From imidazoles to pyrimidines: New inhibitors of cytokine release

Article abstract of DOI:10.1021/jm011098a

On the basis of model imidazole inhibitors of cytokine release, a series of novel pyridinyl pyrimidine derivatives was prepared and tested on their ability to inhibit the release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) from periphera

Full text of DOI:10.1021/jm011098a

J . Med. Chem. 2002, 45, 2733-2740
2733
F r om Im id a zoles to P yr im id in es: New In h ibitor s of Cytok in e Relea se
Stefan A. Laufer* 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
Received November 8, 2001
On the basis of model imidazole inhibitors of cytokine release, a series of novel pyridinyl
pyrimidine derivatives was prepared and 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) and human whole blood. In the pyrimidine series, structure-activity relationships
(SARs) similar to those of the imidazole series were found, although generally pyrimidine
compounds were less potent. Modification of the substituent at the 2 position of the pyrimidine
led to the most active compound 14 which inhibited release of TNF-R (IC50 ) 3.2 µM) and
IL-1â (IC50 ) 2.3 µM) from PBMC as effectively as the model imidazole inhibitor ML 3163
(TNF-R, IC50 ) 3.7 µM; IL-1â, IC50 ) 0.9 µM). Screening in an isolated enzyme assay revealed
both imidazole and pyrimidine compounds as inhibitors of p38 MAP (mitogen-activated protein)
kinase.
In tr od u ction
from human whole blood and peripheral blood mono-
nuclear cells (PBMC). Additional data for inhibition of
p38 MAP kinase by selected compounds are provided.
Antagonizing proinflammatory cytokines such as
tumor necrosis factor-R (TNF-R) and interleukin-1â (IL-
1â) has been recognized as a valid goal in the develop-
ment of new drug candidates for the treatment of
chronic inflammatory conditions such as rheumatoid
arthritis1 and inflammatory bowel disease.2 The scope
of anti-TNF-R strategies has recently been reviewed.3
A number of small molecules lead to reduced cytokine
levels in vitro and in vivo by the inhibition of key
enzymes which are involved in the biosynthesis of
TNF-R and IL-1â.4,5 Among these enzymes, a serine-
threonine kinase termed p38 MAP (mitogen-activated
protein) kinase has become one of the main targets,
because it is required for the biosynthesis and release
of both TNF-R and IL-1â.6,7 Exemplified by SB 203580
(Table 1), several inhibitors of p38 MAP kinase contain
vicinal pyridin-4-yl-/4-fluoro-phenyl groups attached to
a core imidazole ring.8-10 Because of these structural
requirements, SB 203580 binds in the ATP pocket of
p38 MAP kinase11-13 where the pyridin-4-yl moiety
forms a crucial hydrogen bond with the amide NH of
Met109. Various other five-membered heterocycles (e.g.,
pyrrole or pyrazolone) have been reported to efficiently
replace the imidazole system of SB 203580.14 However,
no unambiguous SAR could be established concerning
the interaction of the core heterocycle with p38 MAP
kinase. No data have been published so far on six-
membered heterocycles as suitable scaffolds for the
pyridin-4-yl-/4-fluoro-phenyl pharmacophore. Benzyl-
sulfanyl imidazole ML 3163 combines structural fea-
tures of cytokine release inhibitors SK&F 8600215 and
SB 203580 (Table 1). To elucidate the contribution of
the central ring system to inhibition of cytokine release,
we have prepared a series of pyrimidine analogues of
ML 3163. Herein, we report the synthesis of these
pyrimidines and their ability to inhibit cytokine release
Ch em istr y
4,5-Bisubstituted pyrimidine-2-thiones 7-9, which
served as key intermediates in the synthesis of 2-ben-
zylsulfanyl pyrimidine derivatives, were obtained fol-
lowing the Bredereck methodology16 (Scheme 1): Re-
action of suitably substituted ketones 1-3 with dimethyl-
formamide-dimethylacetale gave enaminones 4-6. Sub-
sequent condensation of pyridin-4-yl- and pyridin-3-yl-
substituted enaminones 4-6 with thiourea afforded the
corresponding 4,5-bisubstituted pyrimidine-2-thiones
7-9. This approach, however, was not suitable for the
synthesis of the pyridin-2-yl-substituted pyrimidine-2-
thione, where reaction of the corresponding enaminone
under basic conditions led to immediate decomposition
of the starting material. 2-Benzylsulfanyl pyrimidine
derivatives 10-12a -c, 13a , and 14 were obtained from
pyrimidine-2-thiones 7-9 by nucleophilic substitution
of the appropriate benzyl chloride derivatives. The
analogue 16, which bears an additional hydrogen donor
at the pyrimidine ring similar to the ring NH of the
imidazole compounds, was prepared from thiouracil 15
(Scheme 2). Condensation of ethyl 2-(4-fluorophenyl)-
3-oxo-3-pyridin-4-yl-propionate14 with thiourea gave
thiouracil 15 which, upon reaction with 1-chloromethyl-
4-methanesulfinyl-benzene, furnished pyrimidin-4-ol
derivative 16. IR and 13C-NMR data show that 16
predominantly exists as the pyrimidin-4-ol and not as
the tautomeric pyrimidin-4-one. Three regioisomeric
benzyl chloride derivatives were applied in the final step
of the general synthesis outlined in Scheme 1. While
1-chloromethyl-2-methanesulfinyl-benzene17 and 1-chlo-
romethyl-4-methanesulfinyl-benzene18 were synthesized
according to literature procedures, 1-chloromethyl-3-
methanesulfinyl-benzene 19 was prepared by the fol-
lowing route (Scheme 3): LiAlH4 reduction of 3-meth-
ylsulfanyl-benzoic acid19 yielded phenyl-methanol deriv-
* Author to whom correspondence should be addressed. Telephone:
++49 7071 2972459. Fax: ++49 7071 295037. E-mail: stefan.laufer@
uni-tuebingen.de.
10.1021/jm011098a CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/17/2002

2734 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Ta ble 1. Inhibition of Cytokine Release by Imidazole Compounds
Laufer and Wagner
IC50 ( SEM (µM)
whole blood
PBMC
compd
X
R
TNF-R
IL-1â
TNF-R
IL-1â
SB 203580
SK&F 86002
ML 3163
21b
bond
-
SCH2
SCH2
SCH2
4-S(O)CH3
-
4-S(O)CH3
3-S(O)CH3
2-S(O)CH3
0.94 ( 0.14 (12)a
12.0 ( 0.5 (6)
20.3 ( 4.8 (2)
33.5 ( 6.4 (4)
47.5 ( 16.5 (2)
0.35 ( 0.09 (12)
27.0 ( 1.4 (6)
2.78 ( 0.13 (2)
4.1 ( 3.0 (3)
0.59 ( 0.09 (21)
5.5 ( 1.0 (2)
3.65 ( 1.73 (6)
23.5 ( 6.5 (2)
15.0 ( 0.5 (2)
0.037 ( 0.006 (20)
0.52 ( 0.03 (2)
0.88 ( 0.34 (6)
1.0 ( 0.4 (2)
22
4.0 ( 2.8 (2)
0.90 ( 0.10 (2)
a
Number in brackets denotes number of experiments.
Sch em e 1a
a
Reagents: (a) DMF-DMA, toluene, reflux; (b) thiourea, NaOC2H5/EtOH, reflux; (c) R3BzCl, Na2CO3, THF/EtOH, reflux; (d) 35% H2O2,
acetic acid, 10 °C then rt.
ative 17. Subsequent chlorination of 17 with SOCl2 gave
1-chloromethyl-3-methylsulfanyl-benzene 18 which was
readily oxidized with H2O2 to yield sulfoxide 19. Nucleo-
philic replacement of 19 with pyrimidine-2-thione 7 did
not, however, provide 5-(4-fluorophenyl)-2-(3-methane-
sulfinyl-benzylsulfanyl)-4-pyridin-4-yl-pyrimidine 13b
in an acceptable state of purity. The synthesis of 13b
went smoothly when 18 was reacted with pyrimidine-
2-thione 7, and the resulting 3-methylsulfanyl-benzyl-
sulfanyl pyrimidine 13a was oxidized to the correspond-
ing sulfoxide 13b by means of 1 equiv of H2O2.
Benzylsulfanyl imidazoles ML 3163, 21b, and 22 were
prepared from 4-(4-fluorophenyl)-5-pyridin-4-yl-1,3-di-
hydro-imidazole-2-thione20 (20) by nucleophilic substitu-
tion of appropriate benzyl chloride derivatives. In the
preparation of 21b, the same synthetic strategy had to
be applied as for the synthesis of 13b (Scheme 3).
Biologica l Resu lts a n d Discu ssion
Imidazole lead compounds SB 203580, SK&F 86002,
and ML 3163 inhibited cytokine release from human
whole blood as well as from PBMC in the sub-micro-

From Imidazoles to Pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2735
Sch em e 2a
a
Reagents: (a) thiourea, neat, 180 °C; (b) 4-methylsulfinyl-benzyl chloride, Na2CO3, THF/EtOH, reflux.
Sch em e 3a,b
a
Reagents: (a) LiAlH4, THF, 0 °C then rt; (b) SOCl2, DCM, reflux; (c) 35% H2O2, acetic acid, 10 °C then rt; (d) Na2CO3, THF/EtOH,
b
reflux. Yields over 2 steps.
formation of a hydrogen bond with the side chain of
Lys53 and the absence of any negative interactions with
this residue have been suggested to explain the efficacy
of different heterocyclic scaffolds for the pyridin-4-yl-/
4-fluoro-phenyl pharmacophore. As a result of the
appropriate orientation of the core heterocycle, a sub-
stituent at the 2 position may interact with the phos-
phate-binding ribbon of the ATP binding site of p38
MAP kinase. Enlargement of the core imidazole hetero-
cycle to a six-membered ring led to a decrease in
biological activity for ML 3163 pyrimidine analogues
10a -c, 11a -c, and 12a -c in an initial whole blood
screen (columns 6 and 7 of Table 2). The compounds
10a , 10c, 11a -c, and 12a -c exhibited less than 50%
inhibition of cytokine release at a concentration of 100
µM. Only sulfoxide 10b exhibited moderate anti-IL-1â
and weak anti-TNF-R activity. These results prompted
us to examine SAR in the PBMC assay (columns 8 and
9 of Table 2). In the pyrimidine series, the oxidation
state of the terminal sulfur atom significantly affected
inhibition of TNF-R release but not that of IL-1â.
Sulfone 10c and sulfoxide 10b were about 4 times more
active than sulfide 10a , demonstrating that only polar
substituents at the 4 position of the benzylsulfanyl
moiety increase inhibitory potency. This correlation has
also been described for other inhibitors of cytokine
release which exert their biological activity by inhibition
F igu r e 1. Sites of interaction between pyridin-4-yl hetero-
cycles and p38 MAP kinase.8
molar to low-micromolar range (Table 1). It has been
demonstrated for several inhibitors which bind in the
ATP site of p38 MAP kinase that a hydrophobic pocket
and the amide NH of Met109 are the main sites of
interaction11-13 (Figure 1). Therefore, a 4-fluoro-phenyl
and a pyridin-4-yl group are regarded as essential for
biological activity in this class of compounds. Both the

2736 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Ta ble 2. Inhibition of Cytokine Release by Pyrimidine Compounds
Laufer and Wagner
IC50 ( SEM (µM)
whole blood
PBMC
compd
R1
R2
R3
R4
TNF-R
>100 (2)a
95.5 ( 64.5 (2)
>100 (2)
>100 (2)
>100 (2)
ndb
IL-1â
>100 (2)
45.5 ( 11.5 (2)
>100 (2)
>100 (2)
>100 (2)
nd
TNF-R
IL-1â
10a
10b
10c
11b
12b
13b
14
4-Pyr
4-Pyr
4-Pyr
3-Pyr
4-F-Ph
4-Pyr
4-Pyr
4-Pyr
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-Pyr
4-F-Ph
4-F-Ph
4-F-Ph
H
H
H
H
H
H
H
HO
4-SCH3
60.0 ( 10.0 (2)
14.1 ( 4.4 (4)
17.8 ( 8.2 (2)
54.0 ( 20.0 (2)
29.9 ( 4.4 (4)
6.5 ( 0.0 (2)
3.2 ( 0.5 (2)
27.0 ( 2.0 (2)
7.4 ( 0.2 (2)
5.6 ( 0.4 (2)
7.7 ( 4.2 (2)
75.5 ( 12.5 (2)
17.8 ( 4.1 (4)
5.4 ( 0.0 (2)
2.3 ( 0.6 (2)
25.5 ( 4.0 (2)
4-S(O)CH3
4-SO2CH3
4-S(O)CH3
4-S(O)CH3
3-S(O)CH3
2-S(O)CH3
4-S(O)CH3
nd
nd
>100 (2)
16
>100 (2)
a
b
Number in parentheses denotes number of experiments. Not determined.
Ta ble 3. p38 MAP Kinase Inhibition Data
IC50 ( SEM
(µM)
IC50
(µM)
compd
compd
SB 203580
ML 3163
0.36 ( 0.07 (7)a
4.0 ( 1.0 (2)
10b
13b
14
36 (1)
19 ( 1.4 (2)
5.1 ( 2.0 (2)
a
Number in parentheses denotes number of experiments.
of p38 MAP kinase.5,21 As can also be observed with
various inhibitors of p38 MAP kinase,22 replacement of
the pyridin-4-yl moiety in 10b with a pyridin-3-yl ring
in 11b resulted in a reduced inhibition of cytokine
release. This loss of potency can be explained by a
disturbance of the pivotal hydrogen bond between the
pyridinyl moiety and Met109. The weaker inhibitory
potency of 12b compared to the regioisomeric pyrimidine
10b underlines the importance of the core heterocycle
in providing an optimum scaffold for both the pyridin-
4-yl- and 4-fluoro-phenyl moiety. This purpose is better
served by the pyrimidine nucleus in 10b than in 12b,
albeit to a lesser extent than by the imidazole in ML
3163. Apart from their different geometries, ML 3163
and 10b also differ in their hydrogen-bonding proper-
ties. Among potent inhibitors of p38 MAP kinase
hydrogen-donating as well as hydrogen-accepting func-
tionalities are found at various positions of the central
heterocycle. In the pyrimidine series, biological activity
decreased when an additional hydrogen-donating sub-
structure was introduced at position 4 of the core
pyrimidine (compound 16) to mimic the ring NH of the
imidazole compounds. This result can be attributed to
an unfavorable interaction with Lys53. Upon examina-
tion of SAR at the methanesulfinyl-benzylsulfanyl
moiety in the pyrimidine series, the 3-methanesulfinyl
isomer 13b and the 2-methanesulfinyl isomer 14 showed
a 2- and 4.5-fold better inhibition, respectively, of TNF-R
release than 10b. The same order of efficacy was
observed when regioisomers 10b, 13b, and 14 were
tested for inhibition of isolated p38 MAP kinase (Table
3). This contrasts remarkably with the corresponding
imidazole derivatives where a reversed correlation was
observed. Placement of the methanesulfinyl substituent
at the 3 or 2 position of the benzylsulfanyl moiety
resulted in reduced bioactivity of compounds 21b and
F igu r e 2. Superposition of SB 203580 (yellow) in complex
with p38 MAP kinase13 and compound 14 (red);23 Thr35,
Met109, and Arg173 are depicted in green.
22 compared to ML 3163 (Table 1). Similar results have
been obtained for the corresponding SB 203580 regioi-
somers.5
Con clu sion
We have shown that 2-benzylsulfanyl imidazole com-
pounds are effective inhibitors of cytokine release from
PBMC and human whole blood. Replacement of the core
imidazole ring with pyrimidine leads to a decrease in
biological activity (ML 3163 vs compound 10b). This is
due to the different geometry which is provided by
imidazole and pyrimidine scaffolds for the pyridin-4-yl-/
4-fluoro-phenyl pharmacophore and the substituent in
position 2. A hydrogen-donating substructure at the core
heterocycle itself is not essential for inhibition of cyto-
kine release (compound 16). In both the imidazole and
pyrimidine series, elongation of the linker between the
core heterocycle and the methylsulfinylphenyl ring
diminished inhibitory potency compared to SB 203580.
As illustrated by the superposition of pyrimidine 14 and
SB 203580 in complex with p38 MAP kinase,13 this can
be attributed to the benzyl moiety being forced out of
the favorable π-π stacking with Tyr35 (Figure 2). The

From Imidazoles to Pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2737
5-(4-F lu or o-p h en yl)-4-p yr id in -4-yl-1H -p yr im id in e-2-
th ion e (7). To a freshly prepared solution of sodium (0.29 g,
12.6 mmol) in ethanol (30 mL) were consecutively added
compound 4 (1.0 g, 3.7 mmol) and thiourea (0.31 g, 4.1 mmol).
The mixture was heated to reflux for 4 h. The solvent was
removed, and the residue was dissolved in H2O (15 mL). The
dark-red aqueous solution was neutralized with 8% aqueous
H3PO4. A precipitate formed which was collected by filtration,
washed with H2O, and dried in vacuo to give 0.92 g (88%) of
7: 1H-NMR (DMSO-d6, δ) 7.16-7.27 (m, 6H, 4-Pyr and 4-F-
Ph), 8.18 (s, 1H, Pyr and CH), 8.55-8.58 (m, 2H, 4-Pyr), NH
not detected; 13C-NMR (DMSO-d6, δ) 115.3, 115.8, 121.1, 123.4,
130.3, 131.3, 131.5, 144.9, 148.1, 149.6, 159.4, 164.3, 164.6,
179.6; IR (KBr) 1509 (HN-CdS), 1218 cm-1 (C-F).
5-(4-F lu or o-p h en yl)-4-p yr id in -3-yl-1H -p yr im id in e-2-
th ion e (8). This compound was prepared from enaminone 5
as described in the synthesis of 7 with a yield of 0.90 g (86%):
1H-NMR (DMSO-d6, δ) 7.13-7.42 (m, 5H, 3-Pyr C5-H and 4-F-
Ph), 7.72 (d, 1H, 3-Pyr C6-H), 8.17 (s, 1H, Pyr CH), 8.45 (m,
1H, 3-Pyr C4-H), 8.57 (d, 1H, 3-Pyr C2-H), NH not detected;
IR (KBr) 1509 (HN-CdS), 1230 cm-1 (C-F).
disadvantageous geometry of the pyrimidine derivatives
can be overcome to some extent if the contribution of
the methanesulfinyl substituent to bioactivity is en-
hanced by its placement at the 3 or 2 position of the
benzylsulfanyl moiety (compounds 13b and 14). We
suggest that the benefits from this regioisomeric modi-
fication in the pyrimidine series may arise from an
additional interaction between Arg173 and the sulfoxide
functionality in 14 which may not be formed by the
corresponding imidazole regioisomer 22 (Figure 2).
Several other SAR in the pyrimidine series parallel
those of known imidazole inhibitors of p38 MAP kinase.
These observations, together with the enzyme data for
selected compounds (Table 3), suggest a mode of action
for pyrimidine inhibitors of cytokine release similar to
those for model imidazole compounds.
Exp er im en ta l Section
4-(4-F lu or o-p h en yl)-5-p yr id in -4-yl-1H -p yr im id in e-2-
th ion e (9). This compound was prepared from enaminone 6
as described in the synthesis of 7 with a yield of 0.84 g (80%):
1H-NMR (DMSO-d6, δ) 7.20-7.24 (m, 4H, 4-Pyr and 4-F-Ph),
7.36-7.43 (m, 2H, 4-F-Ph), 8.22 (s, 1H, Pyr CH), 8.48-8.51
(d, 2H, 4-Pyr), NH not detected; 13C-NMR (DMSO-d6, δ) 115.0,
115.4, 119.1, 123.6, 131.9, 132.0, 132.3, 132.4, 142.4, 148.0,
149.6, 149.8, 164.9, 165.6, 179.8; IR (KBr) 1509 (HN-CdS),
1231 cm-1 (C-F).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Ben zyl-
su lfa n yl P yr im id in es 10a -c, 11a -c, 12a -c, 13a , and 14.
A suspension of the appropriate pyrimidine-2-thione (0.30 g,
1.06 mmol), sodium acetate (0.17 g, 2.1 mmol), and the
appropriate benzyl chloride (1.0 mmol) in 9:1 ethanol/THF (15
mL) was heated to reflux for 2 h. After the mixture was cooled
to room temperature, it was filtered, and the bright-red filtrate
was concentrated in vacuo. The oily residue was purified by
column chromatography or crystallization to give 2-benzylsul-
fanyl pyrimidines 10a -c, 11a -c, 12a -c, 13a , and 14.
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
apparatus and are thermodynamically corrected. 1H- and 13C-
NMR spectra were obtained on a Bruker Spectrospin AC 200
instrument at 200 MHz. Chemical shifts are reported in parts
per million relative to TMS as the 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 analy-
ses were carried out on Merck Hitachi (Darmstadt) equipment,
using a LiChrospher 100 RP-18 column (5 µm) and eluting
with MeCN/NaH2PO4 buffer at pH 3.5 (70:30) and 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 Perkin-Elmer
EA 240 and 2400 instruments. The following compounds were
prepared according to literature procedures: imidazole-2-
thione 20,20 SB 203580,21 SK&F 86002,15 1-chloromethyl-4-
methylsulfanyl-benzene,18 1-chloromethyl-2-/4-methanesulfinyl-
benzene,17,18 1-chloromethyl-4-methanesulfonyl-benzene,18 and
ketones 1-3.5,20
5-(4-Flu or o-ph en yl)-2-(4-m eth ylsu lfan yl-ben zylsu lfan yl)-
4-p yr id in -4-yl-p yr im id in e (10a ). Compound 10a was pre-
pared from 1-chloromethyl-4-methylsulfanyl-benzene and 7
according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, DCM) yielded
1
0.23 g (61%) of 10a : mp 117 °C; H-NMR (CDCl3, δ) 2.47 (s,
3-Dim et h yla m in o-2-(4-flu or o-p h en yl)-1-p yr id in -4-yl-
p r op en on e (4). To a suspension of 2-(4-fluoro-phenyl)-1-
pyridin-4-yl-ethanone 120 (5.0 g, 23.3 mmol) in toluene (30 mL)
was added dimethylformamide-dimethylacetale (5.5 mL, 41
mmol). The mixture was heated to reflux for 2.5 h. Removal
of the solvent and excess dimethylformamide-dimethylacetale
gave a dark-brown solid which, upon trituration with tert-
butylmethyl ether, afforded 5.7 g (91%) of 1: 1H-NMR (CDCl3,
δ) 2.78 (bs, 6H, 2 × CH3), 6.98-7.02 (m, 2H, 4-F-Ph), 7.08-
7.11 (m, 2H, 4-F-Ph), 7.27-7.29 (m, 2H, 4-Pyr), 7.37 (s, 1H,
CH), 8.55-8.57 (m, 2H, 4-Pyr); 13C-NMR (DMSO-d6, δ) 43.2,
108.9, 113.9, 114.4, 122.1, 132.8, 133.6, 133.8, 149.1, 149.3,
154.5, 158.3, 163.2, 190.7; IR (KBr) 1627 (CdO), 1216 cm-1
(C-F).
3-Dim et h yla m in o-2-(4-flu or o-p h en yl)-1-p yr id in -3-yl-
p r op en on e (5). This compound was prepared from ketone 220
as described in the synthesis of 4 with a yield of 1.2 g (94%):
1H-NMR (CDCl3, δ) 2.78 (bs, 6H, 2 × CH3), 6.93-7.02 (m, 2H,
4-F-Ph), 7.09-7.16 (m, 2H, 4-F-Ph), 7.23-7.28 (m, 1H, 3-Pyr
C5-H), 7.39 (s, 1H, CH), 7.71-7.74 (m, 1H, 3-Pyr C6-H), 8.55
(dd, 1H, 1.2/4.8 Hz, 3-Pyr C4-H), 8.62 (d, 1H, 1.2 Hz, 3-Pyr
C2-H); IR (ATR) 1683 (CdO), 1223 cm-1 (C-F).
3H, CH3), 4.43 (s, 2H, CH2), 7.05-7.39 (m, 10H, 4-Pyr, 4-F-
Ph, and 4-MeS-Ph), 8.56-8.59 (m, 3H, 4-Pyr and Pyr CH);
13C-NMR (CDCl3, δ) 15.8, 35.1, 116.0, 116.4, 123.8, 126.6,
128.0, 129.5, 131.0, 131.1, 134.2, 137.4, 144.7, 149.8, 158.9,
160.3, 160.9, 165.5, 171.0; IR (KBr) 1397 (NdC-S), 1224 cm-1
(C-F); HPLC 6.14 min, 98.8%. Anal. (C23H18FN3S2) C, H, N.
5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-4-p yr id in -4-yl-p yr im id in e (10b). Compound 10b was
prepared from 1-chloromethyl-4-methanesulfinyl-benzene and
7 according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, 9:1 DCM/
MeCN) yielded 0.21 g (46%) of 10b: mp 134 °C; 1H-NMR
(CDCl3, δ) 2.71 (s, 3H, CH3), 4.52 (s, 2H, CH2), 7.01-7.17 (m,
4H, 4-F-Ph), 7.26-7.30 (m, 2H, 4-Pyr), 7.56-7.66 (m, 4H,
4-MeS(O)-Ph), 8.57-8.58 (m, 3H, 4-Pyr and Pyr CH); 13C-
NMR (CDCl3, δ) 34.9, 43.9, 116.0, 116.5, 123.7, 128.3, 130.0,
130.8, 130.9, 131.1, 141.2, 144.6, 144.7, 149.8, 159.0, 160.3,
161.1, 165.3, 170.5; IR (KBr) 1398 (NdC-S), 1223 (C-F), 1048
cm-1 (SdO); HPLC 1.96 min, 99.6%. Anal. (C23H18FN3OS2) C,
H, N.
5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu lfa -
n yl)-4-p yr id in -4-yl-p yr im id in e (10c). Compound 10c was
prepared from 1-chloromethyl-4-methanesulfonyl-benzene and
7 according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, 9:1 DCM/
MeCN) yielded 0.14 g (29%) of 10c: mp 142 °C; 1H-NMR
(CDCl3, δ) 3.04 (s, 3H, CH3), 4.53 (s, 2H, CH2), 7.01-7.17 (m,
4H, 4-F-Ph), 7.24-7.28 (m, 2H, 4-Pyr), 7.67 (d, 2H, 8.32 Hz,
3-Dim et h yla m in o-1-(4-flu or o-p h en yl)-2-p yr id in -4-yl-
p r op en on e (6). This compound was prepared from ketone 35
as described in the synthesis of 4 with a yield of 1.17 g (92%):
1H-NMR (CDCl3, δ) 2.80 (s, 6H, 2 × CH3), 6.94-7.07 (m, 4H,
4-Pyr and 4-F-Ph), 7.28-7.43 (m, 3H, CH and 4-F-Ph), 8.48-
8.50 (m, 2H, 4-Pyr); IR (ATR) 1679 (CdO), 1227 cm-1 (C-F).

2738 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Laufer and Wagner
4-MeS(O)2-Ph), 7.88 (d, 2H, 8.33 Hz, 4-MeS(O)2-Ph), 8.58-
8.62 (m, 3H, 4-Pyr and Pyr CH); 13C-NMR (CDCl3, δ) 34.8,
44.5, 116.1, 116.5, 123.7, 127.6, 127.7, 128.4, 130.0, 130.7,
130.8, 131.1, 131.0, 139.4, 144.5, 144.7, 149.8, 159.1, 160.4,
161.2, 165.3, 170.1; IR (KBr) 1399 (NdC-S), 1304 (SO2), 1220
was removed. Trituration of the oily residue with tert-butyl-
methyl ether gave 0.12 g (28%) of 12b: mp 142 °C; H-NMR
1
(CDCl3, δ) 2.72 (s, 3H, CH3), 4.52 (s, 2H, CH2), 7.00-7.06 (m,
2H, 4-F-Ph), 7.15-7.18 (m, 2H, 4-Pyr), 7.33-7.40 (m, 2H,
4-F-Ph), 7.58-7.68 (m, 4H, 4-MeS(O)-Ph), 8.52 (s, 1H, Pyr
CH), 8.59-8.62 (m, 2H, 4-Pyr); 13C-NMR (CDCl3, δ) 35.6, 44.6,
116.1, 116.5, 124.4, 124.5, 126.8, 130.7, 132.5, 132.6, 132.9,
133.0, 141.9, 144.9, 145.3, 150.9, 159.0, 162.0, 163.5, 167.0,
171.93; IR (KBr) 1399 (NdC-S), 1221 (C-F), 1039 cm-1 (Sd
O); HPLC 1.99 min, 97.6%. Anal. (C23H18FN3OS2) C, H, N.
(C-F), 1149 cm-1 (SO2); HPLC 2.15 min, 95.0%. Anal. (C23H18
-
FN3O2S2) C, H, N.
5-(4-Flu or o-ph en yl)-2-(4-m eth ylsu lfan yl-ben zylsu lfan yl)-
4-p yr id in -3-yl-p yr im id in e (11a ). Compound 11a was pre-
pared from 1-chloromethyl-4-methylsulfanyl-benzene and 8
according to the general procedure described above. The crude
oil was extracted with diethyl ether. The organic extract was
washed with H2O and dried over Na2SO4, and the solvent was
removed. Trituration of the oily residue with tert-butylmethyl
4-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu lfa -
n yl)-5-p yr id in -4-yl-p yr im id in e (12c). Compound 12c was
prepared from 1-chloromethyl-4-methanesulfonyl-benzene and
9 according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, 9:1 DCM/
MeCN) yielded 0.25 g (56%) of 12c: mp 151 °C; 1H-NMR
(CDCl3, δ) 3.04 (s, 3H, CH3), 4.53 (s, 2H, CH2), 6.96-7.04 (m,
2H, 4-F-Ph), 7.11-7.17 (m, 2H, 4-Pyr), 7.32-7.39 (m, 2H,
4-F-Ph), 7.67 (d, 2H, 8.28 Hz, 4-MeS(O)2-Ph), 7.88 (d, 2H,
8.32 Hz, 4-MeS(O)2-Ph), 8.51 (s, 1H, Pyr CH), 8.59-8.70 (m,
2H, 4-Pyr); 13C-NMR (CDCl3, δ) 34.7, 44.4, 115.4, 115.8, 123.8,
126.2, 127.5, 129.9, 131.7, 131.9, 132.1, 132.2, 139.3, 144.0,
144.4, 150.2, 158.3, 161.3, 162.9, 166.3, 170.80; IR (KBr) 1402
(NdC-S), 1303 (SO2), 1226 (C-F), 1149 cm-1 (SO2); HPLC
2.21 min, 96.4%. Anal. (C23H18FN3O2S2) C, H, N.
1
ether gave 0.19 g (45%) of 11a : mp 124 °C; H-NMR (CDCl3,
δ) 2.47 (s, 3H, CH3), 4.44 (s, 2H, CH2), 7.06-7.41 (m, 9H, 3-Pyr
C5-H, 4-F-Ph, and 4-MeS-Ph), 7.72-7.80 (m, 1H, 3-Pyr C6-
H), 8.55 (s, 1H, Pyr CH), 8.61 (m, 1H, 3-Pyr C4-H), 8.69 (m,
1H, 3-Pyr C2-H); 13C-NMR (CDCl3, δ) 15.9, 35.1, 116.2, 116.7,
123.4, 126.7, 128.0, 129.5, 131.0, 131.1, 131.2, 133.6, 134.2,
137.5, 138.3, 149.0, 149.3, 158.9, 160.1, 160.3, 165.3, 171.1;
IR (KBr) 1398 (NdC-S), 1222 cm-1 (C-F); HPLC 6.08 min,
98.1%. Anal. (C23H18FN3S2) C, H, N.
5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-4-p yr id in -3-yl-p yr im id in e (11b). Compound 11b was
prepared from 1-chloromethyl-4-methanesulfinyl-benzene and
8 according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, ethyl acetate)
5-(4-Flu or o-ph en yl)-2-(3-m eth ylsu lfan yl-ben zylsu lfan yl)-
4-p yr id in -4-yl-p yr im id in e (13a ). This compound was pre-
pared from 1-chloromethyl-3-methylsulfanyl-benzene (0.18 g,
1.0 mmol) and 7 (0.28 g, 1.0 mmol) according to the general
procedure described above. The crude oil of 13a was used in
the synthesis of 13b without further purification.
1
yielded 0.14 g (40%) of 11b: mp 128 °C; H-NMR (CDCl3, δ)
2.72 (s, 3H, CH3), 4.52 (s, 2H, CH2), 7.02-7.16 (m, 4H, 4-F-
Ph), 7.22-7.32 (m, 1H, 3-Pyr C5-H), 7.61-7.69 (m, 5H, 3-Pyr
C6-H and 4-MeS(O)-Ph), 8.56 (s, 1H, Pyr CH), 8.61 (m, 1H,
3-Pyr C4-H), 8.59-8.70 (m, 1H, 3-Pyr C2-H); 13C-NMR (CDCl3,
δ) 34.0, 43.9, 116.2, 116.7, 123.4, 123.8, 128.3, 130.0, 130.1,
131.0, 131.1, 131.2, 138.0, 141.2, 144.6, 149.2, 149.4, 158.9,
160.3, 160.3, 165.3, 170.5; IR (KBr) 1397 (NdC-S), 1227 (C-
5-(4-F lu or o-p h en yl)-2-(3-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-4-p yr id in -4-yl-p yr im id in e (13b). A suspension of 13a
(0.35 g, 0.83 mmol) in glacial acetic acid (5 mL) was treated
with 35% aqueous H2O2 (0.10 mL, 1.0 mmol). The mixture was
stirred at room temperature for 5.5 h, diluted with H2O (5 mL),
adjusted to pH 9 with 25% aqueous ammonia, and extracted
with DCM (3 times). The combined organic extracts were
washed with saturated brine (3 times) and dried over Na2SO4,
and the solvent was removed to yield an oily residue. The crude
product was purified by column chromatography (silica gel 60,
ethyl acetate) to give 0.15 g (42%) of 13b: 1H-NMR (CDCl3, δ)
2.68 (s, 3H, CH3), 4.53 (s, 2H, CH2), 7.01-7.14 (m, 4H, 4-F-
Ph), 7.27-7.30 (m, 2H, 4-Pyr), 7.46-7.62 (m, 3H, 3-MeS(O)-
Ph C4-/C5-/C6-H), 7.77-7.79 (m, 1H, 3-MeS(O)-Ph C2-H),
8.58-8.60 (m, 3H, 4-Pyr and Pyr CH); 13C-NMR (CDCl3, δ)
35.3, 44.2, 116.4, 116.8, 122.8, 124.2, 124.4, 128.6, 129.8, 131.1,
131.2, 131.3, 131.4, 132.1, 140.0, 145.2, 146.3, 150.1, 159.4,
160.7, 161.4, 165.7, 170.8; IR (ATR) 1396 (NdC-S), 1222 (C-
F), 1044 cm-1 (SdO). Anal. (C23H18FN3OS2) C, H, N.
F), 1042 cm-1 (SdO); HPLC 1.93 min, 99.6%. Anal. (C23H18
-
FN3OS2) C, H, N.
5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfon yl-ben zylsu lfa -
n yl)-4-p yr id in -3-yl-p yr im id in e (11c). Compound 11c was
prepared from 1-chloromethyl-4-methanesulfonyl-benzene and
8 according to the general procedure described above. The
crude oil was extracted with diethyl ether. The organic extract
was washed with H2O and dried over Na2SO4, and the solvent
was removed. Trituration of the oily residue with methanol
gave 0.22 g (48%) of 11c: mp 173 °C; 1H-NMR (CDCl3, δ) 3.05
(s, 3H, CH3), 4.53 (s, 2H, CH2), 7.02-7.18 (m, 4H, 4-F-Ph),
7.22-7.30 (m, 1H, 3-Pyr C5-H), 7.68 (m, 3H, 3-Pyr C6-H and
4-MeS(O)2-Ph), 7.88 (d, 2H, 8.23 Hz, 4-MeS(O)2-Ph), 8.55 (s,
1H, Pyr CH), 8.59-8.62 (m, 2H, 3-Pyr C2-/C4-H); 13C-NMR
(CDCl3, δ) 34.8, 44.5, 116.2, 116.6, 123.1, 127.6, 128.4, 130.0,
130.9, 131.0, 131.1, 133.0, 137.3, 139.3, 144.5, 150.1, 158.9,
160.3, 160.8, 165.3, 170.1; IR (KBr) 1399 (NdC-S), 1301 (SO2),
1223 (C-F), 1151 cm-1 (SO2); HPLC 2.14 min, 99.7%. Anal.
(C23H18FN3O2S2) C, H, N.
4-(4-Flu or o-ph en yl)-2-(4-m eth ylsu lfan yl-ben zylsu lfan yl)-
5-p yr id in -4-yl-p yr im id in e (12a ). Compound 12a was pre-
pared from 1-chloromethyl-4-methylsulfanyl-benzene and 9
according to the general procedure described above. Purifica-
tion by column chromatography (basic alumina, DCM) yielded
0.23 g (61%) of 12a : 1H-NMR (CDCl3, δ) 2.47 (s, 3H, CH3),
4.44 (s, 2H, CH2), 6.98-7.04 (m, 2H, 4-F-Ph), 7.13-7.22 (m,
4H, 4-Pyr and 4-F-Ph), 7.35-7.40 (m, 4H, 4-MeS-Ph), 8.50
(s, 1H, Pyr CH), 8.57-8.62 (m, 2H, 4-Pyr); 13C-NMR (CDCl3,
δ) 15.9, 35.1, 115.4, 115.9, 124.1, 125.6, 126.7, 129.5, 131.8,
132.0, 132.3, 132.4, 134.2, 137.5, 145.3, 149.5, 158.2, 161.4,
162.8, 166.4, 172.1; IR (KBr) 1401 (NdC-S), 1227 cm-1 (C-
F); HPLC 7.31 min, 98.4%. Anal. (C23H18FN3S2) C, H, N.
5-(4-F lu or o-p h en yl)-2-(2-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-4-p yr id in -4-yl-p yr im id in e (14). Compound 14 was
prepared from 1-chloromethyl-2-methanesulfinyl-benzene and
9 according to the general procedure described above. Purifica-
tion by column chromatography (silica gel 60, ethyl acetate)
yielded 0.13 g (56%) of 14: 1H-NMR (CDCl3, δ) 2.84 (s, 3H,
CH3), 4.57 (d, 1H, 13.8 Hz, CH2), 4.68 (d, 1H, 13.8 Hz, CH2),
7.08-7.27 (m, 2H, 4-F-Ph), 7.33-7.37 (m, 2H, 4-Pyr), 7.48-
7.60 (m, 3H, 2-MeS(O)-Ph C4-/C5-/C6-H), 8.07 (dd, 1H, 7.9 Hz,
2-MeS(O)-Ph C3-H), 8.59-8.62 (m, 3H, 4-Pyr and Pyr CH);
IR (ATR) 1397 (NdC-S), 1223 (C-F), 1035 cm-1 (SdO). Anal.
(C23H18FN3OS2) C, H, N.
5-(4-Flu or o-ph en yl)-6-pyr idin -4-yl-2-th ioxo-2,3-dih ydr o-
1H-p yr im id in -4-on e (15). Ethyl 2-(4-fluorophenyl)-3-oxo-3-
(4-pyridyl)-propionate (3.1 g, 10.8 mmol) was reacted with
thiourea (0.62 g, 8.2 mmol) at 180 °C for 20 min. The oily
mixture was cooled to room temperature and taken up in
acetone. Upon trituration, a precipitate formed which was
filtered to yield 0.22 g (9%) of 15: 1H-NMR (DMSO-d6, δ) 6.98-
7.13 (m, 4H, 4-F-Ph), 7.24-7.29 (m, 2H, 4-Pyr), 8.49-8.52
(m, 2H, 4-Pyr), 12.68 (bs, 1H, exchangeable, NH/OH), 12.78
(bs, 1H, exchangeable, NH/OH); 13C-NMR (DMSO-d6, δ) 114.4,
114.8, 116.1, 123.8, 127.9, 128.0, 132.9, 133.0, 139.4, 148.0,
4-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-5-p yr id in -4-yl-p yr im id in e (12b). Compound 12b was
prepared from 1-chloromethyl-4-methanesulfinyl-benzene and
9 according to the general procedure described above. The
crude oil was extracted with diethyl ether. The organic extract
was washed with H2O and dried over Na2SO4, and the solvent

From Imidazoles to Pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2739
149.3, 158.9, 160.6, 163.8, 175.00; IR (KBr) 3408 (NH stretch-
(C-F), 1039 cm-1 (SdO); HPLC 1.92 min, 97.9%. Anal. (C22H18
-
FN3OS2) C, H, N.
ing), 1679 (HN-CdO), 1514 (HN-CdS), 1236 cm-1 (C-F).
5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu lfa -
n yl)-6-p yr id in -4-yl-p yr im id in -4-ol (16). A suspension of 15
(0.10 g, 0.33 mmol), 1-chloromethyl-4-methanesulfinyl-benzene
(0.065 g, 0.34 mmol), and Na2CO3 (0.055 g, 0.52 mmol) in 90%
ethanolic THF (10 mL) was heated to reflux for 4.5 h. After
the mixture was cooled to room temperature, it was filtered,
and the filtrate was concentrated in vacuo. The oily residue
solidified upon trituration with diethyl ether. The crude
product was washed with H2O to yield 0.13 g (90%) of 16: mp
101 °C; 1H-NMR (CD3OD, δ) 2.78 (s, 3H, CH3), 4.48 (s, 2H,
CH2), 6.88-6.96 (m, 2H, 4-F-Ph), 7.08-7.12 (m, 2H, 4-F-Ph),
7.20-7.23 (m, 2H, 4-Pyr), 7.62 (d, 2H, 8.4 Hz, 4-MeS(O)-Ph),
7.71 (d, 2H, 8.4 Hz, 4-MeS(O)-Ph), 8.32-8.35 (m, 2H, 4-Pyr);
13C-NMR (CD3OD, δ) 35.1, 43.6, 115.4, 115.8, 119.6, 124.9,
126.0, 131.4, 133.9, 134.0, 134.2, 134.4, 144.0, 145.5, 149.5,
150.3, 159.6, 160.8, 165.6, 169.7; IR (KBr) 3399 (broad, OH),
1316 (NdC-S), 1220 (C-F), 1033 cm-1 (SdO); HPLC 1.25 min,
99.0%. Anal. (C23H18FN3O2S2) C, H, N.
(3-Meth ylsu lfa n yl-p h en yl)-m eth a n ol (17). To a cooled (0
°C) suspension of 95% LiAlH4 (0.74 g, 18.5 mmol) in THF (20
mL) was slowly added a solution of 3-methylsulfanyl-benzoic
acid (3.7 g, 22 mmol) in THF (35 mL). After the addition was
complete (30 min), the reaction mixture was stirred for 3.5 h
at room temperature. The reaction was quenched by dilution
with cold water (50 mL), and the white precipitate was
dissolved by addition of 10% sulfuric acid. The aqueous
solution was extracted with diethyl ether (3 times). The
combined organic extracts were washed with saturated brine
and dried over Na2SO4, and the solvent was evaporated to yield
3.3 g (97%) of 17 as a yellowish oil: 1H-NMR (CDCl3, δ) 1.72
(s, 1H, exchangeable, OH), 2.49 (s, 3H, CH3), 4.67 (s, 2H, CH2),
7.10-7.32 (m, 4H, ar); IR (ATR) 3343 (OH stretching), 1202
(OH bending), 1013 cm-1 (C-O stretching).
4-[5-(4-F lu or o-p h en yl)-2-(3-m eth ylsu lfa n yl-ben zylsu l-
fa n yl)-3H-im id a zol-4-yl]-p yr id in e (21a ). To a solution of 18
(0.69 g, 4.1 mmol) in ethanol (60 mL) was added compound
20 (1.1 g, 4.1 mmol). The mixture was heated to reflux for 11
h and stirred for another 60 h at room temperature. The yellow
precipitate was filtered off and purified by trituration with
ethanol to yield 1.2 g (73%) of 21a : 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).
4-[5-(4-F lu or o-p h en yl)-2-(3-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-3H-im id a zol-4-yl]-p yr id in e (21b). A suspension of
21a (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 times). The combined organic
extracts were washed with saturated brine (3 times) 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 chromatog-
raphy (RP-18, methanol) to give 0.16 mg (31%) of 13b: 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. (C22H18FN3OS2) C, H, N.
1-Ch lor om eth yl-3-m eth ylsu lfa n yl-ben zen e (18). To a
solution of 17 (3.3 g, 21 mmol) in DCM (20 mL) was added a
solution of SOCl2 (2.5 g, 21 mmol) in DCM (10 mL). The
mixture was heated to reflux for 2 h. The reaction mixture
was cooled to room temperature, consecutively washed with
saturated aqueous NaHCO3 and H2O, and dried over Na2SO4,
and the solvent was removed to yield an oily residue. Purifica-
tion of the crude product by Kugelrohr distillation (130 °C,
0.07 mbar) gave 3.0 g (83%) of 18 as a colorless oil: 1H-NMR
(CDCl3, δ) 2.49 (s, 3H, CH3), 4.55 (s, 2H, CH2), 7.16-7.28 (m,
4H, ar); IR (ATR) 705 cm-1 (C-Cl).
1-Ch lor om et h yl-3-m et h a n esu lfin yl-b en zen e (19). A
cooled (10 °C) solution of 18 (3.0 g, 17.4 mmol) in glacial acetic
acid (25 mL) was treated with a solution of 35% aqueous H 2O2
(1.8 g, 18.5 mmol) in glacial acetic acid (10 mL). After addition
was complete (5 min), the mixture was stirred at room
temperature for 2.25 h. The reaction was quenched by addition
of ice (35 g), neutralized with 25% aqueous ammonia, and
extracted with ethyl acetate (2 times). The combined organic
extracts were washed with H2O and dried over Na2SO4, and
the solvent was removed to yield an oily residue. Purification
of the crude product by Kugelrohr distillation (175 °C, 0.06
mbar) gave 2.4 g (73%) of 19 as a colorless oil: 1H-NMR
(CDCl3, δ) 2.75 (s, 3H, CH3), 4.64 (s, 2H, CH2), 7.52-7.58 (m,
3H, C4-/C5-/C6-H) 7.71 (m, 1H, C2-H); 13C-NMR (CDCl3, δ) 44.0,
45.3, 123.4, 123.5, 129.8, 131.1, 139.2, 146.4; IR (ATR) 1043
(SdO), 707 cm-1 (C-Cl).
4-[5-(4-F lu or o-p h en yl)-2-(2-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-3H-im id a zol-4-yl]-p yr id in e (22). This compound was
prepared from imidazole-2-thione 20 and 1-chloromethyl-2-
methanesulfinyl-benzene as described in the synthesis of ML
1
3163 to yield 0.23 g (54%): mp 205 °C; H-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, 2-MeS(O)-Ph C4-/C5-/C6-H), 7.95 (d, 1H, 7.2 Hz, 2-MeS-
(O)-Ph C3-H), 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. (C22H18
FN3OS2) C, H, N.
-
Biologica l Eva lu a tion : P BMC a n d Wh ole Blood As-
sa y.24 Stock solutions of test compounds were prepared by
serial dilution in DMSO (PBMC assay) or 70:30 Cremophor
EL/ethanol (whole blood assay). Mononuclear cells were
isolated from whole blood of healthy human donors by density
gradient centrifugation, and the resulting suspension was
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) and 1% DMSO
(control samples, PBMC assay) or 1% 70:30 Cremophor EL/
ethanol (control samples, PBMC assay). DMSO (PBMC assay)
or Cremophor EL and ethanol (70:30) (whole blood assay) were
present at a concentration of 1% in all samples. Biosynthesis
of cytokines was induced in all samples by stimulation with 1
µg/mL LPS (from Escherichia coli, serotype 026:B6, Sigma-
Aldrich). All samples were stimulated for 4 h (37 °C, 5% CO2).
The cell reaction was terminated in an ice bath, and the
samples were centrifuged. Concentrations of IL-1â and TNF-R
in supernatants were determined by ELISA (Beckman Coulter
Immunotech). The anti-cytokine activity of each compound was
determined by blotting the percent reduction of cytokine
concentration in test samples compared to the control samples
on semilogarithmic paper over the concentration range of test
4-[5-(4-F lu or o-p h en yl)-2-(4-m eth a n esu lfin yl-ben zylsu l-
fa n yl)-3H-im id a zol-4-yl]-p yr id in e (ML 3163). To a solution
of 1-chloromethyl-4-methanesulfinyl-benzene (0.18 g, 1.0 mmol)
in ethanol (15 mL) was added compound 20 (0.28 g, 1.0 mmol).
The mixture was heated to reflux for 4 h. The solvent was
removed, and the oily residue was extracted with ethyl acetate/
methanol (9:1). When the organic extract was left at room
temperature a precipitate formed which was filtered off to yield
1
0.18 g (41%) of ML 3163: 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

2740 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Laufer and Wagner
compounds (10-4 to 10-8 M). Results are given as IC50 values
(µM). All assays were carried out at least in duplicate.
p 38 MAP Kin a se Assa y.25 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 of BSA, 1
mM 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 transferred 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 then thoroughly washed with
ultrapure H2O and incubated with alkaline phosphatase
conjugated GAR antibody (Santa Cruz Biotechnology) for 1 h
(37 °C). Next, the 96-well plate was thoroughly washed with
ultrapure H2O and blocking buffer containing Tween 20
(0.05%), BSA (0.25%), and NaN3 (0.02%) in TBS; and, finally,
the plate was washed again with ultrapure H2O. 4-Nitro-
phenolphosphate 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 semilogarithmic paper over the concentra-
tion range of test compounds (10-4 to 10-8 M). Results are
given as IC50 values (µM).
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Opin. Ther. Pat. 2000, 10, 25-37.
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B. J .; Adams, J . L. Inhibition of p38 MAP kinase as a therapeutic
strategy. Immunopharmacology 2000, 47, 185-201.
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Foster, M.; Hele, D.; J ayyosi, Z.; Kelley, M.; Maslen, C.; Miller,
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Ack n ow led gm en t . Thanks to Merckle GmbH,
Blaubeuren, Germany, and Fond der Chemischen In-
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eyer for molecular modeling, and C. Greim for develop-
ing the in vitro test assays. We are grateful to R. J aussi
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J M011098A