EP1165504A2 - Inhibiteurs de la liaison de lfa-1 a des icams icams et lieurs utilisations - Google Patents

Inhibiteurs de la liaison de lfa-1 a des icams icams et lieurs utilisations

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Publication number
EP1165504A2
EP1165504A2 EP00921626A EP00921626A EP1165504A2 EP 1165504 A2 EP1165504 A2 EP 1165504A2 EP 00921626 A EP00921626 A EP 00921626A EP 00921626 A EP00921626 A EP 00921626A EP 1165504 A2 EP1165504 A2 EP 1165504A2
Authority
EP
European Patent Office
Prior art keywords
sulfide
chlorophenyl
amino
chloro
dichlorophenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00921626A
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German (de)
English (en)
Inventor
Kerry Fowler
Mark Orme
Donald E. Staunton
Janet Adolphson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Icos Corp
Original Assignee
Icos Corp
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Filing date
Publication date
Application filed by Icos Corp filed Critical Icos Corp
Publication of EP1165504A2 publication Critical patent/EP1165504A2/fr
Withdrawn legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07D207/30—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 two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—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 two 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
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/416—2,5-Pyrrolidine-diones 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 other ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/01—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton
    • C07C323/09—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton having sulfur atoms of thio groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/31—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C07C323/33—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton having at least one of the nitrogen atoms bound to a carbon atom of the same non-condensed six-membered aromatic ring
    • C07C323/35—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton having at least one of the nitrogen atoms bound to a carbon atom of the same non-condensed six-membered aromatic ring the thio group being a sulfide group
    • C07C323/37—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton having at least one of the nitrogen atoms bound to a carbon atom of the same non-condensed six-membered aromatic ring the thio group being a sulfide group the sulfur atom of the sulfide group being further bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
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    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/41—Y being a hydrogen or an acyclic carbon atom
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom 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
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/70—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom 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
    • C07D215/36—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles 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 in position 2
    • C07D277/70—Sulfur atoms
    • C07D277/74—Sulfur atoms substituted by carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/096—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
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    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/87—Benzo [c] furans; Hydrogenated benzo [c] furans
    • C07D307/90—Benzo [c] furans; Hydrogenated benzo [c] furans with an oxygen atom in position 1 and a nitrogen atom in position 3, or vice versa

Definitions

  • LFA- 1 The leukocyte function- associated antigen (LFA- 1 , CD 11 a/CD 18) is a leukocyte-specific ⁇ 2 integrin that participates in cell/cell adhesion. Binding activity of LFA-1 is essential to leukocyte extravasation from circulation to a site of injury in an inflammatory response. Three principle ligands are known to bind LFA-1, ICAM- 1, ICAM-2, and ICAM-3, which are intercellular adhesion molecules that play an important role in localizing leukocyte adhesion to endothelial cells at a site of injury.
  • ICAM-4 and ICAM-5 have also been reported to bind LFA-1.
  • ICAM-1 is normally expressed at low levels on the endothelium, however, injury- induced inflammatory mediators promote enhanced surface expression in cells at the site of the injury which, in turn, promotes localized leukocyte adhesion through binding with activated LFA-1.
  • LFA-1 includes distinct intracellular and extracellular domains that are believed to participate and/or regulate ICAM binding.
  • I domain a region in the ⁇ L chain of approximately 200 amino acids, designated the I domain, that is found in all ⁇ 2 integrins, as well as many other proteins.
  • Evidence suggests that the I domain is essential to LFA-1 binding to ICAM-1 and 3.
  • anti-LFA-1 blocking monoclonal antibodies have been mapped to epitopes within the I domain.
  • recombinant I domain polypeptide fragments have been shown to inhibit integrin-mediated adhesion and bind ICAM-1.
  • LFA-1 Within the I domain of LFA-1 (and other proteins) is a single metal ion dependent adhesion site (MIDAS) that preferentially binds manganese or magnesium ions. Binding of either cation is required for ligand interaction and is believed to induce conformational changes in LFA-1 necessary for binding. Cation binding may therefore be a regulatory mechanism that responds to changes in the extracellular leukocyte environment. This hypothesis is supported by the observation that calcium ion binding actually inhibits LFA-1 interaction with ICAM-1. Indeed, it has been proposed that an inactive LFA-1 conformation results from calcium binding, and that replacement of the calcium ion with a manganese or magnesium ion is a step required for LFA-1 activation [Griggs, et al, J. Biol. Chem. 275:22113-22119 (1998)]. Other factors have also been shown to induce LFA-1 activation, including T cell receptor engagement, cytokine stimulation, and in vitro PMA stimulation.
  • MIDAS metal ion dependent adhe
  • LFA-1 /ICAM binding sites provides targets to modulate leukocyte inflammatory responses.
  • Numerous antibodies have been isolated that are capable of inducing LFA-1 activation [see, for example, Landis, et al, J. Cell Biol. 120:1519-1521 (1993)] or, for example, preventing ICAM-
  • the present invention is directed to compounds that bind to a novel regulatory site in the I domain of LFA-1, and thereby inhibit LFA-1 binding to ICAMs that bind LFA-1.
  • the present invention therefore also provides methods to regulate leukocyte adhesion to endothelial cells.
  • Compounds of the invention are useful for the treatment of pathologies, such as those associated inflammatory diseases, autoimmune diseases, tumor metastasis, allograft rejection and reperfusion injury.
  • the present invention is directed to diaryl sulfides of general structural formula (I), a pharmaceutically acceptable salt, or prodrug thereof, and to the use of diaryl sulfides, and particularly compounds of formula (I), to inhibit LFA-1 binding to an ICAM that binds LF A- 1.
  • a and B independently, are aryl groups selected from the group consisting of
  • 5- and 6-membered aromatic rings including, but not limited to, phenyl, thienyl, furyl, pyrimidinyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrrolyl, and pyridazinyl;
  • R, R 2 and R 3 independently, are selected from the group consisting of
  • R a is hydrogen or an alkyl group containing one to six saturated straight or branched chain carbon atoms (C, .6 alkyl), -O-R a , -halo, wherein halo is Cl, F, Br, or I,
  • R b and R c independently, are H, C,. 6 alkyl, or -CH 2 -aryl, -NO 2 ,
  • n is an integer 1 to 6, a 5- or 6-membered heterocychc ring, either aliphatic or aromatic, containing one or more of O, N, or S, optionally substituted, such as morpholino, and
  • aryl is a 5- or 6-membered aromatic ring, optionally substituted
  • R 4 , R 5 and R 6 independently, are selected from the group consisting of
  • R 4 and R 5 are taken together to form a 5- or 6-membered aromatic ring, optionally containing one or more of O, N, or S in the ring, optionally substituted.
  • novel negative regulators of LFA-1 binding to ICAMs include, but are not limited to the compounds presented in Table I.
  • Table I Exemplary Negative Regulators
  • the compounds as represented by structural formula (I) can be prepared by synthetic methods or by metabolic processes. Preparation of the compounds by metabolic processes include both in vivo and in vitro processes. Pharmaceutical compositions comprising compounds of the invention are also contemplated. The invention also provides methods of inhibiting LFA-1 binding to
  • ICAMs that bind LFA-1 comprising the step of contacting LFA-1 with a diaryl sulfide, and preferably a compound of structural formula I.
  • the invention provides methods of inhibiting leukocyte adhesion to endothelial cells comprising the step of contacting leukocytes expressing LFA-1 with a diaryl sulfide, and preferably a compound of the structural formula (I).
  • the invention also comprehends methods for treating an inflammatory disorder comprising the steps of administering to a mammal an amount of a pharmaceutical composition of the invention sufficient to inhibit binding of LFA-1 to a naturlal ligand thereof that competes with ICAM-1 or ICAM-3 for binding to LFA-1.
  • the invention also comprehends methods for treating an inflammatory disorder arising from LFA-1 binding to a natural ligand thereof that competes with ICAM-1 or ICAM-3 for binding to LFA-1, comprising administering to a mammal in need thereof a compound that competes with 3-chloro-4-(l-chloro- naphthalen-2-ylsulfanyl)-phenylamine for binding to LFA-1 in an amount sufficient to inhibit binding of the natural ligand to LFA-1.
  • the invention provides methods of ameliorating a pathological condition associated with LFA-1 binding to an
  • ICAM that binds LFA-1 comprising administering to an individual in need thereof an effective amount of a diaryl sulfide, and preferably a compound of the structural formula (I) to inhibit LFA-1 binding to the ICAM.
  • inhibitors of the present invention include, but are not limited to, the compounds set out in Table I.
  • the invention also provides for use of a compound of the invention in the production of a medicament for the treatment of pathologies associated with LFA- 1 binding to ICAM-1.
  • the invention also provides methods to identify a negative regulator of LFA-1 binding to a natural ligand thereof that competes with ICAM-1 or ICAM-3 for binding to LFA-1 comprising the steps of: a) contacting LFA-1 with an activator of LFA-1 binding; b) measuring LFA-1 binding with the natural ligand in the presence and absence of a test compound; and c) identifying the test compound as an inhibitor when decreased LFA-1 binding to the ligand is detected in the presence of the test compound.
  • the activator is crystal violet.
  • IC 50 value for a compound is defined as the concentration of the compound required to produce 50% inhibition of a biological activity of interest.
  • a negative regulator is defined as a compound characterized by an IC 50 for inhibition of LFA-1 binding to a natural ligand.
  • Negative regulators of LFA-1 binding are defined to have an IC 50 of less than about 200 ⁇ M, less than about 100 ⁇ M, less than about 50 ⁇ M, and preferably from about 0.05 ⁇ M to 40 ⁇ M.
  • pharmaceutically acceptable carrier refers to those prodrugs of compounds of the invention which are suitable for use in contact with recipient animals and having undue toxicity, irritation, allergic response commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • prodrug refers to compounds which are rapidly transformed in vivo to the parent compound of the above formula, for example, by hydrolysis. A thorough discussion is provided in Higuchi, et al,
  • Prodrugs as Novel Delivery Systems vol. 14 of the A.C.S.D. Symposium Series, and in Roche (ed), Bioreversible Carriers in Drug Design. American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
  • Prodrug design is discussed generally in Hardma, et al, (Eds), Goodman & Gilman's The Pharmacological Basis of Therapeutics. Ninth Edition, New York,
  • prodrugs are therefore pharmacologically inactive compounds which are converted to biologically active metabolites.
  • prodrugs are rendered pharmacologically active through hydrolysis of, for example, and ester or amide linkage, often times introducing or exposing a functional group on the prodrug.
  • the thus modified drug may also react with an endogenous compound to form a water soluble conjugate which further increases pharmacological properties of the compound, for example, as a result of increased circulatory half-life.
  • prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate.
  • the resulting conjugate may be inactivated and excreted in the urine, or rendered more potent than the parent compound.
  • High molecular weight conjugates may also be excreted into the bile, subjected to enzymatic cleavage, and released back into circulation, thereby effectively increasing the biological half life of the originally administered compound.
  • Stereoisomers may exist as stereoisomers where asymmetric or chiral centers are present. Stereoisomers are designated by either “S” or “R” depending on arrangement of substituents around a chiral carbon atom. Mixtures of stereoisomers are contemplated by the invention. Stereoisomers include enantiomers, diastereomers, and mixtures of the two. Individual stereoisomers of compounds of the invention can be prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by separation or resolution techniques well known in the art.
  • Methods of resolution include (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture by recrystallization or chromatography, and liberation of the optically pure product from the auxiliary; (2) salt formation employing an optically active resolving agent, and (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns.
  • Compounds of the present invention include, but are not limited to those embraced by general structural formula (I) above and the compounds set out in Table I.
  • the invention also provides pharmaceutical compositions comprising one or more compounds of the invention, preferably further comprising a pharmaceutically acceptable carrier or diluent.
  • the invention further provides methods for inhibiting LFA-1 binding to an ICAM that binds LFA-1 comprising the step of contacting LFA-1, or an ICAM- binding fragment thereof, with a negative regulator compound; said negative regulator binding the LFA-1 L polypeptide, or a fragment thereof, at a site selected from the group consisting of a conformation that binds a diaryl sulfide or a binding site defined by lie 259 , Leu 298 , lie 235 , Val' 57 , Leu 161 , and He 306 of human LFA-1 L polypeptide and an
  • LFA-1 domain that binds 3-chloro-4-(l-chloro-naphthalen-2-ylsulfanyl)-phenylamine having the structure described above.
  • the negative regulator binding site on LFA-1 is defined by amino acid residues He 259 , Leu 298 , He 235 , Val 157 , Leu 161 , He 306 , Leu 302 ,Tyr 57 , Leu 132 , Val 233 , Val 130 , and Tyr 166 .
  • the negative regulator binding site on LFA-1 is defined by amino acid residues Lys 287 ,
  • Leu 298 lie 259 , Leu 302 , He 235 , Val 157 , Tyr 257 , Lys 305 , Leu 161 , Leu' 32 , Val 233 , He 255 , Val 130 , Tyr 166 , He 306 , Phe 134 , Phe 168 , Phe 153 , Tyr 307 , Val 308 , He 309 , Thr 231 , Glu 284 , Phe 285 , Glu 301 , Met 154 , He 237 , He 150 , and Leu 295 .
  • the LFA-1 regulatory binding site is described in co- pending U.S. patent application entitled "LFA-1 Regulatory Binding Site and Uses Thereof, filed April 2, 1999, attorney docket number 27866/35375, Serial Number
  • methods of the invention include use of cells expressing either LFA-1 or the ICAM.
  • the other binding partner is either purified and isolated, in a fluid sample (purified, partially purified, or crude) taken from an individual, or in a cell lysate.
  • the invention also comprehends methods wherein both LFA-1 and the ICAM are expressed in cells.
  • the LFA-1 and ICAM binding partners may be expressed on the same cell type or different cell types.
  • the LFA-1 polypeptide is expressed on leukocytes, i.e. lymphocytes, monocytes, or granulocytes
  • the ICAM polypeptide is expressed on endothelial cells.
  • the invention also provides methods to inhibit leukocyte adhesion to endothelial cells comprising the step of contacting said leukocyte with a negative regulator of LFA-1 binding to an ICAM that binds LFA-1 , said negative regulator binding an LFA-1 regulatory site selected from the group consisting of a conformation that binds a diaryl sulfide or a binding site defined by He 259 , Leu 298 , He 233 , Val 157 , Leu 161 , and He 306 of human LFA-1 ⁇ L polypeptide or an LFA-1 domain that binds 3- chloro-4-(l-chloro-naphthalen-2-ylsulfanyl)-phenylamine.
  • the diaryl sulfide binding conformation is defined by amino acid residues as described above. In vivo and //; vitro methods are contemplated.
  • the invention also provides methods to ameliorate a pathology arising from LFA-1 binding to an ICAM comprising the step of administering to an individual in need thereof a negative regulator of LFA-1 binding to the ICAM in an amount effective to inhibit LFA-1 binding to the ICAM, said negative regulator binding to an LFA-1 regulatory site selected from the group consisting of a conformation that binds a diaryl sulfide or a site defined by He 259 , Leu 298 , He 235 , Val 157 , Leu 161 , and He 306 of human LFA-1 or an LFA-1 domain that binds compound 3- chloro-4-( 1 -chloro-naphthalen-2-ylsulfanyl)-phenylamine.
  • methods of the invention include use of a diaryl sulfide compound to inhibit binding of LFA-1 to an ICAM.
  • a preferred method includes use of a compound of general structural formula (I), a pharmaceutically acceptable salt, or prodrug thereof as described above.
  • the invention provides methods to ameliorate pathologies associated with accumulation of leukocytes resulting from LFA-1 binding to an ICAM that binds LFA-1 comprising the step of administering to an individual in need thereof an amount of an inhibitor of LFA-1 binding to the ICAM effective to inhibit LFA-1 binding to the ICAM, said inhibitor binding to LFA-1 at a site presented by amino acid residues He 259 , Leu 298 , lie 235 , Val 157 , Leu 161 and He 306 .
  • Exemplary medical conditions include, without limitation, inflammatory diseases, autoimmune diseases, reperfusion injury, myocardial infarction, stroke, hemorrhagic shock, organ transplant, and the like.
  • Methods of the invention provide for amelioration of a variety of pathologies, including, for example, but not limited to adult respiratory distress syndrome, multiple organ injury syndrome secondary to septicemia, multiple organ injury secondary to trauma: reperfusion injury of tissue, acute glomerulonephritis, reactive arthritis, dermatosis with acute inflammatory components, stroke, thermal injury, Crohn's disease; necrotizing enterocolitis, granulocyte transfusion associated syndrome, and cytokine induced toxicity, and T cell mediated diseases.
  • Inflammatory cell activation and excessive or unregulated cytokine (e.g., TNF ⁇ and IL- 1 ⁇ ) production are also implicated in disorders such as rheumatoid arthritis, osteoarthritis, gouty arthritis, spondylitis, thyroid associated ophthalmopathy,
  • Behcet disease sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shock syndrome, asthma, chronic bronchitis, allergic respiratory distress syndrome, chronic pulmonary inflammatory disease, such as chronic obstructive pulmonary disease, silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium, brain, and extremities, fibrosis, cystic fibrosis, keloid formation, scar formation, atherosclerosis, transplant rejection disorders, such as graft vs.
  • chronic pulmonary inflammatory disease such as chronic obstructive pulmonary disease, silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium, brain, and extremities, fibrosis, cystic fibrosis, keloid formation, scar formation, atherosclerosis, transplant rejection disorders, such as graft vs.
  • inflammatory bowel disease such as ulcerative colitis
  • proliferative lymphocyte diseases such as leukemia
  • inflammatory dermatoses such as atopic dermatitis, psoriasis, urticaria, uveitis.
  • cytokine levels include brain injury due to moderate trauma (see J. Neurotrauma, 12, pp. 1035-1043 (1995); J. Clin. Invest., 91, pp. 1421-1428 (1993)), cardiomyopathies, such as congestive heart failure (see Circulation, 97, pp. 1340-1341 (1998)), cachexia, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), ARC (AIDS related complex), fever myalgias due to infection, cerebral malaria, osteoporosis and bone resorption diseases, keloid formation, scar tissue formation, and pyrexia.
  • congestive heart failure see Circulation, 97, pp. 1340-1341 (1998)
  • cachexia cachexia secondary to infection or malignancy
  • ARC AIDS related complex
  • fever myalgias due to infection cerebral malaria
  • osteoporosis and bone resorption diseases keloid formation
  • the ability of the negative regulators to treat asthma can be demonstrated in a murine allergic asthma model according to the method of Wegner, et al, Science, 247:456-459, (1990), or in a murine non-allergic asthma model according to the method of Bloemen, et al, Am. J. Respir. Crit. Care Med. 153:521- 529 (1996).
  • the ability of the negative regulators to treat inflammatory lung injury can be demonstrated in a murine oxygen-induced lung injury model according to the method of Wegner, et al, Lung, 170:261-219, (1992), in a murine immune complex- induced lung injury model according to the method of Mulligan, et al, J. Immunol, 754:1350-1363, (1995), or in a murine acid-induced lung injury model according to the method of Nagase, et al, Am. J. Respir. Crit. Care Med., 154:504-510, (1996).
  • the ability of the negative regulators to treat autoimmune diabetes can be demonstrated in an NOD mouse model according to the method of Hasagawa, et al, Int. Immunol. 6:831-838 (1994), or in a murine streptozotocin-induced diabetes model according to the method of Herrold, et al, Cell Immunol. 757:489-500, (1994).
  • the ability of the negative regulators to treat inflammatory glomerular injury can be demonstrated in a rat nephrotoxic serum nephritis model according to the method of Kawasaki, et al, J. Immunol, 750:1074-1083 (1993).
  • the ability of the negative regulators to treat radiation-induced enteritis can be demonstrated in a rat abdominal irradiation model according to the method of Panes, et al, Gastroenterology, 108: 1161-1169 ( 1995).
  • the ability of the negative regulators to treat pulmonary reperfusion injury can be demonstrated in a rat lung allograft reperfusion injury model according to the method of DeMeester, et al, Transplantation, (52:1477-1485 (1996), or in a rabbit pulmonary edema model according to the method of Horgan, et al, Am. J. Physiol 267 ⁇ 1578-H1584 (1991).
  • the ability of the negative regulators to treat stroke can be demonstrated in a rabbit cerebral embolism stroke model according to the method of Bowes, et al, Exp. Neurol, 119:215-219 (1993), in a rat middle cerebral artery ischemia-reperfusion model according to the method of Chopp, et al, Stroke, 25:869- 875 (1994), or in a rabbit reversible spinal cord ischemia model according to the method of Clark et al, Neurosurg., 75:623-621 (1991).
  • the ability of the negative regulators to treat cerebral vasospasm can be demonstrated in a rat experimental vasospasm model according to the method of Oshiro, et al, Stroke, 25:2031-2038 (1997).
  • the ability of the negative regulators to treat peripheral artery occlusion can be demonstrated in a rat skeletal muscle ischemia reperfusion model according to the method of Gute, et al, Mol. Cell Biochem., 779:169-187 (1998).
  • the ability of the negative regulators to treat graft rejection can be demonstrated in a murine cardiac allograft rejection model according to the method of Isobe, et al, Science, 255:1125-1127 (1992), in a murine thyroid gland kidney capsule model according to the method of Talento, et al, Transplantation, 55:418-422 (1993), in a cynomolgus monkey renal allograft model according to the method of Cosimi, et al, J.
  • GVHD graft-vs.-host disease
  • the present invention also provides pharmaceutical compositions which comprise a diaryl sulfide formulated together with one or more pharmaceutically-acceptable carriers.
  • the pharmaceutical compositions of the invention can be administered to humans and other animals by any suitable route.
  • the compositions can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or nasally.
  • parenteral administration as used herein refers to modes of administration which include intravenous, intraarterial, intramuscular, intraperitoneal, intrasternal, intrathecal, subcutaneous and intraarticular injection and infusion.
  • compositions of this invention for parenteral injection comprise pharmaceutically-acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene, glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oils), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like, Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay abso ⁇ tion such as aluminum monosterate and gelatin.
  • adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents.
  • the abso ⁇ tion of the drug in order to prolong the effect of the drug, it is desirable to slow the abso ⁇ tion of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amo ⁇ hous materials with poor water solubility. The rate of abso ⁇ tion of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed abso ⁇ tion of a parenterally administered drug from is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such a polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial- or viral-retaining filter, or by inco ⁇ orating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with a least one inert, pharmaceutically-acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, gums (e.
  • .alginates, acacia gelatin, polyvinylpyrrolidone, and sucrose
  • humectants such as glycerol
  • disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate
  • disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate
  • e solution retarding agents such a paraffin
  • abso ⁇ tion accelerators such as quaternary ammonium compounds
  • wetting agents such as, for example, cetyl alcohol and glycerol monosterate
  • absorbents such as kaolin and bentonite clay
  • lubricants such as talc, calcium sterate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredients(s) only, or preferentially, in a part of the intestinal tract, optionally, in a delayed manner! Exemplary materials include polymers having pH sensitive
  • solubility such as the materials available as Eudragit .
  • embedding compositions include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi- lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non- toxic, physiologically-acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • compositions of the present invention may be used in the form of pharmaceutically-acceptable salts derived from inorganic or organic acids.
  • pharmaceutically-acceptable salt is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically-acceptable salts are well known in the art. For example, S. M.
  • Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecano
  • Basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialky sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialky sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl, lau
  • Basic addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid- containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or organic primary, secondary or tertiary amine.
  • Pharmaceutically-acceptable basic addition salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.
  • Dosage forms for topical administration of a compound of this invention include powders, sprays, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically-acceptable carrier and any needed preservatives, buffers, or propellants which may be required.
  • Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
  • Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration.
  • the selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.
  • dosage levels of about 0J to about 1000 mg, about 0.5 to about 500 mg, about 1 to about 250 mg, about 1.5 to about 100, and preferably of about 5 to about 20 mg of active compound per kilogram of body weight per day are administered orally or intravenously to a mammalian patent.
  • the effective daily dose may be divided into multiple doses for pu ⁇ oses of administration, e.g., two to four separate doses per day.
  • Example 1 described a high throughput assay to screen for negative regulators of LFA-1 binding to an ICAM that binds LFA-1.
  • Example 2 relates to binding assays to evaluate the ability of various compounds to inhibit LFA-1 binding to an ICAM.
  • Example 3 describe synthesis of negative regulators.
  • Example 4 provides results from cell based assays using the negative regulators.
  • HTS high throughput screening
  • ICAM-1 /IgGl fusion protein (comprising full length ICAM-1) was prepared as described in U.S. Patent Nos. 5,770,686, 5,837,478, and 5,869,262, each of which is inco ⁇ orated herein by reference.
  • the fusion protein was biotinylated using a kit obtained from Pierce Chemical (Rockford, IL). Biotinylated protein (BioIgICAM-1) concentration was determined by measuring absorbance at 280 nm, and serial dilutions were prepared to give a final concentration range of 50 ⁇ g/ml to 0.008 ⁇ g/ml.
  • BioIgICAM-1 Titration of BioIgICAM-1 was carried out with the protein first aliquoted into wells on an assay plate. Recombinant LFA-1 was added to each well at the same concentration and the experiment (as described below) was carried to completion. The amount of binding was determined for each well, and from a subsequent plot of the results, a single concentration of BioIgICAM-1 was selected for subsequent experiments. In a similar manner, LFA-1 was titrated using the BioIgICAM-1 concentration selected as described above.
  • the capture antibody i.e., a non-blocking anti-LFA-1 monoclonal antibody (TS2/4J; ATCC #HB244)
  • plate coating buffer 50 mM sodium carbonate/bicarbonate, 0.05% ProClin 300, pH 9.6
  • Immulon 4 (Dynex
  • BioIgICAM-1 was prepared containing 0J ⁇ g/ml BioIgICAM-1 and 4 ⁇ M crystal violet (found to be an activator of LFA-l/ICAM-1 binding) in Assay Buffer (EG&G Wallac, Gaithersburg, MD). Aliquots (50 ⁇ l) of pooled chemicals (22 compounds/pool in 100% DMSO) from the chemical library were added to the wells, followed by addition of 50 ⁇ l of the 2X stock of BioIgICAM-1 to provide a final assay volume of 100 ⁇ l (containing 2%
  • Controls included both positive and negative wells and 50% binding wells established using blocking antibodies, i.e., anti-LFA-1 monoclonal antibody (TS1/22J, ATCC #HB202) or anti-ICAM-1 monoclonal antibody. Chemical pools in wells showing 50% or greater inhibition of LFA-1 binding to ICAM-1 were identified and the experiment was repeated using individual chemicals from those pools. Inhibitors of LFA-l/ICAM-1 binding were identified, and a further screen was performed to determine dose dependence of the inhibitory activity. Further study of selected compounds was carried out using biochemical and cellular assay techniques. The compounds were grouped according to common structural features, and it was found that a subset (listed below) of the compounds included a characteristic diaryl sulfide structure.
  • diaryl sulfide derivatives were conducted as described below in Example 3. Additional diaryl sulfide derivatives are described in co-pending provisional patent application entitled “Cell Adhesion-Inhibiting Antiinflammatory and Immune Suppressive Compounds” filed April 2, 1999, attorney docket number
  • ICAM-1 /LFA-1 Biochemical Interaction Assay Compounds that antagonize the interaction between ICAM-1 and
  • LFA-1 can be identified, and their activities quantitated, using both biochemical and cell-based assays.
  • a primary biochemical assay measures the ability of the compound in question to block the interaction between LFA-1 and its adhesion partner ICAM-1, as described below.
  • D-PBS Dulbecco's phosphate-buffered saline
  • 100 ⁇ l of anti-LFA-1 antibody at a connection of 5 ⁇ g/ml in Dulbecco's phosphate-buffered saline (D-PBS) was used to coat wells of a 96-well microtiter plate overnight at 4°C. The wells were washed twice with wash buffer (CMF-PBS, 0.05% Tween® 20) and blocked by addition of 200 ⁇ l of D-PBS containing 5% fish skin gelatin.
  • LFA-1 Recombinant LFA-1 (100 ⁇ l of 0.7 ⁇ g ml) in D-PBS was added to each well. Incubation was continued for one hour at room temperature and the wells were washed twice with wash buffer. Serial dilutions of compounds being assayed as LFA-l/ICAM-1 negative regulators, prepared as 10 mM stock solutions in DMSO, were diluted in D-PBS, 2 mM MgCl 2 , 1% fish skin gelatin and 500 ⁇ l of each dilution added to each well, followed by addition of 50 ⁇ l of 0.8 ⁇ g/ml BioIgICAM-1 to the wells, and the plates were incubated at room temperature for one hour.
  • background refers to wells that were not coated with anti-LFA-1 antibody.
  • Biologically relevant activity of the compounds in this invention was confirmed using a cell-based adhesion assay that measures the ability of the compounds to block adherence of JY-8 cells (a human EBV-transformed B cell line expressing LFA-1 on its surface) to immobilized ICAM-1, as follows.
  • This assay may be performed with or without added IL-8.
  • For IL8 stimulation of the standard JY-8 cells 30 ng/ml IL-8 was added in the 30 minute incubation at 37°C with the cells.
  • 96-well microtiter plates were coated with 70 ⁇ l of recombinant ICAM-1/Ig at a concentration of 5 ⁇ g/ml in CMF-PBS overnight at 4°C.
  • JY-8 cells 50 ⁇ l at 2 x 10 6 cells/ml in RPMI- 1640/1 % fetal bovine serum (FBS) were added to the wells.
  • FBS fetal bovine serum
  • 5 x 10° cells washed once in RPMI 1640, were resuspended in 1 ml RPMI-1640 containing 2 ⁇ M Calcein AM (Molecular Probes, OR), were incubated at 37°C for 30 minutes and washed once with RPMI- 1640/1% FBS.
  • Dilutions of compounds to be assayed for LFA-l/ICAM-1 antagonistic activity were prepared in RPMI-1640/1% FBS from 10 mM stock solutions in DMSO and 50 ⁇ l aliquots were added to duplicate wells. Microtiter plates were incubated for 45 min at room temperature and the wells were washed gently once with RPMI-1640/1% FBS. Fluorescence intensity was measured in a fluorescence plate reader with an excitation wavelength at 495 nm and an emission wavelength at 530 nm. The percent inhibition of a candidate compound at a given concentration was calculated using the following equation:
  • 96-well microtiter plates were coated with 50 ⁇ l of recombinant ICAM-3/Ig at a concentration of 10 ⁇ g/ml in CMF-PBS overnight at 4°C. The wells were washed twice with D-PBS, blocked by addition of 100 ⁇ l of D-PBS, 1% bovine serum albumin (BSA) by incubation for one hour at room temperature, and washed once with RPMI- 1640/5% heat-inactivated FBS (adhesion buffer).
  • BSA bovine serum albumin
  • Dilutions of compounds to be assayed for LFA-1 /ICAM-3 antagonistic activity were prepared in adhesion buffer from 10 mM stock solutions in DMSO and 100 ⁇ l aliquots were added to duplicate wells. JY-8 cells (100 ⁇ l at 0.75 x 10 6 cells/ml in adhesion buffer) were then added to the wells (with or without 30 ng/ml IL-8). Microtiter plates were incubated for 30 min at room temperature, the adherent cells were fixed with 50 ⁇ l of 14% glutaraldehyde/D-PBS and incubation carried out for an additional 90 min.
  • the general synthesis method A is diagramed schematically below, wherein X is a halogen, e.g., chlorine.
  • X is a halogen, preferably chloro.
  • the yellow solid product was then collected by filtration and dried at 40 °C in a vacuum oven for 24 hr.
  • the product yield was 66%, and the melting point was 128-130°C.
  • Standard analytical techniques including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chlorofornrhexane (1 :4). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The off-white solid product was collected by filtration and dried at 50°C in a vacuum oven for 24 hr. The product yield was 57%, and the melting point was 180-185 °C (decomposition). Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform:hexane (1 :1). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The white solid product was collected by filtration and dried at 60° C in a vacuum oven for 24 hr. The product yield was 71%, and the melting point was 91-93°C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform :hexane (1:5). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The colorless solid product was collected by filtration and dried at
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform:hexane (1:3). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The white solid product was collected by filtration and dried at 40 °C in a vacuum oven for 24 hr. The product yield was 72%, and the melting point was 109-111 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform :hexane (3:7). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The off-white solid product was collected by filtration and dried at 50°C in a vacuum oven for 24 hr. The product yield was 59%, and the melting point was 86-88 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chlorofor ⁇ uhexane (3:7). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The colorless solid product was collected by filtration and dried at 70 °C in a vacuum oven for 24 hr. The product melting point was 103-105 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chlorofor ⁇ uhexane (3:7). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The off-white solid product was collected by filtration and dried at 60 °C in a vacuum oven for 24 hr. The product yield was 66%, and the melting point was 113-115 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform-hexane (1 :4). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The off-white solid product was collected by filtration and dried at 60° C in a vacuum oven for 24 hr. The product yield was
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform:hexane (2:3). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The pale yellow solid product was collected by filtration and dried at 60° C in a vacuum oven for 24 hr. The product yield was 58%, and the melting point was 133-135 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chlorofor ⁇ uhexane (1 :4). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The brown solid product was collected by filtration and dried at 50° C in a vacuum oven for 24 hr. The product yield was 16%, and the melting point was 65-70 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chloroform:hexane (1 :1). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The product was collected by filtration and dried at 50° C in a vacuum oven for 24 hr. The product melting point was not determined because it was a colorless oil. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • the product was obtained using preparative chromatography by applying the dissolved residue to a small column packed with silica gel (70-230 mesh), followed by elution using a solvent system of chlorofor ⁇ uhexane (3:2). Fractions containing the product were combined and the solvents removed using a rotary vacuum. The yellow solid product was collected by filtration and dried at 50° C in a vacuum oven for 24 hr. The product yield was 62%, and the melting point was 129-131 °C. Standard analytical techniques, including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • 4-Chloro-2-nitro-5-(2,4-dichlorophenylsulfanyl)-phenylamine was prepared by the general procedure with the exception that it was purified as the free amine by flash chromatography. 1J7 g (3.93 mmol) of the free amine was added to 10 ml DMF and 10 ml of EtOH. 4.43 g (5 eq, 19.7 mmol) tin chloride dihydrate was added followed by 10 ml concentrated HCl. This was heated to 60°C for 20 hrs.
  • reaction was allowed to cool to ambient temperature, and was then diluted with 30 ml water, and brought to pH 12 by the addition of 30 ml 5 N NaOH. This mixture was twice extracted with 150 ml ether. The combined organics were washed with 100 ml sat. NaHCO 3 and 2 x 100 ml sat. NaCl, dried over Na 2 SO 4 and filtered.
  • 2-Chloro-4-cyanopheny-(2',4'-dichlorophenyl)-sulfide (1.0 g, 3J8 mmol) was added to 200 ml of THF under nitrogen at 0°C. Lithium aluminum hydride (0J4 g) was then added to the solution in portions. The mixture was then allowed to stand for 2 hr at 0°C, after which 1 ml of 20% NaOH was added to the mixture, followed by 1 ml of dH 2 O. The solution was then filtered, and solvents were removed using a rotary evaporator. The resulting residue was then applied to a small column containing silica gel (70-230 mesh).
  • the column was washed with a 1 : 1 mixture of chloroform and hexane, after which the product was eluted using a rotary evaporator, and the product was obtained as a colorless oil.
  • the product yield was 40%, and the melting point was not determined.
  • Standard analytical techniques including proton NMR spectroscopy, elemental analysis and thin layer chromatography, were employed to characterize the product.
  • Example 4 Cell-Based Assay Results
  • Compounds of the invention displayed activity in the cell-based assays described above as set out in Table in below which provides ⁇ M IC 50 values for inhibition of LFA-1 binding to ICAM-1 and ICAM-3 where tested. Paired values

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Abstract

L'invention porte sur de nouveaux composés qui inhibent la liaison de LFA-1 à un partenaire de liaison d'un ligand naturel, ainsi que sur les utilisations de ces composés.
EP00921626A 1999-04-02 2000-04-03 Inhibiteurs de la liaison de lfa-1 a des icams icams et lieurs utilisations Withdrawn EP1165504A2 (fr)

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US28532599A 1999-04-02 1999-04-02
US285325 1999-04-02
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US6515124B2 (en) * 2000-02-09 2003-02-04 Hoffman-La Roche Inc. Dehydroamino acids
JP2004502681A (ja) 2000-06-29 2004-01-29 アボット・ラボラトリーズ アリールフェニル複素環スルフィド誘導体および細胞接着を阻止する抗炎症性および免疫抑制性物質としてのその使用
US7064180B2 (en) 2000-07-31 2006-06-20 The General Hospital Corporation High affinity integrin polypeptides and uses thereof
US7323552B2 (en) 2000-07-31 2008-01-29 The General Hospital Corporation Variant integrin polypeptides and uses thereof
MXPA03000981A (es) * 2000-07-31 2003-09-05 Gen Hospital Corp Polipeptidos de integrina de alta afinidad y uso de los mismos.
US7153944B2 (en) 2000-07-31 2006-12-26 The General Hospital Corporation High affinity integrin polypeptides and uses thereof
UA81749C2 (uk) 2001-10-04 2008-02-11 Х. Луннбек А/С Фенілпіперазинові похідні як інгібітори зворотного захоплення серотоніну
EP1539744A4 (fr) 2002-07-11 2007-06-06 Vicuron Pharm Inc Derives de n-hydroxyamide ayant une activite anti-bacterienne
ATE406894T1 (de) 2003-04-04 2008-09-15 Lundbeck & Co As H 4-(2-phenylsulfanyl-phenyl)-piperidin-derivate als serotonin-wiederaufnahme-hemmer
RS50589B (sr) 2003-12-23 2010-05-07 H. Lundbeck A/S. 2-(1h-indolilsulfanil)-benzil amin derivati kao ssri
CA2557794A1 (fr) 2004-03-15 2005-10-06 Eli Lilly And Company Antagonistes des recepteurs opioides
AR052308A1 (es) 2004-07-16 2007-03-14 Lundbeck & Co As H Derivados de 2-(1h-indolilsulfanil)-arilamina y una composicion farmaceutica que contiene al compuesto
US7629473B2 (en) 2005-06-17 2009-12-08 H. Lundbeck A/S 2-(1H-indolylsulfanyl)-aryl amine derivatives
AR054393A1 (es) 2005-06-17 2007-06-20 Lundbeck & Co As H Derivados de benzo(b)furano y benzo(b)tiofeno, composiciones farmaceuticas que los contienen y su uso en la fabricacion de un medicamento para el tratamiento de enfermedades mediadas por la inhibicion de la reabsorcion de neurotransmisores de amina biogenicos.
JP2007008877A (ja) * 2005-06-30 2007-01-18 Sato Pharmaceutical Co Ltd ２−ピリドン誘導体を有効成分とする医薬組成物
WO2007045593A2 (fr) * 2005-10-18 2007-04-26 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. Derives de naphtyle inhibiteurs de l'agregation de beta-amyloide
US8436025B2 (en) * 2008-09-19 2013-05-07 CompleGen Partners, Inc. Compounds and methods for PKC theta inhibition
US9884046B1 (en) * 2017-06-26 2018-02-06 Macau University Of Science And Technology Method of treating lung cancer
NL2026714B1 (en) 2020-10-20 2022-06-16 Lumicks Ca Holding B V Improved detection of lymphocyte - target cell interaction

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IL145528A0 (en) 2002-06-30
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NZ515238A (en) 2005-02-25
WO2000059878A3 (fr) 2001-08-09
EA200101017A1 (ru) 2002-04-25
IS6096A (is) 2001-09-28
SK14022001A3 (sk) 2002-03-05
AU4191900A (en) 2000-10-23
HUP0201904A2 (en) 2002-09-28
PL351323A1 (en) 2003-04-07
BR0009421A (pt) 2002-03-26
CA2369005A1 (fr) 2000-10-12
BG106019A (en) 2002-06-28
JP2002541141A (ja) 2002-12-03
CZ20013524A3 (cs) 2002-06-12
CN1351588A (zh) 2002-05-29
AU2004205210A1 (en) 2004-09-23
KR20020003559A (ko) 2002-01-12
MXPA01009915A (es) 2003-08-20
NO20014768D0 (no) 2001-10-01
HUP0201904A3 (en) 2003-04-28
EE200100514A (et) 2002-12-16
HRP20010777A2 (en) 2002-12-31
WO2000059878A2 (fr) 2000-10-12
AU774054B2 (en) 2004-06-17

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