EP0845987A1 - Inhibiteurs de la c-proteinase destines au traitement des affections liees a la surproduction de collagene - Google Patents

Inhibiteurs de la c-proteinase destines au traitement des affections liees a la surproduction de collagene

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Publication number
EP0845987A1
EP0845987A1 EP96930499A EP96930499A EP0845987A1 EP 0845987 A1 EP0845987 A1 EP 0845987A1 EP 96930499 A EP96930499 A EP 96930499A EP 96930499 A EP96930499 A EP 96930499A EP 0845987 A1 EP0845987 A1 EP 0845987A1
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EP
European Patent Office
Prior art keywords
alkyl
mono
group
sulfonyl
compound
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Withdrawn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
EP96930499A
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German (de)
English (en)
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EP0845987A4 (fr
Inventor
Mitch Brenner
Wen-Bin Ho
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Fibrogen Inc
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Fibrogen Inc
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Publication of EP0845987A1 publication Critical patent/EP0845987A1/fr
Publication of EP0845987A4 publication Critical patent/EP0845987A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/022—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07C323/50—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 carboxyl groups bound to the same carbon skeleton
    • C07C323/51—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 carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—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 carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
    • 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
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C07D207/48—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/40—Esters thereof
    • C07F9/4003—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4006—Esters of acyclic acids which can have further substituents on alkyl

Definitions

  • Collagen is integral to, among other things, the proper formation of connective tissue.
  • the over- or under-production of collagen or the production of abnormal collagen has been linked with numerous connective tissue diseases and disorders.
  • C-proteinase is an essential key enzyme for the proper maturation of collagen, and therefore appears to be an ideal target for the inhibition, control and/or modulation of collagen formation.
  • the present invention relates to organic molecules capable of inhibiting C-proteinase activity in order to regulate, modulate and/or inhibit abnormal collagen formation. More specifically, the invention relates to the use of compounds and pharmaceutical compositions thereof for the treatment of various diseases relating to the inappropriate or unregulated production of collagen.
  • Collagen Structure At present nineteen types of collagens have been identified. These collagens, including fibrillar collagen types I, II, III are synthesized as procollagen precursor molecules which contain amino- and carboxy-terminal peptide extensions. These peptide extensions, referred to as “pro-regions, " are designated as N- and C-propeptides, respectively.
  • the pro-regions are typically cleaved upon secretion of the procollagen triple helical precursor molecule from the cell to yield a mature triple helical collagen molecule.
  • the "mature" collagen molecule is capable of association, for example, into highly structured collagen fibers. See e.g. , Fessler and Fessler, 1978, Annu. Rev. Biochem. 47: 129-162; Bornstein and Traub, 1979, in: The Proteins (eds. Neurath, H. and Hill, R.H.), Academic Press, New York, pp. 412-632; Kivirikko et al. , 1984, in: Extracellular Matrix Biochemistry (eds. Piez, K.A.
  • An array of critical diseases has been associated with the inappropriate or unregulated production of collagen, including pathological fibrosis or scarring, including endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers' fibrosis, pericentral fibrosis, hepatitis, dermatof ⁇ broma, biliary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, scleroderma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duch
  • fibrotic disorders may be induced or initiated by surgery, including scar revision plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis.
  • One strategy for the treatment of these diseases is to inhibit the pathological overproduction of collagen.
  • identification and isolation of molecules which control, inhibit and/or modulate the production of collagen are of major medical interest.
  • C-proteinase is the essential key enzyme that catalyzes the cleavage of the C-propeptide of, for example, fibrillar collagens, including type I, type II, and type III collagen. See, U.S. Application Ser. No. 60/002,038, filed August 8, 1995 as a provisional application, and references disclosed therein. " C-proteinase was first observed in the culture media of human and mouse fibroblasts (Goldberg et al , 1975, Cell 4:45-50; Kessler and Goldberg, 1978, Anal. Biochem. 86:463-469), and chick tendon fibroblasts (Duskin et al , 1978, Arch.
  • a partially purified protein having C-proteinase activity was obtained from chick calvaria in 1982. Njieha et al. , 1982, Biochemistry 23:757-764.
  • chicken C-proteinase was isolated, purified and characterized from 0 conditioned media of chick embryo tendons. Hojima et al. , 1985, J. Biol. Chem. 260: 15996-16003.
  • Murine C-proteinase has been subsequently purified from media of cultured mouse fibroblasts. Kessler et al. , 1986, Collagen Relat. Res. 6:249-266; Kessler and Adar, 1989, Eur. J. Biochem. 186:115- 121.
  • the cDNA encoding human C-proteinase has been identified, as 5 set forth in the above-referenced related applications and references disclosed therein.
  • Dithiothreitol, SDS, concanavalin A, Zn 2+ , Cu 2 ' , and Cd 2+ are similarily reported to be inhibitory at low concentrations.
  • some reducing agents, several amino acids, phosphate, and ammonium sulfate were inhibitory at concentrations of 1-lOmM.
  • the enzyme was shown to be inhibited by the basic amino acids lysine and arginine. Leung et al. , supra; Ryhanen et al. , 1982, Arch. Biochem. Biophys. 215:230-236.
  • high concentrations of NaCI or Tris-HCI buffer were found to inhibit C- proteinase's activity.
  • C-proteinase activity and its inhibition have been determined using a wide array of assays. See e.g. , Kessler and Goldberg, 1978, Anal. Biochem. 86:463; Njieha et al. , 1982, Biochemistry 21:757-764. Despite the availability of such assays, large scale review and testing of potential C- proteinase inhibitors has not been performed to date due to the limited availability of human C-proteinase. As articulated in numerous publications, the enzyme is difficult to isolate by conventional biochemical means and the identity of the cDNA sequence encoding such enzyme was not known until reported in the above-referenced and related patent applications.
  • C- proteinase appears to be an ideal target for the treatment of disorders associated with the inappropriate or unregulated production and maturation of collagen.
  • none of the inhibitors so far identified has proven an effective therapeutic for the treatment of collagen-related diseases or even an inhibitor to C-proteinase activity.
  • the present invention relates to organic molecules capable of modulating, regulating and/or inhibiting production and/or maturation of collagen by affecting C-proteinase activity.
  • the compounds of the present invention have the formulae:
  • R is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, 5 acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or * • • " poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • * ⁇ Rj is selected from the group consisting of aryl, heteroaryl, alkyl, aralkyl, heteroaralkyl, alkylamino, arylalky lamin
  • Y is selected from the group consisting of OH, HOHN(hydroxylamine), H 2 N, alkylamino; 2 " Z is a direct bond; methylene, oxygen, sulf or, amino; n is 0 or 1 ;
  • Rj is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl- (thio, sulfmyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl- (thio, sulfmyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 4 is selected from the group consisting of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl;
  • R is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl; j is selected from the group consisting of H, lower alkyl; R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufmyl or sul
  • H is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 4 is selected from the group consisting of H, lower alkyl
  • Rj is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl;
  • Rj is selected from the group consisting of OH, alkoxyl, lower alkyl, alkylamino, peptide
  • X is selected from the group consisting of N, C;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 4 is selected from the group consisting of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl.
  • the present invention is further directed to pharmaceutical compositions comprising a pharmaceutically effective amount of the above- described compounds and a pharmaceutically acceptable carrier or excipient. Such a composition should modulate the production and/or maturation of 3 collagen by inhibiting C-proteinase activity.
  • the present invention is also directed to the use of the disclosed compounds and compositions for the treatment of disorders associated with the inappropriate or unregulated production of collagen by modulating, inhibiting and/or regulating C-proteinase activity.
  • the compositions of the present invention may be included in methods for treating diseases associated with inappropriate or unregulated production of collagen, including, but not limited to, rheumatoid arthritis, scleroderma, pathological fibrosis or scarring.
  • C-proteinase shall be construed to mean an enzyme capable of processing collagen molecules, derivatives or fragments, or their precursors by cleaving through -Ala_l_Asp-Asp- and/or -GlyJ_Asp-Glu-.
  • the term shall include human C-proteinase and derivatives, analogs, fragments and variants
  • “Pharmaceutically acceptable salts” refers to those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,
  • Alkyl refers to a saturated aliphatic hydrocarbon, including straight- chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7
  • Typical alkyl groups includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl
  • the alkyl group may be substituted or unsubstituted.
  • the substituted group(s) is preferably, carboxyl, hydroxyl, mercapto, cycloalkyl, heterocycloalkyl, halo, alkoxyl, alkylamino.
  • Aryl refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocylic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substimted.
  • the aryl is a substimted or unsubstituted phenyl or pyridyl.
  • Preferred aryl substituent(s) preferably phenyl or pydridyl are halogen, trihalomethyl, hydroxyl, SH, NO 2 , amine, thioether, cyano, alkoxy, and groups.
  • the present invention relates to compounds capable of regulating and/or modulating collagen formation by inhibiting C-proteinase activity.
  • the present invention is directed to compounds which inhibit C-proteinase activity as a therapeutic approach to cure or manage various connective tissue disorders, including fibrotic disorders, arthritic disorders, or disorders induced or initiated by surgical or dramatic insults.
  • the invention is generally directed to compounds and/or compositions comprising compounds having the formulae:
  • R[ is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitimted aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitimted aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thk), sulfinyl or sulfonyl)-alkyl;
  • R is selected from the group consisting of aryl, heteroaryl, alkyl, aralkyl, heteroaralkyl, alkylamino, arylalky lamino;
  • Y is selected from the group consistmg of OH, HOHN(hydroxylamine), H 2 N, alkylamino;
  • Z is a direct bond; methylene, oxygen, sulfor, amino; n is 0 or 1 ; or:
  • R is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl- (thio, sulfinyl or sulfonyl )-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl- (thio, sulfinyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R is selected from the group consistmg of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulf ony l)alky 1 , alkoyalkylacylalkyl ; or:
  • R is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;
  • R is selected from the group consisting of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl; or:
  • Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 4 is selected from the group consisting of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl- (thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl; or:
  • R- is selected from the group consisting of OH, alkoxyl, lower alkyl, alkylamino, peptide
  • X is selected from the group consisting of N, C;
  • R 2 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 3 is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
  • R 4 is selected from the group consisting of H, lower alkyl
  • R 5 is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoyalkylacylalkyl.
  • the compounds of the present invention may have the following formulae:
  • the invention is further directed, where applicable, to solvated as well as unsolvated forms of the compounds (e.g. hydrated forms) having the ability to inhibit, regulate and/or modulate the production and/or mamration of collagen by inhibiting C-proteinase activity.
  • the compounds described above may be prepared by any process TM known to be applicable to the preparation of chemically-related compounds. Suitable processes are illustrated by the fallowing representative examples. Necessary starting materials may be obtained by standard procedures of organic chemistry.
  • a compound is subjected to a series of screens to determine the compound's ability to modulate, regulate and/or inhibit the production and mamration of collagen.
  • screens include biochemical assays, cell culmre assays and animal models.
  • Disorders associated with the inappropriate or unregulated production and/or mamration of collagen can be treated with the * J compounds and compositions of the present invention.
  • diseases or disorders include pathological fibrosis or scarring, such as endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers' fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, biliary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic
  • pathological fibrosis or scarring such as endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers' fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, biliary cirrhosis, alcoholic cirrhosis
  • nephropathy 35 nephropathy, scleroderma/ systemic, scleroderma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne's), cardiac fibrosis, muscular fibrosis/retinal separation, esophageal stricture, payronles disease.
  • fibrotic disorders may be induced or initiated by surgery such as scar revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis. Still further fibrotic disorders may be induced l" by chemotherapy, including, for example lung fibrosis and the like.
  • the identified compounds can be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with *•*••*• > suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.
  • a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral 25 delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intra ventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • a local rather than systemic manner for example, via injection of the compound directly into a ** '" arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.
  • the compounds may be administered topically, for example, as eye drops.
  • one may administer the drug in a targeted drug delivery system for example, in a liposome coated with a specific antibody, targeting, 3 for example, arthritic or fibrotic tissue.
  • a targeted drug delivery system for example, in a liposome coated with a specific antibody, targeting, 3 for example, arthritic or fibrotic tissue.
  • the liposomes will be targeted to and taken up selectively by the afflicted tissue.
  • compositions of the present 1" invention may be manufacmred in a manner that is itself known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more l *3 physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous 2 " solutions, preferably in physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by 3 combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, J j optionally grinding a resulting mixmre, and processing the mixmre of granules, after adding suitable auxiliaries, if desired, to obtain tablets or
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
  • polyviny lpyrrolidone PVP
  • disintegrating agents such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally,
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane,
  • the dosage unit may be determined by providing a valve
  • Capsules and cartridges of, e.g. , gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by 3 injection, e.g. , by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi- dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or *•** " dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
  • a suitable vehicle e.g. , sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations
  • the implantation for example subcutaneously or intramuscularly
  • intramuscular injection for example, the
  • 35 compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co-solvent system.
  • VPD is a solution of 3 % w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,
  • VPD VPD co-solvent system
  • VPD:5W VPD diluted 1 : 1 with a 5 % dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be
  • co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.
  • polyvinyl pyrrolidone and other sugars or polysaccharides may substitute for dextrose.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • compositions also may comprise suitable solid or
  • gel phase carriers or excipients examples include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,
  • compositions suitable for use in the 2 " present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the 25 capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated imtially from cell culmre assays.
  • a dose can be formulated in animal models to achieve a • - * -'" circulating concentration range that includes the IC 50 as determined in cell culmre (i. e. , the concentration of the test compound which achieves a half-
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell culmres or experimental animals, e.g. , for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culmre assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g. , Fingl et al. , 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l .
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the C- proteinase inhibiting effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration necessary to achieve 50-90% inhibition of the C-proteinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% .
  • the effective local concentration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be * accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of arthritis or any other fibrotic disorder.
  • the compounds of the present invention may be synthesized according to known techniques. The following represent preferred methods for synthesizing and testing the compounds of the claimed invention:
  • Boc-Glu (OBn)OH and Boc-Asp(OBn)OH have been synthesized using methods readily known in the art.
  • reaction mixmre was allowed to warm to -30°C over 2h, then a 40% aquoeus solution of methyl amine (22 mmol) was add, and the reaction mixmre was allowed to warm to room temperamre. After the reaction had stirred for a further hour, diethyl ether (50 ml) and water (70 ml) were added. The organic layer was separated, washed with 1 M NaHCO 3 , 10% citric acid, and saturated NaCI solution, and dried over MgSO 4 . The solvent was evaporated in vacuo to give a white solid (5.0 g, 72% yield).
  • ester hydroxamate (28) also designated as FG-058, as a white solid.
  • Methoxybenzenesulfonyl)-L-proline hydroxamate also designated as FG054, (Example for Inhibitor A) is as follows:
  • the prefened method for synthesizing N- hydroxy-2-[[N'-(4-methoxybenzenesulfonyl)-N'-(4-methoxybenzyl)]amino]- acetamide (35), also designated as FG-067 (Example for Inhibitor A) is essentially as described for the synthesis of synthesizing N-hydroxy-2-[[N'-(4- methoxybenzenesulfonyl)-N-(4-chlorobenzyl)]amino]-acetamide, using as starting material 4-methoxybenzaldehyde instead of 4-chlorobenzaldehyde.
  • N-(4-Methoxybenzenesulfonyl)- ⁇ -benzyl-(L)-aspartic Acid (40).
  • b-benzyl-(L)-aspartic acid HCl salt (38) (2.00 g, 8.96 mmol)
  • p-methoxybenzenesulfonyl chloride (39) (1.76 g, 8.53 mmol)
  • triethylamine (1.81 g, 17.91 mmol) at room temperamre.
  • the reaction mixmre was quenched with 1 N HCl (60 ml), and extracted with CH 2 C1 2 (3 x 50 ml).
  • N-Benzyloxy-N'-(4-Methoxybenzenesulfonyl)- ⁇ -benzyl-(L)-aspartic amide (41).
  • N-(4-methoxybenzenesulfonyl)-b-benzyl- (L)-aspartic acid 300 mg, 0.76 mmol
  • O- benzylhydroxylamine/HCl in an 0 anhydride solution of (7/3) THF/DMF (10 ml) was added
  • N-hydroxybenzotriazole (HOBT) (103 mg, 0.76 mmol), N-ethy lmorpholine (204 mg, 1.68 mmol) and then diisopropylcarbodiimide (106 mg, 0.84 mmol) at room temperamre. After stirring over the weekend (2.5 days), the reaction mixmre was diluted with (1/1) hexanes/EtOAc (40 ml), washed successively 5 with 1 N HCl (2 x 20 ml), saturated NaHCO3 aqueous solution (2 x 20 mL) and brine. The organic layer was dried over MgSO 4 and concentrated.
  • HOBT N-hydroxybenzotriazole
  • the following assay may be used to determine the level of activity and effect of the different compounds of the present invention on C-proteinase activity.
  • radiolabeled ( 14 C) procollagen is added to 10 units/ml of chicken C-proteinase in a solution of 0.1 M Tris-HCI, 0.1 M NaCI, 0.02%
  • the protein bands are detected by autoradiography.
  • the amount of enzyme activity is based on the disappearance of the band conesponding to uncleaved procollagen.
  • the IC 50 of inhibitors can be determined by plotting the % activity versus inhibitor concentration and estimating the inhibitor concentration which results in 50% activity.
  • TABLE I IC S0 Of Various Identified C-Proteinase Inhibitors.
  • the IC 50 value of the inhibitors can also be determined by a filtration ELISA assay.
  • a filtration ELISA assay In this assay about 25 ng of unlabeled human procollagen I were incubated with the C-Proteinase as, see, section 6.2.1 but for one hour. The reaction was stopped with the addition of 40 ⁇ l precipitation buffer (0.5 X Reaction buffer, 0.1 mg/ml chicken collagen II, 10 ⁇ g/ml BSA, 7.5 mM EDTA). 25 ⁇ l of 75% ethanol was added and the reactions were mixed and incubated on ice for one hour to precipitate the procollagen.
  • 40 ⁇ l precipitation buffer 0.5 X Reaction buffer, 0.1 mg/ml chicken collagen II, 10 ⁇ g/ml BSA, 7.5 mM EDTA
  • the soluble c-propeptide was separated from the precipitated collagen by filtering through a Millipore multiscreen-HV 0.45 ⁇ m hydrophilic plate using a Millipore multiscreen vacuum manifold. 20 ⁇ l of the filtrate was removed and the amount of cleaved c-propeptide was determined by using the procollagen type I C-peptide (PIP) EIA kit from Takara Biomedicals.
  • PIP procollagen type I C-peptide
  • IC 50 of the inhibitors was determined by plotting the % activity * * ⁇ " versus inhibitor concentration and estimating the inhibitor concentration which gives 50 % activity. IC 50 values are shown in TABLE II
  • tissue culture assays by measurement of the production of procollagen and mature collagen in conditioned medium before and after treatment with a compound.
  • the ratio of collagen and procollagen will directly conelate to the cellular conversion of the precursor to the mature collagen product, and as such indicate the C-proteinase activity.
  • the media content of C-propeptide/cell may be determined, and compared for untreated cells and inhibitor-treated cells.
  • animal models which mimic clinical disorders related to unregulated or inappropriate collagen production are known in the art and may be employed to determine the in vivo efficacy of the compounds of the invention. These animal models include a wound chamber model in rats (Schilling et al., 1959, Surgery 46:702-710), an estradiol stimulated uterus expansion model (Mandell et al., 1982, The Journal of Biological Chemistry 257:5268-5273), and an induced angiogenesis model (Matrigel) (Passaniti et al, 1992, Laboratory Investigation 67:519-528).
  • liver fibrosis models include clinical disorder models like liver fibrosis models (Tsukamoto et al., 1990, Seminar in Liver Disease 10:56-65; Kock-Weser, 1952, Laboratory Investigation 1:324-331 ; Manione, 1949, American Journal of Pathology 25:273-285; Tarns, 1957, American Journal of Pathology 33:13-27; Wahl et ai, 1986, Journal of Experimental Medicine 163:884-902), a pulmonary fibrosis model (Kelly et ai, 1980, Journal of Laboratory Clinical Medicine 96:954-964), arterial restenosis models (Jackson, 1994, Trends of Cardiovascular Medicine 4:122-130; Clowes et al., 1983, Laboratory Investigation 49:327-333), a kidney fibrosis model (Yamamoto et al, 1987, Kidney International 3_2:514-525), a tendon repairing model (Franklin et al,
  • cytotoxicity assays are studied in cytotoxicity assays in order to determine whether there is an effect on cell survival or proliferation. These assays may involve the use of rapidly proliferating or quiescent cells. A known number of cells is seeded and exposed for increasing periods of time to a concentration range of potential inhibitors. Cell numbers are determined by cell counting or staining (e.g. Crystal Violet).
  • Cytotoxicity is evaluated as a function of cellular survival and cell proliferation.
  • Cellular survival involves the use of quiescent cells and is determined by cell number (counting or staining). A decrease in cell number indicates cell loss, and thus an effect on cell survival.
  • Cell proliferation involves the use of rapidly proliferating cells and is, as well, determined by cell number. Here a decrease in cell number relative to the untreated controls indicates an effect on cell proliferation.

Abstract

Utilisation nouvelle de molécules organiques capables d'inhiber l'activité de la C-protéinase afin de réguler, moduler et/ou prévenir une formation anormale de collagène.
EP96930499A 1995-08-08 1996-08-08 Inhibiteurs de la c-proteinase destines au traitement des affections liees a la surproduction de collagene Withdrawn EP0845987A4 (fr)

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US203895P 1995-08-08 1995-08-08
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US60120396A 1996-02-14 1996-02-14
US601203 1996-02-14
US60918796A 1996-03-01 1996-03-01
US609187 1996-03-01
PCT/US1996/012876 WO1997005865A1 (fr) 1995-08-08 1996-08-08 Inhibiteurs de la c-proteinase destines au traitement des affections liees a la surproduction de collagene

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EP0845987A4 (fr) 2000-05-24
AU6951296A (en) 1997-03-05
CN1198096A (zh) 1998-11-04
BR9609883A (pt) 1999-03-23
JPH11511137A (ja) 1999-09-28
CA2229098A1 (fr) 1997-02-20
WO1997005865A1 (fr) 1997-02-20
MX9801093A (es) 1998-04-30
KR19990036271A (ko) 1999-05-25

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