Sialyl Lewis X (SLe.sup.x) is a cell surface carbohydrate ligand found on neutrophils, anchored onto the outer membrane thereof by integral membrane glycoproteins and/or glycolipids. SLe.sup.X mediates binding of neutrophils to vascular endothelial cells by binding to E-selectin. (M. Phillips, et al.,. Science. 1990, 250, 1130.; J. Lowe, et al, Cell. 1990, 63, 475; T. Feizi, Trends. Biochem. Sci. 1991, 16, 84; M. Tiemeyer., et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1138; L. Lasky. Science. 1992, 258, 964; and T. Springer, L. A. Lasky, Nature 1991, 349, 196.) E-selectin is a cell surface protein inducibly expressed in endothelial cells in response to inflammatory factors such as interleukin I.beta.(IL-I.beta.) and tumor necrosis factor .alpha.(TNF.alpha.), leukotriene B.sub.4, neurotoxins and bacterial endotoxins, e.g., lipopolysaccharides. These compounds augment polymorphonuclear leukocyte (neutrophil), and monocyte adhesion. Binding of neutrophils to endothelial cells is observed at an early stage after tissue injury and is associated with various acute and chronic inflammations. Neutrophil-mediated inflammatory diseases may be treated by administration of sLe.sup.x. Administration of sLe.sup.x inhibits the sLe.sup.x /E-selectin interaction and blocks adhesion of neutophils to endothelial cells. (M. Buerke, et al., J. Clin. Invest., 1994, 1140.)
In addition to binding to neutrophils, vascular endothelial cells play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the bloodstream. For example, endothelial cells preferentially bind monocytes and granulocytes prior to their migration through the blood vessel wall and into surrounding tissue in an inflammatory response.
Certain inflammation-triggering compounds are known to act directly on the vascular endothelium to promote the adhesion of leukocytes to vessel walls. Cells then move through the walls and into areas of injury or infection.
Cellular adhesion to vascular endothelium is also thought to be involved in tumor metastasis. Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.
Blood platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane fuses with the external plasma membrane. As a result, new cell surface proteins become expressed that confer on the activated platelet new functions, such as the ability to bind both other activated platelets and other cells. Activated platelets are recruited into growing thrombi, or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes that are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.
Specialized cell surface receptors on endothelial cells and platelets, designated E-selectin (endothelial leukocyte adhesion molecule-1; ELAM-1) and P-selectin (granule membrane protein-140; GMP-140), respectively, are involved in the recognition of various circulating cells by the endothelium and platelets. For example, E-selectin has been shown to mediate endothelial leukocyte adhesion, which is the first step in many inflammatory responses. Specifically, E-selectin binds human neutrophils, monocytes, eosinophils, certain T-lymphocytes, NK cells, and the promyelocytic cell line HL-60.
P-selectin (also known as GMP-140 and PADGEM) is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions. Thus, for example, activated platelets that express P-selectin on their surface are known to bind to monocytes and neutrophils, and also to bind monocyte-like cell lines, e.g., HL-60 and U937.
P-selectin is an alpha granule membrane protein of molecular mass 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion. It is also found in megakaryocytes within the Weibel-Palade bodies. Furie et al., U.S. Pat. No. 4,783,330, describe monoclonal antibodies reactive with P-selectin.
A third receptor is the lymphocyte homing receptor, MEL-14 antigen or its human counterpart LAM-1 (L-selectin). In addition to lymphocyte homing, MEL-14 antigen/LAM-1 is believed to function early in neutrophil binding to the endothelium.
The term "selectin" has been suggested for a general class of receptors, which includes E-selectin (ELAM-1), P-selectin (GMP-140) and L-selectin (MEL-14), because of their lectin-like domain and the selective nature of their adhesive functions. The structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors.
The extracellular portion of selectins can be divided into three segments based on homologies to previously described proteins. The N-terminal region (about 120 amino acids) is related to the C-type mammalian lectin protein family as described by Drickamer, J. Biol. Chem., 263:9557-9560 (1988) that induces low affinity IgE receptor CD23. A polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif. Lastly, after the EGF domain are one or more tandem repetitive motifs of about 60 amino acids each, related to those found in a family of complement regulatory proteins.
U.S. Pat. No. 5,079,353 and its divisional Patent No. 5,296,594 teach the synthesis and use of the sialyl Lewis X (sialyl Le.sup.X or SLe.sup.x) and sialyl Lewis A (sialyl Le.sup.a or Sle.sup.a) antigens that are present in cancerous tissues, and are ligands for the before-described selectin receptors. U.S. Pat. No. 5,143,712 teaches the binding interactions between various receptors such as ELAM-1 (E-selectin) and ligands such as sialyl Le.sup.X as well as ligands containing a plurality of N-acetyllactosamine (LacNAc) units along with a terminal sialyl group and one or more fucosyl groups that are bonded to the GlcNAc portion of a LacNAc unit.
Published International application WO 91/19501 and WO 91/19502 disclose that oligosaccharides containing the pentameric and hexameric structures shown below inhibited selective cellular binding between cells containing the ligand (below) and those containing a selectin receptor, and that the penta- and hexasaccharides assayed provided better inhibition than did SLe.sup.x.
NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc.beta. 1,3Gal.beta.-; PA1 NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc.beta. 1,3Gal.beta.1,4Glc-; and PA1 NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc=Sle.s up.x. PA1 R.sub.1 is a radical of formulae (a.sub.1) or (a.sub.2) ##STR2## wherein m is 2 or 3; PA1 n is 2 or 3; PA1 M is a cation; PA1 R.sub.2 is H or a saturated or unsaturated hydrocarbon residue with up to 20 carbon atoms, optionally bearing in .omega. position a formyl or a C.sub.1-4 alcohol acetal or C.sub.2-4 diol acetal group; PA1 R.sub.3 is H, --CH.sub.2 OH or --CH.sub.2 CH.sub.2 OH; and PA1 R.sub.4 is H, C.sub.1-4 alkyl, --CH.sub.2 OH, --CH.sub.2 CH.sub.2 OH or --CH.sub.2 CH.sub.2 CH.sub.2 OH with the provisos that PA1 R.sub.1 is a radical of formula (b) ##STR3## wherein R.sub.5 is ##STR4## wherein p is 1 or 2; PA1 q is 2 or 3; PA1 r is 1 or 2; PA1 R.sub.6 is H, NH.sub.2 or --NHR.sub.x wherein R.sub.x is an amino protecting group; PA1 R.sub.7a is --CH.sub.2 OH, --CH.sub.2 CH.sub.2 OH or --CH(OH)--CH.sub.2 OH and R.sub.7b is H or each of R.sub.7a and R.sub.7b is CH.sub.2 OH; PA1 R.sub.11 is H or --OH; PA1 R.sub.13 is --(CH.sub.2).sub.j --COOM or --SO.sub.3 M wherein j is 1, 2 or 3; and PA1 M is as defined above; PA1 R.sub.1 is a radical of formula (c) ##STR5## wherein R.sub.8 is OM.sub.1, OR.sub.14, R.sub.s -R.sub.p or --NHR.sub.y wherein M.sub.1 is a cation, R.sub.14 is a saturated or unsaturated hydrocarbon residue, R.sub.s is a spacer group, R.sub.p is a phosphatidyl residue and R.sub.y is a saturated or unsaturated lipophilic residue; and PA1 R.sub.9 is ##STR6## wherein s is 1 or 2; PA1 t is 1 or 2; PA1 v is 2 or 3; PA1 M, R.sub.6, R.sub.11 and R.sub.13 are as defined above; and PA1 R.sub.10a is --CH.sub.2 OH, --CH.sub.2 CH.sub.2 OH or --CH(OH)--CH.sub.2 OH and R.sub.10b is H or each of R.sub.10a and R.sub.10b is CH.sub.2 OH; PA1 R is a radical of formula (d) ##STR7## wherein w is 1 or 2; PA1 R.sub.12a is --CH(OH)--(CH.sub.2).sub.x --OH and R.sub.12b is H or each of R.sub.12a and R.sub.12b independently is --CH.sub.2 OH or --H.sub.2 CH.sub.2 OH; PA1 x is 2 or 3; and PA1 R.sub.6 and M are as defined above.
Mulligan et al., Nature, 364; 149-151 (1993) reported upon the in vivo effects of Sle.sup.x and a pentamer such as that above present as a --O(CH.sub.2).sub.5 CO.sub.2 CH.sub.3 glycoside in a neutrophil/P-selection-dependent rat model. Those authors found that intravenous infusion of up to 200 .mu.g of SLe.sup.x or the pentamer dramatically reduced lung injury and diminished tissue accumulation of neutrophils in rats that received an intravenous infusion of cobra venom. Based on the concentrations used, 200 .mu.g, the effective intravenous concentration of SLe.sup.x was calculated to be less than 1 .mu.M.
DeFrees etal., J. Am. Chem. Soc., 117:66-79 (1995) reported on the in vitro inhibition of binding between E-selectin and SLe.sup.x -bearing HL-60 cells for a number of SLe.sup.x -related materials including SLe.sup.x itself, an ethyl glycoside of the above pentamer and a number of bivalent SLe.sup.x analogs. Those authors noted that although the affinity of SLe.sup.x for E-selectin is relatively weak in vitro, the IC.sub.50 value in vivo for protecting against lung injury in rats was in the 1 .mu.M range.
Although SLe.sup.x has been considered to be potentially useful as anti-inflammatory agent and its synthesis on large scales has been developed for clinical evaluation, this natural saccharide can only be used as an injectable form in cases presenting with acute symptoms as it is orally inactive and unstable in the blood stream, because of glycosidase reductions.
The search for novel SLe.sup.x mimetics with simpler structure, higher affinity for the receptor, and better stability against glycosidases, especially fucosidase and sialidase, has been of current interest. A sLe.sup.X mimetic is a compound which includes the functional groups of sLe.sup.X and which mimics the active conformation of sLe.sup.x in space, but which lacks one or more of the glycosidic bonds of sLe.sup.X and/or one or more of the saccharide subunits or analogs thereof. Several active sLe.sup.X mimetics and sLe.sup.X analogs have been designed and synthesized, e.g., a) Allanson, et al., Tetrahedron Lett, 34:3945 (1993), 3945 (30-fold less active than SLe.sup.x); b) Ragan, et al., Bioorg. Med. Chem. Lett, 4:2563 (1994) (a mixture of 4 diastereomers with 40- to 50-fold less activity); c) Hanessian, et al., Synlett, 868 (1993) (inactive); and d) H. Huang and C.-H. Wong. J. Org. Chem. 1995, 60, 3100; J. C. Prodger, et al. Tetrahedron Lett. 1995, 36, 2339; and B. N. Narasinga Rao,. J. Biol. Chem. 1994, 269, 19663. Two sLe.sup.X mimetics synthesized by Uchiyama et al. are of particular note because they exhibit activities similar to sLe.sup.x in the E-selectin binding assay. (T. Uchiyama, et al. J. Am. Chem. Soc. 1995, 117, 5395.) For active natural products inhibiting E-selectin, see Narasinga Rao, et al., J. Biol. Chem., 269:19663 (1994).
The key structural features of sLe.sup.x required for recognition by E-selectin have been determined by structural and conformational studies and by comparative studies of the blocking activity of sLe.sup.X analog families. (B. Brandley, Glycobiology 1993, 3, 633; S. DeFrees, J. Am. Chem. Soc. 1993, 115, 7549; J. Ramphal, J. Med. Chem. 1994, 37, 3459; D. Tyrrell, Proc. Natl. Acad. Sci. USA 1991, 88, 10372; R. Nelson, J. Clin. Invest. 1993, 91, 1157; and A. Giannis, Angew. Chem. Int. Ed. Engl. 1994. 33. 178.) The solution conformation of sLe.sup.x has been characterized using physical methodologies. (Y. C. Lin, et al., J. Am. Chem. Soc. 1992, 114, 5452; Y. Ichikawa, et al. J. Am. Chem. Soc., 1992, 114, 9283; and G. E. Ball et al., J. Am. Chem. Soc., 1992, 114, 5449.) The three-dimensional structure of the human E-selectin has been characterized by X-ray diffraction. (B. J. Graves, et al., Nature, 1994, 367, 532.) It has been found that the L-fucose, D-galactose (Gal) and sialic acid moieties of sLe.sup.x are the major components that interact with E-selectin. N-acetylglucosamine unit appears to act merely as a linker to connect L-fucose and sialyl galactose. The six functional groups of sLe.sup.x molecule including the 2-, 3- and 4-OH groups of L-fucose, the 4- and 6-OH groups of Gal and the --CO.sub.2 -- group of sialic acid are essential for E-selectin recognition, as illustrated in FIG. 1.
Although sLe.sup.x and active sLe.sup.x analogs can be employed as anti-inflammatory agents, these tetrasaccharides can only be used in acute symptoms as they are unstable in the blood and orally inactive. In addition, it is generally difficult to synthesize oligosaccharides on a large-scale. The use of sLe.sup.X mimetics can obviate the above problems associated with sLe.sup.X analogs. Unfortunately, sLe.sup.X mimetics generally have low activity. What are needed are sLe.sup.x mimetics which are more stable as compared to sLe.sup.x and sLe.sup.x analogs; which possess better bioavailability as compared to sLe.sup.x and sLe.sup.x analogs; which are easier to synthesize than sLe.sup.x and sLe.sup.x analogs; and which exhibit greater activity as compared to known sLe.sup.X mimetics.
USPTO Backgrounds
USPTO Backgrounds