WO2001056994A1 - Integrin antagonists - Google Patents

Integrin antagonists Download PDF

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Publication number
WO2001056994A1
WO2001056994A1 PCT/US2001/003347 US0103347W WO0156994A1 WO 2001056994 A1 WO2001056994 A1 WO 2001056994A1 US 0103347 W US0103347 W US 0103347W WO 0156994 A1 WO0156994 A1 WO 0156994A1
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Prior art keywords
aryl
heteroaryl
alkyl
antagonist
alkynyl
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PCT/US2001/003347
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French (fr)
Inventor
Zhongli Zheng
Mary Beth Carter
Julio H. Cuervo
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Biogen, Inc.
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Priority to AU2001234741A priority Critical patent/AU2001234741A1/en
Publication of WO2001056994A1 publication Critical patent/WO2001056994A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/06—1,2,4-Oxadiazoles; Hydrogenated 1,2,4-oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to organic compounds, which are useful for blocking the activity of integ ⁇ n molecules.
  • This invention also relates to compositions containing such compounds and methods of treatment using such compounds.
  • VLA very late antigen
  • Adhesion molecules of the VLA family presently include VLA-1, -2, -3, -4, -5, -6, -9, -10, -11, -v in which each of the molecules comp ⁇ se a ⁇ l chain non- covalently bound to a ⁇ chain, ( ⁇ l, oc2, oc3, oc4, ⁇ 5, oc6, ⁇ 9, al l, av), respectively.
  • the lnteg ⁇ n a4 ⁇ 7 is also intended to be included within the VLA family
  • Such molecules play an essential role in both normal and pathophysiological processes in a wide variety of tissues. For instance, cellular adhesion and trafficking across the vascular interface plays an essential role in both physiological and pathophysiological processes of acute brain injury (Garcia et al 1994, Am. J. Pathol 144: 188; Becker et al, 1997 PNAS 94: 10873). Further, leukocyte migration into glomeru is a typical feature of human glomeruloneph ⁇ tis (GN) and leukocytes are key mediators of kidney damage Therefore, the targeting of specific and relevant molecules in certain disease conditions without interfering with normal cellular functions is essential for an effective and safe therapeutic agent that inhibits cell-cell and cell-mat ⁇ x interactions.
  • GN human glomeruloneph ⁇ tis
  • VLA-1, -2, -4, -6 and ⁇ 4 ⁇ 7 neutralizing antibodies or blocking peptides that inhibit the interaction between these respective VLA moieties and their ligands are known.
  • VLA-4 ( ⁇ 4 ⁇ l) and its ligand VCAM-1 some antibody antagonists have proven efficacious both prophylactic ally and therapeutically in several animal models of disease, including i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis (for example, see T. Yednock et al., "Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha4betal integrin.' Nature, 356, 63 (1993) and E.
  • Keszthelyi et al. Evidence for a prolonged role of alpha4 integrin throughout active experimental allergic encephalomyelitis.” Neurology, 47. 1053 (1996)); ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma (for example, see W. M. Abraham et al., 'alpha4-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.” J. Clin. Invest. 98. 776 (1993) and A. A. Milne and P. P. Piper, Hole of VLA-4 integrin in leukocyte recruitment and bronchial hyperresponsiveness in the guinea-pig.” Eur.
  • One aspect of the invention is a compound having the following structural Formula I:
  • n is an integer from 1 to 2;
  • W is selected from
  • X is selected from S(O) 2 , S(O) 2 NR e , C(O), C(O)O, C(O)NR e , CR f R g
  • Y is selected from N, CR j
  • R° is selected from R 4 and
  • Q is selected from -CR j -, C(O), O, S(O)lock, NH, NXR 1 B is selected from the group consisting of 1 a bond,
  • heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur;
  • alkyl- heteroaryl wherein alkyl, alkenyl. and alkynyl are optionally substituted with one to four substituents independently selected from R a , and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from
  • alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and R x
  • phenyl and Cy are optionally substituted with one to four substituents independently selected from R y R 4 is
  • alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and R ⁇ and phenyl and Cy are optionally substituted with one to four substituents independently selected from R y Or R 4 with either R f or R s forms a mono- or bi-cychc ring containing 0-2 heteroatoms selected from nitrogen, oxygen or sulfur, wherein nitrogen is optionally substituted with R J , C(O)R e , SO 2 R e or SO 2 NR d R e R 6 , R 7 , and R 8 are each independently selected from the group consisting of R d and R x or two of R 6 , R 7 , and R 8 and the atom to which both are attached, or two of R 6 , R 7 , and R 8 and the two adjacent atoms to which they are attached, together form a 5-7 membered saturated or unsaturated monocyclic ⁇ ng containing zero to three heteroatoms selected from N, 0 or S
  • alkyl-heteroaryl wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from R x and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from R y ; or R 12 , R n and the carbon to which they are attached form a 3-7 membered mono- or bicyclic ⁇ ng containing 0-2 heteroatoms selected from N, 0 and S, wherein nitrogen is optionally substitiuted with R J , C(O)R c , SO 2 R c or SO 2 NR d R e R 16 is selected from a bond, NR ⁇ O and S(O)terrorism R a ⁇ s 1 Cy, or
  • alkyl- aryl wherein alkyl, alkenyl, .ilkynyl, aryl, heteroaryl are optionally substituted with a group independently sel scted from R c ; R c is
  • R d and R e are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2- 10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl and Cy is optionally substituted with one to four substituents independently selected from R c ; or R d and R e together with the atoms to which they are attached form a heterocyclic ring of 5 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen; wherein nitrogen is optionally substitiuted with R J , C(O)R e , SO 2 R e or SO 2 NR d R e
  • R and R g are independently selected from R or R and R g together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms mdependently selected from oxygen, sulfur and nitrogen wherein nitrogen is optionally substituted with R J , C(O)R e , SO 2 R e or SO 2 NR d R e R' is
  • alkyl, alkenyl, alkynyl and aryl are each optionally substituted with one to four substituents independently selected from R
  • R J is selected from hydiogen, Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein said alkyl, alkenyl, alkynyl, heteroaryl, and Cy is optionally substituted with one to four substituents independently selected from R c R k and R m are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2-
  • R x is selected from
  • R y is l a group selected from R x ,
  • heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl and aryl are each optionally substituted with one to four substituents independently selected from R x ;
  • Cy is cycloalkyl, heterocycyl, aryl, or heteroaryl
  • R 9 is selected from H and R y R z is selected from
  • the present compounds are biologically active small molecules and are generally composed of several domains: a) an acyl (including sulfonyl) moiety and a heterocycle #1 or a substituted aromatic ⁇ ng, b) a heterocycle #2, and c) acid and a sidechain, and are named in a manner similar to that used to name ohgopeptides.
  • an integrin "antagonist” includes any compound that inhibits a "plurality" (defined below) of integ ⁇ ns from binding with an integrin ligand and/or ligand leceptors.
  • an integrin "antagonist” also refers to agents claimed herein which can inhibit or block integ ⁇ n and/or integ ⁇ n hgand-mediated binding or which can otherwise modulate integ ⁇ n and/or integrin ligand function, e.g., by inhibiting or blocking lnteg ⁇ n-hgand mediated integ ⁇ n signal transduction.
  • Such an antagonist of the integ ⁇ n/integ ⁇ n ligand interaction is an agent which has one or more of the following properties: (1) it coats, or binds to, a plurality of integ ⁇ ns (e.g, ⁇ 4 ⁇ 7 , VLA-1, VLA-9 and VLA-1 ) on the surface of such integrin bea ⁇ ng or secreting cell with sufficient specificity to inhibit an integrin ligand/mteg ⁇ n interaction, e.g., the collagen/VLA-1 interaction; (2) it coats, or binds to, a plurality of integ ⁇ ns on the surface of an mteg ⁇ n-bea ⁇ ng or secreting cell with sufficient specificity to modify, and preferably to inhibit, transduction of an mteg ⁇ n-mediated signal e.g., collagen/VLA-1 -mediated signaling; (3) it coats, or binds to, a plurality of integ ⁇ n receptors, (e.g., collagen only or collagen and V
  • the integrin antagonist has one or both of properties 1 and 2. In other preferred embodiments the antagonist has one or both of properties 3 and 4 Moreover, more than one antagonist can be administered to a patient, e.g., an agent which binds to an integrin can be combined with an agent which binds to its ligand.
  • An antagonist of the invention has "biological activity" if it inhibits a plurality integrins from binding with an integ ⁇ n ligand and/or integ ⁇ n receptor as determined by in vitro and in vivo tests known to workers having ordinary skill in the art.
  • a "pan- ⁇ l antagonist” includes any compound that inhibits a plurality (defined below) of integ ⁇ ns containing the ⁇ l subunit from binding with an integ ⁇ n ligand and/or receptor such as any receptor for the ⁇ l subunit.
  • a "pan- ⁇ l antagonist” also refers to agents claimed herein which can inhibit or block integ ⁇ n and/or integ ⁇ n gand-mediated binding or which can otherwise modulate integrin and/or integ ⁇ n ligand function, e.g., by inhibiting or blocking lnleg ⁇ n-hgand mediated integ ⁇ n signal transduction.
  • Such an antagonist of the lnteg ⁇ n/integ ⁇ n ligand interaction is an agent which has one or more of the properties (1) through (4) as described above.
  • a pan- ⁇ l antagonist of the invention has "biological activity" if it inhibits a plurality of ⁇ l subunit containing integrins (including ⁇ 4 ⁇ 7) from binding with an integrin ligand and/or receptor such as any receptoi for the ⁇ l subunit or, as defined above, any receptor for the ⁇ 4 ⁇ 7 integ ⁇ n.
  • biological activity is determined by in vitro and in vivo tests known to workers having oidinary skill in the art
  • Alkyl as well as other groups having the prefix "alk”, such as alkoxy, alkanoyl, means carbon chains which may be lmeai or branched or combinations thereof Examples ot alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like "Alkenyl” means carbon chains which contain at least one carbon-carbon double bond, and which may be linear or branched or combinations thereof.
  • alkenyl examples include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2- methyl-2- butenyl, and the like
  • alkynyl means carbon chains which contain at least one carbon-carbon t ⁇ ple bond, and which may be linear or branched or combinations thereof.
  • alkynyl examples include ethynyl, propargyl, 3-methyl-l-pentenyl, 2-heptynyl and the like.
  • Cycloalkyl means mono- or bicyclic saturated carbocyc c ⁇ ngs, each of which having from 3 to 10 carbon atoms. The term also includes monocyclic ⁇ ngs fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
  • Aryl means mono- or bicyclic aromatic ⁇ ngs containing only carbon atoms
  • the term also includes aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion.
  • aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3 dihydrobenzofuranyl, benzopyranyl, 1,4-benzod ⁇ oxanyl, and the like
  • Heteroaryl means a mono- or bicyclic aromatic ⁇ ng containing at least one heteroatom selected from N, 0 and S, with each ⁇ ng containing 5 to 6 atoms.
  • heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, py ⁇ dyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, lmidazolyl, t ⁇ azolyl, tetrazolyl, furanyl, t ⁇ azinyl, thienyl, py ⁇ midyl, py ⁇ dazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)py ⁇ dyl, quinolyl, indolyl, isoquinolyl, and the like.
  • Heterocyclyl means mono- or bicyclic saturated ⁇ ngs containing at least one heteroatom selected from N, S and 0, each ot said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen.
  • the term also includes monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion.
  • heterocyclyl examples include pyrro dinyl, pipe ⁇ dinyl, piperazinyl, lmidazohdinyl, 2,3-d ⁇ hydrofuro(2,3-b) pyndyl, benzoxazinyl, tetrahydrohydroquinohnyl, tetrahydroisoquinohnyl, dihydroindolyl, and the like.
  • the term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4 pyndones attached through the nitrogen or ⁇ -substituted- (1H,3H) py ⁇ m ⁇ d ⁇ ne-2,4-d ⁇ ones ( ⁇ -substituted uracils)
  • Halogen includes fluo ⁇ ne, chlorine, bromine and iodine. "Plurality” is intended to mean. (I) any single integrm; or (II) two or more integ ⁇ ns
  • the present methods utilize (l) molecules capable of inhibiting any combination of two or more different integrins such as a molecule capable of antagonizing both VLA-4 ( ⁇ 4 ⁇ l) and ⁇ 4 ⁇ 7 or VLA-2 ( ⁇ 2 ⁇ l), VLA-6 ( ⁇ 6 ⁇ l) and VLA-4, and so on; or (n) molecules capable of selectively inhibiting any one integ ⁇ n, such as ⁇ 4 ⁇ 7 only or VLA-4 only.
  • a pan-betal antagonist inhibits any single integ ⁇ n containing a betal subunit (including ⁇ 4 ⁇ 7) or two or more integ ⁇ ns that contain a betal subunit (such two or more integ ⁇ ns which may include ⁇ 4 ⁇ 7).
  • Polymer has its art recognized meaning as being a molecule constructed from many smaller structural units called “monomers”, bonded together (preferably covalently) in any pattern.
  • the term includes linear molecules and branched molecules.
  • the term also includes homopolymers where only one species of monomer is used to build the molecule, or copolymers where the molecule is composed of two different types of monomers and so on Copolymers also include polymers where the distribution of monomers is random, alternating copolymers, block copolymers and graft copolymers.
  • the polymer is 'biocompatible'
  • a "biocompatible" substance as that term is used herein, is one that has no unacceptable toxic or injurious effects on biological function
  • Antagonists of the invention are 'small molecules' which are organic molecules.
  • a "small molecule”, as defined herein, has a molecular weight generally less than 2000.
  • the term "effective amount” as used herein, means an amount of a compound of the present invention which inhibits a "plurality" (defined herein) of integrins from binding with an integrin ligand and/or integ ⁇ n receptor, as determined by in vitro and in vivo tests known to workers having ordinary skill in the art.
  • Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereome ⁇ c mixtures and individual diastereomers.
  • the present invention is meant to comprehend all such lsome ⁇ c forms of the compounds of Formula I.
  • Some of the compounds desc ⁇ bed herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geomet ⁇ c isomers.
  • Some ot the compounds desc ⁇ bed herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto- enol tautomers.
  • the individual tautomers as well as mixture thereof are encompassed with compounds of Formula I
  • Compounds of the Formula I may be separated into diastereoisome ⁇ c pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof
  • a suitable solvent for example methanol or ethyl acetate or a mixture thereof
  • the pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid as a resolv ng agent.
  • any enantiomer of a compound of the general Formula I may be obtai ⁇ ed by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
  • Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are substituted with R 7 and R 0 .
  • R 9 is selected from H and R y R z is selected from 1 OR d
  • Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are optionally substituted with one to four substituents independently selected from R x and R is selected from H and R y .
  • R z is selected from
  • Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, nd heteroaryl substituted heteroaryl and further wherein
  • aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from R and all other designations and substituents are as recited above.
  • Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from R x , R 9 is selected from H and R y and all other designations and substituents are as recited above
  • Other antagonists of the invention are shown below as Formula VI:
  • Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from R x and all other designations and substituents are as recited above.
  • Further se ⁇ es are those of Formulae VIII, IX, XI, XII, and XIII, where all designations and substituents are as recited above VIII
  • Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from R x , R 9 is selected from H and R y , R 7 is selected from
  • a single polymer molecule may be employed for conjugation with a integrin antagonist, although it is also contemplated that more than one polymer molecule can be attached as well.
  • Conjugated integrin antagonist compositions of the invention may find utility in both in vivo as well as non- in vivo applications.
  • the conjugating polymer may utilize any other groups, moieties, or other conjugated species, as appropriate to the end use application. By way of example, it may be useful in some applications to covalently bond to the polymer a functional moiety imparting UV-degradation resistance, or antioxidation, or other properties or characteristics to the polymer.
  • the polymer may contain any functionality, repeating groups, linkages, or other constitutent structures which do not preclude the efficacy of the conjugated integ ⁇ n antagonist composition for its intended purpose.
  • Illustrative polymers that may usefully be employed to achieve these desirable charactenstics are described herein below in exemplary reaction schemes.
  • the polymer may be coupled to the antagonist to form stable bonds that are not significantly cleavable by human enzymes.
  • bonds that are not 'significantly' cleavable requires that no more than about 20% of the bonds of the compound are cleaved within a 24 hour period, as measured by standard techniques in the art including, but not limited to, high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • Integ ⁇ n antagonists of the invention are conjugated most preferably via a terminal reactive group on the polymer although conjugations can also be branched from non-terminal reactive groups.
  • the polymer with the reactive group(s) is designated herein as "activated polymer".
  • the reactive group selectively reacts with reactive groups on the antagonist molecule.
  • the activated polymer(s) is reacted so that attachment may occur at any available integ ⁇ n antagonist functional group.
  • Ammo, carbon, free carboxylic groups suitably activated carbonyl groups, hydroxyl, guanidyl, oxidized carbohydrate moieties, amino, carbon and mercapto groups of the integ ⁇ n antagonist (if available) can be used as attachment sites.
  • the polyme ⁇ c substanc es included herein are preferably water-soluble at room temperature.
  • a non-limiting list of such polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • polyalkylene glycol residues of C1-C4 alkyl polyalkylene glycols preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycol residues of such glycols are advantageously incorporated in the polymer systems of interest.
  • the polymer to which the antagonist is attached can be a homopolymer of polyethylene glycol (PEG) or is a polyoxyethylated polyol, provided in all cases that the polymer is soluble in water at room temperature.
  • Non- hmiting examples of such polymers include polyalkylene oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated glycols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymer is maintained.
  • polyoxyethylated polyols include, for example, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like.
  • the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, and t ⁇ glyce ⁇ des. Therefore, this branching would not necessarily be seen as a foreign agent in the body.
  • a general formula for PEG and its derivatives is R"-(CH2CH2O)[x]-(CH2)[y]- R', where (x) represents the degree of polymerization or number of repeating units in the polymer chain and is dependent on the molecular weight of the polymer, (y) represents a positive integer, R' is (CHR 1 ), where R 1 is as defined in claim 1 and R" is a capping group (including, without limitation, OH, C[l-4 jalkyl moieties, or va ⁇ ous biologically active and inactive moieties) or is R'.
  • polyethylene glycols PEG's
  • mono-activated, C[l-4 ]alkyl-termmated PAO's such as mono-methyl- terminated polyethylene glycols (mPEG's) are preferred when mono- substituted polymers are desired; bis-activated polyethylene oxides are preferred when disubstituted antagonists are desired.
  • polyalkylene oxides dextran, polyvinyl pyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like may be used.
  • the polymer need not have any particular molecular weight, but it is preferred that the molecular weight be between about 300 and 100,000, more preferably between 10,000 and 40,000. In particular, sizes of 20,000 or more are best at preventing loss of the product due to filtration in the kidneys.
  • Polyalkylene glycol de ⁇ vatization has a number of advantageous properties in the formulation of polymer- integ ⁇ n antagonist conjugates in the practice of the present invention, as associated with the following properties of polyalkylene glycol derivatives- improvement of aqueous solubility, while at the same time eliciting no antigenic or lmmunogenic response; high degrees of biocompatibihty; absence of in vivo bi ode gradation of the polyalkylene glycol derivatives; and ease of excretion by living organisms.
  • PEG Polyethylene glycol
  • PAO's polyalkylene oxides
  • PCT WO 93/24476 PEG has also been conjugated to proteins, peptides and enzymes to increase aqueous solubility and circulating life in vivo as well as reduce antigenicity.
  • U.S. Pat Nos. 5,298,643 and 5,321,095, both to Greenwald, et al PCT WO 93/24476 discloses using an ester linkage to covalently bind an organic molecule to water-soluble polyethylene glycols.
  • an integ ⁇ n antagonist covalently bonded to the polymer component in which the nature of the conjugation involves one or more noncleavable covalent chemical bonds which, preferably, are resistant to degradation by human enzymes.
  • Greenwald et al., supra disclose biologically-active conjugates having substantially hydrolysis-resistant bonds (linkages) between a polyalkylene oxide and the target moiety.
  • a noncleavable linker suitable for the antagonists of the present invention is-
  • R )0 and Rn are independently selected from the group consisting of H, C ⁇ _ 6 alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substituted heteroalkyls and substituted Ci 6 alkyls, q is a positive integer and F is selected from O, NR 1 , S, SO, SO 2 .
  • the linkages between a polymer and the antagonist of the invention is cleavable, allowing for control in terms of the time course over which the polymer may be cleaved from the integrin antagonist. This covalent bond between the integ ⁇ n antagonist and the polymer may be cleaved by chemical or enzymatic reaction.
  • PAO acids can be synthesized by converting mPEG-OH to an ethyl ester. See also Gehrhardt, H., et al. Polymer Bulletin 18: 487 (1987) and Veronese, F. M., et al., J. Controlled Release 10; 145 (1989).
  • the PAO-acid can be synthesized by converting mPEG-OH into a t-butyl ester. Ohya, et al., J. Bioactive and Compatible Polymers Vol.
  • doxorubicin-PEG conjugates which are prepared by linking the two substituents via va ⁇ ous linkages including esters.
  • other polyalkylene oxide de ⁇ vatives of the foregoing such as the polypropylene glycol acids, POG acids, etc., as well as other bifunctional linking groups are also contemplated.
  • the polymer- integrin antagonist product retains an acceptable amount of activity. Concurrently, portions of polyethylene glycol are present in the conjugating polymer to endow the polymer- integ ⁇ n antagonist conjugate with high aqueous solubility and prolonged blood circulation capability.
  • reaction schemes desc ⁇ bed herein are provided for the purposes of illustration only and are not to be limiting with respect to the reactions and structures which may be utilized in the modification of the integ ⁇ n antagonist, e.g , to achieve solubility, stabilization, and cell membrane affinity for parenteral and oral administration.
  • the activity and stability of the integ ⁇ n antagonist conjugates can be va ⁇ ed in several ways by using a polymer of different molecular size. Solubilities of the conjugates can be va ⁇ ed by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition. Salts
  • salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts de ⁇ ved from inorganic bases include aluminum, ammonium, calcium, copper, feme, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
  • Salts de ⁇ ved from pharmaceutically acceptable organic nontoxic bases include salts of pnmary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resms, such as arginine, betaine, caffeine, choline, N, N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-d ⁇ methylam ⁇ noethanol, ethanolamine, ethylenediamine, N-ethyl- morpho ne, N-ethylpipe ⁇ dine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morphohne, piperazine, pipe ⁇ dine, polyamine resins, procaine, pu ⁇ nes, theobromine, t ⁇ ethylamine, t ⁇ methylamine, tnpropylamine, tromethamine,
  • Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfomc, fuma ⁇ c, gluconic, glutamic, hydrobromic, hydrochloric, lsethionic, lactic, maleic, malic, mandehc, methanesulfonic, mucic, nitric, pamoic, pantothenic, phospho ⁇ c, succinic, sulfu ⁇ c, tartaric, p-toluenesulfonic acid, and the like.
  • Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfu ⁇ c, and tartaric acids.
  • references to the compounds of Formula I aie meant to also include the pharmaceutically acceptable salts.
  • Utilities The ability of the compounds of Formula I to antagonize the actions of any VLA integ ⁇ n containing a ⁇ l subunit (including alpha4 beta 7) makes them useful for preventing or reversing the symptoms, disorders or diseases induced by the binding of VLA to its va ⁇ ous hgands.
  • these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation and be useful in conditions such as acute or chronic renal failure or acute brain injury.
  • another aspect of the present invention piovides a method for the treatment (including prevention, alleviation, amelioration or suppression) of diseases or disorders or symptoms, including fibrotic conditions and an inflammatory disorder mediated by integrin binding and cell adhes on activation, which comprises administering to a mammal an effective amount cf a compound of Formula I.
  • an infla nmatory disorder includes, but is not limited to, skin related conditions such as psoriasis, eczema, burns and dermatitis.
  • Other inflammatory disorders contemplated for treatment by the methods of the present invention include, but are not limited to the treatment of asthma, bronchitis, menstrual cramps, tendinitis, bursitis, and the treatment of pain and headaches, or as an antipyretic for the treatment of fever.
  • the methods of the invention also would be useful to treat gastrointestinal conditions such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis and for the prevention of colorectal cancer.
  • the methods of the invention would be useful in treating inflammation in such diseases as vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring after injury, myocardial ischemia, and the like.
  • diseases as vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring after injury, myocardi
  • the methods of the invention would also be useful in the treatment of allergic rhinitis, respiratory distress syndrome, endotoxin shock syndrome, and atherosclerosis as well as asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, organ transplantation rejection, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, tumor metastasis and atherosclerosis.
  • Antagonists of the present invention may also be useful in treating a subject with a fibrotic condition.
  • fibrotic condition refers to, but is not limited to, subjects afflicted with fibrosis of an internal organ, subjects afflicted with a dermal fibrosing disorder, and subjects afflicted with fibrotic conditions of the eye.
  • Fibrosis of internal organs occurs in disorders such as pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, renal interstitial fibrosis, renal fibrosis in patients receiving cyclosporin, and HIV associated nephropathy.
  • Dermal fibrosing disorders include, but are not limited to, scleroderma, morphea, keloids, hypertrophic scars, familial cutaneous collagenoma, and connective tissue nevi of the collagen type.
  • Fibrotic conditions of the eye include conditions such as diabetic retinopathy, postsurgical scarring (for example, after glaucoma filtering surgery and after cross-eye surgery), and prohferative vitreoretinopathy
  • Additional fibrotic conditions which may be treated by the methods of the present invention include: rheumatoid arth ⁇ tis, diseases associated with prolonged joint pain and deteriorated joints; progressive systemic sclerosis, polymyositis, dermatomyositis, eosinophihc fascitis, morphea, Raynaud's syndrome, and nasal polyposis.
  • fibrotic conditions which may be treated the methods of present invention also include inhibiting overproduction of scarring in patients who are known to form keloids or hypertrophic scars, inhibiting or preventing scar ⁇ ng or overproduction of scarring du ⁇ ng healing of various types of wounds including surgical incisions, surgical abdominal wounds and traumatic lacerations, preventing or inhibiting scar ⁇ ng and reclosing of arteries following coronary angioplasty, preventing or inhibiting excess scar or fibious tissue formation associated with cardiac fibrosis after infarction and in hypersensitive vasculopathy
  • the cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of cells expressing integrins to extracellular matrix components such as collagen or fibronectin coated plates In this assay microtiter wells are coated with, for example, collagen.
  • test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells. The cells are allowed to incubate in the wells for at least 30 minutes Following incubation, the wells are emptied and washed Inhibition of binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound
  • Integrin expressing cells that may be utilized in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymphocytes (PBLs). These cells are commercially available and may be fluorescently or radioactively labeled if desired.
  • a direct binding assay may also be employed to quantitate the inhibitory activity of the compounds of this invention (“DBA”)
  • in vitro assays such as the adhesion inhibition and direct binding assays desc ⁇ bed above, substitute the appropnate lnteg ⁇ n-expressing cell and corresponding ligand.
  • PMNs polymorphonuclear cells express integ ⁇ ns on their surface and bind to ICAM. Integ ⁇ ns are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay.
  • VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin. IN VIVO TESTING
  • A. Contact Hypersensitivity An exemplary animal model is desc ⁇ bed by P.L. Chisholm et al., "Monoclonal Antibodies to the Integ ⁇ n ⁇ -4 Subunit Inhibit the Mu ⁇ ne Contact Hypersensitivity Response", Eur J. Immunol., 23, pp. 682-688 (1993) and in "Current Protocols in Immunology", J E. Cohgan, et al., Eds., John Wiley & Sons, New York, 1, pp. 4.2.1- 4.2 5 (1991), the disclosures of which are herein incorporated by reference.
  • the skin of the animal is sensitized by exposure to an irritant, such as dinitrofluorobenzene, followed by light physical imtation, such as scratching the skin lightly with a sharp edge.
  • an irritant such as dinitrofluorobenzene
  • the animals are re-sensitized following the same procedure.
  • one ear of the animal is exposed to the chemical irritant, while the other ear is treated with a non-irntant control solution.
  • the animals are given va ⁇ ous doses of the antagonists by subcutaneous injection.
  • In vivo inhibition of cell adhesion-associated inflammation is assessed by measunng the ear swelling response of the animal in the treated versus untreated ear.
  • mice Delayed hypersensitivity SRBC-induced delayed type hypersensitivity (DTH) responses are adapted from the protocol of Hurtrel et al. 1992 Cell Immunol. 142:252-263.
  • DTH delayed type hypersensitivity
  • antagonists of the invention which are prepared according to the methods described in the Examples is given prior to antigen challenge on d 5.
  • SRBC- induced DTH is a well characterized in vivo model of inflammation, and in particular psoriasis, that has been used to demonstrate the importance of a variety of cytokines and adhesion molecules in inflammation (Tedder et al. 1995 J. Exp. Med. 181:2259- 2264, Terashita et al.1996 J. Immunol. 156:4638-4643).
  • C. Asthma Another in vivo assay that may be employed to test the antagonists of this invention is the sheep asthma assay. This assay is performed essentially as described in W. M. Abraham et al., " ⁇ -Integrins Mediate Antigen-induced Late Bronchial Responses and Prolonged Airway Hyperresponsiveness in Sheep", J. Clin. Invest., 93, pp. 776-87 (1994), the disclosure of which is herein incorporated by reference. This assay measures inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in allergic sheep D. Renal Failure
  • the agents of the present invention also may be tested in animal models of chronic renal failure.
  • Mammalian models of chronic renal failure in, for example, mice, rats, guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and non-human primates, may be created by causing an appropriate direct or indirect injury or insult to the renal tissues of the animal.
  • Animal models of chronic renal failure may, for example, be created by performing a partial (e.g., 5/6) nephrectomy which reduces the number of functioning nephron units to a level which initiates compensatory renal hypertrophy, further nephron loss, and the progressive decline in renal function which characterizes chronic renal failure.
  • the agents of the present invention may be evaluated for their therapeutic efficacy in causing a clinically significant improvement in a standard marker of renal function when administered to a mammalian subject (e.g., a human patient) in, or at risk of, chronic renal failure.
  • a mammalian subject e.g., a human patient
  • markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium (Na+), urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicme, 13th edition, Isselbac ler et al., ed..., McGraw Hill Text, New York; Luke and Strom (1994), in Internal M ;d ⁇ c ⁇ ne, 4th Edition, J H. Stein, ed., Mosby-Year Book, Inc. St. Louis.).
  • E. Acute Brain Injury Male Sprague Dawley (SD) or spontaneously hypertensive rats (SHRS) are anesthetized using isoflurane and the ⁇ ght middle cerebral artery (MCAO) occluded by insertion of a 4-0 nylon monofilament up the internal carotid artery to the ongm of the middle cerebral artery (MCA) (Zea Longa et al, 1989 Stroke 20:84). After lh the filament is retracted, the lschemic temtory reperfused and the animal allowed to recover After 24h the rats are sacrificed, at which time brains were removed and analyzed histologically to quantify infarct volume.
  • SD Sprague Dawley
  • SHRS spontaneously hypertensive rats
  • PBS vehicle
  • antagonist of the invention by continuous subcutaneous infusion via osmotic mini-pumps.
  • Pnmed mini osmotic pumps for example from Alza Corp.
  • the pumps are loaded to release antagonist.
  • Expe ⁇ mental treatments include a se ⁇ es of injections of antagonist given every other day (post- operation). After 14 days post balloon catheter denudation, all rats are anesthetized and the carotid arte ⁇ es fixed by perfusion at 120 mm Hg pressure with 1% paraformaldehyde and 1.25% glutaraldehyde in 0 1M phosphate buffer, pH 7 4 via a large cannula placed retrograde in the abdominal aorta. Ten minutes before fixation, these animals are given an intravenous injection of Evans blue (0 3 ml in 5% saline solution).
  • Intimal areas are measured by determining the area between lumen and internal elastic lamina. Medial areas are derermined by measu ⁇ ng the area beween internal and external elastic lamina. Intimal/medial area ratios are calculated form the measurements
  • chronic respiratory disease- free Golden Syrain hamster weighing 120-130g are purchased (e.g., from Charles River, Boston, MA) and housed in plastic cages in groups of 4 in facilities approved by the American Association for Accreditation of Laboratory Animal Care. The animals are allowed to acclimate for one week to laboratory conditions pnor to starting the expe ⁇ ments.
  • hamsters in appropnate group receive either bleomycin (5.5 un ⁇ ts/kg/4ml) or an equivalent volume (4ml/kg) of pyrogen free isotonic saline through transoral route
  • the antagonists of the invention are administered by lntrape ⁇ toneal (IP) or intratrachial route at a therapeutic dose to hamster in appropriate groups twice a week for 21-28 days post installation Thereafter, the animals in each group are killed by an overdose ot sodium pentobarbital (100-125 mg/kg lp) and their lungs processed tor biochemical and histopathological studies
  • the experimental lesion is acute mesangial pro ferative glomerulonephntis and is characterized by expansion of the mesangial matrix and hypercellulanty.
  • the nephritis reproducibly progresses through glomerular and tubulointerstitial scarring, to end stage renal disease
  • glomerulonephntis is induced in rats with a single injection of anti- glomerular basement membrane nephrotoxm serum (NTS), derived in rabbits.
  • NTS anti- glomerular basement membrane nephrotoxm serum
  • two groups of rats are treated with daily intravenous injections of saline (the negative control group) or antagonists of the invention
  • the animals are sacnficed and slides are made of the kidneys, which are stained with pe ⁇ odic acid- Schiff solution to emphasize the pathological changes.
  • the extent of glomerular injury can be quantitated by performing glomerular cell counts from 30 randomly selected glomeruh from normal animals and nephntic animals in each group.
  • Another measure of the effect of antagonists of the invention on the disease process is to quantitate the amount of extracellular mat ⁇ x accumulation in the glomeruh.
  • the degree of glomerular matnx expansion is determined as the percentage of each glomerulus occupied by the mesangial matnx according to the method of Raij et al. (1984) Kidney Int. 26: 137-43.
  • Joints are clinically monitored by determining the amount of joint erythema, swelling and distortion on a scale of 0 (normal) to 4 (severe inflammation). Radiographs are taken and are evaluated for soft tissue swelling, joint space narrowing, bone erosions and deformity. Tissue specimens are obtained and prepared for histopathologic analysis as desc ⁇ bed in Brandes et al., ibid. Total RNA is isolated from excised synovial tissues according to the method of Allen et al. (1990) J. Exp. Med. 171:231.
  • prophylactic or therapeutic dose of a compound of Formula I will, of course, vary with the nature of the seventy of the condition to be treated and with the particular compound of Formula I and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range he within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.
  • a suitable dosage range is from about 0 001 mg to about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight per day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 1 mg to about 10 mg) of a compound of Formula I per kg of body weight per day.
  • a suitable dosage range is, e.g.
  • compositions which compnses a compound of Formula I and a pharmaceutically acceptable carrier.
  • composition as in pharmaceutical composition, is intended to encompass a product comp ⁇ sing the active ingredients, and the inert ingredients (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly oi indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of Formula I, additional active ingredients, and pharmaceutically acceptable excipients.
  • Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
  • oral, rectal, topical, p. renteral, ocular, pulmonary, nasal, and the like may be employed.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, ae osok, and the like.
  • compositions of the present invention compnse a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable earner and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
  • the compositions include compositions suitable for oral, rectal, topical, parenteral
  • the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressu ⁇ zed packs or nebu sers.
  • the compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device.
  • MDI metered dose inhalation
  • suitable propellants such as fluorocarbons or hydrocarbons
  • DPI dry powder inhalation
  • Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like.
  • the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical earner according to conventional pharmaceutical compounding techniques.
  • the earner may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols,, flavo ⁇ ng agents, preservatives, colo ⁇ ng agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalhne cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent, the most advantageous oral dosage unit form in which case solid pharmaceutical earners are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in- oil liquid emulsion.
  • compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the earner which constitutes one or moie necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid earners or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
  • Compounds of Formula I may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of Formula I are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of Formula I.
  • a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred.
  • the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of Formula I.
  • Examples of other active ingredients that may be combined with a compound of Formula I, either administered separately or in the same pharmaceutical compositions include, but are not limited to: (a) VLA-4 antagonists such as those described in US 5,510,332, W097/03094, W097/02289P W096t4O781P W096/22966, W096/20216, W096101644, W096/06108, W095/15973 and W096131206; (b) steroids such as declomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosponn, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistammes (Hl-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastme,
  • Example 1 Cell Adhesion Assay Protocol
  • Protocol determines the ability of such organic compounds to inhibit and prevent collagen-based cell adhesion. Protocol :
  • BCECF.AM [2',7'-b ⁇ s(2-carboxyethyl)-5-(-6)-carboxyfluoresce ⁇ n, acetoxymethyl ester] at 37°C for 15 min.) each at 25 wl/well.
  • Example 2 Illustrative Synthetic Approaches
  • the artisan identifies the chemical structure of a compound having betal subur it containing integrin activity, such as, for example, the following (A):
  • the amide group adjacent to the acidic moiety in A_is identified and phantom bonds can be formed between the carbonyl of the amide and the ⁇ carbon of the amino acid to form (B) (B)
  • the amide group of (E) adjacent to the acidic moiety is identified and phantom bonds can be formed between the carbonyl of the amide and the ⁇ carbon of the amino acid (F).

Abstract

Novel integrin antagonists of Formula (I) are provided wherein a peptidic amide bond has been replaced, wherein all definitions are as in the claims.

Description

INTEGRIN ANTAGONISTS
FIELD OF THE INVENTION
The present invention relates to organic compounds, which are useful for blocking the activity of integπn molecules. This invention also relates to compositions containing such compounds and methods of treatment using such compounds. BACKGROUND OF THE INVENTION
Many physiological processes require that cells come into close contact with other cells and/or extracellular matrix Such adhesion events may be required for cell activation, migration, proliferation and differentiation Cell-cell and cell-matrix interactions are mediated through several families of cell adhesion molecules including the integπns The lntegπn very late antigen (VLA) superfamily is made up of structurally and functionally related glycoproteins consisting of (alpha and beta) heterodimeπc, transmembrane receptor molecules found in various combinations on nearly every mammalian cell type, (for reviews see: E. C. Butcher, Cell, 67, 1033 (1991); D. Cox et al , "The Pharmacology of the Integπns " Medicinal Research Rev (1994) and V. W. Engleman et al , 'Cell Adhesion Integπns as Pharmaceutical Targets ' in Ann Report in Medicinal Chemistry, Vol 31, J A. Bristol, Ed.; Acad. Press, NY, 1996, p. 191). Adhesion molecules of the VLA family presently include VLA-1, -2, -3, -4, -5, -6, -9, -10, -11, -v in which each of the molecules compπse a βl chain non- covalently bound to a α chain, (αl, oc2, oc3, oc4, α5, oc6, α9, al l, av), respectively. The lntegπn a4β7 is also intended to be included within the VLA family
Such molecules play an essential role in both normal and pathophysiological processes in a wide variety of tissues. For instance, cellular adhesion and trafficking across the vascular interface plays an essential role in both physiological and pathophysiological processes of acute brain injury (Garcia et al 1994, Am. J. Pathol 144: 188; Becker et al, 1997 PNAS 94: 10873). Further, leukocyte migration into glomeru is a typical feature of human glomerulonephπtis (GN) and leukocytes are key mediators of kidney damage Therefore, the targeting of specific and relevant molecules in certain disease conditions without interfering with normal cellular functions is essential for an effective and safe therapeutic agent that inhibits cell-cell and cell-matπx interactions.
VLA-1, -2, -4, -6 and α4β7 neutralizing antibodies or blocking peptides that inhibit the interaction between these respective VLA moieties and their ligands are known. Moreover, in the case of VLA-4 (α4βl) and its ligand VCAM-1, some antibody antagonists have proven efficacious both prophylactic ally and therapeutically in several animal models of disease, including i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis (for example, see T. Yednock et al., "Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha4betal integrin.' Nature, 356, 63 (1993) and E. Keszthelyi et al., Evidence for a prolonged role of alpha4 integrin throughout active experimental allergic encephalomyelitis." Neurology, 47. 1053 (1996)); ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma (for example, see W. M. Abraham et al., 'alpha4-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep." J. Clin. Invest. 98. 776 (1993) and A. A. Milne and P. P. Piper, Hole of VLA-4 integrin in leukocyte recruitment and bronchial hyperresponsiveness in the guinea-pig." Eur. J. Pharmacol., 282, 243 (1995)); ix) tumor metastasis (for examples, see M. Edward, "Integrins and other adhesion molecules involved in melanocytic tumor progression.", Curr. Opin. Oncol., 7, 185 (1995)).
Several peptidyl antagonists specific to VLA-4 have been described (D. Y. Jackson et al., "Potent α4βl peptide antagonists as potential anti-inflammatory agents', J. Med. Chem., 40,3359 (1997); U.S. Patent 5,510,332, PCT Publications W097/03094, W097/02289, W096/40781, W096/22966.W096/20216, W096/01644, W096106108, and W095/15973). These peptide mimetic compounds of necessity contain the peptidic amide bond. See S.P. Adams and Roy R. Lobb, Annual Reports in Medicinal Chemistry, 34: Chapter 18, Academic Press, 1999. The peptidic amide bond is metabolically labile to hydrolysis and this limits the therapeutic utility of such amide bond-containing molecules.
Notwithstanding the fact that peptidyl antagonists to integrins have been prepared, there remains a need for low molecular weight, specific inhibitors of integrins in which the amide bond is modified so that it is no longer susceptible to hydrolysis but contains the features important for integrin antagonist biological activity SUMMARY OF THE INVENTION
We have developed antagonists of integrins with the advantageous property of lacking a peptidic amide bond. We have developed methods of using these antagonists that are useful in inhibiting integrins such as βl subunit containing integrins as well as the integrin α4β7 and, in so doing, are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation. One aspect of the invention is a compound having the following structural Formula I:
Figure imgf000004_0001
wherein
is selected from
1 NRJ-(CRkRm)m;
2 S(O)n-NRJ;
3 NRJ -O;
4 S-S;
5 NRJ -NRJ
6 (CRkRm)m-O;
7 (CRkRm)m - NRJ
8 O-(CRkRm)m
9 (CRkRm)m-( CRkRm)n
10 S(O)n-(CRkRm )m
11 (CRkRm)m-S(O)n
12 NRJ-S(O)n
13 O- NRJ
14 NRJ
15 O;
16 S(O)„
17 C(O);
18 NRJC(O); or
19 C(O)NRJ n is an integer from 0 to 2;
m is an integer from 1 to 2;
W is selected from
1 -(CRfRg)nC(O)ORd
2 -(CRfRg)n5-tetrazolyl or
3 -(CRfRg)nS(O)2NHRd
X is selected from S(O)2, S(O)2NRe, C(O), C(O)O, C(O)NRe, CRfRg
Y is selected from N, CRj
Z is selected from
1 (CRkRm)n
2 NRj
3 S(O)n
4 O; or
5 C(O)
R° is selected from R4 and
Figure imgf000005_0001
A and E are independently selected from -C- and -C-C-, -C=C-; F is selected from N, CR2
Q is selected from -CRj-, C(O), O, S(O)„, NH, NXR1 B is selected from the group consisting of 1 a bond,
2 -C-,
3 -C-C-;
4 -C=C-,
5 a heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur; or
6 -S(O)m - .
R' IS
1 Cl-10 alkyl,
2 C2-10alkenyl,
3 C2-10 alkynyl,
4 Cy,
5 Cl-10 alkyl-Cy,
6 C2-10 alkenyl-Cy, or
7 C2-C10 alkynyl-Cy, wherein alkyl, alkenyl, and alkynyl are optionally substituted with one to four substituents independently selected from Ra; and Cy is optionally substituted with one to four substituents independently selected from Rb;
R2 ιs 1 hydrogen,
2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2-10 alkynyl,
5 aryl, 6 Cl-10 alkyl-aryl,
7 heteroaryl, or
8 Cl-10 alkyl- heteroaryl, wherein alkyl, alkenyl. and alkynyl are optionally substituted with one to four substituents independently selected from Ra, and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from
Rb;
1. hydrogen
2. Cl-10 alkyl, 3. C2- 10 alkenyl,
4. C2-10 alkynyl,
5. Cy,
6. Cl-10 alkyl-Cy, 7. C2-10 alkenyl-Cy or
8. C2-10 alkynyl-Cy
Wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and Rx, and phenyl and Cy are optionally substituted with one to four substituents independently selected from Ry R4 is
1. hydrogen 2 Cl-10 alkyl,
3. C2-10 alkenyl,
4. C2-10 alkynyl, 5. Cy,
6. Cl-10 alkyl-Cy,
7. C2-10 alkenyl-Cy or
8. C2-10 alkynyl-Cy
Wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and R\ and phenyl and Cy are optionally substituted with one to four substituents independently selected from Ry Or R4 with either Rf or Rs forms a mono- or bi-cychc ring containing 0-2 heteroatoms selected from nitrogen, oxygen or sulfur, wherein nitrogen is optionally substituted with RJ, C(O)Re, SO2Re or SO2NRdRe R6, R7, and R8 are each independently selected from the group consisting of Rd and Rx or two of R6, R7, and R8 and the atom to which both are attached, or two of R6, R7, and R8 and the two adjacent atoms to which they are attached, together form a 5-7 membered saturated or unsaturated monocyclic πng containing zero to three heteroatoms selected from N, 0 or S,
R12 ιs
1. hydrogen
2. Cl-10 alkyl,
3. C2- 10 alkenyl,
4. C2- 10 alkynyl, 5. Cy,
6. Cl-10 alkyl-Cy,
7 C2-10 alkenyl-Cy or 8. C2- 10 alkynyl-Cy wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and Rx, and Cy is optionally substituted with one to four substituents independently selected from Ry R,3 ιs
1 hydrogen, 2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2-10 alkynyl,
5 aryl,
6 Cl-10 alkyl-aryl, 7 heteroaryl,
8 Cl-10 alkyl-heteroaryl , wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from Rx and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from Ry ; or R12, Rn and the carbon to which they are attached form a 3-7 membered mono- or bicyclic πng containing 0-2 heteroatoms selected from N, 0 and S, wherein nitrogen is optionally substitiuted with RJ, C(O)Rc, SO2Rc or SO2NRdRe R16 is selected from a bond, NR\ O and S(O)„ Ra ιs 1 Cy, or
2 a group selected from R , wherein Cy is optionally subsituted with one to four substituents independently selected from Rc ;
Rb ιs
1 a group selected from Ra ,
2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2-10 alkynyl, or
5 Cl-10 alkyl- aryl, wherein alkyl, alkenyl, .ilkynyl, aryl, heteroaryl are optionally substituted with a group independently sel scted from Rc ; Rc is
I halogen, 2 N02,
3 C(O)ORf
4 Cl-4 alkyl,
5 Cl-4 alkoxy,
6 aryl, 7 aryl Cl-4 alkyl,
8 aryloxy,
9 heteroaryl,
10 N Rf Rg ,
I I N Rf C(O)Rg, 12 N Rf C(O)NRfRg ,
13 CN,
14 C(O)Cy or
15 C(O)alkyl;
16 C(O)NRfRg 17 alkyloxy, or
18 NRfC(O)ORe wherein aryl, heteroaryl and Cy are optionally substituted with 1 to 4 substitutents independently selected from Rx
Rd and Re are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2- 10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl and Cy is optionally substituted with one to four substituents independently selected from Rc ; or Rd and Re together with the atoms to which they are attached form a heterocyclic ring of 5 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen; wherein nitrogen is optionally substitiuted with RJ, C(O)Re, SO2Re or SO2NRdRe
R and Rg are independently selected from R or R and Rg together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms mdependently selected from oxygen, sulfur and nitrogen wherein nitrogen is optionally substituted with RJ, C(O)Re, SO2Re or SO2NRdRe R' is
1 Cl-10 alkyl, 2 C2-10 alkenyl,
3 C2-10 alkynyl, or
4 aryl; wherein alkyl, alkenyl, alkynyl and aryl are each optionally substituted with one to four substituents independently selected from R , RJ is selected from hydiogen, Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein said alkyl, alkenyl, alkynyl, heteroaryl, and Cy is optionally substituted with one to four substituents independently selected from Rc Rk and Rm are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2-
10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, and heteroaryl
Rx is selected from
1 -O Rd ,
2 -NO2,
3 halogen,
4 -S(O)m Rd
5 -S Rd ,
6 -S(O)2ORd ,
7 -S(O)m N Rd Re ,
8 -N Rd Rc ,
9 -O(C Rf R )n N Rd Re ,
10 -C(O) Rd ,
11 -CO2 Rd ,
12 -CO2(C Rf Rg )n CON Rd RL ,
13 -OC(O) Rd ,
14 -CN,
15 -C(O)N Rd Re ,
16 -N Rd C(O) Rc .
17 -OC(O)N Rd Re , -N Rd C(O)O Re ,
-N Rd C(O)N Rd Re ,
-C Rd (N-O Re ),
-CF3, oxo,
NRdC(O)NRdS02R\
N Rd S(O)m Re ,
-OS(O)2ORd,
-OP(O)(O Rd )2;
Figure imgf000011_0001
-N Rd C(S)N Rd Re , or
Figure imgf000011_0002
Ry is l a group selected from Rx ,
2 Cl-10 alkyl,
3 C2- 10 alkenyl,
4 C2-10 alkynyl,
5 aryl Cl-10 alkyl- aryl, 6 Cl-10 alkyl- heteroaryl,
7 cycloalkyl,
8 heterocyclyl
9 aryl
10 heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl and aryl are each optionally substituted with one to four substituents independently selected from Rx ;
Cy is cycloalkyl, heterocycyl, aryl, or heteroaryl;
A further embodiment of the invention is the compound of the Formula II below:
Figure imgf000012_0001
where Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are substituted with Rzand R9 . R9 is selected from H and Ry Rz is selected from
1 ORd
2 NHRd 3 NRdS(O)mRe
4 NRdC(O)Re
and all other designations are as given above for the first formula. These antagonists are useful in the treatment, prevention and suppression of diseases mediated by any integπn Such diseases include multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthntis, septic arthπtis, type I diabetes, organ transplantation, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, certain types of toxic and immune-based nephπtis, contact dermal hypersensitivity, psonasis, tumor metastasis, atherosclerosis and fibrotic diseases. DETAILED DESCRIPTION OF THE INVENTION
The present compounds are biologically active small molecules and are generally composed of several domains: a) an acyl (including sulfonyl) moiety and a heterocycle #1 or a substituted aromatic πng, b) a heterocycle #2, and c) acid and a sidechain, and are named in a manner similar to that used to name ohgopeptides.
Definitions: An integrin "antagonist" includes any compound that inhibits a "plurality" (defined below) of integπns from binding with an integrin ligand and/or ligand leceptors. For the purposes of the invention, an integrin "antagonist" also refers to agents claimed herein which can inhibit or block integπn and/or integπn hgand-mediated binding or which can otherwise modulate integπn and/or integrin ligand function, e.g., by inhibiting or blocking lntegπn-hgand mediated integπn signal transduction. Such an antagonist of the integπn/integπn ligand interaction is an agent which has one or more of the following properties: (1) it coats, or binds to, a plurality of integπns (e.g, α4β7 , VLA-1, VLA-9 and VLA-1 ) on the surface of such integrin beaπng or secreting cell with sufficient specificity to inhibit an integrin ligand/mtegπn interaction, e.g., the collagen/VLA-1 interaction; (2) it coats, or binds to, a plurality of integπns on the surface of an mtegπn-beaπng or secreting cell with sufficient specificity to modify, and preferably to inhibit, transduction of an mtegπn-mediated signal e.g., collagen/VLA-1 -mediated signaling; (3) it coats, or binds to, a plurality of integπn receptors, (e.g., collagen only or collagen and VCAM-1) in or on cells with sufficient specificity to inhibit the lntegπn/integπn ligand interaction, (4) it coats, or binds to, an integπn ligand (e.g., collagen) in or on cells with sufficient specificity to modify, and preferably to inhibit, transduction of mtegπn-mediated integπn signaling, e.g., collagen-mediated VLA-1 signaling
In preferred embodiments the integrin antagonist has one or both of properties 1 and 2. In other preferred embodiments the antagonist has one or both of properties 3 and 4 Moreover, more than one antagonist can be administered to a patient, e.g., an agent which binds to an integrin can be combined with an agent which binds to its ligand. An antagonist of the invention has "biological activity" if it inhibits a plurality integrins from binding with an integπn ligand and/or integπn receptor as determined by in vitro and in vivo tests known to workers having ordinary skill in the art.
A "pan-βl antagonist" includes any compound that inhibits a plurality (defined below) of integπns containing the βl subunit from binding with an integπn ligand and/or receptor such as any receptor for the βl subunit. For the purposes of the invention only, we specifically include the integrin α4β7 under the definition of a 'βl subunit' containing integrin For the purposes of the invention, a "pan-βl antagonist" also refers to agents claimed herein which can inhibit or block integπn and/or integπn gand-mediated binding or which can otherwise modulate integrin and/or integπn ligand function, e.g., by inhibiting or blocking lnlegπn-hgand mediated integπn signal transduction. Such an antagonist of the lntegπn/integπn ligand interaction is an agent which has one or more of the properties (1) through (4) as described above. A pan-βl antagonist of the invention has "biological activity" if it inhibits a plurality of βl subunit containing integrins (including α4β7) from binding with an integrin ligand and/or receptor such as any receptoi for the βl subunit or, as defined above, any receptor for the α4β7 integπn. Such biological activity is determined by in vitro and in vivo tests known to workers having oidinary skill in the art
"Alkyl", as well as other groups having the prefix "alk", such as alkoxy, alkanoyl, means carbon chains which may be lmeai or branched or combinations thereof Examples ot alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like "Alkenyl" means carbon chains which contain at least one carbon-carbon double bond, and which may be linear or branched or combinations thereof. Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2- methyl-2- butenyl, and the like "Alkynyl" means carbon chains which contain at least one carbon-carbon tπple bond, and which may be linear or branched or combinations thereof. Examples of alkynyl include ethynyl, propargyl, 3-methyl-l-pentenyl, 2-heptynyl and the like.
"Cycloalkyl" means mono- or bicyclic saturated carbocyc c πngs, each of which having from 3 to 10 carbon atoms. The term also includes monocyclic πngs fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
"Aryl" means mono- or bicyclic aromatic πngs containing only carbon atoms The term also includes aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3 dihydrobenzofuranyl, benzopyranyl, 1,4-benzodιoxanyl, and the like
"Heteroaryl" means a mono- or bicyclic aromatic πng containing at least one heteroatom selected from N, 0 and S, with each πng containing 5 to 6 atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyπdyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, lmidazolyl, tπazolyl, tetrazolyl, furanyl, tπazinyl, thienyl, pyπmidyl, pyπdazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyπdyl, quinolyl, indolyl, isoquinolyl, and the like. "Heterocyclyl" means mono- or bicyclic saturated πngs containing at least one heteroatom selected from N, S and 0, each ot said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen. The term also includes monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of "heterocyclyl" include pyrro dinyl, pipeπdinyl, piperazinyl, lmidazohdinyl, 2,3-dιhydrofuro(2,3-b) pyndyl, benzoxazinyl, tetrahydrohydroquinohnyl, tetrahydroisoquinohnyl, dihydroindolyl, and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4 pyndones attached through the nitrogen or Ν-substituted- (1H,3H) pyπmιdιne-2,4-dιones (Ν-substituted uracils)
"Halogen" includes fluoπne, chlorine, bromine and iodine. "Plurality" is intended to mean. (I) any single integrm; or (II) two or more integπns
Thus, the present methods utilize (l) molecules capable of inhibiting any combination of two or more different integrins such as a molecule capable of antagonizing both VLA-4 (α4βl) and α4β7 or VLA-2 (α2βl), VLA-6 (α6βl) and VLA-4, and so on; or (n) molecules capable of selectively inhibiting any one integπn, such as α4β7 only or VLA-4 only. Thus a pan-betal antagonist inhibits any single integπn containing a betal subunit (including α4β7) or two or more integπns that contain a betal subunit (such two or more integπns which may include α4β7).
"Polymer" has its art recognized meaning as being a molecule constructed from many smaller structural units called "monomers", bonded together (preferably covalently) in any pattern. The term includes linear molecules and branched molecules. The term also includes homopolymers where only one species of monomer is used to build the molecule, or copolymers where the molecule is composed of two different types of monomers and so on Copolymers also include polymers where the distribution of monomers is random, alternating copolymers, block copolymers and graft copolymers.
Most preferably, the polymer is 'biocompatible' A "biocompatible" substance, as that term is used herein, is one that has no unacceptable toxic or injurious effects on biological function
Antagonists of the invention are 'small molecules' which are organic molecules. A "small molecule", as defined herein, has a molecular weight generally less than 2000. The term "effective amount" as used herein, means an amount of a compound of the present invention which inhibits a "plurality" (defined herein) of integrins from binding with an integrin ligand and/or integπn receptor, as determined by in vitro and in vivo tests known to workers having ordinary skill in the art. Optical Isomers - Diastereomers - Geometric Isomeis - Tautomers
Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeπc mixtures and individual diastereomers. The present invention is meant to comprehend all such lsomeπc forms of the compounds of Formula I. Some of the compounds descπbed herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometπc isomers. Some ot the compounds descπbed herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto- enol tautomers. The individual tautomers as well as mixture thereof are encompassed with compounds of Formula I
Compounds of the Formula I may be separated into diastereoisomeπc pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid as a resolv ng agent. Alternatively, any enantiomer of a compound of the general Formula I may be obtai αed by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
Other Modifications of the Antagonists
Other species are within the scope of the generic formulae are described herein. For example, an exemplary series of antagonists is found in Formula II :
Figure imgf000017_0001
where Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are substituted with R 7 and R 0 . R9 is selected from H and Ry Rz is selected from 1 ORd
2 NHRd
3 NRdS(O)mRc
4 NRdC(O)Re and all other designations and substituents are as above.
Another series are antagonists of Formula (III):
Figure imgf000018_0001
wherein Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are optionally substituted with one to four substituents independently selected from Rx and R is selected from H and Ry.
Rz is selected from
1 ORd
2 NHRd
3 NRdS(O)mRe
4 NRdC(O)Re
and all other designations and substituents are as previously recited as above.
Further series are those of antagonists of Formula IV:
Figure imgf000019_0001
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, nd heteroaryl substituted heteroaryl and further wherein
said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from R and all other designations and substituents are as recited above.
Further embodiments are found in antagonists having the formula V:
Figure imgf000019_0002
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx , R9 is selected from H and Ry and all other designations and substituents are as recited above Other antagonists of the invention are shown below as Formula VI:
Figure imgf000020_0001
wherein all designations and substituents are as recited above.
Another seπes of antagonists is shown below as Formula VII:
Figure imgf000020_0002
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx and all other designations and substituents are as recited above. Further seπes are those of Formulae VIII, IX, XI, XII, and XIII, where all designations and substituents are as recited above VIII
Figure imgf000021_0001
IX:
Figure imgf000021_0002
XI:
Figure imgf000021_0003
XII:
Figure imgf000022_0001
XIII:
Figure imgf000022_0002
Yet another series of antagonists are those of Formula X:
X:
Figure imgf000023_0001
Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx , R9 is selected from H and Ry , R7 is selected from
1 ORd
2 NHRd 3 NRdS(O)mRe
4 NRdC(O)Re and all other designations and substituents are as recited above.
Specific structures intended to fall within the scope of the present invention are analysed on either a Platform LCZ mass spectrometer (electrospray positive) or a VG Platform II mass spectrometer (electrospray positive or negative). Polymer Conjugate Forms
Within the broad scope of the present invention, a single polymer molecule may be employed for conjugation with a integrin antagonist, although it is also contemplated that more than one polymer molecule can be attached as well. Conjugated integrin antagonist compositions of the invention may find utility in both in vivo as well as non- in vivo applications. Additionally, it will be recognized that the conjugating polymer may utilize any other groups, moieties, or other conjugated species, as appropriate to the end use application. By way of example, it may be useful in some applications to covalently bond to the polymer a functional moiety imparting UV-degradation resistance, or antioxidation, or other properties or characteristics to the polymer. As a further example, it may be advantageous in some applications to functionalize the polymer to render it reactive and enable it to cross-link to a drug molecule, to enhance vaπous properties or characteπstics of the overall conjugated mateπal. Accordingly, the polymer may contain any functionality, repeating groups, linkages, or other constitutent structures which do not preclude the efficacy of the conjugated integπn antagonist composition for its intended purpose. Other objectives and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims. Illustrative polymers that may usefully be employed to achieve these desirable charactenstics are described herein below in exemplary reaction schemes. In one embodiment of a covalently bonded antagonist/polymer conjugate, the polymer may be coupled to the antagonist to form stable bonds that are not significantly cleavable by human enzymes. Generally, for a compound of the invention to contain bonds that are not 'significantly' cleavable requires that no more than about 20% of the bonds of the compound are cleaved within a 24 hour period, as measured by standard techniques in the art including, but not limited to, high pressure liquid chromatography (HPLC). Integπn antagonists of the invention are conjugated most preferably via a terminal reactive group on the polymer although conjugations can also be branched from non-terminal reactive groups. The polymer with the reactive group(s) is designated herein as "activated polymer". The reactive group selectively reacts with reactive groups on the antagonist molecule. The activated polymer(s) is reacted so that attachment may occur at any available integπn antagonist functional group. Ammo, carbon, free carboxylic groups, suitably activated carbonyl groups, hydroxyl, guanidyl, oxidized carbohydrate moieties, amino, carbon and mercapto groups of the integπn antagonist (if available) can be used as attachment sites.
Generally from about 1.0 to about 10 moles of activated polymer per mole of antagonist, depending on antagonist concentration, is employed. The final amount is a balance between maximizing the extent of the reaction while minimizing non-specific modifications of the product and, at the same time, defining chemistnes that will maintain optimum activity, while at the same time optimizing, if possible, the half-life of the antagonist. Preferably, at least about 50% of the biological activity of the antagonist is retained, and most preferably 100% is retained The reactions may take place by any suitable art-recognized method used for reacting biologically active mateπals with inert polymers. Generally the process involves preparing an activated polymer and thereafter reacting the antagonist with the activated polymer to produce the soluble compound suitable for formulation The above modification reaction cai be performed by several methods, which may involve one or more steps.
The polymeπc substanc es included herein are preferably water-soluble at room temperature. A non-limiting list of such polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
In the preferred practice of the present invention, polyalkylene glycol residues of C1-C4 alkyl polyalkylene glycols, preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycol residues of such glycols are advantageously incorporated in the polymer systems of interest. Thus, the polymer to which the antagonist is attached can be a homopolymer of polyethylene glycol (PEG) or is a polyoxyethylated polyol, provided in all cases that the polymer is soluble in water at room temperature. Non- hmiting examples of such polymers include polyalkylene oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated glycols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Examples of polyoxyethylated polyols include, for example, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like. The glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, and tπglyceπdes. Therefore, this branching would not necessarily be seen as a foreign agent in the body.
A general formula for PEG and its derivatives is R"-(CH2CH2O)[x]-(CH2)[y]- R', where (x) represents the degree of polymerization or number of repeating units in the polymer chain and is dependent on the molecular weight of the polymer, (y) represents a positive integer, R' is (CHR1), where R1 is as defined in claim 1 and R" is a capping group (including, without limitation, OH, C[l-4 jalkyl moieties, or vaπous biologically active and inactive moieties) or is R'. In particular, polyethylene glycols (PEG's), mono-activated, C[l-4 ]alkyl-termmated PAO's such as mono-methyl- terminated polyethylene glycols (mPEG's) are preferred when mono- substituted polymers are desired; bis-activated polyethylene oxides are preferred when disubstituted antagonists are desired. As an alternative to polyalkylene oxides, dextran, polyvinyl pyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like may be used.
Those of ordinary skill in the art will recognize that the foregoing list is merely illustrative and that all polymer mateπals having the qualities described herein are contemplated. The polymer need not have any particular molecular weight, but it is preferred that the molecular weight be between about 300 and 100,000, more preferably between 10,000 and 40,000. In particular, sizes of 20,000 or more are best at preventing loss of the product due to filtration in the kidneys. Polyalkylene glycol deπvatization has a number of advantageous properties in the formulation of polymer- integπn antagonist conjugates in the practice of the present invention, as associated with the following properties of polyalkylene glycol derivatives- improvement of aqueous solubility, while at the same time eliciting no antigenic or lmmunogenic response; high degrees of biocompatibihty; absence of in vivo bi ode gradation of the polyalkylene glycol derivatives; and ease of excretion by living organisms.
Polyethylene glycol (PEG) and related polyalkylene oxides (PAO's) are known in the art as being useful adjuncts for the preparation of drugs. See for example, PCT WO 93/24476 PEG has also been conjugated to proteins, peptides and enzymes to increase aqueous solubility and circulating life in vivo as well as reduce antigenicity. See, for example, U.S. Pat Nos. 5,298,643 and 5,321,095, both to Greenwald, et al PCT WO 93/24476 discloses using an ester linkage to covalently bind an organic molecule to water-soluble polyethylene glycols.
In one aspect of the invention, one can utilize an integπn antagonist covalently bonded to the polymer component in which the nature of the conjugation involves one or more noncleavable covalent chemical bonds which, preferably, are resistant to degradation by human enzymes. For instance, Greenwald et al., supra, disclose biologically-active conjugates having substantially hydrolysis-resistant bonds (linkages) between a polyalkylene oxide and the target moiety. One example of a noncleavable linker suitable for the antagonists of the present invention is-
Figure imgf000027_0001
wherein R)0 and Rn are independently selected from the group consisting of H, Cι_6 alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substituted heteroalkyls and substituted Ci 6 alkyls, q is a positive integer and F is selected from O, NR1, S, SO, SO2. In another embodiment, the linkages between a polymer and the antagonist of the invention is cleavable, allowing for control in terms of the time course over which the polymer may be cleaved from the integrin antagonist. This covalent bond between the integπn antagonist and the polymer may be cleaved by chemical or enzymatic reaction. In order to provide a hydrolyzable linkage, mono- or di-acid activated polymers such as PEG acids or PEG diacids are used. Suitable PAO acids can be synthesized by converting mPEG-OH to an ethyl ester. See also Gehrhardt, H., et al. Polymer Bulletin 18: 487 (1987) and Veronese, F. M., et al., J. Controlled Release 10; 145 (1989). Alternatively, the PAO-acid can be synthesized by converting mPEG-OH into a t-butyl ester. Ohya, et al., J. Bioactive and Compatible Polymers Vol. 10 Jan., 1995, 51-66, disclose doxorubicin-PEG conjugates which are prepared by linking the two substituents via vaπous linkages including esters. It will be clear from the foregoing that other polyalkylene oxide deπvatives of the foregoing, such as the polypropylene glycol acids, POG acids, etc., as well as other bifunctional linking groups are also contemplated. The polymer- integrin antagonist product retains an acceptable amount of activity. Concurrently, portions of polyethylene glycol are present in the conjugating polymer to endow the polymer- integπn antagonist conjugate with high aqueous solubility and prolonged blood circulation capability.
It is to be understood that the reaction schemes descπbed herein are provided for the purposes of illustration only and are not to be limiting with respect to the reactions and structures which may be utilized in the modification of the integπn antagonist, e.g , to achieve solubility, stabilization, and cell membrane affinity for parenteral and oral administration. The activity and stability of the integπn antagonist conjugates can be vaπed in several ways by using a polymer of different molecular size. Solubilities of the conjugates can be vaπed by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition. Salts
The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts deπved from inorganic bases include aluminum, ammonium, calcium, copper, feme, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts deπved from pharmaceutically acceptable organic nontoxic bases include salts of pnmary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resms, such as arginine, betaine, caffeine, choline, N, N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dιmethylamιnoethanol, ethanolamine, ethylenediamine, N-ethyl- morpho ne, N-ethylpipeπdine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morphohne, piperazine, pipeπdine, polyamine resins, procaine, puπnes, theobromine, tπethylamine, tπmethylamine, tnpropylamine, tromethamine, and the like When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfomc, fumaπc, gluconic, glutamic, hydrobromic, hydrochloric, lsethionic, lactic, maleic, malic, mandehc, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoπc, succinic, sulfuπc, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuπc, and tartaric acids.
It will be understood that, as used herein, references to the compounds of Formula I aie meant to also include the pharmaceutically acceptable salts. Utilities The ability of the compounds of Formula I to antagonize the actions of any VLA integπn containing a βl subunit (including alpha4 beta 7) makes them useful for preventing or reversing the symptoms, disorders or diseases induced by the binding of VLA to its vaπous hgands. Thus, these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation and be useful in conditions such as acute or chronic renal failure or acute brain injury. Accordingly, another aspect of the present invention piovides a method for the treatment (including prevention, alleviation, amelioration or suppression) of diseases or disorders or symptoms, including fibrotic conditions and an inflammatory disorder mediated by integrin binding and cell adhes on activation, which comprises administering to a mammal an effective amount cf a compound of Formula I.
As used herein, "an infla nmatory disorder", includes, but is not limited to, skin related conditions such as psoriasis, eczema, burns and dermatitis. Other inflammatory disorders contemplated for treatment by the methods of the present invention include, but are not limited to the treatment of asthma, bronchitis, menstrual cramps, tendinitis, bursitis, and the treatment of pain and headaches, or as an antipyretic for the treatment of fever. The methods of the invention also would be useful to treat gastrointestinal conditions such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis and for the prevention of colorectal cancer. The methods of the invention would be useful in treating inflammation in such diseases as vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring after injury, myocardial ischemia, and the like. The methods of the invention would also be useful in the treatment of allergic rhinitis, respiratory distress syndrome, endotoxin shock syndrome, and atherosclerosis as well as asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, organ transplantation rejection, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, tumor metastasis and atherosclerosis.
Antagonists of the present invention may also be useful in treating a subject with a fibrotic condition. The term "fibrotic condition" refers to, but is not limited to, subjects afflicted with fibrosis of an internal organ, subjects afflicted with a dermal fibrosing disorder, and subjects afflicted with fibrotic conditions of the eye. Fibrosis of internal organs (e.g., liver, lung, kidney, heart blood vessels, gastrointestinal tract) occurs in disorders such as pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, renal interstitial fibrosis, renal fibrosis in patients receiving cyclosporin, and HIV associated nephropathy. Dermal fibrosing disorders include, but are not limited to, scleroderma, morphea, keloids, hypertrophic scars, familial cutaneous collagenoma, and connective tissue nevi of the collagen type. Fibrotic conditions of the eye include conditions such as diabetic retinopathy, postsurgical scarring (for example, after glaucoma filtering surgery and after cross-eye surgery), and prohferative vitreoretinopathy Additional fibrotic conditions which may be treated by the methods of the present invention include: rheumatoid arthπtis, diseases associated with prolonged joint pain and deteriorated joints; progressive systemic sclerosis, polymyositis, dermatomyositis, eosinophihc fascitis, morphea, Raynaud's syndrome, and nasal polyposis.
In addition, fibrotic conditions which may be treated the methods of present invention also include inhibiting overproduction of scarring in patients who are known to form keloids or hypertrophic scars, inhibiting or preventing scarπng or overproduction of scarring duπng healing of various types of wounds including surgical incisions, surgical abdominal wounds and traumatic lacerations, preventing or inhibiting scarπng and reclosing of arteries following coronary angioplasty, preventing or inhibiting excess scar or fibious tissue formation associated with cardiac fibrosis after infarction and in hypersensitive vasculopathy
Testing Antagonists of the Invention for Function IN VITRO TESTING
The cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of cells expressing integrins to extracellular matrix components such as collagen or fibronectin coated plates In this assay microtiter wells are coated with, for example, collagen. Once the wells are coated, varying concentrations of the test compound are then added together with appropπately labeled, lntegπn-expressing cells Alternatively, the test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells The cells are allowed to incubate in the wells for at least 30 minutes Following incubation, the wells are emptied and washed Inhibition of binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound
Integrin expressing cells that may be utilized in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymphocytes (PBLs). These cells are commercially available and may be fluorescently or radioactively labeled if desired. A direct binding assay may also be employed to quantitate the inhibitory activity of the compounds of this invention ("DBA")
Generally, in vitro assays such as the adhesion inhibition and direct binding assays descπbed above, substitute the appropnate lntegπn-expressing cell and corresponding ligand. For example, polymorphonuclear cells (PMNs) express integπns on their surface and bind to ICAM. Integπns are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay. VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin. IN VIVO TESTING
Once antagonists are identified, they may be further charactenzed in in vivo assays, non-limit g examples of which are descπbed below: A. Contact Hypersensitivity An exemplary animal model is descπbed by P.L. Chisholm et al., "Monoclonal Antibodies to the Integπn α-4 Subunit Inhibit the Muπne Contact Hypersensitivity Response", Eur J. Immunol., 23, pp. 682-688 (1993) and in "Current Protocols in Immunology", J E. Cohgan, et al., Eds., John Wiley & Sons, New York, 1, pp. 4.2.1- 4.2 5 (1991), the disclosures of which are herein incorporated by reference. In these assays, the skin of the animal is sensitized by exposure to an irritant, such as dinitrofluorobenzene, followed by light physical imtation, such as scratching the skin lightly with a sharp edge. Following a recovery penod, the animals are re-sensitized following the same procedure. Several days after sensitization, one ear of the animal is exposed to the chemical irritant, while the other ear is treated with a non-irntant control solution. Shortly after treating the ears, the animals are given vaπous doses of the antagonists by subcutaneous injection. In vivo inhibition of cell adhesion-associated inflammation is assessed by measunng the ear swelling response of the animal in the treated versus untreated ear. Swelling is measured using calipers or other suitable instrument to measure ear thickness. B. Delayed hypersensitivity SRBC-induced delayed type hypersensitivity (DTH) responses are adapted from the protocol of Hurtrel et al. 1992 Cell Immunol. 142:252-263. Bπefly, mice are immunized s.c. in the back with 2 x 107 SRBC in 100 ul PBS on d 0. The mice are challenged on d 5 by injecting 1 x 108 SRBC in 25 ul PBS s.c into the nght hind footpad. Footpad thickness is measured with an engineer's caliper 20 h after antigen challenge, and the degree of footpad swelling calculated Results are reported as the mean percent increase footpad thickness ± SEM and calculated as % increase = [1- (Right footpad thickness 20 h after antigen challenge/Uninjected left footpad thickness 20 h after antigen challenge)] x 100 To block the effector phase of the SRBC-mduced DTH response, antagonists of the invention which are prepared according to the methods described in the Examples is given prior to antigen challenge on d 5. SRBC- induced DTH is a well characterized in vivo model of inflammation, and in particular psoriasis, that has been used to demonstrate the importance of a variety of cytokines and adhesion molecules in inflammation (Tedder et al. 1995 J. Exp. Med. 181:2259- 2264, Terashita et al.1996 J. Immunol. 156:4638-4643).
In this manner, one may identify those inhibitors of this invention which are best suited for inhibiting inflammation. C. Asthma Another in vivo assay that may be employed to test the antagonists of this invention is the sheep asthma assay. This assay is performed essentially as described in W. M. Abraham et al., "α-Integrins Mediate Antigen-induced Late Bronchial Responses and Prolonged Airway Hyperresponsiveness in Sheep", J. Clin. Invest., 93, pp. 776-87 (1994), the disclosure of which is herein incorporated by reference. This assay measures inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in allergic sheep D. Renal Failure
The agents of the present invention also may be tested in animal models of chronic renal failure. Mammalian models of chronic renal failure in, for example, mice, rats, guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and non-human primates, may be created by causing an appropriate direct or indirect injury or insult to the renal tissues of the animal. Animal models of chronic renal failure may, for example, be created by performing a partial (e.g., 5/6) nephrectomy which reduces the number of functioning nephron units to a level which initiates compensatory renal hypertrophy, further nephron loss, and the progressive decline in renal function which characterizes chronic renal failure. The agents of the present invention may be evaluated for their therapeutic efficacy in causing a clinically significant improvement in a standard marker of renal function when administered to a mammalian subject (e.g., a human patient) in, or at risk of, chronic renal failure. Such markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium (Na+), urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicme, 13th edition, Isselbac ler et al., ed..., McGraw Hill Text, New York; Luke and Strom (1994), in Internal M ;dιcιne, 4th Edition, J H. Stein, ed., Mosby-Year Book, Inc. St. Louis.).
E. Acute Brain Injury Male Sprague Dawley (SD) or spontaneously hypertensive rats (SHRS) are anesthetized using isoflurane and the πght middle cerebral artery (MCAO) occluded by insertion of a 4-0 nylon monofilament up the internal carotid artery to the ongm of the middle cerebral artery (MCA) (Zea Longa et al, 1989 Stroke 20:84). After lh the filament is retracted, the lschemic temtory reperfused and the animal allowed to recover After 24h the rats are sacrificed, at which time brains were removed and analyzed histologically to quantify infarct volume.
Groups of animals are treated with either vehicle (PBS) or antagonist of the invention by continuous subcutaneous infusion via osmotic mini-pumps. Pnmed mini osmotic pumps (for example from Alza Corp.) are implanted into the subcutaneous space at the scruff of the neck immediately pπor to induction of cerebral ischemia. The pumps are loaded to release antagonist.
F. Fibrosis
For vessel injury leading to fibrosis, male Sprague-Dawley rats weighing 400g and about 3-4 months of age (Bantin & Kingman, Edwards, WA) are used. The left common carotid artery is denuded with a 2F balloon catheter by introducing the catheter thiough the external carotid artery The distal left common carotid and external carotid arteries are exposed through a midhne wound in the neck The catheter is passed three times with the balloon distended sufficiently with saline to generate slight resistance; this method produces distension of the carotid itself, the external carotid is ligated ater remoeval of the catherter and the wound colosed. Expeπmental treatments include a seπes of injections of antagonist given every other day (post- operation). After 14 days post balloon catheter denudation, all rats are anesthetized and the carotid arteπes fixed by perfusion at 120 mm Hg pressure with 1% paraformaldehyde and 1.25% glutaraldehyde in 0 1M phosphate buffer, pH 7 4 via a large cannula placed retrograde in the abdominal aorta. Ten minutes before fixation, these animals are given an intravenous injection of Evans blue (0 3 ml in 5% saline solution). After 5 minutes of perfusion, the entire left and πght common carotid arteries are retneved, including the aortic arch The vessels are further fixed by immersion in the same fixative as was used for perfusion Artenal segments are assayed for the presence or absence of endothehum by obtaining three segments from the denuded, blue-sta ed left carotid artery and emdedding them in paraffin for cross sectioning using a microtome. For measuπng mtimal areas, photomicrographs are obtained from 3-4 sections from each animal. The photomicrographs are digitized and anlaysed with image analyusis software (such as NIH Image 1.55 for Macintosh)
Intimal areas are measured by determining the area between lumen and internal elastic lamina. Medial areas are derermined by measuπng the area beween internal and external elastic lamina. Intimal/medial area ratios are calculated form the measurements For testing the effect of the present antagonists on lung fibrosis, chronic respiratory disease- free Golden Syrain hamster weighing 120-130g are purchased (e.g., from Charles River, Boston, MA) and housed in plastic cages in groups of 4 in facilities approved by the American Association for Accreditation of Laboratory Animal Care. The animals are allowed to acclimate for one week to laboratory conditions pnor to starting the expeπments. They have access to food and water ad libitum and housed in a room which gets the filtered air and has 12hr/12hr light/dark cycle. Bleomycin sulfate is dissolved in pyrogen free sterile isotonic saline just before intratraceheal (IT) instillation. Under pentobarbital anesthesia (25-35mg/kg lp) hamsters in appropnate group receive either bleomycin (5.5 unιts/kg/4ml) or an equivalent volume (4ml/kg) of pyrogen free isotonic saline through transoral route The antagonists of the invention are administered by lntrapeπtoneal (IP) or intratrachial route at a therapeutic dose to hamster in appropriate groups twice a week for 21-28 days post installation Thereafter, the animals in each group are killed by an overdose ot sodium pentobarbital (100-125 mg/kg lp) and their lungs processed tor biochemical and histopathological studies
G Glomerulonephntis Model
The experimental lesion is acute mesangial pro ferative glomerulonephntis and is characterized by expansion of the mesangial matrix and hypercellulanty. Of particular intererst, the nephritis reproducibly progresses through glomerular and tubulointerstitial scarring, to end stage renal disease
First, glomerulonephntis is induced in rats with a single injection of anti- glomerular basement membrane nephrotoxm serum (NTS), derived in rabbits. Next, for six days, two groups of rats are treated with daily intravenous injections of saline (the negative control group) or antagonists of the invention On the tenth day, the animals are sacnficed and slides are made of the kidneys, which are stained with peπodic acid- Schiff solution to emphasize the pathological changes. The extent of glomerular injury can be quantitated by performing glomerular cell counts from 30 randomly selected glomeruh from normal animals and nephntic animals in each group. Another measure of the effect of antagonists of the invention on the disease process is to quantitate the amount of extracellular matπx accumulation in the glomeruh. The degree of glomerular matnx expansion is determined as the percentage of each glomerulus occupied by the mesangial matnx according to the method of Raij et al. (1984) Kidney Int. 26: 137-43. H. Arthntis Model Arthntis is induced in pathogen-free female LEW rats (Harlan Sprague Dawley,
Indianapolis, Ind.) weighing about 100 grams. Each receives a dose of cell wall fragments from Group A streptococci (SCW) (30 mu g rhamnose/gm bodyweight), injected intrapentoneally (lp) according to the technique descπbed in Brandes et al. (1991) J. Chn. Invest. 87: 1108 SCW-mjected and control LEW rats are given an intraarticular (IA) injection in one of the hind ankles of antagonists of the invention, earner only, or a control.
Joints are clinically monitored by determining the amount of joint erythema, swelling and distortion on a scale of 0 (normal) to 4 (severe inflammation). Radiographs are taken and are evaluated for soft tissue swelling, joint space narrowing, bone erosions and deformity. Tissue specimens are obtained and prepared for histopathologic analysis as descπbed in Brandes et al., ibid. Total RNA is isolated from excised synovial tissues according to the method of Allen et al. (1990) J. Exp. Med. 171:231.
Other models are available. See Terato et al. 1992 J. Immunol. 148:2103-2108, Terato et al. 1995 Autoimmunity. 22: 137-147. Briefly, arthntis is induced through I. p. injection of a cocktail of 4 anti-collagen type II mAbs ( 1 mg each) on d 0, followed by i.p. injection of 50 ug LPS on d 3 Over the course of the next 3-4 d, the mice develop swollen wnsts, ankles and digits. Therapeutic or control antagonist is administered i.p. 4 h pnor to injection of the anti-collagen mAbs on d 0, and again 4 h pπor to LPS administration on d 3, and then continuing every 3rd day for the length of the expenment. Beginning on d 3, mice are evaluated for the development of arthntis. Seventy of arthntis in each limb is scored using a four point system. 0=normal; l=mιld redness, slight swelling of ankle or wnst; 2=moderate swelling of ankle or wnst; 3=severe swelling including some digits, ankle, and foot; 4=maxιmally inflamed Dose Ranges
The magnitude of prophylactic or therapeutic dose of a compound of Formula I will, of course, vary with the nature of the seventy of the condition to be treated and with the particular compound of Formula I and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range he within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases. For use where a composition for intravenous administration is employed, a suitable dosage range is from about 0 001 mg to about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight per day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 1 mg to about 10 mg) of a compound of Formula I per kg of body weight per day. In the case where an oral composition is employed, a suitable dosage range is, e.g. from about 0.01 mg to about 100 mg of a compound of Formula I per kg of body weight per day, preferably from about 0.1 mg to about 10 mg per kg and for cytoprotective use from 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 10 mg to about 100 mg) of a compound of Formula I per kg of body weight per day.
Pharmaceutical Compositions
Another aspect of the present invention provides pharmaceutical compositions which compnses a compound of Formula I and a pharmaceutically acceptable carrier.
The term "composition", as in pharmaceutical composition, is intended to encompass a product compπsing the active ingredients, and the inert ingredients (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly oi indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of Formula I, additional active ingredients, and pharmaceutically acceptable excipients.
Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, p. renteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, ae osok, and the like.
The pharmaceutical compositions of the present invention compnse a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable earner and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids. The compositions include compositions suitable for oral, rectal, topical, parenteral
(including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and seventy of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressuπzed packs or nebu sers. The compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. The preferred delivery systems for inhalation are metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons and dry powder inhalation (DPI) aerosol, which may be formulated as a dry powder of a compound of Formula I with or without additional excipients
Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like. In practical use, the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical earner according to conventional pharmaceutical compounding techniques. The earner may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In prepanng the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols,, flavoπng agents, preservatives, coloπng agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalhne cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent, the most advantageous oral dosage unit form in which case solid pharmaceutical earners are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
In addition to the common dosage forms set out above, the compounds of Formula I may also be administered by controlled release means and/or delivery devices such as those descnbed in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200 and 4,008,719. Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in- oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the earner which constitutes one or moie necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid earners or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
The following are examples of representative pharmaceutical dosage forms for the compounds of Formula 1:
Injectable Suspension (l.m.) mg/mL Compound of Formula I 10
Methylcellulose 5.0
Tween 80 0.5
Benzyl alcohol 9.0
Benzalkonium chloride 1.0 for injection to a total volume of 1 mL
Tablet mg/tablet
Compound of Formula 1 25
Microcrystalline Cellulose 415
Povidone 14.0
Pregelatinized Starch 43.5
Magnesium Stearate 2.5
500
Capsule mg/capsule
Compound of Formula I 25
Lactose Powder 573.5
Magnesium Stearate L5
600
Aerosol Per canister
Compound of Formula I 24 mg
Lecithin, NF Liquid Concentrate 1.2 mg
Trichlorofluoromethane, NF 4.025 g
Dichlorodifluoromethane, NF 12.15 g
Combination Therapy
Compounds of Formula I may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of Formula I are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of Formula I. When a compound of Formula I is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of Formula I. Examples of other active ingredients that may be combined with a compound of Formula I, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) VLA-4 antagonists such as those described in US 5,510,332, W097/03094, W097/02289P W096t4O781P W096/22966, W096/20216, W096101644, W096/06108, W095/15973 and W096131206; (b) steroids such as declomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosponn, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistammes (Hl-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastme, diphenhydramine, diphenylpyraline, tnpelennamme, hydroxyzine, methdilazine, promethazine, tnmeprazine, azatadine, cyproheptadme, antazo ne, pheniramine pynlamine, astemizole, terfenadine, loratac ne, cetinzine, fexofenadine, descarboethoxyloratadme, and the like; (e) non- steroidal anti-asthmatics such as P2-agonιsts (terbutahne, metaproterenol, EXAMPLES
The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention. Example 1 : Cell Adhesion Assay Protocol
This illustrates the protocol for determining utility of the antagonists herein More particularly, the protocol determines the ability of such organic compounds to inhibit and prevent collagen-based cell adhesion. Protocol :
1. Coat a 96-well plate with Collagen IV (0.5 ug/ml), Collagen I (5 ug/ml), BSA- CS1 (1 ug/ml), or Lammin (20 ug/ml) for adhesion assays, αlβl, α2βl, α4βl, or α6βl, respectively in NaBicarb. pH 9.2 at 4°C overnight.
2. Wash the plate twice with IX PBS, 100 wl/well
3. Block the plate with 1% heat-treated BSA in PBS, 100 wl/well foi 1+ hr.
4. Wash the plate twice with assay buffer (TBS complete + 1 mM MnCl2), 100 wl/well. 5. Add compound (2X desired cone.) and cells (4X10 cells/ml, labeled with
BCECF.AM [2',7'-bιs(2-carboxyethyl)-5-(-6)-carboxyfluoresceιn, acetoxymethyl ester] at 37°C for 15 min.) each at 25 wl/well.
6. Incubate the plate at room temperature for 30 minutes.
7. Before washing the plate, read the total cells input in a fluorescent plate readei 8. Wash the plate three times with assay buffer, 100 wl/well.
9. Read the remaining bound cells in the fluorescent plate reader.
Example 2: Illustrative Synthetic Approaches In certain embodiments, the artisan identifies the chemical structure of a compound having betal subur it containing integrin activity, such as, for example, the following (A):
Figure imgf000041_0001
The amide group adjacent to the acidic moiety in A_is identified and phantom bonds can be formed between the carbonyl of the amide and the α carbon of the amino acid to form (B) (B)
Figure imgf000041_0002
These bonds can then be converted to a heterocycle, such as (C)
(C)
Figure imgf000042_0001
or ffii
Figure imgf000042_0002
This procedure can also be extended to 6 membered ring heterocycles, for example (E)
(E)
Figure imgf000043_0001
The amide group of (E) adjacent to the acidic moiety is identified and phantom bonds can be formed between the carbonyl of the amide and the α carbon of the amino acid (F).
(F)
Figure imgf000043_0002
These bonds can then be converted to a heterocycle, such as (G)
(G)
Figure imgf000043_0003
or (H)
Figure imgf000044_0001

Claims

CLAIMS 1. An antagonist of a plurality of betal subunit containing integrins, the antagonist having the formula (I):wherein D is selected from1 NRJ-(CRkRm)m2 S(O)„-NRJ3 NRJ -O4 S-S5 NRJ -NRJ6 (CRkRm)m-O7 (CRkRm)m - NRJ8 O-(CRkRm)m9 (CRkRm)m-(CRkRm)n10 S(O)n-(CRkRm)m or11 (CRkRm)m-S(O)n12 NRJ-S(O)n13 O- NRJ14 NRJ15 O16 S(O)n17 C(O)18 NR'C(O);or19 C(O)NRJn is an integer from 0 to 2; m is an integer from 1 to 2; W is selected from1 -(CRfRg)nC(O)ORd2 -(CRfRg)n5-tetrazolyl3 -(CRfR )nS(O)2NHRdX is selected from S(O)2, S(O)2NRe, C(O), C(O)O, C(O)NRe, CRfRgY is selected from N, CRJZ is selected from1 (CRkRm)n2 NRJ3 S(O)n4 O; or5 C(O)R° is selected from R4 andA and E are independently selected fiom -C- and -C-C-, -C=C-, F is selected from N, CR2Q is selected from -CRJ-, C(O), O, S(O)n, NH, NXR1 B is selected from the group consisting of1 a bond,2 -C-,3 -C-C-;4 -C=C-,5 a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur; or6 -S(O)m -R' IS 1 Cl-10 alkyl,2 C2-10alkenyl,3 C2-10 alkynyl,4 Cy, 5 Cl-10 alkyl-Cy,6 C2-10 alkenyl-Cy, or7 C2-C10 alkynyl-Cy, wherein alkyl, alkenyl, and alkynyl are optionally substituted with one to four substituents independently selected from Ra; and Cy is optionally substituted with one to four substituents independently selected from Rb;R2 is1 hydrogen,2 Cl-10 alkyl,3 C2-10 alkenyl, 4 C2-10 alkynyl,5 aryl,6 Cl-10 alkyl-aryl,7 heteroaryl, or8 Cl-10 alkyl- heteroaryl, wherein alkyl, alkenyl, and alkynyl are optionally substituted with one to four substituents independently selected from Ra; and aryl and heteroaryl are optionally substituted with one to four substituents independently selected fromKRJ is
1. hydrogen
2. Cl-10 alkyl,
3. C2- 10 alkenyl,
4. C2-10 alkynyl,
5. Cy,
6. Cl-10 alkyl-Cy,
7. C2-10 alkenyl-Cy or
8. C2-10 alkynyl-Cy wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and R ; and phenyl and Cy are optionally substituted with one to four substituents independently selected from Ry R4 is 1. hydrogen
2. Cl-10 alkyl,
3. C2- 10 alkenyl,
4. C2- 10 alkynyl,
5. Cy, 6. Cl-10 alkyl-Cy,
7. C2-10 alkenyl-Cy or
8. C2- 10 alkynyl-Cy wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from phenyl and R"; and phenyl and Cy are optionally substituted with one to four substituents independently selected from Ry or R4 , with either Rf or Rg forming a mono- or bi-cyclic ring containing 0-2 heteroatoms selected from nitrogen, oxygen or sulfur, wherein nitrogen is optionally substituted with Rj, C(O)Re, SO2Re or SO2NRdRe
R , R7, and R are each independently selected from the group consisting of Rd and Rx or two of R6, R7, and R8 and the atom to which both are attached, or two of R6, R7, and
R8 and the two adjacent atoms to which they are attached, together form a 5-7 membered saturated or unsaturated monocyclic ring containing zero to three heteroatoms selected from N, 0 or S,
R,2 is
1. hydrogen
2. Cl-10 alkyl,
3. C2-10 alkenyl,
4. C2-10 alkynyl,
5. Cy,
6. Cl-10 alkyl-Cy,
7. C2-10 alkenyl-Cy or
8. C2-10 alkynyl-Cy Wherein alkyl, alkenj 1 and alkynyl are optionally substituted with one to four substituents selected f "om phenyl and Rx; and Cy is optionally substituted with one to four substituents independently selected from Ry R13 is 1 hydrogen,
2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2-10 alkynyl,
5 aryl, 6 Cl-10 alkyl-aryl,
7 heteroaryl, or
8 Cl-10 alkyl-heteroaryl , wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from Rx and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from Ry ; or
R , R and the carbon to which they are attached form a 3-7 membered mono- or bicyclic ring containing 0-2 heteroatoms selected from N, 0 and S; wherein nitrogen is optionally substitiuted with Rj, C(O)Re, SO2Re or SO2NRdRe R16 is selected from a bond, NRj, O and S(O)n Ra is
1 Cy, or
2 a group selected from Rx ; wherein Cy is optionally subsituted with one to four substituents independently selected from Rc ; Rb is
1 a group selected from Ra ,
2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2-10 alkynyl, or 5 Cl-10 alkyl- aryl, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl are optionally substituted with a group independently selected from Rc ;
Rc is halogen, 2 N02,
3 C(O)ORf
4 Cl-4 alkyl,
5 Cl-4 alkoxy, 6 aryl,
7 aryl Cl-4 alkyl,
8 aryloxy,
9 heteroaryl,
10 N Rf R , 11 N Rf C(O)Rg,
12 N Rf C(O)NRfRg ,
16 CN,
17 C(O)Cy;
18 C(O)alkyl; 16 C(O)NRfRg
17 alkyloxy; or
18 NRfC(O)ORe wherein aryl, heteroaryl and Cy are optionally substituted with 1 to 4 substitutents independently selected from Rx Rd and Re are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl and Cy is optionally substituted with one to four substituents independently selected from Rc , or Rd and Re together with the atoms to which they are attached form a heterocyclic ring of 5 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen; wherein nitrogen is optionally substitiuted with RJ,
C(O)Re, SO2Re or SO2NRdRe
R and Rg are independently selected from R or R and R together with the carbon to which they are attached form a πng of 5 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and nitrogen wherein nitrogen is optionally substitiuted with RJ, C(O)Re, SO2Re or SO2NRdRc
R1 is
1 Cl-10 alkyl, 2 C2-10 alkenyl,
3 C2-10 alkynyl, or
4 aryl; wherein alkyl, alkenyl, alkynyl and aryl are each optionally substituted with one to four substituents independently selected from Rc ;
RJ is selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, Cy and Cy
Cl-10 alkyl, aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl, heteroaryl-substituted heteroaryl wherein alkyl, alkenyl, alkynyl, heteroaryl, and Cy is optionally substituted with one to four substituents independently selected from Rc
Rk and Rm are independently selected from hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2-
10 alkynyl, Cy and Cy Cl-10 alkyl, aryl, and heteroaryl
Rx is
1 -ORd,
2 -NO2,
3 halogen,
4 -S(O)m Rd
5 -SRd,
6 -S(O)2ORd ,
7 -S(O)m N Rd Re ,
8 -N Rd Re ,
9 -O(C Rf R )n N Rd Re ,
10 -C(O) Rd ,
11 -CO2Rd,
12 -CO2(C Rf Rg )n CON Rd Re ,
13 -OC(O) Rd ,
14 -CN,
15 -C(O)N Rd Re ,
16 -N Rd C(O) Re ,
17 -OC(O)N Rd Re ,
18 -N Rd C(O)O Re ,
19 -N Rd C(O)N Rd Re ,
20 -C Rd (N-O Re ),
21 -CF3, 22 oxo,
23 NRdC(O)NRdS02R',
24 NRd S(O)mRe,
25 -OS(O)2ORd,
26 -OP(O)(O Rd )2;
Figure imgf000052_0001
28 -N Rd C(S)N Rd Re , or
Figure imgf000052_0002
Ry is
1 a group selected from Rx ,
2 Cl-10 alkyl,
3 C2-10 alkenyl,
4 C2- 10 alkynyl,
5 aryl Cl-10 alkyl- aryl,
6 Cl-10 alkyl- heteroaryl,
7 cycloalkyl,
8 heterocyclyl
9 aryl, or
10 heteroaryl wherein alkyl, alken '1, alkynyl, heteroaryl and aryl are each optionally substituted with one to four substituents independently selected from Rx ; and
Cy is cycloalkyl, heterocycyl, lryl, or heteroaryl;
2. An antagonist of a plurality of integπns, the antagonist having the formula (II):
Figure imgf000053_0001
where Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are substituted with Rzand R9 . R9 is selected from H and Ry Rz is selected from
1 ORα
2 NHRd
Figure imgf000053_0002
4 NRdC(O)Re and all other designations and substituents are as previously recited in claim 1.
3. An antagonist of a plurality of integrins, the antagonist having the formula (HI):
Figure imgf000053_0003
wherem Ar is aryl, heteroaryl, aryl-substituted aryl, aryl substituted heteroaryl which are optionally substituted with one to four substituents independently selected from Rx and R9 is selected from H and Ry. Rz is selected from
1 ORd
2 NHRα
3 NRdS(O)mRe
Figure imgf000054_0001
and all other designations and substituents are as previously recited in claim 1. 4. An antagonist of a plurality of integrins, the antagonist having the formula (IV):
Figure imgf000054_0002
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx and all other designations and substituents are as recited in claim 1
5. An antagonist of a plurality of integrins, the antagonist having the formula (V):
Figure imgf000054_0003
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx , R9 is selected from H and Ry and all other designations and substituents are as recited in claim 1
6. An antagonist of a plurality of integrins, the antagonist having the formula (VI):
Figure imgf000055_0001
wherein all designations and substituents are as recited in claim 1.
7. An antagonist of a plurality of integrins, the antagonist having the formula (VII):
Figure imgf000055_0002
wherein Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherein said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx and all other designations and substituents are as recited in claim 1.
8. An antagonist of a plurality of integπns, the antagonist having the formula (VIII):
Figure imgf000056_0001
wherem all designations and substituents are as recited in claim 1.
9. An antagonist of a plurality of integπns, the antagonist having the formula (IX):
Figure imgf000056_0002
wherein all designations and substituents are as recited in claim 1.
10. An antagonist of a plurality of integπns, the antagonist having the formula (X):
Figure imgf000056_0003
Ar is selected from aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, and heteroaryl substituted heteroaryl and further wherem said aryl, heteroaryl, aryl substitiuted aryl, aryl substituted heteroaryl, heteroaryl substituted heteroaryl are optionally substituted with 1 to 4 substituents independently selected from Rx , R9 is selected from H and Ry , Rz is selected from 1 ORd
2 NHRα
3 NRdS(O)mRe
Figure imgf000057_0001
and all other designations and substituents are as recited in claim 1.
11. An antagonist of a plurality of integnns, the antagonist having the formula (XI):
Figure imgf000057_0002
wherein all designations and substituents are as recited in claim 1.
12. An antagonist of a plurality of integnns, the antagonist having the formula (XII)-
Figure imgf000057_0003
wherein all designations and substituents are as recited in claim 1.
13. An antagonist of a plurality of integπns, the antagonist having the formula (XIII):
Figure imgf000057_0004
wherem all designations and substituents are as recited in claim 1.
14. The antagonist of claims 1-13, wherein said plurality of integπns is a plurality of beta 1-subunιt containing integπns.
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