CA2224721A1 - Calpain inhibitors for the treatment of neurodegenerative diseases - Google Patents
Calpain inhibitors for the treatment of neurodegenerative diseases Download PDFInfo
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- CA2224721A1 CA2224721A1 CA002224721A CA2224721A CA2224721A1 CA 2224721 A1 CA2224721 A1 CA 2224721A1 CA 002224721 A CA002224721 A CA 002224721A CA 2224721 A CA2224721 A CA 2224721A CA 2224721 A1 CA2224721 A1 CA 2224721A1
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- benzyloxycarbonyl
- ketone
- leucyl
- phenylalanine
- glycine
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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
- C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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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
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Novel amino acid analogs are provided having the formula (I): Z-A3-A2-A1-Q, wherein Z is H or a protecting group; A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either <u>D</u> or <u>L</u>
stereochemistry or a chemical bond; A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine; Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted hereroaryl; and X is CI, Br or F, and a pharmaceutically acceptable salt thereof.
stereochemistry or a chemical bond; A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine; Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted hereroaryl; and X is CI, Br or F, and a pharmaceutically acceptable salt thereof.
Description
CA 0222472l l997-l2-l2 WO 96/4163~ PCT~US95/07463 CALPAIN INHIBITORS FOR THE TREATMENT OF NEURODEGENERATIVE
DISEASES
RACKGROUNI~ OF THE INVENTION
Fiel~ of the Invention 0 This invention relates to a series of novel amino acid analogs whichexhibit s~lective inhibition of Calpain 1, to compositions containing the novel amino acid analogs and methods for therapeutic use. The Calpain I
inhibitors described in this invention comprise novel amino acid derivatives which possess particular utility in treatment of neurodegenerative diseas~s.
Re~orted I )eYeloDments Calpain is a cytosolic protease enzyme found in all mammalian tissue and cell types. There are two forms of the enzyme with different sensitivities to calcium; the high-sensitivity form, calpain 1, is activated by a low calcium concentration (2-75 IlM), and the low-sensitivity form, calpain ll, is activated by a higher calcium concentration (200-800 ,LM).
Although calpain l l is the prominant form, calpain I is concentrated in synaps~s and neuronal cell bodies and is thought to be involved in the phenomenon of long-term synaptic potentiation.
The location of active calpain explain how calpain can promote~
down-regulation of rnembrane-associated active protein kinase C; (2) formation of a calpain-activated soluble kinase; and (3) reorganization of the cytoskeleton (Melloni, E., and Pontremoli, S. (1989), The Calpains, Trencls Neurosci. 12, 438-44). Inactivation of the kinase results in repression of superoxide anion production, a process correlated to the protein kinase C-mediated phosphorylation of membrane proteins. Formation of a soluble, fully active kinase, operating in association with active calpain, results in ~ 35 selective modification in the organization of the cytoskeletal proteins, CA 0222472l l997-l2-l2 W O 96/41638 PCTrUS95/07463 which is correlated with the extracellular discharge of granule contents.
These conclusions have been reached by specific and direct inhibition of the proteinases, which results in: (1 ) a significant increase in superoxide anion production; (2) a marked decrease in the down-regulation of protein kinase C
activity; (3) reduced formation of calpain-activated protein kinase; (4) decreased phosphorylation and phosphorylation-mediated proteolytic degradation of cytoskel~tal proteins; and (5) inhibition of granule exocytosis.
In addition, studies of (Lee, K. S., Frank, S., Vanderklish, P., Arai, A., and Lynch, G. (1991), Inhibition of Proteolysis Protects Hippocampal Neurons from Ischamia, Proc. Nat. Ac~d. Sci. USA, 88, 7233) suggest that the inhibition of calpain may protect from various ischemia induced-neurodegeneration, essential hypertension, and benefits CNS disorders, and stroke.
A wide variety of apeptidylz analogs are reported to inhibit the action of proteases (Mehdi, Shujaath, Cell-Penetrating Inhibitors of Calpain, Tl PS, 16, 150 April 1991). These peptidyl analogs include: epoxisuccinates (E-64), leupeptin (CH3CO-Leu-Leu-ArgH),and ketopeptides. However, these inhibitors suffer from some of the following disadvantages:
weak enzyme specificity, lack of inhibitory potency, inhibit wide variety of proteases in addition to calpain 1, and multi-inhibition of various enzymes limits their therapeutic applicability.
A limited number of peptidyl methyl ketone analogs constitute a well-known class of compounds having enzymatic (papain, cathepsin B) inhibition activity. These analogs, however, are essentially devoid of potency and selectivity in inhibiting calpain 1.
In spite of various known calpain inhibitors, no effective therapy has yet been developed for the majority of ischemia-induced neurodegenerative diseases, CNS disorders, and stroke. Consequently, there is a need for therapeutic agsnts effective in the treatment and prevention of these diseases.
~:UMMARY OF THE INVENTION
Novel amino acid analogs are provided having the formula (I) Z--A3--A2--A1--Q ( ) wherein Z is H or a protecting group;
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either ~ orL stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalanine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH20COL, CH2OL, CH2SL, CH2X, NI~JNHCOCH20COL, NHNHCOCH20L, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, and a pharmaceutically acceptable salt thereof.
As used herein the following terms shall be understood to have the followin~ meanings, unless otherwise indicated.
"Alkyl" means a saturated or an unsaturated aliphatic hydrocarbon which may be either straight- or branched-chain. Preferred groups have no 25 more than about 12 carbon atoms and may be methyl, ethyl and structural isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
"Lower alkylH means an alkyl group as above, having 1 to 7 carbon 30 atoms. Suitable lower alkyl groups are methyl, ethyl, n-propyl, isopropyi, butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and n-heptyl.
"Aryl" means phenyl and substituted phenyl.
W O 96/41638 PCTnJS95/07463 "Substituted phenyl" means a phenyl group in which one or more of the hydrogens has been replaced by the the same or different substituents including halo, lower alkyl, ni~ro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, trifluoromethyl, morpholinoethoxy, morpholino-sulfonyl, and carbobenzoxy-methylsulfamoyl.
~ Heteroaryl" means pyridyl, pyrimidyl, tetrazolyl or thiadiazolyl.
"Substituted heteroaryl" means a heteroaryl group in which one or more 10 of the hydrogens has been replaced by the same or different substituents including halo, lower alkyl, nitro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, trifluoromethyl, morpholinoethoxy, morpholiho-sulfonyl, and carbobenzoxymethylsulfamoyl.
A "protecting group" is a radical attached to an oxygen, sulfur, or nitrogen atom, respectively, which radical serves to protect the oxygen, sulfur, or nitrogen functionally against undesired reaction. Such protecting groups are well known in the art, many are described in "The Peptidesn, E.
Gross and J. Meienhofer, Eds. Vol. 3 Academic Press. NY (1981).
The N-protecting groups can be N-acyl, N-alkoxycarbonyl, N-arylmethoxyGarbonyl and N-arylsulfonyl protecting groups.
Suitable O-protecting groups include benzyl, tert-butyl, methyl, tosyl ad carbobenzoxy groups.
S-protecting groups include methyl, tert-butyl, benzyl and carbobenzoxy groups.
Pharmaceutically acceptable salts include both acid and base addition salts. Pharmaceutically acceptable acid addition salt refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, W O 96/41638 PCT~US95/07463 nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyrubic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, and p-toluenesulfonic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically accceptable organic non-toxic bases include salts of 10 primary, secondary, and tertiary amines, substituted amines including naturally occuring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, dicyclohexylamine, Iysine, arginine, histidine, caffeine, procain, hydrabamine, choline, betaine, ethylendiamine, glucosamine, methylglucamine, theobromine, purines, peperiziner, piperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanol-amine, dicyclohexylamine, choline and caffeine.
This invention also contemplates pharmaceutically acceptable acid-addition salts of the compounds of Formula 1. It is well known in the pharmacological arts that nontoxic addition salts of pharmacologically active amine compounds do not differ in activities from their free base. All stereoisomers as well as optical isomers related to the novel calpain 25 inhibitory amino acid analogs described herein are also considered to be within the scope of this invention.
The amino acid analogs of the present invention are selective calpain inhibitors. More particularly, the amino acid analogs of the present 30 invention bind at the active site of the proteolytic enzyme, specifically calpain 1.
The present invention further provides pharmaceutical compositions comprised of the above-described novel amino acid analog inhibitors and W O 96/41638 PCT~US9S/07463 method of treating ischemia-induced neurode~enerative diseases, stroke, myocardial infarction, CNS disorders, and immunological diseases involving interleukin 1.
nETAlLFD DFSCRIPTION OF THE INVENTION
The compounds of the present invention are prepared by the general synthetic methods described in Schemes 1, 2 and 3.
-~cheme 1 H O ~J~3 (i) Ethyl ~, H :. ' .. "ale H ~ J~ 3 0~ NJI~N~OH 1I!3H3r-HOAC ~3 O~N--I~N~3r Formula 1 / Formula 2 ArOH or ArSH
O~N D~N~OJ~Ar ~3 o~N Il~N~oAr (i) H2; Pd/C Formula 3 Formula 4 (ii) Z-D-AlaOH; N-rr~thyl e; PyBOP
H H~COAr _ CBZ N~NJ~N~
Formula 5 Formula 6 ~:cheme 2 ~Or - HN ~sr (~)A¢X~H~ ~ ~ N ~ N ~O~Ar Formula 7 Formula 8 Scheme 3 Q ~, ~H~CH3 ~r H~OH
Fom~ula 9 Forrnula 10 The first step of this procedure involves the synthesis of N-protected dipeptidic bromomethyl ketone (formula 2). Methods for the preparation of various dipeptides (formula 1 ) are well established in the art. The N-protected dipeptide (formula 1), which in some cases is commercially available, is then converted to the corresponding bromoketone (formula 2) by way of hydrobromination or hydrohalogenation of a diazomethyl ketone intermediate. A displacement reaction of the bromomethyl or chloromethyl ketone by an aromatic carboxylic acid or alcohol (or thiol) then yields the desired arylcarboxymethyl ketone (formula 3) or aryloxy (or aryl-thio)methy ketone (formula 4) of the invention.
The N-protected dipeptidic arylcarboxymethyl ketone (formula 3) is deprotected by conventional hydrogenolysis and the resulting free amino g W O 96/41638 PCTrUS95/07463 dipeptide analog (formula 5) is readily converted to the corresponding tripeptidic arycarboxymethyl ketone (formula 6) under standard peptide coupling conditions as shown in Scheme 1.
The preparation of various amino acid N-arylcarboxyacetyl-hydrazides (for example formula 8) involves the synthesis of amino acid bromoacetyl hydrazide by reacting the corresponding amino acid hydrazide (formula 7) with a haloacyl halide. The resulting haloacyl-hydrazide is then readily converted to the arylcarboxyacetyl-hydrazide (formula 8) or aryloxyacetyl-10 hydrazide by coupling with arylcarboxylic acid or aryl alcohol respectively (Scheme 2).
The peptidic aldehydes (for example formula 10) of this invention are readily prepared by synthesizing the corresponding peptidic N-methoxy-N-15 methylamide analogs (for example formula 9) via standard synthesis followed by LAH reduction of the above amides.
The following examples will further illustrate the compounds of the present invention.
Fxaml~le 1 N-Ben7yloxyc~rhonyl-D-alanyl-L-leucyl-l-~henylalanine ~.6-rlifluorophenyl carboxymethyl ketone (a) ~I-Ren7yloxyc~rhonyl-l-leucyl-l-~henyl~l~nine bromomethyl ketone N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine (10.16 g, 24.63 mmol) was dissolved in dry THF (100 mL) under nitrogen. The solution was cooled to -1 5~C, N-methylmorpholine (2.98 mL 22.1 mmol) was added followed by dropwise addition of isobutyl chloroformate (3.35 mL, 25.86 mmol) over a 5 min period. A solution of dried diazomethane in ether (50 mmol in 100 mL
ether dried over Na2S O 4; from Diazald-Aldrich) was poured into the W O 9~/41638 PCTAUS95/07463 reaction mixture. The reaction mixture (-1 5~C) was allowed to slowly warm to 0~C after 1 hr, and then held 1 hr at room temperature.
The reaction mixture was cooled to 0~C, 47 mL of ~0% HBr/AcOH
added with stirring at 0OC, and the r~sulting mixture was transferred to a separatory funnel with 500 mL of water. The aqueous phase was extracted with ethyl acetate (3x) and the organic layer was washed successiv~ly with water, ~.3N KHSO4, saturated NaHCO3 solution, water, and brine. The organic layer was dried over MgSO4, filtered, and conc~ntrated to yield a 10 white solid which was recrystallized from dichloromethane/hexane to afford 10.35 g (86%) of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone.
(b) N-Ren7yloxyc~rbonyl-l-leucyl-l-,~henyl~l~nine ~.6-difluoro-rhenylcarboxymethyl ketone 2,6-Difluorobenzoic acid (65 mg. 0.41 mmol) was added to a solution of N-b~nzyloxycarbonyl-L-leucyl-L-phenylalanine bromo-methyl ketone 20 (200 mg, 0.41 mmol) and potassium fluoride in dry DMF under nitrogen. The reaction mixture was poured into ether and the organic layer was washed successively with water, 5% NaHCO3, water, and brine. The ether solution was dried over MgSO4 and concentrated lo afford a solid product which was recrystallized from ether/hexane to yield 165 mg (70%) of N-25 benzyloxycarbonyl-L leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, m.p. 1 08-9~C .
(c) I -L eucyl-l -phenyl~l~nine ~.6-difluorophenylc~rboxymethyl ketone To a mixture of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone (670 mg, 1.18 mmol) in anhydrous ethanol under nitrogen was added 10% palladium on carbon (67 mg), and the mixture was cooled to O~C. The nitrogen atmosphere was then replaced with hydrogen gas by equalizing with hydrogen supplied from a balloon.
W O 96/41638 PCT~US95/07463 When the atmosphere was exchanged for hydrogen, 6N HCI solution (0.39 mL) was added and the solution was allowed to stir for 1.5 hr at room temperature. The reaction mixture was filtered through celite and the filtrate was concentrated in vacuo to afford the hydrochloride salt of L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone.
(d) N-Ren7yloxyc~rbonyl-n-~lanyl-l-leucyl-l-Dhenylalanine ~.6-~ifluoroDhenylcarboxymethyl ketone To a mixture of L-leucyl-L-phenylalanine 2,6-difluoro-phenacyloxymethyl ketone hydrochloride (180 mg, 0.394 mmol; azeotroped with toluene), benzyoxycarbonyl-D-alanine (97 mg, 0.43 mmol), benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluoro-phosphate (225 mg, 0.43 mmol) was added under nitrogen 5 mL of dichloromsthane, and the resulting mixture was cooled to 0~C. N-Methylmorpholine (117 rng, 1.06 mmol) was added to the above mixture and the resulting reaction mixture was stirred for 30 min at 0~ C, and then stirred at room temperature overnight. The mixture was poured into water, extracted with ethyl acetate, and the organic layer was washed successively with 0.3N
20 KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2S O4 and concentrated in vacuo and the residue was purified by chromatography eluting with 30-50% ethyl acetate/hexane to afford 111 mg (45%) of N-benzyloxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-difluorophenacyloxymethyl ketone, m.p. 171-2~C.
Empioying the synthetic procedure described in Scheme 1 and Example 1 the following additional calpain inhibitors were synthesized.
W O 96/41638 PCTnUS95/07463 Example 2 Renzyloxycarbonvl-l-leucyl-L-~henylal~nine ~.6-dichloro-3-1~-rnor~?holino)-ethoxy~phenylcarboxymethyl ketone FYamnle 3 Renzyloxycarhonvl-l-leucyl-L-tyrosine ~.6-10~lichlorol~henylcarboxymethyl ketone FYamr~le 4 15R en~yloxycarb onyl-l -~rolyl-L-leucyl-L-~henylalanine 2.6-dichloronhenylcarboxymethyl ketone FYamrle 5 Perllzyloxycarhonyl-~-leucyl-~lvcine 2.6-dichloro-3-(nnor~holinos~!fonyl)~?henylc~rboxymethyl ketone 25Fxample 6 ~enzyloxycarbonyl-L-leucyl-L-~henylalanine 2.6-dichloro-3-(mor~holinosulfonyl)~henylc~rboxymethyl ketone Example 7 Benzyloxycarbonyl-~lycyl~L-leucvl-L-phenylalanine 2~6-difluoro-~henylcarboxymethyl ketone CA 0222472l l997-l2-l2 W O 96/41638 PCTnJS9~/07463 F X a m ple 8 Ren7yloxycarhonyl-~-leucyl-!-tyrosine ~.6-dichloro-3-(mor~holino-sulfonyl)phenvlcarboxymethyl ketone ~ FYam~le 9 10 Ren7yloxycarhonyl-l-leucyl-L-alanine 2.6-dichloro-3-(mor~holino-sulfonyl)phenylcarboxymethyl ketone ~Yz~ml?le 10 15 Ren7yloxycarhonyl-L-Ie~lcyl-L-~henylalanine 2.6-dichlorophenyl-carboxymethyl ketone Example 1 1 Ben7yloxycarbonyl-L-valvl-L-phenylalanine 2.6-dichlorophenyl-carboxymethyl ketone 25 Example 12 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone Exam~le 13 Tert-RI-tyloxycarbonyl-l-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone W O 96/41638 PCT~US95/07463 Fx~ m ~le 14 Renzyloxycarhony~ le~cyl-l -tyrosine ~.6-5~ifluororhenylc~rboxymethyl ketone ., ~Y~m~?le 15 10Renzyloxyc~rhonyl-l-leucyl-l-~lycine 2.6-dichloroDherylcarboxymethvl ketone Ex~mple 1 6 Ren7yloxvcarhonvl-l-le~cyl-L-~lycine 3.6-dichloro-2-~cetAmido-~henylcarboxymethvl ketone Fxaml~le 17 ~?-Toluenesulfonyl-L-leucyl-L-phenylalanine 2.6-~ifluorophenylcarboxymethyl ketone Fxam~le 18 Renzyloxycarbonyl-L-leucyl-L-~henvlalanine 2.6-dimethylphenylcarboxymethyl ketone Fxample 1 9 Renzyl oxyca rbonyl-l -leu cyl- L-g Iyc i ne 2-acetam ido-6-chloroDhenyl-carboxymethyl ketone W O 96/41638 PCT~US95/07463 FYa m ple 20 Ben~yloxycarbonyl-L-leucyl-l-alanine 2-acetamido-6-chlorophenyl-carhoxymethyl ketone FY~mple ~1 10Ren7yloxycarbQnyl-l-N-methylleucyl-L-I?henylalanine 2.6-difluoronhenylcarboxymethyl ketone Exam~le 22 15Ren~yloxycarbonyl-l-N-methylleucyl-L-phenylalanine 2.6-~ichloro-3-~2-(mor~holino)ethoxy]phenylcarboxymethyl ketone FYample 23 ~en7yloxycarbonyl-L-valyl-L-phenylalanine 2-acetamido-6-chlorovhenylcarboxymethyl ketone 25Example 24 Renzyloxycarbonyl-L-N-methylleucyl-L-phenylalanine 2.6-dichloro-3-(morpholinosulfonyl)phenylcarboxymethyl ketone FXa m ple 25 Benzyloxycarbonyl-L-Ie--cyl-l.-Dhenylalanine 2.6-dichloro-3-(c~rbohenzoxymethylsulfamoyl)phenylcarboxymethyl ketone W O 96/41~38 PCT~US95/07463 FYample ?6 ~enzyloxycarhonyl-l -leucyl-L-alanine 2.6-dich loro-3-- 5(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone FYample 27 10Ren7yloxycarbonyl-l-le~3cyl-l-alanine ~.6-dichloro-3-r~-(morpholino)etho~y]Dhenylcarboxymethyl ketone Fxamrle 28 15Renzyloxyc~rhonyl-L-leucyl-!-alanine ~.6-tlimethoxy~henylcarboxymethvl ketone Fxamgle 29 Benzyloxycarbonyl-L-leucyl-L-alanine 2.6-chloroDhenylcarboxymethyl ketone 25F~ample 30 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2-acetamido-6 chlorophenylcarboxymethyl ketone Fxample 31 Renzyloxycarbonyl-l-leucyl-L-glycine 2-acetamido-3.6-dichlorophenyl-carboxymethyl ketone ' 35 -Fxample 32 RenzyloxycarbQnyl-L-leucyl-L-~henylalanine 2-5~yridylcarboxymethyl ketone Exam~le 33 10Benzyloxyc~rbonyl-L-leucyl-L-~lycine 2.6-fluorophenylcarboxymethyl ketone Example 34 15Renzyloxycarbonyl-L-leucyl-L-alanine 2.6-difluoroDhenylcarboxymethyl ketone Fxample 35 13enzyloxycarbonyl-L-valyl-L-alanine 2.6-histrifluoromethylphenyl-carboxymethyl ketone FYa m ~le 36 p-Nitrobenzvloxycarbonyl-L-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone Example 37 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 1-na~hthylcarboxymethyl ketone , Ex~mple 38 Ren7ylo%ycarbonyl-l-leucyl~ henylalanine 2.6-dichloro-3-~ s hen7yloxy~henylcsrboxymethvl ketone FYaml?le 39 N-Benzyloxycarlbonyl-L-leucyl-N-(2.6-dichloro~henvlcarboxyacetyl)hydrazide To a solution of N-benzyloxycarbonyl-L-leucyl-N-(bromoacetyl) hydrazide (50 mg, 0.12 mmol) and 2,6-dichlorobenzoic acid (29 mg, 0.15 mmol) in dry DMF (5 mL) was added potassium fluoride (18 mg) in one portion. The resulting mixture was poured into water, extracted with ether, and the organic layer was washed successively with water, 5% NaHCO3, water, and brine. The organic layer was dried over MgSO4 and concentrated to afford 56 mg (88%) of N-benzyloxycarbonyl-L-leucyl-N-(2,6-20 dichlorophenylcarboxyacetyl) hydrazide, m.p. 1 03-5~C .
Employing the synthetic procedure described in Example 39, the following compounds were made.
Fxam~le 40 N-Benzvloxycarbonyl-L-leucyl-N-methyl. N-(2-acetamido-6-chlorophenycarboxy-acetyl)hydrazide IFxam~le 41 N-Benzvloxycarbonyl-l -leucyl-N-(2-acetamido-6-chloro~henylcarboxy-acetyl)hydrazide -CA 0222472l l997-l2-l2 WO 96/4~638 PCT~US95/07463 Exam~le 4~
Renzyloxycarhonvl-!-leucyl-l-tyrosine 2.6-dichloro-3-r2-(mor~holino)ethoxyl~henylcarboxymethyl ketone FYam~le 43 Methoxycarbonyl-D-alany~ eucy~ henylalanine 2.6-ichloro-3-r2-(morpholino)ethoxy~phenylcarboxymethyl ketone Fxaml?le 44 Ren7yloxycarbonyl-D-~l~nyl-l-leucyl-L-tyrosine ~.6-~ichloro-3-r~-(morpholino)ethoxyll~henylcarboxymethyl ketone Fxample 45 Renzyloxycarbonyl-l-valyl-l-~henylalanine 2.6-dichloro-3-U-(mor~holino)ethoxylphenylcarboxymethyl ketone FYaml~le 46 Benzyloxycarbonyl-L-valyl-plycine 2.6-dichloro-3-(carbobenzoxy-methylsulfamoyl)phenylcarboxymethyl ketone FXa m Dle 47 Benzyloxycarbonyl-L-leucvl-L-alanine 2.6-dichloro-3-r2-(mor~holino)-ethoxylDhenylcarboxymethyl ketone W O 96/41638 PCT~US95/07463 ~Ya m ~le 48 Ben~yloxycarbonyl-l-leucyl-glycine 2.6-dichloro-3-r2-(mor~holino)-ethoxylghenylcarboxymethyl ketone FYam~le 49 Methoxycarbonyl-P-alanyl-3-leucyl-l-~?henylalanine ~.6-difluorophenylcarhoxymethyl ketone FYample 50 en~yloxycarbonyl-l alanyl-L-~lycine 2.6-dichloro-3-(carbohen7oxy-methylsulf~moyl)Dhenylcarboxymethyl ketone Example 51 Renzyloxycarbonyl-glycyl-L-~henylalanine 2.6-dichloro-3-(c~rhohenzoxymethylsulfamoyl)~henylcarboxymethyl ketone 2s Fxample 52 Benzyloxycarbonyl-L-valyl-glycine 2.6-dichlorophenylcarboxymethyl ketone Fxample 53 penzyloxycarbonyl-Qlycvl-l-Dhenylalanine 2.6-dichlororhenYlcarboxymethyl ketone W O 96/41638 PCT~US95/07463 Example 54 E~en7yloxycarbonyl-L-phenylalanyl-L-alanine 2.6-dichloro-3-12-(morl?holino)ethoxylphenylcarboxymethyl ketone ~Yample 55 Renzyloxvcarhonyl-l-~henylalanyl-glycine 2.6-dichlorophenylcarboxymethyl ketone FYam~?le 56 Ren7ylQxyc~rbonyl-D-alanyl-L-leucyl-~lycine 2.6-dichloro-3-r2-15(mor~holino)ethoxyl~henylcarboxymethyl ketone Fxample 57 20Benzyloxycarbonvl-L-leucyl-glycine 2.6-dichlorophenylcarboxymethyl ketone Fxample 58 Benzyloxycarbonyl-L-phenylalanyl-glycine 2.6-dichlorophenylcarboxymethyl ketone FxamPle 59 Benzyloxycarbonvl-L-alanyl-glycine 2.6-dichloro~henylcarboxymethyl ketone W O 96/41638 PCT~US9~/07463 Fxam~le 60 Ren~yloxycarbonyl~ henylalanyl-L-alanine 2.6-~i trifluoromethyl~henylcarboxymethyl ketone Fxaml?le 61 N-Ren7yloxyc~rhonyl-l-lel~cyl-!-c~henylalanine ~.6-~ichlorophelloxymethyl ketone To a solution of benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone (100 m~, 0.204 mmol), 2,6-dichlorophenol 34 mg, 0.204 mmol) and K2CO3 (29 mg, 0.204 mmol) in 8 mL of DMF was added 15 tetra-n-butyl-ammonium iodide (8 mg) and the resulting mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate, washed with water and brine, and the organic layer was dried over Na2SO~. The solvent was concentrated in vacuo to afford 80 mg of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichlorophenoxymethyl ketone, as a white solid, m.p. 102-4~C.
Ernploying the synthetic procedure described in Example 61 and Scheme 1 the following additional calpain inhibitors were synthesized.
FYam6~1e 62 N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-r1-(3-~?yridyl)tetrazolyllthiomethyl ketone Fxample 63 N-~en7yloxycarbonyl-L-leucyl-L-phenylalanine 2-r(4-rnorpholinoethyl)~tetrazolyllthiomethyl ketone W O 96/41638 PCTrUS95/07463 FYamrle 64 N-Penzyloxycarbonyl-!-lel~cyl-l-nhenylalanine 2-r(s-5methylthio)tetra701yllthiomethyl ketone FYamrle 65 10N-Benzyloxycarbonyl-L-leucyl-l-rhenylalanine ~-r(s-methylthio)tetr~zolyllthiomethyl ketone FYS~mple 66 N-~enzyloxycarhonyl-l-leucyl-l-~henylalanine 2.6-~1ifl--orol~henylthiomethyl ketone 20FYample 67 N-Renzyloxyc~rhonvl-l-valyl-l-phenylalanine ~.6-~lifluorophenoxymethyl ketone Example 68 N-Renzyloxycarbonyl-L-leucyl-L-phenvlalanine 2-pyrimidylthiomethyl ketone FYa m ple 69 N-Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2-(1-phenyl)-35tetrazolylthiomethyl ketone W O 96/41638 PCT~US95/07463 To a solution of benzyloxycarbonyl-L-leucyl-L-phanylalanine bromomethyl ketone (150 mg, 0.306 mmol) and 2-mercapto-phenyl tetrazole (57.2 mg, 0.32 mmol) in 2 mL of DMF was added K2C03 ( 42.3 nng, 0.306 mmol) at room temperature and the resulting reaction mixture was stirred overnight. The mixture was poured into 50 mL of water and then extracted with ethyl acetate. The organic layer was washed with 0.3N
KHSO4, 5% NaHCO3, water, and brine and dried over Na2SO4. The solvQnt was concentrated fn vacuo to afford 168 mg (94%) of N-benzyloxycarbonyl-10 L-leucyl-L-phenylalanine 2-(1-phenyl)-tetrazolylthio-methyl ketone, as a white solid, m.p. 1 83-4~C .
Exam~le 70 ~enzyloxycarbonyl-L-leucyl-L-tyrosinal Benzyloxycarbonyl-L-leucyl-L-tyrosyl-N-(methoxy),N-methyl amide (0.182 mmol) was dissoved in 4 mL of ether/THF (1 :1 ) under nitrogen and the solution was cooled to OoC. LAH ether solution (0.182 mmol) was adde by syrin~e to ~he reaction mixture with stirring. The reaction mixture was quenched with 0.3N KHSO4 (0.6 mL) and the mixture was transferred into a separatory funnel containing 50 mL of water and 50 mL of ether/ethyl acetate (1:1). The aqueous layer was extracted with ether/ethyl acetate and the combined organic layer was washed with 0.3N KHSO4, water, and brine. The or~anic solution was dried over Na2SO4 and concentrated in vacuo to afford 53 mg (70.6%) of benzyloxycarbonyl-L-leucyl-L-tyrosinal, m.p. 57-60~C.
Employing the synthetic procedure described in Scheme 1, Scheme 2 and Scherne 3 the following additional calpain inhibitors were prepared.
Fxample 71 Benzyloxycarbonyl-L-valyl-L-tyrosinal -2~-Fxamnle 7~
5IRen~vloxycarbonyl-L-Ieucyl-L-O-methyl-tyrosinal FYam~le 73 10Ren~yloxyc~rhonyl-3-le~cyl-L-~henylalaninal FXa m ~le 74 15Benzyloxycarbonyl-L-isoleucyl-L-tyrosinal Fxample 75 20R e nzylo xyc arbo nyl-L-valyl-~ -2-(2-nap hthylm ethyl)~lycin ~1 Exam p le 76 25B en7ylO xycarh O n yl-L-isole U Cyl-L-p hen ylalaninal Exam~le 77 30R e n~ylo xycarb O n yl-L-valyl-D L-2-(p he nethyl)~ly~i FYa m ple 78 35Benzyloxycarbonyl-L-2-neopentyl-glycyl-L-phenylalaninal CA 0222472l l997-l2-l2 W O 96/~1638 PCTAUS95/07463 FYa m ple 79 R e n~ylo xyc~rb O nyl-l -valyl-DI -2-(1-na D hthylm ethyl)~lyci Ex~m~?le 80 10Ren7yloxycarbonyl-l~-phenylQlycyl-l-phenylal~nin~l IFxam~le 81 15Benzyloxycarbonyl-L-alanyl-L-~henylalaninal ~Ya m ple 82 20R en~ylo xycarbon yl~ henethyl~lycvl-L-~ hen ylalani IFxamr~le 83 25R e nzyloxvcarbon yl-L-rhenylalanyl-L-p hen Yl31ani ~Ya m ~le 84 30P en7y13 xycarb O n yl-L-2Dtert-b utylglycyl-L-p he n ylalani W O 96/41638 PCTnJS95/07463 Exam~le 85 Ren7yloxycarhonyl-~ -2-(1 -na~hthymethyl)~lycyl-nl -~henyl~laninal FYample 86 Ren7yloxycarhonyl-l -leucyl-N-chloroacetyl-hydr~zi~e ~xample 87 Ben~yloxycarbonyl-l -lel~cyl-N-bromoacetyl-hydrazkle Fxaml?le 88 Benzyloxycarbonyl-L-leucine chloromethyl ketone ExamDle 89 Ren7yloxycarbonyl-L-leucyl-L-leucyl-L-~henylalanine 25chloromethyl ketone Exam~le 90 Benzyloxycarbonyl-L-leucyl-l-alanine chloromethyl ketone Example 91 Benzyloxyc:arbonyl-L-lellcyl-l-~henylalanine chloromethvl ketone Fxa m ~le 92 Ren;~yloxycarbonyl-~lycyl-L-leucyl-L-tyrosine chloromethyl ketone FYa m Ple 93 o Ren7vloxvcArhonyl-!-le~cyl-l-rhenylal~nine chloromethyl ketone FYample 94 Rer~7yloxycarbonyl-L-leucyl-~lycine chloromethyl ketone FYarn~le 95 Ren7yloxycarbonyl-L-le~ncyl-L-alanine bromomethyl ketone FYa m ple 96 Renzyloxycarbonyl-L-valyl-l-phenylalanine bromomethyl ketone FYa m ple 97 Benzyloxycarbonyl-L-leucyl-L-leucine bromomethyl ketone FxamPle 98 Benzyloxycarbonyl-L-as~?aragyl-L-phenylalanine chloromethyl keto ne -CA 0222472l l997-l2-l2 FxamDle 99 Renzyloxyc~rhonyl-L-le~cyl-L-~henylalanine bromomethyl ketone FYam~le 1 OQ
Ren7yloxyc~rhonyl-L-~henylalanyl-L-~Ianlne chloromethyl ketone FYam~le 1 01 Renzyloxyc~rbonyl-~lycyl-L-~henylalanine bromomethyl ketone ~xannDle 102 E~enzyloxycarbonyl-L-valyl-glycine bromomethyl ketone FYa m Dle 103 Ren7yloxycarbonyl-L-leucine ch loromethyl ketone Exam~le 1 04 Ren7yloxyc~rbonyl-L-~?henylalanyl-L-alanine bromomethyl ketone Example 1 0~
Benzyloxycarbonyl-L-alanyl-glycine bromomethyl ketone Ex~m~le 1 06 Ren7yloxyc~rhonyl-1-2-~-n~ghthvlmethyl) cllycine chloromethyl keto ne FYaml~le 1 07 Ren7yloxycarbonyl-L-phenylalanyl-~lycine chloromethyl ketone FxamDle 1 08 Ren7yloxyc~rhonyl-l-~?henylal~nyl-L-~henylalanine chloromethyl 15keto ne ExamE~le 1 09 20Renzyloxycarbonyl-L~leucyl-N-(bromoacyl) hydrazide E~xam~le 11 0 25Ren~yloxycarbonyl-L-leuGyl-l-tyrosine bromomethyl ketone Compounds of the present invention were tested for calpain I inhibition activity using the following assay method.
30Cal~ain I InhibitionAssay Isol~tion of Hum~n erythrocyte C~in I
Human red blood cells were obtained from the Northeastern New York Chapter of the American Red Cross. The isolation of calpain from hurnan W O 96/41638 PCT~US95/07463 erythrocytes was similar to that described by Wang et al. (1988). One unit of in-dated packed red cells was diluted with an equal volume of diluting/wash solution and centrifuged. The supernatant was removed and the procedure was repeated. The washed cells were pooled, Iysed with 700 mL of Iysin~ solution and centrifuged to remove cell debris. The membrane-free hemolysate was added to 500 mL DEAE-sephacel and the slurry was stirred gently at 4~C for 1 hour.
Batch elution was done using DEAE-sephacel wash solution to remove a 10 lar~e amount of unwanted protein, most of which was hemoglobin. The slurry was poured into a column connected in tandem to a phenyl-sepharose CL-4B column. Material eluted from the DEAE-sephacel was applied directly to the phenyl-sepharose CL-4B. The phenyl-sepharose CL-4B column was washed first with 75 mM NaCI and then with no salt. Calpain begins to ~is~ssociate from the DEAE-sephacel with the 75 mM NaCI but the majority should adhere to the column until the salt is removed. Fractions were collected (20 mL), assayed for caseinolytic activity with and without c~lp~statin and pooled accordingly. The pooled fractions were concentrated usin~ an Amicon stirred cell equipped with a YK-10 membrane. Calpain was stored at 4~C with 10 mM EDTA and ~ mM 2-mercaptoethanol and is stable for at l~ast 6 months.
W O 96/41638 PCT~US95/07463 A~say Procedl-re The tritated assay is a modification of that described by Gopalakrishna, R. and Barsky, S.H., An~l. Riochem., 148, 413,1985. All r~agents, compound 25 ul, HEPES buffer 25 ul, CaCI2 50 ul, enzyme 50 ul, and 3H-acetyl Casein, were combined in 1 mL polystyrene titer plates. The plates were preincubated at 25~C for 5 min with gentle shaking prior to the addition of substrate. The incubation was continu~d for an additional 2 hours and was terminated with the addition of 0.5 mL ice cold 5% TCA.
Unlabled casein was added, samples were centrifuged and 0.5 mL of the supernatant was counted in 5 rnL of Ready Protein liquid scintillation cocktail for 2 min. This assay measures 3H-acetyl Casein degradation as an endpoint for caipain activity.
Representative assay results are shown in the following tables.
W O 96/41638 PCT~US95/07463 Tsble 1 Acyloxyketone Calpain I Inhibitors Ex. Z A3 A2 Al Q lCso/uM
CBZ D-Ala L-Leu L-Phe 2,6-difluoropheny .046 2 CBZ - L-Leu L-Phe 2,6-dichloro-3-[2- 0.14 0 (morpholino) ethoxy]phenyl 3 CBZ - L-Leu L-Tyr 2,6-dichlorophenyl 0.22 4 CBZ L-Pro L-Leu L-Phe 2,6-fluorophenyl 0.08 CBZ - L-Leu Gly 2,6-dichloro-3- 0. 11 (morpholinosulfonyl)phenyl 6 CBZ - L-Leu L-Phe 2,6-dichloro-3- 0.17 (morpholinosulfonyl)phenyl 7 CBZ Gly L-Leu L-Phe 2,6-difluorophenyl 0.04 8 CBZ - L-Leu L-Tyr 2,6-dichloro-3 0.17 (morpholinosulfonyl)phenyl 9 CBZ - L-Leu L-Ala 2,6-dichloro-3- 0.43 (morpholinosulfonyl)phenyl CBZ - L-Leu L-Phe 2,6-dichlorophenyl 0.33 11 CBZ - L-Val L-Phe 2,6-dichlorophenyl 0.55 12 CBZ - L-Leu L-Phe 2,6-difluorophenyl 0.16 13 CtBu - L-Leu L-Phe 2,6-difluorophenyl 0.42 14 CBZ - L-Leu L-Tyr 2,6-difluorophenyl 0.40 CBZ - L-Leu Gly 2,6-dichlorophenyl 0.29 16 CBZ - L-Leu Gly 3,6-dichloro-2- >10 acetamidophenyl 17 Tos - L-Leu L-Phe 2,6-difluorophenyl 0.16 18 CME - L-Leu L-Phe 2,6-dimethylphenyl 0.63 19 CBZ - L-Leu Gly 2-acetamido-6- 0.78 chlorophenyl CBZ - L-Leu L-Ala 2-acetamido-6- 0.36 chlorophenyl W O 96/41638 PCTrUS95/07463 Table ~
Aryloxyketone Cal~in I Inhibitors ~ 5 Ex. Z A3 A2 Q IC50/u M
61 CBZ L-Leu L-Phe 2,6-dichlorophenoxy 2.3 62 CBZ L-Leu L-Phe 2-[1-(3-pyridyl)tetrazoyl] 0.53 thio 63 CBZ L-Leu L-Phe 2-[(4-morpholinoethyl) 3.8 0 tetr~olyl]thio 64 CBZ L-Leu L-Phe 2-[(5-methylthio)thi~ 7Oyl] 2.0 thio CBZ L-Leu L-Phe 2,6-difluorophenoxy >10 66 CBZ L-Leu L-Phe 2,6-dichlorophenylthio >10 67 CBZ L-Val L-Phe 2,6-difluorophenoxy ~10 68 CBZ L-Leu L-Phe 2-pyrimidylthio >10 69 CBZ L-Leu L-Phe 2-(1 -phenyltetrazoyl)thio >10 Table 3 PeE~tide Aldehyde Cal~ain I Inhibitor~
Z-A2-Al - H
Ex. Z A2 Al IC50/ u M
CBZ L-Leu L-Tyrosinal 0.02 71 CBZ L-Val L-Tyrosinal 0.026 72 CBZ L-Val L-Tyrosinal(O-methyl) 0.03 73 CBZ L-Leu L-Phenylalaninal 0.037 74 CBZ L-lle L-Tyrosinal 0.053 CB Z L-Val DL-2-(2-Naphthy 0.07 methyl)glycinal 76 CBZ L-lle L-Phenylalaninal 0.08 77 CBZ L-Val DL-2-(Phenethyl)glycinal 0.10 78 CBZ L-2-(Neopentyl) L-Phenylalaninal 0.10 Glycyl W O 96/41638 PCT~US95/07463 T~hle 3(contd.) Peptide Aldehyde Cal~ain I Inhibitors Z-A2-Al - H
Ex. Z A2 A1 lCsol u M
79 CBZ L-Val DL-2-(1 -Naphthyl- 0. 11 methyl)glycinal 0 80 CBZ 2-Phenylglycyl L-Phenylalaninal0.11 81 CBZ L-Ala L-Phenylalaninal 0.17 82 CBZ L-2-(Phenethyl) L-Phenylalaninal 0.27 Glycyl 83 CBZ L-Phe L-Phenylalaninal 0.41 T~hle 4 H~loketone Cal~in I Inhibitors Ex. A3 A2 Al X IC50/u M
86 - - L-Leu-NHNHCO Cl 2.2 87 - - L-Leu-NHNHCO Br 6.8 88 - - L-Leu Cl >10 89 L-Leu L-Leu L-Phe Cl >10 - L-Leu L-Ala Cl >10 91 L-Leu L-Phe Cl 43.3 92 Gly L-Leu L-Phe Ci 6.6 93 - L-Leu L-Tyr Cl 40 94 - L-Leu L-Phe Cl >10 - L-Leu L-Ala Br >10 96 - L-Val L-Phe Br >10 97 - L-Leu L-Leu Br >10 98 - L-Asp(NH2) L-Phe Cl >10 99 - L-Leu L-Phe Br 9.1 W O 96/41638 PCT~US95/07463 The present invention includes a calpain inhibitor of this invention formulat~d into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are ~ 5 collectively referred to her~in as carriers, for parentcral injection or oral administration, in solid or liquid form, for rectal or topical administration, or the like.
Th~ compositions can be administered to humans and animals either 10 orally, rectally, parenterally (intravenous, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions ancl sterile powders for reconstitution into steril~ injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and inj~ctable organic esters such as ~thyl oleate. Prop~r fluidity can be maintainHd, for example, by the use of a coating such as lecithin, by the maintenanc~ of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
W O 96/41638 PCT~US95/07463 Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glylcerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates o and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as forexample, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills and ~ranules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
Examples of embedding compositions which can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
W O 96/41638 PCT~US95/07463 Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the ~iquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimathylformamide, oils, in particular, cottonseed oil, ground-nut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid 10 esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, amulsifying and suspending agents, sweet~ning, flavoring and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixh~res of these substances, and the like.
Compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention wit suitable non-irritating excipients or carriers such as cocoa butter, 25 polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
Dosa~e forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Opthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
Actual dosage levels of the active ingredient in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered to 10 a host in single or divided doses may be in amounts, for example, of from about 0.5 mg to about 10 mg per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
-
DISEASES
RACKGROUNI~ OF THE INVENTION
Fiel~ of the Invention 0 This invention relates to a series of novel amino acid analogs whichexhibit s~lective inhibition of Calpain 1, to compositions containing the novel amino acid analogs and methods for therapeutic use. The Calpain I
inhibitors described in this invention comprise novel amino acid derivatives which possess particular utility in treatment of neurodegenerative diseas~s.
Re~orted I )eYeloDments Calpain is a cytosolic protease enzyme found in all mammalian tissue and cell types. There are two forms of the enzyme with different sensitivities to calcium; the high-sensitivity form, calpain 1, is activated by a low calcium concentration (2-75 IlM), and the low-sensitivity form, calpain ll, is activated by a higher calcium concentration (200-800 ,LM).
Although calpain l l is the prominant form, calpain I is concentrated in synaps~s and neuronal cell bodies and is thought to be involved in the phenomenon of long-term synaptic potentiation.
The location of active calpain explain how calpain can promote~
down-regulation of rnembrane-associated active protein kinase C; (2) formation of a calpain-activated soluble kinase; and (3) reorganization of the cytoskeleton (Melloni, E., and Pontremoli, S. (1989), The Calpains, Trencls Neurosci. 12, 438-44). Inactivation of the kinase results in repression of superoxide anion production, a process correlated to the protein kinase C-mediated phosphorylation of membrane proteins. Formation of a soluble, fully active kinase, operating in association with active calpain, results in ~ 35 selective modification in the organization of the cytoskeletal proteins, CA 0222472l l997-l2-l2 W O 96/41638 PCTrUS95/07463 which is correlated with the extracellular discharge of granule contents.
These conclusions have been reached by specific and direct inhibition of the proteinases, which results in: (1 ) a significant increase in superoxide anion production; (2) a marked decrease in the down-regulation of protein kinase C
activity; (3) reduced formation of calpain-activated protein kinase; (4) decreased phosphorylation and phosphorylation-mediated proteolytic degradation of cytoskel~tal proteins; and (5) inhibition of granule exocytosis.
In addition, studies of (Lee, K. S., Frank, S., Vanderklish, P., Arai, A., and Lynch, G. (1991), Inhibition of Proteolysis Protects Hippocampal Neurons from Ischamia, Proc. Nat. Ac~d. Sci. USA, 88, 7233) suggest that the inhibition of calpain may protect from various ischemia induced-neurodegeneration, essential hypertension, and benefits CNS disorders, and stroke.
A wide variety of apeptidylz analogs are reported to inhibit the action of proteases (Mehdi, Shujaath, Cell-Penetrating Inhibitors of Calpain, Tl PS, 16, 150 April 1991). These peptidyl analogs include: epoxisuccinates (E-64), leupeptin (CH3CO-Leu-Leu-ArgH),and ketopeptides. However, these inhibitors suffer from some of the following disadvantages:
weak enzyme specificity, lack of inhibitory potency, inhibit wide variety of proteases in addition to calpain 1, and multi-inhibition of various enzymes limits their therapeutic applicability.
A limited number of peptidyl methyl ketone analogs constitute a well-known class of compounds having enzymatic (papain, cathepsin B) inhibition activity. These analogs, however, are essentially devoid of potency and selectivity in inhibiting calpain 1.
In spite of various known calpain inhibitors, no effective therapy has yet been developed for the majority of ischemia-induced neurodegenerative diseases, CNS disorders, and stroke. Consequently, there is a need for therapeutic agsnts effective in the treatment and prevention of these diseases.
~:UMMARY OF THE INVENTION
Novel amino acid analogs are provided having the formula (I) Z--A3--A2--A1--Q ( ) wherein Z is H or a protecting group;
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either ~ orL stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalanine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH20COL, CH2OL, CH2SL, CH2X, NI~JNHCOCH20COL, NHNHCOCH20L, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, and a pharmaceutically acceptable salt thereof.
As used herein the following terms shall be understood to have the followin~ meanings, unless otherwise indicated.
"Alkyl" means a saturated or an unsaturated aliphatic hydrocarbon which may be either straight- or branched-chain. Preferred groups have no 25 more than about 12 carbon atoms and may be methyl, ethyl and structural isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
"Lower alkylH means an alkyl group as above, having 1 to 7 carbon 30 atoms. Suitable lower alkyl groups are methyl, ethyl, n-propyl, isopropyi, butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and n-heptyl.
"Aryl" means phenyl and substituted phenyl.
W O 96/41638 PCTnJS95/07463 "Substituted phenyl" means a phenyl group in which one or more of the hydrogens has been replaced by the the same or different substituents including halo, lower alkyl, ni~ro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, trifluoromethyl, morpholinoethoxy, morpholino-sulfonyl, and carbobenzoxy-methylsulfamoyl.
~ Heteroaryl" means pyridyl, pyrimidyl, tetrazolyl or thiadiazolyl.
"Substituted heteroaryl" means a heteroaryl group in which one or more 10 of the hydrogens has been replaced by the same or different substituents including halo, lower alkyl, nitro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, trifluoromethyl, morpholinoethoxy, morpholiho-sulfonyl, and carbobenzoxymethylsulfamoyl.
A "protecting group" is a radical attached to an oxygen, sulfur, or nitrogen atom, respectively, which radical serves to protect the oxygen, sulfur, or nitrogen functionally against undesired reaction. Such protecting groups are well known in the art, many are described in "The Peptidesn, E.
Gross and J. Meienhofer, Eds. Vol. 3 Academic Press. NY (1981).
The N-protecting groups can be N-acyl, N-alkoxycarbonyl, N-arylmethoxyGarbonyl and N-arylsulfonyl protecting groups.
Suitable O-protecting groups include benzyl, tert-butyl, methyl, tosyl ad carbobenzoxy groups.
S-protecting groups include methyl, tert-butyl, benzyl and carbobenzoxy groups.
Pharmaceutically acceptable salts include both acid and base addition salts. Pharmaceutically acceptable acid addition salt refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, W O 96/41638 PCT~US95/07463 nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyrubic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, and p-toluenesulfonic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically accceptable organic non-toxic bases include salts of 10 primary, secondary, and tertiary amines, substituted amines including naturally occuring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, dicyclohexylamine, Iysine, arginine, histidine, caffeine, procain, hydrabamine, choline, betaine, ethylendiamine, glucosamine, methylglucamine, theobromine, purines, peperiziner, piperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanol-amine, dicyclohexylamine, choline and caffeine.
This invention also contemplates pharmaceutically acceptable acid-addition salts of the compounds of Formula 1. It is well known in the pharmacological arts that nontoxic addition salts of pharmacologically active amine compounds do not differ in activities from their free base. All stereoisomers as well as optical isomers related to the novel calpain 25 inhibitory amino acid analogs described herein are also considered to be within the scope of this invention.
The amino acid analogs of the present invention are selective calpain inhibitors. More particularly, the amino acid analogs of the present 30 invention bind at the active site of the proteolytic enzyme, specifically calpain 1.
The present invention further provides pharmaceutical compositions comprised of the above-described novel amino acid analog inhibitors and W O 96/41638 PCT~US9S/07463 method of treating ischemia-induced neurode~enerative diseases, stroke, myocardial infarction, CNS disorders, and immunological diseases involving interleukin 1.
nETAlLFD DFSCRIPTION OF THE INVENTION
The compounds of the present invention are prepared by the general synthetic methods described in Schemes 1, 2 and 3.
-~cheme 1 H O ~J~3 (i) Ethyl ~, H :. ' .. "ale H ~ J~ 3 0~ NJI~N~OH 1I!3H3r-HOAC ~3 O~N--I~N~3r Formula 1 / Formula 2 ArOH or ArSH
O~N D~N~OJ~Ar ~3 o~N Il~N~oAr (i) H2; Pd/C Formula 3 Formula 4 (ii) Z-D-AlaOH; N-rr~thyl e; PyBOP
H H~COAr _ CBZ N~NJ~N~
Formula 5 Formula 6 ~:cheme 2 ~Or - HN ~sr (~)A¢X~H~ ~ ~ N ~ N ~O~Ar Formula 7 Formula 8 Scheme 3 Q ~, ~H~CH3 ~r H~OH
Fom~ula 9 Forrnula 10 The first step of this procedure involves the synthesis of N-protected dipeptidic bromomethyl ketone (formula 2). Methods for the preparation of various dipeptides (formula 1 ) are well established in the art. The N-protected dipeptide (formula 1), which in some cases is commercially available, is then converted to the corresponding bromoketone (formula 2) by way of hydrobromination or hydrohalogenation of a diazomethyl ketone intermediate. A displacement reaction of the bromomethyl or chloromethyl ketone by an aromatic carboxylic acid or alcohol (or thiol) then yields the desired arylcarboxymethyl ketone (formula 3) or aryloxy (or aryl-thio)methy ketone (formula 4) of the invention.
The N-protected dipeptidic arylcarboxymethyl ketone (formula 3) is deprotected by conventional hydrogenolysis and the resulting free amino g W O 96/41638 PCTrUS95/07463 dipeptide analog (formula 5) is readily converted to the corresponding tripeptidic arycarboxymethyl ketone (formula 6) under standard peptide coupling conditions as shown in Scheme 1.
The preparation of various amino acid N-arylcarboxyacetyl-hydrazides (for example formula 8) involves the synthesis of amino acid bromoacetyl hydrazide by reacting the corresponding amino acid hydrazide (formula 7) with a haloacyl halide. The resulting haloacyl-hydrazide is then readily converted to the arylcarboxyacetyl-hydrazide (formula 8) or aryloxyacetyl-10 hydrazide by coupling with arylcarboxylic acid or aryl alcohol respectively (Scheme 2).
The peptidic aldehydes (for example formula 10) of this invention are readily prepared by synthesizing the corresponding peptidic N-methoxy-N-15 methylamide analogs (for example formula 9) via standard synthesis followed by LAH reduction of the above amides.
The following examples will further illustrate the compounds of the present invention.
Fxaml~le 1 N-Ben7yloxyc~rhonyl-D-alanyl-L-leucyl-l-~henylalanine ~.6-rlifluorophenyl carboxymethyl ketone (a) ~I-Ren7yloxyc~rhonyl-l-leucyl-l-~henyl~l~nine bromomethyl ketone N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine (10.16 g, 24.63 mmol) was dissolved in dry THF (100 mL) under nitrogen. The solution was cooled to -1 5~C, N-methylmorpholine (2.98 mL 22.1 mmol) was added followed by dropwise addition of isobutyl chloroformate (3.35 mL, 25.86 mmol) over a 5 min period. A solution of dried diazomethane in ether (50 mmol in 100 mL
ether dried over Na2S O 4; from Diazald-Aldrich) was poured into the W O 9~/41638 PCTAUS95/07463 reaction mixture. The reaction mixture (-1 5~C) was allowed to slowly warm to 0~C after 1 hr, and then held 1 hr at room temperature.
The reaction mixture was cooled to 0~C, 47 mL of ~0% HBr/AcOH
added with stirring at 0OC, and the r~sulting mixture was transferred to a separatory funnel with 500 mL of water. The aqueous phase was extracted with ethyl acetate (3x) and the organic layer was washed successiv~ly with water, ~.3N KHSO4, saturated NaHCO3 solution, water, and brine. The organic layer was dried over MgSO4, filtered, and conc~ntrated to yield a 10 white solid which was recrystallized from dichloromethane/hexane to afford 10.35 g (86%) of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone.
(b) N-Ren7yloxyc~rbonyl-l-leucyl-l-,~henyl~l~nine ~.6-difluoro-rhenylcarboxymethyl ketone 2,6-Difluorobenzoic acid (65 mg. 0.41 mmol) was added to a solution of N-b~nzyloxycarbonyl-L-leucyl-L-phenylalanine bromo-methyl ketone 20 (200 mg, 0.41 mmol) and potassium fluoride in dry DMF under nitrogen. The reaction mixture was poured into ether and the organic layer was washed successively with water, 5% NaHCO3, water, and brine. The ether solution was dried over MgSO4 and concentrated lo afford a solid product which was recrystallized from ether/hexane to yield 165 mg (70%) of N-25 benzyloxycarbonyl-L leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, m.p. 1 08-9~C .
(c) I -L eucyl-l -phenyl~l~nine ~.6-difluorophenylc~rboxymethyl ketone To a mixture of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone (670 mg, 1.18 mmol) in anhydrous ethanol under nitrogen was added 10% palladium on carbon (67 mg), and the mixture was cooled to O~C. The nitrogen atmosphere was then replaced with hydrogen gas by equalizing with hydrogen supplied from a balloon.
W O 96/41638 PCT~US95/07463 When the atmosphere was exchanged for hydrogen, 6N HCI solution (0.39 mL) was added and the solution was allowed to stir for 1.5 hr at room temperature. The reaction mixture was filtered through celite and the filtrate was concentrated in vacuo to afford the hydrochloride salt of L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone.
(d) N-Ren7yloxyc~rbonyl-n-~lanyl-l-leucyl-l-Dhenylalanine ~.6-~ifluoroDhenylcarboxymethyl ketone To a mixture of L-leucyl-L-phenylalanine 2,6-difluoro-phenacyloxymethyl ketone hydrochloride (180 mg, 0.394 mmol; azeotroped with toluene), benzyoxycarbonyl-D-alanine (97 mg, 0.43 mmol), benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluoro-phosphate (225 mg, 0.43 mmol) was added under nitrogen 5 mL of dichloromsthane, and the resulting mixture was cooled to 0~C. N-Methylmorpholine (117 rng, 1.06 mmol) was added to the above mixture and the resulting reaction mixture was stirred for 30 min at 0~ C, and then stirred at room temperature overnight. The mixture was poured into water, extracted with ethyl acetate, and the organic layer was washed successively with 0.3N
20 KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2S O4 and concentrated in vacuo and the residue was purified by chromatography eluting with 30-50% ethyl acetate/hexane to afford 111 mg (45%) of N-benzyloxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-difluorophenacyloxymethyl ketone, m.p. 171-2~C.
Empioying the synthetic procedure described in Scheme 1 and Example 1 the following additional calpain inhibitors were synthesized.
W O 96/41638 PCTnUS95/07463 Example 2 Renzyloxycarbonvl-l-leucyl-L-~henylal~nine ~.6-dichloro-3-1~-rnor~?holino)-ethoxy~phenylcarboxymethyl ketone FYamnle 3 Renzyloxycarhonvl-l-leucyl-L-tyrosine ~.6-10~lichlorol~henylcarboxymethyl ketone FYamr~le 4 15R en~yloxycarb onyl-l -~rolyl-L-leucyl-L-~henylalanine 2.6-dichloronhenylcarboxymethyl ketone FYamrle 5 Perllzyloxycarhonyl-~-leucyl-~lvcine 2.6-dichloro-3-(nnor~holinos~!fonyl)~?henylc~rboxymethyl ketone 25Fxample 6 ~enzyloxycarbonyl-L-leucyl-L-~henylalanine 2.6-dichloro-3-(mor~holinosulfonyl)~henylc~rboxymethyl ketone Example 7 Benzyloxycarbonyl-~lycyl~L-leucvl-L-phenylalanine 2~6-difluoro-~henylcarboxymethyl ketone CA 0222472l l997-l2-l2 W O 96/41638 PCTnJS9~/07463 F X a m ple 8 Ren7yloxycarhonyl-~-leucyl-!-tyrosine ~.6-dichloro-3-(mor~holino-sulfonyl)phenvlcarboxymethyl ketone ~ FYam~le 9 10 Ren7yloxycarhonyl-l-leucyl-L-alanine 2.6-dichloro-3-(mor~holino-sulfonyl)phenylcarboxymethyl ketone ~Yz~ml?le 10 15 Ren7yloxycarhonyl-L-Ie~lcyl-L-~henylalanine 2.6-dichlorophenyl-carboxymethyl ketone Example 1 1 Ben7yloxycarbonyl-L-valvl-L-phenylalanine 2.6-dichlorophenyl-carboxymethyl ketone 25 Example 12 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone Exam~le 13 Tert-RI-tyloxycarbonyl-l-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone W O 96/41638 PCT~US95/07463 Fx~ m ~le 14 Renzyloxycarhony~ le~cyl-l -tyrosine ~.6-5~ifluororhenylc~rboxymethyl ketone ., ~Y~m~?le 15 10Renzyloxyc~rhonyl-l-leucyl-l-~lycine 2.6-dichloroDherylcarboxymethvl ketone Ex~mple 1 6 Ren7yloxvcarhonvl-l-le~cyl-L-~lycine 3.6-dichloro-2-~cetAmido-~henylcarboxymethvl ketone Fxaml~le 17 ~?-Toluenesulfonyl-L-leucyl-L-phenylalanine 2.6-~ifluorophenylcarboxymethyl ketone Fxam~le 18 Renzyloxycarbonyl-L-leucyl-L-~henvlalanine 2.6-dimethylphenylcarboxymethyl ketone Fxample 1 9 Renzyl oxyca rbonyl-l -leu cyl- L-g Iyc i ne 2-acetam ido-6-chloroDhenyl-carboxymethyl ketone W O 96/41638 PCT~US95/07463 FYa m ple 20 Ben~yloxycarbonyl-L-leucyl-l-alanine 2-acetamido-6-chlorophenyl-carhoxymethyl ketone FY~mple ~1 10Ren7yloxycarbQnyl-l-N-methylleucyl-L-I?henylalanine 2.6-difluoronhenylcarboxymethyl ketone Exam~le 22 15Ren~yloxycarbonyl-l-N-methylleucyl-L-phenylalanine 2.6-~ichloro-3-~2-(mor~holino)ethoxy]phenylcarboxymethyl ketone FYample 23 ~en7yloxycarbonyl-L-valyl-L-phenylalanine 2-acetamido-6-chlorovhenylcarboxymethyl ketone 25Example 24 Renzyloxycarbonyl-L-N-methylleucyl-L-phenylalanine 2.6-dichloro-3-(morpholinosulfonyl)phenylcarboxymethyl ketone FXa m ple 25 Benzyloxycarbonyl-L-Ie--cyl-l.-Dhenylalanine 2.6-dichloro-3-(c~rbohenzoxymethylsulfamoyl)phenylcarboxymethyl ketone W O 96/41~38 PCT~US95/07463 FYample ?6 ~enzyloxycarhonyl-l -leucyl-L-alanine 2.6-dich loro-3-- 5(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone FYample 27 10Ren7yloxycarbonyl-l-le~3cyl-l-alanine ~.6-dichloro-3-r~-(morpholino)etho~y]Dhenylcarboxymethyl ketone Fxamrle 28 15Renzyloxyc~rhonyl-L-leucyl-!-alanine ~.6-tlimethoxy~henylcarboxymethvl ketone Fxamgle 29 Benzyloxycarbonyl-L-leucyl-L-alanine 2.6-chloroDhenylcarboxymethyl ketone 25F~ample 30 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2-acetamido-6 chlorophenylcarboxymethyl ketone Fxample 31 Renzyloxycarbonyl-l-leucyl-L-glycine 2-acetamido-3.6-dichlorophenyl-carboxymethyl ketone ' 35 -Fxample 32 RenzyloxycarbQnyl-L-leucyl-L-~henylalanine 2-5~yridylcarboxymethyl ketone Exam~le 33 10Benzyloxyc~rbonyl-L-leucyl-L-~lycine 2.6-fluorophenylcarboxymethyl ketone Example 34 15Renzyloxycarbonyl-L-leucyl-L-alanine 2.6-difluoroDhenylcarboxymethyl ketone Fxample 35 13enzyloxycarbonyl-L-valyl-L-alanine 2.6-histrifluoromethylphenyl-carboxymethyl ketone FYa m ~le 36 p-Nitrobenzvloxycarbonyl-L-leucyl-L-phenylalanine 2.6-difluorophenyl-carboxymethyl ketone Example 37 Renzyloxycarbonyl-L-leucyl-L-phenylalanine 1-na~hthylcarboxymethyl ketone , Ex~mple 38 Ren7ylo%ycarbonyl-l-leucyl~ henylalanine 2.6-dichloro-3-~ s hen7yloxy~henylcsrboxymethvl ketone FYaml?le 39 N-Benzyloxycarlbonyl-L-leucyl-N-(2.6-dichloro~henvlcarboxyacetyl)hydrazide To a solution of N-benzyloxycarbonyl-L-leucyl-N-(bromoacetyl) hydrazide (50 mg, 0.12 mmol) and 2,6-dichlorobenzoic acid (29 mg, 0.15 mmol) in dry DMF (5 mL) was added potassium fluoride (18 mg) in one portion. The resulting mixture was poured into water, extracted with ether, and the organic layer was washed successively with water, 5% NaHCO3, water, and brine. The organic layer was dried over MgSO4 and concentrated to afford 56 mg (88%) of N-benzyloxycarbonyl-L-leucyl-N-(2,6-20 dichlorophenylcarboxyacetyl) hydrazide, m.p. 1 03-5~C .
Employing the synthetic procedure described in Example 39, the following compounds were made.
Fxam~le 40 N-Benzvloxycarbonyl-L-leucyl-N-methyl. N-(2-acetamido-6-chlorophenycarboxy-acetyl)hydrazide IFxam~le 41 N-Benzvloxycarbonyl-l -leucyl-N-(2-acetamido-6-chloro~henylcarboxy-acetyl)hydrazide -CA 0222472l l997-l2-l2 WO 96/4~638 PCT~US95/07463 Exam~le 4~
Renzyloxycarhonvl-!-leucyl-l-tyrosine 2.6-dichloro-3-r2-(mor~holino)ethoxyl~henylcarboxymethyl ketone FYam~le 43 Methoxycarbonyl-D-alany~ eucy~ henylalanine 2.6-ichloro-3-r2-(morpholino)ethoxy~phenylcarboxymethyl ketone Fxaml?le 44 Ren7yloxycarbonyl-D-~l~nyl-l-leucyl-L-tyrosine ~.6-~ichloro-3-r~-(morpholino)ethoxyll~henylcarboxymethyl ketone Fxample 45 Renzyloxycarbonyl-l-valyl-l-~henylalanine 2.6-dichloro-3-U-(mor~holino)ethoxylphenylcarboxymethyl ketone FYaml~le 46 Benzyloxycarbonyl-L-valyl-plycine 2.6-dichloro-3-(carbobenzoxy-methylsulfamoyl)phenylcarboxymethyl ketone FXa m Dle 47 Benzyloxycarbonyl-L-leucvl-L-alanine 2.6-dichloro-3-r2-(mor~holino)-ethoxylDhenylcarboxymethyl ketone W O 96/41638 PCT~US95/07463 ~Ya m ~le 48 Ben~yloxycarbonyl-l-leucyl-glycine 2.6-dichloro-3-r2-(mor~holino)-ethoxylghenylcarboxymethyl ketone FYam~le 49 Methoxycarbonyl-P-alanyl-3-leucyl-l-~?henylalanine ~.6-difluorophenylcarhoxymethyl ketone FYample 50 en~yloxycarbonyl-l alanyl-L-~lycine 2.6-dichloro-3-(carbohen7oxy-methylsulf~moyl)Dhenylcarboxymethyl ketone Example 51 Renzyloxycarbonyl-glycyl-L-~henylalanine 2.6-dichloro-3-(c~rhohenzoxymethylsulfamoyl)~henylcarboxymethyl ketone 2s Fxample 52 Benzyloxycarbonyl-L-valyl-glycine 2.6-dichlorophenylcarboxymethyl ketone Fxample 53 penzyloxycarbonyl-Qlycvl-l-Dhenylalanine 2.6-dichlororhenYlcarboxymethyl ketone W O 96/41638 PCT~US95/07463 Example 54 E~en7yloxycarbonyl-L-phenylalanyl-L-alanine 2.6-dichloro-3-12-(morl?holino)ethoxylphenylcarboxymethyl ketone ~Yample 55 Renzyloxvcarhonyl-l-~henylalanyl-glycine 2.6-dichlorophenylcarboxymethyl ketone FYam~?le 56 Ren7ylQxyc~rbonyl-D-alanyl-L-leucyl-~lycine 2.6-dichloro-3-r2-15(mor~holino)ethoxyl~henylcarboxymethyl ketone Fxample 57 20Benzyloxycarbonvl-L-leucyl-glycine 2.6-dichlorophenylcarboxymethyl ketone Fxample 58 Benzyloxycarbonyl-L-phenylalanyl-glycine 2.6-dichlorophenylcarboxymethyl ketone FxamPle 59 Benzyloxycarbonvl-L-alanyl-glycine 2.6-dichloro~henylcarboxymethyl ketone W O 96/41638 PCT~US9~/07463 Fxam~le 60 Ren~yloxycarbonyl~ henylalanyl-L-alanine 2.6-~i trifluoromethyl~henylcarboxymethyl ketone Fxaml?le 61 N-Ren7yloxyc~rhonyl-l-lel~cyl-!-c~henylalanine ~.6-~ichlorophelloxymethyl ketone To a solution of benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone (100 m~, 0.204 mmol), 2,6-dichlorophenol 34 mg, 0.204 mmol) and K2CO3 (29 mg, 0.204 mmol) in 8 mL of DMF was added 15 tetra-n-butyl-ammonium iodide (8 mg) and the resulting mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate, washed with water and brine, and the organic layer was dried over Na2SO~. The solvent was concentrated in vacuo to afford 80 mg of N-benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichlorophenoxymethyl ketone, as a white solid, m.p. 102-4~C.
Ernploying the synthetic procedure described in Example 61 and Scheme 1 the following additional calpain inhibitors were synthesized.
FYam6~1e 62 N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-r1-(3-~?yridyl)tetrazolyllthiomethyl ketone Fxample 63 N-~en7yloxycarbonyl-L-leucyl-L-phenylalanine 2-r(4-rnorpholinoethyl)~tetrazolyllthiomethyl ketone W O 96/41638 PCTrUS95/07463 FYamrle 64 N-Penzyloxycarbonyl-!-lel~cyl-l-nhenylalanine 2-r(s-5methylthio)tetra701yllthiomethyl ketone FYamrle 65 10N-Benzyloxycarbonyl-L-leucyl-l-rhenylalanine ~-r(s-methylthio)tetr~zolyllthiomethyl ketone FYS~mple 66 N-~enzyloxycarhonyl-l-leucyl-l-~henylalanine 2.6-~1ifl--orol~henylthiomethyl ketone 20FYample 67 N-Renzyloxyc~rhonvl-l-valyl-l-phenylalanine ~.6-~lifluorophenoxymethyl ketone Example 68 N-Renzyloxycarbonyl-L-leucyl-L-phenvlalanine 2-pyrimidylthiomethyl ketone FYa m ple 69 N-Renzyloxycarbonyl-L-leucyl-L-phenylalanine 2-(1-phenyl)-35tetrazolylthiomethyl ketone W O 96/41638 PCT~US95/07463 To a solution of benzyloxycarbonyl-L-leucyl-L-phanylalanine bromomethyl ketone (150 mg, 0.306 mmol) and 2-mercapto-phenyl tetrazole (57.2 mg, 0.32 mmol) in 2 mL of DMF was added K2C03 ( 42.3 nng, 0.306 mmol) at room temperature and the resulting reaction mixture was stirred overnight. The mixture was poured into 50 mL of water and then extracted with ethyl acetate. The organic layer was washed with 0.3N
KHSO4, 5% NaHCO3, water, and brine and dried over Na2SO4. The solvQnt was concentrated fn vacuo to afford 168 mg (94%) of N-benzyloxycarbonyl-10 L-leucyl-L-phenylalanine 2-(1-phenyl)-tetrazolylthio-methyl ketone, as a white solid, m.p. 1 83-4~C .
Exam~le 70 ~enzyloxycarbonyl-L-leucyl-L-tyrosinal Benzyloxycarbonyl-L-leucyl-L-tyrosyl-N-(methoxy),N-methyl amide (0.182 mmol) was dissoved in 4 mL of ether/THF (1 :1 ) under nitrogen and the solution was cooled to OoC. LAH ether solution (0.182 mmol) was adde by syrin~e to ~he reaction mixture with stirring. The reaction mixture was quenched with 0.3N KHSO4 (0.6 mL) and the mixture was transferred into a separatory funnel containing 50 mL of water and 50 mL of ether/ethyl acetate (1:1). The aqueous layer was extracted with ether/ethyl acetate and the combined organic layer was washed with 0.3N KHSO4, water, and brine. The or~anic solution was dried over Na2SO4 and concentrated in vacuo to afford 53 mg (70.6%) of benzyloxycarbonyl-L-leucyl-L-tyrosinal, m.p. 57-60~C.
Employing the synthetic procedure described in Scheme 1, Scheme 2 and Scherne 3 the following additional calpain inhibitors were prepared.
Fxample 71 Benzyloxycarbonyl-L-valyl-L-tyrosinal -2~-Fxamnle 7~
5IRen~vloxycarbonyl-L-Ieucyl-L-O-methyl-tyrosinal FYam~le 73 10Ren~yloxyc~rhonyl-3-le~cyl-L-~henylalaninal FXa m ~le 74 15Benzyloxycarbonyl-L-isoleucyl-L-tyrosinal Fxample 75 20R e nzylo xyc arbo nyl-L-valyl-~ -2-(2-nap hthylm ethyl)~lycin ~1 Exam p le 76 25B en7ylO xycarh O n yl-L-isole U Cyl-L-p hen ylalaninal Exam~le 77 30R e n~ylo xycarb O n yl-L-valyl-D L-2-(p he nethyl)~ly~i FYa m ple 78 35Benzyloxycarbonyl-L-2-neopentyl-glycyl-L-phenylalaninal CA 0222472l l997-l2-l2 W O 96/~1638 PCTAUS95/07463 FYa m ple 79 R e n~ylo xyc~rb O nyl-l -valyl-DI -2-(1-na D hthylm ethyl)~lyci Ex~m~?le 80 10Ren7yloxycarbonyl-l~-phenylQlycyl-l-phenylal~nin~l IFxam~le 81 15Benzyloxycarbonyl-L-alanyl-L-~henylalaninal ~Ya m ple 82 20R en~ylo xycarbon yl~ henethyl~lycvl-L-~ hen ylalani IFxamr~le 83 25R e nzyloxvcarbon yl-L-rhenylalanyl-L-p hen Yl31ani ~Ya m ~le 84 30P en7y13 xycarb O n yl-L-2Dtert-b utylglycyl-L-p he n ylalani W O 96/41638 PCTnJS95/07463 Exam~le 85 Ren7yloxycarhonyl-~ -2-(1 -na~hthymethyl)~lycyl-nl -~henyl~laninal FYample 86 Ren7yloxycarhonyl-l -leucyl-N-chloroacetyl-hydr~zi~e ~xample 87 Ben~yloxycarbonyl-l -lel~cyl-N-bromoacetyl-hydrazkle Fxaml?le 88 Benzyloxycarbonyl-L-leucine chloromethyl ketone ExamDle 89 Ren7yloxycarbonyl-L-leucyl-L-leucyl-L-~henylalanine 25chloromethyl ketone Exam~le 90 Benzyloxycarbonyl-L-leucyl-l-alanine chloromethyl ketone Example 91 Benzyloxyc:arbonyl-L-lellcyl-l-~henylalanine chloromethvl ketone Fxa m ~le 92 Ren;~yloxycarbonyl-~lycyl-L-leucyl-L-tyrosine chloromethyl ketone FYa m Ple 93 o Ren7vloxvcArhonyl-!-le~cyl-l-rhenylal~nine chloromethyl ketone FYample 94 Rer~7yloxycarbonyl-L-leucyl-~lycine chloromethyl ketone FYarn~le 95 Ren7yloxycarbonyl-L-le~ncyl-L-alanine bromomethyl ketone FYa m ple 96 Renzyloxycarbonyl-L-valyl-l-phenylalanine bromomethyl ketone FYa m ple 97 Benzyloxycarbonyl-L-leucyl-L-leucine bromomethyl ketone FxamPle 98 Benzyloxycarbonyl-L-as~?aragyl-L-phenylalanine chloromethyl keto ne -CA 0222472l l997-l2-l2 FxamDle 99 Renzyloxyc~rhonyl-L-le~cyl-L-~henylalanine bromomethyl ketone FYam~le 1 OQ
Ren7yloxyc~rhonyl-L-~henylalanyl-L-~Ianlne chloromethyl ketone FYam~le 1 01 Renzyloxyc~rbonyl-~lycyl-L-~henylalanine bromomethyl ketone ~xannDle 102 E~enzyloxycarbonyl-L-valyl-glycine bromomethyl ketone FYa m Dle 103 Ren7yloxycarbonyl-L-leucine ch loromethyl ketone Exam~le 1 04 Ren7yloxyc~rbonyl-L-~?henylalanyl-L-alanine bromomethyl ketone Example 1 0~
Benzyloxycarbonyl-L-alanyl-glycine bromomethyl ketone Ex~m~le 1 06 Ren7yloxyc~rhonyl-1-2-~-n~ghthvlmethyl) cllycine chloromethyl keto ne FYaml~le 1 07 Ren7yloxycarbonyl-L-phenylalanyl-~lycine chloromethyl ketone FxamDle 1 08 Ren7yloxyc~rhonyl-l-~?henylal~nyl-L-~henylalanine chloromethyl 15keto ne ExamE~le 1 09 20Renzyloxycarbonyl-L~leucyl-N-(bromoacyl) hydrazide E~xam~le 11 0 25Ren~yloxycarbonyl-L-leuGyl-l-tyrosine bromomethyl ketone Compounds of the present invention were tested for calpain I inhibition activity using the following assay method.
30Cal~ain I InhibitionAssay Isol~tion of Hum~n erythrocyte C~in I
Human red blood cells were obtained from the Northeastern New York Chapter of the American Red Cross. The isolation of calpain from hurnan W O 96/41638 PCT~US95/07463 erythrocytes was similar to that described by Wang et al. (1988). One unit of in-dated packed red cells was diluted with an equal volume of diluting/wash solution and centrifuged. The supernatant was removed and the procedure was repeated. The washed cells were pooled, Iysed with 700 mL of Iysin~ solution and centrifuged to remove cell debris. The membrane-free hemolysate was added to 500 mL DEAE-sephacel and the slurry was stirred gently at 4~C for 1 hour.
Batch elution was done using DEAE-sephacel wash solution to remove a 10 lar~e amount of unwanted protein, most of which was hemoglobin. The slurry was poured into a column connected in tandem to a phenyl-sepharose CL-4B column. Material eluted from the DEAE-sephacel was applied directly to the phenyl-sepharose CL-4B. The phenyl-sepharose CL-4B column was washed first with 75 mM NaCI and then with no salt. Calpain begins to ~is~ssociate from the DEAE-sephacel with the 75 mM NaCI but the majority should adhere to the column until the salt is removed. Fractions were collected (20 mL), assayed for caseinolytic activity with and without c~lp~statin and pooled accordingly. The pooled fractions were concentrated usin~ an Amicon stirred cell equipped with a YK-10 membrane. Calpain was stored at 4~C with 10 mM EDTA and ~ mM 2-mercaptoethanol and is stable for at l~ast 6 months.
W O 96/41638 PCT~US95/07463 A~say Procedl-re The tritated assay is a modification of that described by Gopalakrishna, R. and Barsky, S.H., An~l. Riochem., 148, 413,1985. All r~agents, compound 25 ul, HEPES buffer 25 ul, CaCI2 50 ul, enzyme 50 ul, and 3H-acetyl Casein, were combined in 1 mL polystyrene titer plates. The plates were preincubated at 25~C for 5 min with gentle shaking prior to the addition of substrate. The incubation was continu~d for an additional 2 hours and was terminated with the addition of 0.5 mL ice cold 5% TCA.
Unlabled casein was added, samples were centrifuged and 0.5 mL of the supernatant was counted in 5 rnL of Ready Protein liquid scintillation cocktail for 2 min. This assay measures 3H-acetyl Casein degradation as an endpoint for caipain activity.
Representative assay results are shown in the following tables.
W O 96/41638 PCT~US95/07463 Tsble 1 Acyloxyketone Calpain I Inhibitors Ex. Z A3 A2 Al Q lCso/uM
CBZ D-Ala L-Leu L-Phe 2,6-difluoropheny .046 2 CBZ - L-Leu L-Phe 2,6-dichloro-3-[2- 0.14 0 (morpholino) ethoxy]phenyl 3 CBZ - L-Leu L-Tyr 2,6-dichlorophenyl 0.22 4 CBZ L-Pro L-Leu L-Phe 2,6-fluorophenyl 0.08 CBZ - L-Leu Gly 2,6-dichloro-3- 0. 11 (morpholinosulfonyl)phenyl 6 CBZ - L-Leu L-Phe 2,6-dichloro-3- 0.17 (morpholinosulfonyl)phenyl 7 CBZ Gly L-Leu L-Phe 2,6-difluorophenyl 0.04 8 CBZ - L-Leu L-Tyr 2,6-dichloro-3 0.17 (morpholinosulfonyl)phenyl 9 CBZ - L-Leu L-Ala 2,6-dichloro-3- 0.43 (morpholinosulfonyl)phenyl CBZ - L-Leu L-Phe 2,6-dichlorophenyl 0.33 11 CBZ - L-Val L-Phe 2,6-dichlorophenyl 0.55 12 CBZ - L-Leu L-Phe 2,6-difluorophenyl 0.16 13 CtBu - L-Leu L-Phe 2,6-difluorophenyl 0.42 14 CBZ - L-Leu L-Tyr 2,6-difluorophenyl 0.40 CBZ - L-Leu Gly 2,6-dichlorophenyl 0.29 16 CBZ - L-Leu Gly 3,6-dichloro-2- >10 acetamidophenyl 17 Tos - L-Leu L-Phe 2,6-difluorophenyl 0.16 18 CME - L-Leu L-Phe 2,6-dimethylphenyl 0.63 19 CBZ - L-Leu Gly 2-acetamido-6- 0.78 chlorophenyl CBZ - L-Leu L-Ala 2-acetamido-6- 0.36 chlorophenyl W O 96/41638 PCTrUS95/07463 Table ~
Aryloxyketone Cal~in I Inhibitors ~ 5 Ex. Z A3 A2 Q IC50/u M
61 CBZ L-Leu L-Phe 2,6-dichlorophenoxy 2.3 62 CBZ L-Leu L-Phe 2-[1-(3-pyridyl)tetrazoyl] 0.53 thio 63 CBZ L-Leu L-Phe 2-[(4-morpholinoethyl) 3.8 0 tetr~olyl]thio 64 CBZ L-Leu L-Phe 2-[(5-methylthio)thi~ 7Oyl] 2.0 thio CBZ L-Leu L-Phe 2,6-difluorophenoxy >10 66 CBZ L-Leu L-Phe 2,6-dichlorophenylthio >10 67 CBZ L-Val L-Phe 2,6-difluorophenoxy ~10 68 CBZ L-Leu L-Phe 2-pyrimidylthio >10 69 CBZ L-Leu L-Phe 2-(1 -phenyltetrazoyl)thio >10 Table 3 PeE~tide Aldehyde Cal~ain I Inhibitor~
Z-A2-Al - H
Ex. Z A2 Al IC50/ u M
CBZ L-Leu L-Tyrosinal 0.02 71 CBZ L-Val L-Tyrosinal 0.026 72 CBZ L-Val L-Tyrosinal(O-methyl) 0.03 73 CBZ L-Leu L-Phenylalaninal 0.037 74 CBZ L-lle L-Tyrosinal 0.053 CB Z L-Val DL-2-(2-Naphthy 0.07 methyl)glycinal 76 CBZ L-lle L-Phenylalaninal 0.08 77 CBZ L-Val DL-2-(Phenethyl)glycinal 0.10 78 CBZ L-2-(Neopentyl) L-Phenylalaninal 0.10 Glycyl W O 96/41638 PCT~US95/07463 T~hle 3(contd.) Peptide Aldehyde Cal~ain I Inhibitors Z-A2-Al - H
Ex. Z A2 A1 lCsol u M
79 CBZ L-Val DL-2-(1 -Naphthyl- 0. 11 methyl)glycinal 0 80 CBZ 2-Phenylglycyl L-Phenylalaninal0.11 81 CBZ L-Ala L-Phenylalaninal 0.17 82 CBZ L-2-(Phenethyl) L-Phenylalaninal 0.27 Glycyl 83 CBZ L-Phe L-Phenylalaninal 0.41 T~hle 4 H~loketone Cal~in I Inhibitors Ex. A3 A2 Al X IC50/u M
86 - - L-Leu-NHNHCO Cl 2.2 87 - - L-Leu-NHNHCO Br 6.8 88 - - L-Leu Cl >10 89 L-Leu L-Leu L-Phe Cl >10 - L-Leu L-Ala Cl >10 91 L-Leu L-Phe Cl 43.3 92 Gly L-Leu L-Phe Ci 6.6 93 - L-Leu L-Tyr Cl 40 94 - L-Leu L-Phe Cl >10 - L-Leu L-Ala Br >10 96 - L-Val L-Phe Br >10 97 - L-Leu L-Leu Br >10 98 - L-Asp(NH2) L-Phe Cl >10 99 - L-Leu L-Phe Br 9.1 W O 96/41638 PCT~US95/07463 The present invention includes a calpain inhibitor of this invention formulat~d into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are ~ 5 collectively referred to her~in as carriers, for parentcral injection or oral administration, in solid or liquid form, for rectal or topical administration, or the like.
Th~ compositions can be administered to humans and animals either 10 orally, rectally, parenterally (intravenous, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions ancl sterile powders for reconstitution into steril~ injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and inj~ctable organic esters such as ~thyl oleate. Prop~r fluidity can be maintainHd, for example, by the use of a coating such as lecithin, by the maintenanc~ of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
W O 96/41638 PCT~US95/07463 Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glylcerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates o and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as forexample, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills and ~ranules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
Examples of embedding compositions which can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
W O 96/41638 PCT~US95/07463 Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the ~iquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimathylformamide, oils, in particular, cottonseed oil, ground-nut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid 10 esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, amulsifying and suspending agents, sweet~ning, flavoring and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixh~res of these substances, and the like.
Compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention wit suitable non-irritating excipients or carriers such as cocoa butter, 25 polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
Dosa~e forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Opthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
Actual dosage levels of the active ingredient in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered to 10 a host in single or divided doses may be in amounts, for example, of from about 0.5 mg to about 10 mg per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
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Claims (42)
1. A compound of the formula (I) Z~A3~A2~A1~Q (I) wherein Z is H or a protecting group;
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either D or L stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, or a pharmaceutically acceptable salt thereof.
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either D or L stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 wherein L is substituted aryl selected from the group consisting of phenyl or naphthyl optionally substituted by 1 to 3 substituents selected from the group consisting of lower alkyl, lower alkoxy, halo, acetyl, acetamido, hydroxy, phenyl, morpholino-lower alkyloxy, morphorino lower alkyl, benzyl, benzyloxy, nitro, amino, loweralkylamino, morpholinosulfonyl, morpholinosulfamoyl, benzyloxycarbonyl-methylsulfamoyl, acetylamino or trifluoromethyl.
3. The compound of claim 1 wherein L is substituted heteroaryl selected from the group consisting of thiazole, furan, thiadiazole, thiophen, tetrazole, pyridyl, pyrimidyl, triazole optionally substituted by 1 to 3 substituents selected from the group consisting of lower alkyl, lower alkoxy, halo, acetyl, acetamido, hydroxy, morpholino-lower alkyloxy, morphorino lower alkyl, benzyl, benzyloxy, nitro, amino, loweralkylamino, morpholinosulfonyl, morpholinosulfamoyl, benzyloxycarbonyl-methylsulfamoyl, acetylamino, phenyl or trofluoromethyl .
4. The compound of claim 1 selected from the group consisting of:
N-Benzyloxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2,6-dichloro3-[(2-morpholino) ethoxy]
phenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-prolyl -L-leucyl-L-phenylalanine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine 2,6-dichloro -3-(morpholinosulfonyl) phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro -3-(morpholinosulfonyl)phenyl carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-phenylalanine 2, 6-difluoro-phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-(morpholinosulfonyl)phenylcarboxymeethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro -3-(morpholinosulfonyl)phenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone.
N-Benzyloxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2,6-dichloro3-[(2-morpholino) ethoxy]
phenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-prolyl -L-leucyl-L-phenylalanine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine 2,6-dichloro -3-(morpholinosulfonyl) phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro -3-(morpholinosulfonyl)phenyl carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-phenylalanine 2, 6-difluoro-phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-(morpholinosulfonyl)phenylcarboxymeethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro -3-(morpholinosulfonyl)phenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone.
5. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-valyl-L-phenylalanine 2, 6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Tert-Butyloxycarbonyl -L-leucyl-L-phenylalanine 2,6-difluorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-glycine 3,6-dichloro-2-acetamido-phenylcarboxymethyl ketone, p-Toluenesulfonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dimethylpheryl carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido -6-chlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L
-leucyl-L- glycine 2-acetamido-6-chlorophenyl-carboxymethyl ketone.
Benzyloxycarbonyl-L-valyl-L-phenylalanine 2, 6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Tert-Butyloxycarbonyl -L-leucyl-L-phenylalanine 2,6-difluorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-glycine 3,6-dichloro-2-acetamido-phenylcarboxymethyl ketone, p-Toluenesulfonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dimethylpheryl carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido -6-chlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L
-leucyl-L- glycine 2-acetamido-6-chlorophenyl-carboxymethyl ketone.
6. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalaninee 2, 6-difluorophenylcarboxymothyl ketone, Benzyloxycarbonyl-L
-N-methylleucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino) ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl -L-phenylalanine 2-acetamido-6-chlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalaninee 2,6-dichloro -3-(morpholinosulfonyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-phenylalanine 2,6-dichloro -3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro -3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3 -[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-alanine 2,6-dimethoxyphenyl carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-acetamido -6-chlorophenylcarboxymethyl ketone.
Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalaninee 2, 6-difluorophenylcarboxymothyl ketone, Benzyloxycarbonyl-L
-N-methylleucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino) ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl -L-phenylalanine 2-acetamido-6-chlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalaninee 2,6-dichloro -3-(morpholinosulfonyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-phenylalanine 2,6-dichloro -3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro -3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3 -[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-alanine 2,6-dimethoxyphenyl carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-acetamido -6-chlorophenylcarboxymethyl ketone.
7. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido-3, 6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2-pyridylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-glycine 2,6-fluorophenylcarboxy-methyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-alanine 2, 6-bistrifluoromethylphenylcarboxymethyl ketone, p-Nitrobenzyloxycarbonyl-L-leucyl-L-phenylalanine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 1-naphthylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-benzyloxyphenylcarboxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-N-(2,6-dichlorophenylcarboxyacetyl)hydrazide and N-Benzyloxycarbonyl-L-leucyl-N-methyl, N-(2-acetamido-6-chlorophenylcarboxy-acetyl)hydrazide.
Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido-3, 6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2-pyridylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-L-glycine 2,6-fluorophenylcarboxy-methyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-alanine 2, 6-bistrifluoromethylphenylcarboxymethyl ketone, p-Nitrobenzyloxycarbonyl-L-leucyl-L-phenylalanine 2, 6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 1-naphthylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-benzyloxyphenylcarboxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-N-(2,6-dichlorophenylcarboxyacetyl)hydrazide and N-Benzyloxycarbonyl-L-leucyl-N-methyl, N-(2-acetamido-6-chlorophenylcarboxy-acetyl)hydrazide.
8. The compound of claim 1 selected from the group consisting of:
N-Benzyloxycarbonyl-L-leucyl-N-(2-acetamido-6-chlorophenyl-carboxy-acetyl)hydrazide, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)-ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)-ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichloro-3-(carbobenzoxy-methylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenyl-carboxymethyl ketone, Benzyioxycarbonyl-L-leucyl-glycine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-alanyl-L-glycine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)-phenylcarboxymethyl ketone.
N-Benzyloxycarbonyl-L-leucyl-N-(2-acetamido-6-chlorophenyl-carboxy-acetyl)hydrazide, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)-ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)-ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichloro-3-(carbobenzoxy-methylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenyl-carboxymethyl ketone, Benzyioxycarbonyl-L-leucyl-glycine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-alanyl-L-glycine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)-phenylcarboxymethyl ketone.
9. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-glycyl-L-phenylalanine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-glycine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-alanyl-glycine 2,6-dichlorophenyl carboxymethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-bistrifluoromethylphenylcarboxymethyl ketone.
Benzyloxycarbonyl-glycyl-L-phenylalanine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-glycine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-alanyl-glycine 2,6-dichlorophenyl carboxymethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-bistrifluoromethylphenylcarboxymethyl ketone.
10. The compound of claim 1 selected from the group consisting of: N-Benzyloxycarbonyl-L-leucyl-L-phanylalanine 2,6-dichlorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[1-(3-pyridyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[(4-morpholinoethyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyi-L-phenylalanine 2-[(5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[(5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylthiomethyl ketone, N-Benzyloxycarbonyl-L-valyl-L-phenylalanine 2,6-difluorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-pyrimidylthiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2(1-phenyl)tetrazolylthiomethyl ketone and Benzyloxycarbonyl-L-leucyl-L-tyrosinal.
11. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-valyl-L-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-O-methyl-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-phenylalaninal, Benzyloxycarbonyl-L-isoleucyl-L-tyrosinal, Benzyloxycarbonyl-L-valyl-DL-2-(2-naphthylmethyl)glycinal, Benzyloxycarbonyl-L-isoleucyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(phenethyl)glycinal, Benzyloxycarbonyl-L-2-neopentyl-glycyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(1-naphthylmethyl)glycinal and Benzyloxycarbonyl-L-2-phenylglycyl-L-phenylalaninal .
Benzyloxycarbonyl-L-valyl-L-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-O-methyl-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-phenylalaninal, Benzyloxycarbonyl-L-isoleucyl-L-tyrosinal, Benzyloxycarbonyl-L-valyl-DL-2-(2-naphthylmethyl)glycinal, Benzyloxycarbonyl-L-isoleucyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(phenethyl)glycinal, Benzyloxycarbonyl-L-2-neopentyl-glycyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(1-naphthylmethyl)glycinal and Benzyloxycarbonyl-L-2-phenylglycyl-L-phenylalaninal .
12. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-alanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-phenethylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-tert-butylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-(1-naphthymethyl)glycyl-DL-phenylalaninal, Benzyloxycarbonyl-L-leucyl-N-chloroacetyl-hydrazide, Benzyloxycarbonyl-L-leucyl-N-bromoacetyl-hydrazide, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanine chloromethyl ketone and Benzyloxycarbonyl-L-leucyl-L-alanine chloromethyl ketone.
Benzyloxycarbonyl-L-alanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-phenethylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-tert-butylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-(1-naphthymethyl)glycyl-DL-phenylalaninal, Benzyloxycarbonyl-L-leucyl-N-chloroacetyl-hydrazide, Benzyloxycarbonyl-L-leucyl-N-bromoacetyl-hydrazide, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanine chloromethyl ketone and Benzyloxycarbonyl-L-leucyl-L-alanine chloromethyl ketone.
13. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-tyrosine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucine bromomethyl ketone, Benzyloxycarbonyl-L-asparagyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl ketone.
Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-tyrosine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucine bromomethyl ketone, Benzyloxycarbonyl-L-asparagyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl ketone.
14. The compound of claim 1 selected from the group consisting of:
Benzyloxycarbonyl-glycyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-alanyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-2-(2-naphthylmethyl)glycine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine chloromethyl ketorle, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-N-(bromoacyl) hydrazide and Benzyloxycarbonyl-L-leucyl-L-tyrosine bromomethyl ketone.
Benzyloxycarbonyl-glycyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-alanyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-2-(2-naphthylmethyl)glycine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine chloromethyl ketorle, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-N-(bromoacyl) hydrazide and Benzyloxycarbonyl-L-leucyl-L-tyrosine bromomethyl ketone.
15. A pharmaceutical composition for the treatment or inhibition of neurodegenerative disease in a mammal comprising an effective amount of a compound of the formula (I) Z--A3--A2--A1--Q (I) wherein Z is H or a protecting group;
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either D or L stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, in a pharmaceutically acceptable vehicle.
A3 and A2 are independently an optionally protected valine, leucine, alanine, isoleucine, phenylalnine, tyrosine, glycine, 2-arylglycine having either D or L stereochemistry or a chemical bond;
A1 is an optionally protected valine, leucine, isoleucine, alanine, phenylalnine, tyrosine, 2-phenyl-glycine, 2-phenethyl-glycine, 2-aryl-glycine;
Q is H, CH2OCOL, CH2OL, CH2SL, CH2X, NHNHCOCH2OCOL, NHNHCOCH2OL, NHNHCOCH2SL, wherein L is an optionally substituted aryl or optionally substituted heteroaryl; and X is Cl, Br or F, in a pharmaceutically acceptable vehicle.
16. The pharmaceutical composition of claim 15 wherein L is substituted aryl selected from the group consisting of phenyl or naphthyl optionally substituted by 1 to 3 substituents selected from the group consisting of lower alkyl, lower alkoxy, halo, acetyl, acetamido, hydroxy, phenyl, morpholino-lower alkyloxy, morpholino lower alkyl, benzyl, benzyloxy, nitro, amino, loweralkylamino, morpholinosulfonyl, morpholinosulfamoyl, benzyloxycarbonylmethylsulfamoyl, acetylamino or trifluoromethyl.
17. The pharmaceutical composition of claim 15 wherein L is substituted heteroaryl selected from the group consisting of thiazole, furan, thiadiazole, thiophen, tetrazole, pyridyl, pyrimidyl, triazole optionally substituted by 1 to 3 substituents selected from the group consisting of lower alkyl, lower alkoxy, halo, acetyl, acetamido, hydroxy, morpholino-lower alkyloxy, morpholino lower alkyl, benzyl, benzyloxy, nitro, amino, loweralkylamino, morpholinosulfonyl, morpholinosulfamoyl, benzyloxycarbonylmethylsulfamoyl, acetylamino, phenyl or trifluoromethyl.
18. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: N-Benzyloxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-[(2-morpholino) ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-prolyl-L-leucyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine 2,6-dichloro-3-(morpholinosulfonyl) phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-(morpholinosulfonyl)phenyl carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-phenylalanine 2,6-difluoro-phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-(morpholinosulfonyl)phenylcarboxymeethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-(morpholinosulfonyl)phenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichlorophenyl carboxymethyl ketone.
19. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-valyl-L-phenylalanine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophanylcarboxymethyl ketone, Tert-Butyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-tyrosinc 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-l-leucyl-L-glycine 3,6-dichloro-2-acetamido-phenylcarboxymethyl ketone, p-Toluenesulfonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dimethylphenyl -carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido-6-chlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido-6-chlorophenyl-carboxymethyl ketone.
20. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino) ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine 2-acetamido-6-chlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-N-methylleucyl-L-phenylalanine 2,6-dichloro-3-(morphorinosulfonyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dimethoxyphenyl- carboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-chlorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-acetamido-6-chlorophenylcarboxymethyl ketone.
21. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-leucyl-L-glycine 2-acetamido-3,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-pyridylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-glycine 2,6-fluorophenylcarboxy-methyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-alanine 2,6-bistrifluoromethylphenylcarboxymethyl ketone, p-Nitrobenzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine 1-naphthylcarboxymethyl ketone, Benzyloxycarbonyl-L-1eucyl-L-phenylalanine 2,6-dichloro-3-benzyloxyphenylcarboxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-N-(2,6-dichlorophenylcarboxyacetyl)hydrazide and N-Benzyloxycarbonyl-L-leucyl-N-methyl-N-(2-acetamido-6-chlorophenylcarboxy-acetyl) hydrazide .
22. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-leucyl-N-(2-acetamido-6-chlorophenyl-carboxy-acetyl)hydrazide, Benzyloxycarbonyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl-L-leucyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-L-tyrosine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichloro-3-(carbobenzoxy-methylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine 2,6-dichloro-3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl -L-leucyl-glycine 2,6-dichloro-3-[2-(morpholino) -ethoxy]phenylcarboxymethyl ketone, Methoxycarbonyl-D-alanyl -L-leucyl-L-phenylalanine 2,6-difluorophenylcarboxymethyl ketone and Benzyloxycarbonyl-L-alanyl-L-glycine 2,6-dichloro -3(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone.
23. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl -glycyl-L-phenylalanine 2,6-dichloro -3-(carbobenzoxymethylsulfamoyl)phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-valyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-glycyl-L-phenylalanine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl -L-phenylalanyl-L-alanine 2,6-dichloro -3-[2-(morpholino)ethoxy]phenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone, Benzyloxycarbonyl-D-alanyl-L-leucyl-glycine 2,6-dichloro-3 -[2-(morpholino)ethoxy]phenyl-carboxymethyl ketone, Benzyloxycarbonyl- L-leucyl-glycine 2,6-dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine 2,6 dichlorophenylcarboxymethyl ketone, Benzyloxycarbonyl-L
-alanyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-bistrifluoromethylphenylcarboxymethyl ketone.
-alanyl-glycine 2,6-dichlorophenyl-carboxymethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine 2,6-bistrifluoromethylphenylcarboxymethyl ketone.
24. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: N-Benzyloxycarbonyl-L
-leucyl-L-phenylalanine 2,6-dichlorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[1-(3-pyridyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2-[(4-morpholinoethyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[(5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[t5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylthiomethyl ketone, N-Benzyloxycarbonyl -L-valyl-L-phenylalanine 2,6-difluorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-pyrimidylthiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-(1-phenyl)tetrazolylthiomethyl ketone and Benzyloxycarbonyl-L-leucyl-L-tyrosinal.
-leucyl-L-phenylalanine 2,6-dichlorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[1-(3-pyridyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl -L-phenylalanine 2-[(4-morpholinoethyl)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[(5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-[t5-methylthio)tetrazolyl]thiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2,6-difluorophenylthiomethyl ketone, N-Benzyloxycarbonyl -L-valyl-L-phenylalanine 2,6-difluorophenoxymethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-pyrimidylthiomethyl ketone, N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine 2-(1-phenyl)tetrazolylthiomethyl ketone and Benzyloxycarbonyl-L-leucyl-L-tyrosinal.
25. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl -L-valyl-L-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-O-methyl-tyrosinal, Benzyloxycarbonyl-L-leucyl-L-phenylalaninal, Benzyloxycarbonyl -L-isoleucyl-L-tyrosinal, Benzyloxycarbonyl-L-valyl-DL
-2-(2-naphthylmethyl)glycinal, Benzyloxycarbonyl-L-isoleucyl -L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(phenethyl)glycinal, Benzyloxycarbonyl-L-2-neopentylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(1-naphthylmethyl)glycinal and Benzyloxycarbonyl-L-2-phenylglycyl-L-phenylalaninal .
-2-(2-naphthylmethyl)glycinal, Benzyloxycarbonyl-L-isoleucyl -L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(phenethyl)glycinal, Benzyloxycarbonyl-L-2-neopentylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-valyl-DL-2-(1-naphthylmethyl)glycinal and Benzyloxycarbonyl-L-2-phenylglycyl-L-phenylalaninal .
26. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-alanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-phenethylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-tert-butylglycyl-L-phenylalaninal, Benzyloxycarbonyl-L-2-(1 -naphthylmethyl)glycyl-DL-phenylalaninal, Benzyloxycarbonyl-L-leucyl-N-chloroacetyl-hydrazide, Benzyloxycarbonyl-L-leucyl-N-bromoacetyl-hydrazide, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanine chloromethyl ketone and Benzyloxycarbonyl-L-leucyl-L-alanine chloromethyl ketone.
27. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-glycyl-L-leucyl-L-tyrosine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-glycine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-leucyl-L-leucine bromomethyl ketone, Benzyloxycarbonyl-L-asparagyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-L-phenylalanine bromomethyl ketone and Benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl ketone.
28. The pharmaceutical composition of claim 15 wherein said compound is selected from the group consisting of: Benzyloxycarbonyl-glycyl-L-phenylalanine bromomethyl ketone, Benzyloxycarbonyl-L-valyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-leucine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-alanine bromomethyl ketone, Benzyloxycarbonyl-L-alanyl-glycine bromomethyl ketone, Benzyloxycarbonyl-L-2-(2-naphthylmethyl)glycine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-glycine chloromethyl ketone, Benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine chloromethyl ketone, Benzyloxycarbonyl-L-leucyl-N-(bromoacyl) hydrazide and Benzyloxycarbonyl-L-leucyl-L-tyrosine bromomethyl ketone.
29. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 15.
30. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 16.
31. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 17.
32. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 18.
33. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 19.
34. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 20.
35. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 21.
36. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 22.
37. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 23.
38. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 24.
39. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 25.
40. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 26.
41. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 27.
42. A method of treating or inhibiting a neurodegenerative disease in a mammal comprising the administration to said mammal an effective amount of a composition according to claim 28.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP95922312A EP0840614A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
| CA002224721A CA2224721A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
| PCT/US1995/007463 WO1996041638A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
| AU27043/95A AU2704395A (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative di seases |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002224721A CA2224721A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
| PCT/US1995/007463 WO1996041638A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2224721A1 true CA2224721A1 (en) | 1996-12-27 |
Family
ID=4161896
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002224721A Abandoned CA2224721A1 (en) | 1995-06-13 | 1995-06-13 | Calpain inhibitors for the treatment of neurodegenerative diseases |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0840614A1 (en) |
| AU (1) | AU2704395A (en) |
| CA (1) | CA2224721A1 (en) |
| WO (1) | WO1996041638A1 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6134498A (en) * | 1997-03-07 | 1998-09-22 | Hoechst Marion Roussel, Inc. | Method of treating trauma associated with brain, spinal cord or peripheral nerveinjury using carbobenzyloxy n-protected di- and tripeptide phenylalaninals |
| DE19718826A1 (en) * | 1997-05-05 | 1998-11-12 | Marion S Dr Eckmiller | Use of biologically active agents to influence the extracellular space of sensory cells and methods for drug administration control |
| JP2002539190A (en) | 1999-03-15 | 2002-11-19 | アクシス・ファーマシューティカルズ・インコーポレイテッド | Novel compounds and compositions as protease inhibitors |
| US7030116B2 (en) | 2000-12-22 | 2006-04-18 | Aventis Pharmaceuticals Inc. | Compounds and compositions as cathepsin inhibitors |
| CA2433520A1 (en) | 2000-12-22 | 2002-07-04 | Axys Pharmaceuticals, Inc. | Novel compounds and compositions as cathepsin inhibitors |
| DE10105040A1 (en) | 2001-02-05 | 2002-08-14 | Tell Pharm Ag Hergiswil | Tripeptide derivatives for the treatment of post-lesion diseases of the nervous system |
| DE10105038B4 (en) | 2001-02-05 | 2005-07-07 | Neurotell Ag | Tripeptide derivatives for the treatment of postläsional diseases of the nervous system |
| DE10105041A1 (en) | 2001-02-05 | 2002-08-14 | Tell Pharm Ag Hergiswil | Tripeptides and tripeptide derivatives for the treatment of neurodegenerative diseases |
| DE10105039A1 (en) * | 2001-02-05 | 2002-08-08 | Tell Pharm Ag Hergiswil | Tripeptide derivatives for the treatment of neurodegenerative diseases |
| MXPA04002282A (en) | 2001-09-14 | 2005-03-07 | Axis Pharmaceutical Inc | Novel compounds and compositions as cathepsin inhibitors. |
| CN1324018C (en) | 2001-11-14 | 2007-07-04 | 安万特药物公司 | Oligopeptides and compositions containing them as cathepsin S inhibitors |
| ES2219187B1 (en) * | 2003-05-14 | 2006-02-16 | Consejo Sup. Investig. Cientificas | CALPAINE INHIBITORS. |
| ES2255848B1 (en) | 2004-12-16 | 2007-07-01 | Consejo Superior Investig. Cientificas | ISOQUINOLINE DERIVATIVES AS CALPAINE INHIBITORS. |
| US9181296B2 (en) | 2008-03-26 | 2015-11-10 | Novozymes A/S | Stabilized liquid enzyme compositions |
| US8236798B2 (en) | 2009-05-07 | 2012-08-07 | Abbott Gmbh & Co. Kg | Carboxamide compounds and their use as calpain inhibitors |
| CN105431546A (en) * | 2013-03-15 | 2016-03-23 | 里兰斯坦福初级大学理事会 | Activity-based probe compounds, compositions, and methods of use |
| CN110023740B (en) | 2016-12-23 | 2023-04-14 | 里兰斯坦福初级大学理事会 | Activity-based probe compounds, compositions, and methods of use thereof |
| WO2018183960A1 (en) | 2017-03-30 | 2018-10-04 | The Board Of Trustees Of The Leland Stanford Junior University | Protease-activated contrast agents for in vivo imaging |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56110661A (en) * | 1980-02-04 | 1981-09-01 | Ajinomoto Co Inc | Preparation of n-benzyloxycarbonyl-l-aspartic acid |
| US5444042A (en) * | 1990-12-28 | 1995-08-22 | Cortex Pharmaceuticals | Method of treatment of neurodegeneration with calpain inhibitors |
| JPH04273826A (en) * | 1991-02-27 | 1992-09-30 | Dainippon Ink & Chem Inc | Anti-rs viral agent |
-
1995
- 1995-06-13 AU AU27043/95A patent/AU2704395A/en not_active Abandoned
- 1995-06-13 EP EP95922312A patent/EP0840614A1/en not_active Withdrawn
- 1995-06-13 WO PCT/US1995/007463 patent/WO1996041638A1/en not_active Application Discontinuation
- 1995-06-13 CA CA002224721A patent/CA2224721A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0840614A1 (en) | 1998-05-13 |
| AU2704395A (en) | 1997-01-09 |
| WO1996041638A1 (en) | 1996-12-27 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |