WO2000032620A1 - Gamma-ketoacid tetrapeptides as inhibitors of caspase-3 - Google Patents

Gamma-ketoacid tetrapeptides as inhibitors of caspase-3 Download PDF

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Publication number
WO2000032620A1
WO2000032620A1 PCT/CA1999/001145 CA9901145W WO0032620A1 WO 2000032620 A1 WO2000032620 A1 WO 2000032620A1 CA 9901145 W CA9901145 W CA 9901145W WO 0032620 A1 WO0032620 A1 WO 0032620A1
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amino
group
aryl
acid
acetylamino
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PCT/CA1999/001145
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French (fr)
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Erich L. Grimm
Johanne Renaud
Renee Aspiotis
Christopher I. Bayly
Robert Zamboni
Shawn Black
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Merck Frosst Canada & Co.
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Priority to JP2000585261A priority Critical patent/JP2002531464A/en
Priority to AU13706/00A priority patent/AU764860B2/en
Priority to CA002353079A priority patent/CA2353079A1/en
Priority to EP99973035A priority patent/EP1135406A1/en
Publication of WO2000032620A1 publication Critical patent/WO2000032620A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Apoptotic cell suicide is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis, however, underlies the etiology of many of the most intractable of human diseases. In only the last few years, many of the molecules that participate in a conserved biochemical pathway that mediates the highly ordered process of apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the 'caspases', that are related to mammalian interleukin-l ⁇ converting enzyme (ICE/caspase-1) and to CED-3, the product of a gene that is necessary for apoptotic suicide in the nematode C.
  • ICE/caspase-1 mammalian interleukin-l ⁇ converting enzyme
  • CED-3 the product of a gene that is necessary for apoptotic suicide in the nematode C.
  • caspase inhibitors would thus be useful for the treatment of human diseases including, but not limited to, acute disorders such as cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, as well as chronic disorders such as neurodegenerative diseases (e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging.
  • acute disorders such as cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, as well as chronic disorders such as neurodegenerative diseases (e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging.
  • caspases have so far been identified in human cells. Each is synthesized as a catalytically dormant proenzyme containing an amino-terminal prodomain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-X junctions (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The strict requirement by caspases for Asp in the Pi position of substrates is consistent with a mechanism whereby proenzyme maturation can be either autocatalytic or performed by other caspases.
  • the three dimensional crystal structures of mature caspase-1 and -3 show that the large subunit contains the principle components of the catalytic machinery, including the active site Cys residue which is harbored within the conserved pentapeptide motif, QACxG,l and residues that stabilize the oxyanion of the tetrahedral transition state (Wilson et al., 1994, Nature 370:270-75; Walker et al., 1994, Cell 78:342-52; Rotonda et al., 1996, Nat Struct Biol 3:619-25).
  • Both subunits contribute residues which stabilize the Pi Asp of substrates while the small subunit appears to contain most of the determinants that dictate substrate specificity and, in particular, those which form the specificity-determining S4 subsite.
  • One distinctive feature of these proteases is the absolute requirement for an aspartic acid residue in the substrate Pi position.
  • the carboxylate side chain of the substrate Pi Asp is tethered by four residues in caspase-1 (Argl79, Gln238 f rom p20 and Arg341, Ser347 f ro plO) that are absolutely conserved in all caspase family members.
  • Catalysis involves a typical cysteine protease mechanism involving a catalytic dyad, composed of His237 and Cys285 (contained within an absolutely conserved QACxG pentapeptide) and an Oxyanion hole' involving Gly238 and Cys285.
  • Inhibitors bind, however, in an unexpected non-transition state configuration (which raises important considerations for inhibitor design) with the oxyanion of the thiohemiacetal being stabilized by the active site His237.
  • Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach.
  • group II caspases which include caspases-2, -3 and -7 as well as C. elegans CED-3) have specificity for [P4]DExD[P ⁇ ], a motif found at the cleavage site of most proteins known to be cleaved during apoptosis.
  • group III caspases caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B
  • group III caspases is [P4](I,V,L)ExD[P ⁇ ] which corresponds to the activation site at the junction between the large and small subunits of other caspase proenzymes including group II (effector) family members.
  • group III caspases function as upstream activators of group II caspases in a proteolytic cascade that amplifies the death signal.
  • group I caspases (caspases-1, -4 and -5) appears to be to mediate cytokine maturation and their role in apoptosis, if any, has not been substantiated.
  • a tetrapeptide corresponding to the substrate P4-P1 residues is sufficient for specific recognition by caspases and as a consequence has formed the basis for inhibitor design.
  • the P4 residue in particular appears to be most important for substrate recognition and specificity.
  • Caspase-1 for example, prefers a hydrophobic residue such as Tyr in P4 (which corresponds to its YVHD cleavage site within proIL-l ⁇ ) whereas caspase-3 (and other group II enzymes) has a preference for an anionic Asp residue (which corresponds to the DXXD cleavage sites within most polypeptides that are cleaved by these enzymes during apoptosis).
  • Peptide aldehydes, nitriles and ketones are potent reversible inhibitors of these proteases while compounds that form thiomethylketone adducts with the active site cysteine (e.g. peptide (acyloxy)methylketones) are potent irreversible inhibitors.
  • the tetrapeptide aldehyde Ac-YVAD-CHO (which was designed to mimic the YVHD caspase-1 recognition sequence within proIL-l ⁇ ) is a potent inhibitor of caspase-1 (Ki ⁇ 1 nM) but
  • the Ac-DEVD-CHO tetrapeptide aldehyde (which was designed to mimic the caspase-3 recognition site) is a very potent inhibitor of caspase-3 (Ki ⁇ 1 nM) although it is also a weaker but reasonable inhibitor of caspase-1, presumably owing to promiscuity in the S4 subsite of this enzyme (Nicholson et al., 1995, Nature 376:37-43).
  • R is selected from the group consisting of:
  • aryl or arylC ⁇ _6alkyl wherein the aryl group is selected from the group consisting of:
  • R3 is selected from the group consisting of:
  • R4 is selected from the group consisting of:
  • R4 and X4 together form a saturated monocyclic ring having the following structure:
  • R5 is selected from the group consisting of:
  • arylC ⁇ _8alkyl wherein the aryl is selected from the group consisting of: (1) phenyl,
  • R6 and R ⁇ are independently selected from the group consisting of:
  • X2, X3 and X4 are independently H or X2 and R2, ⁇ 3 and R3,or X4 and R4 may together form a saturated monocyclic ring having the following structure:
  • the invention also encompasses a pharmaceutical composition comprising a compound of formula I in combination with a pharmacuetically acceptable carrier.
  • the invention also encompasses a method of treating cardiac and cerebral ischemia reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I.
  • the invention encompasses the novel class of compounds represented by formula I, which are caspase-3 inhibitors.
  • R is selected from the group consisting of:
  • R1 is selected from the group consisting of: (a) hydrogen,
  • aryl or arylC ⁇ _6alkyl wherein the aryl group is selected from the group consisting of:
  • R is selected from the group consisting of:
  • R4 is selected from the group consisting of:
  • R5 is selected from the group consisting of:
  • arylCi_8alkyl wherein the aryl is selected from the group consisting of:
  • R and R7 are independently selected from the group consisting of:
  • R and R7 may be joined to form a pyrrolidine, piperidine, morpholine, thiamorpholine or N-R8 substituted piperazine wherein R8 is H or Ci_3alkyl; and X2, X3 and X4 are independently H or X2 and R2, ⁇ 3 and R3,or X4 and R4 may together form a saturated monocyclic ring having the following structure:
  • amino acids from which the structure is constructed are selected from a group consisting of the L- and D- forms of the amino acids including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine and valine.
  • the structures of the L-amino acids are shown below.
  • a preferred embodiment of the invention is that wherein R is Ci_6alkyl or phenyl.
  • R ⁇ is arylCi_8alkyl, wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or disubstituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
  • R is selected from the group consisting of:
  • R5 is arylC ⁇ _8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or disubstituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
  • Ci- 3 alkyl (7) C ⁇ _ 3 fluoroalkyl
  • Rl is selected from the group consisting of:
  • aryl or arylCi- ⁇ alkyl wherein the aryl group is selected from the group consisting of: (1) phenyl and
  • R2 is selected from the group consisting of:
  • R3 is selected from the group consisting of:
  • a preferred embodiment of the present invention is that wherein R4 is isopropyl.
  • R5 is selected from the group consisting of:
  • arylCi_8alkyl wherein the aryl is selected from the group consisting of:
  • R5 is selected from the group consisting of:
  • arylC ⁇ _8alkyl wherein the aryl is selected from the group consisting of:
  • Ci_3fluoroalkyl (8) C ⁇ _3alkylcarbonyl,
  • R5 is selected from the group consisting of:
  • R 1 is selected from the group consisting of:
  • R5 is arylC ⁇ _8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or di- substituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
  • Rl is selected from the group consisting of:
  • R5 is arylC3_5alkyl wherein aryl is selected from the group consisting of phenyl and naphthyl.
  • R9 is selected from the group consisting of: (a) CO 2 H,
  • RlO is selected from the group consisting of:
  • Ar is selected from the group consisting of:
  • R9 is selected from the group consisting of: (a) CO 2 H, (b) SCH3 and
  • RlO is selected from the group consisting of: (a) H and (d) CH2S(0)2CH 3 ; and
  • Ar is phenyl or napthyl.
  • the invention also encompasses the following compound:
  • the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said caspase-3 mediated disease.
  • the invention encompasses a method of treating cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I.
  • cardiac and cerebral ischemia/reperfusion injury e.g. stroke
  • type I diabetes e.g. stroke
  • immune deficiency syndrome including AIDS
  • cerebral and spinal cord trauma injury e.g. stroke
  • organ damage during transplantation e.g., alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders
  • the invention encompasses a method of treating acute disorders, including cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said acute disorder.
  • the invention encompasses a method of treating chronic disorders, including neurodegenerative diseases (e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said chronic disorder.
  • neurodegenerative diseases e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis
  • immunodeficiency e.g. HIV
  • diabetes alopecia and aging
  • the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula II in an amount effective to treat said caspase-3 mediated disease.
  • the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula III in an amount effective to treat said caspase-3 mediated disease.
  • the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula IV in an amount effective to treat said caspase-3 mediated disease.
  • DCC 1,3-dicyclohexylcarbodiimide
  • DIBAL diisobutyl aluminum hydride
  • DIEA N,N-diisoproylethylamine
  • EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • EDTA ethylenediaminetetraacetic acid, tetrasodium salt hydrate
  • FAB fast atom bombardment
  • HMPA hexamethylphosphoramide
  • HATU O-(7-Azabenzotriazol-l-yl)N,N,N',N'- tetramethyluronium hexafluorophosphate
  • alkyl means linear, branched or cyclic structures and combinations thereof, containing one to twenty carbon atoms.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl,
  • SUBST ⁇ UTE SHEET (RULE 26) tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl- 4-propylnonyl, cyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl, 2-ethyl-l- bicyclo[4.4.0]decyl and the like.
  • Alkylcarbonyl signifies groups having the formula - C(O)-alkyl, wherein alkyl is defined as above.
  • Alkylsulfonyl signifies groups having the formula - S(O)2-alkyl, wherein alkyl is defined as above.
  • fluoroalkyl means linear, branched or cyclic alkyl groups and combinations thereof, of one to ten carbon atoms, in which one or more hydrogen but no more than six is replaced by fluorine. Examples are -CF 3 , - CH2CH2F, and -CH2CF3, and the like.
  • Alkoxy means alkoxy groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and the like.
  • Alkoxycarbonyl signifies groups having the formula - C(0)-alkoxy, wherein alkoxy is defined as above.
  • Alkylthio means alkylthio groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkylthio groups include methylthio, propylthio, isopropylthio, etc. By way of illustration, the propylthio group signifies -SCH 2 CH 2 CH 3 .
  • Aryl is, for example, phenyl, naphthyl, pyridyl, furyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl and coumarinyl.
  • Halo includes F, Cl, Br and I.
  • the compounds described typically contain asymmetric centers and may thus give rise to diastereomers and optical isomers.
  • the present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
  • compositions of the present invention comprise a compound of formula I as an active ingredient or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable bases including inorganic bases and organic bases.
  • Representative salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, ammonium, potassium, sodium, zinc and the like. Particularly preferred are the calcium, magnesium, potassium, and sodium salts.
  • Representative salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
  • basic ion exchange resins such as arginine,
  • salts may be prepared from pharmaceutically acceptable non- toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid, and the like.
  • Particularly preferred are
  • SUBST ⁇ UTE SHEET citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
  • cardiac and cerebral ischemia/reperfusion injury e.g. stroke
  • the magnitude of therapeutic dose of a compound of formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of formula I and its route of administration and vary upon the clinician's judgement. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the active component can thus be determined by the clinician after a consideration of all the criteria and using is best judgement on the patient's behalf. A representative dose will range from 0.001 mpk/d to about 100 mpk/d.
  • An ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of formula I in an acceptable ophthalmic formulation may be used. Any suitable route of administration may be employed for providing an effective dosage of a compound of the present invention. For example, oral, parenteral and topical may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
  • compositions include compositions suitable for oral, parenteral and ocular (ophthalmic). They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. In practical use, the compounds of formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, alcohols, oils, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case or oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amound of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into active ingredient with the carrier which constitutes one or more necessary ingredients.
  • compositions are prepared by uniformLy and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • each dosage unit may contain from about 0.01 mg to about 1.0 g of the active ingredient.
  • organo etallic reagent organo etallic reagent
  • a mixed anhydride of N-protected-L-aspartic acid ⁇ - tert-butyl ester (protected-L-Asp (OtBu)-OH)(V) and isobutylchloroformate (IBCF) is formed in the presence of N- methylmorpholine (NMM). This anhydride is reduced to the
  • the ketones IX are loaded onto a solid support using the technology described by Webb et al. (J. Am. Chem. Soc. 114, 3156 (1992)).
  • This method uses the solution synthesis of the complete semicarbazone carboxylic acid linker XI by first reacting ketone IX with semicarbazidyl-£ra-7s-4-methyl cyclohexanecarboxylic acid trifluoroacetate salt (X) to give XI.
  • Coupling of XI to the commercially available Merrifield resin gives the insoluble support XII. This material has all the physical and chemical properties for the automated synthesis of peptides.
  • HATU O-(7-Azabenzotriazol-l-yl)N,N,N',N'- tetramethyluronium hexafluorophosphate
  • DIEA N,N- diisoproylethylamine
  • the uncapped tetrapeptide XIII is acylated and peptide I can be obtained by simultaneous deesterification and cleavage from solid support via exposure to a 9:1 mixture of TFA:H2 ⁇ .
  • melting points are uncorrected and d indicates decomposition; the melting points given are those obtained for the materials prepared as described; polymorphism may result in isolation of materials with different melting points in some preparations;
  • NMR data is in the form of delta ( ⁇ ) values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard, determined at 300 MHz or 400 MHz using the indicated solvent; conventional abbreviations used for signal shape are: s. singlet; d. doublet; t. triplet; m. multiplet; br. broad; etc.: in addition "Ar" signifies an aromatic signal;
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4- hydroxybutanoate
  • N-Fmoc-L-aspartic acid b-tert-butyl ester (19.0 g, 46.2 mmol) in 300 mL of tetrahydrofuran (THF) at -78 °C was added N-methyl morpholine (NMM, 5.9 mL, 53.3 mmol) followed by isobutyl chloroformate (IBCF, 6.9 mL, 53.3 mmol). After 10 minutes this mixture was warmed to 0 °C for 40 minutes and then recooled to -78 °C.
  • NMM N-methyl morpholine
  • IBCF isobutyl chloroformate
  • Step 2 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo r 7 r phenylheptanoate
  • Oxalyl chloride (960 mL, 11 mmol) was added to a solution of DMSO (852 mL, 12 mmol) in 50 mL CH2CI2 at -78°C.
  • the aqueous layer was further extracted with Et2 ⁇ (4 x 100 mL).
  • the combined organic layers were washed with 5% aq. HCl, 5% aq.NaHCO3, brine, and dried over magnesium sulfate.
  • the solvent was removed in vacuo and
  • Step 3 4-[(2-[(#,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l-(3- phenylpropyl)butylidene]hydrazinocarbonyl)amino]m ethyl-1-cyclohexanecarboxylic acid
  • Step 4 (4S)-4-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-5-[(lS)-l-([(l-S)-l-(carboxymethyl)-2- oxopropyl] aminocarbonyl)-2-methylpropyllamino-5-oxopentanoic acid
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-9- phenylnonanoate
  • Step 2 4-[(2-[(£)-l-((lS)-3-(ter*-Butoxy)-l-[(9H-9- fluorenylmethoxy)carbonyl]amino-3-oxopropyl)-6- phenylhexylidene]hydrazinocarbonyl)amino]methyl-l- cyclohexanecarboxylic acid
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenylnonanoic acid
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-4- phenylbutanoate
  • Step 2 4-[([2-((E,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- phenylbutylidene)hydrazino]carbonylamino)methyl]- 1-cyclohexanecarboxylic acid
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxoheptanoate
  • Step 2 4-[([2-((#,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- propylbutylidene)hydrazino]carbonylamino)methyl]-l- cyclohexanecarboxylic acid
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxoheptanoic acid
  • Step 1 tert-Butyl (3S)-S-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-6- phenylhexanoate
  • Step 2 4-[([2-((E,2S)-4-(tert-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- phenethylbutylidene)hydrazino]carbonylamino)methy 11-1-cyclohexanecarboxylic acid
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-6-phenylhexanoic acid
  • Step 1 tert-Butyl (SS)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-8- phenyloctanoate
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-8-phenyloctanoic acid
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyI]amino-4-oxo-5- phenylpentanoate
  • Step 2 4-[([2-(( J B,2S)-l-Benzyl-4-(ter ⁇ -butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4- oxobutylidene)hydrazino]carbonylamino)methyl]-l- cyclohexanecarboxylic acid
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-5-phenylpentanoic acid
  • Step 1 tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl] amino- 7-(4- methoxyphenyl)-4-oxoheptanoate
  • Step 4 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(4-methoxyphenyl)-4- oxoheptanoic acid
  • Step 1 r*-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyllamino-7-(l-naphthyl)-4- oxoheptanoate
  • Step 4 (3-S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid
  • the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), 3,5-dibromobenzoic acid (1.44 g),
  • HATU O-(7-azabenzotriazol-l-yl)N,N,N',N' - tetramethyluronium hexafluorophosphate
  • DIEA N,N- diisopropylethylamine
  • the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), t-Boc-L-Asp (OCH3)-OH (1.79 g),
  • Fmoc-L-Ala-OH (1.59 g), Fmoc-L-Val-OH (1.65 g), O-(7- azabenzotriazol-l-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine
  • Example 104 To the peptide from Example 104 (25 mg, 47 ⁇ mol) in 400 ⁇ L THF was added NaHCO3 (39.5 mg in 500 ⁇ L H2O) and acetyl chloride (33.5 ⁇ L in 500 ⁇ L THF). The mixture was stirred for 16 h and then poured into sat. aq. NH4CI. The layers were separated
  • the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), 4-iodobenzoic acid (1.28 g), Fmoc-L-
  • the above polymer was transferred into a fritted reaction vessel and treated with TFA/H2O (9:1, 2 mL) and agitated 5 for 30 min. The solution was filtered, the solid support was washed with TFA (2 x 0.5 mL) and the filtrate was evaporated.
  • Example 1 Using the resin from Example 1, Step 4, and substituting Fmoc-L-Asp(OtBu)-OH for Fmoc-L-Cys(SMe)-OH(1.56 g) and Fmoc-L-Glu(OtBu)-OH for Fmoc-L-Ala-OH (1.56 g) in Example 1, Step 5, the title compound was obtained as colorless solid:
  • a fluorogenic derivative of the tetrapeptide recognized by caspase-3 and corresponding to the Pi to P4 amino acids of the PARP cleavage site, Ac-DEVD-AMC (AMC, amino-4- methylcoumarin) was prepared as follows: i) synthesis of N-Ac- Asp(OBn)-Glu(OBn)-Val-CO2H, ii) coupling with Asp(OBn)-7- amino-4-methylcoumarin, iii) removal of benzyl groups.
  • Standard reaction mixtures 300 ⁇ L final volume, contained Ac-DEVD-AMC and purified or crude caspase-3 enzyme in 50 mM Hepes/KOH (pH 7.0), 10% (v/v) glycerol, 0.1% (w/v) CHAPS, 2 mM EDTA, 5 mM dithiothreitol, and were incubated at 25°C. Reactions were monitored continuously in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
  • Colorimetric immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone- associated-DNA-fragments (mono- and oligonucleosomes) after induced cell death. This assay was performed using the commercially available kit from Boehringer Mannheim, cat. No. 1 920 685.
  • SUBST ⁇ UTE SHEET (RULE 26) was performed in the region overlying the heart and the pericardium opened, exposing the heart.
  • the origin of the left coronary artery was visualized and a 4.0 suture passed under the artery approximately 2 - 3 mm from its origin.
  • the ends of the suture were passed through a short length of 2 mm id tubing and coronary artery occlusion effected by placing tension on the suture such that the tube compressed the artery.
  • the thoracotomy was closed with a small clamp and opened only to effect occlusion and reperfusion of the artery.
  • a Lead II electrocardiograph (ECG) signal was obtained by placing subdermal platinum leads and continuously monitored.
  • the caspase inhibitor or vehicle was administered as a first bolus 5 minutes before the onset of ischemia and a second bolus was administered again at the onset of reperfusion. Additionally, an infusion was initiated immediately after the first bolus dose. Control animals received the vehicle alone in equal volumes to the caspase inhibitor treated animals.
  • the animals were euthanized and infarct size determined using a dual staining technique (1.5% w/v triphenyltetrazolium chloride to demarcate infarct tissue and 0.25% w/v Evan's blue to demarcate the area at risk of infarct.
  • the heart was subsequently cut transversely into 4 slices of equal thickness, and infarct size and area at risk quantified using planimetry.

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Abstract

The invention encompasses the novel class of compounds represented by formula I, which are caspase-3 inhibitors. The invention also encompasses certain pharmaceutical compositions for treatment of caspase-3 mediated diseases comprising compounds of formula I.

Description

TITLE OF THE INVENTION
GAMMA-KETOACID TETRAPEPTIDES AS INHIBITORS OF
CASPASE-3
BACKGROUND OF THE INVENTION
Apoptotic cell suicide is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis, however, underlies the etiology of many of the most intractable of human diseases. In only the last few years, many of the molecules that participate in a conserved biochemical pathway that mediates the highly ordered process of apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the 'caspases', that are related to mammalian interleukin-lβ converting enzyme (ICE/caspase-1) and to CED-3, the product of a gene that is necessary for apoptotic suicide in the nematode C. elegans (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The role of these proteases in cell suicide is to disable critical homeostatic and repair processes as well as to cleave key structural components, resulting in the systematic and orderly disassembly of the dying cell.
The central importance of caspases in these processes has been demonstrated with both macromolecular and peptide- based inhibitors (which prevent apoptosis from occurring in vitro and in υivo) as well as by genetic approaches. Inhibition of apoptosis via attenuation of caspase activity should therefore be useful in the treatment of human diseases where inappropriate apoptosis is prominent or contributes to disease pathogenesis. Caspase inhibitors would thus be useful for the treatment of human diseases including, but not limited to, acute disorders such as cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, as well as chronic disorders such as neurodegenerative diseases (e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging.
Ten caspases have so far been identified in human cells. Each is synthesized as a catalytically dormant proenzyme containing an amino-terminal prodomain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-X junctions (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The strict requirement by caspases for Asp in the Pi position of substrates is consistent with a mechanism whereby proenzyme maturation can be either autocatalytic or performed by other caspases. The three dimensional crystal structures of mature caspase-1 and -3 show that the large subunit contains the principle components of the catalytic machinery, including the active site Cys residue which is harbored within the conserved pentapeptide motif, QACxG,l and residues that stabilize the oxyanion of the tetrahedral transition state (Wilson et al., 1994, Nature 370:270-75; Walker et al., 1994, Cell 78:342-52; Rotonda et al., 1996, Nat Struct Biol 3:619-25). Both subunits contribute residues which stabilize the Pi Asp of substrates while the small subunit appears to contain most of the determinants that dictate substrate specificity and, in particular, those which form the specificity-determining S4 subsite. One distinctive feature of these proteases is the absolute requirement for an aspartic acid residue in the substrate Pi position. The carboxylate side chain of the substrate Pi Asp is tethered by four residues in caspase-1 (Argl79, Gln238 from p20 and Arg341, Ser347 fro plO) that are absolutely conserved in all caspase family members. Catalysis involves a typical cysteine protease mechanism involving a catalytic dyad, composed of His237 and Cys285 (contained within an absolutely conserved QACxG pentapeptide) and an Oxyanion hole' involving Gly238 and Cys285. Inhibitors bind, however, in an unexpected non-transition state configuration (which raises important considerations for inhibitor design) with the oxyanion of the thiohemiacetal being stabilized by the active site His237. Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach. The principle effectors of apoptosis (group II caspases, which include caspases-2, -3 and -7 as well as C. elegans CED-3) have specificity for [P4]DExD[Pι], a motif found at the cleavage site of most proteins known to be cleaved during apoptosis. On the other hand, the specificity of group III caspases (caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B) is [P4](I,V,L)ExD[Pι] which corresponds to the activation site at the junction between the large and small subunits of other caspase proenzymes including group II (effector) family members. This and other evidence indicates that group III caspases function as upstream activators of group II caspases in a proteolytic cascade that amplifies the death signal. The role of group I caspases (caspases-1, -4 and -5) appears to be to mediate cytokine maturation and their role in apoptosis, if any, has not been substantiated.
A tetrapeptide corresponding to the substrate P4-P1 residues is sufficient for specific recognition by caspases and as a consequence has formed the basis for inhibitor design. In addition to the requirement for a Pi Asp, the P4 residue in particular appears to be most important for substrate recognition and specificity. Caspase-1, for example, prefers a hydrophobic residue such as Tyr in P4 (which corresponds to its YVHD cleavage site within proIL-lβ) whereas caspase-3 (and other group II enzymes) has a preference for an anionic Asp residue (which corresponds to the DXXD cleavage sites within most polypeptides that are cleaved by these enzymes during apoptosis). Peptide aldehydes, nitriles and ketones are potent reversible inhibitors of these proteases while compounds that form thiomethylketone adducts with the active site cysteine (e.g. peptide (acyloxy)methylketones) are potent irreversible inhibitors. For example, the tetrapeptide aldehyde Ac-YVAD-CHO (which was designed to mimic the YVHD caspase-1 recognition sequence within proIL-lβ) is a potent inhibitor of caspase-1 (Ki < 1 nM) but
- 3 -
SUBSTΓΓUTE SHEET (RULE 26) a poor inhibitor of caspase-3 (Ki = 12 μM) (Thornberry et al., 1992, Nature 356:768-74). In contrast, the Ac-DEVD-CHO tetrapeptide aldehyde (which was designed to mimic the caspase-3 recognition site) is a very potent inhibitor of caspase-3 (Ki < 1 nM) although it is also a weaker but reasonable inhibitor of caspase-1, presumably owing to promiscuity in the S4 subsite of this enzyme (Nicholson et al., 1995, Nature 376:37-43).
Several features plague these peptide-derived inhibitors as a platform for drug design. In addition to their metabolic instability and membrane impermeability, the slow- binding time-dependent inhibition of activity (e.g. kon caspase- l:Ac-YVAD-CHO = 3.8 x 105 M-1S-1; kon caspase-3:Ac-DEVD-CHO = 1.3 x 105 M-ls-1) precludes them from the rapid inhibition characteristics that may be necessary to abolish enzymatic activity in vivo. The present patent application describes the resolution of this issue with the discovery of several novel ketones that make highly suitable caspase inhibitors.
SUMMARY OF THE INVENTION The invention encompasses the novel class of compounds represented by formula I:
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from the group consisting of:
(a) H and
(b) C(0)R1; elected from the group consisting of:
(a) hydrogen,
(b) Ci-βalkoxy,
(c) NR6R7, (d) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen, (3) methoxy and
(4) cyano,
(e) Ci-βalkyl or substituted Cι_6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy, (2) halo,
(3) Cι_3alkoxy,
(4) Cι_3alkylthio,
(5) phenyl Cι_3alkoxy,
(6) phenyl Cι_3alkylthio, (7) phenylcarboxy and
(8) carboxy,
(f) aryl or arylCι_6alkyl wherein the aryl group is selected from the group consisting of:
(I) phenyl, (2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl, (7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(II) pyrimidyl, (12) quinolyl, (13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl and
(20) oxazolyl, and
(g) mono and di-substituted aryl as defined above in items
(1) to (20) of (f), wherein the substituents are independently selected from:
(1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano,
(6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci-βalkyl,
(10) Ci-βfluoroalkyl,
(11) C ι_6alkylcarbony 1,
(12) Ci_6alkoxycarbonyl,
(13) Ci-6alkoxy,
(14) Cι_6alkylthio,
(15) Cι_6alkylsulfonyl and
(16) deuterio;
selected from the group consisting of:
(a) H,
(b) CH3 >
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph,
Figure imgf000008_0001
(g) CH2OH, O 00/32620
(h) CH2SH,
(i) CH2CH2SCH3,
0) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(O)NH2,
(q) CH2CH2C(O)NH2,
(r) CH2Cθ2Cι_4alkyl,
(s) CH2SCι_4alkyl,
(t) CH2S(O)2Cι_4alkyl,
Figure imgf000009_0001
or R2 and X2 together form a saturated monocyclic ring having the following structure:
Figure imgf000009_0002
R3 is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
( ) CH2CH(CH3)2,
(e) CH2Ph,
Figure imgf000009_0003
- 7 -
SUBSTΓΓUTE SHEET (RULE 26) (g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
(J) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
( ) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(O)NH2,
(q) CH2CH2C(O)NH2,
(r) CH2CH2CO2Ci_4alkyl,
(s) CH2CH2S(O)2Cι_4alkyl,
Figure imgf000010_0001
or R and X together form a saturated monocyclic ring having the following structure:
Figure imgf000010_0002
R4 is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph, ω CH2PhOH,
- 8 -
SUBSTΓΓUTE SHEET (RULE 26) (g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
(j) CH(CH3)CH2CH3, (k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2, (p) CH2C(0)NH2,
(q) CH2CH2C(O)NH2,
Figure imgf000011_0001
or R4 and X4 together form a saturated monocyclic ring having the following structure:
Figure imgf000011_0002
R5 is selected from the group consisting of:
(a) Cι_6alkyl,
(b) arylCι_8alkyl wherein the aryl is selected from the group consisting of: (1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl, (6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl, (10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl, (15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl, (20) oxazolyl and
(21) coumarinyl and (c) aryl as defined above in items (1) to (21) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from: (1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano, (6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci-βalkyl,
(10) Ci-βfluoroalkyl, (11) Ci-6alkylcarbonyl,
(12) Ci_6alkoxycarbonyl,
(13) Cι_6alkoxy,
(14) Ci_6alkylthio and
(15) Cι_6alkylsulfonyl;
- 10 -
SUBSTΓΓUTE SHEET (RULE 26) R6 and R^ are independently selected from the group consisting of:
(a) Ci- alkyl,
(b) Cι_4fluoroalkyl and (c) benzyl or mono- or disubstituted benzyl wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen,
(3) methoxy and (4) cyano, or R6 and R? may be joined to form a pyrrolidine, piperidine, morpholine, thiamorpholine or N-R8 substituted piperazine wherein R is H or Cι_3alkyl; and
X2, X3 and X4 are independently H or X2 and R2, χ3 and R3,or X4 and R4 may together form a saturated monocyclic ring having the following structure:
Figure imgf000013_0001
The invention also encompasses a pharmaceutical composition comprising a compound of formula I in combination with a pharmacuetically acceptable carrier.
The invention also encompasses a method of treating cardiac and cerebral ischemia reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I. DETAILED DESCRIPTION OF THE INVENTION
The invention encompasses the novel class of compounds represented by formula I, which are caspase-3 inhibitors.
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof, are disclosed, wherein:
R is selected from the group consisting of:
(a) H and
(b) C(O)Rl;
R1 is selected from the group consisting of: (a) hydrogen,
(b) Ci-6alkoxy,
(c) NRβR?,
(d) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen,
(3) methoxy and
(4) cyano, (e) Ci-βalkyl or substituted Cι_6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Ci-3alkoxy, (4) Cι_3alkylthio,
- 12 -
SUBSTΓΓUTE SHEET (RULE 26) (5) phenyl Cι_ alkoxy,
(6) phenyl Cι_ alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(f) aryl or arylCι_6alkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl and
(20) oxazolyl, and
(g) mono and di-substituted aryl as defined above in items
(1) to (20) of (f), wherein the substituents are independently selected from:
(1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano,
(6) carboxy,
(7) formyl, (8) amino carbonyl,
(9) Ci-βalkyl,
(10) Ci-6fluoroalkyl,
(11) Ci-βalkylcarbonyl, (12) Cι_6alkoxy carbonyl,
(13) Cι_6alkoxy,
(14) Ci-βalkylthio,
(15) Ci-βalkylsulfonyl and
(16) deuterio;
elected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2, (d) CH2CH(CH3)2,
(e) CH2Ph,
(f) CH2PhOH,
(g) CH2OH, (h) CH2SH, (i) CH2CH2SCH3,
(j) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH, (n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(p) CH2C(O)NH2,
(q) CH2CH2C(O)NH2,
(r) CH2Cθ2Cι..4alkyl, (S) CH2SCi-4alkyl,
(t) CH2S(O)2Ci-4alkyl,
Figure imgf000016_0001
- 14 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000017_0001
or R and X together form a saturated monocyclic ring having the following structure:
Figure imgf000017_0002
R is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph,
CH2PhOH,
(g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
0) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(O)NH2,
(q) CH2CH2C(O)NH2,
(r) CH2CH2Cθ2Cι_4alkyl,
(s) CH2CH2S(O)2Cι_4alkyl,
Figure imgf000017_0003
- 15 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000018_0001
or R3 and X3 together form a saturated monocyclic ring having the following structure:
Figure imgf000018_0002
R4 is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph,
(0 CH2PhOH,
(g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3, ω CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(0)NH2,
(q) CH2CH2C(O)NH2,
Figure imgf000018_0003
- 1 6 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000019_0001
or R4 and X4 together form a saturated monocyclic ring having the following structure:
Figure imgf000019_0002
R5 is selected from the group consisting of:
(a) Ci_ Salkyl,
(b) arylCi_8alkyl wherein the aryl is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) I isoquinolyl,
(14) benzofuryl,
(15, ) benzothienyl,
(16) > pyrazolyl,
(17 i indolyl,
(18, i purinyl,
(19 i isoxazolyl,
(20 ) oxazolyl and (21) coumarinyl, and (c) aryl as defined above in items (1) to (21) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from: (1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano, (6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci-βalkyl,
(10) Ci-6fluoroalkyl, (11) Ci_6alkylcarbonyl,
(12) C -βalkoxy carbonyl,
(13) Cι_6alkoxy,
(14) Ci-6alkylthio and
(15) Ci-βalkylsulfonyl;
R and R7 are independently selected from the group consisting of:
(a) Cι_4alkyl,
(b) Cι_4fluoroalkyl and (c) benzyl or mono- or disubstituted benzyl wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen,
(3) methoxy and (4) cyano, or R and R7 may be joined to form a pyrrolidine, piperidine, morpholine, thiamorpholine or N-R8 substituted piperazine wherein R8 is H or Ci_3alkyl; and X2, X3 and X4 are independently H or X2 and R2, χ3 and R3,or X4 and R4 may together form a saturated monocyclic ring having the following structure:
Figure imgf000021_0001
In a preferred embodiment, the compounds are represented by formula la
Figure imgf000021_0002
la
or a pharmaceutically acceptable salt thereof, wherein the amino acids from which the structure is constructed, represented in formula la as AAl, AA2 and AA3, are selected from a group consisting of the L- and D- forms of the amino acids including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine and valine. The structures of the L-amino acids are shown below.
L-amiπo acids and abbreviations:
L-Alanino L-Arginiπe L-Asparagine -Aspartic acid L-Cysteine
(Ala, A) (Arg, R) (Asn, N) (Asp, D) (Cys, C)
Figure imgf000022_0001
L-Glutamine L-Glutamic acid Glycme L-Histidine L-lsoleucme
Figure imgf000022_0002
L-Leuαne L-Lvsne L- ethionine L-P enylalanine L-Proltne
(Leu, L) (LyS ) (Met, M) (Phe, F) (Pro, P)
Figure imgf000022_0003
L-Seπnp i πreoniπe - Tryutophane - i yiosine -Valιne (Ser, S) (Thr, T) (Trp, W) (Tyr, Y) (Val, V)
Figure imgf000022_0004
A preferred embodiment of the invention is that wherein R is Ci_6alkyl or phenyl.
Another preferred embodiment of the invention is that wherein R^ is arylCi_8alkyl, wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or disubstituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
- 20 -
SUBSTΓΓUTE SHEET (RULE 26) (1) halo,
(2) amino,
(3) nitro, -
(4) hydroxy,
(5) cyano,
(6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Cι_3alkyl,
(10) C ι_3fluoroalky 1,
(ID C ι_3alkylcarbonyl,
(12) Cι_3alkoxycarbonyl,
(13) Cι_3alkoxy,
(14) Ci_3alkylthio and
(15) Ci_3alkylsulfonyl;
Another preferred embodiment of the present invention is that wherein:
R is selected from the group consisting of:
(a) Ci-βalkoxy,
(b) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from methyl, halogen, methoxy and cyano, (c) Cι_6alkyl or substituted Ci-6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Ci-3alkoxy, (4) Cι_3alkylthio,
(5) phenylCι_3alkoxy,
(6) phenylCi_3alkylthio,
(7) phenylcarboxy and
(8) carboxy, (d) aryl or arylCi_6alkyl wherein the aryl group is selected from the group consisting of: (1) phenyl and
(2) naphthyl, and
(e) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy, (5) amino carbonyl,
(6) Cι_3alkyl,
(7) Cι_3fluoroalkyl,
(8) Ci-3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl, (10) Cι_3alkoxy,
(11) Ci-3alkylthio,
(12) Cι_3alkylsulfonyl and
(13) deuterio; and
R5 is arylCι_8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or disubstituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
(I) halo, (2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Ci-3alkyl, (7) Cι_3fluoroalkyl,
(8) Ci_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl,
(10) Cι_3alkoxy,
(II) Ci-3alkylthio and (12) Cι_3alkylsulfonyl.
- 22 -
SUBSTΓΠJTE SHEET (RULE 26) Another preferred embodiment of the invention is that wherein Rl is selected from the group consisting of:
(a) Cι_6alkyl or substituted Cι_6alkyl, wherein the substituent is selected from the group consisting of: (1) hydroxy,
(2) halo,
(3) Ci-3alkoxy,
(4) Ci-3alkylthio,
(5) phenylCi-3alkoxy, (6) phenylCι_ alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(b) aryl or arylCi-βalkyl wherein the aryl group is selected from the group consisting of: (1) phenyl and
(2) naphthyl, and
(c) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from: (1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl, (6) Cι_3alkyl,
(7) Ci_3fIuoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl,
(10) Ci_3alkoxy, (11) Ci-3alkylthio,
(12) Ci-3alkylsulfonyl and
(13) deuterio.
Another preferred embodiment of the present invention is that wherein R2 is selected from the group consisting of:
- 23 -
SUBSTΓΓUTE SHEET (RULE 26) (a) CH2CO2H,
(b) CH2Cθ2Cι_4alkyl,
(c) CH2SCι_4alkyl and
(d) CH2s(0)2Ci-4alkyl.
Another preferred embodiment of the invention is that wherein R3 is selected from the group consisting of:
(a) CH3,
(b) CH2CH2CO2H, (c) CH2CH2Cθ2Cι_4alkyl,
(d) CH2CH2S(O)2Cι_ alkyl.
A preferred embodiment of the present invention is that wherein R4 is isopropyl.
Another preferred embodiment of the invention is that wherein R5 is selected from the group consisting of:
(a) Ci-βalkyl,
(b) arylCi_8alkyl wherein the aryl is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl and
(4) coumarinyl, and (c) aryl as defined above in items (1) to (4) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from:
(1) halo,
(2) hydroxy, (3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Ci-3alkyl,
(7) Cι_3fluoroalkyl, (8) Cι_3alkylcarbonyl,
(9) Ci_3alkoxycarbonyl,
- 24 -
SUBSTΓΓUTE SHEET (RULE 26) (10) Cι_3alkoxy,
(11) Cι_3alkylthio and
(12) Cι.3alkylsulfonyl.
In one subset that is of particular interest, the compounds are represented by formula II:
Figure imgf000027_0001
including pharmaceutically acceptable salts thereof, wherein:
R5 is selected from the group consisting of:
(a) Ci_6alkyl,
(b) arylCι_8alkyl wherein the aryl is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl and
(4) coumarinyl and
(c) aryl as defined above in items (1) to (4) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Cι.3alkyl,
(7) Ci_3fluoroalkyl, (8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxy carbonyl,
(10) Cι_3alkoxy,
(11) Cι_3alkylthio and
(12) Cι_3alkylsulfonyl.
Another subset of compounds that is of particular interest relates to compounds of formula II wherein:
R5 is selected from the group consisting of:
(a) methyl,
(b) propyl,
(c) phenyl,
(d) phenylCi_5alkyl,
(e) 4-methoxyphenylpropyl,
(f) napthylpropyl and
(g) 4-methylcoumarinyl.
Representative compounds that are of particular interest are the following:
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
In another embodiment that is of particular interest, the compounds are represented by formula III:
Figure imgf000031_0002
III
including pharmaceutically acceptable salts thereof, wherein:
R 1 is selected from the group consisting of:
(a) Ci_6alkyl or substituted Ci_6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Cι_3alkoxy,
(4) Cι_3alkylthio,
(5) phenylCι_3alkoxy,
(6) phenylCi-3alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(b) aryl or arylCi-βalkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl and
(2) naphthyl, and
- 29 -
SUBSTΓΓUTE SHEET (RULE 26) (c) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from:
(I) halo, (2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Cι_3alkyl, (7) Ci_3fluoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl,
(10) Cι_3alkoxy,
(II) Ci-3alkylthio, (12) Cι_3alkylsulfonyl and
(13) deuterio; and
R5 is arylCι_8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or di- substituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
(1) halo,
(2) hydroxy,
(3) cyano, (4) carboxy,
(5) amino carbonyl,
(6) Cι_3alkyl,
(7) Ci-3fluoroalkyl,
(8) Cι_3alkylcarbonyl, (9) Cι_3alkoxy carbonyl,
(10) Ci-3alkoxy,
(11) Cι_3alkylthio and
(12) Cι_3alkylsulfonyl.
A subset of compounds that are of particular interest are defined in accordance with formula III wherein:
- 30 -
SUBSTΓΓUTE SHEET (RULE 26) Rl is selected from the group consisting of:
(a) methyl,
(b) phenyl and (c) mono- or disubstituted phenyl, wherein the substituents are selected from the group consisting of:
(1) halo and
(2) deuterio; and
R5 is arylC3_5alkyl wherein aryl is selected from the group consisting of phenyl and naphthyl.
Representative compounds that are of particular interest are the following:
Figure imgf000033_0001
Figure imgf000034_0001
Yet another embodiment that is of particular interest ed by formula IV:
Figure imgf000034_0002
IV including pharmaceutically acceptable salts thereof, wherein:
R9 is selected from the group consisting of: (a) CO2H,
(b) CO2Ci-4alkyl,
(c) SCι_4alkyl and
(d) S(O)2Cι_4alkyl;
RlO is selected from the group consisting of:
(a) H,
(b) CH2CO2H,
(c) CH2CO2Cι_4alkyl,
(d) CH2S(O)2Cι_4alkyl; and
Ar is selected from the group consisting of:
(a) phenyl and
(b) napthyl.
A subset of compounds that are of particular interest are defined in accordance with formula IV wherein:
R9 is selected from the group consisting of: (a) CO2H, (b) SCH3 and
(c) S(O)2CH3;
RlO is selected from the group consisting of: (a) H and (d) CH2S(0)2CH3; and
Ar is phenyl or napthyl.
Representative compounds that are of particular interest are the following:
Figure imgf000036_0001
The invention also encompasses the following compound:
Figure imgf000037_0001
In a preferred embodiment, the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said caspase-3 mediated disease.
In another embodiment, the invention encompasses a method of treating cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I.
In another embodiment, the invention encompasses a method of treating acute disorders, including cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said acute disorder. In another embodiment, the invention encompasses a method of treating chronic disorders, including neurodegenerative diseases (e.g. Alzheimer's, polyglutamine- repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said chronic disorder.
In another embodiment, the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula II in an amount effective to treat said caspase-3 mediated disease.
In another embodiment, the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula III in an amount effective to treat said caspase-3 mediated disease.
In another embodiment, the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula IV in an amount effective to treat said caspase-3 mediated disease.
For purposes of this specification, the following abbreviations have the indicated meanings:
BOC = t-butyloxycarbonyl
CBZ = carbobenzoxy
DCC = 1,3-dicyclohexylcarbodiimide DIBAL = diisobutyl aluminum hydride
DIEA = N,N-diisoproylethylamine
DMAP = 4-(dimethylamino)pyridine
EDCI = l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDTA = ethylenediaminetetraacetic acid, tetrasodium salt hydrate
FAB = fast atom bombardment
FMOC = 9-fluorenylmethoxycarbonyl
HMPA = hexamethylphosphoramide HATU = O-(7-Azabenzotriazol-l-yl)N,N,N',N'- tetramethyluronium hexafluorophosphate
- 36 -
SUBSTΓΓUTE SHEET (RULE 26) HOBt 1-hydroxybenzotriazole
HRMS high resolution mass spectrometry
ICBF isobutyl chloroformate
KHMDS potassium hexamethyldisilazane
LDA lithium diisopropylamide
MCPBA metachloroperbenzoic acid
Ms methanesulfonyl = mesyl
MsO methanefulfonate = mesylate
NBS N-bromosuccinimide
NMM 4-methylmorpholine
PCC pyridinium chlorochromate
PDC pyridinium dichromate
Ph phenyl
PPTS pyridinium p-toluene sulfonate pTSA p-toluene sulfonic acid r.t. room temperature rac. racemic
TfO trifluoromethanesulfonate = triflate
TLC thin layer chromatography
Alkyl group abbreviations
Me methyl
Et ethyl n-Pr normal propyl i-Pr isopropyl n-Bu normal butyl i-Bu isobutyl s-Bu secondary butyl t-Bu tertiary butyl
For purposes of this specification alkyl means linear, branched or cyclic structures and combinations thereof, containing one to twenty carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl,
- 37 -
SUBSTΓΓUTE SHEET (RULE 26) tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl- 4-propylnonyl, cyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl, 2-ethyl-l- bicyclo[4.4.0]decyl and the like.
Alkylcarbonyl signifies groups having the formula - C(O)-alkyl, wherein alkyl is defined as above.
Alkylsulfonyl signifies groups having the formula - S(O)2-alkyl, wherein alkyl is defined as above.
For purposes of this specification fluoroalkyl means linear, branched or cyclic alkyl groups and combinations thereof, of one to ten carbon atoms, in which one or more hydrogen but no more than six is replaced by fluorine. Examples are -CF3, - CH2CH2F, and -CH2CF3, and the like.
Alkoxy means alkoxy groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and the like.
Alkoxycarbonyl signifies groups having the formula - C(0)-alkoxy, wherein alkoxy is defined as above.
Alkylthio means alkylthio groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkylthio groups include methylthio, propylthio, isopropylthio, etc. By way of illustration, the propylthio group signifies -SCH2CH2CH3.
Aryl is, for example, phenyl, naphthyl, pyridyl, furyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl and coumarinyl.
Halo includes F, Cl, Br and I.
The compounds described typically contain asymmetric centers and may thus give rise to diastereomers and optical isomers. The present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
- 38 -
SUBSTΓΓUTE SHEET (RULE 26) Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
The pharmaceutical compositions of the present invention comprise a compound of formula I as an active ingredient or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable bases including inorganic bases and organic bases. Representative salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, ammonium, potassium, sodium, zinc and the like. Particularly preferred are the calcium, magnesium, potassium, and sodium salts.
Representative salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non- toxic acids, including inorganic and organic acids. Examples of such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid, and the like. Particularly preferred are
- 39 -
SUBSTΓΓUTE SHEET (RULE 26) citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
In the discussion of methods of treatment which follows, reference to the compounds of formula I are meant to also include the pharmaceutically acceptable salts.
The ability of the compounds of formula I to inhibit caspase-3 make them useful research tools in the field of apoptosis. These compounds are also useful to treat, prevent or ameliorate in mammals and especially in humans, diseases including but not limited to:
1. cardiac and cerebral ischemia/reperfusion injury (e.g. stroke)
2. type I diabetes 3. immune deficiency syndrome (including AIDS)
4. cerebral and spinal cord trauma injury
5. organ damage during transplantation
6. alopecia
7. aging 8. Parkinson's disease
9. Alzheimer's disease
10. Down's syndrome
1 1 . spinal muscular atrophy
12. multiple sclerosis 13. neurodegenerative disorders
The magnitude of therapeutic dose of a compound of formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of formula I and its route of administration and vary upon the clinician's judgement. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the active component can thus be determined by the clinician after a consideration of all the criteria and using is best judgement on the patient's behalf. A representative dose will range from 0.001 mpk/d to about 100 mpk/d. An ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of formula I in an acceptable ophthalmic formulation may be used. Any suitable route of administration may be employed for providing an effective dosage of a compound of the present invention. For example, oral, parenteral and topical may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The compositions include compositions suitable for oral, parenteral and ocular (ophthalmic). They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. In practical use, the compounds of formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, alcohols, oils, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case or oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amound of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformLy and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. For example, each dosage unit may contain from about 0.01 mg to about 1.0 g of the active ingredient.
Method of Synthesis
Compounds of the instant invention are conveniently prepared using the procedures described generally below and more explicitly described in the Example section thereafter.
Scheme 1
Figure imgf000045_0001
VI
Figure imgf000045_0002
VII
organo etallic reagent
oxidation
Figure imgf000045_0003
Figure imgf000045_0004
IX VIII
A mixed anhydride of N-protected-L-aspartic acid β- tert-butyl ester (protected-L-Asp (OtBu)-OH)(V) and isobutylchloroformate (IBCF) is formed in the presence of N- methylmorpholine (NMM). This anhydride is reduced to the
- 43 -
SUBSTΓΓUTE SHEET (RULE 26) corresponding alcohol VI using sodium borohydride at -78 °C. The alcohol VI is then oxidized using dimethyl sulfoxide (DMSO), oxalyl chloride, and N,N-diisopropylethylamine to the corresponding aldehyde. The aldehyde (VII) is not isolated but reacted immediately with an organometallic reagent to afford the secondary alcohol (VTII) which can be oxidized to the corresponding ketone (IX).
Scheme 1 Continued
Figure imgf000046_0001
Figure imgf000046_0002
44 -
SUBSTΓΓUTE SHEET (RULE 26) Scheme 1 Continued
Figure imgf000047_0001
Figure imgf000047_0002
I (R=C(O)R' )
The ketones IX are loaded onto a solid support using the technology described by Webb et al. (J. Am. Chem. Soc. 114, 3156 (1992)). This method uses the solution synthesis of the complete semicarbazone carboxylic acid linker XI by first reacting ketone IX with semicarbazidyl-£ra-7s-4-methyl cyclohexanecarboxylic acid trifluoroacetate salt (X) to give XI. Coupling of XI to the commercially available Merrifield resin gives the insoluble support XII. This material has all the physical and chemical properties for the automated synthesis of peptides.
- 45 -
SUBSTΓΓUTE SHEET (RULE 26) Toward this end the 9050 Plus PepSynthesizer from PerSeptive Biosystems is used (Millipore Corporation, 34 Maple Street, Milford, MA 01757, User's Guide 9050 Plus OM 1.0). The synthesis procedure given in the user's guide is followed for the preparation of the tetrapeptide XIII on solid support.
O-(7-Azabenzotriazol-l-yl)N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU) and N,N- diisoproylethylamine (DIEA) are used as coupling reagents instead of of TBTU and HOBt as described in the user's guide. The uncapped tetrapeptide XIII is acylated and peptide I can be obtained by simultaneous deesterification and cleavage from solid support via exposure to a 9:1 mixture of TFA:H2θ.
The invention will now be illustrated by the following non-limiting examples in which, unless stated otherwise:
(i) all operations were carried out at room or ambient temperature, that is, at a temperature in the range 18- 25°C, (ii) evaporation of solvent was carried out using a rotary evaporator under reduced pressure (600-4000 pascals: 4.5- 30 mm. Hg) with a bath temperature of up to 60°C,
(iii) the course of reactions was followed by thin layer chromatography (TLC) and reaction times are given for illustration only;
(iv) melting points are uncorrected and d indicates decomposition; the melting points given are those obtained for the materials prepared as described; polymorphism may result in isolation of materials with different melting points in some preparations;
(v) the structure and purity of all final products were assured by at least one of the following techniques: TLC, mass spectrometry, nuclear magnetic resonance (NMR) spectrometry or microanalytical data; (vi) yields are given for illustration only;
- 46 -
SUBSTΓΠJTE SHEET (RULE 26) (vii) when given, NMR data is in the form of delta (δ) values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard, determined at 300 MHz or 400 MHz using the indicated solvent; conventional abbreviations used for signal shape are: s. singlet; d. doublet; t. triplet; m. multiplet; br. broad; etc.: in addition "Ar" signifies an aromatic signal;
(viii) chemical symbols have their usual meanings; the following abbreviations have also been used v (volume), w (weight), b.p. (boiling point), m.p. (melting point), L (litre(s)), mL (millilitres), g (gram(s)), mg (milligrams(s)), mol (moles), mmol (millimoles), eq (equivalent(s)).
EXAMPLE 1
(3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4- hydroxybutanoate
FmocNH^
^OH
* COOt-Bu
To a solution of N-Fmoc-L-aspartic acid b-tert-butyl ester (19.0 g, 46.2 mmol) in 300 mL of tetrahydrofuran (THF) at -78 °C was added N-methyl morpholine (NMM, 5.9 mL, 53.3 mmol) followed by isobutyl chloroformate (IBCF, 6.9 mL, 53.3 mmol). After 10 minutes this mixture was warmed to 0 °C for 40 minutes and then recooled to -78 °C. A suspension of sodium borohydride (3.85 g, 102 mmol) in 25 mL of methanol was added and the mixture was stirred at -78 °C for 2 h. The reaction was quenched into 400 mL saturated aqueous ammonium chloride and extracted
- 47 -
SUBSTΓΓUTE SHEET (RULE 26) with ethyl acetate (4 x 100 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified on silica gel (50% ethyl acetate/hexane) to give the desired product: IH NMR (400 MHz, CD3COCD3) d 7.85 (d, 2H, J=7.30 Hz), 7.67 (d,
2H, J= 7.37 Hz), 7.40 (t, 2H, J= 7.30 Hz), 7.30 (t, 2H, J= 7.30 Hz), 6.32 (br d, IH), 4.40 - 4.15 (m, 3H), 4.10 - 3.98 (m, IH), 3.92 (t, IH), 3.65 - 3.48 (m, 2H), 2.60 (dd, IH, J= 6.24, 16.80 Hz), 2.41 (dd, IH, J= 6.30, 16.91 Hz), 1.40 (s, 9H).
Step 2: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxor7r phenylheptanoate
Figure imgf000050_0001
COOt-Bu
a) Oxalyl chloride (960 mL, 11 mmol) was added to a solution of DMSO (852 mL, 12 mmol) in 50 mL CH2CI2 at -78°C.
The resulting mixture was stirred at -78°C for 30 minutes and the N-Fmoc-b-tert-butyl aspartic alcohol (3.98 g, 10 mmol) in CH2CI2
(15 mL) was added dropwise. The mixture was stirred at -78°C for 1 h, then i-Pr2NEt (5.20 mL, 30 mmol) was added dropwise. The resulting mixture was stirred at -78°C for 50 min. and at 0°C for 25 min. The mixture was recooled to -78°C and phenylpropylmagnesium bromide (1.0M in Et2θ, 30 mL) was added dropwise. The mixture was stirred at -78°C for 2 h and at 0°C for 75 min. Saturated aqueous NH4CI (100 mL) was added and the two layers were separated. The aqueous layer was further extracted with Et2θ (4 x 100 mL). The combined organic layers were washed with 5% aq. HCl, 5% aq.NaHCO3, brine, and dried over magnesium sulfate. The solvent was removed in vacuo and
- 48 -
SUBSTΠTJTE SHEET (RULE 26) the residue was chromatographed over silica gel (15% EtOAc/toluene) to provide the secondary alcohol (4.48 g, 87%).
b) The secondary alcohol (1.5 g., 2.90 mmol) was dissolved in CH2CI2 (20 mL) and Dess-Martin reagent (1.23 g., 2.90 mmol) was added. The resulting mixture was stirred for 2 h at room temperature and then filtered through a block of silica gel (15% EtOAc/toluene) to provide the phenylpropylketone (1.23 g, 83%): IH NMR (400 MHz, CD3COCD3) δ 7.85 (d, 2H, J = 7.59 Hz), l O 7.68 (d, 2H, J = 7.44 Hz), 7.40 (t, 2H, J = 7.47 Hz), 7.31 (t, 2H, J = 7.86 Hz), 7.25 - 7.08 (m, 5H), 6.85 (br d, IH), 4.55 - 4.35 (m, 3H), 4.22 (t, IH, J = 6.95 Hz), 2.80 (dd, IH, J = 7.1, 16.1 Hz), 2.70 - 2.50 (m, 5H), 1.88 ( q, 2H), 1.40 (s, 9H); MS (+APCI) m/z 514(M+H)+.
5 Step 3: 4-[(2-[(#,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l-(3- phenylpropyl)butylidene]hydrazinocarbonyl)amino]m ethyl-1-cyclohexanecarboxylic acid
Figure imgf000051_0001
The following procedure is an adaption of Webb, T.R. et al., J. Am. Chem. Soc. 114, 3156 (1992); to a suspension of the phenylpropylketone (1.23 g, 2.39 mmol) and semicarbazidyl-trans- 25 4-methyl cyclohexane carboxylic acid trifluoroacetate salt (0.79 g, 2.39 mmol) in EtOH/H2θ (28 mL, 4:1) was added NaOAc (0.22 g, 2.63 mmol) followed by 8.5 mL THF. The clear solution was stirred at room temperature for 21 h. The mixture was poured into 100 mL
- 49 -
SUBSTΓΓUTE SHEET (RULE 26) brine, H2O was added to dissolve the solid and extracted with
EtOAc (4 x 25 mL). The organic layers were washed with saturated aq. NH4CI and dried over MgSO Flash chromatography using 10% EtOAc/toluene, then 5% MeOH/CHCl3 gave 505 mg of recovered ketone and 1.1 g of the desired product: IH NMR (400 MHz, CD3COCD3) δ 9.12 (s, IH), 8.00 (s,lH), 7.85 (d,
2H, J = 7.52 Hz), 7.69 (t, 2H, J = 6.60 Hz), 7.41 (t, 2H, J = 7.50 Hz), 7.31 (t, 2H, J = 7.44 Hz), 7.23 - 7.05 (m, 5H), 6.76 (brd, IH, J = 9.45 Hz), 6.64 (brt, IH, J = 5.69 Hz), 4.85 - 4.75 (m, IH), 4.45 - 4.18 (m, 3H), 3.20 - 3.02 (m, 2H), 2.76 - 2.15 (m, 7H), 2.10 - 1.75 (m, 6H), 1.57 - 1.28 (m, 3H), 1.38 (s, 9H), 1.10 - 0.95 (m, 2H); MS (+APCI) m/z 711 (M+H)+.
Step 4: Resin IXa
Figure imgf000052_0001
Merrifield's amino resin (2.02 g, 1.42 mmol, ca. 0.7 meq/g loading) is placed in a fritted reaction vessel. The resin is washed three times with DMF (10 mL each). After the last wash each of the following are added: carboxylic acid from step 3 (1.41 g, 1.98 mmol) in 36 mL CH2CI2, 1-hydroxybenzotriazole (HOBT)
(0.39 g, 2.55 mmol), l-(3-dimethylaminopropyl) 3-ethycarbodiimide hydrochloride, (EDCI) (0.49 g, 2.55 mmol). The mixture is agitated overnight, filtered and then washed with CH2CI2 (2x), sat. aq. NH4CI (3x), H2O (2x), sat. aq. NaHCO3 (2x), H2O (2x), H2O/THF (1:1, 2x), THF (2x), EtOAc (lx), CH2CI2 (lx) (25 mL each). After drying 3.25 g of resin is obtained.
- 50 -
SUBSTΓΓUTE SHEET (RULE 26) Step 5: (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid
Figure imgf000053_0001
The PerSeptive 9050 Plus PepSynthesizer (Millipore Corporation, 34 Maple Street, Milford, MA 01757, User's Guide
9050 Plus OM 1.0) was used for the preparation of the tetrapeptide on solid support.
Preparation of the tetrapeptide on solid support: Using the synthesis procedure given in the user's guide the tetrapeptide was prepared with the following reagents: 290 mg of polymer from Step 4 (0.7 meq/g loading), Fmoc-L-Val-OH (1.65 g), Fmoc-L-Glu (OtBu)-OH (1.83 g), Fmoc-L-Asp (OtBu)-OH (1.80 g); O-(7-azabenzotriazol-l-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine
(DIEA) were used as coupling reagents instead of TBTU and HOBt as described in the user's guide.
Acetylation:
The uncapped tetrapeptide on solid support was transferred into a fritted reaction vessel and treated with acetylimidazole (550 mg, 5 mmol) in 5 mL DMF. The mixture is agitated for 1.5 h, filtered and then washed with DMF (3x), THF (3x), CH2CI2 (4x) (2 mL each). Cleavage from solid support:
The above polymer was treated with TFA:H2θ (9:1, 2 mL) and agitated for 30 min. The solution was filtered, the solid support washed with TFA (2x 0.5 mL) and the filtrate was evaporated. Trituration from Et2θ gives 58 mg of the tetrapeptide derivative as a colorless solid. H NMR (400 MHz, CD3OD) δ 8.32 (d, IH, = 7.20 Hz), 7.90 (d, IH, J = 8.08 Hz), 7.30 -
7.10 (m, 5H), 4.67 (t, IH, = 6.60 Hz), 4.62 (t, IH, J = 6.23 Hz), 4.40 - 4.32 (m, IH), 4.13 (t, IH, J = 7.60 Hz), 2.90 - 2.80 (m, 2H), 2.78 - 2.35 (m, 8H), 2.20 - 2.05 (m, 2H), 2.02 - 1.90 (m, IH), 1.98 (s, 3H), 1.89 - 1.79 (m, 2H), 0.94 (d, 6H, J = 6.71 Hz); MS (+APCI) m/z 621 (M+H)+.
EXAMPLE 2
(4-S)-4-[(2-S)-2-(Acetylamino)-3-carboxypropanoyl]amino-5-[(l-S)-l- ([(lS)-l-(carboxymethyl)-2-oxopropyl]aminocarbonyl)-2- methylpropyl] amino-5-oxopentanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)car bonyl] amino-4-oxopentanoate
Figure imgf000054_0001
COOt-Bu
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for methylmagnesium bromide the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 7.85 (d, 2H, J= 7.60 Hz), 7.68 (d, 2H,
J= 7.45Hz), 7.41 (t, 2H, J= 7.47 Hz), 7.32 (t, 2H, J= 7.80 Hz), 6.85 (brd, IH), 4.55-4.35 (m, 3H), 4.26 (t, IH, J= 6.95 Hz), 2.80 (dd, IH, J= 7.1, 16.1 Hz), 2.65 (dd, IH), 2.18 (s, 3H), 1.41 (s, 9H).
- 52 -
SUBSTΓΓUTE SHEET (RULE 26) Step 2: 4-[([2-((£',2-S)-4-( r£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-l-methyl-4- oxobutylidene)hydrazino]carbonylamino)methyl]-l- cyclohexanecarboxylic acid
Figure imgf000055_0001
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 8.51 (s, IH), 7.85 (d, 2H, J= 7.50 Hz), 7.69 (d, 2H, J= 6.60 Hz), 7.40 (t,2H, J= 7.50 Hz), 7.31 (t, 2H, J= 7.44 Hz), 6.75 (brd, IH), 6.58 (brt, IH), 4.74- 4.62 (m, IH), 4.45-4.20 (m, 3H), 3.20-3.00 (m, 2H), 2.88-2.55 (m, 3H), 2.28-1.70 (m, 5H), 1.98 (s, 3H), 1.55-1.28 (m, 3H), 1.45 (s, 9H), 1.10-0.95 (m, 2H); MS (+APCI) m/z 607 (M+H)+.
Step 3: Resin C (R5 = Me)
Figure imgf000055_0002
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (4S)-4-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-5-[(lS)-l-([(l-S)-l-(carboxymethyl)-2- oxopropyl] aminocarbonyl)-2-methylpropyllamino-5-oxopentanoic acid
Figure imgf000056_0001
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 8.33 (d, IH, J= 6.95 Hz), 7.92 (d, IH, J= 7.90 Hz), 4.67 (t, IH, J= 6.70 Hz), 4.60 (t, IH, J= 6.20 Hz), 4.40-4.33 (m, IH), 4.14 (t, IH, J= 7.70 Hz), 2.91-2.81 (m, 2H), 2.78-2.65 (m, 2H), 2.50-2.34 (m, 2H), 2.20-2.08 (m, 2H), 2.17 (s, 3H), 2.01-1.89 (m, IH), 1.99 (s, 3H), 0.96 (d, 6H, J= 6.79 Hz); MS (+APCI) m/z 517 (M+H)+.
EXAMPLE 3
(3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenylnonanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-9- phenylnonanoate
Figure imgf000057_0001
'COOt-Bu
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for phenylpentylmagnesium bromide the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 7.85 (d, 2H, = 7.6 Hz), 7.68 (d, 2H, J = 7.4 Hz), 7.40 (t, 2H, J = 7.5 Hz), 7.31 (t, 2H, J = 7.4 Hz), 7.11-7.27 (5H, m), 6.83 (brd, IH, J = 8.4 Hz), 4.35-4.51 (m, 3H), 4.24 (t, IH, J = 6.8 Hz), 2.80 (dd, IH, J = 6.0, 9.0 Hz), 2.46-2.65 (m, 5H), 1.55-1.64 (m, 4H), 1.28-1.41 (m, 2H), 1.41 (s, 9H).
Step 2: 4-[(2-[(£)-l-((lS)-3-(ter*-Butoxy)-l-[(9H-9- fluorenylmethoxy)carbonyl]amino-3-oxopropyl)-6- phenylhexylidene]hydrazinocarbonyl)amino]methyl-l- cyclohexanecarboxylic acid
Figure imgf000057_0002
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 9.66 (s, IH), 7.84 (d, 2H, J = 7.5 Hz), 7.67 (d, 2H, J = 7.5 Hz), 7.39 (t, 2H, J = 7.5 Hz), 7.08-7.32 (m, 8H), 6.72 (brt, IH, J = 4.3 Hz), 4.79 (dd, IH, J =
- 55 -
SUBSTΓΠJTE SHEET (RULE 26) 8.5, 14.7 Hz), 4.33-4.43 (m, 2H), 4.21 (brt, IH, J = 6.9 Hz), 3.03-3.21 (m, 2H), 2.72 (dd, IH, J = 8.7, 14.8 Hz), 2.40-2.65 (m, 4H), 2.17-2.39 (m, 2H), 1.97-2.08 (m, 2H), 1.87 (brd, 2H, J = 10.4 Hz), 1.30-1.68 (m, 5H), 1.40 (s, 9H), 0.95-1.10 (m, 2H).
Step 3: Resin IX (R5 = 5-phenylpent-l-yl)
Figure imgf000058_0001
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenylnonanoic acid
Figure imgf000058_0002
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 7.86 (d, IH, J = 8.4 Hz), 7.09-7.24 (m, 5H), 4.62-4.69 m, 2H), 4.35 (dd, IH, J = 4.8, 8.8 Hz), 4.13-4.18 (m, IH), 2.79- 2.90 (m, 2H), 2.33-2.77 (m, 8H), 2.05- 2.20 (m, 2H), 1.99 (s, 3H), 1.86-2.00 (m, IH), 1.50-1.64 (m, 4H), 1.24- 1.87 (m, 2H), 0.95 (d, 6H, J = 6.7 Hz); MS (-APCI) m/z 647 (M-H) .
EXAMPLE 4
(4S)-4-[(2S)-2-(Acetylamino)-3-carboxypropanoyl]amino-5-[(l-S)-l- ([(l-S)-l-(carboxymethyl)-2-oxo-2-phenylethyl]aminocarbonyl)-2- methylpropyl] amino-5-oxopentanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-4- phenylbutanoate
Figure imgf000059_0001
COOt-Bu
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for phenylmagnesium bromide the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 8.05 (d, 2H, J = 7.6 Hz), 7.83 (d, 2H, J
= 7.6 Hz), 7.61-7.65 (m, 3H), 7.53 (t, 2H, = 7.8 Hz), 7.38 (d, IH, J = 7.5 Hz), 7.27 (dt, 2H, J = 0.9, 7.5 Hz), 7.05 (brd, IH, J = 8.7 Hz), 5.55 (brq, IH, J = 8.6 Hz), 4.32-4.34 (m, 2H), 4.19 (t, IH, J = 7.0 Hz), 2.95 (dd, IH, J = 7.1, 16.1 Hz), 2.66 (dd, IH, J = 6.3, 16.1 Hz), 1.38 (s, 9H).
Step 2: 4-[([2-((E,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- phenylbutylidene)hydrazino]carbonylamino)methyl]- 1-cyclohexanecarboxylic acid
- 57 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000060_0001
Following the procedure of Example 1, step 3, the title compound was obtained: XH NMR (400 MHz, CD3COCD3) δ 8.00 (s, IH), 7.83 (d, 2H, J = 7.6 Hz), 7.59 (t, 2H, J = 7.7 Hz), 7.50 (t, 2H, J = 7.5 Hz), 7.11-7.45 (m, 7H), 6.88 (d, IH, J = 9.3 Hz), 6.76 (brt, IH, J = 5.8 Hz), 5.01-5.64 (m, IH), 4.21-4.91 (m, 2H), 4.13 (brt, IH, J = 7.1 Hz), 3.03-3.22 (m, 2H), 2.85 (dd, IH, J = 7.7 , 14.9 Hz), 2.66 (dd, IH, J = 6.3, 15.1 Hz), 2.20 (brt, IH, J = 3.3 Hz), 1.98 (brd, 2H, = 13.0 Hz), 1.86 (brd, 2H, J = 12.8 Hz), 1.44 (s, 9H), 1.28-1.60 (m, 3H), 0.94-1.06 (m, 2H).
Step 3: Resin IX (R5 = phenyl)
Figure imgf000060_0002
Following the procedure of Example 1, step 4, the title resin was obtained.
58 -
SUBSTΓΓUTE SHEET (RULE 26) Step 4: (4S)-4-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-5~[(l-S)-l-([(l-S)-l- (carboxymethyl)-2-oxo-2-phenylethyl]aminocarbonyl)- 2-methylpropyl]amino-5-oxopentanoic acid
Figure imgf000061_0001
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 7.96 (d, IH, J = 7.2 Hz), 7.59 (t, IH, J = 7.4 Hz), 7.47 (t, 2H, J = 7.8 Hz), 5.70 (dd, IH, J = 5.9, 7.7 Hz), 4.68 (t, IH, J = 7.0 Hz), 4.33 (dd, IH, J = 5.1, 9.1 Hz), 4.06 (d, IH, J = 7.2 Hz), 3.02 (dd, IH, J = 7.8, 16.7 Hz), 2.85 (dd, IH, J = 6.1, 17.0 Hz), 2.71 (dd, IH, J = 7.2, 17.0 Hz), 2.66 (dd, IH, J = 5.8, 16.7 Hz), 2.31-2.40 (m, 2H), 1.83-2.10 (m, 3H), 1.99 (s, 3H), 0.82 (d, 3H, J = 6.8 Hz), 0.77 (d, 3H, J = 6.8 Hz); MS (-APCI) m/z 577 (M-H)-.
EXAMPLE 5
(3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyllamino-4-carboxybutanoyI)aminol-3- methylbutanoylamino)-4-oxoheptanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxoheptanoate
Figure imgf000061_0002
COOt-Bu
- 59 -
SUBSTΓΠJTE SHEET (RULE 26) Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for n- propylmagnesium bromide, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 7.84 (d, 2H, J = 7.5 Hz), 7.68 (d, 2H, J = 7.5 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.31 (t, 2H, J = 7.4 Hz), 6.83 (d, IH, J = 8.3 Hz), 4.36-4.55 (m, 3H), 4.23 (t, IH, J = 6.9 Hz), 2.82 (dd, IH, J = 6.9, 22.1 Hz), 2.48-2.67 (m, 3H), 1.51-1.61 (m, 2H), 1.41 (s, 9H), 0.88 (t, 3H, = 7.4 Hz).
Step 2: 4-[([2-((#,2S)-4-(£er£-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- propylbutylidene)hydrazino]carbonylamino)methyl]-l- cyclohexanecarboxylic acid
Figure imgf000062_0001
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 7.83 (d, 2H, J = 7.5 Hz), 7.63 (d, 2H, J = 7.3 Hz), 7.37 (t, 2H, J = 7.3 Hz), 7.29 (t, 2H, J = 7.4 Hz), 7.19 (d, IH, J = 7.3 Hz), 7.14 (s, IH), 6.75 (brt, IH, J = 6.0 Hz), 4.68 (dd, 1H, J = 6.2, 8.7 Hz), 4.35-4.49 (m, 2H), 4.19 (t, IH, J = 6.5 Hz), 3.01-3.16 (m, 2H), 2.66 (dd, IH, J = 8.8, 14.8 Hz), 2.51 (dd, 1H, J = 7.9, 14.7 Hz), 2.10-2.34 (m, 3H), 1.99 (brd, 2H, J = 12.6 Hz), 1.86 (brd, 2H, J = 14.6 Hz), 1.30-1.58 (m, 5H), 1.39 (s, 9H), 0.86- 1.06 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz). Step 3: Resin IX (R5 = n-Pr)
Figure imgf000063_0001
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxoheptanoic acid
Figure imgf000063_0002
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 4.68 (t, IH, J = 6.7 Hz), 4.63 (t, IH, J = 6.3 Hz), 4.37 (dd, IH, J = 5.0, 9.1 Hz), 4.14 (d, IH, J = 7.2 Hz), ), 2.86 (dd, IH, J = 6.4, 16.9 Hz), 2.85 (dd, IH, = 6.2, 16.9 Hz), 2.72 (dd, IH, J = 7.0, 17.0 Hz), 2.68 (dd, 1H, = 6.4, 18.2 Hz), 2.34-2.54 (m, 4H), 2.08-2.16 (m, 2H), 1.99 (s, 3H), 1.90-1.99 (m, IH), 1.51-1.58 (m, 2H), 0.96 (d, 6H, J = 6.7 Hz), 0.89 (d, 3H, J = 7.4 Hz); MS (-APCI) m z 543 (M-H)-. EXAMPLE 6
(3S)-3-((2-S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-6-phenylhexanoic acid
Step 1: tert-Butyl (3S)-S-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-6- phenylhexanoate
Figure imgf000064_0001
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for phenylethylmagnesium bromide, the title compound was obtained: XH NMR (400 MHz, CD3COCD3) δ 7.84 (d, 2H, J = 7.6 Hz), 7.18 (d, 2H, J = 7.6 Hz), 7.37 (t, 2H, J = 7.5 Hz), 7.14-7.32 (m, 7H), 6.87 (d, IH, J = 8.2 Hz), 4.49-4.58 (m, IH), 4.35-4.46 (m, 3H), 4.23 (t, IH, J = 6.9 Hz), 2.74-3.02 (m, 5H), 2.66 (dd, IH, J = 7.0, 16.1 Hz), 1.41 (s, 9H).
Step 2: 4-[([2-((E,2S)-4-(tert-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-l- phenethylbutylidene)hydrazino]carbonylamino)methy 11-1-cyclohexanecarboxylic acid
Figure imgf000064_0002
62 -
SUBSTΓΓUTE SHEET (RULE 26) Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 9.94 (s, IH), 7.82 (d, 2H, J = 7.6 Hz), 7.67 (t, 2H, J = 7.2 Hz), 7.10-7.39 (m, 9H), 6.75-6.80 (m, 2H), 4.85-4.91 (m, IH), 4.38-4.47 (m, 2H), 4.22 (brt, IH, J = 6.8 Hz), 3.04-3.16 (m, 2H), 2.72-2.92 (m, 2H), 2.54-2.73 (m, 2H), 2.23 (brt, IH, J = 12.1 Hz), 2.02-2.05 (m, 2H), 1.84 (brd, 2H, J = 12.2 Hz), 1.14-1.50 (m, 14H), 0.97-1.03 (m, 2H).
Step 3: Resin IX (R5 = 2-phenylethyl)
Figure imgf000065_0001
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-6-phenylhexanoic acid
Figure imgf000065_0002
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 8.25 (d, IH, J = 7.2 Hz), 7.85 (d, IH, J = 8.1 Hz), 7.11-7.25 (m, 5H), 4.67 (t, IH, = 6.7 Hz), 4.62 (t, IH, J = 6.4 Hz), 4.31-4.36 (m, IH) 4.13 (t, IH, J = 7.4 Hz), 2.64-2.95 (m, 8H), 2.32-2.43 (m, 2H), 2.06-2.14 (m, 2H), 1.98 (s, 3H), 1.89-1.97 (m, IH), 0.93 (d, 6H, J = 6.8 Hz); MS (-APCI) m/z 606 (M-H)-.
EXAMPLE 7 (3-S)-3-((2S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-8-phenyloctanoic acid
Step 1: tert-Butyl (SS)-3-[(9H-9- fluorenylmethoxy)carbonyl]amino-4-oxo-8- phenyloctanoate
Figure imgf000066_0001
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for phenylbutylmagnesium bromide, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 7.84 (d, 2H, J = 7.5 Hz), 7.68 (d, 2H, J = 7.4 Hz), 7.11-7.46 (9H, m), 6.80 (brd, IH, J = 7.9 Hz), 4.32-4.55 (m, 3H), 4.24 (t, IH, J = 6.7 Hz), 2.33-2.84 (m, 6H), 1.23 -1.63 (m, 4H), 1.40 (s, 9H).
- 64 -
SUBSTΓΠJTE SHEET (RULE 26) Step 2: 4-[(2-[(£)-l-((l-S)-3-( r£-Butoxy)-l-[(9H-9- fluorenylmethoxy)carbonyl]amino-3-oxopropyl)-5- phenylpentylidene] hydrazinocarbonyl)amino] methyl- 1-cyclohexanecarboxylic acid
Figure imgf000067_0001
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 9.06 (s, IH), 7.85 (d, 2H, J = 7.4 Hz), 7.69 (d, 2H, J = 7.4 Hz), 7.40 (t, 2H, J = 7.5 Hz), 7.31 (t, 2H, J = 7.5 Hz), 7.09-7.24 (m, 5H), 6.72 (d, IH, J = 9.2 Hz), 6.61 (brt, IH, J = 6.6 Hz), 4.70-4.83 (m, IH), 4.31-4.44 (m, 2H), 4.23 (brt, IH, J = 7.0 Hz), 2.98-3.17 (m, 2H), 2.43-2.88 (m, 5H), 2.13- 2.42 (m, 2H), 1.95-2.05 (m, 2H), 1.87 (brd, 2H, J = 10.4 Hz), 1.30-1.68 (m, 5H), 1.40 (s, 9H), 0.95-1.10 (m, 2H).
Step 3: Resin IX (R5 = 4-phenylbut-l-yl)
Figure imgf000067_0002
- 65 -
SUBSTΓΓUTE SHEET (RULE 26) Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-8-phenyloctanoic acid
Figure imgf000068_0001
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 8.27 (d, IH, J = 7.3 Hz), 7.85 (d, IH, J = 8.0 Hz), 7.08-7.17 (m, 5H), 4.67 (t, IH, = 6.6 Hz), 4.63 (t, IH, J = 6.4 Hz), 4.31-4.40 (m, IH), 4.14 (t, IH, J = 7.9 Hz), 2.79- 2.90 (m, 2H), 2.49-2.76 (m, 6H), 2.30-2.45 (m, 2H), 2.06-2.19 (m, 2H), 1.98 (s, 3H), 1.86-1.98 (m, IH), 1.47-1.64 (m, 4H), 0.95 (d, 6H, J = 6.8 Hz); MS (-APCI) m/z 634 (M-H) .
EXAMPLE 8 (3-S)-3-((2-S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-5-phenylpentanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyI]amino-4-oxo-5- phenylpentanoate
Figure imgf000069_0001
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for benzylmagnesium bromide, the title compound was obtained: lH NMR (400 MHz, CD3COCD3) δ 7.85 (d, 2H, J = 7.4
Hz), 7.71 (d, 2H, J = 7.5 Hz), 7.10-7.46 (m, 9H), 6.87 (brd, IH, J = 7.7 Hz), 4.52-4.65 (m, IH), 4.38-4.50 (m, 2H), 4.26 (m, IH, J = 6.9 Hz), 2.72-2.87 (m, 3H), 2.65 (dd, IH, J = 6.7, 16.2Hz), 1.39 (s, 9H).
Step 2: 4-[([2-((JB,2S)-l-Benzyl-4-(ter<-butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-4- oxobutylidene)hydrazino]carbonylamino)methyl]-l- cyclohexanecarboxylic acid
Figure imgf000069_0002
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 8.93 (s, IH), 7.85 (d, 2H, J = 8.2 Hz), 7.68 (t, 2H, J = 6.5 Hz), 7.40 (t, 2H, J - 7.4 Hz), 7.09-7.35 (m, 7H), 6.76 (d, IH, J = 9.6 Hz), 6.61 (t, IH, J = 5.6 Hz), 4.80-4.86 (m, IH), 4.33-4.45 (m, 2H), 4.22 (t, IH, J = 7.0 Hz), 3.99 (d, IH, J = 15.3 Hz), 3.64 (t, IH, J = 15.5 Hz), 3.01-3.15 (m, 2H), 2.65- 2.83 (m, 2H), 2.61 (dd, IH, J = 5.7, 14.8 Hz), 2.16-2.28 (m, IH), 1.93- 2.04 (m, 2H), 1.80-1.89 (m, 2H), 1.27-1.53 (m, 2H), 1.31 (s, 9H), 0.95- 1.09 (m, 2H).
Step 3: Resin DC (R5 = benzyl)
Figure imgf000070_0001
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-5-phenylpentanoic acid
Figure imgf000070_0002
Following the procedure of Example 1, step 5, the title compound was obtained: *H NMR (400 MHz, CD3OD) δ 7.91 (d, IH, J = 7.3 Hz), 7.15-7.19 (m, 5H), 4.62-4.69 (m, 2H), 4.35-4.42 (m, IH), 4.11-4.17 (m, IH), 3.96 (brs, 2H), 2.81-2.90 (m, 2H), 2.64-2.76 (m, 2H), 2.36-2.45 (m, 2H), 2.07-2.20 (m, 2H), 1.98 (s, 3H), 1.89-2.00 (m, IH), 0.96 (d, 6H, J = 6.8 Hz); MS (-APCI) m/z 591 (M-H)-.
EXAMPLE 9 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(4-methoxyphenyl)-4-oxoheptanoic acid
Step 1: tert-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyl] amino- 7-(4- methoxyphenyl)-4-oxoheptanoate
Figure imgf000071_0001
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for 4- methoxyphenylpropylmagnesium bromide, the title compound was obtained:
*H NMR (400 MHz, CD3COCD3) δ 7.85 (d, 2H, J = 7.5 Hz), 7.69 (d, 2H, J = 7.5 Hz), 7.41 (t, 2H, J = 7.3 Hz), 7.32 (t, 2H, J = 7.5 Hz), 7.08 (d, 2H, J = 8.6 Hz), 6.82 (br d, IH, J = 8.4 Hz), 6.78 (d, 2H, J = 8.6 Hz), 4.32-4.50 (m, 3H), 4.24 (t, IH, J = 6.9 Hz), 2.77-2.82 (m, IH), 2.48-2.66 (m, 5H), 1.79-1.86 (m, 2H), 1.41 (s, 9H).
- 69 -
SUBSTΓΓUTE SHEET (RULE 26) Step 2: 4-([(2-(-E,2-S)-4-(ter/:-Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-l-[3-(4- methoxyphenyl)propyl]-4- oxobutylidenehydrazino)carbonyl]aminomethyl)-l- cyclohexanecarboxylic acid
Figure imgf000072_0001
Following the procedure of Example 1, step 3, the title compound was obtained:
*H NMR (400 MHz, CD3COCD3) δ 9.81 (s, IH), 7.84 (d, 2H, J = 7.6 Hz), 7.68 (t, 2H, J = 7.6 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.30 (t, 2H, J = 7.4 Hz), 7.08 (d, 2H, J = 7.4 Hz), 6.65-6.80 (m, 4H), 4.75-4.85 (m, IH), 4.32-4.44 (m, 2H), 4.21 (brt, IH, J = 7.0 Hz), 3.67 (s, 3H), 3.09- 3.22 (m, 2H), 2.33-2.79 (m, 6H), 2.16-2.28 (m, IH), 1.95-2.05 (m, 2H), 1.70-1.80 (m, 2H), 1.30-1.55 (m, 5H), 1.38 (s, 9H), 0.98-1.10 (m, 2H).
70 -
SUBSTΓΓUTE SHEET (RULE 26) Step 3: Resin IX (R5 = 3-(p-OMe)-phenylprop-l-yl)
Figure imgf000073_0001
Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(4-methoxyphenyl)-4- oxoheptanoic acid
Figure imgf000073_0002
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 8.25 (d, IH, J = 6.7 Hz), 7.84 (d, IH, = 8.0 Hz), 7.06 (d, 2H, J = 8.2 Hz), 6.79 (d, 2H, J = 8.4 Hz), 4.66 (t, IH, J = 6.2 Hz), 4.60 (t, IH, J - 6.3 Hz), 4.32-4.40 (m, IH), 4.12 (brt, IH, J = 7.2 Hz), 3.72 (s, IH), 2.30-2.90 (m, 10H), 2.03-2.18 (m, 2H), 1.85-2.00 (m, IH), 1.97 (s, 3H), 1.72-1.85 (m, 2H), 0.92 (d, 6H, J - 6.6 Hz); MS (-APCI) m/z 650 (M-H)-.
EXAMPLE 10 (3-S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid
Step 1: r*-Butyl (3S)-3-[(9H-9- fluorenylmethoxy)carbonyllamino-7-(l-naphthyl)-4- oxoheptanoate
Figure imgf000074_0001
Following the procedure of Example 1, Step 2, substituting phenylpropylmagnesium bromide for 1- naphthylpropylmagnesium bromide, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 8.16 (d, IH, J = 8.4 Hz), 7.84-7.88 (m, 3H), 7.68-7.73 (m, 3H), 7.29-7.53 (m, 8H), 6.87 (d, IH, J = 8.4 Hz), 4.51-4.57 (m, IH), 4.37-4.47 (m, 2H), 4.23 (t, IH, J = 6.8 Hz), 3.05-3.09 (m, 2H), 2.64-2.87 (m, 4H), 1.96-2.03 (m, 2H), 1.41 (s, 9H).
- 72 -
SUBSTΓΓUTE SHEET (RULE 26) Step 2: 4-([(2-(£,2-S)-4-(£e -Butoxy)-2-[(9H-9- fluorenylmethoxy)carbonyl]amino-l-[3-(l- naphthyl)propyl]-4-oxobutylidenehydrazino) carbon yll aminomethyl)-l-cyclohexanecarboxylic acid
Figure imgf000075_0001
Following the procedure of Example 1, step 3, the title compound was obtained: H NMR (400 MHz, CD3COCD3) δ 9.97 (s, IH), 8.17 (d, IH, J = 8.2 Hz), 7.81-7.84 (m, 3H), 7.81-7.84 (m, 3H), 7.25- 7.50 (m, 8H), 6.73-6.81 (m, 2H), 4.80-4.90 (m, IH), 4.30-4.42 (m, 2H), 4.18 (brt, IH, J = 7.0 Hz), 3.07-3.27 (m, 4H), 2.53-2.79 (m, 3H), 2.14- 2.27 (m, IH), 1.91-2.02 (m, 2H), 1.75-1.88 (m, 2H), 1.23-1.55 (m, 4H), 1.98 (s, 9H), 0.80-1.55 (m, 3H).
Step 3: Resin DC (R5 = 3-(l-naphthyl)prop-l-yl)
Figure imgf000075_0002
73 -
SUBSTΓΓUTE SHEET (RULE 26) Following the procedure of Example 1, step 4, the title resin was obtained.
Step 4: (3-S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid
Figure imgf000076_0001
Following the procedure of Example 1, step 5, the title compound was obtained: H NMR (400 MHz, CD3OD) δ 8.12 (d, IH, J = 8.1 Hz), 7.86 (d, IH, J = 8.1 Hz), 7.82 (d, IH, J = 7.7 Hz), 7.68 (d, IH, J = 8.1 Hz), 7.31-7.50 (m, 4H), 4.60-4.70 (m, 2H), 4.35 (dd, IH, J = 4.7, 8.9 Hz), 4.14 (brt, IH, J = 7.9 Hz), 3.00-3.08 (m, 2H), 2.83-2.92 (m, 2H), 2.55-2.78 (m, 4H), 2.35-2.45 (m, 2H), 2.07-2.19 (m, 2H), 1.90-2.03 (m, 5H), 0.94 (d, 6H, J = 6.8 Hz); MS (-APCI) m/z 670 (M-H)-.
O 00/
EXAMPLE 100 (3-S)-3-((2-S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid
Figure imgf000077_0001
Using the resin from Example 1, Step 4, and substituting Fmoc-L-Glu(OtBu)-OH for Fmoc-L-Ala-OH(1.56 g) in Step 5, the title compound was obtained as colorless solid. τH NMR (400 MHz, CD3OD) δ 8.32 (d, IH, J = 7.20 Hz), 7.82 (d, IH, J = 8.12 Hz), 7.30 - 7.10 (m, 5H), 4.68 (t, IH, J = 6.70 Hz), 4.63 (t, IH, J = 6.21 Hz), 4.30 - 4.10 (m, IH), 4.13 (brt, IH, J = 7.62 Hz), 2.91 - 2.80 (m, 2H), 2.75 - 2.45 (m, 6H), 2.20 - 2.05 (m, IH), 1.97 (s, 3H), 1.90 - 1.78 (m, 2H), 1.37 (d, 3H, J = 7.20 Hz), 0.94 (d, 6H, J = 6.80 Hz); MS (+APCI) m/z 563 (M+H)+.
EXAMPLE 101 (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenyInonanoic acid
Figure imgf000077_0002
Using the resin from Example 3, Step 3, and following the procedure of Example 1, Step 5, substituting Fmoc-L-
75 -
SUBSTΓΓUTE SHEET (RULE 26) Glu(OtBu)-OH for Fmoc-L-Ala-OH (1.56 g), the title compound was obtained as a colorless solid: lH NMR (400 MHz, CD3OD) δ 8.27 (d, IH, J = 6.1 Hz), 7.78 (d, IH, J = 8.4 Hz), 7.09-7.24 (m, 5H), 4.68 (t, IH, J = 6.7 Hz), 4.64 (t, IH, = 6.3 Hz), 4.27-4.31 (m, IH), 4.13 (t, IH, J = 7.9 Hz), 2.42-2.90 (m, 8H), 2.08-2.19 (m, IH), 1.97 (s, 3H), 1.53-1.66 (m, 4H), 1.25-1.38 (m, 2H), 1.36 (d, 3H, J = 7.2 Hz), 0.95 (d, 6H, J = 7.8 Hz); MS (-APCI) m/z 590 (M-H)-.
EXAMPLE 102
(3S)-3-[((2S)-2-[(2S)-2-((2S)-3-Carboxy-2-[(3,5- dibromobenzoyl)amino]propanoylamino)propanoyl]amino-3- methylbutanoyl)aminol-4-oxo-7-phenylheptanoic acid
Figure imgf000078_0001
a) Preparation of the tetrapeptide on solid support
Using the resin from Example 1, Step 4, and following the procedure given in the 9050 PepSynthesizer user's guide, the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), 3,5-dibromobenzoic acid (1.44 g),
Fmoc-L-Val-OH (1.65g,), Fmoc-Ala-OH (1.59 g), Fmoc-L-Asp (OtBu)-
OH (1.80 g); O-(7-azabenzotriazol-l-yl)N,N,N',N' - tetramethyluronium hexafluorophosphate (HATU) and N,N- diisopropylethylamine (DIEA) were used as coupling reagents instead of TBTU and HOBt as described in the user's guide.
76 -
SUBSTΓΓUTE SHEET (RULE 26) b) Cleavage from solid support
The above polymer was transferred into a fritted reaction vessel and treated with TFA/H2O (9:1, 2 mL) and agitated for 30 min. The solution was filtered, the solid support was washed with TFA (2 x 0.5 mL) and the filtrate was evaporated. Trituration from Et2θ gave 83 mg of the title compound as a colorless solid: lH NMR (400 MHz, CD3OD) δ 8.01 (d, 2H, J = 1.67
Hz), 7.90 (t, IH, 1.69 Hz), 7.81 (d, IH, J = 7.85 Hz), 7.28 - 7.05 (m, 5H),
4.87 (t, IH, J = 6.30 Hz) 4.63 (t, IH, J = 6.20 Hz), 4.40 - 4.30 (m, IH),
4,18 - 4.10 (m, IH), 3.00 (dd, IH, 6.12, 16.88 Hz), 2.90 - 2.78 (m, 2H),
2.75 - 2.45 (m, 5H), 2.20 - 2.05 (m, IH), 1.90 - 1.78 (m, 2H), 1.38 (d, 3H,
J = 7.18 Hz), 0.94 (d, 6H, J = 6.73 Hz); MS (+APCI) m z 783 (M+H)+.
EXAMPLE 103
(3S)-3-[((2S)-2-[(2-S)-2-((2S)-3-Carboxy-2-[(3,5- dideuteriobenzoyl)amino]propanoylamino)propanoyl]amino-3- methylbutanoyl)amino]-4-oxo-7-phenylheptanoic acid
Figure imgf000079_0001
To the peptide from Example 103 (10 mg, 12.8 μmol) in 2 mL MeOH and 0.5 mL NEt3 was added Pd/C (10%, 2 mg) and the mixture was stirred under a D2-balloon for 20 h. The mixture was filtered through a pad of Celite, rinsed with MeOH, and evaporated to give 10 mg (100%) of the title compound: H NMR (400 MHz, CD3OD) δ 7.85 (s, 2H), 7.50 (s, IH), 7.25 - 7.05 (m, 5H), ~ 4.8 (m, IH, signal obstructed by CD3OD), 4.60 (t, IH), 4.38 - 4.30 (m, IH), 4.18 - 4.08 (m, IH), 2.90 - 2.45 (m, 8H), 2.21 - 2.08 (m, IH), 1.85 - 1.72 (m, 2H), 1.40 (d, 3H), 0.95 (d, 6H); MS (-APCI) m/z 626 (M-H)".
EXAMPLE 104
(3S)-3-((2S)-2-[((2S)-2-[(2S)-2-Amino-4-methoxy-4- oxobutanoyl]aminopropanoyl)amino]-3-methylbutanoylamino)-4- oxo-7-phenylheptanoic acid
a) Preparation of the tetrapeptide on solid support
Figure imgf000080_0001
Using the resin from Example 1, Step 4, and following the procedure given in the 9050 PepSynthesizer users guide, the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), t-Boc-L-Asp (OCH3)-OH (1.79 g),
Fmoc-L-Ala-OH (1.59 g), Fmoc-L-Val-OH (1.65 g), O-(7- azabenzotriazol-l-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine
(DIEA) were used as coupling reagents instead of TBTU and HOBt as described in the user's guide. b) Cleavage from solid support
Figure imgf000081_0001
The above polymer was transferred into a fritted reaction vessel and treated with TFA/H2O (9:1, 2mL) and agitated for 30 min. The solution was filtered, the solid support was washed with TFA (2 x 0.5 mL) and the filtrate was evaporated. Trituration from Et2θ gave 71 mg of the title compound as a colorless solid: H NMR (400 MHz, CD3OD) δ 7.89 (d, IH, J = 7.87
Hz), 7.28 - 7.08 (m, 5H), 4.59 (t, IH, J = 6.12 Hz), 4.45 - 4.35 (m, IH),
4.25 - 4.10 (m, 2H), 3.73 (s, 3H,), 3.15 - 3.02 (m, IH), 2.92 - 2.78 (m,
2H), 2.75 - 2.45 (m, 5H), 2.15 - 2.00 (m, IH), 1.88 - 1.78 (m, 2H), 1.36 (d,
3H, J = 7.16 Hz), 0.92 (2d, 6H, J = 6.73 Hz); MS (+APCI) m/z 535
(M+H)+.
EXAMPLE 105
(3-S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-4-methoxy-4- oxobutanoyl]aminopropanoyl)amino]-3-methylbutanoylamino)-4- oxo-7-phenylheptanoic acid
Figure imgf000081_0002
To the peptide from Example 104 (25 mg, 47 μmol) in 400 μL THF was added NaHCO3 (39.5 mg in 500 μL H2O) and acetyl chloride (33.5 μL in 500 μL THF). The mixture was stirred for 16 h and then poured into sat. aq. NH4CI. The layers were separated
- 79 -
SUBSTΓΓUTE SHEET (RULE 26) and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over MgSθ4, filtered and evaporated.
The crude material was triturated with Et2θ to give 17 mg (63%) of the product as a colorless solid: H NMR (400 MHz, CD3OD) δ 7.30 - 7.08 (m, 5H), 4.70 (t, IH, J = 6.76 Hz), 4.62 (t, IH, J = 6.12 Hz), 4.35 - 4.25 (m, IH), 4.19 - 4.10 (m, IH), 3.67 (s, 3H), 2.95 - 2.45 (m, 8H), 2.20 - 2.05 (m, IH), 1.96 (s, 3H), 1.90 - 1.78 (m, 2H), 1.37 (d, 3H, J = 7.17 Hz), 0.94 (d, 6H, J = 6.77 Hz); MS (+APCI) m/z 577 (M+H)+.
EXAMPLE 106
(3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid
Figure imgf000082_0001
Using the resin from Example 10, Step 3, and following the procedure of Example 1, Step 5, substituting Fmoc-L- Glu(OtBu)-OH for Fmoc-L-Ala-OH (1.56 g), the title compound was obtained as a colorless solid: H NMR (400 MHz, CD3OD) δ 8.27 (d, IH, J = 6.2 Hz), 8.12 (d, IH, J = 8.3 Hz), 7.82 (d, IH, J = 8.0 Hz), 7.79 (d, IH, = 7.9 Hz), 7.69 (d, IH, J = 8.0 Hz), 7.31-7.50 (m, 4H), 4.61- 4.70 (m, 2H), 4.25-4.35 (m, IH), 4.14 (brt, IH, J = 7.2 Hz), 3.02-3.09 (m, 2H), 2.83-2.92 (m, 2H), 2.53-2.80 (m, 4H), 2.05-2.18 (m, 2H), 1.92- 2.02 (m, 4H), 1.37 (d, 2H, J = 7.2 Hz), 0.94 (d, 6H, J = 6.8 Hz); MS (- APCI) m z 612 (M-H) . EXAMPLE 107
(3-S)-3-[((2-S)-2-[(2-S)-2-((2S)-3-Carboxy-2-[(4- iodobenzoyl)amino] propanoy lamino)propanoy 1] amino-3- 5 methylbutanoyl)amino1-4-oxo-7-phenylheptanoic acid
Figure imgf000083_0001
a) Preparation of the tetrapeptide on solid support
! 0
Using the resin from Example 1, Step 4, and following the procedure given in the 9050 PepSynthesizer user's guide, the tetrapeptide was prepared with the following reagents: 290 mg of polymer (0.7 meq/g loading), 4-iodobenzoic acid (1.28 g), Fmoc-L-
15 Val-OH (1.65g,), Fmoc-Ala-OH (1.59 g), Fmoc-L-Asp (OtBu)-OH (1.80 g); O-(7-azabenzotriazol-l-yl)N,N,N',N' -tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) were used as coupling reagents instead of TBTU and HOBt as described in the user's guide. 0 b) Cleavage from solid support
The above polymer was transferred into a fritted reaction vessel and treated with TFA/H2O (9:1, 2 mL) and agitated 5 for 30 min. The solution was filtered, the solid support was washed with TFA (2 x 0.5 mL) and the filtrate was evaporated. Trituration from Et2θ gave 83 mg of the title compound as a colorless solid: lH NMR (400 MHz, CD3SOCD3) δ 8.71 (d, IH, J = 7.3 Hz), 8.41 (d, IH, J = 7.3 Hz), 8.05 (d, IH, J = 7.5 Hz), 7.84 (d, 2H, J = 30 8.4 Hz), 7.74 (d, IH, J = 8.3 Hz), 7.62 (d, 2H, J = 8.3 Hz), 7.26-7.13 (m, 5H), 4.85-4.75 (m, IH), 4.50-4.45 (m, IH), 4.40-4.28 (m, IH), 4.18-4.08 (m, IH), 2.85-2.60 (m, 3H), 2.55-2.35 (m, 5H), 2.00-1.89 (m, IH), 1.80- 1.65 (m, 2H), 1.18 (d, 3H, J = 6.9 Hz), 0.81 (d, 3H, J = 6.6 Hz), 0.79 (d, 3H, J = 6.7 Hz); MS (-APCI) m z 749 (M-H)".
EXAMPLE 200
(3S)-3-[((2S)-2-[(2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-(methylsulfonyl)butanoyl]amino-3- methylbutanoyI)amino]-4-oxo-7-phenylheptanoic acid
Figure imgf000084_0001
Using the resin from Example 1, Step 4, and substituting Fmoc-L-Glu(OtBu)-OH for Fmoc-L-Met(O2)-OH(1.80 g) in Step 5, the title compound was obtained as colorless solid: H NMR (400 MHz, CD3OD) δ 8.23 (d, IH, = 7.60 Hz), 7.93 (d, IH, J =
7.34 Hz), 7.25-7.05 (m, 5H), 4.68-4.58 (m, 2H), 4.57-4.48 (m, IH), 4.12 (t, IH, J - 7.47 Hz), 3.25-3.10 (m, 2H), 2.95 (s, 3H), 2.90-2.48 (m, 8H), 2.40-2.28 (m, IH), 2.20-2.02 (m, 2H), 1.97 (s, 3H), 1.90-1.78 (m, 2H), 0.93 (d, 6H, J = 6.57 Hz); MS (-APCI) m/z 653 (M-H)".
EXAMPLE 201 (3S)-3-((2S)-2-[((2S)-2-[(2#)-2-(Acetylamino)-3- (methylsulfanyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoyIamino)-4-oxo-7-phenylheptanoic acid
Figure imgf000085_0001
Using the resin from Example 1, Step 4, and substituting Fmoc-L-Asp(OtBu)-OH for Fmoc-L-Cys(SMe)-OH(1.56 g) and Fmoc-L-Glu(OtBu)-OH for Fmoc-L-Ala-OH (1.56 g) in Example 1, Step 5, the title compound was obtained as colorless solid:
XH NMR (400 MHz, CD3OD) δ 7.76 (d, IH, J = 8.02 Hz), 7.25-7.05 (m, 5H), 4.60 (t, IH, J = 6.03 Hz), 4.51 (t, IH, J = 7.28 Hz), 4.40-4.30 (m, IH), 4.15 (t, IH, J = 7.56 Hz), 2.96-2.45 (m, 8H), 2.15-2.08 (m, IH), 2.11 (s, 3H), 1.97 (s, 3H), 1.90-1.88 (m, 2H), 1.35 (d, 3H, J = 7.18 Hz), 0.92 (d, 3H, J = 6.76 Hz), 0.91 (d, 3H, J = 6.74 Hz); MS (- APCI) m/z 563 (M-H) .
EXAMPLE 202 (3S)-3-((2-S)-2-[((2S)-2-[(2Λ)-2-(Acetylamino)-3- (methylsulfonyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid
Figure imgf000085_0002
The title compound from Example 201 (20 mg, 0.035 mmol) in 1 mL MeOH at O°C was treated with OXONE® (22 mg, 0.035 mmol) in 1 mL water. The mixture was allowed to warm to room temperature over 0.5 h and then poured into 30 mL EtOAc. Water was added, the layers were separated, and the aqueous layer was extracted with EtOAc (2x5 mL). The combined organic layers were dried over MgSO4, filtered and evaporated to give 6 mg of the title compound as colorless solid: lH NMR (400 MHz, CD3OD) 8.14 (d, IH, J = 7.81 Hz), 7.75 (d, IH, J = 7.24 Hz), 7.25-7.05
(m, 5H), 4.94 (t, IH, J = 6.59 Hz), 4.62 (t, IH, J = 6.20 Hz), 4.35-4.20 (m, IH), 4.15-4.05 (m, IH), 3.78-3.70 (m, IH), 3.48-3.38 (m, IH), 3.29 (s, 3H), 2.88-2.80 (m, IH), 2.75-2.45 (m, 5H), 2.15-2.03 (m, IH), 1.97 (s, 3H), 1.90-1.80(m, 2H), 1.37 (d, 3H, J = 7.26 Hz), 0.93 (d, 3H, J = 6.77 Hz), 0.92 (d, 3H, J = 6.74 Hz); MS (-APCI) m z 595 (M-H)-.
EXAMPLE 203 (3S)-3-((2S)-2-[((2S)-2-[(2-R)-2-(Acetylamino)-3-
(methylsulfanyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid
Figure imgf000086_0001
Using the resin from Example 10, Step 3, and substituting Fmoc-L-Asp(OtBu)-OH for Fmoc-L-Cys(SMe)-OH(1.56 g) and Fmoc-L-Glu(OtBu)-OH for Fmoc-L-Ala-OH (1.56 g) in Example 10, Step 4, the title compound was obtained as colorless solid: lH NMR (400 MHz, CD3OD) δ 8.38 (d, IH, J = 6.4 Hz), 8.29 (d, IH, J = 8.3 Hz), 8.11 (d, IH, J = 8.0 Hz), 7.82 (d, IH, J = 7.4 Hz), 7.77 (d, IH, J = 8.1 Hz), 7.69 (d, IH, J = 7.9 Hz), 7.31-7.51 (m, 4H), 4.62- 4.70 (m, IH), 4.49-4.57 (m, IH), 4.32-4.40 (m, IH), 4.17 (brt, IH, J = 6.8 Hz), 3.01-3.10 (m, 2H), 2.82-2.94 (m, 2H), 2.56-2.76 (m, 4H), 2.05- 2.16 (m, 4H), 1.92-2.01 (m, 5H), 1.38 (d, 2H, J = 7.2 Hz), 0.93 (dd, 6H, J = 3.9, 6.8 Hz); MS (-APCI) m/z 613 (M-H)-.
- 84 -
SUBSTΓΓUTE SHEET (RULE 26) EXAMPLE 204 (3S)-3-((2S)-2-[((2S)-2-[(2#)-2-(Acetylamino)-3- (methylsulfonyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyI)-4-oxoheptanoic acid
Figure imgf000087_0001
Using the title compound from Example 203, and following the procedure of Example 202, the title compound was obtained: XH NMR (400 MHz, CD3OD) δ 8.48 (br d, IH, J = 6.3 Hz),
8.12 (d, IH, J = 8.2 Hz), 7.83 (d, IH, J = 7.8 Hz), 7.76 (d, IH, J = 7.7 Hz), 7.69 (d, IH, J = 8.3 Hz), 7.31-7.51 (m, 4H), 4.95 (t, IH, J = 6.7 Hz), 4.67 (t, IH, J = 6.3 Hz), 4.28-4.31 (m, IH), 4.14 (brt, IH, J = 7.6 Hz), 3.76 (dd, IH, J = 6.3, 14.7 Hz), 3.41 (dd, IH, J = 7.6, 14.3 Hz), 3.00-3.09 (m, 5H), 2.88 (d, IH, J = 6.0, 16.9 Hz), 2.57-2.80 (m, 3H), 2.05-2.14 (m, IH), 1.93-2.03 (m, 5H), 1.38 (d, 2H, J = 7.2 Hz), 0.93 (d, 6H, J = 6.7 Hz); MS (-APCI) m/z 646 (M-H)-.
Assays for Determining Biological Activity
(a) Measurement of caspase activity by cleavage of a fluorogenic substrate
A fluorogenic derivative of the tetrapeptide recognized by caspase-3 and corresponding to the Pi to P4 amino acids of the PARP cleavage site, Ac-DEVD-AMC (AMC, amino-4- methylcoumarin) was prepared as follows: i) synthesis of N-Ac- Asp(OBn)-Glu(OBn)-Val-CO2H, ii) coupling with Asp(OBn)-7- amino-4-methylcoumarin, iii) removal of benzyl groups.
85 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000088_0001
Standard reaction mixtures (300 μL final volume), contained Ac-DEVD-AMC and purified or crude caspase-3 enzyme in 50 mM Hepes/KOH (pH 7.0), 10% (v/v) glycerol, 0.1% (w/v) CHAPS, 2 mM EDTA, 5 mM dithiothreitol, and were incubated at 25°C. Reactions were monitored continuously in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
(b) Cell Death Detection ELISA (Whole Cell Assay)
Colorimetric immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone- associated-DNA-fragments (mono- and oligonucleosomes) after induced cell death. This assay was performed using the commercially available kit from Boehringer Mannheim, cat. No. 1 920 685.
c) In Vivo Myocardial Ischemia and Reperfusion Injury in Rats Male Sprague-Dawley rats (300-400g) were fasted overnight, and then anesthetized with intraperitoneal administration of sodium pentobarbital (65 mg/kg). To monitor heart rate and aortic pressure the left carotid artery was isolated and a cannula placed in the vessel. The aortic cannula was interfaced with a pressure transducer which was connected to a physiologic recorder. The left jugular vein was isolated and cannulated for administration of a caspase inhibitor compound or vehicle (2 % dimethylsulfoxide in 0.9% NaCl). A left thoracotomy
- 86 -
SUBSTΓΓUTE SHEET (RULE 26) was performed in the region overlying the heart and the pericardium opened, exposing the heart. The origin of the left coronary artery was visualized and a 4.0 suture passed under the artery approximately 2 - 3 mm from its origin. The ends of the suture were passed through a short length of 2 mm id tubing and coronary artery occlusion effected by placing tension on the suture such that the tube compressed the artery. After initial placement of the suture/occluder, the thoracotomy was closed with a small clamp and opened only to effect occlusion and reperfusion of the artery. A Lead II electrocardiograph (ECG) signal was obtained by placing subdermal platinum leads and continuously monitored. After a baseline period of 20-30 minutes the left coronary artery was occluded for 45 minutes. The period of reperfusion was 3 hours. The caspase inhibitor or vehicle was administered as a first bolus 5 minutes before the onset of ischemia and a second bolus was administered again at the onset of reperfusion. Additionally, an infusion was initiated immediately after the first bolus dose. Control animals received the vehicle alone in equal volumes to the caspase inhibitor treated animals. At the end of reperfusion the animals were euthanized and infarct size determined using a dual staining technique (1.5% w/v triphenyltetrazolium chloride to demarcate infarct tissue and 0.25% w/v Evan's blue to demarcate the area at risk of infarct. The heart was subsequently cut transversely into 4 slices of equal thickness, and infarct size and area at risk quantified using planimetry.
Using the above procedure, it is demonstrated that administration of a caspase inhibitor reduces infarct size in the rat subjected to 45 minutes of regional ischemia and 3 hours of reperfusion.
- 87 -
SUBSTΓΓUTE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:
1. A compound of formula I
Figure imgf000090_0001
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from the group consisting of:
(a) H and
(b) C(O)Rl;
R1 is selected from the group consisting of:
(a) hydrogen,
(b) Ci-βalkoxy, (c) NR6R7,
(d) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from the group consisting of:
(1) methyl, (2) halogen,
(3) methoxy and
(4) cyano,
(e) Cι_6alkyl or substituted Cι_6alkyl, wherein the substituent is selected from the group consisting of: (1) hydroxy,
(2) halo,
(3) Cι_3alkoxy,
(4) Cι_3alkylthio,
(5) phenyl Cι_3alkoxy, (6) phenyl Cι_3alkylthio,
- 88 -
SUBSTΓΓUTE SHEET (RULE 26) (7) phenylcarboxy and
(8) carboxy,
(f) aryl or arylCi-βalkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(ID > pyrimidyl,
(12) quinolyl,
(13) > isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16, > pyrazolyl,
(17 ) indolyl,
(18 ) purinyl,
(19 ι isoxazolyl and
(20 i oxazolyl, and
(g) mo no and di-substituted aryl as defined above in items
(1) to (20 ) of (f), wherein the substituents are independently selected τom:
(1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano,
(6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci-βalkyl, (10) Ci-6fluoroalkyl,
(11) Cι_6alkylcarbonyl,
(12) Ci-galkoxy carbonyl,
(13) Ci-βalkoxy,
(14) Ci-βalkylthio,
(15) Ci-βalkylsulfonyl and
(16) deuterio;
selec :ted from the group consistinj
(a) H,
(b) CH3,
(c) CH(CH3)2.
(d) CH2CH(CH3)2,
(e) CH2Ph, ω CH2PhOH,
(g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
(j) CH(CH3)CH2CH3,
( ) CH(CH3)OH,
(1) CH2COOH,
( ) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(0)NH2,
(q) CH2CH2C(O)NH2,
(r) CH2CO2Ci-4alkyl,
(s) CH2SCι_4alkyl,
(t) CH2S(0)2Cι_4alkyl,
Figure imgf000092_0001
Figure imgf000093_0001
or R2 and X together form a saturated monocyclic ring having the following structure:
Figure imgf000093_0002
R3 is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph,
CH2PhOH,
(g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
(j) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(0)NH2,
(q) CH2CH2C(0)NH2,
(r) CH2CH2CO2Ci_4alkyl,
(s) CH2CH2S(0)2Ci. alkyl,
Figure imgf000093_0003
Figure imgf000094_0001
or R3 and X3 together form a saturated monocyclic ring having the following structure:
Figure imgf000094_0002
R4 is selected from the group consisting of:
(a) H,
(b) CH3,
(c) CH(CH3)2,
(d) CH2CH(CH3)2,
(e) CH2Ph,
(f) CH2PhOH,
(g) CH2OH,
(h) CH2SH,
(i) CH2CH2SCH3,
(j) CH(CH3)CH2CH3,
(k) CH(CH3)OH,
(1) CH2COOH,
(m) CH2CH2COOH,
(n) CH2CH2CH2NHCNH(NH2),
(o) CH2CH2CH2CH2NH2,
(P) CH2C(O)NH2,
(q) CH2CH2C(O)NH2,
Figure imgf000094_0003
- 92 -
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000095_0001
or R and X4 together form a saturated monocyclic ring having the following structure:
Figure imgf000095_0002
R is selected from the group consisting of:
(a) Cι_6alkyl,
(b) arylCι_8alkyl wherein the aryl is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl,
(20) oxazolyl and (21) coumarinyl and (c) aryl as defined above in items (1) to (21) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from: (1) halo,
(2) amino,
(3) nitro,
(4) hydroxy,
(5) cyano, (6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci-βalkyl,
(10) Ci_6fluoroalkyl, (11) Cι_6alkylcarbonyl,
(12) Ci_6alkoxycarbonyl,
(13) Cι_6alkoxy,
(14) Ci_6alkylthio and
(15) Ci-βalkylsulfonyl;
R and R^ are independently selected from the group consisting of:
(a) Cι_4alkyl,
(b) Cι_4fluoroalkyl and (c) benzyl or mono- or disubstituted benzyl wherein the substituent is selected from the group consisting of:
(1) methyl,
(2) halogen,
(3) methoxy and (4) cyano, or R and R^ may be joined to form a pyrrolidine, piperidine, morpholine, thiamorpholine or N-R8 substituted piperazine wherein R is H or Ci_3alkyl; and X2, X3 and X4 are independently H or X2 and R2, X3 and R3,or X4 and R4 may together form a saturated monocyclic ring having the following structure:
Figure imgf000097_0001
2. A compound according to Claim 1 represented by formula la:
Figure imgf000097_0002
la
or a pharmaceutically acceptable salt thereof, wherein:
the amino acids represented in formula I as AAl, AA and AA3 are independently selected from a group consisting of:
(a) alanine,
(b) arginine,
(c) asparagine,
(d) aspartic acid,
(e) cysteine,
(f) glutamine,
(g) glutamic acid, (h) glycine,
(i) histidine,
(j) isoleucine,
(k) leucine,
(1) lysine,
(m) methionine,
(n) phenylalanine, (o) proline,
(P) serine,
(q) threonine,
(r) tryptophane,
(s) tyrosine and
(t) valine.
3. A compound according to Claiml wherein Rl is Cι_6alkyl or phenyl.
! θ
4. A compound according to Claim 1 wherein R^ is arylCi-8alkyl, wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or di-substituted derivatives thereof, wherein the substituents are individually 5 selected from the group consisting of:
(1) halo,
(2) amino,
(3) nitro,
(4) hydroxy, 0 (5) cyano,
(6) carboxy,
(7) formyl,
(8) amino carbonyl,
(9) Ci.3alkyl, 5 (10) Cι_3fluoroalkyl,
(11) Cι_3alkylcarbonyl,
(12) Cι_3alkoxycarbonyl,
(13) Cι_3alkoxy,
(14) Cι_3alkylthio and 0 (15) Ci_3alkylsulfonyl;
5. A compound according to Claim 1 wherein:
Rl is selected from the group consisting of: 5 (a) Ci-βalkoxy,
- 96 -
SUBSTΓΓUTE SHEET (RULE 26) (b) benzyloxy or mono- or disubstituted benzyloxy, wherein the substituent is selected from methyl, halogen, methoxy and cyano,
(c) Cι_6alkyl or substituted Cι_6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Cι_3alkoxy,
(4) Ci-3alkylthio, (5) phenylCι_3alkoxy,
(6) phenylCι_3alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(d) aryl or arylCi_6alkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl and
(2) naphthyl, and
(e) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy, (5) amino carbonyl,
(6) Ci-3alkyl,
(7) Cι_3fluoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl, (10) Cι_3alkoxy,
(11) Cι_3alkylthio,
(12) Cι_3alkylsulfonyl and
(13) deuterio; and
R5 is arylCi_8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or di-
- 97 -
SUBSTΓΠJTE SHEET (RULE 26) substituted derivatives thereof, wherein the substituents are individually selected from the group consisting of:
(1) halo,
(2) hydroxy, (3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Ci-3alkyl,
(7) Ci-3fluoroalkyl, (8) Cι_3alkylcarbonyl,
(9) Cχ_3alkoxycarbonyl,
(10) Cι_3alkoxy,
(11) Ci-3alkylthio and
(12) Ci_3alkylsulfonyl.
6. A compound according to Claim 1 wherein
Rl is selected from the group consisting of:
(a) Cι_6alkyl or substituted Ci-galkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Cι_3alkoxy,
(4) Ci_3alkylthio, (5) phenylCι_3alkoxy,
(6) phenylCι_3alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(b) aryl or arylCi-βalkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl and
(2) naphthyl, and
(c) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from:
(1) halo,
- 98 -
SUBSTΓΓUTE SHEET (RULE 6) (2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Cl-3alkyl,
(7) C i_3fluoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl,
(10) Ci-3alkoxy,
,0 (ID Cι_3alkylthio,
(12) Cι_3alkylsulfonyl and
(13) deuterio.
A compound according to Claim 1 wherein 5
R2 is selected from the group consisting of:
(a) CH2CO2H,
(b) CH2Cθ2Cι_4alkyl,
(c) CH2SCι_4alkyl and 0 (d) CH2s(O)2Cι_ alkyl.
8. A compound according to Claim 1 wherein
R3 is selected from the group consisting of: 5 (a) CH3,
(b) CH2CH2CO2H,
(c) CH2CH2CO2Ci_4alkyl and
(d) CH2CH2S(0)2Cι.4alkyl.
0 9. A compound according to Claim 1 wherein
R4 is isopropyl.
10. A compound according to Claim 1 wherein
35
R5 is selected from the group consisting of: (a) Cι.6alkyl,
(b) arylCι_8alkyl wherein the aryl is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl and
(4) coumarinyl, and
(c) aryl as defined above in items (1) to (4) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Cι_3alkyl,
(7) Cι_3fluoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Cι_3alkoxycarbonyl,
(10) Cι_3alkoxy,
(11) Cι_3alkylthio and
(12) Cι_3alkylsulfonyl.
11. A compound of formula II
Figure imgf000102_0001
or a pharmaceutically acceptable salt thereof, wherein:
00 R5 is selected from the group consisting of:
(a) Ci-βalkyl,
(b) arylCι_8alkyl wherein the aryl is selected from the group consisting of: (1) phenyl,
(2) naphthyl,
(3) pyridyl and
(4) coumarinyl, and
(c) aryl as defined above in items (1) to (4) of (b), wherein the aryl portions may be optionally mono- or disubstituted with a substituent independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano, (4) carboxy,
(5) amino carbonyl,
(6) Ci-3alkyl,
(7) Cι_3fluoroalkyl,
(8) Cι_3alkylcarbonyl, (9) Cι_3alkoxycarbonyl,
(10) Ci-3alkoxy,
(11) Ci-3alkylthio and
(12) Ci_3alkylsulfonyl.
12. A compound according to Claim 11 wherein
R5 is selected from the group consisting of:
(a) methyl,
(b) propyl, (c) phenyl,
(d) phenylCi_5alkyl,
(e) 4-methoxyphenylpropyl,
(f napthylpropyl and
(g) 4-methylcoumarinyl.
13. A compound according to Claim 12 selected from the following group:
Figure imgf000104_0001
e
Figure imgf000105_0001
03
Figure imgf000106_0001
14. A compound of formula III
Figure imgf000106_0002
III
or a pharmaceutically acceptable salt thereof, wherein:
Rl is selected from the group consisting of:
(a) Ci-6alkyl or substituted Cl_6alkyl, wherein the substituent is selected from the group consisting of:
(1) hydroxy,
(2) halo,
(3) Cι_3alkoxy,
(4) Ci-3alkylthio, (5) phenylCι_3alkoxy,
- 104 -
SUBSTΓΓUTE SHEET (RULE 26) (6) phenylCι_3alkylthio,
(7) phenylcarboxy and
(8) carboxy,
(b) aryl or arylCι_6alkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl and
(2) naphthyl, and
(c) mono and di-substituted aryl as defined above in items (1) to (2) wherein the substituents are independently selected from:
(1) halo,
(2) hydroxy,
(3) cyano,
(4) carboxy, (5) amino carbonyl,
(6) Cι_3alkyl,
(7) Cι_3fluoroalkyl,
(8) Cι_3alkylcarbonyl,
(9) Ci_3alkoxycarbonyI, (10) Ci_3alkoxy,
(11) Cι_3alkylthio,
(12) Ci_3alkylsulfonyl and
(13) deuterio; and
R5 is arylCι_8alkyl wherein aryl is selected from the group consisting of phenyl, naphthyl, pyridyl, and mono-, or disubstituted derivatives thereof, wherein the substituents are individually selected from the group consisting of: (1) halo, (2) hydroxy,
(3) cyano,
(4) carboxy,
(5) amino carbonyl,
(6) Ci.3alkyl, (7) Ci_3fluoroalkyl,
- 105 -
SUBSTΓΓUTE SHEET (RULE 26) (8) Cι_3alkylcarbonyl,
(9) Cχ_3alkoxycarbonyl,
(10) Cι_3alkoxy,
(11) Ci-3alkylthio and (12) Cι_3alkylsulfonyl.
15. A compound according to Claim 14 wherein:
Rl is selected from the group consisting of: (a) methyl,
(b) phenyl and
(c) mono- or disubstituted phenyl, wherein the substituents are selected from the group consisting of:
(1) halo and (2) deuterio; and
R5 is arylC3_5alkyl wherein aryl is selected from the group consisting of phenyl and naphthyl.
16. A compound according to Claim 15 selected from the following group:
Figure imgf000108_0001
Figure imgf000109_0001
,0
17. A compound of formula IV
- 107-
SUBSTΓΓUTE SHEET (RULE 26)
Figure imgf000110_0001
IV
or a pharmaceutically acceptable salt thereof, wherein:
R9 is selected from the group consisting of:
(a) C02H,
(b) Cθ2Cι_4alkyl,
(c) SCι_4alkyl and (d) S(0)2Ci-4alkyl;
RlO is selected from the group consisting of:
(a) H,
(b) CH2CO2H, (c) CH2Cθ2Cι_4alkyl and
(d) CH2S(O)2Cι_4alkyl; and
Ar is selected from the group consisting of:
(a) phenyl and (b) napthyl.
18. A compound according to Claim 17 wherein:
R9 is selected from the group consisting of: (a) CO2H,
(b) SCH3 and
(c) S(O)2CH3; and
RlO is selected from the group consisting of: (a) H and
(d) CH2S(O)2CH3.
19. A compound according to Claim 18 selected from the following group:
Figure imgf000111_0001
09
20. A compound according to Claim 1 which is
Figure imgf000112_0001
21. A compound selected from the following group:
(a) (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid;
(b) (4S)-4-[(2S)-2-(Acetylamino)-3-carboxypropanoyI]amino-5- [(lS)-l-([(lS)-l-(carboxymethyl)-2-oxopropyl]aminocarbonyl)-2- methylpropyl]amino-5-oxopentanoic acid;
(c) (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenylnonanoic acid;
(d) (4S)-4-[(2-S)-2-(Acetylamino)-3-carboxypropanoy 1] amino-5- [(lS)-l-([(l-S)-l-(carboxymethyl)-2-oxo-2- pheny lethyl] aminocarbonyl)-2-methylpropy 1] amino-5- oxopentanoic acid;
(e) (3S)-3-((2-S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxoheptanoic acid;
(f) (3-S)-3-((2-S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-6-phenylhexanoic acid;
- 1 10 -
SUBSTΓΓUTE SHEET (RULE 26) (g) (3-S)-3-((2-S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-8-phenyloctanoic acid;
(h) (3S)-3-((2-S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-4-oxo-5-phenylpentanoic acid;
(i) (3S)-3-((2S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(4-methoxyphenyl)-4-oxoheptanoic acid;
(j) (3S)-3-((2S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-carboxybutanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid;
(k) (3-S)-3-((2S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid;
(1) (3S)-3-((2S)-2-[((2-S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-9-phenylnonanoic acid;
(m) (3S)-3-[((2S)-2-[(2S)-2-((2S)-3-Carboxy-2-[(3,5- dibromobenzoyl)amino]propanoylamino)propanoyl]amino-3- methylbutanoyl)amino]-4-oxo-7-phenylheptanoic acid;
(n) (3S)-3-[((2S)-2-[(2S)-2-((2-S)-3-Carboxy-2-[(3,5- dideuteriobenzoyl)amino]propanoylamino)propanoyl]amino-3- methylbutanoyl)amino]-4-oxo-7-phenylheptanoic acid;
(o) (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-Amino-4-methoxy-4- oxobutanoyl]aminopropanoyl)amino]-3-methylbutanoylamino)-4- oxo-7-phenylheptanoic acid;
- I l l - (p) (3-S)-3-((2-S)-2-[((2S)-2-[(2S)-2-(Acetylamino)-4-methoxy-4- oxobutanoyl]aminopropanoyl)amino]-3-methylbutanoylamino)-4- oxo-7-phenylheptanoic acid;
(q) (3S)-3-((2S)-2-[((2S)-2-[(2-S)-2-(Acetylamino)-3- carboxypropanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid;
(r) (3-S)-3-[((2S)-2-[(2S)-2-((2S)-3-Carboxy-2-[(4- iodobenzoyl)amino]propanoylamino)propanoyl]amino-3- methylbutanoyl)amino]-4-oxo-7-phenylheptanoic acid;
(s) (3S)-3-[((2S)-2-[(2S)-2-[(2S)-2-(Acetylamino)-3- carboxypropanoyl]amino-4-(methylsulfonyl)butanoyl]amino-3- methylbutanoyl)amino]-4-oxo-7-phenylheptanoic acid;
(t) (3-S)-3-((2-S)-2-[((2-S)-2-[(2JR)-2-(Acetylamino)-3- (methylsulfanyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid;
(u) (3S)-3-((2S)-2-[((2S)-2-[(2-R)-2-(Acetylamino)-3- (methylsulfonyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-4-oxo-7-phenylheptanoic acid;
(v) (3-S)-3-((2S)-2-[((2-S)-2-[(2i?)-2-(Acetylamino)-3-
(methylsulfanyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid; and
(w) (3S)-3-((2-S)-2-[((2S)-2-[(2-R)-2-(Acetylamino)-3- (methylsulfonyl)propanoyl]aminopropanoyl)amino]-3- methylbutanoylamino)-7-(l-naphthyl)-4-oxoheptanoic acid.
22. A pharmaceutical composition comprising a compound of formula I as defined in any one of Claims 1 to 10 or 20, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
- 1 12 -
SUBSTΓΓUTE SHEET (RULE 26)
23. A method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 1 in an amount effective to treat said caspase-3 mediated disease.
24. A method of treating cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound according to Claim 1.
25. A method of treating acute disorders, including cardiac and cerebral ischemia/ reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 1 in an amount effective to treat said acute disorder.
26. A method of treating chronic disorders, including neurodegenerative diseases (e.g. Alzheimer's, polyglutamine-repeat disorders, Down's, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging, in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 1 in an amount effective to treat said chronic disorder.
27. A pharmaceutical composition comprising a compound of formula II as defined in Claim 11, 12 or 13, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
- 1 13 -
SUBSTΓΓUTE SHEET (RULE 26)
28. A method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 11 in an amount effective to treat said caspase-3 mediated disease.
29. A pharmaceutical composition comprising a compound of formula III, as defined in Claim 14, 15 or 16, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
30. A method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 14 in an amount effective to treat said caspase-3 mediated disease.
31. A pharmaceutical composition comprising a compound of formula IV, as defined in Claim 17, 18 or 19, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
32. A method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound according to Claim 17 in an amount effective to treat said caspase-3 mediated disease.
33. A caspase-3 inhibitor pharmaceutical composition comprising an acceptable caspase-3 inhibiting amount of a compound as defined in any one of Claims 1 to 21, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier.
14
34. Use of a compound as defined in any one of Claimsc 1 to 21, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cardiac and cerebral ischemia/reperfusion injury, type I diabetes, immune deficiency syndrome, cerebral and spinal chord trauma injury, organ damage during transplantation, alopecia, aging, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders.
35. A compound as defined in any one of Claims 1 to 21 or a pharmaceutically acceptable salt thereof, for use in treating caspase-3 mediated disease.
- 1 15 -
SUBSTΓΠJTE SHEET (RULE 26)
PCT/CA1999/001145 1998-12-02 1999-11-30 Gamma-ketoacid tetrapeptides as inhibitors of caspase-3 WO2000032620A1 (en)

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