CA2098609A1 - Use of calpain inhibitors in the inhibition and treatment of neurodegeneration - Google Patents
Use of calpain inhibitors in the inhibition and treatment of neurodegenerationInfo
- Publication number
- CA2098609A1 CA2098609A1 CA002098609A CA2098609A CA2098609A1 CA 2098609 A1 CA2098609 A1 CA 2098609A1 CA 002098609 A CA002098609 A CA 002098609A CA 2098609 A CA2098609 A CA 2098609A CA 2098609 A1 CA2098609 A1 CA 2098609A1
- Authority
- CA
- Canada
- Prior art keywords
- leu
- abu
- alpha
- ala
- cooet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
The present invention provides uses for Calpain inhibitor compounds and also provides pharmaceutical compositions containing Calpain inhibitor compounds. One use of these compounds is in the treatment of a neurodegenerative pathology in a human patient. The invention also provides additional uses and pharmaceutical compositions containing Calpain inhibitor compounds, such as Peptide Ketoamides, Peptide Ketoacids and Peptide Ketoesters.
Description
WO 92/118~0 2 ~ 9 8 ~ û 9 P~/U!~i91/097~6 USE OF CALP~IN INHlBl'rORS IN THE ~IIBI'lION
A~D TREATMENT OF NEURODEGENERATII:)N
Baclc~round of the Invention The present invention reiat~s generally to the field of neuroprotectants and more 5specifically to the use of inhibitors of calciuln-sti}nulated proteases, such as ~alpain, as therapeutics for neurodegeneration.
Neural tissues, including brain, are knowT~ to possess a large variety OI proteases, including at least two calcium-stirnulated proteases, termed calpain l and calpain II, v~hich are activated by micromolar and millirnolar Ca2+ concentrations, respectively. Calpains are 10a family of calci~n activated thiol proteases that are present in many tissues. Calpain 11 is the predominant forrn, but calpain I is found at synapses and is thought to be the form involved in long terrn potentiation and synaptic plasticity.
Thiol proteases are distinguished from serine proteases, metalloproteases and other proteases by their mechanism of action and by the amino acid residue (cysteine) that 15participates in substrate attack. Although several thiol proteases are produced by plants, these proteases are not cornrnon in marnmals, ~vith cathepsin B (a lysosomal er~ne), other cathepsins and the calpains being among the few representa~ives of this ~arnily that have been described in mammals. Calpain I and calpain II are the best described of these, but several other members of the calpain ~amily have been reported.
20Other Ca2+-act~vated thiol proteases may e~st, such as those reported by Yoshihara et aL in J. Biol. Chem. 265:5809-5815 (1990). The term "Calpain" is used hereinafter to .. refer to any Ca2+-activated thiol proteases induding the Yoshihara enzyTne and calpa~ns I
and II.
While Calpains degrade a wide ~ariety of protein substrates, cytoskeletal prote~ns 25 -seem to be particularly susceptible to attack. In at least some cases, the products of-the proteolytic digestion of these proteins by Calpain are distinctive and persistent over ~ime.
Since cytoskeletal proteins are major components of certain types of cells, this provides a sirnple method of detecting Calpain activity in cells and tissues. Specifically, the accumulation of the breakdown products ("BDP'sn) of spectrin, a cytoskeletal protein, has 30been associated with the aclivation of Calpain. In neural tissues, act~vation of Calpains, as evidenced by accurnulation of these BDP's, has been obsened in many neurodegenerative conditions, including denervation resulting from focal electrolytic lesions, genetic abnormalities, excitotoxicity, Alzheimer's disease, following ischemia in gerbils and following E:U~S7-1TUTE~ SHEET
,. - . . .
,.. ~ .. .` , , ~ ... .- . . . .
, . `~ ~ .. , ` , . , . . . ` .
.. .. . ~.... . . . .
W092/11850 PCI/US91/097~ ~
A~D TREATMENT OF NEURODEGENERATII:)N
Baclc~round of the Invention The present invention reiat~s generally to the field of neuroprotectants and more 5specifically to the use of inhibitors of calciuln-sti}nulated proteases, such as ~alpain, as therapeutics for neurodegeneration.
Neural tissues, including brain, are knowT~ to possess a large variety OI proteases, including at least two calcium-stirnulated proteases, termed calpain l and calpain II, v~hich are activated by micromolar and millirnolar Ca2+ concentrations, respectively. Calpains are 10a family of calci~n activated thiol proteases that are present in many tissues. Calpain 11 is the predominant forrn, but calpain I is found at synapses and is thought to be the form involved in long terrn potentiation and synaptic plasticity.
Thiol proteases are distinguished from serine proteases, metalloproteases and other proteases by their mechanism of action and by the amino acid residue (cysteine) that 15participates in substrate attack. Although several thiol proteases are produced by plants, these proteases are not cornrnon in marnmals, ~vith cathepsin B (a lysosomal er~ne), other cathepsins and the calpains being among the few representa~ives of this ~arnily that have been described in mammals. Calpain I and calpain II are the best described of these, but several other members of the calpain ~amily have been reported.
20Other Ca2+-act~vated thiol proteases may e~st, such as those reported by Yoshihara et aL in J. Biol. Chem. 265:5809-5815 (1990). The term "Calpain" is used hereinafter to .. refer to any Ca2+-activated thiol proteases induding the Yoshihara enzyTne and calpa~ns I
and II.
While Calpains degrade a wide ~ariety of protein substrates, cytoskeletal prote~ns 25 -seem to be particularly susceptible to attack. In at least some cases, the products of-the proteolytic digestion of these proteins by Calpain are distinctive and persistent over ~ime.
Since cytoskeletal proteins are major components of certain types of cells, this provides a sirnple method of detecting Calpain activity in cells and tissues. Specifically, the accumulation of the breakdown products ("BDP'sn) of spectrin, a cytoskeletal protein, has 30been associated with the aclivation of Calpain. In neural tissues, act~vation of Calpains, as evidenced by accurnulation of these BDP's, has been obsened in many neurodegenerative conditions, including denervation resulting from focal electrolytic lesions, genetic abnormalities, excitotoxicity, Alzheimer's disease, following ischemia in gerbils and following E:U~S7-1TUTE~ SHEET
,. - . . .
,.. ~ .. .` , , ~ ... .- . . . .
, . `~ ~ .. , ` , . , . . . ` .
.. .. . ~.... . . . .
W092/11850 PCI/US91/097~ ~
2~986~9 adn~inistration of the to~ins kainate and colchicine in rats, when adininistered peripherally or centra~ly.
Comrnercially availabie ~ vitro inhibitors of Calpa~n include peptide aldehydes such as leupeptin (Ac-l~u~Leu-Arg-H), as well as epo~ysuccinates such as E~64. These S compounds are not useful in inhibiting Calpain in Central Nervous System (nCNSn) tissue in vivo because they are poorly membrane perrneant and, acs~ordingly, do not cross the blood brain barTier veIy well. Also, many of these in~u~itors are poorly specific and will inhibit a wide var~e~ of protea~ses in addition to Calpain. 'Ihese commercially available compounds are based upon peptide structures that are believed to interact ~nth the substrate binding site of Calpain. Active groups associated with the Calpa~n inhibitors then either block or attacl~ Ihe catalytic moiety of Calpain in order to inhibit the enzyïne.
In addition, other types of compounds thought to possess in vitro Calpain inhibitory activity that are not commercially available have been reported. Examples of such compounds indude the peptide diazomethanes. See Rich, D~., Inhibitors of ~steine1~ proteinases, in Protease Inhibitors, AJ Barrett and G ~alversen, Eds., Elsevier, New York, 1986, ppl53-178, the disclosure of which is hereby incorporated by reference. These peptide diazomethanes are similarly thought to be poorly membrane penneant and non-specific.
There is some evidence that certain particular inhibitors of Calpain have certain therapeutic utilities. For example, leupeptin can facilitate nerve repair in prirnates.
Loxastatin (also known as EST, Ep460 or E-64d), a derivative of E-64, ~s believed to have utiliy in the treatment of muscular dystrophy. E-64d, while not having significant protease inhibitory activity itself, is believed to be converted to more potent fonns, such as to E-64c, inside a marnmalian body.
Evidence from electrophysiological studies suggests that one of the ear~iest factors in the chain of reactions leading to cell death is an increase in intracellular-free calcium as a consequence of Ca2+ channel opening and/or energy depletion. Intracellular calcium is lilcely to produce a large nwnber of consequences, including the activation of a large number of en~yTnes, including p~oteases, such as Calpain, lipases and kinases. An increase in intracellular calcium is also thoudlt to induce changes in gene expression.
Ischenua, head traurna and stroke have all been associated with the rele~ase of glutamate in amounts large enough to lead to excitotoxicity, the to~icity resulting from the actions of certain amino acids on neurons of the CNS. The e~cess glutamate and other factors, such as free radical damaBe of membranes or energy depletion, cause an increase in intracellular Ca2+. It is known that an excess of intraceliular Ca2+ leads to several effects SUBSTITUTI~ S~E~T
.: ..
. :
.' . ' . ~, WO 92/11850 2 ~ ~ 8 ~ ~ ~ PCI`~US91/09786 believed to be associated with neuronal cell damage, including destruction of cell structures through activation of phospholi~ase and Calpain, as well as free radical production resulting from ac~ivation of phospholipase and xanthine oxidase. Many other factors have been - associated with neuroto~city. For example, reductions in action potentials and changes in a wide variety of chemic~l markers are known to be associated wi~h neurons exposed to ischemic conditions.
Notwithstanding the foregoing understanding of certain aspects of neuroto~city, no effecdve therapy has yet been developed for most neurodegenera~ive diseases and conditions of the CNS. Millions of individuals suffer from these diseases and conditions. Thus, there is a need for therapies effective in treadng and preventing these diseases and eonditions.
Summarv of the Tnvention The present invention provide the use of a Calpain inhibitor compound or a pharmaceutically acceptable salt or derivativf~ thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal hanng or likely to experience a neuropathology associated with neurodegeneration. In certain embodiments of this use, the neurodegeneration is occurring due to e~citotoxicity, HIV-induced neuropathy, ischemia following denervation or injury, subarachnoid hemorrhage, stroke, mul~iple infarction dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease. Themediument can comprise a pharmaceutically acceptable carrier and is for parenteral 2û administration, such as for transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or infusion. The mediument can also be in a forrn suitable for oral use. In some embodirnents, the medicarnent is for substantially preventing neurodegeneration in a patient undergoing surgery during and subsequent to the surgery, such as for a patient undergoing neurosurgery, cardiovascular surgery or a surgery using general anesthesia. The Calpain inhibitor compound preferably enters t~ssue of the C~S of the mammaL such as through use of a membrane-permeant Calpin inhl~itor.
The present invention also provides an in vitro method of selecting Calpain Inhibitors for use as Calpain ~hibitor protectants in the in vivo treatment or inhibition of degeneration. This method includes identifying compounds having Calpain inhibitory activity vitro~ and identi~ing those compounds with Calpain inhibito~y activity that are membrane permeant through an vjtro assay for membrane permeance. The irL~i~o assay for membrane permeance can include providing a plurality of tissue portions from a mammal; treating a~ least one, but not a~, of the tissue portions with a Calpain Inhibitor;
WO 92/11850 2 ~ 9 8 ~ 0 9 PCI'/US91/097~
subjecting the tissue portions to an eYent that can cause degeneraeion in untreated eissue;
measuring the amount of degeneration that occurs in ehe tissue poreions; and comparing the arnount of degeneration that occurs in the treated tissue portions with ths amount of degeneration occurring in the untreated tissue portions. The amount of degeneration in the treated tissue portions being less than the amount of degeneration ir~ the untreated tissue portions indicates that the Calpain Inhibitor is membrane-permeant. The tissue portions in some embodiments are brain sli~es, platelets or ceUs in cultllre. The measuring step in some embodirnen~s involves analyzing the tissue portions for the presence of the BDP's of a cytoskeletal eomponent such as spectrin, MAP2, actin binding protein or tau. The measuring step can also include measuring the electncal actiYity of the tissue portions. The in vitro assay for membrane per~neance can b~ performed by measuring the ability of the Calpain Inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the platelets.
Ihe present inYention also proYides the use of a Substituted Hetero~yclic Compound or a pharmaceuticaUy acceptable salt or derIvatiYe ~hereof for the manufacture of a medicament for inhbiting or treating neurodegeneration in a mammal haYing or likely to experience a neuropathology associated with ne~lrodegeneration. The medicarnent is preferabk~r for inhibiting or treating neurodegeneration of the CNS. The Substituted Heterocyclic Compound preferably is a member of the Class I Substituted ~socoumarins, the Class II Substituted Isocournarins or the Class m Heterocyclic Compounds. More preferably, the Substituted Heterocyclic Compound is 3-chloroisocournarin; a 3,4-dichloroisocoumarin; a 3-alkoxy-7-arnino 1-chloroisocoumarin; or a 7-substituted 3-alkoxy~-chloroisocoumarin. The use of Clairn 17, wherein said Substituted Heterocyclic Compound isCl~PrOIC,NH2-Cl~PrOIC,PhCH2NHCONH-C~PrOIC, CH3CONH~CiTPrOIC,L-Phe-NH-ClTPrOIC, PhG~I2NHCONH~Cl~EtOIC, PhCH2CONH-Cs~EtOIC, or D-Phe-NH-' CiTEtOIC.
Another aspect of the present invention provides the use of a Peptide Keto-Compound hav~ng Calpain inhl~itory activity or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for in}~iting or treatingneurodegeneration in a marnmal having or likely to e~perience a neuropathology associated with neurodegeneration. The medicament is preferably for inhibiting or treating neurodegeneration of the GNS. The Peptide Keto-Compound preferably is a peptide a-ketoester, a peptide a-ketoacid or a peptide a-ketoamide. More preferably, the Peptide Keto-Compound is a member of one of the following su~classes: Dipeptide a-Ketoesters SUBS~tTUTE SHEET
.~ .
, . ~ .
;: WO 92/1185û 2 ~ 9 ~ ~ ~ 9 PCI/lUS91/09786 (Subclass A), Dipeptide ~-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide -Ketoesters (Subclass B), Tetrapeptide ~-Ketoesters, Amino Acid Peptide -Ketoesters, Dipeptide a-Ket~acids (Subclass A), Dipeptide -Ketoacids (Subclass B), Tn~eptide -Keeoacids, Tetrapeptide a-Ketoacids ~nd Arnino A~id Peptide ~-Ketoacids, Dipeptide ~-Ketoa~nides (Subclass A), Dipeptide a-Ketoarnides (Subclass B), T~ipeptide c~-Ketoarnides, Tetrapeptide a-Xetoarnides or Amino Acid a-Ketoarnides.
Still another aspect of the present invention provides the use of a Halo-Ketone Peptide having Calpain in~ubitory ac~vity or a pharrnaceutically acceptable salt or derivative thereof Ior the manufacture of a medicament for inh~biting or treating neurodegeneration in a mammal having or ]ikely to experience a neuropathology associated with neurodegeneration. The medicament is preferably for inhibiting or treating neurodegeneration of the CNS. The Halo-Ketone Peptide can be an arnino-halo ketone peptide or a diazo-ketone peptide.
The uses of the present invention of Substituted Heterocrclic Compounds, PeptideKeto-Compounds or Halo-Ketone Peptides can be used in connection with neurodegeneration associated with e~citotoxicity, HIV-induced neuropathy, ischernia, subarachnoid hemorrhage, stroke, bra~ seizure, major heart attack, multiple in~arction dementia, Alzheirner's Disease, Huntington's Disease or Parkinson's Disease. Themedicament of these uses can indude a pharmaceutically acceptable carrier, and be for parenteral administration, such as transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or an infusion technique. The medicament can also be in a form suitable for oral use.
These uses can also be in coMection with neurodegeneration is occurring from ischemia-inducing events, stroke, head injury, major heart attaclc, brain seizure, near drowning, carbon monoxide poison~ng, surgery-related brain damage or another event known to causeneurodegeneration.
Another aspect of the present invention provides a method of minim~ing proteolysis in an in ~o sample containing peptides or proteins during or following the prccessing, production, preparation, isolation, purification, storage or transport of the samples, comprising ehe addition eo the sasnple of a Substituted Hetero~clic Compound or a Peptide Keto-Compound that is a member of one of the following subclasses: Dipeptide a-Ketoesters (Subclass A), Dipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide a-Ketoesters (Subclass 8), Tetrapeptide a-Ketoesters, Arnino Acid Peptide -Ketoesters, Dipeptide a-Ketoacids (Subclass A), Dipeptide a-Ketoacids (Subclass ~VO 92/11850 2 ~ 9 8 6 ~ 9 -6- PCI/US91/~97~ ~
B), Tripeptide ~-Ketoacids, Tetrapeptide a-Ketoacids, Amino Acid Peptide a-Ketoacids, Dipeptide ~-Ketoarnides (Su~class A), Dipeptide a-Ke20arnides (Subclass B), Tripeptide a-Ketoamides, Tetrapeptide a-Ketoarnides or Amino Acid a-Ketoam~des. The presentinvention also provides a method of n~nim~g degradation resul~ting from Calpain act~vity S in a tissue sample during or following preparation of the sample, comprising the addition to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo-Ketone Peptide. The sample can be a whole organ and the addition of compound comprises perfusion of the organ with the compound disso~ved in fluid. Preferably, following the addition step, the tissue sample is used in an assay for neurodegeneratiQn wherein the assay comprises a test for the products of Calpain activity in the tissue samples. The addition of compound in either of these methods can include the addition of a Peptide a-Ketoacid to the sample. Preferably, this Peptide a-Ketoacid comprises a compound that is a member of one of the fo110wing subclasses: Dipeptide a-Ketoacids (Subclass A), Dipeptide a-Ketoacids (Subclass B), Tripeptide -Ketoacids, Tetrapeptide a-Ketoacids or Arnino Acid Peptide a-Ketoacids.
Yet another aspect of the present invention provides pharmaceutical compositionsfor the treatment or inhibition of neurodegeneration. These compositions include a pharmacologically effective neuroprotective amount of a Substituted Hetero~yclicCompound, Peptide Keto-Compound or Halo-Ketone Peptide, or pharmaceutically acceptable salts or derivatives thereof in a pharmaceutically acceptable forrnulation containing a carrier material. In one preferred embodirnent, a Peptide Keto-Compound is included in the composition where~n said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide a-Ketoesters (Subclass A), Dipeptide a-Ketoesters (Subclass B), Tripeptide -Ketoesters (Subclass A), Tripeptide a-Ketoesters (SubclassB), Tetrapeptide a-Ketoesters, orAminoAcid Peptide a-Ketoesters. This Pepdde Keto-Compound is preferabb one of the following compounds: Bz-DL-Ala-COOEt, Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz-DL-Lys-COOEt, PhCO-Abu-COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu-COOEt or Ph(CH2)6CO-Abu-COOEt. Another preferred Peptide Keto-Compound is one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL-Ala-COOBzl, Z-Ala-DL-Ala-COOnBu, Z-Leu-Nva-COOEt, Z-Leu-Me-COOEt, Z-Leu-Abu-COOEt, ~-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, ~-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe-C02Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala~DL-Ala-COOMe, or Z-Leu-Abu-CO2Bzl. Still other SU6~ I tTUl ~ SHFET
. .
WO 92/11850 2 ~ ~ g ~ Q 9 P~/US91/09786 Peptide Keto-Compounds ~r use in ~he preferred compositions are one of ~he followin~
compounds: Z-Ala-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt, Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DL-AbuCOC)CH2-C6H4-CF3 (para), H-Leu-Ala-DL-Lys-COO~t, ZLeu-Leu-Abu-COOEt, ZL~u-Leu-Phe-COOEt, MeO-Suc-Val-Pro-D~Phe-COOMe or 2-NapSO2-IRu-Ieu-Abu-COOEt. A preferred Peptide Keto-Compound could also be MeO-SucAla-Ala-Pro-DI~Abu-COOMe or Z-Ala-Ala-Ala-DI,Ala-COOEt. Still other preferred Peptide Keto-Compouulds would be a compound ~om one of the following subclasses: Dipeptide a-Ketoacids (Subclass A), Dipeptide ~-Ketoacids (Subclass B), Tripeptide a-Ketoacids, Tetrapeptide a-Ketoacids or the Amino Acid Peptide a-Ketoacids.
Thus, preferred Peptide Keto-Compounds could be one of the following compounds:
Bz-DL-Lys-COOH, BznDI~Ala-COOH, ZLeu-Phe-COOH or ZLeu-Abu-COOH. A
compound ~om one of the following subclasses: Dipeptide a-Ketoamides (Subclass A), Dipeptide a-Ketoamides (Subclass B), Tripeptide a-Xetoamides, Tetrapeptide a-Ketoamides or Amino Acid a-Ketoamides, could also be used in the preferred compositions. Thus, another preferred Peptide Ke~o-CompouIld would be one of the~ollow~g compounds: ~Leu-Phe-CC)NH-Et, ~Leu-Phe-CONH-nPr, ~Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-:Bzl, ~Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu~Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, Z-Leu-Abu-CONH-Bzl, ~Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH~2O, ZLeu-Abu-CONH-(C~H2)7CH3, ZLeu-Abu-CONH-(C~I2)20H, ZLeu-Abu-CONH-(CH2)20(CH2)20H, Z-Leu-Abu-CONH (CH2)l7CH3, ZLeu Abu CONH-CH2-C6H3(0CH3)2 or Z-Leu-Abu-CONH-CH2~C4H4N. The composition can also include a Substituted Heterocyclic Compound such as one of the Class I Substi~u~ed Isocoumarins, Class lI Substituted Isocoumarins or Class m Heterocyclic Compounds. Preferred Substituted Hetero~yclic Compounds are 3-chloroisocoumar~n, a 3,4-dichloroisocoumarin, a 3-allco~y-7-amino 1~1oroisocoumarin, a 7-substitu~ed 3-alkoxy-4~chloroisocournarin;
CiTPrOIC, NH2-Cl~TPrOIC, PhCH2NHCONH-ClTPrOIC, CH3CONH-ClTPrOIC, L-Phe-NH-GTPrOIC, PhCH2NIICONH-ClTEtOIC, PhCH2CONH-ClTEtOIC, or D-Phe-NH-CiTEtOIC In these compositions, the composition is preferably in dosage ~orm comprising from 70 llg to 7 g of active ingredient in each dose, and the carrier material includes a liquid, wherein ~he composition is ~n dosage form and wherein each dose comprises ~om 05 ml to 1 liter of said carrier material. The compositions can additionally include at least one of the follo~ing: DMSO or other organic solvent, a lipid carrier, a detergent, a .~
:, ~ ~ ,, '- :
- . . -: . . . . . .
WO 9Z/11850 2 0 9 8 ~ ~ ~ PCr/US91/o97~ ;-surfactant, or an emulsifying agent. These compositions can be su~table for parenteral administration or in a forrn suitable for topical application. The compositions can be in a variety of forms, such as an aqueous solution, a lotion, a jelly, an oily solution, or an oily suspension.
S The present invention also provides the use of a Substituted Heterocyclic Compound as a medicament, the use of a Halo-Ketone Peptide as a medicament, and the use of a Peptide Keto-Compound as a medi~ament, wherein said Peptide Xeto-Compound is a compound from one of the following subclasses: Dipeptide ~-Ketoesters (Subclass A), ~ipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide ~-Ketoesters (Subclass :E3), Tetrapeptide a-~etoesters, or Amino Acid Peptide a -Ketoesters.
Preferred Peptide Keto-Compounds in this use ~nclude any one of the Peptide Keto-Compounds desaibed above in connection with the pharmaceutical compositions.
Brief Summarv of the Fi@lres Figure 1 shows the percentage of i~u~ition of glut~nate-induced cell death through the addition of ~lutamate and various Calpain Inhibitors rela~ve to control where no glu~amate was added.
Figure 2 graphically depic~s the e~ects of ZLeu-Phe-C:ONH-Et (CX269) and ZLeu-A~u-CONH-Et (CX275) on the size of iT~rction produced upon MCA occlusion in malerats.
~igure 3 shows the effects of CX216 (ZLeu-Phe-CO2Et, a Peptide Keto-Compound), and C:I1 (Ac-Leu-Leu-Me-H) rela~ve to ~ontrol slices on survival of hippocampal slices exposed to 10 minutes exposure of anoxic atrnosphere where both of these compounds were added at their optimal inhibitory concentradon at both 1 hour and 2 hour incubation times.
Figure 4 shows the evoked potential amplitude for control, CI1 treated and CX218treated hippocampal slices over a time course during which the slices sre exposed to anoxic atmosphere.
Figure 5 shows the percent recovery of EPSP from severe hypoxia over the course of one hour incubation for ~Leu-Phe-CONH-Et (CX269) and ~Leu-Phe-CO2Et (CX216).
Figure 6 shows a comparison of the ef~ect of the presence of CI1 or CX216 on survlval of hippocampal slices expressed as the duration of anoxia (in minutes) before fiber volley disappearance.
Figure 7 shows the effects of CI1 compared with control on the behavioral and convulsive effects of kainic acid.
~;UBSTITUTE SHEEJ
--.
W(:~ 92/11850 2 0 ~ PC11US91/097~6 "~., ~
Figure 8 shows the amount of spectrin BDP's in rat brains e~posed to kainate forcontrol and CI1 treated rats.
Detailed Description of the Invention A. INTRODUCI ION
We haYe discovered that Calpain activation is an event central to many cases of - bra~n atrophy and degeneration and that inhibition of Calpain alon~ is su~icient to inhibit or prevent cell deterioration and loss. Thus, we have further discovered that inhibition of C~ain provides protertion from neuroto~ncity associated with many neurodegenerative conditions and diseases.
In accordance with the foregoing di~coveries, we believe that the elevation of intracellular ~alcium a~sociated with neuropathological condition~ in neuronal cells activates Calpain and sets in motion the digestion OI neuronal ce11s from within. We believe there may be other mechanisms of activation of Calpains associated with these conditions.
Accordirlgly, o~e aspect of ~he present invention is di~ected to inhibidon and treatment of 1~ the neurodegeneration and o~her diseases associated with this dic,estion through the inhibition of Calpain actiYity. Thus, part of this aspect of the present invention is to prevent the neurodegeneradon and other pathology caused by this diges~ion through the vivo adrninistration of Calpain inhll~itors. By way of example, and not of lirnitation, diseases and conditions which can be treated using this aspect of the present invendon include 20 neurodegeneradon following e~citotoxicity, HIV-induced neuropashy, ischenua, denervation foJlowingischemia orinjury, subarachnoid hemorrhage, stroke, multipleinfarction dementia, Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease, surgery-related brain damage and other neuropathological conditions.
As stated above, spectrin BDP's have been found to be associated with Calpa~n 25 activation in vivo, We have obsened that in each instance of neurodegeneration in which BDP's characteristic of Cal~ain activation are detected, Calpain activation is localized to the brain areas most vulnerabIe to the particular pathogenic manipulation. Isl addition, as judged by histological methods, C:alpain activation precedes overt evidence of neurodegenerat;on. Accordingly, CalpaM activation is spatially and temporally lir~ced to 30 impending or ongoing ce~l death in the brain. Thus, we believe that Calpain activation is an important mechanism of cell damage and death in many pathological conditions,induding neuropathological conditions. Moreover, there is eYidence that the activation of Calpains is an early event in the death of cells including neural cells. This is in contrast to other known proteases which are activated at later stages of cell death. Thus, we believe - ,; , . . . . . ., . ..................... . . . :
-. ~
: ~. - ~ . . . -wo 92/11850 2 0 9 8 6 a 9 pcr/us91~o978 ~
that, advantageously, inhibition of Calpain activity provides intervention at an early stage of cell death, pnor to significant deterioration of ee1lular machinery.
Another aspect of the involvement of Calpains in neurodegeneration is the involvement of these proteins in regenerating systems. It is Icnown that developing or regenerating axons are somehov inhibited from further development in a stabilization process called the "stop pathway.~ This stabilization can occur when axons have reached their targets; however, in some systems stab;lization can also occur at inappropriate places.
One researcher has de~eloped evidence that this stop pathway operates at least in part by the activation of intracellular Calpain and that in11ibition of Calpain can interfere vnth stabilization (Luizi, 1990). We believe that Calpain inhibitors, when used in accordance with the present invPntion, can advantageously aid regeneration and recovery of neural tissue after injury, in addition to inhl~iting neurodegeneration.
Another aspect of the present invention is our diseovery that at least three classes of compounds, the substituted isocournarins, the peptide keto-compounds and the Halo-Ketone Peptides have Calpain inhibitory activity. We have further discovered, as will be described hereinbelow, that these three classes of compounds exhibit additional properties that render them e~pecially useful as therapeutically effective compounds in the ereatment of neurodegenerative conditions and diseases.
B. SUBSTl~TED HEleROCYCIIC COMPOU~DS
One particular class of compounds exhibiting Calpain inhibitory activity, ~hen used in accordance with the present invention, are the substituted heterocyclic compounds. These compounds include the substituted isocoumarins. The substituted heterocyclic compounds are known to be excellent inhl~itors of serine proteases. As discussed hereinbelow, we have now discovered that these compounds are also inhibitors of calpain I and calpa~n I~, and also 2~ of other Calpains. Additionally, as also diuussed below, we have found that, unlLke most ~ known inhibitors of Calpains, these substituted heterocyclic compounds are not effective as ir~ibitors of papain or cathepsin B. Thus, we believe that the sl~bstituted heteros~yclic compounds provide a relatively specific means of inhibiting Calpams while not affecting other thiol proteases.
One particular dass of substituted heterocyclic compounds with Calpain inhibitory ' aceivity are the isocoumarins having cationic substituents. These substituted hetero~yclic compounds are referred to herein as the "Class I Substituted Isocoumanns." The aass I
Substituted Isocoumarins are known to be e~cellent inhibitors of several serine proteases, induding bovine thrombin, human thrombin, hu nan factor Xa, human factor X~a, lluman !E~IUE~STIITU~E SH~T
~ WO92/11~,50 ~ 36~ PC~/US91/0~7f~6 factor X[Ia, bovine trypsin, human plas,ma plasmin, hurnan tissue plasrninogen activator, hurnan lung tryptase, rat skin tryptase, human leukocyte elastase, porcine pancreatic elastase, bovine ch,vmotrypsin and human leuko~yte cathepsin G. The Class I Substituted Isocoumarins inhibit the serine proteases by reaction with the active site serme to form an acyl er~ne, which in some cases may further react with another active site nucleophile to form an additional covalent bond. We have discovered that the Class I Substituted Isocoumarins also react with Calpain. We believe that the mechanism of action of Calpain inllibition is similar to that of the iinh~bition of serine proteases since the reaction mechanism of Calpains is similar to that of the serine proteases.
The Class I Subsdtuted Isocoumarins having Calpain in~u~itory activity have the follo~ing structural forrnula:
~ O
(I) ~2 or a phannaceutically acceptable salt, wherein Z is selected from the group consisdng of C1-6 aLlco~y with an amino group attached to the alkoxy group, C1-6 alkoxy with an isothiureido group attached to the aL~coxy group, Cl-6 aL~coxy with a guanidino group attached to the aL~coxy group, C1-6 allcoxy with an a nidino group attached to the allco:y group, C1-6 alkylwith an arnino group attached to the alkyl group, C1-6 allyl with an isothiureido group attached to the allyl group, C1-6 aLlcyl with an guanidino group attached to the allyl group, C1-6 aLcyl with an an~idino group attached to the alkyl group, R is selected from the g;oup consist~ng of O=C=N-, S= C=N-, AA-NH-, AA-AA-NH~ O,AA-AA-O-, M-NH-, M-A~A-NH, M-AA-AA-NH-, M-O-, M-AA-O-, M-AA-AA-O-, wherein AA repre~,erlts alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, tnreonine, cysteine, tyrosine, beta-alanine, ~BSTITUT SHE.ET
.: , ' : -WO 92/11850 2 a 9 8 6 0 9 PCr/lJS91/0978 norleucine, nonaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine or sarcosine, wherein M represents NH2-CO-, NH2~ , NH2-S02-, X~ CO, X-NH-CS, X-NH-S02, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-, S wherein X represents C1-6 allyl, C1-6 fluoroalkyL C1-6 aLtcyl substituted with K, C1-6 fluoroalkyl substituted wilh K, phenyl, phenyl substituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, naphthyl, naphthyl subsdtuted ~ith J, naphthyl disubstituted with J, naphthyl trisubstituted with J, C1-6 aLkyl with an attached phenyl group, C1-6 allyl with two attached phenyl groups, C1-6 allyl with an attached phenyl group substituted with J, or C1-6 aL~cyl with t~o attached phenyl groups substituted with J, wherein J represents halogen, COOH, OH, CN, N02, C1-6 allyl, C1-6 aL~coxy, C1-6 all~lamine, C1-6 dialkylarnine, or C1-6 alkyl-O-CO-, wherein K represents halogen, COOH, OH, CN, N02, NH2, C1-6 aLkylamine, C1-6 diaL'cylan~ine, or C1-6 alkyl-O-CO-, Y is selected from the group consisting of H, halogen, trifluoromethyL methyl, OH
and metho~y.
The compounds of Formula (I) can also contain on or more substituents at position B as shown in the following structure:
R~ o E3~2 y ` -wherein electronegative substituents such as N02, CN, CI, COOR, and COOH will increase the reactivity of the isocou narin, and electropositive substituents such as NH2, OH, aLcoxy, thioallyL allyl, allylamino, and diaLcylamino will increase its stability. Neutral substituents could also increase the stability of acyl enzgme and improve the effectiveness of she inhibitors.
The following compounds are representative of the Class I Substituted Isocoumarins of ehe present invention:
S~ STITUTE SHEE~ T
`-!W092/ll850 2098~a9 PCr/US91/09786 .
Comrnercially availabie ~ vitro inhibitors of Calpa~n include peptide aldehydes such as leupeptin (Ac-l~u~Leu-Arg-H), as well as epo~ysuccinates such as E~64. These S compounds are not useful in inhibiting Calpain in Central Nervous System (nCNSn) tissue in vivo because they are poorly membrane perrneant and, acs~ordingly, do not cross the blood brain barTier veIy well. Also, many of these in~u~itors are poorly specific and will inhibit a wide var~e~ of protea~ses in addition to Calpain. 'Ihese commercially available compounds are based upon peptide structures that are believed to interact ~nth the substrate binding site of Calpain. Active groups associated with the Calpa~n inhibitors then either block or attacl~ Ihe catalytic moiety of Calpain in order to inhibit the enzyïne.
In addition, other types of compounds thought to possess in vitro Calpain inhibitory activity that are not commercially available have been reported. Examples of such compounds indude the peptide diazomethanes. See Rich, D~., Inhibitors of ~steine1~ proteinases, in Protease Inhibitors, AJ Barrett and G ~alversen, Eds., Elsevier, New York, 1986, ppl53-178, the disclosure of which is hereby incorporated by reference. These peptide diazomethanes are similarly thought to be poorly membrane penneant and non-specific.
There is some evidence that certain particular inhibitors of Calpain have certain therapeutic utilities. For example, leupeptin can facilitate nerve repair in prirnates.
Loxastatin (also known as EST, Ep460 or E-64d), a derivative of E-64, ~s believed to have utiliy in the treatment of muscular dystrophy. E-64d, while not having significant protease inhibitory activity itself, is believed to be converted to more potent fonns, such as to E-64c, inside a marnmalian body.
Evidence from electrophysiological studies suggests that one of the ear~iest factors in the chain of reactions leading to cell death is an increase in intracellular-free calcium as a consequence of Ca2+ channel opening and/or energy depletion. Intracellular calcium is lilcely to produce a large nwnber of consequences, including the activation of a large number of en~yTnes, including p~oteases, such as Calpain, lipases and kinases. An increase in intracellular calcium is also thoudlt to induce changes in gene expression.
Ischenua, head traurna and stroke have all been associated with the rele~ase of glutamate in amounts large enough to lead to excitotoxicity, the to~icity resulting from the actions of certain amino acids on neurons of the CNS. The e~cess glutamate and other factors, such as free radical damaBe of membranes or energy depletion, cause an increase in intracellular Ca2+. It is known that an excess of intraceliular Ca2+ leads to several effects SUBSTITUTI~ S~E~T
.: ..
. :
.' . ' . ~, WO 92/11850 2 ~ ~ 8 ~ ~ ~ PCI`~US91/09786 believed to be associated with neuronal cell damage, including destruction of cell structures through activation of phospholi~ase and Calpain, as well as free radical production resulting from ac~ivation of phospholipase and xanthine oxidase. Many other factors have been - associated with neuroto~city. For example, reductions in action potentials and changes in a wide variety of chemic~l markers are known to be associated wi~h neurons exposed to ischemic conditions.
Notwithstanding the foregoing understanding of certain aspects of neuroto~city, no effecdve therapy has yet been developed for most neurodegenera~ive diseases and conditions of the CNS. Millions of individuals suffer from these diseases and conditions. Thus, there is a need for therapies effective in treadng and preventing these diseases and eonditions.
Summarv of the Tnvention The present invention provide the use of a Calpain inhibitor compound or a pharmaceutically acceptable salt or derivativf~ thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal hanng or likely to experience a neuropathology associated with neurodegeneration. In certain embodiments of this use, the neurodegeneration is occurring due to e~citotoxicity, HIV-induced neuropathy, ischemia following denervation or injury, subarachnoid hemorrhage, stroke, mul~iple infarction dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease. Themediument can comprise a pharmaceutically acceptable carrier and is for parenteral 2û administration, such as for transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or infusion. The mediument can also be in a forrn suitable for oral use. In some embodirnents, the medicarnent is for substantially preventing neurodegeneration in a patient undergoing surgery during and subsequent to the surgery, such as for a patient undergoing neurosurgery, cardiovascular surgery or a surgery using general anesthesia. The Calpain inhibitor compound preferably enters t~ssue of the C~S of the mammaL such as through use of a membrane-permeant Calpin inhl~itor.
The present invention also provides an in vitro method of selecting Calpain Inhibitors for use as Calpain ~hibitor protectants in the in vivo treatment or inhibition of degeneration. This method includes identifying compounds having Calpain inhibitory activity vitro~ and identi~ing those compounds with Calpain inhibito~y activity that are membrane permeant through an vjtro assay for membrane permeance. The irL~i~o assay for membrane permeance can include providing a plurality of tissue portions from a mammal; treating a~ least one, but not a~, of the tissue portions with a Calpain Inhibitor;
WO 92/11850 2 ~ 9 8 ~ 0 9 PCI'/US91/097~
subjecting the tissue portions to an eYent that can cause degeneraeion in untreated eissue;
measuring the amount of degeneration that occurs in ehe tissue poreions; and comparing the arnount of degeneration that occurs in the treated tissue portions with ths amount of degeneration occurring in the untreated tissue portions. The amount of degeneration in the treated tissue portions being less than the amount of degeneration ir~ the untreated tissue portions indicates that the Calpain Inhibitor is membrane-permeant. The tissue portions in some embodiments are brain sli~es, platelets or ceUs in cultllre. The measuring step in some embodirnen~s involves analyzing the tissue portions for the presence of the BDP's of a cytoskeletal eomponent such as spectrin, MAP2, actin binding protein or tau. The measuring step can also include measuring the electncal actiYity of the tissue portions. The in vitro assay for membrane per~neance can b~ performed by measuring the ability of the Calpain Inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the platelets.
Ihe present inYention also proYides the use of a Substituted Hetero~yclic Compound or a pharmaceuticaUy acceptable salt or derIvatiYe ~hereof for the manufacture of a medicament for inhbiting or treating neurodegeneration in a mammal haYing or likely to experience a neuropathology associated with ne~lrodegeneration. The medicarnent is preferabk~r for inhibiting or treating neurodegeneration of the CNS. The Substituted Heterocyclic Compound preferably is a member of the Class I Substituted ~socoumarins, the Class II Substituted Isocournarins or the Class m Heterocyclic Compounds. More preferably, the Substituted Heterocyclic Compound is 3-chloroisocournarin; a 3,4-dichloroisocoumarin; a 3-alkoxy-7-arnino 1-chloroisocoumarin; or a 7-substituted 3-alkoxy~-chloroisocoumarin. The use of Clairn 17, wherein said Substituted Heterocyclic Compound isCl~PrOIC,NH2-Cl~PrOIC,PhCH2NHCONH-C~PrOIC, CH3CONH~CiTPrOIC,L-Phe-NH-ClTPrOIC, PhG~I2NHCONH~Cl~EtOIC, PhCH2CONH-Cs~EtOIC, or D-Phe-NH-' CiTEtOIC.
Another aspect of the present invention provides the use of a Peptide Keto-Compound hav~ng Calpain inhl~itory activity or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for in}~iting or treatingneurodegeneration in a marnmal having or likely to e~perience a neuropathology associated with neurodegeneration. The medicament is preferably for inhibiting or treating neurodegeneration of the GNS. The Peptide Keto-Compound preferably is a peptide a-ketoester, a peptide a-ketoacid or a peptide a-ketoamide. More preferably, the Peptide Keto-Compound is a member of one of the following su~classes: Dipeptide a-Ketoesters SUBS~tTUTE SHEET
.~ .
, . ~ .
;: WO 92/1185û 2 ~ 9 ~ ~ ~ 9 PCI/lUS91/09786 (Subclass A), Dipeptide ~-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide -Ketoesters (Subclass B), Tetrapeptide ~-Ketoesters, Amino Acid Peptide -Ketoesters, Dipeptide a-Ket~acids (Subclass A), Dipeptide -Ketoacids (Subclass B), Tn~eptide -Keeoacids, Tetrapeptide a-Ketoacids ~nd Arnino A~id Peptide ~-Ketoacids, Dipeptide ~-Ketoa~nides (Subclass A), Dipeptide a-Ketoarnides (Subclass B), T~ipeptide c~-Ketoarnides, Tetrapeptide a-Xetoarnides or Amino Acid a-Ketoarnides.
Still another aspect of the present invention provides the use of a Halo-Ketone Peptide having Calpain in~ubitory ac~vity or a pharrnaceutically acceptable salt or derivative thereof Ior the manufacture of a medicament for inh~biting or treating neurodegeneration in a mammal having or ]ikely to experience a neuropathology associated with neurodegeneration. The medicament is preferably for inhibiting or treating neurodegeneration of the CNS. The Halo-Ketone Peptide can be an arnino-halo ketone peptide or a diazo-ketone peptide.
The uses of the present invention of Substituted Heterocrclic Compounds, PeptideKeto-Compounds or Halo-Ketone Peptides can be used in connection with neurodegeneration associated with e~citotoxicity, HIV-induced neuropathy, ischernia, subarachnoid hemorrhage, stroke, bra~ seizure, major heart attack, multiple in~arction dementia, Alzheirner's Disease, Huntington's Disease or Parkinson's Disease. Themedicament of these uses can indude a pharmaceutically acceptable carrier, and be for parenteral administration, such as transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or an infusion technique. The medicament can also be in a form suitable for oral use.
These uses can also be in coMection with neurodegeneration is occurring from ischemia-inducing events, stroke, head injury, major heart attaclc, brain seizure, near drowning, carbon monoxide poison~ng, surgery-related brain damage or another event known to causeneurodegeneration.
Another aspect of the present invention provides a method of minim~ing proteolysis in an in ~o sample containing peptides or proteins during or following the prccessing, production, preparation, isolation, purification, storage or transport of the samples, comprising ehe addition eo the sasnple of a Substituted Hetero~clic Compound or a Peptide Keto-Compound that is a member of one of the following subclasses: Dipeptide a-Ketoesters (Subclass A), Dipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide a-Ketoesters (Subclass 8), Tetrapeptide a-Ketoesters, Arnino Acid Peptide -Ketoesters, Dipeptide a-Ketoacids (Subclass A), Dipeptide a-Ketoacids (Subclass ~VO 92/11850 2 ~ 9 8 6 ~ 9 -6- PCI/US91/~97~ ~
B), Tripeptide ~-Ketoacids, Tetrapeptide a-Ketoacids, Amino Acid Peptide a-Ketoacids, Dipeptide ~-Ketoarnides (Su~class A), Dipeptide a-Ke20arnides (Subclass B), Tripeptide a-Ketoamides, Tetrapeptide a-Ketoarnides or Amino Acid a-Ketoam~des. The presentinvention also provides a method of n~nim~g degradation resul~ting from Calpain act~vity S in a tissue sample during or following preparation of the sample, comprising the addition to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo-Ketone Peptide. The sample can be a whole organ and the addition of compound comprises perfusion of the organ with the compound disso~ved in fluid. Preferably, following the addition step, the tissue sample is used in an assay for neurodegeneratiQn wherein the assay comprises a test for the products of Calpain activity in the tissue samples. The addition of compound in either of these methods can include the addition of a Peptide a-Ketoacid to the sample. Preferably, this Peptide a-Ketoacid comprises a compound that is a member of one of the fo110wing subclasses: Dipeptide a-Ketoacids (Subclass A), Dipeptide a-Ketoacids (Subclass B), Tripeptide -Ketoacids, Tetrapeptide a-Ketoacids or Arnino Acid Peptide a-Ketoacids.
Yet another aspect of the present invention provides pharmaceutical compositionsfor the treatment or inhibition of neurodegeneration. These compositions include a pharmacologically effective neuroprotective amount of a Substituted Hetero~yclicCompound, Peptide Keto-Compound or Halo-Ketone Peptide, or pharmaceutically acceptable salts or derivatives thereof in a pharmaceutically acceptable forrnulation containing a carrier material. In one preferred embodirnent, a Peptide Keto-Compound is included in the composition where~n said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide a-Ketoesters (Subclass A), Dipeptide a-Ketoesters (Subclass B), Tripeptide -Ketoesters (Subclass A), Tripeptide a-Ketoesters (SubclassB), Tetrapeptide a-Ketoesters, orAminoAcid Peptide a-Ketoesters. This Pepdde Keto-Compound is preferabb one of the following compounds: Bz-DL-Ala-COOEt, Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz-DL-Lys-COOEt, PhCO-Abu-COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu-COOEt or Ph(CH2)6CO-Abu-COOEt. Another preferred Peptide Keto-Compound is one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL-Ala-COOBzl, Z-Ala-DL-Ala-COOnBu, Z-Leu-Nva-COOEt, Z-Leu-Me-COOEt, Z-Leu-Abu-COOEt, ~-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, ~-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe-C02Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala~DL-Ala-COOMe, or Z-Leu-Abu-CO2Bzl. Still other SU6~ I tTUl ~ SHFET
. .
WO 92/11850 2 ~ ~ g ~ Q 9 P~/US91/09786 Peptide Keto-Compounds ~r use in ~he preferred compositions are one of ~he followin~
compounds: Z-Ala-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt, Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DL-AbuCOC)CH2-C6H4-CF3 (para), H-Leu-Ala-DL-Lys-COO~t, ZLeu-Leu-Abu-COOEt, ZL~u-Leu-Phe-COOEt, MeO-Suc-Val-Pro-D~Phe-COOMe or 2-NapSO2-IRu-Ieu-Abu-COOEt. A preferred Peptide Keto-Compound could also be MeO-SucAla-Ala-Pro-DI~Abu-COOMe or Z-Ala-Ala-Ala-DI,Ala-COOEt. Still other preferred Peptide Keto-Compouulds would be a compound ~om one of the following subclasses: Dipeptide a-Ketoacids (Subclass A), Dipeptide ~-Ketoacids (Subclass B), Tripeptide a-Ketoacids, Tetrapeptide a-Ketoacids or the Amino Acid Peptide a-Ketoacids.
Thus, preferred Peptide Keto-Compounds could be one of the following compounds:
Bz-DL-Lys-COOH, BznDI~Ala-COOH, ZLeu-Phe-COOH or ZLeu-Abu-COOH. A
compound ~om one of the following subclasses: Dipeptide a-Ketoamides (Subclass A), Dipeptide a-Ketoamides (Subclass B), Tripeptide a-Xetoamides, Tetrapeptide a-Ketoamides or Amino Acid a-Ketoamides, could also be used in the preferred compositions. Thus, another preferred Peptide Ke~o-CompouIld would be one of the~ollow~g compounds: ~Leu-Phe-CC)NH-Et, ~Leu-Phe-CONH-nPr, ~Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-:Bzl, ~Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu~Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, Z-Leu-Abu-CONH-Bzl, ~Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH~2O, ZLeu-Abu-CONH-(C~H2)7CH3, ZLeu-Abu-CONH-(C~I2)20H, ZLeu-Abu-CONH-(CH2)20(CH2)20H, Z-Leu-Abu-CONH (CH2)l7CH3, ZLeu Abu CONH-CH2-C6H3(0CH3)2 or Z-Leu-Abu-CONH-CH2~C4H4N. The composition can also include a Substituted Heterocyclic Compound such as one of the Class I Substi~u~ed Isocoumarins, Class lI Substituted Isocoumarins or Class m Heterocyclic Compounds. Preferred Substituted Hetero~yclic Compounds are 3-chloroisocoumar~n, a 3,4-dichloroisocoumarin, a 3-allco~y-7-amino 1~1oroisocoumarin, a 7-substitu~ed 3-alkoxy-4~chloroisocournarin;
CiTPrOIC, NH2-Cl~TPrOIC, PhCH2NHCONH-ClTPrOIC, CH3CONH-ClTPrOIC, L-Phe-NH-GTPrOIC, PhCH2NIICONH-ClTEtOIC, PhCH2CONH-ClTEtOIC, or D-Phe-NH-CiTEtOIC In these compositions, the composition is preferably in dosage ~orm comprising from 70 llg to 7 g of active ingredient in each dose, and the carrier material includes a liquid, wherein ~he composition is ~n dosage form and wherein each dose comprises ~om 05 ml to 1 liter of said carrier material. The compositions can additionally include at least one of the follo~ing: DMSO or other organic solvent, a lipid carrier, a detergent, a .~
:, ~ ~ ,, '- :
- . . -: . . . . . .
WO 9Z/11850 2 0 9 8 ~ ~ ~ PCr/US91/o97~ ;-surfactant, or an emulsifying agent. These compositions can be su~table for parenteral administration or in a forrn suitable for topical application. The compositions can be in a variety of forms, such as an aqueous solution, a lotion, a jelly, an oily solution, or an oily suspension.
S The present invention also provides the use of a Substituted Heterocyclic Compound as a medicament, the use of a Halo-Ketone Peptide as a medicament, and the use of a Peptide Keto-Compound as a medi~ament, wherein said Peptide Xeto-Compound is a compound from one of the following subclasses: Dipeptide ~-Ketoesters (Subclass A), ~ipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide ~-Ketoesters (Subclass :E3), Tetrapeptide a-~etoesters, or Amino Acid Peptide a -Ketoesters.
Preferred Peptide Keto-Compounds in this use ~nclude any one of the Peptide Keto-Compounds desaibed above in connection with the pharmaceutical compositions.
Brief Summarv of the Fi@lres Figure 1 shows the percentage of i~u~ition of glut~nate-induced cell death through the addition of ~lutamate and various Calpain Inhibitors rela~ve to control where no glu~amate was added.
Figure 2 graphically depic~s the e~ects of ZLeu-Phe-C:ONH-Et (CX269) and ZLeu-A~u-CONH-Et (CX275) on the size of iT~rction produced upon MCA occlusion in malerats.
~igure 3 shows the effects of CX216 (ZLeu-Phe-CO2Et, a Peptide Keto-Compound), and C:I1 (Ac-Leu-Leu-Me-H) rela~ve to ~ontrol slices on survival of hippocampal slices exposed to 10 minutes exposure of anoxic atrnosphere where both of these compounds were added at their optimal inhibitory concentradon at both 1 hour and 2 hour incubation times.
Figure 4 shows the evoked potential amplitude for control, CI1 treated and CX218treated hippocampal slices over a time course during which the slices sre exposed to anoxic atmosphere.
Figure 5 shows the percent recovery of EPSP from severe hypoxia over the course of one hour incubation for ~Leu-Phe-CONH-Et (CX269) and ~Leu-Phe-CO2Et (CX216).
Figure 6 shows a comparison of the ef~ect of the presence of CI1 or CX216 on survlval of hippocampal slices expressed as the duration of anoxia (in minutes) before fiber volley disappearance.
Figure 7 shows the effects of CI1 compared with control on the behavioral and convulsive effects of kainic acid.
~;UBSTITUTE SHEEJ
--.
W(:~ 92/11850 2 0 ~ PC11US91/097~6 "~., ~
Figure 8 shows the amount of spectrin BDP's in rat brains e~posed to kainate forcontrol and CI1 treated rats.
Detailed Description of the Invention A. INTRODUCI ION
We haYe discovered that Calpain activation is an event central to many cases of - bra~n atrophy and degeneration and that inhibition of Calpain alon~ is su~icient to inhibit or prevent cell deterioration and loss. Thus, we have further discovered that inhibition of C~ain provides protertion from neuroto~ncity associated with many neurodegenerative conditions and diseases.
In accordance with the foregoing di~coveries, we believe that the elevation of intracellular ~alcium a~sociated with neuropathological condition~ in neuronal cells activates Calpain and sets in motion the digestion OI neuronal ce11s from within. We believe there may be other mechanisms of activation of Calpains associated with these conditions.
Accordirlgly, o~e aspect of ~he present invention is di~ected to inhibidon and treatment of 1~ the neurodegeneration and o~her diseases associated with this dic,estion through the inhibition of Calpain actiYity. Thus, part of this aspect of the present invention is to prevent the neurodegeneradon and other pathology caused by this diges~ion through the vivo adrninistration of Calpain inhll~itors. By way of example, and not of lirnitation, diseases and conditions which can be treated using this aspect of the present invendon include 20 neurodegeneradon following e~citotoxicity, HIV-induced neuropashy, ischenua, denervation foJlowingischemia orinjury, subarachnoid hemorrhage, stroke, multipleinfarction dementia, Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease, surgery-related brain damage and other neuropathological conditions.
As stated above, spectrin BDP's have been found to be associated with Calpa~n 25 activation in vivo, We have obsened that in each instance of neurodegeneration in which BDP's characteristic of Cal~ain activation are detected, Calpain activation is localized to the brain areas most vulnerabIe to the particular pathogenic manipulation. Isl addition, as judged by histological methods, C:alpain activation precedes overt evidence of neurodegenerat;on. Accordingly, CalpaM activation is spatially and temporally lir~ced to 30 impending or ongoing ce~l death in the brain. Thus, we believe that Calpain activation is an important mechanism of cell damage and death in many pathological conditions,induding neuropathological conditions. Moreover, there is eYidence that the activation of Calpains is an early event in the death of cells including neural cells. This is in contrast to other known proteases which are activated at later stages of cell death. Thus, we believe - ,; , . . . . . ., . ..................... . . . :
-. ~
: ~. - ~ . . . -wo 92/11850 2 0 9 8 6 a 9 pcr/us91~o978 ~
that, advantageously, inhibition of Calpain activity provides intervention at an early stage of cell death, pnor to significant deterioration of ee1lular machinery.
Another aspect of the involvement of Calpains in neurodegeneration is the involvement of these proteins in regenerating systems. It is Icnown that developing or regenerating axons are somehov inhibited from further development in a stabilization process called the "stop pathway.~ This stabilization can occur when axons have reached their targets; however, in some systems stab;lization can also occur at inappropriate places.
One researcher has de~eloped evidence that this stop pathway operates at least in part by the activation of intracellular Calpain and that in11ibition of Calpain can interfere vnth stabilization (Luizi, 1990). We believe that Calpain inhibitors, when used in accordance with the present invPntion, can advantageously aid regeneration and recovery of neural tissue after injury, in addition to inhl~iting neurodegeneration.
Another aspect of the present invention is our diseovery that at least three classes of compounds, the substituted isocournarins, the peptide keto-compounds and the Halo-Ketone Peptides have Calpain inhibitory activity. We have further discovered, as will be described hereinbelow, that these three classes of compounds exhibit additional properties that render them e~pecially useful as therapeutically effective compounds in the ereatment of neurodegenerative conditions and diseases.
B. SUBSTl~TED HEleROCYCIIC COMPOU~DS
One particular class of compounds exhibiting Calpain inhibitory activity, ~hen used in accordance with the present invention, are the substituted heterocyclic compounds. These compounds include the substituted isocoumarins. The substituted heterocyclic compounds are known to be excellent inhl~itors of serine proteases. As discussed hereinbelow, we have now discovered that these compounds are also inhibitors of calpain I and calpa~n I~, and also 2~ of other Calpains. Additionally, as also diuussed below, we have found that, unlLke most ~ known inhibitors of Calpains, these substituted heterocyclic compounds are not effective as ir~ibitors of papain or cathepsin B. Thus, we believe that the sl~bstituted heteros~yclic compounds provide a relatively specific means of inhibiting Calpams while not affecting other thiol proteases.
One particular dass of substituted heterocyclic compounds with Calpain inhibitory ' aceivity are the isocoumarins having cationic substituents. These substituted hetero~yclic compounds are referred to herein as the "Class I Substituted Isocoumanns." The aass I
Substituted Isocoumarins are known to be e~cellent inhibitors of several serine proteases, induding bovine thrombin, human thrombin, hu nan factor Xa, human factor X~a, lluman !E~IUE~STIITU~E SH~T
~ WO92/11~,50 ~ 36~ PC~/US91/0~7f~6 factor X[Ia, bovine trypsin, human plas,ma plasmin, hurnan tissue plasrninogen activator, hurnan lung tryptase, rat skin tryptase, human leukocyte elastase, porcine pancreatic elastase, bovine ch,vmotrypsin and human leuko~yte cathepsin G. The Class I Substituted Isocoumarins inhibit the serine proteases by reaction with the active site serme to form an acyl er~ne, which in some cases may further react with another active site nucleophile to form an additional covalent bond. We have discovered that the Class I Substituted Isocoumarins also react with Calpain. We believe that the mechanism of action of Calpain inllibition is similar to that of the iinh~bition of serine proteases since the reaction mechanism of Calpains is similar to that of the serine proteases.
The Class I Subsdtuted Isocoumarins having Calpain in~u~itory activity have the follo~ing structural forrnula:
~ O
(I) ~2 or a phannaceutically acceptable salt, wherein Z is selected from the group consisdng of C1-6 aLlco~y with an amino group attached to the alkoxy group, C1-6 alkoxy with an isothiureido group attached to the aL~coxy group, Cl-6 aL~coxy with a guanidino group attached to the aL~coxy group, C1-6 allcoxy with an a nidino group attached to the allco:y group, C1-6 alkylwith an arnino group attached to the alkyl group, C1-6 allyl with an isothiureido group attached to the allyl group, C1-6 aLlcyl with an guanidino group attached to the allyl group, C1-6 aLcyl with an an~idino group attached to the alkyl group, R is selected from the g;oup consist~ng of O=C=N-, S= C=N-, AA-NH-, AA-AA-NH~ O,AA-AA-O-, M-NH-, M-A~A-NH, M-AA-AA-NH-, M-O-, M-AA-O-, M-AA-AA-O-, wherein AA repre~,erlts alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, tnreonine, cysteine, tyrosine, beta-alanine, ~BSTITUT SHE.ET
.: , ' : -WO 92/11850 2 a 9 8 6 0 9 PCr/lJS91/0978 norleucine, nonaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine or sarcosine, wherein M represents NH2-CO-, NH2~ , NH2-S02-, X~ CO, X-NH-CS, X-NH-S02, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-, S wherein X represents C1-6 allyl, C1-6 fluoroalkyL C1-6 aLtcyl substituted with K, C1-6 fluoroalkyl substituted wilh K, phenyl, phenyl substituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, naphthyl, naphthyl subsdtuted ~ith J, naphthyl disubstituted with J, naphthyl trisubstituted with J, C1-6 aLkyl with an attached phenyl group, C1-6 allyl with two attached phenyl groups, C1-6 allyl with an attached phenyl group substituted with J, or C1-6 aL~cyl with t~o attached phenyl groups substituted with J, wherein J represents halogen, COOH, OH, CN, N02, C1-6 allyl, C1-6 aL~coxy, C1-6 all~lamine, C1-6 dialkylarnine, or C1-6 alkyl-O-CO-, wherein K represents halogen, COOH, OH, CN, N02, NH2, C1-6 aLkylamine, C1-6 diaL'cylan~ine, or C1-6 alkyl-O-CO-, Y is selected from the group consisting of H, halogen, trifluoromethyL methyl, OH
and metho~y.
The compounds of Formula (I) can also contain on or more substituents at position B as shown in the following structure:
R~ o E3~2 y ` -wherein electronegative substituents such as N02, CN, CI, COOR, and COOH will increase the reactivity of the isocou narin, and electropositive substituents such as NH2, OH, aLcoxy, thioallyL allyl, allylamino, and diaLcylamino will increase its stability. Neutral substituents could also increase the stability of acyl enzgme and improve the effectiveness of she inhibitors.
The following compounds are representative of the Class I Substituted Isocoumarins of ehe present invention:
S~ STITUTE SHEE~ T
`-!W092/ll850 2098~a9 PCr/US91/09786 .
4-chloro 3-(3-isothiureidopropoxy)isocoumarin (CiTPrOIC) 7-(benzylcarbamoylamino)4-chloro-3-(3-isothiureidopropoxy)isocoumarin (PhCH2NHCONH-CiTPrOIC) 7-(phenyl~arbamoylamino)~-chloro-3-(3-isothiureidopropoxy)isocoumarin (PhNHCONH-CiTPrOIC) 7-(acetylarnino)~-chloro-3-(3-10 ~sothiureidopropoxy)isocoumann (CH3CONH-CiTPrOIC) 7-(3-phenylpropionylarnino) 4~hloro-3-(3-isothiureidopropoxy)isocoumarin (PhCH2OE12CONH-CiTPrOIC) 7-(phenylacetylamino)4-chloro-3-(3-isothiureidopropoxy)isocoumarin (PhCH2CONH-CiTRrOIC) 7-(L-phenylalanylamino)~-chloro-3-(3-isothiureidopropoxy)isocoumarin (L-Phe-NH-CiTPrOIC) 7-(N-t-butyloxycarbonyl-I~phenylalanylamino) 1-chloro-3-(3-isothiureidopropoxy)isocoumarin (Boc-L-Phe-NH-CiTRrOIC) 7~ phenylalanylamino) 4-chloro-3-(3-isothiureidoprapoxy)isocoumarin (D-Phe~ CiTPrOIC) 7-(N-t-butyloxycarbonyl-D-phenylalanylam~no)4-chloro-3-(3-isothiureidopropoxy)isocoumarin (Boc-D-Phe~ ClTPrOIC) 7-(benzylcarbamoylamino)4-chloro-3-(2-isothiureidoethoxy)isocoumarin (PhCH2NHCONH-ClTEtOIC) 7-(phenylcarbamoylamino)4-chloro-3-(2-isothiureidoethoxy)isocou~narin (PhNHCONH-CiTEtOIC) 3~ .
7-(isopropylcarbamoylamino)4-chloro-3-(2-isothiureidoethoxy)isocoumarin ((CH3)2CHNHCONH-CiTEtOIC) 7-(phenylacetylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin OEhCH2CONH-CiTEtOIC) 7-(L-phenylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin (L-Phe-NH-Cl~ tOIC) 7-(N-t-butylo~ycarbonyl-L,phenylalanylamino)-4-chloro-3-(2-isothiureidoetho~y)isocoumarin (Boc-L-Phe-NH-Cl~EtOIC) 7-(D-phenylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin (D-Phe-NH-CiTEtOIC~
SlJBSrlllJTE SHE~T
WO 92/11850 2 ~ 9 8 6 0 9 Pcr/usgl/097~! ~
7-(N-t-butylo~ycarbonyl-D-phenylalanyl~nino)4 chlo}o-3-(2-isothiureidoetho~y)isocoumar~n (Boc-D-Phe~ Cl~EtOIC) 7-(N-t-butyloxycarbonyl-L-alanyl-L-alanylamino)-4-chloro-3-(2-isothiureidoethoy~isocoumarin (13Oc-Ala-Ala-NH-CiTEtOIC) 7-(L-alanyl-L-alanylamino)~-chloro-3-(2-isothiureidoethoxy)iso~oumar~n (ala-Ala-NH-ClTEtOIC3 7 (1-naphthylcarbamoylamino)~-chloro-3-(2-isothiureidoethoy)iss)coumarin (:NaphthylNH-CiTEtOIC) 7-((S) a methylben~ylcarbamoylamino)~-chloro-3-(2-isothiureidoetho~y)isocoumann (S-C6H5(CH3)CHNHCONH-C~ tOIC3 15 7~((R)-a-methylbenzylcarbamoylarnino)-4 chloro-3-(2-isothiureidoetho~y)isocoumarin (R-C6H5(CH3~CHNHCONH-Cl~EtOIC) 7-das~sylan~ino~-chloro-3-(2-isothiureidoetho~y)isocoumarin (DansylNH-CiTEtOIC) 7-phenylthiocarbamoylamino~-chloro-3-(2-isothiureidoeth~xy)isocournarin (PhNHCSNH-Cl~EtOIC) 7-(m~arboxyphenylthiocarbamoyl)amino 1-chloro-3-(2-isothiureidoetho~y)isocoumarin (m-COOH-PhNHCSNH-CiTEtOIC) 7-(p~arboxyphenylthiocarbamoyl)amino 4-chloro-3-(2-isothiureidoethoxy)isocoumarin (p-COOH-PhNHCSNH-CiTEtOIC) 7-amino~-chloro-3-(3-isothiureidopropoxy)isocoumarin (AClTIC) Isocoumarins with basic substituents are also known to be effective inhibitors of serine proteases. See Powers et al, U.S. Patent No. 4,845,242, the disclosure of which is hereby incorporated by reference. This class of compounds, referred to herein as the "Class II Substituted Isocoumarins," along with tbe other substituted heterocyclic compounds, is believed to be effective in the use of the present invention.
SI~E'r WO 92/11850 2 ~ ~ ~ 6 a ~ PCI`/US91/09786 ", .
The Class II Substituted Isoc~umarins have the following structural formula:
O
S p~ O
~Z
Y
or a pharmaceutically acceptable salt, wherein R is selected from the group consisting of -N-H-C(=NH)-NH2, -C(=NH)NH2, Cl 6 aL~cyl with an attached amino, and Cl~ alkyl with an attached isothiureido of the formula -s-c(+NH2+)N~2~
Z is selected from the group consisung of H, halogen, Clb allyl, Cl 6 allyl with an attached phenyL Cl 6 ~uorina~ed aLlyL Cl 6 allyl with an attached hydroxyl, Cl 6 allyl with an attached Cl~ alkoxy, Cl.6 aLtco~y, Cl 6 fluorinated alko~y, Cl 6 aL~co~y with an attached phenyL benzyloxy, 4-fluorobenylo~y, -OCH2C~I 4R' (2-substituent), -OCH2C6H4R' (3-substituent), OCH2C6H4R' (4-substituent), -OCH2C6H3R2' (2,3-substituents), -OCH2C6H3R2' (2,4-substi~uents), -OCH2C6H3R2' (2,5-substituents), -OCH2C6H3R2' (2,6-substituents), -OCH2C6H3R2' (3,4-substituents), and OCH2C6H3R2' (3,5-substituents).
R' is selected from the ~oup consisting of H, halogen, trifluoromethyL N02, cyano, methyL methoxy, acetyL carboxyL OH, and amino.
Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy.
Alternately, the Class II Substityted Isocoumarins are represented by structure pI) where, Z is selected from the group consisting of C~ coxy with an attached isothiureido, Cl 6 alkoxy with an a~tached guanidino, Cl 6 aL~coxy with an attached amidino, Cl.6 aLlcyl with an anached amino, Cl 6 allcyl with an attached isothiureido, Cl 6 allyl with an attached guanidino, Cl.6 aLlcyl with an attached asnidino, R is selected from the group consisting of H, OH, NH2, NO2 halogen, C~ coxy, C1.6 fluorinated aL~coxy, Cl 6 alkyl, C1 6 allyl with an attached amino, ~ , M-AA-O-, ~. ~n~ n~rt 1T~ C~FFT
WO 92/11850 2 0 9 8 6 0 9 Pt~r/US91/t)97~i .
wherein AA represents alanine, valine, leucine, ~soleuc~ne, proline, methior~ne,phenylalanine, tryptophan; glycine, ser~ne, threonine, cyste~ne, tyrosine, asparagine, glutamine, aspartic acid, glutan~ic acid, lysine, ar~nine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, ci~rulline, hydroxyproline, on~ithine and sarcosine, wherein M represents H, lower alkanoyl having 1 to 6 cazbons, carbo~ canoyl, hydroxyaLkanoy1 amin-alkanoyL benzene sulfonyl tosyL benzoyl, and lower aLlcyl sulfonyl hav~ng 1 to 6 carbons, Y is selected from the group consist~ng of H, halogen, trifluorome~hyL methyl OHand methoxy.
As a further alternative, the Class II Substituted Isocoumarins are represented by structure (II) where R is selected from the group consisting of -N-H-C( =NH)-NH2, -C( = NH)NH2, allyl with an attached an~ino, Cl 6 allyl with an attached isothiureido, Z is selected ~om the group consisting of Cl 6 alkoxy with an attached amino, Cl 6 alkoxy with an attached isothiureido, Cl 6 alkoxy with an auached guanidino, Cl 6 aL~;ox~
with an attached amidino, C1 6 alkyl with an attached arnino, Cl 6 alkyl with an attacheo guanidino, Cl 6 alkyl with an attached amidino, Y is selected from the group consisting of H, halogen, trifluoromethyL methyL OHand methoxy.
The following compounds are representative of the Class II Substituted Isocoumarins:
3-(3-ammopropoxy)isocoumarin, 3-(3-ammopropoxy)~-chloroisocoumar~n, 3-(2-isothiureidoethoxy)4-chloro~socoumarin, 3-(3-isothiureidopropoxy)4 chloroisocoumarin, 7-amino-3-(3-isothiureidopropoxy)~hloroisocoumarin, 7-g,uanidino-3-methoxyisocoumarin, 7-guanidino-3-methoxy 4-chloroisocoumarin, 7-guar~idino-3-etho~yisocournarin, 7-guanidino-3-ethoxy~hloroisocournarin, 7-guanidino-3 -(2-phenylethoxy)isocoumarin, 7-guanidino-3-(2-phenyletho~y)~-chloroisocoumarin.
S~J71~E SWEET
WO 92/11850 2 ~ 9 ~ PCT/US91/09786 17~
Still another class of susbstituted heterocyclic compounds useful in the presentin~ention ~s referred to herein as the "Class m Heterocyclic Compounds" ~nd have the follow~ng structural formula:
.
~Y
(m) ~ x 1~
wherein Z is selected from the group consisting of CO, SO, SO2, CCl and CF, Y is selected from the group consisting of O, S and NH, X is selected from the group consisting of N and CH, and R is selected from the group consisting of Cl 6 aLtcyl (such as methyl, ethyl and propyl), Cl ~ allyl containing a phenyl (such as benzyl), and Clb fluoroaL~cyl (such a~
trifluoromethyL pentafluoroethyL and hepta~uoropropyl).
The Z group must be electrophilic since it interacts with ~he active site serine OH
group of the serine protease. The R group must be uncharged and hydrophobic. One or more of the carbons in the R group could be replaced by O, S, NH and other such atomic groups as long as the R ~oup maintains its hydrophobic character.
The following compounds are representatire of the Class m Heterocyclic Compounds:
2-trifluorome~hyl 1H-3,1-benzo~azine4-one, 2-pentafluoroethyl-4H-3,1-benzoxazine-4-one, 2-heptafluoropropyl-4H-3,1-benzoxazine-4-one, 2-methyl4H-3,1-benzoa~ine~one, 2-propyl-4H-3,1-benzoa~azine-4-one, 2-benzyl-4H-3,1-be~L~oaxa~ine-4-one, 2-hepta~uoropr3pyl4-quinazolinone, 2-propyW-quina~olinone, 2-benzyl-4-quinazolinone, SU3S~lTU~E SHEET
, '`.. . ` ~ . `
.` ` `
` . ~`. ` ~. ~ . ` .
WO 92/11850 2 ~ 9 g ~ 9 PCI/US91tO978 ;~`
2-(C6HsCCl2)4~hloroql~inazoline, and 2-propyl-4-chloroql~ina~oline.
The Class m Heterocyclic Compounds are disclosed in Powers et al., U.S. Patent No.
4,847,202, the disclssure of which is hereby incorporated by reference.
Other substituted heterocyelic ~ompounds have been prepared earlier for other purposes, such as 3-chloroisocoun~arin, Davies and Poole, J. Chem. Soc., pp. 1616-1629 (1928); 3~hloro and 3,4-dichloroisocoumarin, Milevslcaya, Belins3caya, and Yagupol'sku, Zhur. Org. Xhim. 9, pp. 2145-2149 (1973); 3-methyl and 4 carbo~y-3-methylisocoumarin, Tirodkar and Usgaonkar, Ind. J. Chem. 7, pp. 1114-1116 (1969); 7-nitro and 7-aminoisocoumarin, Cholcsey and Usgaonlcar, Ind. J. Chem. 14B, pp. 596-598 (1976). The disclosures of all of the preceding articles are hereby incorporated by reference. These other substituted isocoumarins are also believed to exhibit Calpain inhibitory activity when used in accordance with the present invention.
Still other substituted isocoumarins which have been prepared recently for inhibition of serine proteases are 3-chloroisocournarin, Harper, Hemrr~i, and Powers, J. A. Chem. Soc.
105, pp. 6518-6S20 (1983); 3,4-dichloroisocournarin, Harper, Hemmi, and Powers, Biochemistly 24, pp. 1831-1841 (1985); 3-alkoxy-7-arnino~chloroisocoumarin, Harper and Powers, J. Am. Chem. Soc. 106, pp. 7618-7619 (1984), ~Iarper and Powers, Biochemistry 24, 7200-7213 (1983); additional substituted isocoumarins with basic groups (aminoalkoxy, guanidino or isothiureidoallcoxy), Kam, Fujikawa and Power~, Biochemistry 27, pp.
2S47-25S7 (1988); 7-substituted 3-~lko~y~^chloroisocoumarins, Powers, K~m, Narasimhan, Olelcsys~yn, Hernandez and Ueda, J. Cell Biochem. 39, pp. 33-46 (1989) and Powers, Oleksyszyn, Narasimhan, Kam, Radhakrishnan and Meyer, Jr. Biochemistry 29, 3108-3118 (1990). The disclosures of all of the preceding articles are herebyincorporated by reference.
We believe that the foregoing compounds, which exhibit serine pro~ease inhibitory activity, also e~hibit Calpa~n inhbitory acti~ity when used in accordance with the pres.ent invention.
All of the ~oregolng isocoumarin compounds, including the Class I and II Substitu~ed Isocoumarins, the Class m Substituted Heterocyclic Compounds and the other substituted heterocyclic compounds useful in the practice of the present invention shall be referred to collectively hereinafter as the "Substituted Heterocyclic Compounds." The term "Substituted Heterocyclic Compound" shall be used to refer to any par~icular species of these compounds.
The preparation of the various Substituted Heterocyclic Compounds is illustrated by Examples SHC1-SHC9.
~:U8~:~TUTE SHEET
. .
.
W0 92/11850 2 0 9 ~ 6 ~ ~ PCl'/US91/09786 EXAMPLE SHCl Preparation of 7-(phenylcarbamoylam~no)~4-chloroisocoumar~n was synthes~zed as previously described (Powers, e~ aL, Biochem~stn~ 29, 3108-3118 (1990)). This compousld (0.32 g 1 mtnole) was mLYed with phenyl isocyanate (0.12g, 1 mmole) in 5 ml of THF and the reaction S rni~ture was st~Ted at r.t. overnight. The product 7-(phenylcarbamoy~am~no)~-chloro-3-(2-bromoethoxy)isocoumarm precipi~ated out, yield 40~o, m.p. 215-217 C, mass spectrum m/e = 437.9 (M~ Anal. Calc. for Cl8Hl4N204ClBr: C, 49.4~; H, 322; N, 6.40; Cl, 8.10.Found: C,49.48; H, 3 25; N,634; Cl, 8.12. The phenylcarbarnoylan~no compound (0.1 g, 0.23 ~slole) was heated with 0.02 g of thiourea (0~6 mmole) in 10 ml of THF at 70C
overmght. The final product prec~pitated out, yield 0.04 g, 36%, m.p. 161-163C (dec.), mass spectrum (FAB+) m/e = 433 (M-Br). Anal. Calc. for ClgHl8N404ClBrS:0.25 THF:C, 45.12; H, 3.86; N, 10.53; Cl, 6.67. Found: C, 44.83; H, 3.92; N, 10.12; Cl, 6.41.
7-(Ethylcarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-(t-butylcarbamoylamino)-4-chloro-3-(2-isothiureidoetho~y)isocoumarin, 7-(benzylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-(ethylthiocarbamoylamino)~-chloro-3-(2-isothiureidoethoxy)isocoumarir~-(4-fluorobenzyl) thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-(2,5-dimethylbenzyl) thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared by the same procedure.
EXAMPLE SHC~2 Preparation of 7-(acetylamino)~-chloro-3-(3-isothiureidopropoxy) isocoumarin:
7-Amino-3(3-bromopropoxy)~chloroisocoumarin was synthesi2ed as previously described (Kam, et al., 1988). This compound (033 g, 1 mmole) was heated with 0.15 g of acetic anhydride (1.5 mmole) in 20 rnl of dry Th~. After a few n~inutes, a yellow solid precipitated out. After 3 hrs, the solution was concentrated to 5 ml, and the solidr was filtered to give 037 g of 7-(acetylamino)-4-chloro-3-(3-bromopropoxy) isocoumarin, m.p.
170-172C; mass spectrum m/e = 375 (M+). The acetylated isocoumarin (0.15 g, 0.4mmole) was treated with thiourea (0.036 g, 0.47 mmole) to ~ive 0.9 E~ o~ the final product, (yield 50%), m.p. 180-181C, maæ spectrum m/e = 370 (M+-Br). Anal. Calc. for Cl5Hl7N304ClBrS: C, 39.97; H, 3.80; N, 932; Cl 7.87. Found: C, 39.86; H 3.83; N, 9.29;
CL 7.~5.
7-trifluoroacetylamino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, 7-hepta~uorobutyroylamino 1-chloro-3-(3-isothiureidopropoxy)isocoumarin,7-succinylamino-~n ~ 8~
.. ,, . ~ .-.
. ~ ... . .
., ~ , -.. . . . ..
WO 92/118~0 2 ~ 9 8 6 0 9 PCI/lJS91/0978~
4-chloro-3-(3-isothiureidopropoxy) isocoumarin, and 7-(o-phthalyl)amino~-chloro-3 (3 isothiureidopropoxy) isocoumarisl can be prepared by the sarne procedur~.
E:XA~'LE SlH~
Preparation of 7 (benzylcarbamoylarnino)~-chloro-3-(3-isothiureidopropO~y) isocouunarin:
7-(benzylcarbamoylamino)-4~chloro-3(3-bromopropo~y) isocoumarin was prepared from the reaction of benzyl isoqanate with 7-amino4-chloro-3-(3-bromopropoxy) isocoumarin as descn~ed above, m.p. 188-189C, mass spectrum. m/e = 359 (M+ -ben~yl).
The final product was obtained ~om the reaction of 7-(ben~ylcarbamoylamino)4-chloro-3-(3-bromopropo~y) isocoumarin with thiourea as described aboYe (yield 74%), m.p.
165-166C; mass spectrum (FAB+) m/e - 461 (M+-Br). AnaL Calc. for C2lH22N404Cl~3rS:0.75 1~: C, 48.36; H, 4.70; N, 9.40; CL 6.56. Found: C, 48.13; H, 4.87;
N, 9.65j Cl, 6.15.
Preparationof7-(phenylacetylamino)~-chloro-3-(2 isothiureidoethoxy)isocoumarin:
7~Amina-4-chloro-3 (2-bromoethoxy) isocoumarin (0.15 g, 0.47 mmole) was first n~Lxed with phenylacetyl chloride (0.09 g, 0.55 mmole) in 10 ml of THF, triethylamine (0.05 g, 0.47 mmole) was then added and the reaction mi~ture was stirred at r.t. overnight. After Et3NHCl salt was removed by filtration, the product 7-(phenylacetylamino)~-chloro-3-(~-bromoethoxy) isocoumarin was crystallized from l~ and Pet. ether (yield, 73%), m.p.
165-169C; mass spectrum; m/e = 436.7 (M+). The phenylacetyamino derivative (0.1 g) was heated with thiourea (0.02 g) to give the product 0.05 g (yield, 40%), m.p. 115-120 C;
mass spectrum (FAB+) m/e - 432 (M+ -Br). Anal. Calc. for C20HlgN304ClBrS 0.5 H20:
C 45.9~; H, 3.83; N, 8.05; Cl, 6.80. Found: C, 46.09; H, 4.17; N, 8.02; Cl, 6.79.
Preparation of 7-(R-~-methylbenzylcarbamoylamino)-4-chloro-3-(2-isothillreidoethoxy) isocoumann:
7-(R a-methylbenzylcarbamoylamino) 1 -chloro-3-(2-bromoelhoxy) isocoumann was synthes~zed in the same manner as described above, m.p. 183-185C; mass spectrum m/e - 464 (M+). This compound (0.1 g) reacted with thiourea (0.02 g) under the same condition de~cribed above to form the finalproduct 7-(R-a-methylben~ylcarbamoylamino) 1 -chloro-3-(2-isothiureidoetho~y) isocoumarin (0.078 g), m.p. 143-150C; mass spectrum (FAB+) m/e 5 461 (M+ -Br). Anal. Calc. for C2lH2;~N404ClBrS0.5H20: C, 45.75; H, 4.35; N, 10.17; Cl, 6.M. Found: C, 44.95; H, 4.31; N, 10.02; CL 6.36.
Sl3BSm~UTE SHE~ET
.
: . :. -. WO 92/118~iO 2 8 ~ ~ ~ 0 9 PC~/US91/09786 EX~MPLE SHC6 P~eparatio!l of 7-(D-phenylalanylamino)-4-chloro-3(2-isothiureidoethoxy) isocousnarin:
- soc-D-phe (0.33 g, 1.2 mrnole) reacted with 1,3-dicyclohexylcarbodiimide (0.13 g, 0.6 - S mmole) in 10 ml THF at 0C for 1 hour ~o forrn the symmetric anhydride, and then 7-amino-4~hloro-3(2-bromoetho~y) isocoumarin (0.2g,0.6 mmole) was added. The reaction was stirred at r.t. overnight and the precipitate 7-(Boc-D-Phe-ammo)~-chloro-3-(2-bromoethoxy) isocoumarin was formed (0.29 g, 71%). TLC one spot, m.p. 180-182 C;
mass spectrum m/3 = 566(M~). Anal. Calc. for C25H2~jN206ClBr: C, 53.07; H, 4.63; N, 4.95; Cl 6.27. Found: C, 53.25: H, 4.66; N, 4.87; CL 6.24. E}oc-D-Phe compound (0.2 g, 0.35 rnmole) was reacted with thiourea (0.027 g, 035 mrnole) in the same maMer to give 7-(Boc-D-phenylalanylamino)-4 chloro-3-(2-isothiureidoethoxy) isocoumarin (0.14 g), yield 62%, mass spectrum (FAB+) m/e = 561 (M -Br). Thi5 compound (Q.1 g) was dissolved in 3 rnl of l~ at 0C and then the solvent was evaporated to dryness. The final product precipitated out after addition of ether, one spot on TLC (CH3CN:H20:AcOH = 8:1:1); mass spectnlm (FAB+) m/e = 462 (M+ -Br -CF3C00).
7-Boc-alanylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-benzoylamino-Ala-4-chloro-3(2-i~.othiureidoetho~y) isocoumarin, 7-benzoylamino-Phe-4-chloro-3-(2-isothiureidoethoxy) isocoumarin and 7-Boc-valylamino4-chloro-3-(2~isothiureidoethoxy) isocoumarin can be prepared by the same procedure.
Preparation of 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoethoxy) isocoumarin:
7-(Boc alanylalanylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin was synthesized2S in the same manner, m.p. 147-151C; mass spectrum m/e - 561 (M ' ). Anal. Calc: C, 47.12: H, 4.85. Found: C, 47.18; H, 4.87. This compound (0.2 g) was reacted with thiourea (0.03 g) by the same procedure to ~orm 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoetho~y) isocoumann (0.04 g), mass spectrwTI m/e = 556 (M+ -Br).
7-(Alanylalanylamino)-4-chloro-3(2-~sothiureidoethoxy) isocoumarin was prepared hy deblocking of 130c-Ala-Ala-NH-CiTEtOIC with trifluoroacetic acid, mass spectrum ` (FAB~ m/e - ~56 (M~ -Br-CF3COO).
Preparation of 7-(phenylthiocarbamoylamino) 1-chloro-3-(2-isothiureidoethoxy) isocoumarin:
~IQ~,~TttTF ~LJI~=T
wo 92/11850 2 ~ ~ 8 6 0 3 PCI/US91/0978~
-2~-7-(Phenylthiocarbamoyl~mino)~chlorG3-(2-bromoethoxy) isocournal~n was prepared from the re~ction of phenyl isothiocyanate with 7-amino~-chloro-3-(2-bromoe~hoxy) isocournarin,yield59%,m.p. 157-158C;massspectrwnm/e = 361 (M~ -PhNH+1). Anal.
Calc.: C, 48.36; H, 3.39. Found: C, 48.26; H, 3.40. The bromoetho~y compound was then reacted with thiourea by the same procedure to give the final product, yield 32%; mass spectrum (FAB+) m/s 449 (M+ -Br~.
Preparationof7-(m-carboxyphenylthiocarbamoylamino)~-chlo~o-3-(2-bromoethoxy) isocoumarin was prepared from the reaction OI m~arboxyphenyl isothiocyanate with 7-amino-4-chloro-3-(2-bromoetho~zy) isocoumarin, yield 64%, m.p. 157-158 C; mass spectrum m/e 361 (M+ -(COOH)PhNH+-Br).
7-(3-Fluorobenzoyl)a~uno 1-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-(3-nitrobenzoyl)amino-4-chloro-3-(2-isothiureidoethoxyysocoumarin,7-diphenylacetylarnino-4-chloro-3-(2-isothiureidoeshoxy) isocoumarin, 7-diph~nylpropionylanuno~-chloro-3-(2 isothiureidoethoxy) isocoumarin, 7-(p-toluenesulfonyl) amino-4-chlo~o-3-(2-isothiureidoethoxy) isocoumarin, and 7-(~-toluenesulfonyl) amino~-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared from the reaction of corresponding 7-substituted-4-chloro-3-(2~bromoethoxy) isocoumarin with thiourea as descnbed above. 7-substituted-4-chloro-3-(2-bromoethoy) isocoumarin can be synthesized by reacting 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin with appropriate acid chloride or sulfonyl chloride in the presence of Et3N.
7-Ethoxycarbonylamino4-chloro-3~(2-isothiureidoethoxy) isocoumarin, 7-benzyloxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-phenoxycarbonylan~ino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin c~n be prepared from T~ 25 the reaction of 7 substituted-4-chloro-3-(2-bromoethoxy) isocoumarin wi~h ~h-iourea. 7-Ethoxycarbonylamino4-chloro-3-(2-bromoethoxy~socownarin,7-benzylo;~ycarbonylan~ino~-chloro-3-(2-bromoethoxy) isocouunarin and 7-phenoxycarbonylamino-4~hloro-3-(~-bromoethoxy) isocoumarin c~n be synthes~zed by reac~ing 7amino-4-chloro-3-(2-bromoethoxy) isocoumarin with the corresponding chloroforrrlate.
30 C. PE~IlPE KETO-CO.MPOUNDS
Peptide a-ketoesters, peptide a-ketoacids, and peptide a-ketoamides are transition state analog inhibitors for serine proteases and cysteine proteases. While these subclasses of compounds are chemically distinguishable, for simplicity, all of these compounds will be referred to collectively herein as the "Peptide Keto-Compol~ndsn.
SU~3~TITVTE S~E~
. . . . .
WO 92/1~850 2 ~ 9 ~ ~ Q 9 Pcr/us91~o9786 The ~nserac~ions of peptides with serine and cysteine proteases are designated herein us~ng the nomenclature of Schechter, I., and Berger, A., 1967, Biochem. Biophys. Res.
Corr~nun. 27: 157-162 (incorporated herein by reference). The individual amino acid residues of a substrate or inhibitor are designated P1, P2, etc. and the corresponding subsites of the enzyme are designated S1, S2, etc The scissile band of the substrate is P1-P1'. The pIimary recognition site of serine proteases is S1. The most important recognition subsites of cysteine proteases are S1 and S2.
Amino acid residues and ~locking groups are designated using standard abbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomenclature rules; incosporated herein by reference]. An ar~ino acid residue (AA) in a peptide or inhibitor structure refers to the part structure -NH-C~IR1-CO-, where R1 is the side chain of the ar~uno acid AA. A
peptide a-ketoester residue would be designated -AA-CO-OR which represents the part structure -NH-CHR1-CO-CO-OR. Thus, the ethyl ketoester derived f~om benzoyl alar~ine would be designated Bz-Ala-CO-OEt which represents C6H5CO-NH-CHMe-CO-CO-O~t.
Likewise, peptide ketoacid residues residues would be designated -AA-CO-OH. Further, peptide ketoamide residues are designated -AA-CO-NH-R l~hus, the ethyl keto amide derived from ZLeu-Phe-OH would be designated ZLeu-Phe-CO~NH-Et which represents C6H5CH20CO NH-CH(CH2CHMe2)-CO-NH-CH(CH2Ph)-C:O-CO-NH Et.
Peptide a-ketoesters containing amino acid residues with hydrophobic side chain at the P1 site have also been found to be excellent inhîbitors of several cysteine proteases including papain, cathepsin B and calpain.
Calpains can be inhibited by peptide inhibitors having several different active groups.
Structure-activity relationships with the commercially available in vitro inhibitors of Calpain, such as peptide aldehydes, have revealed that Calpains strongly prefer Leu or Val in the P2 position. These enymes are inhibited by inhibitors having a wide variety of amino acids ~n the P1 position, but are generally more effectively inhibi`ted by inhibitors ha~ing amino acids with nonpolar or hydrophobic side chains in the P1 position. Thus, we have discovered that another particular class of compounds exhibiting Calpa~n inhibitory activity, vhen used in accordance ~vith the present invention, are the Peptide Keto-Compounds. These are compounds of the general structure:
., O
Il M-~aa)n-C-Q-R
:
~' 8lJB~J'rE S~EE~
': " ' - ` ' ` `
WO92/11850 2~9~603 PCI/US91/097~
or a pharmaceutically acseptable salt, wherein:
M represents NH2-CO-, NH2-CS-, NH~-S02-, X-NH-CO-, X-NH~
X-NH-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-, H, acetyl, carbobenzo~v, succinyL methylo~ysuccinyl, butyloxycarbonyl;
X is selected from the group consisting of C1-6 aL~cyL C1-6 fluoroallyL C1-6 aLtcyl substituted with J, C1-6 fluoroaLlcyl substituted with J, 1-admantyL 9-fluorenyL
phenyl, phenyl substitu~ed with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-6 alkyl with an attached phenyl ~oup, C1-6 allyl with two attached phenyl groups, C1-6 aLkyl with an attached phenyl group substituted with K, and C1-6 allyl with t~o attached phenyl groups substituted with K; .
J is selected from the group consisting of halogen, C(:)OH, OH, CN, N02, NH2, C1-6 aL~coxy, C1-6 alkylamine, Cl-6 diallylarnine, C1-6 alkyl-O-CO-, C1-6 aL'cyl-O-CO-NH, and C1-6 aL~cyl-S-;
K is selected from the group consisting of halogen, C1-6 all~yL C1-6 perfluoroalkyL C:1-6 alko~y, N02, CN, OH, C02H, arnino, C1-6 allylamino, C2-12 diallylarnino, C1-C6 a~yl, and C1-6 allcoxy-CO-, and C1-6 aL~cyl-S-;
aa represents a blocked or unbloclced amino acid of the L or D
configuration, preferably selected from the group consisting of: alanine, valine, leucine, isoleucine, proline, methior~ine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine (nle), no~aline (nva), alpha-asl~inobutyric wid (abu), epsilon-aminocaproic acid, citrulline, hydro~yproline, homoarginine, ornithine or sarcosine;
n is a number ~om 1 to 20;
Q is O or NH, R represents H, C1-6 allyL C1-6 fluoroallyL C1-6 chloroal}yl bei~yl, Cl-6 aLlcyl substituted with phenyl, C1-6 al}yl with an attached phenyl group substituted with K
Thus, the Peptide Keto-Compounds can be divided into the Peptide Ketoesters, Peptide Ketoacids and Peptide Ketoamides. Each of the compounds un also be class ied based on the number of an~ino acids contained within the compound, such as an an~ino acid peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
SUB~3~1TUTE SHE~T
WO 92t11850 ~ ~ 9 ~ ~ ~ 3 PCr/US91/09786 We have ~ound certa~n subelasses of Peptide -Ketoester compounds to be particularl~ useful as Calpain Inhibitors when used in accordance with the present invention These sub~lasses are ~eferred to herein as the Dipeptide ~-Ketoesters (Subclass A), the Dipeptide ~-~etoesters (Subclass B), the Tripeptide a-Ketoesters (Subclass A), the Tripeptide a-Ketoesters (Subc:lass B), the Tetrapeptide a-Ketoesters and the Amino Acid Peptide a-Ketoes~ers. All of these subclasses are considered to be to be within the class of Peptide Keto-Compounds.
The Dipeptide a-Ketoe~ters (Subclass A) are compounds of the formula:
Ml-AA2-AAl-cO'O-Rl or a phannaceutically acceptable s lt, wherein Ml represeDts H, NH2-CO-, NH2-CS-, NH !-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group COnSistiDg of Cl 10 alkyL C1 10 fluoroalkyl, Cl 10 alkyl substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl 1~ substituted with K, phenyl disubstituted with K~ phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 allyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 aL~cyl with two attached phenyl groups substituted ~nth K, Cl 10 allcyl with an attached phenoxy group, and Cl 10 aL~cyl with an attached pheno~r group substituted with K on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C
10 allcoxy, Cl 10 alkylamille, C2 12 dial~ylamine, Cl-10 allyl-O-~O-- Cl-10 a~
and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 aLtcyL Cl 10 perfluoroalkyl, Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 alkylamino, C2 l2 diallylamino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 10 aDyl-S-;
AA~ is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the ~-carbon selected from ~he group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalar~ine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2~rboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-~ I~h'TITl ITF .~lEFr , .
: . .
WO 92/11850 2 ~ 9 ~ ~ 9 PCI/U591/097~
ethyl~ysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-amilloheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-C~I(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohe~yl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, tri~uoroleuc~ne, and hexafluoroleucine;
AA2 iS a side chain blocked or unblocked arnino acid with the L configuration, Dconfiguration, or no ch~rality at the a-carbon selected from the group consisting of leucine, isoleucine, proline, rnethionine, methionine sulfoxide, phenylalamne, tryptophan, glycLne, serine, threor~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, Iysine, arg~nine, histidme, phenylglycine, beta-alanine, norleucine, norvalisie, alpha-arninobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine, homoarg~nine, sarcosine, indoline 2-carboxylic acid, 2- 7etidislecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-e~hylserine, S-methylcysteine, S-ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2C~t2)-COOH, alpha-arninoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH,-cyclohexyl)~COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
Rl is selected from the group consisting of H, Cl 20 allyl, Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 alkyl with an attached phenyl group substituted with K
The Dipeptide a-Ketoesters (Subclass B) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-O-R
or a pharrnaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X~ SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 aLIyL Cl 10 nuoroal~yL C1 iO allyl substituted with J, Cl 10 1uoroalkyl substituted with J, 1-adrnantyl, 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstitutet with Kl naphthyl trisubstituted with K.
Cl 10 allyl with an attached phenyl ~oup, Cl 10 alkyl with two attached phenyl ~roups, Cl 10 aLkyl with an attached phenyl group substituted with K, and Cl 10 al}yl with two attached phenyl groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 aL~cyl with an attached pheno~y group substituted with K on the phenoxy group;
. . .
2~9~9 s WO 92/t1850 PCI/US91/09786 J is selected f~om the group corlsisting of halogen, COOH, OH, CN, N02, NH2, C
10 aL~07cy, Cl 10 aL~cylamine~ 2 diallylar~e, Cl 10 allyl-O-CO-, C1 lo a~ky and Cl 10 allyl-S-;
K is selected from the ~roup consisting of halogen, Cl 10 alky~ Cl 10 perfluoroalkyl, Cl 10 alko~y, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2-12 diallylamino, Cl-C10 acyl, and Cl 10 aL~coxy-CO-~ and Cl 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a carbon selected ~om the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoqcide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutam~c acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, no~valine, alpha-a~unobu~yric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoargi}~ine, sarcosine, indoline 2~arboxylic acid, 2-a~etidinecarboxylic acid, pipecolinic acid (2-piperidine carboxyiic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-e~hylcysteine, S-benzylcysteine, NH2-CH(CH2~;~e2)-COOH, alpha-ars~inoheptanoic acid, NH2-CH(CH2 l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-~yclohexyl)-COOH, ~I2-CH(CH2~yclopentyl)-COO~, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl 8 branched and uulbranched cyclized alkyl; or Cl ~ branched and unbranched ~uoroalkyl;
R is selected from the group consisting of X Cl 20 alkyl, Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 alkyl with an attached phenyl group substituted with K
The Tripeptide a-Ketoesters (Subclass A) are compounds of the structure:
or a phannaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X~ CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyl, Cl 10 ~uoroalkyl, C1 10 a~cyl 30 substituted with J, Cl 10 fluoroallcyl substituted with J, 1-adman~yl, 9-fluorenyl, phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubsti~uted with K, naphthyl, naphthyl substituted with K, naphthyl disubsùtutPd with K, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 1o alkyl with two attached phenyl ~roups, Cl 10 aLlyl with an attached phenyl group substituted with K~ and Cl 10 aL~cyl with two attached SUBSTIT~
-.
WO 92/11850 PCI/US91/097~
2~98~09 phenyl groups substituted with K~ Cl 10 allyl with an attached phenoxy group, and Cl 10 alkyl with an attached pheno~;y group substituted with K on the phenoxy ~oup;
T is selected ~om the group consisting of Cl 10 alkyL Cl 10 ~uoroalkyi Cl 10 alkyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyL 9-~luoreny1 phenyL phenyl S substituted ~ith K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trlsubstituted with K
C2 1o allyl with an atta~hed phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 allcyl with two attached phenyl groups substituted with K;
J is selected from the group cons~ting of halogen, COOH, OH, CN, NO2, NH~, Cl 10 aL1coxy, Cl 10 allyla~e, C~l2 dialkylamLne, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-, and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 perfluoroallyl, Cl 10 aLko~y, N02, CN, OH, C02H, at~.uno, Cl 10 allylamino, C2 l2 dialkylals~no, Cl-C10 acyl, and Cl 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspar~ic acid, glutamic acid, lysine, arguune, histidine, phenyl~ycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-an~inocaproic acid, citrulline, hydroxyproline, ornithine, hornoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-C~I(CH2CH~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH~CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CEl(CH2-cyclopropyl)-COOH, trifluoroleucine~ and hexafluoroleucine;
R is selected frosn the group consisting of H, C2 20 allyl, Cl 20 allyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 a1~cyl with an attached phenyl ~oup substituted with K
The Tripeptide a-Ketoesters (Su~xlass B) are compounds of the structure:
M3-AA-AA-NH-~IR2-CO-CO-O-R
or a pharmaceutically acceptable salt, wherein SUBSTITIJTE SHEE~
., , :
.
, ' ` .~';~.,.WO 9~/11850 2 ~ 9 ~ ~ O ~ PCI`/US9~/09786 M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-(: O-, or X-O-CS-;
X is selected from the g~oup consisting of Cl 10 aL~cyl, Cl 10 fluoroaLlcyL Cl 10 alkyl substituted with J, Cl 10 fluoroaLtcyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl substituted with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 aL~cyl with an attached phenyl group substituted with K, and Cl 10 aLIcyl with two attached phenyl roups substituted wi~h K, Cl 10 aLkyl with an attached phenoxy group, and Cl 10 aLlcyl with an attached phenoxy ~roup substituted with K on the phenoxy group;
T is selected from ~he ~roup consisting of Cl 10 alkyL Cl 10 1uoroalkyL Cl 10 aLtcyl substituted with J, Cl 10 fluoroaLkyl substituted with J, 1-admantyL 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, S:~2 10 aLtcyl with an attached phenyl group, Cl 10 aL~cyl with two attached phenyl g~oups, Cl 10 ~ yl with an attached phenyl group substituted with K, and Cl l0 aLkyl with two attached phenyl groups substituted ~ith K;
J is selected from the group consisting of halogen, COOH, OH, CN~ NO2, NH2, C
10 alkoxy, Cl 10 allylaro~e, Cl lo diallylamme, C1 10 alkyl-O-CO, C1 10 Y
~!0 and Cl lo allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 periluoroaLcyl, 1-10 alkoxy, N02, C'N, OH, C02H, am~no, Cl 10 alkylarnino, C2 l2 dialkylamino, Cl-C10 a~l, and Cl 10 alkoxy-CO-. and Cl.10 a~l-S-;
AA is a side chain blocked or unblocked alTuno acid with the L configuratiorl, Dconfiguration~ or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, prol;ne, methionine, methionine sulfoxide, phenylalanine, tryptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutals~ine, aspartic acid, glutan~ic acid, bsine, argir~ine, histidine, phenyl~ycine, beta-alanine, norleucine, non~aline, alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbc~ylic acid), CJ-methy~senne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-WO 92/11850 8 6 0 9 P~US91/097 ~30-~yclohexyl)-COOH, NH2-CH(ÇH2~yclopentyl)-COOH, NH2-C:H(CH2 cyclobutyl)-COOH, NE2-CH(CH2 cyclopropyl)-COOH, trifluoroleucine, and hexa~luoroleucine;
R2 represents Cl 8 branched and unbranched alkyl, Cl 8 branched and unbranched cyclized allyL or Cl 8 branched and unbranched fluoroallcyl;
S R is selected from the group consisting of H, Cl 20 alkyL Cl 20 alkyl with a phenyl group attached to the Cl 20 al~yL and Cl 20 alkyl with an attached phenyl group substituted with K
The Tetrapeptide a-Ketoesters are compounds of the structure:
M3-AA4-A~ AA-CaO-R
or a phannaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SOr, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 aL~cyl substituted with J, Cl 10 fluoroalkyl substituted with J, l-admantyl, 9-fluorenyL phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 aLlcyl with two attached phenyl groups, Cl 10 alkyl ~ith an attached phenyl group substituted with K, and Cl 10 aLcyl with two attached phenyl groups substi~uted with K, Cl 10 alkyl with an attached phenoxy group, and C~ 10 allcyl with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroaLkyl, Cl 10 a~l substitutedwithJ, Cl 10fluoroalkylsubstitutedwithJ, 1-admantyL 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted ~rith X, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, ~5 C 10 alkyl~nth an attached phenyl group, Cl 10 aL~cylwith two attached phenyl groups, Cl 10 aL~cyl with an attached phenyl group substituted with K, and Cl l0 allyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 alkoxy, Cl 10 aL~ylam~ne, C2 12 dialkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 allyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyL
Cl 10 alko%y, NO2, CN, OH, CO~I, amino, Cl 1o alkylamino, C2 l2 diaL~cylan~ino, Cl-C10 ac~l, and Cl 10 alkoxy-C-, and C1 10 alkyl-S-;
~ttR~TtTlil^ E SHE~ET
, .' . -WO 92/1~850 2 ~ 3 PCl/lJS91/09786 AA is a side chain blocked or unblocked amino acid with the L configuration, D
confi~uration, or no chirality a~ the a~rbon selected from the group consisting of alanine, valine, leucine, isoleucine, pro~ine, methionine, methionine sulfoxid~, phenylalanine, t~ptophan, glycine, serine, threonine, cysteine, fyrosine, asparagine, ~lutars~ine, aspartic acid, S glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, nonaline, alpha-arninobutyric acid, epsilon-aminocaproic acid, cit~ulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2 car~y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~lic acid), O-methylserine, O~ethy3serine, S-methylcysteine, S-ethyl~ysteine, S-benzyl~ysteine, N~I2 CH(CH2C~HEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohe~yl)-COOH, NH2~ H2~yclopentyl)-COOH, NH2-CH(CH2~yclobutyl)-COOH, NH2-CH(CH2-cyclopropyl).COOH, tri~uoroleucine, and hexafluoroleucine;;
AA4 iS a s;de chain blocked or unblocked amino acid ~ith the L corfiguration, D
conf;guration, or no chirality at the a-carbon selected from the group consisting of leucine, isoleucine, methionine, methion~ne sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, aspa~agine, glutamine, aspartic acid, glutamic acid, lysine, arg~nine, histidine, phenylglycine, beta-alanine, norleucine, nor~aline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orni~ne, homoarginine, sarcosine, indoline 2 carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-GEI(CH2C~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2- 1 napthyl)-COOH,NH2-CH(CH2-~-napthyl)-COOH,NH2-CH(CH2~yclohexyl)-COOH,NH2-CH(CH2-cyclopentyl).COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl).COOH, tri~uoroleucine, and hexa~uoroleucine;
2S R is selected from the group consisting of H, Cl 20 aLIcyL Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl-20 allcyl with an attached phenyl group substituted with K
The Amino Acid Peptide a-Ketoessers are compounds of the structure:
Ml-AA-CO-O-R
or a phasmaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-~IH-CO-, X2N CO, X
X2N-CS-, X-NH-SO2-, X2N-S02-, Y-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyL Cl 10 fluoroallyL Cl 10 a~lyl substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl Sl.18S~ITUTE SHEET
` `
WO 92/118~0 2 0 9 g 6 0 9 -32- PCT/U~91/0978 `
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tr~substituted with X, Cl 10 aLIcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 aL1cyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached S phenyl ~oups substituted with K, Cl 10 al~yl with an attached phenoy group, and Cl 10 alkyl with an attached pheno~y group substituted ~ith X on the phenoxy group;
Y is selected from the group consisting of C~10 a~yL Cl 10 ~uoroaL~cyL Cl 10 alkyl substituted ~nth J, Cl-10 fluoroaL~cyl substituted with J, l-admantyL 9-fluorenyL phenyl substituted with ~, phenyl disubstituted with K, phenyl trisubstituted w~th K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with B, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 aLkyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 1~ lo aLkoxy, Cl 10 aL~cylamme, C2 12 diaLkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl-10 perfluoroa~l C~ lo alko~y, N02, CN, OH, ~2~ amino, C1 10 alkylamino, C~ 12 diaLkylan~no, Cl-C10 acyl, and Cl 10 aLcoy-CO-, and Cl l0 allyl-S-;
AA is a side chain ~olocked or unblocked amino acid with ~he L configuration, D
configuration, or no chirali~y at the a-carbon selected from the group consisting of alanine, valinej leucine, isoleucine, proline, methionine, methionine sulfo dde, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutam~ne, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine~ beta-alanine, norleucine, norvaline, 2~ alpha-an~inobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine, homoarg~ni~le, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo4ylic acid, pipecolinic acid (2-piperidme carboylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2C~IEt2)-COO~ alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cydobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trinuoroleucine, and he~ uoroleucine;
R is selected ~om the group consisting of H, Cl 20 alkyL Cl-20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 aL'cyl with an attached phenyl group substituted with K
C~ ~ITI ~TF .S}~F~T
-- , WO 92/11850 2 0 9 8 6 0 3 PCI'/US91/09786 ,-- t ....
The follow~ng Peptide Ketoester compounds are representative of the Peptide Keto-Compounds found to be useful as Calpa~n ;nhibitors Withi~l the conte~t of the present ~nvention:
Bz-DL,Ala-COOEt Bz-DL-Ala-COOBzl Bz-DL,Ala-COOnBu Bz-DL-Phe-COOEt Bz-DL-Ala-COOCH2-C6H4-CF3 (para) Bz-DL-~Arg-COOEt Bz-DL,Lys-COOEt ZAla-DL,Ala-COOEt ZAla-DL,Ala-COOBzl ~AIa-DL-Ala-COOnBu MeO-Suc-Ala-DL,Ala-COOMe ~Leu-N~ra-COOEt Z-Leu-Me-COOEt ~Leu-Phe-COOEt ZLeu-Abu-COOEt ZLeu-Met-COOEt Z-Phe-DL,Phe-COOEt H-Gly-DI~Lys-COOEt H-Ala-DL-Lys-COOEt H-Pro-DL-Lys-COOEt H-Phe-DL-Lys-COOEt Z-Ala-Ala-DL-Ala-COOEt ~AIa-Pro DL Ala COOEt ZAla-Ala-DL,Abu-COOEt ZAla-Ala-DL-Abu-COOBzl Z Ala-Ala-DI,Abu-COOCH2-C6H4-CP3 (para) MeO Suc-Val-Pro-DL Phe COOMe H-Leu-Ala-DL~Lys-COOEt ZAla-Ala-Ala-DL-~la-COOEt MeO-Suc-Ala-Ala-Pro-DL-Abu COOMe.
ZLeu-Phe-COOEt SUlE~;~lT~alTE S~EEr ~;i, :
~"
..
WO92/11850 0986a9 PCI/US~1/097 PhCQ-Abu-COOEt (c~3)2cH(cH2)2co-Abu~coc)Et C$I3CH2CE)2CHCO.Abu-COOEt Ph~CH2)6CO-Abu-COOEt - ZLeu4~ Phe-~OOEt Z-Leu-Leu-Abu-COOEt ~Leu-Leu-Phe-COOEt 2-NapS02-Leu-Abu-COOEt 2-NapS02-Leu-Leu-Abu-COOEt ZLeu-NLeu-C02Et ZLeu-Phe-C02Bu Z-Leu-Abu-C02Bu Z-Leu-Phe-CO2Bzl ZLeu-Abu-C02BzL
1~ We have found certain subclasses of Peptide Ketoacid Compounds to be partieularly useful when used in accordance with the present inVentiQn. These are sul~classes are the Dipeptide a-Ke~oacids (SubclassA), theDipeptide a-Ketoaeids (SubclassB), the Tripeptide -Ketoacids, the Tetrapeptide a-Ketoacids and the Amilio Acid peptide ~-Ketoacids. All of these are considered to be within the c lass of Peptide Xeto-Compounds.
The Dipeptide a-Ketoacids (Subclass A) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-OH
or a phannaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2 CS-, NH2 S02-, X-NH-CO~, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH~S02-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or 2{-O-CS-;
X is selected from the group consisting of Cl 1o allyl, Cl 10 fluoroaL~cyL Cl 10 allyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1~admantyl, 9-iluorenyL phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted ~ith K, Cl ~0 a;lyl with an attached phenyl group, C1 iO alkyl with two attached phenyl groups, Cl 10 30 alkyl with an attacl~ed phenyl group substituted with K, Cl 10 aLlcyl with ~wo attached phenyl groups substituted with K, Cl 10 allyl with an attached pheno~y group, and Cl 10 alkyl with an attached pheno~y group substituted with K on the phenoxy group;
SUBSTITUTE SHEE~
.: :
.. ~,.. ,, . . .,, ~.......... , . , ; , .
WO92/11850 2~98~a~ Pcr/US91/09786 J is selected ~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 aL~cwy, C1 10 allylamille, C2 ~ aLlylamme, Cl 10 allyl~ CO-, Cl 10 allyl-O-CO-NH., and Cl 10 alkyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyl Cl 10 pe~uoroalkyl S Cl 10 alko~y, NO2, CN, OH, CO2H, amino, Cl 10 allylamino, C2-l2 diallyla~ o, Cl-C10 acyL
and Cl 10 alkoxy-CO-, and C1 10 allcyl-S-;
AA is a side chain blocked or unblocked an~ino acid with the L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, t yptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutan~ne, aspar~ic acid, glutan~c acid, lysine, ar~e, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-a~.ûnobutyric acid, epsilon-aminocaproic acid, citrul~ine, hydro~yproline, ornithine, homoargi~une, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-etllylserine, S-methylcysteine, ~-ethylcysteine, S-benzylcysteine, NH2-~l(CH2~Et2)-COOH, alpha-anunoheptanoic acid, NH2-CH(CHrl-napthyl)-COOH, NH2 CH(CH2-2-napthyl)-COOH, NH2-C~(CH2-cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl~-COOH, NH2-C~I(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl ~ branched and unbranched cyclized alkyl, or Cl 8 branched and unbranched fluoroalkyl.
The Dipepdde ~-Ketoacids (Subclass B) are compounds of the structure:
Ml-AA2-AAl-CO-OH
or a pharmaceudcally acceptable salt, Yvherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N-S02-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-~S-;
X is selected from the group consisdng of Cl 10 allyL Cl 10 fluoroalkyL Cl 10 alkyl substi~uted with J, Cl 10 fluoroaDcyl substituted with J, 1-adman~qL 9-nuorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with K, Cl 10 aLcyl with an attached phenyl group, Cl 10 aLcyl with two attached phenyl groups, Cl 10 allyl ~nth an attached phenyl ~oup substituted with K, and Cl 10 allyl with rwo attached phenyl groups substituted with K, Cl 10 alkyl with an attached pheno~y ~oup, and Cl 10 alkyl with an attached pheno~y group substituted with K on the phenoxy group;
S~3BS~7'Ur~ SHEE~
.
. .
, :
wO 92/11850 2 0 9 8 6 0 9 PCI/US91/097B~
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C1 10 aL~co~y, C1 10 aLlyla~e, C~12 dia~lam~e, Cl 10 allyl-O-CO-, Cl 10 al}yl-O-CO-NH, and C1 l0 allcyl-S-;
K is selected from the group consisting of halogen, Cl 10 a~yL cl lo perfluoroallyL
110 aLkoxy, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2 ~2 diallylan~ino, Cl-C10 acyL
and Cl 10 alkoxy-CO~, and C1 10 alXyl-S-;
AAl is a side chain blocked or unblocked amino acid wi~h the L configuration, D
configlaration, or no chirality at the ~-carbon selected f~om ~he group consistirlg of alanine, valine, leuune, isoleucLqe, proline, methionine, methionine sulfo~de, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparag~ne, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylg}ycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminouproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, ~ndoline 2 carbo~y1ic acid, 2-a~etidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbo~y~c acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben2ylc~s~eine,NH2-CH(CH2CHE~ COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2~CH(CH2-2-napthyl)-COOH, NH2-CEI(CH2-cydohe~yl).COOH, NH2 CH(CH2~clopentyl)-COO~ I2-CH(~H2~yclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and he1~afluoroleucine;
M2 is a side chain blacked or unblocked ~o acid with the L configuration, D
configuration, or no chirality at the a carbon selected from the group consLr.ting of alarline, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alaninet norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine, homoarginine, sarcosine, indoline 2 carbo7y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ber2ylcysteine, NH2-C~(GH2CHEt2)-COOH, alpha-am~noheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NHyC~ H2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, tri~uoroleucine, and hexa~uoroleuc~ne.
The Tripeptide a-Ketoacids are compounds of the s~ucture:
Ml-AA-AA-AA-CO-OH
or a pharmaceutically acceptable salt, wherein ' .
SU~3ST~TU~ ~ffEEt , WO 92/118~0 P~/US91/09786 i 209~
Ml represents H, NH2-CO-, N~I2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected fcom the ~oup consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 alkyl subsdtuted with J, Cl 10 fluo~oaL~cyl substituted with J, 1-admantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K~ naphthyL
naphthyl substituted with K, naphthyl L;subsdtuted with K, naphthyl trisubstituted with K, Cl 10 allyl with an attached pheslyl ~oup, Cl 10 allyl with two attached phenyl groups, Cl 10 al}yl with an attached phenyl group substituted ~nth K, and Cl~10 aLlcyl with two attached phenyl groups substituted with K, Cl 10 alkylwith an attached pheno~y group, and Cl 10 alkyl with an attached pheno~y group substituted with K on the pheno~y group;
J is selected ~om the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 10 lko~y, Cl-10 a~ylamme, C2 12 diaLlylamme, Cl 10 alkyl-O-CO, Cl 10 allyl-O-CO-NH, and C1 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 al}yL Cl lo pD-rfluoroalkyL
1-10 xy, N2~ CN, OH, C2H- armno, Cl 10 allylamillo, C2 12 dialkylamino, Cl-C10 acyL
and Cl 10 alko~y-CO-, and Cl 10 aLk~l-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~de, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, ~lutamine, aspar~ic acid, glutamic acid, bsine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydrw~yproline, ornitbine, homoarginine, sarcosine, indoline 2 car~o~ylic acid, 2-azetidinecarbo~ylic acid, pipeco~;nic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-etbylcysteine, S-benzylcysteine, NH2-CH(CH2C~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NHrCH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cycloheyl) COOH, NH2-C~I(CH2-cydopentyl)-COOH, NH2-CH(CH2~:yclobutyl)-COOH, NH2-CH(GEI2 cydopropyl)-COOH, tri~luoroleucine, and he~a~uoroleucine.
The Tetrapeptide a-Ketoacids are compounds of the structure:
Ml-AA-~A-AA-AA-CO-OH
or a pharmaceu~ically acceptable salt, wherein Ml represents H, N~2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-X2N-CS-, X-NH-S02-, X2N-SO2-, Yl-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
WO 92/11850 2 ~ 9 8 6 ~ 9 PCI/US91/0978~
X is selected ~om the group consisting of Cl 10 aLkyl Cl 10 ~uoroalkyL Cl 10 aLI~yl substituted w~th J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyL phenyl substituted w~th K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, ~phthyl disubstituted with K, naphthyl trisubstituted with K, S Cl 10 alkyl w~th an attached phenyl ~oup, Cl 10 all~l with two attached phenyl groups, Cl 10 aLkyl with an attached phenyl group substi~uted with K, and Cl 10 aLkyl ~ith two attached phenyl groups substituted with K, C1 l0 aL~cyl with an attached pheno~y group, and Cl 10 alkyl with an attached pheno.y group substitut d w~th K on the phenoy group;
'Yl is selected f~om the group conslsting of C2 lo aLlcyL Cl lO fluoroalkyL C1 l0 aLlyl substitutedw~thJ, C1 1OfluoroallcylsubstitutedwithJ, 1-a~mantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 allyl wi~h an attached phenyl group, Cl 10 alkyl with two attached phenyl ~oups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached phenyl groups subsdtuted vrith K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 alko~y, Cl 10 alkylamine, C 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-, and Cl 1o alk~l-S-;
K is selected from the group consisting of halogen, Cl 1o alkyL Cl 1o per~uoroalkyl, Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 1O allylamino, C2.12 dialkylarnino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and Cl 10 aLtcyl~;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality a~ the a~on selected from the group consisting of alanine, valine, leuc~ne, ~soleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, glycine, senne, threonine, ~ysteine, tyrosine, asparagine, glutamine, aspartic acid, gluhn~ic acid, }ys3ne, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon~ nocaproic aciL citrulline, hydro~yproline, ornithine, homoargmine, sarcosine, ~ndoline 2-carbo~ylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylsenne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben~ylcysteine, NH2-C~I(CH2C:HEt2)-COOH, alpha-an~inoheptanoic acid, NH2-CH(CH2-l napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-CH(CH2~dobutyl)-COOH, NH2-CH(CH2-cydopropyl)-COOH, trifluoroleucine, and hexafluoroleucine.
The Amino Acid Peptide a-Ketoacids are compounds of the structure:
SU~STITI ITF .~L~r:~T
- .
`,' ~`' '''' ' ~ :, .....
WO 92/11850 ~ ~ ~ 3 ~ o 9 PCI'/US91/09786 M~ CO-OH
or a pharmaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X N~ CO, 2 2 , X N~-S02-, X2N~02-, Y2-C0-7 X-CS-, X-~02-~ X-O-CO-, or X-O-~S-;
S X is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroalkyl, Cl 10 alkyl substituted with J, C 1 10 fluoroalkyl substituted with J, 1-admantyl, 9-~uurenyl, phenyl, phenyl substituted with X, phenyl disubstituted with iK, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disu~sdtuted ~nth K, naphthyl trisubstituted with K, Cl 10 allyl with an attached phenyl group, C1 l0 allyl ~nth two attached phenyl groups, C1 l0 al}yl with an attached phenyl group sllbstituted with K, and Cl 10 allyl with two attached phenyl groups substituted with K, Cl 10 allyl with an at~ached pheno~y group, and Cl~10 allyl with an attached phenoxy group subseituted with K on ~he pheno~y group;
Y2 is selec~ed ~om the group conslsting of Cl 10 aL~cyL C1 10 ~uoroa~yl, C1 10 alkyl subs~ituted ~vith J, Cl 10 fluoroallyl substituted ~nth J, 1-adman~L 9^fluorenyL phenyl substituted wi~h K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted ~th K, naphthyl trisllbs~cituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 alkyl with an attached phenyl group substitu~ed wlth K, and Cl 10 allyl ~nth two attached phenyl groups substituted with K;
J is selected f~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, C
10 allco~y, C1 10 allcylamine, C~12 dialkylamine, C1 10 allykO-CO-, C1 10 aLkyl O
and Cl 1o allyl~-;
K is selected from the group consisting of halogen, Cl-10 alkyl Cl 10 per~uoroalkyl, C1 10 aL~coxy, NO2, CN, O~I, CO2H, amino, Cl 10 allylamino, C2 l2 diaLIcylalruno, Cl-C10 acyl, and Cl 10 alkoxy-CO-, and C1 10 aLlyl~
AA is a side chain blocked or unblocked amino acid with ~he L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ude, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, g~utamic acid, lysine, arginine, histidine, phenylglycine, beta-alunine, norleucine, norvaline, alpha.an~inobutyric acid, epsilon-aminocaproic acid, citrulline, hydro7yproline, ornithine, homoar~pnine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~lic acid, pipecolinic acid (2-piperidine car~oxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-berzylcysteine, NH2-CH(CH2CHEt2)-COOX alpha-aminoheptanoic acid, SW3STlTl3TE SH@ET
. ~ .
WO 92/11850 2 ~ ~ 2 6 ~ ~ PCI`/US91~0978 NH2-CH(CH2-1-napthyl)~COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2 cyclopentyl~-COOH, NH2-CH(CH2 cyclobutyl)-COOH, NH2-CH(CH2 cyclopropyl)-COOH, trifluoroleucine~ and h~afluoroleucine.
The following Peptide ~etoacid compounds are represen~ative of the Peptide Keto-S Compounds found to be useful as Calpain inhibitors withirl the context of the present invention:
:E3z DL,Lys-COOH
Bz-DI,Ala-COOH
Z-Leu-Phe-COOH
i~T eu Abu-COOH.
The peptide -Icetoamides are transition state analogue inhibitors for cysteine proteases, such as Calpain. We have found that Peptide a-ketoarnides containing amino acid residues with hydrophobic side chains at the Pl site are excellent inhibitors of several cysteine proteases including calpain I and calpain IL
We have found fiYe subclasses of the peptide ketoamides to be particularly efEective in inhl~iting Calpain. These subclasses are referred to herein as Di~eptide a-Ketoarnides (SubclassA),Dipeptide a~Ketoamides (SubclassB), Tripeptide a-Ketoan~ides, Tetrapeptide a-Ketoamides and Amino Acid a-Ketoamides. All of these subclasse~ are considered herein to be withn the class of Peptide Keto-Compounds The Dipeptide a-Ketoamides (Subclass A) haYe the following structural formula:
Ml AA NH-CHR2 CO-CO-NR3R4 or a phannaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, N~I2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X O-CS-;
X 3s selected from the group consisting of Cl 10 alkyL Cl 10 fluoroaLIcyL ~ 0 alkyl substituted with J, Cl 10 fluoroaLl~yl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl substituted wi~h K, phenyl disubstituted with K, phenyl tri~ubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with E
Cl 10 aLkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, Cl 10 alkyl with two attached phenyl groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 allyl with an attached pheno~y group subsdtuted with K on the pheno~y group;
5UI~STITUTE SHEET
. .
WO 92/11850 2 ~ P~/US91/09786 J is selected from the group consistirlg of halogen, COOH, OH, CM, NO2, NH2, Cl 10 alkoxy, C1 10 ~ylamine, Cz 12 diallyla~une, C1 10 alkyl-O-CO-, C1 l0 alkyl O CO NH, and Cl 10 alkyl-S-;
K is selected from the group consisti~g of h~logen, Cl 10 a~L cl lo per~UoroalksL
Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 10 allylan~ino, C2 l2 diallylamirlo, Cl-C10 acyl, Cl 10 aL~o~y-CO-, and Cl 10 alkyl-S-;
AA is a side chain blwked or unblocked amino acid with the L coIIfiguration, D
configuration, or no chirality at the a-carbon selested from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspar~ic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, a-arninoblltyric acid, epsilon-a~T~nocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosin, indoline 2-carboylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-beDzylcysteine, NH2-CH(CH2CHEt2)-COOH, -aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-C~H(CH2-2-napthyl)-COOH, NH2-~H(CH2~yclohexyl)-C9X NH2-CH(~E12-cyclopen.yl)-COOH, NH2-CH(CH2 cyclobu~l)-C9OH, NH2-CH(CH2-cyclopropyl)-COOH, tri~uoroleucine, and he~a~uoroleucine;
R2 is selected from the group consisting of Cl 8 branched and unbranched allyl, Cl..8 branched and unbranched cyclized alkyl, and Cl 8 branched and unbranched fluoroa~kyl, R3 and R4 are selected independently from the group consisting of H, Cl 20 aL~cyl, Cl 20 cyclized alkyL Cl 20 allyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized alkyl with an attached phenyl group, Cl 20 a1kyl with an attached phenyl ~oup substitu~ed with K, Cl 20 alkyl with an attached phenyl group disubstituted with K, Cl 20 alkyl with an attached pbenyl group trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with K, Cl 10 allyl with a morpholine-[-N(CH2CH2)O] ring attached through nitrogen to the all~yL Cl 10 allyl with a piperidine ring attached through nitrogen to the alkyL Cl 10 aLlcyl with a pyrrolidine ring attached through nitrogen to the allyL Cl 20 aLlcyl with an OH group attached to the aLlcyL -CH2CH2C)CH2C~2OH, Cl 10 with an attached 4-pyridyl group, Cl 1o with an attached 3-pyridyl group, Cl 10 with an attached 2-pyridyl group, Cl 10 with an attached cyclohe1yl group, -NH-C~H2CH2-(4-hydroyphenyl), and NH-C~2C~2.(3-indolyl).
The Dipeptide a-Ketoamides (Subclass B) have the following structural forrnula:
Ml-AA2-AAl-CO-NR3R4 ~:LI~IM ITF c ~l~
`
:' :
WO 92/11850 2 ~ 9 ~ 6 0 9 P~/US91/097B~. r ~2-or a pharrnaceutically acceptable salt, wherein Ml represents H, NHz-CO-, NH2-C:~S-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N^S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 allyL Cl 10 fluoroalkyL C1 10 alkyl Ssubstituted with J, Cl 10 fluoroaL~cyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl substituted with K, phenyl disubstituted with ~, phenyl trisubstituted with K, naphthyL
naphthyl substituted with ~, naphthyl disubstit~ted with K, naphthyl trisubstituted with K, Cl 10 aL~cyl with an attached phenyl group, C~ 10 allyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted ~nth K, Cl 10 allyl ~vith two attached phenyl 10groups substituted with K, Cl 10 allyl with an attached pheno~ roup, and Cl 10 alkyl with an attached pheno~y group substituted with lE~ on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 10 al~co~y, Cl 10 a~ylamLne, C2 12 diallylamme, Cl 10 a~l-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 allyl~-;
15K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pP.~uoroalX~L
0 alko~, N02, CN, OH, CO2H, amino, Cl 10 allylamino, C2 12 diallylamino, Cl-C10 acyl, and C1 10 alko~y-CO-, and Cl 10 alkyl-S;
AAl is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected f~om the group consisting of alanine, 20valine, leuane, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, serine, threonine, g~steine, tyrosine, asparagine, glu~nine, aspartic acid, glutaïnic acid, lysine, arginine, histidin4 phenylglycin4 beta-alanine, norleucine, norvaline a-an~inobu~yric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orTIitnine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic 25acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-- ethylcysteine, S-benzyl~ystein4 NH2 CH(CH2t;~t2)-COOH, ~-aminoheptanoicacid, NHz-CH(CH2 1 napthyl) COOH, N~2-CH(CH2~2 napthyl) COOH, NH2~CH(CH2-cydohexyl)-COOH, NH2-~I(CH2~clopentyl)-COC)H, NHrC~H(CH2-cyclobutyl)-COOH, NH2-CH(CH2~ydopropyl)-COOH, tri~luoroleucin4 and hexafluoroleucine;
30AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
con~iguration, or no chirality at the a-carbon selected ~om the group consisting of alanine, valine, leucine~ isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glyc~ne, serin4 threonine, cysteine, ~qrosine, asp~ragine, glutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvali, e, SlJBSTiTUT'- S~.EET
.
.
., - ` :
..: wo 92~11850 2 ~ Q ~, pcr/vs91/o9786 ~3-a-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-pi~eridine earbo~ylic acid), O-methylser~ne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2~ )-COOH, ~ -aminoheptanoic acid, NH2-S CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2~CH(C~H2-cyclohexyl)-COOH NH2-CH(CH2~yclopentyl)-C::OOH, NX2-CH(OEI2~yclobutyl) COOH, 2 C~(CH2~yclopropyl)-COC)H, tri1uoroleuane, and he~a~uoroleucine;
R3 and R4 are selected independentl~ f~om the group co~sisting of H, C1 20 alkyLC1 20 cyclized allyL cl 20 aL~yi w~th a phenyl group attached to the C~ kyL C~ 20 cy~zed alkyl with an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group subs~ituted with K, Cl 20 allyl with an attached phenyl group disubstituted with K, C1 20 aL~cyl with an attached phenyl group trisubstituted with K, C~ cycli~ed aIkyl with an attached phenyl ~oup substituted with K, Cl 10 aLlcyl with a morpholine ~-N(C~2C~2)0] ring attached through nitrogen to the aL~cyL C~ 0 allyl with a piperidine ring attached through nitrogen tO the aL~cyL Cl 1o allyl with a pyrrolidine ring attached through r~itrogen to the aLIcyL Cl 20 alkyl with an OH group attached to the a~yL -CH2CH20CH2~H20H, Cl 10 with an attached 4-py~dyl group, Cl 10 with an attached 3-pyndyl group, Ci 10 q ith an attached 2-pyridyl group, Cl-10 with an attached cyclohe1yl group, -NH-CH2~I2 (4-hydro~yphenyl), and -NH-CH2CH2-(3-irldobl~.
The Tripeptide a-Ketoarnides have the follo~ing structural formula:
Ml-AA AA-AA-CO NR3R4 or a pharmaceutically acceptable salt, wherein Ml represents ~ NH2-CO-, NH2-CS-, NH2~O2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N C5-, X-NH SO2-, X2N~02-, X CO-, X-t:~S-, X-SO2-, X-O-CO-, or X-O-CS-;
2~ X ~s selected ~om the group consisting of Cl 10 aLIyL Cl 10 fluoroalkyl~ C1-10 aLIcyl substituted with J, Cl 10 fluoroalkyl substisu~ed with J, 1-admantyl, 9-fluorenyL phenyL phenyl subsdtuted vvith ~, phenyl disubstituted with K, phenyl trisubstituted ~/ith K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl ~risubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 allcyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, Cl 10 allyl with ~o attached phenyl ~oups substituted with K, Cl 10 allyl ~nth an attached pheno~y group, and C 1-10 alkyl with an attached pheno~y group substituted with K on the pheno~y group;
SV8S~ HFF-r . . , ~ ~ , , ......................................................................... .
WO 92/11850~ ~) 9 8 6 0 9 PCr/US91/09786 ~..
J is selected from the g~oup cons~sting of halogen, COOH, C)H, CN, NO2, NH2, Cl 10 aL~co~y, C1 10 allylam~ne, C2 12 dia31ylam~ne, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 aUyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 per~uoroalkyL
S Cl 10 alkoxy, N02, CN, OH, C02H, all~ino, 1-10 allylamino, C2 12 dialkylarl~ino, Cl-C10 acyL
and Cl 10 aLIco~y-CO-~ and Cl 10 ~l~-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
config~ation, or no c~ira~ a~ the a carbon selected from the ~roup consisting nf alanine, valine, leucine"soleucine, proline, methionine, methionine sulfoxide, phenyl~lanine, t~yptophan, glyeine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~arboxylic acid, 2-azetidinecarboxy~ic acid, pipecolinic acid (2-piperidine carb~ylic acid~, O-methylsemle, O-e~hylserine, S-methylcys~eine, S-eshylcysteine, S-benzylcysteine, NEI2-CH(GH2CHEt2)-COOH, a -an~inoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH~2-napthyl)-COOH, NH2-CH(CH2 çyclohe~
COOH, NH2-CH(CH2~clopentyl~COOH, NH2-~I(CH2-cyclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, ~rifluoroleucine, and he~afluordeucine;
R3 and R4 are ~elected ~ndependently from the group consisting of H, Cl 20 aL~cyl, C1 20 cyclized alkyl, C1 ~0 alkyl ~nth a phenyl group attached to the Cl 20 aLIyL cl 20 cyclized allyl ~nth an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group substituted with K, Cl 20 aLlcyl with an attached phenyl group disubstituted with X, C120 allyl with an attached phenyl group trisubstituted with K, ~-20 cyclized alkyl with an attached phenyl group substituted with ~, Cl 10 alkyl with a morpholine [-N(CH2CH2)0] ring attached ~5 through l.utrogen to the allyl, Cl 10 aL~cyl ~nth a piperidine ring attached through nitrogen to the alkyl, Cl 10 allc,vl with a py~olidine ring attached through nitrogen to the allyl, Cl 20 alkyl with an OH group attached to the alkyl, -CH2CH20CH2CH20H, Cl 10 wlth an attached 4-pyridyl group, Cl-10 with an a~tached 3-pyridyl group, Cl 10 with an attached 2-pyridyl group, Cl 10 ~qth an athched cydohe~yl group, -NH-CH2CH2-(4-hydro~yphenyl), and -NH-CH2C~z(3-indolyl).
The Tetrapeptide a-Ketoarnides have the following structural formula:
Ml-AA-AA-AA-AA-CO-NR3R4 or a pharmaceutically acceptable salt, wherein SU~STITUT SHE3ET
. . ~ .~ , . .~ . . .
wo 92/11850 2 ~ 0 9 Pc~/us9l/o97s6 ~5-Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO, X~N CO, X NH CS, X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-~S-, X-S02-, X-O~CO-, or X-O-CS-;
X is selected from the group consisting of Cl,10 alkyL Cl 10 fluoroaL~cyL Cl,10 aLkyl substituted with J, Cl 1(~ fluoroaLlcyl substituted with J, l-admantyL 9 fluorenyL phenyl, phenyl S substitu~ed with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with X, C 1-10 allyl v~,ith an attached phenyl group, Cl 10 aLkyl with two attached phen~l groups, Cl 10 allyl with an attached phenyl group substituted ~rith K, Cl,1o allyl with two attached phenyl groups substituted with K, Cl,10 allyl with an attached pheno~y group, and Cl,10 aLkyl with an athched pheno~y group substituted with K on the pheno~y group;
J is selected from ~he ~oup consisting of halogen, COOH, OH, CN, NO2, NH2, 10 alkoxy, Cl 10 allylamine, C~l2 diallylamine, C~ 0 all~yl-O-CO-, C1 10 alky and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pe~uoroaL~yl, Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 aL~cylamino, C2-12 dialkylan~ino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 l0 alkyl-S-;
AA is a side ~hain blocked or unbloclced asT~no ac~d w~th the L configuration, Dconfiguration, or no chirality at the a-car~on selected from the group consisting of alal~ine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~de, phenyialanine, tryptophan, glycine, serine, threon~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid, ~latamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~ylic acid), O methylserine, O-e~hylserine, S-methylcysteine, S-ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2CHEt2)-COOH, c~-am~noheptanoic acid, NH2-CH(C~I2-l-napthyl)-COOH, NH2-CH(C~H2-2-napthyl)-t:OOH, NH2 CH(CH2 cyclohe~yl)-COOH, NHrCH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH~CH2~yclopropyl)-COOH, trifluoroleucine, and he sa~uoroleucine;
R3 and R4 are selected independently ~om the group consisting of H, Cl 20 allyl,Cl 20 ~yclized alkyl, Cl ~0 alkyl vrith a phenyl group attached to the Cl 20 allyL Cl 20 cyclized aL~cyl with an attached phenyl group, Cl 20 allyl with an attached phenyl group substi~uted with lK, Cl 20 allyl with an attached phenyl group disubstituted with K, Cl 20 allyl with an attached phenyl group trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with K, Cl 10 alkyl with a mo~pholine [-N(GH2C:EI2)O] ring attached SUBSTITUTE SH~ET
2~9g6~
WO 92/118~0 P~/VS91/09786-: -~6-through nitrogen to the alkyL cl lo aLtcyl with a piperidine ring attaohed through nitrogen to the all~L cl lo allyl with a pyrrolidine r~ng attached through nitrogen to the allyL Cl 20 allyl with an OH group a~tached to the allyL -~H2CH20C~2CH20H, Cl 10 with an at~ched 4-pyndyl group, Cl 10 w~th an attached 3-pyridyl ~oup, Cl 10 with an attached 2-S pyridyl group, Cl 10 with an attached ~yclohe~yl group, -NH-C~H[2~2-(4-hydro~yphenyl), and -NH-CH2CH2-(3-indolyl).
I~e Amino Acid ~-Ketoamides have the f~llowing s~ructural fosmula:
Ml-AA-CO-NR3R4 or a pharmaceutically acceptable salt, where~n Ml represents H, NH2-CO-, NH2-C:S-, NH2-SO2-, X-NH-CO-, X2N CO, X NH
X2N~CS-, X-NH~02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O~ O-, or X-O-C~;-;
X is selected ~om the group consistmg of Cl 10 allyL Cl 10 fluoroallyL C1 10 alkyl substitutedwithJ, Cl 1O~uoroallcylsubstitutedwithJ, l-admantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstieuted with K, naphthyl, naphthyl subseituted with K, naphthyl disubstituted with K, naphthyl trisubstituted wiéh K, Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl ~nth two attached phenyl groups, Cl 10 allyl with an attached phenyl group subs~itu~ed ~ith K, Cl 10 aL~cyl wi~h two attached phenyl groups substituted with K, Cl 10 alkyl with an attached pheno~sy group, and Cl 10 aLkyl with an attached pheno~y group substituted with K on the pheno~y ~oup;
J is selected from the group consist~ng of halogen, COOH, OH, CN, NO2, NH2, C
10 aL~o~y, Cl-10 allylamine, C2 12 diaLlcylamine, Cl-10 alkyl-O-CO-, Cl 10 aL~cyl-O-CO~
and Cl 10 allyl~-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyl, C1 10 alka~y, NO~, CN, OH, CO2H, arnino, Cl 10 allylamino, C2 l2 diallylan~ino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 10 alkyl~-;
AA is a side chain blocked or unblocked am~no acid ~th the L configuration, D
configuration, or no chirality at the at-car~on selected from the group consisting of alan~ne, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanirle, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutan~ine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ~-arninobutyric acid, epsilon-aminocaproic acid, ci~rulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~arbo~y]ic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben~ylcysteine, NH2 CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NlH2-S~JBSTITUTE SHEET
--'~ WO 92/1 1850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786 ~7-CH(CH2-1-napthyl)~COOH, NH2~CH(~I2-2-napthyl)-COOH, NH2 CH(CH2-cyclohe~yl)-COOH, NH2-CH(OEI2~yclopentyl)-COOH, NH2-CH(CH2~clobutyl)~COOH, NH2-CH(CH2 cycloprupyl)-COOH, tri~uoroleucine, and he~luordeucine;
R3 and R4 ~re selected independentl~ f~om the ~oup consisting of H, Cl 20 allyl,S C1 20 cyclized allyL C1 20 allyl with a phenyl group a2tached to the C1 20 alkyll C1 20 cyclized al~yl ~ith an attached phenyl group, Cl 20 allyl with an attached phenyl ~oup substituted with K, Cl 20 alkyl ~vith an at2ached phenyl group disubstituted with K, Cl 20 allyl with an attached phenyl ~oup trisubstituted with K, Cl 20 cyclized allyl ~ith an a~tached phenyl group ~ubstituted with K, Cl 10 alkyl ~vith a morpholine [-N(CH2C~2~O] nng attached through nitrogen to the ~yL Cl 1o aLlcyl with a piperidine nng attached through ni~rogen to ~he allyL Cl 10 alkyl with a pq~rolidine nng attached through nitrogen to the aLlcyl, Cl 20 allcyl with an OH group attached to ~he aL~cyL -C~2CH2OCH2CH2OH, Cl 10 with an attached 4-pyridyl group, Cl 10 with an attached 3-pyridyl ~oup, Cl 10 with an attached 2-pyridyl group, Cl 10 with an attached cyclohe;cyl group, NH CH2CH2 (4-hydro~yphenyl), and NH-CH2CH2-(3-indolyl).
The Applic~nts are aware of only a single peptide ketoamide reported in the literature. This compound is Z-Phe-NHOEI2C0-C0-NH-Et (Z-Phe-Gly C0-NH-Et). The compound is reported in Hu and Abeles ~4rch Bioche~L BiopJ~s.. 281, 271-274 (1990)] to be an inhibitor of papain and cathepsin B.
The follo~nng Pep~ide Ketoan~ide compounds are representa~ve of the Peptide Keto-Compounds found to be useful as Calpain inhibitors ~ithin the conte~t of the present invention:
Z-Leu-Phe-CONH-Et ZLeu-Phe-CONH-nPr ZLeu-Phe CONH-nBu Z-Leu-Phe-CONH-iBu ZLeu-Phe-CON~I-13zl Z-Leu-Phe-CONH-(CH~2Ph ZLeu-Abu-CONH-Et ZLeu-Abu-CONH-nPr ZLeu-Abu-CONH-nBu Z-Leu-Abu-CONH-iBu Z-Leu-Abu-CONH-Bzl Z Leu-Abu-CONH-(CH2)2Ph SlJaSTlTUTE SHEET
.
.
~v~u~
WO 92/11B50 P~/US91/09786 -Z Leu-AbU-coNH-(cH2)3 N(cH2c~2)2o ZLeu-Abu-CONH-(CH2~7CH3 Z-Leu-Abu-CONH-(CH~)20H
ZLeu-Abu-CONH~ I2)2o(cH2)2oH
Z-Leu-Abu-CONH~ CH3 ~Leu-Abu-CONH-CH2-c6H3(0cH3)2 Z-I,eu Abu-CONH-CH2-C4~I4N
We studied the inhibition mechanism of the Peptide Keto-Compouulds in both senneand thiol proteases. A cryshl structure of one a-ketcester bound into the active site of the senne protease, porcine pancreatic elastase, has been completed. Ille active site Ser-l9~
o~ygen of the enzyme adds to the carbonyl group of the ketoester to ~onn a te~rahedral ~ntermediate which is stabili~ed by mteractions with the oyanion hole. This structure resembles the tetrahedral intermediate involved in peptide bond hydrolys~s and proves that a-ketoesters are transition-state analogs. His-57 3s hydrogen bonded to the carbonyl group of the ester functional group, the peptide backbone on a section of the polypeptide backbone hydrogen bonds to the inhibitor to forrn a B-sheet, and the ben;cyl ester is directed toward the S' subsites. The side chain of the P1 amino acid residue is located in the S1 pocket of the enzyme. Intsractions with ketoarnides would be similar ~cept ~hat there is the possibility of forming an additional hydrogen bond w~th the NH group of the ketoamide functional group.
~ the case of ketoacids, there would be no R group to interact with the S' subsites~
Therefore, these inhibitors would be e1~pected to be slightly less potent than the ketoesters and ketoamides. However, une~pectedly, certain ketoacid compounds have been found to have surpnsingly high activity when used in the conte~t of the present invention. In particular, Z-Leu-Phe-COOH and ~Leu-Abu COOH have been found to be e~tremely potent inhbiton of Calpains.
The active site of ~ysteine proteases shares ~everal features in common with serir~e proteases including an active site histidine residue. In place of the Ser-19~, ~ysteine proteases have an active site cysteine residue which would add to the ketonic carbonyl group of the peptide ketoacids, ketoesters, or ketoamides to fonn an adduct very similar to the structure described above egcept ~ith a cysteine residue replacing the serine-195 residue.
Additional interactions would occur between the e~ended substrate binding site of ~he ~ysteine protease and the inhl~itor that would increase the binding affini~q and specificity of the inhibitors.
SUBSTITUTE~ SHEET
. . - . . . ... ~ ...... .. .. ..
; . - ~
. . . . - . : ............. .
~, . ~ ` ~ . ...
! ,''.2~ O 92/ll8s0 2 ~ 9 ~ ~ O ~ P~/US91/0978S
~9.
The Peptide Keto-Compounds bind to the proteases inhl~ited thereby using many of the interactions that are found in comple~es of a particular ind~Yidual en~yme wi~h its substrates. In order to design an inhl~itor ~or a particular cysteirle protease, it L'' necessary to: 1) find the amino acid sequences of good peptide substrates for that enzyme, and 2) S place those or si~nilar amino acid sequences into a Peptide Keto-Compound. This design strate~y will also work when other classes of peptide inhibitors are used in place of the peptide substrate to gain infonnation on the appropriate sequence to place in the Peptide Keto-Compound inhibitor. Thu~., we are able to predict the structure of new inhibitors for other proteases based on h~owledge of ~heir substrate specificities. C)nce a good inhibitor structure for a particular enzyme is found, it is then possl~le to change other characteristics such as solubility or hydrophobicity by adding substituents to the M or R groups.
Ln the case of Calpain, a known inhibitor sequence is the peptide aldehyde, Ac-Leu-Leu-Nle-H (also hlown as Calpain Inhl~itor 1 and hereinafter designated as HCIl").
This inhibitor, in addition to a related peptide aldehyde inhl~itor Ac-Leu-Leu-Nme-H (also hlown as Calpan Inhibitor II) are commercially available from Calbiochem of La Jolla, California. We have discovered that peptide ~-ketoesters with aromatic an~ino acid residues in P1 are good inhibitors of the thiol proteases, ca~hepsin B, papain and Calpain.
Additionally, we have disco~vered that peptide a-ketoe~ter and peptide a-ketoamides with either aromatic amino acid residues or small hydrophobic al~yl amino acid residues at P1 are good inhibitors of Calpain.
Our discovery of Peptide Keto-Compounds effective as Calpain Inhibitors was madethrough assay of the Peptide Keto-Compounds as reversible inhibitors. Various concentrations of inhibitors in dimethylsulfoxide (DMSO~ were added to the assay mixture, which contained buffer and substrate. The reaction was started by the addition of the enzyme and the hydrolysis rates were followed spectrophotometrically or fluorimetrically.
88 mM K~I~Pt:)4, 12 mM Na2HPO4, 133 mM EDTA, 2.7 mM cystelne, pH 6.0 was used asa buffer for cathepsin B; and 20 mM Hepes, 10 mM CaC12, 10 mM B-mercaptoethanoL pH
7.2 buf~er was utilized for calpain I and calpain II.
All peptide thioester hydrolysis rates were measured with assay n~i~tures conta~ing 4,4'-dithiodipyridine [e324 = 19800 M-lcm-1; ~asetti & Murray, Arch. Biochem. Biophys.
119, pp 41-49 (1967)]. Papain was assayed with Bz-Arg-AMC or Bz-Arg-NA ~rKanaoka et al., Chem. Pharm. Bull. 25, 3126-3128 (1977)], and the AMC (7-an~ino-4-methylcoumarin) release was followed fluorimetrically (e~citation at 380 nm, and emission at 460 nm).
Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and ~irschke, Methods Enzymol.
SUE~5Tm~ JF~T
~' WO 92/11850 2 ~ 9 ~ 6 0 9 PC~r/US91/0978f-., 80, 535-561 (1981)j, and theAFC (7~amino-4-trifluoromethylcoumarin) releasewas followed ~luorimetrically (e~citation at 400 ~n, and en~ission at 505 nm). Calpain I ~om human erythrocyte~. and calpain II ~om rabbit were assayed using Suc-Leu-~r-AMC [Sasalci et al., J. Bio~ ChenL 259, 12489-12494 (1984), hereby incorporat~ by reference], and the AMC
7-(isopropylcarbamoylamino)4-chloro-3-(2-isothiureidoethoxy)isocoumarin ((CH3)2CHNHCONH-CiTEtOIC) 7-(phenylacetylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin OEhCH2CONH-CiTEtOIC) 7-(L-phenylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin (L-Phe-NH-Cl~ tOIC) 7-(N-t-butylo~ycarbonyl-L,phenylalanylamino)-4-chloro-3-(2-isothiureidoetho~y)isocoumarin (Boc-L-Phe-NH-Cl~EtOIC) 7-(D-phenylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin (D-Phe-NH-CiTEtOIC~
SlJBSrlllJTE SHE~T
WO 92/11850 2 ~ 9 8 6 0 9 Pcr/usgl/097~! ~
7-(N-t-butylo~ycarbonyl-D-phenylalanyl~nino)4 chlo}o-3-(2-isothiureidoetho~y)isocoumar~n (Boc-D-Phe~ Cl~EtOIC) 7-(N-t-butyloxycarbonyl-L-alanyl-L-alanylamino)-4-chloro-3-(2-isothiureidoethoy~isocoumarin (13Oc-Ala-Ala-NH-CiTEtOIC) 7-(L-alanyl-L-alanylamino)~-chloro-3-(2-isothiureidoethoxy)iso~oumar~n (ala-Ala-NH-ClTEtOIC3 7 (1-naphthylcarbamoylamino)~-chloro-3-(2-isothiureidoethoy)iss)coumarin (:NaphthylNH-CiTEtOIC) 7-((S) a methylben~ylcarbamoylamino)~-chloro-3-(2-isothiureidoetho~y)isocoumann (S-C6H5(CH3)CHNHCONH-C~ tOIC3 15 7~((R)-a-methylbenzylcarbamoylarnino)-4 chloro-3-(2-isothiureidoetho~y)isocoumarin (R-C6H5(CH3~CHNHCONH-Cl~EtOIC) 7-das~sylan~ino~-chloro-3-(2-isothiureidoetho~y)isocoumarin (DansylNH-CiTEtOIC) 7-phenylthiocarbamoylamino~-chloro-3-(2-isothiureidoeth~xy)isocournarin (PhNHCSNH-Cl~EtOIC) 7-(m~arboxyphenylthiocarbamoyl)amino 1-chloro-3-(2-isothiureidoetho~y)isocoumarin (m-COOH-PhNHCSNH-CiTEtOIC) 7-(p~arboxyphenylthiocarbamoyl)amino 4-chloro-3-(2-isothiureidoethoxy)isocoumarin (p-COOH-PhNHCSNH-CiTEtOIC) 7-amino~-chloro-3-(3-isothiureidopropoxy)isocoumarin (AClTIC) Isocoumarins with basic substituents are also known to be effective inhibitors of serine proteases. See Powers et al, U.S. Patent No. 4,845,242, the disclosure of which is hereby incorporated by reference. This class of compounds, referred to herein as the "Class II Substituted Isocoumarins," along with tbe other substituted heterocyclic compounds, is believed to be effective in the use of the present invention.
SI~E'r WO 92/11850 2 ~ ~ ~ 6 a ~ PCI`/US91/09786 ", .
The Class II Substituted Isoc~umarins have the following structural formula:
O
S p~ O
~Z
Y
or a pharmaceutically acceptable salt, wherein R is selected from the group consisting of -N-H-C(=NH)-NH2, -C(=NH)NH2, Cl 6 aL~cyl with an attached amino, and Cl~ alkyl with an attached isothiureido of the formula -s-c(+NH2+)N~2~
Z is selected from the group consisung of H, halogen, Clb allyl, Cl 6 allyl with an attached phenyL Cl 6 ~uorina~ed aLlyL Cl 6 allyl with an attached hydroxyl, Cl 6 allyl with an attached Cl~ alkoxy, Cl.6 aLtco~y, Cl 6 fluorinated alko~y, Cl 6 aL~co~y with an attached phenyL benzyloxy, 4-fluorobenylo~y, -OCH2C~I 4R' (2-substituent), -OCH2C6H4R' (3-substituent), OCH2C6H4R' (4-substituent), -OCH2C6H3R2' (2,3-substituents), -OCH2C6H3R2' (2,4-substi~uents), -OCH2C6H3R2' (2,5-substituents), -OCH2C6H3R2' (2,6-substituents), -OCH2C6H3R2' (3,4-substituents), and OCH2C6H3R2' (3,5-substituents).
R' is selected from the ~oup consisting of H, halogen, trifluoromethyL N02, cyano, methyL methoxy, acetyL carboxyL OH, and amino.
Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy.
Alternately, the Class II Substityted Isocoumarins are represented by structure pI) where, Z is selected from the group consisting of C~ coxy with an attached isothiureido, Cl 6 alkoxy with an a~tached guanidino, Cl 6 aL~coxy with an attached amidino, Cl.6 aLlcyl with an anached amino, Cl 6 allcyl with an attached isothiureido, Cl 6 allyl with an attached guanidino, Cl.6 aLlcyl with an attached asnidino, R is selected from the group consisting of H, OH, NH2, NO2 halogen, C~ coxy, C1.6 fluorinated aL~coxy, Cl 6 alkyl, C1 6 allyl with an attached amino, ~ , M-AA-O-, ~. ~n~ n~rt 1T~ C~FFT
WO 92/11850 2 0 9 8 6 0 9 Pt~r/US91/t)97~i .
wherein AA represents alanine, valine, leucine, ~soleuc~ne, proline, methior~ne,phenylalanine, tryptophan; glycine, ser~ne, threonine, cyste~ne, tyrosine, asparagine, glutamine, aspartic acid, glutan~ic acid, lysine, ar~nine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, ci~rulline, hydroxyproline, on~ithine and sarcosine, wherein M represents H, lower alkanoyl having 1 to 6 cazbons, carbo~ canoyl, hydroxyaLkanoy1 amin-alkanoyL benzene sulfonyl tosyL benzoyl, and lower aLlcyl sulfonyl hav~ng 1 to 6 carbons, Y is selected from the group consist~ng of H, halogen, trifluorome~hyL methyl OHand methoxy.
As a further alternative, the Class II Substituted Isocoumarins are represented by structure (II) where R is selected from the group consisting of -N-H-C( =NH)-NH2, -C( = NH)NH2, allyl with an attached an~ino, Cl 6 allyl with an attached isothiureido, Z is selected ~om the group consisting of Cl 6 alkoxy with an attached amino, Cl 6 alkoxy with an attached isothiureido, Cl 6 alkoxy with an auached guanidino, Cl 6 aL~;ox~
with an attached amidino, C1 6 alkyl with an attached arnino, Cl 6 alkyl with an attacheo guanidino, Cl 6 alkyl with an attached amidino, Y is selected from the group consisting of H, halogen, trifluoromethyL methyL OHand methoxy.
The following compounds are representative of the Class II Substituted Isocoumarins:
3-(3-ammopropoxy)isocoumarin, 3-(3-ammopropoxy)~-chloroisocoumar~n, 3-(2-isothiureidoethoxy)4-chloro~socoumarin, 3-(3-isothiureidopropoxy)4 chloroisocoumarin, 7-amino-3-(3-isothiureidopropoxy)~hloroisocoumarin, 7-g,uanidino-3-methoxyisocoumarin, 7-guanidino-3-methoxy 4-chloroisocoumarin, 7-guar~idino-3-etho~yisocournarin, 7-guanidino-3-ethoxy~hloroisocournarin, 7-guanidino-3 -(2-phenylethoxy)isocoumarin, 7-guanidino-3-(2-phenyletho~y)~-chloroisocoumarin.
S~J71~E SWEET
WO 92/11850 2 ~ 9 ~ PCT/US91/09786 17~
Still another class of susbstituted heterocyclic compounds useful in the presentin~ention ~s referred to herein as the "Class m Heterocyclic Compounds" ~nd have the follow~ng structural formula:
.
~Y
(m) ~ x 1~
wherein Z is selected from the group consisting of CO, SO, SO2, CCl and CF, Y is selected from the group consisting of O, S and NH, X is selected from the group consisting of N and CH, and R is selected from the group consisting of Cl 6 aLtcyl (such as methyl, ethyl and propyl), Cl ~ allyl containing a phenyl (such as benzyl), and Clb fluoroaL~cyl (such a~
trifluoromethyL pentafluoroethyL and hepta~uoropropyl).
The Z group must be electrophilic since it interacts with ~he active site serine OH
group of the serine protease. The R group must be uncharged and hydrophobic. One or more of the carbons in the R group could be replaced by O, S, NH and other such atomic groups as long as the R ~oup maintains its hydrophobic character.
The following compounds are representatire of the Class m Heterocyclic Compounds:
2-trifluorome~hyl 1H-3,1-benzo~azine4-one, 2-pentafluoroethyl-4H-3,1-benzoxazine-4-one, 2-heptafluoropropyl-4H-3,1-benzoxazine-4-one, 2-methyl4H-3,1-benzoa~ine~one, 2-propyl-4H-3,1-benzoa~azine-4-one, 2-benzyl-4H-3,1-be~L~oaxa~ine-4-one, 2-hepta~uoropr3pyl4-quinazolinone, 2-propyW-quina~olinone, 2-benzyl-4-quinazolinone, SU3S~lTU~E SHEET
, '`.. . ` ~ . `
.` ` `
` . ~`. ` ~. ~ . ` .
WO 92/11850 2 ~ 9 g ~ 9 PCI/US91tO978 ;~`
2-(C6HsCCl2)4~hloroql~inazoline, and 2-propyl-4-chloroql~ina~oline.
The Class m Heterocyclic Compounds are disclosed in Powers et al., U.S. Patent No.
4,847,202, the disclssure of which is hereby incorporated by reference.
Other substituted heterocyelic ~ompounds have been prepared earlier for other purposes, such as 3-chloroisocoun~arin, Davies and Poole, J. Chem. Soc., pp. 1616-1629 (1928); 3~hloro and 3,4-dichloroisocoumarin, Milevslcaya, Belins3caya, and Yagupol'sku, Zhur. Org. Xhim. 9, pp. 2145-2149 (1973); 3-methyl and 4 carbo~y-3-methylisocoumarin, Tirodkar and Usgaonkar, Ind. J. Chem. 7, pp. 1114-1116 (1969); 7-nitro and 7-aminoisocoumarin, Cholcsey and Usgaonlcar, Ind. J. Chem. 14B, pp. 596-598 (1976). The disclosures of all of the preceding articles are hereby incorporated by reference. These other substituted isocoumarins are also believed to exhibit Calpain inhibitory activity when used in accordance with the present invention.
Still other substituted isocoumarins which have been prepared recently for inhibition of serine proteases are 3-chloroisocournarin, Harper, Hemrr~i, and Powers, J. A. Chem. Soc.
105, pp. 6518-6S20 (1983); 3,4-dichloroisocournarin, Harper, Hemmi, and Powers, Biochemistly 24, pp. 1831-1841 (1985); 3-alkoxy-7-arnino~chloroisocoumarin, Harper and Powers, J. Am. Chem. Soc. 106, pp. 7618-7619 (1984), ~Iarper and Powers, Biochemistry 24, 7200-7213 (1983); additional substituted isocoumarins with basic groups (aminoalkoxy, guanidino or isothiureidoallcoxy), Kam, Fujikawa and Power~, Biochemistry 27, pp.
2S47-25S7 (1988); 7-substituted 3-~lko~y~^chloroisocoumarins, Powers, K~m, Narasimhan, Olelcsys~yn, Hernandez and Ueda, J. Cell Biochem. 39, pp. 33-46 (1989) and Powers, Oleksyszyn, Narasimhan, Kam, Radhakrishnan and Meyer, Jr. Biochemistry 29, 3108-3118 (1990). The disclosures of all of the preceding articles are herebyincorporated by reference.
We believe that the foregoing compounds, which exhibit serine pro~ease inhibitory activity, also e~hibit Calpa~n inhbitory acti~ity when used in accordance with the pres.ent invention.
All of the ~oregolng isocoumarin compounds, including the Class I and II Substitu~ed Isocoumarins, the Class m Substituted Heterocyclic Compounds and the other substituted heterocyclic compounds useful in the practice of the present invention shall be referred to collectively hereinafter as the "Substituted Heterocyclic Compounds." The term "Substituted Heterocyclic Compound" shall be used to refer to any par~icular species of these compounds.
The preparation of the various Substituted Heterocyclic Compounds is illustrated by Examples SHC1-SHC9.
~:U8~:~TUTE SHEET
. .
.
W0 92/11850 2 0 9 ~ 6 ~ ~ PCl'/US91/09786 EXAMPLE SHCl Preparation of 7-(phenylcarbamoylam~no)~4-chloroisocoumar~n was synthes~zed as previously described (Powers, e~ aL, Biochem~stn~ 29, 3108-3118 (1990)). This compousld (0.32 g 1 mtnole) was mLYed with phenyl isocyanate (0.12g, 1 mmole) in 5 ml of THF and the reaction S rni~ture was st~Ted at r.t. overnight. The product 7-(phenylcarbamoy~am~no)~-chloro-3-(2-bromoethoxy)isocoumarm precipi~ated out, yield 40~o, m.p. 215-217 C, mass spectrum m/e = 437.9 (M~ Anal. Calc. for Cl8Hl4N204ClBr: C, 49.4~; H, 322; N, 6.40; Cl, 8.10.Found: C,49.48; H, 3 25; N,634; Cl, 8.12. The phenylcarbarnoylan~no compound (0.1 g, 0.23 ~slole) was heated with 0.02 g of thiourea (0~6 mmole) in 10 ml of THF at 70C
overmght. The final product prec~pitated out, yield 0.04 g, 36%, m.p. 161-163C (dec.), mass spectrum (FAB+) m/e = 433 (M-Br). Anal. Calc. for ClgHl8N404ClBrS:0.25 THF:C, 45.12; H, 3.86; N, 10.53; Cl, 6.67. Found: C, 44.83; H, 3.92; N, 10.12; Cl, 6.41.
7-(Ethylcarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-(t-butylcarbamoylamino)-4-chloro-3-(2-isothiureidoetho~y)isocoumarin, 7-(benzylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-(ethylthiocarbamoylamino)~-chloro-3-(2-isothiureidoethoxy)isocoumarir~-(4-fluorobenzyl) thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-(2,5-dimethylbenzyl) thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared by the same procedure.
EXAMPLE SHC~2 Preparation of 7-(acetylamino)~-chloro-3-(3-isothiureidopropoxy) isocoumarin:
7-Amino-3(3-bromopropoxy)~chloroisocoumarin was synthesi2ed as previously described (Kam, et al., 1988). This compound (033 g, 1 mmole) was heated with 0.15 g of acetic anhydride (1.5 mmole) in 20 rnl of dry Th~. After a few n~inutes, a yellow solid precipitated out. After 3 hrs, the solution was concentrated to 5 ml, and the solidr was filtered to give 037 g of 7-(acetylamino)-4-chloro-3-(3-bromopropoxy) isocoumarin, m.p.
170-172C; mass spectrum m/e = 375 (M+). The acetylated isocoumarin (0.15 g, 0.4mmole) was treated with thiourea (0.036 g, 0.47 mmole) to ~ive 0.9 E~ o~ the final product, (yield 50%), m.p. 180-181C, maæ spectrum m/e = 370 (M+-Br). Anal. Calc. for Cl5Hl7N304ClBrS: C, 39.97; H, 3.80; N, 932; Cl 7.87. Found: C, 39.86; H 3.83; N, 9.29;
CL 7.~5.
7-trifluoroacetylamino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, 7-hepta~uorobutyroylamino 1-chloro-3-(3-isothiureidopropoxy)isocoumarin,7-succinylamino-~n ~ 8~
.. ,, . ~ .-.
. ~ ... . .
., ~ , -.. . . . ..
WO 92/118~0 2 ~ 9 8 6 0 9 PCI/lJS91/0978~
4-chloro-3-(3-isothiureidopropoxy) isocoumarin, and 7-(o-phthalyl)amino~-chloro-3 (3 isothiureidopropoxy) isocoumarisl can be prepared by the sarne procedur~.
E:XA~'LE SlH~
Preparation of 7 (benzylcarbamoylarnino)~-chloro-3-(3-isothiureidopropO~y) isocouunarin:
7-(benzylcarbamoylamino)-4~chloro-3(3-bromopropo~y) isocoumarin was prepared from the reaction of benzyl isoqanate with 7-amino4-chloro-3-(3-bromopropoxy) isocoumarin as descn~ed above, m.p. 188-189C, mass spectrum. m/e = 359 (M+ -ben~yl).
The final product was obtained ~om the reaction of 7-(ben~ylcarbamoylamino)4-chloro-3-(3-bromopropo~y) isocoumarin with thiourea as described aboYe (yield 74%), m.p.
165-166C; mass spectrum (FAB+) m/e - 461 (M+-Br). AnaL Calc. for C2lH22N404Cl~3rS:0.75 1~: C, 48.36; H, 4.70; N, 9.40; CL 6.56. Found: C, 48.13; H, 4.87;
N, 9.65j Cl, 6.15.
Preparationof7-(phenylacetylamino)~-chloro-3-(2 isothiureidoethoxy)isocoumarin:
7~Amina-4-chloro-3 (2-bromoethoxy) isocoumarin (0.15 g, 0.47 mmole) was first n~Lxed with phenylacetyl chloride (0.09 g, 0.55 mmole) in 10 ml of THF, triethylamine (0.05 g, 0.47 mmole) was then added and the reaction mi~ture was stirred at r.t. overnight. After Et3NHCl salt was removed by filtration, the product 7-(phenylacetylamino)~-chloro-3-(~-bromoethoxy) isocoumarin was crystallized from l~ and Pet. ether (yield, 73%), m.p.
165-169C; mass spectrum; m/e = 436.7 (M+). The phenylacetyamino derivative (0.1 g) was heated with thiourea (0.02 g) to give the product 0.05 g (yield, 40%), m.p. 115-120 C;
mass spectrum (FAB+) m/e - 432 (M+ -Br). Anal. Calc. for C20HlgN304ClBrS 0.5 H20:
C 45.9~; H, 3.83; N, 8.05; Cl, 6.80. Found: C, 46.09; H, 4.17; N, 8.02; Cl, 6.79.
Preparation of 7-(R-~-methylbenzylcarbamoylamino)-4-chloro-3-(2-isothillreidoethoxy) isocoumann:
7-(R a-methylbenzylcarbamoylamino) 1 -chloro-3-(2-bromoelhoxy) isocoumann was synthes~zed in the same manner as described above, m.p. 183-185C; mass spectrum m/e - 464 (M+). This compound (0.1 g) reacted with thiourea (0.02 g) under the same condition de~cribed above to form the finalproduct 7-(R-a-methylben~ylcarbamoylamino) 1 -chloro-3-(2-isothiureidoetho~y) isocoumarin (0.078 g), m.p. 143-150C; mass spectrum (FAB+) m/e 5 461 (M+ -Br). Anal. Calc. for C2lH2;~N404ClBrS0.5H20: C, 45.75; H, 4.35; N, 10.17; Cl, 6.M. Found: C, 44.95; H, 4.31; N, 10.02; CL 6.36.
Sl3BSm~UTE SHE~ET
.
: . :. -. WO 92/118~iO 2 8 ~ ~ ~ 0 9 PC~/US91/09786 EX~MPLE SHC6 P~eparatio!l of 7-(D-phenylalanylamino)-4-chloro-3(2-isothiureidoethoxy) isocousnarin:
- soc-D-phe (0.33 g, 1.2 mrnole) reacted with 1,3-dicyclohexylcarbodiimide (0.13 g, 0.6 - S mmole) in 10 ml THF at 0C for 1 hour ~o forrn the symmetric anhydride, and then 7-amino-4~hloro-3(2-bromoetho~y) isocoumarin (0.2g,0.6 mmole) was added. The reaction was stirred at r.t. overnight and the precipitate 7-(Boc-D-Phe-ammo)~-chloro-3-(2-bromoethoxy) isocoumarin was formed (0.29 g, 71%). TLC one spot, m.p. 180-182 C;
mass spectrum m/3 = 566(M~). Anal. Calc. for C25H2~jN206ClBr: C, 53.07; H, 4.63; N, 4.95; Cl 6.27. Found: C, 53.25: H, 4.66; N, 4.87; CL 6.24. E}oc-D-Phe compound (0.2 g, 0.35 rnmole) was reacted with thiourea (0.027 g, 035 mrnole) in the same maMer to give 7-(Boc-D-phenylalanylamino)-4 chloro-3-(2-isothiureidoethoxy) isocoumarin (0.14 g), yield 62%, mass spectrum (FAB+) m/e = 561 (M -Br). Thi5 compound (Q.1 g) was dissolved in 3 rnl of l~ at 0C and then the solvent was evaporated to dryness. The final product precipitated out after addition of ether, one spot on TLC (CH3CN:H20:AcOH = 8:1:1); mass spectnlm (FAB+) m/e = 462 (M+ -Br -CF3C00).
7-Boc-alanylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-benzoylamino-Ala-4-chloro-3(2-i~.othiureidoetho~y) isocoumarin, 7-benzoylamino-Phe-4-chloro-3-(2-isothiureidoethoxy) isocoumarin and 7-Boc-valylamino4-chloro-3-(2~isothiureidoethoxy) isocoumarin can be prepared by the same procedure.
Preparation of 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoethoxy) isocoumarin:
7-(Boc alanylalanylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin was synthesized2S in the same manner, m.p. 147-151C; mass spectrum m/e - 561 (M ' ). Anal. Calc: C, 47.12: H, 4.85. Found: C, 47.18; H, 4.87. This compound (0.2 g) was reacted with thiourea (0.03 g) by the same procedure to ~orm 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoetho~y) isocoumann (0.04 g), mass spectrwTI m/e = 556 (M+ -Br).
7-(Alanylalanylamino)-4-chloro-3(2-~sothiureidoethoxy) isocoumarin was prepared hy deblocking of 130c-Ala-Ala-NH-CiTEtOIC with trifluoroacetic acid, mass spectrum ` (FAB~ m/e - ~56 (M~ -Br-CF3COO).
Preparation of 7-(phenylthiocarbamoylamino) 1-chloro-3-(2-isothiureidoethoxy) isocoumarin:
~IQ~,~TttTF ~LJI~=T
wo 92/11850 2 ~ ~ 8 6 0 3 PCI/US91/0978~
-2~-7-(Phenylthiocarbamoyl~mino)~chlorG3-(2-bromoethoxy) isocournal~n was prepared from the re~ction of phenyl isothiocyanate with 7-amino~-chloro-3-(2-bromoe~hoxy) isocournarin,yield59%,m.p. 157-158C;massspectrwnm/e = 361 (M~ -PhNH+1). Anal.
Calc.: C, 48.36; H, 3.39. Found: C, 48.26; H, 3.40. The bromoetho~y compound was then reacted with thiourea by the same procedure to give the final product, yield 32%; mass spectrum (FAB+) m/s 449 (M+ -Br~.
Preparationof7-(m-carboxyphenylthiocarbamoylamino)~-chlo~o-3-(2-bromoethoxy) isocoumarin was prepared from the reaction OI m~arboxyphenyl isothiocyanate with 7-amino-4-chloro-3-(2-bromoetho~zy) isocoumarin, yield 64%, m.p. 157-158 C; mass spectrum m/e 361 (M+ -(COOH)PhNH+-Br).
7-(3-Fluorobenzoyl)a~uno 1-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-(3-nitrobenzoyl)amino-4-chloro-3-(2-isothiureidoethoxyysocoumarin,7-diphenylacetylarnino-4-chloro-3-(2-isothiureidoeshoxy) isocoumarin, 7-diph~nylpropionylanuno~-chloro-3-(2 isothiureidoethoxy) isocoumarin, 7-(p-toluenesulfonyl) amino-4-chlo~o-3-(2-isothiureidoethoxy) isocoumarin, and 7-(~-toluenesulfonyl) amino~-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared from the reaction of corresponding 7-substituted-4-chloro-3-(2~bromoethoxy) isocoumarin with thiourea as descnbed above. 7-substituted-4-chloro-3-(2-bromoethoy) isocoumarin can be synthesized by reacting 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin with appropriate acid chloride or sulfonyl chloride in the presence of Et3N.
7-Ethoxycarbonylamino4-chloro-3~(2-isothiureidoethoxy) isocoumarin, 7-benzyloxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-phenoxycarbonylan~ino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin c~n be prepared from T~ 25 the reaction of 7 substituted-4-chloro-3-(2-bromoethoxy) isocoumarin wi~h ~h-iourea. 7-Ethoxycarbonylamino4-chloro-3-(2-bromoethoxy~socownarin,7-benzylo;~ycarbonylan~ino~-chloro-3-(2-bromoethoxy) isocouunarin and 7-phenoxycarbonylamino-4~hloro-3-(~-bromoethoxy) isocoumarin c~n be synthes~zed by reac~ing 7amino-4-chloro-3-(2-bromoethoxy) isocoumarin with the corresponding chloroforrrlate.
30 C. PE~IlPE KETO-CO.MPOUNDS
Peptide a-ketoesters, peptide a-ketoacids, and peptide a-ketoamides are transition state analog inhibitors for serine proteases and cysteine proteases. While these subclasses of compounds are chemically distinguishable, for simplicity, all of these compounds will be referred to collectively herein as the "Peptide Keto-Compol~ndsn.
SU~3~TITVTE S~E~
. . . . .
WO 92/1~850 2 ~ 9 ~ ~ Q 9 Pcr/us91~o9786 The ~nserac~ions of peptides with serine and cysteine proteases are designated herein us~ng the nomenclature of Schechter, I., and Berger, A., 1967, Biochem. Biophys. Res.
Corr~nun. 27: 157-162 (incorporated herein by reference). The individual amino acid residues of a substrate or inhibitor are designated P1, P2, etc. and the corresponding subsites of the enzyme are designated S1, S2, etc The scissile band of the substrate is P1-P1'. The pIimary recognition site of serine proteases is S1. The most important recognition subsites of cysteine proteases are S1 and S2.
Amino acid residues and ~locking groups are designated using standard abbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomenclature rules; incosporated herein by reference]. An ar~ino acid residue (AA) in a peptide or inhibitor structure refers to the part structure -NH-C~IR1-CO-, where R1 is the side chain of the ar~uno acid AA. A
peptide a-ketoester residue would be designated -AA-CO-OR which represents the part structure -NH-CHR1-CO-CO-OR. Thus, the ethyl ketoester derived f~om benzoyl alar~ine would be designated Bz-Ala-CO-OEt which represents C6H5CO-NH-CHMe-CO-CO-O~t.
Likewise, peptide ketoacid residues residues would be designated -AA-CO-OH. Further, peptide ketoamide residues are designated -AA-CO-NH-R l~hus, the ethyl keto amide derived from ZLeu-Phe-OH would be designated ZLeu-Phe-CO~NH-Et which represents C6H5CH20CO NH-CH(CH2CHMe2)-CO-NH-CH(CH2Ph)-C:O-CO-NH Et.
Peptide a-ketoesters containing amino acid residues with hydrophobic side chain at the P1 site have also been found to be excellent inhîbitors of several cysteine proteases including papain, cathepsin B and calpain.
Calpains can be inhibited by peptide inhibitors having several different active groups.
Structure-activity relationships with the commercially available in vitro inhibitors of Calpain, such as peptide aldehydes, have revealed that Calpains strongly prefer Leu or Val in the P2 position. These enymes are inhibited by inhibitors having a wide variety of amino acids ~n the P1 position, but are generally more effectively inhibi`ted by inhibitors ha~ing amino acids with nonpolar or hydrophobic side chains in the P1 position. Thus, we have discovered that another particular class of compounds exhibiting Calpa~n inhibitory activity, vhen used in accordance ~vith the present invention, are the Peptide Keto-Compounds. These are compounds of the general structure:
., O
Il M-~aa)n-C-Q-R
:
~' 8lJB~J'rE S~EE~
': " ' - ` ' ` `
WO92/11850 2~9~603 PCI/US91/097~
or a pharmaceutically acseptable salt, wherein:
M represents NH2-CO-, NH2-CS-, NH~-S02-, X-NH-CO-, X-NH~
X-NH-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-, H, acetyl, carbobenzo~v, succinyL methylo~ysuccinyl, butyloxycarbonyl;
X is selected from the group consisting of C1-6 aL~cyL C1-6 fluoroallyL C1-6 aLtcyl substituted with J, C1-6 fluoroaLlcyl substituted with J, 1-admantyL 9-fluorenyL
phenyl, phenyl substitu~ed with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, C1-6 alkyl with an attached phenyl ~oup, C1-6 allyl with two attached phenyl groups, C1-6 aLkyl with an attached phenyl group substituted with K, and C1-6 allyl with t~o attached phenyl groups substituted with K; .
J is selected from the group consisting of halogen, C(:)OH, OH, CN, N02, NH2, C1-6 aL~coxy, C1-6 alkylamine, Cl-6 diallylarnine, C1-6 alkyl-O-CO-, C1-6 aL'cyl-O-CO-NH, and C1-6 aL~cyl-S-;
K is selected from the group consisting of halogen, C1-6 all~yL C1-6 perfluoroalkyL C:1-6 alko~y, N02, CN, OH, C02H, arnino, C1-6 allylamino, C2-12 diallylarnino, C1-C6 a~yl, and C1-6 allcoxy-CO-, and C1-6 aL~cyl-S-;
aa represents a blocked or unbloclced amino acid of the L or D
configuration, preferably selected from the group consisting of: alanine, valine, leucine, isoleucine, proline, methior~ine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine (nle), no~aline (nva), alpha-asl~inobutyric wid (abu), epsilon-aminocaproic acid, citrulline, hydro~yproline, homoarginine, ornithine or sarcosine;
n is a number ~om 1 to 20;
Q is O or NH, R represents H, C1-6 allyL C1-6 fluoroallyL C1-6 chloroal}yl bei~yl, Cl-6 aLlcyl substituted with phenyl, C1-6 al}yl with an attached phenyl group substituted with K
Thus, the Peptide Keto-Compounds can be divided into the Peptide Ketoesters, Peptide Ketoacids and Peptide Ketoamides. Each of the compounds un also be class ied based on the number of an~ino acids contained within the compound, such as an an~ino acid peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
SUB~3~1TUTE SHE~T
WO 92t11850 ~ ~ 9 ~ ~ ~ 3 PCr/US91/09786 We have ~ound certa~n subelasses of Peptide -Ketoester compounds to be particularl~ useful as Calpain Inhibitors when used in accordance with the present invention These sub~lasses are ~eferred to herein as the Dipeptide ~-Ketoesters (Subclass A), the Dipeptide ~-~etoesters (Subclass B), the Tripeptide a-Ketoesters (Subclass A), the Tripeptide a-Ketoesters (Subc:lass B), the Tetrapeptide a-Ketoesters and the Amino Acid Peptide a-Ketoes~ers. All of these subclasses are considered to be to be within the class of Peptide Keto-Compounds.
The Dipeptide a-Ketoe~ters (Subclass A) are compounds of the formula:
Ml-AA2-AAl-cO'O-Rl or a phannaceutically acceptable s lt, wherein Ml represeDts H, NH2-CO-, NH2-CS-, NH !-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group COnSistiDg of Cl 10 alkyL C1 10 fluoroalkyl, Cl 10 alkyl substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl 1~ substituted with K, phenyl disubstituted with K~ phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 allyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 aL~cyl with two attached phenyl groups substituted ~nth K, Cl 10 allcyl with an attached phenoxy group, and Cl 10 aL~cyl with an attached pheno~r group substituted with K on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C
10 allcoxy, Cl 10 alkylamille, C2 12 dial~ylamine, Cl-10 allyl-O-~O-- Cl-10 a~
and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 aLtcyL Cl 10 perfluoroalkyl, Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 alkylamino, C2 l2 diallylamino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 10 aDyl-S-;
AA~ is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the ~-carbon selected from ~he group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalar~ine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2~rboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-~ I~h'TITl ITF .~lEFr , .
: . .
WO 92/11850 2 ~ 9 ~ ~ 9 PCI/U591/097~
ethyl~ysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-amilloheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-C~I(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohe~yl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, tri~uoroleuc~ne, and hexafluoroleucine;
AA2 iS a side chain blocked or unblocked arnino acid with the L configuration, Dconfiguration, or no ch~rality at the a-carbon selected from the group consisting of leucine, isoleucine, proline, rnethionine, methionine sulfoxide, phenylalamne, tryptophan, glycLne, serine, threor~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, Iysine, arg~nine, histidme, phenylglycine, beta-alanine, norleucine, norvalisie, alpha-arninobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine, homoarg~nine, sarcosine, indoline 2-carboxylic acid, 2- 7etidislecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-e~hylserine, S-methylcysteine, S-ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2C~t2)-COOH, alpha-arninoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH,-cyclohexyl)~COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
Rl is selected from the group consisting of H, Cl 20 allyl, Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 alkyl with an attached phenyl group substituted with K
The Dipeptide a-Ketoesters (Subclass B) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-O-R
or a pharrnaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X~ SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 aLIyL Cl 10 nuoroal~yL C1 iO allyl substituted with J, Cl 10 1uoroalkyl substituted with J, 1-adrnantyl, 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstitutet with Kl naphthyl trisubstituted with K.
Cl 10 allyl with an attached phenyl ~oup, Cl 10 alkyl with two attached phenyl ~roups, Cl 10 aLkyl with an attached phenyl group substituted with K, and Cl 10 al}yl with two attached phenyl groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 aL~cyl with an attached pheno~y group substituted with K on the phenoxy group;
. . .
2~9~9 s WO 92/t1850 PCI/US91/09786 J is selected f~om the group corlsisting of halogen, COOH, OH, CN, N02, NH2, C
10 aL~07cy, Cl 10 aL~cylamine~ 2 diallylar~e, Cl 10 allyl-O-CO-, C1 lo a~ky and Cl 10 allyl-S-;
K is selected from the ~roup consisting of halogen, Cl 10 alky~ Cl 10 perfluoroalkyl, Cl 10 alko~y, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2-12 diallylamino, Cl-C10 acyl, and Cl 10 aL~coxy-CO-~ and Cl 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a carbon selected ~om the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoqcide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutam~c acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, no~valine, alpha-a~unobu~yric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoargi}~ine, sarcosine, indoline 2~arboxylic acid, 2-a~etidinecarboxylic acid, pipecolinic acid (2-piperidine carboxyiic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-e~hylcysteine, S-benzylcysteine, NH2-CH(CH2~;~e2)-COOH, alpha-ars~inoheptanoic acid, NH2-CH(CH2 l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-~yclohexyl)-COOH, ~I2-CH(CH2~yclopentyl)-COO~, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl 8 branched and uulbranched cyclized alkyl; or Cl ~ branched and unbranched ~uoroalkyl;
R is selected from the group consisting of X Cl 20 alkyl, Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 alkyl with an attached phenyl group substituted with K
The Tripeptide a-Ketoesters (Subclass A) are compounds of the structure:
or a phannaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X~ CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyl, Cl 10 ~uoroalkyl, C1 10 a~cyl 30 substituted with J, Cl 10 fluoroallcyl substituted with J, 1-adman~yl, 9-fluorenyl, phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubsti~uted with K, naphthyl, naphthyl substituted with K, naphthyl disubsùtutPd with K, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 1o alkyl with two attached phenyl ~roups, Cl 10 aLlyl with an attached phenyl group substituted with K~ and Cl 10 aL~cyl with two attached SUBSTIT~
-.
WO 92/11850 PCI/US91/097~
2~98~09 phenyl groups substituted with K~ Cl 10 allyl with an attached phenoxy group, and Cl 10 alkyl with an attached pheno~;y group substituted with K on the phenoxy ~oup;
T is selected ~om the group consisting of Cl 10 alkyL Cl 10 ~uoroalkyi Cl 10 alkyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyL 9-~luoreny1 phenyL phenyl S substituted ~ith K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trlsubstituted with K
C2 1o allyl with an atta~hed phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 allcyl with two attached phenyl groups substituted with K;
J is selected from the group cons~ting of halogen, COOH, OH, CN, NO2, NH~, Cl 10 aL1coxy, Cl 10 allyla~e, C~l2 dialkylamLne, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-, and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 perfluoroallyl, Cl 10 aLko~y, N02, CN, OH, C02H, at~.uno, Cl 10 allylamino, C2 l2 dialkylals~no, Cl-C10 acyl, and Cl 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspar~ic acid, glutamic acid, lysine, arguune, histidine, phenyl~ycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-an~inocaproic acid, citrulline, hydroxyproline, ornithine, hornoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-C~I(CH2CH~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH~CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CEl(CH2-cyclopropyl)-COOH, trifluoroleucine~ and hexafluoroleucine;
R is selected frosn the group consisting of H, C2 20 allyl, Cl 20 allyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 a1~cyl with an attached phenyl ~oup substituted with K
The Tripeptide a-Ketoesters (Su~xlass B) are compounds of the structure:
M3-AA-AA-NH-~IR2-CO-CO-O-R
or a pharmaceutically acceptable salt, wherein SUBSTITIJTE SHEE~
., , :
.
, ' ` .~';~.,.WO 9~/11850 2 ~ 9 ~ ~ O ~ PCI`/US9~/09786 M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-(: O-, or X-O-CS-;
X is selected from the g~oup consisting of Cl 10 aL~cyl, Cl 10 fluoroaLlcyL Cl 10 alkyl substituted with J, Cl 10 fluoroaLtcyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl substituted with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 aL~cyl with an attached phenyl group substituted with K, and Cl 10 aLIcyl with two attached phenyl roups substituted wi~h K, Cl 10 aLkyl with an attached phenoxy group, and Cl 10 aLlcyl with an attached phenoxy ~roup substituted with K on the phenoxy group;
T is selected from ~he ~roup consisting of Cl 10 alkyL Cl 10 1uoroalkyL Cl 10 aLtcyl substituted with J, Cl 10 fluoroaLkyl substituted with J, 1-admantyL 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, S:~2 10 aLtcyl with an attached phenyl group, Cl 10 aL~cyl with two attached phenyl g~oups, Cl 10 ~ yl with an attached phenyl group substituted with K, and Cl l0 aLkyl with two attached phenyl groups substituted ~ith K;
J is selected from the group consisting of halogen, COOH, OH, CN~ NO2, NH2, C
10 alkoxy, Cl 10 allylaro~e, Cl lo diallylamme, C1 10 alkyl-O-CO, C1 10 Y
~!0 and Cl lo allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 periluoroaLcyl, 1-10 alkoxy, N02, C'N, OH, C02H, am~no, Cl 10 alkylarnino, C2 l2 dialkylamino, Cl-C10 a~l, and Cl 10 alkoxy-CO-. and Cl.10 a~l-S-;
AA is a side chain blocked or unblocked alTuno acid with the L configuratiorl, Dconfiguration~ or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, prol;ne, methionine, methionine sulfoxide, phenylalanine, tryptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutals~ine, aspartic acid, glutan~ic acid, bsine, argir~ine, histidine, phenyl~ycine, beta-alanine, norleucine, non~aline, alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbc~ylic acid), CJ-methy~senne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-WO 92/11850 8 6 0 9 P~US91/097 ~30-~yclohexyl)-COOH, NH2-CH(ÇH2~yclopentyl)-COOH, NH2-C:H(CH2 cyclobutyl)-COOH, NE2-CH(CH2 cyclopropyl)-COOH, trifluoroleucine, and hexa~luoroleucine;
R2 represents Cl 8 branched and unbranched alkyl, Cl 8 branched and unbranched cyclized allyL or Cl 8 branched and unbranched fluoroallcyl;
S R is selected from the group consisting of H, Cl 20 alkyL Cl 20 alkyl with a phenyl group attached to the Cl 20 al~yL and Cl 20 alkyl with an attached phenyl group substituted with K
The Tetrapeptide a-Ketoesters are compounds of the structure:
M3-AA4-A~ AA-CaO-R
or a phannaceutically acceptable salt, wherein M3 represents H, NH2-CO-, NH2-CS-, NH2-SOr, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 aL~cyl substituted with J, Cl 10 fluoroalkyl substituted with J, l-admantyl, 9-fluorenyL phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 aLlcyl with two attached phenyl groups, Cl 10 alkyl ~ith an attached phenyl group substituted with K, and Cl 10 aLcyl with two attached phenyl groups substi~uted with K, Cl 10 alkyl with an attached phenoxy group, and C~ 10 allcyl with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroaLkyl, Cl 10 a~l substitutedwithJ, Cl 10fluoroalkylsubstitutedwithJ, 1-admantyL 9-fluorenyl, phenyl, phenyl substituted with K, phenyl disubstituted ~rith X, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, ~5 C 10 alkyl~nth an attached phenyl group, Cl 10 aL~cylwith two attached phenyl groups, Cl 10 aL~cyl with an attached phenyl group substituted with K, and Cl l0 allyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 alkoxy, Cl 10 aL~ylam~ne, C2 12 dialkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 allyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyL
Cl 10 alko%y, NO2, CN, OH, CO~I, amino, Cl 1o alkylamino, C2 l2 diaL~cylan~ino, Cl-C10 ac~l, and Cl 10 alkoxy-C-, and C1 10 alkyl-S-;
~ttR~TtTlil^ E SHE~ET
, .' . -WO 92/1~850 2 ~ 3 PCl/lJS91/09786 AA is a side chain blocked or unblocked amino acid with the L configuration, D
confi~uration, or no chirality a~ the a~rbon selected from the group consisting of alanine, valine, leucine, isoleucine, pro~ine, methionine, methionine sulfoxid~, phenylalanine, t~ptophan, glycine, serine, threonine, cysteine, fyrosine, asparagine, ~lutars~ine, aspartic acid, S glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, nonaline, alpha-arninobutyric acid, epsilon-aminocaproic acid, cit~ulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2 car~y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~lic acid), O-methylserine, O~ethy3serine, S-methylcysteine, S-ethyl~ysteine, S-benzyl~ysteine, N~I2 CH(CH2C~HEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohe~yl)-COOH, NH2~ H2~yclopentyl)-COOH, NH2-CH(CH2~yclobutyl)-COOH, NH2-CH(CH2-cyclopropyl).COOH, tri~uoroleucine, and hexafluoroleucine;;
AA4 iS a s;de chain blocked or unblocked amino acid ~ith the L corfiguration, D
conf;guration, or no chirality at the a-carbon selected from the group consisting of leucine, isoleucine, methionine, methion~ne sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, aspa~agine, glutamine, aspartic acid, glutamic acid, lysine, arg~nine, histidine, phenylglycine, beta-alanine, norleucine, nor~aline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orni~ne, homoarginine, sarcosine, indoline 2 carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-GEI(CH2C~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2- 1 napthyl)-COOH,NH2-CH(CH2-~-napthyl)-COOH,NH2-CH(CH2~yclohexyl)-COOH,NH2-CH(CH2-cyclopentyl).COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl).COOH, tri~uoroleucine, and hexa~uoroleucine;
2S R is selected from the group consisting of H, Cl 20 aLIcyL Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl-20 allcyl with an attached phenyl group substituted with K
The Amino Acid Peptide a-Ketoessers are compounds of the structure:
Ml-AA-CO-O-R
or a phasmaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-~IH-CO-, X2N CO, X
X2N-CS-, X-NH-SO2-, X2N-S02-, Y-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 alkyL Cl 10 fluoroallyL Cl 10 a~lyl substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl Sl.18S~ITUTE SHEET
` `
WO 92/118~0 2 0 9 g 6 0 9 -32- PCT/U~91/0978 `
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tr~substituted with X, Cl 10 aLIcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10 aL1cyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached S phenyl ~oups substituted with K, Cl 10 al~yl with an attached phenoy group, and Cl 10 alkyl with an attached pheno~y group substituted ~ith X on the phenoxy group;
Y is selected from the group consisting of C~10 a~yL Cl 10 ~uoroaL~cyL Cl 10 alkyl substituted ~nth J, Cl-10 fluoroaL~cyl substituted with J, l-admantyL 9-fluorenyL phenyl substituted with ~, phenyl disubstituted with K, phenyl trisubstituted w~th K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with B, naphthyl trisubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 aLkyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 1~ lo aLkoxy, Cl 10 aL~cylamme, C2 12 diaLkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl-10 perfluoroa~l C~ lo alko~y, N02, CN, OH, ~2~ amino, C1 10 alkylamino, C~ 12 diaLkylan~no, Cl-C10 acyl, and Cl 10 aLcoy-CO-, and Cl l0 allyl-S-;
AA is a side chain ~olocked or unblocked amino acid with ~he L configuration, D
configuration, or no chirali~y at the a-carbon selected from the group consisting of alanine, valinej leucine, isoleucine, proline, methionine, methionine sulfo dde, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutam~ne, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine~ beta-alanine, norleucine, norvaline, 2~ alpha-an~inobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine, homoarg~ni~le, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo4ylic acid, pipecolinic acid (2-piperidme carboylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2C~IEt2)-COO~ alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cydobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trinuoroleucine, and he~ uoroleucine;
R is selected ~om the group consisting of H, Cl 20 alkyL Cl-20 alkyl with a phenyl group attached to the Cl 20 alkyL and Cl 20 aL'cyl with an attached phenyl group substituted with K
C~ ~ITI ~TF .S}~F~T
-- , WO 92/11850 2 0 9 8 6 0 3 PCI'/US91/09786 ,-- t ....
The follow~ng Peptide Ketoester compounds are representative of the Peptide Keto-Compounds found to be useful as Calpa~n ;nhibitors Withi~l the conte~t of the present ~nvention:
Bz-DL,Ala-COOEt Bz-DL-Ala-COOBzl Bz-DL,Ala-COOnBu Bz-DL-Phe-COOEt Bz-DL-Ala-COOCH2-C6H4-CF3 (para) Bz-DL-~Arg-COOEt Bz-DL,Lys-COOEt ZAla-DL,Ala-COOEt ZAla-DL,Ala-COOBzl ~AIa-DL-Ala-COOnBu MeO-Suc-Ala-DL,Ala-COOMe ~Leu-N~ra-COOEt Z-Leu-Me-COOEt ~Leu-Phe-COOEt ZLeu-Abu-COOEt ZLeu-Met-COOEt Z-Phe-DL,Phe-COOEt H-Gly-DI~Lys-COOEt H-Ala-DL-Lys-COOEt H-Pro-DL-Lys-COOEt H-Phe-DL-Lys-COOEt Z-Ala-Ala-DL-Ala-COOEt ~AIa-Pro DL Ala COOEt ZAla-Ala-DL,Abu-COOEt ZAla-Ala-DL-Abu-COOBzl Z Ala-Ala-DI,Abu-COOCH2-C6H4-CP3 (para) MeO Suc-Val-Pro-DL Phe COOMe H-Leu-Ala-DL~Lys-COOEt ZAla-Ala-Ala-DL-~la-COOEt MeO-Suc-Ala-Ala-Pro-DL-Abu COOMe.
ZLeu-Phe-COOEt SUlE~;~lT~alTE S~EEr ~;i, :
~"
..
WO92/11850 0986a9 PCI/US~1/097 PhCQ-Abu-COOEt (c~3)2cH(cH2)2co-Abu~coc)Et C$I3CH2CE)2CHCO.Abu-COOEt Ph~CH2)6CO-Abu-COOEt - ZLeu4~ Phe-~OOEt Z-Leu-Leu-Abu-COOEt ~Leu-Leu-Phe-COOEt 2-NapS02-Leu-Abu-COOEt 2-NapS02-Leu-Leu-Abu-COOEt ZLeu-NLeu-C02Et ZLeu-Phe-C02Bu Z-Leu-Abu-C02Bu Z-Leu-Phe-CO2Bzl ZLeu-Abu-C02BzL
1~ We have found certain subclasses of Peptide Ketoacid Compounds to be partieularly useful when used in accordance with the present inVentiQn. These are sul~classes are the Dipeptide a-Ke~oacids (SubclassA), theDipeptide a-Ketoaeids (SubclassB), the Tripeptide -Ketoacids, the Tetrapeptide a-Ketoacids and the Amilio Acid peptide ~-Ketoacids. All of these are considered to be within the c lass of Peptide Xeto-Compounds.
The Dipeptide a-Ketoacids (Subclass A) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-OH
or a phannaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2 CS-, NH2 S02-, X-NH-CO~, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH~S02-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or 2{-O-CS-;
X is selected from the group consisting of Cl 1o allyl, Cl 10 fluoroaL~cyL Cl 10 allyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1~admantyl, 9-iluorenyL phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted ~ith K, Cl ~0 a;lyl with an attached phenyl group, C1 iO alkyl with two attached phenyl groups, Cl 10 30 alkyl with an attacl~ed phenyl group substituted with K, Cl 10 aLlcyl with ~wo attached phenyl groups substituted with K, Cl 10 allyl with an attached pheno~y group, and Cl 10 alkyl with an attached pheno~y group substituted with K on the phenoxy group;
SUBSTITUTE SHEE~
.: :
.. ~,.. ,, . . .,, ~.......... , . , ; , .
WO92/11850 2~98~a~ Pcr/US91/09786 J is selected ~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 aL~cwy, C1 10 allylamille, C2 ~ aLlylamme, Cl 10 allyl~ CO-, Cl 10 allyl-O-CO-NH., and Cl 10 alkyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyl Cl 10 pe~uoroalkyl S Cl 10 alko~y, NO2, CN, OH, CO2H, amino, Cl 10 allylamino, C2-l2 diallyla~ o, Cl-C10 acyL
and Cl 10 alkoxy-CO-, and C1 10 allcyl-S-;
AA is a side chain blocked or unblocked an~ino acid with the L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, t yptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutan~ne, aspar~ic acid, glutan~c acid, lysine, ar~e, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-a~.ûnobutyric acid, epsilon-aminocaproic acid, citrul~ine, hydro~yproline, ornithine, homoargi~une, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-etllylserine, S-methylcysteine, ~-ethylcysteine, S-benzylcysteine, NH2-~l(CH2~Et2)-COOH, alpha-anunoheptanoic acid, NH2-CH(CHrl-napthyl)-COOH, NH2 CH(CH2-2-napthyl)-COOH, NH2-C~(CH2-cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl~-COOH, NH2-C~I(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl ~ branched and unbranched cyclized alkyl, or Cl 8 branched and unbranched fluoroalkyl.
The Dipepdde ~-Ketoacids (Subclass B) are compounds of the structure:
Ml-AA2-AAl-CO-OH
or a pharmaceudcally acceptable salt, Yvherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N-S02-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-~S-;
X is selected from the group consisdng of Cl 10 allyL Cl 10 fluoroalkyL Cl 10 alkyl substi~uted with J, Cl 10 fluoroaDcyl substituted with J, 1-adman~qL 9-nuorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with K, Cl 10 aLcyl with an attached phenyl group, Cl 10 aLcyl with two attached phenyl groups, Cl 10 allyl ~nth an attached phenyl ~oup substituted with K, and Cl 10 allyl with rwo attached phenyl groups substituted with K, Cl 10 alkyl with an attached pheno~y ~oup, and Cl 10 alkyl with an attached pheno~y group substituted with K on the phenoxy group;
S~3BS~7'Ur~ SHEE~
.
. .
, :
wO 92/11850 2 0 9 8 6 0 9 PCI/US91/097B~
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C1 10 aL~co~y, C1 10 aLlyla~e, C~12 dia~lam~e, Cl 10 allyl-O-CO-, Cl 10 al}yl-O-CO-NH, and C1 l0 allcyl-S-;
K is selected from the group consisting of halogen, Cl 10 a~yL cl lo perfluoroallyL
110 aLkoxy, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2 ~2 diallylan~ino, Cl-C10 acyL
and Cl 10 alkoxy-CO~, and C1 10 alXyl-S-;
AAl is a side chain blocked or unblocked amino acid wi~h the L configuration, D
configlaration, or no chirality at the ~-carbon selected f~om ~he group consistirlg of alanine, valine, leuune, isoleucLqe, proline, methionine, methionine sulfo~de, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparag~ne, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylg}ycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminouproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, ~ndoline 2 carbo~y1ic acid, 2-a~etidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbo~y~c acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben2ylc~s~eine,NH2-CH(CH2CHE~ COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2~CH(CH2-2-napthyl)-COOH, NH2-CEI(CH2-cydohe~yl).COOH, NH2 CH(CH2~clopentyl)-COO~ I2-CH(~H2~yclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and he1~afluoroleucine;
M2 is a side chain blacked or unblocked ~o acid with the L configuration, D
configuration, or no chirality at the a carbon selected from the group consLr.ting of alarline, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alaninet norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine, homoarginine, sarcosine, indoline 2 carbo7y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ber2ylcysteine, NH2-C~(GH2CHEt2)-COOH, alpha-am~noheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NHyC~ H2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, tri~uoroleucine, and hexa~uoroleuc~ne.
The Tripeptide a-Ketoacids are compounds of the s~ucture:
Ml-AA-AA-AA-CO-OH
or a pharmaceutically acceptable salt, wherein ' .
SU~3ST~TU~ ~ffEEt , WO 92/118~0 P~/US91/09786 i 209~
Ml represents H, NH2-CO-, N~I2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected fcom the ~oup consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 alkyl subsdtuted with J, Cl 10 fluo~oaL~cyl substituted with J, 1-admantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K~ naphthyL
naphthyl substituted with K, naphthyl L;subsdtuted with K, naphthyl trisubstituted with K, Cl 10 allyl with an attached pheslyl ~oup, Cl 10 allyl with two attached phenyl groups, Cl 10 al}yl with an attached phenyl group substituted ~nth K, and Cl~10 aLlcyl with two attached phenyl groups substituted with K, Cl 10 alkylwith an attached pheno~y group, and Cl 10 alkyl with an attached pheno~y group substituted with K on the pheno~y group;
J is selected ~om the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 10 lko~y, Cl-10 a~ylamme, C2 12 diaLlylamme, Cl 10 alkyl-O-CO, Cl 10 allyl-O-CO-NH, and C1 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 al}yL Cl lo pD-rfluoroalkyL
1-10 xy, N2~ CN, OH, C2H- armno, Cl 10 allylamillo, C2 12 dialkylamino, Cl-C10 acyL
and Cl 10 alko~y-CO-, and Cl 10 aLk~l-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~de, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, ~lutamine, aspar~ic acid, glutamic acid, bsine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydrw~yproline, ornitbine, homoarginine, sarcosine, indoline 2 car~o~ylic acid, 2-azetidinecarbo~ylic acid, pipeco~;nic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-etbylcysteine, S-benzylcysteine, NH2-CH(CH2C~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH, NHrCH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cycloheyl) COOH, NH2-C~I(CH2-cydopentyl)-COOH, NH2-CH(CH2~:yclobutyl)-COOH, NH2-CH(GEI2 cydopropyl)-COOH, tri~luoroleucine, and he~a~uoroleucine.
The Tetrapeptide a-Ketoacids are compounds of the structure:
Ml-AA-~A-AA-AA-CO-OH
or a pharmaceu~ically acceptable salt, wherein Ml represents H, N~2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-X2N-CS-, X-NH-S02-, X2N-SO2-, Yl-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
WO 92/11850 2 ~ 9 8 6 ~ 9 PCI/US91/0978~
X is selected ~om the group consisting of Cl 10 aLkyl Cl 10 ~uoroalkyL Cl 10 aLI~yl substituted w~th J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyL phenyl substituted w~th K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, ~phthyl disubstituted with K, naphthyl trisubstituted with K, S Cl 10 alkyl w~th an attached phenyl ~oup, Cl 10 all~l with two attached phenyl groups, Cl 10 aLkyl with an attached phenyl group substi~uted with K, and Cl 10 aLkyl ~ith two attached phenyl groups substituted with K, C1 l0 aL~cyl with an attached pheno~y group, and Cl 10 alkyl with an attached pheno.y group substitut d w~th K on the phenoy group;
'Yl is selected f~om the group conslsting of C2 lo aLlcyL Cl lO fluoroalkyL C1 l0 aLlyl substitutedw~thJ, C1 1OfluoroallcylsubstitutedwithJ, 1-a~mantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K, Cl 10 allyl wi~h an attached phenyl group, Cl 10 alkyl with two attached phenyl ~oups, Cl 10 allyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached phenyl groups subsdtuted vrith K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl 10 alko~y, Cl 10 alkylamine, C 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-, and Cl 1o alk~l-S-;
K is selected from the group consisting of halogen, Cl 1o alkyL Cl 1o per~uoroalkyl, Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 1O allylamino, C2.12 dialkylarnino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and Cl 10 aLtcyl~;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality a~ the a~on selected from the group consisting of alanine, valine, leuc~ne, ~soleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, glycine, senne, threonine, ~ysteine, tyrosine, asparagine, glutamine, aspartic acid, gluhn~ic acid, }ys3ne, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon~ nocaproic aciL citrulline, hydro~yproline, ornithine, homoargmine, sarcosine, ~ndoline 2-carbo~ylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylsenne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben~ylcysteine, NH2-C~I(CH2C:HEt2)-COOH, alpha-an~inoheptanoic acid, NH2-CH(CH2-l napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-CH(CH2~dobutyl)-COOH, NH2-CH(CH2-cydopropyl)-COOH, trifluoroleucine, and hexafluoroleucine.
The Amino Acid Peptide a-Ketoacids are compounds of the structure:
SU~STITI ITF .~L~r:~T
- .
`,' ~`' '''' ' ~ :, .....
WO 92/11850 ~ ~ ~ 3 ~ o 9 PCI'/US91/09786 M~ CO-OH
or a pharmaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X N~ CO, 2 2 , X N~-S02-, X2N~02-, Y2-C0-7 X-CS-, X-~02-~ X-O-CO-, or X-O-~S-;
S X is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroalkyl, Cl 10 alkyl substituted with J, C 1 10 fluoroalkyl substituted with J, 1-admantyl, 9-~uurenyl, phenyl, phenyl substituted with X, phenyl disubstituted with iK, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disu~sdtuted ~nth K, naphthyl trisubstituted with K, Cl 10 allyl with an attached phenyl group, C1 l0 allyl ~nth two attached phenyl groups, C1 l0 al}yl with an attached phenyl group sllbstituted with K, and Cl 10 allyl with two attached phenyl groups substituted with K, Cl 10 allyl with an at~ached pheno~y group, and Cl~10 allyl with an attached phenoxy group subseituted with K on ~he pheno~y group;
Y2 is selec~ed ~om the group conslsting of Cl 10 aL~cyL C1 10 ~uoroa~yl, C1 10 alkyl subs~ituted ~vith J, Cl 10 fluoroallyl substituted ~nth J, 1-adman~L 9^fluorenyL phenyl substituted wi~h K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted ~th K, naphthyl trisllbs~cituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 alkyl with an attached phenyl group substitu~ed wlth K, and Cl 10 allyl ~nth two attached phenyl groups substituted with K;
J is selected f~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, C
10 allco~y, C1 10 allcylamine, C~12 dialkylamine, C1 10 allykO-CO-, C1 10 aLkyl O
and Cl 1o allyl~-;
K is selected from the group consisting of halogen, Cl-10 alkyl Cl 10 per~uoroalkyl, C1 10 aL~coxy, NO2, CN, O~I, CO2H, amino, Cl 10 allylamino, C2 l2 diaLIcylalruno, Cl-C10 acyl, and Cl 10 alkoxy-CO-, and C1 10 aLlyl~
AA is a side chain blocked or unblocked amino acid with ~he L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ude, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, g~utamic acid, lysine, arginine, histidine, phenylglycine, beta-alunine, norleucine, norvaline, alpha.an~inobutyric acid, epsilon-aminocaproic acid, citrulline, hydro7yproline, ornithine, homoar~pnine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~lic acid, pipecolinic acid (2-piperidine car~oxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-berzylcysteine, NH2-CH(CH2CHEt2)-COOX alpha-aminoheptanoic acid, SW3STlTl3TE SH@ET
. ~ .
WO 92/11850 2 ~ ~ 2 6 ~ ~ PCI`/US91~0978 NH2-CH(CH2-1-napthyl)~COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2 cyclopentyl~-COOH, NH2-CH(CH2 cyclobutyl)-COOH, NH2-CH(CH2 cyclopropyl)-COOH, trifluoroleucine~ and h~afluoroleucine.
The following Peptide ~etoacid compounds are represen~ative of the Peptide Keto-S Compounds found to be useful as Calpain inhibitors withirl the context of the present invention:
:E3z DL,Lys-COOH
Bz-DI,Ala-COOH
Z-Leu-Phe-COOH
i~T eu Abu-COOH.
The peptide -Icetoamides are transition state analogue inhibitors for cysteine proteases, such as Calpain. We have found that Peptide a-ketoarnides containing amino acid residues with hydrophobic side chains at the Pl site are excellent inhibitors of several cysteine proteases including calpain I and calpain IL
We have found fiYe subclasses of the peptide ketoamides to be particularly efEective in inhl~iting Calpain. These subclasses are referred to herein as Di~eptide a-Ketoarnides (SubclassA),Dipeptide a~Ketoamides (SubclassB), Tripeptide a-Ketoan~ides, Tetrapeptide a-Ketoamides and Amino Acid a-Ketoamides. All of these subclasse~ are considered herein to be withn the class of Peptide Keto-Compounds The Dipeptide a-Ketoamides (Subclass A) haYe the following structural formula:
Ml AA NH-CHR2 CO-CO-NR3R4 or a phannaceutically acceptable salt, wherein Ml represents H, NH2-CO-, NH2-CS-, N~I2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X O-CS-;
X 3s selected from the group consisting of Cl 10 alkyL Cl 10 fluoroaLIcyL ~ 0 alkyl substituted with J, Cl 10 fluoroaLl~yl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl substituted wi~h K, phenyl disubstituted with K, phenyl tri~ubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with E
Cl 10 aLkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, Cl 10 alkyl with two attached phenyl groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 allyl with an attached pheno~y group subsdtuted with K on the pheno~y group;
5UI~STITUTE SHEET
. .
WO 92/11850 2 ~ P~/US91/09786 J is selected from the group consistirlg of halogen, COOH, OH, CM, NO2, NH2, Cl 10 alkoxy, C1 10 ~ylamine, Cz 12 diallyla~une, C1 10 alkyl-O-CO-, C1 l0 alkyl O CO NH, and Cl 10 alkyl-S-;
K is selected from the group consisti~g of h~logen, Cl 10 a~L cl lo per~UoroalksL
Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 10 allylan~ino, C2 l2 diallylamirlo, Cl-C10 acyl, Cl 10 aL~o~y-CO-, and Cl 10 alkyl-S-;
AA is a side chain blwked or unblocked amino acid with the L coIIfiguration, D
configuration, or no chirality at the a-carbon selested from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspar~ic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, a-arninoblltyric acid, epsilon-a~T~nocaproic acid, citrulline, hydroxyproline, ornithine, homoarginine, sarcosin, indoline 2-carboylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-beDzylcysteine, NH2-CH(CH2CHEt2)-COOH, -aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-C~H(CH2-2-napthyl)-COOH, NH2-~H(CH2~yclohexyl)-C9X NH2-CH(~E12-cyclopen.yl)-COOH, NH2-CH(CH2 cyclobu~l)-C9OH, NH2-CH(CH2-cyclopropyl)-COOH, tri~uoroleucine, and he~a~uoroleucine;
R2 is selected from the group consisting of Cl 8 branched and unbranched allyl, Cl..8 branched and unbranched cyclized alkyl, and Cl 8 branched and unbranched fluoroa~kyl, R3 and R4 are selected independently from the group consisting of H, Cl 20 aL~cyl, Cl 20 cyclized alkyL Cl 20 allyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized alkyl with an attached phenyl group, Cl 20 a1kyl with an attached phenyl ~oup substitu~ed with K, Cl 20 alkyl with an attached phenyl group disubstituted with K, Cl 20 alkyl with an attached pbenyl group trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with K, Cl 10 allyl with a morpholine-[-N(CH2CH2)O] ring attached through nitrogen to the all~yL Cl 10 allyl with a piperidine ring attached through nitrogen to the alkyL Cl 10 aLlcyl with a pyrrolidine ring attached through nitrogen to the allyL Cl 20 aLlcyl with an OH group attached to the aLlcyL -CH2CH2C)CH2C~2OH, Cl 10 with an attached 4-pyridyl group, Cl 1o with an attached 3-pyridyl group, Cl 10 with an attached 2-pyridyl group, Cl 10 with an attached cyclohe1yl group, -NH-C~H2CH2-(4-hydroyphenyl), and NH-C~2C~2.(3-indolyl).
The Dipeptide a-Ketoamides (Subclass B) have the following structural forrnula:
Ml-AA2-AAl-CO-NR3R4 ~:LI~IM ITF c ~l~
`
:' :
WO 92/11850 2 ~ 9 ~ 6 0 9 P~/US91/097B~. r ~2-or a pharrnaceutically acceptable salt, wherein Ml represents H, NHz-CO-, NH2-C:~S-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N-CS-, X-NH-S02-, X2N^S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 allyL Cl 10 fluoroalkyL C1 10 alkyl Ssubstituted with J, Cl 10 fluoroaL~cyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl substituted with K, phenyl disubstituted with ~, phenyl trisubstituted with K, naphthyL
naphthyl substituted with ~, naphthyl disubstit~ted with K, naphthyl trisubstituted with K, Cl 10 aL~cyl with an attached phenyl group, C~ 10 allyl with two attached phenyl groups, Cl 10 allyl with an attached phenyl group substituted ~nth K, Cl 10 allyl ~vith two attached phenyl 10groups substituted with K, Cl 10 allyl with an attached pheno~ roup, and Cl 10 alkyl with an attached pheno~y group substituted with lE~ on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl 10 al~co~y, Cl 10 a~ylamLne, C2 12 diallylamme, Cl 10 a~l-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 allyl~-;
15K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pP.~uoroalX~L
0 alko~, N02, CN, OH, CO2H, amino, Cl 10 allylamino, C2 12 diallylamino, Cl-C10 acyl, and C1 10 alko~y-CO-, and Cl 10 alkyl-S;
AAl is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected f~om the group consisting of alanine, 20valine, leuane, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine, tryptophan, serine, threonine, g~steine, tyrosine, asparagine, glu~nine, aspartic acid, glutaïnic acid, lysine, arginine, histidin4 phenylglycin4 beta-alanine, norleucine, norvaline a-an~inobu~yric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orTIitnine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic 25acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-- ethylcysteine, S-benzyl~ystein4 NH2 CH(CH2t;~t2)-COOH, ~-aminoheptanoicacid, NHz-CH(CH2 1 napthyl) COOH, N~2-CH(CH2~2 napthyl) COOH, NH2~CH(CH2-cydohexyl)-COOH, NH2-~I(CH2~clopentyl)-COC)H, NHrC~H(CH2-cyclobutyl)-COOH, NH2-CH(CH2~ydopropyl)-COOH, tri~luoroleucin4 and hexafluoroleucine;
30AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
con~iguration, or no chirality at the a-carbon selected ~om the group consisting of alanine, valine, leucine~ isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glyc~ne, serin4 threonine, cysteine, ~qrosine, asp~ragine, glutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvali, e, SlJBSTiTUT'- S~.EET
.
.
., - ` :
..: wo 92~11850 2 ~ Q ~, pcr/vs91/o9786 ~3-a-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-pi~eridine earbo~ylic acid), O-methylser~ne, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2~ )-COOH, ~ -aminoheptanoic acid, NH2-S CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2~CH(C~H2-cyclohexyl)-COOH NH2-CH(CH2~yclopentyl)-C::OOH, NX2-CH(OEI2~yclobutyl) COOH, 2 C~(CH2~yclopropyl)-COC)H, tri1uoroleuane, and he~a~uoroleucine;
R3 and R4 are selected independentl~ f~om the group co~sisting of H, C1 20 alkyLC1 20 cyclized allyL cl 20 aL~yi w~th a phenyl group attached to the C~ kyL C~ 20 cy~zed alkyl with an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group subs~ituted with K, Cl 20 allyl with an attached phenyl group disubstituted with K, C1 20 aL~cyl with an attached phenyl group trisubstituted with K, C~ cycli~ed aIkyl with an attached phenyl ~oup substituted with K, Cl 10 aLlcyl with a morpholine ~-N(C~2C~2)0] ring attached through nitrogen to the aL~cyL C~ 0 allyl with a piperidine ring attached through nitrogen tO the aL~cyL Cl 1o allyl with a pyrrolidine ring attached through r~itrogen to the aLIcyL Cl 20 alkyl with an OH group attached to the a~yL -CH2CH20CH2~H20H, Cl 10 with an attached 4-py~dyl group, Cl 10 with an attached 3-pyndyl group, Ci 10 q ith an attached 2-pyridyl group, Cl-10 with an attached cyclohe1yl group, -NH-CH2~I2 (4-hydro~yphenyl), and -NH-CH2CH2-(3-irldobl~.
The Tripeptide a-Ketoarnides have the follo~ing structural formula:
Ml-AA AA-AA-CO NR3R4 or a pharmaceutically acceptable salt, wherein Ml represents ~ NH2-CO-, NH2-CS-, NH2~O2-, X-NH-CO-, X2N-CO-, X-NH-CS-, X2N C5-, X-NH SO2-, X2N~02-, X CO-, X-t:~S-, X-SO2-, X-O-CO-, or X-O-CS-;
2~ X ~s selected ~om the group consisting of Cl 10 aLIyL Cl 10 fluoroalkyl~ C1-10 aLIcyl substituted with J, Cl 10 fluoroalkyl substisu~ed with J, 1-admantyl, 9-fluorenyL phenyL phenyl subsdtuted vvith ~, phenyl disubstituted with K, phenyl trisubstituted ~/ith K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl ~risubstituted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 allcyl with two attached phenyl groups, Cl 10 aLlcyl with an attached phenyl group substituted with K, Cl 10 allyl with ~o attached phenyl ~oups substituted with K, Cl 10 allyl ~nth an attached pheno~y group, and C 1-10 alkyl with an attached pheno~y group substituted with K on the pheno~y group;
SV8S~ HFF-r . . , ~ ~ , , ......................................................................... .
WO 92/11850~ ~) 9 8 6 0 9 PCr/US91/09786 ~..
J is selected from the g~oup cons~sting of halogen, COOH, C)H, CN, NO2, NH2, Cl 10 aL~co~y, C1 10 allylam~ne, C2 12 dia31ylam~ne, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-, and Cl 10 aUyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 per~uoroalkyL
S Cl 10 alkoxy, N02, CN, OH, C02H, all~ino, 1-10 allylamino, C2 12 dialkylarl~ino, Cl-C10 acyL
and Cl 10 aLIco~y-CO-~ and Cl 10 ~l~-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
config~ation, or no c~ira~ a~ the a carbon selected from the ~roup consisting nf alanine, valine, leucine"soleucine, proline, methionine, methionine sulfoxide, phenyl~lanine, t~yptophan, glyeine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~arboxylic acid, 2-azetidinecarboxy~ic acid, pipecolinic acid (2-piperidine carb~ylic acid~, O-methylsemle, O-e~hylserine, S-methylcys~eine, S-eshylcysteine, S-benzylcysteine, NEI2-CH(GH2CHEt2)-COOH, a -an~inoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH~2-napthyl)-COOH, NH2-CH(CH2 çyclohe~
COOH, NH2-CH(CH2~clopentyl~COOH, NH2-~I(CH2-cyclobutyl)-COOH, NH2-CH(CH2~yclopropyl)-COOH, ~rifluoroleucine, and he~afluordeucine;
R3 and R4 are ~elected ~ndependently from the group consisting of H, Cl 20 aL~cyl, C1 20 cyclized alkyl, C1 ~0 alkyl ~nth a phenyl group attached to the Cl 20 aLIyL cl 20 cyclized allyl ~nth an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group substituted with K, Cl 20 aLlcyl with an attached phenyl group disubstituted with X, C120 allyl with an attached phenyl group trisubstituted with K, ~-20 cyclized alkyl with an attached phenyl group substituted with ~, Cl 10 alkyl with a morpholine [-N(CH2CH2)0] ring attached ~5 through l.utrogen to the allyl, Cl 10 aL~cyl ~nth a piperidine ring attached through nitrogen to the alkyl, Cl 10 allc,vl with a py~olidine ring attached through nitrogen to the allyl, Cl 20 alkyl with an OH group attached to the alkyl, -CH2CH20CH2CH20H, Cl 10 wlth an attached 4-pyridyl group, Cl-10 with an a~tached 3-pyridyl group, Cl 10 with an attached 2-pyridyl group, Cl 10 ~qth an athched cydohe~yl group, -NH-CH2CH2-(4-hydro~yphenyl), and -NH-CH2C~z(3-indolyl).
The Tetrapeptide a-Ketoarnides have the following structural formula:
Ml-AA-AA-AA-AA-CO-NR3R4 or a pharmaceutically acceptable salt, wherein SU~STITUT SHE3ET
. . ~ .~ , . .~ . . .
wo 92/11850 2 ~ 0 9 Pc~/us9l/o97s6 ~5-Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO, X~N CO, X NH CS, X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-~S-, X-S02-, X-O~CO-, or X-O-CS-;
X is selected from the group consisting of Cl,10 alkyL Cl 10 fluoroaL~cyL Cl,10 aLkyl substituted with J, Cl 1(~ fluoroaLlcyl substituted with J, l-admantyL 9 fluorenyL phenyl, phenyl S substitu~ed with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with X, C 1-10 allyl v~,ith an attached phenyl group, Cl 10 aLkyl with two attached phen~l groups, Cl 10 allyl with an attached phenyl group substituted ~rith K, Cl,1o allyl with two attached phenyl groups substituted with K, Cl,10 allyl with an attached pheno~y group, and Cl,10 aLkyl with an athched pheno~y group substituted with K on the pheno~y group;
J is selected from ~he ~oup consisting of halogen, COOH, OH, CN, NO2, NH2, 10 alkoxy, Cl 10 allylamine, C~l2 diallylamine, C~ 0 all~yl-O-CO-, C1 10 alky and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pe~uoroaL~yl, Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 aL~cylamino, C2-12 dialkylan~ino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 l0 alkyl-S-;
AA is a side ~hain blocked or unbloclced asT~no ac~d w~th the L configuration, Dconfiguration, or no chirality at the a-car~on selected from the group consisting of alal~ine, valine, leucine, isoleucine, proline, methionine, methionine sulfo~de, phenyialanine, tryptophan, glycine, serine, threon~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid, ~latamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, -aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic acid (2-piperidine carba~ylic acid), O methylserine, O-e~hylserine, S-methylcysteine, S-ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2CHEt2)-COOH, c~-am~noheptanoic acid, NH2-CH(C~I2-l-napthyl)-COOH, NH2-CH(C~H2-2-napthyl)-t:OOH, NH2 CH(CH2 cyclohe~yl)-COOH, NHrCH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH~CH2~yclopropyl)-COOH, trifluoroleucine, and he sa~uoroleucine;
R3 and R4 are selected independently ~om the group consisting of H, Cl 20 allyl,Cl 20 ~yclized alkyl, Cl ~0 alkyl vrith a phenyl group attached to the Cl 20 allyL Cl 20 cyclized aL~cyl with an attached phenyl group, Cl 20 allyl with an attached phenyl group substi~uted with lK, Cl 20 allyl with an attached phenyl group disubstituted with K, Cl 20 allyl with an attached phenyl group trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with K, Cl 10 alkyl with a mo~pholine [-N(GH2C:EI2)O] ring attached SUBSTITUTE SH~ET
2~9g6~
WO 92/118~0 P~/VS91/09786-: -~6-through nitrogen to the alkyL cl lo aLtcyl with a piperidine ring attaohed through nitrogen to the all~L cl lo allyl with a pyrrolidine r~ng attached through nitrogen to the allyL Cl 20 allyl with an OH group a~tached to the allyL -~H2CH20C~2CH20H, Cl 10 with an at~ched 4-pyndyl group, Cl 10 w~th an attached 3-pyridyl ~oup, Cl 10 with an attached 2-S pyridyl group, Cl 10 with an attached ~yclohe~yl group, -NH-C~H[2~2-(4-hydro~yphenyl), and -NH-CH2CH2-(3-indolyl).
I~e Amino Acid ~-Ketoamides have the f~llowing s~ructural fosmula:
Ml-AA-CO-NR3R4 or a pharmaceutically acceptable salt, where~n Ml represents H, NH2-CO-, NH2-C:S-, NH2-SO2-, X-NH-CO-, X2N CO, X NH
X2N~CS-, X-NH~02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O~ O-, or X-O-C~;-;
X is selected ~om the group consistmg of Cl 10 allyL Cl 10 fluoroallyL C1 10 alkyl substitutedwithJ, Cl 1O~uoroallcylsubstitutedwithJ, l-admantyL 9-fluorenyL phenyL phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstieuted with K, naphthyl, naphthyl subseituted with K, naphthyl disubstituted with K, naphthyl trisubstituted wiéh K, Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl ~nth two attached phenyl groups, Cl 10 allyl with an attached phenyl group subs~itu~ed ~ith K, Cl 10 aL~cyl wi~h two attached phenyl groups substituted with K, Cl 10 alkyl with an attached pheno~sy group, and Cl 10 aLkyl with an attached pheno~y group substituted with K on the pheno~y ~oup;
J is selected from the group consist~ng of halogen, COOH, OH, CN, NO2, NH2, C
10 aL~o~y, Cl-10 allylamine, C2 12 diaLlcylamine, Cl-10 alkyl-O-CO-, Cl 10 aL~cyl-O-CO~
and Cl 10 allyl~-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyl, C1 10 alka~y, NO~, CN, OH, CO2H, arnino, Cl 10 allylamino, C2 l2 diallylan~ino, Cl-C10 acyl, and Cl 10 alko~y-CO-, and C1 10 alkyl~-;
AA is a side chain blocked or unblocked am~no acid ~th the L configuration, D
configuration, or no chirality at the at-car~on selected from the group consisting of alan~ne, valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanirle, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutan~ine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline, ~-arninobutyric acid, epsilon-aminocaproic acid, ci~rulline, hydro~yproline, ornithine, homoarginine, sarcosine, indoline 2~arbo~y]ic acid, 2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-ben~ylcysteine, NH2 CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NlH2-S~JBSTITUTE SHEET
--'~ WO 92/1 1850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786 ~7-CH(CH2-1-napthyl)~COOH, NH2~CH(~I2-2-napthyl)-COOH, NH2 CH(CH2-cyclohe~yl)-COOH, NH2-CH(OEI2~yclopentyl)-COOH, NH2-CH(CH2~clobutyl)~COOH, NH2-CH(CH2 cycloprupyl)-COOH, tri~uoroleucine, and he~luordeucine;
R3 and R4 ~re selected independentl~ f~om the ~oup consisting of H, Cl 20 allyl,S C1 20 cyclized allyL C1 20 allyl with a phenyl group a2tached to the C1 20 alkyll C1 20 cyclized al~yl ~ith an attached phenyl group, Cl 20 allyl with an attached phenyl ~oup substituted with K, Cl 20 alkyl ~vith an at2ached phenyl group disubstituted with K, Cl 20 allyl with an attached phenyl ~oup trisubstituted with K, Cl 20 cyclized allyl ~ith an a~tached phenyl group ~ubstituted with K, Cl 10 alkyl ~vith a morpholine [-N(CH2C~2~O] nng attached through nitrogen to the ~yL Cl 1o aLlcyl with a piperidine nng attached through ni~rogen to ~he allyL Cl 10 alkyl with a pq~rolidine nng attached through nitrogen to the aLlcyl, Cl 20 allcyl with an OH group attached to ~he aL~cyL -C~2CH2OCH2CH2OH, Cl 10 with an attached 4-pyridyl group, Cl 10 with an attached 3-pyridyl ~oup, Cl 10 with an attached 2-pyridyl group, Cl 10 with an attached cyclohe;cyl group, NH CH2CH2 (4-hydro~yphenyl), and NH-CH2CH2-(3-indolyl).
The Applic~nts are aware of only a single peptide ketoamide reported in the literature. This compound is Z-Phe-NHOEI2C0-C0-NH-Et (Z-Phe-Gly C0-NH-Et). The compound is reported in Hu and Abeles ~4rch Bioche~L BiopJ~s.. 281, 271-274 (1990)] to be an inhibitor of papain and cathepsin B.
The follo~nng Pep~ide Ketoan~ide compounds are representa~ve of the Peptide Keto-Compounds found to be useful as Calpain inhibitors ~ithin the conte~t of the present invention:
Z-Leu-Phe-CONH-Et ZLeu-Phe-CONH-nPr ZLeu-Phe CONH-nBu Z-Leu-Phe-CONH-iBu ZLeu-Phe-CON~I-13zl Z-Leu-Phe-CONH-(CH~2Ph ZLeu-Abu-CONH-Et ZLeu-Abu-CONH-nPr ZLeu-Abu-CONH-nBu Z-Leu-Abu-CONH-iBu Z-Leu-Abu-CONH-Bzl Z Leu-Abu-CONH-(CH2)2Ph SlJaSTlTUTE SHEET
.
.
~v~u~
WO 92/11B50 P~/US91/09786 -Z Leu-AbU-coNH-(cH2)3 N(cH2c~2)2o ZLeu-Abu-CONH-(CH2~7CH3 Z-Leu-Abu-CONH-(CH~)20H
ZLeu-Abu-CONH~ I2)2o(cH2)2oH
Z-Leu-Abu-CONH~ CH3 ~Leu-Abu-CONH-CH2-c6H3(0cH3)2 Z-I,eu Abu-CONH-CH2-C4~I4N
We studied the inhibition mechanism of the Peptide Keto-Compouulds in both senneand thiol proteases. A cryshl structure of one a-ketcester bound into the active site of the senne protease, porcine pancreatic elastase, has been completed. Ille active site Ser-l9~
o~ygen of the enzyme adds to the carbonyl group of the ketoester to ~onn a te~rahedral ~ntermediate which is stabili~ed by mteractions with the oyanion hole. This structure resembles the tetrahedral intermediate involved in peptide bond hydrolys~s and proves that a-ketoesters are transition-state analogs. His-57 3s hydrogen bonded to the carbonyl group of the ester functional group, the peptide backbone on a section of the polypeptide backbone hydrogen bonds to the inhibitor to forrn a B-sheet, and the ben;cyl ester is directed toward the S' subsites. The side chain of the P1 amino acid residue is located in the S1 pocket of the enzyme. Intsractions with ketoarnides would be similar ~cept ~hat there is the possibility of forming an additional hydrogen bond w~th the NH group of the ketoamide functional group.
~ the case of ketoacids, there would be no R group to interact with the S' subsites~
Therefore, these inhibitors would be e1~pected to be slightly less potent than the ketoesters and ketoamides. However, une~pectedly, certain ketoacid compounds have been found to have surpnsingly high activity when used in the conte~t of the present invention. In particular, Z-Leu-Phe-COOH and ~Leu-Abu COOH have been found to be e~tremely potent inhbiton of Calpains.
The active site of ~ysteine proteases shares ~everal features in common with serir~e proteases including an active site histidine residue. In place of the Ser-19~, ~ysteine proteases have an active site cysteine residue which would add to the ketonic carbonyl group of the peptide ketoacids, ketoesters, or ketoamides to fonn an adduct very similar to the structure described above egcept ~ith a cysteine residue replacing the serine-195 residue.
Additional interactions would occur between the e~ended substrate binding site of ~he ~ysteine protease and the inhl~itor that would increase the binding affini~q and specificity of the inhibitors.
SUBSTITUTE~ SHEET
. . - . . . ... ~ ...... .. .. ..
; . - ~
. . . . - . : ............. .
~, . ~ ` ~ . ...
! ,''.2~ O 92/ll8s0 2 ~ 9 ~ ~ O ~ P~/US91/0978S
~9.
The Peptide Keto-Compounds bind to the proteases inhl~ited thereby using many of the interactions that are found in comple~es of a particular ind~Yidual en~yme wi~h its substrates. In order to design an inhl~itor ~or a particular cysteirle protease, it L'' necessary to: 1) find the amino acid sequences of good peptide substrates for that enzyme, and 2) S place those or si~nilar amino acid sequences into a Peptide Keto-Compound. This design strate~y will also work when other classes of peptide inhibitors are used in place of the peptide substrate to gain infonnation on the appropriate sequence to place in the Peptide Keto-Compound inhibitor. Thu~., we are able to predict the structure of new inhibitors for other proteases based on h~owledge of ~heir substrate specificities. C)nce a good inhibitor structure for a particular enzyme is found, it is then possl~le to change other characteristics such as solubility or hydrophobicity by adding substituents to the M or R groups.
Ln the case of Calpain, a known inhibitor sequence is the peptide aldehyde, Ac-Leu-Leu-Nle-H (also hlown as Calpain Inhl~itor 1 and hereinafter designated as HCIl").
This inhibitor, in addition to a related peptide aldehyde inhl~itor Ac-Leu-Leu-Nme-H (also hlown as Calpan Inhibitor II) are commercially available from Calbiochem of La Jolla, California. We have discovered that peptide ~-ketoesters with aromatic an~ino acid residues in P1 are good inhibitors of the thiol proteases, ca~hepsin B, papain and Calpain.
Additionally, we have disco~vered that peptide a-ketoe~ter and peptide a-ketoamides with either aromatic amino acid residues or small hydrophobic al~yl amino acid residues at P1 are good inhibitors of Calpain.
Our discovery of Peptide Keto-Compounds effective as Calpain Inhibitors was madethrough assay of the Peptide Keto-Compounds as reversible inhibitors. Various concentrations of inhibitors in dimethylsulfoxide (DMSO~ were added to the assay mixture, which contained buffer and substrate. The reaction was started by the addition of the enzyme and the hydrolysis rates were followed spectrophotometrically or fluorimetrically.
88 mM K~I~Pt:)4, 12 mM Na2HPO4, 133 mM EDTA, 2.7 mM cystelne, pH 6.0 was used asa buffer for cathepsin B; and 20 mM Hepes, 10 mM CaC12, 10 mM B-mercaptoethanoL pH
7.2 buf~er was utilized for calpain I and calpain II.
All peptide thioester hydrolysis rates were measured with assay n~i~tures conta~ing 4,4'-dithiodipyridine [e324 = 19800 M-lcm-1; ~asetti & Murray, Arch. Biochem. Biophys.
119, pp 41-49 (1967)]. Papain was assayed with Bz-Arg-AMC or Bz-Arg-NA ~rKanaoka et al., Chem. Pharm. Bull. 25, 3126-3128 (1977)], and the AMC (7-an~ino-4-methylcoumarin) release was followed fluorimetrically (e~citation at 380 nm, and emission at 460 nm).
Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and ~irschke, Methods Enzymol.
SUE~5Tm~ JF~T
~' WO 92/11850 2 ~ 9 ~ 6 0 9 PC~r/US91/0978f-., 80, 535-561 (1981)j, and theAFC (7~amino-4-trifluoromethylcoumarin) releasewas followed ~luorimetrically (e~citation at 400 ~n, and en~ission at 505 nm). Calpain I ~om human erythrocyte~. and calpain II ~om rabbit were assayed using Suc-Leu-~r-AMC [Sasalci et al., J. Bio~ ChenL 259, 12489-12494 (1984), hereby incorporat~ by reference], and the AMC
5 (7-amino~-methylcoumarin) rele~e was followed fluorimetrically (e~citation at 380 nm, and emi~sion at ~60 nm). ~atic hydrolysis rates were measured at various substrate and inh~oitor eoncentrations, and Ki value3 were detern~ned by Di~on plot.
Table PKC1 shows the inhibidon constants (Ki) ~or papaint cathepsin B, calpain I, and calpain II.
The inhibition constants for papain shown in Table PKC1 were measured in 0.05 M
Tris-HCL pH 75 buffer, containing 2mM EDTA9 ~mM ~ysteine (freshly prepared), 1%
DMSO, at 25C, using N~-Benzoyl-Arg-AMC as a substrate, ~cept that those values of inhibition constants for papain marked with an ~e~ in Table PKC1 were measured in 50 mM
Tris-HCL pH 7.5 buf~er, containing 2û mM EDTA, S mM qsteine, 9% DMSO, at 25C, using N~-Berlzoyl-Arg-NA as a substrate.
S'Ji3S~lTUTE SHE~T
..
: WOg2/11850 2~609 PCl~l~S9l/09786 l~bition Qf C~ysteine Pro~eases bv Peptide Ketoesters and Ketoacids .
S Ki(mM) Compounds pa GBb ac CIId ZLeU-AbU-COOFt 0.04 0.4 ZLeu-Phe-COOE~t 023 0.4 ZLeu-Me-COOE~t 0.12 0.18 Z-Leu-Nva-COOEt 30 1.2 Bz-DL-Phe-COOEt 500e ~4 Z-Phe-DL,Phe-COOEt 1.8 0.1 ~Phe-DL,Ala-COOEt 3.6 3~
Z-Ala-A.la-DL-Ala-COOEt 15 22 200 :ZAla-Ala-DL,Abu-COOEt 0.9 10 50 200 Z-Ala-Ala-DI~Abu~OOBzl 39 60 ZAla-Ala-DL Nva-COOEt 30 0.1 ~AIa-Pro-D~Ala-COOEt 26 66 MeO Suc Val-Pro-DL~Phe-COOMe 1.1 0.1 2,~e Z-Ala-Ala.Ala.DL-Ala COOEt 2.1 10.0 MeO Suc-Ala-Ala-Pro-Abu-COOMe 0.7 6.0 100 ~' .
aP=Papain Cc~ pain I
bCB=Cathepsin B dC~=Cal~ain 11 ~ O~!TITl IT~ U~Ç:T
. ~ ~ . ... . ~
wo 92/11850 2 0 ~ 8 G 0 3 Pcr/usgl/o97~
It can be seen from the data in Table PKCl that the dipeptide ketoesters with Abu, Phe, or Nle in the Pl site and Leu in the P2 site are potent inhibitors of calpain I and Gllpain ~. Tripeptides with Abu or Ala in the Pl site and Ala in the :P2 site are also seen to be inhibitors of Calpain, albeit somewhat weaker inhibitors than the dipeptides. Thus, S in accordance ~ith the foregoing descri~tion of the design of Peptid~ Keto-Compound inhibitors, we believe that Peptide Keto-Compounds b~sed on these and similar structu~es will ex~ibit Calpain inhibitoly act~ity.
The peptide a-ketoesters are prepared by a two step Dakin-West procedure. This pro~edure can be utilized with either amino acid derivatiYes~ dipeptide derivatives, tripeptide derivatives, or tetrapeptide derivatives as shown in the follo~ing scheme:
o Il M-(AA)n-OH -- ~ Enol Ester ~ > M-(AA)n-CO-R.
l~e precursor peptide ((AA)n) can be prepared using s~ndard peptide chernistry procedures, including those that are well described in publications such as The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, published in 19791987 by Academic Press ("The Peptidesn) and Houben-Weyl Methoden der Organischen Chemie, VoL 15, Parts 1 and 2, Synthese von Peptiden, published by Georg Thieme Verlag, Stuttgart in 1974 (nHouben-Weyln) (both r~ferences hereby incorporated herein by reference).
The M group can be introduced usmg a number OI different reaction schemes. For example, it could be introduced directly on an anuno acid as shown in the following scheme:
H-(AA)n-OH > M-(AA)n-OH.
Alternatively, the M group caD be introduced by reaction ~nth an arnino acid ester, followed by removal of the ester group to g~ve the same product, as shown n the followmg scheme:
~(AA)n-OR'--~ M,-(AA)n-OR'--~ M-(AA)n-OH.
These and other techniques for introduction of the M group are well docurnented in the The Peptides, Houben WeyL and many other tectl?ooks on orgamc synthesis. For example reaction v/ith cyanate or p-nitrophenyl cyanate would introduce a carbamyl group (M = NH;!CO-). Reaction with p-nitrophenyl thiocarbamate ~ould introduce a thio carbamyl group (M = NH2CS-). Reaction with N~I2S402a would introduce the NH2SC)2-group. Reaction with a substituted aLtcyl or aryl isocyanate would introduce ~he X-NH-CO-group where X is a substituted allyl or aryl group. Reaction with a substituted allyl or aryl isothiocyanate would introduce the X~ group where X is a substituted alkyl or aryl group. Reaction with X-S02-Cl would introduce the X-SO2- group. Reaction with a S~JBSTIT~JTE SHEET
. ` : : . . ~ - :
~ .
,A WO 92/11850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786 subslituted allyl or aryl acid chloride would introduce an a~yl group (M = Y-C0-). For e~ample, reaction with MeO-C0-GH~CH2-C0 Cl would gIve ~he Y C0- ~oup when Y is a C2 allyl substituted w~th a C~ yl OC0- group. Reaction with a substituted aL~cyl or aryl thioacid chloride would introduce a thioacyl group (M = Y~ ). Reaction with an asubstituted allyl or aryl sulfonyl chloride would introduce an X-S02- group. For e~ample reaction with dansyl chloride would g~ve the X S02- derIvative where X was a napthyl group monosubstituted with a dimethylamino group. Reaction with a substituted aLl~yl or aryl chlorofonna~e would introduce a X-0-C0- group. Re~ion with a substitu~ed allyl or aryl chloro~iofonnate would introduce a X-O CS-. There are many alternate reaction schemes which could be used to introduce all of the above M groups to gIve either M AA-OH or M~ OR'. The M-AA-OH dernratives could then be used directly in the Dakin-West reaction or could be converted into the dipeptides, tripeptides, and tetrapeptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH which could be be used in the Dakin-West reaction. The substituted peptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH could also be prepared directly from H-AA-AA-OH, H-AA-AA-AA-OH, or H-AA-AA-AA-AA-OH using the reactions described above for introduction of the M group. Alternately1 the M group could be introduced by reaction with carbo~ylblockedpeptides M-AA-AA-OR', M-AA-AA-AA-OR', or M-AA-AA-AA-AA-OR', followed by the removal of the blocking group R'.
The R group in the ketoester structures is introduced during the Dakin-West reaction by reaction with an o~alyl chloride Cl-CO-CO-O-R. For example, reaction of M-AA AA-OH w~th ethyl o~alyl chloride Cl-CO-CO-O-Et gives the keto ester M-AA-AA-CO-O-Et. Reaction of M-AA-AA-AA-AA-OH~ith Cl-CO-CO-O-Bzlwould ~ve the ketoes~er M-AA-AA-AA-AA-CO-O-Bzl. Clearly a wide variety of R groups can be introduced into the ketoester structure by reaction with ,rarious allyl or arylalkyl oxalyl chlorides (Cl-CO-CO-O-R).
The oxalyl chlorides are easily prepared by reaction of an aLlcyl or arylalkyl alcohol with oxalyl chloride Cl CO-CO-CL For e~Eunple, Bzl-O-CO-CO-Cl and n-Bu-O-CO-CO-Cl are prepared by reaction of benzyl alcohol and butanoL respec~vely, ~vith oxalyl chloride in yields of 50% and 80% [Warren, C. B., and Malee, E. J., J. Chmmatograpty 64, 219-222 (1972); incorporated herein by reference].
Ketoacids M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH, M-AA-AA-~A.AA-CO-OH, are generally prepared ~om the corresponding ketoesters M-AA-CO-OR, M-AA-AA-CO-OR, M-AA-~A-AA-CO-OR, M-AA-AA-AA-AA-Co-OR
SlJBSrlTlJ~E SHEET
:.
W O 9 2 / 1 1 8 5 0~ p C r / U S 9 1 t 0 9 7 8 f by aL~caline hydrolysis. In some cases, it may be necessary to use other methods such as hydrogenolysis of a berlzyl ~oup (R = Bzl) or acid cleavage (R = t-butyl) to obtaill the ketoacid. The alternate methods ~vould be used when the M group was labile to aL~caline hydrolysis.
S The various peptide ketoamide subclasses (M-AA-NH-CHR2-CO-CO-NR3R4 (Di~eptide Ketoarnides (Subclass A)), M-AA-AA-CO-NR3R4 (Dipeptide Ketoamides (SubclassB~), M-AA-AA-~A-CO-NR3R4 (InpeptideKetoamides~, M- AA-AA-AA-AA-CO-NR3R4 (tetrapeptide Icetoarnides) and M~ CO-NR3R4 (Amino Acid Ketoamides)) were prepared indirectly f~om the corresponding ketoesters. Ihe ketone carbonyl group was first protected as shown in the following scheme and then the !cetoamide was prepared by reaction with an amine H-NR3R4. The illustrated procedure should also work wi~h other protecting groups.
Ml-AA~ ~ `R ~ -AA2~N~o~R
~ H-NR3R4 Ml-M2~ ~ ~R4 R1 o In addition to the scheme outlined above, a ketoacid could be used as a precursor to produce a corresponding ketoamide. Blocldng the ketone carbonyl group of the ketoacid and then couplingwith an amine H-NR3R4 using standard peptide coupling reagents would yield an intenned~ate ~rhich could then be deblocl~ed to form the ketoamide.
Generol Syn~etfc Method~ for Peptide Reto-Compounds Unless otherwise noted9 materials were obtained from commercial suppliers and used ~nthout further purification. Melting points were taken ~1vith a B~chi capillaly apparatus and are uncorrected. lH NMR spectra were detenT~Lned on a Varian Gemini 300.
Chemical shifts are e~ressed in ppm (~) relative to internal ~etramethylsilane. Flash column chromatography was per~ormed with Universal Scientific Inc. silica gel 0-63.
~:llBSTlTUTE S~IEET
, ~- .
: ; ~ :
WO 92/11850 2 ~ ~ 8 6 ~ 9 P~US9l/09786 Electron-impact mass spectra (MS) of novel compounds were detern~ined with a Varian MAT 112S pe~rometer. The purity of all compounds was checked by thill- laye~
chromatography orl Baker Si250F silica gel plates Usillg th~ following solvent s,vstem: A, CHC13:MeOH = 20:1 v/v; B, CHG13:MeOH = 100:1 v/v; C, AcOEt; D, CHCl3:MeOH =
--10:1 v/v; E, n-BuOH:AcOH;py:H2O = 4:1:1:2 v/v; F, CHC13:MeOH = 5:1 v/v; G, AcOEt:MeOH = 10:1 v/v; H, (i-Pr)20; I, CHC13:MeOH:AcOH = 80:10:5 ~/v; J, CHCl3:MeOH:AcOH = 95:5:3 v/v; K, AcOEt:AcOH = 200:1 v/v; L, C~C13; M, C~IC13:MeOE = 50:1 v/v Amino acid methyl ester hydrochlorides were prepared aecording to M. Brenner e~
al.[~le~. Chen~ Acta 33,568 (1950);36,1109 (1953)] Ln a s~e over 10 rnunol or according to ~achele ~. Ch~. Chen~ 28, 2898 (1963)]in a s~e ofO.1-1.0 n~nol.
Yield (%) mp (~) m.p. (literature) DL-Nva-OCH3HCL 100 113-116 116-117 Ir~e-OC~3 HCL 98 9C-91 98-100 L-Phe-OCH3 HCl, 98 159-161 158-160 DI,Abu-OGH3HCI, 100 148-150 150~151 L-Leu-OCH3 HC1 100 145.S~146.5 147 DL~Me-OCH3 Ha 93 120-121 122-123 4-Cl-Phe-C)C~I3Ha 98 18`4-185 (decomp.) 185-186 N-Acylamino acids was synthesized via Schotten-Baumann reaction lM. Ber~nann, L. Zenas, Chem Ber. 65, 1192 (1932)] in the case when the acyl group was phenylsulphonyl, 2- naphthylsulphonyl or benzoyl.
STITUTE S HE~T
.
~ .
.
.
.
2~9~0~
WO 92/11850 PCr/lJS91/09786 ~5~
Yield (%) mp (C) TLC (R~, eluent) 2-NapSO2-L-Leu-OH 49 115-116 058I
2-NapSO2-D~Abu-OH 51 150-151 050I
2-NapS02-L-Phe-OH 57 148-1485 0.48K
PheSO2-DL-Abu-OH 44 142-143 0.51K
PhCO-DL,Abu-OH 64 141-142 0.64K
N-A~ylamino acids with 4-methylpentanoic, 2-(1- propyl)pentanoic and 7-phenylheptanoic group was synthesized in a two step synthesis. The N-acylamino acid methyl ester was obtained first and then was hydrolysed to the free N-acylan~ino acid.
N-Acyl~ no Acid Met*yl Este~s (Gener~ Pr~cedl~re). To a chi~led (10 C) slurry of the appropriate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was added slowly (temp. 10-15 C) 40 rnmol triethylamine or N- methylmo~pholine and then the reaction mixture ~as stirred for 30 n~inutes at this temperature. Then 18 rnmol of appropriate acid chloride (temp. 10-15 C) was added slowly to the reaction mi~ure and the reaction n~ture was stirred overnight at room temperature. The p~ecipiatatedhydrochloride was filtered, washed on a funnel with 2 ~ 20 ml benzene, and the collected filtrate was washed successivdy with 2 ~ 50 ml 1 M HCI, 2 x 50 rnl 5% NaHC03, 1 x 100 rnl H20, 2 x 50 rnl satd. NaCl and dried over MgSO4. After evaporation of the solvent in vacuo (rotavaporator), the residue was checked for purity (ILC~ and used for the next step (hydrolysis).
Yield (%) mp (C) (cH3)2cH(cH2)2co-DLAbu ocH3 80 oil (CH3CH2CH2)2CHCO-DL-Abu-OCH3 96 117-118 Ph(CH2)6CO-DL,Abu-OCH3 72 oil Hyd~o~is (Get~eral Proced~e). To a solu~ion of 10 mmole of the appropriate N-as~yiamino acid methyl ester in 100 rnl of methanol was added in one portion 11.25 ml of 1 M NaOH (11~ mmol? and the reaction mi~ture was stirred three hours at room temperature. Then the reaction n~Ll~ture was cooled to 0 C (ice- salt bath) and acidified to ~1 = 2 with 1 M Ha aq. To this reaction m~ture was added 100 ml ethyl acetate, transferred to a separatory funnel and organic layer separated. ~he water layer was saturated with solid Naa or (NH4)2SO4 and ree3~tracted with 2 ~ 50 ml AcOEt. Tlse collected organic layer was washed with 2 ~ 50 rnl H20, decolorized with carbon, and dried over MgS04. After evaporation of the solvent in ~vacuo (rotavaporator), the residue was ~ SUBSTITUTE SHlEEt -.. . . . ... ; .
`- ~ .
. ' : . .
~ ` wo 92/11g50 2 ~ 9 3 6 ~ 9 PCr~lJS91/09786 cheeked for puri~ (TLC) and in the case of contan~ination was crystallized ~om an appropriate solvent.
Yield (%) mp (C) (CH3)2CH(CH2)2CO DL-Abu-OH 92 1105-112 (CH3CH2CH2)2 C~HCO-DL-Abu OH 99 126-127 (n-octane) Ph(CH2)5CO-DI~Abu-OH 89 110-112 (n-octane) N~Ac3rldipeptide methyl esters were syllthesized via ~he HOBt-DCC method in a DMF solution [K8 ug and Beiger, C)~em Ber. 103, 788 (1970)].
Yield (%) mp (C) TLC (Rf, eluent) Z-Leu-DL-NVa-OCH3 80 112-113 0.37 B
ZLeu-L-Phe-OCH3 83 86-87 0.85 A
0.39 B
Z-Leu-L-Ile-OCH3 97 oil 0.79 A
0.43 B
Z-Leu-DL Abu-OCH~ 99 86-88 033 B
0.26 H
Z-Leu-L-Leu-OCH3 80 91-92 0.79 G
Z-Leu-DL-NLeu-OCH3 97 111-1115 Z Leu-4 Cl-Phe-OCH3 65 112-132 0.77 J
(liquid c~ystal?) 0.68 K
2-NapSO2-Leu-DL-Abu-OCH3 99 oil 0~9 A
2-NapSO2 Leu L-Leu-OCH3 97-98.5 0.63 A
N-Acyldipeptides were obtained by hydrolysis of the appropriate methyl esters via a generàl hydrolys~s procedure. In the case of N-sulphonyldipeptide methyl esters, 1 equivalent of the methyi.ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH
because of forrn a sulfonamide sodium salt.
SUlBSTlTlJTE SHEIET
WO 92~11850 2 ~ 9 8 ~ ~ 3 I'CTtUS91/09786 r::
Yield (~o) mp (C) TLC (R~, eluent) Z-I.eu DL-~a-OH 100 117-118.5 0.11 A
ZLeu-L-Phe OH 92 105-106.5 0.28 C
Z Leu-L~ OH 79 77-79 0.22A
05~C
~leu-DL-Abu OX 99 gl~ss 0.61 G
~Leu-L~.Leu-OH 97 glass 056I
ZDLeu-DI,NLeu-O~I 98 95~96 ZLeu~-Cl-Phe-OH 87 104-114 0.48 K
(liquid crystal?) 2-NapS02-Leu-DL-Abu-OH
97.4 180-195 (decomp) 0.58 I
2-NapSC)2-Leu-L-Leu~OH
94.0 68-70 052 I
N-Acytripeptide methyl esters were synthesized via HOBt- DCC me~hod in DMF
solution LrKonig and Geiger, Chem. Ber. 103, 78B (1970)].
Yield (%) mp (C) TLC (Rf, eluent) ~Leu-~eu-Abu-0C~I3 87 140-1415 050 A
20~Leu-Leu-Phe-OGH3 76 158-159 0.83 J
2-NapS02-Leu Leu-Abu-OC~I3 97 ~200 0~2 A
N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters via general hydrolysis procedure. Ln the case of N-sulphonyltripeptide me~hyl ester, 1 25 equivalen~ ~f methyl ester was hydrolyzed with 2~5 equivalent of 1 molar NaOH to form the sulfonamide ssdium salt.
Y.ield mp (C) TLC (R~, eluent) ~Leu~Leu-Abu-OII 97 glass 0.69 I
ZLeu-Leu-Phe OH 98 glass 0.44K
302-NapS02-Leu Leu-Abu-OH
- 85 193-195 0.~3 I
(decomp.) 032 J
The following examples, Examples PKC1-PKC65, are given to illus~rate the synthesis 35 of Peptide Keto-Compounds:
SUBSTITVTE SHEET
, . .
, : . -WO 92/11850 2 ~ ~ 8 6 ~ 9 PCTtUS9l/09786 759~
EXAMPI,E PKCl -DL~ COOEt. This compound was s~mthesized by a modi~ied Daki~i-West procedure [Charles et al., J. Chem. Soc. Perlcin L 1139-1146, (1980)~. To a stirred solution of ZAla-Ala-OH (880 mg, 3 mmole), 4 dimethylaminopyridine (15 mg, 0.31 mmole), and pyridine (0.8 mL 10 mmole) in tetrahydrofuran (3 mL) was added ethyl o~alyl chloride (0.7 mL, S rnmole~ at a rate sufficient to ~n~tiate reflu~g. The n~ture was gently reflu~ed for 35 h. The mi~ture was treated ~ith water (3 mL) and stirred vigorously at room ~emperature for 30 n~in. The m~ture was e~tracted w~th ethyl acetate. The organic e~tracts were dried and evaporated to~ ol~tah ~e- residue (1.45 g). The residue was chromatographed on silica gel and eluted with CH2Cl2 to g~ve the enol ster product, oil (500 mg, 37~O); single spot on tlc, R~2 = 0.67 (CHCl3:MeOH = 9:1); MS, m/e = 451(M++1). To a stirred suspension of the enol ester (~10 mg, 0.47 mrnol) ~n anhydrous ethanol (1 mL) at room temperature was added dropwise a solution of sodium ethoxide in ethanol until a dear yello~ soiution resulted. Ihe ethanol was then removed and the residue was trea~ed with ether. The ether solution was washed with water, dried, and evaporated to give a residue. This residue was chromatographed on a silica gel and the product was duted ~nth methylene chloride. The solvent was removed, and the peptide ketoester ZAla-DL,Ala-C02Et was obtained as an sen~i-solid (1~0 mg, 92 %); single spot on tlc, Rf1 058 (C~Ia3:MeOH ~ 5:1); MS, m/e = 351 (M~1). Anal. Calcd. for C17H2206N2-1/3 H20: C7 57.29; H, 622; N, 7.86. Found: C, 57.23; H, 6.36; N, 8.17.
EXAMPI,E PKC2 Z~Ala-Ala-DL~ CO2Et. This compound was prepared ~om ZAla-Ala-Ala-OH
using the same procedure a~ described in E~ample PKC1. The product was crystalli~ed from ethyl ether in 23% yield; sDgle spot on tlc, Rf2 ~ 031 (CHC13:MeOH = 9:1); mp 143-144 ~C; MS, m/e ~ 421 (M+).~AIial. C~llcd. fbr C2~H27O7N3: C, S6.99; X 6-46; N, 9.97. Found: C, 56.96; H, 6.49; N, 9.92.
EXAMPI.E PKC3 Z-Ala-Ala-DL,-Abu-C02Et. This compound was prepared from ZAla-Ala-DL-Abu-OH in 11% yield by the procedure descnbed in Example PKC1; single spot on tlc, Rf2 = 0.60 (CHCl3:MeOH = 9:1); mp 111-113 C; MS, m/e = 436 (M+~1). Anal.
Calcd. for C21H2907N3-1/3 H20: C, 57.13; H, 6.75; N, 9.51. Found: C, 57.38; H, 6.82;
N, 9.62.
~ n~lTl ITr CUE~T
. . .
WO 92/11850 2 ~ 9 8 5 9 ~ PCI/lJS91/09786 E~AMPLE PKC4 ~AIa~Ala-l)L~Nva-CS:)2Et. This compound was prepared from ~AIa-Nva-OH in 20% yield by l:he procedure descri~ed in E~ample PKCl; single spot on tlc, R~l = 0.64 (CHCl3:MeOH = 5:1); MS, m/e = 450 (M~+l). AnaL C~lcd. for C~H3lO7N3H2O: C, 56.51; lH, 7.11; N, 8.99. Found: C, 56.42; H, 7.08; N, 9.06.
EXAMPLE PKC~
Z-Ala-Pro.I)~Ala-CO2Et. ~his compound was prepared ~rom ~AIa-Pro-Ala-OH dicycloh~ylamine ~n 19% yield b~ the proeedLIre descnbed in ~ample PKCl; sing~e spot on tlc, ~2 = 055 (G~CI3:MeOH 8 9:1); MS, m/e = 447 (M~). An~l. Calcd. ~or-C~2H2907N3 1/2 H20: C, 57.88; H, 6.62; N, 9.21. Foumd: C, 57.65; H, 6.68; N, 9.17.
EX~IPLE PKC6 Z-AIa-A~la-~-DL-~la CO2Et~ The compound was prepared from ~AIa-Ala-Ala-Ala-OH in 7% yield by the procedure described in ~ample PKC1; single SpOt on tlc, Rf2 -0.40 (C~Ht:13:MeOH = 9:1); mp. 163-165 C; MS, m/e = 493 (M++1). Anal. Calcd.
for C23H3208N4-1/2 X20: C, 55.08; H, 6.63; N, 11.17. Found: C, 54.85; H, 6.53; N, 11.14.
Bz-DI,Phe-CO2Et. ~ compound was prepared ~om Bz-Phe-OH in 36% yield by the procedure descnbed in E~cample PKC1, oil, single spot on tlc, Rf2 = 0.61 (CHCl3:MeOH = 9:1); MS, m/e ~ 325 (M~). AnaL Calcd. for C~ 904N l/3 H20: C, 68.86; H, 5.98; N, 4 22. Found: C, 69.10; H, 6.09; N, 438.
EX~MPIE PKC8 M~O-Suc~Ala.DL Ala-CO2M~ This compound was prepared from MeO-Suc Ala-Ala OH n 22% yield by the same procedure as descrlbed in E~ample PKC1, e~cept that sodium metho~ide in methanol was used for enol ester bydrolysis, single Spot~orl tlc, - 0.43 (CHC13:MeOH = 9:1); MS, m/e ~ 317 (M~ + 1). AnaL Calcd. for Cl3H2~07N4 1/3 H20: C, 48.44; H, 6.46; N, 8.69. Found: C, 4856; H, 6.39; N, 8.69.
SUBSTITUTE S~5EET
.
wO 92/118S0 2 ~ ~ ~ 6 ~ 9 P~/US91/097~6 ~1-E~MPLE PKC9 MeO Suc~ P~DI~Abu~CO2M[g. This compolmd was prepared ~om MeO-Suc-Ala-Ala-Pro-DL,Abu~OH in 22% yi~ld b~ the proeedure described in ExamylePKC8; foam, single spot on tlc, Rfl = 0.66 (CHCl3:MeOH = 5:1). Anal. Calcd. for S C22H3409N4H20: C, 51.53; H, 7.02; N, 10.85. Found: C. 51.11; H, 7.03; N, 10.88.
MeO-Suc-Val.P~DL-Ph~Ct)2~Me. I~is compound was prepared from MeO-Suc Val-Pro-Phe OH in 42% yield by the same procedure as descnbed in E7~ample PKCB; foam, single spot on ~ 2 057 (~ICl3:MeOH = ~:1); MS, m/e - 517 (M+).
10 Anal. Calcd. ~or C~6H350~N3 2/3 H20: C, 58.96; H, 6.90; N, 7.93. Found: C, 58.92; H, 6.96; N, 7.89.
Bz-DL,Ala-CO2-n-Bu~ This compound was prepared from Bz-Ala-OH in 45%
yield by the procedure descnbed in E~ample PKC1, e~c pt that n-butyl oxalylchloride 15 was used for the Dakin-West reaction and sodi~n n-butoxide in n-butanol was used for enol ester hydrolysis; coloriess oil, single spot on tlc, R~2 = 0.72 (CHCl3:MeOH = 9:1);
MS, m/e = 277 (M+).
Bz DI,Als C02BzL Ihis compound was prepared ~om Bz-Ala-OH in 26% yield 20 by the procedure described in Example PKC1, el~cept that benzyl oxalyl chloride was used in place of ethyl oxayl chloride and sodiurn benzylo~ide in benzyl alcohol was used for enol ester hydrolysis; single spot on tlc, ~2 e0.69 (CHCI3:MeOH = 9:1); mp 95-97 C; MS, m/e - 312 (M-~ +1). AnaL Calcd. for Cl8Hl704N.l/2 H20: C, 67.48; H, 5.66;
N, 437. Found: C, 67.78; H, 5.SS; N, 4.66.
EX~MPLE PKC13 ZAla-DL~Ala-CO2 ~-Bu. This compound was prepared from Z-Ala-Ala-OE in 14% yield by the procedure described in ~ample PKC1, except that n-but~yl oxalylchloride was used in the Daldn-West reaction and s~dium n-butoxide was used for enol ester hydrolysis; oil, single spot on tlc, Rf2 = û.45 (CEIC:13:MeOH = 9:1); MS, m/e =
30 378 (M~). Anal. Calcd. for Cl~H;606Nil/3 H20: C, 5935; H, 7.00; N, 729. Found: C, 59.41; H, 7.03; N, 7.10.
:~Ala-DL,Ala-CO~BzL This compound was prepared ~om ZAla-Ala-OH In 36% yield by the procedure described in Example PKC1, except that benzyl oxalyl ~ . ; . ~. . . .
209~ t WO 92/11850 Pcr/us9l/o9786:
chloride was used in the Dakisl-West reaction and sodiwn be~lo~ide in benzyl alcohol was used for enol ester hydro~sis; single spot on tlc, R~2 = 0.55 (CHCl3:MeOH ~ 9:1);
MS, m/e = 413 (M+ ~1). AnaL Calcd. ~r C22H24O6N2~ C, 64.06; H, 5.87; N, 6.79.
Found: C, 63.79; H, 5.95; N, 6.72.
EXAI~LE PKC15 ~AIa-Ala-DL,Abll-CO2BzL l~is compound was prepared ~om Z~Ala-Ala-Abu-OH in 31~o yield by the proc~dure descnbed in E%ample PXC1, ~s~ept that ben~yl o2a~l chlonde ~as osed in ~he Dal~n-West reaction and sodium benz~ ide in benzyl alcohol ~as used for enol ester hydrolysis; sin~e spot on tk; R~2 = 0 40 (CHa3:MeOH = 9:1);
mp 124-125 C; MS, m/e = 498 (M I + 1). Anal. Calcd. for C2~H3lQ7N3 2/3 H20: C,61.28; H, 639; N, 8.24. Found: C, 61.14; H, 6.65; N, 7.94.
E~AMPLE PKC16 Bz-DI,.ALq-COO}I. The hydrolysis procedure of Tsushima et al. F Org. Chem.
49, 1163-1169 (1984)] was used. Bz-DL,Ala-C02Et (540 mg, 2.2 ;s3~nol) was added to a solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7 mL of H20 and 2-propanol) and stirred at 40 C under n~trogen. After adding ethyl acetate and a saline solu~ion to the reaction mi~ture, the aqueous layer was separated and acidified with 2N HCl and e1~tracted with ethyl acetate. The organic layer was dried over magnesiurn sulfate and the solvent was removed under reduced pressure. The cmde hydrol~sis product was chromatographed on silica gel and eluted with methylene chloride and methanol to obtain an oil (150 mg, 31%); single spot on tlc, R~4 = 0.68 (n-butanol:acetic acid.~yridlne:H2O - 4:1:1:2). Anal. Calcd. for CllH1104N.3/4 H~O: C, 5628; H, 537; N, 5.97. Found: C, 5621; H, ~.46; 5.66.
Eg~MPLE PKC17 Z-Leu-I~L-Nva-COOEt. This compound ~vas prepared ~om Z-Leu-Nva-OH in 60 % yield by the procedure descn~d in E~ample P}~C1; oil, one spot on tlc, Rf = 0.49 (CHGI3:MeOH Y 20:1). NMR ~CDCI3) ~: 0.91 (t, 9H), C~I3; 1.25 (t, 3H), CH3; 1.38 (q, 2H), OCH2C~3; 1.64 (m, 6H), C$I2; 1.85 (m, 1H), CH(CH3)2; 434 (m, 1H) CH2CEI(NHCOOC~I2Ph)CONH; 5.12 (d, 3H) NHGH(CO)CH2 and OCH2Ph; 532 (d, lH) NH; 5.71 (d, 1H) NH; 736 (s, ~H) Ph.
Z-Leu-DI,Nva-end ester, the preeorsor OI Z-I eu-DL-Nva-COOEt was synthesized by the same procedure as descn~ed in Example PKC1 and purified by column chromatography, oiL one spot on tlc. N~IR (CDCl3) ~: 0.96 (t, 9H); 1.25 (t, SVB~;TITUTE~ SH~ET
, . .
2 ~ 3 WO 92~118~;0 PCI`/US91~09786 3H); 1.41 (t, 2H); 1.54 (m, 4H); 1.72 (m, 3H); 2.80 (t, 2H); 4.20 (q, 2H); 4.43 (q, 2H);
.16 (q, 2~I); 5.23 ts, lH); 737 (m, SH); 1133 ~s, lH).
Z L6u DI,Phe-COOEt. This compound was prepared from ~Leu-Phe-OH ~n 30 % yield by the procedure descn~d in E~ample P~Cl; oil~ one spot on tlc, Rf = 0.47 (CHC13:MeOH - 50:13. NMR (CDGI3) ~: 0.88 (d, 9H), OCH2CH3 aIId (CH3)2CH; 1.35 (q, 2H), OCH2CH3; 156 ~q, 2~), (CH3)2CEIGH2 ::H; 3.03 (m, lH), (CH3)2CH; 4.32 (m, 2~I), NHCH(CO)C~H2; 5.08 (s, 4~) GH2Ph; ~.40 (m, lH) NH; 6.61 (d, l~I) NH; 7.31 (s, 5H) Ph; 73S.(s, ~H) Ph.
Z-Leu-DL~Phe enol ester, the precur~or of Z-Leu-DL-Phe~COOEt was synthesized by the same procedure as descnbed in E~ample PKC1 and puri~ied by column chromatography, oil, one spot on tlc. NMR (CDCl3) B: 0.86 (t, 3H); 0.99 (t, 3H); 1.24 (t, 3H); 1.40 (t, 3H); 152 (m, 2H); 1.83 (m, 2H); 4 23 (m, 4H); 4.39 (q, 2H);
5.10 (t, 2H); ~.18 (s, 1H); 7.26 (m, SH); 734 (m, ~H); 8.89 (s, 1~I).
EXAMPLE PKC:l9 2-Leu-DL Abu COOEt. This oompound was prepared from Z-Leu-Abu-OH in 33 % yield by the procedure de~bed in Example P~C1; oiL one spot on tlc, Rf = 0.66 (CHC13 McOH a 20:1). NMR (CDCl3) ~: 0.96 (t, 9H), OG~2CH3 uld (CH3)2CH; 1.26 (t, 3H), CH2CH2CH3; 137 (q, 2~), OGH2S~H3; 1.66 (q, 2H), (C~3)2~CH2CH; 2.00 (m, lH), CH(CH3)2; 4.12 ~q, 2H) GHGH2CH3; 4-34 (m, 1H) NHCH(CONH)CH2CH(C~H3)2; 3.12 (q, 3H) CE[2Ph and CONH~Et)CHCOCOO; ~.29 (t, lH) NH; 6.79 (d, lH) NH; 735 (s~ SH) Ph.
Z Leu-DL-Abu-enol ester, the precursor of Z Leu-DL-Abu-COOEt was ~gnthes~zed ~y the same procedure as descnbed in E%iample PKC1 and puri~ed by column chromatography, o~, one spot on tlc. NMR (CDCI3) ~: a.ss (t~ 6H); 1.12 (t, 3H); 1.24 (t, 3H); 1.41 (t, 3H); 1.73 (m, 4H); 2.86 (q, 2H~; 4.20 (q, 2H); 431 (m, lH);
4.42 (q, 2H); 5.15 (q, 2H); 5.21 (s; 1H); 734 (m, ~H~; 11.29 (s, lH).
EXAMPI,E PKC20 Ala-DI,I,ys-COOEtHCL To a solution OI N-carbobenylo%yalanyl-Ne-,i 30 carbobenzylo~ylysine (1.88 g, 3.9 mmol~, 4-dime~hylan~inopyridine (21 mg, 0.17 mmol), and pyndine (1.0 rnL 12.4 mmol) in l~ (7 mL) was atded ethyl oxalyl chloride (0.9 mL 8.0 mmol) at a rate sufEicien~ to start refluxing. The mi~ture was refllLxed gently for 3 hr, treated with water (4 mL), and stirred vigorously at room temperature for 30 min.
The ~ ture was extracted with ethyl acetate, the organic extracts were washed with SV~TITLIT~ S~FFT
., ~, .
..
. .
.
u u ~J ~
WO 92/~1850 ~CI/US91/09786 ~^
water, dried over MgSO4 and evaporated to give an oily residue (156 g). To a solution of the enol ester (1.56 g, 2.7 snmol) in anhydrous ethanol was added dropwise a solution of sodium etho~de in ethanol at room temperature until the solution turned ~learyellow. Eth~nol w~c remo~ed and the residue was dissolved in ethyl acetate. The S organic soluaion was washed with water, riried over MgSO4, and evaporated to g~ve a residue. This residue was then purifi d b~ colurnn chromatography and the product was eluted with chlorofonn-methanol. The solvent was remo~ed and Z~ DL-Lys(Z)-C02Et was obt~uned as a hygroscopi~ powder (328 mg, 16 %), single spot on tlc, R~2 =
053 (CEIC13:MeOH = 9:1); MS, m/e = 542 (M~ + 1).
N-Carbobenzoxyalanyl-DL,N~carbobenzo~ylysine keto ethyl ester, ZAla-DL-Lys(Z)-CO2Et (328 mg, 0.61 mmol) ~as deprotected with liquid HF contai~ing anisole at O C for 30 min. The HF was removed under reduced pressure. I~e residual oil wasdissolved in absolute ethanol. HCI/ethanol was added to the solution, and ethanol was removed h vacuo. The residue was washed by decantation with ether to give a semisolid (216 mg, 1~0 %); single spot on tlc (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2).
Bz-DL.-Lys-COOES~ICL This compound wasprepared ~rom Bz-DL-Lys(Z)-COOEt in 62% yield by the procedure descnbed in E1~ample PKC20; one spot on tlc, Rf4 = 057 (n-bu~nol:acetic acid pyridine:H20 = 4:1:1:2). The precursor, Bz-DL-Lys(Z)-COOEt w~s prepared ~om Bz-Lys(Z)-OH in 100% yield by the procedure described in Example PKCl; powder, one spot on tlc, E~f2 = 0.75 (CHC13:MeOH = 9:1); MS, m/e =440 (M ~ ). Anal- Calcd. for C24H2806N2~/3 H20: C, 63.70; H, 653; N, 6.19. Found: C, 63.49; H, 651; N, 5.92.
EXAMPLE PKC~2 3~-DL Arg COOEt X~L This compound was prepared from Bz-DL-Arg(Z)-COOEt in 99% yield by the procedure descn~ed in E;~mple PKC20; one spot on tlc, Rf4 = 0.71 (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2), Sakaguchi reagent positive. Bz-DL-Arg(Z)-COOEt was prepared from Bz-DL,Arg(Z)-OH in 19% yield by the procedure described in E~ample PKC20, R~2 = 038 (C:HG13:MeOH = 9:1); mp 140-142 C; MS, m/e = 468 (M+). AnaL Calcd. for C24H2~O6N4: C, 6153; H, 6.02; N, 11.96. Found: C, 61.96; H, 6.48; N, 1234.
8U13ST~UTE SHE~ET
.~ - . WO 92/1 18~0 2 ~ ~ 8 ~ ~ 9 Pcr/US91/09786 ~5-EXAM[PLE PK~3 H-GI~-DL,Lys-COOEt 2HCL l'his compo~nd was prepared from ZC;ly-DL-Lys(Z)-COO:E~ in 92% yield b~ the pro~edure descn~ed in E~ample PKC20; Rf4 = 0.21 (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). ZC;ly-DL-Lys(Z~-COOEt was prepared from ZGly-Lys(Z)-OH in 9% yield by the procedure described in E~ample PKC20, orle spot on tlc, Rf1 = 0.68 (CHC13:MeOH = 5:1); MS, m/e = 528 (M+ + 1).
~LE PKa4 H PFo-DL~Lys-COOEt21HCL Thi~ compound was prepared from Z-Pro-DL-Lys(Z)-COOEt in 100% yield by the procedure descn~ed in E~ample PKC20; one spot on tlc (n-butanol:acetic acid:~dine:H2O = 4:1:1:2). ZPro-DL-Lys(Z)-COOEt was prepared from ZPro Lys(Z) OH in 15% yield by the procedure desoribed in E;~ample PKC20; Rf2 =
0.73 (CHC13:MeOH = 9:1); MS, m/e 568 (M~ + 1).
H-Ph~DL,Lys-COOEt 2HCI. This compound was prepared f~om ~Phe-DL-Lys(Z)-COOEt in 39% yield by the procedure descnbed in E~ample PKC~0; one spot on tlc (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). Z-Phe-DL,Lys(Z)-COOEt was prepared ~om ZPhe-Lys(Z)-OH as previously de~ ed in 9% yield, Rf2 = 0.68 (C~ICl3:MeOH = 9:1); MS, m/e ~ 482 (M~).
H Leu Ala D~L~ COOEt~HCL Thi~ compound was prepared from Z-Leu-Ala-DL Lys(Z)-COOEt in 52% yield by the procedure described in E~ample PKC20; one spot on tlc (n butand:acetic acid:pyridine:H20 2 4:1:1:2).
~Leu Ala DL,Ly~(Z)~COOEt was prepared from Z Leu-Ala DL-Lys(Z)-OH in S~o yield by the pre~iously described Dakin West reaction, R~3 ~ 0.34 (CHCl3:MeOH =
19:1); MS, m/e = 609 (M~-C)CH2~H3)-S~le Am no Acid, Df- and T~fpeptide Enol Esters (Çeneral Proce~re). A
modi~ied Dakir~-West procedure was used [Charles et aL, J. Che~ Soc Per~ , 1139 - 30 (1980)~ and is illustrated ~nth the sgnthesis of Z-IAu-DL~Phe-EE. To a stirred solution of ~Leu-Phe OH (6.19 g, 15.0 mmol), 4- dimethylaminopyridine (0.183 g; 1,5 mmol) and pyridine (4.75 g, 4,85 mL 60 rnmol) in ~etrahydrofuran (45 ml) warmed 50 C was added ethyl oxalyl chloride (430 g, 352 ml, 31.5 rnmol) at a ra~e su~icient to initiate re~uxing.
I~e rnD~ture was then heated at a gentle refl~ for 4 h. After cooling to room ~1 IR~tTI ITF ~ FFT
. . . .
- -: , WO 92/118~0 PCr/US91/0978t temperature the rni~ture vas treated v.rith water (25 rnl) and stirred v;gorously at room temperature for 30 ~in. rhe rn~xture was ~xtracted with ethyl acetate (150 ml) and after separation of the organic layer, the ~vater layer was saturated with solid (NH4)2S04 and re-e~tracted 2-times v~ith 2~ ml ethyl acetate. The combined organic phases were washed 2-tirnes ~ith 75 rnl water, 2-times with 50 rnl of satd. NaCL decolori~ed with ca~bon and dried over MgSO4. After evaporation of the solvent, the crude enol ester (8,36 g, 98%) was Elash-chromatographed on silica gel and the product wa~ eluted with a AcOEt. The solvent was evaporated in vacuo ~rotavaporator~ and the pure enol ester was obtained as a oil (7.22 g, 85%); single spot on TLC, R~ = 0.84, A; 0.68, C.
Z-Leu-Nva-EE. This compound was prepared from :Z-Leu-Nva- OH using the general procedure and purified by flash chromatography on silica gel using CHC13:MeOH - 50:1 v/v as eluent. Yield 95%, single spot on TLC, Rf = 0.92, C; 0.28 ,L.
Z-Leu-Abu-EE. This compound was p~epared from ZLeu-Abu- OH in 78%
yield the general procedure described above. Purifioation by flash obramatography on silica geL $1uent, CHC:13:MeOH = ~0:1 v/Y, sillgle spot on IrLC, Rf = 0,86, A.
PhCO-Abu-EE. This compound was prepared ~om PhCO-Abu-OH in 26% yield by the general procedure as descnbed above. Purification by flash chromatography on silica gel. Eluent CHC13, single spot on TLC, R~ = 0.60, M.
(CH3)2CH(CH2)2CO-Abu~ This compoundwas prepared from (CH3)2CH(CH2)2CO-Abu-OH in 82% yield by the general procedure as described above. Purification by flash chromatography on silica gel. Eluent AcOEt, sin~le spot on TLC, Rf = 0.72, C.
(CH3CH2CH~)2 CH CO-Abu-EE. 'rhis compound was prepared from (CH3GH2CH2)2CH CO-Abu OH in 100% yield by the general praeedure described above. Purification by ~ash chromatography on silica geL Eluent AcOEt, sin~le spot on TLC, Rf = 0.78, C; 0.81, K
Ph(CH~)6CO-Abu-EE. This compound was prepared from Ph(CH2)6CO-Abu-OH in 86% yield by the general procedure descnbed above.
Purification by flash chromatography on silica gel. Eluent AcOEt. Single spot on Tl C, R~ =0.74, C.
Z-Leu4-CI-Phe-EE. T~s compound was prepared from ZLeu- 4-Cl-Phe-OH in 69% yield by the general procedure described above. Purification by flash chromatography on silica geL Eluent AcOEt, single spot on TLC, Rf = 0.77, C; 0.78, K.
!~:U~TlTlJTF ~ IF1~T
` WO92 2~8~9 /11850 PCI'/US~t/09786 Z-Leu-Leu-~bu-EE This compound was prepared from Z2-Leu- Leu-Abu-OH
in 62% yield by the general procedure descIibed above. Puri~cation by flash chroma~ography on silica geL :~lement CHC:13:MeOH = 50:1 v/v. Single spot on TLC, Rf = 0.89, A; 0.75, M.
Z-Leu-Leu-F'he-EE. ~s compound was prepared from ZLeu- Leu-Phe-OH in 60% yield by the general procedure descnbed above. ~ication by fl~sh chromatography on silica geL ~luent CHa3:MeOH = 50:1 v/v. Sin~e spot on TLC, Rf = 0.80, K; 0.70, M.
2-NapSO~ Leu-Abu-E;E;. This compoun~ was prepared from lQ 2-NapSO~-Leu-Abu-OH in 73% yield by the ~eneral procedure descn~ed above.
Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on TLC, Rf =-0.71, K; 05~, C.
2-Nap50~Leu-Leu-Abu-E;E;. ~is compound was prepared 30m 2-NapSO2-Leu-Leu-Abu-OH in 74% yield by the general procedure descnbed above.
Purification by ~ash chromatography Oll silica gel. Eluen~ AcOEt: AcOH = 200: 1 v/v.
Single spot on II~ = 0.69. K
Z-Leu~he-COOEt. Sulgk Arr~ D~nd Tnpeptide- k~tocste~ (Gerleral Procedure). To a stirred solution of B.53 g (15.0 mmol) of ZLeu-Phe-EE in 40 ml anhydrous ethanol at room temperature was added dropwi3e a solution of sodium ethoxide (0.204 g; 3.0 mmol) in 20.0 ml anhydrous ethanol. The c~lor of the reaction n~i~nlre change from colodess or pall yellow to deep yellow or orange dependent on enol-ester. Then the reaction rni~ure was stirred at room temperature for 4-5 hours, the ethanol was then evaporated in va~uo (rohvaporator) and the residue treated with 200 ml ethyl ether (or 200 ml ethyl acetate in the case of the tripeptide). The ether (ethyl acetate) solution was washed with 2 ~ 75 r~ H~O, 2 ~ 75 JTII satd. NaCl, decolorized with carbon and dried over MgS04. After ~vaporation of solvent, the crude product 6.09 g (89.7%) was flash chromatographed on silica gel using CHC13: MeOH - 50:1 v/v.
Evapora~ion of ~olvent give pure Z-Leu-Phe-COOEt (4.08 g, 58.0%) as a thick oil.Single spot on ILC, R~ = 0.60, A; 0.47, M. Mass spectrum, FB-MS [~M+ 1~/Z3 = 469.
EXA~LE PKC28 2.Leu.Nva.COOEt. ~is was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CEICI3: MeOH = 100:1 v/v, yield 86.6%, thick colorless oil, single spot on TLC, Rf = 0.49, A; 037, M. Massspe~rusn F~3-MS ~(M~1)/Z] = 421.
~Tl ITF .
. . .. - ~ .
:
.
~, ',.
wO92~11850 2~6a~ PCI/US91/097~6^--Abu-COOE~ ~s was prepared by the preceding general pr~edure.
Purification by :~ch chromatography on silica gel, eluent CHCl3, yield 82%, thick pale yellow oil, single spot on TLC, R~ = 0.66, A. Mass spectrum, CI-MS [(M+ 1)/Z] = 407.
PhCO-Abu-COOEt. I~is was prepared by the preceding general procedure.
Puri~cation by flilsh chromatography on silica gel, eluent CHM3:MeOH = 50:1 v/v,yield 83%, oiL sin~e spot on TLC, Rf = 0.44, M. Mass spectn~m, M/Z 263 (M~
CI-MS, 264 ((M+ 1~/Z).
(CH3)2CH(~Ia)2CO Abu-CVOEt. ~s was prepared by the preceding general proeedure. Purification by ~ash chromatography on silica geL eluent AcOEt, yield 43%, oil, sin~le spot on TLC, Rf = 056, C. Mass spectmm EI-MS M/7 257 (M+); FB-MS, [(M+1)tZ3 = 258.
13;XAMPLE PKC32 CH3CH2CH)2CHCO-Abu-COOEt. Ihis was prçpared by the preceding general procedure. Purification b~ flash chromatography on silica geL eluent CHC13:MeOH =
50: 1 v/v, thick, yello~h oiL yield 66%, single spot on TLC, R~ = 0.80, C; 0.66, M.
Mass spectrwn EI-MS M/Z = 285 (M+); CI-MS, [(M+ 1)/Z] = 286.
Ph(CH2)6CO-Abu COOE~ This was prepared by the preceding general procedure. Purification by flash chromatography on silica geL eluent CHC13:MeOH =
50:1 v/v, yield 64~o, pale yellow oiL single spot on TLC, Rf = 0.29, M. Mass spectrum EI-MS M/Z - 347 (M~), FB-MS, [(M~1)/Z3 ~ 348-EXM~LE PKC~4 Z~Leu~CI~Phe~COOEt. Ihis was prepared by the preceding gener 1 procedure.
Purification by ~ash chromatography on silica geL eluent AcOEt~ yield 100%, colorless oil, s~ngle spot on TLC, Rf - 0.71, C. Mass spectrum FB-MS M/Z = 503(M ' ).
EX~MPLl: PKC35 Z-Leu-Leu-Abu-COOE~. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica geL eluent C~ICl3:MeOH - 50:1 v/v, yield 79.2%, very thiclc, colorless oiL sDngle spot on TLC, R~ z 0.28. M. Mass spectrum ~3-MS, l(M~ 1)/Z] = 520.
SllBS~TU~E S~E~E~' .
. . . ..
-~ WO 92/11~50 2 ~ 9 PCr/U~;91/09786 .~9 EXAMPLE P~C36 u-Phe-COOEt. Ihis was prepared by the preceding general procedure.
Purificaeion by flash chromatography on silica geL eluent CHC13:MeOH = 50:1 v/v, yield 33%, oiL single spot on TLC, Rf = 056, M. Mass spectrurn, FB-MS, [(M+ 1)/Z] = 582.
S EXAMtPLE PXC:37 2-NapSO2-Leu-Abu-COOEt. This was prepared by the preceding general procedure. Puri~cation by flash chromato~raphy on silica gel, eluent ~C13:MeOH =50:1 v/v, yield 38%, thick oil, sin~ le spot on TLC, Rf = 0.71, K; 054, A. M~LSS spectrum - F~3-MS, [(M+1)/Z] = 463.
EXAMPLE PK~,8 2-NapS02-Leu-Leu-Abu-COOEt. lllis was prepared by the preceding general procedure. Purification by flash chromatography on silica geL eluent AcOEt:AcOH =
200:1 v/v, yield 61%, serni-solid, sMgle spot on rLC, Rf = 0.67, K Mass spec~rumFB-MS, [(M+1)/Z] = 576.
Z-Leu-Met-C02Et. This compound was prepared by the above procedure.
Yellow oil, sin~e spot on TLC, Rf = O.S2 (C~C13:CH30H=50:1), yield 46% (from dipeptide), MS (FAB) 454 (m+ 1).
E~AMPLE PKC40 Z-Leu-NLeu-C02Et. This compound was prepared by the above procedure. Pale yellow oil, single spot on TLC, Rf = 057 (CHCl3:CH3C)H = 50:1), yield 53% (~om dipeptide), MS (FAB) 434 (m+ 1).
EXAMPLE P~41 Syn~hesis of n-Butyl (~ l Chloride. This was prepared by a liserature procedure [Warren and Malee, J. Chromat. 64, 219-æ2 (197~)]. N Butanol (o.i mol. 7.41 g) was ~
added dropwise to o~alyl cblodde (05 mol. 635 g) at -10 C. After the addition was completed, the reaction mi~ure was stirred for 20 Trun. at r.t. and distilled, giving 15.0 g (91.18 moL 91%) of the product n-bu~l o%alyl chloride, bp 58-60 ~C (0.6 mITI Hg).
Z-Leu-Pbe CO2Bu. This compound was prepared f~om ZLeu- Phe-OH and butyl oxalyl chloride in 43% yield by the procedure described for the synthesis of ZLeu-Phe-CO2Et, e~cept that butyl o~alyl chloride was used in place of ethyl oxalyl chloride and sodium butyloxide in butanol was used for enol ester hydrolysis. Single spot on TLC, Rf = 0.54 (CHCl3:CH30H = 50:1) MS(FAB) m/e = 497 (mt 1), lH NMR
(CDC13? ok.
SUBSTITUTE SHEET
, WO 92/11850 2 ~ 9 8 5 ~ 9 Pcr/u5g1/09786 E:~PIE Pl~C42 Z-Leu-Abu-CO2Bu. This compound was prepared by the above procedure.
Single spo~ on TLC, R~ = 0.53 (CHCl3:CH3C)E = 50:1), yield = 36%, pale yellow oiL
MS (F~B) m/e = 435 (M~ H NMR (CDa3) ok.
EX~MPLE PK~3 Synthesfs of Ben~yl O~yl atlorid~ Ben~yl alcohol (0.15 mol. 16 g) was added dropwise to oxalyl chloride (0.75 mol. 95 g) at S-10 C. After the addition was complete, the reaction was st rred for 20 min. at r.t. Ille es:cess ~alyl chloride was distilled and re~,rcled. Then the mi~ture was distilled under vacuo, gIving 26 g (0.12 mol. 86%) of ben~yl o~alyl chloride, bp. 110-112 C (0.6 mm-Hg3. HlNMR (CDCl3) 7.39 (s, SH), 5,33 (s.2H3.
Z-Leu-Phe-CO2BzL This compound was prepared from ZLeu- Phe OH and benzyl oxalyl chloride in 17~o yield by the procedure described in the synthesis of Z Leu-Phe-CO2Et, e~cept that benzyl oxalyl chloride was used in place of ethyl oxalyl lS chloride and sodium benzyloxide in ?oenzyl alcohd was used for enol ester hydrolysis.
Single spot on TLC, Rf = 0.63 (CHCI3:CH3~:)H = 50:1). Pale yellow solid, mp 117-119 C. MS(FAB) m/e = 532 (m~ 1). H1NMR ok ZLeu-Abu-CO2BzL This compound was prepared by the above procedure.
Single spot on TLC. R~ = 051 (CHC:13:CH30H = SO:1), pale yellow oil, MS(FAB) m/e- 469 (m+ 1), yield = 26%.
~Leu-Phe~COOH. D.~peptide Ketoacids (General Procedure). To a stirred solution of 053g (1,13 rnmol) ZLeu-Phe-COOEt in o.O ml methanol was added 1.27 ml ~25 (1.27 mrnol) lM NaOH. The color of the reaction mixture turned dark yellow and a small amount of solid was deposited. The reaction was run at room temperature and progress of the hydrolysis was checlced on TLC. After 24 h. no more substrate was detectet. The reaction mi~ re was chilled in one ice bath at 5 C, acitified with lM
HCl to pH = 3 and e~tracted with AcOEt (2 ~c 50 mL). The organic extract were washed with 2 x 50 ml H20 and if nec~sary, decolorized with carbon and dried over MgSO4. After evaporation of ~he solvent (rotavaporator3, the residue (thick oll) were titurate~ with 2 ~ ml n-he~ane and dried in vacuo. Yield 0.39 g (78%) of colorless, very thick oil. TLC, main spot at Rf = 024, tra e of impurity at Rf = 0.78, I. Mass spectrusn, FB-MS [(M+ l)/Z] = 441.
.
`~- , . - .
WO 92/11~50 2 a 9 ~ 6 0 9 PCI'/US91/09786 E~MPLE PKC46 2-Leu-Abu-COOH. This compound w~c pr~pared from ZL-Leu- Abll-COOEt in 83% yield by the general procedure as descn~d above; TLC, main spot at ~ = 0.14,trace of impur~ty at Rf = 0.73, I. Mass spe~, F~3-MS [(M~ l)/y = 379-S - . EXAMPLE PKC47 ~L~u-Pb~CONH-Et. To a stirred solution of ZLeu-Phe-OH (20 g, 48.5 rnmole), ~dimethylaminopyridine (0587 6 4.8 mmole),and pyridine ~15.7 ml, 194 rnmole) in anhydrous TElF (100 rsll) was added et}lyl o~alyl chloride tll.4 mL 101.8 mmole3 at a rate sufEicient to ~itiate refllmng. The mi~ture was gentb reflu~ced for 4 hours, cooled to room temperature, and water (80 rnl) was added. The reaction mi~ture was stirred vigorously for 30 min, and e~racted with ethyl acetate (3 x 100 ml). The combined or~an~c layers were washed with water (2 ~ 100 ml), sa~urated sodium chloride (2 ~ lOO ml), decolorized with decolorizing car~on, drie~ over magnesium sulfate, and concentrated, leaving a dark orange oil. Chromatography on a silica gel rolumn with CHCl3/CH30H (50:1 v/v) af~orded 14.63 g (y = 53 %) of ~Leu-Phe-enolester. The product was a yellow oil. S~ngle spot on TLC, R~ = 0.77 (CHCL3/CH30H 50:1). NMR
(CDC13) olc.
To a stirred pale yello~v soludon of the ZLcu-Phe-enolester (14.63 g, 25.73 mmole) in anhydrous ethanol (50 ml) was added a solution of sodium ethoxide (0.177 g, 2.6 rnmole) in ethanol (5 rnl). The orange solution was stirred for 3 hours at room temperature, then the ethanol was evaporated and the residue was treated with ethyl ether (300 rnl). The ether layer was washed with water (2 ~ 100 ml), saturated sodium chloride (2 x lOO ml), dried over magnesium sulfa~e, and concentrated, leaving a orange oil. Chromatography on a silica gel column with CHC 13/CH30H (SO:l v/v) af~orded7.76 g (y - 64 %) of the a-l~etoester Z-Leu-Phe-COOEt. The product was a yellow oil.
Single spot on TI,C, R~ - 0.44 (CHa3/CH30H 50:1). NMR (CDC13) ok. MS (FAB, calcd. for C2ÇH32N206: 468.6), m/e = 469 (M+ 1).
The a-carbonyl group of ZLeu~Phe-COOEt was protected by following procedure. A solution of Z-Leu-Phe-COOEt (1 g, 2.13 rnmole) in 5 ml of CH2Ck wasadded 1,2-ethanedithid (0.214 ml, 2.5~ mmole), followed by 05 ml of boron trifluoride etherate. The solution was stirred o~ernight at room tempera~ure. Water (20 ml) and ethyl ether (20 ml) were added. The organic layer was separated, washed with water (2 x 10 ml), saturated sodium chloride (2 ~c 10 ml), dried over magnesium sulfate, and evaporated to a~ord 0.98 g (y = 84 %) yellow semisolid.
.` :
:. :
v v t~ ~J
wo 92/ll~5~ P~r/ussl/097s6 -- -The protec:ted a ketoester (0.98 g, 1.8 mmole) was dissolved in ethanol (5 rnl),cooled to 0-5 C in a ice bath, and ethylalsline was bubbled through the solution Illltil 2.43 g (54 mmole) had been added. The reaction mi~ture was allowed to warm to room temperature slowly, and stirred overnight. The m~ure was filtered, a white precipitate S was removed, leaving a yellow semisolid. Chromato~aphy on a silica gel column with C~ICl3/CH30H (30:1 v/v) afEord 0.63 g (y = 75 %) of ZLeu-Phe-C:ONH-Et. The produc~ was a pale yellow solid. Single spot on TLC, Rf - 0.60 (CHCl3/C~I30H 20~mp 145-147 C. AnaL cal~. for C26H33N30s 46756; ~, 66.79; H, 7.11; N,839; found:C, 6659; H, 7.09; N, 8.95. NMR (CDCl3) ok. MS (~;AB) m/e- - 468-(M+ 1).
Z-Leu-Phe-CONH-~Pr. This compound was synthes~zed ~om the protected a-ke~oester and propylamine in 92 % yield by the prou~dure desc~ibed in Example PKC47.
Single spot on TLC, Rf = O.S0 (CHCl3/CH30H 50:1); mp 152-153 C. Anal. calcd. for C27H3sN30S: 48157; C, 6733; H, 733; M, 8.72. Found: C, 67.21; H, 738; N, 8.64. NMR
(CDCl3) ok. MS (FAB) m/e = 482 (M+l).
~Leu Phe-CONH-nBu. This compound was ~ynthesized f~om the protected a-ketoester and butylan~ine in 67 ~o y~eld by the proeedure descn~d in E~ample PKC47.
Single spot on TLC, R~ = 050.(GHC13/GH30H 50:1); mp 152-153 C. Anal. calcd. forC28H37N30s: 49S59; C, 67.8S; H, 752; N, 8.48. Found: C, 67.70; H, 7~7; N, 8.43. NMR
(GDCl3) ok. MS (P`AB) m/e = 496 (M+ 1).
EX~MPLli~ PKC50 Z-Leu-Phe-CONH-lBlL This compound was synthesi~ed from the protected a ketoester and Isobutylamine in 53 % yield by the procedure descrl~ed in Example PKC47. S~ngle spot on TLC, Rf = 054 (CHC13/CH30H S0:1); mp lg2 ~G ~n~L
calcd. for C28H3~N305: 49S59; C, 67.85; H, 7~2; N, 8.48. Found: C, 67.77; H, 7~6; N, 8.40. NM~ (CDCl3) ok. MS (~ m/e = 496 (M+ 1).
EXAMPLE PKCSl Z-Leu-Phe-CONH.Bzl. This compound was synthesized from the protected a-ketoester and benzylamine in 40 % yield by the procedure described in Example PKC47.
After reacting overnight, ethyl acetate (60 ml) was added. The mD~ture was filtered to remove a white precipitate. The solution was washed with cooled 1 N HCl (3 x 25 ml), water (1 x 20 ml), saturated sodiurn chloride (2 ~ 20 rnl), and dried over ma~esium sulfate. The solution was evaporated leaving a yellow solid. Chromatcgraphy on a silica SUE~STITUTE SHEET
, ~,. WO92/11850 2~ 9 PCr/US91/097~6 gel column with CHC13/CH30H 30:1 v/v) afforded a yellow solid. Single spot on TLC, Rf = 0.45 (CHCl3/CH30H 30:1); mp 16~162 ~C. AnaL calcd. for C3l~I3sN305: 529.61;C, 7030; H, 6.66; N, 7.93. Found: C, 70.18; H, 6.67; N, 7.99. NMR (CDC13) ok. MS(FAB) m/e = 530 (M+ 1).
~IRu-Phe-CONH-(CH2)2Ph ~is compound was synthes~ed from the protected a-ketoester and phenethylamine in S0 % ~neld by the procedure descn~oed ~n ~ample PKC51. Sin~e spot on TLC, Rf = 050 ~GEIC~3/CE30H 30:1); mp 151-153 ~C.
AnaL calcd. for C32H37N305: 543.66; C, 70.70; H, 6.86; N, 7.73. Found: C, 70.54; H, 6.88; N, 7.74. NMR (CDCI3) o~ MS (FAB) m/e = 544 (M+ 1).
~L~u-Abu-CONH-Et. This compound was sgnth~ized ~am protected a-ketoester derived ~om Z-Leu-Abu-C02Et and ethylamine in 64 % y~eld by the procedure descn~ed In E:cample PKC47~ Single spot on TLC, Rf = 0.36 lS (CHCl3/CH30H 50jl); mp 130-132 C. AnaL calcd. for C21H31N305: 405.4~; C, 62.20;
H, 7.71; N, 1036. Found: C, 61.92; X 7.62; N, 1031. NMR (CDC13) ok. MS (FAB) m/e ~ 406 (M+1).
EXAMPL~: PXCS4 Z-Leu-Abu-CONH-IlPr. This compound ~vas sgnthe3ized ~om lhe corresponding protected a-ketoester and propyl~e in 47 % yield by the procedure descnbed in Example PKC47. S~gle spot on TLC, E~f = 0.28 (CEIC13/CH3OH 50:1); mp 134-135 C.
Anal. calcd. for C22H33N30s: 41950; C, 62.98; H, 7.93; N, 10.02. Found: C, 62.84; H, 7.97; N, 9.94. NMR (CDC:13) ok. MS (FAB) m/e ~ 420 (M+ 1).
Z Leu Abu CONH n~lL This co~npound was ~9nthesized ~om the corresponding prote ted a-ketoester and butylamine in 42 ~o yidd by the procedure desc~bed in E~ample PKC47. Single spot on TLC, Rf = 054 (GHC~3/~I3OH 50:1); mp 135-136 C.
Anal. calcd. ~or C23H3sN30s: 433~3; C, 63.71; H, 8.13; N, 9.69. Found: C, 63.48; H, 8.07; N, 9.67. NMR (cDa3) ok. MS (FAB) m/e = 434 (M+ 1).
EXAMPI,E PKC~6 Z-Leu.Abu-CONH.iBu. This compound was synthes~7ed :~om the corresponding protected a-ketoester and isobutylam~ne in 6~ % yield by the pro~edure described in Example PKC47. Single spot on TLÇ, Rf z 0.:;~ (CHC13/CH30H 50:1); mp 133-135 ~C.
~3uBsTlT~lTE S~EET
.: .
, . . . ~ . . . .
W0 92/1 1850 2 ~ 9 8 6 ~ 9 PCT/U~9t/09780~ '`
Anal. calcd. for C23~I3~N30s: 43352; C, 63.72; H, 8.14; N, 9.69. Found: C, 63.46; H, 8.10; N, 9.60. NMR (CDCl3) o~ MS (FA13) m/e = 434 (M+ 1).
E~MPLE PKC57 ~Leu-Abu-CONH-BzL This compound was synthesized from the corresponding protected a-ket~ester and bes~ylamine in 29 % yield by the procedure described in E~ample PKC51. Single spot on TLC, Rf = 056 (CHCl3/CH30H 30:1); mp 140-141 C.
Anal. calcd. for C26H33N305: 46754; C, 66.79; H, 7.11; N, 8.99. Found C, 66.65; H, 7.07; N, 8.93. NMR (CnCl3) ok. MS (FAB) m/e = 468 (M+ 1).
~Leu-Abu-CONH-(CH2)2Ph. This compound was synthesized from the corresponding protected ~-ketoester and phenethyla~ne ~n 51 ~o yield by the procedure descri~ed in Example PXC51. Single spot on TLC, R~ = 0.44 (CHC13/CH30H 30:1); mp156-157 C. AnaL calcd. for ~7H35N30s: 48159; C, 6734; H, 733; N, 8.72. Found: C, 67.38; H, 733; N, 8.78. NMR (C~DC13j ok MS ~A13) m/e = 482 (M+ 1).
Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)20. This compound was synthesized from protected a-ke~oester and 4(3-aminopropyl)morpholine in 33 % yield by the procedure described in E~ample PKC47. After re:acting oven~ight, ethyl acetate (80 ml) was added.
The m~ture wæ filtered so remove a w~ite precipitate. Ihe solusion was washed with water (3 ~ 20 ml), saturated sodium c~loride (2 x 20 ml), and dried over magnesiurn sulfate. The solusion was evaporated leaving a yellow oil. Chromatography on a silica gel column with ~Ha3/CH30H (10:1 v/v) afforded a yellow sen~solid, which was recrystallized from ethyl acetate/h~ane to obtain a pale yellow solid. Sin~le spot on TLC, Rf - 0.42 (CHa3/CH30H 10:1); mp 12S-126 C. Anal, calcd. for C~6H40N406:
504.63; C, 61.88; H, 7.99; N, 11.10. Found: C; 61,69; H, 7.95; N, 11.07. NMR (CDCl3) ok. MS (FAB) m/e - SOS (M+1).
EX~MPLE PKC60 Z Leu Abu-CO~H-(CH2)7CH3 l~is compound was synthesized from the correspondjng protected a-ketoester and ocqlamine in 67 % yield by the proceduredescribed in E.sample PKCS1. It was white solid. S ngle spot on TLC, R~ = 0.55 (CHC13/C~I30H 30:1); mp 134-135 C. AnaL calcd. for C2~H43N305: 489.66; C, 66.23;
H, 8.8S; N, 8.58. Found: C, 66.19; H, 8.81; N, 8.61. NMR (CDCl3) ok. MS (FAB) m/e = 490 (M+ 1)-SUBSTITUTE SffE~
: .
WO 92/11~S0 2 ~ 3 8 ~ ~ 3 PCI/IJS91/0978~
E~LE PKC61 Z-Leu-~bll-CONH-(CH2)20H. Ihis wmpound w~ synthesized ~om the corresponding protected a-ketoes~er and ethaIlolamine in 29 % yield by l:he procedure descnbed in Example PKC59. The product was a whi~e sticky sclid. Single spot on S TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 151-153 C. Anal: calcd. for C21H31N306:
421.49; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11; H, 7.M; N, 9.81. NMR (CDCl3) ok.
MS (FAB) m/e o 4æ (M+ 1).
Leu-~bu-CONH~ I2)20(C~I2)20~. This compound was synthes~ed i~om the corresponding protected a-ketoesser and 2-(2-aminoetho~y)e~hanol in 34 % yield by the procedure descrl~ed in E~ample PKC59. The product was white sticly solid. Single spot on TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 103-105 C. Anal.: calcd. for C23H35N307: 465.35; C~, 5934; H, 758; N, 9.03. Fow~d: S~, ~9.23; H, 758; N, 9.01. NMR
(CDC13) ok. MS (FAB) m/e = 466 (M+1).
L4u Abu-CONH-(CH2)17C~3. ~is compound was synthesized ~om the corresponding protected a-ketoester and oc~adecylamine in 12 % yield by the procedure described in E~nple ~KC~ e product was a pale yellow solid. Single spot on TLC, Rf = 054 (CHCl3/CH30H 30:1); mp 134-136 C. Anal: calcd. for C37~I63N30s: 629.92;
C, 7055; H, 10.08; N, 6.67. Found: C, 70.71; H, 10.14; M, 6.75. NMR (CDa3) ok. MS
(FAB) m/e = 630.2 (M-t 1).
Z L.eu-Abu CONH CH2 C6H3(0CH3)2. Thi~ compound was synthes~zed ~om the corresponding protected e ketoester and 3,5 dime~ho ~ybenzylarsline in 45 % yield by the procedure descnbed in l~ample PKC~1. Ihe product w~ yellow sticky soIid. Sin~le spot on TLC, R~ = 0.44 (CHC~3/CH30H 30:1); mp 153-1~5 C. Anal.: calcd. for C28H37N307: 527.62; C, 63.74; ~, 7.07; N, 7.96. Found: C, 63.66; H, 7.09; N, 7.92. NMR
~CDC13) ok. MS (FAB) m/e = 528.8 (M+ 1).
EXAMPLE PKC6~
2~Leu-Abu-CONH CH~ C4H4N. ~is compound was s5nthesized from the corresponding protected a-ketocster and 4-(aminomethyl)pyridine in 45 % yield by the procedure described in E~ample PKCS9. The product was ~eenish yellow solid. Single spot on TLC, Rf = 05S (CHC13/GH30H 10:1); mp 124-126 C. A~ calcd. for . ,.. ,: . , . ~ . .
wo 9~ 850 2 0 9 ~ ~ Q 9 PCI/US9l/0978~
C25H3~N40s: 468.55; C, 64.08; H, 6.88; N, 11.96. Found: C, 63.88; H, 6.87; N, 11.96.
NMR (CDCl3) ok. MS (FAB) m/e =469 (M[+1).
D. HALO-~TONE P~ES
Halomethyl ketone peptides are irreversl~le inhl~itors for serine proteases and S cysteine proteases. This elass of compounds includes peptides having a variety of halomethyl groups at their C-tern~nus. These halomethyl groups include CH2X CHX2 and CX3, where X represents any halogen. A number of analogous compounds have been synthesized, induding the amino-halo ketones and the diazo-ketone peptides.Although these analogous oompoi~nds are chemically distinguishable, all of thesehaloketone compounds are believed to have a si~slilar mechanism OI aetion. Accordingly, for simplicity, all of the foregoing compounds will be referred to collectively herein as the "Hal~Ketone Peptides."
The reactivity of haloketones has generally been found to be in the order I ~ Br> Cl > F. However, incre~cing the react~ity of the haloketone in this way can lead to acceleration of competing side effects. Thus, it is preferable to incre~se the reactivity of the halomethyl ketone peptides by altering the peptide structure.
In selecting a proper inhibitor for Calpain, the same basic peptide structure selection techniques as used for the Peptide Keto-Compounds can be used. Once a - peptide structure has been identified, the most effecti~e C-tern~inus grouping can be empirically detennined through kinetic inhl~ition studies of each of the compounds with Calpain.
Many of the Halo-Ketone Peptides are available commercially For e~ample, Leu-CH2CL Phe-CH2CL Z-lys-CH2CL Tosyl-LysCH2Cl (TLCK), Tosyl-PheCH2Cl (TPC~), ZGly-Leu-Phe-CH2CL Z-Phe-Ala-CH2CL z-Phe-Phe-CH2Cl, D-Phe-Pro-Arg-CH2Cl, MeoSuc-Phe-Gly-Gly-Ala-CH2CI, MeoSuc-Ala-Ala-Pro-Ala-CH2CL MeoSuc-Ala-Ala-Pro_Val.CH2CL Ala-Ala-Pro-Val-CH2CL Ala-Ala-Phe-CH2CL Suc-Ala-Ala-Pro-Phe-CH2Cl and D-Val-Leu-Lys-GH2CI are all available from suppliers such as En~ne Systems Products of I~vennore, California. From the same suppliers, thz following diazomethyl ketone peptides are available: Leu-CHN2, Z-Phe-Phe-CHN2, Z-Phe-Ala-C~IN2, ZPhe-Pro-CHN2, ZLys-CHN2 and Giy-Phe-CHN2. In addidon, the production of a-amino fluoro ketone pepddes has been described in United States Patent No. 4,518,528 to David W. Rasnick the disclosure of which is hereby incorporated by this reference.
Su~ 1TlJfE 5~EFr ..
A,; WO 92/11850 2 ~ ~ ~ 6 0 9 PCr/US91/09786 l~e preparation of various ~lo-Ketone Pep~ides is reviewed in kIethods in E~, 46:197-208 (1977), the disclosure of whieh is hereby incorporated by reference, Brie~y, halomethyl ke~one derr~a~ves of blocked ~o acids are read~y prepared by the reaction of n~neral acid~ (hydrohalic) with the corresponding 5 diazomethyl ketone. Iodomethyl ketones are prepared by re~ctisn of a halo-ketone with NaI, since reaction with HI with a di~omethyl ketone yields ~he rnethyl ketone. A
3umber of different bl~ng ~oups can be used, including benzylo~ycarbonyl (Z) arld t-butylo~ycarbonyl (BOC). The ~iazomethyl ketone is prepared by reactis~ of diazomethane with the appropriate acid ~a~ed by means of dicyclohe1ylcarbodiiTnide 10 (~ -CI), by the mixed anhydride method.
Unbloc3ced asl~ino acid chloromethyl ketones can be prepared by reaction of benzyloxycarbonyl blocked derrvatives wi~h Br or HOAc, trifluoroacetic acid, or by hydrogenation.
Synthesis of peptide chloromethyl Icetones can be accomplished simply by coupling an appropriate peptide or amino acid with an unblocked an~ino acid chloromethyl ketone. A few dipeptides can be converted directly to the chloromethyl ketone using the n~Lsed anhydride and CH2N2 foll~wed by HC:L
Vanous synthetic problems are encountered in the preparation of chloromethyl ketone derrvat;sves of basic amino acids. I~e side chain usually must be blocked during 20 ~ynthesis, and di~iculties are often encountered dunng removal of the blocking group.
Use of trifluoroacetic acid or HF was eYentually found to give a good conversion ~o product.
A number of exarnples of the preparation of Halo-Ketone Pepddes have been reported in the literature, including a comprehensive re~e v of over 100 amino acid 25 derivatives and appro~natley 60 peptide derivatrves Listed in J.C. Powers, in aChen~istry and Biochernistry of Anuno Acids, Peptide~ and Prote~ns,~ Vol. 4, Dekker, New York (1977), the disclosure of which is hereby incorporated by reference. Those of skill in the art will recognize how to locate a multitude of e~amplæ of the produstion of the Halo-Ketone Peptides. Acoordingly, no addidonal G~nples are pro~rided herein.
30 ~. V~USES
In addition to the foregoing classes of compounds now discovered to possess Calpain inhl~itory activity, we ~elieve that a large number of other such compounds exist. In vie~v of the large number of inhibitors of Calpain of different classes we SuBsr~ u~rr . ~
. ', -;. .. ,.: ~ . -.
W092/11850 2~9~a9 PCr/us91/o97~li disclose herein, all of the h~own, newly discovered and yet undiseovered ~nhibitors of Calpain shall be referred to hereinafter collectively, using the term "Calpain Lnhibitor.~
The Calpain Lnhibitors may be used vi~o for a variety of purposes to inhibit unwanted Calpain ac~vity. For e~ample, the Calpain Inhibitors may be used in vitro to S prevent proteolysis that occurs in the process of production, isolation, purification, storage or transport of peptides and proteins.
The Calpain Inhibitors descnbed herein ca~ also be used vitro to prevent further degradation of ~issue sarnple3 ~om oecurring a~er preparation of the samples.
This vitro prevention of degrad~tion can ~e esp cially useful in the preparation of assays for neurodegenera~ion where~n the ass~y comprises a test for the products of Calpain activity in the tissues, such as assays for breakdown products (BDP's) of cytoskeletal eomponents such as spectrin, MAP2, actin binding protein and tau. P.
Seubert et aL, in Neur~science~ 31:195 (1989), the di~closure of which is herebyincorporated by reference, disclose an exemplary method of quantitating the amount of 1~ spectrin BDP's as an indication of Calpain activity.
The ~alpa~n Is~ubitors of this mvention are also useful in a variety of other experirnental pro~edures where proteolys~s due to Ca4ains is a signifiunt problem. For example, inclusion of the Calpain Inhibitors in radioin~nunoassay exp~riments can resul~
in higher sensiti~nty. The use of the Calpain In}~ibitors in plasma fractionation procedures can result m higher yields of valuable plasma proteins and un make purification of the proteins easier. The C~alpain Inl~ibitors disclosed here can be used ~n clomng experiments utilizing recombinant or transfected bacterial or eukaryo~ic cell cultures in order to increase yield of purified recombinant product.
To use the Calpain ~ibitors in vitro. the Calpain Inhibitors are dissolved in an25 ~ organic acid, such as d~methylsulfo~ide (DMSO) or ethanol, and are added to an aqueous soludon containing the protease which is to be inhl~ited, such that the final concentration of organic so}vent is 25% or less. The Calpain Inhibitors may also be added as solids or in suspension.
F. TREAl'MENT OF ~EURODEGENE~ATIOI~
We have discovered that the Calpain Inh~bitors are use~l in vivo to treat pathologies in which e~cess proteo~sis by Calpains is involved. Such pathologies are believed to include neuropathologies such as neurodegeneration resulting from excitotoxicity, H~V-induced neuropathy, ischernia, denenation, injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD), SU135T~UTE SHEEr . .
wo 92/11850 2 ~ 9 ~ PCl/U591/09786 Hun~ington's Dise~se, surgery-related brain damage, Parkinson's Disease, and other pathological conditions.
1. Identification of Inhibi~ors In order to identify Calpain Inhl~itors that are useful in the practice of the present invention for treatment or inhl~ition of neurodegenera~ve conditions anddiseases, it is important to identi~ ~hose inhl~itors posessing sigrlificant Calpain inhibitory a~vity. It is also import~nt to identi~ those Calpain Inhibitors having a high degree of specificity for inhib n of C~ain, in order to aYoid interference with other biological processes when the Calpain Inhl~itor is introdu~d into a mammal requiring treatment for neurodegeneration. Because all thiol proteases are believed to e~er$ their effect through a similar mechanism ~ action, our prirnary concern ~as to identify those Calpai~ itors having substantial inhl~itory act~ity against Calpain, but relatiYely weak or no ac~vity against other thiol protease~. Accordingly"n order to identify such Calpain Inhibitors, we tested a variety of Calpain Inhl~itors for their ability to inhibit calpains I and II, and compared thls data with the ability of the same Calpa~n Lnhibitors to irlhibit Cathepsin B, another thiol pro~ease. Ihose Calpain Inhibitors with high vitro inh~itory acsivi~r against Calpain and a relative~y lower activiq against S ~athepsin B
are believed to ~e most useful for ~vno therapy. E~arnples lA through lC show the results of these studies for a variety of Calpain Inhl~itor~.
EXAMPLE 1~
InhibitiQn b~ Substitused ~terocyclic C-ompounds The isocownarins are irreve~sible inhl~itors of Calpain. We obtained ICso valuesfor a variety of these Calpa~n Inhibitors as a kinetic analysis of these compounds.
Purified Calpains can be assayed using the fluorogenic substrate succinyl-leucine-2S ~ros~ne-methylarninocownarin (available commercially) or by measuring the release of acid-soluble peptides from casein bec~use we have found that the isocoumarins inhibit casein proteolysis by Calpain.
Calpains I and ~ were purified by the method of (Yoshisnura, et aL 1983).
(Kitahara 1984) provides an altemative puri~cation scheme. Calpain II may alternatively be purchased from Sigma Chemical Co. as "Calcium Activated Neutral Prote se.~ Ln this assay, purified Calpain was incubated with l4C-methylated casein in the presence of various H[eterocyclic Compounds and the amount of acid-soluble radioactivity released by the action of Calpain was measured. The ICso values were determined as the ~ ~o~!~rlT~ S~
WC) 92/11B50 2 0 9 8 6 ~ 9 PCI~/US91/09786~;
concentration of Heterocyclic ~mpound compound at which SO~o of the Calpa~n activity was inhibited. Table ~A shows I~ values for various Isocoumar~n Compounds.
5UB~m~ SH~E~
.:
.
.-, WO 92/1 1~50 2 ~ 9 8 6 ~ 9 PCI~US91/09786 ~ABLE L~
INH~llION OF CALPAINS BY SUlBSTl~D ISOCOUMARINS
~LCs~ (UM! -CalpainI Calpa~n tI
ClTPrOIC 100 70 ~`
NH2-CiTPrOIC (ACl~IC) 10 120 ~12NHCONH-CiTPrOIC 80 30 CH3CONH.Cl~PrOIC 700 80 L-Phe-NH-Cl~PrOIC - 30 BOC-L~Phe-NH-CilPrOICno inhl~ition ~200 PhCH2NHCONH-Cl~EtOIC 90 PhC~I2CONH-CiTEtOIC . 30 D-phe~NH-CiTEtOIC 200 I?lus, it can be seen from Table ~ that a variety of the Isocoumarin C:ompounds have significant ~alpain inhibitory activity at low concentrations.
EX~MP~ lB(I) Prote~se~nhibition by Peptide Keto-Compounds The Peptide Keto-Compounds are reYersl~le inhibitors of Calpa~ns and other 20 thiol proteases. The }4 values for the inhibition of calpain I, calpain Il and Cathepsin B
were detern~ned for several Peptide Keto-Compounds, Inhibition of calpain I ~om human erythro~es and calpain II from rabbit musde were assayed using Suc.Leu.Tyr.amidomethylcoumarin as substrate in an assay buffer of 20mM HEPES
pH=7.2, lOmM CaC12, lOmM ~mercaptoethanol. Cathepsin B from bovine spleen was assayed us~ng Z-Lys-4-nitrophenylphosphate as substrate.
Table lB(i) shows the results of the studies of E:sample lB(i). The Ki value forthe inhibition of Calpains and cathepsin B by several Peptide Keto-Compounds areshown in ~lM (micromolar). The values for leupeptin, which is cornmercially available from Calbiochem of La Jolla, Califonua, are shown for comparison.
C!~ S~E~T
.
.
.. . .
WO 92/1 1850 2 ~ 9 8 ~ o 9 PCr/lJ!i91/0978 Tabl~ lB(i) Kj YALUES FOR PEPTIDE KETO-COMPOUNDS
Inhibitor Calpain I Calpain II Cathepsin B
Leupeptin 032 0.43 6 ~Ala-Ala-Ala-C02Me 200 ~ 1.5 ZAla-Ala-Abu-CO2Et 50 200 0.9 ~Leu-Phe-C02Et 0.23 0.4 ~ 50 Z-Leu-Me-CO2Et 0.12 0.18 i8 ZLeu-Abu-C02Et 0.04 0-4 ld, ZLeu-Nva-COO~t 1.2 30 It can be seen ~om the results in Table lB(i) that the Peptide Keto-Compo mds inhibit C:alpain with Ki values similar or superior to leupeptin. In particular, Z-Leu-Phe-C02Et, ZLeu-Nle-C02Et and Z-Leu-Abu-C02Et were found ~o possess ~eater Calpa~n inhibitory actiYity than leupeptin. In addition, these partiallar compounds were highly specific to Calpain, with lo ver inl~ibitory act~vity toward Cathepsin B than leupeptin.
EX~LE lB(~) otease Ir~ibition o~ Peptide Keto-ComFour~ds We tested the ability of an additional group of Peptide Keto-Compounds to inhibit several proteases in order to e~aluate their specificity for Calpain. The results of these studies are shown ln Table lB(ii).
S~STITUTE SHIE~
.: .
~ wog2/..g50 2~ 3 Pcr/US91/09786 Table lB(i~). Inhibition of Calpa~n I, Ca~ain II, Cathepsin B, PP Elastase ~nd Papain Inhibitor Kl( ~M) . __ . .
Calpain I Calpai~ II CathB Ch~m elasta papa~n ~e ,..... ... .. _ . ~ ..
Z-L~u-Abu-COOEt 45 0.4 30 ~100 ~100 220 . _ . _ . . . . , _ __ .. ___ Z Leu Abu COOnBu 1~8 4 ~ lû0 25 10 _ . ~
Z-Leu-Abu-COOBz . 95 0.47 4 40 > 100 40~ _ _ ............................................. Y . . _ Z-Leu-Lek .~bu-COOEt 1~8 2~6 ~2 > 100 25 .
2-NapSO2-Leu-Leu-Abu-COOEt 16 1.4 25 35 47 _ _ , , . . . . . .
2-N~CO-Le- - _eu-Abu-COOEt 0.09 > 300 28 I _ . _ . .
T~s-Leu-Leu-A~u-COOEt 33 69 25 28 I_ _ _ . _ .
Ph-(CH3)2-CO-Leu-Abu-COOEt 1.2 . _ .- , ..
Z-Leu-Abu-COOH 0.075 0.022 1.5 > 150 > 150 I _ _ _ Z-Leu-Abu-CON~t 05 0.23 2.4 > 150 65 _ ~ __ __ Z I~u~Abu CON~:Pr 0~5 8 >300 2 I... _ . _ __ Z-Leu-Abu-CONHnBu . 0~ 13 ~ 300 5 I .. _ _ _ . ,__ Z-Leu~Abu~ ONHiBu 0.14 4 >300 40 . -I . _ _ _ .
Z-Leu-Abu-CON~ 035 2 > 300 1 . . . ., Z-Leu-Abu-CONH-tC~ 2-Ph 0.022 I - _ _ _ _ _ __ Z-I~-Abu-CONH-(C~I2)3-Mpl 0.041 ~ . . _ _ Z-Leu-Abu-CONH-(CH2)~I3 . 0.019 , ._ _ _ ,, , _ . .
Z-Leu-Abu-CONH-(CH2)20H 0.078 _______ _._ _ SUBSTITUTE SHEET
;. . ~ . .
- ~ .
` , ;~
2~3860~ ,'.`
WO 92/11850 PCl/US91/09786- -, ~ =
¦ Inhibitor ~(I-M) I . . _ . . . . ..
Calpa;n I Ca~ain II Ca~hB Chym elasta papa~n ~ se I . _... . _ _ _ . __ _ _ ___ ¦ Z-Leu-Abu-CONH- 0.16 ¦ (CH2)2t)(~H2)2oH
_ _ __ _ . ~
Z-Leu-Phe-COOEt 1.8 0.4 340 0.025 > 100 75 _~ =.__~ . . ~ . - __ _ _ _.
Z-Leu-Phe-COOnBu 5.0 1.1 15 0.15 > 100 15 Z-L~-Phe-COOBz 3.4 1.6 45 1.6 > 100 4~
: , .,, __ .
Z-Leu-Leu-Phe-COOEt 1.4 _ 42 0.26 > 100 15 Z-Leu-Phe COOH 0.0085 0.0057 45 76 > 150 ~ ....................... . _ Z-Leu-Phe-CON~t 7.0 032 6 73 ~150 ___ . . . _ __ ,, Z~Leu Phe-CON~ 15.0 0.05 3 18 ~300 I _ . _ _ Z-L~-Phe-CON~Bu 0.028 3 8 ~100 I _ . .. _ _ . . ... . .. I
. Z-Leu-Phe-CONHiBu 0.065 4 24 _ . _ _ Z Leu-Phe-CONHBz 0.046 I ......... _ _ . _ _ ~: Z-Leu-Phe CONH(CH2)2Ph 0.024 (2) I _ _ . __ Z-Leu-Me-COOEt 0.18 20 2.2 190 I . ....... _ _ ..
Z.T ~--~va-COOEt 1.4-- - -1.2 25 160 23 150 . I . . .
Z-Leu-Met-COOEt 1.0 15 55 1.75> 100 140 I _ , _ . . -Z-Leu-4-Cl-Phe-COOEt <4.0 0.4 ~0 0.9> 100 150 . !~ ~ _ _. _ _ . _ ., =
SUE~STlTUTE~ JF'FT
. .. ~.. :. , .
, , ' WO 92/11850 2 n ~ 9 PCr/VS91/09786 -8~-Table lB(ii) shows the inhibition Qnstants (KI) for cathepsin B, calpain L and calpain II with peptide ketoamides. Dipeptide Ketoamides with Abu and Phe in the P
site and Leu in the P2 site are pot~nt inhibitors of calpain I and calpain Ir. ZLeu-Abu-CONH-Et is a better inhibitor of calpain I than ZLeu-Phe-CONH-Et by 14 fold.Replacement of the Z group (PhC~I20CO-) by similar groups such as PhCH;2CH2CO-, PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor structures. The best irlllibitor of calpain II is ZLeu-Abu-CONH-(CH2)2-Ph. Changing the R3 and R4 groups significantly improv~s the inllibito~y potency toward calpain II.
I~e ~ Dipeptide Ketoamide inhibitors are those which have long allyl side chains(e.g. ~,Leu-Abu-CONH-(CH2)7CH3), al}yl side chains with phenyl substituted on the alk~ Proup (e.g. Z-Leu-Abu-CONH-(CH~)2-Ph), or a aL~yl groups with a morpholine rir.& 9s,l~stituted on the alkyl group [e.g. Z-Leu-Abu-CONH~ I2)3-Mpl, Mpl = -N(CH2CH2)20]. Dipeptide a-ketoamides with a small aliphatic amino acid residue or a Phe in the Pl site are also good inhi~itors for ca~hepsin B. The best inhibitor is z Leu-Abu-CONH-Et and replacement of the ~ (PhCH20CO-) by PhCH2CH2CO-, PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor structures.
EXAMPLE lB(lil) Stabilitv of Peptide Keto~ompQunds We determined tbe half-life of several Peptide Keto-Compounds in both plasma an~ liver homogenates. The results of the determinations of stabiliq of the compounds in plasma and liver homogenates are sho~n in Table lB(iii).
SUE3STITUTE SI~EET
.: , - . .
.. .
:
~v~ u~
WO 92J11850 pcr/us9l/n9786 -~6-Table 1B(~ii). S~bility 3~ Plasma and in I,i~er o~ Pep~ide Keto-Compolmds.
Inhibitor tl/2 . li~er ~ __ _ S ~Leu-Abu-COOEt 2 . . . ..... _ . . , .. ..... .
2-NaEISO2 Leu Leu-Abu COOEt ~ 60 I - . .
2-NapCO-I,eu-1 eu-Abu~C(:30Et 25 ~__ __ Tos-Leu-Leu-Abu-COOEt 30 Z-Leu-Abu-COOH ~ 60 ~60 ~ __ Z-Leu-Abu-CONHEt ~ 60 ~60 I _ Z-Leu-Abu CONHnPr ~ 60 ~60 l ~Leu-Abu-CONHnBu ~ 60 ~60 ¦
_ __ Z-Leu-Abu-CONHlBu ~ 60 _ ZLeu-Abu-CON}~z ~ 60 ~li0 l I .......... . . . . , .
Z-Leu-Phe-COOEt 7.8 I __ I
Z-I~!u.Phe.COOnBu 7.7 . ............ . , . _ u-Phe-COOBz 1.9 ZLeu-Phg-COOH :- 60 ~60 , _ Z-Leu-Phe-CONHRt ~ 60 ~60 . 11 ~Leu-Phe-CONHnPr ~ CO ~60 ¦
I _ .Il ZLeu-Phe-CONHnBu ~ 60 ~60 . . 11 . LeuRhe-CONHlBu 60 _¦
Z-Leu-Ph~-CONH(CH2)~Ph > 60 I . _ Z-L~u-Nle~COOEt 3.7 5l~13Sl~TE SHEET
... . .
: ~ ' - ~ WO 92/11850 2 ~ 0 9 PCr/US91/09786 Illhibitor tll plasma li~er l . _ . . I
Z-Leu-N~a-COOEt 2.8 __ ..
Z~Leu-Met-COOE;t 8 _= ..
It can be seen from the data in Table 1B(i-i) that ~he Pep~ide Keto-Compounds S are generally quite stable in plasma and l~ver homogenates. However, it is also shown that the Peptide a-ketoamides v ere substantially more stable in both plasma and liver than the corresponding peptide a-ketoesters otease Inhibition bv Halo-Xetone Peptides The Halo-Ketone Peptides, ~ike the s~stituted isocoumarins, are irreversible inhibitors of Calpa n. We detern~ined the X8pp/[I~ ~alues for various members of ~his class of compounds aga~nst Calpains I and IL ~or comparison, we also determined these values against the addi~ional thiol proteases Papain and Cathepsin B for at least one Halo-Ketone Peptide. These Ka~p values are not directly comparable to the Ki or ICSo values determined abo~e for other classes of inhibitors.
We assayed Calpain I and II using Suc-leu~ amidomethylcoumarin. Papain was assayed using benz~yl-arg-4-nitroanilide, and Cathepsin B (bovine) ~as assayed using CBZ-lys 4-nitrophenyl ester. We followed the progress cun~e method of Tian and Tsou, Biochemi~trY~ 21:1028-1032 (1982), the disclosure of which ~s hereby incorporated by reference, to derrve l~netic data. Briefly, this method makes use of the equation:
Vls]/K
(1 + [S]/K)A[~
where [PJ represents the concentration of product formed at a tilTle approachinginf;ni~, A is the ~,p in the presence of substrate (S), K is the Michaelis constant and 2~ ~Y3 is the concentra~ion of the inhibitor. Since lS~ and [Yl are known and V and K can be deterrr~ined, ~,p can be readily deterrnined.
The Kapp/~ for ~arious Halo-Ketone Peptides are shown in Table lC.
c ~ IR.~::TITI ITI~ C!LI''E:T
~: .
WO 92/11850 2 0 9 8 ~ 0 9 PCT/US91/09786 . :^
-8g-TABLE lC
KINETIC P~RAh~RS OF Halo-Keto~ Peptides Inhibitor CI C:~13 P CB
ZGly-Leu-Phe-CH2C1 2840001946000 Boc-Gly-Leu-Phe C~I2C1 902000 ~40000 290000 ZLeu Phe-~I2Cl ~oo02 585000 Z-Gly-Leu-Ala-CH2Cl 210000 Ac Leu-Phe-CH2C1 259001 33400 lQ Z-Val-Phe-CH2Cl 27200 ZAla-Phe-CH2C1 2400 Ac-Ala-Ala-Ala-Ala-CH2C1 1300 CI = Calpain I l - Rat CII = Calpain II 2 Human P - Papain 3 - Rabbi~
CB = Cathepsin B
It can be seen from the results in Table lC that the Hal~Ketone Peptides inhibit Calpain with relatively high Kapp/[I values. In particular, Z-gly-leu-phe-CH2Cl, Boc-gly~leu-phe.C~I2C:I, Z-leu-phe-CH2Cl and ~gly-leu-ala-CEI2a were found to possess significant Calpain inhl~itory ac~vity. In addition, Boc-gly-leu-phe-CH2Cl was shown to be somewhat specific to Calpain, with lower inhibitory acti~.~ity toward Cathepsin B or Papaln than toward Calpain. The results shown in the table reveal that Z gly-leu-phe.CH2Cl and Boc-gly leu-phe-CH2a produce similar inhibitory effec~s. - -Thus, the blocking group is not shown to have a great effect on Calpain inhibitory ac~vity.
The kinetic constants of other irreversl~le Cal~ain Inhl~itors include the following with Kapp/W in parentheses: E 64 (7500), E64-d (23000~ and Zleu-leu-~yr-CHN2 (230000). E-64 is cornrnercially ava~able from Sigma Chemical Co., and is shown here to be a poor inhibitor of Calpain. ~leu-leu-tyr-CH~2 is a diazomethylpeptide compound, here shown to possess significant Calpain inhibitory activity. 2. Inhibition oî C~lpai~ ln Neural Tissues SUBST~TVT S)~EEJ
.
': . ` ` ' "
WO 92/1 1850 ~ 9 PC~/US~l/09786 ,; , In order to evaluate the inhibition of Calpa~n by the various C`alpain Inhibitors in neural tissues, we assayed the Cal~ain Inhibitors using the known ability of Calpain to cleave spe~rin, a proeein component of neuronal and other tissue, into Bl)P's. In this assay, more effective Calpain Inhl~itors will prevent the con~ersion of spectrin into S BDP's. Example 2 is one e~ample of such an assay.
EXA~LE 2 ~bf~
Crude Brais~ l~tracts ~ C~a~pain Inkibitors ~he actIvity of Cal~ain in crude brain e~tracts vas measured by e~amining the Ca 5-su~;ated proteo~sis of the endogenous substrate spectrin. Brain tissue was homogenized in 10mM Tris pH=7.4, 032M sucrose, lmM EGTA, lmM dithiothreitol and the nuclei and debris . - ~ved by low speed centrifugation. Various Calpain Ir, ~itors were added so the supernatant in a DMSO vehicle and a calcium salt (final e~ective concentration about 1.2mM) ad~ed ~o start the reaction. Proteolysis of spectrin was e~aluated by western blot as described by Seubert, et al. (Brain Research, 459:226-232, 1988), the disclosure of which is hereby incorporated by reference.~efly, a known quantity of a spectrin~ontaining sample treated with Calpain is separated by SDS-PAG~ and immunoblotted with anti-spectrin antibody. The amount of spectrin immunoreactiviq found corresponding to the characteristic BDP's is indicative of the amount of spectrin activity present in the sample. An e~amplary methnd for quantitating BDP's is to assay Spectrin BDP's by homogenizin'g brain parts in _umM Tris '?~ =72, 32M sucrose, 50,uM Ac-Leu-Leu-nLeu-H on ice. Homogenates are then mi~ea 1:1 with 10% SDS, S% B-rnercaptoethanoL 10% glyceroL lOmM Tris pH=8.0, 0,5% bromophenolblue, heated to 95C, and subjected to electrophoresis in 4 1/2% polyac~rlamide gels. ~e proteins in the gels are transferred to nitrocellulose and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antibody and es~ablished immunodetection methods. The amount of spec~rin and BDP's in each sample can be quantitated b~ densitrometric scanning of the developed nitrocellulose.
Due to Calpain's requirement for Ca2~, in the absence of Ca2+ little or no - 30 spectrin proteolysis occurred, regardless of the pr~sence of inhibitor, while in the presence of Ca2+ the spe~rin ~as ~ 95% cleaved to BDP's within 40 min. if no Calpain Lnhibitor ~s added.
a~TsTl ITF ~ Ec~
~. .. . . . . " .. -WO 92/ll8s0 2 ~ 9 ~ ~ ~ 3 PCr/US91/0978;; -Both leupep~n and CIl showed inhl~ition in the system of E~ample 2. In addition, the follow~ng compounds of ~he Substituted Heterocyclic Compounds werefound to produce signi~icant inhibition at 100 IlM:
3-chloroisocoumar~n 3,4-dichloroisocoumann .
3-benzyloxy~-chloroi~oco unarin 7-(ace~qlamino)~-chloro-3-(propoy)-isocoumarin 4-chloro-3-(3-isothiureidopropo~y)isocoumarin 7-amino~-chloro-3-(3-isothiureidopropo~y)isocouma~n 7-(benzylcarbamoylam~no) 1~hloro-3-(3-Lsothiureidopropo~y)isocoumarin 7-(phenylcarbamoylan~ino) 1~hloro-3-(3-isothiureidopropo~y)isocoumarin 7-(acetylamino) 1~hloro-3-(3-isothiureidopropo~y)isocoumar~n 7-(3-phenyJpropionylar~uno) 1-chlor~3-(3-isothiureidopropo~y)is~oumarin 7-(phenylacetylamino)~-chloro-3-ZO (3-isothiureidopropoxy)isocoumarin 7-~-phenylalanylamino)-4 chloro-3-(3-isothiureidopropo~y)isocoumar~n 7-(benzylcarbamoylarnino)~-chloro-3-(3-iso~hiureidoetho~y)isocoumarin 7-(phenylcarbamoyl~nino)~ chlor~3-(3-isothiureidoetho~;y)isocoumarin 7-(D-phenylalanylamino)-4~hloro-3-- (3-~sothiureidoetho~y)isocoumarin.
The following compounds of the ~Ialo-Ketone Peptides were also found to produce signifîGant iTl}ùbition at 100 I~M:
ZLeu-Phe-OE12CI
Ac-Leu-Phe-CH2Cl Z-Gly-Leu-Phe-CH2Cl Boc-Gly-Leu-Phe-C~I2Cl SUBST~TUTE S~IFFT
:
. ~ :. WO 92/11850 2 ~ 9 g 6 ~ 9 PClr/US91/09786 Ac-Val-Phe-CH2CI
~Gly-Leu-~la-CH2C 1.
In addition, the ~ollowin~ compDunds of the Peptide Keto-Compounds were found to produce ~i~3nifiGmt inhibition at 100 ~M:
5Bz-I)L-Phe-COOEt ~Leu-Nva-COOEt ZLeu-Nle-COOEt ~Leu-Phe~OC)Et ~-~eu-Abu-COOEt 10_eu-Met-COOEt Z-Ala-Ala-DI~Abu-COOEt MeO-Suc-Val-Pro-DL-Phe-COOMe ~AIa-Ala-Ala-DL-Ala-COOEt MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe.
15ZLeu-Phe-COOEt Thus, the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides, in addition to leupeptin and ~1, provide in~ubition in brain homogenates.
3. V~Q Inhib}tioD o~ N~urodegener~tion ~hrough Infusion Techniques In order to demonstrate that the inhibi~ion of Calpain activ~ty alone is sufficient to inhibit neurodegeneration vivo, we tested the ability of the Calpain In}~ibitor, . ~upeptin, to inhl~it neurodegeneration in gerbils subjected to transient ischemia.
As stated abo~e, leupeptin is poorh~r mcmbrane permeant. Therefore, leupeptin is not e~pected to cross the blood-brain barrier ("BBB") very well. Accordingly, in order to provide the braill with sufficient leupeptin to adequately inl~oit Calpain activation, w~ used brain illfusion techniques. Through thc use of these techniques we were able to subject brain tissues to intimate contact with leupeptin for sustained periods of time. E~ample 3A is provided to show the in vivo protection ~om neurodegeneration found during one such study.
EXA~LE 3A
~Vivo Protectio~ nst~eurode~eneration A small cannula was implanted in the right lateral ventricle of adult gerbils, and secured to the skull with dental cement. An Al~et n~icro-osmotic pwalp was attached to the cannula for intracerebroventri~ular perfusion. The pump was filled with either ~T~ F ~
WO 92/11850 2 0 9 8 G 0 9 r PCltU591/0978~--g2-saline alone (control) or leupeptin (20 mg/ml in saline). After three days perf~sion with ei~her the control solution or with the leupeptin solution, transient ischem~a was induced by b;laterally clamping the c~otid artenes for a period of ten mmutes. Core temperatures were taken dur~ng and follo~rlg ischen~a, with no di~erences noted S between control and leupeptin treated animals. Fourteen days l~ter, the animals were sacriiced by Nembutal overdose and transcardial per~usion of a 105'o solution of paraformaldehyde in PBS. Coronal sections of the brain were stained ~ith cresyl violet and were e~amined for the e~tent of neuronal loss. The control gerbils e~bited the typical damage found in the CA1 field following ischern~a, ~th a 72% loss of neurons.
However, the leupeptin treated ger~ils showed f~r less neurodegeneration, with only a 15% loss of neurons.
The results of E~ample 3A cannot be e~plained by changes in thermoregulation, since core temperatures did not differ between the groups. Accordingly, we believe that the Calpain inhibitors~ act~vity of leupeptin is responsl31e for the observed difEerences in neuronal cell loss. In order to further ~uantitate the differences, and veri~ that leupeptin produced a Calpain inhibitory effect within the observed re~ons of the brain, we performed a related series sf ~per~nents. In this series of e~periments, spectrin BDP's were measured in the leupeptin treated and control animals. As discussed above, these BDP's are indieative of the amount of CalE)ain activity occurring within the tissue. E~ample 3B is provided to demonstrate the results of these experirnents.
In Vivo In}likitior~of C~ ain ~ctivitv Implantation surgeries and clamping of the carotid arteries were performed as 2~ above with a control-ischen~a group (n-4) and a leupeptin-ischernia group (n=5). A
third group of animals (n=4) recesved imp~antation with pumping of s line, but was not subjected to ischen~ia Animals were sacTificed by decapitation 30 n~inutes afterclamping of the arteries. The brains were rapidl~ removed and placed in oold homogenization buffer (0.32 M sucrose, 10 mM Tris-HCL 2 mM ED~A, 1 mM EGTA, 100 ~M leupeptin and 1 ~g/ml of the Halo-Ketone Compound, tos-phe-C~I2Cl (TPCK)). The CA1 region of the hippocampus was then dissected. I~e samples from both control and leupeptin treated anirnal~ were then prepared for SDS-PAGE and ~mmunoblotting with labeled anti-spectrin antibo~r, as described above in connec~ion with in vi~ro uses of the Calpain Inhl~ito~s. lhe wn~rol animals exhl~ited a marked SUBST~ rE~ T
:` , `' ' WO 92/11850 210 9 g ~ 0 3 PCr/lJS91/09786 increase in the levels of BDP's reLatne to the gerbils not subjected to ischemia. These BDP's co-m~grated with BDP's observed after ~ proteolysis of spectr~n with Calpain. The brain t~ssue from the leupeptin treated gerbils e~hl~ited appro~mately 25% of the BDP's observed in the ~ontrol ischenua treated gerbils.
S Another group of gerbils (n=3) were sacrificed ilT~nediately after lschernia without leupeptin treatment in order to obserYe the effects of ischemia wi~hout reo~ygenation. These gerbils e~ ited a similar amoun~ of increase of 13DP's as the control-ischernic gerbils obsened after a 30 minute reperfusion penod.
~;.e -~ results of E~ample 3B indicate that leupeptin ~erts its neuroprotec~ive effect through the inhl~ition of Cal~ain activation. The results also indicate that the observed proteo~sis of spectrin was an efEect of ischemia, and not secondary to the reo~ygenation. Acoordingly, the results indicate that inhl~ition of Calpain activity in VIVO produces a neuroprotective efEect.
Although the foregoing studies demons~ate ~hat leupeptin can inhibit neurodegeneration in vivo, leupeptin is noe ~he therapeutic drug of choice because of .he need to infuse the drug direc~ into the brain for an e stended period of time to exert its neuroprotective ef~ect. This is due to the relative~ poor abili~ of ~his compound to cross the BBB. Accordingly, it is beli~ved that a more therapeutically practical way to inhibit neurodegeneration would be to use more membrane permeant Inhibitor of Calpain.
4. Platelet Pe~eablllty In accordance ~nth our discoveries demonstrated in Examples 3 and 3A, we believe that ha~ing a compound cross the BBB and enter ~::NS tissue is a key characteristic of a therapeutiul~y u~eful approach to treat or inhibit neurodegeneration within the CNS. Use of Calpain inhibitors that have enhanced membrane permeability is one such approach. Thus, wc measured the ability of various Calpain inhl~itors to penetrate the platelet membrane and inhibit Calpain that is normally contained in platelets. As shown beloYv in the following e~amples, our results ind;.cate that particlalar compounds of the Hetero~yclic Compounds, Peptide Keto-Compounds and Halo-3a Ketone Peptide~" in addition to ~he Peptide Aldehyde, CI1, ead~ibis good membrane permeability.
As an indication of the membrane penneability of the various Calpain Inhibitors, we measured the abili~ of various Calpa~n Inhibitors to penetrate platelet . memb~aDes and inhibit the Calpain nonnally found within platelets. I~e membrane of 8VEISTiTUTE SHI~
.
wo 92tll850 2 0 9 ~ G 0 9 PCI/US91/0978t~
platelets is believed to have many simila~ities to the BBB and accordin~ly, such~perirnents are believed to provide a good ~ndication of the ability of the various Calpa~n Inhibitors to cross the BBB. E~ample 4 shows the results of some of these platelet e~periments using the Cal~ain Inhl~itors of the present invention.
- 5 EX~MPLE 4A
Membrane Penneation of Calpain Inhibitors Platelets were isolated b~,r a modification of the method of Ferrell and Mar~in, 1 Biol. Chem.. 264:20723-20729 (1989), ~he disclosure of which is hereby incorporated by refer~nce. Blood (15-20 rnl) was drawn ~om male Sprague-Dawley rats into 100mM
EDTA-citrate contaiII~ng 10 units hepar~ns and centrifuged 30 sn~nutes at 1600 rpm at room temperature. The plasma was resuspended in 15ml bu~er 1 (136mM NaCL
2.7mM KCL 0.42mM NaH2PO4, 12mM NaHCO3, 2mM MgCI2, 2 mg/ml BSA (Sigma), 5.6mM glucose, 22mM Na3Citrate pH 65) and platelets were isolated at 2200 rpm atFoom temperature of 25 minutes. Platelets were resuspended to 107 cells/ml in buffer 2 (136mM NaCL 2.7rnM KCI, 0.42 NaH2PO4, 12mM NaHCO3, 2mM MgCL 1 mg/rnl BSA, 5.6mM glucose, 20mM HEPES pH 7.4) and allowed to "rest~ for a rn~r~murn of 10 minutes at room temperature before use.
Platelets were incubated for ~ r unutes in the presence of in}~ibitor. In order to provide sufficient intracellular calcium to ac~ate Calpain, the calciwn ionophore A23187 was added to a final concentration of 1~ M. After a further 5 minute incubation, the platelets were harvested by centrifugation (1 min 10,000xg) and resuspended in 10% sodium dodecyl sulfa~e, 10mM Tris pH-8.0, 5%
B-mercaptoethanoL 0.02% bromophenol blue, and heated to 95 C for ~ min.
Samples were subjected to SD~PAGE on 6% nuni gels and ~ansferred to nitrocellulose (Schleicher and Schue~l BA83) for 2 hours at 100rnA/gel in an I KR
Novablot. Filters were blocked for 10 rninutes in 0.25% gelatin, 1% BSA, 0.25% Triton X100, 0.9% NaCL 10mM Tns-C:l pH 7.5, incubated overnight in the same solution contairung antibody to rat spe~rin, washed 3 X 10 minutes with 10mM Tris-Cl pH 7.5, 05% TAton X100, incubated 4 hours in wash buffer plus al3c~line phosphatase conjugated goat anti-rabbit antl~o~y (Biorad), and washed as above. Filters weredeveloped using the Biorad AP conjugate substrate Icit. Spectrin ~nunoreact~ity on she filters was quantitated ~y densitometry.
The inhbition of Calpain within platelets as measured by the proteolysis of the endogenous Calpain substrate spectrin in the presence of in}u~itors was assayed for a C!l 1~2CTITI lT_ ~LI ~T
: : :
:. :
... . .
`, .
~ ` ' . ' ` ' WO 92~11850 PCT/US91/09786 _95 Yarie~ of Calpa~n Inhl~itors. Ihe poorly permeant inhibitors leupeptin and E-64 had lit~le effect on intracellular Cal~ain. In contrast, the highly membrane permeant Heterocyclic Compounds, Peptide Ke~Compounds, and Halo-Ketone Peptides effec~ively inhl~ited platelet Calpain.
The following Heteros~yclic Compounds were found to produce significant ition at 100 IlM in the ~stem of ~ample 4:
3~hloroisoeoumarin 4~hlor~3 -(3-~;othiureidopropoy)~umarin 7-an~no~hloro-3-(3-isothiureidopropo~y)isoooumarin 7-(benzylcarbamoylam~no) 1~hloro-3-(3-~othiureidopropo~y)~socoumar~n 7-(phenylcarbamoyl~o)~hloro-3-(3-isothiureidopropo~y)isocoumann 7-(acetylan~ino)-4-chloro-3 (3-isothiureidopropo~sy)~soco~
7-(3-phenylpropionylan~ino)-4-chloro-3-(3 -isothiureidopropo~y)isocournarin 7-(phenylacetylan~ino)-4-chloro-3-(3-isothiureidopropo~y)isocoumarin 7-(L-phenylalanyl~nino)-4~hloro-3-(3-isothiureidopropoxy)isocoumarin 7-(benzylcarbamoylam~no)- 1-chloro-3-(3-1sothiureidoetho~cy)~socoumar~n 2~ 7-(phenylcarbamoylamino)~chloro-3-(3-isothiureidoethoxy)i~ocoumar3n 7-(D-phenylalanylamino) 4-chloro-3-(3-isothilreidoethoxy)isocournar~n.
The fo~lowing Halo-Ketone Pep~ides were fo1md to produce significant inhl~ition at 100 ~M in the system of E~ample 4:
ZLeu-Phe-CH2CI
Ac-Leu-Phe-CH2C:l ZGly-Leu-Phe-CH2Cl Boc-Gly-Leu-Phe-CH2GI.
SUBS~ITU~E SHEEll' WO 92/11X50 2 ~ 9 ~ 6 ~ 9 PCr/US91/09786.~
The following Peptide Ket~Co~npounds were f~u~d to produce s~gn~ficant inhibition at 100 I-M in ~he system of ~ample 4:
~AIa-Ala-D,~Abu-COC)Et ZAla-Ala-Ala-D,~Ala-COOEt MeO-Su~-Ala-Ala-Pro-D,L,Abu-COOMe ZLeu-Phe-COOEt ZLeu-Nle-COOEt ~Leu-Nva-COOEt ~I~u-Abu-COOEt ZLeu-Abu ZLeu 1-CI-Phe-CC)OEt ~IAu-Leu-Abu-COOEs Z-Leu-Leu-Phe-COOEt 2-NapS02-Leu-Abu~COOEt 2-NapSO2-Leu-Leu-Abu-COOEt Z-Leu-Met-C02Et Z-Leu-NLeu-C02Et Z-Leu-Phe-C02Bu Z-Leu-Abu-C02Bu Z-Leu-Phe-C02Bzl Z-Leu-Abu-CO2B~I
Z-Ala-Ala-D,l,A~u-COOBzl 2; Leu-Phe-COOH
ZLeu-Abu-COO~I.
Among those compounds found to exhibit Calpain in}libito~r act~vity in the homogenate system of E~ample 2, we found at least shree compounds which failed to exhibit Calpain in}~ibitory activity in the platdet ~;ystem of E~ample 4. These compounds are leupeptin, MeO-Suc-~al-Pro-D,L,Phe-COOMe and Bz-D,L-Phe-COO~t. Leupeptin is hlown to ~e poorly membrane permeant, thus confi~ning that the platelet assay will e~cclude hlo~n poorl~r membrane permeant compounds.
Aocordingly, ~he two Pep~de Ke~ocompounds found not to provide Calpain inhibitory activity within platelets are also believed to be poor~ membrane permeant, and would not be expected to cross the BBB.
SllE3S~lTUTE SHEf~T
, .
~- :
. . , ~ , , ,... i, . . ..
"~. WO 92/11850 2 ~ 3 ~ ~ 0 9 PCI`/US91/097g6 C2uantitative Studies o~latelet Membrane Permeabilitv We performes~ addi~ional gL~ntita~ve or semi-quantita~ive studies on several Peptide Keto-Compolmds using the assay of E~ample 4A, e~cept that IC50 values were deterrnined as the concentration at ~vhich 50% of the Cal~ain act~vation present in S controls occurred. Results are shown in Table 4B. For the semi-quantitative assays, -~ed with +'s in Table 4B, ~+" indicates detectable inhibition at 100 ~M, "+ +"
inaicates significantly more ir~.ubition than "+~, and "+ + ~" indicates no detectable activation of Calpain detected.
S11~31STITUTE SHE~ET
.
wo 92~1~8~0 2 0 9 ~ p~/us9l/n978~ `
Platelet Assa~ of Peptide Ketoamides9 Ket~sters and Ke~oacids - ~ .
Inhibitor IC
S l _ ~ . I
~Leu-Abu-COOEt 42 . ............ . __ I
Z-Leu-Abu-COOnBu Z-IAu-Abu-COOBz + + l . _ I
Z-Leu-Leu-~bu COOEt 4û l ~ . I
2-NapSO2-Leu-Leu-Abu-COOEt 100 I . . __ ¦ Tos-Leu-Leu-Abu-COO~t 30 ~I~.UrCOON 8 Z-Leu-Abu-CON~t 1.5 . . , , ,, . _ .
ZLeu-Abu-CON~Pr 70 .
Z-Leu-Abu-CON~Bu 2.0 Z-Leu-Abu-CONH~u 28 Z-Leu-Abu-CONBz 15 .
Z-Leu-Phe-COOEt 47 . _ Z-Leu-Phe-COOnBu + + +
_ _ _ _ Z-Leu-~he-COOBz + +
: _ Z-Leu-Leu-Phe-COOEt + +
_~ . .
Z-Leu-Phe-COOH 65 Z-Leu-Phe-CONHEt 1.7 I . _ ¦ Z-Leu-Phe-CONHrlPr SU13~iTUTE Sl JEE t .. . . .
.
I.`Wo92/11850 2a~0~ PCr/US91/0~786 __ _ = .
Inhibitor ICso Z-Leu-Phe-CONHnBu 38 Z-Leu-Phe-CONHiBu 2~!
~ ~r .
Z l t ~-Phe-CONH(CEI2)2Ph 3.0 ,, _ __ _ ~ .
ZLeu-Me-COOEt 5ZLeu-Nva-COOEt 40 ... .. . , _ ~L~u-Met-COOEt +
I .
Z-Leu~ Phe-COOEt +
1, , ~ , Table 4B shows that peptide a-ketoamides and ketoauds were much more ef~ective than corresponding peptide ketoesters in thi~ platelet assay. E~tending the R3 group to an alkyl group or an allyl group substituted with a phenyl group increased the membrane permeability of the inh~itors as indicated by increased potency in the platelet assay. In view of these results, Applicants believe that ectending the 1~ group to indude longer allyl groups or alkyl groups substituted with phenyl groups would increase the membrane permeability of a ~ven inhibitor.
In view of the foregoin& the results of E~amples 4A and 4B support our belief that CI1 and the Substituted Heterocydic Compounds, Peptide Keto Compounds and Halo~Ketone Peptides are believed to be membrane permeant and therefore, are expected to be effective in crossing Ihe BBB subsequent to in vivo administration of the eompounds.
5. Glutamate To~clt~
To further identii y thase Calpain Inhibitors lilcely to possess pharmacologically active neuroprotective ability, we tested the abiliq of the Calpain ~hll~itors to protec~
against glu~nate e~citoto~naty. Excess e~racellular glutamate is ~hought to play a key role in the induction of neuropathology in ischemia, which is acoompanied by Wpain activation. In support of this role for e1~cess glutamate, cultured Nl8~ 105 (a neuroblastoma.retinal hybrid) cells can be killed by the addition of glutamate into the culture medium. This glutamate.mediated cytoto~ici~ is calciurn dependent and can be StlE~STlTlJTE SHEET
. .
.
wo 92~ 50 21~ O ~ P~/US91/09786 reduced ~hrough a number of mechanisms, including free radical scavengers, blockers of the N-type volta~e-sensitive calcium channel, and quisqu~late-subtype glutamate antagonists. Thus, glutamate-mediated killing of N18-~lOS eells is an ~B model for neuropathology.
S According~, we tested She ability of the Calpain Inhibitors to inhibit glutamate-induced cell death in these cells in order to establish that the Calpain ~hibitors can decrease or prevent glutama~e-induced d~ath Of N18-~105 cells. Some of these tests are shown in ~ample 5.
~LE
S~ock cultures of N18-RE 105 cells were maintained in Dulbecco's modified ~agle's medium (DMEM) containing 10% fetal bonne serusn (FBS) and supplemented with hypo~anthine, ~inopterin and thymidine (HA~. Subcos~fluent cultures were split and plated into 96-well plates. Iwenty four hours a~er plating the cells were exposed 15 ~o ~esh media containing glutamate and various concentrations of Calpain inhibitors.
Control cells wer~ not treated with glutamate. The treated cell~ recei~red SmM
glu~nate and leupeptin (5,ug/ml) or the other ~pa~n Inhibitors listed in Figure 1 at 3~g/ml. Conversion of ~IT was measured 19 hours later as descn~ed. Nineteen hours after the onset of e~osure, cell viabiliq was quandtated by measunng the e~ent 20 to which the cells convert 3(4,5-dimethyltJuawl-2 yl)-2-~diphenyltetrazolium bromide (~) to a blue formazan product, which sowrs in the r utochondria of If ving but not dead cells (Pauwels et al., 1988). A higher absorbance is indicatlve of greater cell viability.
Figure 1 sho~. the percent of blue formazan product remail~ng after treatment 25 with glutamate, relative to control where no ~utamate was added. Thus, it can be seen that with vehicle plus glutama~e but no inhl~itor, less than 70% of the mitochondrial activity remains. However, Figure 1 shows that u~eral Calpain i~bitors, including leupeptin, Gll and representa~ves of the Heteroc yclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides protect N18-R~105 cells agaDst glutamate 30 toxicity. Tile Peptide Keto-Compound Calpain inhlbitor, ZAla-Ala-Abu-CO2Et, the Substituted Heterocyclic Compounds, ClTPrOIC and A~IC, and the Halo-Xetone Peptide, TPCK complete~ blocked the toxic effects of glutamate, resulting in 100% or ~eater of the form n product as seen with cells not treated with glutamate. Thus, E~ample ~ shows that these Calpain Inhibitors efEec~ve~r block cell death in an vitro SU8STlTllTE SHEE~
WO ~2/11850 PCT/US91/09786 model for neuropatholo~r. Accordingly, this data further supports our discoYery that Calpain Ir~bitors are neuroprotect~ve ~ n~o.
6. Redu~iosl of I~a~ion o~on MCA O~ ioll Stroke is a significant health problem in the hurnan population. Strokes are S occlusions of cerebr~l arteries produc~ng a decreased blood flow to bra~n re~ons, which cause cell death through oy~en and nutnent dep~vation. I~is type of lesion can be modeled in rats by sur~cal ooclusion of the middle cerebral artery (MCA). Several models for MCA occlusion have beerl developed, and all ~ive substantially similar results.
MCA occlusion produces a large volume of infarcted brain tissue 24 hours after occlusion. Previous studies ha~e shown that the size of the infarct as judged by ITC
sta~ing does not increase after the first 24 hours post-oeclusion. ~us, we used an MCA occlusion model in order to test the ability of Calpain inhibitors to prevent neurodegeneration. lhis model is descn~ed in E~ample 6.
~IYsion Model for ~eurode~eneration Male Sprague-Da~vley albino rat~ weighing appro~mately 250-300 grams were anesthetized with pentobarbital (70 mg/kg, i.p.). The neck region was shaven and a 2 cm incision was rnade. The superficial f~u ia ~vas teased away with tissue forceps and blunt tip tissue scissors using a spread method. The right comrnon carotid artery was isolated away ~om ~he vagus nerve and tied off with a single suture (3.0 siL~;). The external carotid was permanently occluded by sut~ing. Ihe bifurcation of the internal carotid and pterygopalatine arteries was e~osed and a single microaneulysm c lip was placed on the ptenygopalatine. Another microaneutysm dip was placed on the common urotid just pro~mal to the e~ternal/internal bifurution. A suture ~ras placed loosely around the co~Tunon carotid and a lumen was made in the vessel with the tip of a 25g needle. A 40 mm nylon suture was prepared l~y melting the tip ~o smooth the pointed end and marked with a dot ~actb 175 mm from the melted end. The suture was inserted into the lumen of the artery as far a~ the vessel clip, the clip is removed and the suture advanced until the marldng was at the bifurcation of the internal andex~ernal carotid arteries. I~ia places the end of the suture in the circle of Willis just beyond the source of the middle cerebral ar~ery and oecludes this arsery. The loose suture around ~he caro~id is tied lightly to keep the nylon suture in plau. The St~8STlTlllTE SI~EET
2~8~
wo 92/11850 PCr/US91/0978 n~lcroaneurysm clip on the ptPrygopalatine artery was removed, the incision is closed and the animals are allowed to re~over in heated recovery cages.
Twenty-four hours after oeclusion, the brains of the~e animals were removed and sliced into 2rnm sections. The sections were stained using 2,3,5-S triphenyltetrazolium chloride (see Lundy, EF, Solik BS, Frank, RS, Laq, PS, Combs, DJ, Zelenock, GB, and D'Ale~y, LG, Mo~hometric ev luation of brain infarcts in rats and gerbils, J. Pharmaco~ ~eth 16,201-214, 1986). Absence of red color development indicated tissue damage or death. The s~ of the infarcted tissue zone (area with red stain) and impaired zone (area with partial development of red color) were evaluated using quantitative morphome~y.
Drugs or vehicle were administered by infusion into the femoral vein. All animals received the same volume of drug or vehide (20% dimethyl su~fo~ide/80%
propylene ~ycol) via a catheter attached to an Alzet osmo~ic milLipusnp (24 hr pump, 8 ul/hr, 90 ul total volume).
The model of E~a nple 6 was used to determine the size of infarcted area for control (vehicle, i.Y.) and with adrminissration of each of ~wo Calpain inhibitors: ZLeu-Phe-CONH-Et (CX269) and Z Leu-Abu-CONH-Et (CX275). These results are depicsed graphically in Figure 2. It can be ~een that administration of either of the Calpain inhl~itors Z-Leu Phe CONH-Et (CX269) or Z-Leu-Abu-CO~H ~t(CX~7~) produees a reduction in the size of the infarcted area.
7. Inhlbltioo o~ Anoxic and H~ lc Da~nage The CA1 region of hippocampus ~s a brain area par,sicular~ ~ulnerable to ischemic damage and other insults imolving cxcitatory amino acids. The hippoc mpus is also a major focus of cell degeneration in Al~he~mer's disease. Neural cells in slices degenerate following hypoxia throuBI the se chain of events (including reperfusion effects) observed vo dur~ng and after ischemia. We believe that snldies of degeneration of neural slices in the pre~ence of the various Calpain ~h~itors is an effec~ve indicator of the membrane permeance of the Calpain Inhl~itors. Accordingly, we believe that these studies provide a model for the trea~nent and inhl~ition OI
neurodegeneration vo. Similar studies for de~ennining the e~icacy of compounds useful in the treatment of neurodegeneration in accordance ~ith the present invention can be performed using other models, such as protection against degeneration in platelets or cells in culture.
-SUBSTITUTE SHEE~T -~ w~9~/118So 2~ 9 Pcr/V591/09786 .~.
It ~s be~ieved that hypo~a is a major eause of neuroto~icity ~n a variety of neurodegenerat~ve diseases and conditions, such a~ stroke and head injur~1. Thus, we eonducted further studies u~ing hippocampal slices to show that the vanuus Calpain itors, advantageou~b, can ~ncre~se sunmal of hippocampal ne~ve cells during e~posure to hypo~ic or anox~c conditions. An initial screening procedure was first used to qualitatively detennine whether the various Calpain In}h~itors Wl provide neuroprotection from ano~a in hippocampal slices A~ ample of these initial screen~ng procedures is sh~vn by E~ample 7~
E~MPLE 7A
itial Screenfor Inhll?ition of ~o~ic Dama~
Hippocampal slices (400 um) were prepared frorn Sprague Dawley rats ~6 to 7 We~l~) and maintained in an interface chamber at 35C using conventional techniques, i.t~ ~e lower ~u~ce of the slice received a constant peffusion (05 ml/m~zl) of ACSF, while the upper surf~ce was e1~posed to a moist atmosphere f 2:CO2 (gS%:5%) e~changed at a rate of 2 L/min. The AC~;F medium contains (in mM): NaCl ~124), KCl (3), KHPO4 (25), CaCl2 (3.4), NaHCO3 (26~ and D-Glucose (10). Field ~citatory post-~naptic responsss were recorded from stratum radiatu n of CAlb in response to stimulation of Schaffer-cornmissural fi~ers ~n CAla or CAlc. The depth of the recording electrode was optimized and evoked responses were collected at a rate of one evoked response every 30 seconds.
For the initial screening procedure, 14 to 16 slices are harvested from the hippocampus of a single rat and placed in a com non ACSF bath. Each slice is tested in sequence to deterrnine the magnitude of its pre-ano~c evoked response. Five stimulation pulses (each 0.1 ms (millisecond) in duration) were presented over a 15 second ~ntenal. The largest evokcd response was noted and recorded for each slice.
Following this, the slices were incubated for one hour, with either drog or vehicle alone added to the ACSF. After the one hour drug incubation period, the oxygen-enriched atmosphere of the chamber was rnade anoxic by substituting nitrogen for oxygen (N2 = 95%; Co2 = 5%). The slices were retained in this ano~ic environment for 10 n~inutes, following which the a~ygen-enriched a~mosphere (o2 =
95%; C2 = 5%) was reestablished.
Ihe slice~ were given the opportunity to recover for 30 rninutes following reoxygenation whereupon each was stimulated and the ma~nurn evoked po~ential determined, as descn~ed above durin~the pre-ano~a period. Those slices which, after !~1 IR.`t:T~ IT~ ~
.
. .
WO 92/11850 2 ~ 9 ~ ~ O ~ pCI/lJS91/0~7~6'" "
ano~iaa, produced a ma~imLun evolces~ potential of greater than 50% of that observed prior to ano~ia were dei;ned as sun~ing slices.
Results of the studies of E~ample 7A are show~ in Figure 3. Figure 3 shows the effects of CX218 (Z-Leu-Abu-CO2E~t, a Peptide Ket~Compound), and CI1 relative to control slices on sumYal OI hippocampal slices e~osed to 10 minutes esposure of ano~c atmosphere. As ~een ~rl this figure, when the control slices are depr~ed of oxygen for 10 Iminutes ill the absence of drug, virtually all f~il to sur~e, as measured by their ability to elicit SO~o of their pre-anoxia eYoked response. In accordance with this finding, few if any recover upon reoxygenation. Figure 3 also shows that when CI
or CX218 are added to the ACSF, the slices are protected ~om the efEects of ano~ia, evidenced by a substantial proportion of slices eliciting eYoked potentials.
Finally, i~ e seen that CX218 is significantly more effective in protecting against anox~a and preventing degrada~ion of slices at the minimal 1 hour incubation time, and at lower concentrations than CI1. This efEect is believed to be due ~o the superior membrane permeance of the Peptide Keto-Compounds.
Table 7A shows further data from the studies of Example 7A.
SUBSTITUT~ SH~ET
.
! WO 92/11850 2 ~1 9 8 6 ~ 9 PCl[/VS91/097~6 -lOS-PEROEMT OF SLIOES SUBVIVINÇ: TEN M[INImS ~NOXld.
Compound Dose (u ~rubation T~ne Sunnval Control ~ 1 hour <l~o S Leupeptin lOOO . 3 hours 50%
~Il 200 2 hours 53~0 CX13 (SHC) 20 l hour ~O~o CX89 (CKP) SO 1 hour ~O~b CX2l8 ~ C) 1~ l hour 70%
i~, It can be seen from the d~ta in Table 2 that all of the Calpain ~h~ ors tested provide ~ncreased scl~viYaL CX~3 (AClTlC, a Substituted Hetero~yclic Compound (S~ . CX89 (Boe -Cly-Leu-Phe-cH2~1~ a Halo-Ketone Peptide (HKP)) and CX~18 ((Z1~Abu-C02Et, a Peptide Keto-Compound (PKG)), are each shown to ~e highly ef~ective in influencing sun~Ival times. Leupeptin ~s seen to be the least ef~ ve neuroprotective. Thus, we believe that CX13, CX89 and CX218 (ZLeu-Abu-C02Et) are more effec~ve in influencing sumsal be~use of their membrane permeability.
Accordingly, the results shown in Table 7A support our belief that Calpain Inhibitors with membrane permeability are ef~ective neuroprotectants.
To ~rther eluu~idate the ability of Calpain Inhibitors to provide neuroproeection to hippocampal slices, u~d to provide a more quantita~e indica~ion of the membrane perrneability of these Calpain Inhibitors, we measured the effeet of vanous Calpain Inhibitors on the c~oked response on a single neuronsl slice before, during and after anoxia. These srldies are shown in E~ample 7B.
llhiki~iQn of AnQ~UC r)a,Illa8e As ~n Esample 7A, hippocampal sL;ces (400 ym) were prepared from Sprague Davley rats (6-7 veeks3 and main~ained in ~n interf~ce chamber at 3S~C using conventional techniques, ie. the lower eur~ce of the slice recerved a constant perfusion (05 ml/m~n) of an arti~al cerebrospinal fluid (A~SF), while the upper surface was exposed to a moist atmosphcre of 02:C02 (95%:5%) e~changed at a rate of 2 L/min.The ACSF medium con~ains (in m~: NaCI (124), KC:I (3), K~04 (1.25), MgS04 (2.5), CaC12 (3.4), NaHC03 (26) and D-Glucose (10). Field sxcitatory post-synaptic responses ~rere recorded from stratum radiatum of CAlb in response to stimulation of Schaffer co~sural fibers ~ CA1a or CA1c. The depth of the recording ele~rode SUE3STITUTE ~
~U~'3 WO 92/11850 PCI/US91/097B~
was optimized and evoked responses were collected at a rate of one evoked response every 30 seconds.
Af~er establishing a stable baseline of evoked responses (appro~mately 10 rninutes~, ACSF contail~ing ~pain In}~l~itor was washed into the chamber and slices were iT~cubated for a period of one hour. After incubation, evoked responses were again recorded and the change in the amplitude of the responses from base~ine levels was noted. No effect of the inhibitors tes~ed on ba~eline evoked responses was obsened.
For ano7~a ~eriments, incubation in the drug-containing medium was fo~lowed by replacement of the 02:C02 (95%:5%) atmosphere ~nth N2:C02 (95%:5%). Slices were e~posed to this anoac environment until disappearance of the pre-synaptic fiber volley and for two n~inutes (se~ere ano~ia) longer (total time in ano~ic erlviromnent appro~imately 7-8 minutes in control case). Effects of Calpain ~hibitors on the functional recovery of the slices after the anwnc episode were then measured.
Recovery of the evoked po~en~ial (~PSP) slope and arDplitude by the drug treatedslices can be compared to con~rol slices to d~tennine the relative e~icacy of various Calpain Inhibitors.
Figure 4 shows the E~PSP amp~itude in rnill~volts for con~ol, C:[1 treated and CX218 (a Peptide Ket~Compound) ~eated hippocampal slices in the studies of E~ample 7B. It can ~e seen in Figure 4 that the control slices deprived of o~ygen in the absence of drug display a gradual reduction of EPSP and abruptly lose ~ber volley activity about S-6 minute3 after the begin~ing of ano~ia. Reo~ygenation at or before th~s point leads to ~omplete functional recovery after about 20 minuteg of reo~ygenation, but reo~ygenation ~ this point does not. In the latter case the recovered EPSP slope and amplitude become progresslvely reduced as the duration of ano~ post fiber valley disappearance (post-FVD) increases. After severe anoxia (2 minutes post-FVD), slices re~over only 15% of the EPSP ~ope.
In contrast to the control slices, recove~y begins to oceur shortly after the end of anoxia for the treated slices. Figure 4 shows a comparison of the e~ects on EPSPamplitude produced irl the presence of no inhl~itor; the Peptide Keto-Compo~md, CX2lK, (~Leu-Abu-COæt) and CI1. CX218 produces a recovery ~om severe anoxia superior to that seen with CI1.
Figure S sh~ the percent recovery of EPSP ~om seYere hypo~a using the peptide ketoester CX 216 (~Leu-Phe-CO2Et) and its corresponding peptide SlJE3~1TUTE StlEET
.
.--' WO 92~11850 2 ~ 9 8 G O ~ PCI'/US91/09786 ketoan~ide CX269 (Z-Leu-Ph~CONH-Es). These studies were performed ill a manner similar to that of Example 7B, el~cept USiDg a hypo~ic environrnent in place of the a~o~a of ~ample 7B. It can be seen that use of the peptide ketoamide results in essentially complete (near 100%) reeovery ~om hypo~a while the peptide ketoesterS produces a par~ial recovery. I~e control ~lices ~peAenced little or no recovery.
An interesting charactedstic that we have discovered for certain Calpain bitors Is their abili~y to lengthen the period of e1~posure to ano~ia required to produce ~r volley disappearance (FVD). 15pi~, under control ano~a conditions, fiber volley disappearance o:urs in less than s~ minutes (~igure 6). The Peptide 10 Keto-Compound, CX-216, su~stantialb lengthens the period of e~posure to ano;~a required to produce FVD. This is an important advantage of the use of this Peptide Keto Compound for neuroprote~ion be~use slices can be e~pected to recover completely if reo~ygenated before fiber voiley disappearanc ~. Thus, treatment wi~h this Peptide Keto-Compound is e~pected to produce a greater percen~ge of recove~ of cells from incipient neurodegenerative conditions. It is believed that other representatives of the Peptide Keto-Compounds as well as of other classes of ~Ipain itors also provide this effect.
Table 7B shows the perecenhge of recovery of pre-anoxia synaptic transmission (evoked potential amplitude) of slices treated with various Calpain Lnhibitors or of 20 control slices. All of these slices were acposed to ten minutes of ano~a according to the protocol of E~ample 7B.
.:
WO 92/118~i0 ~ 0 9 ~ 6 0 3 PCI'~V~91/~9786~
T~BI~S 7B
Pl~RCE;NT RECOVE;~ OF SYNA~C TRANSMISSION A~R ANOXL9 ÇQm~!lD~ al~ % Recover~
Control -- lS
CX13 (SHC) 20 60 CX89 (a~) 50 30 CX216 (PKC) 100 38 CX218(PKC) 10~ 5~
The results shown in Table 7B provide fu~her eYidence tha~ the peptide aldehyde, C:I1, as well as the Substituted Heteroeyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides are sufficiently membrane permeant to provideneuroprotection through Calpain inhl~itiolL
CIl, which is at least p~y membrane permeant~ produces some effect, 1~ howeYer, does not significantly lengthen the period of ano~ia required to suppress electrical act~ . Thus, compared to controL or even compared to leupeptin and CI1, the Substituted Hetero~yclic Compounds, Pep~ide Keto-Gompounds and Halo-Ketone Peptides can increase the degree of recovery after ano~ic episodes while producing the additional advanhge of ~tending the amount of tirne slices carl tolerate ano~a (and thereby recover completd~
An important efEect of the Peptide Keto-Compounds and other membrane permeant Calpa~n Inhibitors ~s that they are ~ignificantly more effec~ve in lower doses than less permeable Calpain In~bitors such as C:I1. Although C~I1 is sho~m to be at least ~omewhat membrane penneant due to its abili~r to ~ect slice su~iYal, the more 2~ membrane-permeant inkibitors provide significantly increased protection. Thus, the more highly membrane-permeant Calpa~n Inhibitor~ are believed to be especially ef~ective in ~eating and inhlUting neurodegeneration.
The results of the studies of Esamples 7A and 7B show tha~ the Substituted Hetero~yclic Compounds, Peptide Keto-Compounds and Hal~Ke~one Peptides are membrane-permeant CalpaLn ~h~ ors which are believed to ~e especially effective in treating and inhl~iting neuro~egeneration. The results also show that Peptide Keto-Compounds, and perhaps representatives of other classes, can ~end the duration of anoxia required to suppress electlical activity in hippooampal islices.. As discussed above, these effects are ~mportant advantages of these compo~mds.
~1 IR~TIT~ ITF .~F~T
-- ;,; ~ . :- .. ..
WO 92/11850 ~ .5 ~ ,9 PCI/lJS91/09786 8. ~ NeuroprotectioD by Calpa~ I~ibitors As discussed above, therapeutics useful for influencing the function of cells within the CNS must cross the BBB to reach their targets within the CNS. Non-BBBpermeant compounds might, in addition to the brain infusion techniques de~cribedabove~ be administered v~a intraventricular administration, but this also se~erely limits their usefulness in practice. In order to test the vivo effectiveness of ~he Calpain klhl~itors to cross the BBB and become therapeuticalh~r useful, we tested the ability of mtraperitoneal injection of ~he Calp~ Inhl~itors to pro~ec~ a~st ~citoto~ic damage ~YiVQ, Protection w~ measured ~ aluating dlanges in beha~ of rats after inie~ion ~ith Icainate. These studies are sho~m in Example 8A.
EXAM[PLE 8A
Admiristered Calpain Inhibltors Ra~s (male Sprague-Da~ley, 200~5 gms) were injected intraperitone311y with 12mg/kg kainic acid in saline vehicle and ether 200~l DMSO (dirnethylsulfo~ide) or 4.6mg calpain inhibitor dissolYed in the same volwne of DMSO. The ra~s were obserYed for six hours following the injections and the kaina~e-induced behavioral symptoms and convulsions soored on a scale of 0-6 (0=no symptoms; 1-wet dog shakes; 2=salivation and chewing, 3=at least one convulsive episode; 4=repeated or sustained convulsions; 53convul3ions, including rearing and falling; 63convulsions followed by death ~nthin the 6 hr~ post injection).
Figure 7 shows the effects of CI1 on the beha~ioral and con~rulsive effects of l~ainic acid. In the control group, over half the anin~ls showed symptoms greater ~han mild behavi~ral symptoms, and many e1~hibited overt convulsions, presumably reflecting seizure acti~ity within the brain. Une~pectedly, in the inhibitor t~ated group, the incidence and severity of convulsions was reduced. Thu~, this data suggests thatCalpain Inhibitors have an anti-conmlsive effect. Ihis effect is a distinct advantage in the use of Calpain Inh~itors in epilep~-related neurodegenerative conditions and in stroke, which is often accompanied by seizures.
Ln order to rnore clearly demonstrate that the behavioral and anti~on~rulsive effects seen with the Calpain Inhibitors result from inhbition of Calpain we tested the brain tissues of the rats from E~ample gA for accumulation of spectrin BDP's. Asdiscussed above, these BDP's are associated with Calpain activity and with the neurodegeneration a~sociated therewith.
SUBSTITUTE S~ET
, WO 92/118S0 9 8 6 ~ ~ PCI'/US91/0978~' EXA~LE 8B
Protect n ~inst Speçtrin Breakdown ~om E~citoto~ic Damage bv Peri~?herallv ~dministered Calpain Inhibitors Four days follo~ing the inje~ion of kain~te in the rats from 3~ample 8A, the S brains of the rats were removed and assayed for spectrin BDP's. Spectrin BDP's ~ere assayed by homogenizing brain parts in 20mM Tris pH=7.2, 32M sucrose, 50~M Ac-Leu-Leu-nLeu-H on ice. Homogenate~ were mixed lol with 10% SDS, ~%
B-mercaptoethanol, 10% glyceroL lOmM Tris pH=8.0, 05% bromophenolblue, heated to 9~C, and subjeaed to electrophoresLs in 4-1/2% poiyacryl~rnide gels. 'rhe prote~ns in the gels were transferred to l~itrocellulose and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antlbody arld established Dnmunodetection methods.
The amount of spe~trin and BDP's in each sample was quantitated by densitometricscanning of the developed nitrocellulose.
Figure 8 shows the results of ~ample 8B. It can be seen that kainate stimulated the breakdo~n of spectrin in both Cal~ain Inhl~itor treated and con~rol rats.
However, treated rats e~u'oited significant~ less BDP's. These results veri~y that Calpain ac~vity in the brains of the treated rats was reduced. An une~pected observation was that wen those treated animals that ~hibited severe seizures hadsigruficantb less spectrin brea3cdo~n than untreated animals subjected to kainate. Thus Calpain Lnhibitor treatment reduced b~oth the behavioral/convulsive effects of kainate and the activation of calpain in the most severely affected animals.
Table PKC1 shows the inhibidon constants (Ki) ~or papaint cathepsin B, calpain I, and calpain II.
The inhibition constants for papain shown in Table PKC1 were measured in 0.05 M
Tris-HCL pH 75 buffer, containing 2mM EDTA9 ~mM ~ysteine (freshly prepared), 1%
DMSO, at 25C, using N~-Benzoyl-Arg-AMC as a substrate, ~cept that those values of inhibition constants for papain marked with an ~e~ in Table PKC1 were measured in 50 mM
Tris-HCL pH 7.5 buf~er, containing 2û mM EDTA, S mM qsteine, 9% DMSO, at 25C, using N~-Berlzoyl-Arg-NA as a substrate.
S'Ji3S~lTUTE SHE~T
..
: WOg2/11850 2~609 PCl~l~S9l/09786 l~bition Qf C~ysteine Pro~eases bv Peptide Ketoesters and Ketoacids .
S Ki(mM) Compounds pa GBb ac CIId ZLeU-AbU-COOFt 0.04 0.4 ZLeu-Phe-COOE~t 023 0.4 ZLeu-Me-COOE~t 0.12 0.18 Z-Leu-Nva-COOEt 30 1.2 Bz-DL-Phe-COOEt 500e ~4 Z-Phe-DL,Phe-COOEt 1.8 0.1 ~Phe-DL,Ala-COOEt 3.6 3~
Z-Ala-A.la-DL-Ala-COOEt 15 22 200 :ZAla-Ala-DL,Abu-COOEt 0.9 10 50 200 Z-Ala-Ala-DI~Abu~OOBzl 39 60 ZAla-Ala-DL Nva-COOEt 30 0.1 ~AIa-Pro-D~Ala-COOEt 26 66 MeO Suc Val-Pro-DL~Phe-COOMe 1.1 0.1 2,~e Z-Ala-Ala.Ala.DL-Ala COOEt 2.1 10.0 MeO Suc-Ala-Ala-Pro-Abu-COOMe 0.7 6.0 100 ~' .
aP=Papain Cc~ pain I
bCB=Cathepsin B dC~=Cal~ain 11 ~ O~!TITl IT~ U~Ç:T
. ~ ~ . ... . ~
wo 92/11850 2 0 ~ 8 G 0 3 Pcr/usgl/o97~
It can be seen from the data in Table PKCl that the dipeptide ketoesters with Abu, Phe, or Nle in the Pl site and Leu in the P2 site are potent inhibitors of calpain I and Gllpain ~. Tripeptides with Abu or Ala in the Pl site and Ala in the :P2 site are also seen to be inhibitors of Calpain, albeit somewhat weaker inhibitors than the dipeptides. Thus, S in accordance ~ith the foregoing descri~tion of the design of Peptid~ Keto-Compound inhibitors, we believe that Peptide Keto-Compounds b~sed on these and similar structu~es will ex~ibit Calpain inhibitoly act~ity.
The peptide a-ketoesters are prepared by a two step Dakin-West procedure. This pro~edure can be utilized with either amino acid derivatiYes~ dipeptide derivatives, tripeptide derivatives, or tetrapeptide derivatives as shown in the follo~ing scheme:
o Il M-(AA)n-OH -- ~ Enol Ester ~ > M-(AA)n-CO-R.
l~e precursor peptide ((AA)n) can be prepared using s~ndard peptide chernistry procedures, including those that are well described in publications such as The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, published in 19791987 by Academic Press ("The Peptidesn) and Houben-Weyl Methoden der Organischen Chemie, VoL 15, Parts 1 and 2, Synthese von Peptiden, published by Georg Thieme Verlag, Stuttgart in 1974 (nHouben-Weyln) (both r~ferences hereby incorporated herein by reference).
The M group can be introduced usmg a number OI different reaction schemes. For example, it could be introduced directly on an anuno acid as shown in the following scheme:
H-(AA)n-OH > M-(AA)n-OH.
Alternatively, the M group caD be introduced by reaction ~nth an arnino acid ester, followed by removal of the ester group to g~ve the same product, as shown n the followmg scheme:
~(AA)n-OR'--~ M,-(AA)n-OR'--~ M-(AA)n-OH.
These and other techniques for introduction of the M group are well docurnented in the The Peptides, Houben WeyL and many other tectl?ooks on orgamc synthesis. For example reaction v/ith cyanate or p-nitrophenyl cyanate would introduce a carbamyl group (M = NH;!CO-). Reaction with p-nitrophenyl thiocarbamate ~ould introduce a thio carbamyl group (M = NH2CS-). Reaction with N~I2S402a would introduce the NH2SC)2-group. Reaction with a substituted aLtcyl or aryl isocyanate would introduce ~he X-NH-CO-group where X is a substituted allyl or aryl group. Reaction with a substituted allyl or aryl isothiocyanate would introduce the X~ group where X is a substituted alkyl or aryl group. Reaction with X-S02-Cl would introduce the X-SO2- group. Reaction with a S~JBSTIT~JTE SHEET
. ` : : . . ~ - :
~ .
,A WO 92/11850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786 subslituted allyl or aryl acid chloride would introduce an a~yl group (M = Y-C0-). For e~ample, reaction with MeO-C0-GH~CH2-C0 Cl would gIve ~he Y C0- ~oup when Y is a C2 allyl substituted w~th a C~ yl OC0- group. Reaction with a substituted aL~cyl or aryl thioacid chloride would introduce a thioacyl group (M = Y~ ). Reaction with an asubstituted allyl or aryl sulfonyl chloride would introduce an X-S02- group. For e~ample reaction with dansyl chloride would g~ve the X S02- derIvative where X was a napthyl group monosubstituted with a dimethylamino group. Reaction with a substituted aLl~yl or aryl chlorofonna~e would introduce a X-0-C0- group. Re~ion with a substitu~ed allyl or aryl chloro~iofonnate would introduce a X-O CS-. There are many alternate reaction schemes which could be used to introduce all of the above M groups to gIve either M AA-OH or M~ OR'. The M-AA-OH dernratives could then be used directly in the Dakin-West reaction or could be converted into the dipeptides, tripeptides, and tetrapeptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH which could be be used in the Dakin-West reaction. The substituted peptides M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH could also be prepared directly from H-AA-AA-OH, H-AA-AA-AA-OH, or H-AA-AA-AA-AA-OH using the reactions described above for introduction of the M group. Alternately1 the M group could be introduced by reaction with carbo~ylblockedpeptides M-AA-AA-OR', M-AA-AA-AA-OR', or M-AA-AA-AA-AA-OR', followed by the removal of the blocking group R'.
The R group in the ketoester structures is introduced during the Dakin-West reaction by reaction with an o~alyl chloride Cl-CO-CO-O-R. For example, reaction of M-AA AA-OH w~th ethyl o~alyl chloride Cl-CO-CO-O-Et gives the keto ester M-AA-AA-CO-O-Et. Reaction of M-AA-AA-AA-AA-OH~ith Cl-CO-CO-O-Bzlwould ~ve the ketoes~er M-AA-AA-AA-AA-CO-O-Bzl. Clearly a wide variety of R groups can be introduced into the ketoester structure by reaction with ,rarious allyl or arylalkyl oxalyl chlorides (Cl-CO-CO-O-R).
The oxalyl chlorides are easily prepared by reaction of an aLlcyl or arylalkyl alcohol with oxalyl chloride Cl CO-CO-CL For e~Eunple, Bzl-O-CO-CO-Cl and n-Bu-O-CO-CO-Cl are prepared by reaction of benzyl alcohol and butanoL respec~vely, ~vith oxalyl chloride in yields of 50% and 80% [Warren, C. B., and Malee, E. J., J. Chmmatograpty 64, 219-222 (1972); incorporated herein by reference].
Ketoacids M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH, M-AA-AA-~A.AA-CO-OH, are generally prepared ~om the corresponding ketoesters M-AA-CO-OR, M-AA-AA-CO-OR, M-AA-~A-AA-CO-OR, M-AA-AA-AA-AA-Co-OR
SlJBSrlTlJ~E SHEET
:.
W O 9 2 / 1 1 8 5 0~ p C r / U S 9 1 t 0 9 7 8 f by aL~caline hydrolysis. In some cases, it may be necessary to use other methods such as hydrogenolysis of a berlzyl ~oup (R = Bzl) or acid cleavage (R = t-butyl) to obtaill the ketoacid. The alternate methods ~vould be used when the M group was labile to aL~caline hydrolysis.
S The various peptide ketoamide subclasses (M-AA-NH-CHR2-CO-CO-NR3R4 (Di~eptide Ketoarnides (Subclass A)), M-AA-AA-CO-NR3R4 (Dipeptide Ketoamides (SubclassB~), M-AA-AA-~A-CO-NR3R4 (InpeptideKetoamides~, M- AA-AA-AA-AA-CO-NR3R4 (tetrapeptide Icetoarnides) and M~ CO-NR3R4 (Amino Acid Ketoamides)) were prepared indirectly f~om the corresponding ketoesters. Ihe ketone carbonyl group was first protected as shown in the following scheme and then the !cetoamide was prepared by reaction with an amine H-NR3R4. The illustrated procedure should also work wi~h other protecting groups.
Ml-AA~ ~ `R ~ -AA2~N~o~R
~ H-NR3R4 Ml-M2~ ~ ~R4 R1 o In addition to the scheme outlined above, a ketoacid could be used as a precursor to produce a corresponding ketoamide. Blocldng the ketone carbonyl group of the ketoacid and then couplingwith an amine H-NR3R4 using standard peptide coupling reagents would yield an intenned~ate ~rhich could then be deblocl~ed to form the ketoamide.
Generol Syn~etfc Method~ for Peptide Reto-Compounds Unless otherwise noted9 materials were obtained from commercial suppliers and used ~nthout further purification. Melting points were taken ~1vith a B~chi capillaly apparatus and are uncorrected. lH NMR spectra were detenT~Lned on a Varian Gemini 300.
Chemical shifts are e~ressed in ppm (~) relative to internal ~etramethylsilane. Flash column chromatography was per~ormed with Universal Scientific Inc. silica gel 0-63.
~:llBSTlTUTE S~IEET
, ~- .
: ; ~ :
WO 92/11850 2 ~ ~ 8 6 ~ 9 P~US9l/09786 Electron-impact mass spectra (MS) of novel compounds were detern~ined with a Varian MAT 112S pe~rometer. The purity of all compounds was checked by thill- laye~
chromatography orl Baker Si250F silica gel plates Usillg th~ following solvent s,vstem: A, CHC13:MeOH = 20:1 v/v; B, CHG13:MeOH = 100:1 v/v; C, AcOEt; D, CHCl3:MeOH =
--10:1 v/v; E, n-BuOH:AcOH;py:H2O = 4:1:1:2 v/v; F, CHC13:MeOH = 5:1 v/v; G, AcOEt:MeOH = 10:1 v/v; H, (i-Pr)20; I, CHC13:MeOH:AcOH = 80:10:5 ~/v; J, CHCl3:MeOH:AcOH = 95:5:3 v/v; K, AcOEt:AcOH = 200:1 v/v; L, C~C13; M, C~IC13:MeOE = 50:1 v/v Amino acid methyl ester hydrochlorides were prepared aecording to M. Brenner e~
al.[~le~. Chen~ Acta 33,568 (1950);36,1109 (1953)] Ln a s~e over 10 rnunol or according to ~achele ~. Ch~. Chen~ 28, 2898 (1963)]in a s~e ofO.1-1.0 n~nol.
Yield (%) mp (~) m.p. (literature) DL-Nva-OCH3HCL 100 113-116 116-117 Ir~e-OC~3 HCL 98 9C-91 98-100 L-Phe-OCH3 HCl, 98 159-161 158-160 DI,Abu-OGH3HCI, 100 148-150 150~151 L-Leu-OCH3 HC1 100 145.S~146.5 147 DL~Me-OCH3 Ha 93 120-121 122-123 4-Cl-Phe-C)C~I3Ha 98 18`4-185 (decomp.) 185-186 N-Acylamino acids was synthesized via Schotten-Baumann reaction lM. Ber~nann, L. Zenas, Chem Ber. 65, 1192 (1932)] in the case when the acyl group was phenylsulphonyl, 2- naphthylsulphonyl or benzoyl.
STITUTE S HE~T
.
~ .
.
.
.
2~9~0~
WO 92/11850 PCr/lJS91/09786 ~5~
Yield (%) mp (C) TLC (R~, eluent) 2-NapSO2-L-Leu-OH 49 115-116 058I
2-NapSO2-D~Abu-OH 51 150-151 050I
2-NapS02-L-Phe-OH 57 148-1485 0.48K
PheSO2-DL-Abu-OH 44 142-143 0.51K
PhCO-DL,Abu-OH 64 141-142 0.64K
N-A~ylamino acids with 4-methylpentanoic, 2-(1- propyl)pentanoic and 7-phenylheptanoic group was synthesized in a two step synthesis. The N-acylamino acid methyl ester was obtained first and then was hydrolysed to the free N-acylan~ino acid.
N-Acyl~ no Acid Met*yl Este~s (Gener~ Pr~cedl~re). To a chi~led (10 C) slurry of the appropriate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was added slowly (temp. 10-15 C) 40 rnmol triethylamine or N- methylmo~pholine and then the reaction mixture ~as stirred for 30 n~inutes at this temperature. Then 18 rnmol of appropriate acid chloride (temp. 10-15 C) was added slowly to the reaction mi~ure and the reaction n~ture was stirred overnight at room temperature. The p~ecipiatatedhydrochloride was filtered, washed on a funnel with 2 ~ 20 ml benzene, and the collected filtrate was washed successivdy with 2 ~ 50 ml 1 M HCI, 2 x 50 rnl 5% NaHC03, 1 x 100 rnl H20, 2 x 50 rnl satd. NaCl and dried over MgSO4. After evaporation of the solvent in vacuo (rotavaporator), the residue was checked for purity (ILC~ and used for the next step (hydrolysis).
Yield (%) mp (C) (cH3)2cH(cH2)2co-DLAbu ocH3 80 oil (CH3CH2CH2)2CHCO-DL-Abu-OCH3 96 117-118 Ph(CH2)6CO-DL,Abu-OCH3 72 oil Hyd~o~is (Get~eral Proced~e). To a solu~ion of 10 mmole of the appropriate N-as~yiamino acid methyl ester in 100 rnl of methanol was added in one portion 11.25 ml of 1 M NaOH (11~ mmol? and the reaction mi~ture was stirred three hours at room temperature. Then the reaction n~Ll~ture was cooled to 0 C (ice- salt bath) and acidified to ~1 = 2 with 1 M Ha aq. To this reaction m~ture was added 100 ml ethyl acetate, transferred to a separatory funnel and organic layer separated. ~he water layer was saturated with solid Naa or (NH4)2SO4 and ree3~tracted with 2 ~ 50 ml AcOEt. Tlse collected organic layer was washed with 2 ~ 50 rnl H20, decolorized with carbon, and dried over MgS04. After evaporation of the solvent in ~vacuo (rotavaporator), the residue was ~ SUBSTITUTE SHlEEt -.. . . . ... ; .
`- ~ .
. ' : . .
~ ` wo 92/11g50 2 ~ 9 3 6 ~ 9 PCr~lJS91/09786 cheeked for puri~ (TLC) and in the case of contan~ination was crystallized ~om an appropriate solvent.
Yield (%) mp (C) (CH3)2CH(CH2)2CO DL-Abu-OH 92 1105-112 (CH3CH2CH2)2 C~HCO-DL-Abu OH 99 126-127 (n-octane) Ph(CH2)5CO-DI~Abu-OH 89 110-112 (n-octane) N~Ac3rldipeptide methyl esters were syllthesized via ~he HOBt-DCC method in a DMF solution [K8 ug and Beiger, C)~em Ber. 103, 788 (1970)].
Yield (%) mp (C) TLC (Rf, eluent) Z-Leu-DL-NVa-OCH3 80 112-113 0.37 B
ZLeu-L-Phe-OCH3 83 86-87 0.85 A
0.39 B
Z-Leu-L-Ile-OCH3 97 oil 0.79 A
0.43 B
Z-Leu-DL Abu-OCH~ 99 86-88 033 B
0.26 H
Z-Leu-L-Leu-OCH3 80 91-92 0.79 G
Z-Leu-DL-NLeu-OCH3 97 111-1115 Z Leu-4 Cl-Phe-OCH3 65 112-132 0.77 J
(liquid c~ystal?) 0.68 K
2-NapSO2-Leu-DL-Abu-OCH3 99 oil 0~9 A
2-NapSO2 Leu L-Leu-OCH3 97-98.5 0.63 A
N-Acyldipeptides were obtained by hydrolysis of the appropriate methyl esters via a generàl hydrolys~s procedure. In the case of N-sulphonyldipeptide methyl esters, 1 equivalent of the methyi.ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH
because of forrn a sulfonamide sodium salt.
SUlBSTlTlJTE SHEIET
WO 92~11850 2 ~ 9 8 ~ ~ 3 I'CTtUS91/09786 r::
Yield (~o) mp (C) TLC (R~, eluent) Z-I.eu DL-~a-OH 100 117-118.5 0.11 A
ZLeu-L-Phe OH 92 105-106.5 0.28 C
Z Leu-L~ OH 79 77-79 0.22A
05~C
~leu-DL-Abu OX 99 gl~ss 0.61 G
~Leu-L~.Leu-OH 97 glass 056I
ZDLeu-DI,NLeu-O~I 98 95~96 ZLeu~-Cl-Phe-OH 87 104-114 0.48 K
(liquid crystal?) 2-NapS02-Leu-DL-Abu-OH
97.4 180-195 (decomp) 0.58 I
2-NapSC)2-Leu-L-Leu~OH
94.0 68-70 052 I
N-Acytripeptide methyl esters were synthesized via HOBt- DCC me~hod in DMF
solution LrKonig and Geiger, Chem. Ber. 103, 78B (1970)].
Yield (%) mp (C) TLC (Rf, eluent) ~Leu-~eu-Abu-0C~I3 87 140-1415 050 A
20~Leu-Leu-Phe-OGH3 76 158-159 0.83 J
2-NapS02-Leu Leu-Abu-OC~I3 97 ~200 0~2 A
N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters via general hydrolysis procedure. Ln the case of N-sulphonyltripeptide me~hyl ester, 1 25 equivalen~ ~f methyl ester was hydrolyzed with 2~5 equivalent of 1 molar NaOH to form the sulfonamide ssdium salt.
Y.ield mp (C) TLC (R~, eluent) ~Leu~Leu-Abu-OII 97 glass 0.69 I
ZLeu-Leu-Phe OH 98 glass 0.44K
302-NapS02-Leu Leu-Abu-OH
- 85 193-195 0.~3 I
(decomp.) 032 J
The following examples, Examples PKC1-PKC65, are given to illus~rate the synthesis 35 of Peptide Keto-Compounds:
SUBSTITVTE SHEET
, . .
, : . -WO 92/11850 2 ~ ~ 8 6 ~ 9 PCTtUS9l/09786 759~
EXAMPI,E PKCl -DL~ COOEt. This compound was s~mthesized by a modi~ied Daki~i-West procedure [Charles et al., J. Chem. Soc. Perlcin L 1139-1146, (1980)~. To a stirred solution of ZAla-Ala-OH (880 mg, 3 mmole), 4 dimethylaminopyridine (15 mg, 0.31 mmole), and pyridine (0.8 mL 10 mmole) in tetrahydrofuran (3 mL) was added ethyl o~alyl chloride (0.7 mL, S rnmole~ at a rate sufficient to ~n~tiate reflu~g. The n~ture was gently reflu~ed for 35 h. The mi~ture was treated ~ith water (3 mL) and stirred vigorously at room ~emperature for 30 n~in. The m~ture was e~tracted w~th ethyl acetate. The organic e~tracts were dried and evaporated to~ ol~tah ~e- residue (1.45 g). The residue was chromatographed on silica gel and eluted with CH2Cl2 to g~ve the enol ster product, oil (500 mg, 37~O); single spot on tlc, R~2 = 0.67 (CHCl3:MeOH = 9:1); MS, m/e = 451(M++1). To a stirred suspension of the enol ester (~10 mg, 0.47 mrnol) ~n anhydrous ethanol (1 mL) at room temperature was added dropwise a solution of sodium ethoxide in ethanol until a dear yello~ soiution resulted. Ihe ethanol was then removed and the residue was trea~ed with ether. The ether solution was washed with water, dried, and evaporated to give a residue. This residue was chromatographed on a silica gel and the product was duted ~nth methylene chloride. The solvent was removed, and the peptide ketoester ZAla-DL,Ala-C02Et was obtained as an sen~i-solid (1~0 mg, 92 %); single spot on tlc, Rf1 058 (C~Ia3:MeOH ~ 5:1); MS, m/e = 351 (M~1). Anal. Calcd. for C17H2206N2-1/3 H20: C7 57.29; H, 622; N, 7.86. Found: C, 57.23; H, 6.36; N, 8.17.
EXAMPI,E PKC2 Z~Ala-Ala-DL~ CO2Et. This compound was prepared ~om ZAla-Ala-Ala-OH
using the same procedure a~ described in E~ample PKC1. The product was crystalli~ed from ethyl ether in 23% yield; sDgle spot on tlc, Rf2 ~ 031 (CHC13:MeOH = 9:1); mp 143-144 ~C; MS, m/e ~ 421 (M+).~AIial. C~llcd. fbr C2~H27O7N3: C, S6.99; X 6-46; N, 9.97. Found: C, 56.96; H, 6.49; N, 9.92.
EXAMPI.E PKC3 Z-Ala-Ala-DL,-Abu-C02Et. This compound was prepared from ZAla-Ala-DL-Abu-OH in 11% yield by the procedure descnbed in Example PKC1; single spot on tlc, Rf2 = 0.60 (CHCl3:MeOH = 9:1); mp 111-113 C; MS, m/e = 436 (M+~1). Anal.
Calcd. for C21H2907N3-1/3 H20: C, 57.13; H, 6.75; N, 9.51. Found: C, 57.38; H, 6.82;
N, 9.62.
~ n~lTl ITr CUE~T
. . .
WO 92/11850 2 ~ 9 8 5 9 ~ PCI/lJS91/09786 E~AMPLE PKC4 ~AIa~Ala-l)L~Nva-CS:)2Et. This compound was prepared from ~AIa-Nva-OH in 20% yield by l:he procedure descri~ed in E~ample PKCl; single spot on tlc, R~l = 0.64 (CHCl3:MeOH = 5:1); MS, m/e = 450 (M~+l). AnaL C~lcd. for C~H3lO7N3H2O: C, 56.51; lH, 7.11; N, 8.99. Found: C, 56.42; H, 7.08; N, 9.06.
EXAMPLE PKC~
Z-Ala-Pro.I)~Ala-CO2Et. ~his compound was prepared ~rom ~AIa-Pro-Ala-OH dicycloh~ylamine ~n 19% yield b~ the proeedLIre descnbed in ~ample PKCl; sing~e spot on tlc, ~2 = 055 (G~CI3:MeOH 8 9:1); MS, m/e = 447 (M~). An~l. Calcd. ~or-C~2H2907N3 1/2 H20: C, 57.88; H, 6.62; N, 9.21. Foumd: C, 57.65; H, 6.68; N, 9.17.
EX~IPLE PKC6 Z-AIa-A~la-~-DL-~la CO2Et~ The compound was prepared from ~AIa-Ala-Ala-Ala-OH in 7% yield by the procedure described in ~ample PKC1; single SpOt on tlc, Rf2 -0.40 (C~Ht:13:MeOH = 9:1); mp. 163-165 C; MS, m/e = 493 (M++1). Anal. Calcd.
for C23H3208N4-1/2 X20: C, 55.08; H, 6.63; N, 11.17. Found: C, 54.85; H, 6.53; N, 11.14.
Bz-DI,Phe-CO2Et. ~ compound was prepared ~om Bz-Phe-OH in 36% yield by the procedure descnbed in E~cample PKC1, oil, single spot on tlc, Rf2 = 0.61 (CHCl3:MeOH = 9:1); MS, m/e ~ 325 (M~). AnaL Calcd. for C~ 904N l/3 H20: C, 68.86; H, 5.98; N, 4 22. Found: C, 69.10; H, 6.09; N, 438.
EX~MPIE PKC8 M~O-Suc~Ala.DL Ala-CO2M~ This compound was prepared from MeO-Suc Ala-Ala OH n 22% yield by the same procedure as descrlbed in E~ample PKC1, e~cept that sodium metho~ide in methanol was used for enol ester bydrolysis, single Spot~orl tlc, - 0.43 (CHC13:MeOH = 9:1); MS, m/e ~ 317 (M~ + 1). AnaL Calcd. for Cl3H2~07N4 1/3 H20: C, 48.44; H, 6.46; N, 8.69. Found: C, 4856; H, 6.39; N, 8.69.
SUBSTITUTE S~5EET
.
wO 92/118S0 2 ~ ~ ~ 6 ~ 9 P~/US91/097~6 ~1-E~MPLE PKC9 MeO Suc~ P~DI~Abu~CO2M[g. This compolmd was prepared ~om MeO-Suc-Ala-Ala-Pro-DL,Abu~OH in 22% yi~ld b~ the proeedure described in ExamylePKC8; foam, single spot on tlc, Rfl = 0.66 (CHCl3:MeOH = 5:1). Anal. Calcd. for S C22H3409N4H20: C, 51.53; H, 7.02; N, 10.85. Found: C. 51.11; H, 7.03; N, 10.88.
MeO-Suc-Val.P~DL-Ph~Ct)2~Me. I~is compound was prepared from MeO-Suc Val-Pro-Phe OH in 42% yield by the same procedure as descnbed in E7~ample PKCB; foam, single spot on ~ 2 057 (~ICl3:MeOH = ~:1); MS, m/e - 517 (M+).
10 Anal. Calcd. ~or C~6H350~N3 2/3 H20: C, 58.96; H, 6.90; N, 7.93. Found: C, 58.92; H, 6.96; N, 7.89.
Bz-DL,Ala-CO2-n-Bu~ This compound was prepared from Bz-Ala-OH in 45%
yield by the procedure descnbed in E~ample PKC1, e~c pt that n-butyl oxalylchloride 15 was used for the Dakin-West reaction and sodi~n n-butoxide in n-butanol was used for enol ester hydrolysis; coloriess oil, single spot on tlc, R~2 = 0.72 (CHCl3:MeOH = 9:1);
MS, m/e = 277 (M+).
Bz DI,Als C02BzL Ihis compound was prepared ~om Bz-Ala-OH in 26% yield 20 by the procedure described in Example PKC1, el~cept that benzyl oxalyl chloride was used in place of ethyl oxayl chloride and sodiurn benzylo~ide in benzyl alcohol was used for enol ester hydrolysis; single spot on tlc, ~2 e0.69 (CHCI3:MeOH = 9:1); mp 95-97 C; MS, m/e - 312 (M-~ +1). AnaL Calcd. for Cl8Hl704N.l/2 H20: C, 67.48; H, 5.66;
N, 437. Found: C, 67.78; H, 5.SS; N, 4.66.
EX~MPLE PKC13 ZAla-DL~Ala-CO2 ~-Bu. This compound was prepared from Z-Ala-Ala-OE in 14% yield by the procedure described in ~ample PKC1, except that n-but~yl oxalylchloride was used in the Daldn-West reaction and s~dium n-butoxide was used for enol ester hydrolysis; oil, single spot on tlc, Rf2 = û.45 (CEIC:13:MeOH = 9:1); MS, m/e =
30 378 (M~). Anal. Calcd. for Cl~H;606Nil/3 H20: C, 5935; H, 7.00; N, 729. Found: C, 59.41; H, 7.03; N, 7.10.
:~Ala-DL,Ala-CO~BzL This compound was prepared ~om ZAla-Ala-OH In 36% yield by the procedure described in Example PKC1, except that benzyl oxalyl ~ . ; . ~. . . .
209~ t WO 92/11850 Pcr/us9l/o9786:
chloride was used in the Dakisl-West reaction and sodiwn be~lo~ide in benzyl alcohol was used for enol ester hydro~sis; single spot on tlc, R~2 = 0.55 (CHCl3:MeOH ~ 9:1);
MS, m/e = 413 (M+ ~1). AnaL Calcd. ~r C22H24O6N2~ C, 64.06; H, 5.87; N, 6.79.
Found: C, 63.79; H, 5.95; N, 6.72.
EXAI~LE PKC15 ~AIa-Ala-DL,Abll-CO2BzL l~is compound was prepared ~om Z~Ala-Ala-Abu-OH in 31~o yield by the proc~dure descnbed in E%ample PXC1, ~s~ept that ben~yl o2a~l chlonde ~as osed in ~he Dal~n-West reaction and sodium benz~ ide in benzyl alcohol ~as used for enol ester hydrolysis; sin~e spot on tk; R~2 = 0 40 (CHa3:MeOH = 9:1);
mp 124-125 C; MS, m/e = 498 (M I + 1). Anal. Calcd. for C2~H3lQ7N3 2/3 H20: C,61.28; H, 639; N, 8.24. Found: C, 61.14; H, 6.65; N, 7.94.
E~AMPLE PKC16 Bz-DI,.ALq-COO}I. The hydrolysis procedure of Tsushima et al. F Org. Chem.
49, 1163-1169 (1984)] was used. Bz-DL,Ala-C02Et (540 mg, 2.2 ;s3~nol) was added to a solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7 mL of H20 and 2-propanol) and stirred at 40 C under n~trogen. After adding ethyl acetate and a saline solu~ion to the reaction mi~ture, the aqueous layer was separated and acidified with 2N HCl and e1~tracted with ethyl acetate. The organic layer was dried over magnesiurn sulfate and the solvent was removed under reduced pressure. The cmde hydrol~sis product was chromatographed on silica gel and eluted with methylene chloride and methanol to obtain an oil (150 mg, 31%); single spot on tlc, R~4 = 0.68 (n-butanol:acetic acid.~yridlne:H2O - 4:1:1:2). Anal. Calcd. for CllH1104N.3/4 H~O: C, 5628; H, 537; N, 5.97. Found: C, 5621; H, ~.46; 5.66.
Eg~MPLE PKC17 Z-Leu-I~L-Nva-COOEt. This compound ~vas prepared ~om Z-Leu-Nva-OH in 60 % yield by the procedure descn~d in E~ample P}~C1; oil, one spot on tlc, Rf = 0.49 (CHGI3:MeOH Y 20:1). NMR ~CDCI3) ~: 0.91 (t, 9H), C~I3; 1.25 (t, 3H), CH3; 1.38 (q, 2H), OCH2C~3; 1.64 (m, 6H), C$I2; 1.85 (m, 1H), CH(CH3)2; 434 (m, 1H) CH2CEI(NHCOOC~I2Ph)CONH; 5.12 (d, 3H) NHGH(CO)CH2 and OCH2Ph; 532 (d, lH) NH; 5.71 (d, 1H) NH; 736 (s, ~H) Ph.
Z-Leu-DI,Nva-end ester, the preeorsor OI Z-I eu-DL-Nva-COOEt was synthesized by the same procedure as descn~ed in Example PKC1 and purified by column chromatography, oiL one spot on tlc. N~IR (CDCl3) ~: 0.96 (t, 9H); 1.25 (t, SVB~;TITUTE~ SH~ET
, . .
2 ~ 3 WO 92~118~;0 PCI`/US91~09786 3H); 1.41 (t, 2H); 1.54 (m, 4H); 1.72 (m, 3H); 2.80 (t, 2H); 4.20 (q, 2H); 4.43 (q, 2H);
.16 (q, 2~I); 5.23 ts, lH); 737 (m, SH); 1133 ~s, lH).
Z L6u DI,Phe-COOEt. This compound was prepared from ~Leu-Phe-OH ~n 30 % yield by the procedure descn~d in E~ample P~Cl; oil~ one spot on tlc, Rf = 0.47 (CHC13:MeOH - 50:13. NMR (CDGI3) ~: 0.88 (d, 9H), OCH2CH3 aIId (CH3)2CH; 1.35 (q, 2H), OCH2CH3; 156 ~q, 2~), (CH3)2CEIGH2 ::H; 3.03 (m, lH), (CH3)2CH; 4.32 (m, 2~I), NHCH(CO)C~H2; 5.08 (s, 4~) GH2Ph; ~.40 (m, lH) NH; 6.61 (d, l~I) NH; 7.31 (s, 5H) Ph; 73S.(s, ~H) Ph.
Z-Leu-DL~Phe enol ester, the precur~or of Z-Leu-DL-Phe~COOEt was synthesized by the same procedure as descnbed in E~ample PKC1 and puri~ied by column chromatography, oil, one spot on tlc. NMR (CDCl3) B: 0.86 (t, 3H); 0.99 (t, 3H); 1.24 (t, 3H); 1.40 (t, 3H); 152 (m, 2H); 1.83 (m, 2H); 4 23 (m, 4H); 4.39 (q, 2H);
5.10 (t, 2H); ~.18 (s, 1H); 7.26 (m, SH); 734 (m, ~H); 8.89 (s, 1~I).
EXAMPLE PKC:l9 2-Leu-DL Abu COOEt. This oompound was prepared from Z-Leu-Abu-OH in 33 % yield by the procedure de~bed in Example P~C1; oiL one spot on tlc, Rf = 0.66 (CHC13 McOH a 20:1). NMR (CDCl3) ~: 0.96 (t, 9H), OG~2CH3 uld (CH3)2CH; 1.26 (t, 3H), CH2CH2CH3; 137 (q, 2~), OGH2S~H3; 1.66 (q, 2H), (C~3)2~CH2CH; 2.00 (m, lH), CH(CH3)2; 4.12 ~q, 2H) GHGH2CH3; 4-34 (m, 1H) NHCH(CONH)CH2CH(C~H3)2; 3.12 (q, 3H) CE[2Ph and CONH~Et)CHCOCOO; ~.29 (t, lH) NH; 6.79 (d, lH) NH; 735 (s~ SH) Ph.
Z Leu-DL-Abu-enol ester, the precursor of Z Leu-DL-Abu-COOEt was ~gnthes~zed ~y the same procedure as descnbed in E%iample PKC1 and puri~ed by column chromatography, o~, one spot on tlc. NMR (CDCI3) ~: a.ss (t~ 6H); 1.12 (t, 3H); 1.24 (t, 3H); 1.41 (t, 3H); 1.73 (m, 4H); 2.86 (q, 2H~; 4.20 (q, 2H); 431 (m, lH);
4.42 (q, 2H); 5.15 (q, 2H); 5.21 (s; 1H); 734 (m, ~H~; 11.29 (s, lH).
EXAMPI,E PKC20 Ala-DI,I,ys-COOEtHCL To a solution OI N-carbobenylo%yalanyl-Ne-,i 30 carbobenzylo~ylysine (1.88 g, 3.9 mmol~, 4-dime~hylan~inopyridine (21 mg, 0.17 mmol), and pyndine (1.0 rnL 12.4 mmol) in l~ (7 mL) was atded ethyl oxalyl chloride (0.9 mL 8.0 mmol) at a rate sufEicien~ to start refluxing. The mi~ture was refllLxed gently for 3 hr, treated with water (4 mL), and stirred vigorously at room temperature for 30 min.
The ~ ture was extracted with ethyl acetate, the organic extracts were washed with SV~TITLIT~ S~FFT
., ~, .
..
. .
.
u u ~J ~
WO 92/~1850 ~CI/US91/09786 ~^
water, dried over MgSO4 and evaporated to give an oily residue (156 g). To a solution of the enol ester (1.56 g, 2.7 snmol) in anhydrous ethanol was added dropwise a solution of sodium etho~de in ethanol at room temperature until the solution turned ~learyellow. Eth~nol w~c remo~ed and the residue was dissolved in ethyl acetate. The S organic soluaion was washed with water, riried over MgSO4, and evaporated to g~ve a residue. This residue was then purifi d b~ colurnn chromatography and the product was eluted with chlorofonn-methanol. The solvent was remo~ed and Z~ DL-Lys(Z)-C02Et was obt~uned as a hygroscopi~ powder (328 mg, 16 %), single spot on tlc, R~2 =
053 (CEIC13:MeOH = 9:1); MS, m/e = 542 (M~ + 1).
N-Carbobenzoxyalanyl-DL,N~carbobenzo~ylysine keto ethyl ester, ZAla-DL-Lys(Z)-CO2Et (328 mg, 0.61 mmol) ~as deprotected with liquid HF contai~ing anisole at O C for 30 min. The HF was removed under reduced pressure. I~e residual oil wasdissolved in absolute ethanol. HCI/ethanol was added to the solution, and ethanol was removed h vacuo. The residue was washed by decantation with ether to give a semisolid (216 mg, 1~0 %); single spot on tlc (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2).
Bz-DL.-Lys-COOES~ICL This compound wasprepared ~rom Bz-DL-Lys(Z)-COOEt in 62% yield by the procedure descnbed in E1~ample PKC20; one spot on tlc, Rf4 = 057 (n-bu~nol:acetic acid pyridine:H20 = 4:1:1:2). The precursor, Bz-DL-Lys(Z)-COOEt w~s prepared ~om Bz-Lys(Z)-OH in 100% yield by the procedure described in Example PKCl; powder, one spot on tlc, E~f2 = 0.75 (CHC13:MeOH = 9:1); MS, m/e =440 (M ~ ). Anal- Calcd. for C24H2806N2~/3 H20: C, 63.70; H, 653; N, 6.19. Found: C, 63.49; H, 651; N, 5.92.
EXAMPLE PKC~2 3~-DL Arg COOEt X~L This compound was prepared from Bz-DL-Arg(Z)-COOEt in 99% yield by the procedure descn~ed in E;~mple PKC20; one spot on tlc, Rf4 = 0.71 (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2), Sakaguchi reagent positive. Bz-DL-Arg(Z)-COOEt was prepared from Bz-DL,Arg(Z)-OH in 19% yield by the procedure described in E~ample PKC20, R~2 = 038 (C:HG13:MeOH = 9:1); mp 140-142 C; MS, m/e = 468 (M+). AnaL Calcd. for C24H2~O6N4: C, 6153; H, 6.02; N, 11.96. Found: C, 61.96; H, 6.48; N, 1234.
8U13ST~UTE SHE~ET
.~ - . WO 92/1 18~0 2 ~ ~ 8 ~ ~ 9 Pcr/US91/09786 ~5-EXAM[PLE PK~3 H-GI~-DL,Lys-COOEt 2HCL l'his compo~nd was prepared from ZC;ly-DL-Lys(Z)-COO:E~ in 92% yield b~ the pro~edure descn~ed in E~ample PKC20; Rf4 = 0.21 (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). ZC;ly-DL-Lys(Z~-COOEt was prepared from ZGly-Lys(Z)-OH in 9% yield by the procedure described in E~ample PKC20, orle spot on tlc, Rf1 = 0.68 (CHC13:MeOH = 5:1); MS, m/e = 528 (M+ + 1).
~LE PKa4 H PFo-DL~Lys-COOEt21HCL Thi~ compound was prepared from Z-Pro-DL-Lys(Z)-COOEt in 100% yield by the procedure descn~ed in E~ample PKC20; one spot on tlc (n-butanol:acetic acid:~dine:H2O = 4:1:1:2). ZPro-DL-Lys(Z)-COOEt was prepared from ZPro Lys(Z) OH in 15% yield by the procedure desoribed in E;~ample PKC20; Rf2 =
0.73 (CHC13:MeOH = 9:1); MS, m/e 568 (M~ + 1).
H-Ph~DL,Lys-COOEt 2HCI. This compound was prepared f~om ~Phe-DL-Lys(Z)-COOEt in 39% yield by the procedure descnbed in E~ample PKC~0; one spot on tlc (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). Z-Phe-DL,Lys(Z)-COOEt was prepared ~om ZPhe-Lys(Z)-OH as previously de~ ed in 9% yield, Rf2 = 0.68 (C~ICl3:MeOH = 9:1); MS, m/e ~ 482 (M~).
H Leu Ala D~L~ COOEt~HCL Thi~ compound was prepared from Z-Leu-Ala-DL Lys(Z)-COOEt in 52% yield by the procedure described in E~ample PKC20; one spot on tlc (n butand:acetic acid:pyridine:H20 2 4:1:1:2).
~Leu Ala DL,Ly~(Z)~COOEt was prepared from Z Leu-Ala DL-Lys(Z)-OH in S~o yield by the pre~iously described Dakin West reaction, R~3 ~ 0.34 (CHCl3:MeOH =
19:1); MS, m/e = 609 (M~-C)CH2~H3)-S~le Am no Acid, Df- and T~fpeptide Enol Esters (Çeneral Proce~re). A
modi~ied Dakir~-West procedure was used [Charles et aL, J. Che~ Soc Per~ , 1139 - 30 (1980)~ and is illustrated ~nth the sgnthesis of Z-IAu-DL~Phe-EE. To a stirred solution of ~Leu-Phe OH (6.19 g, 15.0 mmol), 4- dimethylaminopyridine (0.183 g; 1,5 mmol) and pyridine (4.75 g, 4,85 mL 60 rnmol) in ~etrahydrofuran (45 ml) warmed 50 C was added ethyl oxalyl chloride (430 g, 352 ml, 31.5 rnmol) at a ra~e su~icient to initiate re~uxing.
I~e rnD~ture was then heated at a gentle refl~ for 4 h. After cooling to room ~1 IR~tTI ITF ~ FFT
. . . .
- -: , WO 92/118~0 PCr/US91/0978t temperature the rni~ture vas treated v.rith water (25 rnl) and stirred v;gorously at room temperature for 30 ~in. rhe rn~xture was ~xtracted with ethyl acetate (150 ml) and after separation of the organic layer, the ~vater layer was saturated with solid (NH4)2S04 and re-e~tracted 2-times v~ith 2~ ml ethyl acetate. The combined organic phases were washed 2-tirnes ~ith 75 rnl water, 2-times with 50 rnl of satd. NaCL decolori~ed with ca~bon and dried over MgSO4. After evaporation of the solvent, the crude enol ester (8,36 g, 98%) was Elash-chromatographed on silica gel and the product wa~ eluted with a AcOEt. The solvent was evaporated in vacuo ~rotavaporator~ and the pure enol ester was obtained as a oil (7.22 g, 85%); single spot on TLC, R~ = 0.84, A; 0.68, C.
Z-Leu-Nva-EE. This compound was prepared from :Z-Leu-Nva- OH using the general procedure and purified by flash chromatography on silica gel using CHC13:MeOH - 50:1 v/v as eluent. Yield 95%, single spot on TLC, Rf = 0.92, C; 0.28 ,L.
Z-Leu-Abu-EE. This compound was p~epared from ZLeu-Abu- OH in 78%
yield the general procedure described above. Purifioation by flash obramatography on silica geL $1uent, CHC:13:MeOH = ~0:1 v/Y, sillgle spot on IrLC, Rf = 0,86, A.
PhCO-Abu-EE. This compound was prepared ~om PhCO-Abu-OH in 26% yield by the general procedure as descnbed above. Purification by flash chromatography on silica gel. Eluent CHC13, single spot on TLC, R~ = 0.60, M.
(CH3)2CH(CH2)2CO-Abu~ This compoundwas prepared from (CH3)2CH(CH2)2CO-Abu-OH in 82% yield by the general procedure as described above. Purification by flash chromatography on silica gel. Eluent AcOEt, sin~le spot on TLC, Rf = 0.72, C.
(CH3CH2CH~)2 CH CO-Abu-EE. 'rhis compound was prepared from (CH3GH2CH2)2CH CO-Abu OH in 100% yield by the general praeedure described above. Purification by ~ash chromatography on silica geL Eluent AcOEt, sin~le spot on TLC, Rf = 0.78, C; 0.81, K
Ph(CH~)6CO-Abu-EE. This compound was prepared from Ph(CH2)6CO-Abu-OH in 86% yield by the general procedure descnbed above.
Purification by flash chromatography on silica gel. Eluent AcOEt. Single spot on Tl C, R~ =0.74, C.
Z-Leu4-CI-Phe-EE. T~s compound was prepared from ZLeu- 4-Cl-Phe-OH in 69% yield by the general procedure described above. Purification by flash chromatography on silica geL Eluent AcOEt, single spot on TLC, Rf = 0.77, C; 0.78, K.
!~:U~TlTlJTF ~ IF1~T
` WO92 2~8~9 /11850 PCI'/US~t/09786 Z-Leu-Leu-~bu-EE This compound was prepared from Z2-Leu- Leu-Abu-OH
in 62% yield by the general procedure descIibed above. Puri~cation by flash chroma~ography on silica geL :~lement CHC:13:MeOH = 50:1 v/v. Single spot on TLC, Rf = 0.89, A; 0.75, M.
Z-Leu-Leu-F'he-EE. ~s compound was prepared from ZLeu- Leu-Phe-OH in 60% yield by the general procedure descnbed above. ~ication by fl~sh chromatography on silica geL ~luent CHa3:MeOH = 50:1 v/v. Sin~e spot on TLC, Rf = 0.80, K; 0.70, M.
2-NapSO~ Leu-Abu-E;E;. This compoun~ was prepared from lQ 2-NapSO~-Leu-Abu-OH in 73% yield by the ~eneral procedure descn~ed above.
Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on TLC, Rf =-0.71, K; 05~, C.
2-Nap50~Leu-Leu-Abu-E;E;. ~is compound was prepared 30m 2-NapSO2-Leu-Leu-Abu-OH in 74% yield by the general procedure descnbed above.
Purification by ~ash chromatography Oll silica gel. Eluen~ AcOEt: AcOH = 200: 1 v/v.
Single spot on II~ = 0.69. K
Z-Leu~he-COOEt. Sulgk Arr~ D~nd Tnpeptide- k~tocste~ (Gerleral Procedure). To a stirred solution of B.53 g (15.0 mmol) of ZLeu-Phe-EE in 40 ml anhydrous ethanol at room temperature was added dropwi3e a solution of sodium ethoxide (0.204 g; 3.0 mmol) in 20.0 ml anhydrous ethanol. The c~lor of the reaction n~i~nlre change from colodess or pall yellow to deep yellow or orange dependent on enol-ester. Then the reaction rni~ure was stirred at room temperature for 4-5 hours, the ethanol was then evaporated in va~uo (rohvaporator) and the residue treated with 200 ml ethyl ether (or 200 ml ethyl acetate in the case of the tripeptide). The ether (ethyl acetate) solution was washed with 2 ~ 75 r~ H~O, 2 ~ 75 JTII satd. NaCl, decolorized with carbon and dried over MgS04. After ~vaporation of solvent, the crude product 6.09 g (89.7%) was flash chromatographed on silica gel using CHC13: MeOH - 50:1 v/v.
Evapora~ion of ~olvent give pure Z-Leu-Phe-COOEt (4.08 g, 58.0%) as a thick oil.Single spot on ILC, R~ = 0.60, A; 0.47, M. Mass spectrum, FB-MS [~M+ 1~/Z3 = 469.
EXA~LE PKC28 2.Leu.Nva.COOEt. ~is was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CEICI3: MeOH = 100:1 v/v, yield 86.6%, thick colorless oil, single spot on TLC, Rf = 0.49, A; 037, M. Massspe~rusn F~3-MS ~(M~1)/Z] = 421.
~Tl ITF .
. . .. - ~ .
:
.
~, ',.
wO92~11850 2~6a~ PCI/US91/097~6^--Abu-COOE~ ~s was prepared by the preceding general pr~edure.
Purification by :~ch chromatography on silica gel, eluent CHCl3, yield 82%, thick pale yellow oil, single spot on TLC, R~ = 0.66, A. Mass spectrum, CI-MS [(M+ 1)/Z] = 407.
PhCO-Abu-COOEt. I~is was prepared by the preceding general procedure.
Puri~cation by flilsh chromatography on silica gel, eluent CHM3:MeOH = 50:1 v/v,yield 83%, oiL sin~e spot on TLC, Rf = 0.44, M. Mass spectn~m, M/Z 263 (M~
CI-MS, 264 ((M+ 1~/Z).
(CH3)2CH(~Ia)2CO Abu-CVOEt. ~s was prepared by the preceding general proeedure. Purification by ~ash chromatography on silica geL eluent AcOEt, yield 43%, oil, sin~le spot on TLC, Rf = 056, C. Mass spectmm EI-MS M/7 257 (M+); FB-MS, [(M+1)tZ3 = 258.
13;XAMPLE PKC32 CH3CH2CH)2CHCO-Abu-COOEt. Ihis was prçpared by the preceding general procedure. Purification b~ flash chromatography on silica geL eluent CHC13:MeOH =
50: 1 v/v, thick, yello~h oiL yield 66%, single spot on TLC, R~ = 0.80, C; 0.66, M.
Mass spectrwn EI-MS M/Z = 285 (M+); CI-MS, [(M+ 1)/Z] = 286.
Ph(CH2)6CO-Abu COOE~ This was prepared by the preceding general procedure. Purification by flash chromatography on silica geL eluent CHC13:MeOH =
50:1 v/v, yield 64~o, pale yellow oiL single spot on TLC, Rf = 0.29, M. Mass spectrum EI-MS M/Z - 347 (M~), FB-MS, [(M~1)/Z3 ~ 348-EXM~LE PKC~4 Z~Leu~CI~Phe~COOEt. Ihis was prepared by the preceding gener 1 procedure.
Purification by ~ash chromatography on silica geL eluent AcOEt~ yield 100%, colorless oil, s~ngle spot on TLC, Rf - 0.71, C. Mass spectrum FB-MS M/Z = 503(M ' ).
EX~MPLl: PKC35 Z-Leu-Leu-Abu-COOE~. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica geL eluent C~ICl3:MeOH - 50:1 v/v, yield 79.2%, very thiclc, colorless oiL sDngle spot on TLC, R~ z 0.28. M. Mass spectrum ~3-MS, l(M~ 1)/Z] = 520.
SllBS~TU~E S~E~E~' .
. . . ..
-~ WO 92/11~50 2 ~ 9 PCr/U~;91/09786 .~9 EXAMPLE P~C36 u-Phe-COOEt. Ihis was prepared by the preceding general procedure.
Purificaeion by flash chromatography on silica geL eluent CHC13:MeOH = 50:1 v/v, yield 33%, oiL single spot on TLC, Rf = 056, M. Mass spectrurn, FB-MS, [(M+ 1)/Z] = 582.
S EXAMtPLE PXC:37 2-NapSO2-Leu-Abu-COOEt. This was prepared by the preceding general procedure. Puri~cation by flash chromato~raphy on silica gel, eluent ~C13:MeOH =50:1 v/v, yield 38%, thick oil, sin~ le spot on TLC, Rf = 0.71, K; 054, A. M~LSS spectrum - F~3-MS, [(M+1)/Z] = 463.
EXAMPLE PK~,8 2-NapS02-Leu-Leu-Abu-COOEt. lllis was prepared by the preceding general procedure. Purification by flash chromatography on silica geL eluent AcOEt:AcOH =
200:1 v/v, yield 61%, serni-solid, sMgle spot on rLC, Rf = 0.67, K Mass spec~rumFB-MS, [(M+1)/Z] = 576.
Z-Leu-Met-C02Et. This compound was prepared by the above procedure.
Yellow oil, sin~e spot on TLC, Rf = O.S2 (C~C13:CH30H=50:1), yield 46% (from dipeptide), MS (FAB) 454 (m+ 1).
E~AMPLE PKC40 Z-Leu-NLeu-C02Et. This compound was prepared by the above procedure. Pale yellow oil, single spot on TLC, Rf = 057 (CHCl3:CH3C)H = 50:1), yield 53% (~om dipeptide), MS (FAB) 434 (m+ 1).
EXAMPLE P~41 Syn~hesis of n-Butyl (~ l Chloride. This was prepared by a liserature procedure [Warren and Malee, J. Chromat. 64, 219-æ2 (197~)]. N Butanol (o.i mol. 7.41 g) was ~
added dropwise to o~alyl cblodde (05 mol. 635 g) at -10 C. After the addition was completed, the reaction mi~ure was stirred for 20 Trun. at r.t. and distilled, giving 15.0 g (91.18 moL 91%) of the product n-bu~l o%alyl chloride, bp 58-60 ~C (0.6 mITI Hg).
Z-Leu-Pbe CO2Bu. This compound was prepared f~om ZLeu- Phe-OH and butyl oxalyl chloride in 43% yield by the procedure described for the synthesis of ZLeu-Phe-CO2Et, e~cept that butyl o~alyl chloride was used in place of ethyl oxalyl chloride and sodium butyloxide in butanol was used for enol ester hydrolysis. Single spot on TLC, Rf = 0.54 (CHCl3:CH30H = 50:1) MS(FAB) m/e = 497 (mt 1), lH NMR
(CDC13? ok.
SUBSTITUTE SHEET
, WO 92/11850 2 ~ 9 8 5 ~ 9 Pcr/u5g1/09786 E:~PIE Pl~C42 Z-Leu-Abu-CO2Bu. This compound was prepared by the above procedure.
Single spo~ on TLC, R~ = 0.53 (CHCl3:CH3C)E = 50:1), yield = 36%, pale yellow oiL
MS (F~B) m/e = 435 (M~ H NMR (CDa3) ok.
EX~MPLE PK~3 Synthesfs of Ben~yl O~yl atlorid~ Ben~yl alcohol (0.15 mol. 16 g) was added dropwise to oxalyl chloride (0.75 mol. 95 g) at S-10 C. After the addition was complete, the reaction was st rred for 20 min. at r.t. Ille es:cess ~alyl chloride was distilled and re~,rcled. Then the mi~ture was distilled under vacuo, gIving 26 g (0.12 mol. 86%) of ben~yl o~alyl chloride, bp. 110-112 C (0.6 mm-Hg3. HlNMR (CDCl3) 7.39 (s, SH), 5,33 (s.2H3.
Z-Leu-Phe-CO2BzL This compound was prepared from ZLeu- Phe OH and benzyl oxalyl chloride in 17~o yield by the procedure described in the synthesis of Z Leu-Phe-CO2Et, e~cept that benzyl oxalyl chloride was used in place of ethyl oxalyl lS chloride and sodium benzyloxide in ?oenzyl alcohd was used for enol ester hydrolysis.
Single spot on TLC, Rf = 0.63 (CHCI3:CH3~:)H = 50:1). Pale yellow solid, mp 117-119 C. MS(FAB) m/e = 532 (m~ 1). H1NMR ok ZLeu-Abu-CO2BzL This compound was prepared by the above procedure.
Single spot on TLC. R~ = 051 (CHC:13:CH30H = SO:1), pale yellow oil, MS(FAB) m/e- 469 (m+ 1), yield = 26%.
~Leu-Phe~COOH. D.~peptide Ketoacids (General Procedure). To a stirred solution of 053g (1,13 rnmol) ZLeu-Phe-COOEt in o.O ml methanol was added 1.27 ml ~25 (1.27 mrnol) lM NaOH. The color of the reaction mixture turned dark yellow and a small amount of solid was deposited. The reaction was run at room temperature and progress of the hydrolysis was checlced on TLC. After 24 h. no more substrate was detectet. The reaction mi~ re was chilled in one ice bath at 5 C, acitified with lM
HCl to pH = 3 and e~tracted with AcOEt (2 ~c 50 mL). The organic extract were washed with 2 x 50 ml H20 and if nec~sary, decolorized with carbon and dried over MgSO4. After evaporation of ~he solvent (rotavaporator3, the residue (thick oll) were titurate~ with 2 ~ ml n-he~ane and dried in vacuo. Yield 0.39 g (78%) of colorless, very thick oil. TLC, main spot at Rf = 024, tra e of impurity at Rf = 0.78, I. Mass spectrusn, FB-MS [(M+ l)/Z] = 441.
.
`~- , . - .
WO 92/11~50 2 a 9 ~ 6 0 9 PCI'/US91/09786 E~MPLE PKC46 2-Leu-Abu-COOH. This compound w~c pr~pared from ZL-Leu- Abll-COOEt in 83% yield by the general procedure as descn~d above; TLC, main spot at ~ = 0.14,trace of impur~ty at Rf = 0.73, I. Mass spe~, F~3-MS [(M~ l)/y = 379-S - . EXAMPLE PKC47 ~L~u-Pb~CONH-Et. To a stirred solution of ZLeu-Phe-OH (20 g, 48.5 rnmole), ~dimethylaminopyridine (0587 6 4.8 mmole),and pyridine ~15.7 ml, 194 rnmole) in anhydrous TElF (100 rsll) was added et}lyl o~alyl chloride tll.4 mL 101.8 mmole3 at a rate sufEicient to ~itiate refllmng. The mi~ture was gentb reflu~ced for 4 hours, cooled to room temperature, and water (80 rnl) was added. The reaction mi~ture was stirred vigorously for 30 min, and e~racted with ethyl acetate (3 x 100 ml). The combined or~an~c layers were washed with water (2 ~ 100 ml), sa~urated sodium chloride (2 ~ lOO ml), decolorized with decolorizing car~on, drie~ over magnesium sulfate, and concentrated, leaving a dark orange oil. Chromatography on a silica gel rolumn with CHCl3/CH30H (50:1 v/v) af~orded 14.63 g (y = 53 %) of ~Leu-Phe-enolester. The product was a yellow oil. S~ngle spot on TLC, R~ = 0.77 (CHCL3/CH30H 50:1). NMR
(CDC13) olc.
To a stirred pale yello~v soludon of the ZLcu-Phe-enolester (14.63 g, 25.73 mmole) in anhydrous ethanol (50 ml) was added a solution of sodium ethoxide (0.177 g, 2.6 rnmole) in ethanol (5 rnl). The orange solution was stirred for 3 hours at room temperature, then the ethanol was evaporated and the residue was treated with ethyl ether (300 rnl). The ether layer was washed with water (2 ~ 100 ml), saturated sodium chloride (2 x lOO ml), dried over magnesium sulfa~e, and concentrated, leaving a orange oil. Chromatography on a silica gel column with CHC 13/CH30H (SO:l v/v) af~orded7.76 g (y - 64 %) of the a-l~etoester Z-Leu-Phe-COOEt. The product was a yellow oil.
Single spot on TI,C, R~ - 0.44 (CHa3/CH30H 50:1). NMR (CDC13) ok. MS (FAB, calcd. for C2ÇH32N206: 468.6), m/e = 469 (M+ 1).
The a-carbonyl group of ZLeu~Phe-COOEt was protected by following procedure. A solution of Z-Leu-Phe-COOEt (1 g, 2.13 rnmole) in 5 ml of CH2Ck wasadded 1,2-ethanedithid (0.214 ml, 2.5~ mmole), followed by 05 ml of boron trifluoride etherate. The solution was stirred o~ernight at room tempera~ure. Water (20 ml) and ethyl ether (20 ml) were added. The organic layer was separated, washed with water (2 x 10 ml), saturated sodium chloride (2 ~c 10 ml), dried over magnesium sulfate, and evaporated to a~ord 0.98 g (y = 84 %) yellow semisolid.
.` :
:. :
v v t~ ~J
wo 92/ll~5~ P~r/ussl/097s6 -- -The protec:ted a ketoester (0.98 g, 1.8 mmole) was dissolved in ethanol (5 rnl),cooled to 0-5 C in a ice bath, and ethylalsline was bubbled through the solution Illltil 2.43 g (54 mmole) had been added. The reaction mi~ture was allowed to warm to room temperature slowly, and stirred overnight. The m~ure was filtered, a white precipitate S was removed, leaving a yellow semisolid. Chromato~aphy on a silica gel column with C~ICl3/CH30H (30:1 v/v) afEord 0.63 g (y = 75 %) of ZLeu-Phe-C:ONH-Et. The produc~ was a pale yellow solid. Single spot on TLC, Rf - 0.60 (CHCl3/C~I30H 20~mp 145-147 C. AnaL cal~. for C26H33N30s 46756; ~, 66.79; H, 7.11; N,839; found:C, 6659; H, 7.09; N, 8.95. NMR (CDCl3) ok. MS (~;AB) m/e- - 468-(M+ 1).
Z-Leu-Phe-CONH-~Pr. This compound was synthes~zed ~om the protected a-ke~oester and propylamine in 92 % yield by the prou~dure desc~ibed in Example PKC47.
Single spot on TLC, Rf = O.S0 (CHCl3/CH30H 50:1); mp 152-153 C. Anal. calcd. for C27H3sN30S: 48157; C, 6733; H, 733; M, 8.72. Found: C, 67.21; H, 738; N, 8.64. NMR
(CDCl3) ok. MS (FAB) m/e = 482 (M+l).
~Leu Phe-CONH-nBu. This compound was ~ynthesized f~om the protected a-ketoester and butylan~ine in 67 ~o y~eld by the proeedure descn~d in E~ample PKC47.
Single spot on TLC, R~ = 050.(GHC13/GH30H 50:1); mp 152-153 C. Anal. calcd. forC28H37N30s: 49S59; C, 67.8S; H, 752; N, 8.48. Found: C, 67.70; H, 7~7; N, 8.43. NMR
(GDCl3) ok. MS (P`AB) m/e = 496 (M+ 1).
EX~MPLli~ PKC50 Z-Leu-Phe-CONH-lBlL This compound was synthesi~ed from the protected a ketoester and Isobutylamine in 53 % yield by the procedure descrl~ed in Example PKC47. S~ngle spot on TLC, Rf = 054 (CHC13/CH30H S0:1); mp lg2 ~G ~n~L
calcd. for C28H3~N305: 49S59; C, 67.85; H, 7~2; N, 8.48. Found: C, 67.77; H, 7~6; N, 8.40. NM~ (CDCl3) ok. MS (~ m/e = 496 (M+ 1).
EXAMPLE PKCSl Z-Leu-Phe-CONH.Bzl. This compound was synthesized from the protected a-ketoester and benzylamine in 40 % yield by the procedure described in Example PKC47.
After reacting overnight, ethyl acetate (60 ml) was added. The mD~ture was filtered to remove a white precipitate. The solution was washed with cooled 1 N HCl (3 x 25 ml), water (1 x 20 ml), saturated sodiurn chloride (2 ~ 20 rnl), and dried over ma~esium sulfate. The solution was evaporated leaving a yellow solid. Chromatcgraphy on a silica SUE~STITUTE SHEET
, ~,. WO92/11850 2~ 9 PCr/US91/097~6 gel column with CHC13/CH30H 30:1 v/v) afforded a yellow solid. Single spot on TLC, Rf = 0.45 (CHCl3/CH30H 30:1); mp 16~162 ~C. AnaL calcd. for C3l~I3sN305: 529.61;C, 7030; H, 6.66; N, 7.93. Found: C, 70.18; H, 6.67; N, 7.99. NMR (CDC13) ok. MS(FAB) m/e = 530 (M+ 1).
~IRu-Phe-CONH-(CH2)2Ph ~is compound was synthes~ed from the protected a-ketoester and phenethylamine in S0 % ~neld by the procedure descn~oed ~n ~ample PKC51. Sin~e spot on TLC, Rf = 050 ~GEIC~3/CE30H 30:1); mp 151-153 ~C.
AnaL calcd. for C32H37N305: 543.66; C, 70.70; H, 6.86; N, 7.73. Found: C, 70.54; H, 6.88; N, 7.74. NMR (CDCI3) o~ MS (FAB) m/e = 544 (M+ 1).
~L~u-Abu-CONH-Et. This compound was sgnth~ized ~am protected a-ketoester derived ~om Z-Leu-Abu-C02Et and ethylamine in 64 % y~eld by the procedure descn~ed In E:cample PKC47~ Single spot on TLC, Rf = 0.36 lS (CHCl3/CH30H 50jl); mp 130-132 C. AnaL calcd. for C21H31N305: 405.4~; C, 62.20;
H, 7.71; N, 1036. Found: C, 61.92; X 7.62; N, 1031. NMR (CDC13) ok. MS (FAB) m/e ~ 406 (M+1).
EXAMPL~: PXCS4 Z-Leu-Abu-CONH-IlPr. This compound ~vas sgnthe3ized ~om lhe corresponding protected a-ketoester and propyl~e in 47 % yield by the procedure descnbed in Example PKC47. S~gle spot on TLC, E~f = 0.28 (CEIC13/CH3OH 50:1); mp 134-135 C.
Anal. calcd. for C22H33N30s: 41950; C, 62.98; H, 7.93; N, 10.02. Found: C, 62.84; H, 7.97; N, 9.94. NMR (CDC:13) ok. MS (FAB) m/e ~ 420 (M+ 1).
Z Leu Abu CONH n~lL This co~npound was ~9nthesized ~om the corresponding prote ted a-ketoester and butylamine in 42 ~o yidd by the procedure desc~bed in E~ample PKC47. Single spot on TLC, Rf = 054 (GHC~3/~I3OH 50:1); mp 135-136 C.
Anal. calcd. ~or C23H3sN30s: 433~3; C, 63.71; H, 8.13; N, 9.69. Found: C, 63.48; H, 8.07; N, 9.67. NMR (cDa3) ok. MS (FAB) m/e = 434 (M+ 1).
EXAMPI,E PKC~6 Z-Leu.Abu-CONH.iBu. This compound was synthes~7ed :~om the corresponding protected a-ketoester and isobutylam~ne in 6~ % yield by the pro~edure described in Example PKC47. Single spot on TLÇ, Rf z 0.:;~ (CHC13/CH30H 50:1); mp 133-135 ~C.
~3uBsTlT~lTE S~EET
.: .
, . . . ~ . . . .
W0 92/1 1850 2 ~ 9 8 6 ~ 9 PCT/U~9t/09780~ '`
Anal. calcd. for C23~I3~N30s: 43352; C, 63.72; H, 8.14; N, 9.69. Found: C, 63.46; H, 8.10; N, 9.60. NMR (CDCl3) o~ MS (FA13) m/e = 434 (M+ 1).
E~MPLE PKC57 ~Leu-Abu-CONH-BzL This compound was synthesized from the corresponding protected a-ket~ester and bes~ylamine in 29 % yield by the procedure described in E~ample PKC51. Single spot on TLC, Rf = 056 (CHCl3/CH30H 30:1); mp 140-141 C.
Anal. calcd. for C26H33N305: 46754; C, 66.79; H, 7.11; N, 8.99. Found C, 66.65; H, 7.07; N, 8.93. NMR (CnCl3) ok. MS (FAB) m/e = 468 (M+ 1).
~Leu-Abu-CONH-(CH2)2Ph. This compound was synthesized from the corresponding protected ~-ketoester and phenethyla~ne ~n 51 ~o yield by the procedure descri~ed in Example PXC51. Single spot on TLC, R~ = 0.44 (CHC13/CH30H 30:1); mp156-157 C. AnaL calcd. for ~7H35N30s: 48159; C, 6734; H, 733; N, 8.72. Found: C, 67.38; H, 733; N, 8.78. NMR (C~DC13j ok MS ~A13) m/e = 482 (M+ 1).
Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)20. This compound was synthesized from protected a-ke~oester and 4(3-aminopropyl)morpholine in 33 % yield by the procedure described in E~ample PKC47. After re:acting oven~ight, ethyl acetate (80 ml) was added.
The m~ture wæ filtered so remove a w~ite precipitate. Ihe solusion was washed with water (3 ~ 20 ml), saturated sodium c~loride (2 x 20 ml), and dried over magnesiurn sulfate. The solusion was evaporated leaving a yellow oil. Chromatography on a silica gel column with ~Ha3/CH30H (10:1 v/v) afforded a yellow sen~solid, which was recrystallized from ethyl acetate/h~ane to obtain a pale yellow solid. Sin~le spot on TLC, Rf - 0.42 (CHa3/CH30H 10:1); mp 12S-126 C. Anal, calcd. for C~6H40N406:
504.63; C, 61.88; H, 7.99; N, 11.10. Found: C; 61,69; H, 7.95; N, 11.07. NMR (CDCl3) ok. MS (FAB) m/e - SOS (M+1).
EX~MPLE PKC60 Z Leu Abu-CO~H-(CH2)7CH3 l~is compound was synthesized from the correspondjng protected a-ketoester and ocqlamine in 67 % yield by the proceduredescribed in E.sample PKCS1. It was white solid. S ngle spot on TLC, R~ = 0.55 (CHC13/C~I30H 30:1); mp 134-135 C. AnaL calcd. for C2~H43N305: 489.66; C, 66.23;
H, 8.8S; N, 8.58. Found: C, 66.19; H, 8.81; N, 8.61. NMR (CDCl3) ok. MS (FAB) m/e = 490 (M+ 1)-SUBSTITUTE SffE~
: .
WO 92/11~S0 2 ~ 3 8 ~ ~ 3 PCI/IJS91/0978~
E~LE PKC61 Z-Leu-~bll-CONH-(CH2)20H. Ihis wmpound w~ synthesized ~om the corresponding protected a-ketoes~er and ethaIlolamine in 29 % yield by l:he procedure descnbed in Example PKC59. The product was a whi~e sticky sclid. Single spot on S TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 151-153 C. Anal: calcd. for C21H31N306:
421.49; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11; H, 7.M; N, 9.81. NMR (CDCl3) ok.
MS (FAB) m/e o 4æ (M+ 1).
Leu-~bu-CONH~ I2)20(C~I2)20~. This compound was synthes~ed i~om the corresponding protected a-ketoesser and 2-(2-aminoetho~y)e~hanol in 34 % yield by the procedure descrl~ed in E~ample PKC59. The product was white sticly solid. Single spot on TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 103-105 C. Anal.: calcd. for C23H35N307: 465.35; C~, 5934; H, 758; N, 9.03. Fow~d: S~, ~9.23; H, 758; N, 9.01. NMR
(CDC13) ok. MS (FAB) m/e = 466 (M+1).
L4u Abu-CONH-(CH2)17C~3. ~is compound was synthesized ~om the corresponding protected a-ketoester and oc~adecylamine in 12 % yield by the procedure described in E~nple ~KC~ e product was a pale yellow solid. Single spot on TLC, Rf = 054 (CHCl3/CH30H 30:1); mp 134-136 C. Anal: calcd. for C37~I63N30s: 629.92;
C, 7055; H, 10.08; N, 6.67. Found: C, 70.71; H, 10.14; M, 6.75. NMR (CDa3) ok. MS
(FAB) m/e = 630.2 (M-t 1).
Z L.eu-Abu CONH CH2 C6H3(0CH3)2. Thi~ compound was synthes~zed ~om the corresponding protected e ketoester and 3,5 dime~ho ~ybenzylarsline in 45 % yield by the procedure descnbed in l~ample PKC~1. Ihe product w~ yellow sticky soIid. Sin~le spot on TLC, R~ = 0.44 (CHC~3/CH30H 30:1); mp 153-1~5 C. Anal.: calcd. for C28H37N307: 527.62; C, 63.74; ~, 7.07; N, 7.96. Found: C, 63.66; H, 7.09; N, 7.92. NMR
~CDC13) ok. MS (FAB) m/e = 528.8 (M+ 1).
EXAMPLE PKC6~
2~Leu-Abu-CONH CH~ C4H4N. ~is compound was s5nthesized from the corresponding protected a-ketocster and 4-(aminomethyl)pyridine in 45 % yield by the procedure described in E~ample PKCS9. The product was ~eenish yellow solid. Single spot on TLC, Rf = 05S (CHC13/GH30H 10:1); mp 124-126 C. A~ calcd. for . ,.. ,: . , . ~ . .
wo 9~ 850 2 0 9 ~ ~ Q 9 PCI/US9l/0978~
C25H3~N40s: 468.55; C, 64.08; H, 6.88; N, 11.96. Found: C, 63.88; H, 6.87; N, 11.96.
NMR (CDCl3) ok. MS (FAB) m/e =469 (M[+1).
D. HALO-~TONE P~ES
Halomethyl ketone peptides are irreversl~le inhl~itors for serine proteases and S cysteine proteases. This elass of compounds includes peptides having a variety of halomethyl groups at their C-tern~nus. These halomethyl groups include CH2X CHX2 and CX3, where X represents any halogen. A number of analogous compounds have been synthesized, induding the amino-halo ketones and the diazo-ketone peptides.Although these analogous oompoi~nds are chemically distinguishable, all of thesehaloketone compounds are believed to have a si~slilar mechanism OI aetion. Accordingly, for simplicity, all of the foregoing compounds will be referred to collectively herein as the "Hal~Ketone Peptides."
The reactivity of haloketones has generally been found to be in the order I ~ Br> Cl > F. However, incre~cing the react~ity of the haloketone in this way can lead to acceleration of competing side effects. Thus, it is preferable to incre~se the reactivity of the halomethyl ketone peptides by altering the peptide structure.
In selecting a proper inhibitor for Calpain, the same basic peptide structure selection techniques as used for the Peptide Keto-Compounds can be used. Once a - peptide structure has been identified, the most effecti~e C-tern~inus grouping can be empirically detennined through kinetic inhl~ition studies of each of the compounds with Calpain.
Many of the Halo-Ketone Peptides are available commercially For e~ample, Leu-CH2CL Phe-CH2CL Z-lys-CH2CL Tosyl-LysCH2Cl (TLCK), Tosyl-PheCH2Cl (TPC~), ZGly-Leu-Phe-CH2CL Z-Phe-Ala-CH2CL z-Phe-Phe-CH2Cl, D-Phe-Pro-Arg-CH2Cl, MeoSuc-Phe-Gly-Gly-Ala-CH2CI, MeoSuc-Ala-Ala-Pro-Ala-CH2CL MeoSuc-Ala-Ala-Pro_Val.CH2CL Ala-Ala-Pro-Val-CH2CL Ala-Ala-Phe-CH2CL Suc-Ala-Ala-Pro-Phe-CH2Cl and D-Val-Leu-Lys-GH2CI are all available from suppliers such as En~ne Systems Products of I~vennore, California. From the same suppliers, thz following diazomethyl ketone peptides are available: Leu-CHN2, Z-Phe-Phe-CHN2, Z-Phe-Ala-C~IN2, ZPhe-Pro-CHN2, ZLys-CHN2 and Giy-Phe-CHN2. In addidon, the production of a-amino fluoro ketone pepddes has been described in United States Patent No. 4,518,528 to David W. Rasnick the disclosure of which is hereby incorporated by this reference.
Su~ 1TlJfE 5~EFr ..
A,; WO 92/11850 2 ~ ~ ~ 6 0 9 PCr/US91/09786 l~e preparation of various ~lo-Ketone Pep~ides is reviewed in kIethods in E~, 46:197-208 (1977), the disclosure of whieh is hereby incorporated by reference, Brie~y, halomethyl ke~one derr~a~ves of blocked ~o acids are read~y prepared by the reaction of n~neral acid~ (hydrohalic) with the corresponding 5 diazomethyl ketone. Iodomethyl ketones are prepared by re~ctisn of a halo-ketone with NaI, since reaction with HI with a di~omethyl ketone yields ~he rnethyl ketone. A
3umber of different bl~ng ~oups can be used, including benzylo~ycarbonyl (Z) arld t-butylo~ycarbonyl (BOC). The ~iazomethyl ketone is prepared by reactis~ of diazomethane with the appropriate acid ~a~ed by means of dicyclohe1ylcarbodiiTnide 10 (~ -CI), by the mixed anhydride method.
Unbloc3ced asl~ino acid chloromethyl ketones can be prepared by reaction of benzyloxycarbonyl blocked derrvatives wi~h Br or HOAc, trifluoroacetic acid, or by hydrogenation.
Synthesis of peptide chloromethyl Icetones can be accomplished simply by coupling an appropriate peptide or amino acid with an unblocked an~ino acid chloromethyl ketone. A few dipeptides can be converted directly to the chloromethyl ketone using the n~Lsed anhydride and CH2N2 foll~wed by HC:L
Vanous synthetic problems are encountered in the preparation of chloromethyl ketone derrvat;sves of basic amino acids. I~e side chain usually must be blocked during 20 ~ynthesis, and di~iculties are often encountered dunng removal of the blocking group.
Use of trifluoroacetic acid or HF was eYentually found to give a good conversion ~o product.
A number of exarnples of the preparation of Halo-Ketone Pepddes have been reported in the literature, including a comprehensive re~e v of over 100 amino acid 25 derivatives and appro~natley 60 peptide derivatrves Listed in J.C. Powers, in aChen~istry and Biochernistry of Anuno Acids, Peptide~ and Prote~ns,~ Vol. 4, Dekker, New York (1977), the disclosure of which is hereby incorporated by reference. Those of skill in the art will recognize how to locate a multitude of e~amplæ of the produstion of the Halo-Ketone Peptides. Acoordingly, no addidonal G~nples are pro~rided herein.
30 ~. V~USES
In addition to the foregoing classes of compounds now discovered to possess Calpain inhl~itory activity, we ~elieve that a large number of other such compounds exist. In vie~v of the large number of inhibitors of Calpain of different classes we SuBsr~ u~rr . ~
. ', -;. .. ,.: ~ . -.
W092/11850 2~9~a9 PCr/us91/o97~li disclose herein, all of the h~own, newly discovered and yet undiseovered ~nhibitors of Calpain shall be referred to hereinafter collectively, using the term "Calpain Lnhibitor.~
The Calpain Lnhibitors may be used vi~o for a variety of purposes to inhibit unwanted Calpain ac~vity. For e~ample, the Calpain Inhibitors may be used in vitro to S prevent proteolysis that occurs in the process of production, isolation, purification, storage or transport of peptides and proteins.
The Calpain Inhibitors descnbed herein ca~ also be used vitro to prevent further degradation of ~issue sarnple3 ~om oecurring a~er preparation of the samples.
This vitro prevention of degrad~tion can ~e esp cially useful in the preparation of assays for neurodegenera~ion where~n the ass~y comprises a test for the products of Calpain activity in the tissues, such as assays for breakdown products (BDP's) of cytoskeletal eomponents such as spectrin, MAP2, actin binding protein and tau. P.
Seubert et aL, in Neur~science~ 31:195 (1989), the di~closure of which is herebyincorporated by reference, disclose an exemplary method of quantitating the amount of 1~ spectrin BDP's as an indication of Calpain activity.
The ~alpa~n Is~ubitors of this mvention are also useful in a variety of other experirnental pro~edures where proteolys~s due to Ca4ains is a signifiunt problem. For example, inclusion of the Calpain Inhibitors in radioin~nunoassay exp~riments can resul~
in higher sensiti~nty. The use of the Calpain In}~ibitors in plasma fractionation procedures can result m higher yields of valuable plasma proteins and un make purification of the proteins easier. The C~alpain Inl~ibitors disclosed here can be used ~n clomng experiments utilizing recombinant or transfected bacterial or eukaryo~ic cell cultures in order to increase yield of purified recombinant product.
To use the Calpain ~ibitors in vitro. the Calpain Inhibitors are dissolved in an25 ~ organic acid, such as d~methylsulfo~ide (DMSO) or ethanol, and are added to an aqueous soludon containing the protease which is to be inhl~ited, such that the final concentration of organic so}vent is 25% or less. The Calpain Inhibitors may also be added as solids or in suspension.
F. TREAl'MENT OF ~EURODEGENE~ATIOI~
We have discovered that the Calpain Inh~bitors are use~l in vivo to treat pathologies in which e~cess proteo~sis by Calpains is involved. Such pathologies are believed to include neuropathologies such as neurodegeneration resulting from excitotoxicity, H~V-induced neuropathy, ischernia, denenation, injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD), SU135T~UTE SHEEr . .
wo 92/11850 2 ~ 9 ~ PCl/U591/09786 Hun~ington's Dise~se, surgery-related brain damage, Parkinson's Disease, and other pathological conditions.
1. Identification of Inhibi~ors In order to identify Calpain Inhl~itors that are useful in the practice of the present invention for treatment or inhl~ition of neurodegenera~ve conditions anddiseases, it is important to identi~ ~hose inhl~itors posessing sigrlificant Calpain inhibitory a~vity. It is also import~nt to identi~ those Calpain Inhibitors having a high degree of specificity for inhib n of C~ain, in order to aYoid interference with other biological processes when the Calpain Inhl~itor is introdu~d into a mammal requiring treatment for neurodegeneration. Because all thiol proteases are believed to e~er$ their effect through a similar mechanism ~ action, our prirnary concern ~as to identify those Calpai~ itors having substantial inhl~itory act~ity against Calpain, but relatiYely weak or no ac~vity against other thiol protease~. Accordingly"n order to identify such Calpain Inhibitors, we tested a variety of Calpain Inhl~itors for their ability to inhibit calpains I and II, and compared thls data with the ability of the same Calpa~n Lnhibitors to irlhibit Cathepsin B, another thiol pro~ease. Ihose Calpain Inhibitors with high vitro inh~itory acsivi~r against Calpain and a relative~y lower activiq against S ~athepsin B
are believed to ~e most useful for ~vno therapy. E~arnples lA through lC show the results of these studies for a variety of Calpain Inhl~itor~.
EXAMPLE 1~
InhibitiQn b~ Substitused ~terocyclic C-ompounds The isocownarins are irreve~sible inhl~itors of Calpain. We obtained ICso valuesfor a variety of these Calpa~n Inhibitors as a kinetic analysis of these compounds.
Purified Calpains can be assayed using the fluorogenic substrate succinyl-leucine-2S ~ros~ne-methylarninocownarin (available commercially) or by measuring the release of acid-soluble peptides from casein bec~use we have found that the isocoumarins inhibit casein proteolysis by Calpain.
Calpains I and ~ were purified by the method of (Yoshisnura, et aL 1983).
(Kitahara 1984) provides an altemative puri~cation scheme. Calpain II may alternatively be purchased from Sigma Chemical Co. as "Calcium Activated Neutral Prote se.~ Ln this assay, purified Calpain was incubated with l4C-methylated casein in the presence of various H[eterocyclic Compounds and the amount of acid-soluble radioactivity released by the action of Calpain was measured. The ICso values were determined as the ~ ~o~!~rlT~ S~
WC) 92/11B50 2 0 9 8 6 ~ 9 PCI~/US91/09786~;
concentration of Heterocyclic ~mpound compound at which SO~o of the Calpa~n activity was inhibited. Table ~A shows I~ values for various Isocoumar~n Compounds.
5UB~m~ SH~E~
.:
.
.-, WO 92/1 1~50 2 ~ 9 8 6 ~ 9 PCI~US91/09786 ~ABLE L~
INH~llION OF CALPAINS BY SUlBSTl~D ISOCOUMARINS
~LCs~ (UM! -CalpainI Calpa~n tI
ClTPrOIC 100 70 ~`
NH2-CiTPrOIC (ACl~IC) 10 120 ~12NHCONH-CiTPrOIC 80 30 CH3CONH.Cl~PrOIC 700 80 L-Phe-NH-Cl~PrOIC - 30 BOC-L~Phe-NH-CilPrOICno inhl~ition ~200 PhCH2NHCONH-Cl~EtOIC 90 PhC~I2CONH-CiTEtOIC . 30 D-phe~NH-CiTEtOIC 200 I?lus, it can be seen from Table ~ that a variety of the Isocoumarin C:ompounds have significant ~alpain inhibitory activity at low concentrations.
EX~MP~ lB(I) Prote~se~nhibition by Peptide Keto-Compounds The Peptide Keto-Compounds are reYersl~le inhibitors of Calpa~ns and other 20 thiol proteases. The }4 values for the inhibition of calpain I, calpain Il and Cathepsin B
were detern~ned for several Peptide Keto-Compounds, Inhibition of calpain I ~om human erythro~es and calpain II from rabbit musde were assayed using Suc.Leu.Tyr.amidomethylcoumarin as substrate in an assay buffer of 20mM HEPES
pH=7.2, lOmM CaC12, lOmM ~mercaptoethanol. Cathepsin B from bovine spleen was assayed us~ng Z-Lys-4-nitrophenylphosphate as substrate.
Table lB(i) shows the results of the studies of E:sample lB(i). The Ki value forthe inhibition of Calpains and cathepsin B by several Peptide Keto-Compounds areshown in ~lM (micromolar). The values for leupeptin, which is cornmercially available from Calbiochem of La Jolla, Califonua, are shown for comparison.
C!~ S~E~T
.
.
.. . .
WO 92/1 1850 2 ~ 9 8 ~ o 9 PCr/lJ!i91/0978 Tabl~ lB(i) Kj YALUES FOR PEPTIDE KETO-COMPOUNDS
Inhibitor Calpain I Calpain II Cathepsin B
Leupeptin 032 0.43 6 ~Ala-Ala-Ala-C02Me 200 ~ 1.5 ZAla-Ala-Abu-CO2Et 50 200 0.9 ~Leu-Phe-C02Et 0.23 0.4 ~ 50 Z-Leu-Me-CO2Et 0.12 0.18 i8 ZLeu-Abu-C02Et 0.04 0-4 ld, ZLeu-Nva-COO~t 1.2 30 It can be seen ~om the results in Table lB(i) that the Peptide Keto-Compo mds inhibit C:alpain with Ki values similar or superior to leupeptin. In particular, Z-Leu-Phe-C02Et, ZLeu-Nle-C02Et and Z-Leu-Abu-C02Et were found ~o possess ~eater Calpa~n inhibitory actiYity than leupeptin. In addition, these partiallar compounds were highly specific to Calpain, with lo ver inl~ibitory act~vity toward Cathepsin B than leupeptin.
EX~LE lB(~) otease Ir~ibition o~ Peptide Keto-ComFour~ds We tested the ability of an additional group of Peptide Keto-Compounds to inhibit several proteases in order to e~aluate their specificity for Calpain. The results of these studies are shown ln Table lB(ii).
S~STITUTE SHIE~
.: .
~ wog2/..g50 2~ 3 Pcr/US91/09786 Table lB(i~). Inhibition of Calpa~n I, Ca~ain II, Cathepsin B, PP Elastase ~nd Papain Inhibitor Kl( ~M) . __ . .
Calpain I Calpai~ II CathB Ch~m elasta papa~n ~e ,..... ... .. _ . ~ ..
Z-L~u-Abu-COOEt 45 0.4 30 ~100 ~100 220 . _ . _ . . . . , _ __ .. ___ Z Leu Abu COOnBu 1~8 4 ~ lû0 25 10 _ . ~
Z-Leu-Abu-COOBz . 95 0.47 4 40 > 100 40~ _ _ ............................................. Y . . _ Z-Leu-Lek .~bu-COOEt 1~8 2~6 ~2 > 100 25 .
2-NapSO2-Leu-Leu-Abu-COOEt 16 1.4 25 35 47 _ _ , , . . . . . .
2-N~CO-Le- - _eu-Abu-COOEt 0.09 > 300 28 I _ . _ . .
T~s-Leu-Leu-A~u-COOEt 33 69 25 28 I_ _ _ . _ .
Ph-(CH3)2-CO-Leu-Abu-COOEt 1.2 . _ .- , ..
Z-Leu-Abu-COOH 0.075 0.022 1.5 > 150 > 150 I _ _ _ Z-Leu-Abu-CON~t 05 0.23 2.4 > 150 65 _ ~ __ __ Z I~u~Abu CON~:Pr 0~5 8 >300 2 I... _ . _ __ Z-Leu-Abu-CONHnBu . 0~ 13 ~ 300 5 I .. _ _ _ . ,__ Z-Leu~Abu~ ONHiBu 0.14 4 >300 40 . -I . _ _ _ .
Z-Leu-Abu-CON~ 035 2 > 300 1 . . . ., Z-Leu-Abu-CONH-tC~ 2-Ph 0.022 I - _ _ _ _ _ __ Z-I~-Abu-CONH-(C~I2)3-Mpl 0.041 ~ . . _ _ Z-Leu-Abu-CONH-(CH2)~I3 . 0.019 , ._ _ _ ,, , _ . .
Z-Leu-Abu-CONH-(CH2)20H 0.078 _______ _._ _ SUBSTITUTE SHEET
;. . ~ . .
- ~ .
` , ;~
2~3860~ ,'.`
WO 92/11850 PCl/US91/09786- -, ~ =
¦ Inhibitor ~(I-M) I . . _ . . . . ..
Calpa;n I Ca~ain II Ca~hB Chym elasta papa~n ~ se I . _... . _ _ _ . __ _ _ ___ ¦ Z-Leu-Abu-CONH- 0.16 ¦ (CH2)2t)(~H2)2oH
_ _ __ _ . ~
Z-Leu-Phe-COOEt 1.8 0.4 340 0.025 > 100 75 _~ =.__~ . . ~ . - __ _ _ _.
Z-Leu-Phe-COOnBu 5.0 1.1 15 0.15 > 100 15 Z-L~-Phe-COOBz 3.4 1.6 45 1.6 > 100 4~
: , .,, __ .
Z-Leu-Leu-Phe-COOEt 1.4 _ 42 0.26 > 100 15 Z-Leu-Phe COOH 0.0085 0.0057 45 76 > 150 ~ ....................... . _ Z-Leu-Phe-CON~t 7.0 032 6 73 ~150 ___ . . . _ __ ,, Z~Leu Phe-CON~ 15.0 0.05 3 18 ~300 I _ . _ _ Z-L~-Phe-CON~Bu 0.028 3 8 ~100 I _ . .. _ _ . . ... . .. I
. Z-Leu-Phe-CONHiBu 0.065 4 24 _ . _ _ Z Leu-Phe-CONHBz 0.046 I ......... _ _ . _ _ ~: Z-Leu-Phe CONH(CH2)2Ph 0.024 (2) I _ _ . __ Z-Leu-Me-COOEt 0.18 20 2.2 190 I . ....... _ _ ..
Z.T ~--~va-COOEt 1.4-- - -1.2 25 160 23 150 . I . . .
Z-Leu-Met-COOEt 1.0 15 55 1.75> 100 140 I _ , _ . . -Z-Leu-4-Cl-Phe-COOEt <4.0 0.4 ~0 0.9> 100 150 . !~ ~ _ _. _ _ . _ ., =
SUE~STlTUTE~ JF'FT
. .. ~.. :. , .
, , ' WO 92/11850 2 n ~ 9 PCr/VS91/09786 -8~-Table lB(ii) shows the inhibition Qnstants (KI) for cathepsin B, calpain L and calpain II with peptide ketoamides. Dipeptide Ketoamides with Abu and Phe in the P
site and Leu in the P2 site are pot~nt inhibitors of calpain I and calpain Ir. ZLeu-Abu-CONH-Et is a better inhibitor of calpain I than ZLeu-Phe-CONH-Et by 14 fold.Replacement of the Z group (PhC~I20CO-) by similar groups such as PhCH;2CH2CO-, PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor structures. The best irlllibitor of calpain II is ZLeu-Abu-CONH-(CH2)2-Ph. Changing the R3 and R4 groups significantly improv~s the inllibito~y potency toward calpain II.
I~e ~ Dipeptide Ketoamide inhibitors are those which have long allyl side chains(e.g. ~,Leu-Abu-CONH-(CH2)7CH3), al}yl side chains with phenyl substituted on the alk~ Proup (e.g. Z-Leu-Abu-CONH-(CH~)2-Ph), or a aL~yl groups with a morpholine rir.& 9s,l~stituted on the alkyl group [e.g. Z-Leu-Abu-CONH~ I2)3-Mpl, Mpl = -N(CH2CH2)20]. Dipeptide a-ketoamides with a small aliphatic amino acid residue or a Phe in the Pl site are also good inhi~itors for ca~hepsin B. The best inhibitor is z Leu-Abu-CONH-Et and replacement of the ~ (PhCH20CO-) by PhCH2CH2CO-, PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor structures.
EXAMPLE lB(lil) Stabilitv of Peptide Keto~ompQunds We determined tbe half-life of several Peptide Keto-Compounds in both plasma an~ liver homogenates. The results of the determinations of stabiliq of the compounds in plasma and liver homogenates are sho~n in Table lB(iii).
SUE3STITUTE SI~EET
.: , - . .
.. .
:
~v~ u~
WO 92J11850 pcr/us9l/n9786 -~6-Table 1B(~ii). S~bility 3~ Plasma and in I,i~er o~ Pep~ide Keto-Compolmds.
Inhibitor tl/2 . li~er ~ __ _ S ~Leu-Abu-COOEt 2 . . . ..... _ . . , .. ..... .
2-NaEISO2 Leu Leu-Abu COOEt ~ 60 I - . .
2-NapCO-I,eu-1 eu-Abu~C(:30Et 25 ~__ __ Tos-Leu-Leu-Abu-COOEt 30 Z-Leu-Abu-COOH ~ 60 ~60 ~ __ Z-Leu-Abu-CONHEt ~ 60 ~60 I _ Z-Leu-Abu CONHnPr ~ 60 ~60 l ~Leu-Abu-CONHnBu ~ 60 ~60 ¦
_ __ Z-Leu-Abu-CONHlBu ~ 60 _ ZLeu-Abu-CON}~z ~ 60 ~li0 l I .......... . . . . , .
Z-Leu-Phe-COOEt 7.8 I __ I
Z-I~!u.Phe.COOnBu 7.7 . ............ . , . _ u-Phe-COOBz 1.9 ZLeu-Phg-COOH :- 60 ~60 , _ Z-Leu-Phe-CONHRt ~ 60 ~60 . 11 ~Leu-Phe-CONHnPr ~ CO ~60 ¦
I _ .Il ZLeu-Phe-CONHnBu ~ 60 ~60 . . 11 . LeuRhe-CONHlBu 60 _¦
Z-Leu-Ph~-CONH(CH2)~Ph > 60 I . _ Z-L~u-Nle~COOEt 3.7 5l~13Sl~TE SHEET
... . .
: ~ ' - ~ WO 92/11850 2 ~ 0 9 PCr/US91/09786 Illhibitor tll plasma li~er l . _ . . I
Z-Leu-N~a-COOEt 2.8 __ ..
Z~Leu-Met-COOE;t 8 _= ..
It can be seen from the data in Table 1B(i-i) that ~he Pep~ide Keto-Compounds S are generally quite stable in plasma and l~ver homogenates. However, it is also shown that the Peptide a-ketoamides v ere substantially more stable in both plasma and liver than the corresponding peptide a-ketoesters otease Inhibition bv Halo-Xetone Peptides The Halo-Ketone Peptides, ~ike the s~stituted isocoumarins, are irreversible inhibitors of Calpa n. We detern~ined the X8pp/[I~ ~alues for various members of ~his class of compounds aga~nst Calpains I and IL ~or comparison, we also determined these values against the addi~ional thiol proteases Papain and Cathepsin B for at least one Halo-Ketone Peptide. These Ka~p values are not directly comparable to the Ki or ICSo values determined abo~e for other classes of inhibitors.
We assayed Calpain I and II using Suc-leu~ amidomethylcoumarin. Papain was assayed using benz~yl-arg-4-nitroanilide, and Cathepsin B (bovine) ~as assayed using CBZ-lys 4-nitrophenyl ester. We followed the progress cun~e method of Tian and Tsou, Biochemi~trY~ 21:1028-1032 (1982), the disclosure of which ~s hereby incorporated by reference, to derrve l~netic data. Briefly, this method makes use of the equation:
Vls]/K
(1 + [S]/K)A[~
where [PJ represents the concentration of product formed at a tilTle approachinginf;ni~, A is the ~,p in the presence of substrate (S), K is the Michaelis constant and 2~ ~Y3 is the concentra~ion of the inhibitor. Since lS~ and [Yl are known and V and K can be deterrr~ined, ~,p can be readily deterrnined.
The Kapp/~ for ~arious Halo-Ketone Peptides are shown in Table lC.
c ~ IR.~::TITI ITI~ C!LI''E:T
~: .
WO 92/11850 2 0 9 8 ~ 0 9 PCT/US91/09786 . :^
-8g-TABLE lC
KINETIC P~RAh~RS OF Halo-Keto~ Peptides Inhibitor CI C:~13 P CB
ZGly-Leu-Phe-CH2C1 2840001946000 Boc-Gly-Leu-Phe C~I2C1 902000 ~40000 290000 ZLeu Phe-~I2Cl ~oo02 585000 Z-Gly-Leu-Ala-CH2Cl 210000 Ac Leu-Phe-CH2C1 259001 33400 lQ Z-Val-Phe-CH2Cl 27200 ZAla-Phe-CH2C1 2400 Ac-Ala-Ala-Ala-Ala-CH2C1 1300 CI = Calpain I l - Rat CII = Calpain II 2 Human P - Papain 3 - Rabbi~
CB = Cathepsin B
It can be seen from the results in Table lC that the Hal~Ketone Peptides inhibit Calpain with relatively high Kapp/[I values. In particular, Z-gly-leu-phe-CH2Cl, Boc-gly~leu-phe.C~I2C:I, Z-leu-phe-CH2Cl and ~gly-leu-ala-CEI2a were found to possess significant Calpain inhl~itory ac~vity. In addition, Boc-gly-leu-phe-CH2Cl was shown to be somewhat specific to Calpain, with lower inhibitory acti~.~ity toward Cathepsin B or Papaln than toward Calpain. The results shown in the table reveal that Z gly-leu-phe.CH2Cl and Boc-gly leu-phe-CH2a produce similar inhibitory effec~s. - -Thus, the blocking group is not shown to have a great effect on Calpain inhibitory ac~vity.
The kinetic constants of other irreversl~le Cal~ain Inhl~itors include the following with Kapp/W in parentheses: E 64 (7500), E64-d (23000~ and Zleu-leu-~yr-CHN2 (230000). E-64 is cornrnercially ava~able from Sigma Chemical Co., and is shown here to be a poor inhibitor of Calpain. ~leu-leu-tyr-CH~2 is a diazomethylpeptide compound, here shown to possess significant Calpain inhibitory activity. 2. Inhibition oî C~lpai~ ln Neural Tissues SUBST~TVT S)~EEJ
.
': . ` ` ' "
WO 92/1 1850 ~ 9 PC~/US~l/09786 ,; , In order to evaluate the inhibition of Calpa~n by the various C`alpain Inhibitors in neural tissues, we assayed the Cal~ain Inhibitors using the known ability of Calpain to cleave spe~rin, a proeein component of neuronal and other tissue, into Bl)P's. In this assay, more effective Calpain Inhl~itors will prevent the con~ersion of spectrin into S BDP's. Example 2 is one e~ample of such an assay.
EXA~LE 2 ~bf~
Crude Brais~ l~tracts ~ C~a~pain Inkibitors ~he actIvity of Cal~ain in crude brain e~tracts vas measured by e~amining the Ca 5-su~;ated proteo~sis of the endogenous substrate spectrin. Brain tissue was homogenized in 10mM Tris pH=7.4, 032M sucrose, lmM EGTA, lmM dithiothreitol and the nuclei and debris . - ~ved by low speed centrifugation. Various Calpain Ir, ~itors were added so the supernatant in a DMSO vehicle and a calcium salt (final e~ective concentration about 1.2mM) ad~ed ~o start the reaction. Proteolysis of spectrin was e~aluated by western blot as described by Seubert, et al. (Brain Research, 459:226-232, 1988), the disclosure of which is hereby incorporated by reference.~efly, a known quantity of a spectrin~ontaining sample treated with Calpain is separated by SDS-PAG~ and immunoblotted with anti-spectrin antibody. The amount of spectrin immunoreactiviq found corresponding to the characteristic BDP's is indicative of the amount of spectrin activity present in the sample. An e~amplary methnd for quantitating BDP's is to assay Spectrin BDP's by homogenizin'g brain parts in _umM Tris '?~ =72, 32M sucrose, 50,uM Ac-Leu-Leu-nLeu-H on ice. Homogenates are then mi~ea 1:1 with 10% SDS, S% B-rnercaptoethanoL 10% glyceroL lOmM Tris pH=8.0, 0,5% bromophenolblue, heated to 95C, and subjected to electrophoresis in 4 1/2% polyac~rlamide gels. ~e proteins in the gels are transferred to nitrocellulose and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antibody and es~ablished immunodetection methods. The amount of spec~rin and BDP's in each sample can be quantitated b~ densitrometric scanning of the developed nitrocellulose.
Due to Calpain's requirement for Ca2~, in the absence of Ca2+ little or no - 30 spectrin proteolysis occurred, regardless of the pr~sence of inhibitor, while in the presence of Ca2+ the spe~rin ~as ~ 95% cleaved to BDP's within 40 min. if no Calpain Lnhibitor ~s added.
a~TsTl ITF ~ Ec~
~. .. . . . . " .. -WO 92/ll8s0 2 ~ 9 ~ ~ ~ 3 PCr/US91/0978;; -Both leupep~n and CIl showed inhl~ition in the system of E~ample 2. In addition, the follow~ng compounds of ~he Substituted Heterocyclic Compounds werefound to produce signi~icant inhibition at 100 IlM:
3-chloroisocoumar~n 3,4-dichloroisocoumann .
3-benzyloxy~-chloroi~oco unarin 7-(ace~qlamino)~-chloro-3-(propoy)-isocoumarin 4-chloro-3-(3-isothiureidopropo~y)isocoumarin 7-amino~-chloro-3-(3-isothiureidopropo~y)isocouma~n 7-(benzylcarbamoylam~no) 1~hloro-3-(3-Lsothiureidopropo~y)isocoumarin 7-(phenylcarbamoylan~ino) 1~hloro-3-(3-isothiureidopropo~y)isocoumarin 7-(acetylamino) 1~hloro-3-(3-isothiureidopropo~y)isocoumar~n 7-(3-phenyJpropionylar~uno) 1-chlor~3-(3-isothiureidopropo~y)is~oumarin 7-(phenylacetylamino)~-chloro-3-ZO (3-isothiureidopropoxy)isocoumarin 7-~-phenylalanylamino)-4 chloro-3-(3-isothiureidopropo~y)isocoumar~n 7-(benzylcarbamoylarnino)~-chloro-3-(3-iso~hiureidoetho~y)isocoumarin 7-(phenylcarbamoyl~nino)~ chlor~3-(3-isothiureidoetho~;y)isocoumarin 7-(D-phenylalanylamino)-4~hloro-3-- (3-~sothiureidoetho~y)isocoumarin.
The following compounds of the ~Ialo-Ketone Peptides were also found to produce signifîGant iTl}ùbition at 100 I~M:
ZLeu-Phe-OE12CI
Ac-Leu-Phe-CH2Cl Z-Gly-Leu-Phe-CH2Cl Boc-Gly-Leu-Phe-C~I2Cl SUBST~TUTE S~IFFT
:
. ~ :. WO 92/11850 2 ~ 9 g 6 ~ 9 PClr/US91/09786 Ac-Val-Phe-CH2CI
~Gly-Leu-~la-CH2C 1.
In addition, the ~ollowin~ compDunds of the Peptide Keto-Compounds were found to produce ~i~3nifiGmt inhibition at 100 ~M:
5Bz-I)L-Phe-COOEt ~Leu-Nva-COOEt ZLeu-Nle-COOEt ~Leu-Phe~OC)Et ~-~eu-Abu-COOEt 10_eu-Met-COOEt Z-Ala-Ala-DI~Abu-COOEt MeO-Suc-Val-Pro-DL-Phe-COOMe ~AIa-Ala-Ala-DL-Ala-COOEt MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe.
15ZLeu-Phe-COOEt Thus, the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides, in addition to leupeptin and ~1, provide in~ubition in brain homogenates.
3. V~Q Inhib}tioD o~ N~urodegener~tion ~hrough Infusion Techniques In order to demonstrate that the inhibi~ion of Calpain activ~ty alone is sufficient to inhibit neurodegeneration vivo, we tested the ability of the Calpain In}~ibitor, . ~upeptin, to inhl~it neurodegeneration in gerbils subjected to transient ischemia.
As stated abo~e, leupeptin is poorh~r mcmbrane permeant. Therefore, leupeptin is not e~pected to cross the blood-brain barrier ("BBB") very well. Accordingly, in order to provide the braill with sufficient leupeptin to adequately inl~oit Calpain activation, w~ used brain illfusion techniques. Through thc use of these techniques we were able to subject brain tissues to intimate contact with leupeptin for sustained periods of time. E~ample 3A is provided to show the in vivo protection ~om neurodegeneration found during one such study.
EXA~LE 3A
~Vivo Protectio~ nst~eurode~eneration A small cannula was implanted in the right lateral ventricle of adult gerbils, and secured to the skull with dental cement. An Al~et n~icro-osmotic pwalp was attached to the cannula for intracerebroventri~ular perfusion. The pump was filled with either ~T~ F ~
WO 92/11850 2 0 9 8 G 0 9 r PCltU591/0978~--g2-saline alone (control) or leupeptin (20 mg/ml in saline). After three days perf~sion with ei~her the control solution or with the leupeptin solution, transient ischem~a was induced by b;laterally clamping the c~otid artenes for a period of ten mmutes. Core temperatures were taken dur~ng and follo~rlg ischen~a, with no di~erences noted S between control and leupeptin treated animals. Fourteen days l~ter, the animals were sacriiced by Nembutal overdose and transcardial per~usion of a 105'o solution of paraformaldehyde in PBS. Coronal sections of the brain were stained ~ith cresyl violet and were e~amined for the e~tent of neuronal loss. The control gerbils e~bited the typical damage found in the CA1 field following ischern~a, ~th a 72% loss of neurons.
However, the leupeptin treated ger~ils showed f~r less neurodegeneration, with only a 15% loss of neurons.
The results of E~ample 3A cannot be e~plained by changes in thermoregulation, since core temperatures did not differ between the groups. Accordingly, we believe that the Calpain inhibitors~ act~vity of leupeptin is responsl31e for the observed difEerences in neuronal cell loss. In order to further ~uantitate the differences, and veri~ that leupeptin produced a Calpain inhibitory effect within the observed re~ons of the brain, we performed a related series sf ~per~nents. In this series of e~periments, spectrin BDP's were measured in the leupeptin treated and control animals. As discussed above, these BDP's are indieative of the amount of CalE)ain activity occurring within the tissue. E~ample 3B is provided to demonstrate the results of these experirnents.
In Vivo In}likitior~of C~ ain ~ctivitv Implantation surgeries and clamping of the carotid arteries were performed as 2~ above with a control-ischen~a group (n-4) and a leupeptin-ischernia group (n=5). A
third group of animals (n=4) recesved imp~antation with pumping of s line, but was not subjected to ischen~ia Animals were sacTificed by decapitation 30 n~inutes afterclamping of the arteries. The brains were rapidl~ removed and placed in oold homogenization buffer (0.32 M sucrose, 10 mM Tris-HCL 2 mM ED~A, 1 mM EGTA, 100 ~M leupeptin and 1 ~g/ml of the Halo-Ketone Compound, tos-phe-C~I2Cl (TPCK)). The CA1 region of the hippocampus was then dissected. I~e samples from both control and leupeptin treated anirnal~ were then prepared for SDS-PAGE and ~mmunoblotting with labeled anti-spectrin antibo~r, as described above in connec~ion with in vi~ro uses of the Calpain Inhl~ito~s. lhe wn~rol animals exhl~ited a marked SUBST~ rE~ T
:` , `' ' WO 92/11850 210 9 g ~ 0 3 PCr/lJS91/09786 increase in the levels of BDP's reLatne to the gerbils not subjected to ischemia. These BDP's co-m~grated with BDP's observed after ~ proteolysis of spectr~n with Calpain. The brain t~ssue from the leupeptin treated gerbils e~hl~ited appro~mately 25% of the BDP's observed in the ~ontrol ischenua treated gerbils.
S Another group of gerbils (n=3) were sacrificed ilT~nediately after lschernia without leupeptin treatment in order to obserYe the effects of ischemia wi~hout reo~ygenation. These gerbils e~ ited a similar amoun~ of increase of 13DP's as the control-ischernic gerbils obsened after a 30 minute reperfusion penod.
~;.e -~ results of E~ample 3B indicate that leupeptin ~erts its neuroprotec~ive effect through the inhl~ition of Cal~ain activation. The results also indicate that the observed proteo~sis of spectrin was an efEect of ischemia, and not secondary to the reo~ygenation. Acoordingly, the results indicate that inhl~ition of Calpain activity in VIVO produces a neuroprotective efEect.
Although the foregoing studies demons~ate ~hat leupeptin can inhibit neurodegeneration in vivo, leupeptin is noe ~he therapeutic drug of choice because of .he need to infuse the drug direc~ into the brain for an e stended period of time to exert its neuroprotective ef~ect. This is due to the relative~ poor abili~ of ~his compound to cross the BBB. Accordingly, it is beli~ved that a more therapeutically practical way to inhibit neurodegeneration would be to use more membrane permeant Inhibitor of Calpain.
4. Platelet Pe~eablllty In accordance ~nth our discoveries demonstrated in Examples 3 and 3A, we believe that ha~ing a compound cross the BBB and enter ~::NS tissue is a key characteristic of a therapeutiul~y u~eful approach to treat or inhibit neurodegeneration within the CNS. Use of Calpain inhibitors that have enhanced membrane permeability is one such approach. Thus, wc measured the ability of various Calpain inhl~itors to penetrate the platelet membrane and inhibit Calpain that is normally contained in platelets. As shown beloYv in the following e~amples, our results ind;.cate that particlalar compounds of the Hetero~yclic Compounds, Peptide Keto-Compounds and Halo-3a Ketone Peptide~" in addition to ~he Peptide Aldehyde, CI1, ead~ibis good membrane permeability.
As an indication of the membrane penneability of the various Calpain Inhibitors, we measured the abili~ of various Calpa~n Inhibitors to penetrate platelet . memb~aDes and inhibit the Calpain nonnally found within platelets. I~e membrane of 8VEISTiTUTE SHI~
.
wo 92tll850 2 0 9 ~ G 0 9 PCI/US91/0978t~
platelets is believed to have many simila~ities to the BBB and accordin~ly, such~perirnents are believed to provide a good ~ndication of the ability of the various Calpa~n Inhibitors to cross the BBB. E~ample 4 shows the results of some of these platelet e~periments using the Cal~ain Inhl~itors of the present invention.
- 5 EX~MPLE 4A
Membrane Penneation of Calpain Inhibitors Platelets were isolated b~,r a modification of the method of Ferrell and Mar~in, 1 Biol. Chem.. 264:20723-20729 (1989), ~he disclosure of which is hereby incorporated by refer~nce. Blood (15-20 rnl) was drawn ~om male Sprague-Dawley rats into 100mM
EDTA-citrate contaiII~ng 10 units hepar~ns and centrifuged 30 sn~nutes at 1600 rpm at room temperature. The plasma was resuspended in 15ml bu~er 1 (136mM NaCL
2.7mM KCL 0.42mM NaH2PO4, 12mM NaHCO3, 2mM MgCI2, 2 mg/ml BSA (Sigma), 5.6mM glucose, 22mM Na3Citrate pH 65) and platelets were isolated at 2200 rpm atFoom temperature of 25 minutes. Platelets were resuspended to 107 cells/ml in buffer 2 (136mM NaCL 2.7rnM KCI, 0.42 NaH2PO4, 12mM NaHCO3, 2mM MgCL 1 mg/rnl BSA, 5.6mM glucose, 20mM HEPES pH 7.4) and allowed to "rest~ for a rn~r~murn of 10 minutes at room temperature before use.
Platelets were incubated for ~ r unutes in the presence of in}~ibitor. In order to provide sufficient intracellular calcium to ac~ate Calpain, the calciwn ionophore A23187 was added to a final concentration of 1~ M. After a further 5 minute incubation, the platelets were harvested by centrifugation (1 min 10,000xg) and resuspended in 10% sodium dodecyl sulfa~e, 10mM Tris pH-8.0, 5%
B-mercaptoethanoL 0.02% bromophenol blue, and heated to 95 C for ~ min.
Samples were subjected to SD~PAGE on 6% nuni gels and ~ansferred to nitrocellulose (Schleicher and Schue~l BA83) for 2 hours at 100rnA/gel in an I KR
Novablot. Filters were blocked for 10 rninutes in 0.25% gelatin, 1% BSA, 0.25% Triton X100, 0.9% NaCL 10mM Tns-C:l pH 7.5, incubated overnight in the same solution contairung antibody to rat spe~rin, washed 3 X 10 minutes with 10mM Tris-Cl pH 7.5, 05% TAton X100, incubated 4 hours in wash buffer plus al3c~line phosphatase conjugated goat anti-rabbit antl~o~y (Biorad), and washed as above. Filters weredeveloped using the Biorad AP conjugate substrate Icit. Spectrin ~nunoreact~ity on she filters was quantitated ~y densitometry.
The inhbition of Calpain within platelets as measured by the proteolysis of the endogenous Calpain substrate spectrin in the presence of in}u~itors was assayed for a C!l 1~2CTITI lT_ ~LI ~T
: : :
:. :
... . .
`, .
~ ` ' . ' ` ' WO 92~11850 PCT/US91/09786 _95 Yarie~ of Calpa~n Inhl~itors. Ihe poorly permeant inhibitors leupeptin and E-64 had lit~le effect on intracellular Cal~ain. In contrast, the highly membrane permeant Heterocyclic Compounds, Peptide Ke~Compounds, and Halo-Ketone Peptides effec~ively inhl~ited platelet Calpain.
The following Heteros~yclic Compounds were found to produce significant ition at 100 IlM in the ~stem of ~ample 4:
3~hloroisoeoumarin 4~hlor~3 -(3-~;othiureidopropoy)~umarin 7-an~no~hloro-3-(3-isothiureidopropo~y)isoooumarin 7-(benzylcarbamoylam~no) 1~hloro-3-(3-~othiureidopropo~y)~socoumar~n 7-(phenylcarbamoyl~o)~hloro-3-(3-isothiureidopropo~y)isocoumann 7-(acetylan~ino)-4-chloro-3 (3-isothiureidopropo~sy)~soco~
7-(3-phenylpropionylan~ino)-4-chloro-3-(3 -isothiureidopropo~y)isocournarin 7-(phenylacetylan~ino)-4-chloro-3-(3-isothiureidopropo~y)isocoumarin 7-(L-phenylalanyl~nino)-4~hloro-3-(3-isothiureidopropoxy)isocoumarin 7-(benzylcarbamoylam~no)- 1-chloro-3-(3-1sothiureidoetho~cy)~socoumar~n 2~ 7-(phenylcarbamoylamino)~chloro-3-(3-isothiureidoethoxy)i~ocoumar3n 7-(D-phenylalanylamino) 4-chloro-3-(3-isothilreidoethoxy)isocournar~n.
The fo~lowing Halo-Ketone Pep~ides were fo1md to produce significant inhl~ition at 100 ~M in the system of E~ample 4:
ZLeu-Phe-CH2CI
Ac-Leu-Phe-CH2C:l ZGly-Leu-Phe-CH2Cl Boc-Gly-Leu-Phe-CH2GI.
SUBS~ITU~E SHEEll' WO 92/11X50 2 ~ 9 ~ 6 ~ 9 PCr/US91/09786.~
The following Peptide Ket~Co~npounds were f~u~d to produce s~gn~ficant inhibition at 100 I-M in ~he system of ~ample 4:
~AIa-Ala-D,~Abu-COC)Et ZAla-Ala-Ala-D,~Ala-COOEt MeO-Su~-Ala-Ala-Pro-D,L,Abu-COOMe ZLeu-Phe-COOEt ZLeu-Nle-COOEt ~Leu-Nva-COOEt ~I~u-Abu-COOEt ZLeu-Abu ZLeu 1-CI-Phe-CC)OEt ~IAu-Leu-Abu-COOEs Z-Leu-Leu-Phe-COOEt 2-NapS02-Leu-Abu~COOEt 2-NapSO2-Leu-Leu-Abu-COOEt Z-Leu-Met-C02Et Z-Leu-NLeu-C02Et Z-Leu-Phe-C02Bu Z-Leu-Abu-C02Bu Z-Leu-Phe-C02Bzl Z-Leu-Abu-CO2B~I
Z-Ala-Ala-D,l,A~u-COOBzl 2; Leu-Phe-COOH
ZLeu-Abu-COO~I.
Among those compounds found to exhibit Calpain in}libito~r act~vity in the homogenate system of E~ample 2, we found at least shree compounds which failed to exhibit Calpain in}~ibitory activity in the platdet ~;ystem of E~ample 4. These compounds are leupeptin, MeO-Suc-~al-Pro-D,L,Phe-COOMe and Bz-D,L-Phe-COO~t. Leupeptin is hlown to ~e poorly membrane permeant, thus confi~ning that the platelet assay will e~cclude hlo~n poorl~r membrane permeant compounds.
Aocordingly, ~he two Pep~de Ke~ocompounds found not to provide Calpain inhibitory activity within platelets are also believed to be poor~ membrane permeant, and would not be expected to cross the BBB.
SllE3S~lTUTE SHEf~T
, .
~- :
. . , ~ , , ,... i, . . ..
"~. WO 92/11850 2 ~ 3 ~ ~ 0 9 PCI`/US91/097g6 C2uantitative Studies o~latelet Membrane Permeabilitv We performes~ addi~ional gL~ntita~ve or semi-quantita~ive studies on several Peptide Keto-Compolmds using the assay of E~ample 4A, e~cept that IC50 values were deterrnined as the concentration at ~vhich 50% of the Cal~ain act~vation present in S controls occurred. Results are shown in Table 4B. For the semi-quantitative assays, -~ed with +'s in Table 4B, ~+" indicates detectable inhibition at 100 ~M, "+ +"
inaicates significantly more ir~.ubition than "+~, and "+ + ~" indicates no detectable activation of Calpain detected.
S11~31STITUTE SHE~ET
.
wo 92~1~8~0 2 0 9 ~ p~/us9l/n978~ `
Platelet Assa~ of Peptide Ketoamides9 Ket~sters and Ke~oacids - ~ .
Inhibitor IC
S l _ ~ . I
~Leu-Abu-COOEt 42 . ............ . __ I
Z-Leu-Abu-COOnBu Z-IAu-Abu-COOBz + + l . _ I
Z-Leu-Leu-~bu COOEt 4û l ~ . I
2-NapSO2-Leu-Leu-Abu-COOEt 100 I . . __ ¦ Tos-Leu-Leu-Abu-COO~t 30 ~I~.UrCOON 8 Z-Leu-Abu-CON~t 1.5 . . , , ,, . _ .
ZLeu-Abu-CON~Pr 70 .
Z-Leu-Abu-CON~Bu 2.0 Z-Leu-Abu-CONH~u 28 Z-Leu-Abu-CONBz 15 .
Z-Leu-Phe-COOEt 47 . _ Z-Leu-Phe-COOnBu + + +
_ _ _ _ Z-Leu-~he-COOBz + +
: _ Z-Leu-Leu-Phe-COOEt + +
_~ . .
Z-Leu-Phe-COOH 65 Z-Leu-Phe-CONHEt 1.7 I . _ ¦ Z-Leu-Phe-CONHrlPr SU13~iTUTE Sl JEE t .. . . .
.
I.`Wo92/11850 2a~0~ PCr/US91/0~786 __ _ = .
Inhibitor ICso Z-Leu-Phe-CONHnBu 38 Z-Leu-Phe-CONHiBu 2~!
~ ~r .
Z l t ~-Phe-CONH(CEI2)2Ph 3.0 ,, _ __ _ ~ .
ZLeu-Me-COOEt 5ZLeu-Nva-COOEt 40 ... .. . , _ ~L~u-Met-COOEt +
I .
Z-Leu~ Phe-COOEt +
1, , ~ , Table 4B shows that peptide a-ketoamides and ketoauds were much more ef~ective than corresponding peptide ketoesters in thi~ platelet assay. E~tending the R3 group to an alkyl group or an allyl group substituted with a phenyl group increased the membrane permeability of the inh~itors as indicated by increased potency in the platelet assay. In view of these results, Applicants believe that ectending the 1~ group to indude longer allyl groups or alkyl groups substituted with phenyl groups would increase the membrane permeability of a ~ven inhibitor.
In view of the foregoin& the results of E~amples 4A and 4B support our belief that CI1 and the Substituted Heterocydic Compounds, Peptide Keto Compounds and Halo~Ketone Peptides are believed to be membrane permeant and therefore, are expected to be effective in crossing Ihe BBB subsequent to in vivo administration of the eompounds.
5. Glutamate To~clt~
To further identii y thase Calpain Inhibitors lilcely to possess pharmacologically active neuroprotective ability, we tested the abiliq of the Calpain ~hll~itors to protec~
against glu~nate e~citoto~naty. Excess e~racellular glutamate is ~hought to play a key role in the induction of neuropathology in ischemia, which is acoompanied by Wpain activation. In support of this role for e1~cess glutamate, cultured Nl8~ 105 (a neuroblastoma.retinal hybrid) cells can be killed by the addition of glutamate into the culture medium. This glutamate.mediated cytoto~ici~ is calciurn dependent and can be StlE~STlTlJTE SHEET
. .
.
wo 92~ 50 21~ O ~ P~/US91/09786 reduced ~hrough a number of mechanisms, including free radical scavengers, blockers of the N-type volta~e-sensitive calcium channel, and quisqu~late-subtype glutamate antagonists. Thus, glutamate-mediated killing of N18-~lOS eells is an ~B model for neuropathology.
S According~, we tested She ability of the Calpain Inhibitors to inhibit glutamate-induced cell death in these cells in order to establish that the Calpain ~hibitors can decrease or prevent glutama~e-induced d~ath Of N18-~105 cells. Some of these tests are shown in ~ample 5.
~LE
S~ock cultures of N18-RE 105 cells were maintained in Dulbecco's modified ~agle's medium (DMEM) containing 10% fetal bonne serusn (FBS) and supplemented with hypo~anthine, ~inopterin and thymidine (HA~. Subcos~fluent cultures were split and plated into 96-well plates. Iwenty four hours a~er plating the cells were exposed 15 ~o ~esh media containing glutamate and various concentrations of Calpain inhibitors.
Control cells wer~ not treated with glutamate. The treated cell~ recei~red SmM
glu~nate and leupeptin (5,ug/ml) or the other ~pa~n Inhibitors listed in Figure 1 at 3~g/ml. Conversion of ~IT was measured 19 hours later as descn~ed. Nineteen hours after the onset of e~osure, cell viabiliq was quandtated by measunng the e~ent 20 to which the cells convert 3(4,5-dimethyltJuawl-2 yl)-2-~diphenyltetrazolium bromide (~) to a blue formazan product, which sowrs in the r utochondria of If ving but not dead cells (Pauwels et al., 1988). A higher absorbance is indicatlve of greater cell viability.
Figure 1 sho~. the percent of blue formazan product remail~ng after treatment 25 with glutamate, relative to control where no ~utamate was added. Thus, it can be seen that with vehicle plus glutama~e but no inhl~itor, less than 70% of the mitochondrial activity remains. However, Figure 1 shows that u~eral Calpain i~bitors, including leupeptin, Gll and representa~ves of the Heteroc yclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides protect N18-R~105 cells agaDst glutamate 30 toxicity. Tile Peptide Keto-Compound Calpain inhlbitor, ZAla-Ala-Abu-CO2Et, the Substituted Heterocyclic Compounds, ClTPrOIC and A~IC, and the Halo-Xetone Peptide, TPCK complete~ blocked the toxic effects of glutamate, resulting in 100% or ~eater of the form n product as seen with cells not treated with glutamate. Thus, E~ample ~ shows that these Calpain Inhibitors efEec~ve~r block cell death in an vitro SU8STlTllTE SHEE~
WO ~2/11850 PCT/US91/09786 model for neuropatholo~r. Accordingly, this data further supports our discoYery that Calpain Ir~bitors are neuroprotect~ve ~ n~o.
6. Redu~iosl of I~a~ion o~on MCA O~ ioll Stroke is a significant health problem in the hurnan population. Strokes are S occlusions of cerebr~l arteries produc~ng a decreased blood flow to bra~n re~ons, which cause cell death through oy~en and nutnent dep~vation. I~is type of lesion can be modeled in rats by sur~cal ooclusion of the middle cerebral artery (MCA). Several models for MCA occlusion have beerl developed, and all ~ive substantially similar results.
MCA occlusion produces a large volume of infarcted brain tissue 24 hours after occlusion. Previous studies ha~e shown that the size of the infarct as judged by ITC
sta~ing does not increase after the first 24 hours post-oeclusion. ~us, we used an MCA occlusion model in order to test the ability of Calpain inhibitors to prevent neurodegeneration. lhis model is descn~ed in E~ample 6.
~IYsion Model for ~eurode~eneration Male Sprague-Da~vley albino rat~ weighing appro~mately 250-300 grams were anesthetized with pentobarbital (70 mg/kg, i.p.). The neck region was shaven and a 2 cm incision was rnade. The superficial f~u ia ~vas teased away with tissue forceps and blunt tip tissue scissors using a spread method. The right comrnon carotid artery was isolated away ~om ~he vagus nerve and tied off with a single suture (3.0 siL~;). The external carotid was permanently occluded by sut~ing. Ihe bifurcation of the internal carotid and pterygopalatine arteries was e~osed and a single microaneulysm c lip was placed on the ptenygopalatine. Another microaneutysm dip was placed on the common urotid just pro~mal to the e~ternal/internal bifurution. A suture ~ras placed loosely around the co~Tunon carotid and a lumen was made in the vessel with the tip of a 25g needle. A 40 mm nylon suture was prepared l~y melting the tip ~o smooth the pointed end and marked with a dot ~actb 175 mm from the melted end. The suture was inserted into the lumen of the artery as far a~ the vessel clip, the clip is removed and the suture advanced until the marldng was at the bifurcation of the internal andex~ernal carotid arteries. I~ia places the end of the suture in the circle of Willis just beyond the source of the middle cerebral ar~ery and oecludes this arsery. The loose suture around ~he caro~id is tied lightly to keep the nylon suture in plau. The St~8STlTlllTE SI~EET
2~8~
wo 92/11850 PCr/US91/0978 n~lcroaneurysm clip on the ptPrygopalatine artery was removed, the incision is closed and the animals are allowed to re~over in heated recovery cages.
Twenty-four hours after oeclusion, the brains of the~e animals were removed and sliced into 2rnm sections. The sections were stained using 2,3,5-S triphenyltetrazolium chloride (see Lundy, EF, Solik BS, Frank, RS, Laq, PS, Combs, DJ, Zelenock, GB, and D'Ale~y, LG, Mo~hometric ev luation of brain infarcts in rats and gerbils, J. Pharmaco~ ~eth 16,201-214, 1986). Absence of red color development indicated tissue damage or death. The s~ of the infarcted tissue zone (area with red stain) and impaired zone (area with partial development of red color) were evaluated using quantitative morphome~y.
Drugs or vehicle were administered by infusion into the femoral vein. All animals received the same volume of drug or vehide (20% dimethyl su~fo~ide/80%
propylene ~ycol) via a catheter attached to an Alzet osmo~ic milLipusnp (24 hr pump, 8 ul/hr, 90 ul total volume).
The model of E~a nple 6 was used to determine the size of infarcted area for control (vehicle, i.Y.) and with adrminissration of each of ~wo Calpain inhibitors: ZLeu-Phe-CONH-Et (CX269) and Z Leu-Abu-CONH-Et (CX275). These results are depicsed graphically in Figure 2. It can be ~een that administration of either of the Calpain inhl~itors Z-Leu Phe CONH-Et (CX269) or Z-Leu-Abu-CO~H ~t(CX~7~) produees a reduction in the size of the infarcted area.
7. Inhlbltioo o~ Anoxic and H~ lc Da~nage The CA1 region of hippocampus ~s a brain area par,sicular~ ~ulnerable to ischemic damage and other insults imolving cxcitatory amino acids. The hippoc mpus is also a major focus of cell degeneration in Al~he~mer's disease. Neural cells in slices degenerate following hypoxia throuBI the se chain of events (including reperfusion effects) observed vo dur~ng and after ischemia. We believe that snldies of degeneration of neural slices in the pre~ence of the various Calpain ~h~itors is an effec~ve indicator of the membrane permeance of the Calpain Inhl~itors. Accordingly, we believe that these studies provide a model for the trea~nent and inhl~ition OI
neurodegeneration vo. Similar studies for de~ennining the e~icacy of compounds useful in the treatment of neurodegeneration in accordance ~ith the present invention can be performed using other models, such as protection against degeneration in platelets or cells in culture.
-SUBSTITUTE SHEE~T -~ w~9~/118So 2~ 9 Pcr/V591/09786 .~.
It ~s be~ieved that hypo~a is a major eause of neuroto~icity ~n a variety of neurodegenerat~ve diseases and conditions, such a~ stroke and head injur~1. Thus, we eonducted further studies u~ing hippocampal slices to show that the vanuus Calpain itors, advantageou~b, can ~ncre~se sunmal of hippocampal ne~ve cells during e~posure to hypo~ic or anox~c conditions. An initial screening procedure was first used to qualitatively detennine whether the various Calpain In}h~itors Wl provide neuroprotection from ano~a in hippocampal slices A~ ample of these initial screen~ng procedures is sh~vn by E~ample 7~
E~MPLE 7A
itial Screenfor Inhll?ition of ~o~ic Dama~
Hippocampal slices (400 um) were prepared frorn Sprague Dawley rats ~6 to 7 We~l~) and maintained in an interface chamber at 35C using conventional techniques, i.t~ ~e lower ~u~ce of the slice received a constant peffusion (05 ml/m~zl) of ACSF, while the upper surf~ce was e1~posed to a moist atmosphere f 2:CO2 (gS%:5%) e~changed at a rate of 2 L/min. The AC~;F medium contains (in mM): NaCl ~124), KCl (3), KHPO4 (25), CaCl2 (3.4), NaHCO3 (26~ and D-Glucose (10). Field ~citatory post-~naptic responsss were recorded from stratum radiatu n of CAlb in response to stimulation of Schaffer-cornmissural fi~ers ~n CAla or CAlc. The depth of the recording electrode was optimized and evoked responses were collected at a rate of one evoked response every 30 seconds.
For the initial screening procedure, 14 to 16 slices are harvested from the hippocampus of a single rat and placed in a com non ACSF bath. Each slice is tested in sequence to deterrnine the magnitude of its pre-ano~c evoked response. Five stimulation pulses (each 0.1 ms (millisecond) in duration) were presented over a 15 second ~ntenal. The largest evokcd response was noted and recorded for each slice.
Following this, the slices were incubated for one hour, with either drog or vehicle alone added to the ACSF. After the one hour drug incubation period, the oxygen-enriched atmosphere of the chamber was rnade anoxic by substituting nitrogen for oxygen (N2 = 95%; Co2 = 5%). The slices were retained in this ano~ic environment for 10 n~inutes, following which the a~ygen-enriched a~mosphere (o2 =
95%; C2 = 5%) was reestablished.
Ihe slice~ were given the opportunity to recover for 30 rninutes following reoxygenation whereupon each was stimulated and the ma~nurn evoked po~ential determined, as descn~ed above durin~the pre-ano~a period. Those slices which, after !~1 IR.`t:T~ IT~ ~
.
. .
WO 92/11850 2 ~ 9 ~ ~ O ~ pCI/lJS91/0~7~6'" "
ano~iaa, produced a ma~imLun evolces~ potential of greater than 50% of that observed prior to ano~ia were dei;ned as sun~ing slices.
Results of the studies of E~ample 7A are show~ in Figure 3. Figure 3 shows the effects of CX218 (Z-Leu-Abu-CO2E~t, a Peptide Ket~Compound), and CI1 relative to control slices on sumYal OI hippocampal slices e~osed to 10 minutes esposure of ano~c atmosphere. As ~een ~rl this figure, when the control slices are depr~ed of oxygen for 10 Iminutes ill the absence of drug, virtually all f~il to sur~e, as measured by their ability to elicit SO~o of their pre-anoxia eYoked response. In accordance with this finding, few if any recover upon reoxygenation. Figure 3 also shows that when CI
or CX218 are added to the ACSF, the slices are protected ~om the efEects of ano~ia, evidenced by a substantial proportion of slices eliciting eYoked potentials.
Finally, i~ e seen that CX218 is significantly more effective in protecting against anox~a and preventing degrada~ion of slices at the minimal 1 hour incubation time, and at lower concentrations than CI1. This efEect is believed to be due ~o the superior membrane permeance of the Peptide Keto-Compounds.
Table 7A shows further data from the studies of Example 7A.
SUBSTITUT~ SH~ET
.
! WO 92/11850 2 ~1 9 8 6 ~ 9 PCl[/VS91/097~6 -lOS-PEROEMT OF SLIOES SUBVIVINÇ: TEN M[INImS ~NOXld.
Compound Dose (u ~rubation T~ne Sunnval Control ~ 1 hour <l~o S Leupeptin lOOO . 3 hours 50%
~Il 200 2 hours 53~0 CX13 (SHC) 20 l hour ~O~o CX89 (CKP) SO 1 hour ~O~b CX2l8 ~ C) 1~ l hour 70%
i~, It can be seen from the d~ta in Table 2 that all of the Calpain ~h~ ors tested provide ~ncreased scl~viYaL CX~3 (AClTlC, a Substituted Hetero~yclic Compound (S~ . CX89 (Boe -Cly-Leu-Phe-cH2~1~ a Halo-Ketone Peptide (HKP)) and CX~18 ((Z1~Abu-C02Et, a Peptide Keto-Compound (PKG)), are each shown to ~e highly ef~ective in influencing sun~Ival times. Leupeptin ~s seen to be the least ef~ ve neuroprotective. Thus, we believe that CX13, CX89 and CX218 (ZLeu-Abu-C02Et) are more effec~ve in influencing sumsal be~use of their membrane permeability.
Accordingly, the results shown in Table 7A support our belief that Calpain Inhibitors with membrane permeability are ef~ective neuroprotectants.
To ~rther eluu~idate the ability of Calpain Inhibitors to provide neuroproeection to hippocampal slices, u~d to provide a more quantita~e indica~ion of the membrane perrneability of these Calpain Inhibitors, we measured the effeet of vanous Calpain Inhibitors on the c~oked response on a single neuronsl slice before, during and after anoxia. These srldies are shown in E~ample 7B.
llhiki~iQn of AnQ~UC r)a,Illa8e As ~n Esample 7A, hippocampal sL;ces (400 ym) were prepared from Sprague Davley rats (6-7 veeks3 and main~ained in ~n interf~ce chamber at 3S~C using conventional techniques, ie. the lower eur~ce of the slice recerved a constant perfusion (05 ml/m~n) of an arti~al cerebrospinal fluid (A~SF), while the upper surface was exposed to a moist atmosphcre of 02:C02 (95%:5%) e~changed at a rate of 2 L/min.The ACSF medium con~ains (in m~: NaCI (124), KC:I (3), K~04 (1.25), MgS04 (2.5), CaC12 (3.4), NaHC03 (26) and D-Glucose (10). Field sxcitatory post-synaptic responses ~rere recorded from stratum radiatum of CAlb in response to stimulation of Schaffer co~sural fibers ~ CA1a or CA1c. The depth of the recording ele~rode SUE3STITUTE ~
~U~'3 WO 92/11850 PCI/US91/097B~
was optimized and evoked responses were collected at a rate of one evoked response every 30 seconds.
Af~er establishing a stable baseline of evoked responses (appro~mately 10 rninutes~, ACSF contail~ing ~pain In}~l~itor was washed into the chamber and slices were iT~cubated for a period of one hour. After incubation, evoked responses were again recorded and the change in the amplitude of the responses from base~ine levels was noted. No effect of the inhibitors tes~ed on ba~eline evoked responses was obsened.
For ano7~a ~eriments, incubation in the drug-containing medium was fo~lowed by replacement of the 02:C02 (95%:5%) atmosphere ~nth N2:C02 (95%:5%). Slices were e~posed to this anoac environment until disappearance of the pre-synaptic fiber volley and for two n~inutes (se~ere ano~ia) longer (total time in ano~ic erlviromnent appro~imately 7-8 minutes in control case). Effects of Calpain ~hibitors on the functional recovery of the slices after the anwnc episode were then measured.
Recovery of the evoked po~en~ial (~PSP) slope and arDplitude by the drug treatedslices can be compared to con~rol slices to d~tennine the relative e~icacy of various Calpain Inhibitors.
Figure 4 shows the E~PSP amp~itude in rnill~volts for con~ol, C:[1 treated and CX218 (a Peptide Ket~Compound) ~eated hippocampal slices in the studies of E~ample 7B. It can ~e seen in Figure 4 that the control slices deprived of o~ygen in the absence of drug display a gradual reduction of EPSP and abruptly lose ~ber volley activity about S-6 minute3 after the begin~ing of ano~ia. Reo~ygenation at or before th~s point leads to ~omplete functional recovery after about 20 minuteg of reo~ygenation, but reo~ygenation ~ this point does not. In the latter case the recovered EPSP slope and amplitude become progresslvely reduced as the duration of ano~ post fiber valley disappearance (post-FVD) increases. After severe anoxia (2 minutes post-FVD), slices re~over only 15% of the EPSP ~ope.
In contrast to the control slices, recove~y begins to oceur shortly after the end of anoxia for the treated slices. Figure 4 shows a comparison of the e~ects on EPSPamplitude produced irl the presence of no inhl~itor; the Peptide Keto-Compo~md, CX2lK, (~Leu-Abu-COæt) and CI1. CX218 produces a recovery ~om severe anoxia superior to that seen with CI1.
Figure S sh~ the percent recovery of EPSP ~om seYere hypo~a using the peptide ketoester CX 216 (~Leu-Phe-CO2Et) and its corresponding peptide SlJE3~1TUTE StlEET
.
.--' WO 92~11850 2 ~ 9 8 G O ~ PCI'/US91/09786 ketoan~ide CX269 (Z-Leu-Ph~CONH-Es). These studies were performed ill a manner similar to that of Example 7B, el~cept USiDg a hypo~ic environrnent in place of the a~o~a of ~ample 7B. It can be seen that use of the peptide ketoamide results in essentially complete (near 100%) reeovery ~om hypo~a while the peptide ketoesterS produces a par~ial recovery. I~e control ~lices ~peAenced little or no recovery.
An interesting charactedstic that we have discovered for certain Calpain bitors Is their abili~y to lengthen the period of e1~posure to ano~ia required to produce ~r volley disappearance (FVD). 15pi~, under control ano~a conditions, fiber volley disappearance o:urs in less than s~ minutes (~igure 6). The Peptide 10 Keto-Compound, CX-216, su~stantialb lengthens the period of e~posure to ano;~a required to produce FVD. This is an important advantage of the use of this Peptide Keto Compound for neuroprote~ion be~use slices can be e~pected to recover completely if reo~ygenated before fiber voiley disappearanc ~. Thus, treatment wi~h this Peptide Keto-Compound is e~pected to produce a greater percen~ge of recove~ of cells from incipient neurodegenerative conditions. It is believed that other representatives of the Peptide Keto-Compounds as well as of other classes of ~Ipain itors also provide this effect.
Table 7B shows the perecenhge of recovery of pre-anoxia synaptic transmission (evoked potential amplitude) of slices treated with various Calpain Lnhibitors or of 20 control slices. All of these slices were acposed to ten minutes of ano~a according to the protocol of E~ample 7B.
.:
WO 92/118~i0 ~ 0 9 ~ 6 0 3 PCI'~V~91/~9786~
T~BI~S 7B
Pl~RCE;NT RECOVE;~ OF SYNA~C TRANSMISSION A~R ANOXL9 ÇQm~!lD~ al~ % Recover~
Control -- lS
CX13 (SHC) 20 60 CX89 (a~) 50 30 CX216 (PKC) 100 38 CX218(PKC) 10~ 5~
The results shown in Table 7B provide fu~her eYidence tha~ the peptide aldehyde, C:I1, as well as the Substituted Heteroeyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides are sufficiently membrane permeant to provideneuroprotection through Calpain inhl~itiolL
CIl, which is at least p~y membrane permeant~ produces some effect, 1~ howeYer, does not significantly lengthen the period of ano~ia required to suppress electrical act~ . Thus, compared to controL or even compared to leupeptin and CI1, the Substituted Hetero~yclic Compounds, Pep~ide Keto-Gompounds and Halo-Ketone Peptides can increase the degree of recovery after ano~ic episodes while producing the additional advanhge of ~tending the amount of tirne slices carl tolerate ano~a (and thereby recover completd~
An important efEect of the Peptide Keto-Compounds and other membrane permeant Calpa~n Inhibitors ~s that they are ~ignificantly more effec~ve in lower doses than less permeable Calpain In~bitors such as C:I1. Although C~I1 is sho~m to be at least ~omewhat membrane penneant due to its abili~r to ~ect slice su~iYal, the more 2~ membrane-permeant inkibitors provide significantly increased protection. Thus, the more highly membrane-permeant Calpa~n Inhibitor~ are believed to be especially ef~ective in ~eating and inhlUting neurodegeneration.
The results of the studies of Esamples 7A and 7B show tha~ the Substituted Hetero~yclic Compounds, Peptide Keto-Compounds and Hal~Ke~one Peptides are membrane-permeant CalpaLn ~h~ ors which are believed to ~e especially effective in treating and inhl~iting neuro~egeneration. The results also show that Peptide Keto-Compounds, and perhaps representatives of other classes, can ~end the duration of anoxia required to suppress electlical activity in hippooampal islices.. As discussed above, these effects are ~mportant advantages of these compo~mds.
~1 IR~TIT~ ITF .~F~T
-- ;,; ~ . :- .. ..
WO 92/11850 ~ .5 ~ ,9 PCI/lJS91/09786 8. ~ NeuroprotectioD by Calpa~ I~ibitors As discussed above, therapeutics useful for influencing the function of cells within the CNS must cross the BBB to reach their targets within the CNS. Non-BBBpermeant compounds might, in addition to the brain infusion techniques de~cribedabove~ be administered v~a intraventricular administration, but this also se~erely limits their usefulness in practice. In order to test the vivo effectiveness of ~he Calpain klhl~itors to cross the BBB and become therapeuticalh~r useful, we tested the ability of mtraperitoneal injection of ~he Calp~ Inhl~itors to pro~ec~ a~st ~citoto~ic damage ~YiVQ, Protection w~ measured ~ aluating dlanges in beha~ of rats after inie~ion ~ith Icainate. These studies are sho~m in Example 8A.
EXAM[PLE 8A
Admiristered Calpain Inhibltors Ra~s (male Sprague-Da~ley, 200~5 gms) were injected intraperitone311y with 12mg/kg kainic acid in saline vehicle and ether 200~l DMSO (dirnethylsulfo~ide) or 4.6mg calpain inhibitor dissolYed in the same volwne of DMSO. The ra~s were obserYed for six hours following the injections and the kaina~e-induced behavioral symptoms and convulsions soored on a scale of 0-6 (0=no symptoms; 1-wet dog shakes; 2=salivation and chewing, 3=at least one convulsive episode; 4=repeated or sustained convulsions; 53convul3ions, including rearing and falling; 63convulsions followed by death ~nthin the 6 hr~ post injection).
Figure 7 shows the effects of CI1 on the beha~ioral and con~rulsive effects of l~ainic acid. In the control group, over half the anin~ls showed symptoms greater ~han mild behavi~ral symptoms, and many e1~hibited overt convulsions, presumably reflecting seizure acti~ity within the brain. Une~pectedly, in the inhibitor t~ated group, the incidence and severity of convulsions was reduced. Thu~, this data suggests thatCalpain Inhibitors have an anti-conmlsive effect. Ihis effect is a distinct advantage in the use of Calpain Inh~itors in epilep~-related neurodegenerative conditions and in stroke, which is often accompanied by seizures.
Ln order to rnore clearly demonstrate that the behavioral and anti~on~rulsive effects seen with the Calpain Inhibitors result from inhbition of Calpain we tested the brain tissues of the rats from E~ample gA for accumulation of spectrin BDP's. Asdiscussed above, these BDP's are associated with Calpain activity and with the neurodegeneration a~sociated therewith.
SUBSTITUTE S~ET
, WO 92/118S0 9 8 6 ~ ~ PCI'/US91/0978~' EXA~LE 8B
Protect n ~inst Speçtrin Breakdown ~om E~citoto~ic Damage bv Peri~?herallv ~dministered Calpain Inhibitors Four days follo~ing the inje~ion of kain~te in the rats from 3~ample 8A, the S brains of the rats were removed and assayed for spectrin BDP's. Spectrin BDP's ~ere assayed by homogenizing brain parts in 20mM Tris pH=7.2, 32M sucrose, 50~M Ac-Leu-Leu-nLeu-H on ice. Homogenate~ were mixed lol with 10% SDS, ~%
B-mercaptoethanol, 10% glyceroL lOmM Tris pH=8.0, 05% bromophenolblue, heated to 9~C, and subjeaed to electrophoresLs in 4-1/2% poiyacryl~rnide gels. 'rhe prote~ns in the gels were transferred to l~itrocellulose and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antlbody arld established Dnmunodetection methods.
The amount of spe~trin and BDP's in each sample was quantitated by densitometricscanning of the developed nitrocellulose.
Figure 8 shows the results of ~ample 8B. It can be seen that kainate stimulated the breakdo~n of spectrin in both Cal~ain Inhl~itor treated and con~rol rats.
However, treated rats e~u'oited significant~ less BDP's. These results veri~y that Calpain ac~vity in the brains of the treated rats was reduced. An une~pected observation was that wen those treated animals that ~hibited severe seizures hadsigruficantb less spectrin brea3cdo~n than untreated animals subjected to kainate. Thus Calpain Lnhibitor treatment reduced b~oth the behavioral/convulsive effects of kainate and the activation of calpain in the most severely affected animals.
9. CoDtluslon All of the foregoing studies support our discovery that Calpain Inhibitors provide ~2 protection against neurodegeneration asso~ated with ano~ia, e~citoto~ci~r and other cause-s. Thu5, these Calpain inhibitors possess neuroprotective activiq against a varie~r of v~vo neurodegenerative di~es and condidons, including ex~itotoxici~ induced neuropathy, ischemia following denenation or injury, subarachnoid hemorrhage, stroke, multiple i~rction dementia, Al~heimer's Disease(AD), Huntington's Disease, Parkinson's D~ease, surgery-related brain damage andother pathological conditions.
Those Calpain Inhibitors which possess significant Calpain Inhibitory ac~vi~ in vitro and also meet at least one of the foregoing or different tests for membrane permeability are e~cellent candidates ~or treatment of neurodegeneration.
G. DRIJG PEI~ -- - -. . .
SUBSTITU~E SH~
, . : ~. ; . . -:
WO 92/11850 2 ~ 0 9 PCr/US91/09786 The ability of ~he various Calpain I~ahibitors to penetrate pl~sma membranes is a si~icant advantage of these compounds from a pharmaceutical perspective. We believe that ~his ability, advantageously, allows the ~ain Inhibitors to provideexcellent permeation of the blood-brain barrier. This is in contrast ~o many phannaceuticals, especially pept;des, which often e~it poor penneatioII of the blood-bra~n banier. Thus, we believe that the Cal~ain In~bitors w~l e~bit e1~cellent results as pha~maceutically neuroprot~e agents.
For tr~tment of neurodegeneration, ~he Calpain Inhibitors can b~ adrninistered orally, topically or parenterally. The term ~parenteral" a3 wed herein includes all non-oral delivery techniques includ~ng transdermal admini~tration, subcutaneous injection, intravenous, intrarnuscular or intrasternal irijection, intrathecal injection (directly into the CNS) or infusion techniques.
The dosage depends primari~y on the specific formulation and on the object of the therapy or prophyla~s. '~e amount of the individual doses a~, well as the administration is best de~ermined by inclividually assessing the particular case.
However, in preferrsd compositions, the dosages of Calpain Inhibitors per day are preferably in the range of 1 ~g/kg ~otal body mass to 100 mg/lcg total body mass, more preferab~r in the range of 10 I g/kg total body mass to 10 mg/kg total body mass.
The pharmaceutical compositions contain~ng the active ingredient may be in a form s~utable for oral use, for ex~nple as tablets, troches, lozen~es, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or eli~drs. The amount of active ingredient that may be combined with carrier materials to produce a sin~le dosage ~orm will vary depending upon the host treated and the pardcular mode of adn~inistration. However, typically, a single dose will 2~ contain sufficient Calpain Inhl~itor to provide a complete dars dosage in a single orally ac eptable form.
For injecdon, the therapeutic amount of the Calpain Inhibitors or their pha~naceutically acceptable salts will normally be made by subcutaneous injection, intravenous, intrarnuscular or intrastemal injection, or by intrathecal injection (directly into th brain). In order to provide a ~ingle dars dose with a single injection, the pharmaceutical compositions for parenteral administration will contain"n a single dosage fonn, from about 70 l~g to about 7 g of ~ain ~ibitor per dose of from about 05 ml to about 1 liter of carrier solution. In addition to the active ingredient, these pharmaceudcal compositions ~vill usuaLly contain a buffer, e.g. a phosphate buffer SuBsTlTuTF.~
, .
' ' W092~1l850 2a9g~0~ Pcr/US9l/09786 that keeps the pH in the range from 35 to 7 and also sodiuun ehloride, and c~n also contain mannitol or sorbitol for adjusting the isotonic pressure. In a preferred form of these compositions, DMSO or other organie so}vent is added in order to assist the introduction of the Calpain Inhibitor across membranes.
Additionally, lipids c~n be introduced in~o the pharmaceutical compositions in order to $acilitate entry of the Calpain inhibi~or compounds into tissue of the CNS.
These compositions are prepared in accorda~ce Yvith methods lalown to those of skill in the art. Briefly, a lipid sueh a~, phosphatidyl ~oline, ch~lesteroL other well-hlown lipid carrier or ~tures thereo~ nth the ac~ve compound along with a solvent, the solvent is d ied off and the material reeonstituted in saline. The compositions can also include other ingredients ~own to those of ordirlary skill ~ the art, such as detergents, surfactants or emulsif~ing agents.
A composition for topical application or infusion can be formulated as an aqueous solution, lotion, jelly or an oily solution or suspension. A composition in the form of an aqueous solution is obtained ~y dissolving the Calpain Inhl~itor in aqueous buf~er solution of pH 4 to 65 and, if desired, adding a polymeric binder. An oily fonnulation for topical application is obtained by suspending the Calpain Inh~itor in an oil, optionally with the addition of a s~elling agent such as aluminium stearate and/or a surfactant. The addition of DMSO to these topical compositions is believed to allow at least partial penetration of the ac~ve Calpain Inhl~itor into the blood stream after application of the composition to the skin of a patient to aUow fortransdermal adn~nistration.
Por treatment of neurodegeneration resulting from e~citotoxici~t HIV-induced neuropathy, ischen~a following dener~ation or injwy, subarachnoid hemorrhage, stroke, multiple infarction dementi~, Alzheimer's Disease (A13),~Huntington's Disease, surgery-related bra~n damage, Parkinson's Disease, and other pathological conditions, the Calpain Inhibitors or pharmaceudcally acceptable salts thereof may be admin~tered orally or parenterally. The dosage depends primarily on the specific fonnulation and on the objec~ of the therapy or prophyla~s. I~e amount of the individual doses as well as the adn~il~istration is best detemuned by individually assessing the particular case.
In many acute neurodegenerative conditions and events, such as stroke and head isljury, it is Lmportant to del~ver the C~ain ~hl~itor as soon after injury as is practicable. Thus, it is preferable to identify those individuals who have suf~ered stroke, head injury or other inju~y in which neurodegen4ration is associated or is likely SIJE~STITUTE SH~ET
, , ... ~.- .. . .
, .. . .
W O 92/11850 2 ~ ~ 8 ~ 0 9 PC~r/US91/09786 ,',.'~ ,t to occur, and to begin administration of a C~apin ~hibl~or within 1 minute to 2 hours after the event, in order to preven~ as much neurodegeneration as possible.
A particular application of ~he Calpain Inhl~itors within the scope of ~he present invention is the application of these compounds dunng surgery to preventS rl~urodegeneration associated therewith. For example, for surgeries perforTned under general anesthesia, hypo~ic conditions can occur through inadequate perfusion of the CNS while under anesthesia. Additionally~ many major surgerie~ of the cardiovascular system require tnat a patient's heart be stopped and that perfusion be maintained through ~icial means In such surgeries, ~here ~s an increased danger of hypo~ia 10 occurring within the C NS, which can also result in r1eurodegeneration, Moreover, during neurosurgeries, there is an inheren~ risk of neurodegeneration resulting from inflammation, bleeding, hemorrhaging and the like. Such neurodegenera~ion can be~nk e~lby infusion with a solution conta~ning Calpain Inhibitor. However, Il . ...generation resulting from neurosurgery can also be reduced prophylactically by adr~inistration of a Calpain Inhl~itor through any of the foregoing adrninistration techniques. Such administration is also believed to inhl~it or prevent neurodegeneration associated with the u~e of anesthesia or with the use of artifical means of perfusion during major surgeries. A surgical pa~ient can also have Calpa~n Inhibitor administered throughout surgery throu~ intravenous drip.
l~e foJlowing e~amples are intended to illustrate certain neuroprotective uses of the Calpain ~}ubitors within the scope of the present invention. As such, they are not .oi lo limit the invention in any way.
~ Neuroprotective Composition fo~Intravenous Inje~tion 500 ~g CH3CONH-ClTPrOIC from E~ample SHC2 4 ml Propylene Glycol 1 rnl DMS--EXAMPLE lO
A Neuroprotective Composition fQr Intrave~ous Drip 250 mg ZLeu-Phe-CONH-Et ~om E~ample PKC 47 1000 rnl Phosphate BufEered Saline (pH 6.0) 10 ml D MSO
5URSTITI ITF .~L1~--:
: .
WO 92/t185(2 1~ 9 8 G ~ 3 P~US91/097~fi EX~MPLE 11 A l~europrotectiv~o~ositio~or Transdermal Application 25 mg~Leu-DL,Abu-COOEt ~rom E~ample PKC19 3 mlPhosphate BufEered Saline (pH 6.0) 2 rnlDMSO
EXAMPLE: 12 eu~o~rote~o~L after ~ead ~ury A first group of p~tient3 who are victims of head trauma is given 2 ml of the injectable composition of E~ample 9 intravenous~ hin ten minutes of the time of injury. A second group of similar~ matche~ patients does not recer~re the composition.
Ille first group of patients e~its marked~ fewer and les5 severe outward symptoms of neurodegeneration, such as dementia, memory loss and parahJsis.
~europrote~n Durin~ Sureerv A patient about to undereo a triple bypass heart surge~y is administered 500 rnlof the composition of E~ample 10 per hour using an intravenous drip system. During surgery, the patient's heart is stopped and perfusion continued through ar~ifiçial means.
Although compLications devdop while re~tarting the heart and disconne~ing the patient from the arti~al means of perfusion, the patient becomes consuous within severalhours of surgery. Within a few days, the patient's mental sta~us is nonnal with no indications of neurodegeneration.
It wDl b~ appreuated that certain variations may ~uggest themselves to those skilled in the art. The foregoing detailed description is to be clearly understood as 8 iven by way of illustration, the spirit and scope of this imention being interpreted 7C throuBh refercnc~ to the appended rJaims.
, SUB~ITUTE SHEE~
. . - , ~ .
: . ::
Those Calpain Inhibitors which possess significant Calpain Inhibitory ac~vi~ in vitro and also meet at least one of the foregoing or different tests for membrane permeability are e~cellent candidates ~or treatment of neurodegeneration.
G. DRIJG PEI~ -- - -. . .
SUBSTITU~E SH~
, . : ~. ; . . -:
WO 92/11850 2 ~ 0 9 PCr/US91/09786 The ability of ~he various Calpain I~ahibitors to penetrate pl~sma membranes is a si~icant advantage of these compounds from a pharmaceutical perspective. We believe that ~his ability, advantageously, allows the ~ain Inhibitors to provideexcellent permeation of the blood-brain barrier. This is in contrast ~o many phannaceuticals, especially pept;des, which often e~it poor penneatioII of the blood-bra~n banier. Thus, we believe that the Cal~ain In~bitors w~l e~bit e1~cellent results as pha~maceutically neuroprot~e agents.
For tr~tment of neurodegeneration, ~he Calpain Inhibitors can b~ adrninistered orally, topically or parenterally. The term ~parenteral" a3 wed herein includes all non-oral delivery techniques includ~ng transdermal admini~tration, subcutaneous injection, intravenous, intrarnuscular or intrasternal irijection, intrathecal injection (directly into the CNS) or infusion techniques.
The dosage depends primari~y on the specific formulation and on the object of the therapy or prophyla~s. '~e amount of the individual doses a~, well as the administration is best de~ermined by inclividually assessing the particular case.
However, in preferrsd compositions, the dosages of Calpain Inhibitors per day are preferably in the range of 1 ~g/kg ~otal body mass to 100 mg/lcg total body mass, more preferab~r in the range of 10 I g/kg total body mass to 10 mg/kg total body mass.
The pharmaceutical compositions contain~ng the active ingredient may be in a form s~utable for oral use, for ex~nple as tablets, troches, lozen~es, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or eli~drs. The amount of active ingredient that may be combined with carrier materials to produce a sin~le dosage ~orm will vary depending upon the host treated and the pardcular mode of adn~inistration. However, typically, a single dose will 2~ contain sufficient Calpain Inhl~itor to provide a complete dars dosage in a single orally ac eptable form.
For injecdon, the therapeutic amount of the Calpain Inhibitors or their pha~naceutically acceptable salts will normally be made by subcutaneous injection, intravenous, intrarnuscular or intrastemal injection, or by intrathecal injection (directly into th brain). In order to provide a ~ingle dars dose with a single injection, the pharmaceutical compositions for parenteral administration will contain"n a single dosage fonn, from about 70 l~g to about 7 g of ~ain ~ibitor per dose of from about 05 ml to about 1 liter of carrier solution. In addition to the active ingredient, these pharmaceudcal compositions ~vill usuaLly contain a buffer, e.g. a phosphate buffer SuBsTlTuTF.~
, .
' ' W092~1l850 2a9g~0~ Pcr/US9l/09786 that keeps the pH in the range from 35 to 7 and also sodiuun ehloride, and c~n also contain mannitol or sorbitol for adjusting the isotonic pressure. In a preferred form of these compositions, DMSO or other organie so}vent is added in order to assist the introduction of the Calpain Inhibitor across membranes.
Additionally, lipids c~n be introduced in~o the pharmaceutical compositions in order to $acilitate entry of the Calpain inhibi~or compounds into tissue of the CNS.
These compositions are prepared in accorda~ce Yvith methods lalown to those of skill in the art. Briefly, a lipid sueh a~, phosphatidyl ~oline, ch~lesteroL other well-hlown lipid carrier or ~tures thereo~ nth the ac~ve compound along with a solvent, the solvent is d ied off and the material reeonstituted in saline. The compositions can also include other ingredients ~own to those of ordirlary skill ~ the art, such as detergents, surfactants or emulsif~ing agents.
A composition for topical application or infusion can be formulated as an aqueous solution, lotion, jelly or an oily solution or suspension. A composition in the form of an aqueous solution is obtained ~y dissolving the Calpain Inhl~itor in aqueous buf~er solution of pH 4 to 65 and, if desired, adding a polymeric binder. An oily fonnulation for topical application is obtained by suspending the Calpain Inh~itor in an oil, optionally with the addition of a s~elling agent such as aluminium stearate and/or a surfactant. The addition of DMSO to these topical compositions is believed to allow at least partial penetration of the ac~ve Calpain Inhl~itor into the blood stream after application of the composition to the skin of a patient to aUow fortransdermal adn~nistration.
Por treatment of neurodegeneration resulting from e~citotoxici~t HIV-induced neuropathy, ischen~a following dener~ation or injwy, subarachnoid hemorrhage, stroke, multiple infarction dementi~, Alzheimer's Disease (A13),~Huntington's Disease, surgery-related bra~n damage, Parkinson's Disease, and other pathological conditions, the Calpain Inhibitors or pharmaceudcally acceptable salts thereof may be admin~tered orally or parenterally. The dosage depends primarily on the specific fonnulation and on the objec~ of the therapy or prophyla~s. I~e amount of the individual doses as well as the adn~il~istration is best detemuned by individually assessing the particular case.
In many acute neurodegenerative conditions and events, such as stroke and head isljury, it is Lmportant to del~ver the C~ain ~hl~itor as soon after injury as is practicable. Thus, it is preferable to identify those individuals who have suf~ered stroke, head injury or other inju~y in which neurodegen4ration is associated or is likely SIJE~STITUTE SH~ET
, , ... ~.- .. . .
, .. . .
W O 92/11850 2 ~ ~ 8 ~ 0 9 PC~r/US91/09786 ,',.'~ ,t to occur, and to begin administration of a C~apin ~hibl~or within 1 minute to 2 hours after the event, in order to preven~ as much neurodegeneration as possible.
A particular application of ~he Calpain Inhl~itors within the scope of ~he present invention is the application of these compounds dunng surgery to preventS rl~urodegeneration associated therewith. For example, for surgeries perforTned under general anesthesia, hypo~ic conditions can occur through inadequate perfusion of the CNS while under anesthesia. Additionally~ many major surgerie~ of the cardiovascular system require tnat a patient's heart be stopped and that perfusion be maintained through ~icial means In such surgeries, ~here ~s an increased danger of hypo~ia 10 occurring within the C NS, which can also result in r1eurodegeneration, Moreover, during neurosurgeries, there is an inheren~ risk of neurodegeneration resulting from inflammation, bleeding, hemorrhaging and the like. Such neurodegenera~ion can be~nk e~lby infusion with a solution conta~ning Calpain Inhibitor. However, Il . ...generation resulting from neurosurgery can also be reduced prophylactically by adr~inistration of a Calpain Inhl~itor through any of the foregoing adrninistration techniques. Such administration is also believed to inhl~it or prevent neurodegeneration associated with the u~e of anesthesia or with the use of artifical means of perfusion during major surgeries. A surgical pa~ient can also have Calpa~n Inhibitor administered throughout surgery throu~ intravenous drip.
l~e foJlowing e~amples are intended to illustrate certain neuroprotective uses of the Calpain ~}ubitors within the scope of the present invention. As such, they are not .oi lo limit the invention in any way.
~ Neuroprotective Composition fo~Intravenous Inje~tion 500 ~g CH3CONH-ClTPrOIC from E~ample SHC2 4 ml Propylene Glycol 1 rnl DMS--EXAMPLE lO
A Neuroprotective Composition fQr Intrave~ous Drip 250 mg ZLeu-Phe-CONH-Et ~om E~ample PKC 47 1000 rnl Phosphate BufEered Saline (pH 6.0) 10 ml D MSO
5URSTITI ITF .~L1~--:
: .
WO 92/t185(2 1~ 9 8 G ~ 3 P~US91/097~fi EX~MPLE 11 A l~europrotectiv~o~ositio~or Transdermal Application 25 mg~Leu-DL,Abu-COOEt ~rom E~ample PKC19 3 mlPhosphate BufEered Saline (pH 6.0) 2 rnlDMSO
EXAMPLE: 12 eu~o~rote~o~L after ~ead ~ury A first group of p~tient3 who are victims of head trauma is given 2 ml of the injectable composition of E~ample 9 intravenous~ hin ten minutes of the time of injury. A second group of similar~ matche~ patients does not recer~re the composition.
Ille first group of patients e~its marked~ fewer and les5 severe outward symptoms of neurodegeneration, such as dementia, memory loss and parahJsis.
~europrote~n Durin~ Sureerv A patient about to undereo a triple bypass heart surge~y is administered 500 rnlof the composition of E~ample 10 per hour using an intravenous drip system. During surgery, the patient's heart is stopped and perfusion continued through ar~ifiçial means.
Although compLications devdop while re~tarting the heart and disconne~ing the patient from the arti~al means of perfusion, the patient becomes consuous within severalhours of surgery. Within a few days, the patient's mental sta~us is nonnal with no indications of neurodegeneration.
It wDl b~ appreuated that certain variations may ~uggest themselves to those skilled in the art. The foregoing detailed description is to be clearly understood as 8 iven by way of illustration, the spirit and scope of this imention being interpreted 7C throuBh refercnc~ to the appended rJaims.
, SUB~ITUTE SHEE~
. . - , ~ .
: . ::
Claims
1. Use of a Calpain inhibitor compound or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
2. The use of Claim 1, wherein the neurodegeneration is occurring due to excitotoxicity, induced neuropathy, ischemia, denervation following ischemia or injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease.
3. The use of Claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier and is for parenteral administration.
4. The use of Claim 1, wherein the medicament is in a form suitable for oral use.
5. The use of Claim 3, wherein the medicament is for transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or infusion.
6. The use of Claim 1, wherein the medicament is for substantially preventing neurodegeneration in a patient undergoing surgery during and subsequent to the surgery.
7. The use Claim 6, wherein the medicament is for a patient undergoing neurosurgery, cardiovascular surgery or a surgery using general anesthesia.
8. The use of Claim 1, wherein said Calpain inhibitor compound enters tissue of the CNS of the mammal.
9. The use of Claim 8, wherein said Calpain inhibitor compound is membrane-permeant.
10. An in vitro method of selecting Calpain inhibitors for use as Calpain Inhibitor protectants in the in vivo treatment or inhibition of neurodegeneration, comprising:
identifying compounds having Calpain inhibitory activity in vitro; and identifying those compounds with Calpain inhibitory activity that are membrane permeant through an in vitro assay for membrane permeance.
11. The method of Claim 10, wherein the in vitro assay for membrane permeance comprises:
providing a plurality of tissue portions from a mammal;
treating at least one, but not all, of the tissue portions with a Calpain Inhibitor;
subjecting the tissue portions to an event that can cause degeneration in untreated tissue; and measuring the amount of degeneration that occurs in the tissue portions;
and comparing the amount of degeneration that occurs in the treated tissue portions with the amount of degeneration occurring in the untreated tissue portions, wherein an amount of degeneration in the treated tissue portions less than the amount of degeneration in the untreated tissue portions indicates that the Calpain Inhibitor is membrane-permeant.
12. The method of Claim 11, wherein the tissue portions comprise brain slices, platelets or cells in culture.
13. The method of Claim 11, wherein the measuring step comprises analyzing the tissue portions for the presence of the BDP's of a cytoskeletal component.
14. The method of Claim 13, wherein the cytoskeletal component is spectrin, MAP2, actin binding protein or tau.
15. The method of Claim 11, wherein the measuring step comprises measuring the electrical activity of the tissue portions.
16. The method of Claim 11, wherein the in vitro assay for membrane permeance comprises measuring the ability of the Calpain inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the platelets.
17. Use of a Substituted Heterocyclic Compound or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
18. The use of Claim 17, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
19. The use of Claim 17, wherein said Substituted Heterocyclic Compound comprises a member of the Class I Substituted Isocoumarins.
20. The use of Claim 17, wherein said Substituted Heterocyclic Compound comprises a member of the Class II Substituted Isocoumarins.
21. The use of Claim 17, wherein said Substituted Heterocydic Compound comprises a member of the Class m Hetercyclic Compounds.
22. The use of Claim 17, wherein said Substituted Hetelocyclic Compound is 3-chloroisocoumarin; a 3,4-dichloroisocoumarin; a 3-alkoxy-7-amino-4.
chloroisocoumarin; or 7-substituted 3-alkoxy-4-chloroisocoumarin.
23. The use of Claim 17, wherein said Substituted Heterocyclic Compound is CiTPrOIC, NH2-CiTPrOIC, PhCH2NHCONH-CiTPrOIC, CH3CONH-CiTPrOIC, L-Phe-NH-CiTPrOIC, PhCH2NHCONH-CiTEtOIC, PhCH2CONH-CiTEtOIC, or D-Phe-????????OIC.
24. Use of a Peptide Keto-Compound having Calpain inhibiting activies or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely experience a neuropathology associated with neurodegeneration.
25. The use of Claim 24, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
26. The use of Claim 24, wherein said Peptide Keto-Compound comprises a peptide .alpha.-ketoester.
27. The use of Claim 24, wherein said Peptide Keto-Compound comprises a peptide .alpha.-ketoacid.
28. The use of Claim 24, wherein said Peptide Keto-Compound comprises a tide .alpha.-ketoamide.
29. The use of Claim 24, wherein said Peptide Keto-Compaund comprises a compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoeisters (Subclass A), Tripepdde .alpha.-Ketoesters (Subdass B), Tetrapeptide .alpha.-Ketoesters, Amino Acid Peptide .alpha.-Ketoesters, Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids and Amino Acid Peptide .alpha.-Ketoacids, Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide a-Ketoamides (Subclass B), Tdpeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
30. Use of a Halo-Ketone Peptide having Calpain inhibitory activity or a pharmaeeutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneradon in a manunal having or likely to experience a neuropathology associated with neurodegeneration.
WO 92/11850 PCT/US91/097?
31. The use of Claim 30, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
32. The use of Claim 30, wherein said Halo-Ketone Peptide eomprises an amino-halo ketone peptide.
33. The use of Claim 30, wherein said Halo-Ketone Peptide comprises a diazo-ketone peptide.
34. The use of any one of Claims 17 through 33 wherein the neurodegeneration is associated with exitotoxicity, HIV-induced neuropathy, ischemia subarachnoid hemorrhage, stroke, brain seizure, major heart attack, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease or Parkinson's Disease.
35. The use of any one of Claims 17 through 33 wherein the medicament comprises a pharmaceutically acceptable carrier, and is for parenteral administration.
36. The method of any one of Claims 17 through 33 wherein the medicament is in a form suitable for oral use.
37. The use of Claim 35, wherein the administration comprises transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or an infusion technique.38. The use of any one of Claims 1 through 9 or 17 through 37, wherein the neurodegeneration is occurring from ischemia-inducing events, stroke, head injury, major heart attack brain seizure, near drowning, carbon monoxide poisoning, surgery-related brain damage or another event hlown to cause neurodegeneration.
39. A method of minimizing proteolysis in an in vivo sample containing peptides or proteins during or following the processing, production, preparation, isolation, purification, storage or transport of the samples, comprising the addition so the sample of a Substituted Heterocyclic Compound or a Peptide Keto-Compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapepdde .alpha.-Ketoesters, Amino Acid Peptide .alpha.-Ketoesters, Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids, Amino Acid Peptide .alpha.-Ketoacids, Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
40. A method of minimizing degradation resulting from Calpain activity in a tissue sample during or following preparation of the sample, comprising the addition to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo-Ketone Peptide.
41. The method of Claim 40, wherein the sample is a whole organ and the addition of compound comprises perfusion of the organ with the compound dissolved in fluid.
42. The method of Claim 40, wherein, following the addition step, said tissue sample is used in an assay for neurodegeneration wherein the assay comprises a test for the products of Calpain activity in the tissue samples.
43. The method of either Claim 39 or 40, wherein the addition of compound comprises addition of a Peptide .alpha.-Ketoacid to the sample.
44. The method of Claim 43, wherein said Peptide .alpha.-Ketoacid comprises a compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or Amino Acid Peptide .alpha.-Ketoacids.
45. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Substituted Heterocyclic Compound or a pharmaceutically acceptable salt or derivative thereof in a pharmaceutically acceptable formulation containing a carrier material.
46. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmaceutically effective neuroprotective amount of a Halo-Ketone Peptide or a pharmaceutically acceptable salt or derivative thereof in a pharmaceutically acceptable formulation containing a carrier material.
47. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapeptide .alpha.-Ketoesters, or Amino Acid Peptide .alpha.-Ketoesters.
48. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz-DL,Lys-COOEt, PhCO-Abu-COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu-COOEt or Ph(CH2)6CO-Abu-COOEt.
WO 92/11850 PCT/US91/0978t 49. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL,Ala-COOBzl, DL-ALa-COOnBu,Z-Leu-Nva-COOEt, Z-Leu-Nle-COOEt, Z-Leu-Phe-COOEt, Z-Leu-Abu-COOEt, Z-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, 2-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe CO2Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala-DL-Ala-COOMe, or Z-Leu-Abu-CO2Bzl.
50. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt. Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DL-Abu-COOOH2-C6H4-CF3 (para), H-Leu-Ala-DL-Lys-COOEt, Z-Leu-Leu-Abu-COOEt, Z-Leu-Leu-Phe-COOEt, MeO-Suc-Val-Pro-DL-Phe-COOMe or 2-NapSO2-Leu-Leu-Abu-COOEt.
51. The composition of Claim 47, wherein said Peptide Keto-Compound comprises MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe or Z-Ala-Ala-Ala-DL-Ala-COOEt.
52. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or the Amino Acid Peptide .alpha.-Ketoacids.
53. The composition of aaim 52, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL-Lys-COOH, Bz-DL-Ala-COOH, Z-Leu-Phe-COOH or Z-Leu-Abu-COOH.
54. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoanudes, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
55. Ihe composition of Claim 54, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Leu-Phe-CONH-Et, Z-Leu-Phe-CONH-nPr, Z-Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-Bzl, Z-Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu-Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, Z-Leu-Abu-CONH-BzL Z-Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O, Z-Leu-Abu-CONH-(C2)7CH3, Z-Leu Abu-CONH-(CH2)2OH, Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH, Z-Leu-Abu-CONH-(CH2)17CH3, Z-Leu-Abu-CONH-CH2 C6H3(OCH3)2 or Z-Leu-Abu CONH-CH2-C4H4N.
56. The composition of Claim 45, wherein said Substituted Heterocyclic Compound comprises a member of the Class I Substituted Isocoumarins, Class II
Substituted Isocoumarins or Class III Heterocyclic Compounds.
57. The composition of Claim 56, wherein said Substituted Heterocyclic compound is 3-chloroisocoumarin, a 3,4 dichloroisocoumarin, a 3-alkoxy-7-amino-4-chloroisocoumarin, a 7-substituted 3-alkoxy-4-chloroisocoumarin; CiTPrOIC, NH2-CiTPrOIC, PhCH2?H2CONH-CiTPrOIC, CH3CONH-CiTPrOIC, L-Phe-NH-CiTPrOIC, PhCH2NHCONH-CiTEtOIC, PhCH2CONH-CiTEtOIC, or D-Phe-NH-CiTEtOIC.
58. The composition of any one of Claims 45 through 57, wherein the composition is in dosage form comprising from 70 µg to 7 g of active ingredient in each dose.
59. The composition of any one of Claims 45 through 57, wherein said carrier material comprises a liquid, wherein the composition is in dosage form and wherein each dose comprises from 05 ml to 1 liter of said carrier material.
60. The composition of any one of Claims 45 through 57, additionally comprising at least one of the following: DMSO or other organic solvent, a lipidcarrier, a detergent, a surfactant or an emulsifying agent.
61. The composition of any one of Claims 45 through 57, wherein the composition is suitable for parenteral administration.
62. The composition of any one of Claims 45 through 57, wherein said composition is in a form suitable for topical application.
63. The composition of any one of Claims 45 through 57, wherein said composition comprises an aqueous solution, a lotion, a jely, an oily solution, or an oily suspension.
64. Use of a Substituted Heterocyclic Compound as a medicament.
65. Use of a Halo-Ketone Peptide as a medicament.
66. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide WO 92/11850 PCT/US91/0978?
.alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapeptide .alpha.-Ketoesters, or Amino Acid Peptide .alpha.-Ketoesters.
67. The use of Claim 66, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL,Ala-COOEt, Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz DL-Lys-COOEt, PhCO-Abu COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu COOEt or Ph(CH2)6CO-Abu-COOEt.
68. The use of aaim 66, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL-Ala-COOBzl, Z-Ala-DL-Ala-COOnBu, Z-Leu-Nva-COOEt, Z-Leu-Nle-COOEt, Z,Leu-Phe-COOEt, Z-Leu-Abu-COOEt, Z-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, Z-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe-CO2Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala-DL-Ala-COOMe, or Z-Leu-Abu CO2Bzl.
69. The use of Claim 66, wherein said Peptide Keto-Compound comprises one of the following compounds Z-Ala-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt, Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DI,Abu-COOOH2-C6H4-CF3(para), H-Leu-Ala-DL-Lys-COOEt, Z-Leu-Leu-Abu-COOEt, Z-Leu-Leu-Phe-COOEt, MeO-Suc-Val-Pro-DL-Phe-COOMe or 2-NapSO2-Leu-Leu-Abu-COOEt.
70. The use of Claim 66, wherein said Peptide Keto-Compound comprises a Tetrapeptide .alpha.-Ketoester.
71. The use of Claim 66, wherein said Peptide Keto-Compound comprises MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe or Z-Ala-Ala-Ala-DL-Ala-COOEt.
72. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or Amino Acid Peptide .alpha.-Ketoacids 73. The use of Claim 72, wherein said Peptide Keto-Compound comprises one of the following compounds Bz-DL-Lys-COOH, Bz-DL-Ala-COOH, Z-Leu-Phe-COOH or Z-Leu-Abu-COOH.
74. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoamides (Subclasses A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides 75. The use of Claim 74, wherein said Peptide Keto-Compound compnses one of the following compounds: Z-Leu-Phe-CONH-Et, Z-Leu-Phe-CONH-nPr, Z-Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-Bzl, Z-Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu-Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, ZLeu-Abu-CONH-Bzl, Z-Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O, Z-Leu-Abu-CONH-(CH2)7CH3, Z-Leu-Abu-CONH-(CH2)2OH, Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH, Z-Leu-Abu-CONH-(CH2)17CH3, Z-Leu-Abu-CONH-CH2-C6H3(OCH3)2 or Z-Leu-Abu-CONH-CH2-C4H4N.
2. The use of Claim 1, wherein the neurodegeneration is occurring due to excitotoxicity, induced neuropathy, ischemia, denervation following ischemia or injury, subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease.
3. The use of Claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier and is for parenteral administration.
4. The use of Claim 1, wherein the medicament is in a form suitable for oral use.
5. The use of Claim 3, wherein the medicament is for transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or infusion.
6. The use of Claim 1, wherein the medicament is for substantially preventing neurodegeneration in a patient undergoing surgery during and subsequent to the surgery.
7. The use Claim 6, wherein the medicament is for a patient undergoing neurosurgery, cardiovascular surgery or a surgery using general anesthesia.
8. The use of Claim 1, wherein said Calpain inhibitor compound enters tissue of the CNS of the mammal.
9. The use of Claim 8, wherein said Calpain inhibitor compound is membrane-permeant.
10. An in vitro method of selecting Calpain inhibitors for use as Calpain Inhibitor protectants in the in vivo treatment or inhibition of neurodegeneration, comprising:
identifying compounds having Calpain inhibitory activity in vitro; and identifying those compounds with Calpain inhibitory activity that are membrane permeant through an in vitro assay for membrane permeance.
11. The method of Claim 10, wherein the in vitro assay for membrane permeance comprises:
providing a plurality of tissue portions from a mammal;
treating at least one, but not all, of the tissue portions with a Calpain Inhibitor;
subjecting the tissue portions to an event that can cause degeneration in untreated tissue; and measuring the amount of degeneration that occurs in the tissue portions;
and comparing the amount of degeneration that occurs in the treated tissue portions with the amount of degeneration occurring in the untreated tissue portions, wherein an amount of degeneration in the treated tissue portions less than the amount of degeneration in the untreated tissue portions indicates that the Calpain Inhibitor is membrane-permeant.
12. The method of Claim 11, wherein the tissue portions comprise brain slices, platelets or cells in culture.
13. The method of Claim 11, wherein the measuring step comprises analyzing the tissue portions for the presence of the BDP's of a cytoskeletal component.
14. The method of Claim 13, wherein the cytoskeletal component is spectrin, MAP2, actin binding protein or tau.
15. The method of Claim 11, wherein the measuring step comprises measuring the electrical activity of the tissue portions.
16. The method of Claim 11, wherein the in vitro assay for membrane permeance comprises measuring the ability of the Calpain inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the platelets.
17. Use of a Substituted Heterocyclic Compound or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely to experience a neuropathology associated with neurodegeneration.
18. The use of Claim 17, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
19. The use of Claim 17, wherein said Substituted Heterocyclic Compound comprises a member of the Class I Substituted Isocoumarins.
20. The use of Claim 17, wherein said Substituted Heterocyclic Compound comprises a member of the Class II Substituted Isocoumarins.
21. The use of Claim 17, wherein said Substituted Heterocydic Compound comprises a member of the Class m Hetercyclic Compounds.
22. The use of Claim 17, wherein said Substituted Hetelocyclic Compound is 3-chloroisocoumarin; a 3,4-dichloroisocoumarin; a 3-alkoxy-7-amino-4.
chloroisocoumarin; or 7-substituted 3-alkoxy-4-chloroisocoumarin.
23. The use of Claim 17, wherein said Substituted Heterocyclic Compound is CiTPrOIC, NH2-CiTPrOIC, PhCH2NHCONH-CiTPrOIC, CH3CONH-CiTPrOIC, L-Phe-NH-CiTPrOIC, PhCH2NHCONH-CiTEtOIC, PhCH2CONH-CiTEtOIC, or D-Phe-????????OIC.
24. Use of a Peptide Keto-Compound having Calpain inhibiting activies or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneration in a mammal having or likely experience a neuropathology associated with neurodegeneration.
25. The use of Claim 24, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
26. The use of Claim 24, wherein said Peptide Keto-Compound comprises a peptide .alpha.-ketoester.
27. The use of Claim 24, wherein said Peptide Keto-Compound comprises a peptide .alpha.-ketoacid.
28. The use of Claim 24, wherein said Peptide Keto-Compound comprises a tide .alpha.-ketoamide.
29. The use of Claim 24, wherein said Peptide Keto-Compaund comprises a compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoeisters (Subclass A), Tripepdde .alpha.-Ketoesters (Subdass B), Tetrapeptide .alpha.-Ketoesters, Amino Acid Peptide .alpha.-Ketoesters, Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids and Amino Acid Peptide .alpha.-Ketoacids, Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide a-Ketoamides (Subclass B), Tdpeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
30. Use of a Halo-Ketone Peptide having Calpain inhibitory activity or a pharmaeeutically acceptable salt or derivative thereof for the manufacture of a medicament for inhibiting or treating neurodegeneradon in a manunal having or likely to experience a neuropathology associated with neurodegeneration.
WO 92/11850 PCT/US91/097?
31. The use of Claim 30, wherein the medicament is for inhibiting or treating neurodegeneration of the CNS.
32. The use of Claim 30, wherein said Halo-Ketone Peptide eomprises an amino-halo ketone peptide.
33. The use of Claim 30, wherein said Halo-Ketone Peptide comprises a diazo-ketone peptide.
34. The use of any one of Claims 17 through 33 wherein the neurodegeneration is associated with exitotoxicity, HIV-induced neuropathy, ischemia subarachnoid hemorrhage, stroke, brain seizure, major heart attack, multiple infarction dementia, Alzheimer's Disease, Huntington's Disease or Parkinson's Disease.
35. The use of any one of Claims 17 through 33 wherein the medicament comprises a pharmaceutically acceptable carrier, and is for parenteral administration.
36. The method of any one of Claims 17 through 33 wherein the medicament is in a form suitable for oral use.
37. The use of Claim 35, wherein the administration comprises transdermal administration, subcutaneous injection, intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the CNS or an infusion technique.38. The use of any one of Claims 1 through 9 or 17 through 37, wherein the neurodegeneration is occurring from ischemia-inducing events, stroke, head injury, major heart attack brain seizure, near drowning, carbon monoxide poisoning, surgery-related brain damage or another event hlown to cause neurodegeneration.
39. A method of minimizing proteolysis in an in vivo sample containing peptides or proteins during or following the processing, production, preparation, isolation, purification, storage or transport of the samples, comprising the addition so the sample of a Substituted Heterocyclic Compound or a Peptide Keto-Compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapepdde .alpha.-Ketoesters, Amino Acid Peptide .alpha.-Ketoesters, Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids, Amino Acid Peptide .alpha.-Ketoacids, Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
40. A method of minimizing degradation resulting from Calpain activity in a tissue sample during or following preparation of the sample, comprising the addition to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo-Ketone Peptide.
41. The method of Claim 40, wherein the sample is a whole organ and the addition of compound comprises perfusion of the organ with the compound dissolved in fluid.
42. The method of Claim 40, wherein, following the addition step, said tissue sample is used in an assay for neurodegeneration wherein the assay comprises a test for the products of Calpain activity in the tissue samples.
43. The method of either Claim 39 or 40, wherein the addition of compound comprises addition of a Peptide .alpha.-Ketoacid to the sample.
44. The method of Claim 43, wherein said Peptide .alpha.-Ketoacid comprises a compound that is a member of one of the following subclasses: Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or Amino Acid Peptide .alpha.-Ketoacids.
45. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Substituted Heterocyclic Compound or a pharmaceutically acceptable salt or derivative thereof in a pharmaceutically acceptable formulation containing a carrier material.
46. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmaceutically effective neuroprotective amount of a Halo-Ketone Peptide or a pharmaceutically acceptable salt or derivative thereof in a pharmaceutically acceptable formulation containing a carrier material.
47. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide .alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapeptide .alpha.-Ketoesters, or Amino Acid Peptide .alpha.-Ketoesters.
48. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz-DL,Lys-COOEt, PhCO-Abu-COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu-COOEt or Ph(CH2)6CO-Abu-COOEt.
WO 92/11850 PCT/US91/0978t 49. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL,Ala-COOBzl, DL-ALa-COOnBu,Z-Leu-Nva-COOEt, Z-Leu-Nle-COOEt, Z-Leu-Phe-COOEt, Z-Leu-Abu-COOEt, Z-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, 2-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe CO2Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala-DL-Ala-COOMe, or Z-Leu-Abu-CO2Bzl.
50. The composition of Claim 47, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt. Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DL-Abu-COOOH2-C6H4-CF3 (para), H-Leu-Ala-DL-Lys-COOEt, Z-Leu-Leu-Abu-COOEt, Z-Leu-Leu-Phe-COOEt, MeO-Suc-Val-Pro-DL-Phe-COOMe or 2-NapSO2-Leu-Leu-Abu-COOEt.
51. The composition of Claim 47, wherein said Peptide Keto-Compound comprises MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe or Z-Ala-Ala-Ala-DL-Ala-COOEt.
52. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or the Amino Acid Peptide .alpha.-Ketoacids.
53. The composition of aaim 52, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL-Lys-COOH, Bz-DL-Ala-COOH, Z-Leu-Phe-COOH or Z-Leu-Abu-COOH.
54. A pharmaceutical composition for the treatment or inhibition of neurodegeneration comprising a pharmacologically effective neuroprotective amount of a Peptide Keto-Compound, wherein said Peptide Keto-Compound comprises a compound from one of the following subclasses: Dipeptide .alpha.-Ketoamides (Subclass A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoanudes, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides.
55. Ihe composition of Claim 54, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Leu-Phe-CONH-Et, Z-Leu-Phe-CONH-nPr, Z-Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-Bzl, Z-Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu-Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, Z-Leu-Abu-CONH-BzL Z-Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O, Z-Leu-Abu-CONH-(C2)7CH3, Z-Leu Abu-CONH-(CH2)2OH, Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH, Z-Leu-Abu-CONH-(CH2)17CH3, Z-Leu-Abu-CONH-CH2 C6H3(OCH3)2 or Z-Leu-Abu CONH-CH2-C4H4N.
56. The composition of Claim 45, wherein said Substituted Heterocyclic Compound comprises a member of the Class I Substituted Isocoumarins, Class II
Substituted Isocoumarins or Class III Heterocyclic Compounds.
57. The composition of Claim 56, wherein said Substituted Heterocyclic compound is 3-chloroisocoumarin, a 3,4 dichloroisocoumarin, a 3-alkoxy-7-amino-4-chloroisocoumarin, a 7-substituted 3-alkoxy-4-chloroisocoumarin; CiTPrOIC, NH2-CiTPrOIC, PhCH2?H2CONH-CiTPrOIC, CH3CONH-CiTPrOIC, L-Phe-NH-CiTPrOIC, PhCH2NHCONH-CiTEtOIC, PhCH2CONH-CiTEtOIC, or D-Phe-NH-CiTEtOIC.
58. The composition of any one of Claims 45 through 57, wherein the composition is in dosage form comprising from 70 µg to 7 g of active ingredient in each dose.
59. The composition of any one of Claims 45 through 57, wherein said carrier material comprises a liquid, wherein the composition is in dosage form and wherein each dose comprises from 05 ml to 1 liter of said carrier material.
60. The composition of any one of Claims 45 through 57, additionally comprising at least one of the following: DMSO or other organic solvent, a lipidcarrier, a detergent, a surfactant or an emulsifying agent.
61. The composition of any one of Claims 45 through 57, wherein the composition is suitable for parenteral administration.
62. The composition of any one of Claims 45 through 57, wherein said composition is in a form suitable for topical application.
63. The composition of any one of Claims 45 through 57, wherein said composition comprises an aqueous solution, a lotion, a jely, an oily solution, or an oily suspension.
64. Use of a Substituted Heterocyclic Compound as a medicament.
65. Use of a Halo-Ketone Peptide as a medicament.
66. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoesters (Subclass A), Dipeptide .alpha.-Ketoesters (Subclass B), Tripeptide WO 92/11850 PCT/US91/0978?
.alpha.-Ketoesters (Subclass A), Tripeptide .alpha.-Ketoesters (Subclass B), Tetrapeptide .alpha.-Ketoesters, or Amino Acid Peptide .alpha.-Ketoesters.
67. The use of Claim 66, wherein said Peptide Keto-Compound comprises one of the following compounds: Bz-DL,Ala-COOEt, Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz DL-Lys-COOEt, PhCO-Abu COOEt, (CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu COOEt or Ph(CH2)6CO-Abu-COOEt.
68. The use of aaim 66, wherein said Peptide Keto-Compound comprises one of the following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL-Ala-COOBzl, Z-Ala-DL-Ala-COOnBu, Z-Leu-Nva-COOEt, Z-Leu-Nle-COOEt, Z,Leu-Phe-COOEt, Z-Leu-Abu-COOEt, Z-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt, Z-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe-CO2Bu, Z-Leu-Abu-CO2Bu, Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala-DL-Ala-COOMe, or Z-Leu-Abu CO2Bzl.
69. The use of Claim 66, wherein said Peptide Keto-Compound comprises one of the following compounds Z-Ala-Ala-DL-Ala-COOEt, Z-Ala-Pro-DL-Ala-COOEt, Z-Ala-Ala-DL-Abu-COOEt, Z-Ala-Ala-DL-Abu-COOBzl, Z-Ala-Ala-DI,Abu-COOOH2-C6H4-CF3(para), H-Leu-Ala-DL-Lys-COOEt, Z-Leu-Leu-Abu-COOEt, Z-Leu-Leu-Phe-COOEt, MeO-Suc-Val-Pro-DL-Phe-COOMe or 2-NapSO2-Leu-Leu-Abu-COOEt.
70. The use of Claim 66, wherein said Peptide Keto-Compound comprises a Tetrapeptide .alpha.-Ketoester.
71. The use of Claim 66, wherein said Peptide Keto-Compound comprises MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe or Z-Ala-Ala-Ala-DL-Ala-COOEt.
72. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoacids (Subclass A), Dipeptide .alpha.-Ketoacids (Subclass B), Tripeptide .alpha.-Ketoacids, Tetrapeptide .alpha.-Ketoacids or Amino Acid Peptide .alpha.-Ketoacids 73. The use of Claim 72, wherein said Peptide Keto-Compound comprises one of the following compounds Bz-DL-Lys-COOH, Bz-DL-Ala-COOH, Z-Leu-Phe-COOH or Z-Leu-Abu-COOH.
74. Use of a Peptide Keto-Compound as a medicament, wherein said Peptide Keto-Compound is a compound from one of the following subclasses:
Dipeptide .alpha.-Ketoamides (Subclasses A), Dipeptide .alpha.-Ketoamides (Subclass B), Tripeptide .alpha.-Ketoamides, Tetrapeptide .alpha.-Ketoamides or Amino Acid .alpha.-Ketoamides 75. The use of Claim 74, wherein said Peptide Keto-Compound compnses one of the following compounds: Z-Leu-Phe-CONH-Et, Z-Leu-Phe-CONH-nPr, Z-Leu-Phe-CONH-nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-Bzl, Z-Leu-Phe-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-Et, Z-Leu-Abu-CONH-nPr, Z-Leu-Abu-CONH-nBu, Z-Leu-Abu-CONH-iBu, ZLeu-Abu-CONH-Bzl, Z-Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)2O, Z-Leu-Abu-CONH-(CH2)7CH3, Z-Leu-Abu-CONH-(CH2)2OH, Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH, Z-Leu-Abu-CONH-(CH2)17CH3, Z-Leu-Abu-CONH-CH2-C6H3(OCH3)2 or Z-Leu-Abu-CONH-CH2-C4H4N.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US635,952 | 1984-07-30 | ||
| US63595290A | 1990-12-28 | 1990-12-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2098609A1 true CA2098609A1 (en) | 1992-06-29 |
Family
ID=24549781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002098609A Abandoned CA2098609A1 (en) | 1990-12-28 | 1991-12-27 | Use of calpain inhibitors in the inhibition and treatment of neurodegeneration |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0564552A1 (en) |
| JP (1) | JPH06504061A (en) |
| AU (2) | AU667463B2 (en) |
| CA (1) | CA2098609A1 (en) |
| WO (1) | WO1992011850A2 (en) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5736520A (en) * | 1988-10-07 | 1998-04-07 | Merrell Pharmaceuticals Inc. | Peptidase inhibitors |
| EP0580161A1 (en) * | 1992-07-22 | 1994-01-26 | THE McLEAN HOSPITAL CORPORATION | Prophylactic and therapeutic treatment of Alzheimer's disease |
| US5371072A (en) * | 1992-10-16 | 1994-12-06 | Corvas International, Inc. | Asp-Pro-Arg α-keto-amide enzyme inhibitors |
| US5541290A (en) * | 1993-06-24 | 1996-07-30 | Harbeson; Scott L. | Optically pure calpain inhibitor compounds |
| US5977074A (en) * | 1993-10-01 | 1999-11-02 | Merrell Pharmaceuticals, Inc. | Inhibitors of β-amyloid protein production |
| US5637589A (en) * | 1993-10-29 | 1997-06-10 | University Of North Carolina At Chapel Hill | Suksdorfin, analogs, compositions thereof, and methods for making and using thereof |
| US5693617A (en) * | 1994-03-15 | 1997-12-02 | Proscript, Inc. | Inhibitors of the 26s proteolytic complex and the 20s proteasome contained therein |
| US6660268B1 (en) | 1994-03-18 | 2003-12-09 | The President And Fellows Of Harvard College | Proteasome regulation of NF-KB activity |
| US5536639A (en) * | 1994-03-25 | 1996-07-16 | Cephalon, Inc. | Methods for detecting calpain activation by detection of calpain activated spectrin breakdown products |
| US6319929B1 (en) | 1994-04-29 | 2001-11-20 | The University Of North Carolina At Chapel Hill | Suksdorfin analogs, compositions thereof, and methods for making and using thereof |
| US6083903A (en) | 1994-10-28 | 2000-07-04 | Leukosite, Inc. | Boronic ester and acid compounds, synthesis and uses |
| AU3935895A (en) * | 1994-11-24 | 1996-06-17 | Takeda Chemical Industries Ltd. | Alpha-ketoamide derivatives as cathepsin l inhibitor |
| US5691368A (en) * | 1995-01-11 | 1997-11-25 | Hoechst Marion Roussel, Inc. | Substituted oxazolidine calpain and/or cathepsin B inhibitors |
| US5723580A (en) * | 1995-09-14 | 1998-03-03 | Cephalon, Inc. | Ketomethylene group-containing aldehyde cysteine and serine protease inhibitors |
| US5827877A (en) * | 1995-09-14 | 1998-10-27 | Cephalon, Inc. | Ketomethylene group-containing cysteine and serine protease inhibitors |
| ES2293651T3 (en) * | 1995-11-28 | 2008-03-16 | Cephalon, Inc. | INHIBITORS OF CYSTEINE AND SERINE PROTEASES DERIVED FROM AMINO ACIDS D. |
| DE19642591A1 (en) * | 1996-10-15 | 1998-04-16 | Basf Ag | New piperidine-ketocarboxylic acid derivatives, their production and use |
| WO1998018485A1 (en) | 1996-10-31 | 1998-05-07 | Alcon Laboratories, Inc. | The use of calpain inhibitors to treat ocular neural pathology |
| DE19650975A1 (en) | 1996-12-09 | 1998-06-10 | Basf Ag | New heterocyclically substituted benzamides and their application |
| SK282680B6 (en) * | 1996-12-11 | 2002-11-06 | Basf Aktiengesellschaft | Ketobenzamides, pharmaceutical preparations containing them and t heir us for disease suppression |
| PL342970A1 (en) | 1998-03-20 | 2001-07-16 | Senju Pharma Co | Pharmaceutical composition containing a cysteine protease inhibitor for prophylaxis and therapy of brain tissue impairment |
| DE19818614A1 (en) | 1998-04-20 | 1999-10-21 | Basf Ag | New benzamide derivatives useful as cysteine protease inhibitors for treating neurodegenerative diseases, neuronal damage, stroke, cranial trauma, Alzheimer's disease, etc. |
| DE19817459A1 (en) | 1998-04-20 | 1999-10-21 | Basf Ag | New heterocyclic amide derivatives useful as cysteine protease inhibitors for treating neurodegenerative diseases, neuronal damage, stroke, cranial trauma, Alzheimer's disease, etc. |
| TR200003004T2 (en) | 1998-04-20 | 2001-02-21 | Basf Aktiengesellschaft | Heterocyclic substituted amides as calpain inhibitors |
| EP1073641B1 (en) | 1998-04-20 | 2004-04-14 | Abbott GmbH & Co. KG | New substituted amides, their production and their use |
| KR20010043799A (en) * | 1998-05-25 | 2001-05-25 | 스타르크, 카르크 | New Heterocyclically Substituted Amides, Their Production and Their Use |
| SK18302000A3 (en) | 1998-06-03 | 2001-06-11 | Gpi Nil Holdings, Inc. | N-linked sulfonamides of n-heterocyclic carboxylic acids or carboxylic acid isosteres |
| WO2000021550A2 (en) * | 1998-10-13 | 2000-04-20 | President And Fellows Of Harvard College | Methods and compositions for treating neurodegenerative diseases |
| US7253169B2 (en) | 1999-11-12 | 2007-08-07 | Gliamed, Inc. | Aza compounds, pharmaceutical compositions and methods of use |
| US6417189B1 (en) | 1999-11-12 | 2002-07-09 | Gpi Nil Holdings, Inc. | AZA compounds, pharmaceutical compositions and methods of use |
| AU1464101A (en) | 1999-12-21 | 2001-07-03 | Gpi Nil Holdings, Inc. | Hydantoin derivative compounds, pharmaceutical compositions, and methods of using same |
| DE60141608D1 (en) | 2000-07-21 | 2010-04-29 | Dendreon Corp | PEPTIDES AS INHIBITORS OF THE NS3 SERINE PROTEASE OF HEPATITIS C VIRUS |
| US7244721B2 (en) | 2000-07-21 | 2007-07-17 | Schering Corporation | Peptides as NS3-serine protease inhibitors of hepatitis C virus |
| MXPA03000626A (en) | 2000-07-21 | 2004-07-30 | Schering Corp | Novel peptides as ns3-serine protease inhibitors of hepatitis c virus. |
| FR2817867B1 (en) * | 2000-12-11 | 2006-06-30 | Trophos | ISOCOUMARINS INHIBITORS OF AMYLOID PEPTIDE PRODUCTION, PREPARATION, COMPOSITIONS CONTAINING SAME AND USES THEREOF |
| DE10105041A1 (en) * | 2001-02-05 | 2002-08-14 | Tell Pharm Ag Hergiswil | Tripeptides and tripeptide derivatives for the treatment of neurodegenerative diseases |
| DE10105038B4 (en) | 2001-02-05 | 2005-07-07 | Neurotell Ag | Tripeptide derivatives for the treatment of postläsional diseases of the nervous system |
| 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 |
| US7282512B2 (en) | 2002-01-17 | 2007-10-16 | Smithkline Beecham Corporation | Cycloalkyl ketoamides derivatives useful as cathepsin K inhibitors |
| US10531655B2 (en) | 2011-12-02 | 2020-01-14 | The Regents Of The University Of California | Reperfusion protection solution and uses thereof |
| US10076384B2 (en) | 2013-03-08 | 2018-09-18 | Symple Surgical, Inc. | Balloon catheter apparatus with microwave emitter |
| CA2963684A1 (en) * | 2014-11-11 | 2016-05-19 | Western University Of Health Sciences | Isoform-specific calpain inhibitors, methods of identification, and uses thereof |
-
1991
- 1991-12-27 WO PCT/US1991/009786 patent/WO1992011850A2/en not_active Application Discontinuation
- 1991-12-27 CA CA002098609A patent/CA2098609A1/en not_active Abandoned
- 1991-12-27 JP JP4503767A patent/JPH06504061A/en active Pending
- 1991-12-27 AU AU91527/91A patent/AU667463B2/en not_active Ceased
- 1991-12-27 EP EP92902904A patent/EP0564552A1/en not_active Withdrawn
-
1996
- 1996-06-11 AU AU55905/96A patent/AU5590596A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU667463B2 (en) | 1996-03-28 |
| EP0564552A1 (en) | 1993-10-13 |
| WO1992011850A2 (en) | 1992-07-23 |
| AU9152791A (en) | 1992-08-17 |
| JPH06504061A (en) | 1994-05-12 |
| AU5590596A (en) | 1996-08-22 |
| WO1992011850A3 (en) | 1992-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2098609A1 (en) | Use of calpain inhibitors in the inhibition and treatment of neurodegeneration | |
| US5444042A (en) | Method of treatment of neurodegeneration with calpain inhibitors | |
| CA2138124A1 (en) | Use of calpain inhibitors in the inhibition and treatment of medical conditions associated with increased calpain activity | |
| DE60119968T2 (en) | HEPATITIS C TRIPEPTIDE INHIBITORS | |
| ES2323159T3 (en) | COMPOUNDS WITH RELEASE PROPERTIES OF THE HORMONE OF GROWTH. | |
| JP3159806B2 (en) | Thrombin inhibitor | |
| EP1506000B1 (en) | Heterocyclicsulfonamide hepatitis c virus inhibitors | |
| KR100611540B1 (en) | Dolastatin 10 derivatives | |
| RU2067585C1 (en) | Derivatives of 5-amino-4-hydroxyhexanoic acid and a method of their synthesis | |
| JPH0789935A (en) | Heterocyclic inhibitor of farnesyl protein transferase | |
| RU2092492C1 (en) | Derivatives of hydrazine or their salts, pharmaceutical agent, aminoalkylhydrazines or their salts | |
| FR2609289A1 (en) | NOVEL COMPOUNDS WITH ACTIVITY OF COLLAGENASE INHIBITORS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAME | |
| EP0356223A2 (en) | HIV protease inhibitors useful for the treatment of aids | |
| HU189531B (en) | Process for producing alanin derivatives | |
| DE10143840A1 (en) | New acylated hydroxamates useful for the treatment of e.g. wound healing | |
| JPH11514330A (en) | Peptide and peptide analog protease inhibitors | |
| WO1998012210A1 (en) | 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamido azetidin-2-one derivatives as cysteine proteinase regulators | |
| US6500802B1 (en) | Peptidyl-2-amino-1-hydroxyalkanesulfonic acid cysteine protease inhibitors | |
| CA2022685A1 (en) | Nitrosothiol derivatives, their production and use | |
| AU2005214388A1 (en) | Propenoyl hydrazides | |
| US7056947B2 (en) | Aza-peptide epoxides | |
| JPS60136595A (en) | Substituted ethylenediamine derivative, manufacture and medicine | |
| KR20000016700A (en) | Depsipeptides and drugs containing the same as the active ingredient | |
| CA2126687A1 (en) | Substituted phosphinic acid-containing peptidyl derivatives as antidegenerative agents | |
| JP4263095B2 (en) | Pyran derivatives as inhibitors of both ACE and NEP |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |