U.S. patent application number 11/147601 was filed with the patent office on 2005-12-08 for anti-inflammatory compositions and methods.
Invention is credited to Pleiss, Michael A., Yednock, Theodore A..
Application Number | 20050272668 11/147601 |
Document ID | / |
Family ID | 34916174 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272668 |
Kind Code |
A1 |
Yednock, Theodore A. ; et
al. |
December 8, 2005 |
Anti-inflammatory compositions and methods
Abstract
The disclosed invention includes pharmaceutical compositions and
methods for treating inflammatory conditions, particularly those
that are characterized by increased binding of alpha-9 integrin to
one or more of its ligands. Also disclosed are methods for
selecting compounds for use in such compositions and methods.
Inventors: |
Yednock, Theodore A.;
(Forest Knolls, CA) ; Pleiss, Michael A.;
(Sunnyvale, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
1530 PAGE MILL ROAD
PALO ALTO
CA
94304
US
|
Family ID: |
34916174 |
Appl. No.: |
11/147601 |
Filed: |
June 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11147601 |
Jun 7, 2005 |
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09127364 |
Jul 31, 1998 |
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6939855 |
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60112020 |
Jul 31, 1997 |
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60054453 |
Aug 1, 1997 |
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Current U.S.
Class: |
514/19.1 ;
514/12.2; 514/21.91; 514/227.5; 514/255.01; 514/447 |
Current CPC
Class: |
A61K 31/381 20130101;
C07K 5/06026 20130101; C07K 5/06139 20130101; C07K 5/06165
20130101; A61K 38/05 20130101; A61K 31/54 20130101; C07K 5/0215
20130101 |
Class at
Publication: |
514/019 ;
514/255.01; 514/447; 514/227.5 |
International
Class: |
A61K 038/04; A61K
031/54; A61K 031/381 |
Claims
1. A pharmaceutical composition effective in treating an
inflammatory condition in mammalian subject, comprising a
pharmaceutically effective dosage of alpha-9 integrin antagonist
compound and a pharmaceutical excipient.
2. A pharmaceutical composition of claim 1, wherein said
inflammatory condition is characterized by increased neutrophil
adhesion.
3. The pharmaceutical composition of claim 1, wherein said alpha-9
antagonist compound inhibits binding between alpha-9 integrin and
an alpha-9 integrin ligand.
4. (canceled)
5. The pharmaceutical composition of claim 3, wherein said alpha-9
integrin antagonist compound is effective in inhibiting binding
between alpha-9 integrin and an alpha-9 integrin ligand as
evidenced by an IC.sub.50 for such inhibition of less than about
100 .mu.M.
6. The pharmaceutical compositon of claim 5, wherein said alpha-9
integrin antagonist compound is selected from a group of compounds
which inhibit alpha-4/beta-1 integrin binding to an alpha-4/beta-a
integrin ligand.
7. The pharmaceutical composition of claim 1, wherein said compound
is selected from the group consisting of compounds having the
formula:
R.sup.1--SO.sub.2--NR.sup.2--CHR.sup.3-Q-CHR.sup.5--CO.sub.2H
wherein R.sup.1 is selected from the group consisting of alkyl,
substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted
cycloalkyl, heterocyclic, substituted heterocyclic, heteroaryl and
substituted heteroaryl; R.sup.2 is selected from the group
consisting of hydrogen, alkyl, cycloalkyl, substituted sycloalkyl,
cycloalkenyl, substituted cylcoalkenyl, heterocyclic, substituted
heterocyclic, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, and R.sup.1 and R.sup.2
together with the nitrogen atom bound to R.sup.2 and the SO.sub.2
group bound to R.sup.1 can form a heterocyclic or a substituted
heterocyclic group; R.sup.3 is selected from the group consisting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic and, when
R.sup.2 does not form a heterocyclic group with R.sup.1, R.sup.2
and R.sup.3 together with the nitrogen atom bound to R.sup.2 and
the carbon atom bound to R.sup.3 can form a heterocyclic or a
substituted heterocyclic group; R.sup.5 is
--(CH.sub.2).sub.x--Ar--R.sup.5' where R.sup.5 is selected from the
group consisting of --O-Z-NR.sup.8R.sup.8' and --O-Z-R.sup.12
wherein R.sup.8R.sup.8 are independently selected from the group
consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, heterocyclic, substituted heterocyclic, and
where R.sup.8 and R.sup.8' are joined to form a heterocycle or a
substituted heterocycle, R.sup.12 is selected from the group
consisting of heterocycle and substituted heterocycle, and Z is
selected from the group consisting of --C(O)-- and --SO.sub.2--, Ar
is aryl, heteroaryl, substituted aryl or substituted heteroaryl, x
is an integer of from 1 to 4; A is --C(X)NR.sup.7-- wherein R.sup.7
is selected from the group consisting of hydrogen and alkyl; and X
is selected from the group consisting of oxygen and sulfur; and
pharmaceutically acceptable salts thereof.
8-17. (canceled)
18. A method of treating an inflammatory condition in mammalian
subject, comprising administering to the subject a pharmaceutically
effective dosage of an alpha-9 integrin antagonist compound.
19. The method of claim 18, wherein said inflammatory condition is
characterized by increased neutrophil adhesion.
20. The method of claim 18, wherein said alpha-9 integrin
antagoinist compound is selected from a group of compounds which
inhibit alpha-4/beta-1 integrin binding to an alpha-4/beta-1
integrin ligand.
21. The method of claim 18, wherein said alpha-9 integrin
antagoinist compound exhibits a potency in inhibiting binding
between alpha-9 integrin and an alpha-9 integrin ligand that is at
least {fraction (1/1000)} as high as an inhibitory potency
exhibited by a compound selected from the group consisting of:
N-(toluene-4-sulfonyl)-L-prolyl-L--
4(4-methylpiperazin-1-ylcarbonyloxy)phenylalanine,
N-(toluene-4-sulfonyl)--
L-prolyl-L-4(N,N-dimethylcarbamyloxy)phenylalanine,
N-(1-methylpyrazole-4-sulfonyl)-L-prolyl-L-4-(N,N-dimethylcarbamyloxy)phe-
nylalanine,
N-(toluene-4-sulfonyl)-L-(1,1-dioxo-5,5-dimethyl)thiaprolyl-L--
4-(N,N-dimethylcarbamyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-N-methyl--
L-alaninyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-L-[(1,1-dioxo)thiamorpholin-3-carbonyl]-L-4-(N,N-d-
imethylcarbamyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)prolyl-4-(piperaz- inoyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)sarcosyl-4-(N,N-dimethylcar-
bamyloxy)phenylalanine, and
N-(toluene-4-sulfonyl)-L-(5,5-dimethyl)thiapro-
lyl-L-4-[3-(N,N-dimethyl)propoxy]phenylalanine.
22. The method of claim 18, wherein said compound is selected from
the group consisting of carbamyl compounds having the formula:
R.sup.1--SO.sub.2--NR.sup.2--CHR.sup.3-Q-CHR.sup.5--CO.sub.2H
wherein R.sup.1 is selected from the group consisiting of alkyl,
substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted
cycloalkyl, heterocyclic, substituted heterocylic, heteroaryl and
substituted heteroaryl; R.sup.2 is selected from the group
consisting of hydrogen, alkyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, heterocyclic, substituted
heterocyclic, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, and R.sup.1 and R.sup.2
together with the nitrogen atom bound to R.sup.2 and the SO.sub.2
group bound to R.sup.1 can form heterocyclic or a substituted
heterocyclic group; R.sup.3 is selected from the group consisiting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic and, when
R.sup.2 does not form a heterocyclic group with R.sup.1, R.sup.2
and R.sup.3 together with the nitrogen atom bound to R.sup.2 and
the carbon atom bound to R.sup.3 can form a heterocyclic or a
substituted heterocyclic group; R.sup.5 is
--(CH.sub.2).sub.x--Ar--R.sup.5' where R.sup.5' is selected from
the group consisting of --O-Z-NR.sup.8R.sup.8 and --O-Z-R.sup.12
wherein R.sup.8 and R.sup.8' are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, heterocyclic, substituted heterocyclic, and
where R.sup.8 and R.sup.8' are joined to form a heterocycle or a
substituted heterocycle, R.sup.12 is selected from the group
consisting of heterocycle and substituted heterocycle, and Z is
selected from the group consisting of --C(O)-- and --SO.sub.2--, Ar
is aryl, heteroaryl, substituted aryl or substituted heteroaryl, x
is an integer of from 1 to 4; Q is --C(X)NR.sup.7-- wherein R.sup.7
is selected from the group consisting of hydrogen and alkyl; and X
is selected from the group consisting of oxygen and sulfur; and
pharmaceutically acceptable salts thereof.
23. The method of claim 18, wherein said alpha-9 integrin
antagonist is selected from the group consisting of:
N-(toluene-4-sulfonyl)-L-prolyl-L--
4(4-methylpiperazin-1-ylcarbonyloxy)phenylalanine,
N-(toluene-4-sulfonyl)--
L-prolyl-L-4(N,N-dimethylcarbamyloxy)phenylalanine,
N-(1-methylpyrazole-4-sulfonyl)-L-prolyl-L-4-(N,N-dimethylcarbamyloxy)phe-
nylalanine,
N-(toluene-4-sulfonyl)-L-(1,1-dioxo-5,5-dimethyl)thiaprolyl-L--
4-(N,N-dimethylcarbamyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-N-methyl--
L-alaninyl-L-4-(N,N-dimethylcarbamyloxy)-phenylalanine,
N-(toluene-4-sulfonyl)-L-[(1,1-dioxo)thiamorpholin-3-carbonyl]-L-4-(N,N-d-
imethylcarbamyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)prolyl-4-(piperaz- inoyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)sarcosyl-4-(N,N-dimethylcar-
bamyloxy)phenylalanine, and
N-(toluene-4-sulfonyl)-L-(5,5-dimethyl)thiapro-
lyl-L-4-[3-(N,N-dimethyl)-propoxy]phenylalanine.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/______, which was converted pursuant to 37
C.F.R. .sctn. 1.53(c)(2)(i) from U.S. patent application Ser. No.
08/904,424, filed Jul. 31, 1997, and of U.S. Provisional
Application No. 60/054,453, filed Aug. 1, 1997, all of which
applications are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The invention relates to compositions that modulate binding
of a specific integrin molecule, .alpha..sub.9.beta..sub.1, to its
receptor(s), to methods of treatment using such compounds, and to
screening assays suitable for identify additional modulatory
compounds for use in such treatment methods. Pharmaceutical
compositions which include such compounds are useful in treating
inflammation and other disorders where modulation of
.alpha..sub.9.beta..sub.1-receptor interactions is desirable.
REFERENCES
[0003] Hynes, R. O. (1987) Cell 48: 549-554.
[0004] Palmer, E. L., et-al. (1993) J. Cell Biol. 123:
1289-1297.
[0005] Smith, et al. (1996) J. Biol. Chem. 271: 28485.
[0006] Yednock, T. A., et al. J. Biol. Chem., 1995, 270:
28740-28750.
[0007] Yokosaki, et al. 1994, J. Biol. Chem. 269: 26691-26696.
[0008] Yokosaki, et al. 1996, J. Biol. Chem. 271: 24144-24150.
BACKGROUND OF THE INVENTION
[0009] The integrins are a group of glycoproteins that are present
on a wide variety of cells, where they mediate cell-cell and
cell-matrix adhesion via interactions with receptors present on
cell membranes or in the extracellular matrix. Known receptors for
the various integrin family members include cell surface
immunoglobulins, extracellular matrix proteins (laminin, collagen,
fibronectin, tenascin), and cadherins.
[0010] All known members of the integrin family are composed of two
subunits, termed alpha and beta. There are currently at least
sixteen recognized alpha subunits and eight different
beta-subunits; integrins containing the .beta..sub.1 form of the
beta subunit are known as the ".beta..sub.1 integrin family."
Members of this family are expressed by a diverse distribution of
tissues and exhibit specific binding specificities. Thus,
.alpha..sub.1.beta..sub.1 integrin is expressed by T-lymphocytes
and fibroblasts and binds to collagen and laminin; in contrast,
.alpha..sub.4.beta..sub.1 integrin (VLA-4) is expressed by several
types of hematopoietic cell and binds to VCAM-1, fibronectin and
madCAM. It is therefore the alpha subunit that apparently confers
receptor binding specificity to the protein.
[0011] A relatively new member of the .beta..sub.1 integrin family,
.alpha..sub.9.beta..sub.1 (also referred to herein as "alpha-9
integrin") has been shown to bind to tenascin and osteopontin, both
of which are components of the extracellular matrix which are
induced at sites of inflammation (Yokosaki; Smith). When sequences
of the various alpha subunits were compared, alpha-9 integrin was
shown to have the closest sequence identity to the alpha-4 subunit;
however this represents only 39% sequence identity (Palmer).
Moreover, the two subunits have different cell and tissue
distributions. While .alpha..sub.9.beta..sub.1 is expressed on
airway smooth muscle cells, and non-intestinal epithelial cells
(Palmer), and diffusely on hepatocytes and basal keratinocytes
(Yokosaki, 1994), .alpha..sub.4.beta..sub.1 integrin is present
mainly on hematopoietic cells.
[0012] Heretofore, there has been no definitive determination of an
in vivo function for .alpha..sub.9.beta..sub.1 integrin, nor has a
physiological consequence of disruption of
.alpha..sub.9.beta..sub.1-rece- ptor interactions been identified,
despite its presence in several tissues, as described above. Nor,
despite its association with osteopontin and tenascin, has there
been any reason to suspect that alpha-9 integrin might play a role
in inflammatory disorders, since the .alpha..sub.9.beta..sub.1
molecule had not been associated with any of the hematopoietic
cells commonly associated with this disorder.
[0013] In studies carried out in support of the present invention,
it is now now found that .alpha..sub.9.beta..sub.1 is present on
neutrophils, a class of phagocytic cells which play an important
role in inflammation. In humans, these cells are notable for their
relative lack of alpha-4/beta-1 integrin. Therefore, the present
invention provides basis for involvement of
.alpha..sub.9.beta..sub.1 in acute inflammatory responses.
[0014] Further differences among the .beta..sub.1-integrins are
associated with their binding specificities or endogenous ligands.
While they all bind one or more proteins or proteoglycans that form
the extracellular matrix, each integrin family member exhibits a
distinct molecular specificity which may dictate, in part, its
physiological specificity. Thus, while alpha-4/beta-1 integrin is
known to bind fibronectin and VCAM-1, alpha-9 integrin has been
characterized as binding the matrix proteins osteopontin and
tenascin (Yokosaki, 1994; Smith, 1996). According to a further
discovery related to the present invention, alpha-9 integrin also
binds VCAM-1, though, as discussed below, it is likely that such
binding occurs at a site that distinct from the alpha-4 binding
site.
[0015] The present invention therefore provides basis for new
therapeutic regimens directed at modulating alpha-9 integrin
binding to its ligand(s), and in particular, those ligands which
are involved in the inflammatory response. In addition, it is a
further discovery of the present invention that many of the
compounds or drugs that modulate (inhibit or enhance)
alpha-4/beta-1 integrin binding also modulate alpha-9 integrin
binding. This discovery therefore provides new pharmaceutical
compositions and methods of treatment for modulating alpha-9
integrin binding, as well as screening methods for identifying new
alpha-9 integrin modulatory compounds.
SUMMARY OF THE INVENTION
[0016] The invention is directed to pharmaceutical compositions and
methods of treatment for disorders that involve binding of alpha-9
integrin, as well as screening assays that are useful in
identifying compounds for use in such compositions and methods.
More particularly, the invention is directed to inflammatory
conditions, particularly those that involve increased adhesion
macrophages or neutrophils, which, according to a discovery of the
present invention, are now known to carry alpha-9 integrin in their
membranes and to exhibit increased expression of alpha-9 integrin
in response to stimulation by a known activator molecule, fMLP, as
described herein.
[0017] A number of inflammatory disorders are therefore susceptible
to treatment in accordance with the present invention, including
but not limited to airway hyper-responsiveness and occlusion that
occur in conjunction 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 artritis, and inflammation and scarring that occur with
the progression of ulcerative colitis, and Crohn's disease.
[0018] In preferred embodiments, pharmaceutical compositions and
methods of treatment of the invention employ alpha-9 antagonist
compounds that inhibit binding between alpha-9 integrin and an
alpha-9 integrin ligand. Preferred ligands in this regard include
any ligand found to specifically bind to alpha-9 integrin, as
exemplified by osteopontin, tenascin, and VCAM-1. Due to its
association with inflammatory reactions, V-CAM-1 is particularly
preferred for a test compound in this regard.
[0019] In one embodiment, pharmaceutical compositions and treatment
methods of the invention contain an alpha-9 integrin antagonist
compound that exhibits a potency in inhibiting binding between
alpha-9 integrin and an alpha-9 integrin ligand that is at least as
high as {fraction (1/1000)}, and preferably at least as high as
{fraction (1/100)} of an inhibitory potency exhibited by a compound
selected from the group consisting of nine reference compounds:
N-(toluene-4-sulfonyl)-L-prolyl-L-
-4(4-methylpiperazin-1-ylcarbonyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-L-prolyl-L-4(N,N-dimethylcarbamyloxy)phenylalanine-
,
N-(1-methylpyrazole-4-sulfonyl)-L-prolyl-L-4-(N,N-dimethylcarbamyloxy)ph-
enylalanine,
N-(toluene-4-sulfonyl)-L-(1,1-dioxo-5,5-dimethyl)thiaprolyl-L-
-4-(N,N-dimethylcarbamyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-N-methyl-
-L-alaninyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine,
N-(toluene-4-sulfonyl)-L-[1,1-dioxo)thiamorpholin-3-carbonyl]-L-4-(N,N-di-
methylcarbamyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)prolyl-4-(piperazi- noyloxy)phenylalanine,
N-(N-p-toluenesulfonyl)sarcosyl-4-(N,N-dimethylcarb- amyloxy)
phenylalanine, and N-(toluene-4-sulfonyl)-L-(5,5dimethyl)thiaprol-
yl-L-4-[3-(N,N-dimethyl)propoxy]phenylalanine.
[0020] The foregoing group of compounds are exemplary in nature,
having been chosen for their relatively high potency in inhibiting
alpha-9 integrin binding to an exemplary ligand, tenascin. The
foregoing compounds also illustrate another aspect of the
invention--that a rich source of candidate compounds for use in the
pharmaceutical compositions and methods of treatment described
herein is compounds known to inhibit binding or activity of
alpha-4/beta-1 integrin (VLA-4). The foregoing 9 reference standard
compounds can also be used in the pharmaceutical compositions and
methods of treatment described above.
[0021] In another embodiment, pharmaceutical compositions and
methods of treatment will employ alpha-9 integrin antagonists that
have a K.sub.i or IC.sub.50 less than about 100 .mu.M, as
determined in an assay which measures inhibition of binding between
alpha-9 integrin and an alpha-9 integrin ligand.
[0022] In another related embodiment, compounds useful in the
pharmaceutical compositions and methods of treatment of the
invention have the formula:
R.sub.1--SO.sub.2--NR.sub.2--CHR.sup.3-Q-CHR.sup.5--CO.sub.2H,
where
[0023] R.sup.1 is selected from the group consisting of alkyl,
substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted
cycloalkyl, heterocyclic, substituted heterocylic, heteroaryl and
substituted heteroaryl;
[0024] R.sup.2 is selected from the group consisting of hydrogen,
alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heterocyclic, substituted heterocyclic,
substituted alkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, and R.sup.1 and R.sup.2 together with the nitrogen atom
bound to R.sup.2 and the SO.sub.2 group bound to R.sup.1 can form a
heterocyclic or a substituted heterocyclic group;
[0025] R.sup.3 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,
substituted heterocyclic and, when R.sup.2 does not form a
heterocyclic group with R.sup.1, R.sup.2 and R.sup.3 together with
the nitrogen atom bound to R.sup.2 and the carbon atom bound to
R.sup.3 can form a heterocyclic or a substituted heterocyclic
group;
[0026] R.sup.5 is --(CH.sub.2).sub.x--Ar--R.sup.5' where R.sup.5'
is selected from the group consisting of
[0027] --O-Z-NR.sup.8R.sup.8' and --O-Z-R.sup.12 wherein R.sup.8
and R.sup.8' are independently selected from the group consisting
of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, heterocyclic, substituted heterocyclic, and where
R.sup.8 and R.sup.8' are joined to form a heterocycle or a
substituted heterocycle, R.sup.12 is selected from the group
consisting of heterocycle and substituted heterocycle, and Z is
selected from the group consisting of --C(O)-- and
[0028] --SO.sub.2--,
[0029] Ar is aryl, heteroaryl, substituted aryl or substituted
heteroaryl,
[0030] x is an integer of from 1 to 4;
[0031] Q is --C(X)NR.sup.7-- wherein R.sup.7 is selected from the
group consisting of hydrogen and alkyl; and X is selected from the
group consisting of oxygen and sulfur, and pharmaceutically
acceptable salts thereof.
[0032] In still another related embodiment, the invention includes
pharmaceutical compositions and methods which employ a small
molecule compound. The compound is selected for its ability to
inhibit binding between alpha-9 integrin and an alpha-9 integrin
ligand, as evidenced by exhibiting a potency in an alpha-9
integrin-alpha-9 integrin ligand binding assay that is at least
{fraction (1/1000)} as high as a potency of a compound selected
from the reference compound group listed above. In a related
embodiment, such a compound is also an inhibitor of inhibitor of
alpha-4/beta-1 integrin binding to VCAM-1, as evidenced by its
ability to inhibit such binding with a potency that is at least
{fraction (1/1000)} as high as a potency exhibited by a compound
selected from the reference standard group listed above. All
pharmaceutical compositions and methods of treatment employ
pharmaceutically effective dosages and are delivered in an
excipient and manner appropriate to the particular treatment
regimen selected by the practitioner.
[0033] According to a related aspect, the invention includes a
method of screening for therapeutic compounds effective in treating
conditions characterized by involvement of alpha-9 integrin, and in
particular, inflammatory conditions, such as those listed above.
The method includes adding test compound to an assay system which
measures an amount of alpha-9 integrin binding to an alpha-9
integrin ligand, and selecting the test compound as an effective
therapeutic drug candidate, if said compound exhibits a binding
inhibitory activity that is at least {fraction (1/1000)} as potent
as an activity exhibited by a compound selected from the group of
reference standard compounds listed above. Candidate compounds
selected in this mode are further tested for safety and toxicity,
according to methods well known in the art, prior to use in the
pharmaceutical compositions and methods of treatment described
herein.
[0034] Selection of test compounds for testing in the screening
method is well within the skill of the practitioner in view of the
wealth of combinatorial libraries now commerically available or
available through the scientific literature. Nonetheless, in
accordance with the present invention, particularly preferred test
compounds are those known to exhibit activity in modulating,
particularly inhibiting, binding between alpha-4/beta-1 integrin
and any of its ligands, but particularly VCAM-1. According to a
preferred embodiment, an compound is selected by the assay if it
exhibits an inhibitory potency that is at least as {fraction
(1/1000)} as high as an inhibitory potency exhibited by a compound
selected from the reference standard group listed above. According
to a related embodiment, preferred compounds are selected from a
group having the formula:
R.sup.1--SO.sub.2--NR.sub.2--CHR.sup.3-Q-CHR.sup.5--CO.sub.2- H,
where the substituent groups and moieties are defined as described
above. These compounds are also described in co-owned parent
applications U.S. patent application Ser. No. 08/904,424, filed
Jul. 31, 1997, and U.S. Provisional Application No. 60/054,453,
filed Aug. 1, 1997, which are incorporated herein by reference.
[0035] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows a bar graph that illustrates expression of
alpha-9 integrin on human neutrophils; and
[0037] FIG. 2 shows a bar graph illustrating selective enhancement
of expression of alpha-9 integrin ("anti-a9 integrin) as compared
to control ("isotype control") on human neutrophils following
activation by fMLP (right-hand bars).
DETAILED DESCRIPTION OF THE INVENTION
[0038] I. Definitions
[0039] This section provides definitions of certain of the terms
used herein. Unless specifically defined, all other scientific and
technical terms used have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. A convenient reference for purposes the present invention
is Stedman's Medical Dictionary 24.sup.th Edition (Williams and
Wilkins, Baltimore)
[0040] The term "alpha-9 integrin" refers to a heterodimeric
protein member of the .beta..sub.1 integrin family which is also
referred to as .alpha..sub.9.beta..sub.1.
[0041] The term "alpha-9 integrin ligand" refers to molecules to
which alpha-9 integrin binds in vitro or preferably in vivo.
Preferably, such binding occurs with a binding affinity or potency
in the range of at least about 10.sup.-4 M and typically between
about 10.sup.-5 to 10.sup.-8 M. The term also refers to fragments
of such compounds that possess such binding affinity
characteristics. Exemplary alpha-9 integrin ligands include, but
are not limited to tenascin, osteopontin, and VCAM-1.
[0042] The term "binding affinity" as used herein refers to the
relative strength with which two or more molecules bind together.
In its usage herein, the term is typically expressed in terms of
the molar amount of compound necessary to observe a desired effect,
such as 1/2 maximal binding or response (EC.sub.50 or K.sub.d), or
inhibition of binding (IC.sub.50 or K.sub.i), but may also be used
in a relative sense to express an amount of compound required to
observe minimal saturation of a ligand, such as in the integrin
saturation assays described herein.
[0043] The term "potency" is generally used to refer to relative
affinities or efficacies; a compound has a "higher potency" than
another if it is more effective than a reference compound when the
two are compared at the same molar concentration, or if it produces
the same effect at a lower concentration. The term may also be used
to compare amounts of different compound needed to observe an
arbitrary effect. By way of example, a test compound is said
exhibits "a potency that is at least as high as {fraction (1/1000)}
of a reference standard potency, if it produces the same effect as
the reference standard at no more than 1000 times the molar
concentration required for the reference standard. Therefore, if
the reference standard produces a given effect at a concentration
of 1 .mu.M, a test compound would be at least {fraction (1/1000)}
as potent if it is capable of producing the same effect at any
concentration less than 1000 .mu.M (1 mM).
[0044] The term "pharmaceutical composition" refers to a
pharmaceutically active preparation of drug or biological which is
prepared in a pharmaceutical excipient, such as buffered saline or
a physiological buffer appropriate for administration to a subject.
Appropriate excipients, including but not limited to diluents,
fillers and the like are formulated based on the anticipated mode
of administration and are readily determined by persons skilled in
the art.
[0045] The term "small molecule" generally refers to an organic
compound having a molecular weight that is less than about 2000 and
preferably less than about 1000; small molecules may include short
peptides and peptidomimetics.
[0046] The term "alpha-4/beta-1 integrin" refers to a heterodimeric
protein which is also referred to as .alpha..sub.4.beta..sub.1 and
as VLA-4.
[0047] The term "alpha-4/beta-1 integrin ligand" refers to
molecules to which alpha-4/beta-1 integrin binds in vitro or
preferably in vivo. Preferably, such binding occurs with a binding
affinity in the range of at least about 10.sup.-4 M and typically
between about 10.sup.-5 to 10.sup.-8 M. The term also refers to
fragments of such compounds that possess such binding affinity
characteristics. Exemplary alpha-4/beta-1 integrin ligands include,
but are not limited to fibronectin (HEPII and CS1 domains), VCAM-1,
osteopontin, and madCAM1. Generally, such ligands include a peptide
binding site having the sequence EILDV.
[0048] The term "condition associated with binding of alpha-9
integrin to an alpha-9 integrin ligand" describes conditions having
attributes consistent with the presence of increased or reduced
alpha-9 integrin bound to an endogenous alpha-9 integrin ligand as
compared to normal. An example of increased alpha-9 integrin
binding is increased adhesion of neutrophils to osteopontin or
tenascin, or another alpha-9 ligand during inflammation. In view of
studies carried out in support of the present invention and
described herein, alpha-9 integrin is likely to be involved in such
increased adherence. Thus inflammation is considered a condition
associated with binding of alpha-9 integrin.
[0049] An "alpha-9 integrin modulatory compound" or an "alpha-9
integrin regulatory compound" is a compound, preferably but not
necessarily a small molecule, which, when added to a mixture which
contains alpha-9 integrin and an alpha-9 integrin ligand, affects
the binding between the integrin molecule and the ligand--for
example, by producing an increase or decrease of such binding. By
way of example, a modulatory compound which inhibits or reduces
alpha-9 integrin binding to tenascin is referred to as an alpha-9
antagonist.
[0050] An effect or response is "significantly different,"
"significantly higher" or "significantly lower" if, when compared
to an appropriate control, the test response shows a statistically
significant change, when analyzed by an appropriate statistical
method. In general, when it is stated that a response or effect is
increased or decreased, it can be inferred that the observed
increase or decrease is statistically significant or is expected to
be statistically significant when subjected to appropriate
experimental analysis.
[0051] Common amino acids are referred to by their one- or
three-letter abbreviations as follows: alanine (A, Ala), cysteine
(C, Cys), aspartic acid (D, Asp), glutamic acid (E, Glu),
phenyalanine (F, Phe), glycine (G, Gly), histidine (H, His),
isoleucine (I, lie), lysine (K, Lys), leucine (L, Leu), methionine
(M, Met), asparagine (N, Asn), proline (P, Pro), glutamine (Q,
Gln), arginine (R, Arg), serine (S, Ser), threonine (T, Thr),
valine (V, Val), tryptophan (W, Trp), tyrosine (Y, Tyr).
[0052] II. Alpha-9 Integrin
[0053] This section provides further background information about
alpha-9 integrin, including means for distinguishing it from other
integrins. Such distinguishing means are important in (i) setting
up assays capable of measuring binding or blockade of binding
between .alpha..sub.9.beta..sub.1 integrin and its ligand(s), and
(ii) identifying compounds that are preferably small molecule
antagonists capable of blocking interactions between
.alpha..sub.9.beta..sub.1 integrin and its ligands(s).
[0054] A. Physical Characteristics
[0055] As mentioned above, .alpha..sub.9.beta..sub.1 integrin is a
heterodimeric protein consisting of an alpha-subunit, termed
.alpha..sub.9 (or "alpha-9"), and a beta-subunit, generally
.beta..sub.1 (or "beta-1"). The two subunits bind to one another
noncovalently, and each consists of a relatively short carboxy
terminal intracellular domain which contains the highly conserved
sequence GFF(R/K)R, a single transmembrane domain and a relatively
large amino terminus extracellular domain which generally projects
on the surface of cells.
[0056] The deduced amino acid sequence of the alpha-9 subunit has
been determined by cloning (Palmer, 1993; GENBANK Accession No.
L24158) The human alpha-9 subunit is a protein of 1006 amino acids.
Studies comparing the various forms of alpha subunits have revealed
that the alpha-9 subunit exhibits only 39% sequence identity with
the alpha-4 integrin subunit.
[0057] B. Tissue Localization and Binding Selectivity of alpha-9
Integrin
[0058] .alpha..sub.9.beta..sub.1 integrin is expressed by a number
of different cell types, as mentioned above. For example,
.alpha..sub.9.beta..sub.1 is found on airway smooth muscle cells,
and non-intestinal epithelial cells, as well as a teratoma cell
line (Palmer), and diffusely on hepatocytes and basal keratinocytes
(Yokosaki, 1994). Heretofore, alpha-9 has not been shown to be
present on any of the hematopoietic cells.
[0059] FIG. 1 shows results of experiments carried out in support
of the present invention that show that, in addition to the
previously known tissue distribution described above, alpha-9
integrin is also expressed by human neutrophils. In this
experiment, alpha-9 subunit, alpha-4 subunit and beta-1 subunit
were measured after reacting human neutrophils with fluorescently
labeled antibodies specifically reactive with each of the foregoing
subunits. This shows that, surprisingly, human neutrophils express
alpha-9 subunit along with beta-1 subunit, and confirms that they
express very little, if any alpha-4 subunit.
[0060] Neutrophils are phagocytic blood cells that are involved in
a number of inflammatory conditions, particularly acute
inflammation, as described below. These cells were previously
distinguished by their lack of alpha-4/beta-1 integrin, which is
expressed by all, or nearly all other circulating leucocytes.
[0061] Further experiments in support of the invention indicated
that alpha-9 integrin is likely involved in inflammatory responses
involving neutrophils. Formyl-Met-Leu-Phe (fMLP) is an activation
factor that is involved in inflammation. FIG. 2 is a bar graph that
shows that alpha-9 integrin expression is significantly and
selectively increased following activation by fMLP (right-hand
bars), as compared to a control idiotype-specific marker. This
increased expression is consistent with alpha-9 involvement in
activation of neutrophils during inflammation.
[0062] Studies on the binding selectivity of alpha-9 integrin have
revealed that the molecule binds to tenascin at a "fibrinogen-like"
type-III repeat (termed "TNfn3"). Although this region of tenascin
contains the characteristic "RGD" (Arg-Gly-Asp) peptide binding
site to which other integrins (.alpha..sub.8.beta..sub.1,
.alpha..sub.v.beta..sub- .3, and .alpha..sub.v.beta..sub.6)
preferentially bind, this is apparently not the site bound by
alpha-9 integrin (Yokosaki, 1994). Rather,
.alpha..sub.9.beta..sub.1 integrin binds preferentially to the B-C
loop which contains the peptide sequence AEIDGIEL, and a peptide
containing this sequence has been shown to disrupt binding between
cells expressing alpha-9 integrin and TNFn3 (Yokosaki, 1998).
[0063] III. Alpha-9 Integrin Modulatory Compounds
[0064] This section provides guidance for identifying compounds for
use as alpha-9 integrin modulatory compounds suitable for use in
pharmaceutical compositions and treatment methods in accordance
with the present invention. Specifically, in studies carried out in
support of the present invention it has been found that, despite
their sequence and ligand binding site dissimilarities noted above,
alpha-4/beta-1 integrin and alpha-9 integrin apparently share
similar binding sites for small molecules, and that binding to such
sites serves to similarly modulate their abilities to bind to
endogenous ligands.
[0065] Therefore, according to a preferred aspect of the present
invention, compounds that modulate alpha-4/beta-1 integrin binding
to its ligands are likely to also have activity in alpha-9 integrin
assays. Accordingly, as described below, a rich source of compounds
for testing in specific alpha-9 integrin modulatory assays consists
of small molecules, including peptides and peptidomimetics, that
have been characterized as alpha-4/beta-1 integrin agonists or
antagonists. Further candidate compounds are provided by a variety
of libraries, including, without limitation, combinatorial
libraries, fermentation broths and lysates, phage libraries, and
the like, such as are well known in the art and/or commercially
available for screening, as discussed in more detail in Part B,
below. Such libraries of compounds, in addition to the
alpha-4/beta-1 modulatory compounds mentioned above and any other
compounds can be conveniently screened in assay formats known in
the art with reference to the exemplary assays described herein.
Such assays can be further modified to accommodate high throughput
screening of compounds according to methods known in the art.
[0066] A. Alpha-4/beta-1 integrin Agonists and Antagonists
[0067] In contrast to alpha-9 integrin, which has only recently
been characterized, alpha-4/beta-1 integrin has been widely studied
and has been the focus of numerous drug development programs.
Alpha-4/beta-1 integrin is expressed by most forms of hematopoeitic
cells, with the exception of neutrophils (e.g.,
.alpha..sub.4.beta..sub.1 is expressed by T- and B-lymphocytes,
monocytes and certain antigen presenting cells). Alpha-4/beta-1
integrin binds endogenous ligands including fibronectin, mucosal
addressin (MadCAM-1), vascular cell adhesion molecule-1 (VCAM-1;
Cd106) and osteopontin. In particular, its interaction with VCAM-1,
which is induced in the vascular epithelium during acute
inflammatory responses, its presence on leukocytes, and its
recognized involvement in the enhanced adhesion of such leukocytes
at sites of inflammation have made .alpha..sub.4.beta..sub.1 a
target for compounds in development for treatment of a variety of
inflammatory diseases, including rheumatoid arthritis, heart
disease and ulcerative colitis, among others.
[0068] Studies carried out in support of the present invention have
revealed that compounds which modulate binding of alpha-4/beta-1
integrin to any of its ligands, including VCAM-1, are generally
also good candidates for modulating binding of alpha-9 integrin to
its ligand(s). More specifically, as described in section IV,
below, specific antagonists of alpha-4/beta-1 integrin also block
alpha-9 integrin binding to tenascin. This discovery therefore
provides a wealth of candidate compounds for use in pharmaceutical
compositions and methods of the present invention. For example,
co-owned, concurrently-filed related application U.S. Ser. No.
______, (PCT ______) incorporated herein by reference, which claims
priority to the same initial U.S. Patent Applications, U.S. patent
application Ser. No. 08/904,424, filed Jul. 31, 1997, and U.S.
Provisional Application No. 60/054,453, filed Aug. 1, 1997, which
are incorporated herein by reference in their entireties, describes
a series of carbamyloxy compounds which have been characterized as
inhibitors of alpha-4/beta-1 integrin binding to its ligands (i.e.,
.alpha..sub.4.beta..sub.1 antagonists). Testing and use of several
exemplary compounds of this series are discussed below. Methods for
making such compounds are detailed in Example 4 herein.
[0069] Similarly, co-owned U.S. patent application Ser. No.
08/904,415, U.S. Ser. No. 08/903,585, U.S. Ser. No. 08/904,423,
U.S. Ser. No. 08/920,353, U.S. Ser. No. 08/904,417, U.S. Ser. No.
08/920,394 and U.S. Ser. No. 08/904,416, all filed on Jul. 31,
1997, describe additional, structurally distinct alpha-4/beta-1
integrin antagonists which inhibit binding of alpha-4/beta-1
integrin to its ligand(s). The foregoing applications are hereby
incorporated herein by reference for their teachings of such
compounds which, in accordance with the present invention, are also
candidates for use in the pharmaceutical compositions and methods
of treatment that are the subjects of the present invention. In
view of the data presented below, is anticipated that many of these
compounds will exhibit approximately equipotent inhibitory
activities in inhibiting alpha-9 integrin as in inhibiting
alpha-4/beta-1 integrin.
[0070] B. Sources of Test Compounds
[0071] Additional sources of candidate alpha-9 integrin modulatory
compounds are therefore apparent, in view of the discovery that at
least a significant subset of alpha-4/beta-1 (VLA-4) inhibitory
compositions may be active as alpha-9 integrin antagonists. That
is, persons skilled in the art will recognize that compounds that
are characterized as inhibiting or enhancing alpha-4/beta-1
integrin binding to its ligand(s) are strong candidates for
modulating alpha-9 integrin binding to its respective ligands.
Therefore, it will be a relatively routine matter, in view of the
teaching of the present invention, to identify candidate compounds,
for example, by conducting database searches of the patent or
chemical literature for alpha-4/beta-1 (VLA-4) inhibitory
compositions. Exemplary methods for further testing such compounds
(e.g., for specificity and selectivity of binding, as well as for
relative binding affinity) are provided in Section IV below.
[0072] With the advent of automated, high throughput screening
procedures and the development of a variety of forms of
combinatorial chemical libraries, persons skilled in the art will
recognize that it will be relatively routine to identify additional
alpha-9 modulatory compounds, in view of the guidance for selecting
such compounds that is provided herein. For example, but not by way
of limitation to the invention, random libraries are a rich source
of materials. Moreover, combinatorial libraries can be produced for
many types of compounds that can be synthesized in a step-by-step
fashion. Such compounds include peptides, beta-turn mimetics,
polysaccharides, phospholipids, hormones, prostaglandins, steroids,
aromatic compounds, heterocyclic compounds, benzodiazepines,
oligomeric N-substituted glycines and oligocarbamates. Large
combinatorial libraries of the compounds can be constructed by the
encoded synthetic libraries (ESL) method described in Affymax, WO
95/12608. Affymax. WP 0306121, Columbia University WO 94/08051,
Pharmacopeia, WO 95/35503, and Scripps WO 95/30642 (each of which
is incorporated herein by reference for all purposes). Peptide
libraries can also be generated by phage display methods. See e.g.,
Devlin, WO 91/18980, incorporated herein by reference.
[0073] Combinatorial libraries and other compounds are initially
screened by testing in an alpha-9 integrin activity assay, such as
one or more of the assays described herein, and a compound is
selected for use in the pharmaceutical compositions and methods of
the invention if it satisfies the criteria set forth, particularly
in Section IV, herein.
[0074] C. Compositions including Alpha-9 Integrin Modulatory
Compounds
[0075] Test compounds, preferably, but not necessarily selected as
described above and tested as described below are further
considered for use in pharmaceutical compositions of the present
invention if they exhibit a potency in an alpha-9 integrin assay
that is comparable to threshold activities determined by certain
reference compounds, as discussed below. Such compositions will be
suitable for further use in pharmaceutical compositions, subject to
testing in an appropriate in vivo model appropriate to the specific
target disorder and subject to appropriate tests of safety for the
mammalian species to be treated. Appropriate dosages to be
delivered will be estimated according to standard pharmacokinetic
analyses, as discussed in Section V, below. As a general guideline,
an effective dosage of a compound will be that amount of compound
which is effective to produce a significant biochemical effect in
the target tissue, with reference to the effective concentrations
determined from in vitro or in vivo assays, such as are discussed
below.
[0076] IV. Screening Assays for Compounds that Regulate Alpha-9
Integrin Binding
[0077] As mentioned above, a highly enriched source of compounds
suitable for inclusion in the pharmaceutical compositions and
methods of treatment of the present invention are compounds that
are identified as alpha-4/beta-1 integrin antagonists. Such
compounds are identified, either with reference to the scientific
and patent literature or by empirical testing in an alpha-4/beta-1
integrin binding assay, as exemplified in Part A below. Part B
describes exemplary alpha-9 integrin activity assays that provide
information on alpha-9 integrin modulatory compounds in accordance
with the present invention.
[0078] A. Alpha-4/beta-1 Integrin Binding and Activity Assays
[0079] Assays and test systems for determining whether a test
compound is active in binding to and modulating activity of
alpha-4/beta-1 integrin are well known in the art. By way of
example, but not limitation, such assays include in vitro assays
which measure the ability of alpha-4/beta-1 integrin present on
cells known to bind to one or more of its ligands, such as VCAM-1.
An exemplary cell-soluble VCAM-1 protein assay suitable for this
purpose is detailed in Example 1 herein, and is also described
further in the parent applications, U.S. Patent Application No.
08/904,424, filed Jul. 31, 1997, and U.S. Provisional Application
No. 60/054,453, filed Aug. 1, 1997, both of which are incorporated
herein in their entireties. These documents also describe results
that identify several hundred effective alpha-4/beta-1 integrin
antagonist compounds in this assay.
[0080] Briefly, in order to test compounds in this assay, compounds
are synthesized, obtained from commercial sources, including a
screening library, as discussed in Section III, above. In
experiments carried out in support of the present invention,
compounds were synthesized, for example, as detailed in Example 4
herein. Test compounds are then added to the screening assay,
incubated, and the amount of binding between alpha-4/beta-1
integrin and its ligand, for example, VCAM-1, is measured as
detailed in Example 1.
[0081] By way of example, an appropriate assay for measuring this
interaction employs an antibody which binds to an
activation/ligand-induc- ed epitope on the beta-1 subunit. It
therefore binds only to ligand activated cells and can therefore be
used as a measure of how much ligand is bound (or, conversely,
displaced) in the presence of test compound.
[0082] Briefly, in experiments carried out in support of the
present invention, the activity of .alpha.4.beta.1 integrin was
measured by the interaction of soluble VCAM-1 with a human T-cell
line (Jurkat) which expresses high levels of .alpha.4.beta.1
integrin. 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 IgG1 heavy chain constant region on
the C-terminus. The detector antibody, termed "15/7" was raised as
a monoclonal antibody against immunopurified
.alpha..sub.4.beta..sub.1 integrin and was selected on the basis of
surface reactivity with U937 cells (ATCC; CRL 1593), then screened
for differential reactivity with Jurkat and THP-1 cells (ATCC;
TIB-202). This antibody was further characterized to have the
reactivity described above (Yednock). Antibodies similar to the
15/7 antibody have been prepared by other investigators (Luque, et
al, 1996, J. Bio. Chem. 271:11067) and may be used in this
assay.
[0083] Jurkat cells were incubated with Mn.sup.2+ and 15/7 antibody
on ice. Mn.sup.+2 activates the receptor to enhance ligand binding,
and 15/7 recognizes an activated/ligand occupied conformation of
.alpha.4.beta.1 integrin and locks the molecule into this
conformation thereby stabilizing the VCAM-1/.alpha.4.beta.1
integrin interaction. Cells were then incubated for 30 minutes at
room temperature with candidate compounds, in various
concentrations using a standard 5-point serial dilution. Soluble
recombinant VCAM-1 fusion protein was then added to Jurkat cells
and incubated for 30 minutes on ice. Cells were then washed two
times and resuspended in PE-conjugated goat F(ab')2 anti-mouse IgG
Fc (Immunotech, Westbrook, Me.) incubated on ice, in the dark, for
30 minutes. Cells were washed twice and analyzed with a standard
fluorescence activated cell sorter ("FACS") analysis as described
in Yednock, et al., supra. Compounds having an IC.sub.50 of less
than about 1 mM, and preferably less than about 100 .mu.M possess
sufficient binding activity to be considered for further
testing.
[0084] Table 1 lists exemplary alpha-4/beta-1 integrin inhibitory
compounds that were found to have activity in the foregoing assay.
Moreover, experiments in support of the present invention revealed
that all 373 compounds described in parent applications U.S. patent
application Ser. No. 08/904,424, filed Jul. 31, 1997, and U.S.
Provisional Application No. 60/054,453, filed Aug. 1, 1997, and/or
in co-owned application PCT/US98/______ filed concurrently
herewith, all of which are incorporated herein by reference,
exhibited sufficient alpha-4-beta-1 integrin inhibitory activity to
be considered candidates for alpha-9 screening, as described in
Part B, below.
1TABLE 1 Com- pound Name 1
N-(toluene-4-sulfonyl)-L-prolyl-L-4(4-methylpiperazin-1-
ylcarbonyloxy)phenylalanine 2 N-(toluene-4-sulfonyl)-L-prolyl-L-4(-
N,N- dimethylcarbamyloxy)phenylalanine 3
N-(1-methylpyrazole-4-sulfonyl)-L-prolyl-L-4-(N,N-
dimethylcarbamyloxy)phenylalanine 4 N-(toluene-4-sulfonyl)-L-(1,1--
dioxo-5,5-dimethyl)thiaprolyl- L-4-(N,N-dimethylcarbamyloxy)phenyl-
alanine 5 N-(toluene-4-sulfonyl)-N-methyl-L-alaninyl-L-4-(N,N-
dimethylcarbamyloxy)phenylalanine 6 N-(toluene-4-sulfonyl)-L-[1-
,1-dioxo)thiamorpholin-3-carbonyl]- L-4-(N,N-dimethylcarbamyloxy)p-
henylalanine 7
N-(N-p-toluenesulfonyl)prolyl-4-(piperazinoyloxy)phe- nylalanine 8
N-(N-p-toluenesulfonyl)sarcosyl-4-(N,N-
dimethylcarbamyloxy)phenylalanine 9 N-(toluene-4-sulfonyl)-L-(5,5--
dimethyl)thiaprolyl-L-4-[3-(N,N- dimethyl)propoxy]phenylalanine
[0085] It is understood that alpha-4/beta-1 integrin modulatory
activity may alternatively be measured in one or more of other
appropriate assays known and available to those skilled in the art.
While absolute inhibitory concentration values may vary from assay
to assay and operator to operator, compounds that inhibit (or
enhance) activity at a concentration no higher than about 1 mM, and
preferably no higher than about 100 .mu.M should be considered as
candidates for alpha-9 integrin modulatory agents.
[0086] B. Alpha-9 Integrin Binding and Activity Assays
[0087] Example 2 describes exemplary assays for measuring alpha-9
integrin binding and activity. A convenient assay is one similar to
the one described above, with reference to binding of
alpha-4-beta-1 integrin to VCAM-1, using the same 15/7 antibody
that recognizes activated beta-1 subunit, but substituting into the
assay cells expressing the alpha-9 subunit. In experiments carried
out in support of the present invention, SW480 cells were
transfected with a plasmid containing an expressable coding region
for the alpha-9 subunit, as described by Yokosaki, et al (1994,
1996), both of which references are incorporated herein by
reference. Compounds were assessed for ability to interfere with
binding of these cells to tenascin.
[0088] Compounds 1-9 described herein potently induced the
ligand-occupied epitope (15/7) on the .alpha..sub.9 transfected
cells, but not in control mock-transfected cells, at concentrations
less than about 1 .mu.M. Furthermore, activity of these compounds
in a binding saturation assay was found to correspond to their
abilities to inhibit .alpha..sub.9-dependent cell adhesion to
tenascin according to the methods set forth in Example 2. Based on
these experiments, alpha-9 inhibitory activity can be defined as
inhibition of alpha-9 integrin binding to tenascin, as evidenced by
an IC.sub.50 or effective concentration of less than about 100
.mu.M, and preferably less than about 20 .mu.M.
[0089] More generally, it is appreciated that active alpha-9
integrin antagonist compounds useful in the pharmaceutical
compositions and methods of the invention will have activities that
are defined relative to the potencies exemplified by the nine
compounds described above. That is, in accordance with a preferred
embodiment of the present invention, active alpha-9 antagonist
compounds have activities reflecting at least {fraction (1/1000)}
and preferably at least {fraction (1/100)} the potency of the
lowest activity compound exemplified herein. Thus, a practitioner
engaged in screening compounds will know to test the above-listed
compounds as reference standards and to compare the activities of
test compounds to the activities of the reference standards
described above. According to this preferred embodiment of the
invention, a test compound will be considered active if it exhibits
an activity that is at least {fraction (1/1000)}, and preferably at
least {fraction (1/100)} that of the lowest activity compound
exemplified above. By way of illustration, if the lowest activity
reference standard compound in a given assay were to exhibit an
IC.sub.50 of 1 .mu.M, test compounds exhibiting IC.sub.50s as high
as 1000 .mu.M (1 mM) and preferably 100 .mu.M would be considered
to be active alpha-9 antagonists in accordance with the present
invention.
[0090] Methods for preparing the above-referenced reference
standard compounds are found, for example in Example 4 herein
(compounds 1-6), in parent application U.S. patent application Ser.
No. 08/904,424, filed Jul. 31, 1997, or U.S. Provisional
Application No. 60/054,453, filed Aug. 1, 1997 (both of which are
concurrently filed with the present application as
PCT/US98/______), or in U.S. patent application Ser. No.
08/920,394, filed Jul. 31, 1997, filed concurrently as
PCT/US98/______), all of which are incorporated herein by
reference.
[0091] C. Selectivity for Alpha-9 Integrin
[0092] A further desirable activity of alpha-9 modulatory compounds
in accordance with the present invention is an ability to
selectively regulate alpha-9 integrin activity. In experiments
carried out in support of the present invention, compounds were
tested for activity in assays measuring activity of various
integrins, including .alpha..sub.4.beta..sub.7 integrin as assessed
by binding to MadCAM1, .alpha..sub.5.beta..sub.1 integrin as
assessed by binding to fibronectin, .alpha.L.beta..sub.2 integrin,
as assessed by binding to ICAM-1, using methods and reagents well
known in the art. As discussed above, the foregoing compounds were
also tested for alpha-4/beta-1 integrin binding to VCAM-1.
[0093] While many of the alpha-4/beta-1 integrin antagonists tested
exhibited equivalent activities in the alpha-4/beta-1 integrin and
alpha-9 integrin activity assays described herein, compounds were
also found which 10-fold or greater selectivity for inhibition in
one of the assays, as compared to the other.
[0094] All nine reference compounds described herein tested
exhibited at least a 100-fold selectivity in the alpha-9 integrin
assay, compared to the .alpha..sub.5.beta..sub.1 integrin and
.alpha..sub.4.beta..sub.7 integrin assays and were inactive in the
.alpha.L.beta..sub.2 integrin assay. Thus, these compounds are
characterized as selective for alpha-9 integrin activity, as
opposed to .alpha..sub.5.beta..sub.1, .alpha..sub.4.beta. or
.alpha.L.beta..sub.2 activity. Such selectivity may be desirable
when specificity of activity is particularly desirable. Guided by
the teachings of the present specification and the particular
therapeutic application for which a particular test compound is to
be used, practitioners skilled in the art will be able to (a)
determine appropriate drug activity assays for purposes of
comparison, and (b) select a selectivity ratio that is acceptable
in the context of such therapeutic application.
[0095] D. Criteria for Selection of Alpha-9 Integrin Modulatory
Compounds
[0096] Active alpha-9 integrin modulatory compounds in accordance
with the present invention increase or decrease binding between
alpha-9 integrin and one or more of its ligands, for example
tenascin, osteopontin or VCAM-1, at a concentration that is of
sufficient potency to provide a pharmaceutical composition.
Generally, it is appreciated that useful drugs will be active in
vitro or in vivo at concentrations less than about 100 .mu.M and
preferably less than about 20 .mu.M. Therefore, in accordance with
the present invention, useful alpha-9 integrin modulatory compounds
will be active in this concentration range in an appropriate
alpha-9 integrin activity, as described herein.
[0097] Exemplified herein are a number of active alpha-9 integrin
antagonist or inhibitory compounds all of which have the requisite
potency to be active. In accordance with the present invention, it
is suggested that these compounds can be used as reference
standards in the exemplified alpha-9 antagonist activity assay or
in any other appropriate alpha-9 activity assay. A compound that is
run in the same assay will be considered active if it exhibits an
activity that is at least {fraction (1/1000)}, and preferably at
least {fraction (1/100)} the potency of the lowest activity
reference compound selected from compounds 1-9 illustrated
herein.
[0098] According to a particularly useful embodiment of the present
invention exemplified herein, it is appreciated that compounds
having alpha-4/beta-1 modulatory activity form a particularly
useful "library" of starting compounds for identifying alpha-9
modulatory compounds. This is a useful, but not essential criterion
for selecting compounds for use in the pharmaceutical compositions
and methods of the present invention.
[0099] Additionally, it is appreciated that it may be advantageous
to select alpha-9 modulatory compounds that are relatively
selective for modulating alpha-9 integrin activity, as compared to
other integrins or other pharmacological activities. Suggested
criteria for selectivity are provided above.
[0100] It is further appreciated that compounds useful in the
pharmaceutical compositions and treatment methods described herein
should conform to acceptable levels of toxicity; persons skilled in
the art will further subject test candidate compounds in toxicity
assays according to standard methods known in the art and/or
mandated by the appropriate regulatory authority.
[0101] V. Utility
[0102] Alpha-9 integrin modulatory compounds selected in accordance
with the present activity have utility in pharmaceutical
compositions, methods of treatments. In addition, the selection
assays described above are useful in identifying compounds for use
in such compositions and methods.
[0103] A. Pharmaceutical Compositions and Treatment of Disorders
Associated with Alpha-9 Integrin Binding
[0104] Alpha-9 integrin modulatory compounds identified and
selected in accordance with the present invention find use in a
number of disorders associated with alpha-9 integrin activity.
Particularly, in view of the discoveries described herein with
respect to the neutrophil localization of alpha-9 integrin, as well
as its ability to interact with VCAM-1, it is appreciated that
alpha-9 integrin inhibitory compounds will find particular utility
in the treatment of a variety of disorders which include an
inflammatory component, particularly those in which the
inflammatory component is associated with VLA-4 binding to alpha-9
integrin.
[0105] 1. Therapeutic Indications
[0106] 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
neutrophils. 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, various lung disorders including 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 Alzheimer's disease, AIDS
dementia, multiple sclerosis (MS), viral meningitis and
encephalitis, as well as stroke (cerebral ischemia) related
disorders.
[0107] More particularly, since alpha-9 integrin binding is
predictive of in vivo utility for inflammatory conditions mediated
by alpha-9 integrin, compositions and methods of the invention can
be used for treating, 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
[0108] In accordance with the present invention, it is appreciated
that alpha-9 integrin modulatory compounds, particularly those
exhibiting inhibitory activity, will find utility in treating the
many, if not all, of the foregoing disorders. Compounds selected
for inhibitory activity in accordance with the methods described
herein are then tested in appropriate animal models, for example,
to determine dosage, volumes of distribution and the like. Efficacy
may also be confirmed in such models, which are well known in the
art.
[0109] For example, appropriate in vivo models for demonstrating
efficacy in treating inflammatory responses include an asthma model
in mice, rats, guinea pigs, goats or primates, as well as other
inflammatory models in which alpha-9 integrins are implicated.
[0110] 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. In accordance with the present invention, it is believed
that airway hyper-responsiveness and occlusion that occur with
chronic asthma are mediated, at least in part, by alpha-9 integrin
binding interactions. This is verified in a standard model such as
described in Example 3, herein.
[0111] 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 mucosa and submucosa. Lymphocytes
and macrophages are numerous in lesions of inflammatory bowel
disease and may contribute to 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. Macrophage and leukocytes are
recruited to atheromas and contribute to inflammatory injury.
[0112] 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).
[0113] 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 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-9 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, 420425 (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).
[0114] A related use for compounds of this invention which bind to
alpha-9 integrin 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.
[0115] A further use of the compounds of this invention is
inhibiting tumor metastasis. Several tumor cells have been reported
to express integrins, such as VLA-4 and 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).
[0116] 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
integrins such as 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.
[0117] 2. Pharmaceutical Compositions
[0118] 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). Such pharmaceutical
compositions are particularly useful in treating diseases having an
inflammatory component, such as those discussed in Part 1,
above.
[0119] Pharmaceutical compositions of the present invention can
also be used in 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 electron spin resonance (ESR), both of which are well
known. In general, any conventional method for visualizing
diagnostic images 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 macrophages
and/or neutrophils expressing alpha-9 integrin it is possible to
determine whether a particular therapeutic regimen aimed at
ameliorating the disease is effective.
[0120] 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 sulfonamide compositions described herein 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(2):83-93).
[0121] 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. Pat. Nos. 4,235,871, 4,501,728 and
4,837,028 each of which is incorporated herein by reference.
[0122] Appropriate treatment dosages and dosage schedules are
determined in accordance with the condition being treated, and a
number of variables, including, but not limited to the intended
mode of administration, the pharmacokinetics of the active
compound, the size of the subject. For example, for intravenous
administration, the dose will typically be in the range of about 20
.mu.g to about 500 .mu.g per kilogram body weight, preferably about
100 .mu.g to about 300 .mu.g per kilogram body weight. Suitable
dosage ranges for intranasal administration are generally about 0.1
.mu.g to 1 mg per kilogram body weight. Dosages can be based on
appropriate estimates or can be determined empirically by persons
skilled in the art. Generally, relative dosages can be estimated
based on comparisons of potencies in one or more of the screening
assays described herein.
[0123] Effective doses can be extrapolated from dose-response
curves derived from in vitro or animal model test systems. In
general, effective dosages can be estimated from the predictive in
vitro assays described herein. That is, an effective dose is calcul
produce at the target tissue(s) in the body, a concentration of
compound that is in range of {fraction (1/10)} to 10-times the
concentration of compound IC.sub.50 in such an assay.
[0124] The compositions administered to a patient are in the form
of pharmaceutical compositions described above. These compositions
may be sterilized by convention sterilization techniques, or may be
sterile filtered. The resulting aqueous solutions packaged for use
as is, or lyophilized, the lyophilized preparation being combined
sterile aqueous carrier prior to administration. The pH of the
compound preparation typically will be between 3 and 11, more
preferably from 5 to 9 and most preferably 7 to 8. It will be
understood that use of certain of the foregoing excipients,
carriers, stabilizers will result in the formation of
pharmaceutical salts.
[0125] The amount administered to the patient will vary depending
upon what is b administered, the purpose of the administration,
such as prophylaxis or therapy, the 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.
[0126] B. Drug Screening Assays
[0127] The screening assays described herein are useful in
identifying new compounds for use in the treatment methods and
pharmaceutical compositions described above. At its most basic, the
screening assay includes testing a candidate compound for its
ability to interfere with (or enhance) binding between alpha-9
integrin and one or more of its ligands, such as osteopontin,
VCAM-1 or tenascin. The test compounds can also be tested for the
ability to competitively inhibit such binding, or between alpha-9
integrin and a labeled compound known to bind alpha-9 integrin such
one of the compounds described herein or antibodies to alpha-9
integrin.
[0128] Preferably, the screening assay is used to identify alpha-9
integrin inhibitory compounds, since such compounds are, in
accordance with the invention, particularly useful in treating
inflammation in a variety of conditions, as discussed above.
According to this aspect of the invention, a test compound is added
to an assay system configured to detect binding between alpha-9
integrin and one or more of its ligands, such as osteopontin,
VCAM-1 or tenascin, and compounds are tested for ability to inhibit
such binding. Particularly useful compounds are those which exhibit
activity which is in the range of, or at most about 100-times less
potent than, the activity ranges defined by the reference standards
provided herein (see Table 1).
[0129] As mentioned above, test compounds can be selected from a
variety of sources, including combinatorial libraries, fermentation
broths and the like. A particularly good source, identified herein,
is the pool of compounds that are known to inhibit or are found to
inhibit binding between alpha-4/beta-1 integrin (VLA-4) and one or
more of its ligands, such as VCAM-1.
[0130] A number of formats can be used for assays for screening for
drugs. In a preferred embodiment, assays will be adapted for high
throughput screening. For example, alpha-9 integrin or membranes
from cells expressing alpha-9 integrin can be immobilized on a
solid surface, such as a microtiter plate or glass fiber filter
optionally adapted with binding aids such as antibodies to a
non-ligand binding portion of the molecule. Such assay formats
generally employ at least one detectably-labeled assay components.
The labeling 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 known in the art. A wide variety of
labels may be used. The component may be labeled by any one of
several methods. The most common method of detection is the use of
autoradiography with .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P labeled compounds and the like. Non-radioactive labels
include ligands which bind to labeled 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.
[0131] In vitro uses of pharmaceutical compositions of the
invention include diagnostic applications such as monitoring
inflammatory responses by detecting the presence of macrophages,
including neutrophils, expressing alpha-9 integrin. Compositions of
this invention can also be used for isolating or labeling such
cells.
[0132] 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,
as adapted to the reagents described herein, according to methods
well within the skill of the practitioner. This application is
incorporated herein by reference in its entirety.
[0133] The following examples illustrate, but in no way are
intended to limit the present invention.
EXAMPLES
Example 1
Binding of Compounds to .alpha..sub.4.beta..sub.1 Integrin
(VLA-4)
[0134] A. 15/7 Antibody Assay
[0135] The cell adhesion assay described below is based on an assay
detailed in a publication by Yednock, et al. (1995), which is
incorporated herein by reference. An in vitro assay was used to
assess binding of candidate compounds to .alpha..sub.4.beta..sub.1
integrin. Compounds which bind in this assay can be used to assess
VCAM-1 levels in biological samples by conventional assays (e.g.,
competitive binding assays). This assay is sensitive to IC.sub.50
values as low as about 1 nM.
[0136] The activity of .alpha.4.beta.1 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;
American Type Culture Collection, Manassas, Va.), a human T-cell
line which expresses high levels of .alpha.4.beta.1 integrin.
VCAM-1 interacts with the cell surface in an .alpha.4.beta.1
integrin-dependent fashion (Yednock).
[0137] 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 IgG1 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 .mu.g/mL 15/7 antibody
for 30 minutes on ice. Mn+2 activates the receptor to enhance
ligand binding, and 15/7 is a monoclonal antibody that recognizes
an activated/ligand occupied conformation of
.alpha..sub.4.beta..sub.1 integrin and locks the molecule into this
conformation thereby stabilizing the VCAM-1/.alpha..sub.4.beta.-
.sub.1 integrin interaction. Yednock, et al., supra. Antibodies
similar to the 15/7 antibody have been prepared by other
investigators (Luque, et al, 1996, J. Bio. Chem. 271:11067) and may
be used in this assay.
[0138] Cells were then incubated for 30 minutes at room temperature
with candidate compounds, in various concentrations ranging from 66
.mu.g/mL to 0.01 .mu.g/mL using a standard 5-point serial dilution.
15 .mu.L 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')2 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 analyzed with a standard
fluorescence activated cell sorter ("FACS") analysis as described
in Yednock, et al., supra.
[0139] When tested in this assay, each of the compounds as
described in Examples 1-373 of parent application U.S. Ser. No.
08/904,424 (PCT/US98/______) or the corresponding carboxylic acids
of the ester compounds, i.e. the prodrugs) exhibited IC.sub.50s of
15 .mu.M or less.
[0140] B. In vitro Saturation Assay For Determining Binding of
Candidate Compounds to .alpha..sub.4.beta..sub.1
[0141] Log-growth Jurkat cells were washed and resuspended in
normal animal plasma containing 20 .mu.g/ml of the 15/7 antibody
(described in the above example). The Jurkat cells were diluted
two-fold into either normal plasma samples containing known
candidate compound amounts in various concentrations ranging from
66 .mu.g/mL to 0.01 .mu.g/mL, 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
were then incubated for 30 minutes at room temperature, washed
twice with phosphate-buffered saline ("PBS") containing 2% fetal
bovine serum and 1 mM each of calcium chloride and magnesium
chloride (assay medium) to remove unbound 15/7 antibody. The cells
were then exposed to phycoerythrin-conjugated goat F(ab')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 were 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. Bio. Chem.,
1995, 270:28740.
[0142] The data were 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.
[0143] This assay may also be used to determine the plasma levels
needed to saturate the binding sites of other integrins, such as
the .alpha.9.beta..sub.1 integrin, using appropriate cells
expressing alpha-9 integrin, as described in Example 2, below.
Example 2
Cell Adhesion to Tenascin
[0144] The following assay was first described by Yokosaki, et al.
(1994) and can be used to estimate serum levels of compounds
required for treating inflammatory diseases related to alpha-9
integrin binding, as exemplified by the asthma model detailed in
Example 3, below.
[0145] Evaluation of cell attachment to extracellular matrix
proteins was performed as follows: Briefly, wells of non-tissue
culture-treated polystyrene 96-well flat-bottom microtiter plates
(Linbro/Titertek, Flow Laboratories, McLean, Va.) were coated by
incubation with intact tenascin (10 .mu.g/ml) or recombinant
tenascin fragments (1 .mu.g/ml to 10 .mu.g/ml) in PBS at 37.degree.
C. for 1 h or at 4.degree. C. for 16 h. Wells were washed with PBS
and then blocked with 1% bovine serum albumin in DMEM. 50,000 cells
(SW480 cell line transfected with alpha-9 integrin, (Yokosaki,
1996) a gift of D. Sheppard, Dept. of Medicine, University of
California, San Francisco) were added to each well in 200 .mu.l of
serum-free DMEM containing 0.05% bovine serum albumin. For blocking
experiments, cells were incubated with the relevant reagent for 30
min at 4.degree. C. before plating. Plates were centrifugated at
10.times.g for 5 min and then incubated for 1 h at 37.degree. C. in
a humidified atmosphere with 5% CO.sub.2. Nonadherent cells were
removed by centrifugation top-side-down at 48.times.g for 5 min.
The attached cells were fixed with 1% formaldehyde and stained with
0.5 crystal violet, and excess dye was washed off with PBS. The
cells were solubilized in 50 .mu.l of 2% Triton X-100 and
quantified by measuring the absorbance at 595 nm in a Microplate
Reader (Bio-Rad). Positive adhesion results in this assay correlate
with increased absorbance.
[0146] Isolated populations of neutrophils can also be used in the
foregoing assay in place of the transfected cells. In addition,
ligands such as fibronectin, VCAM-1 or osteopontin can be
substituted in this assay.
Example 3
Asthma Model
[0147] Inflammatory conditions mediated by alpha-9 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.
[0148] 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 .mu.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 was connected to the inspiratory port of
a piston respirator. The solenoid valve is activated for 1 second
at the 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 was used as
a control.
[0149] To assess bronchial responsiveness, cumulative
concentration-response curves to carbachol are generated according
to Abraham (1994). Bronchial biopsies are taken prior to and
following the initiation of treatment and 24 hours after antigen
challenge. Bronchial biopsies are performed according to Abraham
(1994). An in vitro adhesion study of alveolar macrophages is also
performed according to Abraham (1994), and a percentage of adherent
cells is calculated.
[0150] Aerosol Formulation
[0151] 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:
[0152] A. Preparation of 100 mL of 0.5% Sodium Bicarbonate/Saline
Stock Solution:
[0153] 1. Add 0.5 g sodium bicarbonate into a 100 mL volumetric
flask.
[0154] 2. Add approximately 90.0 mL saline and sonicate until
dissolved.
[0155] 3. Q.S. to 100.0 mL with saline and mix thoroughly.
[0156] B. Preparation of 10.0 mL of 30.0 mg/mL Candidate
Compound:
[0157] 1. Add 0.300 g of the candidate compound into a 10.0 mL
volumetric flask.
[0158] 2. Add approximately 9.7 mL of 0.5% sodium
bicarbonate/saline stock solution.
[0159] 3. Sonicate until the candidate compound is completely
dissolved.
[0160] 4. Q.S. to 10.0 mL with 0.5% sodium bicarbonate/saline stock
solution and mix thoroughly.
Example 4
Synthesis of alpha-9 Integrin-Modulatory Compounds
[0161] This Example provides representative organic syntheses of
pre-cursors and reference standard compounds described herein.
Parent applications U.S. patent application Ser. No. 08/904,424,
filed Jul. 31, 1997, and U.S. Provisional Application No.
60/054,453, filed Aug. 1, 1997, as well as co-owned, concurrently
filed U.S. patent application Ser. No. ______ and corresponding PCT
application PCT/US98/______ all of which applications are
incorporated herein by reference in their entireties, are
referenced specifically for their methods of preparation of these
and many other related compounds.
[0162] A. General Reference Methods
[0163] Method 1: N-Tosylation Procedure
[0164] N-Tosylation of the appropriate amino acid was conducted via
the method of Cupps, Boutin and Rapoport J. Org. Chem. 1985, 50,
3972.
[0165] 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.
[0166] Method 3: BOP Coupling Procedure
[0167] 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 (1.1
equivalent), and DMF. The reaction mixture was stirred at room
temperature overnight. The crude product is purified flash
chromatography to afford the dipeptide ester.
[0168] Method 4: Hydrogenation Procedure I
[0169] 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.
[0170] Method 5: Hydrolysis Procedure I
[0171] To a chilled (0?C) THF/H.sub.2O solution (2:1, 5-10 mL) of
the appropriate ester was added LiOH (or NaOH) (0.95 equivalents).
The temperature was maintained at 0.degree. 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.
[0172] Method 6: Ester Hydrolysis Procedure II
[0173] To a chilled (0.degree. C.) THF/H.sub.2O solution (2:1, 5-10
mL) of the appropriate ester was added LiOH (1.1 equivalents). The
temperature was maintained at 0.degree. C. and the reaction was
complete in 1-3 hours. The reaction mixture was concentrated and
the residue was taken up into H.sub.2O and the pH adjusted to 2-3
with aqueous HCl. The product was extracted with ethyl acetate and
the combined organic phase was washed with brine, dried over MgSO4,
filtered and concentrated to yield the desired acid.
[0174] Method 7: Ester Hydrolysis Procedure III
[0175] The appropriate ester was dissolved in dioxane/H.sub.2O
(1:1) and 0.9 equivalents of 0.5 N NaOH was added. The reaction was
stirred for 3-16 hours and than concentrated. The resulting residue
was dissolved in H.sub.2O and extracted with ethyl acetate. The
aqueous phase was lyophilized to yield the desired carboxylate
sodium salt.
[0176] Method 8: Sulfonylation Procedure I
[0177] To the appropriately protected aminophenylalanine analog
(11.2 mmol), dissolved in methylene chloride (25 ml) 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.times.3), brine (50 mL), and water (100 mL). The organic phase
was dried (MgSO4) and the solvent concentrated to yield the desired
product.
[0178] Method 9: Reductive Amination Procedure
[0179] Reductive amination of Tos-Pro-p-NH2-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.
[0180] Method 10: BOC Removal Procedure
[0181] 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
Et2O and reconcentrated. This procedure was repeated and the
resulting solid was placed under high vacuum overnight.
[0182] Method 11: tert-Butyl Ester Hydrolysis Procedure I
[0183] The tert-butyl ester was dissolved in CH2Cl2 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
H.sub.2O and lyophilized to yield the desired acid.
[0184] B. Preparations
[0185] Prep 1 Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(4-methylpi-
perazin-1-ylcarbonyloxy)phenylalanine Ethyl Ester
[0186] The title compound was prepared following the procedure
outlined for the preparation of Prep 4 and substitution of
appropriate starting materials.
[0187] NMR data were as follows:
[0188] 1H NMR (CD3)2SO): .delta.=8.33 (d, 1H), 7.70 (d, 2H), 7.41
(d, 2H), 7.24 (d, 2H), 7.00 (d, 2H), 4.52-4.44 (m, 1H), 4.09-4.00
(m, 3H), 3.53 (bs, 2H), 3.38-3.31 (m, 3H), 3.11-3.01 (m, 3H), 2.39
(s, 3H), 2.32 (bs, 4H), 2.19 (s, 3H), 1.61-1.50 (m, 3H), 1.43-1.38
(m, 1H), 1.13 (t, 3H). 13C NMR (CD3)2SO): .delta.=171.1, 171.1,
153.9, 149.8, 143.6, 134.1, 133.9, 130.0, 129.8, 127.4, 121.5,
61.2, 60.7, 54.2, 54.1, 53.3, 49.0, 45.7, 44.0, 43.4, 35.8, 30.5,
23.8, 21.0, 14.0.
[0189] Prep 2 Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(N,N-dimeth-
ylcarbamyloxy)phenylalanine Ethyl Ester (Compound 2)
[0190] Into a reaction vial were combined 7.00 g (15.2 mmol, 1.0
eq) Ts-Pro-Tyr(H)-OEt and 1.86 g (15.2 mmol, 1.0 eq) DMAP.
Methylene chloride (50 mL), triethylamine (2.12 mL-1.54 g, 15.2
mmol, 1.0 eq), and dimethylcarbamyl chloride (1.68 mL-1.96 g, 18.2
mmol, 1.2 eq) were then added. The vial was capped tightly, and the
reaction solution swirled to obtain a homogeneous solution. The
reaction solution was then heated to 40.degree. C. After 48 h, TLC
of the resulting colorless solution indicated complete conversion.
The workup of the reaction solution was as follows: add 50 mL EtOAc
and 50 mL hexanes to the reaction mixture, and wash with 3.times.50
mL 0.5 mL hexanes to the reaction mixture, and wash with 3.times.50
mL 0.5 M citric acid, 2.times.50 mL water, 2.times.50 mL 10% K2CO3,
and 1.times.50 mL sat. NaCl. Dry with MgSO4. Filter. Evaporate to
obtain 8.00 g (99%) of the title compound as a clear oil, which
solidifies upon standing. Recrystallize from 5:3:2
heptane/EtOAc/CH.sub.2Cl.sub.2.
[0191] NMR data were as follows:
[0192] 1H NMR (CD3)2SO): .delta.=8.32 (d, 1H), 7.70 (d, 2H), 7.41
(d, 2H), 7.23 (d, 2H), 7.00 (d, 2H), 4.52-4.44 (m, 1H), 4.09-4.02
(m, 3H), 3.37-3.31 (m, 1H), 3.11-2.96 (m, 3H), 3.00 (s, 3H), 2.87
(s, 3H), 2.39 (s, 3H), 1.61-1.50 (m, 3H), 1.43-1.38 (m, 1H), 1.13
(t, 3H).
[0193] 13C NMR (CD3)2SO): .delta.=171.1, 171.1, 154.0, 150.0,
143.6, 133.9, 133.9, 130.0, 129.8, 127.4, 121.5, 61.2, 60.6, 53.3,
49.0, 36.3, 36.1, 35.8, 30.5, 23.8, 21.0, 14.0.
[0194] Prep 3: Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(4-methylp-
iperazin-1-ylcarbonyloxy)phenylalanine Isopropyl Ester
[0195] The title compound was prepared following the procedure
outlined for the preparation of Prep 4 and substitution of
appropriate starting materials.
[0196] NMR data were as follows:
[0197] 1H NMR (CDCl3): .delta.=7.72 (d, 2H), 7.36 (d, 1H), 7.33 (d,
2H), 7.16 (d, 2H), 7.03 (d, 2H), 5.07 (Sept., 1H), 4.78 (dt, 1H),
4.08-4.05 (m, 1H), 3.67 (bs, 2H), 3.57 (bs, 2H), 3.41-3.35 (m, 1H),
3.24 (dd, 1H), 3.15-3.07 (m, 1H), 3.04 (dd, 1H), 3.46-2.43 (m, 7H),
2.34 (s, 3H), 2.05-2.02 (m, 1H). 13C NMR (CDCl3): .delta.=170.9,
170.4, 153.6, 150.5, 144.3, 133.2, 133.1, 130.2, 130.0, 127.9,
121.7, 69.5, 62.2, 54.7, 53.4, 49.6, 46.1, 44.3, 43.7, 37.2, 29.7,
24.1, 21.6, 21.6, 21.4.
[0198] Prep 4 Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(4-methylpi-
perazin-1-ylcarbonyloxy)phenylalanine tert-Butyl Ester
[0199] Combine 41.2 g (84.34 mmol, 1.0 eq) Ts-Pro-Tyr(H)-OtBu and
17.0 g (84.34 mmol, 1.0 eq) 4-nitrophenyl chloroformate. Add 700 mL
CH2Cl2. Cap with a septum. Attach a N2 line. Immerse the flask in a
4:1 water/EtOH+dry ice slurry, and stir to cool to -15.degree. C.
Add 29.38 mL (21.33 g, 210.81 mmol, 2.5 eq) Et3N over five minutes
with stirring. Stir at -10 to -15.degree. C. for 1 h. Add 9.35 mL
(8.45 g, 84.34 mmol, 1.0 eq) N-methyl piperazine over 3 minutes
with stirring. Stir overnight while warming to room temperature.
Dilute with 700 mL hexanes. Wash repeatedly with 10% K2CO3, until
no yellow color (4-nitrophenol) is seen in the aqueous layer. Wash
with sat. NaCl. Dry over anhydrous MgSO4. Filter. Evaporate.
Dissolve in 500 mL EtOH, and evaporate, to remove Et3N. Repeat
once. Dissolve in 400 mL EtOH, and add 600 ml water with stirring,
to precipitate a solid or oil. If an oil, stir vigorously to
solidify. Isolate the solid by filtration. Repeat dissolution,
precipitation, and filtration, once. Rinse with water to remove
traces of yellow color. High vacuum to constant mass yields the
title compound as a white solid.
[0200] NMR data were as follows:
[0201] 1H NMR (CDCl3): .delta.=7.72 (d, 2H), 7.33 (d, 3H), 7.17 (d,
2H), 7.02 (d, 2H), 4.71 (q, 1H), 4.09-4.06 (m, 1H), 3.67 (bs, 2H),
3.57 (bs, 2H), 3.41-3.34 (m, 1H), 3.22 (dd, 1H), 3.16-3.09 (m, 1H),
3.03 (dd, 1H), 2.46-2.43 (m, 7H), 2.34 (s, 3H), 2.05-2.02 (m, 1H),
1.57-1.43 (m, 3H), 1.47 (s, 9H). 13C NMR (CDCl.sub.3):
.delta.=171.8, 169.9, 153.6, 150.4, 144.3, 133.4, 133.1, 130.3,
130.0, 127.9, 121.6, 82.6, 62.3, 54.5, 53.8, 49.6, 46.1, 44.3,
43.7, 37.3, 29.7, 27.8, 24.1, 21.4.
[0202] Prep 5 Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(4-methylpi-
perazin-1-ylcarbonyloxy)phenylalanine (Reference Compound 1)
[0203] The title compound was prepared from the product of Prep 1
using the procedure described in Method 7.
[0204] NMR data were as follows:
[0205] 1H NMR (CD3OD): .delta.=7.74 (d, 2H), 7.42 (d, 2H), 7.26 (d,
2H), 7.04 (d, 2H), 4.58-4.54 (m, 1H), 4.16-4.12 (m, 1H), 3.70 (bs,
2H) 3.53 (bs, 2H), 3.43-3.31 (m, 1H), 3.26-3.13 (m, 7H), 2.82 (s,
3H), 2.43 (s, 3H), 1.98-1.94 (m, 1H), 1.76-1.51 (m, 3H).
[0206] 13C NMR (CD.sub.3OD): .delta.=175.7, 173.6, 154.8, 151.6,
146.1, 136.3, 134.8, 131.9, 131.3, 129.1, 122.7, 63.6, 55.9, 53.9,
50.7, 43.5, 37.6, 31.3, 25.5, 21.5.
[0207] Prep 9 Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-4-(N,N-dimeth-
ylcarbamyloxy)phenylalanine
[0208] The title compound was prepared from the product of Prep 2
using the procedure described in Method 7.
[0209] NMR data were as follows:
[0210] 1H NMR (CD3)2SO: .delta.=8.13 (d, 1H), 7.70 (d, 2H), 7.41
(d, 2H), 7.23 (d, 2H), 6.99 (d, 2H), 4.51-4.44 (m, 1H), 4.11-4.09
(m, 1H), 3.40-3.34 (m, 2H), 3.11-2.94 (m, 3H), 3.00 (s, 3H), 2.87
(s, 3H), 2.39 (s, 3H), 1.59-1.36 (m, 4H). 13C NMR (CD3).sub.2SO:
.delta.=172.7, 171.2, 153.6, 150.2, 143.8, 134.3, 134.0, 130.2,
130.0, 127.6, 121.6, 61.3, 53.2, 49.0, 36.3, 36.1, 35.9, 30.4,
23.8, 21.0.
[0211] Prep 10: Synthesis of
N-(Toluene-4-sulfonyl)-L-prolyl-L-3-(N,N-dime-
thylcarbamyloxy)phenylalanine Ethyl Ester
[0212] The title compound was prepared following the procedure
outlined for the preparation of Prep 2 and substitution of
appropriate starting materials.
[0213] NMR data were as follows:
[0214] 1H NMR (CDCl3): .delta.=7.74 (m, 2H), 7.70-7.36 (m, 4H),
7.24-7.14 (m, 3H), 6.93-4.90 (m, 1H), 4.78-4.27 (m, 3H), 4.05-3.55
(m, 0.5H), 3.48-3.43 (m, 0.5H), 3.37-3.30 (m, 3H), 3.02-3.08 (bs,
3H), 2.99 (bs, 3H), 2.45 (s, 1.5H), 2.43 (s, 1.5H), 2.12 (m, 1H),
198, 1.80 (m, 0.5M), 1.62-1.44 (m, 2.5H), 1.29 (t, 1.5H), 1.24 (t,
1.5H).
[0215] 13C NMR (CDCl3): .delta.=171.1, 171.0, 170.9, 154.9, 154.8,
151.8, 151.6, 144.4, 144.3, 137.6, 137.1, 133.1, 132.9, 130.0,
129.9, 129.5, 129.2, 127.9, 127.9, 126.5, 126.1, 122.9, 122.7,
120.7, 120.5.
[0216] Prep 11: Synthesis of
N-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)thiapr-
olyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine Isopropyl Ester
[0217] The title compound was prepared following the procedure
outlined for the preparation of Prep 2 and substitution of
appropriate starting materials.
[0218] NMR data were as follows:
[0219] 1H NMR (CDCl3): .delta.=7.76 (d, 2H), 7.35 (d, 2H), 7.22 (d,
2H), 7.01 (m, 3H), 5.05 (m, 1H), 4.85 (m, 1H), 4.57 (d, 1H), 4.38
(d, 1H), 3.86 (s, 1H), 3.19-3.00 (m, 2H), 3.09 (s, 3H), 3.01 (s,
3H), 2.45 (s, 3H), 1.24 (t, 6H), 1.16 (s, 3H), 1.09 (s, 3H).
[0220] 13C NMR (CDCl.sub.3): .delta.=170.3, 168.4, 154.9, 150.6,
144.8, 132.9, 132.8, 130.3, 130.0, 128.2, 121.7, 73.4, 69.5, 54.5,
53.2, 50.4, 37.7, 36.5, 36.3, 29.0, 23.8, 21.5, 21.4.
[0221] Prep 12: Synthesis of
N-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)thiapr-
olyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine tert-Butyl
Ester
[0222] The title compound was prepared following the procedure
outlined for the preparation of Prep 2 and substitution of
appropriate starting materials.
[0223] NMR data were as follows:
[0224] 1H NMR (CDCl3): .delta.=7.75 (d, 2H), 7.34 (d, 2H), 7.23 (d,
2H), 7.05-6.98 (m, 3H), 4.76 (m, 1H), 4.56 (d, 1H), 4.40 (d, 1H),
3.85 (s, 1H), 3.09-3.00 (m, 8H), 2.44 (s, 3H), 1.43 (s, 3H), 1.16
(s, 3H), 1.09 (s, 3H).
[0225] 13C NMR (CDCl3): .delta.=169.8, 168.3, 154.9, 150.6, 144.8,
133.2, 132.9, 130.4, 130.0, 128.2, 121.6, 82.6, 73.4, 54.6, 53.8,
50.4, 37.8, 36.5, 36.3, 29.0, 27.7, 23.8, 21.5.
[0226] Prep 13 Synthesis of
N-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)thiapro-
lyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine
[0227] The title compound was prepared from the product of Prep 11
using the procedure described in Method 7.
[0228] NMR data were as follows:
[0229] 1H NMR (CDCl3): .delta.=7.76 (d, 2H), 7.35 (d, 2H), 7.25 (d,
2H), 7.14 (d, 1H), 7.02 (d, 2H), 5.17 (br s, 1H), 4.89 (m, 1H),
4.56 (d, 1H), 4.40 (d, 1H), 3.90 (s, 1H), 3.30-3.00 (m, 8H), 2.43
(s, 3H), 1.09 (s, 6H).
[0230] 13C NMR (CDCl3): .delta.=172.7, 169.3, 155.2, 150.6, 144.9,
133.1, 132.7, 130.5, 130.1, 128.1, 121.9, 73.3, 54.5, 53.3, 50.5,
36.9, 36.6, 36.4, 29.0, 23.7, 21.5.
[0231] Prep 18 Synthesis of
N-(Toluene-4-sulfonyl)sarcosyl-L-4-(N,N-dimeth-
ylcarbamyloxy)phenylalanine Isopropyl Ester
[0232] The title compound was prepared following the procedure
outlined for the preparation of Prep 2 and substitution of
appropriate starting materials. NMR data were as follows:
[0233] 1H NMR (CDCl3): .delta.=7.66 (d, 2H), 7.34 (d, 2H), 7.18 (d,
2H), 7.07 (d, 2H), 6.98 (d, 1H), 5.03 (m, 1H), 4.81 (m, 1H), 3.69
(d, 1H), 3.49 (d, 1H), 3.08 (m, 2H), 3.04 (s, 3H), 2.99 (s, 3H),
2.63 (s, 3H), 2.43 (s, 3H).
[0234] 13C NMR (CDCl3): .delta.=167.4, 154.9, 150.8, 144.4, 132.6,
130.2, 130.1, 127.7, 122.0, 110.9, 69.5, 57.3, 53.9, 53.0, 37.1,
36.6, 21.6, 21.4.
[0235] Prep 19 Synthesis of
N-(Toluene-4-sulfonyl)sarcosyl-L-4-(N,N-dimeth-
ylcarbarnyloxy)phenylalanine tert-Butyl Ester
[0236] The title compound was prepared following the procedure for
the preparation of Prep 2 and substitution of appropriate starting
materials.
[0237] NMR data were as follows:
[0238] 1H NMR (CDCl3): .delta.=7.67 (d, 2H), 7.34 (d, 2H), 7.19 (d,
2H), 7.03 (d, 2H), 6.98 (d, 1H), 4.76 (m, 1H), 3.67 (q, 1H), 3.06
(m, 2H), 3.16 (s, 3H), 2.99 (s, 3H), 2.64 (s, 3H), 2.43 (s, 3H),
1.42 (s, 9H).
[0239] 13C NMR(CDCl3): .delta.=170.0, 137.2, 154.9, 150.7, 144.3,
133.2, 132.9, 130.3, 130.0, 127.7, 121.9, 82.6, 83.9, 53.3, 37.2,
36.6, 36.4, 27.9, 21.4.
[0240] Prep 20 Synthesis of
N-(Toluene-4-sulfonyl)sarcosyl-L-4-(N,N-dimeth-
ylcarbamyloxy)phenylalanine (Reference Compound 8)
[0241] The title compound was prepared from the product of Prep 18
using the procedure described in Method 7.
[0242] NMR data were as follows:
[0243] 1H NMR (CDCl3): .delta.=7.41 (d, 2H), 7.10 (d, 2H), 6.98 (d,
2H), 6.75 (d, 2H), 4.42 (m, 1 H), 3.43 (m, 2H), 3.04 (m, 2H), 2.80
(s, 3H), 2.69 (s, 3H), 2.33 (s, 3H), 2.14 (s, 3H).
[0244] 13C NMR (CDCl3): .delta.=174.2, 170.2, 156.9, 151.9, 145.6,
135.5, 135.2, 131.4, 131.1, 128.9, 123.0, 54.6, 54.0, 37.4, 36.8,
36.7, 21.4.
[0245] Prep 29 Synthesis of
N-(Toluene-4-sulfonyl)-L-(1,1-dioxo-5,5-dimeth-
yl)-thiaprolyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine
tert-Butyl Ester
[0246] The product of Prep 12 was oxidized by the method of Larsson
and Carlson (Acta Chemica Scan. 1994, 48, 517-525), yielding the
title compound as a white solid.
[0247] NMR data were as follows:
[0248] 1H NMR (CDCl3): .delta.=7.73 (d, 2H), 7.36 (d, 2H), 7.21 (d,
2H), 7.06-6.95 (m, 3H), 4.79 (m, 1H), 4.38 (dd, 2H), 4.10 (s, 1H),
3.18-2.95 (m, 8H), 2.43 (s, 3H), 1.45 (s, 9H), 1.33 (s, 3H), 1.08
(s, 3H).
[0249] 13C NMR (CDCl3): .delta.=169.8, 166.2, 154.9, 120.7, 145.8,
133.0, 131.9, 130.2, 128.5, 121.9, 82.9, 68.0, 60.9, 59.3, 53.9,
37.5, 36.6, 36.3, 27.7, 21.6, 19.3, 18.5.
[0250] Prep 30 Synthesis of
N-(1-Methylimidazolyl-4-sulfonyl)-L-prolyl-L-4-
-(N,N-dimethylcarbamyloxy)phenylalanine
[0251] The title compound was prepared from the product of Example
106 using the procedure described in Method 11.
[0252] NMR data were as follows:
[0253] 1H NMR (CDCl3): .delta.=8.07 (d, 1H), 7.75 (s, 1H), 7.71 (s,
1H), 7.25 (d, 2H), 7.01 (d, 2H), 4.71-4.66 (m, 1H), 4.28-4.24 (m,
1H), 3.77 (s, 3H), 3.42-3.05 (m, 3H), 3.09 (s, 3H), 2.96 (s, 3H),
1.84-1.69 (m, 2H), 1.61-1.54 (m, 2H).
[0254] 13C NMR (CDCl3): .delta.=174.4, 174.1, 156.9, 151.9, 141.8,
137.7, 135.6, 131.6, 127.6, 122.9, 63.7, 54.7, 50.8, 37.4, 36.8,
36.7, 34.3, 31.6, 25.4.
[0255] Prep 31 Synthesis of
N-(Toluene-4-sulfonyl)-L-(1,1-dioxo-5,5-dimeth-
yl)-thiaprolyl-L-4-(N,N-dimethylcarbamyloxy)phenylalanine
(Reference Compound 4)
[0256] The title compound was prepared from the product of Prep 29
using the procedure described in Method 11.
[0257] NMR data were as follows:
[0258] 1H NMR (CDCl3): .delta.=7.75 (m, 3H), 7.29 (m, 4H), 7.08 (d,
2H), 4.95 (m, 1H), 4.46-4.20 (m, 3H), 3.17 (s, 3H), 3.30-3.10 (m,
2H), 3.02 (s, 3H), 2.43 (s, 3H), 1.15 (s, 3H), 0.88 (s, 3H).
[0259] 13C NMR (CDCl3): .delta.=127.2, 167.5, 155.8, 150.3, 145.4,
133.6, 132.6, 130.8, 130.2, 128.3, 121.9, 67.9, 65.8, 60.8, 53.9,
36.8, 36.6, 35.8, 21.6, 18.8, 15.0.
[0260] Prep 49 Synthesis of
N-(Toluene-4-sulfonyl)-L-(thiamorpholin-3-carb-
onyl)-L-4-(N,N-dimethylcarbamyloxy)phenylalanine tert-Butyl
Ester
[0261] L-Thiamorpholine-3-carboxylic acid was prepared by the
method of Larsson and Carlson (Acta Chemica Scan. 1994, 48,
517-525). N-(Toluene-4-sulfonyl)-L-thiamorpholine-3-carboxylic acid
was prepared using the procedure described in Method 1. The title
compound was prepared following the procedure for the synthesis of
Prep 2 with substitution of appropriate starting materials.
[0262] NMR data were as follows:
[0263] 1H NMR (CDCl3): .delta.=7.69 (d, 2H), 7.31 (d, 2H), 7.16 (d,
2H), 6.98 (d, 2H), 6.86 (d, 1H), 4.71 (m, 1H), 4.62 (m, 1H), 3.94
(m, 1H), 3.31 (m, 1H), 3.09 (m, 4H), 2.98 (s, 3H), 2.67 (m, 1H),
2.50 (m, 1H), 2.40 (s, 3H), 2.31 (m, 1H), 2.10 (m, 1H), 1.49 (s,
9H).
[0264] 13C NMR (CDCl3): .delta.=169.9, 167.4, 154.8, 150.6, 144.2,
136.8, 132.8, 130.4, 130.2, 127.3, 121.8, 82.6, 55.2, 54.0, 43.3,
36.5, 36.3, 27.8, 25.2, 24.6, 21.4.
[0265] Prep 50 Synthesis of
N-(Toluene-4-sulfonyl)sarcosyl-L-4-(1,1-dioxot-
hiomorpholin-4-ylcarbonyloxy)phenylalanine
[0266] The title compound was prepared from the product of Prep 121
using the procedure described in Method 11.
[0267] NMR data were as follows:
[0268] 1H NMR (CD3OD): .delta.=7.67 (d, 2H), 7.40 (d, 2H), 7.27 (d,
2H), 7.09 (d, 2H), 4.61 (m, 1H), 4.12 (m, 2H), 3.99 (m, 2H), 3.60
(m, 2H), 3.23 (m, 8H), 2.58 (s, 3H), 2.42 (s, 3H).
[0269] 13C NMR (CD3OD): .delta.=174.2, 170.3, 155.0, 151.6, 145.6,
136.1, 135.2, 131.5, 131.1, 128.9, 123.0, 54.6, 54.0, 52.4, 52.2,
44.4, 44.0, 37.4, 36.8, 21.4.
[0270] Prep 51 Synthesis of
N-(Toluene-4-sulfonyl)-L-(1,1-dioxothiamorphol-
in-3-carbonyl)-L-4-(N,N-dimethylcarbamyloxy)phenylalanine
tert-Butyl Ester
[0271] The title compound was prepared from the product of Prep 49
following the procedure described by Larsson and Carlson (Acta
Chemica Scan. 1994, 48, 522).
[0272] NMR data were as follows:
[0273] .sup.1H NMR (CDCl.sub.3): .delta.=7.76 (d, 2H), 7.37 (d,
2H), 7.08 (d, 2H), 6.98 (d, 2H), 6.56 (d, 1H), 4.95 (m, 1H), 4.62
(m, 1H), 3.99 (m, 2H), 3.25 (m, 1H), 3.07 (s, 3H), 2.97 (m, 8H),
2.44 (s, 3H), 1.48 (s, 9H).
[0274] 13C NMR (CDCl3): .delta.=170.0, 164.8, 154.9, 150.7, 145.4,
135.3, 132.6, 130.7, 130.3, 127.5, 122.3, 82.8, 56.1, 53.6, 49.5,
48.6, 41.6, 36.6, 36.4, 27.9, 21.6.
[0275] Prep 60 Synthesis of
N-(Toluene-4-sulfonyl)-L-(1,1-dioxothiamorphol-
in-3-carbonyl)-L-4-(N,N-dimethylcarbamyloxy)phenylalanine
(Reference Compound 6)
[0276] The title compound was prepared from the product of Prep 51
using the procedure described in Method 11.
[0277] NMR data were as follows:
[0278] 1H NMR (CDCl3): .delta.=7.79 (d, 2H), 7.43 (d, 2H), 7.20 (d,
2H), 7.00 (d, 2H), 5.21 (m, 1H), 4.65 (m, 1H), 4.12 (m, 1H), 3.75
(m, 1H), 3.29 (m, 3H), 3.08 (s, 3H), 3.00 (m, 1H), 3.00 (m, 1H),
2.97 (s, 3H), 2.80 (m, 3H), 2.44 (s, 3H).
[0279] 13C NMR(CDCl3): .delta.=165.1, 159.0, 147.9, 143.1, 137.6,
128.6, 126.1, 122.7, 122.6, 119.8, 114.3, 48.3, 45.8, 41.6, 34.0,
28.0, 27.8, 27.7, 12.5.
[0280] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention.
* * * * *