MXPA02009543A

MXPA02009543A - Method of identifying inhibitors of tie 2.

Info

Publication number: MXPA02009543A
Application number: MXPA02009543A
Authority: MX
Inventors: Cornelia Bellamacina
Original assignee: Abbott Gmbh & Co Kg
Priority date: 2000-03-29
Filing date: 2001-03-20
Publication date: 2005-08-26
Also published as: AU2001287268A1; EP1268549A2; WO2001072778A2; JP2004514405A; CA2404532A1; HK1053844A1; WO2001072778A3

Abstract

The present invention relates to polypeptides which comprise the ligand binding domain of Tie 2, crystalline forms of these polypeptides and the use of these crystalline forms to determine the three dimensional structure of the catalytic domain of Tie 2. The invention also relates to the use of the three dimensional structure of the Tie 2 catalytic domain both alone, or in complex with inhibitors, in methods of designing and or identifying potential inhibitors of Tie 2 activity, for example, compounds which inhibit the binding of a native substrate to the Tie 2 catalytic domain.

Description

METHOD TO IDENTIFY TIE-2 INHIBITORS RELATED REQUEST This application claims the benefit of the provisional application for E.U.A. No. 60 / 192,920, filed on March 29, 2000. All the teachings of. the above request are incorporated herein by reference.

BACKGROUND OF THE INVENTION Angiogenesis is a fundamental procedure through which new blood vessels are formed through budding, branching, proliferation and tubule formation by existing vasculature endothelial cells. In healthy human beings, this neovascularization is under severe control, usually occurring during embryonic development, endometrial regulation, lactation and wound repair. However, in many pathological conditions, such as rheumatoid arthritis, solid tumors, Kaposi's sarcoma, blindness due to ocular neovascularization, psoriasis and atherosclerosis, the progression of the disease depends on persistent angiogenesis, the vasculature, which is the conduit for the supply of drugs is one of the most accessible tissues in the body. Each endothelial cell of tumor vessels is estimated to support 100 to 1000 surrounding cells, even in the absence of an endothelial cell of angiogenic stimulus typically divided only once every 1000 days. A number of polypeptide growth factors and their associated endothelial cell-specific receptors have been discovered, which are mainly responsible for the stimulation of endothelial cell growth, differentiation and the establishment of new vasculature. These growth factor receptors include vascular endothelial growth factor (VEGFR) receptors Flk-1 (mouse), KDR / VEG-FR-2 (human), Flt-1 / VEGFR-1, and Flt-4 / VEGFR- 3. The receptors that are responsible for the neu- vasization also include the receptor tyrosine kinases Tie-1 and Tie-2. Due to this role in the regulation of new vascular development, Tie-2 is a potential target for therapies aimed at controlling diseases that depend on persistent angiogenesis. The development of biochemical assays for Tie-2 has allowed drug discovery to proceed along trajectories to identify major Tie-2 inhibitors through the high-throughput classification of composite collections and testing compounds that mimic the structure of the substrate; however, rational structure-based design up to this point has not been possible due to the lack of accurate three-dimensional structural data for Tie-2 receivers.

COMPENDIUM OF THE INVENTION The present invention relates to a polypeptide comprising the catalytic domain of Tie-2, a crystalline form of this polypeptide and the use of structural information derived from the crystalline form of the polypeptide to design and / or identify potential inhibitors of the binding of one or more ligands native to the catalytic domain of Tie-2. In one embodiment, the present invention also relates to a polypeptide comprising the catalytic domain of Tie-2 and having the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the invention relates to a crystalline form of this polypeptide or the polypeptide in complex with the ligand. In another embodiment, the invention provides a method for determining the three-dimensional structure of a crystalline polypeptide comprising the catalytic domain of Tie-2. In one embodiment, the method comprises the steps of, (1) obtaining a crystal of the polypeptide comprising the catalytic domain of Tie-2; (2) obtain X-ray diffraction data for said crystal; and (3) solving the crystal structure of said crystal. The method optionally comprises the additional step of obtaining the polypeptide, with the three-dimensional structure that will be determined, before obtaining the crystal of said peptide. In another embodiment, the method comprises the steps of, (1) obtaining a crystal of the polypeptide comprising the catalytic domain of Tie-2.; (2) obtain X-ray diffraction data for said crystal; and (3) solving the crystal structure of said crystal using X-ray diffraction data and the atomic coordinates for the Tie-2 catalytic domain of a second polypeptide. The method optionally comprises the additional step of obtaining the polypeptide, with the three-dimensional structure that will be determined, before obtaining the crystal of said peptide. The invention further relates to a method for identifying a compound that inhibits the catalytic activity of Tie-2, for example, by inhibiting the binding of natural substrates such as the tyrosyl polypeptide or protein or ATP, to the catalytic domain of Tie-2. Said compound is referred to herein as a "Tie-2 inhibitor". The method comprises the steps of (1) using a three-dimensional Tie-2 structure as defined by the atomic coordinates of the Tie-2 catalytic domain; (2) employing the three-dimensional structure to design or select a potential inhibitor; and (3) determining the ability of the selected compound to inhibit the catalytic amount of Tie-2. The method also includes the step of providing the compound designed or selected in step 2, for example, by synthesizing the compound or obtaining the compound from a collection of compound. In addition, the method can include the step of determining the ability of the identified compound to bind to the Tie-2 catalytic domain and / or determining the ability of the identified compound to inhibit the binding of a natural Tie-2 ligand.

In another embodiment, the method for identifying a compound that inhibits the catalytic activity of Tie-2, comprises the step of determining the ability of one or more functional groups and / or portions of the compound, when present in or linked to the catalytic domain. of Tie-2, to interact with one or more subsites of the Tie-2 catalytic domain. In general, the catalytic domain of Tie-2 is defined by. homologous sequences conserved when compared to other known tyrosine kinases. If the compound is capable of interacting with a preselected number or group of subsites, it obtains a calculated interaction energy within a desired or pre-selected scale, the compound is identified as a potential inhibitor of Tie-2. The invention further provides a method for designing a compound, which is a potential inhibitor of the catalytic activity of Tie-2. The method includes the steps of, (1) identifying one or more functional groups capable of interacting with one or more subsites of the Tie-2 catalytic domain; and (2) identify a scaffold presenting the functional group, or functional groups, identified in step 1 in a suitable orientation to interact with one or more subsites of the Tie-2 catalytic domain. The compound resulting from the union of the functional groups or portions identified to the identified scaffold is a potential inhibitor of Tie-2. The catalytic domain of Tie-2, in general, is defined by the atomic coordinates of a polypeptide comprising the catalytic domain of Tie-2.

In yet another embodiment, the invention provides compounds that inhibit the catalytic activity of Tie-2 and that bind, or bind to the catalytic domain of Tie-2. Such compounds typically comprise one or more functional groups which, when the compound binds in the catalytic domain of Tie-2, interact with one or more subsites of the catalytic domain. In general, the catalytic domain Tie-2 is defined by. the homologous sequence conserved when compared to other known tyrosine kinases. In a particular embodiment, the Tie-2 inhibitor is a compound that is identified or designed by a method of the present invention. The present invention further provides a method for treating a condition mediated by Tie-2 in a patient. The method comprises administering to the patient a therapeutically or prophylactically effective amount of a compound that inhibits the catalytic activity of Tie-2, such as a Tie-2 inhibitor of the invention, for example, a compound identified as a Tie-2 inhibitor. or designed to inhibit Tie-2 through a method of the present invention. The present invention provides several advantages. For example, the invention provides the first detailed three-dimensional structures of the ligand binding domain of a Tie-2 protein. The methods described herein can be used to facilitate the formation of Tie-2 crystals, which have diffraction at high resolution. These structures allow the rational development of Tie-2 inhibitors allowing the design and / or identification of molecular structures that have characteristics that facilitate binding to the binding domain of Tie-2. The methods for using the structures described herein, in this manner, allow the faster discovery of compounds that are potentially useful for the treatment of conditions that are mediated, at least in part, by the activity of Tie-2.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequence of human Tie-2 (SEQ ID NO: 1). Figure 2 presents the amino acid sequence that includes the catalytic domain of human Tie-2 from amino acid 802 to amino acid 1124, and has a catalytically inactive point mutation at amino acid 964 (SEQ ID NO: 2). Figures 3A-300 present the atomic coordinates for the complex of SEQ ID NO 2 / inhibitor I. Figures 4A-400 present the atomic coordinates for the complex of SEQ ID NO 2 / inhibitor II. Figures 5A-5RR present the atomic coordinates for the complex of SEQ ID NO 2 / inhibitor III. Figures 6A-6NN present the atomic coordinates for the complex of SEQ ID NO 2 / inhibitor IV. Figure 7 shows the structure of a prototypic kinase, insulin receptor kinase.

Figure 8 shows identified regions of a pyrrolopyrimidine inhibitor (i.e., inhibitor I), which interacts with the catalytic domain of Tie-2. Figure 9 shows a model of the Tie-2 catalytic domain linked to the inhibitor I. The subsites are shown in different colors.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the X-ray crystallographic study of polypeptides comprising the catalytic domain of Tie-2. The atomic coordinates that result from this study are to be used in the identification of compounds that are fixed in the catalytic domain and, therefore, are potential inhibitors of Tie-2. These Tie-2 inhibitors are each to be used in methods to treat a patient having a condition that is modulated by or that depends on the activity of Tie-2, for example, a condition dependent on persistent angiogenesis. There are at least 400 enzymes identified as protein kinases. These enzymes catalyze the phosphorylation of target protein substrates. Phosphorization is usually a transfer reaction of a phosphate group from ATP to the protein substrate. The specific structure in the target substrate to which the phosphate is transferred is a tyrosine, cerin or threonine residue. Since these amino acid residues are the target structures for phosphoryl transfer, these protein kinase enzymes are commonly referred to as tyrosine kinases or serine / threonine kinases. The phosphorylation reactions, and the phosphatase reactions to counteract, in the tyrosine, serine and threonine residues are involved in innumerable cellular procedures that underline responses to various intracellular signals (typically mediated through cellular receptors), regulation of cellular functions and activation or deactivation of cellular procedures. A cascade of protein kinases usually participate in intracellular signal transduction and is necessary for the performance of these cellular procedures. Because of their ubiquity in these procedures, protein kinases can be found as an integral part of the plasma membrane or as cytoplasmic enzymes or localized in the nucleus, usually as components of enzyme complexes. In many cases, these protein kinases are an essential element of enzyme and structural protein complexes that determine when and where a cellular procedure occurs within a cell. Protein tyrosine kinases. Protein tyrosine kinases (PTKs) are enzymes that catalyze the phosphorylation of specific tyrosine residues in cellular proteins. This post-translational modification of these substrate proteins, usually the same enzymes, acts as a molecular switch regulating cell proliferation, activation or differentiation (for review, see Schlessinger and Ulrich, 1992, Neuron 9: 383-391 ). Abnormal or excessive PTK activity has been observed in many disease states, including benign and malignant proliferative disorders as well as diseases resulting from inappropriate activation of the immune system (e.g., autoimmune disorders), rejection of allograft and graft disease against Guest. In addition, endothelial cell specific receptor PTKs, such as KDR and Tie-2, mediate the angiogenic process, and are thus involved in supporting the progression of cancers and other diseases that involve inappropriate vascularization (eg, diabetic retinopathy, choroidal neovascularization due to macular degeneration related to age, psoriasis, rheumatoid arthritis, retinopathy of prematurity, infantile hemangiomas, psoriasis and atherosclerosis). The tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or of the non-receptor type (being completely intracellular).

Tyrosine Receptor kinases (RTKs). Tyrosine receptor kinases (RTKs) comprise a large family of transmembrane receptors with diverse biological activities. Currently, at least nineteen (19) different subfamilies of RTK have been identified. The receptor tyrosine kinase family (RTK) includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ulrich, Ann.Rev. Biochem., 57: 433-478, 1988; Ulrich and Schlessinger. , Cell 61: 243-254, 1990). The intrinsic function of the RTKs is activated after ligand binding, which results in the phosphorylation of the receptor and multiple cellular substrates, and subsequently a variety of cellular responses (Ulrich &Schlessinger, 1990, Cell 61: 203-212 ). Thus, serial transduction by receptor tyrosine kinase-mediated is initiated by extracellular interaction with a specific growth factor (ligand), typically followed by receptor dimerization, restimulation of intrinsic protein tyrosine kinase activity and trans-phosphorylation of receptor. In this way. binding sites are created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (eg, cell division, differentiation, metabolic effects, changes in the extracellular microenvironment ), see, Schlessinger and Ulrich, 1992, Neuron 9: 1-20. Proteins with SH2 (src-2 homology) or phosphotyrosine (PTB) binding domains bind activated tyrosine kinase receptors and their substrates with high affinity to propagate signals to cells. Both domains recognize phosphotyrosine. (Fantl et al., 1992, Cell 69: 413-423, Songyang et al., 1994, Mol. Cell. Biol. 14: 2777-2785, Songyang et al., 1993, Cell 72: 767-778, and Koch et al. , 1991, Science 252: 668-678, Shoelson, Curr Opin, Chem. Biol. (1997), 1 (2), 227-234, Cowburn, Curr Opin. Struct. Biol. (1997), 7 (6 ), 835-838). Several intracellular substrate proteins that are associated with receptor tyrosine kinases (RTKs) have been identified. These can be divided into two main groups: (1) substrates that have a catalytic domain; and (2) substrates that lack such a domain but serve as adapters and associate with catalytically active molecules (Songyang et al., 1993, Cell 72: 767-778). The specific character of the interactions between receptors or proteins and the SH2 or PTB domains of their substrates is determined a. through the amino acid residues immediately surrounding the phosphorylated tyrosine residue. For example, the differences in the binding affinities between the SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues in particular receptors correlate with the differences observed in their substrate phosphorylation profiles (Songyang et al., 1993, Cell 72 : 767-778). The observation suggests that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability, but also by the arrangement of downstream signal transduction paths that are activated by a particular receptor, as well as the time and duration of these stimuli. In this way, phosphorylation provides an important regulatory step that determines the selectivity of signaling pathways hooked by specific growth factor receptors, as well as differentiation factor receptors. Several receptor tyrosine kinases, such as FGFR-1, PDGFR, TIE-2 and c- et, and growth factors that bind to them, have been suggested to play an important role in angiogenesis, although some may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell, Biol. 129: 895-898, 1995). Tie-2 (TEK) is a member of a newly discovered family of receptor-specific endothelial cell tyrosine kinases, in which is involved in critical angiogenic procedures, such as branching, sprouting, remodeling, maturation and vessel stability. Tie-2 is the first mammalian receptor tyrosine kinase for which both agonist ligands have been identified (eg, angiopoietin 1 ("Ang1"), which stimulates receptor autophosphorylation and signal transduction), as antagonist ligands ( for example, angiopoietin 2 ("Ang2")). The out-of-combat and transgenic manipulation of the expression of Tie-2 and its ligands indicates a hermetic and temporal spatial control of Tie-2 signaling that is essential for the proper development of the new vasculature. The current model suggests that Tie-2 kinase stimulation by the Ang1 ligand is directly involved in branching, budding and new vessel growth, and recruitment and interaction of important periendothelial support cells to maintain vessel integrity and induce immobility . The absence of stimulation of Tie-2 Ang1 or the inhibition of Tie-2 autophosphorylation by Ang2, which occurs at high levels in vascular regression sites, can cause a loss in the vascular structure and the matrix contacts can give as resulting in endothelial cell death. especially in the absence of growth / survival stimuli. However, the situation is more complex since at least two additional Tie-2 ligands (Ang3 and Ang4) have recently been reported, and the capacity for heterooligomerization of the various agonistic and antagonistic angiopoietins has been demonstrated, thus modifying their activity . The activation of Tie-2 ligand receptor interactions as an anti-angiogenic therapeutic aspect is thus less favorable and a kinase inhibitor strategy is preferred. The soluble extracellular domain of Tie-2 ("ExTek") may act to interrupt the establishment of tumor vasculature in a breast tumor xenograft and lung metastasis models and in ocular neovascularization mediated by tumor cell. For adenoviral infection the in vivo production of mg / ml levels of ExTek in rodents can be achieved for 7-10 days without any adverse side effects. These results suggest that the disruption of Tie-2 signaling pathways in normal healthy animals can be well tolerated. These Tie-2 inhibitory responses for ExTek may be a consequence of sequestration of ligand (s) and / or generation of a non-productive heterodimer with full-length Tie-2. Recently, it has been found that significant upregulation of Tie-2 expression within the pannus synovium! vascular arthritic joints of humans, according to an important role in the inappropriate neovascularization. This finding suggests that Tie-2 plays an important role in the progression of rheumatoid arthritis. Point mutations that produce constitutively activated forms of Tie-2 have been identified in association with human malformation disorders. Therefore, Tie-2 inhibitors are useful for treating such disorders, and other situations of inappropriate neovascularization. The examples herein describe the preparation and crystallization of polypeptides comprising the catalytic domain of human Tie-2. As used herein, the term "catalytic domain" refers to a specific module common to all ATP-binding kinases, such as the tyrosyl-binding site, the site where ATP binds, including the metal-binding region. -ion, and the site where the phosphoryl transfer occurs. For Tie-2, the catalytic domain is defined through amino acid residues from about residue 828 to about residue 985 of SEQ ID NO: 1, with residues 828-840, 853-855, 872, 873, 876, 879, 880, 885-888, 900, 902-909, 912, 954, 955. 960. 964, 968-971, and 980-985 included in the catalytic domain. The amino acid sequences of native human Tie-2 (SEQ ID NO: 1) are taken as defined in SWISS-PROT (Ziegler, et al., Oncogene 8: 663 (1993)). As described in the examples, certain of these crystals were examined through X-ray crystallography and atomic coordinates were obtained for peptide. In certain cases, the polypeptide was not ligated, that is, it is not complexed with a ligand. In other cases, the polypeptide complexed with a ligand and the atomic coordinates of the ligand bound to the Tie-2 catalytic domain were also obtained. Tie-2 undergoes autophosphorylation and transphosphorylation through other proteins. The phosphorylation status is a post-translational modification particularly important to consider. A wild-type construct (ie, without the mutation of D964N) having residues 802-1124 of SEQ ID NO: 1 was isolated from an expression system as a single or multiple phosphorylated species. An individually phosphorylated species has its phosphate in either Y897 or Y899. In multiply phosphorylated species, phosphorylation may be in combinations of many Y residues on the protein. A disphosphorylated species was crystallized in the space group P2 (1) 2 (1) 2 (1) with the unit cell dimensions of a = 54,320 Á, b = 75.872 A, c = 78.143 A, and a = ß =? = 90.0 °. The term "space group" is a term in the art that refers to the collection of elements of symmetry of the unitary cell of a crystal. Other phosphorylation sites are described by Jones, N. et al., J. Biol. Chem. (1999), 274 (43): 30896. A catalytically inactive Tie-2 mutant (SEQ ID NO: 2) was also crystallized. The catalytically inactive mutant had the same sequence as residues 802 to 1124 of human Tie-2, except that residue 964, which is aspartic acid in human wild-type Tie-2, was replaced with asparagine. This substitution made the mutant catalytically inactive. SEQ ID NO: 2 crystallized in space group C222 (1), which had the unit cell dimensions of a = 75,195 A, b = 116.28 A, c = 95,060 A, and a = ß =? = 90.0 °. The atomic coordinates for four crystals of the Tie-2 / ligand complexes examined through X-ray crystallization are presented in Figures 3A-300, 4A-400, 5A-5RR and 6A-6NN. The term "atomic coordinates" (or "structural coordinates") refers to mathematical coordinates derived from mathematical equations related to the patterns obtained in the diffraction of X-rays through atoms (centers of diffusion) of a crystalline polypeptide comprising a molecule of the catalytic domain of Tie-2 The diffraction data are used to calculate an electron density map of the crystal repeat unit.The electron density maps are used to establish the positions of the individual atoms within the unit cell The atomic coordinates can be transformed as is known in the art, to different coordinate systems without affecting the relative positions of the atoms, in particular, four high-resolution crystal structures were obtained for SEQ ID NO: 2, in complex with one to four different inhibitors, as shown below: ?? Inhibitor III Inhibitor IV The results of determination of crystal structure of X-rays to SEQ ID NO: 2, the catalytic domain of human tie-2, show the following: The overall structure adopted a fold recognizable kinase with a lobe N-terminal and a stly larger C-terminal lobe. The binding of ATP on the adjoining surface of the two lobes with the inhibitors also uniting in this region. The major secondary structural elements of the N-terminal lobe were a beta sheet of five chain structures and a long alpha helix. The C-terminal lobe was mainly a group of alpha helices with a short beta sheet, of two chain structures, near the surface adjacent to the N-terminal lobe. Figure 7 shows a prototypic receptor tyrosine kinase, an insulin receptor kinase that illuminates the structural features associated with known kinases. The structure of the Tie-2 catalytic domain, shown in Figure 9, has characteristics similar to this. The hinge region connects the N-terminal and C-terminal lobes. The portion of the hinge that is part of the ATP / inhibitor binding region has several hydrogen bonding patterns. The carbonyl-oxygen atoms of E903, A905 and P906 and the chain structure amide protons of A905, H907 and G908 were presented in the cavity. The side chains of L900, I902, Y904 and A905 helped to define the size, shape and nature of the joint cavity. The purine core binding region was the region where the N-terminal and C-terminal lobes of the protein cooperate to form a predominantly hydrophobic, flat binding region, which is the traditional location for the purine ring of ATP in other kinase structures. The residues that contribute to this region included I830, A853, V838, I886, L971 and A981. The side chains of residues in the hinge region, I902, Y904 and A905 also contributed to the hydrophobic character of this region. The carbonyl-oxygen of I830 and the amide proton of V838 also presented an interaction site with this region. Through anología to known kinase structures, the ribose ring of ATP can traditionally occupy an area between G831 in the N-terminal lobe and N909 in the C-terminal lobe called extended cavity sugar. Amide protons base structure G831, E832 and N909, the carbonyl-oxygen R968 and side chains E832, N909 and D912 showed patterns of hydrogen bonding. By analogy to known kinase structures, the α-phosphate of ATP can occupy an area around the side chains of residues D964 (N964 in the catalytically inactive mutant, SEQ ID NO: 2). The side chain of R968 also contributes to this region. The predominant type of interaction available was hydrogen bonding, with some complex coordination. The nucleotide binding loop, or glycine-rich loop, was a fin-like loop in the N-terminal lobe that covered the frontal portion of the ATP binding region. Residues not yet described in other bonding areas include D828, V829, G833, N834, F835, G836, Q837, L839, and K840. Residues I830, G831, E832 and V838 were also part of this structural element, but had already been included in other union regions described above. This loop is usually considered to be very flexible and is capable of altering the shape and size of the ATP binding region. The carbonyl-oxygen atoms, the N834 side chain atoms and the base structure amide protons of G833, N834 and F835 are potentially available for hydrogen bonding. The side chain atoms of D828 and K840 were available for ion / hydrogen bonding interactions. These side chain atoms of V829, 1830, F835 and L839 may contribute to hydrophobic interactions. The previous activation loop was a long flexible loop containing at least one residue, the phosphorylation of which, is generally believed to determine the activation state of the protein. The loop starts at the ATP binding site and ends at the C-terminal lobe in the area that most likely corresponds to the substrate junction. Residues F983, G984 and L985 are part of the ATP binding site and were also at the N-terminal site of the activation loop. The carboxy-oxygen and amide protons of F983 and G984 and the amide proton of L985 were available for hydrogen bonding and the side chains of F983 and L985 can contribute to hydrophobic interactions. K855, by homology to known kinases, is part of the catalytic mechanism of the kinase. The amino group can participate in ionic or hydrogen bonding interactions and the methylene groups can contribute to hydrophobic interactions. The side chain is very mobile.

The distant hydrophobic cavity is characterized by a buried hydrophobic cavity. This portion of the ATP binding region is not occupied by any ATP atom in known kinase structures. The residues that contribute to this cavity include L873, L876, L879, 1885, L888, Y954, L955, F960 and 1980. 1886, A981 and F983 from regions already described also contribute to hydrophobic interactions for this region. In addition, there are a number of base structure hydrogen bonding patterns available in this area. These patterns include the carbonyl-oxygen atoms of? 88T, L879 and G880. With the apparent interaction of the alpha-C helix, the carbonyl-oxygen atoms of E882, L873, and L876 are also available. The base structure amide proton of residues I886 and L888 was also available in this region. Several residues contributed to the ATP / inhibitor binding site but do not appear to be part of a subregion that can be defined. These residues are I854, E872, N887, I970 and 1980. E872 usually forms an ionic interaction with the catalytic lysine in known kinase structures. N887 may contribute to the distant hydrophobic cavity. The 1854 and 1970 side chains look away from the ATP cavity, however, the carbonyl-oxygen atoms of these residues as well as 1980 showed up towards the binding region. The structure of the complex of SEQ ID NO: 2 / inhibitor had the following characteristics: The final resolution of the structure was 2.8 A in space group C2221, with the final coordinates determined for base structure atoms of residues 818- 857, 864-965; 1001-1116. The pyrrolopyrimidine ring of inhibitor I formed hydrogen bonds with the residues in the hinge region and interacts with the purine core region. The inhibitor core presented a hydrogen bond donor in the form of the amino proton of the 4-NH2 substituent, for the carbonyl-oxygen of E903. The N3 atom of the pyrimidine ring accepted a hydrogen bond of N-H from the base structure of A905. This ring-core system presented a flat face to the residues of both the C-terminal and N-terminal lobes. The residues in these areas present a hydrophobic surface that "walls" the flat nucleus of the inhibitor. The residues involved in this hydrophobic sandwich region include I830, V838, I86, I902 and L971. The N1 and N7 atoms of the inhibitor nucleus that looks at the solvent exposed the mouth of the junction cavity. The C6 atom of the inhibitor that looks along the axis of the nucleotide binding loop of the N-terminal lobe of the protein. The cyclopentane ring of N7 of inhibitor I was directed towards the solvent, but still within the protein cavity. This region was previously described as the extended sugar cavity. This region was characterized by hydrophobic interactions mainly with I830 and L971. The methylene groups of E832 also contribute in this way.

The phenyl ring attached to C5 of the pyrrolopyrimidine ring was a predominantly hydrophobic area, generated by residues of the purine core region, the distant hydrophobic cavity and methylene groups of the catalytic Usin, K855. The hydrophobic contacts were with residues V838, 1886, 1902, L971 and A981. The 855 lysine was highly mobile, so that it was also possible that the pyrrolopyrimidine meta ring of the chlorine atom was received as a hydrogen bond. The sulfonamide linker can make a transparent hydrogen bond with an amide proton of D982 and can also make a hydrogen bond with the amide proton of F983.

The terminal phenyl ring was located in the distant hydrophobic cavity. The primary contacts were with L876, I886, L888 and F983. The structure of the complexes of SEQ ID NO: 2 / inhibitor II, SEC IDNO: 2 / inhibitor III and SEQ ID NO: 2 / nibibrator IV had the following characteristics: Residues 818-857, 864-995, 1001-1116 have been modeled to the resolved structure. A space group P42212 was observed. All doubling is a still kinase catalytic domain doubling and the binding regions described above for SEQ ID NO: 2 / inhibitor I still belong. The pyrrolopyrimidine, ring B, linker and C ring of inhibitor II in the complex of SEQ ID NO: 2 / inhibitor was found in the same way as inhibitor I. In addition, the group -7-cyclohexyl-N-methylpiperazinyl occupied the extended sugar cavity and made a strong ionic interaction with D912. The pyrrolopyrimidine of inhibitor II was joined in the same manner in the complex of SEQ ID NO: 2 / inhibitor III as inhibitor I. The group? -7-cyclohexyl-N-methylpiperazinyl occupied the extended sugar cavities and made a strong interaction ion with D912 as in the complex of SEQ ID NO: 2 / inhibitor II. Ring B was joined in a manner similar to inhibitor I, however, hydrogen bonding between halogens, fluorine in this case, and K855 was clearer. Sulfonyloxyloxygens of the sulfonamide linker made two transparent hydrogen bonds for amide protons of chain structure of D983 and F983. Ring C occupied the distant hydrophobic cavity with major interactions coming from L876, I886, L888, L900, I900, I902 and F983. The pyrrolopyrimidine nucleus of the inhibitor IV in the complex of SEQ ID NO: 2 / inhibitor IV was found the same as inhibitor I. The group? -7-cyclohexyl-N-methylpiperazinyl occupies the extended sugar cavity and makes a strong ionic interaction with D912 as in SEQ ID NO: 2 / inhibitor II. Ring B binds in a manner similar to inhibitor I, however, there is no halogen atom that acts as a potential hydrogen bonding pattern in inhibitor IV. The oxygen atom of the linker accepted a hydrogen bond of the catalytic lysine, K855. Ring C of the inhibitor IV occupied the distant hydrophobic cavity with the main interactions coming from L876, I886, I902 and F983.

In analysis of the three-dimensional structure of the Tie-2 catalytic domain it has indicated the presence of a number of subsites, each of which includes molecular functional groups capable of interacting with complementary portions of an inhibitor. Subsites 1 to 9 of the Tie-2 catalytic domain as defined above. A summary of the properties of the chemical portions present in each subsite is presented below. The subsites are further characterized according to the properties of the chemical portions with which they are complementary, or with which they can interact. Said portions may include hydrogen bond acceptors, such as hydroxyl, amino, ether, thioether, carboxyl, P = 0 atoms, and carbonyl groups, halogen atoms such as fluorine, chlorine, bromine and iodine atoms; and other groups including a heterogeneous atom having at least one long pair of electrons such as groups containing trivalent phosphorus atoms, di and tetravalent sulfur atoms, oxygen and nitrogen atoms; hydrogen bond donors, such as hydroxyl, thiol, an amide proton, amine protons, carboxylic acid groups and any of the groups listed under the hydrogen atom acceptors to which a hydrogen atom is attached; hydrophobic groups, such as linear, branched or cyclic alkyl, ether or thioalkyl groups; linear, branched and cyclic alkenyl groups; linear, branched or cyclic alkynyl groups; aryl groups, such as mono and polycyclic aromatic hydrocarbyl groups and monocyclic and polycyclic or heteroaryl heterocyclic groups; positively charged groups, such as primary, secondary and tertiary quaternary ammonium groups, imidazolium and other protonated heteroalkyl and heteroaryl moieties, substituted and unsubstituted guanidinium groups, sulfonium groups and phosphonium groups; and negatively charged groups, such as carboxylate, phenylate, thiolate, sulfonamide, sulfamate, boronate, vanadate, sulfonate ,. sulfinate, phosphinate, tetrasolate, and other heteroaryl anions, heterocyclic N-oxides and phosphonate groups. A chemical portion provided may contain one or more of these groups. Subsitio 1: Region of hinge. Hydrogen acceptors: Carbonyl-oxygen base structure of residues E903, A905 and P906 of the proton acceptors of the present. Hydrogen donors: The amide protons of the base structure of the proton donor residues of the present A905, H907 and G908. Hydrophobic groups: The side chains of L900, 1902, Y904 and A905 have hydrophobic groups.

Subsite 2: The Union Region of the Purina Nucleus. Hydrophobic Groups: Residues 1830, A853, V838, 1886, L971, A981 and the side chains of residues I902. Y904 and A905 represent hydrophobic groups. Hydrogen acceptors: Carbonyl-oxygen of I830 presents a proton acceptor.

Hydrogen donors: The proton amide of V838 presents a proton donor.

Subsite 3: The Extended Sugar Cavity. Hydrogen acceptors: The carbonyl oxygen of the base structure of R968 and the side chain carbonyl oxygen of E832, N909 and D912 have protein acceptors. Hydrogen donors: The amide protons chain structure of proton donors G831, E832 and N909 present.

Subsite 4: Region? -Extended Phosphate. Hydrogen binding groups: residues D964 (N964 in the catalytically inactive mutant), N969 and D982 have both proton donor and proton acceptor groups.

Subsitio 5: Nucleotide Binding Loop. Hydrogen acceptors: The carboni-oxygen of the side chain of N834, residue, has a proton acceptor. Hydrogen donors: The amide protons of the base structure of residues G833, N834 and F835 present proton donors. Positively charged group: The K840 side chain present in a positively charged site. Negatively charged group: The side chain of D828 presents a negatively charged site.

Hydrophobic groups: The residues of V829, 1830, F835 and L839 have hydrophobic groups.

Subsite 6: Early Activation Loop. Hydrogen acceptors: The carbonyl oxygen of the base structure of the base residues of residues F983 and G984 has a proton acceptor. Hydrogen donors: The amide protons of the base structure of residues F983, G984 and L985 present proton donors. Hydrophobic groups: The side chains of F983 and L985 have hydrophobic groups.

Subsite 7: The Catalytic Lysine. Positively charged group: The side chain of K855 presents a positively charged site. Hydrophobic group: The methylene groups of the K855 side chain have a hydrophobic group.

Subsitio 8: The distant hydrophobic cavity. Hydrophobic groups: Residues L873, L876, L879. I885, L888, Y954, L955, F960, I980, I886, A981 and F983 of the present invention. Hydrogen acceptors: Carboni-oxygen base structure of residues I886, L879, G880, E872, L873 and L876 present proton acceptors.

Hydrogen donors: The amide protons of the base structure of residues I886 and L888 have proton donors.

Subsite 9: Several interaction sites that contribute to the ATP binding site. Hydrogen acceptors: The carbonyl-oxogens of the base structure of the waste. I854, I970 and I980 present proton acceptors in the ATP binding region. Negatively charged groups: E872 presents a negatively charged group, which usually forms an ionic bond with the catalytic lysine residue K855.

Figure 9 provides a model of the Tie-2 catalytic domain linked to the inhibitor I. The subsites 1-9 of the catalytic domain are each represented in a different color as follows: the hinge region (dark blue), the purine core (light blue), the extended sugar cavity (light purple), the? -phosphate region (dark yellow), the nucleotide binding loop (red), the early activation loop (dark green), the catalytic lysine ( light green), the distant hydrophobic cavity (dark purple), and the various interaction sites (coffee). The inhibitor is presented in a light yellow color. In one embodiment, the present invention provides polypeptides comprising the catalytic domain of Tie-2. crystalline forms of these polypeptides, optionally in complex with a ligand, and the three-dimensional structure of the polypeptides, including the three-dimensional structure of the Tie-2 catalytic domain. In general, these three-dimensional structures are defined by atomic coordinates derived from X-ray crystallographic studies of the polypeptides. The catalytic domain can be non-phosphorylated, monophosphorylated or multiply phosphorylated. Phosphorylation typically occurs. in tyrosine residues. A monophosphorylated species has a phosphate group in Y897 or Y899. The polypeptides can include the Tie-2 catalytic domain from any species, such as a yeast or other single-celled organism, an invertebrate or a vertebrate. Preferably, the polypeptide includes the catalytic domain of a mammalian Tie-2, such as murine Tie-2. Most preferably, the polypeptide includes the catalytic domain of human Tie-2. The polypeptides of the invention also include polypeptides comprising individual nucleotide polymorphisms of the catalytic domain of human Tie-2 or murine Tie-2. In a modality, the polypeptides of the invention, and their crystalline forms include a sequence having at least 80% identity to the catalytic domain of human Tie-2 or murine Tie-2. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment , and non-homologous (different) sequences can be discarded for comparison purposes). In a preferred embodiment, the length of a first aligned sequence for comparison purposes is at least 30%, preferably at least 40%, most preferably at least 50%, preferably at least 60%, and still very preferably 70%, 80% or 90% of the length of the second sequence. The amino acid residues at the corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical in that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The invention also encompasses polypeptides having a minor degree of identity but having sufficient homology in order to perform one or more of the same functions performed by the Tie-2 polypeptides described herein through the amino acid sequence. Homology for a polypeptide is determined through conservative amino acid substitution. Said substitutions are those that substitute a given amino acid in a polypeptide with another amino acid of similar characteristics. Conservative substitutions can probably be phenotypically silent. Typically seen as conservative substitutions are replacements, one for another, for example, between the aliphatic amino acids Ala, Val, Leu, and He; exchange of the hydroxyl residues Ser and Thr, exchange of the acid residues Asp and Glu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg, or replacement between the aromatic residues Phe, Tyr.and Trp . The guidance regarding which amino acid changes are likely to be silent in phenotypic form is found in Bowie et al., Science 247: 1306-1310 (1990). The comparison of sequences and determination of the percentage of identity and homology between two sequences can be achieved using a mathematical algorithm. . { Computational Molecular Biology, Lesk, A.M., ed. Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W. ed. Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds. Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereaux, J., eds., M. Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Biol. (48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available March 29, 2000 at http://www.gcg.com), using either a Blossom 62 matrix or a PA 250 matrix, and a gap weight of, for example, 14 , 16, 12, 10, 8, 6 or 4 and a length weight, for example, 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12 (1): 387 (1984)) (available March 29, 2000 at http: //www.gcg. com), using the matrix NWSgapdna.CMP and a hole weight of, for example, 40, 50, 60, 70 or 80 and a length weight of, for example, 1, 2, 3, 4, 5 or 6. In another modality, the percentage of identity between two sequences The amino acid nucleotide is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0), using, for example , a weight residue table PAM120, a gap length penalty of 12 and a gap penalty of 4. The protein sequences of the present invention, for example, amino acids 802-1124 of human Tie-2 (SEQ. NO: 1), can also be used as a "question sequence" to perform a search against databases to, for example, identify other family members or related sequences. Such investigations can be carried out using the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (J. Mol. Biol. 215: 403-10 (1990)). Searches of BLAST protein can be performed with the XBLAST program, for example, classification = 50, word length = 2, to obtain amino acid sequences homologous to the proteins of the invention. To obtain hollow alignments for comparison purposes, BLAST with hollow can be used as described by Altschul et al. { Nucleic Acids Res. (25 (17): 3389-3402 (1997)). When using the BLAST and BLAST with hollow programs, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used as given on March 29, 2000 at http: //www.ncbi.nlm. nih.gov The homology for amino acid sequences can be defined in terms of the parameters established by the Advanced Blast research available from NCBI (the National Center for Biotechnology Information; see, for Advanced BLAST, www.ncbi.nlm.nih.Qov/cQ) -bin / BLAST / nph-newblast? Jform = (March 29, 2000) These default parameters recommended by a question molecule of length greater than 85 amino acid or nucleotide residues have been established as follows: cost of gap existence, 11, for waste hole cost, 1; Lambda ratio, 0.85. More explanation of version 2.0 of BLAST can be found on related website pages and in Altschul, S. F. et al., Nucleic Acids Res. 25: 3389-3402 (1997). In one embodiment, the polypeptide includes amino acids 802 a 1124 of SEQ ID NO: 1. the polypeptides can also have amino acids 782 to 1124, 782 to 1124, 772 to 1124, 812 to 1124, 822 to 1124, 832 to 1124, 802 to 1114, 802 to 1104, or 802 to 1094 of SEQ ID NIO.-1 . In another embodiment, the polypeptide can be a catalytically inactive Tie-2 mutant, such as SEQ ID NO: 2, wherein the amino acid asparagine in T64 is replaced with an amino acid of aspartic acid (designated as mutant D964N). Other catalytically inactive mutants include substitution of the amino acid asparagine in 964 with alanine, serine, threonine or glycine. In another embodiment, the catalytic domain, which is crystallized, can have amino acid deletions from the native sequence, for example, a polypeptide that is suitable for crystallization can include amino acids 802 to 918 of SEQ ID NO: 1 fused to the amino acids 934 to 1124 of SEQ ID NO: 1 or other related "kinase insert domain" deletions. The crystalline polypeptide, preferably further includes a ligand bound to the catalytic domain of Tie-2. The ligand, preferably, is a small organic molecule (molecular weight less than about 1500), for example, inhibitor I, II, III or IV. In one embodiment, the invention relates to a method for determining the three-dimensional structure of a first polypeptide comprising the catalytic domain of a Tie-2 protein. The method includes the steps of (1) obtaining a crystal comprising the first polypeptide; (2) obtain X-ray diffraction data for said crystal; and (3) using the X-ray diffraction data and the atomic coordinates of a second polypeptide comprising the catalytic domain of a Tie-2 protein to resolve the crystal structure of the first polypeptide, thereby determining the three-dimensional structure of the first polypeptide. The second polypeptide may include the same catalytic domain Tie-2 as the first polypeptide, or a different Tie-2 catalytic domain. Either or both of the first and second polypeptides can optionally complex with a ligand. That is, the crystal of the first polypeptide can comprise a complex of the first polypeptide with a ligand. The atomic coordinates of the second polypeptide, optionally, may include the atomic coordinates of a ligand molecule linked to the second polypeptide. The atomic coordinates of the second polypeptide, in general, have been previously obtained, for example, through X-ray crystallographic analysis of a crystal comprising the second polypeptide or a complex the second polypeptide with a ligand. The atomic coordinates of the second polypeptide can be used to solve the crystal structure using methods known in the art, for example, molecular replacement or isomorphic replacement. Preferably, the second polypeptide comprises the catalytic domain of a mammalian Tie-2, most preferably, human Tie-2. For example, the atomic coordinates that can be used include the atomic coordinates presented here, preferably the atomic coordinates presented in Figures 3-7. The invention also provides a method for identifying a compound that is a potential inhibitor of Tie-2. The method comprises the steps of (1) obtaining a crystal of a polypeptide comprising the catalytic domain of Tie-2; (2) obtain the atomic coordinates of the polypeptide through X-ray diffraction studies using said crystal; (3) use said atomic coordinates to define the catalytic domain of Tie-2; and (4) identify a compound that conforms to the catalytic domain. The method may further include the steps of obtaining, for example, from a collection of compound, or by synthesizing the compound identified in step 4, and determining the ability of the identified compound to inhibit the enzymatic activity of Tie-2. The polypeptide preferably comprises the catalytic domain of a Tie-2 in a mammal. Very preferably, the polypeptide comprises the catalytic domain of human Tie-2 in a preferred embodiment, the polypeptide being a polypeptide of the present invention, as described above. The polypeptide can be crystallized using methods known in the art, such as the methods described in the examples, to give polypeptide crystals that are suitable for X-ray diffraction studies. A polypeptide / crystalline ligand complex can be produced by soaking the polypeptide crystalline resulting in a solution including the ligand. Preferably, the ligand solution is in a solvent wherein the polypeptide is insoluble. The atomic coordinates of the polypeptide (and ligand) can be determined, for example, by X-ray crystallography using methods known in the art. The data obtained from crystallography can be used to generate atomic coordinates, for example, of the polypeptide and ligand atoms, if present. As is known in the art, the solution and refinement of the X-ray crystal structure can result in the determination of coordinates for some or all of the non-hydrogen atoms. The atomic coordinates can be used, as is known in the art, to generate a three-dimensional structure of the Tie-2 catalytic domain. This structure can also be used to determine the ability of any given compound, preferably using computer-based methods, to look at the catalytic domain. A compound is fixed in the catalytic domain if it is of a size and shape suitable for physically receiving in the catalytic domain, that is, if it has a form that is complementary to the catalytic domain and can reside in the catalytic domain without steric interactions or van der Waals important unfavorable. Preferably, the compound includes one or more functional groups and / or portions that interact with one or more subsites within the catalytic domain. Computational methods for evaluating the ability of the compound to be fixed in the catalytic domain, as defined by the atomic coordinates of the polypeptide, are known in the art, and representative examples are given below. In another embodiment, the method for identifying a potential Tie-2 inhibitor comprises the step of determining the ability of one or more groups and / or functional portions of the compound, when present in the catalytic domain of Tie-2 to interact with one or more subsites of the Tie-2 catalytic domain. Preferably, the catalytic domain of Tie-2 is defined by the atomic coordinates of a polypeptide comprising the catalytic domain of Tie-2. If the compound is capable of interacting with a preselected number or group of subsites, the compound is identified as a potential inhibitor of Tie-2. A functional group or moiety of the compound is said to "interact" with a subsite of the Tie-2 catalytic domain if it participates in an energetically favorable interaction stabilization with one or more complementary portions within the subsite. Two chemical moieties are "complementary" if they are capable of, when conveniently placed, participating in an attractive or stabilizing interaction, such as an electrostatic or van der Waals interaction. Typically, the attractive interaction is an ion-ion (or salt bridge), ion-dipole, dipole-dipole, dihydrogen, or pi-pi or hydrophobic interaction. For example, a negatively charged portion and a positively charged portion are complementary since, if placed properly, it can form a salt bridge. Also, a hydrogen bond donor and a hydrogen bond acceptor are complementary if placed conveniently. Typically, a determination of interactions between the test compound and the Tie-2 catalytic domain can employ computer-based computational methods, such as those known in the art, where possible interactions of a compound with the protein are evaluated, as defined by the atomic coordinates, with respect to the interaction resistance by calculating the interaction energy on the binding of the compound to the protein. Compounds that have interaction energies calculated within a pre-selected scale or which otherwise, in the opinion of the chemical computation that employs the method, have the greatest potential as inhibitors of Tie-2, can be provided, for example , from a collection of compound or through synthesis, and analyzed for the ability to inhibit Tie-2. The interaction energy for a given compound generally depends on the ability of the compound to interact with one or more sites within the protein catalytic domain. In one embodiment, the atomic coordinates used in the method are the atomic coordinates set forth in Figures 3A-300, 4A-400, 5A-5RR and 6A-6NN. It should be understood that the coordinates set forth in Figures 3-6 can be transformed, for example, to a different coordinate system, in ways known to those skilled in the art without substantially changing the three-dimensional structure represented therein. In certain cases, a portion of the compound may interact with a subsite through two or more individual interactions. A portion of the compound and a subsite can interact if they have complementary properties and are placed in sufficient proximity and in a suitable orientation for a stabilization interaction to occur. The possible distance scale for the portion of the compound and the subsite depends on the distance dependency of the interaction, as is known in the art. For example, a hydrogen bond typically occurs when a hydrogen bond donor atom, which carries a hydrogen atom, and an acceptor atom of a hydrogen bond are separated by approximately 2.5 A and approximately 3.5 A. Hydrogen bonds they are well known in the art (Pimentel et al., The Hydrogen Bond, San Francisco: Freeman (1960)). In general, all the interaction, or binding, between the compound and the Tie-2 catalytic domain will depend on the number and strength of these individual interactions. The ability of a test compound to interact with one or more subsites of the Tie-2 catalytic domain can be determined by computationally evaluating interactions between functional groups, or portions, of the test compound and one or more amino acid side chains in a subsite of particular protein, such as subsites 1 to 9 above. Typically, a compound that is capable of participating in stabilization interactions with a preselected number of subsites, preferably without participating simultaneously in significant destabilization interactions, is identified as a potential inhibitor of Tie-2. Said compound will interact with one or more subsites, preferably with two or more subsites and, most preferably, with one or more subsites. The invention further provides a method for designing a compound that is a potential Tie-2 inhibitor. The method includes the steps of (1) identifying one or more functional groups capable of interacting with one or more subsites of the Tie-2 catalytic domain; and (2) identifying a scaffold that presents the functional group or functional groups identified in step 1 in a suitable orientation to interact with one or more subsites of the Tie-2 catalytic domain. The compound resulting from the union of the functional groups or portions identified to the identified scaffold is a potential inhibitor of Tie-2. The catalytic domain of Tie-2, generally, is defined by the conserved homologous sequence when compared to other known tyrosine kinases, for example, the atomic coordinates set forth in Figures 3A-300, 4A-400, 5A-5RR and 6A -6NN Suitable methods, as is well known in the art, can be used to identify chemical portions, fragments or functional groups that are capable of interacting favorably with a particular subsite or groups of subsites. These methods include, but are not limited to: interactive molecular charts; molecular mechanics; conformational analysis; energy evaluation; coupling; search of database; pharmacophore modeling; design and estimation of node property. These methods can also be employed to assemble chemical portions, fragments or functional groups into a single inhibitory molecule. These same methods can be used to determine whether a given chemical portion, fragment or functional group is capable of interacting favorably with a particular subsite or group of subsites. In one embodiment, the design of potential human Tie-2 inhibitors starts from the general perspective of the three-dimensional shape and electrostatic complementarity for the catalytic domain, spanning subsites 1-9, and subsequently, interactive molecular modeling techniques can be applied by anyone skilled in the art to visually inspect the quality of fit of a candidate inhibitor molded to the binding site. Suitable visualization programs include INSIGHTII (Molecular Simulations Inc., San Diego, CA), QUANTA (Molecular Simulations Inc., San Diego, CA), SYBYL (Tripos Inc., St. Louis, MO), RASMOL (Roger Sayle and others, Tends Biochem Sci. 20: 374-376 (1995)), GRASP (Nicholls et al., Proteins 11: 281-289 (1991)), and MIDAS (Ferrin et al., J. Mol. Graphics (6: 13- 27 (1988).) A further embodiment of the present invention utilizes a database search program that is capable of scanning a database of small molecules of known three-dimensional structure for candidates that bind to the target protein binding site. Suitable software programs include CATALYST (Molecular Simulations Inc., San Diego, CA), UNITY (Trypos inc, St. Louis, MO), FLEXX (Rarey et al., J. Mol. Biol. 261: 470-489 ( 1996)), CHEM-3DBS (Oxford Molecular Group, Oxford, UK), DOCK, (Kuntz et al., J. Mol. Biol. 161: 269-288 (1982)), and MACCS-3D (MDL Information Systems Inc. , San Leand rock). It is not expected that the molecules found in the search will necessarily be conducted by themselves, since a full evaluation of all interactions during the initial search will necessarily be made. Rather, it is anticipated that such candidates should act as the structure for an additional design, providing molecular skeletons to which appropriate atomic replacements can be made. Of course, the chemical complementarity of these molecules can be evaluated, but it is expected that the scaffolding, functional groups, linkers and / or monomers can be changed to maximize the electrostatic, hydrogen-binding and hydrophobic interactions with the enzyme. Goodford (Goodford J. Med. Chem. 28: 849-857 (1985)) has produced a computer program, GRID, which seeks to determine regions of high affinity for different chemical groups (called probes) on the molecular surface of the binding site. . The GRID program in this way provides a tool to suggest modifications to known ligands that can improve binding. A scale of factors, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, tension or conformational mobility, chelation and interaction and cooperative movements of ligand and enzyme, all have an influence on the level of union and must be taken into account in attempts to design bioactive inhibitors. In yet another embodiment of a computer-aided molecular design method for identifying inhibitors, it comprises searching for fragments that fit a sub region of attachment and bind to a predefined scaffold. The scaffold itself can be identified in that way. Suitable programs for searching for such functional groups and monomers include LUDI (Boehm, J. Comp.Aid.Mol.Des.d. 6: 61-78 (1992)), CHAVETA (Bartlett et al., In "Molecular Recognition in Chemical and Biological Problems ", special publication of The Royal Chem. Soc, 78: 182-196 (1989)) and MCSS (Miranker et al., Proteins 11: 29-34 (1991)). In another embodiment of a computer-aided molecular design method to identify inhibitors of the main phosphatase, it comprises the synthesis of potential inhibitor nodes through the algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with the active site of the enzyme. The methodology employs a large template group of small molecules that are interactively drilled together in a model of the Tie-2 active site. Suitable programs for this task include GROW (Moon et al., Proteins 11: 314-328 (1991)) and SPROUT (Gillet et al., J. Comp.Aid.Mol.Des. 7: 127 (1993)). In yet another embodiment, the convenience of individual candidates can be determined using an empirical classification function, which can classify the binding affinities for a group of inhibitors. For an example of such a method see Muegge et al., And references (Muegge et al., J. Med. Chem. 42: 791-804 (1999)). Other modeling techniques according to this invention can be used, for example, those described by Cohen et al.,. { J. Med. Chem. 33: 883-894 (1994)); Navia et al. (Current Optlons in Structural Biology 2: 202-210 (1992)); Baldwin et al., (J. Med. Chem. 32: 2510-2513 (1989)); Appelt et al., (J. Med. Chem. 34: 1925-1934 (1991)); and Ealick et al. (Proc. Nat. Acad. Sc / .USA 88: 11540-11544 (1991)). A compound that is identified by one of the above methods as a potential Tie-2 inhibitor can then be obtained, for example, through synthesis or from a collection of compound and determined for the ability to inhibit Tie-2 in vitro. . Said in vitro assay can be performed as is known in the art, for example, by contacting Tie-2 in solution with the test compound in the presence of a substrate for Tie-2. The rate of transformation of the substrate can be determined in the presence of the test compound and compared to the regime in the absence of the test compound. Suitable assays for the biological activity of Tie-2 are described in Example 4. An inhibitor identified or designed by a method of the present invention can be a competitive inhibitor, a non-competitive inhibitor or an inhibitor that is not competitive. A "competitive" inhibitor is one that inhibits Tie-2 activity by binding totally or partially within the same Tie-2 region as ATP, thereby directly competing with ATP for the active Tie-2 site.

A "non-competitive" inhibitor inhibits Tie-2 by binding to a different region of the enzyme than ATP. These inhibitors bind to Tie-2 already bound with ATP and not to the free enzyme. A "non-competitive" inhibitor is one that can bind to either the free or bound ATP form of Tie-2. In some cases, an inhibitor can inhibit the catalytic activity of enzymes by preventing the binding of multiple substrates (eg, ATP and tyrosyl substrates). This can be achieved by totally or partially occluding the multiple substrate binding sites, or by occupying a site that haloesthetically and conformationally reduces affinities for substrates or release of block product. In another embodiment, the present invention provides inhibitors of Tie-2 and methods for using same, which are capable of binding to the catalytic domain of Tie-2, for example, compounds that are identified as inhibitors of at least one activity of Tie-2 or which are designed by the methods described above to inhibit at least one biological activity of Tie-2. For example, the invention includes compounds that interact with one another, preferably two or more, and most preferably three or more of Tie-2 subsites 1 through 9. In one embodiment, the Tie-2 inhibodor of the invention comprises a portion or portions placed to interact with subsite 1, subsite 2 and, optionally with at least one other subsite, when present in the Tie-2 catalytic domain. For example, a functional group that can interact with the subsite 1 can be a hydrogen bond donor, a hydrogen bond acceptor, or a hydrophobic moiety. A functional group that can interact with the subsite 2 can be a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor. In another embodiment, the Tie-2 inhibitor of the invention comprises functional groups positioned to interact with subsites 1, 2 and 3 and, optionally, one or more additional sub-sites. The Tie-2 inhibitors of the invention also include compounds having functional groups placed to interact with subsite 1, subsite 2, subsite 8 and optionally, one or more additional sub sites. In another embodiment, the inhibitor has functional groups placed to interact with subsite 1, subsite 2, subsite 3, subsite 8, and, optionally, one or more additional sub sites. In other embodiments, the Tie-2 inhibitors of the invention include compounds having functional groups positioned to interact with the following groups of subsites, each of which may, optionally, include one or more additional subsites: subsites 1, 4, and 5; subsites 1, 2, 7 and 8; subsites 1, 2, 3, 7 and 8; subsites 1, 2, 3, 7 and 8; subsites 1, 2, 4, 6 and 8; subsites 1. 2, 3, 4 and 8; subsites 1, 2, 3, 4, 6 and 8. A portion of the inhibitor compound is "placed to interact" with a given subsite, if, when placed within the catalytic domain of Tie-2, as defined by the atomic coordinates presented in Figures 3-6, the portion is close to, and appropriately oriented in relation to the appropriate amino acid side chains within the subsite. As indicated in the description of the above sub-sites, several sub-sites 1-9 can potentially interact with two or more types of portions. For each of the subsites listed below, the preferred type of portion to be interacted placed by the potential inhibitor is indicated. Sub-site 1: hydrogen bond donor (E903) and hydrogen bond acceptor (A905). Subsit 2: hydrophobic, preferably aromatic portion (I830, V838, I886, I902 and L971). Sub-site 3: hydrophobic portion, preferably alkyl (I830 and L971) and a positively charged portion (D912). Subsite 4: Hydrogen acceptor portion (D982 and F938). Subsitium 8: hydrophobic, preferably aromatic portion (L876, I886, L888 and F983). A preferred Tie-2 inhibitor of the invention inhibits the enzymatic activity of Tie-2 with a Ki value of at least about 1 mM, preferably at least about 100 μ and most preferably at least about 10 μM ? In another embodiment, a Tie-2 inhibitor selectively binds to a Tie-2 receptor on other tyrosine kinase receptors, such as insulin receptor or Csk, KDR, Ick, or zap. In a preferred embodiment, the inhibitor has an e K value of 0.1 times or less for a Tie-2 receptor than for an insulin or Csk receptor. In a more highly preferred embodiment, the inhibitor has a Kl value of 0.01 times or less for a Tie-2 receptor than for an insulin or Csk receptor. In a highly preferred embodiment, the inhibitor has a K value of 0.001 times less than or less for a Tie-2 receptor than for an insulin or Csk receptor. In a preferred embodiment, the Tie-2 inhibitor for the invention comprises two or more of the following when present in, or bound to the catalytic domain of Tie-2: (a) a hydrogen bond donor placed to interact with Glu 903 of human Tie-2; (b) a hydrogen bond acceptor placed to interact with 905 Ala of human Tie-2; (c) a hydrogen bond donor placed to interact with 905 Ala of human Tie-2; (d) a hydrophobic portion positioned to interact with one or more of He 830, Val 838, Ala 853, Lie 886, Lie 902, Tyr 904, Ala 905 and Leu 971 of human Tie-2; (e) a positively charged hydrogen bond donor or functional group placed to interact with human Tie-2 Asp 912; (f) a hydrogen bond donor or hydrogen bond acceptor placed to interact with Asn 909 of human Tie-2; (g) a hydrophobic portion placed to interact with one or more of Val 838, Lys 855, lie 886, lie 902, Leu 971 and Ala 981 of human Tie-2; (h) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 855 of human Tie-2; (i) a hydrogen bond acceptor placed to interact with Asp982 of human Tie-2; (j) a hydrogen bond acceptor placed to interact with Phe 983 of human Tie-2; (k) a hydrophobic portion positioned to interact with one or more of Leu 873, Leu 876, lie 885, lie 886, Leu 888, Leu 900, Me 902, Ala 981 and Phe 983 of human Tie-2; (I) a positively charged hydrogen bond donor or functional group placed to interact with Asp 982 of human Tie-2; (m) a hydrogen bond donor placed to interact with lie 886 of human Tie-2; (n) a hydrogen bond donor placed to interact with Leu 769 of human Tie-2; (o) a hydrogen bond acceptor placed to interact with Gly 831 of human Tie-2; (p) a positively charged hydrogen bond donor or functional group placed to interact with Glu 832 of human Tie-2; (q) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 840 of human Tie-2; (r) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 916 of human Tie-2; (s) a hydrogen bond acceptor or negatively charged functional group placed to interact with Arg 968 of human Tie-2; (t) a hydrogen bond donor placed to interact with Arg 968 of human Tie-2. In preferred embodiments, the Tie-2 inhibitors of the invention comprise (b) and (d); (d) and at least one of (a), (b) and (c); (d) and at least two of (a), (b) and (c); (d) and at least two of (a), (b) and (c), and at least one of (e) and (f); (d) and (g), and at least two of (a), (b) and (c); (d), (g), and at least two of (a), (b) and (c) and at least one of (e) and (f); (d), (g), (k), and at least two of (a), (b) and (c); (d), (g), (k), and at least one of (e) and (f), at least two of (a), (b), and (c); (d), at least one of (i) and (j), and at least two of (a), (b) and (c); (d) and at least two of (a), (b) and (c), at least one of (e) and (f), and at least one of (i) and (j); (d), (9). (k). at least one of (i) and (j), and at least two of (a), (b) and (c); and (d), (g), (k), at least one of (e) and (f), and at least one of (a), (b) and (c). Preferred Tie-2 inhibitors of the invention comprise a scaffold or molecular structure, to which the portions and / or functional groups that interact with the Tie-2 subsites are linked, either directly or through an intervening portion. The scaffold may be, for example, a peptide or peptide mimetic base structure, a cyclic or polycyclic moiety, such as a monocyclic, bicyclic or tricyclic moiety, and may include one or more hydrocarbon or heterocyclic rings. The molecular scaffold presents the interaction portions linked in the appropriate configuration or orientation for interaction with the appropriate residues of Tie-2. Pyrrolopyrimidines, such as inhibitor I, II, III or IV, are preferred Tie-2 inhibitors. Methods for synthesizing pyrrolopyrimidine are described in PCT Application No. WO 99/21560, the teachings of which are hereby incorporated by reference in their entirety. In one embodiment, the inhibitors of the invention do not include the pyrrolopyrimidines represented by the structural formula V: and their pharmaceutically acceptable salts, wherein: Ring A is a six-membered aromatic ring or a 5- or 6-membered heteroaromatic ring, which is optionally substituted with one or more substituents selected from the group consisting of a substituted aliphatic group or unsubstituted, a halogen, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroarachidium, cyano, nitro, -NR4 5.C (0) 2H, -OH, a substituted or unsubstituted carbonyl alkoxy, -C (0) 2-haloalkyl, a substituted or unsubstituted alkylthioether, a substituted or unsubstituted alkylsulphoxide, a substituted alkylsulfone or not replaced, a substituted or unsubstituted arylthioether, a substituted or unsubstituted arylsulphoxide, a substituted or unsubstituted arylsulfone a, a substituted or unsubstituted alkylcarbonyl, -C (0) -haloalkyl, a substituted or unsubstituted aliphatic ether, a substituted or unsubstituted aromatic ether, carboxamido, tetrasolyl, trifluoromethyl sulfonamido, trifluoromethyl carbonylamino, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkylamino, a substituted or unsubstituted arylamido, -N g5C (0) Rg5l a substituted or unsubstituted styryl, and a substituted or unsubstituted aralkylamido, wherein R95 is an aliphatic group or an aromatic group; L is -O-; -S-; -S (O) -: -S (0) 2-; -N (R) -; -N (C (0) OR) -; -N (C (0) R) -; -N (S02R); -CH20-; -CH2S-; -CH2N (R) -; -CH (NR) -; -CH2N (C (0) R)) -; -CH2N (C (0) OR) -; -CH2N (S02R) -; -CH (NHR) -; -CH (NHC (0) R) -; -CH (NHS02R) -; -CH (NHC (0) OR) -; -CH (OC (0) R) -; -CH (OC (0) NHR) -; -CH = CH; -C (= NOR) -; -CO)-; -CH (OR) -; -C (0) N (R) -; -N (R) C (0) -; -N (R) S (0) -; -N (R) S (0) 2-; -OC (0) N (R) -; -N (R) C (0) N (R) -; -NRC (0) 0-; -S (0) N (R) -; -S (0) 2N (R) -; N (C (0) R) S (0) -; N (C (0) R) S (0) 2-; -N (R) S (0) N (R) -; N (R) S (0) 2N (R) -; -C (0) N (R) C (0) -; -S (0) N (R) C (0) -; -S (0) 2N (R) C (0) -; -OS (0) N (R) -; -OS (0) 2N (R) -; N (R) S (0) 0-; -N (R) S (0) 20-; -N (R) S (0) C (0) -; -N (R) S (0) 2C (0) -; -SON (C (0) R) -; -S02N (C (0) R) -; -N (R) ARE (R) -; -N (R) S02N (R) -; C (0) 0-; -N (R) P (OR ') 0-; -NKRJPÍOR1) -; -N (R) P (0) (0 ') 0-; -N (R) P (0) (OR ') -; -N (C (0) R) P (OR ') 0-; -N (C (0) R) P (OR ') -; -N (C (0) R) P (0) (OR ') 0- or -N (C (0) R) P (OR ') -, wherein R and R' are each, independently, -H, an acyl group, a substituted or unsubstituted aliphatic group, or an aromatic group substituted or not substituted, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted cycloalkyl group; or L is -RbN (R) S (0) 2-, -RbN (R) P (0) -, or -RbN (R) P (0) 0-, where Rb is an alkylene group, which when it is taken together with the sulfonamide, phosphinamide or phosphonamide group to which it is attached, it forms a 5- or 6-membered ring fused to ring A; or L is represented by one of the following structural formulas: wherein R85 taken together with the phosphine or phosphonamide is an aromatic, heteroaromatic or heterocycloalkyl ring system of 5, 6, or 7 members; Ri is a substituted aliphatic group, a substituted cycloalkyl, a substituted bicycloalkyl, a substituted cycloalkenyl, an optionally substituted aromatic group, an optionally substituted heteroaromatic group, an optionally substituted eteroaralkyl, an optionally substituted heterocycloalkyl, an optionally substituted heterobicycloalkyl, an optionally substituted alkylamido , and optionally substituted arylamido, or optionally substituted -S (0) 2 -alkyl or -S (0) 2-cycloalkyl, optionally substituted C (0) -alkyl or C (0) -alkyl, provided that when Ri is an aliphatic group or cycloalkyl group, Ri is not exclusively substituted with one or more substituents selected from the group consisting of hydroxyl and lower alkyl ethers, provided that the heterocycloalkyl is not 2-phenyl-1,3-dioxan-5-yl, and provided that an aliphatic group is not exclusively substituted with one or more aliphatic groups, wherein one or more substituents are selected from the group consisting of a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted heteroaromatic or unsubstituted, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aromatic ether, a substituted or unsubstituted aliphatic ether, an alkoxycarbonyl substituted or unsubstituted, a substituted or unsubstituted alkylcarbonyl, a substituted or unsubstituted arylcarbonyl, a substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted aryloxycarbonyl, -OH, a substituted or unsubstituted aminocarbonyl, an oxime, a substituted azabicycloalkyl or not substituted, heterocycloalkyl , oxo, aldehyde, substituted or unsubstituted alkylsulfonamido group, a substituted or unsubstituted arylsulfonamido group, a substituted or unsubstituted bicycloalkyl, a substituted or unsubstituted heterobicycloalkyl, cyano, NH2, an alkylamino, ureido, thioureido and -B-E; B is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aromatic, a substituted or unsubstituted heteroaromatic, an alkylene, an aminoalkyl, an alkylenecarbonyl, or an aminoalkylcarbonyl; E is a substituted or unsubstituted azacycloalkyl, a substituted or unsubstituted azacycloalkylcarbonyl, a substituted or unsubstituted azacycloalkylsulfonyl, a substituted or unsubstituted azacycloalkylalkyl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroarylcarbonyl, a substituted or unsubstituted heteroarylsulfonyl , a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted alkylsulfonamido, a substituted or unsubstituted arylsulfonamido, a substituted or unsubstituted bicycloalkyl, a substituted or unsubstituted ureido, a substituted or unsubstituted thioureido, or a substituted or unsubstituted aryl; R 2 is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted cycloalkyl, a halogen, -OH, cyano, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted heterocycloalkyl , a substituted or unsubstituted aralkyl, a substituted or unsubstituted heteroaralkyl, -NR4R5 or -C (0) NR4R5; R3 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted heterocycloalkyl group; Whenever L is -SN (R) -, -S (0) N (R) -, -S (0) 2N (R) -, -N (R) S-, -N (R) S (0) -, -N (R) S (0) 2-, -N (R) SN (R ') -. -N (R) S (0) N (R ') -. or -N (R) S (0) 2N (R ') - when R3 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted alkenyl group; Provided that j is 0 when L is -O-, -CH2NR-, -C (0) NR- or -NRC (O) -, and R3 is azacycloalkyl or azaheteroaryl; Always j is 0 when L is -O-, and R3 is phenyl; , R5 and the nitrogen atom together form a substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterobicycloalkyl, or a substituted or unsubstituted heteroaromatic, of 3, 4, 5, 6 or 7 members; or R 4 and R 5 are each independently H, azabicycloalkyl, heterocycloalkyl, a substituted or unsubstituted alkyl group, or Y-Z; Y is selected from the group consisting of -C (O) -, - (CH2) P-, -S (0) 2-, -C (0) 0-, -S02NH-, -CONH-, (CH2) pO -, - (CH2) PNH-, - (CH2) PS-, - (CH2) pS (0) -, and - (CH2) pS (0) 2-; p is an integer from 0 to 6; Z is -H, a substituted or unsubstituted alkyl, a substituted or unsubstituted amino, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocycloalkyl group; and j is an integer from 0 to 6. As used herein, aromatic groups include carbocyclic ring systems (e.g., phenyl and cinnamyl) and polycyclic aromatic ring systems (e.g., naphthyl and 1, 2, 3, 4-tetrahydronaphthyl). Aromatic groups are also referred to herein as aryl groups. Heteroaromatic groups, as used herein, include heteroaryl ring systems (e.g., thienyl, pyridyl, pyrazole, izoxazolyl, thiadiazolyl, oxadiazolyl, indazolyl, furans, pyrroles, imidazoles, pyrazoles, triazoles, pyrimidines, pyrazines, tlazoles, izoxazole, izotiazoles, tetrazoles or oxadiazoles), and heteroaryl ring systems wherein a carbocyclic aromatic ring, a carbocyclic non-aromatic ring or a heteroaryl ring is fused to one or more of the other heteroaryl rings (eg, benzo (b) thienyl , benzimidazole, indole, tetrahydroindole, azaindole, indazole, quinoline, imidazopyridine, purine, pyrrolo [2,3-d] pyrimidine, pyrazolo [3,4-d] pyrimidine) and their N-oxides.

An aralkyl group, as used herein, is an aromatic substituent that is linked to a compound through an aliphatic group having 1 to about 6 carbon atoms. A heteroaralkyl group, as used herein, in a heteroaromatic substituent that is linked to a compound through a group, aliphatic having from 1 to about 6 carbon atoms. A heterocycloalkyl group, as used herein, is a non-aromatic ring system having from 3 to 8 atoms and includes at least one heterogeneous atom, such as nitrogen, oxygen or sulfur. An azyl group, as used herein, is a group -C (0) NRxRz, -C (0) OR ", -C (0) Rx, wherein Rx and Rz are each independently -H, a group aliphatic substituted or unsubstituted, or a substituted or unsubstituted aromatic group. As used herein, the aliphatic groups include hydrocarbons of 1 to 8 straight carbon atoms, branched or cyclic, which are completely saturated or which contain one or more units of unsaturation. A "lower alkyl group" is a saturated aliphatic group having from 1 to 6 carbon atoms. Inhibitor I linked to the catalytically inactive Tie-2 mutant (for sequence and see Figure 3 for atomic coordinates) was crystallized in space group C2221. The X-ray crystallographic structure relieved the following interactions: The pyrrolopyrimidine ring of inhibitor I forms hydrogen bonds for residues in the hinge region and interacts with the purine core region. The core of the inhibitor has a hydrogen bond donor in the form of the amino proton and the 4-NH2 substituent for the carbonyl-oxygen of E903. The N3 atom of the pyrimidine ring accepts a hydrogen bond of the N-H base structure of A905. The core ring system has a flat face for the residues of both the C-terminal and N-terminal nodes. The residues in these areas have a hydrophobic surface that "walls" the flat nucleus of the inhibitor. The residues involved in this hydrophobic sandwich region include I830, V838, I86. I902 and L971. The atoms N1 and N7 of the core look at the solvent exposed in the mouth of the joint cavity. The C6 atom looks at the long axis of the nucleotide binding loop of the N-terminal lobe of the protein. The cyclopentane ring of N7 is directed towards the solvent, but remains within the protein cavity. This region was previously described as the sugar cavity extended after the binding mode of the ribose ring of ATP observed in other kinase structures. This region is characterized by hydrophobic interactions mainly with I830 and L971. The methyl groups of E832 also contribute in this way. The phenyl ring attached to C5 of the pyrrolopyrimidine ring is a predominantly hydrophobic area, generated by residues of the purine core region, the distant hydrophobic quality and methylene groups of the catalytic lysine, K855. The hydrophobic contacts are with residues V838, 1886, 1902, L971 and A981. Lysine 855 is highly mobile, so that it is also possible that the C1 target atom to the pyrroiopyrimidine ring is receiving a hydrogen bond. The sulfonamide linker makes a clear hydrogen bond with an amide proton of D982 and also makes a hydrogen bond to the amide proton of F983. The terminal phenyl ring (marked as ring C) is located in the distant hydrophobic cavity. The primary contacts are with L876, I886, L888 and F983. Inhibitor II bound to the catalytically inactive Tie-2 mutant (see Figure 2 for sequence and Figure 4 for atomic coordinates) was crystallized in space group P42212. The X-ray crystallographic structure revealed the following additional interactions: The pyrroiopyrimidine core, B ring, linker and C ring are joined in the same manner as inhibitor I. The cyclo-N-methyl-pyrazinyl group of N- 7 occupies the extended sugar cavity and makes a strong ionic interaction with D912. Inhibitor III binds to the catalytically inactive Tie-2 mutant (see Figure 2 for sequence and Figure 1 for atomic coordinates) was crystallized in space group P42212. The X-ray crystallographic structure revealed the following additional interactions: The pyrrolopyrimidine core binds in the same way as inhibitor I. The cyclohexyl-N-methyl-piperazinyl group of N-7 occupies the extended sugar cavity and makes a strong ionic interaction with D912 as in Tie-2 / inhibitor II. Ring B binds in the same way to inhibitor I, however, the hydrogen bond between a halogen, fluorine in this case, and K855 is lighter. The linker makes two clear hydrogen bonds to the base structure amide protons of T83 and F983. Ring C occupies the distant hydrophobic cavity with the main interactions coming from L876, I886, L888, L900, I902 and F983. Inhibitor IV binds to the catalytically inactive Tie-2 mutant (see Figure 2 for sequence and see Figure 4 for atomic coordinates) was crystallized in space group P42212. The X-ray crystallographic structure revealed the following additional interactions: The pyrrolopyrimidine core binds in the same way as inhibitor I. The cyclohexyl-N-methyl-piperazinyl group of N-7 occupies the extended sugar cavity and makes a strong ionic interaction with D912 as in Tie-2 / inhibitor II. Ring B binds in a manner similar to inhibitor I, however, there is no chlorine atom to act as a potential hydrogen bonding pattern. In linker in this case it is an oxygen atom, which accepts a hydrogen bond of the catalytic lysine, K855. Ring C occupies the distant hydrophobic cavity with the main interactions coming from L876, 1886, 1902 and F983. In one embodiment, the present invention relates to a method for treating a Tie-2 dependent condition in a patient. The method comprises the step of administering to the patient a therapeutically effective amount of a Tie-2 inhibitor as described above. The patient can be any animal, and preferably, it is a mammal, and most preferably a human being. A "Tie-2 dependent condition" is a disease or medical condition in which the Tie-2 catalytic activity plays an important role, for example, in the development of the disease or condition. For example, in one embodiment, the condition is characterized by excessive vascular proliferation. The Tie-2 inhibitors are useful for the treatment of angiogenesis-dependent disorders and disorders involving aberrant endothelial-periendothelial interactions (eg, restenosis). Tie-2 dependent conditions include hyperproliferative disorders, cancer, a cardiovascular condition, an ocular condition, von Hippel Lindau disease, pemphigoid, psoriasis, Paget's disease, polycystic kidney disease, fibrosis, sarcoidosis, cirrhosis, thyroiditis, disease Osler-Weber-Rendu, chronic inflammation, synabitis, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ulcer and sepsis. In addition, a Tie-2 inhibitor can be used to reduce a patient's fertility. Preferred methods of treatment are where the cancer is a solid tumor, a sarcoma, fibrosarcoma, osteoma, melanoma, retinoblastoma, a radomyosarcoma, gioblastoma, neoblastoma, teratocarcinoma, a hetopoietic malignancy, malignant azitos, Kaposi's sarcoma, Hodgkin's disease, lymphoma, myeloma or leukemia. Another preferred method of treatment is where there is cardiovascular disease, atherosclerosis, restenosis, ischemia / reperfusion injury, chronic occlusive pulmonary disease, vascular occlusion, carotid obstruction disease, Crow-Fukase syndrome (SOP), anemia, ischemia, infarction , vascular effusion disorders. Another preferred preferred method of treatment is when the ocular condition is ocular or macular edema, ocular neovascular disease, scleritis, radial ketotomy, uveitis, vitritis, myopia, optic spots, chronic retinal separation, post-laser treatment complications, conjunctivitis, Stargardt, Eales disease, retinopathy, macular degeneration or microangiopathy. A Tie-2 inhibitor can also be used in a method to promote angiogenesis or vasculogenesis. In addition, a Tie-2 inhibitor can be administered as a pro-angiogenic growth factor. A therapeutically effective amount, as used herein, is an amount that results in partial or complete inhibition of the progression or symptoms of the disease. Said amount will depend, for example, on the size and gender of the patient, the condition to be treated, the severity of the symptoms and the result sought, and can be determined by one skilled in the art. The compound of the invention can, optionally, be administered in combination with one or more additional drugs or therapies which, for example, are known to treat and / or alleviate the symptoms of the Tie-2 mediated condition. The additional drug can be administered simultaneously with the compound of the invention, or sequentially. For example, the Tie-2 inhibitor can be administered in combination with another anticancer agent, as is known in the art. Additional therapies that can be co-administered may include, for example, radiation therapy, ultraviolet irradiation, hyperthermia, laser irradiation, activated radionuclides, and neutron bombardment. The invention further provides pharmaceutical compositions comprising one or more of the above-described Tie-2 inhibitors. Said compositions comprise a therapeutically (or prophylactically) effective amount of one or more Tie-2 binding inhibitors, as described above, and a pharmaceutically acceptable carrier or excipient. Such suitable pharmaceutically acceptable carriers include, but are not limited to, saline, pH regulated saline, dextrose, water, glycerol, ethanol, and combinations thereof. The vehicle and the composition can be sterile. The formulation must adapt to the mode of administration.

Suitable pharmaceutically acceptable carriers include, but are limited to, water, salt solutions (e.g., NaCl), alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, cyclodextrin, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, pH regulators, color substances, flavor substances and / or aromatic, and the like, which do not react in a harmful manner with the active compounds. The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers, or pH regulating agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with binders and traditional vehicles such as triglycerides. The oral formulation may include standard vehicles such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidinone, sodium saccharin, cellulose, magnesium carbonate, etc.

The composition can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in an aqueous, isotonic, sterile pH regulator. When necessary, the composition may include a solubilizing agent and a local anesthetic to alleviate pain at the site of injection. In general, the ingredients are supplied either separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container such as a vial or small bag indicating the amount of active agent. When the composition is to be administered through infusion, it can be dispensed with an infusion bottle containing water, saline or dextrose / pharmaceutical grade water. When the composition is administered by injection, a sterile water vial for injection or exit may be provided so that the ingredients can be mixed before administration. The pharmaceutical compositions of the invention may also include an agent that controls the release of Tie-2 inhibitor compound, thus providing a time-released or sustained release composition. The Tie-2 inhibitor can be administered subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, intraocularly, topically, enterally (for example, orally), rectally, nasally, buccally, sublingually, vaginally, through inhalation spray, through a drug gum, or through a reservoir implanted in dosage formulations containing conventional physiologically acceptable, non-toxic carriers or carriers. The preferred method of administration is through oral administration. The form in which it is administered (for example, syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sun) will depend in part on the route through which it is administered. For example, for administration by glucose (eg, oral mucosa, rectal, ocular mucosa, intestinal mucosa, bronchial mucosa), nasal drops, aerosols, inhalants, nebulizers, eyedrops or suppositories can be used. The compounds and agents of this invention can be administered together with other biologically active agents, such as analgesics, anti-inflammatory agents, anesthetics and other agents that can control one or more symptoms or cause a Tie-2 dependent condition.

In a specific embodiment, it may be desirable to administer the agents of the invention locally to a localized area in need of treatment; this can be achieved, for example, and without limitation, through local infusion during surgery, topical application, transdermal patches, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous material, not porous or gelatinous, including membranes, such as sialastic membranes or fibers.

For example, the agent can be injected into the joints.

EXAMPLES EX EMPLO 1 Protein Purification (His) eTie-2 802-1 124, D964N, which contains a TEV protease cleavage peptide, was expressed recombinantly through baculovirus infection of SF-9 cells. Cells were typed in a pH buffer containing 20 mM Tris pH 8.0, 37 mM NaCl, 1.0% glycerol, 1% Triton X-100, 1 mM ADP. 5 mM Mg CI2, and complete protease inhibitor, EDTA-free cocktail from Boehringer Mannhein. The ADP / Mg ligand was maintained at this concentration in pH regulators of all subsequent purification steps. The cell lysate was centrifuged and the supernatant was applied to a Sepharose column of Ni "chelation, which had been equilibrated in 50 mM H EPES, pH 7.5, 0.3 M NaCl, and the Tie-2 was eluted by competition with 100 mM imidazole The eluted (H is) e Tie-2 was digested with Tev u protease dialyzed against 50 mM H EPES, pH 7.5, 0.25 M NaCl, 5 mM DTT The dialyzed sample was centrifuged to remove any precipitated protein, and Tie-2 bound to a MonoQ anion exchange column and eluted with a linear column volume radius of 0.025-0.2 M NaCl. Typically, the differences in the monodispersity of the Early elution versus late elution fractions can be detected using Dynamic Light Diffusion (DLS) The purity of the sample was determined with SDS-PAGE, native PAGE, and LC / MS total mass analysis. DLS were combined and concentrated more than 2 mg / ml using ultrafracture at -80 ° C. the ultracentrifuged samples were used in crystallographic experiments described below. Table I lists a scale of suitable conditions for crystallization.

EXAMPLE 2 I. Tie-2 802-124 of Diphosphorylate A. Crystallization Conditions: The Tie-2 protein 802-1124 (2P04) was crystallized in a chain or pendant geometry using a vapor diffusion method. The protein concentration was 5 mg / ml and the chain solution was 10% PEG 6000; 0.1 M HEPES, pH 7.5; 5% MPD (2-methyl-2,4-pentanediol). The drops were fixed using equal volumes of protein and the cavity solution containing 500 μ? of inhibitor. The crystals were routinely developed at 0.4 mm x 0.1 mm x 0.1 mm x 0.01 mm in about 1 week. The crystals were from the space group P2 (1) 2 (1) 2 (1) with unit cell dimensions of a = 54.320 A, b = 75.872 A, c = 78.143 A. and a = ß =? = 90.0β. Table I lists a scale of conditions that are suitable for crystallization.

B. Data collection Data on crystals bound to ligands were assembled in a Rigaku RU300 rotating anode generator running at 50 kV 150 mA equipped with an R-Axis II phosphoimage plate detector. The X-rays were monochromatized through large mirrors and filtered with a nickel filter of 0.0065 μp ?. The data were processed and reduced with DENZO and SCALEPACK (Minor, W. 1993). The data was collected at a resolution of 3.5 A.

C. Data Processing The programs were used in CCP4 (Computational Project Collaborative, Number 4, 1994) (tomtz, trunc, cad and ecalc) to format and process the data for molecular replacement. The AMORE molecular replacement program (Navaza, J. 1994) was successfully used to find phases for the fixed data using an initial model. The initial model was composed of a carboxy-terminal portion (residues 566-575 and 592-672) of the FGFR kinase domain cut to poly-alanine (accession number of PDB, 1FGK). A second round of AMORE with a more complete model (residues 464-485, 491-500, 505-575 and 592-762) was also performed to confirm the phase formation solution.

D. Optimization of the Model Several rounds of minimization of least squares using CNS (Brunger, AT et al., 1998) alternating with manual reconstruction, using the graphic program O, version 6.2.1 (Jones, A., 1997; Kleywegt GJ, 1995 ) was also performed iteratively to improve the model while it was purchased with generated electron density maps. for each round with 2fo-fc coefficients found at a level of 1.0 sigma.

II- Tie-2 (D964N) 802-1124 (SEC ID IMO: 2) A. Crystallization Conditions The purified Tie-2 protein (D964N) 802-1124 was crystallized in an etching quality geometry using the vapor diffusion method. The protein concentration was 2.5 mg / ml and the cavity solution was 1.0 to 1.5 M ammonium sulfate, 0.1 M MES, pH 6.5, 5% dioxane (1,4-dioxane). The falls were fixed using equal volumes of protein and cavity solution containing 100-300 μ? of inhibitor. The crystals routinely grew to 0.3 mm x 0.05 mm x 0.01 mm in approximately 2-3 days. The crystals of ??? - 2 / inhibitor I were from the group of space C222 (1) with unit cell dimensions of a = 75,195 A, b = 116,287 A, c = 95,060 A, and a = ß =? = 90.0 °. The crystals of the Tie-2 / inhibitor II, III or IV complex were from the space group P42212 with unit cell dimensions of a = b = 86.0 A, c = 112.0 A, and a = ß =? = 90.0β.

? Data Collection The data in a crystal bound to the ligand (Tie-2 (D964N) 802-1124 in complex with the inhibitors I, II, III or IV were collected in beamline X25 at Brookhaven National Laboratory (Upton, NY), equipped with a Brandis B4, CCD detector, the data were processed and reduced with DENZO and SCALEPACK (Minor, W. 1993) Data for the Tie-2 / inhibitor I complex were collected together at a resolution 2.75 A, with higher resolution reflections visible at a resolution of 2.0 A.

C. Data Processing The programs were used in CCP4 (Computational Project Collaborative, Number 4, 1994) (tomtz, trunc, cad and ecalc) to format and process the data for molecular replacement. The AMORE molecular replacement program (Navaza, J. 1994) was successfully used to find phases for data fixation using an initial model. The initial model consisted of a conservative portion of the FGFR kinase domain (residue numbering of Tie-2 818-830, 841-842, 850-857, 866-890, 900-916, 935-981, 1001-1093 ). The model, mostly poly-alanine, was cut from loop regions which then divided d overlapping five tyrosine kinase structures (IRK, HCK, SRC, FGFR, and LCK).

In addition, this model included only those side chain residues at positions where an identical side chain was found in the FGFR model.

D. Optimization of the Model Several rounds of minimization of least squares using CNS (Brunger, AT et al, 1998) alternating with manual reconstruction, using graphics program O, version 6.2 (Jones A., 1997; Kleywegt GJ, 1995) were performed to iteratively improve the model while comparing with two electron density maps; one generated with 2fo-fc coefficients found at a level of 1.0 sigma and the other generated with fo-fc coefficients found at a level of 1.5 sigma.

E. Inhibitor Coupling Inhibitor I was found to be attached to the active site. Initially it was manually coupled in O by visually inspecting the electron density maps and adjusting the torsion angles of the inhibitor. Parameter and topology files for CNS were generated using the X-util Xplo2d program (Kleywegt G. J. and Jones, T.A. 1997) and were modified slightly to properly model chlorine in the inhibitor.

III. Tie-2 (D964N) 802-1124 (SEQ ID NO 2) A. Crystallization Conditions The protein (construction Tie-2D964N) was provided in a pH buffer containing 25 mM HEPES, pH of 7.5, 50 mM of NaCl, 5 mM of MCI2, 1 mM of ADP and 5 mM of DTT. The protein concentration was approximately 2.3 mg / ml as determined as a Coomassie Plus assay, BSA as standard. Inhibitor III was dissolved in DMSO to give a 50 mM supply solution. The supply solution was added to the protein solution for a minimum inhibitor concentration of 2 mM. The crystallization conditions were classified with the Hampton glass sorting screen, glass screen 2, Membfac, and Natrix and PEG / ion screen at room temperature and at 4 ° C. The crystals were developed with precipitation pH regulator: 20% of PEG3350, 0.2 M of tri-lithium citrate, pH 8.1, (PEG screen / Hampton classification ion, Nr.45) fall of acentamiento or pendant: 750 μ ? of pH regulator in the reservoir in the fall, typically 1 μ I - 2 I of protein and 1 μ? - 2 μ? of tank solution were mixed. The addition of the following additives (10% by volume for the drop) also produced crystals: Classification Ad. I Nr.:01 0.1M Ba chloride Ad Classification I Nr.:03 0.1M Ca chloride Classification Ad. 1 Nr.:06 0.1 M Mg chloride Ad Class. 1 Nr.:16 0.1 M trimetifamine Classification Ad. I Nr.:22 30% ethanol Classification Ad. II Nr.:08 30% xylitol Classification Ad. II Nr.:13 30% 1, 5 diaminopentane dichlorohydrate Classification Ad. II Nr.:14 30% diaminooctane Classification Ad. I Nr,: 17 0.1M hexaaminocobalt trichloride Classification Ad. III Nr.:02 1.0M cesium chloride Classification Ad. III Nr.:04 1.0M lithium chloride Classification Ad. III Nr.:06 0.5M sodium fluoride Classification Ad. III Nr.:16 40% acetonitrile Classification Ad. III Nr.:18 40% n-propanol Classification Ad. III Nr .: 19 5% ethyl acetate Classification Ad. III Nr.:20 40% acetone Classification Ad. III Nr.:21 2.5% dichloromethane Classification Ad. III Nr.:22 7% n-butanol Classification Ad. III Nr.:0.1M 1,4 dithio-DL-treitol B. Data Collection The data were measured in the beam line BW 6 of Max-Planck-Society in DESY, Hamburg. The crystals were cooled by shock at 100 K; The cryo-pH regulator was pH regulator of crystallization plus 20-30% glycerol. 213 frames with delta phi = 0.25 degrees were collected with a MAR CCD detector, at a crystal detector distance of 120 mm and a wavelength of 1072 A. The crystals are from a group of tetragonal space with unitary cell dimensions from a = b = 86.0 A and c = 112 A. All cell dimensions of different crystals vary (for a and b between 85 and 87 A, for c between 97 and 113). Extinctions indicate the space group P42212, which was confirmed through molecular replacement.

TABLE II Crystallization conditions for Tie-2 / inhlbidor complexes Condition Tie-2802-1124 D964N Tie-2802-1124 (diphosphorylated) Concentration of protein 2.5 mg / ml optimal 5 mg / ml optimal scale 1.5-4 mgml scale 2.5-10 ml limits 1.0-5.0 mg / ml Concentration of pH regulator 100 m Optimal MES 100 mM HEPES optimal scale 50-250 mM 50 scale -100 mM limits 20-30 mM limits 20-300 mM PH 6.5 optimum 7.5 optimal scale 5.5-7.5 scale 7.0-7.7 limits 6.5-8.0 pH regulator identity pH regulators capable of (equal) regulating pH on a similar scale of pH expected to give similar results Precipitante (NH,) _ S04 10% PEG 6000 optimal scale 1.0 - .5 M scale of conc.5-15% limits 0.7- 1.8 M limit of conc.1-20% scale of MW 4000 - 8000 MW limits can be wider Parameters of Additive 5% 1, 4-dioxane optimal scale 0- 5% MPD (2-Me-2,4-pentanedi 10% (optimum concentrations optimum attack the plastic cup in range 0 - 10% where the experiment is performed, higher concentrations may be possible in a resistant vessel) 1.3-dioxane, similar molecules s, or mixtures in various ratios should also give similar results Additive identities Examples that have been added (equally) successfully: 1, 5-diaminopentane glycerol (1-10%) ethylene glycol (1-10%) Esperomidine (10-300) mM) Combinations, in various ratios, can give similar results Volumes and fall ratios 2 pL of protein + 2 μ? _ of solution (equal) of optimal cavity Total volume scale: up to 200 pL, assuming a seat geometry for volumes Larger scale of volume ratio: 1 part of protein to 0.5 - 2.0 parts of cavity solution Cavity volume (for 4 pL of Scale 500 - 1000 pL (equal) crystallization drop) Limits 250 - large volume (limited by distance between the drop and the surface of the cavity solution allowed by the cup geometry, see below Distance of the fall solution - optimum 2 cm (equal cavity range 1-4 cm limits 0.1 cm - 5 cm Temperatur a Optimal ambient temperature (22- (equal) 25"C) Limits 17 - 30 ° C DP / Mg" ligands and analogues (equal) Inhibitors: l-IV inhibitors, analogs Expect similar results from ligands that bind reversibly under conditions of crystallization with Kn <values; 1mM Constructions Variants in sequence of (equal) amino acid that crystallizes in the same group of space and cell unit should be considered as equivalent Additional constructions could include the elimination of terms not built as determined by the crystalline structure of this construction. For example, the removal of 24 C-terminal residues (leaving 802-1 00) has been prepared, which is likely to produce similar results Modification after The variants in the modification of 2 forms of phosphate have been translated later translation than crystallizes crystallized. This protein in the same group of space and contains a phosphate in either Y897 unit cell must be either Y899 and one of five T residues, considered equivalent in amino acids 1012, 1024, 1040 and 1048 Other phosphorylated forms can give similar results. A single phosphate species was observed, where the phosphate in either Y897 or Y899 has been isolated. In addition, 3 and 4 species of phosphate have been isolated, which can crystallize. Space group C222 (1) P2 (1) 2 (1) 2 (1) Unit cell a = 75.195 A, b = 116.287 A, c = a = 54.320 A, b = 75.872 A, c = 95.060 A 78.143 A Variations of + 2% should be Variations of ± 2% should be considered equivalents considered equivalent Angles: a = b = c = 90 ° Angles: a = b = c = 90 ° Observed variations of + 1% Observed variations of + 1% should to be considered should be considered equivalent equivalents Other crystallization tricks that Low gravity (equal) should give at least equivalent results Temperature oscillations Presence of cryo protector (15-25% glycerol added before data collection) Variations in tray geometry of crystallization Data collection temperature (scale: -80 to more than 25 ° C) REFERENCES: Brunger A. T., Adams, P. D., Clore, G. M., DeLano, W. L. Gros, P. Grosse-Kunstleve, R. W., Jiang, J-S., Kuszewski. J., Jigels, M., Pannu, N. S., Read, R.J., Rice, L.M., Simonson. T., and Warren, G. L. (1998) Acta Cryst. D54, 905-921. Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50. 760-763. Jones, A. T. and Kjeldgaard, M. (1997) Methods in Enzymology 277, 173-208. Kleywegt G. J. and Jones, T. A. (1997) Methods in Enzymology 277, 525-545. Kleywegt G. J., (1995) ESF / CCP4 Newsletter 31, 45-50. Minor, W. XDISPLAYF program, Purdue University (1993). Navaza, J. (1994) Acta Cryst. A50, 157-163.

EXAMPLE 3 In Vitro Potency Test of Tie-2 Inhibitors The in vitro potency of the compounds to inhibit these protein kinases can be determined through the procedures detailed below. The potency of the compounds can be determined through the amount of inhibition of the phosphorylation of an exogenous substrate (eg, synthetic peptide (Z. Songyang et al., Nature. 373: 536-539) through a test compound. relative to control.

Production and Purification of Human Tie-2 Kinase The coding sequence for the intercellular domain of human Tie-2 (aa775-1124) was generated by PCR using cDNAs isolated from human placenta as a template. A poly-His6 sequence was introduced at the N terminus and this construct was cloned into the transfection vector pVL 1939 at the Xba 1 and Not 1 site. Recombinant BV was generated through co-transfection using the transfection reagent BaculoGoId (PharMingen ). The recombinant BV was purified on the plate and verified by Western analysis. For protein production, SF-9 insect cells were developed in an SF-900-II medium at 2 x 106 / ml, and were infected at an MOI of 0.5. The purification of the His-tagged kinase used in the classification was analogous to that described for KDR.

EGFR Tyrosine Kinase Source The EGFR was purchased from Sigma (Catalog # E-3641; 500 units / 50 I) and the EGF ligand was purchased from Oncogene Research Products / Calbiochem (Catalog # PF011-100).

Enzyme Linked Immunosorbent Assay (ELISA) for PTKs Enzyme-linked immunosorbent assays (ELISA) were used to detect and measure the presence of tyrosine kinase activity. The ELISA assay was conducted according to known protocols, which are described in, for example Voller et al., 1980, "Enzyme-Linked Immunosorbent Assay", In: Manual of Clinical Immunology 2o. ed., edited by Rose and Friedman, p. 359-371 Am. Soc. Of Microbiology, Washington, DC The described protocol was adapted to determine the activity with respect to a specific PTK.For example, the preferred protocols for conducting the ELISA experiments are given below. these protocols for determining the activity of a compound for other members of the PTK receptor family, as well as tyrosine kinases that are not receptor-like, are within the abilities of those skilled in the art. of the inhibitor, a universal substrate of PTK (for example, poly (Glu4 Tyr) random copolymer, 20,000-50,000 MW) was used together with ATP (typically 5 μ?) at concentrations approximately twice the value of apparent Km in this assay The following procedure was used to analyze the inhibitory effect of the compounds of this invention on the tyrosine kinase activity of Tie-2.

PH regulators and Solutions: PGTPoli (Glu, Tyr) 4: 1 Store the powder at -20 °. Dissolve the powder in pH regulated saline with phosphate (PBS) for a 50 mg / ml solution. Store 1 ml aliquots at -20 ° C. When the plates are made dilute to 250 g / ml in Gibco PBS. Reaction pH regulator: 100 mM Hepes, 20 mM MgCl2, 4 mM MnCl2, 5 mM DTT, 0.02% BSA, 200 μ? of NaV04, pH 7.10. ATP: Store 100 mM aliquots at -20 ° C. Dilute to 20 μ? in water Wash pH regulator: PBS with 0.1% Tween 20 Antibody to Dilute pH Regulator: 0.1% bovine serum albumin (BSA) in PBS. TMB substrate: mix the TMB substrate and peroxide solutions, 9: 1 just before use or use Neogen's K-Blue substrate. Stop solution: 1M phosphoric acid.

Procedure 1. Plate Preparation Dilute the PGT supply material (50 mg / ml, frozen) in PBS to 250 μg / ml. Add 125 μ? per cavity of flat bottom high affinity ELISA plates modified with Corning (Corning # 25805-96). Add 125 μ? of PBS to empty cavities. Cover with sealing tape and incubate overnight at 37 ° C. Wash once with 250 μl of washing pH regulator and dry for approximately 2 hours in a 37 ° C drying incubator. Store the covered plates in sealed bags at 4 ° C until they are used. 2. Tyrosine Kinase Reaction Prepare inhibitor solutions at a concentration of 4x in 20% DMSO in water. - Prepare reaction pH regulator. Prepare an enzyme solution so that the desired units are at 50 μ ?, for example, for KDR to make 1 ng / 1 for a total of 50 ng per cavity in the reactions. Store on ice. - Make 4x of ATP solution at 20 μ? from a supply solution of 100 rti in water. Store on ice. Add 50 μ? of the enzyme solution per cavity (typically 5-50 ng of enzyme / cavity depending on the specific activity of the kinase). - Add 25 μ? of 4x inhibitor. Add 25 μ? 4x of ATP for inhibitor assay. Incubate for 10 minutes at room temperature. Stop the reaction by adding 50 μ? 0.05 N HCI per cavity. - Wash the plate.

** Final concentrations for the reaction: 5 μ? of ATP, 5% DMSO. 3. Antibody binding - Dilute a 1 mg / ml aliquot of the PY20-HRP antibody (Pierce) (a phosphotyrosine antibody) at 50 ng / ml in 0.1% BSA in PBS through a two step dilution (100x, after 200x).

Add 100 μ? of Ab per cavity. Incubate for 1 hour at room temperature. Incubate for 1 hour at 4 ° C. - Wash the plate 4x. 4. Color reaction Prepare the TMB substrate and add 100 μ? per cavity. Verify OD 650 nm until reaching a value of 0.6. - Stop with 1M phosphoric acid. Shake on the plate reader. Read OD immediately at 450 nm. The optimal incubation times and the enzyme reaction conditions vary slightly with the enzyme preparations and are determined empirically for each batch.

EXAMPLE 4 Cellular Assay to Determine the Potency of Tle-2 Inhibitors The following cell assay can be used to determine the potency of a Tie-2 inhibitor.

"NIH-3T3 / hTEK Cell Line A retroviral expression vector containing full-length Tie-2 cDNA, LNCX6 h-TEK, was provided by Dr. Kevin Peters A tumorigenic subline of NIH 3T3 cells was infected with 10 ig of LNCX6 h-TEK through the calcium phosphate precipitation method and selected with 400 μg / ml of neomycin, individual clones were isolated and analyzed for the presence of TIE-2 protthrough Western staining. a maximum expression of Tie-2 in clone # 67. The expression of the angiopoietin 1 message has been shown using PCR and an autocrine loop was revealed in the presence of vanadate.

Cell Tie-2 Assay The cell auto-phosphorylation of Tie-2 was measured using the NIH-3T3 / hTEK cell line (hTEK). The cells were seeded in 96-well plates overnight. The medium was removed and the cells were treated with the inhibitor for 20 minutes and with the phosphatase inhibitor NaV03 (2 mM) for a further 15 minutes. The cells were lysed with pH regulator of RIPA and lizatos were immunoprecipitated using a specific a-Tie-2 monoclonal antibody (KP33, provided by Dr. Kevin Peters) and the IP'd protran on SDS-PAGE. The level of phosphotyrosine in the Tie-2 protwas then determined through α-phosphotyrosine antibodies (4G10, Upstate Biotechnology) in Western stains. The films were scanned and the percent inhibition was determined as compared to the untreated control. " EQUIVALENTS Although this invention has been particularly shown and described with references to its preferred embodiments, it will be understood by those skilled in the art that various changes may be made in the form and details herwithout departing from the scope of the invention encompassed by the claims. annexes.

Claims

CLAIMS 1. - A crystalline polypeptide, the polypeptide comprising the catalytic domain of a Tie-2 protein. 2. The crystalline polypeptide according to the claim 1, wherein the polypeptide comprises the catalytic domain of human Tie-2. 3. A crystalline polypeptide-ligand complex, wherein the polypeptide comprises the catalytic domain of a Tie-2 protein. 4. The polypeptide / crystalline ligand complex according to claim 3, wherein the polypeptide comprises the mammalian Tie-2 catalytic domain. 5. The polypeptide / crystalline ligand complex according to claim 4, wherein the mammalian Tie-2 protein is human Tie-2. 6. - The polypeptide / crystalline ligand complex according to claim 5, wherein the polypeptide comprises amino acids 802-1124 of SEQ ID NO: 1. 7. - The polypeptide / crystalline ligand complex according to claim 6, wherein the ligand is of the formula: 8. - The polypeptide / crystalline ligand complex according to claim 7, having the unit cell parameters wherein a is approximately 96 A, b is approximately 118 A, c is approximately 78 A, and a = ß =? = 90 ° . 9. The polypeptide / crystalline ligand complex according to claim 6, wherein the ligand is of the formula: 10. - The polypeptide / crystalline ligand complex according to claim 9, having the unit cell parameters where a and b are approximately 86.0 A, c is approximately 112.0 A and a = IS = y = 90e. 11. - The polypeptide / crystalline ligand complex according to claim 6, wherein the ligand is of the formula: 12. - The polypeptide / crystalline ligand complex according to claim 11, having the unit cell parameters where a and b are approximately 86.0 A, and c is approximately 112.0 A and a = ß =? = 90 °. 13. The polypeptide / crystalline ligand complex according to claim 6, wherein the ligand is of the formula: 14. - The polypeptide / crystalline ligand complex according to claim 13, having the unit cell parameters where a and b are approximately 86.0 A, c is approximately 112.0 A and a = ß =? = 90 °. 15. - A method for determining the three-dimensional structure of a first polypeptide, comprising the catalytic domain of a Tie-2 protein, said method comprising the steps of: (a) obtaining a crystal of the first polypeptide comprising the catalytic domain of Tie -2; (b) obtaining X-ray diffraction data for said crystal; and (c) solving the crystal structure of said crystal using the atomic coordinates of a second polypeptide and said X-ray diffraction data, the second polypeptide comprising the catalytic domain of a Tie-2 protein. 16. - The method according to claim 15, wherein the crystal of the first polypeptide comprises a first polypeptide in complex with a ligand. 17. - The method according to claim 15, wherein the first polypeptide comprises the catalytic domain of a mammalian Tie-2 protein. 18. - The method according to claim 17, wherein the first polypeptide and the second polypeptide, independently, comprise the catalytic domain of a human Tie-2 protein. 19. The method according to claim 18, wherein the first polypeptide comprises the catalytic domain of human wild-type Tie-2 and the second polypeptide comprises the catalytic domain of human wild-type Tie-2. 20. The method according to claim 19, wherein the first polypeptide comprises the catalytic domain of human wild-type Tie-2. 21. - A method for identifying a compound, which is an inhibitor of a Tie-2 protein, said method comprises the steps of: (a) obtaining the atomic coordinates of a crystal of a polypeptide comprising the catalytic domain of a protein of Tie-2; (b) use the atomic coordinates to define the active subsets of Tie-2; and (c) identify a compound that binds to one or more of the active sub-sites; wherein the compound that binds to the subsite or active sites is an inhibitor of a Tie-2 protein. 22. The method according to claim 21, further comprising the step of: (d) determining the ability of the compound identified in step (c) to inhibit Tie-2. 23. - The method according to claim 21, wherein the Tie-2 protein is a mammalian protein. 24. - The method according to claim 22, wherein the Tie-2 protein is a human protein. 25. - The method according to claim 24, wherein the Tie-2 protein is human wild-type Tie-2. 26. The method according to claim 21, wherein the crystal further comprises a ligand bound to said catalytic domain. 27. The method according to claim 24, wherein the polypeptide comprises amino acids 802-1124 of SEQ ID NO: 1. 28. The method according to claim 24, wherein the ligand is of the formula: 29. - The method according to claim 28, wherein the crystal has unitary cell parameters where a is approximately 96 A, b is approximately 118 Á, c is approximately 78 Á and a = β = β = 90 ° ?. 30. The method according to claim 24, wherein the ligand is of the formula: 31. - The method according to claim 30, wherein the crystal has unit cell parameters where a and b are approximately 86.0 A, c is approximately 112.0 A and a = ß =? = 90 °. 32. The method according to claim 24, wherein the ligand is of the formula: 33. - The method according to claim 32, wherein the crystal has unitary cell parameters where a and b are approximately 86.0 A, and c is approximately 112.0 A and a = ß =? = 90 °. 34. The method according to claim 24, wherein the ligand is of the formula: 35. - The method according to claim 34, wherein the crystal has unitary cell parameters where a and b are approximately 86.0 A, c is approximately 112.0 A and a = ß =? = 90 °. 36. - A method for identifying a compound, which is a potential inhibitor of a Tie-2 protein, said method comprises the step of designing a compound that will interact with one or more subsites in the catalytic domain of the Tie-2 protein. 2, based on the coordinates of the crystal structure of a polypeptide comprising the catalytic domain; wherein the compound is identified as a potential inhibitor of the Tie-2 protein. 37. The method according to claim 36, wherein the Tie-2 protein is a mammalian protein. 38. - The method according to claim 37, wherein the Tie-2 protein is a human protein. 39. - The method according to claim 38, wherein the Tie-2 protein is wild-type human Tie-2. 40. The method according to claim 39, wherein the polypeptide comprises amino acids 802-1124 of SEQ ID NO: 1. 41. - The method according to claim 38, wherein the crystal structure coordinates are set forth in Figure 3. 42.- The method according to claim 38, wherein the crystal structure coordinates are set in the Figure 4. 43. - The method according to claim 38, wherein the crystal structure coordinates are set forth in Figure 5. 44. - The method according to claim 38, wherein the crystal structure coordinates are set forth in Figure 6. 45. - The method according to claim 38, wherein the compound interacts with one or more of subsites 1 through 9. 46. - The method according to claim 45, wherein the compound interacts with one or more of subsites 1 to 9. 47. - The method according to claim 46, wherein the compound interacts with one or more of the subsites 1 to 9. 48. - The method according to claim 46, wherein the compound interacts with a group of subsites comprising the subsite 1 and the subsite 2. 49. - The method according to claim 47, wherein the compound interacts with a group of sub-sites comprising sub-site 1, sub-site 2 and sub-site 3. 50. - The method according to claim 47, wherein the compound interacts with a group of sub-sites comprising sub-site 1, sub-site 2 and subsite 8. 51. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 3 and subsite 8. 52. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 4 and subsite 5. 53. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 7 and subsite 8. 54. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 3, subsite 7 and subsite 8. 55. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 3, subsite 7 and subsite 8. 56. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 4, subsite 6 and subsite 8. 57. - The method according to claim 47, wherein the compound interacts with a group of subsites comprising subsite 1, subsite 2, subsite 3, subsite 4, subsite 6 and subsite 8. 58. - The method according to claim 47, wherein the compound interacts with a group of sub sites comprising subsite 1, subsite 2, subsite 3, subsite 4, subsite 6 and subsite 8. 59. - A Tie-2 inhibitor comprising two or more of the following: (a) a hydrogen bond donor placed to interact with Glu 903 Tie 2 human; (b) a hydrogen bond acceptor placed to interact with 905 Ala of human Tie-2; (c) a hydrogen bond donor placed to interact with 905 Ala of human Tie-2; (d) a hydrophobic portion placed to interact with one or more of Me 830, Val 838, Ala 853, Me 886, He 902, Tyr 904, Ala 905 and Leu 971 of human Tie-2; (e) a positively charged hydrogen bond donor or functional group placed to interact with Asp 912 of human T¡e-2; (f) a hydrogen bond donor or hydrogen bond acceptor placed to interact with Asn 909 Human tie-2; (g) a hydrophobic portion placed to interact with one or more of Val 838, Lys 855, Lie 886, Me 902, Leu 971 and Ala 981 of human Tie-2; (h) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 855 of human Tie-2; (i) a hydrogen bond acceptor placed to interact with Asp 982 of human Tie-2; (j) a hydrogen bond acceptor placed to interact with Phe 983 of human Tie-2; (k) a hydrophobic portion placed to interact with one or more of Leu 873, Leu 876, lie 885, lie 886, Leu 888, Leu 900, Me 902, Ala 981 and Phe 983 of human Tie-2; (I) a positively charged hydrogen bond donor or functional group placed to interact with Asp 982 Human Tie-2; (m) a hydrogen bond donor placed to interact with Me 886 of human Tie-2; (n) a hydrogen bond donor placed to interact with Leu 768 from Human-2; (o) a hydrogen bond acceptor placed to interact with Gly 831 of human Tie-2; (p) a positively charged hydrogen bond donor or functional group placed to interact with Glu 832 Human Tie-2; (q) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 840 Human Tie-2; (r) a hydrogen bond acceptor or negatively charged functional group placed to interact with Lys 916 Human Tie-2; (s) a hydrogen bond acceptor or negatively charged functional group placed to interact with Arg 968 Human Tie-2; (t) a hydrogen bond donor placed to interact with Arg 968 of human Tie-2. 60. - The Tie-2 inhibitor according to claim 59, comprising (b) and (d). 61. - The Tie-2 inhibitor according to claim 59, comprising (d) and at least one of (a), (b) and (c). 62. The Tie-2 inhibitor according to claim 59, comprising (d) and at least two of (a), (b) and (c). 63. The Tie-2 inhibitor according to claim 62, further comprising at least one of (e) and (f). 64. - The Tie-2 inhibitor according to claim 62, further comprising (g). 65. The Tie-2 inhibitor according to claim 63, further comprising (g). 66.- The Tie-2 inhibitor according to claim 64, further comprising (k). 67. - The Tie-2 inhibitor according to claim 65, which also comprises (k). 68. The Tie-2 inhibitor according to claim 62, further comprising at least one of (i) and (j). 69. The Tie-2 inhibitor according to claim 63, further comprising at least one of (i) and (j). 70. The Tie-2 inhibitor according to claim 66, further comprising at least one of (i) and (j). 71.- The Tie-2 inhibitor according to claim 67, further comprising one of (i) and (j). 72. The Tie-2 inhibitor according to claim 59, wherein the inhibitor has a Ki value of at least about 1 mM. 73.- The Tie-2 inhibitor according to claim 59, wherein the inhibitor has a Ki value of at least about 100 μ ?. 74.- The Tie-2 inhibitor according to claim 59, wherein the inhibitor has a Ki value of at least about 10 μ ?. 75. - The Tie-2 inhibitor according to claim 59, wherein the inhibitor selectively binds Tie-2 receptors. 76. - A method for treating a Tie-2 dependent condition in a patient, comprising the step of administering to the patient a therapeutically effective amount of a Tie-2 inhibitor of claim 59. 77. - The method in accordance with Claim 76, wherein the patient is a human being. 78. The method according to claim 76, wherein the Tie-2 dependent condition is characterized by excessive vascular proliferation. 79. The method according to claim 78, wherein the Tie-2 dependent condition is a hyperproliferative disorder, cancer, a cardiovascular condition, an ocular condition, von Hippel Lindau disease, pemphigoid, psoriasis, Paget's disease. , polycystic kidney disease, fibrosis, sarcoidosis, cirrhosis, thyroiditis, Osler-Weber-Rendu disease, chronic inflammation, synabitis, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ulcer and sepsis. 80. - The method according to claim 79, wherein the condition is a cancer selected from the group consisting of solid tumors, sarcoma, fibrosarcoma, osteoma, melanoma, retinoblastoma, rhabdomyosarcoma, glioblastoma, neuroblastoma, teratocarcinoma, a hematopoietic malignancy, malignant ascites, Kaposi's sarcoma. Hodgkin's disease, lymphoma, myeloma and leukemia. 81. The method according to claim 79, wherein the condition is a vascular condition selected from the group consisting of atherosclerosis, restenosis, ischemia / reperfusion injury, chronic occlusive pulmonary disease, vascular occlusion, carotid obstructive e.nfermedd , Crow-Fukase syndrome (POEMS), anemia, ischemia, infarction and vascular effusion disorders. 82. The method according to claim 79, wherein the condition is an ocular condition selected from the group consisting of ocular or macular edema, ocular neovacular disease, scleritis, radial keratotomy, uveitis, vitritis, myopia, optical points, separation chronic retinal, complications after laser treatment, conjunctivitis, Stargardt's disease, Eales disease, retinopathy, macular degeneration and microangiopathy. 83. - The method according to claim 76, wherein the disorder involves aberrant endothelial-perioendothelial interactions. 84. A method for reducing fertility in a patient, comprising the step of administering to the patient a therapeutically effective amount of a Tie-2 inhibitor of claim 59. 85.- A method for promoting angiogenesis or vasculogenesis in a patient. , comprising the step of administering to the patient a therapeutically effective amount of a Tie-2 inhibitor of claim 59. 86. - The method according to claim as claimed in claim 85, wherein the Tie-2 inhibitor. it is administered in combination with a pro-angiogenic growth factor. 87. - A method for determining the three-dimensional structure of a polypeptide, comprising the catalytic domain of a Tie-2 protein, the method comprises the steps of: (a) obtaining a crystal of the polypeptide comprising the catalytic domain of Tie-2; (b) obtaining X-ray diffraction data for said crystal; and (c) solving the crystal structure of said crystal. 88. A crystalline polypeptide, the polypeptide comprising a sequence having 80% homology to the catalytic domain of a Tie-2 protein.