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  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4192—1,2,3-Triazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4196—1,2,4-Triazoles
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  • A61P3/04—Anorexiants; Antiobesity agents
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
  • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
  • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
• • C—CHEMISTRY; METALLURGY
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
  • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
  • C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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  • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
  • C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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  • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
  • C07D249/16—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
  • C07D249/22—Naphthotriazoles

Definitions

• This invention relates to a method of inhibiting hMetAP2 by administering compounds with certain structural, physical and spatial characteristics that allow for the interaction of said compounds with specific residues of the active site of the enzyme.
• This interaction between the compounds of this invention and the active site inhibits the activity of hMetAP2 and these compounds are useful for treating conditions mediated by angiogenesis, such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity.
• This invention also relates to the identification of the metalloprotease catalytic active site for this enzyme and methods enabling the design and selection of inhibitors of said active site.
• angiogenesis a process termed angiogenesis (Folkman J. (1974) Adv Cancer Res. 19; 331).
• the new blood vessels induced by tumor cells as their life-line of oxygen and nutrients also provide exits for cancer cells to spread to other parts of the body. Inhibition of this process has been shown to effectively stop the proliferation and metastasis of solid tumors.
• a drug that specifically inhibits this process is known as an angiogenesis inhibitor.
• the anti-angiogenesis therapy (“indirect attack”) has several advantages over the “direct attack” strategies. All the “direct attack” approaches such as using DNA damaging drugs, antimetabolites, attacking the RAS pathway, restoring p53, activating death programs, using aggressive T-cells, injecting monoclonal antibodies and inhibiting telomerase, etc., inevitably result in the selection of resistant tumor cells. Targeting the endothelial compartment of tumors as in the "indirect attack”, however, should avoid the resistance problem because endothelial cells do not exhibit the same degree of genomic instability as tumor cells.
• anti-angiogenic therapy generally has low toxicity due to the fact that normal endothelial cells are relatively quiescent in the body and exhibit an extremely long turnover.
• direct attack target different cell types, there is a great potential for a more effective combination therapy.
• More than 300 angiogenesis inhibitors have been discovered, of which about 31 agents are currently being tested in human trials in treatment of cancers (Thompson, et al., (1999) J Pathol 187, 503).
• TNP-470 a semisynthetic derivative of fumagillin of Aspergillus fuigatus, is among the most potent inhibitors of angiogenesis.
• hMetAP-2-catalyzed cleavage of the initiator methionine of proteins could be essential for releasing many proteins that, after myristoylation, function as important signaling cellular factors involved in cell proliferation.
• Proteins known to be myristoylated include the src family tyrosine kinases, the small GTPase ARF, the HIV protein nef and the ⁇ subunit of heterotrimeric G proteins.
• a recently published study has shown that the myristoylation of nitric oxide synthase, a membrane protein involved in cell apoptosis, was blocked by fumagillin (Yoshida, et al. (1998) Cancer Res. 58(16), 3751).
• MetAP2-catalyzed release of the glycine-terminal myristoylation substrate is proposed to be an indirect outcome of inhibition of MetAP2-catalyzed release of the glycine-terminal myristoylation substrate.
• MetAP enzymes are known to be important to the stability of proteins in vivo according to the " ⁇ -end rule" which suggests increased stability of methionine-cleaved proteins relative to their ⁇ -terminal methionine precursors (Varshavsky, A (1996) Proc. Natl Acad. Sci. U.S.A 93, 12142). Inhibition of hMetAP2 could result in abnormal presence or absence of some cellular proteins critical to the cell cycle.
• Methionine aminopeptidases are ubiquitously distributed in all living organisms. They catalyze the removal of the initiator methionine from newly translated polypeptides using divalent metal ions as cofactors. Two distantly related MetAP enzymes, type 1 and type 2, are found in eukaryotes, which at least in yeast, are both required for normal growth; whereas only one single MetAP is found in eubacteria (type 1) and archaebacteria (type 2). The ⁇ -terminal extension region distinguishes the methionine aminopeptidases in eukaryotes from those in procaryotes.
• a 64-amino acid sequence insertion (from residues 381 to 444 in hMetAP2) in the catalytic C-terminal domain distinguishes the MetAP-2 family from the MetAP- 1 family.
• all MetAP enzymes appear to share a highly conserved catalytic scaffold termed "pita-bread" fold (Bazan, et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2473), which contains six strictly conserved residues implicated in the coordination of the metal cofactors.
• Mammalian type 2 methionine aminopeptidase has been identified as a bifunctional protein implicated by its ability to catalyze the cleavage of N-terminal methionine from nascent polypeptides (Bradshaw, et al (1998) Trends Biochem. Sci. 23, 263) and to associate with eukaryotic initiation factor 2 ⁇ (eEF-2 ⁇ ) to prevent its phosphorylation (Ray, et al. (1992) Proc. Natl Acad. Sci. U.S.A. 89, 539). Both the genes of human and rat MetAP2 were cloned and have shown 92% sequence identity (Wu,. et al. (1993) J Biol. Chem.
• this invention relates to the method of inhibiting hMetAP2 using compounds having the characteristics hereinbelow defined.
• the present invention provides a method for inhibiting hMetAP2 by administering compounds with certain structural, physical and spatial characteristics that allow for the interaction of said compounds with specific residues of the active site of the enzyme.
• This interaction inhibits the activity of hMetAP2 and, thus, enables treatment of diseases in which angiogenesis is a factor, for example, cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity.
• the invention provides a method for identifying inhibitors of the compositions described above which method involves the steps of: providing the coordinates of the metalloprotease structure of hMetAP2 to a computerized modeling system; identifying compounds which will bind to the structure; and screening the compounds or analogs derived therefrom identified for hMetAP2 inhibitory bioactivity.
• this invention provides crystallized molecules and molecular complexes which comprise one or more of the active site binding pockets of hMetAP2 or close structural homologues to the binding pockets.
• this invention provides a data storage medium encoded with the corresponding structure coordinates of those crystallized molecules or molecular complexes.
• Such data storage material is capable of displaying such molecules and molecular complexes as a graphical three-dimensional representation on a computer screen.
• this invention provides methods of using the structure coordinates and atomic details of hMetAP2 to screen, evaluate computationally, design and synthesize inhibitors that bind to hMetAP2, its mutants or homologues thereof, and that avoid the undesirable physical and pharmacologic properties of hMetAP2.
• Figure 1 is a ribbon diagram of hMetAP2. The amino and carboxyl-termini are indicated by N and C. The drawing was produced using the program MOLSCRIPT [Kraulis, P., J. Appl. Crystallogr., 24, 946-950 (1991)].
• Figure 2 is an illustration of the active site of hMetAP2.
• Figure 3 is a stereoview of the active site of hMetAP2. For clarity, no hydrogen atoms or water molecules are shown.
• Figures 4, 6, 8, 10, 12, 14, 16, 18 and 20 are illustrations of the active site of hMetAP2 in complex with novel inhibitors of hMetAP2.
• Figure 4: Inhibitor 4-(4- methylphenyl)-lH-l,2,3-triazole;
• Figure 6: Inhibitor 3-anilino-5-[2-(phenyl)ethyl]- 1,2,4-triazole;
• Figure 8: Inhibitor 3-methylacetate-3-anilino-5-benzylthio- 1,2,4- triazole;
• Figure 10: Inhibitor 3-(2-isopropyl-anilino)-5-(thiophen-2-ylthio)- 1,2,4- triazole;
• Figure 12: Inhibitor 4-(3-methylphenyl)-lH-l,2,3-triazole;
• Figure 14: Inhibitor 4-(2-chlorophenyl)-lH-l,2,3-triazole;
• Figures 5, 7, 9, 11, 13, 15, 17, 19 and 21 are stereoviews of the active site of human methionineaminopeptidase-2 in complex with novel inhibitors of human methionineaminopeptidase-2.
• Figure 5: Inhibitor 4-(4-methylphenyl)-lH- 1,2,3- triazole;
• Figure 7: Inhibitor 3-anilino-5-[2-(phenyl)ethyl]-l,2,4-triazole;
• Figure 9: Inhibitor 3-methylacetate-3-anilino-5-benzylthio-l ,2,4-triazole;
• Figure 13: Inhibitor 4-(3-methylphenyl)-lH-l,2,3-triazole;
• Figure 15: Inhibitor 4-(2-chloropheny
• Table I provides the three dimensional protein coordinates of the human methionineaminopeptidase-2 crystalline structure of the invention.
• Tables II-X provide the three dimensional coordinates for the human methionineaminopeptidase-2 complex with specific inhibitors of the present invention;
• Table II 4-(4-methylphenyl)-lH-l,2,3-triazole;
• Table III: Inhibitor 3- anilino-5-[2-(phenyl)ethyl]-l,2,4-triazole;
• Table IV: Inhibitor 3-methylacetate-3- anilino-5-benzylthio-l,2,4-triazole;
• Table V: Inhibitor 3-(2-isopropyl-anilino)-5- (thio ⁇ hen-2-ylthio)-l,2,4-triazole;
• Table VI: Inhibitor 4-(3-methylphenyl)-lH- 1,2,3- triazole;
• the present invention provides a method of using a crystalline form of the hMetAp2 protein to form an inhibitor complex at the protein's active site with a protein inhibitor, wherein such method utilizes the crystalline form and the active site to identify protein inhibitor compounds.
• binding pocket refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound.
• active site binding pocket or "active site of hMetAP2" as used herein, independently refer to the site where N-terminal methionine cleavage from a peptide substrate occurs.
• hMetAP2 amino acids are situated close enough to a peptide molecule present in the active site (within 5A) to interact with it. It will be readily apparent to those of skill in the art that the numbering of amino acids in other isoforms of hMetAP2 may be different than that isolated from human endothelial cells.
• association with independently refer to a condition of proximity between a chemical entity or compound, or portions thereof, and an hMetAP2 molecule, or portions thereof.
• the association may be non-covalent - wherein the juxtaposition is energetically favored by hydrogen bonding or van deer Walls or electrostatic interactions - or it may be covalent.
• electrophilic atom refers to an electron deficient atom. This term includes, an oxygen, nitrogen or sulfur atom.
• hydrophobic interaction refers to a contact between hydrophobic groups or atoms, such as non-polar or polarizable atoms, e.g., aromatic ring atoms, such as C, N, O or S, or aliphatic non-polar atoms, such as C, H, or S, with a residue where contact defines an interatomic distance between 3.3 - 5.5A.
• Arg Arginine
• the design of compounds that bind to or inhibit hMetAP2 according to this invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with hMetAP2. Non-covalent molecular interactions important in the association of hMetAP2with its substrate include hydrogen bonding, van der Waals and hydropobic interactions.
• the compound must be able to assume a conformation that allows it to associate with hMetAP2. Although certain portions of the compound will not directly participate in this association with hMetAP2, those portions may still influence the overall conformation of the molecule. This may have a significant impact on potency.
• Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding sites, e.g., active site or accessory binding site of hMetAP2, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with hMetAP2.
• the inhibitors of hMetAP2 used in the present invention comprise one or two heteroatoms, wherein the inhibitors interact with any one or more of the following:
• the inhibitors of hMetAP2 used in the present invention interact with any two or more of the above-identified regions of the active site.
• the compounds used in the methods of the present invention include a 5- or 6-membered aromatic heterocyclic ring structure, wherein there is at least one heteroatom in the ring.
• the key structural features of the inhibitors of the present invention include a heterocycle group, preferably a 1,2,3-triazole or a 1,2,4-triazole ring system, and a hydrophobic group. These features must be able to make the appropriate interactions with the hMetAP2 active site.
• the method of inhibiting hMetAP2 of the present invention comprises administering to a mammal, preferably a human, in need thereof a compound that fits spatially into the active site of hMetAP2, said compound comprising one or two heteroatoms, wherein the compound has any one or more of the following interactions.
• a mammal preferably a human
• said compound comprising one or two heteroatoms, wherein the compound has any one or more of the following interactions.
• the heteroatom is 1.7 - 2.4A from the closest metal.
• atom capable of hydrogen bonding such as oxygen, nitrogen or sulfur, that interacts with histidine 231 , such that the distance between the atom and histidine 231 is 2.2 - 4.5A, preferably 2.4 - 3.2A.
• a hydrophobic group that interacts with a residue selected from tyrosine 444, histidine 231, histidine 382, alanine 414, tyrosine 383, phenylalanine 219, proline 220, methionine 384, isoleucine 338, and glycine 222, such that the distance between the hydrophobic group atom and the residue is 2.2 - 4.5A, preferably 2.4 - 3.2 A.
• a group capable of hydrogen bonding such as a carbonyl oxygen or an ether oxygen that interacts with asparagine 315.
• a hydrophobic group such as a methyl group in contact with leucine 328, leucine 447, histidine 231, or alanine 230, such that the distance between the hydrophobic group and the residue is 3.4 - 5. ⁇ A.
• the method of inhibiting hMetAP2 of the present invention comprises administering to a mammal, preferably a human, in need thereof, a compound that fits spatially into the active site of hMetAP2, said compound comprising one or two heteroatoms and which has the following interactions (i) an interaction, singly, or jointly as a pair, to one or both metals in the active site of hMetAP2, wherein the metals are selected from cobalt, zinc, manganese, iron and nickel, and wherein the heteroatoms are 1.5 - 3.5A from the closest metal, or (ii) a hydrophobic interaction with a residue selected from tyrosine 444, histidine 231, histidine 382, alanine 414, tyrosine 383, phenylalanine 219, proline 220, methionine 384, isoleucine 338, and glycine 222, such that the distance between the hydrophobic group atom and the residue is 2.2 - 4.5A,
• the metal in the active site is cobalt or zinc.
• the method of inhibiting hMetAP2 of the present invention comprises administering to a mammal, preferably a human, in need thereof, a compound that fits spatially into the active site of hMetAP2, said compound comprising one or two heteroatoms and which has the following interactions: (1) (i) an interaction, singly, or jointly as a pair, to one or both metals in the active site of hMetAP2, wherein the metals are selected from cobalt, zinc, manganese, iron and nickel, and wherein the heteroatoms are 1.5 - 3.5A from the closest metal, or (ii) a hydrophobic interaction with a residue selected from tyrosine 444, histidine 231 , histidine 382, alanine 414, tyrosine 383, phenylalanine 219, proline 220, methionine 384, isoleucine 338, and glycine 222, such that the distance between
• the method of inhibiting hMetAP2 of the present invention comprises administering to a mammal, preferably a human, in need thereof, a compound that fits spatially into the active site of hMetAP2, said compound comprising one or two heteroatoms and which has the following interactions: (1) (i) an interaction, singly, or jointly as a pair, to one or both metals in the active site of hMetAP2, wherein the metals are selected from cobalt, zinc, manganese, iron and nickel, and wherein the heteroatoms are 1.5 - 3.5A from the closest metal, or (ii) a hydrophobic interaction with a residue selected from tyrosine 444, histidine 231, histidine 382, alanine 414, tyrosine 383, phenylalanine 219, proline 220, methionine 384, isoleucine 338, and glycine 222, such that the distance between the hydrophobic group atom and the residue is 2.2 - 4.5A,
• the method of inhibiting hMetAP2 of the present invention comprises administering to a mammal, preferably a human, in need thereof, a compound that fits spatially into the active site of hMetAP2, said compound comprising one or two heteroatoms and which has the following interactions: (1) (i) an interaction, singly, or jointly as a pair, to one or both metals in the active site of hMetAP2, wherein the metals are selected from cobalt, zinc, manganese, iron and nickel, and wherein the heteroatoms are 1.5 - 3.5A from the closest metal, or (ii) a hydrophobic interaction with a residue selected from tyrosine 444, histidine 231, histidine 382, alanine 414, tyrosine 383, phenylalanine 219, proline 220, methionine 384, isoleucine 338, and glycine 222, such that the distance between the hydrophobic group atom and the residue is 2.2 - 4.5A,
• Compounds used in the method of the present invention include, but are not limited to, the following: 4-(4-methylphenyl)-lH-l,2,3-triazole; 3-anilino-5-[2-(phenyl)ethyl]-l,2,4-triazole; 3-methylacetate-3-anilino-5-benzylthio- 1 ,2,4-triazole; 3-(2-isopropyl-anilino)-5-(thiophen-2-ylthio)- 1 ,2,4-triazole; 4-(3-methylphenyl)- IH- 1 ,2,3-triazole; 4-(2-chlorophenyl)- IH- 1 ,2,3-triazole; 3-Benzyl-5-(benzylthio)-l,2,4-triazole; 3-anilino-5-benzylthio- 1 ,2,4-triazole; and lH-naptho[l,2- ]-l,2,3-tri
• n 1 or 2;
• A, B, D and E are independently selected from C, O, N, and S; R is up to 5 variables selected independently from -H, -OH, Ar-C()-6alkyl-,
• Het'-Co-6 alkyl- optionally substituted C ⁇ _6alkyl, C 2 _6alkenyl, C 2 _galkynyl, optionally substituted Het-Crj- ⁇ alkyl-, C3_7cycloalkyl-Co-6alkyl-, Ci .galkoxy, -S-C ⁇ .galkyl, optionally substituted Ar -C ⁇ - ⁇ alkoxy-, Het'-C ( )-6alkoxy-, -NR4R5 5 Hef-S-Co- ⁇ alkyl-, -(CH 2 )i- 6 OH, -(CH 2 )I _ 6 NR4R5, -O(CH 2 ) ⁇ . 6 NR 4 R 5 , -(CH 2 )o-6CO 2 R 6 , -O(CH 2 ) 1 . 6 CO 2 R6, -(CH 2 )i-6SO 2 R 6 ,
• R is -C 0 -6alkyl-C(O)XAB, -C()-6alkyl-S(O) 2 X'AB, -CQ-6alkyl-X'AB, wherein X' is O, S, C or N, and wherein, A and B are independently selected from H, optionally substituted Ci .galkyl, C3_6alkenyl, C3_6alkynyl, optionally substituted Ar-Co-6 a lkyl-, optionally substituted Het-Co-6 lkyl-, or C3_7cycloalkyl-C ⁇ -6 a
• Q is a 5- or 6-membered monocyclic ring optionally containing up to two heteroatoms selected from N, O, or S, or an 8- to 11-membered fused bicyclic ring optionally containing up to four heteroatoms selected from N, O, or S;
• Ri and R ⁇ are independently selected from H-, Ph-Co-6 a lkyl-, Het-Co-6 alkyl-, Chalky!-, C ⁇ _6alkoxy-, -S-C galkyl-, Ph-C ⁇ - ⁇ alkoxy-, Het-CQ-6alkoxy-, HO-, R 4 R5N-, Het-S-Co_6alkyl-, Ph-S-CQ- ⁇ alkyl-, HO(CH 2 ) ⁇ _6-, R 4 R 5 N(CH 2 ) .
• R R (CH 2 ) 2 . 6 O-, R6cO 2 (CH 2 )o-6-, R 6 CO 2 (CH 2 ) 1 . 6 O-, R6SO 2 (CH 2 )!. 6 -, -CF3, -OCF3, or halogen, and Ph or Het are substituted with up to five of C 2 _6alkyl-, C ⁇ _ 6 alkoxy-, R 4 R 5 N(CH 2 )!_ 6 -, R 4 R 5 N(CH 2 )2-6 0 -> -CO 2 R 6 , -CF3 or, halogen;
• R 4 , R5, and R° are independently selected from H-, optionally substituted Ci .galkyl, C3_galkenyl, C3_6alkynyl, optionally substituted Ar-C()-6 a lkyl-, optionally substituted Het-C()-6alkyl-, or C3_7cycloalkyl-Co-6alkyl-,
• R 7 is optionally substituted C ⁇ _6alkyl, C3_6alkenyl, C3_6alkynyl, optionally substituted Ar-C()-6 a lkyl-, optionally substituted Het-Co_6alkyl-, C3-7cycloalkyl-C ⁇ -6 a lkyl-.
• Ci -6alkyl as used herein at all occurrences means a substituted and unsubstituted, straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof.
• Any Ci-6alkyl group may be optionally substituted independently by one or more of -OR 4 , -R 4 , -NR 4 R5.
• CQalkyl means that no alkyl group is present in the moiety.
• Ar-Cgalkyl- is equivalent to Ar.
• C3_7cycloalkyl as used herein at all occurrences means substituted or unsubstituted cyclic radicals having 3 to 7 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl radicals.
• C 2 -6alkenyl as used herein at all occurrences means an alkyl group of 2 to 6 carbons wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond.
• C 2 -6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included within the scope of this invention.
• Any C 2 -6alkenyl group may be optionally substituted independently by one or more of Ph-Co-6 a lkyl-, Het -Co-6 alkyl-, Cj.galkyl, -S-C ⁇ .6alkyl, Ph-C ⁇ -6 a lkoxy-, Het'-C ⁇ - ⁇ koxy-, -OH, -NR 4 R5, Het'-S-C 0 -6 a lkyl-, -(CH ⁇ j ⁇ OH, (CH 2 )!. 6 NR R5, -O(CH 2 )!. 6 NR 4 R5, -(CH 2 ) 0 . 6 CO 2 R6, - ⁇ (CH 2 ) ⁇ _ 6 CO 2 R 6 , -(CH 2 ) 1 . 6 SO 2 R 7 , -CF 3 , -OCF3 or halogen.
• C2-6 lkynyl as used herein at all occurrences means an alkyl group of 2 to 6 carbons wherein one carbon-carbon single bond is replaced by a carbon-carbon triple bond.
• C -6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
• Ar or "aryl” as used herein interchangeably at all occurrences mean phenyl and naphthyl, optionally substituted by one or more of Ph-C()-6 ai kyl, Het'-Co-6 alkyl-, C ⁇ _6alkyl, Ci . ⁇ alkoxy-, -S-Cj. ⁇ alkyl, Ph-C()-6 a lkoxy-, Het'-C 0 -6alkoxy-, -OH, -NR 4 R 5 , Het -S-C 0 -6alkyl-, -(CH 2 )i_6OH,
• Ph and Het may be optionally substituted with one or more of Ci .galkyl, Ci .galkoxy-, -OH, -(CH 2 ) ⁇ _6NR 4 R 5 , -O(CH 2 )i _6NR 4 R 5 , -CO 2 R 6 , -CF3, or halogen; two C ⁇ _6 a lkyl or Ci .galkoxy groups may be combined to form a 5-7 membered ring, saturated or unsaturated, fused onto the Ar ring (e.g., to form a divalent alkylene or alkylenedioxy moiety attached to adjacent positions on the Ar ring
• Het or "heterocyclic” as used herein interchangeably at all occurrences, mean a stable 5- to 7-membered monocyclic, a stable 7- to 10-membered bicyclic, or a stable 11- to 18-membered tricyclic heterocyclic ring all of which are either saturated or unsaturated, and which consist of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
• the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or more of C ⁇ alkyl, C 1 . 6 alkoxy, -OH, -(CH )!_ 6 NR R 5 , -O(CH 2 ) ⁇ _ 6 NR 4 R 5 , -CO R 6 , -CF3, or halogen.
• heterocycles include, but are not limited to piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridinyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, t
• hetero or “heteroatom” as used herein interchangeably at all occurrences mean oxygen, nitrogen and sulfur.
• halo or halogen as used herein interchangeably at all occurrences mean F, Cl, Br, and I.
• CQ denotes the absence of the substituent group immediately following; for instance, in the moiety ArCQ-6alkyl-, when C is 0, the substituent is Ar, e.g., phenyl. Conversely, when the moiety ArC ⁇ -6alkyl- is identified as a specific aromatic group, e.g., phenyl, it is understood that C is 0.
• a moiety when a moiety is "optionally substituted” the moiety may have one or more optional substituents, each optional substituent being independently selected.
• Compounds of formula (I) include substituted heterocyclic moieties, including, but not limited to, pyrrole, pyrazole, imidazole, oxazole, thiazole, isoxazole, isothiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole,
• An isothiocyanate (such as phenyl isothiocyanate) (1 -Scheme 1) was treated with thiourea and sodium hydroxide in acetonitrile/water to provide 2-Scheme 1, which was treated with iodoethane and triethylamine in DMF to afford 3-Scheme 1.
• 2-Scheme 1 Treatment of 3-Scheme 1 with hydrazine in ethanol provided 4-Scheme 1, which was treated with an alkyl halide (such as benzyl bromide or 4-chlorobenzyl chloride) and potassium carbonate in DMF to give 5-Scheme 1.
• an alkyl halide such as benzyl bromide or 4-chlorobenzyl chloride
• a thiourea (such as phenylthiourea) (8-Scheme 2) may be treated with ethyl iodide and refluxed in EtOH, and the resulting S-ethyl thiourea is then heated in the presence of hydrazine to provide 9-Scheme 2.
• the hydrazine 9-Scheme 2 is treated with carbonyldiimidazole and heated to afford 10-Scheme 2.
• Treatment of 10- Scheme 2 with an alkyl halide such as benzyl bromide or 4-chlorobenzyl chloride
• potassium carbonate in DMF gives 11-Scheme 2.
• Triazole 11-Scheme 2 is protected as the methoxy methylethyl ether to afford 12-Scheme 2.
• Alkylation of 12- Scheme 2 with an alkyl halide (such as methyliodide, ethyliodide, j ' -isobutyl iodide, n- propyliodide, butyliodide, allylbromide, benzylbromide, and methyl bromoacetate) affords the desired tertiary amine 13-Scheme 2.
• Deprotection of the MOM-ether 13- Scheme 2 with trifluoroacetic acid (TFA) provides the desired product 14-Scheme 2.
• a thiourea (such as l-(2-methylphenyl)thiourea, l-(4-methylphenyl)-thiourea, l-(4-fluorophenyl)thiourea, l-(4-methoxyphenyl)thiourea, l-(2-pyridyl)thiourea, l-(2- isopropylphenyl)-thiourea, 3,4-dihydro-2H-quinoline-l-carbothioic acid amide) (1- Scheme 5) was treated with iodoethane and triethylamine in DMF to afford 2-Scheme 5.
• 2-Scheme 5 Treatment of 2-Scheme 5 with an acyl-hydrazide (such as 3-phenyl-propionic hydrazide and thiophen-2-ylsulfanyl-acetic acid hydrazide) in pyridine provided the triazole I-Scheme 5.
• an acyl-hydrazide such as 3-phenyl-propionic hydrazide and thiophen-2-ylsulfanyl-acetic acid hydrazide
• the S-ethylisothiourea 2-Scheme 5 could be acylated with a carboxylic acid (such as 2-methyl-3-phenyl-propanoic acid, 3- cyclohexylpropionic acid, 3-(2-thienyl)propanoic acid, phenylsulfanyl-acetic acid) under standard coupling conditions to afford the amide 3-Scheme 5.
• a carboxylic acid such as 2-methyl-3-phenyl-propanoic
• a carboxylic acid (such as 2-methyl-phenyl acetic acid, 3-methyl-phenyl acetic acid, 2-methoxy-phenyl acetic acid, 4-methoxy-phenyl acetic acid, 4-chloro- phenyl acetic acid, 2-pyridyl acetic acid, 4-dimethylamino-phenyl acetic acid, 1- indancarboxylic acid, and 2-thiophene acetic acid) (1-Scheme 6) was heated in cone. HCl and MeOH to afford the corresponding methyl ester. The ester was subsequently treated with anhydrous hydrazine in MeOH to provide the hydrazide 2-Scheme 6.
• the crystal structure of human MetAP2 has been determined in complex with nine separate inhibitors at resolutions from 3.0 - 2.2A.
• the structures were determined using the method of molecular replacement and refined to R c values ranging from 0.190 - 0.267. Further refinement of the atomic coordinates will change the numbers in Table
• the data are reported in Angstroms with reference to an orthogonal coordinate system in standard format, illustrating the atom, i.e., nitrogen, oxygen, carbon, sulfur (at , ⁇ , ⁇ , ⁇ , or ⁇ , positions in the amino acid residues); the amino acid residue in which the atom is located with amino acid number, and the coordinates X, Y and Z in Angstroms (A) from the crystal structure. Note that each atom in the active site and the entire structure has a unique position in the crystal.
• the data also report the B or Temperature Factor values, which indicate the degree of thermal motion of the atom in root mean square displacement measurements (A ⁇ ).
• the three dimensional structure of hMetAP2 is clearly useful in the structure- based design of enzyme inhibitors, which may be used as therapeutic agents against diseases in which inhibition of angiogenesis is indicated.
• the discovery of the inhibitor-enzyme complexes at the catalytic site permits the design of potent, highly selective protease inhibitors.
• Another aspect of this invention involves a method for identifying inhibitors of hMetAP2 characterized by the crystal structure and the active site described herein, and the inhibitors themselves.
• the novel protease crystal structure of the invention permits the identification of inhibitors of protease activity. Such inhibitors may bind to all or a portion of the active site of hMetAP2; or even be competitive or non- competitive inhibitors. Once identified and screened for biological activity, these inhibitors may be used therapeutically or prophylactically to block protease activity.
• One design approach is to probe the hMetAP2 of the invention with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate hMetAP2 inhibitors and the enzyme. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their hMetAP2 inhibitor activity.
• This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to or with hMetAP2.
• the reaction intermediates of hMetAP2 can also be deduced from the reaction product in co-complex with hMetAP2.
• Such information is useful to design improved analogues of known metallo-protease inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the hMetAP2 enzyme and hMetAP2 inhibitor co-complex. This provides a novel route for designing hMetAP2 inhibitors with both high specificity and stability.
• Another approach made possible by this invention is to screen computationally small molecule databases for chemical entities or compounds that can bind in whole, or in part, to the hMetAP2 enzyme.
• the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity [R. L. DesJarlais et al., J. Med. Chem. 31:722-729 (1988)] or by estimated interaction energy [E. C. Meng et al, J. Comp. Chem., 13:505-524 (1992)].
• shape complementarity R. L. DesJarlais et al., J. Med. Chem. 31:722-729 (1988)
• estimated interaction energy E. C. Meng et al, J. Comp. Chem., 13:505-524 (1992)
• the structure coordinates of hMetAP2, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of hMetAP2.
• hMetAP2 mutants may also be used to solve the structure of hMetAP2 mutants, hMetAP2 co- complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of hMetAP2.
• One method that may be employed for this purpose is molecular replacement.
• the unknown crystal structure whether it is another crystal form of hMetAP2, a hMetAP2 mutant, or a hMetAP2 co-complex, or the crystal of some other protein with significant amino acid sequence homology to any functional domain of hMetAP2, may be determined using the hMetAP2 structure coordinates of this invention as provided in Table I.
• This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
• the hMetAP2 structure permits the screening of known molecules and/or the designing of new molecules which bind to the protease structure, particularly at the active site, via the use of computerized evaluation systems.
• computer modeling systems are available in which the sequence of the protease, and the protease structure (i.e., atomic coordinates of hMetAP2 and/or the atomic coordinate of the active site cavity, bond angles, dihedral angles, distances between atoms in the active site region, etc.) as provided by Table I may be input.
• a machine readable medium may be encoded with data representing the coordinates of Table I in this process.
• the computer then generates structural details of the site into which a test compound should bind, thereby enabling the determination of the complementary structural details of said test compound.
• the design of compounds that bind to or inhibit hMetAP2 according to this invention generally involves consideration of two factors.
• the compound must be capable of physically and structurally associating with hMetAP2.
• Non-covalent molecular interactions important in the association of hMetAP2 with its substrate include hydrogen bonding, van der Waals and hydrophobic interactions.
• the compound must be able to assume a conformation that allows it to associate with hMetAP2. Although certain portions of the compound will not directly participate in this association with hMetAP2, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency.
• conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., active site or accessory binding site of hMetAP2, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with hMetAP2.
• the potential inhibitory or binding effect of a chemical compound with hMetAP2 may be estimated prior to its actual synthesis and testing by using computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and hMetAP2, synthesis and testing of the compound is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to hMetAP2 in a suitable assay. In this manner, synthesis of inoperative compounds may be avoided.
• An inhibitory or other binding compound of hMetAP2 may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding pockets or other areas of hMetAP2.
• One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with hMetAP2 and more particularly with the individual binding pockets of the hMetAP2 active site or accessory binding site. This process may begin by visual inspection of, for example, the active site on the computer screen based on the hMetAP2 coordinates in Table I. Selected fragments or chemical entities may then be position hMetAP2. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
• MCSS Molecular Simulations, Burlington, MA.
• AUTODOCK [D. S. Goodsell and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 8: 195-202 (1990)].
• AUTODOCK is available from Scripps Research Institute, La Jolla, CA.
• DOCK [I. D. Kuntz et al, "A Geometric Approach to Macromolecule- Ligand Interactions", J. Mol. Biol., 161:269-288 (1982)]. DOCK is available from University of California, San Francisco, CA. Additional commercially available computer databases for small molecular compounds includes Cambridge Structural Database and Fine Chemical Database, for a review see Rusinko, A., Chem. Des. Auto. News 8, 44-47 (1993).
• Assembly may be proceeded by visual inspection of the relationship of the fragments to each other on the three- dimensional image displayed on a computer screen in relation to the structure coordinates of hMetAP2. This would be followed by manual model building using software such as Quanta or Sybyl.
• CAVEAT [P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc. 78, pp. 182-196 (1989)].
• CAVEAT is available from the University of California, Berkeley, CA. • 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, CA). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35:2145-2154 (1992).
• inhibitory or other type of binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known inhibitor(s).
• LUDI H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6:61-78 (1992)].
• LUDI is available from Biosym Technologies, San Diego, CA.
• LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)]. LEGEND is available from Molecular Simulations, Burlington, MA. • LeapFrog (available from Tripos Associates, St. Louis, MO).
• the protease inhibitor may be tested for bioactivity using standard techniques.
• structure of the invention may be used in binding assays using conventional formats to screen inhibitors.
• Suitable assays for use herein include, but are not limited to, the hMetAP2 activity assay defined below.
• Other assay formats may be used; these assay formats are not a limitation on the present invention.
• the three dimensional atomic structure can be readily used as a template for selecting potent inhibitors.
• Various computer programs and databases are available for the purpose.
• a good inhibitor should at least have excellent steric and electrostatic complementarity to the target, a fair amount of hydrophobic surface buried and sufficient conformational rigidity to minimize entropy loss upon binding.
• the approach usually comprises several steps:
• hMetAP2 Defining a region to target.
• the active site cavity of hMetAP2 can be selected, but any place that is essential to the protease activity could become a potential target. Since the crystal structure has been determined, the spatial and chemical properties of the target region is known.
• the originally defined target region can be readily expanded to allow further necessary extension.
• a limited number of promising compounds can be selected through the process. They can then be synthesized and assayed for their inhibitory properties.
• Crystals of human methionineaminopeptidase-2 complexed with inhibitor grew to a size of approximately 0.1 - 0.2 mm 3 in about six days at 4°C.
• the concentration of inhibited human methionineaminopeptidase-2 used in the crystallization was approximately 10 - 15 mg./ml.
• the method of vapor diffusion in sitting drops was used to grow crystals from the solution of human methionineaminopeptidase-2. Crystals grew at 4°C from drops containing protein in a solution of 10% glycerol in lOmM Hepes buffer at pH 7.4 containing 0.15M NaCl.
• the crystals contain one molecule in the asymmetric unit and approximately 54% solvent with a V m value of 2.75 A 3 /Dalton.
• X-ray diffraction data were measured from a single crystal for each inhibitor complex using synchrotron radiation provided by beamline 17-ID at the Advance Photon Source, Argonne National Laboratory.
• the protein structures for all complexes were determined by molecular replacement using CNX ( Molecular Simulations Inc).
• the starting model consisted of all protein atoms of the published structure of human methionineaminopeptidase-2 published by LIU et al [S.Liu, J.Widom, C.W.Kemp, CM. Crews, J.Clardy , Structure Of Human Methionine Aminopeptidase-2 Complexed With Fumagillin Science 282 ,1324 (1998)].
• hMetAP2 activity can be measured by direct spectrophotometric assay methods using alternative substrates, L-methionine-/?-nitroanilide (Met-pNA) and L-methionine-7- amido-4-methylcoumarin (Met- AMC).
• Method-pNA L-methionine-/?-nitroanilide
• Method- AMC L-methionine-7- amido-4-methylcoumarin
• the formation of -nitroaniline (pNA) or 7- amido-4-methylcoumarin (AMC) was continuously monitored by increasing absorbance or fluorescence at 405 nm and 460 nm, respectively, on a corresponding plate reader. All assays were carried out at 30°C.
• the fluorescence or spectrophotometric plate reader was calibrated using authentic pNA and AMC from Sigma, respectively.
• each 50 ⁇ L assay solution contained 50 mM Hepes Na + (pH 7.5), 100 mM NaCl, 10-lOOnM purified hMetAP2 enzyme, and varying amounts of Met-AMC (in 3% DMSO aqueous solution) or Met-pNA. Assays were initiated with the addition of substrate and the initial rates were corrected for the background rate determined in the absence of hMetAP2.
• the methionine aminopeptidase activity of hMetAP2 can also be measured spectrophotometrically by monitoring the free L-amino acid formation.
• the release of N- terminal methionine from a tripeptide (Met-Ala-Ser, Sigma) or a tetrapeptide (Met-Gly- Met-Met, Sigma) substrate was assayed using the L-amino acid oxidase (AAO) / horse radish peroxidase (HRP) couple (eq. l-3a,b).
• AAO L-amino acid oxidase
• HRP horse radish peroxidase
• H 2 O 2 hydrogen peroxide
• a typical assay contained 50 mM Hepes Na+, pH 7.5, 100 mM NaCl, 10 ⁇ M CoCl 2 , 1 mM o-Dianisidine or 50 ⁇ M Amplex Red, 0.5 units of HRP (Sigma), 0.035 unit of AAO (Sigma), 1 nM hMetAP2, and varying amounts of peptide substrates. Assays were initiated by the addition of hMetAP2 enzyme, and the rates were corrected for the background rate determined in the absence of hMetAP2.
• v is the initial velocity
• V is the maximum velocity
• K a is the apparent Michaelis constant
• I is the inhibitor concentration
• A is the concentration of variable substrates.
• the nomenclature used in the rate equations for inhibition constants is that of Cleland (1963), in which Kj s and Kjj represent the apparent slope and intercept inhibition constants, respectively.
• hMetAP2 inhibitors The ability of hMetAP2 inhibitors to inhibit cell growth was assessed by the standard XTT microtitre assay.
• XTT a dye sensitive to the pH change of mitochondria in eukaryotic cells, is used to quantify the viability of cells in the presence of chemical compounds. Cells seeded at a given number undergo approximately two divisions on average in the 72 hours of incubation. In the absence of any compound, this population of cells is in exponential growth at the end of the incubation period; the mitochondrial activity of these cells is reflected in the spectrophotometric readout (A450). Viability of a similar cell population in the presence of a given concentration of compound is assessed by comparing the A450 reading from the test well with that of the control well.
• A450 spectrophotometric readout
• DMSO concentration in all wells being 0.2 %.
• Cells plus compound are incubated for an additional 72 hr at 37°C under the normal growth conditions of the cell line used.
• Cells are then assayed for viability using standard XTT/PMS (prepared immediately before use: 8 mg XTT (Sigma X-4251) per plate is dissolved in 100 ul DMSO.
• 3.9 ml H 2 O is added to dissolve XTT and 20 ul of PMS stock solution (30 mg/ml) is added from frozen aliquoted stock solution (10 mg of PMS (phenazine methosulfate, Sigma P-9625) in 3.3 ml PBS without cations. These stocks are frozen at -20°C until use).
• XTT/PMS solution 50 ul of XTT/PMS solution is added to each well and plates incubated for 90 minutes (time required may vary according to cell line, etc.) at 37°C until A 450 is >1.0. Absorbance at 450 nM is determined using a 96-well UV plate reader. Percent viability of cells in each well is calculated from these data (having been corrected for background absorbance). ICso is that concentration of compound that reduces cell viability to 50% control (untreated) viability.
• the compounds of this invention show hMetAP2 inhibitor activity having IC50 values in the range of 0.0001 to 100 uM.
• the full structure/activity relationship has not yet been established for the compounds of this invention.
• one of ordinary skill in the art can utilize the present assays in order to determine which compounds of this invention are inhibitors of hMetAP2 and which bind thereto with an IC50 value in the range of 0.0001 to 100 uM.
• All publications, including, but not limited to, patents and patent applications cited in this specification are herein inco ⁇ orated by reference as if each individual publication were specifically and individually indicated to be inco ⁇ orated by reference herein as though fully set forth.
• H H H H H r H H H H H H H ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ cn cn cn C ⁇ C ⁇ > > > > >
• 03 > 63 03 is LS ⁇ 63 03 > rs ES 03
• O 6 03 IS IS ti ⁇ 03 > ES rs O 63 03 > ⁇ 63 63 03 > 03 >
• Ki Ki Ki ⁇ - ⁇ Ki P P P EC EC EC EC EC EC ts ts LS LS is LS P P P P P EC EC EC EC EC EC EC EC cn cn cn in cn ; Ki Ki 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 i Ki Ki Ki Ki LS ES ts t ts ES
• OJ oo oo OJ OJ Ul Ul Ul Ul Ul Ul Ul Ul OJ 0J 0J lb oo lb lb lb lb lb ⁇ - lb lb lb lb lb lb lb lb lb lb lb lb Ul Ul Ul Ul Ul Ul oo Ui Ul lb lb ib

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