Non-peptidyl inhibitors of VLA-4 dependent cell binding...

Details Non-peptidyl inhibitors of VLA-4 dependent cell binding... Non-peptidyl inhibitors of VLA-4 dependent cell binding...

2002-06-12

2003-12-30

Desai, Rita (Department: 1625)

Harness for working animal

Thill tugs

C514S374000, C548S215000, C548S240000, C546S271400, C546S272100

Reexamination Certificate

active

06668527

ABSTRACT:

The present invention relates to compounds which are non-peptidyl in structure and active as potent inhibitors of the binding of very late antigen-4 (VLA-4; &agr;
4
&bgr;
1
; CD49d/CD29) to proteins such as vascular cell adhesion molecule-1 (VCAM-1), the HepII/IIICS domain (CS-1 region) of fibronectin and osteopontin. As such they are useful in the inhibition of cell adhesion and consequent or associated pathogenic processes subsequently mediated by VLA-4. The compounds and pharmaceutical compositions of this invention may be used in the treatment of many inflammatory, autoimmune and respiratory diseases, especially asthma.
BACKGROUND OF THE INVENTION
One of the most fundamental processes necessary for normal host defence is the regulated trafficking of leukocytes out of the vasculature. This system is designed to allow normal recirculation of leukocytes, yet because it enables the rapid extravasation of leukocytes at sites of injury it is one of the central pathogenic mechanisms of inflammatory, respiratory and autoimmune diseases in mammals. Cell adhesion is a key factor in this process, and it is particularly relevant to the present invention regarding the cell/cell and cell/matrix binding of hematopoietic cells containing VLA-4.
VLA-4 is a member of a superfamily of cell surface macromolecular receptors called integrins, which are non-covalent heterodimeric complexes consisting of an &agr; subunit and a &bgr; subunit (Hemler,
Ann. Rev. Immunol.,
8, p.365, 1990). Eighteen different &agr; subunits have been identified and labeled &agr;
1
-&agr;
10
, &agr;
L
, &agr;
M
, &agr;
x
, &agr;
d
, &agr;
LRI
, &agr;
IIB
, &agr;
V
, and &agr;
E
; while eight different &bgr; subunits have been identified and labeled &bgr;
1
-&bgr;
8
. Each integrin molecule can be categorized into a subfamily based on the type of its &agr; and &bgr; subunits. The &agr;
4
&bgr;
1
integrin, VLA-4, is an integrin constitutively expressed by all leukocytes (e.g., monocytes, lymphocytes, basophils, eosinophils, mast cells and macrophages) except polymorphonuclear leukocytes. The binding of this integrin to one of its ligands has a number of known cell adhesion and activation functions (Hemler,
Ann. Rev. Immunol.,
8, p.365, 1990; Walsh et al.,
Clin. and Exp. Allergy,
25, p. 1128, 1995; Huhtala et al.,
J. Cell Biol.,
129, p. 867, 1995). In particular, it is a receptor for the cytokine-inducible endothelial cell surface protein known as vascular cell adhesion molecule-1 (VCAM-1), for the alternatively spliced forms of the extracellular matrix protein fibronectin (FN) containing the CS-1 domain (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) and for the extracellular matrix protein osteopontin (Bayless, K. I. et al.
J. Cell Science,
111, p. 1165-1174, 1998). The importance of VLA-4 cell adhesion interactions has been established by the use of specific monoclonal antibody (mAb) antagonists of the a subunit of VLA-4, which have demonstrated that inhibitors of VLA-4 dependent cell adhesion prevent or inhibit numerous inflammatory, respiratory and autoimmune pathological conditions (Chisholm et al.,
Eur. J. Immunol.,
23, p. 682, 1993; Lobb et al.,
J. Clin. Invest,
94, p. 1722, 1994; Richards et al.,
Am. J. Respir. Cell Mol. Biol.,
15, p.172, 1996; Soiluhanninen et al.,
J. Neuroimmunol.,
72, p. 95, 1997; Sagara et al.,
Int. Arch. Allergy Immunol.,
112, p.287, 1997; Fryer et al.,
J. Clin. Invest.,
99, p. 2036, 1997). In addition, confirmation that this pathological processes can be inhibited with agents other than antibodies has been observed in animal models following treatment with a synthetic CS-1 peptide or a small molecule peptide inhibitor of VLA-4 (Ferguson et al.,
Proc. Natl. Acad. Sci.,
88, p.8072, 1991; Wahl et al.,
J. Clin. Invest.,
94, p.655, 1994; Molossi et al.,
J. Clin. Invest.,
95, p.2601, 1995; Abraham et al.,
Am. J. Respir. Crit. Care Med.,
156, p. 696, 1997; Jackson et al.,
J. Med. Chem.,
40, p. 3359, 1997).
3.0 Description of the State of the Art
The investigation of mAb and peptide VLA-4 antagonists in the art has already been noted above. In defining the binding site for &agr;
4
&bgr;
1
it has been observed that lymphoid cells can bind to two different sites on fibronectin (Bernardi et al.,
J. Cell Biol.,
105, p. 489, 1987). One component of this cell binding activity has previously been identified as the tripeptide Arg-Gly-Asp (RGD) that binds to the integrin &agr;
5
&bgr;
1
(VLA5). Subsequently, the minimum amino acid sequence required to bind and antagonize the activity of VLA-4 on leukocytes to the alternatively spliced site in fibronectin was determined (Humphries et al.,
J. Biol. Chem.,
266, p.6886, 1987; Garcia-Pardo et al.,
J. Immunol.,
144, p.3361, 1990; Komoriya et al.,
J. Biol. Chem.,
266, p. 15075, 1991). It has been discovered that the VLA-4 binding domain in the CS-1 region of fibronectin (FN) comprises the octapeptide: Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two overlapping pentapeptides: Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser. All of these peptides inhibit FN-dependent cell adhesion, leading to the early conclusion that the minimal amino acid sequence required for inhibition would be Leu-Asp-Val (LDV). In fact the LDV minimal inhibitory sequence has been observed to be equally effective as the full length CS-1 fragment in binding the activated form of VLA-4 (Wayner et al.,
J. Cell Biol.,
116, p. 489, 1992).
Various integrins are believed to bind to extracellular matrix proteins at an Arg-Gly Asp (RGD) recognition site. RGD based cyclic peptides have been made that are said to be able to inhibit both &agr;
4
&bgr;
1
and &agr;
5
&bgr;
1
binding to FN (Nowlin et al.,
J. Biol. Chem.,
268, p. 20352, 1993; PCT/US91/04862) even though the primary recognition on FN for &agr;4&bgr;1 is LDV. The cyclic peptide may be represented by Formula (0.0.1):
where TPro denotes 4-thioproline.
Other peptidyl inhibitors of VLA-4 are those referred to in Arrhenius et al., “CS-1 Peptidomimetics,” WO 95/15973, which is assigned to the Cytel Corporation and is related to the two U.S. patents noted below. A representative compound of the type described is the peptide of Formula (0.0.2):
N-Phenylacetyl-Leu-Asp-Phe-NCy
3
  (0.0.2)
wherein NCy
3
is selected from, inter alia, morpholinamido; thiomorpholinamido; 4-(thiadioxo)piperidinamido; and D-2-(carboxamide)-pyrrolidinamido; piperidinamido; and substituted piperidinamido.
WO 95/15973 . . . U.S. Pat. No. 5,821,231 . . . U.S. Pat. No. 5,936,065 . . . Cytel Corporation
Further work by Arrhenius et al. involving cyclic CS-1 peptidomimetics is described in WO 96/06108 assigned to the Cytel Corporation, which is related to the U.S. patent noted below.
WO 96/06108 . . . U.S. Pat. No. 5,869,448 . . . Cytel Corporation
The Arrhenius group has also discovered non-peptidal inhibitors of VLA-4 dependent cell binding as described in He et al., WO 98/42656. The inhibitors therein described are of the general Formula (0.0.3):
A typical inhibitor is that represented by Formula (0.0.4):
WO 98/42656 . . . Cytel Corporation
The Leu-Asp-Val tripeptide has been used as the core of a group of inhibitors of VLA-4 dependent cell adhesion described in Adams et al. WO 96/22966, which is assigned to Biogen, Inc. These inhibitors may be represented by Formula (0.0.5):
where R
1
may be 4-(N′-(2-methylphenyl)urea)phenylmethyl; Y may be C═O; R
2
may be H; R
3
may be iso-butyl; and R
14
may be 1,3-benzodioxol-5-yl. An example of a typical inhibitor of this type is that of Formula (0.0.6):
WO 96/22966 . . . Biogen, Inc.
The Adams group has also discovered semi-peptidic cell adhesion inhibitors for the treatment of inflammation and autoimmune disease, described in Lin et al., WO 97/03094. These inhibitors may be represented by Formula (0.0.7):
Z-(Y
1
)-(Y
2
)-(Y
3
)
n
-X  (0.0.7)
where Z may be 4-(N′-(2-methylphenyl)urea)phenylacetyl; (

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