Note: Descriptions are shown in the official language in which they were submitted.
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VLA-4 Antagonists
This invention relates to organic compounds which are VLA-4 antagonists, the
preparation of such compounds and their use as pharmaceuticals.
Cell adhesion (i.e., a process by which cells associate with each other,
migrate towards
a specific target, or localize within the extracellular matrix) underlies many
biological
phenomena. Cell adhesion causes adhesion of hemoatopoietic to endothelial
cells and
the subsequent migration of those hemopoietic cells out of blood vessels and
to the site
of injury, thus playing a role in mammalian pathologies such as inflammation
and
immune reactions.
Various cell-surface macromolecules (known as cell adhesion receptors) mediate
cell-
cell and cell-matrix interactions. For example, the integrins are the key
mediators in
adhesive interactions between hematopoietic and other cells. Integrins are non-
covalent heterodimeric complexes consisting of two subunits, a and Vii.
Depending on
the type of its a and (3 subunit components, each integrin molecule is
categorized into
its own subfamily. There are at least 12 different a subunits (al-a6, a-L, a-
M, a-X,
a-IIB, a-V, and a-E) and at least 9 different (i subunits {(31-~i9).
The very late antigen-4 (VLA-4), also known as a4ji1 integrin or CD49dlCD29,
is a
leukocyte cell surface receptor that participates in a variety of cell-cell
and cell-matrix
adhesions. It is a receptor for both the cytokine-inducible endothelial cell
surface
protein, vascular cell adhesion molecule-1 (VCAM-1 ), and the extracellular
matrix
protein fibronectin (FN). Anti-VLA-4 monoclonal antibodies (mAb's) inhibit VLA-
4-
dependent adhesive interactions both in vitro and in vivo. This inhibition of
VLA-4-
dependent cell adhesion may prevent or inhibit several inflammatory and
autoimmune
pathologies.
WO 96122966 describes compounds of the formula
0 /" X
~~ ~I n
R~-Y-N-C~NH CHR4
R3
SUBSTITUTE SHEET (RULE 26)
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as useful for inhibition, prevention, and suppression of VLA-4-mediated cell
adhesion.
It has now been found that certain novel compounds have very good VLA-4
antagonistic activity and useful pharmacological properties.
Accordingly, the present invention provides in one aspect compounds of formula
I
R2
O
R1 ~Y/N\Z II ~X
I
n R
4
R
3
wherein
R~ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl,
cycloalkenyl, aryl, aryl-
substituted alkyl (aralkyl), aryl-substituted alkenyl or alkynyl, cycloalkyl-
substituted
alkyl, cycloalkenyl-substituted cycloalkyl, biaryl, alkoxy, alkenoxy,
alkynoxy, aryl-
substituted alkoxy (aralkoxy), aryl-substituted alkenoxy or alkynoxy,
alkylamino,
alkenylamino or alkynylamino, aryl-substituted alkylamino, aryl-substituted
alkenylamino or alkynylamino; aryloxy, arylamino, N-alkylureido-substituted
alkyl,
N-arylureido-substituted alkyl, alkylcarbonylamino-substituted alkyl,
aminocarbonyl-
substituted alkyl, heterocyclyl, heterocyclyl-substituted alkyl, heterocyclyl-
substituted
amino, carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl, or
heterocyclylalkyl;
RZ is (CHZ)q-V-(CH~)q.-V~-Re;
R3 is H, alkyl, alkenyl, aryl, or heteroaryl;
R4 is H, aryl, alkyl, cycioalkyl, alkenyl, cycloalkenyl, alkynyl and aryl-
substituted
alkyl, heterocyclyl, heterocyclylcarbonyl, aminocarbonyl, amido, mono- or
dialkylaminocarbonyl, mono- or diarylaminocarbonyl, alkylarylaminocarbonyl,
diarylaminocarbonyl, mono- or diacylaminocarbonyl, aromatic or aliphatic acyl,
or
alkyl optionally substituted by substituents selected from the group
consisting of
amino, halo, hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino,
alkylaryiamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy, thioalkoxy,
thioalkenoxy, thioalkynoxy, thioaryloxy, and heterocyclyl;
SUBSTITUTE SHEET (RULE 26)
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RS is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aryl-
substituted alkyl, aryl-
substituted alkenyl, or alkynyl; alkyl optionally substituted by substituents
selected
from the group consisting of amino, halo, hydroxy, mercapto, mono- or
dialkylamino,
mono- or diarylamino, alkylarylamino, mono- or diacylamino, alkoxy, alkenoxy,
aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy, and
heterocyclyl;
R6 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl-
substituted alkenyl
or alkynyl, hydroxy-substituted alkyl, alkoxy-substituted alkyl, araikoxy-
substituted
alkyl, amino-substituted alkyl, (aryl-substituted alkyloxycarbonylamino)-
substituted
alkyl, thiol-substituted alkyl, alkylsulfonyl-substituted alkyl, (hydroxy-
substituted
alkylthio)-substituted alkyl, thioalkoxy-substituted alkyl, acylamino-
substituted alkyl,
alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substituted alkyl,
morpholino-
alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl,
thiomorpholinocarbonyl-substituted alkyl, [N-(alkyl, alkenyl or alkynyl)- or
(N,N-
dialkyl, dialkenyl or dialkynyl)-amino] carbonyl-substituted alkyl, carboxyl-
substituted
alkyl, dialkylamino-substituted acylaminoalkyl; or amino acid side chains
selected
from arginine, asparagine, glutamine, S-methyl cysteine, methionine and
corresponding sulfoxide and sulfone derivatives thereof, glycine, leucine,
isoleucine,
allo-isoleucine, tert-leucine, norleucine, phenylalanine, tyrosine,
tryptophan, proline,
alanine, ornithine, histidine, glutamine, valine, threonine, serine, aspartic
acid, beta-
cyanoalanine, and allothreonine;
R~ and Re are independently H, alkyl, alkenyl, carbocyclic aryl, heteroaryl,
or alkyl,
alkenyl, carbocyclic aryl or heteroaryl substituted by 1-3 substituents
selected from the
group consisting of amino, hydroxy, mercapto, mono- or dialkylamino, mono- or
diarylamino, alkylarylamino, diarylamino, mono- or diacylamino, alkoxy,
alkenoxy,
aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy, and
heterocyclyl;
or RZ and R6 taken together with the atoms to which they are attached may form
a
heterocycle;
v is o, NH, s, so, or sot;
X is COZRS, P03H, SOZRS, S03H, OP03H, COZH, or CON(R4)z;
WisCHorN;
SUBSTITUTE SHEET (RULE 26)
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Y is CO, SO~, or PO~;
Z is (CH~)"~, CHR6, or NR,;
n and n' are independently 0-4;
m is 1-4;
p is 1-4;
q and q' are independently 1-5; and
ris0orl;
or pharmaceutically acceptable salts thereof.
Compounds of the invention, i.e. compounds of formula I and their.
pharmaceutically
acceptable salts, are VLA-4 antagonists and useful to prevent, suppress, or
inhibit cell
adhesions. Thus, they are useful in VLA-4-mediated cell adhesion disease
states,
particularly inflammation and autoimmune diseases. They are particularly
useful in
surgery-induced inflammation, especially transplant surgery. The compounds of
the
invention may be used alone or in combination with other agents active in the
prevention, suppression, or inhibition of cell adhesion.
Another embodiment of the invention is a pharmaceutical composition,
particularly a
composition for VLA-4 antagonism, comprising an effective amount of a compound
of
the invention, optionally together with a pharmaceutically acceptable carrier.
in another aspect, the present invention also provides compounds of the
invention, i.e.
compounds of formula I or pharmaceutically acceptable salts thereof, for use
as
pharmaceuticals, particularly in VLA-4 antagonism.
In a further aspect the invention provides a method of antagonizing VLA-4 in a
mammal which comprises administering to a mammal, preferably man, in need of
such
treatment an effective amount of a compound of the invention.
In a yet further aspect, the invention provides the use of a compound of the
invention
for the preparation of a medicament for the treatment of a disease mediated by
VLA-4.
Preferred compounds of the invention are those of formula Ia
SUBSTITUTE SHEET (RULE 26)
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S-
R2
I O
R1/~C/N~Z
N/
of I ~ R
R 4
3
wherein
RZ is C~_4alkyl-oxy-Cl_8alkyl;
R4 is H, alkyl, alkenyl, carbocyclic aryl or heteroaryl;
X is C02H or CO,_alkyl;
and the other symbols are as defined for formula I; or pharmaceutically
acceptable
salts thereof.
More-preferred compounds of the invention are those of formula Ia wherein R,
is aryl;
RZ is methoxy-n-propyl; R3 is H; R4 is alkenyl or aryl; X is COZH; n is 0; and
W is
CH; or pharmaceutically acceptable salts thereof.
A particular embodiment of the invention is directed to compounds of formula
Ib
R2
O
~X Ib
R~~C ~ ~~N~CH
~R
O ~ a
R3
wherein R1 is N-arylureidophenyl;
Rz is C,-C4-alkyl-oxy-CZ-C4-alkyl;
R3 is H;
R4 is H, C~-C4-alkyl, C2-C4-alkenyl or carbocyclic aryl;
nislor2;
m is 1, 2 or 3;
X is COOH or COZRS; and
RS is optionally substituted lower alkyl;
or pharmaceutically acceptable salts thereof.
SUBSTITUTE SHEET (RULE 28)
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Preferred are the compounds of formula Ib wherein
R~ is N-(optionally substituted phenyl)-ureidophenyl;
RZ is methoxypropyl;
R3 is H;
R4 is C~-C4-alkenyl or optionally substituted phenyl;
nisl;
m is 1; and
X is COON;
or pharmaceutically acceptable salts thereof.
Most-preferred compounds of the invention are those of formula Ic
/COOH
O O \C~~N
H
N ~ O ~ ~ R4 IC
H T O
Ra
wherein
R, is H, CH3, CI or NHS;
RZ 1S (CH~)3OCH3or (CHZ)4OCH3;
R4 is -(CH)=(CH)-CH3, phenyl, 4-methoxyphenyl, or 3,4-dimethoxyphenyl; and T
is
NH or CHZ; '
or a pharmaceutically acceptable salt thereof.
"Alkyl" means a straight-chain or branched-chain alkyl radical containing from
1 to
10, preferably from 1 to 6, and more preferably from 1 to 4, carbon atoms.
Examples
of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl,
tert-butyl, pentyl, iso-amyl, hexyl, and decyl.
"Alkenyl" means a straight-chain or branched-chain alkenyl radical containing
from 2
to 10, preferably from 2 to 6, and more preferably from 2 to 4, carbon atoms.
Examples of such radicals include etheryl, E- and 2-propenyl, isopropenyl, E-
and Z-
butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, and decenyl.
SUBSTITUTE SHEET (RULE 26)
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"Lower" in conjunction with the above terms means a said radical containing up
to 6
carbon atoms.
"Substituted" in conjunction with the above terms means a said radical
substituted by
e.g. amino, halo, hydroxy, mercapto, mono- or dialkylamino, mono- or di-
arylalkylamino, mono- or diarylamino, alkoxy, aryloxy, aryl, thioaryloxy,
thioalkoxy
or heterocyclyl.
"Alkynyl" means a straight-chain or branched-chain alkynyl radical containing
from 2
to 10, preferably from 2 to 6, and more preferably from 2 to 4, carbon atoms.
Examples of such radicals include ethynyl (acetylenyl), propynyl, propargyl,
butynyl,
hexynyl, and decynyl.
"Cycloalkyl" means a cyclic alkyl radical containing from 3 to 8, preferably
from 3 to
6, carbon atoms. Examples of such cycloalkyl radicals include, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cyclopropyl methyl.
"Cycloalkenyl" means a cyclic carbocycle containing from 4 to 8, preferably 5
to 6,
carbon atoms and one or more double bonds. Examples of such cycloalkenyl
radicals
include cyclopentenyl, cyclohexenyl, cyclopentadienyl, and 2-methyl-2-butenyl.
Aryl means carbocyclic or heterocyclic aryl (heteroaryl).
"Aryl" (carbocyclic aryl and heteroaryl) means a 5- or 6-membered carbocyclic
aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N,
and
S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system
containing 0-3
heteroatoms selected from O, N, and S; or a tricyclic 13- or 14-membered
aromatic or
heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and
S;
each of which rings is optionally substituted with 1-3 substituents selected
from e.g.
lower alkyl, alkenyl, alkynyl, substituted lower alkyl, substituted alkenyl,
substituted
alkynyl, =O, N02, halogen, hydroxy, alkoxy, cyano, -NR'R', acylamino, phenyl,
benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy, wherein each of
said
phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy is
optionally
SUBSTITUTE SHEET (RULE 28)
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substituted with 1-3 substituents selected from e.g. lower alkyl, alkenyl,
alkynyl,
halogen, hydroxy, alkoxy, cyano, phenyl, phenoxy, benzyl, benzyloxy, carboxy,
carboalkoxy, carboxamido, heteroaryl, heteroaryloxy, NO~, and -NR' R', wherein
R'
is H or lower alkyl. The carbocyclic aromatic ring systems comprise phenyl,
naphthyl,
indenyl, indanyl, azulenyl, fluorenyl, anthracenyl. The heterocyclic aromatic
ring
systems comprise furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl,
imidazolyl,
pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-
oxadiazolyl,
1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl,
1,3,5-triazinyl,
1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,
benzo[b]furanyl,
2,3-dihydrobenzofuranyl, benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl,
benzthiazolyl, purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl,
cinnolinyl,
phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl,
carbazolyl,
acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.
"Aryl", as it relates in particular to the grouping Rl in the above formulae,
means
carbocyclic or heterocyclic aryl, particularly phenyl optionally substituted
by one to
three substituents which are independently selected from e.g. halo, hydroxyl,
amino,
nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano,
carboxy,
carboalkoxy, Ar'-substituted alkyl, Ar'-substituted alkenyl or alkynyl, 1,2-
dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy, Ar'-
substituted
alkoxy, Ar'-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or
alkynylamino, Ar'-substituted alkylamino, Ar'-substituted alkenylamino or
alkynylamino, Ar'-substituted carbonyloxy, alkylcarbonyloxy, aliphatic or
aromatic
acyl such as alkanoyl, Ar'-substituted alkanoyl or Ar'-substituted carbonyl,
Ar'-
substituted alkylcarbonyloxy, Ar'-substituted carbonylamino, Ar'-substituted
amino,
Ar'-substituted oxy, alkylcarbonylamino, Ar'-substituted alkylcarbonylamino,
Ar'-
substituted aminocarbonylalkyl, alkoxy-carbonylamino, Ar'-substituted
alkoxycarbonyl-amino, Ar'-oxycarbonylamino, alkylsulfonylamino, mono- or bis-
(Ar'-sulfonyl) amino, Ar'-substituted alkyl-sulfonylamino,
morpholinocarbonylamino,
thiomorpholinocarbonylamino, N-alkyl guanidino, N-Ar' guanidino, N-Ar' cyano-
guanidino, N-N- (Ar'-, alkyl) guanidino, N,N- (Ar', Ar') guanidino, N,N-
dialkyi
guanidino, N,N,N-trialkyl guanidino, N-alkyl-ureido, ~ N,N-dialkyl-ureido, N-
Ar'-
ureido, N,N- (Ar',alkyl) ureido and N,N- (Ar')Z ureido; acylcarbonylamino; Ar'-
substituted aryl; aromatic acyl-substituted aromatic or aliphatic acyl; Ar'-
substituted
SUBSTITUTE SHEET (RULE 26)
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heterocyclyl;' Ar'-substituted cycloalkyl or cycloalkenyl; heterocyclylalkoxy;
N,N-
(Ar', hydroxyl) ureido; Ar'-substituted cycloalkyl and cycloalkenyl; Ar'-
substituted
biaryl; Ar'-substituted aminocarbonylamino; Ar'-mercapto-substituted alkyl;
Ar'-
amino-substituted aryl; Ar'-oxy-substituted alkyl; Ar'-substituted
aminocycloalkyl and
cycloalkenyl; aralkylaminosulfonyl; aralkoxyalkyl; N-Ar'-substituted
thioureido; N-
aralkoxyureido; N-hydroxylureido; N-alkenylureido; N,N-(alkyl,
hydroxyl)ureido;
heterocyclyl; thioaryloxy-substituted aryl; N,N-(aryl,alkyl)hydrazino; Ar'-
substituted
sulfonylheterocyclyl; aralkyl-substituted heterocyclyl; cycloaikyl and
cycloalkenyl-
substituted heterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl;
heterocyclylamino; Ar'-substituted arylaminosulfonyl; Ar'-substituted
alkenoyl;
aliphatic or aromatic acylaminocarbonyl; aliphatic or aromatic acyl-
substituted
alkenyl; Ar'-substituted aminocarbonyloxy; Ar',Ar'-disubstituted aryl;
aliphatic or
aromatic acyl-substituted acyl; benzofused-heterocyclylcarbonylamino; Ar'-
substituted
hydrazino; Ar'-substituted aminosulfonyl; Ar'-substituted alkylamino; Ar'-
substituted
heterocyclyl; Ar',Ar'-disubstituted alkanoylamino; Ar'-substituted
cycloalkanoylamino; heterocyclylalkoxy; N,N-Ar',hydroxylureido; N,N'-
Ar',hydroxylureido; heterocyclylcarbonylamino; Ar'-substituted
aminocarbonylheterocyclyl; Ar'-substituted aminocarbonyl; Ar'-substituted
carbonylamino; Ar'-substituted aminosulfonyiamino; Ar'-substituted
mercaptoalkyl;
Ar'-amino substituted biaryl; aralkylaminoalkoxy; alkyl- and aryloxy-
substituted
alkoxy; heterocyclylcarbonyl; Ar'-substituted sulfonylalkyl; Ar'-amino
carbocyclyl;
aralkylsulfonyl; aryl-substituted alkenyl; heterocyclylalkylamino;
heterocyclylalkylaminocarbonyl; Ar'-substituted sulfonylaminoalkyl; Ar'-
substituted
cycloalkyl; thioaryloxyalkyl; thioaryloxymercapto; cycloalkylcarbonylalkyl;
cycloalkyl-
substituted amino; Ar'-substituted arylamino; aryloxycarbonylaIkyl;
phosphorodiamidyl acid or ester; aryloxydimethylsiloxy; 1,3-
indandionylcarbonylalkyl; 1,3-indandionylcarbonyl; oxamidyl;
heterocyclylalkylidenyl; formamidinyl; benzalizinyl; benzalhydrazino;
arylsulfonylureido; benzilylamino; 4-(N- 2-carboxyalkyl-1-(1,3-benzodioxoi-5-
yl)-
amino-N-leucinylalkylamidylarylurea); Ar'-carbamoyloxy and alkyl- and aryloxy-
substituted ureido; wherein "Ar'" is a carbocyclic or heterocyclic aryl group
as defined
above having one to three substituents selected from the group consisting of
hydrogen,
halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl,
alkenyl,
alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy,
SUBSTITUTE SHEET (RULE 26)
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alkylamino, alkenylamino or aikynylamino, alkylcarbonyloxy, aliphatic or
aromatic
acyl, alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or
N,N-
dialkvlureido.
"Alkoxy" means an alkyl ether radical. Examples of alkyl ether radicals
include
methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and
tert-
butoxy. "Alkenoxy" means a radical of formula alkenyl-O-, provided that the
radical
is not an enol ether. Examples of alkenoxy radicals include allyloxy and E-
and Z-3-
methyl-2-propenoxy. "Alkynyloxy" means a radical of formula alkynyl-O-,
provided
that the radical is not an ynol ether. Examples of alkynoxy radicals include
propargyloxy and 2-butynyloxy. "Thioalkoxy" means a thioether radical of
formula
alkyl-S-. "Alkylamino" means a mono- or di-alkyl-substituted amino radical
(i.e., a -
radical of formula alkyl-NH- or (alkyl)2-N-). Examples of alkylamino radicals
include
methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, and N,N-
diethylamino. "Alkenylamino" means a radical of formula alkenyl-NH- or
(alkenyl)2N-, provided that the radical is not an enamine. An example of an
alkenylamino radical is the allylamino radical. "Alkynylamino" means a radical
of
formula alkynyl-NH-or (alkynyl)ZN-, provided that the radical is not an
ynamine. An
example of an alkynylamino radical is the propargyi amino radical. "Aryloxy"
means
a radical of formula aryl-O-. Examples of aryloxy radicals include phenoxy,
naphthoxy, and pyridyloxy. "Arylamino" means a radical of formula aryl-NH-.
Examples of arylamino radicals include phenylamino (anilido), naphthylamino, 2-
, 3-
or 4-pyridylamino. "Biaryl" means a radical of formula aryl-aryl-. "Thioaryl"
means
a radical of formula aryl-S-. An example of a thioaryl radical is the
thiophenyl radical.
"Aryl-fused cycloalkyl" means a cycloalkyl radical which shares two adjacent
atoms
with an aryl radical. An example of an aryl-fused cycloalkyl radical is the
benzofused
cyclobutyl radical. "Aliphatic acyl" means a radical of the formula alkyl-CO-,
alkenyl-CO-, or alkynyl-CO- derived from a carboxylic acid. Examples of
aliphatic
acyl radicals include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl,
acryloyl,
crotyl, propiolyl, and methylpropiolyl. "Aromatic acyl" means a radical of the
formula aryl-CO-. Examples of aromatic acyl radicals include benzoyl, 4-
halobenzoyl,
4-carboxybenzoyl, naphthoyl, and pyridylcarbonyl. "Morpholinocarbonyl" and
"thiomorpholinocarbonyl" mean an N-carbonylated morphoiino and an N-
carbonylated thiomorpholino radical, respectively. "Alkylcarbonylamino" means
a
SUBSTITUTE SHEET (RULE 26)
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radical of formula alkyl-CONH-. "Alkoxycarbonylamino" means a radical of
formula
alkyl-OCONH-. "Alkylsulfonylamino" means a radical of formula alkyl-SO,NH-.
"Arylsulfonylamino" means a radical of formula aryl-SO~NH-. "N-alkylurea" or
"N-
alkylureido" means a radical of formula alkyl-NH-CO-NH-. "N-arylurea" or "N-
arylureido" means a radical of formula aryl-NH-CO-NH-. "Halogen" or "halo"
means fluoro, chloro, bromo, and iodo. "Heterocycle", unless otherwise defined
herein, means a stable 3-7 membered monocyclic heterocyclic ring or an 8-11
membered bicyclic heterocyclic ring which is saturated or unsaturated, and
which may
be optionally benzofused. Each heterocycle consists of one or more carbon
atoms and
from one to four heteroatoms selected from nitrogen, oxygen, and sulfur, any
oxidized
form of nitrogen and sulfur, and the quaternized form of any basic nitrogen.
Any ring
nitrogen may be optionally substituted with a substituent R4, as defined
herein for
compounds of formula I. A heterocycle may be attached at any endocyclic carbon
or
heteroatom which results in the creation of a stable structure. Preferred
heterocycles
include 5-7 membered monocyclic heterocycles and 8-10 membered bicyclic
heterocycles. Heterocycles may be optionally oxo-substituted at 1-3 ring
positions and
may optionally be independently substituted with 1-4 aryl substituents.
Included are
heteroaryl groups as defined herein and saturated heterocycles such as
piperidine,
morpholine, pyrrolidine, thiazolidine, piperazine and the like.
It is intended that the definitions of any substituent or symbol in a
particular molecule
be independent of its definitions elsewhere in the molecule. Thus, for
example, -N(R4)z
represents -NHZ, -NHCH3, -N(CH3)Z etc.
Some of the compounds described herein contain one or more asymmetric centers
and
may thus give rise to enantiomers, diastereomers, and other stereoisomeric
forms
which may be defined in terms of absolute stereochemistry as (R) or (S), or as
(D} or
(L) for amino acids. The present invention is meant to include all such
possible
diastereomers as well as their racemic and optically pure forms. Optically
active (R)
and (S), or (D) and (L), isomers may be prepared using chiral synthons or
chiral
reagents, or resolved using conventional techniques. When the compounds
described
herein contain olefinic double bonds or other centers of geometric asymmetry,
and
unless specified otherwise, it is intended to include both E and Z geometric
isomers.
Likewise, all tautomeric forms are intended to be included.
SUBSTITUTE SHEET (RULE 26)
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In a preferred group of compounds of the invention, where W in formula I is
CH, the
stereochemistry at this carbon atom is (S), i.e. the compounds are of formula
R2
~m
Ry pY/N \2 (~
Id
n
4
R
3
where R~, R,, R3, R4, X, Y, Z, m, n and p are as defined for formula I, and
their
pharmaceutically acceptable salts.
The pharmaceutical compositions of the present invention comprise a compound
of
Formula I or a pharmaceutically acceptable salt thereof as an active
ingredient, and
may also contain a pharmaceutically acceptable carrier and, optionally; other
therapeutic ingredients. The term "pharmaceutically acceptable salts" refers
to salts
prepared from pharmaceutically acceptable non-toxic acids or bases including
organic
and inorganic acids or bases.
When a compound of the present invention is acidic, salts may be prepared from
pharmaceutically acceptable non-toxic bases. Salts derived from all stable
forms of
inorganic bases include aluminum, ammonium, calcium, copper, iron, lithium,
magnesium, manganese, potassium, sodium, zinc, etc. Particularly preferred are
the
ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from
pharmaceutically acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, substituted amines including naturally
occurring
substituted amines, cyclic amines and basic ion-exchange resins such as
arginine,
betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-
diethyi-
aminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-
ethylmorpholine, N-ethylpiperidine, giucamine, glucosamine, histidine,
isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine,
polyamine resins, procaine, purine, theobromine, triethylamine,
trimethylamine,
tripropylamine, etc.
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When a compound of the present invention is basic, salts may be prepared from
pharmaceutically acceptable non-toxic acids. Such acids include acetic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric,
gluconic,
glutamic, hydrobromic, hydrochloric, isethionic, lactic, malefic, mandelic,
methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic,
sulfuric,
tartaric, p-toluenesulfonic, etc. Particularly preferred are citric,
hydrobromic, malefic,
phosphoric, sulfuric, and tartaric acids. Base salts also include ammonium,
alkali
metal, and alkaline earth metal salts, salts with organic bases, such as
dicyclohexylamine salts, and salts with amino acids such as arginine and
lysine. Also,
basic nitrigen-containing groups can be quaternized with such agents as lower
alkyl
halides, such as methyl chloride, dialkyl sulfates, such as dimethyl,
sulfates, long chain
halides such as stearyl chlorides, and aralkyl halides, such as benzyl
chlorides.
The compounds of the invention are particularly useful in mammals as VLA-4
antagonists and as inhibitors of VLA-4 associated cell adhesion.
The ability of the compounds of formula I to inhibit VLA-4-associated cell
adhesions
makes them useful for treating, ameliorating, or preventing a variety of
inflammatory,
immune and autoimmune diseases. Preferably the diseases to be treated with the
methods of this invention are selected from respiratory disorders (such as
asthma),
arthritis, psoriasis, transplantation rejection, multiple sclerosis, type I
diabetes, and
inflammatory bowel disease, stem cell mobilization and engraphment, and sickle
cell
anemia. The compounds of formula I are also useful in transplantation surgery;
specifically, for the treatment of xenograft and allograft rejection; both
chronic and
acute.
As to the respiratory diseases, the compounds of the invention are useful as
agents for
the symptomatic or prophylactic treatment of inflammatory airways diseases.
Such
diseases include asthma of whatever type or genesis including both intrinsic
(non-
allergic) asthma and, especially, extrinsic (allergic) asthma. They are useful
for the
treatment of bronchitic asthma, exercise-induced asthma, occupational asthma,
asthma
induced following bacterial infection and other non-allergic asthmas.
Treatment of
asthma is also to be understood as embracing treatment of patients of less
than 4 or 5
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years of age exhibiting wheezing symptoms, particularly at night and diagnosed
or
diagnosable as "wheezy infants".
Prophylactic efficacy in the treatment of asthma may be manifested by reduced
frequency or reduced severity of symptomatic attack, improvement in lung
function or
improved airways hypereactivity. It may be further evidenced by reduced
requirement
for symptomatic therapy, i.e. therapy for, or intended to restrict or abort,
symptomatic
attack when it occurs, for example for anti-inflammatory therapy using a
corticosteroid.
Other inflammatory airways diseases which may be treated with compounds of the
invention include pneumoconiosis (an inflammatory, commonly occupational,
disease
of the lungs occasioned by repeated inhalation of dusts) including for example
aluminosis, asbestosis, chalicosis, siderosis, silicosis, tabacosis and
byssinosis.
Further inflammatory airways diseases which may be treated with compounds of
the
invention include adult respiratory distress syndrome (ARDS), chronic
obstructive
pulmonary disease (COPD) in the exacerbation phase thereof and exacerbation of
airways hyperactivity consequent to other drug therapy, e.g. aspirin or b-
agonist
bronchodilator therapy.
In view of their anti-inflammatory activity, particularly in relation to
inhibition of
eosinophil activation, compounds of the invention are also useful for the
treatment of
related disorders of the airways, e.g. eosinophilia, hypereosinophilia,
eosinophilic
pneumonia, parasitic infestation (including tropical eosinophilia),
bronchopulmonary
aspergillosis, polyarteritis nodosa, eosinophilic granuloma and eosinophil-
related
disorders affecting the airways caused by drug- reaction.
Compounds of the invention may also be used in the treatment of allergic
inflammatory diseases such as allergic rhinitis.
In accordance with the foregoing, the invention includes:
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(A) the use of a compound of the invention, i.e. a compound of formula I or a
pharmaceutically acceptable salt thereof, as hereinbefore described, for the
preparation
of a medicament for the treatment of inflammatory, immune or autoimmune
diseases,
particularly arthritis, transplant rejection or inflammatory airways diseases,
especially
asthma; and
(B} a method of treating an inflammatory, immune or autoimmune disease,
particularly arthritis, transplant rejection or an inflammatory airways
disease,
especially asthma, which comprises administering to a mammal, particularly a
human,
in need of such treatment a compound of the invention as hereinbefore
described.
The dosage in vitro may range between about 10-6 and 10'1° molar
concentrations,
preferably between about 10-' and 10-9 molar concentrations.
The magnitude of the prophylactic or therapeutic dose of the compounds of the
invention will vary with the nature and severity of the condition to be
treated with the
mammal involved and with the particular compound of the invention and its
route of
administration. In general, the daily dose range lies in the range of 200 to
0.001
mg/kg body weight of a mammal, preferably 50 to 0.05 mg/kg, and most
preferably
1.0 to 0.1 mg/kg, in single or divided doses. In some cases, it may be
necessary to use
doses outside these ranges. When a composition for intravenous administration
is
employed, a suitable daily dosage range is from about 50 to 0.0005 mg
(preferably 20
to 0.01 mg) compound of the invention per kg body weight. When a composition
for
oral administration is employed, a suitable daily dosage range is from about
20 to
0.001 mg (preferably 10 to 0.01 mg) compound of the invention per kg body
weight.
When a composition for ophthalmic administration is employed, a suitable daily
dosage range is from about 10-0.01% (preferably S.0-0.5% compound of the
invention, typically prepared as a 2.0-0.1 % by weight solution or suspension
of the
compound in an acceptable ophthalmic formulation.
The compounds of the invention may also be used in combination with other
pharmaceutically active ingredients. For example, a typical ocular formulation
may
comprise the compound alone or in combination with a b-adrenergic blocking
agent
such as timolol maleate or a parasympathomimetic agent such as pilocarpine.
When
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used in combination, the two active ingredients are present in approximately
equal
parts.
Any suitable route of administration may be employed for providing a mammal,
especially a human, with an effective dosage of a compound of the invention.
For
example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc.
routes may be
employed. Dosage forms include tablets, troches, dispersions, suspensions,
solutions,
capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a compound
of
Formula I, or a pharmaceutically acceptable salt thereof, as an active
ingredient, and
may also contain a pharmaceutically acceptable carrier and, optionally, other
therapeutically active ingredients. The invention includes such compositions
for use in
the treatment of an inflammatory, immune or autoimmune disease, particularly
arthritis, transplant rejection or an inflammatory airways disease, especially
asthma.
The compositions include compositions suitable for oral, rectal, topical
(including
transdermal devices, aerosols, creams, ointments, lotions, and dusting
powders),
parenteral (including subcutaneous, intramuscular, and intravenous), ocular
(ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration;
although the most suitable route in any given case will depend largely on the
nature
and severity of the condition being treated and on the nature of the active
ingredient.
They may be conveniently presented in unit dosage form and prepared by any of
the
methods well known in the art of pharmacy.
For example, in the treatment of airways diseases, compounds of the invention
may be
administered orally, for example in tablet form, or by inhalation, for example
in
aerosol or other atomisable formulations or in dry powder formulations, using
an
appropriate inhalation device such as those known in the art. For use in the
treatment
of allergic rhinitis, the compounds of the invention may also be administered
intranasally.
A compound of the invention may be combined as the active ingredient in
intimate
admixture with a pharmaceutical carrier according to conventional
pharmaceutical
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compounding techniques. The carrier may take a wide variety of forms depending
on
the nature of the preparation desired for administration, i.e., oral,
parenteral, etc. In
preparing oral dosage forms, any of the usual pharmaceutical media may be
used, such
as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring
agents, and
the like in the case of oral liquid preparations (e.g., suspensions, elixirs,
and solutions);
or carriers such as starches, sugars, microcrystalline cellulose, diluents,
granulating
agents, lubricants, binders, disintegrating agents, etc. in the case of oral
solid
preparations such as powders, capsules, and tablets. Solid oral preparations
are
preferred over liquid oral preparations. Because of their ease of
administration, tablets
and capsules are the preferred oral dosage unit form. If desired, capsules may
be coated
by standard aqueous or non-aqueous techniques.
In addition to the dosage forms described above, the compounds of the
invention may
be administered by controlled release means and devices.
Pharmaceutical compositions of the present invention suitable for oral
administration
may be prepared as discrete units such as capsules, cachets, or tablets each
containing
a predetermined amount of the active ingredient in powder or granular form ~r
as a
solution or suspension in an aqueous or nonaqueous liquid or in an oil-in-
water or
water-in-oil emulsion. Such compositions may be prepared by any of the methods
known in the art of pharmacy. In general, the compositions are prepared by
uniformly
and intimately admixing the active ingredient with liquid carriers, finely
divided solid
carriers, or both and then, if necessary, shaping the product into the desired
form. For
example, a tablet may be prepared by compression or molding, optionally with
one or
more accessory ingredients. Compressed tablets may be prepared by compressing
in a
suitable machine the active ingredient in a free-flowing form such as powder
or granule
optionally mixed with a binder, lubricant, inert diluent, or surface active or
dispersing
agent. Molded tablets may be made by molding in a suitable machine, a mixture
of
the powdered compound moistened with an inert liquid diluent. Ophthalmic
inserts
are made from compression molded films which are prepared on a Carver Press by
subjecting the powdered mixture of active ingredient and HPC to a compression
force
of 12,000 lb. (gauge) at 149°C for 1-4 min. The film is cooled under
pressure by
having cold water circulate in the platen. The inserts are then individually
cur from the
film with a rod-shaped punch. Each insert is placed in a vial, which is then
placed in a
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humidity cabinet (88% relative humidity at 30°C) for 2-4 days. After
removal from
the cabinet, the vials are capped and then autoclaved at 121°C for 0.5
hr.
The compositions containing a compound of this invention may also comprise an
additional agent selected from the group consisting of cortiocosteroids,
bronchodilators, antiasthmatics (mast cell stabilizers), anti-inflammatories,
antirheumatics, immunosuppressants, antimetabolites, immunonodulators,
antipsoriatics, and antidiabetics. Specific compounds include theophylline,
sulfasalazine and aminosalicylates (anti-inflammatories); cyclosporin, FK-506,
and
rapamycin (immunosuppressants); cyclophosphamide and methotrexate
(antimetabolites); and interferons (immunomodulators).
The invention includes a compound of the invention as hereinbefore described
in
inhalable form and an inhalable medicament comprising such a compound in
inhalable
form optionally together with a pharmaceutically acceptable carrier in
inhalable form.
The inhalable form may be, for example, an atomisable composition such as an
aerosol comprising the compound of the invention in solution or dispersion in
a
propellant or a nebulizable composition comprising a dispersion of the
compound of
the invention in an aqueous, organic or aqueouslorganic medium, or a finely
divided
particulate form comprising the compound of the invention in finely divided
form
optionally together with a pharmaceutically acceptable carrier in finely
divided form.
An aerosol composition suitable for use as the inhalable form may comprise the
compound of the invention in solution or dispersion in a propellant, which may
be
chosen from any of the propellants known in the art. Suitable such propellants
include
hydrocarbons such as n-propane, n-butane or isobutane or mixtures of two or
more
such hydrocarbons, and halogen-substituted hydrocarbons, for example fluorine-
substituted urethanes, ethanes, propanes, butanes, cyclopropanes or
cyclobutanes,
particularly 1,1,1,2-tetrafluoroethane (HFA134a) and heptafluoropropane
(HFA227),
or mixtures of two or more such halogen-substituted hydrocarbons. Where the
compound of the invention is present in dispersion in the propellant, i.e.
where it is
present in particulate form dispersed in the propellant, the aerosol
composition may
also contain a lubricant and a surfactant, which may be chosen from those
lubricants
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PCT/EP99/003$4
and surfactants known in the art. The aerosol composition may contain up to
about
5% by weight, for example 0.002 to 5%, 0.01 to 3%, 0.015 to 2%, 0.1 to 2%, 0.5
to
2% or 0.5 to 1 %, by weight of the compound of the invention, based on the
weight of
the propellant. Where present, the lubricant and surfactant may be in an
amount up
to 5% and 0.5% respectively by weight of the aerosol composition. The aerosol
composition may also contain ethanol as co-solvent in an amount up to 30% by
weight of the composition, particularly for administration from a pressurised
metered
dose inhalation device.
A finely divided particulate form, i.e. a dry powder, suitable for use as the
inhalable
form may comprise the compound of the invention in finely divided particulate
form,
optionally together with a finely divided particulate carrier, which may be
chosen from
materials known as carriers in dry powder inhalation compositions, for example
saccharides, including monosaccharides, disaccharides and polysaccharides such
as
arabinose, glucose, fructose, ribose, mannose, sucrose, lactose, maltose,
starches or
dextran. As especially preferred carrier is lactose. The dry powder may be in
capsules
of gelatin or plastic, or in blisters, for use in a dry powder inhalation
device, preferably
in dosage units of 5 p.g to 40 mg of the active ingredient. Alternatively, the
dry
powder may be contained as a reservoir in a multi-dose dry powder inhalation
device.
In the finely divided particulate form, and in the aerosol composition where
the
compound of the invention is present in particulate form, the compound of the
invention may have an average particle diameter of up to about 10 ~trn, for
example 1
to 5 ltm. The particle size of the compound of the invention, and that of a
solid carrier
where present in dry powder compositions, can be reduced to the desired level
by
conventional methods, for example by grinding in an air-jet mill, ball mill or
vibrator
mill, microprecipitation, spray-drying, lyophilisation or recrystallisation
from
supercritical media.
The inhalable medicament may be administered using an inhalation device
suitable for
the inhalable form, such devices being well known in the art. Accordingly, the
invention also provides a pharmaceutical product comprising a compound of the
invention in inhalable form as hereinbefore described in association with an
inhalation
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device. In a further aspect, the invention provides an inhalation device
containing a
compound of the invention in inhalable form as hereinbefore described.
Where the inhalable form is an aerosol composition, the inhalation device may
be an
aerosol vial provided with a valve adapted to deliver a metered dose, such as
10 to 100
~tl, e.g. 25 to 50 wl, of the composition, i.e. a device known as a metered
dose inhaler.
Suitable such aerosol vials and procedures for containing within them aerosol
compositions under pressure are well known to those skilled in the art of
inhalation
therapy. Where the inhalable form is a nebulizable aqueous, organic or
aqueous/organic dispersion, the inhalation device may be a known nebulizer,
for
example a conventional pneumatic nebulizer such as an airjet nebulizer, or an
ultrasonic nebulizer, which may contain, for example, from 1 to 50 ml,
commonly 1 to
ml, of the dispersion; or a hand-held nebulizer such as an AERx (ex Aradigm,
US)
or BINEB (Boehringer Ingelheim) nebulizer which allows much smaller nebulized
volumes, e.g. 10 to 100 pl, than conventional nebulizers. Where the inhalable
form is
the finely divided particulate form, the inhalation device may be, for
example, a dry
powder inhalation device adapted to deliver dry powder from a capsule or
blister
containing a dosage unit of the dry powder or a multidose dry powder
inhalation
device adapted to deliver, for example, 25 mg of dry powder per actuation.
Suitable
such dry powder inhalation devices are well known.
The activities and VLA-4 specificities of the compounds of this invention may
be
determined using in vitro and in vivo assays.
The cell adhesion inhibitory activity of these compounds may be measured by
determining the concentration of inhibitor required to block the binding of
VLA-4-
expressing cells to fibronectin-, CSI- or VCAM-I-coated plates. In this assay
microtiter wells are coated with either fibronectin (containing the CS-1
sequence) or
CS-1 or VCAM-I. If CS-1 is used, it must be conjugated to a carrier protein,
such as
bovine serum albumin, in order to bind to the wells. Once the wells are
coated,
varying concentrations of the test compound are then added together with
appropriately labeled, VLA-4-expressing cells. Alternatively, the test
compound may
be added first and allowed to incubate with the coated wells prior to the
addition of
the cells. The cells are allowed to incubate in the wells for at least 30
minutes.
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Following incubation, the wells are emptied and washed. Inhibition of binding
is
measured by quantitating the fluorescence or radioactivity bound to the plate
for each
of the various concentrations of test compound, as well as for controls
containing no
test compound. VLA-4-expressing cells that may be utilized in this assay
include
Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral
blood
lymophocytes (PBLs). The cells used in this assay may be fluorescently or
radioactively
labeled.
A direct binding assay may also be employed to quantitate the inhibitory
activity of
the compounds of this invention. In this assay, a VCAM-IgG fusion protein
containing the first two immunoglobin domains of VCAM (D1D2) attached above
the
hinge region of an IgG1 molecule (VCAM 2D-IgG), is conjugated to a marker
enzyme,
such as alkaline phosphatase (AP). The synthesis of this VCAM-IgG fusion is
described in PCT publication WO 90/13300. The conjugation of that fusion to a
marker enzyme is achieved by well known crosslinking methods. The VCAM-IgG
enzyme conjugate is then placed in the wells of a mufti-well filtration plate,
such as
that contained in the Millipore Multiscreen Assay System (Millipore Corp.,
Bedford,
MA). Varying concentrations of the test inhibitory compound are then added to
the
wells followed by addition of VLA-4-expressing cells. The cells, compound and
VCAM-IgG enzyme conjugate are mixed together and allowed to incubate at room
temperature. Following incubation, the wells are vacuum drained, leaving
behind the
cells and any bound VCAM. Quantitation of bound VCAM is determined by adding
an appropriate colorimetric substrate for the enzyme conjugated to VCAM-IgG
and
determining the amount of reaction cell adhesion inhibitory activity.
Compounds of the Examples have measured IC50 values for VLA-4 binding of an
order as low as 1 nanomolar.
In order to assess the VLA-4 inhibitory specificity of the compounds of this
invention,
assays for other major groups of integrins, i.e., (32 and ~i3, as well as
other ail
integrins, such as VLA-S, VLA-6 and a4(37 are performed. These assays may be
similar to the adhesion inhibition and direct binding assays described above,
substituting the appropriate integrin-expressing cell and corresponding
ligand. For
example, polymorphonuclear cells (PMNs) express ji2 integrins on their surface
and
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bind to ICAM. (33 integrins are involved in platelet aggregation and
inhibition may be
measured in a standard platelet aggregation assay. VLA-5 binds specifically to
Arg-
Gly-Asp sequences, while VLA-6 binds to laminin. Compounds of the Examples are
found to be selective for VLA-4 versus related integrins.
An in vivo assay which tests the inhibition of contact hypersensitivity in an
animal is
described in P.L: Chisholm et al., Eur. J. Immunol., vol. 23, pp. 682-688
(1993).
An assay which measures the inhibition of Ascaris antigen-induced late phase
airway
responses and airway hyperresponsiveness in asthmatic sheep is described in
W.M.
Abraham et al., J. Clin. Invest., vol. 93, pp. 776-87 (1994).
The compounds of the invention may also be tested in the following assay.
Antigen-induced pulmonary eosinophilia in the mouse
Sensitization of mice: Male B6D2F1/J mice are sensitized by i.p. injection of
0.5 mL
alum-precipitated antigen containing 8 p.g of ovalbumin (OVA) adsorbed to 2 mg
of
aluminum hydroxide gel in a saline vehicle. Five days later the mice are given
a
booster injection with OVA/alum. Control animals are sensitized with alum
only.
Ten mice are used for each group.
Challenge and drug administration: Mice are placed in a 12x14x10 inch
plexiglass
chamber and exposed to aerosolized OVA (0.5% in saline) for 1 hour at the
beginning
of the experiment (t = 0), and five hours later. Low molecular weight
antagonists are
dissolved in 2% DMSO and 150 mM TRIS, pH 8.8. A solvent control is included
for
each experiment. Drugs are administered orally 30 min prior to OVA exposure,
and 6
hour after the first OVA exposure.
BAL fluid collection and analysis: Animals are sacrificed by C02 asphyxiation
24 hour
after the first antigen challenge. The tracheas are exposed and cannulated.
The lungs
are lavaged with 0.6 mL buffer (Hanks buffered saline with 10 mM Hepes, 0.5%
BSA
and 10 U/mL heparin). The number of eosinophils in the lavage is assessed by
counting
the total number of leukocytes and the percentage of eosinophils for each
sample.
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The % inhibition is calculated by the formula:
where:
1 - (# Eos with drub in OA~oup - # Eos in no OA eouol X 100%
(#Eos in OA group - # Eos in no OA group)
Eos = average number of eosinophils, OA = challenged and no OA = unchallenged
mice.
In this assay, compounds of the Examples administered at a dosage of 30 mglkg
give
percentage inhibition of eosinophilia values up to 77%.
The compounds of the invention may be synthesized using known techniques. See,
e.g., WO 96/22966, incorporated herein by reference, which teaches the
synthesis of
analogous compounds. The invention is further defined by reference to the
following
examples, which are intended to be illustrative and not limiting. For example,
representative compounds of formula I wherein W is CH are prepared by reacting
a
compound of formula II
R~-(CHZ)P-Y-OH (II)
wherein Rl, p and Y have meaning as defined hereinabove, or a reactive
functional
derivative thereof, with a compound of the formula III
R2
O ",COON
NHS
n \N~CH ~ ill
R4
R3
wherein the carboxyl group is in protected form and wherein R,-R4, Z, n and m
have
meaning as defined hereinabove, and if desired, converting a compound so
obtained to
another compound of the invention. The condensation is carried out according
to
methodology well known in the art for amide formation, e.g. in the presence of
a
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condensing agent such as 1-[3-(dimethylamino)propylJ-3-ethylcarbodiimide
hydrochloride and a base, such as diisopropylethylamine, in an inert solvent
(such as
methylene chloride), preferably at room temperature.
The starting materials of formula II, such as optionally substituted
phenylureidophenylacetic acids, are in turn known in the art or are prepared
according
to methods known in the art, e.g. by, for example, condensing a p-
aminophenylacetic
acid ester with the appropriate aryl isocyanate to obtain the corresponding
phenylureidophenylacetic acid ester and hydrolyzing the resulting ester.
The starting materials of formula III are in turn prepared by reacting a
compound of
the formula IV
R3NH-CH-(CH2)m COOH (IV)
Ra
wherein the carboxyl group is in protected form (e.g. as an alkyl ester) and
R3, R4 and
m have meaning as defined hereinabove, with a compound of the formula V
L-Z-(CH~)"-COOH (V)
preferably as a reactive functional derivative thereof, wherein Z is (CHZ)~.
or CHR6,
and n, n' and R6 have meaning as defined hereinabove and L is a leaving group,
such
as halo or (alkyl or aryl)-sulfonyloxy, in the presence of a base, such as
triethylamine,
to obtain a compound of the formula VI
R3 Ra
L-Z-(CH2)nCON-CH-(CH2)m-COOH (VI)
wherein the carboxylic acid is in protected form (e.g. as an alkyl ester), and
L, R~, RZ
and Z have meaning as defined hereinabove, which is in turn reacted with an
amine of
the formula IX
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R2 NH2 (VII)
wherein Rz has meaning as defined hereinabove under conditions well-known in
the
art, to obtain a starting material of formula III in protected form (e.g. as
an alkyl
ester). Hydrolysis, e.g. with base, such as aqueous lithium hydroxide, gives a
starting
material of formula III.
As noted above in the cited processes, such may be carried out while, if
necessary,
temporarily protecting any interfering reactive group(s), and then liberating
the
resulting compound of the invention. In starting compounds and intermediates
which
are converted to the compounds of the invention in a manner described herein,
functional groups present, such as carboxyl, amino and hydroxy groups, are
optionally
protected by conventional protecting groups that are a common in preparative
organic
chemistry. Well-known protecting groups and their introduction are described,
for
example, in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum
Press, London, New York, T.W. Greene, "Protective Groups in Organic
Synthesis",
Wiley, New York. For example, a hydroxy group is advantageously protected in
the
form of a benzyl ether which can be cleaved by catalytic hydrogenation to
obtain a
hydroxy substituted product.
The resulting compounds of formula I wherein X is esterified carboxyl (COORS)
can
be converted to the corresponding acids e.g. by hydrolysis according to
methods well-
known in the art.
The abbreviations used in the following Examples have the indicated meaning:
conc. - concentrated
DEIA - di-isopropylethylamine
DMSO - dimethyl sulfoxide
EDAC _ 1-[3-(dimethylamino)propylJ-3-ethylcarbodiimide
hydrochloride
HOBT _ hydroxybenzotriazole
HOSu - hydroxysuccinamide
HPLC - high pressure liquid chromatography
MS - mass spectroscopy
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605
-26-
PCT/EP99/00384
NMR - nuclear magnetic resonance
OR - optical rotation
TEA - triethylamine
TLC - thin layer chromatography
TRIS - tris(hydroxymethyl)aminomethane
EXAMPLE 1
(S)-(3-[3-methoxypropyl)[[4-[(2-methylphenylaminocarbonylamino)
phenyl]acetyl]amino]acetylamino-benzenepropanoic acid
Step 1
O O
Br~Br ~Br
TEA
A
To 45 mL CH2C12 1 g (4.5 mmol) 1,1-dimethyl ethyl (3S)-3-amino-3-
phenylpropanoate is added. Then 0.720 mL (5.17 mmol) TEA is added. The mixture
is stirred 10 min., and cooled 0° C. To the mixture is added 0.450 mL
(5.17 mmol)
bromoacetyl bromide in 5 mL CH2CI2 dropwise over 15 min. The mixture is
stirred
over 3 hrs., allowing it to reach room temp. TLC, using 50% ethyl acetate/50%
hexanes, is used to monitor the reaction. The mixture is reduced to dryness
and flash
chromatographed using 30 g silica gel, Merck, grade 9385, 230-400 mesh, 60 A,
using
25% ethyl acetate / 75% hexanes, to yield 1.75 g thick yellow oil, which shows
one
spot on TLC. '~"he product is carried on to next step.
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605
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PCTIEP99/00384
Step 2
O
O
H2N~0''
.A ---"
DMF
TEA g
To 50 mL DMF is added 1.5 g A and 1.0 g (11 mmol) 3-methoxy- propylamine. At
room temp. 0.74 mL (5.3 mmol) triethylamine is added. The mixture is stirred
16 hrs.
at room. temp. TLC, using 10% CH30H/90% CH2C12, is used to monitor the
reaction. The mixture is reduced to dryness and flash chromatographed using 45
g
silica gel, starting with 2% and gradually increasing to 4% CH30H/CHZCl2, to
yield
1:6 g yellow oil, which shows one spot on TLC. The product is carried on to
the next
step.
Step 3
Q ~oH
w i N~NH
I~ ~I
DIEA p ~ ~
EDAC C
CH2C12
To SO mL CH2Cl2 is added 1.5 g B. Then 1.4 g (4.8 mmol) N-(2-methyl)-N'-(4'-
acetic
acid) diphenyl urea (only partially soluble) and 0.74 mL (5.3 mmol) DIEA is
added.
The mixture is stirred 15 min. at room temp. to give a clear yellow solution.
0.98 g
(4.8 mmol) EDAC is added and the mixture stirred 3 hrs. TLC, using 10%
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
PCT/EP99/00384
WO 99!37605 .
- Z8
CH30H/90% CHZCh, is used to monitor the reaction. The mixture is reduced to
dryness and flash chromatographed using 90 g silica gel, starting with 1 % and
increasing to 5% CH30H/CH~CIZ, to yield 1.93 g white foam.
St_, ep 4
C 20% TFA/CH2CI2
'o
I~
O
To 35 ml CHzCl2 at room temp is added 1.7 g C. Then 8 mL TFA acid is added
dropwise with 5 mL CHZC12. The mixture is stirred 2 hrs. TLC, using 10% CH30H
/
90% CHZCL~, is used to monitor the reaction. The mixture is reduced to
dryness.
Fresh CHZCIZ is added several times to remove all TFA. The product is flash
chromatographed using SO g silica gel and Z% to 5% CH30H/CH,CIZ to yield 1.5 g
of
the title compound as a white powder.
mp: 125-127°C
OR: -27.4°, DMSO (10 mg/mL)
EXAMPLE 2
(S)-[3-methoxypropyl)[[4-[(2-methylphenylaminocarbonylamino)
phenyl~acetyl~aminolacetvlamino-4-hexanoic acid
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605 .
-29-
St_ ep 1
PCT/EP99/00384
O
Br~
NH2 ~Br NH
Br
~o ~ ~o
o TEA o
CH2C12
A
Following the procedure of Example 1, step 1, but starting with 0.834 g (4.5
mmol)
1,1-dimethyl ethyl(3S)-3-amino-4-hexeneoate there is obtained 01.46 g thick
yellow
oil, which shows one spot on TLC. The product is carried on to the next step.
Sten 22
O'
H2N~0~ O
,i H N' v H
A _B
DMF ~ O O
TEA
To 10 mL DMF is added 0.31 g (1 mmol) A. Then 0.18 g (2 mmol) 3-
methoxypropylamine is added. At room temp. 0.23 mL (2 mmol) TEA is added. The
mixture is stirred 16 hrs. at room. temp. TLC, using 10% CH30H190% CHZCI2, is
used to monitor the reaction. The mixture is reduced to dryness and flash
chromatographed using 12 g silica gel, starting with 2% and gradually
increasing to
4% CH30H/CH~C12, to yield 0.1g yellow oil, which shows one spot on TLC. The
product is carried on to the next step.
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
PCT/EP99/00384
WO 99/37605
-30-
St. e-p 3
d
~ OH
_B wl~l~ O
i ~ N~NH
DIEA
DEAC C
CH2C12
To 50 mL CH~CI~ is added 1.4 g (4.4 mrriol) B. Then 1.4 g (4.8 mmol) N-(2-
methyl)-
N'-(4'-acetic acid) diphenyl uiea, only partially soluble, and 0.74 mL (5.3
mmol) DIEA
are added and the mixture stirred 15 min. to give a clear yellow solution.
0.98 g (4.8
mmol) EDAC is added and the mixture stirred 3 hrs. TLC, using 10% CH30H in
CHZCIZ, is used to monitor the reaction. The mixture is reduced to dryness,
flashed
chromatographed using 90 g silica gel, starting with 1 % and increasing to 5 %
CH30H
in CH~CI2, to yield 1.8 g white foam.
Step 4
C
20% TFA/CH2C12
~O
w ~ N~NH
~ ~o
HO
To 35 ml CH~CIZ at room temp is added 1.7 g C. Then 8 mL TFA is added dropwise
with S mL CHZC12. The mixture is stirred 2 hrs. TLC, using 10% CH3OH / 90%
CHZCL~, is used to monitor the reaction. The mixture is reduced to dryness.
Fresh
CHZCl2 is added several times to remove all TFA. The product is flash
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605
-32-
PCT/EP99100384
chromatographed using 50 g silica gel and 2% to 5% CH30H/CHZCl2 to yield 1.5 g
of
the title compound as a white powder.
mp: 88-90°C
EXAMPLE 3
(S)-(i-(3-methoxypropyl)[(4-[(2-methylphenylaminocarbonylamino)
phenyl]acetyl]amino]acetylamino-3,4-dimethoxy-benzenepropanoic acid
Steo 11
O H2S04 ~ O
O / I \ OH O / I \ Oi
w0 \ CH30H w0 \
A
To 300 mL CH30H is 30 g (144.2 mmol) 3,4-dimethoxycinnamic acid. Four drops
HZS04 is added and the mixture refluxed for 4 hrs. TLC, using 70/30, ethyl
acetate/
hexanes, is used to monitor the reaction. The mixture is reduced .to dryness
and flash
chromatographed, using 20% ethyl acetate 80% hexanes, on 350 g silica gel,
grade
60, 70-230 mesh, to yield 14.14 g A.
Step 2
I O.
A
I
THF
O.
CH3(CH2)3Li
B
To 200 mL THF is added 11.8 g (55.8 mmol) (R)-(+)-N-benzyl a-
methylbenzylamine.
The mixture is cooled to 0° C and 34.9 mL (55.8 mmol) n-buLi (1.6 M in
hexanes)
added dropwise over 30 min. The mixture is stirred for an 30 additional min.
The
reaction is cooled to -78°C. Then 6.2 g (27.9 mmol) methyl 3,4-
dimethoxycinnamate,
SUBSTITUTE SHEET (RULE 2B)
CA 02318639 2000-07-18
WO 99/37605 _
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PCT/EP99/00384
dissolved in 1S0 mL THF, is added dropwise over 1 hr. The mixture is stirred
for 30
min. at -78°C and slowly, maintaining -78°C, 2S mL saturated
NH4CI solution is
added and the mixture warmed to room temp., washed with brine, and reduced to
dryness. TLC, using SO/S0, ethyl acetate/ hexanes, is used to monitor the
reaction.
The mixture is flashed chromatographed on 180 g silica gel, Merck, grade 9385,
230-
400 mesh, 60A, to yield 10.5 g thick yellow oil.
Step, 3
H O
~O~
_g Pd(OH)2
H2N ~
O
CH30H H20
HOAc C
S.0 g (11.5 mmol) B is added to 2S0 mL CH30H, 2S mL H20, and 7.S mL HOAc. 1
g Pearlman's catalyst (Pd (OH)Z) is added. Using a ballon, the mixture is
refluxed in
an H2 atmosphere for 16 hrs. at room temp. TLC, using S% CH30H/ CH2ClZ, is
used
to monitor the reaction. The mixture is filtered through celite, washed with
CH30H,
and reduced to dryness. To the dry product is added CHZCIz and it is washed
with
brine made basic with sat'd NaHC03. The mixture is reduced to dryness and
flash
chromatographed using 1S0 g silica gel, 230-400 mesh, 1 to 4% CH30H/ CH2C1Z,
to
yield 1.54 g yellow oil.
Step 4
O
~Br
Br
C
TEA
CH2C12
I
D
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 9913?605 _
-33-
PCT/EP99/00384
To 9 mL CH~CI~ is added 0.2 g (0.8 mmol) _C and 0.13 mL (0.9 mmol) TEA. The
mixture is stirred 10 min. and the mixture cooled to 0° C. 0.08 mL (0.9
mmol)
bromoacetyl bromide in 1 mL CHZCh is added dropwise over 15 min. The mixture
is
stirred over 3 hrs. allowing the mixture to reach room temp. TLC, using 50%
ethyl
acetate/50% hexanes, is used to monitor the reaction. The mixture is reduced
to
dryness and flash chromatographed using 30 g silica gel, Merck, grade 9385,
230-400
mesh, 60 A, using 25% ethyl acetate/75% hexanes, to yield 0.237 g thick yellow
oil,
which shows one spot on TLC. The product is carried on to next step.
Step 5
~O
0
Oi Hv 'NH
'-O
D M F _-p w. I O
TEA
E
To 10 mL -DMF are added 0.36 g (1 mmol) D and 0.18 g (2 mmol) 3-
methoxypropylamine. At room temp. 0.23 mL TEA is added. The mixture is stirred
16 hrs. at room. temp. TLC, using 10% CH30H/90% CH~C12, is used to monitor the
reaction. The mixture is reduced to dryness and flash chromatographed using 12
g
silica gel, starting with 2% and gradually increasing to 4% CH30H/CH2Clz, to
yield
0.1 g yellow oil, which shows one spot on TLC. The product is carried on to
the next
step.
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605
-34-
Step 6
PCT/EP99/00384
O ~OH O
H ~
i N~NH
DIEA
0
EDAC ~' O
_F ,
CH2C12
To 5 mL _CHZCl2 is added 0.1 g (0.27 mmoi) E and 0.0853 g (0.30 mmol) N-(2-
methyl)-N'-(4'-acetic acid) diphenyl urea, which is only partially soluble.
0.056 mL
(0.34 mmol) DIEA is added and the mixture stirred 15 min. at room temp. to
give a
clear yellow solution. 0.058 g (0.30 mmol) EDAC is added and the mixture
stirred 3
hrs. TLC, using 10% CH30H/90% CH2CI2, is used to monitor the reaction. The
mixture is reduced to dryness, flash chromatographed using 90 g silica gel,
using 1%
increasing to 5 % CH30H/CHZC12, to yield 0.113 g white foam.
Ste~7
LiOH H20
_F
THF/ H20 ~O
Room temp.
1 1/2 hrs.
N~NH
Ii ~ ~I i
I
HO O
-U
To 21 mL -THF and 8 mL H20 is added 0.36 g (0.57 mmol) F. 0.36 g (0.86 mmol)
LiOH dissolved in 1 mL H20 is added dropwise over 5 min. and the mixture
stirred
for two hrs. at room temp. TLC, using 10% CH30H/ CHZCl2, is used to monitor
the
SUBSTITUTE SHEET (RULE 2B)
CA 02318639 2000-07-18
WO 99/37605
-35-
PCT/EP99/00384
reaction. The mixture is reduced to dryness and flash chromatographed on 20 g
silica
gel, using 100% CH~Ch to S% CH30H/CHZC12, to yield 0.36 g of the title
compound
as a white powder.
mp: 118-120° C
OR: _ -33.6°, DMSO (10 mg / mL)
EXAMPLE 4
(S)-[i-[3-methoxypropyl)[[4-[(2-methylphenylaminocarbonylamino)
phenyl]acetyl]amino]acetylamino--4- methoxy
benzene propanoic acid, sodium salt acid
Step 1
O H2S04 O
i ~ w OH ~ i ~ ~. O
w0 ~ CH30H w0 W
A
To 250 mL CH30H is added 50 g (280.8 mmol) 4-dimethoxycinnamic acid and 2 mL
conc. HZS04. The mixture is refluxed for 6 hrs. TLC using 70/30, ethyl
acetate/hexanes, is used to monitor the reaction. About 30 mL CH30H is
removed.
The mixture is cooled to r.t, and then crystalized, filtered, washed with H20,
and -dried
to yield 49.23 g of the desired product.
Step 2
O,
THF O.
CH3(CH2)3Li
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
PCT/EP99/00384
WO 99137605 _
-36-
To 300 mL THF is added 10.99 g (52 mmol) (R)-(+)-N-benzyl-a-
methylbenzylamine.
The mixture is cooled to 0° C. and 32.5 mL (52 mmol) n-buLi {1.6 M in
hexanes)
added dropwise over 30 min. The mixture is stirred for an 30 additional min.
The
reaction is cooled to -78° C. Then 5 g (26 mmol) methyl 4-
methoxycinnamate,
dissolved in 100 mL THF, is added dropwise over 1 hr. The mixture is stirred
for 3U
min. at -78°C and slowly, maintaining -78°C, 25 mL saturated
NH4Cl solution is
added and the mixture warmed to room temp., washed with brine, and reduced to
dryness. TLC, using 50/50, ethyl acetate/ hexanes, is used to monitor the
reaction.
The mixture is flashed chromatographed on 180 g silica gel, Merck, grade 9385,
230-
400 mesh, 60A to yield 9.738g thick pale-yellow oil (recrystalized from
EtOAdhexanes
to give white crystals).
St_ ep 3
Hi O
~O~'
Pd(OH)2
g H2N \
CH30H, H20 ~ O
HOAc C
7.74 g (19.2 mmol) B is added to 250 mL CH30H, 25 mL H20, and 7.5 mL HOAc.
1 g Pearlman's catalyst is added. Using a hydrgen ballon, the mixture is
refluxed in an
HZ atmosphere for 16 hrs. at room temp. TLC, using 5% CH30H/ CHZCIz, is used
to
monitor the reaction. The mixture is filtered through celite, washed with
CH30H, and
reduced to dryness. To the dry product added CHZC12 and it is washed with
brine
made basic with sat'd NaHC03. The mixture is reduced to dryness and flash
chromatographed using 150 g silica gel, used 230-400 mesh, using 1 to 4%
CH30H/
CHZC12, to yield 3.4 g thick pale-yellow oil (recrystalized from EtOAdhexanes
to give
white crystals).
SUBSTITUTE SHEET (RULE 28)
CA 02318639 2000-07-18
WO 99/37605
_37_
St~ ep 4
O
~Br
Br
_C
TEA
CH2C12
O
i
D
PCT/EP99/00384
To 25 mL _CH~C12. is added 0:8 g (3.82 mmol) C and 0.62 mL (4.4 mmol) TEA. The
mixture is stirred 10 min. and the mixture cooled to 0° C. 0.38 mL (4.4
mmol)
bromoacetyl bromide in 5 mL CH~C12 is added dropwise over 15 min. The mixture
is
stirred over 3 hrs. allowing the mixture to reach room temp. TLC; using SO%
ethyl
acetate/SO% hexanes, is used to monitor the reaction. The mixture is reduced
to
dryness and flash chromatographed using 30 g silica gel, Merck, grade 9385,
230-400
mesh, 60 ~, using 25% ethyl acetate/75% hexanes, to yield 0.1.3 g thick yellow
oil,
which shows one spot on TLC. The product is carried on to next step.
Step 5
H2N~4~
D
DMF
TEA
E
0
To 70 mL _DMF are added 1.3 g (3.94 mmol) D and 0.667 g (7.49 mmol) 3-
methoxypropylamine. At room temp. 0.1.05 mL (7.49 mmol) TEA is added. The
mixture is stirred 16 hrs. at room. temp. TLC, using 10% CH30H/90% CHZCl2, is
used to monitor the reaction. The mixture is reduced to dryness and flash
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
WO 99/37605
-38-
PCT/EP99/00384
chromatographed using 7~ g silica gel, starting with 2% and gradually
increasing to
4% CH30H/CH,Ch, to yield 1.29 g yellow oil which shows one spot on TLC. The
product is carried on to the next step.
St_ eg 6
E O ~OH ~O
~ O ~ N~NH
DIEA
o t ~o
EDAC
F
CH2C12
To 30 mL _CH2Cl2 are added 0.72 g {2.1 mmol) E and 0.739 g (2.6 mmol) N-{2-
methyl)-N'-(4'-acetic acid) diphenyl urea, which is only partially soluble.
0.46 mL (2.6
mmol) DIEA is added and the mixture stirred 15 min. at room temp. to give a
clear
yellow solution. 0.499 g (2.6 mmol) EDAC is added and the mixture stirred 3
hzs.
TLC, using 10% CH30H/90% CHZCIZ, is used to monitor the reaction. The mixture
is reduced to dryness, flash chromatographed using 90 g silica gel, using 1 %
increasing
to 5% CH30H / CH,Cl2, to yield 0.920 g white foam. -
Step 7
NaOH, H20
CH30H
Room temp. o
31/2 hrs. _ ~
\ ~ / Nv 'NH
/ ~ \
~O
1'O Na0
SUBSTITUTE SHEET (RULE 26)
CA 02318639 2000-07-18
PCT/EP99/00384
WO 99/37605
-39-
To 30 mL EtOH and 8 mL H20 is added 0.90 g (1.49 mmol) _F. To the mixture is
added 0.057 g (1.42 mmol) NaOH in 1 mL HBO. The mixture is stirred 3.5 hrs. at
r.t.
The mixture is filtered and dried to yield 0.720 g of the title compound as a
white
solid.
mp: 216-118°C (dec)
OR: -21.069° in DMSO (5.3 mg/mL)
EXAMPLE 5
Prepared similarly to the previous examples are the compounds of the formula
R2 COOH
I O
/ N v _N
/ O ~ H Ra
O
~N T
H
Ra
Comwound R T R R~ (
C)
(a) C~-I3 NH (CH2)40CH3 3,4-dimethoxyphenyl114-117
(dec)
CH3 ~ (CHz)44CH3 Phenyl 116-118
(dec)
(c) CH3 CHZ (CHz)30CH3 4-methoxyphenyl 127-130
(dec)
(d) H NH (CHi)30CH3 Phenyl 107-111
(dec)
(e) C1 NH (CH2)aOCH3 Phenyl 118-122
( NHz NH (CHZ)30CH3 Phenyl 105-109
f)
(dec)
SUBSTITUTE SHEET (RULE 26)
