Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
  • C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the present invention relates to the discovery of novel, low molecular weight, non- peptide inhibitors of matrix metalloproteinases (e.g. gelatinases, stromelysins and collagenases) and TNF- ⁇ converting enzyme (TACE, tumor necrosis factor- ⁇ converting enzyme) which are useful for the treatment of diseases in which these enzymes arc implicated such as arthritis, tumor metastasis, tissue ulceration, abnormal wound healing, periodontal disease, bone disease, proteinuria, aneurysmal aortic disease, degenerative cartilage loss following traumatic joint injury, demyelinating diseases of the nervous system and HIV infection.
• matrix metalloproteinases e.g. gelatinases, stromelysins and collagenases
• TACE tumor necrosis factor- ⁇ converting enzyme
• MMPs Matrix metalloproteinases
• endopeptidases consist of several subsets of enzymes including collagenases, stromelysins and gelatinases. Of these classes, the gelatinases have been shown to be the MMPs most intimately involved with the growth and spread of tumors, while the collagenases have been associated with the pathogenesis of osteoarthritis [Howell, D.S.; Pelletier, J.-P. In Arthritis and Allied Conditions; McCarthy, D.J.; Koopman, W.J., Eds.; Lea and Febiger: Philadelphia, 1993; 12th Edition Vol. 2, pp. 1723; Dean, D.D. Sem. Arthritis Rheum.
• Angiogenesis required for the growth of solid tumors, has also recently been shown to have a gelatinase component to its pathology [Crawford, H.C; Matrisian, L.M. Invasion Metast. 1994-95, 14, 234; Ray, J.M.; Stetler- Stevenson, W.G. Exp. Opin. Invest. Drugs, 1996, 5, 323.].
• MMPs diseases mediated by MMPs
• Other conditions mediated by MMPs are restenosis, MMP-mediated osteopenias, inflammatory diseases of the central nervous system, skin aging, tumor growth, osteoarthritis, rheumatoid arthritis, septic arthritis, corneal ulceration, abnormal wound healing, bone disease, proteinuria, aneurysmal aortic disease, degenerative cartilage loss following traumatic joint injury, demyelinating diseases of the nervous system, cirrhosis of the liver, glomerular disease of the kidney, premature rupture of fetal membranes, inflammatory bowel disease, periodontal disease, age related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, ocular inflammation, keratoconus, Sjogren's syndrome, myopia, ocular tumors, ocular angiogenesis/neovascularization and corneal graft rejection.
• MMPs are important mediators of the tissue destruction that occurs in arthritis.
• these enzymes are capable of degrading collagens and proteoglycans which are the major structural components of cartilage [Sapolsky, A.I.; Keiser, H.; Howell, D.S.; Woessner, J.F., Jr.; J. Clin. Invest. 1976, 58, 1030; Pelletier, J.-P.; Martel-Pelletier, J.; Howell, D.S.; Ghandur- Mnaymneh, L.; Enis, J.E.; Woessner, J.F., Jr., Arthritis Rheum.
• MMP-13 collagenase-3
• MMP-13 is produced by chondrocytes, and elevated levels of MMP-13 has been found in human osteoarthritic tissues [Reboul, P.; Pelletier, J-P.; Hambor, J.; Magna, H.; Tardif, G.; Cloutier, J-M.; Martel-Pelletier, J. Arthritis Rheum. 1995, 38 (Suppl. 9), S268;Shlopov, B.V.; Mainardi, C.L.; Hasty, K.A. Arthritis Rheum. 1995, 38 (Suppl.
• TNF- ⁇ converting enzyme catalyzes the formation of TNF- ⁇ from membrane bound TNF- ⁇ precursor protein.
• TNF- ⁇ is a pro-inflammatory cytokine that is now thought to have a role in rheumatoid arthritis, septic shock, graft rejection, insulin resistance and HIV infection in addition to its well documented antitumor properties.
• anti-TNF- ⁇ antibodies and transgenic animals have demonstrated that blocking the formation of TNF- ⁇ inhibits the progression of arthritis [Rankin, E.C.; Choy, E.H.; Kassimos, D.; Kingsley, G.H.; Sopwith, A.M.; Isenberg, D.A.; Panayi, G.S. Br. J.
• TNF- ⁇ thelial growth factor- ⁇
• Other conditions mediated by TNF- ⁇ are congestive heart failure, cachexia, anorexia, inflammation, fever, inflammatory disease of the central nervous system, and inflammatory bowel disease.
• patents 5,455,258, 5,506,242 and 5,552,419, as well as European patent application EP606,046A1 and WIPO international publications WO96/00214 and WO97 22587 disclose non-peptide matrix metalloproteinase inhibitors of which the compound CGS27023A is representative. The discovery of this type of MMP inhibitor is further detailed by MacPherson, et. al. in J. Med. Chem., (1997),40, 2525.
• German patent application DE 19,542, 189- A 1 discloses additional examples of cylic sulfonamides as MMP inhibitors.
• the sulfonamide-containing ring is fused to a phenyl ring to form an isoquinoline.
• TACE and MMP inhibiting ortho-sulfonamido aryl hydroxamic acids of the present invention are represented by the formula
• hydroxamic acid moiety and the sulfonamido moiety are bonded to adjacent carbons of group A where:
• A is a 5-6 membered heteroaryl having from 1 to 2 heteroatoms independently selected from N, O, and S, and substituted by R 1 and R 2 on adjacent carbons wherein R 1 and R 2 together with the carbons to which they are attached form a fused phenyl ring or a 5-6 membered heteroaryl ring having from 1 to 3 heteroatoms selected independently from N, O and S, wherein either ring can be substituted by one or more substituents selected from R 4 ;
• Z is aryl, heteroaryl, or heteroaryl fused to a phenyl, where aryl is phenyl or naphthyl optionally substituted by R 1 , R 2 , R 3 and
• heteroaryl is a 5-6 membered heteroaromatic ring having from 1 to 3 heteroatoms independently selected from N, O, and S, and optionally substituted by R 1 , R 2 , R 3 and R 4 ; and when heteroaryl is fused to phenyl, either or both of the rings can be optionally substituted by R 1 , R 2 , R 3 and R 4 ;
• R 1 , R 2 , R 3 and R 4 are independently -H, -COR 5 , -F,-Br, -Cl, -I,
• R 5 and R 6 are independently defined as H, aryl and heteroaryl as defined above, -C3-C6-cycloalkyl as defined above, -C3-C6-cycloheteroalkyl as defined above, -C ⁇ -C4-perfluoroalkyl, or straight chain or branched -Ci- alkyl, -C2-C6-alkenyl, or -C2-C6-alkynyl each optionally substituted with -OH, -COR 8 , -CN, -C(O)NR8QR 9 , -C 2 -C 6 -alkenyl, -C 2 -C6-alkynyl, -OR 8 , -C ⁇ -C4-perfluoroalkyl, -S(O) x R 8 where x is 0-2, -OPO(OR )OR 9 , -PO(OR 8 )R9, -OC(O)NR R 9 ,
• R 7 is hydrogen, straight chain or branched -C ⁇ -C6-alkyl, -C2-C6-alkenyl, or -C2- C ⁇ -alkynyl each optionally substituted with -OH, -COR 5 , -CN, -C2-C6- alkenyl, -C 2 -C 6 -alkynyl, -OR 5 , -Ci-Q-perfluoroalkyl, -S(O) x R 5 where x is 0-2, -OPO(OR 5 )OR 6 , -PO(OR 5 )R 6 , -OC(O)NR 5 R 6 , - COOR 5 , -CONR 5 R 6 , -SO3H, -NR 5 R 6 ,-NR 5 COR 6 , -NR 5 COOR 6 , - SO NR 5 R6, -NO2, -N(R )SO 2 R 6 , -NR 5 CONR 5 R 6
• R 7 CH 2 -N-A- where A is as defined above, can form a non-aromatic 7-12 membered heterocyclic ring optionally containing an additional heteroatom selected from O, S and N wherein said heterocyclic ring may be optionally fused to another benzene ring;
• R 8 and R 9 are independently H, aryl or heteroaryl as defined above, -C3-C7- cycloalkyl or cycloheteroalkyl as defined above, -Ci-C perfluoroalkyl, straight chain or branched -C ⁇ -C6-alkyl, -C2-C6-alkenyl, or -C2-C6- alkynyl, each optionally substituted with hydroxy, alkoxy, aryloxy, -Ci- -perfluoroalkyl, amino, mono- and di-Ci- -alkylamino, carboxylic acid, carboalkoxy and carboaryloxy, nitro, cyano, carboxamido primary, mono- and di-Ci-C ⁇ -alkylcarbamoyl; and the pharmaceutically acceptable salts thereof and the optical isomers and diastereomers thereof.
• Preferred compounds are those wherein both of the carbons of A adjacent to the carbon bearing the sulfonamido group have a substituent other than hydrogen. Also preferred are compounds where Z is 4-alkoxyphenyl, 4-aryloxyphenyl or 4- heteroaryloxyphenyl.
• heteroaryl as defined hereinabove includes, but is not limited to, pyrrole, furan, thiophene, pyridine, pyrimidine, pyridazine, pyrazine,triazole, pyrazole, imidazole, isothiazole, thiazole, isoxazole and oxazole.
• heteroaryl fused to a phenyl includes, but is not limited to, indole, isoindole, benzofuran, benzothiophene, quinoline, isoquinoline, quinoxaline, quinazoline, benzotriazole, indazole, benzimidazole, benzothiazole, benzisoxazole, and benzoxazole.
• Compound V a) Denzel, T; Hoehn, H J. Heterocyclic Chem. (1977), 14, 813-817. b) Al-Shaar, AHM; Chambers, RK; Gilmour, DW; Lythgoe, DJ; McClenaghan, I; Ramsden, CA J. Chem. Soc; Perkin Trans. I (1992) 21, 2789-2812. c) Elworthy, T.R.; Ford, A.P.D.; et.al. J. Med. Chem. (1997), 40(17), 2674-2687.
• Compound VI a) Forbes, IT; Johnson, CN; Jones, GE; Loudon, J; Nicholass, JM J. Med.
• the compounds of this invention are shown to inhibit the enzymes MMP-1, MMP-
• MMP-13 and TNF- ⁇ converting enzyme are therefore useful in the treatment of arthritis, tumor metastasis, tissue ulceration, abnormal wound healing, periodontal disease, graft rejection, insulin resistance, bone disease and HTV infection.
• the invention compounds are prepared using conventional techniques known to those skilled in the art of organic synthesis.
• the following scheme (Scheme I) illustrates the reaction sequence employed.
• the bicyclic heteroaryl group A shown is a quinoline, 4-chloro-7-trifluoromethylquinoUne-3-carboxyUc acid ethyl ester, prepared from the corresponding aniline, is reacted with N-benzyl-p- methoxybenzenesulfonamide, wherein Z is p-methoxybenzene, to provide the requisite N ⁇ V-disubstituted sulfonamido-ester which is then converted into the corresponding hydroxamic acid in two steps.
• the 4-chloroquinoline carboxylic acid ester could be first reacted with R 7 -NH 2 and the resulting 4-(R 7 -amino)quinoline carboxylic acid ester then reacted with the appropriate Z-SO 2 -Cl. Hydrolysis of the ester and reaction with hydroxylamine hydro- chloride would then give the invention compound.
• haloquinolines may also be accomplished via palladium catalyzed couplings of alkynes, as illustrated in Scheme HI. Hydrogenation of the alkynes accesses the olefins and alkanes as well.
• Schemes IV and V illustrate two methods for incorporating amino groups into the substituent attached to the sulfonamide nitrogen of the compounds of the invention.
• the NH- sulfonamide is alkylated with propargyl bromide to provide the propargyl sulfonamide.
• This alkyne is reacted with paraformaldehyde in the presence of a primary or secondary amine and cuprous chloride to give the propargyl amine which is converted, as before, to the desired hydroxamic acid.
• Schemes VI through VIII Methods for synthesizing variations of substituents on the sulfonyl aryl group are shown in Schemes VI through VIII.
• biaryl sulfonyl groups are synthesized by Suzuki couplings on a bromo- substituted benzene sulfonamide.
• the starting bromo-substituted benzene sulfonamide is synthesized from the commercially available bromobenzenesulfonyl chloride and the amino-acid or amino-ester, H 2 N-A-CO 2 R, followed by alkylation of the resulting NH- sulfonamide.
• the bromo aryl sulfonamide is converted into the corresponding boronic acid by the method of Ishiyama, et.al. [J. Org. Chem. (1995), 60, 7508] followed by coupling with an appropriate aryl halide.
• Schemes VII through IX Methods for synthesizing sulfonyl aryl ethers are shown in Schemes VII through IX.
• biaryl ethers, or aryl heteroaryl ethers are synthesized starting from the known sulfonyl chlorides (see for example: Zook SE; Dagnino, R; Deason, ME, Bender, SL; Melnick, MJ WO 97/20824). 8/16514
• the biaryl ethers may be prepared from the corresponding boronic acids or via the sulfonyl phenols as shown in Scheme VIH
• Aryl ethers may also be prepared via displacement of the fluorine from a para- fluorobenzene sulfonamide, as shown in Scheme DC.
• Aryl or alkyl ethers may be prepared in this manner.
• Scheme X illusstrates the synthesis of pyrazolopyridines of the invention.
• a substituted amino-pyrazole is condensed with ethoxymethylene malonate to provide the pyrazolylamino methylene malonate, B.
• This compound is converted into the pyrazolopyridine, C, by heating at 240°C.
• Compound C is then converted into the chloro- ester, D, via reaction with phosphorus oxychloride.
• Displacement of the chloro substituent with a sulfonamide then gives compound E.
• Hydrolysis of the ester and conversion of the carboxylate into the hydroxamate then gives compound G.
• Scheme X illusstrates the synthesis of pyrazolopyridines of the invention.
• Example 15 4-[Benzyl-(4-methoxy-benzenesulfonyl)-amino]-6-trifluoromethyl- quinoline-3-carboxylic acid hydroxyamide
• Example 31 4-[Methyl-(4-methoxy-benzenesulfonyl)-amino]- 8-bromo-quinoline-3- carboxylic acid hydroxyamide
• the mixture was heated in an oil bath at 50°C overnight and then was heated in an oil bath at 100° C for 1.5 days.
• the mixture was poured into 800 ml of water and extracted with ethyl acetate.
• the extract was washed with water, 2N citric acid, water, brine and dried (Na 2 SO_j).
• the solvent was removed and the residue chromatographed on sihca gel with hexane-ethyl acetate (2:1) as eluent to give 0.64 g of product as a solid, mp 170-172°.
• the mixture was stirred in a sealed tube under nitrogen in an oil bath at 90°C for 3 days.
• the mixture was cooled, poured into water and extracted with ethyl acetate.
• the extract was washed with H 2 0, brine and dried (Na 2 S0 ).
• the solution was filtered through a thin pad of hydrous magnesuim silicate and the filter pad washed with ethyl acetate.
• the filtrate was concentrated to dryness under vacuum to give 1.3 g of solid. Chromatography on silica gel with ethyl acetate as solvent gave 0.35 g of product as a solid, mp 152-154°C.
• the solvent was removed under vacuum and the residue diluted with H 2 O, acidified with 2 N citric acid and extracted with two 30-ml portions of CH 2 C1 2 .
• the aqueous layer was neutrallized with solid NaHCO 3 to bring the pH to 7.
• the soUd which precipitated was filtered and washed with H 2 O to give 0.610 g of product as a solid, mp. 202-204°C.
• the CH 2 C1 2 extract was extracted with 2 N citric acid and the aqueous layer neutralized with soUd NaHCO 3 .
• the precipitated solid was filtered off and washed with water to give 0.226 g of product, mp 196-198°C. (mass spectrum (ES) 483.5 (M+l).
• Example 47 Following the procedure of Example 49, the product of Example 47 is reacted with benzyl-(4-methoxybenzenesulfonyl)amine and sodium hydride to provide ethyl 4-[benzyl- (4-methoxybenzenesulfonyl)amino]-l-phenyl-lH-pyrazolo[3,4-b]pyridine-5-carboxylate. m.p. 124°-126°C.
• Example 47 Following the procedure of Example 53, the product of Example 47 is reacted with (4-methoxybenzenesulfonyl) (3-pyridinylmethyl) amine and sodium hydride to provide ethyl 4-[(4-methoxybenzenesulfonyl)pyridin-3-ylmethylamino]-l-phenyl-lH-pyrazolo[3,4- b]pyridine-5-carboxylate. m.p. 89°-91°C.
• Example 65 Ethyl 4-chloro-l-phenyI-3-methyI-lH-pyrazolo [3,4-b]pyridine-5- carboxylate
• Example 65 Following the procedure of Example 49, the product of Example 65 is reacted with benzyl-(4-methoxybenzenesulfonyl)amine and sodium hydride to provide ethyl 4-[benzyl- (4-methoxybenzenesulfonyl)amino]-l-phenyl-3-methyl-lH-pyrazolo[3,4-b]pyridine-5- carboxylate. m.p. 164°-166°C.
• Example 65 Following the procedure of Example 53, the product of Example 65 is reacted with (4-methoxybenzenesulfonyl) (3-pyridinylmethyl) amine and sodium hydride to provide ethyl-4-[(4-methoxybenzenesulfonyl)pyridin-3-ylmethylamino]- 1 -phenyl-3- methyl-lH-pyrazolo[3,4-b]pyridine-5-carboxylate. m.p. 148°-150°C.
• Example 54 Following the procedure of Example 54, the above ester is hydrolyzed to provide 4- [(4-methoxybenzenesulfonyl)pyridin-3-ylmethylamino]-l-phenyl-3-methyl-lH- pyrazolo[3,4-b]pyridine-5-carboxylic acid. m.p. 235°-236°C.
• the carboxyUc acid is converted into the corresponding hydroxamic acid, 4-[(4-memoxybenzenesulfonyl)pyridin-3-ylmethylarnino]- l-phenyl-3-methyl-lH-pyrazolo[3,4-b]pyridine-5-carboxylic acid, hydroxyamide. m.p. 192°-194°C.
• Example 67 Following the procedure of Example 61, the product of Example 67 is converted into the corresponding hydrochloride salt. m.p.225°-226°C.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• Example 82 4-[(4-Methoxybenzenesulfonyl)pyridin-3-ylmethylamino]-l-methyI-3- phenyl-lH-pyrazoIo[3,4-b]pyridine-5-carboxylic acid, hydroxyamide
• the tide compound may be prepared.
• the tide compound may be prepared.
• the tide compound may be prepared.
• thiopeptide substrates such as Ac-Pro- Leu-Gly(2-mercapto-4-methyl-pentanoyl)-Leu-Gly-OEt by the matrix metalloproteinases MMP-1, MMP-13 (collagenases) or MMP-9 (gelatinase), which results in the release of a substrate product that reacts colorimetrically with DTNB (5,5'-dithiobis(2-nitro-benzoic acid)).
• DTNB 5,5'-dithiobis(2-nitro-benzoic acid)
• the thiopeptide substrate is made up fresh as a 20 mM stock in 100% DMSO and the DTNB is dissolved in 100% DMSO as a 100 mM stock and stored in the dark at room temperature. Both the substrate and DTNB are diluted together to 1 mM with substrate buffer (50 mM HEPES pH 7.5, 5 mM CaCl2) before use. The stock of enzyme is diluted with assay buffer (50 mM HEPES, pH 7.5, 5 mM CaCl2, 0.02% Brij) to the desired final concentration.
• substrate buffer 50 mM HEPES pH 7.5, 5 mM CaCl2
• assay buffer 50 mM HEPES, pH 7.5, 5 mM CaCl2, 0.02% Brij
• the assay buffer, enzyme, vehicle or inhibitor, and DTNB/substrate are added in this order to a 96 weU plate (total reaction volume of 200 ⁇ l) and the increase in color is monitored spectrophotometrically for 5 minutes at 405 nm on a plate reader and the increase in color over time is plotted as a linear line.
• a fluorescent peptide substrate is used.
• the peptide substrate contains a fluorescent group and a quenching group.
• MMP Upon cleavage of the substrate by an MMP, the fluorescence that is generated is quantitated on the fluorescence plate reader.
• the assay is run in HCBC assay buffer (50mM HEPES, pH 7.0, 5 mM Ca +2 , 0.02% Brij, 0.5% Cysteine), with human recombinant MMP-1, MMP-9, or MMP- 13.
• the substrate is dissolved in methanol and stored frozen in 1 mM aliquots.
• substrate and enzymes are diluted in HCBC buffer to the desired concentrations.
• the slope of the line is calculated and represents the reaction rate.
• the linearity of the reaction rate is confirmed (r >0.85).
• the mean (x ⁇ sem) of the control rate is calculated and compared for statistical significance (p ⁇ 0.05) with drug-treated rates using Dunnett's multiple comparison test. Dose-response relationships can be generated using multiple doses of drug and IC50 values with 95% Cl are estimated using linear regression.
• a 2 cm piece of dialysis tubing (molecular weight cut-off 12-14,000, 10 mm flat width) containing matrix metalloproteinase enzyme (stromelysin, coUagenase or gelatinase in 0.5 mL of buffer) is implanted either ip or sc (in the back) of a rat (Sprague-Dawley, 150-200g) or mouse (CD-I, 25-50g) under anesthesia.
• Drugs are administered PO, IP, SC or IV through a canula in the jugular vein. Drugs are administered in a dose volume of 0.1 to 0.25 mL/animal. Contents of the dialysis tubing is collected and enzyme activity assayed.
• Enzyme reaction rates for each dialysis tube are calculated. Tubes from at least 3 different animals are used to calculate the mean ⁇ sem. Statistical significance (p ⁇ 0.05) of vehicle-treated animals versus drug- treated animals is determined by analysis of variance. (Agents and Actions 21: 331, 1987).
• each well receives a solution composed of 10 ⁇ L TACE (Immunex, final concentration l ⁇ g/mL), 70 ⁇ L Tris buffer, pH 7.4 containing 10% glycerol (final concentration 10 mM), and 10 ⁇ L of test compound solution in DMSO (final concentration l ⁇ M, DMSO concentration ⁇ 1%) and incubated for 10 minutes at room temperature.
• the reaction is initiated by addition of a fluorescent peptidyl substrate (final concentration 100 ⁇ M) to each well and then shaking on a shaker for 5 sec.
• the reaction is read (excitation 340 nm, emission 420 nm) for 10 min. and the increase in fluorescence over time is plotted as a linear line. The slope of the Une is calculated and represents the reaction rate.
• the Unearity of the reaction rate is confirmed (r 2 >0.85).
• the mean (x ⁇ sem) of the control rate is calculated and compared for statistical significance (p ⁇ 0.05) with drug- treated rates using Dunnett's multiple comparison test. Dose-response relationships can be generate using multiple doses of drug and IC50 values with 95% Cl are estimated using linear regression.
• Compounds of this invention may be administered neat or with a pharmaceutical carrier to a patient in need thereof.
• the pharmaceutical carrier may be solid or liquid.
• Applicable solid carriers can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents or an encapsulating material.
• the carrier is a finely divided solid which is in admixture with the finely divided active ingredient.
• the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain up to 99% of the active ingredient.
• Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• Liquid carriers may be used in preparing solutions, suspensions, emulsions, syrups and elixirs.
• the active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fat.
• a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fat.
• the liquid carrier can contain other suitable pharmaceutical additives such a solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabiUzers or osmo-regulators.
• liquid carriers for oral and parenteral administration include water (particularly containing additives as above, e.g., ceUulose derivatives, preferable sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil).
• the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utiUzed by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. Oral administration may be either liquid or soUd composition form.
• the compounds of this invention may be administered rectally in the form of a conventional suppository.
• the compounds of this invention may be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol.
• the compounds of this invention may also be administered transdermally through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non-toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin.
• the carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices.
• the creams and ointments may be viscous liquid or semi-solid emulsions of either the oil in water or water in oil type.
• Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable.
• a variety of occlusive devices may be used to release the active ingredient into the blood stream such as a semipermeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient. Other occlusive devices are known in the literature.
• the dosage to be used in the treatment of a specific patient suffering a MMP or TACE dependent condition must be subjectively determined by the attending physician.
• the variables involved include the severity of the dysfunction, and the size, age, and response pattern of the patient.
• Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached.
• Precise dosages for oral, parenteral, nasal, or intrabronchial administration will be determined by the administering physician based on experience with the individual subject treated and standard medical principles.
• the pharmaceutical composition is in unit dosage form, e.g., as tablets or capsules.
• the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient;
• the unit dosage form can be packaged compositions, for example packed powders, vials, ampoules, prefilled syringes or sachets containing Uquids.
• the unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.

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