Investigations of SCIO-469-like compounds for the inhibition of p38 MAP kinase

Article abstract of DOI:10.1016/j.bmcl.2009.01.023

The p38 MAP kinase is implicated in the release of the pro-inflammatory cytokines TNFα and IL-1b. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn's disease. A new lead structure for p38 MAP kinase inhibition was identified. Herein, we report the SAR of this new class of p38 inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.01.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1461–1464
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigations of SCIO-469-like compounds for the inhibition of p38 MAP kinase
*
Stefan Laufer , Frank Lehmann
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The p38 MAP kinase is implicated in the release of the pro-inflammatory cytokines TNF
a and IL-1b.
Received 3 December 2008
Revised 8 January 2009
Accepted 9 January 2009
Available online 14 January 2009
Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid
arthritis and Crohn’s disease. A new lead structure for p38 MAP kinase inhibition was identified. Herein,
we report the SAR of this new class of p38 inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
p38 MAPK
SAR
Indoles
The p38 mitogen-activated protein (MAP) kinase is a member of
the serine/threonine kinase superfamily that includes extracellular
signal regulated kinase-2 (ERK2) and c-Jun N-terminal kinase
(JNK). Activation of p38 under a variety of conditions (such as
external stimuli, inflammatory cytokines, heat, and UV light) re-
sults in the bis-phosphorylation of the Ser-Thr amino acids in the
activation loop.1 Subsequently, activation of other downstream ki-
nases and transcription factors leads to mRNA stabilisation and an
increase or decrease in the expression of certain target genes.2 To
p38
LCK.
a
inhibitor with >1000-fold selectivity versus ERK2, JNK1, and
A distinctive feature of the indole amide class of p38 inhibitors
relative to others (e.g., pyridinylimidazoles, diarly ketones) is the
ability to achieve high selectivity toward the p38
the p38b isoform. SX-011 has 10-fold p38 /b selectivity whereas
the azaindole derivative of SX-011 shows 249-fold p38 /b selectiv-
inhibitors reported
a isoform versus
a
a
ity and is one of the most highly selective p38
a
(Fig. 1).
date, four splice variants of p38 MAPK are known (p38
p38 , and p38d). Although the role of the ubiquitously expressed
isoform p38 during inflammation has been defined, the functions
a
, p38b,
The proposed binding mode for SCIO-469 involves interaction of
the C5 carbonyl interacting with p38 Met109 via a hydrogen
bond.4 The lipophilic benzylpiperazine is proposed to occupy the
selectivity pocket of the kinase. The ortho substituent on the indole
c
a
of the other isoforms are not well understood.
The discovery that p38 MAPK is a member of the stress-acti-
vated signal transduction pathway and experiments with small-
molecule p38 inhibitors (SB-203580) validated this kinase as an
important anti-inflammatory therapeutic target. Numerous ana-
logues of SB-203580, as well as a variety of alternate scaffolds,
have been reported as potent and selective inhibitors of p38
MAPK.3 In addition, several of these inhibitors were shown to be
effective as anti-inflammatory agents when evaluated in animal
models of acute and chronic diseases.
Several companies have reported preliminary human clinical
results for p38 MAPK inhibitors. Scios Inc. is a leader in this field,
with the advancement of SCIO-469 into Phase II human clinical tri-
als for the treatment of pain, multiple myeloma, and rheumatoid
ring may be instrumental in achieving the high p38a selectivity.
This prediction is based on the proposal that the ortho substituent
occupies a hydrophobic pocket that is near the Ala40 residue in
N
O
O
O
F
F
N
Cl
N
N
SCIO-469
N
O
O
O
arthritis. SCIO-469 modestly inhibits LPS-induced TNFa production
in human whole blood with an IC50 = 300 nM. SCIO-469 is a potent
N
Cl
N
SX-011
* Corresponding author.
E-mail address: stefan.laufer@uni-tuebingen.de (S. Laufer).
Figure 1. p38 inhibitors SCIO-469 and SX-011.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.023

1462
S. Laufer, F. Lehmann / Bioorg. Med. Chem. Lett. 19 (2009) 1461–1464
p38a. Because p38b contains a larger, more hydrophilic serine res-
idue at the analogous position, this isoform was proposed to be less
H2N
N
H
likely to accommodate the ortho substituent, which leads to in-
creased selectivity towards p38a versus p38b. The proposed bind-
a
ing orientation of this class of inhibitors was confirmed recently.5
Scios Inc. reported structure–activity relationship (SAR) studies
of a series of indole-based heterocyclic inhibitors.6 The inhibitory
activities of indole-3-, indole-4-, indole-5-, indole-6-, and indole-
7-carboxamides were studied. In order to close the gaps of these
study and based on modeling results, we investigated some syn-
thetic modifications of the Scios template. Here, we report a new
lead structure of p38 inhibitors derived from SCIO-469.
O
F
N
N
H
N
N
Boc
6
Scheme 2. Synthesis of tert-butyl 5-((4-(4-fluorobenzyl)piperazine-1-carboxa-
mido)methyl)-1H-indole-1-carboxylate (6). Reagents and conditions: (a) 1—
(Boc2)O, DMAP, CH2Cl2, rt, 20 min; 2—4-fluorobenzylpiperazine, 40 °C, 14 h, 38%.
The first attempted synthetic approach to obtain the urea deriv-
ative is outlined in Scheme 1. 5-Amino-1-methyl-1H-indole (3)
was prepared in good yields with previously described methods.7
For the urea formation, we adopted the procedure from Knölker
et al. Therefore, 3 was reacted with di-tert-butydicarbonate, with
4-dimethylaminopyridine (DMAP) as catalyst, to form in situ an
isocyanate, which was treated with 4-fluorobenzylpiperazine to
COOEt
N
H
b
a
obtain
(4-(4-fluorobenzyl)-N-(1-methyl-1H-indole-5-yl)pipera-
O
Cl
zine-1-carboxamide (4) in excellent yields. The introduction of
the oxalylamide residue was accomplished by standard proce-
dures. Because urea has a fixed binding angle, we wished to syn-
thesize the urea with a benzylic CH2 group before the indole
nucleus. Starting from commercially available 5-(aminomethyl)-
1H-indole, we could only obtain the Boc-protected urea (6)
(Scheme 2). Attempts to gently cleave the protecting group re-
sulted also in cleavage at the benzylic group of the molecule.
Subsequently, we prepared a set of heterocyclic 2-amides. The
synthesis of the amides was done under standard conditions with
N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDAC) and DMAP in dichloromethane to activate the carboxylic
acids (Scheme 4).8 The acetyl-substituted indoles were obtained
by Friedel–Crafts acylation of ethyl 1H-indole-2-carboxylate or
ethyl 3-chloro-1H-indole-2-carboxylate (10) (Scheme 3). In this
manner, the different isomers were generated in the same ratio
and could be separated via column chromatography (silica gel) with
dichloromethane as eluent. The produced esters (7–9, 11, 12) were
saponified with sodium hydroxide in ethanol and subsequently con-
verted to the corresponding amides using the standard procedure
(Scheme 4). 4-Bromo-1H-indole-2-carboxylic acid, which is re-
quired to obtain the amide 16, was synthesized accordingly to the
described procedure.9 Benzofuran-2-carboxylic acid, which is re-
quired for amides 20 and 21, was prepared analogous to the method
from Ashram.10 The precursor for amide 19 was obtained by previ-
COOEt
COOEt
COOEt
N
H
N
H
7: 3-Ac
8: 5-Ac
9: 7-Ac
10
c
O
Cl
N
H
11: 5-Ac
12: 7-Ac
Scheme 3. Synthesis of the acetyl-substituted indole-2-carboxylates. Reagents and
conditions: (a) AcCl, AlCl3, CH2Cl2, reflux, 10 h; (b) NCS, acetone, rt, 1 h; (c) AcCl,
AlCl3, CH2Cl2, reflux, 10 h.
O
R1
a
COOH
R2
R1
N
X
Scheme 4. Synthesis of heterocyclic 2-amides. Reagents and conditions: (a) EDAC,
DMAP, 4-fluorobenzylpiperazine or benzylpiperazine or benzylpiperidine, CH2Cl2,
rt, 14 h.8
N
O
O2N
O2N
H2N
F
a
b
c
d
N
N
H
N
N
H
N
N
1
2
3
4
N
O
O
N
O
F
N
N
H
N
5
Scheme 1. Synthesis of the urea derivatives 4 and 5. Reagents and conditions: (a) dimethyl carbonate, potassium carbonate, DMF, reflux, 2 h, 96%; (b) Pd/C 10%, EtOH, 40 °C,
4 h, 85%; (c) 1—(Boc2)O, DMAP, CH2Cl2, rt, 20 min; 2—4-fluorobenzylpiperazine, 40 °C, 14 h, 77%; (d) 1—oxalyl chloride, CH2Cl2, rt, 3 h; 2—dimethylamine, CH2Cl2, rt, 30 min,
94%.

S. Laufer, F. Lehmann / Bioorg. Med. Chem. Lett. 19 (2009) 1461–1464
1463
O
N
X
N
a
28: X = C
29: X = N
N
N
O
N
X
F
F
X
N
H
b
c
N
H
N
30: X = C
N
F
27: X = N
R
O
N
X
31: X = C, R = Cl
32: X = C, R = Br
33: X = C, R = I
N
H
N
N
F
d
R
O
N
X
N
34: X = C, R = Br
F
Scheme 5. Derivatization of amides. Reagents and conditions: (a) 1—NaH, THF, 0 °C, 1 h; 2—MeI, rt, 12 h, 83% (28), 96% (29); (b) LiAlH4, THF, reflux, 3 h, 49%; (c) NCS or NBS or
NIS, acetone, rt, 1 h, 95–97%; (d) 1—NaH, THF, 0 °C, 1 h; 2—MeI, rt, 12 h, 90%.
ously described procedures (Table 1).11 As outlined in Scheme 5,
some derivatizations of the obtained amides were executed.
N-Methylation of (4-(4-fluorobenzyl)piperazine-1-yl)(1H-in-
dole-2-yl)methanone (13) was accomplished with sodium hydride
and methyl iodide in tetrahydrofuran to yield compound 28. The
synthesis for the 1H-benzo[d]imidazole amide (27) began with
benzene-1,2-diamine, which was reacted with methyl 2,2,2-tri-
chloroacetimidate to obtain 2-(trichloromethyl)-1H-benzo[d]imid-
azole according to the procedure of Venable et al.12
Subsequent reaction with 4-fluorobenzylpiperazine resulted in
the amide formation. The N-methylation of 27 was accomplished
analogous to the indole amide. The reduction of the carbonyl func-
tion was carried out with lithium aluminum hydride in refluxing
tetrahydrofuran to obtain 2-((4-(4-fluorobenzyl)piperazin-1-yl)-
methyl)-1H-indole (30). We were able to chlorinate, brominate,
and iodinate the amides in high yields with a simple and fast syn-
thesis using N-chlorosuccinimide, N-bromosuccinimide, and N-
iodosuccinimide, respectively, in acetone at room temperature.
According to the developed method,13 25 compounds were
tested for anti-p38 activity at concentrations ranging from 10ꢀ5
to 10ꢀ8 M. Pyridinyl-imidazole SB-203580 was used as a reference
compound, and the optimized ATP concentration at which the test
factor-2) on microtiter plates, addition of the kinase reaction mix-
ture, and measurement of substrate phosphorylation by a two-step
antigen-antibody reaction in which primary antibody binds to the
doubly phosphorylated (Thr69 and Thr71) ATF-2 and acts as antigen
for the secondary antibody. The secondary antibody is conjugated
to alkaline phosphatase, which is able in the last step to dephos-
phorylate 4-nitrophenolphosphate disodium salt (4-NPP). 4-Nitro-
phenol was detected by an ELISA reader at 405 nm.
The biological test results (Table 2) show that the ureas 4 and 5
were inactive against p38 MAPK. This result arose from the fixed
binding angle of the urea group. The urea 6, which was extended
with a CH2 group to make the indole nucleus freely rotatable, also
showed no inhibition. This finding can be explained by the pres-
ence of a bulky Boc-group that cannot be cleaved synthetically.
In the heterocyclic 2-amide series, the following SAR can be
concluded. The p-fluoro substituent improved the inhibitory activ-
ity (e.g., compounds 13 and 14). Comparison of indole-2-amides 14
and 15 reveals that the piperidine-amides are more potent than
the corresponding piperazine-amides. Another improvement in
the inhibitory activity was achieved through N-methylation. The
N-methylated derivative (28) was three times more potent than
the unmethylated compound (13). In general, substituents at the
3-position (compounds 22, 31, 32, and 33) were not preferred com-
pared with the unsubstituted analogues. All 2-furanyl- (17, 18),
was performed was 100 lM. Briefly, the assay involved the immo-
bilization of the kinase substrate ATF-2 (activating transcription
Table 2
Inhibition of p38 MAPK by heterocyclic 2-amides
Table 1
Synthesized heterocyclic 2-amides
Compound
IC50 SEMa
[lM]
Compound
IC50 SEMa
[lM]
28%b
23
24
25
26
27
28
29
30
31
32
33
34
45%b
Compound
R1
X
R2
Yield (%)
4
5
6
12%b
3.61 0.98
6.51 0.35
2.46 0.18
24%b
13
14
15
16
17
18
19
20
21
22
23
24
25
26
2-Indolyl
2-Indolyl
2-Indolyl
4-Bromo-2-indolyl
2-Furanyl
N
N
C
N
N
N
N
N
C
N
N
N
N
N
F
61
31
80
72
44
42
56
16
37
27
64
94
30
63
8%b
H
H
F
F
H
F
F
H
F
F
F
13
14
15
16
17
18
19
20
21
22
3.20 1.04
5.77 1.56
1.09 0.28
34%b
1.36 0.34
32%b
2-Furanyl
35%b
25%b
Imidazo[1,2-a]pyridine-2-yl
2-Benzofuranyl
2-Benzofuranyl
3-Acetyl-2-indolyl
5-Acetyl-2-indolyl
7-Acetyl-2-indolyl
5-Acetyl-3-chloro-2-indolyl
7-Acetyl-3-chloro-2-indolyl
20%b
7.57 0.13
6.46 0.37
7.47 0.88
2.24 0.35
33%b
8.42 1.21
1.75 0.32
24%b
F
F
a
% Number of determinations were three.
% Inhibition at 10 lM.
b

1464
S. Laufer, F. Lehmann / Bioorg. Med. Chem. Lett. 19 (2009) 1461–1464
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.023.
References and notes
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Figure. 2. Proposed binding mode for 15 (pink) in overlay with the ligand from
pdb-ID
2qd9
(grey;
1-[5-[[3-(2,4-difluorophenyl)-6,8-dihydro-5H-imidazo
[5,1-c]pyrazin-7-yl]carbonyl]-6-methoxy-3aH-pyrrolo[5,4-b]pyridin-3-yl]-2-[(3R)-
3-hydroxypyrrolidin-1-yl]ethane-1,2-dione. After geometric optimization in the
MMFF94 force field, the molecule has been docked in the p38 binding pocket by
using the software AutoDock 4.0.14 The picture was generated using PyMOL.
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8. General synthesis of heterocyclic 2-amides: To a solution of 1.00 equiv of the
corresponding carboxylic acid in 15 mL absolute dichloromethane was added
imidazo[1,2-a]pyridin-2-yl- (19), and 1H-benzo[d]imidazol-2-yl-
(27, 29) derivatives were inactive against p38 MAPK. Whereas 2-
benzofuranyl-derivatives (20, 21) were equipotent to the indole-
amides. The acetyl-group on the indole nucleus was best tolerated
at the 7-position (compare compounds 22, 23, and 24) A chloro-
substituent at the 3-position of an indole further improved the
inhibitory activity (compound 26).
In Figure 2, the suggested binding mode of compound 15 (pink)
is displayed. Noteworthy is the similarity with the binding mode of
the ligand from pdb-ID 2qd9 (grey). Compound 15 forms hydrogen
bonds to the hinge region (Met109 and Gly110) of p38 MAPK. The
position is equally to the ligand from the X-ray structure and
shows that either the 4- or the 7-position of our heterocyclic 2-
amides should be substituted with the oxalic amide residue, which
improve the inhibitory activity in SCIO-469.
at
room
temperature
1.40 equiv
N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride, 1.40 equiv triethylamine, 0.20 equiv 4-
dimethylaminopyridine and 1.00 equiv 4-fluorobenzylpiperazine. The
mixture was stirred for 14 h at room temperature. Following it was
quenched with water and extracted three times with dichloromethane. The
organic layer was dried over sodium sulfate and evaporated. If necessary the
raw product was purified through column chromatography. The identity and
purity of the target compounds were evaluated by 1H, 13C NMR, MS, IR, mp and
HPLC. All experimental procedures and analytical data are given in Supporting
Information.
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First investigations of the inhibitory activities and structure–
activity relationships of heterocyclic 2-amide inhibitors for p38
MAPK were made. Novel inhibitors for p38 were identified that
should be optimized in further studies.