Novel ATP competitive MK2 inhibitors with potent biochemical and cell-based activity throughout the series

Article abstract of DOI:10.1016/j.bmcl.2011.10.071

Optimization of our previously described pyrrolopiperidone series led to the identification of a new benzamide sub-series, which exhibits consistently high potency in biochemical and cell-based assays throughout the series. Strong inhibition of LPS-induce

Full text of DOI:10.1016/j.bmcl.2011.10.071

Bioorganic & Medicinal Chemistry Letters 22 (2012) 613–618
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel ATP competitive MK2 inhibitors with potent biochemical
and cell-based activity throughout the series
Arthur Oubrie, Allard Kaptein, Edwin de Zwart, Niels Hoogenboom, Rianne Goorden, Bas van de Kar,
Maaike van Hoek, Vera de Kimpe, Ruud van der Heijden, Judith Borsboom, Bert Kazemier, Jeroen de Roos,
⇑
Michiel Scheffers, Jos Lommerse, Carsten Schultz-Fademrecht, Tjeerd Barf
Merck Research Laboratories, MSD, PO Box 20, 5340 BH Oss, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of our previously described pyrrolopiperidone series led to the identification of a new benz-
amide sub-series, which exhibits consistently high potency in biochemical and cell-based assays
Received 25 August 2011
Revised 20 October 2011
Accepted 21 October 2011
Available online 3 November 2011
throughout the series. Strong inhibition of LPS-induced production of the cytokine TNF
a is coupled to
the regulation of HSP27 phosphorylation, indicating that the observed cellular effects result from the
inhibition of MK2. X-ray crystallographic and computational analyses provide a rationale for the high
potency of the series.
Keywords:
MAPKAPK2
MK2
Ó 2011 Elsevier Ltd. All rights reserved.
Anti-TNF
HSP27
a
Rheumatoid arthritis
Modulation of the p38/MK2 pathway is an attractive approach to
interfere with the production of pro-inflammatory cytokines and
thus towards control of inflammatory disease conditions like rheu-
matoid arthritis and psoriasis. Several p38 inhibitors have been
tested in clinical development, but so far overall progress has been
hampered by potential adverse effects such as hepatotoxicity. MK2
is downstream of p38 in this pathway and appears to be the central
switch in the production of pro-inflammatory components. MK2
knockout mice show strong reduction in disease incidence and dis-
ease severity scores in the arthritic CIA model, and this reduction
was also observed for the MK2 heterozygote mice.1 With the ab-
sence of feedback control via TAK1 as observed for p38 inhibition,
selective inhibition of MK2 may not be compromised by the safety
limitations observed with p38 inhibitors. Consequently, MK2 inhi-
bition could present a valid alternative in this pathway.
optimized by the introduction of a 3-piperidyl moiety in the ribose
pocket so as to generate 2, an orally available ATP-competitive
MK2 inhibitor (Fig. 1).3
In view of the high potency and selectivity reported for several
clinical p38 inhibitors, we aimed to further improve these properties
of 2. In biochemical and cell-based assays, compound 2 has inhibi-
tion values of EC50-IMAP = 7.4 nM, EC50-TNFa = 850 nM, and EC50-
PBMC = 450 nM, and so improvement of cell-based potency was
one main objective.
A new scaffold modification was made by the introduction of
benzamide groups directed at the hinge and front pocket regions
of MK2. Choice of position and direction of the benzamides
was guided by computational chemistry and inspired on the
binding mode of pyrazinoindolone MK2 inhibitors reported
We have recently reported on a structure-based lead identifica-
tion project culminating in the identification of derivative 1
(Fig. 1).2 This compound showed potency in biochemical and
cell-based assays, and was also efficacious in the acute in vivo
O
O
NH
NH
NH
N
N
N
H
N
H
N
N
model of LPS-induced TNFa production in rats after s.c. administra-
tion. The PK properties of compound
1 were subsequently
O
O
N
H
O
O
1
2
⇑
Corresponding author.
E-mail address: tjeerd.barf@covalution.com (T. Barf).
Figure 1. Pyrrolopyridinone compounds 1 and 2.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.071

614
A. Oubrie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 613–618
previously.4 Construction of the pyrrololactam scaffold was ef-
fected essentially as described previously, via the Paal–Knorr con-
densation reaction of bromoketones A1 & A2 and piperidinediones
B1–B3 (Scheme 1).5 A range of substituents was introduced via a
Buchwald–Hartwig coupling reaction using several substituted
benzamides (Scheme 1). In the last synthesis step, acidic removal
of protective TMB and Boc groups by TFA gave the final com-
pounds. When desired, the right hand 4-piperidyl group was N-
methylated via reductive amination with formaldehyde.
Inspired by our previous success with improving membrane
permeability and oral availability through variation of the spiro-
rings,3 we expanded the available spiro-ring repertoire to include
pyrrolidines in addition to the already available non-substituted
and N-methylated 4-piperidyl, and non-substituted 3-piperidyl
derivatives.
hydrogen atmosphere. Acylation of the methylamine side chain
with ethyl (chloroformyl)acetate, followed by a one-pot N-benzy-
lation and protection with Boc gave G. A Dieckmann condensation
and decarboxylation yielded pyrrolidine-piperidinedione B3.
Introduction of the amide linker resulted in highly potent com-
pounds exhibiting low nanomolar or even high picomolar inhibi-
tion values, as exemplified by 1- and 2-naphthyl analogues 4b
and 5b (Table 1). The data in Table 1 show that the front pocket
of MK2 can accommodate large lipophilic moieties, consistent with
previous SAR and the relatively planar and open front pocket of
MK2.2–4 Comparison of pyrimidine and pyridine analogues reveals
that the latter are 3–15-fold more potent, compare for example,
the pairs 3a and 3b (3.1 vs 1.0 nM), 5a and 5b (14 vs 0.96 nM)
and 6a and 6b (15 vs 1.6 nM). The potency differences between
the pyridine and pyrimidine analogues in the IMAP assay are trans-
The five-membered pyrrolidine ring was constructed via a
1,3-dipole addition on cyanoacrylate D as outlined in Scheme 2.
Reduction of the nitrile group was effected with Raney-Nickel in
lated into similar differences in LPS-induced TNF
THP1 cells. Furthermore, the inhibition levels of TNF
are coupled to values for pHSP27, the target engagement bio-
a
production by
production
a
marker for MK2 activity. As this chemotype does not inhibit p38,
O
O
this observation further supports the hypothesis that the TNF
inhibition observed can be attributed to MK2 inhibition.
a
R
R
Br
N
N
a
N
N
The potency difference between the pyridine and pyrimidine
derivatives can be explained by a strong, intrinsic conformational
+
N
H
X
X
O
O
Cl
Cl
preference for the
s
= 0o rotamer for the pyridine derivatives
Y
Y
(Fig. 2), which allows the N–H to interact with the hinge region
of MK2 (Fig. 3). Gas phase ab initio geometry optimizations at 6-
31G(d) basis set level (Gaussian 03)6 show that the conformational
A1: X = CH
A2: X = N
B1: Y = -NR'-CH2- (R' = Boc)
B2: Y = -CH2-NR'- (R' = Boc)
B3: Y = NR' (R' = Boc)
C1 - C3
energy of the
s
= 0o rotamer is 9.3 kcal/mol more favorable than
= 180o rotamer (Fig. 2). The strong preference for the
s
= 0o
O
the
s
R
rotamer is caused by electrostatically attractive N–Hꢀ ꢀ ꢀN(pyridine)
and C@Oꢀ ꢀ ꢀH–C(pyridine) interactions, whereas the N–Hꢀ ꢀ ꢀH–
C(pyridine) and C@Oꢀ ꢀ ꢀN(pyridine) interactions are repulsive for
N
b and c
N
N
H
O
X
N
H
the
s
= 180o rotamer. This preference is confirmed by the 113
Ar
Y
benzamide substructures present in crystal structures retrieved
from the CSD (version 5.31).7 All rotamers have dihedral angles
of less than 45o (Fig. 2). Stabilization of the conformation with
2 - 3 (R' = H)
4 ( R' = Me)
d
s
= 0o is much reduced in the pyridimine-containing compounds
due to the absence of the relevant CH(pyridine). There is no confor-
mational preference for the pyrimidine-containing compounds and
binding of such compounds to MK2 thus occurs at the expense of
loss of entropy. This is energetically unfavorable and results in
compromised potencies compared to pyridine-containing
compounds.
The binding mode of benzamide compounds to MK2 was deter-
mined via soaking experiments using MK3 crystals. This system
was described previously as a suitable, more accessible surrogate
for MK2.2,8 The crystal structure of an MK3:3-trifluorobenzamide
13d complex was solved at 2.25 Å resolution (Fig. 3). Consistent
with the computational analysis, the pyridine-containing 13d has
O
N
O
O
TMB
TMB
N
N
N
NH
O
O
O
N
Boc
Boc
Boc
B1
B2
B3
Scheme 1. Reagents and conditions: (a) NH4OAc, EtOH (35%); (b) arylbenzamide,
4,5-bis(diphenylphosphono)-9,9-dimethylxanthene, Pd(OAc)2, tBuONa, DMF, MW;
(c) TFA, rt (4–48% in two steps); (d) formaldehyde, NaCNBH3, ACN, pH 5, 15 h, rt.
a low dihedral angle
s
(of ꢁ4°). The benzamide group forms hydro-
O
O
NH2
gen bonds with the main chain carbonyl of Met141/121 (MK2/MK3
numbering) and with a structural water molecule that also inter-
acts with the pyrrole of the central pyrrolopyridinone core. These
interactions stabilize the binding of 13d in its low-energy confor-
mation, thereby contributing to binding energy and high potency
of this series. The pyridine nitrogen of 13d is hydrogen-bonded
to the main chain amide of Met141/121. The cyclic amide of 13d
makes hydrogen bonds to Lys93/73 and Asp207/187 and interacts
with His108/88 and Glu104/84 in the back pocket through water-
mediated hydrogen bonds. Consistent with vibrational circular
dichroism data for 3-piperidyl-aryl compounds published previ-
ously, the spiro-piperidyl moiety has been built in the S-configura-
tion.3 The basic amine of the 3-piperidyl does not make direct ion
pair or hydrogen bond interactions with either Glu145/125,
Glu190/170, or Asp207/187, but longer range electrostatic interac-
tions with these side chains are likely to contribute to binding
N
O
N
EtO
MeO
a
b
EtO
N
N
Bn
Bn
D
E
F
O
O
O
O
N
H
OEt
NH
c and d
e and f
MeO
O
N
N
Boc
Boc
G
B3
Scheme 2. Reagents and conditions: (a) N-Benzyl-1-methoxy-N-((trimethyl-
silyl)methyl)methanamine, TFA, DCM 0 °C–rt (94%); (b) 50% Ra-Ni, H2 (g), MeOH,
2 h, rt (48%); (c) ClCOCH2COOMe, Et3N, DCM, rt (94%); (d) Boc2O, Pd/C 10%, H2 (g),
EtOAc, 21 h, rt (e) NaOMe, MeOH, 60 °C (91%); (f) ACN/H2O (1/1) 80 °C (46%).

A. Oubrie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 613–618
615
Table 1
Inhibitory MK2 activity of compounds 1, 3–6
O
NH
N
N
H
A
O
N
H
Ar
N
H
-
3
6
a
b
b
Compound
Ar
A
MK2 IMAP EC50 (nM)
TNF-THP1 EC50
(
lM)
pHSP27 EC50 (lM)
1
—
—
N
CH
N
CH
N
CH
N
CH
4.3
3.1
1.0
5.1
0.72
14
0.96
15
1.6
0.91
3.4
0.23
3.4
0.32
>10
0.49
7.9
0.62
NTc
0.35
NT
0.28
NT
0.52
NT
3.7
3a
3b
4a
4b
5a
5b
6a
6b
3,4-di-MeC6H3
3,4-di-MeC6H3
Naphth-2-yl
Naphth-2-yl
Naphth-1-yl
Naphth-1-yl
4-F-C6H4
4-F-C6H4
1.3
a
Values are means of two independent experiments for each dilution per experiment. For further details see Ref. 2.
THP1 cells are pre-incubated for 30 min (TNFa) or 60 min (pHSP27) with MK2 inhibitors (duplicates for each dilution) prior to stimulation with LPS. For further details see
b
Ref. 2.
c
NT means Not Tested.
N occurences
Rel. energy (kcal/mol)
50
40
8.0
6.0
N
N
H
H
N
O
O
N
H
30
20
10
H
4.0
2.0
0.0
= 0o
= 180o
0
20 40 60 80 100 120 140 160 180
(degrees)
Figure 2. Distribution of dihedral angles
s of pyridylbenzamide substructures
retrieved from the CSD (in blue) as well as relative conformational energies of
geometry optimized pyridylbenzamides calculated at 6-31(d)G basis set level (in
red).
Figure 3. Crystal structure of 13d in human MK3 at a resolution of 2.25 Å (PDB
code: 3SHE). The numbering is according to MK3. The glycine-rich loop has been
deleted for clarity.
energy. Previous work demonstrated that the presence of a basic
amine in the spiro-ring improved biochemical potency
significantly.2
compared to their non-substituted analogues. The N-methylated
Having established and understood on a molecular level the
pyridine ring was favored in combination with the benzamide
groups in the front pocket, the left and right hand side substituents
were explored simultaneously. Right-hand side variations included
non-substituted and N-methylated 4-piperidyl, non-substituted 3-
piperidyl, and pyrrolidyl derivatives. Left-hand side variations
were mostly hydrophobic and aromatic, consistent with SAR and
aimed at maintaining favorable physicochemical parameters.
Although only a few data points are given for the N-methylated
4-piperidyl sub-series, it appears to exhibit somewhat lower
potencies than its non-substituted counterpart: Comparison of di-
rect analogs in Table 2 shows that the substituted compounds are
consistently less potent in the IMAP and THP1-based assays, even
though differences are often small; relevant analog pairs include
3b and 3c, 4b and 4c, 7b and 7c, and 8b and 8c. In contrast, the
substituted compounds are 3–5-fold more potent in PBMC’s
compounds 4c, 7c and 8c exhibit EC50-PBMC values in the range
of 0.03–0.07 lM, and thereby belong to the eight most potent com-
pounds out of 31 tested in this assay. The molecular basis for this
unexpectedly high potency in PBMC’s is not well understood. N-
methylation results in relatively high clogP (3.2 for both 4c and
8c) and low pKa values (8.3 and 8.4 for 4c and 8c, respectively)
compared to the non-substituted compounds 4b and 8b—clogP
values of 2.7 and 2.8, and pKa values of 9.2 and 9.1, respectively.
Even though these parameters in theory would result in relatively
improved membrane permeability, determined PAMPA values are
very low throughout the series. Unlike in THP1 cells, we were
not able to corroborate the role of MK2 in PBMCs with a target
engagement marker, and therefore off-target inhibition in PBMC
cells is another explanation.
For the racemic 3-piperidyl analogs, the biochemical potencies
ranged from 2.5 to 12 nM, and this sub-series is thus somewhat

616
A. Oubrie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 613–618
less potent than the 4-piperidyl series in the IMAP assay. Compar-
ison of direct analogs shows that the 3-piperidyl-containing 3d, 4d,
and 8d, are all less potent in IMAP, TNFa and pHSP27 assays than
their respective 4-piperidyl analogs 3b, 4d and 8d. In contrast, the
3-piperidyl derivatives appear more potent than their 4-piperidyl
equivalents in PBMC’s and they generally show stronger inhibition
in PBMC’s than in THP1 cells. The relatively high potency in PBMC’s
is a shared feature of the 3-piperidyl and N-methyl-4-piperidyl
sub-series: Five of a total of eight compounds with strong activity
in PBMC’s have a 3-piperidyl moiety, the other three have an
N-methylated 4-piperidyl. Furthermore, the 3-piperidyl series is
unique among the benzamide series in that some analogs show
membrane permeability in the PAMPA model, as exemplified by
7d (19 nm/s) and 12d (63 nm/s). The presence of the amide NH
generally undermines permeability in the PAMPA model of the
whole benzamide series. These data indicate that introduction of
fluorines on the phenyl meta-positions is beneficial for this prop-
erty. A number of enantiomerically pure 3-piperidyl analogs were
prepared via chiral separation of a common chloropyrimidine
intermediate.5 The difference in IMAP inhibition values for the
Table 2
Inhibitory MK2 activity of compounds 1–13
O
O
O
NH
NH
NH
NH
NH
N
N
N
N
H
N
H
N
H
O
O
O
N
H
N
H
N
H
Ar
Ar
Ar
N
R'
I
II
III
a
a
b
b
c
Compound Ar
Type R0
MK2 IMAP EC50
(nM)
pHSP27 EC50
M)
TNF-THP1 EC50
M)
TNF-PBMC EC50
M)
clogP PAMPA
pKa
hPPB
(%)
(
l
(
l
(
l
(nm/s)
1
3b
—
3,4-Di-
—
I
—
H
4.3
1.0
0.62
0.23
0.91
0.35
0.26
0.89
1.7
2.5
0
0
MeC6H3
3,4-di-
3c
I
Me 2.3
0.92
0.72
NTd
2.9
NT
MeC6H3
4b
4c
5b
6b
7b
7c
8b
8c
9b
10b
11b
3d
Naphth-2-yl
Naphth-2-yl
Naphth-1-yl
4-F-C6H4
3-F-C6H4
3-F-C6H4
3-OCF3-C6H4
3-OCF3-C6H4
4-CF3-C6H4
Pyridin-2-yl
Pyridin-4-yl
3,4-Di-
I
I
I
I
I
I
I
I
I
I
I
II
H
0.72
0.32
0.73
0.49
3.7
0.68
0.64
0.13
0.83
1.3
0.28
1.1
0.52
1.3
0.78
0.54
0.08
1.0
0.66
0.72
>10
0.46
0.23
0.07
0.65
1.4
0.15
0.03
0.16
0.05
1.6
2.7
3.2
2.7
1.8
1.8
2.2
2.8
3.2
2.6
0.7
0.4
2.9
0
0
0
0
6
0
0
0
0
9.2
8.3
99.7
99.5
Me 2.6
H
H
H
0.96
1.6
1.1
Me 2.5
1.5
Me 2.7
H
9.1
8.4
95.8
97.3
H
H
H
H
1.6
1.4
2.6
8.1
0.55
>10
0.69
0.26
2.6
0.04
0
0
NT
MeC6H3
3d.S1
3d.S2
3,4-di-
MeC6H3
3,4-di-
II
II
H
H
5.5
32
NT
5.3
0.65
8.5
NT
1.2
2.9
2.9
NT
10
MeC6H3
4d
Naphth-2-yl
Naphth-2-yl
Naphth-2-yl
Naphth-1-yl
4-F-C6H4
4-F-C6H4
4-F-C6H4
3-F-C6H4
II
II
II
II
II
II
II
II
H
H
H
H
H
H
H
H
H
H
H
H
7.6
2.5
129
5.1
8.9
2.4
31
6.2
11
9.1
4.2
2.5
1.1
0.53
NT
0.78
0.51
NT
0.22
0.32
0.31
>10
0.56
0.62
1.3
0.07
0.17
NT
0.39
0.07
0.07
1.6
0.03
0.20
0.48
0.13
0.14
3.1
3.1
3.1
3.1
2.1
2.1
2.1
2.1
3.1
2.9
1.1
2.3
NT
NT
0
1
5
11
0
19
0
0
2
4d.S1
4d.S2
5d
8.2
8.2
99.6
98.7
1.1
6d
0.87
0.32
7.4
NT
0.61
1.2
6d.S1
6d.S2
7d
8d
9d
3-OCF3-C6H4 II
4-CF3-C6H4
Pyridin-2-yl
3,5-Di-F-
C6H3
3-CF3-C6H4
3,4-Di-
II
II
II
10d
12d
0.66
0.59
0.48
1.1
63
13de
3e
II
III
H
H
3.3
0.71
0.59
0.24
0.85
0.34
0.14
0.15
2.9
2.3
0
2
MeC6H3
4e
6e
7e
8e
Naphth-2-yl
4-F-C6H4
3-F-C6H4
3-OCF3-C6H4 III
Pyridin-2-yl III
III
III
III
H
H
H
H
H
0.62
1.3
1.9
1.6
4.1
0.39
0.84
0.48
0.40
0.66
1.7
1.1
1.1
0.69
3.9
NT
2.5
1.6
1.6
2.6
0.5
0
0
6
0
8.5
8.4
99.8
98.3
0.20
0.17
0.65
NT
10e
NT
a
See Table 1.
b
hPBMC’s are incubated for 30 min with MK2 inhibitors (duplicates for each dilution) prior to stimulation with LPS. Four hours following LPS induction culture medium is
measurements. For further details see Ref. 3.
Experimentally determined pKa values at pH 7.4.
NT means Not Tested.
withdrawn from the cells for TNF
a
c
d
e
The X-ray structure of this compound bound to MK3 is presented in Figure 3.

A. Oubrie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 613–618
617
two enantiomers was at least 5–50-fold, always in favor of the ste-
reoisomer eluting from the HPLC columns first. The most active
stereo-isomers were 4d.S1 (MK2 EC50 = 2.5 nM; pHSP27 0.53
l
l
M;
M;
PBMC 0.17
PBMC 0.07
lM), and 6d.S1 (MK2 EC50 = 2.4 nM; pHSP27 0.32
lM); both compounds have very good potencies in
THP1 cells and PBMC’s.
Comparison of the pyrrolidine sub-series to the other sub-series
shows that this series has similar overall biochemical and cell-
based activity to the 4-piperidyl series. Across all four benzamide
sub-types, two pyrrolidine-containing compounds show the high-
est biochemical activity, that is, 3e and 4e exhibited high picomo-
lar inhibition values of 0.71 and 0.62 nM. Within the sub-series, the
choice of substituted benzamide did not affect the biochemical po-
tency much and so this sub-series shows the most promise in rela-
tion to biochemical potency. Comparison of cell-based inhibition
values between, for example, 4-piperidyl and pyrrolidyl sub-series
does not show structure-related differences that are as clear as for
the other sub-series. Pyridin-2-yl analogue 10e is an outlier in the
entire benzamide series due to its reduced cellular potency, which
appears coupled to its relatively low clogP of 0.5.
Across all benzamide derivatives, it appears that the biochemi-
cal MK2 potency has little predictive power for cell-based activities
in THP cells and PBMC’s. To analyze this further, the difference, in
Figure 4. Shift in biochemical and THP1 cellular potency (ꢁlogEC50 MK2
log units, between inhibition values in IMAP and THP1-TNF
a as-
IMAP ꢁ ꢁlogEC50 TNF
a) binned by front pocket substitution patterns. Compounds
are colored by front pocket substitutions as indicated. The figure was generated
using Spotfire.
says was calculated for each compound (Table 2), and compounds
were binned according to the substitution pattern on the benzam-
ide group (Fig. 4). The analysis was done with THP1 cells due to the
good overall correlation between the TNF
a and pHSP27 readouts,
these series and it is therefore not possible to judge whether or
not the cellular effect can be attributed to MK2 inhibition. A series
of tetracyclic inhibitors represented the first series with consistent
underpinning the MK2-mediated inhibition in these cells. Fig. 4
illustrates very clearly that the potency loss observed in cellular as-
says is in general much smaller for the 3-piperidyl analogues than
for the other groups. For this analysis, only racemates of pyrroli-
dine and 3-piperidyl analogues were included. For the 3-piperidyl
series, sub-micromolar cell-based EC50 values may be predicted
when the biochemical inhibition is better than 10 nM, whereas
potencies for example, the 4-piperidyl series need to be 3–4-fold
higher for a similar cellular effect. We previously established that
the lower pKa of 3-piperidyl analogues improved the intrinsic
membrane permeability properties.3 Similarly, a lower pKa will
present a smaller population of ionized compounds to cells, lead-
ing to higher cell permeation and higher cellular efficacy.
It is important to note that the biochemical and cell-based
potencies were consistently high across the entire benzamide ser-
ies. Of a total of 36 reported compounds, 32 compounds (89%)
showed single-digit nanomolar inhibition levels in the IMAP assay,
or better. Phosphorylation of HSP27 was inhibited at sub-micro-
molar levels by 25 out of 33 compounds tested (76%). Sub-micro-
submicromolar inhibition of LPS-induced TNF
a production and
phosphorylation of HSP27 in THP1 cells.13 Of the 30 compounds
disclosed, respectively 21 (70%) and 11 (37%) inhibited LPS-in-
duced TNFa production and phosphorylation of HSP27 with sub-
micromolar potencies. Interestingly, these tetracycles combine
pyridinone and basic spiro-amines in one chemotype, which is
very similar to our own compounds. Finally, our own chemical ser-
ies did initially not show strong cell-based inhibition, and inhibi-
tion of TNF
a production was not always coupled to HSP27
phosphorylation.2,3
As discussed above, introduction of an amide linker dramati-
cally improved this situation, resulting in one of the most potent
MK2 inhibitor series reported to date.
In summary, we have identified a new pyrrolopiperidone sub-
series by introduction of substituted benzamides binding to the
front pocket region of MK2. Simultaneous variation of benzamide
substitutions and the spiroheterocyclyl ring in terms of ring size
and position of the basic amine in the ribose pocket yielded inhib-
itors that demonstrated consistently potent MK2-mediated cellu-
lar responses throughout the sub-series.
molar inhibition of TNF
achieved by 26 out of 31 compounds (84%), while eight compounds
(26%) showed very high inhibition levels (0.03–0.1 M) in this as-
say. Furthermore, pHSP27 target engagement and TNF release
a production/release in PBMC’s was
l
a
data from THP1 cells correlated well across the series, implying
that inhibition of cytokine production is an on-target effect medi-
ated by inhibition of the p38-MK2 pathway. As our compounds do
not inhibit p38, the effects should be driven by inhibition of MK2.
From the early pyrrolopyridine series reported a few years ago,9
it has been difficult to identify MK2 inhibitors with potent activity
in cellular assays throughout the chemical series. For example,
squarate and 4-anilino-6-phenyl-quinoline series showed only
Acknowledgments
We thank Mercachem for the synthesis of the pyridine building
block C2, and chiral separation of the enantiomers.
References and notes
1. Hegen, M.; Gaestel, M.; Nickerson-Nutter, C. L.; Lin, L.-L.; Telliez, J.-B. J.
Immunol. 2006, 177, 1913.
micromolar inhibition of LPS-induced production of TNF
a in
THP1 cells.10 Pyrrolopyridine and 3-aminopyrazole series addition-
ally exhibited micromolar inhibition of phosphorylation of HSP27
in THP1 cells.9,11 Several other inhibitor series including pyrrolo-
pyrimidones, benzothiophene, and 2,4 diaminopyrimidines,
2. Barf, T.; Kaptein, A.; de Wilde, S.; van der Heijden, R.; van Someren, R.; Demont,
D.; Schultz-Fademrecht, C.; Versteegh, J.; van Zeeland, M.; Seegers, N.;
Kazemier, B.; van de Kar, B.; van Hoek, M.; de Roos, J.; Klop, H.; Smeets, R.;
Hofstra, C.; Hornberg, J.; Oubrie, A. Bioorg. Med. Chem. Lett. 2011, 21, 3818.
3. Kaptein, A.; Oubrie, A.; de Zwart, E.; Hoogenboom, N.; de Wit, J.; van de Kar, B.;
van Hoek, M.; Vogel, G.; de Kimpe, V.; Schultz-Fademrecht, C.; Borsboom, J.;
van Zeeland, M.; Versteegh, J.; Kazemier, B.; de Roos, J.; Wijnands, F.; Dulos, J.;
Jaeger, M.; Leandro-Garcia, P.; Barf, T. Bioorg. Med. Chem. Lett. 2011, 21, 3823.
showed potent submicromolar inhibition of TNF
a production in
cells.12 Unfortunately, no pHSP27 data were reported for any of

618
A. Oubrie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 613–618
4. Goldberg, D. R.; Choi, Y.; Cogan, D.; Corson, M.; DeLeon, R.; Gao, A.; Gruenbaum,
7. Allen, F. H. Acta Cryst. 2002, B58, 380.
L.; Hao, M. H.; Joseph, D.; Kashem, M. A.; Miller, C.; Moss, N.; Netherton, M. R.;
Pargellis, C. P.; Pelletier, J.; Sellati, R.; Skow, D.; Torcellini, C.; Tseng, Y. C.; Wang,
J.; Wasti, R.; Werneburg, B.; Wu, J. P.; Xiong, Z. Bioorg. Med. Chem. Lett. 2008, 18,
938.
8. Cheng, R.; Felicetti, B.; Palan, S.; Toogood-Johnson, I.; Scheich, C.; Barker, J.;
Whittaker, M.; Hesterkamp, T. Protein Sci. 2010, 19, 168.
9. 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.; Reitz, R. J. J. Med. Chem.
2007, 50, 2647.
5. Separation of enantiomers was effected at the chloropyridine stage (C2:
R = PMB, A = CH, Y = –CH2–NBoc–) on
a
Chiralpack AD-H column
10. (a) Lovering, F.; Kirincich, S.; Wang, W.; Combs, K.; Resnick, L.; Sabalski, J. E.;
Butera, J.; Liu, J.; Parris, K.; Telliez, J. B. Bioorg. Med. Chem. 2009, 17, 3342; (b)
Olsson, H.; Sjö, P.; Ersoy, O.; Kristoffersson, A.; Larsson, J.; Nordén, B. Bioorg.
Med. Chem. Lett. 2010, 20, 4738.
(20 ꢂ 250 mm, particle size
5 l
m), eluting with heptane/IPA (90/10 v/v).
Stereoisomer 1 eluted at 25.6 min, and stereoisomer 2 at 32.5 min. Ee’s were
>99% as determined with analytical chiral chromatography.
6. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, J. A.; Vreven Jr., T. Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
2004, Gaussian, Inc. Wallingford, CT.
11. Velcicky, J.; Feifel, R.; Hawtin, S.; Heng, R.; Huppertz, C.; Koch, G.; Kroemer, M.;
Moebitz, H.; Revesz, L.; Scheufler, C.; Schlapbach, A. Bioorg. Med. Chem. Lett.
2010, 20, 1293.
12. Schlapbach, A.; Feifel, R.; Hawtin, S.; Heng, R.; Koch, G.; Moebitz, H.; Revesz, L.;
Scheufler, C.; Velcicky, J.; Waelchli, R.; Huppertz, C. Bioorg. Med. Chem. Lett.
2008, 18, 6142; 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.; Parikh, M. D.
Bioorg. Med. Chem. Lett. 2009, 19, 4882; (b) Harris, C. M.; Ericsson, A. M.;
Argiriadi, M. A.; Barberis, C.; Borhani, D. W.; Burchat, A.; Calderwood, D. J.;
Cunha, G. A.; Dixon, R. W.; Frank, K. E.; Johnson, E. F.; Kamens, J.; Kwak, S.; Li,
B.; Mullen, K. D.; Perron, D. C.; Wang, L.; Wishart, N.; Wu, X.; Zhang, X.; Zmetra,
T. R.; Talanian, R. V. Bioorg. Med. Chem. Lett. 2010, 20, 334.
13. Revesz, L.; Schlapbach, A.; Aichholz, R.; Dawson, J.; Feifel, R.; Hawtin, S.;
Littlewood-Evans, A.; Koch, G.; Kroemer, M.; Möbitz, H.; Scheufler, C.; Velcicky,
J.; Huppertz, C. Bioorg. Med. Chem. Lett. 2010, 20, 4719.