Note: Descriptions are shown in the official language in which they were submitted.
F E F: - 1 ' -~ w ~~ l~l E Z:: 1 ~I ~ 1 1 FW h I h-.I F_ ~Wf I C=: C: 1-I Y~I
r_'. le:: I E
SLDCKAGE t7F Ct7ENZ'~ME A-~lIN~3EP'ENLDEi~T
TRANSACYLASE ACTI'~I~''~ AS A METHOD
Tt~ xNI~IIEI~' LIPID MEDIA1~OR PFtGDIICTI~N
FIELD ~QF 'rHE Il~'~'E
The invention relates to the area of infiarnmatary mediators.
~"he invention is bayed on the discovery that blocking a key enzyme
responsible for araoialdanie aoid rennadelling, Coenzyme A
independent tcansacylase (CoA~~Ta, inhibits the production o% Iipid
mediators (arachidanic acid, araehidanie acid metabolites, and
platelet aetivatin~ factor (PAF)). It has been disaavered that CoA-IT
is rewired in tiza synthesis of arachidonic acid, arachidonie acid
metabolites and FAF.
~AsC%~~Y~f.~UND t~F ~'IIJS I~T''~'EIdTICIN
An early event in the response of mast inilannmatory cells to
immunalo~ia aotivation and other stimuli is the release of newly
formed pratfucts which alter the function and biochemistry of
surrpundin~ cells and %issu~s. The ensuing biola~ioal responses, as
well as mush of the patho~enes~ which is attributed to inilammaticn
and allergy, are thought to be dependent on the effects these products
have on adjacent cells within the inilaminatory re~ian.
FED:-1~-.c.~ 4JE=: 14 : 1 :l F:fah~Ih~IEF: L:IF:n:I-I hhG~F:'tE F'. ~7E.
_Z_
Althougta many inflammatory mediators are produced, two
ela5se~ of naturally oCCUrring lipids almost always appear during cell
activation. One class eonststs of an endogenously formed
phospholipid, PAF. The seoond class consists of eicosanoid products
which are endogenously ~ormad metabolites o~ arachidonie acid (AA).
L L'~own' 9The eicosanoids include lauicotri~n~ LTB4, LTC~g and
LTD, HBT~~s, thrombo~canes and prostaglandins. Both PAS' and
eleosanoids are potent lipid mediators whioh induce signs and
symptatns ol~erv~d in the pathogen~l~ of various in~lammatary
disorders. for example, these mediators have been implicated as
having an important rate in allergy, asthma, anaphylaxis, adult
respiratory distress syndrome, reperPusion injury, inflammatory bowel
disease, rheumatoid arthritis, andotaxie shook, and cardiovascular
disuse. Salmon and Higgs ~~r, Med. dull, (19?S) ~3a~8b-298,; Piper
et al, Ann. NY Acad. Sci. (1891) 6as;112-119a; ~oltxrnan tAm. ~~ .
Respir. iJis. (3891) i43a188°037. Snyder (Am. J, Physiol. Cell Physlol.
(1990) 259aC897°C?081; Prescott et al. t~. viol. Chem. (1990)
265ai738i°17884, Each ox the cell typws involved in the
inflammatory response produce and secrete a unic~u~ subset o~ lipid
nnediator~. The quantities and nature of the metabolites depend on
which enzymes and preoursar peals are available to the cell.
Studies ire the last few years have indicated that the
bioehexnistry at araehidonie acid and PAF overlap at a number of key
~roints, xn particular, it was shown in i98~ that araehidonie aoid and
lyso-PA~ (an intermediate in PAP biosynth~ts) could be derived Rrom
a~ common greeursor phospholipid, i-alkyl-2-araehidonoyl-sn°glyeero-
FEE-..., ~ '-~~~' w~L. i a. : i w ~;Hrur~~~~: F; i ~:mm rar.:r::: z ~ F~ . ,-
;;
~. ..s
m$_ ,
3-phosphocholine (CPC) (Chiltan et al., J. Biol. Chem. (1984)
25:12019-iz019). The association of arachidonic acid mobilisation
with PAF production was supported by studies in which arachidonic
acid depletion of cells is coupled to a loss of PAF production and
refeeding araolxidanie acid to these cells restores PAS' production.
Suggs et al, LJ. Biol. Chew. (1990) 285:12363-°123711; Ramoeha and
Plckett ~J.18ia1. Chem. (1986) 261:7592-9895'.
i-alkyl°2-arachidonoyl-OPC, the proposed common precursor
%or PAF and eicasanoids, belongs to a family of phasphalipids termed
1-ether phasphoiipids, This term indicates that there is an ether
linkage at the sn-i position of the glycerol backbone of the
phospholipid. Over the last years, it has been determined that several
inflammatory cells have high Content of ether lipfds. In addition,
arachidonate is preferentially located in i-ether linked phosphalipids
of these cells. Moreover, these 1-ether linked pools are the primary
phosphollpfds from which araehidonic acid is mobilized during
inflammatory cell activation, Therefore, inflammatory cells, in
general, contain large pools of 1-ether linked phespholipids which
have the large reservoirs of arachidonate. During cell activation,
these 1-ether linked phospholipids release arachidanic acid which
forms eicosanaids; and the i-ether phosphalipid lyso PAF is acetylated
to form pAF,
SUMiVIAR'~' ~~' ?'HR IN'l~~~d'~I~l~
It is an abject of this invention to provide a method of reducing
allergy and inflammation,
F E F: - 1 ~ - v~,'_..,., ~'.'a E =; J. =E : 1 :._ E; W r-~ ha E F: G: I F~: y
r~ r~ ~ ;_ t<:: z E P . 4i
-
It !s also an object of this invention to inhibit undesir2.bie lipid
mediator production.
This invention is based on the discovery that blocking Co-A
independent transaeylase, using selective pharmaCOlogie tools,
prevents the movement of arachidonic acid into and from i-ether
linked pho5pholipids and the ooncomistant formation of PAF,
araohidonla acid and its metabolites such as eieosanoids.
The invention relates to a method of treating disease or
disorders mediated by araohidonic acid, its metabolites and/or pA~k' by
admiz~isterlng to a patient in need thereof, an effective amount of a
compound which inhibits the production, activation or action of
~oA-IT. Inhibition of ~oA-IT inhibits iipid mediator produotfon as
well as signs and symptoms of disease and disorders induced by lipid
mediators.
The pretrcise of this invention is that blocking the movement of
araehidonio acid into and from i-ether phospholipids inhibits lipid
mediator (PAF as~d eicosanoid) production by inflammatory cells.
Mor~ preciseiy, when arachidonie acid is prevented from entering key
common precuPSOr phospholipicl9, precursor malecules will not be
formed and therefore 1P,~~ as weh as eicosanoid, formation are
blocked, Similarly, prevention of the removal of arachidonie acid
from salient precursors ~i,e., i-ether linked phospholipids) mear~ that
araehidonic acid and lyso PAF will be not be mobilized and therefore
PA>r' as well as eiccrsanoids will not be produced.
Mill another aspect of the invention relates to a method of
screening chemical compounds %or potential anti-inflammatory
1
CA 02061061 2003-02-28
-5-
action. In this way, chemical compounds can be rapidly and easily screened
for the ability to inhibit CoA-IT and be useful as an anti-inflammatory agent.
According to an aspect of the present invention, there is provided use
of an effective amount of a compound which inhibits the production, activation
or action of Coenzyme A-independent transacylase (CoA-IT) for the treatment
of disease or disorders mediated by arachidonic acid, its metabolities andlor
by platelet activating factor (PAF).
According to another aspect of the present invention, there is provided
a composition useful for the treatment of adult respiratory distress syndrome,
wherein the composition comprises a therapeutically effective amount of a
compound which inhibits the production, activation or action of Coenzyme A-
independent transacylase (CoA-I~T), and a pharmaceutically compatible carrier
for the active compound.
According to a further aspect of the present invention, there is provided
the use of a therapeutically effective amount of a compound which inhibits the
production, activation or action of Coenzyme A-independent transacylase
(CoA-IT), and a pharmaceutically compatible carrier for the active compound
in the treatment of adult respiratory distress syndrome.
According to another aspect of the present invention, there is provided
a composition useful for the treatment of reperfusion injury, wherein the
composition comprises a therapeutically effective amount of a compound
which inhibits the production, activation or action of Coenzyme A-independent
transacylase (CoA-IT), and a pharmaceutically compatible carrier for the
active compound.
According to a further aspect of the present invention, there is provided
the use of a therapeutically effective amount of a compound which inhibits the
production, activation or action of Coenzyme A-independent transacylase
(CoA-IT), and a pharmaceutically compatible carrier for the active compound
in the treatment of repertusion injury.
According to another aspect of the present invention, there is provided
a composition used for the treatment of inflammatory bowel disease wherein
the composition comprises a therapeutically effective amount of a compound
i i
CA 02061061 2003-02-28
-5a-
which inhibits the production, activation or action of Coenzyme A-independent
transacylase (CoA-IT), and a pharmaceutically compatible carrier for the
active compound.
According to a further aspect of the present invention, there is provided
the use of a therapeutically effective amount of a compound which inhibits the
production, activation or action of Coenzyme A-independent transacylase
(CoA-IT), and a pharmaceutically compatible carrier for the active compound
in the treatment of inflammatory bowel disease.
According to another aspect of the present invention, there is provided
a composition used for the treatment of disease mediated by arachidonic acid,
its metabolites and by platelet activating factor (PAF), wherein the
composition comprises a therapeufiically effective amount of a compound
which inhibits the production, activation or action of Coenzyme A-independent
transacylase (CoA-IT), and a pharmaceutically compatible carrier for the
active compound.
According to a further aspect of the present invention, there is provided
the use of a therapeutically effective amount of a compound which inhibits the
production, activation or action of Coenzyme A-independent transacylase
(CoA-IT), and a pharmaceutically compatible carrier for the active compound
in the treatment of a disease mediated by arachidonic acid, its metabolites
and by platelet activating factor (PAF).
DETAILED DESCRIPTION OF THE INVENTION
This invention concerns and encompasses the enzyme CoA-IT, its
unique characteristics, its requirement for the production of PAF, free
arachidonic acid and metabolites of arachidonic acid, and the therapeutic
utility of inhibiting CoA-IT.
It has now been discovered that CoA-IT activity is required for lipid
mediator production. Specifically, it has been discovered that CoA-IT activity
is required for the movement of arachidonic acid into phospholipid pools from
which it can be released to form free arachidonic acid and for the production
of lyso PAF needed for PAF synthesis. Further, CoA-IT has been shown to
CA 02061061 2002-09-11
-5b-
be crucial in the mobilization of lyso-PAF and arachidonic acid during
inflammatory cell activation. Inhibition of CoA-IT activity will result in a
decreased production of PAF and a decreased release of arachidonic acid
from cellular phospholipids.
1. Characteristics of CoA-IT Activity
CoA-IT activity had been defined to have the following characteristics.
A. Co-factors
CoA-IT activity is independent of the presence of Coenzyme A.
In addition, no other co-factors required for activity or that modulate
activity
have been discovered. CoA-IT activity is not
~i
CA 02061061 2002-09-11
-6-
altered by the absence or presence of Ca2+ (0-10 mM), Mg2+ (0-10
mM), EGTA (0-2 mM), EDTA (0-10 mM), ATP, CoA or CoA-fatty
acids.
B. J~H
CoA-IT activity over a wide range of pH levels was
determined. The results demonstrate that the enzyme is active over
a broad pH range of 6.5 - 9. The activity of the enzyme rapidly
decreases below pH 6.5 and above pH 10.
C. din ti
The kinetics of the CoA-IT reaction were studied with
various concentrations of 1-alkyl-2-lyso-GPC. CoA-IT activity
increases as a function of the concentration of substrate, 1-alkyl-2-
lyso-GPC. The enzyme exhibits an apparent Km for 1-alkyl-2-lyso-
GPC of 0.1 - 2 pM.
D. Other Characteristics
CoA-IT is stable when treated with dithiothreitol or 2-
mercaptoethanol (1-10 mM). CoA-IT is inactivated by exposure to
heat or acid and is inhibited by addition of detergents such as 8-
octyl glucoside, deoxycholate, cholate, TritonT"" X-100, C~2E8,
CHAPS and hexadecyltrimethyl ammonium bromide.
E. S_aecificitX
A key characteristic of CoA-IT is the exquisite
specificity of this enzyme for polyunsaturated fatty acids and
especially arachidonic acid. Sugiura et al. (J. Biol. Chem. (1987)
202:1199-1205); Chilton et al. (J. Biol. Chem. (1983) 2~$:7268-
7271 ); Kramer and Deykin (J. Biol. Chem. (183) X5$:13806-13811 ).
F E F: - 1 '~ - '_: '~ G,.~ E T; 1 ~4 : 1 ~1. E; ri h~I t-I E ~' F; :I F: r_,:
t-I t~i n: t=: :L E F' . 1 1
F. Location
Within the cell, CaA-IT activity is completely and tightly
associated with mierosomal membranes, Ti reatrnent of these
membranes with 2 M ICCI fails to extract more than '1596 of the
CoA-I'T activity, suggesting that CoA-I'1' is an Integral rriombrane
component. The subGallular location of CoA-lx activity remains to be
determined.
Evidence of CaA-i'F activity exista in a variety of in%lammatory
cells, including human PMN, nronocyt~, lung mast cells, guinea pig
eosi~nophil$ and ~hu~nan X93? monoaytic and I~IL-60 granuloayte ceps
lines. There is also preliminary evidence that somewhat less CoA°1~'
activity is found in tissues such as lung, liver and kidney. Lass
activity yet is found in heart, skeletal muscle and brain,
C. ComaarLSOn with other enzymes
CoA°xx has eharaetaristfes vrhieh distinguish its activity from
the activities of other enzymes involved in lipid metabolism, such as
phosphalipasa A2, lypoxygenasas, cyeloaxygenases, CoA dependent
acyltransierases and 1~AF acetyl transferasa. This distinction of
CoA°IT from the other enzym,~.s based on eharacte~ristics ~ impartant
far several reasons, First, the data indicate that CoA-IT activity is a
novel enzyme activity. Second, even though a microsomal
preparation is used to assess CoA-IT activity, the distinct
characteristics of CoA-I'f assure that the assays znaasure only CoA°xT
activity. Finally, the characteristics of CoAmx~' demonstrate that the
pharmacologioai utility of inhibition of CoA~i~ is unique.
FEF:- 1 W-~-~',_~ IJEW 1 ~4 : :L ~ F:yhlh-IEF'. ~: I F: C:f-I f~ln:h::: I E F'
. 1 W
-g-
Z. Role of CaA-IT in PAF Praduetior~ and AA Release
The molecule 1-alkyl-z-araohidonoy,l-~GPC has been spawn to be
a beoe~ssary precursor far PAF production. ~oA-lx activity plays two
pivotal rotes in PAF production, eenterir~g on this molecule. First,
CoA-IT activity is required for the specific ~navernent of araehidonic
acid into the alkyl PC poet to produce this precursor molecule,
Second, CaA-iT activity has been shown to promote the breakdown of
1-alkyl-z-arachidonoyl-GPC into lysa PAF to allow FAF production.
'This CoA-X~' mediated production to lyso BAF can be differentiated
from p~A2 activity. CaA-IT activity plays a central aed necessary
role in the production of PAF.
There is strong evidence that, in activated inilamcnatory sells,
arachidonic acid is released from specitie phaspholipid peals. ~'ar
axam~ple, in neutrophils and mast cells the primary source of free
arachidanic said is I,-al4cenyi-2-arachidonoyl-GPE. Due to its unidue
properties, GoA-IT activity can replenish this pool with arachidonic
acid to allow and maintain the release of araehidonie acid, tt has new
been discovered that OoA-IT activity is necessary and essential xor
the release of free arachidanic acid and the subs~~uen~t formation of
biaactive lipid mediators.
S. OoA-IT inhibition
Inhibitors of ~oA-IT activity have now been dissevered and
Characterized. Suitable inhibitors can readily be identified employing
assay tai described below, Often, inhibitors will include an imida2ole
structure,
F E F: _. 1 ~ _ .a .' LJ E =~ 1 ~-~. : 1 .W F: ha t-1 ha E F: F: I F='. i '. f-
I t' I C: H:: I E H~ . 1 .;
-C~.
Represontative eornpounds which inhibit CoA-XT activity in a
mierosomal Cod,-iT essay (assay a) at So uM are:
1. 8thy1 &-(3,9,5-txSphany'1-2-oxa-'2,3-dihydroimidazoL-1-y1)haxanoata
2. Sodium 7(3,4,5-txiphenyl~2-oxo-2,3-dihydroimidazol-1-yi)heptanosulphona
3, Diothyl 7-(3.4,5-txiphAnyl-2-a><o-2,3-dihydroimidazoi-1-yi)heptano
phoaphonato
4. 8-(1,9,5,~Trlphanyilmldaaol°2-yloxy)octanoia acid
5. 8-(2,3-Diph~nyimaiwlmldo)oetania aald
6. ~1-(2,3-Diphonylmalaimido)undaoanoia acid
7, ~thyi 3-(3,4,x-trlphenyl'2-oxo-2,3-dihydroimldaaol-1-yl)propionato
0, mthyi 5-(3,4,5-triphonyi-2-oxo-2,3-dlhydroimldazol-1-yi)valerate
9. ethyl 5-(1,4,9-triphanyLlmidazol-1-yloxy)Valerata
10, 2-(9-Carboxyhoptyi)-4,5-diphonyloxaaola
li. ethyl 6-(3'mothyl-9,5-Aiphanyi~2~oxo-2,3-dihydrolmidazol-1-yl)hoxanoAto
12. ethyl-A-(4,S-Biphenyl-2°oxo-2,3-dihydroimidazal-1-yi)oatanoato
13. 8-1(1,4,5-Ttiphgnyllmidaaol-2-yi-oxy)petanoio acid, ammpnlum salt
14. 1-(7-M~thoxycarbonyihop~tyi)~4,B-diphonyl-1,2,3-txiaaoio
15. 8-(1.4,5-Txiphanylimidazol-2-yloxy)-ootanbmida
16. 1-(7-Cerboxyhaptyi)-2r3r4-triph~nylimldazola
17. 6-(4,9-Diphonyllmldaaoi-2-ylthio)ootanoic aoid
18. 4-tZ-(3,4,5-Txiph~nyl-2-oMO-2,3-dihydrolmldazolyl)nonanoic acid
19. 2-(9-fiydxoxynonyl)-4,3-diphanyl-1.2.3-txiazolo
20. Diothyl 7-(1,4'S~0xiphenylimidazol-2-yloxy)haptano phoophanate
21. 1-(6-$thoxyoaxbonyih~xyl)°2,4,5-triph~nylimidazola
22: Bthy1 8--(4,5-Oiphanylimidaaol-1-yi)octanoato
23. 11-(3.4.5-Txiph~nyl-2-oxa-1,2-dihydeoimidazol-1'y1)undaadnolc acid
34. 7-(3,4,d-Txiphanyl-2-axo-1,2-dihydroimldaaol-1-yi)hoptanitYiS.a
FE~o- 1 :;_- __~~; 0.~JELr 1 -~ ~ 1 c. E~F,hIhdEF' E~ I F'.WI-i Y~1 C: H:: C E
- ~o -
25. 7-(3,4r9-Triphanyllmtdaaol-L-yloxy)heptanikxilA
Z6. i~(6°CaCboxyhs~xyl)-2,4.9~tslphenyLimidazolo
27. 2-(6-Carboxyh~ptyl)-4,5-diphonyi-1,2r3-triazoio
28. i-(8-~romoootyl)-4,8-dlphenyl-1,2,3-txlazol~
29. 1-(8-Cnrboxyockyl)-2,4,5-trlph~nylimidazole
30. RGhy1 (7-(3,A,g-tslphanyl-x-oxo-2,3-dihydroimidamol-1-yl)m~khyl
phonphonako
31. Z-(2-MAthnxyothoxy)ekhyl, 8-(4,5-dlphonyllmidaxal~L-yl)actanoata
3t. 1-(8-Cyandoatyi)-4,3-diphanyL-1,2r3-Cri.axolA
33. 1-( 7-Carboxyhoptyl )-3-( 4-mathoxyphanyl ) ~A r 5-d lphanyl lmidazolo
34. 1-(7-~Ghoxycarbonyihapkyl)-a-methyl-4r5-d(phanylimidaaolo
39. MAChyi 7-(3r4,5-kriphanyl-2-oxo-2,3-Aihydrolmida,zol-1-yi)-3-hApkynoaGo
39. 2-eonzyl-1-(7-carboxyheptyl)-4,3-dlphanyllmldazalo
37. ~thy1 0-IDh~nanthroE9,14-d)lmldazol-1-yl)oatanoato
38. 1-(7-Catboxyheptyl)~2°(4-hydroxyphony1)-4r5-diph~nyllmidazolo
39. ~khyl 7~t1.4,9-trlphonylimidnzal-2-yloxy)hAptano n~mthylphoephinakA
46. 2-(4-(3-CdriSaxypropaxy)phenyl)-4,5-diphonyilmldaaol9
4L. 1-(7-Caxbaxoyhepkyl)-6,9,-bla(2-chlorophpnyi)-2-phonyllmidazole
42. 1-(?-Casboxyh~ptyl)-3~(4-hydroxy-3.5-d11o8ophonyl)-4r5-diphonyiimidazolo
43. 1-(7-CatbdxyhoptyL)-2-phenyl-4,9-bi9(4-makhoxyphonyi)imidsaclo
44. 1-(10-CarboxyAocyl)-x,4,g-trlphonylimldazolo
45. 1-(7-Casboxyheptyl)-2-phenylimldazole
46. 1-(7-~thoxyearboriyl)-4-ph~nylimidazolA
47. 8-(3,4-Diph~nyLpyxazoi~~L-yl)oobanoic said
48. 1-(8-Carboxy~6,8-dim~thylackyl)~3r4.5-Crlph~nyi lmldaaolo
49, 1-(7-Caxboxyhopkyl)~2-aetylkhia-4.9-dlphanylimidazaie
50. 4~(9-(2,4,9~-'rrSphenyilmidazol-1-~yl)butyloxyabonxoic a=ld
CA 02061061 2002-09-11
-11-
51. 1-(Carboxyheptyl)-2-heptyl-4, 5-diphenylimidazole
52. 1-[7-(5-Tetrazolyl)heptyl]-2,4,5-triphenylimidazole
53. Sodium 7-(2,4,5-triphenylimidazole-1-yl)heptane sulphonate
54. 2-[5-(1,3-dioxalan-2-yl)pentylthio]-1-(7-ethoxycarbonylheptyl)-4,5-
diphenylimidazole
55. 7-(2,4,5-Triphenylimidazol-1-yl)heptane phosphonic acid
Methods of making the above compounds are within the skill of the art.
Methods relevant to the preparation of suitable compounds and relevant
activity data are presented in copending U.S. Patent Nos. 5,648,373 and
5,663,053.
To further demonstrate the utility of inhibiting CoA-IT, compounds 1-3
were shown to inhibit the production of PAF (assay c) and the release of
arachidonic acid (assay b) from human neutrophils. The methods of synthesis
of these compounds and their structural formulas are set forth below. These
compounds inhibited PAF production and arachidonic acid release completely
and in a concentration dependent fashion. The specificity for inhibition of
CoA-
IT activity for these compounds and not the activity of other enzymes, such as
PLA2 and PAF acetyl transferase, has been demonstrated. This data
demonstrate the inhibition of CoA-IT can and will inhibit the production of
PAF
and the release of arachidonic acid.
F E ~: - 1 ~ - ~~ 2 lJ E W ~ <E = a r E; Fn r-i r-i ~ ~: F; x F::: m Nt r~1 ~
_: f::: x ~ F . t :__,
..lz_
~xaro~pl~ 1
iath~rl 5-(3,4.5-triphenvi-2-oxo-2.~-dih dx.~.~irnidazol-1-vi hexanoat~
A mixture of 1,4,8 triphenylin~iidazole t6.24g), ethyl 6°bromohexanaat~
d13.83g), potassium earbanate d13.2~) and 2~butanone was stirred at
refiux for 6 hours. The mixture waa filtered, and tho tiltrat~ was
~vaporat~d. The residue was ehromato~raph~d on siltea gel otut~dd
with ~tt~anol-hexane to give tire title compound d8.61~) zn.p.
104-106 ° C. ,
found: C.?6:35; 'x,6.561 N,6.079~
(C2g~Ig~~2C3)
g3,equires: C,?8.63;11,6.69; N,6.16~
F' E E: - 1 ::. - ~ '~ 4~.I E _: 1 ~* : 1 ;=: F: f-"H h~a h~ I E F~ E: I F~.
n: I-i Y~ 1 C h::: I F-_" F~ . 1 i'
example 2
3odlum 7-(5.4.5-triuhenYi~a-oxow2,~°dih~ydroimidazoi-l~vi~,-
h~n~an~u~honate
ar4
a) A, mixture of 1,4,5-trlphenyllmidaxol°2-one (15-3g),
dibromaheptane (50.6g) and potassium carbonate (13.50 was heated at
reflux temperature in dry butanone (750rn1) for 30 hours. ~'h~ mixture
was cooled, filtered and the filtrate evaporated to an oil which was
ohromato~raphed era silica gel (heXane/ethyl sestets) to giue
1,~,5~~triphenyl°3-d?-bromoheptyl)imlda%ole°°2-~ne
(ll.lg,. 4696) as an
ail s
PIMIEt d(~D~.",13)1.2°l.d(l~l~,tri,5XC1~2)r3y(~I$,t;
~I°i~~r),5.7(2EI,t,
-CH~id),8.5-7.~( t5I~,1s~,3xph)pplm.
b) A solution of 1,4,5°triphenyl-8°(7-bromohEptyl)-
imidazol°~-one (3.a~D in ethanol (lOxnl) was refluxed with a solution
of
sodium sulphite (0.58g) in water 45m1) 8or 2Q hours. Mare sodium
sulphite (0.2g) was added and refluxin~ continued for a further 2~
hours. 3'he nnl~tture was evaporated to dryness, boiled in ethanol,
Illtered hot and a aparated to an oil. This was taken up in ~a small
volume of ethanol, a%aess diethyl ether added and the precipitated
solid filtered off and et~romato~raphed on silica gel (diehloro-
F E F: - 1 ' _' - ~=. _' G~.) ~ L: 1 4 : 1 c; F: i-: twl h-1 E F~: ~: T F: n:
hl 1'I n: k: I F F~ . :l c:
- ~4 .,
methanelmect~anat ~;i). 'rlm resulting oil in aneth~nnl/w~t8r 1:1 'Wad
passed down an Amberlyst i5 ion exchange rein (~1a form) and
evaporated to a solid. ~'hIs was taken up In ethanol and precipitated
with diethyl ether giving Sodium '~-(3,4,5-triphenyl-2°oxo-2,3-
dihydr~imidazol-1-yl)°he~tanesua~hanate (0.49g) as a white solid, m.p.
160°C.
Found: 0,63.47; I~,5.69; N,S.04; S,6,659~
C~gl3zgNzNaoq,S+s.59G water
~i,e~uires: 0,63,31; ~I,5.89, N,5.23; 8,6.0496
~xamule3
,P~ solution of i,4,5-tri~henylm3-(7-bromoheptyl?-imidazol-2-one
(l.Og?, p~fuced in Exarnple 1~, step (a) above, and triethyiphosphite
(1.66g) in xylene (5 ml) waa heated at reilux temperature for 40 hears.
The Solution was evaporated to an oil and ohramatographed on silica
gel (ethyl acerate/ethanol).
F E Ff - 1 :c: - W ::G~., E tf 1 ~. : 1 '=~ ~f f_, hd hd E F: F7 I F:: C: E-I
f' I C: f : I E F~ ~ d ~-,
.
-
The resulting oli was taken up in diethyl ether, filtered and
evaporated to give diethyl 7-(3,~,5-triphenyl-2-oxo-2,3-dihydro-
imldazoi-1-yi)°heptanesulphonate as a clear oil (0.8g, 7596).
Found 0,70.11; H,7.3~; N,~,9496
~32~38~2~4p
~,equires: x,70,31; H,7,19; N,5.1296
.~ssavs
(a) Asst, f~A~1T ~.etivity
~Cel.l Preparatir~n w
11937 cells were obtained from ~me~iaan Type Culturo
Collection and grown in itPMI-1640 media 4Cibco, brand Island, New
York) supplemented with 1096 fetal bovine serum (Hyalone, Logan,
13T) at 37°C, S96C02. Cells were grown without differentiation (basal
state) by any agent, such as dimethyi sulfoxide.
Mlerosorz~al ~~r~uaration
C~1~ were washed with a buffer of 250 ,mM sucrose, 10 mM
Tris, i, mM E~T,~, 1 rnM MgCl2, pH 7.4 and ruptured by N2 Gavitation
(790 psi, 10 minutes). The ruptured c~iis were centrifuged 1000 ~ g, 5
minutes. The resulting supernatant was aentri~uged at 20,000 X g, 20
minutes. Mierosomes were prepared from this supernatant bay
aentri~ugation at 9,00,000 x g, 60 minutes. The resulting pellet was
washed ono with assay buffer (150 rnM Nazi, 10 mM Na2lKPQ~, 1 mM
EG'~,A, pH 7.4), r~:centrituged and tixe pellet resuspended in assay
buffer (4-20 mg protein/a~l) and was stored a,t -80°C until assayed,
F E ~: - 1 '~ -- ~~ '~: t~J E Li 1 ~~ : 1 ~=~ L: Fn h~1 hd E F: F: I F: C: f-I
h1 C: h:: T E F~ , ~_ 4_~
..
-16-
CoA-IT aotLwitY
CoA-1't activity was measured in 1.5 ml centrifuge tubes in a
total volume of 100 ~1. Mierosomes wars diluted in assay buffer to
the desired protein concentration (6-20 ug/tube). ~'he reaction was
initiated by addition at ~ 8171-alkyl-2-lyso~sn-glyeero-3-
phosphocholine (CpC) (0.I ~Ci/tube) and 1 uM tinal cold
1-alkyl-2-lyso-GPC in assay nutter with 0.25 mg/ml catty aoid-poor
bovine serum albumin (BSA) (Calbioehem, La Jolla, CA).
(9Ft~1-alkyl-2-lysa-GPC, approximately 50 Ci/mmol, was from
MIEN-Dupont (Boston, Massachusetts) and oold 1-alkyl-2-lyso-GpC was
from 'Biomoi (Plymouth Meeting, Pennsylvania). Nlicrosomes were
pretreated with desired agents for 10 minutes before the additian of
~ 81,1-alkyl-2-lyso-GPC. The reaction was run for 10 minutes at
97~C. 'The reaction was stopped and the lipids extracted by addition
of 100 pl of chloroform:methanol (1:2, v/v) tollowed by 100 ~f3 of
ehlorpform and 100 lei of 1. M KCI. The samples were vortexed and
centrifuged at higlx spe~;d in a microfuge for 2-8 minutes. An aliquot
of the ehlorotoem-extracted materials w~re separated by TJGC in
chlorotor~n/methanol/acetic aoid/water (50:25:8:4, v/v), visualized by
radioseanning (Bioscan) and the product, I aII y Z-alkyl-2-aryl-GPC,
was scraped and quanti%ied by liquid scintillation spaetrosoopy. 'With
thls 'TLC System, the ~f values for synthetic standards of
1-alkyl°2-lyso-GPI: and k-alkyl-2°aoyl-GPC were approximately
0,25
and O.SS, respectively.
FEFt-1 W ~_~'~ 4.lELi 1=E :..r'4y k:W hlh~IE_F: E:IF:C:F-I f~YC:h::IE P. ~::1
-17-
Protein
Protein concentration were assesssad using the protein assay
yeagents horn Bio-Rad (itiehmond, ~aiiforr~ia)a
~, variety of compounds have been tested in this assay and
inhibitors o! 5-lipoxygenasse (S-1.0) and eyeloo~sygenase (CO), such as
indomethiein, naproxen, g-(~1~°~'luorophenyi)-5°(~'-
pye°ldyl)-2,8-
dihydroimidzo-~ 2, i-b a thiazole and 0-(4~-1' Iuorophenyl)-
5~(~'°pyridyl)-
2,8-dihydroimldzo-'~,1-b~thiazole-dioxide had no effect an ~o,~-IT
activity at concentrations up to 100 uM. The anti-oxidant HIT also
has na eitect at concentrations up to 100 ~rM. Compounds whiot~
complex with phospholipids and inhibit P1,A~ activity, such as
quinaerine and aristolvchic acid have no effect on GoA-IT activity at
concentrations up to 500 uM, Doxepine, a compound reported to
inhibit PAF release did not inhibit Cop-IT at concentrations up to 100
uM. Sodium diclofenac, reported to deerea5e leukotriene production
by altering arachidonic acid metabolism, had no effect on CoA-IT
activity at concentrations up to 500 ~M.
(b) Assay for Arachidonic Acid release
Preparation of hung.,an n~utroohils
Human neutrophi>s were obtained in the lahoratory using three
diiierent methods. Clne meth! used ieukophoresis packs irorn normal
humans and neutrophi>s were isolated using the histopaque-i077
technique. The blood was centrifuged at X00 ~c g for 10 minutes. The
cell pellets were resuspended in PAS composed of 13T mM NaGI, 8.8
mM Na~HPO~, 1.5 nttVl K~I~PO~, 2.~ mM 1101 (Dulbeceo~g Gibeo
Laboratories, Lotlg Island, New York) and layered over
F E F. - 1 w - ~. ~ w G~.i a r.; t ~~ : ::~ a m m r~n r-i ~ r=: F; x ~: ;w t-i
r~y r: r::; x E
I~~~.~~~~.
histopaque°1077 (Sigma, 5t. Louis, Missouri), The pellets were
eolleoted after centrifugation (300 x g for 30 minutes) and washed
once in PBS. '~ha cell pellets were ea~pase~A briefly to lionized water
to lyre any erythrocytes. The remaining cells were collected by
centrifugation, suspended in PBS, counted and identified after
Cytaspinning and staining. The final leuicaCyte prepac°ation was of
greater than 9596 purity and viability.
The second method isolated human neutrophils iroan fresh
heparinized normal blood using the Histopaque-i077 technique. The
blood was layered over Fiistopaque°10?7 (Sigma, St. Louis Missouri)
and centrifuged at X00 x g for 80 minutes. The a~ll pellets were
resuspended !n 35 rnl of ;CBS and 12 rnl of 69b Dextran, followed by
Dextran sedimentation at room temperature for 45 minutes, The
upper layer was Collected and further Centrifugated for 10 minutes at
1000 rpm. The cell pellets were exposed brief ly to deionized water to
lyre erythrocytes. The remaining calls were collected by
centrifugation, suspended fn PHS, Counted and identified after
cytaspinning and staining, The final leukocyte preparation was of
greater tkaan 9596 purity and viability.
The third method isolated human neutrophiis from freshly
drawn heparinized normal blood using the Percoll teohniqu~. The
blood was first treated witty 696 Dextran at room temperature for a i
hour sedmination. The upper layers of plasma were collected and
centrifuged at X00 x g for 10 minutes. The cell pellets were
resuspended in PerCOl1 1.070 g/ml supplemented with 5~ fetal bovine
serum and layered on discontinuous gradients (1.650, 1.085, 1.090,
.. . . . . ._ .~ '__.__4~~ ~ L~ 1 ~4 : ~ 1 F: r, t~d t~a E 'F: F: I F: r_': Ni
f~1 n: h=: 't ~ F' . ~ _
oi~_
1.095 g/ml) ioiiowed by centriiugatlon at 400 x g for 45 minutes, The
neutrophils were collected from interfaces of 1,090 and 1,.085 and the
1.095 and 1.090 Pereoll der:sities, followed by a centrfiugation at 400
~c g for 45 minutes, 'The neutrophils were suspended in PBS, counted
and identified sitar cytospinning a;nd stai~~ning, The final leuicoeyte
preparation was of greater than 9596 purity and viability,
There was no difference noted In the response di the
neutrophils nor in the etteots of test compounds in neutrophils
isolated by the three diifer~ant techniques.
~xeatm,ent of human neutr.., aahils
Neutrophils were suspended in pBS with i mM Ca2* and i.i
mM Mg2* at eaneentratlor~s of S to 20 x lAS cells per ml, Cells were
added to test tubes and treated with the desired compounds for 5 to 10
minutes, then challenged with calcium ionophere A29187, 2 ~M, or
vehicle control, PAS containing 0.25-i ang/ml 1~SA. Alter S to 20
minutes, the reactioa~ were terminated by addition ax an equal
volume of ehlorotorm:nnethanol (1:2, v/v) to the samples.
'2l~glAraehidonic said (SQ, i00 or 200 ng) was added as an internal
standard and the lipids were extracted by addition ox equal volumes of
chloroform and dLstilled water. The samples were vorteaced and
centrifuged at high speed and the chloroform ia~yer removed to a
alear~ tub~.
Assay for tree araehidonic acid
'The chloroform extract far each sample was evaporated to
dt°yness and the material resuspended in hexane, The hexane was
passed through a Silica solid phase column (500 mg), washed 2x with
F'EF7-1'~-~n:<: 4.IEr~ 1=E:.._.._ WWhlh~IEF: WxF:C:H f~ln:Er:'tE'. F'.~:4
~~~3~ '~~i~
-Za-
hexane az~d a fatty acid enriched fraction eluted with he~cane:ethy!
ether (1:1,, v/v). Solvents wer~ removed from the samples under a
stream o~f nitrogen then the samples were eanverted to
pentafluorobesa~yi esters using pentafluorobenzyl bromide and
dilsopropylethylamine in aoetronitrile. Solvents were removed and
samples w8re suspended in hexane. GC/M8i analysis was performed on
a suitable instrument, such as a Finnigan ~YAT Tack ?Oa GC/MS/1~S/DS
(Sass Jose, California) operated as a single stage quadruple system or a
Hewlett~Packard 5$9a
(c) Assa fo Product on of P atelet~ tivat n F'aetor 1PA
Preparation of human neutrons '
Blond was obtained f rom normal humans and neutrophils were
isolated as described for the araehidonie acid release assay, above,
The final leukocyte preparation was of greater than 9596 purity and
viaaiilty.
Treatment of human neutroohils
Neutrophils were suspended in FIBS at concentrations of S to 20
x ~aS sails per ml. Cells were added to test tubes and treated with
tlze.desired compounds for 5 to iU minutes, thcxi challenged with
calcium ionophore A23~19?, 2 ~M and 2a-30 uCi of (sH7aoetio acid
(NEN-lDupont, Boston, Massachusetts). ar the vehicle a8 1PE5 with
0,25-1 mg/ml of the. A~f tar 9 to 20 minutes, the reaetiorrss were
terminated by addition of an equal volume of ehioroform:methanol
(1:2, v/v) to the samples and the lipids were extraetecl by addition of
equal volumes of ehlorofarm azrd distilled water. The samples were
F a ~: - 1 ~y - ._--.: ~~ G.a E =. i ~x : ~ .__ F: ~: r~ i r..i ~ r: ~, z ~:
:_: Hi r~ i :~: r.:: z ~ ~~ . _ ._.
~21~
vortexed and centrifuged at high speed and the chlorotorxn layer
removed to a clean tubE.
Assay ior_ P.A,
The chloraiorm from each tube was evaporated to drynea and
the material suspended in a small volume of ohloroiorm or
ehloroiorm:methanol (2S°100 ul) and the total material spotted on a
Silica TLC plate. The plates were deveidped inn c:hloroinrm/methanoi/
acetic acid/water (SO:ZS:B:~;r v/v) visualfzeef by radiogcannin~
(BioSean) and the product, C 3~ 1 PAF, was scraped and quantified try
liquid scintillation spectroscopy. 'With this T1,C system, the Rf value
for a syntk~etio standard of PAF was approximately o.33.
5. Assa or se Benin ehe iaal eo ounds far ten ia1
anti-initammator~r a~e_tion
An assay method for determining the inhibitory activity of
compounds for !?AF and araohidocaie acid production is also
eneompa~sed by the invention, The method eornpris~ (1) measuring
the inhibition of the CoA independent acylation of lysophospholipids
in broken cell preparations of said compounds; (2) measuring the
inhibition of PA1F production its aetiv&;ted ingla:nmatory cells of Said
compounds; and/or (3) measuring the inhibition of araohidonie acid
reXease ire activated intlaanmatory cells of said compounds, The
activity a~d the eornpound is determined by inhibition of at least 209b
of the activities of CoA-1~, PAF or arachidomic said r~leas~, This
gay method provides a means wherein chemical compounds can be
easily screened for CoA-T"1' inhibiting activity.
FEF:- 1'c'.-W.' L.IEL~ 1 ~1. :.._ _ ~'i=,hlh~IEF: F~ I Fi W'1-I 1~l n:F:.'. I
E F'. 'i=.
~zzr
Therapeutic use
inhibition of CoA°1T and the simultaneous reduction of PAF and
eicosanoid release from inflammatory cells according to this
invention i5 0% therapeutic benefit in a broad range of diseases or
disorders. The invention is use%ul to trs:at disease states both in
humans and in other mammals.
PAF is a very potent gasteric uloerogen whose endogenous
release may underlie or contribute to certain forms of gastric
ulceration. Intravenous infusion of PAF at doses of 20°200 pmol
kga°l,~rnin<°l,a~ into rata has been reported to result in the
formation
of extensive haernorrhagie erc~ions in the gastric mueosa.
Psoriasis is an inflammatory and proli%erative dis~ase
characterized by skin lions. PAF is pro°inflammatory and has been
isolated from lesioned scale or psoriatic patients indicating PAS' has a
role is the disease of psoriasis, There is also increasing evidence for a
potential patho°physiological role for PAk' in cardiovascular disease.
In this regard, recent studies in angina patients show PAF is released
during atrial pacing. antraeoronary injection ox PAF in pigs induces a
prolonged decrease in coronary flow and, in guineas pig hearts, it
induces regional shunting and ischaemia. in addition, PAF has been
shown to initiate thrombus formation in a mesenteric artery
preparation, both when administered exogenousiy and when released
endogenously, More recently PAF has been shown to play a role in
brain ischeania induced to animal m~els of stroke.
disease states which benefit xrom the inhibition of CoA~IT
include adult respiratory distress syndrome, asthma, arthritis,
F E E. - 1 '~: - ~ '._ 4J E =: -* ~ .: -6 E: F: h-I by E F='. E: T F='. :_: f-
I Y~ I :;: h::: I E F' . ' i
-z3- ~~~ ~~~ r.
reperfusian injury, endotoxie shock, inflammatory bowel disease and
various inflammatory skin disorders. Compounds which inhibit
~oA-1T, by virtue of their ability to antagor~iz~ the actions of IaAF, as
well as CoA-TT, are of value in the treatment of any of these
conditions.
xn therapeutic use, the CoA~tT inhibitors are usually
administered in a standard pharmaceutical composition. ~oA-Ix
inhibitors and their pharmaaeuticaily acceptable salt9 which are
active when given orally, can be formulated as llquids,,for wrxample
syrups, suspensions ar emulsions, tablets, cagsuleg and Lozenges. The
choice of form for administration as well as effective dosages will
vary depending, inter ai , on the conditiar~ being treated. The choice
of mode of administration and dosage is within the skill of the art.
A liquid formulation w111 generally consist of a suspension or
solution of the inhibitor or pharmaceutically acceptable sail in a
suitable liquid carriers) for exa,mpie, ethanol, glycerine, non-aqueous
solvent, for example polyethylene glycol, oils, or water with a
suspending agent, preservative, flavoring or colouring agent.
A composition in the form of a tablet can be prepared using
any suitable pharmaceutical aarrierts) routinely used for preparing
solid formulations. Exampi~ of such carriers include magnesium
stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using
routine encapsulation procedures. For example, pellets containing
the active tng~redient can be prepared using standard carriers and then
filled iota a hard gelatin capsule; alternatively, a dispersion or
F E F: - 1 ~,~ y t 4~.~ E L: 1 4 : ~ -1. F: ~, N h-1 E F°. 7E: I F: C:
y r~1 r_: k: I E F' o ~: .~
suspension can be prepared using any suitable pharmaceutical
earrler(s), far example aqueous gums, cellulases, silicates or oils and
the dispersion or suspension then filled into a sof t gelatin capsule.
Typical parenteral eompositians consist ~f a solution nor
suspension of the inhibitor ar pharmaceuticaiiy acceptable salt in a
sterile aqueous carrier or parenterally adceptabie oil, for exampio
polyethylene glycol, polyvinyl pyrrolidone, lecithin, araehis oil or
sesame oil, Alternatively, the solution can be lyophfl)sed and then
reeor~titute with a suitable solvent dust prier to administration.
A typical suppository formulation comprises a eo~mpound of
structure El) or a pharmaoeutiealiy acceptable salt thereof which is
active when administered in this way, with a binding and/or
lubricating agent such as polymeric glyeols, gelatins or cocoa butter
or ether low melting vegetable or synthetic waxes ar fats,
Preferably the eornpositian is in unit dace form such as a tablet
4r d:ap8u18o
Each dosage unit for oral administration contains preferably
from 1 to 250 mg land for parenteral administration contains
preferably from 0.1 to 25 mg) of the ~oA-1T inhibitors or a
pharrnaeeuticaily aCeeptable salt thereof calculated as the tree bas~,
The pharmaceutically acceptable compounds will normally be
administered to a sub~eet in a daily dosage regimen. For an adult
patient tlzts may be, for example, an oral dose of l~tween 1 mg and
900 mg, preferably between 1 mg and 250 rng, or an intravenous,
subcutaneous, or intramuseular does of between 0.1 mg and 100 mg,
pret~rabiy between 0.1 mg and 25 mg, of 'the CoA°1T inhibitor or a
. ~ ~. . ~ < ~ ~ 4.~ ~ :~~ ~ ~~ : < ~ F~ H r-i ~n ~ ~: ~ t ~~ c: Hi r~ i n:
f::: i ~ r~ . w ~a
pharrna~coutically acceptable salt thereof calculated as the free base,
the compound being administered 1 to 4 times per d,~y.
'the shave description fully disatos~es nrie invention im;iuding
preferred embodiments thereof. Modil~ca~tions and improve;nents of
the emlaodim~nts specifically disclosed herein are within teh seopu of
the following eta~3ms. ~tith 5ut further elak~rar'~m, it is belie~~ed that
one skilled in the art can, using the prececf,~.~~ desc~riiation, utilize the
present invention to its fullest extent. Th~>re'ure, the Examples
herein 2~re to be construed as merely 'ttustrativ%; and not a limitation
of the scope of the present invenrl;;n in a~~~ gray. The ernbodirnents
of the invention in which an exolu~i~~~: propa:~ y or privilege is claimed
are defined as follows.
