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

Article abstract of DOI:10.1021/jm0611004

A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC₅₀ values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFα production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

Full text of DOI:10.1021/jm0611004

J. Med. Chem. 2007, 50, 2647-2654
2647
Pyrrolopyridine Inhibitors of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2
(MK-2)
David R. Anderson,* Marvin J. Meyers,* William F. Vernier, Matthew W. Mahoney, Ravi G. Kurumbail, Nicole Caspers,
Gennadiy I. Poda, John F. Schindler, David B. Reitz, and Robert J. Mourey
Pfizer Global Research and DeVelopment, St. Louis Laboratories, 700 Chesterfield Parkway W, Chesterfield, Missouri 63017
ReceiVed September 20, 2006
A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase
2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These
inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a
number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress
TNFR production in U397 cells and to be efficacious in an acute inflammation model. The structure-
activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo
models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.
Introduction
Tumor necrosis factor-R (TNFR) is a cytokine that is
overproduced in inflammatory disease states such as rheumatoid
arthritis (RA).1 Clinical efficacy and FDA approval of anti-
TNFR biologics such as etanercept, infliximab, and adalimumab
demonstrate the importance of TNFR in inflammatory disease.2-5
Results and Discussion
Similarly, small molecule inhibitors of TNFR production would
be expected to be effective as disease modifying treatments for
RA and other inflammatory diseases. A number of potential
biological targets for inhibition of TNFR biosynthesis have been
identified. Inhibitors of p38 MAP kinase have been shown to
play an important role in the regulation of the production of
inflammatory cytokines such as TNFR.6,7 Furthermore, p38
MAP kinase inhibitors have been shown to be efficacious in
RA patients in clinical trials. For example, VX-745 demonstrated
efficacy in a 12-week clinical study,8 but development was
suspended due to adverse CNS effects observed at high doses
in animal models.9 More recently, VX-702 was reported as being
efficacious for the treatment of RA in a phase II trial in
combination with methotrexate.10
Chemistry. This series of compounds, originating from
modification of a hit obtained from high-throughput screening,
can be made via the general synthesis of compounds as shown
in Scheme 1. Commercially available 2-chloro-4-cyanopyridine
2 was converted to the methyl ketone 3 by treatment with
methylmagnesium bromide followed by hydrolysis.15 Ketone 3
was brominated with bromine and HBr to give bromoketone 4.
Bromoketones 4 and 5 (commercially available) were reacted
with piperidine-2,4-dione (6) and ammonium acetate to generate
pyrroles 7 and 8 via a one-pot three-component pyrrole
condensation reaction. Chloropyridine 7 was readily scaled and
served as a useful late-stage intermediate for rapid analog
preparation. Facile Suzuki coupling of aromatic and heteroaro-
matic boronic acids or their pinacol esters to 7 allowed access
to a variety of analogs in short order using solution-phase
parallel synthesis techniques.
To broaden the scope of the structure-activity relationships
(SAR), a variety of amides (32-37) were prepared. The amides
were either prepared via Suzuki coupling of the corresponding
commercially available boronic acids to chloropyridine 7
(Scheme 1) or by amide bond coupling to carboxylic acid 31
(Scheme 2).
In Vitro Potency and Selectivity. Tables 1 and 2 summarize
the in vitro potency found in this series of compounds. The
inhibitors discovered were found to be competitive with ATP.
Increasing ATP concentrations in the enzyme assay to levels
above the Km for MK-2 (30 µM) induced a right shift in the
apparent IC50 to higher values. Detailed kinetic analysis of
compound 23, for example, showed a classic ATP competitive
mechanism in a Lineweaver-Burke plot of the data (see
Supporting Information Figure S1). Most compounds prepared
showed good potency against the isolated MK-2 enzyme while
exhibiting some SAR trends. The unadorned phenyl ring (9)
provided improved potency relative to H (8) or Cl (7; Table 1).
In fact, a wide variety of aryl and heteroaryl rings (9-16) were
Mitogen-activated protein kinase-activated protein kinase-2
(MAPKAP-K2 or MK-2) is a direct substrate of p38 and
regulates cytokine production through a post-transcriptional
mechanism.11-13 MK-2 knockout mice express TNFR when
challenged with lipopolysaccharide (LPS), but only to about
10-20% of the levels expressed by wild-type mice.11 Impor-
tantly, while MK-2 knockout mice are healthy and have a
normal phenotype, deletion of the p38R gene is embryonic
lethal. In addition, MK-2 knockout mice have been shown to
be resistant to developing arthritis in animal models.14 These
observations suggest that a selective MK-2 inhibitor may
demonstrate efficacy equal to that of a p38 inhibitor without
affecting additional cellular pathways governed by p38 that may
lead to undesirable side effects. In this report, we disclose the
preparation of a series of potent, selective inhibitors of MK-2
and their effectiveness in blocking the production of TNFR.
These compounds have the general structure shown in 1.
* To whom correspondence should be addressed. Phone: 636-247-
7651 (D.R.A.); 636-247-7672 (M.J.M.). Fax: 636-247-5400 (D.R.A.); 636-
247-6953 (M.J.M.). E-mail: david.r.anderson@pfizer.com (D.R.A.);
marvin.j.meyers@pfizer.com (M.J.M.).
10.1021/jm0611004 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/05/2007

2648 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Anderson et al.
Table 1. Inhibition of MK-2 and TNFR Production in U937 Cells by
2-Aryl Pyridine Analogs
Table 2. Inhibition of MK-2 and TNFR Production in U937 Cells by
2-Aryl Pyridine Analogs
a
b
a
b
MK-2 IC50
U937 IC50
MK-2 IC50
U937 IC50
cmpd
R
(µM)
(µM)
cmpd
R
(µM)
(µM)
8
7
9
10
11
12
13
14
15
16
H
Cl
Ph
4-pyridine
3-pyridine
5-pyrimidine
3-thienyl
3-naphthyl
2-benzothienyl
3-quinoline
0.171 ( 0.082
0.608 ( 0.263
0.066 ( 0.029
0.056 ( 0.026
0.048 ( 0.005
0.083 ( 0.026
0.076 ( 0.020
0.052 ( 0.012
0.030 ( 0.0001
0.0085 ( 0.0016
19
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
2-OH
3-OH
4-OH
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
4-CF3
4-CN
4-Ac
4-MeO
4-NH2
4-CO2H
0.410 ( 0.058
0.025 ( 0.012
0.021 ( 0.001
0.606 ( 0.004
0.037 ( 0.003
0.032 ( 0.006
0.126 ( 0.038
0.030 ( 0.005
0.050 ( 0.030
0.071 ( 0.0004
0.062 ( 0.013
0.051 ( 0.009
0.055 ( 0.015
0.041 ( 0.007
0.022 ( 0.006
0.015 ( 0.003
0.008 ( 0.002
0.017 ( 0.007
0.008 ( 0.003
0.046 ( 0.005
0.056 ( 0.004
47
36
35
1.4
23
9.0
4.6
2.7
4.8
7.4
2.4
3.8
>100
18
2.2
12
>100
>100
23
9.9
>100
6.8
4.2
5.7
4.4
a Values are reported as the mean ( standard deviation (n g 2). b IC50
values are reported as the mean of 2-3 experiments with a standard
deviation less than 3-fold.
Scheme 1. Synthesis of 2-Arylpyridyl Pyrroles
4-CONH-c-Pr
4-CONH-c-pentyl
4-CONH-c-Hex
4-CONHCH2Ph
4-CONH(CH2)2Ph
4-CON(Me)CH2Ph
10
20
11
15
a Values are reported as the mean ( standard deviation (n g 2). b IC50
values are reported as the mean of 2-3 experiments with a standard
deviation less than 3-fold.
Table 3. Kinase Selectivity of Potent MK-2 Inhibitors
cmpd
8
9
16
23
aryl group
H
Ph
3-quinolinyl
2-F-Ph
kinase IC50a (µM)
MK-2
0.171
57.7
0.50
2.67
2.34
3.01
0.17
0.066
0.66
0.14
16
146
148
18
0.0085
0.21
0.081
5.7
>200
>200
>200
3.44
0.126
1.1
0.21
>10
>200
58
MK-3
PRAK (MK-5)
MNK1
MSK1
MSK2
CDK2
ERK2
30
4.6
JNK2
IKK2
p38a
>200
>200
>100
21
>200
87
Scheme 2. Synthesis of Aryl Amides
>10
>200
a IC50 values are reported as the mean of 2-3 experiments with a standard
deviation of less than 3-fold.
amide, as demonstrated by 36. Tertiary amides (37) were
somewhat less potent than secondary amides (35).
Selectivity against other related MAPKAP kinases, CDK2,
and kinases associated with TNFR production for several of
the more potent analogs is recorded in Table 3. To utilize IC50
values to compare the selectivity of compounds between kinase
assays, Km values for ATP with each kinase was determined
and all kinase selectivity reactions were run at Km values of
ATP (see Supporting Information). While the unsubstituted
analog (8) had no selectivity against CDK2, most 2-aryl
substituted compounds (e.g., 9, 16, and 23) demonstrated
exceptional selectivity against CDK2, ERK2, JNK2, IKK2, as
well as MNK1, MSK1, and MSK2. The MAPKAP kinases MK-
3, PRAK (MK-5), MNK1, MSK1, and MSK2 are all direct
substrates of p38, with MK-3 and PRAK sharing the highest
degree of sequence identity with MK-2. Therefore, it is not
surprising that most compounds show only modest selectivity
versus PRAK and MK-3. The fact that this series of compounds
shows some selectivity against these related kinases is indeed
not only tolerated, but enhanced enzyme potency. The 3-quino-
line substitution (16) represents one of the most potent
compounds identified in this series.
A wide variety of substituents were tolerated on the phenyl
ring as well (Table 2). In general, ortho substituents were poorly
tolerated (17, 20, 23), while both meta (18, 21, 24) and para
(19, 22, 25) substitutions were preferred. This suggests that the
dihedral angle between the pyridine and the aryl ring may be
important for potency. The enzyme is also quite tolerant of a
diverse array of substituents, regardless of size or electron-
donating or electron-withdrawing character (26-31), which had
little effect on measured potencies. Additionally, a variety of
secondary amides were found to be especially potent MK-2
inhibitors (32-37). There is, however, a limit to the size of the

Pyrrolopyridine Inhibitors of Protein
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2649
noteworthy. Importantly, these selected compounds show little
activity on any pathway known to be implicated in TNFR
production (e.g., p38R, ERK2, JNK2, and IKK2) except for their
inhibition of MK-2 or its close homologues, MK-3 and PRAK.
Crystallography and Rationale for Selectivity. To under-
stand the details of the molecular interactions of MK-2 inhibitors
with protein kinases, we undertook crystallographic analysis of
the inhibitors complexed with MK-2 and CDK2. Prior to the
availability of these crystal structures, we had obtained the
structure of MK-2 complexed with AMPPNP, a nonhydrolyzable
analog of ATP.16 Docking studies of early lead compounds into
the MK-2 crystal structure suggested that substitution at the
2-position of the pyridyl group could potentially enhance the
potency of these compounds for MK-2. Moreover, an overlay
of MK-2 and CDK2 crystal structures revealed two key
structural differences between these two kinases: (1) the
presence of a large hydrophobic pocket in MK-2 in the hinge
region of the ATP site due to the presence of an additional amino
acid relative to CDK2 and (2) substitution of a key tyrosine
residue in the hinge region of CDK2 by a smaller cysteine
residue in MK-2. The latter results in a more expanded pocket
in MK-2 near the protein surface. We hypothesized that
analogues containing large, rigid groups from the 2-position of
the pyridyl moiety of our inhibitors could have decreased
binding affinity for CDK2 relative to MK-2 because of the above
structural features.
A crystal structure of compound 23 complexed with MK-2
was determined at 3.8 Å resolution (Figure 1a,b). Although the
modest resolution of the crystal structure prevents us from
making definite conclusions regarding hydrogen bonds and other
intermolecular interactions, the pyridyl nitrogen of the inhibitor
is within a reasonable distance (3.4 Å) of forming a hydrogen
bond with the peptide nitrogen of Leu141. The lactam moiety
appears to form two hydrogen bonds in the phosphate binding
region of MK-2. While the oxygen of lactam forms a hydrogen
bond to the terminal nitrogen of the conserved lysine (Lys93),
the lactam nitrogen engages the side chain carboxylate of the
conserved aspartic acid, Asp207. In addition, the ligand forms
close van der Waals interactions with several protein residues
in the active site including the gatekeeper residue, Met138. Clear
electron density is visible for most of the glycine-rich loop,
which is a relatively flexible segment in many protein kinase
structures. The 2-fluorophenyl group of the inhibitor is sand-
wiched between the glycine loop and the hinge region of MK-
2, with good lipophilic interactions with the side chain of Leu70
of the glycine loop. The fluorine atom is located near the side
chain of Leu193 of the carboxy-terminal domain of MK-2. The
terminal aryl group of the ligand is rotated by ∼37 degrees
relative to the rest of the inhibitor.
Figure 1. Crystal structure of MK-2 with compound 23. (a) Edge view
showing the MK-2 binding pocket with compound 23. (b) Top view
showing H-bonding network of 23 with Leu141, Lys93, and Asp207
in the MK-2 binding pocket. (c) Overlay of compound 23 bound to
MK-2 (gray) and the ATP binding site of CDK2 (orange). In Figure
1a,b, the molecular surface of MK-2 is color-coded by hydrophobicity,
with brown representing the most hydrophobic region of the protein
and blue indicating the most hydrophilic region.
Superposition of the crystal structure of CDK2 on the MK-2
structure reveals the structural basis for the specificity of the
aryl analogues of the pyrrole lactam series of inhibitors (Figure
1c). As shown in Table 3, compounds that contain large aromatic
substituents from the 2-position of the pyridyl moiety are highly
selective for MK-2, MK-3, and PRAK compared to other
kinases. The side chain of Cys140 is located near the terminal
aryl group of compound 23 in the crystal structure. Most kinases
possess an amino acid with a large side chain at a position
corresponding to Cys140 of MK-2. It is very likely that the
presence of bulky side chains in these kinases could form a
steric clash with the terminal aryl group of compound 23 and
related analogues. In fact, there are only eight other kinases in
the entire human kinome that contain a cysteine or a smaller
amino acid at a position corresponding to Cys140 of MK-2.
While this would explain the high binding affinity of compound
23 for MK-3, which also contains a cysteine at this position, it
is unclear why this compound is also a good inhibitor of PRAK,
which contains a methionine at the corresponding position. One
explanation is that the methionine side chain of PRAK could
adopt an alternate rotamer conformation that would result in a
similar pocket in PRAK as that observed in the MK-2 structure.
In addition, the hinge region is shorter in many kinases, such
as cyclin-dependent kinase family, relative to MK-2. Conse-
quently, MK-2 has a larger pocket at the hinge region compared
to CDK2, which is exploited by the large substitent from the
2-position of the pyridyl ring. The combination of a large amino
acid side chain (phenylalanine) at the position corresponding
to Cys140 of MK-2 and one amino acid deletion in the hinge
region in CDK2 results in a crowded and smaller binding pocket.

2650 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Anderson et al.
Figure 2. Western Blot analysis of MAPK signaling in U937 cells treated with MK-2 inhibitor 23.
This smaller pocket in the hinge region of CDK2 precludes the
binding of the terminal aryl group of compound 23. This
observation presents a general strategy for the design of specific
inhibitors for MK-2.
levels may be a contributing factor.18,19 The source of the
discrepancy between the enzyme and the cell potencies may be
explained by an examination of the affinity of MK-2 for ATP.
The measured in vitro Km value of ATP for MK-2 is 30 µM.
Because the inhibitors described are ATP competitive and ATP
concentrations in the cell are 1-5 mM, one can use the Cheng-
Prusoff equation20 to calculate the theoretical shift in potencies
from the enzyme assay to cell assay due to increased ATP
concentrations. For MK-2, a shift of approximately 33-fold can
be expected. Analog 23, which has a moderate to high perme-
ability, 69% unbound fraction in cell media, and high solubility,
has a cell to enzyme ratio of 38, which is nearing this theoretical
minimum based on differences in ATP concentrations in the
assays. Deficiencies in physical properties that limit cell
permeability or free drug concentration would be expected to
exacerbate this discrepancy even further. We therefore conclude
that unfavorable ATP competition is the most likely source of
the discrepancy between enzyme and cell potencies.
Because the compounds described appear to be inactive
against other kinases in signal transduction pathways known to
affect the production of TNFR, but there is a shift in observed
potency between enzyme and cell assays, we sought to
determine whether the observed activity in the cell assay was
solely due to inhibition of MK-2, or possibly a combination of
pathways and mechanisms. We sought to demonstrate that
blockade of MK-2 activity in a cell-based system correlated
directly with inhibition of TNFR production. In Western blot
analysis of LPS-stimulated U937 cells treated with various
concentrations of compound 23, we see dose-dependent inhib-
itition of phospho-Ser78 on HSP27, a site shown to be
specifically phosphorylated by MK-221 (Figure 2A). The ratio
of phospho-HSP27 to total HSP27 protein was quantified from
the Western blot and expressed as percent control of the phospho
levels detected after 30 min of LPS stimulation in the absence
or presence of compound 23 (Figure 2B). Inhibition of HSP27
phosphorylation at 30 min correlated directly with TNFR
inhibition measured at 4 h, with an IC50 value of approximately
4 µM. Furthermore, compound 23 failed to inhibit upstream of
MK-2 or within the JNK or ERK MAPK pathways, as measured
by the lack of cellular inhibition of phospho-p38, phospho-
JNK2, and phospho-ERK2, respectively. Because HSP27 phos-
phorylation and TNFR production are attenuated at roughly
the same inhibitor concentrations and given the evidence
linking MK-2 to TNFR production, it is reasonable to conclude
that the activity in the cellular assay is the result of MK-2
inhibition.
In Vitro Cellular Potency and Selectivity. MK-2 inhibitor
compounds were tested for activity in a U937 cellular TNFR
production assay,17 and the results for selected analogs are
summarized in Tables 1 and 2. As can be seen, many of these
compounds are active in suppressing cellular production of
TNFR, albeit with low micromolar potencies. Unfortunately,
there is very little correlation between increased MK-2 potency
and potency in the U937 cell assay. For example, the phenyl
analog 9 is 66 nM in the enzyme assay and 1.4 µM in the cell
assay, while the 3-quinoline derivative is about 8-fold more
potent (8 nM) in the enzyme assay but only 4.4 µM in the cell.
Sub-optimal physical properties may contribute to the at-
tenuated potencies of these inhibitors in the cellular assay. Many
of the compounds have low aqueous solubilities. For example,
quinoline analog 16 has an aqueous solubility of <0.4 µM. Some
compounds have higher solublities. For example, 2-fluorophenyl
analog 23 has an aqueous solubility of 160 µM. The improved
solubility of 23 may be related to the decreased planarity of
the compound due to the slightly larger fluorine atom in the
ortho position. Unfortunately, improving solubility by twisting
the two aromatic groups from planarity often leads to weaker
MK-2 potency (vide supra). When compared to the unsubstituted
phenyl analog, the 2-fluorophenyl analog is approximately 2-fold
less potent, with an IC50 value of 0.126 µM. While this
difference may not be statistically significant and within the
error of the assay, it does follow the general trend that ortho-
substitution is detrimental to enzyme potency. The 3- and
4-fluoro-substituted analogs 24 and 25 have solubilities of 130
and 20 µM, respectively, and have improved potencies for the
recombinant enzyme (0.034 µM and 0.05 µM, respectively),
but do not show markedly improved potency in the cellular assay
(3.4 µM and 2.4 µM) when compared to 23. This suggests that
solubility alone cannot account for the attenuation in potency
observed in the U937 cell for this series. Analog 23 has
moderate to high permeability, as judged by CACO-2 (A-B )
9.45 × 10-6 cm/sec), and also has reasonably low rat plasma
protein binding (92%). Protein binding of 23 in the media used
in the cell assay containing 10% fetal calf serum was determined
to be 31%.
Lack of correlation between isolated biochemical assays for
kinases and cell-based assays are well documented, and it has
been suggested that unfavorable competition with cellular ATP

Pyrrolopyridine Inhibitors of Protein
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2651
Table 4. Efficacy of MK-2 Inhibitors an In Vivo Acute Model of
Inflammation (rLPS)
and the solid was collected by filtration. The solid was washed
with ethyl ether and dried under vacuum to give 4 (6.51 g, 93%)
as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, 1H),
7.96 (s, 1H), 7.83 (d, 1H), 4.99 (s, 2H). MS m/z (M + H): 234,
236.
TNFRa (% inhibition)
[compound]b (µg/mL)
cmpd
R
2.5 h
3.5 h
2.5 h
3.5 h
9
16
22
29
23
24
25
Ph
44
20
38
<0.1
73
55
57
<0.1
<0.1
<0.1
<0.1
87
4.54
blq
2.18
blq
3.87
ndd
nd
blqc
blq
blq
2,4-Dioxopiperidine (6). Sodium 3-(methoxycarbonyl)-4-oxo-
1,4,5,6-tetrahydropyridin-2-olate (Degussa; 50 g, 259 mmol) was
partitioned between 2 N aqueous hydrogen chloride and dichlo-
romethane. The aqueous layer was extracted two additional times
with dichloromethane. The organic extracts were dried over sodium
sulfate, filtered, and evaporated. The residue was suspended in
acetonitrile (500 mL) and water (100 mL) and heated to reflux for
3 h. The reaction mixture was cooled and evaporated. The residue
was recrystallized from 1:1 ethyl acetate/hexane to provide 6 (19.5
3-quinolinyl
4-Cl-Ph
4-MeO-Ph
2-F-Ph
3-F-Ph
4-F-Ph
blq
3.13
5.80
nd
83
49
a Percent inhibition of TNFR production relative to control, 1.5 h after
LPS challenge. Compounds were dosed orally at 20 mpk, 1 or 2 h prior to
LPS challenge. b Compound concentrations in the plasma samples for TNFR
levels were measured by LC-MS and correspond to 2.5 and 3.5 h after
compound dosing. c blq ) below limits of quantitation. d nd ) not
determined.
1
g, 67%) as a white solid. H NMR (400 MHz, CDCl3) δ 7.05 (s,
1H), 3.58 (td, 2H), 3.34 (s, 2H), 2.64 (t, 2H). MS m/z (M + H):
114.
2-(2-Chloropyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-
c]pyridin-4-one (6a). Bromoketone 4 (6.5 g, 20.6 mmol) was
combined in absolute ethanol (65 mL) with ammonium acetate (6.35
g, 82.4 mmol) and dioxopiperidine 6 (2.57 g, 22.7 mmol). After
30 min, the mixture was diluted with water (130 mL) and the
mixture was filtered. The resulting solid was washed with water
and ethyl ether and dried under vacuum to give 7 (3.15 g, 62%) as
a white solid. H NMR (400 MHz, DMSO-d6) δ 12.00 (s, 1H),
8.27 (d, 1H), 7.73 (s, 1H), 7.63, (d, 1H), 7.12 (s, 1H), 7.08 (s, 1H),
3.40 (td, 2H), 2.83 (t, 2H). MS m/z (M + H): 248.
In Vivo Efficacy. Analogs with potencies less than 5 µM in
the cellular assay were tested in vivo. It was found that a number
of the compounds tested were orally active in an acute model
of inflammation, the rat LPS (rLPS) model.17 The results for a
select group of analogs are shown in Table 4. While many potent
MK-2 inhibitors had little or no oral activity in the rLPS assay,
the fluorophenyl analogs (23-25) were found to be efficacious,
presumably due to improved physical and pharmacokinetic
properties. For example, analogs 9 and 22 showed low to
moderate activity in the rLPS model at 20 mpk 1 h prior to
LPS challenge with measurable levels of compound in the
plasma. However, if dosed 2 h prior to the LPS challenge, no
TNFR inhibition was observed and plasma compound concen-
trations were undetectable at 3.5 h, presumably due to high
clearance or poor bioavailability of the compounds. Compounds
23 and 24, on the other hand, inhibited >80% of TNFR
production when dosed 2 h prior to LPS challenge, with good
levels of compound detected in the plasma, most likely due to
reduced clearance or other improved pharmacokinetic properties.
In addition to potent TNFR inhibition, compound 23 has many
desirable drug-like properties that will be explored in a
forthcoming pharmacology publication.
1
2-Pyridin-4-yl-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-
one Trifluoroacetate (8). A mixture of 4-(bromoacetyl)pyridine
hydrobromide (250 mg, 0.89 mmol), dioxopiperidine 6 (151 mg,
1.33 mmol, 1.5 equiv), and ammonium acetate (411 mg, 5.34 mmol,
4.0 equiv) in ethanol (3.0 mL) was stirred at room temperature for
1 h. The reaction mixture was purified by preparative reverse phase
HPLC to give 8 (207 mg, 0.63 mmol, 71%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 12.82 (s, 1H), 8.66 (d, J ) 7.0 Hz, 2H), 8.16 (d,
J ) 7.0 Hz, 2H), 7.52 (d, J ) 2.4 Hz, 1H), 7.26 (s, 1H), 3.37-
3.43 (m, 3H), 2.89 (t, J ) 6.8 Hz, 2H). HRMS calcd for C12H12N3O
(MH+), 214.0975; found, 214.0949. Anal. (C12H11N3O‚CF3CO2H‚
0.2H2O) C, H, N.
General Procedure for the Suzuki Coupling of Chloropyri-
dine 6a with Aryl Boronic Acids. A suspension of chloropyridine
7 (1.0 equiv) in dimethylformamide (2.0 mL/100 mg 7) was treated
with an aryl boronic acid (1.5 equiv) and 2.0 M aqueous cesium
carbonate (3.0 equiv). The reaction was purged with nitrogen and
then tetrakistriphenylphosphinepalladium (0.05 equiv) was added.
The reaction was then heated to 80 °C for 8 to 16 h and then cooled
to room temperature. The reaction mixture was acidified with
trifluoroacetic acid and purified by preparative reverse phase HPLC
(acetonitrile/water/0.05% trifluoroacetic acid). The desired fractions
were lyophilized or concentrated under a stream of nitrogen to give
the product. Compounds 9-35 were obtained in this manner.
2-(2-Phenylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-
c]pyridin-4-one Trifluoroacetate (9): Yellow solid, 208 mg, 85%.
1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 8.63 (d, J ) 5.9
Hz, 1H), 8.36 (s, 1H), 8.08 (dd, J ) 7.6, 1.8 Hz, 2H), 7.86 (d, J )
5.7 Hz, 1H), 7.64-7.56 (m, 3H), 7.48 (s, 1H), 7.20 (s, 1H), 3.43
(t, J ) 6.7 Hz, 2H), 2.91 (t, J ) 6.8 Hz, 2H). HRMS calcd for
C18H16N3O(MH+),290.1288;found,290.1319.Anal.(C18H15N3O‚CF3-
CO2H‚H2O) C, H, N.
In conclusion, we have prepared a set of potent small
molecule inhibitors selective for MK-2 kinase. These compounds
were found to be effective in reducing TNFR production in both
U937 cells and in vivo. These results demonstrate that selective,
small molecule MK-2 inhibitors have the potential to be effective
treatments for RA and other inflammatory diseases mediated
by TNFR.
Experimental Section
Chemistry. General Methods. All reagents were purchased from
commercial sources and were used without further purification.
Solvents were of analytical or anhydrous grade (Sigma-Aldrich).
Reactions were monitored by HPLC. Reverse phase preparative
HPLC was performed using a Gilson preparative HPLC system.
1H NMR spectra were recorded on a Varian Unity Innova 400
instrument. Chemical shifts (δ) are reported in ppm downfield from
solvent references. Mass spectra were obtained on a Finnigan LCQ
Duo LCMS ion trap electrospray ionization (ESI) mass spectrom-
eter, a PerSeptive Biosystems Mariner TOF HPLC-MS (ESI), or a
Waters ZQ mass spectrometer (ESI). Elemental analyses were
performed by Atlantic Microlabs, Norcross, GA, and are within
(0.4% of theoretical values.
2-Bromo-1-(2-chloropyridin-4-yl)ethanone Hydrobromide (4).
4-Acetyl-2-chloropyridine (3)15 was dissolved in glacial acetic acid
(100 mL) and treated with bromine (1.26 mL, 24.6 mmol), followed
by HBr/AcOH (30% w/v, 4.4 mL, 22.3 mmol). After 15 min of
stirring, a precipitate formed, and the reaction was complete after
2-3 h. The reaction mixture was diluted with ethyl ether (100 mL),
2-(2,4′-Bipyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]-
pyridin-4-one Trifluoroacetate (10): Yellow solid, 18 mg, 31%.
1H NMR (400 MHz, DMSO-d6) δ ppm 12.05 (s, 1H), 8.84-8.92
(m, 2H), 8.67 (d, J ) 5.4 Hz, 1H), 8.50 (d, J ) 1.1 Hz, 1H), 8.44
(d, J ) 6.2 Hz, 2H), 7.74 (dd, J ) 5.2, 1.7 Hz, 1H), 7.31 (d, J )
2.7 Hz, 1H), 7.07 (s, 1H), 3.41 (td, J ) 6.8, 1.2 Hz, 2H), 2.86 (t,
J ) 6.8 Hz, 2H). HRMS calcd for C17H15N4O (MH+), 291.1240;
found, 291.1240. Anal. (C17H14N4O‚2.6CF3CO2H) C, H, N.
2-(2,3′-Bipyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]-
pyridin-4-one Trifluoroacetate (11): Yellow solid, 169 mg, 90%.
1H NMR (400 MHz, DMSO-d6) δ ppm 12.13 (s, 1H), 9.37 (s, 1H),
8.79 (d, J ) 4.6 Hz, 1H), 8.73 (d, J ) 8.3 Hz, 1H), 8.65 (d, J )

2652 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Anderson et al.
5.6 Hz, 1H), 8.41 (d, J ) 1.1 Hz, 1H), 7.80 (dd, J ) 8.1, 5.1 Hz,
1H), 7.76 (dd, J ) 5.5, 1.5 Hz, 1H), 7.36 (d, J ) 2.4 Hz, 1H), 7.12
(s, 1H), 3.41 (t, J ) 6.7 Hz, 2H), 2.87 (t, J ) 6.8 Hz, 2H). HRMS
calcd for C17H15N4O (MH+), 291.1240; found, 291.1219. Anal.
(C17H14N4O‚2CF3CO2H‚0.8H2O) C, H, N.
2H), 2.91 (t, J ) 6.4 Hz, 2H). HRMS calcd for C18H15N3O2 (MH+),
306.1237; found, 306.1219. Anal. (C18H15N3O2‚CF3CO2H‚1.9H2O)
C, H, N.
2-[2-(2-Chlorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (20): Yellow solid,
1
107 mg, 40%. H NMR (400 MHz, DMSO-d6) δ ppm 12.24 (s,
2-(2-Pyrimidin-5-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (12): Yellow solid, 67
1H), 8.68 (d, J ) 5.9 Hz, 1H), 8.05 (s, 1H), 7.90 (d, J ) 5.4 Hz,
1H), 7.64-7.71 (m, 2H), 7.49-7.62 (m, 2H), 7.35 (s, 1H), 7.19
(s, 1H), 3.41 (td, J ) 6.7, 2.4 Hz, 2H), 2.87 (t, J ) 6.8 Hz, 2H).
HRMS calcd for C18H15ClN3O (MH+), 324.0898; found, 324.0892.
Anal. (C18H14ClN3O‚CF3CO2H‚2H2O) C, H, N.
1
mg, 27%. H NMR (300 MHz, DMSO-d6) δ 12.03 (s, 1H), 9.49
(s, 2H), 9.27 (s, 1H), 8.65 (d, J ) 5.4 Hz, 1H), 8.40 (s, 1H), 7.71
(dd, J ) 5.4, 1.6 Hz, 1H), 7.31 (d, J ) 2.2 Hz, 1H), 7.10 (s, 1H),
3.42 (t, J ) 6.7 Hz, 2H), 2.88 (t, J ) 6.8 Hz, 2H). HRMS calcd
forC16H14N5O(MH+),292.1193;found,292.1179.Anal.(C16H13N5O‚CF3-
CO2H) C, H, N.
2-[2-(3-Chlorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (21): Yellow solid,
31 mg, 48%. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.18 (s, 1H),
8.60 (d, J ) 5.6 Hz, 2H), 8.33 (s, 1H), 8.17 (s, 1H), 8.07 (t, J )
3.6 Hz, 1H), 7.76 (d, J ) 5.4 Hz, 1H), 7.58 (d, J ) 4.8 Hz, 1H),
7.39 (d, J ) 1.1 Hz, 1H), 7.13 (s, 1H), 3.41 (t, J ) 6.7 Hz, 2H),
2.88 (t, J ) 6.7 Hz, 2H). HRMS calcd for C18H15ClN3O (MH+),
324.0898; found, 324.0886. Anal. (C18H14ClN3O‚1.3CF3CO2H‚
0.4H2O) C, H, N.
2-(2-Thien-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo-
[3,2-c]pyridin-4-one Trifluoroacetate (13): Yellow solid, 280 mg,
1
57%. H NMR (300 MHz, DMSO-d6) δ 12.33 (s, 1H), 8.56 (d, J
) 6.0 Hz, 1H), 8.45 (s, 1H), 8.37 (s, 1H), 7.89 (d, J ) 5.0 Hz,
1H), 7.80 (m, 2H), 7.54 (s, 1H), 7.21 (s, 1H), 3.44 (m, 2H), 2.91
(t, J ) 6.7 Hz, 2H). HRMS calcd for C16H13N3OS (MH+),
296.0852; found, 296.0869. Anal. (C16H13N3OS‚CF3CO2H‚1.4H2O)
C, H, N.
2-[2-(4-Chlorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (22): Yellow solid,
2-[2-(2-Naphthyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyrrolo-
[3,2-c]pyridin-4-one Trifluoroacetate (14): Yellow solid, 22 mg,
1
218 mg, 80%. H NMR (400 MHz, DMSO-d6) δ ppm 12.18 (s,
1H), 8.58 (d, J ) 5.6 Hz, 1H), 8.30 (d, J ) 1.3 Hz, 1H), 8.05-
8.17 (m, 2H), 7.74 (d, J ) 5.4 Hz, 1H), 7.58-7.65 (m, 2H), 7.36
(s, 1H), 7.13 (s, 1H), 3.41 (t, J ) 6.4 Hz, 2H), 2.87 (t, J ) 6.8 Hz,
2H). HRMS calcd for C18H15ClN3O (MH+), 324.0898; found,
324.0897. Anal. (C18H14ClN3‚CF3CO2H‚H2O) C, H, N.
1
32%. H NMR (400 MHz, DMSO-d6) δ ppm 12.28 (s, 1H), 8.68
(d, J ) 1.6 Hz, 1H), 8.65 (d, J ) 5.6 Hz, 1H), 8.48 (s, 1H), 8.21
(dd, J ) 8.6, 1.9 Hz, 1H), 8.11 (d, J ) 8.6 Hz, 1H), 8.05 (dd, J )
6.3, 3.6 Hz, 1H), 8.00 (dd, J ) 6.2, 3.5 Hz, 1H), 7.58-7.64 (m,
2H), 7.44-7.48 (m, 1H), 7.16 (s, 1H), 3.42 (td, J ) 6.7, 1.6 Hz,
2H), 2.90 (t, J ) 6.8 Hz, 2H). HRMS calcd for C22H18N3O (MH+),
340.1444; found, 340.1417. Anal. (C22H17N3O‚1.3CF3CO2H‚
0.5H2O) C, H, N.
2-[2-(2-Fluorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (23): Yellow solid, 168
1
mg, 70%. H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 8.67
(d, J ) 5.7 Hz, 1H), 8.13 (s, 1H), 7.88-7.84 (m, 2H), 7.63-7.57
(m, 1H), 7.46-7.38 (m, 2H), 7.31 (s, 1H), 7.17 (s, 1H), 3.42 (t, J
) 6.7 Hz, 2H), 2.88 (t, J ) 6.6 Hz, 2H). HRMS calcd for C18H15-
FN3O (MH+), 308.1194; found, 308.1224. Anal. (C18H14FN3O‚CF3-
CO2H‚1.2H2O) C, H, N.
2-[2-(1-Benzothien-2-yl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (15): Yellow solid,
1
139 mg, 38%. H NMR (300 MHz, DMSO-d6) δ ppm 12.07 (s,
1H), 8.52 (d, J ) 5.4 Hz, 1H), 8.43 (d, J ) 1.0 Hz, 1H), 8.28 (s,
1H), 7.98-8.08 (m, 1H), 7.85-7.97 (m, 1H), 7.62 (dd, J ) 5.4,
1.8 Hz, 1H), 7.37-7.49 (m, 2H), 7.28 (d, J ) 2.4 Hz, 1H), 7.12
(br s, 1H), 3.45 (t, J ) 6.7 Hz, 2H), 2.91 (t, J ) 6.8 Hz, 2H).
HRMS calcd for C20H16N3OS (MH+), 346.1009; found, 346.1043.
Anal. (C20H15N3OS‚CF3CO2H‚0.3H2O) C, H, N.
2-[2-(3-Fluorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (24): Yellow solid, 127
1
mg, 51%. H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.62
(d, J ) 5.6 Hz, 1H), 8.35 (s, 1H), 7.99-7.94 (m, 2H), 7.78 (d, J
) 4.8 Hz, 1H), 7.63 (td, J ) 8.1, 6.4 Hz, 1H), 7.40 (s, 1H), 7.35
(td, J ) 8.3, 2.2 Hz, 1H), 7.16 (s, 1H), 3.43 (t, J ) 6.8 Hz, 2H),
2.90 (t, J ) 6.8 Hz, 2H). HRMS calcd for C18H15FN3O (MH+),
308.1194; found, 308.1162. Anal. (C18H14FN3O‚CF3CO2H‚1.4H2O)
C, H, N.
2-(2-Quinolin-3-ylpyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo-
1
[3,2-c]pyridin-4-one (16): Yellow solid, 51 mg, 75%. H NMR
(300 MHz, DMSO-d6) δ ppm 12.06 (s, 1H), 9.72 (d, J ) 2.2 Hz,
1H), 9.12 (d, J ) 2.0 Hz, 1H), 8.68 (d, J ) 5.2 Hz, 1H), 8.49 (s,
1H), 8.12 (t, J ) 7.9 Hz, 2H), 7.84 (ddd, J ) 8.5, 6.8, 1.6 Hz, 1H),
7.64-7.75 (m, 2H), 7.29 (d, J ) 2.4 Hz, 1H), 7.10 (br s, 1H), 3.46
(t, J ) 6.9 Hz, 2H), 2.92 (t, J ) 7.0 Hz, 2H). HRMS calcd for
C21H17N4O(MH+),341.1397;found,341.1352.Anal.(C21H16N4O‚2.8CF3-
CO2H) C, H, N.
2-[2-(4-Fluorophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (25): Yellow solid, 229
1
mg, 67%. H NMR (400 MHz, DMSO-d6) δ ppm 12.18 (s, 1H),
8.58 (d, J ) 5.9 Hz, 1H), 8.28 (s, 1H), 8.11-8.17 (m, 2H), 7.75
(d, J ) 5.1 Hz, 1H), 7.35-7.45 (m, J ) 8.9, 8.9 Hz, 3H), 7.13 (s,
1H), 3.38-3.44 (m, 2H), 2.87 (t, J ) 6.8 Hz, 2H). HRMS calcd
for C18H15FN3O (MH+), 308.1194; found, 308.1155. Anal. (C18H14-
FN3O‚CF3CO2H‚1.1H2O) C, H, N.
2-[2-(2-Hydroxyphenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (17): Yellow solid,
1
165 mg, 67%. H NMR (300 MHz, DMSO-d6) δ 12.31 (s, 1H),
2-{2-[4-(Trifluoromethyl)phenyl]pyridin-4-yl}-1,5,6,7-tetrahy-
dro-4H-pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (26): Yel-
8.55 (d, J ) 6.0 Hz, 1H), 8.37 (s, 1H), 7.95 (d, J ) 8.1 Hz, 1H),
7.85 (d, J ) 5.8 Hz, 1H), 7.47-7.35 (m, 2H), 7.19 (s, 1H), 7.04-
6.96 (m, 2H), 3.43 (t, J ) 6.2 Hz, 2H), 2.91 (t, J ) 6.6 Hz, 2H).
HRMS calcd for C18H15N3O2 (MH+), 306.1237; found, 306.1250.
Anal. (C18H15N3O2‚CF3CO2H‚2.3H2O) C, H, N.
1
low solid, 27 mg, 38%. H NMR (400 MHz, DMSO-d6) δ ppm
12.11 (s, 1H), 8.61 (d, J ) 5.4 Hz, 1H), 8.26-8.41 (m, 2H), 7.89
(d, J ) 8.3 Hz, 2H), 7.72 (dd, J ) 5.6, 1.6 Hz, 1H), 7.29 (d, J )
2.4 Hz, 1H), 7.09 (s, 1H), 3.41 (t, J ) 6.7 Hz, 1H), 2.87 (t, J ) 6.8
Hz, 1H). HRMS calcd for C19H15N3OF3 (MH+), 358.1162; found,
358.1162. Anal. (C19H14N3OF3‚CF3CO2H‚H2O) C, H, N.
2-[2-(3-Hydroxyphenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (18). 1H NMR (300
MHz, DMSO-d6) δ 12.35 (s, 1H), 8.59 (d, J ) 6.0 Hz, 1H), 8.31
(s, 1H), 7.87 (d, J ) 5.6 Hz, 1H), 7.43 (m, 4H), 7.20 (s, 1H), 6.99
(d, J ) 7.8 Hz, 2H), 3.43 (m, 2H), 2.90 (t, J ) 6.6 Hz, 2H). HRMS
calcd for C18H15N3O2 (MH+), 306.1237; found, 306.1224. Anal.
(C18H13N3O2‚CF3CO2H‚2H2O) C, H, N.
2-[2-(4-Hydroxyphenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one (19): Yellow solid, 109 mg, 59%. 1H
NMR (300 MHz, DMSO-d6) δ 12.42 (s, 1H), 8.54 (d, J ) 6.0 Hz,
1H), 8.31 (s, 1H), 7.93 (d, J ) 8.6 Hz, 2H), 7.85 (d, J ) 5.8 Hz,
1H), 7.54 (s, 1H), 7.23 (s, 1H), 6.98 (d, J ) 8.6 Hz, 2H), 3.43 (m,
4-[4-(4-Oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl)-
pyridin-2-yl]benzonitrile Trifluoroacetate (27): Yellow solid, 56
1
mg, 7%. H NMR (300 MHz, DMSO-d6) δ ppm 12.12 (s, 1H),
8.65 (d, J ) 5.4 Hz, 1H), 8.32-8.42 (m, 3H), 8.04 (d, J ) 8.7 Hz,
2H), 7.74 (dd, J ) 5.4, 1.6 Hz, 1H), 7.33 (d, J ) 2.2 Hz, 1H), 7.13
(br s, 1H), 3.45 (t, J ) 6.6 Hz, 2H), 2.90 (t, J ) 6.8 Hz, 2H).
HRMS calcd for C19H15N4O (MH+), 315.1240; found, 315.1235.
Anal. (C19H14N4O‚CF3CO2H‚1.5H2O) C, H, N.
2-[2-(4-Acetylphenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (28): Yellow solid, 15

Pyrrolopyridine Inhibitors of Protein
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2653
1
mg, 22%. H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.59
followed by DIEA (0.38 mL, 2.3 mmol). Heated to 50 °C for 16 h,
cooled, filtered through a syringe filter (0.2 micron), purified by
reverse phase HPLC, and lyophilized to give the title compound
as a yellow solid (118 mg, 0.27 mmol, 35%). 1H NMR (300 MHz,
DMSO-d6) δ 12.25 (s, 1H), 8.72 (t, J ) 5.6 Hz, 1H), 8.64 (d, J )
5.6 Hz, 1H), 8.39 (s, 1H), 8.20 (d, J ) 8.4 Hz, 2H), 8.00 (d, J )
8.4 Hz, 2H), 7.80 (d, J ) 5.6 Hz, 1H), 7.42 (s, 1H), 7.34-7.13 (m,
6H), 3.56-3.38 (m, 4H), 2.94-2.83 (m, 4H). HRMS calcd
for C27H24N4O2 (MH+), 437.1972; found, 437.1991. Anal.
(C27H24N4O2‚CF3CO2H‚0.7H2O) C, H, N.
N-Benzyl-4-(4-(4,5,6,7-tetrahydro-4-oxo-1H-pyrrolo[3,2-c]py-
ridin-2-yl)pyridin-2-yl)-N-methylbenzamide (37). The title com-
pound was prepared as described for compound 36 using N-me-
thylbenzyl amine (0.12 mL, 0.92 mmol). The title compound was
obtained as yellow solid (112 mg, 0.25 mmol, 32%). 1H NMR (300
MHz, DMSO-d6) δ 12.26 (s, 1H), 8.62 (s, 1H), 8.37 (s, 1H), 8.17
(m, 2H), 7.82 (s, 1H), 7.67 (m, 2H), 4.70 (br s, 1H), 4.51 (br s,
1H), 3.43 (t, J ) 6.2 Hz, 1H), 2.91 (m, 5H). HRMS calcd for
C27H24N4O2(MH+),437.1972;found,437.1935.Anal.(C27H24N4O2‚CF3-
CO2H‚1.6H2O) C, H, N.
Enzyme, U937 Cell, and Rat LPS Assays. The MK-2 enzyme
assay, U937-TNFR cell, and rLPS-TNFR assays were performed
as described previously.17 The MK-2 assay conditions were
modified to include 15 nM MK-2 enzyme, 30 µM ATP (∼Km
levels), and 200 µM HSP27 peptide (KKKALSRNLSVAA).
Compound selectivity toward kinases was evaluated for inhibition
of peptide phosphorylation using in-house recombinant enzymes
and the anion exchange resin capture assay method as described.17
Specific selectivity assay conditions are described in the Supporting
Information.
U937 Cell Signaling Studies. PMA-differentiated U937 cells
were incubated with test compound in DMSO (1% final conc) for
1 h prior to stimulation with LPS (100 ng/mL final conc). After 30
min, media was removed and cells were solubilized at 8 million
cells/mL using 2× SDS-PAGE sample buffer (Invitrogen). Boiled
samples (5-10 µL/lane) were resolved on a 10-20% SDS-PAGE
Tris-glycine gradient gel and transferred to a nitrocellulose mem-
brane. The membrane was blocked for 1 h at 25 °C in Odyssey
blocking buffer (OBB; LiCor Biosciences, Lincoln, NE) and then
incubated overnight at 4 °C with primary antibody diluted in OBB/
0.1% Tween 20 buffer. Primary antibodies and dilutions used were
1:750 rabbit anti-pT180/pY182 p38 (cell signaling #9211S), 1:1000
goat anti-p38 total (Santa Cruz #SC-535G), 1:2000 rabbit anti-pS78
HSP27 (in-house generated), 1:1000 goat anti-HSP27 total (Santa
Cruz #SC1048), 1:1000 rabbit anti-pT182/pY185 JNK (Biosource
#44-682G), 1:1000 goat anti-JNK2 total (Santa Cruz #SC-7345),
1:2000 rabbit anti-pT185/pY185 ERK (Biosource #44-680G), and
1:3000 mouse anti-ERK2 total (Santa Cruz #SC-1647). Membranes
were washed 3 × 10 min with 25 mL Tris-buffered saline/0.1%
Tween 20 (TBST) and then incubated for 1 h at 25 °C with
secondary antibodies diluted 1:10 000 in OBB/0.1% Tween 20:
donkey anti-rabbit IgG-fluor 700 (Rockland #611-730-127), donkey
anti-goat IgG-IR dye 800 (Rockland #605-730-125), and donkey
anti-mouse IgG-IR dye 800 (Rockland #610-732-124). Membranes
were washed as above followed by a 25 mL PBS wash, and imaging
was done on a LI-COR Odyssey Infrared Imaging System (LiCore
Biosciences). Dye-labeled target bands were digitized and quanti-
fied, and IC50’s were determined using Grafit (Erithacus Software
Limited, Surrey, U.K.).
(d, J ) 5.6 Hz, 1H), 8.33 (s, 1H), 8.26 (d, J ) 8.4 Hz, 2H), 8.08
(d, J ) 8.4 Hz, 2H), 7.69 (d, J ) 4.8 Hz, 1H), 7.28 (s, 1H), 7.09
(s, 1H), 3.46-3.38 (m, 4H), 2.87 (t, J ) 6.8 Hz, 2H), 2.62 (s, 3H).
HRMS calcd for C20H18N3O2 (MH+), 332.1394; found, 332.1394.
Anal. (C20H17N3O2‚1.4CF3CO2H‚0.9H2O) C, H, N.
2-[2-(4-Methoxyphenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-
pyrrolo[3,2-c]pyridin-4-one Trifluoroacetate (29): Yellow solid,
33 mg, 52%. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.35 (s, 1H),
8.55 (d, J ) 6.2 Hz, 1H), 8.32 (s, 1H), 7.98-8.07 (m, 2H), 7.83
(d, J ) 5.6 Hz, 1H), 7.52 (s, 1H), 7.10-7.26 (m, 3H), 3.85 (s,
3H), 3.42 (td, J ) 6.8, 2.1 Hz, 2H), 2.89 (t, J ) 6.8 Hz, 2H). HRMS
calcd for C19H18N3O2 (MH+), 320.1394; found, 320.1390. Anal.
(C19H17N3O2‚1.3CF3CO2H‚H2O) C, H, N.
2-[2-(4-Aminophenyl)pyridin-4-yl]-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one Trifluoroacetate (30): Yellow solid, 140
1
mg, 76%. H NMR (300 MHz, DMSO-d6) δ 12.48 (s, 1H), 8.43
(d, J ) 6.4 Hz, 1H), 8.30 (s, 1H), 7.82 (m, 3H), 7.61 (s, 3H), 7.26
(s, 1H), 6.75 (d, J ) 8.6 Hz, 2H), 3.43 (m, 2H), 2.92 (t,J ) 6.8
Hz, 2H). HRMS calcd for C18H16N4O (MH+), 305.1397; found,
305.1385. Anal. (C18H16N4O‚1.2CF3CO2H‚1.3H2O) C, H, N.
4-[4-(4-Oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl)-
pyridin-2-yl]benzoic Acid Trifluoroacetate (31): Yellow solid,
1
110 mg, 42%. H NMR (300 MHz, DMSO-d6) δ 12.25 (s, 1H),
8.64 (d, J ) 5.4 Hz, 1H), 8.39 (s, 1H), 8.23 (d, J ) 8.5 Hz, 2H),
8.10 (d, J ) 8.3 Hz, 2H), 7.81 (d, J ) 5.2 Hz, 1H), 7.40 (s, 1H),
7.16 (s, 1H), 3.43 (t, J ) 6.1 Hz, 2H), 2.90 (t, J ) 6.6 Hz, 2H).
HRMS calcd for C19H15N3O3 (MH+), 334.1186; found, 334.1188.
Anal. (C19H15N3O3‚1.2CF3CO2H‚1.6H2O) C, H, N.
N-Cyclopropyl-4-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-
c]pyridin-2-yl)pyridin-2-yl]benzamide (32): Yellow solid, 230
1
mg, 76%. H NMR (300 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.63
(d, J ) 5.8 Hz, 1H), 8.59 (d, J ) 4.2 Hz, 1H), 8.38 (s, 1H), 8.18
(d, J ) 8.4 Hz, 2H), 7.99 (d, J ) 8.5 Hz, 2H), 7.81 (d, J ) 4.6 Hz,
1H), 7.43 (s, 1H), 7.17 (s, 1H), 3.43 (t, J ) 6.7 Hz, 2H), 2.95-
2.81 (m, 3H), 0.77-0.55 (m, 4H). HRMS calcd for C22H20N4O2
(MH+), 373.1659; found, 373.1638. Anal. (C22H20N4O2‚CF3CO2H‚
1.6H2O) C, H, N.
N-Cyclopentyl-4-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-
c]pyridin-2-yl)pyridin-2-yl]benzamide (33): Yellow solid, 210
1
mg, 65%. H NMR (300 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.64
(d, J ) 5.6 Hz, 1H), 8.46-8.36 (m, 2H), 8.19 (d, J ) 8.5 Hz, 2H),
8.03 (d, J ) 8.7 Hz, 2H), 7.85-7.74 (m, 1H), 7.44 (s, 1H), 7.17
(s, 1H), 4.33-4.18 (m, 1H), 3.43 (t, J ) 6.7 Hz, 2H), 2.90 (t, J )
6.8 Hz, 2H), 1.98-1.47 (m, 8H). HRMS calcd for C24H24N4O2
(MH+), 401.1972; found, 401.1982. Anal. (C24H24N4O2‚1.2CF3-
CO2H‚0.6H2O) C, H, N.
N-Cyclohexyl-4-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-
c]pyridin-2-yl)pyridin-2-yl]benzamide Trifluoroacetate (34):
Yellow solid, 90 mg, 36%. 1H NMR (300 MHz, DMSO-d6) δ 12.19
(s, 1H), 8.57 (d, J ) 5.8 Hz, 1H), 8.29 (m, 2H), 8.13 (d, J ) 8.4
Hz, 2H), 7.96 (d, J ) 8.4 Hz, 2H), 7.74 (s, 1H), 7.36 (s, 1H), 7.11
(s, 1H), 3.73 (m, 1H), 3.37 (t, J ) 6.7 Hz, 2H), 2.84 (t, J ) 6.8
Hz, 2H), 1.77 (m, 2H), 1.69 (m, 2H), 1.56 (m, 1H), 1.26 (m, 4H),
1.09 (m, 1H). HRMS calcd for C25H26N4O2 (MH+), 415.2129;
found, 415.2156. Anal. (C25H26N4O2‚CF3CO2H‚2.1H2O) C, H, N.
N-Benzyl-4-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]py-
ridin-2-yl)pyridin-2-yl]benzamide Trifluoroacetate (35): Yellow
1
solid, 240 mg, 70%. H NMR (300 MHz, DMSO-d6) δ 12.25 (s,
1H), 9.20 (t, J ) 6.4 Hz, 1H), 8.63 (d, J ) 5.6 Hz, 1H), 8.39 (s,
1H), 8.22 (d, J ) 8.4 Hz, 2H), 8.08 (d, J ) 8.4 Hz, 2H), 7.86-
7.78 (m, 1H), 7.42 (s, 1H), 7.37-7.14 (m, 6H), 4.52 (m, 2H),
3.43 (t, J ) 6.7 Hz, 2H), 2.90 (t, J ) 6.8 Hz, 2H). HRMS calcd
for C26H22N4O2 (MH+), 423.1816; found, 423.1811. Anal.
(C26H22N4O2‚CF3CO2H‚H2O) C, H, N.
4-(4-(4,5,6,7-Tetrahydro-4-oxo-1H-pyrrolo[3,2-c]pyridin-2-
yl)pyridin-2-yl)-N-phenethylbenzamide (36). A solution of 4-[4-
(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl)pyridin-2-
yl]benzoic acid trifluoroacetate 31 (200 mg, 0.77 mmol), EDCI
(164 mg, 0.86 mmol), and HOBt (116 mg, 0.86mmol) in 4.0 mL
of DMF was treated with phenethylamine (0.115 mL, 0.92 mmol)
Crystallization of MAPKAP Kinase-2 (MK-2). Human MAP-
KAP kinase-2 (MK-2) spanning amino acids 45-371 was expressed
in E. coli with an N-terminal hexa-histidine tag and purified by
immobilized metal affinity chromatography, followed by ion
exchange chromatography. Crystallization experiments of MK-2/
inhibitor complex (5 mg/mL of protein along with 1 mM of
compound 23 dissolved in DMSO) were set up at room temperature
by hanging drop vapor diffusion against a well solution of 1.6-
2.0 M sodium malonate, pH 5.4. Larger crystals with square-
bipyramidal morphology were grown by microseeding into clear
drops at lower precipitant concentrations. These crystals belong to
face-centered cubic lattice and have the space group, F4132, with

2654 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Anderson et al.
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lattice edges of 253.9 Å, with one molecule per asymmetric unit.
Complete diffraction data was collected to 3.5 Å resolution at
Advanced Photon Source IMCA CAT (sector 17). The structure
was solved by difference Fourier methods using the coordinates of
MK-2/AMP-PNP complex.16 Clear electron density was visible for
the ligand at the ATP site. The ligand was included in the final
rounds of refinement with final crystallographic R-factor 30.3% to
3.8 Å resolution. Coordinates for the complex of MK-2 with
compound 23 have been deposited in the Protein Data Bank under
PDB ID 2P3G.
Acknowledgment. We thank John E. Baldus and Ingrid P.
Buchler for chemical synthesis contributions; Robert P. Comp-
ton, Jeffrey L. Hirsch, Heidi M. Morgan, and Matthew J. Saabye
for enzymology support; Heidi R. Hope, Shelia C. Short, and
Jian Zhang for cell assay and signaling studies; Richard M.
Broadus for protein production; and Stephen J. Mnich, Sarah
A. South, Mark A. Thiede, and Elizabeth G. Webb for in vivo
model support. Use of the IMCA-CAT beamline 17-ID (or 17-
BM) at the Advanced Photon Source was supported by the
companies of the Industrial Macromolecular Crystallography
Association through a contract with the Center for Advanced
Radiation Sources at the University of Chicago. Use of the
Advanced Photon Source was supported by the U.S. Department
of Energy, Office of Science, Office of Basic Energy Sciences,
under Contract No. W-31-109-Eng-38.
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Supporting Information Available: Combustion analysis data,
kinase selectivity assay protocols, CACO-2 assay protocols, ATP
competition experiments, and Km determinations. This material is
available free of charge via the Internet at http://pubs.acs.org.
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