The discovery of carboline analogs as potent MAPKAP-K2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.05.101

The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with IC₅₀s as low as 10 nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the p-lo

Full text of DOI:10.1016/j.bmcl.2007.05.101

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4664–4669
The discovery of carboline analogs as potent
MAPKAP-K2 inhibitors
Jiang-Ping Wu,* Ji Wang, Asitha Abeywardane, Denise Andersen, Michel Emmanuel,
Elda Gautschi, Daniel R. Goldberg, Mohammed A. Kashem, Susan Lukas, Wang Mao,
Leslie Martin, Tina Morwick, Neil Moss, Christopher Pargellis, Usha R. Patel,
Lori Patnaude, Gregory W. Peet, Donna Skow, Roger J. Snow, Yancey Ward,
Brian Werneburg and Andre White
Research and Development, Boehringer-Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877, USA
Received 9 April 2007; revised 21 May 2007; accepted 22 May 2007
Available online 7 June 2007
Abstract—The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with
IC50s as low as 10 nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the
p-loop and the hinge region of MK2a.
Ó 2007 Elsevier Ltd. All rights reserved.
Mitogen activated protein kinases (MAPKs) are signal
transduction molecules that control the expression of a
variety of gene products involved in inflammation, cell
proliferation, and apoptosis.1 Of the four major classes
of MAPKs,2 the p38 MAPK family activates a wide
range of regulatory proteins upon stimulation by exter-
nal signals. The p38 pathway has long been a drug dis-
covery target for pharmaceutical companies.3
inflammatory bowel disease.5 In fact, a number of phar-
maceutical companies have disclosed drug discovery
programs targeting this kinase.6
Our program to develop small molecule MK2 inhibitors
began with high throughput screening that identified
two carboline analogs, 1 and 2, as moderate MK2 inhib-
itors (Fig. 1).7 A simple combination of the structural
features of 1 and 2 produced a more potent analog 3.
This prompted us to start a chemistry program to fur-
ther explore this class of compounds. In this paper, we
report our SAR studies that led to a series of potent
carboline-based MK2 inhibitors.
MAPKAP-K2 (MK2), a substrate of p38 MAPKs, plays
central roles in p38 mediated signal transduction. In
mice genetically deficient in MK2, the production of
pro-inflammatory cytokines, such as tumor necrosis fac-
tor-a (TNF-a), interleukin-1b (IL-1b), IL-6, and inter-
feron-c (IFN-c), is inhibited significantly following
stimulation of splenocytes with lipopolysaccharide
(LPS). Moreover, this phenotype cannot be rescued by
the expression of a kinase dead MK2 mutant, indicating
that the kinase function of MK2 is required for pro-
inflammatory cytokine production.4 Thus, inhibitors of
MK2 kinase activity are potentially useful for treatment
of inflammatory diseases such as toxic shock syndrome,
rheumatoid arthritis, osteoarthritis, diabetes, and
Compounds in Tables 1–6 were prepared according to
Schemes 1 and 2. Reaction of methylacrylonitrile (4)
and diethyl malonate (5) under basic conditions provided
H
H
N
H
N
8
9
N
O
O
1
7
6
O
O
NH
S
NH
O
NH
2
5
4
3
1
2
3
IC50 = 30 µM
IC50 = 8 µM
IC50 = 2.7µM
Keywords: MAPKAP-K2; MK2; MK2a; Carboline.
*
Figure 1. MK2 inhibitors discovered via high throughput screening.
IC50s were obtained in a DELFIA assay.7
Corresponding author. Tel.: +1 203 798 4982; fax: +1 203 791
6072; e-mail: jwu@rdg.boehringer-ingelheim.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.101

J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
4665
Table 1. SAR at 6-position
Me
Me
CN
H
a
NH
b
N
+
O
EtO2C CO2Et
EtO2C
CN
O
EtO2C CO2Et
R6
NH
4
5
6
7
H
Me
R6
IC50 (lM)
N
O
Compound
R
+
R
c
d
20
21
22
23
24
17
25
26
27
H
>40
>40
R
NH
7
NH
NH2
N N
Me
Ph
H
O
Me
10
8
>40
15 13
>40
9
Br
Scheme 1. Reagents and conditions: (a) NaOEt, EtOH, rt, 88%; (b)
1—H2, Pt2O, EtOH, 2—100 °C, 70–80%; (c) 1—NaNO2, HCl, 0–
10 °C, 2—KOH, 70–80%; (d) formic acid, reflux, 50–70%.
HO
MeO
BnO
4.5 2.9
>40
>40
AcNH
H2N(CO)
5.4 2.9
H
H
N
O
N
O
R
N
Table 1). Removal of the carbonyl group at C1 diminished
potency (13). Methylation of either N2 or N9 gave inac-
tive compounds (14 and 15). At C4, replacement of the
gem-dimethyl group with a cyclobutyl group or removal
of one of the methyl groups did not show significant
effects (16 and 17). However, larger alkyl substituents
or substituents containing polar groups were not tolerated
(18 and 19) (Fig. 2). On the left side of the structure,
replacement of the C6–OMe group in compound 178
with H, Ph, Me, OH or AcNH abolished activity (20–
26). The larger benzyl ether analog 25 was also inactive.
The only C6–OMe replacement tolerated was an amide
group: compound 27 displayed potency comparable to
that of compound 17 (Table 1).
a
HO
NH RR'NH
+
R'
NH
O
O
Me
Me
11
12
Scheme 2. Reagents and conditions: (a) EDC, HOBT, DMAP (cat.),
DMF, rt to 60 °C, overnight 50–90%.
H
H
H
N
9
N
N
O
O
1
O
O
NH
N Me
O
NH
2
6
4
14
13
3
IC50 >40 µM
IC50 >40 µM
IC50 = 2.7 1.4 µM
The C6 amide moiety in 27 was further derivatized. A
series of aliphatic and aromatic amides were synthesized
(Table 2). Aliphatic amides were not active (29–31, 33,
and 34), the only exception being the methyl amide 28,
which was weakly active (IC50 = 12 lM). On the other
hand, aromatic or heteroaromatic amides mostly dis-
played better potency compared to the unsubstituted
amide 27 (32 and 35–42). Among the more potent ana-
logs were pyridyl amides 35, 36 and thiazole amide 37,
showing IC50 values below 1 lM.
H
N
Me
N
H
N
O
O
O
O
NH
O
NH
O
NH
17
IC50=4.5 2.9 µM
16
15
IC50=1.1 0.2 µM
IC50 >40 µM
H
N
H
N
O
O
O
NH
O
NH
Following up on these findings, we next examined the
effects of substitution on the aromatic amides. A series
of substituted phenyl amides were initially synthesized
(Table 3). Substitutions at C20 were not tolerated (43–
47). At C30, large groups reduced potency (49 and 50),
while smaller substitutions had no significant effect (51–
53 and 55–57). An exception was the 30-CN group, which
lowered the IC50 to 770 nM (54). At C40, most substitu-
tions showed no significant effects (58–64 and 66). How-
ever, amide and ester groups at this position improved
compound potency moderately (65, 67, IC50 < 1 lM).
AcO
19
18
IC50 > 40 µM
IC50 > 40 µM
Figure 2. Preliminary SAR.
compound 6. Hydrogenation of compound 6 followed by
heating gave a cyclic lactam 7. Compound 7 was next
reacted with a diazonium intermediate derived from
aniline 8 to generate hydrazone 9, which was refluxed
in formic acid to provide a carboline analog 10 (Scheme 1).
Amide analogs 12 were prepared via EDC mediated
coupling of carboline 6-carboxylic acid 11 and corre-
sponding amines (Scheme 2).
Encouraged by the beneficial effects of amide and ester
groups, we next incorporated these groups onto pyridyl
and thiazole amides 35 and 37. As shown in Table 4,
neither ester nor amide moiety improved the potency
of pyridyl amide 35 (68 and 69). However, an amide
substitution increased the potency of thiazole analog
37 significantly: compounds 71 and 73 gave IC50 values
of 83 and 120 nM, respectively.
Our initial SAR studies were aimed at identifying
structural features exploitable for improving compound
potency. Thus, the major functional groups in
compound 3 were individually modified (Fig. 2 and

4666
J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
Table 3. Effects of substitution on phenyl amide
Table 2. C6-amide analogs
H
H
N
R2'
N
O
O
H
R3'
N
R'
NH
NH
R4'
O
O
0
0
0
R2
R3
R4
IC50 (lM)
Compound
R0
IC50 (lM)
Compound
27
H2N
5.4 2.9
32
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.9 0.1
>20
Me
F
H
H
9.7 1.9
>10
>10
Br
CN
H
28
29
30
31
32
33
34
35
36
37
38
39
MeHN
Me2N
12
1
H
CONH2
H
H
3.0 (n = 1)
3.4
>20
Me
i-Pr
t-Bu
F
>20
>20
>20
H
H
>20
H
1.7 (n = 1)
1.3 0.4
2.7 (n = 1)
0.77 0.57
7.4 0.2
4.4 (n = 1)
2.7 (n = 1)
3.0 0.7
7.7 0.2
>20
N
H
Cl
Br
H
H
H
CN
CO2Et
AcNH
CONH2
H
H
N
H
H
H
Me
i-Pr
2.9 0.1
>20
N
H
H
H
H
H
t-Bu
F
H
H
3.0 (n = 1)
1.7 0.6
7.0 (n = 1)
1.7 0.5
0.93 0.02
1.8 (n = 1)
0.62 0.13
N
H
H
H
Cl
Br
H
H
H
H
CN
N
>20
H
H
CO2Me
AcNH
CONH2
H
H
H
H
H
N
0.52 0.15
0.76 0.17
0.82 0.26
1.50 (n = 1)
1.50 0.14
N
H
N
N
H
amide nitrogen lowered potency a few fold (75), sug-
gesting that one of the amide N–H might be engaged
in a binding interaction with MK2. Several analogs
containing polar groups were also prepared, leading
to compounds with improved potency (76–81,
IC50 ꢀ 20–30 nM). Larger lipophilic groups, such as
cyclohexyl group in 83, also had beneficial effect.
Incorporation of an N-methyl amino group into the
6-membered ring further improved potency, providing
compound 85 with an IC50 of 10 nM. Note that com-
pound 84, which contains a polar group and a 6-mem-
bered moiety but lacks an amide N–H, was less active
than the parent compound 71, again demonstrating
the importance of the amide N–H. Similar modifica-
tions to compound 73 did not show beneficial effects.
In fact, substitution of the amides with polar groups
reduced activity (86 and 87).
N
N
H
S
O
N
N
N
H
N
N
H
N
40
0.44 0.11
N
N
H
N
41
42
>20
N
N
H
N
1.5 0.8
An X-ray crystal structure of MK2a—compound 76
N
S
9
H
˚
complex was obtained (2.9 A resolution, Fig. 3).
The structure revealed that compound 76 binds be-
tween the p-loop and residues V118, T206, L193,
and Y194 (all not shown), and interacts with the hinge
region M138–D142. The oxygen atom of the internal
carbonyl amide (at C6) is in close proximity to the
Further derivatization of the thiazole amides followed
(Table 5). For analogs of 71, addition of one methyl
group to the amide nitrogen gave a compound of sim-
ilar potency (74). However, dimethylation of the
˚
NH of L141 (2.6 A) for H-bonding. The thiazole aryl
is nearly co-planar with the carboline scaffold, with

J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
4667
Table 4. Substituted pyridyl and thiazole amides
Table 5. Substituted thiazole amides
H
H
N
N
O
O
H
H
N
N
N
R4'
R5'
NH
R"
NH
S
O
O
Compound
R00
IC50 (lM)
0
0
Compound R4
R5
IC50 (lM)
71
74
75
76
77
78
79
80
NH2(CO)–
MeNH(CO)–
Me2NC(O)–
H
H
H
H
H
H
H
H
0.089 0.027
0.072 0.014
0.333 0.085
0.034 0.014
0.017 0.002
0.035 0.010
0.124 0.054
0.021 0.009
35
0.52 0.15
N
O
Me2N(CH2)2NHC(O)–
H2N(CH2)2NHC(O)–
Me2N(CH2)3NHC(O)–
H2N(CO)CH2NHC(O)–
H2N(CO)(CH2)2
68
69
37
70
71
72
73
0.76 0.13
0.62 0.27
0.82 0.26
0.75 (n = 1)
0.083 0.028
0.77 0.33
0.12 0.01
O
N
H2N
O
N
NHC(O)–
HO(CH2)2NHC(O)–
Me(CH2)2NHC(O)–
81
82
H
H
0.031 (n = 1)
0.045 0.018
N
S
O
O
O
N
83
84
85
H
H
H
0.044 0.004
0.25 0.10
N
H
S
O
O
N
N
N
S
H2N
O
N
N
O
O
0.010 0.001
N
H
S
N
73
86
H
H
H2NC
(O)–
0.13 0.02
H2N
S
O
Me2N
(CH2)2
NHC
1.3 (n = 1)
(O)–
87
H
Me2N
(CH2)3
NHC(O)–
0.48 0.07
dihedral angle of ꢀ17°. The external amide (at 40 of
thiazole moiety) is nearly co-planar to the thiazole
ring and presents its NH proton inward, forming a
˚
second H-bond with L70 (3.1 A). However, the por-
tion beyond the external amide bond, which appar-
ently contributes to compound potency, is not
shown to engage in binding interactions. Additionally,
the carboline amide oxygen [C1@O] and nitrogen
(N2), shown by SAR to be essential for compound
potency, are not within bonding distances to nearby
amino acid residues or peptide backbones.
Table 6. Selectivity and cellular activities
Compound MK2 IC50
(lM)
MK1 IC50 ERK2 IC50 THP-1
(lM) (lM) IC50 (lM)
71
76
78
79
83
85
35
0.089 0.027 0.75 0.01 0.56 0.15 >5
0.034 0.014 0.66 0.14
0.017 0.002
0.035 0.010 0.80 0.21
1.9 0.0
1.3 0.1
1.5 0.4
>5
>5
Selected compounds were tested for cellular activity
and kinase selectivity (Table 6). In an assay that mea-
sures LPS-stimulated TNF-a production from THP-1
cells,10 the majority of these compounds did not show
measurable activity (IC50 > 10 lM). Compound 83, an
analog without a terminal polar group, was among
the few that were active in the assay.11 In the MK1
and ERK2 kinase assays,12,13 these compounds showed
moderate activity (IC50 ꢀ 1 lM). Improving cellular
activity and kinase selectivity thus became the focus
of our subsequent chemistry efforts, which will be re-
ported in future publications.
—
>5
1.60 0.00
>5
0.044 0.004
0.011 0.001
0.52 0.15
—
—
—
—
1.2 0.9
0.21 0.06 >5
in the low nanomolar range. A crystal structure revealed
that this compound class binds between p-loop and res-
idues V118, T206, L193, and Y194, and interact with the
hinge region of MK2a (M138–D142). These compounds
are moderately selective against MK1 and ERK2. How-
ever, most of these analogs are not active in a cellular
(THP-1) assay.
In conclusion, we have discovered a series of potent
carboline-based MK2 inhibitors. In a DELFIA, the
most active compounds inhibit MK2 with IC50 values

4668
a
J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
quenching with excess DTT and purification by Sephadex
b
G-25 gel filtration. A mouse monoclonal anti-phospho-
LSP1 IgG1 antibody was generated to mcKLH (Pierce)
conjugated phospho-peptide antigen, CRTPKLARQA-
pS-IELPSM, from AnaSpec. The anti-phospho LSP1
IgG1 antibody was labeled with the Eu-chelate of N1-(p-
isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tet-
raacetic acid (Perkin Elmer Life Sciences) for the DELFIA
of MK2 catalysis and inhibition. Compounds and acti-
vated GST-MK2a 1-400 (1 nM) were incubated in neutra-
vidin coated plates (Pierce) at 25 °C in the following 40 lL
reaction mixture for 30 min: 50 mM HEPES (pH 7.6),
50 mM KCl, 10 mM MgCl2, 100 lM Na3VO4, 0.01%
CHAPS, 1 mM DTT, 10 lg/mL bovine serum albumin, 1%
DMSO, 500 nM biotinylated GST-LSP1 179–339, and
2 lM ATP. The plates were washed with 25 mM Tris–HCl
(pH 7.5), 150 mM NaCl, and 0.05% Tween 20. Eu-chelated
anti-phospho-LSP1 IgG1 antibody (0.5 mg/mL) was
diluted 1:20,000 in 50 mM Tris–HCl (pH 7.5), 150 mM
NaCl, 10 lM DTPA, 0.05% Tween 40, 0.2% bovine serum
albumin, and 0.05% BGG; 40 lL of the diluted antibody
solution was added to the plates. After 1-h incubation at
25 °C, the plate was washed prior to the addition of
DELFIA enhancement solution (Wallac #4001-0010), and
read after 15 min at exk = 360 nm and emk = 620 nm.
8. The synthesis of the C4-monomethyl analog 17 is
considerably easier than that of C4-gem-dimethyl analog
3. Since compounds 17 and 3 are comparable in potency,
we chose compound 17 as the reference compound for
initial SAR studies. Later studies (to be published in
separate papers) showed that in more advanced struc-
tures, the C4-gem-dimethyl moiety improved potency
moderately.
M138
76
H108
K93
E104
D207
P-loop
(red)
L141
2.6 Å
D142
L70
3.2 Å
N191
E145
Figure 3. X-ray structure of MK2a [41–364] co-crystallized with
compound 76.
Supplementary data
Experimental procedures for crystal complex formation
and X-ray data collection; synthetic schemes for com-
pounds in Figure 2. This material is available free of
charge via internet. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.bmcl.2007.05.101.
References and notes
1. Robinson, M. J.; Cobb, M. H. Curr. Opin. Cell Biol. 1997,
9, 180.
2. There are four major families of MAPKs: (1) the
archetypal extracellular regulated kinases (ERKs), (2)
the c-jun N-terminal kinases (JNKs), (3) the p38 MAPKs,
and (4) the ERK5 or BigMAPKs, see: Tibbles, L. A.;
Woodgett, J. R. Cell. Mol. Life Sci. 1999, 55, 1230.
3. (a) Jackson, P. F.; Bullington, J. L. Curr. Topics Med.
Chem 2002, 2, 1009; (b) Cirillo, P. F.; Pargellis, C. A.;
Regan, J. Curr. Top. Med. Chem. 2002, 2, 1021, and
references cited therein.
9. (a) The X-ray coordinates have been deposited with RCSB
Protein Data Bank, deposition #2PZY; The protein used
in this study is a segment of MK2a containing residues 41-
364. For a detailed crystal structure of a different MK2
complex, in which MK2 is bound to its activator p38, see:
(b) White, A.; Pargellis, C. A.; Studts, J. M.; Werneburg,
B. G.; Farmer, B. T., II Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 6353.
10. For the THP-1 assay protocol, see: Regan, J.; Breitfelder,
S.; Cirillo, P.; Gilmore, T.; Graham, A. G.; Hickey, E.;
Klaus, B.; Madwed, J.; Moriak, M.; Moss, N.; Pargellis,
C.; Pav, S.; Proto, A.; Swinamer, A.; Tong, L.; Torcellini,
C. J. Med. Chem. 2002, 45, 2994.
4. Kotlyarov, A.; Yannoni, Y.; Fritz, S.; Laaß, K.; Telliez,
J.-B.; Pitman, D.; Lin, L.-L.; Gaestel, M. Mol. Cell. Biol.
2002, 22, 4827.
5. Dinarello, C. A. Nutrition 1995, 11, 492; . Rev. Infect. Dis.
1984, 6, 51; Curr. Opin. Pharmacol. 2004, 4, 378.
6. (a) A selection of patent and patent applications claiming
MK2 inhibitors is listed below Meyers, M. et al.
WO2005009370, US20050143371, US20050137220, US
20050101623; (b) Anderson, D. R. et al. WO2004055015;
WO2004054505; WO2004054504; US2004127519, US2004
127511, US2004 142978; (c) Vazquez, M. L. et al. US20
04127492; (d) Buzon, R. A. et al. WO2004020440; WO
2004020438; (e) Sato, H. et al. WO2005007092; (f)
Kataoka, K. et al. WO2004076458; (g) Aronov, A. et al.
WO2004037814; (h) Hodge, C. N. et al. WO2005/033102;
(i) Wyatt, P. et al. WO2005014554
7. A dissociation-enhanced lanthanide fluorescent immuno-
assay (DELFIA) of MK2 catalysis was employed to
determine the potency of MK2 inhibition, see: (a) Lukas,
S. M.; Kroe, R.; Wildeson, J.; Peet, G. W.; Frego, L.;
Davidson, W.; Ingraham, R. H.; Pargellis, C. A.; Labadia,
M. E.; Werneburg, B. G. Biochemistry 2004, 43, 9950; (b)
Assay protocol: The GST-MK2a 1–400 variant of MK2
was activated with p38a MAPK by methods similar to
those described previously7 and re-purified by GSH
affinity chromatography. The GST-LSP1 179–339 sub-
strate of MK2 was biotinylated with a 70-fold molar
excess of PEO-iodoacetyl biotin (Pierce), followed by
11. It is conceivable that poor membrane permeability
contributed to the lack of cellular potency for compounds
with terminal polar groups. This was investigated subse-
quently, the results of which will be published separately.
12. The selectivity against MK1a and ERK2 was determined
in an IMAP assay, see: (a) Sportsman, J.; Daijo, J.;
Gaudet, E. A. Comb. Chem. High Throughput Screening
2003, 6, 195; (b) Assay protocol: Compounds were diluted
with DMSO in kinase assay buffer (10 mM Tris–HCl (pH
7.2), 10 mM MgCl2, 0.01% Tween 20, and 1 mM DTT)
such that the final concentration of DMSO was 1%. The
diluted compounds were incubated with kinase, ATP and
peptide substrate in Corning 96-well half-area, black non-
binding surface microtiter plates in kinase assay buffer for
90 min at room temperature. The MK1a reaction con-
sisted of 250 pM MK1a (Upstate Catalog #14-509), 1 lM
ATP, and 100 nM FAM-S6 ribosomal protein-derived
peptide. The ERK2 reaction consisted of 100 pM ERK2
(Upstate Catalog #14-173), 2.5 lM ATP, and 100 nM
FAM-Erktide. The kinase reactions were halted by
addition of IMAP progressive binding reagent in each
well. After 30 min of incubation at room temperature, the
plates were read for fluorescence polarization on an

J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
4669
Analyst Plate Reader using exk = 485 nm, emk = 530 nm,
and FL 505 dichroic mirror.
13. MK1 and ERK2 were selected as primary targets
for kinase selectivity testing. This was mainly due
to our desire to avoid inhibiting kinases involved in
growth differentiation. The close homology of MK1
to MK2 was another reason for screening against
this kinase.