Imidazole inhibitors of cytokine release: Probing substituents in the 2 position

Article abstract of DOI:10.1021/jm020873z

Novel 2,4,5-trisubstituted imidazole derivatives were prepared as potential anticytokine agents. Thirty-seven compounds were tested on their ability to inhibit the release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) from peripheral blood

Full text of DOI:10.1021/jm020873z

J . Med. Chem. 2002, 45, 4695-4705
4695
Im id a zole In h ibitor s of Cytok in e Relea se: P r obin g Su bstitu en ts in th e 2
P osition
Stefan A. Laufer,*,† Hans-Gu¨nther Striegel,‡ and Gerd K. Wagner†
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tu¨bingen, Auf der
Morgenstelle 8, 72076 Tu¨bingen, Germany, and Merckle GmbH, Dr. Georg Spohn Strasse, 89143 Blaubeuren, Germany
Received March 12, 2002
Novel 2,4,5-trisubstituted imidazole derivatives were prepared as potential anticytokine agents.
Thirty-seven compounds were tested on their ability to inhibit the release of tumor necrosis
factor-R (TNF-R) and interleukin-1â (IL-1â) from peripheral blood mononuclear cells (PBMC)
or human whole blood. SARs (structure activity relationships) for substituents at the 4 and 5
position of the imidazole core were similar to those described for other inhibitors of cytokine
release and p38 MAP (mitogen-activated protein) kinase. Starting from benzylsulfanyl imidazole
2b (IC50 p38, 4.0 µM; TNF-R, 1.1 µM; IL-1â, 0.38 µM), the contribution of substituents at the
2 position to enzyme inhibitory and cellular activity of test compounds was investigated. This
strategy led to the identification of compound 2q (IC50 p38, 0.63 µM; TNF-R, 0.90 µM; IL-1â,
0.04 µM), which was 6-10 times more potent than the initial lead 2b with respect to inhibition
of p38 and IL-1â release and equipotently inhibited TNF-R release.
In tr od u ction
forms a crucial hydrogen bond from the backbone NH
of Met 109 to the pyridin N atom.13 The adjacent
4-fluorophenyl substituent provides inhibitor selectivity
for p38 over other kinases, filling a hydrophobic pocket
which is left unoccupied by ATP.13-15 On the other hand,
substituents at the 2 position of the core imidazole have
mostly served to adjust the physicochemical properties
of p38 inhibitors.16 A number of amino acids in p38 are,
however, known as possible sites of interaction if appro-
priate side chains are attached to the 1 or 2 position of
the imidazole nucleus, e.g., the efficacy of an aromatic
moiety at the 2 position of SB 203580, has been
attributed to its stacking interaction with Tyr 35.13 In
the present study, we investigated whether inhibition
of cytokine release in a series of novel pyridinylimida-
zoles depends on the nature of substituents at the 2
position of the imidazole core. 1H imidazoles SB 203580
and ML 3163 (2b), a known inhibitor of cytokine release,
were both derived from the early lead SK&F 86002
(Figure 1).17-19 Opening the imidazothiazoline system
through incision of bonds a and c yielded SB 203580
after insertion of a 4-methanesulfinylphenyl moiety,
while dissection of bonds c and d and subsequent inser-
tion of a 4-methanesulfinylphenyl moiety led to 2b.
Superposition of SB 203580 and ATP shows the meth-
anesulfinyl moiety of SB 203580 in close proximity to
the phosphates of ATP.10 For a number of kinases, it
has been found that upon binding of ATP, the triphos-
phate group is coordinated by one or two metal ions
which are ligated by amino acids located in the phos-
phate binding ribbon.20 The suitability of phenolic
structures as metal chelators has been demonstrated.21
Therefore, we reasoned that the methanesulfinyl part
of 2b interacts with the phosphate binding ribbon of p38
and that analogues of 2b bearing an additional phenolic
functionality at the benzylsulfanyl moiety should sub-
sequently be better suited for interaction with this
region of the enzyme. As part of our program to find
novel and potent inhibitors of cytokine release, we have
With the current drug therapy of chronic inflamma-
tory conditions such as rheumatoid arthritis (RA) and
inflammatory bowel disease (IBD) often being limited
by severe side effects, there is a continuous medical need
for new efficient and safe anti-inflammatory drugs. The
antibody Infliximab and the fusion protein Etanercept
were successfully launched in 1999 and have shown
efficacy in the treatment of RA and IBD ever since by
reducing the synovial and circulating blood levels of
tumor necrosis factor-R (TNF-R), a pro-inflammatory
cytokine.1,2 Therapeutic application of both Infliximab
and Etanercept on a large scale is, however, hampered
by high costs of therapy3 and the general disadvantages
of protein drugs such as the lack of oral availability and
a time-dependent loss of activity. Therefore, attention
in inflammation research has focused on the develop-
ment of small molecular inhibitors of cytokine release.4,5
An important drug target in this respect is provided by
p38 MAP (mitogen-activated protein) kinase, a Ser/Thr
kinase involved in the biosynthesis of pro-inflammatory
cytokines interleukin-1â (IL-1â) and TNF-R.6 It has been
demonstrated that small molecular inhibitors of p38
significantly reduce the release of both of these cyto-
kines from human monocytes,6 a highly valuable feature
with the synergistic effects of anti-IL-1â and anti-TNF-R
therapy becoming increasingly clear.7 Several potent
inhibitors of p38 like SB 203580 (Figure 1, Table 2)
compete with ATP at the ATP binding site, which is
located in the cleft between the two domains of p38.8-10
Rigid structure activity relationships (SAR) have been
established for these trisubstituted imidazoles.11,12
pyridin-4-yl moiety corresponding to the aminopyrimi-
dine fragment of the ATP purin ring system is essential
for inhibitory potency in this class of compounds as it
A
* Corresponding author. Telephone: ++49-7071-2972459. Telefax:
++49-7071-295037. E-mail: stefan.laufer@uni-tuebingen.de.
† Eberhard-Karls-University Tu¨bingen.
‡ Merckle GmbH.
10.1021/jm020873z CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/13/2002

4696 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Laufer et al.
F igu r e 1. Dissection of the SK&F 86002 imidazo[2,1-b]thiazoline system. Insertion of a 4-methanesulfinylphenyl moiety gives
SB 203580 after incision of bonds a and c and ML 3163 after incision of bonds c and d.
Ta ble 1. Inhibition of Cytokine Release by Benzylsulfanyl
dazoles (Scheme 1). Thus compounds 2a -c, 2e, and
Imidazolesa
2g-q were obtained from 1a in good to moderate yields
under basic conditions (Scheme 1, pathway a). The
analogues 3a -c, 4a -c, and 5a -c were prepared from
imidazole thiones 1b-d according to the same synthetic
procedures. However, application of this same method-
ology to benzyl chlorides bearing a phenolic functionality
yielded an unseparable mixture of products. The pro-
nounced reactivity of benzyl alcohols toward nucleo-
philic reagents24 was exploited in the preparation of
IC50 ( SEM (µM) PBMC
compounds 6a -d , 6f, and 6h under relatively mild con-
ditions. Acid catalyzed reaction of appropriately sub-
stituted benzyl alcohols with 1a led to compounds 6a -
d , 6f, and 6h (Scheme 1, pathway b). Sulfides 2e, 6d ,
6f, and 6h were converted into the corresponding sul-
foxides 2f, 6e, 6g, and 6i upon treatment with 1 equiv
of 35% H2O2. 1H/13C NMR data for the methylene group
indicate that the oxidation state of the sulfur atom
proximal to the imidazole nucleus is not altered under
these conditions. Benzyl alcohols 10a -c containing a
phenolic as well as a methylsulfanyl substituent were
prepared from the corresponding benzoic acids 9a -c by
LiAlH4 reduction (Scheme 2). The required benzoic acids
were obtained by modification of Stewart’s methodol-
ogy25 for the preparation of methylsulfanyl salicylic
acids, which has been previously applied to the synthe-
sis of 9b,c.26 We extended this strategy to the synthesis
of the regioisomeric 4-hydroxy-3-methylsulfanyl-benzoic
acid 9a starting from 4-methoxybenzoic acid. Initial
attempts to introduce the chlorosulfonyl moiety on the
corresponding phenolic compound failed. Moreover,
chlorosulfonation of 4-methoxybenzoic acid to 7 was
achieved only when carried out in CCl4. Subsequent
triphenylphosphine reduction27 of 7 and alkaline me-
thylation of the intermediate thiol was accompagnied
by saponification of the benzoic acid ester to yield
methylsulfanyl benzoic acid 8. After cleavage of the
protecting group under standard reaction conditions,
phenol 9a was obtained and subsequently converted into
benzyl alcohol 10a . Compounds 12a and 12b, which
bear an alkyl or alkenyl substituent at the 2 position of
the imidazole, were prepared by triethyl phosphite
reduction of the corresponding N-hydroxyimidazoles 11a
and 11b according to literature procedures (Scheme 3).12
compd
X
R
TNF-R
IL-1â
2a
F- 4-SCH3
F- 4-SO2CH3
4.9 ( 0.1
1.1 ( 0.4 (4) 0.38 ( 0.13 (4)
2.5 ( 0.4 (4) 0.33 ( 0.10 (4)
0.67 ( 0.19
2b (ML 3163) F- 4-S(O)CH3
2c
2d b
2e
2f
F- 4-S(O)CH3 >100
98 ( 23
0.55 ( 0.03
1.0 ( 0.4
6.7 ( 4.4
0.90 ( 0.10
2.2 ( 0.4
0.32 ( 0.06
0.32 ( 0.07
2.4 ( 0.6
0.82 ( 0.12
0.64 ( 0.45
72.5 ( 32.5
0.84 ( 0.46
1.7 ( 0.2
0.37 ( 0.14
0.31 (1)
0.44 ( 0.00
F- 3-SCH3
12.0 ( 4.4
F- 3-S(O)CH3 23.5 ( 6.5
2g
2h
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
F- 2-SCH3
18.3 ( 1.3
F- 2-S(O)CH3 15.0 ( 0.5
Cl- 4-SCH3
Cl- 4-S(O)CH3
Cl- 4-SO2CH3
Br- 4-SCH3
Br- 4-S(O)CH3
Br- 4-SO2CH3
H- 4-SCH3
H- 4-S(O)CH3
H- 4-SO2CH3
F- 4-HO
14.0 ( 4.0
3.1 ( 1.2
6.1 ( 4.0
12.9 ( 4.2
7.8 ( 0.3
7.3 ( 0.5
17.1 ( 10.0
4.3 ( 1.8
8.0 ( 1.0
6.8 ( 0.2
5.4 ( 0.6
24.8 ( 5.3
F- 3-HO
F- 2-HO
a
Results are given as the mean of two independent experi-
ments, except where otherwise stated. b3-pyr.
prepared a series of 2b analogues differing in their
respective substituents at the 2 position. Much to our
surprise and in contradiction to our initial concept, SAR
for these compounds in peripheral blood mononuclear
cells (PBMC) revealed the optimum substituent at the
2 position to be a simple methylsulfanyl group. This
result contrasts remarkably with published data which
claim large substituents at the 1 or 2 position of the
imidazole core to be required for cellular activity in this
class of compounds.16,22
Ch em istr y
Nucleophilic substitution of appropriate halogenoal-
kanes with 4-(4-fluorophenyl)-5-pyridin-4-yl-1,3-dihy-
dro-imidazole-2-thione 1a 17,23 represents the key step
in the straightforward synthesis of alkylsulfanyl imi-
Biologica l Resu lts a n d Discu ssion
In a first series of 2b analogues, we confirmed that
SAR concerning the pyridinyl-, 4-fluorophenyl- and

Imidazole Inhibitors of Cytokine Release
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4697
Ta ble 2. Spacera
IC50 ( SEM (µM) PBMC
compd
X
R
TNF-R
IL-1â
SB 203580
2i
2j
2k
2l
2m
12a
12b
bond
-S(O)CH3
-S(O)CH3
-S(O)CH3
-H
0.59 ( 0.09 (21)
7.3 ( 0.3
0.037 ( 0.006 (20)
0.50 ( 0.09
-S-(CH2)2-
-S-(CH2)3-
-S-CH2-
-S-(CH2)2-
-S-(CH2)3-
-CH2-CH2-
3.6 ( 1.4 (4)
3.5 ( 0.5
0.18 ( 0.04 (4)
0.30 ( 0.01
-H
23.5 ( 1.5
16.0 ( 4.0
5.4 ( 0.1
0.64 ( 0.12
-H
0.47 ( 0.17
-H
0.14 ( 0.01
-H
6.5 ( 0.7 (4)
0.13 ( 0.07
a
Results are given as the mean of 2 independent experiments, except where otherwise stated.
Sch em e 1a
a
Reagents: (a) R-hal, Na2CO3, THF/EtOH, reflux; (b) benzyl alcohol derivatives, 10% HCl, acetic acid, rt; (c) 1 aeq 35% H2O2, acetic
acid, 10 °C then rt.
methanesulfinyl-moiety correspond to those which have
been established for SB 203580 and its analogues.11,12
The importance of the hydrogen bond between the
pyridin-4-yl moiety of 2b and Met 109 of p38 for
inhibition of cytokine release from PBMC is underlined
by the poor bioactivity of the pyridin-3-yl analogue 2d
(Table 1). Replacement of the fluoro atom in 2b with
other halogeno atoms in compounds 3b and 4b affected
neither inhibition of p38 (Table 5) nor inhibition of
cytokine release from PBMC significantly (Table 1).
However, a simple phenyl group at the 5 position of the
core imidazole led to reduced activity for compound 5b
in the enzyme but not in the cellular assay. Within each
halogenoaryl series as well as in the aryl series, a
remarkable difference in inhibitory potency of the
sulfides 2a , 3a , 4a and 5a compared to the more polar
sulfoxides/sulfones 2b,c, 3b,c, 4b,c and 5b,c was ob-
served in the PBMC (Table 1) and whole blood assay
(Table 6, compounds 2a -c). Placement of the methane-
sulfinyl functionality of 2b at the 3 or 2 position of the
benzyl moiety provided the less potent regioisomers 2f
and 2h . Moreover, the said difference in bioactivity
between sulfides and sulfoxides disappeared in the case
of the regioisomeric analogues 2e-h . All these results
concerning substituents at the 2, 4, and 5 position of
the imidazole core were in good agreement with data
reported for SB 203580 analogues.11,12 In conclusion we
assumed a similar binding mode to p38 for 2b as for
SB 203580. 2b differs from the more active SB 203580
only in the distance between the imidazole nucleus and
the 4-methanesulfinylphenyl moiety (Table 2). There-
fore, we investigated how the nature and chain length
of the spacer connecting these two parts of the molecule
influence biological activity. Elongation of the spacer by
one (compound 2i) or two (compound 2j) methylene
units resulted in enhanced inhibition of p38 compared
to the parent compound 2b. (Table 5). However, this
improved inhibitory potency did not show in the cellular
assay (Table 2). Likewise, biological activity of com-
pounds lacking the 4-methanesulfinylphenyl moiety

4698 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Laufer et al.
Sch em e 2a
a
Reagents: (a) SO3HCl, CCl4, -5 °C then 50 °C; (b) PPh3, toluene, rt; (c) 10% NaOH, SO2(OCH3)2, rt then reflux; (d) acetic acid/HBr
48%, reflux; (e) LiAlH4, THF, rt then 60 °C.
Sch em e 3a
a
Reagents: (a) RCHO, ammonium acetate, acetic acid; (b) triethyl phosphite, DMF.
Ta ble 3. Substitution Patterns at the Benzylsulfanyl Moietya
Ta ble 4. Replacement of the Benzylsulfanyl Moietya
IC50 ( SEM (µM) PBMC
compd
n
R
X
TNF-R
IL-1â
6d
6e
6f
6g
6h
6i
0
1
0
1
0
1
4-HO
4-HO
2-HO
2-HO
6-HO
6-HO
H-
H-
H-
H-
Cl-
Cl-
1.1 ( 0.3
5.0 ( 0.3
0.28 ( 0.14
2.3 ( 0.48
0.74 ( 0.26
1.0 ( 0.3 (4)
0.64 ( 0.22
4.4 ( 1.35
5.1 ( 2.6
5.8 ( 1.5 (4)
13.3 ( 6.8
28.0 ( 1.0
a
Results are given as the mean of two independent experi-
ments, except where otherwise stated.
a
Results are given as the mean of two independent experi-
(compounds 2k , 2l, 2m ) depended on the length of the
side chain attached to the 2 position. Elongation of the
spacer again led to notably decreased inhibition of
cytokine release (Table 2). A 7-fold better inhibition of
p38 was achieved by replacement of the sulfur in 2k
with a carbon atom in 12a (Table 5). As with compounds
2i and 2j, the increase in potency was also not main-
tained in the cellular assay (Table 2). No difference was
observed whether the spacer contained sp3 or sp2 hybrid
ments, except where otherwise stated.
carbon atoms (compounds 12a and 12b respectively).
Potent inhibition of p38 is not the sole parameter
affecting the activity of test compounds in the cellular
assay. The different trends found for compounds 2b ,
2i-k , and 12a in the enzyme assay and in the PBMC
assay may therefore be ascribed to differences in

Imidazole Inhibitors of Cytokine Release
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4699
Ta ble 5. Inhibition of p38 MAP Kinase by Selected
ring (compound 2o) without subsequent loss of activity
(Table 4). Biological results comparable to those of 2k
were provided only by compound 2p with a nonaromatic
π-electron system serving as a suitable substitute for
the phenyl ring. Following an incremental approach, we
formally divided the benzyl moiety of compound 2k into
its C1 and phenyl part. Subsequently we sought to
determine the contribution of the phenyl ring to inhibi-
tion of cytokine release by preparation of methanesulfa-
nyl imidazole 2q. Surprisingly, 2q exceeded parent
compound 2k in potency, being the only compound in
this series to inhibit release of TNF-R from PBMC in
the nanomolar range and to equal SB 203580 in anti-
IL-1â activity (Table 4). Moreover, the improved inhibi-
tion of cytokine release from PBMC was also reflected
by p38 kinase (Table 5) and whole blood (Table 6)
results.
Compoundsa
IC50 ( SEM
(µM) p38
IC50
(µM) p38
compd
compd
SB 203580
2b (ML 3163)
2e
2f
2i
0.29 ( 0.03 (7)
3b
4b
5b
6a
6d
1.40
3.8
12
5.7
3.8
4.0 ( 1.0 (2)
2.30
2.10
1.50
2j
0.75
6e
3.4
0.69
2k
2q
35
12a
0.63 ( 0.08 (2)
a
Results are of one experiment, except where otherwise stated.
transport, membrane penetration or intracellular dis-
tribution. In general it appears that the presence of the
4-methanesulfinyl moiety (2b vs 2k ), the elongation of
the spacer (2i and 2j vs 2b), and the replacement of the
sulfur with a carbon atom (12a vs 2k ) though favorable
for p38 inhibition have a disadvantageous effect on these
properties. In a third independent study, we put the
concept of an additional polar functionality at the
benzylsulfanyl moiety to the test. Results in the PBMC
assay for phenols 6a -c showed that, preferably at the
3 and 4 position of the benzyl substituent, replacement
of the methanesulfinyl moiety with a hydroxyl group is
tolerated (Table 1). We speculated that combination of
both polar functionalities should result in enhanced
similarity between inhibitor and ATP and may subse-
quently lead to improved bioactivity. While sulfoxide 6e
indeed reduced the release of TNF-R from PBMC more
efficiently than sulfoxide 2f and phenol 6a , a reversed
result was obtained concerning the release of IL-1â
(Table 3). Combination of hydroxyl and sulfide func-
tionalities provided an unambiguous result with com-
pound 6d exceeding both 2e and 6a in potency with
respect to inhibition of both cytokines. The same order
of efficacy for the sulfide and sulfoxide was displayed
by regioisomers 6f,g and 6h ,i, albeit on a lower level
than that by 6e and 6f. However, no such differences
in potency between compounds containing either a
sulfide or sulfoxide moiety were found in the p38 assay.
Within the series of compounds 2e,f and 6d ,e, neither
the oxidation state of the sulfur nor the presence of an
additional phenolic group at the 4 position considerably
altered inhibition of p38 kinase activity (Table 5). We
attributed these differences between p38 and PBMC
results to the possibility that mainly at the 4 position
of the benzyl moiety do suitable substituents contribute
to enhanced binding properties of p38 inhibitors. A polar
functionality is most favorable for this purpose and its
benefit is illustrated by the 10-fold better inhibition of
TNF-R release from whole blood by 4-OH derivative 6e
compared to 2-OH derivative 6g (Table 6). Introduction
of additional substituents at the 3 or 2 position of the
benzyl moiety considerably alters the physicochemical
properties of benzylsulfanyl imidazole compounds. In
this respect, less polar substituents are better tolerated.
The moderate potency of compounds 6h and 6i demon-
strates that a large additional substituent such as the
chloro atom is detrimental for bioactivity. In a final
series of compounds, we elucidated the influence of the
aromatic ring system as part of the substituent attached
to the 2 position on bioactivity. The benzyl moiety of
compound 2k was not replaced neither by an extended
aromatic system (compound 2n ) nor by a nonaromatic
Con clu sion
These findings prove that suitably substituted ben-
zylsulfanyl imidazoles are efficient inhibitors of p38 and
cytokine release, displaying characteristics comparable
to those of model compound SB 203580. In conclusion
we suggest for our initial lead 2b and its analogues a
binding mode to p38 similar to that of SB 203580.
Substituents at the 2 position of the imidazole nucleus
influence the physicochemical as well as the binding
properties in this class of compounds. Enzyme data for
benzylsulfanyl imidazole 2b, for phenylalkyl analogues
2i and 2j, and for the sulfur-free compound 12a indicate
that the -S-CH2- chain between core imidazole and
phenyl moiety of 2b does not constitute the optimum
spacer with respect to inhibition of p38. The inhibitory
superiority of imidazole derivatives containing better
suited spacer molecules (compounds 2i, 2j, 12a ) disap-
pears, however, in the cellular assay, which is probably
due to the reduced ability of these compounds to
penetrate the cellular membrane. Substituents at the
3 and 4 position of the benzylsulfanyl moiety contribute
considerably to inhibition of p38 by compounds 2b ,
2e-f, and 6a compared to the simple benzylsulfanyl
imidazole 2k , which is about 10 times less active. The
benzylsulfanyl group itself therefore seems to be rather
unfavorable for inhibition of p38 and to need substitu-
ents for additional interactions with the enzyme. Polar
substituents at the benzyl moiety enhance inhibition of
cytokine release most effectively if attached to the 4
position. In a series of polyfunctionalized benzylsulfanyl
imidazoles, combination of a polar and a more lipophilic
substituent at the benzyl ring gives better results in the
cellular assay than combination of two polar function-
alities. On the basis of these results, we suggest that
additional substituents at the 2 and 3 position of the
benzyl moiety may point outside the ATP cleft, affecting
the physicochemical properties of test compounds rather
than their interaction with p38, if a polar substituent
at the 4 position is already present (Figure 2). The latter
may indeed be crucial for enzyme inhibition, interacting
with residues on the fringe of the ATP binding site (e.g.,
in the phosphate binding ribbon or the activation loop).
The cellular activity of 6d may then be attributed to its
combination of substituents serving both the purpose
of good membrane penetration and tight binding to p38.
However, the benefits of completely omitting the aro-
matic part of the benzyl moiety (compound 2q) seem to

4700 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Laufer et al.
Ta ble 6. Inhibition of Cytokine Release from Whole Blood by Selected Compoundsa
IC50 ( SEM (µM)
IC50 ( SEM (µM)
compd
TNF-R
IL-1â
compd
TNF-R
IL-1â
SB 203580
2a
2b (ML 3163)
2c
2f
0.94 ( 0.14 (12)
>100
0.35 ( 0.09 (12)
28.5 ( 1.5
2h
2q
6e
6g
47.5 ( 16.5
4.2 ( 0.7
9.0 ( 2.1
103 ( 27
4.0 ( 2.8
1.2 ( 0.6
12.1 (1)
20.3 ( 4.8
2.78 ( 0.13
2.9 ( 1.7
28.8 ( 10.3
33.5 ( 6.4 (4)
13.0 ( 0.0
4.1 ( 3.0 (3)
a
Results are given as the mean of two independent experiments, except where otherwise stated.
NaH2PO4 buffer pH 3.5 (70:30) at a flow rate of 1.00 mL/min
at 25 °C (UV detection at 254 nm). HPLC results are presented
as retention times (min) and relative purity (%). Microanalyses
were carried out on a Perkin-Elmer EA 2400 instrument. The
following compounds were prepared according to literature
procedures: SB 203580,12 imidazole-2-thiones 1a -d ,23 and
benzoic acids 9b,c.26 Alkylhalides employed in General Pro-
cedure A were either commercially available or synthesized
as previously reported.28 1-(4-Fluorophenyl)-2-pyridin-4-yl-
ethane-1,2-dione 1-oxime was prepared from 2-(4-fluorophen-
yl)-1-pyridin-4-yl-ethanone23 using the standard nitrosation
protocol of Gallagher and co-workers.12
P r ep a r a tion of 2-Alk ylsu lfa n yl Im id a zoles 2a -e, g-q,
3a -c, 4a -c, 5a -c, Gen er a l P r oced u r e A. A suspension of
an appropriate imidazole-2-thione (1 equiv), Na2CO3 (1.2 equiv)
and the appropriate arylalkyl- or alkyl chloride (1 equiv) in
ethanol/THF 8”2 was heated to reflux for the given time. After
cooling to room temperature, the mixture was filtered and the
filtrate concentrated in vacuo. The residue was purified by
column chromatography or trituration to give 2-alkylsulfanyl
imidazoles 2a -e, g-q, 3a -c, 4a -c, 5a -c.
F igu r e 2. Weighting of interaction sites in the ATP cleft of
p38 MAP kinase. Though an appropriately substituted benzyl
moiety at the 2 position of the core imidazole may contribute
moderately to biological activity, it is suggested that small
substituents at this position allow for deeper penetration into
the ATP binding cleft and subsequently much tighter binding
of the pyridine and 4-fluorophenyl ring.
4-[5-(4-F lu or op h en yl)-2-(4-m eth ylsu lfa n yl-ben zylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (2a ). This compound was
prepared from imidazole-2-thione 1a (1.0 g, 3.7 mmol) and
1-chloromethyl-4-methylsulfanyl-benzene according to gen-
eral procedure A (4 h). Recrystallization from ethyl acetate
contradict this model and to rule out any significant
contribution to bioactivity through the benzylsulfanyl
moiety. The superior potency of 2q in all three assays
can, however, be reconciled with our initial concept if
the importance of the hydrogen bond between the
pyridine nitrogen and Met 109 of p38 is taken into
account. The absence of any steric hindrance by sub-
stituents at the 2 position may allow compound 2q to
penetrate deeper into the ATP cleft of p38 with the
pyridin-4-yl ring subsequently binding much tighter to
the backbone NH of Met 109 (Figure 2). A strength-
ened binding at this predominant site of interaction may
as a result overcompensate the loss of the comparatively
weak interaction between any known substituent at the
2 position and the phosphate binding region or the
activation loop of p38. Molecular modeling studies to
further substantiate this preliminary concept are cur-
rently carried out.
1
yielded 0.11 g (7%) of 2a : mp 243 °C; H NMR (DMSO-d6) δ
2.44 (s, 3H, CH3), 4.38 (s, 2H, CH2), 7.18-7.55 (m, 10H, 4-Pyr,
4-F-Ph and 4-MeS-Ph), 8.41-8.52 (m, 2H, 4-Pyr), 12.80 (bs,
1H, exchangeable, NH); IR (ATR) 1226 cm-1 (C-F); HPLC 3.97
min, 97.4%. Anal. (C22H18FN3S2) C, H, N.
4-[5-(4-F lu or op h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (2b). This compound was
prepared from imidazole-2-thione 1a (1.0 g, 3.7 mmol) and
1-chloromethyl-4-methanesulfinyl-benzene according to gen-
eral procedure A (3 h). Trituration with ethanol yielded 0.62
1
g (41%) of 2b: mp 232 °C; H NMR (DMSO-d6) δ 2.71 (s, 3H,
CH3), 4.48 (s, 2H, CH2), 7.22-7.53 (m, 6H, 4-Pyr and 4-F-Ph),
7.57-7.65 (m, 4H, 4-MeS(O)-Ph), 8.45-8.48 (m, 2H, 4-Pyr),
12.75 (bs, 1H, exchangeable, NH); IR (ATR) 1226 (C-F), 1039
cm-1 (SdO); HPLC 1.92 min, 97.9%. Anal. (C22H18FN3OS2) C,
H, N.
4-[5-(4-F lu or op h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (2c). This compound was
prepared from imidazole-2-thione 1a (1.0 g, 3.7 mmol) and
1-chloromethyl-4-methanesulfonyl-benzene according to gen-
eral procedure A (6.5 h). Trituration with hot ethyl acetate
yielded 0.59 g (36%) of 2c: mp 249 °C; 1H NMR (DMSO-d6) δ
3.19 (s, 3H, CH3), 4.52 (s, 2H, CH2), 7.25-7.51 (m, 6H, 4-Pyr
and 4-F-Ph), 7.67 (d, 2H, 8.2 Hz, 4-MeSO2-Ph), 7.89 (d, 2H,
8.3 Hz, 4-MeSO2-Ph), 8.46-8.49 (m, 2H, 4-Pyr), 12.85 (bs, 1H,
exchangeable, NH); IR (ATR) 1304 (SO2), 1220 (C-F), 1145
cm-1 (SO2); HPLC 2.10 min, 99.7%. Anal. (C22H18FN3O2S2) C,
H, N.
Exp er im en ta l Section
Gen er a l. All reagents and solvents were of commercial
quality and used without further purification. All reactions
were carried out under an inert atmosphere of argon. Melting
points were determined on a Buechi Melting Point B-545
1
apparatus and are thermodynamically corrected. H and 13C
NMR spectra were obtained on a Bruker Spectrospin AC 200
at 200 MHz. Chemical shifts are reported in parts per million
relative to TMS as internal standard. Infrared spectra were
recorded using KBr pellets on a Nicolet Impact 410 or by ATR
technique on a Perkin-Elmer Spectrum One spectrometer. TLC
analyses were performed on fluorescent silica gel 60 plates
(Macherey-Nagel Art.-Nr. 805021). Spots were visualized
under 254-nm UV illumination. HPLC analyses were car-
ried out on Merck Hitachi (Darmstadt) equipment, using a
LiChrospher 100 RP-18 column (5µm) and eluting with MeCN/
3-[5-(4-F lu or op h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (2d ). This compound was
prepared from 4-(4-fluoro-phenyl)-5-pyridin-3-yl-1,3-dihydro-
imidazole-2-thione (0.42 g, 1.5 mmol) and 1-chloromethyl-4-
methanesulfinyl-benzene according to general procedure A (8
h). Purification by column chromatography (SiO2, DCM/
1
ethanol 9:1) yielded 0.05 g (8%) of 2d : mp 127 °C; H NMR
(DMSO-d6) δ 3.19 (s,3H, CH3), 4.46 (s, 2H, CH2), 7.16-7.46
(m, 5H, 3-Pyr and 4-F-Ph), 7.56-7.66 (m, 4H, 4-MeS(O)-Ph),

Imidazole Inhibitors of Cytokine Release
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4701
7.72-7.81 (m, 1H, 3-Pyr), 8.41-8.62 (m, 2H, 3-Pyr), 12.77 (bs,
1H, exchangeable, NH); IR (ATR) 1222 (C-F), 1027 cm-1
(SdO). Anal. (C22H18FN3OS2) C, H, N.
pound was prepared from imidazole-2-thione 1a (0.25 g, 0.9
mmol) and 1-(3-chloropropyl)-4-methanesulfinyl-benzene ac-
cording to general procedure A (40 h) upon treatment with a
catalytic amount of NaI. Purification by column chroma-
tography (SiO2, DCM/ethanol 9:1) yielded 0.11 g (27%) of 2j:
mp 142 °C; 1H NMR (DMSO-d6) δ 1.95-2.09 (m, 2H, CH2),
2.71 (s, 3H, CH3), 2.82 (t, 2H, 7.4 Hz, CH2), 3.15 (t, 2H, 7.0
Hz, CH2), 7.25-7.62 (m, 10H, 4-Pyr, 4-F-Ph and 4-MeS(O)-
Ph), 8.46-8.49 (m, 2H, 4-Pyr), 12.86 (bs, 1H, exchangeable,
4-[5-(4-F lu or op h en yl)-2-(3-m eth ylsu lfa n yl-ben zylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (2e). This compound was
prepared from imidazole-2-thione 1a (1.1 g, 4.1 mmol) and
1-chloromethyl-3-methylsulfanyl-benzene according to general
procedure A (11 h). Recrystallization from ethanol yielded 1.21
g (73%): mp 218 °C; 1H NMR (DMSO-d6) δ 2.40 (s, 3H, CH3),
4.46 (s, 2H, CH2), 7.16-7.43 (m, 6H, 4-F-Ph and 3-MeS-Ph),
7.56-7.63 (m, 2H, 4-F-Ph), 7.90-7.93 (m, 2H, 4-Pyr), 8.66-
8.69 (m, 2H, 4-Pyr), NH not detected; 13C NMR (DMSO-d6) δ
14.5, 36.0, 116.0, 116.5, 121.4, 124.7, 125.4, 126.2, 129.0, 130.6,
131.1, 131.3, 136.9, 138.2, 138.7, 141.3, 143.3, 148.9, 160.1,
165.1; IR (KBr) 1225 cm-1 (C-F); HPLC 4.22 min, 98.3%. Anal.
(C22H18FN3S2) C, H, N.
4-[5-(4-F lu or op h en yl)-2-(3-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (2f). A suspension of 2e
(0.50 g, 1.2 mmol) in glacial acetic acid (7 mL) was treated
with 35% aqueous H2O2 (0.13 mL, 1.3 mmol). The mixture was
stirred at room temperature for 20.5 h, diluted with H2O (5
mL), adjusted to pH 9 with 25% aqueous ammonia, and
extracted with ethyl acetate (3×). The combined organic
extracts were washed with brine (3×) and dried over Na2SO4,
and the solvent was removed to yield an oily residue, which
was crystallized from diethyl ether/ethyl acetate (1:1). The
crude product was purified by column chromatography (RP-
18, methanol) to give 0.16 mg (31%) of 2f: mp 171 °C; 1H NMR
(CD3OD) δ 2.67 (s, 3H, CH3), 4.37 (s, 2H, CH2), 7.13-7.21 (m,
2H, 4-F-Ph), 7.37-7.58 (m, 8H, 4-Pyr, 4-F-Ph and 3-MeS(O)-
Ph), 8.40-8.43 (m, 2H, 4-Pyr); 13C NMR (CD3OD) δ 39.6, 43.7,
116.9, 117.3, 123.0, 123.9, 125.1, 130.9, 131.9, 132.0, 133.2,
141.4, 146.6, 149.8, 150.2, 162.0, 167.0; IR (KBr) 1228 (C-F),
1019 cm-1 (SdO); HPLC 2.28 min, 95.8%. Anal. (C22H18FN3-
OS2) C, H, N.
4-[5-(4-F lu or op h en yl)-2-(2-m eth ylsu lfa n yl-ben zylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (2g). This compound was
prepared from imidazole-2-thione 1a (0.28 g, 1.0 mmol) and
1-chloromethyl-2-methylsulfanyl-benzene according to general
procedure A (5.5 h). Purification by column chromatography
(SiO2, ethyl acetate) yielded 0.24 g (59%) of 2g: mp 223 °C;
1H NMR (CD3OD) δ 2.51 (s, 3H, CH3), 4.44 (s, 2H, CH2), 7.13-
7.48 (m, 10H, 4-Pyr, 4-F-Ph and 2-MeS-Ph), 8.43-8.46 (m, 2H,
4-Pyr); IR (ATR) 1228 cm-1 (C-F). Anal. (C22H18FN3S2) C, H,
N.
4-[5-(4-F lu or op h en yl)-2-(2-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (2h ). This compound was
prepared from imidazole-2-thione 1a (0.28 g, 1.0 mmol) and
1-chloromethyl-2-methanesulfinyl-benzene according to gen-
eral procedure A (4 h). Recrystallization from methanol/
ethyl acetate yielded 0.23 g (54%) of 2h : mp 205 °C; 1H NMR
(CD3OD) δ 2.87 (s, 3H, CH3), 4.50 (d, 1H, 13.6 Hz, CH2), 4.62
(d, 1H, 13.6 Hz, CH2), 7.24-7.33 (m, 2H, 4-F-Ph), 7.47-7.62
(m, 5H, 4-F-Ph, C4-/C5-/C6-H 2-MeS(O)-Ph), 7.95 (d, 1H, 7.2
Hz, C3-H 2-MeS(O)-Ph), 7.99-8.03 (m, 2H, 4-Pyr), 8.55-8.58
(m, 2H, 4-Pyr); 13C NMR (CD3OD) δ 35.1, 43.5, 117.5, 117.9,
123.5, 125.0, 126.9, 130.7, 132.0, 132.4, 132.5, 132.8, 133.4,
136.7, 138.9, 142.3, 143.8, 145.3, 152.3, 162.7, 167.6; IR (KBr)
1213 (C-F), 1033 cm-1 (SdO); HPLC 2.11 min, 98.7%. Anal.
(C22H18FN3OS2) C, H, N.
NH); IR (ATR) 1222 (C-F), 1043 cm-1 (SdO). Anal. (C24H22
-
FN3OS2) C, H, N.
4-[2-Ben zylsu lfa n yl-5-(4-flu or op h en yl)-1H-im id a zol-4-
yl]-p yr id in e (2k ). This compound was prepared from imida-
zole-2-thione 1a (0.28 g, 1.0 mmol) and 1-bromomethyl-
benzene according to general procedure A (6 h). Trituration
with methanol yielded 0.17 g (47%) of 2k : mp 223 °C; 1H NMR
(DMSO-d6) δ 4.41 (s, 2H, CH2), 7.23-7.51 (m, 11H, 4-Pyr, 4-F-
Ph and Bz), 8.44-8.47 (m, 2H, 4-Pyr), 12.82 (bs, 1H, exchange-
able, NH); IR (ATR) 1233 cm-1 (C-F). Anal. (C21H16FN3S) C,
H, N.
4-[5-(4-F lu or op h en yl)-2-p h en eth ylsu lfa n yl-1H-im id a -
zol-4-yl]-p yr id in e (2l). This compound was prepared from
imidazole-2-thione 1a (0.5 g, 1.9 mmol) and (2-chloroethyl)-
benzene according to general procedure A (70 h) upon treat-
ment with a catalytic amount of NaI. Trituration with ethanol
1
yielded 0.30 g (42%) of 2l: mp 257 °C; H NMR (DMSO-d6) δ
2.99 (t, 2H, 7.4 Hz, CH2), 3.40 (t, 2H, 7.5 Hz, CH2), 7.17-7.53
(m, 11H, 4-Pyr, 4-F-Ph and Bz), 8.44-8.46 (m, 2H, 4-Pyr), NH
not detected; IR (ATR) 1223 cm-1 (C-F). Anal. (C22H18FN3S)
C, H, N.
4-[5-(4-F lu or op h en yl)-2-(3-p h en yl-p r op ylsu lfa n yl)-1H-
im id a zol-4-yl]-p yr id in e (2m ). This compound was prepared
from imidazole-2-thione 1a (0.5 g, 1.9 mmol) and (3-chloro-
propyl)-benzene according to general procedure A (70 h) upon
treatment with a catalytic amount of NaI. Trituration with
ethanol yielded 0.32 g (43%) of 2m : mp 183 °C; 1H NMR
(DMSO-d6) δ 1.90-2.04 (m, 2H, CH2), 2.72 (t, 2H, 7.4 Hz, CH2),
3.12 (t, 2H, 7.0 Hz, CH2), 7.18-7.51 (m, 11H, 4-Pyr, 4-F-Ph
and Bz), 8.37-8.44 (m, 2H, 4-Pyr), 12.82 (bs, 1H, exchangeable,
NH); IR (ATR) 1226 cm-1 (C-F). Anal. (C23H20FN3S) C, H, N.
4-[5-(4-F lu or op h en yl)-2-(n a p h th a len -1-ylm eth ylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (2n ). This compound was
prepared from imidazole-2-thione 1a (0.28 g, 1.0 mmol) and
1-chloromethyl-naphthalene according to general procedure A
(6.5 h). Purification by column chromatography (SiO2, ethyl
acetate) yielded 0.18 g (44%) of 2n : mp 364 °C; 1H NMR
(DMSO-d6) δ 4.90 (s, 2H, CH2), 7.25-7.62 (m, 10H, 4-Pyr, 4-F-
Ph and Naphthyl), 7.80-7.98 (m, 2H, Naphthyl), 8.20-8.23
(m, 1H, Naphthyl), 8.48-8.52 (m, 2H, 4-Pyr), 12.86 (bs, 1H,
exchangeable, NH); IR (ATR) 1225 cm-1 (C-F). Anal. (C25H18
FN3S) C, H, N.
-
4-[2-Cycloh exylm eth ylsu lfa n yl-5-(4-flu or op h en yl)-1H-
im id a zol-4-yl]-p yr id in e (2o). This compound was prepared
from imidazole-2-thione 1a (0.25 g, 0.9 mmol) and chloro-
methyl-cyclohexane according to general procedure A (47 h)
upon treatment with a catalytic amount of NaI. Trituration
with ethanol yielded 0.25 g (76%) of 2o: mp 235 °C; 1H NMR
(DMSO-d6) δ 0.95-1.23 (m, 5H, c-Hex), 1.51-1.85 (m, 6H,
c-Hex), 3.06 (d, 2H, 6.7 Hz, CH2), 7.22-7.51 (m, 6H, 4-Pyr and
4-F-Ph), 8.43-8.45 (m, 2H, 4-Pyr), 12.76 (bs, 1H, exchangeable,
NH); IR (ATR) 2922, 2852 (c-Hex), 1222 cm-1 (C-F). Anal.
(C21H22FN3S) C, H, N.
4-{5-(4-Flu or op h en yl)-2-[2-(4-m eth a n esu lfin yl-p h en yl)-
eth ylsu lfa n yl]-1H-im id a zol-4-yl}-p yr id in e (2i). This com-
pound was prepared from imidazole-2-thione 1a (0.25 g, 0.9
mmol) and 1-(2-chloroethyl)-4-methanesulfinyl-benzene ac-
cording to general procedure A (50 h) upon treatment with a
catalytic amount of NaI. Purification by column chromatog-
raphy (SiO2, DCM/ethanol 9:1) yielded 0.06 g (15%) of 2i: mp
177 °C; 1H NMR (DMSO-d6) δ 2.71 (s, 3H, CH3), 3.06-3.13
(m, 2H, CH2), 3.42-3.49 (m, 2H, CH2), 7.25-7.65 (m, 10H,
4-Pyr, 4-F-Ph and 4-MeS(O)-Ph), 8.40-8.58 (m, 2H, 4-Pyr),
12.80 (bs, 1H, exchangeable, NH); IR (ATR) 1221 (C-F), 1032
cm-1 (SdO). Anal. (C23H20FN3OS2) C, H, N.
[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H -im id a zol-2-yl-
su lfa n yl]-a ceton itr ile (2p ). This compound was prepared
from imidazole-2-thione 1a (1.1 g, 4.0 mmol) and chloroaceto-
nitrile according to general procedure A (18 h). Purification
by column chromatography (SiO2, ethyl acetate) yielded 0.32
1
g (26%) of 2p : mp 219 °C; H NMR (DMSO-d6) δ 4.32 (s, 2H,
CH2), 7.34-7.57 (m, 6H, 4-Pyr and 4-F-Ph), 8.50-8.52 (m, 2H,
4-Pyr), 13.20 (bs, 1H, exchangeable, NH); IR (ATR) 2243 (CN),
1226 cm-1 (C-F). Anal. (C16H11FN4S) C, H, N.
4-[5-(4-F lu or op h en yl)-2-m eth ylsu lfa n yl-1H-im id a zol-4-
yl]-p yr id in e (2q). This compound was prepared from imida-
zole-2-thione 1a (0.41 g, 0.5 mmol) and iodomethane according
4-{5-(4-Flu or op h en yl)-2-[3-(4-m eth a n esu lfin yl-p h en yl)-
p r op ylsu lfa n yl]-1H-im id a zol-4-yl}-p yr id in e (2j). This com-

4702 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Laufer et al.
to general procedure A (8 h). Trituration with ethanol yielded
0.11 g (26%) of 2q: mp 263 °C; 1H NMR (DMSO-d6) δ 2.61 (s,
3H, CH3), 7.22-7.51 (m, 6H, 4-Pyr and 4-F-Ph), 8.42-8.45 (m,
2H, 4-Pyr), NH not detected; IR (ATR) 1226 cm-1 (C-F). Anal.
(C15H12FN3S) C, H, N.
(SiO2, DCM/ethanol 9:1) yielded 0.26 g (45%) of 5a : mp 213
°C; 1H NMR (DMSO-d6) δ 2.44 (s, 3H, CH3), 4.38 (s, 2H, CH2),
7.18-7.58 (m, 11H, 4-Pyr, Ph and 4-MeS-Ph), 8.44-8.47 (m,
2H, 4-Pyr), 12.82 (bs, 1H, exchangeable, NH). Anal. (C22H19N3S2)
C, H, N.
4-[5-(4-Ch lor op h en yl)-2-(4-m eth ylsu lfa n yl-ben zylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (3a ). This compound was
prepared from imidazole-2-thione 1b (0.26 g, 0.9 mmol) and
1-chloromethyl-4-methylsulfanyl-benzene according to general
procedure A (6.5 h). Purification by column chromatography
(SiO2, DCM/ethanol 9:1) yielded 0.25 g (66%) of 3a : mp 236
°C; 1H NMR (DMSO-d6) δ 2.44 (s, 3H, CH3), 4.38 (s, 2H, CH2),
7.18-7.56 (m, 10H, 4-Pyr, 4-Cl-Ph and 4-MeS-Ph), 8.45-8.55
(m, 2H, 4-Pyr), 12.86 (bs, 1H, exchangeable, NH); IR (ATR)
684 cm-1 (C-Cl). Anal. (C22H18ClN3S2) C, H, N.
4-[2-(4-Meth a n esu lfin yl-ben zylsu lfa n yl)-5-p h en yl-1H-
im id a zol-4-yl]-p yr id in e (5b). This compound was prepared
from imidazole-2-thione 1d (0.38 g, 1.5 mmol) and 1-chloro-
methyl-4-methanesulfinyl-benzene according to general pro-
cedure A (5.5 h). Purification by column chromatography (SiO2,
1
DCM/ethanol 9:1) yielded 0.15 g (25%) of 5b: mp 189 °C; H
NMR (DMSO-d6) δ 2.71 (s, 3H, CH3), 4.48 (s, 2H, CH2), 7.32-
7.52 (m, 7H, 4-Pyr and Ph), 7.57-7.67 (m, 4H, 4-MeS(O)-Ph),
8.45-8.54 (m, 2H, 4-Pyr), 12.84 (bs, 1H, exchangeable, NH);
IR (ATR) 1051 cm-1 (SdO). Anal. (C22H19N3OS2) C, H, N.
4-[5-(4-Ch lor op h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (3b). This compound was
prepared from imidazole-2-thione 1b (0.26 g, 0.9 mmol) and
1-chloromethyl-4-methanesulfinyl-benzene according to gen-
eral procedure A (6.5 h). Purification by column chromatog-
raphy (SiO2, DCM/ethanol 9:1) yielded 0.23 g (62%) of 3b: mp
4-[2-(4-Meth a n esu lfon yl-ben zylsu lfa n yl)-5-p h en yl-1H-
im id a zol-4-yl]-p yr id in e (5c). This compound was prepared
from imidazole-2-thione 1d (0.38 g, 1.5 mmol) and 1-chloro-
methyl-4-methanesulfonyl-benzene according to general pro-
cedure A (4.25 h). Purification by column chromatography
(SiO2, DCM/ethanol 9:1) yielded 0.36 g (57%) of 5c: mp 247
°C; 1H NMR (DMSO-d6) δ 3.21 (s, 3H, CH3), 4.54 (s, 2H, CH2),
7.31-7.58 (m, 7H, 4-Pyr and Ph), 7.70 (d, 2H, 8.3 Hz, 4-MeSO2-
Ph), 7.91 (d, 2H, 8.3 Hz, 4-MeSO2-Ph), 8.45-8.59 (m, 2H,
4-Pyr), 12.87 (bs, 1H, exchangeable, NH); IR (ATR) 1298, 1145
cm-1 (SO2). Anal. (C22H19N3O2S2) C, H, N.
1
224 °C; H NMR (DMSO-d6) δ 2.70 (s, 3H, CH3), 4.47 (s, 2H,
CH2), 7.31-7.65 (m, 10H, 4-Pyr, 4-Cl-Ph, 4-MeS(O)-Ph), 8.44-
8.54 (m, 2H, 4-Pyr), 12.87 (bs, 1H, exchangeable, NH); IR
(ATR) 1033 (SdO), 677 cm-1 (C-Cl). Anal. (C22H18ClN3OS2)
C, H, N.
4-[5-(4-Ch lor op h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (3c). This compound was
prepared from imidazole-2-thione 1b (0.26 g, 0.9 mmol) and
1-chloromethyl-4-methanesulfonyl-benzene according to gen-
eral procedure A (6.5 h). Purification by column chromatog-
raphy (SiO2, DCM/ethanol 9:1) yielded 0.22 g (54%) of 3c: mp
P r epar ation of 2-Ben zylsu lfan yl Im idazoles 6a-i, Gen -
er a l P r oced u r e B. 1a (1 equiv) was dissolved in glacial acetic
acid (5 mL) by addition of 10% hydrochloric acid (10-15 drops).
To the bright yellow solution was added the appropriate benzyl
alcohol (1 equiv). After complete consumption of the starting
materials (time/temperature), 35% aqueous H2O2 (1 equiv) was
added for the preparation of sulfoxides 6e, 6g, and 6i, and the
mixture was stirred for another 4 h at room temperature. The
reaction was diluted with H2O (5 mL) and adjusted to pH 8
with 25% aqueous ammonia. A precipitate formed which was
collected by filtration and washed with H2O. The crude product
was purified by column chromatography or trituration to give
imidazol-2-yl-sulfanylmethyl-phenols 6a -i.
4-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-p h en ol (6a ). This compound was prepared
from imidazole-2-thione 1a (0.20 g, 0.7 mmol) and 4-hy-
droxymethyl-phenol according to general procedure B (14 h,
rt). Purification by column chromatography (SiO2 60, DCM/
ethanol 9:1) yielded 0.08 g (31%) of 6a : mp 250 °C (decomp.);
1H NMR (DMSO-d6) δ 4.32 (s, 2H, CH2), 6.69 (d, 2H, 7.5 Hz,
4-HO-Ph), 7.19 (d, 2H, 7.9 Hz, 4-HO-Ph), 7.27-7.51 (m, 6H,
4-Pyr and 4-F-Ph), 8.43-8.53 (m, 2H, 4-Pyr), 9.41 (s, 1H,
exchangeable, OH), 12.79 (bs, 1H, exchangeable, NH); IR
(ATR) 1271 (OH bending), 1232 (C-F), 1004 cm-1 (C-O). Anal.
(C21H16FN3OS) C, H, N.
1
232 °C; H NMR (DMSO-d6) δ 3.19 (s, 3H, CH3), 4.52 (s, 2H,
CH2), 7.32-7.58 (m, 6H, 4-Pyr and 4-Cl-Ph), 7.67 (d, 2H, 8.2
Hz, 4-MeSO2-Ph), 7.88 (d, 2H, 8.3 Hz, 4-MeSO2-Ph), 8.45-8.55
(m, 2H, 4-Pyr), 12.89 (bs, 1H, exchangeable, NH); IR (ATR)
1300, 1141 (SO2), 681 cm-1 (C-Cl). Anal. (C22H18ClN3O2S2) C,
H, N.
4-[5-(4-Br om op h en yl)-2-(4-m eth ylsu lfa n yl-ben zylsu lfa -
n yl)-1H-im id a zol-4-yl]-p yr id in e (4a ). This compound was
prepared from imidazole-2-thione 1c (0.25 g, 0.75 mmol) and
1-chloromethyl-4-methanesulfanyl-benzene according to gen-
eral procedure A (5 h). Purification by column chromatography
(SiO2, DCM/ethanol 9:1) yielded 0.14 g (40%) of 4a : 1H NMR
(DMSO-d6) δ 2.43 (s, 3H, CH3), 4.36 (s, 2H, CH2), 7.16-7.87
(m, 10H, 4-Pyr, 4-Br-Ph and 4-MeS-Ph), 8.45-8.55 (m, 2H,
4-Pyr), 12.90 (bs, 1H, exchangeable, NH). Anal. (C22H18BrN3S2)
C, H, N.
4-[5-(4-Br om op h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (4b). This compound was
prepared from imidazole-2-thione 1c (0.25 g, 0.75 mmol) and
1-chloromethyl-4-methanesulfinyl-benzene according to gen-
eral procedure A (10 h). Purification by column chromatogra-
phy (SiO2, DCM/ethanol 9:1) yielded 0.13 g (36%) of 4b: mp
3-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-p h en ol (6b). This compound was prepared
from imidazole-2-thione 1a (0.20 g, 0.7 mmol) and 3-hy-
droxymethyl-phenol according to general procedure B (9 h,
reflux). Purification by column chromatography (SiO2 60,
1
222 °C; H NMR (DMSO-d6) δ 2.71 (s, 3H, CH3), 4.48 (s, 2H,
CH2), 7.40-7.62 (m, 20H, 4-Pyr, 4-Br-Ph and 4-MeS(O)-Ph),
8.49-8.57 (m, 2H, 4-Pyr), 12.90 (bs, 1H, exchangeable, NH);
IR (ATR) 1035 cm-1 (SdO). Anal. (C22H18BrN3OS2) C, H, N.
4-[5-(4-Br om op h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu l-
fa n yl)-1H-im id a zol-4-yl]-p yr id in e (4c). This compound was
prepared from imidazole-2-thione 1c (0.25 g, 0.75 mmol) and
1-chloromethyl-4-methanesulfonyl-benzene according to gen-
eral procedure A (5 h). Purification by column chromatography
(SiO2, DCM/ethanol 9:1) yielded 0.12 g (32%) of 4c: mp 226
°C; 1H NMR (DMSO-d6) δ 3.18 (s, 3H, CH3), 4.50 (s, 2H, CH2),
7.33-7.89 (m, 10H, 4-Pyr, 4-Br-Ph and 4-MeSO2-Ph), 8.45-
8.54 (m, 2H, 4-Pyr), 12.91 (bs, 1H, exchangeable, NH); IR
(ATR) 1303, 1145 cm-1 (SO2). Anal. (C22H18BrN3O2S2) C, H,
N.
1
DCM/ethanol 9:1) yielded 0.14 g (54%) of 6b: mp 230 °C; H
NMR (DMSO-d6) δ 4.34 (s, 2H, CH2), 6.65 (dd, 1H, 1.4/8.0 Hz,
3-HO-Ph C4-H), 6.79-6.82 (m, 2H, 3-HO-Ph C2-/C6-H),
7.07-7.15 (m, 1H, 3-HO-Ph C5-H), 7.27-7.53 (m, 6H, 4-Pyr
and 4-F-Ph), 9.45 (s, 1H, exchangeable, OH), 12.83 (bs, 1H,
exchangeable, NH); IR (ATR) 1287 (OH bending), 1241
(C-F), 1007 cm-1 (C-O). Anal. (C21H16FN3OS) C, H, N.
2-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-p h en ol (6c). This compound was prepared
from imidazole-2-thione 1a (0.20 g, 0.7 mmol) and 2-hy-
droxymethyl-phenol according to general procedure B (23 h,
rt). Trituration with ethanol yielded 0.09 g (35%) of 6c: mp
200 °C (decomp.); 1H NMR (DMSO-d6) δ 4.37 (s, 2H, CH2),
6.70-6.85 (m, 2H, 2-HO-Ph), 7.05-7.14 (m, 1H, 2-HO-Ph),
7.23-7.53 (m, 7H, 4-Pyr, 4-F-Ph and 2-HO-Ph), 8.46-8.49 (m,
2H, 4-Pyr), 9.95 (bs, 1H, exchangeable, OH), 12.81 (bs, 1H,
exchangeable, NH); IR (ATR) 1266 (OH bending), 1222
(C-F), 1005 (C-O). Anal. (C21H16FN3OS) C, H, N.
4-[2-(4-Met h ylsu lfa n yl-b en zylsu lfa n yl)-5-p h en yl-1H -
im id a zol-4-yl]-p yr id in e (5a ). This compound was prepared
from imidazole-2-thione 1d (0.38 g, 1.5 mmol) and 1-chloro-
methyl-4-methanesulfanyl-benzene according to general pro-
cedure A (5.75 h). Purification by column chromatography

Imidazole Inhibitors of Cytokine Release
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4703
4-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-2-m eth ylsu lfa n yl-p h en ol (6d ). This com-
pound was prepared from 1a (0.20 g, 0.7 mmol) and 10a
according to general procedure B (2 h, rt). Trituration with
methanol yielded 0.14 g (47%) of 6d : mp 230 °C (decomp.);
1H NMR (CD3OD) δ 2.21 (s, 3H, CH3), 4.17 (s, 2H, CH2), 6.69
(d, 1H, 8.0 Hz, 4-HO-Ph C3-H), 6.90-7.01 (m, 2H, 4-HO-Ph
C2-/C6-H), 7.12-7.21 (m, 2H, 4-F-Ph), 7.32-7.53 (m, 4H,
4-Pyr and 4-F-Ph), 8.39-8.43 (m, 2H, 4-Pyr); IR (KBr) 1227
(SdO), 1005 cm-1 (C-O); HPLC 2.59 min, 91.9%. Anal.
(C22H17ClFN3O2S2) C, H, N.
3-Ch lor osu lfon yl-4-m eth oxy-ben zoic a cid eth yl ester
(7). Chlorosulfonic acid (17.5 mL, 263 mmol) was added
dropwise to a solution of 4-methoxy-benzoic acid ethyl ester
(15.7 g, 87.2 mmol) in CCl4 while carefully maintaining the
temperature below -5 °C. After addition of chlorosulfonic acid
was complete the solution was stirred at room temperature
for 2 h and finally heated to 50 °C until all of the starting
material was consumed. The reaction was cooled to room
temperature and poured unto ice/CCl4 (100 mL). After separa-
tion of the organic layer the aqueous phase was extracted with
DCM. The combined organic extracts were washed with brine
(2x), dried over Na2SO4 and the solvent was evaporated. The
brown oily residue was crystallized from diethyl ether to yield
7.6 g (31%) of 7 as a white powder: 1H NMR (CDCl3) δ 1.41
(t, 3H, 7.1 Hz, CH3), 4.14 (s, 3H, CH3), 4.42 (q, 2H, 7.1 Hz,
CH2), 7.18 (d, 1H, 8.8 Hz, C5-H), 8.37 (dd, 1H, 2.1/8.8 Hz, C6-
H), 8.63 (d, 1H, 2.1 Hz, C2-H).
4-Meth oxy-3-m eth ylsu lfa n yl-ben zoic a cid (8). To a so-
lution of 7 (5.1 g, 18.3 mmol) in toluene (50 mL) was added
triphenylphosphine (20.5 g, 78.2 mmol) in small portions. The
reaction was stirred at room temperature for 4.5 h. The
precipitate was filtered off and the yelllow filtrate was
extracted with 10% aqueous NaOH (4x). To the combined
alkaline extracts was added sulfuric acid dimethyl ester (2 mL)
and the reaction was stirred at room temperature for 2 h. A
precipitate formed which was dissolved upon heating to
reflux. The aqueous solution was cooled to room tempera-
ture and acidified with 20% hydrochloric acid. The precipi-
tate was collected by filtration to yield 2.83 g of 8 (74%) as a
white solid: 1H NMR (CD3OD) δ 2.43 (s, 3H, S-CH3), 3.93 (s,
3H, O-CH3), 6.98 (d, 1H, 8.4 Hz, C5-H), 7.79-7.86 (m, 2H,
C2-/C6-H).
4-Hyd r oxy-3-m eth ylsu lfa n yl-ben zoic a cid (9a ). A sus-
pension of 8 (0.5 g, 2.5 mmol) in glacial acetic acid/hydrobromic
acid 48% 1:1 (7 mL) was heated to reflux for 6 h. The reaction
was cooled to room temperature, diluted with H2O (20 mL),
adjusted to pH 2 with 10% aqueous Na2CO3 and extracted with
diethyl ether (4x). The combined organic extracts were dried
over Na2SO4 and the solvent was evaporated. The residue was
washed with H2O to give 0.24 g (52%) of 9: 1H NMR (CDCl3)
δ 2.38 (s, 3H, CH3), 7.05 (d, 1H, 8.5 Hz, C5-H), 8.02 (dd, 1H,
2.2/8.5 Hz, C6-H), 8.29 (d, 1H, 2.2 Hz, C2-H), OH not detected.
4-Hyd r oxym eth yl-2-m eth ylsu lfa n yl-p h en ol (10a ). Un-
der cooling (5-10 °C) a solution of 9a (1.37 g, 7.4 mmol) in
THF (15 mL) was slowly added to LiAlH4 (0.6 g, 15 mmol) in
THF (25 mL). The reaction was stirred at room temperature
for 0.5 h until the evolution of H2 had ceased and stirred at
60 °C for another 17 h. The reaction was quenched by addition
of ice, the precipitate dissolved with 10% aqueous H2SO4 and
the solution extracted with diethyl ether (3x). The combined
organic extracts were extracted with 10% aqueous NaOH (2x)
and the combined alkaline extracts neutralized with 10%
hydrochloric acid. The precipitate was collected by filtration
and the aqueous filtrate extracted with diethyl ether (3x). The
combined organic extracts were washed with brine (2x), dried
over Na2SO4 and the solvent was evaporated to yield 10 as a
white solid, combined yield 0.67 g (57%): 1H NMR (CDCl3) δ
2.34 (s, 3H, CH3), 4.60 (s, 2H, CH2), 6.97 (d, 1H, 8.3 Hz, C6-
H), 7.24 (dd, 2.0/8.4 Hz, C5-H), 7.50 (d, 1H, 2.0 Hz, C3-H),
OH not detected.
(C-F), 1019 cm-1 (C-O); HPLC 2.41 min, 88.5%. Anal. (C22H18
-
FN3OS2) C, H, N.
4-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-2-m eth a n esu lfin yl-p h en ol (6e). This com-
pound was prepared from 1a (0.20 g, 0.7 mmol), 10a and 35%
aqueous H2O2 according to general procedure B (2.5 h, rt).
Trituration with acetone yielded 0.17 g (55%) of 6e: mp 185
1
°C (decomp.); H NMR (CD3OD) δ 2.70 (s, 3H, CH3), 4.28 (s,
2H, CH2), 6.78 (d, 1H, 8.3 Hz, 4-HO-Ph C3-H), 7.12-7.21 (m,
2H, 4-F-Ph), 7.28 (dd, 1H, 2.2/8.3 Hz, 4-HO-Ph C2-H), 7.39-
7.46 (m, 5H, 4-Pyr, 4-F-Ph and 4-HO-Ph C6-H), 8.40 (m, 2H,
4-Pyr); IR (KBr) 1296 (OH bending), 1230 (C-F), 1062
(SdO), 1013 cm-1 (C-O); HPLC 1.76 min, 98.7%. Anal.
(C22H18FN3O2S2) C, H, N.
2-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-4-m eth ylsu lfa n yl-p h en ol (6f). This com-
pound was prepared from 1a (0.50 g, 2.9 mmol) and 10b
according to general procedure B (1 h, rt). Trituration with
methanol yielded 0.90 g (72%) of 6f: mp 243 °C; 1H NMR
(DMF-d7) δ 2.36 (s, 3H, CH3), 4.46 (s, 2H, CH2), 6.90 (d, 1H,
8.4 Hz, 2-HO-Ph C3-H), 7.13 (dd, 1H, 2.3/8.3 Hz, 2-HO-Ph
C4-H), 7.27-7.35 (m, 3H, 4-F-Ph and 2-HO-Ph C6-H), 7.51-
7.53 (m, 2H, 4-Pyr), 7.58-7.65 (m, 2H, 4-F-Ph), 8.52-8.55 (m,
2H, 4-Pyr), 10.30-10.70 (bs, 1H, exchangeable, NH), OH not
detected; 13C NMR (DMF-d7) δ 17.5, 32.5, 116.2, 116.6, 117.5,
120.0, 121.6, 126.5, 127.6, 129.8, 131.2, 131.4, 131.6, 143.5,
150.7, 155.0, 160.7, 162.9, 165.6; IR (KBr) 1275 (OH bending),
1230 (C-F), 1005 cm-1 (C-O); HPLC 3.27 min, 91.7%. Anal.
(C22H18FN3OS2) C, H, N.
2-[5-(4-F lu or op h en yl)-4-p yr id in -4-yl-1H-im id a zol-2-yl-
su lfa n ylm eth yl]-4-m eth a n esu lfin yl-p h en ol (6g). This com-
pound was prepared from 1a (0.27 g, 1.0 mmol), 10b and 35%
aqueous H2O2 according to general procedure B (1 h, rt).
Recrystallization from toluene/THF 1:1 yielded 0.10 g (23%)
of 6g: mp 216 °C; 1H NMR (CD3OD) δ 2.60 (s, 3H, CH3), 4.33
(s, 2H, CH2), 6.96 (d, 1H, 8.2 Hz, 2-HO-Ph C3-H), 7.11-7.21
(m, 2H, 4-F-Ph), 7.41-7.47 (m, 6H, 4-Pyr, 4-F-Ph and 2-HO-
Ph C4-/C6-H), 8.39-8.42 (m, 2H, 4-Pyr); 13C NMR (CD3OD)
δ 34.6, 43.5, 116.8, 117.2, 117.6, 123.0, 126.4, 127.7, 127.8,
128.6, 131.9, 132.0, 134.9, 143.0, 150.3, 160.1, 162.0, 166.9;
IR (KBr) 1278 (OH bending), 1232 (C-F), 1031 (SdO), 1003
cm-1 (C-O); HPLC 1.79 min, 97.7%. Anal. (C22H18FN3O2S2)
C, H, N.
4-Ch lor o-2-[5-(4-flu or op h en yl)-4-p yr id in -4-yl-1H -im i-
d a zol-2-ylsu lfa n ylm eth yl]-6-m eth ylsu lfa n yl-p h en ol (6h ).
This compound was prepared from 1a (0.80 g, 3.0 mmol) and
10c according to general procedure B (1.5 h, 75 °C). Trituration
with methanol yielded 0.80 g (62%) of 6h : mp 220 °C
(decomp.); 1H NMR (DMSO-d6) δ 2.34 (s, 3H, CH3), 4.38 (s,
2H, CH2), 6.97 (d, 1H, 2.3 Hz, 3-Cl-Ph C2-H), 7.17 (d, 1H, 2.3
Hz, 3-Cl-Ph C4-H), 7.23-7.51 (m, 6H, 4-Pyr and 4-F-Ph),
8.48-8.50 (m, 2H, 4-Pyr), 12.74 (bs, 1H, exchangeable, NH),
OH not detected; IR (KBr) 1259 (OH bending), 1225 (C-F),
1007 cm-1 (C-O); HPLC 5.19 min, 85.0%. Anal. (C22H17ClFN3-
OS2) C, H, N.
2-Hyd r oxym eth yl-4-m eth ylsu lfa n yl-p h en ol (10b). This
compound was prepared from 2-hydroxy-5-methylsulfanyl-
benzoic acid as described in the synthesis of 10a , yield 1.18 g
(86%): 1H NMR (CDCl3) δ 2.42 (s, 3H, CH3), 4.81 (s, 2H, CH2),
6.82 (d, 1H, 8.4 Hz, C6-H), 7.01 (d, 1H, 2.1 Hz, C3-H), 7.17
(dd, 1H, 2.3/8.4 Hz, C5-H), OH not detected.
4-Ch lor o-2-h yd r oxym et h yl-6-m et h ylsu lfa n yl-p h en ol
(10c). This compound was prepared from 5-chloro-2-hydroxy-
3-methylsulfanyl-benzoic acid as described in the synthesis of
10a , yield 1.55 g (74%): 1H NMR (DMSO-d6) δ 2.38 (s, 3H,
CH3), 4.52 (s, 2H, CH2), 5.4 (bs, 1H, exchangeable, OH), 7.03
4-Ch lor o-2-[5-(4-flu or op h en yl)-4-p yr id in -4-yl-1H -im i-
d a zol-2-ylsu lfa n ylm eth yl]-6-m eth a n esu lfin yl-p h en ol (6i).
This compound was prepared from 1a (0.27 g, 1.0 mmol), 10c
and 35% aqueous H2O2 according to general procedure B (1.5
h, 75 °C). Purification by column chromatography (SiO2 60,
1
acetone) yielded 0.04 g (8%) of 6i: mp 175 °C (decomp.); H
NMR (CD3OD) δ 2.72 (s, 3H, CH3), 4.39 (s, 2H, CH2), 7.14-
7.23 (m, 2H, 4-F-Ph), 7.39 (d, 1H, 2.6 Hz, 3-Cl-Ph C2-H), 7.42-
7.49 (m, 6H, 4-Pyr, 4-F-Ph and 3-Cl-Ph C4-H), 8.43-8.46 (m,
2H, 4-Pyr); IR (KBr) 1265 (OH bending), 1236 (C-F), 1051

4704 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Laufer et al.
(d, 1H, 2.6 Hz, C3-H), 7.11 (d, 1H, 2.4 Hz, C5-H), 9.02 (bs,
p 38 MAP Kin a se Assa y.30 Inhibitor dilutions were made
in DMSO. Final DMSO concentrations did not exceed 1%. KB2
buffer was prepared by dissolution of p38 MAP kinase (16 µL)
in KB1 buffer (7984 µL) containing 50 mM Tris (pH 7.5), 10
mM MgCl2, 10 mM â-glycerolphosphate, 100 µg/mL BSA, 1mM
DTT, 100 µM ATP and 0.1 mM Na3VO4. Samples of KB2 buffer
were preincubated for 5 min with test compounds in different
concentrations (37 °C). The preincubated samples were trans-
ferred to a 96-well Immulon 4HBX plate coated with ATF-2
and incubated for 1 h (37 °C). Control samples were included
containing only KB2 buffer. The 96-well plate was thoroughly
washed with ultrapure H2O and incubated with phospho-ATF-
2-antibody (Cell Signaling Technology) for 1 h (37 °C). The 96-
well plate was thoroughly washed with ultrapure H2O and
incubated with alkaline phosphatase conjugated GAR-antibody
(Santa Cruz Biotechnology) for 1 h (37 °C). The 96-well plate
was thoroughly washed with ultrapure H2O, blocking buffer
containing Tween 20 (0.05%), BSA (0.25%) and NaN3 (0.02%)
in TBS and again with ultrapure H2O. 4-Nitrophenolphosphate
was added to all wells and the optical density was measured
at 405 nm. Inhibition of p38 MAP kinase was determined by
blotting the percent reduction of phospho-ATF-2 concentration
in test samples compared to control samples on semilogarith-
mic paper over the concentration range of test compounds
(10-4-10-8 M). Results are given as IC50-values (µM).
1H, exchangeable, OH).
5-(4-F lu or op h en yl)-2-p h en eth yl-4-p yr id in -4-yl-im id a -
zol-1-ol (11a ). To a suspension of 1-(4-fluorophenyl)-2-pyridin-
4-yl-ethane-1,2-dione 1-oxime (1.0 g, 4.1 mmol) in glacial acetic
acid (15 mL) were added ammonium acetate (0.76 g, 10 mmol)
and 3-phenyl-propionaldehyde (0.83 g, 6.2 mmol). The reaction
was heated to reflux for 6 h, cooled to room temperature, and
diluted with H2O. The dark brown solution was neutralized
with 10% aqueous Na2CO3. Upon extraction with DCM, a
precipitate formed between aqueous and organic layer which
was collected by filtration to give 1.05 g (71%) of 11a as a gray
powder: 1H NMR (DMSO-d6) δ 2.55-2.63 (m, 2H, CH2), 2.82-
2.90 (m, 2H, CH2), 7.08-7.17 (m, 7H, 4-F-Ph and Ph), 7.24-
7.30 (m, 4H, 4-Pyr and 4-F-Ph), 8.29-8.32 (m, 2H, 4-Pyr), OH
not detected; IR (ATR) 1229 cm-1 (C-F).
5-(4-F lu or op h en yl)-4-p yr id in -4-yl-2-styr yl-im id a zol-1-
ol (11b). This compound was prepared from 1-(4-fluorophenyl)-
2-pyridin-4-yl-ethane-1,2-dione 1-oxime (2.2 g, 9.0 mmol),
ammonium acetate (1.7 g, 22 mmol), and 3-phenyl-propenal
(1.8 g, 13.2 mmol) as described in the synthesis of 11a , yield
1.3 g (41%): 1H NMR (DMSO-d6) δ 7.20 (d, 1H, 16.3 Hz, CH),
7.30-7.72 (m, 12H, 4-Pyr, 4-F-Ph, Ph and CH), 8.43-8.46 (m,
2H, 4-Pyr), 11.95 (bs, 1H, exchangeable, OH); IR (ATR) 1227
cm-1 (C-F).
4-[5-(4-F lu or op h en yl)-2-p h en et h yl-1H -im id a zol-4-yl]-
p yr id in e (12a ). Triethyl phosphite (1.0 mL, 5.7 mmol) was
added dropwise to a solution of 11a (0.95 g, 2.6 mmol) in DMF
(20 mL). The reaction was stirred at 100 °C for 20 h. After
cooling to room temperature, the mixture was diluted with H2O
(60 mL), and the aqueous solution was extracted with ethyl
acetate (3×). The combined organic extracts were washed with
brine (3×) and dried over Na2SO4, and the solvent was
removed to yield a dark orange, semisolid residue, which
solidified upon trituration with diethyl ether/ethanol (19:1) to
give 0.22 g (25%) of 12a as a cream-colored powder: mp 225
Ack n ow led gm en t. Financial support by Merckle
GmbH, Blaubeuren, Germany, and Fonds der Chemis-
chen Industrie is gratefully acknowledged. We thank
Dr. W. Zimmermann for helpful discussions and C.
Greim for establishing the p38 test assay.
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1
°C; H NMR (DMSO-d6) δ 2.96-3.10 (m, 4H, 2x CH2), 7.23-
7.53 (m, 13H, 4-Pyr, 4-F-Ph and Ph), 8.43-8.45 (m, 2H, 4-Pyr),
12.40 (bs, 1H, exchangeable, NH); IR (ATR) 1230 cm-1
(C-F). Anal. (C22H18FN3) C, H, N.
4-[5-(4-F lu or op h en yl)-2-styr yl-1H-im id a zol-4-yl]-p yr i-
d in e (12b). This compound was prepared from 11b (1.2 g, 3.4
mmol) as described in the synthesis of 12a , yield 0.66 g
(52%): mp 270 °C; 1H NMR (DMSO-d6) δ 7.10 (d, 1H, 16.5
Hz, CH), 7.29-7.63 (m, 12H, 4-Pyr, 4-F-Ph, Ph and CH), 8.45-
8.56 (m, 2H, 4-Pyr), NH not detected; IR (ATR) 1229 (C-F),
963 cm-1 (trans db: C-H out of plane). Anal. (C22H16FN3) C,
H, N.
Biologica l Eva lu a tion : P BMC a n d Wh ole Blood As-
sa y.29 Stock solutions of test compounds were prepared by
serial dilution in DMSO (PBMC assay) or Cremophor EL/etha-
nol 70:30 (whole blood assay). Mononuclear cells were isolated
from whole blood of healthy human donors by density gradient
centrifugation and the resulting suspension adjusted to an
approximate cell count of 106 mL-1(PBMC assay). Cell samples
(PBMC assay) or whole blood samples (whole blood assay) were
preincubated for 15 min (37 °C, 5% CO2) with test compounds
(test samples), 1% DMSO (control samples, PBMC assay) or
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an ice bath, and the samples were centrifuged. Concentrations
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percent reduction of cytokine concentration in test samples
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