Design, synthesis, and biological evaluation of novel tri- and tetrasubstituted imidazoles as highly potent and specific ATP-mimetic inhibitors of p38 MAP kinase: Focus on optimized interactions with the enzyme's surface-exposed front region

Article abstract of DOI:10.1021/jm701529q

The synthesis, biological testing, and SAR of novel 2,4,5- and 1,2,4,5-substituted 2-thioimidazoles are described. Amino, oxy, or thioxy substituents at the 2-position of the pyridinyl moiety were evaluated for their contributions to inhibitor potency and

Full text of DOI:10.1021/jm701529q

4122
J. Med. Chem. 2008, 51, 4122–4149
Design, Synthesis, and Biological Evaluation of Novel Tri- and Tetrasubstituted Imidazoles as
Highly Potent and Specific ATP-Mimetic Inhibitors of p38 MAP Kinase: Focus on Optimized
Interactions with the Enzyme’s Surface-Exposed Front Region
Stefan A. Laufer,* Dominik R. J. Hauser, David M. Domeyer, Katrin Kinkel, and Andy J. Liedtke
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard-Karls-UniVersity Tuebingen, Auf der Morgenstelle 8,
72076 Tuebingen, Germany
ReceiVed December 7, 2007
The synthesis, biological testing, and SAR of novel 2,4,5- and 1,2,4,5-substituted 2-thioimidazoles are
described. Amino, oxy, or thioxy substituents at the 2-position of the pyridinyl moiety were evaluated for
their contributions to inhibitor potency and selectivity against p38 mitogen activated protein kinase (p38
MAPK) as well as for the ability to minimize cytochrome P450 (CYP450) inhibition. Incorporation of
polar substituted (cyclo)aliphatic amino substituents (e.g., tetrahydropyranylamino), which positively interacted
with the surface-exposed front region (hydrophobic region II) of the enzyme led to the identification of
extremely potent p38 MAPK inhibitors with p38 IC50 values in the low nanomolar range. Approximately
90 pyridinylimidazole-based compounds with a range of potencies against p38R MAP kinase were further
investigated for their ability to inhibit the release of tumor necrosis factor-R (TNFR) and/or interleukin-1ꢀ
(IL-1ꢀ) from human whole blood. Some of the most promising drug candidates underwent selectivity profiling
against a panel of 17 different kinases besides p38R and/or were tested for their interaction potential toward
a number of metabolically relevant CYP450 isozymes.
Introduction
Protein kinases are critical enzymes in cellular signal trans-
duction cascades. Numerous diseases including inflammatory
and autoimmune diseases such as rheumatoid arthritis (RA) or
inflammatory bowel disease IBD), neurodegenerative conditions,
cardiovascular events, and even cancer could be associated with
atypical regulation of protein kinase-mediated cell signaling.1
The progress of chronic inflammation is driven by an amplified
systemic occurrence of several proinflammatory cytokines like
tumor necrosis factor-R (TNFR) and interleukin-1ꢀ (IL-1ꢀ),
whose biosynthesis and release is elevated by the activity of
serine/threonine/tyrosine kinases, e.g., the signal molecule p38
mitogen-activated protein kinase (p38, p38 MAPKa).2,3 While
p38 is mainly activated by ambient cell stress (e.g., UV
radiation, osmotic shock, mechanic stress, lipopolysaccharide
(LPS) exposure), this kinase can also be phosphorylated upon
TNFR or IL-1ꢀ receptor binding and functions in the cell
signaling network responsible for the up-regulation of these
inflammatory mediators both at the transcriptional and at the
translational level.4 Several small molecule p38 MAPK inhibi-
tors have been shown to effectively block the production of
Figure 1. Essential interactions of SB203580 with p38R MAP kinase.
The base of the hydrophobic back pocket defined by the residue of
IL-1, TNF, and other cytokines in vitro and in animal tests.5–8
Many of these “ATP mimetics” were derived from the proto-
typical pyridin-4-ylimidazole SB203580 (Figures 1 and 2),
which decisively contributed to the identification and charac-
terization of the p38 MAPK signaling pathway as a valid
therapeutic target in inflammation, and the structural require-
ments for p38 inhibition have been reported.8–10
Thr106 (gatekeeper) is more effectively occupied by selective 4-fluo-
rophenyl ring containing inhibitors. Sufficient contacts between the side
chain of the respective gatekeeper residue and this 4-fluorophenyl
substituent are not possible within other MAPK (family members), e.g.,
p38δ, p38γ, or ERK2, thus gaining selectivity.
Figure 4):11–13 (1) hydrogen donor/acceptor functions of the
2-aminopyridyl residues within the hinge region (mainly gaining
activity); (2) space-filling lipophilic aryl residues binding to the
hydrophobic back pocket (also hydrophobic region I, mainly
gaining selectivity); (3) interactions with the hydrophobic front
region (also hydrophobic region II, Figure 3, gaining both
activity and selectivity); (4) further interactions with both the
sugar pocket and the phosphate binding region (importance less
clear, preferred positions to modify physicochemical properties).
The essential interactions of pyridinylimidazole inhibitors with
the ATP-binding cleft are briefly summarized as follows (see
* To whom correspondence should be addressed. Telephone: +49-7071-
2972459. Fax: +49-7071-295037. E-mail: stefan.laufer@uni-tuebingen.de.
a
Abbreviations: p38 MAP kinase, p38 mitogen activated protein kinase;
ATP, adenosine triphosphate; CYP450, cytochrome P450; LPS, lipopolysac-
caride; TNF-R, tumor necrosis factor-R; IL-1ꢀ, interleukin-1ꢀ; SAR,
structure-activity relationship.
10.1021/jm701529q CCC: $40.75
2008 American Chemical Society
Published on Web 06/26/2008

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4123
characteristic associated with the electron density of these
heterocycles, and related compounds possessing these substruc-
tures are often potent inhibitors of P450 enzymes.18–20 However,
the exposed pyridin-4-yl ring of the diaryl heterocyclic inhibi-
tors, which is obligatory to promote strong binding to the
backbone Met109 within the linker region of p38 than the better
shielded core heterocycle (imidazole), is regarded as the ligand
more likely to coordinate to the CYP450 system.18 Inhibitors
of human hepatic cytochrome P450 can potentially cause
drug-drug interactions or lead to other hepatic changes such
as P450 enzyme induction. Furthermore SB-like small molecule
p38 inhibitors occasionally possess genotoxic potential.
The selectivity profile, i.e., the extent of p38 inhibition versus
inhibition of other protein kinases, represents a critical feature
of inhibitor design as well. Until now 518 different kinases have
been identified in the human genome ()kinome).21 Nearly one-
fourth of the druggable kinome consists of kinases involved in
signal transduction.22–24 All these kinases possess a catalytic
domain, which is highly conserved in sequence and structure
and wherein the native cosubstrate ATP binds in a very similar
manner.21 The general architecture of the ATP binding site in
protein kinases has been sufficiently reviewed.25–28 The design
of a small, highly specific synthetic inhibitor directed against
the ATP-binding site of a single kinase is hampered by the
remarkable consistencies in the catalytic mechanisms of the
kinases, the high degree of sequence homology, identical protein
folding topologies, and the common cosubstrate ATP.17,23,29
Figure 2. Pyridinyl- and pyrimidinylimidazole-based p38 MAP kinase
inhibitors, which have been advanced to preclinical or clinical studies
for therapeutic intervention of acute and chronic inflammatory diseases.
Nonetheless, many selective p38 MAPK inhibitor candidates
are based upon the imidazole scaffold and bind at the ATP site
located in the cleft between the N- and the C-terminal domains
of p38 (Figure 2).30 This class of compounds is exemplified by
the p38 inhibitors SB203580, SB202190, SB242235, L-790070,
RWJ67657, and SB202190 (Figure 2) and targets all major
interaction sites.31
Because first generation SB-like p38 inhibitors suffered from
nonmechanistic side effects, more structural modification will
be necessary. Thus, attempts to optimize newer pyridinylimi-
dazole inhibitors have included incorporating different substit-
uents on the imidazole core itself and/or by introducing
additional (amino) substituents at the ortho position of the
pyridin-4-yl moiety.7,32 Particularly, the substituents at the
imidazole N1 and C2 positions have been varied extensively to
improve physicochemical properties and to reduce toxicity.33,34
The different design strategies have led to several inhibitors
with high potency and enhanced selectivity among closely
related kinases.8,35 Here, 2-sulfanylimidazoles and their active
in vivo metabolic transformation products (sulfoxides, sulfones)
demonstrated decisive advantages over prototype SB203580-
like 2-arylimidazoles, e.g., fewer interactions with metabolic
enzymes like CYP450 and better kinetic and metabolic
properties.36–39
Figure 3. View into the ATP-binding site of p38 with the bound
pyridinylimidazole prototype inhibitor SB203580 (brownish formula).
Corresponding amino acids (differently colored) of both areas of the
hydrophobic region II, stretching above as well as beneath the ring
plane of the imidazole scaffold, are highlighted. The hydrophobic region
II is widely opened to the solvent area (in the front). In the case of p38
this hydrophobic region seems to be capable of accommodating large
and electronically diverse substituents linked to the 2-position of the
pyridine of the respective pyridinylimidazole inhibitor (figure generated
with Sybyl on the basis of PDB structure 2ewa).
Although almost 20 compounds under investigation as
anticytokine agents entered clinical studies, only six p38 MAPK
inhibitor candidates (BIRB796, SCIO469, VX702, pamapimod
(Roche), SB-681323, and GW-856553)9,14 for the treatment of
rheumatoid arthritis reached phase II clinical trials.15 Poor kinetic
properties and unacceptable safety profiles including side effects
in the liver and in the CNS were mainly responsible for this
failure. Infections, dermatological diseases, or renal problems
have also been observed.16,17 Although the toxicity of the p38
inhibitor class is believed to be primarily mechanism-derived,
there are also strong indications for nonmechanistic, CYP450-
negotiated, harmful effects.
The 2-alkylsulfanyl-5-(pyridin-4-yl)imidazoles III and IV
(Figure 5) have been recently recognized as strong inhibitors
of both the isolated p38 MAP kinase and cytokine release in
PBMCs as well as in human whole blood, with IC50 values in
the low micromolar range, comparable to those of the potent
model compound SB203580.36 Furthermore, CYP450-dependent
toxic side effects were decisively reduced, thus showing a
general advantage of 2-thioimidazoles compared to prototypical
SB-like 2-aryl-subtituted derivatives. Compounds III and IV
can be viewed as open chain analogues of the early lead
compound SK&F86002 (Figure 5) and are derived from their
parent by opening of the imidazothiazole ring system via the
biologically active derivatives I (starting point for trisubstituted
First generation pyridinylimidazoles, e.g., SB203580, are
liver-selective and have been associated with CYP450 inhibition.
Both imidazoles and pyridines coordinate well to the heme iron
of the human cytochrome P450 (CYP450) system, a common

4124 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Figure 4. Binding mode of ATP (left) and proposed binding mode of an exemplary inhibitor molecule (compound 35i, right) to the ATP-binding
site of p38R MAP kinase. The hinge part of the kinase polypeptide backbone is shown on the left. The hydrophobic back pocket and the surface-
exposed front region are not utilized for interactions with the enzyme’s native cosubstrate ATP.
Figure 5. Structural deduction of the general target molecule V from the early lead compound SK&F86002. The partial structures that are to be
varied and optimized in order to improve relevant parameters like activity, selectivity, toxicity, or physicochemical properties are encircled in blue.
The fundamental vicinal diaryl system of this compound class is highlighted as well (bold). Reported p38 IC50 data for compounds I-IV descend
from an old in-house test model, where the average test values were about 1 order of magnitude (10-fold) higher than those of the newly synthesized
and here presented pyridnylimidazoles, which were tested using a new, partly automated ELISA assay.
2-alkylsulfanylimidazoles) and II (starting point for trisubstituted
2-alkylsulfanylimidazoles) and placement of additional phenyl-
ethylamino substituents at the pyridine component.
In response to the need for promising development candidates
and to overcome the lack of specificity as well as the CYP450-
dependent side effects (hepatotoxicity) of the diaryl-substituted
heterocyclic class of p38 inhibitors, we aimed to synthesize
highly substituted 2-thioimidazoles targeting all relevant regions
within the binding site of the kinase. By exploitation of less-
conserved surrounding kinase areas, those not or only marginally
involved in ATP binding, it was proposed to improve the
pharmacological profile of the small molecule inhibitors (Figure

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4125
Scheme 1. Summary of the Synthetic Approaches toward the 2-Halogenopyridinylimidazole Scaffolds 5 and 731,41,a
a Reagents and conditions: (a) NaNO2, glacial acetic acid, room temp; (b) H2, Pd-C 10%, 1 atm, 2-propanolic hydrogen chloride, room temp; (c) DMF,
KSCN, reflux; (d) iodomethane, ethanol/THF 8 + 2, reflux;36 (e) DMF, methyl-/ethyl-/benzylthiocyanate, reflux;41 (f) 50% HBF4 (aq), NaNO2, 0 °C then
45 °C;74 (g) NaNO2, 70% HF in pyridine (Olah’s reagent), -15 to -10 °C f room temp; (h) NaNO2, glacial acetic acid, room temp; (i) (H2CNR2)3, ethanol,
reflux; (j) 2,2,4,4-tetramethylcyclobutane-1,3-dithione, dichloromethane, room temp; (k) Hal-R3, K2CO3, methanol, room temp; (l) Br2, glacial acetic acid,
room temp;45 (m) excess H2N-R2, DCM (+MeOH), -5 to 0 °C, then 1.25% HCl/EtOH, diethyl ether/acetone 1 + 1; (n) KSCN, DMF, reflux; (o) R3-SCN,
DMF, reflux; (p) Hal-R3, K2CO3, methanol, room temp; (q) NBS, CCl4, 0 °C then room temp; (r) Pd(PPh3)4 resp. PdCl2(PPh3)2/PPh3, argon, 2 M Na2CO3
or K-acetate, toluol or DMF, 120 or 80 °C.75
4).17,26 These sites of incomplete interaction with the bound
cosubstrate ATP vary widely among the 518 identified human
kinases and thus offer great capacity for selective inhibitor
binding (Figure 4).23,40 Here, the often neglected surface-
exposed front region (Figures 3 and 4) seemed to be best suited
for further improvements in potency and selectivity of known
inhibitor molecules.23 In the present work, the 2-alkylsulfa-
nylimidazole derivatives (III and IV) served as starting com-
pounds for additional structural optimizations. Moreover, in-
troduction of substituents at the pyridine ring that are sterically
demanding or electronically shielding, but at the same time
metabolically stable, should reduce interferences with the
CYP450 system and reduce in vivo toxicity.
5-position (depending on the present tautomeric form) was
performed starting from ethanones 1 via the intermediates 2
and 3 (and 4) and pursuing either a previously published four-
step nitrosation/reduction/cyclization strategy36 or a newly
optimized and fully regioselective three-step variant with
generally high overall yields (lower part of Scheme 1,
Table 1).41
Alternatively, 5 could be obtained in only two steps but in
lower yields by direct cyclization of an R-bromoketone with
S-methylisothiourea (Scheme 2).42–44 To achieve the 2-chloro-
pyridin-4-ylimidazole target compound 5d, the appropriate
1-aryl-2-heteroaryl-substituted ethanone 1b was subjected to acid
catalyzed R-monofunctionalization reaction with Br2 at ambient
temperature.45 The resulting unstable R-bromoethanone inter-
mediate 13c then was immediately reacted with S-methylisothi-
uronium sulfate in DMF under reflux conditions to obtain a
mixture of cyclization products from which the imidazole-2-
Chemistry
Synthesis of the 2,4,5-trisubstituted imidazole scaffolds 5
bearing a substitutable 2-halogenopyridin-4-yl at the 4- or

4126 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Table 1. Substitution of Compounds 1-20 According to Scheme 1
Laufer et al.
regioselective syntheses,31 which are outlined in Scheme 1
(upper part) starting from 5-(2-aminopyridin-4-yl)imidazoles
6,33,46 1-(2-aminopyridin-4-yl)ethanone 8,33 or 2-(2-fluoropy-
ridin-4-yl)ethanones 136,41 and acetophenones 15,47 respectively.
Table 1 summarizes the substitution pattern of all intermediates
as well as of the target compounds 7.
The most elegant access to compounds 7 with moderate
overall yields was accomplished from 2-(2-fluoropyridin-4-
yl)ethanones 1 via the obligatory intermediates 13 and 14 and
the optional imidazole-2-thiones 12 by a novel and extremely
flexible synthetic methodology that allowed the regioselective
introduction and variation of all relevant substituents in only
three (or four) steps.31
compd
R1
R2
R3
n
X
1a
1b
1c
1d
4-F
4-F
4-F
3-CF3
4-F
4-F
4-F
3-CF3
4-F
1
1
1
1
F
Cl
Br
F
2a, 3a, 4a
2b, 3b, 4b
2c, 3ca
2d, 3d
3e
F
Cl
Br
F
H
F
5a
5b
4-F
4-F
Me
Et
F
F
5c
5d
5e
4-F
4-F
4-F
Bn
Me
Et
Cl
Cl
Cl
H
F
2-Fluoropyridin-4-yl compounds 7 were well suited for the
synthesis of target compounds 35 (Table 8) upon substitution
with all kinds of nucleophiles (Scheme 3).
5f
5g
5i
5j
5k
6a
6b
7a
7b
7c
7d
7e
4-F
4-F
3-CF3
3-CF3
3-CF3
Bn
Me
Me
Et
Bn
Me
Me
Me
Me
Bn
F
F
In a final step, a variety of different (hetero)aryl, (hetero)ary-
lalkyl, and (cyclo)alkyl residues could be introduced at the
pyridin-4-yl moiety of the obtained halogenopyridinylimidazoles
via known SNAr methods using suitable amines, alcohols, or
thioalcohols as the corresponding nucleophiles, giving p38
inhibitors with maximum structural diversity.32,48 Starting points
for further derivatization primarily were the methylsulfanylimi-
dazoles 5a, 5d, 5i, 7a, and 7b (Scheme 1, Table 1) with a
2-chloro- or a 2-fluoropyridinyl moiety. The functional require-
ment for the reactivity of the pyridine component was achieved
by the above-mentioned syntheses. C2 of the pyridine is
activated for SNAr reactions. Substituents are directed ortho (or
para) by the -M effect of the heterocyclic sp2-N. The SNAr
mechanism allows the introduction of chiral building blocks (N-,
O-, or S-nucleophiles) and even tolerates aniline-like substituents
[e.g., 33c, 34a (Table 7), 35e (Table 8)], which generally cannot
be coupled when using aminopyridines 6 and substituted alkyl
halides as reactants and executing a SN reaction.31 Commercial
availability of several primary or secondary amines allows rapid
variation leading to a broad scope of substitution patterns.
Me
(CH2)2OMe
Me
(CH2)2OMe
(CH2)2OMe
(CH2)2OMe
Me
4-F
4-F
4-F
4-F
4-F
F
F
F
F
H
CH2CH(OH)CH2OH
Me
11a
11b
12a
12b
12c
13a
13b
14a
14b
14c
15a
15b
16a
16b
17a
17b
17c
17d
18a
18b
18c
18d
19a
19b
19c
19d
20a
20b
Me
(CH2)2OMe
Me
(CH2)2OMe
(CH2)2OMe
4-F
4-F
4-F
4-F
3-CF3
4-F
4-F
3-CF3
H
4-F
H
4-F
H
1
1
1
1
1
0
0
0
0
0
0
0
0
F
F
F
F
F
Me
(CH2)2OMe
(CH2)2OMe
(CH2)2OMe
Bn
Me
(CH2)2OMe
(CH2)2OMe
Bn
H
The chloropyridinylimidazoles 5d-f proved to be rather
inactive toward conventional nucleophilic aromatic substitution
reactions, with several model primary amines (e.g., benzylamine)
requiring harsh conditions (excess of amine, high temperatures,
long reaction times, solvent-free operation) that led to multiple
side products. Our initial intention was to reinforce the coupling
tendency of such 2-chloropyridine intermediates (5d) toward
amines (here, 1,2-diphenylethylamine) with the aid of Pd
catalysts conducting a cross-coupling-like C-N formation
method. However, an attempted literature procedure using a
chelate complex of either (()-2,2′-bis(diphenylphosphino)-1,1′-
binaphthyl [(()BINAP] or 1,3-bis(diphenyl-phosphino)propane
(dppp) with Pd2(DBA)3 as the catalytic system, NaO-t-Bu as
base, and toluene (or DMF) as solvent at 70-80 °C under argon
was not successful.49 None of the desired substitution product
was detected. Furthermore, attempts to deprotonate some amines
with NaH in DMF giving the putatively more reactive ionic
nitrogen nucleophiles also failed, regularly leaving unstirrable
gels that prevented completion of this reaction. Dimethylfor-
mamide (DMF) reacts as an equivalent of dimethylamine in
nucleophilic substitution of active halogen atoms implicating
dimethylaminopyridines (exemplified by 32a) as side products.50
Therefore, the points of origin for further derivatization at the
pyridyl moieties of the starting imidazole-2-thioethers were the
presumably more reactive 2-fluoropyridines 5a, 5i as well as
7a and 7b. Here, the reactivity is additionally enhanced by the
high electronegativity of the fluorine atom strongly polarizing
the pyridine-C2-F bond, making it the preferred leaving group.
4-F
4-F
H
H
4-F
4-F
H
Me
(CH2)2OMe
(CH2)2OMe
Bn
Me
(CH2)2OMe
Me
Me
Me
Me
Me
Me
Me
H
4-F
4-F
4-F
4-F
(CH2)2OMe
a As hydrogen sulfate.
thioether 5d could be isolated in 12% yield by column
chromatography.42 As side products (SP), the related 2-ami-
nothiazole derivative (SP1, m/z ) 305), an obvious S-oxidation
artifact of 5d (SP2, m/z ) 335), and higher amounts of
debrominated starting material (ethanone 1b) were identified
(GC-MS) and separated.
We also applied the same reaction conditions to the reaction
of 13c with N,S-dimethylisothiourea in an attempted regiose-
lective preparation of 1,2,4,5-tetrasubstituted imidazoles. How-
ever, contrary to the results of Dodson et al. for a structurally
similar compound, no product accumulation was observed.44
Instead, decomposition of the starting material(s) took place.
The required 1,2,4,5-tetrasubstituted fluoropyridinylimidazole
precursors 7, which allow the subsequent SNAr-like introduction
of chiral N-, O- or S-(aryl)alkyl or aryl substituents at the
2-position of the pyridine, could be obtained via four different

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4127
Scheme 2. Condensation with S-Methylisothiuronium Sulfate or N,S-Dimethylisothiuronium Iodidea
a (a) Br2, glacial acetic acid, room temp; (b) DMF, reflux.
Scheme 3. Nucleophilic Aromatic Displacement of the Fluorine at the Pyridine Moietiesa
a Reagents and conditions: (a) 5- to 20-fold excess of neat primary or secondary amine (aliphatic or aromatic), reflux or 150-170 °C, argon; (b) NaH,
DMF, excess of aliphatic or aromatic alcohol or thioalkohol, 160 °C, argon.
For the SNAr with N-nucleophiles these fluoropyridine reactants
were either directly dissolved or suspended in a 5- to 10-fold
excess of the respective liquid amino reactant and the mixture
was stirred (refluxed) under argon at 150-170 °C until the
starting material completely disappeared, an augmented appear-
ance of side products was observed, or the product accumulation
ceased. In the case of solid but low melting amines, the
substitution reaction was carried out in the molten material under
otherwise identical conditions. Amines with low boiling points
(,100 °C), on the other hand, only could be effectively coupled
under elevated pressure in a glass or metal bomb. When the
2-fluoropyridines were used, the nucleophilicity of the intro-
ducible and mostly primary amines or amino alcohols was
sufficient for SNAr and the latter did not have to be activated
by basic deprotonation in advance. When amino alcohols were
reacted, possible concomitant ether formation at the specified
position of the pyridnylimidazole was never detected and only
the respective aminopyridine derivatives were isolated.51 In

4128 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Table 2. Biological Activity of 2-Halogen-Substituted Pyridin-4-yl- and Quinolin-4-ylimidazole Derivatives with a 4-Fluorophenyl Moiety at Imidazole
C4 or C5a
a n: number of experiments. n.t.: not tested.
contrast, less nucleophilic alcohols and thioalcohols merely
reacted as the corresponding anionic salts (alcoholates or
thioalcoholates, respectively). Consequently, quite a few N-, O-
and S-nucleophiles could be readily introduced at the 2-position
of the respective pyridine moiety using known SNAr methods
(Tables 1–10). In the case of 2,4,5-trisubstituted imidazoles,
nucleophilic aromatic substitutions frequently could not be
driven to completion leaving unreacted starting material and
traces of undefined side products. In contrast, when the
corresponding 1,2,4,5-tetrasubstituted imidazoles were produced,
reaction times were even shorter and yields were significantly
higher (up to 100%). Usually no side products could be
observed, as both the starting materials (7a, 7b) and the resulting
products 35a-o (Table 8) proved to be surprisingly thermo-
stable. Because of steric hindrance, primary amines having an
additional R-(aryl)alkyl branch on their (hetero)(aryl)alkyl side
chain on average gave lower product yields than those with a
linear aliphatic residue. Attempts to couple, for example, R,R-
dimethylpropylamine with the 2-fluoropyridinyl of 5a using
comparable reaction conditions completely failed and could not
be accomplished, even with attempts to accelerate the reaction
time drastically by microwave radiation.
We also proposed to undertake pyridine amination of
analogous 2-halogenopyridin-4-ylimidazole-2-thioethers with an
exocyclic S-ethyl or S-benzyl substituent instead of the small
methyl group. Because of the obligatory long reaction times,
initial attempts to specifically react the chloropyridinyl analogue
5f by means of SNAr with 1-phenylethylamine resulted in the
concomitant nucleophilic attack at the benzylic position of the
benzylsulfanyl substituents by the excessive amine. The un-
wanted cleavage of the benzyl residue at the exocyclic sulfur
combined with the actually expected amination at the fluoro-
pyridine ring inevitably resulted in the production of the
imidazol-2-thione 24 (Scheme 4), as the single isolated and
detected product.36 To avoid this problem, we chose to prepare
the 2-aminopyridin-4-yl imidazole-2-thione 24 first as a main
product that should be consequently S-alkylated in a final step
(Scheme 4). This inverse process then would probably tolerate
the subsequent introduction of functionalized S-residues as well.
However, reacting the starting 2-fluoropyridin-4-ylimidazol-2-
thione 4a with a 10-fold excess of 1-phenylethylamine yielded
a mixture of several products, from which 24 could not be
isolated as a pure solid by any accepted method (LC/MS purity:
∼50-60%).

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4129
Table 3. Biological Activity of 2-Halogen-Substituted Pyridin-4-ylimidazole Derivatives with a 3-Trifluoromethylphenyl Moiety at Imidazole C4 or C5
1H (and 13C NMR) spectra of the prepared (and in general
relatively hydrophilic) amino or (thi)oxypyridinylimidazole
compounds 22 and 25-35 (see Scheme 3) were preferably
established that small (methyl) substituents located at the
exocyclic sulfur atom (ML3375) significantly improved inhibi-
tion of activity in both the isolated kinase assay and of cytokine
release from human whole blood. This greater inhibitory
potential was attributed to better entry of the inhibitor molecule
into the binding cleft of p38, leading to stronger enzyme-drug
interactions.34 Furthermore, diverse C2-S-substituents were
investigated as ligands possibly targeting the kinase phosphate
binding region, in close analogy to the triphosphate end of
ATP.60 Attempts to develop compounds that bind at positions
within the p38 kinase that are unoccupied by first generation
inhibitors like ML3375 or SB203580 were conducted mainly
by regioselective introduction and structural variation of ad-
ditional functionalities, e.g., at the imidazole nitrogen that is
located next to the pyridyl part and/or at the 2-position of the
pyridine ring.33,36 These novel interactions included the forma-
tion of a second hydrogen bond between the backbone Met109
inside the linker region and a primary or secondary amino
function ortho to the pyridine ring nitrogen, hydrophilic interac-
tions with less-conserved areas of the ribose pocket via
appropriate imidazole N1 substituents, and finally, lipophilic
vdW contacts and H-bonds between kinase residues or backbone
atoms around the hydrophobic front region or its surrounding
solvent accessible area by pyridine-C2 adducts. As the structural
modifications at the imidazole N1 had been already extensively
reported, this article will now focus on the introduction and
wide variation of substituents at the pyridine part of these
2-thioimidazoles bridged by different heteroatoms (NH, O, S).
1
collected in deuteromethanol. Whenever the H NMR spectra
were collected in the more viscous DMSO at ambient temper-
ature, we observed two sets of signals (in nearly 1:1 ratio),
indicating a slow (on the NMR time scale) equilibrium between
two isomers (atropisomers).52 This effect could be attributed to
the sterically restricted free rotation of the substituted pyridinyl
ring around its σ bond with the imidazole core, particularly at
compounds with bulky (aryl)alkyl residues [for example, at
compounds 5h (spectrum is in the Supporting Information), 22e,
26a, c, 28a, 30c, and 33d]. The appearance of different
atropisomers, however, repeatedly complicated the interpretation
of the spectra. In deuteromethanol the interconversion of the
two isomers is fast on the NMR time scale and the resonances
are averaging to a single signal.
Biological Testing
All compounds were primarily screened in an isolated p38R
MAP kinase assay.53,54 Inhibition of cytokine release (TNFR,
IL-1ꢀ) from immune cells was accomplished using diluted fresh
human whole blood of at least two anonymized donors.55–57
CYP interactions were investigated by BDGentest Corp.58 The
selectivity profiling of selected compounds against a panel of
18 different kinases (including p38R) was performed by
Upstate.59
Biological Discussion
The strength of the H-bonding between the pyridine-N of
the inhibitor and the amide-NH of Met109 is influenced by the
electron donating or withdrawing capacity of the substituents.
The p38 inhibitory potency has been correlated with the electron
density at the respective heterocyclic ring nitrogen.36 With
reference to the parent compound ML3375 (Table 2), an
electronegative halogen atom located at the 2-position of the
pyridin-4-yl moiety significantly decreased the biological activ-
ity. Fluoropyridine 5a, for example, exhibited a 2.8-fold76 worse
inhibitory potency toward the isolated enzyme, and a similar
3.5-fold decrease in the suppression of cytokine release (TNFR)
2,4,5-Trisubstituted Imidazoles. Since the objective 2-thio-
imidazole derivatives are structurally similar to SB203580, they
may bind similarly to the p38 MAP kinase through comparable
interactions with active site residues, with the exception of π-π
stacking with Tyr35.34 Structure-activity-relationships (SAR)
comparable to those of SB203580 or analogous literature
examples have been described for different series of variable
tri- or tetrasubstituted 2-(aryl)alkylsulfanylimidazoles whose
structures are based on the pyridinylimidazole p38 inhibitors
ML3163 and ML3375 (Figure 6).8,33,35,36 These earlier studies

4130 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Table 4. Biological Activity of Different (Hetero)arylalkylaminopyridines (2-Thioimidazole Derivatives with a 4-Fluorophenyl ring)a
a Key for the symbols. $: last step (nucleophilic aromatic substitution). †: The 1-benzyl-2-phenyl-ethylamine used for the SNAr reaction was produced by (catalytic)
reductive amination (Leuckart-Wallach reaction) of 1,3-diphenylpropan-2-one according to Allegretti.70 Yield: 53% (free base) over two steps. ‡: The C-benzo[b]thiophen-
2-yl-methylamine used for the SNAr reaction was produced in advance by reduction of benzo[b]thiophen-2-carboxamide with LiAlH4 in 41% yield.
was also observed. For compound 5f, the large benzylsulfanyl
p38 inhibitory potency in contrast to its sterically less demanding
moiety at the 2-position of the imidazole core diminished the
ethylsulfanyl analogue 5e (3.8-fold) and particularly with respect

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4131
Table 5. Biological Activity of Different (Hetero)arylalkylaminopyridines, (Cyclo)alkylaminopyridines and (Cyclo)alkylaminopyridines with an
Additional Polar Residue (2-Thioimidazole Derivatives with a 3-Trifluoromethylphenyl Ring)
to the “small” methylsulfanyl derivatives ML3375, 5a, and 5d
(17.8-, 6.4- and 2.8-fold, respectively). Moreover, comparison
of TNFR-inhibition data of 5f and ML3375 shows that the lesser
p38R inhibitory activity of 5f is largely translated into a
diminished activity in the whole blood model (9.5-fold decrease
compared to ML3375). The presence of the less electronegative
chlorine atom (instead of a fluorine atom) in compounds 5d,
5e, and 5f was not found to have significant influence on the
electron-withdrawing properties at the pyridine, as the inhibition
toward the isolated enzyme was comparable to the fluoropyri-
dine derivatives (5a-c). These findings suggest that for the
imidazole-2-thioethers the size of the substituent at the proximal
sulfur and the resulting (in)ability of the inhibitor molecule to
enter into the ATP cleft deeply have greater influence than the
nature of the halogen atom at the pyridine-C2 (Figure 7). In
the kinase activity assay, the ethylsulfanyl substituent of 5e
proved to be nearly bioisosteric to the well-defined methylsul-
fanyl substituent (e.g., at compound 5a, divergence factor of
1.7), additionally confirming the idea of stronger binding of the
4-fluorophenyl/pyridin-4-yl pharmacophore inside the ATP site,
when substituents at the exocyclic sulfur are not bulky. The
slight difference (1.6-fold) in TNFR-release between alkylsul-
fanyls 5a and 5e implied similar cell penetration behavior and
plasma protein binding for both compounds. The ethyl vs benzyl
functionalities of 5e and 5f, respectively, caused significant
differences in p38 inhibition, which were completely canceled
out in the cell-based assay system. Perhaps the benzylsulfanyl
derivative 5f penetrated the cellular membrane lipid bilayers
more efficiently and/or other meaningful influencing factors
adherent to whole blood systems (i.e., enzymatic metabolism

4132 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Table 6. Biological Activity of Different (Cyclo)alkylaminopyridines and (Cyclo)alkylaminopyridines with an Additional Polar Group (2-Thioimidazole
Derivatives with a 4-Fluorophenyl Ring)a
a Key for the symbols. /: The cis-28f and the trans-28g isomers were not especially produced but separated from the cis/trans mixture (28h) by column
chromatography. †: The tetrahydropyran-4-ylamine used for the SNAr reaction was produced by (catalytic) reductive amination (Leuckart-Wallach reaction)
of tetrahydropyran-4-one according to the protocol of Allegretti.70 Yield: 46% (free base) over two steps.

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4133
Table 7. Biological Activity of Different 2-(Thi)oxypyridinylimidazolesa
a Asterisk (/) indicates not determined.
reactions, plasma protein binding, cell-cell interaction) are
responsible for these effects.
system probably led to a suboptimal H-bond formation between
the quinoline-N and the amide-NH of Met109.
Comparable SARs were found within the series of 2-(aryl)-
alkylsulfanyls having a congruent 5-(2-halogeno)pyridine-4-yl
moiety but bearing a 3-trifluoromethylphenyl system instead of
the classical 4-fluorophenyl ring at the 4-position of the
imidazole core (Table 3). For these 3-trifluoromethylphenylimi-
dazole derivatives, p38 inhibition is generally less efficient (see
later, 2-sulfanylimidazole derivatives with additional amino
substituents at the pyridine ring). In the p38 assay, compounds
5i and 5j (Table 3) with a methylsulfanyl and an ethylsulfanyl
substituent, respectively, at the imidazole-C2, were ∼2.5-fold
better than the benzylsulfanyl analogue 5k, with both 5i and 5j
exhibiting similar IC50 values. However, in the whole blood
model, differences in inhibition of cytokine release were less
drastic among all three representatives.
Introduction of Arylalkylamino Substituents at the
Pyridine C2. Hydrogen bonding between the pyridine ring N
of the inhibitor and the backbone amide-NH of Met109 in the
otherwise lipophilic linker region (Ala51, Thr106, His107,
Leu108, and Met109) is essential for the biological activity of
the vicinal diaryl-substituted heterocyclic compound class.11–13
Appropriate amino substituents at the 2-position of the pyridine
can enhance activity toward unsubstituted (e.g., ML3375) and
toward halogenated pyridine derivatives (Tables 1 and 2), as
they are able to form a second H-bond with the linker region
next to the existing one (here, between the exocyclic 2-amino
group as H-donor and the main chain carbonyl oxygen of
Met109 as H-acceptor).13,36 On the other hand, an electron
donating amino group should increase the electron density at
the pyridine, thereby intensifying the affinity of the inhibitor
for the enzyme.32,61,62 Moreover, by adequate lipophilic side
chains, further interactions with the surface-exposed front region
(hydrophobic region II) can be achieved.36 Therefore, we
prepared a series of analogues by introducing a number of
primary arylalkylamines at the pyridinyl ring of ML3375.
Replacement of the essential 4-pyridine ring in ML3375 by
a 4-quinoline ring (5h) did not improve the inhibitory efficacy
in the kinase assay or the inhibition of cytokine release from
immune cells. Compound 5h exhibited ∼25% of the p38 kinase
inhibitory activity of ML3375. Differences in the inhibition of
TNFR release were even less significant, with ML3375 being
1.6-fold more effective than 5h. The restricted rotation of the
bulkier quinolin-4-yl moiety around its σ bond to the imidazole
C5 combined with a planar alignment of this condensed aromatic
The biological results of these novel arylalkylamino-
substituted pyridinylimidazole analogues 22a-m are sum-
marized (Table 4). Structural analyses of the more potent

4134 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Table 8. Biological Activity of Different Tetrasubstituted Aminopyridinylimidazoles
inhibitor candidates demonstrated a certain tolerance for dif-
ferently constituted and configurated arylalkyl moieties; how-
ever, no clear tendency for optimum SAR could be found. In
general, sufficiently lipophilic arylalkylamino residues favorably
affected biological activity (compounds 22b-g,l). For con-
densed aromatic ring systems (22d,e,m), the spatial geometry
seemed to play a crucial role; however, multiple separate
aromatic cycles in the aliphatic side chain resulted in increased
IC50 values (22h,i). The most potent representatives of this series
(22b-g) commonly possess an R- or ꢀ-methyl or ethyl group
at the introduced amino substituent (22b,c,f,g) and/or a more
flexible spacer of up to three carbon atoms between the
exocyclic amino-N and the terminal phenyl ring (22e,f,k, Figure
8). Alternatively, the R-methyl branch can be ring-closed to
either a penta- or a hexaycyclic indane or a tetrahydronaphtha-
lene structure element with the adjacent phenyl ring (compounds

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4135
Table 9. Kinase Selectivity Profile of Selected Compoundsa
kinases71 78
BIRB796
SB203580
SB202190
22c
22d
22g
26b
28o
30c
35g
CaMK-II
c-Raf
ERK1
MAPK2/ERK2
JNK1R1/SAPK1c
MEK1
no data
0
no data
no data
no data
11
0
0
0
0
0
7
28
6
86
2
91
99
94
96
6
61
0
18
8
46
20
86
96
95
96
8
12
0
0
5
85
0
99
89
100
100
0
(91)59 79
(14)
28
85
no data
15
0
31
23
21
13
4
36
28
0
62
1
79
100
98
90
9
19
32
17
88
3
94
99
97
96
0
53
46
31
72
1
90
99
99
99
0
1
(14)
50
9
97
97
97
89
21
7
0
no data
no data
no data
100
97
20
13
34
JNK2R2
JNK3
32
86
98
90
4
4
63
99
98
17
0
SAPK2a/p38R
SAPK2b/p38ꢀ2
SAPK3/p38γ
SAPK4/p38δ
PKA
7
4
0
0
11
0
9
4
2
0
8
1
n.t.
0
0
PKCR
30
0
0
22
11
38
34
23
68
8
47
39
39
0
14
0
0
25
0
12
0
0
26
0
5
0
0
0
1
1
0
0
6
0
0
0
0
6
7
7
PKBR
12
10
0
GSK3ꢀ
ROCK-II
LCK
82
63
30
28
47
26
25
a The inhibitory potency was evaluated by the Upstate Kinase Profiler carried out at 100 µM ATP concentration. Values are reported as %-inhibition at
a inhibitor concentration of 10 µM.
Table 10. Inhibition of the Activity of Relevant CYP450 Isozymes at
10 µM Inhibitor Concentrationa
front area of the enzyme, resulting in increased activity as well
as selectivity, the (halogenated) aryl ring in the 4-position of
the imidazole is mostly responsible for gaining selectivity to
p38R over other (related) kinases. This important structural
element occupies the deep hydrophobic back pocket of p38,
comprising the residues Val38, Ala51, Val52, Lys53, Leu75,
Ile85, Leu86, Leu104, Val105, and Thr106 that correspond to
the hydrophobic region I (Figures 3 and 4).7 Small (halogen or
alkyl) substituents are tolerated in the 2-, 3-, and 4-position of
the aryl system, and other researchers have varied these aryl
substituents in an effort to improve the selectivity profile of
these diaryl heterocyclic p38 inhibitors.7,10 The formal introduc-
tion of a 3-trifluoromethylphenyl group instead of the para
halogen atom at the phenyl ring of 2,4,5-trisubstituted imidazoles
only slightly affected the p38 inhibitory activity but led to a
30-fold selectivity toward the c-raf kinase (vs equipotent
inhibition of p38 and c-raf with the analogous 4-chlorophenyl
derivative (Figure 11, left-hand side).7 Combination of individu-
ally optimized substituents at the N1- and C2-position of the
imidazole and the introduction of adequately substituted 2-amino
functions at the pyridine or the pyrimidine ring resulted in
additive increases in p38 potency. Simultaneously, a more than
100-fold selectivity toward both the c-raf and JNKa1 resulted
(Figure 11, right-hand side).7
compd
1A2
2C9
2C19
2D6
3A4
22c
55
68
38
83
83
76
53
67
62
45
23
29
70
94
52
60
25
5
80
84
88
82
93
71
52
77
73
75
76
39
47
81
78
66
71
85
73
40
87
88
79
89
78
94
63
47
81
80
76
84
25
51
86
87
76
83
95
80
58
92
73
68
78
51
88
66
9
54
63
53
66
5
81
76
95
83
89
81
53
93
80
59
71
33
52
95
85
87
87
81
81
80
77
22d
22g
26a
26b
26d
26f
26g
28a
28l
28o
30b
30c
22
0
33c
35a
35b
35c
35d
35g
35j
53
27
64
25
54
24
73
0
23
38
SB203580
a Results in % inhibition. Each case originated from a single-point
detection using a standardized method.59
22d and 22e, Figure 9). A possible binding model using
compound 22f was generated, illustrating the possible orientation
of the branched (ꢀ-methyl) arylalkyl side chain within the
hydrophobic region II (Figure 10). Here, the 2-phenylpropyl
residue is more directed toward the lipophilic subarea that is
located above the imidazole ring plane (area A). The best
compound in this series, the 1-methyl-3-phenylpropylaminopy-
ridine 22g, proved to be 5 times as effective as the standard
SB203580 with respect to the inhibition of isolated p38 MAPK
and was also significantly superior to the prototype in suppres-
sion of TNFR release. Contrary to our expectations, introduction
of sterically demanding (and rather hydrophilic) arylalkyl
residues at the pyridin-4-yl ring could not reduce interactions
of the inhibitor with the heme iron of the CYP450 system
compared to SB203580. This modification was inadequate to
satisfactorily prevent the inhibition of metabolically essential
CYP isozymes, with the exception of CYP450 1A2 (22c,g;
compare Table 10) and consequently was not qualified for
decreasing in vivo liver toxicity.
Against this background, we replaced the classical 4-fluorophe-
nyl component of our objective alkylsulfanylimidazoles by a
3-trifluoromethylphenyl ring. To attain synergistic effects caused
by the concurrent location of key substituents at the relevant
positions of the inhibitor molecules, we varied the already tested
arylalkylamino residues at the 2-position of the pyridine ring while
retaining the small methylsulfanyl substituent at the 2-position of
the imidazole.
The 4-(3-trifluoromethylphenyl) groups of the explored
compounds 27a,b (Table 5) are supposed to target the hydro-
phobic region I composed of the ꢀ-strands 3, 4, and 5 as well
as the R-helix C, in close analogy to the 4-fluorophenyl residue
of SB203580 (Figure 12).25 The selectivity of those pyridin-4-
yl-/3-trifluoromethylphenyl-substituted imidazole analogues is
assumed to depend on the presence of a Thr in position 106 of
the p38R MAPK (“gatekeeper residue”).12,13,63 In other kinases,
this explicit position is occupied by sterically more demanding
amino acid side chains (Met, Glu) that do not tolerate such bulky
3-CF3-aryl substituents of putatively specific p38 inhibitors. The
arylalkyl residues introduced at the pyridine part lie in the
Besides the positive interactions of the substituted 5-pyridin-
4-yl moiety with both the linker region and the surface-exposed

4136 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Scheme 4. Amination of Imidazol-2-thione 23a
Laufer et al.
a (a) 10-fold excess of 1-phenylethylamine, 150-160 °C, 22 h; (b) 10-fold excess of 1-phenylethylamine, 160 °C, 4 h; (c) iodomethane (3 equiv), EtOH/
THF 1 + 1, reflux, 40 h; (d) 10-fold excess of 1-phenylethylamine, 160 °C, 15 h; (/) LC/MS purity: ∼50-60%.
Figure 6. (Aryl)Alkylsulfanylimidazole lead structures from Merckle. The asterisk (/) indicates that the given p38 IC50 data come from a precursor
ELISA assay, where test values generally proved to be about 10-fold higher than with the new procedure mentioned at the beginning of this section,
which was used for all newly tested compounds in the present work. The corresponding biological activities of the reference SB203580 are p38
0.29, TNFR 0.59, IL-1ꢀ 0.037.
hydrophobic region II within the vdW distances of the side chain
atoms of Val30, Ile108, Asp112 and the main chain atoms of
Ala111 (Figure 12).25 This pocket, which is quite spacious in
the inactive p38 MAPK, could function as a major selectivity
determinant for such compounds bearing bulky NH-alkylphenyl-
like residues at the pyridine for p38 compared to other kinases.
The 3-trifluoromethylphenyl analogues 25a-e (Table 5) can
form up to two hydrogen bonds with residues that also contribute
to the ATP binding in the kinase linker (hinge) region (His107-
Ala110). Here, the 2-aminopyridine ring interacts with the main
chain nitrogen and the main chain oxygen atoms of Met109 (H
donor-acceptor interactions, Figure 12). In a structurally related
inhibitor molecule with a 3-trifluoromethylphenyl system, the
conformation of this peptide region in the bound p38R was
identical to that in the apo enzyme.10 In contrast to SB203580
and similar compounds,13 the side chain nitrogen of the con-
served Lys53 does not interact with the N3 of the imidazole
ring of this representative compound (Figure 13, right).10The crys-
tal structure shows the differing spatial orientation of the present
imidazole compound within the ATP site in comparison to the
exemplified 4-fluorophenyl derivative (Figure 13, left). The
imidazole-N3 of this inhibitor is separated from the side chain
amino group of Lys53 by 4 Å, too far to allow the formation
of an H-bond. Thus, this excessive distance may explain the
poorer p38 inhibition potency of the 4-(3-trifluoromethylphenyl)-
2-thioimidazole compounds in Table 5 compared to the analo-
gous 4-fluorophenyl derivatives (Table 4) having similar
substitution patterns. Calculated binding distances of the respec-
tive aminopyridine moieties to the backbone residues of the
kinase linker region (field of strong interactions) in the cocrys-
tallized structures 1BL7 and 1OUK (Figure 13) should have
promised stronger enzyme-drug interactions for the 3-trifluo-
romethylphenyl-substituted imidazoles.

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4137
Figure 7. Electron-withdrawing halogen atoms at the 2-position of the pyridine ring of the investigated 2-thioimidazoles as well as short arylalkyl
residues at the proximal sulfur lead to a reduced p38 inhibition potency, weakening the H-bond of the pyridine ring N and the backbone amide NH
of Met109.
Figure 8. Binding model for 2-alkylsulfanyl-4(5)-pyridin-4-yl-imidazole p38 inhibitors having additional arylalkyl substituents at pyridine C2.
Each of the compounds 25a-e (Table 5) on average exhibited
only a fifth of the p38 inhibitory potency of the corresponding
pyridinylimidazole derivatives with a 4-fluorophenyl residue (see
Table 4). SAR of the introduced arylalkylamino substituents at
the pyridinyl moiety of these 3-trifluoromethylphenyl-substituted
imidazoles generally correlated with the SAR of the 4-fluo-
rophenyl analogues. For both series of inhibitors, a qualitative
as well as a reasonably quantitative effect was caused by the
introduction of the same arylalkylamino substituent. Thus, the
abolition of the H-bond between the imidazole-N and the amino
group of Lys53 was more important for inhibitor potency than
the nature of the pyridine substituents. Consequently, the least
displaced orientation of those modified “bulkier” drug molecules
within the active site was responsible for the consistently
reduced activity of the 3-trifluoromethylphenyl-substituted imi-
dazoles. In contrast, the most favorable arylalkylaminopyridine
residues among the 4-fluorophenylimidazole derivatives (Table
4) led to a relatively significant enhanced kinase inhibition
compared to the fluorinated pyridinylimidazole precursor 5i
(Table 3) and were also able to (over)compensate for this loss
in potency. As a result, compound 25e, bearing the already
proven and tested 1-methyl-3-phenylpropylamino substituent at
the pyridine, is at least as potent as the reference SB203580.
The elevated lipophilicity of the 3-trifluoromethylphenylimi-
dazoles 25a-e promotes good plasma protein binding with
probable accumulation in the lipophilic cell membranes, qualify-
ing these compounds as relatively poor inhibitors of cytokine
release (TNFR, IL-1ꢀ) in an almost double-digit micromolar
concentration array (25a,c,d).
In a related consideration, the increased lipophilicity resulting
in presumed low water solubility makes these 3-CF3-phenyl
derivatives less feasible for peroral administration.64
Introduction of Heteroarylalkylamino Substituents at
the Pyridine C2. Beside the wide-ranging modifications at the
aliphatic spacer bearing the (terminal) phenyl ring(s) (Tables 3
and 4) another promising route was to replace the six-membered
aromatic system by diverse heteroaromatic cyclic structures.
Moreover, suitably located O- or N-containing ring systems (H-
acceptors) probably would make additional electrostatic interac-
tions with polar backbone residues or with the solvent area

4138 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Figure 9. Binding model for 2-alkylsulfanyl-4(5)-pyridin-4-ylimidazole p38 inhibitors with a (rigid) phenylethylamino substituent in the 2-position
of the pyridine.
increase potency up to 2.5-fold of the activity of SB203580
(compound 27b) as was required with the arylalkylamino
residues (compare Tables 3 and 4).
Substitution of the sulfur in the terminal thiophene ring by a
more electronegative oxygen atom led to a marked loss in
activity (26d, 27b). The position of the heteroatom within the
heterocycle was also important. In a series of pyridinyl position
isomers [5-(pyridin-2-, 3-, 4-ylmethylaminopyridin-4-yl)imida-
zoles 26f-h] a clear preference was identified for the derivative
in which the terminal pyridyl ring is bound in ortho position to
the methylene spacer (26f) whereas the 4-pyridyl analogue 26h
was notably less active (Table 5).
For the specified examples 27a and 27b (Table 5) of the series
of 3-trifluoromethylphenyl derivatives, identical binding modes
and thus equal binding energies of the matching heteroaryla-
lkylamino substituents in the hydrophobic region II can be
predicted, as they also existed for the 4-fluorophenyl analogues.
Figure 10. Binding model of 22f (S-enantiomer) in the ATP-site of
p38 based on the PDB structure 1a9u. Colors correspond with the grade
of lipophilicity of the side chains of the amino acids (blue ) hydrophilic,
red ) lipophilic). The spatial position of 22f in the kinase was received
by docking with FlexX (version 1.13).72 Arrows indicate the subareas
of the hydrophobic region II (framed in white color) above (area A,
Val30, Ala40, Leu108) and beneath (area B, Ile84, Met109, Ala157,
Leu167) the imidazole plane of the inhibitor in proximity to the linker
region. Between these two hydrophobic surfaces the 2-phenylpropyl
residue, bound in the 2-position of the pyridine via a nitrogen atom, is
optimally buried. The figure was provided with Insight II.73
Once more, this prediction is reinforced because the furfury-
laminopyridine 27b is 1.4-fold discriminated by its sulfur
pendant 27a and this difference in activity is almost identical
to that between 26a and 26d (Table 4). Furthermore, as already
found for the related arylalkylaminopyridines (Tables 3 and 4),
the p38 inhibitory potency of each of these two 3-trifluorom-
ethylphenyl-substituted imidazoles proved to be ∼5-fold lower
than its identical pyridine substituted analogue from the 4-fluo-
rophenyl series. In turn, the comparatively reduced activity can
be attributed to the lack of the H-bond between the sp2-
hybridized imidazole-N and Lys53, the quantitatively different
lipophilic interactions of the fluorinated aryl ring at C4 of the
imidazole, and the deviating orientation of the inhibitor molecule
in the adenine region of the kinase.
surrounding the hydrophobic region II. Hypothetical SARs for
the heteroarylalkylamino substituents of compounds 26a-h
(Table 4), partly corresponding to those of the arylalkylami-
nopyridines, are schematized in Figure 14.
Despite the good inhibition in the p38 assay, prevention of
TNFR (and IL-1ꢀ) release by these manufactured heteroaryla-
lkylaminopyridines lagged behind that of the SB prototype. The
still relatively high lipophilicity of the introduced heteroarylalkyl
residues may be mainly responsible for this failure. As expected,
the comparatively more hydrophilic heteroarylalkylamino de-
rivatives (26a) 26d and 26f-h exhibited the lowest TNFR IC50
values in this series.
Placement of flexible amino substituents bearing a side chain
heteroaromatic ring led to a clearly enhanced inhibitory potency
(p38 IC50 values) in the isolated kinase assay comparable in
magnitude of that of the unsubstituted pyridinylimidazole
ML3375 (e.g., 26e,g,h) or even to the potent reference
SB203580 (26a,d,f) except for compound 26c with a condensed
heterocycle and the 3-trifluoromethylphenyl compounds 27a and
27b (Table 5). A 2-thienyl ring one or two carbon atoms distant
from the exocyclic NH group in the shape of a flexible linear
aliphatic chain gave the best results (Tables 5 and 6). There
was no need for an R-methyl branch in this linear spacer to
Introduction of (Cyclo)alkylamino Substituents at the
Pyridine C2. With regard to a future oral bioavailability of these
drug candidates, we also aimed to improve the expected in vivo

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4139
Figure 11. Several published Merck compounds with a 3-trifluoromethylphenyl ring at the imidazole-C4.7,10
Figure 12. Binding model of substituted 5-pyridin-4-yl-2-thioimidazoles in which the 4-fluorophenyl component at the 4-position of the imidazole
is replaced by an analogous 3-trifluoromethylphenyl residue.
absorption or permeation behavior of the more potent aryl(a-
lkyl)aminopyridinylimidazoles by enhancing their hydrophilicity
(solubility) to meet the Lipinski rule criteria.64 In particular,
the putatively more selective but quite lipophilic derivatives with
a 3-trifluoromethylphenyl moiety are anticipated to be poorly
soluble in aqueous media and become worse resorbed in the
blood circuit and furthermore are expected to distribute better
to more lipophilic compartiments and thus will possibly exhibit
lower plasma levels after oral gavage [e.g., calculated log P
values: 25e (Table 5), 6.03 ( 0.64; 22g (Table 4) 5.08 ( 0.65;
SB203580, 4.19 ( 0.67].7 Structurally similar inhibitors of p38
MAPK have been previously prepared in which the arylalky-
lamino residues at the pyridine (or at the bioisosteric pyrimidine)
were replaced by “smaller” (cyclo)aliphatic amino residues.32
These modified compounds showed suitable hydrophilicity
(solubility) and occupied better log P values32 but also claimed
a higher metabolical stability upon further substitution compared
to analogous diaryl heterocyclic inhibitors bearing methylben-
zylamine-like residues at the pyridine/pyrimidine C2.37,38 Some
of these derivatives exhibited highly potent inhibition of cytokine
release, particularly the suppression of the TNFR release.32
2-Thioimidazoles in Table 6 were modified in an analogous
way with either an aliphatic or a cycloaliphatic amino substituent
at the 2-position of their pyridine-4-yl moiety. With the
exception of compounds 28a,c,j,k, substances out of this series
exhibited a good to excellent p38 inhibitory activity, as clearly
reflected by IC50 concentrations ranging from the submicromolar
to the single-digit nanomolar range (e.g., 28b,l). The most potent
representatives 28b and 28l were 4- to 4.5-fold superior to
SB203580. The disintegration of the planarity of the aromatic
system introduced with the alkylamino side chain at the pyridine
moiety of the substituted imidazoles (Figure 15) may be
considered as a cause of the general increase in activity of these
arylalkylaminopyridines (Tables 3 and 4). Starting from com-
pound 28a with a cyclohexylmethylamino substituent, it was
again very effective to position an additional R-methyl branch
in the aliphatic spacer next to the exocyclic NH-nitrogen. The
resulting asymmetry of the residue was of great importance, as

4140 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Figure 13. Cocrystal structures of the p38R MAPK with different ATP-competitive diaryl-substituted imidazole inhibitors: (left) compound from
SmithKlineBeecham with a 4-fluorophenyl ring (PDB entry 1BL7);13 (right) compound from Merck with a 3-trifluoromethylphenyl ring (PDB
entry 1OUK).10 Pictured are the spatial distances of the possible H-bonds between the respective drug molecule and the backbone amino acids
Met109 as well as Lys53. Because of the differing orientation of the MERCK compound (with a bulkier 3-CF3 residue) in the adenine-binding
region of the kinase and the resulting larger distance of the imidazole-N3 to the side chain of Lys53, the corresponding ligand-enzyme H-bond is
missing.
Figure 14. Binding model for heteroarylalkylaminopyridines.
both enantiomers differently inhibited the p38, whereas in the
case of the objective cyclohexylethyl residue, the preference
clearly was in favor of the R-enantiomer (28b vs 28c, 16-fold
difference in activity). This chiral residue seems to be best
orientated between the two hydrophobic subareas of the
hydrophobic region II (Figure 3), while the more flexible
cyclohexylmethyl substituent of 28a probably cannot optimally
orientate at either the upper (area A) or the lower (area B)
lipophilic surface of this solvent-exposed front region (compare
ML3403 vs 22a, Table 4). Decisive benefits could be also
achieved by either shortening the spacer (28d-h) or lengthening
this side chain for one to three CH2 units (28i,j,n,o) and also
by a formal ring opening and narrowing of the aliphatic terminus
to a dimethyl group (at compounds 28i,j,l,n,o). Placement of
an additional methyl group in the ortho position of the directly
NH-linked cyclohexyl ring in 28e further improved the p38
inhibitory activity compared to the corresponding nor compound
28d. In contrast, additional para-placed methyl groups offered
only small advantages for the activity (28f-h). The trans-
configured 4-methylcyclohexyl residues gave better results than
their cis-configured counterparts (28f vs 28g). Inhibition data
for the suppression of TNFR release were more diverse than
the values for inhibition of the kinase, varying over 2 decimal
powers from the 2-digit micromolar to the 3-digit nanomolar
range. The smaller and less flexible the alkyl residues at the
exocyclic NH groups were, the lower the cytokine IC50 values
tended to be. The best results were obtained with the compounds
whose alkylamino substituents at the pyridine formally resemble
the biologically interesting 1,2-dimethylpropyl structure of
compound 28l, as proved by the analogues 28d,e,l,m and
partially by 28i and 28n. These derivatives dominate with their
cytokine inhibitory potency elevated up to 1 order of magnitude
(factor 10) over the model compound SB203580. The corre-
sponding SARs were also found for the less active analogues
from Table 5 bearing a 3-trifluoromethylphenyl ring at their
imidazole-C4 (29a,b), which exhibited decreased inhibition of
TNFR release owing to their higher lipophilicity. In addition,
calculated log P values of the most potent inhibitors 28 (Table

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4141
values of the remainder of the compounds in Tables 5 and 6
were in the submicromolar sector as well (between 61 and 10
nM). For a better understanding of the binding mode, a binding
model for compound 31b in the p38R MAPK was calculated
by molecular computer modeling based on the PDB structure
2ewa (Figure 18). The substituted trans-cyclohexyl ring fits
optimally between the two lipophilic surfaces of the hydrophobic
region II. Beside the positive vdW contacts within this region
an additional hydrogen bond could be identified linking the
exposed 4-hydroxy group at the cyclohexyl ring and the carbonyl
side chain of Asn115.
This series of relatively hydrophilic compounds (30a-i, Table
6), including the two derivatives with a 3-trifluoromethylphenyl
residue (31a,b, Table 5), consistently proved to be excellent
inhibitors of cytokine release from human whole blood, as
documented by the corresponding low micromolar IC50 con-
centrations for the inhibition of TNFR release. Here, the
inhibitory constants vary from 5.2 µM for compound 31a to
0.4 µM for 30h and 30i, which denotes, in the worst case, an
equipotent inhibitor and, at best (30i), a more than 8-fold
increased activity with respect to the reference SB203580. Better
water solubility, a strongly reduced plasma protein binding
behavior, and a possibly accelerated cell penetration with only
minor accumulation in the lipid membrane may have contributed
to this improvement. Furthermore, some of these more “hydro-
philic” pyridinylimidazoles were characterized by a significantly
diminished CYP450 interaction.
Besides the general positive effect on the physicochemical
properties, functionalized (polar) (cyclo)alkylresidues (e.g., with
additional hydroxy groups) in the 2-position of the pyridine may
further improve the activity profile by providing sites for an
even more flexible derivatization, e.g., via the subsequent
introduction of additional substituents in the already installed
side chain of the amino residues, etherification, oxidation, etc.
Figure 15. Possible binding model for the alkylaminopyridinylimi-
dazole derivatives with superimposed alkylamino substituents of all
compounds from Table 6. Highlighted is the 1,2-dimethylpropyl residue
of the most potent compound 28l, which formally represents a central
structure part in the other potent derivatives.
6, e.g., 28l, log P ) 3.93 ( 0.65) and 29 (Table 5, e.g., 29b,
log P ) 4.87 ( 0.64) fit much better with the Lipinski criteria
than those of the analogous arylalkylaminopyridine compounds
(Tables 4 and 5), which exhibited similar p38 activities,
qualifying them (28 and 29) as the preferred candidates for
potential oral application.
Introduction of (Cyclo)alkylamino Substituents with an
Additional Polar Group at the Pyridine C2. We considered
that placement of an additional polar group in the (cyclo)alky-
lamino residue may permit the formation of a further H-bond
with polar atoms of the protein backbone proximal to the
hydrophobic region II or its surrounding solvent-exposed area.
This change may improve the affinity of the inhibitor for p38
and increase the inhibitory potency compared to analogues with
simple alkyl substituents. Moreover, unwanted interactions with
the CYP450 system may be minimized by changing the
electronic properties of the substituted (cyclo)alkyl residues.
Indeed, even more biologically active 5-pyridin-4-yl-2-
thioimidazoles were achieved by the introduction of different
(cyclo)aliphatic primary amines with additional polar groups
(Tables 5 and 6). On the basis of the hydroxyethylaminopyridine
30a (Table 6), which had already been proved to be equipotent
to SB203580, the efficacy in the p38 assay was further improved
by extending the spacer (30g) or by the successful placement
of a small R- or ꢀ-alkyl group (next to the nitrogen atom) in
the aliphatic chain. However, the most potent representatives
generally possessed an R-methyl or ethyl branch in the side
chain of the amino substituent, as illustrated by the comparison
of 30d and 30e relative to 30b and 30f (Figure 16). The p38
IC50 value of 30d averaged 6 nM. The functionalities giving
the greatest advantage for the p38 activity are residues that can
be formally derived from cyclohexylamine, e.g., tetrahydropy-
ran-4-ylamine (at 30h) or 4-hydroxycyclohexylamine (at 30i).
For the latter substituent, as the trans form, the inhibitory
potency of 30i could be increased more than 20-fold as opposed
to SB203580 (Figure 17). Compound 30h exhibited a p38 IC50
of 5 nM, while compound 30i averaged an IC50 of only 3 nM.
Accordingly, the identical substituent, introduced at the fluori-
nated pyridinyl moiety in the 3-trifluoromethylphenyl-substituted
imidazoles series, led to a 5-fold boost of the activity over the
reference compound (31b, Table 5, IC50 ) 11 nM) and
overcame the lost binding energy arising from the absence of
the H-bond between the imidazole-N and Lys53. The p38 IC50
Introduction of (Hetero)(aryl)alkyloxy or Thioxy Sub-
stituents at the Pyridine C2. In the (hetero)(aryl)alkylamino-
pyridines presented here, the contribution of the exocyclic NH
function to the interaction with the p38 MAP kinase could be
quantitatively assessed by replacing the nitrogen atom in the
side chain of the pyridine with an oxygen or a sulfur or by
substituting the exocyclic amino group with an additional methyl
group (dimethylaminopyridine). Because of a disproportionate
steric demand within the targeted enzyme region, the latter
modification led in the case of 32 (Figure 19) only to a moderate
p38 inhibition, emphasizing the importance of the “free” NH
function in making an additional H-bond in the linker region.36
Because the benzyloxypyridine 33a (Table 7) and the 1-phe-
nylethyloxypyridine 33b, each with concurrent arylalkyl residues
complementary to their analogous aminopyridines ML3403,
exhibited a strongly decreased biological activity (5.1- and 16-
fold, respectively), it became evident that the nitrogen atom in
the side chain could not be replaced by an oxygen without
accepting losses of affinity and, consequently, of the activity
(Figure 19).
The (hetero)arylalkyl- and (cyclo)alkyloxy- or thioxypyridines
(Table 7), except compound 33c which most likely had an
alternative binding mode, generally exhibited worse biological
activity profiles in the isolated p38 MAPK assay as well as in
the whole blood assay, compared to the standard SB203580
(here about 1 magnitude, factor 10) or to the “unsubstituted”
pyridinylimidazole ML3375. The reduced activity can be
attributed to the abolition of the second hydrogen bond to
Met109 within the linker region (Figure 20). On the other hand,
factors like the atom size and the electronegativity as it affects

4142 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Figure 16. Common binding model for substituted pyridinylimidazoles with an additional polar group at the aliphatic or cyclicoaliphatic amino
substituent.
Figure 17. Possible interactions of compound 33i with the p38 MAPK.
polarizability of the exocyclic heteroatom at the 2-position of
the pyridine as well as differing binding angles of the linked
residues and subsequent changes in the spatial geometry play a
role. Small, short chain, and less voluminous but flexible
(hetero)arylalkyl (33a,b) or, even better, simple alkyl substit-
uents (34b, Table 7) still achieved the best results, whereas long
chain or branched (chiral) residues (e.g., with R-methyl groups)
at compounds 33b and 33g were counterproductive. In the series
of (hetero)(aryl)alkylaminopyridines, the development of an
asymmetric center by introduction of an R-methyl group in the
alkyl chain of the substituent at the pyridine always led to a
significant increase of the inhibitory activity of the respective
derivative in comparison to the underlying nor derivative.
Presumably, the resulting unfavorable conformation of com-
pounds 33b and 33g averted a deep entry of the drug molecule
into the active site of the kinase. Finally, interpretation of the
biological data led to the conclusion that there is no correlation
in the binding behavior of identical residues within the
hydrophobic region II between these (thi)oxypyridines and the
corresponding aminopyridine analogues. Compound 34b is
almost as active as the reference SB203580, indicating that
promising results could be also reached without the implied
H-bond by positive interactions (or no negative interactions)
with suitable but sterically less demanding substituents. For the
more potent p-tolyloxypyridine derivative 33c [and optionally,
for its untested phenylthioxy analogue 34a (Table 7)] a different

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4143
likely to occur. Thus, the potency of the inhibitors is largely
codetermined by the nature of the N1 residue.33
Inhibition concentrations of compounds 35a-o (Table 8) in
the kinase test assay vary from 960 to 3 nM. A modestly reduced
p38 activity, a general weakness for the N1-substituted meth-
ylsulfanylimidazole derivatives compared to the corresponding
N1 unsubstituted imidazoles (with identical or similar pyridine
moieties), was acceptable, as additional substituents at this
position of the imidazole core can further modify physicochem-
ical (hydrophilicity, solubility, electron density of the core
heterocycle, stability) and toxic properties33,36 (see Table 10).
Nevertheless, in some cases (35b,d,g,i,j), the introduction of
structurally diverse but sterically capable amino substituents at
the pyridine part in addition to the 2-methoxyethyl substituent
on the imidazole N1-position implied very effective interactions
with p38, leading to biological activities that clearly discrimi-
nated the reference SB203580. The chirality of introduced
amines is also relevant, as evidenced by the IC50 values of
enantiomers 35a and 35b as well as 35c and 35d. Here, the
S-enantiomers (35a and 35d) are identified as the predominant
stereoisomers in clear contrast to amino residues at the pyridine
moieties in the 2,4,5-trisubstituted imidazole series, where the
R-enantiomer was the more active form (see Table 6, 28b vs
28c).36 This finding is also consistent with the disappointing
results of the tetrasubstituted imidazole 35f (Table 8) bearing
an R-configured amino substituent at its pyridine. The inverted
activities can be most likely attributed to steric discrepancies
of the introduced (chiral) 2-aminopyridine substituents with the
adjacent imidazole N1 residues (2-methoxyethyl) causing a
“switch” in the conformation of the more extended inhibitor
molecules, leading to a change in the binding mode of the
(aryl)alkyl side chain at the exocyclic NH group in the
hydrophobic region II. In the presence of the 2-methoxyethyl
group at position N1, bulkier pyridine substituents are less well
accepted. At this point, the R-methyl group in the side chain of
the aliphatic spacer at the 2-aminopyridine sometimes seemed
to be rather harmful, as can be inferred from the biological
results of 35l and 35o. If the N1-methoxyethyl residue makes
a constant contibution to the affinity with p38, a different binding
mode for each of these pyridine substituents inside the hydro-
phobic region II (compared to the N-unsubstituted imidazole
analogues) can be postulated. This reasoning may explain the
lack of correlation in the IC50 values between analogous tri-
and tetrasubstituted imidazoles having identical (chiral) sub-
stituents at the pyridine.
Figure 18. Hypothetical binding mode of compound 31b (differently
colored) within the active site of p38 superposed by the reference
compound SB203580 (brownish). The docking is based on the PDB
structure 2ewa and was generated by FlexX implemented in Sybyl 7.2.
The 4-hydroxy functionality at the cyclohexyl moiety can form a
hydrogen bond with the carbonyl group of the side chain of Asn115,
which is located marginal the linker region.
binding mode inducing a peptide flip between Met109 and
Gly110 in the linker region was speculated.10,65 In turn, an
additional H-bond (here, between the oxygen atom in position
2 of the pyridine and the amide-NH of Gly110) led to a much
stronger binding of the investigated molecule in the ATP site
(Figure 21). Gly110 is an amino acid that is specific for the R-,
ꢀ-, and γ-isoforms of p38.10 Bulkier residues, which predomi-
nate in other MAP kinases, would make this peptide flip
energetically unfavorably, possibly determining the high selec-
tivity of this compound class (33c). In the isolated kinase, 33c
is 2-fold as active as the prototype SB203580. However, all of
the compounds from Table 7 were identified only as very poor
inhibitors of TNFR release compared with their aminopyridine
analogues, presumably because of their higher lipophilicity.
1,2,4,5-Tetrasubstituted Imidazoles. Introduction of
Different Amino Substituents at the Pyridine C2. For the
investigated pyridinylimidazoles, by the final combination of
optimized amino substituents at the 2-position of the pyridine
(deduced from the trisubstituted imidazole series discussed
above) with recently approved33,36,46,66 N1 (particularly methyl
and methoxyethyl) and C2 (preferably methylsulfanyl) substit-
uents, we sought to gain synergistic effects that would result in
improvements in affinity and activity, the physicochemical
properties, the metabolical stability, and the reduction of harmful
interactions with different CYP450 isozymes. Introduction of
large substituents at the 1 and 2 positions of the core imidazole
resulted in a general loss of activity. This effect could be
overcome by introducing appropriate substituents at the pyridinyl
moiety. Besides the well-known H-donor/acceptor functions of
the 2-aminopyridin-4-yl pharmacophore part with the p38 hinge
region, the NH-linked aryl, arylalkyl or (substituted) (cyclo)alkyl
side chains can make further hydrophobic and/or electrostatic
interactions with the surface-exposed front region and its
bordering areas leading to an even stronger binding of the small
molecule. For the N1 substituent, particularly of the 2-meth-
oxyethyl residue, which was found to have the most favorable
effects on the biological activity, the exact mode of binding
remains unclear.33 Because of steric interferences especially with
(bulkier) pyridine substituents, differing binding modes of the
flexible 2-methoxyethyl (perhaps within the ribose pocket) are
To clarify the binding mode of the more potent compounds
in this series, an energetically minimized binding mode was
generated for the tetrahydropyran-4-ylaminopyridine 35i by
molecular modeling (Figure 22). Analogue 35i is nearly
equipotent to the cyclohexylaminopyridine 35g both in the p38
test assay and in the whole blood test model. Among the listed
1,2,4,5-tetrasubstituted imidazoles (Table 8) compound 35j
exhibited the best results, with a relative p38 IC50 value of only
0.048 (IC50 ) 3 nM!). Here, the substituents at all relevant
positions of the pyridinylimidazole scaffold seem to be best
possibly combined. In analogy to 35i, the introduced trans-4-
hydroxycyclohexyl residue of 35j optimally fits into the surface-
exposed front region making hydrophobic as well as additional
electrostatic interactions and significantly increasing the activity
and probably the selectivity. Furthermore, 35j also maintained
its high potency in the cytokine test, whereas TNFR results of
the other compounds were more widespread. Substituents placed
at the 2 position of the imidazole (here, 2-methoxyethyl)
contribute to bioactivity only to a lesser extent. By steric

4144 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Figure 19. Influence of the exocyclic NH functionality to the inhibition of p38.
Figure 20. SAR of the (hetero)(aryl)alkyloxypyridines.
potential inhibitors, however, cannot be simply predicted on the
basis of present sequence or structure information, requiring
extensive testing of the compounds against multiple kinases.
The specificity among many of the known kinase inhibitor
molecules that were either advanced to clinical trials or admitted
for therapeutic use widely varies, and the specificity could not
be strongly correlated with the chemical structure or the identity
of the intended target structure.30 Of 20 inhibitor molecules that
had been profiled against a panel of 113 different kinases, the
pyridinylimidazoles SB203580 and SB202190 were among the
most selective compounds, binding only to a few other kinases
apart from their primary target.
Selectivity profilings of a selection of our substituted pyridi-
nyl-2-thioimidazole inhibitors, summarized in Table 9, also
indicate a clear preference for p38 compared to other relevant
human kinases selected from different categories of the phy-
logenetic kinase tree. In this test (of 17 kinases beside p38R)
compounds 22c,d,g, 26b, 28o, 30c, and 35g exhibited excellent
p38R IC50 values between 13 and 68 nM. The p38ꢀ form was
on average 96% inhibited at an inhibitor concentration of 10
µM, whereas the γ- and the δ-forms were not inhibited by these
compounds. The documented tri- and tetrasubstituted imidazole
derivatives only exhibited an inhibitory activity toward the very
closely related JNKs. However, while compounds 22d,g, 26b,
28o, and 35g inhibited all three JNK subtypes (JNK 1R1, JNK
2R2, and JNK3) nearly equipotently, the most selective 1-phe-
nylethylaminopyridine derivative 22c of this series shows only
a dominant inhibition toward JNK3, whereas both other isoforms
Figure 21. Possible binding model of the aryloxypyridine 33c, which
gives consideration to the experimentally detected high p38 inhibitory
activity compared to the less potent (hetero)(aryl)alkyloxypyridine
derivatives.
shielding and/or changing of the electronical properties of the
critical imidazole core, these N1 substituents reduced negative
CYP450 interactions.
Many kinase inhibitors that are currently in clinical or
preclinical development states are directed to the ATP binding
site of the enzyme. Many of these ATP-competitive inhibitors
lack of selectivity. Binding specificity and affinity of the

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4145
imidazole series of the present work. To determine if the
introduced residues actually can reduce the affinity of these
compounds with the CYP450 system, some of the more potent
representatives were additionally subjected to a screening assay
with relevant CYP 450 isozymes. Among the 20 inhomogeneous
pyridinyl-2-thioimidazoles (Table 10), there are differences in
the inhibition of the tested CYP isozymes. These differences
are particularly apparent when examining the metabolically
relevant isozymes 1A2 and 2D6. Compounds 22c,g, 26f, 28l,o,
30b, 35a, and 35c-j inhibited CYP450 1A2 less than 55%.
Moreover, the isoform 2D6 is also less inhibited by compounds
26a,f,g, 28l, 30b,c, 33c, and 35a,b,d,g,j compared with the
model inhibitor SB203580, whereas the other candidates were
stronger inhibitors of these two isoforms. Of the remaining tested
CYP450 isozymes, only slight improvements could be achieved
over SB203580 by the introduction of the respective substituents
at the pyridine ring (inhibition of 2C9, 2C19, and 3A4 less than
55% by 26f, 30b,c as well as by 35j). It is remarkable that the
investigated CYP450 enzymes are commonly better inhibited
by compounds with nonpolar substituents (e.g., 22d, 26b,d,g,
28a, or 33c). Accordingly, the relatively more hydrophilic
compounds 26f, 30b,c as well as 35b and 35j exhibited the
lowest affinity (inhibition less than 55%) compared to almost
all tested CYP isozymes (except CYP450 1A2 at 30c and 3A4
at 35b,j). These compounds would be expected to have the
lowest pharmacological toxicity in vivo too and therefore
represent the most promising developmental candidates. This
result is gratifying, as the most potent inhibitors presented here
were gained by the introduction of polar amino alcohols at
the pyridine. Furthermore, as indicated by the examples in Table
10, a reduction of the CYP450-mediated toxicity can be
achieved only by a sterically shielded and electronically
modified pyridine ring without additional N1 substituents, thus
making the pyridine the more critical structural determinant,
with respect to toxicology, of this inhibitor class.
Figure 22. Proposed binding mode of compound 35i in the active
site of the 38R MAPK. The docking was performed with FlexX
implemented in Sybyl 7.2 based on the PDB structure 1BL7. One
possible H-bond is detected between the basic imidazole N (sp2) and
Lys53. Moreover, the 2-aminopyridine ring can execute the essential
H-donor/acceptor functions with backbone Met109 in the linker region.
The tetrahydropyran-4-yl side chain in the 2-position of the pyridine
ring is optimally located in the hydrophobic region II. The ring oxygen
atom of the pyrane can possibly make further electrostatic interactions
(e.g., with or via a water molecule of the solvent area surrounding
the surface-exposed front region) leading to a significant increase in
the activity. Because of the inability to form an H-bond with the
(deprotonated) Asp168 under physiological conditions, the impor-
tance of the flexible 2-methoxyethyl substituent at the N1 position for
the inhibitor potency remains less clear. A sterical antagonism with
the respective substituent at the pyridine ring (here, tetrahydropyran-
4-ylamino) is most likely leading to a “conformational switch” of the
compound compared to analogous 2,4,5-trisubstituted imidazoles, which
lack an additional substituent at the imidazole N1. However, adequate
aliphatic substituents at the N1 position of the imidazole can possibly
further improve the physicochemical properties (e.g., log P values) of
the inhibitor molecule.
Conclusions
(JNK2 and JNK3) were not addressed. Similarly, the tetrahy-
drofuranylmethylaminopyridine 30c exhibited a clearly reduced
inhibition of the JNK1 subtype.
The optimized and, on the whole, extremely flexible synthetic
concepts illustrated herein allowed us to regioselectively produce
numerous highly (2,4,5-tri- and 1,2,4,5-tetra-) substituted and
structurally diverse 2-thioimidazole derivatives starting from
comparatively few ethanone structures. In the p38-MAPK test
assay, many of the represented (amino)pyridinylimidazole
analogues were superior to the current standard SB203580 with
respect to the inhibitory activity. Because of their suitable cell
penetration properties, the most potent compounds also inhibited
the release of relevant cytokines (TNFR, IL-1ꢀ) from human
whole blood with high efficacy.
In this study the vast importance of the kinase’s surface-
exposed front region (hydrophobic region II) as an important
binding area of protein kinases (here, the p38R MAPK) for
gaining additional activity and selectivity was impressively
demonstrated.
By introduction of additional substituents at the pyridine ring
of the 2-thioimidazole derivatives, several inhibitors could be
generated whose specificity for p38 and whose toxic pharma-
cological profile may suggest further developments to anti-
inflammatory drugs.4,67,68 On the basis of these biological
primary screenings, a number of compounds were selected for
follow-up investigation (in particular, 22c,g, 26b,f, 28l, 29a,
30c,d,h,i, 31b, 33c, 35b,d,g,i, j).
Despite the fact that the shown inhibitors (Table 9) quasi-
mimic the enzymatic cosubstrate and thus compete with ATP
for binding to the active site of the kinase(s), they are much
more selective to p38R and ꢀ than to the p38γ and the δ
isoforms (e.g., in comparison to the “allosteric” DFGout binder
BIRB796) as well as toward most of the other members of the
MAPK family. In addition, these compounds have virtually no
activity against other kinases including the tyrosine kinases.7
Most of the interactions of these exemplified compounds
(22c,d,g, 26b, 28o, 30c, and 35g) with p38R occur with areas
of the protein that are not directly involved in the binding of
ATP and thus are less highly conserved among the kinases but
that decisively contribute to the recognized selectivity. By
introduction of suitable substituents at the 2-position of the
pyridine of the diaryl heterocyclic imidazoles, which can
effectively interact with the kinase’s surface-exposed front
region (hydrophobic region II), not only the inhibitory potency
can be significantly enhanced but also additional selectivity to
p38 with regard to other kinases can be gained.
In order to minimize the toxic effects arising from unwanted
CYP interactions, future generations of pyridinylimidazoles will
have to be modified by introducing sterically demanding and
electronically modifying substituents at the imidazole and/or
the pyridine ring. These variations could be successfully
accomplished at both the tri- and the tetrasubstituted 2-thio-
With regard to the potent prototype SB203580, the most
promising inhibitory properties useful for a further preclinical
development are mainly offered by those compounds that

4146 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
Experimental Section
Chemistry. All reagents and solvents were of commercial quality
and used without further purification. General procedures A and B
were followed for the preparation of target compounds via
nucleophilic aromatic substitution of 2-halogenopyridines 5 and 7.
General Procedure A for the Preparation of N-Substituted
2-Aminopyridines. Variant 1. Under an inert atmosphere of argon,
the respective 5-(2-halogenopyridin-4-yl)imidazole (1 equiv) was
suspended in an excess volume of the respective (primary) amine
(5-10 equiv). The mixture was stirred at the appropriate temper-
ature until the imidazole starting material had been consumed
completely and no further product formation took place or increas-
ing amounts of any side products were detected (TLC, SiO2 60,
DCM/ethyl acetate, 1/2). The reaction was cooled to room tem-
perature and suspended in an aqueous solution of citric acid (10%),
which had been brought to pH 5 with an aqueous solution of NaOH
(20%). The emulsion was extracted with ethyl acetate (3×). The
combined organic extract was washed with citric acid (10%, pH
5), an aqueous solution of Na2CO3 (10%), and saturated brine, dried
over Na2SO4, and concentrated in vacuo. The crude oily residue
was mostly purified by column chromatography or preparative TLC
and seldom by recrystallization from ethanol. Crystallization/
solidification of the pure compound occurred upon evaporation with
little diethyl ether.
Variant 2. After the 5-(2-halogenopyridin-4-yl)imidazole was
reacted with an excess of the respective amine according to variant
1, the cold reaction mixture was taken up/dissolved in the required
volume of ethyl acetate (6-90 mL/mmol starting material) and the
remaining amine was removed by vacuum distillation (amines with
relatively low boiling points) or by multiple washing of the organic
solution with water (4-10 mL/mmol starting material) or brine.
The pH of the final aqueous extraction phase should be almost
neutral (pH 6-7). The pooled aqueous extract then was once again
extracted with ethyl acetate. The entire organic extract was dried
over Na2SO4 and the solvent rotary evaporated in vacuo. In some
cases only (e.g., with less hydrophilic amino nucleophiles), the
resulting oily residue had to be additionally purified by a chro-
matographic method. Otherwise, the concentrated and commonly
analytically pure compound was either crystallized from n-hexane
or solidified upon evaporation with diethyl ether as an amorphous
or a glasslike substance.
Figure 23. Selectivity of 22c for p38 toward other kinases (left) in
comparison with SB203580 (right), modified according to Fabian et
al.30 (both compounds were tested against a panel of 17 different kinases
of the human genome).
optimally combine excellent enzyme-inhibitory concentrations
(in the low nanomolar range) and cell penetration promoting
characteristics. This is in particular realized for compounds 22c
and 22g, 26f, 28l, 30b,c, 30h, 35b,d, and 35g. However, for
compounds 26b, 29b, 30d, 33c, 35b, and 35h-k the good
inhibitory potencies in the p38 kinase test assay are offset by
unfavorable cell permeation properties, and so effective inhibi-
tion is not expressed in the cellular (whole blood) model.
Furthermore, with regard to the molecular weights, the calcu-
lated log P values, and the number of available H-bond donors
and acceptors, the Lipinski rule of five criteria have been fulfilled
by all selected imidazole compounds, additionally qualifying
them as promising drug candidates for an oral administration.64
Moreover, cytochrome P450 interaction was very efficiently
reduced through introduction of sterically and also electronically
shielding (amino)substituents at the 2-position of the pyridine
moiety particularly in compounds 26f, 30b,c, 35b, and 35j.18
Other derivatives also exhibited a clearly decreased CYP450
interaction potential, especially with the metabolically relevant
isozymes 1A2 and 2D6.
Many first generation p38 inhibitors, which imitate the
cosubstrate ATP, are limited by insufficient specificity because
of the sequential and structural similarities of the ATP-binding
sites of a multiplicity of the 518 known protein kinases. Thus,
the design of specific ATP site directed kinase inhibitors (ATP
mimetics) is still considered a major challenge to be met by
medicinal chemistry.23 Here, the prototypical pyridinylimida-
zoles SB203580 and SB202190 still rank among the most
selective inhibitor representatives30 in their class, whose selec-
tivity profile is even better than, for example, that of the
“allosteric” urea-based inhibitor BIRB796 binding in the so-
called DFG out region hindering ATP from entering into the
ATP site.69
General Procedure B for the Preparation of O- and S-Sub-
stituted 2-Oxy- or Thioxypyridines. To a suspension of NaH
(moistened with oil, 55-65%, 1.05 equiv) in dry DMF under argon
was dropwise added the respective liquid alcohol or thioalcohol (1
equiv). The mixture was stirred at room temperature until gas release
(H2) ceased and deprotonation of the nucleophilic reagent was
complete (1-2 h, bubble counter). Subsequently the respective 5-(2-
halogenopyridin-4-yl)imidazole (0.1 equiv) was added in one
portion to this suspension and the mixture was heated to reflux
temperature and left stirring at this temperature for the indicated
time. Then the mixture was cooled to room temperature and was
hydrolyzed with the respective volume of water or 10% citric acid
(pH 4-5). The aqueous/organic mixture was extracted with ethyl
acetate (4 × 50 mL), and after it was optionally dried over Na2SO4,
the solvent of the combined organic phases was evaporated as far
as possible (removal of residual DMF!). To obtain the pure
substitution product, the resulting dark oily residue was purified
by using a chromatographic method. Further purification techniques
could follow (e.g., recrystallization procedures or washing steps).
The initially sticky product was finally dried and solidified by
treatment with diethyl ether.
By considering and exploiting the less conserved areas at the
active site of p38 that are not occupied by ATP, we have
attempted to design inhibitors that target hydrophobic region II
with the aim to improve p38 MAPK selectivity. The novel
2-alkylsulfanyl-5-pyridin-4-ylimidazole compounds of the present
work having additional (amino-) substituents in the 2-position
of the pyridine ring, which make efficient interactions with
residues of the hydrophobic region II and beyond, show a clearly
enhanced specificity for p38 over other kinases that exceeds
the reference SB203580 (Figure 23, exemplary for 22c).
Typical Procedures. {4-[4-(4-Fluorophenyl)-2-methylsulfanyl-
1H-imidazol-5-yl]pyridin-2-yl}-(2-phenylpropyl)amine (22f). Ac-
cording to general procedure A (variant 1) the title compound was
obtained from 2-fluoro-4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-
imidazol-4-yl]pyridine 5a (0.5 g, 1.6 mmol) and (RS)-2-phenyl-
propylamine (2.23 g, 16.5 mmol) after 8 h at 160 °C. The crude
product was purified by column chromatography (SiO2 60, CH2Cl2/
EA 1 + 2) to yield 0.45 g (65.2%) of 22f: C24H23FN4S (Mr 418.54);

Substituted Imidazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4147
mp 107 °C; 1H NMR (CD3OD) δ (ppm) 1.18-1.27 (m, 3H, -CH3),
2.60 (s, 3H, -SCH3), 2.89-3.00 (m, 1H, methine-H), 3.25-3.34
(m, 2H, N-CH2-, superposed by the CD3OD signal), 6.50-6.56
(m, 2H, C3-/C5-H 2-amino-Pyr), 7.06-7.26 (m, 7H, C3-/C5-H
4-F-Phe and Phe), 7.40-7.47 (m, 2H, C2-/C6-H 4-F-Phe), 7.80
(dd, 1H, J ) 0.71/5.48 Hz, C6-H 2-amino-Pyr); IR (ATR) 1220
(C-F) cm-1; GC (method 2) 23.3 min; MS m/z (%) 418 (6, M+),
313 (100), 298 (8), 105 (5, C8H9+), 91 (1, tropylium+), 77 (2), 65,
51; HRMS (ESI, pos mode) m/z [M + H]+ calcd for C24H24FN4S,
419.1700; found, 419.1690.
Furan-2-ylmethyl-{4-[2-methylsulfanyl-4-(3-trifluorometh-
ylphenyl)-1H-imidazol-5-yl]pyridin-2-yl}amine (27b). According
to general procedure A (variant 1), the title compound was obtained
from 2-fluoro-4-[2-methylsulfanyl-5-(3-trifluoromethyl-phenyl)-3H-
imidazol-4-yl]pyridine 5i (0.3 g, 0.8 mmol) and furfurylamine (1.0
g, 10.3 mmol) after 8.5 h under reflux. The crude product was
purified by column chromatography (SiO2 60, CH2Cl2/EA 1 + 2)
to yield 164 mg (44.9%) of 27b: C21H17F3N4OS (Mr 430.46); mp
105 °C; 1H NMR (CD3OD) δ (ppm) 2.64 (s, 3H, -SCH3), 4.41 (s,
2H, -CH2-), 6.14-6.16 (m, C3-H furyl-), 6.29-6.31 (m, 1H,
C4-H furyl-), 6.56-6.62 (m, 2H, C3-/C5-H 2-amino-Pyr),
7.37-7.39 (m, 1H, C5-H furyl-), 7.51-7.71 (m, 3H, C4-/C5-/
C6-H 3-trifluoromethyl-Phe), 7.78 (s, 1H, C2-H 3-trifluoromethyl-
Phe), 7.90 (d, 1H, J ) 5.43 Hz, C6-H 2-amino-Pyr); IR (ATR)
1122 (CF3) cm-1; GC (method 2) 12.2 min; MS m/z (%) 430 (100,
M+), 415 (3, M+ - CH3), 401 (71), 375 (12), 335 (6), 262 (2),
205 (13), 171 (3), 145 (2, 3-trifluoromethylphenyl+), 96 (7,
furfurylamino+), 81 (25, furfuryl+), 53 (11); HRMS (ESI, pos
mode) m/z [M + H]+ calcd for C21H18F3N4OS, 431.1148; found,
431.1150.
4-[4-(4-Fluorophenyl)-2-methylsulfanyl-1H-imidazol-5-yl]-2-
(1-phenylethoxy)pyridine (33b). According to general procedure
B, the title compound was obtained from NaH (0.79 g, 18.1 mmol,
moistened with oil, 55-65%), (RS)-1-phenylethanol (2.1 g, 17.2
mmol), and 2-fluoro-4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-
imidazol-4-yl]pyridine 5a (0.5 g, 1.6 mmol) in absolute DMF (7
mL) after 4.5 h at 155 °C. The crude product was prepurified by
column chromatography (SiO2 60, CH2Cl2/EA 4 + 1), and after
complete evaporation of the organic solvent(s) the residue dissolved
in methanol. Upon dropwise addition of water, pure 33b precipitated
from the alcoholic solution as fine white crystals. The suspension
was ultracentrifugated and the surmounting solvent mixture (includ-
ing residual phenylethanol) decanted. The sediment was dried by
multiple evaporation with little diethyl ether. Yield, 0.4 g (59.8%)
1
of 33b; C23H20FN3OS (Mr 405.50); mp 62-64 (82) °C; H NMR
(CD3OD) δ (ppm) 1.56 (d, 3H, J ) 6.50 Hz, -CH3), 2.61 (s, 3H,
-SCH3), 5.96 (q, 1H, J ) 6.48 Hz, methine-H), 6.82-6.93 (m,
2H, C3-/C5-H 2-oxy-Pyr), 7.08-7.44 (m, 9H, 4-F-Phe and Phe),
7.92 (d, 1H, J ) 5.30 Hz, C6-H 2-oxy-Pyr); IR (ATR) 1222 (C-F)
cm-1; GC (method 2) 17.8 min; MS m/z (%) 405 (85, M+), 390
(22, M+ - CH3), 328 (6), 300 (100, M+ - phenylethyl), 285 (39),
268 (28), 228 (9), 172 (8), 121 (4), 105 (76, C8H9+), 79 (12), 77
(11), 51 (2); HRMS (ESI, pos mode) m/z [M + H]+ calcd for
C23H21FN3OS, 406.1384; found, 406.1371.
2-Ethylsulfanyl-4-[4-(4-fluorophenyl)-2-methylsulfanyl-1H-
imidazol-5-yl]pyridine (34b). According to general procedure B,
the title compound was obtained from 2-fluoro-4-[5-(4-fluorophe-
nyl)-2-methylsulfanyl-3H-imidazol-4-yl]pyridine 5a (0.4 g, 1.3
mmol) and sodium ethanthiolate (1.11 g, 13.2 mmol) in absolute
DMF (5 mL) after 7 h at 155-160 °C. The cold reaction mixture
was combined with ice-water (∼150 mL) and acidified with 20%
HCl to pH 3-4. From the cloudy solution a fluffy yellow-beige
precipitate dropped down, which was filtered off and dried in vacuo.
The crude product was two times purified by column chromatog-
raphy (SiO2, CH2Cl2/EA 1 + 2 and SiO2, CH2Cl2/EA 1 + 1) to
finally yield 0.17 g (37.3%) of 34b. Crystallization of 34b occurred
upon multiple evaporation with little diethyl ether: C17H16FN3S2
((R)-1-Cyclohexylethyl)-{4-[4-(4-fluorophenyl)-2-methylsulfa-
nyl-1H-imidazol-5-yl]pyridin-2-yl}amine (28b). According to
general procedure A (variant 1), the title compound was obtained
from 2-fluoro-4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-
4-yl]pyridine 5a (0.5 g, 1.6 mmol) and (R)-(-)-1-cyclohexylethy-
lamine (2.1 g, 16.5 mmol) after 8.5 h at 155-160 °C. The crude
product was purified by column chromatography (SiO2 60, CH2Cl2/
EA 1 + 2) to yield 180 mg (26.6%) of 28b: C23H27FN4S (Mr
1
(Mr 345.46); mp 60 °C; H NMR (CD3OD) δ (ppm) 1.23-1.37
1
(m, 3H, -CH2-CH3), 2.63 (s, 3H, -SCH3), 2.94-3.05 (m, 2H,
-S-CH2-), 7.09-7.27 (m, 3H, C3-/C5-H 4-F-Phe and C5-H
2-thioxy-Pyr), 7.37 (s, 1H, C3-H 2-thioxy-Pyr), 7.43-7.50 (m, 2H,
C2-/C6-H 4-F-Phe), 8.22 (d, 1H, J ) 5.46 Hz, C6-H 2-thioxy-
Pyr); IR (ATR) 1223 (C-F) cm-1; GC (method 2) 10.5 min; MS
m/z (%) 345 (100, M+), 330 (48, M+ - CH3), 312 (71), 285 (24),
269 (9), 212 (7), 121 (6), 95 (4, 4-fluorophenyl+); HRMS (ESI,
pos mode) m/z [M + H]+ calcd for C17H17FN3S2, 346.0842; found,
346.0850.
410.56); mp 117 °C; H NMR (CD3OD) δ (ppm) 0.93-1.40 (m,
9H, C3-/C4-/C5-H2 cyclohexyl and -CH3), 1.72-1.82 (m, 5H,
C2-/C6-H2 and C1-H cyclohexyl), 2.62 (s, 3H, -SCH3), 3.50
(quint, 1H, J ) 6.51 Hz, NCH<), 6.48-6.52 (m, 2H, C3-/C5-H
2-amino-Pyr), 7.09-7.18 (m, 2H, C3-/C5-H 4-F-Phe), 7.41-7.50
(m, 2H, C2-/C6-H 4-F-Phe), 7,78 (d, 1H, J ) 5.80 Hz, C6-H
2-amino-Pyr); IR (ATR) 1221 (C-F) cm-1; GC (method 2) 18.7
min; MS m/z (%) 410 (13, M+), 395 (3, M+ - CH3), 327 (100,
M+-cyclohexyl), 311 (12), 300 (28), 285 (3), 267 (3), 163 (5),
147 (15), 95 (1, 4-fluorophenyl+), 55 (4); HRMS (ESI, pos mode)
m/z [M + H]+ calcd for C23H28FN4S, 411.2013; found, 411.2009.
2-{4-[4-(4-Fluorophenyl)-2-methylsulfanyl-1H-imidazol-5-
yl]pyridin-2-ylamino}propan-1-ol (30d). According to general
procedure A (variant 1), the title compound was obtained from
2-fluoro-4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-
yl]pyridine 5a (0.5 g, 1.6 mmol) and (RS)-2-amino-1-propanol (2.48
g, 33.0 mmol) after 8 h at 155 °C. The reaction mixture was
combined with 10% citric acid (pH 4-5, 15 mL) and subsequently
extracted with ethyl acetate (5 × 30 mL). Washing the organic
extract conformable to the specifications with 10% NaCO3(aq), 10%
citric acid (pH 4-5) and saturated NaCl(aq) left the analytically
pure product that could be solidified as amorphous foam upon
multiple evaporating with diethyl ether. A chromatographic puri-
fication was not necessary. Yield: 0.47 g (79.6%) of 30d;
C18H19FN4OS (Mr 358.44); mp 92 °C; 1H NMR (CD3OD) δ (ppm)
1.17 (d, 3H, J ) 6.57 Hz, -CH3), 2.63 (s, 3H, -SCH3), 3.51-3.55
(m, 2H, -CH2-), 3.82 (sext, 1H, J ) 6.58 Hz, methine-H), 6.53
(dd, 1H, J ) 1.52/5.51 Hz, C5-H 2-amino-Pyr), 6.60 (s, 1H, C3-H
2-amino-Pyr), 7.09-7.19 (m, 2H, C3-/C5-H 4-F-Phe), 7.42-7.50
(m, 2H, C2-/C6-H 4-F-Phe), 7.82 (d, 1H, J ) 5.53 Hz, C6-H
2-amino-Pyr); IR (ATR) 1221 (C-F) cm-1; LC 11.0 min, 99.9%;
HRMS (ESI, pos mode) m/z [M + H]+ calcd for C18H20FN4OS,
359.1336; found, 359.1324.
{4-[4-(4-Fluorophenyl)-3-(2-methoxyethyl)-2-methylsulfanyl-
1H-imidazol-5-yl]pyridin-2-yl}(tetrahydro-pyran-4-yl)amine (35i).
Starting from 0.3 g (0.8 mmol) of 2-fluoro-4-[5-(4-fluorophenyl)-
3-(2-methoxyethyl)-2-methylsulfanyl-3H-imidazol-4-yl]pyridine 7b
and 0.84 g (8.3 mmol) tetrahydropyran-4-ylamine, an amount of
345 mg (93.9%) of compound 35i was obtained according to the
general procedure A (variant 2). Reaction time, 16 h; C23H27FN4O2S
(Mr 442.56); mp 59-62 °C; 1H NMR (CDCl3) δ (ppm) 1.40-1.60
(m, 2H, C3-/C5-H tetrahydropyranyl-), 1.93-2.00 (m, 2H, C3-/
C5-H tetrahydropyranyl-), 2.72 (s, 3H, -SCH3), 3.27 (s, 3H,
-OCH3), 3.42-3.56 (m, 4H, C2-/C6-H tetrahydropyranyl- and
-CH2-O-), 3.65-3.81 (m, 1H, methine-H), 3.93-4.07 (m, 4H,
C2-/C6-H tetrahydropyranyl- and >N-CH2-), 4.64 (d, 1H,
exchangeable, J ) 7.65 Hz, >NH), 6.36 (s, 1H, C3-H 2-amino-
Pyr), 6.57 (dd, 1H, J ) 1.36/5.21 Hz, C5-H 2-amino-Pyr),
6.88-6.96 (m, 2H, C3-/C5-H 4-F-Phe), 7.43-7.50 (m, 2H, C2-/
C6-H 4-F-Phe), 8.14 (dd, 1H, J ) 0.55/5.22 Hz, C6-H 2-amino-
Pyr); IR (ATR) 1219 (C-F) cm-1; GC (method 2) 16.1 min; MS
m/z (%) 442 (100, M+), 427 (4, M+ - CH3), 413 (8), 397 (9), 385
(50), 370 (8), 357 (64, M+ - tetrahydropyranyl), 342 (3, M+
-
tetrahydropyranylamino+), 325 (11), 299 (8), 267 (16), 146 (4),
121 (4), 100 (2, tetrahydropyran-4-ylamino+), 59 (4, methoxy-
ethyl+), 55 (5); HRMS (ESI, pos mode) m/z [M + H]+ calcd for
C23H28FN4O2S, 443.1912; found, 443.1909.

4148 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Laufer et al.
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The syntheses and analytical properties of compounds 1a-d,
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Acknowledgment. This work was supported financially by
Merckle GmbH, Blaubeuren, Germany, and the Fonds der
Chemischen Industrie, Germany. We thank S. Linsenmaier, S.
Luik, C. Klein, M. Goettert,and K. Bauer for the assistance in
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Supporting Information Available: Synthetic procedures,
routine spectroscopic and HRMS data of all target compounds, and
(HP)LC and 13C NMR data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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