Note: Descriptions are shown in the official language in which they were submitted.
2~~2253
124/GL49
-1- 18060Y
15
TITLE 0~ INVENTION
5-LIPOXYGENASE ACTIVATING PROTEIN
EACK~ROUND OF THE INVENTION
Several inflammatory diseases, including
asthma, arthritis and psoriasis are associated With
the production of leukotrienes (LT) by neutrophils,
mast cells and macrophages. The initial enzymatic
step in the formation of leukotrienes is the
oxidation of arachidonic acid by 5-lipoxygenase
(5-LO) to leukotriene A4. 0steosarcoma cells
transfected with 5-LO express active enzyme in broken
cell preparations, but no leukotriene metabolites are
produced by these cells when stimulated with the
calcium ionophore A23187, indicating that an
3o additional component is necessary for cellular 5-LO
2~~~~~~
124/GL49 -2- 18060IA
activity. Indole leukotriene inhibitors have been
described (EP 275,667, Gillard ~ ~.) that inhibit
the formation of cellular leukotrienes but have no
direct inhibitory effect on soluble 5-LO activity.
We have now used these potent agents to identify and
isolate a novel membrane protein of relative
molecular mass 18,000 which is necessary for cellular
leukotriene synthesis.
An lBkD protein (FLAP) has now been isolated
from mammalian (rat and human) cells which is
necessary for the cellular production of leukotrienes
from arachidonic acid.
The rat and human genes (cDNAs) encoding FLAP
have now been isolated. The rat gene (cDNA) has been
cloned and used to express FLAP in osteosarcoma cell
lines.
DETAILED DESCRIPTION OF THE INVENTION
The indole MX-886 is a known leukotriene
biosynthesis inhibitor, described in EP 275,667,
Gillard g~ ~,1~. The compound is 3-[1-(4-chloro-
benzyl)-3-~-butylthio-5-isopropylindol-2-yl]-2,2-dimet
hYlpropanoic acid. '
125I_L_669,083 is radiolabeled 3-(1-(4-
hydroxy-3-iodobenzyl)-3-(4-azidophenylsulfonyl)-5-
isopropylindol-2-yl)-2,2-dimethylpropanoic acid.
L-583,916, 2-(1-p-chlorobenzyl-5-methoxy-2-
methylindol-3-yl)propionic acid, is known (U.S.P.
3,161,654).
L-615,919, 4-chloro-3H-phenothiazin-3-one,
is described in U.S. Patent 4,677,032 (Lau ~. al..).
It is a direct inhibitor of 5-L0.
~fl~~2~3
124/GL49 -3- ~ 18060IA
FLAP can be used as an antigen to raise
antibodies (polyclonal and monoclonal) which would
inhibit FLAP-activated production of leukotrienes.
Because of their activity as leukotriene
biosynthesis inhibitors, these antibodies are useful
as anti-asthmatic, anti-allergic, and
anti-inflammatory agents and are useful in treating
allergic rhinitis and chronic bronchitis and for
amelioration of skin diseases like psoriasis and
1o atopic eczema. The antibodies are also useful to
inhibit the pathologic actions of leukotrienes on the
cardiovascular and vascular systems for example,
actions such as result in angina or endotoxin shock
and also in the treatment of inflammatory and
is allergic diseases of the eye, including allergic
conjunctivitis. They could also be used as
cytoprotective agents and for the treatment of
migraine headache.
The antibodies may also be used to treat or
2o Prevent mammalian (especially, human) disease states
such as erosive gastritis; erosive esophagitis;
inflammatory bowel disease; ethanol-induced
hemorrhagic erosions; hepatic ischemia; noxious
agent-induced damage or necrosis of hepatic,
25 Pancreatic, renal, or myocardial tissue; liver
parenchyma) damage caused by hepatoxic agents such as
CC14 and D-galactosamine; ischemic renal failure;
disease-induced hepatic damage; bile salt induced
pancreatic or gastric damage; trauma- or
3o stress-induced cell damage; and glycerol-induced
renal failure.
124/GL49 -4- 18060IA
FLAP promotes the biosynthesis of
>-lipoxygenase metabolites of arachidonic acid, such
as 5-HPETE, 5-HETE and the leukotrienes.
Leukotrienes B4, C4, D4 and E4 are known to
contribute to various disease conditions such as
asthma, psoriasis, pain, ulcers and systemic
anaphylaxis. Thus, inhibition of the synthesis of
such compounds will alleviate these and other
leukotriene-related disease states.
io Antibodies to FLAP can also be used in
~ vitro diagnostic assays to determine FLAP levels
in biological samples.
The gene (cDNA) encoding for FLAP can be
used f or large-scale production of FLAP.
The cellular target of MK-886 was identified
by its specific labelling with an 1125 radio-
labelled photoaffinity probe (125I_L-669,083) and by
its retention on agarose gels to which analogues of
MK-886 had been bound. Incubation of 1251-L_669,083
with neutrophil extracts followed by irradiation with
ultraviolet light resulted in the labelling of
several proteins; but labelling was only specifically
inhibited by MK-886 in the case of a membrane protein
of relative molecular mass (Mr) 18,000 (l8kD). When
solubilized extracts of neutrophil membranes (100,000 ..
g-centrifugation pellets) Were chromatographed over
affinity columns prepared from analogues of MK-886,
an lBkD protein found in both rat and human
neutrophils was bound and was subsequently eluted by
x-886. This protein co-migrated on SDS-polyacryl-
amide gels with the photoaffinity-labelled protein.
4
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The l8kD protein comprised a minor portion of the
10,OOOg-centrifugation pellet protein and was
essentially absent from other cellular fractions.
The photoaffinity labelling of the l8kD protein was
inhibited by MK-886 in a concentration-dependent
manner comparable to the inhibition of leukotriene
synthesis by this compound; the 50~ inhibitory
concentration (IC50) for photoaffinity labelling by
I~-886 is 80 nM, whereas it is 100 nM for inhibition
of leukotriene synthesis inhuman leukocytes at
similar protein concentrations. Indole analogues
that do not inhibit leukotriene biasyrrth'esis, such as
L-583,916, did not inhibit photoaffinity labelling of
the l8kD protein, nor did they elute this protein
from affinity columns. Several other compounds that
have inhibitory effects on leukotriene production
(such as nordihydroguaiaretic acid, eicosatetraynoic
acid, methylisobutylxanthine, and calmodulin
antagonists (WF, trifluoroperazine, and
calmidazolium)), including direct inhibitors of 5-LO
(such as L-651,392, described in Guindon, Y.
Adv. in Prostaglandin Thromboxane and Leukotriene
Res. 554-557 (Raven, N.Y., 1987)), neither eluted the
l8kD protein from the affinity gels nor_~nhibited
Photoaffinity labelling of the l8kD protein.
To purify the l8kD protein, the fraction
released from the affinity column by I~-886 was
chromatographed on a Superose-12*column followed by a
subsequent separation on two TSK-300 columns linked
in tandem. After reduction and alkylation, the
sample was~repurified a second time over the TSK
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columns, resulting in a single protein peak as shown
by silver staining after SDS-PAGE. The total amount
of protein isolated from rat peritoneal neutrophils
was N1.5 ~,g/1010 cells. N-terminal sequence
g analysis of the purified rat l8kD protein revealed
the single hydrophobic sequence MDQEAVGNWLLAIVTLIS-
WQNAFFAXKVELESKAQSG (single-letter amino-acid
code). Cyanogen bromide cleavage of the purified
l8kD protein followed by fractionation over a C4
l0 microbore column identified two additional
sequences: SLAGILNHYLIFFFGSDFENY and XLFVXQKYFVGYL-
GEXTQ, where X represents an unidentified amino
acid. Tryptic cleavage of the l8kD protein blotted
onto nitrocellulose followed by separation on a C8
15 microbore column revealed one unique sequence,
XQSTPGYIFGKXIILF. With over half of the predicted
number of residues of the l8kD protein contained
within the four essentially nonoverlapping peptides
described above, there is little sequence homology of
2o this protein to other known proteins.
In the sequences herein, the capital letters
have their conventional meaning and represent the
following amino acids:
25 A Ala Alanine
C Cys Cysteine
D Asp Aspartic acid
E Glu ~ Glutamic acid
Phe Phenylalanine
3o G G1Y Glycine
His Histidine
1.24/GL49 -7- 18060IA
I Ile Isoleucine
I~ Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
P Pro Proline
Q G1n Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
A polyclonal rabbit antibody was prepared to
the N-terminal 39 amino acids of the lBkD protein.
The peptide immunogen was prepared by fusion of the
DNA sequence coding for the peptide to the N-terminus
of Che Y protein, and its expression in and
Purification from ~. coli (Gan,,Z.R., Gene 79,
159-166 (1989)). Immunoblots with this antibody on
rat or human neutrophil 100,000 g-centrifugation
pellets or supernatants detected comparable amounts
of a single lBkD protein in the membrane fractions.
I~unoblots also demonstrated the presence of this
protein in membranes of a variety of leukocyte cell
lines which make leukotrienes, whereas it was present
in only trace or undetectable amounts in other cell
lines which lack the ability to synthesize
leukotrienes. When human leukocyte membranes
labelled with the 1251-L_669,083 photoaffinity
~~3~~j3
124/GL49 -8- 18060IA
probe were immunoprecipitated with the antipeptide
antibody, a single labelled protein of Mr 18,000 was
observed which was absent in membranes labelled in
the presence of competing MK-886. These results
confirm that the protein identified by the
photoaffinity probe is identical to that isolated by
the affinity columns, and that the N-terminal
sequence is derived from that protein.
Based on the amino acid sequence of purified
rat FLAP, cDNA clones approximately 1 kb in length
were isolated from rat RBL-1 and human HL60 cell cDNA
libraries (Fig. 1). Both the rat and human cDNA
clones encode 161 amino acid proteins which are 92~
identical and contain all of the peptide sequences
i5 derived from purified rat FLAP. Hybridization
analysis of RNA from HL60 cells with the cDNA
identified a single 1 kb species suggesting that the
isolated clones are near full length. Hydropathicity
analysis (using the methods of Hopp, T.P. g~
~-~- Natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981)
and Kyte, J. ~, ~.., J. Molec. B Col. 157, 105-132
(1982)) of the predicted rat or human FLAP amino acid
sequences demonstrated that the proteins are very
hydrophobic, consistent with their membrane
localization. Of particular interest were three
hydrophobic regions of 20-30 residues in length (Fig.
1) which are predicted by hydrophobic moment analysis
(Eisenberg, D. ~.t, ~,.. , J. Molec. Biol. 179, 125-142
(1982)) to form membrane spanning alpha helices.
Based on these data, a model for the topology of the
protein can be proposed in which the 3 transmembrane
~~~~~~3
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domains are connected by 2 hydrophilic loops, with
the N-terminus and C-terminus of the protein located
on opposite sides of the membrane. Comparison of the
sequence of FLAP with sequences of other proteins
revealed that while FLAP is not identical to any
other known protein, a general similarity with
several integral membrane proteins was observed.
This similarity appears to result from the
hydrophobic residues contained within the putative
transmembrane regions, however, rather than from any
significant primary sequence identity. No concensus
sequences for glycosylation, myristyolation, or
phosphorylation were identified in the FLAP sequence.
To determine whether FLAP is required for
5-LO function in cells, human osteosarcoma 143 cell
lines transf ected with the DNA for either FLAP, 5-L0,
or FLAP and 5-LO (5-L0/FLAP) were prepared. The
expression of the relevent proteins was determined by
immunoblots of these cell lines with antisera
directed toward FLAP or 5-L0. Parental osteosarcoma
143 cells did not contain detectable levels of either
5-L0 protein or FLAP, while the rat neutrophils
contained both proteins. The level of 5-LO protein
expressed in the transfected 5-LO or 5-LO/FLAP cells
was comparable to or slightly higher than that
observed in neutrophils. However, the amount of FLAP
expressed in the transfected FLAP or 5-LO/FLAP cells
was only 20~ of the level detected in neutrophils.
When the parental osteosarcoma 143 cells or
3o the cells expressing 5-LO or FLAP alone were treated
with the Ca2+ ionophore A23187, no arachidonic acid
metabolites were detected. In contrast, A23187
124/GL49 -10- 18060IA
treatment of the cell line expressing both 5-LO and
FLAP resulted in significant production of 5-LO
products, including LTB4 and the LTA4 hydrolysis
products 6-traps LTB4 and 6-traps-12-epi LTB4.
A23187-treated rat neutrophils produced these
products, as well as 5-HETE and the LTB4 metabolites
20-hydroxy LTB4 and 20-carboxy LT84. The 5-LO/FLAP
cell line produced only 14°l° of the neutrophil level
of LTB4 (0.15 nmol/mg protein versus 1.I nmol/mg
Protein), while it produced 41% as much of the LTA4
hydrolysis products (0.064 nmol 6-traps LTB4 and
0.059 nmol 6-traps-12-epi LTB4/mg protein versus 0.18
nmol 6-traps LTB4 and 0.12 nmol 6-traps-12-epi
LTB4/mg protein). These data are consistent with the
observation (Evans, J.A. gt~ ~., ~io~hem. Bioph3rs.
~ 840, 43-50 (1985)) that the osteosarcoma cells
contain only a fraction of the amount of LTA4
hydrolase that is present in the rat neutrophils.
1~c-886 and L-615,919 blocked LT synthesis both in the
5-LO/FLAP cell line and in neutrophils.
These experiments clearly demonstrate that
the expression of FLAP, together with 5-L0, is
essential for cellular LT synthesis and that the
activity of the expressed protein is inhibited by
~-886~ Although the mechanism by which FLAP causes
the activation of 5-LO is not fully understood, FLAP
may serve as a membrane anchor for activated 5-L0.
According to this model, a stable complex would be
required to form at the membrane between activated
5-L0, FLAP, and possibly other components of the LT
124/GL49 -11- 18060IA
synthetic pathway, such as phospholipase A2 or LTA4
hydrolase. The formation of this complex could
regulate the interaction of 5-LO with its substrate,
arachidonic acid. However, the invention is not
intended to be limited by this theory of mechanism.
The invention is further defined by
reference to the following examples, which are
intended to be illustrative and not limiting.
EXAMPLE 1
A. Affinity Column Purification
Rat peritoneal neutrophils (Ham, E.A. gt, $,1,.
Proc. Natn. Acad. Sci. USA 80, 4349-4353 (1983)) and
human peripheral neutrophils (Boyum, A. Scand. J.
Clin. Lab. Invest. (suppl. 97) 21, 77-89 (1968)) at
108 celis/mi were sonicated in 50 mM Tris-HC1 buffer,
pH 7.4, 140 mM NaCI, 2 mM EDTA, 1 mM DTT
(dithiotreitol), 10% glycerol (homogenization
buffer), together with the protease inhibitors 1 mM
phenylmethylsulphonyl fluoride, 2 mg/ml pepstatin, 2
mg/ml leupeptin, and 10 mM furyl saccharine. After
removal of 1000 g and 10,000 g-centrifugation
pellets, the 100,000 g pellet was resuspended (3
mg/ml) in the homogenization buffer.
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To purify FLAP, the microsome suspension was
solubilized on ice for 30 minutes in 50 mM Tris-HC1
buffer, pH 7.4, with 140 mM NaCl, 0.5 mM DTT, 5~
glycerol and 1% CHAPS detergent (3-[(3-cholamido-
propyl)-dimethylammonio]-1-propanesulfonate). After
centrifugation for 10 minutes at 30,000 g, the
supernatant was applied to 3 ml columns <6 X 109 cell
equivalents per column) containing the affinity gel
which was prepared by coupling MK-886 in THF
i0 (tetrahydrofuran) to pre-activated Affi-Gel 10,
capping with ethanolamine, and washing with
isopropanol. The columns were washed'ri~h N150 ml
solubilization buffer for 16 hours at 5°C and eluted
for 1 hour with four column volumes of buffer
containing 100 ~.M MK-886. To remove detergent and
MK-886, samples were vacuum dialysed overnight at 5°C
with a 10 mM Tris-HCI, pH 7.4, 0.25 mM DTT buffer
followed by SDS-PAGE (10-20% gradient gels) and
visualization by silver staining to show the presence
of FLAP.
B. Further Purification
Fluent from Step A was dried by speed-vac
;.
(combined centrifugation and lyophilizati~en under
vacuum), dissolved in 0.05 SDS-0.1 M NH4HC03, and
separated on a Superose-12 column eluted with the
same buffer at 0.25 ml/min. The fractions containing
FLAP were pooled and dried by speed-vac* and then
dissolved in 0.05% SDS-0.1 M NH4HC03. The pool was
fractionated over two TSK-3000 columns, linked in
tandem and eluted with 0.05% SDS-0.1 M NH4HC03 at
0.25 m1/min. The pooled fractions were concentrated
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124/GL49 -13- 18060IA
and dried by speed-vac. For reduction and
alkylation, the sample was suspended in 150 ~t1 of a
solution containing 3 ~tmol DTT in 67 mM Tris-HC1, pH
8.3, incubated for 6 hours at 50°C, and alkylated for
1 hour at 37°C, With l9 Nano1 iodoacetic acid brought
to pH 8.3 with 1 M Tris base. DTT was then added to
a final concentration of 1 M, and the sample
incubated overnight at 37°C, dried, and
rechromatographed on the TSK-3000 columns to yield
1o Pure FLAP as evidenced by a single band on a
silver-stained SDS gel and a single sequence on a
protein sequenator. '
EXAMPLE 2
FLAP SEnUENCING
All cells were grown in Dulbecco~s modified
minimal essential medium (GIBCO) containing 10% fetal
bovine serum. HL60 cells were differentiated by the
addition of 1% dimethylsulfoxide and 10 ~M
2o dexamethasone and incubation at 37° for 5 days
according to the method of Dixon, R.A.F.
Proc. Natn. Acad. Sci. U.S.A. 85, 416-420 (1988).
Poly(A)+ RNA was isolated from RBL-1 cells and
differentiated HL60 cells by the guanidinium
isothiocyanate-CsCI method (Chargwin, J.M. g~ ~ .
BiQchemistrv 18, 5294-5299 (1979)) followed by
oligo(dT) cellulose chromatography (Aviv, g. gt, ~,.,
Proc. ~latn. Acad. Sci. U.S.A. 69, 1408-1412 (1972)).
Double stranded cDNA was prepared from the poly(A)+
~A and ligated to lambda ZAP arms (Stratagene) as
described in Dixon gt. 8.~... (1988, ~t~pra) and Dixon,
R.A.F. g~ ~,., Nature 321, 75-79 (1986). The DNA was
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124/GL49 -14- 18060IA
packaged using Gigapack gol~ (Stratagene) and the
resulting phage screened unamplified on E.coli strain
LE392 as described in Dixon ~. ~. (1988, supra) and
Mamiatis, T. ,g~.~t ~., Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratories, N.Y.
- (1982). Pairs of complementary oligonucleotides with
5' extensions (oligonucleotides 5'-ATG GAC CAG GAG
GCT GTG GGC AAT GTG GTG CTG CTG GCC ATT GTG ACC CTG
ATC TCT GTG GTG CAG and 5'-GCC AGA CTG GGC CTT GGA
CTC CAG GGC CAC CTT GTA GGC AAA GAA GGC ATT CTG CAC
CAC AGA GAT) corresponding to a sequence capable of
encoding each of the rat FLAP derived~peptide
sequences (N-terminus =
MDQEAVGNWLLAIVTLISWQNAFFAXKVELESKAQSG;
CNBr peptide fragments = YLFVXQKYFVGYLGEXTQS and
SLAGILNHYLIFFFGSDFENY; tryptic peptide fragment =
XQSTPPGYIFGKXIILF) were synthesized using an Applied
Biosystems 380A DNA synthesizer, annealed, radio-
labeled using Klenow DNA polymearse and all four
~32P~dNTPs, and used to screen the RBL-1 cell cDNA
library as described in Dixon ~ ~. (1988) and
Maniatis ~ ~., both supra Phage which hybridized
to probes corresponding to all of the peptide
sequences were purified and the inserts rescued per
the manufacturer's (Stratagene) instructions. The
cDNA inserts were sequenced completely on both
strands as described in Dixon ~ ~. (1988, supra)
and Hattori, M. ~ ~1.., Anal. Biochem. 152, 232-238
(1983). Nucleotide sequence analysis of several of
3o the clones revealed a 161 amino acid open reading
frame following the first ATG in the sequence. All
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124/GL49 -15- 18060IA
of the peptide sequences derived from purified rat
FLAP were present in the predicted protein sequence.
The open reading frame was followed by 408 by of
untranslated sequence ending in a poly(A) tail. The
human FLAP cDNA was isolated by using the rat cDNA to
probe the dimethylsulfoxide-differentiated HL60 cell
cDNA library. Several clones with inserts of
approximately 1 kb in length were isolated which
contained an open reading frame similar to the one
found in rat FLAP cDNA. Similarity searches were
performed on the GenBank*and EMBL databases and no
sequence of primary structure homology was found.
EXAMPLE 3
EXPRESSION OF 5-LO AND FLAP IN TRANSFECTED CELL
LINES
Normal rat peritoneal neutrophils were
prepared as described by Ham, E.A. ~ ~. (1983,
)~ Osteosarcoma 143 cell lines expressing 5-LO
were isolated by the method of Rouzer, C.A. g~ ~.,
~. Biol. Chem. 263, 10135-10140 (1988). The
expression vector used contains the CMV immediate
early promoter which drives the expression of the
5-LO gene, a hygromycin resistance gene as a
selectable marker, and the EBV origin of replication
(Rouzer ~t ~., ~pra). An EcoRI fragment derived
from the RBL-1 cDNA clone containing the cDNA insert
for rat FLAP was cloned into the EcoRI site of the
vector pSVLneo (Dixon, R.A.F. g~ ~1., Nature 326,
73-79 (1987)). This vector contains an SV40 origin
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of replication and utilizes the SV40 early promoter
for expression of the FLAP gene. The vector also
contains a neomycin resistance gene as a selectable
marker. The FLAP vector was transf ected into
osteosarcoma 143 cells which were previously
transf ected with the human 5-LO vector and colonies
resistant to both hygromycin and neomycin were
isolated according to the methods of Rouzer and Dixon
~ ~7. (1987), both supra. The various cell lines
io were screened for expression of 5-LO and FLAP by
immunoblotting with antibodies directed against each
protein (Gan and Rouzer, both supra). Because the
5-LO vector is episomal in this cell line and had
previously been found to be unstable, (Rouzer,
)~ the cell line expressing both 5-LO and FLAP
was grown only in the presence of neomycin to allow
for loss of the 5-LO gene. Upon passage, a revertant
cell line was isolated which retained expression of
FLAP, but failed to express 5-L0.
EXAMPLE 4
LT SYN'r'RESTS BY CELLS EXPRE~~rNr 5-LO AND FLA_p
Confluent monolayers of osteosarcoma cells
(1x107 cells) or a suspension of rat peritoneal
neutrophils (Ix108) were incubated with 4 ml of Hanks
phosphate buff ered saline containing 15 mM HEPES
buffer and 5 ~g/ml A23187 (Ca2+ ionophore) for 10 min
at 37°C. The extracellular fluids were removed,
3o Prostaglandin PGB2 was added as an internal standard,
and the fluids were acidified with 150 ~.1 of 1 N
acetic acid. The samples were loaded on to a Sep-Pak*
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Clg column, washed with 1570 methanol and with water,
eluted with methanol and dried. The samples were
dissolved in 657. methanol/35~ 820/0.059° acetic
acid/0.5mM oxalic acid, pH 5.7, and isocratically
separated by chromatography on a Bondpak*C18 column.
The identity of each of the major ultraviolet
absorbing species, which included LTB4, was confirmed
by coelution with known standards and by spectral
analysis. The identity of LTB4 was also confirmed by
radioimmune assay.
EXAMPLE 5
SYNTHESIS OF 1251-L-669 083
The methodology herein is similar to that in
EP 275,667.
N-Trifluoroacetamido-4-aminothiophenol (I)
is reacted with 2,2-dimethyl-4-oxo-5-bromopentanoic
acid methyl ester (II) in solvents such as THF with a
base such as diisopropylcyclohexylamine to provide
2,2-dimethyl-4-oxo-5(4-N-trifluoroacetamidophenolthio)
-pentanoic acid methyl ester (III). Parahydroxybenz-
aldehyde (IV) is reacted with pivaloyl chloride and
the aldeyde is reduced with sodium borohydride to
Provide the corresponding 4-pivaloyloxybenzyl alcohol
(V) which is converted to the benzyl chloride (VI)
with triphenylphosphine and carbon tetrachloride.
Reaction of this benzyl chloride with
4-isopropylphenylhydrazine hydrochloride salt in
30_ toluene and triethylamine provides the N-benzylated
hydrazine (VII). This hydrazine (VII) is condensed
*Trad~-marks
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124/GL49 -18- 18060IA
with the previously prepared ketone (III) in acetic
acid/toluene under reflux to provide the indole
(VIII). The trifluoroacetamide group is hydrolyzed
With methanol and potassium carbonate to provide the
aminophenol (IX) which is then converted to the azide
(X) by the action of hydrochloric acid and sodium
nitrite followed by the addition of sodium azide.
Iodination of this resulting phenolic indole (X) with
sodium iodide and chloramine T in dimethylformamide
provides the corresponding iodo compound (XI).
Oxidation, and hydrolysis of the ester with sodium
hydroxide and THF/methanol/water, gives the free acid
compound, 3-(1-((4-Hydroxy-3-iodophenyl)methyl)-3-
(4-azidophenyl-sulfonyl)-5-isopropylindol-2-yl)-2,2-
dimethylprogionic acid, L-669,083 (XII).
The radiolabeled analog is prepared by
replacing Na125I for NaI.
An alternative partial synthesis is shown in
Method B, where treatment of indole VIII with 2.5
2a equivalents of m-chloroperbenzoic acid (mCPBA) in
CH2C12 at room temperature for 10 hours oxidizes the
sulfide to the sulfone (IXa), which is then treated
similarly to method A.
30
~0~~~~3
124/GL49 -19- 180607A
METHOD A
O
1. pivaloyl
chloride o ~ ( OH Ph3P
i ~~
2. NeHFl4 J( O CC14
Iy ~ V
N F3 O ' ~ C1
O
I
isopropyl- Toluene
phenylhydra-~ Et9N
zinc IjCl
(Hr
,~ ~( THF'
O!~COaMs ''~.." ~ N Fs
II
O
III -.
Cii3CO=H/t oluene
reFlux
H
N II Fa
_ Ma
2~3~~~3
124/GL49 -20- ~ 18060IA
METHOD A (cont~d)
H
w N~Fa
~/
MsOH
COMB K=COa ~ CO~t~
00 w f FD w'
VIII _IX
NeI
1. HCl 6N ' f ~ Chlorerrine T
3. T e~s J C~~ DME
60 rrln.
HD
X
/I
N_eoH( ~ N)
Tr~~ot~H.,o
~I
2 5 H°
I ~ I
~~~2~~3
124/GL49 -21- 18060IA
METHOD B
1 ~ r,cesa
~naa 2)M~oH
x,co,
axa
rya
1. HCl 6N Chlorerrine T
----.
2. NaN09 Dl~'
3. NetJ3 15 Mn.
Xe
25
a
a ~1
NeorX 1 rn
T~n~saotwx=o
124/GL49 -22- 18060IA
The sequences of rat and human FLAP are
shown With a comgarison of the nucleotide and
predicted protein sequences of the rat and human FLAP
open reading frames. Numbering begins with the first
case of the initiation codon. The termination codon
is represented by an asterisk. Nucleotide identity
is indicated with the vertical lines. Amino acid
identity is indicated with capital letters for
identical residues. The hydrophobic regions of the
FLAP amino acid sequence which are predicted to be
membrane spanning domains are indicated by~~~-~-~~ ,
20
30
