Note: Descriptions are shown in the official language in which they were submitted.
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WO 97t25046 PCT~USg7~00~2
NOVEL SUB~il'll U 1~ IMIDAZOLE COMPOUNDS
This invention relates to a novel group of imi~7ole compounds, processes for thepr~3~alion thereof, the use thereof in treating cytokine m~ te-l ~i~e~es and
S ph~rm~elltic~l compositions for use in such therapy.
p,ACKGROUND OF THE INVENTION
Interleukin-l (IL-l) and Tumor Necrosis Factor (TNF) are biological sub~lances
produced by a variety of cells, such as monocytes or macrophages. IL-l has been
10 demon~tr~t~rl to mP-1i~t- a variety of biological activities thought to be i~ u~L~lL in
;mmlm- regulation and other physiological co~Titions such as in 11~,, ....~1 ion [See, e.g.,
Dinarello et al., Rev. Tnfect. Disease~ 6, S1 (1984)]. The myriad of known biological
activities of IL-l include the activation of T helper cells, induction of fever, s~imlll~tion of
prost~gl~n-lin or collagenase production, neutrophil chemotaxis, induction of acute phase
lS proteins and the suppression of plasma iron levels.
There are many disease states in which excessive or unregulated IL- 1 production is
jmrli~tt~d in exacerbating and/or c~ in~ the disease. These include rh~llm~toid arthritis,
osteoarthritis, endotoxemia and/or toxic shock syndrome, other ~ute or chronic
infi~mm~tory disease states such as the inT~ y reaction in~ e-l by endotoxin or
20 infl~mm~tory bowel ~ e~e; tuberculosis, atherosclerosis, muscle degelleldliûn, cachexia,
psoriatic arthritis, Reiter's syndrome, rhellm~t~ "iLis, gout, traumatic arthritis, rubella
arthritis, and acute synovitis. Recent evidence also links IL-1 activity tO diabetes and
pancreatic B cells.
Dinarello, J. C1;T1;t'~1 Immunolo~y~ 5 (5), 287-297 (1985), reviews the biological
2~ activities which have been attributed to IL- 1. It should be noted that some of these effects
have been descAbed by others as indirect effects of IL-l.
Fx-~e~cive or unregulated TNF production has been impli~t.ofl in m~ tin~ or
exacell,alillg a llUllll of ~ice~ces including rh~lTm~foid arthritis, rh~llm~toid spondylitis,
osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic
30 shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome,
cerebral m~l~ri~, chronic pulmonary infl~mm~tory flice~se, silicosis, pulmonary sarcoisosis,
bone resorption dice~ces, reperfusion injury, graft vs. host reaction, allograft rejections,
fever and myalgias due to infection, such as infll~ 7~ cachexia secondary to infection or
m~lig~n~y, cachexia, secondary to acquired immllne deficiency syndrome (AIDS), AIDS,
35 ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease,
ulcerative colitis, or pyresis.
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AIDS results from the infection of T lymphocytes with Human Immunodeficien.~y
Virus (HIV). At }east three types or strains of HIV have been identified, i.e., HIV-1, HIV-
2 and HIV-3. As a consequence of HIV infection, T-cell m~ ted immnnity is impaired
a~nd infected individuals manifest severe opportunistic infections and/or Imll~u~l neoplasrns.
5 EIIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as
HIV- I, HIV-2 infect T lymphocytes after T Cell activation and such virus protein
expression and/or replication is m~o~iAt~A or mAintAined by such T cell activation. Once an
activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be
mAintAined in an activated state to permit HIV gene e~ll,ssion and/or HIV replication.
10 Monokines, ~perifir~1y TNF, are impli~t~cl in activated T-cell m~Ai~te-l HIV protein
e~ es~.ion and/or virus replication by playing a role in m~int~ining T lymphocyte
activation. Therefore, hltG,rGlGllce with monokine activity such as 'r.y inhibition of
monokine production, notably TNF, in an HIV-infected individual aids in limitin~ the
~Ai~.t~nAnr~e of T cell activation, thereby re~ ing the progression of HIV infectivity to
15 previously uninfected cells which results in a slowing or elimin~tion of the progression of
immllne dysfunction caused by HIV infection. Monocytes, macrophages, and related cells,
such as kupffer and glial cells, have also been implicated in ~ An~e of the HIV
infection. These cells, like T-cells, are targets for viral replication and the level of viral
replication is (lepenf~nt upon the activation state of the cells. Monokines, such as TNF,
20 have been shown to activate EIIV replication in monocytes and/or macrophages [See Poli, et
~L. Proc. Natl. Acad. Sci., 87:782-784 (1990)], Illeref~e, inhibition of monokine
production or activity aids in limiting HIV progression as stated above for T-cells.
TNF has also been implicated in various roles with other viral infections, such as the
cyt- ~nt-~Ali~ virus (CMV), inflllen~ virus, and the herpes virus for sirnilar reasons as those
25 noted.
Interleukin-8 (IL-8) is a chemotactic factor first id~ntified and characterized in
1987. IL-8 is produced by several cell types in- lu-lin~ mononllcl~r cells, fibroblasts,
endothelial cells, and keratinocytes. Its production from endothelial cells is in~lllce(l by IL-
1, TNF, or lipopolysachharide (LPS). Human IL 8 has been shown to act on Mouse,
30 Guinea Pig, Rat, and Rabbit NeuLI~hils. Many dirr~r~ l names have been applied to IL-8,
such as neutrophil attractant/activation protein-l (NAP-1), monocyte derived neutrophil
chemotactic factor (MDNCF), neutrophil activating factor (NAF), and T-cell lymphocyte
chemotactic factor.
IL-8 stim~ tes a number of functions in vitro. It has been shown to have
3~ chemoattractant plo~ellies for neutrophils, T-lymphocytes~ and basophils. In addition it
induces hic~mine release from basophils from both normal and atopic individuals as well
as lysozomal enzyme release and ~t;spilalory burst from neutrophils. IL-8 has also been
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WO 97125046 PCT/US97/00529
shown to increase the surface exples~ion of Mac-1 (CD1 lb/CD18) on neutrophils without
de novo protein synth~si~, this may con~ uL~ to increased adhesion of the n~ u,~hils to
vascular endothelial cells. Many (li~e~ces are char~rto,ri7ecl by massive neull~hi
infiltration. Conditions associated with an increased in IL-8 production (which is
5 responsible for chemotaxis of neul ~ u~l into the infi~mm~tory site) would benefit by
compounds which are suppressive of IL-8 production.
IL-l and TNF affect a wide variety of cells and tissues and these cytokines as well
as other leukocyte derived cytokines are illl~.)Ull~ . and critical inflAmm~tory m~A;~tor.s of a
wide variety of disease states and conditions. The inhibition of these cytokines is of benefit
10 in controlling, r~A~lcin,~ and alleviating many of these disease states.
There remains a need for l~ t, in this field, for colll~ou~ds which are cytokine
suppressive anti-infl~mm~tnry drugs, i.e. compounds which are capable of inhibiting
cytokines, such as IL-1, IL-6, IL-8 and TNF.
15 SUMMARY OF T~F. IN~IENTIC~N
This invention relates to the novel compounds of Formula (I) and rh~rm:~çeut~
compositions corn~)ri~in~ a compound of Formula (I) and a ph~rm~relltic~lly acceptable
diluent or carrier.
This invention relates to a metnod of treating a CS~P/RK/p38 kinase m~Ai~ted
Ai~e~e, in a Ill~ 1 in need thereof, which comprises ~Amini~t~,ring to said ".~.. ~1 an
erre-;live ~mount of a compound of Formula (I).
This invention also relates to a method of inhibiting cytokines and the tre~tm~t of a
cytokine m~Ai~ted Ai~e~e, in a m~mm~l in need thereof, which comprises ~mini~tering to
said m~mm~l an effective amount of a compound of Formula (I).
This invention more specifically relates to a method of inhibiting the production of
IL-l in a m~mm~l in need thereof which compri~es ~tlmini~tPring to said m~mm~l an
effective amount of a compound of Formula (I).
This invention more speçific~lly relates to a method of inhibiting the production of
IL-8 in a ~ 1 in need thereof which comprises ~Amini~terin~ to said m~mm~l an
effective amount of a compound of Formula (I).
This invention more specifically relates to a method of inhibiting the production of
TNF in a m~mm~l in need thereof which comprises ~-l...it~ e, ;..g to said m~mm~l an
effective amount of a compound of Formula (I).
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Accordingly, the present invention provides a compound of Pormula (I):
~2
N
I ~>
R4~ (I)
Rl is a 4-pyridyl, or 4-pyrimidinyl ring which is substituted with a C1~ alkoxy or a C1-4
alkylthio group, and is additionally optionally sllbstitllt~sl indeFçnl1~ntly by Cl 4 aL~cyl,
halogen, hydroxyl, Cl 4 alkoxy, Cl 4 alkylthio, Cl 4 alkylsulfinyl, CH20R12, .amino,
mono and di- C1 6 alkyl substituted amino, N~Rlo)C(O)Rc or an N-heterocyclyl ring
which ring has from 5 to 7 melllbe-~ and optionally contains an additional heteluato
selected from oxygen, sulfur or NR1s;
R4 is phenyl, naphth- 1 -yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by
one or two subsli~ . ,Ix, each of which is indepçn~ntly S~leCtefl, and which, for a
4-phenyl, ~naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl snbstit~lP-nt is halogen, cyano,
nitro, -C(Z)NR7R17, -C(Z)OR16, -(CRloR2o)vcoRl2~ -SR5, -SORs, -OR12, halo-
s~lbstitlltt~l-cl~ alkyl, Cl~ aLkyl, -ZC(Z)R12, -NRloC(Z)R16, or
-(CRloR20)vNRloR20 and which, for other positions of substitution, is halogen,
cyano, -C(Z)NR13R14, -C(Z)OR3, -(CRloR20)m~COR3, -S(O)mR3, -OR3, halo-
~ub~ ul~d-C l 4 a1kyl, -Cl 4 aLkyl, -(cRloR2o)m~NRloc(z)R3~ -NRlos(o)mlR8
-NRlos(o)m~NR7Rl7~ -ZC(Z)R3 or-~cRloR2o)ml~NRl3Rl4;
v is 0, or an integer having a value of 1 or 2;
m is 0, or the integer 1 or 2;
20 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 an optionally substituted heterocyclyl, or an optionally substituted heterocyclylC 1-10
alkyl moiety;
n is an integer having a value of 1 to 10;
25 Z is oxygen or sulfur;
Rc is hydrogen, C1 6 alkyl, C3 7 cycloaLkyl, aryl, arylCl ~ alkyl, helelu~
heteroarylCl 4alkyl, h~tc~u.:yclyl, or heterocyclylCl~alkyl Cl 4 alkyl;
R3 is heterocyclyl, heterocyclylcl-lo alkyl or R8;
Rs is hydrogen, C 1-4 aLkyl, C2 4 alkenyl, C2 4 alkynyl or NR7R17, eYch~lin~ the moeities
-SR5 being -SNR7R17 and -SOR5 being -SOH;
R7 and R17 is each independently selected from hydrogen or Cl 4 alkyl or R7 and R17
together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7
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m.ombe~c which ring optionally contains an ~ ition~l heteroatom 5~ ct~fl from
oxygen, sulfur or NR15;
R8 is Cl lo alkyl, halo-~ub~liluLed Cl lo alkyl, C2 10 alkenyl, C2 10 alkynyL C3-7
cycloaLkyl, C5 7 cycloalkenyl, aryl, arylCl lo alkyl, heteroaryl, ht~telu~ylCl 10 alkyl,
(CRloR2o)nORll, (cRloR2o)ns(o)mRl8~ (CRl0R20)nNHs(O)2Rl8~
(CRloR20)nNR13R14; wherein the aryl, atylalkyl, heteroaryl, heteroaryl alkyl may be
optionally substituted;
Rg is hydrogen, -C(Z)Rll or optionally ~,ul;~LiLuLed Cl lo alkyl, S(O)2Rlg, optionally
substituted aryl or optionally substituted aryl-Cl 4 alkyl;
10 R1o and R20 is each independently sel~cte~ from hydrogen or C1 4 alkyl;
Rl 1 is hydrogen, Cl 10 alkyl, C3 7 cycloaL~yl, heterocyclyl, heterocyclyl Cl loalkyl, aryl,
arylCl 10 alkyl, heteroatyl or heteroarylC1 10 alkyl;
R12 is hydrogen or R16;
R13 and R14 is each indepe~lently sel~ct~d from hydrogen or optionally substituted Cl 4
alkyl, optionally substituted aryl or optionally substituted aryl-Cl 4 alkyl, or together
with the nitrogen which they are attached form a heterocyclic ring of 5 to 7 members
which ring optionally cont~inc an additional heteroatom selected from oxygen, sulfur or
NRg;
Rls is Rlo or C(Z)-Cl ~1 alkyl;
20 R16 is Cl~ allyl, halo-substituted-Cl 4 alkyl, or C3 7 cycloaLkyl;
Rlg is Cl lo alkyl, C3 7 cycloalkyl, heterocyclyl, aryl, aryll loalkyl, heterocyclyl,
heterocyclyl-Cl 1oalkyl, heteroaryl or heteroaryl1 1oalkyl;
or a ph~rm~ euti~lly ~cep~hle salt thereof.
25 Dl~TAILED DESCRIPTION OF THE INVENTION
The novel compounds of Formula (I) may also be used in association with the
veterinary tre~tm~nt of m~mm~l~, other than hllm~n~, in need of inhibition of cytokine
inhibition or production. In particular, cytokine m~ te~ diseases for tre~tm~nt
th~ ea Lically or plopllyl~rti(~lly, in ~nim~l~ include disease states such as those noted
30 herein in the Methods of Treatment section, but in particular viral infections. Examples of
such viruses inclll~e, but are not limited to, lentivirus infections such as, equine infectious
anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections,
such as but not limited to feline irnmuno-leficien~y virus (FIV), bovine irnrnunodeficiency
virus, or canine immuno~1efi~iency virus or other retroviral infections.
In Formula ~I), suitable R1 moieties include a 4-pyridyl or a 4-pyrimidinyl ring.
The R1 moieties are substituted at least one time by a Cl 4 alkoxy or C1 4alkylthio moiety.
Preferably the R1 moiety is a C1 4 alkoxy group, such as n-butyl, isoproxy, ethoxy or
-5 -
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methoxy. A preferred ring pl~em~nt of the R~ substituent on the 4-pyridyl derivative is in
the 2-position, such as 2-methoxy4-pyridyl. A pl~rc~led ring pl~ren~nt on the
4-pyrimidinyl ring is also at the 2-position, such as in 2-methoxy-pyrirnidinyl.Suitable additional ~ùb~Liluents for the R1 heteroaryl rings are Cl 4 alkyl, halo, OH,
S Cl 4 alkoxy, Cl 4 alkylthio, Cl 4 aL~ylsulfinyl, CH20R12, amino, mono and di-Cl 6
alkyl substihlt~l amino, N(Rl~)C(O)RC, or an N-heterocyclyl ring which ring has from 5 to
7 members and optionally contains an additional heteroatom selected from oxygen, sulfur
or NR15. The alkyl group in the mono- and di-Cl 6 aLkylsubsLiluled moiety may be halo
~ub~Liluled, such as in trifluoro- i.e., trifluoromethyl or trifluroethyl.
When the Rl optional substituent is N(Rlo)c(o) Rc, wll~lchl Rc is hydrogen, C1-6alkyl, C3 7 cycloalkyl, aryl, arylCl~ alkyl, heteroaryl, heteroarylCl 4aL~yl, heterocyclyl, or
heterocyclylCl 4alkyl Cl~ alkyl, Rc is preferably C~1-6 alkyl; pr~relably Rlo is hydrogen.
It is also recognized that the Rc moieties, in particular the C1 6 alkyl group may be
optionally s-lhstitute-l, preferably from one to three times, preferably with halogen, such as
1~ fluorine, as in trifluoromethyl or trifluroethyl.
Suitably, R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is
optionally substituted by one or two subsfitl~entc~ More preferably R4 is a phenyl or
na~hLllyl ring. Suitable ~lbsLiLuLions for R4 when this is a 4-phenyl, 4-naphth-1-yl, 5-
naphth-2-yl or 6-naphth-2-yl moiety are one or two ~ubs~ .ent~ each of which sre20 independently selected from halogen, -SR5, -SOR5, -OR12, CF3, or
-(CRloR2o)vNRl~)R2o~ and for other positions of substit~tion on these rings ~ d
~ubsLiLution is halogen, -s(o)mR3~ -OR3, CF3, -(cRIoR2o)ml~NRl3Rl4~ -NRlOC(Z)~3
and -NR~S(O)m~Rg. Preferred sub~ for the 4-position in phenyl and naphth-l-yl
and on the 5-position in naphth-2-yl include halogen, especially fluoro and chloro and -SR5
25 and -SOR5 whel~in R5 is preferably a C1 2 alkyl, more preferably methyl; of which the
fluoro and chloro is more preferred, and most especially preferred is fluoro. ~Gfelr~d
~iub.~ .nt~ for the 3-position in phenyl and naphth-l-yl rings include: halogen, especially
fluoro and chloro; -OR3, especially Cl 4 alkoxy; CF3, NRloR2o~ such as amino; -
NRloC(Z)R3, especially -NHCO(Cl l() alkyl); -NRlos(o)m~Rg~ especially -
30 NHS02(Cl lo alkyl), and -SR3 and -SOR3 wherein E;~3 is ~lert;~Lbly a C1 2 allcyl, more
preferably methyl. When the phenyl ring is disubstituted preferably it is two independent
halogen moieties, such as fluoro and chloro, preferably di-chloro and more preferably in the
3,4-position. It is also plert;l,ed that for the 3-position of both the -OR3 and -ZC(Z)R3
moietitf~s, R3 may also include hydrogen.
3~ Preferably, the R4 moiety is an un~llb.stitute~l or substituted phenyl moiety. More
preferably, R4 is phenyl or phenyl substituted at the 4-position with fluoro and/or
~ub~liLuLed at the 3-position with fluoro, chloro, C 14 alkoxy, methane-sulfo~mitlQ or
-- 6 -
.
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~ce~ ln, or R4 is a phenyl di-substituted at the 3,4-position independently with chloro or
fluoro, more preferably chloro. Most preferably, R4 is a 4-fluorophenyl.
In Formula (I), Z is oxygen or sulfur, preferably oxygen.
Suitably, R2 is an optionally s~ stitl-te~ heL~,u-;y~;lyl, or a heterocyclylCl lo alkyl
5 moiety.
When R2 is an optionally substituted heterocyclyl the ring is preferably a
morpholino, pyrrolidinyl, or a piperidinyl group. When the ring is optionally substituted
the substit~ t~ may be directly ~ eh.~cl 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,
10 more preferably piperillinç The heterocyclyl ring may be optionally ~l~h~ one to four
times independently by halogen; C14 alkyl; aryl, such as phenyl; arylaL~yl, such as benzyl,
wherein the aryl or aryl alkyl moiet;t~s themselves may be optionally ~ub~LiluLt;d (as in the
definition section below~; C(O)ORl 1, such as the C(O)Cl 4 aL~yl or C(O)OH moieties,
C(O)H; C(C))Cl 4 aLkyl, hydroxy substituted Cl 4 alkyl, Cl~ alkoxy, S~O)mC1 4 alkyl
(wherein m is 0, 1, or 2), NRloR20 (wherein Rlo and R20 are indepçn~ 7tly hydrogen or
C1 ~lalkyl).
Preferably if tne ring is a piperidine, the ring is attached to the imi~ e at the 4-
position, and tne substitu~onts are ~I.,e~,Lly on the available nitrogen, i.e. al-Formyl-4-pipçriclin~o7 1-benzyl-4-pirç~i~inP 1-methyl4-piperidine, 1-etho~yc~lJollyl4-
20 pir~oriciin~. If the ring is ~.u~sliLuLed by an aLkyl group and the ring is ,7tt~7~h~d in the4-position, it is preferably ~.ul~..LiLuLed in tne 2- or 6- position or both, such as 2,2,6,6-
tetramethyl-4-piperidine. Similarly, if the ring is a pyrrole, tne ring is attached to the
imin~7Ole at the 3-position, and the substituents are all directly on the available nitrogen.
When R2 is an optionally substituted heterocyclyl Cl lo alkyl group, tne ring ispreferably a morpholino, pyrrolidinyl, or a piperidinyl group. Preferably the alkyl moiety
is from 1 to 4 carbons, more preferably 3 or 4, and most preferably 3, such as in a propyl
group. Prcfell~d heterocyclic alkyl groups include but are not limited to, morpholino etnyl,
morpholino propyl, pyrrolidinyl propyl, and piperidinyl propyl moieties. The heterocyclic
ring herein is also optionally ~.ul~liLul~d in a similar manner to that inf~ tÇCl above for the
direct ;.~ h.~ t of the heterocyclyl.
In all in.ct~n~çs herein where there is an alkenyl or alkynyl moiety as a substituent
g;oup, the unsaturated linkage, i.e., the vinylene or acetylene linkage is preferably not
directly ,.tt .C~hPt1 to tne nitrogen, oxygen or sulfur moieties, for in~t~nee in OR3, or for
certain R2 moieties.
As used herein, "optionally substituted" unless specifically defined shall mean such
groups as halogen, such as flllorine, chlorine, bromine or iodine, ~lyd~OXy; hydroxy
.ubsLiLuLed Cl 1oaLI~yl; Cl lo alkoxy, such as me~oxy or ethoxy; S(~)m a~kyl, wherein m
-- 7 --
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W O 97/25046 PCT~US97/005~9
is 0, 1 or 2, such as methyl tnio, methylsulfinyl or methyl sulfonyl; amino, mono & di-
~.uI,~.LiluLcd amino, such as in the NR7R17 group; or where the 1~7R17 may together with the
nitrogen to which they are ~tt~rhP(1 cyclize to form a 5 to 7 membered ring which optionally
inchlAes an ~flAition~l heteroatom selected from O/N/S; Cl lo alkyl, cycloalkyl, or
5 cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopluL,yl, t-butyl, etc. or cyclopropyl
methyl; halosl1bstitlltçd Cl lo alkyl, such CF2CF2H, or CF3; halo~.ulr.!LiLuled Cl lo aLkoxy,
such OCF2CF2H; an optionally substituted aryl, such as phenyl, or an optionally ~b..~ d
arylalkyl, such as benzyl or ~h~ ~yl, W~ ll these aryl moieties may also be substituted
one to two times by halogen; hyd~o~y; hydroxy substituted alkyl; Cl lo alkoxy; S(O)m
10 alkyl; amino, mono & di-sllbstitllt~ amino, such as in the NR7R17 group; alkyl, or CF3.
In a ~lcÇcllcd subgenus of compounds of Formula (I), Rl is 2-alkoxy-4-pyridyl or2-alkoxy-4-pyrirnidinyl; R2 is morpholinyl propyl, pipçriflinyl, N-benzyl~-piperidinyl, or
N-methyl~-piperidinyl; and R4 is phenyl or phenyl substh~lte~ one or two times by fluoro,
chloro, Cl 4 alkoxy, -S(O)m alkyl, mpth~npslllfonamido or ~ret~mitlo.
Suitable ph~n~relltically 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, sl-lrhllrir acid, phosphoric acid, m~th~.nP snlphonic acid, ethane sulphonic
acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, sllrrinic acid,
film~r~r acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and m~n~1~1ic acid.
20 In addition, ph~rm~reutir~lly acceptable salts of com~oul,ds of Formula (I) may also be
formed with a ph~....A~e~ll;r,~lly acceptable cation, for in~t~n~e, if a ~.ub~ t group
co-ll~ es a carboxy moiety. Suitable ~ ""~rentir~lly acceptable cations are well known to
t_ose skilled in the art and include zllk5~1in~, ~lk~lin~ earth, ammonium and qn~t.orn~ry
ammonium cations.
The following terms, as used herein, refer to:
~ "halo" or "halogens", include the halogens: chloro, fluoro, bromo and iodo.
~ "Cl loaLI~yl" or "aLkyl" - both straight and branched chain radicals of 1 to 10
carbon atoms, unless the chain length is otherwise limite~, including, but not limited to,
methyl, ethyl, n-propyl, zso-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 bond in~ ing but not lirnited to cyclopentenyl,
cyclohexenyl, and the like.
~ The term "aL~enyl" 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, inclll~ing, but
-- 8 --
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WO 97/25046 PCT~US97/00529
not limited to ethenyl, 1~ ellyl, 2-propenyl, 2-methyl~ lu~,nyl, 1-butenyl, 2-butenyl
and the like.
~ "aryl" - phenyl and naphthyl;
~ "hel~ alyl" (on its own or in any co,llbhl~lion, such as "h~ Lyloxy", or
"hc;lel~o~yl aLkyl") - a 5-10 m~mhered aromatic ring system in which one or more rings
contain one or more heteroatoms selected from the group con~ ting of N, O or S, such as,
but not limitt~.-l, to pyrrole, pyrazole, furan, thiophene, quinoline, isoq-linolin~.7 quinazolinyl,
pyridine, pyrimirlin~, oxazole, thiazole, thi~ 7.ole, triazole, imidazole, or l~ç,-7.i~ 7Ole.
~ "heterocyclic" (on its own or in any combination, such as "heterocyclylaL'cyl") - a
saturated or partially lln~tllr~qted 4-10 lllelllb~lt;d ring system in which one or more rings
contain one or more heteroatoms selected from the group co~ ting of N, O, or S; such as,
but not limited to, pyrrolidine, pir~eri~lin~ e, morpholine, tetrahydro pyran, or
imi~ 01i~1inP.
~ The term "aralkyl" or "h~L~loalylalkyl" or "heterocyclicalkyl" is used herein to
mean Cl 4 alkyl as defined above ~tt~h~ to an aryl, heteroaryl or heterocyclic moiety as
also fl~fin~-l herein unless otherwise inrlirslt~.
~ "sulfinyl" - the oxide S ~O) of the corresponding sulfide, the term "thio" refers to
the sulfide, and the te~n "sulfonyl" refers to the fully oxidized S (~)2 moiety.~ "aroyl" - a C(O)Ar, wherein Ar is as phenyl, naphthyl, or aryl aLkyl deliv~live
such as defined above, such group include but are not limited to benzyl and phenethyl.
~ "aLkanoyl" - a C(O)Cl lo alkyl ~hen,ill the aL~cyl is as defined above.
For the purposes herein the "core" 4-pyrimidinyl moiety for R1 or R2 is referred to
N~
as the forrnula:
The compounds of the present invention may contain one or more asymm~tric
carbon atoms and may exist in racemic and optically active forrns. All of these compounds
are ;nslnd~l within the scope of the present invention.
Exemplifil-~l compounds of Formula (I) include:
1-(4-Piperidinyl)-4-(4-Flourophenyl)-5-(2-isopropoxy-4-pyrimidinyl) imi~l~7ole
1-(4-Piperidinyl)-4-(4-Fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imi~l~7.ole
5-(2-Hydroxy-4-pyrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imi-1~7.ole
~-(2-Methoxy-4-pyridinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imirl~7Qle
5-(2-iso-Propoxy~pyridinyl)-4-(4-fluorophenyl) -1-(4-piperidinyl)imitl~7O1e
5-(2-Methylthio4-pyrimidinyl)-4-(4-fluorophenyl)- 1-(4-piperidinyl)imifi~7ole
g
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WO 97/25046 PCT~US97/00529
5-(2-Methylthio4-pynrnidinyl)-4-(4-fluorophenyl)-1-[(1-methyl4-
piperidinyl~imi(l~ole
5-(2-Ethoxy~-pyrimi(1inyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imi~1~7ole 1-(1-
Ethylcarboxylpiperidin~yl)-3-(4-thiolll(,lhylphenyl)-5-r2-(thiomethyl)-
S pyrimidin-4-yl]-im~ ole
I-(1-Ethylcarbo~-yl~ idine~-yl)-4-(4-methylsulfinylphenyl)-5-[2-methylsulfinyl-
pyrimidin-4-yl] imid~7ole
A prefered grouping of compounds of Formula (I) have the structure:
R2
Rt N
~r ,,
R4 N (Ia)
wllGlehl
Rl is pyrirnidinyl substituted with a C1 4 alkoxy, and is additionally optionally substituted
independently one or more times by Cl 4 alkyl, halogen, hydroxyl, C1_4 alkoxy, Cl 4
alkylthio, Cl 4 alkylsulfinyl, CH20R12, amino, mono and di- C1 6 alkyl ~u~LiLuled
amino, N(Rlo)C(O)Rc or an N-heterocyclyl ring which ring has from S to 7 membersand optionally contains an additional hetelo~olll selected from oxygen, sulfur or NR15;
R2 is an optionally ~lb~LiLuL~d heterocyclyl, or an optionally substituted heterocyclylCl lo
alkyl moiety;
R4 is phenyl, which is optionally substituted by halogen;
R1o is indep~n~ntly selected from hydrogen or C1 4 alkyl;
Rc is hydrogen, C1 6 alkyl, C3 7 cycloaLkyl, aryl, arylCI~ alkyl, heteroaryl,
heteroarylCl~alkyl, heLe~ ;yclyl, or hc;L~ L;yclylCl 4alkyl Cl~ alkyl, all of which
may be optionally substit lt~
R12 is hydrogen or Rl6;
R16 is Cl 4 allyl, halo-substituted-C14 alkyl, or C3 7 cycloalkyl;
R15 is hydrogen, C14 alkyl or C(Z)-C1 4 alkyl;
Z is oxygen or sulfur;
or a ph~ re~ltically acceptable salt thereof.
Preferably, R2 is piperidine, 1-Forrnyl-4-piperidine, l-benzyl~-piperidine, l-methyl-
4-piperidine, 1-ethoxycarbonyl-4-piperidine, 2,2,6,6-tetramethyl~-piperidine, morpholino
ethyl, morpholino propyl, pyrrolidinyl propyl, or piperidinyl propyl.
Another prefered grouping of c~ll.poul.ds of Formula (I) have the structure:
- 10-
-
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WO 97/25046 PCT~US97~00529
R
R
t~ _N
I ~>
R4~ (Ib)
wherein
Rl is pyridyl substihlte-l with a Cl 4 alkoxy, and is ~ tions-lly optionally substituted
indepç~eIltly one or more times by Cl 4 alkyl, halogen, hydroxyl, Cl ~ alkoxy, Cl 4
alkylthio, C1 4 alkylsulfinyl, CH20R12, amino, mono and di- C1 6 alkyl substituted
amino, N(Rlo)c(o)Rc or an N-h~ u~yclyl ring which ring has from 5 to 7 members
and optionally co~ s an additional hel~roa~ûll, se.lect~l from oxygen, sulfur or NR15;
R2 is an optionally ~u~Lilul~d heterocyclyl, or an optionally substituted heterocyclylCl lo
alkyl moiety;
R4 is phenyl, which is optionally sul~sLiLuled by halogen;
Rlo is indeper rlently sel~ctt-~l from hydrogen or Cl~ alkyl;
Rc is hydrogen, C1 6 alkyl, C3 7 cycloalkyl, aryl, arylCl 1 alkyl, het~loa,yl,
heteroarylCl 4alkyl, heterocyclyl, or heterocyclylCl 4alkyl Cl 4 alkyl, all of which
may be optionally ~b~ e~;
R12 is hydrogen or R16;
R16 is Cl 4 aLkyl, halo-~ùbsliluLed-Cl 4 alkyl, or C3 7 cycloalkyl;
R1s is hydrogen, Cl~ alkyl or C(Z)-Cl~ aLkyl;
Z is oxygen or sulfur;
or a ph~nn~ellti~lly acc~L~le salt thereof.
Preferably, R2 is pipçri~linP, 1-Formyl-4-pipPri~lin~, 1-benzyl-4-piperidine, l-methyl-
4-piperidine, 1-etho~yca,l~ollyl-4-piperidine, 2,2,6,6-tetramethyl~-pipen~linlo, morpholino
ethyl, morpholino propyl, pyrrolidinyl propyl, or piperidinyl propyl.
l~he co~ oullds of Formula (I) may be obtained by applying synthetic procedures,some of which are illustrated in Schemes I to XI herein. The synthesis provided for in
these Schemes is applicable for the producing compounds of Formula (I) having a variety
of Lrr.,.~,nt Rl, R2, and R4 groups which are reacted, employing optional substituents
which are suitably ~lote~;led~ to achieve colll~alibility with the reactions outlined herein.
Subsequent deprotection, in those cases, then affords compounds of the nature generally
disclosed. Once the imi~7ole nucleus has been established, further compounds of Formula
(I) may be prepared by applying standard techniques ~or functional group interconversion~
well known in the art.
For instance: -C(O)NR13R14 from -C02CH3 by heating with or without catalytic
~ metal cyanide, e.g. NaCN, and HNR13R14 in CH30H; -OC(O)R3 from -OH with e.g.,
ClC(O)R3 in pyridine; -NRlo-C(S)NR13R14 from -NHRlo with an alkylisothiocyante or
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thiocyanic acid; NR6C(O)OR6 from -NHR6 with the aLkyl chlolofc..lllate; -
NRloC(O)NR13R14 from -NHRlo by t-e~ t with an isocyanate, e.g. HN=C=O or
R1oN=C=O; -NR10-c(o)R8 from -NHRlo by tre~tm~nt with Cl-C(O)R3 in pyridine;
C(=NRlo)NR13R14 from -C(NR13R14)SR3 with H3NR3~0Ac- by heating in alcohol;
C(NR13R14)SR3 from -C(S)NR13R14 with R6-I in an inert solvent, e.g. S~cetr~n~;
C(S)NR13R14 (where R13 or R14 is not hydrogen) from -C(S~NH2 with
HNR13R14-C(=NCN)-NR13R14 from -C(=NR13R14)-SR3 with NH2CN by heating in
anhydrous alcohol, ~ VCly from -C(=NH3-NR13R14 ~y tre~tm~nt with BrCN and
NaOEt in EtOH; -NRlo-C(=NCN)SRg from -NHRlo by lle~ nt with (RgS)2C=NCN;
NR1oS02R3 from -NHRlo by tre~tm~nt with ClS02R3 by heating in pyridine;
NRloC(S)R3 from -NRloC(O)Rg by tre~tn~nt with Lawesson's reagent ~2,4-bis(4-
methoxyphenyl)-1,3,2,4-(1ithi~iphosrhet~ne-2,4-~ ulfiflel; -NRloS02CF3 from -NHR6
with triflic anhydride and base wherein R3, R6, Rlo, R13 and R14 are as defined in
Formula (~) herein.
Precurso~s of the groups Rl, R2 and R4 can be other R~!, R2 and R4 groups which
can be i..tc~cc,.~vcl~ed by applying standard techniques for functional group interconversion.
For ex~mple a compound of the formula (I) wherein 1~2 is halo -substituted Cl lo alkyl can
be converted to the corresponding Cl lo aLkylN3 de~;v~Live by reacting with a suitable
azide salt, and thereafter if desired can be reduced to the corresponding Cl loaL~ylNH2
20 compound, which in turn can be reacted with RlgS(0)2X wll~Gin X is halo (e.g., chloro) to
yield the corresponding Cl 1oaLkylNHS(0)2Rlg colllpoul-d.
~lt~ tively a compound of the formula (I) where R2 is halo-substituted
Cl lo-aL~cyl can be reacted with an amine R13R14NH to yield the corresponding
Cl lo-aL~cylNR13Rlq compound, or can be reacted with an alkali metal salt of RlgSH to
25 yield the cullcs~ ding Cl loalkylSE;: 18 compound.
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R4CHO ( V) + Ar S(O)p H R4CH2NH2 (Vm)
/ \ r~ lau~,gagent
H~CHO C~CI3 ~ ~
NaOH R4CH2NHCHO
CH2Cl2
H20, PTC \~ ~ dch~JlàLil~s agent
Ar--S~O)p R4CH2NC (VI)
~1~ A~ S(0)2 L~ (V~)/
R4 NHCHO
wherem p =~
RlCHO + R2NH2
d~h~a~g agent
Ar--S(O)p P~1~NR2
~ H ~)
R4 NC
\/
R4
SCElEME I
Referring to Scheme I the compounds of Formula (I) are suitably l~l~cd by
S reacting a compound of the Formula (II) with a compound of the Formula (III) whe}ein p is
O or 2, Rl, R2 and R4 are as ~e~ln~-l herein, for Formula (I), or are precursors of the groups
Rl, R2 and R4, and Ar is an optionally substituted phenyl group, and thereafter if necessary
converting a precursor of R1, R2 and R4 to a group R1, R2 and R4.
Suitably, the reaction is performed at ambient le~ el~tule or with cooling (e.g. -50~
10 to 10~) or heating in an inert solvent such as methylene chloride, DMF, tetrahydrofuran,
toluene, acelul~ ile, or dimethoxyethane in the presence of an a~l~liate base such as 1,8-
- 13-
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W O 97/2S046 PCT~US97/OOS29
diazabicyclo ~.4Ø] undec-7-ene (DBU) or a guanidine base such as 1,5,7-tri~a-bicyclo
[4.4.0] dec-5-ene (TBD). The int~rm.oAi~t~s of forrnula (II) have been found to be very
stable and capable of storage for a long time. Preferably, p is 2. PTC is defined as a phase
transfer catalyst.
Compounds of the Formula (II) have the structure:
Ar--S(O)p
(II)
R4/~\ NC
wherein p is 0, or 2; R4 is as defined for Formula (I) and Ar is an optionally
substituted aryl as Ae~ln~A herein. Suitably, Ar is phenyl optionally S~ ?~ tG~ by
10 Cl 4allcyl, Cl 4 aLkoxy or halo. I~e~Gl~ly Ar is phenyl or 4-methyl~hen~l, i.e. a tosyl
de~ivali~
Reaction of a compound of the Formula (II) wherein p = 2, with a compound of thePormula (m) in Scheme I gives con~ictçntly higher yields of compounds of Formula (I)
than when p=0. In addition, the reaction of Pormula (II) com~oullds wherein p = 2 is more
15 envirorlm~nt~lly and economically attractive. When p=0, the preferred solvent used is
methylene cloride, which is envirt nmP~t~lly unattractive for large scale procçs~ing, and
the ~r~f~l~d base7 TBD, is also GXl~en~ive, and produces some byproducts and illl~u~iLies,
than when using the culll~llcrcially attractive synthesis (p=2) as further described herein.
As noted, Sch~ o I utilizes the 1,3-dipolar cyclo~A-liti~ns of an anion of a
20 substituted aryl thiol~letl-ylisocyanide (when p=0) to an imine. More specifically, this
reaction requires a strong base, such as an amine base, to be used for the dep~-3tvllation step.
The co-~ cially available TBD is preferred although t-bllto~ide, Li~ or Naf, or K~
hP.x~methylAi~ 7i~1e may also be used. While methylene chloride is the prefered solvent,
other halogenated solvents, such as chloroform or carbon tetrachloride; ethers, such as
25 T~, DME, DMP, diethylether, t-butyl methyl ether; as ~,vell as acGLv.,il,ile, toluene or
LIlr~s thereof can be ~tilti7~ll The reaction may take place from about -20~C to about;
40~C, preferably from about 0~C to about 23~C, more preferably from about 0~C to about
10~C, and most preferably about 4~C for reactions involving an R1 group of pyrimidine.
For compounds wherein Rl is pyridine, it is recognized that varying the reations conditions
30 of both tell~ dtulG and solvent may be n~ces~ry, such as decreasing temperatures to about
-50~C or eh~ngin~ the solvent to THF.
- 14-
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WO 97/25046 PCTJIJS97JOD529
In a further process, compounds of Formula (I) may be prepared by coupling a
suitable delivaLive of a compound of Formula (IX):
, 2
Tl N
11 /~
~--N
T4 (IX)
wherein Tl is hydrogen and T4 is R4, or ~lf~-rn~tively Tl is Rl and T4 is H m which Rl,
R2 and R4 are as hereinbefore defined; with: (i) when T1 is hydrogen, a suitable derivative
of the helel~yl ring R1H, under ring coupling conditions, to effect coupling of the
heteroaryl ring Rl to the imi~l~7Ole nucleus at position 5; (ii) when T4 is hydrogen, a
10 suitable d~,liv~tive of the aryl ring R4H, under ring coupling conditions, to effect coupling
of the aryl ring R4 to the imi~ ol~ nucleus at position 4.
Such aryl/heteroaryl coupling reactions are well known to those skilled in the art.
In general, an organomPt~llic synthetic equivalent of an anion of one component is coupled
with a reactive d,.iv~live of the second component, in the presence of a suitable catalyst.
15 The anion equivalent may be formed from either the imi-l~7Ole of Formula (IX), in which
case the aryl/h~ 1 compound provides the reactive de~ivalivc, or the aryl/heL~
compound in which case the imicl~7.o]e. provides the reactive dcliv~l~ve. Accordingly,
suitable deliv~Lives of the compound of Formula (~) or the aryl/heteroaryl rings include
organomPt~,llic d~,liv~ves such as organrJm~ .,., organozinc, org~nc-st~nn~n~ and
20 boronic acid deliv~-ivcs and suitable reactive derivatives include the bromo, iodo,
fluorosulfonate and trifluor~m~-th~neslllrhonate delivativ-~s. Suitable procedures are
described in WO 91/19497, the disclosure of which is incorporated by reference herein.
Suitable organom~gn~ lm and org~no7inr d~ivalives of a compound of Formula
~IX) may be reacted with a halogen, fluorosulfonate or triflate de~iv~l;ve of the heteroaryl
25 or aryl ring, in the presence of a ring coupling catalyst, such as a p~ lm (O) or
p~ Aillm (II) catalyst, following the procedure of Kllm~ et aL, Tetrahedron T .etters, 22,
5319 (1981). Suitable such catalysts include tetrakis-(triphenylphosphine)p~ Aillm and
PdC12rl,4-bis-(diphenylphosphino)-butane], optionally in the presence of lithium chloride
and a base, such as triethylamine. In z~klitio~ a nickel (II) catalyst, such as M(II)C12(1,2-
30 biphenylphosphino)ethane, may also be used for coupling an aryl ring, following theprocedure of Pridgen et al., J. Org. Chem, 1982, 47, 4319. Suitable reaction solvents
include h~-~c:~m~.thylphosphor-amide. When the heteroaryl ring is ~pyridyl, suitable
derivatives include 4-bromo- and 4-iodo-pyridine and the fluorosulfonate and triflate esters
of 4-hydroxy pyridine. Similarly, suitable derivatives for when the aryl ring is phenyl
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W O 97/25046 PCT~US97/00529
include the bromo, fluorosulfonate, triflate and, preferably, the iodo-derivatives. Suitable
org~nom~gn~sJIlm and organozinc derivatives may be obtai.~ed by treating a compound of
Formula (IX) or the bromo derivative thereof with an alkyllithium compound to yield the
corresponding lithium reagent by deprotonation or tr~n~m~t~llation~ respectively. This
lithium il~t~ di~t~ may then be treated with an excess of a m~necillm halide or zinc
halide to yield the co~ ollding organom~t~l]ic reagent.
A triaLkyltin deliv~live of the compound of Formula (IX) may be treated with a
bromide, fluorosulfonate, triflate, or, pr,fe-d~ly, iodide d~,l;vaLive of an aryl or heteroaryl
ring compound, in an inert solvent such as tetrah~ r~all, preferably cont~inin~ 10~o
10 hPxamlothylphosphoramide, in the presence of a suitable coupling catalyst, such as a
p~lla(1illm (O) catalyst, for inct~nce tetrakis-(triphenylphosphine)-p~ lm~ by the method
described in by Stille, J. Amer. Chem. Soc, 1987, 109, 5478, US Patents 4,719,218 and
5,002,942, or by using a p~lla-lillm (Il) catalyst in the presence of lithium chloride
optionally with an added base such as triethylamine, in an inert solvent such as dimethyl
15 fs,. ".~",it1~. Trialkyltin Llelivatives may be conveniently obtained by m~ot~ tion of the
ujlLc;~onding compound of Formula (IX) with a lithi~ting agent, such as s-butyl-lithium or
n-butyllithillm, in an ethereal solvent, such as tetrahy~vfu.~, or Llc~ of the bromo
deliv~live of the corresponding compound of Formula (IX) with an alkyl lithinm, followed,
in each case, by tre~tmtont with a triaLlcyltin halide. ~ltt~rn~tively, the bromo- delivalivt; of
20 a compound of Formula (IX) may be treated with a suitable heteroaryl or aryl tnaL~yl tin
compound in the presence of a catalyst such as tetrakis4~iphellyl-phosphine)-p~ rlillm,
under conditions similar to t_ose fiPst~ribe~l above.
Boronic acid derivatives are also useful. Hence, a suitable de~iv~liv~ of a
compound of Forrnula (IX), such as the bromo, iodo, triflate or fluorosulphonate derivative,
25 may be reacted with a heteroaryl- or aryl-boronic acid, in the presence of a p~ rn
catalyst such as tetrakis-(lli~hel yl~hosphine)-p~ inm or PdC12rl,4-bis-(diphenyl-
phosphino)-butane] in the presence of a base such as sodium bicarbonate, under reflux
conditions, in a solvent such as dimetho~y~lh~e (see Fischer and Haviniga, Rec. Trav.
Chim. Pays Bas, 84, 439, 196~, Sni~ s, V., Tetrahedron Lett., 29, 2135, 1988 and30 Ter~chimi~ M., Chem. Pharm. Bull., 11, 4755, 198~). Non-aqueous conditions, for
inSt~nre, a solvent such as DMF, at a tenl~e~alulc; of about 100~C, in the presence of a
Pd(II) catalyst may also be employed (see Thompson W J et al, J Org Chem, 49, 5237,
1984). Suitable boronic acid de.ivalives may be prepared by treating the magnesium or
lithium de.ivaliv~ with a trialkylborate ester, such as triethyl, tri-iso-propyl or
35 tributylborate, according to standard procedures.
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In such coupling reactions, it will be readily appreciated that due regard must be
exercised with respect to functional groups present in the compounds of Forrnula (IX)
Thus, in general, amino and sulfur ~ubsl ;l, IP,I~t.c: should be non-oxidised or protected
Compounds of Formula (IX) are im~ 7~oles and may be obtained by any of the
S procedures herein before t1es( rihe(1 for ~l~ing compounds of Formula (I) In particular,
an oc-halo-ketone or other suitably activated ketones R4COC~I2Hal (for compounds of
Formula (IX) in which Tl is hydrogen) or RlCOCH2Hal (for co~ ullds of Formula (IX)
in which T4 is hydrogen) may be reacted with an ~mi-lint- of the formula R2NH-C=NH,
wherein R2 is as (lef1n~A in Formula (I), or a salt thereof, in an inert solvent such as a
10 halogenated hydrocarbon solvent, for in~t~n-~e chloroform, at a moderately elevated
lelll~e~dluie~ and, if n~cec~ry, in the presence of a suitable contl~ns~tion agent such as a
base The preparation of suitable a-halo-ketones is described in WO 91/19497. Suitable
reactive esters include esters of strong organic acids such as a lower aL~cane sulphonic or
aryl sulphonic acid, for in~t~n~e, m~th~ne orp-toluene sulphonic acid The ~mitlin~ is
15 preferably used as the salt, suitably the hydroçhloride salt, which may then be converted
into the free ~mi~in~ in situ, by employing a two phase system in which the reactive ester
is in an inert organic solvent such as chloroform, and the salt is in an aqueous phase tO
which a solution of an aqueous base is slowly added, in dimolar amount, with vigorous
stirrin~ Suitable ~miflin~s may be obtained by standard mPtho-1~, see for in~t~n~e,
20 Garigipati R, Tetra'nedron T ~tt~r~, 190, 31, 1989
C(i..-~ou lds of Formula (I) may also be prepared by a process which comprises
reacting a compound of Formula (IX), wllel~ Tl is hydrogen, with an N-acyl heteroaryl
salt, according to the method disclosed in US patent 4,803,279, US patent 4,719,218 and
US patent 5,002,942, to give an i~ I~" t~ t.o in which the heteroaryl ring is ~ft~Ch~l to the
25 imi(l~7olP nucleus and is present as a 1,4-dihydro derivative thereof, which interm~ tt~
may then be subjected to oxidative-deacylation conditions (Scheme II) The hetelu~yl salt,
for in~t~n~e a pyri~inillm salt, may be either preformed or, more preferably, prepared in
situ by adding a substituted carbonyl halide (such as an acyl halide, an aroyl halide, an
arylaL~yl haloformate ester, or, ~lefeldbly, an aLkyl haloformate ester, such as acetyl
30 bromide, benzoy}chloride, benzyl chlororol.,ldt~, or, preferably, ethyl chloroformate) to a
solution of the compound of Formula (IX) in the heteroaryl compound RlH or in an inert
solvent such as methylene chloride to which the hete-~3~yl compound has been added
Suitable deacylating and oXi~ ng conditions are described in U S Patent Nos 4,803,279,
4,719,218 and 5,0()2,942, which references are hereby incorporated by reference in their
35 entirety. Suitable oxidizing ~y~lellls include sulfur in an inert solvent or solvent lui~lul~,
such as (l~c~lin~ decalin and diglyme, p-cymene, xylene or mesitylene, under reflux
conditions, or, preferably, pot~ m t-butoxide in t-butanol with dry air or oxygen
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~2 R RQJ~N
RX4l0 NH ~ N,~ p~mdine/R~COCI ~N,~ S/deralin,~ or 1~N~
SCHEMl~ II
S In a further process, illustrated in ~ch~m~ m below, compounds of Formula (I)
may be prepared by treating a compound of Formula (X) th~rm~lly or with the aid of a
cyclising agent such as pho..~hc~ s oxychk)ri~le or phosphorus pentachloride (see Engel and
Steglich, Liebigs Ann Chem, 1978, 1916 and SLI~yl~liy et al., J Org Chem, 1963, 28, 3381).
Compounds of Formula (X) may be obtained, for in.ct~nt~e, by acylating the collG~onding
10 a-keto-amine with an activated formate derivative such as the corresponding anhydride,
under standard acylating conditions followed by formation of tne imine with R2NH2. The
a ninoketone may be derived from the parent ketone by o~min~tion and reduction and the
requisite ketone may in turn be L~lc~ ,d by decarboxylation of the beta-ketoester obtained
from the conden~tion of an aryl (heteroaryl) acetic ester with the RlCOX co~ ol1ent.
Rl~C~
X l.)NaOMe R 0 R 0 1-) ~ ~ R,2 ,R2
+ 2.) HC~1~ 1 .)NaNO2~ HCI, H20 ~'F HlOJI Me ~N O poC13 Rl~f N~
R~OR R~ 2.) redure R4~NH2 2-)NH2R2~ -H20 R4~--H~H R4 N
fcrmula (X)
SCHEM~ III
In Scheme IV illustrated below, two (2) different routes which use ketone (fo~nula
20 XI) for ~repali~g a compound of Formula (I). A heterocyclic ketone (XI) is ~ alcd by
adding the anion of the allcyl heterocycle such as 4-methyl-quinoline (prepared by tre~tm~nt
thereof with an alkyl 1ithinm, such as n-butyl lithium) to an N-aLkyl-O-alkoxybel-7~mi~
ester, or any other suitably activated deliv~ e of the same oxidation state. ~lt~rn~tJvely~
the anion may be con-len~ed with a be~7~lclehyde, to give an alcohol which is then oxidised
2~ to the ketone (XI).
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WO 97~25046 PCT~US97/OOS29
Rl NHR2 R~ rH
R4~0 R ~
~X ) HN ~ r
~ R4 ~ R4 ~ R4~0 R~
SCHEME IV
In a further process, N-substituted compounds of Formula (I) may be prepared by
5 treating the anion of an amide of Formula (XII):
RlCH2NR2COH (XII)
wll~ Rl and R2 with:
(a) a nitrile of the Forrnula (Xm):
R4CN
10 wh~ R4 is as hereinbefore defin~l, or
(b) an excess of an acyl halide, for in~t~n~e an acyl chloride, of the Forrnula (XIV):
R4COHal (XIV)
wl,.~eill R4 is as helGi~ rul~ defined and Hal is halogen, or a corresponding anhydride, to
give a bis-acylated int~rm~ te which is then treated with a source of ~mmoni~, such as
1~ ~mm~niilm acetate.
R2HN base R1 CI R R2 1.) LiI -N(i-Pr~ ~ R1 N
o/~H o R4~CN R ~N
(Xll)
SCE~ME V
One variation ûf this approach is illustrated in Scheme V above. A ~illl~y amine(R2NH2) is treated with a halomethyl heterocycle of Pormula RlCH2X to give the
secondary amine which is then converted to the amide by standard techniques.
~lt~ fively the amide may be prepared as illustrated in scheme V by alkylation of the
form~mide with RlCH2X. Deprotonation of this amide with a strong arnide base, such as
lithium di-iso-propyl amide or sodium bis-(trimethylsilyl)amide, followed by addition of an
excess of an aroyl chloride yields the bis-acylated compound which is then closed to an
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WO 97125046 PCT/US97/00529
imidazole compound of Formula (I), by heating in acetic acid cv..l~ini..g ammonium
acetate. ~ltPrn~tively, the anion of the amide may be reacted with a s~ liLuLt;d a~l nitrile
to produce the im~ 7ole of Forrnula (I) directly.
The following description and schemes are further exemplification of the process as
S previously described above in Scheme I. Various pyrimi~1ine aldehyde derivatives 6 and 7
as depicted in scheme VI below, can be prepared by modification of the procedures of
Bredereck et al. (Chem. Ber. 1964, 97, 3407) whose disclosure is incc"~olated by reference
herein. These pyrimi~linP. aldehydes are then utilized as ;nt~Prm~ te!s in the synthesis as
further described.
MeO
o >--NMe2
MeO>~ MeO MeO>~ N
MeO 1 ~MeO 2
1. thiourea
NaOEt tEtOI~
2. RI
SR ~ ~ RO~
3 N~ O(n) NaORt ROH~ ~j 5
~ R ~3 / MeO OMe
HCI MeO OMe HCI
THF / H20 n = 1 or 2 THF / H2O
6 SR ~N~3 R7~~N~
H O H O
Scl~
The reaction of imines with tosylmethyl isonitriles was first reported by van Leusen
(van T ~lls~.n ~ et al., J. Org. Chem. 1977, 42, 11 53.) Reported were the following
conditions: tert butyl arnine(tBuNH2) in dimethoxyethane (DME), K2C03 in MeOH, and
NaH in DME. Upon re-exzlmin~tion of these conditions each was found produce low
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yields. A second pathway involving amine exchange to produce the t-butyl imine followed
by reaction with the isocyanide to produce a l-tBu imi~l~7OIe was also Opeld~illg. This will
likely occur using any ~ ll~ y amine as a base. The secondary ~mines~ while not ~lefe~,~,d
may be used, but may also decompose the isonitrile slowly. ~ ction~ will likely re~uire
S about 3 equivalents of amine to go to completion, reslllting in appr xim~t~oly 50% isolated
yields. Hindered secondary amines (diisopropylamine) while usable are very slow and
generally not too effective. Use of tertiary and aromatic amines, such as pyridine, and
triethylamine gave no reaction under certain test conditions, but more basic types such as
DBU, and 4-dimethylamino pyridine (DMAP) while slow, did produce some yields and10 hence may be sllit~hle for use herein.
As depicted in Sch~mes VII and VIII below, the pyrimidine aldehydes of Scheme
VI, can be con-l~n~e-l with a plimal y amine, to generate an imine, which may suitably be
isolated or reacted in situ, with the desired isonitrile in the presence of a variety of suitable
bases, and solvents as described herein to afford the 5-(4-pyrimidinyl)-substituted
15 imicl~7oles, wherein R2 and E~4 are as defined herein for Formula (I) compounds.
One preferred method for ~lG~JOlil~g compounds of Formula (I) is shown below in
Scheme VII. The imines, prepared and isolated in a separate step were often tars, which
were hard to handle. The black color was also often carried over into the final product.
The yield for m~king the imines varied, and envil~,.,..~.,l~lly less-acceptable solvents, such
20 as CH~Cl2 were often used in their preparation.
This reaction, wherein p=2. requires a suitable base for the reaction to proceed. The
reaction requires a base strong enough to deprotonate the isonitrile. Suitable bases include
an amine, a carbonate, a hydride, or an alkyl or aryl lithium reagent; or ~ ul~,s thereof.
Bases inc.lt~ , but are not limited to, pot~ m carbonate, sodium carbonate, primary and
2~ secondary ~mine~, such as morpholine, piperidine, pyrrolidine, and other non-nucleophilic
bases.
Suitable solvents for use herein, include but are not lirnited to N,N-dimethyl-
forrn~mi~le (DMP), MeCN, halogenated solvents, such as methylene chloride or
chloroform, tetrahydl~)rul~l (THF), dimethylsulfoxide (DMSO), alcohols, such as methanol
30 or ethanol, benzene, or toluene, or DME. Preferably the solvent is DMF, DME, THF, or
MeCN, more preferably DMF. Product isolation may generally be accompli~h~-l by adding
water and filtering the product as a clean compound.
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N~' Me ~ Me
S~TOI ~ N
N-C RX 1
X = O, S
SCHEl~E VII
While not convenient for large scale work, addition of NaH to the isonitrile, perhaps
with Lelllyelalulc;s lower than 25 ~C (in THF) are likely nPefle~ tionAlly, BuLi has
also been reported to be an ~rr~cLi~e base for d~..,l(~llating tosyl benzylisonitriles at -50~C.
(DiSanto,et al., Synth. Commun. 1995, 25, 795).
Various tel~l~e.dlu,~ conditions may be utili7erl depending upon the preferred base.
For in.ct~nf~e, tBuNH2/DME, K2CO3/MeOH, K2C03 in DMF, at telll~e-aLu.Gs above 40~C, the yields may drop to about 20% but little ~lirÇ~,lGllce is e~pected between 0~C and 25
10 ~C. Consequently, Iclllpl l~tul~, ranges below 0 ~C, and above 80 ~C are con~ te~l as
also being within the scope of this invention. Preferably, the temperature ranges are from
about 0 ~C to about 25 ~C.
As shown in Scheme VIII below, the imine is preferably formed in situ in a solvent.
This ~rGfGlled synthf~sic, is a process which occurs as a one-pot synthesis. Suitably, when
1~ the primary amine is utilized as a salt, the reaction may further include a base, such as
potassium carbonate prior to the addition of the isonitrile. ~ltern~tively, the piperidine
nitrogen may be required to be protected as shown below. Reaction conditions, such as
solvents, bases, LG111~)e1~lU1GS, etc. are similar to those illustrated and discussed above for
the i~ol~t~ll imine as shown in Scheme VII. One skilled in the art would readily recogr~ize
20 that under some e~ u ~ es, the in situ formation of the imine may require dehydrating
conditions, or may require acid catalysis.
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N~ BOC
BOC
,,CHO ~ ~ DMF
r , 5 h ~
OR N~2 N~ N
OR
BOC N
KD~CMOF3 _RO ~N~ 3N HCI N~
SO~Tol ~ N
NC ~ F
S~F,lV~, VIII
Scheme IX, describes an ai: .:rnative process for making compounds of formula (I).
S In this particular in~t~n~e, the alkylthio moiety is oxifli7e~l to the alkylsulfinyl or sulfonly
moiety which is reacted with a suitable alkoxy moiety.
RS ~ N R(~n)S~ N RO~ N
OXONE ~ ~3 ,R2 NaOR/ROH 1~3 R2
Scheme IX
Another embodiment of the present invention is the novel hydrolysis of 2-
thiomethylpyrimirline acetal to 2-thiom~Lhyl~y~ i(1in~ aldehyde, as shown in Scheme X
below. Hydrolysis of the acetal to aldehyde using various known reaction conditions, such
as formic acid, did not produce a s~ti~f~c~Qry yield of the aldehyde, <13%) was obtained.
15 The preferred synthesis involves the use of AcO~I (fresh) as solvent and con-centrated
H2SO4 under heating conditions, preferably a catalytic amount of sulfuric acid. ~ting
conditions include tGll~eldLUl~s from about 60 to 85~C, preferably from about 70 to about
80~C as higher te~ GldlulGs show a ~1~rk~ning of the reaction mixture. After the reaction
- 23 -
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PCTrUS97/00529
W O 97/25046
is complete the ~ Lul~,is cooled to about room ~Clll~J~d~lllC and the acetic acid is removed.
A more ~l~f~,~r~,d ~ltPrn~t;ve procedure to this involves heating the acetal in 3N HCl at
40~C for about 18 hours, cooling and extracting the bicarbonate neutralized solution into
EtOAc.
S
OMe O
OMe AcoEvconc H2so4 ~ H
SMe
SMe
Ss ~ - ~ X
While these schP~Ps herein are presçntPrl, for in~t~nce, with an optionally
substituted piperidine moiety for the res--lt~nt R2 position, or a 4-fluoro phenyl for R4, any
10 suitable R2 moiety or R4 moiety may be added in this manner if it can be prepared on the
pl;lnal y amine. Similarly, any suitable R4 can be added via the isonitrile route.
The compounds of Formula (II), in Scheme I, may be ~r~,d by the methods of
van Leusen et al., supra. For çx~mrle a compound of the Formula (II) may be ~.c~alcd by
del,y~Ldti,lg a compound of the Formula (IV)-Scheme I, wherein Ar, R4 and p are as
15 defined herein.
Suitable delly~dLillg agents include phosphorus oxychloride, oxalyl chloride,
thionyl chloride, phosgene, or tosyl chloride in the presence of a suitable base such as
triethylamine or diiscpl.,~ylethylamine, or similar bases, etc. such as pyridine. Suitable
solvents are rlimPthoxy ether, tetrahydrofuran, or halogen~teA solvents, preferably THF.
20 The reaction is most effi~ent when the reaction temperatures are kept beL~vv~en 10~C and
0~C. At lower telll~eldlu-~,s in~omrlPte reaction occurs and at higher telll~cl~lulc;s, the
solution turns dark and the product yield drops.
The compounds of formula (IV)-Scheme I may be ~ ~ed by reacting a compound
of the formula (V)-Scheme L R4CHO where R4 is as defined herein, with ~rS(0)pH and
2~ ~. "".",i~l.o with or without water removal, preferably under dehydrating conditions, at
ambient or elevated temperature e.g. 30~ to 150~, conveniently at reflux, optionally in the
presence of an acid catalyst. ~lt~rn~tively trimethysilylchloride can be used in place of the
acid catalyst. E~xarnples of acid catalysts include c~mrhor-10-sulphonic acid, formic acid,
p-toluenesulphonic acid, hydrogen chloride or slllphllri~ acid.
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An optimal method of making an isonitrile of Formula (II~ is i~ tr~ted below, inScheme XI.
rV",.d",i.l~ NHCHO SO2Tol
PhMe. ~ hlHCHO TolSO2H ~e~ NHCHO
50 ~C 2 3
SO2Tol Q.5 MTHF SO2Tol
~~ NHCHO Et,qN ~ NC
10toO ~C F
70% yield 4
SCHEME XI
S The conversion of the substituted aldehyde to the tosylbenzyl fonn~mi~ may be
accomr li.~h~ by heating the aldehyde, l-Scheme XI, with an acid, such as p-toluene-
sulfonic acid, formic acid or c~mrhorsulfonic acid; with fo""~lllicle and ~toluene-slllfinic
acid [under reaction conditions of about 60~C for about 24 hours]. Preferably, no solvent is
used. The reaction, may give poor yields (< 30%) when solvents, such as DMF, DMSO,
toluene, acetonitrile, or excess form~mi-lç are used. TG111~1alU~CS less than 60~C are
generally poor at producing the desired product, and tel-l~e.;~lul~s in excess of 60~C may
produce a product which decomposes, or obtain a benzylic bis-fc.. ".~ le, 2-Scheme XI.
Another embo~1imt-~t of the present invention is the ~ylll~esis of the tosyl benzyl
form~mi-lP. compound, achieved by reacting the bi~r~. ".i...,ill~. int~rm.o.rli~t~, 2-Scheme XI
15 with p-tol~le~s~llfinic acid. In this ~l~,fe~cd route, ~lGp~dlion of the bis-fo....~.icle from
the aldehyde is accomplished by heating the aldehyde with form~mi(le7 in a suitable solvent
with acid catalysis. Suitable solvents are toluene, aceton,~lile, DMF, and DMSO or
ules thereof. Acid catalysts, are those well known in the art, and include but are not
limited to hydrogen chloride, p-tol~lçnesulfonic acid, c~mrhors~-lfonic acid, and other
20 anhydrous acids. The reaction can be cond~-~ te~1 at t~ el~lu.~s ranging from about 25~C
to 110~C, preferably about 50~C, suitably for about 4 to about 5 hours, longer reaction
times are also acceptable. Product decomposition and lower yields may be observed at
higher telllpeldlulcs (~70~C) at prolonged reactions times. Complete conversion of the
product generally requires water removal from the reaction n~i~lulc.
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Preferred conditions for con~e~ g a bis-form~mi-l~ deliv~tive to the tosyl benzyl
rO. IIIAIIIitle are accomplished by heating the bisform~micle in a suitable solvent with an acid
catalyst and p-tol~en~s~llfinic acid. Solvents for use in this reaction include but are not
limited to toluene, and acetonitrile or ll~u~L~lleS thereof. Additional mixtures of these
5 solvents with DMF? or DMSO may also be used but may result in lower yields.
Temp-,.alu~._s may range from about 30~C to about 100~C. Te,ll~eralulc;s lower than 40~C
and higher than 60~C are not ~l~fe~l~d as the yield and rate decreases. Preferably the range
is from about 40 to 60~C, most preferably about 50~C. The optimal time is about 4 to 5
hours, although it may be longer. Preferably, acids used include but are not limited to,
10 tc~ on~sulfonic acid, c~mphcrslllfonic acid, and hydrogen chloride and other anhydrous
acids. Most preferably the bisform~micle is heated in toluene:~etonitrile in a 1:1 ratio,
with p-t lllen~os~ nic acid and hydrogen chloride.
Another embodiment of the present invention is the ~lefelled synthetic route forsynthesis of the tosylbenzyl r~" .,.;...,~ compound which is accomplished using a one-pot
procedure. This process first converts the aldehyde to the bis-fortn~mi-l~ de.iv~Live and
subsequently reacts the biS-form~mi~lç dc~ivaLi~re with tQluen~osulfinic acid. This procedure
comhin~s the optimi7~ conditions into a single, efficient process. High yields, >90% of
the aryl benzylf ). " ~ may be obtained in such a manner.
PlGfc,led reaction conditions employ a catalyst, such as LlhllcLhylsilyl chloride
(TMSCl), in a ~e~ll~d solvent, toluene:acetonitrile, preferably in a 1:1 ratio. A reagent,
such as TMSCl, is preferred which reacts with water produced therein and at the same time
produces hydrogen chloride to catalyze the reaction. Also preferred is use of hydrogen
chloride and p-tolll~-n~--slllfonic acid. Therefore, three suitable reaction conditions for use
herein include 1) use of a dehydrating agent which also provides hydrogen chloride, such as
TMSCl; or by 2) use of a suitable delly~ g agent and a suitable source of acid source,
such as but not limited to, c~mphor.sulfonic acid, hydrogen chloride or t ~hlenes~l~fonic acid;
and 3) ~It~rn~tive dehydrating conditions, such as the ~eotropic removal of water, and
using an acid catalyst and p-toluene sulfinic acid.
Compounds of the formula (II) where p is 2 may also be prepared by reacting in the
presence of a strong base a compound of the formula (VI) -Scheme I, R4CH2NC with a
compound of the formula (VII)-Scheme I, ArS02Ll wL~ ll R4 and Ar are as defined
herein and Ll is a leaving group such as halo, e.g. fluoro. Suitable strong bases include,
but are not limited to, alkyl lithillm~ such as butyl lithium or lithium diisopropylamide (Van
Leusen çt al., Tetrahedron Letters. No. 23, 2367-68 (1972)).
The compounds of formula (VI)-Scheme I may be p~ ed by reacting a conl~,oulld
of the formula (VIII)-Scheme I, R4CH2NH2 with an aLkyl formate (e.g. ethylformate) to
yield an interm~ t~ amide which can be converted to the desired isonitrile by reacting
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with well known dehydlalillg agent, such as but not limited to oxalyl chloride, phosphorus
oxychloride or tosyl chloride in the presence of a suitable base such as triethylamine.
~ lt~rnz~tively a colll~uul-d of the formula (VIII) - Scheme I may be converted to a
compound of the formula ~ Scheme I by reaction with chloroform and sodium
S hydroxide in aqueous dichlorom~-th~ne under phase transfer catalysis.
The compounds of the formula (m) - Scheme I may be prepared by reacting a
compound of the formula RlCHO with a primary amine R2N~2.
The arnino compounds of the formula (VII~ çh.ome I are known or can be
~lc~a,ed from the co.l~s~ollding alcohols, oximes or amides using standard functional
10 group interconversions.
Suitable protecting groups for use with hydroxyl groups and the imi~1~7ole nitrogen
are well known in the art and described in many references, for in.~t~nce, Protecting Groups
in Organic Synthesis, Greene T W, Wiley-lnterscience, New York, 1981. Suitable
exarnples of hydroxyl protecting groups include silyl ethers, such as t-buty1ciim.-thyl or t-
15 butyl-lirh~nyl, and allyl ethers, such as methyl connected by an aL~yl chain of variable link,
(CRloR2o)n. Suitable e~c~rnrl~s of imit1~7.ole nitrogen ~ te~ g groups include
tetrahydr~,~y,~lyl.
ph~ cGulir~lly acid addition salts of compounds of For-m--ula (I) may be obtained
in known manner, for ex~mrl~ by tre~tm~nt thereof with an a~r~ ;ate amount of acid in
20 the presence of a suitable solvent.
MF.THODS OF I~EATMENT
The compounds of Pormula (I) or a ph~rm~r~lltic~lly acceptable salt thereof can be
used in t_e m~nllfactl-re of a m~-lic~m~nt for the pl~llylactic or therapeutic tre~tnn~nt of
25 any disease state in a human, or other m~mm~l, which is exacerbated or caused by
excessive or unreg~ t~-~l cytokine prod~ ti-~n by such ~ s cell, such as but not
limited to monocytes and/or macrophages.
Compounds of Formula (I) are capable of inhibiting proinfl~mm~t-~ry cytokin~s,
such as IL-l, IL-6, IL-8 and TNF and are therefore of use in therapy. IL-l, IL-6, IL-8 and
30 TNF affect a wide variety of cells and tissues and these cytokin~, as well as other
leukocyte-derived cyt--kinPs, are important and critical infl~mm~tory m~ tcrc of a wide
variety of disease states and conditions. The inhibition of these pro-infl~mm~tQry cytokines
is of benefit in controlling, re~ ing and alleviating many of these disease states.
Accordingly, the present invention provides a method of treating a cytokine-
m~-.Ai~te-l disease which comprises z~-lmini~t~rin~ an effective cytokine-interfering amount
of a compound of Formula (I) or a ph~rm~reutically acceptable salt thereo~.
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Compounds of Formula (I) are capable of inhihitin~ in~1llcihle proinfl~mm~tQry
proteins, such as COX-2, also referred to by many other names such as prost~lan~lin
endoperoxide synthase~2 (PGHS-2) and are therefore of use in therapy. These
proinflamm~tory lipid m.~ t~,rs of the cyclooxygenase (CO) pathway are produced by the
S in~lnr.il~le COX-2 enzyme. Regulation, therefore of COX-2 which is responsibIe for the
these products derived from aracnidonic acid, such as prost~ nflin.c affect a wide variety
of cells and tissues are inl~G Lallt and critical infl~ rJI y m~ tors of a wide variety of
disease states and conditions. Expression of COX- 1 is not effected by compounds of
Formula (I). This selective inhibition of COX-2 may alleviate or spare ulcerogenic Iiability
10 associated with inhibition of COX-1 thereby inhibiting prosto~lan(1in.s çssçnti~l for
~;yLopl~tective effects. Thus inhibition of tnese pro-infl~mm~tory m~ tr)r.c is of benefit in
controlling, retlllcing and alleviating many of these disease states. Most notably these
infl~.. ~1ul y m~.Ai~tor.s, in particular prost~ n~lin.c, have been implicated in pain, such as
in the senciti7~tion of pain receptors, or edema. This aspect of pain m~n~em~nt therefore
15 inrlucle.c ~ .nt of neuromuscular pain, h~ he, cancer pain, and arthritis pain.
Compounds of Formula (I) or a ph~rm~elltie~lly acceptable salt thereof, are of use in the
u~hylaxis or therapy in a human, or other m~mm~l, by inhibition of the synthesis of the
COX-2 enzyme.
Accordingly, the p;esent invention provides a metnod of inhibiting the synthesis of
20 COX-2 which comprises ~clmini.cterin~ an effective amount of a compound of Formula (I)
or a ph~rm~ce~ltic~lly acceptable salt thereof. The present invention also provides for a
method of prophylaxis ll~dllllen~ in a human, or other m~mm~l, by inhibition of the
synthesis of the COX-2 enzyme.
In particular, compounds of Formula (I) or a ph~rm~r~eutically acceptable sait
25 thereof are of use in the prophylaxis or therapy of any disease state in a human, or other
m~mm~l, which is exacerbated by or caused by excessive or unregulated IL 1, IL-8 or TNF
production by such ~ s cell, such as, but not limited to, monocytes and/or
macrophages.
Accordingly, in another aspect, this invention relates to a method of inhibiting the
30 production of IL-1 in a m~mm~l in need thereof which comprises ~lmini.cte.ring to said
m~mm~31 an effective amount of a compound of Formula (I) or a rh~rn ~-~e~ltic~lly
acceptable salt thereof.
There are many disease states in which excess}ve or unregulated IL- 1 production is
implic~t~d in exacerbating and/'or c~nsing the ~lice~ce These include rhellm~toid arthritis,
3~ osteoarthritis, stroke, endotoxemia and/or toxic shock syndrome, other acute or chronic
inflamm~tory disease states such as the infl~mm~tnry reaction intll~ce~l by endotoxin or
infl~mm~to;y bowel tlic~.~ce7 tuberculosis, atherosclerosis, muscle degeneration, multiple
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sclerosis, c~c}-Pxi~ bone resorption, psoriatic arthritis, Reiter's syndrome, rheum~tQid
arthritis, gout, traumatic arthritis, rubella arthritis and acute synovitis. Recent evidence also
links IL-l activity to diabetes, pancreatic ~ cells and Alzheimer's disease.
In a further aspect, this invention relates to a method of inhibiting the production of
5 TNP in a rn~mm~l in need thereof which comI)ri~C ~ ";ni~le, I--g to said m~mm~l an
effective amount of a compound of Formula (I) or a ph~rrn~eutir~lly acceptable salt
thereof.
Excessive or unregulated TNF production has been implicated in mt~ tin~ or
exacell,aL~llg a number of diseases inrh~(1;n~ rh~llm~toid arthritis, rhe!-m~tQi~l spondylitis,
10 osteoarthritis, gouty art_ritis and other arthritic conditions, sepsis, septic shock, endotoxic
shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome,
stroke, cerebral m~l~ri~, ch~onic pulmonary infl;~ e~ce~ silicosis, p~llmon~ry
sarcoisosis, bone resorption ~lice.~c, such as osteoporosis, reperfusion injury, graft vs. host
reaction, allograft rejections, fever and myalgias due to infection, such as i~ n~,
15 c~ch~ secondary to infection or m~lign~ncy, cachexia secondary to acquired immnnP
deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid form~tiQIl, scar
tissue formation, Crohn's disease, ulcerative colitis and pyresis.
Compounds of Formula (I) are also useful in the L~ "Irl~ of viral infections, where
such viruses are sensitive to upregulation by TNF or will elicit TNF production fn vivo.
20 The viruses cont~mpl~t~ for tre~trn~nt herein are those that produce TNF as a result of
infection, or those which are sensitive to inhibition, such as by decreased replication,
dheclly or indhc~;Lly, by the TNF inhibiting-compounds of Formula (1). Such viruses
in~lu~le, but are not lirnited to HIV-l, HIV-2 and HIV-3, Cytomegalovirus (CMV),Tnfll-lon7~ adenovirus and the Herpes group of viruses, such as but not lirnited to, Herpes
2S Zoster and Herpes Simplex. Accordingly, in a further aspect, this invention relates to a
method of treating a m5~rnm~1 ~ffli~t~-l with a human immunodeficiency virus (HI~) which
comrri~es ~lmini~terin~ to such m~mm~l an effective TNF inhibiting amount of a
compound of Pormula (I) or a ph~ reuti~lly acceptable salt thereof.
Compounds of Formula (I) may also ~e used in association with the veterinary
30 tre~tmt~nt of mzlmm~l~, other tnan in hllm~n~, in need of inhibition of TNF production.
TNF mr~ t~ e~es for treatment, therapelltir~lly or prophylactically, in ~nim~l~ include
disease states such as those noted above, but in particular viral infections. Fxamples of
such viruses include, but are not limited to, lentivil~s infections such as, equine infectious
~n~r~ virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections,
35 such as but not limited to feline imm~ln~defIciency virus (FIV), bovine immunodeficiency
virus, or canine imrnunodeficiency virus or other retroviral infections.
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The compounds of Formula (I) may also be used topically in the tre~tm~nt or
prophylaxis of topical disease states m~ tçcl by or exacerbated by excessive cytokine
production, such as by rL-1 or TNF respectively, such as infl~mt~.cl joints, eczema, psoriasis
and other infl~Tnm~tory skin conditions such as sunburn; in~ oly eye conditions
S including conjunctivitis; pyresis, pain and other conditions associated with infl~mm~tion.
Compounds of Formula (I) have also been shown to inhibit the production of IL-8
(Interleukin-8, NAP). Accordingly, in a further aspect, this invention relates to a method of
inhibiting the production of IL-8 in a mAmm~l in need thereof which comprises
?~lrnini~terin~ to said mATnm~l an effective amount of a compound of Formula (I) or a
10 ph~rm~-~euti~lly acceptable salt thereof.
There are many disease states in which excessive or unregulated IL-8 production is
implicated in exacerbating and/or c~llsinE the disease. These ~ e~çs are characterized by
massive neutrophil infiltration such as, psoriasis, inflAmm~tory bowel ~i~c~ce~ A~thm~,
cardiac and renal reperfusion inJury, adult respilatoly distress syndrome, thrombosis and
15 glomerulone~hliLis. All of these diseases are associated with increased IL-8 production
which is responsible for the chemotaxis of ncuLl~hils into the inflAmm~tory site. In
contrast to other inf~AmmAtQry cytokines (IL- 1, TNF, and IL-6), IL-8 has the unique
. ly of promoting neutrophil chemotaxis and activation. Therefore, the inhibition of
IL-8 production would lead to a direct reduction in the neulluphil infiltration.The compounds of Formula (I) are ~Amini~tered in an amount sllffic;ent to inhibit
cytokine, in particular IL-1, IL-6, IL-8 or TNP, production such that it is regulated down to
norrnal levels, or in some case to subnormal levels, so as to ameliorate or prevent the
disease state. Abnormal levels of IL-1, II,-6, IL-8 or TNF, for in-~t~nre in the context of the
present invention, consLilule: (i) levels of free (not cell bound) IL-I, IL-6, 11,-8 or TNF
2~ greater than or equal to 1 picogram per ml; (ii) any cell associated IL-l, IL-6, II,-8 or
TNF; or (iii) the presence of IL-l, IL-6, IL-8 or INF mRNA above basal levels in cells or
tissues in which IL-1, IL-6, IL-8 or TNF, lc*~e~;livcly~ is produced.
The discovery that the compounds of Formula (I) are inhibitors of cytokines,
specifically IL- 1, IL-6, IL-8 and TNP is based upon the effects of the compounds of
30 Formulas (I) on the production of the IL-l, IL-8 and TNF in in vitro assays which are
described herein.
As used herein, the term "inhibiting the production of IL-l (IL-6, IL-8 or TNF)"refers to:
a) a decrease of excessive in vivo levels of the cytokine (IL-l, IL-6, IL 8 or TNF)
35 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;
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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- l, IL-6, IL-8 or TNT~- ~ in a human to normal or sub-normal levels.
As used herein, the term "TNF meAiplte~l disease or disea~se state" refers to any and
all disease states in which TNF plays a role, either by production of TNF itself, or by TNF
c~ in~ another monokine to be released, such as but not lirnited to IL-1, IL-6 or IL-8. A
10 disease state in which, for inst~nee~ IL-1 is a major component, and whose production or
action, is exacerbated or secreted in response to TNF, would therefore be considered a
disease stated m~ t~l by TNF.
As used herein, the term "cytokine" refers to any secreted polypeptide that affects
the functions of cells and is a molec~ which modulates interactions between cells in the
15 immllne7 infl~mm~tory or hematopoietic response. A cytokine inrl~ es, but is not limited
to, monokin~s and lymph-)kin~s, regardless of which cells produce them. For in~t~nr,e, 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,
20 brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-lymphocytes.
Lymphokin~s are generally referred to as being produced by lymphocyte cells. Examples
of cytokines incllltle, but are not limited to, Interleukin-l (IL-l), TntPrlellkin-6 (IL-6),
Tnt~rlPukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-oc) and Tumor Necrosis Factor
beta (TNF-B).
As used herein, the term "cytokine interfering" or "cytokine ~.u~ ssive amount"
refers to an effective ~mollnt of a compound of Formula (I) which will cause a decrease in
the in vivo levels of the cytokine to normal or sub-norrnal 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.
As used herein, the cytokine referred to in the phrase "inhibition of a cytokine, for
use in the tre~tment of a HIV-infected human" is a cytokine which is implicated in (a) the
initiation and/or . . .~ e of T cell activation and/or activated T cell-m~ t~l HIV
gene expression and/or replication and/or (b) any cytokine-mediated disease associated
problem such as cachexia or muscle degeneration.
As TNF-~ (also known as lymphotoxin) has close structural homology with TNF-a
(also known as cachectin) and since each induces similar biologic responses and binds to
the same cellular receptor, both TNF-a and TNF-~ are inhibited by the compounds of the
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present invention and thus are herein referred to collectively as "TNF" unless specifirs.lly
delinP~S7tPd otherwise.
A new member of the MAP kinase family, alternatively termed CSBP, p38, or RK,
has been identified independently by several laboratories recently [See Lee et al., Nature,
Vol. 300 n(72), 739-746 (1994)]. Activation of this novel protein kinase via dual
phosphorylation has been observed in diff~G.It cell systems upon stim--lS~tion by a wide
~Ct;llulll of stimuli, such as physicochemical stress and Ll~,alll~~t with lipopolyss rchs ri~l~
or proinflS7mmS7tory cytokines such as intPrieukin-l and tumor necrosis factor. The
cytokine biosynthesis inhihitors, of the present invention, compounds of Formula (I), have
10 been clet~ Pd to be potent and selective inhibitors of C~BP/p38/RK kinase activit,v.
These inhibitors are of aid in dete! ...i";l~ the ~i~ns71ing ~Jal~lW~5; involvement in
inlls7mms7tory responses. In particular, for the first time a definitive signal tr;~n~duction
pathway can be prescribed to the action of lipopolyss7c~çhs7ri(1e in cytokine production in
macrophages. In addition to those ~lice~es already noted, treatment of stroke, neulvLIdullla,
cardiac and renal lepel~usion injury, thrombosis, glomerulonephritis, ~ hetrs and
pancreatic ,B cells, mnltirle sclerosis, muscle d~genPration, ec7Pm~ psoriasis, sunburn, and
col~ lclivilis are also inr.l~lrlrrl
The cytokine inhibitors were subsequently tested in a number of animal models
for anti-inflS~mmS~tory activity. Model systems were chosen that were relativelyin.cPncitive to cyclooxygenase inhihitor.c in order to reveal the unique activities of
cytokine ~u~lGs.sive agents. The inhihitors exhibited ~jgnifics7nt activity in many such
in vivo studies. Most notable are its effe~ ,ness in the collagen-in~uce-l arthritis model
and inhibition of TNF production in the endotoxic shock model. In the latter study, the
reduction in plasma level of TNF correlated with survival and protection from endotoxic
shock related mortality. Also of great i.~ oll~ce are the colllpuullds effectiveness in
inhibiting bone resorption in a rat fetal long bone organ culture system. Griswold et al.,
(1988) Arthritis Rheum. 31:1406-1412; Badger, et al., (1989) Circ. Shock 27, 51-61;
Votta et al., (1994)in vitro. Bone 15, 533-538; Lee et al., (1993). B Ann. N. Y. Acad.
Sci. 696, 149-170.
In order to use a compound of Formula (I) or a phs7rm~reutics711y acceptable salt
thereof in therapy, it will normally be Forml-ls tr~ into a phs7n~7s7relltical composition in
accordance with standard phs7rms7re~ltics71 practice. This invention, therefore, also relates to
a pharn-~renticS~l colll~osilion coll~ .ing an effective, non-toxic amount of a compound of
Pormula (I) and a pharrnS7re~ltirS7lly acceptable carrier or diluent.
Compounds of Formula (I), phS7rn7~reutirs7lly acceptable salts thereof and
phs7rms7reutical compositions incorporating such may conveniently be s7~mini~tered by any
of the routes conventionally used for drug s7~1mini~tration, for inctS7nce. orally, topically,
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pa~ f,ldlly or by inhalation. l'he compounds of Formula (I) may be ~lmini~tf~red in
conventional dosage forms plGpa.~,d by combining a compound of Formula (I) with
standard ph~rn ~relltical carriers according to conventional procedures. The compounds of
Pormula (I) may also be ~flmini~tf-.red in conventional dosages in combination with a
5 known, second therapentif ~lly active compound. These procedures may involve mixin~,
gr~n~ tin~ and colll~lessing or dissolving the ingredients as ~ ~iate to the desired
cpalaLion. It will be appreciated that the form and çh~r~rtPr of the ph~rm~f e~ltic"lly
acceptable character or diluent is f~ t~tf~rl by the amount of active ingredient with which it
is to be combined, the route of ~1mini~tration and other well-known variables. The
10 carrier(s) must be "acceptable" in the sense of being comr~tihle with the other ingredients
of the Formulation and not ~el~tf-rious to the recipient thereof.
The ph~rm~el~tical carrier employed may be, for example, either a solid or liquid.
F.xempl~ry of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,
nPcium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup,
15 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 phs~rm~f elltic ~1 forms can be employed. Thus, if a solid carrier is
used, the ~lG~aLion can be t~hif~tf ~l placed in a hard gelatin capsule in powder or pellet
20 form or in the form of a troche or lozenge. The amount of solid calTier 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 forrn of a syrup, emulsion, soft gelatin c~rslllf, sterile injectable
liquid such as an ampule or nonaqueous liquid suspension.
Co~ ou~ds of Formula (I) may be ~Amini~tered topically, that is by non-systemic
2~ ~flmini~tration. This inf lllf'f s the application of a co~"po~nd of Formula (I) externally to
the epidermis or the buccal cavity and the inctill~fion of such a compound into the ear, eye
and nose, such that the co~ oulld does not .~i~nifi(~"ntly enter the blood stream. In contrast,
systemic ~flmini~tration refers to oral, intravenous, inl~ ol~~~1 and il~ uscular
Z~f~ on.
Form~ tif)ns suitable for topical "flmini~tration include liquid or semi-liquid
~alations suitable for penc;l~dlion through the skin to the site of ;nfl~mm~tion such as
li"i...~ , lotions, creams, ointmf nt~ or pastes, and drops snit~ble for ~lmini~tration to the
eye, ear or nose. The active ingredient may comprise, for topical ~-lmini~tration, from
~ 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 conlprise less than 5% w/w,
more preferably from 0.1% to 1% w/w of the Form~ tion.
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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
ctmt~ining a bactericide and may be prepared by methods similar to those for the~lc~alation of drops. Lotions or liniment~ for application to the skin may also include an
agent to hasten drying and to cool the skin, such as an alcohol or ~reton~, and~or a
moictnri7P~ such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointm~-.nt~s or pastes according to the present invention are semi-solidForm~ tions of the active ingredient for e~tf~rn~1 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 m~rhin~.ry, with a
greasy or non-greasy base. The base may comrrice hydrocarbons such as hard, soft or
liquid pdld~rlll, glycerol, beeswax, a m~ot~11iC 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
F )rm~ tinn may incolpc.ldlc any suitable surface active agent such as an anionic, cationic
or non-ionic surf~t~nt such as a sorbitan ester or a polyoxyethylene delival ive thereof.
Suspending agents such as natural gums, cellulose derivatives or inorganic m~t~ri~l~ such
as ~ci1ic~ceous silicas, and other ingredients such as l~nolin, may also be inc~ rle-1
Drops according to the present invention may co~ ise sterile aqueous or oily
solutions or sucperlcions and may be plc~aLcd by dissolving the active ingredient in a
suitable aqueous solution of a bact-rici~l~1 and/or f~1n~ici~l~1 agent and/or any other suitable
preservative, and preferably including a surface active agent. The res111ting solution may
then be cl~rifi~cl by filtration, transferred to a suitable container which is then sealed and
steri1i~t~cl by autoclaving or ~ i"i"g at 98-lO0~ C. for half an hour. ~l~rn~tively~ the
~25 solution may be steri1i7~d by filtration and transferred to the container by an aseptic
technique. Examples of bactericidal and fungicidal agents suit~hle for inclusion in the
drops are phenylmercuric nitrate or acetate (0.002%), ben7~1k~.nium chloride (0.01%) and
chlorh~iflin~ acetate (0.01%). Suitable solvents for the plG~dL~Lion of an oily solution
include glycerol, diluted alcohol and propylene glycol.
Compounds of formula (I) may be ~-l, l .i l);.ciL.~,I ed ~a Gl,teldlly, tnat is by iL~L avGllous,
h~Lld~ scular, ~.ub~;uLaneous intranasal, intrarectal, intravaginal or illLLd~Glilolleal
~lmini~tration. The subcutaneous and inL-,.111~~sc~ r forms of palGlltt;ldl ~imini~tration are
generally preferred. Appropriate dosage forms for such ~lmini.ctration may be prepared by
conventional techniques. Compounds of Fo~nula (I) may also be ~-lmini~tered by
inh~1~tion, that is by intranasal and oral inh~1~tion ~rimini~tration Appropriate dosage
forms for such ~rlminictration, such as an aerosol Formulation or a metered dose inhaler,
may be prepared by conventional techniques.
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For all methods of use disclosed herein for the co~ ollllds of Formula (I), 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 ~ lcl~l dosage regimen about 0.1 to about 80 mg/kg of total bodyS weight, preferably from about 0.2 to about 30 mg/kg, and more preferably from about 0.5
mg to l~mg/kg. The daily topical dosage regimen will preferably be from 0.1 mg to 150
mg, ~lmini.ctPred one to four, preferably two or three times daily. The daily inh~l~tion
dosage regimen will pre~erably be ~rom 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 a compound of Formula (I) or a rh~rm~ceutically acceptable salt
thereof will be ~l~t.-rmin~fl by the nature and extent of the condition being treated, the form,
route and site of z~-1mini~tration, and the particular patient being treated, and that such
o~ can be ~1ete~ 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 of a
co~ oul~d of Formula (I) or a ph~m~ce~ltic~lly acceptable salt thereof given per day for a
defined number of days, can be ascertained by those skilled in the art using convel~lional
course of IlGdL~ ,nt ~l~;t~ ",;,~ on tests.
The novel compounds of Forrnula (I) may also be used in association with the
vele~ll.~y tre~tmPnt of m~mm~l~, other than hllm~n~, in need of inhihition of cytokine
inhibition or production. In particular, cytokine mt~rli~tecl ~ e~eS for tre~tm~.n~,
t~c.,~ lly or ~r~llyl~ti~ ~lly, in ~nim~lc include disease states such as those noted
herein in the Methods of Tr~tm~-nt section, but in particular viral infections. Fx~mrl~s of
such viruses inf.luAe, but are not limited to, lelllivi~us infections such as, equine infectious
~n~ ni~ virus, caprine arthritis virus, visna virus, or maedi virus or l~,t~O~ us infections,
such as but not limited to feline imrnuno~efteienry virus (FIV), bovine immlln~ ficiency
virus, or canine i~ n~orlefie~ip~nry virus or other reL~vilal infections.
The invention will now be ~lescri~e(l by reference to the following biological
exarnples which are merely illLl~lldliv~ and are not to be co~sllued as a limit~fi~ n of the
scope of the present invention.
BIOLOGICAL EXAMPLES
The cytokine-inhibiting effects of compounds of the present invention were
deterrnine-l by the following in vitro assays:
I~ ,,.lc ~l~in ~ 1 (IL-1)
Human peripheral blood monocytes are isolated and purified from either fresh blood
alations from volunteer donors, or from blood bank buffy coats, according to theprocedure of Colotta et al, J Immunol, 132, 936 (1984). These monocytes (lx106) are
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plated in 2~well plates at a concentration of 1-2 million/rnl 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 lipopolys~cch~n-l~
(50 ng/rnl), and the cultures are incubated at 37~C for an additional 24h. At the end of this
S period, culture supern~t~nt~ are removed and cl~rifiçcl of cells and all debris. Culture
sl~pern~t~ntC are then im mt~ tely assayed for IL-l biological activity, either by the method
of Simon et al., J. Tmm~lnol Methods, 84, 85, (1985) (based on ability of IL-l to stimnl~te a
Tnterlt~.ukin 2 producing cell line (EL-4) to secrete IL-2, in concert with A23 187 ionophore)
or the method of Lee et al., J. ImmunoTherapy, 6 (1), 1-12 (1990) (ELISA assay).~ represP.~ ive compound of Formula (I), F~r~mrle 1, demonstrated positive
inhibition in this assay.
Tumour Nc~ ~ Factor (TNF):
Hurnan peripheral blood monocytes are isolated and purified from either blood bank
buffy coats or pl~tç~ .heresis re~iAIlces, acco,~lulg to the procedure of Colotta, R. et al., J
~nunol, 132(2), 936 (1984). The monocytes are plated at a density of lx106 cells/ml
n Pdinm/well in 24-well multi-dishes. The cells are allowed to adhere for 1 hour after
which time the sup~",~tSlnt is as~i,a~d and fresh m~tljurn (lml, RPMI-1640, Whitaker
Biomedical Products, Whitaker, CA) cont;~ g 1% fetal calf serum plus pçni(~.illin and
~llG~Lolllycin (10 ul~its/rnl) added. The cells are in~ b~t~l for 45 minntto.s in the presence or
20 absence of a test compound at lnM-lOmM dose ranges (co~ oullds are solubilized in
dimethyl sulfoxide/eth~nol, such that the final solvent concentration in the culture m~ lm
is 0.5% dimethyl sulfoxide/0.5% ethanol). Bacterial lipopoly-s~csh~ride (E. coli 055:B5
LPS~ from Sigrna ~h~mir~l~ Co.) is then added (100 ng/ml in 10 rnl phosphate buffered
saline) and cultures incubated for 16-18 hours at 37~C in a 5% C02 incubator. At the end
25 of the incubation period, culture sup~rn~t~nt~ are removed from the cells, ce~ ; r~ ~ged at
3000 rpm to remove cell debris. The ~u~ t~nt is then assàyed for TNF activity using
either a radio-~ll~o or an ELISA assay, as described in WO 92/10190 and by Becker et
al., J Tmmllnol, 1991, 147, 4307.
IL-l and TNF inhibitory activity does not seem to correlate with the l~lu~ Ly of the
30 compounds of Formula (I) in m~ ting arachidonic acid metabolism inhibition. Further
the ability to inhibit production of prost~ nflin and/or leukotriene synth~ , bynonsteroidal anti-infl~mm~tory drugs with potent cyclooxygenase and/or lipoxygenase
inhibitory activity does not mean that the compound will n.ocess~rily also inhibit TNF or
IL-l production, at non-toxic doses.
35 In vivo TNF ~ssay:
While the above jn~ t~c~ assay in an in vitro assay, the compounds of
Formula (I) may also be tested in an in vivo system such as described in:
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WO 97/25046 PCT/US97/00529
(1) Griswold et al., Drugs Under Exp. and Clir~ical Res..XIX (6), 243-248 (1993); or
(2) Boehm, et al., Journal Of Medicinal ~hemist~y 39, 3929-3937 (1996) whose
disclosures are incol~ulaled by reference herein in their entirety.
IL ~lrin -8 ~IL,-8 ):
Primary human umbilical cord e~ othPlial cells (HUVEC) (Cell Systems, Kirl~nn',
Wa) are ms~int~inP-l in culture mPrlillm supplPm~tPtl with 15% fetal bovine serum and 1%
CS-HBGF c~.n~ tin~ of aFGF and hPp .rjn The cells are then diluted 20-fold before being
plated (250~1) into gelating coated 96-well plates. Prior tû use, culture m~r'.inm are
replaced with fresh mPf'illm (200,ul). l3uffer or test compound (25~, at con-~Pntrations
10 between 1 and lO,uM) is then added to each well in quadr lpliç~t~ wells and the plates
in~,llb~t.o(l for 6h in a hnmi~;fi.o.~l in.-l-b~lol at 37~C in an ~tm~sph~e of 5% C02. At the
end of the in~llb~til n period, ~u~e .~t~nt is removed and assayed for IL-8 concentration
using an IL-8 ELISA kit obtained from R~D Systems ~Minneapolis, MN). All data is~lesr~ rl as mean value (ng/ml) of mllltirlP. s~mrl~P.s based on the standard curve. IC50's
15 where a~p~ iate are generated by non-linear regression analysis.
Cyto~ Specific Binding r~vlei~ Assay
A radioco...pel ;l i v~ bin~ ng assay was developed to provide a highly reproducible
~'lilll~ screen for structure-activity studies. This assay provides many advantages over
the con~ Lional bioassays which utilize freshly i~ok~te(~ human monocytes as a source of
cytokines and ELISA assays to quantify them. Re~i-1Ps being a much more facile assay,
the binding assay has been extensively v~ t~cl to highly correlate with the results of the
bioassay. A specific and reproducible cytokine inhibitor binding assay was developed
using soluble cystosolic fraction from TEIP. 1 cells and a radiolabeled cc,nl~o~ d. Patent
Application USSN 08/123175 Lee et al., filed September 1993, USSN; Lee et al., PCT
94/10529 filed 16 September 1994 and Lee et al., Nature 300, n(72), 739-746 (Dec. 1994)
whose disclosures are incol~ol~ted by reference herein in its c~ GIy describes the above
noted method for screening drugs to identify compounds which illt~,.a;l with and bind to
the cytokine specific binding protein (hereinafter CSBP). However, for purposes herein
the bin-lin~ protein may be in isolated form in solution, or in immobilized form, or may be
g~n~tic~lly e~ginPered to be expressed on the surface of recomhin~nt host cells such as in
phage display system or as fusion proteins. ~ltPrn~tively, 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 sllfflcient to forrn a compoundl binding protein comple~c and
compound capable of forming, enhancing or illtelre~ g with said complexes are detected.
Represent~tive final compounds of Pormula (I), Examples 1 to 4, and 6 have all
demonstrated positive inhibitory activity of an IC~o of < SOuM in this binding assay.
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5; SRP KINASE ASSA~:
This assay measures the CSBP-catalyzed transfer Of 32p from [a-32P3ATP to
threonine residue in an epi(1~-rrn~l growth factor receptor (EGFR)-derived peptrde
(T669) with the following sequence: KRELVEPLTPSGEAPNQALLR (residues
S G61-681). (See ~T~ gh~.r et al., "Regulation of Stress Tn~ red Cytokine Production
by Pyridinyl Tm~ 7oles: Inhibition of CSPB Kinase", BioOrganic & Meflir,in~l
Chemistry, to be published 1996).
Kinase reactions (total volume 30 ul) contain: 25 mM Hepes buffer, pH 7.5;
10 mM MgC12; 170 uM ATP(l); 10 uM Na ortho vanadate; 0.4 mM T669 peptide;
and 20-80 ng of yeast-e~icss~d purified CSBP2 (see Lee et al., Na~ure 300, n(72),
739-746 (Dec. 1994)). Colllpoullds (5 ul from [6X3 stock(2)) are pre-incllb~tefl with
the enzyme and peptide for 20 min on ice prior to starting the reactions with
32P/MgATP. Reactions are incub~t~-~ at 30 oc for 10 min and stopped by adding 10ul of 0.3 M phosphoric acid. 32P-labeled peptide is separated on phosphocellulose
(Wattman, p81) filters by spotting 30 ul reaction ~ LuLe~ Filters are washed 3 times
with 75 mM ph~sphoric acid followed by 2 washes with H20, and counted for 32P.
(1) The Km of CSBP for ATP was t~ e~ l to be 170 uM. Therefore,
compounds screened at the Km value of ATP.
(2) Compounds are usually dissolved in DMSO and are diluted in 25 mM
20 Hepes buffer to get final co,lcel,t,dlion of DMSO of 0.17%.
Re~,;,e~ live final compounds of Pormula (1), Examples 1,5 8, and 9 have all
demonstrated ~o~iLive inhibitory activity of an ICso of < 50uM in this binding assay.
Fx~mple 10 demonstrated an IC50 of > 50uM in this assay.
25 Pl v~l~J~ c' ~ e .r oxide ~.y~lhas~-2 (PGHS-2) assay:
The following assay describes a method for ~ele....i~ g the inhibitory effects of
compounds of Formula (I) on human PGHS-2 protein expression in LPS stim~ t~d human
monocytes.
Method: Human periph~l blood monocytes were isolated from buffy coats by
30 centrifugation through Ficoll and Percoll gradients. Cells were seeded at 2 X 106/well in
24 well plates and allowed to adhere ~or 1 hour in RPMI supplemented with 1% human AB
serum, 20mM L-~ , ptonicillin-Streptomycin and 10mM HEPES. Compounds were
added at various concentrations a~d incubated at 37~C for 10 minnte~. LPS was added at
50 ng/well (to induce enzyme expression) and in~l~b~tP-l overnight at 37~C. The
3~ supernatant was removed and cells washed once in cold PBS. The cells were lysed in
lOO,ul of cold lysis buffer(50mM Tris/HCl pH 7.5, 150mM NaCl, 1% NP40, 0.5% sodium
deoxycholate, 0.1% SDS, 300ug/ml DNAse, 0.1% Tl~ITON X-100, lmM PMS~, lrnM
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leupeptin, lmM ~ atill). The lysate was centrif~ged (10,000 X g for 10 min. at 4~C) to
remove debns and the soluble fraction was subjected to SDS PAGE. analysis (12% gel).
Protein separated on the gel were transferred onto nitrocellulose ,lw.)l~ e by
electrophoretic means for 2 hours at 60 volts. The membrane was pretreated for one hour
S in PBS/0. 1% Tween 20 with 5% non-fat dry miLk. After washing 3 times in PBS/Tween
buffer, the membrane was inr~llb~tP~ with a 1:2000 dilution of a monospecific antiserum to
PGHS-2 or a 1:1000 dilution of an antiserum to PGHs-l in PBS/Tween with 1% BSA for
one hour with continuous ~h~kin~ The membrane was washed 3~ in PBS/Tween and then
incubated with a 1:3000 dilution of horseradish peroxidase conjugated donkey antiserum to
10 rabbit Ig (~mF r.ch~m) in PBS/l ween with 1% BSA for one hour with continuous sh~king.
The m~..-.l-l,.,lF was then washed 3X in PBSlTween and the ECL imrnunodetection system
(AmPr.ch~m) was used to detect the level of expression of prost~gl~nclin endoperoxide
synthases-2.
Results: The following compounds were tested and found to be active in this assay (i.e.,
inhibited LPS in~ e-l PGHS-2 protein e~ ssion in rank order potency similar to that for
inhibiting cytokine production as noted in assays in-lic~tFed):
4-(4-Fluc luphc.lyl)-2-(4-methylsulrll,ylpllenyl)-5-(4-pyridyl)imi-1~7Qle
6-(4-Fluo-v~h~lyl)-2,3-dihydro-5-(4-pyridinyl)imitl~7Ot2,1-b~thiazole; and De~mPth~one
Several compounds were tested and found to be inactive (up to 10uM):
2-(4-Methylsulfinylphenyl)-3-(~pyridyl)-6,7-dihydro-(5H)-pyrrolo[ 1,2-
a]imi~1~7ole;rolipram, ph~-ni~lone and NDGA.
None of the compounds tested was found to inhibit PGHS- 1 or cPLA2 protein
levels in si~lar e.~ F~
TNF-a in T-~ Brain Injury Assay
The present assay provides for e~min~tion of the expression of tumor
necrosis factor mRNA in specific brain regions which follow e~ t~lly in~ ced
lateral fluid-percussion tr~l~m~tiC brain injury (TBI) in rats. Adult Sprague-Dawley
rats (n=42) are ~n~ F~.d with sodium pentobarbital (60 mg/~g, i.p.) and subjected
to lateral fluid-pe,.;~lssion brain iniury of moderate severity (2.4 atm.) centered over
the left te~lpo~ uietal cortex (n=18), or "sham" treatment (~npsthp~ia and surgery
without injury, n=18). Animals are s~c-rifiçe~l by decapitation at 1, 6 and 24 hr. post
injury, brains removed, and tissue samples of left (injured) parietal cortex (LC),
corresponding area in the contralateral right cortex (RC), cortex adjacent to injured
parietal cortex (LA), corresponding adjacent area in the right cortex (RA), lefthippocampus (LEI) and right hippocampus (RH) are prepared. Total RNA is isolated
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and Northern blot hybri~li7~tion is pel~~ ed and qllAntitAt~l relative to an TNF-oc
~o~ilive control RNA (macrophage = 100%). A mArk~ll increase of TNF- a mRNA
e~lGssion is observed in LH (104+17% of positive control, p < 0.05 co~ d with
sham), LC (105+21%, p< 0.05) and LA (69+8%, p c 0.01) in the tr~nm~ti~ed
hemisphere 1 hr. following injury. An increased TNF- a mRNA expression is also
observed in I,H (46+8%, p < 0.05), LC (30+3%, p < 0.01) and LA (32+3%, p < 0.01)at 6 hr. which resolves by 24 hr. following injury. In the contralateral h~mi~ph~o~re~
e~lession of TNF- a rnRNA is increased in RH (46~2%, p < 0.01), RC (4+3%) and
RA (22+8%) at 1 hr. and in RH (28+11%), RC (7+5%) and RA (26+6%, p < 0.05) at
10 6 hr. but not at 24 hr. following injury. In sham (surgery without injury) or naive
AnimAl~, no consistent changes in expression of TNF- a mRNA is observed in any of
the 6 brain areas in either hemisphere at any times. These results in~ ate that
following par~cAgitt~l fluid-pel~;u~sion brain injury, the t~ ol~l expression of TNF-
a rnRNA is altered in specific brain regions, inrlu-1inf~ those of the non-trAnm15 hemisphere. Since TNF- a is able to induce nerve growth factor (NGF) and
stimlllAt~ the release of other cytokines from activated a~,o~;yl~s, this post-trA--mAtir
alteration in gene e~lcssion of TNF- a plays an important role in both the acute and
regenerative response to CNS trauma.
20 ~NS Tn~iury model ~or IL-~ mRNA
This assay characterizes the regional expression of interleukin-1~ 1B)
mRNA in specific brain regions following e7crerim~nt~l lateral fluid-percussion
tr~ tic brain injury (IBI) in rats. Adult Sprague-Dawley rats (n=42) are
~n~sthtoti7.o(l with sodium pentobarbital (60 mg/kg, i.p.) and subjected to lateral
25 fluid-~c~;ussion brain injury of moderate severity (2.4 atm.) centered over the left
temporAp~riPtAl cortex (n=18), or "sham" L.~ .l (Anesth~ci~ and surgery without
injury). Animals are s~crifi~eA at 1, 6 and 24 hr. post injury, brains removed, and
tissue s~n-ples of left (injured) parietal cortex (L,C), co,le~onding area in the
contralateral right cortex (RC), cortex ~ A~ent to injured parietal cortex (LA),30 corresponding ~-1jAr~nt area in the right cortex (RA), left hippocarnpus (LH) and
right hippoc~n-rl~ (RH) were ~rep~ed. Total RNA is isolated and Northern blot
hy7Wdization is pe,~.,lled and the quantity of brain tissue IL-l~ mRNA is
presented as percent relative radioactivity of IL- ~ ~ positive macrophage RNA
which is loaded on same gel. At 1 hr. following brain injury, a m~rk~fl and
35 ~ignifir~nt increase in e~ ssion of IL-lB rnRNA is observed in LC (20.0_0.7% of
positive control, n=6, p < 0.05 compared with sham animal), LH (24.5_0.9%,
p < 0.05) and LA (21.5+3.1%, p < 0.05) in the injured hemisphere, which remained
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elevated up to 6 hr. post injury in the LC (4.0~0.4%, n=6, p < 0.05) and LH
(5.0+1.3%, p < 0.05). In sham or naive ~nim~l~, no expression of IL-l~ mRNA is
observed in any of the respective brain areas. These results indicate that following
TBI, the L~ dl expression of IL-l~ mRNA is regionally stim~ t~d in specific
brain regions. These regional changes in cy~okin~, such as IL-113 play a role in the
post-tr~llm~ti~ pathologic or regellGld~ivG sequelae of brain injury.
SY~I l'H~':llC EXAMPLES
The invention will now be described by reference to the following examples whichare merely illu~lldlive and are not to be construed as a limitation of the scope of the present
invention. All l~m~er~Lulcs are given in degrees centigrade, all solvents are highest
available purity and all reactions run under anyllluus conditions in an argon atmosphere
unless othenvise intlic~t~rl Mass spectra were ~ rolllled upon a VG Zab mass
spectrometer using fast atom bombardment, unless otherwise in(1ir~t~(1 lH-NMR
1~ (hereinafter "NMR") spectra were recorded at 250 MHz using a Bruker AM 250 or Am 400
spectrometer. Mlllti~ ti~s in~ t~(l are: s=singlet, d=doublet, t=triplet, q=quartet,
m=multiplet and br indicates a broad signal. Sat. inf~ t~:S a saturated solution, eq indicates
the plupulLion of a molar equivalent of reagent relative to the principal re~t~nt
Plash cllrolllatography is run over Merck Silica gel 60 (230 - 400 mesh).
Example 1
5-(2-Methoxy-4-pvrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinvl)irnidazole
a) 2-N-Methylthio~li."iclin~carboxaldehyde dimethyl acetal
Pyruvic aldehyde d1I11GI11Y1 acetal (60 rnL, 459 mmol) and N,N-dimethyl form~mifie
dilllGLllyl acetal (60 rnL, 459 rnmol) were stirred together at 100 for 18 h. The ll~iAIulG
was cooled.
Methanol (300 mL), thiourea (69.6 g) and sodium methoxide (231 rnL, 25 wt% in
MeOH) were added to the above mixture and stirred at 70~ for 2 h. After cooling,iodom~th~nlo (144 mL) was added dropwise and the lllix.LulG was stirred 3 h. at room temp.
After ~lilntin~ with PtOAc and H20, the organic phase was sep~r~tefl, dried (Na2S04),and
concelllldted to yield the title compound as a brown oil (75.~ g, 82% yield). lH NMR
(CDC13): d 8.17 (d, lH), 6.77 (d, lH), 5.15 ~s, lH), 3.40 (s, 6H).
b) 2- Metho~y~ylhnidine-4-carboxaldehyde dimethyl acetal
The product of the prece~ling example (5.0 g, 25 mmol) was dissolved in m~th~nol~ (100 mL), cooled to 4~and a solution of oxone (9.21g), in H20 (100 mL) was added
dropwise (T < 15~). Warmed to 23~, stirred 2h, poured into 10 % aq NaOH (250 mL) and
- extracted with EtOAc. The extracts were washed with 10% aq NaOH, dried (Na2SO4),
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filte.~d, concentrated, and flash cnromatographed (70% hexanelEtOAc) to afford 1.66g
(36%) of the title compound. ESP~ (Mass Spec) m/z 185 (MH+3.
c) 2- Metho~y~y~ line~-carboxaldehyde
The product of the prece~ling example (0.54 g, 2.93 mmol), was dissolved in 3 M
S HCl (2.17 mL, 6.5 mmol) and stirred at 23~ for 3 days, cooled to 4~, layered with EtOAc
and made slightly basic by the addition of solid Na2CO3. Extraction with EtOAc (5 x 40
mL) afforded 0.309 g (76%) of the title compound as a white solid. lH NMR (CDC13): d
9.96 (s,1), 8.78 (d,l), 7.46 (d, 1), 4.10 (s, 3).
d) 1-t-Bulo~yc~bullyl4-aminopiperidine
1- t-Buto~yc~l,oliyl piperidine~-one (cornmercially available from T ~n~eter
Chem) (39.9 g, 0.20 mol), THF (150 mL), EI2O (300 rnL), and H2NOH HCl (55.2, 0.80
mol) were dissolved together and Na2CO3 (55.2 g, 0.53 mol) was added in small portions.
The llLi~luie was stirred at 23~ for 14 h, most of the THF was evaporated in vacuo, adjusted
to pH > 10 with 50% aq NaOH, extracted with EtOAc(5 x 50 mL) and concentrated to a
15 white foam. Triturated with hexane, filtered and the solid was dried in vacuo to afford
40.31 g of the title co~ oulld.
The above residue was dissolved in EtOH (absolute, 1 L) and Raney Ni (50 mL of aslurry in EtOH) was added and the mi~ule was reduced under H2 (50 psi) for 3.5 h. The
catalyst was filtered off and washed with EtOH to afford. Concentration afforde 38.44g
20 (96% overall) of the title compound as a colorless oil which solidified to a white solid upon
sts~n~1in~ at-20~.
e) 4-Fluulul~rlcllyl-tolylsulfonolll~ ylro~ icle
To a ~u~ ion of p-tol~ n~s~ nic acid sodium salt (30 g) in H20 (100 mL) was added
methyl t-butyl ether (50 mL) followed by dropwise addition of conc HCl (15 rnL). After
25 stirring 5 min, the organic phase was removed and the aqueous phase was extr~-~teA with
methyl t-butyl ether. The organic phase was dried (Na2S04) and conce,lllated to near
dryness. Hexane was added and the resllltin~ preCirit~t~ collected to afford p-
tolll~neslllfinic acid; yield 22 g.
p-T~ e~ s--lfinie acid (22 g, 140.6 ~nol), p-fluorobt~n7~kl.-rlyde (22 mL, 206 mmol),
30 fnrrn~mide (20 nL, 503 mmol) and camphor sulphonic acid (4 g, 17.3 mmol) were combined
and stirred at 60 ~ 18 h. The resulting solid was broken up and stirred with a l~ lul~ of
MeOH (35 mL) and hexane (82 mL) then filtered. The solid was resuspended in MeOH /
hexanes (1:3, 200 mL) and stirred vigorously to break up the rem~ining chunks. Filtration
af~orded the title compound (27 g, 62 % yield): lH NMR (400 MHz, CDC13) d 8.13 (s, lH),
35 7.71 (d, 2H), 7.43 (dd~ 2H), 7.32 (d, 2H), 7.08 (t, 2H), 6.34 (d, lH), 2.45 (s, 3H).
f) 4-Fluorophenyl-tolylsul~ûnomethylisocyanide
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WO 97/25046 PCT~US97/00529
4-Fluorophenyl-tolylsulfonomelhylro~ mi~ (2.01g, 6.25 mmol~ in DME (32 mL) was
cooled to -10 ~C. POC13 (1.52 m~, 16.3 mmol) was added followed by the dropwise
addition of triethylamine (4.6 rnL, 32.6 mmol) in DME (3mL) keeping the internalte~ eldLul~, below -5 ~. The ll~h-Lule was gradually warmed to ambient temperature over 1
S h., poured into H20 and ~ ed with EtOAc. The orga~uc phase was washed with sat aq
NaHCO3, dried (Na2S04), and concel~ t~d. The rçs~ ing residue was ~liLuldled with
petroleum ether and filtered to afford the title col~uLIlld (1.7 g, 90% yield): lH NMR
(CDC13) d 7.63 (d, 2H), 7.33 (m, 4H), 7.10 (t, 2H), 5.60 (s, IH), 2.50 (s, 3H).
g) 2- Metho~y~yl; " ~;~ine-4-carboxaldehyde rl-t-blllo~syc~l,ollyl-4-aminopiperidinel
10 i ~e
The product of example l(d) (0.308 g, 2.23 mmol), and the product of example l(c)
(0.468 g, 2.34 mrnol) were combined in CH2C12 (50 mL) and stirred at 23~ for 16h.
Concentration afforded the title compound as a light orange foam. lH NMR (CDC13): d
8.56 (d, 1), 8.26 (s, 1), 7.57 (d, 1), 4.05 (s and m, 4), 3.5 (m, 2), 3.0 (m, 2), 1.75 (m, 4),
1.46 (s, 9).
h) 5-(2-Methoxy-4-pyrimidinyl)-4-(4-ffuorophenyl)-1-~(1-t-butoxycarbonyl)-4-
piperidinyllimi-l~7Ole
The product of the prece(1in~ example, DMF (5 rnL), the product of e~mple l(f)
(0.708 g, 2.23 mmol) and K2C03 (0.308 g, 2.23 mmol) were combined and stirred for 2
20 days, diluted with Et20 and filtered. The filtrate was co~ . d~d under high vacuum to a
brown solid. Trituration witb Et~O and hexane (1: 1, 200 mL) afforded the title compound
as a tan solid. Cryst~11i7~tinn from ?-~etorl~/hexane afforded 0.50~g (64% from the product
of example 4(c). ESP+ (Mass Spec) m/z 453 (MH~).
i) 5-(2-Methoxy-4-pyrimidinyl)-4-(4-fluolo~hellyl)- l-(4-piperidinyl)imi~1~7.( 1e
The product of the prece~l;ng example (0.505g, 1.43 mmol), was added to ice coldTFA, under Ar. The resulting solution was warmed to 23~ and stirred 1.5 h. The TFA was
removed in vacuo the residue was dissolved in EtOAc and extracted into H2O (2 x 20 mL).
The com~ined aqueous phases were layered with EtOAc and cooled to 4~, made basic by
the addition of 10% aqueous NaOH and the aqueous was extracted with EtOAc (4 x 25
mL). The combined extracts were dried (Na2SO4) and concentrated to a white crystalline
solid. Trituration of the solid with hexane afforded 16~ mg of white solid. Lvaporation of
the above filtrate afforded an additional 133mg of slightly yellow crystals. Total yield 298
mg (~9%). For the first crop of crystals: mp 1~9-160~.
Fxample 2
5-(2-Iso-Propoxy~-pyrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imi~7Ole _
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a) ~-Methylthi~ h~idine-4-carbo~Aldehyde
The product of example l(a) (9.96 g, 50 rnmol), and 3 N HCl (42 mL,
126 mmol) were combined and sti~red at 48~ for 16h, cooled to 23~, combined withEtOAc (200rnL) and made basic by the addition of solid Na2C03 (12.6 g, 150 mmol).
The aqueous phase was extracted with EtOAc (4 x 150 mL, dried ~f~Na2S04),
conc~lLl~ted and the residue was filtered through a pad of silica (ca 150 rnL) with
CH2C12 to afford 7.49 g (97%) of the title c~3ll~0ulld lH NMR (CDCl3): ~ 9.96 (s, l),
8.77 (d, 1), 7.44 (d, 1), 2.62 (s, 3).
b) 2-Methylthic,~ ine-4-carboxaldehyde 1-t-l~ulo~y~;~bollyl~
10 ~minopiperidine imine
The product of the previous step (4.84 g, 31.4 mmol), MgSO4 (ca 2 g), the
product of example l(d) (6.51 g, 32.6 mmol) and CH2Cl2 (100 ml) were combined
and stirred at 23 ~ for 16 h. Filtration and concentration of the filtrate afforded the title
compound as a yellow oil. lH NMR (CDCl3): ~ 8.57 (d, 1), 8.27 (s, 1), 7.58 (d, 1),
15 4.05 (m, 2), 3.55 (m, 1), 3.00 (m, 2), 2.60 (s, 3), 1.75 (m, 4), 1.48 (s, 9).
c) 5-(2-Methylthio-4-pyrimi-linyl)-4-(4-fluorophenyl)-1-r(l-t-butoxycarbonyl)~-
piperidinyllimid~ole
T~he product of the previous çx~mrle and the product of example l(f~ (9.41 g, 32.6
mmol), DMF (64 mL) and K2CO3 (4.43 g, 32.4 mmol) were reacted by the procedure of
20 e~mple l(h) to afford 9.07 g of product (62% from the product of example l(a). MS ES+
rn/z = 470 f~MH+).
d) 5-(2-M~-tl~ylsulfinyl-4-pyrimidinyl)~(4-fluorophenyl)- 1 -rt 1 -t-butoxycarbonyl~4-
piperidinyllimidazole
The product of the previous example (4.69g, 10 mmol) was dissolved in THF,
25 cooled to -10~ and oxone (6.14g, 10 ~nol) in H2O (50 rnL) was added dropwise (T < 5~).
The resllltin~ , was warmed to 20~ over ca 50 min, poured into a vigorously stirred
Ul~ of 10% aq NaOH (300 mL), ice (100 rnL), and EtOAc (300 mT ). The EtOAc was
separated, dried (Na2SO4), and concenl~dl~d to a yellow oil. Flash ehlolllaLography (0-2%
MeOH in CH2C12) afforded 3.58g (74%). ESP+ (Mass Spec) m/z 486 (MH+).
30 e) 5-f2-iso-Propoxy~-pyrirnidinyl)4-(4-fluorophenyl)- 1 --r~ 1 -t-butoxycarbonyl)-4-
piperidinyllimitl~7ole NaH (60% in mineral oil) was washed with dry THF and layered
with more THF (5 mL) and anhydrous iso-propanol (1.15 mL) was added. When the
bubbling subsided the reslllting soln was recooled to 23~ and the product of the previous
example (0.58 g, 1.19 mmol) in THF (5 mL) was added dropwise. After 5 min the reaction
35 was shaken with EtOAc (ca 100 mL) and H20 (50 mT ~) and the phases were separated and
-- 44 --
CA 02242974 1998-07-10
W O 97~250~6 PCTAU597~00529
the EtOAc was dried and conce"l~ d. The residue was cryst~lli7~-1 from ~cetQn~rhexane
to afford 335 mg of the title compound (58~). MS ES+ m/z = 482 (MH~).
f) 5-(2-iso-Propoxy-4-pyrir~udinyl)4-(4-fluorophenyl)- 1 -(4-piperidinyl)irnidazole
The product of the preceeding example (325 mg, 0.68 mmol) was treated with TFA
S by the procedure of example l(i). The crude product was crystallized from 13t20/hexane to
afford 106 mg (41%) of white crystals. mp = 121 - 122~.
Example 3
5-(2-Hydroxy-4-pyrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imicl~7-)le
10 trifln- roacetate _
a) 5-(2-Methylsulfonyl~-pyrimidinyl)-4-(~fluorophenyl~- 1 -r( l -t-butoxycarbonyl)-4-
piperidinyllimi~l~7~1e
The product of example 2(c) (9.07 g, 19.3 mmol), dissolved in 1~ was cooled to
-10~ and oxone (28.5g, 46.4 mmol)in H20 (250 rnL)was added dropwise. The resulting
15 lLUYlwc was stirred at 23~ for 24h, combined with ice (100 mL) and CH2C12 and washed
with brine (l00 mL), dried (Na2S04), concelllLdled and dried in vacuo to afford 8.27 g
(85%). MS ES+ m/z = 502 (MH+).
b) 5-(2-Hydroxy4-pyrimidinyl) 4-(4-fluorophenyl)-1-r(l-t-butoxycarbonyl)~
piperidinyl~imitl~zole
The product of the previous example (141 mg, 0.28 rnmol) was dissolved in THF ~5rnL) to which was added 50~o aq NaOH (150 uL, ca 1.8 mmol). The soln was stirred for 3
days and a precipitate foImed. The solid was filtered off, washed with THF, and dried in
vacuo to afford the title compound. MS ES~ m/z = 440 (MlI+).
5-(2-Hydroxy4-pyrimidinyl)-4-(4-fluorophenvl)- 1 -(4-piperidinyl)imi~1~7olP
25 c) The product of the previous example and TFA (3 mL) were combined and stirred
for 30 rnin, conc~ ed and the residue was triturated with ~t20 and filtered and the white
solid was washed with Et2O, dried in vacuo to afford 114 mg (90% of monoTFA salt from
the product of çx~mple 3(a). mp = 80 - l 10~ (dec).
Example 4
5-(2-Methoxy~-pyridinyl~l (4-fluorophenvl)-1-(4-piperidinyl)imitl~7Ole
a) 2-Chlo~ ylidine4-carboxaldehyde 1-t-l,ulokyca,l~onyl~aminopiperidine ;min~,
2-Chloropyridine-4-carboxaldehyde was yle~al~d as described in the patent
liLcldlulc (VVPI Acc. No. 88-258820/37) whose disclosure is incorporated by reference in
35 its entirety herein. This aldehyde was reacted with the product of example l(d) by the
procedure of example l(g) to afford the title compound as a yellow oil, d~alenlly a
lulc of imine isomers based on NMR. . lH NMR (CD3Cl): o 8.49, 8.35 (2d, lH),
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W O 97/25046 PCTrUS97/00529
8.22, 8,21 (2s, 1), 7.57, 7.29 (2s, lH), 7.45,7.12 (2d, lH), 2.93 (m, 1), 2.70 (m, 3), 1.64
(m, 3), 1.42, 1.40 (2s, 9), 1.17 (m, 2).
b) 5-(2-Chloro-4-pyridinyl) 4-(4-fluorophenyl)- 1 -(1 -t-buto~yc~l,onylpiperidin-
4-yl)irnidazole
The product of examp}e 4(a) was reacted with the product of example 1 (f~
by the procedure of example 1 (h). The crude product was filtered through silicaeluting with 0 - 2% MeOH in CH2C12 to afford the title compound as a light yellow
solid. MS ES+ m/z = 457, 459 (MH+).
c) 5-(2-Methoxv-4-pyridinyl) 4-(4-fluorophenyl~- 1 -(1 -t-butoxycarbonyl
10 piperidin4-yl)irnidazole
The product of the precee~in~ example (l.Og, 2.19 rnrnol) was dissolved in
25% NaOMe in MeOH (20 mL) and heated to reflux for 1 h, cooled and combined
with H20 and extracted with EtOAc (2x). The extracts were dried (Na2S04) and
concentrated. The residue was flash cl~omalographed (0 - 30% EtOAc in hexane)
15 afforded 300 mg (32%) of the title co~ oulld as a brown solid.
Crystals from acetone/h~Y~nP- MS ES~ mlz = 453 (MH+).
d) 5-(2-Methoxy~pyridinyl~-4-14-fluorophenyl) - 1 -(4-piperidinyl)irnidazole
The product of the previous example was reacted by the procedure of
ex~mrlel(i). The crude product was l~ dled with 1:10 Et20/hexane filtered and
20 dried in vacuo to afford the title compound as a white solid. mp=136 - 137.
Px~ S
5-(2-iso-PropQxv-4-pyridinyl)-4-(4-fluolo~he~ (4-piperidinyl)imi-1~7,ole
The product was ~lc~ared by the procedure of Example 4 sub~ ; . ,g sodium
25 isopropoxide and is.,~r~allol for sodium methoxide and methanol. MS ES~ m/z = 381 (MH+).
F.y~m~le 6
5-~2-Methylthio-4-pyrimi~linyl)-4-(4-fluorophpnyl)- 1 -(4-piperidinyl)imidai!;ole
The product of example 2(c) was reacted by the procedure of example l(i) to
afford the title compound. mp = 182 - 183~.
Ex~m~1e 7
5-(2-Methylthio-4-pyrimi~1inyl)-4-(4-fluorophenyl)- 1 -r(1 -methyl)-4-
3~ piperidinyl]imi~l~7Ole
a) 2-Methylthiopvrimicline-4-carboxaldehyde 1-methyl-4-arninopiperidine irnine
- 46 -
_
CA 02242974 1998-07-10
W~ 97~25046 PCTAUS97/aO529
The product of example 2(a) was reacted with 1-methyl 4-amino piperidine
by the procedure of ç~mple 2(b) to afford the title co.,-~ound
b) 5-(2-Methylthio4-pyrirnidinyl)-4-~4-fluu~ yl)- 1 -r( 1 -methyl)-4-
piperidinvll;mi-1~7,Qle
The product of the previous example was reacted with the product of
example 1 (f) by the procedure of example 1 ~i) to afford the title compound. mp =
181 - 182~.
Fx~m,l?le g
5-(2-Ethoxv4-pyrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imidazole
10 a) 5-(2-Fthoxy~-pyrimidinyl)-4-(4-fluoropheny~ rf l-t-b~lL~yc~lJollyl)~-
piperi~ yllimi~l~7ole The title colll~uulld was ~ ,d by the method of example 2(e)
except that anhydrous EtOH was used in place of 2-propanol.
b) 5-(2-Ethoxy-4-pyrimi~linyl)~-(4-fluoroph~nyl)-1-(4-piperidinyl~imi~ ole
The product of the precee~lin~ example was treated with TFA by the procedure of
e~c~mrle 1 (i) to afford the title compound as white crystals. mp - 128 - 129.
F.x~mple 9
1-(1 -Ethylcarbo~ylyiyGlidin-4-yl)-3-(4-thiomethylphenyl)-5-r2-~thiomethyl)pyri-m--idin
4-yll-imi~ .7Ole
20 a) 4-Thiom~f hyl~he~lyl-tolysulfon~ Iylisocyanide
The titled compound was prepared using the proceedures 1 (e) & (f) sll~s~ .li.,g 4-
thiomethylben7~k1hyde for 4-fluorob~n7~ hyde.
b) 2-Thiomethylpyrimi-lin~-carboxaldehyde[1-ethoxycarbonyl-4-aminopiperidine~imine
The titled co~ oulld was prepared using the proccedure of example 2 (b) subslill.lillg the
25 commercially available 1-ethoxycarbonyl4-aminopireri~lin~ for 1-t-bulo~ycalbonyl4-
aminopiperidine
c) 1-( l-Ethylcarbo~yl~i~elidin-4-yl)-3-(4-thiomethylphenyl)-5-[2-(thiomethyl)pyrimidin-
4-yl]-imi(1~7Ole4-thiomethylphenyl-tolysulfonomethylisocyanide (9.0g, 29.2 mmol)and2-thiomrll.yl~y,;l..i~lin~-4-carboxaldehyde[l-ethoxycarbonyl-4-aminopireri~line] imine
(7.0g, 22.1 mmol) allowed to react ~cording to the proceedure of example 1 (h). Upon
completion of the reaction most of the DMF was eva~olal~d in high vacuo, the lGIII~ i"g
solution poured into water and extracted with EtOAc. The extracts were washed with water,
brine, dried (Na2SO4), filtered, concentrated, and flash chromatographed (60%
EtOAc/hexane) to yield the titled compound (4.0g, 38.5% yield). ESP+ (Mass Spec) m/z 471
(MH+).
-47-
CA 02242974 1998-07-10
W O 97/25046 PCT~US97/00529
Example 10
Ethylcarbonyl~ ,idine~-yl)-4-(4-methylsul~lllyl~llenyl)-5-r2-methylsulfinyl-
pyrimi~1in~-yll imidazole
The product of the previous example (2g, 4.26mmol) was dissolved in TH~ cooled to
-10~ and OXONE (3.3g, 8.52mmol) in water (10 ml) was added dropwise (T < 5~). The
r~,slllting mixture was warmed to 20~ over ~0 mins, poured into a vigorously stirred
e of 10% aq NaOH (1~0 ml), ice (100 ml), and E~tOAc was separated, dried
(Na2S04), and concentrated to a yellow solid. ~Pçrict~lli7erl from EtOAc/h~xh~nP
(1:10) to afford the titled compound (80mg). ESP+ (Mass Spec~ m/z 502 (M~+).
All publications, inr,ll-rling but not limited to patents and patent applications, cited
in this specific~tion are herein incol~G,ated by lefelcnce as if each individual publication
were specifically and individually intlic~tecl to be incol~ol~ted by reference herein as
though fully set forth.
The above description fully discloses the invention inrlllrlin~ preferred
emborlim~nt~ thereof. Mo-lific~tinn~ and improv~"lents of the embor~ tSnl!i specifically
disclosed herein are within the scope of the following claims. Without further elaboration,
it is believed that one skilled in the are can, using the prece~ing description, utilize the
present invention to its fullest extent. Therefore the P,x~mI)le,c herein are to be construed as
20 merely illusLI~tive and not a lirnitation of the scope of the present invention in any way.
The embo~ of the i,l~.,lion in which an exclusive plu~clly orprivilege is claimed
are defined as follows.
- 48 -
