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WO1997010231A1 - Inhibiteurs de la cysteine protease et de la serine protease, contenant un groupe cetomethylene - Google Patents

Inhibiteurs de la cysteine protease et de la serine protease, contenant un groupe cetomethylene Download PDF

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Publication number
WO1997010231A1
WO1997010231A1 PCT/US1996/014719 US9614719W WO9710231A1 WO 1997010231 A1 WO1997010231 A1 WO 1997010231A1 US 9614719 W US9614719 W US 9614719W WO 9710231 A1 WO9710231 A1 WO 9710231A1
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Prior art keywords
compound
carbons
groups
alkyl
nmr
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PCT/US1996/014719
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English (en)
Inventor
Sankar Chatterjee
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Cephalon, Inc.
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Filing date
Publication date
Priority claimed from US08/646,513 external-priority patent/US5723580A/en
Priority claimed from US08/646,071 external-priority patent/US5827877A/en
Application filed by Cephalon, Inc. filed Critical Cephalon, Inc.
Priority to AU69770/96A priority Critical patent/AU6977096A/en
Publication of WO1997010231A1 publication Critical patent/WO1997010231A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/74—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/02—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines
    • C07D217/06—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines with the ring nitrogen atom acylated by carboxylic or carbonic acids, or with sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9

Definitions

  • Novel ketomethylene group-containing inhibitors of cysteine or serine proteases methods for making these novel compounds, and methods for using the same are disclosed.
  • cysteine and serine proteases Numerous cysteine and serine proteases have been identified in human tissues.
  • a "protease” is an enzyme which degrades proteins into smaller components (peptides) .
  • the terms "cysteine protease” and “serine protease” refer to proteases which are distinguished by the presence therein of a cysteine or serine residue which plays a critical role in the catalytic process.
  • Mammalian systems, including humans normally degrade and process proteins via a variety of enzymes including cysteine and serine proteases. However, when present at elevated levels or when abnormally activated, cysteine and serine proteases may be involved in pathophysiological processes. For example, calcium-activated neutral proteases
  • calpain comprise a family of intracellular cysteine proteases which are ubiquitously expressed in mammalian tissues. Two major calpains have been identified; calpain I and calpain II. While calpain II is the predominant form in many tissues, calpain I is thought to be the predominant form in pathological conditions of nerve tissues. The calpain family of cysteine proteases has been implicated in many diseases and disorders, including neurodegeneration, stroke, Alzheimer's, amyotrophy, motor neuron damage, acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation.
  • Calpain I has been implicated in excitatory amino-acid induced neurotoxicity disorders including ischemia, hypoglycemia, Huntington's Disease, and epilepsy.
  • the lysosomal cysteine protease cathepsin B has been implicated in the following disorders: arthritis, inflammation, myocardial infarction, tumor metastasis, and muscular dystrophy.
  • Other lysosomal cysteine proteases include cathepsins C, H, L and S.
  • Interleukin-l ⁇ converting enzyme (“ICE") is a cysteine protease which catalyzes the formation of interleukin-l ⁇ .
  • Interleukin-l ⁇ is an immunoregulatory protein implicated in the following disorders: inflammation, diabetes, septic shock, rheumatoid arthritis, and Alzheimer's disease.
  • ICE has also been linked to apoptotic cell death of neurons, which is implicated in a variety of neurodegenerative disorders including Parkinson's disease, ischemia, and amyotrophic lateral sclerosis (ALS) .
  • ALS amyotrophic lateral sclerosis
  • Cysteine proteases are also produced by various pathogens.
  • the cysteine protease clostripain is produced by Clostridium histolvticum.
  • Other proteases are produced by Trypanosoma cruzi, malaria parasites Plasmodium falciparum and P.vinckei and Streptococcus .
  • Hepatitis A viral protease HAV C3 is a cysteine protease essential for processing of picornavirus structural proteins and enzymes.
  • Exemplary serine proteases implicated in degenerative disorders include thrombin, human leukocyte elastase, pancreatic elastase, chymase and cathepsin G.
  • thrombin is produced in the blood coagulation cascade, cleaves fibrinogen to form fibrin and activates Factor VIII; thrombin is implicated in thrombophlebitis, thrombosis and asthma.
  • Human leukocyte elastase is implicated in tissue degenerative disorders such as rheumatoid arthritis, osteoarthritis, atherosclerosis, bronchitis, cystic fibrosis, and emphysema.
  • Pancreatic elastase is implicated in pancreatitis.
  • Chymase an enzyme important in angiotensin synthesis, is implicated in hypertension, myocardial infarction, and coronary heart disease.
  • Cathepsin G is implicated in abnormal connective tissue degradation, particularly in the lung.
  • the present invention is directed to novel cysteine and serine protease inhibitors which contain a ketomethylene group adjacent to the P2 position. They are represented by the following Formula I:
  • Q is aryl having from about 6 to about 14 carbons, heteroaryl having from about 6 to about 14 ring atoms, aralkyl having from about 7 to about 15 carbons, alkyl having from 1 to about 10 carbons, said alkyl groups being optionally substituted with one or more J groups, heteroalkyl having from 2 to about 7 carbons, arylheteroalkyl wherein the aryl portion can be unfused or fused with the heteroalkyl ring, alkoxy having from 1 to about 10 carbons, aralkyloxy having from about 7 to about 15 carbons, a carbohydrate moiety optionally containing one or more alkylated hydroxyl groups, xanthene-9-yl, CH(i- C 4 H 9 )NHCbz, CH 2 N(i-C 4 H 9 )Cbz, or Formula II or III:
  • Y has the formula:
  • R 1 and R 2 are independently H, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, or a natural or unnatural side chain of an L- amino acid, said alkyl and cycloalkyl groups being optionally substituted with one or more J groups;
  • R 3 and R 4 are each independently hydrogen, alkyl having from 1 to about 10 carbons, said alkyl groups being optionally substituted with one or more J groups, aryl having from about 6 to about 14 carbons, and aralkyl having from about 7 to about 15 carbons; and
  • R 5 is halogen.
  • said J is an ⁇ -amino group that is tertiary (that is, trisubstituted with other than hydrogen) .
  • Q is -CH(R 6 ) -NH (R 7 ) where R 7 is a protecting group and R 6 is H, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, or a natural or unnatural side chain of an L-amino acid, said alkyl and cycloalkyl groups being optionally substituted with one or more J groups.
  • R 7 is a protecting group and R 6 is H
  • alkyl having from one to about 14 carbons cycloalkyl having from 3 to about 10 carbons, or a natural or unnatural side chain of an L-amino acid
  • the compounds of the invention are useful for the inhibition of cysteine and serine proteases.
  • the compounds find utility in a variety of settings.
  • the claimed compounds can be used, for example, as standards to screen for natural and synthetic cysteine protease and serine protease inhibitors which have the same or similar functional characteristics as the disclosed compounds.
  • our compounds can be used to alleviate, mediate, reduce and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases and/or serine proteases.
  • Q is aryl having from about 6 to about 14 carbons, heteroaryl having from about 6 to about 14 ring atoms, aralkyl having from about 7 to about 15 carbons, alkyl having from 1 to about 10 carbons, said alkyl groups being optionally substituted with one or more J groups, heteroalkyl having from 2 to about 7 carbons, arylheteroalkyl wherein the aryl portion can be unfused or fused with the heteroalkyl ring, alkoxy having from 1 to about 10 carbons, aralkyloxy having from about 7 to about 15 carbons, a carbohydrate moiety optionally containing one or more alkylated hydroxyl groups, xanthene-9-yl, CH(i- C 4 H 9 )NHCbz, CH 2 N(i-C diligentH 9 )Cbz, or Formula II or III:
  • R 1 and R 2 are independently H, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, or a natural or unnatural side chain of an L- amino acid, said alkyl and cycloalkyl groups being optionally substituted with one or more J groups;
  • J is halogen, lower alkyl, aryl, heteroaryl, amino optionally substituted with one to three aryl or lower alkyl groups, guanidino, alkoxycarbonyl, aralkoxycarbonyl, alkoxy, hydroxy, or carboxy; and
  • R 3 and R 4 are each independently hydrogen, alkyl having from 1 to about 10 carbons, said alkyl groups being optionally substituted with one or more J groups, aryl having from about 6 to about 14 carbons, and aralkyl having from about 7 to about 15 carbons; and
  • R 5 is halogen.
  • said J is an a-amino group that is tertiary.
  • G is hydrogen
  • Q is -CH(R 6 ) -NH(R 7 ) where R 7 is a protecting group and R 6 is H, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, or a natural or unnatural side chain of an L-amino acid, said alkyl and cycloalkyl groups being optionally substituted with one or more J groups.
  • R 2 is isobutyl.
  • R 1 is isobutyl, benzyl, or ethyl.
  • Q is - ( CH 2 ) 4 - C 6 H 5 , diphenylmethyl , xanthen- 9 -yl or Q has the Formula I I or I I I :
  • R 2 is isobutyl
  • R 1 is isobutyl or benzyl
  • Q is - (CH 2 ) 4 -C 6 H 5 , diphenylmethyl, xanthen-9- yl, - (S) -CH(C 2 H S ) (C 6 H 5 ) , - (S) -CH (i-C 4 H 9 )NHCbz or Q has the Formula II or III above.
  • alkyl is meant to include straight-chain, branched and cyclic hydrocarbon groups such as, for example, ethyl, isopropyl and cydopropyl groups. Preferred alkyl groups have 1 to about 10 carbon atoms. "Cycloalkyl” groups are cyclic alkyl groups. "Aryl” groups are aromatic cyclic compounds including but not limited to phenyl, tolyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl. Preferred aryl groups include phenyl and naphthyl.
  • Carbocyclic refers to cyclic groups in which the ring portion is composed solely of carbon atoms.
  • heterocyclic refers to cyclic groups in which the ring portion includes at least one heteroatom such as O, N or S.
  • Heteroalkyl groups are heterocycles containing solely single bonds within their ring portions, i.e. saturated heteroatomic ring systems.
  • lower alkyl refers to alkyl groups of 1-4 carbon atoms.
  • halogen refers to F, Cl, Br, and I atoms.
  • aralkyl denotes alkyl groups which bear aryl groups, for example, benzyl groups.
  • aralkyloxy denotes an aralkyl group linked through an oxygen atom.
  • heteroaryl denotes aryl groups having one or more heteroatoms contained within an aromatic ring.
  • Heteroaralkyi groups are aralkyl groups which have one or more heteroatoms in their aromatic ring portion.
  • carbbohydrate includes monosaccharides, disaccharides, and polysaccharides, as well as their protected derivatives, such as, for example, mono- and diisopropylidine, and benzylidene derivatives.
  • amino acid denotes a molecule containing both an amino group and a carboxyl group.
  • L-amino acid denotes an ⁇ -amino acid having the L configuration around the - carbon, that is, a carboxylic acid of general formula CH(COOH) (NH 2 ) - (sidechain) , having the L-configuration.
  • Sidechains of L-amino acids include naturally occurring and non-naturally occurring moieties.
  • Nonnaturally occurring amino acid sidechains are moieties that are used in place of naturally occurring amino acid sidechains in, for example, amino acid analogs. See, for example, Lehninger, Biochemistry, Second Edition, Worth Publishers, Inc, 1975, pages 73-75. Representative ⁇ -amino acid sidechains are shown below on Table 1.
  • Functional groups present on the compounds of Formula I may contain protecting groups.
  • Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention.
  • One such protecting group is the benzyloxycarbonyl (Cbz; Z) group.
  • protecting groups include the phthalimido arylcarbonyls, alkylcarbonyls, alkoxycarbonyls, aryloxycarbonyls, aralkyloxycarbonyls, alkyl- and aralkylsulfonyls, and arylsulfonyl groups such as those whic h
  • ketomethylene group-containing components of the invention inhibit cysteine proteases and serine proteases, they can be used in both research and therapeutic settings.
  • preferred compounds having defined attributes can be used to screen for natural and synthetic compounds which evidence similar characteristics in inhibiting protease activity.
  • the compounds can also be used in the refinement of in vi tro and in vivo models for determining the effects of inhibition of particular proteases on particular cell types or biological conditions.
  • compounds of the invention can be utilized to alleviate, mediate, reduce and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases and/or serine proteases.
  • compositions are provided for inhibiting a serine protease or a cysteine protease comprising a compound of the invention.
  • methods are provided for inhibiting serine proteases or cysteine proteases comprising contacting a protease selected from the group consisting of serine proteases and cysteine proteases with an inhibitory amount of a compound of the invention.
  • the disclosed compounds of the invention are useful for the inhibition of cysteine proteases and serine proteases.
  • the terms “inhibit” and “inhibition” mean having an adverse effect on enzymatic activity.
  • An inhibitory amount is an amount of a compound of the invention effective to inhibit a cysteine and/or serine protease.
  • compositions as disclosed herein.
  • pharmaceutically acceptable salts as used herein means an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate.
  • pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt.
  • pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine.
  • compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers.
  • pharmaceutically acceptable nontoxic excipients and carriers may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, transdermal patches; or prepared in other suitable fashions for these and other forms of administration as will be apparent to those skilled in the art.
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington ' s Pharmaceutical Sci ences (Mack Pub. Co., Easton, PA, 1980) .
  • Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds.
  • parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene- -lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration.
  • Formulations for transdermal patches are preferably lipophilic emulsions.
  • the materials for this invention can be employed as the sole active agent in a pharmaceutical or can be used in combination with other active ingredients which could facilitate inhibition of cysteine and serine proteases in diseases or disorders.
  • concentrations of the compounds described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration.
  • the compounds of this invention may be provided in effective inhibitory amounts in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. Typical dose ranges are from about l ⁇ g/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day.
  • Such formulations typically provide inhibitory amounts of the compound of the invention.
  • the preferred dosage of drug to be administered is likely, however, to depend on such variables as the type or extent of progression of the disease or disorder, the overall health status of the - 14 - particular patient, the relative biological efficacy of the compound selected, and formulation of the compound excipient, and its route of administration.
  • the term "contacting" means directly or indirectly causing at least two moieties to come into physical association with each other. Contacting thus includes physical acts such as placing the moieties together in a container, or administering moieties to a patient.
  • administering a compound of the invention to a human patient evidencing a disease or disorder associated with abnormal and/or aberrant activity of such proteases falls within the scope of the definition of the term "contacting".
  • the faster moving isomer was a white solid, mp 150-160 °C (softening to melt) ; R f (5% methanol in methylene chloride) : 0.37; 'H-NMR (300MHz, CDC1 3 ) ⁇ 7.40-7.10 (m, 5H) , 7.00-6.80 (m, IH) , 6.80-6.70 (m, IH) , 4.30-3.60 (m, 3H) , 3.60-3.50 (m, IH) , 3.40-3.20 ( , 3H) , 2.80-2.60 (m, 2H) , 2.60-2.40 (m, IH) , 2.10-1.90 (m, 2H) , 1.80-1.60 (m, 2H) , 1.50-1.30 (m, 2H) , 1.25-1.10 (m, 3H) , 1.00-0.70 (m, 12H) ; The slower moving isomer was a white solid, mp
  • the faster moving isomer was a white solid, mp 134-154 °C (softening to melt) ; R f (5% methanol in methylene chloride) : 0.44; 'H-NMR (300MHz, CDC1 3 ) ⁇ 7.40-7.10 (m, 10H) , 6.95-6.85 (broad, IH) , 6.20 (d, IH) , 5.75-5.55 (broad, IH) , 5.10 (s, IH) , 4.15 (s, IH) , 3.90-3.75 (m, IH) , 3.40-3.25 (m, IH) , 3.20-3.05 (m, IH) , 3.05-2.95 (dd, IH) , 2.85-2.70 (m, IH) , 2.65-2.50 (dd, IH) , 1.90-1.60 (m, 3H) , 1.60-1.40 (m, 2H) , 1.20-1.05 (t, 3
  • the slower moving isomer was a white solid, mp 124-144 °C (softening to melt) ; R £ (5% methanol in methylene chloride) : 0.38; 'H-NMR (300MHz, CDC1 3 ) ⁇ 7.40-7.10 (m, 10H) , 6.95-6.85 (broad, IH) , 6.20 (d, IH) , 5.75-5.55 (broad, IH) , 5.10 (s, IH) , 4.15 (s, IH) , 3.90-3.75 (m, IH) , 3.40-3.25 (m, IH) , 3.20-3.05 (m, IH) , 3.05-2.95 (dd, IH) , 2.85-2.70 (m, IH) , 2.65-2.50 (dd, IH) , 1.90-1.60 (m, 3H) , 1.60-1.40 (m, 2H) , 1.20-1.05 (t, 3H
  • Acetonitrile was removed in the rotavapor, base was added to pH 9-10 and the layer was washed several times with a mixture of ether and hexane (1:1) . The aqueous layer was then acidified (solid citric acid) to pH 3-4 and extracted into ethyl acetate (3 x 40mL) . Drying (MgS0TON) and solvent evaporation gave compound 27 which was used without further purification.
  • Inhibition of calpain I activity was calculated as the percent decrease in the rate of substrate (S) hydrolysis in the presence of inhibitor (-) relative to the rate in its absence ( 0 ) . Comparison between the inhibited and control rates was made within the linear range for substrate hydrolysis. For screening, compounds were tested at 10 ⁇ M. Compounds having 50% inhibition at 10 ⁇ M were considered active. The IC50s of inhibitors (concentration yielding 50% inhibition) were determined from the percent decrease in the rates of substrate hydrolysis in the presence of five to seven different concentrations of the test compound. The results were plotted as % inhibition versus log inhibitor concentration and the IC50 was calculated from linear regression of the data. Apparent second order rate constants were determined from analysis of reaction progress curves under pseudo-first order conditions. Each determination represents the means of three or more independent single cuvette analyses continually monitored via a Perkin-Elmer LS50B spectrofluorimeter. The rate of inhibition of hydrolysis was obtained by fitting the curve to the exponential equation (1) :
  • K obs is the pseudo- first order rate constant for inactivation.
  • a and B are constants.
  • the apparent second order rate constant k app was determined as K obg /[I] . This was corrected for the presence of substrate to give the second order rate constant k 2 according to equation (2) :
  • cathepsin B (Caibiochem, cat#219364) and cathepsin L (Caibiochem, cat#219402)
  • assays were performed substantially the same as outlined above except that the cathepsin B and cathepsin L were diluted into a different assay buffer consisting of 50mM sodium acetate (pH 6.0) /ImM EDTA/lmM dithiothreitol and the substrate used was Cbz-Phe-Arg-AMC (Bachem cat# 1-1160; 0. ImM for cathepsin B; 0.006mM for cathepsin L) .
  • Example 22A To demonstrate activity against the serine protease ⁇ -chymotrypsin (Sigma Chem. Co. Cat. #C-3142) the protocol of Example 22A was followed except that the enzyme was diluted into assay buffer consisting of 50mM Hepes (pH

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Abstract

La présente invention concerne de nouveaux inhibiteurs de la cystéine ou de la sérine protéase, contenant un groupe cétométhylène. On décrit également des procédés d'utilisation de ces inhibiteurs de protéases.
PCT/US1996/014719 1995-09-14 1996-09-13 Inhibiteurs de la cysteine protease et de la serine protease, contenant un groupe cetomethylene WO1997010231A1 (fr)

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AU69770/96A AU6977096A (en) 1995-09-14 1996-09-13 Ketomethylene group-containing cysteine and serine protease inhibitors

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US367895P 1995-09-14 1995-09-14
US60/003,678 1995-09-14
US08/646,513 US5723580A (en) 1995-09-14 1996-05-07 Ketomethylene group-containing aldehyde cysteine and serine protease inhibitors
US08/646,071 US5827877A (en) 1995-09-14 1996-05-07 Ketomethylene group-containing cysteine and serine protease inhibitors
US08/646,513 1996-05-07
US08/646,071 1996-05-07

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029435A1 (fr) * 1996-12-27 1998-07-09 Boehringer Ingelheim (Canada) Ltd. Inhibiteurs peptidomimetiques de protease du cytomegalovirus humain
US6448254B1 (en) 1998-04-20 2002-09-10 Abbott Laboratories Substituted amides, their production and their use
US6489308B1 (en) 1999-03-05 2002-12-03 Trustees Of University Of Technology Corporation Inhibitors of serine protease activity, methods and compositions for treatment of nitric-oxide-induced clinical conditions
US7704958B1 (en) 1999-03-05 2010-04-27 Bio Holding, Inc. Methods and compositions for inhibiting apoptosis using serine protease inhibitors
US7935478B2 (en) 2004-02-02 2011-05-03 Core Dynamics Limited Biological material and methods and solutions for preservation thereof
EP2520654A1 (fr) 2003-08-26 2012-11-07 The Regents of the University of Colorado Inhibiteurs de l'activité de sérine protéase et leur utilisation dans des procédés et compositions pour le traitement des infections bactériennes

Citations (3)

* Cited by examiner, † Cited by third party
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WO1998029435A1 (fr) * 1996-12-27 1998-07-09 Boehringer Ingelheim (Canada) Ltd. Inhibiteurs peptidomimetiques de protease du cytomegalovirus humain
US6291640B1 (en) 1996-12-27 2001-09-18 Boehringer Ingelheim Ltd. Peptidomimetic inhibitors of the human cytomegalovirus protease
US6448254B1 (en) 1998-04-20 2002-09-10 Abbott Laboratories Substituted amides, their production and their use
US6489308B1 (en) 1999-03-05 2002-12-03 Trustees Of University Of Technology Corporation Inhibitors of serine protease activity, methods and compositions for treatment of nitric-oxide-induced clinical conditions
US7704958B1 (en) 1999-03-05 2010-04-27 Bio Holding, Inc. Methods and compositions for inhibiting apoptosis using serine protease inhibitors
US8071551B2 (en) 1999-03-05 2011-12-06 BioHolding, Inc. Methods and compositions for treating diabetes
EP2520654A1 (fr) 2003-08-26 2012-11-07 The Regents of the University of Colorado Inhibiteurs de l'activité de sérine protéase et leur utilisation dans des procédés et compositions pour le traitement des infections bactériennes
EP3192872A1 (fr) 2003-08-26 2017-07-19 The Regents of the University of Colorado, a body corporate Inhibiteurs de l'activité de sérine protéase et leur utilisation dans les procédés et compositions pour le traitement des infections bactériennes
US7935478B2 (en) 2004-02-02 2011-05-03 Core Dynamics Limited Biological material and methods and solutions for preservation thereof

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