Cell adhesion inhibitors

Details Cell adhesion inhibitors Cell adhesion inhibitors

1998-08-10

2001-05-29

Davenport, Avis M. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S016700, C514S017400, C514S018700, C530S326000, C530S327000, C530S328000

Reexamination Certificate

active

06239108

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel compounds that are useful for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. This invention also relates to pharmaceutical formulations comprising these compounds and methods of using them for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. The compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well-suited for treatment of many inflammatory and autoimmune diseases.
BACKGROUND OF THE INVENTION
Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. As such, cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hemopoietic cells out of blood vessels and to the site of injury. As such, cell adhesion plays a role in pathologies such as inflammation and immune reactions in mammals.
Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromolecules—collectively known as cell adhesion molecules or receptors—mediate cell-cell and cell-matrix interactions. For example, proteins of the superfamily called “integrins” are key mediators in adhesive interactions between hematopoietic cells and their microenvironment (M. E. Hemler, “VLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.”,
Ann. Rev. Immunol.,
8, p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called &agr; and &bgr;. There are at least 12 different &agr; subunits (&agr;
1
-&agr;
6
, &agr;-L, &agr;-M, &agr;-X, &agr;-IIB, &agr;-V and &agr;-E) and at least 9 different &bgr; (&bgr;1-&bgr;9) subunits. Based on the type of its &agr; and &bgr; subunit components, each integrin molecule is categorized into a subfamily.
&agr;4&bgr;1 integrin, also known as very late antigen-4 (“VLA-4”) or CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions (M. E. Hemler,
Ann. Rev. Immunol.,
8, p. 365 (1990)). It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (“VCAM-1”), as well as to the extracellular matrix protein fibronectin (“FN”) (Ruegg et al.,
J. Cell Biol.,
177, p. 179 (1991); Wayner et al.,
J. Cell Biol.,
105, p. 1873 (1987); Kramer et al.,
J. Biol. Chem.,
264, p. 4684 (1989); Gehlsen et al.
Science,
24, p. 1228 (1988)). Anti-VLA4 monoclonal antibodies (“mAb's”) have been shown to inhibit VLA4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al.
Proc. Natl. Acad. Sci.,
88, p. 8072 (1991); Ferguson et al.,
J. Immunol.,
150, p. 1172 (1993)). Results of in vivo experiments suggest that this inhibition of VLA-4-dependent cell adhesion may prevent or inhibit several inflammatory and autoimmune pathologies (R. L. Lobb et al., “The Pathophysiologic Role of &agr;4 Integrins In Vivo”,
J. Clin. Invest.,
94, pp. 1722-28 (1994)).
In order to identify the minimum active amino acid sequence necessary to bind VLA-4, Komoriya et al. (“The Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valine”,
J. Biol. Chem.,
266 (23), pp. 15075-79 (1991)) synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. They identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr [SEQ ID NO: 1], as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2] and Leu-Asp-Val-Pro-Ser [SEQ ID NO: 3], that possessed inhibitory activity against FN-dependent cell adhesion. These results suggested the tripeptide Leu-Asp-Val as a minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus bringing into question the utility of such a peptide in vivo (E. A. Wayner et al., “Activation-Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectin”,
J. Cell. Biol.,
116(2), pp. 489-497 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo [T. A. Ferguson et al., “Two Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Vivo”,
Proc. Natl. Acad. Sci. USA,
88, pp. 8072-76 (1991); and S. M. Wahl et al., “Synthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitment”,
J. Clin. Invest.,
94, pp. 655-62 (1994)].
A cyclic pentapeptide,
(wherein TPro denotes 4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described (D. M. Nowlin et al. “A Novel Cyclic Pentapeptide Inhibits &agr;4&bgr;1 and &agr;5&bgr;1 Integrin-mediated Cell Adhesion”,
J. Biol. Chem.,
268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This peptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins.
Despite these advances, there remains a need for small, specific inhibitors of VLA-4-dependent cell adhesion. Ideally, such inhibitors would be semi-peptidic or non-peptidic so that they may be orally administered. Such compounds would provide useful agents for treatment, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding. Co-pending U.S. patent application Ser. No. 08/376,372 describes &bgr;-amino acid containing linear peptidyl compounds with cell adhesion inhibitory activity. International patent applications WO 94/15958 and WO 92/00995 describe cyclic peptide and peptidomimetic compounds with cell adhesion modulating activity. International patent applications WO 93/08823 and WO 92/08464 describe guanidinyl-, urea- and thiourea-containing cell adhesion modulating compounds. U.S. Pat. No. 5,260,277 describes guanidinyl cell adhesion modulation compounds.
SUMMARY OF THE INVENTION
The present invention solves this problem by providing novel semi-peptidic compounds that inhibit the binding of ligands to VLA-4. These compounds are useful for inhibition, prevention and suppression of VLA-4-mediated cell adhesion and pathologies associated with that adhesion, such as inflammation and immune reactions. The compounds of this invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, prevent or suppress cell adhesion. This invention also provides pharmaceutical formulations containing these VLA-4-mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention for inhibition of cell adhesion.
According to one embodiment of this invention, these novel compounds, compositions and methods are advantageously used to treat inflammatory and immune diseases. The present invention also provides methods for preparing the compounds of this invention and intermediates useful in those methods.
DETAILED DESCRIPTION OF THE INVENTION
The following abbreviations are used in the description:
Designation
Reagent or Fragment
Ac
acetyl
Bn
benzyl
Boc
tert-butoxycarbonyl
Bu
butyl
Cbz
carbobenzyloxy
Cy
cyclohexyl
CyM
cyclohexylmethyl
DIPEA
diisopropylethylamine
EDC
1-(3-diethylaminopropyl)-3-
ethylcarbodiimide
HOBT
1-hydroxybenzotriazole hydrate
i-amyl
isoamyl
i-Pn
isopentyl
i-Pr
isopropyl
Me
methyl
2-MPUBA
4-(N′-(2-methylphenyl)urea)-
phenylmethylamino
2-MPUPA
4-(N′-(2-methylphenyl)urea)-
phenylacetyl
NMP
N-methylpyrrolidinone
NMM
N-methylmorpholine
Ph
phenyl
PUPA
4-(N-phenylurea)phenyl

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