fluorostyryl derivative 9 potently
were introduced into clinical practice in the late 1990s. Since then
they became very successful in the treatment of several autoim-
mune diseases like rheumatoid arthritis (RA), Crohn’s disease or
psoriasis.1 Anti-TNF therapeutics meanwhile became the ‘gold-
standard’ in RA therapy, which fostered the search for new anti-
cytokine targets which are amenable for modulation using low-
molecular weight compounds. Considerable efforts have focused
on the discovery and development of p38 inhibitors,2 yet, the first
compound of this class has still not reached the market. While p38
is still an attractive target, the search for new, potentially more
selective components in the p38 signaling cascade is ongoing.3
Such a target could be mitogen-activated protein kinase activated
protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase
downstream of p38 which has been shown to play a critical role
inhibited MK2 with an IC50 value of 0.20
displayed cellular activity, that is, inhibition of TNF
LPS-stimulated hPBMCs10 in the same activity range.
l
M and more importantly
a
release from
Variation of R2 did not yield any major improvements. Small
substituents like an amino or methyl group are tolerated. Larger al-
kyl groups (CF3, n-butyl) reduce potency, but interestingly further
increase in size as in the case of the benzyl-derivative 14, gener-
ated a more potent inhibitor of MK2, albeit without activity in
our cellular assay.
in TNF
a
signaling, both in vitro and in vivo.4 We now disclose
our efforts to identify and optimize low-molecular weight
inhibitors of MK2, their structure–activity relationships and cellu-
lar profile.
To further explore the styrene motif and to improve physico-
chemical properties, for example, solubility, a series of substituted
styrene derivatives were synthesized and evaluated (Table 2). The
Recently, several reports of MK2 inhibitors from a variety of
structural classes were published, amongst them aminocyanopyri-
dines5, tetrahydro-carbolines6, indole derivatives,7 and pyrrolo-
pyridones.8 In this communication we will present our results on
a related compound class, namely pyrrolo-pyrimidones, exempli-
fied by the general structure 1 (Fig. 1)
O
NH
N
R2
N
N
R1
H
1
* Corresponding author. Tel.: +41 61 324 4308; fax: +41 61 324 8847.
E-mail address: achim.schlapbach@novartis.com (A. Schlapbach).
Figure 1. Pyrrolo-pyrimidone core structure.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.039

A. Schlapbach et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6142–6146
6143
Table 1
In vitro activity of pyrrolo-pyrimidones
Compound
R1
R2
MK2a IC50
[lM]
hPBMCb IC50
[lM]
2
3
4
5
6
7
8
9
10
11
12
13
14
4-Fluorophenyl
3-Fluorophenyl
H
H
H
H
H
H
H
H
1.30
1.23
0.55
5.20
1.45
0.19
0.17
0.20
0.29
1.70
0.35
1.26
0.34
n.d.
n.d.
>10
n.d.
n.d.
10
2.30
0.35
1.50
n.d.
1.10
n.d.
>10
4-Methoxyphenyl
3-Methoxyphenyl
4-Morpholino-methylphenyl
4-Cyclopentyl-benzamido
Phenylethenyl
p-Fluorostyryl
Phenylethenyl
Phenylethenyl
Phenylethenyl
NH2
CF3
CH3
n-C4H9
Benzyl
Phenylethenyl
Phenylethenyl
IC50 values are reported as the mean of P2 experiments.
a
MK2 enzyme assay is performed as described.9
b
Inhibition of LPS stimulated release of TNFa
from hPBMCs.10
Table 2
Effect of styrene substitution on in vitro activity of pyrrolo-pyrimidones
O
N
NH
N
N
H
R
Compound
R
MK2a IC50
0.20
[lM]
hPBMCb IC50
0.35
[lM]
Solubilityc [g/l]
0.003
9
F-
O
N
15
16
0.042
0.051
0.15
0.11
0.009
0.041
O
O
N
17
18
0.082
0.150
0.04
0.38
0.41
n.d.
N
O
N
IC50 values are reported as the mean of P2 experiments.
a
MK2 enzyme assay is performed as described.9
Inhibition of LPS stimulated release of TNF
Thermodynamic solubility at pH 6.8.
a
from hPBMCs.10
b
c
addition of substituents to the styrene para position did not only
influence solubility but also improved potency up to ca. 5-fold.
Compounds 15–17 now display high MK2 activity (40–80 nM)
Takai-olefination12/metalation sequence (Scheme 2). Thus, amina-
tion of ethynylbenzyl alcohol with morpholine under Zaragoza
conditions13 yielded 22, which in turn could be hydroborated
smoothly with pinacolborane using Wilkinson’s catalyst to give
the boronate 23. Compounds 25 and 27 were synthesized in the
same manner. Alternatively p-hydroxybenzaldehyde was treated
with iodoform and chromium chloride to give vinyl iodide 28.
The desired boronate 30 was then obtained after alkylation of
the phenol, iodine lithium exchange and transmetalation with
2-isopropoxy-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane.
and show strong inhibition of TNFa release from LPS-stimulated
hPBMCs.
The synthesis of pyrrolo-pyrimidones started with bromo-
ketone 19. Cyclo-condensation with ethyl 3,3-diamino-acrylate11
in methanol yielded the desired amino-pyrrole 20, which when
treated with formamidine under forcing conditions provided the
pyrrolo-pyrimidone core structure 21. The pyridyl-2-substituent
was finally introduced by Suzuki-coupling with the corresponding
boronates (Scheme 1).
To further explore features which may be necessary for potent
MK2 inhibition, derivatives lacking either the pyrrole or the pyrim-
idine N-H were synthesized. N-Methyl derivative 31 was obtained
simply by alkylation of 16 with methyliodide. The synthesis of 34
Boronates which were not commercially available were synthe-
sized either by hydroboration of the corresponding alkynes, or by a

6144
A. Schlapbach et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6142–6146
O
O
a
O
N
N
Br
N
H
NH2
Cl
Cl
19
20
O
N
O
N
c
b
NH
NH
N
N
N
N
H
H
Cl
R
2 - 18
21
Scheme 1. Synthesis of pyrrolo-pyrimidone derivatives. Reagents and conditions: (a) ethyl 3,3-diamino-acrylate, NaHCO3, methanol, rt 16 h; (b) formamidine, 1-butanol,
reflux, 48 h; (c) boronate, Pd(PPh3)2Cl2, 2 N NaHCO3/1-propanol, reflux, 16 h (or microwave, 160 °C, 15 min), 10–75% (over three steps).
O
B
a)
b)
O
O
O
HO
N
N
22
23
O
B
c)
b)
O
O
O
HO
N
N
O
O
O
24
25
27
O
B
d)
b)
O
O
N
N
26
I
I
O
f)
e)
N
O
HO
HO
28
29
O
B
O
g)
N
O
30
Scheme 2. Synthesis of styryl-boronates. Reagents and conditions: (a) Morpholine, cyanomethyl-trimethyl-phosphonium iodide, ethyl-diisopropyl-amine, propionitrile,
reflux, 16 h, 95%; (b) 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane, Rh(PPh3)3Cl, dichloromethane, rt, 24 h, 65%; (c) morpholine, EDC, dichloromethane, rt, 24 h, 73%; (d)
diethylamine, NaCNBH3, methanol/acetic acid, 93:7, rt, 16 h, 62%; (e) CHI3, CrCl2, 0 °C, 1 h, 73%; (f) dimethylaminoethyl chloride, K2CO3, acetone, 50 °C, 16 h, 68%; (g) 2-
isopropoxy-4,4,5,5-tetramethyl-[1,3,2]dioxaboro lane, t-BuLi, THF, À78 °C, 1 h; then rt, 2 h, 60%.
proceeded via the amino-imidazole 32, which was condensed with
ethyl acetoacetate to provide imidazolo-pyrimidone 33. Finally,
Suzuki-coupling with boronate 23 led to compound 34 (Scheme
3). Compounds 31 and 34 were both significantly less potent
Styrene derivatives 16 and 17 which were found to be both
potent MK2 inhibitors and active in the cellular assay were further
evaluated for their selectivity towards other kinases. Testing in a
panel of 28 kinases revealed a modest degree of selectivity (Table
3). Kinases which showed strongest inhibition were the closely re-
lated kinase MK5 in addition to Aurora A, GSK3b, Jak3, JNK2, Kdr,
MK2 inhibitors, with IC50 values of ca. 1.3
lM. This demonstrates
the importance of both H-donors for potent MK2 inhibition.

A. Schlapbach et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6142–6146
6145
O
O
N
NH
NH
N
N
N
N
N
a)
H
O
N
O
N
16
31
O
O
d)
b,c)
N
N
N
N
N
N
Br
N
N
NH2
H
Cl
Cl
H
Cl
19
32
33
O
N
N
e)
N
N
H
34
O
N
Scheme 3. Synthesis of 31 and 34. Reagents and conditions: (a) NaH, MeI, DMF, rt, 16 h, 15%; (b) acetyl guanidine, acetonitrile reflux, 16 h, 50%; (c) cat H2SO4, MeOH/water,
reflux, 16 h, 45%; (d) ethyl acetoacetate, ammonium acetate, neat 140 °C; (e) 23, Pd(PPh3)2Cl2, 2 N NaHCO3/1-propanol, microwave, 160 °C, 15 min, 17% over two steps.
and c-met. Kinases which would be predicted to interfere with our
cellular readout (TNFa release) were not (p38) or only moderately
16 [μM]
17 [μM]
5
1
0.5
5
1
0.5
(JNK) inhibited. Furthermore, both compounds showed no major
effect on cytotoxicity as assessed on the proliferation of THP-1 cells
and mouse bone marrow derived cells.
LPS
-
-
+
+
+
+
+
+
+
+
p-hsp27
It was important to demonstrate that the inhibition of TNFa re-
lease from PBMCs seen with 16 and 17 directly results from inhibi-
total hsp27
Figure 2. Inhibition of hsp27 phosphorylation in LPS-stimulated THP-1 cells.15
Table 3
Kinase selectivity of compounds 16 and 17
Kinase
Compound 16 IC50
[l
M]
Compound 17 IC50 [lM]
tion of MK2. Thus, we determined if the phosphorylation of hsp27,
a direct downstream target of MK214 was also inhibited. Phosphor-
ylated hsp27 is hardly detectable under basal conditions, and
strongly induced upon stimulation with LPS (Fig. 2). Compounds
16 and 17 inhibited the LPS-stimulated phosphorylation of hsp27
dose dependently, while total hsp27 was not affected.15 This sug-
gests that perturbation of cytokine release is indeed mediated via
inhibition of MK2.16
In summary, we have discovered a new class of MK2 inhibitors:
Pyrrolo-pyrimidones. Essential features for potent inhibition of
MK2 within this compound class are a styrene substituent, opti-
mally substituted with a polar head group as well as the pyrrole-
and pyrimidone NH donors. Compounds 16 and 17 represent
derivatives, which potently inhibit MK2 with modest selectivity
in a panel of 28 kinases and exert cellular activity by inhibition
of both TNF release from LPS-stimulated PBMCs and hsp27
phosphorylation.
Alk
Aurora A
Btk
>10
0.093
>10
>10
1.1
4.1
0.28
>10
8.2
2.6
5.6
1.4
6.8
0.53
3.3
9.5
6.4
7.8
3.6
1.0
0.96
0.24
0.19
4.3
0.028
3.4
CDK2
ERK2
EphA4
EphB4
FGFR-4
GSK3b
HER1
HER2
IGF1R
Ins1R
Jak2
>10
1.7
>10
0.30
>10
>10
>10
>10
1.3
0.31
4.0
0.95
0.10
6.0
0.024
2.2
2.0
6.7
>10
6.2
Jak3
JNK1
JNK2
Kdr
Lck
MK5
PDK1
PDGFRa
PKA
PKB
Syk
3.3
Acknowledgments
>10
>10
2.5
6.1
0.14
>10
We are very grateful to our colleagues from the NIBR kinase
platform for providing in vitro kinase and selectivity data as
well as the NIBR Prep Labs for the syntheses of valuable
intermediates.
c-Kit
c-Met
p38
5.2
0.085
>10

6146
A. Schlapbach et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6142–6146
final) or vehicle control, 250 nM hsp27 peptide biotinyl-AYSRALSRQLSSGVSEI-
R-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 (Ser82
antibody (2.5 nM, 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. Individual IC50 values of
compounds were determined by nonlinear regression after fitting of curves to
the experimental data using Excel XL fit 4.0 (Microsoft).
References and notes
l
l
lM
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Moss, N.; Pargellis, C.; Patel, U. R.; Patnaude, L.; Peet, G. W.; Skow, D.; Snow, R.
J.; Ward, Y.; Werneburg, B.; White, A. Bioorg. Med. Chem. Lett. 2007, 17, 4664;
Trujillo, J. I.; Meyers, M. J.; Anderson, D. R.; Hegde, S.; Mahoney, M. W.; Vernier,
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l
l
l
l
)
10. Human peripheral blood mononuclear cells (hPBMCs) were prepared from the
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)
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) samples from each well
are measured against a TNF calibration curve using a HTRF TNF kit (CisBio).
c (10 ng/ml)
l
l
a
a
11. Meyer, H.; Bossert, F.; Horstmann, H. Liebigs Ann. Chem. 1977, 3, 1895.
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14. Clifton, A. D.; Young, P. R.; Cohen, P. FEBS Lett. 1996, 392, 209.
15. THP-1 cells were pre-incubated for 1 h with or without compound at the
indicated concentrations and then stimulated with LPS or vehicle (basal
condition) for 15 min. Total lysates were prepared and analyzed by Western
blot, using an anti-phospho-hsp27 antibody (Upstate #05-645, dilution 1:
5000) and an anti-hsp27 antibody (Upstate #06-478, dilution 1: 1000). Bands
were visualized with enhanced chemoluminescence (Amersham Pharmacia
Biotech).
8. Anderson, D. R.; Meyers, M. J.; Vernier, W. F.; Mahoney, M. W.; Kurumbail, R.
G.; Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz, D. B.; Mourey, R. J. J. Med. Chem.
2007, 50, 2647.
9. MK2 was pre-activated in kinase buffer (25 mM Tris–HCl, pH 7.5, 25 mM 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
16. Some contribution of kinases upstream of p38 as well as involvement of JNK2
in the case of compound 17 cannot be fully excluded.
5
l
l
l
a
for 30 min at
l; 0.5% DMSO
l