Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

• the present invention relates generally to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it relates to novel heteroaryl compounds and their physiologically acceptable salts and prodrugs, which modulate the activity of protein tyrosine enzymes related to cellular signal transduction and, therefore, are expected to exhibit a salutary effect against disorders associated with abnormal protein tyrosine enzyme related cellular signal transduction.
• Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells.
• the biochemical pathways through which signals are transmitted within cells comprise a circuitry of directly or functionally connected interactive proteins.
• One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins.
• the phosphorylation state of a protein may affect its conformation and/or enzymic activity as well as its cellular location.
• the phosphorylation state of a protein is modified through the reciprocal actions of protein tyrosine kinases (PTKS) and protein tyrosine phosphatases (PTPS) at various specific tyrosine residues.
• PTKS protein tyrosine kinases
• PTPS protein tyrosine phosphatases
• a common mechanism by which receptors regulate cell function is through an inducible tyrosine kinase activity which is either endogenous to the receptor or is imparted by other proteins that become associated with the receptor.
• tyrosine kinases comprise a large family of transmembrane receptor and intracellular enzymes with multiple functional domains (Taylor et al., 1992, Ann. Rev. Cell Biol. 8:429-62).
• RPTKs receptor protein tyrosine kinase
• EGFR epidermal growth factor receptor
• PDGFR platelet-derived growth factor receptor
• Cytoplasmic protein tyrosine kinases such as Janus kinases (e.g., JAK1, JAK2, TYK2) and Src kinases (e.g., src, lck, fyn) are associated with receptors for cytokines (e.g., IL-2, IL-3, IL-6, erythropoietin), interferons and antigens. These receptors also undergo oligomerization, and have tyrosine residues that become phosphorylated during activation, but the receptor polypeptides themselves do not possess kinase activity.
• cytokines e.g., IL-2, IL-3, IL-6, erythropoietin
• the protein tyrosine phosphatases comprise a family of transmembrane and cytoplasmic enzymes, possessing at least an approximately 230 amino acid catalytic domain containing a highly conserved active site with the consensus motif [I/V]IHCXNGXXR[S/T]G.
• the substrates of PTPs may be PTKs which possess phosphotyrosine residues or the substrates of PTKS.
• Transmembrane or receptor-like PTPs possess an extracellular domain, a single transmembrane domain, and one or two catalytic domains followed by a short cytoplasmic tail.
• the extracellular domains of these RPTPs are highly divergent, with small
• glycosylated segments e.g., RPTP ⁇ , RPTP ⁇
• fibronectin type III domains e.g., LAR
• carbonic anhydrase like domains e.g., RPTP ⁇
• Intracellular or cytoplasmic PTPs such as PTP1C and PTP1D, typically contain a single catalytic domain flanked by several types of modular conserved domains.
• CPTPs cytoplasmic PTPs
• PTP1C a hemopoietic cell CPTP is characterized by two Src homo logy 2 (SH2) domains that recognize short peptide motifs bearing phosphotyrosine (pTyr).
• SH2 Src homo logy 2
• SH2-containing proteins are able to bind pTyr sites in activated receptors and cytoplasmic phosphoproteins.
• Another conserved domain known as SH3 binds to proteins with proline-rich regions.
• a third type known as the pleckstrin-homology (PH) domain has also been identified.
• PH pleckstrin-homology
• Multiprotein signaling complexes comprising receptor subunits, kinases, phosphatases and adapter molecules are assembled in subcellular compartments through the specific and dynamic interactions between these domains with their binding motifs.
• Such signaling complexes integrate the extracellular signal with the ligand-bound receptor and relay the signal to other downstream signaling proteins or complexes in other locations inside the cell or in the nucleus (Koch et al., 1991. Science. 252:668-674; Pawson, 1994, Nature. 373:573-580; Mauro et al., 1994, Trends Biochem. Sci.. 19:151-155; Cohen et al., 1995, Cell. 80:237-248).
• the levels of tyrosine phosphorylation required for normal cell growth and differentiation at any time are achieved through the coordinated action of PTKs and PTPS.
• these two types of enzymes may either antagonize or cooperate with each other during signal transduction.
• An imbalance between these enzymes may impair normal cell functions leading to metabolic disorders and cellular transformation.
• insulin binding to the insulin receptor which is a PTK
• triggers a variety of metabolic and growth promoting effects such as glucose transport, biosynthesis of glycogen and fats, DNA synthesis, cell division and differentiation.
• Diabetes mellitus which is characterized by insufficient or a lack of insulin signal transduction, can be caused by any abnormality at any step along the insulin signaling pathway.
• chromosome 3p21 which is a segment frequently altered in renal and small lung carcinoma.
• Mutations may occur in the extracellular segment of RPTP ⁇ which renders the RPTP no
• PTPIC also known as HCP or SHP
• PTP1D has been shown to bind through SH2 domains to sites of phosphorylation in PDGFR, EGFR and insulin receptor substrate 1 (IRS-1).
• Vanadates and pervanadates are known to be non-specific phosphatase inhibitors.
• this class of compounds is toxic because each compound contains a heavy metal (U.S. Patent No. 5,155,031; Fantus et al., 1989, Biochem... 28:8864-71; Swamp et al., 1982, Biochem. Biophvs. Res. Commun.. 107:1104-9).
• the present invention relates generally to novel heteroaryl compounds which modulate the activity of protein tyrosine enzymes which are related to cellular signal transduction; namely, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).
• the compounds of this invention are expected to modulate protein tyrosine phosphatase activity.
• the present invention relates to the preparation and use of pharmaceutical compositions of the disclosed compounds and their physiologically acceptable salts and prodrugs for the treatment or prevention of disorders associated with abnormal protein tyrosine enzyme related cellular signal tranduction including, but not limited to, cancer and diabetes.
• a “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• a “prodrug” refers to an agent which is converted into the parent drug in vivo.
• Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not.
• the prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
• An example, without limitation, of a prodrug would be a compound of the present invention wherein it is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is not beneficial, but then it is metabolically hydrolyzed to the carboxylic acid once inside the cell where water solubility is beneficial.
• an “ester” is a C-carboxy group, as defined herein, wherein R" is any of the listed groups other than hydrogen.
• a “physiologically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• this invention relates to heteroaryl compounds having the general chemical structure shown in Formula 1 :
• r and s are independently 0 or 1.
• A, B, D, E, F, G, J, K, L, and M are independently selected from the group consisting of carbon and nitrogen, it being understood that the six-member nitrogen heteroaryl rings so formed are those known in the chemical arts; it being further understood that when A, B, D, E, F, G, J, K, L or M is nitrogen, R 1 , R 2 , R ⁇ R 4 , R 5 , R 6 , R 7 , R 8 R 9 or R 10 , respectively, do not exist. At least one of A, B, D, E or F and at least one of G, J, K, L and M must be nitrogen.
• A, B, D, and F or G, K, L and M are independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur, it being understood that the five-member heteroaryl rings so formed are those known in the chemical arts; it being further understood that when A, B, D, F, G, K, L or M is oxygen or sulfur or A, B, D, F, G, K, L or M is nitrogen and that nitrogen is participating in a heteroaryl ring double bond, R 1 , R 2 , R ⁇ R 5 , R 6 , R ⁇ R 9 or R 10 do not exist.
• R 1 , R 2 , R ⁇ R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are independently selected from the group consisiting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicychc, alkoxy, aryloxy, thioalkoxy, thioaryloxy, heteroaryloxy, heteroahcycloxy, sulfinyl, sulfonyl, S-sulfonamido, N-Sulfonamido, trihalomethanecarbonyl, trihalomethanesulfonyl, carbonyl, C-carboxy, O-carboxy, C-amido, C-thioamido, N-amido, hydrazino, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thioc
• R 11 and R 12 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, carbonyl, C-carboxy, sulfonyl, trihalomethanesulfonyl, trihalomethanecarbonyl and, combined, a five- or six-member heteroalicychc ring.
• R 1 , R 2 , R 3 , R 5 , R 6 , R 8 , R 9 and R 10 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, trihalomethanecarbonyl, sulfonyl, trihalomethane- sulfonyl, cyano, C-carboxy, O-carboxy, C-amido, C-thioamido and guanyl;
• Q is selected from the group consisting of oxygen, sulfur, sulfinyl, sulfonyl and -NR 13 .
• R 13 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, cyano, trihalomethanecarbonyl, sulfonyl, trihalomethanesulfonyl, C-carboxy, O-carboxy, C-amido, C-thioamido and guanyl.
• Any two adjacent R groups may combine to form an additional aryl, cycloalkyl, heteroaryl or heteroalicychc ring fused to the ring initially bearing those R groups.
• A is sulfur
• F is nitrogen
• Q is sulfur
• R 2 is nitro
• R 6 , R 7 , R 8 , R 9 and R 10 are selected so as to afford the compounds of Table 1.
• alkyl group refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
• the alkyl group has 1 to 20 carbon atoms (whenever a numerical range; e.g. "1-20", is stated herein, it means that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms.
• the alkyl group may be substituted or unsubstituted.
• the substituent group(s) is preferably one or more individually selected from trihaloalkyl, cycloalkyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroahcycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C- thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethane- sulfonamido, trihalomethanesulfonyl, silyl, gu
• a "cycloalkyl” group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
• examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene and adamantane.
• a cycloalkyl group may be substituted or unsubstituted.
• the substituent group(s) is preferably one or more individually selected from alkyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroahcycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl, thiocarbonyl, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalo- methanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ure
• alkenyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond.
• alkynyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
• aryl refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi- electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.
• the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroahcycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C- amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethane- sulfonyl, silyl, guanyl, guani
• heteroaryl group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
• heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole.
• the heteroaryl group may be substituted or unsubstituted.
• the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroahcycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O- carboxy, sulfinyl, sulfonyl, sulfonamido, nitro, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino
• a “heteroalicychc” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur.
• the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
• the heteroalicychc ring may be substituted or unsubstituted.
• the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicychc, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroahcycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, nitro, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guani
• a "hydroxy” group refers to an -OH group.
• alkoxy refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
• aryloxy refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.
• heteroaryloxy refers to a heteroaryl-O- group with heteroaryl as defined herein.
• heteroahcycloxy refers to a heteroalicyclic-O- group with heteroalicychc as defined herein.
• thiohydroxy refers to an -SH group.
• a “thioalkoxy” group refers to both an S-alkyl and an -S-cycloalkyl group, as defined herein.
• a “thioaryloxy” group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein.
• a “thioheteroaryloxy” group refers to a heteroaryl-S- group with heteroaryl as defined herein.
• a “thioheteroalicycloxy” group refers to a heteroalicyclic-S- group with heteroalicychc as defined herein.
• aldehyde refers to a carbonyl group where R" is hydrogen.
• a “carboxylic acid” group refers to a C-carboxy group in which R" is hydrogen.
• halo refers to fluorine, chlorine, bromine or iodine.
• a “trihalomethyl” group refers to a -CX 3 group wherein X is a halo group as defined herein.
• amino refers to an -NH 2 group.
• a “cyano” group refers to a -C ⁇ N group.
• sil refers to a -Si(R") 3 , with R" as defined herein.
• a “hydrazino” group refers to a -NR ⁇ NR 12 R 14 group with R", R 12 and R 14 as defined herein.
• Preferred structural features of this invention are those in which r and s are 0 and Q is sulfur.
• the present invention is directed to the use of compounds capable of modulating or regulating signal transduction in normal or diseased cells.
• the present invention is also directed to the use of compounds capable of inhibiting the activity of protein tyrosine enzymes, in particular protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), to modulate or trigger signal transduction.
• the invention is further directed to the regulation of cellular processes that are controlled by signal transduction through the inhibition of the activity of PTKs and PTPs by the compounds.
• the invention further provides for the use of such compounds in the treatment of a subject having a disorder caused by dysfunctional signal transduction.
• the compounds of the invention are capable of inhibiting the activity of protein tyrosine phosphatases, that are transmembrane or intracellular, and that may have one or more characteristic catalytic domains.
• the amino acid sequences of the PTPs in the catalytic domains may include but are not limited to [I/V]HCXAGXXR(S/T]G (single-letter amino acid code; X is any amino acid).
• the PTPs may possess one or more modular conserved domains, which include but are not limited to, SH2, SH3 and PH domains.
• the compounds of the invention can be used to inhibit the phosphatase activity of PTP1B (Charbonneau, et al., 1989, Proc. Natl. Acad. Sci.. USA. 86: 5252-5256), T-cell PTP (Cool, et al., 1989, Proc. Natl. Acad. Sci.. USA. 86: 52575261, PTPIC (Shen, et al., 1991, Nature.
• PTP1B Charge Bonneau, et al., 1989, Proc. Natl. Acad. Sci.. USA. 86: 5252-5256
• T-cell PTP Cool, et al., 1989, Proc. Natl. Acad. Sci.. USA. 86: 52575261
• PTPIC Shen, et al., 1991, Nature.
• the PTKs and PTPs preferred in the invention are of human origin. Inhibition of phosphatase activity that is substantially specific to a PTP or a set of PTPs in a signaling pathway is preferred. While the inhibition of phosphatase activity is believed to be the mechanism of action of the compounds of the present invention with respect to their ability to modulate and/or regulate signal transduction, additional mechanisms have not been ruled out.
• signal transduction is not limited to transmembrane signaling, and includes the multiple pathways that branch off throughout the cell and into the nucleus.
• signaling pathways may include but are not limited to the Ras pathway (Schlessinger, 1994, Curr. Onin. Genet. Dev.. 4:25-30), the JAK/STAT pathways (Sadowski, et al., 1994, Science. 261:1739-1744), the phosphoinositide 3-kinase pathway and
• Cellular processes under the control of signal transduction may include, but are not limited to, transcription of specific genes; normal cellular functions, such as metabolism, proliferation, differentiation, adhesion, apoptosis and survival; as well as abnormal processes, such as transformation, blocking of differentiation and metastasis.
• a signal may be triggered by the binding of a ligand to its receptor on the cell surface, and the signal is transduced and propagated by the phosphorylation or dephosphorylation of specific tyrosine residues on various substrates inside the cell.
• the specific interactions between the PTKs, PTPs and their substrates may involve the formation of a transient or stable multimolecular complex on the inner face of the plasma membrane or in other subcellular compartments including the nucleus.
• a substrate may contain one or more tyrosine residues that are phosphorylated or dephosphorylated by PTKs or PTPs in the signaling pathway.
• Such substrates may include the receptor and its subunits, molecules associated with or recruited to the receptor such as cytoplasmic kinases, cytoplasmic phosphatases, adapter molecules, cytoskeletal proteins and transcription factors.
• the term receptor as used herein may include, but is not limited to, insulin receptor, members of the insulin-like growth factor receptor family, epidermal growth factor receptor family, fibroblast growth factor receptor family, hepatocyte growth factor receptor family, vascular endothelial growth factor receptor family, neurotrophin receptor (trk) family, the T-cell receptor, the B cell receptor and members of the Type I-IV cytokine receptor families (Heldin, 1995, Cell. 80: 213-223; Taniguchi, 1995, Science. 268: 251-255).
• Adapter molecules that are substrates may include the Grb proteins, IRS-1, Zap-70 and She (Pawson, et al., 1995, Nature. 373: 573-580). Cytoskeletal proteins such as actin and transcription factors such as the STAT proteins (Ihle, et al., Trends Biochem. Sci.. 19:222-227) may also serve as substrates.
• ligand is synonymous with extracellular signaling molecules, and includes but is not limited to growth factors such as insulin, EGF, PDGF, fibroblast growth factors, vascular endothelial growth factor, and neurotrophins; and cytokines such as growth hormone, erythropoietin, tumor necrosis factor, interleukins and interferons.
• growth factors such as insulin, EGF, PDGF, fibroblast growth factors, vascular endothelial growth factor, and neurotrophins
• cytokines such as growth hormone, erythropoietin, tumor necrosis factor, interleukins and interferons.
• the term ligand is not limited to soluble molecules, and includes, for example, extracellular matrix proteins, cell adhesion molecules as well as antigenic peptides associated with the major histocompatibility complex proteins on the surface of an antigen-presenting cell.
• the compounds of the invention can be used to trigger or upregulate signal transduction in cells so that the effect of ligand binding to a receptor is enhanced, or mimicked if the ligand is not present.
• the compounds exert the effect by inhibiting or diminishing the activity of a phosphatase in the signaling pathway which normally acts negatively toward signaling.
• PTPs normally downregulate signal transduction involves the dephosphorylation of specific phosphotyrosine residues (pTyr) on PTKs and their substrates since many PTKs require phosphorylation of some of its own tyrosine residues in order to become optimally active in the signaling pathway.
• the compounds of the invention can be used to prevent the dephosphorylation of pTyr residues on receptors or their subunits which normally becomes phosphorylated upon ligand binding, thereby enhancing the extent and duration of PTK phosphorylation.
• the compounds of the invention can also be used to prevent the dephosphorylation of PTKs in which the tyrosine residues become autophosphorylated or transphosphorylated due to its basal activity.
• a signal may be triggered by the compounds of the invention in the absence of ligand binding since the basal activity of PTKs is sufficient to promote a signal if constitutive PTP activity is inhibited or diminished by the compounds.
• a preferred embodiment of the invention is directed to a method of triggering, enhancing or sustaining insulin receptor signal transduction by inhibiting the constitutive, dephosphorylation of the pTyr sites on the activated insulin receptor. This would allow the insulin receptor to remain phosphorylated, thus enhancing or sustaining the insulin signal. Furthermore, since it has been shown that insulin receptor is phosphorylated at a low level even in the absence of insulin (Goldstein, 1992, J. Cell Biol.. 48:33-42), the compounds of the invention can be used to trigger a signal, even in the absence of insulin, by allowing the tyrosine residues on the receptor to become self-phosphorylated.
• the compounds of the invention can be used to upregulate or prolong signal transduction by preventing the dephosphorylation of pTyr sites on substrate proteins that normally serve as binding sites for SH2-containing proteins which promote signaling.
• the compounds of the invention may be used to prevent the dephosphorylation of specific pTyr residues on any substrate, which pTyr residues are essential to the transmissions or propagation of the signal. Furthermore, the compounds of the invention may be used to prevent the dephosphorylation of specific pTyr residues on any substrate, which pTyr residues are inhibitory to signal transduction.
• the compounds of the invention can also be used to suppress or downregulate signal transduction in cells so that the effect of ligand binding to a receptor is abolished or attenuated.
• the compounds can inhibit a phosphatase in a signaling pathway which normally acts positively toward signaling.
• PTPs promote signaling through the activation of members of the Src family of PTKs.
• Src family PTKs have an inhibitory site of phosphorylation in their carboxy termini which by dephosphorylation activates kinase activity.
• the compounds of the invention can be used to prevent the dephosphorylation of the inhibitory pTyr in the carboxy termini of kinases which function normally to promote signal fransductions.
• Src family PTKs may include Src, Fyn, Lck, Lyn, Blk, Hck, Fgr and Yrk.
• Other kinases which may be similarly regulated by a phosphatase may include Fak and Csk (Taniguchi, 1995, Science. 268: 251-255).
• pharmaceutically acceptable salt refers to those salts which retain the biological effectiveness and properties of the compound and which are obtained by reaction with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• the present invention is further directed, where applicable, to solvated as well as unsolvated forms of the compounds (e.g., hydrated forms) having the ability to regulate and/or modulate phosphatase activity.
• the compounds described above may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Suitable processes are illustrated by the representative examples provided, infra. Necessary starting materials may be obtained by standard procedures of organic chemistry.
• a compound of this invention can be administered to a human patient as such or in pharmaceutical compositions in which a therapeutically effective dose is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders, including solid cell tumor growth, including Kaposi's sarcoma, glioblastoma, and melanoma and ovarian, lung, mammary, prostate, pancreatic, colon and epidermoid carcinoma, diabetes, diabetic retinopathy, hemangioma and rheumatoid arthritis.
• a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms of uncontrolled vasculogenesis and angiogenesis.
• the formulations of the present invention normally will consist of at least one compound of formula I mixed with a carrier, or diluted by a carrier, or enclosed or encapsulated by an ingestible carrier in the form of a capsule, sachet, cachet, paper or other container or by a disposable container such as an ampoule.
• a carrier or diluent may be a solid, semi-solid or liquid material, which serves as a vehicle, excipient or medium for the active therapeutic substance.
• diluents or carriers which may be employed in the pharmaceutical compositions of the present invention are lactose, dextrose, sucrose, sorbitol, mannitol, propylene glycol, liquid paraffin, white soft paraffin, kaolin, microcrystalline cellulose, calcium silicate, silica polyvinylpyrrolidone, cetostearyl alcohol, starch, gum acacia, calcium phosphate, cocoa butter, oil of theobroma, arachis oil, alginates, tragacanth, gelatin, syrup B.P., methyl cellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate and propylhydroxybenzoate, sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol. Routes Of Administration
• administer refers to the delivery of a compound, salt or prodrug of the present invention or of a pharamaco logical composition containing a compound, salt or prodrug of this invention to an organism for the purpose of prevention or treatment of a disorder associated with an abnormal protein tyrosine enzyme related cellular signal transduction.
• a "disorder associated with an abnormal protein tyrosine enzyme related cellular signal transduction” refers to a condition characterized by inappropriate; i.e., under or, more commonly, over, catalytic activity on the part of a protein tyrosine enzyme. Inappropriate catalytic activity can arise as the result of either: (1) protein tyrosine enzyme expression in cells which normally do not express protein tyrosine enzymes; (2) increased protein tyrosine enzyme expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased protein tyrosine enzyme expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
• Over-activity of protein tyrosine enzymes refers to either amplification of the gene encoding a particular protein tyrosine enzyme or production of a level of protein tyrosine enzyme activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the protein tyrosine enzyme increases, the severity of one or more of the symptoms of the cellular disorder increases). Underactivity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the protein tyrosine enzyme decreases.
• the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from in the first place acquiring a disorder associated with abnormal protein tyrosine enzyme related cellular signal transduction.
• the terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating the abnormal protein tyrosine enzyme related cellular signal transduction disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.
• the term “organism” refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukariotic cell or as complex as a mammal, including a human being.
• Suitable routes of administration include, without limitation, oral, rectal, fransmucosal, or intestinal administration; intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections; transdermal, topical and vaginal application, and the like.
• Dosage forms include but are not limited to tablets, troches, dispersions, suspensions, suppositories, solutions, capsules, creams, patches, minipumps and the like.
• one may administer the compound in a local rather than systemic manner for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
• the liposomes will be targeted to and taken up selectively by the tumor.
• compositions of the present invention may be manufactured by processes well known in the art, for example and without limitation by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
• physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Pharmaceutical preparations for oral use can, for instance, be prepared by adding a compound of this invention to a solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
• the compositions may take the form of tablets or lozenges formulated in conventional manner.
• the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
• the compounds of this invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a suitable vehicle e.g., sterile pyrogen-free water
• the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives such as, for example, a sparingly soluble salt.
• N pharmaceutical carrier for the hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
• the cosolvent system may be the VPD co-solvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD co-solvent system (VPD: 05 W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied, for example, other low
• toxicity nonpolar surfactants may be used instead of polysorbate 80®; the fraction size of
• hydrophobic pharmaceutical compounds may be employed.
• Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
• Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
• the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Numerous sustained release products are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
• additional strategies for protein stabilization may be employed.
• compositions also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• the compounds of the present invention may also be administered by controlled release means and/or delivery devices
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose.
• therapeutically effective amount refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
• a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or preferably eliminating) one or more symptoms associated with the cancer. Determination of the therapeutically effective amount of a compound of this invention is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTP activity). Such information can be used to more accurately determine useful doses in humans.
• a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in or a prolonged survival of a patient.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the ratio of toxic does to therapeutic effective, LD50/ ED50 is the therapeutic index.
• Compounds which exhibit high therapeutic indices are preferred.
• the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
• a dosage preferably lies within a range of circulating concentrations that include the ED5O and exhibits little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1, p.l).
• Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the tyrosine enzyme modulating effects, known as the minimal effective concentration (MEC).
• MEC minimal effective concentration
• the MEC will vary for each compound but can be estimated from in vitro data; for example, without limitation, the concentration necessary to achieve a 50-90% inhibition of the tyrosine enzyme using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using the MEC value.
• Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90%, preferably between 30-90% and most preferably between 50-90% of the time.
• Usual patient dosages for systemic administration range from 1 to 2000 mg/day, commonly from 1 to 250 mg/day, and typically from 10 to 150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02 to 25 mg/kg/day, commonly from 0.02 to 3 mg/kg/day, typically from 0.2 to 1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5 to 1200 mg/m 2 /day, commonly from 0.5 to 150 Mg/m 2 /day, typically from 5 to 100 Mg/ m 2 /day. Usual average plasma levels should be
• the effective local concentration of the drug may not be related to plasma concentration.
• the amount of a particular composition administered will, of course, be dependent on the subject being freated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
• Desirable blood levels may be maintained by a continuous infusion of the compound; plasma level can be monitored by HPLC. It should be noted that the attending physician would know how and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response is not adequate and toxicity os not a problem.
• a prophylactic or therapeutic dose of a compound in the acute or chronic management of disease will vary with the severity of the condition to be treated and the route of administration. Again, it should be noted that the clinician or physician would know when to interrupt and/or adjust the treatment dose due to toxicity or bone marrow, liver or kidney dysfunctions.
• the dose, and perhaps the dosage frequency will also vary according to the age, body weight, and response of the individual patient.
• the total daily dose ranges for the compounds for the majority of the disorders described herein is from about 0.02 to about 25 mg/kg patient.
• a daily dose range should be between about 0.02 to about 3 mg/kg, while most preferably a daily dose range should be between about 0.2 to about 1.5 mg/kg per day.
• compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
• Such notice for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
• any compound of the invention which inhibits or diminishes protein tyrosine enzyme activity in a signaling pathway may be used in the therapeutic methods of the invention.
• the activity of the compound is sufficiently specific for the paticular protein tyrosine enzyme pathway so that the compound does not interfere with the function of other enzymatic activity, including other tyrosine enzyme activity, in the cell.
• the compounds and pharmaceutical compositions of the invention can be used for treating diabetes mellirus.
• the pathogenesis of diabetes generally involves insufficient or a total lack of insulin signal transduction.
• the paucity or absence of the insulin signal may be caused by a variety of factors such as a lack of insulin, loss of binding affinity, defective receptor or under expression of receptor.
• Insulin receptor activity can be modulated by inhibiting tyrosine phosphatases in the signaling using the compounds of the invention.
• the insulin signal may be restored or stimulated in cells through the inhibition of dephosphorylating activity, even in the absence of insulin.
• the example of diabetes mellirus illustrates the principles of therapeutic applications of the compounds of this invention which may be applied to other disorders that implicate signal transduction by tyrosine enzymes, in particular, phosphotyrosine phosphatases.
• the compounds and pharmaceutical compositions of the invention may be used to treat immune disorders in which cytokine signal transduction is deficient. Cytokines play a crucial role in hemopoiesis as well as coordinating immune and inflammatory responses.
• the compounds may be used to replace or enhance the activity of a cytokine in signaling the differentiation and proliferation of hemopoietic cells, as well as B and T cells in response to antigenic stimulation, and thus be useful for treating anemia and immunodeficiency.
• the compounds may also be used as an antiinflammatory agent to treat disorders such as rheumatoid arthritis.
• the compounds may also be therapeutically useful in treating neurodegenerative diseases by stimulating the growth and differentiation of neuronal cells which is regulated by neurotrophin-mediated signal transduction.
• the compounds and pharmaceutical compositions of the invention may be used to treat cancer, such as glioma, melanoma, Kaposi's sarcoma, hemangioma and ovarian, breast, lung, pancreatic, liver, prostate, colon and epidermoid cancer, in which the malignant cells proliferate and or metastasize as a result of uncontrolled signal transduction mediated by growth factors.
• a PTK such as HER2 has been shown to correlate with the aberrant growth characteristics of tumor cells.
• Vasculogenesis and/or angiogenesis that facilitates tumor growth may also be inhibited by the compounds of this invention.
• the compounds may modulate signal transduction in these tumor cells so that normal growth characteristics are restored.
• the compounds may also be useful in treating psoriasis which is caused by excessive epidermal growth factor mediated signal transduction.
• the starting material 2-brono-5- nitrothiazol was prepared by treating 2-amino-5- nitrothiazol (Aldrich) with sodium nitrite and hydrogen bromide (Fr. Demande 2,015,4 34, 1970).
• 3-Phenyl-l,2,4-triazole-5-thione (E. Hogarth, J. Chem. Soc. (1949) 1163) was prepared by first reacting benzoyl chloride with thiosemicarbazide in pyridine at 0 °C to give benzoyl thiosemicarbazide.
• Benzoyl thiosemicarbazide was treated with potassium hydroxide in ethanol to give 3phenyl-l,2,4-triazole-5-thione.
• 3-Phenyl-l,2,4-triazole-5thione (1.77 g) was then dissolved in 50 mL of methanol and treated with 0.57 g of 95% sodium methoxide, and then with 2-bromo-5-nitrothiazole (2.09 g). The mixture was stirred at room temperature for 2 hours and the precipitated sodium bromide was removed by filtration. The methanol was evaporated and the product crystallized from ethanol and watre to give 1.5 g of 3-[(5-nifrothiazol-2-yl)mercapto]-5-phenyl 1,2,4-triazole, a white solid, MP 155-157°C.
• Example 1 The title compound was prepared in the manner described in Example 1. Substituting pivaloyl chloride for the benzoyl chloride in Example 1 gave pivaloyl thiosemicarbazide and then 3-t-butyl-l,2,4-triazole-5-thione. Reaction of 1.79 g of the sodium salt of the thione with 2.09 g of 2-bromo-5-nitrothiazole as in Example 1 yielded 1 g of 2-[(5-nitro- thiazol-2-yl)- mercapto]-5-t-butyl- 1,2,4-triazole, a yellow solid, MP 219-221° C.
• Example 3 3-[(5-nitrothiazol-2-yl)mercapto]-5-(thien-2-yI)-l,2,4-triazole
• the title compound was prepared in the manner described in Example 1. Substituting the acid chloride of thiophene-2-carboxylic acid (prepared from the acid and oxalyl chloride) for the benzoyl chloride in Example 1 gave the thiosemicarbazide of thiophene-2-carboxylic acid and then 3-(thien-2-yl)-l,2,4-triazole-5-thione.
• Example 7 4-benzy_-3-hydroxy-5-[(5-nitrothien-2-y_)mercapto[-l,2,4-triazole
• the title compound was prepared in a manner similar to that described in Example 5 starting with benzyl isothiocyanate.
• the intermediate 4-benzyl-3-thiosemicarbazide (1.81 g) was treated with ethyl chloroformate (1.09 g) as in Example 5.
• the reaction product, 4-benzyl-3-hydroxy-5-mercapto- 1,2,4-triazole (1.04 g) was reacted with 1.05 g of 2-bromo-5-mtrothiazole as in Example 5. Crystallization from ethanol and water gave 0.3 g of 4benzyl-3-hydroxy-5-[(5-nitrothien-2-yl)mercapto]-l,2,4-triazole, a yellow solid, MP 221-224° C.
• Example 8 3-hydroxy-5-[(5-nitrothiazoI-2-yl)mercapto]-4-[2-(trifluoromethyl) phenyl]-l,2,4-triazole
• the title compound was prepared in a manner similar to that described in Example 5 starting with 2-(trifluoromethyl) phenyl isothiocyanate.
• the intermediate 4-[2- (trifluoromethyl)- phenyl]-3-thiosemicarbazide (2.04 g) was treated with ethyl chloroformate (1.09 g) as in example 5.
• 3-thiosemicarbazide (1.64 g) was reacted with l-ethyl-3-methylpyrazole-5-carboxylic acid chloride (1.73 g, prepared from the acid and oxalyl chloride) to give 2 g of 1 -(1-ethyl- 3-methylpyrazole-5-carbonyl)-4-(3-methoxy-n-propyl)-3-thiosemicarbazide.
• Example 10 3-(4-chlorophenyl)-5-[(5-nitrothiazo_-2-y_)amino]-l,2,4-triazole
• the title compound was prepared in a similar manner to that described in Example 1 by heating 3-amino-5-(4-chlorophenyl)- 1,2,4-triazole with 2-bromo-5-nitrothiazole in refluxing tetrahydrofuran followed by silica gel column chromatograph using a mixture of dichloromethane and methanol as the eluent to yield 3-(4-chlorophenyl)-5-((5-nitrothiazo- 2-yl)amino]- 1 ,2,4-triazole.
• Butanoyl hydrazide is prepared according to the general method of A. I. Vogel, Practical Organic Chemistry, 3rd edition, 1956 (Longman Group, London) p 395. Ten grams of ethyl butanoate is refluxed in 10 mL of hydrazine hydrate for 15 minutes. Absolute ethanol is added, reflux is continued for 3 hours, and the ethanol distilled. The solution is cooled and the crystalline hydrazide isolated by vacuum filtration and dried to give 8 g of butanoyl hydrazide. By substituting 1.7 g of butanoyl hydrazide for the formic acid hydrazide in Example 14, 0.6 g of the desired triazole compound is obtained as an off-white solid.
• 2-Mercapto-5-methyl-l,3,4-thiadiazole is prepared according to the general method described by S. G. Boots and C. C. Cheng, 1967, J. Hetercvclic Chemistrv. 4: 272-283.
• Acetic acid hydrazide (7.4 g) 160 mL of methanol, 5.0 g of 85 % potassium hydroxide pellets, and 10 mL of carbon disulfide is stirred at room temperature for 4 hours.
• Ether 400 mL
• the mixture is cooled in an ice bath to give 10 g of solid potassium acetyldithiocabazate, which is collected by vacuum filtration, dried, and used immediately.
• the crude potassium acetyldithiocarbazate in a mixture of 300 mL of dichloromethane and 54 mL of boron trifluoride etherate is stirred under nitrogen for 18 hours.
• the orange solution is poured onto ice and extracted with ether.
• the ether extract is washed with 10 % potassium hydroxide solution and the aqueous phase acidified to pH 2 with cold 10 % hydrochloric acid.
• 2-[(5-nitrothiazol-2-yl)mercapto]-5-methy-l,3,4-thiadiazole is prepared by the general method of J. Bourdais, et al., 1981, Eur. J. Chem. Chim. Ther.. 16: 233-240.
• 2Bromo-5-nitrothiazole (2.09 g) and 1.32 g of 2-mercapto-5methyl-l,3,4-thiadiazole in 10 mL of ethanol and 10 mL of 1 N potassium hydroxide are stirred at room temperature overnight.
• the precipitated solid is collected by vacuum filtration, washed with water and crystallized from ethyl acetate/hexane to give 1 g of the title compound as an off-white solid.
• 2-[(5-nitrothiazol-2-yl)mercapto]-5-methyl-l,3,4-thiadiazole is prepared by forming and isolating the sodium salt of 2-mercapto-5-methyl-l,3,4-thiadiazole, reacting it with 2-bromo-5-nitrothiazole in a suitable inert solvent at room temperature and isolating the product as described above.
• 2-bromo-5-nitrothiazole in a suitable inert solvent at room temperature
• Butanoyl hydrazide is prepared according to the general method of A. I. Vogel, Practical Organic Chemistry, 3rd edition, 1956 (Longman Group, London) p 395. Ten grams of ethyl butanoate is refluxed in 10 mL of hydrazine hydrate for 15 minutes. Absolute ethanol is added, reflux is continued for 3 hours, and the ethanol distilled. The solution is cooled and the crystalline hydrazide isolated by vacuum filtration and dried to give 8 g of propanoyl hydrazide.
• 2-Mercapto-5-propyl-l,3,4-thiadiazole is prepared as for 2 ⁇ mercapto-5-methyl-l,3,4-thiadiazole by substituting propanoyl hydrazide for acetic hydrazide in Example 16.
• 2-mercapto-5-propyl-l,3,4-thiadiazole is substituted for 2 -mercapto-5-methyl-
• Table 1 shows preferred chemical structures which are within the scope of this invention. The compounds shown are in no way to be construed as limiting the scope of this invention.
• BIOLOGICAL EVALUATION It will be appreciated that, in any given series of compounds, a spectrum of biological activity will be observed.
• the present invention relates to novel heteroaryl compounds demonstrating the ability to modulate protein tyrosine enzymes related to cellular signal transduction, most preferrably, protein tyrosine phosphatase.
• the assays described below are employed to select those compounds demonstrating the optimal degree of the desired activity .
• the phrase "optimal degree of desired activity" refers to the highest therapeutic index, defined above, against a protein tyrosine enzyme which mediates cellular signal transduction and which is related to a particular disorder so as to provide a patient, preferably a human, suffering from such disorder with a therapeutically effective amount of a compound of this invention at the lowest possible dosage.
• phosphatases such assays involve exposing target cells in culture to the compounds and a) biochemically analyzing cell lysates to assess the level and/or identity of tyrosine phosphorylated proteins; or (b) scoring phenotypic or functional changes in treated cells as compared to control cells that were not exposed to the test substance.
• the cells are exposed to the compound of the invention and compared to positive controls which are exposed only to the natural ligand, and to negative controls which were not exposed to either the compound or the natural ligand.
• the assay may be carried out in the absence of the ligand.
• the cells are exposed to the compound of the invention in the presence of the natural ligand and compared to controls which are not exposed to the compound of the invention.
• the assays described below may be used as a primary screen to evaluate the ability of the compounds of this invention to inhibit phosphatase activity of the compounds of the invention.
• the assays may also be used to assess the relative potency of a compound by
• Biochemical Assays Target cells having a substrate molecule that is phosphorylated or dephosphorylated on a tyrosine residue during signal transduction are exposed to the compounds of the invention and radiolabelled phosphate, and thereafter, lysed to release cellular contents, including the substrate of interest.
• the substrate may be analyzed by separating the protein components of the cell lysate using a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique, in either one or two dimensions, and detecting the presence of phosphorylated proteins by exposing to X-ray film.
• SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
• the substrate of interest be first isolated by incubating the cell lysate with a substrate-specific anchoring antibody bound to a solid support, and thereafter, washing away non-bound cellular components, and assessing the presence or absence of pTyr on the solid support by an anti-pTyr antibody.
• This preferred method can readily be performed in a microtiter plate format by an automated robotic system, allowing for testing of large numbers of samples within a reasonably short time frame.
• the anti-pTyr antibody can be detected by labeling it with a radioactive substance which facilitates its detection by autoradiography.
• the anti-pTyr antibody can be conjugated with an enzyme, such as horseradish peroxidase, and detected by subsequent addition of an appropriate substrate for the enzyme, the choice of which would be clear to one skilled in the art.
• an enzyme such as horseradish peroxidase
• a further alternative involves detecting the anti-pTyr antibody by reacting with a second antibody which recognizes the anti-pTyr antibody, this second antibody being labelled with either a radioactive substance or an enzyme as previously described. Any other methods for the detection of an antibody known in the art may be used.
• the above methods may also be used in a cell-free system wherein cell lysate containing the signal-transducing substrate molecule and phosphatase is mixed with a compound of the invention and a kinase.
• the substrate is phosphorylated by initiating the kinase reaction by the addition of adenosine triphosphate (ATP).
• ATP adenosine triphosphate
• the reaction mixture may be analyzed by the SDS-PAGE technique or it may be added to a substrate-specific anchoring antibody bound to a solid support, and a detection procedure as described above is performed on the separated or captured substrate to assess the presence or absence of pTyr. The results are compared to those obtained with reaction mixtures to which the compound is not added.
• the cell-free system does not require the natural ligand or knowledge of its identity.
• Posner et al. U.S. Patent No. 5,155,031 describes the use of insulin receptor as a substrate and rat adipocytes as target cells to demonstrate the ability of pervanadate to inhibit PTP activity.
• Such cellular processes may include, but are not limited to, anabolic and catabolic processes, cell proliferation, cell differentiation, cell adhesion, cell migration and cell death.
• the techniques that have been used for investigating the various biological effects of vanadate as a phosphatase inhibitor may be adapted for use with the compounds of the invention.
• vanadate has been shown to activate an insulin-sensitive facilitated transport system for glucose and glucose analogs in rat adipocytes (Dubyak, et al., 1980, J__ Biol. Chem.. 256:5306-5312).
• the activity of the compounds of the invention may be assessed by measuring the increase in the rate of transport of glucose analog such as 2-deoxy- 3 Hglucose in rat adipocytes that have been exposed to the compounds.
• Vanadate also mimics the effect of insulin on glucose oxidation in rat adipocytes (Shechter, et al., 1980, Nature. 284:556-558).
• the compounds of this invention may be tested for stimulation of glucose oxidation by measuring the conversion of 14 C-glucose to 14 CO 2 .
• the effect of sodium orthovanadate on erythropoietin-mediated cell proliferation has been measured by cell cycle analysis based on DNA content as estimated by incorporation of tritiated thymidine during DNA synthesis (Spivak, et al., 1992, Exp. Hematol.. 20:500-504).
• the activity of the compounds of this invention toward phosphatases that play a role in cell proliferation may be assessed by cell cycle analysis.
• the activity of the compounds of this invention can also be assessed in animals using experimental models of disorders caused by or related to dysfunctional signal transduction.
• the activity of a compound of this invention may be tested for its effect on insulin receptor signal transduction in non-obese diabetic mice (Lund et al., 1990, Nature. 345:727-729), B B Wistar rats and streptozotocin-induced diabetic rats (Solomon et al., 1989, Am. J. Med. Sci.. 297:372-376).
• the activity of the compounds may also be assessed in animal carcinogenesis experiments since phosphatases can play an important role in dysfunctional signal transduction leading to cellular transformation.
• okadaic acid a phosphatase inhibitor
• okadaic acid has been shown to promote tumor formation on mouse skin (Suganuma et al., 1988, Proc. Natl. Acad. Sci.. 85:1768-1771).
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
• the dosage of the compounds of the invention should lie within a range of circulating concentrations with little or no toxicity.
• the dosage may vary within this range depending on the dosage form employed and the route of administration.
• the present invention encompasses compounds capable of regulating and/or modulating signal transduction by, including but not limited to, inhibiting the activity of protein tyrosine phosphatases. More specifically, the present invention encompasses compounds capable of inhibiting protein tyrosine phosphatase activity. These compounds will be referred to herein generically as "phosphatase inhibitors", even though these compounds either upregulate or downregulate cellular processes that are controlled by signal transduction.
• Phosphotyrosine Enzyme Linked Immunosorbent Assay This assay may be used to test the ability of the compounds of the invention to inhibit dephosphorylation of phosphotyrosine (ptyr) residues on insulin receptor (IR).
• IR insulin receptor
• substrate molecules such as platelet derived growth factor receptor
• the activities of the compounds of this invention toward different protein tyrosine enzymes may be assessed.
• an endogenous kinase activity is active at low level even in the absence of insulin binding. Thus, no insulin is needed to stimulate phosphorylation of IR.
• cell lysates can be prepared and added to microtiter plates coated with anti-insulin receptor antibody.
• the level of phosphorylation of the captured insulin receptor is detected using an anti-pTyr antibody and an enzyme-linked secondary antibody.
• the cell line used for the IR assay is NIH3T3 cells (ATCC# CRL 1658) engineered to over-express the human IR (H25 cells). Growth media for these cells is DMEM (Gibco) containing 10% fetal bovine serum, 1% L-glutamine, and 20 mM Hepes. 2.
• the anchoring antibody used was BBE which recognizes the extracellular domain of human IR (Enzymology Laboratories, Sugen Inc.).
• PBS Gibco: KH 2 PO 4 (0.20 g/1), K 2 HP0 4 (2.16 g/1), KC1 (0.20 g/1), NaCl (8.0 g/1), pH 7.2.
• Goat anti-rabbit IgG POD conjugate (Tago, Burlingame, CA, Cat.No. 6430) is used as the secondary antibody.
• TBST buffer 50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, adjusted to pH 7.2 with ION HC1.
• Blocking buffer PBS plus 5% milk (Carnation instant non-fat dry milk).
• 5X HNTG buffer 100 mM HEPES, 750 mM NaCl, 50% glycerol, 0.5% Triton X-100, pH 7.5.
• ABTS solution 100 mM citric acid, 250 mM Na ⁇ O ⁇ 0.5 mg/ml ABTS (2,2'-azinobis(3-ethylbenzthiazlinesulfonic acid), adjusted to pH 4.0 with IN HC1.
• Cell lysis buffer HNTG containing lmM Na ⁇ O ⁇ (0.5M solution kept as a 100X stock at -80°C in aliquots), 5mM NaP 2 0 7 and 5mM EDTA prepared fresh and keep on ice until ready for use.
• the coating buffer is replaced with 100 ⁇ l
• Cells are grown in a 15cm culture dish (Corning 25020-100) in DMEM media containing 10% fetal bovine serum (FES) until 80-90% confluent. The cells are harvested with trypsin-EDTA (0.25%, 0.5ml, Gibco), resuspended in fresh medium containing 10% FBS, 1%) L-glutamine and Hepes, and transferred to round bottom 96-well tissue culture
• the assay is set up in the 96-well tissue culture plate. Before adding the compounds
• Positive control wells receive 80 ⁇ l DMEM.
• Negative controls received 90 ⁇ l DMEM.
• test compound is diluted 1:10 with DMEM and 10 ⁇ l/well of the diluted test substances are
• DMSO dimethyl sulfoxide
• tissue culture plate is shaken for 1 minute before incubation at 37°C, 5% C02. After 90 minutes of incubation, the media is
• the plate is shaken for 5 minutes and then placed on ice for 10 minutes.
• the cells are homogenized by repeated aspirating and dispensing, and the lysate is then transferred to the corresponding wells of a precoated assay plate.
• the substrate in the cell lysates is allowed to bind to the anchoring antibody for 1 hour with shaking at room temperature.
• the lysate is then removed, and the assay plate is washed. All ELISA plate washings are done by rinsing in water 3 times followed by one rinse with TBST.
• the plate is dried by tapping it on paper towels. Phosphotyrosine is detected by
• reaction is stopped after 10 minutes by adding 100 ⁇ l/well of 0.2M HCl, and incubating
• Glucose Transport Assay This assay is used to assess the ability of the compounds of the invention to inhibit phosphatase activity that is involved in the signaling pathway that regulates the insulin-induced transport of glucose into adipocytes. It has been shown that incubation of isolated adipocytes with vanadate resulted in a dose-dependent increase in the rate of glucose uptake.
• the cell line used for the glucose transport assay is 3T3-L1, a preadipocyte cell line
• 3T3-L1 cells are first differentiated by treating the cells under confluent growth conditions in
• DMEM fetal bovine serum
• FBS fetal bovine serum
• Cells for use in the assay are first grown overnight in DMEM media and 1% FBS at 37°C at 5% CO 2 . Two hours before use, the overnight media is replaced with serum free DMEM containing 5mM glucose. After washing the cells twice with phosphate buffered saline (PBS), serial dilutions of the test compound 1 : 100 into DMEM are added to the wells
• PBS phosphate buffered saline
• This assay is used to assess the effect of the compounds of the invention on insulin-mediated signal transduction of primary adipocytes as determined by glucose uptake by the cells.
• adipocytes used in the assay are obtained from euthanized male rats (Sprague-Dawley or other appropriate strains) with a body weight of 200-250 grams. Old and heavier rats are not used as these rats may be resistant to insulin and do not provide a good response. Each animal is expected to yield 1-1.5 g of fat. Approximately 2.5 g of fat is required to run 40 reactions, with 20 samples in the glucose uptake assay and a matching set of 20 LDH samples. Using sterile techniques, a midline abdominal incision is made through the skin followed by a 4-6 cm incision through the peritoneum. The fat body adjoining the testes is identified by tracing the vas deferens to the testes.
• the fat pads are carefully cut away from the epididymis and testes, and the innervating blood vessels.
• the excised fat pads are weighed, finely chopped, and digested with 5 ml of collagenase buffer at 37°C for 1 hour.
• the digested material was then strained through a 250-micron nylon mesh sieve.
• the cells float to the top, and are collected and washed three times with transport buffer.
• the cell concentration is determined by one of the following methods:
• Cells as percentage of solution The cells in suspension are centrifuged at 500x g for 5-10 minutes in a hematocrit tube. The total length of the column of liquid and the length of column of "white" cellular material at the bottom of the tube is measured in millimeters. The cell concentration is estimated as a percentage of the length of cell column to the total length. For the glucose uptake assay, approximately 2-3% of the cells in the final reaction
• the fat cell stock solution is diluted to 25%
• Cells are first fixed with osmium tetroxide in collidine buffer so that the adipocytes sink in suspension. The fixed cells are centrifuged to remove the osmium tetroxide, and then counted with a Coulter counter. Once the cell number is determined, the
• cell concentration is adjusted and 50 ⁇ l aliquots of the cells are added to each sample.
• Assay Adipocytes collected from rats are exposed to a test compound in the absence or presence of saturation levels of insulin. l4 C-labelled glucose, which would normally be taken up by the cells via an insulin-induced mechanism, are added to the cells. The amount of radioactive glucose retained by the treated cells is determined and compared to that of untreated cells to assess the activity of the test compounds.
• a typical assay can be set up as follows:
• DMSO dimethyl sulfoxide
• the cells are separated from the reaction buffer by centrifugation.
• the amount of glucose taken up by the cells is determined by standard scintillation counting.
• the cells in duplicates can be separated by centrifugation in narrow bore microcentrifuge tubes (5.8 x
• reaction sample is then stirred to ensure even distribution of cells and 200 ⁇ l is transferred to
• the mixture is centrifuged at 13000 rpm for 10 minutes. After centrifugation, the cells float to the top and are separated at the silicon fluid interface. The top layer is then transferred to a borosilicate vial with 7-10 ml scintillation fluid and counted
• Cellular Insulin Receptor Activation Assay This assay is used to provide a consistent method for determination of catalytic insulin receptor activity in intact cells in a 96 well ELISA format.
• a EY-20mer peptide is used as the IR substrate in vitro for determination of the activation state of IR in an enzyme-linked- immunosorbent-assay (ELISA).
• ELISA enzyme-linked- immunosorbent-assay
• DMEM Dulbecco's Modified Eagle Medium
• FBS Fetal Bovine Serum
• H25 cells (NIH 3T3c7 cells fransfected with a plasmid expressing the human insulin receptor) grown in growth media containing 5% CO 2 at 37° C.
• EY-Tide Biotin linked peptide (sequence biotin
• ABTS/H 2 O 2 formulation: 15 mL ABTS solution 2 ⁇ L H 2 O 2
• Kinase Buffer formulation 25 mM HEPES/CL, pH 7.0 150 mM NaCl 0.1% Triton X-100 lO mM MnCl, 17) TBST Buffer (Tris-buffered Saline with Triton X0199) formulation: 50 mM Tris, pH 7.2
• Blocking buffer 5% powdered non-fat milk in PBS (w/v).
• Block plate with 150 ⁇ l blocking buffer per well. Incubate while shaking on a microtiter plate shaker at room temperature for 30 minutes.
• Results are calculated as an IC50.
• the compounds, methods and pharmaceutical compositions of the present invention are expected to modulate the activity of protein tyrosine enzymes which mediate cellular signal transduction, in particular, protein tyrosine phosphatase, and therefore are expected to be effective as therapeutic agents against disorders associated with protein tyrosine enzyme related cellular signal transduction.

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