WO1999027921A2

WO1999027921A2 - Compositions and methods for modulating the activity of fibroblast growth factor

Info

Publication number: WO1999027921A2
Application number: PCT/JP1998/005440
Authority: WO
Inventors: Takatoshi Kawai; Ramnarayan Kalyanaraman
Original assignee: Eisai Co., Ltd.
Priority date: 1997-12-03
Filing date: 1998-12-02
Publication date: 1999-06-10
Also published as: AU1350699A; WO1999027921A3

Abstract

Methods for using bis-sulfonamide compounds of formula (I) and pharmaceutical compositions containing the compounds of formula (I) or pharmaceutically acceptable derivatives thereof for modulating the activity of the FGF family of peptides are provided. Methods for inhibiting the binding of an FGF peptide to an FGF receptor by contacting the receptor with a bis-sulfonamide compound of formula (I) are provided. Methods for treating FGF-mediated disorders by administering effective amounts of one or more of these bis-sulfonamides or prodrugs thereof that inhibit the activity of one or more FGF peptide are also provided.

Description

DESCRIPTION

COMPOSITIONS AND METHODS FOR MODULATING THE ACTIVITY OF

FIBROBLAST GROWTH FACTOR

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions and methods for modulating the activity of the fibroblast growth factor (FGF) family of peptides. BACKGROUND OF THE INVENTION

During the last thirty years, a great deal of attention has been directed towards the identification and characterization of factors that stimulate the growth, proliferation and differentiation of specific cell types. Numerous growth factors and families of growth factors that share structural and functional features have been identified. Many of these factors have multifunctional activities and affect a wide spectrum of cell types. Fibroblast growth factors and fibroblast growth factor receptors One family of growth factors that has a broad spectrum of activities is the fibroblast growth factor (FGF) family [e.g., see Johnson et al. . Advan. Cancer Res. 60: 1 -41 ( 1 993)] . This family of proteins includes FGFs designated FGF- 1 through FGF- 1 0 (or acidic FGF (aFGF), basic FGF (bFGF) , int-2, hst/K-FGF, FGF-5, FGF-6, keratinocyte growth factor (KGF), FGF-8, FGF-9 and FGF-1 0, respectively) . Acidic and basic FGF, which were the first members of the FGF family that were characterized, are about 55 % identical at the amino acid level and are highly conserved among species. Basic FGF has a molecular weight of approximately 1 6 kD, is basic and temperature sensitive and has a high isoelectric point [pi = 9.6; e.g . , see in The Cytokine acfsBook, Callard and Gearing, eds. , p.1 21 , Academic Press, Inc., London ( 1 994)] . Acidic FGF has an acidic isoelectric point with a pi of about 5.4. The other members of the FGF family have subseguently been identified on the basis of amino acid sequence homologies with aFGF and bFGF and common physical and biological properties. These proteins are widely distributed in tissues, such as the central and peripheral nervous system, retina, kidney and myocardium. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. They are also important in differentiation and development. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such effects have stimulated considerable interest in FGFs as therapeutic agents, for example, as pharmaceuticals for wound healing, neovascularization, nerve regeneration and cartilage repair.

In addition to potentially useful proliferative effects, FGF-induced mitogenic stimulation may, in some instances, be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes. Because FGFs are associated with many disease states, they are therapeutic targets. For example, antagonists of bFGF activity and/or aFGF or other FGFs should have a therapeutic use in treatment of tumorigenic conditions, restenosis, and other such conditions in which an FGF peptide plays a pathogenic role.

The effects of FGFs are mediated by high affinity receptor tyrosine kinases on the cell surface membranes or FGF-responsive cells [e.g . , see Lee et al. , ( 1 989) Science 245, 57-60; Imamura et al. , B.B.R.C. 1 55 , 583-590 ( 1 989) ; Huang and Huang, ( 1 986) J . Biol. Chem. , 261 , 9568- 9571 ; Moscatelli, ( 1 987) J . Cell . Phvsiol.. 1 31 , 1 23-1 30; U .S. Patent No. 5,288,855] . Lower affinity receptors also play a role in mediating FGF activities. The high affinity receptor proteins constitute a family of structurally related FGF receptors. Four FGF receptor genes have been identified and at least two of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript [e.g . , see U.S. Patent No. 5,288,855; Kiefer et aL., ( 1 991 ) Growth Factors 5: 1 1 5- 1 271. This splicing potentially creates a large number of different molecular forms that can interact with FGF family members, thereby permitting cells to respond to different FGF family members. For example, alternative splicing of a single gene results in the receptor FGFR2, which has high affinity for acidic and basic FGFs but no detectable affinity for KGF, and the KGF receptor, which has high affinity for KGF but reduced affinity for basic FGF. Similarly, alternative splicing of FGFR1 produces variants that have about a 50-fold decreased the affinity for basic FGF, but unchanged acidic FGF binding .

Receptor expression is altered by physical, chemical, and hormonal injury as well as in certain pathological conditions such as restenosis, tumors and selected proliferative diseases. Receptor messenger RNA and protein is expressed in melanoma cells (see, e.g . , Becker et a_L ( 1 992) Oncogene 7 : 2303-231 3) . The receptor message is not normally expressed in the palmar fascia, but is present in the proliferative hand disease Dupuytren's Contracture, (see, e.g . , Gonzales et a_L ( 1 992)

Amer. J . Pathol. 1 41 : 61 -671 ) . Quiescent smooth muscle cells (SMCs) do not respond to bFGF, but proliferating SMCs, in a model of restenosis after balloon angioplasty, strongly respond to exogenous bFGF (see, e.g . , Casscells et aL ( 1 992) Proc. Natl . Acad . Sci. U.S.A. 89:71 59-71 63) . Thus, there is a need to develop FGF-specific pharmacological products that modulate the activity of one or more FGF peptides. Therefore it is an object herein to provide compounds and compositions containing the compounds that modulate the activity of FGF peptides. It also an object herein to provide methods for modulating the activity of FGF peptides. It is also an object herein to provide methods ameliorating conditions, such as restenosis, tumoπgenesis and other conditions involving angiogenesis and undesirable proliferation of fibroblasts. SUMMARY OF THE INVENTION Pharmaceutical compositions and methods for modulating the activity of an FGF peptide using the compounds of formula (I) are provided . In particular, the pharmaceutical compositions contain and the methods use the bis-sulfonamide compounds of formula (I) : Ar SO- N — Y — N — SO- Ar, I I

R, R₂

or a pharmaceutically acceptable derivative of a compound of formula (I), wherein:

Ar, and Ar₂ are each independently selected from cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that is unsubstituted or substituted with one or more substituents designated Q; each Q is selected from halogen, hydroxy, nitπle, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, ammo, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkyla ino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diary laminosulfonyl;

R, and R₂ are each independently H or lower alkyl; and Y is selected from hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene group.

The compositions are formulated for local and systemic administration. In particular, compositions formulated for oral, intravenous, local and topical application are provided . Methods of modulating the activity of an FGF peptide, particularly basic FGF (FGF-2) . Such modulation provides a means for treatment of FGF-mediated disorders. In particular methods of treatment of restenosis, tumors, rheumatoid arthritis, ophthalmic disorders, proliferative diabetic retinopathies and other complications of diabetes, proliferative disorders and other diseases in which FGF-mediated physiological responses are implicated, using compositions containing compounds of formula (I) are provided.

The methods provided herein use compositions containing therapeutically effective concentrations of the compounds of formula (I) formulated for oral, intravenous, local and topical application. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference herein.

As used herein, an effective amount of a compound for treating a disorder is an amount that is sufficient to ameliorate, or in some manner reduce a symptom or stop or reverse progression of a condition. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.

As used herein, an FGF-mediated disorder is a disorder in which FGF causes or contributes to the pathology. Such disorders include, but are not limited to: restenosis, diabetic retinopathies, tumorigenesis, ophthalmic disorders and other proliferative disorders, including dermatological disorders, such as psoriasis.

As used herein, treatment means any manner in which the symptoms or pathology of a condition, disorder or disease are ameliorated or otherwise beneficially altered . Treatment also encompasses any pharmaceutical use of the compositions herein.

As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition .

As used herein, local application or administration refers to administration of an anti-hyperalgesic agent to the site, such as an inflamed joint, that exhibits the hyperalgesic condition and that does not exert central analgesic effects. Such local application includes intrajoint, such as intra-articular application, via injection, application via catheter or delivery as part of a biocompatible device.

As used herein, topical application refers to application to the surface of the body, such as to the skin, eyes, mucosa and lips, which can be in or on any part of the body, including but not limited to the epidermis, any other dermis, or any other body tissue. Topical administration or application means the direct contact of the anti- hyperalgesic with tissue, such as skin or membrane, particularly the cornea, or oral, vaginal or buccal mucosa. Topical administration also includes application to hardened tissue such as teeth and appendages of the skin such as nails and hair. A composition formulated for topical administration is generally liquid or semi-liquid carriers such a gel, lotion, emulsion, cream, plaster, or ointment, a spray or aerosol, or a "finite" carrier, L^, a non-spreading substance that retains its form, such as a patch, bioadhesive, dressing and bandage. It may be aqueous or non- aqueous; it may be formulated as a solution, emulsion or a suspension.

As used herein, biological activity refers to the in. vivo activities of a compound or physiological responses that result upon |n vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activity may be detected by in vitro assays, such as those described herein. As used herein, pharmaceutically acceptable derivatives of the compounds include any salts, esters, acids, bases, hydrates, solvates or prodrugs that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, hydroxy groups can be esterified or etherified. Pharmaceutically-acceptable salts, include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, 1 -para-chlorobenzyl-2-pyrrolidin-1 '-ylmethyl- benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography [TLC] , gas chromatography [GC], gel electrophoresis and high performance liquid chromatography [HPLC], used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound .

As used herein, adequately pure or "pure" per se means sufficiently pure for the intended use of the adequately pure compound .

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound . To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, once a pharmaceutically active compound is identified, those of skill in the pharmaceutical art generally can design prodrugs of the compound [see, e.g . , Nogrady ( 1 985)

Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392] .

As used herein, the IC₅₀ refers to an amount, concentration or dosage of a particular compound that achieves a 50% inhibition of a maximal response.

As used herein, EC₅₀ refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound. As used herein, an FGF antagonist is a compound, such as a drug or an antibody, that inhibits FGF-mediated physiological responses. The antagonist may act by interfering with the interaction of FGF with the FGF-specific receptor or by interfering with the physiological response to or bioactivity of an FGF peptide, such as cell proliferation. The effectiveness of a potential antagonist can be assessed using methods known to those of skill in the art. For example, the properties of a potential antagonist may be assessed as a function of its ability to compete with radiolabelled bFGF to bind to one or more FGF receptor using a purified FGF receptor binding assay or a cell-based receptor assay.

As used herein, alkyl, alkenyl and alkynyl carbon chains, if not specified contain from 1 to 20 carbons, preferably 1 to 1 2 carbons, and are straight or branched . Alkenyl carbon chains of from 1 to 20 carbons preferably contain 1 to 8 double bonds, and the alkenyl carbon chains of 1 to 1 2 carbons preferably contain 1 to 4 double bonds. Alkynyl carbon chains of from 1 to 20 carbons preferably contain 1 to 8 triple bonds, and the alkynyl carbon chains of 1 to 1 2 carbons preferably contain 1 to 4 triple bonds. The alkyl, alkenyl and alkynyl groups may be substituted, with one or more Z substituents.

As used herein, "Z" is amino, mercapto, halogen, haloalkyl, hydroxy, alkoxy, alkylthio, arylthio, aralkyloxy, aralkylthio and carboxy alkoxycarbonyl.

As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having one to about 6 carbons. Preferred embodiments of the compounds provided herein that include alkyl, alkenyl, or alkynyl portions include lower alkyl, lower alkenyl, and lower alkynyl portions. Preferred among lower carbon chains are those having 4-6 carbons.

As used herein, aryl refers to an mono- or multicyclic aromatic ring system, preferably containing up to about 1 6 carbon atoms, more preferably from about 5 to about 1 4 carbon atoms. The aryl group may be substituted with one or more substituents designated Q.

As used herein, each Q is independently selected from among halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to

2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diarylaminosulfonyl. Exemplary Q-substituted aryl groups include substituted phenyl, substituted indenyl, substituted naphthyl and substituted anthracenyl.

As used herein, cycloalkyl refers to a saturated mono- or multi- cyclic ring system, preferably of 3 to 1 0 carbon atoms, more preferably 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond . Cycloalkenyl and cycloalkynyl groups may preferably contain 3 to 1 0 carbon atoms, with cycloalkenyl groups more preferably containing 4 to 7 carbon atoms and cycloalkynyl groups more preferably containing 8 to 1 0 carbon atoms. The ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion, and may be unsubstituted or substituted with one or more Z substituents.

As used herein, heteroaryl refers to a monocyclic or multicyclic ring system, preferably of about 5 to about 1 4 members where one or more, more preferably 1 to 5 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms. The heteroaryl may be unsubstituted or substituted with one or more, preferably 1 to 3, substituents. Exemplary heteroaryl groups include, for example, furyl, thienyl, pyridyl, pyrrolyl, N- methylpyrrolyl, pyranyl, indolyl, quinolinyl and isoquinolinyl, cinnolinyl, quinoxalinyl, naphthridinyl, benzothiazolyl, dibenzothiazoyl and xanthenyl, with indolyl, quinolinyl and isoquinolinyl, cinnolinyl, quinoxalinyl, naphthridinyl and xanthenyl being preferred. As used herein, heterocyclic refers to a monocyclic or multicyclic ring system, preferably of 3 to 14 members, more preferably 4 to 1 2 members, where one or more, preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms. The heterocycle may be substituted with one or more substituents designated Q. Preferred Q groups of the heterocyclic group include hydroxy, alkoxy containing 1 to 4 carbon atoms, halo lower alkyl, including trihalomethyl, such as trifluoromethyl, alkylamino, such as dimethylamino and halogen. As used herein, the term heterocycle may include reference to heteroaryl . Exemplary heterocycles include, for example, pyrrolidinyl, piperidinyl, alkylpiperidinyl, 1 ,4-dioxanyl, 1 ,4-dithianyl, 1 ,4-morpholinyl, 1 ,4- thiomorpholinyl, 1 ,2, 3-oxadiazolyl, 1 , 3,5-trithianyl or 1 ,2,5-triazolyl.

As used herein, wherever Ar, and Ar₂, and other groups in which ring systems are among alternative embodiments, when such embodiments are specified, among the preferred selections therefor are those in which each is independently selected from a ring system, preferably a 5- to 1 4-membered ring system, more preferably an aryl ring system, even more preferably a multicyclic aryl ring system, most preferably a bicyclic ring system or a heteroatom-containing ring system, preferably a 5- to 1 4-membered heteroatom-containing ring system, more preferably a heteroaryl ring system, containing 1 or more heteroatoms, preferably 1 to 3 heteroatoms, such as oxygen, sulfur or nitrogen atoms, wherein the aryl and heteroaryl ring systems are unsubstituted or substituted with one or more substituents.

Ar, and Ar₂ are each preferably independently phenyl, benzyl, naphthyl, azulenyl, indolyl, fluorenyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, 1 ,6-naphthridinyl, benzothiazolyl, dibenzothiazoyl, xanthenyl, pteridinyl, acridinyl, phenazinyl, phenothiazinyl and anthracenyl that are unsubstituted or substituted with one or more Q substituent.

As used herein, "halogen" or "halide" or "halo" refers to F, Cl, Br or I, and also pseudohalides. In preferred embodiments halo refers to F, Cl, Br and I .

As used herein, pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X^', in which X is a halogen, such as Cl or Br) . Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, azide and trifluoromethyl.

Haloalkyl embraces such moieties as CF₃, -CF₂H, -CFH₂, CH₂CI and CH₂Br and other halo substituted lower aikyls. Exemplary of aryloxyalkenyl and aryloxyalkynyl moieties of R_A are phenoxymethyl, CF₃- substituted phenoxymethyl, benzyloxymethyl, phenoxybutyr-2-ene, 1 - phenyl- 1 -propene, CF₃-phenoxybutyr-2-ene, CF₃-benzyloxymethyl.

Preferred aralkyl and aryl moieties are phenyl, benzyl, phenethyl, 1 - and 2-naphthalmethyl, 1 - and 2-naphthyl, 2-, 3-, 4-pyridyl, 2- and 3- furyl, benzofuryl, dibenzofuryl, 1 - and 2-indenyl, 1 - and 2-thienyl, imidazolyl, benzimidazolyl, indolyl, indolinyl, 2- and 3-thienyl, indole-3- ethyl, carbazolyl, quinolinyl, isoquinolinyl, acridinyl, naphthyridinyl, benzothiazolyl, dibenzothiazoyl, phenazinyl, xanthenyl, tosyl, and phenanthridinyl. Other carbocycles are such fused moieties as pentalenyl, indenyl, naphthaleneyl, naphthylmethyl, azulenyl, heptalenyl, acenaphthylenyl, 9-fluorenyl, phenalenyl, phenanthrenyl, anthracenyl, triphenylenyl, pyrenyl, chryrsenyl, and naphthacenyl.

As used herein, "alkylene" refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 1 2 carbons, even more preferably lower alkylene. The alkylene group is optionally substituted with one or more Z groups. The alkylene group may contain one or more heteroatoms. Exemplary alkylene groups include methylene (-CH₂-), ethylene (-CH₂CH₂-) , propylene ( — (CH₂)₃-) , cyclohexylene (-C₆H₁₀-), -(CH₂)_n-N(R)-(CH₂)_m-, where each of m and n is independently an integer from 0 to 30 and R is hydrogen or alkyl, methylenedioxy (-O-CH₂-O-) and ethylenedioxy (-O-(CH₂)₂-O-) . The term "lower alkylene" refers to alkylene groups having 1 to 6 carbons. Preferred alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred .

As used herein, "alkenylene" refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms and at least one double bond, more preferably 1 to 1 2 carbons, even more preferably lower alkenylene. The alkenylene group is optionally substituted with one or more "alkyl group substituents. " The alkenylene group may contain one or more heteroatoms. Exemplary alkenylene groups include - CH = CH - CH = CH - and -CH = CH-CH₂-. The term "lower alkenylene" refers to alkenylene groups having 2 to 6 carbons. Preferred alkenylene groups are lower alkenylene, with alkylene of 3 to 4 carbon atoms being particularly preferred.

As used herein, a carbocyclic group is a ring containing at least three carbons; a heterocyclic group is a ring containing at least one carbon and one or more heteroatoms, preferably selected from among O, S, and N, more preferably N and O. A heteroaryl group is an unsaturated ring structure containing 1 or more, preferably 1 to 3 heteroatoms in the ring . The rings may be single rings or two or more fused rings. Heteroaryl is used interchangeably with heterocycle.

As used herein, heteroatoms are selected from O, N or S. As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. are used as is generally understood by those of skill in this art. For example, as used herein alkyl refers to non-aromatic carbon chains that contain one or more carbons; the chains may be straight or branched or include cyclic portions or be cyclic. As used herein, alicyclic refers to aryl groups that are cyclic.

As used herein, "haloalkoxy" refers to RO- in which R is a haloalkyl group.

As used herein, "aminocarbonyl" refers to -C(O)NH₂. As used herein, "alkylaminocarbonyl" refers to -C(O)NHR in which

R is hydrogen, alkyl, preferably lower alkyl or aryl, preferably lower aryl.

As used herein, "dialkylaminocarbonyl" refers to -C(O)NR R in which R and R are independently selected from alkyl or aryl, preferably lower alkyl or lower aryl; "carboxamide" refers to groups of formula NR^'COR.

As used herein, "alkoxycarbonyl" as used herein refers to -C(O)OR in which R is alkyl, preferably lower alkyl or aryl, preferably lower aryl.

As used herein, "alkoxy" and "thioalkoxy" refer to RO- and RS-, in which R is alkyl, preferably lower alkyl or aryl, preferably lower aryl. As used herein, when particular group, such as phenyl or naphthyl, is specified, this means that the group is unsubstituted or is substituted . Preferred substituents where not specified are halo, halo lower alkyl, alkylamino, dialkylamino and lower alkyl.

As used herein, the abbreviations for any group or other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the lUPAC-IUB Commission on Biochemical Nomenclature (see, ( 1 972) Biochem. 1 1 :942-944) . A. Compounds for use in treating FGF-mediated diseases Pharmaceutical compositions and methods using bis-sulfonamide compounds that antagonize the in vivo or vitro activity of at least one

FGF peptide, preferably FGF-2, are provided . More particularly, the methods provided use compounds of formula (I) :

Ar SO- N — Y - N - SO- Ar₂

I I

R , ^R ₂

and pharmaceutically acceptable derivatives thereof .

Preferred compounds for use in the methods herein include those of formula (I) in which Ar, and Ar₂ are each independently phenyl, benzyl, naphthyl, azulenyl, indolyl, fluorenyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazoiinyl, benzothiazolyl, dibenzothiazoyl, 1 ,6- naphthridinyl, xanthenyl, pteridinyl, acridinyl, phenazniyl, phenothiazinyl and anthracenyl optionally substituted with halo, hydroxy, haloalkyl, preferably halo lower alkyl, particularly trifluoromethyl, alkyl, alkyloxy, aminosulfonyl, alkylcarbonyl, nitro, alkylamino, preferably dimethylamino, aminocarbonyl, phenylcarbonyl which is optionally substituted with one or more, preferably up to three, substituents selected from halo, haloalkyl and alkyl, or thienyl which is optionally substituted with halo, haloalkyl or alkyl, where the alkyl groups are straight or branched chain and preferably contain from 1 to 6 carbons, more preferably 1 to 3 carbons; more preferably are phenyl, benzyl, azulenyl, naphthyl, 5- dimethylaminonaphthyl, isoquinolinyl, cinnolinyl, quinoxalinyl, naphthridinyl and xanthenyl, and most preferably naphthyl and 5- dimethylaminonaphthyl. Preferred moieties for Y are hydroxycarbonylalkylenes, preferably 1 -hydroxycarbonylbutylene, 1 -hydroxycarbonylpentylene or 1 -hydroxy- carbonylhexylene, lower alkylene, preferably butylene, pentylene or hexylene, and lower alkenylene. Most preferred compounds for use in the methods are di-dansyl-L- lysine, di-naphthalenesulfonyl-L-lysine, di-dansyl-L-ornithine, di- naphthalenesulfonyl-L-ornithine, di-dansylcadaverine, di- naphthalenesulfonaylcadaverine, di-dansylputrescine and di- naphthalenesulfonylputrescine. B. Preparation of the compounds

Intermediates useful for the preparation of compounds of formula (I) are available commercially or may be prepared from known starting materials or intermediates using the methods described below or other methods known to those of skill in the art [see, e.g., Winterbottom ( 1 940) J . Am. Chem. Soc. 62: 1 60; Fossbinder ( 1 939) J. Am. Chem. Soc. 61 :2032: Leitch et al. ( 1 945) Can. J. Res. 23B: 1 39; Foster et al. ( 1 973) Biochem. Biophys. Res. Comm. 53:70; Gray et al. ( 1 963) Biochem. J . 89: 59P; U .S. Patent Nos. 4,959,500 and 5,292,737] . Also, certain compounds, such as di-dansyl lysine are commercially available [Sigma Chemical Co, St. Louis , Mo.] 1 . Preparation of precursors a. Ar₁ and Ar₂ precursors Presently preferred Ar, and Ar₂ precursors for the synthesis of compounds of formula (I) are aryl- and heteroarylsulfonyl halides, which may be purchased commercially or synthesized using methods known to those of skill in the art [see, e.g . , Morrison & Boyd, Organic Chemistry (6^thed), Prentice Hall, NJ ( 1 992)] . For example, sulfonyl halide precursors may be synthesized by sulfonation of aryl, heteroaryl and aromatic amines followed by formation of the corresponding sulfonyl halide, or by forming the corresponding sulfonyl halide from variety of known substituted and unsubstituted aryl- and heteroarylsulfonic acids. Alternatively, the sulfonyl chlorides may be directly synthesized from the corresponding unsubstituted aryl, heteroaryl or aromatic compounds by treatment with chlorosulfonic acid. i. Sulfonation

A wide variety of Ar, and Ar₂ precursors may be prepared by sulfonation of aryl and heteroaryl groups using methods generally known to those of skill in the art [see, e.g . , Morrison & Boyd, pp. 862-863] . In this reaction, an electrophilic sulfur trioxide is formed using sulfuric acid containing an excess of SO₃. The corresponding sulfonic acid is formed by an attack of the electrophilic sulfur trioxide upon the ring of the selected aromatic substrate to form a carbocation. The addition of a suitable base, e.g. , sodium hydroxide, results in abstraction of the proton from the carbocation to form the corresponding sulfonic acid. The sulfonation reaction occurs predominately at the 2-position using unsubstituted heteroaryls, such as furans, pyrroles and thiophenes. However, by selecting the appropriate aryl or heteroaryl moiety substituted with one or more aryl group substituents, the position of the sulfonic acid substitution on the ring may be varied.

Alternatively, aryl- and heteroarylsulfonic acid precursors used for the preparation of compounds of formula (I) may be purchased commercially. For example, benzenesulfonic acid, benzaidehyde-2- sulfonic acid, anthraquinone-2-suifonic acid, 1 ,2-naphthoquinone-4- sulfonic acid, 5-diaminomethylnaphthylene- 1 -sulfonic acid, 5- diaminobutylnaphthylene- 1 -sulfonic acid, 8-anilinonaphthylene-1 -sulfonic acid, 8-amino-2-naphthylene-sulfonic acid, 8-amino-1 -naphthol-5-sulfonic acid, 8-(2-aminoethylamino)- 1 -naphthylene-sulfonic acid, 2,3- dihydroxynaphthylene-6-sulfonic acid, 2,5-diphenyloxazole-4-sulfonic acid, 8-hydroxyquinoline-5-sulfonic acid, dodecylbenzene-sulfonic acid, p-xylene-2-sulfonic acid, benzoxazole-2-sulfonic acid, 4-amino-3-hydroxy- 1 -naphthalene-sulfonic acid, 5-(2-aminoethylamino)- 1 -naphthylene- sulfonic acid, 1 -(2-hydroxy-1 -naphthylazo)-2-naphthol-4-sulfonic acid, 2- nitroso- 1 -naphthol-4-sulfonic acid, 1 ~amino-2-naphthol-4-sulfonic acid, toluene-4-sulfonic acid are commercially available from either Sigma Chemical Corp. (St. Louis, MO.), Aldrich Chemical Corp. (Milwaukee, Wl .) or Fluka Chemical Corp. (Milwaukee, Wl) . ii. Sulfonyl halide formation Methods for the preparation aromatic sulfonyl halides, particularly sulfonyl chlorides, from aromatic sulfonic acids are well known [e.g., Morrison & Boyd, p. 857] . A sulfonyl chloride is prepared by the substitution of -Cl for the -OH group of the sulfonic acid. For example, the sulfonated aryl or heteroaryl group is treated with water and an alkali metal hydroxide (e.g., sodium hydroxide) to form the sulfonic acid salt. The sulfonic acid salt is treated with a halogenating reagent in an anhydrous organic solvent at about 0°C to 80°C. Halogenating reagents commonly used for generating acid halides are a halosulfonic acid solution [e.g ., chloro-sulfonic acid] , thionyl chloride, oxalyl chloride, phosgene, phosphorus oxychloride, chlorine gas, phosphorus trichloride and phosphorus pentachloride. Thionyl chloride is advantageous because the by-products of the reaction are gases and thionyl chloride is easily removed by distillation (b.p. 79°C) .

Alternatively, the sulfonyl halide derivatives of aryl and heteroaryl moieties may be purchased commercially. For example, benzenesulfonyl chloride, 4-methylphenylsulfonyl chloride (tosyl chloride) , 5- dimethylamino- 1 -naphthylenesulfonyl chloride ( e^, dansyl chloride) are commercially available from Sigma Chemical Co. or Aldrich Chemical Co. iii. Aromatic amines

Ar, and Ar₂ sulfonyl halide precursors may also be prepared from aromatic amines. For example, an aromatic amine is treated with an acid, e.g ., hydrochloric acid, and sodium nitrite in an aqueous acid solution, e.g . , water-acetic acid, at about -20°C to 0°C. To this mixture, containing the corresponding diazonium salt, sulfur dioxide is added, followed by a copper (I) salt in an appropriate solvent, such as copper (I) chloride in acetic acid, at about 5°C to 30°C to form the aryl sulfonyl halide [see, e.g . , European Patent Application No. EP 569 1 93] . Furthermore, aryl and heteroaryl amines used as precursors for generating sulfonyl halide precursors, as described above, may be formed by reduction of the corresponding nitro compounds. For example, the nitro group may be reduced to the corresponding amine using hydrogen gas and appropriate catalyst, such as platinum, or by using an appropriate acid, e.g., hydrochloric acid, and an appropriate metal, e.g. , tin. In an acidic solution, the amine is obtained as a salt and the free amine is liberated by the addition of an appropriate base, e.g . , sodium hydroxide, and steamed distilled from the reaction mixture. b. Y precursors A wide variety of Y precursor are provided herein and in light of this disclosure may be prepared by those of skill in the art. Presently preferred Y precursors are aminocarboxylic acids and diamines, such as 5-aminohexanoic acid, 4-aminopentanoic acid, 3-aminobutanoic acid, 2- aminopropionic acid, 6-aminohexanoic acid, 5-aminopentanoic acid, 4- aminobutanoic acid, 3-aminopropionic acid, 2, 3-diaminopropionic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid and 2,6- diaminohexanoic acid .

An amino group may be added, for example, to the alpha carbon of an aliphatic halocarboxylic acid by nucleophilic substitution (ammonolysis) . For example, alanine (σ-aminopropionic acid) may be formed by reacting σ-bromopropionic acid with ammonia at ambient temperature or under conditions using heat and pressure. Displacement of the halogen by NH₃ yields the amine salt from which the free amine may be liberated by treatment with a suitable base, e.g., sodium hydroxide [e.g ., Morrison & Boyd, p.761 ] .

It is to be appreciated that substituting σ-bromopropionic acid with another suitable halogenated carboxylic acid, e.g. , β- or y-halobutanoic acid, β-, y- or Δ-halopentanoic acid or ?-, y-, Δ- or ω-halohexanoic acid, results in the formation of Y precursors in which the length of the carbon backbone is varied.

Alternatively, halogenated and amino substituted carboxylic acids may be purchased from a commercial supplier.

In addition, aminocarboxylic acids may be prepared from cycloalkanones and cycloalkenones by addition of, e.g., hydroxylamine followed by, e.g., phosphoric acid/acetic anhydride (Beckmann Rearrangement) ; hydrazoic acid (Schmidt Rearrangement) ; or hydrazine followed by nitrous acid (Curtius Rearrangement) [see, e.g. , Beckmann Rearrangement: Tetrahed. , 37: 1 283 ( 1 981 ) , Schmidt Rearrangement: Wolff Orq. React. 3:307 ( 1 946) , Curtius Rearrangement: Smith Org . React. 3:337 ( 1 947), Banthorpe, in Patai, The Chemistry of the Azido Group, pp. 397-405 ( 1 971 )] . The resulting lactam is then hydrolyzed under acidic conditions to provide the desired Y precursor (aminoalkanoic and aminoalkenoic acids) . Aminoalkynoic acids may be prepared, for example, from amino-1 - alkynes using the method described below or other methods known to those of skill in the art. For example, the dianion of an amino- 1 -alkyne, generated with a strong base (e.g ., butyllithium) , may be added to a halocarboxylic acid or carbon dioxide to provide the corresponding aminoalkynoic acid . Alternatively, the dianion may be added to an alkyl or aryl chloroformate. Subsequent basic hydrolysis of the resulting ester would afford the desired aminoalkynoic acids.

Diaminoalkanoic acids may be prepared by σ-amination of the corresponding cc-aminoalkanoic acids, whose synthesis is described above. σ-Amination may be accomplished by reaction of the σ-anion of the acid group (or a derivative thereof, e.g., an ester) with, e.g ., tosyl azide. Reduction of the resulting σ-azιdo-α/-amιnoalkanoιc acid under conditions known to those of skill in the art (e.g., hydrogen and a metal catalyst, stannous halide, etc.) provides the desired diaminoalkanoic acid . Use of other aminoalkanoic acids in this preparation affords other diaminoalkanoic acids of this class.

2. Preparation of compounds a. General synthetic methods In general, most of the syntheses involve the condensation of a sulfonyl chloride with an appropriate Y-precursor in dry pyπdine or in tetrahydrofuran (THF) and sodium hydride. Alternatively, the reaction may be performed in aqueous solution using a base such as NaOH, Na₂CO₃, or NaHCO₃. As described above, the sulfonyl chloride precursors and Y precursors can be obtained commercially or in light of this disclosure may be prepared by those of skill in the art.

For example, the ώ/s-sulfonamide compounds of formula (I) can be prepared by condensing an Ar, or Ar₂ sulfonyl chloride with a Y precursor, an Ar,-Y-NH₂ intermediate or NH₂-Y-Ar₂ intermediate in tetrahydrofuran solution containing a base, such as sodium hydride.

Following the reaction, the THF is removed under pressure, the residue dissolved in water, acidified and extracted with methylene chloride. The organic layer is washed and then dried over anhydrous magnesium sulfate, the solvents are evaporated and the residue is purified by recrystallization from ethyl acetate/hexanes, to yield the pure product.

Alternatively, the b/s-sulfonamide compounds of formula (I) , such as didansyl-L-lysine, may be prepared by condensing the NH₂-Y-Ar₂ intermediate, Ne-dansyl-L-lysine, with a sulfonyl chloride, e.g. , dansyl chloride, in dry pyridine with or without the catalyst 4- (dimethylamino)pyridine. Following the reaction, the pyridine is removed under reduced pressure and the residue is partitioned between water and ethyl acetate. The organic layer is washed and then dried over anhydrous magnesium sulfate, the solvents are evaporated and the residue is purified by column chromatography over silica gel (e.g ., 1 % methanol in chloroform as a eluent) to yield a solid . Further purification is achieved by recrystallization from an suitable solvent, e.g. , ethyl acetate/hexanes, to yield the pure product. In some cases, the N,N-6/s-sulfonyl compound is obtained as the major or exclusive product. The 6/s-sulfonated products can be readily hydrolyzed to the sulfonamide using aqueous sodium hydroxide and a suitable co-solvent, such as methanol or tetrahydrofuran, generally at room temperature. b. Preparation of compounds wherein Y is a hydroxycarbonylalkylene

Following the aforementioned methods, compounds of formula (I) where Ar, and Ar₂ are the same may be prepared in a single reaction vessel by adding a suitable diaminocarboxylic acid Y-precursor to an excess of the desired sulfonyl chloride. It is to be appreciated by those of skill in the art that when Ar, and Ar₂ are different, the coupling of one free amine of Y to the sulfonyl chloride of Ar, or Ar₂ may require protection of the other free amine. For example, the epsilon nitrogen of 2,6-diaminohexanoic acid may be protected from reacting with a suitable sulfonyl chloride by prior coupling to a protecting group, such as t-butyloxycarbonyl (Boc) group, benzoxycarbonyl or substituted benzoxycarbonyl group, preferably Boc or 2-chlorobenzoxycarbonyl group, during the formation of Ar,-Y [see, e.g . ,

Stewart and Young, in Solid Phase Peptide Synthesis, Pierce Chemical

( 1 984)] . After formation of the sulfonamide, the protecting group is removed, e.g ., using TFA, and the second amine may be regenerated using a suitable base, e.g. , NaOH, and reacted with an Ar₂ sulfonyl chloride precursor, as defined above, to form compounds of formula (I) . c. Preparation of compounds wherein Y is lower alkylene, alkenylene, or alkynylene

Compounds of formula (I) wherein Y is lower alkylene, alkenylene or alkynylene may be prepared from aminocarboxylic acids using the method described below or other methods known to those of skill in the art [see, e.g . , Curtius Rearrangement: Smith Org . React. 3:337 ( 1 947) , Banthorpe, in Patai, The Chemistry of the Azido Group, pp. 397-405 ( 1 971 ) ; Hoffman Rearrangement: Wallis et al. Org . React. 3:267 ( 1 946) ; Lossen Rearrangement: Yale Chem. Rev. 33:242 ( 1 943) ; Schmidt Rearrangement: Wolff Org . React. 3:307 ( 1 946)] .

For example, the addition of an Ar, or Ar₂ sulfonyl chloride, as defined above, to 6-aminohexanoic acid forms the corresponding aryl sulfonamide. The carboxylic acid group is converted to an acid chloride using thionyl chloride, and the corresponding acyl hydrazide is afforded by addition of hydrazine. To this mixture nitrous acid is added to form the corresponding acyl azide. Alternatively, addition of sodium azide to the acid chloride affords the acyl azide directly. Upon heating, the acyl azide undergoes a Curtius rearrangement to generate the free amine. Alternatively, the acid chloride may be converted to a primary amide by addition of ammonia. Treatment of this primary amide with a hypohalite salt (e.g., sodium hypochlorite), iodosobenzene, or lead tetraacetate results in a Hoffman Rearrangement to provide the desired free amine.

As another alternative, the carboxylic acid, following conversion of the amine to the corresponding aryl sulfonamide, may be converted to a hydroxamic acid by reaction with hydroxylamine. Subjection of this hydroxamic acid to thionyl chloride, acetic anhydride, phosphorus pentoxide, or polyphosphoric acid results in a Lossen Rearrangement to afford the free amine.

A further alternative involves reaction of the carboxylic acid, following conversion of the amine to the corresponding aryl sulfonamide, with hydrazoic acid at elevated temperature to provide the desired free amine directly (Schmidt Rearrangement) .

The free amine, prepared by any of the aforementioned methods, is then reacted with an appropriate Ar, or Ar₂ sulfonyl chloride to form the 6/s-sulfonamide compounds of formula (I) . d. Protection of reactive functionalities during synthesis

For compounds of formula (I) wherein Ar, , Ar₂ and Y include reactive functionalities, the reactants may also be treated with protecting groups prior to coupling . The amine portion of the sulfonamide core may also need protecting when, for example, different substitutions are introduced . Suitable protecting groups for and procedures for use thereof are generally known in the art. Among the classes of amino protecting groups contemplated are: ( 1 ) acyl type protecting groups such as formyl, trifluoroacetyl, phenyl carbonyl, 4-[(4- chlorophenyl)sulfonylaminocarbonyl]phenyl carbonyl, 4-[(4- bromophenyl)sulfonylaminocarbonyl]phenyl carbonyl, phthalyl, p_- toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfonyl, tritylsulfonyl, -nitrophenoxyacetyl, and σ-chlorobutryl; (2) aromatic urethane type protecting groups such as phenyloxycarbonyl, benzyloxycarbonyl and substituted benzyloxycarbonyls such as p_- chlorobenzyloxycarbonyl, jo-methoxybenzyloxycarbonyl, p_- nitrobenzyloxycarbonyl, jD-bromobenzyloxycarbonyl, 1 -(p_-biphenylyl)-1 - methylethoxycarbonyl, σ,σ-dimethyl-3, 5-dimethoxy benzyloxycarbonyl, and benzhydryloxycarbonyl; (3) aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, methoxycarbonyl, isobutyloxycarbonyl, ethoxycarbonyl, and allyloxycarbonyl; (4) cycloalkyl urethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; (5) thiourethane type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups such as triphenylmethyl (trityl) and benzyl (Bn) ; and (7) trialkylsilane protecting groups such as trimethylsilyl, tert- butyldimethylsilyl, tert-butyldiphenylsilyl, and triisopropylsilyl . e. Compounds where R, or R₂ is lower alkyl Compounds where R, or R₂ is lower alkyl may be synthesized from the sulfonamides, whose synthesis is described above. For example, treatment of the sulfonamide with a base, such as NaOH, lithium di-iso- propylamide, or n-butyllithium, generates the sulfonamide N-anion . Subsequent reaction of this anion with a lower alkyl halide provides the desired compounds.

C. Evaluation of the bioactivity of the compounds

Standard physiological, pharmacological and biochemical procedures are available for testing the compounds to identify those that possess any biological activities of compounds that interfere with or inhibit FGF peptides. Numerous assays are known to those of skill in the art for evaluating the ability of compounds to modulate the activity of one or more FGF peptide. For example, the properties of a potential antagonist may be assessed as a function of its ability to inhibit FGF activity including the ability in vitro to compete for binding to FGF receptors present on the surface of tissues or recombinant cell lines, cell- based competitive assays [see, e.g., Mostacelli et al. (1987) J. Cell. Physiol. 131 :123-130]; mitogenic assays [Gospardarowicz et al. (1984) Proc. Natl. Acad. Sci. U.S.A.81:6963-6967; Thomas et al. (1984) Proc. Natl. Acad. Sci. U.S.A.81 :357]; stimulation of angiogenesis |n vitro [see, e.g., European Patent Application No. EP 645451]; cell proliferation assays or heparin binding assays [see, e.g., International Application Publication No. WO 92/12245]; assays measuring the release of cellular proteases [Mostacelli et al. (1986) Proc. Natl. Acad. Sci. U.S.A.83:2091-2095: Phadke (1987) Biochem. Biophvs. Res.

Comm. 142:448-453]; and, assays for the promotion of FGF-mediated neurite outgrowth and neuron survival [Togari et al. (1983) Biochem. Biophvs. Res. Comm. 114:1189-1193; Wagner et al. (1986) J. Cell Biol. 103:1363-1367]. In addition, FGF isotype specific antagonists may be identified by the ability of a test compound to interfere with one or more FGF peptide binding to different tissues or cells expressing different FGF receptor subtypes, or to interfere with the biological effects of an FGF peptide [see, e.g.. International Patent Application Publication No. WO 95/24414].

Using such assays, the relative affinities of the compounds for FGF receptors have been and can be assessed. Those that possess the desired iri vitro properties, such as specific inhibition of the binding of bFGF, are selected. The selected compounds that exhibit desirable activities may be therapeutically useful in the methods described herein and are tested for such uses employing the above-described assays from which the vivo effectiveness may be evaluated [Gospodarowicz et al. ( 1 987) Endocrin . Rev. 8:95- 1 1 4; Buntrock et al. ( 1 982) Exp. Pathol . 2J_:62-67; International Patent Application Publication No WO

92/08473] . Compounds that exhibit the jπ vitro activities that correlate with the in vivo effectiveness will then be formulated in suitable pharmaceutical compositions and used as therapeutics.

An assay that has been used to assess interaction of bFGF with its native receptor is exemplified herein (see, also, Zhu et a_L ( 1 995) J . Biol. Chem. 270:21 869-21 874) . This assay can be used to identify compounds provided herein that may be therapeutically useful for treating FGF mediated disorders. D. Formulation of pharmaceutical compositions Compositions are provided for use in the methods herein that contain therapeutically effective amounts of the compounds of formula (I) or pharmaceutically acceptable derivatives thereof . The compounds are preferably formulated into suitable pharmaceutical preparations such as tablets, capsules or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation. Typically the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

About 1 0 to 500 mg of a compound or mixture of compounds of formula (I) or pharmaceutically acceptable derivatives thereof is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc. , in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained .

To prepare compositions, one or more compounds of formulae (I) or a pharmaceutically acceptable salt, ester or acid thereof, are mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined . Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action or have other action. The compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used . Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as tween, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts of the compounds or prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.

The concentrations of the compounds or pharmaceutically acceptable derivatives thereof are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the disorder for which the compounds are administered . Typically, the compositions are formulated for single dosage administration.

The compounds of formula (I) may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems,

The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated . The therapeutically effective concentration may be determined empirically by testing the compounds in known in. vitro and vivo model systems for the treated disorder.

The compositions can be enclosed in ampules, disposable syringes or multiple or single dose vials made of glass, plastic or other suitable material. Such enclosed compositions can be provided in kits.

The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated . It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. If oral administration is desired, the compound could be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as, but not limited to, gum tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose, starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc. or a synthetic fatty vehicle like ethyl oleate or the like, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid

(EDTA) ; buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material. Buffers, preservatives, antioxidants and the like can be incorporated as required.

For ophthalmic indications, the compositions are formulated in an opthalmically acceptable carrier. For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred. Time release formulations are also desirable. Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.

Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. If necessary, pharmaceutically acceptable salts or other derivatives of the compounds may be prepared.

The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. It is understood that number and degree of side effects depends upon the condition for which the compounds are administered . For example, certain toxic and undesirable side effects are tolerated when treating life- threatening illnesses, such as tumors, that would not be tolerated when treating disorders of lesser consequence. The concentration of compound in the composition will depend on absorption, inactivation and excretion rates thereof, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

Ophthamologically effective concentrations or amounts of one or more of the compounds are mixed with a suitable pharmaceutical carrier or vehicle. The concentrations or amounts of the conjugates that are effective requires delivery of an amount, upon administration, that prevents or substantially reduces the effects of FGF-mediated ophthamological conditions, including, but not limited to, diabetic retinopathy, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery and the recurrence of pterygii.

The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 million) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, e.g . , U.S. Patent Nos. 5,292,362, 5,282,851 , 5,273,056, 5,229, 1 27, 4,51 7,295 and 4,328,803) , VISCOAT (fluorine-containing (meth)acrylates, such as,

1 H, 1 H,2H,2H-heptadecafluorodecylmethacrylate; see, e.g . , U.S. Patent Nos. 5,278, 1 26, 5,273,751 and 5,21 4,080; commercially available from Alcon Surgical, Inc.), ORCOLON (see, e^, U.S. Patent Nos. 5,273,056; commercially available from Optical Radiation Corporation) , methylcellulose, methyl hyaluronate, polyacrylamide and polymethacrylamide (see, e.g ., U.S. Patent No. 5,273,751 ) . The viscoelastic materials are present generally in amounts ranging from about 0.5 to 5.0%, preferably 1 to 3% by weight of the conjugate material and serve to coat and protect the treated tissues. The compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.

The ophthalmologic indications herein are typically treated locally either by the application of drops to the affected tissue(s) , contacting with a biocompatible sponge that has absorbed a solution of the conjugates or by injection of a composition . For the indications herein, the composition will be applied during or immediately after surgery in order to prevent closure of the trabeculectomy, prevent a proliferation of keratocytes following excimer laser surgery, prevent the proliferation of lens epithelial cells following cataract surgery or to prevent a recurrence of pterygii. The composition may also be injected into the affected tissue following surgery and applied in drops following surgery until healing is completed . For example, to administer the formulations to the eye, it can be slowly injected into the bulbar conjunctiva of the eye.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) , and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof . Liposomal suspensions, including tissue-targeted iiposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,81 1 . The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of such formulations are known to those skilled in the art.

The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, may be formulated as 0.01 % - 100% (weight to volume) isotonic solutions, pH about 5-7, with appropriate salts. The compounds may be formulated as aerosols for topical application, such as by inhalation [see, e.g . , U.S. Patent Nos. 4,044, 1 26, 4,41 4,209, and 4,364,923] .

Finally, the compounds may be packaged as articles of manufacture containing packaging material, an acceptable composition containing a compound of formula (I) or pharmaceutically acceptable derivatives thereof provided herein, which is effective for treating the particular disorder, and a label that indicates that the compound or derivative thereof is used for treating FGF mediated disorders or inhibiting the binding of one or more FGF peptides to their receptors. E. Methods of treating of FGF-mediated disorders

Methods using compositions containing therapeutically effective concentrations of the compounds of formula (I) for treating disorders, particularly proliferative disorders, in which FGF causes or contributes to the pathology are provided herein. In particular, compounds may be used to prevent the undesired growth and proliferation of FGF-sensitive cells occurring in vascular disorders characterized by accelerated smooth muscle cell proliferation, such as rheumatoid arthritis, tumor angiogenesis, Kaposi's sarcoma, restenosis, certain ophthalmic disorders and dermatological disorders, such as psoriasis, are provided herein. Preferably, the medicament containing the compound or pharmaceutically acceptable derivative thereof is administered intravenously (IV), although treatment by localized administration of the may be tolerated in some instances. Generally, the medicament containing the compound is injected into the circulatory system of a subject in order to deliver a dose to the targeted cells. Targeting may be effected by linking the compound to a targeting agent specific for FGF receptors, particularly bFGF receptors. Dosages may be determined empirically, but will typically be in the range of about 0.01 mg to about 1 00 mg of the compound per kilogram of body weight are expected to be employed as a daily dosage.

Restenosis and vascular injury Methods for treating vascular injury, particularly, restenosis by contacting the vascular wall with an effective amount of a compound of formula (I) are provided.

Atherosclerosis, also referred to as arteriosclerosis, results from the development of an intimal lesion and the subsequent narrowing of the vessel lumen. Frequently, atherosclerosis originally appears as a result of the buildup of plaque which lines the interior of blood vessels, particularly the arteries. Whereas bypass surgery is sometimes employed to replace such clogged arteries, in recent years, a number of surgical procedures have been developed so as to interarterially remove such plaque, often by balloon catheterization or other such treatments in which the plaque is either compressed against or scraped away from the interior surface of the artery. This scraping of the interior wall removes endothelial cells, which constitute the lining of the blood vessel. As a result of this removal, the smooth muscle cells (SMCs) , which are normally located exterior of the endothelial cells (ECs) and form the blood vessel structure, begin to grow and multiply causing a narrowing of the vessel lumen. Not infrequently, the patient so treated finds a recurrence of such narrowing of the vessel lumen in a relatively short period thereafter as a result of this proliferation, generally referred to as restenosis, requiring a repetition of the surgical procedure to again remove the increasing blockage Angioplasty can also result in injury to SMCs.

Proliferating SMCs express functional FGF receptors and are responsive to bFGF. By inhibiting proliferation of migrating smooth muscle cells (SMCs) , it is possible to prevent the undesirable growth and ultimate clogging which occurs following vascular injury, and which is generally referred to as restenosis [e.g., see Kearney et al. Circul. 95: 1 998-2002 ( 1 997)] . Basic FGF appears to play a pivotal role in the subsequent responses of the vascular wall [e.g ., see Lindner et al. Proc. Natl. Acad . Sci. U.S.A. 88:3739-3743 ( 1 991 )] . Basic FGF is known to be synthesized by endothelial and smooth muscle cells (SMCs) and is thought to be stored in the subendothelial matrix, and in some instances, this growth factor is released from cells after injury. Therefore, compounds that inhibit FGF-mediated proliferation of SMCs may be used in methods for treating restenosis by preventing the proliferation that causes the narrowing of the vessel lumen.

Treatment is effected by administering a therapeutically effective amount of a medicament containing the compound in a physiologically acceptable carrier or recipient, in a manner so that the compound reaches regions in a human or other mammal where the compound will inhibit the proliferation of the target cells. For restenosis, intraarterial infusion will be among the preferred methods. Although a single dose should inhibit neointimal proliferation, IV administration over a period of time is preferred.

The compounds for treating restenosis as well as In-Stent restenosis may be formulated for intravenous or local administration. Alternatively, compounds may be conjugated to an agent that specifically targets proliferating SMCs, such as antibodies, hormones, ligands or the like to improve delivery and uptake of the compound . The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems (see, e.g. , Mostacelli et al. ( 1 987) J. Cell . Phvsiol . 1 31 : 1 23-1 30] ; mitogenic assays [Gospardarowicz et al. ( 1 984) Proc. Natl . Acad . Sci. U.S.A. 81 :6963- 6967; Thomas et al. ( 1 984) Proc. Natl . Acad . Sci. U.S.A. 81 :357]; stimulation of angiogenesis in vitro [see, e.g. , European Patent Application No. EP 645 451 ]; cell proliferation assays or heparin binding assays [see, e.g.. International Application Publication No. WO 92/1 2245]; assays measuring the release of cellular proteases

[Mostacelli et al. ( 1 986) Proc. Natl. Acad. Sci. U.S.A. 83:2091 -2095; Phadke ( 1 987) Biochem. Biophvs. Res. Comm. 1 42:448-4531 ; and, assays for the promotion of FGF-mediated neurite outgrowth and neuron survival [Togari et al. ( 1 983) Biochem. Biophvs. Res. Comm. 1 1 4: 1 1 89- 1 1 93; Wagner et al. ( 1 986) J . Cell Biol. 1 03: 1 363- 1 3671) and then extrapolated therefrom for dosages for humans. Rheumatoid arthritis

Rheumatoid arthritis is a systemic, chronic inflammatory disease, that is characterized by the destruction of the joint cartilage and inflammation of the synovium. The hallmark feature of rheumatoid arthritis is the production circulating autoantibodies, also referred to as rheumatoid factors, which are reactive with the Fc portions of the patients own IgG molecules [e.g . , see Abbas et al. , Cellular and Molecular Immunology, W.B. Saunders Co., Philadelphia, PA ( 1 991 )] . One of the systemic complications of rheumatoid arthritis is the formation of injurious immune complexes in the synovial fluid of the joints that initiates vascular inflammation by activation of the complement cascade. T-cells, activated B-cells, plasma cells and macrophages are often found in synovial fluid of affected joints as well as a variety of soluble proteins, such as cytokines [e.g. , interleukin- 1 , IFN- and tumor necrosis factor (TNF)] and growth factors, such as bFGF. It has been suggested that cytokines act in concert with the inflammatory mediators, e.g. , bFGF, to cause local tissue destruction. Chronically, cytokines and bFGF stimulate fibroblast and collagen proliferation resulting in angiogenesis, and prolonged exposure can result in hyperproliferation of epithelial cells that form fibrous tissue, referred to as fibrosis.

Thus, compounds that inhibit the FGF-mediated hyperproliferation of epithelial cells may be used to treat rheumatoid arthritis. The compounds or pharmaceutically acceptable derivatives thereof for treating rheumatoid arthritis may be formulated for oral administration or intravenous injection and an effective concentration may be administered . The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined .

Tumor Angiogenesis

Angiogenesis plays a critical role in embryonic development and in several physiologic and pathologic conditions, including wound healing, ovulation, diabetic retinopathy and malignancy. In particular, without the nutrients and oxygen provided via this neovascularization, solid tumors would be unable to grow beyond about 2 mm in diameter.

Evidence exists that several cancers, including melanomas, ovarian, pancreatic and some colon carcinomas and Kaposi's sarcomas, have receptors for bFGF. Testing with radioactive binding assays on a number of human carcinogenic cell lines isolated from human cancers demonstrated that many but not all of these cell lines bind ^{, 25}I-FGF. Tumor growth may be inhibited by modulating FGF receptor activity in the components of a blood vessel, e.g . , vascular endothelial cells or vascular SMCs [see e.g . , Haberman Angiogenesis :98-1 -98-20 ( 1 996) ; Coville-Nash et al. Molec. Med . Today : 1 4-23 ( 1 997) ; Shawver et al. Drug Discov. Today 2:50-63] . Thus, compounds that inhibit the activity of FGF may be used to treat tumorigenic pathophysiological conditions caused by a proliferation of cells which are sensitive to FGF mitogenic stimulation.

The compounds or pharmaceutically acceptable derivatives thereof may be specifically targeted to tumorigenic tissues by linking the compound to an agent that specifically binds to the surface of the tumorigenic ceil, e.g ., an anti-tumor antigen antibody, or linking the compound to an agent that is preferentially interacts with or taken up by targeted tumor. In addition, compounds may be encapsulated in tissue- targeted liposomal suspensions for targeted delivery of the compound . The compounds for treating tumor angiogenesis may be formulated for topical application and administered to the skin, e.g . , for treatment of melanoma, or may be formulated for intravenous administration for treatment of solid tumors, such as carcinomas. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro (see, e.g ., inhibition of angiogenesis in vitro [see, e.g., European Patent Application No. EP 645 451 ]) and then extrapolated therefrom for dosages for humans. Ophthalmic Disorders

Pharmaceutical compositions provided herein may be used in methods of treating ophthalmic disorders resulting from FGF-mediated hyper-proliferation of lens epithelial cells, fibroblasts or keratinocytes [e.g . , see Dell Drug Discov. Today 1:221 -222 ( 1 996)] . In particular, ophthalmic disorders that may be treated using the methods and compositions provided herein include, but are not limited to, diabetic retinopathy, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery and the recurrence of pterygii.

The compounds or pharmaceutically acceptable derivatives thereof for treating ophthalmic disorders may be formulated for local or topical application and administered by topical application of an effective concentration to the skin and mucous membranes, such as in the eye. The compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined .

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention . EXAMPLE 1

Assays for identifying compounds that exhibit FGF antagonistic activity A. Soluble FGF receptor assay

Compounds of formula (I) that exhibit FGF antagonist activity were and can be identified by testing their ability to compete with ¹²⁵l- bFGF for binding to one or more FGF receptor or FGF-binding fragment thereof . In one embodiment, a recombinant FGF receptor fusion protein was used in which the extracellular domain of a human FGF receptor, FGFR1 , was fused to the amino terminal fragment of tissue plasminogen activator (tPA) protein. This fusion protein retains the ability to bind FGF, such as bFGF [Zhu et al. ( 1 995) J . Biol . Chem. 270:21 869-21 874] .

(i) Isolation of DNA encoding the shorter form of human fibroblast growth factor receptor 1 (FGFR1 )

The nucleotide sequence of the DNA encoding the shorter form of human basic fibroblast growth factor receptor 1 (FGFR1 ) has been determined [e.g. , N . Itoh et al. , ( 1 990) Biochem. Biophvs. Res. Comm

1 69:680-6851. This shorter form of FGFR1 is a 731 amino acid polypeptide that has a signal peptide, two extracellular immunoglobulin- like domains, a transmembrane domain and an intracellular tyrosine kinase domain. Based on the reported sequence, two oligonucleotides complementary to sequences flanking the FGFR1 coding region were synthesized and used as primers in polymerase chain reactions (PCR) to isolate a DNA encoding a full-length human FGFR1 from a human aorta cDNA library (Quickclone, Clontech, Palo Alto, CA) . PCR amplification was performed using a commercially available PCR kit according to manufacturer's instructions (Perkin Elmer Cetus, Norwalk, CT) . An oligonucleotide corresponding to nt -20 to + 5, relative to the A of the ATG initiation codon of FGFR1 , [e.g . , N . Itoh et ai , ( 1 990) Biochem. Biophvs. Res. Comm. 1 69:680-685] and an oligonucleotide complementary to nt 221 8-2243 were used as primers to amplify a 2,243 bp PCR product encoding the entire FGRF 1 coding region.

The full-length FGFR1 -encoding DNA was used as a template for a subsequent PCR reaction, performed as described above, to amplify a 869 bp DNA fragment encoding only the FGFR1 extracellular domain. Simultaneously, a Hindi 11 restriction endonuclease site was introduced upstream of the FGFR1 initiation codon and a Sail site was introduced downstream of the second immunoglobulin-like extracellular domain (Igll) to facilitate cloning of the amplified product. The Hindlll site was introduced at nt -8 to -3 during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt - 1 2 to + 22 that introduced nucleotide changes at three positions in the FGFR1 sequence: nt -3 (G to T) , nt -6 (A to G) and nt -8 (G to A) . The Sail site was introduced at nt 849 to nt 854 by synthesizing an oligonucleotide primer complementary to nt 823 to 857 containing nucleotide substitutions at three positions in the FGFR1 sequence: nt 849 (C to G) , nt 851 (G to C) and nt 854 (G to C) . The 857 bp PCR fragment was incubated with Hindlll and SaM and purified by agarose gel electrophoresis according to the standard procedures [Sambrook et al. , ( 1 989) Molecular Cloning, 2nd ed ., Cold Spring Harbor Laboratory Press,

New York] . The DNA was isolated from gel by electroelution and recovered by precipitation with ethanol.

Thus, the resulting Hindlll to Sa|l DNA fragment consists of nt -7 to nt 849 of the FGFR1 cDNA described by Itoh et al. and encodes ammo acid residues 1 to 284 of the shorter form of the bFGF receptor.

(ii) Isolation of DNA encoding human tissue plasminogen activator

The nucleotide sequence of the DNA encoding human tissue plasminogen activator (tPA) has been determined [e.g ., see Pennica et al ( 1 983) Nature 301 :21 4-221 ] . Human tPA is a 562 ammo acid polypeptide which is processed during secretion to its mature form by cleavage of a 35 ammo acid signal peptide. Several regions of the primary structure of mature tPA have a high degree of homology to known structural domains of other proteins, such as homology to the finger and growth factor domains, the Kπngle 1 and Kπngle 2 domains of plasminogen and prothrombin and the C-termmal seπne protease domain [e.g . , see Ny et al. Proc. Natl . Acad . Sci. U .S.A. 81 : 5355-5359] .

Based on the reported sequence, ohgonucleotides complementary to sequences flanking the tPA coding region were synthesized and used as primers in PCR reactions to isolate a full-length cDNA encoding human tPA from a human placenta cDNA library (Clontech, Palo Alto, CA) . An oligonucleotide corresponding to nt -6 to + 21 , relative to the A of the initiation codon of the of human tPA prepro polypeptide [e.g . , see Pennica er a/ ( 1 983) Nature 301 :21 4-221 ] and an oligonucleotide complementary to nt 1 558 to nt 1 584 were used to amplify a 1 591 bp DNA encoding the entire human tPA prepro polypeptide.

The full-length DNA was used as a template for a subsequent PCR reaction to amplify a 599 bp DNA encoding the a portion of the signal peptide-finger-growth factor-first Kringle domains of tPA, and which also to introduce an in-frame amber stop codon [i.e. , UGA] at amino acid codon 1 80 of mature tPA sequence. Concurrently, a Sjall restriction endonuclease site and a mutation substituting a Pro for an Arg at position -6 were introduced upstream of the first Ser codon of mature tPA and a BamHI site was introduced downstream of newly introduced translational stop codon to allow for convenient subcloning of the amplified product. The substitution of Pro for Arg at amino acid residue position -6 introduces a proteolytic cleavage site for thrombin in the linker sequence (Le^ , Phe-Pro-Arg-Gly at positions -7 to -4) .

The Sail site and the amino acid substitution were introduced at nt 76 to 81 and 91 and 92 (nt -30 to -25 and -1 5 and -1 4, respectively, relative to the first nucleotide of mature tPA) during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt 72 to nt 1 1 1 containing nucleotide substitutions at six positions in the tPA sequence: nt 76 (A to G), nt 79 (C to G) , nt 81 (T to C) , nt 91 (A to C) and nt 92 (G to C) . The BamHI site at nt 652 to nt 657 and translational stop codon at amino acid codon 1 80 (nt 642-644) were introduced by synthesizing an oligonucleotide primer complementary to nt 623 to 661 containing nucleotide substitutions at three positions in the tPA sequence: nt 644 (C to A), nt 655 (A to T) and nt 657 (G to C) .

The amplified PCR fragment was incubated with SjaH and BamHI and subjected to agarose gel electrophoresis according to the standard procedures [Sambrook et al. , ( 1 989) Molecular Cloning, 2nd ed. , Cold Spring Harbor Laboratory Press, New York] . The 585 bp DNA was isolated from gel by electroelution and recovered by precipitation with ethanol. (iii) Construction of a vector for expressing human

FGFR1 -tPA fusion protein

The isolated Sail to BamHI fragment encoding the portion of human tPA was ligated into the SaN and BamHI sites of pUC 1 8 to generate plasmid HTPA3/4-pUC 1 8. HTPA3/4-pUC 1 8 was then digested with Hindlll and Sail into which the isolated Hindlll to Sail FGFR1 - encoding fragment was inserted. The plasmid carrying the FGFR1 -tPA chimeric DNA was digested with Hindlll and BamHI, subjected to agarose gel electrophoresis and the 1 ,426 bp DNA fragment was excised from the gel and isolated as described above. The resulting DNA encodes a 472 amino acid peptide comprised of amino acids 1 -284 of human FGFR1 , a 1 0 amino acid linker sequence VDARFPRGAR, derived from the human tPA signal peptide, and amino acids 1 -1 78 from human tPA. The resulting DNA encoding the FGFR1 -tPA fusion protein is shown in SEQ ID No: 1 and the deduced amino acid is shown in SEQ ID No: 2.

The DNA of SEQ ID No. 1 was digested with Hindlll to BamHI and the 1 ,434 bp fragment (nt 2-1 435 of SEQ ID No: 1 ) was isolated and ligated into the mammalian expression vector pK4K for recombinant expression of the FGFR1 -tPA fusion protein (Niidome, T. et al. ( 1 994) Biochem. Biophvs. Res. Commun. 203, 1 821 - 1 827) . The plasmid pK4K is a pBR322-based vector that has unique Hindlll and BamHI sites for directional cloning of heterologous DNAs whose expression is under the control of the SV40 early promoter. This plasmid also contains the β- lactamase and DHFR genes for use as selectable markers in prokaryotes and eukaryotic organisms, respectively.

(iv) Expression of FGFR1 -tPA chimeric protein in mammalian cells

Baby hamster kidney cells (BHK cells; Waechter, D.E., et al.

( 1 982) Proc. Natl. Acad . Sci. , USA:79: 1 1 06) were transfected with 5 μg of the FGFR1 -tPA-containing expression plasmid using the CellPhect calcium phosphate method according to manufacturer's instructions (Pharmacia, Sweden) . Transfectants were selected for the presence of the DHFR gene by selecting resistance to methotrexate and maintained in Dulbecco's Eagle medium containing 10% fetal bovine serum and 250 nM methotrexate.

Upon expression, the recombinant FGFR1 -tPA fusion protein is secreted into the surrounding culture medium. Recombinant FGFR1 -tPA fusion protein expression in BHK cells was monitored by sandwich enzyme-linked immunosorbent assays (sandwich ELISAs) . A mouse IgG monoclonal antibody specific for human tPA, designated 1 4-6, was used as the capture antibody and a polyclonal, rabbit anti-lgG antibody conjugated to horseradish peroxidase was used as the secondary-labelled antibody.

(v) Purification of FGFR1 -tPA chimeric protein The recombinant FGFR1 -tPA fusion protein was purified from conditioned medium of BHK-expressing cells by affinity chromatography. Transfected cells were grown as described above and the condition medium was harvested. The osmolarity of the conditioned medium was adjusted to a final concentration of 0.5 M NaCI by the addition of 5 M NaCI solution. The sample was applied onto a column of Cellulofine (Seikagaku Kogyo, Tokyo, Japan) conjugated with anti-tPA 1 4-6 monoclonal antibody previously equilibrated in column buffer [50 mM Tris-HCI, pH 7.5, and 0.5 M NaCI] . The column was then washed with 1 0 column volumes of column buffer and bound fusion protein was eluted from the column by the addition of 0.2 M glycine-HCI, pH 2.5. Fractions (O.5 ml) were collected into a tube containing 0.5 ml of 1 M Tris-HCI, pH 8.0 to neutralize the acidic eluate. Eluted fractions were monitored for the presence of FGFR1 -tPA protein by measuring the absorbance of each fraction at 280 nm. The FGFR1 -tPA-containing fractions were dialyzed against PBS and concentrated to a final concentration of 1 .5-2.0 mg/ml using Centriprep filters (AMICON) . (vi) Analysis of bFGF-FGFR1 interaction The soluble, recombinant FGFR1 -tPA fusion protein was immobilized to a solid support by attachment to the surface of the wells of an enzyme-linked immunosorborbent assay plate (High binding plates, COSTAR) . A 0. 1 ml aliquot of a 1 0/ g/ml solution of rFGFR1 -tPA in PBS was added and the plate was incubated for approximately 1 6 hr at 4°C. Unbound fusion protein was removed by washing three times with an equal volume of cold PBS.

To each well, a 0.1 ml aliquot of blocking buffer (25 mM HEPES, pH 7.5, 1 00 mM NaCI and 0.5% gelatin) was added, and the samples incubated for 1 hr at ambient temperature to prevent non-specific binding of reagents. The wells were washed three times with binding buffer (25 mM HEPES, pH 7.5, 100 mM NaCI and 0.3% gelatin) followed by addition of 0.1 ml of binding buffer supplemented with 2 μg/ml heparin and a range of 1 -20ng/ml of labelled ¹²⁵l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and incubated in the absence or presence of 2.5 /vg/ml unlabeiled bFGF or a test compound for 3 hr at ambient temperature. The buffer was removed by aspiration and the wells were washed twice each with PBS and a solution of 25 mM HEPES, pH 7.5, containing 2 M NaCI. Bound bFGF was dissociated from the immobilized fusion protein by the addition of two aliquots of a solution of 25 mM sodium acetate, pH 4.0, containing 2 M NaCI. The two sodium acetate washes were combined and the amount of radioactivity present was determined using a gamma counter.

The amount of bound radiolabelled bFGF in each well was calculated and the specificity of bFGF binding was analyzed according to Scatchard [Scatchard ( 1 949) Ann . N .Y. Acad . Sci. 51 :6601. From this analysis, a 280 pM dissociation constant (K_D) for the binding of bFGF to the recombinant FGFR1 -tPA fusion protein of was calculated . This value correlates well with 1 30 pM K_D value reported for bFGF binding to native FGFR1 receptors expressed in smooth muscle cells [Saltis et al. ( 1 995) Arteriosclerosis 1 1 8:77-87] ,

B. Membrane-bound FGF receptor assays

(i) Competitive inhibition of FGF binding The rabbit aortic smooth muscle cell line, Rb-1 , expresses high and low affinity FGF receptors [e.g . , see Nachtigal et al. ( 1 989) In Vitro Cell. & Develop . Biol . 25:892-897] . Compounds of formula (I) that have FGF antagonist activity were and can be identified by their ability to compete with ¹²⁵l-bFGF for binding to the FGF receptors expressed on cell surface of such cells [e.g ., see, Mostacelli et al. ( 1 987) vλ Cell. Phvsiol. 1 31 : 1 23-1 301. Rb-1 cells were grown in 24-well plates to near-confluence in

Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) supplemented with 1 0% fetal bovine serum, penicillin ( 1 00 unit/ml) and streptomycin ( 1 00 ug/ml) . The culture medium was removed by aspiration and the cells were incubated in binding buffer [serum-free DMEM supplemented with 20 mM HEPES (pH 7.5) and 0.1 % BSA] containing 2.5 ng/ml recombinant human ¹²⁵l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and varying concentrations of test compound, for 2 hr at ambient temperature. The nonspecific binding of iodinated bFGF to Rb-1 cells was estimated in parallel reactions performed in the presence of an excess of unlabeled bFGF.

The cells were washed twice with cold phosphate-buffered saline (PBS) and the bFGF bound to low affinity heparan sulfate proteoglycan (HSPG) receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM HEPES (pH 7.5) containing 2 M NaCI. Following removal of the low affinity sample, the bFGF bound to high affinity FGF receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM sodium acetate (pH 4.0) containing 2 M NaCI. A 1 ml aliquot from each well was transferred to a polypolyene tube and the amount of radioactivity present in the high affinity samples was determined using a gamma counter.

(ii) Competitive inhibition of EGF binding The specificity of identified FGF antagonists was examined by measuring the ability of compounds to inhibit the binding of epidermal growth factor (EGF) to the surface of Rb-1 cells. Rb-1 cells were grown as described above and incubated in binding buffer containing 2 ng/ml of ¹²⁵I-EGF ( > 750Ci/mmol; Amersham) under similar conditions. Nonspecific binding of radiolabelled EGF was estimated in parallel reactions performed in an excess of unlabeled EGF. After washing the cells twice with cold PBS, specifically bound

EGF was dissociated from the cells by addition of a solution of 0.1 % Triton-X- 1 00 and 5 min incubation at ambient temperature. The amount of radioactivity in each supernatant was measured using a gamma counter. C. Inhibition of ³H-thymidine incorporation

The incorporation of radiolabelled nucleotides into newly synthesized cellular DNA may be used as an indicator of cell proliferation [] . SMCs, such as rat aortic SMCs, incorporate tritiated thymidine into DNA upon stimulation with bFGF, PDGF or EGF. The effectiveness of compounds of formula (I) as FGF antagonists was and can be assessed by measuring the inhibition of tritiated thymidine incorporation into the DNA of cultured SMCs incubated in the presence of bFGF, PDGF or EGF. An inoculum of approximately 2 X 1 0⁴ Rb- 1 cells was added to a plurality of wells and the cells cultured for three days as described in EXAMPLE 1 B(i) . The cells were washed twice with serum-free medium [DMEM supplemented with 0.1 % BSA, 5 μg/ml transferrin, penicillin ( 1 00 unit/ml) and streptomycin ( 1 00 ug/ml)] and cultured for an additional three days in serum-free DMEM medium. After washing twice in serum-free DMEM medium, the follow was added to each well: 400 μl of serum-free DMEM, 50 μl of 3ng/ml of unlabelled bFGF in DMEM and 50 μl of known concentration test compound in DMEM 1 0% DMSO for 23 hr at 37°C in a 5% CO₂ atmosphere. To each well, 1 0μl of tritiated thymidine (³H-thymidine, 50μCi/ml) was added and cells were incubated for 1 hour at 37°C. The medium was removed and the cells were washed twice with cold PBS. An 500 μl aliquot of a cold 1 0% TCA solution was added to each well and the cells incubated at 4°C overnight. After washing three times in cold PBS, the cells were incubated in 500 μl of 0.5 N NaOH for 30 min and the pH of the sample was neutralized by the addition of an equal volume of 0.5 N HCl. The amount of radioactivity present the supernatant of each well was determined using a liquid scintillation counter.

EXAMPLE 2 Identification of compounds of formula (I) useful as bFGF antagonists Rb- 1 cells were grown as described in EXAMPLE 1 B(i) and incubated in binding buffer containing 2ng/ml recombinant human ¹²⁵l- bFGF in the presence of 10, 30, 100, 300 and 1000 μM didansyl-L- lysine (final concentration) . The amount of specifically bound ¹²⁵l-bFGF was measured for each sample and an IC₅₀ was calculated . Under these conditions, the IC₅₀ for didansyl-L-lysine is about 300 μM.

On the contrary, Rb- 1 cells were grown as described in EXAMPLE 1 B(ii) and incubated in binding buffer containing 2ng/ml recombinant human ^{1 25}I-EGF in the presence of 1 0, 30, 1 00, 300 and 1 000 μM didansyl-L-lysine (final concentration) . The amount of specifically bound ¹²⁵I-EGF was measured for each sample. No inhibition of EGF binding was detected, even at the highest concentrations tested . Thus, didansyl-L-lysine specifically inhibits bFGF binding to the FGFR1 receptor.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION

(i) APPLICANT:

(A) NAME: Eisai Co., Ltd.

(B) STREET: 4-6-10 Koishikawa

(C) CITY: Bunkyo-ku Tokyo

( D ) STATE :

(E) COUNTRY: Japan

(F) POSTAL CODE (ZIP): 112-8088

(i) INVENTOR:

(A) NAME: Takatoshi Kawai

(B) STREET: 25-3-101, Matsushiro 3-chome

(C) CITY: Tsukuba-shi, Ibaraki

( D ) STATE :

(D) COUNTRY: Japan

(E) POSTAL CODE (ZIP): 305-0035

(i) INVENTOR:

(A) NAME: Ramnarayan Kalyanaraman

(B) STREET: 11674 Springside Rd.

(C) CITY: San Diego

(D) STATE: California

(D) COUNTRY: USA

(E) POSTAL CODE (ZIP): 92128

(ii) TITLE OF THE INVENTION: COMPOSITIONS AND METHODS FOR MODULATING THE ACTIVITY OF FIBROBLAST GROWTH FACTOR

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: KAWAGUCHI Yoshio

(B) STREET: Yamada Bldg., 1-14, Shinjuku 1-Chome

(C) CITY: Shinjuku-ku

( D ) STATE :

(E) COUNTRY: Japan

(F) ZIP: 160-0022

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 1.5

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING REFERENCE: SAP-666-PCT

(C) FILING DATE: 02-DEC-1998 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/984,462

(B) FILING DATE: 03-DEC-1997

(2) INFORMATION FOR SEQ ID NO : 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1440 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 9...1427 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

AAGCTTGG ATG TGG AGC TGG AAG TGC CTC CTC TTC TGG GCT GTG CTG GTC 50 Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val 1 5 10

ACA GCA ACA CTC TGC ACC GCT AGG CCG TCC CCG ACC TTG CCT GAA CAA 98 Thr Ala Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gin 15 20 25 30

GAT GCT CTC CCC TCC TCG GAG GAT GAT GAT GAT GAT GAT GAC TCC TCT 146 Asp Ala Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser 35 40 45

TCA GAG GAG AAA GAA ACA GAT AAC ACC AAA CCA AAC CCC GTA GCT CCA 194 Ser Glu Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val Ala Pro 50 55 60

TAT TGG ACA TCC CCA GAA AAG ATG GAA AAG AAA TTG CAT GCA GTG CCG 242 Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro 65 70 75

GCT GCC AAG ACA GTG AAG TTC AAA TGC CCT TCC AGT GGG ACC CCA AAC 290 Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn 80 85 90

CCC ACA CTG CGC TGG TTG AAA AAT GGC AAA GAA TTC AAA CCT GAC CAC 338 Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His 95 100 105 110

AGA ATT GGA GGC TAC AAG GTC CGT TAT GCC ACC TGG AGC ATC ATA ATG 386 Arg lie Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser lie lie Met 115 120 125

GAC TCT GTG GTG CCC TCT GAC AAG GGC AAC TAC ACC TGC ATT GTG GAG 434 Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys lie Val Glu 130 135 140

AAT GAG TAC GGC AGC ATC AAC CAC ACA TAC CAG CTG GAT GTC GTG GAG 482 Asn Glu Tyr Gly Ser lie Asn His Thr Tyr Gin Leu Asp Val Val Glu 145 150 155

CGG TCC CCT CAC CGG CCC ATC CTG CAA GCA GGG TTG CCC GCC AAC AAA 530 Arg Ser Pro His Arg Pro lie Leu Gin Ala Gly Leu Pro Ala Asn Lys 160 165 170

ACA GTG GCC CTG GGT AGC AAC GTG GAG TTC ATG TGT AAG GTG TAC AGT 578 Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser 175 180 185 190

GAC CCG CAG CCG CAC ATC CAG TGG CTA AAG CAC ATC GAG GTG AAT GGG 626 Asp Pro Gin Pro His lie Gin Trp Leu Lys His lie Glu Val Asn Gly 195 200 205

AGC AAG ATT GGC CCA GAC AAC CTG CCT TAT GTC CAG ATC TTG AAG ACT 674 Ser Lys lie Gly Pro Asp Asn Leu Pro Tyr Val Gin lie Leu Lys Thr 210 215 220

GCT GGA GTT AAT ACC ACC GAC AAA GAG ATG GAC GTG CTT CAC TTA AGA 722 Ala Gly Val Asn Thr Thr Asp Lys Glu Met Asp Val Leu His Leu Arg 225 230 235

AAT GTC TCC TTT GAG GAC GCA GGG GAG TAT ACG TGC TTG GCG GGT AAC 770 Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn 240 245 250

TCT ATC GGA CTC TCC CAT CAC TCT GCA TGG TTG ACC GTT CTG GAA GCC 818 Ser lie Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu Glu Ala 255 260 265 270

CTG GAA GAG AGG CCG GCA GTG ATG ACC TCG CCC CTG TAC GTC GAC GCC 866 Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Val Asp Ala 275 280 ^' 285

CGA TTC CCA AGA GGA GCC AGA TCT TAC CAA GTG ATC TGC AGA GAT GAA 914 Arg Phe Pro Arg Gly Ala Arg Ser Tyr Gin Val lie Cys Arg Asp Glu 290 295 300

AAA ACG CAG ATG ATA TAC CAG CAA CAT CAG TCA TGG CTG CGC CCT GTG 962 Lys Thr Gin Met lie Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val 305 310 315

CTC AGA AGC AAC CGG GTG GAA TAT TGC TGG TGC AAC AGT GGC AGG GCA 1010 Leu Arg Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala 320 325 330

CAG TGC CAC TCA GTG CCT GTC AAA AGT TGC AGC GAG CCA AGG TGT TTC 1058 Gin Cys His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe 335 340 345 350

AAC GGG GGC ACC TGC CAG CAG GCC CTG TAC TTC TCA GAT TTC GTG TGC 1106 Asn Gly Gly Thr Cys Gin Gin Ala Leu Tyr Phe Ser Asp Phe Val Cys 355 360 365

CAG TGC CCC GAA GGA TTT GCT GGG AAG TGC TGT GAA ATA GAT ACC AGG 1154 Gin Cys Pro Glu Gly Phe Ala Gly Lys Cys Cys Glu lie Asp Thr Arg 370 375 380

GCC ACG TGC TAC GAG GAC CAG GGC ATC AGC TAC AGG GGC ACG TGG AGC 1202 Ala Thr Cys Tyr Glu Asp Gin Gly He Ser Tyr Arg Gly Thr Trp Ser 385 390 395

ACA GCG GAG AGT GGC GCC GAG TGC ACC AAC TGG AAC AGC AGC GCG TTG 1250 Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu 400 405 410

GCC CAG AAG CCC TAC AGC GGG CGG AGG CCA GAC GCC ATC AGG CTG GGC 1298 Ala Gin Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala He Arg Leu Gly 415 420 425 430

CTG GGG AAC CAC AAC TAC TGC AGA AAC CCA GAT CGA GAC TCA AAG CCC 1346 Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro 435 440 445

TGG TGC TAC GTC TTT AAG GCG GGG AAG TAC AGC TCA GAG TTC TGC AGC 1394 Trp Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser 450 455 460

ACC CCT GCC TGC TCT GAG GGA AAC AGT GAC TGA TACTTTGGGA TCC 1440

Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp * 465 470

(2) INFORMATION FOR SEQ ID NO : 2 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 472 amino acids

(C) STRANDEDNESΞ : single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION. SEQ ID NO : 2.

Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala

1 5 10 15

Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gin Asp Ala

20 25 30

Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser Ser Glu

35 40 45

Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val Ala Pro Tyr Trp

50 55 60

Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro Ala Ala 65 70 75 80

Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn Pro Thr

85 90 95

Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His Arg He

100 105 ^' 110

Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser He He Met Asp Ser

115 120 125

Val Val Pro Ser Asp Lys Glv Asn Tvr Thr Cys He Val Glu Asn Glu

130 135 ^' 140

Tyr Gly Ser He Asn His Thr Tyr Gin Leu Asp Val Val Glu Arg Ser 145 150 155 160

Pro His Arg Pro He Leu Gin Ala Gly Leu Pro Ala Asn Lys Thr Val 165 ^' 170 175 Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro

180 185 190

Gin Pro His He Gin Trp Leu Lys His He Glu Val Asn Gly Ser Lys

195 200 205

He Gly Pro Asp Asn Leu Pro Tyr Val Gin He Leu Lys Thr Ala Gly

210 215 220

Val Asn Thr Thr Asp Lys Glu Met Asp Val Leu His Leu Arg Asn Val 225 230 235 240

Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser He

245 250 255

Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu Glu Ala Leu Glu

260 265 270

Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Val Asp Ala Arg Phe

275 280 285

Pro Arg Gly Ala Arg Ser Tyr Gin Val He Cys Arg Asp Glu Lys Thr

290 295 300

Gin Met He Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val Leu Arg 305 310 315 320

Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gin Cys

325 330 335

His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly

340 345 350

Gly Thr Cys Gin Gin Ala Leu Tyr Phe Ser Asp Phe Val Cys Gin Cys

355 360 365

Pro Glu Gly Phe Ala Gly Lys Cys Cys Glu He Asp Thr Arg Ala Thr

370 375 380

Cys Tyr Glu Asp Gin Gly He Ser Tyr Arg Gly Thr Trp Ser Thr Ala 385 390 395 400

Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gin

405 410 415

Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala He Arg Leu Gly Leu Gly

420 425 430

Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys

435 440 445

Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro

450 455 460

Ala Cys Ser Glu Gly Asn Ser Asp 465 470

Claims

1 . A method of treating an FGF-mediated disorder, comprising administering to a mammal an effective amount of a compound of formula (I) :

Ar SO- N - Y - N - SO- Ar,

I I

^R 1 ^R ₂ or a pharmaceutically acceptable derivative thereof, wherein:

Ar, and Ar₂ are each independently selected from the group consisting of cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that are unsubstituted or substituted with one or more substituents Q; each Q is independently selected from the group consisting of halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylaikyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diarylaminosulfonyl; R, and R₂ are each independently H or lower alkyl;

Y is selected from the group consisting of hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene; and the effective amount is sufficient to ameliorate the symptoms of the FGF-mediated disorder.

2. The method of claim 1 , wherein Ar, and Ar₂ are each independently selected from the group consisting of phenyl, benzyl, naphthyl, azulenyl, indolyl, fluorenyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazoiinyl, benzothiazolyl, dibenzothiazoyl, naphthridinyl, xanthenyl, pteridinyl, acridinyl, phenazinyl, phenothiazinyl and anthracenyl that are unsubstituted or substituted with one or more Q substituents.

3. The method of any of claims 1 -2, wherein each Q is selected from the group consisting of amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkylsufonyl, arylsulfonyl, aminosulfonyl and alkylaminosulfonyl.

4. The method of any of claims 1 -3, wherein Ar, and Ar₂ are each independently selected from naphthyl and Q-substituted naphthyl.

5. The method of any of claims 1 -4, wherein Ar, or Ar₂ is 5- dimethylamino-1 -naphthyl.

6. The method of any of claims 1 -5, wherein Y is selected from the group consisting of 1 -hydroxycarbonylethylene, 1 - hydroxycarbonylpropylene, 1 -hydroxycarbonylbutylene and 1 - hydroxycarbonylpentylene.

7. The method of any of claims 1 -5, wherein Y is selected from the group consisting of propylene, butylene, pentylene and hexylene.

8. The method of any of claims 1 -6, wherein

Ar, is 5-dimethylamino-1 -naphthyl or naphthyl; Y is 1 -hydroxycarbonylbutylene or 1 - hydroxycarbonylpentylene; and Ar₂ is 5-dimethylamino-1 -naphthyl or naphthyl.

9. The method of any of claims 1 -8, wherein the compound is di-dansyl lysine.

1 0. The method of any of claims 1 -9, wherein the FGF-mediated disorder is selected from the group consisting of restenosis, tumors, rheumatoid arthritis, ophthalmic disorders, proliferative diabetic retinopathies, dermatoiogical disorders and cellular proliferative disorders.

1 1 . The method of claim 9, wherein the FGF-mediated disorder is selected from the group consisting of restenosis, tumors, rheumatoid arthritis, ophthalmic disorders, proliferative diabetic retinopathies, dermatoiogical disorders and cellular proliferative disorders.

1 2. A pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, an effective amount of a compound of formula (I) : Ar SO- N Y - N - SO- Ar

or a pharmaceutically acceptable derivative thereof, wherein: Ar, and Ar₂ are each independently selected from the group consisting of cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that are unsubstituted or substituted with one or more substituents Q; each Q is independently selected from the group consisting of halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to

2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylaikyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diarylaminosulfonyl;

R, and R₂ are each independently H or lower alkyl; and Y is selected from the group consisting of hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene, wherein the effective amount is sufficient for treatment of an FGF-mediated disorder.

1 3. The pharmaceutical composition of claim 1 2, wherein Ar, and Ar₂ are each independently selected from the group consisting of phenyl, benzyl, naphthyl, azulenyl, indolyl, fluorenyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazoiinyl, benzothiazolyl, dibenzothiazoyl, naphthridinyl, xanthenyl, pteridinyl, acridinyl, phenazinyl, phenothiazinyl and anthracenyl that are unsubstituted or substituted with one or more Q substituents.

1 4. The pharmaceutical composition of any of claims 1 2-1 3, wherein the Q substituents are selected from the group consisting of amino, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkylsufonyl, arylsulfonyl, aminosulfonyl and alkylaminosulfonyl.

1 5. The pharmaceutical composition of any of claims 1 2-14, wherein Ar, and Ar₂ are each independently selected from naphthyl and Q-substituted naphthyl.

1 6. The pharmaceutical composition of any of claims 1 2-1 5, wherein Ar, or Ar₂ is 5-dimethylamino-1 -naphthyl.

1 7. The pharmaceutical composition of any of claims 1 2-1 6, wherein Y is selected from the group consisting of 1 - hydroxycarbonylethylene, 1 -hydroxycarbonylpropylene, 1 - hydroxycarbonylbutylene and 1 -hydroxycarbonylpentylene.

1 8. The pharmaceutical composition of any of claims 1 2-1 6, wherein Y is selected from the group consisting of propylene, butylene, pentylene and hexylene.

1 9. The pharmaceutical composition of any of claims 1 2-1 7, wherein

Ar, is 5-dimethylamino-1 -naphthyl or naphthyl;

Y is 1 -hydroxycarbonylbutylene or 1 - hydroxycarbonylpentylene; and Ar₂ is 5-dimethylamino- 1 -naphthyl or naphthyl.

20. The pharmaceutical composition of any of claims 1 2-1 9, wherein the compound is di-dansyl lysine.

21 . The composition of any of claims 1 2-20 that is formulated for topical or local application to the eye.

22. The composition of any of claims 1 2-20 that is formulated for topical or local application to the skin.

23. The composition of any of claims 1 2-20 that is formulated for intravenous, intramuscular or parenteral administration.

24. The pharmaceutical composition of any of claims 1 2-23 that is formulated for single dosage administration.

25. An article of manufacture, comprising packaging material and a pharmaceutical composition of any of claims 1 2-24 contained within the packaging material, wherein the pharmaceutical composition is effective for antagonizing the effects of FGF, ameliorating the symptoms of an FGF-mediated disorder, or inhibiting the binding of an FGF peptide to an FGF receptor, and the packaging material includes a label that indicates that the pharmaceutical composition is used for antagonizing the effects of FGF, inhibiting the binding of an FGF peptide to an FGF receptor or treating an FGF-mediated disorder.

26. A method for treating an FGF-mediated ophthalmic disorders, comprising topically or locally administering to an affected area of the eye an effective amount of a compound of formula (I):

Ar: SO- N Y N ΓÇö SO- Ar₂

R , ^R2 or a pharmaceutically acceptable derivative thereof, wherein :

Ar, and Ar₂ are each independently selected from the group consisting of cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that are unsubstituted or substituted with one or more substituents Q; each Q is independently selected from the group consisting of halogen, hydroxy, nitrite, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylaikyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy. perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diarylaminosulfonyl;

R, and R₂ are each independently H or lower alkyl; Y is selected from the group consisting of hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene; and the amount is effective to ameliorate the symptoms of the FGF- mediated ophthalmic disorder.

27. The method of claim 26, wherein the composition comprises an effective amount of didansyl lysine.

28. A method of treating a mammal having an FGF-mediated tumorigenic pathophysiological condition comprising, administering a therapeutically effective amount of a compound of formula (I) :

Ar SO- N Y N ΓÇö SO- Ar₂

^R 1 ^R2 or a pharmaceutically acceptable derivative thereof, wherein:

Ar, and Ar₂ are each independently selected from the group consisting of cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that are unsubstituted or substituted with one or more substituents Q; each Q is independently selected from the group consisting of halogen, hydroxy, nitrile, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylaikyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkyiaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl and diarylaminosulfonyl;

R, and R₂ are each independently H or lower alkyl; Y is selected from the group consisting of hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene; and the amount is effective for inhibiting proliferation of tumorigenic cells.

29. The method of claim 28, wherein the composition comprises an effective amount of didansyl lysine.

30. A method for inhibiting the binding of an FGF peptide to an FGF receptor, comprising contacting the receptor with an FGF peptide and with a compound of formula (I) :

Ar SO- N Y ΓÇö N SO- Ar

₁ ^R ₂ or a pharmaceutically acceptable derivative thereof, wherein: Ar, and Ar₂ are each independently selected from the group consisting of cycloalkyl, heterocycle, aryl, heteroaryl and heteroaralkyl that are unsubstituted or substituted with one or more substituents Q; each Q is independently selected from the group consisting of halogen, hydroxy, nitrite, nitro, formyl, mercapto, carboxy, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylaikyl, heteroarylalkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylamino- carbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, alkoxycarbonylamino, arylcarbonylamino, aryloxycarbonylamino, azido, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsufonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyi and diarylaminosulfonyl; R, and R₂ are each independently H or lower alkyl;

Y is selected from the group consisting of hydroxycarbonylalkylene, lower alkylene, lower alkenylene and lower alkynylene, wherein the contacting is effected prior to, simultaneously with, or subsequent to contacting the receptor with the peptide.

31 . The method of claim 30, wherein the composition comprises di-dansyl lysine.

32. The method of any of claims 30-31 , wherein the FGF peptide is bFGF.