Note: Descriptions are shown in the official language in which they were submitted.
WO 92/10190 Pcr/ussl/094l6
20~77
TlTLE
NOVEL CSA~S
FIELD OF THE I~VENTION
This invention relates to the novel compounds of Formula (I), phanrlaceutical
compositions and various methods of use of the compounds of Formulas (1).
15 BACKGROUND OF THE INVENTION
The cyclooxygenase (CO) mediated pathway oxidizes arachidonic acid to
produce PGH2 which is in turn metabolized to the prostanoids (PGE2, TxA2, and
prostacyclin). These products are produced by various cells including polymorpho-nuclear
leukocytes, mast cells and monocytes. The 5-lipoxygenase (5-LO) mediated pathway2 0 oxidizes arachidonic acid initially to 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) which is
further metabolized to LTA4, the precursor to the pepddoleukotrienes (LTC4, LTD4, and
LTE4) and LTB4. Additionally 5-HPETE is converted to 5-hydroxyeicosatetraenoic acid (5-
HETE).
The arachidonic acid oxygenated products, as noted above, have been identified
2 s as mediators of various inflammatory conditions. The various inflammatory disease states
caused by these mediators and many other conditions, as discussed herein, are all conditions
in which a dual inhibitor of both CO and 5-LO would be indicated.
Interleukin-l (IL-l) and Tumor Necrosis Factor ('I NF) are biological substancesproduced by a variety of cclls, such as monocytes or macrophages. IL l and TNF affect a
3 0 wide variety of cells and tissucs and these cytokines as well as other leukocyte derived
cytokines are irnportant and critical inflammatory mediators of a wide variety of disease states
and conditions. The inhibilion of these cytokines is of benefit in controlling, reducing and
alleviating many of these disease states.
There remains a need for treatment, in this field, for compounds which are
3 5 cytokine suppresive anti-inflammatory drugs (hereinafter CSAID's), i.e. compounds which
are capable of inhibiting cytokines, such as IL-1, IL-6 and TNF; and compounds which are
also capable of inhibiting the oxygenation of arachidonic acid by inhibition of enzymes such
as lipoxygenase, specifically 5-lipoxygenase (5-LO) and cyclooxygenase (CO) thereby
preventing the fonnation of various leukotrienes and prostaglandins.
WO 92/10190 PCI/US91/09416
-2-
209~177
SUMMARY OF THE INVENTION
This invention relales to the novel compounds of Formula (I) and
pharmaceutical compositions comprising a compound of Formula (I) and a pharmaceutically
5 acceptable diluent or carrier.
This invention relates to a method of treating an oxygenated polyunsaturated
fatty acid mediated disease (hereinafter OPUFA) in an animal in need thereof which comprises
administering to such animal, an effective amount of a compound of Formula (I).
This invention also relates to a method of treating a cytokine mediated disease,10 in an animal in need thereof, which comprises administering to such animal an effective
amount of a compound of Formula (II).
This invention specifically relates to a method of inhibiting the production of
interleukin- 1 (hereinafter L- 1) in an animal in need thereof which comprises adminis~ering to
such animal an effective amount of a compound of Forrnula (II) sufficient to inhibit IL- 1.
15 More specifically the inhibition of the production of ~-1 is useful in the treatment,
prophylactically or therapeutically, of any disease state in a mammal which is exacerbated or
caused by excessive or unregulated L-1 production.
This invention specifically relates IO a method of inhibiting the production of
Tumor Necrosis Factor (hereinafter TNF) in an animal in need thereof which comprises
2 0 adrnhistcrhg to such animal, an cffecdve amount of a compound of Formula (II) sufficient to
inhibit TNF. More specifically the inhibition of the production of TNF is useful in the
treatment, prophylactically or therapeutically, of any disease state in a mammal which is
exacerbated or caused by excessive or unregulated TNF production.
2 5 pETALED DESCRIPI ION OF THE INVENTION
The compounds of Formula (II) are also useful in the treatment of viral
infections, where such viruses are sensitive to upregulation by TNF or will elicit TNF
production in vivo. The viruses contemplated for treatment herein are those that produce TNF
as a result of infection, or those which are sensitive to inhibition, such as by decreased
3 0 replication, directly or indirectly, by the TNF inhibitors of Formula (II). Such viruses
include, but are not limited to; HIV- 1, HIV-2 and HIV-3, Cytomegalovirus (CMV),Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes
Zoster and Herpes Simplex.
This invention more specifically relates to a method of treating a mammal,
3 5 afflicted with a human immunodeficiency virus (HIV), which comprises administering to such
marnmal an effective INF inhibiting amount of a compound of Formula (1).
The compounds of Formula (II) may also be used in association with the
veterinary treatment of mammals, other than in humans, in need of inhibition of TNF
wo 92/10190 PCr/US9l/09416
~3~ 2098~7
production. TNF mediated diseases for treatment, therapeutically or prophylactically, in
animals include disease states such as those noted above, but in par~icular viral infections.
Examples of such viruses include, but are not limited to, feline immunodeficiency virus (FIV)
or other retroviral infection such as equine infectious anaemia virus, caprine arthritis virus,
s visna virus, maedi virus and other lentiviruses.
A preferred method of this invention is the treatment, therapeutically or
prophylactically, of viral infections, in particular where such viruses are sensitive to
upregulation by TNF or IL- l will elicit TNF or IL-l production in vivo by administering an
effective arnount of a compound of Formula (II).
The compounds of Formula (I) are represented by the structure:
R3 R~
R2 ~Rg
R, ~,~ N yW
~N
Ro (I)
wherein
W l is -(CR4R5)-, or -(CR4Rs)-(cR6R7)
R2 R3, R6, R7, Rg, and Rg are hydrogen; or one or two of R2, R3, R6, R7,
Rg, and Rg are, independently, hydrogen or C1 2 alkyl;
one of R4 and Rs is ORlo and thc other is selected from H, alkyll.6, optionally
substituted alkyll.6, aryl, optionally substituted aryl, heteroaryl, or optionally substituted
heteroaryl;
Rlo is hydrogen, opdonally substituted Cl.6 alkyl, or optionally substituted
aryl; provided that when Rlo is hydrogen the other of R4 or Rs is other than hydrogen;
one of R1 and Ro is 4-pyridyl or Cl 4 alkyl-4-pyridyl; and the other of Rl and
Ro is
(a) phenyl;
(b) mono- or di-substituted phenyl wherein said substituents are
2 5 independently selected from Cl 4 alkyl, halo, hydroxy, Cl 4 alkoxy,
aryloxy, heteroaryloxy, Cl 3 alkylthio, C1 3 alkylsulfinyl, C2 5 1-alkenyl-1-
thio, C2 5 2-alkenyl-1-thio, C2 5 l-alkenyl-l-sulfinyl, C2 5 2-aLlcenyl-1-
sulfinyl, C1 3 alkylamino, C1 3 dialkylamino, CF3, N-(C1 3alkanamido), N-
(C1 3 alkyl)-N-(C1 3alkanamido), N-pyrrolidino, N-piperidino, prop2-ene-
3 0 1-oxy, 2,2,2-trihaloethoxy, thiol, acylthio, dithioacyl, thiocarbamyl,
dithiocarbamyl, alkylcarbonylalkylthio, carbalkoxyalkylthio,
alkoxycarbonylthio, alkoxythionothio, phenylthio, phenylsulfinyl,
alkoxyalkylthio, alkoxyalkyl-sulfmyl alkylthioaL~ylthio, acyloxyalkylthio,
acyloxyaLkylsulfinyl or Z; or
" ' .: ' ' , ' ' :,
. - .
wo 92/lolso PCr/U~91/09416
2098177 ~4~
(c) a moiety of the forrnulae:
~ S--(CRaR,,),--5~
wherein Y is selected from
s
j~3 j~3
R3 Ra
R2 ~Rg
~N
R
whcrcin t is 0 or l; W, Rl, R2, R3, R4, Rs, R6, R7, Rg, and Rg are as
defined above;
A is -CRs=CR7-, -N=CR7-, -S- or -O-;
Ra and Rb are independently selected from hydrogen, opdonally subsdtuted
Cl g aLIcyl, optionally substituted aryl or optionally substituted heteroaryl;
Z is -s-(cRaRb)rs-zl;
Zl is a functional moiety;
1 S or a pharmaceutically acceptable salt thereof.
Preferred mono-substitution of the phenyl ring for compounds of Formula (I)
is C14 alkyl, C14 alkyl S(O)m, m is 0 or l; C~4 alkoxy, halo, N-(Cl 3 alkyl) alkanamido, or
N-(Cl 3 alkanamido).
2 0 Prefe~ed di-substitution of the phenyl ring for compounds of Formula (I) is:
(b) disubstituted phenyl wherein said substituents are,
independently, Cl 3 aLtcylthio, Cl 3 alkoxy, halo, Cl 4 alkyl, Cl 3
alkylarnino, N- (Cl 3alkyl)-N-(Cl 3 alkanamido), Cl 3 dialkylamino, amino,
N-pyrrolidino or N-piperidino; or
2 5 (c) disubstituted phenyl wherein one of said substituents is C1 3
aLlcoxy, halo, Cl 4 aL~cyl or CP3, and the other substituent is thiol, alkylthio,
alkylsulfinyl, acylthio, dithioacyl, thiocarbamyl, dithiocarbamyl,
alkylcarbonylalkylthio, carbalkoxyalkylthio, alkoxycarbonylthio,
wo 92/10190 PCr/US91/09416
~5~ 2~8177
alkoxythionothio, arylthio, arylsulfinyl, alkoxyaLtcylthio, alkoxyalkylsulfinyl,alkylthioalkylthio, acyloxyalkylsulfinyl, acyloxyalkyl~hio or Z; or
(d) disubstituted phenyl wherein one of said substituents is arnino,
Cl 3 alkylamino or Cl 3 dialkylarnino; and the other substituent is Cl 3
S alkylthio, Cl 3 alkylsulfinyl, C2 5 -l-alkenyl-l-thio, C2 5 l-aLlcenyl-l-
sulfinyl, C3 5 2-alkenyl-l-thio, C3 5 2-alkenyl-l- sulfinyl, thiol, acylthio,
dithioacyl, thiocarbarnyl, dithiocarbarnyl, alkylcarbonylalkylthio,
carbalkoxyalkylthio, alkoxycarbonylthio, alkoxythionothio, phenylthio,
phenylsulfinyl, alkoxyalkylthio, alkoxyallcylsulfinyl, alkylthioalkylthio,
acyloxyalkylthio, acyloxyalkylsulfinyl or Z; or
(e) disubstituted phenyl wherein said substituents are the same and
are selected from halo, Cl 3 alkoxy, Cl 3 aLtcylamino, Cl 3 dialkylarnino, N-
pyrrolidino, N-piperidino, 2.2,2-tnhaloethoxy, prop-2-ene-1-oxy, hydroxy,
C1 3 alkylthio, C1 3 alkyl-sulfonyl, thiol, acylthio, dithioacyl, thiocarbamyl,
dithiocarbamyl, alkylcarbonylalkylthio, carbalkoxyalkylthio,
alkoxycarbonylthio, alkoxythionothio, phenylthio, phenylsulfinyl,
alkoxyalkylthio, alkoxyalkylsulfinyl, alkylthioalkylthio, acyloxyalkylthio,
acyloxyalkylsulfinyl or Z;
Preferably, for all the compounds of Formulas (I) when R1 is C1 4 alkyl-4-
pyridyl the alkyl substituent is located at the 2-position of the pyridine ring. More prefcrably
the alkyl substituent is methyl.
Zl is a functional moiety that does not interferc with breakagc of the disulfidebond in-vivo to yield the SH moiety. Preferable Zl moieties are aryl, optionally substituted
25 aryl, Cl g alkyl, optionally substituted aLkyl, heteroaryl, an opdonally substituted heteroaryl,
cystiene or glutathione. The optional substituents may be the sarne as the Ro or R1 phenyl
moieties noted above for Formula (r).
Ra and Rb are independently selected from hydrogen, optionally substituted
Cl 9 alkyl, optionally subsdtuted aryl, or optionally substituted heteroaryl. The opdonal
3 0 substituents for the aryl and heteroaryl ring are the same as the Ro and Rl phenyl moieties
noted above for Formula (I), other than Z. Preferably Ra and Rb are unsubstituted or
substituted with C14 alkyl.
Preferably, one of R4 or Rs is hydroxyl, i. e. Rlo is hydrogen. When the
other of R4 or R5 is aryl, it is preferably an optionally substituted phenyl moiety. Optional
3 5 substdtuents groups for R4 or R5 as aryl or heteroaryl are halogen, Cl g alkyl, halo-substituted
Cl g alkyl, hydroxy-substituted Cl galkyl, C1 6 alkoxy, S(o)nallcyll-6~ (cH2)m C2H,
(CH2)mNRI IR12, wherein Rl I and R12 are independently selected from hydrogen, allcyll4,
aryl, or Rl 1 and R12 together forrn a heterocyclic ring of 5 to 7 members, wherein one or two
..
,
wO 92/1019~ PCr/US91/09416 ~-
2 098177 -6-
of the ring members of the heurocyclic ring may additionally be O, N or S and may contain
additional unsaturation; n is 0 to 2, and m is 0 to 4. For 5-LO or CO inhibitory activity the
R4 or Rs moiety is not substituted with a carboxylic acid moiety.
When R4 or Rs is an optionally substituted aL~cyl the substituents are selected
5 from halogen, hydroxy, alkoxy, aL~cylS(O)n, aryl, heteroaryl, C02H, or NRIIR12. For all
R4 and Rs substitutions the halogen substituted alkyl moiety may contain more than one
halogen selected independently from fluorine, chloride, iodine or bromine; the hydroxy
substituted alkyl may also be polyhydroxy substituted.
Preferably when one of R4 or Rs is a substituted phenyl, the substituents are
10 halo, methoxy, carboxylic acid (and salts thereof), or a mono- or di-alkyl substituted
methylamine. Preferred heterocyclic rings when Rll and R12 cyclize are a pyrrole,
pyrrolidine, piperidine, or morpholino ring.
The optional substituents for Rlo moieties is the same for the R4 and Rs terms
described above.
The compounds of Formula (II), a subgenus of the compounds of Formula (I)
are also useful in the treatment of an OPUFA mediated diseases and are preferably useful as
cytokine inhibitors. The compounds of Formula (Il) are represented by the structure:
R3 R3
R2~Rg
R, ~NyW2
~N
R~ (Il)
wherem
2 0 W2 is -(CR4R5)-, or -(CR4Rs)-(CR6R7);
R2 R3, R6, R7, Rg, and Rg are hydrogen; or one or two of R2 R3, R6, R7,
Rg, and Rg are, independently, hydrogen or C1 2 alkyl;
one of R4 and Rs is ORlo and the other is selected from H, alkyll~, halogen
subsdtuted alkyll~, aryl, opdonally substituted aryl;
Rlo is hydrogen or Cl~ alkyl; provided that when Rlo is hydrogen the other
of R4 or Rs is other than hydrogen;
Rl is 4-pyridyl or C1 4 alkyl-4-pyridyl;
Rois
(a) phenyl;
(b) mono or di-substituted phenyl wherein said substituents are
independently selected from Cl 4 alkyl, halo, halosubstituted alkyl, Cl 4
alkoxy, C1 3 alkylthio, Cl 3 alkylsulfinyl, C2 5 l-aL~cenyl-l-thio, C2 5 2-
alkenyl-l-thio, C2 5 l-aLkenyl-l-sulfinyl, C2 5 2-aLkenyl-l-sulfinyl, Cl 3
alkylarnino, Cl 3 diaLkylamino, CF3, N-pyrrolidino, N-piperidino, 2,2,2-
wo 92/10190 Pcr/lJs9l/o94l6
209~t 77
trihaloethoxy, thiol, acylthio, dithioacyl, thiocarbamyl, dithiocarbamyl,
alkylcarbonylaL~cylthio, carbalkoxyalkylthio, alkoxycarbonylthio,
aL~coxythionothio, phenylthio, phenylsulfinyl, alkoxyalkylthio,
aLkoxyalkylsulfinyl, alkylthioalkylthio, acyloxyaL~cylthio, acyloxyalkylsulfinylS or Z; provided that is the phenyl is substituted with a C34 alkoxy it is in other
than the 4-position;
(c) a moiety of the formulae:
~ S--~CRaRb)l--S~
wherein Y is selected from
~, R2~R8
~A R,~_~W2
whereintisOorl; W2,RI,R2,R3,R4,Rs,R6,R7,Rg,andRgareas
15 defined above;
A is -CRs=CR7-, -N=CR7-, -S- or -0-;
Ra and Rb are independently selected from hydrogen, op~ionally substituted
C1 9 alkyl, optionally substituted aryl or optionally substituted heteroaryl;
Z is -S-(CRaRb)rS-ZI;
2 0 Z1 is a functional moiety;
or a pharmaceutically acceptable salt thçreof.
Preferable R4 or Rs groups are a substituted aryl or allcyl with halogen,
tCH2)m C02H, or a (CH2)mNRllRl2 moiety, and m is O to 4. As in Formula (I) when Rl is
2 5 a Cl.4 alkyl-4-pyridyl it preferably substituted in the 2-position of the pyridyl ring and the
alkyl substituent is preferably methyl. As also the instancein Formula (I) or (Il) compounds,
the W or W2 term is preferably -(CR4Rs)-.
Preferable Ro mono-substitution of a compound of Formula (II) is a Cl.3
alkyl, Cl 2 alkyl S(O)n, halogen, or CF3 moiety, and n is O or l. If Ro is substituted with a
3 0 C14 alkoxy moiety it is preferably a methoxy or ethoxy derivative, or if C34 alkoxy it is in
other than the para position.
Preferable di-substitu~ion of a compound of Formula (II) is:
wo 92/10190 PCr/US91/09416
2~98177 -8-
(a) disubstituted phenyl wherein said subs~ituents are,
independently, Cl 3 aLkylthio, Cl 3 alkoxy, halo, Cl 4 alkyl, Cl 3
aLkylarnino, Cl 3 diaLkylarnino, amino, N-pyrrolidino or N-piperidino; or
(b) disubstituted phenyl wherein one of said substituents is C1 3
aLkoxy, halo, Cl 4 aLkyl or CF3, and the other substituent is thiol, aL~cylthio,
alkylsulfinyl, acylthio, dithioacyl, thiocarbarnyl, dithiocarbarnyl,
alkylcarbonylalkylthio, carbalkoxyalkylthio, alkoxycarbonylthio,
alkoxythionothio, phenylthio, phenylsulfinyl, alkoxyaLkylthio,
alkoxyalkylsulfinyl, alkylthioalkylthio, Z, or acyloxyaL~cylthio; or
l o (c) disubstituted phenyl wherein one of said substituents is arnino,
Cl 3 alkylamino or C1 3 dialkylarnino; and the other substituent is C1 3
alkylthio, C1 3 alkylsulfinyl, C2 5 -l-alkenyl-l-thio, C2 5 I-alkenyl-l-
sulfinyl, C3 5 2-alkenyl-l-thio, C3 5 2-alkenyl-l- sulfinyl, thiol, acylthio,
dithioacyl, thiocarbarnyl, dithiocarbamyl, alkylcarbonylalkylthio,
carbalkoxyalkylthio, alkoxycarbonylthio, alkoxythionothio, phenylthio,
phenylsulfinyl, alkoxyalkylthio, alkoxyalkylsulfinyl, alkylthioalkylthio,
acyloxyalkylthio, acyloxyalkylsulfinyl, or Z; or
(d) disubstituted phenyl wherein said substituents are the same and
are selected from halo, Cl 3 alkoxy, Cl 3 alkylamino, Cl 3 dialkylamino, N-
pyrrolidino, N-piperidino, 2,2,2-trihaloethoxy, Cl.3 alkylthio, thiol, acylthio,
dithioacyl, thiocarbarnyl, dithiocarbatnyl, alkylcarbonylalkylthio,
carbalkoxyalkylthio, alkoxycarbonylthio, alkoxythionothio, phenylthio,
phenylsulfinyl, alkoxyalkylthio, alkoxyalkylsulfinyl, alkylthioalkylthio,
acyloxyalkylthio, acyloxyalkylsulfinyl or Z.
It should be noted that the compounds of F~rmula (I) where Rl or Ro may be a
C1 3 alkylsulfinyl, C2 5 l-alkenyl-l-sulfinyl, C2 5 -2-alkenyl-l-sulfinyl, alkoxyalkyl-
sulfinyl, and phenylsulfinyl moiety, may act as prodrugs which are reductively converted in
vjvo to the corresponding alkylthio or aLkenylthio form.
It should be noted that the compounds of Formula (I) where Rl or Ro may be a
phenyl substituted with an acylthio, dithioacyl, thiocarbarnyl, dithiocarbamyl,
alkylcarbonylalkylthio, carbalkoxyalkylthio, alkoxycarbonylthio, alkoxythionothio, or
acyloxyalkylthio may act as prodrugs which are hydrolytically converted m vivo to the
corresponding sulfhydryl form.
It should be noted that the compounds of Formula (I) where Rl or Ro may be a
phenyl substituted with any of the disulfide moieties described herein may act as prodrugs
which are oxidatively converted in vivo to the corresponding sulfhydryl form.
wo 92/1OIgo PCr/US91/09416
20~ 77
By the term "halo" as used herein is meant all halogens, i.e., chloro, fluoro,
bromo and iodo.
By the term "C1 galkyl" or "aLkyl" groups as used herein is meant to include
both straight or branched chain radicals of 1 to 9 carbon atoms, unless the chain length is
5 limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-
butyl, isobutyl, tert-butyl, and the like.
By the term "allcenyl" as used herein is meant to include both straight or
branched chain radicals of 1 to 9 carbon atoms, unless the chain length is limited thereto, but
not limited to vinyl, 1-propenyl, 2-propenyl, or 3-methyl-2-propenyl.
By the term "aryl" as used herein, in any combination, such as "aryloxy", is
meant phenyl, or naphthyl.
By the term "heteroaryl" as used herein, in any combination, such as
"heteroaryloxy", is meant a 5-10 membered aromatic ring system in which one or more rings
contain one or more heteroatoms selected from the group consisting of N, O or S; such as, bu~
15 not limited, to quinoline, isoquinoline, pyridine, pyrimidine, oxazole, thiazole, thiadiazole,
triazole, imidazole.
By the term "sulfinyl" as used herein is meant the oxide of the corresponding
sulfide. By the term "thio" as used herein is meant the sulfide. For further clarification, the
following table outlines the structural attachment of the atoms of the Rl and Ro substituents of
20 thc compounds of Formula (I):
Table I
Bo substituents S~uctu~al Attachment
C1 3 a~ylsulfinyl [AS(O)-~
2 5 C2 5 1-allcenyl-1-~io [AAlC=CHS-]
C2 5 l-alkenyl-l-sulfinyl [AAlC~HS(O)-]
C3 5 2-alkenyl-1-thio [ACH=CAlCH2S-]
C3 5 2-alkenyl-1-sulfinyl [ACH=CAlCH2S(O)-]
1 acyloxy- 1 -a~ylthio [AC(O)OCH(Al)S-~
NOTE:A and Al are hydrogen or alkyl;
Table 2
~dditional Rl or Rsl SubstiNents StrucNral Attachments:
3 5 acylthio [DC(O)S-]
dithioacyl [DC(S)S-]
thiocarbamyl [DDlNC(O)S-]
dithiocarbamyl [DD1NC(S)S-]
WO 92/10190 Pcr/us9l/o94l6
20~177 -lO-
aLtcylcarbonylalkylthio [DC(O)CH2S-]
carba1~coxyalkylthio [BOC(O)CH2S-]
aLtcoxycarbonylthio [BOC(O)S-]
aLkoxythionothio [BOC(S)S-]
s alkoxyalkylthio [BOCH2S-]
alkoxyalkylsulfinyl [BOCH2s(o)]
alkylthioalkylthio [BSCH2S-]
disulfide [Z] [-s(cRaRb)t-s-zl]
Note: D and D1 are hydrogen, Cl 9 alkyL or phenyl; t is 0 or 1
B is Cl g alkyl or aryl; Ra, Rb and Z1 is aryl, heteroaryl or Cl 9
alkyl (optionally substituted). The hydrogen atoms in the CH2
groups described in Table 2 are, independently, opdonally
substituted by a Cl 4 allcyl moiety.
5
By the term "lipoxygenase" as used herein is meant the 5-lipoxygenase, 12-
lipoxygenase or 15-lipoxygenase enzymes.
By the term "inhibiting the production of L-1" is meanl
a) a decrease of excessive in vivo L- 1 levels in a human to normal levels or
2 0 below no~mal levels by inhibition of the in vivo release of L- I by all cclls, including but not
limited to monocytes or macrophages;
b) a down regulation, at the genomic level, of excessive m vivo L-1 levels in a
human to normal levels or below normal levels; or
c) a down regulation, by inhibition of the direct synthesis of L- 1 as a
2 5 postranslational event.
By the terrn "inhibiting the production of TNF" is meant
a) a decrease of excessive in vivo TNF levels in a human to normal levels or
below normal levels by inhibition of the m vivo release of TNF by all cells, including but not 0 limited to monocytes or macrophages;
b) a down regulation, at the genomic level, of excessive in vivo TNF levels in
a human to normal levels or below normal levels; or
c) a down regulation, by inhibidon of the direct synthesis of TNF as a
postranslational evcnt.5
By the term "TNF mediated disease or disease state" is meant any and all
disease states in which INF plays a role, either by production of TNF itself, or by TNF
causing another monokine to be released, such as but not limited to L- 1, or IL-6. A disease
,
WO 92/10190 PCI/US91/09416
-11- 2~81 7~
state in which IL- 1, for instance is a major component, and whose production or action, is
exacerbaoed or secreted in response to TNF, would therefore be considered a disease stated
mediated by TNF.
By the term "cytokine" as used herein is meant any secreted polypepdde that
affects the functions of other cells, and is a molecule which modulates interacdons between
cells in the immune or inflammatory response. A cytokine includes, but is not limited to
monokines and Iymphokines regardless of which cells produce them. For instance, a
monokine is generally referred to as being produced and secreted by a mononuclear cell, such
as a macrophage andlor monocyte but many other cells produce monokines, such as natural
killer eells, fibroblasts, basophils, neutraphils, endothelial cells, brain asttocytes, bone
marrow stromal eells, epideral keratinoeytes, and B- lymphocytes. Lymphokines are
generally referred to as being produeed by Iymphoetye eells. Examples of cytokines include,
but are not limited to, lnterleukin-1 (lL-l), Interleukin-6 (IL-6), Tumor Neerosis Faetor-alpha
(TNFa) and Tumor Necrosis Faetor beta (TNFB).
By the term "eytokine interfering or eytokine suppresive amount" is meant an
effective amount of a eompound of Formula (I) to (m) which will, when given for the
treatment, prophylaedealy or therapeudcally, of any disease state which is exacerbated or
eaused by exeessive or unregulated cytokine producdon, cause a deerease the ~n vivo levels of
the eytokine to normal or below normal levels.
2 0 The inhibition of a eytokine, contemplated by the present invendon, for use in
the treatment of a ~V-infected human, must be a eytokine whieh is implieated in (a) the
initiation and/or maintenanee of T cell aetivation and/o~ aetivated T eell-mediated HIV gene
expression and/or replieation, and/or (b) any eytokine-media~ed disease assoeiated problem
sueh as eaehexia or musele degeneradon.
2 5 As TNF-B (also known as Iymphotoxin) has elose struetural homology with
TNF-a (also kno vn as eaeheetin) and sinee eaeh induces similar biologie responses and binds
to the same eellular reeeptor, both TNF-~ and TNF-B are inhibited by the eompounds of the
present invention and thus are herein referred to eolleetively as "TNF" unless speeifieally
delineated otherwise.
3 0 By the term "OPUFA mediated disease or disease state" is meant any disease
state whieh is mediated (or modulated) by oxidation of polyunsaturated fatty aeids,
speeifieally the araehidonie aeid metabolie pathway. The oxidation of araehidonie aeid by
sueh enzymes as the lipoxygenase enzyrnes or eyelooxgenase enzyme is speeifieally targeted
by the present invention. Sueh enzymes inelude, but are not limited to, 5-LO, 12-LO, 15-LO,
3 5 and CO; whieh produee the following mediators,ineluding but not limited to, PGE2, LTB4,
LTC4, LTD4, prostaglandins, thromboxane, and prostocyelin.
...
wo 92/10190 2 0 9 8 ~ 7 7 -12- PCI/US91/09416
By the tenn "OPllFA interfering amount" is meant an effec~ive amount of a
compound of Fonnula (I) which shows a reduction of the in vivo levels of an oxgyenated
arachidonic acid metabolite.
The compounds of Formula (I) may be prepared from the known intennediates
of Formula (A), as shown below. The compounds of Formula (A) are known compoundsand are prepared in Bender et al., U.S. Patent Application Serial Number 07/255,816, filed
October 11, 1988; Bender et al., U.S. Patent Number 4,175,127, issued November 20,
1979; Bender et aL U.S. Patent Applicadon Serial Number 07/106,199 filed on July 10,
1987; Bender çt al., U.S. Patent Number 4,803,279, issued February 9, 1989, Bender et al.,
U.S. Patent Number 4,719,218, issued January 12, 1988; Bender et a]., U.S. Patent
Number 4,715,310, issued January 14, 1988 the entire disclosures of all of which are hereby
incorporated by referencç.
Compounds of Formula (A) wherein Ro or Rl is a phenyl subsdtuted with a
substituted disulfide moiety are prepared by mild air oxidation of the compounds of Formula
(A) wherein the R or R1 is a phenyl substituted with a sulfhydryl group. The non-
symmetrical disulfides (Z) wherein Z is -S-S-Zl and Zl is aryl, heteroaryl or alkyl, the
compounds may be prepared by reaction of the sulfhydryl compound wi~h the appropriate
sulfenyl halide in an ethereal solvent to afford compounds of Formula (A) wherein one of Ro
or Rl is a phenyl substituted with one or more alkyldithio or aryl-dithio groups.
The method of Mukaiyama.et al., Tetrahedron 1,etters,56:5907-08 (1968)
allows for use of the desired aryl-SH or alkyl-SH reagent treated with diethylazodicarboxylate
in 1: 1 equivalence at room temperature in a solvent, yielding an adduct which is then treated
with 1: 1 rado of the mercaptan of a Formula (A) compound. This process will also yield the
disulfide dimer of the compounds of Forrnula (A). Preferably the disulfide linkage is on the
2 5 Ro position of the compounds of Forrnula (A).
Compounds of Forrnula (A) wherein R or Rl is phenyl substituted with an
alkylthioalkylthio group are prepared by reacting the analogous sulfhydryl compound,
prepared as described above, with the appropriate carbonyl component, such as
formaldehyde, acetone, or acetaldehyde, using either mineral or Lewis acid catalysis
3 0 conditions to yield the symmetrical dithioketal. The interrnediate hydroxylalkylthio derivative
reacts with another sulhydryl containing compound under the acid catalysis conditions to yield
what is essentially a "bis" type compound, differing only by the alkyl chain insertion. This
process produces the bis disulfide moieties of part (c) Claim l, for instance, i.e. Formula (A)-
S-CRRl-S-Formula (A). The substitution of the alkyl, R or Rl, is deterrnined by the reactive
3 5 carbonyl functional group, wherein R or R1 may be Cl 9 alkyl, aryl or heteroaryl, all
optionally substituted.
The nonsymmetrical thioketals can be prepared by the reaction of the metal
mercaptan salt, prepared as described above, with a halomethyl thioether to yield compounds
WO 92/10190 Pcl/ussl/o9416
-13- 209~177
of Formula (A) wherein one of R or R1 is phenyl substituted with one or more
alkylthioalkylthio groups. The metal salt reacts with an independent and varying alkyl chain
length halomethyl-[CRR1]-thioalkyl[aryVheteroaryl] compound to yield the "non-bis" type
compounds, [Formula (A)-S-CRR1-S-R2], wherein R and R1 are as defined above for the
5 "bis" compounds, and R2 is a C1 9 alkyl, aryl or heteroaryl group which may be optionally
substituted. A mixture of Ro and R1 linkages is contemplated, as part of the present
invention, however, preferably the linkage is on both Ro positions of the compounds of
Formula (A).
An alternate method of preparation of the nonsymmetrical disulfide compound,
10 wherein only one component is a compound of Formula (A), and the other half of the
disulfide link is an alkyl, aryl or heteroaryl derivative, may be prepared by reaction of a
sulfhydryl compound of Formula (A), with the appropriate sulfenyl halide, in an ethereal
solvent to afford compounds of Formula (A) wherein one of R or Rl is phenyl substituted
wit~ ~ne or more [alkyl]- dithio groups, i.e. [Formula (A)-S-S-R2], wherein R-R2 are as
15 defined in the above paragraph. The contemplated sulfenyl halide derivatives of alkyl, aryl, or
heteroaryl groups may be optionally substituted.
The disulfide compound(s) may also be prepared from the corresponding alkyl
sulfoxide compounds, such as methylsulfinyl, propylsulfinyl, iso-propylsulfinyl, wherein the
alkyl can be a straight chain or branched derivativc having from 1 to 9 carbon atoms, in a
2 0 solvent, preferably a chlorinated one such as chloroethylcne, methylene chloride or
chlorofonn, to which is added a carboxcylic acid anhydride, such as trifluroacedc anhydride,
or acetic anhydride. The Pummerer rearrangement reacdon may require some heating prior to
addition of an alkali metal hydroxide, such as sodium hydroxide. If acedc anhydride is used
than heating is also likely to be needed during the hydroxide treatment, before addition of
2 5 iodine solid (I2), which then affords the symmetrical disulfide compound as is noted above.
Mixtures of the sulfoxide compounds may be present in the solution to yield "symmetrical"
compounds but with varying substituent groups on the di-heteroaryl-imidazole ring system of
the present invention.
The compounds of Formula (A) are represented by the structure:
R3 Ra
27~ 9
Rl yN~W
~l~
3 0 Ro (A)
wherein
W1 is -(CR4R5)-, or-(CR4Rs)-(CR6R7)-;
R2, R3, R4, Rs, R6, R7, Rg, and R9 are, independently, -H or Cl 2 aL~cyl;
. .: . .
-
WO 92/10190 PCI/US91/09416
one of.R~l Qn9 ~olis~4-pyridyl or C1 4 alkyl-4-pyridyl; and the other of Rl and
Rois
(a) phenyl or monosubstituted phenyl wherein said substituent is
Cl 4 alkyl, halo, hydroxy, C1 4 alkoxy, Cl 3 aLcylthio, Cl 3 alkylsulfinyl,
C1 3 alkylsulfonyl, C2 5 1-alkenyl-1-thio, C2 5 2-alkenyl-1-thio, C2 5 1-
alkenyl-l-sulfinyl, C2 5 2-alkenyl-1-sulfinyl, C2 5 1-alkenyl-1-sulfonyl, C3
5 2-alkenyl-1-sulfonyl, C1 3 alkylarnino, C1 3 dialkylamino, CF3, N-(C
3alkanamido), N-(Cl 3 alkyl)-N-(Cl 3aL~canarnido), N-pyrrolidino, N-
piperidino, prop-2-ene-1-oxy, 2,2,2-trihaloethoxy, thiol, acylthio, dithioacyl,
thiocarbarnyl, dithiocarbarnyl, alkylcarbonylalkylthio, carbalkoxyalkylthio,
alkoxycarbonylthio, alkoxythionothio, phenylthio, phenylsulfinyl,
aL`~oxyalkylthio, alkoxyalkylsulfinyl alkyl~hioalkylthio, Z, or
acyloxyalkylthio;
(b) disubstituted phenyl wherein said substituents are,
independently, Cl 3 alkylthio, C1 3 alkoxy, halo, C1 4 alkyl, C1 3
alkylarnino, N- (Cl 3alkyl)-N-(Cl 3 alkanarnido), Cl 3 dialkylarnino, arnino,
N-pyrrolidino or N-piperidino;
(c) disubstituted phenyl wherein one of said substituents is C 1-3
alkoxy, halo, Cl 4 alkyl or CF3, and the other substituent is thiol,
alkylsulfinyl, acylthio, dithioacyl, LhiocarbarnyL dithiocarbamyl,
allcylcarbonylalkylthio, carbalkoxyalkylthio, alkoxycarbonylthio,
alkoxythionothio, phenylthio, phenylsulfinyl, alkoxyalkylthio,
alkoxyalkylsulfinyl, alkylthioalkylthio, Z, or acyloxyalkylthio; or
(d) disubstitutcd phenyl wherein one of said substituents is arnino,
2 5 Cl 3 alkylamino or C1 3 dialkylamino; and the other substituent is Cl 3
alkylsulfinyl, C2 5 -l-alkenyl-l-thio, C2 5 l-alkenyl-l-sulfinyl, C3 5 2-
alkenyl-l-thio, C3 5 2-alkenyl-1- sulfinyl, thiol, acylthio, dithioacyl,
thiocarbamyl, dithiocarbamyl, alkylcarbonylalkylthio, carbalkoxyalkylthio,
alkoxycarbonylthio, alkoxythionothio, phenyl~hio, phenylsulfinyl,
3 0 alkoxyalkylthio, alkoxyalkylsulfinyl, aL~cylthioalkylthio, Z, or
acyloxyalkylthio; or
(e) disubstituted phenyl wherein said substituents are the same and
are selected from halo, C1 3 alkoxy, C1 3 alkylamino, C1 3 dialkylarnino, N-
pyrrolidino, N-piperidino, 2,2,2-trihaloethoxy, prop-2-ene-1-oxy, hydroxy,
Cl 3 alkylthio, Cl 3 alkyl-sulfonyl, thiol, acylthio, dithioacyl, thiocarbamyl,
dithiocarbamyl, alkylcarbonylaLkylthio, carbaL~coxyaLkylthio,
alkoxycarbonylthio, aL~coxythionothio, phenylthio, phenylsulfinyl,
alkoxyaLkylthio, aLkoxyalkylsulfinyl, aLlcylthioalkylthio, or Z,
wo 92/lolgo 2 0 9 8 1 Pcr/ussl/l)94l6
(f) a moiety of one of the Formulae:
l3 is
R2 / \ Rg
~N yWa
S-- (CH2),--S~N
or
R3 R8
R2~Rg
~3S--(CH2),--S~Y
wherein t is O or 1;
wherein Wa, and R1 - Rg are as defined above;
or a pharmaceutically acceptable salt thereof.
Alternatively the compounds of Formula (A) can be preferably be prepa~d as
outlined in the schematic below. While only a five membered pyrrole is shown the synthesis
10 is also applicablc to the six membered nitrogen containing ring. The desired R2-Rg alkyl
substituted compounds of Formula (A) are prepared from the correspondingly R2-Rgsubstituted compounds of Formula (3).
This process comprises cyclizing a compound of Formula (3):
~A
R~ 60
11
R~ NH2
Fornwla (3)
wherein A is (CH2)n and n is 1 or 2; R 1 and Ro are as dcfined for Fonnula (I)
herein. Preferably Ro is a phenyl substituted by a Cl 4 alkylthio, halogen, Cl 4 aLtcyl, or C
4 alko~cy.
The compounds of Formula (3) are prepared by reacting thc compounds of
Formula (1) and (2):
~A base R N~
R~`CHN--60 D 1 ~ 0
Ro NH2
2 0Forrnula (1 ) Fonnula (2) Formula (3)
.
WO 92tlOl90 PCI/US91/09416
2 0 9 81 7 7 -16-
Suitable bases include alkyl lithiums such as but not limited to, n-butyl lithium,
potassium t-butoxide, lithium diisopropylamide, lithium hexamethylsilylazide, sodium or
potassium hydride or potassium hydroxide optionally with a phase transfer catalyst such as
tetraethylammonium bromide, or a suitable mixture thereof, e.g. n-butyl-lithium and
5 potassium t-butoxide. Conveniently a compound of Formula (1) is reacted with 1 to 2 mole
equivalents, preferably 1.4 to 1.7 mole equivalents of the base be~ore treatment with a
compound of Formula (2).
The reaction to form a compound of Formula (3) is in an organic solvent, such
as but not limitcd to, THF, dialkylether, dimethylformamide, toluene, aimcthylethylideneurea
10 or tetramethylethylencdiamine or a suitable mixturc thereof. The reaction should be pcrformed
within a temperature range of about -80C to about 100C. Prcferably the reaction is cooled
initially and the temperature is raised to optimize the reaction time of the process.
The compounds of Formula (3) may be isolated on workup and then cyclized
to a compound of the Formula (A) with a suitable base as hereinbefore described. An
15 cxample of such preparation can be found in synthetic Example 3.
Preferably, the compound of the Formula (3) is not isolated, but is forrned in
situ and cyclized directly to a compound of the Formula (A) under the basic condltions of the
reacdon mixture. An example of such preparation can bc found in synthedc Example 4.
Compounds of Formula (1) are prepared by reacting in the presence of a base a
2 0 compound of Formula (4), or an acid salt thereof:
RlCH2L
Formula (4)
wherein R1 is as hereinbefore defined, and L is a suitable leaving group, with
a compound of Formula (5)
~A
N~
o
2 5 Fomwla (5)
wherein A is as defined above for Formula (3).
Examples of suitable bases include but are no~ limited to, potassium carbonate,
sodium hydride, sodium hydroxide or lithium diisopropylamide. Suitable leaving groups (L)
are well known to those skilled in the art, and include halogens, such as bromine or chloride,
3 0 or a tosylate or mesylate moiety.
The reaction is performed in a solvent, preferably THF, DMF, or mixtures
thereof. The reaction may optionally be performed in the presence of water in appropriate
cases, where for example when using solid potassium hydroxide together with a phase
transfer catalyst as the base. The reaction is conveniently performed at ambient or slightly
3 5 elevated temperatures. Preferably an aqueous solution of an acid addition salt of a compound
wo 92/10190 Pcr/uS91/09416
-17- 2()98~.77
of the Formula (4) is gradually added to a solution of a compound of the Formula (S) and the
base.
The compounds of Formula (A) may be themselves used as intermediates to
s produce other compounds of Forrnula (A) and such preparations are well described in Bender
et al., U.S. Patent Application Serial Number 07/255,816, filed October 11, 1988; Bender et
ak, U.S. Patent Number 4,175,127, issued November 20, 1979; Bender et al., U.S. Patent
Application Serial Number 07/106,199 filed on July 10, 1987; Bender et al., U.S. Patent
Number 4,803,279, issued February 9, 1989, Bender et al.. U.S. Patent Number
10 4,719,218, issued January 12, 1988; Bender et al., U.S. Patent Number 4,715,310, issued
January 14, 1988 the entire disclosures of all of which arc hereby incorporated by reference.
Compounds of Formula (A) wherein Ro or Rl is a mono- or di-subsdtuted
phenyl having a Cl 3alkylsulfinyl, or Cl 3 alkenylsulfinyl; or wherein R or Rl is a di-
substituted phenyl having at least one Cl 3alkylsulfinyl, or Cl 3alkenyl-sulfinyl; or wherein R
15 or Rl is a mono- or di-substituted phenyl having at least one acyloxyalkylsulfinyl,
alkoxyalkylsulfinyl or phenyl-sulfinyl S~! ~ituent are prepared by treatment with appropriate
oxidative procedures well known to thos illed in the art and additionally can be found in
Bender et al., U.S. Patent Applicadon Serial Number 07/2S5,816, filed October 11, 1988;
Bender ~~L. U.S. Patent Number 4,175,127, issued November 20, 1979; Bender et al.,
2 0 U.S. Patent Applicadon Serial Number 07/106,199, filed on July 10, 1987; Bender et al.,
U.S. Patent Number 4,803,279, issued February 9, 1989, Bender ~aL. U.S. Patent
Number 4,719,218, issued January 12, 1988; Bender ç~.aL. U.S. Patent Number
4,715,310, issued January 14, 1988; and in Adams et al., US Patent Application Serial
Number 07/537,195, filed June 12, 1990, Attorney's Docket Number SB 14506. Preferably
2 5 the oxidadon is by use of potassium persulfate procedure as described in Adams et al., USSN
07/537,195, filed June 12, 1990, Attorney's Docket Number SB 14506, whose disclosure is
herein incorporated by reference.
The compounds of Formula (A) arc used as intcrmediatcs to form a 7-hydroxyl
30 or 7 kcto moiety by analogous preparadon to the methods disclosed in Gallagher et al.,
Tetrahedron Lctters, Vol. 30, No. 48, pp. 6599-6602 (1989) the entire disclosure of which is
hereby incorporated by reference. The 7-OH and 7-keto compounds are then used asintermediates to makc the final compounds`of Fo~mula (I).
3 5 The 7-posidon compounds of Fo~mula (A) containing a 7-hydroxyl or 7-oxo
are converted to the protected hydroxyls of Formula (I) or (II) by the schemadc illustrated
below. The ketal, and 7-position di-subsubsdtuted compounds are illustrated in the schematic
below as well.
.,
.. , . : .
wo 92/10190 Pcr/US91/09416
20g~ 77 -18-
Nl~_ N NaH, DMF; R~ N
~OH [3J~ N~
NaBH4, CH2CI2/MeOH
N~ N~ X(OH)2, mineral or ~_ N
Lewis acld cat. ~ O
3--N1 [3 N X
RsMgX
N~ ,N NaH, DMF; R,oX ~ ~_N~
5 0J~N~>~R OR1o
SCHEME I
Compounds 1 and 2 are prepared, as stated above, by the methods outlined in
Teorahedron Letters, Gallagher ~1~ . In scheme I above, the conversion of compound
1 to compound 4 can bc accomplished by reaction of compound 1 with an appropriate diol,
herein referred to as X(OH)2, using acid catalysts well known to those skilled in the art.
Catalysis is preferably conducted with a Lewis acid, for cxample boron ~ifluoride etherate, a
mineral acid such as HC1, p-toluene suphonic acid, or titanium tetrachloride; see Greene,T.,
Protective Groups in Organic Synthesis, Wiley Publishers, p. 11~128 for addidonal agents.
The diol X(OH)2 is a generic formula for X which preferably contains 2 to 3
carbons in a chain which may additionally aL~cyl substituted, thereby providing for branched
diols. Suitable examples would be a 1,2-ethanediol o~ a 1,3-(2-methyl)propandiol moiety.
The conversion of compound 1 to 5 can be accomplished using a variety of
organometallic reagenss which are known to undergo nucleophilic additions to the carbonyl
WO 92/10190 PCr/US91/09416
,9 2093177
containing compounds. Examples of such reagents are a suitably substiluted organo-
magnesium (grignard reagents), -titanium, or -cerium reagents.
The requisite organometallic reagents are either known or are readily available
by adaptation of published procedures. The ether compounds 3 and 6 are prepared from
s compound 2 or 5 respectively using a base catalyzed allcylation (known as the Williamson
ether synthesis) when Rlo is alkyl or an (hetero)aryl substituted alkyl. Typical alkylation
conditions would employ an alkali metal hydridc in a dipolar aprotic solvent or an ethereal
solvent which is added to an alkyl halide although other properly activated alkylating agents
such as mesylates or tosylates may also suffice. The usc of an alkali metal alkoxide of a
10 sterically hindered alcohol, for example potassium t-butoxidc in t-butanol is also commonly
employed. In cascs whcrc Rlo is aryl the use of a mctal catalysis such as cupric oxide, is
commonly cmployed to effect the Ullman reaction for the synthesis of aryl ethers.
More particularly the synthesis of compounds 5 and 6 whcrein Rs is a
functionalized aryl containing a halo, amino, cyano or carboxy group of Formula (I) and (II)
15 may be prcpared from the corresponding benzostabase protected halo aniline (Bonar-Law et
al., Tetrahedron Letters, Vol. 31, p 6721 (1990)), see scheme II below. After forrnadon of
the organomettalic (illustrated below by lithiation and addition of CeC13 to form the
organoccrium) the addition of compound 1 affords the corresponding carbonyl addidon
product 7. This product may thcn be convened to the ether moiety, compound 8, using a
2 0 base initiated alkylation reacion.
Treatmcnt of either compound 7 or 8 with dilute mineral acid or an appropriate
source of nucleophilic flouride ion affords thc dcprotected anilincs. These anilines may bc
diazodzed unda standard condidons to produce diazonium salts which arc thcn reacted with
eithcr halo anions, cyanide or carbon monoxide and the requisite catalyst to afford the halo,
2 5 cyano or carboxy subsdtution products rcspectively. For additional procedural informadon,
see March, J., Advanced Organic Chemistrv, 3rd Ed., (1985), pages 646 to 649 (Wi1ey-
lnterscience Publishers).
The anilines may also be convertcd to mono- or di-alkyl amines by reaction
with an alkyl halide or by acyladon to the amide. Altcrnadvely, whcn the dialkyl arnine is the
30 desired product the preferred route would be to begin with corresponding halo dialkylaniline,
forming the organometallic reagent directly without the use of the benzostabasc protecting
group. Similarly when compound 6 is an alkoxy substituted aryl, thc aLlcoxy aryl bromide or
iodide may bc used to prepare the organometallic reagent which is then added to compound 1.
Thc preparadon of all thc remaining compounds of Fonnula (I) and aI) not
3s described herein can be readily achieved as the techniques are well known and can be caIIied
out by one of skill in the art acco~ling to the procedures outlined above or in the Exarnples,
infra.
. :
WO 92/10190 Pcr/US9l/09416
2~9~177 -20-
Si ~ Li, EtO2 --Si ~ ~ NSBS
Li ~ Br
1.) CeC13 THF
2.) add compound 1
3.) NaHCO3
N~, N NaH, DMF; R10X ~ N~L
OH ~N ~3
7 NSBS 8 NSBS
(Bu)4N~F, THF
N~NN'~30H N~N'~NH302R1o
NH2
1.) diazotize
2.) react with appropriate
nucleophile and catalyst i
~ N~
N~NN'~R OH ~R~30R,o
R4 = CO2H, Halo, cyano
SCHEME II
.. . . . - .
WO 92~10l90 Pcr/us9l/o94l6
-21- 2~9~177
Pharmaceutically acceptable salts and their preparation are well known to those
skilled in pharmaceuticals. Pharmaceutically acceptable salts of the compounds of Formula (I)
which are useful in the present invention include, but are not lirnited to hydrochloIide,
hydrobromide, sulfate or phosphate salts. Preferred pharmaceutically acceptable salts of the
5 compounds of Formula (I) and (II) can be prepared by known techniques such as the method
of Bender et al., U.S. Patent 4,175,127, issued November 20, 1979 the disclosure of which
is hereby incorporated by reference.
The compounds of the present invendon may contain one or more asymmetric
carbon atoms and may exist in racemic and optically acdve forms. All of these compounds are
10 contemplated to be within the scope of the present invention.
IvlETHOD OF TREAT~ENT
All of the compounds of Formulas (I) are useful in the methods of the subject
invention, i.e. methods of treating an OPUFA disease state, specifically by inhibition of the 5-
15 LO and CO enzymes, and the compounds of Formula (~) are useful for inhibidng cytokines,specifically the produc~ion of the IL-1 or TNF in an animal, including humans, in need
thercof.
The oxidadon of OPUFA's, specifically the arachidonic acid metabolic
pathway leading to inflammatory mediators, can be controlled by the 5-LO enzyme, amongst
2 0 others. That the compounds of Formulas (I) are inhibitors of the 5-lipoxygenase pathway is
based on the effects of said compounds on the production of 5-lipoxygenase products in
blood ç~ vivo and on the 5-lipoxygenase in vitro assays, some of which are described
hereinafter. The 5-lipoxygenase pathway inhibitory action of the compounds of Formulas (I)
may be confirmed by showing that they impair the producdon of 5-lipoxygenase products
2 5 such as leukotriene B4 producdon by RB~1 cell supernatants.
The pathophysiological role of arachidonic acid metabolites has been the focus
of ~ecent intensive studies. In addidon to the well-described phlogisdc acdvity (i.e. general
inflammatory acdvity) of prostaglandins, the more recent descripdon of similar acdvity for
eicosanoids has broadened the interest in these products as mediators of inflammation. These
30 mediators produce inflammatory condidons such as rheumatoid arthritis, osteoarthrids,
br~nchial inflarnmadon, inflammatory bowel disease, ulcNative colids, asthma,
cardiovascular disorders, glaucoma, emphysema, acute respiratory distress syndrome, lupus,
gout, psoriasis, dermadds, pyresis, pain and other allergic oriented disorders such as allergic
rhinitis, allergic conjuncdvitis, food allergies, and uticaria.
3 5 Additional condidons such as blood platelet aggregadon, and notably
condidons resulting from thrombosis, including total or partial thrombosis, coronary
thrombosisS phlebitis and phlebothrombosis are also implicated in the arachidonic acid
pathway. Other disease states for which a 5-LO inhibitor would be useful is in the treatment
: :
' ~
WO 92/101~0 2 0 9 8 1 7 7 -22- Pcr/US91/09416
of myocardial infarctions, rejection of organ transplan~s, tissue trauma, multiple sclerosis,
atherosclerosis, vasculitis, glomerulo-nephritis, and immune complex disease, as wcll as use
in the optical areas, particularly for general inflammation of the corneal anterior and posterior
segments due to disease or surgery, such as post surgical inflammation or uveitis.
s The compounds of Formula (I) are also useful for treating disease states mediated
by the cyclooxygenase pathway metabolism of arachidonic acid in an animal, including
humans, in need thereof. That the compounds of Formula (I) are inhibitors of
cyclooxygenase products is based upon assays which effect the production of the PGE2
products, and assays with human monocytes, the assays of which are described herein.
The disease states associated with the CO metabolic pathway are typically those
considered for the non-steroidal antiinflammatory drugs (nsaids), whose primary mode of
action is by CO inhibition. The primary diseases of interest, but not limited thereto, are the
various arthritic conditions, pyresis and pain.
Interleukin-l (L-l) has been demonstrated to mediate a variety of biological
activities thought to be important in immunoregulation and other physiological conditions such
as inflammation [See, e.g., Dinarello et al., Rev. Infect. Disease, 6, 51 (1984)]. The myriad
of known biological activities of L- 1 include the activadon of T helper cells, inducdon of
fever, stimuladon of prostaglandin or collagenase production, neutrophil chemotaxis,
induetion of acute phase proteins and the suppression of plasma iron levels.
2 0 The compounds of Formulas tlI) are useful as inhibitors ~f cytokines,
speeif~cally L- 1. The inhibitory activity of a eompound of Formula (II) on the produetion of
the IL-l tn vitro, on the human monocyte, may be deterrnined as described in an assay
herein.
There are many disease states in which excessive or unregulated L- l
2 5 produetion is implieated in exacerbating and/or causing the disease. These inelude rheumatoid
arthrids, osteoarthritis, endotoxemia and/or toxic shock syndrome, other aeute or chronie
inflammatory disease states such as the inflammatory reaction indueed by endotoxin or
inflammatory bowel disease; tubereulosis, atheroselerosis, musele degeneration, eaehexia,
psoriatie arthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumatie arth~itis, rubella
3 0 arthritis, and aeute synovitis. Reeent evidenee also links L- l aetivity to diabetes and
panereatie B eells.
Dinarello, J. Clinieal Immunologv, 5 (5), 287-297 (l985), reviews the
biologieal aetivities whieh have been attributed to L-l. It should be noted that some of these
effeets have been deseribed by others as indireet effeets of L- 1.
3 5 The diseovery of a eompound whieh speeifically inhibits ~NF production will
not only contribute to the understanding of how this molecule is synthesized, processed and
secreted, but will also provide a therapeutic approach for diseases in which excessive or
unregulated TNF production is implicated.
wo 92~10190 Pcr/ussl/094l6
-23- 2~177
Excessive or unregulated TNF production is implicated in mediating or
exacerbadng a number of diseases including rheu oid arthritis, rheumatoid spondylitis,
osteoarthrids, gouty arthrids and other arthritic coi. lidons; sepsis, septic shock, endotoxic
shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome,
s cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis,
bone resorption diseases, reperfusion injury, graft vs. host reaction, allograft rejections, fever
and myalgias due to infection, such as influenza, cachexia secondary to infection or
malignancy, cachexia, secondary to acquired immune deficiency syndrome (AIDS), AIDS,
ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease,
10 ulcérative colitis, or pyresis.
AlDS results from the infecdon of T Iymphocytes with Human
Immunodeficiency Virus (HIV). At least 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 mediated
immunity is impaired and infected individuals manifest severe opportunistic infections and/or
15 unusual neoplasms. HIV entry into the T Iymphocyte requires T Iymphocyte activation.
Other viruses, such as HIV- 1, HIV-2 infect T Iymphocytes after T Cell activation and such
virus protein expression and/or replication is mediated or maintained by such T cell activation.
Once an acdvated T lymphocyte is infected with HIV, the T Iymphocyte must condnue to be
maintained in an acdvated state to perrnit HIV gene expression and/or HlV replicadon.
2p Monokines, specifically TNF, are implicated in activated T-cell mediatcd HIV protein
expression andlor virus replication by playing a role in maintaining T Iymphocyte activation.
Thercfore, inurference with monokine activity such as by inhibition of monokine production,
notably TNF, in an HIV-infected individual aids in limidng the maintenance of T cell
activadon, thereby reducing the progression of HIV infecdvity to previously uninfected cells
2 5 which results in a slowing or elimination of the progression of immune dysfuncdon caused by
HIV infection. Monocyus, macrophages, and related cells, such as kupffer and glial cells,
have also been implicated in maintenance of the HIV infection. These cells, like T-cells, are
targets for viral rcplication and the level of viral replication is dependent upon the acdvation
state of the cells. [See Rosenberg ~., The Immunopathogenesis of HIV Infection,
30 Advances in Immunology, Vol. 57, (1989)]. Monokincs, such as TNF, have been shown to
activate ~IV replication in monocytes and/or macrophages [See Poli, g~L, Proc. Natl. Acad.
Sci., 87:782-784 (1990)], therefore, inhibition of monokine production or activity aids in
limiting HIV progrcssion as stated above for T-cells. Additional studies have identified TNF-
a as a common factor in the activadon of HIV in vitro and has provided a clear mechanism of
3 s action via the nuclear factor ~B, a nuclear regulatory protein found in the cytoplasm of cells
(Osborn, et al., PNAS (86) 233~2340). This evidence suggests that a reduction of TNF
synthesis may have an antiviral effect in HIV infections, by reducing the transcription and
thus virus production.
WO 92~101gO 2 0 9 ~ 1 7 7 -24- Pcr/ussl/o94l6
TNF has also been irnplicated in various roles with other viral infections, suchas the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes farnily of
viruses, such as Herpes Zoster and Herpes Simplex I and II, for similar reasons as those
noted above.
TNF also alters the properties of endothelial cells and has various pro-
coagulant activities, such as producing an increase in tissue factor pro-coagulant activity and
suppression of the anticoagulant protein C pathway as well as down-regulating the expression
of thrombomodulin. TNF also has pro-inflammatory activities which together with its early
production (during the initial stage of an inflammatory event) make it a likely mediator of
tissue injury in several important disorders including but not limited to, myocardial infarction,
stroke and circulatory shock. Of specific importance may be TNF-induced expression of
adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte
adhesion molecule (ELAM) on endothelial cells.
TNF is also believed to be an important mediator of many other inflammatory
states or diseases. Therefore, inhibitors of TNF producdon would have utility in any
inflammatory state or disease in which abnormal levels of TNF are produced. Abnormal
levels of TNF constitute levels of l ) free (not cell bound) TNF, greater than or equal to l
picogram per ml; 2) any cell associated TNF; or 3) the presence of TNF mRNA above basal
levels in cells or tissues in which TNF is produced. In addition, the present invendon
20 attributes many biological disease states noted herein to IL l acdvity. These disease states are
also considered appropriate disease states of TNF activity and hencc compounds of Forrnulas
(II) are also useful in their treatment as well, and should not be considacd solely a limitation
to IL-l activity alone.
It has also been discovered that the compounds of Formulas (Il) are useful for
treating disease states mediated by the cytokine TNF in an animal, including mammals, in
necd thereof. The hhibitory effect of a compound of Formulas (II) on the production of the
TNF in - vitr~, on the human monocyte, may be determined by the assay which is described
herein.
PHARMACEIIII~ALCOMPOSITTONS
This invention further relates to the use of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment
of prophylactically or therapeutically, any disease state in an animal, including humans, which
is caused by or exacerbated by OPUFA metabolizing enzymes, such as 5-LO or CO.
3 5 This invention further relates to the use of a compound of Formula (II), or
pharmaceutically acceptable salts then~of in the manufacture of a medicament for the treatrnent
of prophylactically or therapeutically, any disease state in an animal, including humans, which
is exacerbated or caused by excessive or unregulated IL-l, or TNF p~ducdon.
WO 92/10190 2 0 ~ ~17 7 pcr/us9l/o94l6
This invention also relates to a pharrnaceutical composition comprising an
effective, non-toxic amount of a compound of Forrnulas (I) or (II) and a pharrnaceutically
acceptable carrier or diluent. The compounds of Fo~mula (I) and (Il) are administered in
conventional dosage forms prepared by combining a compound of Forrnula (I) and (~) with
s standard pharmaceutical carriers according to conventional procedures. The compounds of
Formula (I) and (II) may also be administered in conventional dosages in combination with a
known, second therapeutically active compound. These procedures may involve mixing,
granulating and compressing or dissolving the ing~dients as appropriate tO the desired
preparadon.
The pharmaceutical carrier employed may be, for example, either a solid or
liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup,
peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time
delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate
15 alone or with a wax.
A wide variety of pharmaceutical forrns can be employed Thus, if a solid
carrier is used, the preparadon can be tableted, placed in a hard gelatin capsule in powder or
pellet forrn or in the form of a trochc or lozenge. Thc amount of solid carrier will vary widely
but prefcrably will be from about 25 mg. to about 1 g. When a liquid ca~ier is used, the
20 pteparadon will be in the fonn of a syrup, emulsion, soft gelatin capsule, stcrile injectable
liquid such as an ampule or nonaqueous liquid suspension.
To obtain a stable water soluble dose forrn of an insoluble Formula (I) or (II)
compound, a pharmaceudcally acceptable salt of the Formula (I) or (Il) compound is
dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of
2 5 succinic acid or citric acid.
All applicable dosage ranges, formulations, applications, i.e. topical, oral,
parenteral, etc. apply equally to the compounds of Formulas (I) and (11).
The compounds of Forrnula (I) may be administered topically. Thus, the
compounds of Formula (I) may be adrninistered topically in the treatment or prophylaxis of
3 0 inflammation in an animal, including man and other mammals, and may be used in the relief
or prophylaxis of 5-lipoxygenase pathway mediated diseases such as rheumatoid arthrids,
rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, inflamed
joints, eczema, psoriasis or other inflammatory skin condidons such as sunburn;
inflammatory eye conditions including conjunctivitis; pyresis, pain and other conditions
3 5 associated with inflammation. For those disease states indicated above which arc also
mediated by a cytokine the compounds of Formula (II) may be administered topically.
The amount of a compound of Formula (I) or (II), for all methods of use
disclosed herein, required for therapeutic effect on topical administration will, of course, vary
wo 92/10190 Pcr/Us91/09416
209~177 -26-
with the compound chosen, the nature and severi~y of the inflammatory condition, whether
eicosanoid or cytoldne mediated, and the animal undergoing treatment, and is ultimately at the
discretion of the physician. A suitable, topical, anti-inflammatory dose of an active
ingredient, i.e., a compound of Forrnula (I) or (II) is O.l mg to 150 mg, administered one to
four, preferably two or three times daily.
By topical administration is meant non-systemic administration and includes the
application of a compound of Forrnula (I) or (II) extemally to the epidermis, to the buccal
cavity and instillation of such a compound into the ear, eye and nose, and where the
compound does not significantly enter the blood stream. By systemic adrninistration is meant
oral, intravenous, intraperitoneal and intrarnuscular administration.
While it is possible for an active ingredient to be administered alone as the raw
chemical, it is preferable to present it as a pharmaceutical formulation. The active ingredient
may comprise, for topical administration, from 0.001% to 10% w/w, e.g. from l % to 2% by
weight of the formulation although it may comprise as much as 10% w/w but preferably not in
excess of 5% w/w and more preferably from 0.1% to l % w/w of the formulation.
The topical formulations of the present invenion comprise an active ingredient
together with one or more acceptable carrier(s) therefor and optionally any other therapeutic
ingredient(s). The carria(s) must be 'acceptable' in the sense of being compatible with the
other ingredients of the formuladon and not deleterious to the recipient thcreof.
2 0 Formulations suitable for topical adrninistration include liquid or semi-liquid
preparations suitable for penetration through the skin to the site of inflammation such as
liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the
eye, ear or nose.
Drops according to the present invention may comprise sterile aqueous or oily
2 5 solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable
aqueous solution of a bactericidal andlor fungicidal agent and/or any other suitable
preservadve, and preferably including a surface active agent. The resulting solution may then
be clarified by filtration, transferred to a suitable container which is then sealed and sterilized
by autoclaving or maintaining at 98-lOOC. for half an hour. Alternatively, the solution may
3 0 be sterilized by filtration and transferred to the container by an aseptic technique. Examples of
bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate
or acetate (0.002%), benzalkonium chloride (0.0l%) and chlorhexidine acetate (0.01%).
Suitable solvents for the preparadon of an oily solution include glycerol, diluted alcohol and
propylene glycol.
3 5 Lotions according to the present invention include those suitable for application to
the skin or eye. An eye lotion may compIise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the preparation of drops.
Lotions or liniments for application to the skin may also include an agent to hasten drying and
wO 92/lolgo Pcr/usg1/os416
-27- 209~.1 77
to cool the skin, such as an alcohol or acetone, and/or a moisturiær such as glycerol or an oil
such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid
formulations of the active ingredient for external application. They may be made by mixing
s the active ingredient in finely-divided or powdered forrn, alone or in solution or suspension in
an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-
greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, com,
arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as steric or oleic acid
10 together with an alcohol such as propylene glycol or macrogels. The formulation may
incorporate any suitable surface active agent such as an anionic, cationic or non-ionic
surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents
such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas,
and other ingredients such as lanolin, may also be included.
The methods of the subject invenion may be carried out by delivering the
monokine activity inteRering a~ent parenterally. The term 'parenteral' as used herein includes
intravenous, intramuscular, sub~utaneous intranasal, in~arectal, intravaginal or intraperitoneal
adrninistration. The subcutaneous and intramuscular forms of parenteral administration are
gcnerally preferred. Appropriate dosage forms for such administration may bc prcparcd by
20 conventional techniques.
For all mcthods of use disclosed hercin, for thc compounds of Formulas (I)
and (r[), thc daily oral dosage redmen will preferably bc from about .l to about 80
mg/kilogram of total body weight, prefcrably from about .5 to 30 mglkg, more preferably
from about lmg to l5mg. The daily parenteral dosage regimen will preferably be from about
2 5 . l to about 80 mg per ldlogram (kg) of total body weight, preferably from about .5 to about
30 mg/kg, and more preferably from about lmg to lSmgllcg.
The compounds of Formula (I) and (II) may also be administered by
inhalation. By "inhalation" is meant intranasal and oral inhalation administration. Appropriate
dosage forms for such adrninistration, such as an aerosol formulation or a metered dose
30 inhaler, may be prepared by conventional techniques. The preferred daily dosage amount of a
compound of Fonnula (I) administered by inhaladon for all methods discloscd herein, is from
about .Ol mg/kg to about 1 mg/kg per day.
It will be recognized by one of skill in the art that the form and character of the
pharmaceutically acceptable carrier or diluent is dictatcd by the amount of active ingledicnt
5 with which it is to be combined, the route of administration and other well-known variables.
It will also be recogmzed by one of skill in the art that the optimal quantity and
spacing of individual dosages of a compound of Formula (I) or (I[), or . pharmaceutically
acceptable salt thereof will be determined by the nature and extent of the condition being
wo 92/10190 Pcr/usg1/os416
2~9817~ -28-
treated, the form, route and site of administration, and the particular patient being treated, and
that such optimums can be determined by conventional techniques.
It will also be appreciated by one of skill in the art that the optimal course of
treatment, i.e., the number of doses of a compound of Forrnula (I) and (II) or the
s pharmaceuically acceptable salts thereof given per day for a defined number of days, can be
ascertained by those skilled in the art using conventional course of treatment determination
tests.
EXAMPLES
Without further elaboration, it is believed that one skilled in the art can, using
the preceding description, utilize the present invention to its fullest extent. The following
Exarnples are, therefore, to be construed as merely illustrative and not a limitation of the scope
of the present invention in any way.
EXAMPLE A
Inhibitory Effect of compounds of Forrnula (I) on
m vitro IL- 1 Production by Human Monocytes
The effects of compounds of Formula (I) on the ~ vitro production of L- 1 by
human monocytes are examined using the following protocol..
Bacterial lipopolysaccharide (L PS) is used to induce IL- 1 producdon by human
2 0 peripheral blood monocytes. IL 1 activity is measured by its ability to stimulate a Interleukin
2 (IL-2) producing cell line (E~4) to secrete IL-2, in concert with A23187 ionophore,
according to the method of Simon ~L. I~mmunol. Methods, 84, 85, (1985). Human
peripheral blood monocytes is isolated and purified from either fresh blood preparations from
volunteer donors, or from blood bank buffy coats, according to the procedure of Colotta et
2 5 al., J. Immunol., 132, 936 (1984). 1 X 106 of such monocytes were plated in 24-well plates
at a concentration of 1-2 million/ml per well. The cells were allowed to adhere for 2 hours,
after which time non-adherent cells is removed by gentle washing. Test compounds were
then added to the cells for 1 hour (hr) before the addition of lipopolysaccharide (50 ng/ml),
and the cultures are incubated at 37C for an additional 24 hours. At the end of the incubation
30 period, culture supernatants were removed and clarified of cells and all debris. Culture
supernatants were immediatdy assayed for IL- I biological activity in the manner described
above, as well as for prostaglandin and/or leukotriene concentrations by radioimmunoassay.
The compound of Example 7 demonstrated an ICso of 0.2911M.
3 5 I~ IILITY EXAMPLE B
In the tests used to detennine activity as 5-lipoxygenase pathway inhibitors,
male Balb/c mice (2~28 g), are used. All mice were obtained from Charles River Breeding
Laboratories, Kingston, N.Y. Within a single experiment, mice were age matched.
wo 92/ 10190 Pcr/us91 /09416
-29-
Reagents were employed as follows 9 8 7 7
Compounds of Formula (I) are used as the free base. The compounds were
dissolved in acid saline. Compounds are administered by lavage at the indicated dose in a
final volume of 10 mUkg.
For in vitro experiments, compounds are dissolved at appropriate
concentrations in ethanol (final concentration 1.0%) and then diluted to final concentrations
using the buffers indicated in the text.
Arachidonic Acid-Induced Mouse Ear Inflammation
Arachidonic acid in acetone (2 mg/20 ml) is applied to the inner surface of the
left ear. The thickness of both ears is then measured with a dial micrometer one hour after
treatment, and Ihe data is expressed as the change in thickness (10-3 cm) between treated and
untreated ears.
Test compounds are given orally in acid/saline at the times indicated prior to
the topical application of arachidonic acid.
Assav of S-Lipoxveenase Activities
The S-lipoxygenase (S-LO) is isolued from extracts of RBL-I cells. These
cells are obtained from the American Type Culture Collection (#CRL 1378) and are grown at
37 with 5% C2 in spinner culture using Eagles essential medium (MEM) supplemented
medium with 10% heat inactivated fetal calf serum. The cells were collccted from culture by
centrifugadon at 2,000xg for 20 minutes and then washcd twice with SOmM sodium
phosphate (pH 7.0) which contains ImM EDTA and 0.1% geladn. After this wash, the cells
wex resuspended in fresh phosphate buffer to achieve a concentration of 5X107 cells/ml.
This suspension is disrupted by nitrogen cavitadon using the Parr bomb at 750psi for 10
2 s minutes. The broken cells are then centrifuged at lO,OOOxg for 20 minutes. The supernatant
was collected and centrifuged at 100,000 xg for 60 minutes. This supernatant was collected
and stored at -70C until assayed.
The inhibition of 5-1ipoxygcnase activity is measured by one of two assays,
the radiotracer extent assay either measured after 90 seconds at 20C or measured according to
3 0 the method of G. K. Hogaboom et al., Molecular Pharmacol. 30, 510-S 19 (1986) or the
. continuous 2 consumpdon assay. The results from either assay are comparablc if not
identical. Compounds were dissolved in ethanol with the final concentration of ethanol being
1 % in the assay.
The radiotracer extent assay examines the 5-lipoxygenase products ltransLTB4
(DI-HETE),5HETE and SHPETE] produced after a 90 second incubation at 20C. Aliquots
(40mL) of the supernatant are preincubated with the inhibitor or vehicle fo~ 10 minutes in
25mM BisTris buffer (pH 7.0) which also contains lmM EDTA, lmM ATP,50mM NaCI,
5% ethylene gylcol and 100 mg/ml of sonicated phosphatidylcholine (total volume 0.238 ml).
wO 92/10190 2 0 9 81 7 7 -30- pcr/us91/o94l6
The 5-lipoxygenase reaction is initiaeed by the addition of CaC12 (2mM) and 1-C14-
arachidonic acid (25mM; 100,000dpm))(final volume 0.25ml). After 90 seconds, the reaction
is terminated by the addition of two volurnes (0.5ml) of ice chilled acetone. The sample is
allowed to deproteinize on ice for 10 minutes prior to centrifuging at 1,000 xg for 10 rninutes.
5 The deproteinized supernatants are dried under argon and then redissolved in 200 mL of
e~hanol. These samples are then analyzed by reverse phase HPLC as described by G.K.
Hogaboom et al., Molecular Pharrnacol. 30: 510-519 (1986), herein incorporated by
reference. The compound-mediated inhibition of 5-lipoxygenase activity is described as the
concentradon of compound causing a 50% inhibidon of product synthesis.
The second assay for assessing inhibition of the 5-lipoxygenase activity is a
contdnuous assay which monitors the consumpdon Of 2 as the reacdon progresses. The 5-
lipoxygenase enzyme t200mL) is preincubated with the inhibitor or its vehicle in 25mM
BisTris buffer (pH 7.0) which contains lmM EDTA, lmM ATP,5mM NaCI and 5% ethylene
glycol for 2 minutes at 20C (total volume 2.99 ml). Arachidonic acid (lOmM) and CaC12
15 (2mM) are added to start the reacdon, and the decxase in 2 concentradon follows with time
using a Clark-type electrode and the Yellow Spnng 2 monitor (type 53)(Yellow Springs,
OH). The opdmum velocity is calculated from progress curves. The compound mediated
inhibidon of 5-lipoxygenase acdvity is described as the concentradon of compound causing a
50% inhibidon of opdmum velocity for the vehicle-treated sample. The compound of2D Example 7 demonstrated a 6ffb inhibition at 20~ml.
LTC-4 / PGE~2 Production from Human Monocvtes in vitro
a) Cell Preparadon: Human monocytes are prepared from leukosource packs suppliedby the American Red Cross (Philadelphia,Pa). The leukosouree paeks are &aetionated by a
25 two-step proeedure deseribed by F. Colatta et al., J. Immunol. 132, 936 (1984), herein
ineorporated by referenee, that uses sequendal sedimentation on Fieoll followed by
sedimentadon on Pereoll. The monocyte fraedon which results from this technique is
eomposed of greater than 85% monoeytes (with the remainder being neutrophils andIymphoeytes). The monoeytes (1.5 X 106) are plaeed into polypropylene tubes and used as a
30 suspended eulture. The assay buffer eonsisted of RPMI 1640 buffer, [Moore, G. E. et al.,
JAMA, !99,519 (1967) herein ineorporated by referenee] 1% human AB serum,2mM
glutamine,100 U/ml Penicillin/Streptomycin,25 mM BPES [4-(2-hydroxyethyl)-1-
pipaazine-ethanesulfonie aeid], and lmM CaC12.
b) LTC4/PGE2 Producdon: Monocytes (0.9mUtube) were dispensed into 12 X 75
35 mm polypropylene tubes (as a suspended culture). Compounds (100ul of a 10X stock of the
compound of inoerest) dissolved in the assay media are added per tube (performed in
duplicaoe). The cells are incubaoed for about 45 minutes at about 7C with constant agitation in
a humidified incubator. A23187 calcium ionophore t2UM final concentration) used to
: . ,
wo 92/10190 Pcr/ussl/o94l6
-31- 209~1~7
stimulate the cells, is added and the monocytes are incubated an additional 15 minutes.
Supernatants are then collected from each tube, clarified by centrifugation, divided into two
aliquots and stored at -70C until assayed.
c) Radio-immunoassay: Supernatants are assayed for LTC4 production and PGE2 by
5 radioimmunassay; which is performed using a New England Nuclear Leukotriene [3Hl-LTC4
and [125Il-PGE2 RIA Kit according to the manufacturer's (New England Nuclear, Boston
Massachusetts) instructions. The compound-mediated inhibition of LTC4 is described as the
concentration of compound causing a 50% inhibition of LTC4 production.
10rI ILlTY EXAMPLE C
Inhibitory Effect of compounds of Formula (I) on
m vitro TNF production by Human Monocytes
Section 1: Assav set-up
1 5The effects of compounds of Formula (Il) on the in vitro production of TNF by human monocytes are examined using the following protocol.
Hu nan peripheral blood monocytcs are isolated and purified from either blood bank
buffy coats or platcletpheresis residues, according to the procedure of Colotta, R. et al., L
lmmunol., 132(2):936 (1984). The monocytes are plated at a density of 1 X 106 cells/ml
20 medium/well in 24-well multi-dishes. The cells are allowed to adhere for 1 hour after which
time the supernatant is aspirated and 1 ml fresh medium (RPMI-1640 (Whitaker Biomedical
Products, Whitaker, CA) conta~ning 1 % fetal calf serum and penicillin and streptomycin at 10
units/ml is added. The cells are incubated for 45 minutes in the presence or absence of test
compounds at lnM-lOuM dose ranges (compounds were solubilized in Dimethyl-
25 sulfoxide/Ethanol such that the final solvent concentration in the culture medium is 0.5%Dimethyl sulfoxide/0.5% Ethanol). Bacterial lipopolysacchande (E. coli 055:B5 tLPS] from
Sigma Chemicals Co.) is then added at 100 ng/ml in 10 ml Phosphate Buffered Saline (PBS)
and cultures incubated for 1~18 hours at 37C in a 5% C02 incubator. At the end of the
incubation period, culture supematants are removed from the cells, centrifuged at 3000
3 0 revoludons per minute (rpm) to remove cell debris and .05 ml of the supernatant is assayed
for TNF activity using the radioimmunoassay described below.
Section II: RadioimmunoassaY procedure for TNF activitv
The assay buffer consists of O.OlM NaP04, 0.15M NaCI, 0.025M EDTA and 0.1%
3 5 sodium azide at pH 7.4. Human recombinant TNF (rhTNF) obtained using the procedure of
Chen et al., Nature, 330:581-583 (1987) is iodinated by a modified Chlor~nine-T method
described in Section m below. To samples (50 ~11 culture supernatants) or rhTNF standards,
a 1/9000 dilution of polyclonal rabbit anti-rhTNF (Genzyme, Boston, MA) and 8000 cpm of
WO 92/10190 2 0 9 817 7 -32- pcr/us91/o94l6
125I-TNF is added in a final volume of 400 lal buffer and incubated overnight (18 hours) a~
4C. Normal rabbit serum and goat and-rabbit IgG (Calbiochem) are titered against each other
for maximum precipitation of the anti-rhTNF. The appropriate dilutions of carrier normal
rabbi~ serum (1/200), goat and-rabbit IgG (1/4) and 25 Units heparin (Calbiochem) are
s allowed to precipitate and 200 ~1 of this complex was added per assay tube and incubated
overnight at 4C. Tubes are centrifuged for 30 minutes at 2000 rpm, supematants are
carefully aspirated, and radioactivity associated with the pellets is measured in a Beckman
Gamma 5500 counter. The logit-log linear transformatdon curve is used for the calculations.
The concentradons of TNF in the samples are read from a standard curve of rhTNF that is
10 linear in the 157 to 20,000 pg/ml range.
Section m Radioiodinadon of rhTNF
lodinadon of rhTNF is performed using a modified chloramine-T method of Frolik et
al., J. Biol. Chem., 259:10995-11000 (1984). Briefly, 5 mg of rhTNF in 5 ml of 20MM
15 Tris ph 7.5, is diluted with 15 ml of 0.5M KPO4 and 10 ml of carrier free
125I(100mCUml;ICN). To inidate the reacdon, a 5ml aliquot of a 100mg/ml (aqueous)
chloramine-T soludon is added. After 2 minutes at room temperature, an addidonal 5 ml
aliquot is added followed 1.5 minutes later by a final 5 ml addition of chloramine-T. The
reaction is stopped I minute later by sequendal addition of 20 ml of 50mM Sodium20 Metabisulfite,100 ml of 120mM Potassium Iodide and 200 ml of 1.2 mg/ml Urea. The
contents are mixed and the reacdon mixture is passed over a pre-packed Sephadex G-25
column (PD 10 Phatmacia), equilibrated and eluted with Phosphate Buffered Saline pH 7.4
containing 0.25% gelatin. The peak radioactivity containing fractions are pooled and stored at
-20C. Specific activity of 125I-TNF is 80-100 mCi/mg protein. Biological activity of
2 5 iodina~ed TNF is measured by the L929 cytotoxicity assay of Neale, M.L. et al., Eur. J. Can.
Clin. Oncol., 25(1):133-137 (1989) and has been found to be 80% that of unlabeled TNF.
Section IV: Measurement of TNF- ELISA:
Levels of TNF are also measured using a modification of the basic sandwich
30 ELISA assay method described in Winston et al., Current Protocols in Molecular Biolo~v.
Page 11.2.1, Ausubel et al., Ed. (1987) John Wiley and Sons, New York, USA The ELISA
employed a murine monoclonal anti-human TNF antibody, described below, as the capture
antibody and a polyclonal rabbit anti-human TNF, descTibed below, as the second antibody.
For detection, a peroxidase-conjugated goat anti-rabbit antibody (Boehringer Mannheim,
3 5 Indianopolis, Indiana, USA, Catalog # 605222) is added followed by a substrate for
peroxidase ( lmg/ml orthophenylenediamine with 0.1 % urea peroxide). TNF levels in
samples are calculated from a standard curve generated with recombinant human TNF
..
wO 92/l0190 PCr/US9l/09416
~33~ 2093177
produced in E. Coli (obtained from SmithKline Beecham Pharmaceuticals, King of Prussia,
PA, USA).
Sec~on V: Production of anti-human TNF antibodies:
Monoclonal antibodies to human TNF are prepared from spleens of BALB/c mice
immunized with recombinant human TNF using a modification of the method o~ ;ohler and
Millstein, Nature 256:495 (1975), the entire disclosure of which is hereby incorporated by
reference. Polyclonal rabbit anti-human TNF antibodies are prepared by repeated
immunization of New Zealand White (NZW) rabbits with recombinant human TNF emulsified
in complete Freund's adjuvant (DIFCO, IL., USA).
SYNTHETIC EXAMPLES
EXAMPLE I
5.6-Dihvdro-2-(4 methvlthiophenvl)-3-(4 ~vridinvl)-7H-pvTrolor l .2-alimidazol-7-ol
(Intermediate 7-ol compound)
a) 5.6-Dihvdro-2-(4-methvlthio~henvl)-3-(4-pvridinvl)-7H-~vr~olo~1.2-al-
imidazol-7-ol.
To a soludon of 5,6-dihydro-2-(4-fluorophenyl)-3-(4-pyridinyl)-7H-pyrrolo[1,2-
2 0 a]imidazol-7-ol (0.85 grams (hereinafter g), 2.9 millimoles(hereinafter mmol)) in DMF (10
milliLiters (hereinafter mL)) was added sodium thiomethoxide (0.30 g,4.4 mmol). The
resulting mixture was heated at 120C for 48 h, then allowed to cool. The mixture was
concentrated under reduced pressure, and the residue was partitioned benveen H2O and
CH2C12. The organic extract was washed with saturated a~gueous NaCl and dried (MgSO4).
2 5 The solvent was removed in vacuo, and the residue was recrystallizcd t vice from MeOH to
provide a light tan solid (0.19 g, 20%). m.p. 229 - 230C
lH Nk~ (CDC13): o 8.59 (d, 2H); 7.40 (d, 2H); 7.19 (2 overlapping d, 4H); 6.18 (br d,
lH); 5.27 (m, lH); 4.27 (m, lH); 3.94 (m, lH); 2.90 (m, lH); 2.63 (m, lH); 2.50 (s,
3H).
30 CIMS (NH3); m/e (rel. int.): 324 [(M+H)+, 100], 308 (11).
,~n~l. Calc. for Cl8Hl7N3os: C 66.85, H 5.30, N 12.99, S 9.91; found: C 66.78, H 5.55, N 12.95, S 9.58.
EXAMPLE 2
3 5 5.6-Dihvdro-2-(4-fluorophenvl)-3-(4-pvridinvl)-7H-pvrrolorl .2-alimidazol-7-ol
(Intermediate 7-ol compound)
a) I - l 5.6-Dihvdro-2-(4-fluorophenvl)-3-(4-pvridinvl)-7H-pvrrolor1.2-al-
.
~ . .
wo 92/10190 Pcr/ussl/094l6
2098177
imidazol-7-vll-1-(4 nitrophenvl)methanol.
To a solution of 5,~dihydro-2-(4-fluorophenyl)-3-(4-pyridinyl)-7H-pyrrolo[1,2-
a]imidazole (15.0 g, 0.054 moles (hereinafter mol)) in CH2C12 (50 mL) at 0C was added
methoxyethoxymethyl chlo~ide (30 mL, 0.26 mol). The resulting mixture was allowed to
5 warm to room temperature and stirred for 1 hour (hereinafter h). Ether was added, and the
mixture was decanted (3x). The residue was dissolved in EtOH (400 mL), and to this
solution were added triethylamine (40 mL, 0.29 mol) and 4-nitrobenzaldehyde (15.0 g, 0.10
mol). The resulting mixture was heated at reflux for 48 h, then allowed to cool and
concentrated under reduced pressure. The residue was partiioned between H2O and
10 CH2C12. The organic extract was washed with saturated aqueous NaCI and dried (MgSO4).
The solvent was removed in vacu~, and the residue was triturated with EtOAc. The orange
solid which formed was collected by filtration to afford the title compound (8.0 g, 34%)
which was used without further purification.
b) 5~6-Dihvdro-2-(4-fluoropheml)-3-(4-pvridinvl)-7H-pvlTolorl.2-alimidazol-7-one.
To a solution of Jones reagent (25 mL) in acetone (250 mL) was added 1-{5,6-dihydro-2-(4-
fluorophenyl)-3-(4-pyridinyl)-7H-pyrrolo[ 1 ,2-a]imidazol-7-yl ) -1 -(4-nitrophenyl)methanol
(5.0 g, 12 mmol). Thc rcsulting mixture was stirred at room tcmperature for 30 min, thcn the
pH was adjustcd to 7 - 8 with 2.5 N NaOH. Thc solid matcrial was rcmovcd from thc
2 0 acctonc soludon by dccantadon and parddoned betwcen 2.5 N NaOH and 1: 2 CH2C12/
Et20. This mixturc was filtered, and thc layers were scparated. Thc organic cxtract was
combined with thc acctone solution and cvaporatcd undcr reduccd prcssurc. Thc rcsiduc was
partidoncd bctween 2.5 N NaOH and CH2C12, and the organic cxtract was washed with
saturated aqueous NaCI and dried (MgSO4). The solvcnt was removed in vacuo, and the
2 5 residuc was triturated with Et2O to provide the title compound as an orange solid (1.5 g,
43%), which was used without further purification.
c) 5.6-DihYdro-2-(4-fluorophenYI)-3-(4-pvridinYl)-7H-pyrrolo-rl~2-alimidazol-7
To a soludon of 5,~dihydro-2-(4-fluorophenyl)-3-(4-pyridinyl)-7H-pyrrolo[1,2-
30 a]imidazol-7-one (crude product prepared above) in MeOH (15 mL) was added sodium
borohydride (1.5 g, 40 mmoi), and the resulting mixture was stirred at room tcmperature foq
15 min. The mixture was concentrated under reduced pressure, and the residue waspartitioned between H20 and CH2C12. The organic extract was washcd with saturated
aqucous NaCI and dricd (MgSO4). The solvent was removed in vacuo, and the residue was
3 5 triturated sparingly with EtOAc and copiously with Et20. The solid which formed (0.90 g,
26%) was recrystallized from MeOH to afford the title compound as a white solid.lH NMR (DMSO~ 8.58 (d, 2H); 7.45 (dd, 2H); 7.36 (d, 2H); 7.16 (apparent t, 2H);
5.76 (d, lH); 4.99 (m, lH); 4.16 (m, lH); 3.95 (m, lH); 2.82 (m, lH); 2.30 (m, lH).
WO 92/10190 PCI`/US9l/09416
- - 2098~77
CIMS (NH3); m/e (rel. int.): 296 [(M+H)+, 100].
EXAMPLE 3
6~7-Dihvdro-2-(4-methvlthiophenvl)-3-(4-pvridinYl)-5H-pvrrolorl 2-alimidazole
5 (Intermediate compound of Formula (A)
a) To a vigorously stirred suspension of potassium hydroxide (341.0g,6.09 mol) and
tetraethylammonium bromide (51.2g, 0.24mol) in tetrahydrofuran (THF) 2.0 l) was added 2-
pyrrolidinone (97.2 ml,1.28 mol) at 20C. A thick white slurry formed and the temperature
rose to 27C within 30 minutes.
The reaction mixture was stirred mechanically for a total of 100 minutes between 20-
30C before 4-picolyl chloride hydrochloride (200.0g, 1.22mol) in demineralized water
(120ml) was added over 25 minutes. The temperature rose to 40C and was not allowed to
rise above this. The reaction mixture was stirred for 120 minutes after this addition and was
then filtered through Celite. The reaction flask and filtered solids were washed with THF
15 (400ml) and the washings combined with the filtrate. Any aqueous material carried over
during the filtradon was separated before the organic soludon was concentrated to a volume of
800ml by atmospheric distilladon of the THF. The solution was cooled to 20C at which
point 60-80 pctrol (500ml) was added. The solution was stirred for 10 minutes when a
further 500ml quantity of 60-80 petrol was addcd. This mixturc was stirred for a further 10
20 minutes when a final 600ml quantity of 60-80 petrol was addcd. Thc mixn~re was cooled to
5C for 16 hours before the product was isolated by filtration, washed with 60-80 petrol
(400ml), and dried at 40C, 100 mmHg for 24 hours. Hence 1-(4-picolyl)-2-pyrrolidinone
186.0g (86%) was obtained as a pale brown granular crystalline solid; m.p. 82-84C; HPLC
assay 96.1%; M+, 176.0947. C10Hl2N2o requires 176.0950; m/z 176, (M+), 147 (M+ -
25 C2Hs), 119 (147-CO) and 903 (119 - HCN); v maximum (KBr) 2950, 1690 (C=O), 1600,
1450, 1420, 1300 and 1280 cm~1; oH(270 MHz, CDC13) 1.85 (2H, m, -CH2CH2CH2-).
2.20 (2H, t, -C_2C(0), 3.10 (2H, t, -CH2CH2NRRI), 4.25 (2H, s, PyCH2-), 6.95 (2H,
m, Ar(3,5)) and 8.30 (2H, m, Ar(2,6).
b) To a solution of 1-(4-picolyl)-2-pyrrolidinone (20.0g, 0.114mol) in dry THF
30 (260ml) was added n-butyllithium (50.Oml of a 2.5 M solution in hexane 0.125mol) at O to -
10C. The addition required 10 minutes. Potassium tertbutoxide (12.7g,0.114mol) in THF
(65ml) was then added at O to 10C over 5 minutes and the resultant golden yellow suspension
stiITed for 10 minutes. At this point 4 methylthiobenzonitrile (18.6g, 0.125mol) in THF
(31ml) was added over 5 minutes at 0 to -10C. When the addition was complete thereaction
3 s mixture was allowed to warm to ambient temperature over 30 minutes. After this period the
reaction mixture was heated underreflux for 120 minutes and the cooled to 30C before
demineralised water (80snl) was added. The resultant mobile solution was stirred for 30
minutes and the aqueous layer then allowed 30 minutes to separate before it was removed.
wo 92/10190 2 0 9 817 7 -36- PCI /US9l/09416
The solvent was exchanged with ethyl acetate via a put and take distillation where
140ml solvent was removed and the replaced with 140ml ethyl acetate. This process was
continued until the base temperature reached 77C. A further 45ml ethyl acetate was added
and the solution cooled to 50C before 60-80 petrol t87ml) was added. The product
5 crystallized on cooling to room temperature and after stilTing for 3 hours the suspension was
cooled to 0-5C and stiIred for a further 2 hours. The product was then isolated by filtration,
washed with 60-80 petrol (40ml) and then dried at 40C,100mmHg for 24 hours. Hence
6,7-dihydro-2-(4-methylthiophenyl)-3-(4pyridinyl)-5H-pyrrolo [1,2-a] imidazole was
obtained as a pale yellow crystalline solid; 17.6g, 50%; m.p. 172C; HPLC assay 95.6%; ~H
10 (270MHz, CDCl3) 2.50 (3H, S, -S~), 2.70 (2H, m, -CH2C_2, CH2-) 3.00 (2H, t, - C_2CH2CH2NRRl), 4.05 (2H, t, -CH2CH2C_2NRR1), 7.20 (2H, m, MeS ~), 7.30
(2H, m, 3,5-Py), 7.50 (2H, m, Me S ,~) and 8.60 (2H, m, 2,6-Py).
EXAMPLE 4
15 6.7-Dihvdro-2-(4methvlthiophenvl)-3-(4-pvridinvl)-5H-pvrrolo ~1.2-al imidazole
(Intermediate compound of Formula (A)
a) To a solution of 1-(4-picolyl)-2-pyrrolidinone (2.01g, 11.4mmol) in THF (285ml)
was added n-butyllithium (8.70ml of a 2.5M soludon in hexane, 21.7mmol) at -70C. The
resultant yellow suspension was stirred for 90 minutcs between -30 to -70C before 4-
20 methylthiobenzonitrilc (2.72g, 18.3mmol) in THF (40ml) was added at 65C. The reacdonmixture was st~ned with warming to room tcmperature over 15 minutes and was then stirred
for a further 21 hours. After this time ammonia (720,ul of a 35% w/w aqueous solution) was
added which caused the reaction mixture to change from blood red to yellow in color. This
soludon was st~rred for 30 minutes before the solvent was removed in vacuo and the residue
2 5 chromatographed on silica gel using ethyl acetate: triethylamine - 96:4 as elutant. Hence Z I -
amino-1-(4-methylthiophenyl)-2-(4-pyridyl)-2-(1-(2-pyrrolidinoyl))ethene (1.2g, 32%) was
obtained as a free flowing yellow powder, m.p. 220-222C (from ethyl acetate) M+325.1271.
C1gH1gN3OS requires 325.1249. v maximum (nujol mull) 3500-3300 (N-H), 1669 (C-O),
30 1632 (C-C) and 1566 cm~1; ~H t270 MHz, d6-DMSO) 2.10 (2H, m, -CH2CH2CH2-), 2.40
(2H, t, -C_2CH2CH2C(0)-), 2.50 (3H, s, -S~), 3.50 (2H, t, -CH2CH2CH2C(0)-), 5.70(2H, s, -NH2), 6.50 (2H, m, 3,5-Py), 7.25 (4H, m, MeS ~) and 8.05 (2H, m, 2,6-Py);
m/z 325(M+), 308 (M-NH3), 268 (M-C3H50) and 150 (CgHgNS).
b) To a suspension of Z-1-amino-1-(4-methylthiophenyl)-2-(4-pyridyl)-2-~ 1-(2-
35 pyrrolidinoyl) ~ethene (114mg, 0.35 lmmol) in THF (8.8ml) was added n-butyllithium (249111
of a 2.5M solution in hexane, 0.491mmol) at -40C. The resultant dark red solution was
allowed to warm to room temperature over 30 minutes and was then stirred at this temperature
. ~ .
wo 92/10190 Pcr/Us91/09416
~37~ 2098177
for 19 hours. After this time the color changed to light yellow. At this point the reaction
mixture was assayed by HPLC and found to contain the title compound 88mg, 82%.
EXAMPLE S
5 2-(fluorophenvl)-6.7-dihvdro-3-(4-~YridinYI)-5H-pYrrolorl~2-alimidazole (Interrnediate
compound of Formula (A)
To a soludon of 1-(4-picolyl)-2-pyrrolidinone (56mg, 0.318mmol) in dry T~; (8ml)was added n-butyllithium (472111 of a l.OM solution in hexane, 0.477mmol) at -80C. The
resultant cloudy bright yellow solution was stirred between -50 to -80C for 50 minutes before
10 p-fluorobenzonitrile (61mg, 0.8097mmol) was added in THF 93ml) at -80C. The reacdon
mixture was then allowed to warm to room temperature when it became dark red. It was
stirred for 18 hours before the solvent was removed in vacuo and the residue
chromatographed on silica gel using ethyl acetate:methanol - 4: 1 as elutant. Hence the dtle
compound was obtained (7mg, 7%).
EXAMPLE 6
2-(4-Bromo~nYI~-6.7-dihvdro-3-(4-~Yridinvl)-SH-pvrrolorl .2-alimidazole
ntermediate com~ound of Formula (A)
To a soludon of 1-(4-picolyl)-2-pyrrolidinone (2.66g, lS.lmmol) in dry T~ (76ml)20 was added n-butyllithium (7.26ml of a 2.5M soludon in hexane, 18.1mmol) at -40C. A
soludon of potassium tert butoxide (1.69g, lS.lmmol) in TE~ (8.5ml) was then added and
the tesultant golden yellow suspension sdrred at -40C for 10 minutes. At this point a
solution of 4-bromobenzonitrile (5.50g, 30.2mmol) in l~IF (50rnl) was added at -40C and
the reaction mixture then allowed to warrn to room temperature. After stirring for 18 hours
2 5 the reaction mixture was concentrated to dryness and the residue chromatographed on silica
gel using ethyl acetate:methanol - 5: 1 as elutant. Hence the dtle compound was obtained as a
yellow crystalline solid (0.71g, 14%); M+ 339.0371. C17H14N3 79Brrequires 339.0371
~+ 341.0387. C17H14N3 81Br requires 341.0351. oH (270MHz, CDC13) 2.65 (2H, m, -
C~2,CH2CH2-), 3.00 (2H, t, -CH2CH2CH2NRR'), 4.00 (2H, t, -CH2CH2C~;2NRR'),
30 7.25 (2H, m, 3,5-Py), 7.40 (4H, m, Br-Ar) and 8.60 (2H, m, 2,6-Py); m/z 339 (a5l ), 341
(~+) 259 (M-HBr), 310 (M-C2Hs) and 312 (M-C2Hs).
3 5 EXAMPLE 7
5.6-Dihvdro-2-f4-fluorophenvl)-7-methvl-3-(~pYridinyl)-7H-pylrolor1.2-alimidazol-7-ol.
(A compound of Forrnula I and II)
wo 92/10190 PCrtUS91/09416
2 0 9 ~ 38-
5.6-Dihvdr~2-(~fluoroDhenvl)-3-(4-pvndvl)-7-meIkvl-7H-pvrrolo~1.2-alimidazol-7-ol. To
a stining solution of 5,6-dihydro-2-(4-fluorophenyl)-3-(4-pyridyl)-7H-pyrrolo[1,2-a]-
imidazol-7-one (100 mg,0.34 mmol) in toluene (10 mL) under an argon atrnosphere was
added trimethylaluminum (1.7 mL of 2 M soludon in toluene, 3.41 mmol). The resuldng
mixture was heated at reflux for 4 h, then cooled to 0C. To the cooled mixture was slowly
added MeOH, followed by CH2C12. The mixture was washed successively with H20,
saturated aqueous NaCI and dried (MgS04). The solvent was removed in vacuo, and the
residue was purified by flash chromatography, eluting with 2% MeOH/ CH2CI2 to afford a
light tan solid (47 mg,45%) which was recrystalliæd from MeOH and dried in vacuo. m.p.
246- 248C
lH NMR (CDC13): 8.60 (m, 2H); 7.48 (apparent dd, 2H); 7.20 (m, 2H); 7.02 (apparent
t, 2H); 4.19 (m, lH); 3.95 (m, lH); 2.81 - 2.58 (m, 2H); 1.80 (s, 3H).
CIMS; m/e (rel. int.): 310 (M+H)+.
Anal. Calc. for ClgHl6FN3O-1/2 H2O: C 67.91, H 5.38, N 13.19; found: C 67.95, H
5.09, N 13.05.
EXAMPLE 8
5.6-Dihvdro-2-(4-fluorophenvl)-7-methoxv-7-methvl-3-(4-pvridinvl)-7H-pvrrolo-
~1.2-alimidazol-7-ol. (A compound of Formula I and II)
To a stirring solution of the 5,6-Dihydro-2-(4-fluorophenyl)-7-methyl-3-(4-
pyridinyl)-7H-pyrrolo[1,2-a]imidazol-7-ol (1.0 g,3.4mmol) pT~pared in Example 7, in DMF
(2ml) is added NaH (16.8mg, 0.35mmol of a 50% suspension in oil). After the gas evolution
ceases MeI (0.022ml, 0.32mmol) is added and sdrring is condnued for 1 h at which point the
reaction mixNre is evaporated to dryness under reduced pressure and chromatographed on
2 5 silica gel to yield the desired methyl ether derivative.
The above description fully discloses the invendon including preferred
embodiments thereof. Modifications and improvemenLs of the embodiments specifically
disclosed herein are within the scope of the following claims. Without further elaboration, it is
3 0 believed that one skilled in the art can, using the preceding descripdon, udlize the present
invention to its fullest extenL Therefore the Examples herein are to be construed as merely
illustrative and not a limitation of the scope of the present invention in any way. The
embodiments of the invendon in which an exclusive properry or privilege is claimed are
defined as follows.
