Note: Descriptions are shown in the official language in which they were submitted.
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COMPOUNDS WHICH INHIBIT LEUKOCYTE ADHESION
MEDIATED BY VLA-4
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to compounds which inhibit leukocyte adhesion
and, in particular, leukocyte adhesion mediated by VLA-4.
References
The following publications, patents and patent applications are cited
in this application as superscript numbers:
' Hemler and Takada, European Patent Application Publication
No. 330,506, published August 30, 1989
Elices, et al., Cell, x:577-584 (1990)
Springer, Nature, x:425-434 (1990)
Osborn, Cell, X2:3-6 (1990)
Vedder, et al., Surgery, 106:509 (1989)
6 Pretolani, et al. , J. Exp. Med. , 1~( :795 ( 1994)
' Abraham, et al. , J. Clin. Invest. , 23 :776 ( 1994)
Mulligan, et al., J. Immunology, 15 :2407 (1993)
Cybulsky, et al., Science, 2:788 (1991)
'o Li, et al., Arterioscler. Thromb., x:197 (1993)
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__ 2 __
~' Sasseville, et al., Am. J. Path., 144:27 (1994)
lz yang, et al.; Proc. Nat. Acad. Science (USA),
90:10494
(1993)
Burkly, et al., Diabetes, 43:529 (1994)
'4 Baron, et al., J. Clin. Invest., 23:1700
(1994)
15 Hamann, et al., J. Immunology, 152:3238
(1994)
'6 Yednock, et al., Nature, 356:63 (1992)
" Baron, et al., J. Exp. Med., 177:57 (1993}
'g van Dinther-Janssen, et al., J. Immunology,
147:4207 (1991)
van Dinther-Janssen, et al., Annals. Rheumatic
Dis., x:672
(1993)
zo Elices, et al., J. Clin. Invest., 2:405
(1994)
z1 Postigo, et al., J. Clin. Invest., $x:1445
(1991)
zz Paul, et al., Transpl. Proceed., 2:813 (1993)
23 Okarhara, et al. , Can. Res. , 54:3233 (
1994)
za paavonen, et al. , Int. J. Can. , 5 $:298
( 1994)
zs Schadendorf, et al. , J. Path. , l 7 :429
( 1993)
zb Bao, et al., Diff., x:239 (1993)
z' Lauri, et al., British J. Cancer, 68:862
(1993)
z$ Kawaguchi, et al. , Japanese J. Cancer Res.
, 83 :1304 ( 1992)
z9 Kogan, et al., U. S. Patent No. 5,510,332,
issued April 23,
1996
3o International Patent Appl. Publication No.
WO 96/01644
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All of the above publications, patents and patent applications are
herein incorporated by reference in their entirety to the same extent as if
each individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.
State of the Art
VLA-4 (also referred to as a4~i1 integrin and CD49d/CD29), first
identified by Hemler and Takadal is a member of the ail integrin family of
cell surface receptors, each of which comprises two subunits, an a chain and
a (3 chain. VLA-4 contains an a4 chain and a (31 chain. There are at least
nine X31 integrins, all sharing the same X31 chain and each having a distinct
a
chain. These nine receptors all bind a different complement of the various
cell matrix molecules, such as fibronectin, laminin, and collagen. VLA-4,
for example, binds to fibronectin. VLA-4 also binds non-matrix molecules
that are expressed by endothelial and other cells. These non-matrix
molecules include VCAM-l, which is expressed on cytokine-activated
human umbilical vein endothelial cells in culture. Distinct epitopes of VLA-
4 are responsible for the fibronectin and VCAM-1 binding activities and
each activity has been shown to be inhibited independently.z
Intercellular adhesion mediated by VLA-4 and other cell surface
receptors is associated with a number of inflammatory responses. At the site
of an injury or other inflammatory stimulus, activated vascular endothelial
cells express molecules that are adhesive for leukocytes. The mechanics of
leukocyte adhesion to endothelial cells involves, in part, the recognition and
binding of cell surface receptors on leukocytes to the corresponding cell
surface molecules on endothelial cells. Once bound, the leukocytes migrate
across the blood vessel wall to enter the injured site and release chemical
mediators to combat infection. For reviews of adhesion receptors of the
immune system, see, for example, Springer3 and Osborn4.
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Inflammatory brain disorders, such as experimental autoimmune
encephalomyelitis (EAE), multiple sclerosis (MS) and meningitis, are
examples of central nervous system disorders in which the
endothelium/leukocyte adhesion mechanism results in destruction to
otherwise healthy brain tissue. Large numbers of leukocytes migrate across
the blood brain barrier (BBB) in subjects with these inflammatory diseases.
The leukocytes release toxic mediators that cause extensive tissue damage
resulting in impaired nerve conduction and paralysis.
In other organ systems, tissue damage also occurs via an adhesion
mechanism resulting in migration or activation of leukocytes. For example,
it has been shown that the initial insult following myocardial ischemia to
heart tissue can be further complicated by leukocyte entry to the injured
tissue causing still further insult (Vedder et a1.5). Other inflammatory
conditions mediated by an adhesion mechanism include, by way of example,
asthma~-g, Alzheimer's disease, atherosclerosis9-'°, AIDS demential',
diabetes'2-'a (including acute juvenile onset diabetes), inflammatory bowel
disease's (including ulcerative colitis and Crohn's disease), multiple
sclerosis'-", rheumatoid arthritis'$-21, tissue transplantation2z, tumor
metastasises-is, meningitis, encephalitis, stroke, and other cerebral traumas,
nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia and
acute leukocyte-mediated lung injury such as that which occurs in adult
respiratory distress syndrome.
In view of the above, assays for determining the VLA-4 level in a
biological sample containing VLA-4 would be useful, for example, to
diagnosis VLA-4 mediated conditions. Additionally, despite these advances
in the understanding of leukocyte adhesion, the art has only recently
addressed the use of inhibitors of adhesion in the treatment of inflammatory
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brain diseases and other inflammatory conditions29.so. The present invention
addresses these and other needs.
SUM1VIARY OF THE INVENTION
This invention provides compounds which bind to VLA-4. Such
compounds can be used, for example, to assay for the presence of VLA-4 in
a sample and in pharmaceutical compositions to inhibit cellular adhesion
mediated by VLA-4, for example, binding of VCAM-1 to VLA-4. The
compounds of this invention have a binding affinity to VLA-4 as expressed
by an ICso of about 15 ,uM or less (measured as described in Example A
below) which compounds are defined by formula I below:
3 5
1 O- - -
R -S N-C Q-C(H) C~OH I
O RZ Ra
wherein
RI is selected from the group consisting of alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocylic,;
R2 is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and R' and RZ together with the
nitrogen atom bound to RZ and the SOZ group can form a heterocyclic or a
substituted heterocyclic group;
R3 is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
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cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and, when RZ does not form a
heterocyclic group with R', RZ and R3 together with the nitrogen atom bound
to RZ and the carbon atom bound to R3 can form a heterocyclic group or a
substituted heterocyclic group;
R4 is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and, when R3 does not form a
heterocyclic or a substituted heterocyclic group with R2, then R3 and R4
together with the carbon atom to which they are attached can form a
cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic
group;
RS is selected from the group consisting of isopropyl, -CHZ-W and
=CH-W, where W is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted
alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxyaryl,
substituted aryloxyaryl, heteroaryl, substituted heteroaryl, heterocyclic,
substituted heterocyclic, acylamino, carboxyl, carboxylalkyl, carboxyl-
substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl,
carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-
substituted heteroaryl, carboxyheterocyclic, carboxy-substituted
heterocyclic, and hydroxyl with the proviso that when RS is =CH-W then
(H) is removed from the formula and W is not hydroxyl;
Q is selected from the group consisting of:
(i)
X
-C-N-
I_
O
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__
wherein X is selected from the group consisting of oxygen, sulfur and
NH;
(ii)
R$
-CH-N°
R~
wherein R' is selected from the group consisting of hydrogen, alkyl
and substituted alkyl;
R8 is selected from the group consisting of hydrogen, alkyl and
substituted alkyl; or R' and R8 together with the nitrogen atom bound to R'
and the carbon bound to R8 can form a heterocyclic or substituted
heterocyclic ring;
(iii)
X
-CH-C-N
R~o Rs
wherein R9 is selected from the group consisting of hydrogen, alkyl
and substituted alkyl;
Rl° is selected from the group consisting of hydrogen, alkyl and
substituted alkyl; or R9 and R'° together with the nitrogen atom bound
to R9,
the carbon atom bound to R'° and the -C(X)- group can form a
heterocyclic
or substituted heterocyclic group;
X is selected from the group consisting of oxygen, sulfur and NH;
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(iv)
R12
N
R11
-C~N/
wherein R" and R12 together with the nitrogen atom bound to R" and
the > C=N- group bound to R'2 form a heterocyclic, substituted
heterocyclic, heteroaryl, or substituted heteroaryl ring; and
(v)
X
-N-C-N
R13 R14
wherein R'3 is selected from the group consisting of hydrogen, alkyl,
and substituted alkyl;
R'4 is selected from the group consisting of hydrogen, alkyl, and
substituted alkyl;
X is selected from the group consisting of oxygen, sulfur and NH;
and pharmaceutically acceptable salts thereof.
In another embodiment, the compounds of this invention can also be
provided as prodrugs which convert (e.g., hydrolyze, metabolize, etc.) in
vivo to a compound of formula I above. In a preferred example of such an
embodiment, the carboxylic acid in the compound of formula I is modified
into a group which, in vivo, will convert to the carboxylic acid (including
salts thereof). In a particularly preferred embodiment, such prodrugs are
represented by compounds of formula IA:
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__ 9 __
3 5
1-O- - 0
R S N C-Q-C(H) C~ 6 IA
R
O Rz Ra
wherein
R' is selected from the group consisting of alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocylic,;
RZ is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and Rl and RZ together with the
nitrogen atom bound to RZ and the SOZ group can form a heterocyclic or a
substituted heterocyclic group;
R3 is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and, when RZ does not form a
heterocyclic group with Rl, RZ and R3 together with the nitrogen atom bound
to RZ and the carbon atom bound to R3 can form a heterocyclic group or a
substituted heterocyclic group;
R4 is selected from the group consisting of hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, and, when R3 does not form a
heterocyclic or a substituted heterocyclic group with RZ, then R3 and R4
together with the carbon atom to which they are attached can form a
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cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic
group;
RS is selected from the group consisting of isopropyl, -CHZ-W and
=CH-W, where W is selected from the group consisting of hydrogen, alkyl,
S substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted
alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxyaryl,
substituted aryloxyaryl, heteroaryl, substituted heteroaryl, heterocyclic,
substituted heterocyclic, acylamino, carboxyl, carboxylalkyl, carboxyl-
substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl,
carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-
substituted heteroaryl, carboxyheterocyclic, carboxy-substituted
heterocyclic, and hydroxyl with the proviso that when RS is =CH-W then
(H) is removed from the formula and W is not hydroxyl;
R6 is selected from the group consisting of amino, alkoxy, substituted
alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy, substituted aryloxy,
heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted
heterocyclyloxy, -NHOY where Y is hydrogen, alkyl, substituted alkyl,
aryl, or substituted aryl, and -NH(CHZ)pCOOY' where Y' is hydrogen,
alkyl, substituted alkyl, aryl, or substituted aryl, and p is an integer of
from
1 to 8;
Q is selected from the group consisting of:
(i)
X
-C-N-
I_
O
wherein X is selected from the group consisting of oxygen, sulfur and
NH;
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(ii)
R$
-CH-N-
R~
wherein R' is selected from the group consisting of hydrogen, alkyl
and substituted alkyl;
R$ is selected from the group consisting of hydrogen, alkyl and
substituted alkyl; or R' and Rg together with the nitrogen atom bound to R'
and the carbon bound to R8 can form a heterocyclic or substituted
heterocyclic ring;
(iii)
-X
-CH-C-N
R~o Rs
wherein R9 is selected from the group consisting of hydrogen, alkyl
and substituted alkyl;
R'° is selected from the group consisting of hydrogen, alkyl and
substituted alkyl; or R9 and R'° together with the nitrogen atom bound
to R9,
the carbon atom bound to RI° and the -C(X)- group can form a
heterocyclic
or substituted heterocyclic group;
X is selected from the group consisting of oxygen, sulfur and NH;
and
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(iv)
R12
N
I I R11
-C~N/
wherein R" and R12 together with the nitrogen atom bound to R" and
the > C=N- group bound to R'2 form a heterocyclic, substituted
heterocyclic, heteroaryl, or substituted heteroaryl ring; and
(v)
X
-N-C-N
R13 R14
wherein R'3 is selected from the group consisting of hydrogen, alkyl,
and substituted alkyl;
R'4 is selected from the group consisting of hydrogen, alkyl, and
substituted alkyl;
X is selected from the group consisting of oxygen, sulfur and NH;
and pharmaceutically acceptable salts thereof.
Preferably, in the compounds of formula I and IA above, R' is
selected from the group consisting of alkyl, substituted alkyl, aryl,
substituted aryl, heterocyclic, substituted heterocylic, heteroaryl and
substituted heteroaryl. Even more preferably R' is selected from the group
consisting of methyl, isopropyl, n-butyl, benzyl, phenethyl, phenyl, 4-
methylphenyl, 4-t-butylphenyl, 2,4,6-trimethylphenyl, 2-fluorophenyl, 3-
fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 3,5-
difluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-
dichlorophenyl, 3,5-dichlorophenyl, 3-chloro-4-fluorophenyl, 4-
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bromophenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,4-
dimethoxyphenyl, 4-t-butoxyphenyl, 4-(3'-dimethylamino-n-propoxy)-
phenyl, 2-carboxyphenyl, 2-(methoxycarbonyl)phenyl, 4-(HZNC(O)-)phenylr
4-(HZNC(S)-)phenyl, 4-cyanophenyl, 4-trifluoromethylphenyl, 4-
trifluoromethoxyphenyl, 3,5-di-(trifluoromethyl)phenyl, 4-nitrophenyl, 4-
aminophenyl, 4-(CH3C(O)NH-)phenyl, 4-(PhNHC(O)NH-)phenyl, 4-
amidinophenyl, 4-methylamidinophenyl, 4-(CH3SC(=NH)-)phenyl, 4-
chloro-3-(HZNS(O)2-)phenyl, 1-naphthyl, 2-naphthyl, pyridin-2-yl, pyridin-3-
yl, pyrimidin-2-yl, quinolin-8-yl, 2-(trifluoroacetyl)-1,2,3,4-
tetrahydroisoquinolin-7-yl, morpholin-4.-yl, 2-thienyl, S-chloro-2-thienyl,
2,5-dichloro-4-thienyl, 1-N-methylimidazol-4-yl, 1-N-methylpyrazol-3-yl, 1-
N-methylpyrazol-4-yl, 1-N-butylpyrazol-4-yl, 1-N-methyl-3-methyl-5-
chloropyrazol-4-yl, 1-N-methyl-5-methyl-3-chloropyrazol-4-yl, 2-thiazolyl
and 5-methyl-1,3,4-thiadiazol-2-yl.
Preferably, in the compounds of formula I and IA above, RZ is
hydrogen, methyl, phenyl, benzyl, -(CHZ)z-2-thienyl, and -(CHZ)2-~.
In one embodiment, R' and RZ together with the nitrogen atom bound
to RZ and the SOZ group bound to R' are joined to form a heterocyclic group
or substituted heterocyclic group. Preferred heterocyclic and substituted
heterocyclic groups include those having from 5 to 7 ring atoms having 2 to
3 heteroatoms in the ring selected from the group consisting of nitrogen,
oxygen and sulfur which ring is optionally fused to another ring such as a
phenyl or cyclohexyl ring to provide for a fused ring heterocycle of from 10
to 14 ring atoms having 2 to 4 heteroatoms in the ring selected from the
group consisting of nitrogen, oxygen and sulfur. Specifically preferred
R'/Rz joined groups include, by way of example, benzisothiazolonyl
(saccharin-2-yl), N-2,10-camphorsultamyl and 1,1-dioxo-2,3-dihydro-3,3-
dimethyl-1,2-benzisothiazol-2-yl.
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In one preferred embodiment, RZ and R3 together with the nitrogen
atom bound to RZ substituent and the carbon bound to the R3 substituent
form a heterocyclic group or a substituted heterocyclic group of 4 to 6 ring
atoms having 1 to 2 heteroatoms in the ring selected from the group
consisting of nitrogen, oxygen and sulfur which ring is optionally substituted
with 1 to 2 substituents selected from the group consisting of fluoro, methyl,
hydroxy, oxo (=O), amino, phenyl, thiophenyl, thiobenzyl, (thiomorpholin-
4-yl)C(O)O- , CH3S(O)2- and CH3S(O)20-, or can be fused to another ring
such as a phenyl or cycloalkyl ring to provide for a fused ring heterocycle of
from 10 to 14 ring atoms having 1 to 2 heteroatoms in the ring selected from
the group consisting of nitrogen, oxygen and sulfur. Such heterocyclic rings
include azetidinyl (e.g., L-azetidinyl), thiazolidinyl (e.g., L-
thiazolidinyl),
piperidinyl (e.g., L-piperidinyl), piperazinyl (e.g., L-piperazinyl),
dihydroindolyl (e.g., L-2,3-dihydroindol-2-yl), tetrahydroquinolinyl (e.g.,
L-1,2,3,4-tetrahydroquinolin-2-yl), thiomorpholinyl (e.g., L-thiomorpholin-
3-yl), pyrrolidinyl (e.g., L-pyrrolidinyl), substituted pyrrolidinyl such as
4-hydroxypyrrolidinyl (e.g., 4-a-(or ~i-)hydroxy-L-pyrrolidinyl), 4-
oxopyrrolidinyl (e.g., 4-oxo-L-pyrolidinyl), 4-fluoropyrrolidinyl (e.g., 4-a-
(or ~i-)fluoro-L-pyrrolidinyl), 4,4-difluoropyrrolidinyl (e.g., 4,4-difluoro-L-
pyrrolidinyl), 4-(thiomorpholin-4-y1C(O)O-)pyrrolidinyl (e.g., 4-a-(or ~i-)-
(thiomorpholin-4-y1C(O)O-)-L-pyrrolidinyl, 4-(CH3S(O)20-)pyrrolidinyl
(e.g., 4-a-(or ~i-)(CH3S(O)ZO-)-L-pyrrolidinyl, 3-phenylpyrrolidinyl (e.g., 3-
a-(or (3-)phenyl-L-pyrrolidinyl), 3-thiophenylpyrrolidinyl (e.g., 3-a-(or ~3-)-
thiophenyl-L-pyrrolidinyl), 4-aminopyrrolidinyl (e.g., 4-a-(or (3-)amino-L-
pyrrolidinyl), 3-methoxypyrrolidinyl (e.g., 3-a-(or (3-)methoxy-L-
pyrrolidinyl), 4,4-dimethylpyrrolidinyl, substituted piperazinyl such as 4-N-
Cbz-piperazinyl and 4-(CH3S(O)2-)piperazinyl, substituted thiazolidinyl such
as 5,5-dimethylthiazolindin-4-yl, 1,1-dioxo-thiazolidinyl (e.g., L-1,1-dioxo-
thiazolidin-2-yl), substituted 1,1-dioxo-thiazolidinyl such as L-1,1-dioxo-5,5-
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dimethylthiazolidin-2-yl, l,l-dioxothiomorpholinyl (e.g., L-1,1-dioxo-
thiomorpholin-3-yl) and the like.
Preferably, in the compounds of formula I and IA above, R3 includes
all of the isomers arising by substitution with hydrogen, methyl, phenyl,
benzyl, diphenylmethyl, -CHZCHZ-COOH, -CHZ-COOH, 2-amidoethyl, iso-
butyl, t-butyl, -CHzO-benzyl and hydroxymethyl. Additionally, in another
preferred embodiment, R3 and RZ together with the nitrogen atom bound to
RZ can form a heterocyclic group or substituted heterocyclic group.
Preferably, in the compounds of formula I and IA above, R4 is
selected from the group consisting of hydrogen, methyl, ethyl, phenyl and
where R3 and R4 are joined together with the carbon atom to which they are
attached to form a cycloalkyl group of from 3 to 6 carbon atoms or a
heterocyclic group of from 3 to 8 ring atoms. Preferred cycloalkyl alkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Q is preferably -C(O)N(O)-, -CHzNH-, -CH(OH)C(O)NH-,
-NHC(O)NH-, or tetrazol-1,5-diyl.
RS is preferably selected from all possible isomers arising by
substitution with the following groups:
4-methylbenzyl,
4-hydroxybenzyl,
4-methoxybenzyl,
4-t-butoxybenzyl,
4-benzyloxybenzyl,
4-[~-CH(CH3)O-]benzyl,
4-[~-CH(COOH)O-]benzyl,
4-[BocNHCH2C(O)NH-]benzyl,
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4-chlorobenzyl,
4-[NHZCHZC(O)NH-]benzyl,
4-carboxybenzyl,
4-[CbzNHCH2CH2NH-]benzyl,
3-hydroxy-4-(~-OC(O)NH-)benzyl,
4-[HOOCCHZCHZC(O)NH-]benzyl,
benzyl,
4-[2'-carboxylphenoxy-]benzyl,
4-[~-C(O)NH-]benzyl,
3-carboxybenzyl,
4-iodobenzyl,
4-hydroxy-3,5-diiodobenzyl,
4-hydroxy-3-iodobenzyl,
4-[2'-carboxyphenyl-]benzyl,
~-CHZCHZ-,
4-nitrobenzyl,
2-carboxybenzyl,
4-[dibenzylamino]-benzyl,
4-[(1'-cyclopropylpiperidin-4'-yl)C(O)NH-]benzyl,
4-[-NHC(O)CHZNHBoc]benzyl,
4-carboxybenzyl,
4-hydroxy-3-nitrobenzyl,
4-[-NHC(O)CH(CH3)NHBoc]benzyl,
4-[-NHC(O)CH(CH2~)NHBoc]benzyl,
isobutyl,
methyl,
4-[CH3C(O)NH-]benzyl,
-CH2-(3-indolyl),
n-butyl,
t-butyl-OC(O)CHZ-,
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t-butyl-OC(O)CHZCHZ-,
HZNC(O)CHZ-,
HZNC(O)CHZCHZ-,
BocNH-(CHZ)a-,
t-butyl-OC (O)-(CHZ)a-,
HOOCCHZ-,
HOOC(CHZ)2-,
HZN(CHZ)a-,
isopropyl,
(1-naphthyl)-CHZ-;
(2-naphthyl)-CHZ-,
(2-thiophenyl)-CHZ-,
(~-CHZ-OC(O)NH-(CHZ)a-,
cyclohexyl-CHZ-,
benzyloxy-CH2-,
HOCHZ-,
5-(3-N-benzyl)imidazolyl-CHZ-,
2-pyridyl-CHZ-,
3-pyridyl-CHZ-,
4-pyridyl-CHZ-,
5-(3-N-methyl)imidazolyl-CHZ-,
N-benzylpiperid-4-yl-CHZ-,
N-Boc-piperidin-4-yl-CHZ-,
N-(phenyl-carbonyl)piperidin-4-yl-CHZ-,
H3CSCHZCH2-,
1-N-benzylimidazol-4-yl-CHZ-,
iso-propyl-C(O)NH-(CHZ)a-,
iso-butyl-C(O)NH-(CHZ)a-,
phenyl-C(O)NH-(CHZ)a-,
benzyl-C(O)NH-(CHZ)a-,
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__ 1 g __
allyl-C(O)NH-(CHZ)a-,
4-(3-N-methylimidazolyl)-CHZ ,
4-imidazolyl,
4-[(CH3)ZNCHZCHZCHZ-O-]benzyl,
4-[(benzyl)ZN-]-benzyl,
4-aminobenzyl,
allyloxy-C(O)NH(CHZ)a-,
allyloxy-C(O)NH(CHZ)3-,
allyloxy-C(O)NH(CHZ)z-,
NHZC(O)CHZ-,
~-CH=,
2-pyridyl-C(O)NH-(CHZ)a-,
4-methylpyrid-3-yl-C(O)NH-(CHZ)a-,
3-methylthien-2-yl-C(O)NH-(CHZ)a-,
2-pyrrolyl-C(O)NH-(CHZ)a-,
2-furanyl-C(O)NH-(CHZ)a-,
4-methylphenyl-SOZ-N(CH3)CHZC(O)NH(CHZ)a-,
4-[cyclopentylacetylenyl]-benzyl,
4-(-NHC(O)-(N-Boc)-pyrrolidin-2-yl)]-benzyl-,
1-N-methylimidazol-4-yl-CHZ-,
1-N-methylimidazol-5-yl-CHZ-,
imidazol-5-yl-CHZ-,
6-methylpyrid-3-yl-C(O)NH-(CHZ)a-,
4-(2'-carboxymethylphenyl]-benzyl,
4-[-NHC(O)NHCHzCH2CH2 ~]-benzyl,
4-[-NHC(O)NHCHZCHZ-~]-benzyl,
-CH2C(O)NH(CHZ)a~,
4-[~(CHZ)40-]-benzyl,
4-[-C =C-~-4' ~]-benzyl,
4-[-C=C-CH2-O-S(O)2-4'-CH3-~]-benzyl,
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4-[-C---C-CHZNHC(O)NHZ]-benzyl,
4-[-C---C-CHZ-O-4'-COOCHZCH3-~]-benzyl,
4-[-C---C-CH(NHZ)-cyclohexyl]-benzyl,
-(CHz)aNHC(O)CHZ-3-indolyl,
-(CHZ)aNHC(O)CHZCHZ-3-indolyl,
-(CHZ)aNHC(O)-3-(5-methoxyindolyl),
-(CHZ)aNHC(O)-3-(1-methylindolyl),
-(CHZ)aNHC(O)-4-(-SOZ(CH3)-~),
-(CHZ)aNHC(O)-4-(C(O)CH3)-phenyl,
-(CHZ)aNHC(O)-4-fluorophenyl,
-(CHZ)aNHC(O)CHZO-4-fluorophenyl,
4-[-C---C-(2-pyridyl)]benzyl,
4-[-C---C-CHZ-O-phenyl]benzyl,
4-[-C=C-CHZOCH3]benzyl,
4-[-C---C-(3-hydroxyphenyl)]benzyl,
4-[-C---C-CHZ-O-4'-(-C(O)OCZHS)phenyl]benzyl,
4-[-C---C-CHZCH(C(O)OCH3)2]benzyl,
4-[-C ---- C-CH2NH-(4, 5-dihydro-4-oxo-5-phenyl-oxazol-2-yl),
3-aminobenzyl,
4-[-C---C-CHZCH( NHC(O)CH3)C(O)OH]-benzyl,
-CHZC(O)NHCH(CH3)~,
-CHIC(O)NHCHZ-(4-dimethylamino)-~,
-CHZC(O)NHCHZ-4-nitrophenyl,
-CHZCHzC(O)N(CH3)CHZ-~,
-CHZCHZC(O)NHCHZCHZ-(N-methyl)-2-pyrrolyl,
-CHZCHZC(O)NHCHZCHZCHZCH3,
-CHZCHZC(O)NHCHZCHZ-3-indolyl,
-CHZC(O)N(CH3)CHzphenyl,
-CHIC(O)NH(CHZ)2-(N-methyl)-2-pyrrolyl,
-CHZC(O)NHCHZCHZCHZCH3,
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-CHZC(O)NHCHZCHZ-3-indolyl,
_(CHZ)zC(O)NHCH(CH3)~~
-(CH2)ZC(O)NHCHZ-4-dimethylaminophenyl,
-(CHZ)ZC(O)NHCHZ-4-nitrophenyl,
-CHIC(O)NH-4-[-NHC(O)CH3-phenyl],
-CHZC(O)NH-4-pyridyl,
-CHIC(O)NH-4-[dimethylaminophenyl],
-CHZC(O)NH-3-methoxyphenyl,
-CHZCHZC(O)NH-4-chlorophenyl,
-CHzCH2C(O)NH-2-pyridyl,
-CHZCHZC(O)NH-4-methoxyphenyl,
-CHZCHZC(O)NH-3-pyridyl,
4-[(CH3)ZNCHZCH20-]benzyl,
-(CHZ)3NHC(NH)NH-S02-4-methylphenyl,
4-[(CH3)ZNCHZCH20-]benzyl,
-(CHZ)4NHC(O)NHCHZCH3,
-(CHZ)4NHC(O)NH-phenyl,
-(CHz)4NHC(O)NH-4-methoxyphenyl,
4-[4'-pyridyl-C(O)NH-]benzyl,
4-[3'-pyridyl-C(O)NH-]benzyl,
4-[-NHC(O)NH-3'-methylphenyl)benzyl,
4-[-NHC(O)CHZNHC(O)NH-3'-methylphenyl)benzyl,
4-[-NHC(O)-(2',3'-dihydroindol-2-yl)]benzyl,
4-[-NHC(O)-(2',3'-dihydro-N-Boc-indol-2-yl)]benzyl,
p-[-OCHZCHZ-1'-(4'-pyrimidinyl)-piperazinyl]benzyl,
4-[-OCHZCHZ-(1'-piperidinyl)benzyl,
4-[-OCHZCHZ-(1'-pyrrolidinyl)]benzyl,
4-[-OCHZCHZCHZ-(1'-piperidinyl)]benzyl-,
-CHZ-3-(1,2,4-triazolyl),
4-[-OCHZCHZCHZ-4-(3'-chlorophenyl)-piperazin-1-yl]benzyl,
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4-[-OCHZCHZN(~)CHZCH3]benzyl,
4-[-OCHZ-3'-(N-Boc)-piperidinyl]benzyl,
4-[di-n-pentylamino]benzyl,
4-[n-pentylamino]benzyl,
4-[di-iso-propylamino-CHZCH20-]benzyl,
4-[-OCHZCHZ-(N-morpholinyl)]benzyl,
4-[-O-(3'-(N-Boc)-piperidinyl]benzyl,
4-[-OCHZCH(NHBoc)CHZCyclohexyl]benzyl,
p-(OCHZCHZ-(N-piperidinyl]benzyl,
4-[-OCHzCH2CH2-(4-m-chlorophenyl)-piperazin-1-yl]benzyl,
4-[-OCHZCHZ-(N-homopiperidinyl)benzyl,
4-[-NHC(O)-3'-(N-Boc)-piperidinyl]benzyl,
4-[-OCHZCHZN(benzyl)2]benzyl,
-CHZ-2-thiazolyl,
3-hydroxybenzyl,
4-[-OCHZCHZCHZN(CH3)~]benzyl,
4-[-NHC(S)NHCHZCHZ-(N-morpholino)]benzyl,
4-[-OCHZCHzN(CZHS)2]benzyl,
4-[-OCHZCHZCHZN(CZHS)2]benzyl,
4-[CH3(CHZ)4NH-]benzyl,
4-(N-n-butyl,N-n-pentylamino-]benzyl,
4-[-NHC(O)-4'-piperidinyl]benzyl,
4-[-NHC(O)CH(NHBoc)(CHZ)4NHCbz]benzyl,
4-[-NHC(O)-(1',2',3',4'-tetrahydro-N-Boc-isoquinolin-1'-yl]benzyl,
p-[-OCHZCHZCHZ-1'-(4'-methyl)-piperazinyl]benzyl,
-(CHZ)4NH-Boc,
3-[-OCHZCHZCHZN(CH3)Z]benzyl,
4-[-OCHZCHZCHZN(CH3)2]benzyl,
3-[-OCHZCHZ (1'-pyrrolidinyl)]benzyl,
4-[-OCHZCHZCHZN(CH3)benzyl]benzyl,
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4-[-NHC(S)NHCHZCHZCHZ-(N-morpholino)]benzyl,
4-[-OCHZCH2-(N-morpholino)]benzyl,
4-[-NHCHZ-(4'-chlorophenyl)]benzyl,
4-[-NHC(O)NH-(4'-cyanophenyl)]benzyl,
4-[-OCHZCOOH]benzyl,
4-(-OCHZCOO-t-butyl]benzyl,
4-(-NHC(O)-5'-fluoroindol-2-yl]benzyl,
4-[-NHC(S)NH(CHZ)Z-1-piperidinyl]benzyl,
4-[-N(SOZCH3)(CHZ)3-N(CH3)2]benzyl,
4-[-NHC(O)CHZCH(C(O)OCH2~)-NHCbz]benzyl,
4-[-NHS(O)ZCF3]benzyl,
3-[-O-(N-methylpiperidin-4.'-yl]benzyl,
4-[-C(=NH)NHZ]benzyl,
4-[-NHSOZ-CHZCI]benzyl,
4-[-NHC (O)-( 1 ' , 2' , 3' ,4'-tetrahydroisoquinolin-2' -yl] benzyl,
4-[-NHC(S)NH(CHZ)3-N-morpholino]benzyl,
4-[-NHC(O)CH(CH2CHzCH2CH2NH2)NHBoc]benzyl,
4-(-C(O)NHz]benzyl,
4-[-NHC(O)NH-3'-methoxyphenyl]benzyl,
4-[-OCHZCHZ-indol-3'-yl]benzyl,
4-[-OCH2C(O)NH-benzyl]benzyl,
4-[-OCHZC(O)O-benzyl]benzyl,
4-[-OCHZC(O)OH]benzyl,
4-[-OCHZ-2'-(4',5'-dihydro)imidazolyl]benzyl,
-CHIC(O)NHCHZ-(4-dimethylamino)phenyl,
-CHIC(O)NHCHZ-(4-dimethylamino)phenyl,
4-[-NHC(O)-L-2'-pyrrolidinyl-N-SOZ-4'-methylphenyl]benzyl,
4-[-NHC(O)NHCHZCHZCH3]benzyl,
4-aminobenzyl]benzyl,
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4-[-OCHZCHZ-1-(4-hydroxy-4-(3-methoxypyrrol-2-yl)-
piperazinyl]benzyl,
4-[-O-(N-methylpiperidin-4'-yl)] benzyl,
3-methoxybenzyl,
4-[-NHC(O)-piperidin-3'-yl]benzyl,
4-[-NHC(O)-pyridin-2'-yl]benzyl,
4-[-NHCHZ-(4'-chlorophenyl)]benzyl,
4-[-NHC(O)-(N-(4'-CH3-~-SOZ)-L-pyrrolidin-2'-yl)]benzyl,
4-[-NHC(O)NHCHZCHZ-~]benzyl,
4-[-OCHZC(O)NHZ]benzyl,
4-[-OCHZC(O)NH-t-butyl]benzyl,
4-[-OCHZCH2-1-(4-hydroxy-4-phenyl)-piperidinyl] benzyl,
4-[-NHS02-CH=CHZ]benzyl,
4-[-NHSOZ-CHZCHZCI]benzyl,
-CHZC(O)NHCHZCHZN(CH3)2,
4-[(1'-Cbz-piperidin-4'-yl)C(O)NH-]benzyl,
4-[(1'-Boc-piperidin-4'-yl)C(O)NH-]benzyl,
4-[(2'-bromophenyl)C(O)NH-]benzyl,
4-[-NHC(O)-pyridin-4'-yl]benzyl,
4-[(4'-(CH3)ZNC(O)O-)phenyl)-C(O)NH-]benzyl,
4-[-NHC(O)-1'-methylpiperidin-4'-yl-]benzyl,
4-(dimethylamino)benzyl,
4-[-NHC(O)-(1'-N-Boc)-piperidin-2'-yl]benzyl,
3-[-NHC(O)-pyridin-4'-yl]benzyl,
4-[(tent-butyl-O(O)CCHZ-O-benzyl)-NH-]benzyl,
[BocNHCH2C(O)NH-]butyl,
4-benzylbenzyl,
2-hydroxyethyl,
4-[(Et)ZNCHZCHZCHzNHC(S)NH-]benzyl,
4-[(1'-Boc-4.'-hydroxypyrrolidin-2'-yl)C(O)NH-]benzyl,
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4-[~CHZCHZCHZNHC(S)NH-]benzyl,
4-[(perhydroindolin-2'-yl)C(O)NH-]benzyl,
2-[4-hydroxy-4-(3-methoxythien-2-yl)piperidin-1-yl]ethyl,
4-[(1'-Boc-perhydroindolin-2'-yl)-C(O)NH-]benzyl,
4-[N 3-methylbutyl-N trifluoromethanesulfonyl)amino]benzyl,
4-[N vinylsulfonyl)amino]benzyl,
4-[2-(2-azabicyclo [3 .2. 2] octan-2-yl)ethyl-O-] benzyl,
4-[4'-hydroxypyrrolidin-2'-yl)C(O)NH-]benzyl,
4-(~NHC(S)NH)benzyl,
4-(EtNHC(S)NH)benzyl,
4-(~CHZNHC(S)NH)benzyl,
3-[(1'-Boc-piperidin-2'-yl)C(O)NH-]benzyl,
3-[piperidin-2'-yl-C(O)NH-]benzyl,
4-[(3'-Boc-thiazolidin-4'-yl)C(O)NH-]benzyl,
4-(pyridin-3'-yl-NHC(S)NH)benzyl,
4-(CH3-NHC(S)NH)benzyl,
4-(HZNCHZCHZCHZC(O)NH)benzyl,
4-(BocHNCH2CHZCHZC(O)NH)benzyl,
4-(pyridin-4'-yl-CHZNH)benzyl,
4-[(N,N di(4-N,N dimethylamino)benzyl)amino]benzyl,
4-[(1-Cbz-piperidin-4-yl)C(O)NH-]butyl,
4-[~CHZOCHZ(BocHN)CHC(O)NH]benzyl,
4-[(piperidin-4'-yl)C(O)NH-]benzyl,
4-[(pyrrolidin-2'-yl)C(O)NH-]benzyl,
4-(pyridin-3'-yl-C(O)NH)butyl,
4-(pyridin-4.'-yl-C(O)NH)butyl,
4-(pyridin-3'-yl-C(O)NH)benzyl,
4-[CH3NHCHZCHZCHZC(O)NH-]benzyl,
4-[CH3N(Boc)CHZCHZCHZC(O)NH-]benzyl,
4-(aminomethyl)benzyl,
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4-[c~CH20CH2(HZN)CHC(O)NH]benzyl,
4-[(1',4'-di(Boc)piperazin-2'-yl)-C(O)NH-]benzyl,
4-[(piperazin-2'-yl)-C(O)NH-]benzyl,
4-[(N toluenesulfonylpyrrolidin-2'-yl)C(O)NH-]butyl,
4-[-NHC(O)-4'-piperidinyl]butyl,
4-[-NHC(O)-1'-N-Boc-piperidin-2'-yl]benzyl,
4-[-NHC(O)-piperidin-2'-yl]benzyl,
4-[(1'-N-Boc-2',3'-dihydroindolin-2'-yl)-C(O)NH]benzyl,
4-(pyridin-3'-yl-CHZNH)benzyl,
4-[(1'-Cbz-piperidin-4'-yl)C(O)NH-]benzyl,
4-[(piperidin-1'-yl)C(O)CHZ-O-]benzyl,
4-[(CH3)ZCH)ZNC(O)CHZ-O-]benzyl,
4-[HO(O)C(Cbz-NH)CHCHZCHZ-C(O)NH-]benzyl,
4-[~CH20(O)C(Cbz-NH)CHCHZCHZ-C(O)NH-]benzyl,
4-[-NHC(O)-2'-methoxyphenyl] benzyl,
4-[(pyrazin-2'-yl)C(O)NH-]benzyl,
4-[HO(O)C(NHZ)CHCHZCHZ-C(O)NH-]benzyl,
4-(2'-formyl-1',2',3',4'-tetrahydroisoquinolin-3'-yl-CHZNH-)benzyl,
N Cbz-NHCHZ-,
4-[(4'-methylpiperazin-1'-yl)C(O)O-]benzyl,
4-[CH3(N Boc)NCHZC(O)NH-]benzyl,
4-[-NHC (O)-( 1' , 2' , 3' , 4'-tetrahydro-N-B oc-isoquinolin-3'-yl]-benzyl,
4-[CH3NHCHzC(O)NH-]benzyl,
(CH3)ZNC(O)CHZ-,
4-(N methylacetamido)benzyl,
4-(1',2',3',4'-tetrahydroisoquinolin-3'-yl-CHzNH-)benzyl,
4-[(CH3)ZNHCHZC(O)NH-]benzyl,
( 1-toluenesulfonylimidizol-4-yl)methyl,
4-[(1'-Boc-piperidin-4'-yl)C(O)NH-]benzyl,
4-trifluoromethylbenzyl,
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4-[(2'-bromophenyl)C(O)NH-]benzyl,
4-[(CH3)ZNC(O)NH-]benzyl,
4-[CH30C(O)NH-]benzyl,
4-[(CH3)ZNC(O)O-]benzyl,
4-[(CH3)ZNC(O)N(CH3)-]benzyl,
4-[CH30C(O)N(CH3)-]benzyl,
4-(N methyltrifluoroacetamido)benzyl,
4-[(1'-methoxycarbonylpiperidin-4'-yl)C(O)NH-]benzyl,
4-[(4'-phenylpiperidin-4'-yl)C(O)NH-]benzyl,
4-[(4'-phenyl-1'-Boc-piperidin-4'-yl)-C(O)NH-]benzyl,
4-[(piperidin-4'-yl)C(O)O-]benzyl, 4-[(1'-methylpiperidin-4'-yl)-
O-]benzyl,
4-[(1'-methylpiperidin-4'-yl)C(O)O-]benzyl,
4-[(4'-methylpiperazin-1'-yl)C(O)NH-]benzyl,
3-[(CH3)ZNC(O)O-]benzyl,
4-[(4'-phenyl-1'-Boc-piperidin-4.'-yl)-C(O)O-]benzyl,
4-(N toluenesulfonylamino)benzyl,
4-[(CH3)3CC(O)NH-]benzyl,
4-[(morpholin-4'-yl)C(O)NH-]benzyl,
4-[(CH3CH2)ZNC(O)NH-]benzyl,
4-[-C(O)NH-(4'-piperidinyl)]benzyl,
4-[(2'-trifluoromethylphenyl)C(O)NH-]benzyl,
4-[(2'-methylphenyl)C(O)NH-]benzyl,
4-[(CH3)zNS(O)20-]benzyl,
4-[(pyrrolidin-2'-yl)C(O)NH-]benzyl,
4-[-NHC(O)-piperidin-1'-yl]benzyl,
4-[(thiomorpholin-4'-yl)C(O)NH-]benzyl,
4-[(thiomorpholin-4'-yl sulfone)-C(O)NH-]benzyl,
4-[(morpholin-4'-yl)C(O)O-]benzyl,
3-nitro-4-(CH30C(O)CH20-)benzyl,
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(2-benzoxazolinon-6-yl)methyl-,
(2H 1,4-benzoxazin-3(4F~-one-7-yl)methyl-,
4-[(CH3)ZNS(O)ZNH-]benzyl,
4-[(CH3)zNS(O)ZN(CH3)-]benzyl,
4-[(thiomorpholin-4'-yl)C(O)O-]benzyl,
4-[(thiomorpholin-4.'-yl sulfone)-C(O)O-]benzyl,
4-[(piperidin-1'-yl)C(O)O-]benzyl,
4-[(pyrrolidin-1'-yl)C(O)O-]benzyl,
4-[(4'-methylpiperazin-1'-yl)C(O)O-]benzyl,
4-[(2'-methylpyrrolidin-1'-yl)-,
(pyridin-4-yl)methyl-,
4-[(piperazin-4'-yl)-C(O)O-]benzyl,
4-[(1'-Boc-piperazin-4'-yl)-C(O)O-]benzyl,
4-[(4'-acetylpiperazin-1'-yl)C(O)O-]benzyl,
p-[(4'-methanesulfonylpiperazin-1'-yl)-benzyl,
3-nitro-4-[(morpholin-4'-yl)-C(O)O-]benzyl,
4-{[(CH3)ZNC(S)]ZN-}benzyl,
N Boc-2-aminoethyl-,
4-[(1,1-dioxothiomorpholin-4-yl)-C(O)O-]benzyl,
4-[(CH3)zNS(O)2-]benzyl,
4-(imidazolid-2'-one-1'-yl)benzyl,
4-[(piperidin-1'-yl)C(O)O-]benzyl,
1-N-benzyl-imidazol-4-yl-CHZ-,
3,4-dioxyethylenebenzyl,
3,4-dioxymethylenebenzyl,
4-[-N(SOZ)(CH3)CHZCHZCHZN(CH3)2]benzyl,
4-(3'-formylimidazolid-2'-one-1'-yl)benzyl,
4-(NHC(O)CH(CHZCHZCHZCHZNHZ)NHBoc]benzyl,
[2'-[4"-hydroxy-4"-(3 "'-methoxythien-2"'-yl)piperidin-2"-
yl]ethoxy]benzyl, and
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p-[(CH3)ZNCHZCHZN(CH3)C(O)O-]benzyl.
In a preferred embodiment, RS is preferably selected from all possible
isomers arising by substitution with the following groups:
3-[(CH3)ZNC(O)O-]benzyl,
4-[(CH3)ZNC(O)O-]benzyl,
4-[(CH3)ZNS(O)20-]benzyl,
4-[(piperidin-1'-yl)C(O)O-]benzyl,
4-[(piperidin-4'-yl)C(O)O-]benzyl,
4-[(1'-methylpiperidin-4'-yl)C(O)O-]benzyl,
4-[(4'-hydroxypiperidin-1'-yl)C(O)O-]benzyl,
4-[(4'-formyloxypiperidin-1'-yl)C(O)O-]benzyl,
4-[(4'-ethoxycarbonylpiperidin-1'-yl)C(O)O-]benzyl,
4-[(4'-carboxylpiperidin-1'-yl)C(O)O-]benzyl,
4-[(3'-hydroxymethylpiperidin-1'-yl)C(O)O-]benzyl,
4-[(4'-hydroxymethylpiperidin-1'-yl)C(O)O-]benzyl,
4-[(4'-phenyl-1'-Boc-piperidin-4.'-yl)-C(O)O-]benzyl,
4-[(4'-piperidon-1'-yl ethylene ketal)C(O)O-]benzyl,
4-[(piperazin-4'-yl)-C(O)O-]benzyl,
4-[(1'-Boc-piperazin-4'-yl)-C(O)O-]benzyl,
4-[(4'-methylpiperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-methylhomopiperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(2-hydroxyethyl)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-phenylpiperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(pyridin-2-yl)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(4-trifluoromethylpyridin-2-yl)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(pyrimidin-2-yl)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-acetylpiperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(phenylC(O)-)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(pyridin-4-y1C(O)-)piperazin-1'-yl)C(O)O-]benzyl,
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4-[(4'-(phenylNHC(O)-)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-(phenylNHC(S)-)piperazin-1'-yl)C(O)O-]benzyl,
4-[(4'-methanesulfonylpiperazin-1'-yl-C(O)O-)benzyl,
4-[(4'-trifluoromethanesulfonylpiperazin-1'-yl-C(O)O-)benzyl,
4-[(morpholin-4'-yl)C(O)O-]benzyl,
3-vitro-4-[(morpholin-4'-yl)-C(O)O-]benzyl,
4-[(thiomorpholin-4'-yl)C(O)O-]benzyl,
4-[(thiomorpholin-4'-yl sulfone)-C(O)O-]benzyl,
(alternative nomenclature 4-[(1,1-dioxothiomorpholin-4-yl)-
C(O)O-]benzyl),
4-[(pyrrolidin-1'-yl)C(O)O-]benzyl,
4-[(2'-methylpyrrolidin-1'-yl)C(O)O-]benzyl,
4-[(2'-(methoxycarbonyl)pyrrolidin-1'-yl)C(O)O-]benzyl,
4-[(2'-(hydroxymethyl)pyrrolidin-1'-yl)C(O)O-]benzyl,
4-[(2'-(N,N-dimethylamino)ethyl)(CH3)NC(O)O-]benzyl,
4-[(2'-(N-methyl-N-toluene-4-sulfonylamino)ethyl)(CH3)N-
C(O)O-]benzyl,
4-[(2'-(morpholin-4'-yl)ethyl)(CH3)NC(O)O-]benzyl,
4-[(2'-(hydroxy)ethyl)(CH3)NC(O)O-]benzyl,
4-[bis(2'-(hydroxy)ethyl)NC(O)O-]benzyl,
4-[(2'-(formyloxy)ethyl)(CH3)NC(O)O-]benzyl,
4-[(CH30C(O)CHZ)HNC(O)O-]benzyl,
4-[2'-(phenylNHC(O)O-)ethyl-]HNC(O)O-]benzyl,
3-chloro-4-[(CH3)zNC(O)O-]benzyl,
3-chloro-4.-[(4'-methylpiperazin-1'-yl)C(O)O-]benzyl,
3-chloro-4-[(4'-(pyridin-2-yl)piperazin-1'-yl)C(O)O-]benzyl,
3-chloro-4.-[(thiomorpholin-4'-yl)C(O)O-]benzyl, and
3-fluoro-4-[(CH3)ZNC(O)O-]benzyl.
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__ 30 __
In the compounds of formula IA, R6 is preferably 2,4-dioxo-
tetrahydrofuran-3-yl (3,4-enol), methoxy, ethoxy, iso-propoxy, n-butoxy,
t-butoxy, cyclopentoxy, neo-pentoxy, 2-a-iso-propyl-4.-~3-
methylcyclohexoxy, 2-~i-isopropyl-4-~i-methylcyclohexoxy,
-NH2, benzyloxy, -NHCHZCOOH, -NHCHzCH2COOH, -NH-adamantyl,
-NHCHZCHZCOOCHzCH3, -NHSOZ p-CH3-~, -NHOR$ where R8 is
hydrogen, methyl, iso-propyl or benzyl, O-(N-succinimidyl),
-O-cholest-5-en-3-(3-yl, -OCH2-OC(O)C(CH3)3, -O(CHZ)ZNHC(O)W where z
is 1 or 2 and W is selected from the group consisting of pyrid-3-yl, N-
methylpyridyl, and N-methyl-1,4-dihydro-pyrid-3-yl, -NR"C(O)-R' where
R' is aryl, heteroaryl or heterocyclic and R" is hydrogen or
-CHZC(O)OCHZCH3.
Preferred compounds within the scope of formula I and IA above
include by way of example:
N [N-(toluene-4-sulfonyl)-L-pyrrolidin-2-ylmethyl]-L-phenylalanine
N-[N-(toluene-4-sulfonyl)-L-prolinyl]-N-hydroxy-L-phenylalanine
N-[N-(toluene-4-sulfonyl)-L-prolinyl]-N-hydroxy-D-phenylalanine
N-[2-(N-(toluene-4-sulfonyl)-L-pyrrolidinyl)-2-hydroxyacetyl]-L-4-
(N benzyloxycarbonyl-isonipecotamido)phenylalanine
N-[2-(N-(toluene-4-sulfonyl)-L-pyrrolidinyl)-2-hydroxyacetyl]-L-4-
(isonipecotamido)phenylalanine
(2S)-2-[5-(N-(toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-
nitrobenzyl)propionic acid
(2S)-2-[5-(N-(toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-
(N-tent-butoxycarbonylisonipecotamido)benzyl)propionic acid methyl
ester
(2S)-2-[5-(N-(toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-
(N-tent-butoxycarbonylisonipecotamido)benzyl)propionic acid
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N-[N-(toluene-4-sulfonyl)pyrrolidin-2-yl]aminocarbonyl)-L-
phenylalanine
and pharmaceutically acceptable salts thereof as well as any of the
ester compounds recited above wherein one ester is replaced with another
ester selected from the group consisting of methyl ester, ethyl ester, n-
propyl ester, isopropyl ester, n-butyl ester, isobutyl ester, sec-butyl ester
and tent-butyl ester.
This invention also provides methods for binding VLA-4 in a
biological sample which method comprises contacting the biological sample
with a compound of formula I or IA above under conditions wherein said
compound binds to VLA-4.
Certain of the compounds of formula I and IA above are also useful
in reducing VLA-4 mediated inflammation in vivo.
This invention also provides pharmaceutical compositions comprising
a pharmaceutically acceptable carrier and a therapeutically effective amount
of one or more of the compounds of formula I or IA above with the
exception that R3 and RS are derived from L-amino acids or other similarly
configured starting materials. Alternatively, racemic mixtures can be used.
The pharmaceutical compositions may be used to treat VLA-4
mediated disease conditions. Such disease conditions include, by way of
example, asthma, Alzheimer's disease, atherosclerosis, AIDS dementia,
diabetes (including acute juvenile onset diabetes), inflammatory bowel
disease (including ulcerative colitis and Crohn's disease), multiple
sclerosis,
rheumatoid arthritis, tissue transplantation, tumor metastasis, meningitis,
encephalitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic
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dermatitis, psoriasis, myocardial ischemia and acute leukocyte-mediated lung
injury such as that which occurs in adult respiratory distress syndrome.
Accordingly, this invention also provides methods for the treatment
of an inflammatory disease in a patient mediated by VLA-4 which methods
comprise administering to the patient the pharmaceutical compositions
described above.
Preferred compounds of formula I and IA above include those set
forth in Table I below:
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--33--
, ,
x x
, , z z
0 0 0 0 '
.. ,. ...
z z U U
T O O x ~C
0
U U U O O
x z
U U
.~ x x x x x x
0 0 0 0 0 0
, , , , ,
d
O-U z ,;,
x
,
.. z
~
Z U_
,~ x ~ ~ Z E >,
v
V U o o , G.
U U .
~ ~ ~ ~ z
a" " A ' ~
..a
V- ~ 'c
I
g
a
V
O= =O a.
x x x x x x
, , , ,
M , , , , , ,
, , , ,
Vi ~ H ~ N .Ur y-Uy ..V.n
.-,-~.,~~.. .-~., VJ Vl fn
.-~ ~ ..~~
U ~ U ~ U ~ U ~ U ~ U ~
.d .d .d ,d ,d ,b
M ~ M ~ M .~ M .D M ~ M w~
~ ~ ~"~ ~ ~
C~ ~ cG ~' CC ttJ CC ~"
, CL G1. A" CL CC
U n N U ,..~ H U N U N U
~,M'~ n U , , , n
~M"~, ~M~ ~.M'~ RiM~ QiMa
, , , , , ,
LL p. C.S. ~L 0, Cs,
, , , n , ,
M M M M M M
x x x x x x
U U U U U U
, , , , , ,
a. a, 4 ~, a. a,
~n O v1
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WO 00/43415 __34__ PCT/US00/01603
x
b z
0
.
.
, , .
z
.~
' x
o 0
0
_,
x x
z z
0 0
~r v
U U ,
N
~, ~, x
b b
m
, ~ -z
.. .r
z
i a
,
x x x
,
, , ,
, , ,
...,
'
~
O v O v O C
~ I
U cC U c0 U ~
b ;d .
N
H H 1
W: ~ ~ i ~: i o
a n.~',
U U
~ ~ ~ '~ i
. M . M Q M
, i ,
W Q, fs~
i i i
M n M
x x x
U U U
, ,
4
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DETAILED DESCRIPTION OF THE INVENTION
As above, this invention relates to compounds which inhibit
leukocyte adhesion and, in particular, leukocyte adhesion mediated by VLA-
4. When describing the compounds, compositions and methods of this
invention, the following terms have the following meanings, unless
otherwise indicated.
Definitions
As used herein, "alkyl" refers to alkyl groups preferably having from
1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is
exemplified by groups such as methyl, t-butyl, n-heptyl, octyl and the like.
"Substituted alkyl" refers to an alkyl group, preferably of from 1 to
10 carbon atoms, having from 1 to 5 substituents selected from the group
consisting of alkoxy, substituted alkoxy, acyl, acylamino,
thiocarbonylamino, acyloxy, amino, amidino, alkyl amidino,thioamidino,
aminoacyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,
aryloxylaryl, substituted aryloxyaryl, cyano, halogen, hydroxyl, vitro,
carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl,
carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl,
carboxylheteroaryl, carboxyl-substituted heteroaryl, carboxylheterocyclic,
carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,
guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,
thioaryl,
substituted thioaryl, thiocycloalkyl, substituted thiocycloalkyl,
thioheteroaryl, substituted thioheteroaryl, thioheterocyclic, substituted
thioheterocyclic, heteroaryl, substituted aryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, cycloalkoxy, substituted cycloalkoxy,
heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted
heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino, -OS(O)Z alkyl, -
OS(O)z-substituted alkyl, -OS(O)2-aryl, -OS(O)2-substituted aryl, -OS(O)2-
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heteroaryl, -OS(O)Z substituted heteroaryl, -OS(O)2-heterocyclic, -OS(O)z-
substituted heterocyclic, -OSOZ-NRR where R is hydrogen or alkyl,
-NRS(O)2-alkyl, -NRS(O)Z substituted alkyl, -NRS(O)Z-aryl, -NRS(O)2-
substituted aryl, -NRS(O)2-heteroaryl, -NRS(O)Z-substituted heteroaryl,
-NRS(O)2-heterocyclic, -NRS(O)2-substituted heterocyclic, -NRS(O)2-NR-
alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl, -NRS(O)2-NR-
substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)z-NR-substituted
heteroaryl, -NRS(O)2 NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and substituted alkyl groups having amino groups blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
alkyl/substituted alkyl groups substituted with -SOZ-alkyl, -SOz-substituted
alkyl, -SOZ-alkenyl, -SOZ-substituted alkenyl, -SOZ-cycloalkyl, -SOZ-
substituted cycloalkyl, -SOZ-aryl, -SO~-substituted aryl, -SOZ-heteroaryl, -
SOZ-substituted heteroaryl, -SOZ-heterocyclic, -SOZ-substituted heterocyclic
and -SOZNRR where R is hydrogen or alkyl.
"Alkoxy" refers to the group "alkyl-O-" which includes, by way of
example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tent-butoxy,
sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
"Substituted alkoxy" refers to the group "substituted alkyl-O-".
"Aryl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-
C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted
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alkynyl-C(O)- cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-,
substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O),
heterocyclic-C(O)-, and substituted heterocyclic-C(O)- wherein alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
S cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Acylamino" refers to the group -C(O)NRR where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,
substituted heteroaryl, heterocyclic, substituted heterocyclic and where each
R is joined to form together with the nitrogen atom a heterocyclic or
substituted heterocyclic ring wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
"Thiocarbonylamino" refers to the group -C(S)NRR where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,
substituted heteroaryl, heterocyclic, substituted heterocyclic and where each
R is joined to form, together with the nitrogen atom a heterocyclic or
substituted heterocyclic ring wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
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"Acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-
C(O)O-, alkenyl-C(O)O-, substituted alkenyl-C(O)O-, alkynyl-C(O)O-,
substituted alkynyl-C(O)O-, aryl-C(O)O-, substituted aryl-C(O)O-,
cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, heteroaryl-C(O)O-,
substituted heteroaryl-C(O)O-, heterocyclic-C(O)O-, and substituted
heterocyclic-C(O)O- wherein alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
aryl, substituted aryl; heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocyclic are as defined herein.
"Alkenyl" refers to alkenyl group preferably having from 2 to 10
carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1
and preferably from 1-2 sites of alkenyl unsaturation.
"Substituted alkenyl" refers to alkenyl groups having from 1 to 5
substituents selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,
alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy,
substituted aryloxy, aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl,
cyano, vitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl-
cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-
substituted aryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,
carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,
substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,
substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,
substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,
thioheterocyclic, substituted thioheterocyclic, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy, substituted
cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,
substituted heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino,
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-OS(O)Z-alkyl, -OS(O)2-substituted alkyl, -OS(O)2-aryl, -OS(O)Z-substituted
aryl, -OS(O)2-heteroaryl, -OS(O)2-substituted heteroaryl, -OS(O)2-
heterocyclic, -OS(O)2-substituted heterocyclic, -OSOZ-NRR where R is
hydrogen or alkyl, -NRS(O)2-alkyl, -NRS(O)2-substituted alkyl, -NRS(O)Z-
aryl, -NRS(O)2-substituted aryl, -NRS(O)2-heteroaryl, -NRS(O)~substituted
heteroaryl, -NRS(O)2-heterocyclic, -NRS(O)z-substituted heterocyclic,
-NRS(O)2-NR-alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl,
-NRS(O)Z-NR-substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-
substituted heteroaryl, -NRS(O)Z-NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and substituted alkenyl groups having amino groups blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
alkenyl/substituted alkenyl groups substituted with -SOZ-alkyl, -SOZ-
substituted alkyl, -SOZ-alkenyl, -SOZ-substituted alkenyl, -SOZ-cycloalkyl,
-SOZ-substituted cycloalkyl, -SOZ-aryl, -S02-substituted aryl, -S02-
heteroaryl, -SOZ-substituted heteroaryl, -SOZ-heterocyclic, -SOZ-substituted
heterocyclic and -SOZNRR where R is hydrogen or alkyl.
"Alkynyl" refers to alkynyl group preferably having from 2 to 10
carbon atoms and more preferably 3 to 6 carbon atoms and having at least 1
and preferably from 1-2 sites of alkynyl unsaturation.
"Substituted alkynyl" refers to alkynyl groups having from 1 to 5
substituents selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,
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alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy,
substituted aryloxy, aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl,
cyano, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl-
cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-
substituted aryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,
carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,
substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,
substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,
substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,
thioheterocyclic, substituted thioheterocyclic, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy, substituted
cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,
substituted heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino,
-OS(O)2-alkyl, -OS(O)2-substituted alkyl, -OS(O)2-aryl, -OS(O)2-substituted
aryl, -OS(O)Z-heteroaryl, -OS(O)2-substituted heteroaryl, -OS(O)2-
heterocyclic, -OS(O)2-substituted heterocyclic, -OSOZ-NRR where R is
hydrogen or alkyl, -NRS(O)2 alkyl, -NRS(O)2-substituted alkyl, -NRS(O)2-
aryl, -NRS(O)2-substituted aryl, -NRS(O)2-heteroaryl, -NRS(O)2-substituted
heteroaryl, -NRS(O)z heterocyclic, -NRS(O)2-substituted heterocyclic,
-NRS(O)2-NR-alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl,
-NRS(O)Z-NR-substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-
substituted heteroaryl, -NRS(O)2-NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and substituted alkynyl groups having amino groups blocked by
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conventional blocking groups such as Boc, Cbz, formyl, and the like or
alkynyl/substituted alkynyl groups substituted with -SOZ-alkyl, -SOZ-
substituted alkyl, -SOZ-alkenyl, -SOZ-substituted alkenyl, -SOZ-cycloalkyl,
-SOZ-substituted cycloalkyl, -SOZ-aryl, -SOZ-substituted aryl, -SOZ-
heteroaryl, -SOZ-substituted heteroaryl, -SOZ-heterocyclic, -SOZ-substituted
heterocyclic and -SOZNRR where R is hydrogen or alkyl.
"Amidino" refers to the group HZNC(=NH)- and the term
"alkylamidino" refers to compounds having 1 to 3 alkyl groups (e.g.,
alkylHNC(=NH)-).
"Thioamidino" refers to the group RSC(=NH)- where R is hydrogen
or alkyl.
"Aminoacyl" refers to the groups -NRC(O)alkyl,
-NRC(O)substituted alkyl, -NRC(O)cycloalkyl, -NRC(O)substituted
cycloalkyl, -NRC(O)alkenyl, -NRC(O)substituted alkenyl, -NRC(O)alkynyl,
-NRC(O)substituted alkynyl, -NRC(O)aryl, -NRC(O)substituted aryl,
-NRC(O)heteroaryl, -NRC(O)substituted heteroaryl, -NRC(O)heterocyclic,
and -NRC(O)substituted heterocyclic where R is hydrogen or alkyl and
wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted
aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic
are as defined herein.
"Aminocarbonyloxy" refers to the groups -NRC(O)O-alkyl,
-NRC(O)O-substituted alkyl, -NRC(O)O-alkenyl, -NRC(O)O-substituted
alkenyl, -NRC(O)O-alkynyl, -NRC(O)O-substituted alkynyl, -NRC(O)O-
cycloalkyl, -NRC(O)O-substituted cycloalkyl, -NRC(O)O-aryl, -NRC(O)O-
substituted aryl, -NRC(O)O-heteroaryl, -NRC(O)O-substituted heteroaryl,
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-NRC(O)O-heterocyclic, and -NRC(O)O-substituted heterocyclic where R is
hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocyclic are as defined herein.
"Oxycarbonylamino" refers to the groups -OC(O)NH2, -OC(O)NRR,
-OC(O)NR-alkyl, -OC(O)NR-substituted alkyl, -OC(O)NR-alkenyl,
-OC(O)NR-substituted alkenyl, -OC(O)NR-alkynyl, -OC(O)NR-substituted
alkynyl, -OC(O)NR-cycloalkyl, -OC(O)NR-substituted cycloalkyl,
-OC(O)NR-aryl, -OC(O)NR-substituted aryl, -OC(O)NR-heteroaryl,
-OC(O)NR-substituted heteroaryl,- OC(O)NR-heterocyclic, and
-OC(O)NR-substituted heterocyclic where R is hydrogen, alkyl or where
each R is joined to form, together with the nitrogen atom a heterocyclic or
substituted heterocyclic ring and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
"Oxythiocarbonylamino" refers to the groups -OC(S)NH2,
-OC(S)NRR, -OC(S)NR-alkyl, -OC(S)NR-substituted alkyl, -OC(S)NR-
alkenyl, -OC(S)NR-substituted alkenyl, -OC(S)NR-alkynyl, -OC(S)NR-
substituted alkynyl, -OC(S)NR-cycloalkyl, -OC(S)NR-substituted cycloalkyl,
-OC(S)NR-aryl, -OC(S)NR-substituted aryl, -OC(S)NR-heteroaryl, -
OC(S)NR-substituted heteroaryl, -OC(S)NR-heterocyclic, and
-OC(S)NR-substituted heterocyclic where R is hydrogen, alkyl or where
each R is joined to form together with the nitrogen atom a heterocyclic or
substituted heterocyclic ring and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
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"Aminocarbonylamino" refers to the groups -NRC(O)NRR,
-NRC(O)NR-alkyl, -NRC(O)NR-substituted alkyl, -NRC(O)NR-alkenyl,
-NRC(O)NR-substituted alkenyl, -NRC(O)NR-alkynyl,
-NRC(O)NR-substituted alkynyl, -NRC(O)NR-aryl, -NRC(O)NR-substituted
aryl, -NRC(O)NR-cycloalkyl, -NRC(O)NR-substituted cycloalkyl, -
NRC(O)NR-heteroaryl, and -NRC(O)NR-substituted heteroaryl,
NRC(O)NR-heterocyclic, and -NRC(O)NR-substituted heterocyclic where
each R is independently hydrogen, alkyl or where each R is joined to form
together with the nitrogen atom a heterocyclic or substituted heterocyclic
ring as well as where one of the amino groups is blocked by conventional
blocking groups such as Boc, Cbz, formyl, and the like and wherein alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminothiocarbonylamino" refers to the groups -NRC(S)NRR,
-NRC(S)NR-alkyl, -NRC(S)NR-substituted alkyl, -NRC(S)NR-alkenyl,
-NRC(S)NR-substituted alkenyl, -NRC(S)NR-alkynyl, -NRC(S)NR-
substituted alkynyl, -NRC(S)NR-aryl, -NRC(S)NR-substituted aryl,
-NRC(S)NR-cycloalkyl, -NRC(S)NR-substituted cycloalkyl, -NRC(S)NR-
heteroaryl, and -NRC(S)NR-substituted heteroaryl, -NRC(S)NR-
heterocyclic, and -NRC(S)NR-substituted heterocyclic where each R is
independently hydrogen, alkyl or where each R is joined to form together
with the nitrogen atom a heterocyclic or substituted heterocyclic ring as well
as where one of the amino groups is blocked by conventional blocking
groups such as Boc, Cbz, formyl, and the like and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.
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__
"Aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group
of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple
condensed rings (e.g., naphthyl or anthryl) which condensed rings may or
may not be aromatic (e.g., 2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-
one-7y1, and the like). Preferred aryls include phenyl and naphthyl.
Substituted aryl refers to aryl groups which are substituted with from
1 to 3 substituents selected from the group consisting of hydroxy, acyl,
acylamino, thiocarbonylamino, acyloxy, alkyl, substituted alkyl, alkoxy,
substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl,
amidino, alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,
aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,
aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,
heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted
heterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,
carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-
substituted aryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,
carboxylheterocyclic, carboxyl-substituted heterocyclic, carboxylamido,
cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl, substituted
thioaryl,
thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substituted
thiocycloalkyl, thioheterocyclic, substituted thioheterocyclic, cycloalkyl,
substituted cycloalkyl, guanidino, guanidinosulfone, halo, vitro, heteroaryl,
substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,
substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,
heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,
oxythiocarbonylamino, -S(O)2 alkyl, -S(O)z substituted alkyl, -S(O)2-
cycloalkyl, -S(O)Z-substituted cycloalkyl, -S(O)Z-alkenyl, -S(O)2-substituted
alkenyl, -S(O)2-aryl, -S(O)Z-substituted aryl, -S(O)2-heteroaryl, -S(O)2-
substituted heteroaryl, -S(O)2-heterocyclic, -S(O)2-substituted heterocyclic, -
OS(O)2-alkyl, -OS(O)2-substituted alkyl, -OS(O)z-aryl, -OS(O)z substituted
aryl, -OS(O)2-heteroaryl, -OS(O)2-substituted heteroaryl, -OS(O)2
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heterocyclic, -OS(O)Z-substituted heterocyclic, -OSOZ-NRR where R is
hydrogen or alkyl, -~1RS(O)2-alkyl, -NRS(O)2-substituted alkyl, -NRS(O)Z-
aryl, -NRS(O)2-substituted aryl, -NRS(O)2-heteroaryl, -NRS(O)2-substituted
heteroaryl, -NRS(O)Z heterocyclic, -NRS(O)2-substituted heterocyclic,
NRS(O)2-NR-alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl, -
NRS(O)2-NR-substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-
substituted heteroaryl, -NRS(O)z-NR-heterocyclic, -NRS(O)Z-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and amino groups on the substituted aryl blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
substituted with -SOZNRR where R is hydrogen or alkyl.
"Aryloxy" refers to the group aryl-O- which includes, by way of
example, phenoxy, naphthoxy, and the like.
"Substituted aryloxy" refers to substituted aryl-O- groups.
"Aryloxyaryl" refers to the group -aryl-O-aryl.
"Substituted aryloxyaryl" refers to aryloxyaryl groups substituted
with from 1 to 3 substituents on either or both aryl rings selected from the
group consisting of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy,
alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, amidino, alkylamidino, thioamidino,
amino, aminoacyl, aminocarbonyloxy, aminocarbonylamino,
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aminothiocarbonylamino, aryl, substituted aryl, aryloxy, substituted aryloxy,
cycloalkoxy, substituted cycloalkoxy, heteroaryiexy, substituted
heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy, carboxyl,
carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-
substituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl,
carboxylheteroaryl, carboxyl-substituted heteroaryl, carboxylheterocyclic,
carboxyl-substituted heterocyclic, carboxylamido, cyano, thiol, thioalkyl,
substituted thioalkyl, thioaryl, substituted thioaryl, thioheteroaryl,
substituted thioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,
thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substituted
cycloalkyl, guanidino, guanidinosulfone, halo, nitro, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy, substituted
cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,
substituted heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino,
-S(O)2-alkyl, -S(O)2-substituted alkyl, -S(O)2-cycloalkyl, -S(O)Z-substituted
cycloalkyl, -S(O)2-alkenyl, -S(O)2-substituted alkenyl, -S(O)2-aryl, -S(O)2-
substituted aryl, -S(O)2-heteroaryl, -S(O)Z-substituted heteroaryl, -S(O)Z-
heterocyclic, -S(O)2-substituted heterocyclic, -OS(O)Z-alkyl, -OS(O)2-
substituted alkyl, -OS(O)2-aryl, -OS(O)2-substituted aryl, -OS(O)Z-heteroaryl,
-OS(O)Z-substituted heteroaryl, -OS(O)2-heterocyclic, -OS(O)2-substituted
heterocyclic, -OSOZ-NRR where R is hydrogen or alkyl, -NRS(O)2-alkyl,
-NRS(O)2-substituted alkyl, -NRS(O)2-aryl, -NRS(O)2-substituted aryl,
-NRS(O)2-heteroaryl, -NRS(O)2-substituted heteroaryl, -NRS(O)2-
heterocyclic, -NRS(O)Z-substituted heterocyclic, -NRS(O)2-NR-alkyl,
-NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl, -NRS(O)2-NR-
substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-substituted
heteroaryl, -NRS(O)Z-NR-heterocyclic, -NRS(O)Z-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
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di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and amino groups on the substituted aryl blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
substituted with -SOZNRR where R is hydrogen or alkyl.
"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 8 carbon
atoms having a single cyclic ring including, by way of example,
cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Excluded
from this definition are mufti-ring alkyl groups such as adamantanyl, etc.
"Cycloalkenyl" refers to cyclic alkenyl groups of from 3 to 8 carbon
atoms having single or multiple unsaturation but which are not aromatic.
"Substituted cycloalkyl" and "substituted cycloalkenyl" refer to a
cycloalkyl and cycloalkenyl groups, preferably of from 3 to 8 carbon atoms,
having from 1 to 5 substituents selected from the group consisting of oxo
(=O), thioxo (=S), alkoxy, substituted alkoxy, acyl, acylamino,
thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, thioamidino,
aminoacyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,
aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, vitro,
carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl,
carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl,
carboxylheteroaryl, carboxyl-substituted heteroaryl, carboxylheterocyclic,
carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,
guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,
thioaryl,
substituted thioaryl, thiocycloalkyl, substituted thiocycloalkyl,
thioheteroaryl, substituted thioheteroaryl, thioheterocyclic, substituted
thioheterocyclic, heteroaryl, substituted heteroaryl, heterocyclic,
substituted
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heterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,
substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,
oxycarbonylamino, oxythiocarbonylamino, -OS(O)Z-alkyl, -OS(O)z-
substituted alkyl, -OS(O)2-aryl, -OS(O)2-substituted aryl, -OS(O)2-heteroaryl,
-OS(O)Z-substituted heteroaryl, -OS(O)2-heterocyclic, -OS(O)2-substituted
heterocyclic, -OSOZ-NRR where R is hydrogen or alkyl,
-NRS(O)2-alkyl, -NRS(O)2-substituted alkyl, -NRS(O)2-aryl, -NRS(O)2-
substituted aryl, -NRS(O)z-heteroaryl, -NRS(O)2-substituted heteroaryl,
-NRS(O)Z-heterocyclic, -NRS(O)2-substituted heterocyclic,
-NRS(O)2-NR-alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)Z-NR-aryl,
-NRS(O)2-NR-substituted aryl, -NRS(O)2 NR-heteroaryl, -NRS(O)2 NR-
substituted heteroaryl, -NRS(O)2-NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and substituted alkynyl groups having amino groups blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
alkynyl/substituted alkynyl groups substituted with -SOZ-alkyl, -SOZ-
substituted alkyl, -SOZ-alkenyl, -SOZ-substituted alkenyl, -SOZ-cycloalkyl,
-SOZ-substituted cycloalkyl, -SOz-aryl, -SOZ-substituted aryl, -SOZ_
heteroaryl, -SOZ-substituted heteroaryl, -SOZ-heterocyclic, -SOZ-substituted
heterocyclic and -S02NRR where R is hydrogen or alkyl.
"Cycloalkoxy" refers to -O-cycloalkyl groups.
"Substituted cycloalkoxy" refers to -O-substituted cycloalkyl groups.
"Guanidino" refers to the groups -NRC(=NR)NRR,
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-NRC(=NR)NR-alkyl, -NRC(=NR)NR-substituted alkyl, -NRC(=NR)NR-
alkenyl. -NRC(=NR)NR-substituted alkenyl, -NRC(=NR)NR-alkynyl,
-NRC(=NR)NR-substituted alkynyl, -NRC(=NR)NR-aryl,
-NRC(=NR)NR-substituted aryl, -NRC(=NR)NR-cycloalkyl,
-NRC(=NR)NR-heteroaryl, -NRC(=NR)NR-substituted heteroaryl,
-NRC(=NR)NR-heterocyclic, and -NRC(=NR)NR-substituted heterocyclic
where each R is independently hydrogen and alkyl as well as where one of
the amino groups is blocked by conventional blocking groups such as Boc,
Cbz, formyl, and the like and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined herein.
"Guanidinosulfone" refers to the groups -NRC(=NR)NRSOZ-alkyl,
-NRC(=NR)NRSOZ-substituted alkyl, -NRC(=NR)NRSOZ-alkenyl,
-NRC(=NR)NRS02-substituted alkenyl, -NRC(=NR)NRS02-alkynyl,
-NRC(=NR)NRSOZ-substituted alkynyl, -NRC(=NR)NRSOZ-aryl,
-NRC(=NR)NRSOZ-substituted aryl, -NRC(=NR)NRSOZ-cycloalkyl,
-NRC(=NR)NRSOZ-substituted cycloalkyl, -NRC(=NR)NRSOZ-heteroaryl,
and -NRC(=NR)NRSOZ-substituted heteroaryl, -NRC(=NR)NRSOZ-
heterocyclic, and -NRC(=NR)NRS02-substituted heterocyclic where each R
is independently hydrogen and alkyl and wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and
preferably is either chloro or bromo.
"Heteroaryl" refers to an aromatic carbocyclic group of from 2 to 10
carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and
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sulfur within the ring. Such heteroaryl groups can have a single ring (e.g.,
pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or
benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl, indolyl and
furyl.
"Substituted heteroaryl" refers to heteroaryl groups which are
substituted with from 1 to 3 substituents selected from the group consisting
of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, amidino, alkylamidino, thioamidino, amino, aminoacyl,
aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl,
substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substituted
cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,
substituted heterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted
alkyl, carboxyl-cycloalkyl, ~ carboxyl-substituted cycloalkyl, carboxylaryl,
carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substituted
heteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,
carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,
substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,
thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic, substituted
thioheterocyclic, cycloalkyl, substituted cycloalkyl, guanidino,
guanidinosulfone, halo, nitro, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, cycloalkoxy, substituted cycloalkoxy,
heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted
heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino, -S(O)Z alkyl,
-S(O)2-substituted alkyl, -S(O)2-cycloalkyl, -S(O)2-substituted cycloalkyl,
-S(O)2-alkenyl, -S(O)2-substituted alkenyl, -S(O)2-aryl, -S(O)2-substituted
aryl, -S(O)2-heteroaryl, -S(O)2-substituted heteroaryl, -S(O)2-heterocyclic,
-S(O)Z-substituted heterocyclic, -OS(O)2-alkyl, -OS(O)2-substituted alkyl,
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-OS(O)z-aryl, -OS(O)Z-substituted aryl, -OS(O)2-heteroaryl, -OS(O)2-
substituted heteroaryl, -OS(O)z-heterocyclic, -OS(O)2-substituted
heterocyclic, -OSOZ-NRR where R is hydrogen or alkyl, -NRS(O)2-alkyl,
-NRS(O)2-substituted alkyl, -NRS(O)Z-aryl, -NRS(O)2-substituted aryl,
-NRS(O)2-heteroaryl, -NRS(O)2-substituted heteroaryl, -NRS(O)2-
heterocyclic, -NRS(O)z substituted heterocyclic, -NRS(O)Z-NR-alkyl,
-NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl, -NRS(O)z-NR-
substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-substituted
heteroaryl, -NRS(O)2-NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and amino groups on the substituted aryl blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
substituted with -S02NRR where R is hydrogen or alkyl.
"Heteroaryloxy" refers to the group -O-heteroaryl and "substituted
heteroaryloxy" refers to the group -O-substituted heteroaryl.
"Heterocycle" or "heterocyclic" refers to a saturated or unsaturated
group having a single ring or multiple condensed rings, from 1 to 10 carbon
atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen
within the ring wherein, in fused ring systems, one or more of the rings can
be aryl or heteroaryl.
"Substituted heterocyclic" refers to heterocycle groups which are
substituted with from 1 to 3 substituents selected from the group consisting
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of oxo (=O), thioxo (=S), alkoxy, substituted alkoxy, acyl, acylamino,
thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, thioamidino,
aminoacyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,
aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,
carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl,
carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl,
carboxylheteroaryl, carboxyl-substituted heteroaryl, carboxylheterocyclic,
carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,
guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,
thioaryl,
substituted thioaryl, thiocycloalkyl, substituted thiocycloalkyl,
thioheteroaryl, substituted thioheteroaryl, thioheterocyclic, substituted
thioheterocyclic, heteroaryl, substituted heteroaryl, heterocyclic,
substituted
heterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,
substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,
oxycarbonylamino, oxythiocarbonylamino, -OS(O)2-alkyl, -OS(O)2-
substituted alkyl, -OS(O)Z-aryl, -OS(O)Z-substituted aryl, -OS(O)2-heteroaryl,
-OS(O)2-substituted heteroaryl, -OS(O)2-heterocyclic, -OS(O)2-substituted
heterocyclic, -OSOz NRR where R is hydrogen or alkyl,
-NRS(O)Z-alkyl, -NRS(O)2-substituted alkyl, -NRS(O)2-aryl, -NRS(O)2-
substituted aryl, -NRS(O)2-heteroaryl, -NRS(O)Z-substituted heteroaryl,
-NRS(O)2-heterocyclic, -NRS(O)2-substituted heterocyclic,
-NRS(O)2-NR-alkyl, -NRS(O)2-NR-substituted alkyl, -NRS(O)2-NR-aryl,
-NRS(O)Z-NR-substituted aryl, -NRS(O)2-NR-heteroaryl, -NRS(O)2-NR-
substituted heteroaryl, -NRS(O)2-NR-heterocyclic, -NRS(O)2-NR-substituted
heterocyclic where R is hydrogen or alkyl, mono- and di-alkylamino, mono-
and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-
substituted arylamino, mono- and di-heteroarylamino, mono- and di-
substituted heteroarylamino, mono- and di-heterocyclic amino, mono- and
di-substituted heterocyclic amino, unsymmetric di-substituted amines having
different substituents selected from alkyl, substituted alkyl, aryl,
substituted
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aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic and substituted alkynyl groups having amino groups blocked by
conventional blocking groups such as Boc, Cbz, formyl, and the like or
alkynyl/substituted alkynyl groups substituted with -SOZ-alkyl, -SOZ-
S substituted alkyl, -SOZ-alkenyl, -SOZ substituted alkenyl, -SOZ-cycloalkyl,
-S02-substituted cycloalkyl, -SOZ-aryl, -SOZ-substituted aryl, -SOZ-
heteroaryl, -SOZ-substituted heteroaryl, -SOZ-heterocyclic, -SOZ-substituted
heterocyclic and -SOZNRR where R is hydrogen or alkyl.
Examples of heterocycles and heteroaryls include, but are not limited
to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,
pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine,
quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-
tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,
benzo[b]thiophene, morpholino, thiomorpholino, piperidinyl, pyrrolidine,
tetrahydrofuranyl, and the like.
"Heterocyclyloxy" refers to the group -O-heterocyclic and
"substituted heterocyclyloxy" refers to the group -O-substituted heterocyclic.
"Thioi" refers to the group -SH.
"Thioalkyl" refers to the groups -S-alkyl
"Substituted thioalkyl" refers to the group -S-substituted alkyl.
"Thiocycloalkyl" refers to the groups -S-cycloalkyl.
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"Substituted thiocycloalkyl" refers to the group -S-substituted
cycloalkyl .
"Thioaryl" refers to the group -S-aryl and "substituted thioaryl"
refers to the group -S-substituted aryl.
"Thioheteroaryl" refers to the group -S-heteroaryl and "substituted
thioheteroaryl" refers to the group -S-substituted heteroaryl.
"Thioheterocyclic" refers to the group -S-heterocyclic and
"substituted thioheterocyclic" refers to the group -S-substituted
heterocyclic.
"Pharmaceutically acceptable salt" refers to pharmaceutically
acceptable salts of a compound of Formula I which salts are derived from a
variety of organic and inorganic counter ions well known in the art and
include, by way of example only, sodium, potassium, calcium, magnesium,
ammonium, tetraalkylammonium, and the like; and when the molecule
contains a basic functionality, salts of organic or inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate
and the like.
Compound Pre amation
The compounds of this invention can be prepared from readily
available starting materials using the following general methods and
procedures. It will be appreciated that where typical or preferred process
conditions (i.e., reaction temperatures, times, mole ratios of reactants,
solvents, pressures, etc.) are given, other process conditions can also be
used unless otherwise stated. Optimum reaction conditions may vary with
the particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization procedures.
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Additionally, as will be apparent to those skilled in the art,
conventional protecting groups may be necessary to prevent certain
functional groups from undergoing undesired reactions. Suitable protecting
groups for various functional groups as well as suitable conditions for
protecting and deprotecting particular functional groups are well known in
the art. For example, numerous protecting groups are described in T. W.
Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second
Edition, Wiley, New York, 1991, and references cited therein.
Furthermore, the compounds of this invention will typically contain
one or more chiral centers. Accordingly, if desired, such compounds can be
prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or
diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers
(and enriched mixtures) are included within the scope of this invention,
unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may
be prepared using, for example, optically active starting materials or
stereoselective reagents well-known in the art. Alternatively, racemic
mixtures of such compounds can be separated using, for example, chiral
column chromatography, chiral resolving agents and the like.
In a preferred embodiment, the compounds of formula I and IA
wherein Q is a tetrazol-1,5-diyl moiety are readily prepared by contacting
the corresponding amide, i.e., where Q is -C(O)NH-, with an equimolar
amount of phosphorus pentachloride (PCIs) in an anhydrous inert solvent,
such as benzene, followed by treatment of the resulting imino chloride with
hydrazoic acid (HN3). Typically, this reaction is conducted by contacting
the amide with PCIs at ambient temperature for about 90 minutes. The
resulting imino chloride is typically not isolated, but is reacted in situ
with
an equimolar amount of hydrazoic acid at ambient temperature for about 2
hours to afford the tetrazole.
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The amide intermediates, i.e. where Q is -C(O)NH-, employed in
this reaction are readily prepared by first coupling an amino acid of formula
II:
R3
R2-N-CH-COOH II
H
wherein RZ and R3 are as defined herein (e.g., in formula I and IIA), with a
sulfonyl chloride of formula III:
O
I
I
R~-S-CI III
I
I
O
wherein R' is as defined herein, to provide an N sulfonyl amino acid of
formula IV:
R3
O
R~-S- ~ -CH-COOH IV
O
R2
wherein R1-R3 are as defined herein.
This reaction is typically conducted by reacting the amino acid of
formula II with at least one equivalent, preferably about 1.1 to about 2
equivalents, of sulfonyl chloride III in an inert diluent such as
dichloromethane and the like. Generally, the reaction is conducted at a
temperature ranging from about -70 ° C to about 40 ° C for about
1 to about
24 hours. Preferably, this reaction is conducted in the presence of a suitable
base to scavenge the acid generated during the reaction. Suitable bases
include, by way of example, tertiary amines, such as triethylamine,
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diisopropylethylamine, N methylmorpholine and the like. Alternatively, the
reaction can be conducted under Schotten-Baumann-type conditions using
aqueous alkali, such as sodium hydroxide and the like, as the base. Upon
completion of the reaction, the resulting N sulfonyl amino acid IV is
recovered by conventional methods including neutralization, extraction,
precipitation, chromatography, filtration, and the like.
The amino acids of formula II employed in the above reaction are
either known compounds or compounds that can be prepared from known
compounds by conventional synthetic procedures. Examples of suitable
amino acids for use in this reaction include, but are not limited to, L-
proline, traps-4-hydroxyl-L-proline, cis-4-hydroxyl-L-proline, traps-3-
phenyl-L-proline, cis-3-phenyl-L-proline, L-(2-methyl)proline, L-pipecolinic
acid, L-azetidine-2-carboxylic acid, L-indoline-2-carboxylic acid, L-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid,
L-(5,5-dimethyl)thiazolidine-4-carboxylic acid, L-thiamorpholine-3-
carboxylic acid, glycine, 2-tent-butylglycine, D,L-phenylglycine, L-alanine,
a-methylalanine, N methyl-L-phenylalanine, L-diphenylalanine, sarcosine,
D,L-phenylsarcosine, L-aspartic acid (3-tert-butyl ester, L-glutamic acid y-
tent-butyl ester, L-(O-benzyl)serine, 1-aminocyclopropanecarboxylic acid, 1-
aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid
(cycloleucine) 1-aminocyclohexanecarboxylic acid, L-serine and the like. If
desired, the corresponding carboxylic acid esters of the amino acids of
formula II, such as the methyl esters, ethyl esters and the like, can be
employed in the above reaction with the sulfonyl chloride III. Subsequent
hydrolysis of the ester group to the carboxylic acid using conventional
reagents and conditions, i.e., treatment with an alkali metal hydroxide in an
inert diluent such as methanol/water, then provides the N sulfonyl amino
acid IV.
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Similarly, the sulfonyl chlorides of formula III employed in the above
reaction are either known compounds or compounds that can be prepared
from known compounds by conventional synthetic procedures. Such
compounds are typically prepared from the corresponding sulfonic acid, i.e.,
from compounds of the formula R'-S03H where R' is as defined above,
using phosphorous trichloride and phosphorous pentachloride. This reaction
is generally conducted by contacting the sulfonic acid with about 2 to 5
molar equivalents of phosphorous trichloride and phosphorous
pentachloride, either neat or in an inert solvent, such as dichloromethane, at
temperature in the range of about 0 ° C to about 80 ° C for
about 1 to about 48
hours to afford the sulfonyl chloride. Alternatively, the sulfonyl chlorides
of formula III can be prepared from the corresponding thiol compound, i.e.,
from compounds of the formula R'-SH where Rl is as defined herein, by
treating the thiol with chlorine (C12) and water under conventional reaction
conditions.
Examples of sulfonyl chlorides suitable for use in this invention
include, but are not limited to, methanesulfonyl chloride, 2-propanesulfonyl
chloride, 1-butanesulfonyl chloride, benzenesulfonyl chloride, 1-
naphthalenesulfonyl chloride, 2-naphthalenesulfonyl chloride, p-
toluenesulfonyl chloride, a-toluenesulfonyl chloride, 4-
acetamidobenzenesulfonyl chloride, 4-amidinobenzenesulfonyl chloride, 4-
tert-butylbenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 2-
carboxybenzenesulfonyl chloride, 4-cyanobenzenesulfonyl chloride, 3,4-
dichlorobenzenesulfonyl chloride, 3,5-dichlorobenzenesulfonyl chloride,
3,4-dimethoxybenzenesulfonyl chloride, 3,5-
ditrifluoromethylbenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride,
4-methoxybenzenesulfonyl chloride, 2-methoxycarbonylbenzenesulfonyl
chloride, 4-methylamidobenzenesulfonyl chloride, 4-nitrobenzenesulfonyl
chloride, 4-thioamidobenzenesulfonyl chloride, 4-
trifluoromethylbenzenesulfonyl chloride, 4-trifluoromethoxybenzenesulfonyl
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chloride, 2;4,6-trimethylbenzenesulfonyl chloride, 2-phenylethanesulfonyl
chloride, 2-thiophenesulfonyl chloride, 5-chloro-2-thiophenesulfonyl
chloride, 2,5-dichloro-4-thiophenesulfonyl chloride, 2-thiazolesulfonyl
chloride, 2-methyl-4-thiazolesulfonyl chloride, 1-methyl-4-imidazolesulfonyl
chloride, 1-methyl-4-pyrazolesulfonyl chloride, 5-chloro-1,3-dimethyl-4-
pyrazolesulfonyl chloride, 3-pyridinesulfonyl chloride, 2-pyrimidinesulfonyl
chloride and the like. If desired, a sulfonyl fluoride, sulfonyl bromide or
sulfonic acid anhydride may be used in place of the sulfonyl chloride in the
above reaction to form the N sulfonyl amino acids of formula IV.
The intermediate N sulfonyl amino acids of formula IV can also be
prepared by reacting a sulfonamide of formula V
O
I I
R~-S-H- R2 V
O
wherein R' and RZ are as defined herein, with a carboxylic acid derivative of
the formula L(R3)CHCOOR where L is a leaving group, such as chloro,
bromo, iodo, mesylate, tosylate and the like, R3 is as defined herein and R is
hydrogen or an alkyl group. This reaction is typically conducted by
contacting the sulfonamide V with at least one equivalent, preferably 1. l to
2 equivalents, of the carboxylic acid derivative in the presence of a suitable
base, such as triethylamine, in an inert diluent, such as DMF, at a
temperature ranging from about 24°C to about 37°C for about 0.5
to about
4 hours. This reaction is further described in Zuckermann et al., J. Am.
Chem. Soc. , 1992, 114, 10646-10647. Preferred carboxylic acid derivatives
for use in this reaction are a-chloro and a-bromocarboxylic acid esters such
as tent-butyl bromoacetate and the like. When a carboxylic acid ester is
employed in this reaction, the ester group is subsequently hydrolyzed using
conventional procedures to afford an N sulfonyl amino acid of formula IV.
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The amide intermediates where Q is -C(O)NH- are then prepared by
coupling the intermediate N sulfonyl amino acid of formula IV with an
amino acid derivative of formula VI:
O
H N-CH-IC-R6 VI
R5
wherein RS and R6 are as defined herein. This coupling reaction is typically
conducted using well-known coupling reagents such as carbodiimides, BOP
reagent (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophosphonate) and the like. Suitable carbodiimides include, by
way of example, dicyclohexylcarbodiimide (DCC), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide (EDC) and the like. If desired, polymer
supported forms of carbodiimide coupling reagents may also be used
including, for example, those described in Tetrahedron Letters, 34(48), 7685
(1993). Additionally, well-known coupling promoters, such as N-
hydroxysuccinimide, 1-hydroxybenzotriazole and the like, may be used to
facilitate the coupling reaction.
This coupling reaction is typically conducted by contacting the N
sulfonylamino acid IV with about 1 to about 2 equivalents of the coupling
reagent and at least one equivalent, preferably about 1 to about 1.2
equivalents, of amino acid derivative VI in an inert diluent, such as
dichloromethane, chloroform, acetonitrile, tetrahydrofuran, N,N
dimethylformamide and the like. Generally, this reaction is conducted at a
temperature ranging from about 0°C to about 37°C for about 12 to
about 24
hours. Upon completion of the reaction, the amide is recovered by
conventional methods including neutralization, extraction, precipitation,
chromatography, filtration, and the like.
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Alternatively, the N sulfonyl amino acid IV can be converted into an
acid halide and the acid halide coupled with amino acid derivative VI to
provide an amide. The acid halide of VI can be prepared by contacting VI
with an inorganic acid halide, such as thionyl chloride, phosphorous
trichloride, phosphorous tribromide or phosphorous penta-chloride, or
preferably, with oxalyl chloride under conventional conditions. Generally,
this reaction is conducted using about 1 to 5 molar equivalents of the
inorganic acid halide or oxalyl chloride, either neat or in an inert solvent,
such as dichloromethane or carbon tetrachloride, at temperature in the range
of about 0°C to about 80°C for about 1 to about 48 hours. A
catalyst, such
as DMF, may also be used in this reaction.
The acid halide of N sulfonyl amino acid IV is then contacted with at
least one equivalent, preferably about 1.1 to about 1.5 equivalents, of amino
acid derivative VI in an inert diluent, such as dichloromethane, at a
temperature ranging from about -70°C to about 40°C for about 1
to about
24 hours. Preferably, this reaction is conducted in the presence of a suitable
base to scavenge the acid generated during the reaction. Suitable bases
include, by way of example, tertiary amines, such as triethylamine,
diisopropylethylamine, N methylmorpholine and the like. Alternatively, the
reaction can be conducted under Schotten-Baumann-type conditions using
aqueous alkali, such as sodium hydroxide and the like. Upon completion of
the reaction, the amide is recovered by conventional methods including
neutralization, extraction, precipitation, chromatography, filtration, and the
like.
Alternatively, the amide intermediates where Q is -C(O)NH- can be
prepared by first forming a diamino acid derivative of formula VII:
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II II VII
R2-N-CH-C-N-CH-C-R6
H I H I
R3 R5
wherein R2, R3, R5, and R6 are as defined herein. The diamino acid
derivatives of formula VII can be readily prepared by coupling an amino
acid of formula II with an amino acid derivative of formula VI using
conventional amino acid coupling techniques and reagents, such
carbodiimides, BOP reagent and the like, as described above. Diamino acid
VII can then be sulfonated using a sulfonyl chloride of formula III and using
the synthetic procedures described above to provide an amide.
The amino acid derivatives of formula VI employed in the above
reactions are either known compounds or compounds that can be prepared
from known compounds by conventional synthetic procedures. For
example, amino acid derivatives of formula VI can be prepared by C
alkylating commercially available diethyl 2-acetamidomalonate (Aldrich,
Milwaukee, Wisconsin, USA) with an alkyl or substituted alkyl halide. This
reaction is typically conducted by treating the diethyl 2-acetamidomalonate
with at least one equivalent of sodium ethoxide and at least one equivalent of
an alkyl or substituted alkyl halide in refluxing ethanol for about 6 to about
12 hours. The resulting C-alkylated malonate is then deacetylated,
hydrolyzed and decarboxylated by heating in aqueous hydrochloric acid at
reflux for about 6 to about 12 hours to provide the amino acid, typically as
the hydrochloride salt.
Examples of amino acid derivatives of formula VI suitable for use in
the above reactions include, but are not limited to, L-tyrosine methyl ester,
L-3,5-diiodotyrosine methyl ester, L-3-iodotyrosine methyl ester, ~3-(4-
hydroxy-naphth-1-yl)-L-alanine methyl ester, ~3-(6-hydroxy-naphth-2-yl)-L-
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alanine methyl ester, and the like. If desired, of course, other esters or
amides of the above-described compounds may also be employed.
In another preferred embodiment, the compounds of formula I/IA
wherein Q is -CH(OH)C(O)NH- are readily prepared by coupling an a-
hydroxy carboxylic acid of formula IVa:
R3
O
R~°S-N-CH~CH-COOH IVa
O R2 O H
with an amino acid derivative of formula VI using the coupling reagents and
procedures described above. The a-hydroxy carboxylic acids of formula
IVa are either commercially available or can be prepared from readily
available starting materials using conventional reagents and reaction
conditions well-known to those skilled in the art.
Additionally, in another preferred embodiment, the compounds of
formula I/IA where Q is -C(O)N(O)- are readily prepared by coupling an N
sulfonyl amino acid of formula IV with an N hydroxy amino acid of formula
IVb:
O
HO-N-CH-C-R6
H I IVb
Rs
where RS and R6 are as defined herein, using the coupling reagents and
procedures described above. The N hydroxy amino acids of formula IVb
can be prepared from the corresponding amino acid derivative of formula
IV, where R6 is OH, by reaction with sodium nitrite and potassium bromide
in dilute aqueous sulfuric acid (typically 2.5 N), followed by treatment with
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hydroxylamine. Typically, this reaction is conducted by first contacting a
solution IV and excess potassium bromide in 2.5 N sulfuric acid with excess
sodium nitrite at 0°C for about 0.25 to 0.5 hours and then at ambient
temperature for 1 hour. The product is then esterified using thionyl chloride
and methanol under standard conditions to form the methyl ester. The
methyl ester is then immediately contacted with hydroxylamine in methanol
and the reaction mixture is refluxed for 6 to 24 hours to afford the N
hydroxy amino acid of formula IVb.
In still another preferred embodiment, the compounds of formula
I/IA where Q is -NHC(O)NH- are readily prepared by contacting an N
sulfonyl amino acid of formula IV with an equimolar amount of
diphenylphosphoryl azide in an inert solvent, such as toluene, followed by
treatment with a trialkylamine, such as triethylamine, to form the
corresponding isocyanate. The intermediate isocyanate is generally not
isolated, but is reacted in situ with an amino acid derivative of formula VI
to
afford the urea. This reaction is typically conducted at about 80°C for
about
6 to about 24 hours to provide the urea.
In yet another preferred embodiment, the compounds of formula I/IA
where Q is -CH(R8)NR'- are conveniently prepared by reductive alkylation
of an amino acid derivative of formula VI using a carbonyl compound of
formula IVc:
R~-S-N-CH-C-R$ IVc
O
R2
using conventional reductive alkylation reagents and conditions. Typically,
this reaction is conducted by contacting the amino acid derivative VI with an
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excess of IVc, preferably with 1.1 to 2 equivalents of IVc, and an excess,
preferably 1.1 to 1.5 equivalents, of a reducing agent, such as sodium
cyanoborohydride. Generally, this reaction is conducted in an essentially
inert diluent, such as methanol, at a temperature ranging from about
0°C to
about 50°C, preferably at ambient temperature, for about 0.5 to 3 hours
to
afford the desired product.
For ease of synthesis, the compounds of formula I are typically
prepared as an ester, i.e., where R6 is an alkoxy or substituted alkoxy group
and the like. If desired, the ester group can be hydrolysed using
conventional conditions and reagents to provide the corresponding
carboxylic acid. Typically, this reaction is conducted by treating the ester
with at least one equivalent of an alkali metal hydroxide, such as lithium,
sodium or potassium hydroxide, in an inert diluent, such as methanol or
mixtures of methanol and water, at a temperature ranging about 0°C to
about 24°C for about 1 to about 12 hours. Alternatively, benzyl esters
may
be removed by hydrogenolysis using a palladium catalyst, such as palladium
on carbon. The resulting carboxylic acids may be coupled, if desired, to
amines such as ~3-alanine ethyl ester, hydroxyamines such as hydroxylamine
and N hydroxysuccinimide, alkoxyamines and substituted alkoxyamines such
as O-methylhydroxylamine and O-benzylhydroxylamine, and the like, using
conventional coupling reagents and conditions as described above.
As will be apparent to those skilled in the art, other functional groups
present on any of the substituents of the compounds of formula I can be
readily modified or derivatized either before or after the above-described
coupling reactions using well-known synthetic procedures. For example, a
nitro group present on a substituent of a compound of formula I or an
intermediate thereof may be readily reduced by hydrogenation in the
presence of a palladium catalyst, such as palladium on carbon, to provide the
corresponding amino group. This reaction is typically conducted at a
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temperature of from about 20 ° C to about 50 ° C for about 6 to
about 24 hours
in an inert diluent, such as methanol. Compounds having a nitro group on
the RS substituent can be prepared, for example, by using a 4-
nitrophenylalanine derivative and the like in the above-described coupling
reactions.
Similarly, a pyridyl group can be hydrogenated in the presence of a
platinum catalyst, such as platinum oxide, in an acidic diluent to provide the
corresponding piperidinyl analogue. Generally, this reaction is conducted
by treating the pyridine compound with hydrogen at a pressure ranging from
about 20 psi to about 60 psi, preferably about 40 psi, in the presence of the
catalyst at a temperature of about 20°C to about 50°C for about
2 to about
24 hours in an acidic diluent, such as a mixture of methanol and aqueous
hydrochloric acid. Compounds having a pyridyl group can be readily
prepared by using, for example, ~3-(2-pyridyl)-, ~i-(3-pyridyl)- or ~3-(4-
pyridyl)-L-alanine derivatives in the above-described coupling reactions.
Additionally, when the RS substituent of a compound of formula I or
an intermediate thereof contains a primary or secondary amino group, such
amino groups can be further derivatized either before or after the above
coupling reactions to provide, by way of example, amides, sulfonamides,
ureas, thioureas, carbamates, secondary or tertiary amines and the like.
Compounds having a primary amino group on the RS substituent may be
prepared, for example, by reduction of the corresponding nitro compound as
described above. Alternatively, such compounds can be prepared by using
an amino acid derivative of formula VI derived from lysine, 4-
aminophenylalanine and the like in the above-described coupling reactions.
By way of illustration, a compound of formula I or an intermediate
thereof having a substituent containing a primary or secondary amino group,
such as where RS is a (4-aminophenyl)methyl group, can be readily N
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acylated using conventional acylating reagents and conditions to provide the
corresponding amide. This acylation reaction is typically conducted by
treating the amino compound with at least one equivalent, preferably about
1.1 to about 1.2 equivalents, of a carboxylic acid in the presence of a
coupling reagent such as a carbodiimide, BOP reagent (benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphonate) and the like, in an
inert diluent, such as dichloromethane, chloroform, acetonitrile,
tetrahydrofuran, N,N dimethylformamide and the like, at a temperature
ranging from about 0 ° C to about 37 ° C for about 4 to about 24
hours .
Preferably, a promoter, such as N hydroxysuccinimide, 1-hydroxy-
benzotriazole and the like, is used to facilitate the acylation reaction.
Examples of carboxylic acids suitable for use in this reaction include, but
are
not limited to, N tent-butyloxycarbonylglycine, N tent-butyloxycarbonyl-L-
phenylalanine, N tent-butyloxycarbonyl-L-aspartic acid benzyl ester, benzoic
acid, N ten-butyloxycarbonylisonipecotic acid, N methylisonipecotic acid,
N tent-butyloxycarbonylnipecotic acid, N tent-butyloxycarbonyl-L-
tetrahydroisoquinoline-3-carboxylic acid, N (toluene-4-sulfonyl)-L-proline
and the like.
Alternatively, a compound of formula I or an intermediate thereof
containing a primary or secondary amino group can be N acylated using an
acyl halide or a carboxylic acid anhydride to form the corresponding amide.
This reaction is typically conducted by contacting the amino compound with
at least one equivalent, preferably about 1.1 to about 1.2 equivalents, of the
acyl halide or carboxylic acid anhydride in an inert diluent, such as
dichloromethane, at a temperature ranging from about of about -70°C to
about 40 ° C for about 1 to about 24 hours. If desired, an acylation
catalyst
such as 4-(N,N dimethylamino)pyridine may be used to promote the
acylation reaction. The acylation reaction is preferably conducted in the
presence of a suitable base to scavenge the acid generated during the
reaction. Suitable bases include, by way of example, tertiary amines, such
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as triethylamine, diisopropylethylamine, N methylmorpholine and the like.
Alternatively, the reaction can be conducted under Schotten-Baumann-type
conditions using aqueous alkali, such as sodium hydroxide and the like.
Examples of acyl halides and carboxylic acid anhydrides suitable for
use in this reaction include, but are not limited to, 2-methylpropionyl
chloride, trimethylacetyl chloride, phenylacetyl chloride, benzoyl chloride,
2-bromobenzoyl chloride, 2-methylbenzoyl chloride, 2-trifluoro-
methylbenzoyl chloride, isonicotinoyl chloride, nicotinoyl chloride,
picolinoyl chloride, acetic anhydride, succinic anhydride; and the like.
Carbamyl chlorides, such as N,N dimethylcarbamyl chloride, N,N
diethylcarbamyl chloride and the like, can also be used in this reaction to
provide ureas. Similarly, Bicarbonates, such as di-tert-butyl Bicarbonate,
may be employed to provide carbamates.
In a similar manner, a compound of formula I or an intermediate
thereof containing a primary or secondary amino group may be N sulfonated
to form a sulfonamide using a sulfonyl halide or a sulfonic acid anhydride.
Sulfonyl halides and sulfonic acid anhydrides suitable for use in this
reaction
include, but are not limited to, methanesulfonyl chloride,
chloromethanesulfonyl chloride, p-toluenesulfonyl chloride,
trifluoromethanesulfonic anhydride, and the like. Similarly, sulfamoyl
chlorides, such as dimethylsulfamoyl chloride, can be used to provide
sulfamides (e.g., > N-SOZ-N < ).
Additionally, a primary and secondary amino group present on a
substituent of a compound of formula I or an intermediate thereof can be
reacted with an isocyanate or a thioisocyanate to give a urea or thiourea,
respectively. This reaction is typically conducted by contacting the amino
compound with at least one equivalent, preferably about 1.1 to about 1.2
equivalents, of the isocyanate or thioisocyanate in an inert diluent, such as
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toluene and the like, at a temperature ranging from about 24°C to about
3?°C for about 12 to about 24 hours. The isocyanates and
thioisocyanates
used in this reaction are commercially available or can be prepared from
commercially available compounds using well-known synthetic procedures.
For example, isocyanates and thioisocyanates are readily prepared by
reacting the appropriate amine with phosgene or thiophosgene. Examples of
isocyanates and thioisocyanates suitable for use in this reaction include, but
are not limited to, ethyl isocyanate, n-propyl isocyanate, 4-cyanophenyl
isocyanate, 3-methoxyphenyl isocyanate, 2-phenylethyl isocyanate, methyl
thioisocyanate, ethyl thioisocyanate, 2-phenylethyl thioisocyanate, 3-
phenylpropyl thioisocyanate, 3-(N,N diethylamino)propyl thioisocyanate,
phenyl thioisocyanate, benzyl thioisocyanate, 3-pyridyl thioisocyanate,
fluorescein isothiocyanate (isomer I) and the like.
Furthermore, when a compound of formula I or an intermediate
thereof contains a primary or secondary amino group, the amino group can
be reductively alkylated using aldehydes or ketones to form a secondary or
tertiary amino group. This reaction is typically conducted by contacting the
amino compound with at least one equivalent, preferably about 1.1 to about
1.5 equivalents, of an aldehyde or ketone and at least one equivalent based
on the amino compound of a metal hydride reducing agent, such as sodium
cyanoborohydride, in an inert diluent, such as methanol, tetrahydrofuran,
mixtures thereof and the like, at a temperature ranging from about 0°C
to
about 50°C for about 1 to about 72 hours. Aldehydes and ketones
suitable
for use in this reaction include, by way of example, benzaldehyde, 4-
chlorobenzaldehyde, valeraldehyde and the like.
In a similar manner, when a compound of formula I or an
intermediate thereof has a substituent containing a hydroxyl group, the
hydroxyl group can be further modified or derivatized either before or after
the above coupling reactions to provide, by way of example, ethers,
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carbamates and the like. Compounds having a hydroxyl group on the RS
substituent, for example, can be prepared using an amino acid derivative of
formula VI derived from tyrosine and the like in the above-described
reactions .
By way of example, a compound of formula I or an intermediate
thereof having a substituent containing a hydroxyl group, such as where RS
is a (4-hydroxyphenyl)methyl group, can be readily O-alkylated to form
ethers: This O-alkylation reaction is typically conducted by contacting the
hydroxy compound with a suitable alkali or alkaline earth metal base, such
as potassium carbonate, in an inert diluent, such as acetone, 2-butanone and
the like, to form the alkali or alkaline earth metal salt of the hydroxyl
group.
This salt is generally not isolated, but is reacted in situ with at least one
equivalent of an alkyl or substituted alkyl halide or sulfonate, such as an
alkyl chloride, bromide, iodide, mesylate or tosylate, to afford the ether.
Generally, this reaction is conducted at a temperature ranging from about
60°C to about 150°C for about 24 to about 72 hours. Preferably,
a catalytic
amount of sodium or potassium iodide is added to the reaction mixture when
an alkyl chloride or bromide is employed in the reaction.
Examples of alkyl or substituted alkyl halides and sulfonates suitable
for use in this reaction include, but are not limited to, tent-butyl
bromoacetate, N tent-butyl chloroacetamide, 1-bromoethylbenzene, ethyl a-
bromophenylacetate, 2-(N ethyl-N phenylamino)ethyl chloride, 2-(N,N
ethylamino)ethyl chloride, 2-(N,N diisopropylamino)ethyl chloride, 2-(N,N
dibenzylamino)ethyl chloride, 3-(N,N ethylamino)propyl chloride, 3-(N
benzyl-N methylamino)propyl chloride, N (2-chloroethyl)morpholine, 2-
(hexamethyleneimino)ethyl chloride, 3-(N methylpiperazine)propyl chloride,
1-(3-chlorophenyl)-4-(3-chloropropyl)piperazine, 2-(4-hydroxy-4-
phenylpiperidine)ethyl chloride, N tent-butyloxycarbonyl-3-piperidinemethyl
tosylate, and the like.
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Alternatively, a hydroxyl group present on a substituent of a
compound of formula I or an intermediate thereof can be O-alkylating using
the Mitsunobu reaction. In this reaction, an alcohol, such as 3-(N,N
dimethylamino)-1-propanol and the like, is reacted with about 1.0 to about
1.3 equivalents of triphenylphosphine and about 1.0 to about 1.3 equivalents
of diethyl azodicarboxylate in an inert diluent, such as tetrahydrofuran, at a
temperature ranging from about -10°C to about 5°C for about 0.25
to about
1 hour. About 1.0 to about 1.3 equivalents of a hydroxy compound, such as
N tent-butyltyrosine methyl ester, is then added and the reaction mixture is
stirred at a temperature of about 0 ° C to about 30 ° C for
about 2 to about 48
hours to provide the O-alkylated product.
In a similar manner, a compound of formula I or an intermediate
thereof containing a aryl hydroxy group can be reacted with an aryl iodide to
provide a diaryl ether. Generally, this reaction is conducted by forming the
alkali metal salt of the hydroxyl group using a suitable base, such as sodium
hydride, in an inert diluent such as xylenes at a temperature of about -25
° C
to about 10°C. The salt is then treated with about 1.1 to about 1.5
equivalents of cuprous bromide dimethyl sulfide complex at a temperature
ranging from about 10°C to about 30°C for about 0.5 to about 2.0
hours,
followed by about 1. l to about 1.5 equivalents of an aryl iodide, such as
sodium 2-iodobenzoate and the like. The reaction is then heated to about
70 ° C to about 150 ° C for about 2 to about 24 hours to provide
the diaryl
ether.
Additionally, a hydroxy-containing compound can also be readily
derivatized to form a carbamate. In one method for preparing such
carbamates, a hydroxy compound of formula I or an intermediate thereof is
contacted with about 1.0 to about 1.2 equivalents of 4-nitrophenyl
chloroformate in an inert diluent, such as dichloromethane, at a temperature
ranging from about -25°C to about 0°C for about 0.5 to about 2.0
hours.
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Treatment of the resulting carbonate with an excess, preferably about 2 to
about 5 equivalents, of a trialkylamine, such as triethylamine, for about 0.5
to 2 hours, followed by about 1.0 to about 1.5 equivalents of a primary or
secondary amine provides the carbamate. Examples of amines suitable for
using in this reaction include, but are not limited to, piperazine, 1-
methylpiperazine, 1-acetylpiperazine, morpholine, thiomorpholine,
pyrrolidine, piperidine and the like.
Alternatively, in another method for preparing carbamates, a
hydroxy-containing compound is contacted with about 1.0 to about 1.5
equivalents of a carbamyl chloride in an inert diluent, such as
dichloromethane, at a temperature ranging from about 25°C to about
70°C
for about 2 to about 72 hours. Typically, this reaction is conducted in the
presence of a suitable base to scavenge the acid generated during the
reaction. Suitable bases include, by way of example, tertiary amines, such
as triethylamine, diisopropylethylamine, N methylmorpholine and the like.
Additionally, at least one equivalent (based on the hydroxy compound) of 4-
(N,N dimethylamino)pyridine is preferably added to the reaction mixture to
facilitate the reaction. Examples of carbamyl chlorides suitable for use in
this reaction include, by way of example, dimethylcarbamyl chloride,
diethylcarbamyl chloride and the like.
Likewise, when a compound of formula I or an intermediate thereof
contains a primary or secondary hydroxyl group, such hydroxyl groups can
be readily converted into a leaving group and displaced to form, for
example, amines, sulfides and fluorides. For example, derivatives of 4-
hydroxy-L-proline can be converted into the corresponding 4-amino, 4-thio
or 4-fluoro-L-proline derivatives via nucleophilic displacement of the
derivatized hydroxyl group. Generally, when a chiral compound is
employed in these reactions, the stereochemistry at the carbon atom attached
to the derivatized hydroxyl group is typically inverted.
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These reactions are typically conducted by first converting the
hydroxyl group into a leaving group, such as a tosylate, by treatment of the
hydroxy compound with at least one equivalent of a sulfonyl halide, such as
p-toluenesulfonyl chloride and the like, in pyridine. This reaction is
generally conducted at a temperature of from about 0 ° C to about 70
° C for
about 1 to about 48 hours. The resulting tosylate can then be readily
displaced with sodium azide, for example, by contacting the tosylate with at
least one equivalent of sodium azide in an inert diluent, such as a mixture of
N,N dimethylformamide and water, at a temperature ranging from about
0°C
to about 37°C for about 1 to about 12 hours to provide the
corresponding
azido compound. The azido group can then be reduced by, for example,
hydrogenation using a palladium on carbon catalyst to provide the amino (-
NH2) compound.
Similarly, a tosylate group can be readily displaced by a thiol to form
a sulfide. This reaction is typically conducted by contacting the tosylate
with at least one equivalent of a thiol, such as thiophenol, in the presence
of
a suitable base, such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), in an
inert diluent, such as N,N dimethylformamide, at a temperature of from
about 0°C to about 37°C for about 1 to about 12 hours to provide
the
sulfide. Additionally, treatment of a tosylate with morpholinosulfur
trifluoride in an inert diluent, such as dichloromethane, at a temperature
ranging from about 0°C to about 37°C for about 12 to about 24
hours
affords the corresponding fluoro compound.
Furthermore, a compound of formula I or an intermediate thereof
having a substituent containing an iodoaryl group, for example, when RS is a
(4-iodophenyl)methyl group, can be readily converted either before or after
the above coupling reactions into a biaryl compound. Typically, this
reaction is conducted by treating the iodoaryl compound with about 1.1 to
about 2 equivalents of an arylzinc iodide, such as 2-
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(methoxycarbonyl)phenylzinc iodide, in the presence of a palladium catalyst,
such as palladium tetra(triphenylphosphine), in an inert diluent, such as
tetrahydrofuran, at a temperature ranging from about 24 ° C to about 30
° C
until reaction completion. This reaction is further described, for example, in
Rieke, J. Org. Chem. 1991, 56, 1445.
In some cases, the compounds of formula I or intermediates thereof
may contain substituents having one or more sulfur atoms. Such sulfur
atoms will be present, for example, when the amino acid of formula II
employed in the above reactions is derived from L-thiazolidine-4-carboxylic
acid, L-(5,5-dimethyl)thiazolidine-4-carboxylic acid, L-thiamorpholine-3-
carboxylic acid and the like. When present, such sulfur atoms can be
oxidized either before or after the above coupling reactions to provide a
sulfoxide or sulfone compound using conventional reagents and reaction
conditions. Suitable reagents for oxidizing a sulfide compound to a
sulfoxide include, by way of example, hydrogen peroxide, 3-
chloroperoxybenzoic acid (MCPBA), sodium periodate and the like. The
oxidation reaction is typically conducted by contacting the sulfide compound
with about 0.95 to about 1.1 equivalents of the oxidizing reagent in an inert
diluent, such as dichloromethane, at a temperature ranging from about -
50°C to about 75°C for about 1 to about 24 hours. The resulting
sulfoxide
can then be further oxidized to the corresponding sulfone by contacting the
sulfoxide with at least one additional equivalent of an oxidizing reagent,
such as hydrogen peroxide, MCPBA, potassium permanganate and the like.
Alternatively, the sulfone can be prepared directly by contacting the sulfide
with at least two equivalents, and preferably an excess, of the oxidizing
reagent. Such reactions are described further in March, "Advanced Organic
Chemistry", 4th Ed., pp. 1201-1202, Wiley Publisher, 1992.
As described above, the compounds of formula I having an RZ
substituent other an hydrogen can be prepared using an N substituted amino
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acid of formula II, such as sarcosine, N methyl-L-phenylalanine and the like,
in the above-described coupling reactions. Alternatively, such compounds
can be prepared by N alkylation of a sulfonamide of formula I or IV (where
RZ is hydrogen) using conventional synthetic procedures. Typically, this N
alkylation reaction is conducted by contacting the sulfonamide with at least
one equivalent, preferably 1.1 to 2 equivalents, of an alkyl or substituted
alkyl halide in the presence of a suitable base, such as potassium carbonate,
in an inert diluent, such as acetone, 2-butanone and the like, at a
temperature
ranging from about 25°C to about 70°C for about 2 to about 48
hours.
Examples of alkyl or substituted alkyl halides suitable for use in this
reaction
include, but are not limited to, methyl iodide, and the like.
Additionally, the sulfonamides of formula I or IV wherein R2 is
hydrogen and R' is a 2-alkoxycarbonylaryl group can be intramolecularly
cyclized to form 1,2-benzisothiazol-3-one derivatives or analogues thereof.
This reaction is typically conducted by treating a sulfonamide, such as N (2-
methoxycarbonylphenylsulfonyl)glycine-L-phenylalanine benzyl ester, with
about 1.0 to 1.5 equivalents of a suitable base, such as an alkali metal
hydride, in a inert diluent, such as tetrahydrofuran, at a temperature ranging
from about 0°C to about 30°C for about 2 to about 48 hours to
afford the
cyclized 1,2-benzisothiazol-3-one derivative.
Lastly, the compounds of formula I where Q contains a thiocarbonyl
group, (C = S) can be prepared by using an amino thionoacid derivative in
place of amino acid II in the above described synthetic procedures. Such
amino thionoacid derivatives can be prepared by the procedures described in
Shalaky, et al. , J. Org. Chem. , 61:9045-9048 ( 1996) and Brain, et al . , J.
Org. Chem., 62:3808-3809 (1997) and references cited therein.
Pharmaceutical Formulations
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When employed as pharmaceuticals, the compounds of formula I and
IA are usually administered in the form of pharmaceutical compositions.
These compounds can be administered by a variety of routes including oral,
rectal, transdermal, subcutaneous, intravenous, intramuscular, and
intranasal. These compounds are effective as both injectable and oral
compositions. Such compositions are prepared in a manner well known in
the pharmaceutical art and comprise at least one active compound.
This invention also includes pharmaceutical compositions which
contain, as the active ingredient, one or more of the compounds of formula I
and IA above associated with pharmaceutically acceptable carriers. In
making the compositions of this invention, the active ingredient is usually
mixed with an excipient, diluted by an excipient or enclosed within such a
carrier which can be in the form of a capsule, sachet, paper or other
container. When the excipient serves as a diluent, it can be a solid, semi-
solid, or liquid material, which acts as a vehicle, carrier or medium for the
active ingredient. Thus, the compositions can be in the form of tablets,
pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,
solutions, syrups, aerosols (as a solid or in a liquid medium), ointments
containing, for example, up to 10 % by weight of the active compound, soft
and hard gelatin capsules, suppositories, sterile injectable solutions, and
sterile packaged powders.
In preparing a formulation, it may be necessary to mill the active
compound to provide the appropriate particle size prior to combining with
the other ingredients. If the active compound is substantially insoluble, it
ordinarily is milled to a particle size of less than 200 mesh. If the active
compound is substantially water soluble, the particle size is normally
adjusted by milling to provide a substantially uniform distribution in the
formulation, e.g. about 40 mesh.
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Some examples of suitable excipients include lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents such as talc,
magnesium stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and propylhydroxy-
benzoates; sweetening agents; and flavoring agents. The compositions of the
invention can be formulated so as to provide quick, sustained or delayed
release of the active ingredient after administration to the patient by
employing procedures known in the art.
The compositions are preferably formulated in a unit dosage form,
each dosage containing from about 5 to about 100 mg, more usually about
10 to about 30 mg, of the active ingredient. The term "unit dosage forms"
refers to physically discrete units suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined quantity
of active material calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
The active compound is effective over a wide dosage range and is
generally administered in a pharmaceutically effective amount. It, will be
understood, however, that the amount of the compound actually
administered will be determined by a physician, in the light of the relevant
circumstances, including the condition to be treated, the chosen route of
administration, the actual compound administered, the age, weight, and
response of the individual patient, the severity of the patient's symptoms,
and the like.
For preparing solid compositions such as tablets, the principal active
ingredient is mixed with a pharmaceutical excipient to form a solid
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preformulation composition containing a homogeneous mixture of a
compound of the present invention. When referring to these preformulation
compositions as homogeneous, it is meant that the active ingredient is
dispersed evenly throughout the composition so that the composition may be
readily subdivided into equally effective unit dosage forms such as tablets,
pills and capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for example, 0.1
to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the present invention may be coated or
otherwise compounded to provide a dosage form affording the advantage of
prolonged action. For example, the tablet or pill can comprise an inner
dosage and an outer dosage component, the latter being in the form of an
envelope over the former. The two components can be separated by an
enteric layer which serves to resist disintegration in the stomach and permit
the inner component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and mixtures
of polymeric acids with such materials as shellac, cetyl alcohol, and
cellulose acetate.
The liquid forms in which the novel compositions of the present
invention may be incorporated for administration orally or by injection
include aqueous solutions suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as cottonseed oil,
sesame oil, coconut oil, or peanut oil, as well as elixirs and similar
pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic solvents, or
mixtures thereof, and powders. The liquid or solid compositions may
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contain suitable pharmaceutically acceptable excipients as described supra.
Preferably the compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
pharmaceutically acceptable solvents may be nebulized by use of inert gases.
Nebulized solutions may be breathed directly from the nebulizing device or
the nebulizing device may be attached to a face masks tent, or intermittent
positive pressure breathing machine. Solution, suspension, or powder
compositions may be administered, preferably orally or nasally, from
devices which deliver the formulation in an appropriate manner.
The following formulation examples illustrate the pharmaceutical
compositions of the present invention.
Formulation Exam In a 1
Hard gelatin capsules containing the following ingredients are
prepared:
Quantity
I r ie t (mg/ca sn ule)
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin capsules
in 340 mg quantities.
Formulation Example 2
A tablet formula is prepared using the ingredients below:
Quantity
n r di n ~m /tg ablet~
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
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The components are blended and compressed to form tablets, each
weighing 240 mg.
Formulation Example 3
A dry powder inhaler formulation is prepared containing the
following components:
Ingredient W i h
Active Ingredient 5
Lactose 95
The active mixture is mixed with the lactose and the mixture is added
to a dry powder inhaling appliance.
Formulation Exam In a 4
Tablets, each containing 30 mg of active
ingredient, are prepared as
follows:
Quantity
In reg diem (m /tablet)
Active Ingredient 30.0 mg
Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone
(as 10 % solution in water) 4.0 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1. m
Total 120 mg
The active ingredient, starch and cellulose are passed through a No.
20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinyl-
pyrrolidone is mixed with the resultant powders, which are then passed
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through a 16 mesh U.S. sieve. The granules so produced are dried at 50°
to
60°C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl
starch, magnesium stearate, and talc, previously passed through a No. 30
mesh U.S. sieve, are then added to the granules which, after mixing, are
compressed on a tablet machine to yield tablets each weighing 150 mg.
Formulation Example 5
Capsules, each containing 40 mg of medicament are made as follows:
Quantity
In reg diem ~mg/ca~sule~
Active Ingredient 40.0 mg
Starch 109.0 mg
Magnesium stearate 1.0 m~
Total 150.0 mg
The active ingredient, cellulose, starch, an magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled into hard
gelatin capsules in 150 mg quantities.
Formulation Example 6
Suppositories, each containing 25 mg of active ingredient are made as
follows:
In redient unt
Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
The active ingredient is passed through a No. 60 mesh U.S. sieve and
suspended in the saturated fatty acid glycerides previously melted using the
minimum heat necessary. The mixture is then poured into a suppository
mold of nominal 2.0 g capacity and allowed to cool.
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Formulation Example 7
Suspensions, each containing 50 mg of medicament per 5.0 ml dose are
made as follows:
In reg diem unt
Active Ingredient 50.0 mg
Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (11 %
)
Microcrystalline cellulose (89 % ) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v.
Purified water to 5.0 ml
The medicament, sucrose and xanthan gum are blended, passed through
a No. 10 mesh U.S. sieve, and then mixed with a previously made solution
of the microcrystalline cellulose and sodium carboxymethyl cellulose in
water. The sodium benzoate, flavor, and color are diluted with some of the
water and added with stirring. Sufficient water is then added to produce the
required volume.
Formulation Example 8
Quantity
n red' nt lmg/capsule)
Active Ingredient 15.0 mg
Starch 407.0 mg
Magnesium stearate 3.0 mg
Total 425.0 mg
The active ingredient, cellulose, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled into hard
gelatin capsules in 560 mg quantities.
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Formulation Example 9
An intravenous formulation may be prepared as follows:
In reg diem ti
Active Ingredient 250.0 mg
Isotonic saline 1000 ml
Formulation Example 10
A topical formulation may be prepared as follows:
Ingredient uanti
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 g
The white soft paraffin is heated until molten. The liquid paraffin and
emulsifying wax are incorporated and stirred until dissolved. The active
ingredient is added and stirring is continued until dispersed. The mixture is
then cooled until solid.
Another preferred formulation employed in the methods of the present
invention employs transdermal delivery devices ("patches"). Such
transdermal patches may be used to provide continuous or discontinuous
infusion of the compounds of the present invention in controlled amounts.
The construction and use of transdermal patches for the delivery of
pharmaceutical agents is well known in the art. See, e. g. , U. S. Patent
5,023,252, issued June 11, 1991, herein incorporated by reference. Such
patches may be constructed for continuous, pulsatile, or on demand delivery
of pharmaceutical agents.
Direct or indirect placement techniques may be used when it is desirable
or necessary to introduce the pharmaceutical composition to the brain.
Direct techniques usually involve placement of a drug delivery catheter into
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the host's ventricular system to bypass the blood-brain barrier. One such
implantable delivery system used for the transport of biological factors to
specific anatomical regions of the body is described in U.S. Patent
5,011,472 which is herein incorporated by reference.
Indirect techniques, which are generally preferred, usually involve
formulating the compositions to provide for drug latentiation by the
conversion of hydrophilic drugs into lipid-soluble drugs. Latentiation is
generally achieved through blocking of the hydroxy, carbonyl, sulfate, and
primary amine groups present on the drug to render the drug more lipid
soluble and amenable to transportation across the blood-brain barrier.
Alternatively, the delivery of hydrophilic drugs may be enhanced by
intra-arterial infusion of hypertonic solutions which can transiently open the
blood-brain barrier.
Utility
The compounds of this invention can be employed to bind VLA-4 (a4~31
integrin) in biological samples and, accordingly have utility in, for example,
assaying such samples for VLA-4. In such assays, the compounds can be
bound to a solid support and the VLA-4 sample added thereto. The amount
of VLA-4 in the sample can be determined by conventional methods such as
use of a sandwich ELISA assay. Alternatively, labeled VLA-4 can be used
in a competitive assay to measure for the presence of VLA-4 in the sample.
Other suitable assays are well known in the art.
In addition, certain of the compounds of this invention inhibit, in vivo,
adhesion of leukocytes to endothelial cells mediated by VLA-4 and,
accordingly, can be used in the treatment of diseases mediated by VLA-4.
Such diseases include inflammatory diseases, in mammalian patients
such as asthma, Alzheimer's disease, atherosclerosis, AIDS dementia,
diabetes (including acute juvenile onset diabetes), inflammatory bowel
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disease (including ulcerative colitis and Crohn's disease), multiple
sclerosis,
rheumatoid arthritis, tissue transplantation, tumor metastasis, meningitis,
encephalitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic
dermatitis, psoriasis, myocardial ischemia and acute leukocyte-mediated lung
injury such as that which occurs in adult respiratory distress syndrome.
The biological activity of the compounds identified above may be
assayed in a variety of systems. For example, a compound can be
immobilized on a solid surface and adhesion of cells expressing VLA-4. can
be measured. Using such formats, large numbers of compounds can be
screened. Cells suitable for this assay include any leukocytes. known to
express VLA-4 such as T cells, B cells, monocytes, eosinophils, and
basophils. A number of leukocyte cell lines can also be used, examples
include Jurkat and U937.
The test compounds can also be tested for the ability to competitively
inhibit binding between VLA-4 and VCAM-1, or between VLA-4 and a
labeled compound known to bind VLA-4 such as a compound of this
invention or antibodies to VLA-4. In these assays, the VCAM-1 can be
immobilized on a solid surface. VCAM-1 may also be expressed as a
recombinant fusion protein having an Ig tail (e.g., IgG) so that binding to
VLA-4 may be detected in an immunoassay. Alternatively, VCAM-1
expressing cells, such as activated endothelial cells or VCAM-1 transfected
fibroblasts can be used. For assays to measure the ability to block adhesion
to brain endothelial cells, the assays described in International Patent
Application Publication No. WO 91/05038 are particularly preferred. This
application is incorporated herein by reference in its entirety.
Many assay formats employ labelled assay components. The labelling
systems can be in a variety of forms. The label may be coupled directly or
indirectly to the desired component of the assay according to methods well
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known in the art. A wide variety of labels may be used. The component
may be labelled by any one of several methods. The most common method
of detection is the use of autoradiography with 3H, 'ZSI, 3sS, laC, or 32p
labelled compounds or the like. Non-radioactive labels include ligands
which bind to labelled antibodies, fluorophores, chemiluminescent agents,
enzymes and antibodies which can serve as specific binding pair members
for a labelled ligand. The choice of label depends on sensitivity required,
ease of conjugation with the compound, stability requirements, and available
instrumentation.
Appropriate in vivo models for demonstrating efficacy in treating
inflammatory responses include EAE (experimental autoimmune
encephalomyelitis) in mice, rats, guinea pigs or primates, as well as other
inflammatory models dependent upon a4 integrins.
Compounds having the desired biological activity may be modified as
necessary to provide desired properties such as improved pharmacological
properties (e.g., in vivo stability, bio-availability), or the ability to be
detected in diagnostic applications. For instance, inclusion of one or more
D-amino acids in the sulfonamides of this invention typically increases in
vivo stability. Stability can be assayed in a variety of ways such as by
measuring the half life of the proteins during incubation with peptidases or
human plasma or serum. A number of such protein stability assays have
been described (see, e.g., Verhoef et al., Eur. J. Drug Metab.
Pharmacokinet., 1990, 15(21:83-93).
For diagnostic purposes, a wide variety of labels may be linked to the
compounds, which may provide, directly or indirectly, a detectable signal.
Thus, the compounds of the subject invention may be modified in a variety
of ways for a variety of end purposes while still retaining biological
activity.
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In addition, various reactive sites may be introduced at the terminus for
linking to particles, solid substrates, macromolecules, or the like.
Labeled compounds can be used in a variety of in vivo or in vitro
applications. A wide variety of labels may be employed, such as
radionuclides (e.g., gamma-emitting radioisotopes such as technetium-99 or
indium-111), fluorescers (e.g., fluorescein), enzymes, enzyme substrates,
enzyme cofactors, enzyme inhibitors, chemiluminescent compounds,
bioluminescent compounds, and the like. Those of ordinary skill in the art
will know of other suitable labels for binding to the complexes, or will be
able to ascertain such using routine experimentation. The binding of these
labels is achieved using standard techniques common to those of ordinary
skill in the art.
In vitro uses include diagnostic applications such as monitoring
inflammatory responses by detecting the presence of leukocytes expressing
VLA-4. The compounds of this invention can also be used for isolating or
labeling such cells. In addition, as mentioned above, the compounds of the
invention can be used to assay for potential inhibitors of VLA-4./VCAM-1
interactions.
For in vivo diagnostic imaging to identify, e.g., sites of inflammation,
radioisotopes are typically used in accordance with well known techniques.
The radioisotopes may be bound to the peptide either directly or indirectly
using intermediate functional groups. For instance, chelating agents such as
diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic
acid (EDTA) and similar molecules have been used to bind proteins to
metallic ion radioisotopes.
The complexes can also be labeled with a paramagnetic isotope for
purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or
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electron spin resonance (ESR), both of which are well known. In general,
any conventional method for visualizing diagnostic imaging can be used.
Usually gamma- and positron-emitting radioisotopes are used for camera
imaging and paramagnetic isotopes are used for MRI. Thus, the compounds
can be used to monitor the course of amelioration of an inflammatory
response in an individual. By measuring the increase or decrease in
lymphocytes expressing VLA-4 it is possible to determine whether a
particular therapeutic regimen aimed at ameliorating the disease is effective.
The pharmaceutical compositions of the present invention can be used to
block or inhibit cellular adhesion associated with a number of diseases and
disorders. For instance, a number of inflammatory disorders are associated
with integrins or leukocytes. Treatable disorders include, e.g.,
transplantation rejection (e.g., allograft rejection), Alzheimer's disease,
atherosclerosis, AIDS dementia, diabetes (including acute juvenile onset
diabetes), retinitis, cancer metastases, rheumatoid arthritis, acute
leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome),
asthma, nephritis, and acute and chronic inflammation, including atopic
dermatitis, psoriasis, myocardial ischemia, and inflammatory bowel disease
(including Crohn's disease and ulcerative colitis). In preferred embodiments
the pharmaceutical compositions are used to treat inflammatory brain
disorders, such as multiple sclerosis (MS), viral meningitis and encephalitis.
Inflammatory bowel disease is a collective term for two similar diseases
referred to as Crohn's disease and ulcerative colitis. Crohn's disease is an
idiopathic, chronic ulceroconstrictive inflammatory disease characterized by
sharply delimited and typically transmural involvement of all layers of the
bowel wall by a granulomatous inflammatory reaction. Any segment of the
gastrointestinal tract, from the mouth to the anus, may be involved, although
the disease most commonly affects the terminal ileum and/or colon.
Ulcerative colitis is an inflammatory response limited largely to the colonic
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mucosa and submucosa. Lymphocytes and macrophages are numerous in
lesions of inflammatory bowel disease and may contribute to inflammatory
injury.
Asthma is a disease characterized by increased responsiveness of the
tracheobronchial tree to various stimuli potentiating paroxysmal constriction
of the bronchial airways. The stimuli cause release of various mediators of
inflammation from IgE-coated mast cells including histamine, eosinophilic
and neutrophilic chemotactic factors, leukotrines, prostaglandin and platelet
activating factor. Release of these factors recruits basophils, eosinophils
and
neutrophils, which cause inflammatory injury.
Atherosclerosis is a disease of arteries (e.g., coronary, carotid, aorta
and iliac). The basic lesion, the atheroma, consists of a raised focal plaque
within the intima, having a core of lipid and a covering fibrous cap.
Atheromas compromise arterial blood flow and weaken affected arteries.
Myocardial and cerebral infarcts are a major consequence of this disease.
Macrophages and leukocytes are recruited to atheromas and contribute to
inflammatory injury.
Rheumatoid arthritis is a chronic, relapsing inflammatory disease that
primarily causes impairment and destruction of joints. Rheumatoid arthritis
usually first affects the small joints of the hands and feet but then may
involve the wrists, elbows, ankles and knees. The arthritis results from
interaction of synovial cells with leukocytes that infiltrate from the
circulation into the synovial lining of the joints. See e.g., Paul, Immunology
(3d ed., Raven Press, 1993).
Another indication for the compounds of this invention is in treatment of
organ or graft rejection mediated by VLA-4. Over recent years there has
been a considerable improvement in the efficiency of surgical techniques for
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transplanting tissues and organs such as skin, kidney, liver, heart, lung,
pancreas and bone marrow. Perhaps the principal outstanding problem is
the lack of satisfactory agents for inducing immunotolerance in the recipient
to the transplanted allograft or organ. When allogeneic cells or organs are
transplanted into a host (i.e., the donor and donee are different individuals
from the same species), the host immune system is likely to mount an
immune response to foreign antigens in the transplant (host-versus-graft
disease) leading to destruction of the transplanted tissue. CD8+ cells, CD4
cells and monocytes are all involved in the rejection of transplant tissues.
Compounds of this invention which bind to alpha-4 integrin are useful, inter
alia, to block alloantigen-induced immune responses in the donee thereby
preventing such cells from participating in the destruction of the
transplanted
tissue or organ. See, e. g., Paul et al. , Transplant International 9, 420-425
(1996); Georczynski et al., Immunology 87, 573-580 (1996); Georcyznski
et al., Transplant. Immunol. 3, 55-61 (1995); Yang et al., Transplantation
60, 71-76 (1995); Anderson et al., APMIS 102, 23-27 (1994).
A related use for compounds of this invention which bind to VLA-4 is
in modulating the immune response involved in "graft versus host" disease
(GVHD). See e.g., Schlegel et al., J. Immunol. 155, 3856-3865 (1995).
GVHD is a potentially fatal disease that occurs when immunologically
competent cells are transferred to an allogeneic recipient. In this situation,
the donor's immunocompetent cells may attack tissues in the recipient.
Tissues of the skin, gut epithelia and liver are frequent targets and may be
destroyed during the course of GVHD. The disease presents an especially
severe problem when immune tissue is being transplanted, such as in bone
marrow transplantation; but less severe GVHD has also been reported in
other cases as well, including heart and liver transplants. The therapeutic
agents of the present invention are used, inter alia, to block activation of
the
donor T-cells thereby interfering with their ability to lyse target cells in
the
host.
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A further use of the compounds of this invention is inhibiting tumor
metastasis. Several tumor cells have been reported to express VLA-4 and
compounds which bind VLA-4 block adhesion of such cells to endothelial
cells. Steinback et al., Urol. Res. 23, 175-83 (1995); Orosz et al., Int. J.
Cancer 60, 867-71 (1995); Freedman et al., Leuk. Lymphoma 13, 47-52
(1994); Okahara et al., Cancer Res. 54, 3233-6 (1994).
A further use of the compounds of this invention is in treating multiple
sclerosis. Multiple sclerosis is a progressive neurological autoimmune
disease that affects an estimated 250,000 to 350,000 people in the United
States. Multiple sclerosis is thought to be the result of a specific
autoimmune reaction in which certain leukocytes attack and initiate the
destruction of myelin, the insulating sheath covering nerve fibers. In an
animal model for multiple sclerosis, murine monoclonal antibodies directed
against VLA-4. have been shown to block the adhesion of leukocytes to the
endothelium, and thus prevent inflammation of the central nervous system
and subsequent paralysis in the animals'6.
Pharmaceutical compositions of the invention are suitable for use in a
variety of drug delivery systems. Suitable formulations for use in the present
invention are found in Remington's Pharmaceutical Sciences, Mace
Publishing Company, Philadelphia, PA, 17th ed. (1985).
In order to enhance serum half life, the compounds may be
encapsulated, introduced into the lumen of liposomes, prepared as a colloid,
or other conventional techniques may be employed which provide an
extended serum half life of the compounds. A variety of methods are
available for preparing liposomes, as described in, e.g., Szoka, et al., U.S.
Patent Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is
incorporated herein by reference.
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The amount administered to the patient will vary depending upon what
is being administered, the purpose of the administration, such as prophylaxis
or therapy, the state of the patient, the manner of administration, and the
like. In therapeutic applications, compositions are administered to a patient
already suffering from a disease in an amount sufficient to cure or at least
partially arrest the symptoms of the disease and its complications. An
amount adequate to accomplish this is defined as "therapeutically effective
dose." Amounts effective for this use will depend on the disease condition
being treated as well as by the judgment of the attending clinician depending
upon factors such as the severity of the inflammation, the age, weight and
general condition of the patient, and the like.
The compositions administered to a patient are in the form of
pharmaceutical compositions described above. These compositions may be
sterilized by conventional sterilization techniques, or may be sterile
filtered.
The resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a sterile
aqueous carrier prior to administration. The pH of the compound
preparations typically will be between 3 and 11, more preferably from 5 to 9
and most preferably from 7 to 8. It will be understood that use of certain of
the foregoing excipients, carriers, or stabilizers will result in the
formation
of pharmaceutical salts.
The therapeutic dosage of the compounds of the present invention will
vary according to, for example, the particular use for which the treatment is
made, the manner of administration of the compound, the health and
condition of the patient, and the judgment of the prescribing physician. For
example, for intravenous administration, the dose will typically be in the
range of about 20 ,ug to about 500 ~cg per kilogram body weight, preferably
about 100 ,ug to about 300 ,ug per kilogram body weight. Suitable dosage
ranges for intranasal administration are generally about 0.1 pg to 1 mg per
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kilogram body weight. Effective doses can be extrapolated from
dose-response curves derived from in vitro or animal model test systems.
The following synthetic and biological examples are offered to illustrate
this invention and are not to be construed in any way as limiting the scope of
this invention. Unless otherwise stated, all temperatures are in degrees
Celsius.
EXAMPLES
In the examples
below, the following
abbreviations
have the following
meanings. If an viation is not defined, it has its
abbre generally accepted
meaning.
aq or aq. - aqueous
AcOH - acetic acid
bd - broad doublet
bm - broad multiplet
bs - broad singlet
Bn - benzyl
Boc - N tent-butoxylcarbonyl
Boc20 - di-tent-butyl dicarbonate
BOP - benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium
hexafluorophosphate
Cbz - carbobenzyloxy
CHC13 - chloroform
CHzCl2 - dichloromethane
(COCI)Z - oxalyl chloride
d - doublet
dd - doublet of doublets
dt - doublet of triplets
DBU - 1,8-diazabicyclo[5.4.0]undec-7-ene
DCC - 1,3-dicyclohexylcarbodiimide
DMAP - 4-N,N dimethylaminopyridine
DME - ethylene glycol dimethyl ether
DMF - N,N dimethylformamide
DMSO - dimethylsulfoxide
EDC - 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride
Et3N - triethylamine
Et20 - diethyl ether
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EtOAc - ethyl acetate
EtOH - ethanol
eq or eq. - equivalent
Fmoc - N (9-fluorenylmethoxycarbonyl)
FmocONSu - N (9-fluorenylmethoxycarbonyl)-
succinimide
g - grams
h - hour
H20 - water
HBr - hydrobromic acid
HCl - hydrochloric acid
HOBT - 1-hydroxybenzotriazole hydrate
hr - hour
KZCO3 - potassium carbonate
L - liter
m - multiplet
MeOH - methanol
mg - milligram
MgS04 - magnesium sulfate
mL - milliliter
mm - millimeter
mM - millimolar
mmol - millimol
mp - melting point
N - normal
NaCI - sodium chloride
Na2C03 - sodium carbonate
NaHC03 - sodium bicarbonate
NaOEt - sodium ethoxide
NaOH - sodium hydroxide
NH4C1 - ammonium chloride
NMM - N methylmorpholine
Phe - L-phenylalanine
Pro - L-proline
psi - pounds per square inch
PtOz - platinum oxide
q - quartet
quint. - quintet
rt - room temperature
s - singlet
sat - saturated
t - triplet
t-BuOH - tert-butanol
TFA - trifluoroacetic acid
THF - tetrahydrofuran
TLC or tlc - thin layer chromatography
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Ts - tosyl
TsCI - tosyl chloride
TsOH - tosylate
~L - microliter
In the examples below, all temperatures are in degrees Celcius (unless
otherwise indicated). The following Methods were used to prepare the
compounds set forth below as indicated.
Method 1
N Tosvlation Procedure
N Tosylation of the appropriate amino acid was conducted via the
method of Cupps, Boutin and Rapoport J. Org. Chem. 1985, S0, 3972.
Method 2
Methyl Ester Preparation Procedure
Amino acid methyl esters were prepared using the method of Brenner
and Huber Helv. Chim. Acta 1953, 36, 1109.
Method 3
BOP Coupling Procedure
The desired dipeptide ester was prepared by the reaction of a suitable N-
protected amino acid (1 equivalent) with the appropriate amino acid ester or
amino acid ester hydrochloride (1 equivalent), benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate [BOP] (2.0
equivalent), triethylamine (l.l equivalent), and DMF. The reaction mixture
was stirred at room temperature overnight. The crude product is purified
flash chromatography to afford the dipeptide ester.
Method 4
Hydrogenation Procedure I
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Hydrogenation was performed using 10% palladium on carbon (10% by
weight) in methanol at 30 psi overnight. The mixture was filtered through a
pad of Celite and the filtrate concentrated to yield the desired amino
compound.
Method S
H,~ysis Procedure I
To a chilled (0 ° C) THF/H20 solution (2:1, 5 - 10 mL) of the
appropriate ester was added LiOH (or NaOH) (0.95 equivalents). The
temperature was maintained at 0°C and the reaction was complete in 1-3
hours. The reaction mixture was extracted with ethyl acetate and the
aqueous phase was lyophilized resulting in the desired carboxylate salt.
Method 6
Ester Hydrolysis Procedure II
To a chilled (0°C) THF/H20 solution (2:1, 5 - 10 mL) of the
appropriate ester was added LiOH (1.1 equivalents). The temperature was
maintained at 0°C and the reaction was complete in 1-3 hours. The
reaction
mixture was concentrated and the residue was taken up into H20 and the pH
adjusted to 2-3 with aqueous HCI. The product was extracted with ethyl
acetate and the combined organic phase was washed with brine, dried over
MgS04, filtered and concentrated to yield the desired acid.
Method 7
Ester Hydrolysis Procedure III
The appropriate ester was dissolved in dioxane/HZO (1:1) and 0.9
equivalents of 0.5 N NaOH was added. The reaction was stirred for 3-16
hours and then concentrated. The resulting residue was dissolved in HZO
and extracted with ethyl acetate. The aqueous phase was lyophilized to yield
the desired carboxylate sodium salt.
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Method 8
Sulfonylation Procedure I
To the appropriately protected aminophenylalanine analog (11.2 mmol),
dissolved in methylene chloride (25m1) and cooled to -78°C was added
the
desired sulfonyl chloride (12 mmol) followed by dropwise addition of
pyridine (2 mL). The solution was allowed to warm to room temperature
and was stirred for 48 hr. The reaction solution was transferred to a 250
mL separatory funnel with methylene chloride (100 mL) and extracted with
1N HCl (50 mL x 3), brine (50 mL), and water (100 mL). The organic
phase was dried (MgS04) and the solvent concentrated to yield the desired
product.
Method 9
Reductive Amination Procedure
Reductive amination of Tos-Pro-p-NHz-Phe with the appropriate
aldehyde was conducted using acetic acid, sodium triacetoxyborohydride,
methylene chloride and the combined mixture was stirred at room
temperature overnight. The crude product was purified by flash
chromatography.
Method 10
BOC Removal Procedure
Anhydrous hydrochloride (HCl) gas was bubbled through a methanolic
solution of the appropriate Boc-amino acid ester at 0°C for 15 minutes
and
the reaction mixture was stirred for three hours. The solution was
concentrated to a syrup and dissolved in Et,O and reconcentrated. This
procedure was repeated and the resulting solid was placed under high
vacuum overnight.
Method 11
Tert-Butvl Ester H,~lvsis Procedure I
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The tent-butyl ester was dissolved in CHZC12 and treated with TFA. The
reaction was complete in 1-3 hr at which time the reaction mixture was
concentrated and the residue dissolved in HZO and lyophilized to yield the
desired acid.
Method 12
EDC Cou~li~ Procedure I
To a CHZC12 solution (5-20 mL,) of N-(toluene-4-sulfonyl)-L-proline (1
equivalent), the appropriate amino acid ester hydrochloride (1 equivalent),
N-methylmorpholine (1.1-2.2 equivalents) and 1-hydroxybenzotriazole (2
equivalents) were mixed, placed in an ice bath and 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide (l.l equivalents) added. The
reaction was allowed to rise to room temperature and stirred overnight. The
reaction mixture was poured into H20 and the organic phase was washed
with sat. NaHC03, brine, dried (MgS04 or Na2S04), filtered and
concentrated. The crude product was purified by column chromatography.
Method 13
EDC Coupling Procedure II
To a DMF solution (5-20 mL) of the appropriate N-protected amino
acid (1 equivalent), the appropriated amino acid ester hydrochloride (1
equivalent), Et3N (1.1 equivalents) and 1-hydroxybenzotriazole (2
equivalents) were mixed, placed in an ice bath and 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide (l.l equivalents) added. The
reaction was allowed to rise to room temperature and stirred overnight. The
reaction mixture was partitioned between EtOAc and H20 and the organic
phase washed with 0.2 N citric acid, H20, sat. NaHC03, brine, dried
(MgS04 or Na~SO~), filtered and concentrated. The crude product was
purified by column chromatography or preparative TLC.
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Method 14
Sulfo~lation Procedure II
The appropriate sulfonyl chloride was dissolved in CHzCl2 and placed in
an ice bath. L-Pro-L-Phe-OMe ~ HCl (1 equivalent) and Et3N (1.1
equivalent) was added and the reaction allowed to warm to room
temperature and stirred overnight under an atmosphere of nitrogen. The
reaction mixture was concentrated and the residue partitioned between
EtOAc and HZO and the organic phase washed with sat. NaHC03, brine,
dried (MgS04 or Na2S04), filtered and concentrated. The crude product was
purified by column chromatography or preparative TLC.
Method 15
Sulfonvlation Procedure III
To a solution of L-Pro-L-4-(3-dimethylaminopropyloxy)-Phe-OMe
[prepared using the procedure described in Method 10] (1 equivalent) in
CHZC12 was added Et3N (5 equivalents) followed by the appropriate sulfonyl
chloride (1.1 equivalent). The reaction was allowed to warm to room
temperature and stirred overnight under an atmosphere of nitrogen. The
mixture was concentrated, dissolved in EtOAc, washed with sat. NaHC03
and 0.2 N citric acid. The aqueous phase was made basic with solid
NaHC03 and the product extracted with EtOAc. The organic phase was
washed with brine, dried (MgS04 or Na2S04), filtered and concentrated.
The crude methyl ester was purified by preparative TLC. The
corresponding acid was prepared using the procedure described in Method 7.
Method 16
H,~genation Procedure II
To a methanol (10 -15 mL) solution of the azlactone, was added NaOAc
(1 equivalent) and 10%o Pd/C. This mixture was placed on the hydrogenator
at 40 psi H2. After 8 - 16 hours, the reaction mixture was filtered through a
pad of Celite and the filtrate concentrated to yield the dehydrodipeptide
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methyl ester. The ester was dissolved in dioxane/H20 (S- 10 mL), to which
was added 0.5 N NaOH (1.05 equivalents). After stirring for 1- 3 hours,
the reaction mix was concentrated and the residue was redissolved in H20
and washed with EtOAc. The aqueous phase was made acidic with 0.2 N
HCl and the product was extracted with EtOAc. The combined organic
phase was washed with brine (1 x 5 mL), dried (MgS04 or Na2S04), filtered
and concentrated to yield the acid as approximately a 1:1 mixture of
diastereomers.
Method 17
Tert-Butvl Ester H, drolysis Procedure I
The tent-butyl ester was dissolved in CHZC12 (5 mL) and treated with
TFA (5 mL). The reaction was complete in 1-3 hours at which time the
reaction mixture was concentrated and the residue dissolved in H20 and
concentrated. The residue was redissolved in H20 and lyophilized to yield
the desired product.
Example 1
Synthesis of
N [N-(Toluene-4-sulfonyl)-L-pyrrolidin-2-ylmethyl]-L-phenylalanine
N (Toluene-4-sulfonyl)-L-prolinol (358 mg, 1.41 mmol) was taken into
7 mL of dry DMSO, with Et3N (3.0 eq, 590 mL) at room temperature,
under NZ. In a separate flask, pyridine sulfur trioxide complex (3.0 eq, 660
mg) in 3 mL of DMSO, was cooled to 15°C under NZ. The prolinol
solution was syringed into the latter reaction mixture and allowed to warm
up to room temperature. The reaction, after a couple of hours, was poured
into cold brine (10 mL), then extracted with ether (2x 50 mL). The organic
layer was washed with 10% citric acid (25 mL), water, saturated NaHC03
(25 mL), and brine. Upon filtration and evaporation of the solvent under
reduced pressure, the desired prolinal was isolated as a fluffy solid. This
was then taken up in MeOH (4 mL) with L-phenylalanine benzyl ester, and
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NaCNBH3 (1.0 eq). The pH was maintained at 6.0, with 1 drop of AcOH.
The reaction mixture was stirred overnight at room temperature. EtOAc
was added as well as water and brine. The organic layer was washed
several times, and dried over MgS04. Upon filtration and evaporation of the
solvents under reduced pressure, the crude material was eluted on column
chromatography (silica gel) with EtOAc/hexanes l:l. The ester was diluted
in EtOAc/EtOH/water 1:0.5 :0.5 , with a catalytic amount of 10 % Pd on C
and hydrogenated. After filtration over Celite, the title product was isolated
as a white foam.
Physical data were as follows:
1H NMR (300 MHz, CDC13): 8 = 7.76 (m, 2H), 7.39-7.26 (m, 7H),
4.05 (m, 1H), 3.92 (m, 1H), 3.55-3.12 (m, SH), 2.82 (m, 1H), 2.38 (s,
3H), 1.55 (m, 3H), 1.25 (m, 2H).
Mass Spectroscopy: (FAB) 403 (M+H).
Example 2
Synthesis of
N-[N-(Toluene-4-sulfonyl)-L-prolinyl]-N-hydroxy-L-phenylalanine
Powdered sodium nitrite (1.27 g) was added portion-wise at 0°C to
a
solution of potassium bromide (4.896 g) and D-phenylalanine (2 g) in 2.5 N
sulfuric acid (24.2 mL). The reaction mixture was stirred 20 minutes at
0°C
followed by 1 hour at ambient temperature. The product was extracted with
ether (3 x 15 mL), the combined organic extract washed with water, brine,
dried over MgS04, filtered and evaporated to dryness in vacuo. The
product (2.4 g) was used without further purification in the next reaction.
The methyl ester was prepared using thionyl chloride and methanol. This
product (2 g) was added at once to a solution of hydroxylamine in methanol
(10 mL). The free hydroxylamine was prepared by treating hydroxylamine
HCl (1.15 g) in methanol (10 mL) with Na (380 mg). The reaction mixture
was refluxed overnight, evaporated in vacuo and the residue partitioned
between water and chloroform. The separated organic layer was dried and
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evaporated. The residue was re-partitioned between 2N HCl and ether. The
separated aqueous layer was made basic with NaHC03, extracted with
chloroform, dried over MgS04, filtered and evaporated in vacuo furnishing
400 mg of N-hydroxy-L-phenylalanine methyl ester. A solution of N-
(toluene-4-sulfonyl)-L-proline carbonyl chloride (412 mg) in THF (10 mL)
was added dropwise at 0°C to as solution of the product from the last
reaction (280 mg) in THF (20 mL) containing triethylamine (145 mg). The
mixture was stirred for one hour at 0°C, one hour at ambient
temperature,
then evaporated in vacuo and the residue partitioned between water and ethyl
acetate. The separated organic layer was dried over MgS04, filtered and
evaporated in vacuo. The residue was flash chromatographed on 30 g silica
gel. Elution with chloroform furnished 360 mg of N-[N-(toluene-4-
sulfonyl)-L-prolinyl]-N-hydroxy-L-phenylalanine methyl ester. The title
compound was prepared from the product of the last reaction (1150 mg) in
THF (80 mL) which was treated at once with 1N NaOH (2.57 mL). This
mixture was stirred at ambient temperature overnight. The crystallized
product was filtered, washed with ether and dried at 0.1 Torr to yield 750
mg of the title compound, mp 202-204°C.
Example 3
Synthesis of
N-[N-(Toluene-4-sulfonyl)-L-prolinyl]-N-hydroxy-D-phenylalanine
The title compound was prepared following the procedure described for
the preparation of Example 2, substituting L-phenylalanine for D-
phenylalanine.
Physical data were as follows:
MS: [(+)FAB], [M+H]+ 433.
Example 4
Synthesis of
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N-[2-(N-(Toluene-4-sulfonyl)-L-pyrrolidinyl)-2-hydroxyacetyl]-L-4-(N
benzyloxycarbonyl-isonipecotamido)phenylalanine
2-(Hydroxyacetyl)pyrrolidine (J. Org. Chem. (1991), 56, 1624) was N-
tosylated and the resultant product coupled to the appropriately substituted
phenylalanine utilizing standard peptide synthesis methodology. The title
compound was obtained after basic hydrolysis of the ester, mp 92-95°C.
Physical data were as follows:
MS (+)FAB [M+H]+ 707:
Example 5
Synthesis of
N-[2-(N-(Toluene-4-sulfonyl)-L-pyrrolidinyl)-2-hydroxyacetyl]-L-4
(isonipecotamido)phenylalarune
The product from Example 4 was deprotected by hydrogenation over
Pd/C in ethanol for 4 hours. The catalyst was filtered and the filtrate was
taken up in water and lyophilized to give 240 mg of a white solid.
Physical data were as follows:
MS (+)FAB [M+H]+ 573.
Calcd. for: C28H36N4O7S ~ CF3C02H ~ 3H20. C: 48.71; H: 5.73;
N: 7.57. Found: C: 48.88; H: 5.35; N: 7.45.
Example 6
Synthesis of
(2S)-2-(5-(N-(Toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-
nitrobenzyl)propionic Acid
N-(Toluene-4-sulfonyl-L-prolyl-L-(4-nitro)phenylalanine methyl ester
was mixed with an equimolar amount of PC15 in dry benzene and stirred for
90 minutes. The yellow solution was treated with a solution of hydrazoic
acid in benzene and the mixture was stirred at room temperature for 2 hours,
then it was diluted with benzene and washed with satd. NaHC03 and brine.
The organic layer was dried and evaporated to give a light yellow foam.
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The title compound was prepared from the product of the last reaction using
standard basic hydrolysis techniques, yielding an off white fluffy solid, mp
150-160°C.
Physical data were as follows:
MS (+)FAB [M+H]+ 487.
Example 7
Synthesis of
(2S)-2-[5-(N-(Toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-(N-
tert butoxycarbonyl isonipecotamido)benzyl)propionic Acid Methyl Ester
The vitro intermediate described in Example 6 was reduced, dissolved
in methylene chloride and mixed with N-Boc-isonipecotic acid, HOBT,
chilled to 5°C and treated with DCC. The reaction mixture was allowed
to
reach ambient temperature overnight, filtered, evaporated to dryness and the
residue was taken up in ethyl acetate. The organic phase was washed as
usual, dried and evaporated to provide the title compound, mp 92-102°C.
Physical data were as follows:
MS (+)FAB [M+H]+ 682.
Example 8
Synthesis of
(2S)-2-[5-(N-(Toluene-4-sulfonyl)pyrrolidin-2-yl)tetrazol-1-yl]-2-(4-(N
tent-butoxycarbonyl isonipecotamido)benzyl)propionic Acid
The title compound was prepared from the product of Example 7 using
standard basic hydrolysis techniques, mp 180-189°C.
Physical data were as follows:
MS (+)FAB [M+Li]+ 674.
Example 9
Synthesis of
N-[N-(toluene-4-sulfonyl)pyrrolidin-2-yl]aminocarbonyl]-L-phenylalanine
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N-Tosylproline (1 g, 3.71 mmol) was added to a solution of
diphenylphosphoryl azide (1.13 g, 3.71 mmol) in 50 mL toluene under
argon atmosphere. Triethylamine (394 mg, 3.9 mmol) was added, then the
mixture was heated at 80°C for 2 hrs. L-Phenylalanine benzyl ester
(1.75 g,
4.1 mmol) and triethylamine (394 mg, 3.9 mmol) was added and heated at
80°C overnight. The toluene was removed under reduced pressure and the
residue purified by flash column chromatography (silica, hexane:EtOAc,
2:1) to give the benzyl ester as a white solid (540 mg, 28 % ). The benzyl
ester was removed by hydrogenation (10% Pd/C, EtOH, 40 psi Hz). The
catalyst was removed and the product recrystallized from EtOH/Et20 to give
the title compound as a white solid (75 mg, 40 % ).
Physical data were as follows:
CZ1H~N305S ~ 0.75 Hz0 . Calcd C: 56.68; H: 6.00; N: 9.44. Found
C: 56.94; H: 5.68; N: 9.05.
Mass Spec [FAB] [M-H]- ~a M/Z 430 (100 % ).
Example A
In vitro Assay For Determining Binding of
Candidate Compounds to VLA-4
An in vitro assay was used to assess binding of candidate compounds to
a4~ii integrin. Compounds which bind in this assay can be used to assess
VCAM-1 levels in biological samples by conventional assays (e.g.,
competitive assays). This assay is sensitive to ICSO values as low as about
lnM.
The activity of a4(3, integrin was measured by the interaction of soluble
VCAM-1 with Jurkat cells (e.g., American Type Culture Collection Nos.
TIB 152, TIB 153, and CRL 8163), a human T-cell line which expresses
high levels of a4~i, integrin. VCAM-1 interacts with the cell surface in an
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a4~3, integrin-dependent fashion (Yednock, et al. J. Biol. Chem., 1995,
270:28740).
Recombinant soluble VCAM-1 was expressed as a chimeric fusion
protein containing the seven extracellular domains of VCAM-1 on the N-
terminus and the human IgGI heavy chain constant region on the C-terminus.
The VCAM-1 fusion protein was made and purified by the manner described
by Yednock, supra.
Jurkat cells were grown in RPMI 1640 supplemented with 10 % fetal
bovine serum, penicillin, streptomycin and glutamine as described by
Yednock, supra.
Jurkat cells were incubated with 1.5 mM MnCl2 and 5 ~,g/mL 15/7
antibody for 30 minutes on ice. Mn+Z activates the receptor to enhance
ligand binding, and 15/7 is a monoclonal antibody that recognizes an
activated/ligand occupied conformation of a4~i, integrin and locks the
molecule into this conformation thereby stabilizing the VCAM-1/a4~i,
integrin interaction. Yednock, et al., supra. Antibodies similar to the 15/7
antibody have been prepared by other investigators (Luque, et al, 1996, J.
Biol. Chem. 271:11067) and may be used in this assay.
Cells were then incubated for 30 minutes at room temperature with
candidate compounds, in various concentrations ranging from 66 ~.M to 0.01
~cM using a standard 5-point serial dilution. 15 ~cL soluble recombinant
VCAM-1 fusion protein was then added to Jurkat cells and incubated for 30
minutes on ice. (Yednock et al., supra.).
Cells were then washed two times and resuspended in PE-conjugated
goat F(ab')z anti-mouse IgG Fc (Immunotech, Westbrook, ME) at 1:200 and
incubated on ice, in the dark, for 30 minutes. Cells were washed twice and
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analyzed with a standard fluorescence activated cell sorter ("FACS")
analysis as described in Yednock, et al., supra.
Compounds having an ICSO of less than about lS~cM possess binding
affinity to a4~31.
When tested in this assay, each of the compounds in Examples 1-9 has
an ICSo of 15 ~cM or less.
Example B
In vitro Saturation Assay For Determining Binding of
Candidate Compounds to a4~i,
The following describes an in vitro assay to determine the plasma levels
needed for a compound to be active in the Experimental Autoimmune
Encephalomyelitis ("EAE") model, described in the next example, or in
other in vivo models.
Log-growth Jurkat cells are washed and resuspended in normal animal
plasma containing 20 ~.g/ml of the 15/7 antibody (described in the above
example).
The Jurkat cells are diluted two-fold into either normal plasma samples
containing known candidate compound amounts in various concentrations
ranging from 66 ~,M to 0.01 ~.M, using a standard 12 point serial dilution
for a standard curve, or into plasma samples obtained from the peripheral
blood of candidate compound-treated animals.
Cells are then incubated for 30 minutes at room temperature, washed
twice with phosphate-buffered saline ("PBS") containing 2 %o fetal bovine
serum and 1mM each of calcium chloride and magnesium chloride (assay
medium) to remove unbound 15/7 antibody.
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The cells are then exposed to phycoerythrin-conjugated goat Flab' )2
anti-mouse IgG Fc (Immunotech, Westbrook, ME), which has been
adsorbed for any non-specific cross-reactivity by co-incubation with 5 %
serum from the animal species being studied, at 1:200 and incubated in the
dark at 4°C for 30 minutes.
Cells are washed twice with assay medium and resuspended in the same.
They are then analyzed with a standard fluorescence activated cell sorter
("FACS") analysis as described in Yednock et al. J. Biol. Chem., 1995,
270:28740.
The data is then graphed as fluorescence versus dose, e.g., in a normal
dose-response fashion. The dose levels that result in the upper plateau of the
curve represent the levels needed to obtain efficacy in an in vivo model.
This assay may also be used to determine the plasma levels needed to
saturate the binding sites of other integrins, such as the a9~31 integrin,
which
is the integrin most closely related a4~i, (Palmer et al, 1993, J. Cell Bio.,
123:1289). Such binding is predictive of in vivo utility for inflammatory
conditions mediated by a9~31 integrin, including by way of example, airway
hyper-responsiveness and occlusion that occurs with chronic asthma, smooth
muscle cell proliferation in atherosclerosis, vascular occlusion following
angioplasty, fibrosis and glomerular scarring as a result of renal disease,
aortic stenosis, hypertrophy of synovial membranes in rheumatoid arthritis,
and inflammation and scarring that occur with the progression of ulcerative
colitis and Crohn's disease.
Accordingly, the above-described assay may be performed with a
human colon carcinoma cell line, SW 480 (ATTC #CCL228) transfected
with cDNA encoding a9 integrin (Yokosaki et al., 1994, J. Biol. Chem.,
269:26691), in place of the Jurkat cells, to measure the binding of the x9(31
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integrin. As a control, SW 480 cells which express other a and (31 subunits
may be used.
Accordingly, another aspect of this invention is directed to a method for
treating a disease in a mammalian patient, which disease is mediated by
a9(3,, and which method comprises administering to said patient a
therapeutically effective amount of a compound of this invention. Such
compounds are preferably administered in a pharmaceutical composition
described herein above. Effective daily dosing will depend upon the age,
weight, condition of the patient which factors can be readily ascertained by
the attending clinician. However, in a preferred embodiment, the
compounds are administered from about 20 to 500 ~,g/kg per day.
Example C
In vivo Evaluation
The standard multiple sclerosis model, Experimental Autoimmune (or
Allergic) Encephalomyelitis ("EAE"), was used to determine the effect of
candidate compounds to reduce motor impairment in rats or guinea pigs.
Reduction in motor impairment is based on blocking adhesion between
leukocytes and the endothelium and correlates with anti-inflammatory
activity in the candidate compound. This model has been previously
described by Keszthelyi et al., Neurology, 1996, 47:1053-1059, and
measures the delay of onset of disease.
Brains and spinal cords of adult Hartley guinea pigs were homogenized
in an equal volume of phosphate-buffered saline. An equal volume of
Freund's complete adjuvant (100 mg mycobacterium tuberculosis plus 10 ml
Freund's incomplete adjuvant) was added to the homogenate. The mixture
was emulsified by circulating it repeatedly through a 20 ml syringe with a
peristaltic pump for about 20 minutes.
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Female Lewis rats (2-3 months old, 170-220 g) or Hartley guinea pigs
(20 day old, 180-200 g) were anesthetized with isoflurane and three
injections of the emulsion, 0.1 ml each, were made in each flank. Motor
impairment onset is seen in approximately 9 days.
Candidate compound treatment began on Day 8, just before onset of
symptoms. Compounds were administered subcutaneously ("SC"), orally
("PO") or intraperitoneally ("IP"). Doses were given in a range of lOmg/kg
to 200 mg/kg, bid, for five days, with typical dosing of 10 to 100 mg/kg
SC, 10 to 50 mg/kg PO, and 10 to 100 mg/kg IP.
Antibody GGS/3 against a4~i1 integrin (Keszthelyi et al., Neurology,
1996, 47:1053-1059), which delays the onset of symptoms, was used as a
positive control and was injected subcutaneously at 3 mg/kg on Day 8 and
11.
Body weight and motor impairment were measured daily. Motor
impairment was rated with the following clinical score:
0 no change
1 tail weakness or paralysis
2 hindlimb weakness
3 hindlimb paralysis
4 moribund or dead
A candidate compound was considered active if it delayed the onset of
symptoms, e.g., produced clinical scores no greater than 2 or slowed body
weight loss as compared to the control.
Example D
Asthma Model
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Inflammatory conditions mediated by a4~31 integrin include, for example,
airway hyper-responsiveness and occlusion that occurs with chronic asthma.
The following describes an asthma model which can be used to study the in
vivo effects of the compounds of this invention for use in treating asthma.
Following the procedures described by Abraham et al, J. Clin. Invest,
93:776-787 (1994) and Abraham et al, Am J. Respir Crit Care Med,
156:696-703 (1997), both of which are incorporated by reference in their
entirety, compounds of this invention are formulated into an aerosol and
administered to sheep which are hypersensitive to Ascaris suum antigen.
Compounds which decrease the early antigen-induced bronchial response
and/or block the late-phase airway response, e.g., have a protective effect
against antigen-induced late responses and airway hyper-responsiveness
("AHR"), are considered to be active in this model.
Allergic sheep which are shown to develop both early and late bronchial
responses to inhaled Ascaris suum antigen are used to study the airway
effects of the candidate compounds. Following topical anesthesia of the
nasal passages with 2 % lidocaine, a balloon catheter is advanced through
one nostril into the lower esophagus. The animals are then intubated with a
cuffed endotracheal tube through the other nostril with a flexible fiberoptic
bronchoscope as a guide.
Pleural pressure is estimated according to Abraham (1994). Aerosols
(see formulation below) are generated using a disposable medical nebulizer
that provides an aerosol with a mass median aerodynamic diameter of 3.2
~,m as determined with an Andersen cascade impactor. The nebulizer is
connected to a dosimeter system consisting of a solenoid valve and a source
of compressed air (20 psi). The output of the nebulizer is directed into a
plastic T-piece, one end of which is connected to the inspiratory port of a
piston respirator. The solenoid valve is activated for 1 second at the
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beginning of the inspiratory cycle of the respirator. Aerosols are delivered
at VT of 500 ml and a rate of 20 breaths/minute. A 0.5 % sodium
bicarbonate solution only is used as a control.
To assess bronchial responsiveness, cumulative concentration-response
curves to carbachol can be generated according to Abraham (1994).
Bronchial biopsies can be taken prior to and following the initiation of
treatment and 24 hours after antigen challenge. Bronchial biopsies can be
preformed according to Abraham (1994).
An in vitro adhesion study of alveolar macrophages can also be
performed according to Abraham (1994), and a percentage of adherent cells
is calculated.
Aerosol Formulation
A solution of the candidate compound in 0.5 % sodium
bicarbonate/saline (w/v) at a concentration of 30.0 mg/mL is prepared using
the following procedure:
A Preparation of 0 5 % Sodium Bicarbonate / Saline Stock Solutiom
1 m
Ingredient Gram / 100.0 mL Final Concentration
Sodium Bicarbonate0.5 g 0.5 %
Saline q.s. ad 100.0 q.s. ad 100%
mL
Procedure:
1. Add O.Sg sodium bicarbonate into a 100 mL volumetric flask.
2. Add approximately 90.0 mL saline and sonicate until dissolved.
3. Q.S. to 100.0 mL with saline and mix thoroughly.
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B. Preparation of 30.0 mg/mL Candidate Compound: 10.0 mL
Ingredient Gram / 10.0 mL Final Concentration
Candidate Compound0.300 g 30.0 mg/mL
0.5% Sodium q.s. ad 10.0 mL q.s ad 100%
Bicarbonate /
Saline
Stock Solution
Procedure:
1. Add 0.300 g of the candidate compound into a 10.0 mL
volumetric flask.
2. Add approximately 9.7 mL of 0.5 % sodium bicarbonate / saline
stock solution.
3. Sonicate until the candidate compound is completely dissolved.
4. Q.S. to 10.0 mL with 0.5% sodium bicarbonate / saline stock
solution and mix thoroughly.
Using a conventional oral formulation, compounds of this invention
would be active in this model.
