Tri- and tetrasubstituted imidazoles as p38α mitogen-activated protein kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.012

The synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles as potent p38α mitogen-activated protein kinase inhibitors is described. The trisubstituted imidazole series was found to be more potent than the tetrasubstituted imidazole seri

Full text of DOI:10.1016/j.bmcl.2010.09.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6671–6675
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Bioorganic & Medicinal Chemistry Letters
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Tri- and tetrasubstituted imidazoles as p38
kinase inhibitors
a mitogen-activated protein
Stefan Laufer a, , Dominik Hauser a,b, Thomas Stegmiller a, Claudia Bracht a, Kathrin Ruff a, Verena Schattel a,
⇑
Wolfgang Albrecht b, Pierre Koch a
a Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
b c-a-i-r biosciences GmbH, Paul Ehrlich Str. 15, 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 synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles as potent p38
activated protein kinase inhibitors is described. The trisubstituted imidazole series was found to be more
potent than the tetrasubstituted imidazole series. Many of these compounds show low-nanomolar activ-
a mitogen-
Received 13 August 2010
Revised 1 September 2010
Accepted 1 September 2010
Available online 9 September 2010
ities in the isolated p38
a MAP kinase inhibition assay. The structure–activity relationships between these
two series are different and not comparable.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
p38
MAP kinase
Pyridinylimidazoles
ATP competitive inhibitors
F
The p38a mitogen-activated protein (MAP) kinase, a serine/thre-
onine kinase, is a key enzyme of the cascade leading to pro-inflam-
F
CH3
N
N
N
N
S
matory cytokines such as interleukin-1b and tumor necrosis
S
factor-a
.1 Thesecytokinesare knowntobeinvolvedinthepathogen-
R2
N
esis of inflammatory disorders such as rheumatoid arthritis, inflam-
N
HN
matory bowel disease, and psoriasis.2–4 In peripheral mononuclear
R1
SKF86002
A
blood cells, the p38
of external stress stimuli like osmotic shock, heat, lipopolysaccha-
ride and other cytokines.5 Inhibition of p38
MAP kinase is therefore
a MAP kinase pathway is activated by a variety
R1 = acyl or alkyl
R
2 = alkyl
a
a promising therapeutic strategy to block the biosynthesis of these
pro-inflammatory cytokines.
Figure 1. Vicinal 4-fluorophenyl/pyridin-4-yl-substituted imidazoles A, derived
from the early lead SKF86002 [IC50(p38 ): 0.26
M],8 under investigation as
inhibitors of p38 MAP kinase.
a
l
As part of our lead optimization program, different 1,2,4,5-tetra-
substituted imidazoles (A) were identified which can be regarded as
open chain analogues of the prototypical dihydrothiazoline
SKF86002 (Fig. 1).6,7 These pyridinylimidazoles inhibit the kinase
by competing for the binding site of the substrate ATP. For optimal
interaction with the kinase, this class of inhibitors must possess a
4-pyridinyl moiety, a 4-fluorophenyl group, and an unsubstituted
N-atom in the imidazole ring adjacent to the 4-fluorophenyl group.
The 4-pyridinyl moiety interacts with the amino group on the main
chain of Met109 of the hinge region via a hydrogen bond while the
4-fluorophenyl ring is located at the hydrophobic region I (selectiv-
ity pocket) which is mediated by the gatekeeper amino acid Thr106
(Fig. 2). The imidazole N-3 accepts a hydrogen bond from the
a
1-alkyl-4-(4-fluorophenyl)-2-methylsulfanyl-5-pyridin-4-ylimi-
dazoles A possess an acylated or alkylated amino function at the
pyridine-C2 position which can act as a hydrogen bond donor to
the carbonyl function of Met109 and the moiety R1 addresses the
hydrophobic region II.
We recently reported that the carbonyl function of 2-acylami-
nopyridin-4-ylimidazoles contributes to the metabolic stability of
this structural class of compounds, in contrast to the analogous
2-alkylaminopyridin-4-ylimidazoles.7
Our initial approach was to investigate the structure–
activity relationship (SAR) of 2-acylaminopyridin-4-ylimidazoles
(A, R1 = acyl) targeting the moieties R1 and R2.
e
-amino group of Lys53 side chain. In contrast to SKF86002, the
The general synthesis to the 1,2,4,5-tetrasubstituted imidazoles
A (R1 = acyl) has been already described by our group.7,9 As shown
in Scheme 1, the putative inhibitors 7–29 and 37–51 were
⇑
Corresponding author. Tel.: +49 70701 72459; fax: +49 7071 5037.
E-mail address: stefan.laufer@uni-tuebingen.de (S. Laufer).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.012

6672
S. Laufer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6671–6675
For all compounds prepared in this Letter, we evaluated inhib-
itory potency of p38 MAP kinase with the immunosorbent-based
a
assay of Laufer et al.12 The p38
a MAP kinase activities for
compounds 7–29 are summarized in Table 1. Since R2 is directed
Table 1
Tetrasubstituted imidazoles: SAR of modifications to R2
F
CH3
S
N
N
R2
N
HN CH3
O
Compd
R2
p38a IC50 (l
M)a
Figure 2. p38
(hydrogen bonds are drawn in dashed lines).
a MAP kinase binding schematic for tetrasubstituted imidazoles A
7
8
OMe
OH
OH
0.11 0.01
0.11 0.04
0.15 0.05
0.14 0.01
9
F
F
F
10
OH
NOH
O
OH
c
a
b
11
12
1.49 0.64
6.28 1.50
OH
OH
OH
R2
O
O
N
N
N
N
4
1
2
3
NH2
HN CH3
O
HN CH3
N
N
R2
R2
OH
O
13
14
15
16
17
18
6.14 1.59
2.26 0.87
0.44 0.07
0.33 0.08
1.62 0.28
0.77 0.17
OH
F
F
F
O-
OH
O
H
N+
N
N
CH3
O
d
e
H
S
S
CH3
N
N
N
R2
R2
R2
N
N
N
O
O
O
HN CH3
HN CH3
HN CH3
CH3
O
5
O
6
O
7-29
7,30,31
O
F
F
OH
OH
g
f
CH3
CH3
N
N
N
N
S
S
O
R2
R2
N
N
N
N
N
N
19
20
1.14 0.12
1.20 0.15
NH2
HN R1
O
32-34
37-51
O
O
O
CH3
CH3
Scheme 1. Synthetic pathway toward 1,2,4,5-tetrasubstituted imidazoles 7–29 and
37–51. Reagents and conditions: (a) Ac2O, DMAP, reflux; (b) isoamyl nitrite, NaOMe,
MeOH, rt; (c) EtOH, reflux; (d) 2,2,4,4-tetramethylcyclobutane-1,3-dithione, DCM,
rt; (e) iodomethane, Na2CO3, EtOH, rt; (f) 10% HCl aq, reflux; (g) R1-COCl 35, NEt3,
THF, 0 °C or R1-COOH 36, CDI, N-methylpyrrolidinone, rt, then 120 °C.
21
22
2.24 0.69
1.57 0.19
O
synthesized starting from substituted ethanone 1. The amino func-
tion at the pyridine-C2 position was protected with an acetyl group
by heating 1 in acetic anhydride to reflux temperature. Compound
3 was prepared by reacting 2 with isoamyl nitrite and sodium
methoxide in methanol at room temperature. Different functional
groups at the imidazole-N1 position (R2) were introduced by using
several substituted triazinanes 4 which were synthesized accord-
ing to literature procedures.10,11 The imidazole N-oxides 5 were
converted into the thiones 6 by using 2,2,4,4-tetramethylcyclobu-
tane-1,3-dithione as the sulfur donor. The target compounds
7–29 were obtained by S-methylation with iodomethane. To vary
the acyl moiety (R1), the acetyl protecting group of compounds 7,
30,31 (R2 = methyl, ethyl, or methoxyethyl) was removed using
an excess of 10% aqueous hydrochloric acid at reflux temperature
to generate compounds 32–34.
O
N
O
O
CH3
23
24
0.060 0.006
0.28 0.03
CH3
N
O
O
25
26
0.78 0.25
0.32 0.09
OH
27
28
0.79 0.35
0.40 0.04
Starting from these compounds, different coupling methods
yielded the 2-acylaminopyridin-4-ylimidazole derivatives 37–51.
The target compounds were synthesized using the carboxylic acid
chlorides 35 or the corresponding carboxylic acids 36 after activa-
tion with N,N0-carbonyldiimidazole (CDI).
CH3
CH3
29
0.57 0.07
a
Mean SEM of three experiments.

S. Laufer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6671–6675
6673
toward the sugar pocket of the ATP binding site, we especially
F
F
chose flexible hydroxylated aliphatic moieties (8–13), which could
imitate the ribose part of ATP. The hydroxylated moieties (8–10)
show no improvement in inhibitory activity compared to 7. Modi-
CH3
N
N
CH3
N
N
S
a
S
N
n
OH
fication of R2 to
a-branched diols 12–13 resulted in only modest
N
n
Cl
n = 1,2,3
activity with IC50 values in the micromolar range. Docking results13
of compound 13 show that although the hydroxy groups can form
hydrogen bonds with the carbonyl functions of Ala111 and Ser154,
the bulky moiety at the imidazole-N1 position causes a steric hin-
drance within the ligand (Fig. 3). The pyridine moiety becomes
twisted, which may interfere with the entry of the inhibitor mole-
HN CH3
HN CH3
O
O
52-54
8-10
F
F
CH3
S
N
N
CH3
N
N
b
S
N
N
cule into the binding cleft of p38
enzyme–ligand interactions.
a MAP kinase and lead to weaker
OH
F
Br
55
HN CH3
O
HN CH3
O
9
Substitution of the imidazole-N1 position with propanoic acid
or propanoic acid derivatives (esters and amides) led to decreased
c
d
F
p38
derivatives 15 and 16 were superior to the carboxylic acid 14 in
terms of p38 MAP kinase potency. The introduction of cyclic moi-
a MAP kinase activity compared to compound 7. The ester
CH3
S
N
CH3
S
N
N
a
N
eties (compounds 25–28) at this position resulted in a weaker inhi-
bition compared to 7. The most potent inhibitor of this series,
compound 23, featured a one-carbon spacer between the N,N-
disubstituted amide group and the imidazole-N1 atom and possess
N
N
I
HN CH3
O
F
HN CH3
57
O
56
no substituent in a-position to the imidazole-N1 position. Inhibitor
23 is the only compound of this series with an IC50 value in the
double-digit nanomolar range.
Scheme 2. Synthetic pathway toward 1,2,4,5-tetrasubstituted imidazoles 52–57.
Reagents and conditions: (a) PPh3, hexachloroacetone, DCM; (b) CBr4, PPh3; (c) NaI,
acetone; (d) N,N-diethylaminosulfur trifluoride, DCM.
We then synthesized compounds 52–57 which bear an alkylha-
lide substituent at the imidazole-N1 position and tested their abil-
Table 2
ity to inhibit p38a MAP kinase. These compounds can serve as
Tetrasubstituted imidazoles: SAR of modifications to R2
intermediates for further derivatives and may interact via halogen
bonding14 with the enzyme. The synthetic pathway to this series is
depicted in Scheme 2. Compounds 8–10, which all bear a terminal
hydroxy group at the imidazole-N1 moiety but differ in chain
length, were used as starting materials for the synthesis of test
F
CH3
S
N
N
R2
N
compounds 52–57. The introduction of
a
chlorine atom
HN CH3
O
(compounds 52–54) was achieved using a modified Appel reac-
tion.15 Compounds 8–10 were treated with triphenylphosphine
and hexachloroacetone. The bromo compound 55 was synthesized
using the classic Appel reaction,16 using tetrabromomethane as the
bromine source. Compound 56 was synthesized by fluorination of
9 using N,N-diethylaminosulfur trifluoride17 in dichloromethane.
Finkelstein-reaction18 of 55 yielded the iodo compound 57.
The chlorine compounds 52–54 were first used to define the
favored chain length (Table 2). The inhibitory potency improved
Compd
R2
p38
a IC50 (lM)
a
52
53
54
55
56
57
0.15 0.04
Cl
Cl
0.017 0.002
0.18 0.002
0.027 0.012
0.18 0.09
Cl
Br
F
I
0.019 0.009
a
Mean SEM of three experiments.
when R2 was modified to incorporate three carbons. In the next
step, the influence of the halogen atom was investigated. However,
there were no significant differences in inhibitory activity among
the chloro, iodo, and bromo compounds. The fluoro derivative 56
showed a clear decrease in inhibitory activity compared to 53,
55, and 57.
The SARs of compounds 37–51 are listed in Table 3 and show
the influence of modifications at R1 and R2. The acetyl moiety of
7 was replaced in compounds 37–40 by an acrylamide, a propio-
nylamide, a trifluoropropionylamide, and a thiophene-2-carbox-
amide, respectively. Introduction of
a larger moiety at the
pyridine-C2 position (R1) resulted in a decrease of inhibitory activ-
ity, compare 7 versus 37–40. The methoxyethyl moiety at R2 may
cause a steric hindrance with larger moieties at R1. Thus, we short-
ened the moiety at the imidazole-N1 position. Compounds 41–51
bear only a methyl or an ethyl group at this position. Previous
experiments in a similar series identified moieties at R1 with a car-
bon spacer between the aminoacyl group and a phenyl ring as
favorable substituents at this position.7 Therefore, we chose
Figure 3. Proposed binding mode of 13 in the ATP binding site of p38a MAP kinase.
As the protein model, the X-ray structure 1YWR.pdb was used.

6674
S. Laufer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6671–6675
Table 3
F
F
F
Tetrasubstituted imidazoles: SAR of modifications to R1 and R2
H
N
O
3 steps
CH3
S
N
a
F
S
N
H
N
H
CH3
S
N
N
N
N
N
58
59
60
F
F
F
R2
N
F
F
HN
R1
CH3
N
c
CH3
S
N
a
Compd
37
R1
R2
p38a IC50 (lM)
b
S
N
H
N
H
OMe
N
CH2
CH3
F
0.17 0.05
0.18 0.04
N
O
HN R1
62-76
NH2
61
OMe
OMe
38
O
O
O
Scheme 3. Synthetic pathway to 2,4,5-trisubstituted imidazoles 62–76. Reagents
and conditions: (a) CH3I, Na2CO3, EtOH, rt; (b) NH4OH, 180 °C; (c) R1-COOH, CDI,
N-methylpyrrolidinone, rt, then 120 °C.
39
40
0.49 0.03
0.24 0.04
F
F
S
OMe
O
O
O
However, attempts to clarify the role of substituents at the pyr-
Cl
CH3
idine-C2 position (R1) in p38
a MAP kinase inhibition through SAR
41
42
0.10 0.01
comparisons between the imidazole N1-ethyl (41–44) and the
imidazole N1-methyl (45–51) series were not so straightforward.
While the dichloro, dimethoxy, and fluoro pyridine-C2 analogues
in the N1-ethyl series were generally better inhibitors than their
counterparts in the N1-methyl series, the aforementioned p-meth-
oxy compound 47 of the methyl series contradicted this trend.
In the next step of optimization, we synthesized compounds
62–76, which bear no substituent at the imidazole-N1 position.
The general synthesis to the 2,4,5-trisubstituted imidazole deriva-
tives 62–76 is depicted in Scheme 3. Methylation of the exocyclic
Cl
OMe
CH3
CH3
CH3
0.038 0.010
OMe
OMe
43
44
45
0.075 0.015
0.093 0.023
0.17 0.04
O
O
F
Cl
CH3
CH3
CH3
Table 4
Trisubstituted imidazoles: SAR of modifications to R1
Cl
O
O
F
OMe
CH3
S
N
46
47
0.28 0.06
N
H
OMe
OMe
N
HN
0.018 0.006
R1
O
O
a
Compd
R1
p38a IC50 (lM)
CH3
CH3
CH3
48
49
0.13 0.03
CH3
F
62
0.032 0.008
0.010 0.003
0.022 0.002
O
O
O
O
Cl
OMe
50
51
0.17 0.02
0.15 0.01
63
64
O
O
CH3
Cl
0.003 0.0004
a
Mean SEM of three experiments.
Cl
F
different substituted cinnamide and 2-phenylactamide moieties at
the pyrdine-C2 position.
65
66
0.027 0.002
0.009 0.0004
O
O
The introduction of different substituted 2-phenylamides and
cinnamides was well tolerated. Within the series of compounds
bearing an ethyl moiety at the imidazole-N1 position (41–44),
compound 42 with an (E)-3-(2,4-dimethoxyphenyl)acrylamide at
the pyridine-C2 position exhibited an IC50 value of 38 nM. In the
series of N1-methylated compounds (45–51), two compounds
could be identified with IC50 values in the low double-digit
F
CH3
F
67
68
0.014 0.002
0.10 0.01
O
O
nanomolar range. Compound 47 having
a (E)-3-(4-methoxy-
Cl
phenyl)acrylamide and 49 having a 2-(4-chlorophenyl)acetamide
moiety at the pyridine-C2 position exhibit IC50 values of 18 and
22 nM, respectively.
O

S. Laufer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6671–6675
6675
As shown in Table 4, the trisubstituted imidazoles were potent
inhibitors with IC50 values as low as the single-digit nanomolar
range. Compounds bearing a substituted 2-phenoxyacetamide
moiety (68–70) or a 3-phenylpropionamide moiety (74) at the
pyridine-C2 position show less inhibitory activity in comparison
to compounds having the rigid cinnamide moieties (62–65). Of
particular interest were compounds 64 and 66 bearing an (E)-3-(2,
6-dichlorophenyl)acrylamide and a 2-(4-fluorophenyl)acetamide
moiety at the pyridine-C2 position, respectively. Both compounds
Table 4 (continued)
a
Compd
R1
p38a IC50 (lM)
Cl
Cl
Cl
69
70
71
0.16 0.01
0.16 0.03
O
O
O
O
O
O
Cl
inhibited the p38a MAP kinase with IC50 values in the single-digit
O
nanomolar range.
0.053 0.017
0.027 0.004
0.090 0.014
In Figure 4 an overlay of the proposed binding modes of
compounds 44, 48 and 66 is depicted. All these compounds bear
a 2-(4-fluorophenyl)acetamide moiety at the pyridine-C2 position
but differ in the substitution pattern of the imidazole-N1 position.
The substituent at the imidazole-N1 position causes a rotation of
the pyridine moiety compared to the imidazole-N1 unsubstituted
compound. This may result in a weaker enzyme–inhibitor interac-
tion of the pyridine-C2 moiety and therefore in a decrease of inhib-
itory activity of the tetrasubstituted imidazoles.
72
73
O
O
CH3
CH3
CH3
H3C
CH3
CH3
In summary, we have reported p38a MAP kinase inhibition data
74
75
0.13 0.04
0.013 0.005
0.10 0.01
for different series of 1,2,4,5-tetrasubstituted and 2,4,5-trisubsti-
tuted imidazoles. The SARs between these two compound classes
are different and not readily comparable. The trisubstituted imid-
azole series was found to be more potent than the tetrasubstituted
imidazole series. Potent inhibitors with IC50 values down to 3 nM
were identified.
O
O
O
76
Acknowledgments
O
a
Mean SEM of three experiments.
We thank M. Goettert, Dr. S. Luik, Dr. S. Linsenmeier and K. Bauer
for providing the p38
a MAP kinase inhibition data. S. Bühler,
M. Gehringer, J. Schlosser, and R. Seelig are gratefully acknowledged
for helpful discussions.
References and notes
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Figure 4. Overlay of the proposed binding modes of inhibitors 44 (yellow), 48
(cyan) and 66 (gold) in the ATP binding site of p38
a MAP kinase. As the protein
model, the X-ray structure 1YWR.pdb was used.
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