WO1998025949A1 - 5 amino-1,3,4-thiadiazole-2-thiones substituees - Google Patents

5 amino-1,3,4-thiadiazole-2-thiones substituees Download PDF

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WO1998025949A1
WO1998025949A1 PCT/US1997/022534 US9722534W WO9825949A1 WO 1998025949 A1 WO1998025949 A1 WO 1998025949A1 US 9722534 W US9722534 W US 9722534W WO 9825949 A1 WO9825949 A1 WO 9825949A1
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substituted
thiadiazole
compound
thione
group
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PCT/US1997/022534
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English (en)
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Jozef Oleksyszyn
Alan R. Jacobson
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Proscript, Inc.
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Priority to AU56923/98A priority Critical patent/AU5692398A/en
Publication of WO1998025949A1 publication Critical patent/WO1998025949A1/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
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Matrix metalloproteinases are a class of zinc-dependent, proteolytic enzymes. These enzymes play a role in a number of disease processes.
  • Matrix metalloproteinases also contribute to cartilage degradation by cleaving ,-antiproteinase inhibitor- 1, thereby removing its ability to inactivate human neutrophil elastase. Furthermore, it has been shown in vivo that inhibitors of matrix metalloproteinases are able to inhibit angiogenesis (Garlardy, et al, Cancer Research, 54:4715 (1994)), i.e. the formation of new blood vessels. Although angiogenesis occurs in normal processes, such as ovulation, placental development and wound healing, it is also involved in pathological processes such as arthritis and inflammation (D'Armore, et al, Ann. Rev. Physiol, 49:453 (1987)).
  • Matrix metalloproteinases play critical roles in other pathological processes such as periodontal disease (Birkedal-Hansen, et al, Crit. Rev. Oral Biol. Med, 4:197-250, (1993)), various ulcerated conditions (Brown, et al, Arch. Opthal, 57:370-373 (1969)), and epidermolysis bullosa, (Johnson, et al, Enzyme
  • the present invention is broadly directed to a method for inhibiting matrix metalloproteinases by contacting a matrix metalloproteinase with an effective amount of an N5-substituted 5-amino-l,3,4-thiadiazole-2-thione, 5- amino-l,3,4-thiadiazole-2-one or 5-amino-l,3,4-oxadiazole-2-thione having Formula /, as described herein.
  • compositions useful for inhibiting a matrix metalloproteinase comprise a compound having the Formula /, and a carrier or diluent.
  • Yet another aspect of the present invention is a method of treating a disease in an individual or animal which can be ameliorated by inhibiting at least one matrix metalloproteinase.
  • the method comprising administering to the individual or animal a therapeutically effective amount of a compound having the structure of Formula /.
  • Substituted 5-amino- 1, 3, 4-thiadiazole-2-thiones and their analogs of the present invention are useful for treating individuals and animals with diseases resulting from over activity of matrix metalloproteinases, such as osteoarthritis, rheumatoid arthritis, cancer and the inflammation associated with many of these diseases.
  • the thiadiazoles of the present invention have other in vivo uses, such as aiding in identifying the location of matrix metalloproteinases in an individual or animal. These thiadiazole compounds are also useful in vitro for preventing the degradation of tissue and proteins present in biological samples containing matrix metalloproteinases, as an aid in identifying new drug targets for the treatment of these diseases and in isolating matrix metalloproteinases.
  • the present invention relates to novel compositions comprising inhibitors of matrix metalloproteinases.
  • Known inhibitors of matrix metalloproteinases comprise an oligopeptide bound to a functional group such as a hydroxamic acid or thiol which can chelate the zinc atom in the active site of the matrix metalloproteinase. It has now been found that 5-substituted l,3,4-thiadiazol-2- thiones and their analogs, as defined by Formula / which are capable of chelating zinc can also inhibit matrix metalloproteinases.
  • the 5-amino position of one of 5-amino- 1,3, 4-thiadiazole-2-thione, 5- amino-l,3,4-thiadiazole-2-one, or 5-amino- 1, 3, 4-oxadiazole-2-thione can be covalently attached to a variety of organic moieties and the resulting compounds have the ability to bind to and inhibit matrix metalloproteinases. Additionally, Applicants have discovered that the 2-thiol substituent of the tautomeric N5- substituted, 5-amino-l,3,4-thiadiazole-2-thiols of the present invention can form disulfide linkages with one another, thereby forming dimers.
  • disulfide linked dimers also exhibit metalloproteinase inhibition activity when screened in the assays described herein.
  • the 1 ,3,4-thiadiazole-2-thione, and/or its tautomers interact with the zinc atom that is present at the active site of metalloproteinases.
  • diverse organic radicals can be attached to the 5-amino-l,3,4-thiadiazole-2-thione, as long as the radical does not diminish the ability of the l,3,4-thiadiazole-2-thione to interact with the active site of matrix metalloproteinases.
  • the structural requirements to bind to the active site of matrix metalloproteinases are not very stringent.
  • a first aspect of the present invention is directed to a method for inhibiting matrix metalloproteinases, comprising contacting a matrix metalloproteinase with a compound of Formula /:
  • Q and A are each independently selected from the group consisting of sulfur and oxygen and one of Q and A is sulfur;
  • Rl is one of hydrogen, lower alkyl or acyl
  • Z represents an organic radical that does not substantially interfere with
  • R4 is hydrogen
  • Preferred compounds include compounds where A and Q are both sulfur.
  • a further aspect of the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier or diluent, and an amount effective to inhibit a matrix metalloproteinase of a compound of
  • the present invention provides the use of a compound of Formula/, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of conditions, in particular disorders, such as collagen degradation or abnormal angiogenesis, which require the administration of a selective inhibitor of matrix metalloproteinases.
  • the compounds of the present invention means that a compound resulting from the attachment of a particular organic radical to an oxa- or thia- diazole ring (Formula IV) exhibits matrix metalloproteinase inhibition activity, as measured in one of the biological assays described hereinbelow (Examples 38-41).
  • the compounds of the invention have an IC 50 of 50 ⁇ M or less, more preferably 5 ⁇ M or less, as measured in one of the assays described in Examples 38-41.
  • Useful organic radicals "Z" include straight-chained, branched and cyclic groups, including heterocyclic groups, containing up to 25 carbon atoms, suitably up to 15 carbon atoms, and conveniently up to 12 carbon atoms, wherein said heterocyclic groups can include from 1 to 5, preferably, 1 to 3 heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, and wherein said radicals can be optionally substituted.
  • Suitable organic radicals include C, .12 alkyl, C 2.10 alkenyl, C 2-10 alkynyl, C 3 . 8 cycloalkyl, C 3.7 cycloalkyl(C, .6 )alkyl, C 3.8 cycloalkyl(C 2 .
  • cyclic and heterocyclic groups can also be part of a fused or spiro ring system having from one to four rings.
  • the organic radical may in turn, be optionally substituted by one or more groups selected from hydroxy, nitro, trifluoromethyl, halogen, halo(C,. 6 )alkyl, C,. 6 alkoxy, C,_ 6 alkoxy(C,. 6 )alkoxy, adamantyl, aryl, benzyl, aryloxy, heteroaryl, heterocycloalkyl, keto, C, .3 alkylenedioxy, cyano, carboxy, C 2 . 6 alkoxycarbonyl, C 2 . 6 alkoxycarbonyl(C,. 6 )alkyl, C 2 . 6 alkylcarbonyl, optionally substituted arylcarbonyl, C,.
  • alkanoyl C 2.6 alkoxycarbonyl, phenyl(C 2.6 )alkoxycarbonyl, carbamoyl, C 2 . 6 alkylcarbamoyl, hydroxy(C 2 . 6 )alkylcarbamoyl, di((C 2 . 6 )alkyl)carbamoyl, amino(C 2.6 )alkylcarbamoyl, cycloalkylcarbamoyl, cycloalkyl(C 2.6 )alkylcarbamoyl, N-hydroxycarbamoyl, N-(C 2 . 6 )alkenyloxy carbamoyl, phosphono, C,.
  • alkylphosphono di((C ! . 6 )alkyl)phosphono, tri((C, .6 )alkyl)alkylphosphono, C ⁇ alkylsulfonyl, C 2.6 alkenylsulfonyl, C 2 . 6 alkynylsulfonyl, C,. 6 alkylsulfmyl, arylthio, C,. 6 alkylthio, C,.
  • the organic radical can be covalently linked to the 5-amino nitrogen through an amide, amine, sulfonamide, urea, thiourea, carbamate, thiocarbamate, sulfmamide (-S(O)NRl-), sulfenamide (-SNR1-), phosphonamide or phosphinamide bond.
  • Preferred compounds have an amide, amine, sulfonamide, carbamate, thiocarbamate, sulfinamide, phosphonamide or phosphinamide with an amide linkage between the 5-amino moiety of the heterocyclic thiadiazole group and the organic radical being most preferred.
  • most preferred organic radicals include C,. 12 alkanoyl, C 3 . 10 alkenoyl,
  • C 3 . I0 alkynoyl C 3 . 7 cycloalkyl(C,. 6 )alkanoyl, aryl(C 2 . 6 )alkanoyl, C 3 . 8 heterocycloalkyl(C,. 8 )alkanoyl, and heteroaryl(C 1.6 )alkanoyl.
  • the cyclic and heterocyclic groups can also be part of a fused or spiro ring system having from one to four rings. Examples of useful radicals that can be employed as "Z” include: steroids, amino acids, oligopeptides, and moieties commonly referred to as amino protecting groups.
  • amino protecting groups include, but are not limited to, 9-fluorenylmethoxycarbonyl, t-butoxycarbonyl, (4-phenyl)phenylacetyl, 8-quinolinesulfonyl, 2-methylthionicotyl, xanthene-9-carbonyl, hydrocinamoyl, phenylbenzoyl, nonanoyl,
  • X is O or S
  • R 2 is -O-R 5 , or -NR ⁇ R,;
  • R 3 is H, C,. 6 alkyl, -(CH 2 ),-aryl, -(CH 2 ) r cycloalkyl, -(C alkyl)-O-R 4 ,
  • R 4 is H, C,. 6 alkyl, or -(CH 2 ),-aryl;
  • R 5 is H, C 1-6 alkyl, or aryl
  • Het is a 5-, 6-, 9-, or 10-membered heteroaromatic moiety having one or more atoms selected from the group consisting of N, O, and S;
  • Q is a saturated 5-, or 6-membered heterocyclic moiety having 1-2 atoms selected from the group consisting of N, O, and S; i is 0, 1, 2, 3, or 4; j is 1, 2, 3, or 4; n is 0, or 1.
  • compounds of Formula / are other than thiadiazolyl(thio)urea derivatives compounds of Formula VII.
  • Rg is benzyl or 2-phenylethyl.
  • compounds of Formula / are other than compounds of Formulae Viand VII.
  • thiadiazole derivatives are matrix metalloproteinase inhibitors that are useful as preventatives and therapeutics for diseases related to connective tissue degradation.
  • the present invention further provides a composition for inhibiting matrix metalloproteinases, comprising a compound of Formula /, wherein said compound is present in an amount effective to inhibit matrix metalloproteinases; and a carrier or diluent.
  • a carrier or diluent is a pharmaceutically acceptable carrier or diluent.
  • the matrix metalloproteinase inhibitors of the present invention include amino acid amides of 5-amino-l,3,4-thiadiazole-2-thiol represented by Structural Formula //:
  • matrix metalloproteinase inhibitors of the present invention can be represented by Formula ///: n
  • Q and A are each independently selected from the group consisting of sulfur and oxygen and one of Q and A is sulfur. It is preferred that Q and A are both sulfur.
  • n is a positive integer and is chosen such that the compound inhibits a matrix metalloproteinase. Preferably, n is an integer from 1 to about 10. More preferably, n is an integer from 1 to about 4. In a separate embodiment of the invention, the value of n can be zero for compounds of Formula III.
  • Rl is selected from the group consisting of -H, lower alkyl and acyl.
  • Lower alkyl includes Cl to about C6 straight or branched chain hydrocarbons.
  • the hydrocarbon can be saturated or can have one or more units of unsaturation.
  • Suitable acyl groups include -CO-(lower alkyl), wherein lower alkyl is defined above.
  • Rl is -H.
  • Each R4 is hydrogen, or is taken together with the R2 on the carbon atom adjacent to the nitrogen to which R4 is bound to form a 3 or 4 carbon atom carbocyclic bridge, that is optionally substituted or optionally fused to a benzene ring.
  • Preferred groups formed by the combination of R4 and R2 include proline (azacyclopentane) and hydroxyproline.
  • R4 is preferably other than hydrogen.
  • Each R2, when not taken with R4 to form a carbocyclic bridge, is independently selected from the group consisting of C1-C10 straight or branched alkyl, C1-C10 straight or branched substituted alkyl, C3-C8 cyclic alkyl, substituted C3-C8 cyclic alkyl, C1-C10 straight or branched alkenyl, Cl- C10 straight or branched substituted alkenyl, C1-C10 straight or branched alkynyl, C1-C10 straight or branched substituted alkynyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.
  • the R2 in each amino acid subunit in Formulae // and /// can be the same or different.
  • Suitable substituents on a substituted alkyl, alkenyl or alkynyl group include halo, -COOH, -COO(X), -CHO, -OH, -CN, -NO 2 , -NH 2 , -O(M), -SH,
  • a substituted alkyl, alkenyl and alkynyl group can optionally have more than one substituent.
  • An alkyl, alkenyl or alkynyl group can also be completely substituted, e.g. perfluorinated.
  • An alkenyl or alkynyl group can have more than one double or triple bond.
  • M is selected from the group consisting of -X, X-CO-, X-CS-, X-SO 2 -, X-O-CO- and X-O-CS-.
  • X is selected from the group consisting of C1-C10 alkyl, C1-C10 substituted alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. X can also be heterocycloalkyl and substituted heterocycloalkyl
  • An aryl group can be monocylic (e.g. phenyl) or polycyclic.
  • a polycyclic aromatic group includes fused polycyclic structures, e.g. naphthyl, tetrahydronaphthyl or anthracyl.
  • a polycyclic aromatic group also includes structures with two or more aromatic rings connected by a linker containing one or more single bonds, carbon atoms, and/or heteroatoms, e.g. biphenyl, xanthenyl and fluorenyl.
  • Suitable aryl substituents include halo, -COOH, -COO(M), -CHO, -OH, -CN, -NO 2 , -NH 2 , -O(M), -SH, -S(M), -NH(M), -N(M 2 ), aryl, substituted aryl, heteroaryl and substituted heteroaryl.
  • M is as defined above.
  • a substituted aryl group can optionally have more than one substituent.
  • Suitable heteroaryl groups include monocyclic or polycyclic aromatic groups containing one or more heteroatoms such as oxygen, nitrogen or sulfur.
  • Suitable monocyclic heterocyclic groups include imidazolyl, thienyl, pyridyl, furanyl, oxazoyl, pyrollyl, pyrimidinyl, furanyl, pyrazolyl, pyrrolyl, thiazolyl and the like.
  • a polycyclic heteroaryl group includes fused structures such as quinonyl, indoyl, benzimidazoyl, benzothiazolyl, benzothiophenyl, benzofuranyl and benzopyranyl.
  • a polycyclic heteroaromatic group can also include structures with a heteroaromatic ring and one or more aromatic or heteroaromatic rings connected by a linker containing one or more single bonds. Examples include phenylthienyl, thienylthienyl, phenylfuranyl, phenyloxazoyl, thienyloxazoyl and the like.
  • Suitable heteroaryl substituents include halo, -COOH, -COO(alkyl), -OH, -CN, -NO 2 , -NH 2 , -O(M), -SH, -S(M), -NH(M), -N(M 2 ), aryl, substituted aryl heteroaryl and substituted heteroaryl.
  • M is as defined above.
  • a substituted heteroaryl group can optionally have more than one substituent.
  • R2 is selected from the group consisting of cyclohexyl, cyclopentyl, (substituted phenyl)-CH 2 -, naphthyl, naphthyl-CH 2 -, the side chain of a naturally occurring amino acid, and the side chain of a naturally occurring amino acid having a derivatized heteroatom-containing functional group.
  • R2 is also preferably phenyl.
  • a substituted phenyl can have the same substituents as described above for aryl.
  • 2-Fluoro, 3,4-diiodo, 4-nitro 4-benzyloxycarbonylamino 4-dibenzylamino and 4-fluoro are examples.
  • Naphthyl can be either 1 -naphthyl or 2-naphthyl.
  • amino acid has the general structure NH 2 -CHR-COOH, wherein R is the side chain.
  • Naturally occurring amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, homophenylalanine, tryptophan, glycine, serine, homoserine, threonine, cysteine, homocysteine, tyrosine, aminoadipic acid, asparagine, glutamine, aspartic acid, glutamic acid, lysine, histidine, proline, ornithine, homocysteine, hydroxyproline, phenylglycine and tryptophan.
  • heteroatom-containing functional groups which can be derivatized.
  • heteroatom-containing functional groups include the thiol of cysteine, the hydroxyl of serine, hydroxyproline and threonine, the carboxylic acid of glutamic acid, adipic acid and aspartic acid, the phenol of tyrosine, the amine of lysine, ornithine, arginine and histidine and the amide of asparagine and glutamine.
  • Suitable derivatizing groups include -X, X-CO-, X-CS-, X-SO 2 -, X-O-CO- and X-O-CS-, wherein X is as described above.
  • suitable derivatizing groups include O-benzyl for tryosyl, seryl, glutamoyl; S-benzyl for cysteinyl; N-trityl for glutamoyl; O-methylene-2-naphthyl for tryosyl; N-trityl for glutamyl; N,N-dibenzyl for glutamyl; e-N-t-butoxycarbonyl for lysyl; and N-2-phenylethyl for glutamyl.
  • R3 is an amine derivatizing group such as an amine protecting group.
  • An "amine protecting group” is a functional group which can be bonded to a primary amine, which can be cleaved from the primary amine without causing undesired side reactions in other parts of the molecule and which results in a matrix metalloproteinase inhibitor.
  • suitable amine derivatizing groups include X-CO-, X-CS-, X-SO 2 -, X-O-CO- and X-O-CS-, wherein X is as defined above.
  • Preferred amine derivatizing groups include 9-fluorenylmethoxycarbonyl, t-butoxycarbonyl, (4-phenyl)phenylacetyl, 8-quinolinesulfonyl, 2-methylthionicotyl, xanthene-9-carbonyl, hydrocinamoyl, phenylbenzoyl, nonanoyl, (4-benzyloxy)benzoyl, acetyl and (4-(4-t-butylphenylsulfonamino)benzoyl.
  • a preferred subgenus within the scope of Formula //which encompasses compounds that exhibit potent and selective activity as matrix metalloproteinases includes compounds of Formula V:
  • Y is one of-CO- or -SO 2 -;
  • Q and A are each independently selected from the group consisting of sulfur and oxygen and one of Q and A is sulfur;
  • R 21 is one of hydrogen, lower alkyl or acyl
  • R 22 is one of C1-C10 straight or branched alkyl, C1-C10 straight or branched substituted alkyl, C3-C8 cyclic alkyl, substituted C3-C8 cyclic alkyl, C2-C10 straight or branched alkenyl, C2-C10 straight or branched substituted alkenyl, C2-C10 straight or branched alkynyl, C2-C10 straight or branched substituted alkynyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and
  • R 23 is C1-C10 alkyl, C1-C10 substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroraryl;
  • R 21 is hydrogen
  • R 22 is selected from the group consisting of cyclohexyl, cyclopentyl, phenyl, (substituted phenyl)-CH 2 -, naphthyl, naphthyl-CH 2 -, the side chain of a naturally occurring amino acid, and the side chain of a naturally occurring amino acid having a derivatized heteroatom-containing functional group.
  • R 22 is also preferably phenyl.
  • a substituted phenyl can have the same substituents as described above for aryl.
  • 2-Fluoro, 3,4-diiodo, 4-nitro 4-benzyloxycarbonylamino 4-dibenzylamino and 4-fluoro are examples.
  • Naphthyl can be either 1 -naphthyl or 2-naphthyl.
  • An amino acid has the general structure NH 2 -CHR-COOH, as defined above for R2.
  • the side chains of many of these naturally occurring amino acids have heteroatom-containing functional groups which can be derivatized as described above for R2.
  • Preferred combinations of R 23 -Y- include
  • Physiologically acceptable salts include a hydrochloride salt, a hydrobromide salt and an acetic acid salt.
  • matrix metalloproteinase inhibitors of the present invention include the compounds whose structures are depicted in
  • Additional examples include:
  • the compounds of the present invention are formed by reacting (A) 5- amino-l,3,4-thiadiazole-2-thione, 5-amino- 1, 3, 4-thiadiazole-2-one, or 5-amino- l,3,4-oxadiazole-2-thione with (B) an organic molecule possessing a functional group capable of reacting with the amino group of (A).
  • Suitable functional groups capable of reacting with the 5-amino group of (A) include: carboxylic acids, acid halides and other activated acid groups as are known in the art; sulfonyl chlorides; aldehydes; alcohols; and alkyl, aryl and aralkyl halides.
  • the compounds of Formula / are generally synthesized by reacting 5- amino-l,3,4-thiadiazole-2-thione, or an analog thereof (where one of Q or A is oxygen), with an organic compound having a functional group capable of reacting with the 5-amino group.
  • 5-Amino-l,3,4-thiadiazole-2-thione is prepared according to known methods (Cho and Kim, J. Heterocyclic Chem., 30:397
  • the 5-amino group may be reacted with a carboxylic acid, an activated carboxylic acid or acid chloride to form an amide linkage to the 1 ,3,4-thiadiazole- 2-thione.
  • Direct reaction of the 5-amino group with a carboxylic acid is suitably carried out by methods that are typically employed for peptide synthesis, for example by dicyclohexylcarbodiimide mediated coupling in the presence of 1- hydroxybenzotriazole (see Examples 1-37).
  • the 5-amino group can be reacted with a sulfonyl chloride to form a sulfonamide linkage to the 1,3,4- thiadiazole-2 -thione.
  • the 5-amino group can also be reacted with an isocyanate to form a urea or with a thioisocyanate to form a thiourea.
  • the extra functional groups are first selectively reacted with an appropriate protecting group.
  • Protecting groups are well known in the art. See, Greene and Wuts, Protecting Groups in Organic Synthesis, John Wiley and Sons, (1991).
  • the 5-amino group is first derivatized to form another reactive functional group, that is capable of further reaction.
  • the 5-amino group is converted to an isocyanate or isothiocyanate group.
  • Thiourea compounds can be formed by reacting 5 -amino- 1,3,4- thiadiazole-2-thione with thiophosgene and triethylamine to give a 5- isothiocyanate group. Reaction of the isothiocyanate with an amine will give a thiourea compound.
  • Urea compounds can be prepared by reacting 5-amino-l ,3,4- thiadiazole-2-thione with triphosgene and triethylamine to give the 5-isocyanate derivative, followed by reaction with an appropriate amine.
  • Carbamate compounds can be prepared by reacting the isocyanate intermediate formed above with an appropriate anhydrous alcohol.
  • 5-Amino-l,3,4-thiadiazole-2-thione is prepared according to known methods (Cho and Kim, J. Heterocyclic Chem., 30:397 (1993)). Methods of protecting the N-terminus of amino acids or oligopeptides are also well known.
  • Coupling is carried out by known methods of peptide synthesis, for example by dicyclohexylcarbodiimide mediated coupling in the presence of 1- hydroxybenzotriazole (see Examples 1-36).
  • Synthesis of compounds having formula V is further illustrated in Example 37. Additional amino acids or oligopeptides can be added to the N-terminus by cleavage of the amino protecting group and then performing a second coupling with an N-terminus protected amino acid or oligopeptide. The process can be repeated as often as required to synthesize a thiadiazole having an oligopeptide of desired length and sequence which is bound to the 5-amino group of the thiadiazole.
  • the methods of the present invention comprise contacting the matrix metalloproteinase with an inhibitory amount of a compound represented by Formulae /, //, /// or V.
  • Matrix metalloproteinases are a class of zinc-dependent, proteolytic enzymes which bind and cleave peptides having a specific amino acid sequence.
  • An inhibitory amount of the compound is the quantity of the compound which results in reduced cleavage of matrix metalloproteinase substrates in the presence of the compound compared with in its absence. An inhibitory amount depends on several factors, including the inhibitor used, the pH of the solution, other constituents in the solution and temperature. The skilled artisan is able to vary the amount of inhibitor used, depending on the application.
  • a concentration from about 1 nanomolar or less to about 10,000 nanomolar is used, preferably about 1 nanomolar or less to about 1000 nanomolar and more preferably about 1 nanomolar or less to about 500 nanomolar.
  • Specific examples where at least one matrix metalloproteinase is inhibited in vitro with an amino acid amide of 5-amino-l,3,4-thiadiazole-2-thione are provided in Examples 38-41.
  • amino acid amides of 5-amino- l,3,4-thiadiazole-2-thiones are tested in vitro for their ability to inhibit stromelysin, 92 kDa human gelatinase, 72 kDa human gelatinase and human neutrophil collagenase.
  • Inhibition data are provided in Tables I-IV and VI as the
  • Another embodiment of the present invention is a method of treating an individual with a disease that can be ameliorated by inhibiting at least one matrix metalloproteinase enzyme.
  • the method comprises administering a therapeutically effective amount of a compound of Formulae /, //, /// or V.
  • the method can also be used to treat an animal with a disease that can be ameliorated by inhibiting at least one matrix metalloproteinase enzyme.
  • Animals which can be treated by this method include, dogs, cats, farm animals, guinea pigs and the like.
  • a disease is "ameliorated" when the development or progression of a disease process associated with the disease is slowed, arrested or reversed as a result of a treatment.
  • osteoarthritis and rheumatoid arthritis can be ameliorated by slowing the cartilage degradation that occurs as a result of the disease.
  • "amelioration" can include alleviating pain and inflammation in the afflicted joints of an individual with osteoarthritis or rheumatoid arthritis.
  • Another example of disease "amelioration” includes increasing the life expectancy of individual with the disease, for example an individual with cancer, or increasing the quality of life of the individual, e.g. by increasing the mobility of an individual with osteoarthritis.
  • a disease process is ameliorated by the administration of amino acid amides of 5-amino- 1,3, 4-thiadiazole-2-thi one are provided in Example 41.
  • compounds are tested for their ability to inhibit the degradation of extracellular matrix in tissue culture. Extracellular cartilage degradation occurs in osteoarthritis and rheumatoid arthritis. Inhibition data for the compounds tested in the tissue culture assay are provided in Table V as the percent inhibition of cartilage degradation at the given concentration.
  • metalloproteinase inhibitors of the present invention include tumor cell metastasis in cancer, ulcerations and infections resulting from periodontal disease or epidermolysis bullosa.
  • these compounds can be used to treat inflammation in diseases in which inflammation is caused by the overactivity of at least one matrix metalloproteinase enzyme.
  • a therapeutically effective amount of the compound is the quantity which brings about an amelioration of the disease without causing unacceptable side effects.
  • the amount of compound which is administered to the individual or animal depends on many factors, including the age, sex, weight and general health of the individual as well the severity of the disease with which the individual is afflicted. The skilled artisan will be able to vary the amount of compound administered to the individual, depending on these and other factors.
  • a therapeutically effective amount ranges from about 0.1 mg/kg per day or less to about 100 mg/kg per day, preferably from about 0.1 mg/kg per day or less to about 20.0 mg/kg per day.
  • the compound can be administered orally, for example, in capsules, suspensions or tablets.
  • Other modes of administration which can be used include systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.
  • the compound is preferably administered intraarticularly into the afflicted joint, for example by intraarticular injection.
  • Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound. Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • Suitable pharmaceutical carriers for intraarticular and other parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin of cyclodextran) are known in the art (Baker, et al, Controlled Release of Biological Active Agents, John Wiley and Sons, 1986).
  • the composition in addition to the compound, additionally comprises another pharmacologically active agent.
  • Osteoarthritis and rheumatoid arthritis are characterized by pain in the afflicted joints. Individuals with cancer often suffer from pain resulting from tumors contacting organs or other body tissue. Consequently, when treating osteoarthritis, rheumatoid arthritis or cancer it can be advantageous to co- administer the compound with an analgesic or other pain-killing medication.
  • Suitable analgesics include acetominophen, acetyl salicylic acid and the like.
  • Osteoarthritis and rheumatoid arthritis are also characterized by inflammation in the afflicted joints.
  • an anti-inflammatory agent such as a non-steroidal anti- inflammatory drug or steroid (e.g. triamcinolone, aminodide and the like) when treating osteoarthritis and rheumatoid arthritis.
  • an anti-inflammatory agent such as a non-steroidal anti- inflammatory drug or steroid (e.g. triamcinolone, aminodide and the like) when treating osteoarthritis and rheumatoid arthritis.
  • a non-steroidal anti- inflammatory drug or steroid e.g. triamcinolone, aminodide and the like
  • the metalloproteinase inhibitors of the present invention have useful applications in vitro. Because matrix metalloproteinases have protease activity and are present in a wide variety of tissue, the isolation of useful tissue and biological fluids is often hampered by undesired proteolysis of useful proteins by these enzymes. Destruction of useful tissue and proteins by these matrix metalloproteinases can be prevented by adding an inhibitory amount of
  • Matrix metalloproteinases as discussed earlier, are involved in a wide variety of disease processes. Consequently, inhibitors of matrix metalloproteinase are useful in disease research, for example to study the structure activity requirements for designing new and better inhibitors of these enzymes.
  • a substituted l,3,4-thiadiazole-2-thione, or analog thereof of the present invention can be coupled to a radiolabel, such as the Te" or I 131 scintigraphic labels, using standard coupling methods.
  • a radiolabeled amino acid amide of a l,3,4-thiadiazole-2-thione is then administered to a subject to determine any locations of excess amounts of one or more metalloproteinase in vivo.
  • the ability of a thiadiazole compound to selectively bind to a metalloproteinase is then used to map the distribution of these enzymes in situ.
  • the techniques can also, of course, be employed in the histological procedures, and the labeled compounds can be used in competitive immunoassays.
  • At least one amino acid amide of a 1, 3, 4-thiadiazole-2 -thione can also be coupled to a solid support, such as a separation membrane, a chromatographic support, for example agarose, sepharose, polyacrylamide, or the like, or to a microtiter plate to provide an affinity support which is useful in purifying a matrix metalloproteinase enzyme.
  • a solid support such as a separation membrane, a chromatographic support, for example agarose, sepharose, polyacrylamide, or the like
  • a microtiter plate to provide an affinity support which is useful in purifying a matrix metalloproteinase enzyme.
  • the selective binding of the matrix metalloproteinase to the thiadiazole compound permits the adsorption of the desired enzyme and its subsequent elution using, for example, altered ionic strength and/or pH conditions.
  • Dicyclohexylcarbodiimide (0.42 grams) was dissolved in 5 mL of anhydrous dimethylformamide (DMF), followed by the addition of N- fluorenylmethoxycarbonyl-trytophan (N-(9-fluorenylmethoxycarbonyl is referred to as "Fmoc") (0.85 grams) and 1-hydroxybenzotriazole (HBT) (0.36 grams).
  • DMF dimethylformamide
  • HBT 1-hydroxybenzotriazole
  • Example 3 5-(N-benzyloxycarbonyl-tryptonylamino)-l,3,4-thiadiazole-2-thione N-Benzyloxycarbonyl-tryptophan (1.7 grams), 1-hydroxybenzotriazole (1 grams), dicyclohexylcarbodiimide (1.1 grams) and 5-amino- 1,3, 4-thiadiazole- 2-thione (2.5 grams) were reacted according to the procedure described in Example 1. The resulting product was recrystallized from ethanol/ethyl acetate/pentane to give a white powder. M.P. 128-132°C. NMR spectrum
  • reaction was then allowed to warm to room temperature over 1 hour.
  • the reaction mixture was washed with 5% HCI (2 x 30 mL), 10% NaHCO 3 (2 x 30 mL) and brine (2 x 30 mL).
  • the organic layer was dried (Na ⁇ O , evaporated to dryness, and used without further purification.
  • Example 9 except that Boc-2-naphthalanine was used in place of Boc- naphthalanine.
  • An analytically pure sample was obtained by preparative TLC using
  • N-Benzyloxycarbonyl-(O-benzyl)tyrosine (6.35, 1.5 mmol), valine methyl ester hydrochloride (250 mg, 1.1 eq), diisopropylethylamine (260 mL, 1.1 eq) and
  • HOBt 230 mg were added to 15 mL of DMF. The reaction was then allowed to equilibrate for 15 minutes, followed by the addition of dicyclohexylcarbodiimide (340 mg 1.1 eq). The reaction was allowed to stir overnight at room temperature, after which the precipitated dicyclohexylurea was filtered off. The supernatant was extracted with 5% HCI (2 x 30 mL), 10%
  • Example 11 The procedure described in Example 11, using Cbz-4-nitrophenylalanine in place of Cbz-(O-benzyl)tyrosine, was used to complete the synthesis.
  • N-Benzyloxycarbonyl-(p-benzyloxycarbonylamino)-phenylalanine compound was prepared using Schotten-Baumann conditions (Bodanszky, "Principles of Peptide Synthesis") as described in Examples 14 and 15 starting with (L)-4-amino-phenylalanine (Bachem Bioscience, King of Prussia, PA) and using two equivalents of Cbz-Cl.
  • N-Benzyloxycarbonyl-(O-benzyl)glutamate 200 mg, 0.54 mmol was added to 5 mL of anhydrous DMF under argon. To this mixture was added 5- valyamino-l,3,4-thiadiazole-2-thione (144 mg, 1.1 eq), diisopropylethylamine
  • Example 11 using N-benzyloxycarbonyl-(O-benzyl)serine in place of N- benzyloxycarbonyl-(O-benzyltyrosine).
  • An analytical sample was prepared by preparative TLC using
  • This compound was prepared according the method described in Example 11 using N-benzyloxycarbonyl-(N-trityl)glutamine in place of N- benzyloxycarbonyl-(O-benzyltyrosine).
  • methyl 2-aminoisobutyrate (AibOMe) was accomplished by esterifying 2-aminoisobutyric acid in methanolic HCI.
  • the HCI salt was crystallized from methanol/ether. The remainder of the synthesis was carried out according to the procedure described in Example 11 , using AibOMe in place of valine methyl ester hydrochloride.
  • This compound was prepared according to the procedure described in Example 11 using phenylglycine methyl ester hydrochloride in place of valine methyl ester hydrochloride.
  • Example 11 using N-benzyloxycarbonyl-(( ⁇ -N-trityl)glutamine and phenylgly cine methyl ester hydrochloride in place of N-benzyloxycarbonyl-(O-benzyl)tyrosine) and valine methyl ester hydrochloride.
  • An analytical sample was prepared by preparative TLC using CH 2 Cl 2 :MeOH (95:5), R 0.46. Melting point 158.4-167.4°C. 'H-NMR (d ⁇ DMSO, int.
  • N,N-dibenzyl)glutamine-phenylglycine (0.48 grams) and the 1-hydroxybenzotriazole (0.15 grams) were dissolved in 5 mL of anhydrous DMF. The solution was kept in room temperature until dicyclohexylurea precipitation was completed (about 40 minutes). 5-Amino-l,3,4- thiadiazole-2-thione (0.39 grams) was added and solution was left for 2 days. An excess of ethyl acetate was added (50mL) and dicyclohexylurea was filtered off.
  • Example 25 using N-benzyloxycarbonyl-(( ⁇ -N-phenylethyl)glutamine- phenylglycine in place of N-benzyloxycarbonyl-(( ⁇ -N,N-dibenzyl)glutamine- phenylglycine.
  • N-Benzyloxycarbonyl-leucyl-valine (.419 grams), 1 -hydroxybenzotriazole (0.186 grams) and 5-amino- 1,3, 4- thiadiazole-2-thione (0.497 grams) were added to 6 mL of anhydrous DMF, followed by dicyclohexylcarbodiimide (0.277 grams). The reaction mixture was allowed to stir for three days at room temperature. The DMF was partially removed on a rotary evaporator and the residue was diluted with 100 mL of ethyl acetate.
  • This compound was prepared by the procedure described in Example 27 except that N-(benzyloxycarbonyl)-(2-fluoro)phenylalanyl-valine was used in place of N-(benzyloxycarbonyl)leucyl-valine.
  • the final product was purified from preparative TLC and gave one spot by TLC analysis (Rf 0.81 : eluent;
  • This compound was prepared by the procedure described in Example 27 except that N-(benzyloxycarbonyl)-(O-benzyl)tyrosyl-glycine was used in place of N-(benzyloxycarbonyl)leucyl-valine and the reaction was stirred for six days.
  • This compound was prepared by the procedure described in Example 27 except that N-(benzyloxycarbonyl)-(O-benzyl)tyrosyl-(t-butyl)glycine was used in place of N-(benzyloxycarbonyl)leucyl-valine and the reaction was stirred for six days.
  • the final product was purified from preparative TLC and gave one spot by TLC analysis (Rf 0.84: eluent; Methylene chloride: methanol 95:5). M.P.146°C.
  • This compound was prepared by the procedure described in Example 27 except that N-(benzyloxycarbonyl)-(O-benzyl)tyrosyl-(cyclohexyl)glycine was used in place of N-(benzyloxycarbonyl)leucyl- valine and the reaction was stirred for four days.
  • the final product was purified from preparative TLC and gave one spot by TLC analysis (Rf 0.64: eluent; Methylene chloride: methanol 95:5).
  • the dicyclohexylurea was filtered off and the filtrate was extracted with 10%> citric acid (3 x 20 mL), 10% sodium carbonate (3 x 20 mL), and brine solution (3 x 20 mL).
  • the ethyl acetate was dried over sodium sulfate and evaporated to dryness on the rotary evaporator.
  • the final product was purified by preparative TLC and gave one spot by TLC (Rf 0.80: eluent; Methylene chloride: methanol 95:5). M.P. 197-199 °C.
  • Dicyclohexylcarbodiimide (0.47 grams) was dissolved in 5 mL of anhydrous DMF, followed by N-(benzyloxycarbonyl)phenyalanyl-tryptonyl- valine (1.34 grams) and 1-hydroxybenzotriazole (0.45 grams). The solution was kept at room temperature until dicyclohexylurea was completely precipitated (about 1 hour). 5-Amino-l,3,4-thiadiazole-2-thione (1.1 grams) was added and the mixture was allowed to stir for 2 days.
  • Phenylglycine methyl ester hydrochloride (1.8 grams), triethylamine (1.3 mL) and HBT (1.09 grams) were dissolved in 30 mL CH 2 C1 2 .
  • N-t-Boc-(O- benzyl)tyrosine (3.0 grams) was then added and the solution was stirred at room temperature for 15 minutes.
  • Dicyclohexylcarbodiimide (1.83 grams) was then added and the solution stirred for 6 hours.
  • An excess of methylene chloride 250 mL was added and the resulting solution was washed until TLC analysis showed one spot. (Rf 0.71: eluent; methanolxhloroform 5:95).
  • Stromelysin was first activated by trypsin. This was done by preparing a reaction mixture in H-150 (H-150 consists of 10 mM CaCl 2 , 150 mM NaCI and 100 mM HEPES at pH 7.4) containing a final concentration of 25 ⁇ g/mL trypsin and 2.2 ⁇ M of stromelysin (Marcy et al, Biochemistry, 30:6476 (1991); Koklitis et al., Biochem. J., 376:217 (1991). The reaction was incubated for 30 minutes at 37 °C and then quenched by adding trypsin inhibitor agarose to the reaction mixture at a 20-fold excess with respect to trypsin.
  • H-150 consists of 10 mM CaCl 2 , 150 mM NaCI and 100 mM HEPES at pH 7.4
  • the reaction was incubated for 30 minutes at 37 °C and then quenched by adding trypsin inhibitor agarose to the
  • the reaction mixture was centrifuged at 14,000 rpm (16,000 x g) for 30 minutes using an Eppendorf Centrifuge 5415C.
  • the supernatant, which contains activated stromelysin, was concentrated with a Centricon 10 (5000 g, 1 hour).
  • a sample was analyzed by Bradford total protein assay and 12% SDS polyacrylamide gel electrophoresis. All steps of the stromelysin inhibition assay were performed at room temperature.
  • Assay solutions were prepared for each inhibitor tested.
  • Activated stromelysin was added to a stirred cuvette to a final concentration of 2 nM in 2 mL of H-150 buffer. 4 ⁇ L 5 mM coumarin peptide substrate was dissolved in DMSO and the initial rate of hydrolysis was measured for 100-200 seconds. Substrate hydrolysis was assessed by fluorescence using a slit width of
  • H-150 buffer pH 7.4, was prepared by adding CaCl 2 »H 2 O (1.47 grams), NaCI (8.77 grams) and N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (23.83 grams) to distilled and deionized water and then bringing the final volume of the solution to 1 liter.
  • a reaction mixture with a final volume of 2.0 mL was prepared from H- 150 buffer, human neutrophil collagenase (Schnierer, et al, Biochem. Biophys. Res. Comm., 191:319 (1993); Knight et al, Federation of European Biochemical Societies 296:263 (1992)) (2 nM).
  • Coumarin peptide substrate (4 ⁇ L of a 5 mM solution in dimethyl sulfoxide) was added to the reaction mixture. The rate of hydrolysis was determined for about 100-200 seconds by measuring the fluorescence of the reaction at ex328 nanometers and em329 nanometers using a Hitachi F-2000 Fluorescence Spectrophotometer.
  • H-150 buffer pH 7.4, was prepared by CaCl 2 » H 2 O (1.47 grams), NaCI (8.77 grams) andN-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (23.83 grams) to distilled and deionized water and then bring the final volume of the solution to 1 liter.
  • An reaction mixture having a final volume of 250 ⁇ L was prepared with H-150 buffer, 40 ⁇ grams of Pro-72 KD gelatinase (Strongin, et al, J. Biol. Chem. 265:14033 (1993); Goldberg, et al, J. Biol.
  • reaction mixture was applied to a NAP-5 column (G-25 SEPHADEXTM, Pharmacia). Fractions containing 92KD gelatinase were identified using 12% SDS polyacrylamide gel electrophoresis and concentrated to about 100-200 ⁇ liters by centrifugation (5000 g, 15 minutes).
  • H-150 buffer pH 7.4, was prepared by CaCl 2 -H 2 O (1.47 grams), NaCI (8.77 grams) andN-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (23.83 grams) to distilled and deionized water and then bring the final volume of the solution to 1 liter.
  • a reaction mixture having a final volume of 500 ⁇ L was prepared with H-150 buffer, 200 ⁇ grams of Pro-92KD gelatinase (Strongin et al, J. Biol. Chem., 268:14033 (1993); Goldberg, et al, J. Biol. Chem., 267:4583 (1992); Okada et al, J. Biol. Chem. 267:21712 (1992)), p-aminophenyl mercuric acetate (Sigma Chemical Co., St. Louis, MO) (1 mM) and Brij (dodexylpoly
  • reaction mixture was applied to a NAP-5 column (G-25 SEPHADEXTM, Pharmacia). Fractions containing 92KD gelatinase were identified using 12% polyacrylamide gel electrophoresis and concentrated to about 100-200 ⁇ liters by centrifugation (5000 g, 1 hour).
  • Coumarin peptide substrate (4 ⁇ L of a 5 mM solution in dimethyl sulfoxide) was added to the reaction mixture. The rate of hydrolysis was determined for about 100-200 seconds by measuring the fluorescence of the reaction at ex328 nanometers and em329 nanometers using a Hitachi F-2000 Fluorescence Spectrophotometer.
  • 2-thione 5- (N- (4- (4-t-butylphenylsulfonylamino) benzoyl) phenylalanyl-valylamino) -1,3,4- 0.31 thiadiazole-2-thione 5- (N-benzyloxycarbonyl- ( (p-benzyloxycarbonylamino (phenylalanyl) -valylamino-1, 3,4- 0.145 thiadiazole-2-thione 5- (N-benzyloxycarbonyl-phenylalanyl-alanylamino) -1, 3, 4-thiadiazole-2-thione 3.6
  • tissue culture assay was used to measure the ability of the compounds of the present invention to slow the degradation of the extracellular matrix by metalloproteinases. This assay measured the amount of 35 S-glycosaminoglycan
  • Knee joints from a 1 to 3 week old calf were obtained immediately after sacrifice from the Abattoir and then transported on ice.
  • the intact joints were washed well with tap water and soaked in 50%> (v/v) Povidine iodine solution, obtained from Burre National, Inc., Baltimore, MD. All subsequent steps were performed in a laminar flow tissue culture hood using standard sterile technique.
  • the joint was immobilized in a shank holder and the joint capsule was cut open to expose the articular cartilage.
  • Cartilage explant plugs approximately 15 mg wet weight, were removed from the flat articulating surfaces in the lower-most region of the knee joint using a sterile steel cork-borer and collected in a 250 mL roller bottle containing about 100 mL fresh Delbecco's minimum essential medium (DMEM), obtained from Gibco BRC, Life Technologies, Gaithersburg, MD, containing 4.5 g/1 (D)-glucose and (L)-glutamine, without sodium pyruvate.
  • the fresh media also contained enough Hepes buffer and sodium bicarbonate such that the pH was about 7.4.
  • the media was then further supplemented just before use with 100 units Penicillin, 100 ⁇ g Streptomycin, and 50 ⁇ g (L)-ascorbic acid per mL of medium.
  • the explant plugs were washed four times with 50 mL fresh DMEM. The plugs were then placed in the incubator for a minimum of 1 hour to equilibrate, before proceeding to make disks from the articulating surface of each plug.
  • a 1 mm thick disk was sliced from individual plugs from the end that was the articulating surface of the joint.
  • the plug was held steady in the sterile template (4 mm diameter x 1.5 mm deep) using sterile forceps.
  • a scalpel blade was used to carefully slice off the disk. Only the superficial articulating surface responded well in culture. Individual disks obtained were transferred to a tissue culture flask containing about 100 mL fresh media.
  • the flask containing the disks was placed in an incubator at 37 °C (with 5% CO 2 , 95% air) and allowed to equilibrate overnight and at least one additional day before labeling.
  • the old media was replaced with 50 mL fresh media containing about 500 ⁇ Ci
  • test media consisted of the desired concentration of a compound being tested for its ability to inhibit extracellular matrix degradation and concomitant recombinant human Interleukin rhIL- 1 (5 ng/mL) in fresh DMEM solution.
  • control media were identical to the test media, except that the first control media lacked rhIL-l and the second control media lacked the test compound. 250 ⁇ L of each of the test and control media were transferred to separate 96-well TC plates. Flamed forceps were used to transfer a disk from the incubator to each 96-well TC plates that had been filled with either the test media or one of the two control media.
  • TC plates were then placed in the incubator and cultured for 3-4 days (initial incubation with rhIL-l alpha takes at least 3 days to stimulate endogenous metalloproteinases).
  • a 50 ⁇ L aliquot of media from each TC plate was saved and counted. The rest of the media was removed with a suction device.
  • the cartilage disks from each TC plate were also saved for counting.
  • the disks were removed with forceps and placed in microcentrifuge tubes and then dissolved in 250 ⁇ L of full strength formic acid.
  • the tubes were capped and placed at 65-70 °C in a block-heater for 4-6 hours. A 50 ⁇ L aliquot was then counted.
  • the percent 35 S-GAG release is calculated as follows:
  • the percent inhibition of extracellular matrix damage in cartilage explant was calculated as follows:
  • A % GAG release induced by rhIL-l ⁇
  • 2-thione 5- N- (4-phenyl) phenylacetyl-phenylalanyl-valyla ino) -1,3,4- 37% (25) thiadiazole-2-thione 5- (N-benzyloxycarbonyl- ( (NH-trityl) lutamyl) -valylamino) -1,3,4- 56% (25) thiadiazole-2-thione 5- (N- (4-phenyl)phenylacetyl-valylamino) -1, 3, 4-thiadiazole-2-thione 47% (25) 5- (N-benzyloxycarbonyl- ( (O-benzyl) tyrosyl) -phenylglycylamino) - 20% (25)
  • w/o det refers to without detergent. In certain instances, a particular detergent and concentration will appear in parentheses. In the chemical diagrams, the abbreviation “Bz” refers to a benzyl group.

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Abstract

L'invention concerne l'utilisation de 5-amino-1,3,4-thiadiazole-2-thiols à substitution N5 utilisés en tant que métalloprotéinases pour inhiber les enzymes métalloprotéinases matricielles et empêcher la dégradation du cartilage. L'invention concerne également des méthodes de traitement de maladies provoquées par une suractivité de métalloprotéinases matricielles, telles que l'ostéoarthrite et la polyarthrite rhumatoïde.
PCT/US1997/022534 1996-12-09 1997-12-09 5 amino-1,3,4-thiadiazole-2-thiones substituees WO1998025949A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003514904A (ja) * 1999-11-23 2003-04-22 メチルジーン インコーポレイテッド ヒストン脱アセチル酵素の抑制剤
US6592902B2 (en) 1998-12-09 2003-07-15 Shilpa S. Thosar Controlled release eplerenone compositions
WO2007010885A1 (fr) * 2005-07-19 2007-01-25 Daiichi Sankyo Company, Limited Dérivé de propanamide substitué et composition pharmaceutique contenant celui-ci
EP2855467A4 (fr) * 2012-06-04 2015-12-30 Dow Agrosciences Llc Procédés de production de certains 2-(pyridine-3-yl)thiazoles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3407505A1 (de) * 1984-03-01 1985-09-05 A. Nattermann & Cie GmbH, 5000 Köln Neue benzothiazin-carbonsaeureamide mit antiarthritischer wirksamkeit
JPS61161281A (ja) * 1985-01-10 1986-07-21 Grelan Pharmaceut Co Ltd １，２−ベンゾチアジン−３−カルボキサミド誘導体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3407505A1 (de) * 1984-03-01 1985-09-05 A. Nattermann & Cie GmbH, 5000 Köln Neue benzothiazin-carbonsaeureamide mit antiarthritischer wirksamkeit
JPS61161281A (ja) * 1985-01-10 1986-07-21 Grelan Pharmaceut Co Ltd １，２−ベンゾチアジン−３−カルボキサミド誘導体

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6592902B2 (en) 1998-12-09 2003-07-15 Shilpa S. Thosar Controlled release eplerenone compositions
JP2003514904A (ja) * 1999-11-23 2003-04-22 メチルジーン インコーポレイテッド ヒストン脱アセチル酵素の抑制剤
USRE43343E1 (en) 1999-11-23 2012-05-01 Methylgene Inc. Inhibitors of histone deacetylase
WO2007010885A1 (fr) * 2005-07-19 2007-01-25 Daiichi Sankyo Company, Limited Dérivé de propanamide substitué et composition pharmaceutique contenant celui-ci
US8344029B2 (en) 2005-07-19 2013-01-01 Daiichi Sankyo Company, Limited Substituted propanamide derivative and pharmaceutical composition containing the same
JP5311822B2 (ja) * 2005-07-19 2013-10-09 第一三共株式会社 置換プロパンアミド誘導体及びそれを含む医薬組成物
EP2855467A4 (fr) * 2012-06-04 2015-12-30 Dow Agrosciences Llc Procédés de production de certains 2-(pyridine-3-yl)thiazoles
RU2647853C2 (ru) * 2012-06-04 2018-03-21 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Способы получения определенных 2-(пиридин-3-ил)тиазолов

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