EP1012150A1 - Composes d'heteroarylcarboxamide actifs contre les etats pathologiques lies aux proteine tyrosine kinases - Google Patents

Composes d'heteroarylcarboxamide actifs contre les etats pathologiques lies aux proteine tyrosine kinases

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Publication number
EP1012150A1
EP1012150A1 EP98924794A EP98924794A EP1012150A1 EP 1012150 A1 EP1012150 A1 EP 1012150A1 EP 98924794 A EP98924794 A EP 98924794A EP 98924794 A EP98924794 A EP 98924794A EP 1012150 A1 EP1012150 A1 EP 1012150A1
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European Patent Office
Prior art keywords
cancer
compound
group
cells
salt
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EP98924794A
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German (de)
English (en)
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EP1012150A4 (fr
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Gerald Mcmahon
Peng Cho Tang
Laura Kay Shawver
Klaus Peter Hirth
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Sugen LLC
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Sugen LLC
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Publication of EP1012150A1 publication Critical patent/EP1012150A1/fr
Publication of EP1012150A4 publication Critical patent/EP1012150A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members 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
    • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates generally to organic chemistry, biochemistry, pharmacology, medicine and cancer therapy. More particularly, it relates to heteroarylcarboxamide compounds and their physiologically acceptable salts which modulate the activity of protein tyrosine kinases and, as a result, are expected to exhibit a salutary effect against disorders related to abnormal protein tyrosine kinase activity including cancer.
  • Novel chemotherapeutices comprising a heteroarylcarboxamide in combination with known chemotherapeutic agents such as the nitrosoureas are also described herein.
  • Gfrs Growth factor receptors
  • Gfrs are cell-surface proteins. When bound by a growth factor ligand, Gfrs are converted to an active form which interacts with proteins on the inner surface of a cell membrane. As the result of this interaction, one of the key biochemical mechanisms of signal transduction is initiated; i.e., the reversible phosphorylation of various proteins within the cell.
  • PKs protein kinases
  • the RTKs are comprised of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins.
  • CTKs are entirely intra-cellular and do not contain extracellular and transmembrane domains.
  • PTKs play an important role in the control of cellular processes including proliferation, differentiation, migration and survival. It is clear from numerous studies (q.v., infra) that the activity of PTKs must be tightly controlled in normal cells and healthy tissue, as mutations resulting in overactivity and/or overexpression of PTKs cause diseases that are associated with excessive cell growth and proliferation such as, significantly, cancer. At the other end of the spectrum, mutations which result in reduction or loss of activity can cause embryonal lethality or developmental disorders .
  • the RTKs comprise one of the larger families of PTKs and have diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been identified.
  • HER family of RTKs, which include EGFR (epithelial growth factor receptor), HER2 , HER3 and HER4.
  • EGFR epidermal growth factor receptor
  • HER2 epidermal growth factor receptor
  • HER3 epidermal growth factor receptor
  • HER4 epidermal growth factor receptor
  • RTKs consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins.
  • PTKs/RTKs One well-known example of the apparent involvement of PTKs/RTKs in cellular disorders is the association of Her2 over-expression with breast cancer (Slamon, et al, Science, 244:707 (1989).
  • RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and the insulin receptor related receptor (IRR) .
  • IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer of two entirely extracellular glycosylated ⁇ subunits and two ⁇ subunits which cross the cell membrane and which contain the tyrosine kinase domain.
  • a third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFR ⁇ , PDGFR ⁇ , CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.
  • flk fetus liver kinase
  • KDR/FLK-1 kinase insert domain-receptor fetal liver kinase-1
  • flk-lR fetal liver kinase-1
  • flt-1 fetus liver kinase 1
  • fibroblast growth factor contains at least four distinct members: fibroblast growth factor receptor 1 (FGFR1, also called Fig and Cekl), FGFR2 (also called Bek, Ksam, KsamI and Cek3) , FGFR3 (also called Cek2) and FGFR4. They share a common structure consisting of, in the mature protein, one or more immunoglobulin-like (IgG-like) loops flanked by characteristic cysteines, a hydrophobic transmembrane domain and a intracellular domain containing a catalytic region that is split by a short insert; See Ullrich and Schlessinger, Cell, 61:203 (1990).
  • IgG-like loops flanked by characteristic cysteines
  • cysteines a hydrophobic transmembrane domain
  • intracellular domain containing a catalytic region that is split by a short insert
  • Fibroblast growth factors and their receptors play an important role in angiogenesis, wound healing, embryonic development, and malignant transformation (Basilic and Moscatelli, Adv. Cancer Res . 59:115, 1992) .
  • FGFRs have been implicated in the induction of angiogensis (Folkman and Klagsbrun, Science 235:442, 1987; Klagsbrun and D'Amore, Annu. Rev. Physiol . 53:217, 1991), suggesting that inappropriate FGFR activity may contribute to human disorders such as diabetic retinopathy, macular degeneration, rheumatoid arthritis, atherosclerosis and tumor neovascularization .
  • FGFRs have been directly associated with a number of cancers including oral and salivary gland cancer (Myoken, et al . , Int . J. Cancer 65(5) :650, 1996) prostate cancer (Nakahara, et al., Nishinihon J. Urology 58(4) :310, 1996; Nakamoto, et al . , Cancer Res. 52(3) :571, 1992), breast cancer (Penault-Llorca, et al . , Int. J.
  • Mutations in human FGFR genes have been also implicated in a variety of human skeletal disorders. For example, mutations resulting in the constitutive (ligand-independent ) activity of FGFR3 play a role in long bone disorders and cause several clinically related forms of dwarfism including achondroplasia, hypochondroplasia and the neonatal-lethal thanatophoric dysplasia (Shiang, et al., Cell 78:3,35, 1994; Rousseau et al . , Nature 371:252, 1994; Bellus et al . , Nature Genet. 10:357, 1995; Tavormina, et al . , Nature Genet.
  • RTKs and CTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, arterial restenosis, wound scarring, kidney sclerosis and a variety of other renal disorders .
  • RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al . , Mol . Bio. Cell 4:358A (1993); Kinsella, et al . , Exp . Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys . , 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No.
  • the brain cancers in particular primary intra-axial brain cancers such as glioblastoma multiforme, astrocytoma, anaplastic astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma, hemangioblastoma and pineal parenchymal cancer.
  • primary intra-axial brain cancers such as glioblastoma multiforme, astrocytoma, anaplastic astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma, hemangioblastoma and pineal parenchymal cancer.
  • a malignancy within the brain presents particular difficulties not associated with other cancers. That is, the "blood-brain barrier" prevents penetration of many types of molecules into the brain parenchyma. Other complications involve the lack of lymphatic drainage within the brain to effect the removal of cellular wastes and toxins and the fixed space (the cranium) within which the tumor is located, which can give rise to increased intracranial pressure due to the fact that some tumors swell when they are subjected to chemotherapy. Charette, ibid.
  • the drugs generally regarded as having the best activity against brain tumors are alkylating agents which have been shown to display a directly proportional dose- response relationship.
  • the alkylating agents are cytotoxic; that is, they kill cancer cells, usually by affecting DNA synthesis or function.
  • the nitrosoureas are the agents of choice for treatment of brain tumors due to their ability to cross the blood-brain barrier.
  • N,N'-bis(2- chloroethyl) -N-nitrosourea or BCNU is the most active of the nitrosoureas against malignant gliomas, especially at high dosages.
  • BCNU at high dosage e.g., in excess of 800 mg/m 2 (cancer drug dosages are generally reported in either mg/kg (milligrams of drug per kilogram body weight) or mg/m 2
  • HWA 486 or A10 (and which shall be referred to herein as MCTA)
  • HWA 486 or A10 is an effective modulator of select PK, notably the RTKs PDGFR and FGFR, activity.
  • the present invention relates to a method for the treatment of diseases related to inappropriate FGFR activity by administering to a patient in need of such treatment a therapeutically effective amount of the heteroarylcarboxamide, N- (4-trifluoromethylanilino) -5- methylisoxazole-4-carboxamide ("MCTA”) in a pharmacological composition.
  • the FGFR-related disease is cancer .
  • the present invention relates to novel heteroarylcarboxamides which modulate the activity of PKs, in particular RTKs, especially PDGFR and FGFR.
  • the present invention relates to the preparation and use of pharmacological compositions of the disclosed compounds and their physiologically acceptable salts in the treatment and prevention of PTK driven disorders such as, significantly, cancer .
  • the present invention relates to the treatment of solid tumor cancers, especially glioblastomas, using combinations of MCTA with known chemotherapeutic agents such as the nitrosoureas, exemplified herein by BCNU.
  • heteroarylcarboxamide refers to a compound having the general structure shown in Formula 3.
  • a “pharmacological composition” refers to a mixture of one or more of the compounds described herein, or a physiologically acceptable salt thereof, with other chemical components, such as pharmaceutically acceptable carriers and excipients .
  • the purpose of a pharmacological composition is to facilitate administration of a compound to an organism.
  • physiologically acceptable carrier or “pharmaceutically acceptable carrier” are used interchangeably to refer 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 pharmacological composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • MCTA 4- (N- ( 4 ' -trifluoromethylphenyl) carboxamido) -5-methyl- isoxazole
  • MCTA 4- (N- ( 4 ' -trifluoromethylphenyl) carboxamido) -5-methyl- isoxazole
  • MCTA 4- (N- ( 4 ' -trifluoromethylphenyl) carboxamido) -5-methyl- isoxazole
  • MCTA acts only as a prodrug; i.e., a compound which is inactive in and of itself but is biochemically converted to an active specie i_n vivo .
  • MCTA is but a prodrug
  • MCTA itself is an extremely effective modulator of PDGFR and FGFR activity and is an effective chemotherapeutic agent, especially against solid tumor cancers, both alone and in combination with known chemotherapeutic agents such as BCNU.
  • novel analogs of MCTA which are described herein demonstrate activity which is similar to that of unmetabolized MCTA and, like unmetabolized MCTA, is different from that of A771726.
  • the ability of A771726 to inhibit pyrimidine biosynthesis is overcome by the addition of uridine which is characteristic of pyrimide biosynthesis inhibitors .
  • the compounds of the present invention while structurally similar to MCTA, are capable of inhibiting cellular growth by a mechanism not affected by the addition of uridine.
  • the heteroaryl group of the claimed compounds do not metabolize to an open form at all and therefore exhibit their activity in their native configuation, or, if they do metabolize to an open form, the chemical composition of the open form to which they are converted is either inactive or active, but not as an inhibitor of pyrimidine biosynthesis (as evinced by the fact that uridine addition has no effect) .
  • the compounds of the present invention comprise a new family of compounds capable of modulating protein tyrosine kinase activity.
  • the compounds of this invention affect cell proliferation by modulating PTK signaling.
  • modulate refers to the alteration of the catalytic activity of PTKs.
  • modulating refers to the activation of the catalytic activity of PTKs, more preferably the activation or inhibition of the catalytic activity of PTKs, depending on the concentration of the compound administered or, more preferably still, the inhibition of the catalytic activity of PTKs.
  • Modulation may be effected by direct interaction with a PTK or through intervention at some other point in the biochemical process controlled by the particular PTK, the observable result of which appears as a modulation of PTK catalytic activity.
  • catalytic activity refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect of PTKs.
  • the present invention relates to heteroarylcarboxamide compounds having the chemical structure shown in Formula 3 :
  • A is selected from the group consisting of oxygen, nitrogen and sulfur.
  • B is selected from the group consisting of nitrogen and sulfur and it is understood that when B is sulfur and A is nitrogen, the nitrogen is participating in both a single bond and a double bond within the ring so that it cannot be bonded to any atom outside the ring; that is, when B is sulfur, R 2 cannot exist.
  • D, E, F, G, and J are independently selected from the group consisting of carbon and nitrogen such that the six- member monocylic heteroaryl ring formed is one known in the chemical arts and, furthermore, when D, E, F, G or J is nitrogen, R 5 , R 6 , R 7 , R 8 or R 9 , respectively, does not exist.
  • R 1 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heteroalicyclic.
  • R 2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, carbonyl, C-carboxy, S-sulfonamido, sulfonyl, hydroxy, alkoxy, trihalomethanesulfonyl, halo, guanyl, C-amido and C-thioamido.
  • R 3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heteroalicyclic .
  • Z is selected from the group consisting of oxygen and sulfur.
  • R 4 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl heteroaryl, heteroalicyclic, sulfonyl, trihalomethanesulfonyl, hydroxy, alkoxy and C-carboxy.
  • R 5 , R 6 , R 7 , R 8 and R 9 are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, alkenyl, alkynyl, cycycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, cycloalkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkyoxy, thiocycloalkoxy, thioheteraryloxy, thioheteralicycloxy, halo, nitro, cyano, 0- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, silyl, phosphonyl, C-carboxy, O-carboxy, N-amido, C-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, tri
  • R 13 and R 14 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, C- carboxy, sulfonyl, trihalomethanesulfonyl and, combined, a five- or six-member heteroalicyclic ring containing at least one nitrogen.
  • alkyl 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, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, 0- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, 0- carboxy, nitro, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino,
  • R 13 and R 14 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, C- carboxy, sulfonyl, trihalomethysulfonyl and, combined, a five- or six-member heteroalicyclic ring.
  • 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.
  • cycloalkyl groups examples, without limitation, are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane and, cycloheptatriene .
  • a cycloalkyl group may be substituted or unsubstituted.
  • the substituent group (s) is preferably one or more individually selected from alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl,
  • 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 group 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, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, nitro, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido,
  • 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, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, nitro, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido,
  • heteroalicyclic 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 heteroalicyclic ring may be substituted or unsubstituted.
  • the substituted group (s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ureid
  • alkoxy refers to both an -O-alkyl and an -0- cycloalkyl group, as defined herein.
  • aryloxy refers to both an -O-aryl and an -0- heteroaryl group, as defined herein.
  • heteroaryloxy refers to a heteroaryl-O- group with heteroaryl as defined herein.
  • heteroalicycloxy refers to a heteroalicyclic-O- group with heteroalicyclic 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 heteroalicyclic as defined herein.
  • aldehyde refers to a carbonyl group where R" is hydrogen.
  • a “carboxylic acid” group refers to a C-carboxyl group in which R" is hydrogen.
  • halo refers to fluorine, chlorine, bromine or iodine .
  • trihalomethyl refers to a -CX 3 group wherein X is a halo group as defined herein.
  • a “cyano” group refers to a -C ⁇ N group.
  • a Ann ""NN--tthhiiooccaarrbbaammyyll"" ggrroouupp rreeffers to a R 13 0C ( S) NR 14 - group, with R 13 and R 14 as defined herein
  • amino refers to an -NR 13 R 14 group in which both R 13 and R 14 are hydrogen.
  • a "nitro” group refers to a -N0 2 group.
  • sil refers to a -Si(R") 3 , with R" as defined herein.
  • a “methylenedioxy” group refers to a -0CH 2 0- group where the two oxygens are covalently bonded to two adjacent carbon atoms of an aryl or heteroaryl ring with aryl and heteroaryl as defined herein.
  • a "1, 3-dioxano” group refers to a -CH 2 OCH 2 0- group where the -CH 2 and the oxygen are covalently bonded to two adjacent carbon atoms of an aryl or heteroaryl ring with aryl and heteroaryl as defined herein.
  • six-member monocyclic heteroaryl rings known in the chemical arts include, but are not limited to the following :
  • A is oxygen and B is nitrogen;
  • R 1 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl and alkynyl; and, R 3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl and aryl; and, R 4 is hydrogen.
  • R 2 is selected from the group consisting of hydrogen, alkyl and cycloalkyl; Z is oxygen;
  • R 5 , R 8 and R 9 are hydrogen; R 6 is selected from the group consisting or hydrogen and alkyl; and,
  • R 7 is selected from the group consisting of hydrogen, trihalomethyl and trihalomethanesulfonyl.
  • R 6 and R 7 combined, form a methylenedioxy or a 1,3- dioxano group.
  • a preferred structure for the claimed compounds is that in which J is nitrogen.
  • this invention relates to a method for the treatment or prevention of a disorder characterized by inappropriate PK activity comprising administering to a patient afflicted with such a disorder a therapeutically effective amount of one or more of the disclosed compounds, or a physiologically acceptable salt thereof, alone or in a pharmaceutically acceptable composition.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts .
  • the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from acquiring a PTK mediated cellular disorder in the first place.
  • the terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a PTK mediated cellular disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy or an individual afflicted with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.
  • a "disorder characterized by inappropriate PTK activity” includes, but is not limited to, cell proliferative disorders, cell differentiation disorders, cell growth disorders and metastatic disorders. Such disorders include, but are not limited to, cancer, as described below and, in addition, the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis) , fibrotic disorders and metabolic disorders.
  • neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis) , fibrotic disorders and metabolic disorders.
  • cancer refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites, as defined by Stedman's Medical Dictionary 25th edition (Hensyl ed. 1990).
  • Examples of cancers which may be treated by the present invention include, but are not limited to, brain, ovarian, colon, prostate, kidney, bladder, breast, lung, oral and skin cancers which exhibit inappropriate PTK activity. These types of cancers can be further characterized.
  • brain cancers include glioblastoma ultiforme, anaplastic astrocytoma, astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma, hemangioblastoma, and pineal parenchymal .
  • Skin cancers include melanoma and Kaposi's sarcoma .
  • Unwanted cell proliferation can result from inappropriate PTK activity occurring n different types of cells including cancer cells, cells surrounding a cancer cell (stromal cells) , endothelial cells and smooth muscle cells.
  • stromal cells cells surrounding a cancer cell
  • endothelial cells cells surrounding a cancer cell
  • smooth muscle cells cells surrounding a cancer cell
  • an increase in FGFR and/or PDGFR activity of endothelial cells surrounding cancer cells may lead to an increased vascula ⁇ zation (angiogenesis) of the tumor, thereby facilitating growth of the cancer cells.
  • Inappropriate PTK activity may also contribute to the proliferation of cancer cells by direct mitogenic stimulation.
  • Cell proliferative disorders refer to disorders wherein unwanted cell proliferation of one or more subset of cells m a multicellular organism occurs resulting in harm (e.g., discomfort or decreased life expectancy) to the multicellular organism.
  • Cell proliferative disorders can occur in different types of animals and in humans .
  • Cell proliferative disorders include cancers, skeletal disorders, angiogenic or blood vessel proliferative disorders, fibrotic disorders and mesangial cell proliferative disorders.
  • Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels.
  • blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
  • ocular diseases like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
  • disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis are also implicated.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrices.
  • fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders.
  • Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
  • An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis.
  • Lipocytes appear to play a major role in hepatic cirrhosis.
  • Other fibrotic disorders implicated include atherosclerosis.
  • Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
  • Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well as disorders such as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies .
  • PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47S-54S.
  • Other examples of cell proliferative disorders are disclosed in the following references which are incorporated as if fully set forth herein.
  • the EGFR Tuzi et al . , 1991, Br. J.
  • EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer.
  • HER2 has been associated with breast, ovarian, gastric, lung, pancreatic and bladder cancer.
  • PDGFR has been associated with glioblastoma, lung cancer, ovarian cancer, prostate cancer, and melanoma.
  • the RTK c-met has been associated with hepatocarcinogenesis . Additionally, c-met has been linked to other malignant tumor formations. More specifically, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over- expression of the c-met gene has been detected in patients with Hodgkins disease and Burkitts disease.
  • IGF-IR in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers.
  • IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517).
  • IGF-I while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas.
  • IGF-IR insulin growth factor-IR
  • fibroblasts epithelial cells, smooth muscle cells, T- lymphocytes, myeloid cells, chondrocytes, osteoblasts (the stem cells of the bone marrow)
  • IGF-I the stem cells of the bone marrow
  • Baserga suggests that IGF-IR plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol . Cell. Biol., 14:4588- 4595.
  • 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.
  • 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.
  • a “therapeutically effective amount”, in reference to the treatment of a cell proliferative disorder other than a cancer refers to an amount sufficient to bring about one or more of the following results: inhibit the growth of cells causing the disorder, relieve discomfort due to the disorder, or prolong the life of a patient suffering from the disorder.
  • RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases.
  • EGF-R is indicated in corneal and dermal wound healing.
  • Defects in the Insulin-R and the IGF-IR are indicated in type-II diabetes mellitus.
  • RTKs As noted previously, not only RTKs but CTKs as well including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al . , FASEB J . , 6:3403-3409 (1992)) are involved in the proliferative and metabolic signal transduction pathway and thus were expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60 v_src ) in chicken.
  • v-src mutated src
  • pp60 c_src transmits oncogenic signals of many receptors.
  • over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60 c_src , which is characteristic of malignant cells but absent from normal cells.
  • mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
  • Zap70 is implicated in T-cell signaling.
  • a further feature of this invention is a method for treating a disorder characterized by inappropriate FGFR activity comprising administering MCTA in a pharmaceutical composition to a patient in need of such treatment.
  • the disorder comprises cancer.
  • Still another feature of this invention is a method for treating a disorder characterized by inappropriate PDGFR activity likewise comprising administering MCTA or a compound of this invention in a pharmaceutical composition to a patient in need of such treatment.
  • the disorder comprises cancer.
  • Unwanted cell proliferation can result from inappropriate FGFR activity occurring in different types of cells including cancer cells, cells surrounding a cancer cell (stromal cells), endothelial cells and smooth muscle cells.
  • stromal cells cells surrounding a cancer cell
  • endothelial cells cells surrounding a cancer cell
  • smooth muscle cells For example, an increase in FGFR activity of endothelial cells surrounding cancer cells may lead to an increased vascularization (angiogenesis) of the tumor, thereby facilitating growth of the cancer cells .
  • Inappropriate FGFR activity may also contribute to the proliferation of cancer cells by direct mitogenic stimulation.
  • Inappropriate FGFR activity refers to either 1) FGFR expression in cells which normally do not express FGFR; 2) FGF ligand expression by cells which normally do not express FGF; 3) increased FGFR expression leading to a disorder; 4) increased FGF expression leading to a disorder; or 5) mutations leading to constitutive activation of FGFR.
  • the existence of inappropriate or abnormal FGF and FGFR levels or activities is determined by procedures well known in the art and are discussed in greater detail below. Inappropriate FGFR activity can also result in metastatic disorders. Metastasis is the term used to describe the process by which a primary tumor spreads to distant sites in the body, forming secondary tumors. Metastatic disease is often more deadly than a primary cancer. The process and consequences of metastasis are described more fully below.
  • compositions refers to pharmaceutical compositions of MCTA or any of the other compounds of this invention useful in the claimed method.
  • compositions can be used to treat a disorder characterized by inappropriate FGFR activity by administering a therapeutically effective amount of the composition to a patient (i.e. a human or an animal) having a disorder.
  • a patient i.e. a human or an animal
  • MCTA and the other heteroarylcarboxamides of this invention may also be used in in vitro studies of the mechanism of action of FGFR or FGF itself.
  • “Significant” inhibition of receptor tyrosine kinase activity refers to an IC50 of less than or equal to 100 ⁇ M using one or more of the assays described in the Examples infra.
  • the compound can inhibit FGFR activity with an IC50 of less than or equal to 85 ⁇ M.
  • a lower IC50 is preferred because the IC50 provides an indication of the in vivo effectiveness of the compound.
  • Other factors known in art, such as compound half-life, biodistribution, and toxicity should also be considered for therapeutic uses. Such factors may enable a compound with a higher IC50 to have greater iri vivo efficacy than a compound having a lower IC50.
  • the term "significant" can also refer to comparisons between tumor growth in animals treated with a compound of the invention and a control animal wherein the reduction in growth is found to be statistically different.
  • MCTA and the compounds of this invention are receptor specificity. This is not to say that the compounds will have absolute specificity, however they show selective inhibition of a limited number of RTKs and, therefore, will be useful only in disorders characterized by inappropriate activity of that limited set of RTKs, i.e. FGFR. Selective inhibition of the FGFR family is achieved by significantly inhibiting FGFR activity, while having an insignificant effect on PTK activity from a disparate family (e.g., an IC50 greater than 100 ⁇ M on EGFR).
  • MCTA inhibits signaling by PDGFR (See WO 95/19169, published July 20, 1995) it would not be assumed that it would also be effective at inhibiting the activity of FGFR, which belongs to a distinct family of receptors. And, of course, it follows that MCTA would therefore not be expected to be useful in treating a distinct set of disorders, i.e., those characterized by inappropriate FGFR activity.
  • MCTA is preferably administered in a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a formulation to which the compound can be added to dissolve or otherwise facilitate administration of the compound.
  • Examples of pharmaceutically acceptable carriers include water, saline, physiologically buffered saline, cyclodextrins and
  • VPD:D5W Hydrophobic compounds such as MCTA are preferably administered using a carrier such as VPD:D5W.
  • An important factor in choosing an appropriate pharmaceutically acceptable carrier is choosing a carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound.
  • the compound may also be administered in a continuous fashion using a slow release formulation or a pump to maintain a constant or varying drug level in a patient.
  • FGFR1 also called Fig and Cekl
  • FGFR2 FGFR2
  • FGFR3 also called Cek2
  • FGFR4 also called Cek2
  • the degree of homology varies between them, with the highest homology found in the catalytic domain. Additional diversity in the family is created through splice variants that vary the number and character of the IgG-like regions in the extracellular domain. At least nine FGFR ligands have been identified including FGF1 (acidic FGF), FGF2 (basic FGF), FGF3 (int-2),
  • FGF4 Keratinocyte growth factor
  • KGF kinase-like growth factor
  • FGF8 androgen-induced growth factor
  • FGF9 FGF9
  • Multiple members of the FGF ligand family can bind to the same receptor species.
  • FGFs and FGFRs see Johnson and Williams, Adv. in Cancer Res . 60:1, 1993.
  • the PDGF receptor family contains only the two isoforms PDGFR- ⁇ and PDGFR- ⁇ , which are known to heterodimerize .
  • PDGF ligand is a pleiotropic factor that exists as a homo- or heterodimer of two polypeptides, the A- and B- chains (Habenicht, et al, Klin. Kli-Schrift 68:53, 1990; Heldin, EMBO J. 11:4251, 1992) .
  • tyrosine kinase receptors structurally and functionally related to FGFR and PDGFR include Fit (de Vries, et al, Science 255:989, 1992) and KDR (Ter an, et al, BBRC 187;1579, 1992), both of which are activated by the ligand VEGF (Rosenthal, et al . , Growth Factors, 4:53-59, 1990; Conn, et al., Proc. Natl. Acad. Sci. (USA), 87:1423-1427, 1990; Houck, et al., Mol. Endocrinol. , 5:1806-1814, 1991).
  • VEGF expression is known to be increased by hypoxia (such as would be found in growing tumors) and is known to stimulate endothelial cells and to be involved in angiogenesis (Plate et al . , Nature, 359:845-848, 1992; Shweike, et al . , Nature 359:843-845, 1992).
  • PDGF- and FGF-dependent signaling is initiated immediately following binding of ligand to a receptor.
  • Ligand binding induces receptor dimerization, either homodimers or heterodimers, leading to activation of receptor tyrosine kinase activity and autophosphorylation .
  • Activation of the receptor leads to increased tyrosine phosphorylation on a number of cellular proteins, although many of their identities and functions are still largely unknown.
  • PDGFR and/or FGFR activation ultimately leads to proliferation, differentiation, inhibition of differentiation, motility, etc.
  • the use of the present invention is facilitated by first determining whether a disorder is related to inappropriate PDGFR and/or FGFR activity. Once such disorders are identified as such, patients suffering from the disorder can be identified by analysis of their symptoms by procedures well known in the medical arts . Such patients can then be treated as described herein. Many well known techniques exist for determining whether a disorder is related to inappropriate PDGFR and/or FGFR activity. For example, comparisons can be made in the level of expression of either FGF and/or PDGF ligand or FGFR and/or PDGFR, in a tumor biopsy with levels in similar normal tissues or tumor cells known to be unrelated to FGFR and/or PDGFR activity (such as A431 cells, Yaish, et al, Science 242:933, 1988).
  • Such comparisons can be done by immunostaining with FGFR and/or PDGFR specific antibodies or binding and detection of FGF and/or PDGF ligand using techniques well known in the art, by Northern blot analysis for the presence of ligand or receptor RNA, or by transcript imaging (Plowman, W096/34985, published 7 November 1996, and incorporated by reference herein) .
  • samples can be analyzed for level of receptor phosphorylation, which is indicative of activity, compared to normal tissues.
  • Receptor phosphorylation is readily detected by means well known in the art such as by using anti-phosphotyrosine antibodies.
  • cancer cells have a higher level of FGFR and/or PDGFR activity or expression than non-FGFR and/or PDGFR driven cancers or normal tissues, preferably equal to or greater than previously identified FGFR and/or PDGFR driven cancers, then they are candidates for treatment using the described inhibitors.
  • the level of receptor activity is compared to that level occurring in the general population (e.g., the average level occurring in the general population of people or animals excluding those people or animals suffering from a cell proliferative disorder) . If the unwanted cell proliferation disorder is characterized by a higher receptor level then occurring in the general population then the disorder is a candidate for treatment using the described inhibitors.
  • the unwanted cell proliferation disorder is characterized by a higher receptor level then occurring in the general population then the disorder is a candidate for treatment using the described inhibitors.
  • PDGFR are cell proliferative disorders. As discussed above, all proliferative disorders result in unwanted cell proliferation of one or more subsets of cells in a multicellular organism resulting in harm to the organism. Two ways in which inappropriate PTK/FGFR/PDGFR activity can stimulate unwanted cell proliferation of a particular type of cell are by directly stimulating growth of the particular cell, or by increasing vascularization of a particular area
  • angiogenesis such as tumor tissue, thereby facilitating growth of the tissue.
  • Angiogenesis also plays a significant role in metastasis, a complex disorder which is discussed in more detail below.
  • Cell proliferative disorders include cancers, blood vessel proliferation disorders, skeletal malformations and fibrotic disorders. These disorders are not necessarily independent.
  • fibrotic disorders may be related to, or overlap with, blood vessel disorders.
  • Moyamoya disease which is characterized herein as a blood vessel disorder results in the abnormal formation of fibrous tissue in the intracranial arteries.
  • Blood vessel proliferation disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. Examples of such disorders besides cancer include Moyamoya disease and macular degeneration.
  • FGFR and KDR have been recognized has having a regulatory role in angiogensis, along with other factors, due to their role in both endothelial cell proliferation and migration (Friesel and Maciag, FASEB J. 9:919, 1995; Folkman and Klagsbrun, Science 235:442, 1987; Mullins and Rifkin, J.
  • FGFR activity has been suggested to play a role specifically in the angiogenesis associated with macular degeneration (Amin, et al, Invest. Ophthal. and Vis. Sci. 35(8):3178, 1994).
  • PDGF and VEGF expression is associated with angiogeneis and metastasis in breast cancer (Anan, et al . , Surgery : 119 : 333, 1996).
  • PDGF expression has been correlated with increased blood vessel count in colon cancers (Hsu, et al, J. Cell . Physol . 165:239, 1995) .
  • Moyamoya disease is characterized by intracranial carotid artery stenosis and occlusions and a fine network of vessels at the base of the brain and may be described as both an angiogenic and fibrotic disorder (Suzuki and Kodama, Stroke 14:104, 1983; Suzuki and Takaku, Arch Neurol 20:288, 1969).
  • Suzui, et al, (Neurosurgery 35(1):20, 1994) have found that both FGF ligand and FGFR are increased in the superficial temporal artery of patients with Moyamoya disease.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrix.
  • fibrotic disorders include those found in the liver (hepatic cirrhosis) , kidney (glomerular sclerosis, interstitial nephritis), lung (interstitial pulmonary fibrosis) , arteries (restenosis, atherosclerosis) and skin (wound scarring, scleroderma) .
  • Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
  • An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis.
  • Lipocytes appear to play a major role in hepatic cirrhosis. Inappropriate FGFR activity can stimulate lipocyte proliferation .
  • Liotta, et al describe invasion and metastasis as "the most life-threatening aspects of the oncogenic process" (See review in Liotta, et al, Cell 64:327, 1991) .
  • Invasion and metastasis are complex events mediated by a group of coordinated cellular processes. They are facilitated by proteins that stimulate tumor cell attachment to extracellular matrix, proteolysis of barriers such as the basement membrane, migration into the circulatory system and attachment to and colony formation in distant organs. There is also a significant correlation between metastatic potential and angiogeneic potential, and the two processes may have many factors in common. (See Claffey, et al, Cancer Res . 56:172, 1996; Takahashi, et al., Cancer Res. 55:3964, 1995.)
  • FGF FGF
  • PDGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • FGFR and PDGFR expression has been associated with increased aggressiveness and metastasis of a number of cancers.
  • the invasive tumor showed a significant increase in FGFR at both the protein and RNA level, with the non-invasive tumor (RT4) showing almost no FGFR present.
  • the FGFR was also shown to be biologically active by receptor phosphorylation in response to ligand.
  • H gh FGFR expression was significantly associated with shorter post-operative survival and extent of malignancy.
  • Hasegawa, et al. (BBRC 200(3) :1435, 1994) showed that FGF ligand stimulated basement membrane invasion by human pancreatic cancer cells in an in vitro model compared to other growth factors (EGF, PDGF) and that invasion could be significantly inhibited by the addition of an anti-FGFR antibody.
  • this invention is directed to compounds which modulate PTK signal transduction by affecting the enzymatic activity of the PTKs and thereby interfering with the signals transduced by such proteins. More particularly, an aspect of the present invention is directed to compounds which modulate the PTK-mediated signal transduction pathways as a therapeutic approach to treat many kinds of solid tumors, including but not limited to carcinoma, sarcoma, erythroblastoma, glioblastoma, menmgioma, astrocytoma, melanoma and myoblastoma.
  • Leukemias may also be susceptible to treatment using the compounds of this invention. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreatic cancers, colon cancers, blood cancers, lung cancers, bone cancers and leukemias.
  • administering refers to a method of contacting a compound of this invention with a PTK both _ ⁇ n vitro, i.e. in a test tube, and in vivo, i.e. m cells or tissues of a living organism.
  • the PTK mediated disorders which are the object of this invention can be studied, prevented or treated by the methods set forth herein whether the cells or tissues of the organism exist within the organism or outside the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • the ability of a particular compound to affect a PTK related disorder can be determined; i.e., the IC50 of the compound, defined below, before the use of the compounds in more complex living organisms is attempted.
  • PTK related disorder As used herein, "PTK related disorder,” “PTK driven disorder”, “abnormal PTK activity” and “inappropriate PTK activity” all refer to a disorder characterized by inappropriate or over-activity of PTKs, which can be either RTKs or CTKs. Inappropriate activity refers to either: (1) PTK expression in cells which normally do not express PTKs; (2) increased PTK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PTK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
  • Overactivity of PTKs refers to either amplification of the gene encoding a particular PTK or production of a level of PTK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PTK increases, the severity of one or more of the symptoms of the cellular disorder increases) .
  • the methods and compositions of the invention are designed to inhibit unwanted cell proliferation or metastasis by altering the activity of PTKs.
  • inhibition of the activity of PTKs may occur by inhibiting tyrosine phosphorylation of a RTK, by inhibiting substrate or adaptor protein binding to the receptor, or by inhibiting other downstream signaling events, thereby inhibiting the activity of the RTK.
  • the use of the claimed methods and compositions are not limited to this particular theory.
  • MCTA also known as leflunomide and HWA 486, has been previously discussed in various publications as having a variety of uses including as an antirheumatic, antiphlogistic, antipyretic or analgesic and in the treatment of multiple sclerosis (Kommerer F-J, et al . , U.S. Pat. No. 4,284,786 (1981) and Ltderer F-J, et al . , U.S. Pat. No. 4,351,841 (1982). It has been suggested as an anti-inflammatory agent (Bartlett, et al., Agents and Actions 32:10-21, 1991; EP 607 775, EP 607 776,
  • EP 607 777 European Application 0 414 329 A2 suggests the use of MCTA for the treatment of ocular disorders associated with an inflammatory response, and (EP 665 014, US 5,519,042) suggests its use in treating vascular disorders.
  • PDGFR Platelet Derived Growth Factor Receptor
  • Bartlett, et al. (PCT/EP90/01800, US 5,532,259 issued July 2, 1996) assert that MCTA can be used to treat cancers, in particular cancers associated with inappropriate activity of the Epidermal Growth Factor Receptor (EGFR).
  • EGFR Epidermal Growth Factor Receptor
  • MCTA inhibits FGFR activity while having little if any effect on EGF-receptor activity in cells.
  • MCTA inhibited growth of tumors characterized by inappropriate FGFR activity, it did not significantly inhibit the growth of cells shown in PCT/EP90/01800 (A431 and KB) when tested in in vivo models.
  • This data is surprising in view of the results described by Bartlett et al . , supra, (Agents and Actions) in which MCTA was ostensibly shown to inhibit EGF induced EGF receptor autophosphorylation and cell proliferation, and Mattar et al . , supra, in which a metabolite of MCTA inhibited the _in. vitro growth of A431 cells.
  • neither Bartlett or Mattar suggest that MCTA can be used to inhibit metastasis.
  • the present disclosure demonstrates the ability of MCTA to inhibit the cellular response to FGFR activity and unwanted cell proliferation in vivo, such as that found in cancers characterized by inappropriate FGFR activity. Moreover, the present invention demonstrates that MCTA is useful for inhibiting metastasis.
  • the present invention relates to a method for inhibiting tumorigenic activity in a cell comprising administering to the cell a combination of drugs including a chemotherapeutic nitrosourea and a cytostatic signal transduction inhibitor.
  • the cytostatic signal transduction inhibitor may be MCTA or one or more of the novel compounds described herein; preferably, it is MCTA. It is understood, however, that reference to MCTA in the description that follows applies also to the novel compounds of this invention.
  • MCTA might perform effectively with any nitrosourea; e.g., chlorozoticin, fotemustine, lomustine, nimustine and ranimustine, at present BCNU, also known as carmustine or, formally, N,N' -bis (2-chloroethyl) -N-nitrosourea, is considered the most active of the group; thus, this invention is directed in particular toward the combination of MCTA with BCNU.
  • BCNU also known as carmustine or, formally, N,N' -bis (2-chloroethyl) -N-nitrosourea
  • “Comprising” as used herein in connection with “administering” is intended to mean that drugs being administered pursuant to the present invention may be administered as simply a combination of MCTA and BCNU only or may be expanded to include additional drugs which are known or expected to offer additional beneficial characteristics to the combination.
  • “combination” refers to two or more compounds or drugs which are administered simultaneously, sequentially, continuously, intermittently, etc. in accordance with a regimen calculated to take maximum advantage of the characteristics of each of the component drugs.
  • the combination comprises MCTA and BCNU.
  • combinations of BCNU with two, three and more additional compounds are well known to those skilled in the art and it would be within the spirit and scope of this invention to include one or more of these additional compounds together with the BCNU and MCTA.
  • “Tumorigenic” activity as it relates to a cell, refers to both intracellular and extracellular biochemical activity which contributes to the formation of a neoplasm.
  • a "neoplasm” is an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow even after the stimuli that initiated the new growth cease.
  • a neoplasm partially or completely lacks structural organization and functional coordination with the normal tissue and usually forms a distinct mass of tissue. Such masses may be benign (benign tumors) or malignant (solid tumor cancer) .
  • Malignant neoplasms are locally invasive and destructive and in many cases metastasize (spread to and invade and destroy tissues in areas of the affected organism remote from the site of origin) .
  • the process of neoplasm formation is generally referred to as "neoplasia"; i.e., neoplasia is the biochemical process by which a neoplasm forms and grows.
  • MCTA and BCNU are used together to inhibit the formation of malignant neoplasms; i.e., to inhibit neoplasia.
  • inhibitor refers to eliminating, reducing, containing, impeding, preventing, slowing, retarding and/or restricting neoplasia .
  • malignant neoplasm malignant neoplasm
  • cancer tumor-containing neoplasm
  • tumors solid tumor cancer
  • brain tumors are classified as primary, originating in cells found in the brain itself; secondary, metastatic from sites outside the central nervous system; or developmental, arising from displaced midline epithelium or germ cells. Such tumors are further described as intra-axial or extra-axial.
  • Intra-axial brain tumors are those which originate within the brain parenchyma; that is, the interstitial tissues of the brain itself or closely associated tissues such as the pineal gland, the posterior pituitary gland, the spinal cord and the retina.
  • Extra-axial tumors originate in tissues not directly associated with the brain but still within its vicinity; e.g., in the skull, the cranial nerves or in brain appendages such as the pituitary gland.
  • About half of primary intra-axial brain tumors are gliomas which include astrocytoma, particularly anaplastic astrocytoma, glioblastoma multiforme and oligodendroglioma . Both glioblastoma and astrocytoma are expected to be particularly vulnerable to the combination of drugs claimed herein.
  • glioblastoma glioblastoma multiforme
  • Glioblastoma consist chiefly of undifferentiated anaplastic cells of glial origin that show marked nuclear pleomorphism, necrosis and vascular endothelial proliferation. They grow extremely rapidly and invade extensively. They occur most frequently in the cerebrum of adults. While survival depends on numerous factors, generally speaking, most patients survive less than 9 months after diagnosis of glioblastoma.
  • a method for treating solid tumors (malignant neoplasms) in a patient by administering to the patient a therapeutically effective amount of MCTA and a therapeutically effective amount of a chemotherapeutic nitrosourea, in particular, BCNU.
  • MCTA solid tumors
  • BCNU chemotherapeutic nitrosourea
  • Individual pharmacological compositions of both MCTA and of BCNU, which compositions facilitate the administration of each of the drugs, are an additional feature of this invention.
  • the use of the pharmacological compositions of MCTA and BCNU for the treatment of brain tumors, especially glioblastoma and astrocytoma, is a particularly preferred aspect of this invention .
  • a “patient” refers to any higher organism which is susceptible to the formation of neoplasms.
  • patient refers to mammals, especially, of course, human beings .
  • chemotherapeutic refers to a chemical substance or drug used to treat a disease; the term is most often applied to such substances or drugs which are used primarily for the treatment of cancer.
  • the nitrosoureas appear to inhibit changes necessary for repair of damaged DNA, perhaps by virtue of the fact that, as well as being alkylating agents, the nitrosoureas are carbamoylating agents since isocyanates are formed during their decomposition at physiological pHs; proteins are known to be carbamoylated by isocyanates derived from BCNU.
  • BCNU has been used to treat a number of different solid tumor cancers; e.g., lymphomas, malignant melanoma, breast cancer and multiple myeloma. It has been employed in combination with other drugs such as cyclophosphamide, cisplatin, vincristine, melphalan, dexamethasone, vinblastine, methotrexate, daunorubicin, etoposide, procarbazine, prednisone, cytarabine, thioguanine and hydroxyurea. In addition, because of its ability to cross the blood-brain barrier, BCNU is presently the chemotherapeutic of choice for the treatment of brain cancers, including glioblastoma.
  • other drugs such as cyclophosphamide, cisplatin, vincristine, melphalan, dexamethasone, vinblastine, methotrexate, daunorubicin, etoposide, procarbazin
  • BCNU has been combined with cyclophosphamide, cisplatin, thiotepa, procarbazine, vincristine, etoposide and taxol .
  • BCNU is known to cause myelosuppresion (resulting in leukopenia and thrombocytopenia) , pulmonary fibrosis (lung disease) , hepatotoxicity (liver damage) and nephrotoxicity (kidney damage) .
  • the effects of BCNU are dose dependent, progressive and cumulative.
  • BCNU itself has been shown to cause tumors in rats and mice at doses approximating those clinically employed (Bristol Laboratories Oncology Products, FDA disclosure enclosure accompanying commercial BCNU packaging) .
  • MCTA affects tumors by a mode of action which to the inventors' knowledge is completely different from that of the known chemotherapeutic agents and, therefore, it was unknown whether it would have any beneficial effect n conjunction with BCNU. That is, as stated previously, BCNU acts primarily as an alkylating agent, attacking and damaging a number of key enzymatic reactions involved m DNA synthesis and DNA itself. Virtually all of the reported chemotherapeutic combinations with BCNU likewise interfere directly m one way or another with DNA synthesis or activity. MCTA, on the other hand, does not interact directly with DNA synthesis/activity. Rather, MCTA intervenes at an earlier stage of the cell cycle.
  • MCTA has been shown to inhibit signaling events which are necessary for a cell to initially enter into the active stage of DNA synthesis and cell proliferation where BCNU and the other alkylating agents exhibit their predominant activity. Specifically, MCTA inhibits platelet derived growth factor (PDGF) mediated cell signaling events which are necessary for the inception of cell growth, cell cycle progression and cell proliferation.
  • PDGF platelet derived growth factor
  • MCTA inhibits cell activity but does not kill cells.
  • tumor cells have been shown to over-express PDGF receptors and to be particularly dependent on the abnormally high PDGF signaling events arising from this over- expression for their continued proliferation.
  • PDGF ligands and receptors have been detected in many cell lines or tissues derived from, without limitation, glioma, melanoma, esophageal tumors, gastric tumors, colorectal adenocarcinoma, basal cell cancer, choriocarcinoma, breast cancer, Kaposi's sarcoma, ovarian cancer, non-small cell lung carcinoma and prostate adenocarcinoma.
  • Some cancer cells have even been shown to co-express PDGF itself as well as the receptor for it.
  • tumor cells may be self-stimulating; i.e., employ an autocrine as well as a paracrine growth mechanism which could account for the previously noted propensity for neoplasms to continue growing and proliferating even when the external stimuli are removed.
  • MCTA is a blocker of PDGF-mediated activity and thereby is capable of inhibiting the uncontrolled growth and proliferation of tumors.
  • the therapeutic index that is, the ratio of the LD 50 (the dose lethal to 50% of the population) to the ED 50 (the dose therapeutically effective in 50% of the population)
  • the therapeutic index is higher that for either drug alone which translates into a substantial benefit to patients in need of as much chemotherapy as possible without a concomitant increase in intolerable side effects requiring cessation of treatment.
  • MCTA is known to metabolize in mammals to N- ( 4-trifluoromethylphenyl) -2-cyano-3-hydroxy- crotonamide.
  • MCTA tends to metabolize most readily when subjected to acid conditions such as those encountered in the stomach when a drug is administered orally.
  • the preferred mode of administration of the combination claimed herein is more direct; e.g., without limitation, intravenously or intraarterially .
  • a portion of the MCTA administered to a patient may metabolize to N- (4-trifluoro- methylphenyl) -2-cyano-3-hydroxy- crotonamide prior to reaching the target tumor.
  • some of the activity of the claimed combination may in fact be due to a combination which includes not only MCTA and BCNU but N-(4- trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide as well.
  • the mode of action of N- ( 4-trifluoromethylphenyl) -2-cyano-3- hydroxycrotonamide has been determined to be inhibition of pyrimidine biosynthesis and therefore subsequent cell growth and/or survival which may in fact provide additional synergy of the claimed combination.
  • Physiologically acceptable salts can be acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.
  • Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • Physiologically acceptable salts can be prepared by standard techniques. For example, the free base form of the compound is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In a another example, the salt is prepared by reacting the free base and acid in an organic solvent. Carriers or excipient can be used to facilitate administration of the compound, for example, to increase the solubility of the compound. Examples of carriers and excipient include calcium carbonate, calcium phosphate, various sugars or starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
  • compositions or pharmaceutical composition can be administered by different routes including intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, or transmuccosally .
  • the specific delivery route of any selected agent depends on the use of the agent. Generally, a specific delivery program for each agent focuses on agent uptake with regard to intracellular localization, followed by demonstration of efficacy. Alternatively, delivery to these same cells in an organ or tissue of an animal can be pursued. Uptake studies include uptake assays to evaluate, e . g. , cellular nucleic acid or protein uptake, regardless of the delivery vehicle or strategy.
  • Such assays also determine the intracellular localization of the agent following uptake, ultimately establishing the requirements for maintenance of steady-state concentrations within the cellular compartment containing the target sequence (nucleus and/or cytoplasm) . Efficacy and cytotoxicity can then be tested. Toxicity not only includes cell viability but also cell function. Generally, the dosages of the mutated protein and nucleic acid is as described above for the featured compounds.
  • Drug delivery vehicles are effective for both systemic and topical administration. They can be designed to serve as a slow release reservoir, or to deliver their contents directly to the target cell.
  • An advantage of using direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles increase the circulation half-life of drugs which would otherwise be rapidly cleared from the blood stream.
  • Some examples of such specialized drug delivery vehicles falling into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres . Pumps can also be used for this purpose.
  • liposomes are preferred. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion to those lipids making up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Antibodies can be attached to liposomes to target particular cells.
  • Topical administration of the featured compound is advantageous, particularly when treating skin disorders such as Kaposi's sarcoma, since it allows localized concentration at the site of administration with minimal systemic adsorption.
  • MCTA MCTA
  • MCTA MCTA
  • Systemic absorption refers to the accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic absorption include: intravenous, subcutaneous, intraperitoneal, intranasal and intrathecal. Each of these administration routes expose the drug to an accessible diseased tissue.
  • Subcutaneous administration drains into a localized lymph node which proceeds through the lymphatic network into the circulation. The rate of entry into the circulation has been shown to be a function of molecular weight or size.
  • MCTA is hydrophobic and thus not very soluble in water. It is expected that the novel compounds of this invention may also be hydrophobic. Effective doses of hydrophobic compounds for systemic administration can be obtained using the pharmaceutical formulations described in US Patent No. 5,610,173, issued March 11, 1997. A particularly preferred formulation is obtained using a combination of the compound and VPD:D5W.
  • VPD is diluted 1:22 in a diluent. Preferred diluents are 0.45% saline, and 0.9% saline. A particularly preferred diluent is 5% dextrose in water (D5W) .
  • the composition can be administered at short time intervals, preferably the composition can be administered using a pump to control the time interval or achieve continuously administration.
  • Suitable pumps are commercially available (e.g., the ALZET® pump sold by Alza corporation, and the BARD ambulatory PCA pump sold by Bard MedSystems) .
  • Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a 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, alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, 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.
  • compositions 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.
  • 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.
  • the proper dosage depends on various factors such as the type of disease being treated, the particular composition being used, the dosing regimen and the size and physiological condition of the patient.
  • the minimal plasma concentration in a patient be greater than 5 ⁇ g/ml, more preferably greater than 25 ⁇ g/ml, most preferably greater than 50 ⁇ g/ml.
  • the compound can be delivered daily or less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. Plasma levels may be reduced if pharmacological effective concentrations of the drug are achieved at the site of interest. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disruption of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex component) .
  • IC 50 as determined in cell culture
  • levels in plasma may be measured, for example, by HPLC.
  • 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 pi). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) .
  • the magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • MCTA and BCNU can be administered to a human patient per se, or in pharmacological compositions wherein they are mixed with suitable carriers or excipient (s) .
  • suitable carriers or excipient s
  • suitable carriers or excipient s
  • Suitable routes of administration may, for example, include transmucosal, intramuscular, subcutaneous, intra- medullary, intrathecal, intraventricular , intravenous, intra- arterial or intraperitoneal .
  • one may administer MCTA and BCNU 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.
  • Other means of administering the drugs of this invention which are well within the knowledge of those skilled in the art are within the scope and spirit of this invention.
  • Pharmacological compositions of MCTA and BCNU are preferred embodiments of this invention.
  • Pharmacological compositions of the present invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmacological compositions for use in accordance with the present invention 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.
  • BCNU for injection, preferred formulation for BCNU are provided with the commercial compound.
  • this includes Dehydrated Alcohol injection USP in which the BCNU is dissolved and then the alcoholic solution is diluted aseptically with sterile water to a final concentration of 3.3 mg/ml of BCNU in 10% ethanol, pH 5.6 to 6.0 (BCiNU® from Bristol Laboratories Oncology Products).
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents.
  • a preferred formulation for injection comprises ethanol/water containing a surfactant.
  • the specifics of such formulation are disclosed in Schwartz, et al . , U. S. Patent No.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation
  • the compounds may be formulated with suitable polymeric or hydrophobic materials
  • the pharmacological compositions herein 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.
  • 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. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein and, with regard to BCNU, existing literature.
  • the dosage and interval should be adjusted individually to provide plasma levels of the compound at or above the amount necessary to maintain the PDGF-mediated signal transduction inhibiting effects of the compound.
  • HPLC assays or bioassays can be used to determine plasma concentrations .
  • Dosage intervals for MCTA can also be determined using plasma concentrations.
  • MCTA should be administered using a regimen which maintains plasma levels above, preferably well above, the level necessary to elicit its therapeutic effect for 10-90% of the time, preferably between 50-90% and most preferably between 70-90%.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • the amount of both MCTA and BCNU administered will, of course, be dependent on the subject being treated, on the subject's weight (from which body surface area is calculated), the severity of the cancer, the manner of administration and the judgment of the prescribing physician. Since MCTA is cytostatic rather than cytotoxic, it is a feature of this invention that MCTA be administered to a patient throughout all or a substantial portion of a course of treatment with the combination of MCTA and BCNU.
  • a "course of treatment” is defined as a time period over which a complete cycle of a pre-determined regimen of administration of MCTA and BCNU is performed.
  • such regime may entail simultaneous administration of MCTA and BCNU or sequential administration of the drugs in such order, dosages and intervals as are deemed most appropriate with regard to a particular patient by the treating physician.
  • MCTA may be administered prior to administration of BCNU.
  • BCNU can be administered before treatment with MCTA begins.
  • MCTA is administered to the patient in a "loading dose" prior to administration of BCNU.
  • a "loading dose” refers to an initial dose or doses of a drug given to create an initial level of the drug in the body of the patient prior to the administration of another drug.
  • the loading dose may be administered as a single dose, as a continuous infusion over time or as several discrete administrations of equal or unequal doses at discrete intervals.
  • the time period over which the loading dose is administered may vary as seen fit by the prescribing physician.
  • the loading dose is administered over a period of 3 to 6 days either as a continuous infusion or as equal doses given on each day of the loading dose period.
  • the loading dose may be from 100 to 550 mg/m 2 /day.
  • the loading dose is 200 to 500 mg/m 2 /day. Most preferably, it is from 350 - 450 mg/m 2 /day.
  • the exact amount of the loading dose would, of course, be determined by the treating physician on a case-by-case basis.
  • BCNU is administered on the day immediately following completion of the loading dose.
  • the BCNU may be administered in a single dose or in smaller doses given over a period of several days.
  • the amount of BCNU administered may be from 75 to 200 mg/m 2 . Actual choice of dosage is likewise within the province of the treating physician. Preferable, however, the BCNU is administered in a single dose of from 150 to 200 mg/m 2 .
  • the overall course of treatment may vary extensively depending on the judgment of the treating physician. Preferable it is from 6 to 8 weeks.
  • maintenance doses of MCTA are administered either for the entire duration of the course of treatment or for any portion thereof.
  • Such maintenance doses may be administered at intervals of from 2 to 10 days apart.
  • the maintenance doses are administered 6-7 days apart and the number of such maintenance doses is from 5 to 10, preferable at a rate of one maintenance dose per week for the entire course of treatment; e.g., if an 8 week course of treatment is selected, a maintenance dose of MCTA is administered during each of those weeks at approximately one-week intervals.
  • the amount of the maintenance dose would be selected by the treating physician but is preferably in the range of 100 to 555 mg/m 2 /week, more preferable 200 to 450 mg/m 2 /week and most preferably, from 350 to 450 mg/m 2 /week.
  • a “maintenance dose” refers to an additional dose or doses of the drug given initially as a loading dose, the purpose being to maintain the level of the drug or its metabolite achieved by the loading dose for an extended period of time.
  • the patient is evaluated and, if the indications, including but not limited to, positive response to the therapy and no signs of serious side effects, are appropriate in the eyes of the treating physician, the course of treatment may be repeated.
  • the number of repeat courses of treatment is left to the discretion of the treating physician and should be limited only by the determination by the treating physician that continued treatment could be detrimental to the patient.
  • dosages different from those described above may be administered and still be within the scope and spirit of this invention.
  • different routes of administration such as direct injection into a tumor or interstitial implantation would be expected to permit even lower doses than those discussed herein due to less likelihood of dilution and metabolic decomposition when the target is more directly addressed.
  • alkylating agents could include, without limitation, the alkyl sulfonates; e.g., busulfan (used for treatment of chronic granulocytic leukemia) , improsulfan and piposulfan; the aziridines; e.g., benzodepa, carboquone, meturedepa, and uredepa; the ethyleneimines and methylmelamines; e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine and the nitrogen mustards; e.g., chlorambucil (used in treatment of chronic lymphocytic leukemia, primary macroglobulinemia and non-Hodgkin ' s lymphoma) , cyclophosphamide (used in treatment of Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, Wilm's tumor and rhab
  • MCTA could have a beneficial effect in combination with the antimetabolite chemotherapeutic agents such as, without limitation, the folic acid analogs (e.g., methotrexate (used in treating acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, neck and head and lung cancer, osteogenic sarcoma) and pteropterin) ; the pyrimidine analogs (fluorouracil (5-FU) used in treatment of breast, colon, stomach, pancreatic, ovarian, head and neck, and urinary cancers as well as topically for premalignant skin lesions and cytarabine (acute granulocytic and acute lymphocytic leukemias) and the purine analogs such at mercaptopurine and thioguanine which find use in the treatment of acute granulocytic, acute lymphocytic and chronic granulocytic leukemias) .
  • the folic acid analogs
  • MCTA could also prove effective in combination with natural product chemotherapeutic agents such as, without limitation, the vinca alkaloids (vinblastine (used for breast and testicular cancer) , vincristine and vindesine) , the epipodophylotoxins (etoposide, teniposide (both used in the treatment of testicular cancer and Kaposi's sarcoma)), the antibiotic chemotherapeutic agents (daunorubicin, doxorubicin, bleomycin mitomycin (used for stomach, cervix, colon, breast, bladder and pancreatic cancer) ) , dactinomycin, plicamycin, bleomycin (used for skin, esophagus and genitourinary tract cancer) and the enzymatic chemotherapeutic agents such as L- Asparaginase .
  • the vinca alkaloids vinblastine (used for breast and testicular cancer) , vincristine and vindesine
  • MCTA might also benefit the activity of chemotherapeutic agents such as platinum coordination complexes (cisplatin, etc.), substituted ureas (hyroxyurea) , methylhydrazine derivatives (procarbazine) , adrenocortical suppressants (mitotane, aminoglutethimide) as well as hormones and antagonists such as adrenocorticosteroids
  • chemotherapeutic agents such as platinum coordination complexes (cisplatin, etc.), substituted ureas (hyroxyurea) , methylhydrazine derivatives (procarbazine) , adrenocortical suppressants (mitotane, aminoglutethimide) as well as hormones and antagonists such as adrenocorticosteroids
  • progestins hydroxyprogesterone caproate
  • estrogens diethylstilbestrol
  • antiestrogens tamoxifen
  • androgens testosterone propionate
  • Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect.
  • a measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index: IC50/LD50.
  • IC50 the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein.
  • LD 50/ the dosage which results in 50% toxicity can also be measured by standard techniques (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.
  • Compounds with a large therapeutic index are preferred.
  • the therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.
  • the compounds of this invention may be readily synthesized using techniques well known in the chemical arts. The following syntheses are shown by way of example only and are not to be construed as limiting in any way. In fact, it will be appreciated by those skilled in the art that other synthetic pathways for forming the compounds of the invention are available and that the following are but a few of the possible routes to the claimed compounds.
  • Ethyl propiolate (2.8 g) and pyrrolidine (1.4 g) in 5 ml of acetonitrile are mixed at room temperature for 1 hour, the solvent evaporated and the ethyl 3-pyrrolidin-l-acrylate used as isolated or distilled under vacuum.
  • Triethylamine (0.25 ml) is added to a mixture of 1.8 g of ethyl 3-pyrrolidin-l- acrylate, 0.9 g of nitroethane and 2.5 g of phenyl isothiocyanate in 10 ml of toluene at room temperature and stirred overnight. The mixture is then refluxed for 0.5 hour, cooled, and the diphenylurea removed by filtration.
  • Ethyl 3-methyl-4-isoxazolecarboxylate (1.3 g) is stirred at room temperature overnight in 5 ml of ethanol and 10 ml of 2.5 N sodium hydroxide. Dilution with water, cooling in ice, and acidification to pH 2 with 6 N hydrochloric acid precipitates an off-white solid which is collected by vacuum filtration, washed with ethanol/water , and dried under vacuum to give 1.0 g of 3-methyl-4-isoxazolecarboxylic acid.
  • 3-Methyl-4-isoxazolecarboxylic acid (0.9 g) is stirred with 5 ml of thionyl chloride at room temperature for one hour and the mixture evaporated to dryness.
  • N- [4- (Trifluoromethyl) phenyl] -2- (ethoxymethylene) - acetoacetamide (4 g) is suspended in 10 ml of ethanol treated with 1.5 g of hydroxylamine hydrochloride in 10 ml of water which has been adjusted to pH 5 with sodium hydroxide. The mixture is stirred and warmed to 40 °C for 2 hours, then cooled to room temperature and the precipitate collected by vacuum filtration. Sodium hydroxide (1 g) is added to the filtrate which is stirred for 30 minutes. The mixture is acidified to pH 2 with 6 N hydrochloric acid and the precipitate collected by vacuum filtration.
  • the filtrate is then diluted with 100 ml of water and allowed to stand at 4 °C overnight.
  • the precipitate is collected by vacuum filtration, washed with ethanol : ater 2:1 and dried to give 300 mg of crude 3-methyl-4- [4- (trifluoromethyl) phenylaminocarbonyl] isoxazole .
  • the crude is purified on a column of silica gel eluting with ethyl acetate : hexane 1:4 to give 100 mg of 3-methyl-4- [4- (trifluoromethyl) phenylaminocarbonyl] isoxazole, an off-white solid.
  • Table 1 is a comparison of the activity of MCTA, its metabolite and a compound of this invention as inhibitors of FGF induced DNA synthesis alone or with added uridine.
  • Table 2 shows the results of the ability of several of the compounds of this invention to inhibit DNA synthesis induced by FGF, PDGF and EGF.
  • Table 3 shows the results of a subcutaneous xenograft experiment testing the ability of Cmpd. 1 to inhibit tumor growth in vivo and, in addition, testing the toxic effects of Cmpd. 1 at the dose used.
  • Table 4 demonstrates that MCTA inhibits the specific biological response of cells to FGF while having no effect on the response of the cells to EGF. MCTA also inhibits the specific biological responses to PDGF. The results are shown as IC50s, the concentration at which 50% of the maximal BrdU incorporation response is inhibited.
  • Table 5 shows the results of subcutaneous xenograft experiments wherein administration of MCTA significantly inhibited the growth of tumors associated with FGFR activity with no significant effect on a non-FGFR expressing tumor thus demonstrating MCTA's effectiveness as a treatment for cancers characterized by inappropriate FGFR activity.
  • Figure 1 graphically depicts the effect over time of 20, 10, 5.0 and 2.5 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide alone on tumor weight compared to that in the untreated controls.
  • Figure 2 is a graph of the effect over time of 27, 18, 12 and 8.0 mg/kg BCNU alone on tumor weight compared to the untreated control .
  • Figure 3 is a graphical comparison of the effect over time of 10.0 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide alone, 18 mg/kg BCNU alone and the combination of 10.0 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide with 18 mg/kg BCNU, administered as described in Section 4, below, compared to the control.
  • Figure 4 is a graph of the effect over time on tumor weight of 10 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide alone, 12 mg/kg BCNU alone and the combination of 10 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide with 12 mg/kg BCNU, again, compared to the untreated control .
  • Figure 5 graphically compares the effect over time on tumor weight of 5 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide alone, 18 mg/kg BCNU alone, the combination, in the manner described in Section 4, below, of 5 mg/kg N- (4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide with 18 mg/kg BCNU and the untreated control.
  • Figure 6 is a graph depicting the effect over time on tumor weight of 5.0 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide alone, 12 mg/kg BCNU alone, the combination of 5.0 mg/kg N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide with 12 mg/kg BCNU, compared as before with the untreated control.
  • this invention relates to novel heteroarylcarboxamides demonstrating the ability to modulate PTK activity.
  • the following assays are employed to select those compounds demonstrating the optimal degree of the desired activity.
  • the phrase "optimal degree of the desired activity" refers to the lowest IC50, defined elsewhere herein, against a PTK related to a particular disorder so as to provide an organism, preferably a human, with a therapeutically effective amount of a compound of this invention at the lowest possible dosage.
  • vi tro assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the RTKs. Similar assays can be designed along the same lines for any PTK using techniques well known in the art.
  • the cellular/catalytic assays described herein are performed in an ELISA format.
  • the general procedure is a follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor's activity is added. The cells are lysed and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody recognizing the substrate of the enzymatic phosphorylation reaction. Non-substrate components of the cell lysate are washed away and the amount of phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine compared with control cells that were not contacted with a test compound.
  • the cellular/biologic assays described herein measure the amount of DNA made in response to activation of a test kinase, which is a general measure of a proliferative response.
  • the general procedure for this assay is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor's activity is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or 3H-thymidine is added. The amount of labeled DNA is detected with either an anti-BrdU antibody or by measuring radioactivity and is compared to control cells not contacted with a test compound.
  • a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or 3H-thymidine is added.
  • Enzyme linked immunosorbent assays may be used to detect and measure the presence of PTK activity.
  • the ELISA may be conducted according to known protocols which are described in, for example, Voller, et al . , 1980, "Enzyme-Linked Immunosorbent Assay," In: Manual of Clinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C.
  • the disclosed protocol may be adapted for determining activity with respect to a specific RTK.
  • the preferred protocols for conducting the ELISA experiments for specific RTKs is provided below. Adaptation of these protocols for determining a compound's activity for other members of the RTK family, as well as for CTKs, is well within the scope of knowledge of those skilled in the art.
  • An ELISA assay can be conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1.
  • a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96) ; b. Cappel goat anti-rabbit IgG (catalog no. 55641); c. PBS (Gibco Catalog No. 450-1400EB) ; d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20) ; e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4°C) ; f.
  • HNTG buffer (20mM HEPES buffer (pH 7.5), 150mM NaCl, 0.2% Triton X-100, and 10% glycerol) ; g. EDTA (0.5 M (pH 7.0) as a 100X stock); h. Sodium ortho vanadate (0.5 M as a 100X stock); i. Sodium pyro phosphate (0.2M as a 100X stock); j . NUNC 96 well V bottom polypropylene plates (Applied Scientific Catalog No. AS-72092); k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);
  • DMEM with IX high glucose L Glutamine (catalog No. 11965-050) ; m. FBS, Gibco (catalog no. 16000-028); n. L-glutamine, Gibco (catalog no. 25030-016); o. VEGF, PeproTech, Inc. (catalog no. 100-20) (kept as 1 ⁇ g/lOO ⁇ l stock in Milli-Q dH 2 0 and stored at -20°C; p. Affinity purified anti-FLK-1 antiserum; q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et al . , 1990, Cancer Research 50:1550-1558) ; r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011); s. 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid
  • ABTS ABTS solution
  • ABTS lOOmM citric acid (anhydrous), 250 mM Na 2 HP0 4 (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A-1888)
  • solution should be stored in dark at 4°C until ready for use; t. H 2 0 2 (30% solution) (Fisher catalog no. H325); u. ABTS/H 2 0 2 (15ml ABTS solution, 2 ⁇ l H 2 0 2 ) prepared 5 minutes before use and left at room temperature; v. 0.2 M HC1 stock in H 2 0; w. dimethylsulfoxide (100%) (Sigma Catalog No. D-8418); and y.
  • HNTG formulation includes sodium orthovanadate, sodium pyrophosphate and EDTA.
  • Assay 1 EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells.
  • HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described below: Materials and Reagents .
  • b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain) .
  • Anti-phosphotyrosine antibody (anti-Ptyr) poly- clonal) ⁇ see, Fendley, et al . , supra) .
  • Detection antibody Goat anti-rabbit IgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
  • Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 g per well in PBS, 100 ⁇ l final volume/well, and store overnight at 4°C. Coated plates are good for up to 10 days when stored at 4°C.
  • An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER2 kinase domain can be used for this assay.
  • 2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, at room temperature for 5 minutes . 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable.
  • EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 ⁇ l dilute EGF (1:12 dilution), 100 nM final concentration is attained.
  • HNTG * (10 ml) HNTG stock 2.0 ml milli-Q H 2 0 7.3 ml
  • the maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.
  • All cell culture media, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37°C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
  • cells (U1242, obtained from Joseph Schlessinger, NYU) are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium, cells are changed to serum-free medium and treated with test compound for 2 hr in a 5% C0 2 , 37 °C incubator.
  • Cells are then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 V0 4 , 0.2% Triton X-100, and 2 mM NaPyr) .
  • Cell lysates (0.5 mg/well in PBS) are transferred to ELISA plates previously coated with receptor-specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) at room temperature for 30 min. Lysates are incubated with shaking for 1 hour at room temperature.
  • the plates are washed with TBST four times and then incubated with polyclonal anti- phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody was added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times.
  • the following protocol may be used to measure phosphotyrosine level on IGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity.
  • Materials And Reagents The following materials and reagents are used: a. The cell line used in this assay is 3T3/IGF-1R, a cell line genetically engineered to overexpresses IGF-1 receptor. b. NIH3T3/IGF-1R is grown in an incubator with 5% C0 2 at 37°C. The growth media is DMEM + 10% FBS (heat inactivated) + 2mM L-glutamine. c. Affinity purified anti-IGF-lR antibody 17-69. d. D-PBS: KH 2 P0 4 0.20 g/1
  • Blocking Buffer TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk) .
  • TBST buffer 5% Milk (Carnation Instant Non-Fat Dry Milk) .
  • EDTA/HC1 0.5 M pH 7.0 (NaOH) as 100X stock.
  • Na 3 V0 4 0.5 M as 100X stock and aliquots are kept in -80°C.
  • Na 4 P 2 0 7 0.2 M as 100X stock, k.
  • ABTS solution should be kept in dark and 4°C. The solution should be discarded when it turns green.
  • Hydrogen Peroxide 30% solution is kept in the dark and at 4°C. Procedure. All the following steps are conducted at room temperature unless it is specifically indicated. All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. Pat plate dry with paper towels.
  • the cells grown in tissue culture dish (Corning 25020-100) to 80-90% confluence, are harvested with Trypsin-
  • EDTA 0.25%, 0.5 ml/D-100, GIBCO.
  • the drugs are tested in serum-free condition.
  • EGF Receptor kinase activity in cells genetically ⁇ engineered to express human EGF-R is measured as described below:
  • UBI 05-101
  • Anti-pnosphotyosine antibo ⁇ y anti-Ptyr
  • Detection antibody Goat anti-rabbit IgG horse 5 radish peroxidase conjugate, TAGO, Inc., Burlmgame,
  • Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 ⁇ g per well in PBS, 150 ⁇ l final volume/well, and store overnight at 4°C. Coated plates are good for up to 10 days when stored at 4°C.
  • NIH 3T3/C7 cell line (Honegger, et al . , Cell 51:199- 209, 1987) can be use for this assay.
  • DMEM seeding medium
  • seeding medium 0.5% bovine serum
  • seed cells m DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 ⁇ l per well, in a 96 well microtiter plate. Incubate seeded cells in 5% C0 2 at 37°C for about 40 hours.
  • EGF ligand dilute stock EGF m DMEM so that upon transfer of 10 ⁇ l dilute EGF (1:12 dilution), 25 nM final concentration s attained.
  • HNTG * comprises: HNTG stock (2.0 ml), milli-Q H 2 0
  • EGF ligand to cells, 10 ⁇ l per well, to yield a final concentration of 25 nM .
  • Control wells receive DMEM alone. IncuDate, shaking, at room temperature, for 5 minutes. 6.
  • the maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.
  • PDGF human PDGF B/B; 1276-956, Boehringer Mannheim, Germany
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • PBS Washing Solution IX PBS, pH 7.4, made in house .
  • Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gin in a 96 well plate. Cells are incubated overnight at 37 °C in 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the blocking solution is removed by decanting and the wells are washed once with PBS.
  • Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
  • the antibody con ugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • the absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at
  • EGF mouse EGF, 201; Toyobo,Co., Ltd. Japan
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidme (TMB), ready to use, Cat. No. 1 647 229, Boehringer
  • PBS Washing Solution IX PBS, pH 7.4, made in house .
  • Cells are seeded at 8000 cells/well in 10% CS, 2mM Gin in DMEM, n a 96 well plate. Cells are mcuDated overnight at 37°C in 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
  • Test compounds are prepared m serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added
  • EGF mouse EGF, 201; Toyobo,Co., Ltd. Japan
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannneim, Germany.
  • TMB Substrate Solution tetramethylbenzidme (TMB) , ready to use, Cat. No. 1 647 229, Boehringer
  • PBS Washing Solution IX PBS, pH 7.4, made in house .
  • 3T3 cell line engineered to express a chimeric receptor having the extra-cellular domain of EGF-R and the mtra-cellular domain of Her2. Protocol :
  • Cells are seeded at 8000 cells/well m DMEM, 10% CS, 2mM Gin in a 96- well plate. Cells are incubated overnight at 37° m 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then are serum starved m serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
  • Test compounds are prepared m serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the medium is removed by decanting and tapping the inverted plate on a paper towel.
  • FixDenat solution is added (50 ⁇ l/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
  • the FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.
  • the blocking solution is removed by decanting and the wells are washed once with PBS.
  • Anti-BrdU-POD solution (1:100 dilution m PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
  • the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • the absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at
  • IGF1 Ligand human, recombinant; G511, Promega Corp, USA.
  • TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer
  • Cells are seeded at 8000 cells/well m DMEM, 10% CS, 2mM Gin in a 96-well plate. Cells are incubated overnight at 37°C in 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
  • test compounds are added to the cells simultaneously.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the following protocol may also be used to measure a compound's activity against PDGF-R, FGF-R or Flk-1/KDR, all of which are naturally expressed by HUV-EC cells.
  • HUV-EC-C cells human umbilical ve n endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL) . Wash with Dulbecco's pnosphate- buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at aoout 1 ml/10 cm 2 of tissue culture flask. Trypsinize with 0.05% trypsm-EDTA n non-enzymatic cell dissociation solution (Sigma Chemical Company; catalogue no. C-1544) .
  • D-PBS Dulbecco's pnosphate- buffered saline
  • the 0.05% trypsm was made by diluting 0.25% tryps ⁇ n/1 mM EDTA (Gibco; catalogue no. 25200-049) in the cell dissociation solution. Trypsinize w th about 1 ml/25-30 cm 2 of tissue culture flask for about 5 minutes at 37°C. After cells nave detached from the flask, add an equal volume of assay medium and transfer to a 50 ml sterile centrifuge tube (Fisher Scientific; catalogue no. 05-539-6).
  • diluent made up to 2% DMSO in assay medium (F12K + 0.5% fetal bovine serum) is used as diluent for the drug titrations in order to dilute the drug but keep the DMSO concentration constant.
  • VEGF vascular endothelial cell growth factor
  • aFGF acidic fibroblast growth factor
  • RPMI heat-inactivated fetal bovine serum
  • Day 4 Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester 96 (R) ) onto filter mats (Wallac; catalogue no. 1205-401); read counts on a Wallac Betaplate (TM) liquid scintillation counter.
  • Tomtec Harvester 96 R
  • filter mats Wallac; catalogue no. 1205-401
  • TM Wallac Betaplate
  • Xenograft Animal Models The ability of human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful A. vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice.
  • human tumor cell lines e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas
  • the following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention.
  • Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and i ⁇ vivo .
  • the object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell.
  • the object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell.
  • the object of the Al vivo assay is to determine the effect of a compound in an animal model of a particular disorder such as cancer.
  • Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619) , A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435) and NIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-IR or any other test kinase.
  • the following protocol can be used to perform xenograft experiments :
  • mice Female athymic mice (BALB/c, nu/nu) are obtained from BALB/c, nu/nu.
  • Alpha-dri bedding They receive sterile rodent chow and water ad libitum.
  • Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM glutamine (GLN) ) . All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37°C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
  • appropriate medium for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)
  • All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in
  • Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) to a particular concentration and the cells are implanted into the hindflank of the mice (8 - 10 mice per group, 2 - 10 x 10 6 cells/animal) . Tumor growth is measured over 3 to 6 weeks using venier calipers. Tumor volumes are calculated as a product of length x width x height unless otherwise indicated. P values are calculated using the Students' t-test. Test compounds in 50 - 100 ⁇ L excipient (DMSO, or VPD:D5W) was delivered by IP injection at different concentrations generally starting at day one after implantation.
  • DMSO DMSO, or VPD:D5W
  • Tumor Invasion Model The following tumor invasion model has been developed and maybe used for the evaluation of therapeutic value and efficacy of the compounds identified to selectively inhibit KDR/FLK-1 receptor . Procedure
  • 8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells was performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg) are administered intraperitoneally . A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10 7 tumor cells in a volume of 100 ⁇ l medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin was closed by using would clips. Animals are observed daily .
  • mice After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry and iri situ hybridization) .
  • the following example illustrates the ability of the compounds of the invention to inhibit FGFR-stimulated and PDGFR-stimulated DNA synthesis in cells.
  • DNA synthesis is required for many of the activities of FGFR and PDGFR including, but not limited to, cell proliferation.
  • Uridine is added in one set of samples to overcome any contribution made by inhibition of DHOD and just evaluate the inhibition of PDGFR or FGFR signaling. (See Greene, et al . , Biochem. Pharmacol., 50(6) :861 (1995), Nair, et al., Immunology Letters, 47:171 (1995) ) .
  • EGF mouse EGF, 201; Toyobo,Co., Ltd. Japan; PDGF, Boehringer Mannheim, Germany; FGF, Gibco.
  • BrdU Labeling Reagent 10 mM, in PBS (pH7.4),Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • PBS Washing Solution IX phosphate buffered saline, pH 7.4
  • NIH3T3 clone C7 ( 3T3/EGFRc7 ) (Honegger et al., Cell 51:199-209, 1987) engineered to over-express human
  • EGF receptor EGF receptor. These cells natively express FGFR and PDGFR.
  • 3T3/EGFRc7 cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gin in a 96 well plate. Cells are incubated overnight at 37° C in 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.
  • test compound (final concentration) uridine) and test compound is added to the cells simultaneously.
  • the negative control wells received serum free DMEM with 0.1% BSA only; the positive control cells received ligand but no test compound.
  • Test compound is prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate was incubated for 30 minutes at room temperature on a plate shaker.
  • the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate dried by inverting and tapping oh a paper towel.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development was sufficient for photometric detection.
  • the following example demonstrates the ability of Cmpd. 1 to inhibit the A. vivo growth of tumors characterized by inappropriate FGFR and/or PDGFR activity.
  • the FGFR expressing cells are from two glioblastomas (C6, ATCC CRL 107, Powell and Klagsbrun, Exp. Cell Res., 209:224 (1993); for PDGFR, see Strawn, et al . , J. Biol. Chem., 269:21215 (1995).
  • mice Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories (Gilroy, CA) . All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They received sterile rodent chow and water ad. libitum.
  • Cell lines are grown in Ham's F10 plus 5% fetal bovine serum (FBS) and 2 mM glutamine (GLN) . All cell culture media, glutamine, and fetal bovine serum is purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37 °C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
  • Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) and the cells are implanted into the hindflank of the mice (8 - 10 mice per group, 3 x 10 6 cells/animal) . Tumor growth was measured over 3 weeks using venier calipers. Tumor volumes are calculated as a product of length x width x height unless otherwise indicated. P values are calculated using the Students' t-test. Cmpd. 1 in 50 ⁇ L excipient (DMSO) was delivered by IP bolus injection daily.
  • DMSO excipient
  • the following example can be used to test the ability of the compounds of the invention to inhibit growth and metastasis of a tumor cell line expressing FGFR and PDGFR (C6 cells) .
  • mice Ten to 12 week old athymic Balb/c nu/nu mice are obtained from Simonsen Laboratory (Gilroy, CA) and maintained in a pathogen-free environment throughout the experiments.
  • C6 cells (ATCC CCL 107) are grown and maintained in F-10 medium (Life Technologies, Inc. Grand Island, NY) supplemented with 10% fetal bovine serum, 2mM glutamine in a 5% C02 environment. Approximately 80% confluent cultures are harvested by brief trypsinization (0.0625% trypsin-0.25 mM EDTA in Cell Dissociation Medium) (Life Technologies) and resuspended at a final concentration of 8 x 10 7 cells per ml in magnesium and calcium free phosphate buffered saline for implantation. Cell viability is determined by Trypan blue exclusion and found to be >95% .
  • compositions herein described inhibit the activity of FGFR in cells and can be used to reduce tumor growth and inhibit metastasis.
  • the following example illustrates the ability of MCTA to inhibit ligand-stimulated DNA synthesis in cells, specifically when stimulated with FGF or PDGF but not when stimulated with
  • FGFR including, but not limited to, cell proliferation.
  • Uridine is added to remove the effect of a metabolite of MCTA.
  • EGF mouse EGF, 201; Toyobo,Co., Ltd. Japan, PDGF, Boehringer Mannheim, Germany, FGF, Gibco, USA
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • 3T3/EGFRc7 cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gin in a 96 well plate. Cells are incubated overnight at 37 °C in 5% C0 2 .
  • test compound (final concentration) uridine) and test compound was added to the cells simultaneously.
  • the negative control wells received serum free DMEM with 0.1% BSA only; the positive control cells received ligand (EGF, PDGF or FGF) but no test compound.
  • Test compound was prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations .
  • FixDenat solution was thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate was incubated for 30 minutes at room temperature on a plate shaker.
  • the antibody conjugate was thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate dried by inverting and tapping on a paper towel.
  • TMB substrate solution was added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development was sufficient for photometric detection.
  • the following example demonstrates the ability of MCTA to inhibit the _in vivo growth of tumors characterized by inappropriate FGFR activity while having no inhibitory effect on a tumor that does not express FGFR.
  • the FGFR expressing cells are from a prostate cancer (PC3, Nakamoto, et al . , Cancer Res . 52:571, 1992).
  • the non-FGFR-expressing cell line was obtained from an epidermoid carcinoma (A431, ATTC CRL 1555, Takahashi, et al . , supra) .
  • mice Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories (Gilroy, CA) . All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They received sterile rodent chow and water ad libitum.
  • Cell lines are grown in appropriate medium (A431 - DMEM with 10% FBS and 2 mM GLN; PC3 - HAM'S F12 with 7% FBS and 2 mM GLN) . All cell culture media, glutamine, and fetal bovine serum is purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37°C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
  • Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) to a particular concentration and the cells are implanted into the hindflank of the mice (8 - 10 mice per group) . Tumor growth was measured over 3 to 6 weeks using venier calipers . Tumor volumes are calculated as a product of length x width x height unless otherwise indicated. P values are calculated using the Students' t-test. MCTA in 50 - 100 ⁇ L excipient . (DMSO, or VPD:D5W) was delivered by IP injection at different concentrations . Re sult s
  • the following example illustrates the ability of MCTA to inhibit growth and metastasis of a tumor cell line expressing FGFR (C6 cells) .
  • mice Ten to 12 week old athymic Balb/c nu/nu mice are obtained from Simonsen Laboratory (Gilroy, CA) and maintained in a pathogen-free environment throughout the experiments.
  • C6 cells (ATCC CCL 107) are grown and maintained in F-10 medium (Life Technologies, Inc. Grand Island, NY) supplemented with 10% fetal bovine serum, 2mM glutamine in a 5% C02 environment. Approximately 80% confluent cultures are harvested by brief trypsinization (0.0625% trypsin-0.25 mM EDTA in Cell Dissociation Medium) (Life Technologies) and resuspended at a final concentration of 8 x 10 7 cells per ml in magnesium and calcium free phosphate buffered saline for implantation. Cell viability was determined by Trypan blue exclusion and found to be >95%.
  • BCNU is commercially available.
  • One source is Bristol Laboratories Oncology Products, Princeton, NJ, under the trade name BiCNU G .
  • SF763T cells derived from human glioblastoma cells, are used in all of the examples shown below. 1X10 7 cells per mouse are implanted in 100 ⁇ l PBS subcutaneously into the hindflanks of female BALC/c nu/nu mice. Tumors are allowed to establish before dosing was initiated.
  • N- ( 4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide was administered IP in VDP:D5W (VDP: 3% benzyl alcohol (w/v), 8% Tween 80 (v/v) , 65% PEG 300 (w/v), QS to 100% with 100% ethanol; D5W: 5% dextrose (w/v) in water) at 2.5, 5.0, 10 and 20 mg/kg from day 8 to day 29 post implantation.
  • BCNU was administered IV in 2% ethanol/saline at 8 , 12, 18 and 27 mg/kg on days 8, 12 and 16 post implantation.
  • Combinations of N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide at 2.5, 5.0 and 10 mg/kg and BCNU at 8, 12 andl ⁇ mg/kg were also administered with the same regimens.
  • Tumor weight is determined twice per week until day 43 post implantation.
  • the size of the tumor is determined by measuring two of its dimensions, length and width, by deducing its depth using procedures well known to those skilled in the art, and calculating its weight based on standard assumptions regarding the density of such tumors.
  • Example 1 the results of which can be seen in graphical form as the CONTROL in each of Figures 1-6 shows tumor growth in untreated mice. All subsequent examples are compared to this control for determination of whether statistically significant inhibition is being shown by either N-(4- trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide or BCNU alone or by various combinations of these two drugs.
  • N-(4- trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide is in fact cytostatic rather than cytotoxic; i.e., N-(4- trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide temporarily stops cell activity but does not kill the cell .
  • Example 9 This example, the results of which are also shown graphically in Figure 1, shows the results of N-(4- trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide alone at 10 mg/kg. Again, statistical analysis of the raw data, not shown, revealed that the percentage inhibition is less than with 20 mg/kg but inhibition continued throughout the time period during which N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide is being actively administered and then, when treatment with N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide ceased, tumor growth resumed.
  • Fig. 1 shows the effect of 5 mg/kg N-(4- trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide . Again, the percentage inhibition is less but, when the raw data is statistically analyzed using the Student's T Test, still statistically significant.
  • Example 14 At 12 mg/kg BCNU is found to be ineffective; i.e., no statistically significant percentage inhibition is observed from the raw data. Graphically, it can be seen in Fig. 2 that the 12 mg/kg curve shows some inhibition in the days immediately post treatment but as the days post-treatment increassed, the curve approached that of the control.
  • Example 16 When N- (4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide is administered at 10 mg/kg and BCNU at 18 mg/kg, the result is very positive.
  • Statistical analysis of the raw data showed that the overall percentage inhibition is substantially higher than treatment with BCNU alone at 18 mg/kg or N- (4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide alone at 10 mg/kg. Furthermore, the time period over which statistically significant inhibition occurred is much longer than that observed with either drug alone.
  • Fig. 3 graphically displays this result.
  • the compounds, methods and pharmacological compositions of the present invention modulate PTK activity and therefore are expected to be effective as therapeutic agents against PTK-related disorders.
  • the heteroarylcarboxamide, N- ( 4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide (“MCTA”) is particularly useful for the treatment of disorders characterized by inappropriate FGFR activity and is expected to be especially useful in the treatment of invasive cancers noted for their aggressiveness and tendency to metastatize.
  • N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide together with a chemotherapeutic nitrosourea, in particular BCNU, is an effective tumor suppressant for human glioblastoma tumor cells at dosages substantially lower than that necessary when either of the compounds is used alone and that it is therefore expected that this combination will have similarly positive results in human subjects.

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Abstract

Nouveaux hétéroarylcarboxamides qui modulent l'activité des protéine tyrosine kinases et dont on pense qu'ils sont utiles dans le traitement d'états pathologiques dus à l'activité anormale des protéine tyrosine kinases, méthodes de traitement d'états pathologiques liés l'activité inappropriée des récepteurs de facteur croissance des fibroblastes (FGFR) à l'aide de l'hétéroarylcarboxamide N-(4-trifluorométhyl-phényl)-5-méthylisoxazole-4-carboxamide et traitement de cancers à tumeurs solides, en particulier le glioblastome et l'astrocytome, avec une combinaison de nitrosourée, de préférence BCNU (carmustine), et de N-(4-trifluorométhyl-phényl)-5-méthylisoxazole-4-carboxamide.
EP98924794A 1997-05-19 1998-05-18 Composes d'heteroarylcarboxamide actifs contre les etats pathologiques lies aux proteine tyrosine kinases Withdrawn EP1012150A4 (fr)

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US7125875B2 (en) 1999-04-15 2006-10-24 Bristol-Myers Squibb Company Cyclic protein tyrosine kinase inhibitors
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US7977333B2 (en) 2000-04-20 2011-07-12 Bayer Healthcare Llc Substituted pyridines and pyridazines with angiogenesis inhibiting activity
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EP1990048A3 (fr) * 2007-05-08 2009-05-06 Ulrike Wiebelitz Thérapie pour l'hyperplasie bénigne de la prostate
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US20140179712A1 (en) 2012-12-21 2014-06-26 Astrazeneca Ab Pharmaceutical formulation of n-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2h-pyrazol-3-yl]-4-[(3r,5s)-3,5-dimethylpiperazin-1-yl]benzamide
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