Note: Descriptions are shown in the official language in which they were submitted.
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Use of CSAIDT"' Compounds for the Management of Uterine Contractions
FIELD OF THE INVENTION
The present invention relates to the treatment of inappropriate, excessive or
undesirable uterine contractions, in a mammal.
BACKGROUND OF THE INVENTION
Preterm labour is of major concern to both obstetricians and
neonatologists. It affects approximately 10% of pregnancies and results in 30%
of long term neonatal handicap and 85% of perinatal deaths. The pathogenesis
of preterm labour is incompletely understood at present, it is therefore
difficult
to both predict and treat effectively. The underlying biochemical pathways
involved in the initiation of parturition have not yet been fully elucidated.
There
are clear links between inflammatory processes and activation of the immune
system in the maternal decidua. Prostaglandins, which have a well documented
role in the initiation of labour, (Mitchell, MD., (1984) Journal of
Developmental
Physiology, 6, 107-118) and the inflammatory cytokines (Mitchell, MD., et al.
(1993) Clinical Obs. and Gynaecol, 3, 553-575) have therefore both been
implicated in the pathogenesis of labour. Interleukin-1(3 (IL-1(3) has been
demonstrated to be the pivotal cytokine in the biochemical pathway leading to
labour (Mitchell, MD., et al.(1993), supra). It has been shown to directly
stimulate the production of prostaglandins which are the terminal products of
the
inflammatory response and direct initiators of labour (Romero, R., et al.
(1992)
Am. J. Reprod. Immunol, 33, 117-123; and Kent A.S.H., et al. (1993)
Prostaglandins 46, 51-59). Levels of IL-1(3 have been shown to rise following
stimulation with bacterial endotoxin (Romero, R., et al. (1989) Am. J. Obstet
and Gynaecol., 160, 1117) which implies a mediating role for this cytokine in
the induction of labour in the presence of infection.
The production of prostaglandins is under the influence of two enzymes,
phospholipase A2 and cyclo-oxygenase (COX) alternatively known as
prostaglandin H synthase. COX has been found to be present in two forms;
type-1 COX is the constitutive form of the enzyme present in all tissues. Type-
2
COX is the inducible form of the enzyme that has been shown to be up-regulated
near term (Macchia L., et al. (1997) Biochemical and Biophysical Research
-1-
SUBSTiTUTE SHEET (RULE 26)
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Communications, 233,496-501). IL- l(3 is able to increase the production of
COX-2 in amnion (Trautman M.S., et al. (1996) Placenta, 17, 239-245).
By modulating the production of prostaglandin E2 (PGE2) the contractile
activity of the uterus may be controlled. Tocolytic agents that completely
suppress contractions are not yet available. There has been very little recent
progress in the therapeutic modalities available to treat preterm labour.
Currently a therapeutic cocktail consisting of betasypathomimetics, Non-
Steroidal Anti-inflammatory Drugs (NSAID's), and oxytocin antagonists is used
to try and suppress unwanted uterine activity.
The therapeutic cocktail does not fully address the management of preterm
labor in patients, therefore a need still exists to find suitable small
molecule
inhibitors which will help modulate the contactile activity of the uterus in a
mammal
in need thereof.
SUMMARY OF THE INVENTION
The present invention is to the novel use of a cytokine inhibitor for the
prophylatic treatment or management of excessive, undesired or inappropriate
uterine
activity in a mammal, which method comprises administering to said mammal an
effective amount of a compound which inhibits the production, transcription or
translation of a cytokine. Preferably, the cytokine is inhibited by inhibition
of the
kinase CSBP/p38/RK.
The preferred compounds for use as cytokine inhibitors are those compounds
of Formula (I) as noted herein. The preferred method of inhibition is the
inhibition of
the CSBP/p38/RK kinase pathway.
DETAILED DESCRIPTION OF THE INVENTION
The current application teaches the novel finding that CSAIDT"' compounds,
i.e. compounds that block cytokine production, can be a therapeuticially
effective
agent in modulating uterine contractile activity. Such modulation will help
treat
preterm labour in patients in need thereof.
The present invention is to the novel use of a cytokine inhibitor, in
particular
that of cytokine CSBP/p38, for the prophylactic treatment or management of
excessive, undesired or inappropriate uterine activity in a mammal.
As noted above, by modulating the production of prostaglandin E2
(PGE2) the contractile activity of the uterus may be controlled. Tocolytic
agents
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that completely suppress contractions are not yet available and there has been
very little recent progress in the therapeutic modalities available to treat
preterm
labour. The current treatment for preterm labour is a therapeutic cocktail
consisting of betasypathomimetics, Non-Steroidal Anti-inflammatory Drugs
(NSAID's), and oxytocin antagonists. This cocktail is used to try and suppress
unwanted uterine activity.
PGE2 production by fetal membranes has been shown to be inhibited by
the use of NSAID's. The efficacy in controlling uterine activity is however
dependent on the specificity of the agent used and the treatments are not
without
their side-effects. It has now been found that small molecule inhibitors which
act on cytokine synthesis may also have a similar inhibitory effect on the
production PGE2 by inhibiting cytokine driven prostaglandin production and
could therefore be useful in the treatment and prevention of preterm labour.
Therefore, another aspect of the present invention is the use of cytokine
inhibitors of the p38/CSPB/RK pathway in the treatment of both preterm and
term labour.
As will be demonstrated herein in the Example section, the CSAIDTM
compound SKF86002 has effectively been used to modulate prostaglandin and
interleukin production from gestational tissues. The compound SKF 86002 has
previously been shown to decrease the production of IL-1(3 from
lipopolysaccharide (LPS) stimulated isolated human macrophages (Perregaux D.
G., et al. (1995) Molecular pharmacology, 48, 433-442) and the production of
prostaglandin H synthase from rat basophilic leukaemic cells (Griswold D.E.,
et
al. (1987) Biochemical Pharmacology, 36, 3463-3470).
The term "inappropriate uterine activity "as used herein includes, but is not
limited to, diseases which are characterized by the presence of unwanted or
excessive uterine activity prior to the completion of normal gestational
period in a
mammal. In the case of a human, for instance, this would be approximately a 40
week gestation.
The term "excessive or increased uterine activity" as used herein is
characterized by the presence of abnormal uterine action during labour.
Abnormal
uterine action during labour is characterized by the excessive frequency,
amplitude
or duration of uterine contractions.
The term "pre-term labour" as used herein refers to the onset of labour before
37
weeks gestation. However, it is recognized that pre-term labour is a complex
syndrome due
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to many aetiological factors. See for instance, Romero, et al., Ann NY Acad.
Sci., 734,
page 414 (1994).
Labour and pre-term labour are characterized by the presence of 1)
sustained uterine contractions; 2) dilatation of the cervix; and 3) rupture of
the
fetal membranes
All three components of labour are mediated in part by the action of
inflammatory cytokines, consequently use of a CSBP inhibitor would be of use
in all these areas.
In particular, dilatation, or ripening of the cervix is affected by the
reorganization of collagen bundles in the cervix. This is believed to be under
the
control of the enzyme collagenase, produced by neutrophils (Osmers, et al.,
American Journal of Obstetrics & Gynecology, 166, pp 1455-1460 (1992)).
Neutrophils migrate into the cervix from the surrounding tissues under the
influence of chemotactic inflammatory cytokines such as IL-8. They are then
stimulated by II.-8 to release collagenase which breaks down the collagen
bundles in the cervix. IL-8 has also been shown to soften the cervix after
topical
application. See, Chwalisz, et al., Human Reproduction, 9, pp 2173-2181
(1994); and El Maradny et al., American Journal of Obstetrics & Gynecology,
171, pp 77-83 (1994). Therefore, use of a CSBP inhibitor should inhibit
unwanted cervical ripening in a mammal.
In preterm rupture of the fetal membrane (PROM) there is significant
evidence which implicates infection and inflammation in the pathogenesis of
fetal membrane rupture (French et al., Seminars in Perinatology, 20 pp 344-368
(1996)). Proteases produced in response to infection have been shown to be
involved in a reduction in fetal membrane tensile strength (McGregor, et al.,
Obstet and Gynceol, 69, pp 167-174 (1987)). These proteases are under the
influence of inflammatory cytokines such as IL-1 a and IL-1 P, and TNF-a
(Woessner, J. FASEB J, 5, pp 2145-2154 (1991). An inhibitor of these
cytokines, such as CSAIDT"' inhibitor, would therefore be able to prevent the
unwanted rupture of the fetal membranes by preventing the release of
inflammatory cytokines which stimulate the release of proteases, etc., which
in
turn destroy the integrity of the fetal membranes.
Eclampsia and pre-eclampsia are thought to result from the defective
penetration of maternal blood vessels (the spiral arteries) into the
trophoblast.
Inflammatory cytokines are important in mediating this process. Many studies
have implicated a variety of cytokine in the pathogenesis of pre-eclampsia. IL-
4
has been shown to be elevated in the sera of pre-eclamptic women (Omu-Ae et
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al., Nutrition, 11(SSuppl), pp 688-91 (1995 -Sep-Oct). Thus, pre-eclampsia/
intrauterine dystrophy is characterized by reduction of some cytokines within
the
amniotic fluid compartment and concomitant reactive augmentations of other
cytokines within the maternal and fetal organism. Stallmach et al., Reprod-
Fertil-Dev. 1995; 7(6): 1573-80 (1995). IL-6levels have also been shown to be
increased in pre-eclamptic patients (Greer et al., Obstet-Gynecol., 84(6)
(1994).
A CSAIDT"I inhibitor, may therefore favourably modify the cytokine profile in
pre-eclamptic and eclamptic patients by decreasing the severity of the
disease.
Other cytokine inhibitory compounds for use in the present invention
include, but are not limited to, those described in USSN 08/091,491, published
as
W095702575; WO95/02591; US 5,593,992; US 5,663,334; US 5,670,527;
W096/21452; W096/21654; US 5,658,903 and W096/40143; US 5,739,143;
W096/21654; W093/14081; US Patent 5,656,644; USSN 08/095,234; US
5,686,455; US 5,559,137; US 5,656,644 and W095/03297; USSN 08/481,671;
W097/25048; W097/25047; US 5,756,499 and W097/25045; US 5,716,955 and
W097/25046; W097/33883; W092/10190; W092/10498; W098/06715;
W093/14082; W095/13067; W095/31451; WO 98/07425; PCT/US98/12387;
W097/05877; PCT/US98/12828; PCT/US98/13808; PCT/US97/23638; USSN
60/068,178; USSN 60/061351; W092/12154; EP 0531901; US 5,670,503 and
W094/19350; W097/05878; W097/05877; WO97/05878; W097/16441;
W097/16426; W097/12876; US 5,717,100; W097/45412; W097/36587; and
W097/16442. Each of these references are incorporated by reference herein in
their
entirety.
Preferred compounds for use as cytokine inhibitors are those compounds of
Formula (I) as noted herein. Synthetic chemistry and methods of pharmaceutical
formulations thereof are also contained within each noted patent application.
A
description of the assay for inhibition of the cytokine specific binding
protein
(CSBP) is also found in W095/07922, whose disclosure is incorporated by
reference
in its entirety.
A preferred group of compounds for use herein are those compounds of the
formula (I):
R2
R I
,
N
R
a N
(1)
-5-
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wherein:
Rl is a pyrid-4-yl, pyrimidin-4-yl, pyridazin-4-yl, 1,2,4-triazin-5-yl, quinol-
4-yl,
isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or 1-benzimidazolyl ring, which
ring is
optionally substituted independently one to three times with Y, NHRa,
optionally
substituted C 1-4 alkyl, halogen, hydroxyl, optionally substituted C 1-4
alkoxy,
optionally substituted C 1-4 alkylthio, C 1-4 alkylsulfinyl, CH2OR 12, amino,
mono and
di- C1-6 alkyl substituted amino, or N(R10)C(O)Rb;
Y is O-Ra;
R4 is phenyl, naphth-1-yl or naphth-2-yl, or heteroaryl, which is optionally
substituted by
one or two substituents, each of which is independently selected, and which,
for a
4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is
halogen, cyano,
nitro, C(Z)NR7R17, C(Z)OR16, (CR10R20)vCOR12, SR5, SOR5, OR12, halo-
substituted-C 1-4 alkyl, C 1-4 alkyl, ZC(Z)R 12, NRlOC(Z)R16, or
(CR10R20)vNRlOR20, and which, for other positions of substitution, is halogen,
cyano, C(Z)NR13R14, C(Z)OR3, (CR10R20)m"COR3, S(O)mR3, OR3, halo-
substituted-C 1-4 alkyl, C 1-4 alkyl, (CR 10R20)m"NR l OC(Z)R3, NR 1 OS
(O)mRg,
NR10S(O)m'NR7R17, ZC(Z)R3 or (CR10R20)m"NR13R14;
v is 0, or an integer having a value of 1 or 2;
n is an integer having a value of 1 to 10;
n' is 0, or an integer having a value of 1 to 10;
m is 0, or an integer having a value of 1 or 2;
m' is an integer having a value of 1 or 2,
m" is 0, or an integer having a value of 1 to 5;
R2 is hydrogen, (CR l OR20)n' OR9, heterocyclyl, heterocyclylC 1-10 alkyl, C 1-
10 alkyl,
halo-substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl,
C3-7
cycloalkylC 1-10 alkyl, C5-7 cycloalkenyl, C5-7 cycloalkenylC 1-10 alkyl,
aryl, arylC 1_
10 alkyl, heteroaryl, heteroarylC 1-10 alkyl, (CR l OR20)nOR 11.
(CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, (CR10R20)nNR13R14,
(CR 10R20)nNO2, (CR i 0R20)nCN, (CR 10R20)n'SO2R 1 g,
(CR10R20)nS(O)m'NR13R14. (CR10R20)nC(Z)R11, (CR10R20)nOC(Z)R11,
(CR10R20)nC(Z)OR11, (CR10R20)nC(Z)NR13R14, (CR10R20)nC(Z)NR11 OR9,
(CR10R20)nNR10C(Z)R11, (CR10R20)nNR10C(Z)NR13R14,
(CR 10R20)nN(OR6)C(Z)NR 13R 14, (CR 10R20)nN(OR6)C(Z)R 11,
(CR 10R20)nC(=NOR6)R 11. (CR 10R20)nNR 1 pC(=NR 19)NR 13R 14,
(CR 10R20)nOC(Z)NR 13R 14, (CR 10R20)nNR 1 pC(Z)NR 13R 14.
(CR 10R20)nNR 1 pC(Z)OR 10, 5-(Rl g)-1,2,4-oxadizaol-3-yl or 4-(R 12)-5-(R 1
gRl9)-4,5-
dihydro-1,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, cycloalkyl,
cycloalkylalkyl,
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heteroaryl, heteroaryl alkyl, heterocyclyl and heterocyclyl alkyl groups may
be
optionally substituted;
Z is oxygen or sulfur;
Ra is a C 1_6 alkyl, aryl, ary1C 1_6 alkyl, heterocyclyl, heterocyclylC 1_6
alkyl, heteroaryl, or
heteroarylC 1_6 alkyl moiety, and wherein each of these moieties may be
optionally
substituted;
Rb is hydrogen, C 1_6 alkyl, C3_7 cycloalkyl, aryl, ary1C 1..4 alkyl,
heteroaryl,
heteroarylC 1-4 alkyl, heterocyclyl, or heterocyclylC 1-4 alkyl;
R3 is heterocyclyl, heterocyc1y1C1-10 alkyl or R8;
R5 is hydrogen, C 1-4 alkyl, C2_4 alkenyl, C2_4 alkynyl or NR7R 17, excluding
the
moieties SR5 being SNR7R17 and SOR5 being SOH;
R6 is hydrogen, a pharmaceutically acceptable cation, C1-10 alkyl, C3_7
cycloalkyl, aryl,
arylC 1-4 alkyl, heteroaryl, heteroarylC 1-4 alkyl, heterocyclyl, aroyl, or C
1-10
alkanoyl;
R7 and R 17 is each independently selected from hydrogen or C 1-4 alkyl or R7
and R 17
together with the nitrogen to which they are attached form a heterocyclic ring
of 5 to 7
members which ring optionally contains an additional heteroatom selected from
oxygen, sulfur or NR 15 ;
R8 is a C 1_ 10 alkyl, halo-substituted C 1-10 alkyl, C2-10 alkenyl, C2-10
alkynyl, C3-7
cycloalkyl, C5_7 cycloalkenyl, aryl, ary1C 1-10 alkyl, heteroaryl, heteroarylC
1-10 alkyl,
(CR10R20)nOR11, (CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, or
(CR10R20)nNR13R14 moiety; wherein the aryl, arylalkyl, heteroaryl, heteroaryl
alkyl
moieties may be optionally substituted;
R9 is hydrogen, C(Z)R 11 or optionally substituted C 1-10 alkyl, S(O)2R 18,
optionally
substituted aryl or optionally substituted arylC 1_4 alkyl;
R 10 and R20 is each independently selected from hydrogen and C 1_4 alkyl;
R 11 is hydrogen, C 1-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylC 1-
10 alkyl,
aryl, ary1C 1-10 alkyl, heteroaryl or heteroarylC 1-10 alkyl;
R12 is hydrogen or R16;
R 13 and R 14 is each independently selected from hydrogen, optionally
substituted C 1-4
alkyl, optionally substituted aryl and optionally substituted arylC 1-4 alkyl;
or together
with the nitrogen to which they are attached R13 and R14 form a heterocyclic
ring of 5
to 7 members which ring optionally contains an additional heteroatom selected
from
oxygen, sulfur or NR9;
R15 is R10 or C(Z)-C1-4 alkyl;
R16 is C1-4 alkyl, halo-substituted-C1-4 alkyl, or C3-7 cycloalkyl;
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R 18 is C 1-10 alkYl, C3-7 cycloalkyl, heterocyclyl, aryl, ary1C 1-10 alkyl,
heterocyclyl,
heterocyclylC 1-10 alkyl, heteroaryl or heteroarylC 1-10 alkyl;
R19 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl;
or a pharmaceutically acceptable salt thereof.
Suitably, RI is a substituted 4-pyridyl or 4-pyrimindyl. More suitably R1 is
substituted by an optionally substituted alkoxy, alkylthio, amino,
methylamino, NHRa, or
Y. A preferred ring placement of the R1 substituent on the 4-pyridyl
derivative is the 2-
position, such as in 2-methoxy-4-pyridyl. A preferred ring placement on the 4-
pyrimidinyl
ring is also at the 2-position, such as in 2-methoxy-pyrimidinyl.
Suitably, Ra is a C 1-6 alkyl, aryl, arylC 1-6 alkyl, heterocyclyl,
heterocyclylC 1-6
alkyl, heteroaryl, or heteroarylC 1-6 alkyl moiety, wherein each of these
moieties may be
optionally substituted.
When the substituent is Y, and Ra is aryl, it is preferably an optionally
substituted
phenyl or naphthyl. When Ra is an arylalkyl, it is preferably an optionally
substituted
benzyl or naphthylmethyl. When Ra is a heterocyclyl or heterocyclyl alkyl
moiety, the
heterocyclic portion is preferably an optionally substituted ring which is
pyrrolindinyl,
piperidine, morpholino, tetrahydropyran, tetrahydrothiopyranyl,
tetrahydrothipyran-sulfinyl,
tetrahydrothio-pyransulfonyl, pyrrolindinyl, indole, or piperonyl. It is noted
that the
heterocyclic rings herein may contain unsaturation, such as in a tryptamine
ring.
The Ra moieties, in particular the aryl, may be optionally substituted,
preferably 1
to 3 times, independently with halogen; C1-4 alkyl, such as methyl, ethyl,
propyl,
isopropyl, or t-butyl; halosubstituted alkyl, such as CF3; hydroxy; hydroxy
substituted C 1-
4 alkyl; C1-4 alkoxy, such as methoxy or ethoxy; S(O)malkyl and S(O)m aryl
(wherein m
is 0, 1, or 2); C(O)OR11, such as C(O)C1-4 alkyl or C(O)OH moieties; C(O)R11;
OC(O)Rc; -O-(CH2)s-O-, such as in a ketal or dioxyalkylene bridge; amino; mono-
and di-
C1-6 alkylsubstituted amino; N(R10)C(O)Rb; C(O)NR10R20; cyano; nitro; or an N-
heterocyclyl ring which ring has from 5 to 7 members and optionally contains
an additional
heteroatom selected from oxygen, sulfur or NR 15; optionally substituted aryl,
such as
phenyl; optionally substituted arylalkyl, such as benzyl or phenethyl;
optionally substituted
aryloxy, such as phenoxy; or optionally substituted arylalkyloxy such as
benzyloxy. The
aryl, arylalkyl, aryloxy, or arylalkyloxy optional substituents are as defined
in the "optional
substituent" definition herein.
Suitably, Rc is an optionally substituted C1-6 alkyl, optionally substituted
C3-7
cycloalkyl, optionally substituted aryl, optionally substituted arylC 1-4
alkyl, optionally
substituted heteroaryl, optionally substituted heteroarylC 1 -4 alkyl,
optionally substituted
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WO 99/18942 PCT/GB98/03015
heterocyclyl, or optionally substituted heterocyclylC 1-4 alkyl moieties; and
wherein the
optional substituents are as defined in the "optional substituent" definition
herein.
Preferably, the Ra groups include C 1-4 alkyl, benzyl, halosubstituted benzyl,
naphthylmethyl, phenyl, halosubstituted phenyl, aminocarbonylphenyl,
alkylphenyl,
cyanophenyl, alkylthiophenyl, hydroxyphenyl, alkoxyphenyl, morpholinopropyl,
piperonyl, piperidin-4-yl, alkyl substituted piperidine, such as 1-
methylpiperidine, or
2,2,6,6-tetramethylpiperidin-4-yl.
Preferably, when the substituent is NHRa then Ra is aryl, arylalkyl,
halosubstituted
arylalkyl, halosubstituted aryl, heterocyclyl alkyl, hydroxy alkyl, alkyl-l-
piperidine-
carboxylate, heterocyclyl, alkyl substituted heterocyclyl, halosubstituted
heterocyclyl, or
aryl substituted heterocyclyl. More preferably, Ra is benzyl, halosubstituted
benzyl,
naphthylmethyl, phenyl, halosubstituted phenyl, morpholinopropyl, 2-hydroxy
ethyl, ethyl-
1-piperidinecarboxylate, piperonyl, piperidin-4-yl, alkyl substituted
piperidine,
chlorotryptamine, or tetrathiohydropyranyl.
Preferably, when the R I substituent is an optionally substituted C 1-4 alkoxy
or
C1-4 alkylthio, it is preferably a methoxy group. If the alkyl chain in these
moieties is
optionally substituted it is preferably substituted by halogen, such as
fluorine, chlorine,
bromine or iodine; hydroxy, such as hydroxyethoxy; C 1-10 alkoxy, such as a
methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2; amino, mono and di-
substituted
amino, such as in the NR7R 17 group, i.e. tert-butylaminoethoxy; or where the
R7R 17 may
together with the nitrogen to which they are attached cyclize to form a 5 to 7
membered
ring which optionally includes an additional heteroatom selected from O/N/S;
C1-10 alkyl,
cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl,
isopropyl, t-butyl, etc.
or cyclopropyl methyl; or halosubstituted C1-10 alkyl, such as CF3.
Suitably, R4 is an optionally substituted phenyl. Preferably the phenyl is
substituted one or more times independently by halogen, SR5, S(O)R5, OR12,
halo-
substituted-C 1-4 alkyl, or C 1-4 alkyl, preferably in the 4-position of the
ring. More
preferably R4 is a halosubstituted phenyl, more preferably in the 4-position;
and most
preferably by fluorine.
Suitably, R2 is hydrogen, (CR l OR20)n' OR9, heterocyclyl, heterocyclylC 1-10
alkyl, C I-10 alkyl, halo-substituted C i-10 alkyl, C2-10 alkenyl, C2-10
alkynyl, C3-7
cycloalkyl, C3-7 cycloalkylCl-10 alkyl, C5-7 cycloalkenyl, C5-7 cycloalkenylCl-
10 aikyl,
aryl, ary1C 1- 10 alkyl, heteroaryl, heteroarylC 1-10 alkyl, (CR 10R20)nOR 11,
(CR10R20)nS(O)mR18, (CR10R20)nNHS(O)2R18, (CR10R20)nNR13R14.
(CR10R20)nNO2, (CR10R20)nCN. (CR10R20)n'S02R18, (CR10R20)nS(O)m'NR13R14,
(CR10R20)nC(Z)R11, (CR10R20)nOC(Z)R11, (CR10R20)nC(Z)OR11,
(CR l OR20)nC(Z)NR 13R 14, (CR 10R20)nC(Z)NR 11 OR9, (CR 10R20)nNR l OC(Z)R
11,
-9-
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(CR 10R20)nNR l OC(Z)NR 13R 14, (CR 10R20)nN(OR6)C(Z)NR 13R 14,
(CR 10R20)nN(OR6)C(Z)R 11, (CR 10R20)nC(=NOR6)R l 1.
(CR10R20)nNRIOC(=NR19)NR13R14, (CR10R20)nOC(Z)NR13R14,
(CR 10R20)nNR lOC(Z)NR 13R 14, (CR 10R20)nNR l OC(Z)OR 10, 5-(R18)-1,2,4-
oxadizaol-
3-yl or 4-(R12)-5-(R18R19)-4,5-dihydro-1,2,4-oxadiazol-3-yl group, and wherein
the aryl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaryl alkyl,
heterocyclyl and
heterocyclyl alkyl groups may be optionally substituted.
Suitably, R2 is hydrogen, optionally substituted heterocyclyl, optionally
substituted heterocyclylC 1-10 alkyl, optionally substituted C 1-10 alkyl,
optionally
substituted C3-7 cycloalkyl, optionally substituted C3-7 cycloallcylCl-10
alkyl,
(CR10R20)nC(Z)OR11, (CR10R20)nNR13R14, (CR10R20)nNHS(O)2R18.
(CR 1 UR20)nS(O)mR 18, optionally substituted aryl, optionally substituted
arylC 1-10
alkyl, (CR l OR20)nOR 11, (CR 1OR20)nC(Z)R 11, or (CR l OR20)nC (=NOR6)R 11.
More suitably, R2 is selected from hydrogen, optionally substituted C 1-10
alkyl,
optionally substituted heterocyclyl, optionally substituted heterocyclylC i-10
alkyl,
(CR10R20)nNS(O)2R18. (CR10R20)nS(O)mR18, arylCl-10 alkyl, (CR10R20)nNR13Rl4,
optionally substituted C3-7 cycloalkyl, or optionally substituted C3-7
cycloalkylC 1-10
alkyl.
Preferably R2 is hydrogen, morpholino propyl, piperidine, N-methylpiperidine,
N-benzylpiperidine, 2,2,6,6-tetramethylpiperidine, 4-aminopiperidine, 4-amino-
2,2,6,6-
tetramethylpiperidine, 4-hydroxycyclohexyl, 4-methyl-4-hydroxycyclohexyl,
4-pyrrolinindylcyclohexyl, 4-methyl-4-aminocyclohexyl, 4-methyl-4-
acetamidocyclohexyl,
4-ketocyclohexyl, 4-oxiranyl, or 4-hydroxy-4-(1-propynyl)cyclohexyl.
More preferably R2 is an optionally substituted heterocyclyl ring, optionally
substituted heterocyclylC 1-10 alkyl, optionally substituted aryl, (CR
10R20)nNR 13R 14, or
(CR10R20)nC(Z)OR11 group. Most preferably an optionally substituted
heterocyclyl ring,
or optionally substituted heterocyclylCl-10 alkyl.
When R2 is optionally substituted heterocyclyl, the ring is preferably a
morpholino,
pyrrolidinyl, or a piperidinyl group. When the ring is optionally substituted,
the
substituents may be directly attached to the free nitrogen, such as in the
piperidinyl group
or pyrrole ring, or on the ring itself. Preferably the ring is a piperidine or
pyrrole, more
preferably piperidine. The heterocyclyl ring may be optionally substituted one
to four
times independently by halogen; C 1-4 alkyl; aryl, such as phenyl; aryl alkyl,
such as benzyl
- wherein the aryl or aryl alkyl moieties themselves may be optionally
substituted (as in the
"optionally substituted" definition below); C(O)ORl 1, such as the C(O)C1-4
alkyl or
C(O)OH moieties; C(O)H; C(O)C 1-4 alkyl; hydroxy substituted C 1-4 alkyl; C 1-
4 alkoxy;
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S(O)mC 1-} alkyl (wherein m is 0, 1, or 2); and NR 1 pR20 (wherein R lO and
R20 are
independently hydrogen or Ci-4alkyl).
Preferably if the ring is a piperidine, the ring is attached to the imidazole
at the
4-position, and the substituents are directly on the available nitrogen, i.e.
a
1-formyl-4-piperidine, 1-benzyl-4-piperidine, 1-methyl-4-piperidine, or 1-
ethoxycarbonyl-
4-piperidine. If the ring is substituted by an alkyl group and the ring is
attached in the
4-position, it is preferably substituted in the 2- or 6- position or both,
such as 2,2,6,6-
tetramethyl-4-piperidine. Similarly, if the ring is a pyrrole, the ring is
attached to the
imidazole at the 3-position, and the substituents are all directly on the
available nitrogen.
When R2 is an optionally substituted heterocyclylCl-10 alkyl group, the ring
is
preferably a morpholino, pyrrolidinyl, or a piperidinyl group. Preferably the
alkyl linking
moiety is from 1 to 4, more preferably 3 or 4, and most preferably 3 carbons,
such as in a
propyl group. Preferred heterocyclyl alkyl groups include but are not limited
to,
morpholino ethyl, morpholino propyl, pyrrolidinyl propyl, and piperidinyl
propyl moieties.
The heterocyclic ring herein is also optionally substituted in a similar
manner to that
indicated above for the direct attachment of the heterocyclyl ring.
When R2 is an optionally substituted C3-7 cycloalkyl, or an optionally
substituted C3-7 cycloalkylCl-10 alkyl, the cycloalkyl group is preferably a
C4 or C6
ring, most preferably a C6 ring, which ring is optionally substituted.
The cycloalkyl ring may be optionally substituted one to three times
independently by halogen, such as fluorine, chlorine, bromine or iodine;
hydroxy;
C1-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1, or 2,
such
as methyl thio, methylsulfinyl or methyl sulfonyl; S(O)m aryl; cyano; nitro;
amino;
mono and di-C 1-10 alkyl substituted amino, such as in the NR7R17 group,
wherein
R7 and R 17 are as defined in Formula (I), or where the R7R 17 may cyclize
together
with the nitrogen to which they are attached to form a 5 to 7 membered ring
which
optionally includes an additional heteroatom selected from oxygen, sulfur or
NR 15 ;
N(R 10)C(O)X 1, wherein X l is C 1-4 alkyl, aryl or arylC 14 alkyl; C 1-10
alkyl, such
as methyl, ethyl, propyl, isopropyl, or t-butyl; optionally substituted alkyl
wherein the
substituents are halogen, (such as CF3), hydroxy, nitro, cyano, amino, mono &
di-
C1-10 alkyl substituted amino; NR7R17; S(O)m alkyl and S(O)m aryl, wherein m
is
0, 1 or 2; optionally substituted alkylene, such as ethylene or propylene;
optionally
substituted alkyne, such as ethyne; C(O)OR11; the group Re; C(O)H; =0; =N-
OR11;
N(H)-OH (or substituted alkyl or aryl derivatives thereof on the nitrogen or
the oxime
moiety); N(ORd)-C(O)-R6 ; an optionally substituted aryl, such as phenyl; an
optionally substituted ary1C 14 alkyl, such as benzyl or phenethyl; an
optionally
substituted heterocyclyl or heterocyclylC 1-4 alkyl, and further wherein these
aryl,
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arylalkyl, heterocyclyl, and heterocyclyl alkyl moieties are optionally
substituted one
to two times by halogen, hydroxy, C 1-10 alkoxy, S(O)m alkyl, cyano, nitro,
amino,
mono and di-C 1-10 alkyl substituted amino, alkyl, or halosubstituted alkyl.
Suitably Rd is hydrogen, a pharmaceutically acceptable cation, aroyl or a C1-
10
alkanoyl group.
Suitably Re is a 1,3-dioxyalkylene group of the formula -O-(CH2)s-O-, wherein
s is
1 to 3, preferably s is 2 yielding a 1,3-dioxyethylene moiety, or ketal
functionality.
Suitably R6'is NR19,R20 -9 C1-6 alkyl; halosubstituted CI-6 alkyl; hydroxy
substituted C 1-6 alkyl; C2-6 alkenyl; or an aryl or heteroaryl optionally
substituted by
halogen, C 1_6 alkyl, halosubstituted C 1-6 alkyl, hydroxyl, or C 1-6 alkoxy.
Suitably R19, is H or C1-6 alkyl.
Suitably R20, is H, C 1-6 alkyl, aryl, benzyl, heteroaryl, C 1-6 alkyl
substituted by
halogen or hydroxyl, or phenyl substituted by a member selected from the group
consisting
of halo, cyano, C 1-12 alkyl, C 1-6 alkoxy, halosubstituted C 1-6 alkyl, C 1-6
alkylthio, C 1-6
alkylsulphonyl, or C 1-6 alkylsulfinyl; or R 19, and R20'may together with the
nitrogen to
which they are attached form a ring having 5 to 7 members, which members of
the ring
may be optionally replaced by a heteroatom selected from oxygen, sulfur or
nitrogen. The
ring may be saturated or contain more than one unsaturated bond. Preferably
R6= is
NR19,R20, and R19, and R20, are preferably hydrogen.
When the R2 cycloalkyl moiety is substituted by a NR7R 17 group, or NR7R 17
C1-10 alkyl group, and the R7 and R17 are as defined in Formula (I), the
substituent is
preferably an amino, amino alkyl, or an optionally substituted pyrrolidinyl
moiety.
A preferred ring placement on the cycloalkyl moiety is the 4-position, such as
in a
C6 ring. When the cycloalkyl ring is di-substituted it is preferably di-
substituted at the 4
position, such as in:
R" 82'
wherein R 1' and R2' are independently the optional substituents indicated
above for R2:
Preferably, Rl' and R2' are hydrogen, hydroxy, alkyl, substituted alkyl,
optionally
substituted alkyne, aryl, arylalkyl, NR7R17, and N(RIO)C(O)R11. Suitably, the
alkyl is a
C 1-4 alkyl, such as methyl, ethyl, or isopropyl; NR7R17 and NR7R 17 alkyl,
such as
amino, methylamino, aminomethyl, aminoethyl; substituted alkyl such as in
cyanomethyl,
cyanoethyl, nitroethyl, pyrrolidinyl; aryl such as in phenyl; arylalkyl, such
as in benzyl;
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optionally substituted alkyne, such as ethyne or propynyl; or together R 1'
and R2' are a
keto functionality.
Preferably R4 is an optionally substituted phenyl; R I is an optionally
substituted 4-
pyridyl or 4-pyrimidinyl; and R2 is an optionally substituted heterocyclyl,
heterocyclyl C 1-
4 alkyl, a cycloalkyl or a cycloalkyl alkyl. More preferably R2 is an
optionally substituted
C4 or C6 cycloalkyl, cyclopropyl methyl, morpholinyl butyl, morpholinyl
propyl,
morpholinyl ethyl, cyclohexyl substituted by methyl, phenyl, benzyl, amino,
acetamide,
aminomethyl, aminoethyl, cyanomethyl, cyanoethyl, hydroxy, nitroethyl,
pyrrolidinyl,
ethynyl, 1-pr.opynyl, =0, -O-(CH2)20-, =NOR11, wherein R1 I is hydrogen, alkyl
or aryl,
NHOH, or N(OH)-C(O)-NH2; or R2 is morpholinyl propyl, aminopropyl,
piperidinyl, N-
benzyl-4-piperidinyl, N-methyl-4-piperidinyl, 2,2,6,6-tetramethypiperidinyl,
substituted
piperidine, such as 1-formyl-4-piperidine, or a 1-ethoxycarbonyl-4-piperidine.
More
preferably R 1 is a 4-pyridyl or 4-pyrimidinyl subtituted by Y, NHRa, or C 1-4
alkoxy.
In all instances herein where there is an alkenyl or alkynyl moiety as a
substituent group, the unsaturated linkage, i.e., the vinylene or acetylene
linkage is
preferably not directly attached to the nitrogen, oxygen or sulfur moieties,
for instance
in OR3, or for certain R2 moieties.
As used herein, "optionally substituted", unless specifically defined, shall
mean
such groups as halogen, such as fluorine, chlorine, bromine or iodine;
hydroxy;
hydroxy substituted C1-10 alkyl; C1-10 alkoxy, such as methoxy or ethoxy;
S(O)m
alkyl, wherein m is 0, 1 or 2, such as methylthio, methylsulfinyl or
methylsulfonyl;
amino, mono and di-C 1-10 alkyl substituted amino, NR7R 17 wherein the R7R 17
may
together with the nitrogen to which they are attached cyclize to form a 5 to 7
membered ring which optionally includes an additional heteroatom selected from
O/N/S; C1-10 alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl,
ethyl,
propyl, isopropyl, t-butyl, etc. or cyclopropyl methyl; halosubstituted C 1-10
alkyl,
such CF3; an optionally substituted aryl, such as phenyl, or an optionally
substituted
arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties may also
be
substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; C
1-10
alkoxy; S(O)m alkyl; amino, mono and di- C1-10 alkyl substituted amino, such
as in
the NR7R 17 group; C 1-10 alkyl; or halosubstituted alkyl, such as CF3.
Suitable pharmaceutically acceptable salts are well known to those skilled in
the art
and include basic salts of inorganic and organic acids, such as hydrochloric
acid,
hydrobromic acid, sulfuric acid, phosphoric acid, methane sulphonic acid,
ethane sulphonic
acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic
acid, succinic acid,
fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and
mandelic acid.
In addition, pharmaceutically acceptable salts of compounds of Formula (I) may
also be
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formed with a pharmaceutically acceptable cation, for instance, if a
substituent group
comprises a carboxy moiety. Suitable pharmaceutically acceptable cations are
well knowri
to those skilled in the art and include alkaline, alkaline earth, ammonium and
quaternary
ammonium cations.
The following terms, as used herein, refer to:
= "halo" or "halogens", include the halogens: chloro, fluoro, bromo and iodo.
="C1-10alkyl" or "alkyl" - both straight and branched chain radicals of 1 to
10
carbon atoms, unless the chain length is otherwise limited, including, but not
limited to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-
butyl, n-pentyl and
the like.
= The term "cycloalkyl" is used herein to mean cyclic radicals, preferably of
3 to 8
carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl,
and the like.
= The term "cycloalkenyl" is used herein to mean cyclic radicals, preferably
of 5 to
8 carbons, which have at least one double bond including but not limited to
cyclopentenyl,
cyclohexenyl, and the like.
= The term "alkenyl" is used herein at all occurrences to mean straight or
branched
chain radical of 2-10 carbon atoms, unless the chain length is limited
thereto, including, but
not limited to ethenyl, I-propenyl, 2-propenyl, 2-methyl-l-propenyl, I-
butenyl, 2-butenyl
and the like.
= "aryl" - phenyl and naphthyl;
="heteroaryl" (on its own or in any combination, such as "heteroaryloxy", or
"heteroarylalkyl") - a 5-10 membered aromatic ring system in which one or more
rings
contain one or more heteroatoms selected from the group consisting of N, 0 or
S, such as,
but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline,
isoquinoline,
quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole,
imidazole, or
benzimidazole.
="heterocyclyl" (on its own or in any combination, such as
"heterocyclylalkyl") - a
saturated or partially unsaturated 4-10 membered ring system in which one or
more rings
contain one or more heteroatoms selected from the group consisting of N, 0, or
S; such as,
but not limited to, pyrrolidine, piperidine, piperazine, morpholine,
tetrahydropyran, or
imidazolidine.
= The term "aralkyl" or "heteroarylalkyl" or "heterocyclylalkyl" is used
herein to
mean C1-4 alkyl as defined above attached to an aryl, heteroaryl or
heterocyclyl moiety as
also defined herein unless otherwise indicated.
="sulfinyl" - the oxide S(O) of the corresponding sulfide, the term "thio"
refers to
the sulfide, and the term "sulfonyl" refers to the fully oxidized S(0)2
moiety.
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="aroyl" - a C(O)Ar, wherein Ar is a phenyl, naphthyl, or aryl alkyl
derivative
such as defined above, such groups include but are not limited to benzyl and
phenethyl.
="alkanoyl" - a C(O)C1-10 alkyl wherein the alkyl is as defined above.
For the purposes herein the "core" 4-pyrimidinyl moiety for R 1 or R2 is
referred to
as the formula:
N
~
N--
It is recognized that the compounds for use in the present invention may exist
as stereoisomers, regioisomers, or diastereiomers. These compounds may contain
one or more asymmetric carbon atoms and may exist in racemic and optically
active
forms. All of these compounds are included within the scope of the present
invention.
As noted previously, methods of making these compounds can be found in their
respective patent applications as noted above.
Specifically exemplified compounds of Formula (I) include:
1-[3-(4-Morpholinyl)propyl]-4-(4-fluorophenyl )-5-(4-pyridyl)imidazole;
1-(3-Chloropropyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Azidopropyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Aminopropyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Methylsulfonamidopropyl)-4-(4-fluorophenyl)-5-(4-pyridy l)imidazole;
1-[3-(N-Phenylmethyl)aminopropyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(N-Phenylmethyl-N-methyl)aminopropyl]-4-(4-fluorophenyl)-5-(4-
pyridyl)imidazole;
1-[3-(1-Pyrrolidinyl)propyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Diethylaminopropyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(1-Piperidinyl)propyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(Methylthio)propyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[2-(4-Morpholinyl)ethyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(4-Morpholinyl)propyl]-4-(3-methylthiophenyl)-5-(4-pyridyl)imidazole;
(+/-)-1-[3-(4-Morpholinyl)propyl]-4-(3-methylsulfinylphenyl)-5-(4-
pyridyl)imidazole;
1-[3-(N-Methyl-N-benzyl)aminopropyl]-4-(3-methylthiophenyl)-5-(4-
pyridyl)imidazole;
1-[3-(N-Methyl-N-benzyl)aminopropyl]-4-(3-methylsulfinylphenyl)-5-(4-
pyridyl)imidazole;
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1-[4-(Methylthio)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[4-(Methylsulfinyl)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(Methylthio)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
(+/-)-1-[3-(Methylsulfinyl)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[2-(Methylthio)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[2-(Methylsulfinyl)phenyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[4-(4-Morpholinyl)butyl]-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Cyclopropyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Isopropyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Cyclopropylmethyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1 -tert-B utyl-4-(4-fluorophenyl )-5-(4-pyridyl)imidazole;
1-(2,2=Diethoxyethyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Formylmethyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Hydroxyiminylmethyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-Cyanomethyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(4-Morpholinyl)propyl)-4-(4-fluorophenyl)-5-(2-methylpyridin-4-
yl)imidazole;
4-(4-Fluorophenyl)-1-[3-(4-morpholinyl)propyl]-5-(2-chloropyridin-4-
yl)imidazole;
4-(4-Fluorophenyl)-1-[3-(4-morpholinyl)propyl]-5-(2-amino-4-pyridyl)imidazole;
1-(4-Carboxymethyl)propyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(4-Carboxypropyl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Carboxymethyl )ethyl-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
1-(3-Carboxy)ethyl-4-(4-fluorophenyl )-5-(4-pyridyl)imidazole;
1-(1-Benzylpiperidin-4-yl)-4-(4-fluorophenyl)-5-(4-pyridyl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-[3-(4-
morpholinyl)propyl]imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(1-benzylpiperidin-4-
yl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(2-propyl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(cyclopropylmethyl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(1-carboxyethyl-4-
piperidinyl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
1-Methyl-4-phenyl-5-(4-pyridyl)imidazole;
1-Methyl-4-(3-chlorophenyl)-5-(4-pyridinyl)imidazole;
1-Methyl-4-(3-methylthiophenyl)-5-(4-pyridyl)imidazole;
(+/-)-1-Methyl-4-(3-methylsulfinylphenyl)-5-(4-pyridyl)imidazole;
(+/-)-4-(4-Fluorophenyl)-1-[3-(methylsulfinyl)propyl]-5-(4-
pyridinyl)imidazole;
4-(4-Fluorophenyl)-1-[(3-methylsulfonyl)propyl]-5-(4-pyridinyl)imidazole;
1-(3-Phenoxypropyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
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1- [3-(Phenylthio)propyl]-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
1-[3-(4-Morpholinyl)propyl]-4-(4-fluorophenyl)-5-(4-quinolyl)imidazole;
(+/-)-1-(3-Phenylsulfinylpropyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
1-(3-Ethoxypropyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
1-(3-Phenylsulfonylpropyl-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
1-[3-(4-Morpholinyl)propyl]-4-(3-chlorophenyl)-5-(4-pyridyl)imidazole;
1-[3-(4-Morpholinyl)propyl]-4-(3,4-dichlorophenyl)-5-(4-pyridyl)imidazole;
4-[4-(4-Fluorophenyl)-1-[3-(4-morpholinyl)propyl]-5-(pyrimid-2-one-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[2-(methylthio)-4-pyrimidinyl]-1-[3-(4-morpholinyl)propyl
]-
imidazole;
(+/-)-4-(4-Fluorophenyl)-5-[2-(methylsulfinyl)-4-pyrimidinyl]-1-[3-(4-
morpholinyl)-
propyl]imidazole;
(E)-1-(1-Propenyl)-4(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
1-(2-Propenyl )-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
5-[(2-N,N-Dimethylamino)pyrimidin-4-yl]-4-(4-fluorophenyl)-1-[3-(4-
morpholinyl)-
propyl]imidazole;
1-[3-(4-Morpholinyl)propyl]-5-(4-pyridinyl)-4-[4-
(trifluoromethyl)phenyl]imidazole;
1-[3-(4-Morpholinyl)propyl]-5-(4-pyridinyl)-4-[3-
(trifluoromethyl)phenyl]imidazole;
1-(Cyclopropylmethyl)-4-(3,4-dichlorophenyl)-5-(4-pyridinyl)imidazole;
1-(Cyclopropylmethyl)-4-(3-trifluoromethylphenyl)-5-(4-pyridinyl)imidazole;
1-(Cyclopropylmethyl)-4-(4-fluorophenyl)-5-(2-methylpyrid-4-yl)imidazole;
1-[3-(4-Morpholinyl)propyl]-5-(4-pyridinyl)-4-(3,5-bistrifluoromethylphenyl)-
imidazole;
5-[4-(2-Aminopyrimidinyl)]-4-(4-fluorophenyl)-1-(2-carboxy-2,2-
dimethylethyl)imidazole;
1-(1-Formyl-4-piperidinyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(1-methyl-4-
piperidinyl)imidazole;
1-(2,2-Dimethyl-3-morpholin-4-yl)propyl-4-(4-fluorophenyl)-5-(2-amino-4-
pyrimidinyl)imidazole;
4-(4-Fluorophenyl)-5-(4-pyridyl)-1-(2-acetoxyethyl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(1-benzylpyrrolin-3-
yl)imidazole;
5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyi)-1-(2,2,6,6-tetramethylpiperidin-4-
yl)imidazole;
5-[4-(2-N-Methylamino)pyrimidinyl]-4-(4-fluorophenyl)-1-(4-N-methylpiperidine)-
imidazole;
5-[4-(2-N-Methylamino)pyrimidinyl]-4-(4-fluorophenyl)-1-(4-N-morpholino-l-
propyi)imidazole;
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5-[4-(2-N-Methylamino)pyrimidinyl]-4-(4-fluorophenyl)-1-(4-
piperidine)imidazole;
5-[(2-Ethylamino)pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-methylpiperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[2-(isopropyl)aminopyrimidiny-4-yl]-1-(1-methylpiperidin-
4-
yl)imidazole;
5-(2-Acetamido-4-pyrimidinyl)-4-(4-fluorophenyi)-1-(4-N-morpholino-l-propyl)-
imidazole;
5-(2-Acetamido-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(1-methyl-4-
piperidinyl)imidazole;
5-[4-(2-N-Methylthio)pyrimidinyl]-4-(4-fluorophenyl )-1-(4-
piperidine)imidazole;
4-(Fluorophenyl)-1-(methyl-4-piperidinyl)-5-(2-methylthio-4-
pyrimidinyl)imidazole;
4-(Fluorophenyl)-1-(methyl-4-piperidinyl)-5-(2-methysulfinyl-4-
pyrimidinyl)imidazole;
1-tert-Butyl-4-(4-fluorophenyl)-5-(2-methysulfinyl-4-pyrimidinyl)imidazole;
5-[4-(2-Aminopyrimidinyl)]-4-(4-fluorophenyl)-1-(2,2,6,6-tetramethyl-4-
piperidinyl)imidazole;
5-[4-(2-N-Methylamino-4-pyrimidinyl)]-4-(4-fluorophenyl)-1-(2,2,6,6-
tetramethyl-4-
piperidine)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(tetrahydro-4-
thiopyranyl)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(tetrahydro-4-
pyranyl)imidazole;
5-(2-Methylamino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(2-cyanoethyl)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(tetrahydro-4-
sulfinylpyranyl)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(tetrahydro-4-sulfonylpyranyl)-
imidazole;
5-(2-Methylamino-4-pyrimidinyl)-4-(4-fluorophenyl)- 1-(2,2,2-trifluoroethyl-4-
piperidinyl)imidazole;
5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(trifluoroacetyl-4-piperidinyl)-
imidazole;
5-(4-Pyridyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)irnidazole;
5-(4-Pyridyl)-4-(4-fluorophenyl)-1-(1-t-butoxy carbonyl-4-
piperidinyl)imidazole;
4-(4-Fluorophenyl)-5-(4-pyridyl)imidazole;
4-(4-Fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole;
4-(4-Fluorophenyl)-5-(2-methylthio-pyrimidin-4-yl)imidazole;
5-[(2-Benzylamino)pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-methylpiperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-1-(1-methylpiperidin-4-yl)-5-[ 2-(4-tetrahydrothio-
pyranyl)aminopyrimidin-4-yl]imidazole;
5-[(2-(3-Chlorobenzylamino))pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-methyl-
piperidin-
4-yl)imidazole;
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5-[(2-(1-Naphthylmethylamino))pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-
methylpiperidin-4-yl)imidazole;
5-[(2-(1-Benzyl-4-piperidinylamino))pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-
methylpiperidin-4-yl)imidazole;
4-(4-Fluorophenyl)-1-(1-methylpiperdin-4-yl)-5-[2-[3-(morpholino)propyl]-
aminopyrimidiny-4-yl]imidazole;
5-[2-[(3-Bromophenyl)amino]pyrimidin-4-ylJ-4-(4-fluorophenyl)-1-(1-
methylpiperidin-
4-yl)imidazole;
5-[(2-(Piperonylamino)pyrimidin-4-ylJ-4-(4-fluorophenyl)-1-(1-methylpiperidin-
4-
yl)imidazole;
5-[(2-(4-Piperdinylamino))pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-
methylpiperidin-4-
yl)imidazole;
5-[(2-(5-Chlorotryptamino))pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(1-
methylpiperidin-4-
yl)imidazole;
5-[(2-(2,2,6,6-Tetramethylpiperidin-4-yl)amino)pyrimidin-4-yl]-4-(4-
fluorophenyl)-1-(1-
methylpiperidin-4-yl)imidazole;
5-[(2-(1-Ethoxycarbonyl))piperdin-4-yl]aminopyrimidin-4-yl]-4-(4-fluorophenyl)-
1-(1-
methylpiperidin-4-yl)imidazole;
5-[2-(Phenylamino)pyrimidin-4-yl]=4-(4-fluorophenyl)-1-(4-
oxocyclohexyl)imidazole;
5-[4-(2-Phenylamino)pyrimidin-4-yl]-4-(4-fluorophenyl)-1-(4-hydroxycyclohexyl)-
imidazole;
4-(4-Fluorophenyl)-1-(1-methylpiperdin-4-yl)-5-[(2-phenylamino)pyrimidin-4-yl]-
imidazole;
4-(4-Fluorophenyl)- 1-(2,2,6,6-tetramethylpiperidin-4-yl)-5-[(2-
phenylamino)pyrimidin-
4-yl]imidazole;
4-(4-Fluorophenyl)-5-[(2-phenylamino)pyrimidin-4-yl ]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[2-[3-(imidazol-l-yl)propyl]aminopyrimidin-4-yl]-1-[(1-t-
butoxycarbonyl)piperidin-4-yl]imidazole;
4-(4-Fluorophenyl)-5-[2-[3-(imidazol-l-yl)propyl]aminopyrimidin-4-yl]-1-
(piperidin-4-
yl)imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-anilino-4-pyridinyl)imidazole;
4-(4-Thiomethylphenyl)-5-[(2-phenylamino)pyrimidin-4-yl]-1-(1-ethoxy
carbonylpiperidin-4-yl)imidazole;
4-(4-Thiomethylphenyl)-5-[(2-phenylamino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Methylsulfinylphenyl)-5-[(2-phenylamino)pyrimidin-4-ylJ-1-(piperidin-4-
yl)imidazole;
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4-(4-Fluorophenyl)-5-[(2-(4-fluorophenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-fluorophenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(2-fluorophenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(4-benzyloxyphenyl)amino)pyrimidin-4-yl]-1-(1-
ethoxycarbonylpiperidin-4-yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-benzyloxyphenyl)amino)pyrimidin-4-yi]-1-(1-
ethoxycarbonylpiperidin-4-yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-trifluoromethylphenyl)amino)pyrimidin-4-yl]-1-
(piperidin-
4-yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3,4-difluorophenyl)amino)pyrimidin-4-yl]-1-
(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(4-hydroxyphenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-hydroxyphenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(4-methoxyphenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-methoxyphenyl)amino)pyrimidin-4-yl]-1-(piperidin-4-
yl)imidazole;
4-(.4-Fluorophenyl)-5-[(2-(2-methoxyphenyl)amino)pyrimidin-4-yl]-1-(piperidin-
4-
yl)imidazole;
4-(4-Fluorophenyl)-5-[(2-(3-fluoro-2-methylphenyl)amino)pyrimidin-4-yl]-1-
(piperidin-4-yl)imidazole;
1-(4-Oxocyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl]
imidazole;
cis -1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5- [(2-methoxy)pyrimidin-4-
yl]imidazole;
trans-l-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-
yl]imidazole;
1-(4-Oxocyclohexyl)-4-(4-fluorophenyl)-5-[(2-methylthio)pyrimidin-4-yl]-
imidazole;
trans-l-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methylthio)pyrimidin-4-
yllimidazole;
1-(4-Oxocyclohexyl)-4-(4-fluorophenyl)-5-[(2-hydroxy)pyrimidin-4-yl]imidazole;
1-(4-Oxocyclohexyl )-4-(4-fluorophenyl )-5-[(2-isopropoxy)pyrimidi n-4-
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yl]imidazole;
1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-isopropoxy)pyrimidin-4-
yl]imidazole;
trans-l-(4-Hydroxy-4-methylcyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)-
pyrimidin-4-yl]imidazole;
cis- 1-(4-Hydroxy-4-methylcyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)-
pyrimidin-4-yl]imidazole;
trans-1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-ethoxy)pyrimidine-4-
yl]imidazole;
1-Cycioheptyl-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole;
1-Cyclopropyl-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole;
1-Cyciobutyl-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole;
1-Cyclopentyl-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole;
1-Cyclohexyl-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole;
trans-5-[4-(2-Methoxy)pyrimidinyl]-4-(4-fluorophenyl)-1-[4-(2-
tetrahydropyranyl)-
oxycyclohexyl] imidazole;
1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-hydroxy)pyrimidin-4-
yl]imidazole
cis-1-[(4-Hydroxy-4-methylcyclohexyl)]-4-(4-fluorophenyl)-5-(2-methoxy-4-
pyrimidinyl)imidazole;
trans- 1-[(4-Hydroxy-4-methylcyclohexyl)]-4-(4-fluorophenyl)-5-(2-methoxy-4-
pyrimidinyl)imidazole;
trans-l-(4-Aminocyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-
pyrimidinyl)imidazole;
trans-4-(4-Fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl]-1-[4-
((methylthio)methoxy)cyclohexyl]imidazole;
cis- 1-(4-Aminocyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-
pyrimidinyl)imidazole;
trans-l-[(4-Butyryloxy)cyclohexyl]-4-(4-fluorophenyl)-5-[(2-methoxypyrimidin)-
4-
yl]imidazole;
trans-4-(4-Fluorophenyl)-1-[4-(2-(N,N-dimethylamino)ethoxy)cyclohexyl]-5-[(2-
methoxy)pyrimidin-4-yl]imidazole hydrochloride;
cis/trans-l-(4-Hydroxy-4-hydroxymethylcyclohexyl)-4-(4-fluorophenyl)-5-[(2-
methoxy)pyrimidin-4-yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-phenoxypyridin-4-yl)imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(4-methoxyphenoxy)pyridin-4-
yl]imidazole;
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1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(4-fluorophenoxy)pyridin-4-y1
]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-methoxyphenoxy)pyrimidin-4-yl]-
imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-fluorophenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5- [2-(4-aminocarbonylphenoxy)pyrimidin-
4-yl]-
imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-ethylphenoxy)pyrimidin-4-yl]
imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-benzyloxyphenoxy)pyrimidin-4-yl]-
imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-cyanophenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-hydroxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[2-(phenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(2,6-dimethylphenoxy)pyridin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-methylphenoxy)pyridin-4-yl]
imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-chlorophenoxy)pyridin-4-
yl]imidazole;
1-[3-(N-Morpholino)propyl]-4-(4-fluorophenyl)-5-[2-(phenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(3-methoxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-phenylphenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-phenoxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5- [2-(3-hydroxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(3-(N-Morpholino)propyl)-4-(4-fluorophenyl )-5-[2-(4-fluorophenoxy)pyrimidin-
4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(2-hydroxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl )-5-[2-(3,4-methylenedioxyphenoxy)-
pyrimidin-4-yl ] imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(3-fluorophenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(2-fluorophenoxy)pyrimidin-4-
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yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(2-methoxyphenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(3-trifluoromethylphenoxy)pyrimidin-
4-
yl]imidazole;
1-(Piperidin-4-yl)-4(4-fluorophenyl)-5-[2-(3,4-difluorophenoxy)pyrimidin-4-
yl]imidazole;
1-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-methylsulfonylphenoxy)pyrimidin-
4-
yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-thiophenoxypyrimidin-4-yl)imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(1-methyltetrazol-5-ylthio)pyridin-4-
yl]imidazole
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[(2-acetamidophenoxy)pyrimidin-4-
yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[(3-propionamidophenoxy)pyrimidin-4-
yl]imidazole;
1-Cyclohexyl-4-(4-fluorophenyl)-5-[(2-phenoxy)pyrimidin-4-yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(2,6-dimethylphenoxy)pyrimidin-4-
yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(2-methylphenoxy)pyrimidin-4-
yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(2,6-dimethyl-4-chlorophenoxy)-
pyrimidin-4-yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(indol-4-yloxy)pyrimidin-4-
yl]imidazole;
1-Cyclopropyl-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yi)imidazole;
1-Isopropyl-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)imidazole;
1-Cyclopentyl-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)imidazole;
1-(1-Hydroxyprop-2-yl)-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)-
imidazole;
3-[4-(4-Fluorophenyl)-5-[(2-phenoxy)pyrimidin-4-yl]imidazol-l-
yl]propionitrile;
(R)-(1-Hydroxy-3-phenylprop-2-yl)-4-(4-fluorophenyl)-5-(2-phenoxy)pyrimidin-4-
yl)imidazole;
(S)-(1-Hydroxy-3-phenylprop-2-yl)-4-(4-fluorophenyl)-5-(2-phenoxy)pyrimidin-4-
yl)imidazole;
1-(1-Phenoxyprop-2-yl)-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)-
imidazole;
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1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(3-piperazin-1-ylacetamido)phenoxy-
pyrimidin-4-yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-[2-(3-piperazin-1-ylamidophenoxy)-
pyrimidin-4-yl]imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-isopropoxy-4-pyrimidinyl)imidazole;
1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)imidazole;
5-(2-Hydroxy-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
5-(2-Methoxy-4-pyridinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
5-(2-iso-Propoxy-4-pyridinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
5-(2-Methylthio-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
5-(2-Methylthio-4-pyrimidinyl)-4-(4-fluorophenyl)-1-[(1-methyl-4-
piperidinyl]imidazole;
5-(2-Ethoxy-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole;
1-(1-Ethylcarboxylpiperidin-4-yl)-3-(4-thiomethylphenyl)-5-[2-(thiomethyl)-
pyrimidin-4-yl]imidazole;
1-(1-Ethylcarbonylpiperidin-4-yl)-4-(4-methylsulfinylphenyl)-5-[2-
methylsulfinyl-
pyrimidin-4-yl]imidazole; or
pharmaceutically acceptable salts thereof.
METHODS OF TREATMENT
The cytokine inhibitor compounds, in particular those of Formula (I) or a
pharmaceutically acceptable salt, can be used in the manufacture of a
medicament for the
prophylactic treatment or management of excessive, undesired or inappropriate
uterine
activity in a mammal, preferably a human, which activity is exacerbated or
caused by
excessive or unregulated cytokine production by such mammal.
The cytokine inhibitors of the p38/CSPB pathway are capable of inhibiting
proinflammatory cytokines, such as II.-1, IL-6, IL-8 and TNF.
The cytokine inhibitors of the p38/CSPB pathway are administered in an amount
sufficient to inhibit the cytokine, in particular IL-1, IL-6, IL-8 or TNF,
production such that
it is regulated down to normal levels, or in some cases to subnormal levels,
so as to
ameliorate or prevent the disease state. Abnormal levels of IL-1, IL-6, IL-8
or TNF, for
instance in the context of the present invention, constitute: (i) levels of
free (not cell bound)
IL-1, IL-6, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) any
cell associated
IL-1, IL-6, IL-8 or TNF; or (iii) the presence of IL- 1, IL-6, IL-8 or TNF
mRNA above
basal levels in cells or tissues in which IL-1, IL-6, IL-8 or TNF,
respectively, is produced.
As used herein, the term "inhibiting the production of IL-1 (IL-6, IL-8 or
TNF)"
refers to:
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a) a decrease of excessive in vivo levels of the cytokine (IL-1, IL-6, IL-8 or
TNF) in
a human to normal or sub-normal levels by inhibition of the in vivo release of
the cytokine -
by all cells, including but not limited to monocytes or macrophages;
b) a down regulation, at the genomic level, of excessive in vivo levels of the
cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels;
c) a down regulation, by inhibition of the direct synthesis of the cytokine
(IL-1,
IL-6, IL-8 or TNF) as a postranslational event; or
d) a down regulation, at the translational level, of excessive in vivo levels
of the
cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels.
As =used herein, the term "cytokine" refers to any secreted polypeptide that
affects
the functions of cells and is a molecule which modulates interactions between
cells in the
immune, inflammatory or hematopoietic response. A cytokine includes, but is
not limited
to, monokines and lymphokines, regardless of which cells produce them. For
instance, a
monokine is generally referred to as being produced and secreted by a
mononuclear cell,
such as a macrophage and/or monocyte. Many other cells, however, also produce
monokines, such as natural killer cells, fibroblasts, basophils, neutrophils,
endothelial cells,
brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-
lymphocytes.
Lymphokines are generally refenred to as being produced by lymphocyte cells.
Examples
of cytokines include, but are not limited to, Interleukin-1 (IL-1),
Interleukin-6 (IL-6),
Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-(x) and Tumor Necrosis
Factor
beta (TNF-B).
As used herein, the term "cytokine interfering" or "cytokine suppressive
amount"
refers to an effective amount of a compound of Formula (I) which will cause a
decrease in
the in vivo levels of the cytokine to normal or sub-normal levels, when given
to a patient
for the prophylaxis or treatment of a disease state which is exacerbated by,
or caused by,
excessive or unregulated cytokine production.
The MAP kinase family, alternatively termed CSBP, p38, or RK, has been
identified
independently by several laboratories. Activation of this novel protein kinase
via dual
phosphorylation has been observed in different cell systems upon stimulation
by a wide
spectrum of stimuli, such as physiochemical stress and treatment with
lipopolysaccharide or
proinflammatory cytokines such as IL-1 and TNF. The cytokine biosynthesis
inhibitors for
use in the present invention, for instance compounds of Formula (I), have been
determined
to be potent and selective inhibitors of this CSBP/p38/RK kinase activity.
These inhibitors
are of aid in determining the signaling pathways involvement in inflammatory
responses. In
particular, for the first time a definitive signal transduction pathway can be
prescribed to the
action of lipopolysaccharide in cytokine production in macrophages. The CSBP
protein is
described in detail in Patent Application USSN 08/123175 Lee et al., filed
September 1993;
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WO 99/18942 PCT/GB98/03015
Lee et al., PCT/US94/10529 filed 16 September 1994; USSN 08/605002, filed 15
April
1996; USSN 08/469421, USSN 08/468902; and Lee et al., Nature 300, n(72), 739-
746
(Dec. 1994). Inhibitors of the variants and homologs of the CSBP protein are
also
considered as another aspect of the present invention. One such variant is the
p38 beta
protein, as described in Jiang, Y., et al., J. Biol. Chem., 271, pp 17920-26
(1996); and
variants in USSN 08/746788, filed 15 November 1996; whose disclosures are
incorporated
by reference in their entirety herein.
Intracellular signal transduction is the means by which cells respond to
extracellular stimuli. Regardless of the nature of the cell surface receptor
(e.g. protein
tyrosine kinase or seven-transmembrane G-protein coupled), protein kinases and
phosphatases along with phopholipases are the essential machinery by which the
signal
is further transmitted within the cell [Marshall, J. C. Cell , 80, 179-278
(1995)].
Protein kinases can be categorized into five classes with the two major
classes being,
tyrosine kinases and serine / threonine kinases depending upon whether the
enzyme
phosphorylates its substrate(s) on specific tyrosine(s) or serine /
threonine(s) residues
[Hunter, T., Methods in Enzvmolog,Y(Protein Kinase Classification) p. 3,
Hunter, T.;
Sefton, B. M.; eds. vol. 200, Academic Press; San Diego, 1991].
For most biological responses, multiple intracellular kinases are involved and
an individual kinase can be involved in more than one signaling event. These
kinases
are often cytosolic and can translocate to the nucleus or the ribosomes where
they can
affect transcriptional and translational events, respectively. The involvement
of
kinases in transcriptional control is presently much better understood than
their effect
on translation as illustrated by the studies on growth factor induced signal
transduction
involving MAP/ERK kinase [Marshall, C. J. Cell , 80, 179 (1995); Herskowitz,
I. Cell,
80, 187 (1995); Hunter, T. Cell , 80, 225 (1995); Seger, R., and Krebs, E. G.
FASEB
J., 726-735 (1995)].
While many signaling pathways are part of cell homeostasis, numerous
cytokines (e.g., IL-1 and TNF) and certain other mediators of inflammation
(e.g.,
COX-2, and iNOS) are produced only as a response to stress signals such as
bacterial
lippopolysaccharide (LPS). The first indications suggesting that the signal
transduction pathway leading to LPS-induced cytokine biosynthesis involved
protein
kinases came from studies of Weinstein [Weinstein, et al., J. Immunol. 151,
3829(1993)] but the specific protein kinases involved were not identified.
Working
from a similar perspective, Han [Han, et al., Science, 265, 808(1994)]
identified
murine p38 as a kinase which is tyrosine phosphorylated in response to LPS.
Definitive proof of the involvement of the p38 kinase in LPS-stimulated signal
transduction pathway leading to the initiation of proinflammatory cytokine
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biosynthesis was provided by the independent discovery of p38 kinase by Lee
[Lee; et
al., Nature, 372, 739(1994)] as the molecular target for a novel class of anti-
inflammatory agents. The discovery of p38 (termed by Lee as CSBP 1 and 2)
provided
a mechanism of action of a class of anti-inflammatory compounds for which SK&F
86002 was the prototypic example. These compounds inhibited IL-1 and TNF
synthesis in human monocytes at concentrations in the low uM range [Lee, et
al., Int.
J. Immunopharmac. 10(7), 835(1988)] and exhibited activity in animal models
which
are refractory to cyclooxygenase inhibitors [Lee; et al., Annals N. Y. Acad.
Sci., 696,
149(1993)].
MITOGEN AND STRESS ACTIVATED PROTEIN KINASE CASCADES
mito9ens, growth
factors hormones
+ + + oxidative
F PROINFLAMMATORY CYTOKINES / STRESS STRESS
? ? ?
:?irlAt~IP~"! '~`M!~ SiitIIC4 : ;SKK~ :SKK2 :$Kim
ds SARKS HSP27
Figure 1
It is now firmly established that CSBP/p38 is a one of several kinases
involved
in a stress-response signal transduction pathway which is parallel to and
largely
independent of the analogous mitogen-activated protein kinase (MAP) kinase
cascade
(Figure 1). Stress signals, including LPS, pro-inflammatory cytokines,
oxidants, UV
light and osmotic stress, activate kinases upstream from CSBP/p38 which in
turn
phosphorylate CSBP/p38 at threonine 180 and tyrosine 182 resulting in CSBP/p38
activation. MAPKAP kinase-2 and MAPKAP kinase-3 have been identified as
downstream substrates of CSBP/p38 which in turn phosphorylate heat shock
protein
Hsp 27 (Figure 2). It is not yet known whether MAPKAP-2, MAPKAP-3, Mnk 1 or
Mnk2 are involved in cytokine biosynthesis or alternatively that inhibitors of
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CSBP/p38 kinase might regulate cytokine biosynthesis by blocking a yet
unidentified
substrate downstream from CSBP/p38 [Cohen, P. Trends Cell Biol., 353-
361(1997)].
p38 Kinase Pathway
LPS/IL-1/TNF
stress/UV
SKK2(MKK3) SKK3 (MKK6)
1hIYTY
p38a p38(3
(SAPK2a, CSBP2) (SAPK2b) ? cytokine and other
proinflammatory protein
= er/Thr p38 inhibitor synthesis
Mnkl/Mnk2 MAPKAP3 MAPKAP2
HSP27
Figure 2
What is known, however, is that in addition to inhibiting II.-i and TNF,
CSBP/p38 kinase inhibitors (SK&F 86002 and SB 203580) also decrease the
synthesis
of a wide variety of pro-inflammatory proteins including, IL-6, IL-8, GM-CSF
and
COX-2. Inhibitors of CSBP/p38 kinase have also been shown to suppress the TNF-
induced expression of VCAM-1 on endothelial cells, the TNF-induced
phosphorylation
and activation of cytosolic PLA2 and the IL-1-stimulated synthesis of
collagenase and
stromelysin. These and additional data demonstrate that CSBP/p38 is involved
not
only cytokine synthesis, but also in cytokine signaling [CSBP/P38 kinase
reviewed in
Cohen, P. Trends Cell Biol., 353-361(1997)].
Pharamaceutical Formulations
The cytokine biosynthesis inhibitors for use in the present invention will
normally
be formulated into a pharmaceutical composition in accordance with standard
pharmaceutical practice, for example they will be formulated with a
pharmaceutically
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acceptable diluent or carrier. Cytokine biosynthesis inhibitors, and
pharmaceutically
acceptable salts thereof, and pharmaceutical compositions incorporating such
may
conveniently be administered by any of the routes conventionally used for drug
administration, for instance, orally, topically, parenterally or by
inhalation. The cytokine
biosynthesis inhibitors may be administered in conventional dosage forms
prepared by
combining a cytokine biosynthesis inhibitor with standard pharmaceutical
carriers
according to conventional procedures. Cytokine biosynthesis inhibitors may
also be
administered in conventional dosages in combination with a known, second
therapeutically
active compound. These procedures may involve mixing, granulating and
compressing or
dissolving the ingredients as appropriate to the desired preparation. It will
be appreciated
that the form and character of the pharmaceutically acceptable character or
diluent is
dictated by the amount of active ingredient with which it is to be combined,
the route of
administration and other well-known variables. The carrier(s) must be
"acceptable" in the
sense of being compatible with the other ingredients of the formulation and
not deleterious
to the recipient thereof.
The phanmaceutical carrier employed may be, for example, either a solid or
liquid.
Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin,
agar, pectin, acacia,
magnesium stearate, stearic acid and the like. Exemplary of liquid carriers
are syrup,
peanut oil, olive oil, water and the like. Similarly, the carrier or diluent
may include time
delay material well known to the art, such as glyceryl mono-stearate or
glyceryl distearate
alone or with a wax.
A wide variety of pharmaceutical forms can be employed. Thus, if a solid
carrier is
used, the preparation can be tableted, placed in a hard gelatin capsule in
powder or pellet
form or in the form of a troche or lozenge. The amount of solid carrier will
vary widely
but preferably will be from about 25mg to about lg. When a liquid carrier is
used, the
preparation will be in the form of a syrup, emulsion, soft gelatin capsule,
sterile injectable
liquid such as an ampule or nonaqueous liquid suspension.
The cytokine biosynthesis inhibitors may be administered topically, that is by
non-
systemic administration. This includes the application of an inhibitor
externally to the
epidermis or the buccal cavity and the instillation of such a compound into
the ear, eye and
riose, such that the compound does not significantly enter the blood stream.
In contrast,
systemic administration refers to oral, intravenous, intraperitoneal and
intramuscular
administration.
Formulations suitable for topical administration include liquid or semi-liquid
preparations suitable for penetration through the skin to the site of
inflammation such as
liniments, lotions, creams, ointments or pastes, and drops suitable for
administration to the
eye, ear or nose. The active ingredient may comprise, for topical
administration, from
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0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It
may
however comprise as much as 10% w/w but preferably will comprise less than 5%
w/w,
more preferably from 0.1% to 1% w/w of the formulation.
Lotions according to the present invention include those suitable for
application to
the skin or eye. An eye lotion may comprise a sterile aqueous solution
optionally
containing a bactericide and may be prepared by methods similar to those for
the
preparation of drops. Lotions or liniments for application to the skin may
also include an
agent to hasten drying and to cool the skin, such as an alcohol or acetone,
and/or a
moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid
formulations of the active ingredient for external application. They may be
made by
mixing the active ingredient in finely-divided or powdered form, alone or in
solution or
suspension in an aqueous or non-aqueous fluid, with the aid of suitable
machinery, with a
greasy or non-greasy base. The base may comprise hydrocarbons such as hard,
soft or
liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of
natural origin such
as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or
a fatty acid such
as steric or oleic acid together with an alcohol such as propylene glycol or a
macrogel. The
formulation may incorporate any suitable surface active agent such as an
anionic, cationic
or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene
derivative thereof.
Suspending agents such as natural gums, cellulose derivatives or inorganic
materials such
as silicaceous silicas, and other ingredients such as lanolin, may also be
included.
Drops according to the present invention may comprise sterile aqueous or oily
solutions or suspensions and may be prepared by dissolving the active
ingredient in a
suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any
other suitable
preservative, and preferably include a surface active agent. The resulting
solution may
then be clarified by filtration, transferred to a suitable container which is
then sealed and
sterilized by autoclaving or maintaining at 98-100 C for half an hour.
Aiternatively, the
solution may be sterilized by filtration and transferred to the container by
an aseptic
technique. Examples of bactericidal and fungicidal agents suitable for
inclusion in the
drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride
(0.0 1%) and
chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an
oily solution
include glycerol, diluted alcohol and propylene glycol.
The cytokine biosynthesis inhibitors may be administered parenterally, that is
by
intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal
or
intraperitoneal administration. The subcutaneous and intramuscular forms of
parenteral
administration are generally preferred. Appropriate dosage forms for such
administration
may be prepared by conventional techniques. The cytokine biosynthesis
inhibitors may
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also be administered by inhalation, that is by intranasal and oral inhalation
administration.
Appropriate dosage forms for such administration, such as an aerosol
formulation or a
metered dose inhaler, may be prepared by conventional techniques.
For all methods of use disclosed herein for the cytokine biosynthesis
inhibitors,
including compounds of Formula (I), the preferred, dosage regimen would be
parenteral
until contractions cease, and then as long as necessary to optimise fetal well-
being prior to
delivery, i.e. as near to term (37 weeks gestation) as is necessary. In light
of this, daily
parenteral dosage regimen will be from about 0.1 to about 80 mg/kg of total
body weight,
preferably from about 0.2 to about 30 mg/kg, and more preferably from about
0.5 mg to
15mg/kg. The daily oral dosage regimen will preferably be from about 0.1 to
about 80
mg/kg of total body weight, preferably from about 0.2 to 30 mg/kg, more
preferably from
about 0.5 mg to 15mg. The daily topical dosage regimen, such as may be applied
to
prevent cervical ripening, could be administered topically to the cervix to
prevent cervical
softening and delay fetal membrane rupture. Such as topical dosage will
preferably be in a
formulation containing from 0.1 mg to 150 mg, administered one to four,
preferably two or
three times daily. The daily inhalation dosage regimen, if applicable, will
preferably be
from about 0.01 mg/kg to about 1 mg/kg per day.
It will also be recognized by one of skill in the art that the optimal
quantity and
spacing of individual dosages of cytokine biosynthesis inhibitor will be
determined by the
nature and extent of the condition being treated, the form, route and site of
administration,
and the particular patient being treated, and that such optimums can be
determined by
conventional techniques. It will also be appreciated by one of skill in the
art that the
optimal course of treatment, i.e., the number of doses given per day for a
defined number
of days, can be ascertained by those skilled in the art using conventional
course of
treatment determination tests.
BIOLOGICAL EXAMPLES
The cytokine-inhibiting effects of compounds for use in the present invention,
such as those noted above, may be determined by the following in vitro assays:
Assays for Interleukin-1 (IL-1), Interleukin-8 (IL-8 ), and Tumour Necrosis
Factor (TNF) are well known in the art, and may be found in a number of
publications,
and patents. Representative suitable assays for use herein are described in
Adams et
al., US 5,593,992, whose disclosure is incorporated by reference in its
entirety.
Interleukin -1(IL-1)
Human peripheral blood monocytes are isolated and purified from either fresh
blood preparations from volunteer donors, or from blood bank buffy coats,
according to the
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procedure of Colotta et al, J Immunol, 132, 936 (1984). These monocytes (1 x
106) are
plated in 24-well plates at a concentration of 1-2 million/ml per well. The
cells are allowed
to adhere for 2 hours, after which time non-adherent cells are removed by
gentle washing.
Test compounds are then added to the cells for lh before the addition of
lipopolysaccharide
(50 nghnl), and the cultures are incubated at 370C for an additional 24h. At
the end of this
period, culture supernatants are removed and clarified of cells and all
debris. Culture
supernatants are then immediately assayed for IL-1 biological activity, either
by the
method of Simon et al., J. Immunol. Methods, 84, 85, (1985) (based on ability
of IL-1 to
stimulate a Interleukin 2 producing cell line (EL-4) to secrete IL-2, in
concert with A23187
ionophore) or the method of Lee et al., J. ImmunoTherapy, 6 (1), 1-12 (1990)
(ELISA
assay).
In vivo TNF assay:
(1) Griswold et al., Drugs Under Exp. and Clinical Res.,XIX (6), 243-248
(1993); or
(2) Boehm, et al., Journal Of Medicinal Chemistry 39, 3929-3937 (1996)
whose disclosures are incorporated by reference herein in their entirety.
LPS-induced TNFa Production in Mice and Rats
In order to evaluate in vivo inhibition of LPS-induced TNFa production in
rodents, both mice and rats are injected with LPS.
Mouse Method
Male Balb/c mice from Charles River Laboratories are pretreated (30
minutes) with compound or vehicle. After the 30 min. pretreat time, the mice
are
given LPS (lipopolysaccharide from Esherichia coli Serotype 055-85, Sigma
Chemical Co., St Louis, MO) 25 ug/mouse in 25 ul phosphate buffered saline (pH
7.0) intraperitoneally. Two hours later the mice are killed by CO2 inhalation
and
blood samples are collected by exsanguination into heparinized blood
collection
tubes and stored on ice. The blood samples are centrifuged and the plasma
collected
and stored at -200C until assayed for TNFa by ELISA.
Rat Method
Male Lewis rats from Charles River Laboratories are pretreated at various
times with compound or vehicle. After a determined pretreat time, the rats are
given
LPS (lipopolysaccharide from Esherichia coli Serotype 055-85, Sigma Chemical
Co., St Louis, MO) 3.0 mg/kg intraperitoneally. The rats are killed by CO2
inhalation and heparinized whole blood is collected from each rat by cardiac
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puncture 90 minutes after the LPS injection. The blood samples are centrifuged
and
the plasma collected for analysis by ELISA for TNFa levels.
ELISA Method
TNFa levels were measured using a sandwich ELISA, as described in Olivera
et al., Circ. Shock, 37, 301-306, (1992), whose disclosure is incorporated by
reference
in its entirety herein, using a hamster monoclonal antimurine TNFa (Genzyme,
Boston, MA) as the capture antibody and a polyclonal rabbit antimurine TNFa
(Genzyme) as the second antibody. For detection, a peroxidase-conjugated goat
antirabbit antibody (Pierce, Rockford, IL) was added, followed by a substrate
for
peroxidase (1 mg/ml orthophenylenediamine with 1% urea peroxide). TNFa levels
in
the plasma samples from each animal were calculated from a standard curve
generated
with recombinant murine TNFa (Genzyme).
LPS-Stimulated Cytokine Production in Human Whole Blood
Assav: Test compound concentrations were prepared at 10 X concentrations and
LPS prepared at 1 ug/mI (final conc. of 50 ng/ml LPS) and added in 50 uL
volumes
to 1.5 mL eppendorf tubes. Heparinized human whole blood was obtained from
healthy volunteers and was dispensed into eppendorf tubes containing compounds
and LPS in 0.4 mL volumes and the tubes incubated at 37 C. Following a 4 hour
incubation, the tubes were centrifuged at 5000 rpm for 5 minutes in a TOMY
microfuge, plasma was withdrawn and frozen at -80 C.
Cytokine measurement: IL-I and/or TNF were quantified using a standardized
ELISA technology. An in-house ELISA kit was used to detect human IL-1 and TNF.
Concentrations of IL-1 or TNF were determined from standard curves of the
appropriate cytokine and IC50 values for test compound (concentration that
inhibited
50% of LPS-stimulated cytokine production) were calculated by linear
regression
analysis.
Cytokine Specific Binding Protein Assay
A radiocompetitive binding assay was developed to provide a highly
reproducible primary screen for structure-activity studies. This assay
provides many
advantages over the conventional bioassays which utilize freshly isolated
human
monocytes as a source of cytokines and ELISA assays to quantify them. Besides
being a much more facile assay, the binding assay has been extensively
validated to
highly correlate with the results of the bioassay. A specific and reproducible
cytokine
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inhibitor binding assay was developed using soluble cystosolic fraction from
THP.1
cells and a radiolabeled compound. Patent Application USSN 08/123175 Lee et
al.,
filed September 1993, US 5,783,644 and US 5,777,097 Lee et al., W094/10529
filed
16 September 1994 and Lee et al., Nature 300, n(72), 739-746 (Dec. 1994) whose
disclosures are incorporated by reference herein in its entirety describes the
above
noted method for screening drugs to identify compounds which interact with and
bind
to the cytokine specific binding protein (hereinafter CSBP). However, for
purposes
herein the binding protein may be in isolated form in solution, or in
immobilized form,
or may be genetically engineered to be expressed on the surface of recombinant
host
cells such as in phage display system or as fusion proteins. Alternatively,
whole cells
or cytosolic fractions comprising the CSBP may be employed in the screening
protocol. Regardless of the form of the binding protein, a plurality of
compounds are
contacted with the binding protein under conditions sufficient to form a
compound/
binding protein complex and compound capable of forming, enhancing or
interfering
with said complexes are detected.
CSBP/p38 Kinase Assay:
This assay measures the CSBP/p38-catalyzed transfer of 32P from [a-
32P]ATP to threonine residue in an epidermal growth factor receptor (EGFR)-
derived
peptide (T669) with the following sequence: KRELVEPLTPSGEAPNQALLR
(residues 661-681). (See Gallagher et al., "Regulation of Stress Induced
Cytokine
Production by Pyridinyl Imidazoles: Inhibition of CSBP Kinase", BioOrganic &
Medicinal Chemistry, 1997, 5, 49-64).
Reactions were carried in round bottom 96 well plate (from Corning) in a 30
ml volume. Reactions contained (in final concentration): 25 mM Hepes, pH7.5; 8
mM MgCl2; 0.17 mM ATP (the Km[ATp] of p38 (see Lee et al., Nature 300, n72 pg
639-746 (Dec. 1994)); 2.5 uCi of [g-32P]ATP; 0.2 mM sodium orthovanadate; 1
mM DTT; 0.1% BSA; 10% glycerol; 0.67 mM T669 peptide; and 2-4 nM of yeast-
expressed, activated and purified p38. Reactions were initiated by the
addition of
[gamma-32P]Mg/ATP, and incubated for 25 min. at 37 C. Inhibitors (dissolved
in
DMSO) were incubated with the reaction mixture on ice for 30 minutes prior to
adding the 32P-ATP. Final DMSO concentration was 0.16%. Reactions were
terminated by adding 10 ul of 0.3 M phosphoric acid, and phosphorylated
peptide
was isolated from the reactions by capturing it on p81 phosphocellulose
filters.
Filters were washed with 75 mM phosphoric acids, and incorporated 32P was
quantified using beta scintillation counter. Under these conditions, the
specific
activity of p38 was 400-450 pmol/pmol enzyme, and the activity was linear for
up to
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2 hr of incubation. The kinase activity values were obtained after subtracting
values
generated in the absence of substrate which were 10-15% of total values.
Modulation of PGE2 and IL-10 production
The Examples below determine the effects of the CSAID" compounds on the
ability to modulate both PGE2 and II.-10 production from fetal membranes.
Fetal membranes were obtained from term elective caesarean sections
from uncomplicated pregnancies and washed in phosphate buffered saline
containing 10% penicillin, streptomycin and L-glutamine. Whole membrane
discs consisting of adherent amnion and chorio-decidua each measuring 1.5 cm
were then cut and incubated overnight in multiwell tissue culture plates at 37
C,
95% air, 5% CO2 in serum free medium 199 (Sigma) supplemented with insulin,
transferrin and selenium. Following the addition of fresh medium 199 four
experimental groups (A to D) were set up. Each experimental group consisted
of identical triplicate tissue culture wells.
Groun Medium
A Control (Unstimulated)
B Control (Unstimulated) + SKF86002
C LPS
D LPS + SKF 86002
LPS (Sigma-E.coli Serotype 0114:B4) was used at a concentration of 10-9 g/dI.
SKF 86002 was dissolved in ethanol to provide a final concentration of 10 M.
Group A consisted of control discs incubated in medium alone. Incubations
were carried out for four, eight and twelve hours following which the
supernatants were harvested and stored at -20 C for subsequent estimation of
IL-
1P and PGE2 levels by ELISA (enzyme linked immunosorbent assay
(Amersham) } .
Control wells consisting of ethanol and medium 199 with and without LPS were
also included. Fetal membrance viability was assessed using the diaphorase
histochemical method, see Aldred L.F., et al. (1983) J. Steroid Biochem., 18,
411-414.
RESULTS: IL-1D production:
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Mixed model analysis of variance was used to analyse the data. The
assumptions of the analysis of variance were checked and a log transformation
was found to be the most appropriate. Data is presented on an untransformed
original scale for ease of interpretation.
Grup:
Group A v Group B p=0.007
Difference = 25.5 95% CI (-2.3, 53.3)
Group C v Group D p=0.002
Difference 45.5 95% CI (17.9, 73.2)
Time:
4 hours v 12 hours p=0.08
Difference 22.7 95% CI (-3.0, 48.4)
PGEs nroduction:
Mixed model analysis of variance was used to analyse the data. The
assumptions of the analysis of variance were checked and a log transformation
was found to be the most appropriate. Data is presented on an untransformed
scale for ease of interpretation.
Groun:
Group A v Group B p<0.001
Difference 109 95% CI (78.96, 139.9)
Group C v Group D p<0.001
Difference 161 95% CI (130.6, 191.9)
Tissue viability was not compromised by the addition of the drug as tested by
the diaphorase method. Ethanol alone did not significantly inhibit production
of
either PGE2 or IL-1(3 from the fetal membranes.
Conclusions
The results indicate that following treatment with the prototype CSAIDT"'
compound SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-
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pyridinyl)imidazo[2, 1 -b]thiazole] there was a significant decrease in the
production of both PGE2 and IL-1 J3 from fetal membranes.
SKF 86002 reduced IL-10 production from both control (p=0.07) and
LPS stimulated wells (p=0.002). The effects of the agent appeared to diminish
after 12 hours incubation. Maximal production of IL-1(3 from all treatment
groups occurred after 8 hours incubation. IL-1(3 production was significantly
lower at 12 hours compared to 4 hours in all treatment groups (p=0.08).
SKF86002 reduced PGEZ production from both control (p<0.001) and LPS
stimulated wells (p<0.001). The effects did not seem to diminish throughout
the
incubation period.
This data indicates that CSAIDT" compounds have utility to modulate
prostaglandin and interleukin production from gestational tissues. As the
compound decreased basal PGE2 and IL-1(3 production as well as production in
response to LPS, this provides a basis for treatment of both infection driven
and
idiopathic pre-term labour.
All publications, including but not limited to patents and patent
applications,
cited in this specification are herein incorporated by reference as if each
individual
publication were specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
The above description fully discloses the invention including preferred
embodiments thereof. Modifications and improvements of the embodiments
specifically disclosed herein are within the scope of the following claims.
Without
further elaboration, it is believed that one skilled in the art can, using the
preceding
description, utilize the present invention to its fullest extent. Therefore
the
Examples herein are to be construed as merely illustrative and not a
limitation of the
scope of the present invention in any way. The embodiments of the invention in
which an exclusive property or privilege is claimed are defined as follows.
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